An approximate line attractor in the hypothalamus encodes an aggressive state.

The hypothalamus regulates innate social behaviors, including mating and aggression. These behaviors can be evoked by optogenetic stimulation of specific neuronal subpopulations within MPOA and VMHvl, respectively. Here, we perform dynamical systems modeling of population neuronal activity in these nuclei during social behaviors. In VMHvl, unsupervised analysis identified a dominant dimension of neural activity with a large time constant (>50 s), generating an approximate line attractor in neural state space. Progression of the neural trajectory along this attractor was correlated with an escalation of agonistic behavior, suggesting that it may encode a scalable state of aggressiveness. Consistent with this, individual differences in the magnitude of the integration dimension time constant were strongly correlated with differences in aggressiveness. In contrast, approximate line attractors were not observed in MPOA during mating; instead, neurons with fast dynamics were tuned to specific actions. Thus, different hypothalamic nuclei employ distinct neural population codes to represent similar social behaviors.

Fluorescence imaging of large-scale neural ensemble dynamics.

Recent progress in fluorescence imaging allows neuroscientists to observe the dynamics of thousands of individual neurons, identified genetically or by their connectivity, across multiple brain areas and for extended durations in awake behaving mammals. We discuss advances in fluorescent indicators of neural activity, viral and genetic methods to express these indicators, chronic animal preparations for long-term imaging studies, and microscopes to monitor and manipulate the activity of large neural ensembles. Ca2+ imaging studies of neural activity can track brain area interactions and distributed information processing at cellular resolution. Across smaller spatial scales, high-speed voltage imaging reveals the distinctive spiking patterns and coding properties of targeted neuron types. Collectively, these innovations will propel studies of brain function and dovetail with ongoing neuroscience initiatives to identify new neuron types and develop widely applicable, non-human primate models. The optical toolkit's growing sophistication also suggests that "brain observatory" facilities would be useful open resources for future brain-imaging studies.

A neural circuit state change underlying skilled movements.

In motor neuroscience, state changes are hypothesized to time-lock neural assemblies coordinating complex movements, but evidence for this remains slender. We tested whether a discrete change from more autonomous to coherent spiking underlies skilled movement by imaging cerebellar Purkinje neuron complex spikes in mice making targeted forelimb-reaches. As mice learned the task, millimeter-scale spatiotemporally coherent spiking emerged ipsilateral to the reaching forelimb, and consistent neural synchronization became predictive of kinematic stereotypy. Before reach onset, spiking switched from more disordered to internally time-locked concerted spiking and silence. Optogenetic manipulations of cerebellar feedback to the inferior olive bi-directionally modulated neural synchronization and reaching direction. A simple model explained the reorganization of spiking during reaching as reflecting a discrete bifurcation in olivary network dynamics. These findings argue that to prepare learned movements, olivo-cerebellar circuits enter a self-regulated, synchronized state promoting motor coordination. State changes facilitating behavioral transitions may generalize across neural systems.

Shared Cortex-Cerebellum Dynamics in the Execution and Learning of a Motor Task.

Throughout mammalian neocortex, layer 5 pyramidal (L5) cells project via the pons to a vast number of cerebellar granule cells (GrCs), forming a fundamental pathway. Yet, it is unknown how neuronal dynamics are transformed through the L5→GrC pathway. Here, by directly comparing premotor L5 and GrC activity during a forelimb movement task using dual-site two-photon Ca2+ imaging, we found that in expert mice, L5 and GrC dynamics were highly similar. L5 cells and GrCs shared a common set of task-encoding activity patterns, possessed similar diversity of responses, and exhibited high correlations comparable to local correlations among L5 cells. Chronic imaging revealed that these dynamics co-emerged in cortex and cerebellum over learning: as behavioral performance improved, initially dissimilar L5 cells and GrCs converged onto a shared, low-dimensional, task-encoding set of neural activity patterns. Thus, a key function of cortico-cerebellar communication is the propagation of shared dynamics that emerge during learning.

Ultrafast Two-Photon Imaging of a High-Gain Voltage Indicator in Awake Behaving Mice.

Optical interrogation of voltage in deep brain locations with cellular resolution would be immensely useful for understanding how neuronal circuits process information. Here, we report ASAP3, a genetically encoded voltage indicator with 51% fluorescence modulation by physiological voltages, submillisecond activation kinetics, and full responsivity under two-photon excitation. We also introduce an ultrafast local volume excitation (ULoVE) method for kilohertz-rate two-photon sampling in vivo with increased stability and sensitivity. Combining a soma-targeted ASAP3 variant and ULoVE, we show single-trial tracking of spikes and subthreshold events for minutes in deep locations, with subcellular resolution and with repeated sampling over days. In the visual cortex, we use soma-targeted ASAP3 to illustrate cell-type-dependent subthreshold modulation by locomotion. Thus, ASAP3 and ULoVE enable high-speed optical recording of electrical activity in genetically defined neurons at deep locations during awake behavior.

Neuronal Representation of Social Information in the Medial Amygdala of Awake Behaving Mice.

The medial amygdala (MeA) plays a critical role in processing species- and sex-specific signals that trigger social and defensive behaviors. However, the principles by which this deep brain structure encodes social information is poorly understood. We used a miniature microscope to image the Ca2+ dynamics of large neural ensembles in awake behaving mice and tracked the responses of MeA neurons over several months. These recordings revealed spatially intermingled subsets of MeA neurons with distinct temporal dynamics. The encoding of social information in the MeA differed between males and females and relied on information from both individual cells and neuronal populations. By performing long-term Ca2+ imaging across different social contexts, we found that sexual experience triggers lasting and sex-specific changes in MeA activity, which, in males, involve signaling by oxytocin. These findings reveal basic principles underlying the brain's representation of social information and its modulation by intrinsic and extrinsic factors.

Cell-Type-Specific Optical Recording of Membrane Voltage Dynamics in Freely Moving Mice.

Electrophysiological field potential dynamics are of fundamental interest in basic and clinical neuroscience, but how specific cell types shape these dynamics in the live brain is poorly understood. To empower mechanistic studies, we created an optical technique, TEMPO, that records the aggregate trans-membrane voltage dynamics of genetically specified neurons in freely behaving mice. TEMPO has >10-fold greater sensitivity than prior fiber-optic techniques and attains the noise minimum set by quantum mechanical photon shot noise. After validating TEMPO's capacity to track established oscillations in the delta, theta, and gamma frequency bands, we compared the D1- and D2-dopamine-receptor-expressing striatal medium spiny neurons (MSNs), which are interspersed and electrically indistinguishable. Unexpectedly, MSN population dynamics exhibited two distinct coherent states that were commonly indiscernible in electrical recordings and involved synchronized hyperpolarizations across both MSN subtypes. Overall, TEMPO allows the deconstruction of normal and pathologic neurophysiological states into trans-membrane voltage activity patterns of specific cell types.

Distinct Hippocampal Pathways Mediate Dissociable Roles of Context in Memory Retrieval.

Memories about sensory experiences are tightly linked to the context in which they were formed. Memory contextualization is fundamental for the selection of appropriate behavioral reactions needed for survival, yet the underlying neuronal circuits are poorly understood. By combining trans-synaptic viral tracing and optogenetic manipulation, we found that the ventral hippocampus (vHC) and the amygdala, two key brain structures encoding context and emotional experiences, interact via multiple parallel pathways. A projection from the vHC to the basal amygdala mediates fear behavior elicited by a conditioned context, whereas a parallel projection from a distinct subset of vHC neurons onto midbrain-projecting neurons in the central amygdala is necessary for context-dependent retrieval of cued fear memories. Our findings demonstrate that two fundamentally distinct roles of context in fear memory retrieval are processed by distinct vHC output pathways, thereby allowing for the formation of robust contextual fear memories while preserving context-dependent behavioral flexibility.

Dopamine-mediated interactions between short- and long-term memory dynamics.

In dynamic environments, animals make behavioural decisions on the basis of the innate valences of sensory cues and information learnt about these cues across multiple timescales1-3. However, it remains unclear how the innate valence of a sensory stimulus affects the acquisition of learnt valence information and subsequent memory dynamics. Here we show that in the Drosophila brain, interconnected short- and long-term memory units of the mushroom body jointly regulate memory through dopamine signals that encode innate and learnt sensory valences. By performing time-lapse in vivo voltage-imaging studies of neural spiking in more than 500 flies undergoing olfactory associative conditioning, we found that protocerebral posterior lateral 1 dopamine neurons (PPL1-DANs)4 heterogeneously and bidirectionally encode innate and learnt valences of punishment, reward and odour cues. During learning, these valence signals regulate memory storage and extinction in mushroom body output neurons (MBONs)5. During initial conditioning bouts, PPL1-γ1pedc and PPL1-γ2α'1 neurons control short-term memory formation, which weakens inhibitory feedback from MBON-γ1pedc>α/ß to PPL1-α'2α2 and PPL1-α3. During further conditioning, this diminished feedback allows these two PPL1-DANs to encode the net innate plus learnt valence of the conditioned odour cue, which gates long-term memory formation. A computational model constrained by the fly connectome6,7 and our spiking data explains how dopamine signals mediate the circuit interactions between short- and long-term memory traces, yielding predictions that our experiments confirmed. Overall, the mushroom body achieves flexible learning through the integration of innate and learnt valences in parallel learning units sharing feedback interconnections. This hybrid physiological-anatomical mechanism may be a general means by which dopamine regulates memory dynamics in other species and brain structures, including the vertebrate basal ganglia.

Neural circuit basis of placebo pain relief.

Placebo effects are notable demonstrations of mind-body interactions1,2. During pain perception, in the absence of any treatment, an expectation of pain relief can reduce the experience of pain-a phenomenon known as placebo analgesia3-6. However, despite the strength of placebo effects and their impact on everyday human experience and the failure of clinical trials for new therapeutics7, the neural circuit basis of placebo effects has remained unclear. Here we show that analgesia from the expectation of pain relief is mediated by rostral anterior cingulate cortex (rACC) neurons that project to the pontine nucleus (rACC→Pn)-a precerebellar nucleus with no established function in pain. We created a behavioural assay that generates placebo-like anticipatory pain relief in mice. In vivo calcium imaging of neural activity and electrophysiological recordings in brain slices showed that expectations of pain relief boost the activity of rACC→Pn neurons and potentiate neurotransmission in this pathway. Transcriptomic studies of Pn neurons revealed an abundance of opioid receptors, further suggesting a role in pain modulation. Inhibition of the rACC→Pn pathway disrupted placebo analgesia and decreased pain thresholds, whereas activation elicited analgesia in the absence of placebo conditioning. Finally, Purkinje cells exhibited activity patterns resembling those of rACC→Pn neurons during pain-relief expectation, providing cellular-level evidence for a role of the cerebellum in cognitive pain modulation. These findings open the possibility of targeting this prefrontal cortico-ponto-cerebellar pathway with drugs or neurostimulation to treat pain.

Emergent reliability in sensory cortical coding and inter-area communication.

Reliable sensory discrimination must arise from high-fidelity neural representations and communication between brain areas. However, how neocortical sensory processing overcomes the substantial variability of neuronal sensory responses remains undetermined1-6. Here we imaged neuronal activity in eight neocortical areas concurrently and over five days in mice performing a visual discrimination task, yielding longitudinal recordings of more than 21,000 neurons. Analyses revealed a sequence of events across the neocortex starting from a resting state, to early stages of perception, and through the formation of a task response. At rest, the neocortex had one pattern of functional connections, identified through sets of areas that shared activity cofluctuations7,8. Within about 200 ms after the onset of the sensory stimulus, such connections rearranged, with different areas sharing cofluctuations and task-related information. During this short-lived state (approximately 300 ms duration), both inter-area sensory data transmission and the redundancy of sensory encoding peaked, reflecting a transient increase in correlated fluctuations among task-related neurons. By around 0.5 s after stimulus onset, the visual representation reached a more stable form, the structure of which was robust to the prominent, day-to-day variations in the responses of individual cells. About 1 s into stimulus presentation, a global fluctuation mode conveyed the upcoming response of the mouse to every area examined and was orthogonal to modes carrying sensory data. Overall, the neocortex supports sensory performance through brief elevations in sensory coding redundancy near the start of perception, neural population codes that are robust to cellular variability, and widespread inter-area fluctuation modes that transmit sensory data and task responses in non-interfering channels.

Fundamental bounds on the fidelity of sensory cortical coding.

How the brain processes information accurately despite stochastic neural activity is a longstanding question1. For instance, perception is fundamentally limited by the information that the brain can extract from the noisy dynamics of sensory neurons. Seminal experiments2,3 suggest that correlated noise in sensory cortical neural ensembles is what limits their coding accuracy4-6, although how correlated noise affects neural codes remains debated7-11. Recent theoretical work proposes that how a neural ensemble's sensory tuning properties relate statistically to its correlated noise patterns is a greater determinant of coding accuracy than is absolute noise strength12-14. However, without simultaneous recordings from thousands of cortical neurons with shared sensory inputs, it is unknown whether correlated noise limits coding fidelity. Here we present a 16-beam, two-photon microscope to monitor activity across the mouse primary visual cortex, along with analyses to quantify the information conveyed by large neural ensembles. We found that, in the visual cortex, correlated noise constrained signalling for ensembles with 800-1,300 neurons. Several noise components of the ensemble dynamics grew proportionally to the ensemble size and the encoded visual signals, revealing the predicted information-limiting correlations12-14. Notably, visual signals were perpendicular to the largest noise mode, which therefore did not limit coding fidelity. The information-limiting noise modes were approximately ten times smaller and concordant with mouse visual acuity15. Therefore, cortical design principles appear to enhance coding accuracy by restricting around 90% of noise fluctuations to modes that do not limit signalling fidelity, whereas much weaker correlated noise modes inherently bound sensory discrimination.

Diametric neural ensemble dynamics in parkinsonian and dyskinetic states.

Loss of dopamine in Parkinson's disease is hypothesized to impede movement by inducing hypo- and hyperactivity in striatal spiny projection neurons (SPNs) of the direct (dSPNs) and indirect (iSPNs) pathways in the basal ganglia, respectively. The opposite imbalance might underlie hyperkinetic abnormalities, such as dyskinesia caused by treatment of Parkinson's disease with the dopamine precursor L-DOPA. Here we monitored thousands of SPNs in behaving mice, before and after dopamine depletion and during L-DOPA-induced dyskinesia. Normally, intermingled clusters of dSPNs and iSPNs coactivated before movement. Dopamine depletion unbalanced SPN activity rates and disrupted the movement-encoding iSPN clusters. Matching their clinical efficacy, L-DOPA or agonism of the D2 dopamine receptor reversed these abnormalities more effectively than agonism of the D1 dopamine receptor. The opposite pathophysiology arose in L-DOPA-induced dyskinesia, during which iSPNs showed hypoactivity and dSPNs showed unclustered hyperactivity. Therefore, both the spatiotemporal profiles and rates of SPN activity appear crucial to striatal function, and next-generation treatments for basal ganglia disorders should target both facets of striatal activity.

The relationship between birth timing, circuit wiring, and physiological response properties of cerebellar granule cells.

Cerebellar granule cells (GrCs) are usually regarded as a uniform cell type that collectively expands the coding space of the cerebellum by integrating diverse combinations of mossy fiber inputs. Accordingly, stable molecularly or physiologically defined GrC subtypes within a single cerebellar region have not been reported. The only known cellular property that distinguishes otherwise homogeneous GrCs is the correspondence between GrC birth timing and the depth of the molecular layer to which their axons project. To determine the role birth timing plays in GrC wiring and function, we developed genetic strategies to access early- and late-born GrCs. We initiated retrograde monosynaptic rabies virus tracing from control (birth timing unrestricted), early-born, and late-born GrCs, revealing the different patterns of mossy fiber input to GrCs in vermis lobule 6 and simplex, as well as to early- and late-born GrCs of vermis lobule 6: sensory and motor nuclei provide more input to early-born GrCs, while basal pontine and cerebellar nuclei provide more input to late-born GrCs. In vivo multidepth two-photon Ca2+ imaging of axons of early- and late-born GrCs revealed representations of diverse task variables and stimuli by both populations, with modest differences in the proportions encoding movement, reward anticipation, and reward consumption. Our results suggest neither organized parallel processing nor completely random organization of mossy fiber→GrC circuitry but instead a moderate influence of birth timing on GrC wiring and encoding. Our imaging data also provide evidence that GrCs can represent generalized responses to aversive stimuli, in addition to recently described reward representations.

Cerebellar granule cells encode the expectation of reward.

The human brain contains approximately 60 billion cerebellar granule cells, which outnumber all other brain neurons combined. Classical theories posit that a large, diverse population of granule cells allows for highly detailed representations of sensorimotor context, enabling downstream Purkinje cells to sense fine contextual changes. Although evidence suggests a role for the cerebellum in cognition, granule cells are known to encode only sensory and motor context. Here, using two-photon calcium imaging in behaving mice, we show that granule cells convey information about the expectation of reward. Mice initiated voluntary forelimb movements for delayed sugar-water reward. Some granule cells responded preferentially to reward or reward omission, whereas others selectively encoded reward anticipation. Reward responses were not restricted to forelimb movement, as a Pavlovian task evoked similar responses. Compared to predictable rewards, unexpected rewards elicited markedly different granule cell activity despite identical stimuli and licking responses. In both tasks, reward signals were widespread throughout multiple cerebellar lobules. Tracking the same granule cells over several days of learning revealed that cells with reward-anticipating responses emerged from those that responded at the start of learning to reward delivery, whereas reward-omission responses grew stronger as learning progressed. The discovery of predictive, non-sensorimotor encoding in granule cells is a major departure from the current understanding of these neurons and markedly enriches the contextual information available to postsynaptic Purkinje cells, with important implications for cognitive processing in the cerebellum.

Social behaviour shapes hypothalamic neural ensemble representations of conspecific sex.

All animals possess a repertoire of innate (or instinctive) behaviours, which can be performed without training. Whether such behaviours are mediated by anatomically distinct and/or genetically specified neural pathways remains unknown. Here we report that neural representations within the mouse hypothalamus, that underlie innate social behaviours, are shaped by social experience. Oestrogen receptor 1-expressing (Esr1+) neurons in the ventrolateral subdivision of the ventromedial hypothalamus (VMHvl) control mating and fighting in rodents. We used microendoscopy to image Esr1+ neuronal activity in the VMHvl of male mice engaged in these social behaviours. In sexually and socially experienced adult males, divergent and characteristic neural ensembles represented male versus female conspecifics. However, in inexperienced adult males, male and female intruders activated overlapping neuronal populations. Sex-specific neuronal ensembles gradually separated as the mice acquired social and sexual experience. In mice permitted to investigate but not to mount or attack conspecifics, ensemble divergence did not occur. However, 30 minutes of sexual experience with a female was sufficient to promote the separation of male and female ensembles and to induce an attack response 24 h later. These observations uncover an unexpected social experience-dependent component to the formation of hypothalamic neural assemblies controlling innate social behaviours. More generally, they reveal plasticity and dynamic coding in an evolutionarily ancient deep subcortical structure that is traditionally viewed as a 'hard-wired' system.

Neural ensemble dynamics underlying a long-term associative memory.

The brain's ability to associate different stimuli is vital for long-term memory, but how neural ensembles encode associative memories is unknown. Here we studied how cell ensembles in the basal and lateral amygdala encode associations between conditioned and unconditioned stimuli (CS and US, respectively). Using a miniature fluorescence microscope, we tracked the Ca2+ dynamics of ensembles of amygdalar neurons during fear learning and extinction over 6 days in behaving mice. Fear conditioning induced both up- and down-regulation of individual cells' CS-evoked responses. This bi-directional plasticity mainly occurred after conditioning, and reshaped the neural ensemble representation of the CS to become more similar to the US representation. During extinction training with repetitive CS presentations, the CS representation became more distinctive without reverting to its original form. Throughout the experiments, the strength of the ensemble-encoded CS-US association predicted the level of behavioural conditioning in each mouse. These findings support a supervised learning model in which activation of the US representation guides the transformation of the CS representation.

Kilohertz two-photon brain imaging in awake mice.

Two-photon microscopy is a mainstay technique for imaging in scattering media and normally provides frame-acquisition rates of ~10-30 Hz. To track high-speed phenomena, we created a two-photon microscope with 400 illumination beams that collectively sample 95,000-211,000 µm2 areas at rates up to 1 kHz. Using this microscope, we visualized microcirculatory flow, fast venous constrictions and neuronal Ca2+ spiking with millisecond-scale timing resolution in the brains of awake mice.

Fast, in vivo voltage imaging using a red fluorescent indicator.

Genetically encoded voltage indicators (GEVIs) are emerging optical tools for acquiring brain-wide cell-type-specific functional data at unparalleled temporal resolution. To broaden the application of GEVIs in high-speed multispectral imaging, we used a high-throughput strategy to develop voltage-activated red neuronal activity monitor (VARNAM), a fusion of the fast Acetabularia opsin and the bright red fluorophore mRuby3. Imageable under the modest illumination intensities required by bright green probes (<50 mW mm-2), VARNAM is readily usable in vivo. VARNAM can be combined with blue-shifted optical tools to enable cell-type-specific all-optical electrophysiology and dual-color spike imaging in acute brain slices and live Drosophila. With enhanced sensitivity to subthreshold voltages, VARNAM resolves postsynaptic potentials in slices and cortical and hippocampal rhythms in freely behaving mice. Together, VARNAM lends a new hue to the optical toolbox, opening the door to high-speed in vivo multispectral functional imaging.

Three-photon imaging of mouse brain structure and function through the intact skull.

Optical imaging through the intact mouse skull is challenging because of skull-induced aberrations and scattering. We found that three-photon excitation provided improved optical sectioning compared with that obtained with two-photon excitation, even when we used the same excitation wavelength and imaging system. Here we demonstrate three-photon imaging of vasculature through the adult mouse skull at >500-µm depth, as well as GCaMP6s calcium imaging over weeks in cortical layers 2/3 and 4 in awake mice, with 8.5 frames per second and a field of view spanning hundreds of micrometers.