Probes for INS

Impairments in the function of the hypothalamic-pituitary-adrenal (HPA) axis and enhanced glucocorticoid receptor (GR) activity in the central amygdala (CeA) are critical mechanisms in the pathogenesis of alcohol use disorder (AUD). The GR antagonist mifepristone attenuates craving in AUD patients, alcohol consumption in AUD models, and decreases CeA γ-aminobutyric acid (GABA) transmission in alcohol-dependent rats. Previous studies suggest elevated GR activity in the CeA of male alcohol-preferring Marchigian-Sardinian (msP) rats, but its contribution to heightened CeA GABA transmission driving their characteristic post-dependent phenotype is largely unknown. We determined Nr3c1 (the gene encoding GR) gene transcription in the CeA in male and female msP and Wistar rats using in situ hybridization and studied acute effects of mifepristone (10 μM) and its interaction with ethanol (44 mM) on pharmacologically isolated spontaneous inhibitory postsynaptic currents (sIPSCs) and electrically evoked inhibitory postsynaptic potentials (eIPSPs) in the CeA using ex vivo slice electrophysiology. Female rats of both genotypes expressed more CeA GRs than males, suggesting a sexually dimorphic GR regulation of CeA activity. Mifepristone reduced sIPSC frequencies (GABA release) and eIPSP amplitudes in msP rats of both sexes, but not in their Wistar counterparts; however, it did not prevent acute ethanol-induced increase in CeA GABA transmission in male rats. In msP rats, GR regulates CeA GABAergic signaling under basal conditions, indicative of intrinsically active GR. Thus, enhanced GR function in the CeA represents a key mechanism contributing to maladaptive behaviors associated with AUD.

Alcohol use disorder (AUD) is a chronically relapsing disease characterized by loss of control in seeking and consuming alcohol (ethanol) driven by recruitment of brain stress systems. However, AUD differs among the sexes: men are more likely to develop AUD, but women progress from casual to binge drinking and heavy alcohol use more quickly. The central amygdala (CeA) is a hub of stress and anxiety, with corticotropin releasing factor (CRF)-CRF1 receptor and GABAergic signaling dysregulation occurring in alcohol dependent male rodents. However, we recently showed that GABAergic synapses in female rats are less sensitive to the acute effects of ethanol. Here, we used patch clamp electrophysiology to examine the effects of alcohol dependence on the CRF-modulation of rat CeA GABAergic transmission of both sexes. We found that GABAergic synapses of naïve female rats were unresponsive to CRF application compared males, although alcohol dependence induced a similar CRF responsivity in both sexes. In situ hybridization revealed that females had less CeA neurons containing mRNA for the CRF1 receptor (Crhr1) than males, but in dependence, the percentage of Crhr1-expressing neurons in females increased, unlike males. Overall, our data provide evidence for sexually dimorphic CeA CRF system effects on GABAergic synapses in dependence.

The endogenous opioid system plays a crucial role in stress-induced analgesia. Mu-opioid receptors (MORs), one of the major opioid receptors, are expressed widely in subpopulations of cells throughout the CNS. However, the potential roles of MORs expressed in glutamatergic (MORGlut) and γ-aminobutyric acidergic (MORGABA) neurons in stress-induced analgesia remain unclear. By examining tail-flick latencies to noxious radiant heat of male mice, here we investigated the contributions of MORGABA and MORGlut to behavioral analgesia and activities of neurons projecting from periaqueductal gray (PAG) to rostral ventromedial medulla (RVM) induced by a range of time courses of forced swim exposure. The moderate but not transitory or prolonged swim exposure induced a MOR-dependent analgesia, although all of these three stresses enhanced β-endorphin release. Selective deletion of MORGABA but not MORGlut clearly attenuated analgesia and blocked the enhancement of activities of PAG-RVM neurons induced by moderate swim exposure. Under transitory swim exposure, in contrast, selective deletion of MORGlut elicited an analgesia behavior via strengthening the activities of PAG-RVM neurons. These results indicate that MOR-dependent endogenous opioid signaling participates in nociceptive modulation in a wide range, not limited to moderate, of stress intensities. Endogenous activation of MORGABA exerts analgesia, whereas MORGlut produces antianalgesia. More importantly, with an increase of stress intensities, the efficiencies of MORs on nociception shifts from balance between MORGlut and MORGABA to biasing toward MORGABA-mediated processes. Our results point to the cellular dynamic characteristics of MORs expressed in excitatory and inhibitory neurons in pain modulation under various stress intensities.

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.