AB133 Sigma-AldrichAnti-Glutamate Antibody

The basal ganglia are critical for selecting actions and evaluating their outcome. Although the circuitry for selection is well understood, how these nuclei evaluate the outcome of actions is unknown. Here, we show in lamprey that a separate evaluation circuit, which regulates the habenula-projecting globus pallidus (GPh) neurons, exists within the basal ganglia. The GPh neurons are glutamatergic and can drive the activity of the lateral habenula, which, in turn, provides an indirect inhibitory influence on midbrain dopamine neurons. We show that GPh neurons receive inhibitory input from the striosomal compartment of the striatum. The striosomal input can reduce the excitatory drive to the lateral habenula and, consequently, decrease the inhibition onto the dopaminergic system. Dopaminergic neurons, in turn, provide feedback that inhibits the GPh. In addition, GPh neurons receive direct projections from the pallium (cortex in mammals), which can increase the GPh activity to drive the lateral habenula to increase the inhibition of the neuromodulatory systems. This circuitry, thus, differs markedly from the "direct" and "indirect" pathways that regulate the pallidal (e.g., globus pallidus) output nuclei involved in the control of motion. Our results show that a distinct reward-evaluation circuit exists within the basal ganglia, in parallel to the direct and indirect pathways, which select actions. Our results suggest that these circuits are part of the fundamental blueprint that all vertebrates use to select actions and evaluate their outcome.

Slow wave oscillations in the brain are essential for coordinated network activity but have not been shown to self-organize in vitro. Here, the development of dissociated hippocampal neurons into an active network with oscillations on multi-electrode arrays was evaluated in the absence and presence of chronic external stimulation. Significant changes in signal power were observed in the range of 1-400 Hz with an increase in amplitude during bursts. Stimulation increased oscillatory activity primarily in the theta (4-11 Hz) and slow gamma (30-55 Hz) bands. Spikes were most prominently phase-locked to the slow gamma waves. Notably, the dissociated network self-organized to exhibit sustained delta, theta, beta and gamma oscillations without input from cortex, thalamus or organized pyramidal cell layers.

Increased extracellular brain glutamate has been implicated in the pathophysiology of human refractory temporal lobe epilepsy (TLE), but the cause of the excessive glutamate is unknown. Prior studies by us and others have shown that the glutamate degrading enzyme glutamine synthetase (GS) is deficient in astrocytes in the epileptogenic hippocampal formation in a subset of patients with TLE. We have postulated that the loss of GS in TLE leads to increased glutamate in astrocytes with elevated concentrations of extracellular glutamate and recurrent seizures as the ultimate end-points. Here we test the hypothesis that the deficiency in GS leads to increased glutamate in astrocytes. Rats were chronically infused with methionine sulfoximine (MSO, n=4) into the hippocampal formation to induce GS deficiency and recurrent seizures. A separate group of rats was infused with 0.9% NaCl (saline) as a control (n=6). At least 10days after the start of infusion, once recurrent seizures were established in the MSO-treated rats, the concentration of glutamate was assessed in CA1 of the hippocampal formation by immunogold electron microscopy. The concentration of glutamate was 47% higher in astrocytes in the MSO-treated vs. saline-treated rats (p=0.02), and the ratio of glutamate in astrocytes relative to axon terminals was increased by 74% in the MSO-treated rats (p=0.003). These data support our hypothesis that a deficiency in GS leads to increased glutamate in astrocytes. We additionally propose that the GS-deficient astrocytes in the hippocampal formation in TLE lead to elevated extracellular brain glutamate either through decreased clearance of extracellular glutamate or excessive release of glutamate into the extracellular space from these cells, or a combination of the two.

Sea anemones have a structurally simple nervous system that controls behaviors like feeding, locomotion, aggression, and defense. Specific chemical and tactile stimuli are transduced by ectodermal sensory cells and transmitted via a neural network to cnidocytes and epithelio-muscular cells, but the nature of the neurotransmitters operating in these processes is still under discussion. Previous studies demonstrated an important role of peptidergic transmission in cnidarians, but during the last decade the contribution of conventional neurotransmitters became increasingly evident. Here, we used immunohistochemistry on light and electron microscopical preparations to investigate the localization of glutamate and GABA in tentacle cross-sections of the sea anemone Phymactis papillosa. Our results demonstrate strong glutamate immunoreactivity in the nerve plexus, while GABA labeling was most prominent in the underlying epithelio-muscular layer. Immunoreactivity for both molecules was also found in glandular epithelial cells, and putative sensory cells were GABA positive. Under electron microscopy, both glutamate and GABA immunogold labeling was found in putative neural processes within the neural plexus. These data support a function of glutamate and GABA as signaling molecules in the nervous system of sea anemones.

In the tiger salamander retina, visual signals are transmitted to the inner retina via six morphologically distinct types of photoreceptors: large/small rods, large/small single cones, and double cones composed of principal and accessory members. The objective of this study was to determine the morphology of these photoreceptors and their synaptic interconnection with bipolar cells and horizontal cells in the outer plexiform layer (OPL). Here we showed that glutamate antibodies labeled all photoreceptors and recovering antibodies strongly labeled all cones and weakly labeled all rods. Antibodies against calbindin selectively stained accessory members of double cones. Antibodies against S-cone opsin stained small rods, a subpopulation of small single cones, and the outer segments of accessory double cones and a subtype of unidentified single cones. On average, large rods and small S-cone opsin positive rods accounted for 98.6% and 1.4% of all rods, respectively. Large/small cones, principle/accessory double cones, S-cone opsin positive small single cones, and S-cone opsin positive unidentified single cones accounted for about 66.9%, 23%, 4.5%, and 5.6% of the total cones, respectively. Moreover, the differential connection between rods/cones and bipolar/horizontal cells and the wide distribution of AMPA receptor subunits GluR2/3 and GluR4 at the rod/cone synapses were observed. These results provide anatomical evidence for the physiological findings that bipolar/horizontal cells in the salamander retina are driven by rod/cone inputs of different weights, and that AMPA receptors play an important role in glutamatergic neurotransmission at the first visual synapses. The different photoreceptors selectively contacting bipolar and horizontal cells support the idea that visual signals may be conveyed to the inner retina by different functional pathways in the outer retina.

The fundamental network for locomotion in all vertebrates contains a central pattern generator or CPG that produces the required motor output in the spinal cord. In the lamprey spinal cord different classes of interneuron's forming the core CPG circuitry have been characterized based on their morphological and electrophysiological features. The commissural interneuron's (C-INs) represent one essential component of CPG that have been implicated in controlling left-right alternation of the motor activity during swimming. However, it is still unclear if the C-INs displays a homogenous neurotransmitter phenotype and how they are distributed. In this paper we investigated the segmental distribution of glycine, glutamate and GABA-immunoreactive (ir) C-INs by combining retrograde Neurobiotin tracing with specific antibodies for these transmitters. The C-INs were more abundant in caudal and rostral segments adjacent to the injection site and their number gradually decreased in more distal segments, suggesting that these interneurons project over a short distance. The glycine-ir neurons represented around 50% of the total C-INs, while glutamate-ir neurons represented only 29%. Both types of C-INs were homogenously distributed over different segments along the spinal cord. Finally, no Neurobiotin labeled C-INs displayed GABA-ir, although many interneurons were ir to GABA, suggesting that GABAergic interneurons are not directly responsible for controlling left-right alternation of activity during locomotion in lamprey. Overall, these results show that the C-INs display a gradual rostrocaudal distribution and consist of both glycine- and glutamate-ir neurons. The difference in the proportion of inhibitory and excitatory C-INs represents an anatomical substrate that can ensure the predominance of alternating activity during locomotion.

In prior studies that used transneuronal transport of isogenic recombinants of pseudorabies virus, we established that medial medullary reticular formation (MRF) neurons sent collateralized projections to both diaphragm and abdominal muscle motoneurons. Furthermore, inactivation of MRF neurons in cats and ferrets increased the excitability of diaphragm and abdominal motoneurons, suggesting that MRF neurons controlling respiratory activity are inhibitory. To test this hypothesis, the present study determined the neurochemical phenotypes of MRF premotor respiratory neurons in the ferret by using immunohistochemical procedures. Dual-labeling immunohistochemistry combining pseudorabies virus injections into respiratory muscles with the detection of glutamic acid decarboxylase-like immunoreactive and glutamate-like immunoreactive cells showed that both GABAergic and glutamatergic MRF neurons project to respiratory motoneurons, although the latter are more common. These data suggest that the role of the MRF in respiratory regulation is multifaceted, as this region provides both inhibitory and excitatory influences on motoneuron activity.

Cajal-Retzius cells are reelin-secreting neurons found in the marginal zone of the mammalian cortex during development. Recently, it has been proposed that Cajal-Retzius cells may be generated both early and late in corticogenesis, and may migrate into the cortex from proliferative zones in the subpallium (lateral ganglionic eminence and medial ganglionic eminence) or cortical hem. In the present study, we used reelin as a marker to study the properties of Cajal-Retzius cells, including their likely origins, neurotransmitters, and birthdates. In double labeling experiments, Cajal-Retzius cells (reelin(+)) expressed transcription factors characteristic of pallial neurons (Tbr1 and Emx2), contained high levels of glutamate, were usually calretinin(+), and were born early in corticogenesis, on embryonic days (E)10.5 and E11.5. Tbr1(+) cells in the marginal zone were almost always reelin(+). The first Cajal-Retzius cells (Tbr1(+)/reelin(+)) appeared in the preplate on E10.5. In contrast, interneurons expressed a subpallial transcription factor (Dlx), contained high levels of GABA, were frequently calbindin(+), and were born throughout corticogenesis (from E10.5 to E16.5). Interneurons (Dlx(+)) first appeared in the cortex on E12.5. Our results suggest that the marginal zone contains two main types of neurons: Cajal-Retzius cells derived from the pallium, and migrating interneurons derived from the subpallium.

PURPOSE: Retinal detachment induces neurochemical changes in the neural retina over a span of days to weeks. However, little information is available on the acute response in the retina to detachment. METHODS: Distribution of the neurotransmitters glutamate, glycine, and gamma-aminobutyric acid (GABA) and the metabolic amino acids aspartate and glutamine was examined immunocytochemically from 5 to 30 minutes and at 3 hours after retinal detachment in a salamander eyecup preparation. RESULTS: Glutamate showed a rapid depletion from neuronal cell bodies in detached retina, whereas Müller cells, which normally sequester and metabolize glutamate, showed increased immunolabeling for glutamine. Changes occurred exclusively in detached retinal regions of the eyecup. Aspartate, a precursor for glutamate synthesis, also showed decreased labeling in neuronal cell bodies in detached retinal regions, although these changes were not as striking as those observed for glutamate. In contrast, the distributions of the inhibitory amino acid neurotransmitters glycine and GABA were not affected appreciably by acute retinal detachment. CONCLUSIONS: These results indicate that retinal detachment induces rapid, localized alterations in the glutamatergic system of the neural retina that are consistent with a massive efflux of neuronal glutamate and concomitant alterations in glutamate metabolism. An acute efflux of neuronal glutamate in detached retina could contribute to excitotoxicity and to the initiation of structural alterations and changes in gene expression; it is also consistent with reported neurochemical changes associated with longer term retinal detachment.