Specific GABA Receptor Research Articles

A conditionally immortalized glial cell line that expresses mature myelin proteins and functional GABA(A) receptors.

We have isolated and characterized a conditionally immortalized glial cell line that expresses mature myelin proteins, as well as functional GABA(A) receptors. Glial cells were isolated from postnatal day 1 H-2Kb-tsA58 transgenic mice that contain the temperature-sensitive SV40 large T antigen oncogene under the control of an interferon-gamma-inducible promoter. A clonal line was isolated that grew rapidly under permissive conditions (33 degrees C in the presence of interferon-gamma), but not under nonpermissive conditions (37 degrees C in the absence of interferon-gamma). Cells expressed mRNAs of mature myelin proteins (myelin basic proteins and proteolipid protein) when grown under either permissive or nonpermissive conditions, but myelin basic proteins were detected only when cells were maintained at 37 degrees C. GABA induced an increase in intracellular calcium concentration, indicating the presence of functional receptors for this transmitter. Characterization of the GABA-evoked calcium response and whole-cell currents indicated that these responses were mediated by GABA(A) receptors. Reverse transcription-polymerase chain reaction revealed differential expression of mRNAs for specific GABA(A) receptor subunits. Messages for the alpha2, alpha4, beta1, and delta subunits were readily detected by reverse transcription-polymerase chain reaction, whereas messages for the alpha1, alpha3, alpha5, alpha6, beta2, beta3, and gamma1-3 subunits were not. As this cell line can be easily propagated and differentiated, it should provide a valuable tool in studying not only the development of oligodendroglia, but also the function of their transmitter receptors and myelin proteins.

Biochemical actions of chronic ethanol exposure in the mesolimbic dopamine system.

In previous studies, we have demonstrated that chronic administration of morphine or cocaine produces some common biochemical adaptations in the ventral tegmental area (VTA) and nucleus accumbens (NAc), components of the mesolimbic dopamine system implicated in the reinforcing actions of these and other drugs of abuse. Since this neural pathway is also implicated in the reinforcing actions of ethanol, it was of interest to determine whether chronic ethanol exposure results in similar biochemical adaptations. Indeed, as seen for chronic morphine and cocaine treatments, we show here that chronic ethanol treatment increased levels of tyrosine hydroxylase and glial fibrillary acidic protein immunoreactivity, and decreases levels of neurofilament protein immunoreactivity, in the VTA. Also like morphine and cocaine, ethanol increases levels of cyclic AMP-dependent protein kinase activity in the NAc. These actions of ethanol required long-term exposure to the drug, and were in most cases not seen in the substantia nigra or caudateputamen, components of the nigrostriatal dopamine system studied for comparison. Altered levels of tyrosine hydroxylase in catecholaminergic cells frequently reflect altered states of activation of the cells. Moreover, increasing evidence indicates that ethanol produces many of its acute effects on the brain by regulating NMDA glutamate and GABAA receptors. We therefore examined the influence of chronic ethanol treatment on levels of expression of specific glutamate and GABA receptor subunits in the VTA. It was found that long-term, but not short-term, ethanol exposure increased levels of immunoreactivity of the NMDAR1 subunit, an obligatory component of NMDA glutamate receptors, and of the GluR1 subunit, a component of many AMPA glutamate receptors; but at the same time, long-term ethanol exposure decreased immunoreactivity levels of the alpha 1 subunit of the GABAA receptor complex. These changes are consistent with an increased state of activation of VTA neurons inferred from the observed increase in tyrosine hydroxylase (TH) expression. These results demonstrate that chronic ethanol exposure results in several biochemical adaptations in the mesolimbic dopamine system, which may underlie prominent changes in the structural and functional properties of this neural pathway related to alcohol abuse and alcoholism.

Chapter 4 Structural organization of GABAergic circuitry in ectotherm retinas

This chapter discusses visualizing gamma-aminobutyric acid–releasing (GABAergic) neurons, the scope of GABAergic organization throughout all vertebrates, and the general principles of GABAergic control throughout the retina. The first step in describing GABAergic circuits is to provide compelling evidence that GABAergic neurons themselves can be characterized with high functional and structural fidelity. There are three main cell biological mechanisms underlying the rapidity, sustainability, and potency of GABAergic neurotransmission: (1) the abilities of neurons to synthesize and mobilize GABA for release, (2) the precise and rapid control of extracellular neurotransmitter levels by potent pre-synaptic neurotransmitter/Na + symporters, and (3) the mediation of rapid postsynaptic conductance or synaptic efficacy changes by specific GABA receptors. Each of these features can theoretically be associated with visualization techniques. GABA immunoreactivity has been mapped to many vertebrate retinas, but the general patterns of organization in non-mammalians/ectotherms are easily demonstrated. A clear population of GABA immunoreactive horizontal cells can be seen in most species.

Evidence that genetic differences in habituation and GABAergic mechanisms may be related to sensitivity to ethanol and development of ethanol tolerance in mice

Habituation to a test environment following daily exposure for 5 days was examined in three genetically different strains of mice. C57 animals showed significant habituation to the new environment already on the second day. The habituation of NMRI mice was significant on the third day, whereas CBA mice showed no habituation at all during the experimental period. There was no difference between the animal strains in learning capacity in a passive avoidance test, but CBA mice displayed a significant increase in latency in their performance. When tested for sensitivity to the convulsant actions of GABAergic antagonists, picrotoxin produced seizures at lower doses in CBA as compared to NMRI and C57 mice, whereas there was no difference between the strains in the seizure activity produced by the specific GABA receptor antagonist bicuculline. When the animals were tested for sensitivity to ethanol in a horizontal wire test, ethanol (2 g/kg, IP) produced muscle relaxation in CBA mice whereas the performance of NMRI and C57 was not affected. A large dose of ethanol (4 g/kg, IP) produced a significantly longer sleeping time in CBA mice as compared to NMRI and C57 animals. Ethanol-produced hypothermia was, however, similar in all animals. Environment-dependent development of tolerance to ethanol following daily injections of ethanol for 4 days was examined. C57 mice showed the most rapid development of tolerance towards ethanol's hypnotic actions, whereas CBA mice showed no tolerance to this effect of ethanol. No difference between the strains to the development of tolerance to ethanol's hypothermic effects was observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of the GABA receptor agonist, progabide, upon local cerebral glucose utilization

The effects of a specific GABA receptor agonist, progabide, have been examined on local cerebral glucose utilization through the use of the autoradiographic [ 14C]2-deoxyglucose technique in conscious rats. The intraperitoneal administration of progabide (80–320 mg·kg −1) resulted in a heterogeneous pattern of significantly reduced glucose utilization throughout the 50 discrete regions of the brain that were studied. Insignificant decreases in local cerebral glucose use were noted following the low dose of progabide (80 mg·kg −1). Following progabide (160 mg·kg −1), reductions of approximately 20% in glucose utilization were observed in most of the extrapyramidal system (substantia nigra, pars compacta and reticulata; globus pallidus; caudate nucleus), some thalamic nuclei (lateral geniculate body, anterior and ventrolateral thalamic nuclei), a number of areas related to the limbic system (cingulate cortex; amygdala; hypothalamus; lateral habenula; nucleus accumbens; dorsal hippocampus as well as the CA 3 field) and the raphénuclei. In contrast, layer IV of the neocortex, the cerebellum and cerebellar nuclei, displayed only minimal (10–15%) reductions in glucose utilization. At the highest dose (320 mg·kg −1) examined, progabide effected widespread though heterogeneously distributed decreases in glucose use. The overall pattern of decreased glucose use seen with progabide was different from that noted with previously studied GABA-mimetic drugs, such as muscimol, except for those changes observed in the extrapyramidal and sensory-motor areas for which similar dose-response relationships occurred. A significant correlation was observed between progabide-induced decreases in glucose utilization and in serotonin synthesis in a number of brain areas. The pattern of progabide-induced changes in integrated functional activity is compatible with its neurochemical spectrum and could indicate the loci at which its anticonvulsant, antidepressant and extrapyramidal activities are initiated.

Biochemistry and neurotoxicology of guanidino compounds. History and recent advances.

Guanidino compounds are known to have important biological roles, such as the participation of arginine in ureagenesis, and of creatine in muscular contraction. On the other hand, the high toxicity of guanidino compounds, such as methylguanidine and guanidine, has been under study for quite a long time in the biochemical as well as clinical fields. In this review, the author summarizes the experimental results of neurophysiological and neurochemical studies on guanidino compound-induced seizures, conducted by his colleagues since 1966, and introduces several topics arising from their recent investigations on guanidino compounds and seizure mechanism, i.e., (1) alpha-guanidino-glutaric acid in the cobalt epileptic focus and its convulsive activity; (2) guanidino-ethanesulfonic acid and epilepsy; (3) delta-guanidinovaleric acid, and endogenous and specific GABA receptor antagonist; and (4) guanidino compounds as radical generators.

δ-Guanidinovaleric acid as an endogenous and specific GABA-receptor antagonist: electroencephalographic study

Sporadic spike discharges recorded on EEGs from epidural electrodes appeared 5–10 min after topical application of 0.3 nmol ·-guanidinovaleric acid (DGVA) on the pia mater of the sensorimotor cortex, on the same side as the application. Spike discharges induced by DGVA were completely suppressed within 10 min of supplementary application of GABA (50 nmol), (3R)-(-)-4-amino-3-hydroxybutanoic acid ( l-GABOB) (5 nmols) or muscimol (5 nmols) on the pia mater, but the discharges were not affected by supplementing with 500 nmol of α-amino-DGVA i.e., arginine (Arg). Whereas spike discharges were not induced by DGVA together with L-GABOB or muscimol, DGVA applied together with Arg induced spike discharges. Neither phenobarbital (PB) (20 mg/kg i.m.), diazepam (DZ) (10 mg/kg i.p.), sodium valproate (200 mg/kg i.p.) nor diphenylhydantoin (20 mg/kg i.p.) showed any suppressive effects on spike discharges induced by DGVA. DGVA induced spike discharges 20 min after pre-injection of PB or DZ. These electroencephalographic findings suggest that DGVA, which has one more carbon in its chain than N-amidino-GABA, might act directly on the GABA-receptor to induce spike discharges and might be a specific GABA-receptor antagonist.

GABA-agonists induce the formation of low-affinity GABA-receptors on cultured cerebellar granule cells via preexisting high affinity GABA receptors.

The kinetics of specific GABA-binding to membranes isolated from cerebellar granule cells, cultured for 12 days from dissociated cerebella of 7-day-old rats was studied using [3H]GABA as the ligand. The granule cells were cultured in the presence of the specific GABA receptor agonist 4, 5, 6, 7-tetrahydroisoxazolo [5,4-c]pyridin-3-ol (THIP, 150 microM) or THIP plus the antagonist bicuculline methobromide (150 microM of each) or in the absence of the agonist or antagonist. Membranes isolated from granule cells cultured in a medium without the GABA agonist revealed a single binding site for GABA with a binding constant (KD) of 7.9 +/- 0.4 nM and a Bmax of 3.42 +/- 0.08 pmol X mg-1 protein. Membranes from cells cultured in the presence of THIP had two binding sites for GABA with KD-values of 6.8 +/- 0.9 nM and 476 +/- 311 nM, respectively. The corresponding Bmax values were 4.41 +/- 0.42 pmol X mg-1 and 5.81 +/- 1.20 pmol X mg-1. The effect of culturing the cells in THIP was antagonized by the simultaneous presence of bicuculline in the culture media, i.e. no significant low-affinity binding for GABA was found on the membranes from granule cells cultured in both THIP and bicuculline. The KD value (14.3 +/- 1.4 nM) for the high affinity binding site was, however, slightly increased compared to the non-treated cells. These findings suggest that the ability of THIP to induce formation of low-affinity GABA receptors is mediated by preexisting high-affinity GABA-receptors on the granule cells.

3H-muscimol binding sites within guinea pig ovary: a histoautoradiographic study.

The distribution of 3H-muscimol within guinea pig ovary was studied using combined radioreceptor binding studies and histoautoradiographic technique performed on frozen sections of ovary mounted on microscope slides. 3H-muscimol was bound by sections of ovary in a manner consistent with the existence of specific GABA receptors. The binding was reversible, saturable, of high affinity (KD was 38 +/- 6 nmol/l and Bmax was about 378 +/- 61 fmol/mg protein, and inhibited by GABA receptor interfering drugs. 3H-muscimol binding sites were found in the blood vessel wall, in the follicle and in the oocytes. The number of oocyte and follicular receptors gradually decreases during the development of ovarian follicles.

Evaluation of different GABA receptor agonists in the kindled amygdala seizure model in rats

The effects of three specific GABA receptor agonists, muscimol, progabide, and gaboxadol, on kindled seizures were evaluated in amygdala-kindled rats. The only compound that exerted significant anticonvulsant effects at nonsedative doses was progabide. Thus, after i.p. administration of 100 mg/kg progabide, a significant increase in seizure latency and significant decreases in duration of motor seizures and amygdala afterdischarges were determined. A decrease in severity of motor seizures was found only after 200 mg/kg progabide which, however, gave rise to marked sedation and muscle relaxation. Muscimol and gaboxadol were almost inactive in attenuating kindled seizures even at doses that produced pronounced side effects. Assuming that the amygdala-kindled rat is a useful model of complex partial seizures with secondary generalization in the human, the data suggest that GABA receptor agonists are not effective against this type of epilepsy (muscimol, gaboxadol) or effective only at large doses (progabide).

Involvement of a GABAergic mechanism in the anticonvulsant effect of pentobarbital against maximal electroshock-induced seizures in rats

The interaction between pentobarbital and other modulators of GABAergic transmission (diazepam, ethanol and progabide) was investigated on maximal electroshcok seizures and on the loss of righting reflexes in rats. Pentobarbital, diazepam and ethanol produced a dose-dependent protection against electroshock seizures, with pentobarbital being more potent (3- and 50-times) than diazepam and ethanol. Progabide neither provided protection nor caused loss of righting reflex. Subprotective doses of pentobarbital and diazepam, together or when combined with a single ineffective dose of ethanol or progabide, caused protection against seizures and loss of righting reflex for variable durations, while ethanol and progabide combination did not provide protection. The protective effect of diazepam was antagonized by RO15-1788, picrotoxin and bicuculline pretreatments. The antagonism of pentobarbital protection by a specific GABA receptor antagonist, bicuculline suggests involvement of the GABAergic system in the anticonvulsant effect of pentobarbital. These results indicate that, like diazepam, the anticonvulsant effect of pentobarbital appears to be mediated through a GABAergic mechanism.

An analysis of the actions of progabide, a specific GABA receptor agonist, on kindling and kindled seizures

The anticonvulsant properties of the specific GABA receptor agonist, progabide, were evaluated in the kindled amygdaloid seizure model in rats. Progabide attenuated afterdischarge durations and the severity of the accompanying convulsive responses in previously kindled rats. This effect occurred only at doses that also produced sedation and ataxia. When administered daily during kindling acquisition, progabide increased the number of trials necessary to complete kindling. The duration and severity of responses induced by stimulations during the acquisition period were reduced in a dose-dependent manner. In spite of these changes during the acquisition period, all subjects exhibited kindled behavior comparable to that of controls when they had accrued the same total afterdischarge experience. In light of these and other data regarding the GABA system and its influences on kindling, we conclude that GABA acts nonspecifically to attenuate various seizure states. It appears that GABA plays no major role in the mechanisms actually responsible for kindling development.

Pharmacodynamic effects and possible therapeutic uses of THIP, a specific GABA-agonist.

THIP (4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol) is a potent and specific GABA receptor agonist which does not influence the GABA uptake system or GABA metabolizing enzymes. The specificity for the GABA receptor is also demonstrated by lack of action on monoaminergic, cholinergic, histaminergic or opiate receptors. Since in recent years GABA receptor stimulants-among others THIP--have become available many have speculated as to what clinical indication GABA-ergic stimulation might be an important element. The first suggestion was that GABA-ergic drugs by an inhibitory effect on the dopamine neurons would improve the antischizophrenic effect of neuroleptics and improve tardive dyskinesia. Furthermore, studies on brains of deceased Parkinson and Huntington's chorea patients have demonstrated a low level of GABA and its synthesizing enzyme glutamic acid decarboxylase (GAD) in the basal ganglia. Also in epilepsy and diseases with dementia a deficit in the GABA system has been proposed. Therefore a therapeutic strategy for these diseases may be supplementary treatment with drugs which increase GABA receptor activity. Furthermore, recent results in humans have shown that GABA agonists perhaps also could be of benefit in mania and depressions. When considering the neurophysiological elements of nociception and muscle tone it is also reasonable to suggest that GABA-ergic stimulation may reduce pain perception and muscle tone.

GABA and muscimol inhibit the release of prolactin from dispersed rat anterior pituitary cells.

Prolactin release from columns of dispersed rat anterior pituitary cells was inhibited by GABA and the GABA agonist muscimol. The log dose-response curves for GABA and muscimol appeared to be parallel, but were not parallel to that for dopamine. Two GABA antagonists, bicuculline and picrotoxin, blocked the action of both GABA and muscimol, but the dopamine antagonists metoclopramide and perphenazine did not. The data suggest that there are specific GABA receptors on pituitary lactotrophs.

Specific cerebrovascular localization of GABA-related receptors and enzymes

A role for GABA in cerebral vascular function is supported by data indicating that specific GABA receptors and GABA-related enzymes are associated with cerebral blood vessels. 3H-Muscimol, which labels GABA receptor sites in brain, was found to bind to a crude membrane fraction prepared from pial blood vessels. Specific 3H-muscimol binding was saturable, of high affinity ( K D = 41 nM), and was selectively inhibited by known GABA agonists. The relative potencies for the agonists in competing for the GABA binding site correlated well with the effectiveness of these compounds in dilating pial arteries in vitro (see Edvinsson et al., this volume). No specific 3H-muscimol binding was detected in aorta and mesenteric arteries. An association of GABA with cerebral blood vessels is further indicated by the presence of the enzyme which synthesizes GABA, glutamate decarboxylase (GAD) in pial arteries and intraparenchymal microvessels. GABA transaminase (GABA-T), the degradative enzyme for GABA, also has been demonstrated histochemically in large concentrations around brain vessels, but not around other, e.g. kidney, vessels. It is suggested that the GABA system in cerebral blood vessels is involved in the control of cerebral vascular tone.

GABA of CNS origin in the rat anterior pituitary inhibits prolactin secretion.

Hypothalamic hormones synthesised in nuclei within the medial basal hypothalamus (MBH) and extrahypothalamic regions and released from nerve endings at the level of the median eminence (ME) into the hypophysial portal capillary system control anterior pituitary (AP) function1. The median eminence is a key area for neuroendocrine regulation as it receives projections of neurotransmitter pathways which modulate functions such as synthesis and release of hypothalamic hormones2. In addition, transmitters released from the ME into the stalk portal blood may act directly at the AP level to control hormone secretion—this has been demonstrated for dopamine (DA) with respect to prolactin (PRL) secretion3 and it has been suggested that DA itself may represent the so far elusive PRL-inhibiting factor (PIF)2,4,5. Recently, evidence has been presented which suggests that γ-aminobutyric acid (GABA) may also have an important inhibitory role in regulating PRL secretion in the rat. Both GABA and muscimol, a powerful agonist at GABA receptors6, inhibited PRL secretion when injected into urethane anaesthetised female7 or freely moving male rats8. In view of the poor penetrability of the blood-brain barrier (BBB) by both amino acids9 and the recently described specific GABA receptor sites in rat AP10, the above data support the notion that GABA may act directly at the level of the AP to inhibit PRL secretion. This proposition is reinforced by the demonstration that both GABA11 and muscimol12 inhibit PRL secretion from rat isolated APs, an effect which is blocked by the antagonist picrotoxin. We report here that GABA, derived from the central nervous sytem (CNS), is present in the rat anterior pituitary, and we provide evidence that GABA has a functional role in the control of PRL secretion.

Modulation of the GABA shunt based on the knowledge of the enzymes involved

A role for GABA in cerebral vascular function is supported by data indicating that specific GABA receptors and GABA-related enzymes are associated with cerebral blood vessels. 3H-Muscimol, which labels GABA receptor sites in brain, was found to bind to a crude membrane fraction prepared from pial blood vessels. Specific 3H-muscimol binding was saturable, of high affinity (KD = 41 nM), and was selectively inhibited by known GABA agonists. The relative potencies for the agonists in competing for the GABA binding site correlated well with the effectiveness of these compounds in dilating pial arteries in vitro (see Edvinsson et al., this volume). No specific 3H-muscimol binding was detected in aorta and mesenteric arteries. An association of GABA with cerebral blood vessels is further indicated by the presence of the enzyme which synthesizes GABA, glutamate decarboxylase (GAD) in pial arteries and intraparenchymal microvessels. GABA transaminase (GABA-T), the degradative enzyme for GABA, also has been demonstrated histochemically in large concentrations around brain vessels, but not around other, e.g. kidney, vessels. It is suggested that the GABA system in cerebral blood vessels is involved in the control of cerebral vascular tone.

Gamma-Aminobutyric acid receptor function in rat anterior pituitary: evidence for control of prolactin release.

The rise in serum PRL concentration induced by administration of haloperidol (0.05-0.5 mg/kg, iv) or morphine (2–15 mg/kg, sc) to male rats and during estrogen treatment (1 mg/day for 15 days) in ovariectomized rats was reduced by systemic injection of the γ-aminobutyric acid (GABA) receptor agonist, muscimol (1 mg/kg, iv). Intraventricular or intrahypothalamic injection of muscimol (100–500 ng) did not alter PRL release or block the PRL release induced by haloperidol or morphine; rather, a slight stimulation of PRL release was observed after intraventricular injection of a dose of 800 μl muscimol. Since the concentration of muscimol in different brain areas after central administration was similar to that after systemic injection, these observations suggested that peripheral rather than central GABA receptors were involved in blocking haloperidol- or morphine-induced PRL release. Proof that peripheral GABA receptors were involved was provided by the experiments with bicuculline methiodide. This specific GABA receptor agonist, which does not enter the brain, counteracted the action of muscimol. The high levels of muscimol in the anterior pituitary after systemic injection and its absence after central administration suggested that blockade of PRL release by muscimol might result from a stimulation of GABA receptors located in the pituitary. Indeed, [3H]muscimol and [3H]GABA receptor sites with characteristics similar to those of brain were present in rat anterior pituitary. In addition, muscimol (10-8–10-6 M) reduced the spontaneous release of PRL from anterior pituitary halves in vitro. This effect was reversed by the GABA receptor antagonist, bicuculline, but not by the dopamine antagonist, sulpiride. The results provide evidence that GABA receptor stimulation in the anterior pituitary can act to regulate PRL release.

Studies on the mechanism of the GABA-mediated inhibition of prolactin secretion.

SummaryGABA has a direct inhibiting effect on prolactin release by the pituitary in vitro and this effect is not blocked by coin-cubation with the specific GABA-receptor antagonist picrotoxin. GABA appears to be at least 100-fold less potent than dopamine as an inhibitor of prolactin secretion. Systemic administration of GABA did not affect basal serum prolactin levels, but inhibited the 5-hydroxytryptophan-induced secretion of prolactin. It is concluded that the direct effect of GABA on prolactin secretion by the pituitary is not mediated by well-defined neurotransmitter mechanisms.

A comparison of the effects of GABA, 3-aminopropanesulphonic acid and imidazoleacetic acid on the frog spinal cord

The effects of GABA, 3-aminopropanesulphonic acid (3APS) or imidazoleacetic acid (IMA) on the isolated spinal cord of the frog were studied by means of extracellular recording techniques. In the presence of tetrodotoxin (6.2 μm), GABA, 3APS or IMA produced dose-dependent dorsal root depolarizations. From the analysis of the dose-response relationships the order of depolarizing potency for these substances was 3APS > GABA > IMA. The effects of IMA were antagonized by either strychnine or picrotoxin whereas those of GABA or 3APS were only antagonized by picrotoxin. None of the observed antagonisms appeared competitive. The action of GABA was dependent on the Na + and Cl − content of the bathing solution while that of 3APS and IMA was dependent only on external Cl −. By stepwise increases in the extracellular K + content it was found that the polarity of 3APS or IMA responses could be inverted at a d.c. level where GABA still produced depolarizations. It is suggested that the mode of action of 3APS and IMA on the frog spinal cord is somewhat different from that of GABA; therefore 3APS and IMA could not be regarded as specific GABA receptor agonists on this preparation.