Hee Chul Han

Excitatory actions of GABA in the suprachiasmatic nucleus.

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+-K+-2Cl− 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.

NMDA receptors are important for both mechanical and thermal allodynia from peripheral nerve injury in rats.

PREVIOUS studies showed that heat-hyperalgesia and mechanical allodynia produced by chronic constrictive injury of the sciatic nerve were differentially sensitive to the NMDA receptor antagonist dextrorphan and to morphine and other opioid receptor agonists. These results support the hypothesis that different kinds of neuropathic pain symptoms are caused by different pathological mechanisms. In the present study we determined whether mechanical and thermal allodynia produced by unilateral transection of the ‘superior’ caudal trunk which innervates the tail in rats were differentially sensitive to the non-competitive NMDA receptor antagonist MK-801. Injection of MK-801 (0.3 mg/kg, i.p.) prior to nerve injury delayed the emergence of both types of allodynia; the antagonist-treated rats exhibited neither mechanical nor thermal allodynia at least for 4 days after the injury, whereas untreated control rats exhibited clear signs of allodynia from the first day after the injury. MK-801 injection on post-injury day 14, when the allodynia was near peak severity, suppressed temporarily both the mechanical and thermal allodynia. These results suggest that the mechanical and thermal allodynia from partial denervation of the tail are both dependent on NMDA receptors in their induction and maintenance. Thus, our results do not support the notion that different pathological mechanisms underlie different modalities of neuropathic pain from partial peripheral nerve injury.

Chronic Hyperosmotic Stress Converts GABAergic Inhibition into Excitation in Vasopressin and Oxytocin Neurons in the Rat

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 GABAA 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 (EGABA) in MNCs. This EGABA shift was associated with increased expression of Na+–K+–2Cl− 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 GABAA 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− gradients.

Voltage‐gated calcium channels play crucial roles in the glutamate‐induced phase shifts of the rat suprachiasmatic circadian clock

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 Ca2+ 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 ω‐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) ω‐conotoxin MVIIC blocked largely, if not entirely, both delays and advances. Subsequent whole‐cell recordings revealed that T‐type Ca2+ currents in neurons in the ventrolateral, not dorsomedial, region of the SCN were larger during early than late night, whereas L‐type Ca2+ 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 Ca2+ current in retinorecipient neurons is related to the differential involvement of T‐type VGCC in phase delays and advances.

Intraarticular pretreatment with ketamine and memantine could prevent arthritic pain: relevance to the decrease of spinal c-fos expression in rats.

To determine whether intraarticular pretreatment with N-methyl-d-aspartic (NMDA) receptor antagonist ketamine or memantine currently used in humans has prophylactic analgesia in arthritic pain, we examined the effects of their intraarticular injection before carrageenan injection into the knee joint on pain-related behavior and spinal c-Fos expression in rats. Injection of ketamine (0.2 mg and 1 mg) or memantine (0.1 mg, 0.2 mg, and 1 mg) into the knee joint, but not the abdominal cavity, immediately before carrageenan injection (2%, 40 μL) significantly prevented pain-related behavior. The intraarticular injection of ketamine (1 mg) or memantine (0.2 mg) also suppressed c-Fos expression in the laminae I-II and laminae V-VI at the L3-4 spinal level. Subsequent statistical analyses revealed that the degree of the spinal c-Fos expression was correlated with the extent of the pain-related behavior. These results suggest that peripheral administration of NMDA receptor antagonists has prophylactic analgesic effects in arthritic pain, which might be associated with the decrease of central nociceptive signaling. Because ketamine and memantine are currently used in humans and considered clinically safe, they may have therapeutic value in the treatment of joint pain.

GABAergic Excitation of Vasopressin Neurons Possible Mechanism Underlying Sodium-Dependent Hypertension

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 GABAA 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 (GABAA 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 GABAA 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. # Novelty and Significance {#article-title-52}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 GABAA 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 (GABAA 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 GABAA 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.

The peripheral role of group I metabotropic glutamate receptors on nociceptive behaviors in rats with knee joint inflammation

We examined whether the mGluR1 and mGluR5 were involved in development and maintenance of behavioral signs of non-evoked pain and secondary mechanical hyperalgesia induced by knee joint inflammation. Selective mGluR1 antagonist, (RS)-1-aminoindan-1,5-dicarboxylic acid (AIDA: 50, 100, 200 microM/25 microl, n=10 per group) and selective mGluR5 antagonist, 2-methyl-6-(phenylethynyl)-pyridine (MPEP: 50, 100, 200 nM/25 microl, n=10 per group) was intra-articularly (i.a.) injected 30 min before and 4h after carrageenan injection and behavioral tests were conducted. In the pre-treatment, only a higher dose (200 nM) of MPEP significantly prevented the magnitude of weight load reduction, whereas AIDA (200 microM) and MEPE (50, 100 and 200 nM) significantly reduced the development of mechanical hyperalgesia compared to saline treated group. In the post-treatment, AIDA (200 microM) and MPEP at 100 and 200 nM partially reversed the reduction of weight load induced by carrageenan. MPEP significantly increased the withdrawal threshold to mechanical stimulation in a dose-dependent manner, whereas AIDA had significantly reversed the decreased the paw withdrawal threshold only at 200 microM. The present study demonstrated that i.a. MPEP, selective mGluR5 antagonist is more effective than selective mGluR1 antagonist, AIDA on non-evoked pain as well as mechanical hyperalgesia in both induction and maintenance phase in knee joint inflammation. It is suggested that peripheral mGlu5 receptors play a more prominent role in inflammatory pain including evoked and spontaneous pain. Thus, selective mGluR5 antagonist could be effective therapeutic tools in clinical setting.

GABAergic inhibition is weakened or converted into excitation in the oxytocin and vasopressin neurons of the lactating rat

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.

Histamine resets the circadian clock in the suprachiasmatic nucleus through the H1R‐CaV1.3‐RyR pathway in the mouse

Histamine, a neurotransmitter/neuromodulator implicated in the control of arousal state, exerts a potent phase‐shifting effect on the circadian clock in the rodent suprachiasmatic nucleus (SCN). In this study, the mechanisms by which histamine resets the circadian clock in the mouse SCN were investigated. As a first step, Ca2+‐imaging techniques were used to demonstrate that histamine increases intracellular Ca2+ concentration ([Ca2+]i) in acutely dissociated SCN neurons and that this increase is blocked by the H1 histamine receptor (H1R) antagonist pyrilamine, the removal of extracellular Ca2+ and the L‐type Ca2+ channel blocker nimodipine. The histamine‐induced Ca2+ transient is reduced, but not blocked, by application of the ryanodine receptor (RyR) blocker dantrolene. Immunohistochemical techniques indicated that CaV1.3 L‐type Ca2+ channels are expressed mainly in the somata of SCN cells along with the H1R, whereas CaV1.2 channels are located primarily in the processes. Finally, extracellular single‐unit recordings demonstrated that the histamine‐elicited phase delay of the circadian neural activity rhythm recorded from SCN slices is blocked by pyrilamine, nimodipine and the knockout of CaV1.3 channel. Again, application of dantrolene reduced but did not block the histamine‐induced phase delays. Collectively, these results indicate that, to reset the circadian clock, histamine increases [Ca2+]i in SCN neurons by activating CaV1.3 channels through H1R, and secondarily by causing Ca2+‐induced Ca2+ release from RyR‐mediated internal stores.

Peripheral group II and III metabotropic glutamate receptors in the knee joint attenuate carrageenan-induced nociceptive behavior in rats

This study sought to evaluate whether peripheral group II and III metabotropic glutamate receptors (mGluRs) in the knee joint have inhibitory effects on carrageenan-induced nociceptive behavior. To this end, changes in weight load and hind paw withdrawal threshold were measured in rats following the administration of specific peripheral group II and III mGluR agonists 30min before (induction phase) and 4h after (maintenance phase) the injection of carrageenan (1%, 50μl). During the induction phase of arthritic pain, a significant recovery of reduced weight load occurred after the administration of 500μM APDC ((2R, 4R)-4-aminopyrrolidine-2,4-dicarboxylate; group II agonist) and 100 and 500μM L-AP4 (l-2-amino-4-phosphonobutylate; group III agonist). Similarly, 100 and 500μM APDC and 500μM L-AP4 significantly reduced mechanical hyperalgesia during the induction phase. In the maintenance phase, APDC at all doses (10, 100 and 500μM) and 100 and 500μM L-AP4 significantly counteracted the reduction in weight load, and APDC and L-AP4 at all doses (10, 100 and 500μM) inhibited mechanical hyperalgesia. The current study suggests that peripheral group II and III mGluRs in the knee joint negatively modulates nociceptive behavior during both the induction and maintenance phases of carrageenan-induced arthritic pain.