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

Abstract

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.

Cervicothalamic tract terminals are enriched in glutamate-like immunoreactivity: an electron microscopic double-labeling study in the cat

“…The immunogold labeling pattern for Glu seen in the present and previous studies (e.g., Somogyi et al, 1986;Ottersen, 1987Ottersen, , 1989aMontero and Wenthold, 1989;Maxwell et al, 1990;Montero, 1990;Ji et al, 199 1) is much less differentiated than that seen, for example, with GABA (Somogyi and Hodgson, 1985;De Biasi et al, 1988;Ottersen, 1989a). Specifically at sites where the gold particle density is low, such as over astrocytes and PSDs, it is important to evaluate whether the low density of gold particles represents unspecific binding to resin or cross-reacting substances in the tissue or if it represents low levels of fixed Glu.…”

“…The procedure used for postembedding immunogold staining of ultrathin sections was based on that of Somogyi and Hodgson (1985) and modified to include the following steps: (1) 1% HIO, in distilled water (10 min); (2) 5% NaIO, in distilled water (60 min); (3) 1% human serum albumin (HSA) in distilled water (15 min); (4) Glu antiserum 03 [Glu03; raised by Storm-Mathisen Although the Glu antiserum was treated with glutaraldehyde fixation complexes of Gln before use, slight cross-reactivity with Gln still remained in the present experiments (see Results). To evaluate to what extent the cross-reactivity may have influenced the immunogold labeling for Glu, sections adjacent to those incubated in Glu antiserum were in some cases processed with Gln antiserum 34 [Gln34; raised by Laake et al, 1986; characterized at the EM immunogold level by Zhang et al (199 1) and Ottersen et al (199 l)].…”

Redistribution of Glutamate and Glutamine in Slices of Human Neocortex Exposed to Combined Hypoxia and Glucose Deprivation in vitro

“…The first protocol was modified from Somogyi and Hodgson (1985) and has been described in detail previ ously (Ottersen, 1987(Ottersen, , 1989a. Following treatment with 1% HI04 (7 min) and 9% NaHI04 (15 min) to alleviate the masking effect of OS04' the sections were incubated for 2 h in the primary antiserum to glutamate (code no.…”

Columnar Organization of Dendrites and Axons of Single and Synaptically Coupled Excitatory Spiny Neurons in Layer 4 of the Rat Barrel Cortex

“…The immunogold staining procedure was performed as described by Somogyi and Hodgson (1985), using a commercially available antiserum against GABA (Sigma). The immunostaining was performed on droplets of Millipore-filtered solutions in humid Petri dishes.…”