Jeffrey G. Tasker

Physiological Mapping of Local Inhibitory Inputs to the Hypothalamic Paraventricular Nucleus

Local inhibitory synaptic inputs to neurons of the rat hypothalamic paraventricular nucleus (PVN) were studied by using glutamate microstimulation and conventional intracellular and whole-cell patch-clamp recordings in coronal, horizontal, and parasagittal slices of rat hypothalamus. PVN cells were classified as magnocellular or parvocellular neurons on the basis of electrophysiological andpost hoc immunohistochemical analyses; GABA-producing neurons were localized with in situ hybridization. Glutamate microstimulation of different sites around the PVN evoked volleys of postsynaptic potentials in 43% of the PVN neurons tested. Some responses to stimulation at each site were blocked by bicuculline, suggesting that they were mediated by the activation of presynaptic GABA neurons. In the coronal plane, presynaptic inhibitory sites were located lateral to the PVN and ventral to the fornix, corresponding to the lateral hypothalamic area and the posterior bed nucleus of the stria terminalis (BNST). In the horizontal plane, presynaptic inhibitory sites were found rostral, lateral, and caudal to the nucleus, corresponding to parts of the anterior hypothalamic area, the posterior BNST, the medial preoptic area, and the dorsomedial hypothalamus. In the parasagittal plane, presynaptic inhibitory neurons were revealed at sites rostral and caudal to the nucleus, corresponding to the medial preoptic area and the dorsomedial hypothalamus, and in a site dorsal to the optic chiasm that included the suprachiasmatic nucleus. These presynaptic sites each contained GABA-producing neurons based on in situ hybridization with a glutamic acid decarboxylase riboprobe and together formed a three-dimensional ring around the PVN. Unexpectedly, both magnocellular and parvocellular neurons received inhibitory synaptic inputs from common sites.

Local circuit regulation of paraventricular nucleus stress integration Glutamate: GABA connections

Limbic neurocircuits play a central role in regulation of the hypothalamic-pituitary-adrenocortical (HPA) axis. Limbic influences on adrenocortical hormone secretion are mediated by transynaptic activation or inhibition of hypophysiotrophic neurons in the medial parvocellular paraventricular nucleus (PVN). Projections from the ventral subiculum, prefrontal cortex, medial amygdala, lateral septum, paraventricular thalamus and suprachiasmatic nucleus (SN) terminate in the immediate surround of the PVN, an area heavily populated by GABAergic interneurons. As such, these regions are positioned to modulate paraventricular output via excitation or inhibition of interneuronal projections into the PVN. In addition, the same limbic and diencephalic regions have projections to local PVN-projecting hypothalamic and basal telencephalic nuclei, including the dorsomedial and medial preoptic nuclei and the bed nucleus of the stria terminalis. These regions are involved in both inhibitory and excitatory regulation of the stress axis, indicating that they contain heterogeneous neuronal populations whose relative impact on the PVN is determined by the nature of afferent stimuli. Thus, limbic modulation of the pituitary-adrenocortical system appears to be a multisynaptic process integrated at the level of local PVN-projecting neurocircuits. Local circuits are likely the primary integrators of anticipatory stress responses, and may indeed be the focus of HPA dysfunction seen with aging or affective disease.

Fast Feedback Inhibition of the HPA Axis by Glucocorticoids Is Mediated by Endocannabinoid Signaling

Glucocorticoid hormones are secreted in response to stimuli that activate the hypothalamo-pituitary-adrenocortical (HPA) axis and self-regulate through negative feedback. Negative feedback that occurs on a rapid time scale is thought to act through nongenomic mechanisms. In these studies, we investigated fast feedback inhibition of HPA axis stress responses by direct glucocorticoid action at the paraventricular nucleus of the hypothalamus (PVN). Local infusion of dexamethasone or a membrane-impermeant dexamethasone-BSA conjugate into the PVN rapidly inhibits restraint-induced ACTH and corticosterone release in a manner consistent with feedback actions at the cell membrane. The dexamethasone fast feedback response is blocked by the cannabinoid CB1 receptor antagonist AM-251, suggesting that fast feedback requires local release of endocannabinoids. Hypothalamic tissue content of the endocannabinoid 2-arachidonoyl glycerol is elevated by restraint stress, consistent with endocannabinoid action on feedback processes. These data support the hypothesis that glucocorticoid-induced fast feedback inhibition of the HPA axis is mediated by a nongenomic signaling mechanism that involves endocannabinoid signaling at the level of the PVN.

Endocannabinoid signaling, glucocorticoid-mediated negative feedback, and regulation of the hypothalamic-pituitary-adrenal axis.

The hypothalamic-pituitary-adrenal (HPA) axis regulates the outflow of glucocorticoid hormones under basal conditions and in response to stress. Within the last decade, a large body of evidence has mounted indicating that the endocannabinoid system is involved in the central regulation of the stress response; however, the specific role endocannabinoid signaling plays in phases of HPA axis regulation, and the neural sites of action mediating this regulation, were not mapped out until recently. This review aims to collapse the current state of knowledge regarding the role of the endocannabinoid system in the regulation of the HPA axis to put together a working model of how and where endocannabinoids act within the brain to regulate outflow of the HPA axis. Specifically, we discuss the role of the endocannabinoid system in the regulation of the HPA axis under basal conditions, activation in response to acute stress, and glucocorticoid-mediated negative feedback. Interestingly, there appears to be some anatomical specificity to the role of the endocannabinoid system in each phase of HPA axis regulation, as well as distinct roles of both anandamide and 2-arachidonoylglycerol in these phases. Overall, the current level of information indicates that endocannabinoid signaling acts to suppress HPA axis activity through concerted actions within the prefrontal cortex, amygdala, and hypothalamus.

Functional Interactions between Stress and the Endocannabinoid System: From Synaptic Signaling to Behavioral Output

Endocannabinoid signaling is distributed throughout the brain, regulating synaptic release of both excitatory and inhibitory neurotransmitters. The presence of endocannabinoid signaling within stress-sensitive nuclei of the hypothalamus, as well as upstream limbic structures such as the amygdala, suggests it may play an important role in regulating the neuroendocrine and behavioral effects of stress. The evidence reviewed here demonstrates that endocannabinoid signaling is involved in both activating and terminating the hypothalamic-pituitary-adrenal axis response to both acute and repeated stress. In addition to neuroendocrine function, however, endocannabinoid signaling is also recruited by stress and glucocorticoid hormones to modulate cognitive and emotional processes such as memory consolidation and extinction. Collectively, these data demonstrate the importance of endocannabinoid signaling at multiple levels as both a regulator and an effector of the stress response.

Glucocorticoids Regulate Glutamate and GABA Synapse-Specific Retrograde Transmission via Divergent Nongenomic Signaling Pathways

Glucocorticoids exert an opposing rapid regulation of glutamate and GABA synaptic inputs to hypothalamic magnocellular neurons via the activation of postsynaptic membrane-associated receptors and the release of retrograde messengers. Glucocorticoids suppress synaptic glutamate release via the retrograde release of endocannabinoids and facilitate synaptic GABA release via an unknown retrograde messenger. Here, we show that the glucocorticoid facilitation of GABA inputs is due to the retrograde release of neuronal nitric oxide and that glucocorticoid-induced endocannabinoid synthesis and nitric oxide synthesis are mediated by divergent G-protein signaling mechanisms. While the glucocorticoid-induced, endocannabinoid-mediated suppression of glutamate release is dependent on activation of the Gαs G-protein subunit and cAMP–cAMP-dependent protein kinase activation, the nitric oxide facilitation of GABA release is mediated by Gβγ signaling that leads to activation of neuronal nitric oxide synthase. Our findings indicate, therefore, that glucocorticoids exert opposing rapid actions on glutamate and GABA release by activating divergent G-protein signaling pathways that trigger the synthesis of, and glutamate and GABA synapse-specific retrograde actions of, endocannabinoids and nitric oxide, respectively. The simultaneous rapid stimulation of nitric oxide and endocannabinoid synthesis by glucocorticoids has important implications for the impact of stress on the brain as well as on neural-immune interactions in the hypothalamus.

Role of the paraventricular nucleus microenvironment in stress integration

The hypothalamic paraventricular nucleus is the primary controller of hypothalamo‐pituitary–adrenocortical glucocorticoid release. In performing this function, the paraventricular nucleus summates a variety of information from both external and internal sources into a net secretory signal to the adrenal cortex. In this review, we will provide an overview of neuronal circuit mechanisms governing activation and inhibition of hypophysiotrophic neurons, highlight recent developments in our understanding of nonsynaptic mechanisms regulating paraventricular cellular activity, including dendritic neuropeptide release, direct steroid feedback, cytokine cascades and gaseous neurotransmission, and illustrate the capacity for hypophysiotrophic, neurohypophysial and preautonomic paraventricular effector pathways to work together in control of glucocorticoid release. The current state of knowledge reveals the paraventricular nucleus to be a dynamic entity, capable of integrating diverse classes of signals into control of adrenocortical activation.

Mechanisms of rapid glucocorticoid feedback inhibition of the hypothalamic–pituitary–adrenal axis

Stress activation of the hypothalamic–pituitary–adrenal (HPA) axis culminates in increased circulating corticosteroid concentrations. Stress-induced corticosteroids exert diverse actions in multiple target tissues over a broad range of timescales, ranging from rapid actions, which are induced within seconds to minutes and gene transcription independent, to slow actions, which are delayed, long lasting, and transcription dependent. Rapid corticosteroid actions in the brain include, among others, a fast negative feedback mechanism responsible for shutting down the activated HPA axis centrally. We provide a brief review of the cellular mechanisms responsible for rapid corticosteroid actions in different brain structures of the rat, including the hypothalamus, hippocampus, amygdala, and in the anterior pituitary. We propose a model for the direct feedback inhibition of the HPA axis by glucocorticoids in the hypothalamus. According to this model, glucocorticoids activate membrane glucocorticoid receptors to induce endocannabinoid synthesis in the hypothalamic paraventricular nucleus (PVN) and retrograde cannabinoid type I receptor-mediated suppression of the excitatory synaptic drive to PVN neuroendocrine cells. Rapid corticosteroid actions in the hippocampus, amygdala, and pituitary are mediated by diverse cellular mechanisms and may also contribute to the rapid negative feedback regulation of the HPA neuroendocrine axis as well as to the stress regulation of emotional and spatial memory formation.

Voltage‐gated currents distinguish parvocellular from magnocellular neurones in the rat hypothalamic paraventricular nucleus

Magnocellular and parvocellular neurones of the hypothalamic paraventricular nucleus (PVN) differentially regulate pituitary hormone secretion and autonomic output. Previous experiments have suggested that magnocellular, or type I neurones, and parvocellular, or type II neurones, of the PVN express different electrophysiological properties. Whole‐cell patch‐clamp recordings were performed in hypothalamic slices to identify the voltage‐gated currents responsible for the electrophysiological differences between type I and type II PVN neurones. Type I neurones, which display transient outward rectification and lack a low‐threshold spike (LTS), generated a large A‐type K+ current (IA) (mean ± s.e.m.: 1127.5 ± 126.4 pA; range: 250–3600 pA; voltage steps to −25 mV) but expressed little or no T‐type Ca2+ current (IT). Type II neurones, which lack transient outward rectification but often display an LTS, expressed a smaller IA (360.1 ± 56.3 pA; range: 40–1100 pA; voltage steps to −25 mV), and 75 % of the type II neurones generated an IT (‐402.5 ± 166.9 pA; range: −90 to −2200 pA; at peak). The voltage dependence of IA was shifted to more negative values in type I neurones compared to type II neurones. Thus, the activation threshold (‐53.5 ± 0.9 and −46.1 ± 2.6 mV), the half‐activation potential (‐25 ± 1.9 and −17.9 ± 2.0 mV), the half‐inactivation potential (‐80.4 ± 9.3 and −67.2 ± 3.0 mV), and the potential at which the current became fully inactivated (‐57.4 ± 2.1 and −49.8 ± 1.5 mV) were more negative in type I neurones than in type II neurones, respectively. I T in type II neurones activated at a threshold of −59.2 ± 1.2 mV, peaked at −32.6 ± 1.7 mV, was half‐inactivated at −66.9 ± 2.2 mV, and was fully inactivated at −52.2 ± 2.2 mV. Both cell types expressed a delayed rectifier current with similar voltage dependence, although it was smaller in type I neurones (389.7 ± 39.3 pA) than in type II neurones (586.4 ± 76.0 pA). In type I neurones IA was reduced by 41.1 ± 7.0 % and the action potential delay caused by the transient outward rectification was reduced by 46.2 ± 10.3 % in 5 mm 4‐aminopyridine. In type II neurones IT was reduced by 66.8 ± 10.9 % and the LTS was reduced by 76.7 ± 7.8 % in 100 μM nickel chloride, but neither IT nor LTS was sensitive to 50 μM cadmium chloride. Thus, differences in the electrophysiological properties between type I, putative magnocellular neurones and type II, putative parvocellular neurones of the PVN can be attributed to the differential expression of voltage‐gated K+ and Ca2+ currents. This diversity of ion channel expression is likely to have profound effects on the response properties of these neurosecretory and non‐neurosecretory neurones.

Activity‐dependent release and actions of endocannabinoids in the rat hypothalamic supraoptic nucleus

Exogenous cannabinoids have been shown to significantly alter neuroendocrine output, presaging the emergence of endogenous cannabinoids as important signalling molecules in the neuroendocrine control of homeostatic and reproductive functions, including the stress response, energy metabolism and gonadal regulation. We showed recently that magnocellular and parvocellular neuroendocrine cells of the hypothalamic paraventricular nucleus and supraoptic nucleus (SON) respond to glucocorticoids by releasing endocannabinoids as retrograde messengers to modulate the synaptic release of glutamate. Here we show directly for the first time that both of the main endocannabinoids, anandamide (AEA) and 2‐arachidonoyl glycerol (2‐AG), are released in an activity‐dependent fashion from the soma/dendrites of SON magnocellular neurones and suppress synaptic glutamate release and postsynaptic spiking. Cannabinoid reuptake blockade increases activity‐dependent endocannabinoid levels in the region of the SON, and results in the inhibition of synaptically driven spiking activity in magnocellular neurones. Together, these findings demonstrate an activity‐dependent release of AEA and 2‐AG that leads to the suppression of glutamate release and that is capable of shaping spiking activity in magnocellular neurones. This activity‐dependent regulation of excitatory synaptic input by endocannabinoids may play a role in determining spiking patterns characteristic of magnocellular neurones under stimulated conditions.