Anat Embryol (1989) 179 : 221-226 Anatomy and Embryology 9 Springer-Verlag 1989 Original articles GABA-immunoreactive cells in the rat gastrointestinal epithelium Svend Davanger, Ole Petter Ottersen, and Jon Storm-Mathisen Anatomical Institute, University of Oslo, Karl Johans Gate 47, N-0162 Oslo 1, Norway Summary. Frozen sections of the corpus ventriculi, antrum pyloricum, duodenum, jejunum, ileum and colon from ani- mals perfusion fixed with glutaraldehyde were treated with an antiserum specific for glutaraldehyde-fixed GABA and processed by the peroxidase antiperoxidase method. Semi- thin plastic sections from the antrum pyloricum were treated similarly. Stained cells appeared in the epithelium of all segments examined except the corpus ventriculi. The highest density of cells was observed along the major curva- ture of the antrum pyloricum. Here they were located in the bottom half of the gastric glands. Many of the cells showed a process extending towards the glandular lumen. No significant staining in the epithelium appeared when the antiserum was preincubated with glutaraldehyde- GABA complexes, nor when the anti-GABA serum was exchanged with anti-glycine or preimmune serum. The pres- ent findings and previous physiological data suggest that GABA may play a role in gut endocrine regulation. Key words: GABA - Immunocytochemistry - Gastrointes- tinal tract - Epithelium Enteroendocrine cells Rat Introduction y-Aminobutyric acid (GABA) is now considered as a likely neurotransmitter in the mammalian gastrointestinal tract. Evidence from physiological and biochemical studies that GABA may be involved in neuronal control of intestinal motility (Hobbiger 1958; Jessen etal. 1979; rev.: Jessen et al. 1987) has recently been corroborated by immunocyto- chemical studies demonstrating the presence of GABA in neurons and varicose fibres of myenteric ganglia and in varicose nerve fibres projecting into the circular muscle layer (Jessen et al. 1986; Saito and Tanaka 1986; Davanger et al. 1987a; Hills et al. 1987). Previous studies on the immunocytochemical localiza- tion of GABA in the stomach and gut have not focused on the mucosa, and in the submucosa few immunopositive fibres and no immunopositive perikarya have been observed (Hills et al. 1987). Nevertheless, GABA seems to affect gas- tric endocrine functions: application of GABA to isolated rat antral mucosa results in an increase in gastrin release and a reduction in somatostatin release (Harty and Frank- lin 1983). These effects were inhibited by application of the specific GABAA receptor antagonist, bicuculline. An- Offprint requests to: S. Davanger other study indicates that GABA stimula~oes secretin release from canine duodenal organ culture (Harry and Murthy 1986). Gastric acid production is influenced by i.p. injection of both GABAA and GABAB receptor agonists, and ulcer reducing effects of GABA receptor stimulation have been shown (Lloyd et al. 1986). The origin and site of action of GABA involved in these biochemical processes are not known. Harty and Murthy (1986) have suggested a model whereby GABAergic neurons within mucosal and submu- cosal plexus modulate cholinergic neurotransmission within antral submucosal plexus and thereby affect endocrine cell function. However, we here present immunocytochemical evidence that GABA is not restricted to neurons in the gastrointestinal nervous system, but also exists in epithelial cells in the mucosa of most regions of the rat alimentary canal. The observation of GABA-immunoreactive cells in the gastrointestinal epithelium has previously been reported in abstract form (Davanger et al. 1987b; Erd6 et al. 1987), and has been mentioned in a discussion (Hills et al. 1987). Also, immunoreactivity of the GABA synthesizing enzyme glutamate decarboxylase (GAD) in antral endocrine cells has been described in abstract form (HSkanson et al. 1978). Materials and methods The anti-GABA serum used (serum 26) was produced, puri- fied and tested as reported elsewhere (Storm-Mathisen et al. 1983; Ottersen and Storm-Mathisen 1984). The purified se- rum 26 did not show significant crossreactivity when tested with a large series of amino acids, amines (including putres- cine and spermidine) and peptides conjugated by glutaralde- hyde (G) to a total macromolecular extract from rat brain (Ottersen and Storm-Mathisen 1984; Ottersen et al. 1986). In the present study a selection of similar conjugates spotted on cellulose ester filters was incubated together with free floating tissue sections. The specifity of tissue staining was further checked by adding amino acid-G complexes (300 pM final concentration with respect to the amino acid) to the serum in order to absorb specific antibodies. Rats (n=8, weight=210-360 g, of either sex), deeply anesthetized with pentobarbital (50 mg/kg), were fixed by transcardial perfusion (0.5 1) with 5% G, or with a mixture of 1% formaldehyde (depotymerized from paraformalde- hyde) and 2.5% G, in 0.1 M sodium phosphate buffer (pH 7.4, 37 ~ C), preceded by a brief flush (15 30 s) of 2% dextran (MW 70 000) in the same buffer. Five of the animals received an intraperitoneal injection of the GABA transa- minase inhibitor 7-vinyl-GABA (1500 mg/kg, dissolved in