scholarly journals Antisera to gamma-aminobutyric acid. III. Demonstration of GABA in Golgi-impregnated neurons and in conventional electron microscopic sections of cat striate cortex.

1985 ◽  
Vol 33 (3) ◽  
pp. 249-257 ◽  
Author(s):  
P Somogyi ◽  
A J Hodgson
Keyword(s):  
Striate Cortex ◽  
Protein A ◽  
Three Dimensional ◽  
Aminobutyric Acid ◽  
Gamma Aminobutyric Acid ◽  
Electron Microscopic ◽  
Golgi Impregnation ◽  
Second Procedure ◽  
Unlabeled Antibody ◽  
Layer Vi

Two methods are described for the immunocytochemical demonstration of immunoreactive gamma-aminobutyric acid (GABA) in the visual cortex of the cat, an area that contains several types of GABAergic neurons and requires combined methods for their characterization. The first method is illustrated by a representative example of a Golgi-impregnated and gold-toned interneuron of the "bitufted" type situated in layer VI and having an ascending axon. After recording the three-dimensional features of the cell, semithin (0.5 micron) sections of the perikaryon were cut and GABA was demonstrated in the cell body by the unlabeled antibody enzyme method. While immunocytochemistry was used to determine the probable transmitter of the neuron, Golgi-impregnation of the same cell was used to identify its neuronal type. Since aldehyde-osmium fixation was used, further electron microscopic (EM) analysis of the neuron's synaptic connections was possible. The second procedure demonstrated GABA in EM sections of aldehyde-osmium-fixed cortex using protein A-gold as an immunocytochemical marker. Immunoreactivity was found in certain neurons, dendrites, axons, and boutons forming type II synaptic contacts that from previous studies have been thought to be GABAergic. Thus ultrastructural analysis using optimal conditions can now be supplemented with the identification of the transmitter in the same section.

The Lectin Vicia Villosa Labels a Distinct Subset of GABAergic Cells in Macaque Visual Cortex

1989 ◽  
Vol 2 (1) ◽  
pp. 63-72 ◽  
Author(s):  
K. A. Mulligan ◽  
J. F. M. Van Brederode ◽  
A. E. Hendrickson
Keyword(s):  
Cortical Neurons ◽  
Striate Cortex ◽  
Aminobutyric Acid ◽  
Size Analysis ◽  
Clear Correlation ◽  
Gamma Aminobutyric Acid ◽  
Vicia Villosa ◽  
Double Labeling ◽  
Distinct Subset ◽  
Soma Size

AbstractThe morphology and distribution of neurons labeled specifically by the lectin, Vicia villosa (VVA), were examined in striate cortex of adult macaque monkeys. Following incubation with VVA conjugated to histochemical markers, fine punctate reaction product appears to cover the surface of the soma and proximal dendrites of a population of cortical neurons. Although a small number of VVA-labeled cells are located in layers 2, 3A, 5, and 6, approximately 75% are located in a strip of cortex overlying layers 3B through 4Ca. Layers 1 and 4Cβ are virtually devoid of labeled cells. The morphology of labeled cells varies throughout the layers. In the supragranular layers, the labeled cells generally display a round or multipolar soma with a small number of radially disposed dendrites. In deeper layers, labeled cells are multipolar or horizontal, and their proximal dendrites are often more densely labeled. There is no clear correlation between the distribution of labeled cells and the pattern of cytochrome oxidase staining in supragranular layers.Double labeling of single sections for VVA and for GABA (gamma-aminobutyric acid) immunoreactivity revealed that most VVA-labeled cells are also immunoreactive for GABA. The double-labeled cells comprise approximately 30% of all GABA immunoreactive cells. Soma size analysis of double-labeled cells shows that medium-to-large GABA cells in each layer are labeled by VVA. The soma size, laminar distribution, and morphology of the VVA-labeled GABA cells suggest that they include the large basket cells originally observed in Golgi preparations.

scholarly journals gamma-Aminobutyric acid-containing terminals can be apposed to glycine receptors at central synapses

1987 ◽  
Vol 104 (4) ◽  
pp. 947-956 ◽  
Author(s):  
A Triller ◽  
F Cluzeaud ◽  
H Korn
Keyword(s):  
Gaba Receptors ◽  
Glycine Receptor ◽  
Cross Reactivity ◽  
Aminobutyric Acid ◽  
Electron Microscopic Level ◽  
Acid Decarboxylase ◽  
Gamma Aminobutyric Acid ◽  
Glycine Receptors ◽  
Electron Microscopic ◽  
Double Labeling

The distributions of terminals containing gamma-aminobutyric acid (GABA) and of endings apposed to glycine receptors were investigated cytochemically in the ventral horn of the rat spinal cord. For this purpose, a polyclonal antibody raised to recognize glutamic acid decarboxylase (GAD), a synthetic enzyme for GABA, and three monoclonal antibodies (mAb's) directed against the glycine receptor were used. Double immunofluorescence showed that, surprisingly, GAD-positive terminals are closely associated in this system with glycine receptors at all the investigated cells, most of which were spinal motoneurons. Furthermore, double labeling was performed with immunoenzymatic recognition of GAD and indirect marking of mAb's with colloidal gold. With this combined approach, it was found, at the electron microscopic level, that all GAD-positive terminals are in direct apposition with glycine receptors while, on the other hand, not all glycine receptors are in front of GABA-containing boutons. This result is not due to a cross-reactivity of mAb's with GABA receptors as shown by using as a control synapses known to use GABA as a neurotransmitter in the cerebellar cortex. Indeed, no glycine receptor immunoreactivity was detected on Purkinje cells facing basket axon terminals. However, Purkinje neurons can express glycine receptor immunoreactivity at other synaptic contacts. Assuming that the presence of postsynaptic receptors for glycine indicates that this amino acid is used for neurotransmission at a given synapse, our results strongly support the notion that GABA and glycine, two classical inhibitory transmitters, coexist at some central connections. However, such is not always the case; in the cerebellum, Golgi terminals impinging on the dendrites of granule cells are either GAD-positive or face glycine receptors, in a well-segregated manner.

scholarly journals Demonstration of antigens at both sides of plasma membranes in one coincident electron microscopic image: a double-immunogold replica study of virus-infected cells.

1988 ◽  
Vol 36 (8) ◽  
pp. 1015-1021 ◽  
Author(s):  
G Rutter ◽  
W Bohn ◽  
H Hohenberg ◽  
K Mannweiler
Keyword(s):  
High Resolution ◽  
Measles Virus ◽  
Plasma Membranes ◽  
Protein A ◽  
Three Dimensional ◽  
Electron Microscopic ◽  
Cytoplasmic Surface ◽  
Critical Point Drying ◽  
Electron Microscopic Image ◽  
Infected Cells

We present here a procedure for obtaining high-resolution topographical information about the spatial distribution of antigens at both sides of isolated plasma membranes. HeLa cells grown on coverslips and infected with measles virus served as a model system. Virus glycoproteins appearing at the cell surface were demonstrated by tagging them with rabbit anti-measles antibodies and protein A-gold probes. Cells were stabilized with tannic acid, covered with a cationized coverslip, and then split in potassium-containing buffer. Membranes adherent to the cationized coverslip were fixed in formaldehyde-glutaraldehyde and reacted with mouse monoclonal antibodies against various structural proteins of measles virus. Antibody binding sites at the cytoplasmic surface were visualized either by the antibody bridge method, using normal mouse Ig coupled to gold colloid of different sizes, or by the peroxidase-antiperoxidase procedure. After osmication and critical point-drying, the cytoplasmic surfaces were replicated by platinum-carbon evaporation and examined by TEM without prior cleaning from biological material. This new method permits concomitant localization of antigens present at the inner and outer leaflets of the plasma membrane, and provides high-resolution information about the three-dimensional organization of the cytoplasmic surface.

Gamma-aminobutyric acid-immunoreactivity in the rat hippocampus. A light and electron microscopic study with anti-GABA antibodies

1986 ◽  
Vol 364 (1) ◽  
pp. 30-38 ◽  
Author(s):  
Halima Gamrani ◽  
Brigitte Onteniente ◽  
Philippe Seguela ◽  
Michel Geffard ◽  
Andre Calas
Keyword(s):  
Microscopic Study ◽  
Electron Microscopic Study ◽  
Rat Hippocampus ◽  
Aminobutyric Acid ◽  
Gamma Aminobutyric Acid ◽  
Electron Microscopic

scholarly journals Antisera to gamma-aminobutyric acid. I. Production and characterization using a new model system.

1985 ◽  
Vol 33 (3) ◽  
pp. 229-239 ◽  
Author(s):  
A J Hodgson ◽  
B Penke ◽  
A Erdei ◽  
I W Chubb ◽  
P Somogyi
Keyword(s):  
Detection System ◽  
High Specificity ◽  
Aminobutyric Acid ◽  
Gamma Aminobutyric Acid ◽  
Fixed Tissue ◽  
Beta Alanine ◽  
Tissue Sections ◽  
Beta Hydroxybutyrate ◽  
Unlabeled Antibody ◽  
Related Compounds

Antisera to the amino acid gamma-aminobutyric acid (GABA) have been developed with the aim of immunohistochemical visualization of neurons that use it as a neurotransmitter. GABA bound to bovine serum albumin was the immunogen. The reactivities of the sera to GABA and a variety of structurally related compounds were tested by coupling these compounds to nitrocellulose paper activated with polylysine and glutaraldehyde and incubating the paper with the unlabeled antibody enzyme method, thus simulating immunohistochemistry of tissue sections. The antisera did not react with L-glutamate, L-aspartate, D-aspartate, glycine, taurine, L-glutamine, L-lysine, L-threonine, L-alanine, alpha-aminobutyrate, beta-aminobutyrate, putrescine, or delta-aminolevulinate. There was cross-reaction with gamma-amino-beta-hydroxybutyrate, 1-10%, and the homologues of GABA: beta-alanine, 1-10%, delta-aminovalerate, approximately 10%, and epsilon-amino-caproate, approximately 10%. The antisera reacted slightly with the dipeptide gamma-aminobutyrylleucine, but not carnosine or homocarnosine. Immunostaining of GABA was completely abolished by adsorption of the sera to GABA coupled to polyacrylamide beads by glutaraldehyde. The immunohistochemical model is simple, amino acids and peptides are bound in the same way as in aldehyde-fixed tissue and, in contrast to radioimmunoassay, it uses an immunohistochemical detection system. This method has enabled us to define the high specificity of anti-GABA sera and to use them in some novel ways. The model should prove useful in assessing the specificity of other antisera.

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