The Journal of Neuroscience. June 1993, 13(6): 2651-2661 Mechanisms of Nitric Oxide-mediated Neurotoxicity in Primary Brain Cultures Valina L. Dawson,’ Ted M. Dawson ,2.3 Duane A. Bartley,2 George R. Uhl,1.2.3 and Solomon H. SnydeC4 ‘National Institute on Drug Abuse, Addiction Research Center, Laboratory of Molecular Neurobiology, Baltimore, Maryland 21224 and Departments of *Neuroscience, 3Neurology, 4Pharmacology and Molecular Sciences, and Psychiatry, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205 In addition to mediating several physiological functions, ni- of macrophagecytotoxicity (Hibbs et al., 1987; Marietta, 1989; tric oxide (NO) has been implicated in the cytotoxicities ob- Ignarro, 1990), nitric oxide (NO) appearsto be a neuronal mes- sewed following activation of macrophages or excess stim- sengerin the brain and PNS, satisfying many criteria of a neu- ulation of neurons by glutamate. We extend our previous rotransmitter (Garthwaite, 199 1; Snyder and Bredt, 1991; Daw- observations of glutamate-stimulated, NO-mediated neuro- son et al., 1992; Snyder, 1992). NO synthase(NOS) is localized toxicity in primary cultures of rat fetal cortical, striatal, and to the samediscrete neuronal populations in the brain that stain hippocampal neurons. Neurotoxicity elicited by either NMDA for NADPH-diaphorase (T. M. Dawson et al., 1991; Hope et or sodium nitroprusside (SNP) exhibits a similar concentra- al., 199l), a histochemical marker for neurons that resistcertain tion-effect relationship and time course. The concentration- forms of neurotoxicity (Beal ct al., 1986; Koh et al., 1986; Koh effect curve of NMDA-induced neurotoxicity is shifted to the and Choi, 1988). NOS catalytic activity accounts for NADPH- right in the presence of nitro-L-arginine and farther to the diaphorase staining, as transfection of human kidney 293 cells right in arginine-free media. The rank order of potency of with NOS cDNA provides NADPH-diaphorase and NOS stain- several NO synthase (NOS) inhibitors in preventing neuro- ing in the sameproportions as in neurons (T. M. Dawson et al., toxicity is the same as the rank order of these compounds 1991). in inhibiting NOS, and this inhibition is stereospecific. NMDA Glutamate neurotoxicity elicited via NMDA subtypesofglu- neurotoxicity is also prevented by flavoprotein Inhibitors and tamate receptors appearsto mediate much of the neurotoxicity calmodulin inhibitors, fitting with the roles of flavoproteins in focal ischemia, as NMDA antagonistsblock such neurotox- and calmodulin as NOS regulators. 8-Bromo-cGMP and icity (Choi, 1988, 1990). Glutamate ncurotoxicity may also play guanylyl cyclase inhibitors do not affect neurotoxicity, while a role in neurodegenerative diseasessuch as Alzheimer’s and superoxide dismutase attenuates neurotoxicity. NOS neu- Huntington’s discascs(Choi, 1988; Meldrum and Garthwaite, rons appear to be the source of neurotoxic NO in culture, as 1990). At NMDA receptors, glutamate triggers the opening of lesions of these neurons with 20 CAM quisqualate diminish cation-permeable channels. The entry of calcium through these subsequent NMDA neurotoxicity. Moreover, NMDA neuro- channels into cells stimulates NOS activity (Bredt and Snyder, toxicity develops over time in culture coincident with the 1989) by binding to calmodulin, which is a cofactor for NOS expression of NOS. lmmunohistochemical localization of NOS (Bredt and Snyder, 1990). in cultures and intact brain demonstrates widespread dis- Recently we demonstrated that NMDA neurotoxicity is at- tribution of the cell processes suggesting that NOS neurons tenuated in primary cerebral cortical cultures by the coappli- contact the majority of cortical neurons and so could mediate cation of NOS inhibitors or removal of the precursor of NO, widespread neurotoxicity. L-arginine (V. L. Dawson et al., 1991; T. M. Dawson et al., [Key words: NADPH-diaphorase, glutamate, NMDA, nitric 1993). These protective effectsare reversed by addition ofexcess oxide synthase, excitotoxicity, neurodegeneration] L-argininc. In addition, sodium nitroprusside (SNP), which gen- erates NO, mimics NMDA-induced neurotoxicity. Both SNP Besidesits roles as endothelial-derived relaxing factor (Furch- and NMDA neurotoxicities are also blocked by hemoglobin, gott, 1990; Ignarro, 1990; Moncada et al., 1991) and a mediator which binds NO. Together, these results implicate NO as a potential mediatorofNMDA neurotoxicity. In the presentstudy we examine the detailed mechanismsregulating NO mediation Received Oct. 5, 1992; revised Jan. 4, 1993; accepted Jan. I I, 1993. of glutamate neurotoxicity. We thank Dr. Charles E. Spivak for performing the electrophysiologic experi- men& This work was supported by USPHS Grants MH- 1850 1 and DA-00266, Materials and Methods Contract DA 271-90-7408, and Research Scientist Award DA-00074 to S.H.S., and NlGMS Pharmacology Research Associate Training Program and an Intra- Cell cultures mural Research Training Award (IRTA) from the NIH to V.L.D. T.M.D. is a Primary cell cultures were prepared from fetal rats (gestation day 14 for Pfizer postdoctoral fellow and is supported by grants from the American Academy of Neurology, the French Foundation for Alzheimer’s Research, the DANA Foun- cortex andcaudate-putamen cultures, gestation day I7 for hippocampal dation, and USPHS CIDA NS 01578-01. We gratefully acknowledge support of cultures). The various brain regions were dissected under a microscope, the W. M. Keck Foundation. incubated for 20 min in 0.027% trypsin/saline solution (5% phosphate- Correspondence should he addressed to Solomon H. Snyder, Departments of buffered saline, 40 mM sucrose, 30 mM glucose, IO mM HEPES, pH Neuroscienceand Pharmacology and Molecular Sciences,725 North Wolfe Street, 7.4). and transferred to modified Eagle’s medium (MEM), 10% horse Baltimore, MD 2 1205. serum,10% fetal bovine serum, 2 mM glutamine. Cells were dissociated Copyright 0 I993 Society for Neuroscience 0270-6474193113265 I-I I $05.00/O by trituration, counted, and plated in I5 mm multiwell (Nunc) plates 2652 Dawson et al. - Mechanisms of Nitric Oxide Neurotoxicity coated with polyomithine at a density of u x 10’ cells per well. Four NOS immunohistochemistry. days after plating, the cells were treated with IO pg/ml of 5-Buoro-2’- deoxyuridine for 3 d to inhibit proliferation of non-neuronal cells. Cells Cells were washed three times with CSS and fixed for 30 min at 4°C in were maintained in MEM, 5% horse serum, 2 mM glutamine in 8% a 4% PF, 0.1 M PB. The cells were then washed with TBS. The cells CO,, humidified, 37°C atmosphere. The medium was changed twice a were then pcrmeabilized with 0.2% TX-100 in TBS for 5 min, followed week. Mature neurons (more than 21 d in culture) were used in all by blocking with 4% normal goat serum (NGS), 0.1% TX-100 in TBS experiments except for the experiments examining the ontogeny and for I hr. This was followed by-incubating the cells with affinity-purified development of NMDA neurotoxicity in relation to NMDA currents anti-NOS antibodies overnight at 4°C (Bredt et al.. 1990. 1991: T. M. and expression of NOS. In the mature cultures the percentage ofneurons Dawson et al., I99 I). The c&s were then rinsed three times in TBS for is approximately 70-90% of the total number of ceils as assessed by IO min each. After rinsing, the cells were incubated with biotin-con- neuron-specific enolase (NSE) and glial fibrillary acidic protein (GFAP) jugated secondary antibody (goat anti-rabbit; Vector Laboratories) for immunocytochemical staining of neurons and astrocytes, respectively 1 hr at room temperature in 1.5% NGS, TBS, 0.1% TX-100. After an (V. L. Dawson, T. M. Dawson, D. A. Bartley, G. R. Uhl, and S. H. additional three washes in TBS, the cells were incubated with an avidin- Snyder, unpublished observations). biotin-peroxidase complex (1:50; Vector Elite, Vector Laboratories) in TBS for 45 min at room temperature. The cells were again rinsed three times for IO min each in TBS and were developed with a substrate Cytotoxicity solution consisting of 0.01% H,O, and 0.5 mg/ml diaminobenzidine in Cells were exposed to test solutions as previously described (V. L. Daw- TBS. Cells were then rinsed in TBS containing 0.02% sodium azide. son et al., 1991). The cells were washed three times with a Tris-buffered All NOS-positive cells in each well were counted utilizing an inverted control salt solution (CSS) containing I20 rnM NaCI, 5.4 mM KCI, 1.8 microscope mM CaCI,, 25 mM Tris-HCI, and 15 mM glucose, pH 7.4 at room lmmunohistochemistry for NSE (Incstar) and GFAP (Incstar) was temperature. Except for exposures to kainate, all other drug solutions performed as described above with substitution of NSE antiserum or were applied to the cells for 5 min and then washed away with CSS GFAP antiserum for anti-NOS antibody. replaced with MEM containing 21 mM glucose and the cells were put NOS immunohistochemistry of rat brain was performed as described back in the incubator. Exposures to kainate were performed in MEM, (Bredt et al., 1990, 1991; T. M. Dawson et al., 1991). Briefly, male 21 mM glucose for 24 hr in the incubator. Twenty to twenty-four hours Sprague-Dawley rats (I 50-250 pm) were perfused with phosphate-buf- after exposure to drug solutions the cells were exposed to 0.4% trypan fered saline (PBS; 50 mM PB, 0.9% NaCI, pH 7.4, 4°C) followed by blue’in CSS to stain the residue of nonviable cells. Two to four pho- perfusion with 4% PF in 0. I M PB, pH 7.4,4”C. The brains were removed toprints at I 0-20x were made of each well. Viable versus nonviable and postfixed for 2 hr in 4% PF in 0.1 M PB, pH 7.4, 4°C. This was cells were counted, with approximately 500-l 500 cells counted per well.