AB1570W Sigma-AldrichAnti-GABA Transporter-1 Antibody

The region that surrounds the central canal (CC) in the turtle spinal cord is a neurogenic niche immersed within already functional circuits, where radial glia expressing brain lipid binding protein (BLBP) behave as progenitors. The behaviour of both progenitors and neuroblasts within adult neurogenic niches must be regulated to maintain the functional stability of the host circuit. In the brain, GABA plays a major role in this kind of regulation but little is known about GABAergic signalling in neurogenic niches of the postnatal spinal cord. Here we explored the action of GABA around the CC of the turtle spinal cord by combining patch-clamp recordings of CC-contacting cells, immunohistochemistry for key components of GABAergic signalling and Ca(2+) imaging. Two potential sources of GABA appeared around the CC: GABAergic terminals and CC-contacting neurones. GABA depolarized BLBP(+) progenitors via GABA transporter-3 (GAT3) and/or GABA(A) receptors. In CC-contacting neurones, GABA(A) receptor activation generated responses ranging from excitation to inhibition. This functional heterogeneity appeared to originate from different ratios of activity of the Na(+)-K(+)-2Cl(-) co-transporter (NKCC1) and the K(+)-Cl(-) co-transporter (KCC2). In both progenitors and immature neurones, GABA induced an increase in intracellular Ca(2+) that required extracellular Ca(2+) and was blocked by the selective GABA(A) receptor antagonist gabazine. Our study shows that GABAergic signalling around the CC shares fundamental properties with those in the embryo and adult neurogenic niches, suggesting that GABA may be part of the mechanisms regulating the production and integration of neurones within operational spinal circuits in the turtle.

The γ-aminobutyric acid (GABA) transporters (GATs) are located in the plasma membrane of neurons and astrocytes and are responsible for termination of GABAergic transmission. It has previously been shown that brain derived neurotrophic factor (BDNF) modulates GAT-1-mediated GABA transport in nerve terminals and neuronal cultures. We now report that BDNF enhances GAT-1-mediated GABA transport in cultured astrocytes, an effect mostly due to an increase in the V(max) kinetic constant. This action involves the truncated form of the TrkB receptor (TrkB-t) coupled to a non-classic PLC-γ/PKC-δ and ERK/MAPK pathway and requires active adenosine A(2A) receptors. Transport through GAT-3 is not affected by BDNF. To elucidate if BDNF affects trafficking of GAT-1 in astrocytes, we generated and infected astrocytes with a functional mutant of the rat GAT-1 (rGAT-1) in which the hemagglutinin (HA) epitope was incorporated into the second extracellular loop. An increase in plasma membrane of HA-rGAT-1 as well as of rGAT-1 was observed when both HA-GAT-1-transduced astrocytes and rGAT-1-overexpressing astrocytes were treated with BDNF. The effect of BDNF results from inhibition of dynamin/clathrin-dependent constitutive internalization of GAT-1 rather than from facilitation of the monensin-sensitive recycling of GAT-1 molecules back to the plasma membrane. We therefore conclude that BDNF enhances the time span of GAT-1 molecules at the plasma membrane of astrocytes. BDNF may thus play an active role in the clearance of GABA from synaptic and extrasynaptic sites and in this way influence neuronal excitability.

The mouse gamma-aminobutyric acid (GABA) transporter mGAT1 was expressed in neuroblastoma 2a cells. 19 mGAT1 designs incorporating fluorescent proteins were functionally characterized by [(3)H]GABA uptake in assays that responded to several experimental variables, including the mutations and pharmacological manipulation of the cytoskeleton. Oligomerization and subsequent trafficking of mGAT1 were studied in several subcellular regions of live cells using localized fluorescence, acceptor photobleach Förster resonance energy transfer (FRET), and pixel-by-pixel analysis of normalized FRET (NFRET) images. Nine constructs were functionally indistinguishable from wild-type mGAT1 and provided information about normal mGAT1 assembly and trafficking. The remainder had compromised [(3)H]GABA uptake due to observable oligomerization and/or trafficking deficits; the data help to determine regions of mGAT1 sequence involved in these processes. Acceptor photobleach FRET detected mGAT1 oligomerization, but richer information was obtained from analyzing the distribution of all-pixel NFRET amplitudes. We also analyzed such distributions restricted to cellular subregions. Distributions were fit to either two or three Gaussian components. Two of the components, present for all mGAT1 constructs that oligomerized, may represent dimers and high-order oligomers (probably tetramers), respectively. Only wild-type functioning constructs displayed three components; the additional component apparently had the highest mean NFRET amplitude. Near the cell periphery, wild-type functioning constructs displayed the highest NFRET. In this subregion, the highest NFRET component represented approximately 30% of all pixels, similar to the percentage of mGAT1 from the acutely recycling pool resident in the plasma membrane in the basal state. Blocking the mGAT1 C terminus postsynaptic density 95/discs large/zona occludens 1 (PDZ)-interacting domain abolished the highest amplitude component from the NFRET distributions. Disrupting the actin cytoskeleton in cells expressing wild-type functioning transporters moved the highest amplitude component from the cell periphery to perinuclear regions. Thus, pixel-by-pixel NFRET analysis resolved three distinct forms of GAT1: dimers, high-order oligomers, and transporters associated via PDZ-mediated interactions with the actin cytoskeleton and/or with the exocyst.

GABA is known to produce membrane depolarization and secretion in adrenal medullary (AM) cells in various species. However, whether the GABAergic system is intrinsic or extrinsic or both in the adrenal medulla and the role that GABA plays are controversial. Therefore, these issues were addressed by combining a biochemical and functional analysis. Glutamic acid decarboxylase (GAD), a GABA synthesizing enzyme, and vesicular GABA transporter (VGAT) were expressed in rat AM cells at the mRNA and protein levels, and the adrenal medulla had no nerve fibre-like structures immunoreactive to an anti-GAD Ab. The double staining for VGAT and chromogranin A indicates that GABA was stored in chromaffin granules. The alpha1, alpha3, beta2/3, gamma2 and delta subunits of GABA(A) receptors were identified in AM cells at the mRNA and protein levels. Pharmacological properties of GABA-induced Cl(-) currents, immunoprecipitation experiments and immunocytochemistry indicated the expression of not only gamma2-, but also delta-containing GABA(A) receptors, which have higher affinities for GABA and neurosteroids. Expression of GATs, which are involved in the clearance of GABA at GABAergic synapses, were conspicuously suppressed in the adrenal medulla, compared with expression levels of GABA(A) receptors. Increases in Ca(2+) signal in AM cells evoked trans-synaptically by nerve stimulation were suppressed during the response to GABA, and this suppression was attributed to the shunt effect of the GABA-induced increase in conductance. Overall Ca(2+) responses to electrical stimulation and GABA in AM cells were larger or smaller than those to electrical stimulation alone, depending on the frequency of stimulation. The results indicate that GABA functions as a paracrine in rat AM cells and this function may be supported by the suppression of GAT expression and the expression of not only gamma2-, but also delta-GABA(A) receptors.

The uptake of neurotransmitter by plasma membrane transporters is a principal method for regulating extracellular transmitter levels. Neurotransmitter-mediated signals in turn are able to regulate transporter expression and function. Thus, there is a continual interplay between transporters and the transmitters they transport. Previously we showed that extracellular gamma-aminobutyric acid (GABA) increases the expression of the GABA transporter 1 (GAT1) on a time scale of minutes by acting via the transporter to slow transporter internalization. This mechanism requires in part direct tyrosine phosphorylation of the transporter. In the present study we show that the presence of GABA on a longer time scale causes a net decrease in GAT surface expression. The decrease in expression represents the contributions of transporter-mediated up-regulation and a more substantial GABA-receptor-mediated down-regulation. This receptor-mediated down-regulation is the result of both changes in the rates of transporter trafficking and in the number of transporters available for trafficking. As with transporter-mediated regulation of GAT1, the receptor-mediated regulation is associated with changes in the direct phosphorylation of GAT1. These data suggest that multiple pathways, perhaps converging upon mechanisms involving protein phosphorylation, act to regulate GAT1 expression in neurons.

The acquisition of the dynamic balance between excitation and inhibition in developing Purkinje cells, necessary for their proper function, is analyzed. Newborn (P0) mouse cerebellum contains glutamatergic (VGLUT2-IR) and gamma-aminobutyric acid (GABA)-ergic (VIAAT-IR) axons. The former prevail and belong to climbing fibers, whereas the latter neither colabel with calbindin-expressing fibers nor belong to axons of the cortical GABAergic interneurons. During the first postnatal week, VIAAT-IR axons in the Purkinje cell neighborhood remains very low, and the first synapses with basket fibers are formed at P7, when climbing fibers have already established dense pericellular nets. The descending basket fibers reach the Purkinje cell axon initial segment by P9, immediately establishing axoaxonic synapses. The pinceaux appear as primitive vortex-like arrangements by P12, and by P20 interbasket fiber septate-like junctions, typical of fully mature pinceaux, are still missing. The climbing fiber's somatodendritic translocation occurs later than expected, after the regression of the multiple innervation, and follows the ascending collaterals of the basket axons, which are apparently the optimal substrate for the proper subcellular targeting of the climbing fibers. These results emphasize that chemical transmission in the axon initial segment precedes the electrical inhibition generated by field effects. In addition, GABAergic Purkinje cells, as opposed to glutamatergic projection neurons in other cortical structures, do not begin to receive their excitation to inhibition balance until the end of the first postnatal week, despite the early presence of potentially functional GABAergic axons that possess the required vesicular transport system.

The formation and maturation of gamma-aminobutyric acid (GABA)-ergic synapses was studied in cultured hippocampal pyramidal neurons by both performing immunocytochemistry for GABAergic markers and recording miniature inhibitory postsynaptic currents (mIPSCs). Nascent GABAergic synapses appeared between 3 and 8 days in vitro (DIV), with GABAA receptor subunit clusters appearing first, followed by GAD-65 puncta, then functional synapses. The number of GABAergic synapses increased from 7 to 14 DIV, with a corresponding increase in frequency of mIPSCs. Moreover, these new GABAergic synapses formed on neuronal processes farther from the soma, contributing to decreased mIPSC amplitude and slowed mIPSC 19-90% rise time. The mIPSC decay quickened from 7 to 14 DIV, with a parallel change in the distribution of the alpha5 subunit from diffuse expression at 7 DIV to clustered expression at 14 DIV. These alpha5 clusters were mostly extrasynaptic. The alpha1 subunit was expressed as clusters in none of the neurons at 7 DIV, in 20% at 14 DIV, and in 80% at 21 DIV. Most of these alpha1 clusters were expressed at GABAergic synapses. In addition, puncta of GABA transporter 1 (GAT-1) were localized to GABAergic synapses at 14 DIV but were not expressed at 7 DIV. These studies demonstrate that mIPSCs appear after pre- and postsynaptic elements are in place. Furthermore, the process of maturation of GABAergic synapses involves increased synapse formation at distal processes, expression of new GABAA receptor subunits, and GAT-1 expression at synapses; these changes are reflected in altered frequency, kinetics, and drug sensitivity of mIPSCs.

High-affinity gamma-aminobutyric (GABA) plasma membrane transporters (GATs) influence the action of GABA, the main inhibitory neurotransmitter in the human cerebral cortex. In this study, the cellular expression of GAT-1, the main cortical GABA transporter, was investigated in the human cerebral cortex by using immunocytochemistry with affinity-purified polyclonal antibodies directed to the C-terminus of rat GAT-1. In temporal and prefrontal association cortex (Brodmann's areas 21 and 46) and in cingulofrontal transition cortex (area 32), specific GAT-1 immunoreactivity (ir) was localized to numerous puncta and fibers in all cortical layers. GAT-1+ puncta were distributed homogeneously in all cortical layers, although they were slightly more numerous in layers II-IV, and appeared to have a preferential relationship to the somata and proximal dendrites of unlabeled pyramidal cells, even though, in many cases, they were also observed around nonpyramidal cells. Electron microscopic observations showed that GAT-1+ puncta were axon terminals that formed exclusively symmetric synapses. In addition, some distal astrocytic processes also contained immunoreaction product. Analysis of the patterns of GAT-1 labeling in temporal and prefrontal association areas (21 and 46), in cingulofrontal transition areas (32), and in somatic sensory and motor areas (1 and 4) of the monkey cortex revealed that its distribution varies according to the type of cortex examined and indicated that the distribution of GAT-1 is similar in anatomically corresponding areas of different species. The present study demonstrates that, in the human homotypical cortex, GAT-1 is expressed by both inhibitory axon terminals and astrocytic processes. This localization of GAT-1 is compatible with a major role for this transporter in GABA uptake at GABAergic synapses and suggests that GAT-1 may contribute to determining GABA levels in the extracellular space.

We have examined the pattern of immunostaining for the high-affinity GABA transporter GAT-1 in the human temporal neocortex. Immunocytochemistry for GAT-1 labels terminal-like puncta in the neuropil and around unstained cell bodies. The characteristic terminal portions of chandelier cell axons (Ch-terminals, which form multiple inhibitory GABAergic synaptic contacts with the axon initial segments of pyramidal cells) were among the strongest immunocytochemically stained elements for GAT-1. Since Ch-terminals are immunoreactive for the calcium-binding protein parvalbumin, experiments were carried out to study the co-localization of GAT-1 and parvalbumin in Ch-terminals. These experiments showed that the vast majority of Ch-terminals immunoreactive for GAT-1 were also immunoreactive for PV. We concluded that GAT-1 transporter may have an important functional role in controlling pyramidal cell activity.