Publications Leveraging Our Tools
A dual-mode, image-enhanced, miniaturized microscopy system for incubator-compatible monitoring of live cells
Imaging live cells under stable culture conditions is essential to investigate cell physiological activities and proliferation. To achieve this goal, typically, a specialized incubation chamber that creates desired culture conditions needs to be incorporated into a microscopy system to perform cell monitoring. However, such imaging systems are generally large and costly, hampering their wide applications. Recent advances in the field of miniaturized microscopy systems have enabled incubator cell monitoring, providing a hospitable environment for live cells. Although these systems are more cost-effective, they are usually limited in imaging modalities and spatial temporal resolution. Here, we present a dual-mode, image-enhanced, miniaturized microscopy system (termed MiniCube) for direct monitoring of live cells inside incubators. MiniCube enables both bright field imaging and fluorescence imaging with single-cell spatial resolution and sub-second temporal resolution. Moreover, this system can also perform cell monitoring inside the incubator with tunable time scales ranging from a few seconds to days. Meanwhile, automatic cell segmentation and image enhancement are realized by the proposed data analysis pipeline of this system, and the signal-to-noise ratio (SNR) of acquired data is significantly improved using a deep learning based image denoising algorithm. Image data can be acquired with 5 times lower light exposure while maintaining comparable SNR. The versatility of this miniaturized microscopy system lends itself to various applications in biology studies, providing a practical platform and method for studying live cell dynamics within the incubator.
Temporal Dynamics of Nucleus Accumbens Neurons in Male Mice During Reward Seeking
The nucleus accumbens (NAc) regulates reward-motivated behavior, but the temporal dynamics of NAc neurons that enable “free-willed” animals to obtain rewards remain elusive. Here, we recorded Ca2+ activity from individual NAc neurons when mice performed self-paced lever-presses for sucrose. NAc neurons exhibited three temporally-sequenced clusters, defined by times at which they exhibited increased Ca2+ activity: approximately 0, -2.5 or -5 sec relative to the lever-pressing. Dopamine D1 receptor (D1)-expressing neurons and D2-neurons formed the majority of the -5-sec versus -2.5-sec clusters, respectively, while both neuronal subtypes were represented in the 0-sec cluster. We found that pre-press activity patterns of D1- or D2-neurons could predict subsequent lever-presses. Inhibiting D1-neurons at -5 sec or D2-neurons at -2.5 sec, but not at other timepoints, reduced sucrose-motivated lever-pressing. We propose that the time-specific activity of D1- and D2-neurons mediate key temporal features of the NAc through which reward motivation initiates reward-seeking behavior.
MiniXL: An open-source, large field-of-view epifluorescence miniature microscope for mice capable of single-cell resolution and multi-brain region imaging
Capturing the intricate dynamics of neural activity in freely behaving animals is essential for understanding the neural mechanisms underpinning specific behaviors. Miniaturized microscopy enables investigators to track population activity at cellular level, but the field of view (FOV) of these microscopes have been limited and does not allow multiple-brain region imaging. To fill this technological gap, we have developed the eXtra Large field-of-view Miniscope (MiniXL), a 3.5g lightweight miniaturized microscope with an FOV measuring 3.5 mm in diameter and an electrically adjustable working distance of 1.9 mm ± 200 μm. We demonstrated the capability of MiniXL recording the activity of large neuronal population in both subcortical area (hippocampal dorsal CA1) and deep brain regions (medial prefrontal cortex, mPFC and nucleus accumbens, NAc). The large FOV allows simultaneous imaging of multiple brain regions such as bilateral mPFCs or mPFC and NAc during complex social behavior and tracking cells across multiple sessions. As with all microscopes in the UCLA Miniscope ecosystem, the MiniXL is fully open-source and will be shared with the neuroscience community to lower the barriers for adoption of this technology.
Miniscope Recording Calcium Signals at Hippocampus of Mice Navigating an Odor Plume
Mice navigate an odor plume with a complex spatiotemporal structure in the dark to find the source of odorants. This article describes a protocol to monitor behavior and record Ca2+ transients in dorsal CA1 stratum pyramidale neurons in hippocampus (dCA1) in mice navigating an odor plume in a 50 cm x 50 cm x 25 cm odor arena. An epifluorescence miniscope focused through a GRIN lens imaged Ca2+ transients in dCA1 neurons expressing the calcium sensor GCaMP6f in Thy1-GCaMP6f mice. The paper describes the behavioral protocol to train the mice to perform this odor plume navigation task in an automated odor arena. The methods include a step-by-step procedure for the surgery for GRIN lens implantation and baseplate placement for imaging GCaMP6f in CA1. The article provides information on real-time tracking of the mouse position to automate the start of the trials and delivery of a sugar water reward. In addition, the protocol includes information on using of an interface board to synchronize metadata describing the automation of the odor navigation task and frame times for the miniscope and a digital camera tracking mouse position. Moreover, the methods delineate the pipeline used to process GCaMP6f fluorescence movies by motion correction using NorMCorre followed by identification of regions of interest with EXTRACT. Finally, the paper describes an artificial neural network approach to decode spatial paths from CA1 neural ensemble activity to predict mouse navigation of the odor plume. SUMMARY This protocol describes how to investigate the brain-behavior relationship in hippocampal CA1 in mice navigating an odor plume. This article provides a step-by-step protocol, including the surgery to access imaging of the hippocampus, behavioral training, miniscope GCaMP6f recording and processing of the brain and behavioral data to decode the mouse position from ROI neural activity.
Simultaneous dual-color calcium imaging in freely-behaving mice
Miniaturized fluorescence microscopes (miniscopes) enable imaging of calcium events from a large population of neurons in freely behaving animals. Traditionally, miniscopes have only been able to record from a single fluorescence wavelength. Here, we present a new open-source dual-channel Miniscope that simultaneously records two wavelengths in freely behaving animals. To enable simultaneous acquisition of two fluorescent wavelengths, we incorporated two CMOS sensors into a single Miniscope. To validate our dual-channel Miniscope, we imaged hippocampal CA1 region that co-expressed a dynamic calcium indicator (GCaMP) and a static nuclear signal (tdTomato) while mice ran on a linear track. Our results suggest that, even when neurons were registered across days using tdTomato signals, hippocampal spatial coding changes over time. In conclusion, our novel dual-channel Miniscope enables imaging of two fluorescence wavelengths with minimal crosstalk between the two channels, opening the doors to a multitude of new experimental possibilities. Teaser Novel open-source dual-channel Miniscope that simultaneously records two wavelengths with minimal crosstalk in freely behaving animals.
Constraints on the subsecond modulation of striatal dynamics by physiological dopamine signaling
Dopaminergic neurons play a crucial role in associative learning, but their capacity to regulate behavior on subsecond timescales remains debated. It is thought that dopaminergic neurons drive certain behaviors by rapidly modulating striatal spiking activity; however, a view has emerged that only artificially high (that is, supra-physiological) dopamine signals alter behavior on fast timescales. This raises the possibility that moment-to-moment striatal spiking activity is not strongly shaped by dopamine signals in the physiological range. To test this, we transiently altered dopamine levels while monitoring spiking responses in the ventral striatum of behaving mice. These manipulations led to only weak changes in striatal activity, except when dopamine release exceeded reward-matched levels. These findings suggest that dopaminergic neurons normally play a minor role in the subsecond modulation of striatal dynamics in relation to other inputs and demonstrate the importance of discerning dopaminergic neuron contributions to brain function under physiological and potentially nonphysiological conditions.
CADENCE — Neuroinformatics Tool for Supervised Calcium Events Detection
CADENCE is an open Python 3-written neuroinformatics tool with Qt6 graphic user interface for supervised calcium events detection. In neuronal ensembles recording during calcium imaging experiments, the output of instruments such as Celena X, Zeiss LSM 5 Live confocal microscope and Miniscope is a movie showing flashing cells somata. There are few pipelines to convert video to relative fluorescence ΔF/F, from simplest ImageJ plugins to sophisticated tools like MiniAn (Dong et al. in Elife 11, https://doi.org/10.7554/eLife.70661, 2022). Minian, an open-source miniscope analysis pipeline. Elife, 11.). While in some areas of study relative fluorescence ΔF/F may be the desired result in itself, researchers of neuronal ensembles are typically interested in a more detailed analysis of calcium events as indirect proxy of neuronal electrical activity. For such analyses, researchers need a tool to infer calcium events from the continuous ΔF/F curve in order to create a raster representation of calcium events for later use in analysis software, such as Elephant (Denker, M., Yegenoglu, A., & Grün, S. (2018). Collaborative HPC-enabled workflows on the HBP Collaboratory using the Elephant framework. Neuroinformatics, 19.). Here we present such an open tool with supervised calcium events detection.
Integration of Single-Photon Miniature Fluorescence Microscopy and Electrophysiological Recording Methods for in vivo Studying Hippocampal Neuronal Activity
The miniature single-photon fluorescent microscope (miniscope)enables the visualization of calcium activity in vivo in freelymoving laboratory animals, providing the capability to track cellularactivity during the investigation of memory formation, learning,sleep, and social interactions. However, the use of calcium sensorsfor in vivo imaging is limited by their relatively slow (millisecond-scale)kinetics, which complicates the recording of high-frequency spikeactivity. The integration of methods from single-photon miniaturefluorescent microscopy with electrophysiological recording, whichpossesses microsecond resolution, represents a potential solutionto this issue. Such a combination of techniques allows for the simultaneousrecording of optical and electrophysiological activity in a singleanimal in vivo. In this study, a flexible polyimide microelectrodewas developed and integrated with the gradient lens of the miniscope.The in vivo tests conducted in this research confirmed that themicroelectrode combined with the gradient lens facilitates simultaneoussingle-photon calcium imaging and local field potential recordingin the hippocampus of an adult mouse.
Protocol for calcium imaging and analysis of hippocampal CA1 activity evoked by non-spatial stimuli
The hippocampus has a major role in processing spatial information but has been found to encode non-spatial information from multisensory modalities in recent studies. Here, we present a protocol for recording non-spatial stimuli (visual, auditory, and a combination) that evoked calcium activity of hippocampal CA1 neuronal ensembles in C57BL/6 mice using a miniaturized fluorescence microscope. We describe steps for experimental apparatus setup, surgical procedures, software development, and neuronal population activity analysis. For complete details on the use and execution of this protocol, please refer to Sun et al.1
Hippocampal and orbitofrontal neurons contribute to complementary aspects of associative structure
The ability to establish associations between environmental stimuli is fundamental for higher-order brain functions like state inference and generalization. Both the hippocampus and orbitofrontal cortex (OFC) play pivotal roles in this, demonstrating complex neural activity changes after associative learning. However, how precisely they contribute to representing learned associations remains unclear. Here, we train head-restrained mice to learn four ‘odor-outcome’ sequence pairs composed of several task variables—the past and current odor cues, sequence structure of ‘cue-outcome’ arrangement, and the expected outcome; and perform calcium imaging from these mice throughout learning. Sequence-splitting signals that distinguish between paired sequences are detected in both brain regions, reflecting associative memory formation. Critically, we uncover differential contents in represented associations by examining, in each area, how these task variables affect splitting signal generalization between sequence pairs. Specifically, the hippocampal splitting signals are influenced by the combination of past and current cues that define a particular sensory experience. In contrast, the OFC splitting signals are similar between sequence pairs that share the same sequence structure and expected outcome. These findings suggest that the hippocampus and OFC uniquely and complementarily organize the acquired associative structure.
Months-long stability of the head-direction system
Spatial orientation is a universal ability that allows animals to navigate their environment. In mammals, the head-direction (HD) system is an essential component of the brain’s navigation system, yet the stability of its underlying neuronal code remains unclear. Here, by longitudinally tracking the activity of the same HD cells in freely moving mice, we show that the internal organization of population activity in the HD system was preserved for several months. Furthermore, the HD system developed a unique mapping between its internal organization and spatial orientation in each environment. This was not affected by visits to other environments and was stabilized with experience. These findings demonstrate that stable neuronal code supports the sense of direction and forms long-lasting orientation memories.
Social valence dictates sex differences in identity recognition
Social valence is the directional emotional significance affiliated with social experiences. Maladaptive processing of negative social valence (NSV) has been linked to mood disorder susceptibility, which is more prevalent in women. To determine whether there are sex differences in NSV processing, we developed social valence tasks where the identity recognition of conspecifics with distinct valences served as the readout. Male mice demonstrated identity recognition regardless of social valence. Conversely, female mice did not show identity recognition following the NSV task. In vivo calcium imaging of the dorsal CA1 further revealed sex differences in NSV processing with reduced hippocampal representation of social information in female mice. These results suggest the imprecise encoding of NSV may contribute to the heightened vulnerability to social stress-related mood disorders in women.
Decoding the Role of Secondary Motor Cortex Neuronal Ensembles during Cocaine Self-Administration: Insights from Longitudinal in vivo Calcium Imaging via Miniscopes
Recent findings in our lab demonstrated that the risk of cocaine relapse is closely linked to the hyperexcitability of cortical pyramidal neurons in the secondary motor cortex (M2), noticeable 45 days after cocaine intravenous self-administration (IVSA). The present study was designed to explore the underlying mechanisms of neuronal alterations in M2. Our hypothesis was that M2 neurons were affected directly by cocaine taking behaviors. This hypothesis was tested by monitoring individual neuronal activity in M2 using MiniScopes for in vivo Ca2+ imaging in C57BL/6J mice when they had access to cocaine IVSA as a reinforcement (RNF) contingent to active lever press (ALP) but not to inactive lever press (ILP). With support of our established pipeline to processing Ca2+ imaging data, the current study was designed to monitor M2 neuronal ensembles at the single-neuron level in real time with high temporal resolution and high throughput in each IVSA session and longitudinally among multiple IVSA sessions. Specifically, five consecutive 1-hr daily IVSA sessions were used to model the initial cocaine taking behaviors. Besides detailed analyses of IVSA events (ALP, ILP, and RNF), the data from Ca2+ imaging recordings in M2 were analyzed by (1) comparing neuronal activation within a daily IVSA session (i.e., the first vs. the last 15 min) and between different daily sessions (i.e., the first vs. the last IVSA day), (2) associating Ca2+ transients with individual IVSA events, and (3) correlating Ca2+ transients with the cumulative effects of IVSA events. Our data demonstrated that M2 neurons are exquisitely sensitive to and significantly affected by concurrent operant behaviors and the history of drug exposure, which in turn sculpt the upcoming operant behaviors and the response to drugs. As critical nodes of the reward loop, M2 neurons appear to be the governing center orchestrating the establishment of addiction-like behaviors.
Hidden state inference requires abstract contextual representations in ventral hippocampus
The ability to form and utilize subjective, latent contextual representations to influence decision making is a crucial determinant of everyday life. The hippocampus is widely hypothesized to bind together otherwise abstract combinations of stimuli to represent such latent contexts, and to allow their use to support the process of hidden state inference. Yet, direct evidence for this remains limited. Here we show that the CA1 area of the ventral hippocampus is necessary for mice to perform hidden state inference during a 2-armed bandit task. vCA1 neurons robustly differentiate between the two abstract contexts required for this strategy in a manner similar to the differentiation of spatial locations, despite the contexts being formed only from past probabilistic outcomes. These findings offer insight into how latent contextual information is used to optimize decision-making processes, and emphasize a key role of the hippocampus in hidden state inference.
Distinct functional classes of CA1 hippocampal interneurons are modulated by cerebellar stimulation in a coordinated manner
There is mounting evidence that the cerebellum impacts hippocampal functioning, but the impact of the cerebellum on hippocampal interneurons remains obscure. Using miniscopes in freely behaving animals, we find optogenetic stimulation of Purkinje cells alters the calcium activity of a large percentage of CA1 interneurons. This includes both increases and decreases in activity. Remarkably, this bidirectional impact occurs in a coordinated fashion, in line with interneurons’ functional properties. Specifically, CA1 interneurons activated by cerebellar stimulation are commonly locomotion-active, while those inhibited by cerebellar stimulation are commonly rest-active interneurons. We additionally find that subsets of CA1 interneurons show altered activity during object investigations, suggesting a role in the processing of objects in space. Importantly, these neurons also show coordinated modulation by cerebellar stimulation: CA1 interneurons that are activated by cerebellar stimulation are more likely to be activated, rather than inhibited, during object investigations, while interneurons that show decreased activity during cerebellar stimulation show the opposite profile. Therefore, CA1 interneurons play a role in object processing and in cerebellar impacts on the hippocampus, providing insight into previously noted altered CA1 processing of objects in space with cerebellar stimulation. We examined two different stimulation locations (IV/V Vermis; Simplex) and two different stimulation approaches (7Hz or a single 1s light pulse) – in all cases, the cerebellum induces similar coordinated CA1 interneuron changes congruent with an explorative state. Overall, our data show that the cerebellum impacts CA1 interneurons in a bidirectional and coordinated fashion, positioning them to play an important role in cerebello-hippocampal communication. Significance Statement Acute manipulation of the cerebellum can affect the activity of cells in CA1, and perturbing normal cerebellar functioning can affect hippocampal-dependent spatial processing, including the processing of objects in space. Despite the importance of interneurons on the local hippocampal circuit, it was unknown how cerebellar activation impacts CA1 inhibitory neurons. We find that stimulating the cerebellum robustly affects multiple populations of CA1 interneurons in a bidirectional, coordinated manner, according to their functional profiles during behavior, including locomotion and object investigations. Our work also provides support for a role of CA1 interneurons in spatial processing of objects, with populations of interneurons showing altered activity during object investigations.
Cholecystokinin facilitates motor skill learning by modulating neuroplasticity in the motor cortex
Cholecystokinin (CCK) is an essential modulator for neuroplasticity in sensory and emotional domains. Here, we investigated the role of CCK in motor learning using a single pellet reaching task in mice. Mice with a knockout of Cck gene (Cck−/−) or blockade of CCK-B receptor (CCKBR) showed defective motor learning ability; the success rate of retrieving reward remained at the baseline level compared to the wildtype mice with significantly increased success rate. We observed no long-term potentiation upon high-frequency stimulation in the motor cortex of Cck−/− mice, indicating a possible association between motor learning deficiency and neuroplasticity in the motor cortex. In vivo calcium imaging demonstrated that the deficiency of CCK signaling disrupted the refinement of population neuronal activity in the motor cortex during motor skill training. Anatomical tracing revealed direct projections from CCK-expressing neurons in the rhinal cortex to the motor cortex. Inactivation of the CCK neurons in the rhinal cortex that project to the motor cortex bilaterally using chemogenetic methods significantly suppressed motor learning, and intraperitoneal application of CCK4, a tetrapeptide CCK agonist, rescued the motor learning deficits of Cck−/− mice. In summary, our results suggest that CCK, which could be provided from the rhinal cortex, may surpport motor skill learning by modulating neuroplasticity in the motor cortex.
Toward a brighter constellation: multiorgan neuroimaging of neural and vascular dynamics in the spinal cord and brain
SignificancePain comprises a complex interaction between motor action and somatosensation that is dependent on dynamic interactions between the brain and spinal cord. This makes understanding pain particularly challenging as it involves rich interactions between many circuits (e.g., neural and vascular) and signaling cascades throughout the body. As such, experimentation on a single region may lead to an incomplete and potentially incorrect understanding of crucial underlying mechanisms.AimWe aimed to develop and validate tools to enable detailed and extended observation of neural and vascular activity in the brain and spinal cord. The first key set of innovations was targeted to developing novel imaging hardware that addresses the many challenges of multisite imaging. The second key set of innovations was targeted to enabling bioluminescent (BL) imaging, as this approach can address limitations of fluorescent microscopy including photobleaching, phototoxicity, and decreased resolution due to scattering of excitation signals.ApproachWe designed 3D-printed brain and spinal cord implants to enable effective surgical implantations and optical access with wearable miniscopes or an open window (e.g., for one- or two-photon microscopy or optogenetic stimulation). We also tested the viability for BL imaging and developed a novel modified miniscope optimized for these signals (BLmini).ResultsWe describe “universal” implants for acute and chronic simultaneous brain–spinal cord imaging and optical stimulation. We further describe successful imaging of BL signals in both foci and a new miniscope, the “BLmini,” which has reduced weight, cost, and form-factor relative to standard wearable miniscopes.ConclusionsThe combination of 3D-printed implants, advanced imaging tools, and bioluminescence imaging techniques offers a coalition of methods for understanding spinal cord–brain interactions. Our work has the potential for use in future research into neuropathic pain and other sensory disorders and motor behavior.
Excitatory neurons of the anterior cingulate cortex encode chosen actions and their outcomes rather than cognitive state
The anterior cingulate cortex (ACC) causally influences cognitive control of goal-directed behaviour. However, it is unclear whether ACC directly encodes cognitive variables like attention or impulsivity, or implements goal-directed action selection mechanisms that are modulated by them. We recorded ACC activity with miniature endoscopic microscopes in mice performing the 5-choice-serial-reaction time task, and applied decoding and encoding analyses. ACC pyramidal cells represented specific actions before and during the behavioural response, whereas the response type (e.g. correct/incorrect/premature) – indicating the state of attentional and impulse control – could only be decoded during and after the response with high reliability. Devaluation and extinction experiments further revealed that action encoding depended on reward expectation. Our findings support a role for ACC in goal-directed action selection and monitoring, that is modulated by cognitive state, rather than in tracking levels of attention or impulsivity directly in individual trials.
Targeted micro-fiber arrays for measuring and manipulating localized multi-scale neural dynamics over large, deep brain volumes during behavior
Distinct changes to hippocampal and medial entorhinal circuits emerge across the progression of cognitive deficits in epilepsy
Temporal lobe epilepsy (TLE) causes pervasive and progressive memory impairments, yet the specific circuit changes that drive these deficits remain unclear. To investigate how hippocampal-entorhinal dysfunction contributes to progressive memory deficits in epilepsy, we performed simultaneous in vivo electrophysiology in hippocampus (HPC) and medial entorhinal cortex (MEC) of control and epileptic mice 3 or 8 weeks after pilocarpine-induced status epilepticus (Pilo-SE). We found that HPC synchronization deficits (including reduced theta power, coherence, and altered interneuron spike timing) emerged within 3 weeks of Pilo-SE, aligning with early-onset, relatively subtle memory deficits. In contrast, abnormal synchronization within MEC and between HPC-MEC emerged later, by 8 weeks after Pilo-SE, when spatial memory impairment was more severe. Furthermore, a distinct subpopulation of MEC layer 3 excitatory neurons (active at theta troughs) was specifically impaired in epileptic mice. Together, these findings suggest that hippocampal-entorhinal circuit dysfunction accumulates and shifts as cognitive impairment progresses in TLE.
Neural coding in gustatory cortex reflects consumption decisions: Evidence from conditioned taste aversion
Taste-responsive neurons in the gustatory cortex (GC) have been shown to encode multiple properties of stimuli, including whether they are palatable or not. Previous studies have suggested that a form of taste-involved learning, conditioned taste aversion (CTA), may alter the cortical representation of taste stimuli in a number of ways. We used miniscopes to image taste responses from a large population of neurons in the gustatory cortex of mice before and after CTA to NaCl, comparing taste responses in control and conditioned mice. Following conditioning, no significant effects on the number of responsive cells, or the magnitude of response to either NaCl or other taste stimuli were found. However, population-level analyses showed that in mice receiving a CTA, the representation of NaCl diverged from other appetitive stimuli in neural space and moved closer to that of aversive quinine. We also tracked extinction of the CTA in a subset of animals and showed that as NaCl became less aversive, the neural pattern reverted to match the behavior. These data suggest that the predominant function of the taste representation in GC is palatability; the neuronal response pattern to stimuli at the population level reflects the decision of the animal to consume or not consume the stimulus, regardless of quality or chemical identity.
Subicular neurons encode concave and convex geometries
Animals in the natural world constantly encounter geometrically complex landscapes. Successful navigation requires that they understand geometric features of these landscapes, including boundaries, landmarks, corners and curved areas, all of which collectively define the geometry of the environment1–12. Crucial to the reconstruction of the geometric layout of natural environments are concave and convex features, such as corners and protrusions. However, the neural substrates that could underlie the perception of concavity and convexity in the environment remain elusive. Here we show that the dorsal subiculum contains neurons that encode corners across environmental geometries in an allocentric reference frame. Using longitudinal calcium imaging in freely behaving mice, we find that corner cells tune their activity to reflect the geometric properties of corners, including corner angles, wall height and the degree of wall intersection. A separate population of subicular neurons encode convex corners of both larger environments and discrete objects. Both corner cells are non-overlapping with the population of subicular neurons that encode environmental boundaries. Furthermore, corner cells that encode concave or convex corners generalize their activity such that they respond, respectively, to concave or convex curvatures within an environment. Together, our findings suggest that the subiculum contains the geometric information needed to reconstruct the shape and layout of naturalistic spatial environments.
Stable sequential dynamics in prefrontal cortex represents subjective estimation of time
Time estimation is an essential prerequisite underlying various cognitive functions. Previous studies identified “sequential firing” and “activity ramps” as the primary neuron activity patterns in the medial frontal cortex (mPFC) that could convey information regarding time. However, the relationship between these patterns and the timing behavior has not been fully understood. In this study, we utilized in vivo calcium imaging of mPFC in rats performing a timing task. By aligning long-term time-lapse datasets, we discovered that sequential patterns of time coding were stable over weeks, while cells with ramping activity patterns showed constant dynamism. Furthermore, with a novel behavior design that allowed the animal to determine individual trial interval, we were able to demonstrate that real-time adjustment in the sequence procession speed closely tracked the trial-to-trial interval variations. And errors in the rats’ timing behavior can be primarily attributed to the premature ending of the time sequence. Together, our data suggest that sequential activity might be a more relavent coding regime than the ramping activity in representing time under physiological conditions. Furthermore, our results imply the existence of a unique cell type in the mPFC that participates in the time-related sequences. Future characterization of this cell type could provide important insights in the neural mechanism of timing and related cognitive functions.
A population code for idiothetic representations in the hippocampal-septal circuit
The hippocampus is a higher-order brain structure responsible for encoding new episodic memories and predicting future outcomes. In absence of external stimuli, neurons in the hippocampus express sequential activities which have been proposed to support path integration by tracking elapsed time, distance traveled, and other idiothetic variables. On the other hand, with sufficient external sensory inputs, hippocampal neurons can fire with respect to allocentric cues. Previously, these idiothetic codes have been described in conditions where running speed is clamped experimentally. To this day, the exact determinants of idiothetic representations during free navigation remain unclear. Additionally, whether CA1 and CA3 temporal and distance codes are transmitted downstream to the lateral septum has not been established. Here, we develop an unsupervised model trained to compress neural information with minimal loss, and find that we can efficiently decode elapsed time and distance traveled from low-dimensional embeddings of neural activity in freely moving mice. We also developed unbiased information metrics that are minimally sensitive to quantization parameters and enable comparisons across modalities and brain regions. In more than 30,000 CA1 pyramidal neurons, we show that spatiotemporal information is represented as a mixture of idiothetic and allocentric information, the balance of which is dictated by task demand and environmental conditions. In particular, we find that a subset of CA1 pyramidal neurons encode the spatiotemporal distance to rewards. Single cell and population statistics across the hippocampal-septal circuit reveal that idiothetic variables emerge in CA1 and are integrated postsynaptically in the lateral septum. Finally, we implement a computational model trained to replicate real world neural activity, and find that grid cells could provide a plausible input for CA1 representations of time and distance. Altogether, our results suggest that hippocampal CA1 continuously integrates both idiothetic and allocentric signals depending on task demand and available cues, and these high-level representations are effectively transmitted to downstream regions.
Correlated signatures of social behavior in cerebellum and anterior cingulate cortex
The cerebellum has been implicated in the regulation of social behavior. Its influence is thought to arise from communication, via the thalamus, to forebrain regions integral in the expression of social interactions, including the anterior cingulate cortex (ACC). However, the signals encoded or the nature of the communication between the cerebellum and these brain regions is poorly understood. Here, we describe an approach that overcomes technical challenges in exploring the coordination of distant brain regions at high temporal and spatial resolution during social behavior. We developed the E-Scope, an electrophysiology-integrated miniature microscope, to synchronously measure extracellular electrical activity in the cerebellum along with calcium imaging of the ACC. This single coaxial cable device combined these data streams to provide a powerful tool to monitor the activity of distant brain regions in freely behaving animals. During social behavior, we recorded the spike timing of multiple single units in cerebellar right Crus I (RCrus I) Purkinje cells (PCs) or dentate nucleus (DN) neurons while synchronously imaging calcium transients in contralateral ACC neurons. We found that during social interactions a significant subpopulation of cerebellar PCs were robustly inhibited, while most modulated neurons in the DN were activated, and their activity was correlated with positively modulated ACC neurons. These distinctions largely disappeared when only non-social epochs were analyzed suggesting that cerebellar-cortical interactions were behaviorally specific. Our work provides new insights into the complexity of cerebellar activation and co-modulation of the ACC during social behavior and a valuable open-source tool for simultaneous, multimodal recordings in freely behaving mice.
Experience and behavior modulate piriform cortex odor representation in freely moving mice
In rodents, activity in the piriform cortex (PC) has been shown to reliably encode the identity of olfactory information within single sessions of odor delivery. However, recent evidence from chronic PC recordings found significant unreliability in this ensemble code over longer periods. The causes of this phenomenon, termed representational drift, are still being investigated across multiple sensory systems, but prior work has suggested a role for animal behavior in this observed unreliability of coding. To explore this possibility in PC, we recorded from the same populations of neurons in freely-moving, awake mice using micro-endoscopic calcium imaging as they gained passive experience with a panel of odorants over 5 consecutive days. As in prior studies, PC odor responses within a single session could be used to accurately decode odor identity. However, responses became less consistent across days of experience as odor-evoked response properties of the neurons shifted with experience. During these recordings, within and across sessions, decreases in olfactory investigative behavior correlated with decreased odor-evoked response from PC neurons. Similarly, decreases in odor investigation correlated with a decrease in representational consistency, and trials with greater odor investigation could be used to decode odor identity from PC neurons more accurately over time. Overall, this data supports recent evidence of long-term shifts in the ensembles of PC neurons encoding odor-identity (drift) but supports a role for behavioral modulation of overall PC activity and ensemble response consistency.
On Optimizing Miniscope Data Analysis with Simulated Data: A Study of Parameter Optimization in the Minian Analysis Pipeline
In vivo calcium imaging is widely used in neuroscience to assess the activity of neuronal ensembles. The advent of the single-photon miniature fluorescence microscope (miniscope) has made it possible to use intravital calcium imaging in freely moving animals. Various algorithms and analysis packages have been developed to analyze miniscope data. The present work uses model data with different noise levels as an example to examine the relationship between the accuracy of neuron detection and the values of parameters in Minian, a package for analyzing miniscope data. On the basis of the results obtained, recommendations are given for changing the values of the Minian parameters depending on the noise level in the processed data. The results obtained here provide preliminary guidance for selecting appropriate values for Minian parameters for processing experimental data. The results of this study are expected to be relevant to neuroscientists using intravital calcium imaging in freely moving animals.
Cortical regulation of helping behaviour towards others in pain
Humans and animals exhibit various forms of prosocial helping behaviour towards others in need1–3. Although previous research has investigated how individuals may perceive others’ states4,5, the neural mechanisms of how they respond to others’ needs and goals with helping behaviour remain largely unknown. Here we show that mice engage in a form of helping behaviour towards other individuals experiencing physical pain and injury—they exhibit allolicking (social licking) behaviour specifically towards the injury site, which aids the recipients in coping with pain. Using microendoscopic imaging, we found that single-neuron and ensemble activity in the anterior cingulate cortex (ACC) encodes others’ state of pain and that this representation is different from that of general stress in others. Furthermore, functional manipulations demonstrate a causal role of the ACC in bidirectionally controlling targeted allolicking. Notably, this behaviour is represented in a population code in the ACC that differs from that of general allogrooming, a distinct type of prosocial behaviour elicited by others’ emotional stress. These findings advance our understanding of the neural coding and regulation of helping behaviour.
Prefrontal multistimulus integration within a dedicated disambiguation circuit guides interleaving contingency judgment learning
Understanding how cortical network dynamics support learning is a challenge. This study investigates the role of local neural mechanisms in the prefrontal cortex during contingency judgment learning (CJL). To better understand brain network mechanisms underlying CJL, we introduced ambiguity into associative learning after fear acquisition, inducing a generalized fear response to an ambiguous stimulus sharing nontrivial similarities with the conditioned stimulus. Real-time recordings at single-neuron resolution from the prelimbic (PL) cortex using miniature microscopes revealed distinct PL network dynamics across CJL phases. Fear acquisition triggered PL network reorganization, led by a disambiguation circuit managing spurious and predictive relationships during cue–danger, cue–safety, and cue–neutrality contingencies. Subjects with PL-targeted memory deficiency showed malfunctioning disambiguation circuit function, while naive subjects lacking unconditioned stimulus exposure lacked a CJL-specific disambiguation circuit. This study uncovers that fear conditioning induces prefrontal cortex cognitive map reorganization, and subsequent CJL relies on the disambiguation circuit's ability to learn predictive relationships.
Hippocampal cognitive and relational map paradigms explored by multisensory encoding recording with wide-field calcium imaging
Syntalos: A software for precise simultaneous multi-modal data acquisition and closed-loop interventions
Complex experimental protocols often require multi-modal data acquisition with precisely aligned timing, as well as state- and behavior-dependent interventions. Tailored solutions are mostly restricted to individual experimental setups and lack flexibility and interoperability. We present an integrated software solution, called ‘Syntalos’, for simultaneous acquisition of data from an arbitrary number of sources, including multi-channel electrophysiological recordings and different live imaging devices, as well as closed-loop, real-time interventions with different actuators. Precisely matching timestamps for all inputs are ensured by continuous statistical analysis and correction of individual devices’ timestamps. New data sources can be integrated with minimal programming skills. Data is stored in a comprehensively structured format to facilitate pooling or sharing data between different laboratories. Syntalos enables precisely synchronized multi-modal recordings as well as closed-loop interventions for multiple experimental approaches. Preliminary experiments with different research questions show the successful performance and easy-to-learn structure of the software suite.
Top-down control of flight by a non-canonical cortico-amygdala pathway
Survival requires the selection of appropriate behaviour in response to threats, and dysregulated defensive reactions are associated with psychiatric illnesses such as post-traumatic stress and panic disorder1. Threat-induced behaviours, including freezing and flight, are controlled by neuronal circuits in the central amygdala (CeA)2; however, the source of neuronal excitation of the CeA that contributes to high-intensity defensive responses is unknown. Here we used a combination of neuroanatomical mapping, in vivo calcium imaging, functional manipulations and electrophysiology to characterize a previously unknown projection from the dorsal peduncular (DP) prefrontal cortex to the CeA. DP-to-CeA neurons are glutamatergic and specifically target the medial CeA, the main amygdalar output nucleus mediating conditioned responses to threat. Using a behavioural paradigm that elicits both conditioned freezing and flight, we found that CeA-projecting DP neurons are activated by high-intensity threats in a context-dependent manner. Functional manipulations revealed that the DP-to-CeA pathway is necessary and sufficient for both avoidance behaviour and flight. Furthermore, we found that DP neurons synapse onto neurons within the medial CeA that project to midbrain flight centres. These results elucidate a non-canonical top-down pathway regulating defensive responses.
MiniMounter: A low-cost miniaturized microscopy development toolkit for image quality control and enhancement
Head-mounted miniaturized fluorescence microscopy (Miniscope) has emerged as a significant tool in neuroscience, particularly for behavioral studies in awake rodents. However, the challenges of image quality control and standardization persist for both Miniscope users and developers. In this study, we propose a cost-effective and comprehensive toolkit named MiniMounter. This toolkit comprises a hardware platform that offers customized grippers and four-degree-of-freedom adjustment for Miniscope, along with software that integrates displacement control, image quality evaluation, and enhancement of 3D visualization. Our toolkit makes it feasible to accurately characterize Miniscope. Furthermore, MiniMounter enables auto-focusing and 3D imaging for Miniscope prototypes that possess solely a 2D imaging function, as demonstrated in phantom and animal experiments. Overall, the implementation of MiniMounter effectively enhances image quality, reduces the time required for experimental operations and image evaluation, and consequently accelerates the development and research cycle for both users and developers within the Miniscope community.
Understanding Fear and Beyond in Neuronal Networks with Tensor and Graph Methods: An Interdisciplinary End-to-End Data Science Approach
Understanding fear and where it is generated, modulated, and interpreted is paramount when trying to develop our knowledge of anxiety and stressor type disorders and how they develop. We use calcium imaging as a method to record real-time neuronal network activity from a precise region of interest in freely behaving mice. However, a continual challenge with calcium imaging methods is (1) the background noise and artifacts within the video recordings, (2) the large amounts of data that need to be processed and analyzed for underlying structure, and (3) the inability to label our data to provide an interpretable output. To address the problem of video quality and processing, we employ several algorithms to denoise, demix, and extract valid data. Using tensor decomposition and community analysis in independent analyses, we found three non-orthogonal (tensor decomposition) and orthogonal (community analysis) populations of neurons that encoded information for different aspects of the behavioral paradigms, including a habituation population, a discrimination population responsive to the novel safe and threat environment presentations, and a familiar control environment exposure population. Overall, these results are informative of the neuronal activity modulation occurring within the recorded region of interest and provide valuable insight into the different populations of neurons that are selectively co-active during different trials and environment presentations.
Vagal interoception of microbial metabolites from the small intestinal lumen
The vagus nerve is proposed to enable communication between the gut microbiome and brain, but activity-based evidence is lacking. Herein, we assess the extent of gut microbial influences on afferent vagal activity and metabolite signaling mechanisms involved. We find that mice reared without microbiota (germ-free, GF) exhibit decreased vagal afferent tone relative to conventionally colonized mice (specific pathogen-free, SPF), which is reversed by colonization with SPF microbiota. Perfusing non-absorbable antibiotics (ABX) into the small intestine of SPF mice, but not GF mice, acutely decreases vagal activity, which is restored upon re-perfusion with bulk lumenal contents or sterile filtrates from the small intestine and cecum of SPF, but not GF, mice. Of several candidates identified by metabolomic profiling, microbiome-dependent short-chain fatty acids, bile acids, and 3-indoxyl sulfate stimulate vagal activity with varied response kinetics, which is blocked by co-perfusion of pharmacological antagonists of FFAR2, TGR5, and TRPA1, respectively, into the small intestine. At the single-unit level, serial perfusion of each metabolite class elicits more singly responsive neurons than dually responsive neurons, suggesting distinct neuronal detection of different microbiome- and macronutrient- dependent metabolites. Finally, microbial metabolite-induced increases in vagal activity correspond with activation of neurons in the nucleus of the solitary tract, which is also blocked by co-administration of their respective receptor antagonists. Results from this study reveal that the gut microbiome regulates select metabolites in the intestinal lumen that differentially activate chemosensory vagal afferent neurons, thereby enabling microbial modulation of interoceptive signals for gut-brain communication.
Vagal interoception of microbial metabolites from the small intestinal lumen
The vagus nerve is proposed to enable communication between the gut microbiome and brain, but activity-based evidence is lacking. Herein, we assess the extent of gut microbial influences on afferent vagal activity and metabolite signaling mechanisms involved. We find that mice reared without microbiota (germ-free, GF) exhibit decreased vagal afferent tone relative to conventionally colonized mice (specific pathogen-free, SPF), which is reversed by colonization with SPF microbiota. Perfusing non-absorbable antibiotics (ABX) into the small intestine of SPF mice, but not GF mice, acutely decreases vagal activity, which is restored upon re-perfusion with bulk lumenal contents or sterile filtrates from the small intestine and cecum of SPF, but not GF, mice. Of several candidates identified by metabolomic profiling, microbiome-dependent short-chain fatty acids, bile acids, and 3-indoxyl sulfate stimulate vagal activity with varied response kinetics, which is blocked by co-perfusion of pharmacological antagonists of FFAR2, TGR5, and TRPA1, respectively, into the small intestine. At the single-unit level, serial perfusion of each metabolite class elicits more singly responsive neurons than dually responsive neurons, suggesting distinct neuronal detection of different microbiome- and macronutrient-dependent metabolites. Finally, microbial metabolite-induced increases in vagal activity correspond with activation of neurons in the nucleus of the solitary tract, which is also blocked by co-administration of their respective receptor antagonists. Results from this study reveal that the gut microbiome regulates select metabolites in the intestinal lumen that differentially activate chemosensory vagal afferent neurons, thereby enabling microbial modulation of interoceptive signals for gut-brain communication. HIGHLIGHTSMicrobiota colonization status modulates afferent vagal nerve activityGut microbes differentially regulate metabolites in the small intestine and cecumSelect microbial metabolites stimulate vagal afferents with varied response kineticsSelect microbial metabolites activate vagal afferent neurons and brainstem neurons via receptor-dependent signaling
Cortical processing of pain and itch information by distinct neuronal populations
Pain and itch perception both evoke aversive but qualitatively different feelings. The transmission pathways and brain areas that process pain and itch are related, with the anterior cingulate cortex (ACC) being important for the affective dimension of both sensations. The cellular mechanisms by which these two somatosensory stimuli are processed in the same brain area, however, remain largely unknown. Here we identified distinct neuronal populations related to pain and itch processing in layer II/III of the ACC. These include neurons activated by both itch and pain stimuli separated by a short time interval and modality-specific neurons activated only by either itch or pain stimuli regardless of the interval between them. Using the dual-eGRASP (enhanced green fluorescent protein reconstitution across synaptic partners) technique, we found that pain- and itch-specific neurons preferentially receive synaptic connections from mediodorsal thalamic neurons activated by pain and itch stimuli, respectively. Using an inhibitory designer receptor exclusively activated by a designer drug (DREADD), we found that although suppressing itch- or pain-specific neurons reduced pruriception or nociception, respectively, neither type of inhibition affected the opposite modality. Together, these results indicate that the processing of itch and pain information in the ACC involves activity-dependent and modality-specific neuronal populations, and that pain and itch are processed by functionally distinct ACC neuronal subsets.
Current Status and Future Strategies for Advancing Functional Circuit Mapping In Vivo
The human brain represents one of the most complex biological systems, containing billions of neurons interconnected through trillions of synapses. Inherent to the brain is a biochemical complexity involving ions, signaling molecules, and peptides that regulate neuronal activity and allow for short- and long-term adaptations. Large-scale and noninvasive imaging techniques, such as fMRI and EEG, have highlighted brain regions involved in specific functions and visualized connections between different brain areas. A major shortcoming, however, is the need for more information on specific cell types and neurotransmitters involved, as well as poor spatial and temporal resolution. Recent technologies have been advanced for neuronal circuit mapping and implemented in behaving model organisms to address this. Here, we highlight strategies for targeting specific neuronal subtypes, identifying, and releasing signaling molecules, controlling gene expression, and monitoring neuronal circuits in real-time in vivo. Combined, these approaches allow us to establish direct causal links from genes and molecules to the systems level and ultimately to cognitive processes.
Advances in cellular resolution microscopy for brain imaging in rats
Rats are used in neuroscience research because of their physiological similarities with humans and accessibility as model organisms, trainability, and behavioral repertoire. In particular, rats perform a wide range of sophisticated social, cognitive, motor, and learning behaviors within the contexts of both naturalistic and laboratory environments. Further progress in neuroscience can be facilitated by using advanced imaging methods to measure the complex neural and physiological processes during behavior in rats. However, compared with the mouse, the rat nervous system offers a set of challenges, such as larger brain size, decreased neuron density, and difficulty with head restraint. Here, we review recent advances in in vivo imaging techniques in rats with a special focus on open-source solutions for calcium imaging. Finally, we provide suggestions for both users and developers of in vivo imaging systems for rats.
A manifold neural population code for space in hippocampal coactivity dynamics independent of place fields
Hippocampus place cell discharge is temporally unreliable across seconds and days, and place fields are multimodal, suggesting an “ensemble cofiring” spatial coding hypothesis with manifold dynamics that does not require reliable spatial tuning, in contrast to hypotheses based on place field (spatial tuning) stability. We imaged mouse CA1 (cornu ammonis 1) ensembles in two environments across three weeks to evaluate these coding hypotheses. While place fields “remap,” being more distinct between than within environments, coactivity relationships generally change less. Decoding location and environment from 1-s ensemble location-specific activity is effective and improves with experience. Decoding environment from cell-pair coactivity relationships is also effective and improves with experience, even after removing place tuning. Discriminating environments from 1-s ensemble coactivity relies crucially on the cells with the most anti-coactive cell-pair relationships because activity is internally organized on a low-dimensional manifold of non-linear coactivity relationships that intermittently reregisters to environments according to the anti-cofiring subpopulation activity.
Identifying representational structure in CA1 to benchmark theoretical models of cognitive mapping
Decades of theoretical and empirical work have suggested the hippocampus instantiates some form of a cognitive map, yet tests of competing theories have been limited in scope and largely qualitative in nature. Here, we develop a novel framework to benchmark model predictions against observed neuronal population dynamics as animals navigate a series of geometrically distinct environments. In this task space, we show a representational structure that generalizes across brains, effectively constrains biologically viable model parameters, and discriminates between competing theories of cognitive mapping.
A Novel Design Method of Uniformity Energy Distribution Lens for Miniscopes
Objective: Calcium imaging is an essential tool for obtaining neuroactivities to understand the complex function of the brain. The one-photon miniscope, the most widely used endoscope in neuroscience, is employed to record neuronal calcium activities in vivo. However, the current half ball lens overlooks the energy distribution in the brain field, resulting in non-uniform fluorescence imaging and the omission of important signals, leading to misunderstanding in brain science. The main flaw in the existing lens is the lack of controlled energy distribution, where the center fluorescence is much stronger than the edge area, causing the fluorescence noisy background to overlap with numerous neuron signals. To address this issue, we propose a novel approach to simplify the optical path by setting a medial target. By combining the Ray Mapping Method and Energy Feedback Method, we design and optimize the lens based on the energy mapping mesh. Simulation results demonstrate a 2.1-fold enhancement in energy distribution uniformity in the brain field compared to the original half-ball lens, with edge neurons receiving 6.95 times more energy. This novel design is theoretically proved to be an innovative improvement of the one-photon miniscope method, being a potential breakthrough of this mainstream method lasting more than ten years.
Representational similarity supports rapid context retrieval during fear discrimination in ventral hippocampus
The dorsal and ventral regions of the hippocampus (dCA1 and vCA1) are critical for contextual fear conditioning tasks, yet the dynamics of hippocampal neuronal representations during memory acquisition, retrieval, and extinction processes have yet to be fully elucidated. The canonical theory is that the hippocampus generates and retrieves spatial maps of contexts during memory acquisition and retrieval. It is hypothesized the hippocampus prevents memory interference by generating context representations that are dissimilar and the prediction follows that representation dissimilarity facilitates discrimination between contexts. Here, we developed a context fear memory retrieval task and combined it with 1-photon neuronal imaging in dCA1 and vCA1 of freely behaving mice to test this prediction. We identified several phenomena specific to vCA1. First, fear conditioning induced an immediate and strong representational change that was predictive of freezing behavior. During context discrimination, vCA1 representations of the threatening and neutral contexts became more similar. Third, during threatening context memory retrieval, vCA1 expressed rapid and strong context representations. These unexpected results suggest that representational similarity in vCA1 facilitates faster and more efficient network state transitions. In further support of this view, both phenomena of representational similarity and rapid context recall were no longer observed in vCA1 once fear behavior was extinguished. Together, these results reveal that vCA1 unexpectedly generates similar population codes, promoting faster transitions between network states essential for contextual fear memory retrieval. HighlightsWe established a novel context teleportation task that allows population level hippocampal recordings during key moments of threat memory acquisition, retrieval, and extinction.The ventral region of CA1 (vCA1) exhibited the greatest change in neural representation during fear memory acquisition, compared to dorsal CA1 (dCA1).After fear conditioning, representations for threatening and neutral contexts were more similar in vCA1 compared to dCA1, yet representational similarity in vCA1 supported rapid context memory retrieval.Context fear memory extinction reversed the context representation in vCA1 to patterns observed prior to fear conditioning. Download figureOpen in new tab
Unraveling the mechanisms of deep-brain stimulation of the internal capsule in a mouse model
Deep-brain stimulation (DBS) is an effective treatment for patients suffering from otherwise therapy-resistant psychiatric disorders, including obsessive-compulsive disorder. Modulation of cortico-striatal circuits has been suggested as a mechanism of action. To gain mechanistic insight, we monitored neuronal activity in cortico-striatal regions in a mouse model for compulsive behavior, while systematically varying clinically-relevant parameters of internal-capsule DBS. DBS showed dose-dependent effects on both brain and behavior: An increasing, yet balanced, number of excited and inhibited neurons was recruited, scattered throughout cortico-striatal regions, while excessive grooming decreased. Such neuronal recruitment did not alter basic brain function such as resting-state activity, and only occurred in awake animals, indicating a dependency on network activity. In addition to these widespread effects, we observed specific involvement of the medial orbitofrontal cortex in therapeutic outcomes, which was corroborated by optogenetic stimulation. Together, our findings provide mechanistic insight into how DBS exerts its therapeutic effects on compulsive behaviors.
A unified open-source platform for multimodal neural recording and perturbation during naturalistic behavior
Behavioral neuroscience faces two conflicting demands: long-duration recordings from large neural populations and unimpeded animal behavior. To meet this challenge, we developed ONIX, an open-source data acquisition system with high data throughput (2GB/sec) and low closed-loop latencies (<1ms) that uses a novel 0.3 mm thin tether to minimize behavioral impact. Head position and rotation are tracked in 3D and used to drive active commutation without torque measurements. ONIX can acquire from combinations of passive electrodes, Neuropixels probes, head-mounted microscopes, cameras, 3D-trackers, and other data sources. We used ONIX to perform uninterrupted, long (∼7 hours) neural recordings in mice as they traversed complex 3-dimensional terrain. ONIX allowed exploration with similar mobility as non-implanted animals, in contrast to conventional tethered systems which restricted movement. By combining long recordings with full mobility, our technology will enable new progress on questions that require high-quality neural recordings during ethologically grounded behaviors.
Learning-prolonged maintenance of stimulus information in CA1 and subiculum during trace fear conditioning
Aversive experience drives offline ensemble reactivation to link memories across days
Memories are encoded in neural ensembles during learning and stabilized by post-learning reactivation. Integrating recent experiences into existing memories ensures that memories contain the most recently available information, but how the brain accomplishes this critical process remains unknown. Here we show that in mice, a strong aversive experience drives the offline ensemble reactivation of not only the recent aversive memory but also a neutral memory formed two days prior, linking the fear from the recent aversive memory to the previous neutral memory. We find that fear specifically links retrospectively, but not prospectively, to neutral memories across days. Consistent with prior studies, we find reactivation of the recent aversive memory ensemble during the offline period following learning. However, a strong aversive experience also increases co-reactivation of the aversive and neutral memory ensembles during the offline period. Finally, the expression of fear in the neutral context is associated with reactivation of the shared ensemble between the aversive and neutral memories. Taken together, these results demonstrate that strong aversive experience can drive retrospective memory-linking through the offline co-reactivation of recent memory ensembles with memory ensembles formed days prior, providing a neural mechanism by which memories can be integrated across days.
Neural Networks Are Tuned Near Criticality During a Cognitive Task and Distanced from Criticality In a Psychopharmacological Model of Alzheimer’s Disease
Dynamical systems that exhibit transitions between ordered and disordered states are described as “critical” when the system is at the borderline between these states. The ability of criticality to explain a variety of brain properties, including optimal information processing, makes it of considerable interest to investigate whether these in vivo networks display critical behaviour, and whether some forms of cognitive impairment such as dementia might display altered critical behaviour. To investigate these questions, the activity of several hundred hippocampal CA1 neurons in freely-behaving mice was studied with miniscope widefield calcium imaging during rest, a novel object recognition task, and novel object recognition after administration of the amnesic drug scopolamine which acts as a psychopharmacological model of Alzheimer’s disease. Utilizing rigorous metrics of criticality including the Deviation from Criticality Coefficient and Branching Ratio, the ensemble neural activity in the hippocampus was observed to display evidence of near-critical behaviour during rest periods, but moved significantly closer to a critical state when engaged in a cognitive task. The dynamics were observed to move significantly away from criticality during the cognitive task after scopolamine administration. In contrast to previous theoretical predictions, our results indicate that hippocampus neural networks move closer to criticality under cognitive load, and that critical dynamical regimes produce a near-optimal state for cognitive operations.
Accelerated social representational drift in the nucleus accumbens in a model of autism
Impaired social interaction is one of the core deficits of autism spectrum disorder (ASD) and may result from social interactions being less rewarding. How the nucleus accumbens (NAc), as a key hub of reward circuitry, encodes social interaction and whether these representations are altered in ASD remain poorly understood. We identified NAc ensembles encoding social interactions by calcium imaging using miniaturized microscopy. NAc population activity, specifically D1 receptor-expressing medium spiny neurons (D1-MSNs) activity, predicted social interaction epochs. Despite a high turnover of NAc neurons modulated by social interaction, we found a stable population code for social interaction in NAc which was dramatically degraded in Cntnap2-/- mouse model of ASD. Surprisingly, non-specific optogenetic inhibition of NAc core neurons increased social interaction time and significantly improved sociability in Cntnap2-/- mice. Inhibition of D1- or D2-MSNs showed reciprocal effects, with D1 inhibition decreasing social interaction and D2 inhibition increasing interaction. Therefore, social interactions are preferentially, specifically and dynamically encoded by NAc neurons and social representations are degraded in this autism model.
Multi-functional accessory toolkit for Miniscope prototyping and image enhancement
During the last decade, Miniaturized microscopy, or Miniscope, has gained popularity in neuroscience, particularly for behavioral studies in awake rodents. However, image quality control and standardization remain challenging for both users and developers. To address these challenges, we present MiniMounter, a cost-effective and multi-functional accessory toolkit that includes a hardware platform with customized grippers and four-degree-of-freedom adjustment for Miniscope, as well as software for displacement control and image quality evaluation. Our toolkit enables auto-focusing and accurate measurement of spatial resolution and field of view (FOV). We have demonstrated the effectiveness of such a toolkit through comprehensive phantom and animal experiments.
Hippocampal place cell remapping occurs with memory storage of aversive experiences
Aversive stimuli can cause hippocampal place cells to remap their firing fields, but it is not known whether remapping plays a role in storing memories of aversive experiences. Here, we addressed this question by performing in vivo calcium imaging of CA1 place cells in freely behaving rats (n = 14). Rats were first trained to prefer a short path over a long path for obtaining food reward, then trained to avoid the short path by delivering a mild footshock. Remapping was assessed by comparing place cell population vector similarity before acquisition versus after extinction of avoidance. Some rats received shock after systemic injections of the amnestic drug scopolamine at a dose (1 mg/kg) that impaired avoidance learning but spared spatial tuning and shock-evoked responses of CA1 neurons. Place cells remapped significantly more following remembered than forgotten shocks (drug-free versus scopolamine conditions); shock-induced remapping did not cause place fields to migrate toward or away from the shocked location and was similarly prevalent in cells that were responsive versus non-responsive to shocks. When rats were exposed to a neutral barrier rather than aversive shock, place cells remapped significantly less in response to the barrier. We conclude that place cell remapping occurs in response to events that are remembered rather than merely perceived and forgotten, suggesting that reorganization of hippocampal population codes may play a role in storing memories for aversive events.
Conjunctive vector coding and place coding in hippocampus share a common directional signal
Vector coding is becoming increasingly understood as a major mechanism by which neural systems represent an animal’s location in both a global reference frame and a local, item-based reference frame. Landmark vector cells (LVCs) in the hippocampus complement classic place cells by encoding the vector relationship (angle and distance) between the individual and specific landmarks in the environment. How these properties of hippocampal principal cells interact is not known. We simultaneously recorded the activities of place cells and LVCs via in vivo calcium imaging of the CA1 region of freely moving rats during classic, cue-card rotation studies. The firing fields of place cells rotated relative to the center of the platform to follow the cue card rotation, whereas the firing fields of simultaneously recorded LVCs rotated by the same amount as the place cells, but the axis of rotation was the nearby local landmarks, not the environmental center. We identified a novel type of place cell that exhibited conjunctive coding of the classic place field properties and LVC properties. These results demonstrate the capacity of CA1 neurons to encode both world-centered spatial information and animals’ location relative to the local landmarks, with a common directional input presumably provided by the head direction cell system.
Open-MAC: A low-cost open-source motorized commutator for electro- and opto-physiological recordings in freely moving rodents
In vivo electro- and optophysiology experiments in rodents reveal the neural mechanisms underlying behavior and brain disorders but mostly involve a cable connection between an implant in the animal and an external recording device. Standard tethers with thin cables or non-motorized commutators require constant monitoring and often manual interference to untwist the cable. Motorized commutators offer a solution, but those few that are commercially available are expensive and often not adapted to widely used connector standards of the open-source community like 12-channel SPI. Here we introduce an open-source motorized all-in-one commutator (Open-MAC): a low-cost (240–390 EUR), low-torque motorized commutator that can operate with minimal audible noise in a torque-based mode relying on dual magnetic Hall sensors. It further includes electronics to operate in a torque-free, online pose-estimation-based mode, with future developments. Operation is controlled by an onboard microcontroller (XIAO SAMD21) powered by a USB-C cable or DC power supply. The body and movable parts are 3D-printed. Different Open-MAC versions can support electrophysiology with up to 64 recording channels using the Open-Ephys / IntanTM recording systems as well as miniature endoscope (miniscope) recordings using the UCLA Miniscope v3/4, and can host a fibre for optogenetic modulation.
CaliAli, a tool for long-term tracking of neuronal population dynamics in calcium imaging
Neuron-tracking algorithms exhibit suboptimal performance in calcium imaging when the same neurons are not consistently detected, as unmatched features hinder intersession alignment. CaliAli addresses this issue by employing an alignment-before-extraction strategy that incorporates vasculature information to improve the detectability of weak signals and maximize the number of trackable neurons. By excelling in neural remapping and high spatial overlap scenarios, CaliAli paves the way toward further understanding long-term neural network dynamics.
The subiculum encodes environmental geometry
Corners are a cardinal feature of many of the complex environmental geometries found in the natural world but the neural substrates that could underlie the perception of corners remain elusive. Here we show that the dorsal subiculum contains neurons that encode corners across environmental geometries in an allocentric reference frame. Corner cells changed their activity to reflect concave corner angles, wall height and the degree of wall intersection. A separate population of subicular neurons encoded convex corners. Both concave and convex corner cells were non-overlapping with subicular neurons that encoded environmental boundaries, suggesting that the subiculum contains the geometric information needed to re-construct the shape and layout of naturalistic spatial environments. One Sentence Summary Separate neural populations in the subiculum encode concave and convex environmental corners.
The temporal and contextual stability of activity levels in hippocampal CA1 cells
Recent long-term optical imaging studies have demonstrated that the activity levels of hippocampal neurons in a familiar environment change on a daily to weekly basis. However, it is unclear whether there is any time-invariant property in the cells’ neural representations. In this study, using miniature fluorescence microscopy, we measured the neural activity of the mouse hippocampus in four different environments every 3 d. Although the activity level of hippocampal neurons fluctuated greatly in each environment across days, we found a significant correlation between the activity levels for different days, and the correlation was higher for averaged activity levels across multiple environments. When the number of environments used for averaging was increased, a higher activity correlation was observed. Furthermore, the number of environments in which a cell showed activity was preserved. Cells that showed place cell activity in many environments had greater spatial information content and more stable spatial representation, and thus carried more abundant and stable information about the current position. In contrast, cells that were active only in a small number of environments provided sparse representation for the environment. These results suggest that each cell has not only an inherent activity level but also play a characteristic role in the coding of space.
Miniscope-LFOV: A large-field-of-view, single-cell-resolution, miniature microscope for wired and wire-free imaging of neural dynamics in freely behaving animals
Imaging large-population, single-cell fluorescent dynamics in freely behaving animals larger than mice remains a key endeavor of neuroscience. We present a large-field-of-view open-source miniature microscope (MiniLFOV) designed for large-scale (3.6 mm × 2.7 mm), cellular resolution neural imaging in freely behaving rats. It has an electrically adjustable working distance of up to 3.5 mm ± 100 μm, incorporates an absolute head orientation sensor, and weighs only 13.9 g. The MiniLFOV is capable of both deep brain and cortical imaging and has been validated in freely behaving rats by simultaneously imaging >1000 GCaMP7s-expressing neurons in the hippocampal CA1 layer and in head-fixed mice by simultaneously imaging ~2000 neurons in the dorsal cortex through a cranial window. The MiniLFOV also supports optional wire-free operation using a novel, wire-free data acquisition expansion board. We expect that this new open-source implementation of the UCLA Miniscope platform will enable researchers to address novel hypotheses concerning brain function in freely behaving animals.
Elucidating a locus coeruleus-dentate gyrus dopamine pathway for operant reinforcement
Animals can learn to repeat behaviors to earn desired rewards, a process commonly known as reinforcement learning. While previous work has implicated the ascending dopaminergic projections to the basal ganglia in reinforcement learning, little is known about the role of the hippocampus. Here, we report that a specific population of hippocampal neurons and their dopaminergic innervation contribute to operant self-stimulation. These neurons are located in the dentate gyrus, receive dopaminergic projections from the locus coeruleus, and express D1 dopamine receptors. Activation of D1 + dentate neurons is sufficient for self-stimulation: mice will press a lever to earn optogenetic activation of these neurons. A similar effect is also observed with selective activation of the locus coeruleus projections to the dentate gyrus, and blocked by D1 receptor antagonism. Calcium imaging of D1 + dentate neurons revealed significant activity at the time of action selection, but not during passive reward delivery. These results reveal the role of dopaminergic innervation of the dentate gyrus in supporting operant reinforcement.
Somatic Calcium Signals from Layer II/III Motor Cortex for Continuous Neural Decoding
The latest research shows that calcium signals can provide a new signal source for brain-machine interfaces (BMI). However, it remains a question whether the calcium signals from layer 2/3 motor cortex can be used for continuous neural decoding. And how they are involved in movement coding is also worth investigating. Here we collect the somatic signals in M1 layer 2/3 while mice performed a lever-press task under the one-photon imaging. We first present the potential of somatic calcium signals from layer 2/3 in continuous neural decoding through an improved recurrent neural network. Layer 2/3 neurons exhibit three types of calcium dynamics with distinct spatiotemporal coding patterns involved in the movement. Pre-pressing and pressing neurons enable sparse coding of movement through complementary spatiotemporal information. While post-pressing neurons predict the lever movement most accurately through the calcium dynamics with higher fidelity. These results demonstrate the capability of calcium signals from layer 2/3 neurons as a motor BMI driver and underscore their diversity in motor coding, opening a new avenue for studying the motor cortex and designing optical BMIs.
Distributed processing for value-based choice by prelimbic circuits targeting anterior-posterior dorsal striatal subregions in male mice
Fronto-striatal circuits have been implicated in cognitive control of behavioral output for social and appetitive rewards. The functional diversity of prefrontal cortical populations is strongly dependent on their synaptic targets, with control of motor output mediated by connectivity to dorsal striatum. Despite evidence for functional diversity along the anterior-posterior striatal axis, it is unclear how distinct fronto-striatal sub-circuits support value-based choice. Here we found segregated prefrontal populations defined by anterior/posterior dorsomedial striatal target. During a feedback-based 2-alternative choice task, single-photon imaging revealed circuit-specific representations of task-relevant information with prelimbic neurons targeting anterior DMS (PL::A-DMS) robustly modulated during choices and negative outcomes, while prelimbic neurons targeting posterior DMS (PL::P-DMS) encoded internal representations of value and positive outcomes contingent on prior choice. Consistent with this distributed coding, optogenetic inhibition of PL::A-DMS circuits strongly impacted choice monitoring and responses to negative outcomes while inhibition of PL::P-DMS impaired task engagement and strategies following positive outcomes. Together our data uncover PL populations engaged in distributed processing for value-based choice.
Implementation of miniaturized modular-array fluorescence microscopy for long-term live-cell imaging
Fluorescence microscopy imaging of live cells has provided consistent monitoring of dynamic cellular activities and interactions. However, because current live-cell imaging systems are limited in their adaptability, portable cell imaging systems have been adapted by a variety of strategies, including miniaturized fluorescence microscopy. Here, we provide a protocol for the construction and operational process of miniaturized modular-array fluorescence microscopy (MAM). The MAM system is built in a portable size (15c m×15c m×3c m) and provides in situ cell imaging inside an incubator with a subcellular lateral resolution (∼3µm). We demonstrated the improved stability of the MAM system with fluorescent targets and live HeLa cells, enabling long-term imaging for 12 h without the need for external support or post-processing. We believe the protocol could guide scientists to construct a compact portable fluorescence imaging system and perform time-lapse in situ single-cell imaging and analysis.
A one-photon endoscope for simultaneous patterned optogenetic stimulation and calcium imaging in freely behaving mice
Optogenetics and calcium imaging can be combined to simultaneously stimulate and record neural activity in vivo. However, this usually requires two-photon microscopes, which are not portable nor affordable. Here we report the design and implementation of a miniaturized one-photon endoscope for performing simultaneous optogenetic stimulation and calcium imaging. By integrating digital micromirrors, the endoscope makes it possible to activate any neuron of choice within the field of view, and to apply arbitrary spatiotemporal patterns of photostimulation while imaging calcium activity. We used the endoscope to image striatal neurons from either the direct pathway or the indirect pathway in freely moving mice while activating any chosen neuron in the field of view. The endoscope also allows for the selection of neurons based on their relationship with specific animal behaviour, and to recreate the behaviour by mimicking the natural neural activity with photostimulation. The miniaturized endoscope may facilitate the study of how neural activity gives rise to behaviour in freely moving animals.
An optical design enabling lightweight and large field-of-view head-mounted microscopes
Here we present a fluorescence microscope light path that enables imaging, during free behavior, of thousands of neurons in mice and hundreds of neurons in juvenile songbirds. The light path eliminates traditional illumination optics, allowing for head-mounted microscopes that have both a lower weight and a larger field of view (FOV) than previously possible. Using this light path, we designed two microscopes: one optimized for FOV (~4 mm FOV; 1.4 g), and the other optimized for weight (1.0 mm FOV; 1.0 g).
Distinct neural mechanisms for heading retrieval and context recognition in the hippocampus during spatial reorientation
Reorientation, the process of regaining one’s bearings after becoming lost, requires identification of a spatial context (context recognition) and recovery of heading direction within that context (heading retrieval). We previously showed that these processes rely on the use of features and geometry, respectively. Here, we examine reorientation behavior in a task that creates contextual ambiguity over a long timescale to demonstrate that mice learn to combine both featural and geometric cues to recover heading with experience. At the neural level, most CA1 neurons persistently align to geometry, and this alignment predicts heading behavior. However, a small subset of cells shows feature-sensitive place field remapping, which serves to predict context. Efficient heading retrieval and context recognition require integration of featural and geometric information in the active network through rate changes. These data illustrate how context recognition and heading retrieval are coded in CA1 and how these processes change with experience.
Ensemble remodeling supports memory-updating
Memory-updating is critical in dynamic environments because updating memories with new information promotes versatility. However, little is known about how memories are updated with new information. To study how neuronal ensembles might support memory-updating, we used a hippocampus-dependent spatial reversal task to measure hippocampal ensemble dynamics when mice switched navigational goals. Using Miniscope calcium imaging, we identified neuronal ensembles (co-active neurons) in dorsal CA1 that were spatially tuned and stable across training sessions. When reward locations were moved during a reversal session, a subset of these ensembles decreased their activation strength, correlating with memory-updating. These “remodeling” ensembles were a result of weakly-connected neurons becoming less co-active with their peers. Middle-aged mice were impaired in reversal learning, and the prevalence of their remodeling ensembles correlated with their memory-updating performance. Therefore, we have identified a mechanism where the hippocampus breaks down ensembles to support memory-updating.
Ensemble-specific deficit in neuronal intrinsic excitability in aged mice
With the prevalence of age-related cognitive deficits on the rise, it is essential to identify cellular and circuit alterations that contribute to age-related memory impairment. Increased intrinsic neuronal excitability after learning is important for memory consolidation, and changes to this process could underlie memory impairment in old age. Some studies find age-related deficits in hippocampal neuronal excitability that correlate with memory impairment but others do not, possibly due to selective changes only in activated neural ensembles. Thus, we tagged CA1 neurons activated during learning and recorded their intrinsic excitability 5 hours or 7 days post-training. Adult mice exhibited increased neuronal excitability 5 hours after learning, specifically in ensemble (learning-activated) CA1 neurons. As expected, ensemble excitability returned to baseline 7 days post-training. In aged mice, there was no ensemble-specific excitability increase after learning, which was associated with impaired hippocampal memory performance. These results suggest that CA1 may be susceptible to age-related impairments in post-learning ensemble excitability and underscore the need to selectively measure ensemble-specific changes in the brain.
Ensemble-specific deficit in neuronal intrinsic excitability in aged mice
With the prevalence of age-related cognitive deficits on the rise, it is essential to identify cellular and circuit alterations that contribute to age-related memory impairment. Increased intrinsic neuronal excitability after learning is important for memory consolidation, and changes to this process could underlie memory impairment in old age. Some studies find age-related deficits in hippocampal neuronal excitability that correlate with memory impairment but others do not, possibly due to selective changes only in activated neural ensembles. Thus, we tagged CA1 neurons activated during learning and recorded their intrinsic excitability 5 hours or 7 days post-training. Adult mice exhibited increased neuronal excitability 5 hours after learning, specifically in ensemble (learning-activated) CA1 neurons. As expected, ensemble excitability returned to baseline 7 days post-training. In aged mice, there was no ensemble-specific excitability increase after learning, which was associated with impaired hippocampal memory performance. These results suggest that CA1 may be susceptible to age-related impairments in post-learning ensemble excitability and underscore the need to selectively measure ensemble-specific changes in the brain.
Population dynamics of head-direction neurons during drift and reorientation
The head direction (HD) system functions as the brain’s internal compass1,2, classically formalized as a one-dimensional ring attractor network3,4. In contrast to a globally consistent magnetic compass, the HD system does not have a universal reference frame. Instead, it anchors to local cues, maintaining a stable offset when cues rotate5–8 and drifting in the absence of referents5,8–10. However, questions about the mechanisms that underlie anchoring and drift remain unresolved and are best addressed at the population level. For example, the extent to which the one-dimensional description of population activity holds under conditions of reorientation and drift is unclear. Here we performed population recordings of thalamic HD cells using calcium imaging during controlled rotations of a visual landmark. Across experiments, population activity varied along a second dimension, which we refer to as network gain, especially under circumstances of cue conflict and ambiguity. Activity along this dimension predicted realignment and drift dynamics, including the speed of network realignment. In the dark, network gain maintained a ‘memory trace’ of the previously displayed landmark. Further experiments demonstrated that the HD network returned to its baseline orientation after brief, but not longer, exposures to a rotated cue. This experience dependence suggests that memory of previous associations between HD neurons and allocentric cues is maintained and influences the internal HD representation. Building on these results, we show that continuous rotation of a visual landmark induced rotation of the HD representation that persisted in darkness, demonstrating experience-dependent recalibration of the HD system. Finally, we propose a computational model to formalize how the neural compass flexibly adapts to changing environmental cues to maintain a reliable representation of HD. These results challenge classical one-dimensional interpretations of the HD system and provide insights into the interactions between this system and the cues to which it anchors.
Calcium imaging and analysis of the jugular-nodose ganglia enables identification of distinct vagal sensory neuron subsets
Objective. Sensory nerves of the peripheral nervous system (PNS) transmit afferent signals from the body to the brain. These peripheral nerves are composed of distinct subsets of fibers and associated cell bodies, which reside in peripheral ganglia distributed throughout the viscera and along the spinal cord. The vagus nerve (cranial nerve X) is a complex polymodal nerve that transmits a wide array of sensory information, including signals related to mechanical, chemical, and noxious stimuli. To understand how stimuli applied to the vagus nerve are encoded by vagal sensory neurons in the jugular-nodose ganglia, we developed a framework for micro-endoscopic calcium imaging and analysis. Approach. We developed novel methods for in vivo imaging of the intact jugular-nodose ganglion using a miniature microscope (Miniscope) in transgenic mice with the genetically-encoded calcium indicator GCaMP6f. We adapted the Python-based analysis package Calcium Imaging Analysis (CaImAn) to process the resulting one-photon fluorescence data into calcium transients for subsequent analysis. Random forest classification was then used to identify specific types of neuronal responders. Results. We demonstrate that recordings from the jugular-nodose ganglia can be accomplished through careful surgical dissection and ganglia stabilization. Using a customized acquisition and analysis pipeline, we show that subsets of vagal sensory neurons respond to different chemical stimuli applied to the vagus nerve. Successful classification of the responses with a random forest model indicates that certain calcium transient features, such as amplitude and duration, are important for encoding these stimuli by sensory neurons. Significance. This experimental approach presents a new framework for investigating how individual vagal sensory neurons encode various stimuli on the vagus nerve. Our surgical and analytical approach can be applied to other PNS ganglia in rodents and other small animal species to elucidate previously unexplored roles for peripheral neurons in a diverse set of physiological functions.
Distinct reward processing by subregions of the nucleus accumbens
Characterization of ventromedial hypothalamus activity during exposure to innate and conditioned threats
In the face of imminent predatory danger, animals quickly detect the threat and mobilize key survival defensive actions, such as escape and freezing. The dorsomedial portion of the ventromedial hypothalamus (VMH) is a central node in innate and conditioned predator-induced defensive behaviours. Prior studies have shown that activity of steroidogenic factor 1 (sf1)-expressing VMH cells is necessary for such defensive behaviours. However, sf1-VMH neural activity during exposure to predatory threats has not been well characterized. Here, we use single-cell recordings of calcium transients from VMH cells in male and female mice. We show this region is activated by threat proximity and that it encodes future occurrence of escape but not freezing. Our data also show that VMH cells encoded proximity of an innate predatory threat but not a fear-conditioned shock grid. Furthermore, chemogenetic activation of the VMH increases avoidance of innate threats, such as open spaces and a live predator. This manipulation also increased freezing towards the predator, without altering defensive behaviours induced by a shock grid. Lastly, we show that optogenetic VMH activation recruited a broad swath of regions, suggestive of widespread changes in neural defensive state. Taken together, these data reveal the neural dynamics of the VMH during predator exposure and further highlight its role as a critical component of the hypothalamic predator defense system.
Imaging neuro-urodynamics of mouse major pelvic ganglion with a micro-endoscopic approach
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Mechanism of kisspeptin neuron synchronization for pulsatile hormone secretion in male mice
Optogenetic frequency scrambling of hippocampal theta oscillations dissociates working memory retrieval from hippocampal spatiotemporal codes
The precise temporal coordination of activity in the brain is thought to be fundamental for memory function. Inhibitory neurons in the medial septum provide a prominent source of innervation to the hippocampus and play a major role in controlling hippocampal theta (~8 Hz) oscillations. While pharmacological inhibition of medial septal neurons is known to disrupt memory, the exact role of septal inhibitory neurons in regulating hippocampal representations and memory is not fully understood. Here, we dissociate the role of theta rhythms in spatiotemporal coding and memory using an all-optical interrogation and recording approach. We find that optogenetic frequency scrambling stimulations abolish theta oscillations and modulate a portion of neurons in the hippocampus. Such stimulation decreased episodic and working memory retrieval while leaving hippocampal spatiotemporal codes intact. Our study suggests that theta rhythms play an essential role in memory but may not be necessary for hippocampal spatiotemporal codes.
A hardware system for real-time decoding of in vivo calcium imaging data
Epifluorescence miniature microscopes (‘miniscopes’) are widely used for in vivo calcium imaging of neural population activity. Imaging data are typically collected during a behavioral task and stored for later offline analysis, but emerging techniques for online imaging can support novel closed-loop experiments in which neural population activity is decoded in real time to trigger neurostimulation or sensory feedback. To achieve short feedback latencies, online imaging systems must be optimally designed to maximize computational speed and efficiency while minimizing errors in population decoding. Here we introduce DeCalciOn, an open-source device for real-time imaging and population decoding of in vivo calcium signals that is hardware compatible with all miniscopes that use the UCLA Data Acquisition (DAQ) interface. DeCalciOn performs online motion stabilization, neural enhancement, calcium trace extraction, and decoding of up to 1024 traces per frame at latencies of <50 ms after fluorescence photons arrive at the miniscope image sensor. We show that DeCalciOn can accurately decode the position of rats (n = 12) running on a linear track from calcium fluorescence in the hippocampal CA1 layer, and can categorically classify behaviors performed by rats (n = 2) during an instrumental task from calcium fluorescence in orbitofrontal cortex. DeCalciOn achieves high decoding accuracy at short latencies using innovations such as field-programmable gate array hardware for real-time image processing and contour-free methods to efficiently extract calcium traces from sensor images. In summary, our system offers an affordable plug-and-play solution for real-time calcium imaging experiments in behaving animals.
Development and Dissemination of Novel Open-Source Miniscope Technology
The ability to measure neural activity patterns and relate them to behavior is fundamental to the advancement of neuroscience. Miniature microscope (miniscope) technology has made a significant contribution to this endeavor by allowing researchers to record neural activity from thousands of neurons with single-cell resolution in freely behaving animals across days to months. However, while this technology has allowed scientists to readily acquire immense and theoretically illuminating datasets, data analysis remains a bottleneck for adopting miniscope technology. In addition, currently miniscopes can only image one fluororphore at a time, limiting the scope of applications. Hence, development of novel software and hardware that expand the existing capabilities of miniscopes and make the technology more accessible will be of great value to neuroscience. One of the biggest challenges in adopting Miniscope technology is data analysis. We developed an open-source calcium imaging analysis pipeline, Minian, with interactive visualizations and reduced memory demand. By providing interactive visualization, detailed documentation, and guidance for each step in the pipeline, Minian helps users build intuitive understanding of the underlying algorithms and readily perform accurate parameter selection. This greatly improved the accessibility of calcium imaging analysis to neuroscience researchers. Moreover, Minian supports out-of-core computation, allowing data larger than the RAM of the computer to be processed. This enables researchers to analyze data with arbitrary recording length without specialized computer hardware. Taken together, these features made Minian more transparent, accessible and user-friendly. Traditionally, Miniscopes were only capable to image one fluorophore. However, with the advancement of new genetic tools, there is a growing need to image multiple fluorophores simultaneously at single-cell resolution. Here, we describe a novel open-source dual-channel Miniscope developed to fill this gap in current technologies. Our design can correct for focal plane mismatch due to chromatic aberration and is light enough to be carried by adult mice, enabling dual-channel calcium imaging in behaving animals. Furthermore, we have demonstrated a use case where our dual-channel Miniscope can be used to track the same population of neurons across long time and study the stability of hippocampal spatial representation in an unbiased way. Taken together, we have described the development and dissemination of novel open-source Miniscope software and hardware that fill gaps in the field and make the technology more impactful.
Calcium activity is a degraded estimate of spikes
Place cells are nonrandomly clustered by field location in CA1 hippocampus
A challenge in both modern and historic neuroscience has been achieving an understanding of neuron circuits, and determining the computational and organizational principles that underlie these circuits. Deeper understanding of the organization of brain circuits and cell types, including in the hippocampus, is required for advances in behavioral and cognitive neuroscience, as well as for understanding principles governing brain development and evolution. In this manuscript, we pioneer a new method to analyze the spatial clustering of active neurons in the hippocampus. We use calcium imaging and a rewarded navigation task to record from 100 s of place cells in the CA1 of freely moving rats. We then use statistical techniques developed for and in widespread use in geographic mapping studies, global Moran's I, and local Moran's I to demonstrate that cells that code for similar spatial locations tend to form small spatial clusters. We present evidence that this clustering is not the result of artifacts from calcium imaging, and show that these clusters are primarily formed by cells that have place fields around previously rewarded locations. We go on to show that, although cells with similar place fields tend to form clusters, there is no obvious topographic mapping of environmental location onto the hippocampus, such as seen in the visual cortex. Insights into hippocampal organization, as in this study, can elucidate mechanisms underlying motivational behaviors, spatial navigation, and memory formation.
A circuit from lateral septum neurotensin neurons to tuberal nucleus controls hedonic feeding
Feeding behavior is regulated by both the homeostatic needs of the body and hedonic values of the food. Easy access to palatable energy-dense foods and the consequent obesity epidemic stress the urgent need for a better understanding of neural circuits that regulate hedonic feeding. Here, we report that neurotensin-positive neurons in the lateral septum (LSNts) play a crucial role in regulating hedonic feeding. Silencing LSNts specifically promotes feeding of palatable food, whereas activation of LSNts suppresses overall feeding. LSNts neurons project to the tuberal nucleus (TU) via GABA signaling to regulate hedonic feeding, while the neurotensin signal from LSNts→the supramammillary nucleus (SUM) is sufficient to suppress overall feeding. In vivo calcium imaging and optogenetic manipulation reveal two populations of LSNts neurons that are activated and inhibited during feeding, which contribute to food seeking and consumption, respectively. Chronic activation of LSNts or LSNts→TU is sufficient to reduce high-fat diet-induced obesity. Our findings suggest that LSNts→TU is a key pathway in regulating hedonic feeding.
The impact of familiarity on cortical taste coding
Neural circuit dynamics of drug-context associative learning in the mouse hippocampus
The environmental context associated with previous drug consumption is a potent trigger for drug relapse. However, the mechanism by which neural representations of context are modified to incorporate information associated with drugs of abuse remains unknown. Using longitudinal calcium imaging in freely behaving mice, we find that unlike the associative learning of natural reward, drug-context associations for psychostimulants and opioids are encoded in a specific subset of hippocampal neurons. After drug conditioning, these neurons weakened their spatial coding for the non-drug paired context, resulting in an orthogonal representation for the drug versus non-drug context that was predictive of drug-seeking behavior. Furthermore, these neurons were selected based on drug-spatial experience and were exclusively tuned to animals’ allocentric position. Together, this work reveals how drugs of abuse alter the hippocampal circuit to encode drug-context associations and points to the possibility of targeting drug-associated memory in the hippocampus.
Amygdalostriatal transition zone neurons encode sustained valence to direct conditioned behaviors
In order to respond appropriately to threats in the environment, the brain must rapidly determine whether a stimulus is important and whether it is positive or negative, and then use that information to direct behavioral responses. Neurons in the amygdala have long been implicated in valence encoding and in fear responses to threatening stimuli, but show transient firing responses in response to these stimuli that do not match the timescales of associated behavioral responses. For decades, there has been a logical gap in how behavioral responses could be mediated without an ensemble representation of the internal state of valence that has rapid onset, high signal-to-noise, and is sustained for the duration of the behavioral state. Here, we present the amygdalostriatal transition zone (ASt) as a missing piece of this highly conserved process that is of paramount importance for survival, which does exactly this: represents an internal state (e.g. fear) that can be expressed in multiple motor outputs (e.g. freezing or escape). The ASt is anatomically positioned as a “shortcut” to connect the corticolimbic system (important for evaluation) with the basal ganglia (important for action selection) with the inputs of the amygdala and the outputs of the striatum – ideally poised for evaluating and responding to environmental threats. From in vivo cellular resolution recordings that include both electrophysiology and calcium imaging, we find that ASt neurons are unique in that they are sparse coding, extremely high signal-to-noise, and also maintain a sustained response for negative valence stimuli for the duration of the defensive behavior – a rare but essential combination. We further show that photostimulation of the ASt is sufficient to drive freezing and avoidance behaviors. Using single-nucleus RNA sequencing and in situ RNA labelling we generate a comprehensive profile of cell types and gene expression in the ASt, and find the ASt is genetically distinct from adjacent striatal and amygdalar structures. We also find that the ASt has a greater proportion of neurons expressing Drd2 than neurons expressing Drd1a, a unique feature compared to other regions of the striatum. Using in vivo calcium imaging, we show that that this Drd2+ population robustly encodes stimuli of negative valence, and in loss-of-function experiments find that optogenetic inhibition of Drd2+ ASt neurons causes a striking reduction in cue-conditioned fear responses. Together, our findings identify the ASt as a previously-unappreciated critical missing link for encoding learned associations and directing ongoing behavior.
Dissociable effects of oxycodone on behavior, calcium transient activity, and excitability of dorsolateral striatal neurons
Opioids are the most common medications for moderate to severe pain. Unfortunately, they also have addictive properties that have precipitated opioid misuse and the opioid epidemic. In the present study, we examined the effects of acute administration of oxycodone, a μ-opioid receptor (MOR) agonist, on Ca2+ transient activity of medium-sized spiny neurons (MSNs) in freely moving animals. Ca2+ imaging of MSNs in dopamine D1-Cre mice (expressing Cre predominantly in the direct pathway) or adenosine A2A-Cre mice (expressing Cre predominantly in the indirect pathway) was obtained with the aid of miniaturized microscopes (Miniscopes) and a genetically encoded Cre-dependent Ca2+ indicator (GCaMP6f). Systemic injections of oxycodone (3 mg/kg) increased locomotor activity yet, paradoxically, reduced concomitantly the number of active MSNs. The frequency of Ca2+ transients was significantly reduced in MSNs from A2A-Cre mice but not in those from D1-Cre mice. For comparative purposes, a separate group of mice was injected with a non-Cre dependent Ca2+ indicator in the cerebral cortex and the effects of the opioid also were tested. In contrast to MSNs, the frequency of Ca2+ transients in cortical pyramidal neurons was significantly increased by oxycodone administration. Additional electrophysiological studies in brain slices confirmed generalized inhibitory effects of oxycodone on MSNs, including membrane hyperpolarization, reduced excitability, and decreased frequency of spontaneous excitatory and inhibitory postsynaptic currents. These results demonstrate a dissociation between locomotion and striatal MSN activity after acute administration of oxycodone.
New imaging instrument in animal models: Two-photon miniature microscope and large field of view miniature microscope for freely behaving animals
Over the past decade, novel optical imaging tools have been developed for imaging neuronal activities along with the evolution of fluorescence indicators with brighter expression and higher sensitivity. Miniature microscopes, as revolutionary approaches, enable the imaging of large populations of neuron ensembles in freely behaving rodents and mammals, which allows exploring the neural basis of behaviors. Recent progress in two-photon miniature microscopes and mesoscale single-photon miniature microscopes further expand those affordable methods to navigate neural activities during naturalistic behaviors. In this review article, two-photon miniature microscopy techniques are summarized historically from the first documented attempt to the latest ones, and comparisons are made. The driving force behind and their potential for neuroscientific inquiries are also discussed. Current progress in terms of the mesoscale, i.e., the large field-of-view miniature microscopy technique, is addressed as well. Then, pipelines for registering single cells from the data of two-photon and large field-of-view miniature microscopes are discussed. Finally, we present the potential evolution of the techniques.
SpecSeg is a versatile toolbox that segments neurons and neurites in chronic calcium imaging datasets based on low-frequency cross-spectral power
A locus coeruleus-dorsal CA1 dopaminergic circuit modulates memory linking
Advances of optical miniscopes for in vivo imaging of neural activity in freely moving animals
To study neural mechanisms of ethologically relevant behaviors including many social behaviors and navigations, optical miniscopes, which can be carried by the model animals, are indispensable. Recently, a variety of optical miniscopes have been developed to meet this urgent requirement, and successfully applied in the study of neural network activity in free-moving mice, rats, and bats, etc. Generally, miniature fluorescence microscopes can be classified into single-photon and multi-photon fluorescence miniscopes, considering their differences in imaging mechanisms and hardware setups. In this review, we introduce their fundamental principles and system structures, summarize technical advances, and discuss limitations and future trends, for
Vagus nerve stimulation drives selective circuit modulation through cholinergic reinforcement
BehaviorDEPOT is a simple, flexible tool for automated behavioral detection based on markerless pose tracking
Quantitative descriptions of animal behavior are essential to study the neural substrates of cognitive and emotional processes. Analyses of naturalistic behaviors are often performed by hand or with expensive, inflexible commercial software. Recently, machine learning methods for markerless pose estimation enabled automated tracking of freely moving animals, including in labs with limited coding expertise. However, classifying specific behaviors based on pose data requires additional computational analyses and remains a significant challenge for many groups. We developed BehaviorDEPOT (DEcoding behavior based on POsitional Tracking), a simple, flexible software program that can detect behavior from video timeseries and can analyze the results of experimental assays. BehaviorDEPOT calculates kinematic and postural statistics from keypoint tracking data and creates heuristics that reliably detect behaviors. It requires no programming experience and is applicable to a wide range of behaviors and experimental designs. We provide several hard-coded heuristics. Our freezing detection heuristic achieves above 90% accuracy in videos of mice and rats, including those wearing tethered head-mounts. BehaviorDEPOT also helps researchers develop their own heuristics and incorporate them into the software’s graphical interface. Behavioral data is stored framewise for easy alignment with neural data. We demonstrate the immediate utility and flexibility of BehaviorDEPOT using popular assays including fear conditioning, decision-making in a T-maze, open field, elevated plus maze, and novel object exploration.
Neuronal activity under transcranial radio-frequency stimulation in metal-free rodent brains in-vivo
As the use of Radio Frequency (RF) technologies increases, the impact of RF radiation on neurological function continues to receive attention. Whether RF radiation can modulate ongoing neuronal activity by non-thermal mechanisms has been debated for decades. However, the interactions between radiated energy and metal-based neural probes during experimentation could impact neural activity, making interpretation of the results difficult. To address this problem, we modified a miniature 1-photon Ca2+ imaging device to record interference-free neural activity and compared the results to those acquired using metal-containing silicon probes. We monitored the neuronal activity of awake rodent-brains under RF energy exposure (at 950 MHz) and in sham control paradigms. Spiking activity was reliably affected by RF energy in metal containing systems. However, we did not observe neuronal responses using metal-free optical recordings at induced local electric field strengths up to 230 V/m. Our results suggest that RF exposure higher than levels that are allowed by regulatory limits in real-life scenarios do not affect neuronal activity.
Simultaneous Cellular Imaging, Electrical Recording and Stimulation of Hippocampal Activity in Freely Behaving Mice
Hippocampal sharp-wave ripple activity (SWRs) and the associated replay of neural activity patterns are well-known for their role in memory consolidation. This activity has been studied using electrophysiological approaches, as high temporal resolution is required to recognize SWRs in the neuronal signals. However, it has been difficult to analyze the individual contribution of neurons to task-specific SWRs, because it is hard to track neurons across a long time with electrophysiological recording. In this study, we recorded local field potential (LFP) signals in the hippocampal CA1 of freely behaving mice and simultaneously imaged calcium signals in contralateral CA1 to leverage the advantages of both electrophysiological and imaging approaches. We manufactured a custom-designed microdrive array and targeted tetrodes to the left hippocampus CA1 for LFP recording and applied electrical stimulation in the ventral hippocampal commissure (VHC) for closed-loop disruption of SWRs. Neuronal population imaging in the right hippocampal CA1 was performed using a miniature fluorescent microscope (Miniscope) and a genetically encoded calcium indicator. As SWRs show highly synchronized bilateral occurrence, calcium signals of SWR-participating neurons could be identified and tracked in spontaneous or SWR-disrupted conditions. Using this approach, we identified a subpopulation of CA1 neurons showing synchronous calcium elevation to SWRs. Our results showed that SWR-related calcium transients are more disrupted by electrical stimulation than non-SWR-related calcium transients, validating the capability of the system to detect and disrupt SWRs. Our dual recording method can be used to uncover the dynamic participation of individual neurons in SWRs and replay over extended time windows.
An Open-Source Real-Time Motion Correction Plug-In for Single-Photon Calcium Imaging of Head-Mounted Microscopy
Single-photon-based head-mounted microscopy is widely used to record the brain activities of freely-moving animals. However, during data acquisition, the free movement of animals will cause shaking in the field of view, which deteriorates subsequent neural signal analyses. Existing motion correction methods applied to calcium imaging data either focus on offline analyses or lack sufficient accuracy in real-time processing for single-photon data. In this study, we proposed an open-source real-time motion correction (RTMC) plug-in for single-photon calcium imaging data acquisition. The RTMC plug-in is a real-time subpixel registration algorithm that can run GPUs in UCLA Miniscope data acquisition software. When used with the UCLA Miniscope, the RTMC algorithm satisfies real-time processing requirements in terms of speed, memory, and accuracy. We tested the RTMC algorithm by extending a manual neuron labeling function to extract calcium signals in a real experimental setting. The results demonstrated that the neural calcium dynamics and calcium events can be restored with high accuracy from the calcium data that were collected by the UCLA Miniscope system embedded with our RTMC plug-in. Our method could become an essential component in brain science research, where real-time brain activity is needed for closed-loop experiments.
Cable-free brain imaging with miniature wireless microscopes
The invention of the miniaturized microscope has enabled neuroscientists to investigate neural mechanisms in freely moving mice. A lot of efforts have been made to optimize performance of the miniaturized microscope. However, the tethered cables limit the ability of mini-microscope systems to record neural activity from multiple mice simultaneously. Here, we present a wireless mini-microscope (wScope) that enables both real-time remote control and data preview during animal behavior; this design also supports simultaneous recording from up to 8 mice. The wScope has a mass of 2.7 g and a maximum frame rate of 25 Hz at 750 μm by 450 μm field of view with 1.8 μm resolution. We validated the wScopes in video-recording of the cerebral blood flow (CBF) and the activity of neurons in the primary visual cortex (V1) of different mice. The wScope provides a powerful tool for brain imaging of free moving animals, including large primates, in their much larger spaces and more naturalistic environments.
GABAergic CA1 neurons are more stable following context changes than glutamatergic cells
The CA1 region of the hippocampus contains both glutamatergic pyramidal cells and GABAergic interneurons. Numerous reports have characterized glutamatergic CAMK2A cell activity, showing how these cells respond to environmental changes such as local cue rotation and context re-sizing. Additionally, the long-term stability of spatial encoding and turnover of these cells across days is also well-characterized. In contrast, these classic hippocampal experiments have never been conducted with CA1 GABAergic cells. Here, we use chronic calcium imaging of male and female mice to compare the neural activity of VGAT and CAMK2A cells during exploration of unaltered environments and also during exposure to contexts before and after rotating and changing the length of the context across multiple recording days. Intriguingly, compared to CAMK2A cells, VGAT cells showed decreased remapping induced by environmental changes, such as context rotations and contextual length resizing. However, GABAergic neurons were also less likely than glutamatergic neurons to remain active and exhibit consistent place coding across recording days. Interestingly, despite showing significant spatial remapping across days, GABAergic cells had stable speed encoding between days. Thus, compared to glutamatergic cells, spatial encoding of GABAergic cells is more stable during within-session environmental perturbations, but is less stable across days. These insights may be crucial in accurately modeling the features and constraints of hippocampal dynamics in spatial coding.
Minian, an open-source miniscope analysis pipeline
Miniature microscopes have gained considerable traction for in vivo calcium imaging in freely behaving animals. However, extracting calcium signals from raw videos is a computationally complex problem and remains a bottleneck for many researchers utilizing single-photon in vivo calcium imaging. Despite the existence of many powerful analysis packages designed to detect and extract calcium dynamics, most have either key parameters that are hard-coded or insufficient step-by-step guidance and validations to help the users choose the best parameters. This makes it difficult to know whether the output is reliable and meets the assumptions necessary for proper analysis. Moreover, large memory demand is often a constraint for setting up these pipelines since it limits the choice of hardware to specialized computers. Given these difficulties, there is a need for a low memory demand, user-friendly tool offering interactive visualizations of how altering parameters at each step of the analysis affects data output. Our open-source analysis pipeline, Minian (miniscope analysis), facilitates the transparency and accessibility of single-photon calcium imaging analysis, permitting users with little computational experience to extract the location of cells and their corresponding calcium traces and deconvolved neural activities. Minian contains interactive visualization tools for every step of the analysis, as well as detailed documentation and tips on parameter exploration. Furthermore, Minian has relatively small memory demands and can be run on a laptop, making it available to labs that do not have access to specialized computational hardware. Minian has been validated to reliably and robustly extract calcium events across different brain regions and from different cell types. In practice, Minian provides an open-source calcium imaging analysis pipeline with user-friendly interactive visualizations to explore parameters and validate results.
Ubiquitous proximity to a critical state for collective neural activity in the CA1 region of freely moving mice
Using miniscope recordings of calcium fluorescence signals in the CA1 region of the hippocampus of mice, we monitor the neural activity of hippocampal regions while the animals are freely moving in an open chamber. Using a data-driven statistical modeling approach, the statistical properties of the recorded data are mapped to spin-glass models with pairwise interactions. Considering the parameter space of the model, the observed system is generally near a critical state between two distinct phases. The close proximity to the criticality is found to be robust against different ways of sampling and segmentation of the measured data. By independently altering the coupling distribution and the network structure of the statistical model, the network structures are found to be vital to maintain the proximity to the critical state. We further find the observed assignment of the coupling strengths makes the net coupling at each site more balanced with slight variation, which likely helps the maintenance of the critical state. Network analysis on the connectivity obtained by thresholding the coupling strengths find the connectivity of the networks to be well described by a random network model. These results are consistent across different experiments, sampling and segmentation choices in our analysis. A new result of our analysis is that the proximity to critical state and all the network properties are largely maintained even if random subsamples with a fraction of neurons are selected from the dataset as long as the number of neurons is more than 30 to 40. Thus the relevant degrees of freedom of CA1 region in the collective state we studied is not as large as one would expect.
In Vivo Multi-Day Calcium Imaging of CA1 Hippocampus in Freely Moving Rats Reveals a High Preponderance of Place Cells with Consistent Place Fields
Calcium imaging using GCaMP indicators and miniature microscopes has been used to image cellular populations during long timescales and in different task phases, as well as to determine neuronal circuit topology and organization. Because the hippocampus (HPC) is essential for tasks of memory, spatial navigation, and learning, calcium imaging of large populations of HPC neurons can provide new insight on cell changes over time during these tasks. All reported HPC in vivo calcium imaging experiments have been done in mouse. However, rats have many behavioral and physiological experimental advantages over mice. In this paper, we present the first (to our knowledge) in vivo calcium imaging from CA1 HPC in freely moving male rats. Using the UCLA Miniscope, we demonstrate that, in rat, hundreds of cells can be visualized and held across weeks. We show that calcium events in these cells are highly correlated with periods of movement, with few calcium events occurring during periods without movement. We additionally show that an extremely large percent of cells recorded during a navigational task are place cells (77.3 ± 5.0%, surpassing the percent seen during mouse calcium imaging), and that these cells enable accurate decoding of animal position and can be held over days with consistent place fields in a consistent spatial map. A detailed protocol is included, and implications of these advancements on in vivo imaging and place field literature are discussed. SIGNIFICANCE STATEMENT In vivo calcium imaging in freely moving animals allows the visualization of cellular activity across days. In this paper, we present the first in vivo Ca2+ recording from CA1 hippocampus (HPC) in freely moving rats. We demonstrate that hundreds of cells can be visualized and held across weeks, and that calcium activity corresponds to periods of movement. We show that a high percentage (77.3 ± 5.0%) of imaged cells are place cells, and that these place cells enable accurate decoding and can be held stably over days with little change in field location. Because the HPC is essential for many tasks involving memory, navigation, and learning, imaging of large populations of HPC neurons can shed new insight on cellular activity changes and organization.
Tracking longitudinal population dynamics of single neuronal calcium signal using SCOUT
Environmental enrichment leads to behavioral circadian shifts enhancing brain-wide functional connectivity between sensory cortices and eliciting increased hippocampal spiking
Environmental enrichment induces widespread neuronal changes, but the initiation of the cascade is unknown. We ascertained the critical period of divergence between environmental enriched (EE) and standard environment (SE) mice using continuous infrared (IR) videography, functional magnetic resonance imaging (fMRI), and neuron level calcium imaging. Naïve adult male mice (n = 285, C57BL/6J, postnatal day 60) were divided into SE and EE groups. We assessed the linear time-series of motion activity using a novel structural break test which examined the dataset for change in circadian and day-by-day motion activity. fMRI was used to map brain-wide response using a functional connectome analysis pipeline. Awake calcium imaging was performed on the dorsal CA1 pyramidal layer. We found the preeminent behavioral feature in EE was a forward shift in the circadian rhythm, prolongation of activity in the dark photoperiod, and overall decreased motion activity. The crepuscular period of dusk was seen as the critical period of divergence between EE and SE mice. The functional processes at dusk in EE included increased functional connectivity in the visual cortex, motor cortex, retrosplenial granular cortex, and cingulate cortex using seed-based analysis. Network based statistics found a modulated functional connectome in EE concentrated in two hubs: the hippocampal formation and isocortical network. These hubs experienced a higher node degree and significant enhanced edge connectivity. Calcium imaging revealed increased spikes per second and maximum firing rate in the dorsal CA1 pyramidal layer, in addition to location (anterior-posterior and medial-lateral) effect size differences between EE and SE. The emergence of functional-neuronal changes due to enrichment consisted of enhanced hippocampal-isocortex functional connectivity and CA1 neuronal increased spiking linked to a circadian shift during the dusk period. Future studies should explore the molecular consequences of enrichment inducing shifts in the circadian period.
The impact of pitolisant, an H3 receptor antagonist/inverse agonist, on perirhinal cortex activity in individual neuron and neuronal population levels
Histamine is a neurotransmitter that modulates neuronal activity and regulates various brain functions. Histamine H3 receptor (H3R) antagonists/inverse agonists enhance its release in most brain regions, including the cerebral cortex, which improves learning and memory and exerts an antiepileptic effect. However, the mechanism underlying the effect of H3R antagonists/inverse agonists on cortical neuronal activity in vivo remains unclear. Here, we show the mechanism by which pitolisant, an H3R antagonist/inverse agonist, influenced perirhinal cortex (PRh) activity in individual neuron and neuronal population levels. We monitored neuronal activity in the PRh of freely moving mice using in vivo Ca2+ imaging through a miniaturized one-photon microscope. Pitolisant increased the activity of some PRh neurons while decreasing the activity of others without affecting the mean neuronal activity across neurons. Moreover, it increases neuron pairs with synchronous activity in excitatory-responsive neuronal populations. Furthermore, machine learning analysis revealed that pitolisant altered the neuronal population activity. The changes in the population activity were dependent on the neurons that were excited and inhibited by pitolisant treatment. These findings indicate that pitolisant influences the activity of a subset of PRh neurons by increasing the synchronous activity and modifying the population activity.
The representation of context in mouse hippocampus is preserved despite neural drift
The hippocampus is thought to mediate episodic memory through the instantiation and reinstatement of context-specific cognitive maps. However, recent longitudinal experiments have challenged this view, reporting that most hippocampal cells change their tuning properties over days even in the same environment. Often referred to as neural or representational drift, these dynamics raise questions about the capacity and content of the hippocampal code. One such question is whether and how these long-term dynamics impact the hippocampal code for context. To address this, we image large CA1 populations over more than a month of daily experience as freely behaving mice participate in an extended geometric morph paradigm. We find that long-timescale changes in population activity occur orthogonally to the representation of context in network space, allowing for consistent readout of contextual information across weeks. This population-level structure is supported by heterogeneous patterns of activity at the level of individual cells, where we observe evidence of a positive relationship between interpretable contextual coding and long-term stability. Together, these results demonstrate that long-timescale changes to the CA1 spatial code preserve the relative structure of contextual representation.
Highly unstable heterogeneous representations in VIP interneurons of the anterior cingulate cortex
A hallmark of the anterior cingulate cortex (ACC) is its functional heterogeneity. Functional and imaging studies revealed its importance in the encoding of anxiety-related and social stimuli, but it is unknown how microcircuits within the ACC encode these distinct stimuli. One type of inhibitory interneuron, which is positive for vasoactive intestinal peptide (VIP), is known to modulate the activity of pyramidal cells in local microcircuits, but it is unknown whether VIP cells in the ACC (VIPACC) are engaged by particular contexts or stimuli. Additionally, recent studies demonstrated that neuronal representations in other cortical areas can change over time at the level of the individual neuron. However, it is not known whether stimulus representations in the ACC remain stable over time. Using in vivo Ca2+ imaging and miniscopes in freely behaving mice to monitor neuronal activity with cellular resolution, we identified individual VIPACC that preferentially activated to distinct stimuli across diverse tasks. Importantly, although the population-level activity of the VIPACC remained stable across trials, the stimulus-selectivity of individual interneurons changed rapidly. These findings demonstrate marked functional heterogeneity and instability within interneuron populations in the ACC. This work contributes to our understanding of how the cortex encodes information across diverse contexts and provides insight into the complexity of neural processes involved in anxiety and social behavior.
Longitudinal dynamics of microvascular recovery after acquired cortical injury
Acquired brain injuries due to trauma damage the cortical vasculature, which in turn impairs blood flow to injured tissues. There are reports of vascular morphological recovery following traumatic brain injury, but the remodeling process has not been examined longitudinally in detail after injury in vivo. Understanding the dynamic processes that influence recovery is thus critically important. We evaluated the longitudinal and dynamic microvascular recovery and remodeling up to 2 months post injury using live brain miniscope and 2-photon microscopic imaging. The new imaging approaches captured dynamic morphological and functional recovery processes at high spatial and temporal resolution in vivo. Vessel painting documented the initial loss and subsequent temporal morphological vascular recovery at the injury site. Miniscopes were used to longitudinally image the temporal dynamics of vascular repair in vivo after brain injury in individual mice across each cohort. We observe near-immediate nascent growth of new vessels in and adjacent to the injury site that peaks between 14 and 21 days post injury. 2-photon microscopy confirms new vascular growth and further demonstrates differences between cortical layers after cortical injury: large vessels persist in deeper cortical layers (> 200 μm), while superficial layers exhibit a dense plexus of fine (and often non-perfused) vessels displaying regrowth. Functionally, blood flow increases mirror increasing vascular density. Filopodia development and endothelial sprouting is measurable within 3 days post injury that rapidly transforms regions devoid of vessels to dense vascular plexus in which new vessels become increasingly perfused. Within 7 days post injury, blood flow is observed in these nascent vessels. Behavioral analysis reveals improved vascular modulation after 9 days post injury, consistent with vascular regrowth. We conclude that morphological recovery events are closely linked to functional recovery of blood flow to the compromised tissues, which subsequently leads to improved behavioral outcomes.
Prefrontal pyramidal neurons are critical for all phases of working memory
Electrophysiological correlates of sound processing in the limbic pathways and implications for tinnitus-related anxiety
This thesis investigates the electrophysiological correlates of the hippocampus and medial prefrontal cortex to auditory activity, either in an animal model of tinnitus induced by highdose salicylate or in the response to loud broadband noise in mice during locomotion. The results are organized into four chapters with three experimental articles (chapter 1-3, with 2 published articles and one manuscript in preparation) and one book protocol (chapter 4, preprint). In the first article (Winne et al. 2019), we found that a high dose of salicylate (300mg/kg) induced type 2 theta (4-6Hz) oscillation in the ventral hippocampus (VHipp) as well as elicits anxiety-like behavior in mice. In addition, high dose salicylate administration abolished dorsal hippocampus (DHipp) type 1 theta (7-10Hz) correlation with running speed. In the second article (Winne et al. 2020), we showed that after pretreatment with salicylate, only young mice with preserved hearing exhibited anxiety-like behavior, compared to old mice. We hypothesize that old C57BL/6 mice might already have altered hearing due to agerelated hearing loss, and thus are less likely to present salicylate-induced tinnitus and tinnitusrelated anxiety. In young mice, an increase in type 2 theta oscillations and slow gamma (30- 60Hz) was observed in the VHipp, as well as the medial prefrontal cortex (mPFC), after salicylate treatment during the open field and in the elevated plus-maze. Furthermore, we also noticed an increased theta 2 coherence between VHipp and mPFC during the tasks. Lastly, pretreatment of mice with a single dose of the psychedelic substance 5-MeO-DMT prevented the emergence of anxiety-associated behaviors and the induction of type 2 theta and slow gamma after salicylate injection. Based on electrophysiological and behavioral evidence, articles 1 and 2 indicate that anxiety-related mechanisms are triggered in the salicylate-model of tinnitus. Chapter 3 focuses on mice with normal hearing, specifically how loud noise stimuli modulates limbic circuits, and the Reticular-limbic auditory pathway. Using combined approaches (silence/noise, chemo- and optogenetics), type 1 theta oscillations (7- 10Hz) of the DHipp were shown to be accelerated by loud broadband noise. Specifically, noise input relayed from the entorhinal cortex, and medial septum could modulate DHipp theta 1 oscillations while decreasing the activity of the auditory cortex did not affect this noncanonical pathway of loud noise processing. We also verified that the activation of this pathway increased coherence between the DHipp and medial prefrontal cortex. We hypothesize that increasing the activity of DHipp neurons through loud auditory stimuli could be a biological strategy to quickly identify dangerous environments and increase the alertness of animals in response to possible surrounding threats. Lastly, in Chapter 4 (Winne et al., 2021, pre-print), we discuss a technique of calcium imaging of freely moving animals, using miniaturised microendoscopes (‘miniscopes’) and describe in detail the lens and baseplate implant surgery for the UCLA Miniscope V3 and V4. We used a gradient index (GRIN) lens for the hippocampus and a combination of a prism and GRIN lens for the auditory and motor cortex, with adaptations from published work for an improved lens fixation (GRIN-relayPrism). Finally, we comment on the differences between analysis software. In conclusion, this thesis demonstrates electrophysiological correlates of salicylate-induced tinnitus that strengthen the neurobiological link between tinnitus and anxiety. More so, limbic pathways relaying loud noise information were also shown to modulate the activity of the hippocampus and the medial prefrontal cortex. Together these results evidence new electrophysiological signatures of sound processing in the limbic system.
A longitudinal CA2/3 to CA1 circuit for initiating context-dependent associative learning
Associating environmental context with emotional experience is a vital brain function that requires the activity in both dorsal and ventral hippocampus. While only ventral hippocampus connects with the amygdala, a hub for fear learning, it remains unclear how the two hippocampal areas interact during contextual fear conditioning (CFC). We found that projections from dorsal CA2/CA3 (dCA2/3) to the dorsal part of ventral CA1 (vCA1d) contributed significantly to CFC. Deep-brain Ca2+ imaging revealed a CFC-enhanced difference in neuronal activities evoked by conditioned vs. neutral environmental context in both dCA2/3 and vCA1d areas. Notably, contextual fear retrieval correlated with changes in conditioned context-evoked activity in vCA1d, but not in dCA2/3. Furthermore, slice recordings showed that CFC potentiated dCA2/3-to-vCA1d projection strength, in line with coordinated elevation of monosynaptic excitation and reduction of disynaptic inhibition mediated by somatostatin-expressing interneurons. Thus, vCA1d represents a critical site for initiating emotional association with contextual information from dCA2/3.
Rapid deep widefield neuron finder driven by virtual calcium imaging data
Widefield microscope provides optical access to multi-millimeter fields of view and thousands of neurons in mammalian brains at video rate. However, calcium imaging at cellular resolution has been mostly contaminated by tissue scattering and background signals, making neuronal activities extraction challenging and time-consuming. Here we present a deep widefield neuron finder (DeepWonder), which is fueled by simulated calcium recordings but effectively works on experimental data with an order of magnitude faster speed and improved inference accuracy than traditional approaches. The efficient DeepWonder accomplished fifty-fold signal-to-background ratio enhancement in processing terabytes-scale cortex-wide recording, with over 14000 neurons extracted in 17 hours in workstation-grade computing resources compared to nearly week-long processing time with previous methods. DeepWonder circumvented the numerous computational resources and could serve as a guideline to massive data processing in widefield neuronal imaging.
Spatial coding defects of hippocampal neural ensemble calcium activities in the triple-transgenic Alzheimer's disease mouse model
Alzheimer's disease (AD) causes progressive age-related defects in memory and cognitive function and has emerged as a major health and socio-economic concern in the US and worldwide. To develop effective therapeutic treatments for AD, we need to better understand the neural mechanisms by which AD causes memory loss and cognitive deficits. Here we examine large-scale hippocampal neural population calcium activities imaged at single cell resolution in a triple-transgenic Alzheimer's disease mouse model (3xTg-AD) that presents both amyloid plaque and neurofibrillary pathological features along with age-related behavioral defects. To measure encoding of environmental location in hippocampal neural ensembles in the 3xTg-AD mice in vivo, we performed GCaMP6-based calcium imaging using head-mounted, miniature fluorescent microscopes (“miniscopes”) on freely moving animals. We compared hippocampal CA1 excitatory neural ensemble activities during open-field exploration and track-based route-running behaviors in age-matched AD and control mice at young (3–6.5 months old) and old (18–21 months old) ages. During open-field exploration, 3xTg-AD CA1 excitatory cells display significantly higher calcium activity rates compared with Non-Tg controls for both the young and old age groups, suggesting that in vivo enhanced neuronal calcium ensemble activity is a disease feature. CA1 neuronal populations of 3xTg-AD mice show lower spatial information scores compared with control mice. The spatial firing of CA1 neurons of old 3xTg-AD mice also displays higher sparsity and spatial coherence, indicating less place specificity for spatial representation. We find locomotor speed significantly modulates the amplitude of hippocampal neural calcium ensemble activities to a greater extent in 3xTg-AD mice during open field exploration. Our data offer new and comprehensive information about age-dependent neural circuit activity changes in this important AD mouse model and provide strong evidence that spatial coding defects in the neuronal population activities are associated with AD pathology and AD-related memory behavioral deficits.
Custom-Built Operant Conditioning Setup for Calcium Imaging and Cognitive Testing in Freely Moving Mice
Visual Abstract Download figureOpen in new tabDownload powerpoint Operant chambers are widely used in animal research to study cognition, motivation, and learning processes. Paired with the rapidly developing technologies for brain imaging and manipulations of brain activity, operant conditioning chambers are a powerful tool for neuroscience research. The behavioral testing and imaging setups that are commercially available are often quite costly. Here, we present a custom-built operant chamber that can be constructed in a few days by an unexperienced user with relatively inexpensive, widely available materials. The advantages of our operant setup compared with other open-source and closed-source solutions are its relatively low cost, its support of complex behavioral tasks, its user-friendly setup, and its validated functionality with video imaging of behavior and calcium imaging using the UCLA Miniscope. Using this setup, we replicate our previously published findings showing that mice exposed to social defeat stress in adolescence have inhibitory control impairments in the Go/No-Go task when they reach adulthood. We also present calcium imaging data of medial prefrontal cortex (mPFC) neuronal activity acquired during Go/No-Go testing in freely moving mice and show that neuronal population activity increases from day 1 to day 14 of the task. We propose that our operant chamber is a cheaper alternative to its commercially available counterparts and offers a better balance between versatility and user-friendly setup than other open-source alternatives.
Optogenetic inhibition of indirect pathway neurons in the dorsomedial striatum reduces excessive grooming in Sapap3-knockout mice
Excessive grooming of Sapap3-KO mice has been used as a model of obsessive-compulsive disorder (OCD). Previous studies suggest that dysregulation of cortico-striatal circuits is critically important in the generation of compulsive behaviors, and it has been proposed that the alteration in the activity patterns of striatal circuitry underlies the excessive grooming observed in Sapap3-KO mice. To test this hypothesis, we used in-vivo calcium imaging of individual cells to record striatal activity in these animals and optogenetic inhibition to manipulate this activity. We identified striatal neurons that are modulated during grooming behavior and found that their proportion is significantly larger in Sapap3-KO mice compared to wild-type littermates. Inhibition of striatal cells in Sapap3-KO mice increased the number of grooming episodes observed. Remarkably, the specific inhibition of indirect pathway neurons decreased the occurrence of grooming events. Our results indicate that there is striatal neural activity related to excessive grooming engagement in Sapap3-KO mice. We also demonstrate, for the first time, that specific inhibition of striatal indirect pathway neurons reduces this compulsive phenotype, suggesting that treatments that alleviate compulsive symptoms in OCD patients may exert their effects through this specific striatal population.
Examining Hippocampal Activity During Behaviour in a Mouse Model of Alzheimer’s Disease
Although Alzheimer’s disease was first identified in 1906 by Alois Alzheimer the extensive pathophysiology that produces the disease is still poorly understood. It is a neurodegenerative disorder characterised by a marked reduction in the volume of the cerebral cortex and hippocampus, which produces general mental decline including memory loss and confusion, inability to create new memories, behavioural changes, and eventual death. Therefore, the primary aim of this project was to investigate neuronal calcium network dynamics responsible for memory deficits with simultaneous behavioural in transgenic mice [APPswe/PS1dE9] through the utilisation of head-mountable miniaturised microscopes. To image activity in the hippocampus mice were virally injected with the calcium activity reporter (GCaMP7) into the CA1, a GRIN lens was implanted superiorly for optical access, and a baseplate was secured to the skull to allow the future attachment of a miniscope. Male transgenic and non-transgenic littermate mice aged 15-17 months underwent open field, y-maze, and novel object recognition behaviour tests. These cognitive assessments query anxiety and fear (time spent in the exterior region and freezing in open field), short-term spatial memory (alternations in y-maze and novel object location), and short-term and long-term object memory (novel object recognition). Neuronal dynamics were simultaneously recorded through in vivo miniscope imaging of GCaMP7 expressing hippocampal CA1 neurons. Neuronal dynamics (mean amplitude, frequency, spike width, participation, and location specific firing) were examined, correlated with behaviour, and compared across genotypes. When examining two separate cohorts there were significant phenotypical differences between transgenic and wildtype regarding ambulation, open field anxiety and freezing, ymaze and novel object location short-term spatial memory measures, and novel object shortterm and long-term object memory measures. There were also significant differences between genotypes in mean spike width and participation – with aged transgenic mice exhibiting neuronal hyperactivity. Additionally, when staining for amyloid-β plaques (a hallmark histology marker) were witnessed in the transgenic hippocampus and cortex but not wildtype. This project in conjunction with previous work has established a protocol that permits queries into the underlying neuropathology to link behavioural deficits to neuronal changes.
A Semi-Supervised Deep Learning Solution to Cell Registration in Video Data from Calcium Imaging Studies
Calcium imaging is a technique that detects the transient changes of calcium ions during an action potential. It enables researchers to record thousands of neurons simultaneously. However, researchers require a method of tracking individual neurons across different recordings for analyses; this problem is known as cell registration. Current approaches rely on image alignment and statistical correlations to perform cell registration. However, these approaches are often slow and require tuning when used on novel datasets. Our end-to-end approach begins with the generation of semi-synthetic data from a few real calcium recordings examples. This allows the option of creating a dataset of any size to train a deep learning model that performs cell registration. We tested three network architectures that are specialized for image patch comparisons to perform cell registration: Siamese, 2-channel, and center-surround networks. These networks were trained once using only semi-synthetic data and were tested on real held-out datasets. The three models were evaluated on a hand-annotated Chronic Social Defeat Stress dataset and were compared to an existing cell registration approach, CellReg. Our best model, center-surround, achieved an average accuracy of 80.17% while maintaining a precision score of 0.8982. Unlike previous methods, our end-to-end method introduces a new approach of generating synthetic neuronal data that mimics real world data. Next, we show that the cell registration problem can be structured as a binary classification problem to be solved by a deep learning model. Therefore, with our generated synthetic data, we train deep learning models that reliably map neurons across recordings in a scalable manner while requiring minimal parameter tuning. Our contribution includes a versatile approach to cell registration that introduces a novel way of generating semi-synthetic data which is used to train a deep learning model to reliably match cells from raw imaging data. Alternate abstract: LUimagerie calcigue est une techniğue gui dötecte les changements transitoires des ions calcium pendant un potentiel d'action. Il permet aux chercheurs d'enregistrer simultanöment des milliers de neurones. Cependant, les chercheurs ont besoin d'une möthode de suivi des neurones individuel â travers difförents enregistremenis pour les analyses; ce problâme est connu sous le nom d'enreğistrement cellulaire. Les approches actuelles reposent sur Valignement des images et les corrölations statistigues pour effectuer Valignement des cellules. Cependant, ces approches sont souvent lentes et doivent ötre ajustdes lorsgu'elles sont utilisdes sur de nou- veaux ensembles de donndes. Notre approche de bout en bout commence par la gönöration de donndes semi-synth&tigues â partir de guelgues exemples rdels d'enregistrements calcigues. Cela permet de cröer un ensemble de donndes de toutes tailles pour former un modöle d'apprentissage en profondeur gui effectue Venregistrement des cellules. Nous avons mis A İ'essai trois architectures de röseau spöcialisdes dans la comparaison de correctifs d'image pour Venregistrement de cellules : röseaux siamois, â deux canaux et â surround central. Ces röseaux ont ete formes une fois en utilisant seulement des donndes semi-synthstigues et ont ete testis sur des ensembiles de donndes relles. Les trois modâles ont 6t8 övaluds a Vaide d'un ensemble de donnâes annotdes A la main sur le stress social chronigue et ont öt comparös â une approche d'enreğistrement cellulaire existante, Cell- Reg. Notre meilleur modöle, centre, a atteint une precision moyenne de 80,179 tout en maintenant un score de pröcision de 0,8982. Contrairement aux möthodes pröcödentes, notre mâthode de bout en bout introduit une nouvelle approche de gönsration de donndes neuronales synthötigtes gui imitent les donndes du monde röel. Ensuite, nous maontrons gue le problöme d'enregistremeni des cellules peut dtre siructur& comme un problöme de classification binaire â rösoudre par un modöle d'apprentissage profond. Par consöguenit, avec nas donndes synthâtigues göndrdes, nous formons des modöles d'apprentissage profond gui cartographient de maniğre fiable les neurones â travers les enregistremenis de maniöre öyolutive bout en ex- igeant un röglage minimal des paramötres. Notre contribution comprend une ap- proche polyvalente de Venregistrement des cellules güi introduit une nouvelle façon de gönerer des donndes semi-synthâtigues gui est utilisde pour former un modöle d'apprentissage profond pour apparier de façon fiable les cellules â partir de danndes d'imagerie brutes.
Coordination of Escape Circuits Orchestrates Versatile Flight and Controls Escape Vigor from Multimodal Threats
Naturalistic escape requires versatile context-specific flight with rapid evaluation of local geometry to identify and use efficient escape routes. It is unknown how spatial navigation and escape circuits are recruited to produce context-specific flight. Using mice, we show activity in cholecystokinin-expressing hypothalamic dorsal premammillary cells (PMd-cck) is sufficient and necessary for context-specific escape that adapts to each environment’s layout. Contrastingly, numerous other nuclei implicated in flight only induced stereotyped panic-related escape. We reasoned the PMd can induce context-specific escape because it projects to both escape and spatial navigation nuclei. Indeed, activity in PMd-cck projections to thalamic spatial navigation circuits are only necessary for context-specific escape induced by moderate threats, but not panic-related stereotyped escape caused by perceived asphyxiation. Conversely, the PMd projection to the escape-inducing dorsal periaqueductal gray projection is necessary for all escapes tested. Thus, PMd-cck controls versatile flight, engaging spatial navigation and escape circuits. It is additionally unknown if a single circuit controls escape vigor from innate and conditioned threats. We further demonstrate that PMd-cck cells are activated during escape, but not other defensive behaviors. PMd-cck ensemble activity can also predict future escape. Furthermore, PMd inhibition decreases escape speed from both innate and conditioned threats. Inhibition of the PMd-cck projection to the dlPAG also decreased escape speed. Intriguingly, PMd- cck and dlPAG activity in mice showed higher mutual information during exposure to innate and conditioned threats. In parallel, human fMRI data show that a posterior hypothalamic-to-dlPAG pathway increased activity during exposure to aversive images, indicating that a similar pathway may possibly have a related role in humans. Our data identify the PMd-dlPAG circuit as a central node, controlling escape vigor elicited by both innate and conditioned threats.
Miniature Microscopy of Hippocampal CA1 to Identify Engram Cells and Record Calcium Transients for Analyses of Ensemble Activities
In the brain, memories are thought to be stored by neurons termed engram cells that are activated during learning. Engram cells in the hippocampus can be targeted in c-Fos-tTA mice and have been identified as those that are activated during the acquisition and retrieval of a specific memory. Notably, the recent application of a genetically encoded calcium (Ca2+) indicator (GECI) combined with miniature microscopy (i.e., Ca2+ imaging) enables us to observe neuronal activity within deep brain regions of rodents. Here, we explain the technical details of systems for engram cell targeting and Ca2+ imaging with a miniature microscope specifically applied for hippocampal CA1. In addition, we introduce a method to combine engram identification and Ca2+ imaging, which provides the ability to analyze the ensemble activity of engram cells during memory processes.
Distributed processing for action control by prelimbic circuits targeting anterior-posterior dorsal striatal subregions
Fronto-striatal circuits have been extensively implicated in the cognitive control of behavioral output for both social and appetitive rewards. The functional diversity of prefrontal cortical populations is strongly dependent on their synaptic targets, with control of motor output strongly mediated by connectivity to the dorsal striatum. Despite evidence for functional diversity along the anterior-posterior axis of the dorsomedial striatum (DMS), it is unclear how distinct fronto- striatal sub-circuits support neural computations essential for action selection. Here we identify prefrontal populations targeting distinct DMS subregions and characterize their functional roles. We first performed neural circuit tracing to reveal segregated prefrontal populations defined by anterior/posterior dorsomedial striatal target. We then probed the functional relevance of these parallel circuits via in vivo calcium imaging and temporally precise causal manipulations during a feedback-based 2-alternative choice task. Single-photon imaging revealed circuit-specific representations of task-relevant information with prelimbic neurons targeting anterior DMS (PL::A- DMS) uniquely encoded choices and responses to negative outcomes, while prelimbic neurons targeting posterior DMS (PL::P-DMS) encoded internal representations of value and positive outcomes contingent on prior choice. Consistent with this distributed coding, optogenetic inhibition of PL::A-DMS circuits strongly impacted choice monitoring and behavioral control in response to negative outcomes while perturbation of PL::P-DMS signals impaired task engagement and strategies following positive outcomes. Di-synaptic retrograde tracing uncovered differences in afferent connectivity that may underlie these pathways functional divergence. Together our data uncover novel PL populations engaged in distributed processing for action control. SUMMARYPrelimbic cortex engages A- and P-DMS via distinct circuitsPL::A-DMS and PL::P-DMS pathways encode divergent aspects of a simple goal-directed taskPL::A-DMS pathways shape responding to negative outcomes via multiple mechanismsPL::P-DMS pathways guide engagement and choices in response to positive outcomesAfferent connectomes of PL neurons defined by A-P DMS target are distinct
Cognitive control persistently enhances hippocampal information processing
Could learning that uses cognitive control to judiciously use relevant information while ignoring distractions generally improve brain function, beyond forming explicit memories? According to a neuroplasticity hypothesis for how some cognitive behavioural therapies are effective, cognitive control training (CCT) changes neural circuit information processing1–3. Here we investigated whether CCT persistently alters hippocampal neural circuit function. We show that mice learned and remembered a conditioned place avoidance during CCT that required ignoring irrelevant locations of shock. CCT facilitated learning new tasks in novel environments for several weeks, relative to unconditioned controls and control mice that avoided the same place during reduced distraction. CCT rapidly changes entorhinal cortex-to-dentate gyrus synaptic circuit function, resulting in an excitatory–inhibitory subcircuit change that persists for months. CCT increases inhibition that attenuates the dentate response to medial entorhinal cortical input, and through disinhibition, potentiates the response to strong inputs, pointing to overall signal-to-noise enhancement. These neurobiological findings support the neuroplasticity hypothesis that, as well as storing item–event associations, CCT persistently optimizes neural circuit information processing.
Cognitive control persistently enhances hippocampal information processing
Could learning that uses cognitive control to judiciously use relevant information while ignoring distractions generally improve brain function, beyond forming explicit memories? According to a neuroplasticity hypothesis for how some cognitive behavioural therapies are effective, cognitive control training (CCT) changes neural circuit information processing1–3. Here we investigated whether CCT persistently alters hippocampal neural circuit function. We show that mice learned and remembered a conditioned place avoidance during CCT that required ignoring irrelevant locations of shock. CCT facilitated learning new tasks in novel environments for several weeks, relative to unconditioned controls and control mice that avoided the same place during reduced distraction. CCT rapidly changes entorhinal cortex-to-dentate gyrus synaptic circuit function, resulting in an excitatory–inhibitory subcircuit change that persists for months. CCT increases inhibition that attenuates the dentate response to medial entorhinal cortical input, and through disinhibition, potentiates the response to strong inputs, pointing to overall signal-to-noise enhancement. These neurobiological findings support the neuroplasticity hypothesis that, as well as storing item–event associations, CCT persistently optimizes neural circuit information processing.
Vagus nerve stimulation accelerates motor learning through cholinergic modulation
Vagus nerve stimulation (VNS) is a neuromodulation therapy for a broad and rapidly expanding set of neurologic conditions. Classically used to treat epilepsy and depression, VNS has recently received FDA approval for stroke rehabilitation and is under preclinical and clinical investigation for other neurologic indications. Despite benefits across a diverse range of neurological disorders, the mechanism through which VNS influences central nervous system circuitry is not well described, limiting therapeutic optimization. A deeper understanding of the influence of VNS on neural circuits and activity is needed to maximize the use of VNS therapy across a broad range of neurologic conditions. To investigate how VNS can influence the neurons and circuits that underlie behavior, we paired VNS with upper limb movement in mice learning a skilled motor task. We leveraged genetic tools to perform optogenetic circuit dissection, as well as longitudinal in vivo imaging of calcium activity in cortical neurons to understand the effect of VNS on neural function. We found that VNS robustly enhanced motor learning when temporally paired with successful movement outcome, while randomly applied VNS impaired learning. This suggests that temporally-precise VNS may act through augmenting outcome cues, such as reinforcement signals. Within motor cortex, VNS paired with movement outcome selectively modulates the neural population that represents outcome, but not other movement-related neurons, across both acute and behaviorally-relevant timescales. Phasic cholinergic signaling from basal forebrain is required both for VNS-driven improvements in motor learning and the effects on neural activity in M1. These results indicate that VNS enhances motor learning through precisely-timed phasic cholinergic signaling to reinforce outcome, resulting in the recruitment of specific, behaviorally-relevant cortical circuits. A deeper understanding of the mechanisms of VNS on neurons, circuits and behavior provides new opportunities to optimize VNS to treat neurologic conditions.
Neural control of affiliative touch in prosocial interaction
The ability to help and care for others fosters social cohesiveness and is vital to the physical and emotional well-being of social species, including humans1–3. Affiliative social touch, such as allogrooming (grooming behaviour directed towards another individual), is a major type of prosocial behaviour that provides comfort to others1–6. Affiliative touch serves to establish and strengthen social bonds between animals and can help to console distressed conspecifics. However, the neural circuits that promote prosocial affiliative touch have remained unclear. Here we show that mice exhibit affiliative allogrooming behaviour towards distressed partners, providing a consoling effect. The increase in allogrooming occurs in response to different types of stressors and can be elicited by olfactory cues from distressed individuals. Using microendoscopic calcium imaging, we find that neural activity in the medial amygdala (MeA) responds differentially to naive and distressed conspecifics and encodes allogrooming behaviour. Through intersectional functional manipulations, we establish a direct causal role of the MeA in controlling affiliative allogrooming and identify a select, tachykinin-expressing subpopulation of MeA GABAergic (γ-aminobutyric-acid-expressing) neurons that promote this behaviour through their projections to the medial preoptic area. Together, our study demonstrates that mice display prosocial comforting behaviour and reveals a neural circuit mechanism that underlies the encoding and control of affiliative touch during prosocial interactions.
A synaptic temperature sensor for body cooling
Calcium imaging and analysis of the mouse hippocampus or neocortex using miniature microendoscopes
Calcium imaging using miniscopes are becoming increasingly popular in the neuroscience community with a multitude of microendoscope versions, lens systems and packages for analysis available as open source. Here we describe in detail how to implant lenses and baseplates for the University of California at Los Angeles (UCLA) miniscope V3 and V4 using a Gradient Index (GRIN) lens for the hippocampus or a combination of prism and GRIN lens for recording in the neocortex. Our protocols contain adaptations from several published protocols however the GRIN-relay-Prism lens system protocol is developed in-house and not previously described. This chapter also suggests how lenses may be re-used and how to test the quality of a reused lens. Lastly, we comment on the difference between analysis software and the computational needs for different packages.
Fast Calcium Trace Extraction for Large-Field-of-View Miniscope
Recent advancement in miniaturized calcium imaging microscope enables monitoring the cell activity from hundreds of neurons simultaneously in vivo. However, extracting calcium traces from a large population of cells in real time under a strict resource and energy constraint remains a challenge. To overcome it, we design a customized accelerator on a low-power FPGA for fast calcium trace extraction. This enables us to extract calcium traces from hundreds of cells in real time with a short and deterministic latency. In addition, we propose a series of techniques, including the region segmentation, the double buffering and the fast forward mechanisms, to further reduce the latency with minimal overhead. Applying these techniques, our implementation can achieve real-time calcium trace extraction for a maximum of 1024 cells from a 512×512 calcium video with sub-ms latency, which is promising in support of closed loop neurofeedhack applications.
Neural circuit dynamics of drug-context associative learning in the hippocampus
The environmental context associated with previous drug consumption serves as a potent trigger for relapse to drug use. The mechanism by which existing neural representations of context are modified to incorporate information associated with a given drug however, remains unknown. Using longitudinal calcium imaging in freely behaving mice, we reveal that drug-context associations for psychostimulants and opioids are encoded in a subset of hippocampal neurons. In these neurons, drug context pairing in a conditioned place preference task weakened their spatial coding for the nondrug-paired context, with drug-induced changes to spatial coding predictive of drug-seeking behavior. Furthermore, the dissociative drug ketamine blocked both the drug-induced changes to hippocampal coding and corresponding drug-seeking behavior. Together, this work reveals how drugs of abuse can alter the hippocampal circuit to encode drug-context associations and points to the hippocampus as a key node in the cognitive process of drug addiction and context-induced drug relapse.
Dorsal premammillary projection to periaqueductal gray controls escape vigor from innate and conditioned threats
Escape from threats has paramount importance for survival. However, it is unknown if a single circuit controls escape vigor from innate and conditioned threats. Cholecystokinin (cck)-expressing cells in the hypothalamic dorsal premammillary nucleus (PMd) are necessary for initiating escape from innate threats via a projection to the dorsolateral periaqueductal gray (dlPAG). We now show that in mice PMd-cck cells are activated during escape, but not other defensive behaviors. PMd-cck ensemble activity can also predict future escape. Furthermore, PMd inhibition decreases escape speed from both innate and conditioned threats. Inhibition of the PMd-cck projection to the dlPAG also decreased escape speed. Intriguingly, PMd-cck and dlPAG activity in mice showed higher mutual information during exposure to innate and conditioned threats. In parallel, human functional magnetic resonance imaging data show that a posterior hypothalamic-to-dlPAG pathway increased activity during exposure to aversive images, indicating that a similar pathway may possibly have a related role in humans. Our data identify the PMd-dlPAG circuit as a central node, controlling escape vigor elicited by both innate and conditioned threats.
Conjunctive spatial and self-motion codes are topographically organized in the GABAergic cells of the lateral septum
The hippocampal spatial code’s relevance for downstream neuronal populations—particularly its major subcortical output the lateral septum (LS)—is still poorly understood. Here, using calcium imaging combined with unbiased analytical methods, we functionally characterized and compared the spatial tuning of LS GABAergic cells to those of dorsal CA3 and CA1 cells. We identified a significant number of LS cells that are modulated by place, speed, acceleration, and direction, as well as conjunctions of these properties, directly comparable to hippocampal CA1 and CA3 spatially modulated cells. Interestingly, Bayesian decoding of position based on LS spatial cells reflected the animal’s location as accurately as decoding using the activity of hippocampal pyramidal cells. A portion of LS cells showed stable spatial codes over the course of multiple days, potentially reflecting long-term episodic memory. The distributions of cells exhibiting these properties formed gradients along the anterior–posterior and dorsal–ventral axes of the LS, directly reflecting the topographical organization of hippocampal inputs to the LS. Finally, we show using transsynaptic tracing that LS neurons receiving CA3 and CA1 excitatory input send projections to the hypothalamus and medial septum, regions that are not targeted directly by principal cells of the dorsal hippocampus. Together, our findings demonstrate that the LS accurately and robustly represents spatial, directional as well as self-motion information and is uniquely positioned to relay this information from the hippocampus to its downstream regions, thus occupying a key position within a distributed spatial memory network.
Opposing roles for striatonigral and striatopallidal neurons in dorsolateral striatum in consolidating new instrumental actions
Comparatively little is known about how new instrumental actions are encoded in the brain. Using whole-brain c-Fos mapping, we show that neural activity is increased in the anterior dorsolateral striatum (aDLS) of mice that successfully learn a new lever-press response to earn food rewards. Post-learning chemogenetic inhibition of aDLS disrupts consolidation of the new instrumental response. Similarly, post-learning infusion of the protein synthesis inhibitor anisomycin into the aDLS disrupts consolidation of the new response. Activity of D1 receptor-expressing medium spiny neurons (D1-MSNs) increases and D2-MSNs activity decreases in the aDLS during consolidation. Chemogenetic inhibition of D1-MSNs in aDLS disrupts the consolidation process whereas D2-MSN inhibition strengthens consolidation but blocks the expression of previously learned habit-like responses. These findings suggest that D1-MSNs in the aDLS encode new instrumental actions whereas D2-MSNs oppose this new learning and instead promote expression of habitual actions.
A Modified Miniscope System for Simultaneous Electrophysiology and Calcium Imaging in vivo
The miniscope system is one of the calcium (Ca2+) imaging tools with small size and lightweight and can realize the deep-brain Ca2+ imaging not confined to the cerebral cortex. Combining Ca2+ imaging and electrophysiology recording has been an efficient method for extracting high temporal-spatial resolution signals in the brain. In this study, a particular electrode probe was developed and assembled on the imaging lens to modify the miniscope system. The electrode probe can be tightly integrated into the lens of the miniscope without increasing the volume, weight, and implantation complexity.
Large-scale calcium imaging with a head-mounted axial scanning 3D fluorescence microscope
Miniaturized fluorescence microscopes are becoming more important for deciphering the neural codes underlying various brain functions. With gradient index (GRIN) lenses, these devices enable recording neuronal activity in deep brain structures. However, to minimize any damage to brain tissues and local circuits, the diameter of the GRIN lens should be 0.5–1 mm, resulting in a small field of view. Volumetric imaging capability might increase the number of neurons imaged through the lenses considering the three-dimensional (3D) structure of neural circuits in the brain. To observe 3D calcium dynamics, we developed a miniaturized microscope with an electrically tunable lens and a novel CNMF-based neural signal extraction algorithm for wide-field 3D imaging data. By combining the hardware and software, approximately 1000 neurons were imaged from the cortices of freely behaving mice. Compared with the state-of-the-art 2D imaging technique, the proposed 3D method imaged 1.7–2.6 times more cells with a higher separation of cellular signals.
Shared Dorsal Periaqueductal Gray Activation Patterns during Exposure to Innate and Conditioned Threats
The brainstem dorsal periaqueductal gray (dPAG) has been widely recognized as being a vital node orchestrating the responses to innate threats. Intriguingly, recent evidence also shows that the dPAG mediates defensive responses to fear conditioned contexts. However, it is unknown whether the dPAG displays independent or shared patterns of activation during exposure to innate and conditioned threats. It is also unclear how dPAG ensembles encode and predict diverse defensive behaviors. To address this question, we used miniaturized microscopes to obtain recordings of the same dPAG ensembles during exposure to a live predator and a fear conditioned context in male mice. dPAG ensembles encoded not only distance to threat, but also relevant features, such as predator speed and angular offset between mouse and threat. Furthermore, dPAG cells accurately encoded numerous defensive behaviors, including freezing, stretch-attend postures, and escape. Encoding of behaviors and of distance to threat occurred independently in dPAG cells. dPAG cells also displayed a shared representation to encode these behaviors and distance to threat across innate and conditioned threats. Last, we also show that escape could be predicted by dPAG activity several seconds in advance. Thus, dPAG activity dynamically tracks key kinematic and behavioral variables during exposure to threats, and exhibits similar patterns of activation during defensive behaviors elicited by innate or conditioned threats. These data indicate that a common pathway may be recruited by the dPAG during exposure to a wide variety of threat modalities. SIGNIFICANCE STATEMENT The dorsal periaqueductal gray (dPAG) is critical to generate defensive behaviors during encounters with threats of multiple modalities. Here we use longitudinal calcium transient recordings of dPAG ensembles in freely moving mice to show that this region uses shared patterns of activity to represent distance to an innate threat (a live predator) and a conditioned threat (a shock grid). We also show that dPAG neural activity can predict diverse defensive behaviors. These data indicate the dPAG uses conserved population-level activity patterns to encode and coordinate defensive behaviors during exposure to both innate and conditioned threats.
Using Baseplating and a Miniscope Preanchored with an Objective Lens for Calcium Transient Research in Mice
Neuroscientists use miniature microscopes (miniscopes) to observe neuronal activity in freely behaving animals. The University of California, Los Angeles (UCLA) Miniscope team provides open resources for researchers to build miniscopes themselves. The V3 UCLA Miniscope is one of the most popular open-source miniscopes currently in use. It permits imaging of the fluorescence transients emitted from genetically modified neurons through an objective lens implanted on the superficial cortex (a one-lens system), or in deep brain areas through a combination of a relay lens implanted in the deep brain and an objective lens that is preanchored in the miniscope to observe the relayed image (a two-lens system). Even under optimal conditions (when neurons express fluorescence indicators and the relay lens has been properly implanted), a volume change of the dental cement between the baseplate and its attachment to the skull upon cement curing can cause misalignment with an altered distance between the objective and relay lenses, resulting in the poor image quality. A baseplate is a plate that helps mount the miniscope onto the skull and fixes the working distance between the objective and relay lenses. Thus, changes in the volume of the dental cement around the baseplate alter the distance between the lenses. The present protocol aims to minimize the misalignment problem caused by volume changes in the dental cement. The protocol reduces the misalignment by building an initial foundation of dental cement during relay lens implantation. The convalescence time after implantation is sufficient for the foundation of dental cement to cure the baseplate completely, so the baseplate can be cemented on this scaffold using as little new cement as possible. In the present article, we describe strategies for baseplating in mice to enable imaging of neuronal activity with an objective lens anchored in the miniscope.
Coordination of escape and spatial navigation circuits orchestrates versatile flight from threats
Dorsal periaqueductal gray ensembles represent approach and avoidance states
Animals must balance needs to approach threats for risk assessment and to avoid danger. The dorsal periaqueductal gray (dPAG) controls defensive behaviors, but it is unknown how it represents states associated with threat approach and avoidance. We identified a dPAG threatavoidance ensemble in mice that showed higher activity farther from threats such as the open arms of the elevated plus maze and a predator. These cells were also more active during threat avoidance behaviors such as escape and freezing, even though these behaviors have antagonistic motor output. Conversely, the threat approach ensemble was more active during risk assessment behaviors and near threats. Furthermore, unsupervised methods showed that avoidance/approach states were encoded with shared activity patterns across threats. Lastly, the relative number of cells in each ensemble predicted threat avoidance across mice. Thus, dPAG ensembles dynamically encode threat approach and avoidance states, providing a flexible mechanism to balance risk assessment and danger avoidance.
Behavioral clusters revealed by end-to-end decoding from microendoscopic imaging
In vivo calcium imaging using head-mounted miniature microscopes enables tracking activity from neural populations over weeks in freely behaving animals. Previous studies focus on inferring behavior from a population of neurons, yet it is challenging to extract neuronal signals given out-of-focus fluorescence in endoscopic data. Existing analysis pipelines include regions of interest (ROIs) identification, which might lose relevant information from false negatives or introduce unintended bias from false positives. Moreover, these methods often require prior knowledge for parameter tuning and are time-consuming for implementation. Here, we develop an end-to-end decoder to predict the behavioral variables directly from the raw microendoscopic images. Our framework requires little user input and outperforms existing decoders that need ROI extraction. We show that neuropil/background residuals carry additional behaviorally relevant information. Video analysis further reveals an optimal decoding window and dynamics between residuals and cells. Critically, saliency maps reveal the emergence of video-decomposition across our decoder, and identify distinct clusters representing different behavioral aspects. Together, we present a framework that is efficient for decoding behavior from microendoscopic imaging, and may help discover functional clustering for a variety of imaging studies.
Miniaturized head-mounted microscope for whole-cortex mesoscale imaging in freely behaving mice
The advent of genetically encoded calcium indicators, along with surgical preparations such as thinned skulls or refractive-index-matched skulls, has enabled mesoscale cortical activity imaging in head-fixed mice. However, neural activity during unrestrained behavior substantially differs from neural activity in head-fixed animals. For whole-cortex imaging in freely behaving mice, we present the ‘mini-mScope’, a widefield, miniaturized, head-mounted fluorescence microscope that is compatible with transparent polymer skull preparations. With a field of view of 8 × 10 mm2 and weighing less than 4 g, the mini-mScope can image most of the mouse dorsal cortex with resolutions ranging from 39 to 56 µm. We used the mini-mScope to record mesoscale calcium activity across the dorsal cortex during sensory-evoked stimuli, open field behaviors, social interactions and transitions from wakefulness to sleep.
Scopolamine Impairs Spatial Information Recorded With “Miniscope” Calcium Imaging in Hippocampal Place Cells
The hippocampus and associated cholinergic inputs have important roles in spatial memory in rodents. Muscarinic acetylcholine receptors (mAChRs) are involved in the communication of cholinergic signals and regulate spatial memory. They have been found to impact the memory encoding process, but the effect on memory retrieval is controversial. Previous studies report that scopolamine (a non-selective antagonist of mAChR) induces cognitive deficits on animals, resulting in impaired memory encoding, but the effect on memory retrieval is less certain. We tested the effects of blocking mAChRs on hippocampal network activity and neural ensembles that had previously encoded spatial information. The activity of hundreds of neurons in mouse hippocampal CA1 was recorded using calcium imaging with a miniaturised fluorescent microscope and properties of place cells and neuronal ensemble behaviour in a linear track environment were observed. We found that the decoding accuracy and the stability of spatial representation revealed by hippocampal neural ensemble were significantly reduced after the administration of scopolamine. Several other parameters, including neural firing rate, total number of active neurons, place cell number and spatial information content were affected. Similar results were also observed in a simulated hippocampal network model. This study enhances the understanding of the function of mAChRs on spatial memory impairment.
Neuregulin signaling mediates the acute and sustained antidepressant effects of subanesthetic ketamine
Subanesthetic ketamine evokes rapid antidepressant effects in human patients that persist long past ketamine’s chemical half-life of ~2 h. Ketamine’s sustained antidepressant action may be due to modulation of cortical plasticity. We find that ketamine ameliorates depression-like behavior in the forced swim test in adult mice, and this depends on parvalbumin-expressing (PV) neuron-directed neuregulin-1 (NRG1)/ErbB4 signaling. Ketamine rapidly downregulates NRG1 expression in PV inhibitory neurons in mouse medial prefrontal cortex (mPFC) following a single low-dose ketamine treatment. This NRG1 downregulation in PV neurons co-tracks with the decreases in synaptic inhibition to mPFC excitatory neurons for up to a week. This results from reduced synaptic excitation to PV neurons, and is blocked by exogenous NRG1 as well as by PV targeted ErbB4 receptor knockout. Thus, we conceptualize that ketamine’s effects are mediated through rapid and sustained cortical disinhibition via PV-specific NRG1 signaling. Our findings reveal a novel neural plasticity-based mechanism for ketamine’s acute and long-lasting antidepressant effects.
Midbrain dopamine neurons provide teaching signals for goal-directed navigation
In naturalistic environments, animals navigate in order to harvest rewards. Successful goal-directed navigation requires learning to accurately estimate location and select optimal state-dependent actions. Midbrain dopamine neurons are known to be involved in reward value learning1–13. They have also been linked to reward location learning, as they play causal roles in place preference14,15 and enhance spatial memory16–21. Dopamine neurons are therefore ideally placed to provide teaching signals for goal-directed navigation. To test this, we imaged dopamine neural activity as mice learned to navigate in a closed-loop virtual reality corridor and lick to report the reward location. Across learning, phasic dopamine responses developed to visual cues and trial outcome that resembled reward prediction errors and indicated the animal’s estimate of the reward location. We also observed the development of pre-reward ramping activity, the slope of which was modulated by both learning stage and task engagement. The slope of the dopamine ramp was correlated with the accuracy of licks in the next trial, suggesting that the ramp sculpted accurate location-specific action during navigation. Our results indicate that midbrain dopamine neurons, through both their phasic and ramping activity, provide teaching signals for improving goal-directed navigation. We investigated midbrain dopamine activity in mice learning a goal-directed navigation task in virtual realityPhasic dopamine signals reflected prediction errors with respect to subjective estimate of reward locationA slow ramp in dopamine activity leading up to reward location developed over learning and was enhanced with task engagementPositive ramp slopes were followed by improved performance on subsequent trials, suggesting a teaching role during goal-directed navigation
Large-scale cellular-resolution imaging of neural activity in freely behaving mice
Miniaturized microscopes for head-mounted fluorescence imaging are powerful tools for visualizing neural activity during naturalistic behaviors, but the restricted field of view of first-generation ‘miniscopes’ limits the size of neural populations accessible for imaging. Here we describe a novel miniaturized mesoscope offering cellular-resolution imaging over areas spanning several millimeters in freely moving mice. This system enables comprehensive visualization of activity across entire brain regions or interactions across areas.
Calcium Imaging of Cortical and Hippocampal Neurons During Learning and Decision Making
Studies of the neural basis of memory and decision making have utilized rats as a model organism for decades. Place cells in the hippocampus were first described fifty years ago, and our understanding of the cellular mechanisms of episodic memory and spatial navigation have been primarily based on the rat as a model organism. Modern calcium imaging techniques now allow researchers to monitor hundreds of cells simultaneously, and this has been a boon to brain research. However, until my first publication, calcium imaging in the freely behaving rat had not been demonstrated, and emerging research using calcium imaging in the mouse hippocampus conflicted with decades of rat literature. An intellectual gap was widening, and it was unclear whether differing results stemmed from differences in species or methodology. Therefore, we sought to apply calcium imaging to the rat to demonstrate its feasibility and utility. The research presented in this dissertation is a demonstration of both my efforts developing rat calcium imaging within the Miniscope open-science project, as well as applying these methods to specific hypotheses. Chapter 2 details our most recent experiment, studying the effect of aversive learning on hippocampal place cells and the role of cholinergic signaling. Chapter 3 summarizes collaborative work on the technical development and imaging capabilities of the Large Field of View miniature microscope utilized in the previous chapter. Chapter 4 presents my first co-authored publication where we demonstrated the first usage of single photon calcium imaging in the freely behaving rat, studying how cortical neurons reflect choice utility during effortful decision making.
Encoding Distinct Representations in the Hippocampus
It is believed that the brain creates representations of the world that are elaborated and refined through experience. Yet, it is unknown how such representations are created and altered by experience. The hippocampus has been demonstrated to be crucial for memory processes but relationships between the activity of hippocampal principal neurons and memory representations have been difficult to describe. In this thesis, we examine hippocampal activity using different neurophysiological and theoretical tools to address this question. Using local-field potential recording following pathway-specific stimulations, we demonstrate that spatial learning can cause long-lasting changes to hippocampal circuit function. These changes are limited to specific dendritic compartments which are important for normal cognition, are the support of a general improvement in cognition rather than specific to memories, and rely on changes to inhibitory circuits. Furthermore, we assessed how specific representations can be encoded in the activity of CA1 hippocampal neurons. Using calcium-imaging in freely behaving mice, we recorded large ensembles of neurons and demonstrated that coactivity dynamics are important for encoding distinct environments. These temporal dynamics rely disproportionately on anti-coactive cell pairs, display scale-free properties, and are naturally represented on a 2-D non-linear manifold. Preliminary results indicate that spatial memory learning also alters temporal dynamics. We conclude that experience-dependent alterations can be directly measured and that temporal coactivity constitutes an advantageous code for their description.
Encoding of Space and Self-Motion by GABAergic Cells in the Lateral Septum
The relevance of the hippocampal spatial code for downstream neuronal populations – in particular its main subcortical output, the lateral septum (LS) - is still poorly understood. A large body of evidence has underlined the role of the hippocampus in constructing a detailed internal spatial representation of the environment and its function in spatial navigation and memory. However, there is little evidence on how this hippocampal spatial map is processed in downstream regions such as the LS for goal directed navigation. The present work is designed to elucidate the functional significance of the descending output from the hippocampus, by examining the structural organization of the input and output pathways of the LS. The goal of the proposed research is to investigate the exact role of the LS neurons in place coding, with a particular focus on the integrative role of the LS in the septohippocampal network. To this end, we first clarify the organization of LS afferents and efferents via retrograde and anterograde trans-synaptic tracing, respectively. We found that murine LS receives inputs from hippocampal subregions CA1, CA3, and subiculum, and in turn sends long-range inhibitory projections to a large number of downstream regions, including the lateral hypothalamus, ventral tegmental area, and medial septum. We functionally characterized the spatial tuning properties of LS GABAergic cells, which represent the vast majority of cells in the LS, using calcium imaging combined with unbiased analytical methods. In contrast to what was previously shown, we find that the LS actually carries a highly reliable spatial code, comparable to that of the hippocampus. We identified a significant number of cells that are modulated by place, speed, acceleration, and head-direction, and conjunctions of these properties, with spatial tuning qualities comparable to those of hippocampal CA1 and CA3 place cells. Bayesian decoding of position on the basis of LS place cell activity accurately reflected the location of the animal. The distribution of cells exhibiting these properties formed gradients along the anterior-posterior axis of the LS, directly reflecting the organization of hippocampal inputs to the LS. A portion of LS place cells showed stable fields over the course of multiple days, potentially reflecting long-term episodic memory expression. Together, our findings demonstrate that the LS accurately and robustly represents spatial and idiothetic information and is uniquely positioned to relay this information from the hippocampus to downstream regions, thus occupying a key position within this distributed spatial memory network Alternate abstract: L'importance du code spatial hippocampique pour les populations neuronales en aval - et en particulier sa cible sous-corticale principale, le septum latéral (SL), reste à élucider. De nombreuses études soulignent le rôle de l'hippocampe dans la construction d'une représentation interne de l'environnement et sa fonction dans la navigation spatiale et la mémoire. Cependant, peu de données permettent d'expliquer comment ces représentations spatiales sont traitées dans les régions en aval, comme le SL dans la navigation avec une composante motivationnelle. Ce travail de thèse a pour but de comprendre la signification fonctionnelle des projections de l'hippocampe, en examinant l'organisation structurelle des entrées et sorties du SL. L'objectif proposé est de comprendre le rôle exact des neurones du SL dans l'encodage spatial, avec un intérêt particulier pour le rôle intégratif du SL dans le réseau septo-hippocampique. Dans cette optique, nous clarifions tout d'abord l'organisation des entrées et sorties du SL en utilisant une technique de traçage trans-synaptique rétrograde et antérograde, respectivement. Nous trouvons que le SL murin reçoit des projections des sous-régions hippocampiques CA1, CA3, ainsi que du subiculum, et envoie d'autre part de longues projections inhibitrices à un large nombre de régions en aval, incluant l'hypothalamus latéral, la zone tegmentale ventrale, ainsi que le septum médian. Nous proposons une caractérisation fonctionnelle de l'encodage spatial des cellules GABAergiques du SL, qui représentent la vaste majorité des cellules dans le SL, en utilisant l'imagerie calcique combinée avec des méthodes analytiques impartiales. Contrairement à ce qui a été précédemment démontré, nous observons que le SL possède un code spatial très fiable, comparable à celui de l'hippocampe. Nous avons identifié un nombre significatif de cellules qui sont modulées par l'espace, la vitesse, l'accélération, et la direction de la tête, ainsi qu'une conjonction de ces propriétés, avec un qualité de d'encodage spatial comparable à celle observée dans les cellules des sous-régions hippocampiques CA1 et CA3. Le décodage Bayésien de la position sur la base de l'activité des cellules du SL correspond précisément à la localisation de l'animal. La distribution des cellules qui présentent ces propriétés forme des gradients le long de l'axe antéro-postérieur du SL, reflétant directement l'organisation des entrées hippocampiques à cette structure. Une portion des cellules de lieu du SL présente des champs d'activité stables au cours de plusieurs jours, reflétant potentiellement l'expression d'une mémoire épisodique à long-terme. Ensemble, nos résultats démontrent que le SL représente de manière précise et robuste les informations spatiales et idiothétiques et est situé de manière unique pour relayer ces informations de l'hippocampe aux régions en aval, occupant de ce fait une position clé dans ce réseau distribué de mémoire spatiale.
The McGill-Mouse-Miniscope platform: A standardized approach for high-throughput imaging of neuronal dynamics during behavior
Understanding the rules that govern neuronal dynamics throughout the brain to subserve behavior and cognition remains one of the biggest challenges in neuroscience research. Recent technical advances enable the recording of increasingly larger neuronal populations to produce increasingly more sophisticated datasets. Despite bold and important open-science and data-sharing policies, these datasets tend to include unique data acquisition methods, behaviors, and file structures. Discrepancies between experimental protocols present key challenges in comparing data between laboratories and across different brain regions and species. Here, we discuss our recent efforts to create a standardized and high-throughput research platform to address these issues. The McGill-Mouse-Miniscope (M3) platform is an initiative to combine miniscope calcium imaging with standardized touchscreen-based animal behavioral testing. The goal is to curate an open-source and standardized framework for acquiring, analyzing, and accessing high-quality data of the neuronal dynamics that underly cognition throughout the brain in mice, marmosets, and models of disease. We end with a discussion of future developments and a call for users to adopt this standardized approach.
MiniFAST: A sensitive and fast miniaturized microscope for in vivo neural recording
Observing the activity of large populations of neurons in vivo is critical for understanding brain function and dysfunction. The use of fluorescent genetically-encoded calcium indicators (GECIs) in conjunction with miniaturized microscopes is an exciting emerging toolset for recording neural activity in unrestrained animals. Despite their potential, current miniaturized microscope designs are limited by using image sensors with low frame rates, sensitivity, and resolution. Beyond GECIs, there are many neuroscience applications which would benefit from the use of other emerging neural indicators, such as fluorescent genetically-encoded voltage indicators (GEVIs) that have faster temporal resolution to match neuron spiking, yet, require imaging at high speeds to properly sample the activity-dependent signals. We integrated an advanced CMOS image sensor into a popular open-source miniaturized microscope platform. MiniFAST is a fast and sensitive miniaturized microscope capable of 1080p video, 1.5 µm resolution, frame rates up to 500 Hz and high gain ability (up to 70 dB) to image in extremely low light conditions. We report results of high speed 500 Hz in vitro imaging of a GEVI and ∼300 Hz in vivo imaging of transgenic Thy1-GCaMP6f mice. Finally, we show the potential for a reduction in photobleaching by using high gain imaging with ultra-low excitation light power (0.05 mW) at 60 Hz frame rates while still resolving Ca2+ spiking activity. Our results extend miniaturized microscope capabilities in high-speed imaging, high sensitivity and increased resolution opening the door for the open-source community to use fast and dim neural indicators.
Long-Term Characterization of Hippocampal Remapping during Contextual Fear Acquisition and Extinction
Hippocampal CA1 place cell spatial maps are known to alter their firing properties in response to contextual fear conditioning, a process called “remapping.” In the present study, we use chronic calcium imaging to examine remapping during fear retrieval and extinction of an inhibitory avoidance task in mice of both sexes over an extended period of time and with thousands of neurons. We demonstrate that hippocampal ensembles encode space at a finer scale following fear memory acquisition. This effect is strongest near the shock grid. We also characterize the long-term effects of shock on place cell ensemble stability, demonstrating that shock delivery induces several days of high fear and low between-session place field stability, followed by a new, stable spatial representation that appears after fear extinction. Finally, we identify a novel group of CA1 neurons that robustly encode freeze behavior independently from spatial location. Thus, following fear acquisition, hippocampal CA1 place cells sharpen their spatial tuning and dynamically change spatial encoding stability throughout fear learning and extinction. SIGNIFICANCE STATEMENT The hippocampus contains place cells that encode an animal's location. This spatial code updates, or remaps, in response to environmental change. It is known that contextual fear can induce such remapping; in the present study, we use chronic calcium imaging to examine inhibitory avoidance-induced remapping over an extended period of time and with thousands of neurons and demonstrate that hippocampal ensembles encode space at a finer scale following electric shock, an effect which is enhanced by threat proximity. We also identify a novel group of freeze behavior-activated neurons. These results suggest that, more than merely shuffling their spatial code following threat exposure, place cells enhance their spatial coding with the possible benefit of improved threat localization.
Miniscope3D: optimized single-shot miniature 3D fluorescence microscopy
Miniature fluorescence microscopes are a standard tool in systems biology. However, widefield miniature microscopes capture only 2D information, and modifications that enable 3D capabilities increase the size and weight and have poor resolution outside a narrow depth range. Here, we achieve the 3D capability by replacing the tube lens of a conventional 2D Miniscope with an optimized multifocal phase mask at the objective’s aperture stop. Placing the phase mask at the aperture stop significantly reduces the size of the device, and varying the focal lengths enables a uniform resolution across a wide depth range. The phase mask encodes the 3D fluorescence intensity into a single 2D measurement, and the 3D volume is recovered by solving a sparsity-constrained inverse problem. We provide methods for designing and fabricating the phase mask and an efficient forward model that accounts for the field-varying aberrations in miniature objectives. We demonstrate a prototype that is 17 mm tall and weighs 2.5 grams, achieving 2.76 μm lateral, and 15 μm axial resolution across most of the 900 × 700 × 390 μm3 volume at 40 volumes per second. The performance is validated experimentally on resolution targets, dynamic biological samples, and mouse brain tissue. Compared with existing miniature single-shot volume-capture implementations, our system is smaller and lighter and achieves a more than 2× better lateral and axial resolution throughout a 10× larger usable depth range. Our microscope design provides single-shot 3D imaging for applications where a compact platform matters, such as volumetric neural imaging in freely moving animals and 3D motion studies of dynamic samples in incubators and lab-on-a-chip devices.
Subanesthetic Ketamine Reactivates Adult Cortical Plasticity to Restore Vision from Amblyopia
Cortical Representations of Conspecific Sex Shape Social Behavior
HippoBellum: Acute Cerebellar Modulation Alters Hippocampal Dynamics and Function
Here we examine what effects acute manipulation of the cerebellum, a canonically motor structure, can have on the hippocampus, a canonically cognitive structure. In male and female mice, acute perturbation of the cerebellar vermis (lobule 4/5) or simplex produced reliable and specific effects in hippocampal function at cellular, population, and behavioral levels, including evoked local field potentials, increased hippocampal cFos expression, and altered CA1 calcium event rate, amplitudes, and correlated activity. We additionally noted a selective deficit on an object location memory task, which requires objection-location pairing. We therefore combined cerebellar optogenetic stimulation and CA1 calcium imaging with an object-exploration task, and found that cerebellar stimulation reduced the representation of place fields near objects, and prevented a shift in representation to the novel location when an object was moved. Together, these results clearly demonstrate that acute modulation of the cerebellum alters hippocampal function, and further illustrates that the cerebellum can influence cognitive domains. SIGNIFICANCE STATEMENT The cerebellum, a canonically motor-related structure, is being increasingly recognized for its influence on nonmotor functions and structures. The hippocampus is a brain region critical for cognitive functions, such as episodic memory and spatial navigation. To investigate how modulation of the cerebellum may impact the hippocampus, we stimulated two sites of the cerebellar cortex and examined hippocampal function at multiple levels. We found that cerebellar stimulation strongly modulates hippocampal activity, disrupts spatial memory, and alters object-location processing. Therefore, a canonically cognitive brain area, the hippocampus, is sensitive to cerebellar modulation.
Loud noise exposure differentially affects subpopulations of auditory cortex pyramidal cells
Loud noise-exposure generates tinnitus in both humans and animals. Macroscopic studies show that noise exposure affects the auditory cortex; however, cellular mechanisms of tinnitus generation are unclear. Here we compare membrane properties of layer 5 (L5) pyramidal cells (PCs) of the primary auditory cortex (A1) from control and noise-exposed mice. PCs were previously classified in type A or type B based on connectivity and firing properties. Our analysis based on a logistic regression model predicted that afterhyperpolatization and afterdepolarization following the injection of inward and outward current are enough to predict cell type and these features are preserved after noise trauma. One week after a noise-exposure (4-18kHz, 90dB, 1.5 hr, followed by 1.5hr silence) no passive membrane properties of type A or B PCs were altered but principal component analysis showed greater separation between control/noise-exposure recordings for type A neurons. When comparing individual firing properties, noise exposure differentially affected type A and B PC firing frequency in response to depolarizing current steps. Specifically, type A PCs decreased both initial and steady state firing frequency and type B PCs significantly increased steady state firing frequency following noise exposure. These results show that loud noise can cause distinct effects on type A and B L5 auditory cortex PCs one week following noise exposure. As the type A PC electrophysiological profile is correlated to corticofugal L5 neurons, and type B PCs correlate to contralateral projecting PCs these alterations could partially explain the reorganization of the auditory cortex observed in tinnitus patients.
Robust Population Single Neuronal Calcium Signal Extraction Using SCOUT Allows for Longitudinal Analysis of Behavior-associated Neural Ensemble Dynamics
In vivo calcium imaging enables simultaneous recording of large neuronal ensembles while engaged in operations such as learning and memory. However, such in vivo optical recordings are typically subject to motion artifact and background contamination from neurons and blood vessels. Further, population cell tracking across multiple recordings is complicated by non-rigid transformation induced by cell movements and field shifts. We introduce the novel method SCOUT for Single-Cell SpatiOtemporal LongitUdinal Tracking, consisting of two crucial parts: (1) imposition of spatial constraints on neuronal footprints extracted from individual optical recordings to improve ROI selection and eliminate false discoveries, and (2) application of a predictor-corrector, using spatiotemporal correlation of extracted neurons across sessions, for population cell tracking across multiple sessions. SCOUT empirically outperforms current methods for cell extraction and tracking in long-term multi-session imaging experiments across multiple brain regions. Application of this method allows for robust longitudinal analysis of contextual discrimination associated neural ensemble dynamics in the hippocampus up to 60 days.
Deciphering Brain Function by Miniaturized Fluorescence Microscopy in Freely Behaving Animals
Animal behavior is regulated by environmental stimuli and is shaped by the activity of neural networks, underscoring the importance of assessing the morpho-functional properties of different populations of cells in freely behaving animals. In recent years, a number of optical tools have been developed to monitor and modulate neuronal and glial activity at the protein, cellular, or network level and have opened up new avenues for studying brain function in freely behaving animals. Tools such as genetically encoded sensors and actuators are now commonly used for studying brain activity and function through their expression in different neuronal ensembles. In parallel, microscopy has also made major progress over the last decades. The advent of miniature microscopes (mini-microscopes also called mini-endoscopes) has become a method of choice for studying brain activity at the cellular and network levels in different brain regions of freely behaving mice. This technique also allows for longitudinal investigations while animals carrying the microscope on their head are performing behavioral tasks. In this review, we will discuss mini-endoscopic imaging and the advantages that these devices offer to research. We will also discuss current limitations of and potential future improvements in mini-endoscopic imaging.
Chemogenetic Modulation and Single-Photon Calcium Imaging in Anterior Cingulate Cortex Reveal a Mechanism for Effort-Based Decisions
The ACC is implicated in effort exertion and choices based on effort cost, but it is still unclear how it mediates this cost-benefit evaluation. Here, male rats were trained to exert effort for a high-value reward (sucrose pellets) in a progressive ratio lever-pressing task. Trained rats were then tested in two conditions: a no-choice condition where lever-pressing for sucrose was the only available food option, and a choice condition where a low-value reward (lab chow) was freely available as an alternative to pressing for sucrose. Disruption of ACC, via either chemogenetic inhibition or excitation, reduced lever-pressing in the choice, but not in the no-choice, condition. We next looked for value coding cells in ACC during effortful behavior and reward consumption phases during choice and no-choice conditions. For this, we used in vivo miniaturized fluorescence microscopy to reliably track responses of the same cells and compare how ACC neurons respond during the same effortful behavior where there was a choice versus when there was no-choice. We found that lever-press and sucrose-evoked responses were significantly weaker during choice compared with no-choice sessions, which may have rendered them more susceptible to chemogenetic disruption. Together, findings from our interference experiments and neural recordings suggest that a mechanism by which ACC mediates effortful decisions is in the discrimination of the utility of available options. ACC regulates these choices by providing a stable population code for the relative value of different options. SIGNIFICANCE STATEMENT The ACC is implicated in effort-based decision-making. Here, we used chemogenetics and in vivo calcium imaging to explore its mechanism. Rats were trained to lever press for a high-value reward and tested in two conditions: a no-choice condition where lever-pressing for the high-value reward was the only option, and a choice condition where a low-value reward was also available. Inhibition or excitation of ACC reduced effort toward the high-value option, but only in the choice condition. Neural responses in ACC were weaker in the choice compared with the no-choice condition. A mechanism by which ACC regulates effortful decisions is in providing a stable population code for the discrimination of the utility of available options.
Miniaturized Devices for Bioluminescence Imaging in Freely Behaving Animals
In vivo fluorescence miniature microscopy has recently proven a major advance, enabling cellular imaging in freely behaving animals. However, fluorescence imaging suffers from autofluorescence, phototoxicity, photobleaching and non- homogeneous illumination artifacts. These factors limit the quality and time course of data collection. Bioluminescence provides an alternative kind of activity-dependent light indicator. Bioluminescent calcium indicators do not require light input, instead generating photons through chemiluminescence. As such, limitations inherent to the requirement for light presentation are eliminated. Further, bioluminescent indicators also do not require excitation light optics: the removal of these components should make a lighter and lower cost microscope with fewer assembly parts. While there has been significant recent progress in making brighter and faster bioluminescence indicators, the advances in imaging hardware have not yet been realized. A hardware challenge is that despite potentially higher signal-to-noise of bioluminescence, the signal strength is lower than that of fluorescence. An open question we address in this report is whether fluorescent miniature microscopes can be rendered sensitive enough to detect bioluminescence. We demonstrate this possibility in vitro and in vivo by implementing optimizations of the UCLA fluorescent miniscope v3.2. These optimizations yielded a miniscope (BLmini) which is 22% lighter in weight, has 45% fewer components, is up to 58% less expensive, offers up to 15 times stronger signal and is sensitive enough to capture spatiotemporal dynamics of bioluminescence in the brain with a signal-to-noise ratio of 34 dB.
Novel Virtual Reality System for Auditory Tasks in Head-fixed Mice
An emerging corpus of research seeks to use virtual realities (VRs) to understand the neural mechanisms underlying spatial navigation and decision making in rodents. These studies have primarily used visual stimuli to represent the virtual world. However, auditory cues play an important role in navigation for animals, especially when the visual system cannot detect objects or predators. We have developed a virtual reality environment defined exclusively by free-field acoustic landmarks for head-fixed mice. We trained animals to run in a virtual environment with 3 acoustic landmarks. We present evidence that they can learn to navigate in our context: we observed anticipatory licking and modest anticipatory slowing preceding the reward region. Furthermore, we found that animals were highly aware of changes in landmark cues: licking behavior changed dramatically when the familiar virtual environment was switched to a novel one, and then rapidly reverted to normal when the familiar virtual environment was re-introduced, all within the same session. Finally, while animals executed the task, we performed in-vivo calcium imaging in the CA1 region of the hippocampus using a modified Miniscope.org system. Our experiments point to a future in which auditory virtual reality can be used to expand our understanding of the neural bases of audition in locomoting animals and the variety of sensory cues which anchor spatial representations in a new virtual environment.
DG–CA3 circuitry mediates hippocampal representations of latent information
Survival in complex environments necessitates a flexible navigation system that incorporates memory of recent behavior and associations. Yet, how the hippocampal spatial circuit represents latent information independent of sensory inputs and future goals has not been determined. To address this, we image the activity of large ensembles in subregion CA1 via wide-field fluorescent microscopy during a novel behavioral paradigm. Our results demonstrate that latent information is represented through reliable firing rate changes during unconstrained navigation. We then hypothesize that the representation of latent information in CA1 is mediated by pattern separation/completion processes instantiated upstream within the dentate gyrus (DG) and CA3 subregions. Indeed, CA3 ensemble recordings reveal an analogous code for latent information. Moreover, selective chemogenetic inactivation of DG–CA3 circuitry completely and reversibly abolishes the CA1 representation of latent information. These results reveal a causal and specific role of DG–CA3 circuitry in the maintenance of latent information within the hippocampus.
A Probabilistic Framework for Decoding Behavior From in vivo Calcium Imaging Data
Understanding the role of neuronal activity in cognition and behavior is a key question in neuroscience. Previously,
Latent learning drives sleep-dependent plasticity in distinct CA1 subpopulations
Latent learning allows the brain the transform experiences into cognitive maps, a form of implicit memory, without reinforced training. Its mechanism is unclear. We tracked the internal states of the hippocampal neural ensembles and discovered that during latent learning of a spatial map, the state space evolved into a low-dimensional manifold that topologically resembled the physical environment. This process requires repeated experiences and sleep in-between. Further investigations revealed that a subset of hippocampal neurons, instead of rapidly forming place fields in a novel environment, remained weakly tuned but gradually developed correlated activity with other neurons. These ‘weakly spatial’ neurons bond activity of neurons with stronger spatial tuning, linking discrete place fields into a map that supports flexible navigation.
Breakdown of spatial coding and interneuron synchronization in epileptic mice
NINscope, a versatile miniscope for multi-region circuit investigations
Miniaturized fluorescence microscopes (miniscopes) have been instrumental to monitor neural signals during unrestrained behavior and their open-source versions have made them affordable. Often, the footprint and weight of open-source miniscopes is sacrificed for added functionality. Here, we present NINscope: a light-weight miniscope with a small footprint that integrates a high-sensitivity image sensor, an inertial measurement unit and an LED driver for an external optogenetic probe. We use it to perform the first concurrent cellular resolution recordings from cerebellum and cerebral cortex in unrestrained mice, demonstrate its optogenetic stimulation capabilities to examine cerebello-cerebral or cortico-striatal connectivity, and replicate findings of action encoding in dorsal striatum. In combination with cross-platform acquisition and control software, our miniscope is a versatile addition to the expanding tool chest of open-source miniscopes that will increase access to multi-region circuit investigations during unrestrained behavior.
Hippocampal Encoding of Space Induced by Novel Auditory VR System Using One-Photon Miniaturized Microscope
In virtual reality settings, spatial navigation in animal models has traditionally been studied using primarily visual cues. However, auditory cues play an important role in navigation for animals, especially when the visual system cannot detect objects or predators in the dark. We have developed a virtual reality system defined exclusively by auditory landmarks for head-fixed mice performing a navigation task. We report behavioral evidence that mice can learn to navigate in our task. Namely, we observed anticipatory licking and modest anticipatory slowing preceding the reward region. Furthermore, we found that the animal’s licking behavior changes when switching from a familiar virtual environment to a novel virtual environment, followed by reverting to normal licking behavior after the familiar virtual environment is re-introduced within the same session. While animals carried out the task, we performed in-vivo calcium imaging in the CA1 region of the hippocampus using a modified Miniscope system. We envision that this approach has the potential to provide new insight into how animals respond to stimuli using spatial aspects of sound in an environment. (abstract adapted from Gao et al., EMBC’20, forthcoming).
Studies on pain and itch signal processing in the anterior cingulate cortex with head-mounted fluorescent microscope
통증과 가려움은 서로 다른 정동감각이며 전달 경로와 두 감각을 처리하는 뇌 영역이 긴밀하게 관련되어 있으며, 그 중에서도 전두대상피질은 통증과 가려움 모두를 처리하는 것으로 잘 알려져 있다. 뇌가 여러 체감각들을 서로 다른 것으로 인지하는지에 관해 여러 가설들이 제시되었으나 지금까지 한 영역에서 두 체감각이 어떻게 처리되는지에 관해서는 연구된 바 없었다. 본 연구에서는 쥐에 6시간 간격으로 가려움과 통증 자극을 주었을 때 두 자극 모두에 반응하는 전두대상피질영역의 뉴런들의 비율이 72시간 간격으로 자극이 주어졌을 때보다 높은 것을 확인하였다. 또한 통증 또는 가려움 자극에 반응하는 뉴런 집단에 GiDREADD를 발현시킴으로써 억제하였을 때 통증 또는 가려움 관련 행동이 감소하는 것을 확인하였다. 머리탑재형 초소형 형광현미경인 미니스콥은 UCLA에서 개발되었으며, 움직이는 쥐에서 실시간으로 뉴런의 활성을 촬영하기 위해 사용된다. 본 연구에서는 미니스콥 부품들을 개선하고 수술 도구를 대체함으로써 촬영 시스템의 안정성을 높이고 수술로 인한 뇌조직의 염증을 줄임으로써 깨끗한 영상을 얻을 수 있었으며 최적화된 미니스콥의 촬영 시스템을 통해 뉴런 활성 영상의 질을 높이고 데이터의 신뢰도를 높였다. 또한, 새로운 칼슘 이미징 분석 프로그램인 CaImAn이 미니스콥으로 얻어진 데이터에 성공적으로 적용되는 것을 확인하였다. 이렇게 구축된 시스템을 이용하여 활성화된 뉴런 내부로 유입되는 칼슘과 결합했을 때 형광을 발현하는 단백질의 일종인 GCaMP6f 단백질의 형광 신호를 촬영하였다. 미니스콥을 통해 얻어진 결과 또한 면역조직화학법으로 얻어진 결과와 마찬가지로 6시간 간격으로 통증 또는 가려움 자극이 주어질 경우 두 자극 모두에 반응한 뉴런들의 수가 72시간 간격이었을 때 보다 많은 경향을 보이는 것을 확인하였다. 이러한 결과들은 일부 전두대상피질 뉴런들은 흥분성 주기에 영향을 받아 통증 또는 가려움에 반응하는 뉴런 집단에 속하는 경향이 있으며 다른 일부 전두대상피질 뉴런들은 통증 또는 가려움에 특이적으로 반응하는 고유의 뉴런 집단을 형성하여 통증 또는 가려움 행동 반응 유도에 필요하다는 것을 제시한다.
CA1-projecting subiculum neurons facilitate object–place learning
Recent anatomical evidence suggests a functionally significant back-projection pathway from the subiculum to the CA1. Here we show that the afferent circuitry of CA1-projecting subicular neurons is biased by inputs from CA1 inhibitory neurons and the visual cortex, but lacks input from the entorhinal cortex. Efferents of the CA1-projecting subiculum neurons also target the perirhinal cortex, an area strongly implicated in object–place learning. We identify a critical role for CA1-projecting subicular neurons in object-location learning and memory, and show that this projection modulates place-specific activity of CA1 neurons and their responses to displaced objects. Together, these experiments reveal a novel pathway by which cortical inputs, particularly those from the visual cortex, reach the hippocampal output region CA1. Our findings also implicate this circuitry in the formation of complex spatial representations and learning of object–place associations.
Precise Coordination of Three-Dimensional Rotational Kinematics by Ventral Tegmental Area GABAergic Neurons
Miniaturized microscope with flexible light source input for neuronal imaging and manipulation in freely behaving animals
Simultaneous imaging and manipulation of a genetically defined neuronal population can provide a causal link between its activity and function. Here, we designed a miniaturized microscope (or ‘miniscope’) that allows fluorescence imaging and optogenetic manipulation at the cellular level in freely behaving animals. This miniscope has an integrated optical connector that accepts any combination of external light sources, allowing flexibility in the choice of sensors and manipulators. Moreover, due to its simple structure and use of open source software, the miniscope is easy to build and modify. Using this miniscope, we demonstrate the optogenetic silencing of hippocampal CA1 neurons using two laser light sources—one stimulating a calcium sensor (i.e., jGCaAMP7c) and the other serving as an optogenetic silencer (i.e., Jaws). This new miniscope can contribute to efforts to determine causal relationships between neuronal network dynamics and animal behavior.
Correlated Neural Activity and Encoding of Behavior across Brains of Socially Interacting Animals
Imaging and analysis of genetically encoded calcium indicators linking neural circuits and behaviors
A striatal interneuron circuit for continuous target pursuit
Most adaptive behaviors require precise tracking of targets in space. In pursuit behavior with a moving target, mice use distance to target to guide their own movement continuously. Here, we show that in the sensorimotor striatum, parvalbumin-positive fast-spiking interneurons (FSIs) can represent the distance between self and target during pursuit behavior, while striatal projection neurons (SPNs), which receive FSI projections, can represent self-velocity. FSIs are shown to regulate velocity-related SPN activity during pursuit, so that movement velocity is continuously modulated by distance to target. Moreover, bidirectional manipulation of FSI activity can selectively disrupt performance by increasing or decreasing the self-target distance. Our results reveal a key role of the FSI-SPN interneuron circuit in pursuit behavior and elucidate how this circuit implements distance to velocity transformation required for the critical underlying computation.
A Two-Step GRIN Lens Coating for In Vivo Brain Imaging
The complex spatial and temporal organization of neural activity in the brain is important for information-processing that guides behavior. Hence, revealing the real-time neural dynamics in freely-moving animals is fundamental to elucidating brain function. Miniature fluorescence microscopes have been developed to fulfil this requirement. With the help of GRadient INdex (GRIN) lenses that relay optical images from deep brain regions to the surface, investigators can visualize neural activity during behavioral tasks in freely-moving animals. However, the application of GRIN lenses to deep brain imaging is severely limited by their availability. Here, we describe a protocol for GRIN lens coating that ensures successful long-term intravital imaging with commercially-available GRIN lenses.
Circuit Investigations With Open-Source Miniaturized Microscopes: Past, Present and Future
The ability to simultaneously image the spatiotemporal activity signatures from many neurons during unrestrained vertebrate behaviors has become possible through the development of miniaturized fluorescence microscopes, or miniscopes, sufficiently light to be carried by small animals such as bats, birds and rodents. Miniscopes have permitted the study of circuits underlying song vocalization, action sequencing, head-direction tuning, spatial memory encoding and sleep to name a few. The foundation for these microscopes has been laid over the last two decades through academic research with some of this work resulting in commercialization. More recently, open-source initiatives have led to an even broader adoption of miniscopes in the neuroscience community. Open-source designs allow for rapid modification and extension of their function, which has resulted in a new generation of miniscopes that now permit wire-free or wireless recording, concurrent electrophysiology and imaging, two-color fluorescence detection, simultaneous optical actuation and read-out as well as wide-field and volumetric light-field imaging. These novel miniscopes will further expand the toolset of those seeking affordable methods to probe neural circuit function during naturalistic behaviors. Here, we will discuss the early development, present use and future potential of miniscopes.
All the light that we can see: a new era in miniaturized microscopy
One major challenge in neuroscience is to uncover how defined neural circuits in the brain encode, store, modify, and retrieve information. Meeting this challenge comprehensively requires tools capable of recording and manipulating the activity of intact neural networks in naturally behaving animals. Head-mounted miniature microscopes are emerging as a key tool to address this challenge. Here we discuss recent work leading to the miniaturization of neural imaging tools, the current state of the art in this field, and the importance and necessity of open-source options. We finish with a discussion on what the future may hold for miniature microscopy.
Imaging Cortical Dynamics in GCaMP Transgenic Rats with a Head-Mounted Widefield Macroscope
Reducing Astrocyte Calcium Signaling In Vivo Alters Striatal Microcircuits and Causes Repetitive Behavior
Enhanced Image Sensor Module for Head-Mounted Microscopes
Several research groups have developed head-mounted fluorescence microscopes as a modality for recording neural activity in freely behaving mice. The current designs have shown exciting results from in vivo imaging of the bright dynamics of genetically encoded calcium indicators (GECI). However, despite their potential, head-mounted microscopes are not in use with genetically encoded voltage indicators (GEVI) or bioluminescence indicators. Due to its ability to match the temporal resolution of neuron spiking, GEVIs offer great benefits to experiments designed to provide feedback after real-time detection of specific neural activity such as the less than 250ms replay events that can occur in the hippocampus. Orthogonally, the emerging bioluminescence activity reporters have the potential to eliminate autofluorescence and photobleaching that can occur in fluorescence imaging. There are two important properties of the head-mounted microscope’s image sensor affecting the ability to image GEVIs and bioluminescence indicators. First, the low signal to noise ratio (SNR) characteristics of GEVIs and bioluminescent indicators make signal detection difficult. Second, in order to take advantage of the GEVIs faster fluorescence kinetics, the image sensor must be capable of matching frame rates. Here, we present the design of a new imaging module for head-mounted microscopes incorporating the latest CMOS sensor technology aimed at increasing image sensor sensitivity and frame rates for use in real-time detection experiments. The design builds off an existing open-source project and can integrate into the existing data acquisition hardware and microscope housing.
High-speed volumetric imaging of neuronal activity in freely moving rodents
Thus far, optical recording of neuronal activity in freely behaving animals has been limited to a thin axial range. We present a head-mounted miniaturized light-field microscope (MiniLFM) capable of capturing neuronal network activity within a volume of 700 × 600 × 360 µm3 at 16 Hz in the hippocampus of freely moving mice. We demonstrate that neurons separated by as little as ~15 µm and at depths up to 360 µm can be discriminated.
Implementing simultaneous calcium imaging and optogenetics in freely moving rodents to investigate the role of local inhibition in place field stability
The role of the medial prefrontal cortex in reward seeking
Miniaturized two-photon microscope: seeing clearer and deeper into the brain
A shared neural ensemble links distinct contextual memories encoded close in time
A similar neural ensemble participates in the encoding of two distinct memories, resulting in the recall of one memory increasing the likelihood of recalling the other, but only if those memories occur very closely in time—within a day rather than across a week.