BIOCHEMICAL PROPERTIES OF GABA (B) RECEPTORS (BACLOFEN, ADENYLATE CYCLASE, CYCLIC-AMP, PHOSPHOLIPASE, PROTEIN KINASE C)

(gamma)-Aminobutyric acid (GABA), a neurotransmitter in the mammalian central nervous system, influences neuronal activity by interacting with at least two pharmacologically and functionally distinct receptors. GABA(,A) receptors are sensitive to blockade by bicuculline, are associated with benzodiazepine and barbiturate binding sites, and mediate chloride flux. The biochemical and pharmacolocal properties of GABA(,B) receptors, which are stereoselectively activated by (beta)-p-chlorophenyl GABA (baclofen), are less well understood. The aim of this study was to define these features of GABA(,B) receptors, with particular emphasis on their possible relationship to the adenylate cyclase system in brain.^ By themselves, GABA agonists have no effect on cAMP accumulation in rat brain slices. However, some GABA agonists markedly enhance the cAMP accumulation that results from exposure to norepinephrine, adenosine, VIP, and cholera toxin. Evidence that this response is mediated by the GABA(,B) system is provided by the finding that it is bicuculline-insensitive, and by the fact that only those agents that interact with GABA(,B) binding sites are active in this regard. GABA(,B) agonists are able to enhance neurotransmitter-stimulated cAMP accumulation in only certain brain regions, and the response is not influenced by phosphodiesterase inhibitors, although is totally dependent on the availability of extracellular calcium. Furthermore, data suggest that inhibition of phospholipase A(,2), a calcium-dependent enzyme, decreases the augmenting response to baclofen, although inhibitors of arachidonic acid metabolism are without effect. These findings indicate that either arachidonic acid or lysophospholipid, products of PLA(,2)-mediated degradation of phospholipids, mediates the augmentation. Moreover, phorbol esters, compounds which directly activate protein kinase C, were also found to enhance neurotransmitter-stimulated cAMP accumulation in rat brain slices. Since this enzyme is known to be stimulated by unsaturated fatty acids such as arachidonate, it is proposed that GABA(,B) agonists enhance cAMP accumulation by fostering the production of arachidonic acid which stimulates protein kinase C, leading to the phosphorylation of some component of the adenylate cyclase system. Thus, GABA, through an interaction with GABA(,B) receptors, modulates neurotransmitter receptor responsiveness in brain. The pharmocological manipulation of this response could lead to the development of therapeutic agents having a more subtle influence than current drugs on central nervous system function. ^

Effect of the GABA B agonist baclofen on dipyrone-induced delayed gastric emptying in rats

Dipyrone administered intravenously (iv) or intracerebroventricularly (icv) delays gastric emptying (GE) in rats. Gamma-aminobutyric acid (GABA) is the most potent inhibitory neurotransmitter of the central nervous system. The objective of the present study was to determine the effect of icv baclofen, a GABA B receptor agonist, on delayed GE induced by dipyrone. Adult male Wistar rats received a saline test meal containing phenol red as a marker. GE was indirectly evaluated by determining the percent of gastric retention (%GR) of the meal 10 min after orogastric administration. In the first experiment, the animals were injected iv with vehicle (Civ) or 80 mg/kg (240 µmol/kg) dipyrone (Dp iv), followed by icv injection of 10 µl vehicle (bac0), or 0.5 (bac0.5), 1 (bac1) or 2 µg (bac2) baclofen. In the second experiment, the animals were injected icv with 5 µl vehicle (Cicv) or an equal volume of a solution containing 4 µmol (1333.2 µg) dipyrone (Dp icv), followed by 5 µl vehicle (bac0) or 1 µg baclofen (bac1). GE was determined 10 min after icv injection. There was no significant difference between control animals from one experiment to another concerning GR values. Baclofen at the doses of 1 and 2 µg significantly reduced mean %GR induced by iv dipyrone (Dp iv bac1 = 35.9% and Dp iv bac2 = 26.9% vs Dp iv bac0 = 51.8%). Similarly, baclofen significantly reduced the effect of dipyrone injected icv (mean %GR: Dp icv bac1 = 30.4% vs Dp icv bac0 = 54.2%). The present results suggest that dipyrone induces delayed GE through a route in the central nervous system that is blocked by the activation of GABA B receptors.

A transcriptional study in mice with different ethanol-drinking profiles: Possible involvement of the GABA(B) receptor

Previous studies have suggested that gamma-aminobutyric acid-B (GABA(B)) receptor agonists effectively reduce ethanol intake. The quantification using real-time polymerase chain reaction of Gabbr1 and Gabbr2 mRNA from the prefrontal cortex, hypothalamus, hippocampus, and striatum in mice exposed to an animal model of the addiction developed in our laboratory was performed to evaluate the involvement of the GABAB receptor in ethanol consumption. We used outbred, Swiss mice exposed to a three-bottle free-choice model (water, 5% v/v ethanol, and 10% v/v ethanol) that consisted of four phases: acquisition (AC), withdrawal (W), reexposure (RE), and quinine-adulteration (AD). Based on individual ethanol intake, the mice were classified into three groups: "addicted" (A group; preference for ethanol and persistent consumption during all phases), "heavy" (H group; preference for ethanol and a reduction in ethanol intake in the AD phase compared to AC phase), and "light" (L group; preference for water during all phases). In the prefrontal cortex in the A group, we found high Gabbr1 and Gabbr2 transcription levels, with significantly higher Gabbr1 transcription levels compared with the C (ethanol-naive control mice). L, and H groups. In the hippocampus in the A group, Gabbr2 mRNA levels were significantly lower compared with the C, L, and H groups. In the striatum, we found a significant increase in Gabbr1 transcription levels compared with the C, L, and H groups. No differences in Gabbr1 or Gabbr2 transcription levels were observed in the hypothalamus among groups. In summary, Gabbr1 and Gabbr2 transcription levels were altered in cerebral areas related to drug taking only in mice behaviorally classified as "addicted" drinkers, suggesting that these genes may contribute to high and persistent ethanol consumption. (C) 2012 Elsevier Inc. All rights reserved.

GABA(B) receptor agonist only reduces ethanol drinking in light-drinking mice

Baclofen, a GABA(B) agonist, reduces ethanol intake in animals and humans, but the contrary or no effect was also reported. Our previous study demonstrated that mice characterized as "loss of control over ethanol intake" had different Gabbr1 and Gabbr2 transcription levels, which express, respectively, the GABA(B1) and GABA(B2) subunits in brain areas related to addictive behavior. In the present study, we tested baclofen on ethanol intake in mice exposed to the free-choice paradigm. Adult male Swiss mice, individually housed, had free access to three bottles: ethanol (5% and 10%) and water. The protocol had four phases: acquisition (AC, 10 weeks), withdrawal (W, 4 cycles during 2 weeks of 2 day-free-choice and 2 day-only-water), reexposure (RE, 2 weeks), and adulteration of ethanol solutions with quinine (AD, 2 weeks). Mice characterized as "loss of control" (A, n = 11, preference for ethanol in AC and maintenance of ethanol intake levels in AD), heavy (H, n = 11, preference for ethanol in AC and reduction of ethanol intake levels in AD), and light (L n = 16, preference for water in all phases) drinkers were randomly distributed into two subgroups receiving either intraperitoneal injections of all doses of baclofen (1.25, 2.5, and 5.0 mg/kg, given each dose twice in consecutive days) or saline, being exposed to free-choice. Fluid consumption was measured 24 h later. Baclofen reduced ethanol intake in group L In group H a reduction compared to AC was observed. Group A maintained their high ethanol intake even after baclofen treatment. Activation of the GABA(B) receptor depends on the precise balance between the GABA(B1) and GABA(B2) subunits, so the disproportionate transcription levels, we reported in group A, could explain this lack of response to baclofen. These data highlight the importance to test baclofen in individuals with different ethanol drinking profiles, including humans. (C) 2012 Elsevier Inc. All rights reserved.

GABAergic mediation of stress-induced secretion of corticosterone and oxytocin, but not prolactin, by the hypothalamic paraventricular nucleus

The hypothalamus-pituitary-adrenal axis (HPA) participates in mediating the response to stressful stimuli. Within the HPA, neurons in the medial parvocellular region of paraventricular nucleus (PVN) of the hypothalamus integrate excitatory and inhibitory signals triggering secretion of corticotropin-releasing hormone (CRH), the main secretagogue of adrenocorticotropic hormone (ACTH). Stressful situations alter CRH secretion as well as other hormones, including prolactin and oxytocin. Most inputs to the PVN are of local origin, half of which are GABAergic neurons, and both GABA-A and GABA-B receptors are present in the PVN. The objective of the present study was to investigate the role of GABA-A and GABA-B receptors in the PVN`s control of stress-induced corticosterone, oxytocin and prolactin secretion. Rats Were microinjected with saline or different doses (0.5, 5 and 50 pmol) of GABA-A (bicuculine) or GABA-B (phaclofen) antagonists in the PVN. Ten minutes later, they were subjected to a stressor (ether inhalation) and blood samples were collected 30 min before and 10, 30, 60, 90 and 120 min after the stressful stimulus to measure hormone levels by radioimmunoassay. Our results indicate that GABA acts in the PVN to inhibit stress-induced corticosterone secretion via both its receptor subtypes, especially GABA-B. In contrast, GABA in the PVN stimulates oxytocin secretion through GABA-B receptors and does not alter prolactin secretion. (C) 2008 Elsevier Inc. All rights reserved.

Differential effects of GABAB receptor subtypes, {gamma}-hydroxybutyric Acid, and Baclofen on EEG activity and sleep regulation.

The role of GABA(B) receptors in sleep is still poorly understood. GHB (γ-hydroxybutyric acid) targets these receptors and is the only drug approved to treat the sleep disorder narcolepsy. GABA(B) receptors are obligate dimers comprised of the GABA(B2) subunit and either one of the two GABA(B1) subunit isoforms, GABA(B1a) and GABA(B1b). To better understand the role of GABA(B) receptors in sleep regulation, we performed electroencephalogram (EEG) recordings in mice devoid of functional GABA(B) receptors (1(-/-) and 2(-/-)) or lacking one of the subunit 1 isoforms (1a(-/-) and 1b(-/-)). The distribution of sleep over the day was profoundly altered in 1(-/-) and 2(-/-) mice, suggesting a role for GABA(B) receptors in the circadian organization of sleep. Several other sleep and EEG phenotypes pointed to a more prominent role for GABA(B1a) compared with the GABA(B1b) isoform. Moreover, we found that GABA(B1a) protects against the spontaneous seizure activity observed in 1(-/-) and 2(-/-) mice. We also evaluated the effects of the GHB-prodrug GBL (γ-butyrolactone) and of baclofen (BAC), a high-affinity GABA(B) receptor agonist. Both drugs induced a state distinct from physiological sleep that was not observed in 1(-/-) and 2(-/-) mice. Subsequent sleep was not affected by GBL whereas BAC was followed by a delayed hypersomnia even in 1(-/-) and 2(-/-) mice. The differential effects of GBL and BAC might be attributed to differences in GABA(B)-receptor affinity. These results also indicate that all GBL effects are mediated through GABA(B) receptors, although these receptors do not seem to be involved in mediating the BAC-induced hypersomnia.

GABA receptors inhibited by benzodiazepines mediate fast inhibitory transmission in the central amygdala

The amygdala is intimately involved in emotional behavior, and its role in the generation of anxiety and conditioned fear is well known. Benzodiazepines, which are commonly used for the relief of anxiety, are thought to act by enhancing the action of the inhibitory transmitter GABA. We have examined the properties of GABA-mediated inhibition in the amygdala. Whole-cell recordings were made from neurons in the lateral division of the central amygdala. Application of GABA evoked a current that reversed at the chloride equilibrium potential. Application of the GABA antagonists bicuculline or SR95531 inhibited the GABA-evoked current in a manner consistent with two binding sites. Stimulation of afferents to neurons in the central amygdala evoked an IPSC that was mediated by the release of GABA. The GABA(A) receptor antagonists bicuculline and picrotoxin failed to completely block the IPSC. The bicuculline-resistant IPSC was chloride-selective and was unaffected by GABA(B)-receptor antagonists. Furthermore, this current was insensitive to modulation by general anesthetics or barbiturates. In contrast to their actions at GABA(A) receptors, diazepam and flurazepam inhibited the bicuculline-resistant IPSC in a concentration-dependent manner. These effects were fully antagonized by the benzodiazepine site antagonist Ro15-1788. We conclude that a new type of ionotropic GABA receptor mediates fast inhibitory transmission in the central amygdala. This receptor may be a potential target for the development of new therapeutic strategies for anxiety disorders.

The bradycardic and hypotensive responses to serotonin are reduced by activation of GABA A receptors in the nucleus tractus solitarius of awake rats

We investigated the effects of bilateral injections of the GABA receptor agonists muscimol (GABA A) and baclofen (GABA B) into the nucleus tractus solitarius (NTS) on the bradycardia and hypotension induced by iv serotonin injections (5-HT, 2 µg/rat) in awake male Holtzman rats. 5-HT was injected in rats with stainless steel cannulas implanted bilaterally in the NTS, before and 5, 15, and 60 min after bilateral injections of muscimol or baclofen into the NTS. The responses to 5-HT were tested before and after the injection of atropine methyl bromide. Muscimol (50 pmol/50 nl, N = 8) into the NTS increased basal mean arterial pressure (MAP) from 115 ± 4 to 144 ± 6 mmHg, did not change basal heart rate (HR) and reduced the bradycardia (-40 ± 14 and -73 ± 26 bpm at 5 and 15 min, respectively, vs -180 ± 20 bpm for the control) and hypotension (-11 ± 4 and -14 ± 4 mmHg, vs -40 ± 9 mmHg for the control) elicited by 5-HT. Baclofen (12.5 pmol/50 nl, N = 7) into the NTS also increased basal MAP, but did not change basal HR, bradycardia or hypotension in response to 5-HT injections. Atropine methyl bromide (1 mg/kg body weight) injected iv reduced the bradycardic and hypotensive responses to 5-HT injections. The stimulation of GABA A receptors in the NTS of awake rats elicits a significant increase in basal MAP and decreases the cardiac Bezold-Jarisch reflex responses to iv 5-HT injections.

The bradycardic and hypotensive responses to serotonin are reduced by activation of GABA A receptors in the nucleus tractus solitarius of awake rats

We investigated the effects of bilateral injections of the GABA receptor agonists muscimol (GABA A) and baclofen (GABA B) into the nucleus tractus solitarius (NTS) on the bradycardia and hypotension induced by iv serotonin injections (5-HT, 2 µg/rat) in awake male Holtzman rats. 5-HT was injected in rats with stainless steel cannulas implanted bilaterally in the NTS, before and 5, 15, and 60 min after bilateral injections of muscimol or baclofen into the NTS. The responses to 5-HT were tested before and after the injection of atropine methyl bromide. Muscimol (50 pmol/50 nl, N = 8) into the NTS increased basal mean arterial pressure (MAP) from 115 ± 4 to 144 ± 6 mmHg, did not change basal heart rate (HR) and reduced the bradycardia (-40 ± 14 and -73 ± 26 bpm at 5 and 15 min, respectively, vs -180 ± 20 bpm for the control) and hypotension (-11 ± 4 and -14 ± 4 mmHg, vs -40 ± 9 mmHg for the control) elicited by 5-HT. Baclofen (12.5 pmol/50 nl, N = 7) into the NTS also increased basal MAP, but did not change basal HR, bradycardia or hypotension in response to 5-HT injections. Atropine methyl bromide (1 mg/kg body weight) injected iv reduced the bradycardic and hypotensive responses to 5-HT injections. The stimulation of GABA A receptors in the NTS of awake rats elicits a significant increase in basal MAP and decreases the cardiac Bezold-Jarisch reflex responses to iv 5-HT injections.

Multiple GABA receptors in rabbit retina

The task of encoding and processing complex sensory input requires many types of transsynaptic signals. This requirement is served in part by an extensive group of neurotransmitter substances which may include thirty or more different compounds. At the next level of information processing, the existence of multiple receptors for a given neurotransmitter appears to be a widely used mechanism to generate multiple responses to a given first messenger (Snyder and Goodman, 1980). Despite the wealth of published data on GABA receptors, the existence of more than one GABA receptor was in doubt until the mid 1980's. Presently there is still disagreement on the number of types of GABA receptors, estimates for which range from two to four (DeFeudis, 1983; Johnston, 1985). Part of the problem in evaluating data concerning multiple receptor types is the lack of information on the number of gene products and their subsequent supramolecular organization in different neurons. In order to evaluate the question concerning the diversity of GABA receptors in the nervous system, we must rely on indirect information derived from a wide variety of experimental techniques. These include pharmacological binding studies to membrane fractions, electrophysiological studies, localization studies, purification studies, and functional assays. Almost all parts of the central and peripheral nervous system use GABA as a neurotransmitter, and these experimental techniques have therefore been applied to many different parts of the nervous system for the analysis of GABA receptor characteristics. We are left with a large amount of data from a wide variety of techniques derived from many parts of the nervous system. When this project was initiated in 1983, there were only a handful of pharmacological tools to assess the question of multiple GABA receptors. The approach adopted was to focus on a single model system, using a variety of experimental techniques, in order to evaluate the existence of multiple forms of GABA receptors. Using the in vitro rabbit retina, a combination of pharmacological binding studies, functional release studies and partial purification studies were undertaken to examine the GABA receptor composition of this tissue. Three types of GABA receptors were observed: Al receptors coupled to benzodiazepine and barbiturate modulation, and A2 or uncoupled GABA-A receptors, and GABA-B receptors. These results are evaluated and discussed in light of recent findings by others concerning the number and subtypes of GABA receptors in the nervous system. ^

Phasic, nonsynaptic GABA-A receptor-mediated inhibition entrains thalamocortical oscillations.

GABA-A receptors (GABA-ARs) are typically expressed at synaptic or nonsynaptic sites mediating phasic and tonic inhibition, respectively. These two forms of inhibition conjointly control various network oscillations. To disentangle their roles in thalamocortical rhythms, we focally deleted synaptic, γ2 subunit-containing GABA-ARs in the thalamus using viral intervention in mice. After successful removal of γ2 subunit clusters, spontaneous and evoked GABAergic synaptic currents disappeared in thalamocortical cells when the presynaptic, reticular thalamic (nRT) neurons fired in tonic mode. However, when nRT cells fired in burst mode, slow phasic GABA-AR-mediated events persisted, indicating a dynamic, burst-specific recruitment of nonsynaptic GABA-ARs. In vivo, removal of synaptic GABA-ARs reduced the firing of individual thalamocortical cells but did not abolish slow oscillations or sleep spindles. We conclude that nonsynaptic GABA-ARs are recruited in a phasic manner specifically during burst firing of nRT cells and provide sufficient GABA-AR activation to control major thalamocortical oscillations.

Couplage du récepteur à sept domaines transmembranaires GABA-B1 aux voies intracellulaires de signalisation en absence de GABA-B2

Le GABA est le principal neurotransmetteur inhibiteur du SNC et est impliqué dans le développement du cerveau, la plasticité synaptique et la pathogénèse de maladies telles que l’épilepsie, les troubles de l’anxiété et la douleur chronique. Le modèle actuel de fonctionnement du récepteur GABA-B implique l’hétérodimérisation GABA-B1/B2, laquelle est requise au ciblage à la surface membranaire et au couplage des effecteurs. Il y est cependant des régions du cerveau, des types cellulaires et des périodes du développement cérébral où la sous-unité GABA-B1 est exprimée en plus grande quantité que GABA-B2, ce qui suggère qu’elle puisse être fonctionnelle seule ou en association avec des partenaires inconnus, à la surface cellulaire ou sur la membrane réticulaire. Dans le cadre de cette thèse, nous montrons la capacité des récepteurs GABA-B1 endogènes à activer la voie MAPK-ERK1/2 dans la lignée dérivée de la glie DI-TNC1, qui n’exprime pas GABA-B2. Les mécanismes qui sous-tendent ce couplage demeurent mal définis mais dépendent de Gi/o et PKC. L’immunohistochimie de récepteurs endogènes montre par ailleurs que des anticorps GABA-B1 dirigés contre la partie N-terminale reconnaissent des protéines localisées au RE tandis des anticorps C-terminaux (CT) marquent une protéine intranucléaire. Ces données suggèrent que le domaine CT de GABA-B1 pourrait être relâché par protéolyse. L’intensité des fragments potentiels est affectée par le traitement agoniste tant en immunohistochimie qu’en immunobuvardage de type western. Nous avons ensuite examiné la régulation du clivage par le protéasome en traitant les cellules avec l’inhibiteur epoxomicine pendant 12 h. Cela a résulté en l’augmentation du marquage intranucléaire de GABA-B1-CT et d’un interacteur connu, le facteur de transcription pro-survie ATF-4. Dans des cellules surexprimant GABA-B1-CT, l’induction et la translocation nucléaire d’ATF-4, qui suit le traitement epoxomicine, a complètement été abolie. Cette observation est associée à une forte diminution du décompte cellulaire. Étant donné que les trois derniers résidus de GABA-B1-CT (LYK) codent un ligand pseudo-PDZ et que les protéines à domaines PDZ sont impliquées dans la régulation du ciblage nucléaire et de la stabilité de protéines, en complément de leur rôle d’échaffaud à la surface cellulaire, nous avons muté les trois derniers résidus de GABA-B1-CT en alanines. Cette mutation a complètement annulé les effets de GABA-B1-CT sur l’induction d’ATF-4 et le décompte cellulaire. Cette deuxième série d’expériences suggère l’existence possible de fragments GABA-B1 intranucléaires régulés par le traitement agoniste et le protéasome dans les cellules DI-TNC1. Cette régulation d’ATF-4 dépend des résidus LYK de GABA-B1-CT, qui modulent la stabilité de GABA-B1-CT et favorisent peut-être la formation d’un complexe multiprotéique incluant GABA-B1-CT, ATF-4, de même qu’une protéine d’échaffaudage inconnue. En somme, nous démontrons que les sous-unités GABA-B1 localisées au RE, lorsque non-hétérodimérisées avec GABA-B2, demeurent capables de moduler les voies de signalisation de la prolifération, la différentiation et de la survie cellulaire, via le couplage de protéines G et possiblement la protéolyse régulée. Les mécanismes de signalisation proposés pourraient servir de nouvelle plate-forme dans la compréhension des actions retardées résultant de l’activation des récepteurs 7-TMs.

Gabaergic and opioid receptors mediate the facilitation of NaCl intake induced by alpha(2)-adrenergic activation in the lateral parabrachial nucleus

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)