Coexistence of glutamic acid decarboxylase- and somatostatin-like immunoreactivity in neurons of the feline nucleus reticularis thalami

Abstract

The presence of somatostatin-like (SOM) immunoreactivity within GABAergic neurons of the nucleus reticularis thalami (NRT) was demonstrated by immunocytochemical methods in the cat using a two-color, double immunoperoxidase method with antisera raised in different species. GABAergic neurons were identified by means of a sheep antiserum to glutamic acid decarboxylase, the biosynthetic enzyme for γ-aminobutyric acid (GABA). SOM immunoreactivity was visualized with a rabbit antiserum to synthetic somatostatin. Vibratome sections of perfusion-fixed tissue were processed according to the pre-embedding unlabeled peroxidase antiperoxidase (PAP) method for light and electron microscopy. Single sections through the NRT were first processed for glutamic acid decarboxylase-like (GAD) or SOM immunoreactivity. With either antiserum, most neurons of the NRT were immunoreactive. The intracellular sites recognized by the two antisera had only partially overlapping distributions. SOM immunoreactivity appeared largely restricted to perinuclear structures which were identified by electron microscopy as the Golgi apparatus and multivesicular bodies. GAD immunoreactivity also appeared in the Golgi apparatus but was broadly dispersed throughout the cytoplasm of the cell body and dendrites. Intensely GAD-immunoreactive dots in the neuropil of the NRT were shown by electron microscopy to be immunoreactive boutons. Coexistence of the SOM and GAD antigens was demonstrated in sections sequentially incubated with rabbit anti-somatostatin, swine anti-rabbit IgG, rabbit PAP, and 4-chloro-1-naphthol (blue color), followed by sheep anti-rat glutamic acid decarboxylase, donkey anti-goat IgG, goat PAP, and 3,3'-diaminobenzidine (brown color). Many neurons contained blue-black perinuclear skeins (SOM and GAD immunoreactivity) and brown reaction product in the cytoplasm (GAD immunoreactivity) and were contacted by dark brown punctate profiles (GAD immunoreactivity). Control experiments showed that a weak cross-reactivity of the two linking antisera did not impair the simultaneous visualization method. This study provides direct evidence for the existence, within GABAergic neurons of the cat NRT, of a substance immunocytochemically indistinguishable from somatostatin. The data suggest that neurons of the feline NRT compromise a 'SOM-positive subtype' of GABAergic neurons.