BRIEF COMMUNICATIONS
Relative Sparing of Nitric Oxide Sy nthase-Containing Neurons in the Hippocampal Formation in Alzheimer's Disease Bradley T. Hyman, MD, PhD,* Kristin Marzloff, BA," Julia J. Wenniger, BA," Ted M. Dawson, MD, PhD,tl: David S. Bredt, BA,f§ and Solomon H. Snyder, MDtO"
Nitric oxide (NO) is an endogenous neuromodulator that may mediate neurotoxic effects of glutamate. NOsynthesizing neurons are, however, resistant to NO- and glutamate-induced neurotoxicity. We now show that NO synthase neurons are selectively spared in patients with Alzheimer's disease, even in a severely affected region of the brain such as the hippocampal formation. Hyman BT, Marzloff K, Wenniger JJ, Dawson TM, Bredt DS, Snyder SH. Relative sparing of nitric oxide synthase-containing neurons in the hippocampal formation in Alzheimer's disease. Ann Neurol 1992;32:818-820
Alzheimer's disease (AD) is characterized by neurofibrillary tangles (NFT), senile plaques (SP), and neuronal loss. The consistently severe pathological changes in the hippocampal formation may underlie the profound learning impairment of patients with Alzheimer's disease El-31. The mechanism of this neuronal destruction is unknown, but has been suggested to involve N-methybaspartate (NMDA)-type excitotoxicity [4}.Recent evidence suggests that the novel free radical neuromodulator, nitric oxide (NO) [ S , 61, mediates NMDA receptor-linked excitotoxicity, and that neurons that contain NO synthase (NOS) (identical to "NADPH-diaphorase" activity [7-91) are themselves spared from NMDA and NO toxic effects [lo, 111. Diaphorase neurons preferentially survive in the striatum in patients with Huntington's disease [123, and are relatively spared in experimental models of brain damage due to ischemia [13] or NMDA-mediated neurotoxins [14-161.
From the *Neurology Service, Massachusetts General Hospital and Harvard Medical School, Boston, MA, and Departments of tNeuroscience, $Neurology, $Pharmacology and Molecular Sciences, and "Psychiatry,Johns Hopkins University, Baltimore, MD. Received Apr 10, 1992, and in revised form Jun 5. Accepted for publication Jun 5 , 1992. Address correspondence to Dr Hyman, Neurology Service, Massachusetts General Hospital, 32 Fruit Street, Boston, MA 02114.
If NO toxicity is involved in neuronal damage in patients with AD, and NOS neurons are resistant to NO toxicity, ope would predict relative sparing of NOS-containing neurons in patients with AD. We therefore examined NOS immunocytochemistry in the hippocampal formation and temporal neocortex of 11 patients with dementia and the neuropathological diagnosis of AD (ages, 70-90 yr; mean +. SEM, 80.8 +2.0 yr) and 9 control subjects (ages, 24-79 yr; mean t SEM, 60.4 k 5.9 yr) who did not have AD pathological changes. Neuropathological examination of one AD individual also showed cortical Lewy bodies. One control subject had amyotrophic lateral sclerosis, 1 had the diagnosis of progressive supranuclear palsy, and 3 others had been noted to have infarcts in remote brain areas. The other control subjects had no specific neurological or neuropathological diagnosis.
Methods Tissue blocks were fixed for 24 hours in paraformaldehyde/ lysine/metaperiodate,cryoprotected in 15% glycerol/O. 1 M phosphate-buffered saline (pH 7.4), and cut on a freezing microtome. Fifty-micrometer-thick sections were immunostained for NOS immunoreactivity using an affinity-purified rabbit polyclonal antibody [7, 91 (1:50) and visualized with a peroxidase-linked secondary antibody (JacksonImmunoresearch, West Grove, PA). Sections were counterstained with thionin for Nissl substance or thioflavine S for NIT and SP, and viewed under bright-field and epifluorescence conditions. Video images of immunoreactive neurons were obtained with a Dage-MTI CCD72 video camera and number and area of neurons assessed with Bioquant Microquant (Michigan City, IN) Image Analysis software. The area of each cytoarchitectural field was also assessed, and NOS neuron number is expressed as the number per square centimeter.
Results We examined the following 6 cytoarchitectural fields: dentate gyrus, CA4, CA3, CA1, and subiculum, and inferior temporal gyrus cortex (area 2O), and inferior temporal gyrus white matter. NOS neurons were not present in the dentate gyrus in 18 of the 20 patients and subjects, so that further analysis was not performed in this field. In both patients with AD and control subjects, a variety of morphological types and sizes of neurons were visualized, and a very extensive plexus of NOS-positive neurites was present in the neuropil. Fusiform neurons within the deep white matter were strongly NOS positive. The distribution and morphological types of neurons that were NOS positive were the same in control subjects and patients with AD. There was no difference in total number of NOS neurons in patients with A D compared with control subjects in any of these fields (Fig 1). Ball [3] has quantitated the degree of neuronal loss in the hippocampus of patients with AD, and found an
818 Copyright 0 1992 by the American Neurological Association
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Fig I . The number and area of nitric oxide synthaseimmunoreactive neurons were measured using Bioquant Microp a n t Image Analrfis software. The mean and standard error are displayed. CA = cornu ammonis; A20C = cortex in Brodmann area 20; A20S = subcortical white matter in Brodmann area 20. *p < 0.05, t test.
overall 47% loss combining all the pyramidal subfields of the hippocampal formation. By contrast to this pronounced overall loss of neurons, the A D hippocampus contained 3.2 -+ 0.9, and the control hippocampus 2.9 ? 0.7 NOS neurons/cm2 (mean k SEM). Based on the estimate of neuronal loss by Ball C31, there is significant sparing of NOS neurons in the A D hippocampus ( p < 0.02, one-tailed t test). To confirm this, NOS-stained sections were counterstained with thioflavine S. More than 300 NFT were examined in each patient with AD, and in no instance was an NFT found to colocalize with NOS immunostaining. In the cortex of experimental animals, approximately 2% of neurons are NOS positive C71. Assuming that this is the case in the human hippocampus, the identification of >3,300 NFT without a single NOS neuron is highly statistically significant using the Poisson approximation to the binomial distribution ( p < 0.0001). Thus, either neurons that contain NOS are
not prone to develop NFT, or NOS neurons lose this phenotype if they develop NFT. That the total number of NOS neurons is unchanged suggests that the former explanation is more likely. In addition, 20 to 30 NOS-positive neurons were identified in each patient, and again none were found to colocalize with NFT. Thioflavine S also stains neuropil threads and dystrophic neurites surrounding plaques. NOS staining was found in dystrophic neurites in only rare instances (el in 1,000 SP), although given the density of NOS fibers and of senile plaques, it was not unusual to find the two juxtaposed. Measurement of the size of individual neurons in each field using the Microquant Image Analysis software showed a 35% decrease in average size of NOS neurons in area CA1 in patients with A D ( p < 0.02, t test), but no quantitative differences in other fields (see Fig 1). Nonetheless, a qualitative change in the morphology of NOS neurons was generally present in patients with A D (Fig 2). The neurons' dendritic arborization appeared foreshortened or blunted, especially in regions that had substantial neuropathological change and neuronal loss. The normally fine, evenly beaded appearance of NOS axons became distorted and swollen, without associated thioff avine S amyloid. Many examples of NOS fibers were noted surrounding the soma of neurons in both patients with AD and control subjects. Discussion N O is a potent biological effector molecule in the vasculature, immune system, and central nervous system C17-207. In the brain, a functional role for NO has been suggested in activation of NMDA receptors and in mediating glutamate-associated increases in cyclic GMP { 5 , 6, 171. NO synthesis is also important in the induction of long-term potentiation in the Schaeffer collateral projection in the hippocampus {2 1-24]. Our data show that NOS neurons in the hippocampus and temporal neocortex are relatively spared from neuronal loss, NET, and SP formation in patients with AD. The localization of NOS-diaphorase to selected neuronal populations that resist NMDA neurotoxicity 17, 9, 11, 14-16] as well as ischemic destruction 1131 and loss in neurodegenerative disease { 111 fits with the preservation of NOS neurons in patients with AD. Our results extend a previous study suggesting relative preservation of diaphorase neurons in cortical areas in 2 patients with AD {25]. NOS neurons appear to release NO and destroy other neurons in NMDA toxicity in primary brain cultures [lo, 111 and hippocampal slices 126). It is possible that the relative sparing of NOS neurons in areas that have pronounced neuronal loss in patients with A D such as CA1 and subiculum [1-3] may lead to an imbalance of input to remaining neurons, and contribute to a cascade of events leading
Brief Communication: Hyman et al: NO Synthase Neurons in AD
819
3. B d MJ. Neuronal loss, neurofibrillary tangles and granulova-
Fig 2. Photomicrograph of nitric oxide synthaseimmunoreactive neurons in control subject (A)and patient with Alzheimer’s disease (B) in area 20s. Magnificationbar = 20 Pm.
to further neuronal loss in patients with neurodegenerative disease. Supported by N I H AG08487, the American Federation for Aging Research, and the Brookdale Foundation. T.M.D. is a Pfizer Postdoctoral Fellow and is supported by the American Academy of Neurology and the French Foundation for Alzheimer’s Research. Supported by US Public Health Service Grants DA-00266 and MH-18501, contract DA 271-90-7408, and Research Scientist Award DA-00074 to S.H.S. D.S.B. is supported by training grant GM-07309. We thank the Massachusetts Alzheimer Disease Research Center Brain Bank (Dr E. T. Hedley Whyte, Director, N I H P50 AGO5134) for tissue resources. We thank S. Melanson for assistance with the manuscript and Dr Joseph Locasio for assistance with statistical analysis.
References 1. Hyman BT, Damasio AR, Van Hoesen GW, Barnes CL. Cell specific pathology isolates the hippocampal formation in Alzheimer’s disease. Science 1984;225:1168-1170 2. Hyman BT, Van Hoesen GW, Damasio AR. Memory-related neural systems in Alzheimer’s disease: an anatomic study. Neurology 1990;40: 1721-1 730
cuolar degeneration in the hippocampus with ageing and dementia. A quantitative study. Acta Neuropathol 1977;37:111-118 4. Greenamyre JT, Young AB. Excitatory amino acids and Alzheimer’s disease. Neurobiol Aging 1989;10:593-602 5. Garthwaite J. Glutamate nitric oxide and cell-cell signaling in the nervous system. Trends Neurosci 1991;14:60-67 6. Bredt DS, Snyder SH. Nitric oxide, a novel neuronal messenger. Neuron 1992;8:3-11 7. Dawson TM, Bredt DS, Fotuhi M, et al. Nitric oxide synthase and neuronal NADPH diaphorase are identical in brain and peripheral tissue. Proc Natl Acad Sci USA 1991;88:7797-7801 8. Hope BT, Michael GJ, Knigge KM, Vincent SR. Neuronal NADPH diaphorase is a nitric oxide synthase. Proc Natl Acad Sci USA 1991;88:2811-2814 9. Bredt DS, Glatt CE, Hwang PM, et al: Nitric oxide synthase protein and mRNA are discretely localized in neuronal populations of the mammalian CNS together with NADPH diaphorase. Neuron 1991;7:615-624 10. Dawson VL, Dawson TM, London ED, Bredt DS. Nitric oxide mediates glutamate neurotoxicity in primary cortical cultures. Proc Natl Acad Sci USA 1991;88:6368-6371 11. Dawson TM, Dawson VL, Bredt DS, et al. Nitric oxide synthase/NADPH diaphorase neurons: role in neurotoxicity. Neurosci Abstr 1991;17:784 (Abstract) 12. Ferrante RJ, Kowall NW, Beal MF, et al. Selective sparing o f a class of striatal neurons in Huntington’s disease. Science 1985; 2301561-563 13. Uemura Y, Kowall NW, Beal MF. Selective sparing of NADPH-diaphorase-somatostatin-neuropeptide Y neurons in ischemic gerbil striatum. Ann Neurol 1990;27:620-625 14. Beal MF, Kowall NW, Swartz KJ, et al. Differential sparing of somatostatin-neuropeptide Y and cholinergic neurons following striatal excitotoxin lesions. Synapse 1989;3:38-47 15. Beal MF, Swartz KJ, Hyman BT, et al. Aminooxyacetic acid results in excitotoxin lesions by a novel indirect mechanism. J Neurochem 1991;57:1068-107 3 16. Koh JY, Choi DW. Vulnerability of cultured cortical neurons to damage by excitotoxins: differential susceptibility of neurons containing NADPH-diaphorase. J Neurosci 1988;8:2153-2 163 17. Moncada S, Palmer RMJ, H i a s EA. Nitric oxide, physiology, pathophysiology and pharmacology. Pharmacol Rev 1991;43: 109-142 18. Furchgott RF, Vanhoutte PM. Endothelium-derived relaxing and contracting factors. FASEB J 1990;3:2007-2018 19. Ignarro LJ. Signal transduction mechanisms involving nitric oxide. Biochem Pharmacol 1991;41:485-490 20. Marletta MA. Nitric oxide: biosynthesis and biological significance. Trends Biol Sci 1989;14:488-492 21. Haley JE, Wilcox GL, Chapman PF. The role of nitric oxide in hippocampal long-term potentiation. Neuron 1992;8:2 11-2 I 6 22. ODell TJ, Hawkins RD, Kandel ER, Arancio 0. Tests on the roles of two diffusible substances in LTP: evidence for nitric oxide as a possible early retrograde messenger. Proc Natl Acad Sci USA 1991;88:11285-11289 23. Bohme GA, Bon C, Stutzman JM, et al. Possible involvement of nitric oxide in long-term potentiation. Eur J Pharmacol 1991;199:379-38 1 24. Schuman EM, Madison DV. A requirement for the intercellular messenger nitric oxide in long-term potentiation. Science 1991; 254: 1503-1506 25. Kowall NW, Beal MF. Cortical somatostatin, neuropeptide Y, and NADPH diaphorase neurons: normal anatomy and alterations in Alzheimer’s disease. Ann Neurol 1988;23:105-114 26. Izumi Y, Benz AM, Clifford DB, Zorumski CF. Nitric oxide inhibitors attenuate N-methybaspartate excitotoxicity in rat hippocampal slice. Neurosci Lett 1992;135:227-230
820 Annals of Neurology Vol 32 No 6 December 1992
Relative Sparing of Nitric Oxide Sy nthase-Containing Neurons in the Hippocampal Formation in Alzheimer's Disease Bradley T. Hyman, MD, PhD,* Kristin Marzloff, BA," Julia J. Wenniger, BA," Ted M. Dawson, MD, PhD,tl: David S. Bredt, BA,f§ and Solomon H. Snyder, MDtO"
Nitric oxide (NO) is an endogenous neuromodulator that may mediate neurotoxic effects of glutamate. NOsynthesizing neurons are, however, resistant to NO- and glutamate-induced neurotoxicity. We now show that NO synthase neurons are selectively spared in patients with Alzheimer's disease, even in a severely affected region of the brain such as the hippocampal formation. Hyman BT, Marzloff K, Wenniger JJ, Dawson TM, Bredt DS, Snyder SH. Relative sparing of nitric oxide synthase-containing neurons in the hippocampal formation in Alzheimer's disease. Ann Neurol 1992;32:818-820
Alzheimer's disease (AD) is characterized by neurofibrillary tangles (NFT), senile plaques (SP), and neuronal loss. The consistently severe pathological changes in the hippocampal formation may underlie the profound learning impairment of patients with Alzheimer's disease El-31. The mechanism of this neuronal destruction is unknown, but has been suggested to involve N-methybaspartate (NMDA)-type excitotoxicity [4}.Recent evidence suggests that the novel free radical neuromodulator, nitric oxide (NO) [ S , 61, mediates NMDA receptor-linked excitotoxicity, and that neurons that contain NO synthase (NOS) (identical to "NADPH-diaphorase" activity [7-91) are themselves spared from NMDA and NO toxic effects [lo, 111. Diaphorase neurons preferentially survive in the striatum in patients with Huntington's disease [123, and are relatively spared in experimental models of brain damage due to ischemia [13] or NMDA-mediated neurotoxins [14-161.
From the *Neurology Service, Massachusetts General Hospital and Harvard Medical School, Boston, MA, and Departments of tNeuroscience, $Neurology, $Pharmacology and Molecular Sciences, and "Psychiatry,Johns Hopkins University, Baltimore, MD. Received Apr 10, 1992, and in revised form Jun 5. Accepted for publication Jun 5 , 1992. Address correspondence to Dr Hyman, Neurology Service, Massachusetts General Hospital, 32 Fruit Street, Boston, MA 02114.
If NO toxicity is involved in neuronal damage in patients with AD, and NOS neurons are resistant to NO toxicity, ope would predict relative sparing of NOS-containing neurons in patients with AD. We therefore examined NOS immunocytochemistry in the hippocampal formation and temporal neocortex of 11 patients with dementia and the neuropathological diagnosis of AD (ages, 70-90 yr; mean +. SEM, 80.8 +2.0 yr) and 9 control subjects (ages, 24-79 yr; mean t SEM, 60.4 k 5.9 yr) who did not have AD pathological changes. Neuropathological examination of one AD individual also showed cortical Lewy bodies. One control subject had amyotrophic lateral sclerosis, 1 had the diagnosis of progressive supranuclear palsy, and 3 others had been noted to have infarcts in remote brain areas. The other control subjects had no specific neurological or neuropathological diagnosis.
Methods Tissue blocks were fixed for 24 hours in paraformaldehyde/ lysine/metaperiodate,cryoprotected in 15% glycerol/O. 1 M phosphate-buffered saline (pH 7.4), and cut on a freezing microtome. Fifty-micrometer-thick sections were immunostained for NOS immunoreactivity using an affinity-purified rabbit polyclonal antibody [7, 91 (1:50) and visualized with a peroxidase-linked secondary antibody (JacksonImmunoresearch, West Grove, PA). Sections were counterstained with thionin for Nissl substance or thioflavine S for NIT and SP, and viewed under bright-field and epifluorescence conditions. Video images of immunoreactive neurons were obtained with a Dage-MTI CCD72 video camera and number and area of neurons assessed with Bioquant Microquant (Michigan City, IN) Image Analysis software. The area of each cytoarchitectural field was also assessed, and NOS neuron number is expressed as the number per square centimeter.
Results We examined the following 6 cytoarchitectural fields: dentate gyrus, CA4, CA3, CA1, and subiculum, and inferior temporal gyrus cortex (area 2O), and inferior temporal gyrus white matter. NOS neurons were not present in the dentate gyrus in 18 of the 20 patients and subjects, so that further analysis was not performed in this field. In both patients with AD and control subjects, a variety of morphological types and sizes of neurons were visualized, and a very extensive plexus of NOS-positive neurites was present in the neuropil. Fusiform neurons within the deep white matter were strongly NOS positive. The distribution and morphological types of neurons that were NOS positive were the same in control subjects and patients with AD. There was no difference in total number of NOS neurons in patients with A D compared with control subjects in any of these fields (Fig 1). Ball [3] has quantitated the degree of neuronal loss in the hippocampus of patients with AD, and found an
818 Copyright 0 1992 by the American Neurological Association
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Fig I . The number and area of nitric oxide synthaseimmunoreactive neurons were measured using Bioquant Microp a n t Image Analrfis software. The mean and standard error are displayed. CA = cornu ammonis; A20C = cortex in Brodmann area 20; A20S = subcortical white matter in Brodmann area 20. *p < 0.05, t test.
overall 47% loss combining all the pyramidal subfields of the hippocampal formation. By contrast to this pronounced overall loss of neurons, the A D hippocampus contained 3.2 -+ 0.9, and the control hippocampus 2.9 ? 0.7 NOS neurons/cm2 (mean k SEM). Based on the estimate of neuronal loss by Ball C31, there is significant sparing of NOS neurons in the A D hippocampus ( p < 0.02, one-tailed t test). To confirm this, NOS-stained sections were counterstained with thioflavine S. More than 300 NFT were examined in each patient with AD, and in no instance was an NFT found to colocalize with NOS immunostaining. In the cortex of experimental animals, approximately 2% of neurons are NOS positive C71. Assuming that this is the case in the human hippocampus, the identification of >3,300 NFT without a single NOS neuron is highly statistically significant using the Poisson approximation to the binomial distribution ( p < 0.0001). Thus, either neurons that contain NOS are
not prone to develop NFT, or NOS neurons lose this phenotype if they develop NFT. That the total number of NOS neurons is unchanged suggests that the former explanation is more likely. In addition, 20 to 30 NOS-positive neurons were identified in each patient, and again none were found to colocalize with NFT. Thioflavine S also stains neuropil threads and dystrophic neurites surrounding plaques. NOS staining was found in dystrophic neurites in only rare instances (el in 1,000 SP), although given the density of NOS fibers and of senile plaques, it was not unusual to find the two juxtaposed. Measurement of the size of individual neurons in each field using the Microquant Image Analysis software showed a 35% decrease in average size of NOS neurons in area CA1 in patients with A D ( p < 0.02, t test), but no quantitative differences in other fields (see Fig 1). Nonetheless, a qualitative change in the morphology of NOS neurons was generally present in patients with A D (Fig 2). The neurons' dendritic arborization appeared foreshortened or blunted, especially in regions that had substantial neuropathological change and neuronal loss. The normally fine, evenly beaded appearance of NOS axons became distorted and swollen, without associated thioff avine S amyloid. Many examples of NOS fibers were noted surrounding the soma of neurons in both patients with AD and control subjects. Discussion N O is a potent biological effector molecule in the vasculature, immune system, and central nervous system C17-207. In the brain, a functional role for NO has been suggested in activation of NMDA receptors and in mediating glutamate-associated increases in cyclic GMP { 5 , 6, 171. NO synthesis is also important in the induction of long-term potentiation in the Schaeffer collateral projection in the hippocampus {2 1-24]. Our data show that NOS neurons in the hippocampus and temporal neocortex are relatively spared from neuronal loss, NET, and SP formation in patients with AD. The localization of NOS-diaphorase to selected neuronal populations that resist NMDA neurotoxicity 17, 9, 11, 14-16] as well as ischemic destruction 1131 and loss in neurodegenerative disease { 111 fits with the preservation of NOS neurons in patients with AD. Our results extend a previous study suggesting relative preservation of diaphorase neurons in cortical areas in 2 patients with AD {25]. NOS neurons appear to release NO and destroy other neurons in NMDA toxicity in primary brain cultures [lo, 111 and hippocampal slices 126). It is possible that the relative sparing of NOS neurons in areas that have pronounced neuronal loss in patients with A D such as CA1 and subiculum [1-3] may lead to an imbalance of input to remaining neurons, and contribute to a cascade of events leading
Brief Communication: Hyman et al: NO Synthase Neurons in AD
819
3. B d MJ. Neuronal loss, neurofibrillary tangles and granulova-
Fig 2. Photomicrograph of nitric oxide synthaseimmunoreactive neurons in control subject (A)and patient with Alzheimer’s disease (B) in area 20s. Magnificationbar = 20 Pm.
to further neuronal loss in patients with neurodegenerative disease. Supported by N I H AG08487, the American Federation for Aging Research, and the Brookdale Foundation. T.M.D. is a Pfizer Postdoctoral Fellow and is supported by the American Academy of Neurology and the French Foundation for Alzheimer’s Research. Supported by US Public Health Service Grants DA-00266 and MH-18501, contract DA 271-90-7408, and Research Scientist Award DA-00074 to S.H.S. D.S.B. is supported by training grant GM-07309. We thank the Massachusetts Alzheimer Disease Research Center Brain Bank (Dr E. T. Hedley Whyte, Director, N I H P50 AGO5134) for tissue resources. We thank S. Melanson for assistance with the manuscript and Dr Joseph Locasio for assistance with statistical analysis.
References 1. Hyman BT, Damasio AR, Van Hoesen GW, Barnes CL. Cell specific pathology isolates the hippocampal formation in Alzheimer’s disease. Science 1984;225:1168-1170 2. Hyman BT, Van Hoesen GW, Damasio AR. Memory-related neural systems in Alzheimer’s disease: an anatomic study. Neurology 1990;40: 1721-1 730
cuolar degeneration in the hippocampus with ageing and dementia. A quantitative study. Acta Neuropathol 1977;37:111-118 4. Greenamyre JT, Young AB. Excitatory amino acids and Alzheimer’s disease. Neurobiol Aging 1989;10:593-602 5. Garthwaite J. Glutamate nitric oxide and cell-cell signaling in the nervous system. Trends Neurosci 1991;14:60-67 6. Bredt DS, Snyder SH. Nitric oxide, a novel neuronal messenger. Neuron 1992;8:3-11 7. Dawson TM, Bredt DS, Fotuhi M, et al. Nitric oxide synthase and neuronal NADPH diaphorase are identical in brain and peripheral tissue. Proc Natl Acad Sci USA 1991;88:7797-7801 8. Hope BT, Michael GJ, Knigge KM, Vincent SR. Neuronal NADPH diaphorase is a nitric oxide synthase. Proc Natl Acad Sci USA 1991;88:2811-2814 9. Bredt DS, Glatt CE, Hwang PM, et al: Nitric oxide synthase protein and mRNA are discretely localized in neuronal populations of the mammalian CNS together with NADPH diaphorase. Neuron 1991;7:615-624 10. Dawson VL, Dawson TM, London ED, Bredt DS. Nitric oxide mediates glutamate neurotoxicity in primary cortical cultures. Proc Natl Acad Sci USA 1991;88:6368-6371 11. Dawson TM, Dawson VL, Bredt DS, et al. Nitric oxide synthase/NADPH diaphorase neurons: role in neurotoxicity. Neurosci Abstr 1991;17:784 (Abstract) 12. Ferrante RJ, Kowall NW, Beal MF, et al. Selective sparing o f a class of striatal neurons in Huntington’s disease. Science 1985; 2301561-563 13. Uemura Y, Kowall NW, Beal MF. Selective sparing of NADPH-diaphorase-somatostatin-neuropeptide Y neurons in ischemic gerbil striatum. Ann Neurol 1990;27:620-625 14. Beal MF, Kowall NW, Swartz KJ, et al. Differential sparing of somatostatin-neuropeptide Y and cholinergic neurons following striatal excitotoxin lesions. Synapse 1989;3:38-47 15. Beal MF, Swartz KJ, Hyman BT, et al. Aminooxyacetic acid results in excitotoxin lesions by a novel indirect mechanism. J Neurochem 1991;57:1068-107 3 16. Koh JY, Choi DW. Vulnerability of cultured cortical neurons to damage by excitotoxins: differential susceptibility of neurons containing NADPH-diaphorase. J Neurosci 1988;8:2153-2 163 17. Moncada S, Palmer RMJ, H i a s EA. Nitric oxide, physiology, pathophysiology and pharmacology. Pharmacol Rev 1991;43: 109-142 18. Furchgott RF, Vanhoutte PM. Endothelium-derived relaxing and contracting factors. FASEB J 1990;3:2007-2018 19. Ignarro LJ. Signal transduction mechanisms involving nitric oxide. Biochem Pharmacol 1991;41:485-490 20. Marletta MA. Nitric oxide: biosynthesis and biological significance. Trends Biol Sci 1989;14:488-492 21. Haley JE, Wilcox GL, Chapman PF. The role of nitric oxide in hippocampal long-term potentiation. Neuron 1992;8:2 11-2 I 6 22. ODell TJ, Hawkins RD, Kandel ER, Arancio 0. Tests on the roles of two diffusible substances in LTP: evidence for nitric oxide as a possible early retrograde messenger. Proc Natl Acad Sci USA 1991;88:11285-11289 23. Bohme GA, Bon C, Stutzman JM, et al. Possible involvement of nitric oxide in long-term potentiation. Eur J Pharmacol 1991;199:379-38 1 24. Schuman EM, Madison DV. A requirement for the intercellular messenger nitric oxide in long-term potentiation. Science 1991; 254: 1503-1506 25. Kowall NW, Beal MF. Cortical somatostatin, neuropeptide Y, and NADPH diaphorase neurons: normal anatomy and alterations in Alzheimer’s disease. Ann Neurol 1988;23:105-114 26. Izumi Y, Benz AM, Clifford DB, Zorumski CF. Nitric oxide inhibitors attenuate N-methybaspartate excitotoxicity in rat hippocampal slice. Neurosci Lett 1992;135:227-230
820 Annals of Neurology Vol 32 No 6 December 1992
