Tabela com principais espec.
|Species Reactivity||Key Applications||Host||Format||Antibody Type|
|H, M, Mk, R, Rb, Fe||IHC||Rb||Affinity Purified||Polyclonal Antibody|
|Description||Anti-GABA Transporter-1 Antibody, CT (a.a. 588-599)|
|Presentation||Affinity purified antibody. Lyophilized from in 0.1 M PBS with 1% BSA and 0.1% sodium azide. Reconstitute with 100 μL of sterile distilled water.|
|Safety Information according to GHS|
|Storage and Shipping Information|
|Storage Conditions||Maintain lyophilized material at 2-8°C for up to 12 months. After reconstitution maintain at -20°C in undiluted aliquots for up to 6 months. Avoid repeated freeze/thaw cycles.|
|Material Size||10 µg|
Referências | 21 Disponível | Ver todas as referências
|Visão geral das referências||Aplicação||Espécies||Pub Med ID|
|Molecular identity of axonal sodium channels in human cortical pyramidal cells. |
Tian, C; Wang, K; Ke, W; Guo, H; Shu, Y
Frontiers in cellular neuroscience 8 297 2014
Studies in rodents revealed that selective accumulation of Na(+) channel subtypes at the axon initial segment (AIS) determines action potential (AP) initiation and backpropagation in cortical pyramidal cells (PCs); however, in human cortex, the molecular identity of Na(+) channels distributed at PC axons, including the AIS and the nodes of Ranvier, remains unclear. We performed immunostaining experiments in human cortical tissues removed surgically to cure brain diseases. We found strong immunosignals of Na(+) channels and two channel subtypes, NaV1.2 and NaV1.6, at the AIS of human cortical PCs. Although both channel subtypes were expressed along the entire AIS, the peak immunosignals of NaV1.2 and NaV1.6 were found at proximal and distal AIS regions, respectively. Surprisingly, in addition to the presence of NaV1.6 at the nodes of Ranvier, NaV1.2 was also found in a subpopulation of nodes in the adult human cortex, different from the absence of NaV1.2 in myelinated axons in rodents. NaV1.1 immunosignals were not detected at either the AIS or the nodes of Ranvier of PCs; however, they were expressed at interneuron axons with different distribution patterns. Further experiments revealed that parvalbumin-positive GABAergic axon cartridges selectively innervated distal AIS regions with relatively high immunosignals of NaV1.6 but not the proximal NaV1.2-enriched compartments, suggesting an important role of axo-axonic cells in regulating AP initiation in human PCs. Together, our results show that both NaV1.2 and NaV1.6 (but not NaV1.1) channel subtypes are expressed at the AIS and the nodes of Ranvier in adult human cortical PCs, suggesting that these channel subtypes control neuronal excitability and signal conduction in PC axons.
|Distinct neurochemical and functional properties of GAD67-containing 5-HT neurons in the rat dorsal raphe nucleus. |
Shikanai, H; Yoshida, T; Konno, K; Yamasaki, M; Izumi, T; Ohmura, Y; Watanabe, M; Yoshioka, M
The Journal of neuroscience : the official journal of the Society for Neuroscience 32 14415-26 2012
The serotonergic (5-HTergic) system arising from the dorsal raphe nucleus (DRN) is implicated in various physiological and behavioral processes, including stress responses. The DRN is comprised of several subnuclei, serving specific functions with distinct afferent and efferent connections. Furthermore, subsets of 5-HTergic neurons are known to coexpress other transmitters, including GABA, glutamate, or neuropeptides, thereby generating further heterogeneity. However, despite the growing evidence for functional variations among DRN subnuclei, relatively little is known about how they map onto neurochemical diversity of 5-HTergic neurons. In the present study, we characterized functional properties of GAD67-expressing 5-HTergic neurons (5-HT/GAD67 neurons) in the rat DRN, and compared with those of neurons expressing 5-HTergic molecules (5-HT neurons) or GAD67 alone. While 5-HT/GAD67 neurons were absent in the dorsomedial (DRD) or ventromedial (DRV) parts of the DRN, they were selectively distributed in the lateral wing of the DRN (DRL), constituting 12% of the total DRL neurons. They expressed plasmalemmal GABA transporter 1, but lacked vesicular inhibitory amino acid transporter. By using whole-cell patch-clamp recording, we found that 5-HT/GAD67 neurons had lower input resistance and firing frequency than 5-HT neurons. As revealed by c-Fos immunohistochemistry, neurons in the DRL, particularly 5-HT/GAD67 neurons, showed higher responsiveness to exposure to an open field arena than those in the DRD and DRV. By contrast, exposure to contextual fear conditioning stress showed no such regional differences. These findings indicate that 5-HT/GAD67 neurons constitute a unique neuronal population with distinctive neurochemical and electrophysiological properties and high responsiveness to innocuous stressor.
|Inhibition of Activity of GABA Transporter GAT1 by δ-Opioid Receptor. |
Pu, L; Xu, N; Xia, P; Gu, Q; Ren, S; Fucke, T; Pei, G; Schwarz, W
Evidence-based complementary and alternative medicine : eCAM 2012 818451 2012
Analgesia is a well-documented effect of acupuncture. A critical role in pain sensation plays the nervous system, including the GABAergic system and opioid receptor (OR) activation. Here we investigated regulation of GABA transporter GAT1 by δOR in rats and in Xenopus oocytes. Synaptosomes of brain from rats chronically exposed to opiates exhibited reduced GABA uptake, indicating that GABA transport might be regulated by opioid receptors. For further investigation we have expressed GAT1 of mouse brain together with mouse δOR and μOR in Xenopus oocytes. The function of GAT1 was analyzed in terms of Na(+)-dependent [(3)H]GABA uptake as well as GAT1-mediated currents. Coexpression of δOR led to reduced number of fully functional GAT1 transporters, reduced substrate translocation, and GAT1-mediated current. Activation of δOR further reduced the rate of GABA uptake as well as GAT1-mediated current. Coexpression of μOR, as well as μOR activation, affected neither the number of transporters, nor rate of GABA uptake, nor GAT1-mediated current. Inhibition of GAT1-mediated current by activation of δOR was confirmed in whole-cell patch-clamp experiments on rat brain slices of periaqueductal gray. We conclude that inhibition of GAT1 function will strengthen the inhibitory action of the GABAergic system and hence may contribute to acupuncture-induced analgesia.
|The synaptic proteome during development and plasticity of the mouse visual cortex. |
Dahlhaus, M; Li, KW; van der Schors, RC; Saiepour, MH; van Nierop, P; Heimel, JA; Hermans, JM; Loos, M; Smit, AB; Levelt, CN
Molecular & cellular proteomics : MCP 10 M110.005413 2011
During brain development, the neocortex shows periods of enhanced plasticity, which enables the acquisition of knowledge and skills that we use and build on in adult life. Key to persistent modifications of neuronal connectivity and plasticity of the neocortex are molecular changes occurring at the synapse. Here we used isobaric tag for relative and absolute quantification to measure levels of 467 synaptic proteins in a well-established model of plasticity in the mouse visual cortex and the regulation of its critical period. We found that inducing visual cortex plasticity by monocular deprivation during the critical period increased levels of kinases and proteins regulating the actin-cytoskeleton and endocytosis. Upon closure of the critical period with age, proteins associated with transmitter vesicle release and the tubulin- and septin-cytoskeletons increased, whereas actin-regulators decreased in line with augmented synapse stability and efficacy. Maintaining the visual cortex in a plastic state by dark rearing mice into adulthood only partially prevented these changes and increased levels of G-proteins and protein kinase A subunits. This suggests that in contrast to the general belief, dark rearing does not simply delay cortical development but may activate signaling pathways that specifically maintain or increase the plasticity potential of the visual cortex. Altogether, this study identified many novel candidate plasticity proteins and signaling pathways that mediate synaptic plasticity during critical developmental periods or restrict it in adulthood.Texto completo do artigo
|Differential distribution of neurons in the gyral white matter of the human cerebral cortex. |
V García-Marín,L Blazquez-Llorca,J R Rodriguez,J Gonzalez-Soriano,J DeFelipe
The Journal of comparative neurology 518 2010
The neurons in the cortical white matter (WM neurons) originate from the first set of postmitotic neurons that migrates from the ventricular zone. In particular, they arise in the subplate that contains the earliest cells generated in the telencephalon, prior to the appearance of neurons in gray matter cortical layers. These cortical WM neurons are very numerous during development, when they are thought to participate in transient synaptic networks, although many of these cells later die, and relatively few cells survive as WM neurons in the adult. We used light and electron microscopy to analyze the distribution and density of WM neurons in various areas of the adult human cerebral cortex. Furthermore, we examined the perisomatic innervation of these neurons and estimated the density of synapses in the white matter. Finally, we examined the distribution and neurochemical nature of interneurons that putatively innervate the somata of WM neurons. From the data obtained, we can draw three main conclusions: first, the density of WM neurons varies depending on the cortical areas; second, calretinin-immunoreactive neurons represent the major subpopulation of GABAergic WM neurons; and, third, the somata of WM neurons are surrounded by both glutamatergic and GABAergic axon terminals, although only symmetric axosomatic synapses were found. By contrast, both symmetric and asymmetric axodendritic synapses were observed in the neuropil. We discuss the possible functional implications of these findings in terms of cortical circuits.
|Localization and function of GABA transporters in the globus pallidus of parkinsonian monkeys. |
Adriana Galvan,Xing Hu,Yoland Smith,Thomas Wichmann
Experimental neurology 223 2010
The GABA transporters GAT-1 and GAT-3 are abundant in the external and internal segments of the globus pallidus (GPe and GPi, respectively). We have shown that pharmacological blockade of either of these transporters results in decreased neuronal firing, and in elevated levels of extracellular GABA in normal monkeys. We now studied whether the electrophysiologic and biochemical effects of local intra-pallidal injections of GAT-1 and GAT-3 blockers, or the subcellular localization of these transporters, are altered in monkeys rendered parkinsonian by the administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). The subcellular localization of the transporters in GPe and GPi, studied with electron microscopy immunoperoxidase, was similar to that found in normal animals: i.e., GAT-3 immunoreactivity was mostly confined to glial processes, while GAT-1 labeling was expressed in unmyelinated axons and glial processes. A combined injection/recording device was used to record the extracellular activity of single neurons in GPe and GPi, before, during and after administration of small volumes (1microl) of either the GAT-1 inhibitor, SKF-89976A hydrochloride (720ng), or the GAT-3 inhibitor, (S)-SNAP-5114 (500ng). In GPe, the effects of GAT-1 or GAT-3 blockade were similar to those seen in normal monkeys. However, unlike the findings in the normal state, the firing of most neurons was not affected by blockade of either transporter in GPi. These results suggest that, after dopaminergic depletion, the functions of GABA transporters are altered in GPi; without major changes in their subcellular localization.Texto completo do artigo
|Pericellular innervation of neurons expressing abnormally hyperphosphorylated tau in the hippocampal formation of Alzheimer's disease patients. |
Blazquez-Llorca, L; Garcia-Marin, V; Defelipe, J
Frontiers in neuroanatomy 4 20 2010
Neurofibrillary tangles (NFT) represent one of the main neuropathological features in the cerebral cortex associated with Alzheimer's disease (AD). This neurofibrillary lesion involves the accumulation of abnormally hyperphosphorylated or abnormally phosphorylated microtubule-associated protein tau into paired helical filaments (PHF-tau) within neurons. We have used immunocytochemical techniques and confocal microscopy reconstructions to examine the distribution of PHF-tau-immunoreactive (ir) cells, and their perisomatic GABAergic and glutamatergic innervations in the hippocampal formation and adjacent cortex of AD patients. Furthermore, correlative light and electron microscopy was employed to examine these neurons and the perisomatic synapses. We observed two patterns of staining in PHF-tau-ir neurons, pattern I (without NFT) and pattern II (with NFT), the distribution of which varies according to the cortical layer and area. Furthermore, the distribution of both GABAergic and glutamatergic terminals around the soma and proximal processes of PHF-tau-ir neurons does not seem to be altered as it is indistinguishable from both control cases and from adjacent neurons that did not contain PHF-tau. At the electron microscope level, a normal looking neuropil with typical symmetric and asymmetric synapses was observed around PHF-tau-ir neurons. These observations suggest that the synaptic connectivity around the perisomatic region of these PHF-tau-ir neurons was apparently unaltered.Texto completo do artigo
|Glutamate uptake triggers transporter-mediated GABA release from astrocytes. |
Héja, L; Barabás, P; Nyitrai, G; Kékesi, KA; Lasztóczi, B; Toke, O; Tárkányi, G; Madsen, K; Schousboe, A; Dobolyi, A; Palkovits, M; Kardos, J
PloS one 4 e7153 2009
Glutamate (Glu) and gamma-aminobutyric acid (GABA) transporters play important roles in regulating neuronal activity. Glu is removed from the extracellular space dominantly by glial transporters. In contrast, GABA is mainly taken up by neurons. However, the glial GABA transporter subtypes share their localization with the Glu transporters and their expression is confined to the same subpopulation of astrocytes, raising the possibility of cooperation between Glu and GABA transport processes.Here we used diverse biological models both in vitro and in vivo to explore the interplay between these processes. We found that removal of Glu by astrocytic transporters triggers an elevation in the extracellular level of GABA. This coupling between excitatory and inhibitory signaling was found to be independent of Glu receptor-mediated depolarization, external presence of Ca(2+) and glutamate decarboxylase activity. It was abolished in the presence of non-transportable blockers of glial Glu or GABA transporters, suggesting that the concerted action of these transporters underlies the process.Our results suggest that activation of Glu transporters results in GABA release through reversal of glial GABA transporters. This transporter-mediated interplay represents a direct link between inhibitory and excitatory neurotransmission and may function as a negative feedback combating intense excitation in pathological conditions such as epilepsy or ischemia.Texto completo do artigo
|A novel type of interplexiform amacrine cell in the mouse retina. |
Karin Dedek,Tobias Breuninger,Luis Pérez de Sevilla Müller,Stephan Maxeiner,Konrad Schultz,Ulrike Janssen-Bienhold,Klaus Willecke,Thomas Euler,Reto Weiler
The European journal of neuroscience 30 2009
Mammalian retinas comprise an enormous variety of amacrine cells with distinct properties and functions. The present paper describes a new interplexiform amacrine cell type in the mouse retina. A transgenic mouse mutant was used that expressed the gene for the enhanced green fluorescent protein (EGFP) instead of the coding DNA of connexin45 in several retinal cell classes, among which a single amacrine cell population was most prominently labelled. Staining for EGFP and different marker proteins showed that these amacrine cells are interplexiform: they stratify in stratum S4/5 of the inner plexiform layer and send processes to the outer plexiform layer. These cells were termed IPA-S4/5 cells. They belong to the group of medium-field amacrine cells and are coupled homologously and heterologously to other amacrine cells by connexin45. Immunostaining revealed that IPA-S4/5 cells are GABAergic and express GAT-1, a plasma-membrane-bound GABA transporter possibly involved in non-vesicular GABA release. To characterize the light responses of IPA-S4/5 cells, patch-clamp recordings in retinal slices were made. Consistent with their stratification in the ON sublamina of the inner plexiform layer, cells depolarized in response to light ON stimuli and transiently hyperpolarized in response to light OFF. Responses of cells to green (578 nm) and blue (400 nm) light suggest that they receive input from cone bipolar cells contacting both M- and S-cones, possibly with reduced S-cone input. A new type of interplexiform ON amacrine cell is described, which is strongly coupled and uses GABA but not dopamine as its neurotransmitter.
|An automated segmentation methodology for quantifying immunoreactive puncta number and fluorescence intensity in tissue sections. |
Fish, Kenneth N, et al.
Brain Res., 1240: 62-72 (2008) 2008
A number of human brain diseases have been associated with disturbances in the structure and function of cortical synapses. Answering fundamental questions about the synaptic machinery in these disease states requires the ability to image and quantify small synaptic structures in tissue sections and to evaluate protein levels at these major sites of function. We developed a new automated segmentation imaging method specifically to answer such fundamental questions. The method takes advantage of advances in spinning disk confocal microscopy, and combines information from multiple iterations of a fluorescence intensity/morphological segmentation protocol to construct three-dimensional object masks of immunoreactive (IR) puncta. This new methodology is unique in that high- and low-fluorescing IR puncta are equally masked, allowing for quantification of the number of fluorescently-labeled puncta in tissue sections. In addition, the shape of the final object masks highly represents their corresponding original data. Thus, the object masks can be used to extract information about the IR puncta (e.g., average fluorescence intensity of proteins of interest). Importantly, the segmentation method presented can be easily adapted for use with most existing microscopy analysis packages.
|GABA uptake by purified avian Müller glia cells in culture. |
Clarissa De Sampaio Schitine, Regina Célia Cussa Kubrusly, Ricardo Augusto De Melo Reis, Edna Nanami Yamasaki, Maria Christina Fialho De Mello, Fernando Garcia De Mello
Neurotoxicity research 12 145-53 2007
GABA is the main inhibitory aminoacid transmitter present in neurons and glial cells. Its uptake is carried out by specific high-affinity Na(+)/Cl (-) dependent transporters (GATs). It has been reported in the past that, in the avian retina, [(3)H]GABA appears to be exclusively accumulated by horizontal and amacrine cells in the inner nuclear layer, and also by ganglion cells. Purified chick Müller glia cultures were able to take up [(3)H]GABA in a Na(+) and Cl(+) dependent way. Increasing GABA concentration increases GABA uptake by these cells, reaching half-maximal transport efficiency (EC50) around 0.3 mM. [(3)H]GABA uptake by Müller glia neuronal-free cultures was not mediated by neuronal transporters since it was not blocked by NNC-711, but was inhibited by beta-alanine, a specific glial transporter inhibitor. Chick Müller glia in culture express both GAT-1 and GAT-3 GABA transporters. Although mixed neuron-glial dense cultures released GABA upon glutamate, high K[(+) or veratridine stimulation, Müller glial cells did not release [(3)H]GABA upon treatment with these agents, suggesting that different from neurons, transporter mediated GABA release is not a common mechanism operating in these cells. The data also suggest that Müller cells take up GABA unidirectionally, which may constitute an important mechanism of inactivating GABA activity mediated by neurons.
|Voltage-gated ion channels in the axon initial segment of human cortical pyramidal cells and their relationship with chandelier cells. |
Inda, Maria Carmen, et al.
Proc. Natl. Acad. Sci. U.S.A., 103: 2920-5 (2006) 2006
The axon initial segment (AIS) of pyramidal cells is a critical region for the generation of action potentials and for the control of pyramidal cell activity. Here we show that Na+ and K+ voltage-gated channels, together with other molecules involved in the localization of ion channels, are distributed asymmetrically in the AIS of pyramidal cells situated in the human temporal neocortex. There is a high density of Na+ channels distributed along the length of the AIS together with the associated proteins spectrin betaIV and ankyrin G. In contrast, Kv1.2 channels are associated with the adhesion molecule Caspr2, and they are mostly localized to the distal region of the AIS. In general, the distal region of the AIS is targeted by the GABAergic axon terminals of chandelier cells, whereas the proximal region is innervated, mostly by other types of GABAergic interneurons. We suggest that this molecular segregation and the consequent regional specialization of the GABAergic input to the AIS of pyramidal cells may have important functional implications for the control of pyramidal cell activity.
|Expression of the gamma-aminobutyric acid (GABA) plasma membrane transporter-1 in monkey and human retina. |
Casini, G; Rickman, DW; Brecha, NC
Investigative ophthalmology & visual science 47 1682-90 2006
To determine the expression pattern of the predominant gamma-aminobutyric acid (GABA) plasma membrane transporter GAT-1 in Old World monkey (Macaca mulatta) and human retina.GAT-1 was localized in retinal sections by using immunohistochemical techniques with fluorescence and confocal microscopy. Double-labeling studies were performed with the GAT-1 antibody using antibodies to GABA, vasoactive intestinal polypeptide (VIP), tyrosine hydroxylase (TH), and the bipolar cell marker Mab115A10.The pattern of GAT-1 immunostaining was similar in human and monkey retinas. Numerous small immunoreactive somata were in the inner nuclear layer (INL) and were present rarely in the inner plexiform layer (IPL) of all retinal regions. Medium GAT-1 somata were in the ganglion cell layer in the parafoveal and peripheral retinal regions. GAT-1 fibers were densely distributed throughout the IPL. Varicose processes, originating from both the IPL and somata in the INL, arborized in the outer plexiform layer (OPL), forming a sparse network in all retinal regions, except the fovea. Sparsely occurring GAT-1 processes were in the nerve fiber layer in parafoveal regions and near the optic nerve head but not in the optic nerve. In the INL, 99% of the GAT-1 somata contained GABA, and 66% of the GABA immunoreactive somata expressed GAT-1. GAT-1 immunoreactivity was in all VIP-containing cells, but it was absent in TH-immunoreactive amacrine cells and in Mab115A10 immunoreactive bipolar cells.GAT-1 in primate retinas is expressed by amacrine and displaced amacrine cells. The predominant expression of GAT-1 in the inner retina is consistent with the idea that GABA transporters influence neurotransmission and thus participate in visual information processing in the retina.
|Axo-axonic structures in the medial prefrontal cortex of the rat: reduction by prenatal exposure to cocaine. |
Bret A Morrow, John D Elsworth, Robert H Roth
The Journal of neuroscience : the official journal of the Society for Neuroscience 23 5227-34 2003
The cognitive deficits associated with prenatal exposure to cocaine have been hypothesized to be the results of changes in the anatomy and function of the frontal cortex. In this study, pregnant dams were treated with cocaine (3 mg/kg i.v. twice a day) and the resulting adolescent (postnatal day, approximately 45) male offspring were killed for immunocytochemical determination of the total linear measure, number, location, and lengths of inhibitory GABA transporter-1 immunoreactive axo-axonic structures commonly called candles or cartridges in the medial prefrontal cortex. These inhibitory structures are the axon terminals of GABAergic cells that impinge on the initial axon segments of excitatory pyramidal neurons. We report that prenatal cocaine exposure decreased the number of these inhibitory candles. The greatest reduction of candles was observed in the ventral prelimbic cortex. Additionally, there was a subtle difference in the pattern of distribution of candles, namely the depth of the initial candle in the ventral portions of the prefrontal cortex was greater in rats exposed to prenatal cocaine. However, there was no overt change in the number of cells that were immunoreactive for the calcium-binding protein parvalbumin, an indicator of a subset of GABAergic interneurons that includes axo-axonic chandelier cells. We conclude that exposure to cocaine in utero disrupts the development of the axo-axonic cells in the prefrontal cortex and this disruption could contribute to the cognitive deficits reported with prenatal cocaine exposure.
|Heterotopic neurons with altered inhibitory synaptic function in an animal model of malformation-associated epilepsy. |
Calcagnotto, Maria Elisa, et al.
J. Neurosci., 22: 7596-605 (2002) 2002
Children with brain malformations often exhibit an intractable form of epilepsy. Although alterations in cellular physiology and abnormal histology associated with brain malformations has been studied extensively, synaptic function in malformed brain regions remains poorly understood. We used an animal model, rats exposed to methylazoxymethanol (MAM) in utero, featuring loss of lamination and distinct nodular heterotopia to examine inhibitory synaptic function in the malformed brain. Previous in vitro and in vivo studies demonstrated an enhanced susceptibility to seizure activity and neuronal hyperexcitability in these animals. Here we demonstrate that inhibitory synaptic function is enhanced in rats exposed to MAM in utero. Using in vitro hippocampal slices and whole-cell voltage-clamp recordings from visualized neurons, we observed a dramatic prolongation of GABAergic IPSCs onto heterotopic neurons. Spontaneous IPSC decay time constants were increased by 195% and evoked IPSC decay time constants by 220% compared with age-matched control CA1 pyramidal cells; no change in IPSC amplitude or rise time was observed. GABA transport inhibitors (tiagabine and NO-711) prolonged evoked IPSC decay kinetics of control CA1 pyramidal cells (or normotopic cells) but had no effect on heterotopic neurons. Immunohistochemical staining for GABA transporters (GAT-1 and GAT-3) revealed a low level of expression in heterotopic cell regions, suggesting a reduced ability for GABA reuptake at these synapses. Together, our data demonstrate that GABA-mediated synaptic function at heterotopic synapses is altered and suggests that inhibitory systems are enhanced in the malformed brain.
|Neuronal and glial localization of GAT-1, a high-affinity gamma-aminobutyric acid plasma membrane transporter, in human cerebral cortex: with a note on its distribution in monkey cortex. |
Conti, F, et al.
J. Comp. Neurol., 396: 51-63 (1998) 1998
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.
|Chandelier cell axons are immunoreactive for GAT-1 in the human neocortex. |
DeFelipe, J and González-Albo, M C
Neuroreport, 9: 467-70 (1998) 1998
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.
|Temporal correlations between functional and molecular changes in NMDA receptors and GABA neurotransmission in the superior colliculus. |
Shi, J, et al.
J. Neurosci., 17: 6264-76 (1997) 1997
Activation of the NMDA subtype of glutamate receptor is required for activity-dependent structural plasticity in many areas of the young brain. Previous work has shown that NMDA receptor currents decline approximately at the time that developmental synaptic plasticity ends, and in situ hybridization studies have suggested that receptor subunit changes may be occurring during the same developmental interval. To establish a system in which the relationship between these properties of developing synapses can be explored, we have combined patch-clamp recordings with mRNA- and protein-level biochemical analyses to study the developmental regulation of NMDA receptors in the superficial layers of the rat superior colliculus. These experiments document an abrupt decrease in the NMDA receptor contribution to synaptic currents that occurs before eye opening and is closely associated with changes in NR1 protein, rapidly rising levels of the NMDA receptor subunit NR2A, and decreasing levels of NR2B. The functional and molecular changes also are correlated with the developmental decline in structural plasticity in these layers. In addition, both physiological and biochemical methods show evidence of GABA-mediated inhibition in the superficial collicular layers beginning after eye opening. This may provide an additional heterosynaptic mechanism for controlling excitation and plasticity in this neuropil by pattern vision. Thus our findings lend support to the idea that high levels of NMDA receptor function are associated with the potential for structural rearrangement in CNS neuropil and that the functional downregulation of this molecule results, at least partially, from changes in its subunit composition.
|GABA plasma membrane transporters, GAT-1 and GAT-3, display different distributions in the rat hippocampus. |
Ribak, C E, et al.
J. Comp. Neurol., 367: 595-606 (1996) 1996
This study evaluates the distribution of two high affinity gamma-aminobutyric acid (GABA) transporters (GAT-1 and GAT-3) in the rat hippocampus using immunocytochemistry and affinity purified antibodies. GAT-1 immunoreactivity was prominent in punctate structures and axons in all layers of the dentate gyrus. In Ammon's horn, immunoreactive processes were concentrated around the somata of pyramidal cells, particularly at their basal regions. The apical and basal dendritic fields of pyramidal cells also displayed numerous GAT-1 immunoreactive punctate structures and axons. The zone of termination of the mossy fibers that includes both the hilus of the dentate gyrus and stratum lucidum of the CA3 area was the lightest immunolabeled region of the hippocampal complex. Electron microscopic preparations demonstrated that GAT-1 immunoreactive axon terminals form symmetric synapses with somata, axon initial segments, and dendrites of granule and pyramidal cells in the dentate gyrus and Ammon's horn, respectively. Immunoreactivity was localized to the plasma membrane and the cytoplasm of axon terminals. The somata of previously described local circuit neurons in the dentate gyrus and Ammon's horn contained GAT-1 immunoreactivity associated with the Golgi complex. Light, diffuse GAT-3 immunoreactivity was present throughout the hippocampal formation. Thin, astrocytic glial processes displayed GAT-1 and GAT-3 immunoreactivity. This localization of GAT-1 and GAT-3 indicates that they are involved in the uptake of GABA from the extracellular space into GABAergic axon terminals and astrocytes.
|Multiple gamma-Aminobutyric acid plasma membrane transporters (GAT-1, GAT-2, GAT-3) in the rat retina. |
Johnson, J, et al.
J. Comp. Neurol., 375: 212-24 (1996) 1996
gamma-Aminobutyric acid (GABA) plasma membrane transporters (GATs) influence synaptic neurotransmission by high-affinity uptake and release of GABA. The distribution and cellular localization of GAT-1, GAT-2, and GAT-3 in the rat retina have been evaluated by using affinity-purified polyclonal antibodies directed to the C terminus of each of these GAT subtypes. Small GAT-1-immunoreactive cell bodies were located in the proximal inner nuclear layer (INL) and ganglion cell layer (GCL), and processes were distributed to all laminae of the interplexiform layer (IPL). Varicose processes were in the optic fiber layer (OFL) and the outer plexiform layer (OPL). Weak GAT-1 immunostaining surrounded cells in the INL and GCL, and it was found in the OFL and OPL and in numerous processes in the outer nuclear layer (ONL) that ended at the outer limiting membrane. GAT-1 is therefore strongly expressed by amacrine, displaced amacrine, and interplexiform cells and weakly expressed by Müller cells. GAT-2 immunostaining was observed in the retina pigment epithelium and the nonpigmented ciliary epithelium. GAT-3 immunoreactivity was distributed to the OFL, to all laminae of the IPL, GCL and INL, and to processes in the ONL that ended at the outer limiting membrane. Small GAT-3-immunoreactive cell bodies were in the proximal INL and GCL. GAT-3 is therefore strongly expressed by Müller cells, and by some amacrine and displaced amacrine cells. Together, these observations demonstrate a heterologous distribution of GATs in the retina. These transporters are likely to take up GABA from, and perhaps release GABA into, the synaptic cleft and extracellular space. This suggests that GATs regulate GABA levels in these areas and thus influence synaptic neurotransmission.
|GAT-1, a high-affinity GABA plasma membrane transporter, is localized to neurons and astroglia in the cerebral cortex. |
Minelli, A, et al.
J. Neurosci., 15: 7734-46 (1995) 1995
High affinity, GABA plasma membrane transporters influence the action of GABA, the main inhibitory neurotransmitter. The cellular expression of GAT-1, a prominent GABA transporter, has been investigated in the cerebral cortex of adult rats using in situ hybridization with 35S-labeled RNA probes and immunocytochemistry with affinity purified polyclonal antibodies directed to the C-terminus of rat GAT-1. GAT-1 mRNA was observed in numerous neurons and in some glial cells. Double-labeling experiments were performed to compare the pattern of GAT-1 mRNA containing and GAD67 immunoreactive cells. The majority of neurons expressing GAT-1 mRNA also contained GAD67 immunoreactivity (ir), but GAT-1 mRNA was also observed in a few pyramidal neurons. GAT-1-ir was localized to numerous puncta and fibers and to astrocytic processes, was not observed in sections incubated in GAT-1 antibodies preadsorbed with rat GAT-1 C-terminal peptide, and was observed in sections incubated in GAT-1 antibodies preadsorbed with the C-terminal portion of the related peptides rat GAT-3(607-627) or rat glycine transporter-1(625-633). The highest number of GAT-1-ir puncta was in layer IV, followed by layers II-III. GAT-1 positive puncta appeared to have a preferential relationship to the soma and proximal dendrites of unlabeled pyramidal cells. All GAT-1 positive axon terminals formed symmetric synapses. This study demonstrates that (1) GAT-1 is expressed by both neurons and astrocytes, (2) the majority of GAT-1 expressing neurons contain GAD67, and (3) GAT-1 uptake system is more extensive than the GABA synthetizing system. These observations support the hypothesis that, in addition to its role in terminating GABA action by uptake into GABAergic axon terminals, GAT-1 influences both excitatory and inhibitory transmission by modulating the "paracrine" spread of GABA (Isaacson et al., 1993), and suggest that astrocytes may play an important role in this process.
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|RABBIT ANTI-GABA TRANSPORTER-1 (GAT-1)|