English (United Kingdom)

https://curated-unify.zendy.io/wp-json/zendy-region/v1/featured_content/oa?rat=en

https://curated-unify.zendy.io/wp-json/zendy-region/v1/highlighted_journal/

Global: Zendy Plus annual

Presents the access of premium content as premium feature

Premium Content

Presents the keyphrase highlighting as premium feature

Keyphrase Highlighting

Presents the summarisation as premium feature

Summarisation

Insights

Presents the pdf analysis as premium feature

PDF Analysis

Presents the zaia usage as premium feature

ZAIA

Global: Zendy Tools annual

Global: Zendy Free Plan

Global: Zendy Tools monthly

Global: Zendy Plus monthly

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 y-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. Immunohistochemical evidence strongly suggests that central, peripheral, autonomic, and enteric nervous sysneuropeptides coexist with classical and nonclassical neutems. The production of low molecular weight polypeprotransmitters in endocrine cells and in neurons of the tides by amine-producing endocrine and enteric cells was ’ This work was supported by Grant Oe 95/l, Deutsche Forschungsgemeinschaft (W. H. 0.); by National Science Foundation Grant BNS I-81-12125, National Institutes of Health Grant 5-ROl-EY02866-03, and National Aeronautics and Space Administration Grant NAG2-124 (A. M. G.); by United States Public Health Service Grant 09904 (E. M.); and by NADA Grant 02148 and the McKnight Foundation (R. P. E.). We wish to thank Virginia K. Weise, Elaine Yoneoka, and AnneLise Dahl for their excellent technical assistance and Henry F. Hall and Larry Osby for help with the photography. ‘To whom correspondence should be sent at his current address: Neurologische Klinik (Leiter: Prof. Dr. A. Struppler), Technische Universitaet Muenchen, Moehlstrasse 28, D-8000 Muenchen 80, Federal Republic of Germany. The Journal of Neuroscience Coexistence of GAD and SOM Immunoreactivity 1323 first studied systematically by Pearse (1968, 1969), who formalized the concept of cellular co-production of amines and polypeptides in his characterization of the APUD (amine and precursor uptake and decarboxylation) series. Since this pioneering work, a number of examples of co-localization of biogenic amines and neuropeptides have been studied in central and peripheral neurons (Hokfelt et al., 1977, 1978a, 1980a, b, c, d; ChanPalay et al., 1978, 1981; Schultzberg et al., 1980). In addition, neuropeptides have been demonstrated in presumed cholinergic neurons (Lundberg et al., 1979; Fex and Altschuler, 1981; Erichsen et al., 1982) and in neurons that contain other neuropeptides (Watson et al., 1978; Schultzberg et al., 1980; Hunt et al., 1981; Erichsen et al., 1982. For review, see Osborne, 1981, and Potter et al., 1981.). A striking absence in the list of neurotransmitters coexisting with neuropeptides has been y-aminobutyric acid (GABA), a major inhibitory neurotransmitter in the brain. Only recently, Chan-Palay et al. (1981) and Nilaver et al. (1982) have reported light microscopic evidence for the presence of motilin-like immunoreactivity in some GABAergic cerebellar Purkinje cells. In the present study we applied two-color immunohistochemical methods to examine the possibility that the neuropeptide, somatostatin (Elde and Parsons, 1975), coexists with a specific endogenous marker for GABAergic neurons, namely, glutamic acid decarboxylase, the biosynthetic enzyme for GABA (McLaughlin et al., 1974; Saito et al., 1974; Barber and Saito, 1976; Wu, 1976; Ribak et al., 1976; Roberts, 1979; Oertel et al., 1981a). We chose to study the thalamic reticular nucleus (NRT) because this nucleus has been shown to contain glutamic acid decarboxylase-like (GAD) -immunoreactive neurons in the rat (Houser et al., 1980) and somatostatin-like (SOM)-immunoreactive neurons in the cat (Graybiel et al., 1981) and monkey (Graybiel and Elde, 1983). This work was summarized at the Conference of Cytochemical Methods in Neuroanatomy (June 10 to 12, 1981, Bethesda, MD) and at the 11th Annual Meeting of the American Society for Neuroscience (Oertel et al., 1981c). Materials and Methods Observations were made on the brains of 10 young domestic cats (1000 to 2000 gm) and 10 adult OsbornMendal and Sprague-Dawley rats (150 to 200 gm). Two rats received an intraventricular injection of 50 pg of colchicine dissolved in 10 ~1 of saline 36 hr prior to perfusion; in three rats 10 to 25 pg of colchicine in 1 to 2.5 ~1 of saline was injected stereotaxically in the thalamic relay nuclei 30 hr prior to perfusion. Animals were perfused under deep sodium pentobarbital anesthesia with 4% formaldehyde in 0.12 M sodium phosphate buffer (pH 7.4). Coronal and sag&al (20to 25-pm) sections through the thalamus were cut on a Vibratome and collected in phosphate-buffered saline (PBS; pH 7.4). For the immunohistochemical localization of glutamic acid decarboxylase, we employed sheep antiserum S3 (second or 3 Abbreviations: DAB, 3,3’-diaminobenzidine; GABA, y-aminobutyric acid; GAD, glutamic acid decarboxylase-like; NRT, nucleus reticularis thalami; PAP, peroxidase-antiperoxidase; PBS, phosphatebuffered saline; RT, room temperature; SOM, somatostatin-like. fourth bleed), and for somatostatin, rabbit antisera R176C and R176D. These antisera have been characterized elsewhere (Elde and Parsons, 1975; Oertel et al., 1981b) and seem to be specific for their respective antigens. Single-antigen immunohistochemistry was carried out on floating sections according to the pre-embedding unlabeled peroxidase-antiperoxidase (PAP) method of Sternberger (1979). The two-color double PAP technique represented a simple sequential staining procedure for the two antigens (Fig. 6). As linking antisera we used swine anti-rabbit IgG and donkey anti-goat IgG for localizing SOM and GAD immunoreactivity, respectively. Normal donkey serum was employed for blocking of unspecific binding sites. GAD immunocytochemistry. Sections from rat and cat brains were stained for GAD immunoreactivity at the light and electron microscopic level. All solutions were prepared in PBS and the sera were diluted in 1% normal donkey serum, 0.01 M monohydrochloric lysine, and 0.2% Triton X-100 dissolved in PBS. For light microscopic investigations the sections were pretreated with 3% hydrogen peroxide (Hz02)/10% methanol at room temperature (RT) for 5 min, preincubated in 10% normal donkey serum containing 0.1 M monohydrochloric lysine and 0.2% Triton X-100 for 1 hr at RT, and incubated for 16 hr at RT in sheep glutamic acid decarboxylase antiserum S3 diluted 1:2000. Control sections were incubated with preimmune serum S3 diluted 1:1800 (16 hr at RT). All sections were then exposed to donkey anti-goat IgG antiserum (1:50; Miles Laboratories, Elkhard, IN) for 30 min at RT, rinsed, and incubated for 30 min at RT in goat PAP complex (Sternberger-Meyer, Garretsville, MD) diluted 1:70 to 1:lOO. After being washed in Trisbuffered saline (0.05 M Tris, 0.9% NaCl, pH 7.6; four to five times, 5 min each), the sections were carried through the 3,3’-diaminobenzidine (DAB) procedure of Graham and Karnovsky (1966) or the 4-chloro-1-naphthol procedure of Dacheux and Dubois (1976). Some DAB-treated sections were counterstained with toluidine blue or cresyl violet. Sections were mounted from PBS onto subbed slides and coverslipped with Permount or a glycerol/PBS (3:1, v/v) mixture for bright-field photography. For electron microscopic immunocytochemistry, 50pm thick sections were reacted according to the preembedding method, as detailed above, with minor modifications: the HzOn/methanol pretreatment was omitted and subsequent steps were performed in the absence of Triton X-100. Following DAB incubation, the sections were fixed with 1% 0~01, treated with uranyl acetate, dehydrated, and embedded for electron microscopic analysis as detailed elsewhere (Oertel et al., 1981a). Semithin sections, lightly counterstained with toluidine blue, were used to determine the localization of immunoreactive neurons. SOM immunocytochemistry. Sections were treated with primary incubations in rabbit somatostatin antiserum R176C or R176D (1:500 to 1:700) for 48 hr at 4°C and for 5 hr at RT, and with control incubations in antiserum R176C (1:500 to 1:700) absorbed for 30 min prior to the incubation at RT with 10 PM somatostatin 16.4 pg/ml). Sect

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