ORIGINAL PAPER Eur. 1. Histochcm. 42,71-76,1998 © Luigi Ponzio e tiglio - Editori in Pavia Changes in NADPH-diaphorase expression induced by excitotoxicity in the goldfish retina: relation with some morphological and biochemical aspects I L. Villani and T. Guamieri / \ Department of Biology, University of Bologna, Via Selmi 3, 1-40126 Bologna, Italy. Accepted 26/ I /98 Key words: NADPH-diaphorase, nitric oxide, excitotoxicity, retina, goldtish SUMMARY We report alterations in the pattern of NADPH- diaphorase staining, a marker of nitric oxide, in the goldfish retina after kainic acid administration. The heavily stained ellipsoids of the photorecep- tors, the heavily stained neurons of the inner plexiform layer and the labeled neurons of the gan- glion cell layer are spared by excitotoxic insult, while the faintly medium sized neurons of the inner plexiform layer disappear after kainic acid administration. Furthermore, in the bipolar and in the horizontal retinal neurons we observe an in- duction of NADPH-diaphorase expression. The kainic acid-induced neurotoxicity evaluated by morphological observations and by measuring the levels of choline acetyltranferase in retinal homo- genates, is not prevented by the administration of a nitric oxide synthase inhibitor. INTRODUCTION Nitric oxide (NO) acts in the nervous system as a messenger molecule (Bredt and Snyder, 1992). The distribution of this gaseous molecule may be revealed by the histochemical reaction for NADPH-diaphorase (Hope and Vincent, 1989). This enzyme is co-localized in the neuronal cells with NO synthase (NOS) (Dawson et al., 1991 a), the enzyme responsible for NO synthesis from L- arginine. Recent reports on the distribution of NADPH-diaphorase and NOS positive structures (Djamgoz et al., 1996; Liepe et ai., 1994; Ostholm et al., 1994; Villani and Guarnieri, 1996; Weiler et al., 1993), suggest a physiological role for NO in the different retinal layers of fish. A key role for NO in the nervous tissue includes its neurotoxic effect (Choi, 1993). Strong NADPH-diaphorase positive cells are notably resistant to the neuro- toxicity of excitatory amino acids (Beal et al., 1989; Lafon-Cazal et al., 1993; Koh and Choi, 1988; Koh et al., 1986); other reports (Dawson et al., 1991b; Dwyer et al., 1991; Przegalinski et ai., 1994; Schulz et al., 1995) demonstrate that gluta- mate neurotoxicity is prevented by inhibitors of NOS. Recently, Zeevalk and Nicklas (1994) repor- ted that NO pathways present in chick retina do not play a role in NMDA neurotoxicity. Kainic acid (KA), a structural analogue of glutamate, is a potent neurotoxin that severely damages selected populations of vertebrate neurons. In fish retina the excitotoxic insult of KA, caused by the activation of non-NMDA glutamate receptors, induces vacuolization of both outer and inner plexiform layers (Kleinschrnidt et al., 1986) and decrease of 71