Selective modulation of GABAA receptors by aluminum

Aluminum has been implicated in several neurodegenerative conditions including Alzheimer's disease. Because the mammalian olfactory system has an unusual capacity for the uptake and transneuronal spread of inhaled substances such as aluminum, whole cell recording techniques were used to examine the actions of aluminum on basic membrane properties and amino acid receptors on rat olfactory bulb mitral/tufted (M/T) neurons in culture. Aluminum had little direct effects on M/T neurons. Aluminum (100 microM) did not evoke a membrane current or alter action-potential shape or duration. Aluminum also had no marked effects on the family of voltage-gated membrane currents evoked by a series of 10-mV, 50-ms depolarizing steps. However, aluminum dramatically potentiated the current evoked by 30 microM gamma-aminobutyric acid (GABA) at concentrations <100 microM. Conversely, higher concentrations of aluminum blocked the GABA-evoked current. The effects of aluminum on GABA-evoked currents were not voltage dependent. Aluminum (100 microM) equally potentiated both inward currents at -30 mV and outward currents at + 30 mV. At 300 microM, aluminum blocked both inward and outward currents to a similar extent. In some neurons, aluminum only blocked the current and potentiation was not observed. The biphasic action of aluminum on GABA-evoked currents suggests separate binding sites: a high-affinity potentiating site and a low-affinity inhibiting site. Despite its effects on GABA-evoked currents, aluminum did not alter membrane currents evoked by glutamate, N-methyl-D-aspartate, kainate, or glycine. Aluminum also did not reduce spontaneous excitatory synaptic activity, suggesting little, if any, effect on glutamate release. Although a causal role for aluminum in Alzheimer's disease and other neuropathological conditions remains controversial, it is clear that elevated aluminum concentrations in the brain are associated with a variety of cognitive impairments. The present results indicate that aluminum can alter the function of GABAA receptors and may suggest that aluminum can contribute to cognitive impairment through disruption of inhibitory circuits.     

Glutamate inhibits GABA excitatory activity in developing neurons

In contrast to the mature brain, in which GABA is the major inhibitory neurotransmitter, in the developing brain GABA can be excitatory, leading to depolarization, increased cytoplasmic calcium, and action potentials. We find in developing hypothalamic neurons that glutamate can inhibit the excitatory actions of GABA, as revealed with fura-2 digital imaging and whole-cell recording in cultures and brain slices. Several mechanisms for the inhibitory role of glutamate were identified. Glutamate reduced the amplitude of the cytoplasmic calcium rise evoked by GABA, in part by activation of group II metabotropic glutamate receptors (mGluRs). Presynaptically, activation of the group III mGluRs caused a striking inhibition of GABA release in early stages of synapse formation. Similar inhibitory actions of the group III mGluR agonist L-AP4 on depolarizing GABA activity were found in developing hypothalamic, cortical, and spinal cord neurons in vitro, suggesting this may be a widespread mechanism of inhibition in neurons throughout the developing brain. Antagonists of group III mGluRs increased GABA activity, suggesting an ongoing spontaneous glutamate-mediated inhibition of excitatory GABA actions in developing neurons. Northern blots revealed that many mGluRs were expressed early in brain development, including times of synaptogenesis. Together these data suggest that in developing neurons glutamate can inhibit the excitatory actions of GABA at both presynaptic and postsynaptic sites, and this may be one set of mechanisms whereby the actions of two excitatory transmitters, GABA and glutamate, do not lead to runaway excitation in the developing brain. In addition to its independent excitatory role that has been the subject of much attention, our data suggest that glutamate may also play an inhibitory role in modulating the calcium-elevating actions of GABA that may affect neuronal migration, synapse formation, neurite outgrowth, and growth cone guidance during early brain development.     

Astrocytes regulate amino acid receptor current densities in embryonic rat hippocampal neurons

Embryonic rat hippocampal neurons were cultured in a serum-free defined medium (MEM/N3) either directly on poly-D-lysine (PDL) or on a confluent monolayer of postnatal cortical astrocytes, C6 glioma cells, or Rat2 fibroblasts. Neurons on PDL were grown in MEM/N3 or in MEM/N3 conditioned for 24 h by astrocytes or C6 cells. Membrane capacitance (Cm) and gamma-aminobutyric acid (GABA)-, glycine-, kainate-, and N-methyl-D-aspartate (NMDA)-induced currents were quantified using whole-cell patch-clamp recordings. Cm as well as the amplitude and the density of these currents in neurons cultured on astrocytes were significantly greater than those in neurons grown on PDL after 24 and 48 h. C6 cells mimicked astrocytes in promoting Cm and GABA-, glycine-, and NMDA-evoked, but not kainate-evoked, currents. Cm and currents in neurons grown on Rat2 cells were comparable to those in neurons on PDL. Astrocytes maintained in culture for 3 months were noticeably less effective than freshly prepared ones just grown to confluence. Suppression of spontaneous cytoplasmic Ca2+ (Ca[c]2+) elevations in astrocytes by 1,2-bis(2-aminophenoxy) ehane-N, N, N, N-tetraacetic acid acetoxymethyl ester (BAPTA-AM) loaded intracellularly blocked the observed modulatory effects. Medium conditioned by either astrocytes or C6 cells mimicked the effects of direct coculture of neurons on these cells in promoting Cm and amino acid-evoked currents. Inclusion of antagonists at GABA and glutamate receptors in coculture experiments blocked the observed effects. Thus, diffusible substances synthesized and/ or secreted by astrocytes in a Ca(c)2+-dependent manner can regulate neuronal growth and aminoacid receptor function, and these effects may involve neuronal GABA and glutamate receptors.     

The GABAA receptor is expressed in human neurons derived from a teratocarcinoma cell line

NT2 cells, a human teratocarcinoma cell line, are shown to be differentiated in neuron-like cells (NT2-N cells) by treatment with retinoic acid. The present study identified the neurotransmitter receptors expressed in NT2-N cells using patch-clamp recording. Voltage-sensitive Na+ currents, which are specific for neurons, were observed in NT2-N cells but not in NT2 cells, suggesting that NT2-N cells actually function as neurons. Glutamate receptor agonists, N-methyl-D-aspartate (NMDA) and kainate, evoked whole-cell currents. In addition, gamma-aminobutyric acid (GABA) evoked currents and the currents were inhibited by the selective GABAA receptor antagonist, bicuculline. In outside-out patches, GABA elicited single channel currents with two classes of the slope conductance (26 and 50 pS). No current, however, was induced by ACh, serotonin, or dopamine NT2-N cells, thus, express at least two types of the major excitatory and inhibitory neurotransmitter receptor in the central nervous system, the glutamate and GAGAA receptors, suggesting that these receptors have a crucial role in neurotransmission from the earlier stage of the brain development.     

GABAergic neurons in the embryonic olfactory pit/vomeronasal organ: maintenance of functional GABAergic synapses in olfactory explants

In previous work, we showed a robust gamma-aminobutyric acid (GABAergic) synaptic input onto embryonic luteinizing hormone-releasing hormone (LHRH) neurons maintained in olfactory explants. In this study, we identify GABAergic neurons in olfactory pit (OP) of embryonic mice in vivo and study, using patch-pipet whole-cell current and voltage clamp techniques, synaptic interactions of these neurons in explant cultures. In vivo, glutamate decarboxylase (GAD, the enzyme which synthesizes GABA) mRNA was first detected in nasal regions on Embryonic Day (E) 11.5. From E12.5 to E13.5, robust GAD expression was localized to cells primarily in the ventral aspect of the OP. GAD mRNA was not detected over dorsally located cells in olfactory sensory or respiratory epithelium. In addition, GAD mRNA was not observed in cells along olfactory axons. GAD mRNA was dramatically reduced in the OP/vomeronasal organ by E16.5. Using antibodies against both GABA and GAD, immunopositive axonal-like tracts were detected in the nasal septum on E12.5. GABAergic staining decreased by E13.5. To examine synaptic interactions of these GABAergic cells, embryonic olfactory explants were generated and maintained in serum-free media. As explants spread, neuron-like cells migrated into the periphery, sometimes forming ganglion-like clusters. Cells were recorded, marked intracellularly with Lucifer Yellow and post-fixation, immunocytochemically examined. Forty-six cells, typically multipolar, were GABAergic, had resting potentials around -50 mV, and exhibited spontaneous action potentials which were generated by spontaneous depolarizing GABAergic (GABAA) synaptic activity. OP neurons depolarized in response to GABA by increasing Cl- conductance. The biophysical properties of OP-derived GABAergic neurons were distinct from those reported for olfactory receptor neurons but similar to embryonic LHRH neurons. However, unlike LHRH neurons, GABAergic neurons did not migrate large distances in olfactory explants or appear to leave the olfactory pit in vivo.     

Carnosine: its properties, functions and potential therapeutic applications

Carnosine and related dipeptides such as anserine are naturally-occurring histidine-containing compounds. They are found in several tissues most notably in muscle where they represent an appreciable fraction of the total water-soluble nitrogen-containing compounds. The biological role of these dipeptides are conjectural but they are believed to act as cytosolic buffering agents. Numerous studies have demonstrated, both at the tissue and organelle level, that they possess strong and specific antioxidant properties. Carnosine and related dipeptides have been shown to prevent peroxidation of model membrane systems leading to the suggestion that they represent water-soluble counterparts to lipid-soluble antioxidants such as alpha-tocopherol in protecting cell membranes from oxidative damage. Other roles ascribed to these dipeptides include actions as neurotransmitters, modulation of enzymic activities and chelation of heavy metals. Many claims have been made in respect of therapeutic actions of carnosine and histidine-containing dipeptides. These include antihypertensive effects, actions as immunomodulating agents, wound healing and antineoplastic effects. Many of these claims have not been convincingly documented nor subject to rigorous clinical evaluation. Nevertheless, there are examples where studies have shown considerable promise. One is the treatment of senile cataract in dogs and another is in acceleration of healing of surface wounds and burns to the skin. It is clear from this review that many of the effects of these histidine-containing dipeptides, especially in regard to claims for their therapeutic effects, need to be subjected to critical experimental and clinical examination. Several applications do, however, show clear evidence of being useful therapeutic agents.     

Formation of olfactory memories mediated by nitric oxide

Sheep learn to recognize the odours of their lambs within two hours of giving birth, and this learning involves synaptic changes within the olfactory bulb. Specifically, mitral cells become increasingly responsive to the learned odour, which stimulates release of both glutamate and GABA (gamma-aminobutyric acid) neurotransmitters from the reciprocal synapses between the excitatory mitral cells and inhibitory granule cells. Nitric oxide (NO) has been implicated in synaptic plasticity in other regions of the brain as a result of its modulation of cyclic GMP levels. Here we investigate the possible role of NO in olfactory learning. We find that the neuronal enzyme nitric oxide synthase (nNOS) is expressed in both mitral and granule cells, whereas the guanylyl cyclase subunits that are required for NO stimulation of cGMP formation are expressed only in mitral cells. Immediately after birth, glutamate levels rise, inducing formation of NO and cGMP, which potentiate glutamate release at the mitral-to-granule cell synapses. Inhibition of nNOS or guanylyl cyclase activity prevents both the potentiation of glutamate release and formation of the olfactory memory. The effects of nNOS inhibition can be reversed by infusion of NO into the olfactory bulb. Once memory has formed, however, inhibition of nNOS or guanylyl cyclase activity cannot impair either its recall or the neurochemical release evoked by the learned lamb odour. Nitric oxide therefore seems to act as a retrograde and/or intracellular messenger, being released from both mitral and granule cells to potentiate glutamate release from mitral cells by modulating cGMP concentrations. We propose that the resulting changes in the functional circuitry of the olfactory bulb underlie the formation of olfactory memories.     

Dipeptide uptake and transport characteristics in rabbit tracheal epithelial cell layers cultured at an air interface

PURPOSE: To determine the functional presence ofa H+/peptide cotransport process in rabbit tracheal epithelial cell layers cultured at an air-interface and its contribution to transepithelial dipeptide transport. METHODS: Rabbit tracheocytes were isolated, plated on Transwells, and cultured at an air-interface. After 5 or 6 days in culture, uptake and transepithelial transport of carnosine were examined. RESULTS: Carnosine uptake by tracheocytes was pH-dependent and was saturable with a Michaelis-Menten constant of 170 microM. Moreover, carnosine uptake was inhibited 94% by Gly-L-Phe, 28% by beta-Ala-Gly, but not at all by Gly-D-Phe or by the amino acids beta-Ala and L-His. Unexpectedly. transepithelial carnosine transport at pH 7.4 (i.e., in the absence of a transepithelial pH gradient) was similar in both the apical-to-basolateral (ab) and basolateral-to-apical (ba) directions. Lowering the apical fluid pH to 6.5 reduced ab transport 1.6 times without affecting ba transport, consistent with predominantly paracellular diffusion of carnosine under an electrochemical potential gradient. CONCLUSIONS: The kinetic behavior of carnosine uptake into cultured tracheal epithelial cell layers is characteristic of a H+-coupled dipeptide transport process known to exist in the small intestine and the kidney. Such a process does not appear to be rate-limiting in the transport of carnosine across the tracheal epithelial barrier.     

Receptor subtype specific effects of GABA agonists on neurons receiving aortic depressor nerve inputs within the nucleus of the solitary tract

The inhibitory amino acid gamma amino butyrate (GABA) has been shown to profoundly alter the integration of arterial baroreceptor inputs within the nucleus of the solitary tract (NTS). However, the relative roles of the major GABA receptor subtypes, the GABA(A) and the GABA(B) receptors, in the modulation of monosynaptic compared to polysynaptic afferent transmission within the NTS remain uncharacterized. In anesthetized rats, three types of NTS neuron were identified by their responses to aortic depressor nerve (ADN) stimulation; monosynaptic neurons (MSNs), polysynaptic neurons (PSNs) and ADN non-evoked neurons (NENs). Selective GABA(A) and GABA(B) agonists were applied to these neurons using iontophoretic techniques. The endogenous ligand GABA (2 mM), the selective GABA(A) agonist muscimol (0.04 and 0.02 mM) and the GABA(B) agonist baclofen (10 mM) all inhibited the spontaneous discharge of MSNs, PSNs and NENs (P < 0.01 for each group). In addition, GABA, muscimol and baclofen also inhibited ADN evoked discharge in both MSNs and PSNs (P < 0.05 for each group). Both GABA and baclofen significantly inhibited ADN evoked discharge in PSNs to a greater extent than in MSNs (P < 0.05 for each comparison). Muscimol at both doses, however, similarly inhibited ADN evoked discharge in both MSNs and PSNs. Examination of action potential amplitude and co-iontophoretic application of glutamate and GABA agonists suggested that GABA and muscimol induced inhibition were likely to be post-synaptic in origin, while baclofen produced both pre-synaptic and post-synaptic inhibition, depending upon the cell. In conclusion, GABA can influence baroreceptor afferent integration through both pre-synaptic and post-synaptic mechanisms. Furthermore, the effects of GABA(B) agonists are variable depending upon the level of afferent integration, with MSNs being generally less sensitive than PSNs.     

Ethanol modulation of excitatory and inhibitory synaptic interactions in cultured cortical neurons

The effects of ethanol on spontaneous excitatory and inhibitory postsynaptic currents (EPSCs and IPSCs) were studied in a culture of embryonic rat cortical neurons. In these experiments, EPSCs and IPSCs were recorded concurrently as inward and outward currents, respectively. These spontaneous currents were dominated by a slow (<1 Hz) repetitive pattern of prolonged N-methyl D-aspartate (NMDA)-EPSCs and co-occurring IPSCs when Mg2+ was left out of the perfusate. A 3- to 5-min bath perfusion of 100 mM ethanol reduced the average integrated EPSC by 65%, while simultaneously potentiating IPSCs by about 3-fold. EPSC frequency was also reduced by about one-third. NMDA-mediated EPSCs were inhibited more than non-NMDA currents. A perfusion of 30 mM ethanol was less effective and probably represents a threshold concentration for these effects. The ethanol inhibition of currents evoked by directly applied glutamate or NMDA to these cells was much less than that observed for spontaneous EPSCs. Currents evoked by exogenous gamma-aminobutyric acid (GABA) application were never potentiated by ethanol. When spontaneous NMDA-EPSCs were blocked with an NMDA antagonist, ethanol no longer potentiated the IPSCs. However, benzodiazepine treatment increased these IPSCs 2-fold. In other experiments, spontaneous IPSCs were blocked by a GABA(A) antagonist. Here, the EPSCs occurred as groups of repetitive bursts. Ethanol decreased the total number of EPSCs per burst but did not decrease their overall amplitude, as in the control recordings. Thus, the way in which ethanol affects concurrently recorded spontaneous EPSCs and IPSCs appears different from the way in which it affects isolated GABA- and NMDA-evoked currents. In addition, the antagonist studies show that concurrently activated NMDA and GABA channels each tend to limit the responses of the other. Thus, the overall effect of ethanol on spontaneous activity may result, in part, by a modification of this synaptic interaction.     

Inhibition of the growth of transformed and neoplastic cells by the dipeptide carnosine

Human diploid fibroblasts growth normally in medium containing physiological concentrations of the naturally occurring dipeptide carnosine (beta-alanyl-L-histidine). These concentrations are cytotoxic to transformed and neoplastic cells lines in modified Eagle medium (MEM), whereas these cells grow vigorously in Dulbecco's modified Eagle medium (DMEM) containing carnosine. This difference is due to the presence of 1 mM sodium pyruvate in DMEM. Seven human cell lines and two rodent cell lines were tested and all are strongly inhibited by carnosine in the absence of pyruvate. Experiments with HeLa cells show that anserine is similar to carnosine, but D-carnosine and homocarnosine are without effect. Also, the non-essential amino acids alanine and glutamic acid contribute to the effect of pyruvate in preventing carnosine toxicity, and oxaloacetate and alpha-ketoglutarate can substitute for pyruvate. We have used mixtures of normal MRC-5 fibroblasts and HeLa cells to demonstrate that 20 mM carnosine can selectively eliminate the tumour cells. This has obvious implications which might be exploited in in vivo and in vitro studies. Carnosine is known to react strongly with aldehyde and keto groups of sugars by Amadori reaction, and we propose that it depletes certain glycolysis intermediates. It is well known that tumour cells are more dependent on glycolysis than normal cells. A reduction of glycolysis intermediates by carnosine may deplete their energy supply, but this effect is totally reversed by pyruvate.     

Distribution of GABA, glycine, and glutamate immunoreactivities in the vestibular nuclear complex of the frog

This study describes the localization of gamma-aminobutyric acid (GABA), glycine, and glutamate immunoreactive neurons, fibers, and terminal-like structures in the vestibular nuclear complex (VNC) of the frog by using a postembedding procedure with consecutive semithin sections at the light microscopic level. For purposes of this study, the VNC was divided into a medial and lateral region. Immunoreactive cells were observed in all parts of the VNC. GABA-positive neurons, generally small in size, were predominantly located in the medial part of the VNC. Glycine-positive cells, more heterogeneous in size than GABA-positive cells, were scattered throughout the VNC. A quantitative analysis of the spatial distribution of GABA glycine immunoreactive cells revealed a complementary relation between the density of GABA and glycine immunoreactive neurons along the rostrocaudal extent of the VNC. In about 10% of the immunolabeled neurons, GABA and glycine were colocalized. Almost all vestibular neurons were, to a variable degree, glutamate immunoreactive, and colocalization of glutamate with GABA and/or glycine was typical. GABA, glycine, or glutamate immunoreactive puncta were found in close contact to somata and main dendrites of vestibular neurons. A quantitative analysis revealed a predominance of glutamate-positive terminal-like structures compared to glycine or GABA containing profiles. A small proportion of terminal-like structures expressed colocalization of GABA and glycine or glycine and glutamate. The results are compared with data from mammals and discussed in relation to vestibuloocular and vestibulo-spinal projection neurons, and vestibular interneurons. GABA and glycine are the major inhibitory transmitters of these neurons in frogs as well as in mammals. The differential distribution of GABA and glycine might reflect a compartmentalization of neurons that is preserved to some extent from the early embryogenetic segmentation of the hindbrain.     

Metabotropic glutamate receptor inhibition of visceral afferent potassium currents

The effects of metabotropic glutamate receptor activation (mGluR) on voltage-gated potassium currents have been characterized in visceral sensory afferent neurons. L-Glutamate is known to be a primary neurotransmitter in visceral afferents which terminate at the level of the nucleus of the solitary tract (NTS). Synaptic communication between these afferents and the NTS has been shown to involve both postsynaptic ionotropic and presynaptic metabotropic glutamate receptor activation. The purpose of the present study was to determine the effects of mGluR activation on voltage-gated potassium currents in visceral sensory neurons. Application of mGluR agonist t-ACPD inhibited both the peak and the steady state voltage-gated potassium current in 39 out of 56 visceral afferent neurons tested (70%) by 22.0 +/- 3 and 22.8 +/- 2%, respectively. Voltage and pharmacological protocols were utilized to isolate the potassium current affected by mGluR activation. Increasing the holding potential from -100 mV to -30 mV only partially attenuated the inhibitory effects of t-ACPD (decreased effect by 11%), suggesting that t-ACPD modulates both a voltage insensitive and a voltage-sensitive potassium current. In addition, 4-aminopyridine (5 microM) was applied to eliminate the 4-AP sensitive transient current. Also, this protocol only partially attenuated the inhibitory effects of t-ACPD (decreased effect by 6.3%), suggesting that mGluR activation inhibits both a 4-AP-sensitive and 4-AP-insensitive potassium current in visceral afferent neurons. Results from this study suggest that mGluRs may regulate visceral sensory afferent neuronal activity through inhibition of voltage-gated potassium channels.     

P19 cells differentiate into glutamatergic and glutamate-responsive neurons in vitro

The neurotransmitter L-glutamate has been associated with a number of developmental events within the central nervous system including synaptogenesis and the refinement of topographically ordered neural maps. As a model for studying such events at the molecular level, we have examined the expression of glutamate and glutamate receptors in neurons that develop from P19 cells in response to retinoids. We report here that many P19-derived neurons do contain glutamate in secretory vesicles and that this glutamate appears to function as a neurotransmitter. The neurotransmitter GABA is also present in these cultures and both glutamate and GABA appeared to co-localize in some neuronal processes. Both neurotransmitters were released from the neurons in response to membrane depolarization. These neurons also express various glutamate receptor subunits including GluR1, GluR4 and NMDAR1 as detected by immunological methods. Using whole-cell patch-clamping, we have recorded spontaneous postsynaptic potentials which increase in both amplitude and frequency with time in culture and which are sensitive to the glutamate antagonist kynurenic acid Thus, P19-derived neurons mature in culture and form electrically active neural networks involving glutamate and glutamate receptors.     

An AP-2 binding sequence within exon 1 of human and porcine choline acetyltransferase genes enhances transcription in neural cells

It has been established that GABAA and GABAB receptors can exist separately and/or co-exist in the membrane of dorsal root ganglion neurons. In our previous investigation it has been shown that co-existence of these two kinds of receptors is about 80% of the neurons examined (20/25). The present study was aimed to explore whether the activation of these two kinds of receptors could interact with each other using intracellular and whole-cell patch-clamp recordings. Baclofen, a specific GABAB receptor agonist, was found to exert negative modulatory effects on the responses mediated by GABAA receptor. In experiments with intracellular recording, GABA (0.3-1000 microM)- and muscimol (100-1000 microM)-induced depolarization was attenuated markedly and reversibly by preapplication of baclofen (100 microM) (15/21 and 17/21, respectively). In whole-cell patch-clamp recordings GABA (100 microM) and two specific GABAA receptor agonists, muscimol (10 microM) and isoguvacine (50 microM), activated currents were inhibited markedly by preapplication of baclofen 30 s or more and the inhibition was concentration dependent (1-100 microM baclofen) and reversible. The possible mechanisms underlying the inhibition by baclofen of the responses mediated by GABAA receptor and the physiological significance implicated are discussed.     

Luteinizing hormone releasing hormone (LHRH) neurons maintained in nasal explants decrease LHRH messenger ribonucleic acid levels after activation of GABA(A) receptors

Inhibition of the LHRH system appears to play an important role in preventing precocious activation of the hypothalamic-pituitary-gonadal axis. Evidence points to gamma-aminobutyric acid (GABA) as the major negative regulator of postnatal LHRH neuronal activity. Changes in LHRH messenger RNA (mRNA) levels after alterations of GABAergic activity have been reported in vivo. However, the extent to which GABA acts directly on LHRH neurons to effect LHRH mRNA levels has been difficult to ascertain. The present work evaluates the effect of GABAergic activity, via GABA(A) receptors, on LHRH neuropeptide gene expression in LHRH neurons maintained in olfactory explants generated from E11.5 mouse embryos. These explants maintain large numbers of primary LHRH neurons that migrate from bilateral olfactory pits in a directed manner. Using in situ hybridization histochemistry and single cell analysis, we report dramatic alterations in LHRH mRNA levels. Inhibition of spontaneous synaptic activity by GABA(A) antagonists, bicuculline (10(-5) M) or picrotoxin (10(-4) M), or of electrical activity by tetrodotoxin (TTX, 10(-6) M) significantly increased LHRH mRNA levels. In contrast, LHRH mRNA levels decreased in explants cultured with the GABA(A) receptor agonist, muscimol (10(-4) M), or KCl (50 mM). The observed responses suggest that LHRH neurons possess functional pathways linking GABA(A) receptors to repression of neuropeptide gene expression and indicate that gene expression in embryonic LHRH neurons, outside the CNS, is highly responsive to alterations in neuronal activity.     

Quantification of the GABA shunt and the importance of the GABA shunt versus the 2-oxoglutarate dehydrogenase pathway in GABAergic neurons

We investigated the activity of the cerebral GABA shunt relative to the overall cerebral tricarboxylic acid (TCA) cycle and the importance of the GABA shunt versus 2-oxoglutarate dehydrogenase for the conversion of 2-oxoglutarate into succinate in GABAergic neurons. Awake mice were dosed with [1-(13)C]glucose, and brain extracts were analyzed by 13C NMR spectroscopy. The percent enrichments of GABA C-2 and glutamate C-4 were the same: 5.0 +/- 1.6 and 5.1 +/- 0.2%, respectively (mean +/- SD). This, together with previous data, indicates that the flux through the GABA shunt relative to the overall cerebral TCA cycle flux equals the GABA/glutamate pool size ratio, which in the mouse is 17%. It has previously been shown that under the experimental conditions used in this study, the 13C labeling of aspartate from [1-(13)C]-glucose specifically reflects the metabolic activity of GABAergic neurons. In the present study, the reduction in the formation of [13C]aspartate during inhibition of the GABA shunt by gamma-vinyl-GABA indicated that not more than half the flux from 2-oxoglutarate to succinate in GABAergic neurons goes via the GABA shunt. Therefore, because fluxes through the GABA shunt and 2-oxoglutarate dehydrogenase in GABAergic neurons are approximately the same, the TCA cycle activity of GABAergic neurons could account for one-third of the overall cerebral TCA cycle activity in the mouse. Treatment with gamma-vinyl-GABA, which increased GABA levels dramatically, caused changes in the 13C labeling of glutamate and glutamine, which indicated a reduction in the transfer of glutamate from neurons to glia, implying reduced glutamatergic neurotransmission. In the most severely affected animals these alterations were associated with convulsions.     

Multiple NPY receptors coexist in pre- and postsynaptic sites: inhibition of GABA release in isolated self-innervating SCN neurons

Although NPY has been shown to influence the action of many transmitters in the brain, modulation of GABA, the primary inhibitory transmitter, has not been detected with electrophysiology. Using whole-cell patch-clamp recording, we found that NPY has a large modulatory effect on GABAergic neurons of the suprachiasmatic nucleus (SCN) that act as the circadian clock in the mammalian brain. NPY, acting at both Y1- and Y2-like receptors, reduced the frequency of spontaneous miniature inhibitory postsynaptic currents while having little effect on the postsynaptic GABA receptors, suggesting a presynaptic mechanism of NPY action. In single self-innervating neurons, application of either Y1 or Y2 agonists to the same neuron significantly inhibited the evoked autaptic GABA release. The use of single-neuron microcultures has allowed the demonstration that a single peptide, NPY, has two different receptors coded for by different genes in the same axon terminal. The Y1 and Y2 agonists also inhibited whole-cell calcium currents when applied to the same neuron, indicating a coexistence of Y1- and Y2-like receptors in the postsynaptic cell body. The self-innervating cell model we use here may be applicable generally for discriminating presynaptic versus postsynaptic actions of other neurotransmitters and neuromodulators and locating their subtype receptors.     

The role of an alpha subtype M2-M3 His in regulating inhibition of GABAA receptor current by zinc and other divalent cations

Sensitivity of GABAA receptors (GABARs) to inhibition by zinc and other divalent cations is influenced by the alpha subunit subtype composition of the receptor. For example, alpha6beta3gamma2L receptors are more sensitive to inhibition by zinc than alpha1beta3gamma2L receptors. We examined the role of a His residue located in the M2-M3 extracellular domain (rat alpha6 H273) in the enhanced zinc sensitivity conferred by the alpha6 subtype. The alpha1 subtype contains an Asn (N274) residue in the equivalent location. GABA-activated whole-cell currents were obtained from L929 fibroblasts after transient transfection with expression vectors containing GABAA receptor cDNAs. Mutation of alpha1 (alpha1(N274H)) or alpha6 (alpha6(H273N)) subtypes did not alter the GABA EC50 of alphabeta3gamma2L receptors. alpha1(N274H)beta3gamma2L receptor currents were as sensitive to zinc as alpha6beta3gamma2L receptor currents, although alpha6(H273N)beta3gamma2L receptor currents had the reduced zinc sensitivity of alpha1beta3gamma2L receptor currents. We also examined the activity of other inhibitory divalent cations with varying alpha subtype dependence: nickel, cadmium, and copper. alpha6beta3gamma2L receptor currents were more sensitive to nickel, equally sensitive to cadmium, and less sensitive to copper than alpha1beta3gamma2L receptor currents. Studies with alpha1 and alpha6 chimeric subunits indicated that the structural dependencies of the activity of some of these cations were different from zinc. Compared with alpha6beta3gamma2L receptor currents, alpha6(H273N)beta3gamma2L receptor currents had reduced sensitivity to cadmium and nickel, but the sensitivity to copper was unchanged. Compared with alpha1beta3gamma2L receptor currents, alpha1(N274H)beta3gamma2L receptor currents had increased sensitivity to nickel, but the sensitivity to cadmium and copper was unchanged. These findings indicate that H273 of the alpha6 subtype plays an important role in determining the sensitivity of recombinant GABARs to the divalent cations zinc, cadmium, and nickel, but not to copper. Our results also suggest that the extracellular N-terminal domain of the alpha1 subunit contributes to a regulatory site(s) for divalent cations, conferring high sensitivity to inhibition by copper and cadmium.     

Differential response of cortical plate and ventricular zone cells to GABA as a migration stimulus

A microdissection technique was used to separate differentiated cortical plate (cp) cells from immature ventricular zone cells (vz) in the rat embryonic cortex. The cp population contained >85% neurons (TUJ1(+)), whereas the vz population contained approximately 60% precursors (nestin+ only). The chemotropic response of each population was analyzed in vitro, using an established microchemotaxis assay. Micromolar GABA (1-5 microM) stimulated the motility of cp neurons expressing glutamic acid decarboxylase (GAD), the rate-limiting enzyme in GABA synthesis. In contrast, femtomolar GABA (500 fM) directed a subset of GAD- vz neurons to migrate. Thus, the two GABA concentrations evoked the motility of phenotypically distinct populations derived from different anatomical regions. Pertussis toxin (PTX) blocked GABA-induced migration, indicating that chemotropic signals involve G-protein activation. Depolarization by micromolar muscimol, elevated [K+]o, or micromolar glutamate arrested migration to GABA or GABA mimetics, indicating that migration is inhibited in the presence of excitatory stimuli. These results suggest that GABA, a single ligand, can promote motility via G-protein activation and arrest attractant-induced migration via GABAA receptor-mediated depolarization.