Ž . Molecular Brain Research 70 1999 159–163 www.elsevier.comrlocaterbres Short communication Caspase-3-dependent neuronal death in the hippocampus following kainic acid treatment Ciaran J. Faherty a , Stephen Xanthoudakis b , Richard J. Smeyne a, ) a Department of DeÕelopmental Neurobiology, St. Jude Children’s Research Hospital, 332 North Lauderdale, Memphis, TN 38105, USA b Department of Biochemistry and Molecular Biology, Merck-Frosst Center for Therapeutic Research, Kirkland, Quebec, Canada Accepted 6 April 1999 Abstract Ž . In this study, we examined the levels of activated caspase-3 in the kainic acid KA model of hippocampal degeneration in both Ž . Ž . sensitive FVBrN and resistant 129rSvEMS strains of mice. At 30 h, 2 and 4 days following KA administration, animals were sacrificed and brains examined for pyknosis, TUNEL labeling, and activated caspase-3 immunoreactivity. Catalytically active caspase-3 was first detected 30 h following KA treatment in the sensitive, FVBrN strain. This was 18 h before the appearance of pyknosis or TUNEL labeling. The expression of activated caspase-3 continues up to 4 days post-injection. No activated caspase-3 immunoreactivity was detected in the resistant, 129rSvEMS strain, neither was there evidence of pyknosis or TUNEL staining. This suggests that activation of caspase-3 is a necessary component of KA-induced cell death. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Seizure; Strain; Apoptosis; TUNEL; Excitotoxicity Excitatory amino acid neurotoxicity is thought to be a contributing factor in a number of central nervous system Ž . w x CNS disorders 6,31 . One model of excitotoxic cell death is the administration of the glutamate mimetic, kainic Ž . acid KA , a potent agonist at the a-amino-3-hydroxy-5- Ž . w x methyl-4-isoxaolepropionic AMPA receptor 17 . In sen- w x sitive strains of mice 23 , systemic administration of KA induces a period of generalized seizures, followed by a w x defined pattern of neuronal death 20,25 . This cell death w x exhibits some of the characteristics of apoptosis 11,24 . Several well-defined apoptosis pathways have been identified in mammals, that involves members of the Bcl-2 w x and caspase gene families 2,30 . One member of the caspase family, caspase-3 is a cysteine protease that cleaves specific aspartate residues in proteins in a variety of w structural, housekeeping and regulatory proteins 5,18,29, x 30,32 . These proteolytic events lead to death. Caspase-3 is synthesized as a latent proenzyme and is proteolytically processed to the active form in response to a number of w x apoptotic stimuli 5,30 . Caspase-3 protease activity is thought to be a contributing factor in a number of neurode- ) Corresponding author. Fax: q1-901-495-3143; E-mail: richard.smeyne@stjude.org generative diseases and has recently been shown, in vitro, w x to contribute to KA-induced cell death 21 . In order to ascertain if the cell death was dependent on caspase-3 activation in vivo, we examined the distribution of the activated form of the protein in sensitive and resistant strains of mice following administration of KA. Male FVBrN and 129rSvEMS mice aged between 60 Ž . and 90 days were injected subcutaneously s.c. with a Ž . Ž single dose of either 0.9% NaCl saline or KA 30 .Ž . mgrkg Sigma, MO . Animals were continually moni- tored for 2 h for the onset of seizure activity. Only animals w x attaining a Racine stage 5 seizure 23 were included in the study. At 30 h, 2 and 4 days following treatment, animals Ž . were anesthetized with tribromoethanol 250 mgrkg i.p. and transcardially perfused with 4% paraformaldehyde in 0.1 M phosphate buffered saline. Brains were removed, post-fixed overnight and processed for paraffin embedding. Serial sections were cut at 5 mm and mounted on three Ž . series of polyionic slides Superfrost-plus, Fisher . The first series was stained with Cresyl violet to identify cells with pyknotic nuclei. The second series was processed for TUNEL staining in order to assess the degree of DNA w x Ž . fragmentation 12 Intergen, NY . A third series was w x immunolabeled for activated caspase-3 3,22 . Immunocy- tochemistry was carried out by dewaxing the slides in 0169-328Xr99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. Ž . PII: S0169-328X 99 00143-6