Methamphetamine-evoked depression of GABA(B) receptor signaling in GABA neurons of the VTA

Psychostimulants induce neuroadaptations in excitatory and fast inhibitory transmission in the ventral tegmental area (VTA). Mechanisms underlying drug-evoked synaptic plasticity of slow inhibitory transmission mediated by GABA(B) receptors and G protein-gated inwardly rectifying potassium (GIRK/Kir(3)) channels, however, are poorly understood. Here, we show that 1 day after methamphetamine (METH) or cocaine exposure both synaptically evoked and baclofen-activated GABA(B)R-GIRK currents were significantly depressed in VTA GABA neurons and remained depressed for 7 days. Presynaptic inhibition mediated by GABA(B)Rs on GABA terminals was also weakened. Quantitative immunoelectron microscopy revealed internalization of GABA(B1) and GIRK2, which occurred coincident with dephosphorylation of serine 783 (S783) in GABA(B2), a site implicated in regulating GABA(B)R surface expression. Inhibition of protein phosphatases recovered GABA(B)R-GIRK currents in VTA GABA neurons of METH-injected mice. This psychostimulant-evoked impairment in GABA(B)R signaling removes an intrinsic brake on GABA neuron spiking, which may augment GABA transmission in the mesocorticolimbic system.     

GABA(A) receptor activation at medullary sympathetic neurons contributes to postexercise hypotension

A single bout of exercise results in a postexercise hypotension (PEH) that is accompanied by a reduced baroreflex function. Based on the role of rostral ventrolateral medulla (RVLM) neurons in controlling sympathetic nerve activity (SNA) and blood pressure, the role of gamma-aminobutyric acid (GABA) in controlling RVLM neuronal activity, and the reduced baroreflex-SNA relationship during PEH, we determined whether: 1) RVLM neuronal activity is decreased during PEH, 2) GABA(A)-receptor mechanisms mediate the decrease, and 3) baroreflex control of RVLM activity is reduced. Spontaneously hypertensive rats (SHR) were subjected to 40 min of treadmill or sham exercise (Sham PEH). PEH lasted 10 h in conscious and anesthetized SHR, indicating that the anesthetics did not affect the expression of PEH. Extracellular RVLM neuronal activity having a cardiac and sympathetic rhythm, lumbar SNA, and blood pressure were recorded at rest and during baroreflex function curves. Resting RVLM neuronal activity was lower and was increased to a greater extent by GABA(A)-receptor antagonism in PEH versus Sham PEH (P < 0.05). Baroreflex control of RVLM neuronal activity operated with a reduced gain (P < 0.05). Thus increased GABA signaling at RVLM neurons may contribute to PEH.     

GABARAP and GABA(A) receptor clustering.

Coyle et al. (2002), in this issue of Neuron, reveal the crystal structure for the GABA(A) receptor binding protein, GABARAP. They show GABARAP can switch from a monomer to an extended linear polymer form that may function to assemble microtubules during the intracellular trafficking or postsynaptic clustering of GABA(A) receptors.     

Mechanism by which GABA, through its GABA(A) receptor, modulates glutamate release from rat cortical neurons in culture

In cortical neurons, the GABA(A) agonist, muscimol, increases: (a) basal glutamate release (with a EC50 of 99 +/- 7 microM); (b) intracellular calcium and (c) membrane potential, all of these in a dose-dependent manner. These muscimol effects were specific since they were reversed by bicuculline, a GABA(A) antagonist. When the action of muscimol was measured at different KCl concentrations, an increase or decrease of the glutamate secretion was observed, depending on the KCl concentration in the medium. At low KCl concentration (5.6 mM of KCl), it depolarized, at 20 mM of KCl it had no effect, but at higher KCl concentrations (30-100 microM of KCl), it produced a hyperpolarization in these cells. The mechanism by which the GABA-Cl(-)-channel permits Cl- fluxes, inward or outward, depending on the membrane potential.     

GABA(A)-mediated toxicity of hippocampal neurons in vitro

In the present study, we examined whether the elevation of GABA by gamma-vinyl-GABA protects cultured rat fetal hippocampal neurons against toxicity induced by a 20-min incubation with 100 microM L-glutamate. Neither a 24-h pretreatment nor posttreatment with gamma-vinyl-GABA (100 microM) had any neuroprotective effects, as determined by counting microtubule-associated protein-2 positive cells and lactate dehydrogenase assay 24 h after the glutamate treatment. Unexpectedly, gamma-vinyl-GABA alone induced a 20% loss of microtubule-associated protein-2-positive cells in a culture that was grown in medium containing 25 mM KCl. The toxic effect of gamma-vinyl-GABA was mimicked by a 24-h treatment with GABA (100 microM) and the GABA(A) receptor agonist, muscimol (10 microM), but not the GABA(B) receptor agonist, baclofen (10 microM). The GABA(A) receptor antagonist, bicuculline (10 microM), protected against gamma-vinyl-GABA and GABA-evoked toxicity. Neither gamma-vinyl-GABA nor GABA was toxic in culture medium containing 15 mM KCl. These data indicate that, under depolarizing conditions, an increased GABA level is toxic for a subpopulation of developing hippocampal neurons in vitro. The effect is GABA(A) receptor-mediated. These data provide a new view for understanding neurodegenerative processes, and raise a question of the safety of therapies aimed at increasing GABA concentration following brain insults, especially in immature brains.     

Changes of GABA(A)receptor activation kinetics in hippocampal neurons cultured for different periods of time

Cell culture is a convenient model for pharmacokinetic studies, but during the culture period, GABA(A)receptors are likely to undergo different modulatory processes. In this study, the current responses to ultrafast GABA applications were recorded from patches excised from neurons cultured for either up to two days (short-term culture) or for more than two weeks (long-term culture). The dose-dependencies of the current rising phases revealed significant differences between the two groups. In the short-term cultures, the responses to both saturating and non-saturating GABA concentrations were slower than in the case of the long-term cultures. We conclude that the GABA(A)receptors in cultured neurons undergo profound kinetic changes involving the modulation of the binding reaction and transitions between bound states.     

Antisense suppression of GABA(A) receptor beta subunit levels in cultured cerebellar granule neurons demonstrates their importance in receptor expression

GABA(A) receptors in the CNS are pentameric molecules composed of alpha, beta, gamma, delta, epsilon and theta subunits. Studies on transfected cells have shown that GABA(A) receptor beta subunit isoforms can direct alpha1 subunit localization within the cell. To examine the role of selected subunits in governing GABA(A) receptor expression in neurons, cultures of rat cerebellar granule cells were grown with antisense or sense oligodeoxynucleotides (ODNs) specific for the alpha 1, beta 2 or gamma 2 subunits. These subunits are all expressed in granule neurons where they are thought to contribute to an abundant receptor type. Following ODN treatment, subunit expression and distribution were examined by western blotting, immunocytochemistry and RT-PCR. Treatment of the cultures with the antisense, but not the corresponding sense, ODNs reduced the levels of the targeted subunit polypeptides. In addition, the beta 2 antisense ODN reduced the level of the alpha1 subunit polypeptide without altering the level of its mRNA. In contrast, treatment with the beta 2 subunit antisense ODN did not alter gamma 2 subunit polypeptide expression, distribution or mRNA level. These findings suggest that the alpha1 subunit requires a beta subunit for assembly into GABA(A) receptors in cerebellar granule neurons.     

GABAergic neurons and GABA(A)-receptors in temporal lobe epilepsy.

Mesial temporal lobe epilepsy (MTLE) is the most prevalent form of epilepsy, characterized by recurrent complex partial seizures and hippocampal sclerosis. The pathophysiology underlying this disorder remains unidentified. While a loss of benzodiazepine binding sites is a key diagnostic feature of MTLE, experimental studies have shown enhanced inhibitory transmission and increased expression of GABA(A)-receptors, suggesting that compensatory mechanisms are operative in epileptic hippocampus. In the present study, changes in the expression and cellular distribution of major GABA(A)-receptor subunits were investigated in the hippocampus of pilocarpine-treated rats during the phase of spontaneous recurrent seizures. A uniform decrease in GABA(A)-receptor subunit-immunoreactivity was observed in regions of extensive neuronal death (i.e. CA1, CA3, hilus). whereas a prominent increase occurred in the dentate gyrus (DG). Most strikingly, the increase was largest for the alpha3- and alpha5-subunits, which are expressed at very low levels in the DG of control rats, suggesting the formation of novel GABA(A)-receptor subtypes in epileptic tissue. Furthermore, an extensive loss of interneurons expressing the alpha1-subunit, representing presumptive basket cells, was seen in the DG. These changes were very similar to those reported in a novel mouse model of MTLE, based on the unilateral injection of kainic acid into the dorsal hippocampus (Bouilleret et al., 1999). This indicates that the regulation of GABA(A)-receptor expression is related to chronic recurrent seizures, and is not due to the extrahippocampal neuronal damage affecting pilocarpine-treated rats. These results allow causal relationships in the induction and maintenance of chronic recurrent seizures to be distinguished. The loss of a critical number of interneurons in the DG is a possible cause of seizure initiation, whereas the long-lasting upregulation of GABA(A)-receptors in granule cells represents a compensatory response to seizure activity.     

GABA(A) receptor alpha-subunit proteins in human chronic alcoholics

Antibodies were raised against specific peptides from N-terminal regions of the alpha1 and alpha3 isoforms of the GABA(A) receptor, and used to assess the relative expression of these proteins in the superior frontal and primary motor cortices of 10 control, nine uncomplicated alcoholic and six cirrhotic alcoholic cases were matched for age and post-mortem delay. The regression of expression on post-mortem delay was not statistically significant for either isoform in either region. In both cortical areas, the regression of alpha1 expression on age differed significantly between alcoholic cases, which showed a decrease, and normal controls, which did not. Age had no effect on alpha3 expression. The alpha1 and alpha3 isoforms were found to be expressed differentially across cortical regions and showed a tendency to be expressed differentially across case groups. In cirrhotic alcoholics, alpha1 expression was greater in superior frontal than in motor cortex, whereas this regional difference was not significant in controls or uncomplicated alcoholics. In uncomplicated alcoholics, alpha3 expression was significantly lower in superior frontal than in motor cortex. Expression of alpha1 was significantly different from that of alpha3 in the superior frontal cortex of alcoholics, but not in controls. In motor cortex, there were no significant differences in expression between the isoforms in any case group.     

Role of the GABA(A)beta2, GABA(A)alpha6, GABA(A)alpha1 and GABA(A)gamma2 receptor subunit genes cluster in drug responses and the development of alcohol dependence

gamma-Aminobutyric acid (GABA) is the major inhibitory neurotransmitter of the central nervous system and it acts at the GABA(A) and GABA(B) receptors. A possible role for the GABA(A) receptors in alcohol action has been derived from in vitro cell models, animal studies and human research. GABA(A) subunit mRNA expression in cell models has suggested that the long form of the gamma2 subunit is essential for ethanol enhanced potentiation of GABA(A) receptors, by phosphorylation of a serine contained within the extra eight amino acids. Several animal studies have demonstrated that alterations in drug and alcohol responses may be caused by amino-acid differences at the GABA(A)alpha6 and GABA(A)gamma2 subunits. An Arg(100)/Glu(100) change at the GABA(A)alpha6 subunit conferring altered binding efficacy of the benzodiazepine inverse agonist Ro 15-4513, was found between the AT (alcohol tolerance) and ANT (alcohol non-tolerance) rats. Several loci related to alcohol withdrawal on mouse chromosome 11 which corresponds to the region containing four GABA(A) subunit (beta2, alpha6, alpha1 and gamma2) genes on human chromosome 5q33-34, were also identified. Gene knockout studies of the role of GABA(A)alpha6 and GABA(A)gamma2 subunit genes in mice have demonstrated an essential role in the modulation of other GABA(A) subunit expression and the efficacy of benzodiazepine binding. Absence of the GABA(A)gamma2 subunit gene has more severe effects with many of the mice dying shortly after birth. Disappointingly few studies have examined the effects of response to alcohol in these gene knockout mice. Human genetic association studies have suggested that the GABA(A)beta2, alpha6, alpha1 and gamma2 subunit genes have a role in the development of alcohol dependence, although their contributions may vary between ethnic group and phenotype. In summary, in vitro cell, animal and human genetic association studies have suggested that the GABA(A)beta2, alpha6, alpha1 and gamma2 subunit genes have an important role in alcohol related phenotypes (300 words).     

A trafficking checkpoint controls GABA(B) receptor heterodimerization

Surface expression of GABA(B) receptors requires heterodimerization of GB1 and GB2 subunits, but little is known about mechanisms that ensure efficient heterodimer assembly. We found that expression of the GB1 subunit on the cell surface is prevented through a C-terminal retention motif RXR(R); this sequence is reminiscent of the ER retention/retrieval motif RKR identified in subunits of the ATP-sensitive K+ channel. Interaction of GB1 and GB2 through their C-terminal coiled-coil alpha helices masks the retention signal in GB1, allowing the plasma membrane expression of the assembled complexes. Because individual GABA(B) receptor subunits and improperly assembled receptor complexes are not functional even if expressed on the cell surface, we conclude that a trafficking checkpoint ensures efficient assembly of functional GABA(B) receptors.     

Nova regulates GABA(A) receptor gamma2 alternative splicing via a distal downstream UCAU-rich intronic splicing enhancer

Nova is a neuron-specific RNA binding protein targeted in patients with the autoimmune disorder paraneoplastic opsoclonus-myoclonus ataxia, which is characterized by failure of inhibition of brainstem and spinal motor systems. Here, we have biochemically confirmed the observation that splicing regulation of the inhibitory GABA(A) receptor gamma2 (GABA(A)Rgamma2) subunit pre-mRNA exon E9 is disrupted in mice lacking Nova-1. To elucidate the mechanism by which Nova-1 regulates GABA(A)Rgamma2 alternative splicing, we systematically screened minigenes derived from the GABA(A)Rgamma2 and human beta-globin genes for their ability to support Nova-dependent splicing in transient transfection assays. These studies demonstrate that Nova-1 acts directly on GABA(A)Rgamma2 pre-mRNA to regulate E9 splicing and identify an intronic region that is necessary and sufficient for Nova-dependent enhancement of exon inclusion, which we term the NISE (Nova-dependent intronic splicing enhancer) element. The NISE element (located 80 nucleotides upstream of the splice acceptor site of the downstream exon E10) is composed of repeats of the sequence YCAY, consistent with previous studies of the mechanism by which Nova binds RNA. Mutation of these repeats abolishes binding of Nova-1 to the RNA in vitro and Nova-dependent splicing regulation in vivo. These data provide a molecular basis for understanding Nova regulation of GABA(A)Rgamma2 alternative splicing and suggest that general dysregulation of Nova's splicing enhancer function may underlie the neurologic defects seen in Nova's absence.     

Positive feedback regulation between gamma-aminobutyric acid type A (GABA(A)) receptor signaling and brain-derived neurotrophic factor (BDNF) release in developing neurons

During the early development of the nervous system, γ-aminobutyric acid (GABA) type A receptor (GABA(A)R)-mediated signaling parallels the neurotrophin/tropomyosin-related kinase (Trk)-dependent signaling in controlling a number of processes from cell proliferation and migration, via dendritic and axonal outgrowth, to synapse formation and plasticity. Here we present the first evidence that these two signaling systems regulate each other through a complex positive feedback mechanism. We first demonstrate that GABA(A)R activation leads to an increase in the cell surface expression of these receptors in cultured embryonic cerebrocortical neurons, specifically at the stage when this activity causes depolarization of the plasma membrane and Ca(2+) influx through L-type voltage-gated Ca(2+) channels. We further demonstrate that GABA(A)R activity triggers release of the brain-derived neurotrophic factor (BDNF), which, in turn by activating TrkB receptors, mediates the observed increase in cell surface expression of GABA(A)Rs. This BDNF/TrkB-dependent increase in surface levels of GABA(A)Rs requires the activity of phosphoinositide 3-kinase (PI3K) and protein kinase C (PKC) and does not involve the extracellular signal-regulated kinase (ERK) 1/2 activity. The increase in GABA(A)R surface levels occurs due to an inhibition of the receptor endocytosis by BDNF, whereas the receptor reinsertion into the plasma membrane remains unaltered. Thus, GABA(A)R activity is a potent regulator of the BDNF release during neuronal development, and at the same time, it is strongly enhanced by the activity of the BDNF/TrkB/PI3K/PKC signaling pathway.     

Regulation of the subcellular distribution and gene expression of GABA(A) receptor by microtubules and microfilaments in cultured brain neurons

Mechanisms underlying the intracellular transport of gamma-aminobutyric acid(A) receptor (GABA(A)R) were examined in the cultured neurons derived from chicken embryo brains. In situ trypsinization of the cultures and (3)H-flunitrazepam (FNZ) binding assay were employed to determine the cell surface and intracellular distribution of the receptor. A 3-h treatment of the cells with 1 microM of colchicine, a microtubule depolymerizer, reversibly raised the proportion of intracellular GABA(A)R density by about 36% and decreased that of the cell surface receptors by 18% from respective control values, whereas the 3-h incubation with 2 microM of cytochalasin D, a microfilament disrupter, did not cause significant changes. These treatments failed to alter the total number of the (3)H-FNZ binding sites of the neurons and the affinity of the ligand. Moreover, the exposure to colchicine seemed to produce a stronger cytoplasmic immunostaining of the GABA(A)R alpha subunits in many neurons without affecting the total cellular level of the proteins, in accordance with the increased fraction of intracellular (3)H-FNZ binding. However, in the neurons exposed to cytochalasin D, there was an increase of around 28% in the total content of alpha(1)+51kDa proteins. In addition, the colchicine or cytochalasin D treatment inhibited approximately 21 or 18% of the rate of general protein synthesis in the culture. Notably, in situ hybridization assay showed that the GABA(A)R alpha(1) or alpha(2) mRNA was present in 92 +/- 2% or 94 +/- 2% of the cytochalasin D-treated neurons, both of which were higher than 71 +/- 2-74 +/- 3% of the control and colchicine-treated cells. The data suggest that by regulating the intracellular transport, the microtubular system participates in the maintenance of normal subcellular distribution of GABA(A)R in the neurons. By contrast, the organization of microfilaments may play a role in modulating the gene expression of GABA(A)R subunits.     

Insulin receptor signaling regulates actin cytoskeletal organization in developing photoreceptors

The insulin receptor (IR) and IR signaling proteins are widely distributed throughout the CNS. IR signaling provides a trophic signal for transformed retinal neurons in culture and we recently reported that deletion of IR in rod photoreceptors by Cre/ lox system resulted in stress-induced photoreceptor degeneration. These studies suggest a neuroprotective role of IR in rod photoreceptor cell function. However, there are no studies available on the role of insulin-induced IR signaling in the development of normal photoreceptors. To examine the role of insulin-induced IR signaling, we analyzed cultured neuronal cells isolated from newborn rodent retinas. In insulin-lacking cultures, photoreceptors from wild-type rat retinas exhibited an abnormal morphology with a wide axon cone and disorganization of the actin and tubulin cytoskeleton. Photoreceptors from IR knockout mouse retinas also exhibited a similar abnormal morphology. A novel finding in this study was that addition of docosahexaenoic acid, a photoreceptor trophic factor, restored normal axonal outgrowth in insulin-lacking cultures. These data suggest that IR signaling pathways regulate actin and tubulin cytoskeletal organization in photoreceptors; they also imply that insulin and docosahexaenoic acid activate at least partially overlapping signaling pathways that are essential for the development of normal photoreceptors.     

Evolution of GABA(A) receptor diversity in the human genome

Nowhere is the record of receptor evolution more accessible than in the organization of the 19 vertebrate genes coding for subunits of the major inhibitory neurotransmitter receptor in the central nervous system, the gamma-aminobutyric acid receptor (GABAAR). Co-expression of alpha, beta, and gamma subunit genes is necessary for the formation of a GABAAR that is potentiated by widely used anxiolytics, anticonvulsants, and hypnotics. The identification of alpha, beta, and gamma genes on chromosomes 4, 5, and 15 suggests that co-localization of a gamma gene with an alpha and beta may be important for brain function. We have now directly examined the organization of GABAAR subunit genes on human chromosomes. Estimates of physical distance using in situ hybridization to cells in interphase, and gene localization using hybridization to cells in metaphase demonstrate the existence of beta-alpha-alpha-gamma gene clusters in cytogenetic bands on chromosomes 4(p12) and 5(q34). Sequencing of PAC clones establishes intercluster conservation of a unique head-to-head configuration for alpha and beta genes on chromosomes 4, 5, and 15. Remarkably, phylogenetic tree analysis predicts the existence of a beta-alpha-gamma ancestral gene cluster in which internal duplication of an ancestral alpha was followed by cluster duplication, resulting in the relative chromosomal positions of modern GABAAR subunit genes in the human genome.     

Synthesis and GABA(A) receptor activity of oxygen-bridged neurosteroid analogs

Three analogs of neuroactive steroids were prepared (4-6) in which 1,11- or 11,19-oxygen bridges give a constrained conformation. Their 3D structures were obtained by ab initio calculations and in the case of 3alpha-hydroxy-11,19-epoxypregn-4-ene-20-one (4), confirmed by X-ray analysis. Biological activity of the synthetic steroids was assayed in vitro using t-[(3)H]butylbicycloorthobenzoate as radiolabeled ligand for the GABA(A) receptor. The activity of compound 4 was similar to that of allopregnanolone (1). 1alpha,11alpha-Epoxypregnanolone (6) was more active than pregnanolone (2).     

Detection and binding properties of GABA(A) receptor assembly intermediates

Density gradient centrifugation of native and recombinant gamma-aminobutyric acid, type A (GABA(A)) receptors was used to detect assembly intermediates. No such intermediates could be identified in extracts from adult rat brain or from human embryonic kidney (HEK) 293 cells transfected with alpha(1), beta(3), and gamma(2) subunits and cultured at 37 degrees C. However, subunit dimers, trimers, tetramers, and pentamers were found in extracts from the brain of 8-10-day-old rats and from alpha(1)beta(3)gamma(2) transfected HEK cells cultured at 25 degrees C. In both systems, alpha(1), beta(3), and gamma(2) subunits could be identified in subunit dimers, indicating that different subunit dimers are formed during GABA(A) receptor assembly. Co-transfection of HEK cells with various combinations of full-length and C-terminally truncated alpha(1) and beta(3) or alpha(1) and gamma(2) subunits and co-immunoprecipitation with subunit-specific antibodies indicated that even subunits containing no transmembrane domain can assemble with each other. Whereas alpha(1)gamma(2), alpha(1)Ngamma(2), alpha(1)gamma(2)N, and alpha(1)Ngamma(2)N, combinations exhibited specific [(3)H]Ro 15-1788 binding, specific [(3)H]muscimol binding could only be found in alpha(1)beta(3) and alpha(1)beta(3)N, but not in alpha(1)Nbeta(3) or alpha(1)Nbeta(3)N combinations. This seems to indicate that a full-length alpha(1) subunit is necessary for the formation of the muscimol-binding site and for the transduction of agonist binding into channel gating.