Enhanced fear memory after social defeat in mice is dependent on interleukin-1 receptor signaling in glutamatergic neurons.

Chronic stress is associated with increased anxiety, cognitive deficits, and post-traumatic stress disorder. Repeated social defeat (RSD) in mice causes long-term stress-sensitization associated with increased microglia activation, monocyte accumulation, and enhanced interleukin (IL)-1 signaling in endothelia and neurons. With stress-sensitization, mice have amplified neuronal, immune, and behavioral responses to acute stress 24 days later. This is clinically relevant as it shares key aspects with post-traumatic stress disorder. The mechanisms underlying stress-sensitization are unclear, but enhanced fear memory may be critical. The purpose of this study was to determine the influence of microglia and IL-1R1 signaling in neurons in the development of sensitization and increased fear memory after RSD. Here, RSD accelerated fear acquisition, delayed fear extinction, and increased cued-based freezing at 0.5 day. The enhancement in contextual fear memory after RSD persisted 24 days later. Next, microglia were depleted with a CSF1R antagonist prior to RSD and several parameters were assessed. Microglia depletion blocked monocyte recruitment to the brain. Nonetheless, neuronal reactivity (pCREB) and IL-1ß RNA expression in the hippocampus and enhanced fear memory after RSD were microglial-independent. Because IL-1ß RNA was prominent in the hippocampus after RSD even with microglia depletion, IL-1R1 mediated signaling in glutamatergic neurons was assessed using neuronal Vglut2+/IL-1R1-/- mice. RSD-induced neuronal reactivity (pCREB) in the hippocampus and enhancement in fear memory were dependent on neuronal IL-1R1 signaling. Furthermore, single-nuclei RNA sequencing (snRNAseq) showed that RSD influenced transcription in specific hippocampal neurons (DG neurons, CA2/3, CA1 neurons) associated with glutamate signaling, inflammation and synaptic plasticity, which were neuronal IL-1R1-dependent. Furthermore, snRNAseq data provided evidence that RSD increased CREB, BDNF, and calcium signaling in DG neurons in an IL-1R1-dependent manner. Collectively, increased IL-1R1-mediated signaling (monocytes/microglia independent) in glutamatergic neurons after RSD enhanced neuronal reactivity and fear memory.

Antagonism of the brain P2X7 ion channel attenuates repeated social defeat induced microglia reactivity, monocyte recruitment and anxiety-like behavior in male mice.

Chronic stress is linked to increased anxiety. Repeated social defeat (RSD) in mice causes anxiety that is dependent on activated neurons, reactive microglia, and accumulation of monocytes in the brain. This response requires interactions between the immune system and central nervous system (CNS). Neuronal activation within threat appraisal regions is a key response to RSD, however, it is unclear how microglia become activated. One potential explanation is that microglia express a purinergic non-selective ligand gated adenosine-triphosphate (ATP) receptor 7 (P2X7). Activation of P2X7 promotes the release of chemokines and cytokines, and recruitment of monocytes to the brain. Thus, the purpose of this study was to determine if a novel P2X7 antagonist blocked neuronal and microglia interactions and the corresponding anxiety following RSD. Male mice were administered (i.p.) a P2X7 antagonist, JNJ-54471300, prior to each cycle of RSD. Fourteen hours after RSD, behavioral deficits including social avoidance and anxiety-like were determined. Moreover, several immune parameters were assessed. RSD caused neuronal activation in stress-responsive regions, monocyte production and release, splenomegaly, and social avoidance. These parameters were unaffected by P2X7 antagonism. RSD-associated proportional area of Iba-1+ microglia, monocyte accumulation in the brain, IL-1ß mRNA expression in enriched myeloid cells, plasma IL-6, and anxiety-like behavior were ameliorated by P2X7 antagonism. Gene expression analysis in the hippocampus and amygdala showed regional specific responses to RSD and some were reversed with P2X7 antagonism. Overall, blocking P2X7 activation attenuated RSD-induced microglia reactivity with corresponding reduction in neuroinflammation, monocyte accumulation, and anxiety-like behavior in male mice.

Unique brain endothelial profiles activated by social stress promote cell adhesion, prostaglandin E2 signaling, hypothalamic-pituitary-adrenal axis modulation, and anxiety.

Chronic stress may precipitate psychiatric disorders including anxiety. We reported that Repeated Social Defeat (RSD) in mice increased accumulation of inflammatory monocytes within the brain vasculature, which corresponded with increased interleukin (IL)-1 Receptor 1-mediated activation of endothelia, and augmented anxiety-like behavior. One unknown, however, is the role of immune-activated endothelia in regulating the physiological and behavioral responses to social stress. Thus, we sought to determine the RNA profile of activated endothelia and delineate the pathways by which these endothelia communicate within the brain to influence key responses to social stress. First, endothelial-specific RiboTag mice were exposed to RSD and brain endothelial mRNA profiles from the whole brain and prefrontal cortex were determined using RNAseq. RSD increased expression of cell adhesion molecules (Icam1), inflammatory genes (Lrg1, Lcn2, Ackr1, Il1r1), and cyclooxygenase-2 (Ptgs2/COX-2). In studies with IL-1R1KO mice, there was clear dependence on IL-1R1 on endothelia-associated transcripts including Lrg1, Icam1, Lcn2. Moreover, prostaglandin (PG)E2 was increased in the brain after RSD and Ptgs2 was localized to endothelia, especially within the hypothalamus. Next, a selective COX-2 inhibitor, Celecoxib (CCB), was used with social stress. RSD increased PGE2 in the brain and this was abrogated by CCB. Moreover, CCB reduced RSD-induced Hypothalamic-Pituitary-Adrenal (HPA) axis activation with attenuation of hypothalamic paraventricular neuron activation, hypothalamic Crh expression, and corticosterone in circulation. Production, release, and accumulation of inflammatory monocytes after RSD was COX-2 independent. Nonetheless, CCB blocked anxiety-like behavior in response to RSD. Collectively, social stress stimulated specific endothelia RNA profiles associated with increased cell adhesion, IL-1 and prostaglandin signaling, HPA axis activation, and anxiety.

Effect of Aggressive Experience in Female Syrian Hamsters on Glutamate Receptor Expression in the Nucleus Accumbens.

Our social relationships determine our health and well-being. In rodent models, there is now strong support for the rewarding properties of aggressive or assertive behaviors to be critical for the expression and development of adaptive social relationships, buffering from stress and protecting from the development of psychiatric disorders such as depression. However, due to the false belief that aggression is not a part of the normal repertoire of social behaviors displayed by females, almost nothing is known about the neural mechanisms mediating the rewarding properties of aggression in half the population. In the following study, using Syrian hamsters as a well-validated and translational model of female aggression, we investigated the effects of aggressive experience on the expression of markers of postsynaptic structure (PSD-95, Caskin I) and excitatory synaptic transmission (GluA1, GluA2, GluA4, NR2A, NR2B, mGluR1a, and mGluR5) in the nucleus accumbens (NAc), caudate putamen and prefrontal cortex. Aggressive experience resulted in an increase in PSD-95, GluA1 and the dimer form of mGluR5 specifically in the NAc 24 h following aggressive experience. There was also an increase in the dimer form of mGluR1a 1 week following aggressive experience. Aggressive experience also resulted in an increase in the strength of the association between these postsynaptic proteins and glutamate receptors, supporting a common mechanism of action. In addition, 1 week following aggressive experience there was a positive correlation between the monomer of mGluR5 and multiple AMPAR and NMDAR subunits. In conclusion, we provide evidence that aggressive experience in females results in an increase in the expression of postsynaptic density, AMPARs and group I metabotropic glutamate receptors, and an increase in the strength of the association between postsynaptic proteins and glutamate receptors. This suggests that aggressive experience may result in an increase in excitatory synaptic transmission in the NAc, potentially encoding the rewarding and behavioral effects of aggressive interactions.

Caveolin-1 regulates medium spiny neuron structural and functional plasticity.

RATIONALE: Caveolin-1 (CAV1) is a structural protein critical for spatial organization of neuronal signaling molecules. Whether CAV1 is required for long-lasting neuronal plasticity remains unknown. OBJECTIVE AND METHODS: We sought to examine the effects of CAV1 knockout (KO) on functional plasticity and hypothesized that CAV1 deficiency would impact drug-induced long-term plasticity in the nucleus accumbens (NAc). We first examined cell morphology of NAc medium spiny neurons in a striatal/cortical co-culture system before moving in vivo to study effects of CAV1 KO on cocaine-induced plasticity. Whole-cell patch-clamp recordings were performed to determine effects of chronic cocaine (15 mg/kg) on medium spiny neuron excitability. To test for deficits in behavioral plasticity, we examined the effect of CAV1 KO on locomotor sensitization. RESULTS: Disruption of CAV1 expression leads to baseline differences in medium spiny neuron (MSN) structural morphology, such that MSNs derived from CAV1 KO animals have increased dendritic arborization when cultured with cortical neurons. The effect was dependent on phospholipase C and cell-type intrinsic loss of CAV1. Slice recordings of nucleus accumbens shell MSNs revealed that CAV1 deficiency produces a loss of neuronal plasticity. Specifically, cocaine-induced firing rate depression was absent in CAV1 KO animals, whereas baseline electrophysiological properties were similar. This was reflected by a loss of cocaine-mediated behavioral sensitization in CAV1 KO animals, with unaffected baseline locomotor responsiveness. CONCLUSIONS: This study highlights a critical role for nucleus accumbens CAV1 in plasticity related to the administration of drugs of abuse.

Palmitoylation of caveolin-1 is regulated by the same DHHC acyltransferases that modify steroid hormone receptors.

Palmitoylation is a reversible post-translational addition of a 16-carbon lipid chain involved in trafficking and compartmentalizing target proteins. It is important for many cellular functions, including signaling via membrane-localized estrogen receptors (ERs). Within the nervous system, palmitoylation of ERα is necessary for membrane surface localization and mediation of downstream signaling through the activation of metabotropic glutamate receptors (mGluRs). Substitution of the single palmitoylation site on ERα prevents its physical association with the integral membrane protein caveolin-1 (CAV1), required for the formation of the ER/mGluR signaling complex. Interestingly, siRNA knockdown of either of two palmitoyl acyltransferases, zinc finger DHHC type-containing 7 (DHHC7) or DHHC21, also eliminates this signaling mechanism. Because ERα has only one palmitoylation site, we hypothesized that one of these DHHCs palmitoylates CAV1. We investigated this possibility by using an acyl-biotin exchange assay in HEK293 cells in conjunction with DHHC overexpression and found that DHHC7 increases CAV1 palmitoylation. Substitution of the palmitoylation sites on CAV1 eliminated this effect but did not disrupt the ability of the DHHC enzyme to associate with CAV1. In contrast, siRNA-mediated knockdown of DHHC7 alone was not sufficient to decrease CAV1 palmitoylation but rather required simultaneous knockdown of DHHC21. These findings provide additional information about the overall influence of palmitoylation on the membrane-initiated estrogen signaling pathway and highlight the importance of considering the influence of palmitoylation on other CAV1-dependent processes.