Histochemistry(1991) 96: 355-365 030155649100107R Histochemis © Springer-Verlag 1~991t9~ Localization of GAD-like immunoreactivity in the pancreas and stomach of the rat and mouse P. Gilon 1, M. Tappaz 2, and C. Remacle 3. 1 Unit6 de Diab~tologieet Nutrition, Universit6 Catholiquede Louvain,B-1200 Bruxelles,Belgium 2 Laboratoirede Neurochimiefonctionnelle (INSERM U 171), Centre Hospitalier-Lyon Sud, F-69310 Pierre-B6nite, France 3 Laboratoirede BiologicCellulaire(CELL),Universit6Catholique de Louvain,Place Croix du Sud 5, B-1348 Louvain-la-Neuve,Belgium Received December 28, 1990 / Accepted May 2, 1991 Summary. The aim of this study was to localize cells immunoreactive for glutamate decarboxylase (GAD), the enzyme of GABA synthesis, in pyloric and oxyntic regions of the rat stomach as well as in the rat and mouse pancreas. GAD immunocytochemistry was car- ried out on polyethylene glycol or cryostat sections of alkaline paraformaldehyde fixed tissue, with simulta- neous immunolabelling of various gastro-pancreatic hor- mones for topographical comparison. In the rat stom- ach, nerve fibers displaying intense GAD-like immuno- reactivity were seen in the myenteric plexus, the circular muscular layer, the submucosa and the lamina propria of the mucosa. But, they were absent from the submu- cous plexus. Colchicine treatment of the rats allowed to detect some labelled perikarya in the myenteric plexus suggesting that the GABAergic innervation is at least partly intrinsic to the stomach. In the oxyntic and pyloric mucosa, endocrine cells appeared immunostained for GAD. However, the nature of their hormones remained unknown since double immunodetections revealed that they were immunoreactive neither for gastrin nor for somatostatin. In the rat and mouse pancreas, GAD-like immunoreactivity was found in islet cells which corre- sponded only to insulin-secreting cells. Somatostatin-, glucagon- and pancreatic polypeptide-immunopositive cells were devoid of GAD immunolabelling. No GAD- like immunoreactivity was detected in the exocrine tissue and innervation. These results strenghten the hypothesis that GABA is not only a neurotransmitter in the stom- ach but that it could also be an endocrine or paracrine factor in the stomach and pancreas. Introduction Gamma-aminobutyric acid (GABA), an established in- hibitory neurotransmitter in the brain, is now recognized as a neuromodulator in the gastro-enteric nervous sys- * Offprint requests to: C. Remacle tem (Jessen et al. 1987). It mainly affects the gut muscu- lar contraction by controlling the neurotransmitter re- lease from neighbouring effector fibers. The majority of the physiological studies have focussed on the gut motility since GABA immunodetections revealed GABA-immunoreactive nerves located largely in the myenteric plexus and the circular muscular layer but very rarely in the submucosa and mucosa (Davanger etal. 1987; Hills etal. 1987). However, a few studies showed that GABA can also affect the endocrine func- tions by altering gastrin and somatostatin secretion by rat stomach (Harty and Franklin 1983, 1986; Koop and Arnold 1986; Guo et al. 1989) and secretin release from canine duodenal mucosa (Harty and Murthy 1986). Moreover, GABA immunoreactivity and 3H-GABA up- take were shown in endocrine cells of the rat stomach and gut (Gilon et al. 1987b, 1988, 1990; Jessen et al. 1988; Davanger et al. 1989). But, immunodetection of the enzyme of GABA synthesis, glutamate decarboxy- lase (GAD), was never extensively described in the stom- ach. In the pancreas, another entity of the diffuse endo- crine system, biochemical and immunocytochemical stu- dies have clearly revealed that the insulin-secreting Cells (B-cells) were the only cells to contain high amounts of GABA, GAD and its degradation enzyme, GABA- transaminase (GABA-T) (Vincent et al. 1983; Okada 1986; Garry etal. 1987a, 1987b; Sakaue etal. 1987; Gilon et al. 1988). However recently, Garry et al. (1988) have also reported an immunoreactivity of GABA and GAD in the zymogen granules of the acini suggesting that GABA could play a role in exocrine functions. The present study reports immunocytochemistry of GAD in pyloric and oxyntic regions of the rat stomach and mainly focusses on the mucosa. It also includes fur- ther investigations on the pancreas, in the rat and especially in the mouse, a species for which no informa- tion is available so far. The coupling of immunodetec- tions of GAD and various hormones (insulin, glucagon, pancreatic polypeptide, somatostatin or gastrin) allowed to determine whether or not the corresponding immuno- reactive cells synthesize GABA by GAD.