Control of feeding by a bottom-up midbrain-subthalamic pathway.

Investigative exploration and foraging leading to food consumption have vital importance, but are not well-understood. Since GABAergic inputs to the lateral and ventrolateral periaqueductal gray (l/vlPAG) control such behaviors, we dissected the role of vgat-expressing GABAergic l/vlPAG cells in exploration, foraging and hunting. Here, we show that in mice vgat l/vlPAG cells encode approach to food and consumption of both live prey and non-prey foods. The activity of these cells is necessary and sufficient for inducing food-seeking leading to subsequent consumption. Activation of vgat l/vlPAG cells produces exploratory foraging and compulsive eating without altering defensive behaviors. Moreover, l/vlPAG vgat cells are bidirectionally interconnected to several feeding, exploration and investigation nodes, including the zona incerta. Remarkably, the vgat l/vlPAG projection to the zona incerta bidirectionally controls approach towards food leading to consumption. These data indicate the PAG is not only a final downstream target of top-down exploration and foraging-related inputs, but that it also influences these behaviors through a bottom-up pathway.

Endocannabinoids modulate fear extinction controlled by a cortical-amygdala projection.

The projection from the medial prefrontal cortex to the amygdala is a key node in fear extinction. In this issue of Neuron, Gunduz-Cinar et al.1 show that extinction induction by this projection requires recruitment of endocannabinoids (eCBs) in the amygdala.

A dual-pathway architecture enables chronic stress to promote habit formation.

Chronic stress can change how we learn and, thus, how we make decisions by promoting the formation of inflexible, potentially maladaptive, habits. Here we investigated the neuronal circuit mechanisms that enable this. Using a multifaceted approach in male and female mice, we reveal a dual pathway, amygdala-striatal, neuronal circuit architecture by which a recent history of chronic stress shapes learning to disrupt flexible goal-directed behavior in favor of inflexible habits. Chronic stress inhibits activity of basolateral amygdala projections to the dorsomedial striatum to impede the action-outcome learning that supports flexible, goal-directed decisions. Stress also increases activity in direct central amygdala projections to the dorsomedial striatum to promote the formation of rigid, inflexible habits. Thus, stress exerts opposing effects on two amygdala-striatal pathways to promote premature habit formation. These data provide neuronal circuit insights into how chronic stress shapes learning and decision making, and help understand how stress can lead to the disrupted decision making and pathological habits that characterize substance use disorders and other psychiatric conditions.

A pilot study of closed-loop neuromodulation for treatment-resistant post-traumatic stress disorder.

The neurophysiological mechanisms in the human amygdala that underlie post-traumatic stress disorder (PTSD) remain poorly understood. In a first-of-its-kind pilot study, we recorded intracranial electroencephalographic data longitudinally (over one year) in two male individuals with amygdala electrodes implanted for the management of treatment-resistant PTSD (TR-PTSD) under clinical trial NCT04152993. To determine electrophysiological signatures related to emotionally aversive and clinically relevant states (trial primary endpoint), we characterized neural activity during unpleasant portions of three separate paradigms (negative emotional image viewing, listening to recordings of participant-specific trauma-related memories, and at-home-periods of symptom exacerbation). We found selective increases in amygdala theta (5-9 Hz) bandpower across all three negative experiences. Subsequent use of elevations in low-frequency amygdala bandpower as a trigger for closed-loop neuromodulation led to significant reductions in TR-PTSD symptoms (trial secondary endpoint) following one year of treatment as well as reductions in aversive-related amygdala theta activity. Altogether, our findings provide early evidence that elevated amygdala theta activity across a range of negative-related behavioral states may be a promising target for future closed-loop neuromodulation therapies in PTSD.

Regulation of social interaction in mice by a frontostriatal circuit modulated by established hierarchical relationships.

Social hierarchies exert a powerful influence on behavior, but the neurobiological mechanisms that detect and regulate hierarchical interactions are not well understood, especially at the level of neural circuits. Here, we use fiber photometry and chemogenetic tools to record and manipulate the activity of nucleus accumbens-projecting cells in the ventromedial prefrontal cortex (vmPFC-NAcSh) during tube test social competitions. We show that vmPFC-NAcSh projections signal learned hierarchical relationships, and are selectively recruited by subordinate mice when they initiate effortful social dominance behavior during encounters with a dominant competitor from an established hierarchy. After repeated bouts of social defeat stress, this circuit is preferentially activated during social interactions initiated by stress resilient individuals, and plays a necessary role in supporting social approach behavior in subordinated mice. These results define a necessary role for vmPFC-NAcSh cells in the adaptive regulation of social interaction behavior based on prior hierarchical interactions.

Orchestration of innate and conditioned defensive actions by the periaqueductal gray.

The midbrain periaqueductal gray (PAG) has been recognized for decades as having a central role in the control of a wide variety of defensive responses. Initial discoveries relied primarily on lesions, electrical stimulation and pharmacology. Recent developments in neural activity imaging and in methods to control activity with anatomical and genetic specificity have revealed additional streams of data informing our understanding of PAG function. Here, we discuss both classic and modern studies reporting on how PAG-centered circuits influence innate as well as learned defensive actions in rodents and humans. Though early discoveries emphasized the PAG's role in rapid induction of innate defensive actions, emerging new data indicate a prominent role for the PAG in more complex processes, including representing behavioral states and influencing fear learning and memory. This article is part of the Special Issue on "Fear, Anxiety and PTSD".

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.

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.

Sparse genetically defined neurons refine the canonical role of periaqueductal gray columnar organization.

During threat exposure, survival depends on defensive reactions. Prior works linked large glutamatergic populations in the midbrain periaqueductal gray (PAG) to defensive freezing and flight, and established that the overarching functional organization axis of the PAG is along anatomically-defined columns. Accordingly, broad activation of the dorsolateral column induces flight, while activation of the lateral or ventrolateral (l and vl) columns induces freezing. However, the PAG contains diverse cell types that vary in neurochemistry. How these cell types contribute to defense remains unknown, indicating that targeting sparse, genetically-defined populations may reveal how the PAG generates diverse behaviors. Though prior works showed that broad excitation of the lPAG or vlPAG causes freezing, we found in mice that activation of lateral and ventrolateral PAG (l/vlPAG) cholecystokinin-expressing (CCK) cells selectively caused flight to safer regions within an environment. Furthermore, inhibition of l/vlPAG-CCK cells reduced predator avoidance without altering other defensive behaviors like freezing. Lastly, l/vlPAG-CCK activity decreased when approaching threat and increased during movement to safer locations. These results suggest CCK cells drive threat avoidance states, which are epochs during which mice increase distance from threat and perform evasive escape. Conversely, l/vlPAG pan-neuronal activation promoted freezing, and these cells were activated near threat. Thus, CCK l/vlPAG cells have opposing function and neural activation motifs compared to the broader local ensemble defined solely by columnar boundaries. In addition to the anatomical columnar architecture of the PAG, the molecular identity of PAG cells may confer an additional axis of functional organization, revealing unexplored functional heterogeneity.

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.

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.

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.

Coordination of escape and spatial navigation circuits orchestrates versatile flight from 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 that activity in cholecystokinin-expressing hypothalamic dorsal premammillary nucleus (PMd-cck) cells is sufficient and necessary for context-specific escape that adapts to each environment's layout. In contrast, numerous other nuclei implicated in flight only induced stereotyped panic-related escape. We reasoned the dorsal premammillary nucleus (PMd) can induce context-specific escape because it projects to escape and spatial navigation nuclei. Indeed, activity in PMd-cck projections to thalamic spatial navigation circuits is 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 tested escapes. Thus, PMd-cck cells control versatile flight, engaging spatial navigation and escape circuits.

Estrogen-sensitive medial preoptic area neurons coordinate torpor in mice.

Homeotherms maintain a stable internal body temperature despite changing environments. During energy deficiency, some species can cease to defend their body temperature and enter a hypothermic and hypometabolic state known as torpor. Recent advances have revealed the medial preoptic area (MPA) as a key site for the regulation of torpor in mice. The MPA is estrogen-sensitive and estrogens also have potent effects on both temperature and metabolism. Here, we demonstrate that estrogen-sensitive neurons in the MPA can coordinate hypothermia and hypometabolism in mice. Selectively activating estrogen-sensitive MPA neurons was sufficient to drive a coordinated depression of metabolic rate and body temperature similar to torpor, as measured by body temperature, physical activity, indirect calorimetry, heart rate, and brain activity. Inducing torpor with a prolonged fast revealed larger and more variable calcium transients from estrogen-sensitive MPA neurons during bouts of hypothermia. Finally, whereas selective ablation of estrogen-sensitive MPA neurons demonstrated that these neurons are required for the full expression of fasting-induced torpor in both female and male mice, their effects on thermoregulation and torpor bout initiation exhibit differences across sex. Together, these findings suggest a role for estrogen-sensitive MPA neurons in directing the thermoregulatory and metabolic responses to energy deficiency.

To Approach or Avoid: An Introductory Overview of the Study of Anxiety Using Rodent Assays.

Anxiety is a widely studied phenomenon in behavioral neuroscience, but the recent literature lacks an overview of the major conceptual framework underlying anxiety research to introduce young researchers to the field. In this mini-review article, which is aimed toward new undergraduate and graduate students, we discuss how researchers exploit the approach-avoidance conflict, an internal conflict rodents face between exploration of novel environments and avoidance of danger, to inform rodent assays that allow for the measurement of anxiety-related behavior in the laboratory. We review five widely-used rodent anxiety assays, consider the pharmacological validity of these assays, and discuss neural circuits that have recently been shown to modulate anxiety using the assays described. Finally, we offer related lines of inquiry and comment on potential future directions.

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.

Integration of optogenetics with complementary methodologies in systems neuroscience.

Modern optogenetics can be tuned to evoke activity that corresponds to naturally occurring local or global activity in timing, magnitude or individual-cell patterning. This outcome has been facilitated not only by the development of core features of optogenetics over the past 10 years (microbial-opsin variants, opsin-targeting strategies and light-targeting devices) but also by the recent integration of optogenetics with complementary technologies, spanning electrophysiology, activity imaging and anatomical methods for structural and molecular analysis. This integrated approach now supports optogenetic identification of the native, necessary and sufficient causal underpinnings of physiology and behaviour on acute or chronic timescales and across cellular, circuit-level or brain-wide spatial scales.

Wiring and Molecular Features of Prefrontal Ensembles Representing Distinct Experiences.

A major challenge in understanding the cellular diversity of the brain has been linking activity during behavior with standard cellular typology. For example, it has not been possible to determine whether principal neurons in prefrontal cortex active during distinct experiences represent separable cell types, and it is not known whether these differentially active cells exert distinct causal influences on behavior. Here, we develop quantitative hydrogel-based technologies to connect activity in cells reporting on behavioral experience with measures for both brain-wide wiring and molecular phenotype. We find that positive and negative-valence experiences in prefrontal cortex are represented by cell populations that differ in their causal impact on behavior, long-range wiring, and gene expression profiles, with the major discriminant being expression of the adaptation-linked gene NPAS4. These findings illuminate cellular logic of prefrontal cortex information processing and natural adaptive behavior and may point the way to cell-type-specific understanding and treatment of disease-associated states.

Basomedial amygdala mediates top-down control of anxiety and fear.

Anxiety-related conditions are among the most difficult neuropsychiatric diseases to treat pharmacologically, but respond to cognitive therapies. There has therefore been interest in identifying relevant top-down pathways from cognitive control regions in medial prefrontal cortex (mPFC). Identification of such pathways could contribute to our understanding of the cognitive regulation of affect, and provide pathways for intervention. Previous studies have suggested that dorsal and ventral mPFC subregions exert opposing effects on fear, as do subregions of other structures. However, precise causal targets for top-down connections among these diverse possibilities have not been established. Here we show that the basomedial amygdala (BMA) represents the major target of ventral mPFC in amygdala in mice. Moreover, BMA neurons differentiate safe and aversive environments, and BMA activation decreases fear-related freezing and high-anxiety states. Lastly, we show that the ventral mPFC-BMA projection implements top-down control of anxiety state and learned freezing, both at baseline and in stress-induced anxiety, defining a broadly relevant new top-down behavioural regulation pathway.

Natural neural projection dynamics underlying social behavior.

Social interaction is a complex behavior essential for many species and is impaired in major neuropsychiatric disorders. Pharmacological studies have implicated certain neurotransmitter systems in social behavior, but circuit-level understanding of endogenous neural activity during social interaction is lacking. We therefore developed and applied a new methodology, termed fiber photometry, to optically record natural neural activity in genetically and connectivity-defined projections to elucidate the real-time role of specified pathways in mammalian behavior. Fiber photometry revealed that activity dynamics of a ventral tegmental area (VTA)-to-nucleus accumbens (NAc) projection could encode and predict key features of social, but not novel object, interaction. Consistent with this observation, optogenetic control of cells specifically contributing to this projection was sufficient to modulate social behavior, which was mediated by type 1 dopamine receptor signaling downstream in the NAc. Direct observation of deep projection-specific activity in this way captures a fundamental and previously inaccessible dimension of mammalian circuit dynamics.