The microbiota regulate neuronal function and fear extinction learning.

Multicellular organisms have co-evolved with complex consortia of viruses, bacteria, fungi and parasites, collectively referred to as the microbiota1. In mammals, changes in the composition of the microbiota can influence many physiologic processes (including development, metabolism and immune cell function) and are associated with susceptibility to multiple diseases2. Alterations in the microbiota can also modulate host behaviours-such as social activity, stress, and anxiety-related responses-that are linked to diverse neuropsychiatric disorders3. However, the mechanisms by which the microbiota influence neuronal activity and host behaviour remain poorly defined. Here we show that manipulation of the microbiota in antibiotic-treated or germ-free adult mice results in significant deficits in fear extinction learning. Single-nucleus RNA sequencing of the medial prefrontal cortex of the brain revealed significant alterations in gene expression in excitatory neurons, glia and other cell types. Transcranial two-photon imaging showed that deficits in extinction learning after manipulation of the microbiota in adult mice were associated with defective learning-related remodelling of postsynaptic dendritic spines and reduced activity in cue-encoding neurons in the medial prefrontal cortex. In addition, selective re-establishment of the microbiota revealed a limited neonatal developmental window in which microbiota-derived signals can restore normal extinction learning in adulthood. Finally, unbiased metabolomic analysis identified four metabolites that were significantly downregulated in germ-free mice and have been reported to be related to neuropsychiatric disorders in humans and mouse models, suggesting that microbiota-derived compounds may directly affect brain function and behaviour. Together, these data indicate that fear extinction learning requires microbiota-derived signals both during early postnatal neurodevelopment and in adult mice, with implications for our understanding of how diet, infection, and lifestyle influence brain health and subsequent susceptibility to neuropsychiatric disorders.

Role of BDNF in the development of an OFC-amygdala circuit regulating sociability in mouse and human.

Social deficits are common in many psychiatric disorders. However, due to inadequate tools for manipulating circuit activity in humans and unspecific paradigms for modeling social behaviors in rodents, our understanding of the molecular and circuit mechanisms mediating social behaviors remains relatively limited. Using human functional neuroimaging and rodent fiber photometry, we identified a mOFC-BLA projection that modulates social approach behavior and influences susceptibility to social anxiety. In humans and knock-in mice with a loss of function BDNF SNP (Val66Met), the functionality of this circuit was altered, resulting in social behavioral changes in human and mice. We further showed that the development of this circuit is disrupted in BDNF Met carriers due to insufficient BDNF bioavailability, specifically during a peri-adolescent timeframe. These findings define one mechanism by which social anxiety may stem from altered maturation of orbitofronto-amygdala projections and identify a developmental window in which BDNF-based interventions may have therapeutic potential.

Ventral hippocampus interacts with prelimbic cortex during inhibition of threat response via learned safety in both mice and humans.

Heightened fear and inefficient safety learning are key features of fear and anxiety disorders. Evidence-based interventions for anxiety disorders, such as cognitive behavioral therapy, primarily rely on mechanisms of fear extinction. However, up to 50% of clinically anxious individuals do not respond to current evidence-based treatment, suggesting a critical need for new interventions based on alternative neurobiological pathways. Using parallel human and rodent conditioned inhibition paradigms alongside brain imaging methodologies, we investigated neural activity patterns in the ventral hippocampus in response to stimuli predictive of threat or safety and compound cues to test inhibition via safety in the presence of threat. Distinct hippocampal responses to threat, safety, and compound cues suggest that the ventral hippocampus is involved in conditioned inhibition in both mice and humans. Moreover, unique response patterns within target-differentiated subpopulations of ventral hippocampal neurons identify a circuit by which fear may be inhibited via safety. Specifically, ventral hippocampal neurons projecting to the prelimbic cortex, but not to the infralimbic cortex or basolateral amygdala, were more active to safety and compound cues than threat cues, and activity correlated with freezing behavior in rodents. A corresponding distinction was observed in humans: hippocampal-dorsal anterior cingulate cortex functional connectivity-but not hippocampal-anterior ventromedial prefrontal cortex or hippocampal-basolateral amygdala connectivity-differentiated between threat, safety, and compound conditions. These findings highlight the potential to enhance treatment for anxiety disorders by targeting an alternative neural mechanism through safety signal learning.

Elevating levels of the endocannabinoid 2-arachidonoylglycerol blunts opioid reward but not analgesia.

Converging findings have established that the endocannabinoid (eCB) system serves as a possible target for the development of new treatments for pain as a complement to opioid-based treatments. Here we show in male and female mice that enhancing levels of the eCB, 2-arachidonoylglycerol (2-AG), through pharmacological inhibition of its catabolic enzyme, monoacylglycerol lipase (MAGL), either systemically or in the ventral tegmental area (VTA) with JZL184, leads to a substantial attenuation of the rewarding effects of opioids in male and female mice using conditioned place preference and self-administration paradigms, without altering their analgesic properties. These effects are driven by CB1 receptors (CB1Rs) within the VTA as VTA CB1R conditional knockout, counteracts JZL184's effects. Conversely, pharmacologically enhancing the levels of the other eCB, anandamide (AEA), by inhibition of fatty acid amide hydrolase (FAAH) has no effect on opioid reward or analgesia. Using fiber photometry with fluorescent sensors for calcium and dopamine (DA), we find that enhancing 2-AG levels diminishes opioid reward-related nucleus accumbens (NAc) activity and DA neurotransmission. Together these findings reveal that 2-AG counteracts the rewarding properties of opioids and provides a potential adjunctive therapeutic strategy for opioid-related analgesic treatments.

The role of BDNF in mediating the prophylactic effects of (R,S)-ketamine on fear generalization and extinction.

Fear generalization is a conserved survival mechanism that can become maladaptive in the face of traumatic situations, a feature central to certain anxiety disorders including posttraumatic stress disorder (PTSD). However, the neural circuitry and molecular mechanisms underlying fear generalization remain unclear. Recent studies have shown that prophylactic treatment with (R,S)-ketamine confers protective effects in stress-induced depressive behaviors and enhances contextual fear discrimination, but the extent to which these effects extend to fear generalization after auditory fear conditioning remains unclear. Here, we build on this work by using a behavioral model of fear generalization in mice involving foot shocks with differential intensity levels during auditory fear conditioning. We find that prophylactic (R,S)-ketamine treatment exerts protective effects that results in enhanced fear discrimination in wild type mice. As the growth factor, brain-derived neurotrophic factor (BDNF), has been shown to mediate the rapid antidepressant actions of (R,S)-ketamine, we used a loss-of-function BDNF mouse line (BDNF Val66Met) to determine whether BDNF is involved in (R,S)-ketamine's prophylactic effects on fear generalization. We found that BDNF Val66Met mice were resistant to the protective effects of prophylactic (R,S)-ketamine administration on fear generalization and extinction. We then used fiber photometry to parse out underlying neural activity and found that in the ventral hippocampus there were significant fear generalization-dependent patterns of activity for wild type and BDNF Val66Met mice that were altered by prophylactic (R,S)-ketamine treatment. Overall, these findings indicate a role for the ventral hippocampus and BDNF signaling in modulating the mitigating effects of prophylactic (R,S)-ketamine treatment on generalized fear.

Endocannabinoid genetic variation enhances vulnerability to THC reward in adolescent female mice.

Adolescence represents a developmental period with the highest risk for initiating cannabis use. Little is known about whether genetic variation in the endocannabinoid system alters mesolimbic reward circuitry to produce vulnerability to the rewarding properties of the exogenous cannabinoid Δ9-tetrahydrocannabinol (THC). Using a genetic knock-in mouse model (FAAHC/A) that biologically recapitulates the human polymorphism associated with problematic drug use, we find that in adolescent female mice, but not male mice, this FAAH polymorphism enhances the mesolimbic dopamine circuitry projecting from the ventral tegmental area (VTA) to the nucleus accumbens (NAc) and alters cannabinoid receptor 1 (CB1R) levels at inhibitory and excitatory terminals in the VTA. These developmental changes collectively increase vulnerability of adolescent female FAAHC/A mice to THC preference that persists into adulthood. Together, these findings suggest that this endocannabinoid genetic variant is a contributing factor for increased susceptibility to cannabis dependence in adolescent females.

Immune function in PTSD.

Disturbed regulation of both the hypothalamic-pituitary-adrenal (HPA) axis and sympathoadrenomedullary system in posttraumatic stress disorder (PTSD) suggests that immune function, which is modulated by these systems, may also be dysregulated. Two dermatologic, in vivo measures of immune function, delayed-type hypersensitivity (DTH) and skin barrier function recovery, were examined in female subjects with PTSD and compared to measures in healthy female comparison subjects. In addition, at the time of DTH test placement, circulating numbers of lymphocyte subtypes were assessed. In separate studies, the effects of acute psychological stress on DTH and skin barrier function recovery were examined in healthy volunteer subjects. Both DTH and barrier function recovery were enhanced in women with PTSD. These findings contrast with the effects of acute stress in healthy control subjects, which was associated with suppression of DTH responses and skin barrier function recovery. There was no difference between subjects with PTSD and healthy control subjects in proportions of circulating lymphocyte subsets or in expression of the lymphocyte markers CD62, CD25, and CD45RO/CD45RA. These results suggest that cell-mediated immune function is enhanced in individuals with PTSD, a condition that imposes chronic physiologic and mental stress on sufferers. These findings contrast with suppression of DTH and skin barrier function recovery in healthy volunteers in response to acute psychological stress.

FAAH genetic variation enhances fronto-amygdala function in mouse and human.

Cross-species studies enable rapid translational discovery and produce the broadest impact when both mechanism and phenotype are consistent across organisms. We developed a knock-in mouse that biologically recapitulates a common human mutation in the gene for fatty acid amide hydrolase (FAAH) (C385A; rs324420), the primary catabolic enzyme for the endocannabinoid anandamide. This common polymorphism impacts the expression and activity of FAAH, thereby increasing anandamide levels. Here, we show that the genetic knock-in mouse and human variant allele carriers exhibit parallel alterations in biochemisty, neurocircuitry and behaviour. Specifically, there is reduced FAAH expression associated with the variant allele that selectively enhances fronto-amygdala connectivity and fear extinction learning, and decreases anxiety-like behaviours. These results suggest a gain of function in fear regulation and may indicate for whom and for what anxiety symptoms FAAH inhibitors or exposure-based therapies will be most efficacious, bridging an important translational gap between the mouse and human.

Changes in cerebrospinal fluid neurochemistry during pregnancy.

BACKGROUND: Little is known about changes in brain function that may occur during pregnancy. Studies in rodents and sheep suggest that several brain neurotransmitter and neurohormonal systems known to modulate anxiety may be altered during pregnancy. METHODS: Cerebrospinal fluid (CSF) and plasma samples were obtained from 21 women (during weeks 38-39 of pregnancy) who were undergoing elective cesarean section and from 22 healthy nonpregnant women. RESULTS: The CSF levels of g-aminobutyric acid (GABA) and 3-methoxy-4-hydroxyphenylglycolwere reduced in pregnant women. There were no changes in CSF glutamate, 5-hydroxyindoleactic acid, and homovanillic acid. There was a large increase in CSF prolactin in pregnant women and also a trend toward an elevation in CSF oxytocin. Levels of prolactin, but not oxytocin, in CSF and plasma were correlated in pregnant women. CONCLUSIONS: These results suggest that pregnancy alters regulation of brain GABA, norepinephrine, and prolactin, which may play a role in changes in vulnerability to anxiety and depression during pregnancy and postpartum. Prolactin circulating in the bloodstream seems to be the major source of CSF prolactin during pregnancy.