Gamma-aminobutyric Acid Responses Research Articles

Heterogeneous subpopulations of GABAAR-responding neurons coexist across neuronal network scales and developmental stages in health and disease

Gamma-aminobutyric acid (GABA) is the main inhibitory neurotransmitter in adults. Depolarizing GABA responses have been well characterized at neuronal-population average level during typical neurodevelopment and partially in brain disorders. However, no investigation has specifically assessed whether a mosaicism of cells with either depolarizing or hyperpolarizing/inhibitory GABAergic responses exists in animals in health/disease at diverse developmental stages, including adulthood. Here, we showed that such mosaicism is present in wild-type (WT) and down syndrome (DS) neuronal networks, as assessed at increasing scales of complexity (cultures, brain slices, behaving mice). Nevertheless, WT mice presented a much lower percentage of cells with depolarizing GABA than DS mice. Restoring the mosaicism of hyperpolarizing and depolarizing GABA-responding neurons to WT levels rescued anxiety behavior in DS mice. Moreover, we found heterogeneous GABAergic responses in developed control and trisomic human induced-pluripotent-stem-cells-derived neurons. Thus, a heterogeneous subpopulation of GABA-responding cells exists in physiological/pathological conditions in mouse and human neurons, possibly contributing to disease-associated behaviors.

GABA and glutamate response to social processing: a functional MRS feasibility study.

Several studies have suggested that atypical social processing in neurodevelopmental conditions (e.g. autism) is associated with differences in excitation and inhibition, through changes in the levels of glutamate and gamma-aminobutyric acid (GABA). While associations between baseline metabolite levels and behaviours can be insightful, assessing the neurometabolic response of GABA and glutamate during social processing may explain altered neurochemical function in more depth. Thus far, there have been no attempts to determine whether changes in metabolite levels are detectable using functional MRS (fMRS) during social processing in a control population. We performed Mescher-Garwood point resolved spectroscopy edited fMRS to measure the dynamic response of GABA and glutamate in the superior temporal sulcus (STS) and visual cortex (V1) while viewing social stimuli, using a design that allows for analysis in both block and event-related approaches. Sliding window analyses were used to investigate GABA and glutamate dynamics at higher temporal resolution. The changes of GABA and glutamate levels with social stimulus were largely non-significant. A small decrease in GABA levels was observed during social stimulus presentation in V1, but no change was observed in STS. Conversely, non-social stimulus elicited changes in both GABA and glutamate levels in both regions. Our findings suggest that the current experimental design primarily captures effects of visual stimulation, not social processing. Here, we discuss the feasibility of using fMRS analysis approaches to assess changes in metabolite response.

Dynamical changes in the expression of GABAergic and purinergic components occur during the polarization of THP-1 monocytes to proinflammatory macrophages

The monocytes are key components of innate immunity, as they can differentiate into phagocytic cells or macrophages with proinflammatory or anti-inflammatory phenotypes. The gamma-aminobutyric acid (GABA) and adenosine triphosphate (ATP), two known neurotransmitters, are two environmental signals that contribute to the differentiation of monocytes into macrophages and their subsequent polarization into proinflammatory M1 and anti-inflammatory M2 macrophages. Although monocytes and macrophages express proteins related to GABA and ATP-mediated response (GABAergic and purinergic systems, respectively), it is unknown whether changes in their expression occur during monocyte activation or their differentiation and polarization into macrophages. Therefore, we evaluated the expression levels of GABAergic and purinergic signaling components in the THP-1 monocyte cell line and their changes during monocyte activation, differentiation, and polarization to M1 proinflammatory macrophages. Our results showed that activated monocytes are characterized by increased expression of two GABAergic components, the GABA transporter 2 (GAT-2) and the glutamic acid decarboxylase (GAD)-67, an enzyme involved in GABA synthesis. Also, monocytes showed a pronounced expression of the purinergic receptors P2X4 and P2X7. Interestingly, during differentiation, monocytes increased the expression of the β2 subunit of GABA A-type receptor (GABA-AR), while the purinergic receptors P2X1 and P2X1del were reduced. In contrast, proinflammatory M1 macrophages showed a reduced expression in the α4 subunit of GABA-AR and GAD67, while P2X4 and P2X7 were overexpressed. These results indicate that dynamical changes in the GABAergic and purinergic components occur during the transition from monocytes to macrophages. Since GABA and ATP are two neurotransmitters, our results suggest that monocytes and macrophages respond to neurotransmitter-induced stimulation and may represent a path of interaction between the nervous and immune systems during peripheral inflammation and neuroinflammation development.

Comprehensive transcriptional and metabolomic analysis reveals the neuroprotective mechanism of dietary gamma-aminobutyric acid response to hypoxic stress in the Chinese mitten crab (Eriocheir sinensis)

Hypoxia is one of the serious stress challenges that aquatic animals face throughout their life. Our previous study found that hypoxia stress could induce neural excitotoxicity and neuronal apoptosis in Eriocheir sinensis, and observed that gamma-aminobutyric acid (GABA) has a positive neuroprotective effect on juvenile crabs under hypoxia. To reveal the neuroprotective pathway and metabolic regulatory mechanism of GABA in E. sinensis exposed to hypoxia stress, an 8-week feeding trial and acute hypoxia challenge were performed. Subsequently, we performed a comprehensive transcriptomic and metabolomic analysis of the thoracic ganglia of juvenile crabs. Differential genes and differential metabolites were co-annotated to 11 KEGG pathways, and further significant analysis showed that only the sphingolipid signaling pathway and the arachidonic acid metabolism pathway were significantly enriched. In the sphingolipid signaling pathway, GABA treatment significantly increased long-chain ceramide content in thoracic ganglia, which exerted neuroprotective effects by activating downstream signals to inhibit hypoxia-induced apoptosis. Moreover, in the arachidonic acid metabolism pathway, GABA could increase the content of neuroprotective active substances and reduce the content of harmful metabolites by regulating the metabolism of arachidonic acid for inflammatory regulation and neuroprotection. Furthermore, the decrease of glucose and lactate levels in the hemolymph suggests the positive role of GABA in metabolic regulation. This study reveals the neuroprotective pathways and possible mechanisms of GABA in juvenile E. sinensis exposed to hypoxia stress and inspires the discovery of new targets for improving hypoxia tolerance in aquatic animals.

Consolidation and maintenance of long-term memory involve dual functions of the developmental regulator Apterous in clock neurons and mushroom bodies in the Drosophila brain.

Memory is initially labile but can be consolidated into stable long-term memory (LTM) that is stored in the brain for extended periods. Despite recent progress, the molecular and cellular mechanisms underlying the intriguing neurobiological processes of LTM remain incompletely understood. Using the Drosophila courtship conditioning assay as a memory paradigm, here, we show that the LIM homeodomain (LIM-HD) transcription factor Apterous (Ap), which is known to regulate various developmental events, is required for both the consolidation and maintenance of LTM. Interestingly, Ap is involved in these 2 memory processes through distinct mechanisms in different neuronal subsets in the adult brain. Ap and its cofactor Chip (Chi) are indispensable for LTM maintenance in the Drosophila memory center, the mushroom bodies (MBs). On the other hand, Ap plays a crucial role in memory consolidation in a Chi-independent manner in pigment dispersing factor (Pdf)-containing large ventral–lateral clock neurons (l-LNvs) that modulate behavioral arousal and sleep. Since disrupted neurotransmission and electrical silencing in clock neurons impair memory consolidation, Ap is suggested to contribute to the stabilization of memory by ensuring the excitability of l-LNvs. Indeed, ex vivo imaging revealed that a reduced function of Ap, but not Chi, results in exaggerated Cl− responses to the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) in l-LNvs, indicating that wild-type (WT) Ap maintains high l-LNv excitability by suppressing the GABA response. Consistently, enhancing the excitability of l-LNvs by knocking down GABAA receptors compensates for the impaired memory consolidation in ap null mutants. Overall, our results revealed unique dual functions of the developmental regulator Ap for LTM consolidation in clock neurons and LTM maintenance in MBs.

Dietary GABA enhances hypoxia tolerance of a bottom-dwelling carp, Cirrhinus mrigala by modulating HIF-1α, thyroid hormones and metabolic responses.

The Indian major carp, mrigal (Cirrhinus mrigala), is a bottom-dwelling fish that can survive hypoxic episodes in its natural environment. We hypothesise that it can better survive hypoxic conditions by altering metabolic responses through GABA (Gamma-aminobutyric acid) supplementation. In the first experiment, the hypoxia tolerance time of the fishes was evaluated under extreme anoxic conditions after feeding with GABA, which showed that GABA had improved survival time under hypoxia. To study the response of dietary GABA in hypoxia-exposed fish, the branchial HIF-1α expression levels, serum thyroid hormone levels and hepatic metabolic responses were assessed in the subsequent experiment. The treatment groups were fed for 60 days with experimental diets containing 4 levels of GABA (0.00% G, 0.50% G, 0.75% G and 1.0%G) and were subjected to 72-h hypoxia exposure (0.5 ± 0.02 mg L-1 dissolved oxygen (DO)) whereas a control group was maintained under normoxic conditions (6.0 ± 0.21 mg L-1 DO). The five treatment groups with three replicates were C0 (0% G + normoxia), H0 (0% G + hypoxia), H0.5 (0.50% G + hypoxia), H0.75 (0.75% G + hypoxia) and H1.0 (1.00% G + hypoxia). The results indicated that GABA supplementation triggered downregulation of HIF 1 alpha expression. When compared with the control group, decreased thyroxine (T4) and triiodothyronine (T3) levels were observed in the GABA-fed hypoxic groups. However, TSH (thyroid stimulating hormone) level remained unchanged in all the treatments. The LDH (lactate dehydrogenase) level in hypoxia-exposed groups was decreased by GABA supplementation. Our study demonstrated that GABA supplementation restores acute hypoxia-induced HIF-1α expression, thyroid hormone levels and LDH activities. On the other hand, it enhanced the citrate synthase (CS) activities at 0.5-1.00%, which showed a sharp decline in hypoxia. Hypoxia caused increase in the serum metabolites such as glucose, lactate, cholesterol and triglycerides. However, GABA supplementation was partially effective in reducing glucose and lactate level while triglycerides and cholesterol values remained unchanged. Overall, our results suggested a potential role of GABA in suppressing metabolism during hypoxia exposure, which can increase the chances of survival of the species Cirrhinus mrigala during hypoxia.

Tolerance potential of Chilo suppressalis larvae to fipronil exposure via the modulation of detoxification and GABA responses

The Asiatic rice borer, Chilo suppressalis Walker, is one of the most destructive rice pests in the world; its physiological adaptability to the sustained application of fipronil was investigated. After topical application, fipronil showed strong lethal effect with LD20 and LD80 values on the fourth instar larvae as 0.00159 and 0.00906μg per larvae at 48h- post treatment, and also revealed slight inhibition on the eclosion of pupae and the hatching of eggs. LD20 dosage of fipronil significantly enhanced the activities of carboxylesterase (CarE), glutathione-S-transferase (GST) and cytochrome P450-linked p-nitro O-demethylase (PNOD) in the borers. LD80 dosage of fipronil showed a mild decrease on the activity of CarE, GST and PNOD, and a slight suppression on the activity of glutamate dehydrogenase (GDH), but strongly induced the transform of glutamate to gamma-aminobutyric acid (GABA) and the activity of glutamate decarboxylase (GAD), thereby improved GABA level in the bores. Results suggested that the borer possessed positive tolerance potential to firponil via the modulation of detoxification and GABA responses and provided the basis for further studies on reducing the tolerance of the borer in the control strategies.

Acute inflammation reveals GABAA receptor-mediated nociception in mouse dorsal root ganglion neurons via PGE2 receptor 4 signaling.

Gamma‐aminobutyric acid (GABA) depolarizes dorsal root ganglia (DRG) primary afferent neurons through activation of Cl− permeable GABAA receptors but the physiologic role of GABAA receptors in the peripheral terminals of DRG neurons remains unclear. In this study, we investigated the role of peripheral GABAA receptors in nociception using a mouse model of acute inflammation. In vivo, peripheral administration of the selective GABAA receptor agonist muscimol evoked spontaneous licking behavior, as well as spinal wide dynamic range (WDR) neuron firing, after pre‐conditioning with formalin but had no effect in saline‐treated mice. GABAA receptor‐mediated pain behavior after acute formalin treatment was abolished by the GABAA receptor blocker picrotoxin and cyclooxygenase inhibitor indomethacin. In addition, treatment with prostaglandin E2 (PGE 2) was sufficient to reveal muscimol‐induced licking behavior. In vitro, GABA induced sub‐threshold depolarization in DRG neurons through GABAA receptor activation. Both formalin and PGE 2 potentiated GABA‐induced Ca2+ transients and membrane depolarization in capsaicin‐sensitive nociceptive DRG neurons; these effects were blocked by the prostaglandin E2 receptor 4 (EP4) antagonist AH23848 (10 μmol/L). Furthermore, potentiation of GABA responses by PGE 2 was prevented by the selective Nav1.8 antagonist A887826 (100 nmol/L). Although the function of the Na+‐K+‐2Cl‐ co‐transporter NKCC1 was required to maintain the Cl‐ ion gradient in isolated DRG neurons, NKCC1 was not required for GABAA receptor‐mediated nociceptive behavior after acute inflammation. Taken together, these results demonstrate that GABAA receptors may contribute to the excitation of peripheral sensory neurons in inflammation through a combined effect involving PGE 2‐EP4 signaling and Na+ channel sensitization.

Comparative proteomic analysis of gamma-aminobutyric acid responses in hypoxia-treated and untreated melon roots

Hypoxia is one of the main environmental stresses that accounts for decreasing crop yield. To further investigate the mechanisms whereby exogenous GABA alleviates hypoxia injury to melon seedlings, a comparative proteomic analysis was performed using roots subjected to normal aeration and hypoxia conditions with or without GABA (5mM). The results indicated that protein spots on gels after hypoxia and hypoxia+GABA treatment were significantly changed. Three “matched sets” were analyzed from four treatments, and 13 protein spots with large significant differences in expression were identified by MALDI-TOF/TOF mass spectrometry. Exogenous GABA treatment enhanced the expression of protein in cytosolic phosphoglycerate kinase 1, exaA2 gene product, dnaJ and myb-like DNA-binding domain-containing proteins, as well as elongation factor-1 alpha and hypothetical proteins in hypoxia-induced roots. However, the hypoxia+GABA treated roots had a significantly lower expression of proteins including malate dehydrogenase, nucleoside diphosphate kinase, disease resistance-like protein, disulfide isomerase, actin, ferrodoxin NADP oxidoreductase, glutathione transferase, netting associated peroxidase. This paper describes the effect of GABA on melon plants under hypoxia-induced stress using proteomics, and supports the alleviating function of GABA in melon plants grown under hypoxic conditions.

Enhanced GABA action on the substantia gelatinosa neurons of the medullary dorsal horn in the offspring of streptozotocin-injected mice

Peripheral neuropathy is a frequent complication of diabetes mellitus and a common symptom of neuropathic pain, the mechanism of which is complex and involves both peripheral and central components of the sensory system. The lamina II of the medullary dorsal horn, called the substantia gelatinosa (SG), is well known to be a critical site for processing of orofacial nociceptive information. Although there have been a number of studies done on diabetic neuropathy related to the orofacial region, the action of neurotransmitter receptors on SG neurons in the diabetic state is not yet fully understood. Therefore, we used the whole-cell patch clamp technique to investigate this alteration on SG neurons in both streptozotocin (STZ)-induced diabetic mice and offspring from diabetic female mice. STZ (200mg/kg)-injected mice showed a small decrease in body weight and a significant increase in blood glucose level when compared with their respective control group. However, application of different concentrations of glycine, gamma-aminobutyric acid (GABA) and glutamate on SG neurons from STZ-injected mice did not induce any significant differences in inward currents when compared to their control counterparts. On the other hand, the offspring of diabetic female mice (induced by multiple injections of STZ (40mg/kg) for 5 consecutive days) led to a significant decrease in both body weight and blood glucose level compared to the control offspring. Glycine and glutamate responses in the SG neurons of the offspring from diabetic female mice were similar to those of control offspring. However, the GABA response in SG neurons of offspring from diabetic female mice was greater than that of control offspring. Furthermore, the GABA-mediated responses in offspring from diabetic and control mice were examined at different concentrations ranging from 3 to 1,000μM. At each concentration, the GABA-induced mean inward currents in the SG neurons of offspring from diabetic female mice were larger than those of control mice. These results demonstrate that SG neurons in offspring from diabetic mice are more sensitive to GABA compared to control mice, suggesting that GABA sensitivity may alter orofacial pain processing in offspring from diabetic female mice.

An evolutionarily conserved switch in response to GABA affects development and behavior of the locomotor circuit of Caenorhabditis elegans.

The neurotransmitter gamma-aminobutyric acid (GABA) is depolarizing in the developing vertebrate brain, but in older animals switches to hyperpolarizing and becomes the major inhibitory neurotransmitter in adults. We discovered a similar developmental switch in GABA response in Caenorhabditis elegans and have genetically analyzed its mechanism and function in a well-defined circuit. Worm GABA neurons innervate body wall muscles to control locomotion. Activation of GABAA receptors with their agonist muscimol in newly hatched first larval (L1) stage animals excites muscle contraction and thus is depolarizing. At the mid-L1 stage, as the GABAergic neurons rewire onto their mature muscle targets, muscimol shifts to relaxing muscles and thus has switched to hyperpolarizing. This muscimol response switch depends on chloride transporters in the muscles analogous to those that control GABA response in mammalian neurons: the chloride accumulator sodium-potassium-chloride-cotransporter-1 (NKCC-1) is required for the early depolarizing muscimol response, while the two chloride extruders potassium-chloride-cotransporter-2 (KCC-2) and anion-bicarbonate-transporter-1 (ABTS-1) are required for the later hyperpolarizing response. Using mutations that disrupt GABA signaling, we found that neural circuit development still proceeds to completion but with an ∼6-hr delay. Using optogenetic activation of GABAergic neurons, we found that endogenous GABAA signaling in early L1 animals, although presumably depolarizing, does not cause an excitatory response. Thus a developmental depolarizing-to-hyperpolarizing shift is an ancient conserved feature of GABA signaling, but existing theories for why this shift occurs appear inadequate to explain its function upon rigorous genetic analysis of a well-defined neural circuit.

Eugenol inhibits the GABAA current in trigeminal ganglion neurons.

Eugenol has sedative, antioxidant, anti-inflammatory, and analgesic effects, but also serves as an irritant through the regulation of a different set of ion channels. Activation of gamma aminobutyric acid (GABA) receptors on sensory neurons leads to the stabilization of neuronal excitability but contributes to formalin-induced inflammatory pain. In this study, we examined the effect of eugenol on the GABA-induced current in rat trigeminal ganglia (TG) neurons and in human embryonic kidney (HEK) 293 cells expressing the GABAA receptor α1β2γ2 subtype using the whole-cell patch clamp technique. RT-PCR and Western blot analysis were used to confirm the expression of GABAA receptor γ2 subunit mRNA and protein in the TG and hippocampus. Eugenol decreased the amplitude ratio of the GABA-induced current to 27.5 ± 3.2% (p < 0.05) in TG neurons, which recovered after a 3-min washout. In HEK 293 cells expressing the α1β2γ2 subtype, eugenol inhibited GABA-induced currents in a dose-dependent manner. Application of eugenol also decreased the GABA response in the presence of a G-protein blocker. Eugenol pretreatment with different concentrations of GABA resulted in similar inhibition of the GABA-induced current in a non-competitive manner. In conclusion, eugenol inhibits the GABA-induced current in TG neurons and HEK 293 cells expressing the GABAA receptor in a reversible, dose-dependent, and non-competitive manner, but not via the G-protein pathway. We suggest that the GABAA receptor could be a molecular target for eugenol in the modulation of nociceptive information.

Sweetened-Fat Intake Sensitizes Gamma-Aminobutyric Acid—Mediated Feeding Responses Elicited from the Nucleus Accumbens Shell

There is much interest in exploring whether reward-driven feeding can produce druglike plasticity in the brain. The gamma-aminobutyric acid (GABA) system in the nucleus accumbens (Acb) shell, which modulates hypothalamic feeding systems, is well placed to "usurp" homeostatic control of feeding. Nevertheless, it is unknown whether feeding-induced neuroadaptations occur in this system. Separate groups of ad libitum-maintained rats were exposed to daily bouts of sweetened-fat intake, predator stress, or intra-Acb shell infusions of either d-amphetamine (2 or 10 μg) or the μ-opioid agonist D-[Ala2, N-MePhe4, Gly-ol]-enkephalin (DAMGO, 2.5 μg), then challenged with intra-Acb shell infusion of the GABAA agonist, muscimol (10 ng). Exposure to sweetened fat robustly sensitized muscimol-induced feeding. Sensitization was present 1 week after cessation of the palatable feeding regimen but had abated by 2 weeks. Rats exposed to sweetened fat did not show an altered feeding response to food deprivation. Repeated intra-Acb shell infusions of DAMGO (2.5 μg) also sensitized intra-Acb shell muscimol-driven feeding. However, neither repeated intra-Acb shell d-amphetamine infusions (2 or 10 μg) nor intermittent exposure to an aversive stimulus (predator stress) altered sensitivity to muscimol. Palatable feeding engenders hypersensitivity of Acb shell GABA responses; this effect may involve feeding-induced release of opioid peptides. Heightened arousal, aversive experiences, or increased catecholamine transmission alone are insufficient to produce the effect, and a hunger-induced feeding drive is insufficient to reveal the effect. These findings reveal a novel type of food-induced neuroadaptation within the Acb; possible implications for understanding crossover effects between food reward and drug reward are discussed.

Noradrenergic Control of Neuronal Firing in Cerebellar Nuclei: Modulation of GABA Responses

The effects of noradrenaline (NA) on inhibitory responses to gamma aminobutyric acid (GABA) in neurones of the deep cerebellar nuclei were studied in vivo in rats, using extracellular single-unit recordings and microiontophoretic drug application. NA application altered GABA-evoked responses in 95% of the neurones tested, but the effects differed between nuclei. Application of NA depressed GABA responses in the medial (MN) and posterior interpositus (PIN) nuclei, but enhanced GABA responses in the anterior interpositus nucleus (AIN). Comparable proportions of enhancing (57%) and depressive (43%) effects were found in the lateral nucleus (LN). The alpha2 noradrenergic receptor agonist clonidine mimicked the depressive effect of NA on GABA responses in MN and PIN and its enhancing effects in AIN and LN, while the alpha2 antagonist yohimbine partially blocked these effects. The beta-adrenergic agonist isoproterenol and antagonist timolol respectively induced and partially blocked enhancements of GABA responses in all nuclei except for LN, where isoproterenol had a weak depressive effect. It is concluded that NA modulates GABA responses by acting on both alpha2 and beta receptors. Activation of these receptors appears to be synergistic in the AIN and opposite in the remaining deep nuclei. These results support the hypothesis that the noradrenergic system participates in all the regulatory functions involving the cerebellum in a specific and differential manner, and suggest that any change in NA content, as commonly observed in ageing or stress, could influence cerebellar activity.

The anesthetic urethane blocks excitatory amino acid responses but not GABA responses in isolated frog spinal cords

The anesthetic urethane is commonly used in physiological experiments. We tested urethane's actions on GABA receptors on the primary afferents in the spinal cord, which are one of the few areas in the adult central nervous system (CNS) that are depolarized by GABA, and on ligand-gated excitatory amino acid (EAA) receptors located on motoneurons. Both receptor types are critically important during anesthetic immobilization. We used the isolated hemisected spinal cord of the frog in a sucrose gap chamber to record glutamate-, N-methyl-D-aspartate (NMDA)-, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-, kainate-, and gamma-aminobutyric acid (GABA)-induced depolarizations of the dorsal root (DR) and ventral root (VR). DR potentials (DRPs) and VR potentials (VRPs) evoked by single supramaximal afferent stimuli were also studied. Urethane (10-80 mM) was applied for 10-30 min. Urethane depressed EAA responses on the motoneurons in a dose-dependent manner. At a clinical anesthetic concentration (10 mM), EAA-induced depolarizations were reduced by 8.1 ± 2.2 % (n = 7, P = 0.025), but increasing the concentration to 40 mM revealed a larger, 24.7 ± 3 % (n = 53, P = 0.0001) depressing effect of urethane on all EAA responses in the motoneurons. However, GABA and K(+) responses recorded in the DR were not altered by the presence of 10 or 40 mM urethane. Evoked DRPs and VRPs were reduced by urethane and spontaneous DR and VR potentials were suppressed by 10 or blocked by 40 mM urethane. Urethane appears to be selective for EAA-, sparing GABA responses at a clinical anesthetic concentration. Only a 10 % reduction of EAA activity seems to be necessary to induce anesthesia.

Hypothermic effect of GABA in conscious stressed rats: its modification by cholinergic agonists and antagonists.

gamma-Aminobutyric acid (GABA) intraperitoneally injected (i.p.) produced a dose-dependent hypothermia in restrained rats. GABA-induced hypothermia (1000 mg kg-1) was antagonized by pretreatment with atropine (2.5 and 10 mg kg-1 i.p.), hyoscine butylbromide (2.5 mg kg-1 i.p.), hexamethonium (0.75 mg kg-1 i.p.) or physostigmine (0.2 mg kg-1 s.c.). Hexamethonium (7.5 mg kg-1 i.p.) did not influence the hypothermia induced by GABA. The antagonism by physostigmine of GABA-induced hypothermia was attenuated by pretreatment of the rats with either alpha-methyl-p-tyrosine (200 mg kg-1 i.p.) or hexamethonium (7.5 mg kg-1 i.p.), but it was potentiated by either atropine (5 mg kg-1 i.p.) or hexamethonium (0.75 mg kg-1 i.p.). The data indicate that GABA-induced hypothermia may be partly mediated by acetylcholine release. Muscarinic receptors may play an important role in the effect of GABA. The results support the hypothesis of nicotinic presynaptic receptors modulating noradrenergic nerve endings that play a part in the hypothermic response of GABA.

GABAA Receptors Mediate Excitation in Adult Rat GnRH Neurons1

Gonadotropin-releasing hormone (GnRH) neurons form the final common pathway for the central regulation of reproduction. Gamma-amino butyric acid (GABA), the main inhibitory neurotransmitter in the adult brain, has long been implicated in playing key roles in the regulation of GnRH neurons. Two groups reported recently that GABA depolarizes GnRH neurons, although one group reported a hyperpolarizing action of GABA. In this study, we investigated the GABA-induced changes in [Ca(2+)](i) of GnRH neurons from GnRH-enhanced green fluorescent protein (GnRH-EGFP) rats both to confirm the depolarizing action of GABA and to further examine the developmental and estrous cycle-dependent modulations of GABA action. GABA increased [Ca(2+)](i) in GnRH neurons at all developmental stages of both sexes. GABA also increased [Ca(2+)](i) in adult female GnRH neurons prepared in the afternoon at each estrous cycle stage. The percentages of neurons with increased [Ca(2+)](i) were 90% in proestrus, 59% in estrus, 84% in diestrus I, and 89% in diestrus II. In GnRH neurons prepared from adult females in the morning, however, the percentage was significantly lower than in those prepared in the afternoon, except in estrus. The percentage was also lower in adult males than in adult females. GABA responses were mimicked by muscimol and blocked by bicuculline. In addition, removal of extracellular Ca(2+) completely suppressed the GABA action, and bumetanide attenuated the response. These results indicate that GABA depolarizes GnRH neurons by activating GABA(A) receptors, thereby activating voltage-gated Ca(2+) channels and facilitating Ca(2+) influx. In addition, the response to GABA is modulated according to the estrous cycle stage, diurnal rhythm, and sex.

Tibolone rapidly attenuates the GABAB response in hypothalamic neurones.

Tibolone is primarily used for the treatment of climacteric symptoms. Tibolone is rapidly converted into three major metabolites: 3 alpha- and 3beta-hydroxy (OH)-tibolone, which have oestrogenic effects, and the Delta 4-isomer (Delta 4-tibolone), which has progestogenic and androgenic effects. Because tibolone is effective in treating climacteric symptoms, the effects on the brain may be explained by the oestrogenic activity of tibolone. Using whole-cell patch clamp recording, we found previously that 17beta-oestradiol (E(2)) rapidly altered gamma-aminobutyric acid (GABA) neurotransmission in hypothalamic neurones through a membrane oestrogen receptor (mER). E(2) reduced the potency of the GABA(B) receptor agonist baclofen to activate G-protein-coupled, inwardly rectifying K(+) (GIRK) channels in hypothalamic neurones. Therefore, we hypothesised that tibolone may have some rapid effects through the mER and sought to elucidate the signalling pathway of tibolone's action using selective inhibitors and whole cell recording in ovariectomised female guinea pigs and mice. A sub-population of neurones was identified post hoc as pro-opiomelanocortin (POMC) neurones by immunocytochemical staining. Similar to E(2), we have found that tibolone and its active metabolite 3 beta OH-tibolone rapidly reduced the potency of the GABA(B) receptor agonist baclofen to activate GIRK channels in POMC neurones. The effects were blocked by the ER antagonist ICI 182 780. Other metabolites of tibolone (3 alpha OH-tibolone and Delta 4-tibolone) had no effect. Furthermore, tibolone (and 3 beta OH-tibolone) was fully efficacious in ER alpha knockout (KO) and ER beta KO mice to attenuate GABA(B) responses. The effects of tibolone were blocked by phospholipase C inhibitor U73122. However, in contrast to E(2), the effects of tibolone were not blocked by protein kinase C inhibitors or protein kinase A inhibitors. It appears that tibolone (and 3 beta OH-tibolone) activates phospholipase C leading to phosphatidylinositol bisphosphate metabolism and direct alteration of GIRK channel function. Therefore, tibolone may enhance synaptic efficacy through the G(q) signalling pathways of mER in brain circuits that are critical for maintaining homeostatic functions.

Resting intracellular calcium concentration, depolarizing Gamma-Aminobutyric Acid and possible role of local estradiol synthesis in the developing male and female hippocampus

The maturation of the hippocampus is impacted by a multitude of factors, including the regulation of intracellular calcium levels. Depolarizing actions of Gamma-Aminobutyric Acid (GABA) can profoundly alter intracellular calcium in immature hippocampal neurons via influx through voltage-gated calcium channels. We here report fundamental sex differences in properties of depolarizing GABA responses and in resting intracellular calcium in neonatal cultured hippocampal neurons. The effects of the estrogen receptor antagonist, ICI 182,780, and the estradiol-synthesis inhibitor, formestane, indicate the sex differences in depolarizing GABA responses are at least in part due to de novo estradiol synthesis by female neurons, whereas a sex difference in resting calcium is independent of steroids. We postulate that local estradiol synthesis in cultured female hippocampal neurons affects the kinetics of either the GABAA receptor or voltage sensitive calcium channels. These data highlight the fact that immature hippocampal neurons exhibit fundamentally different physiological properties in males versus females. Elucidating how and where immature male and female neurons differ is essential for a complete understanding of normal rodent brain development.

Postnatal changes in somatic γ‐aminobutyric acid signalling in the rat hippocampus

During postnatal development of the rat hippocampus, gamma-aminobutyric acid (GABA) switches its action on CA3 pyramidal cells from excitatory to inhibitory. To characterize the underlying changes in the GABA reversal potential, we used somatic cell-attached recordings of GABA(A) and N-methyl-D-aspartate channels to monitor the GABA driving force and resting membrane potential, respectively. We found that the GABA driving force is strongly depolarizing during the first postnatal week. The strength of this depolarization rapidly declines with age, although GABA remains slightly depolarizing, by a few millivolts, even in adult neurons. Reduction in the depolarizing GABA driving force was due to a progressive negative shift of the reversal potential of GABA currents. Similar postnatal changes in GABA signalling were also observed using the superfused hippocampus preparation in vivo, and in the hippocampal interneurons in vitro. We also found that in adult pyramidal cells, somatic GABA reversal potential is maintained at a slightly depolarizing level by bicarbonate conductance, chloride-extrusion and chloride-loading systems. Thus, the postnatal excitatory-to-inhibitory switch in somatic GABA signalling is associated with a negative shift of the GABA reversal potential but without a hyperpolarizing switch in the polarity of GABA responses. These results also suggest that in adult CA3 pyramidal cells, somatic GABAergic inhibition takes place essentially through shunting rather than hyperpolarization. Apparent hyperpolarizing GABA responses previously reported in the soma of CA3 pyramidal cells are probably due to cell depolarization during intracellular or whole-cell recordings.