Glutamic Acid Decarboxylase Immunoreactive Neurons Research Articles

Catecholaminergic Innervation of Luteinizing Hormone-Releasing Hormone and Glutamic Acid Decarboxylase Immunopositive Neurons in the Rat Medial Preoptic Area

Catecholaminergic innervation of luteinizing hormone-releasing hormone (LHRH) and glutamic acid decarboxylase (GAD) immunoreactive neurons in the rat medial preoptic area (MPO) was studied using electron-microscopic (EM) double-label immunostaining and combinations of single- and double-label immunostaining with acute axonal degeneration. The EM double-immunostaining experiments included double staining for either tyrosine hydroxylase (TH) and LHRH, or TH and GAD. Analysis of TH and LHRH double-immunostained material revealed synaptic connections between TH immunoreactive axons and LHRH immunopositive neurons. The TH and GAD double-staining experiments also demonstrated synaptic connections between axons immunoreactive for TH and GAD immunopositive neurons. Two days following unilateral surgical transection of the ventral and dorsal noradrenergic bundles, synaptic connections were found between degenerated boutons and GAD immunoreactive neurons in the ipsilateral MPO. However, no synapses could be observed in the same area between degenerated axons and the LHRH immunopositive neurons. Following the same operation and immunostaining for TH, a moderate number of degenerating TH axons as well as a large number of nondegenerated TH immunoreactive boutons were observed. Double immunostaining for TH and GAD in MPO sections ipsilateral to the operation revealed synaptic connections between the degenerating TH immunopositive axons and GAD immunoreactive neurons. These results suggest that there are direct synaptic connections between catecholaminergic axons and GAD and LHRH immunoreactive neurons in the medial preoptic area of the rat. Some of the connections between TH immunopositive afferents and GAD immunoreactive neurons may represent connections from noradrenergic neurons in the brain stem, while the majority of TH-GAD and TH-LHRH connections may represent innervation of GABA and LHRH neurons from local dopamine-containing cells.

Distribution of GABAergic neurons and axon terminals in the macaque striate cortex.

Antisera to glutamic acid decarboxylase (GAD) and gamma-aminobutyric acid (GABA) have been used to characterize the morphology and distribution of presumed GABAergic neurons and axon terminals within the macaque striate cortex. Despite some differences in the relative sensitivity of these antisera for detecting cell bodies and terminals, the overall patterns of labeling appear quite similar. GABAergic axon terminals are particularly prominent in zones known to receive the bulk of the projections from the lateral geniculate nucleus; laminae 4C, 4A, and the cytochrome-rich patches of lamina 3. In lamina 4A, GABAergic terminals are distributed in a honeycomb pattern which appears to match closely the spatial pattern of geniculate terminations in this region. Quantitative analysis of axon terminals that contain flat vesicles and form symmetric synaptic contacts (FS terminals) in lamina 4C beta and in lamina 5 suggest that the prominence of GAD and GABA axon terminal labeling in the geniculate recipient zones is due, at least in part, to the presence of larger GABAergic axon terminals in these regions. GABAergic cell bodies and their initial dendritic segments display morphological features characteristic of nonpyramidal neurons and are found in all layers of striate cortex. The density of GAD and GABA immunoreactive neurons is greatest in laminae 2-3A, 4A, and 4C beta. The distribution of GABAergic neurons within lamina 3 does not appear to be correlated with the patchy distribution of cytochrome oxidase in this region; i.e., there is no significant difference in the density of GAD and GABA immunoreactive neurons in cytochrome-rich and cytochrome-poor regions of lamina 3. Counts of labeled and unlabeled neurons indicate that GABA immunoreactive neurons make up at least 15% of the neurons in striate cortex. Layer 1 is distinct from the other cortical layers by virtue of its high percentage (77-81%) of GABAergic neurons. Among the other layers, the proportion of GABAergic neurons varies from roughly 20% in laminae 2-3A to 12% in laminae 5 and 6. Finally, there are conspicuous laminar differences in the size and dendritic arrangement of GAD and GABA immunoreactive neurons. Lamina 4C alpha and lamina 6 are distinguished from the other layers by the presence of populations of large GABAergic neurons, some of which have horizontally spreading dendritic processes. GABAergic neurons within the superficial layers are significantly smaller and the majority appear to have vertically oriented dendritic processes.(ABSTRACT TRUNCATED AT 400 WORDS)

Glutamic acid decarboxylase-like immunoreactivity in brainstem auditory nuclei of the rat.

The distribution of GABA-producing neurons in the brainstem auditory nuclei of the rat was investigated immunohistochemically by using an antibody to glutamic acid decarboxylase (GAD). In the cochlear nuclei, GAD immunoreactive neurons are present only in the superficial granular and molecular layers, whereas terminals are found in all subdivisions of the nuclei and are particularly dense surrounding large spherical cells and one type of stellate cell. In the superior olivary complex, GAD immunoreactive neurons are located in the lateral olivary nucleus and throughout the periolivary region. Immunoreactive terminals are distributed along dendrites of principal cells of the medial and lateral olivary nuclei and are clustered around somata of globular neurons of the nucleus of the trapezoid body. An extremely dense band of immunoreactive somata and terminals is present along the ventral edge of the olivary complex. The ventral, intermediate, and dorsal nuclei of the lateral lemniscus contain small fusiform GAD-immunoreactive neurons and a moderately dense plexus of immunoreactive terminals. The inferior colliculus contains a large population of GAD-immunoreactive perikarya and an extremely dense accumulation of immunoreactive terminals in the central, dorsomedial, and external nuclei. These observations indicate that GABA systems are involved in function at all levels of the brainstem auditory pathway.

Times of generation of glutamic acid decarboxylase immunoreactive neurons in mouse somatosensory cortex.

The birth dates of neurons showing glutamic acid decarboxylase (GAD) immunoreactivity have been determined in mouse somatosensory cortex. Pregnant C57Bl mice received pulse injections of (3H)thymidine from E10 through E17 (E0 being the day of mating). The distributions of thymidine-labeled, GAD-positive and nonimmunoreactive (non-GAD) cells as a function of depth under the pial surface were recorded in adult animals. The maximum rate of generation of GAD-positive neurons occurred at E14, whereas the generation of non-GAD neurons reached its maximum rate at E13. Except for those in layer I, GAD-positive neurons followed an inside-out sequence of positioning. GAD-positive neurons born at E12 and E13 were located in layers VI-IV. GAD-positive neurons born at E14 were found throughout the cortical thickness, with a maximum in layer IV. The GAD-positive neurons labeled after pulses at E15 or E16 or E17 were limited to the superficial strata, forming a band that became narrower as it moved toward the pial surface with increase in age of pulse labeling. GAD-positive neurons in layer I were generated at a constant rate during the whole embryonic period analyzed. Non-GAD neurons also followed an inside-out spatiotemporal gradient. Two partially overlapping phases were distinguished in non-GAD neurogenesis. During the first phase (from E12 to E14) neurons populating adult layers VI and V originated, while neurons located in layers IV through I were generated during the second phase (from E13 to E17). Since GAD-immunoreactive neurons form a heterogeneous population, we envisage further studies in order to test whether differences exist in birth dates among the classes.

Tyrosine hydroxylase immunoreactive neurons throughout the hypothalamus receive glutamate decarboxylase immunoreactive synapses: a double pre- embedding immunocytochemical study with particulate silver and HRP

Silver-intensified gold (SIG) particles were used for light- and electron-microscopic immunocytochemical localization of neuronal antigens, and the SIG method was compared with related heavy-metal methods for the purpose of dual ultrastructural localization of neurotransmitter-related antigens. SIG immunostaining was combined with peroxidase immunostaining to allow simultaneous study of differentially labeled tyrosine hydroxylase and glutamate decarboxylase immunoreactive neurons in the medial hypothalamus. A number of electron-dense markers that might be of use in double immunostaining for light and electron microscopy were examined, either with a simple nitrocellulose dot-blot method or on Formvar-coated slot grids. Of these, silver-intensified 5 nm colloidal gold was the most effective. Silver intensification of colloidal silver and of peroxidase reaction product also showed promise for combined LM and EM double-immunolabeling studies. Since the silver-intensification procedure used here intensifies both gold and peroxidase, in experiments involving, double staining, the silver-intensified gold procedure should be used for the first antigen and nonintensified HRP for the second. Presumptive dopaminergic neurons containing the enzyme tyrosine hydroxylase were located throughout the hypothalamus with SIG immunostaining. In the same areas where frequent tyrosine hydroxylase immunoreactive neurons were found, many axons and bouton terminals were also found with antisera against GABA or against the GABA-synthesizing enzyme glutamate decarboxylase. Areas containing cells immunoreactive for tyrosine hydroxylase and stained with SIG and axons immunoreactive for glutamate decarboxylase and stained with peroxidase included the periventricular area (A14), the arcuate nucleus (A12), the dorsomedial hypothalamus/zona incerta area (A13), the posterior hypothalamus (A11), the medial paraventricular nucleus, and dorsal to the supraoptic nucleus, in addition to the preoptic area near the third ventricle and dorsally adjacent to the anterior commissure. For comparison, the SIG procedure was also used to stain dopaminergic neurons outside the hypothalamus in the substantia nigra and ventral tegmental area. Double immunocytochemical staining of two different neurotransmitter-related antigens allowed examination with both light and electron microscopy. By virtue of a large silver shell formed around the colloidal gold particle and its adsorbed immunoglobulin or protein A, cross-reactivity of the first set of immunoreagents stained with particulate silver and a second set stained with peroxidase could be reduced or eliminated.(ABSTRACT TRUNCATED AT 400 WORDS)

Substance P-immunoreactive neurons in the neocortex of the rat: A subset of the glutamic acid decarboxylase-immunoreactive neurons

Substance P-immunoreactive (SP-IR) neurons are found throughout the layers of the rat neocortex. Within the somatosensory (SI) cortex these cells make up about 2% of all neurons and within area 17 about 3%. Colocalization studies reveal that the SP-IR neurons are a subset of the population immunoreactive for glutamic acid decarboxylase.

Silver-intensified gold and peroxidase as dual ultrastructural immunolabels for pre- and postsynaptic neurotransmitters.

An ultrastructural immunostaining method that uses silver-intensified gold was combined with another procedure that uses biotin peroxidase conjugates to allow simultaneous identification of two neurotransmitter-related antigens in the central nervous system. Tyrosine hydroxylase-immunoreactive neurons labeled with silver-intensified gold could be differentiated at both light and electron microscopic levels from glutamate decarboxylase-immunoreactive neurons labeled with peroxidase. Cross reactivity of the second group of immunoreagents with the first group was reduced by the heavy metal silver shell formed around the colloidal gold immunoglobulin complex. With this dual pre-embedding method, peroxidase-stained axons containing the inhibitory neurotransmitter gamma-aminobutyric acid were found to synapse directly on silver-stained dopamine neurons in the rat dorsomedial hypothalamus. This approach can be used in combination with a post-embedding immunocytochemical colloidal gold procedure, allowing ultrastructural identification of three neurotransmitter-related antigens in the same tissue section.

Glutamic acid decarboxylase-immunoreactive neurons and terminals in the lateral geniculate nucleus of the cat

We have examined the distribution of neurons and terminals that are immunoreactive for glutamic acid decarboxylase (GAD), the synthesizing enzyme for the inhibitory neurotransmitter gamma-aminobutyric acid within the lateral geniculate nucleus of the cat. We estimate that GAD-positive neurons constitute approximately one-fourth of the neurons in all layers of the lateral geniculate nucleus and in the medial interlaminar nucleus (MIN). In addition, almost all of the neurons within the perigeniculate nucleus are GAD-positive. The mean size of GAD-positive cell bodies is significantly smaller than the mean size of unlabeled neurons in all subdivisions of the lateral geniculate nucleus. GAD-positive neurons have thick primary dendrites which are associated with thin lightly immunoreactive processes that give rise to clusters of GAD-positive terminals. Clusters of GAD-positive terminals are prominent in lamina A, A1, magnocellular C, and MIN but are rare in the parvocellular C laminae. Within the A laminae, GAD immunoreactivity is found within vesicle-containing profiles of the synaptic glomerulus lying postsynaptic to optic axon terminals and presynaptic to unlabeled dendritic profiles. GAD-positive neurons in the A laminae are distinguished from other small to medium-sized neurons by their failure to label following injections of HRP into visual cortex and by their lack of cytoplasmic laminated body. These results support the idea that GAD-positive neurons constitute a distinct population of neurons in the lateral geniculate nucleus of the cat; a population which has a number of features in common with previous descriptions of presumed local circuit neurons based on Golgi staining.