MAB316 Sigma-AldrichAnti-GABA Antibody, clone 5A9

The 53 kDa insulin receptor substrate protein (IRSp53) is highly enriched in the brain. Despite evidence that links mutations of IRSp53 with autism and other neuropsychiatric problems, the functional significance of this protein remains unclear. We used light and electron microscopic immunohistochemistry to demonstrate that IRSp53 is expressed throughout the adult rat brain. Labeling concentrated selectively in dendritic spines, where it was associated with the postsynaptic density (PSD). Surprisingly, its organization within the PSD of spiny excitatory neurons of neocortex and hippocampus differed from that within spiny inhibitory neurons of neostriatum and cerebellar cortex. The present data support previous suggestions that IRSp53 is involved in postsynaptic signaling, while hinting that its signaling role may differ in different types of neurons.

Starburst amacrine cells (SACs), the only acetylcholine (ACh)-releasing amacrine cells (ACs) in adult rabbit retina, contain GABA and are key elements in the retina's directionally selective (DS) mechanism. Unlike many other GABAergic ACs, they use glutamic acid decarboxlyase (GAD)(67), not GAD(65), to synthesize GABA. Using immunocytochemistry, we demonstrate the apoptosis at birth (P0) of transitory putative ACs that exhibit immunoreactivity (IR) for the ACh-synthetic enzyme choline acetyltransferase (ChAT), GAD(67), and the GABA transporter, GAT1. Only a few intact, displaced ChAT-immunoreactive SAC bodies are detected at P0. At P2, ChAT-IR is detected in the two narrowly stratified substrata of starburst dendrites in the inner plexiform layer (IPL). Quantitative analysis reveals that in the first postnatal week, only a small fraction of SACs cells express ChAT- and GABA-IR. Not until the end of the second week are they expressed in all SACs. At P0, a three-tiered stratification of GABA-IR is present in the IPL, entirely different from the adult pattern of seven substrata, emerging at P3-P4, and optimally visualized at P13. At P0, GAD(65) is detectable in normally placed AC bodies. At P1, GAD(65)-IR appears in dendrites of nonstarburst GABAergic ACs, and by P5 is robust in the adult pattern of four substrata in the IPL. GAD(65)-IR never co-localizes with ChAT-IR. In a temporal comparison of our data with physiological, pharmacological, and ultrastructural studies, we suggest that transitory ChAT-immunoreactive cells share with SACs production of stage II (nicotinic) waves of previsual synchronous activity in ganglion cells (GCs). Further, we conclude that (1) GAD(65)-immunoreactive, non-SAC GABAergic ACs are the most likely candidates responsible for the suppression of stage III (muscarinic/AMPA-kainate) waves and (2) DS responses first appear in DS GCs, when about 50% of SACs express ChAT- and GABA-IR, and in 100% of DS GCs, when expression occurs in all SACs.

Early in development spontaneous activity modulates survival and connectivity of neurons and thus plays a crucial role in the formation of neural networks. The emergence of synchronous activity in cultured neocortical networks initially is driven by large GABAergic interneurons. Here we studied the impact of thyroid hormone on early network development and especially on the development of large GABAergic neurons. Triiodothyronine enhances the frequency of early spontaneous synchronous network activity and an overall increase in network connectivity is indicated by the increased density of glutamatergic and GABAergic synapses. The hormone-induced increase of activity parallels cell type-specific changes in neuronal soma size and cell density, with strong effects on somatic and axonal growth of large GABAergic interneurons. Interestingly, large GABAergic neuron growth is both activity- and hormone-regulated. Blocking neuronal activity by tetrodotoxin or the glutamate receptor blockers D-2-amino-5-phosphonopentanoic acid and 6-cyano-7-nitroquinoxaline-2,3-dione disodium reveals a direct contribution of triiodothyronine to somatic growth, which also precedes the formation of synchronous network activity. The hormone-mediated effects on spontaneous activity and on large GABAergic neurons growth can be blocked by the nuclear thyroid hormone receptors antagonist 1-850. Thus, our data suggest that triiodothyronine actions result in functional maturation of early cortical networks and cell type-specific structural alterations. The increase in spontaneous activity might initially follow the growth of the large GABAergic neurons, which show an exquisite sensitivity to the presence of thyroid hormones. For the most part, however, the hormone-mediated growth of the GABAergic neurons relies strongly on the maturation of glutamatergic synaptic activity. 2010 IBRO. Published by Elsevier Ltd. All rights reserved.

Nitrosative stress has been implicated in the pathophysiology of several CNS disorders, including multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE). We have recently shown that protein nitrosothiols (PrSNOs) accumulate in the brain of MS patients, and there is indirect evidence that PrSNO levels are also increased in EAE. In this study we sought to identify the major PrSNOs in the spinal cord of EAE animals prepared by active immunization of C57/BL6 mice with MOG(35-55) peptide. For this purpose, PrSNOs from control and EAE mice at various disease stages were derivatized with HPDP-biotin, and the biotinylated proteins were isolated with streptavidin-agarose. Proteins from total and streptavidin-bound fractions were then analyzed by Western blotting using antibodies against the major S-nitrosylated substrates of CNS tissue. With this approach we found that the proportion of S-nitrosylated neurofilament proteins, NMDA receptors, alpha/beta-tubulin, beta-actin, and GAPDH is increased in EAE. Other potential substrates either were not S-nitrosylated in vivo (HCN3, HSP-72, CRMP-2, gamma-actin, calbindin) or their S-nitrosylation levels were unaltered in EAE (Na/K ATPase, hexokinase, glycogen phosphorylase). We also discovered that neuronal specific enolase is the major S-nitrosylated protein in acute EAE. Given that S-nitrosylation affects protein function, it is likely that the observed changes are significant to the pathophysiology of inflammatory demyelination.

Although neocortical GABAergic (gamma-aminobutyric acidergic) interneurons have been the focus of intense study, especially in the rat, a consensus view of the functional diversity and organization of inhibitory cortical neurons has not yet been achieved. To better analyze GABAergic neurons in the rat, we used a bacterial artificial chromosome (BAC) construct and established 2 lines of transgenic rats that coexpress Venus, a yellow fluorescent protein, with the vesicular GABA transporter. The brain GABA content from both transgenic lines was similar to the level found in wild-type rats. In the frontal cortex, Venus was expressed in greater than 95% of GABAergic neurons, most of which also expressed at least one of 6 biochemical markers, including alpha-actitin-2, which preferentially labeled late-spiking neurogliaform cells. Taking advantage of the fact that Venus expression allows for targeted recording from all classes of nonpyramidal cells, irrespective of their somatic morphologies, we demonstrated that fast-spiking neurons, which were heterogeneous in somatic size as well as vertical dendritic projection, had relatively uniform horizontal dimensions, suggesting a cell type-specific columnar input territory. Our data demonstrate the benefits of VGAT-Venus rats for investigating GABAergic circuits, as well as the feasibility of using BAC technology in rats to label subsets of specific, genetically defined neurons.

The inferior colliculus is a critical structure for processing auditory information and receives ascending and descending synaptic auditory projections. In addition to GABAergic and glutamatergic innervations, other neurotransmitter systems are also reported in the inferior colliculus, including opioid peptides. In the present study, the relative distribution of each type of opioid receptor, mu (MOR), delta (DOR) and kappa (KOR) within GABAergic neurons in the inferior colliculus was examined. GABA immunoreactivity was expressed by small, medium and large neurons and distributed in the central nucleus and the pericentral nucleus of the inferior colliculus. Immunostaining for MOR, DOR and KOR receptors was found in both disc-shaped cells and stellate cells. Punctiform beta-endorphin immunolabelling was observed in the proximity of GABA-positive neurons. Co-localization of GABA and MOR receptors was observed in neurons and nerve terminals in the central nucleus, dorsal cortex and external cortex of the inferior colliculus. Quantification of the co-localization patterns determined that a higher proportion of GABA neurons was associated with MOR receptors compared with KOR or DOR receptors.

The neuronal localization of alpha-amino-3-hydroxyl-5-methyl-4-isoxazole propionic acid (AMPA) glutamate receptor (GluR) subunits is vital as they play key roles in the regulation of calcium permeability. We have examined the distribution of the calcium permeable AMPA glutamate receptor subunit GluR1 in the mouse visual cortex immunocytochemically. We compared this distribution to that of the calcium-binding proteins calbindin D28K, calretinin, and parvalbumin, and of GABA. The highest density of GluR1-immunoreactive (IR) neurons was found in layers II/III. Enucleation appeared to have no effect on the distribution of GluR1-IR neurons. The labeled neurons varied in morphology; the majority were round or oval and no pyramidal cells were labeled by the antibody. Two-color immunofluorescence revealed that 26.27%, 10.65%, and 40.31% of the GluR1-IR cells also contained, respectively, calbindin D28K, calretinin, and parvalbumin. 20.74% of the GluR1-IR neurons also expressed GABA. These results indicate that many neurons that express calcium-permeable GluR1 also express calcium binding proteins. They also demonstrate that one fifth of the GluR1-IR neurons in the mouse visual cortex are GABAergic interneurons.

gamma-Aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the mammalian central nervous system and exerts its actions via both ionotropic (GABA(A)/GABA(C)) and metabotropic (GABA(B)) receptors (R). In addition to their location on neurons, GABA and functional GABA(B) receptors have been detected in nonneuronal cells in peripheral tissue. Although the GABA(B)R has been shown to function as a prejunctional inhibitory receptor on parasympathetic nerves in the lung, the expression and functional coupling of GABA(B) receptors to G(i) in airway smooth muscle itself have never been described. We detected the mRNA encoding multiple-splice variants of the GABA(B)R1 and GABA(B)R2 in total RNA isolated from native human and guinea pig airway smooth muscle and from RNA isolated from cultured human airway smooth muscle (HASM) cells. Immunoblots identified the GABA(B)R1 and GABA(B)R2 proteins in human native and cultured airway smooth muscle. The GABA(B)R1 protein was immunohistochemically localized to airway smooth muscle in guinea pig tracheal rings. Baclofen, a GABA(B)R agonist, elicited a concentration-dependent stimulation of [(35)S]GTPgammaS binding in HASM homogenates that was abrogated by the GABA(B)R antagonist CGP-35348. Baclofen also inhibited adenylyl cyclase activity and induced ERK phosphorylation in HASM. Another GABA(B)R agonist, SKF-97541, mimicked while pertussis toxin blocked baclofen's effect on ERK phosphorylation, implicating G(i) protein coupling. Functional GABA(B) receptors are expressed in HASM. GABA may modulate an uncharacterized signaling cascade via GABA(B) receptors coupled to the G(i) protein in airway smooth muscle.

Aberrant sprouting and synaptic reorganization of the mossy fiber (MF) axons are commonly found in the hippocampus of temporal lobe epilepsy patients and result in the formation of excitatory feedback loops in the dentate gyrus, a putative cellular basis for recurrent epileptic seizures. Using ex vivo hippocampal cultures, we show that prolonged hyperactivity induces MF sprouting and the resultant network reorganizations and that brain-derived neurotrophic factor (BDNF) is necessary and sufficient to evoke these pathogenic plasticities. Hyperexcitation induced an upregulation of BDNF protein expression in the MF pathway, an effect mediated by L-type Ca2+ channels. The neurotrophin receptor tyrosine kinase (Trk)B inhibitor K252a or function-blocking anti-BDNF antibody prevented hyperactivity-induced MF sprouting. Even under blockade of neural activity, local application of BDNF to the hilus, but not other subregions, was capable of initiating MF axonal remodeling, eventually leading to dentate hyperexcitability. Transfecting granule cells with dominant-negative TrkB prevented axonal branching. Thus, excessive activation of L-type Ca2+ channels causes granule cells to express BDNF, and extracellularly released BDNF stimulates TrkB receptors present on the hilar segment of the MFs to induce axonal branching, which may establish hyperexcitable dentate circuits.