The presence of GABA in gastropod mucus and its role in inducing larval settlement

Chemical substances that induce larval settlement have been the focus of many gastropod studies due to the importance of wild stock recruitment and production within aquaculture facilities. Gamma-aminobutyric acid (GABA), GABA analogs, and GABA-mimetics associated with certain crustose coralline algae (CCA), are known to induce larval settlement in commercial abalone (Haliotis) species, and other gastropods. Furthermore, mucus secreted from these gastropods has been shown to induce larval settlement, but the stimulatory components of mucus have not been thoroughly investigated. We now present data confirming that GABA is the settlement-inducing effector molecule contained within abalone mucus. To do this, we initially generated anti-GABA for use in immunoenzyme and immunofluorescent microscopy. Using these techniques GABA was identified in the nerves and epithelial cells of the foot, including mucus. Dried mucus samples subject to HPLC analysis revealed a mean concentration of 0.68 mM GABA after sample rehydration. The presence of GABA in these samples was confirmed by time-of-flight mass spectroscopy (TOF-MS). In addition, GABA was detected in the mucus of several abalone species and other gastropods by immunocytochemistry. Subsequent bioassays using both dry and fresh mucus strongly promoted induction of larval settlement.

Distribution of Gaba in the nerve ganglia of haliotis asinina linnaeus

Gamma-aminobutyric acid (GABA) is a major neurotransmitter and effective settlement inducer in abalone aquaculture. This study aimed to explore the distribution of GABA within neural tissues of Haliotis asinina. Gamma-aminobutyric acid was found in neuronal cell type 1 of 3 major ganglia (i.e., cerebral, pleuropedal, and visceral ganglia) of both sexes. The distribution of GABA-immunoreactive (-ir) cells in the cerebral ganglion was concentrated mostly in the cortex region of the dorsal horn, whereas it was scattered throughout the pleuropedal ganglion, with more in the upper half. Gamma-aminobutyric acid-ir nerve fibers were found throughout the neuropils of the ganglia. The visceral ganglion had the least numbers of GABA-ir neurons compared with the other 2 ganglia. The cells were distributed mainly in the dorsal horn. We also observed GABA to be colocalized with 2 other neurotransmitters: serotonin (5-HT) and dopamine (DA). In the cerebral ganglion, fluorescence double staining of GABA and 5-HT, and GABA and DA showed immunoreactivity in separate cells and was also colocalized in the same cells. In the pleuropedal ganglion, the staining pattern was similar to the cerebral ganglion, but positive-staining cells were less numerous. In the visceral ganglion, GABA and DA, and GABA and 5-HT were colocalized in the same cell types. Overall, we found that GABAergic cells were most numerous in the cerebral ganglion of H. asinina. Further studies are required to determine the functions of these neurotransmitters in relation to their distribution.

Efeito da desnervação periférica sobre o padrão de imunorreatividade ao GABA, neuropeptídeo Y, somatostatina e CGRP na medula espinal de rãs Rana catesbeiana

A sensação de dor é mediada por diferentes sistemas de transmissão, os quais estão continuamente sendo integrados e modulados por diversos mecanismos neurais, agindo em diferentes períodos de tempo. Para o estudo da dor neuropática, um dos modelos mais empregados é a lesão nervosa periférica, sendo que a maioria desses estudos é realizada em mamíferos. Apesar da ausência de um arranjo laminar, a medula espinal de anfíbios apresenta muitas similaridades anatômicas e funcionais com a dos mamíferos. Por isso, o estudo desses animais pode fornecer subsídios adicionais para compreensão dos mecanismos da transmissão nociceptiva, além de esclarecer os aspectos evolutivos envolvidos na mesma. No presente trabalho foi analisado o padrão de imunorreatividade ao neuropeptídeo Y (NPY), peptídeo relacionado ao gene da calcitonina (CGRP), somatostatina (SOM) e ácido γ-aminobutírico (GABA) em medula espinal lombossacral de rãs Rana catesbeiana em condições basais e após a secção do nervo ciático. Para isso, foram utilizados animais adultos, de ambos os sexos, os quais foram divididos em grupos controle (animais em condições basais) e experimental (animais submetidos à secção do nervo ciático). Para o estudo da imunorreatividade ao NPY, os animais desnervados foram sacrificados 3, 7 e 15 dias após a secção do nervo ciático. Para CGRP, SOM e GABA os intervalos de tempo considerados foram de 3, 5, 8 e 15 dias após a axotomia. A técnica imunoistoquímica utilizada foi a de Sternberger (1979), sendo utilizados anticorpos primários do tipo policlonal nas concentrações de 1:1000 (GABA e neuropeptídeo Y), 1:500 (somatostatina) e 1:100 (CGRP). A imunorreação foi semi-quantificada através de densitometria óptica. A intensidade dos produtos de reação foi comparada entre os lados ipsilateral e contralateral à lesão e com o grupo controle. Os resultados obtidos nos animais controle foram semelhantes aos descritos anteriormente para os anfíbios. A maior intensidade de imunorreação ocorreu na parte dorsal do funículo lateral para todas as substâncias neuroquímicas consideradas. Imunorreatividade ao GABA, NPY e SOM ainda foram observadas ao longo do funículo lateral e no funículo ventral. Na substância cinzenta, o corno dorsal apresentou maior imunorreatividade quando comparado ao ventral, sendo esta uma característica comum entre as substâncias neuroquímicas consideradas no presente estudo. Neurônios bitufted imunorreativos para GABA, NPY e SOM foram detectados na banda mediolateral. No corno ventral, neurônios motores apresentaram imunorreação à SOM, ao CGRP e ao GABA, sendo neste último de fraca intensidade. Após a desnervação periférica não houve variação no padrão de distribuição da imunorreatividade à SOM e ao CGRP. Entretanto, a axotomia causou uma redução significativa na imunorreatividade ao GABA na parte dorsal do funículo lateral no lado ipsilateral à lesão. Essa diminuição foi evidenciada 3 dias após a desnervação, persistindo aos 5, 8 e 15 dias após a secção do nervo ciático. A imunorreatividade ao NPY apresentou inicialmente (3 e 7 dias após a axotomia) um aumento bilateral na intensidade de reação. Porém, 15 dias após a desnervação periférica, houve uma queda na imunorreatividade ao NPY, a qual também foi evidenciada bilateralmente. Esses resultados sugerem o envolvimento das substâncias neuroquímicas abordadas neste estudo no processamento das informações sensoriais de rãs Rana catesbeiana. Todavia, ainda é especulativa a participação das mesmas nos mecanismos de transmissão e codificação da nocicepção nesses animais. Estudos complementares são necessários para o esclarecimento dessas questões. Todavia, pode-se afirmar que o corno dorsal desses animais apresenta uma circuitaria complexa, onde diferentes sistemas de neurotransmissores e/ou neuromoduladores interagem para a modulação dos sinais nociceptivos, semelhante ao que é descrito para os mamíferos.

Involvement of GABA(A)-benzodiazepine receptor in the anxiolytic effect induced by hexanic fraction of Rubus brasiliensis

To investigate the ability of hexanic ethanolic fraction of Rubus brasiliensis Martius (Roseceae), to induce anxiolytic effect and also the possible involvement of the GABA(A)-benzodiazepine receptor complex, male Wistar rats and Swiss mice behaviour were tested in the elevated plus maze (EPM). All the doses of the extract, 50, 100 and 150 mg/kg, administered per gavage (vo), 30 min before the behavioural evaluation, induced an anxiolytic effect expressed by: increased number of entries in and time spent in the open arms and percentage of open arm entries: and decreased number of entries and time spent in the closed arms. The treatment of mice with flumazenil (Ro 15-1788), 0.5, 1.0 and 1.5 mg/kg, i.p., 15-min before the administration of hexanic fraction, 100 mg/kg, vo, blocked the hexanic fraction-induced anxiolytic effect. The LD50 for the hexanic fraction was 1512 mg/kg. In conclusion, it was shown that the hexanic fraction of R. brasiliensis induced an anxiolytic effect in rats and mice. This effect can be attributed to a liposoluble principle with low toxicity which may be acting as an agonist on GABA(A)-benzodiazepine receptor complex. (C) 1998 Elsevier B.V. Ireland Ltd. All rights reserved.

GABA(A) receptor activation in the lateral parabrachial nucleus induces water and hypertonic NaCl intake

Inhibitory serotonergic and cholecystokinergic mechanisms in the lateral parabrachial nucleus and central GABAergic mechanisms are involved in the regulation of water and NaCl intake. In the present study we investigated if the GABA(A) receptors in the lateral parabrachial nucleus are involved in the control of water, NaCl and food intake in rats. Male Holtzman rats with stainless steel cannulas implanted bilaterally into the lateral parabrachial nucleus were used. Bilateral injections of muscimol (0.2 nmol/0.2 mu l) into the lateral parabrachial nucleus strongly increased 0.3 M NaCl (20.3 +/- 7.2 vs. saline: 2.6 +/- 0.9 ml/180 min) without changing water intake induced by the treatment with the diuretic furosemide combined with low dose of the angiotensin converting enzyme inhibitor captopril s.c. In euhydrated and satiated rats, bilateral lateral parabrachial nucleus injections of muscimol (0.2 and 0.5 nmol/0.2 0) induced 0.3 M NaCl intake (12.1 +/- 6.5 and 32.5 +/- 7.3 ml/180 min, respectively, vs. saline: 0.4 +/- 0.2 ml/180 min) and water intake (5.2 +/- 2.0 and 7.6 +/- 2.8 ml/ 180 min, respectively, vs. saline: 0.8 +/- 0.4 ml/180 min), but no food intake (2 +/- 0.4 g/240 min vs. saline: 1 +/- 0.3 g/240 min). Bilateral lateral parabrachial nucleus injections of the GABAA antagonist bicuculline (1.6 nmol/0.2 mu l) abolished the effects of muscimol (0.5 nmol/0.2 mu l) on 0.3 M NaCl and water intake. Muscimol (0.5 nmol/0.2 mu l) into the lateral parabrachial nucleus also induced a slight ingestion of water (4.2 +/- 1.6 ml/240 min vs. saline: 1.1 +/- 0.3 ml/240 min) when only water was available, a long lasting (for at least 2 h) increase on mean arterial pressure (14 +/- 4 mm Hg, vs. saline: -1 +/- 1 mm Hg) and only a tendency to increase urinary volume and Na+ and K+ renal excretion. Therefore the activation of GABAA receptors in the lateral parabrachial nucleus induces strong NaCl intake, a small ingestion of water and pressor responses, without changes on food intake. (c) 2005 Published by Elsevier Ltd on behalf of IBRO.

Sodium intake by hyperosmotic rats treated with a GABA(A) receptor agonist into the lateral parabrachial nucleus

Inhibitory mechanisms in the lateral parabrachial nucleus (LPBN) and central GABAergic mechanisms are involved in the regulation of water and NaCl intake. Besides increasing fluid depletion-induced sodium intake, the activation of GABA(A) receptors with muscimol into the LPBN also induces ingestion of 0.3 M NaCl in normonatremic, euhydrated rats. It has been suggested that inhibitory mechanisms activated by osmotic signals are blocked by GABAA receptor activation in the LPBN, thereby increasing hypertonic NaCl intake. Therefore, in the present study we investigated the effects of muscimol injected into the LPBN on water and 0.3 M NaCl intake in hyperosmotic cell-dehydrated rats (rats treated with an intragastric load of 2 M NaCl). Male Wistar rats with stainless steel cannulas implanted bilaterally into the LPBN were used. In euhydrated rats, muscimol (0.5 nmol/0.2 mu l), bilaterally injected into the LPBN, induced ingestion of 0.3 M NaCl (24.6 +/- 7.9 vs. vehicle: 0.5 +/- 0.3 ml/180 min) and water (6.3 +/- 2.1 vs. vehicle: 0.5 +/- 0.3 ml/180 min). One hour after intragastric 2 M NaCl load (2 ml), bilateral injections of muscimol into the LPBN also induced 0.3 M NaCl intake (22.1 +/- 5.2 vs. vehicle: 0.9 +/- 0.8 ml/210 min) and water intake (16.5 +/- 3.6 vs. vehicle: 7.8 +/- 1.8 ml/210 min). The GABAA antagonist bicuculline (0.4 nmol/0.2 mu l) into the LPBN reduced the effect of muscimol on 0.3 M NaCl intake (7.1 +/- 2.1 ml/210 min). Therefore, the activation of GABAA receptors in the LPBN induces ingestion of 0.3 M NaCl by hyperosmotic cell-dehydrated rats, suggesting that plasma levels of renin or osmolarity do not affect sodium intake after the blockade of LPBN inhibitory mechanisms with muscimol. (c) 2007 Elsevier B.V. All rights reserved.

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.

Hypnotic, anticonvulsant and muscle relaxant effects of Rubus brasiliensis. Involvement of GABA(A)-system

Rubus brasiliensis hexanic fraction induced anxiolysis in rodents, which was reversed by flumazenil, a specific GABA(A)-benzodiazepine receptor antagonist (Nogueira et al., 1998a,b). Then, we investigated if this hexanic fraction was able to induce hypnotic, anticonvulsant and muscle relaxant effects, and the involvement of GABA(A)-system. The hexanic fraction (50, 100, 150 and 300 mg/kg, vo) was administered to male Swiss mice, 30 min before the tests. Only the dose of 300 mg/kg of this fraction decreased the latency and increased sleeping time in the barbituric-hypnosis test (sodium pentobarbital, 30 mg/kg, ip), prevented the pentylenetetrazol seizures (70 mg/kg, ip) and induced muscle relaxant (inclined plane) in 100% of animals. These effects were reversed by flumazenil (3 mg/kg, ip). In conclusion: (1) R. brasiliensis hexanic fraction induced hypnotic, anticonvulsant and muscle relaxant effects, in mice, and the GABA(A)-benzodiazepine receptor may play an important role in the effects of this fraction; (2) it is strongly suggested that this fraction contains a benzodiazepine-like principle. (C) 2000 Elsevier B.V. Ireland Ltd. All rights reserved.

Efeito modulador da glutationa na liberação de gaba induzida por glutamato em retinas de embrião de galinha

O ácido γ-aminobutírico (GABA) e o glutamato são, respectivamente, os principais neurotransmissores inibitório e excitatório no Sistema Nervoso Central (SNC) e são fundamentais para o processamento visual. Estudos revelam que o glutamato induz liberação de GABA na retina. Trabalhos prévios também apontam que compostos tióis regulam a liberação de GABA, mas ainda não são totalmente esclarecidos os efeitos de tióis (-SH) sobre os níveis endógenos deste neurotransmissor na retina. Neste intermédio, a glutationa (GSH) além de ser o mais importante dos compostos tióis, vem demonstrando exercer um papel neuromodulador na liberação de neurotransmissores. Desta forma, o objetivo deste trabalho foi avaliar um possível efeito modulador de GSH sobre a liberação de GABA mediada por glutamato em retinas de embrião de galinha. Para isso, utilizamos como modelo experimental tecido retiniano íntegro de embrião de galinha, com sete ou oito dias de desenvolvimento. Nos ensaios de liberação de GABA, as retinas foram tratadas com GSH (100 e 500 μM); glutamato (50 e 500 μM) e Butionina Sulfoximina (BSO), inibidor da síntese de glutationa, (50 μM) por 15 minutos, e os níveis de GABA liberado para o meio extracelular foram quantificados por Cromatografia Líquida de Alta Eficácia (CLAE). Para experimentos de liberação de compostos tióis (–SH), as retinas foram incubadas com glutamato (100 μM) com ou sem Na+ por 15 minutos, e os seus níveis extracelulares foram determinados pela reação com DTNB e quantificados por espectrofotometria (412 nm). Os resultados revelam que o glutamato, assim como GSH, liberam GABA. Nossos dados também demonstram que BSO atenua a liberação de GABA promovida por glutamato. Além disso, demonstramos que glutamato induz liberação de compostos tióis independentemente de sódio. Sendo assim, é sabido que glutamato é capaz de liberar GABA e tióis; dentre estes, GSH é o mais abundante e responsável por também liberar GABA. Sabe-se também que uma vez inibida a síntese de GSH por BSO, a liberação de GABA induzida por glutamato é atenuada. Então, se sugere uma possível modulação de GSH na liberação de GABA induzida por glutamato, em retinas íntegras de embrião de galinha.

GABA and glutamate transporters: new events and function in the vertebrate retina

The neural retina is a highly complex tissue composed of excitatory and inhibitory neurons and glial cells. Glutamate, the main excitatory neurotransmitter, mediates information transfer from photoreceptors, bipolar cells, and ganglion cells, whereas interneurons, mainly amacrine and horizontal cells, use γ-aminobutyric acid (GABA), the main inhibitory neurotransmitter. In this review we place an emphasis on glutamate and GABA transporters as highly regulated molecules that play fundamental roles in neurotransmitter clearance, neurotransmitter release, and oxidative stress. We pharmacologically characterized glutamate transporters in chicken retina cells and identified two glutamate transporters: one Na+-dependent transporter and one Na+-independent transporter. The Na+-dependent uptake system presented characteristics related to the high-affinity xAG- system (EAAT1), and the Na+-independent uptake system presented characteristics related to the xCG- system, which highly contributes to glutamate transport in the retina. Glutamate shares the xCG- system with another amino acid, L-cysteine, suggesting the possible involvement of glutathione. Both transporter proteins are present mainly in Müller glial cells. GABA transporters (GATs) mediate high-affinity GABA uptake from the extracellular space and terminate the synaptic action of GABA in the central nervous system. GABA transporters can be modulated by molecules that act on specific sites to promote transporter phosphorylation and dephosphorylation. In addition to a role in the clearance of GABA, GATs may also release GABA through a reverse transport mechanism. In the chicken retina, a GAT-1 blocker, but not GAT2/3 blocker, was shown to inhibit GABA uptake, suggesting that GABA release from retina cells is mainly mediated by a GAT-1-like transporter.

Cocaine effects on mouse incentive-learning and human addiction are linked to alpha 2 subunit-containing GABA(A) receptors

Because GABA(A) receptors containing alpha 2 subunits are highly represented in areas of the brain, such as nucleus accumbens (NAcc), frontal cortex, and amygdala, regions intimately involved in signaling motivation and reward, we hypothesized that manipulations of this receptor subtype would influence processing of rewards. Voltage-clamp recordings from NAcc medium spiny neurons of mice with alpha 2 gene deletion showed reduced synaptic GABA(A) receptor-mediated responses. Behaviorally, the deletion abolished cocaine`s ability to potentiate behaviors conditioned to rewards (conditioned reinforcement), and to support behavioral sensitization. In mice with a point mutation in the benzodiazepine binding pocket of alpha 2-GABA(A) receptors (alpha 2H101R), GABAergic neurotransmission in medium spiny neurons was identical to that of WT (i.e., the mutation was silent), but importantly, receptor function was now facilitated by the atypical benzodiazepine Ro 15-4513 (ethyl 8-amido-5,6-dihydro-5-methyl-6-oxo-4H-imidazo [1,5-a] [1,4] benzodiazepine-3-carboxylate). In alpha 2H101R, but not WT mice, Ro 15-4513 administered directly into the NAcc-stimulated locomotor activity, and when given systemically and repeatedly, induced behavioral sensitization. These data indicate that activation of alpha 2-GABA(A) receptors (most likely in NAcc) is both necessary and sufficient for behavioral sensitization. Consistent with a role of these receptors in addiction, we found specific markers and haplotypes of the GABRA2 gene to be associated with human cocaine addiction.

Maternal exposure to picrotoxin modifies the response of the GABA(A) receptor during sexual behavior of adult male rat offspring

This study investigated whether perinatal exposure to picrotoxin, a GABA(A) antagonist, modifies the effect of muscimol, a GABA(A) agonist, on the sexual behavior of adult male rats. Two hours after birth and then once daily during the next 9 days of lactation, dams received picrotoxin (0.75 mg/kg subcutaneously) or saline (1 ml/kg subcutaneously). The adult male offspring from the picrotoxin and saline groups received saline (1 ml/kg intraperitoneally) or muscimol (1 mg/kg intraperitoneally), and 15 min later, their sexual behavior was assessed. Muscimol treatment in the saline-exposed group increased the mount and intromission latencies. However, these effects were absent in the picrotoxin-exposed groups. The latencies to first ejaculation, postejaculatory mount, and intromission were decreased in both picrotoxin-exposed groups relative to the saline-exposed groups. The picrotoxin + muscimol-treated rats required more intromissions to ejaculate and the picrotoxin-exposed groups made more ejaculations than the saline-exposed groups. Thus, muscimol treatment did not increase the mount and intromission latencies following picrotoxin exposure, but increased the ejaculation frequency, which did not differ between the picrotoxin + muscimol and the picrotoxin + saline groups. These data indicate that perinatal picrotoxin treatment interfered with GABA(A) receptor development Behavioural Pharmacology 23:703-709 (c) 2012 Wolters Kluwer Health vertical bar Lippincott Williams & Wilkins.