Biomedical subjects

D R Grayson

Publications and source records attributed to D R Grayson.

At least 37 records · Page 2Linked to original sources

PubMed1998

Differentially expressed GABAA-receptor subunits result in structurally and functionally receptor assemblies following excitatory afferent synaptic transmission.

Cerebellar granule cells isolated from postnatal day 7 rat pups are ideal for studying epigenetic events associated with the regulation of neuronal gene expression. These cultures contain from 90 to 95% glutamatergic granule cells and express mRNAs encoding a variety of ionotropic and metabotropic glutamate receptors as well as virtually all of the GABAA-receptor subunit mRNAs to different extents. A unique feature of this culture system is that the neurons undergo time-dependent maturation changes in vitro that mimic many of the characteristics of these receptors occurring in vivo. Granule cell cultures in vitro require depolarizing concentrations of KCl for long-term growth and survival. Both N-methyl-D-aspartate (NMDA) and GABA have been reported to exert trophic actions on these cells replacing the requirement for maintaining the cultures in high KCl. Cerebellar granule cells maintained under different conditions in vitro can be induced to alter their patterns of maturation, as indicated by the different temporal changes in gene expression of receptor subunit mRNAs and proteins. The focus of the current studies is the effect of NMDA afferent synaptic signaling on the changes in mRNA content and functional properties of GABAA receptors and how this may relate to comparable changes shown to occur in vivo.

Animals↗

PubMed1997

Alterations of GABA(A)beta2/3 immunoreactivity in the dentate gyrus after perforant pathway lesion.

Immunocytochemical techniques were employed to examine the changes in the GABA receptor subunits beta2/3 within the dentate gyrus of the rat brain 1, 3, 7, 14, 30 and 90 days after a unilateral perforant pathway lesion. Three days post-lesion we observed a decrease in beta2/3 immunolabeling in the inner molecular layer of the dentate gyrus followed by a comparable decrease in the outer molecular layer 7 days post-lesion. These decreases were transient; 30 and 90 days post-lesion, beta2/3 immunolabeling appeared similar to controls in the inner portion of the molecular layer, while in the outer region the labeling was increased. In this latter region we also observed a dense band of AChE fibers. Following survival times of 3 days we observed a diffuse staining of the neuropil in the hilar region, and a dense amorphous accumulation of peroxidase reaction product in the polymorphic region. These responses were transient and by 14 days the hilar/polymorphic region appeared indistinguishable from controls. These data suggest a unique pattern of immunoabeling in the molecular and polymorphic region in response to perforant pathway lesion. A putative explanation for this response is discussed.

Acetylcholinesterase↗

PubMed1997

Regulation of GABA(A) receptor subunit mRNA expression by the pesticide dieldrin in embryonic brainstem cultures: a quantitative, competitive reverse transcription-polymerase chain reaction study.

Cyclodiene organochlorine pesticides, such as dieldrin, inhibit gamma-aminobutyric acid (GABA)ergic neurotransmission by blocking the Cl- channel of GABA(A) receptors. This action may make the developing nervous system especially vulnerable to these neurotoxins, which could interfere with the trophic actions of GABA on developing neurons and alter expression of GABA(A) receptors. We have used an in vitro model to determine whether exposure to dieldrin alters developmental expression of GABA(A) receptor subunit mRNA transcripts. Dissociated cell cultures were prepared from embryonic day 14 (E14) brainstem and cultured in serum-containing medium for 1 day in vitro (DIV), then treated for 2 DIV with 10 microM dieldrin in serum-free medium. This dose was based on preliminary experiments and previous studies (Nagata et al.: Brain Res 645:19-26, 1994; Pomes et al.: J Pharmacol Exp Ther 271:1616-1623, 1994). Absolute amounts of alpha1, beta3, gamma1, gamma2S and gamma2L mRNA transcripts were quantified in these cultures by quantitative, competitive reverse transcription-polymerase chain reaction (RT-PCR) using subunit-selective internal standards. The most abundant GABA(A) subunit transcript was beta3, which was much more highly expressed than gamma2S, gamma1, gamma2L, or alpha1 subunit mRNAs. Dieldrin differentially regulated expression of these transcripts. Levels of beta3 subunit transcripts were significantly increased (by 300%) by dieldrin, whereas expression of gamma2S and gamma2L transcripts were decreased (by 50% and 40%, respectively). However, dieldrin did not alter the ratio of gamma2S to gamma2L transcripts, indicating that it did not affect alternative splicing of gamma2 transcripts. Dieldrin appeared to increase expression of alpha1 subunit transcripts, but this effect was not statistically significant. Dieldrin did not significantly alter expression of gamma1 subunit transcripts. These results support the hypothesis that in utero exposure to cyclodiene pesticides could pose a risk to the developing brain by virtue of their ability to alter gene expression of GABA(A) receptor subunits, which could produce GABA(A) receptors with altered functional properties.

Animals↗

PubMed1997

GABAA receptors mediate trophic effects of GABA on embryonic brainstem monoamine neurons in vitro.

The inhibitory neurotransmitter GABA may act as a trophic signal for developing monoamine neurons in embryonic rat brain, because GABA neurons and their receptors appear in brainstem during generation of monoamine neurons. To test this hypothesis, we used dissociated cell cultures from embryonic day 14 rat brainstem, which contains developing serotonin (5-HT), noradrenaline (tyrosine hydroxylase; TH), and GABA neurons. Immunocytochemistry and reverse transcription-PCR (RT-PCR) revealed the presence of multiple alpha, beta, gamma, and delta subunits in these cultures. Competitive RT-PCR demonstrated high levels of beta3 subunit transcripts. Expression of functional GABAA receptors was demonstrated using 36Cl- flux assays. To investigate GABAergic regulation of neuronal survival and growth, cultures were treated for 1-3 d in vitro with 10 microM GABA and/or GABAA antagonist (bicuculline or the pesticide dieldrin). The effects of treatments were quantified by analysis of immunoreactive 5-HT, TH, and GABA neurons. GABAA receptor ligands differentially regulated neuronal survival and growth depending on neurotransmitter phenotype. GABA exerted positive effects on monoamine neurons, which were countered by bicuculline (and dieldrin, 5-HT neurons only). By itself, bicuculline produced inhibitory effects on both 5-HT and TH neurons, whereas dieldrin potently inhibited 5-HT neurons only. GABA neurons responded positively to both antagonists, but more strongly to bicuculline. Taken together, these results demonstrate that the activation/inhibition of GABAA receptors produces opposite effects on the development of embryonic monoamine and GABA neurons. This suggests that these neurotransmitter phenotypes may express GABAA receptors that differ in fundamental ways, and these differences determine the developmental responses of these cells to GABAergic stimuli.

Animals↗

PubMed1997

Immunohistochemical study of GABA(A) receptor beta2/3 subunits in the hippocampal formation of aged brains with Alzheimer-related neuropathologic changes.

In AD, it is hypothesized that one factor contributing to the vulnerability of neurons is a delicate balance of excitatory and inhibitory inputs. To examine this hypothesis we have initiated a number of studies examining the role of the excitatory neurotransmitter glutamate and the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) in the neurodegeneration of AD. As an initial investigation into the GABAergic system in AD, we employed immunocytochemical techniques and examined the distribution and density of the GABAA receptor subunits beta2/3 within the hippocampus of 13 subjects with a clinical diagnosis of AD and 6 nondemented elderly subjects. Collectively, these 19 subjects presented with a broad range of pathologic severity (i.e., Braak stages I-VI). Density measurements of nine hippocampal regions demonstrated highest levels of beta2/3 immunolabeling in the inner molecular layer of the dentate gyrus > CA1 > CA2, while the lowest levels were found in the granular layer of the dentate gyrus < or = CA4 < CA3 field. Despite these regional variations no significant difference in the mean density of beta2/3 immunolabeling was observed when comparing the pathologically mild (stages I and II), moderate (stages III and IV), and severe (stages V and VI) groups. These data suggest that in the hippocampus receptor subunits associated with GABAergic neurotransmission are relatively maintained even until the terminal stages of the disease.

Aged↗

PubMed1997

GABA and NMDA in the prevention of apoptotic-like cell death in vitro.

We have shown recently that cerebellar granule neurons die in the absence of depolarizing concentrations of KCl through an apoptosis-like process. To study the contributions of inhibitory (gamma-aminobutyric acid; GABA) and excitatory (glutamate) neurotransmitters in the prevention of apoptotic-like cell death in cultures grown in the presence of reduced concentrations of KCl (12.5 mM), we treated these cultures either acutely or chronically with GABA, bicuculline methiodide, a GABAA receptor antagonist, N-methyl-D-aspartate (NMDA) and/or the NMDA receptor antagonist, MK-801. Cell viability was measured with fluorescein diacetate/propidium iodide (FDA/PI) and trypan blue exclusion tests. In addition, DNA fragmentation was assessed quantitatively using an in situ terminal deoxynucleotidyl transferase assay. Our results demonstrate that treatment of cerebellar granule cell cultures maintained in 12.5 mM KCl with the glutamate receptor agonist NMDA and/or bicuculline protects against cell death and reduces DNA fragmentation. In contrast, GABA potentiated cerebellar granule cell apoptosis mediated by KCl deprivation. These data indicate that signal transduction pathways activated following NMDA receptor stimulation mimic the anti-apoptotic action of high potassium in primary cultures of cerebellar granule neurons. Also, our data support an inhibitory (hyperpolarizing) role for GABA in these cultures. Collectively, the results suggest that the neurotrophic actions of NMDA on granule cells maintained in low KCl and GABA on granule cells cultured in high KCl are due to the necessity for maintaining appropriate intraneuronal calcium concentrations.

Animals↗

PubMed1997

N-acetylaspartylglutamate stimulates metabotropic glutamate receptor 3 to regulate expression of the GABA(A) alpha6 subunit in cerebellar granule cells.

We have shown that the vertebrate neuropeptide N-acetylaspartylglutamate (NAAG) meets the criteria for a neurotransmitter, including function as a selective metabotropic glutamate receptor (mGluR) 3 agonist. Short-term treatment of cerebellar granule cells with NAAG (30 microM) results in the transient increase in content of GABA(A) alpha6 subunit mRNA. Using quantitative PCR, this increase was determined to be up to 170% of control values. Similar effects are seen following treatment with trans-1-aminocyclopentane-1,3-dicarboxylate and glutamate and are blocked by the mGluR antagonists (2S,3S,4S)-2-methyl-2-(carboxycyclopropyl) glycine and (2S)-alpha-ethylglutamic acid. The effect is pertussis toxin-sensitive. The increase in alpha6 subunit mRNA level can be simulated by activation of other receptors negatively linked to adenylate cyclase activity, such as adenosine A1, alpha2-adrenergic, muscarinic, and GABA(B) receptors. Forskolin stimulation of cyclic AMP (cAMP) levels abolished the effect of NAAG. The change in alpha6 levels induced by 30 microM NAAG can be inhibited in a dose-dependent manner by simultaneous application of increasing doses of the beta-adrenergic receptor agonist isoproterenol. The increase in alpha6 mRNA content is followed by a fourfold increase in alpha6 protein level 6 h posttreatment. Under voltage-clamped conditions, NAAG-treated granule cells demonstrate an increase in the furosemide-induced inhibition of GABA-gated currents in a concentration-dependent manner, indicating an increase in functional alpha6-containing GABA(A) receptors. These data support the hypothesis that NAAG, acting through mGluR3, regulates expression of the GABA(A) alpha6 subunit via a cAMP-mediated pathway and that cAMP-coupled receptors for other neurotransmitters may similarly influence GABA(A) receptor subunit composition.

Animals↗

PubMed1997

Gene knockout of the alpha6 subunit of the gamma-aminobutyric acid type A receptor: lack of effect on responses to ethanol, pentobarbital, and general anesthetics.

The alpha6 subunit of the gamma-aminobutyric acid type A receptor (GABA(A)-R) has been implicated in mediating the intoxicating effects of ethanol and the motor ataxic effects of general anesthetics. To test this hypothesis, we used gene targeting in embryonic stem cells to create mice lacking a functional alpha6 gene. Homozygous mice are viable and fertile and have grossly normal cerebellar cytoarchitecture. Northern blot and reverse transcriptase-polymerase chain reaction analyses demonstrated that the targeting event disrupted production of functional alpha6 mRNA. Autoradiography of histological sections of adult brains demonstrated that diazepam-insensitive binding of [3H]Ro15-4513 to the cerebellar granule cell layer of wild-type mice was completely absent in homozygous mice. Cerebellar GABA(A)-R density was unchanged in the mutant mice; however, the apparent affinity for muscimol was markedly reduced. Sleep time response to injection of ethanol after pretreatment with vehicle or Ro15-4513 did not differ between genotypes. Sleep time response to injection of pentobarbital and loss of righting reflex and response to tail clamp stimulus in mice anesthetized with volatile anesthetics also did not differ between genotypes. Thus, the alpha6 subunit of the GABA(A)-R is not required for normal development, viability, and fertility and does not seem to be a critical or unique component of the neuronal pathway mediating the hypnotic effect of ethanol and its antagonism by Ro15-4513 in mice. Similarly, the alpha6 subunit does not seem to be involved in the behavioral responses to general anesthetics or pentobarbital.

Anesthetics, Inhalation↗

PubMed1996

Developmental and mature expression of full-length and truncated TrkB receptors in the rat forebrain.

The neurotrophins brain-derived neurotrophic factor (BDNF) and NT-4/5 exert their trophic effects on the nervous system via signaling through trkB receptors. These receptors occur as splice variants of the trkB gene that encodes a full-length receptor containing the signal transducing tyrosine kinase domain as well as truncated forms lacking this domain. Because the importance of the trkB isoforms for development and maturation of the nervous system is unknown, we have examined the expression of trkB receptor isoforms during development of the rat forebrain using 1) a sensitive ribonuclease protection assay to distinguish full-length and truncated trkB transcripts, 2) western blot analysis to characterize developmental changes in trkB proteins, and 3) immunohistochemistry to determine the cellular localization of trkB receptors. In the rat forebrain, adult mRNA levels for full-length trkB are reached by birth, whereas truncated trkB message does not peak until postnatal days 10-15. Western blot analysis indicates that full-length trkB protein is the major form during early development, whereas truncated trkB protein predominates in all forebrain regions of late postnatal and adult rats. These data also suggest that the glycosylation state of these receptors changes during postnatal maturation. TrkB immunoreactivity is present predominately within neurons, where it is localized to axons, cell soma, and dendrites. Strong dendritic immunostaining is particularly evident in certain neuronal populations, such as pyramidal neurons in the hippocampus and in layer V of the neocortex. The dendritic localization of trkB receptors supports the hypothesis that dendrites, as well as axons, are important sites for neurotrophin actions in the central nervous system.

Animals↗

PubMed1996

Chronic flumazenil alters GABA(A) receptor subunit mRNA expression, translation product assembly and channel function in neuronal cultures.

Flumazenil competitively blocks the pharmacological effects of both positive and negative allosteric modulators acting at the benzodiazepine binding sites of gamma-aminobutyric acid (GABA(A)) receptors. Using quantitative reverse transcription polymerase chain reaction, label-fracture immunocytochemistry and whole-cell patch-clamp recordings, we determined changes in the contents of selected GABA(A) receptor subunit mRNA(s), in their translation products and in the electrophysiological characteristics of the receptor channels in cultured cerebellar granule cells treated daily with flumazenil (10 microM) for 4 days in vitro. The contents of the alpha1 and alpha6 receptor subunit mRNAs were significantly increased in the flumazenil-treated group as compared with the dimethyl sulfoxide vehicle-treated control group, whereas there were no significant differences in the absolute amounts of the beta2, beta3, gamma2S, gamma2L++ + and delta receptor subunit mRNAs. The gold immunolabeling densities of the alpha1 and delta receptor subunits were significantly increased, whereas those of the alpha6, beta2/beta3 and gamma2 receptor subunits were decreased. Double-immunolabeling experiments using 5- and 10-nm gold particles suggest that after chronic flumazenil treatment, receptor subunit assemblies containing the alpha1/gamma2 and alpha6/delta subunits may be replaced by a receptor assembly containing the alpha1/delta subunits. The GABA potency in eliciting Cl- channel activity decreased significantly, as indicated by the elevated EC50 values, and the positive modulation of GABA action by diazepam also decreased. These results suggest that flumazenil, perhaps by blocking the action of endogenous allosteric modulators of GABA(A) receptors, may trigger a change in the expression and assembly of the subunits of the GABA(A) receptor. This implies that there might be a dynamic state in the regulation of GABA(A) receptor structure.

Animals↗

PubMed1996

Alpha 6 and gamma 2 subunit antisense oligodeoxynucleotides alter gamma-aminobutyric acid receptor pharmacology in cerebellar granule neurons.

To characterize the role of the alpha 6 subunit in gamma-aminobutyric acid (GABA) receptors in cerebellar granule cells, primary cerebellar cultures were treated with antisense oligodeoxynucleotides (ODNs) complementary to and overlapping the initial codon of the alpha 6 subunit cDNA. The specific reduction in the expression of the alpha 6 receptor subunit protein after a 48-hr antisense ODN treatment was assessed with the use of immunoblot assays. Sister cultures were treated in parallel with mismatched (scrambled) ODNs. Inhibition of GABA-gated currents by furosemide, a selective inhibitor of GABAA receptors containing alpha 6 subunits, was attenuated after the alpha 6 antisense treatment. Furosemide was tested in parallel in transfected cells expressing various combinations of the alpha 1 and alpha 6 subunits, which showed that the relative abundance of these subunit mRNAs determines the extent of furosemide-induced inhibition of GABA-gated currents. Compared with control or mismatched ODN-treated cell cultures, treatment of granule neurons with alpha 6 antisense ODNs caused a decrease in GABA-induced maximal current density and increased the half-maximal concentration derived from GABA dose-response curves. Furthermore, the depletion of alpha 6 subunits from cerebellar granule cells enhanced flunitrazepam-induced potentiation of GABA-activated currents. In contrast, gamma 2 antisense ODN treatments of cell cultures increased the receptor sensitivity to GABA and potently decreased the response to flunitrazepam. Our results show that alpha 6 and gamma 2 subunit expression can be blocked with the use of synthetic ODNs and that these subunits are crucial determinants of the pharmacological properties of native GABAA receptors in cerebellar granule cells.

Allosteric Regulation↗

PubMed1995

Intrauterine cocaine exposure of rabbits: persistent elevation of GABA-immunoreactive neurons in anterior cingulate cortex but not visual cortex.

The effects of prenatal cocaine exposure on the development of the rabbit cerebral cortex were studied. Two cortical areas were compared: primary visual cortex (VC) and anterior cingulate cortex (ACC). ACC was selected because behavioral deficits observed in cocaine-exposed infants suggest the involvement of ACC. In addition, ACC receives dense dopaminergic innervation and cocaine's action in inhibiting the re-uptake of dopamine is believed to underly the rewarding properties of cocaine. VC was selected as a control area because there is no evidence of behavioral deficits associated with visual perception in cocaine-exposed infants, and because VC receives minimal dopaminergic innervation. Two aspects of cortical development were studied: (i) cortical morphology, growth and cytoarchitectonic organization; and (ii) the development of the GABAergic neurotransmitter system. Measures of postnatal cortical growth, including cortical lamination, cell number and soma size, were compared in cocaine-exposed or control (saline) rabbits aged P5-P60. There was no difference between cocaine and saline animals in any of these parameters, and cortical cytoarchitecture appeared normal. However, despite the absence of major abnormalities in cortical development, we found that the number of GABA-immunoreactive neurons in cocaine-exposed animals was significantly higher than normal in ACC. This effect was highly consistent, was present in all laminae and at all ages studied, and persisted into maturity (P60). In contrast, in VC, the number of GABA-immunoreactive neurons in cocaine-exposed animals did not differ from normal. We suggest that increased GABA immunoreactivity may reflect a compensatory response to excessive excitatory input to ACC. A change in the balance of excitation and inhibition in ACC, reflecting 'noisy' or dysfunctional intracortical circuitry, may underly the emotional lability and attentional deficits characteristically described in infants exposed in utero to cocaine.

Animals↗

PubMed1995

Neuronal apoptosis in an in vitro model of photochemically induced oxidative stress.

In neurons, oxidative stress can be triggered by neurotransmitter-linked mechanisms and may lead to apoptotic cell death. A simple and reproducible model of inducing oxidative stress is needed to elucidate mechanisms which link oxidative stress and neuronal apoptosis. We report here a method of inducing apoptosis in cell cultures by loading them with a photosensitive dye, rose bengal, and exposing the cultures to light, a procedure which generates reactive singlet oxygen. We used this model in primary culture of rat cerebellar granule neurons, and in a nonneuronal human embryonic kidney 293 cell line. We have measured the following: (a) metabolic activity of the mitochondria by quantitative staining with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), (b) DNA fragmentation by quantitative in situ terminal deoxynucleotidyl transferase assay, and (c) cell viability by a trypan blue exclusion test. The oxidative stress caused an early impairment of mitochondrial function (MTT assay). This was followed by DNA fragmentation and ultimately by cell death. Protection was obtained with an inhibitor of macromolecular synthesis, anisomycin, and with antioxidant, vitamin E. This model can be used to study the mechanism of oxidative stress-triggered neuronal apoptosis, and it may help in discovering new targets for neuroprotective drugs.

Animals↗

PubMed1995

Exposure of neuronal cultures to K+ depolarization or to N-methyl-D-aspartate increases the transcription of genes encoding the alpha 1 and alpha 5 GABAA receptor subunits.

The transcription rates of the alpha 1, alpha 5, and alpha 6 gamma-aminobutyric acidA receptor subunit genes were analyzed in cultures maintained in low KCl (12.5 mM), in low KCl treated with NMDA (10 microM), and in high KCl (25 mM). alpha 1 and alpha 5 transcription rates were significantly increased in response to NMDA or high KCl treatment, while alpha 6 and cyclophilin transcription rates were not changed by either condition. These data suggest that following NMDA or high KCl treatment of granule cells, changes in alpha 1 and alpha 5 mRNA content are a consequence of a specific transcriptional rate increase of the corresponding subunit genes.

Animals↗

PubMed1995

Characterization of NMDA receptor subunit-specific antibodies: distribution of NR2A and NR2B receptor subunits in rat brain and ontogenic profile in the cerebellum.

Selective antisera for NMDA receptor subunits NR2A and NR2B have been developed. Each antiserum identifies a single band on an immunoblot at approximately 175 kDa that appears to be the appropriate subunit of the NMDA receptor. Using these antisera the relative densities of the subunits in eight areas of adult rat brain have been determined. The NR2A subunit was found to be at its highest level in hippocampus and cerebral cortex, to be at intermediate levels in striatum, olfactory tubercle, mid-brain, olfactory bulb, and cerebellum, and to be at lowest levels in the pons-medulla. The NR2B subunit was found to be expressed at its highest levels in the olfactory tubercle, hippocampus, olfactory bulb, and cerebral cortex. Intermediate levels were expressed in striatum and mid-brain, and low levels were detected in the pons-medulla. No signal for NR2B was found in the cerebellum. These regional distributions were compared with that for [3H]MK-801 binding sites. It was found that although the distribution of the NR2A subunit corresponds well with radioligand binding, the distribution of the NR2B subunit does not. The ontogenic profiles of NR2A and NR2B subunits in the rat cerebellum were also determined. Just following birth [postnatal day (P) 2] NR2A subunits are undetectable, whereas NR2B subunits are expressed at amounts easily measurable. Beginning at about P12 the levels of NR2A rise rapidly to reach adult levels by P22. At the same time (P12), levels of NR2B protein begin to decline rapidly to reach undetectable levels by 22 days after birth.(ABSTRACT TRUNCATED AT 250 WORDS)

Aging↗

PubMed1995

Opposite effect of protein synthesis inhibitors on potassium deficiency-induced apoptotic cell death in immature and mature neuronal cultures.

Typically, primary cultures of rat cerebellar granule neurons are grown in the presence of 25 mM KCl and are considered to mature by approximately 7 days in vitro. Potassium deficiency was created by growing the neurons from days 1 to 4 in the presence of 12.5 mM KCl (immature cultures) or by switching the mature neurons grown with 25 mM KCl to 12.5 mM KCl. In both conditions we observed neuronal death that bears the signs of apoptosis, i.e., DNA fragmentation determined qualitatively by agarose gel electrophoresis of DNA and quantitatively by in situ terminal deoxynucleotidyl transferase assay. The protein synthesis inhibitors cycloheximide and anisomycin provided neuroprotection in the mature cultures but potentiated the toxic effect of KCl deprivation in the immature neurons. The results suggest that a prudent use of protein synthesis inhibitors is critical in experiments with primary neuronal cultures.

Animals↗

PubMed1995

NMDA-mediated modulation of gamma-aminobutyric acid type A receptor function in cerebellar granule neurons.

GABAA receptors are ligand-gated CI- ion channels with multiple clinically relevant drug-recognition sites. We have previously shown that stimulation of N-methyl-D-aspartic acid (NMDA)-specific glutamate receptors quantitatively alters selected GABAA receptor subunit mRNAs and proteins in primary cultures of rat cerebellar granule neurons. We used whole-cell recordings of GABA-elicited CI- currents and flunitrazepam binding experiments in granule cell cultures maintained in low K+ (12.5 mM), cells maintained in low K+ and treated with a single dose of NMDA (10 microM), and cell cultures maintained in depolarizing concentrations of K+ (25 mM). The EC50 obtained from the dose-response curves for GABA in eliciting a maximal response was comparable in neurons maintained in high K+ or in low K+ and treated with a single dose of NMDA, but that it increased significantly in cells maintained in low K+. The potentiation of GABA-gated CI- currents by flunitrazepam increased significantly, while the negative allosteric modulator methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM) was significantly more effective in cultures either maintained in high K+ or treated with NMDA. This was coincident with a twofold increase in the Bmax associated with flunitrazepam binding. To further characterize the receptor assemblies present in the depolarization and NMDA induced paradigms, the Zn(2+)-induced inhibition of GABA-gated CI- currents was reduced as was the inhibition mediated by furosemide. Our data indicate that GABAA receptor assemblies alter their composition in response to excitatory afferent receptor stimulation.

Animals↗

PubMed1995

Chronic ethanol consumption differentially alters the expression of gamma-aminobutyric acidA receptor subunit mRNAs in rat cerebral cortex: competitive, quantitative reverse transcriptase-polymerase chain reaction analysis.

The molecular mechanisms that underlie ethanol dependence appear to involve alterations in GABAA receptor function and gene expression. In rat cerebral cortex, chronic exposure to ethanol alters many functional properties of GABAA receptors, including reduction of GABAA receptor-mediated chloride uptake. These functional alterations occur without a concomitant alteration in total receptor density or affinity. Previous investigations have shown that chronic ethanol exposure elicits alterations in mRNA and polypeptide levels for several abundant GABAA receptor subunits. For example, alpha 1 and alpha 2 subunit mRNA and polypeptide levels have been shown to decrease with chronic ethanol exposure. The present study was undertaken to further investigate the effects of chronic ethanol consumption on GABAA receptor subunit mRNA levels in rat cerebral cortex by using a competitive, quantitative reverse transcriptase-polymerase chain reaction assay that incorporates subunit-specific internal standards and allows for the absolute quantification of mRNA levels. We find that chronic ethanol consumption elicits a significant increase in alpha 4 subunit mRNA levels that is equal, in absolute amount, to a decrease in alpha 1 subunit mRNA levels. There is a small (30%) increase in gamma 2S but not gamma 2L subunit mRNA levels after chronic ethanol consumption. In addition, gamma 1 subunit mRNA levels are increased by 70%, whereas alpha 5, beta 1, beta 2, beta 3, gamma 3, and delta subunit mRNA levels do not change. We also reproduced results obtained previously by Northern blot analysis showing a 40% reduction in alpha 1 mRNA levels with no change in beta 2 subunit mRNA levels after chronic ethanol consumption. These results are consistent with the hypothesis that chronic ethanol consumption alters the function of GABAA receptors by eliciting changes in receptor subunit assembly. These changes may underlie the development of ethanol dependence.

Animals↗