PREFRONTAL CORRELATES OF FEAR GENERALIZATION DURING ENDOCANNABINOID DEPLETION.

Maladaptive fear generalization is one of the hallmarks of trauma-related disorders. The endocannabinoid 2-arachidonoylglycerol (2-AG) is crucial for modulating anxiety, fear, and stress adaptation but its role in balancing fear discrimination versus generalization is not known. To address this, we used a combination of plasma endocannabinoid measurement and neuroimaging from a childhood maltreatment exposed and non-exposed mixed population combined with human and rodent fear conditioning models. Here we show that 2-AG levels are inversely associated with fear generalization at the behavioral level in both mice and humans. In mice, 2-AG depletion increases the proportion of neurons, and the similarity between neuronal representations, of threat-predictive and neutral stimuli within prelimbic prefrontal cortex ensembles. In humans, increased dorsolateral prefrontal cortical-amygdala resting state connectivity is inversely correlated with fear generalization. These data provide convergent cross-species evidence that 2-AG is a key regulator of fear generalization and suggest 2-AG deficiency could represent a trauma-related disorder susceptibility endophenotype.

Administration of the glutamate-modulating drug, riluzole, after stress prevents its delayed effects on the amygdala in male rats.

Extracellular glutamate levels are elevated across brain regions immediately after stress. Despite sharing common features in their genesis, the patterns of stress-induced plasticity that eventually take shape are strikingly different between these brain areas. While stress causes structural and functional deficits in the hippocampus, it has the opposite effect on the amygdala. Riluzole, an FDA-approved drug known to modulate glutamate release and facilitate glutamate clearance, prevents stress-induced deficits in the hippocampus. But whether the same drug is also effective in countering the opposite effects of stress in the amygdala remains unexplored. We addressed this question by using a rat model wherein even a single 2-h acute immobilization stress causes a delayed expression of anxiety-like behavior, 10 days later, alongside stronger excitatory synaptic connectivity in the basolateral amygdala (BLA). This temporal profile-several days separating the acute stressor and its delayed impact-allowed us to test if these effects can be prevented by administering riluzole in drinking water after acute stress. Poststress riluzole not only prevented the delayed increase in anxiety-like behavior on the elevated plus maze but also blocked the increase in spine density on BLA neurons 10 days later. Further, stress-induced increase in the frequency of miniature excitatory postsynaptic currents recorded in BLA slices, 10 days later, was also blocked by the same poststress riluzole administration. Together, these findings underscore the importance of therapeutic strategies, aimed at glutamate uptake and modulation, in correcting the delayed behavioral, physiological, and morphological effects of stress on the amygdala.

Endocannabinoids at the synapse and beyond: implications for neuropsychiatric disease pathophysiology and treatment.

Endocannabinoids (eCBs) are lipid neuromodulators that suppress neurotransmitter release, reduce postsynaptic excitability, activate astrocyte signaling, and control cellular respiration. Here, we describe canonical and emerging eCB signaling modes and aim to link adaptations in these signaling systems to pathological states. Adaptations in eCB signaling systems have been identified in a variety of biobehavioral and physiological process relevant to neuropsychiatric disease states including stress-related disorders, epilepsy, developmental disorders, obesity, and substance use disorders. These insights have enhanced our understanding of the pathophysiology of neurological and psychiatric disorders and are contributing to the ongoing development of eCB-targeting therapeutics. We suggest future studies aimed at illuminating how adaptations in canonical as well as emerging cellular and synaptic modes of eCB signaling contribute to disease pathophysiology or resilience could further advance these novel treatment approaches.

Neuronal activity drives pathway-specific depolarization of peripheral astrocyte processes.

Astrocytes are glial cells that interact with neuronal synapses via their distal processes, where they remove glutamate and potassium (K+) from the extracellular space following neuronal activity. Astrocyte clearance of both glutamate and K+ is voltage dependent, but astrocyte membrane potential (Vm) is thought to be largely invariant. As a result, these voltage dependencies have not been considered relevant to astrocyte function. Using genetically encoded voltage indicators to enable the measurement of Vm at peripheral astrocyte processes (PAPs) in mice, we report large, rapid, focal and pathway-specific depolarizations in PAPs during neuronal activity. These activity-dependent astrocyte depolarizations are driven by action potential-mediated presynaptic K+ efflux and electrogenic glutamate transporters. We find that PAP depolarization inhibits astrocyte glutamate clearance during neuronal activity, enhancing neuronal activation by glutamate. This represents a novel class of subcellular astrocyte membrane dynamics and a new form of astrocyte-neuron interaction.

Riluzole prevents stress-induced spine plasticity in the hippocampus but mimics it in the amygdala.

Stress elicits divergent patterns of structural plasticity in the amygdala and hippocampus. Despite these contrasting effects, at least one of the immediate consequences of stress - elevated levels of extracellular glutamate - is similar in both brain areas. This raises the possibility that the contrasting effects of stress on neuronal plasticity is shaped by differences in astrocytic glutamate clearance in these two brain areas. Although astrocytes play a key role in glutamate reuptake, past analyses of, and interventions against, stress-induced plasticity have focused largely on neurons. Hence, we tested the impact of riluzole, which potentiates glutamate clearance by astrocytic glutamate transporters, on principal neurons and astrocytes in the basal amygdala (BA) and hippocampal area CA1. Chronic immobilization stress reduced spine-density on CA1 pyramidal neurons of male rats. Riluzole, administered in the drinking water during chronic stress, prevented this decrease; but, the drug by itself had no effect. In contrast, the same chronic stress enhanced spine-density on BA principal neurons, and this effect, unlike area CA1, was not reversed by riluzole. Strikingly, riluzole treatment alone also caused spinogenesis in the BA. Thus, the same riluzole treatment that prevented the effect of stress on spines in the hippocampus, mimicked its effect in the amygdala. Further, chronic stress and riluzole alone decreased the neuropil volume occupied by astrocytes in both the BA and CA1 area. Riluzole treatment in stressed animals, however, did not reverse or further add to this reduction in either region. Thus, while the effects on astrocytes were similar, neuronal changes were distinct between the two areas following stress, riluzole and the two together. Therefore, similar to the impact of repeated stress, pharmacological potentiation of glutamate clearance, with or without stress, also leads to differential effects on dendritic spines in principal neurons of the amygdala and hippocampus. This highlights differences in the astrocytic glutamate reuptake machinery that are likely to have important functional consequences for stress-induced dysfunction, and its reversal, in two brain areas implicated in stress-related psychiatric disorders.

Stress Elicits Contrasting Effects on the Structure and Number of Astrocytes in the Amygdala versus Hippocampus.

Stress causes divergent patterns of structural and physiological plasticity in the hippocampus versus amygdala. However, a majority of earlier studies focused primarily on neurons. Despite growing evidence for the importance of glia in health and disease, relatively little is known about how stress affects astrocytes. Further, previous work focused on hippocampal astrocytes. Hence, we examined the impact of chronic immobilization stress (2 h/d, 10 d), on the number and structure of astrocytes in the rat hippocampus and amygdala. We observed a reduction in the number of glial fibrillary acidic protein (GFAP)-positive astrocytes in the basal amygdala (BA), 1 d after the end of 10 d of chronic stress. Detailed morphometric analysis of individual dye-filled astrocytes also revealed a decrease in the neuropil volume occupied by these astrocytes in the BA, alongside a reduction in the volume fraction of fine astrocytic protrusions rather than larger dendrite-like processes. By contrast, the same chronic stress had no effect on the number or morphology of astrocytes in hippocampal area CA3. We also confirmed previous reports that chronic stress triggers dendritic hypertrophy in dye-filled BA principal neurons that were located adjacent to astrocytes that had undergone atrophy. Thus, building on earlier evidence for contrasting patterns of stress-induced plasticity in neurons across brain areas, our findings offer new evidence that the same stress can also elicit divergent morphological effects in astrocytes in the hippocampus versus the amygdala.