The neutral CB1 antagonist AM6527 reduces ethanol seeking, binge-like consumption, reinforcing, and withdrawal effects in male and female mice.

RATIONALE: Alcohol use disorder (AUD) is a debilitating physiological and psychiatric disorder which affects individuals globally. The current pharmacological interventions to treat AUD are limited, and hence there is an urgent need for a novel pharmacological therapy which can be effective and safe across the population. OBJECTIVE: We aimed to investigate a novel neutral cannabinoid receptor-1 (CB1R) antagonist, AM6527, in several preclinical models of ethanol consumption using male and female C57BL6/J mice. METHODS: Independent groups of male and female mice were subjected to repeated cycles of drinking in the dark (DID), or intermittent access to alcohol (IAA) procedures. Twenty minutes prior to ethanol access in each procedure, animals were treated with intraperitoneal injections of either 1, 3, and 10 mg/kg of AM6527 or its respective vehicle. Acamprosate (100, 200, 300, and 400 mg/kg) or its respective vehicle was used as a positive control. Separate groups of male mice were subjected to a chain schedule of ethanol reinforcement to gain access to ethanol wherein completion of a fixed interval (FI; 5 min) schedule (link 1: "Seeking") was reinforced with continuous access to ethanol (fixed ratio; FR1) for up to 1.8 g/kg (link 2: "consumption"). All the animals were treated with 1, 3, and 10 mg/kg of AM6527 or its respective vehicle 20 mins prior to the start of the FI chain of the procedure. Separately, AM6527 was also evaluated in male and female mice undergoing acute ethanol withdrawal following 8 weeks of intermittent or continuous access to 20% ethanol drinking. RESULTS: In both DID and IAA procedures, AM6527 reduced ethanol consumption in a dose-related manner in both male and female mice. AM6527 produced no tolerance in the DID procedure; mice treated with 3 mg/kg of AM6527 for 3 weeks continuously drank significantly smaller amounts of ethanol as compared to vehicle-treated mice over a period of three DID cycles. Moreover, in the IAA procedure, AM6527 caused an increase in water intake over the 24-h period. Acamprosate transiently reduced ethanol intake in male mice in both the DID and the IAA procedures but failed to produce any significant effect in female mice. AM6527 also produced a decrease in the FI responding ("ethanol seeking") in animals trained to self-administer ethanol. Lastly, AM6527 mitigated neurological withdrawal signs, i.e., handling induced convulsions (HIC) in mice undergoing acute ethanol withdrawal. CONCLUSIONS: Current findings support previous studies with CB1R neutral antagonist in reducing voluntary ethanol intake and seeking behavior. Based on results shown in this work, AM6527 can be developed as a first in class CB1R neutral antagonist to treat AUD in both males and females.

PACAP-mediated gating of anxiety-controlling circuits.

Combining the use of ex vivo and in vivo optogenetics, viral tracing, electrophysiology and behavioral testing, we show that the neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) gates anxiety-controlling circuits by differentially affecting synaptic efficacy at projections from the basolateral amygdala (BLA) to two different subdivisions of the dorsal subdivision of the bed nucleus of the stria terminalis (BNST), modifying the signal flow in BLA-ovBNST-adBNST circuits in such a way that adBNST is inhibited. Inhibition of adBNST is translated into the reduced firing probability of adBNST neurons during afferent activation, explaining the anxiety-triggering actions of PACAP in BNST, as inhibition of adBNST is anxiogenic. Our results reveal how innate, fear-related behavioral mechanisms may be controlled by neuropeptides, PACAP specifically, at the level of underlying neural circuits by inducing long-lasting plastic changes in functional interactions between their different structural components.

Genome-wide transcriptomics of the amygdala reveals similar oligodendrocyte-related responses to acute and chronic alcohol drinking in female mice.

Repeated excessive alcohol consumption is a risk factor for alcohol use disorder (AUD). Although AUD has been more common in men than women, women develop more severe behavioral and physical impairments. However, relatively few new therapeutics targeting development of AUD, particularly in women, have been validated. To gain a better understanding of molecular mechanisms underlying alcohol intake, we conducted a genome-wide RNA-sequencing analysis in female mice exposed to different modes (acute vs chronic) of ethanol drinking. We focused on transcriptional profiles in the amygdala including the central and basolateral subnuclei, brain areas previously implicated in alcohol drinking and seeking. Surprisingly, we found that both drinking modes triggered similar changes in gene expression and canonical pathways, including upregulation of ribosome-related/translational pathways and myelination pathways, and downregulation of chromatin binding and histone modification. In addition, analyses of hub genes and upstream regulatory pathways revealed that voluntary ethanol consumption affects epigenetic changes via histone deacetylation pathways, oligodendrocyte and myelin function, and the oligodendrocyte-related transcription factor, Sox17. Furthermore, a viral vector-assisted knockdown of Sox17 gene expression in the amygdala prevented a gradual increase in alcohol consumption during repeated accesses. Overall, these results suggest that the expression of oligodendrocyte-related genes in the amygdala is sensitive to voluntary alcohol drinking in female mice. These findings suggest potential molecular targets for future therapeutic approaches to prevent the development of AUD, due to repeated excessive alcohol consumption, particularly in women.

Fighting Females: Neural and Behavioral Consequences of Social Defeat Stress in Female Mice.

BACKGROUND: Despite the twofold higher prevalence of major depressive and posttraumatic stress disorders in women compared with men, most clinical and preclinical studies have focused on male subjects. We used an ethological murine model to study several cardinal symptoms of affective disorders in the female targets of female aggression. METHODS: Intact Swiss Webster (CFW) female resident mice were housed with castrated male mice and tested for aggression toward female intruders. For 10 days, aggressive CFW female residents defeated C57BL/6J (B6) female intruders during 5-minute encounters. Measures of corticosterone, c-Fos activation in hypothalamic and limbic structures, and species-typical behaviors were collected from defeated and control females. Ketamine (20 mg/kg) was tested for its potential to reverse stress-induced social deficits. RESULTS: Housed with a castrated male mouse, most intact resident CFW females readily attacked unfamiliar B6 female intruders, inflicting >40 bites in a 5-minute encounter. Compared with controls, defeated B6 females exhibited elevated plasma corticosterone and increased c-Fos activation in the medial amygdala, ventral lateral septum, ventromedial hypothalamus, and hypothalamic paraventricular nucleus. Chronically defeated females also showed vigilance-like behavior and deficits in social interactions, novel object investigation, and nesting. The duration of social interactions increased 24 hours after chronically defeated female mice received a systemic dose of ketamine. CONCLUSIONS: These findings demonstrate that CFW female mice living with male conspecifics can be used as aggressive residents in an ethological model of female social defeat stress. These novel behavioral methods will encourage further studies of sex-specific neural, physiological, and behavioral adaptations to chronic stress and the biological bases for interfemale aggression.

Brain circuit dysfunction in post-traumatic stress disorder: from mouse to man.

Post-traumatic stress disorder (PTSD) is a prevalent, debilitating and sometimes deadly consequence of exposure to severe psychological trauma. Although effective treatments exist for some individuals, they are limited. New approaches to intervention, treatment and prevention are therefore much needed. In the past few years, the field has rapidly developed a greater understanding of the dysfunctional brain circuits underlying PTSD, a shift in understanding that has been made possible by technological revolutions that have allowed the observation and perturbation of the macrocircuits and microcircuits thought to underlie PTSD-related symptoms. These advances have allowed us to gain a more translational knowledge of PTSD, have provided further insights into the mechanisms of risk and resilience and offer promising avenues for therapeutic discovery.

Common Biological Mechanisms of Alcohol Use Disorder and Post-Traumatic Stress Disorder.

Post-traumatic stress disorder (PTSD) and alcohol use disorder (AUD) are highly comorbid. Although recent clinical studies provide some understanding of biological and subsequent behavioral changes that define each of these disorders, the neurobiological basis of interactions between PTSD and AUD has not been well-understood. In this review, we summarize the relevant animal models that parallel the human conditions, as well as the clinical findings in these disorders, to delineate key gaps in our knowledge and to provide potential clinical strategies for alleviating the comorbid conditions.

Conjunctive input processing drives feature selectivity in hippocampal CA1 neurons.

Feature-selective firing allows networks to produce representations of the external and internal environments. Despite its importance, the mechanisms generating neuronal feature selectivity are incompletely understood. In many cortical microcircuits the integration of two functionally distinct inputs occurs nonlinearly through generation of active dendritic signals that drive burst firing and robust plasticity. To examine the role of this processing in feature selectivity, we recorded CA1 pyramidal neuron membrane potential and local field potential in mice running on a linear treadmill. We found that dendritic plateau potentials were produced by an interaction between properly timed input from entorhinal cortex and hippocampal CA3. These conjunctive signals positively modulated the firing of previously established place fields and rapidly induced new place field formation to produce feature selectivity in CA1 that is a function of both entorhinal cortex and CA3 input. Such selectivity could allow mixed network level representations that support context-dependent spatial maps.

Successful execution of working memory linked to synchronized high-frequency gamma oscillations.

Neuronal oscillations have been hypothesized to play an important role in cognition and its ensuing behavior, but evidence that links a specific neuronal oscillation to a discrete cognitive event is largely lacking. We measured neuronal activity in the entorhinal-hippocampal circuit while mice performed a reward-based spatial working memory task. During the memory retention period, a transient burst of high gamma synchronization preceded an animal's correct choice in both prospective planning and retrospective mistake correction, but not an animal's incorrect choice. Optogenetic inhibition of the circuit targeted to the choice point area resulted in a coordinated reduction in both high gamma synchrony and correct execution of a working-memory-guided behavior. These findings suggest that transient high gamma synchrony contributes to the successful execution of spatial working memory. Furthermore, our data are consistent with an association between transient high gamma synchrony and explicit awareness of the working memory content.

Island cells control temporal association memory.

Episodic memory requires associations of temporally discontiguous events. In the entorhinal-hippocampal network, temporal associations are driven by a direct pathway from layer III of the medial entorhinal cortex (MECIII) to the hippocampal CA1 region. However, the identification of neural circuits that regulate this association has remained unknown. In layer II of entorhinal cortex (ECII), we report clusters of excitatory neurons called island cells, which appear in a curvilinear matrix of bulblike structures, directly project to CA1, and activate interneurons that target the distal dendrites of CA1 pyramidal neurons. Island cells suppress the excitatory MECIII input through the feed-forward inhibition to control the strength and duration of temporal association in trace fear memory. Together, the two EC inputs compose a control circuit for temporal association memory.

Cell type-specific genetic and optogenetic tools reveal hippocampal CA2 circuits.

The formation and recall of episodic memory requires precise information processing by the entorhinal-hippocampal network. For several decades, the trisynaptic circuit entorhinal cortex layer II (ECII)→dentate gyrus→CA3→CA1 and the monosynaptic circuit ECIII→CA1 have been considered the primary substrates of the network responsible for learning and memory. Circuits linked to another hippocampal region, CA2, have only recently come to light. Using highly cell type-specific transgenic mouse lines, optogenetics and patch-clamp recordings, we found that dentate gyrus cells, long believed to not project to CA2, send functional monosynaptic inputs to CA2 pyramidal cells through abundant longitudinal projections. CA2 innervated CA1 to complete an alternate trisynaptic circuit, but, unlike CA3, projected preferentially to the deep, rather than to the superficial, sublayer of CA1. Furthermore, contrary to existing knowledge, ECIII did not project to CA2. Our results allow a deeper understanding of the biology of learning and memory.

Impaired hippocampal ripple-associated replay in a mouse model of schizophrenia.

The cognitive symptoms of schizophrenia presumably result from impairments of information processing in neural circuits. We recorded neural activity in the hippocampus of freely behaving mice that had a forebrain-specific knockout of the synaptic plasticity-mediating phosphatase calcineurin and were previously shown to exhibit behavioral and cognitive abnormalities, recapitulating the symptoms of schizophrenia. Calcineurin knockout (KO) mice exhibited a 2.5-fold increase in the abundance of sharp-wave ripple (SWR) events during awake resting periods and single units in KO were overactive during SWR events. Pairwise measures of unit activity, however, revealed that the sequential reactivation of place cells during SWR events was completely abolished in KO. Since this relationship during postexperience awake rest periods has been implicated in learning, working memory, and subsequent memory consolidation, our findings provide a mechanism underlying impaired information processing that may contribute to the cognitive impairments in schizophrenia.

Creating a false memory in the hippocampus.

Memories can be unreliable. We created a false memory in mice by optogenetically manipulating memory engram-bearing cells in the hippocampus. Dentate gyrus (DG) or CA1 neurons activated by exposure to a particular context were labeled with channelrhodopsin-2. These neurons were later optically reactivated during fear conditioning in a different context. The DG experimental group showed increased freezing in the original context, in which a foot shock was never delivered. The recall of this false memory was context-specific, activated similar downstream regions engaged during natural fear memory recall, and was also capable of driving an active fear response. Our data demonstrate that it is possible to generate an internally represented and behaviorally expressed fear memory via artificial means.

NMDA signaling in CA1 mediates selectively the spatial component of episodic memory.

Recent studies focusing on the memory for temporal order have reported that CA1 plays a critical role in the memory for the sequences of events, in addition to its well-described role in spatial navigation. In contrast, CA3 was found to principally contribute to the memory for the association of items with spatial or contextual information in tasks focusing on spatial memory. Other studies have shown that NMDA signaling in the hippocampus is critical to memory performance in studies that have investigated spatial and temporal order memory independently. However, the role of NMDA signaling separately in CA1 and CA3 in memory that combines both spatial and temporal processing demands (episodic memory) has not been examined. Here we investigated the effect of the deletion of the NR1 subunit of the NMDA receptor in CA1 or CA3 on the spatial and the temporal aspects of episodic memory, using a behavioral task that allows for these two aspects of memory to be evaluated distinctly within the same task. Under these conditions, NMDA signaling in CA1 specifically contributes to the spatial aspect of memory function and is not required to support the memory for temporal order of events.

Entorhinal cortex layer III input to the hippocampus is crucial for temporal association memory.

Associating temporally discontinuous elements is crucial for the formation of episodic and working memories that depend on the hippocampal-entorhinal network. However, the neural circuits subserving these associations have remained unknown. The layer III inputs of the entorhinal cortex to the hippocampus may contribute to this process. To test this hypothesis, we generated a transgenic mouse in which these inputs are specifically inhibited. The mutant mice displayed significant impairments in spatial working-memory tasks and in the encoding phase of trace fear-conditioning. These results indicate a critical role of the entorhinal cortex layer III inputs to the hippocampus in temporal association memory.