MAB351R Sigma-AldrichAnti-Glutamate Decarboxylase Antibody, 65 kDa isoform, clone GAD-6

The NMDAR plays a unique and vital role in subcellular signaling. Calcium influx initiates signaling cascades important for both synaptic plasticity and survival; however, overactivation of the receptor leads to toxicity and cell death. This dichotomy is partially explained by the subcellular location of the receptor. NMDARs located at the synapse stimulate cell survival pathways, while extrasynaptic receptors signal for cell death. Thus far, this interplay between synaptic and extrasynaptic NMDARs has been studied exclusively in cortical (CTX) and hippocampal neurons. It was unknown whether other cell types, such as GABAergic medium-sized spiny projection neurons of the striatum (MSNs), which bear the brunt of neurodegeneration in Huntington's disease, follow the same pattern. Here we report synaptic versus extrasynaptic NMDAR signaling in striatal MSNs and resultant activation of cAMP response element binding protein (CREB), in rat primary corticostriatal cocultures. Similarly to CTX, we found in striatal MSNs that synaptic NMDARs activate CREB, whereas extrasynaptic NMDARs dominantly oppose CREB activation. However, MSNs are much less susceptible to NMDA-mediated toxicity than CTX cells and show differences in subcellular GluN2B distribution. Blocking NMDARs with memantine (30 μm) or GluN2B-containing receptors with ifenprodil (3 μm) prevents CREB shutoff effectively in CTX and MSNs, and also rescues both neuronal types from NMDA-mediated toxicity. This work may provide cell and NMDAR subtype-specific targets for treatment of diseases with putative NMDAR involvement, including neurodegenerative disorders and ischemia.

The mesocortical pathway projecting from the ventral tegmental area (VTA) to the prefrontal cortex (PFC) plays a critical role in a number of cognitive and emotional processes. While this pathway has been traditionally viewed as dopaminergic, recent data indicate that a considerable proportion of rostromedial VTA neurons possess markers for glutamate transmission. However, the relative density of the glutamatergic projection to the PFC from these rostromedial regions is unknown. In the present study, anterograde tracer injections into 4 ventral midbrain subregions were coupled with immunohistochemical analysis of labeled axons in PFC for markers of dopamine (DA; tyrosine hydroxylase [TH]) and glutamate (vesicular glutamate transporter 2; VGLUT2). We found that while tracer injections into the interfascicular nucleus produced labeled fibers in the PFC that were mainly TH positive, tracer injections into the rostral linear nucleus, rostral VTA, and parabrachial pigmented nucleus produced labeled fibers in PFC that contained mainly VGLUT2-positive rather than TH-positive varicosities. When viewed in the light of the previously documented strong γ-aminobutyric acidergic component, it would seem that the rostromedial mesocortical projection is actually an amino acid pathway that in addition has a DA component.

The mammalian cochlea is under direct control of two groups of cholinergic auditory brainstem neurons, the medial and the lateral olivocochlear neurons. The former modulate the electromechanical amplification in outer hair cells and the latter the transduction of inner hair cells to auditory nerve fibers. The lateral olivocochlear neurons express not only acetylcholine but a variety of co-transmitters including urocortin, which is known to regulate homeostatic responses related to stress; it may also be related to the ontogeny of hearing as well as the generation of hearing disorders. In the present study, we investigated the distribution of urocortin-expressing lateral olivocochlear neurons and their connectivity and distribution of synaptic terminals in the cochlea of juvenile and adult gerbils. In contrast to most other rodents, the gerbil's audiogram covers low frequencies similar to humans, although their communication calls are exclusively in the high-frequency domain. We confirm that in the auditory brainstem urocortin is expressed exclusively in neurons within the lateral superior olive and their synaptic terminals in the cochlea. Moreover, we show that in adult gerbils urocortin expression is restricted to the medial, high-frequency processing, limb of the lateral superior olive and to the mid and basal parts of the cochlea. The same pattern is present in juvenile gerbils shortly before hearing onset (P 9) but transiently disappears after hearing onset, when urocortin is also expressed in low-frequency processing regions. These results suggest a possible role of urocortin in late cochlear development and in the processing of social calls in adult animals.

Activation of neuropeptide Y (NPY) Y1 receptors (Y1r) in the rat basolateral nuclear complex of the amygdala (BLA) produces anxiolysis and interferes with the generation of conditioned fear. NPY is important in regulating the output of the BLA, yet the cell types involved in mediating this response are currently unknown. The current studies employed multiple label immunocytochemistry to determine the distribution of Y1r-immunoreactivity (-ir) in glutamatergic pyramidal and GABAergic cell populations in the BLA using scanning laser confocal stereology. Pyramidal neurons were identified by expression of calcium-calmodulin dependent kinase II (CaMKII-ir) and functionally distinct interneuron subpopulations were distinguished by peptide (cholecystokinin, somatostatin) or calcium-binding protein (parvalbumin, calretinin) content. Throughout the BLA, Y1r-ir was predominately on soma with negligible fiber staining. The high degree of coexpression of Y1r-ir (99.9%) in CaMKII-ir cells suggests that these receptors colocalize on pyramidal cells and that NPY could influence BLA output by directly regulating the activity of these projection neurons. Additionally, Y1r-ir was also colocalized with the interneuronal markers studied. Parvalbumin-ir interneurons, which participate in feedforward inhibition of BLA pyramidal cells, represented the largest number of Y1r expressing interneurons in the BLA ( approximately 4% of the total neuronal population). The anatomical localization of NPY receptors on different cell populations within the BLA provides a testable circuit whereby NPY could modulate the activity of the BLA via actions on both projection cells and interneuronal cell populations.

In animal models of temporal lobe epilepsy (TLE), neurosteroid sensitivity of GABA(A) receptors on dentate granule cells (DGCs) is diminished; the molecular mechanism underlying this phenomenon remains unclear. The current study investigated a mechanism for loss of neurosteroid sensitivity of synaptic GABA(A) receptors in TLE. Synaptic currents recorded from DGCs of epileptic animals (epileptic DGCs) were less frequent, larger in amplitude, and less sensitive to allopregnanolone modulation than those recorded from DGCs of control animals (control DGCs). Synaptic currents recorded from epileptic DGCs were less sensitive to diazepam and had altered sensitivity to benzodiazepine inverse agonist RO 15-4513 (ethyl-8-azido-6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5alpha][1,4]benzodiazepine-3-carboxylate) and furosemide than those recorded from control DGCs. Properties of synaptic currents recorded from epileptic DGCs appeared similar to those of recombinant receptors containing the alpha4 subunit. Expression of the alpha4 subunit and its colocalization with the synaptic marker GAD65 was increased in epileptic DGCs. Location of the alpha4 subunit in relation to symmetric (inhibitory) synapses on soma and dendrites of control and epileptic DGCs was examined with postembedding immunogold electron microscopy. The alpha4 immunogold labeling was present more commonly within the synapse in epileptic DGCs compared with control DGCs, in which the subunit was extrasynaptic. These studies demonstrate that, in epileptic DGCs, the neurosteroid modulation of synaptic currents is diminished and alpha4 subunit-containing receptors are present at synapses and participate in synaptic transmission. These changes may facilitate seizures in epileptic animals.

Autoantibodies to glutamic acid decarboxylase (GADAs), specifically the 65-kDa isoform GAD65, and autoantibodies to the protein tyrosine phosphatase-like molecule IA-2 (IA-2As) predict development of diabetes. Our aim was to develop a time-resolved immunofluorometric (TR-IFMA) dual-label assay method for the simultaneous detection of these autoantibodies and to evaluate the diagnostic sensitivity of the method compared with single-label TR-IFMA and fluid-phase radiobinding assay (RBA) in screening children with type 1 diabetes.We incubated combined biotinylated GAD65 and IA-2 proteins, glutathione S-transferase (GST)-IA-2, europium-labeled GAD65, terbium-labeled anti-GST antibody, and serum sample or calibrator and transferred aliquots to a streptavidin-coated 96-well microtiter plate for a second incubation. After washing, we added Delfia Enhancement solution to each well and measured the fluorescence of Eu. We developed the Tb fluorescence signal by use of the Delfia Enhancer solution and measured it. We analyzed serum samples from a cohort of 100 children with newly diagnosed type 1 diabetes.The correlation coefficients between the autoantibody concentrations measured by dual- and single-label TR-IFMA assays were 0.962 for GADA and 0.874 for IA-2A. Among 100 children with newly diagnosed diabetes, 65 of them were GADA positive in the dual-label assay, 64 in the single-label assay, and 66 in the RBA GADA assay. Seventy-four of the children tested positive for IA-2A in both TR-IFMA assay types, and 79 in the RBA IA-2A assay.The novel dual-label immunofluorometric assay performed comparably to the separate, single-label GADA and IA-2A assays in screening for beta-cell autoimmunity in children with newly diagnosed type 1 diabetes.

The 29/30 amino acid neuropeptide galanin has been implicated in pain processing at the spinal level and local dorsal horn neurons expressing the Gal(1) receptor may play a critical role. In order to determine the transmitter identity of these neurons, we used immunohistochemistry and antibodies against the Gal(1) receptor and the three vesicular glutamate transporters (VGLUTs), as well as in situ hybridization, to explore a possible glutamatergic phenotype. Gal(1) protein, which could not be demonstrated in Gal(1) knockout mice, colocalized with VGLUT2 protein, but not with glutamate decarboxylase, in many nerve endings in lamina II. Moreover, Gal(1) and VGLUT2 transcripts were often found in the same cell bodies in laminae I-IV. Gal(1)-protein and galanin-peptide showed an overlapping distribution but were not colocalized. Gal(1) staining did not appear to be affected by dorsal rhizotomy. Taken together, these findings provide strong evidence that Gal(1) is a heteroreceptor expressed on excitatory glutamatergic dorsal horn interneurons. Activation of such Gal(1) receptors may thus decrease the inhibitory tone in the superficial dorsal horn, and possibly cause antinociception.

The type of vesicular transporter expressed by a neuron is thought to determine its neurotransmitter phenotype. We show that inactivation of the vesicular inhibitory amino acid transporter (Viaat, VGAT) leads to embryonic lethality, an abdominal defect known as omphalocele, and a cleft palate. Loss of Viaat causes a drastic reduction of neurotransmitter release in both GABAergic and glycinergic neurons, indicating that glycinergic neurons do not express a separate vesicular glycine transporter. This loss of GABAergic and glycinergic synaptic transmission does not impair the development of inhibitory synapses or the expression of KCC2, the K+ -Cl- cotransporter known to be essential for the establishment of inhibitory neurotransmission. In the absence of Viaat, GABA-synthesizing enzymes are partially lost from presynaptic terminals. Since GABA and glycine compete for vesicular uptake, these data point to a close association of Viaat with GABA-synthesizing enzymes as a key factor in specifying GABAergic neuronal phenotypes.

GABAA receptors are pentamers composed of subunits derived from the alpha, beta, gamma, delta, theta, epsilon, and pi gene families. alpha1, alpha4, gamma2, and delta subunits are expressed in the dentate gyrus of the hippocampus, but their subcellular distribution has not been described. Hippocampal sections were double-labeled for the alpha1, alpha4, gamma2, and delta subunits and GAD65 or gephyrin, and their subcellular distribution on dentate granule cells was studied by means of confocal laser scanning microscopy (CLSM). The synaptic versus extrasynaptic localization of these subunits was inferred by quantitative analysis of the frequency of colocalization of various subunits with synaptic markers in high-resolution images. GAD65 immunoreactive clusters colocalized with 26.24+/-0.86% of the alpha1 subunit immunoreactive clusters and 32.35+/-1.49% of the gamma2 subunit clusters. In contrast, only 1.58+/-0.13% of the alpha4 subunit immunoreactive clusters and 1.92+/-0.15% of the delta subunit clusters colocalized with the presynaptic marker GAD65. These findings were confirmed by studying colocalization with immunoreactivity of a postsynaptic marker, gephyrin, which colocalized with 27.61+/-0.16% of the alpha1 subunit immunoreactive clusters and 23.45+/-0.32% of the gamma2 subunit immunoreactive clusters. In contrast, only 1.90+/-0.13% of the alpha4 subunit immunoreactive clusters and 1.76+/-0.10% of the delta subunit clusters colocalized with gephyrin. These studies demonstrate that a subset of alpha1 and gamma2 subunit clusters colocalize with synaptic markers in hippocampal dentate granule cells. Furthermore, all four subunits, alpha1, alpha4, gamma2, and delta, are present in the extrasynaptic locations.

Previous studies have reported the presence of migrating and dividing neuronal progenitors in the subventricular zone (SVZ) and rostral migratory stream (RMS) of the postnatal mammalian brain. Although the behaviour of these progenitors is thought to be influenced by local signals, the nature and mode of action of the local signals are largely unknown. One of the signalling molecules known to affect the behaviour of embryonic neurons is the neurotransmitter GABA. In order to determine whether GABA affects neuronal progenitors via the activation of specific receptors, we performed cell-attached, whole-cell and gramicidin perforated patch-clamp recordings of progenitors in postnatal mouse brain slices containing either the SVZ or the RMS. Recorded cells displayed a morphology typical of migrating neuronal progenitors had depolarized zero-current resting potentials, and lacked action potentials. A subset of progenitors contained GABA and stained positive for glutamic acid decarboxylase 67 (GAD-67) as shown by immunohistochemistry. In addition, every neuronal progenitor responded to GABA via picrotoxin-sensitive GABAA receptor (GABAAR) activation. GABAARs displayed an ATP-dependent rundown and a low sensitivity to Zn2+. GABA responses were sensitive to benzodiazepine agonists, an inverse agonist, as well as a barbiturate agonist. While GABA was hyperpolarizing at the zero-current resting potentials, it was depolarizing at the cell resting potentials estimated from the reversal potential of K+ currents through a cell-attached patch. Thus, our study demonstrates that neuronal progenitors of the SVZ/RMS contain GABA and are depolarized by GABA, which may constitute the basis for a paracrine signal among neuronal progenitors to dynamically regulate their proliferation and/or migration.