GABABR Modulation of Electrical Synapses and Plasticity in the Thalamic Reticular Nucleus.

Two distinct types of neuronal activity result in long-term depression (LTD) of electrical synapses, with overlapping biochemical intracellular signaling pathways that link activity to synaptic strength, in electrically coupled neurons of the thalamic reticular nucleus (TRN). Because components of both signaling pathways can also be modulated by GABAB receptor activity, here we examined the impact of GABAB receptor activation on the two established inductors of LTD in electrical synapses. Recording from patched pairs of coupled rat neurons in vitro, we show that GABAB receptor inactivation itself induces a modest depression of electrical synapses and occludes LTD induction by either paired bursting or metabotropic glutamate receptor (mGluR) activation. GABAB activation also occludes LTD from either paired bursting or mGluR activation. Together, these results indicate that afferent sources of GABA, such as those from the forebrain or substantia nigra to the reticular nucleus, gate the induction of LTD from either neuronal activity or afferent glutamatergic receptor activation. These results add to a growing body of evidence that the regulation of thalamocortical transmission and sensory attention by TRN is modulated and controlled by other brain regions. Significance: We show that electrical synapse plasticity is gated by GABAB receptors in the thalamic reticular nucleus. This effect is a novel way for afferent GABAergic input from the basal ganglia to modulate thalamocortical relay and is a possible mediator of intra-TRN inhibitory effects.

Anti-spastic effect induced by waggle needling correlates with KCC2-GABAA pathway in post-stroke spasticity rats.

Although clinical efficacy of waggle needling has been confirmed, therapeutic mechanisms still remain poorly understood. Reduction of GABA was involved in the etiology of spasticity. Recently, accumulated evidences suggest that the inhibitory effect of GABA is determined by low intracellular chloride concentration, which is predominantly mediated by KCC2. This study was designed to investigate whether KCC2-GABAA pathway was involved in the mechanism underlying acupuncture intervention in rats with middle cerebral artery occlusion (MCAO). Three days after modeling, the rats received waggle needling, routine needling and placebo needling for 7 consecutive days. After treatment, the muscle spasticity, motor function and infarct volumes were tested. KCC2 and GABAAγ2 levels were detected via western blotting, RT-PCR and immunofluorescence. KCC2 antagonist and agonist were administered after the last intervention. We found that acupuncture, particularly waggle needling, could remarkably alleviate muscle spasticity, reverse motor deficits and reduce cerebral infraction in MCAO rats, possibly due to its effects on up-regulating expressions of KCC2 and GABAAγ2 in the cortical infarct regions. However, the effects were blocked by KCC2 antagonist. In summary, this study suggests that improvements in muscle spasticity and motor function induced by waggle needling correlates with the activation of KCC2-GABAA pathway.

Role of leptin in the regulation of food intake in fasted mice.

Leptin is well acknowledged as an anorexigenic hormone that plays an important role in feeding control. Hypothalamic GABA system plays a significant role in leptin regulation on feeding and metabolism control. However, the pharmacological relationship of leptin and GABA receptor is still obscure. Therefore, we investigated the effect of leptin or combined with baclofen on the food intake in fasted mice. We detected the changes in hypothalamic c-Fos expression, hypothalamic TH, POMC and GAD67 expression, plasma insulin, POMC and GABA levels to demonstrate the mechanisms. We found that leptin inhibit fasting-induced increased food intake and activated hypothalamic neurons. The inhibitory effect on food intake induced by leptin in fasted mice can be reversed by pretreatment with baclofen. Baclofen reversed leptin's inhibition on c-Fos expression of PAMM in fasted mice. Therefore, these results indicate that leptin might inhibit fasting-triggered activation of PVN neurons via presynaptic GABA synaptic functions which might be partially blocked by pharmacological activating GABA-B. Our findings identify the role of leptin in the regulation of food intake.

Amino acid substitutions in the human homomeric ß3 GABAA receptor that enable activation by GABA.

GABAA receptors (GABAARs) are pentameric ligand-gated ion channels that mediate synaptic inhibition throughout the central nervous system. The α1ß2γ2 receptor is the major subtype in the brain; GABA binds at the ß2(+)α1(-) interface. The structure of the homomeric ß3 GABAAR, which is not activated by GABA, has been solved. Recently, four additional heteromeric structures were reported, highlighting key residues required for agonist binding. Here, we used a protein engineering method, taking advantage of knowledge of the key binding residues, to create a ß3(+)α1(-) heteromeric interface in the homomeric human ß3 GABAAR that enables GABA-mediated activation. Substitutions were made in the complementary side of the orthosteric binding site in loop D (Y87F and Q89R), loop E (G152T), and loop G (N66D and A70T). The Q89R and G152T combination enabled low-potency activation by GABA and potentiation by propofol but impaired direct activation by higher propofol concentrations. At higher concentrations, GABA inhibited gating of ß3 GABAAR variants containing Y87F, Q89R, and G152T. Reversion of Phe87 to tyrosine abolished GABA's inhibitory effect and partially recovered direct activation by propofol. This tyrosine is conserved in homomeric GABAARs and in the Erwinia chrysanthemi ligand-gated ion channel and may be essential for the absence of an inhibitory effect of GABA on homomeric channels. This work demonstrated that only two substitutions, Q89R and G152T, in ß3 GABAAR are sufficient to reconstitute GABA-mediated activation and suggests that Tyr87 prevents inhibitory effects of GABA.

GABAB receptor subtypes differentially regulate thalamic spindle oscillations.

Following the discovery of GABAB receptors by Norman Bowery and colleagues, cloning and biochemical efforts revealed that GABAB receptors assemble multi-subunit complexes composed of principal and auxiliary subunits. The principal receptor subunits GABAB1a, GABAB1b and GABAB2 form two heterodimeric GABAB(1a,2) and GABAB(1b,2) receptors that can associate with tetramers of auxiliary KCTD (K+ channel tetramerization domain) subunits. Experiments with subunit knock-out mice revealed that GABAB(1b,2) receptors activate slow inhibitory postsynaptic currents (sIPSCs) while GABAB(1a,2) receptors function as heteroreceptors and inhibit glutamate release. Both GABAB(1a,2) and GABAB(1b,2) receptors can serve as autoreceptors and inhibit GABA release. Auxiliary KCTD subunits regulate the duration of sIPSCs and scaffold effector channels at the receptor. GABAB receptors are well known to contribute to thalamic spindle oscillations. Spindles are generated through alternating burst-firing in reciprocally connected glutamatergic thalamocortical relay (TCR) and GABAergic thalamic reticular nucleus (TRN) neurons. The available data implicate postsynaptic GABAB receptors in TCR cells in the regulation of spindle frequency. We now used electrical or optogenetic activation of thalamic spindles and pharmacological experiments in acute slices of knock-out mice to study the impact of GABAB(1a,2) and GABAB(1b,2) receptors on spindle oscillations. We found that selectively GABAB(1a,2) heteroreceptors at TCR to TRN cell synapses regulate oscillation strength, while GABAB(1b,2) receptors control oscillation frequency. The auxiliary subunit KCTD16 influences both oscillation strength and frequency, supporting that KCTD16 regulates network activity through GABAB(1a,2) and GABAB(1b,2) receptors. This article is part of the "Special Issue Dedicated to Norman G. Bowery".

Opposing needling promotes behavior recovery and exerts neuroprotection via the cAMP/PKA/CREB signal transduction pathway in transient MCAO rats.

The aim of the present study was to investigate whether the cyclic adenosine 3',5'­monophosphate (cAMP)/protein kinase A(PKA)/cAMP­responsive element binding protein (CREB) signal transduction pathway triggered by γ­aminobutyric acid class B (GABA(B)) receptor activation is involved in neuroprotection against ischemia and behavioral recovery induced by opposing needling (ON). A total of 80 rats were randomly divided into four groups: A sham operation group, an ischemia group, an ON group and an ON group effectively inhibited by the GABA(B) receptor antagonist, CGP35384 (n=20/group). The behavior of the rats was assessed by their neurological deficit score, whereas the impairment of gait was examined using the CatWalk system. The volume of cerebral infarction was examined upon treatment with 2,3,5­triphenyltetrazolium chloride. The expression levels of CREB, GABA(B1) and GABA(B2) were examined by western blotting and reverse transcription­quantitative polymerase chain reaction, and the activity of adenylyl cyclase (AC), cAMP and PKA in the serum was detected using an enzyme­linked immunosorbent assay. In the present study, in comparison with other groups, the ON group exhibited a reduced score for the neurological deficit, the stride length and swing speed were improved, and the volume of infarction was reduced. However, these effects were reversed upon administration of CGP35384. Additionally, the expression levels of CREB, GABA(B1) and GABA(B2) were increased in the ON group. The levels of AC, cAMP and PKA in the serum were also increased in the ON group, whereas the addition of CGP35384 reversed these effects. The results of the present study demonstrated that ON markedly protected the brain against transient cerebral ischemic injury, and this effect was possibly mediated by the activation of the GABAB/cAMP/PKA/CREB signal transduction pathway. These findings implied that ON may be a potential therapeutic method for treating stroke.

Gua Lou Gui Zhi decoction attenuates post­stroke spasticity via the modulation of GABAB receptors.

The aim of the present study was to investigate the mechanisms underlying the neuroprotective and antispastic effects of Gua Lou Gui Zhi decoction (GLGZD) in a rat model of middle cerebral artery occlusion (MCAO). The MCAO rats were treated with GLGZD (14.3 g/kg body weight) once a day for a period of seven days. Neurological deficit scores and screen tests were analyzed every other day. Following treatment with GLGZD for 7 days, the ischemic infarct volume of the rat brains was measured using 2,3,5­triphenyl tetrazolium chloride staining. Reverse transcription­polymerase chain reaction was performed in order to determine the mRNA expression levels of γ­amino butyric acid B (γ­GABAB) receptor (R) in the cortical infarct region. Furthermore, the protein expression levels of GABAB R were detected in the cortical infarct region by western blot analysis. Following 7 days, treatment with GLGZD significantly ameliorated the neurological defects and cerebral infarction in the MCAO rats. In addition, treatment with GLGZD ameliorated motor performance in the MCAO rats, as determined by screen tests. Furthermore, GLGZD was able to upregulate the mRNA and protein expression levels of GABAB1 R and GABAB2 R in the ischemic cerebral cortex. The results of the present study suggested that GLGZD may exert neuroprotective and antispastic effects in a cerebral ischemia model, through upregulating the expression of GABAB R.

Thymoquinone and vitamin C attenuates pentylenetetrazole-induced seizures via activation of GABAB1 receptor in adult rats cortex and hippocampus.

Epilepsy is a common neurological disorder that leads to neuronal excitability and provoke various forms of cellular reorganization in the brain. In this study, we investigate the anti-convulsant and neuroprotective effects of thymoquinone (TQ) and vitamin C against pentylenetetrazole (PTZ)-induced generalized seizures. Epileptic seizures were induced in adult rats using systemic intraperitoneal injections of PTZ (50 mg/kg) for 7 days. Animals pretreated with either TQ or vitamin C or in combination attenuated PTZ-induced seizures and mortality in rats as well neurodegeneration in the cells. Compared to PTZ, TQ and vitamin C significantly prolonged the onset of seizures (p > 0.05) as well decrease the high-grade seizures. Analysis of electroencephalogram (EEG) recordings revealed that TQ or vitamin C supplementation significantly reduced polyspike and epileptiform discharges. Epileptic seizures caused a decline in expression of gamma-aminobutyric acid B1 receptor (GABAB1R) (p > 0.05), unchanged expression of protein kinase A (PKA), decreased calcium/calmodulin-dependent protein kinase II (CaMKII) (p > 0.05) and inhibit the phosphorylation of cAMP response element-binding protein (CREB) (p > 0.05) in cortex and hippocampus, respectively, compared with control. Changes in expression of GABAB1R, CaMKII and CREB by PTZ were reversed by TQ and vitamin C supplementation. Moreover, PTZ significantly increased Bax, decreased Bcl-2 expression and finally the activation of caspase-3. TQ and vitamin C pretreatment reversed all these deleterious effects induced by PTZ. TQ and vitamin C showed anticonvulsant effects via activation of GABAB1R/CaMKII/CREB pathway and suggest a potential therapeutic role in epilepsy.

Influence of JuA in evoking communication changes between the small intestines and brain tissues of rats and the GABAA and GABAB receptor transcription levels of hippocampal neurons.

ETHNOPHARMACOLOGICAL RELEVANCE: Jujuboside A (JuA) is a main active ingredient of semen ziziphi spinosae, which can significantly reduce spontaneous activity in mammals, increase the speed of falling asleep, prolong the sleeping time as well as improve the sleeping efficiency. In this study, the mechanism and the pathway of the sedative and hypnotic effect of JuA were investigated. MATERIALS AND METHODS: After being treated with JuA (in vitro), the rat׳s small intestine tissues cultures were used to stimulate the brain tissues. Then 27 cytokine levels were detected in the two kinds of tissue culture via liquid protein chip technology; In addition, the cultured hippocampal neurons of rat were treated with JuA, and γ-aminobutyric acid (GABA) receptor subunits (GABAAα1, GABAAα5, GABAAß1 and GABABR1) mRNAs were evaluated by Real-time PCR. RESULTS: The levels of IL-1α, MIP-1α, IL-1ß and IL-2 were reduced significantly after 3h of treating the small intestine tissues with JuA (200µl/ml), and the concentration change rates, in order, were -59.3%, -3.59%, -50.1% and -49.4%; these cytokines were transmitted to brain tissues 2h later, which could lead to significant levels of reduction of IL-1α, IFN-γ, IP-10 and TNF-α; the concentration change rates were -62.4%, -25.7%, -55.2% and -38.5%, respectively. Further, the intercellular communication network diagram was mapped out, which could suggest the mechanism and the pathway of the sedative and hypnotic effect of JuA. The results also indicated that JuA (50µl/ml) increased significantly GABAAα1 receptor mRNAs and reduced GABABR1, mRNAs in hippocampal neurons after 24h of stimulation; however, all the mRNA transcription levels of GABAAα1,GABAAα5, GABAAß1 and GABABR1 receptors increased significantly after 48h. CONCLUSION: JuA performed its specific sedative and hypnotic effect through not only adjusting GABA receptors subunit mRNAs expression, but also down-regulating the secretion of relevant inflammation cytokines on the intestinal mucosal system to affect the intercellular cytokine network between nerve cells in the brain. This mechanism is similar to that of melatonin.

Impaired GABAB receptor signaling dramatically up-regulates Kiss1 expression selectively in nonhypothalamic brain regions of adult but not prepubertal mice.

Kisspeptin, encoded by Kiss1, stimulates reproduction and is synthesized in the hypothalamic anteroventral periventricular and arcuate nuclei. Kiss1 is also expressed at lower levels in the medial amygdala (MeA) and bed nucleus of the stria terminalis (BNST), but the regulation and function of Kiss1 there is poorly understood. γ-Aminobutyric acid (GABA) also regulates reproduction, and female GABAB1 receptor knockout (KO) mice have compromised fertility. However, the interaction between GABAB receptors and Kiss1 neurons is unknown. Here, using double-label in situ hybridization, we first demonstrated that a majority of hypothalamic Kiss1 neurons coexpress GABAB1 subunit, a finding also confirmed for most MeA Kiss1 neurons. Yet, despite known reproductive impairments in GABAB1KO mice, Kiss1 expression in the anteroventral periventricular and arcuate nuclei, assessed by both in situ hybridization and real-time PCR, was identical between adult wild-type and GABAB1KO mice. Surprisingly, however, Kiss1 levels in the BNST and MeA, as well as the lateral septum (a region normally lacking Kiss1 expression), were dramatically increased in both GABAB1KO males and females. The increased Kiss1 levels in extrahypothalamic regions were not caused by elevated sex steroids (which can increase Kiss1 expression), because circulating estradiol and testosterone were equivalent between genotypes. Interestingly, increased Kiss1 expression was not detected in the MeA or BNST in prepubertal KO mice of either sex, indicating that the enhancements in extrahypothalamic Kiss1 levels initiate during/after puberty. These findings suggest that GABAB signaling may normally directly or indirectly inhibit Kiss1 expression, particularly in the BNST and MeA, and highlight the importance of studying kisspeptin populations outside the hypothalamus.

Biphasic effects of cannabinoids in anxiety responses: CB1 and GABA(B) receptors in the balance of GABAergic and glutamatergic neurotransmission.

Biphasic effects of cannabinoids have been shown in processes such as feeding behavior, motor activity, motivational processes and anxiety responses. Using two different tests for the characterization of anxiety-related behavior (elevated plus-maze and holeboard), we first identified in wild-type C57BL/6N mice, two doses of the synthetic CB1 cannabinoid receptor agonist CP-55,940 with anxiolytic (1 µg/kg) and anxiogenic properties (50 µg/kg), respectively. To clarify the role of CB1 receptors in this biphasic effect, both doses were applied to two different conditional CB1 receptor knockout (KO) mouse lines, GABA-CB1-KO (CB1 receptor inactivation in forebrain GABAergic neurons) and Glu-CB1-KO (CB1 receptor inactivation in cortical glutamatergic neurons). We found that the anxiolytic-like effects of the low dose of cannabinoids are mediated via the CB1 receptor on cortical glutamatergic terminals, because this anxiolytic-like response was abrogated only in Glu-CB1-KO mice. On the contrary, the CB1 receptor on the GABAergic terminals is required to induce an anxiogenic-like effect under a high-dose treatment because of the fact that this effect was abolished specifically in GABA-CB1-KO mice. These experiments were carried out in both sexes, and no differences occurred with the doses tested in the mutant mice. Interestingly, the positive allosteric modulation of GABA(B) receptor with GS-39783 was found to largely abrogate the anxiogenic-like effect of the high dose of CP-55,940. Our results shed new light in further understanding the biphasic effects of cannabinoids at the molecular level and, importantly, pave the way for the development of novel anxiolytic cannabinoid drugs, which may have favorable effect profiles targeting the CB1 receptor on glutamatergic terminals.

[Effects of electroacupuncture of "Futu" (LI 18), etc. on pain behavior and expression of GABA receptor subunit genes in cervical spinal cord in rats with thyroid regional pain].

OBJECTIVE: To observe the effect of electroacupuncture (EA) at different acupoints on the expression of gamma aminobutyric (GABA) receptor (R) subunit genes in the cervical spinal cord in rats with thyroid regional inflammatory pain so as to analyze its analgesic mechanism for thyroid surgery. METHODS: A total of 50 Wistar rats were randomized into control, model, Futu (LI 18), Hegu (LI 4)-Neiguan (PC 6, LI 4-PC 6), Zusanli (ST 36)-Yanglingquan (GB 34, ST 36-GB 34) groups, with 10 rats in each group. Thyroid regional pain model was established by subcutaneous injection of 2.5% formalin (100 microL). Ten minutes after modeling, EA (2 Hz/15 Hz, 1 mA) was applied to LI 18, LI 4-PC 6 and ST 36-GB 34 for 30 min, respectively. The animals' face-grooming (FG) times in 5 min and thermal pain threshold (paw withdrawal latency, PWL) were recorded. The expression of GABA(A) R, GABA(B) R1, and GABA(B) R2 genes in the cervical 1-3 segments of the spinal cord 80 min after modeling was detected by using reversed transcription-polymerase chain reaction (RT-PCR). Histological changes of the tissues of the thyroid region were observed by using H. E. staining. RESULTS: After subcutaneous injection of formalin, the animals' FG times in 5 min were increased considerably and the thermal pain threshold was decreased obviously in the model group (P < 0.05). Concomitantly, the expression levels of GABA(B) R1, and GABA(B) R2 genes of the cervical spinal cord were upregulated significantly in the model group (P < 0.05), and GABA(A) R expression was increased slightly (P > 0.05). Compared with the model group, the FG times in 5 min in the LI 18 and LI 4-PC 6 groups at 40 min and 70 min after modeling were decreased significantly (P < 0.05), and their thermal pain threshold values were increased markedly (P < 0.05). The expression levels of GABA(B) R1 mRNA, GABA(B) R2 mRNA and GABAA R mRNA were significantly higher in the LI 18 and LI 4-PC 6 groups than in the model group (P < 0.05). The expression level of GABA(B) R1 mRNA of the ST 36-GB 34 group was obviously higher than that of the model group (P < 0.05). The inflammatory cells in the formalin-injected thyroid region were relatively fewer in the LI 18 and LI 4-PC 6 groups than in the model group. CONCLUSION: Both EA of LI 18 and LI 4-PC 6 can significantly suppress formalin-injection induced pain reactions in the thyroid region, which may be closely associated with its effects in upregulating expression levels of cervico-spinal GABA(B) R1 mRNA, GABA(B) R2 mRNA and GABA(A) R mRNA, and reduce regional inflammatory reactions in the thyroid region.

In vitro and in vivo characterization of the novel GABAB receptor positive allosteric modulator, 2-{1-[2-(4-chlorophenyl)-5-methylpyrazolo[1,5-a]pyrimidin-7-yl]-2-piperidinyl}ethanol (CMPPE).

There is preclinical evidence supporting the finding that the GABA(B) receptor orthosteric agonist, baclofen, has significant effects on eating behavior suggesting the potential therapeutic application of this compound for the treatment of eating related disorders. However, the wide clinical use of baclofen might be limited by the appearance of sedative and motor impairment effects. The identification of positive allosteric modulators (PAMs) of GABA(B) receptors represents a novel therapeutic approach to reduce the centrally-mediated adverse effects typical of the GABA(B) receptor orthosteric agonist. In the present work, we report the in vitro profile of a novel chemical structure, 2-{1-[2-(4-chlorophenyl)-5-methylpyrazolo[1,5-a]pyrimidin-7-yl]-2-piperidinyl}ethanol (CMPPE) identified by screening the GSK compound collection. CMPPE potentiates GABA-stimulated [(35)S]GTPγS binding to membranes of human recombinant cell line and of rat brain cortex. GABA concentration-response curves (CRC) in the presence of fixed concentrations of CMPPE, in rat native tissue, revealed an increase of both the potency and maximal efficacy of GABA. A similar modulatory effect was observed in GABA(B) receptor-mediated activation of inwardly rectifying potassium channels in hippocampal neurons. CMPPE (30-100 mg/kg) and GS39783 (100 mg/kg) significantly decreased food consumption in rat without impairment on the animal locomotor activity. On the contrary, baclofen (2.5 mg/kg) decreased both food intake and motor performance. All together these findings confirm the role of GABA(B) system in controlling animal food intake and for the first time demonstrate that GABA(B) receptor PAMs may represent a novel pharmacological approach to treat eating disorders without unwanted sedative effects.

[Rat orbital frontal (orbital frontal cortex, OFC) GABA B receptor mechanisms in stress and depression].

Stress-induced depression is a kind of functional and structural disability of the brain and involves many neurotransmitters and regions of the brain. A number of studies suggest involvement of γ-Aminobutyric acid (GABA) in the orbital frontal cortex (OFC) in the mechanism of stress-associated depression-like behavior in rodents. However, little work has been done on the relationship between GABA and neural plasticity of the OFC under stress. Here we examine the effect of the GABA in the OFC during acute forced swim stress (FSS). We found remarkable depression-like behavior in FSS and an open field test (OFT), and we observed a marked decrease in Kalirin-7 expression and the basal dendritic spine density of layer V pyramidal neurons in OFC after FSS. GABA administration reversed these changes, which were inhibited by CGP35348, an antagonist of GABA-B receptors. These results suggest an anti-depression effect of GABA in the OFC, which may be mediated by GABA-B receptor. The anti-depression effect of GABA is related to the plasticity of the dendritic spine density. This discovery may be helpful in the development of new therapies to treat depression.

Decreased GABAB receptor function in the cerebellum and brain stem of hypoxic neonatal rats: role of glucose, oxygen and epinephrine resuscitation.

BACKGROUND: Hypoxia during the first week of life can induce neuronal death in vulnerable brain regions usually associated with an impairment of cognitive function that can be detected later in life. The neurobiological changes mediated through neurotransmitters and other signaling molecules associated with neonatal hypoxia are an important aspect in establishing a proper neonatal care. METHODS: The present study evaluated total GABA, GABAB receptor alterations, gene expression changes in GABAB receptor and glutamate decarboxylase in the cerebellum and brain stem of hypoxic neonatal rats and the resuscitation groups with glucose, oxygen and epinephrine. Radiolabelled GABA and baclofen were used for receptor studies of GABA and GABAB receptors respectively and Real Time PCR analysis using specific probes for GABAB receptor and GAD mRNA was done for gene expression studies. RESULTS: The adaptive response of the body to hypoxic stress resulted in a reduction in total GABA and GABAB receptors along with decreased GABAB receptor and GAD gene expression in the cerebellum and brain stem. Hypoxic rats supplemented with glucose alone and with oxygen showed a reversal of the receptor alterations and changes in GAD. Resuscitation with oxygen alone and epinephrine was less effective in reversing the receptor alterations. CONCLUSIONS: Being a source of immediate energy, glucose can reduce the ATP-depletion-induced changes in GABA and oxygenation, which helps in encountering hypoxia. The present study suggests that reduction in the GABAB receptors functional regulation during hypoxia plays an important role in central nervous system damage. Resuscitation with glucose alone and glucose and oxygen to hypoxic neonatal rats helps in protecting the brain from severe hypoxic damage.

Distinct α subunit variations of the hypothalamic GABAA receptor triplets (αßγ) are linked to hibernating state in hamsters.

BACKGROUND: The structural arrangement of the γ-aminobutyric acid type A receptor (GABAAR) is known to be crucial for the maintenance of cerebral-dependent homeostatic mechanisms during the promotion of highly adaptive neurophysiological events of the permissive hibernating rodent, i.e the Syrian golden hamster. In this study, in vitro quantitative autoradiography and in situ hybridization were assessed in major hypothalamic nuclei. Reverse Transcription Reaction-Polymerase chain reaction (RT-PCR) tests were performed for specific GABAAR receptor subunit gene primers synthases of non-hibernating (NHIB) and hibernating (HIB) hamsters. Attempts were made to identify the type of αßγ subunit combinations operating during the switching ON/OFF of neuronal activities in some hypothalamic nuclei of hibernators. RESULTS: Both autoradiography and molecular analysis supplied distinct expression patterns of all α subunits considered as shown by a strong (p < 0.01) prevalence of α1 ratio (over total α subunits considered in the present study) in the medial preoptic area (MPOA) and arcuate nucleus (Arc) of NHIBs with respect to HIBs. At the same time α2 subunit levels proved to be typical of periventricular nucleus (Pe) and Arc of HIB, while strong α4 expression levels were detected during awakening state in the key circadian hypothalamic station, i.e. the suprachiasmatic nucleus (Sch; 60%). Regarding the other two subunits (ß and γ), elevated ß3 and γ3 mRNAs levels mostly characterized MPOA of HIBs, while prevalently elevated expression concentrations of the same subunits were also typical of Sch, even though this time during the awakening state. In the case of Arc, notably elevated levels were obtained for ß3 and γ2 during hibernating conditions. CONCLUSION: We conclude that different αßγ subunits are operating as major elements either at the onset of torpor or during induction of the arousal state in the Syrian golden hamster. The identification of a brain regional distribution pattern of distinct GABAAR subunit combinations may prove to be very useful for highlighting GABAergic mechanisms functioning at least during the different physiological states of hibernators and this may have interesting therapeutic bearings on neurological sleeping disorders.

The circuitry of atypical absence seizures in GABA(B)R1a transgenic mice.

The objective of the current study was to determine the origin of the slow spike and wave discharges (SSWD) in the transgenic mouse with postnatal over-expression of the GABA(B) receptor subunit R1a (GABA(B)R1a(tg)), a mutant animal with a characteristic phenotype consisting of atypical absence seizures and cognitive dysfunction. Using simultaneous electrocorticographic (ECoG) recordings from cortical and depth electrodes in freely moving GABA(B)R1a(tg) mice, we showed that the SSWD in this model of atypical absence seizures arise exclusively from midline thalamus (MT), reticular nucleus of the thalamus (nRT), and the CA1 region of the hippocampus. Lesioning of the MT and nRT with ibotenic acid abolished SSWD. Microinjection of the GABA(B) receptor agonist, (-) baclofen, into MT and nRT exacerbated, and the GABA(B)R antagonist, CGP 35348 abolished, SSWD in the GABA(B)R1a(tg) mice. These data suggest that the nRT and MT are necessary for the generation of SSWD in the GABA(B)R1a(tg) model of atypical absence seizures, and indicate that GABA(B)R-mediated mechanisms within thalamus are necessary for the genesis of SSWD in atypical absence seizures. A putative cortico-thalamo-hippocampal circuit is proposed to explain the unique electrographic findings, ictal behavior, pharmacology, and impairment of cognition that characterize atypical absence seizures.

Ischaemia differentially regulates GABA(B) receptor subunits in organotypic hippocampal slice cultures.

Reduced synaptic inhibition due to dysfunction of ionotropic GABA(A) receptors has been proposed as one factor in cerebral ischaemia-induced excitotoxic cell death. However, the participation of the inhibitory metabotropic GABA(B) receptors in these pathological processes has not been extensively investigated. We used oxygen-glucose deprivation (OGD) and NMDA-induced excitotoxicity as models to investigate whether ischaemia-like challenges alter the protein levels of GABA(B1) and GABA(B2) receptor subunits in rat organotypic hippocampal slice cultures. Twenty-four hours after the insult both OGD and NMDA produced a marked decrease in the total levels of GABA(B2) (approximately 75%), while there was no significant change in the levels of GABA(B1) after OGD, but an increase after NMDA treatment (approximately 100%). The GABA(B) receptor agonist baclofen (100 microM) was neuroprotective following OGD or NMDA treatment if added before or during the insult. GABA(B) receptors comprise heterodimers of GABA(B1) and GABA(B2) subunits and our results suggest that the separate subunits are independently regulated in response to extreme neuronal stress. However, because GABA(B2) is required for functional surface expression, down-regulation of this subunit removes an important inhibitory feedback mechanism under pathological conditions.

Increased GABA B receptor subtype expression in the nucleus of the solitary tract of the spontaneously hypertensive rat.

Expression of GABA(B) receptor messenger RNA (mRNA) in the central nervous system was compared between the spontaneously hypertensive (SHR) and normotensive Wistar Kyoto (WKY) rat. Polymerase chain reaction (PCR) revealed all the isoforms except B1e in cortex, hypothalamus, and medulla oblongata. In the nucleus of the solitary tract (NTS) and ventrolateral medulla (VLM), the B1a-c and 1 g isoforms were present as well as B2. Real-time PCR detected significantly higher levels of B1a (p < 0.01) and B2 (p < 0.05) mRNA in the NTS of SHR compared to WKY. A significant increase in B1a expression (p < 0.05) was detected in VLM. Immunolabeling suggested presynaptic and postsynaptic expression of B1a, B1b, and B2 subtypes throughout the NTS, with significant differences in distribution patterns and labeling between subtypes and between SHR and WKY. These findings suggest that GABA(B) receptors expressed by neurones in NTS may be involved in cardiovascular regulation and that changes in GABA(B) mRNA expression levels may contribute to the hypertensive state in SHR.

Direct detection of the interaction between recombinant soluble extracellular regions in the heterodimeric metabotropic gamma-aminobutyric acid receptor.

The gamma-aminobutyric acid, type B (GABAB) receptor is a heterodimeric receptor consisting of two complementary subunits, GABAB1 receptor (GBR1) and GABAB2 receptor (GBR2). GBR1 is responsible for GABA binding, whereas GBR2 is considered to perform a critical role in signal transduction toward downstream targets. Therefore, precise communication between GBR1 and GBR2 is thought to be essential for the proper signal transduction process. However, biochemical data describing the interaction of the two subunits, especially for the extracellular regions, are not sufficient. Thus we began by developing a protein expression system of the soluble extracellular regions. One of the soluble recombinant GBR1 proteins exhibited a ligand binding ability, which is similar to that of the full-length GBR1, and thus the ligand-binding domain was determined. Direct interaction between GBR1 and GBR2 extracellular soluble fragments was confirmed by co-expression followed by affinity column chromatography and a sucrose density gradient sedimentation. In addition, we also found homo-oligomeric states of these soluble extracellular regions. The interaction between the two soluble extracellular regions caused the enhancement of the agonist affinity for GBR1 as previously reported in a cell-based assay. These results not only open the way to future structural studies but also highlight the role of the interaction between the extracellular regions, which controls agonist affinity to the heterodimeric receptor.