Opioid-driven disruption of the septal complex reveals a role for neurotensin-expressing neurons in withdrawal.

Because opioid withdrawal is an intensely aversive experience, persons with opioid use disorder (OUD) often relapse to avoid it. The lateral septum (LS) is a forebrain structure that is important in aversion processing, and previous studies have linked the lateral septum (LS) to substance use disorders. It is unclear, however, which precise LS cell types might contribute to the maladaptive state of withdrawal. To address this, we used single-nucleus RNA-sequencing to interrogate cell type specific gene expression changes induced by chronic morphine and withdrawal. We discovered that morphine globally disrupted the transcriptional profile of LS cell types, but Neurotensin-expressing neurons (Nts; LS-Nts neurons) were selectively activated by naloxone. Using two-photon calcium imaging and ex vivo electrophysiology, we next demonstrate that LS-Nts neurons receive enhanced glutamatergic drive in morphine-dependent mice and remain hyperactivated during opioid withdrawal. Finally, we showed that activating and silencing LS-Nts neurons during opioid withdrawal regulates pain coping behaviors and sociability. Together, these results suggest that LS-Nts neurons are a key neural substrate involved in opioid withdrawal and establish the LS as a crucial regulator of adaptive behaviors, specifically pertaining to OUD.

VMHvllCckar cells dynamically control female sexual behaviors over the reproductive cycle.

Sexual behavior is fundamental for the survival of mammalian species and thus supported by dedicated neural substrates. The ventrolateral part of ventromedial hypothalamus (VMHvl) is an essential locus for controlling female sexual behaviors, but recent studies revealed the molecular complexity and functional heterogeneity of VMHvl cells. Here, we identify the cholecystokinin A receptor (Cckar)-expressing cells in the lateral VMHvl (VMHvllCckar) as the key controllers of female sexual behaviors. The inactivation of VMHvllCckar cells in female mice diminishes their interest in males and sexual receptivity, whereas activating these cells has the opposite effects. Female sexual behaviors vary drastically over the reproductive cycle. In vivo recordings reveal reproductive-state-dependent changes in VMHvllCckar cell spontaneous activity and responsivity, with the highest activity occurring during estrus. These in vivo response changes coincide with robust alternation in VMHvllCckar cell excitability and synaptic inputs. Altogether, VMHvllCckar cells represent a key neural population dynamically controlling female sexual behaviors over the reproductive cycle.

PACAP-expressing neurons in the lateral habenula diminish negative emotional valence.

The lateral habenula (LHb) is a small, bilateral, epithalamic nucleus which processes aversive information. While primarily glutamatergic, LHb neurons express genes coding for many neuropeptides, such as Adcyap1 the gene encoding pituitary adenylate cyclase-activating polypeptide (PACAP), which itself has been associated with anxiety and stress disorders. Using Cre-dependent viral vectors, we targeted and characterized these neurons based on their anatomical projections and found that they projected to both the raphe and rostromedial tegmentum but only weakly to ventral tegmental area. Using RiboTag to capture ribosomal-associated mRNA from these neurons and reanalysis of existing single cell RNA sequencing data, we did not identify a unique molecular phenotype that characterized these PACAP-expressing neurons in LHb. In order to understand the function of these neurons, we conditionally expressed hM3 Dq DREADD selectively in LHb PACAP-expressing neurons and chemogenetically excited these neurons during behavioral testing in the open field test, contextual fear conditioning, sucrose preference, novelty suppressed feeding, and conditioned place preference. We found that Gq activation of these neurons produce behaviors opposite to what is expected from the LHb as a whole-they decreased anxiety-like and fear behavior and produced a conditioned place preference. In conclusion, PACAP-expressing neurons in LHb represents a molecularly diverse population of cells that oppose the actions of the remainder of LHb neurons by being rewarding or diminishing the negative consequences of aversive events.

Transcriptional and functional divergence in lateral hypothalamic glutamate neurons projecting to the lateral habenula and ventral tegmental area.

The lateral hypothalamic area (LHA) regulates feeding- and reward-related behavior, but because of its molecular and anatomical heterogeneity, the functions of defined neuronal populations are largely unclear. Glutamatergic neurons within the LHA (LHAVglut2) negatively regulate feeding and appetitive behavior. However, this population comprises transcriptionally distinct and functionally diverse neurons that project to diverse brain regions, including the lateral habenula (LHb) and ventral tegmental area (VTA). To resolve the function of distinct LHAVglut2 populations, we systematically compared projections to the LHb and VTA using viral tracing, single-cell sequencing, electrophysiology, and in vivo calcium imaging. LHAVglut2 neurons projecting to the LHb or VTA are anatomically, transcriptionally, electrophysiologically, and functionally distinct. While both populations encode appetitive and aversive stimuli, LHb projecting neurons are especially sensitive to satiety state and feeding hormones. These data illuminate the functional heterogeneity of LHAVglut2 neurons, suggesting that reward and aversion are differentially processed in divergent efferent pathways.

Transcriptional and Spatial Resolution of Cell Types in the Mammalian Habenula.

The habenula complex is appreciated as a critical regulator of motivated and pathological behavioral states via its output to midbrain nuclei. Despite this, transcriptional definition of cell populations that comprise both the medial habenular (MHb) and lateral habenular (LHb) subregions in mammals remain undefined. To resolve this, we performed single-cell transcriptional profiling and highly multiplexed in situ hybridization experiments of the mouse habenula complex in naive mice and those exposed to an acute aversive stimulus. Transcriptionally distinct neuronal cell types identified within the MHb and LHb, were spatially defined, differentially engaged by aversive stimuli, and had distinct electrophysiological properties. Cell types identified in mice also displayed a high degree of transcriptional similarity to those previously described in zebrafish, highlighting the well-conserved nature of habenular cell types across the phylum. These data identify key molecular targets within habenular cell types and provide a critical resource for future studies.

Paraventricular Thalamus Projection Neurons Integrate Cortical and Hypothalamic Signals for Cue-Reward Processing.

The paraventricular thalamus (PVT) is an interface for brain reward circuits, with input signals arising from structures, such as prefrontal cortex and hypothalamus, that are broadcast to downstream limbic targets. However, the precise synaptic connectivity, activity, and function of PVT circuitry for reward processing are unclear. Here, using in vivo two-photon calcium imaging, we find that PVT neurons projecting to the nucleus accumbens (PVT-NAc) develop inhibitory responses to reward-predictive cues coding for both cue-reward associative information and behavior. The multiplexed activity in PVT-NAc neurons is directed by opposing activity patterns in prefrontal and lateral hypothalamic afferent axons. Further, we find that prefrontal cue encoding may maintain accurate cue-reward processing, as optogenetic disruption of this encoding induced long-lasting effects on downstream PVT-NAc cue responses and behavioral cue discrimination. Together, these data reveal that PVT-NAc neurons act as an interface for reward processing by integrating relevant inputs to accurately inform reward-seeking behavior.

Rapid, biphasic CRF neuronal responses encode positive and negative valence.

Corticotropin-releasing factor (CRF) that is released from the paraventricular nucleus (PVN) of the hypothalamus is essential for mediating stress response by activating the hypothalamic-pituitary-adrenal axis. CRF-releasing PVN neurons receive inputs from multiple brain regions that convey stressful events, but their neuronal dynamics on the timescale of behavior remain unknown. Here, our recordings of PVN CRF neuronal activity in freely behaving mice revealed that CRF neurons are activated immediately by a range of aversive stimuli. By contrast, CRF neuronal activity starts to drop within a second of exposure to appetitive stimuli. Optogenetic activation or inhibition of PVN CRF neurons was sufficient to induce a conditioned place aversion or preference, respectively. Furthermore, conditioned place aversion or preference induced by natural stimuli was significantly decreased by manipulating PVN CRF neuronal activity. Together, these findings suggest that the rapid, biphasic responses of PVN CRF neurons encode the positive and negative valences of stimuli.

The Neural Mechanisms of Sexually Dimorphic Aggressive Behaviors.

Aggression is a fundamental social behavior that is essential for competing for resources and protecting oneself and families in both males and females. As a result of natural selection, aggression is often displayed differentially between the sexes, typically at a higher level in males than females. Here, we highlight the behavioral differences between male and female aggression in rodents. We further outline the aggression circuits in males and females, and compare their differences at each circuit node. Lastly, we summarize our current understanding regarding the generation of sexually dimorphic aggression circuits during development and their maintenance during adulthood. In both cases, gonadal steroid hormones appear to play crucial roles in differentiating the circuits by impacting on the survival, morphology, and intrinsic properties of relevant cells. Many other factors, such as environment and experience, may also contribute to sex differences in aggression and remain to be investigated in future studies.

Esr1+ cells in the ventromedial hypothalamus control female aggression.

As an essential means of resolving conflicts, aggression is expressed by both sexes but often at a higher level in males than in females. Recent studies suggest that cells in the ventrolateral part of the ventromedial hypothalamus (VMHvl) that express estrogen receptor-α (Esr1) and progesterone receptor are essential for male but not female mouse aggression. In contrast, here we show that VMHvlEsr1+ cells are indispensable for female aggression. This population was active when females attacked naturally. Inactivation of these cells reduced female aggression whereas their activation elicited attack. Additionally, we found that female VMHvl contains two anatomically distinguishable subdivisions that showed differential gene expression, projection and activation patterns after mating and fighting. These results support an essential role of the VMHvl in both male and female aggression and reveal the existence of two previously unappreciated subdivisions in the female VMHvl that are involved in distinct social behaviors.

Ventromedial Hypothalamus and the Generation of Aggression.

Aggression is a costly behavior, sometimes with severe consequences including death. Yet aggression is prevalent across animal species ranging from insects to humans, demonstrating its essential role in the survival of individuals and groups. The question of how the brain decides when to generate this costly behavior has intrigued neuroscientists for over a century and has led to the identification of relevant neural substrates. Various lesion and electric stimulation experiments have revealed that the hypothalamus, an ancient structure situated deep in the brain, is essential for expressing aggressive behaviors. More recently, studies using precise circuit manipulation tools have identified a small subnucleus in the medial hypothalamus, the ventrolateral part of the ventromedial hypothalamus (VMHvl), as a key structure for driving both aggression and aggression-seeking behaviors. Here, we provide an updated summary of the evidence that supports a role of the VMHvl in aggressive behaviors. We will consider our recent findings detailing the physiological response properties of populations of VMHvl cells during aggressive behaviors and provide new understanding regarding the role of the VMHvl embedded within the larger whole-brain circuit for social sensation and action.

The neural circuits of mating and fighting in male mice.

Tinbergen proposed that instinctive behaviors can be divided into appetitive and consummatory phases. During mating and aggression, the appetitive phase contains various actions to bring an animal to a social target and the consummatory phase allows stereotyped actions to take place. Here, we summarize recent advances in elucidating the neural circuits underlying the appetitive and consummatory phases of sexual and aggressive behaviors with a focus on male mice. We outline the role of the main olfactory inputs in the initiation of social approach; the engagement of the accessory olfactory system during social investigation, and the role of the hypothalamus and its downstream pathways in orchestrating social behaviors through a suite of motor actions.

Blockade of stimulus convergence in amygdala neurons disrupts taste associative learning.

Humans and non-human animals learn associations of temporally contingent stimuli to better cope with the changing environment. In animal models of classical conditioning, a neutral conditioned stimulus (CS) predicts an aversive unconditioned stimulus (US). Several lines of indirect evidence indicate that this learning may rely on stimulus convergence in a subset of neurons, but this hypothesis has not been directly tested. In the current study, we tested this hypothesis using a pharmacogenetic approach, the cAMP response element-binding protein (CREB)/Allatostatin Receptor system, to target a subset of amygdala neurons receiving convergent stimuli in mice during conditioned taste aversion. Virally infected basolateral amygdala neurons with higher CREB levels were predominantly active during CS presentation. Blocking stimulus convergence in infected neurons by silencing them during US disrupted taste associative memory. Moreover, silencing infected neurons only during CS also disrupted associative memory formation. These results provide support for the notion that convergent inputs of CS and US in a subpopulation of neurons are critical for associative memory formation.

Off-line Arc transcription in active ensembles during fear memory retrieval.

Neural activity and de novo protein synthesis during a rest period following memory retrieval in the amygdala is necessary for stabilization of reactivated fear memory. Arc/Arg3.1 (Arc) expression is regulated by neural activity and is a critical protein for memory reconsolidation. However, it remains unclear whether memory retrieval alters Arc transcription during subsequent rest. In this study, the populations of mouse lateral amygdala neurons that transcribe Arc during memory retrieval and at rest were detected using Arc cellular compartment analysis of temporal activity by fluorescence in situ hybridization (Arc catFISH). Results demonstrated that memory retrieval alters the composition of neuronal populations, which activate Arc transcription during subsequent rest. Approximately 50% of neurons that transcribe Arc at subsequent rest, transcribed Arc during memory retrieval, whereas only approximately 10% of neurons that transcribed Arc during a rest period prior to memory retrieval transcribe Arc during memory retrieval. In contrast, re-exposure to the chamber induced less preferential Arc transcription in latent inhibited mice that received shocks but recalled less conditioned fear. Taken together, these findings indicate that neuronal subpopulations activated during fear memory retrieval preferentially transcribe Arc during subsequent rest in the lateral amygdala. This preferential Arc transcription may contribute to memory reconsolidation.

Memory coding in plastic neuronal subpopulations within the amygdala.

Specific neuronal subpopulations within specific brain areas are responsible for learning and memory. A fear memory engages a subset of lateral amygdala neurons, but whether multiple contextual fear memories engage the same or different subsets of lateral amygdala neurons remains unclear. Here, we demonstrate the representation of multiple contextual fear memories in the amygdala with cellular and temporal resolution using a large-scale imaging method. Mice were conditioned with a footshock in 2 separate chambers. They were then re-exposed to either the same conditioning chamber twice or 2 different conditioning chambers. The activities of individual neurons related to the re-exposures were determined by the subcellular distribution of Arc/Arg3.1 RNA. Reactivation of different memories activated partially (about 50%) overlapping neurons, whereas reactivation of the same memory activated more overlapping (about 65%) neurons. These findings indicate that lateral amygdala neurons related to different fear memories are partly common, and that a small but significant neuronal population (2.7% of total lateral amygdala neurons) encodes differences in individual fear memories. Moreover, memory retrieval increased the size of the neuronal subpopulation activated during subsequent retrieval. Taken together, our findings indicate that small plastic subsets of neurons encode fear memories from individual contexts.

Preferential Arc transcription at rest in the active ensemble during associative learning.

Information processing in the central nervous system (CNS) during periods of rest is crucial for lasting memories but the precise off-line neuronal population activity that contributes to long-term memory formation remains unclear. This pattern of neuronal activity during rest triggers transcription of immediate early genes such as activity regulated cytoskeletal gene (Arc). We compared the active neuronal population in the lateral amygdala of C57BL/6J mice during fear conditioning and rest periods using a large scale imaging technique, Arc cellular compartment analysis of temporal activity by fluorescence in situ hybridization (catFISH). We found that the neuronal population transcribing Arc during fear conditioning was more similar to that the population transcribing Arc after fear conditioning than before fear conditioning. The overlapping population was larger in conditioned mice that acquired associative memory than in unshocked mice and in latent inhibited mice that received shocks but did not form associative memory. Moreover, these results were confirmed using Arc/Homer 1a catFISH. Our findings indicate that Arc is preferentially transcribed in neurons that are active during fear conditioning after associative learning. This preferential transcription may contribute to the formation of long-lasting memory.

Persistent neural activity regulates Arc/Arg3.1 transcription in the dentate gyrus.

The activity-regulated cytoskeleton-associated gene (Arc, also known as Arg3.1) is an effector immediate-early gene rapidly induced by strong neural activity. Although a number of studies have revealed significant functions of Arc and Arc has come into widespread use as a neural activity marker in behavioral studies, the mechanisms regulating Arc transcription remain unclear. Here, we examined the conditions of Arc transcription in acute slices of dentate gyrus. Surprisingly, kainic acid (1 µM to 10 mM) application to slices did not induce Arc transcription, although intraperitoneal injection of kainic acid (20 mg/kg) induced robust Arc transcription. No types of high-frequency stimulation examined induced Arc transcription in acute slices. These findings indicate that Arc transcription is dramatically suppressed in acute slices of the dentate gyrus, in which background neural activity is markedly reduced. Burst stimulation increased the number of Arc-expressing cells in the presence of picrotoxin, in which excitation was maintained even after the end of stimulation. Moreover, the involvement of background neural activity in Arc transcription was tested by application of carbachol, a muscarinic receptor agonist. Carbachol also increased the number of Arc-expressing cells, which was blocked by atropine, a muscarinic receptor antagonist. Taken together, these findings suggest that persistent background activity is critical for Arc transcription.

Hyperactivity in novel environment with increased dopamine and impaired novelty preference in apoptosis signal-regulating kinase 1 (ASK1)-deficient mice.

Apoptosis signal-regulating kinase 1 (ASK1) is a mitogen-activated protein (MAP) kinase kinase kinase family member, which induces apoptosis in various cells through JNK and p38 MAP kinase cascades. In addition to apoptosis signaling, a number of recent in vitro studies have suggested that ASK1 may play roles in neural function. However, the behavioral significance of ASK1 has remained unclear. Here, we subjected ASK1 (-/-) mice to a battery of behavioral tests and found that they displayed temporary hyperactivity in an open-field test. Activities in the familiar field were normal, indicating that the hyperactivity observed was specific to the novel environment. ASK1 (-/-) mice also exhibited impairment of novelty preference 24h after training and superior performance on the rotarod test. Brain tissue contents of dopamine and 4-dihydroxyphenylacetic acid (DOPAC) were elevated in ASK1 (-/-) mice. Our findings thus demonstrate novel behavioral functions of ASK1, including regulation of locomotor activity, novelty preference, and motor coordination with dopaminergic transmission.