Stress and glucocorticoids stimulate the rapid mobilization of endocannabinoids in the basolateral amygdala (BLA). Cannabinoid receptors in the BLA contribute to anxiogenesis and fear-memory formation. We tested for rapid glucocorticoid-induced endocannabinoid regulation of synaptic inhibition in the rat BLA. Glucocorticoid application to amygdala slices elicited a rapid, nonreversible suppression of spontaneous, but not evoked, GABAergic synaptic currents in BLA principal neurons; the effect was also seen with a membrane-impermeant glucocorticoid, but not with intracellular glucocorticoid application, implicating a membrane-associated glucocorticoid receptor. The glucocorticoid suppression of GABA currents was not blocked by antagonists of nuclear corticosteroid receptors, or by inhibitors of gene transcription or protein synthesis, but was blocked by inhibiting postsynaptic G-protein activity, suggesting a postsynaptic nongenomic steroid signaling mechanism that stimulates the release of a retrograde messenger. The rapid glucocorticoidinduced suppression of inhibition was prevented by blocking CB1 receptors and 2-arachidonoylglycerol (2-AG) synthesis, and it was mimicked and occluded by CB1 receptor agonists, indicating it was mediated by the retrograde release of the endocannabinoid 2-AG. The rapid glucocorticoid effect in BLA neurons in vitro was occluded by prior in vivo acute stress-induced, or prior in vitro glucocorticoidinduced, release of endocannabinoid. Acute stress also caused an increase in anxiety-like behavior that was attenuated by blocking CB1 receptor activation and inhibiting 2-AG synthesis in the BLA. Together, these findings suggest that acute stress causes a long-lasting suppression of synaptic inhibition in BLA neurons via a membrane glucocorticoid receptor-induced release of 2-AG at GABA synapses, which contributes to stress-induced anxiogenesis.

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150 words ABSTRACT Noradrenergic afferents to corticotropin releasing hormone (CRH) neurons of the hypothalamic paraventricular nucleus (PVN) provide a major excitatory drive to the hypothalamic-pituitaryadrenal (HPA) axis via α 1 adrenoreceptor activation. The ascending noradrenergic afferents are recruited preferentially by physiological, rather than psychological, stress modalities.Glucocorticoids secreted in response to HPA activation feed back onto the hypothalamus to negatively regulate the HPA axis, providing a critical autoregulatory constraint that prevents glucocorticoid overexposure. Whether differential negative feedback mechanisms target stress modality-specific HPA activation is not known. Here, we reveal a desensitization of the α 1 adrenoreceptor activation of the HPA axis following acute stress that is mediated by rapid glucocorticoid regulation of adrenoreceptor trafficking. Prior stress desensitized the HPA axis to subsequent physiological, but not psychological, stress. Our findings demonstrate rapid glucocorticoid suppression of adrenoreceptor signaling in CRH neurons that is specific to physiological stress activation, and reveal, therefore, a rapid, modality-selective glucocorticoid feedback mechanism.

Several membrane proteins function as ion pumps/transporters that work to maintain the relative ionic concentrations necessary for neuronal signaling. The potassium-chloride co-transporter two (KCC2) is a symporter that actively pumps K + and Cl - out of the cell in order to maintain a low intracellular [Cl - ], while the sodium-potassium-chloride cotransporters one and two (NKCC1/NKCC2) are symporters that actively pump Na + , K + , and Cl - into the cell. KCC2 and NKCC1/2, therefore, have opposing actions on Cl - transport. In the adult, high KCC2 expression leads to low intracellular [Cl - ], which causes GABA A receptor Cl- channels to flux Cl - into the neuron, causing a membrane hyperpolarization. Early in development however, low KCC2 expression reverses the Cl - gradient and causes GABA A receptors to flux Cl - out of the neuron, leading to membrane depolarization and neuronal excitation. The change in the polarity of GABA signaling during development is, in part, due to a shift in KCC2 expression from low to high with the maturation of synaptic circuits. Our previous studies have demonstrated an excitatory GABA A receptor-mediated response due to a GABA equilibrium potential (E GABA ) that is shifted positive, like that seen early in development, in adult vasopressin (VP)-secreting neurons of the rat hypothalamus. In the current study, we used immunohistochemistry to compare expression levels of the main C l- transporters, KCC2, NKCC1, and NKCC2, between VP- and oxytocin (OT)-secreting neurons of the hypothalamic paraventricular and supraoptic nuclei. We found that, in adult male Wistar rats, expression of all three transporters is uniformly lower in VP neurons than in adjacent OT neurons in the hypothalamus. Because the phosphorylation state of KCC2 affects membrane trafficking and stabilization, we are also using Western blot to analyze phosphorylation of KCC2 under different osmotic conditions. Finally, we are using Cre-dependent viral transduction to express a light-activatable Cl - channel conditionally in vasopressin-secreting neurons to test for the effect of Cl - flux on vasopressin secretion.

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