Differential Postreceptor Signaling Events Triggered by Excitotoxic Stimulation of Different Ionotropic Glutamate Receptors in Retinal Neurons Armanda E. Santos, 1,2 Ana L. Carvalho, 1 Maria C. Lopes, 1,2 and Arse ´lio P. Carvalho 1 * 1 Center for Neuroscience of Coimbra, Department of Zoology, University of Coimbra, Coimbra, Portugal 2 Faculty of Pharmacy, University of Coimbra, Coimbra, Portugal The aim of this work was to investigate whether excito- toxicity induced by overstimulation of different ionotropic glutamate receptors could trigger different intracellular signaling cascades. Cultured chick neuronal retina cells, essentially amacrine-like, were particularly sensitive to the toxicity induced by non-NMDA glutamate receptor agonists. One hour stimulation with 100 M kainate in- duced a reduction of cell viability of about 44%, as assessed by the MTT test 24 hr after stimulation. Kainate-induced toxicity was mediated through AMPA receptors. Glutamate (100 M, 1 hr) reduced cell viability by 26%, essentially acting through N-methyl-D- aspartate receptors. Five hours after stimulation, neuro- nal retina cells had an apoptotic-like nuclear morphol- ogy. In retinal neurons, the excitotoxic stimulation, with either glutamate or kainate, induced a calcium- dependent enhancement of the DNA-binding activity of the activating protein-1 (AP-1) transcription factor, which was maximal 2 hr after stimulation. Glutamate induced a greater increase in the AP-1 DNA-binding activity than did kainate. Supershift assays using antibodies directed against different members of the Fos and Jun protein families showed that the AP-1 complex in retinal neurons includes proteins of the Fos family, namely, Fra-2, c-Jun, and Jun D. The DNA-binding activity of the nuclear factor-B transcription factor was not significantly changed upon excitotoxic stimulation with any agonist. Stimulation of glutamate receptors with 100 M kainate or 100 M glutamate for 2 min was sufficient to induce the activation of the extracellular signal-regulated kinase (ERK). Inhibition of the ERK activation with the MEK inhibitors U 0126 and PD 98059 increased the toxicity induced by kainate but was without effect on the toxicity induced by glutamate. These results indicate that, al- though stimulation with both glutamate receptor agonists increased ERK phosphorylation, only kainate-induced ERK activation correlates with the activation of a survival signaling pathway. Our results suggest that, in chick embryo retinal neurons, the signaling pathways that me- diate excitotoxic cell death and neuroprotection are stim- ulus specific. J. Neurosci. Res. 66:643– 655, 2001. © 2001 Wiley-Liss, Inc. Key words: AMPA receptors; AP-1 transcription factor; extracellular signal regulated-kinase; neuroprotection; apoptosis Excitotoxic neurodegeneration involves overactiva- tion of ionotropic glutamate receptors (Olney, 1978; Choi, 1988), which consist of the N-methyl-D-aspartate (NMDA) and non-NMDA receptors, including the -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and kainic acid (KA) receptors (Hollmann and Heinemann, 1994; Bettler and Mulle, 1995). Excitotox- icity has been implicated in several neuronal disorders, such as cerebral ischemia, epilepsy, and Huntington’s, Parkinson’s, and Alzheimer’s diseases (Bittigau and Ikono- midou, 1997; Martin et al., 1998; Doble, 1999; White et al., 2000). Ischemia or excitotoxic insults to the rabbit or chick retina point to a high sensitivity of retinal amacrine cells to overstimulation of the ionotropic glutamate recep- tors (Osborne and Herrera, 1994; Zeevalk and Nicklas, 1994; Osborne et al., 1995, 1996; Chen et al., 1999; for review see Duarte et al., 1998). The overactivation of glutamate receptors can in- duce apoptotic cell death by a mechanism involving cal- cium influx (Ankarcrona et al., 1995; Glazner et al., 2000), and such toxicity may occur in acute neurodegenerative conditions, such as ischemia, trauma, and severe epileptic seizures, as well as in Alzheimer’s disease and motor system disorders (Choi, 1996; Mattson, 2000). Often, the inten- sity of the same initial insult decides the prevalence of either apoptosis or necrosis (Bonfoco et al., 1995). Apoptosis can be attenuated, in some cases, by in- hibitors of protein synthesis, suggesting that transcription and translation of new products could be important during Contract grant sponsor: Portuguese Science and Technology Foundation; Contract grant number: SAU/14120/98. *Correspondence to: Arse ´lio Pato de Carvalho, Center for Neuroscience of Coimbra, Department of Zoology, University of Coimbra, 3004-517 Co- imbra, Portugal. E-mail: carvalho@cnc.uc.pt Received 8 May 2001; Revised 13 August 2001; Accepted 21 August 2001 Journal of Neuroscience Research 66:643– 655 (2001) © 2001 Wiley-Liss, Inc.