Hyperalgesic effect induced by barbiturates, midazolam and ethanol: pharmacological evidence for GABA-A receptor involvement

The involvement of GABA-A receptors in the control of nociception was studied using the tail-flick test in rats. Non-hypnotic doses of the barbiturates phenobarbital (5-50 mg/kg), pentobarbital (17-33 mg/kg), and thiopental (7.5-30 mg/kg), of the benzodiazepine midazolam (10 mg/kg) or of ethanol (0.4-1.6 g/kg) administered by the systemic route reduced the latency for the tail-flick response, thus inducing a 'hyperalgesic' state in the animals. In contrast, non-convulsant doses of the GABA-A antagonist picrotoxin (0.12-1.0 mg/kg) administered systemically induced an increase in the latency for the tail-flick response, therefore characterizing an 'antinociceptive' state. Previous picrotoxin (0.12 mg/kg) treatment abolished the hyperalgesic state induced by effective doses of the barbiturates, midazolam or ethanol. Since phenobarbital, midazolam and ethanol reproduced the described hyperalgesic effect of GABA-A-specific agonists (muscimol, THIP), which is specifically antagonized by the GABA-A antagonist picrotoxin, our results suggest that GABA-A receptors are tonically involved in the modulation of nociception in the rat central nervous system

Acute phenobarbital administration induces hyperalgesia: pharmacological evidence for the involvement of supraspinal GABA-A receptors

The aim of the present study was to determine if phenobarbital affects the nociception threshold. Systemic (1-20 mg/kg) phenobarbital administration dose dependently induced hyperalgesia in the tail-flick, hot-plate and formalin tests in rats and in the abdominal constriction test in mice. Formalin and abdominal constriction tests were the most sensitive procedures for the detection of hyperalgesia in response to phenobarbital compared with the tail-flick and hot-plate tests. The hyperalgesia induced by systemic phenobarbital was blocked by previous administration of 1 mg/kg ip picrotoxin or either 1-2 mg/kg sc or 10 ng icv bicuculline. Intracerebroventricular phenobarbital administration (5 µg) induced hyperalgesia in the tail-flick test. In contrast, intrathecal phenobarbital administration (5 µg) induced antinociception and blocked systemic-induced hyperalgesia in this test. We suggest that phenobarbital may mediate hyperalgesia through GABA-A receptors at supraspinal levels and antinociception through the same kind of receptors at spinal levels.

Cardiovascular responses to microinjections of GABA or anesthetics into the rostral ventrolateral medulla of conscious and anesthetized rats

The rostral ventrolateral medulla (RVLM) contains neurons involved in tonic and reflex control of arterial pressure. We describe the effects of gamma-aminobutyric acid (GABA) and anesthetics injected into the RVLM of conscious and urethane (1.2 g/kg, iv) anesthetized Wistar rats (300-350 g). In conscious rats, bilateral microinjection of GABA (50 nmol/200 nl) induced a small but significant decrease in blood pressure (from 130 ± 3.6 to 110 ± 5.6 mmHg, N = 7). A similar response was observed with sodium pentobarbital microinjection (24 nmol/200 nl). However, in the same animals, the fall in blood pressure induced by GABA (from 121 ± 8.9 to 76 ± 8.8 mmHg, N = 7) or pentobarbital (from 118 ± 4.5 to 57 ± 11.3 mmHg, N = 6) was significantly increased after urethane anesthesia. In contrast, there was no difference between conscious (from 117 ± 4.1 to 92 ± 5.9 mmHg, N = 7) and anesthetized rats (from 123 ± 6.9 to 87 ± 8.7 mmHg, N = 7) when lidocaine (34 nmol/200 nl) was microinjected into the RVLM. The heart rate variations were not consistent and only eventually reached significance in conscious or anesthetized rats. The right position of pipettes was confirmed by histology and glutamate microinjection into the RVLM. These findings suggest that in conscious animals the RVLM, in association with the other sympathetic premotor neurons, is responsible for the maintenance of sympathetic vasomotor tone during bilateral RVLM inhibition. Activity of one or more of these premotor neurons outside the RVLM can compensate for the effects of RVLM inhibition. In addition, the effects of lidocaine suggest that fibers passing through the RVLM are involved in the maintenance of blood pressure in conscious animals during RVLM inhibition.

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.

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.

Agonistic-like responses from the torus semicircularis dorsalis elicited by GABA A blockade in the weakly electric fish Gymnotus carapo

Findings by our group have shown that the dorsolateral telencephalon of Gymnotus carapo sends efferents to the mesencephalic torus semicircularis dorsalis (TSd) and that presumably this connection is involved in the changes in electric organ discharge (EOD) and in skeletomotor responses observed following microinjections of GABA A antagonist bicuculline into this telencephalic region. Other studies have implicated the TSd or its mammalian homologue, the inferior colliculus, in defensive responses. In the present study, we explore the possible involvement of the TSd and of the GABA-ergic system in the modulation of the electric and skeletomotor displays. For this purpose, different doses of bicuculline (0.98, 0.49, 0.245, and 0.015 mM) and muscimol (15.35 mM) were microinjected (0.1 µL) in the TSd of the awake G. carapo. Microinjection of bicuculline induced dose-dependent interruptions of EOD and increased skeletomotor activity resembling defense displays. The effects of the two highest doses showed maximum values at 5 min (4.3 ± 2.7 and 3.8 ± 2.0 Hz, P < 0.05) and persisted until 10 min (11 ± 5.7 and 8.7 ± 5.2 Hz, P < 0.05). Microinjections of muscimol were ineffective. During the interruptions of EOD, the novelty response (increased frequency in response to sensory novelties) induced by an electric stimulus delivered by a pair of electrodes placed in the water of the experimental cuvette was reduced or abolished. These data suggest that the GABA-ergic mechanisms of the TSd inhibit the neural substrate of the defense reaction at this midbrain level.

GABA in the central amygdaloid nucleus modulates the electrolyte excretion and hormonal responses to blood volume expansion in rats

We investigated the involvement of GABAergic mechanisms of the central amygdaloid nucleus (CeA) in unanesthetized rats subjected to acute isotonic or hypertonic blood volume expansion (BVE). Male Wistar rats bearing cannulas unilaterally implanted in the CeA were treated with vehicle, muscimol (0.2 nmol/0.2 µL) or bicuculline (1.6 nmol/0.2 µL) in the CeA, followed by isotonic or hypertonic BVE (0.15 or 0.3 M NaCl, 2 mL/100 g body weight over 1 min). The vehicle-treated group showed an increase in sodium excretion, urinary volume, plasma oxytocin (OT), and atrial natriuretic peptide (ANP) levels compared to control rats. Muscimol reduced the effects of BVE on sodium excretion (isotonic: 2.4 ± 0.3 vs vehicle: 4.8 ± 0.2 and hypertonic: 4.0 ± 0.7 vs vehicle: 8.7 ± 0.6 µEq·100 g-1·40 min-1); urinary volume after hypertonic BVE (83.8 ± 10 vs vehicle: 255.6 ± 16.5 µL·100 g-1·40 min-1); plasma OT levels (isotonic: 15.3 ± 0.6 vs vehicle: 19.3 ± 1 and hypertonic: 26.5 ± 2.6 vs vehicle: 48 ± 3 pg/mL), and ANP levels (isotonic: 97 ± 12.8 vs vehicle: 258.3 ± 28.1 and hypertonic: 160 ± 14.6 vs vehicle: 318 ± 16.3 pg/mL). Bicuculline reduced the effects of isotonic or hypertonic BVE on urinary volume and ANP levels compared to vehicle-treated rats. However, bicuculline enhanced the effects of hypertonic BVE on plasma OT levels. These data suggest that CeA GABAergic mechanisms are involved in the control of ANP and OT secretion, as well as in sodium and water excretion in response to isotonic or hypertonic blood volume expansion.

In vivo photorelease of GABA in the mouse cortex

Electrical stimulation has been used for more than 100 years in neuroscientific and biomedical research as a powerful tool for controlled perturbations of neural activity. Despite quickly driving neuronal activity, this technique presents some important limitations, such as the impossibility to activate or deactivate specific neuronal populations within a single stimulation site. This problem can be avoided by pharmacological methods based on the administration of receptor ligands able to cause specific changes in neuronal activity. However, intracerebral injections of neuroactive molecules inherently confound the dynamics of drug diffusion with receptor activation. Caged compounds have been proposed to circumvent this problem, for spatially and temporally controlled release of molecules. Caged compounds consist of a protecting group and a ligand made inactive by the bond between the two parts. By breaking this bond with light of an appropriate wavelength, the ligand recovers its activity within milliseconds. To test these compounds in vivo, we recorded local field potentials (LFPs) from the cerebral cortex of anesthetized female mice (CF1, 60-70 days, 20-30 g) before and after infusion with caged γ-amino-butyric-acid (GABA). After 30 min, we irradiated the cortical surface with pulses of blue light in order to photorelease the caged GABA and measure its effect on global brain activity. Laser pulses significantly and consistently decreased LFP power in four different frequency bands with a precision of few milliseconds (P < 0.000001); however, the inhibitory effects lasted several minutes (P < 0.0043). The technical difficulties and limitations of neurotransmitter photorelease are presented, and perspectives for future in vivo applications of the method are discussed.

GABA-induced inactivation of Cebus apella V2 neurons: effects on orientation tuning and direction selectivity

We investigated the GABA-induced inactivation of V2 neurons and terminals on the receptive field properties of this area in an anesthetized and paralyzedCebus apella monkey. Extracellular single-unit activity was recorded using tungsten microelectrodes in a monkey before and after pressure-injection of a 0.25 or 0.5 M GABA solution. The visual stimulus consisted of a bar moving in 8 possible directions. In total, 24 V2 neurons were studied before and after blocker injections in 4 experimental sessions following GABA injection into area V2. A group of 10 neurons were studied over a short period. An additional 6 neurons were investigated over a long period after the GABA injection. A third group of 8 neurons were studied over a very long period. Overall, these 24 neurons displayed an early (1-20 min) significant general decrease in excitability with concomitant changes in orientation or direction selectivity. GABA inactivation in area V2 produced robust inhibition in 80% and a significant change in directional selectivity in 60% of the neurons examined. These GABA projections are capable of modulating not only levels of spontaneous and driven activity of V2 neurons but also receptive field properties such as direction selectivity.

GABA (γ-aminobutyric acid) production, antioxidant activity in some germinated dietary seeds and the effect of cooking on their GABA content

Abstract Germinated grains have been known as sources of Gamma-aminobutyric acid (GABA) that provide beneficial effects for human health. This study was aimed to investigate GABA production, dietary fiber, antioxidant activity, and the effect of cooking on GABA loss in germinated legumes and sesame. The highest GABA content was found in germinated mung bean, (0.8068 g kg-1, 24 h incubation) followed by germinated soybean, germinated black bean and soaked sesame. Beside GABA, dietary fiber content also increased in all grains during germination where the insoluble dietary fiber fractions were always found in higher proportions to soluble dietary fiber fractions. Our results also confirmed that germinated mung bean is a rich source of GABA and dietary fibers. Microwave cooking resulted in the smallest loss of GABA in mung bean and sesame, while steaming led to the least GABA content loss in soybean and black bean. Therefore microwave cooking and steaming are the most recommended cooking processes to preserve GABA in germinated legumes and sesame.

Is GABA involved in regulating plant growth and development? /

Rapid and large accumulation of GABA (y-aminobutyric acid) in response to a number of plant stresses has been well documented. But the role(s) of GABA in plants is not well defined. In recent years, the possibility of GABA involvement in regulating plant growth and development has been raised. In the present study, this possibility was examined. First, to rapidly and accurately determine GABA levels in plant tissues, a spectrometric method for GABA determination was developed based on a commercially available enzyme Gabase. Seventy mM LaCb almost completely removed water-soluble pigments from plant tissues which greatly interfere with the absorbance reading at 340nm. Inactivation of GAD (glutamate decarboxylase) by immediately adding methanol to a frozen plant tissue powder was suggested to prevent GABA production during extraction. The recovery of GABA with this method was approximately 100%. Second, the relationship between GABA levels and hypocotyl elongation in soybean seedlings was analyzed using different approaches to regulate in vivo GABA levels and the elongation of hypocotyls. The following major observations were made. (1) Mechanical stimulation by stroking elevated GABA levels and concurrently induced a rapid and significant reduction in hypocotyl elongation. (2) External GABA was demonstrated to penetrate into the hypocotyls using '*C-GABA. Application of external GABA elevated in vivo GABA levels, but failed to inhibit hypocotyl elongation. (3) LaCla and blue light irradiation caused an inhibition in the elongation of dark-grown hypocotyls, whereas GABA levels were not significantly affected. (4) Ca^was suggested to be involved in the signal transduction pathway leading from mechanical stimulation to GABA production, as indicated by the ability of La'* to inhibit GABA production in stimulated hypocotyls. (5) Bicuculline, saclofen and baclofen (agonists and antagonists of GABA receptors in animals) had no effect on hypocotyl elongation. It might indicate that GABA-binding components which are structurally similar to animal GABA receptors and functionally capable of regulating plant growth may not exist in plants. Therefore, the conclusion was drawn that GABA alone is not sufficient to inhibit hypocotyl elongation. Third, chloride influx in isolated Asparagus cells was enhanced by lOmM GABA during a 3 hour incubation, but the effect was not specific for GABA. Chloride efflux was not influenced by GABA. Both influx and efflux of chloride were significantly inhibited by NPPB, a chloride channel blocker. These results suggest that GABA does not influence the activity of plant chloride channels.

GABA accumulation and the hypersensitive response in isolated mesophyll cells treated with the G protein activator mastoparan

GABA (y-amino butyric acid) is a non-protein amino acid synthesized through the a-decarboxylation of L-glutamate. This reaction is catalyzed by L-glutamate decarboxylase (EC 4.1.1.15), a cytosolic Ca2+/calmodulin-stimulated enzyme. The purpose of this study is to determine whether or not GABA accumulation is associated with the hypersensitive response of isolated Asparagus sprengeri mesophyll cells. The addition of 25 J.lM mastoparan, a G protein activator, to suspensions of isolated asparagus mesophyll cells significantly increased GABA synthesis and cell death. Cell death was assessed using Evan's blue dye and fluorescein diacetate tests for cell viability. In addition, mastoparan stimulated pH-dependent alkalinization of the external medium, and a rapid and large 02 consumption followed by a loss of photosynthetic activity. The rate of 02 consumption and the net decrease in 02 in the dark was enhanced by light. The inactive mastoparan analogue Mas17 was ineffective in stimulating GABA accumulation, medium alkalinization, 02 uptake and cell death. Accumulation of H202 in response tomastoparan was not detected, however, mastoparan caused the cell-dependent degradation of added H202. The pH dependence of mastoparan-stimulated alkalinization suggests cellular electrolyte leakage, while the consumption of 02 corresponds to the oxidative burst in which 02 at the cell surface is reduced to form various active oxygen species. The results are indicative of the "hypersensitive response" of plants to pathogen attack, namely, the death of cells in the locality of pathogen invasion. The data are compatible with a model in which mastoparan triggers G protein activity, subsequent intracellular signal transduction pathway/s, and the hypersensitive response. It is postulated that the physiological elicitation of the hypersensitive response involves G protein signal transduction. The synthesis of GABA during the hypersensitive response has not been documented previously; however the role/s of GABA synthesis in the hypersensitive response, if any, remain unclear.

L-Glutamate uptake, decarboxylation to -aminobutyric acid and GABA efflux in isolated Asparagus sprengeri mesophyll cells

Addition of L-glutamate caused alkalinization of the medium surrounding Asparagus spreng.ri mesophyll cells. This suggests a H+/L-glutmate symport uptake system for L-glutamate. However stoichiometries of H+/L-glutamate symport into Asparagus cells were much higher than those in other plant systems. Medium alkalinization may also result from a metabolic decarboxylation process. Since L-glutmate is decarboxylated to r-amino butyric acid (SABA) in this system, the origin of medium alkalinization was reconsidered. Suspensions of mechanically isolated and photosyntheically competent Asparagus sprengeri mesophyll cells were used to investigate the H+/L-glutamate symport system, SABA production, GABA transport, and the origin of L-glutamate dependent medium alkalinization. The major results obtained are summarized as follows: 1. L-Glutamate and GABA were the second or third most abundant amino acids in these cells. Cellular concentrations of L-glutamate were 1.09 mM and 1.31 mM in the light and dark, respectively. Those of SABA were 1.23 mM and 1.17 mM in the light and dark, respectively. 2. Asparagine was the most abundant amino acid in xylem sap and comprised 54 to 68 1. of the amino acid pool on a molar basis. GABA was the second most abundant amino acid and represented 10 to 11 1. of the amino acid pool. L-Slutamate was a minor component. 3. A 10 minute incubation with 1 mM L-glutamate increased the production of GABA in the medium by 2,743 7. and 2,241 7. in the light and dark, respectively. 4. L-Glutamate entered the cells prior to decarboxylation. 5. There was no evidence for a H+/GABA symport process • 6. GABA was produced by loss of carbon-1 of L-glutamate. 7. The specific activity of newly synthesized labeled GABA suggests that it is not equilibrated with a storage pool of GABA. 8. The mechanism of GABA efflux appears to be a passive process. 9. The evidence indicates that the origin of L-glutamate dependent medium alkalinization is a H+/L-glutamate symport not an extracellular decarboxylation. The possible role of GABA production in regulating cytoplasmic pH and L-glutamate levels during rapid electrogenic H+/L-glutamate symport is discussed.

Expresión funcional del receptor GABA p1 humano en levaduras

Tesis (Doctorado en en Ciencias con Especialidad en Biología Molecular e Ingeniería Genética) UANL

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.