Neurons in the suprachiasmatic nucleus (SCN) are responsible for the generation of circadian oscillations, and understanding how these neurons communicate to form a functional circuit is a critical issue. The neurotransmitter GABA and its receptors are widely expressed in the SCN where they mediate cell-to-cell communication. Previous studies have raised the possibility that GABA can function as an excitatory transmitter in adult SCN neurons during the day, but this work is controversial. In the present study, we first tested the hypothesis that GABA can evoke excitatory responses during certain phases of the daily cycle by broadly sampling how SCN neurons respond to GABA using extracellular single-unit recording and gramicidin-perforated-patch recording techniques. We found that, although GABA inhibits most SCN neurons, some level of GABA-mediated excitation was present in both dorsal and ventral regions of the SCN, regardless of the time of day. These GABA-evoked excitatory responses were most common during the night in the dorsal SCN region. The Na ϩ -K ϩ -2Cl Ϫ cotransporter (NKCC) inhibitor, bumetanide, prevented these excitatory responses. In individual neurons, the application of bumetanide was sufficient to change GABA-evoked excitation to inhibition. Calcium-imaging experiments also indicated that GABA-elicited calcium transients in SCN cells are highly dependent on the NKCC isoform 1 (NKCC1). Finally, Western blot analysis indicated that NKCC1 expression in the dorsal SCN is higher in the night. Together, this work indicates that GABA can play an excitatory role in communication between adult SCN neurons and that this excitation is critically dependent on NKCC1.

In mammals, the increased secretion of arginine-vasopressin (AVP) (antidiuretic hormone) and oxytocin (natriuretic hormone) is a key physiological response to hyperosmotic stress. In this study, we examined whether chronic hyperosmotic stress weakens GABA A receptor-mediated synaptic inhibition in rat hypothalamic magnocellular neurosecretory cells (MNCs) secreting these hormones. Gramicidin-perforated recordings of MNCs in acute hypothalamic slices prepared from control rats and ones subjected to the chronic hyperosmotic stress revealed that this challenge not only attenuated the GABAergic inhibition but actually converted it into excitation. The hyperosmotic stress caused a profound depolarizing shift in the reversal potential of GABAergic response (E GABA ) in MNCs. This E GABA shift was associated with increased expression of NaϪ cotransporter 1 (NKCC1) in MNCs and was blocked by the NKCC inhibitor bumetanide as well as by decreasing NKCC activity through a reduction of extracellular sodium. Blocking central oxytocin receptors during the hyperosmotic stress prevented the switch to GABAergic excitation. Finally, intravenous injection of the GABA A receptor antagonist bicuculline lowered the plasma levels of AVP and oxytocin in rats under the chronic hyperosmotic stress. We conclude that the GABAergic responses of MNCs switch between inhibition and excitation in response to physiological needs through the regulation of transmembrane Cl Ϫ gradients.

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The resetting of the circadian clock based on photic cues delivered by the glutamatergic retinohypothalamic tract is an important process helping mammals to function adaptively to the daily light-dark cycle. To see if the photic resetting relies on voltage-gated Ca(2+) channels (VGCCs), we examined the effects of VGCC blockers on the glutamate-induced phase shifts of circadian firing activity rhythms of suprachiasmatic nucleus (SCN) neurons in hypothalamic slices. First, we found that a cocktail of amiloride, nimodipine and omega-conotoxin MVIIC (T-, L- and NPQ-type VGCC antagonists, respectively) completely blocked both phase delays and advances, which were, respectively, induced by glutamate application in early and late night. Next, we discovered that: (i) amiloride and another T-type VGCC antagonist, mibefradil, completely obstructed the delays without affecting the advances; (ii) nimodipine completely blocked the advances while having less impact on delays; and (iii) omega-conotoxin MVIIC blocked largely, if not entirely, both delays and advances. Subsequent whole-cell recordings revealed that T-type Ca(2+) currents in neurons in the ventrolateral, not dorsomedial, region of the SCN were larger during early than late night, whereas L-type Ca(2+) currents did not differ from early to late night in both regions. These results indicate that VGCCs play important roles in glutamate-induced phase shifts, T-type being more important for phase delays and L-type being so for phase advances. Moreover, the results point to the possibility that a nocturnal modulation of T-type Ca(2+) current in retinorecipient neurons is related to the differential involvement of T-type VGCC in phase delays and advances.

Rationale: Increased arginine-vasopressin (AVP) secretion is a key physiological response to hyperosmotic stress and may be part of the mechanism by which high-salt diets induce or exacerbate hypertension. Objective: Using deoxycorticosterone acetate-salt hypertension model rats, we sought to test the hypothesis that changes in GABA A receptor–mediated inhibition in AVP-secreting magnocellular neurons contribute to the generation of Na + -dependent hypertension. Methods and Results: In vitro gramicidin-perforated recordings in the paraventricular and supraoptic nuclei revealed that the GABAergic inhibition in AVP-secreting neurons was converted into excitation in this model, because of the depolarization of GABA equilibrium potential. Meanwhile, in vivo extracellular recordings in the supraoptic nuclei showed that the GABAergic baroreflexive inhibition of magnocellular neurons was transformed to excitation, so that baroreceptor activation may increase AVP release. The depolarizing GABA equilibrium potential shift in AVP-secreting neurons occurred progressively over weeks of deoxycorticosterone acetate-salt treatment along with gradual increases in plasma AVP and blood pressure. Furthermore, the shift was associated with changes in chloride transporter expression and partially reversed by bumetanide (Na + -K + -2Cl – cotransporter inhibitor). Intracerebroventricular bumetanide administration during deoxycorticosterone acetate-salt treatment hindered the development of hypertension and rise in plasma AVP level. Muscimol (GABA A agonist) microinjection into the supraoptic nuclei in hypertensive rats increased blood pressure, which was prevented by previous intravenous V1a AVP antagonist injection. Conclusions: We conclude that the inhibitory-to-excitatory switch of GABA A receptor–mediated transmission in AVP neurons contributes to the generation of Na + -dependent hypertension by increasing AVP release. We speculate that normalizing the GABA equilibrium potential may have some utility in treating Na + -dependent hypertension.

In this report, we present a novel virtual high-throughput screening methodology to assist in computer-aided drug discovery. Our method, designated as SLIM, involves ligand-free shape and chemical feature matching. The procedure takes advantage of a negative image of a binding pocket in a target receptor. The negative image is a set of virtual atoms representing the inner shape and chemical features of the binding pocket. Using this image, SLIM implements a shape-based similarity search based on molecular volume superposition for the ensemble of conformers of each molecule. The superposed structures, prioritized by shape similarity, are subjected to comparison of chemical feature similarities. To validate the merits of the SLIM method, we compared its performance with those of three distinct widely used tools ROCS, GLIDE, and GOLD. ROCS was selected as a representative of the ligand-centric methods, and docking programs GLIDE and GOLD as representatives of the receptor-centric methods. Our data suggest that SLIM has overall hit ranking ability that is comparable to that of the docking method, retaining the high computational speed of the ligand-centric method. It is notable that the SLIM method offers consistently reliable screening quality against conformational variations of receptors, whereas the docking methods have limited screening performance.

BackgroundIncreased secretion of oxytocin and arginine vasopressin (AVP) from hypothalamic magnocellular neurosecretory cells (MNCs) is a key physiological response to lactation. In the current study, we sought to test the hypothesis that the GABAA receptor-mediated inhibition of MNCs is altered in lactating rats.ResultsGramicidin-perforated recordings in the rat supraoptic nucleus (SON) slices revealed that the reversal potential of GABAA receptor-mediated response (EGABA) of MNCs was significantly depolarized in the lactating rats as compared to virgin animals. The depolarizing EGABA shift was much larger in rats in third, than first, lactation such that GABA exerted an excitatory, instead of inhibitory, effect in most of the MNCs of these multiparous rats. Immunohistochemical analyses confirmed that GABAergic excitation was found in both AVP and oxytocin neurons within the MNC population. Pharmacological experiments indicated that the up-regulation of the Cl− importer Na+-K+-2Cl− cotransporter isotype 1 and the down-regulation of the Cl− extruder K+-Cl− cotransporter isotype 2 were responsible for the depolarizing shift of EGABA and the resultant emergence of GABAergic excitation in the MNCs of the multiparous rats.ConclusionWe conclude that, in primiparous rats, the GABAergic inhibition of MNCs is weakened during the period of lactation while, in multiparous females, GABA becomes excitatory in a majority of the cells. This reproductive experience-dependent alteration of GABAergic transmission may help to increase the secretion of oxytocin and AVP during the period of lactation.

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