Changes in succinate dehydrogenase activity of rats after intraventricular injections of GABA, muscimol and picrotoxin

Effects of intraventricular injections of GABA, and a GABA agonist, muscimol and an antagonist, picrotoxin on succinate dehydrogenase (SDH) enzyme activity in plasma and a few hypothalamic nuclei of brain of rats have been investigated using biochemical, histochemical and cytophotometric techniques. Results show that SDH decreased by GABA and muscimol treatment, and increased after picrotoxin injection. From the above findings, it is apparent that GABA, muscimol and picrotoxin influence SDH activity of plasma and hypothalamic nuclei.     

Qualitative and quantitative changes in monoamine oxidase activity after acute third ventricle treatment of GABA, muscimol, and picrotoxin in rat

The effect of acute IIIrd ventricle injection of GABA, muscimol, and picrotoxin on the activity of monoamine oxidase (MAO) has been investigated in serum and a few hypothalamic nuclei of the rat brain using biochemical, histochemical, and cytophotometric techniques. Biochemical estimation demonstrated a significant reduction in MAO enzyme activity after GABA and muscimol injection, whereas picrotoxin produced pronounced increase in the enzyme activity. Histochemical and cytophotometric studies confirmed the biochemical findings. Even in brain, GABA and muscimol inhibited and picrotoxin stimulated the MAO activity. From the above findings, it may be concluded that GABA, muscimol, and picrotoxin regulate the MAO activity, possible mechanisms for which are being discussed.     

Centrally administered GABA and GABA-selective agonist and antagonist in rats: histoenzymological cytophotometric study

In the present study, the effect of intracerebroventricular (icv) injection of GABA, its agonist--muscimol, and antagonist--picrotoxin, has been studied on histoenzymological alterations of acetylcholinesterase (AChE). butyrylcholinesterase (BuChE), monoamine oxidase (MAO), and succinic dehydrogenase (SDH) by cytophotometric technique. This study was conducted on medial preoptic area (mPOA), nucleus paraventricularis hypothalami (PVH), area lateralis hypothalami (LHA), nucleus dorsomedialis hypothalami (DMH), and nucleus ventromedialis hypothalami (VMH). Results showed that GABA and muscimol inhibited AChE, BuChE, MAO, and SDH in all the areas while picrotoxin stimulated these enzymes. These changes in enzyme activity by GABA, muscimol, and picrotoxin and their possible mode of action are discussed.     

Nociceptive responses to altered GABAergic activity at the spinal cord

GABA agonists and antagonists were injected intrathecally at the spinal cord, to determine their effect on nociceptive thresholds. Tactile stimulation, applied against the flank by a medium diameter von Frey fiber (5.5 g force), elicited distress vocalizations after, but not before injection of the GABA antagonists, bicuculline MI or picrotoxin (0.25 and 1 microgram dosages). Vocalization threshold to tail shock was significantly reduced by bicuculline MI or picrotoxin. Tail flick withdrawal latency from radiant heat was not altered by GABA antagonists. The GABA agonist, muscimol, significantly elevated vocalization threshold to tail shock at a 5 micrograms dose. At a lower dose level (1 microgram), muscimol significantly reduced vocalization threshold to tail shock. Tail flick latency was significantly prolonged by the 5 micrograms dose of muscimol; however, flaccid paralysis of the hind limbs was also evident. Nociceptive thresholds were not altered by GABA or saline injection. These findings indicate that GABAergic activity contributes to the tonic modulation of nociception at the spinal cord.     

Acute administration of alcohol modulates pyroglutamyl amino peptidase II activity and mRNA levels in rat limbic regions

Released TRH is inactivated by an ectopeptidase, pyroglutamyl aminopeptidase II (PPII). PPII expression and activity are stringently regulated in adenohypophysis, and in rat brain, during kindling stimulation that activates TRHergic neurons. To gain further insight into the possible regulation of PPII, we studied the effect of an acute intraperitoneal ethanol administration that affects TRH content and expression. PPII activity was determined by a fluorometric assay and PPII mRNA levels by semi-quantitative RT-PCR. Activity decreased in frontal cortex 1 h after ethanol injection and, after 6 h, in hippocampus, amygdala and n. accumbens. PPII mRNA levels decreased at 30 and 60 min in frontal cortex and n. accumbens while increased at longer times in these regions and, in hippocampus and hypothalamus. NMDA and GABA(A) receptors' agonists and antagonists were tested at 1 h (+/-ethanol) on PPII activity and mRNA levels, as well as on TRH content and its mRNA. In n. accumbens, PPII mRNA levels decreased by ethanol, MK-801, and muscimol while picrotoxin or NMDA reversed ethanol's inhibition. Ethanol decreased TRH content and increased TRH mRNA levels as MK-801 or muscimol did (NMDA or picrotoxin reverted the effect of ethanol). In frontal cortex, PPII activity was inhibited by ethanol, NMDA and MK-801 with ethanol; its mRNA levels were reduced by ethanol, MK-801 and muscimol (NMDA and picrotoxin reverted ethanol's inhibition). These results show that PPII expression and activity can be regulated in conditions where TRHergic neurons are modulated. Effects of ethanol on PPII mRNA levels as well as those of TRH and its mRNA may involve GABA or NMDA receptors in n. accumbens. Changes observed in frontal cortex suggest combined effects with stress. The response was region-specific in magnitude, tendency and kinetics. These results give further support for brain PPII regulation in conditions that modulate the activity of TRHergic neurons.     

GABA-mediated inhibition of breathing in the late gestation sheep fetus

The effects of the GABA antagonist picrotoxin, and the GABA agonist muscimol, have been studied in chronically instrumented unanaesthetized fetal sheep of 115-132 days gestation. Picrotoxin (300-400 micrograms/kg intravenous bolus injection) induced a period of stimulated breathing (40-112 min) which was associated with high voltage electrocortical activity, but inhibited by hypoxia. Muscimol (4 mg infused) had the opposite effect and caused a prolonged period of apnoea (85-418 mins) which was followed by a rebound period of increased breathing. These observations suggest that the GABA-ergic system may be involved in the apnoea of high voltage sleep states in the late gestation fetal sheep, but not in the apnoea associated with hypoxaemia in the fetus.     

大鼠尾壳核的GABA能传递在条件性行为调控中的作用

本工作采用了行为和脑内注射相结合的方法研究了大鼠尾壳核的 GABA 能传递在条件性行为调控中的作用。在分辨学习的基础上训练大鼠完成条件性回避任务,以比较药物对分辨学习和条件性回避的不同效应。实验结果表明,于大鼠双侧尾壳核内分别注入 γ-氨基丁酸(GABA)(每侧100μg/μl)和 GABA 受体激动剂蝇蕈醇(Muscimol)(每侧0.1μg/μl)后可暂时抑制条件性回避反应的出现,但分辨学习无明显影响。作为对照,于尾壳核内注入等量的生理盐水则既不影响条件性回避反应,也不影响分辨学习。在条件性回避反应被 Muscimol抑制后于尾壳核内再注入 GABA 受体阻断剂印防己毒素(PTX)(每侧0.1μg/μl)则可拮抗Muscimol 的行为抑制效应,即条件反应的出现率可恢复到或接近注射前水平。实验结果表明,大鼠尾壳核的 GABA 能传递在条件性行为调控中的重要作用。     

GABA in the entopeduncular nucleus and the subthalamic nucleus participates in mediating dopaminergic striatal output functions

The bilateral intracerebral injection of the specific GABA agonists muscimol (25, 100 ng) and THIP (500 ng) into the pallido-entopeduncular nucleus (EP) and the subthalamic nucleus (STN) of rats induced a behavioural stimulation closely resembling the syndrome evoked by direct stimulation of dopamine receptors in the striatum or by the systemic injection of dopamine agonists. The rats showed strong locomotor and rearing activity followed by characteristic stereotyped behaviour consisting of sniffing and gnawing activity. The stimulation induced by muscimol (25 ng) was found independent of dopamine, since the dopamine antagonist haloperidol (1 mg/kg s.c.) induced no blockade. Injection of the GABA antogonist picrotoxin (100 ng) into the EP or STN induced sedation and catalepsy. The unilateral injection of muscimol and picrotoxin provoked contraversive and ipsiversive postural changes. Related behavioral effects were induced by GABAergic drugs injected in substantia nigra, zona reticulata (SNR). These data provide support for the new hypothesis that GABA in the EP, SNR and STN is important for the expression of behavior related to stimulation of dopamine receptors in the striatum. The effects may be induced by a dopamine activation of the descending striato-EP, striato-SNR GABAergic pathways and possibly also the pallido-STN GABAergic pathway. The findings suggest that in addition to a pathology of the dopamine system there may also be a GABAergic dysfunction in the efferent system of the basal ganglia localized to the EP, SNR and STN in diseases, such as parkinsonism, Huntington's chorea and possibly schizophrenia.     

Evidence that GABA in the nucleus dorsalis raphé induces stimulation of locomotor activity and eating behavior

Recent electrophysiological studies have provided evidence that GABA controls inhibitorily the activity of the serotonin containing cell bodies in nucleus dorsalis raphé (NDR). The present investigation shows that local injection of baclofen or the GABA agonist muscimol (25–100 ng) into the NDR strongly increased locomotor activity and stimulated eating in satiated rats. These effects are antagonized by the GABA antagonists bicuculline or picrotoxin given systemically or locally. Muscimol injected in NDR also decreased serotonin and 5-hydroxyindole acetic acid in hypothalamus but not in striatum. These findings support a transmitter role of GABA in NDR and may be interpreted related to a decreased activity of serotonin.     

Modulation of [3H]Muscimol Binding in Rat Cerebellar and Cerebral Cortical Membranes by Picrotoxin, Pentobarbitone, and Etomidate

Modulation of [3H]muscimol binding by picrotoxin, pentobarbitone, and etomidate was investigated in rat cerebellar and cerebral cortical membranes. In cerebellum, at 37 degrees C in the presence of chloride ions (150 mM), picrotoxin and picrotoxinin decreased specific [3H]muscimol binding to 43 +/- 3% of control, with an EC50 of 1.2 +/- 0.1 microM. [3H]Muscimol saturation experiments in the presence and absence of picrotoxin indicated that the picrotoxin effect was primarily due to a loss of high-affinity muscimol sites with KD approximately equal to 10 nM. Pentobarbitone enhanced specific [3H]muscimol binding to 259 +/- 3% of control, with EC50 = 292 +/- 37 microM, and etomidate increased binding to 298 +/- 18%, with EC50 = 7.1 +/- 1.0 microM. The influence of temperature and chloride ion concentration on these effects was investigated by comparing experiments at 37 and 0 degrees C in the presence or absence of chloride at constant ionic strength. The results indicate that studies at 0 degrees C underestimate the coupling between GABA receptors and barbiturate sites and that they greatly overestimate the importance of chloride ions in this phenomenon. In cerebral cortical membranes (37 degrees C, 150 mM Cl-), the effect of picrotoxin was similar to that observed in cerebellum, whereas the effects of pentobarbitone and etomidate were greater, but occurred at higher concentrations.     

GABA and the regulation of serotonin N-acetyltransferase activity in amphibian retina—II. The role of dopamine

Retinal melatonin biosynthesis is regulated in part by the activity of serotonin N-acetyltransferase (NAT), which increases in dark-adapted, but not light-exposed, retinas at night. Using an in vitro eye cup preparation from the African clawed frog (Xenopus laevis), we have obtained evidence indicating that dopamine and gamma-aminobutyric acid (GABA) interact in the regulation of the nocturnal rise in NAT activity. Increases of NAT activity induced by the GABA agonist muscimol were suppressed by dopamine. Spiperone, a D2 dopamine receptor antagonist, and muscimol separately increased NAT activity, but were not additive in their effects. Inhibition of NAT activity by the GABA antagonist picrotoxin was blocked by spiperone. Additionally, muscimol decreased concentrations of dopamine and its principle metabolite, 3,4-dihydroxyphenylacetic acid (DOPAC), in light exposed retinas, while picrotoxin increased retinal DOPAC levels in darkness. These data suggest that in darkness, activation of GABA receptors inhibits dopamine secretion, consequently releasing NAT-synthesizing cells from a tonic inhibitory influence.     

In Vivo Release of Endogenous γ-Aminobutyric Acid from Rat Striatum: Effects of Muscimol, Oxotremorine, and Morphine

Abstract: A push-pull cannula technique was used to study the in vivo release of endogenous GABA from the striaturn of chloral hydrate anaesthesized rats. The GABA in the perfusate was isolated with hplc and fluorimetrically detected (detection limit 0.6 pmol, signalhoke = 3). The mean resting release of GABA under steady state conditions was 1.62 ± 0.09 pmo/min (n = 180, ± s.E.M.). GABA release was increased after addition of depolarizing amounts of potassium to the perfusion medium. Inhibition of GABA synthesis with 3-mercaptopropionic acid (MPA, 0.5 mM) or blockade of the neuronal activity with tetrodotoxin (TTX, 0.2 μM) diminished the spontaneous release of GABA. MPA, but not TTX, reduced the potassium-induced increase in GABA release. Striatal GABA release was decreased by local application of muscimol (l0 μM) but enhanced by picrotoxin (100 μM); the latter counteracted the effect of muscimol. Intrastriatally applied serotonin (100 μM) did not affect the rate of endogenous GABA release. Oxotremorine (25 μM) added to the perfusion medium slightly increased the striatal GABA release. This effect was blocked both by locally applied atropine (100 μM) and haloperidol (5 μM). The latter two drugs did not themselves affect the rate of GABA release. Perfusion with morphine (100 μM) inhibited striatal GABA release. This effect was not influenced by haloperidol, but was no longer observed in the presence of nalorphine (10 μM) which itself did not alter GABA release. These results indicate that GABA released from the striatum is, at least in part, of neuronal origin and that the spontaneous GABA release can be affected by various neuromodulators (including GABA, enkephalins, and acetylcholine).     

GABAA Receptor Blockade in the Anterior Ventral Tegmental Area Increases Extracellular Levels of Dopamine in the Nucleus Accumbens of Rats

Abstract: Previously, it was shown that microinfusion of the GABA_A antagonist picrotoxin into the anterior ventral tegmental area (VTA) is reinforcing. It was hypothesized that this reinforcing effect of picrotoxin in the anterior VTA is mediated, at least in part, by the activation of the mesoaccumbens dopamine (DA) system. The objective of the present study was to determine if blockade of GABA_A receptors in the anterior VTA can increase extracellular levels of DA in the nucleus accumbens (ACB), using an in vivo microdialysis technique in freely moving rats. Concentrations of picrotoxin (40, 80, and 160 µ M ) that had previously been shown to produce a reinforcing effect increased the extracellular levels of DA and its major metabolites in the ACB. The increased extracellular DA levels induced by intra-VTA injection of picrotoxin was markedly attenuated by coadministration with the GABA_A agonist muscimol, whereas intra-VTA injection of muscimol alone did not have an apparent effect on extracellular DA levels in the ACB. Microinjection of another GABA_A antagonist, bicuculline, into the anterior VTA also increased the extracellular release of DA in the ACB. These results suggest that DA neurons projecting from the anterior VTA to the ACB are tonically inhibited by GABA through its actions at the GABA_A receptors.     

Modulation of GABA binding sites by CNS depressants and CNS convulsants

[³H]Muscimol binding at 23°C and muscimol stimulated [³H]flunitrazepam binding at 37°C to membranes of rat cerebral cortex have been investigated. In washed membrane preparations, 2 apparent populations of [³H]muscimol binding sites can be observed. At 23°C [³H]muscimol binding is more sensitive to inhibition by NaCl and by other salts than at 0°C. The CNS depressants etazolate and pentobarbital reversibly enhance [³H]muscimol binding and they increase the affinity of muscimol as a stimulator of [³H]flunitrazepam binding. Conversely the CNS convulsants picrotoxin, picrotoxinin and isopropylbicyclophosphate (IPTBO) reversibly interfere with [³H]muscimol binding when NaCl is present and these drugs antagonize the effects of etazolate. In the presence of NaCl, picrotoxin, picrotoxinin and IPTBO also decrease the apparent affinity of muscimol or GABA as stimulator of [³H]flunitrazepam binding. Binding of [³H]muscimol to GABA recognition sites of rat cerebral cortex is enhanced by Ag⁺, Hg⁺ and Cu²⁺ in μM concentrations, Ag⁺ being most potent. The effects of 100 μM AgNO₃ persist after repeated washing of the membranes. When membranes are pretreated with AgNO₃ only one apparent population of [³H]muscimol binding sites with high affinity (K_d: 6–8 nM) is found. In AgNO₃ pretreated membranes, the affinity of muscimol as stimulator of [³H]flunitrazepam binding is increased 18 times (EC₅₀ 14 nM) when compared to control membranes, (EC₅₀ 253 nM). In AgNO₃ pretreated membranes, etazolate, pentobarbital and IPTBO fail to perturb either [³H]muscimol binding or baseline and muscimol stimulated [³H]flunitrazepam binding. The results demonstrate that the apparent sensitivity of GABA binding sites of the GABA-benzodiazepine-picrotoxin receptor complex can be increased by etazolate and pentobarbital and decreased by picrotoxin and IPTBO. These drugs have in common that they interfere with [³H]dihydropicrotoxinin binding.     

The binding of 3H-Isoguvacine to mouse brain synaptic membranes

³H-Isoguvacine, a gamma aminobutyric acid (GABA) agonist, has been shown to bind to a mouse forebrain synaptic membrane preparation. The specific binding is displaceable by GABA, muscimol and bicuculline but not by picrotoxin or diaminobutyric acid. Kinetic data suggest two binding affinities. Highest levels of binding are observed in the cerebellum, cortex and hippocampus. It is suggested that isoguvacine binds to GABA binding sites and therefore represents a new ligand for measuring GABA receptor binding.     

Characterization of GABAA receptor-mediated 36chloride uptake in rat brain synaptoneurosomes

gamma-Aminobutyric acid (GABA) receptor-mediated 36chloride (36Cl-) uptake was measured in synaptoneurosomes from rat brain. GABA and GABA agonists stimulated 36Cl- uptake in a concentration-dependent manner with the following order of potency: Muscimol greater than GABA greater than piperidine-4-sulfonic acid (P4S)greater than 4,5,6,7-tetrahydroisoxazolo-[5,4-c]pyridin-3-ol (THIP) = 3-aminopropanesulfonic acid (3APS) much greater than taurine. Both P4S and 3APS behaved as partial agonists, while the GABAB agonist, baclofen, was ineffective. The response to muscimol was inhibited by bicuculline and picrotoxin in a mixed competitive/non-competitive manner. Other inhibitors of GABA receptor-opened channels or non-neuronal anion channels such as penicillin, picrate, furosemide and disulfonic acid stilbenes also inhibited the response to muscimol. A regional variation in muscimol-stimulated 36Cl- uptake was observed; the largest responses were observed in the cerebral cortex, cerebellum and hippocampus, moderate responses were obtained in the striatum and hypothalamus and the smallest response was observed in the pons-medulla. GABA receptor-mediated 36Cl- uptake was also dependent on the anion present in the media. The muscimol response varied in media containing the following anions: Br- greater than Cl- greater than or equal to NO3- greater than I- greater than or equal to SCN- much greater than C3H5OO- greater than or equal to ClO4- greater than F-, consistent with the relative anion permeability through GABA receptor-gated anion channels and the enhancement of convulsant binding to the GABA receptor-gated Cl- channel.     

Effects of GABA receptor stimulation on the release of [3H]GABA from slices of rat neostriatum.

Slices of rat neostriatum were incubated in Krebs-Henseleit medium. Modulation of [3H]GABA release by GABA agonists and antagonists was investigated. The GABAA receptor agonists muscimol (0.1 microM) and isoguvacine (5 microM) enhanced the stimulated release of [3H]GABA. The antagonists picrotoxin (1 microM) and bicuculline (50 microM) prevented the effects of the agonists. In the presence of naloxone (1 microM), which blocked the effects of enkephalinergic neurons within the slice preparation, muscimol (1 microM) no longer affected the release of [3H]GABA.     

REDUCTION OF γ-AMINOBUTYRIC ACID LEVEL IN THE CAT CEREBRAL CORTEX BY AFFERENT ELECTRICAL STIMULATION

—Electrical stimulation for 30 s of one brachial plexus in cat (afferent electrical stimulation = AES) produced a 20% decrease in GABA level of the stimulated (contralateral) cerebral cortex as compared to the non-stimulated (ipsilateral) cortex in the same animal. This change in GABA was reversed within a few seconds after cessation of stimulation. Inhibition of GABA catabolism by aminooxyacetic acid elevated considerably the cortical level of GABA but failed to prevent lowering GABA by AES. When AES was performed in preconvulsive condition induced by administration of picrotoxin, the decrease in GABA was negligible, while similar treatment with pentylenetetrazol had no influence on the decrease in GABA produced by AES. The observed lowering cortical GABA by AES is interpreted as being associated with some mechanism of the inhibitory transmitter inactivation.     

Growth hormone secretion of the neonatal rat pituitaries is stimulated by gamma-aminobutyric acid in vitro

Growth hormone secretion from pituitaries of neonatal rats was stimulated by gamma-aminobutyric acid (GABA) and the GABA agonist muscimol $\text{in vitro}$. This response to GABA was absent after the 9th postnatal day. The stimulation of growth hormone secretion by GABA was antagonized by bicuculline-methiodide and by picrotoxin. Diazepam stimulated while baclophen had no effect on growth hormone secretion. This stimulatory GABA effect might be related to a certain developmental stage of the pituitary GABA receptors or to the lack of hypothalamic regulatory influence(s) in the newborn.     

GABA and the behavioral effects of anxiolytic drugs

Much recent research has shown that benzodiazepine binding sites in the central nervous system are associated with GABA receptors. It is therefore possible that the pharmacological and therapeutic effects of benzodiazepines and drugs with similar profiles are mediated through GABAergic mechanisms. In this paper the evidence is considered for a possible involvement of GABA in the behavioral effects of anxiolytic drugs. There are a number of reports that the behavioral actions of anxiolytics can be antagonised by GABA antagonists such as bicuculline or picrotoxin but there are many contradictory findings and these drugs are difficult to use effectively in behavioral studies. In general, GABA agonists do not exert anxiolytic-like behavioral effects after systemic injection but intracerebral administration of muscimol has been shown to produce benzodiazepine-like actions. Although a number of questions remain unanswered, current evidence does not provide strong support for a role for GABA in the behavioral effects of anxiolytic drugs.