γ‐Aminobutyric acid‐mediated regulation of the activity‐dependent olfactory bulb dopaminergic phenotype

γ‐Aminobutyric acid (GABA) regulates the proliferation and migration of olfactory bulb (OB) interneuron progenitors derived from the subventricular zone (SVZ), but the role of GABA in the differentiation of these progenitors has been largely unexplored. This study examines the role of GABA in the differentiation of OB dopaminergic interneurons using neonatal forebrain organotypic slice cultures prepared from transgenic mice expressing green fluorescent protein (GFP) under the control of the tyrosine hydroxylase (Th) gene promoter (ThGFP). KCl‐mediated depolarization of the slices induced ThGFP expression. The addition of GABA to the depolarized slices further increased GFP fluorescence by inducing ThGFP expression in an additional set of periglomerular cells. These findings show that GABA promoted differentiation of SVZ‐derived OB dopaminergic interneurons and suggest that GABA indirectly regulated Th expression and OB dopaminergic neuron differentiation through an acceleration of the maturation rate for the dopaminergic progenitors. Additional studies revealed that the effect of GABA on ThGFP expression required activation of L‐ and P/Q‐type Ca²⁺ channels as well as GABA_A and GABA_B receptors. These voltage‐gated Ca²⁺ channels and GABA receptors have previously been shown to be required for the coexpressed GABAergic phenotype in the OB interneurons. Together, these findings suggest that Th expression and the differentiation of OB dopaminergic interneurons are coupled to the coexpressed GABAergic phenotype and demonstrate a novel role for GABA in neurogenesis. © 2009 Wiley‐Liss, Inc.     

Increased neuronal survival in the brainstem during liver injury: Role of γ‐aminobutyric acid and serotonin chitosan nanoparticles

γ‐Aminobutyric acid (GABA)‐ and serotonin (5‐HT)‐mediated cell signaling, neuronal survival enhancement, and reduced neuronal death in brainstem during liver injury followed by active liver regeneration have a critical role in maintaining routine bodily functions. In the present study, GABA_B and 5‐HT_2A receptor functional regulation, interrelated actions of neuronal survival factors, and expression of apoptotic factors in the brainstem during GABA and 5‐HT chitosan nanoparticles‐induced active liver regeneration in partially hepatectomized rats were evaluated. Partially hepatectomized rats were treated with the nanoparticles, and receptor assays and confocal microscopic studies of GABA_B and 5‐HT_2A receptors, gene expression studies of GABA_B and 5‐HT_2A receptors, nuclear factor‐κB (NF‐κB), tumor necrosis factor‐α (TNF‐α), Akt‐1, phospholipase C, Bax, and caspase‐8 were performed with the brainstems of experimental animals. A significant decrease in GABA_B and 5‐HT_2A receptor numbers and gene expressions denoted a homeostatic adjustment by the brain to trigger the sympathetic innervations during elevated DNA synthesis in the liver. The neuronal apoptosis resulting from the loss of liver function after partial hepatectomy was minimized by nanoparticle treatment in rats compared with rats with no treatment during regeneration. This was confirmed from the gene expression patterns of NF‐κB, TNF‐α, Akt‐1, phospholipase C, Bax, and caspase‐8. The present study revealed the potential of GABA and 5‐HT chitosan nanoparticles for increasing neuronal survival in the brainstem during liver injury following regeneration, which avoids many neuropsychiatric problems. © 2013 Wiley Periodicals, Inc.     

Experimental early‐life febrile seizures induce changes in GABAAR‐mediated neurotransmission in the dentate gyrus

Purpose: Febrile seizures (FS), the most frequent seizure type during childhood, have been linked to temporal lobe epilepsy (TLE) in adulthood. Yet, underlying mechanisms are still largely unknown. Altered γ‐aminobutyric acid (GABA)ergic neurotransmission in the dentate gyrus (DG) circuit has been hypothesized to be involved. This study aims at analyzing whether experimental FS change inhibitory synaptic input and postsynaptic GABA_AR function in dentate granule cells. Methods: We applied an immature rat model of hyperthermia (HT)–induced FS. GABA_AR‐mediated neurotransmission was studied using whole‐cell patch‐clamp recordings from dentate granule neurons in hippocampal slices within 6–9 days post‐HT. Key Findings: Frequencies of spontaneous inhibitory postsynaptic currents (sIPSCs) were reduced in HT rats that had experienced seizures, whereas sIPSC amplitudes were enhanced. Whole‐cell GABA responses revealed a doubled GABA_AR sensitivity in dentate granule cells from HT animals, compared to that of normothermic (NT) controls. Analysis of sIPSCs and whole‐cell GABA responses showed similar kinetics in postsynaptic GABA_ARs of HT and NT rats. quantitative real‐time polymerase chain reaction (qPCR) experiments indicated changes in DG GABA_AR subunit expression, which was most pronounced for the α3 subunit. Significance: The data support the hypothesis that FS persistently alter neuronal excitability.     

Effects of the volatile anesthetic sevoflurane on tonic GABA currents in the mouse striatum during postnatal development

The volatile anesthetic sevoflurane, which is widely used in pediatric surgery, has proposed effects on GABA_A receptor‐mediated extrasynaptic tonic inhibition. In the developing striatum, medium‐sized spiny projection neurons have tonic GABA currents, which function in the excitatory/inhibitory balance and maturation of striatal neural circuits. In this study, we examined the effects of sevoflurane on the tonic GABA currents of medium spiny neurons in developing striatal slices. Sevoflurane strongly increased GABA_A receptor‐mediated tonic conductance at postnatal days 3–35. The antagonist of the GABA transporter‐1, 1‐[2‐[[(diphenylmethylene)imino]oxy]ethyl]‐1,2,5,6‐tetrahydro‐3‐pyridinecarboxylic acid hydrochloride further increased tonic GABA conductance during the application of sevoflurane, thereby increasing the total magnitude of tonic currents. Both GABA (5 μm ) and 4,5,6,7‐tetrahydroisoxazolo[5,4‐c]pyridine‐3‐ol hydrochloride, the δ‐subunit‐containing GABA_A receptor agonist, induced tonic GABA currents in medium spiny neurons but not in cholinergic neurons. However, sevoflurane additively potentiated the tonic GABA currents in both cells. Interestingly, 4,5,6,7‐tetrahydroisoxazolo[5,4‐c]pyridine‐3‐ol hydrochloride‐sensitive neurons made a large current response to sevoflurane, indicating the contribution of the δ‐subunit on sevoflurane‐enhanced tonic GABA currents. Our findings suggest that sevoflurane can affect the tone of tonic GABA inhibition in a developing striatal neural network.     

GABAB receptor‐dependent modulation of network activity in the rat prefrontal cortex in vitro

GABA (γ‐aminobutyric acid) can mediate inhibition via pre‐ and post/extrasynaptic GABA receptors. In this paper we demonstrate potentially post/extrasynaptic GABA_B receptor‐dependent tonic inhibition in L2/3 pyramidal cells of rat medial prefrontal cortex (mPFC) in vitro. First, we show via voltage‐clamp experiments the presence of a tonic GABA_B receptor‐dependent outward current in these neurons. This GABA_Bergic current could be induced by ambient GABA when present at sufficient concentrations. To increase ambient GABA levels in the usually silent slice preparation, we amplified network activity and hence synaptic GABA release with a modified artificial cerebrospinal fluid. The amplitude of tonic GABA_B current was similar at different temperatures. In addition to the tonic GABA_B current, we found presynaptic GABA_B effects, GABA_B‐mediated inhibitory postsynaptic currents and tonic GABA_A currents. Second, we performed current‐clamp experiments to evaluate the functional impact of GABA_B receptor‐mediated inhibition in the mPFC. Activating or inactivating GABA_B receptors led to rightward (reduction of excitability) or leftward (increase of excitability) shifts, respectively, of the input–output function of mPFC L2/3 pyramidal cells without effects on the slope. Finally, we showed in electrophysiological recordings and epifluorescence Ca²⁺‐imaging that GABA_B receptor‐mediated tonic inhibition is capable of regulating network activity. Blocking GABA_B receptors increased the frequency of excitatory postsynaptic currents impinging on a neuron and prolonged network upstates. These results show that ambient GABA via GABA_B receptors is powerful enough to modulate neuronal excitability and the activity of neural networks.     

γ‐aminobutyric acid‐mediated neurotransmission in cerebellar–hypothalamic circuit attenuates gastric mucosal injury induced by ischemia‐reperfusion

Background Excessive greater splanchnic nerve (GSN) activation contributes to the progression of gastric ischemia‐reperfusion (GI‐R) injury. This study was designed to investigate the protective mechanism of cerebellar fastigial nucleus (FN) stimulation against GI‐R injury. Methods The GI‐R injury model was induced in rats by clamping the celiac artery for 30 min, and then reperfusion for 30 min, 1, 3, 6, or 24 h, respectively. Key Results Microinjection of l ‐Glu (3, 6, 12 μg) into the FN dose‐dependently attenuated GI‐R injury and GSN activity. In addition, there was an enhancement of gastric mucosal blood flow in GI‐R rats. Pretreatment with the glutamic acid decarboxylase antagonist into the FN, the GABA_A receptor antagonist into the lateral hypothalamic area or lesion of superior cerebellar peduncle all reversed the protective effects of the FN stimulation. Furthermore, the FN stimulation reduced the TUNEL‐positive gastric mucosal cell and Bax‐positive gastric mucosal cell in GI‐R rats. Conclusions & Inferences These results indicate that the protective effects of the FN stimulation against GI‐R injury may be mediated by attenuation of the excessive GSN activation, gastric mucosal cell apoptosis, and Bax expression in GI‐R rats.     

Activity‐dependent plasticity of presynaptic GABAB receptors at parallel fiber synapses

Parallel fiber synapses in the cerebellum express a wide range of presynaptic receptors. However, presynaptic receptor expression at individual parallel fiber synapses is quite heterogeneous, suggesting physiological mechanisms regulate presynaptic receptor expression. We investigated changes in presynaptic GABA_B receptors at parallel fiber‐stellate cell synapses in acute cerebellar slices from juvenile mice. GABA_B receptor‐mediated inhibition of excitatory postsynaptic currents (EPSCs) is remarkably diverse at these synapses, with transmitter release at some synapses inhibited by >50% and little or no inhibition at others. GABA_B receptor‐mediated inhibition was significantly reduced following 4 Hz parallel fiber stimulation but not after stimulation at other frequencies. The reduction in GABA_B receptor‐mediated inhibition was replicated by bath application of forskolin and blocked by application of a PKA inhibitor, suggesting activation of adenylyl cyclase and PKA are required. Immunolabeling for an extracellular domain of the GABA_B2 subunit revealed reduced surface expression in the molecular layer after exposure to forskolin. GABA_B receptor‐mediated inhibition of action potential evoked calcium transients in parallel fiber varicosities was also reduced following bath application of forskolin, confirming presynaptic receptors are responsible for the reduced EPSC inhibition. These data demonstrate that presynaptic GABA_B receptor expression can be a plastic property of synapses, which may compliment other forms of synaptic plasticity. This opens the door to novel forms of receptor plasticity previously confined primarily to postsynaptic receptors.     

Roles for γ‐aminobutyric acid in the development of the paraventricular nucleus of the hypothalamus

The development of the hypothalamic paraventricular nucleus (PVN) involves several factors that work together to establish a cell group that regulates neuroendocrine functions and behaviors. Several molecular markers were noted within the developing PVN, including estrogen receptors (ER), neuronal nitric oxide synthase (nNOS), and brain‐derived neurotrophic factor (BDNF). By contrast, immunoreactive γ‐aminobutyric acid (GABA) was found in cells and fibers surrounding the PVN. Two animal models were used to test the hypothesis that GABA works through GABA_A and GABA_B receptors to influence the development of the PVN. Treatment with bicuculline to decrease GABA_A receptor signaling from embryonic day (E) 10 to E17 resulted in fewer cells containing immunoreactive (ir) ERα in the region of the PVN vs. control. GABA_BR1 receptor subunit knockout mice were used to examine the PVN at P0 without GABA_B signaling. In female but not male GABA_BR1 subunit knockout mice, the positions of cells containing ir ERα shifted from medial to lateral compared with wild‐type controls, whereas the total number of ir ERα‐containing cells was unchanged. In E17 knockout mice, ir nNOS cells and fibers were spread over a greater area. There was also a significant decrease in ir BDNF in the knockout mice in a region‐dependent manner. Changes in cell position and protein expression subsequent to disruption of GABA signaling may be due, in part, to changes in nNOS and BDNF signaling. Based on the current study, the PVN can be added as another site where GABA exerts morphogenetic actions in development. J. Comp. Neurol. 518:2710–2728, 2010. © 2010 Wiley‐Liss, Inc.     

Effects of the conformationally restricted GABA analogues,Cis‐and Trans‐4‐aminocrotonic acid,on GABA neurotransmission in primary neuronal cultures

The effects of the GABA analogues, cis‐ and trans‐4‐aminocrotonic acid (ACA) on GABA_A receptor function and GABA uptake, together with the presence of ρ‐1 subunit mRNA and putative GABA_C receptors, were studied in primary cultures of neocortical neurons and cerebellar granule cells. Both isomers induced a Cl^‐ influx, which was inhibited by bicuculline, t‐butylbicyclophosphorothionate (TBPS), picrotoxinin (PTX), and γ‐hexachlorocyclohexane (γ‐HCH or lindane). [³H]‐flunitrazepam binding was also increased by both isomers and this increase was inhibited by bicuculline. In neocortical neurons, the trans‐isomer completely inhibited the [³H]GABA uptake, whereas the cis‐isomer produced only a 25% inhibition at the highest concentration used. The possible presence of GABA_C receptors was investigated only in neocortical cultures by using RT‐PCR in order to detect the presence of the mRNA encoding the ρ‐1 subunit which assembles to form homooligomeric Cl^‐ channels. The results presented here show that ρ‐1 subunits, and thus GABA_C receptors, may represent a very minor population of GABA receptors in these neuronal preparations. We conclude that both GABA analogues may act as agonists at the GABA_A receptors, although with very different potencies. J. Neurosci. Res. 57:95–105, 1999. © 1999 Wiley‐Liss, Inc.     

Role of GABAA receptors in the GABA attenuation of ethanol neurotoxicity

We have previously reported that GABA reverses the neuronotoxic effects of ethanol in neuroblast-enriched cultures derived from 3-day-old whole chick embryo (E3WE). In the present study, we examined the effects of GABA agonists and antagonists on morphological growth patterns and on cholinergic neuronal phenotypic expression, using choline acetyltransferase (ChAT) activity as a marker. E3WE neuroblast-enriched cultures showed positive immunoreactivity for neurofilament and as previously reported, control cultures exhibited the characteristic pattern of outgrowth of neurites of varying thickness radiating from the aggregates. In contrast, cultures grown in ethanol consisted of neuronal aggregates lacking fasciculation but having a complex network of individual thin neurites. Both GABA and GABA_A agonist muscimol enhanced neuritic fasciculation and arborization in control and ethanol-treated cultures, and this growth enhancement was inhibited by GABA_A antagonist bicuculline. No effects were noted with GABA_B agonist baclofen. GABA increased ChAT activity in E3WE control cultures, as previously reported. A similar effect was seen with GABA_A agonist muscimol, but not with GABA_B agonist baclofen. However, the GABA effect was not apparent in the presence of GABA_B antagonist phaclofen. Thus, it appears that the cholinotrophic effects of GABA are mediated by both GABA_A and GABA_B receptors. In ethanol-treated cultures the already-reported ChAT decline was reversed by GABA and muscimol, but not by baclofen. Moreover, the GABA effect in ethanol-treated cultures was not antagonized by GABA_B antagonist phaclofen, suggesting that the GABA effect was mediated by a GABA_A receptor. We conclude from these findings that the cholinotrophic effects of GABA are mediated by GABA_A and GABA_B receptors, while the rescuing effects of GABA in the ethanol-treated cultures are mediated via GABA_A receptors. © 1996 Wiley-Liss, Inc.     

The α1, α2, α3, and γ2 subunits of GABAA receptors show characteristic spatial and temporal expression patterns in rhombencephalic structures during normal human brain development

γ‐Aminobutyric acid (GABA) is the most abundant inhibitory neurotransmitter in adult mammalian brain, mediating its actions chiefly via a pentameric chloride ion channel, the GABA_A receptor. Nineteen different subunits (α1‐6, β1‐3, γ1‐3, δ, ε, π, θ, ρ1‐3) can give rise to multiple receptor subtypes that are the site of action of many clinically important drugs. In the developing brain, however, GABA_A receptors mediate excitatory actions due to an increased chloride concentration within neurons and seem to control cell proliferation, migration, differentiation, synapse maturation, and cell death. Little is known about the distribution of single subunits in the human brain. Here we describe developmental changes in the immunohistochemical distribution of four subunits (α1, α2, α3, and γ2) in the human rhombencephalon. The γ2 was the most abundant subunit in all rhombencephalic structures during development and in adults, whereas α subunits showed a structure‐ and age‐characteristic distribution. The α1 was expressed prenatally in the molecular and Purkinje cell layer, but only postnatally in the granule cell layer and the dentate nucleus. Expression was completely absent in the inferior olivary nucleus. The α2 gradually increased during development, showing some layer specificity in the cerebellar cortex. The α3‐immunoreactivity in the cerebellar cortex was relatively weak, but it was abundantly observed in different cell populations in the subcortical cerebellar structures. Structure‐ and age‐characteristic colocalization between subunits during development suggests differences in GABA_A receptor composition. Interestingly, subunit expression in several instances differed between human and rodent brain, underlining the importance of immunohistochemical studies in humans. J. Comp. Neurol. 524:1805–1824, 2016. © 2015 Wiley Periodicals, Inc.     

Cocaine withdrawal reduces GABABR transmission at entopeduncular nucleus – lateral habenula synapses

Lateral habenula (LHb) hyperactivity plays a pivotal role in the emergence of negative emotional states, including those occurring during withdrawal from addictive drugs. We have previously implicated cocaine‐driven adaptations at synapses from the entopeduncular nucleus (EPN) to the LHb in this process. Specifically, ionotropic GABA_A receptor (R)‐mediated neurotransmission at EPN‐to‐LHb synapses is reduced during cocaine withdrawal, due to impaired vesicle filling. Recent studies have shown that metabotropic GABA_BR signaling also controls LHb activity, although its role at EPN‐to‐LHb synapses during drug withdrawal is unknown. Here, we predicted that cocaine treatment would reduce GABA_BR‐mediated neurotransmission at EPN‐to‐LHb synapses. We chronically treated mice with saline or cocaine, prepared brain slices after two days of withdrawal and performed voltage‐clamp recordings from LHb neurons whilst optogenetically stimulating EPN terminals. Compared with controls, mice in cocaine withdrawal exhibited reduced GABA_AR‐mediated input to LHb neurons, and a reduced occurrence of GABA_BR‐signaling at EPN‐to‐LHb synapses. We then assessed the underlying mechanism of this decrease. Application of GABA_BR agonist baclofen evoked similar postsynaptic responses in EPN‐innervated LHb neurons in saline‐ and cocaine‐treated mice. Release probability at EPN‐to‐LHb GABAergic synapses was also comparable between groups. However, incubating brain slices in glutamine to facilitate GABA vesicle filling, normalized GABA_BR‐currents at EPN‐to‐LHb synapses in cocaine‐treated mice. Overall, we show that during cocaine withdrawal, together with reduced GABA_AR transmission, also GABA_BR‐mediated inhibitory signaling is diminished at EPN‐to‐LHb synapses, likely via the same presynaptic deficit. In concert, these alterations are predicted to contribute to the emergence of drug withdrawal symptoms, facilitating drug relapse.     

Early continuous white noise exposure alters l‐α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazole propionic acid receptor subunit glutamate receptor 2 and γ‐aminobutyric acid type a receptor subunit β3 protein expression in rat auditory cortex

Auditory experience during the postnatal critical period is essential for the normal maturation of auditory function. Previous studies have shown that rearing infant rat pups under conditions of continuous moderate‐level noise delayed the emergence of adult‐like topographic representational order and the refinement of response selectivity in the primary auditory cortex (A1) beyond normal developmental benchmarks and indefinitely blocked the closure of a brief, critical‐period window. To gain insight into the molecular mechanisms of these physiological changes after noise rearing, we studied expression of the AMPA receptor subunit GluR2 and GABA_A receptor subunit β3 in the auditory cortex after noise rearing. Our results show that continuous moderate‐level noise rearing during the early stages of development decreases the expression levels of GluR2 and GABA_Aβ3. Furthermore, noise rearing also induced a significant decrease in the level of GABA_A receptors relative to AMPA receptors. However, in adult rats, noise rearing did not have significant effects on GluR2 and GABA_Aβ3 expression or the ratio between the two units. These changes could have a role in the cellular mechanisms involved in the delayed maturation of auditory receptive field structure and topographic organization of A1 after noise rearing. © 2009 Wiley‐Liss, Inc.     

GluN2B‐containing N‐methyl‐D‐aspartate receptors compensate for the inhibitory control of synaptic plasticity during the early critical period in the rat visual cortex

In the visual cortex, synaptic plasticity is very high during the early developmental stage known as the critical period and declines with development after the critical period. Changes in the properties of N‐methyl‐D‐aspartate receptor (NMDAR) and γ‐aminobutyric acid type A receptor (GABA_AR) have been suggested to underlie the changes in the characteristics of plasticity. However, it is largely unknown how the changes in the two receptors interact to regulate synaptic plasticity. The present study investigates the changes in the properties of NMDAR and GABA_AR from 3 to 5 weeks of age in layer 2/3 pyramidal neurons of the rat visual cortex. The impact of these changes on the characteristics of long‐term potentiation (LTP) is also investigated. The amplitude and decay time constant of GABA_AR‐mediated currents increased during this period. However, the decay time constant of NMDAR‐mediated currents decreased as a result of the decrease in the proportion of the GluN2B subunit‐mediated component. Induction of NMDAR‐dependent LTP at 3 weeks depended on the GluN2B subunit, but LTP at 5 weeks did not. Enhancement of GABA_AR‐mediated inhibition suppressed the induction of LTP only at 5 weeks. However, partial inhibition of the GluN2B subunit with a low concentration of ifenprodil allowed the GABA_AR‐mediated suppression of LTP at 3 weeks. These results suggest that changes in the properties of NMDAR‐ and GABA_AR‐mediated synaptic transmission interact to determine the characteristics of synaptic plasticity during the critical period in the visual cortex. © 2015 Wiley Periodicals, Inc.     

Immunohistochemical Localization of GABA, GAD65, and the Receptor Subunits GABAAα1 and GABAB1 in the Zebrafish Cerebellum

Gamma-aminobutyric acid (GABA) is the principal inhibitory neurotransmitter in the central nervous system. Its role is especially prominent in the cerebellum, where most neuron types are GABAergic. However, little is known about its function in the cerebellum of teleost fish, which is only partly homologous to its mammalian counterpart. Here, we investigated the expression and distribution of GABA, the GABA-synthesizing enzyme glutamic acid decarboxylase 65 (GAD65), and the receptor subunits GABA_Aα1 and GABA_B1 in the cerebellum of adult zebrafish. GABA and GAD65 presented a similar expression pattern that comprised the molecular layer, Purkinje cells and groups of presumed Golgi cells in the granular layer, both in the cerebellar corpus and valve. GABA_A receptor subunits are principally found on fine radial fibers in the molecular layer, while GABA_B receptor subunits localized prominently to the cell bodies of Purkinje cells in the ganglionic layer, and to their dendrites that span the molecular layer. These results are compared to the expression of the GABAergic system in the mammalian cerebellum.     

Rapid use‐dependent down‐regulation of γ‐aminobutyric acid type A receptors in rat mesencephalic trigeminal neurons

Rundown is ubiquitously seen in response to repetitive activation of receptor or ion channels as a use‐dependent down‐regulation through various mechanisms. In contrast to AMPA receptors, γ‐aminobutyric acid type A receptor (GABA_AR) are believed to display no rapid use‐dependent down‐regulation. We report here a rapid use‐dependent down‐regulation of GABA_AR in primary sensory neurons of rat mesencephalic trigeminal nucleus (MTN), which express synaptic GABA_ARs in addition to extrasynaptic ones, unlike other primary sensory neurons. When muscimol was repetitively puff‐applied to an MTN neuron every 2 min before, during, and after the muscimol bath application for 5 min, both the GABA_A responses obtained under both current‐ and voltage‐clamp conditions were almost completely depressed during the bath application. However, the former and latter GABA_A responses recovered to 26% ± 7% and 36% ± 7% of their control amplitudes, respectively, 15 min after washout of the bath‐applied muscimol. By contrast, when examined in the presence of chelerythrine, a protein kinase C (PKC) inhibitor, together with a stringent chelation of intracellular Ca²⁺, the puff responses were almost completely recovered, whereas those were recovered to 40–60% of the control by either chelerythrine or EGTA alone. A phosphatidylinositol 3‐kinase inhibitor (PI3K), wortmannin, which blocks various signal transductions, including vesicular trafficking, significantly enhanced the rundown of the puff responses examined every 2 min. These findings indicate that the rundown of GABA_A response in MTN neurons is mediated by the use‐dependent down‐regulation of GABA_AR, which is reversed by PKC inhibition together with intracellular Ca²⁺ chelation, while being facilitated by PI3K inhibition. © 2009 Wiley‐Liss, Inc.     

Attenuated Benzodiazepine‐Sensitive Tonic GABAA Currents of Supraoptic Magnocellular Neuroendocrine Cells in 24‐h Water‐Deprived Rats

In supraoptic nucleus (SON) magnocellular neurosecretory cells (MNCs), γ‐GABA, via activation of GABA_A receptors (GABA_ARs), mediates persistent tonic inhibitory currents (I_tonic), as well as conventional inhibitory postsynaptic currents (IPSCs, I_phasic). In the present study, we examined the functional significance of I_tonic in SON MNCs challenged by 24‐h water deprivation (24WD). Although the main characteristics of spontaneous IPSCs were similar in 24WD compared to euhydrated (EU) rats, I_tonic, measured by bicuculline (BIC)‐induced I_holding shifts, was significantly smaller in 24WD compared to EU rats (P < 0.05). Propofol and diazepam prolonged IPSC decay time to a similar extent in both groups but induced less I_tonic in 24WD compared to EU rats, suggesting a selective decrease in GABA_A receptors mediating I_tonic over I_phasic in 24WD rats. THIP (4,5,6,7‐tetrahydroisoxazolo[5,4‐c]pyridin‐3‐ol), a preferential δ subunit agonist, and L‐655,708, a GABA_A receptor α₅ subunit selective imidazobenzodiazepine, caused a significantly smaller inward and outward shift in I_holding, respectively, in 24WD compared to EU rats (P < 0.05 in both cases), suggesting an overall decrease in the α₅ subunit‐containing GABA_ARs and the δ subunit‐containing receptors mediating I_tonic in 24WD animals. Consistent with a decrease in 24WD I_tonic, bath application of GABA induced significantly less inhibition of the neuronal firing activity in 24WD compared to EU SON MNCs (P < 0.05). Taken together, the results of the present study indicate a selective decrease in GABA_ARs functions mediating I_tonic as opposed to those mediating I_phasic in SON MNCs, demonstrating the functional significance of I_tonic with respect to increasing neuronal excitability and hormone secretion in 24WD rats.