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Changes in functional GABAAR signaling in hippocampus have previously been evaluated using pre-clinical animal models of either diffuse brain injury or extreme focal brain injury that precludes measurement of cells located ipsilateral to injury. As a result, there is little information about the status of functional GABAAR signaling in dentate granule cells (DGCs) located ipsilateral to focal brain injury, where significant cellular changes have been documented. We used whole-cell patch-clamp recordings from hippocampal slices to measure changes in GABAARs in dentate granule cells (DGCs) at 1-2, 3-5, and 8-13 weeks after controlled cortical impact (CCI) brain injury. Synaptic and tonic GABAAR currents (ITonicGABA) were measured in DGCs at baseline conditions and during application of the GABAAR agonist 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridine-3-ol hydrochloride (THIP) to assess in the function of δ subunit-containing GABAARs. DGCs ipsilateral to CCI exhibited no changes in the amplitude of resting ITonicGABA relative to DGCs after sham-injury or contralateral to CCI. In contrast, there was a significant reduction in the THIP-evoked ITonicGABA in DGCs ipsilateral to CCI at both time-points. Tonic GABAergic inhibition of DGCs ipsilateral to injury also exhibited reduced responsiveness to the neurosteroid THDOC. ITonicGABA in DGCs ipsilateral to CCI did not exhibit a change in sensitivity to L655,708, an inverse agonist with selectivity for α5 subunit-containing GABAARs, suggesting a lack of functional change in GABAARs containing this subunit. At the 8-13 week time-point, gene expression of GABAAR subunits expected to contribute to ITonicGABA (i.e., α4, α5 and δ) was not significantly altered by CCI injury in isolated dentate gyrus. Collectively, these results demonstrate enduring functional changes in ITonicGABA in DGCs ipsilateral to focal brain injury that occur independent of altered gene expression., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Glucose concentration changes in the nucleus tractus solitarius (NTS) affect visceral function and metabolism by influencing central vagal circuits, especially inhibitory, GABAergic NTS neurons. Acutely elevated glucose can alter NTS neuron activity, and prolonged hyperglycemia and hypoinsulemia in animal models of type 1 diabetes results in plasticity of neural responses in the NTS. NTS neurons contributing to metabolic regulation therefore act as central glucose sensors and are functionally altered in type 1 diabetes. Glucokinase (GCK) mediates cellular utilization of glucose, linking increased glucose concentration to excitability changes mediated by ATP-sensitive K(+) channels (KATP). Using quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), Western blot, and in vitro electrophysiology, we tested the hypothesis that changes in GCK expression in the NTS accompany the development of diabetes symptoms in the streptozotocin (STZ)-treated mouse model of type 1 diabetes. After several days of hyperglycemia in STZ-treated mice, RNA expression of GCK, but not Kir6.2 or SUR1, was decreased versus controls in the dorsal vagal complex. Electrophysiological recordings in vitro indicated that neural responses to acute hyperglycemia, and synaptic responsiveness to blockade of GCK with glucosamine, were attenuated in GABAergic NTS neurons from STZ-treated mice, consistent with reduced molecular and functional expression of GCK in the vagal complex of hyperglycemic, STZ-treated mice. Altered autonomic responses to glucose in type 1 diabetes may therefore involve reduced functional GCK expression in the dorsal vagal complex., (Copyright © 2015 IBRO. Published by Elsevier Ltd. All rights reserved.)

Autonomic dysregulation accompanies type-1 diabetes, and synaptic regulation of parasympathetic preganglionic motor neurons in the dorsal motor nucleus of the vagus (DMV) is altered after chronic hyperglycemia/hypoinsulinemia. Tonic gamma-aminobutyric acid A (GABAA) inhibition prominently regulates DMV neuron activity, which contributes to autonomic control of energy homeostasis. This study investigated persistent effects of chronic hyperglycemia/hypoinsulinemia on GABAA receptor-mediated inhibition in the DMV after streptozotocin-induced type-1 diabetes using electrophysiological recordings in vitro, quantitative (q)RT-PCR, and immunohistochemistry. Application of the nonspecific GABAA receptor agonist muscimol evoked an outward current of significantly larger amplitude in DMV neurons from diabetic mice than controls. Results from application of 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol hydrochloride (THIP), a δ-subunit agonist, suggested that GABAA receptors containing δ-subunits contributed to the enhanced inducible tonic GABA current in diabetic mice. Sensitivity to THIP of inhibitory postsynaptic currents in DMV neurons from diabetic mice was also increased. Results from qRT-PCR and immunohistochemical analyses indicated that the altered GABAergic inhibition may be related to increased trafficking of GABAA receptors that contain the δ-subunit, rather than an expression change. Overall these findings suggest increased sensitivity of δ-subunit containing GABAA receptors after several days of hyperglycemia/hypoinsulinemia, which dramatically alters GABAergic inhibition of DMV neurons and could contribute to diabetic autonomic dysregulation., (Copyright © 2015 the American Physiological Society.)

A variety of metabolic disorders, including complications experienced by diabetic patients, have been linked to altered neural activity in the dorsal vagal complex. This study tested the hypothesis that augmentation of N-Methyl-D-Aspartate (NMDA) receptor-mediated responses in the vagal complex contributes to increased glutamate release in the dorsal motor nucleus of the vagus nerve (DMV) in mice with streptozotocin-induced chronic hyperglycemia (i.e., hyperglycemic mice), a model of type 1 diabetes. Antagonism of NMDA receptors with AP-5 (100 μM) suppressed sEPSC frequency in vagal motor neurons recorded in vitro, confirming that constitutively active NMDA receptors regulate glutamate release in the DMV. There was a greater relative effect of NMDA receptor antagonism in hyperglycemic mice, suggesting that augmented NMDA effects occur in neurons presynaptic to the DMV. Effects of NMDA receptor blockade on mEPSC frequency were equivalent in control and diabetic mice, suggesting that differential effects on glutamate release were due to altered NMDA function in the soma-dendritic membrane of intact afferent neurons. Application of NMDA (300 μM) resulted in greater inward current and current density in NTS neurons recorded from hyperglycemic than control mice, particularly in glutamatergic NTS neurons identified by single-cell RT-PCR for VGLUT2. Overall expression of NR1 protein and message in the dorsal vagal complex were not different between the two groups. Enhanced postsynaptic NMDA responsiveness of glutamatergic NTS neurons is consistent with tonically-increased glutamate release in the DMV in mice with chronic hyperglycemia. Functional augmentation of NMDA-mediated responses may serve as a physiological counter-regulatory mechanism to control pathological disturbances of homeostatic autonomic function in type 1 diabetes.

Behavioral and physiological coupling between energy balance and fluid homeostasis is critical for survival. The orexigenic hormone ghrelin has been shown to stimulate the secretion of the osmoregulatory hormone vasopressin (VP), linking nutritional status to the control of blood osmolality, although the mechanism of this systemic crosstalk is unknown. Here, we show using electrophysiological recordings and calcium imaging in rat brain slices that ghrelin stimulates VP neurons in the hypothalamic paraventricular nucleus (PVN) in a nutritional state-dependent manner by activating an excitatory GABAergic synaptic input via a retrograde neuronal-glial circuit. In slices from fasted rats, ghrelin activation of a postsynaptic ghrelin receptor, the growth hormone secretagogue receptor type 1a (GHS-R1a), in VP neurons caused the dendritic release of VP, which stimulated astrocytes to release the gliotransmitter adenosine triphosphate (ATP). ATP activation of P2X receptors excited presynaptic GABA neurons to increase GABA release, which was excitatory to the VP neurons. This trans-neuronal-glial retrograde circuit activated by ghrelin provides an alternative means of stimulation of VP release and represents a novel mechanism of neuronal control by local neuronal-glial circuits. It also provides a potential cellular mechanism for the physiological integration of energy and fluid homeostasis.

Neuronal excitability in the adult brain is controlled by a balance between synaptic excitation and inhibition mediated by glutamate and GABA, respectively. While generally inhibitory in the adult brain, GABA(A) receptor activation is excitatory under certain conditions in which the GABA reversal potential is shifted positive due to intracellular Cl(-) accumulation, such as during early postnatal development and brain injury. However, the conditions under which GABA is excitatory are generally either transitory or pathological. Here, we reveal GABAergic synaptic inputs to be uniformly excitatory in vasopressin (VP)-secreting magnocellular neurons in the adult hypothalamus under normal conditions. The GABA reversal potential (E(GABA)) was positive to resting potential and spike threshold in VP neurons, but not in oxytocin (OT)-secreting neurons. The VP neurons lacked expression of the K(+)-Cl(-) cotransporter 2 (KCC2), the predominant Cl(-) exporter in the adult brain. The E(GABA) was unaffected by inhibition of KCC2 in VP neurons, but was shifted positive in OT neurons, which express KCC2. Alternatively, inhibition of the Na(+)-K(+)-Cl(-) cotransporter 1 (NKCC1), a Cl(-) importer expressed in most cell types mainly during postnatal development, caused a negative shift in E(GABA) in VP neurons, but had no effect on GABA currents in OT neurons. GABA(A) receptor blockade caused a decrease in the firing rate of VP neurons, but an increase in firing in OT neurons. Our findings demonstrate that GABA is excitatory in adult VP neurons, suggesting that the classical excitation/inhibition paradigm of synaptic glutamate and GABA control of neuronal excitability does not apply to VP neurons.

Chronic dehydration induces structural changes in the hypothalamic supraoptic nucleus (SON), including increased glutamate synapses and retraction of astroglial processes. We performed whole-cell recordings in acute hypothalamic slices to determine whether these changes increase tonic activation of presynaptic metabotropic glutamate receptors (mGluRs) by increasing ambient glutamate in the SON. Activation of presynaptic group III mGluRs caused a decrease in the frequency of miniature excitatory postsynaptic currents (mEPSCs) in SON neurones that was significantly attenuated in slices from dehydrated rats (-27.8 %) compared with untreated rats (-41.7 %), suggesting a higher basal occupancy of mGluRs by ambient glutamate during dehydration. Blocking group III mGluRs caused an increase in the frequency of mEPSCs that was significantly higher in slices from dehydrated rats (+42.8 %) than untreated rats (+31.4 %), suggesting greater tonic activation of presynaptic mGluRs by ambient glutamate during dehydration. Increasing ambient glutamate levels by inhibiting astrocyte glutamate uptake resulted in a decrease in mEPSC frequency due to increased activation of presynaptic mGluRs. This was attenuated in slices from dehydrated rats (-35.4 %) compared with slices from untreated rats (-48.8 %), suggesting diminished astrocytic glutamate uptake during dehydration. Immunochemical analyses revealed a robust expression of the GLT-1 transporter protein in the SON, which was diminished in SON punches from dehydrated rats compared with untreated controls. Thus, dehydration leads to increased tonic activation of presynaptic mGluRs on glutamate terminals, consistent with a decrease in glutamate buffering capacity. The resulting reduction in glutamate release probability may compensate for the increase in glutamate release sites that occurs during dehydration.

Glucocorticoid negative feedback in the brain controls stress, feeding, and neural-immune interactions by regulating the hypothalamic-pituitary-adrenal axis, but the mechanisms of inhibition of hypothalamic neurosecretory cells have never been elucidated. Using whole-cell patch-clamp recordings in an acute hypothalamic slice preparation, we demonstrate a rapid suppression of excitatory glutamatergic synaptic inputs to parvocellular neurosecretory neurons of the hypothalamic paraventricular nucleus (PVN) by the glucocorticoids dexamethasone and corticosterone. The effect was maintained with dexamethasone conjugated to bovine serum albumin and was not seen with direct intracellular glucocorticoid perfusion via the patch pipette, suggesting actions at a membrane receptor. The presynaptic inhibition of glutamate release by glucocorticoids was blocked by postsynaptic inhibition of G-protein activity with intracellular GDP-beta-S application, implicating a postsynaptic G-protein-coupled receptor and the release of a retrograde messenger. The glucocorticoid effect was not blocked by the nitric oxide synthesis antagonist N(G)-nitro-L-arginine methyl ester hydrochloride or by hemoglobin but was blocked completely by the CB1 cannabinoid receptor antagonists AM251 [N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide] and AM281 [1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-4-morpholinyl-1H-pyrazole-3-carboxamide] and mimicked and occluded by the cannabinoid receptor agonist WIN55,212-2 [(beta)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone mesylate], indicating that it was mediated by retrograde endocannabinoid release. Several peptidergic subtypes of parvocellular neuron, identified by single-cell reverse transcripton-PCR analysis, were subject to rapid inhibitory glucocorticoid regulation, including corticotropin-releasing hormone-, thyrotropin-releasing hormone-, vasopressin-, and oxytocin-expressing neurons. Therefore, our findings reveal a mechanism of rapid glucocorticoid feedback inhibition of hypothalamic hormone secretion via endocannabinoid release in the PVN and provide a link between the actions of glucocorticoids and cannabinoids in the hypothalamus that regulate stress and energy homeostasis.

Parvocellular neurones of the hypothalamic paraventricular nucleus (PVN) comprise neurosecretory and non-neurosecretory subpopulations. We labelled neurosecretory neurones with intravenous injection of the retrograde tracer, fluoro-gold, and recorded from fluoro-gold-positive and negative PVN parvocellular neurones in hypothalamic slices. Non-neurosecretory parvocellular neurones generated a low-threshold spike (LTS) and robust T-type Ca2+ current, whereas neurosecretory neurones showed no LTS and a small T-current. LTS neurones were located in non-neurosecretory regions of the PVN, and non-LTS neurones were located in neurosecretory regions of the PVN. These findings indicate that neurosecretory and non-neurosecretory subtypes of parvocellular PVN neurones express distinct membrane electrical properties.

Type I putative magnocellular neurosecretory cells of the hypothalamic paraventricular nucleus (PVN) express a prominent transient outward rectification generated by an A-type potassium current. Described here is a slow transient outward current that alters cell excitability and firing frequency in a subset of type I PVN neurons (38%). Unlike most of the type I neurons (62%), the transient outward current in these cells was composed of two kinetically separable current components, a fast activating, fast inactivating component, resembling an A-type potassium current, and a slowly activating [10-90% rise time: 20.4 +/- 12.8 (SE) ms], slowly inactivating component (time constant of inactivation: tau = 239.0 +/- 66.1 ms). The voltage dependence of activation and inactivation and the sensitivity to block by 4-aminopyridine (5 mM) and tetraethylammonium chloride (10 mM) of the fast and slow components were similar. Compared to the other type I neurons, the neurons that expressed the slow transient outward current were less excitable when hyperpolarized, requiring larger current injections to elicit an action potential (58.5 +/- 13.2 vs. 15.4 +/- 2.4 pA; 250-ms duration; P < 0.01), displaying a longer delay to the first spike (184.9 +/- 15.7 vs. 89.7 +/- 8.8 ms with 250- to 1,000-ms, 50-pA current pulses; P < 0.01), and firing at a lower frequency (18. 7 +/- 4.6 vs. 37.0 +/- 5.5 Hz with 100-pA current injections; P < 0. 05). These data suggest that a distinct subset of type I PVN neurons express a novel slow transient outward current that leads to a lower excitability. Based on double labeling following retrograde transport of systemically administered fluoro-gold and intracellular injection of biocytin, these cells are neurosecretory and are similar morphologically to magnocellular neurosecretory cells, although it remains to be determined whether they are magnocellular neurons.