Abstract
In order to establish an in vitro model of Huntington's disease, we prepared slice cultures of striatal tissue from newborn rats. The striatal cultures were grown for 12–39 days in the absence of any other brain tissue. The presence of specific cell markers was shown by immunocytochemistry, histochemistry and in situ hybridization with alkaline-phosphatase-labeled oligonucleotide probes. We focused on (1) the medium-sized, aspiny interneurons, which in vivo express the neuropeptides somatostatin and neuropeptide Y and the nitric oxide synthesizing enzyme nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase, and which are spared in Huntington's disease and (2) the enkephalinergic, medium-sized projection neurons, which are particularly vulnerable in Huntington's disease. Similar basic morphologies of the presumed interneurons and double staining of NADPH-diaphorase positive and somatostatin immunoreactive neurons suggest that the two neuropeptides and NADPH-diaphorase are extensively colocalized in the cultures, as in vivo. In the newborn rats, included as controls, a patch-matrix distribution of the NADPH-diaphorase staining is described for the first time. In the striatal slices the distribution of the NADPH-diaphorase staining stayed uneven after 3–5 weeks in culture, with areas almost devoid of staining alternating with more heavily stained areas. This pattern may represent an intermediate stage between the patch-matrix distribution in the newborn and the homogeneous staining in the adult rat striatum. From quantitative estimates we found the same mutual rank order of the numbers of neuropeptide Y- and somatostatin-immunoreactive neurons and NADPH-diaphorase positive neurons in vivo and in vitro. Both in the slice cultures and in the brain, the number of enkephalin mRNA-containing neurons significantly exceeded that of neuropeptide Y- and somatostatin mRNA-containing neurons. This implies that the mutual distribution of presumed interneurons and projection neurons was preserved in the slice cultures. Comparison of cell numbers per unit volume showed that, in the cultures, the number of presumed interneurons, with the exception of NPY mRNA-containing neurons, significantly exceeded that in vivo. In contrast, the enkephalin mRNA-containing neurons, which in vivo are projection neurons, were significantly fewer in the cultures. The relative loss of projection neurons and preservation of interneurons in single slice cultures of striatal tissue apparently mimick some of the neurodegenerative changes of Huntington's disease. From the finding that the number of neuropeptide Y mRNA-containing neurons both in vitro and in vivo was significantly higher than the number of neuropeptide Y-immunoreactive neurons — unlike the number of somatostatin mRNA containing neurons — it is suggested that somatostatin and neuropeptide Y are differentially regulated at the level of translation. The peptides are also suggested to be differentially regulated at the level of transcription because the number of somatostatin mRNA-containing neurons increased significantly in the slice cultures compared with in vivo, while the number of neuropetide Y mRNA-containing neurons was unchanged.
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References
Abercrombie M (1946) Estimation of the nuclear population from microtome sections. Anat Rec 94:239–247
Albin RL, Young AB, Penney JB (1989) The functional anatomy of basal ganglia disorders. Trends Neurosci 12:366–375
Aoki C, Pickel VM (1988) Neuropeptide Y-containing neurons in the rat striatum: ultrastructure and cellular relations with tyrosine hydroxylase-containing terminals and with astrocytes. Brain Res 459:205–225
Arentsen R, Baldini F Jr, Davis LG, Higgins GA, Lin Y, Manning RW, Wolfson B (1985) In situ hybridization of putative somatostatin mRNA within hypothalamus of the cat using synthetic oligonucleotide probes. J Cell Biochem 27:415–422
Aronin N, DiFiglia M, Graveland GA, Schwartz WJ, Wu JY (1984) Localization of immunoreactive enkephalins in GABA synthesizing neurons of the rat neostriatum. Brain Res 300:376–380
Beal MF, Kowall NW, Swartz KJ, Ferrante RJ, Martin JB (1989) Differential sparing of somatostatin-neuropeptide Y and cholinergic neurons following striatal excitotoxin lesions. Synapse 3:38–47
Bredt DS, Hwang PM, Snyder SH (1990) Localization of nitric oxide synthase indicating a neural role for nitric oxide. Nature 347:768–770
Butcher LL, Hodge GK (1976) Postnatal development of acetylcholinesterase in the caudate-putamen nucleus and substantia nigra of rats. Brain Res 106:223–240
Chang HT, Wilson CJ, Kitai ST (1982) A Golgi study of rat neostriatal neurons: light microscopic analysis. J Comp Neurol 208:107–126
Chesselet MF, Graybiel AM (1986) Striatal neurons expressing somatostatin-like immunoreactivity: evidence for a peptidergic interneuronal system in the cat. Neuroscience 3:547–571
Dawson TM, Bredt DS, Fotuhi M, Hwang PM, Snyder SH (1991) Nitric oxide synthase and neuronal NADPH diaphorase are identical in brain and peripheral tissues. Proc Natl Acad Sci USA 88:7797–7801
De Quidt ME, Emson PC (1986) Distribution of neuropeptide Y-like immunoreactivity in the rat central nervous system. II. Immunohistochemical analysis. Neuroscience 18:545–618
DiFiglia M (1990) Excitotoxic injury of the neostriatum: a model for Huntington's disease. Trends Neurosci 13:286–289
DiFiglia M, Aronin N (1982) Ultrastructural features of immunoreactive somatostatin neurons in the rat caudate nucleus. J Neurosci 2:1267–1274
Dimova R, Vuillet J, Seite R (1980) Study of the rat neostriatum using a combined Golgi-electron microscope technique and serial sections. Neuroscience 5:1581–1596
Ferrante RJ, Kowall NW, Beal MF, Richardson EP Jr, Bird ED, Martin JB (1985) Selective sparing of a class of striatal neurons in Huntington's disease. Science 230:561–563
Ferriero DM, Arcavi LJ, Sagar SM, IcIntosh TK, Simon RP (1988) Selective sparing of NADPH-diaphorase neurons in neonatal hypoxia-ischemia. Ann Neurol 24:670–676
Finsen BR, Sørensen T, Castellano B, Pedersen EB, Zimmer J (1991) Leucocyte infiltration and glial reaction in zenografts of mouse brain tissue undergoing rejection in the adult rat brain. A light and electronmicroscopical immunocytochemical study. J Neuroimmunol 32:159–183
Finsen BR, Tønder N, Augood S, Zimmer J (1992) Somatostatin and neuropeptide Y in organotypic slice cultures of the rat hippocampus: an immunocytochemical and in situ hybridization study. Neuroscience 47:105–113
Gerfen CR (1984) The neostriatal mosaic: compartmentalization of corticostriatal input and striatonigral output systems. Nature 311:461–464
Gerfen CR (1985) The neostriatal mosaic. I. Compartmental organization of projections from the striatum to the substantia nigra in the rat. J Comp Neurol 236:454–476
Gerfen CR, McGinty JF, Young WS III (1991) Dopamine differentially regulates dynorphin, substance P, and enkephalin expression in striatal neurons: in situ hybridization histochemical analysis. J Neurosci 11:1016–1031
Goodman RH, Aron DC, Roos BA (1983) Rat pre-prosomatostatin. J Biol Chem 258:5570–5574
Graybiel AM (1984) Correspondence between the developing islands and striosomes of the mammalian striatum. Neuroscience 13:1157–1187
Higuchi H, Yang H-YT, Sabol SL (1988) Rat neuropeptide Y precursor gene expression. J Biol Chem 263:6288–6295
Howells RD, Kilpatrick DL, Bhatt R, Monahan JJ, Poonian M, Udenfriend S (1984) Molecular cloning and sequence determination of rat preproenkephalin cDNA: sensitive probe for studying transcriptional changes in rat tissues. Proc Natl Acad Sci USA 81:7651–7655
Jablonski E, Moomaw EW, Tullis RH, Ruth JL (1986) Preparation of oligodeoxynucleotide-alkaline phosphatase conjugates and their use as hybridization probes. Nucleic Acids Res 14:6115–6128
Kerkerian L, Bosler O, Pelletier G, Nieoullon A (1986) Striatal neuropeptide Y neurones are under the influence of the nigrostriatal dopaminergic pathway: immunohistochemical evidence. Neurosci Lett 66:106–112
Kiyama H, Emson PC (1990) Distribution of somatostatin mRNA in the rat nervous system as visualized by a novel non-radioactive in situ hybridization histochemistry procedure. Neuroscience 38:223–244
Kowall NW, Beal MF, Ferrante RJ, Martin J B (1985) Topography of nicotinamide adenine dinucleotide phosphate-diaphorase staining neurons in rat striatum. Neurosci Lett 59:61–66
Kubota Y, Inagaki S, Kito S, Shimada S, Okayama T, Hatanaka H, Tohyama M (1988) Neuropeptide Y-immunoreactive neurons receive synaptic inputs from dopaminergic axon terminals in the rat neostriatum. Brain Res 458:389–393
Larhammar D, Ericsson A, Persson H (1987) Structure and expression of the rat neuropeptide Y gene. Proc Natl Acad Sci USA 84:2068–2072
Leigh PN, Connick JH, Stone TW (1990) Distribution of NADPH-diaphorase positive cells in the rat brain. Comp Biochem Physiol 97:259–264
Lindefors N, Brené S, Herrera-Marschitz M, Persson H (1990) Neuropeptide gene expression in brain is differentially regulated by midbrain dopamine neurons. Exp Brain Res 80:489–500
Minth CD, Bloom SR, Polak JR, Dixon JE (1984) Cloning, characterization and DNA sequence of a human cDNA encoding neuropeptide tyrosine. Proc Natl Acad Sci USA 51:4577–4581
Olson L, Seiger Å, Fuxe K (1972) Heterogeneity of striatal and limbic innervation: highly fluorescent islands in developing and adult rats. Brain Res 44:283–288
Osterberg KA, Wattenberg LW (1962) Oxidative histochemistry of reactive astrocytes. Arch Neurol 7:211–218
Østergaard K (1993) Organotypic slice cultures of the rat striatum. I. A histochemical and immunocytochemical study of acetylcholinesterase (AChE), choline acetyltransferase (ChAT), glutamic acid decarboxylase (GAD) and gamma-aminobutyric acid (GABA). Neuroscience 53:679–693
Østergaard K, Schou JP, Zimmer J (1990) Rat ventral mesencephalon grown as organotypic slice cultures and co-cultured with striatum, hippocampus, and cerebellum. Exp Brain Res 82:547–565
Østergaard K, Schou JP, Gähwiler BH, Zimmer J (1991) Tyrosine hydroxylase immunoreactive neurons in organotypic slice cultures of the rat striatum and neocortex. Exp Brain Res 83:357–365
Pickel VM, Sumal KK, Beckley SC, Miller RJ, Reis DJ (1980) Immunocytochemical localization of enkephalin in the neostriatum of rat brain: a light and electron microscopic study. J Comp Neurol 189:721–740
Rubinstein LJ, Klatzo I, Miquel J (1962) Histochemical observations on oxidative enzyme activity of glial cells in a local brain injury. J Neuropath Exp Neurol 21:116–137
Salin P, Kerkerian-LeGoff L, Heidet V, Epelbaum J, Nieoullon A (1990) Somatostatin-immunoreactive neurons in the rat striatum: effects of corticostriatal and nigrostriatal dopaminergic lesions. Brain Res 521:23–32
Sandell JH, Graybiel AM, Chesselet M-F (1986) A new enzyme marker for striatal compartmentalization: NADPH diaphorase activity in the caudate nucleus and putamen of the cat. J Comp Neurol 243:326–334
Scherer-Singler U, Vincent SR, Kimura H, McGeer EG (1983) Demonstration of a unique population of neurons with NADPH-diaphorase histochemistry. J Neurosci Methods 9:229–234
Smith Y, Parent A (1986) Neuropeptide Y-immunoreactive neurons in the striatum of cat and monkey: morphological characteristics, intrinsic organization and co-localization with somatostatin. Brain Res 372:241–252
Soghomonian J-J, Chesselet M-F (1991) Lesions of the dopaminergic nigrostriatal pathway alter preprosomatostatin messenger RNA levels in the striatum, the entopeduncular nucleus and the lateral hypothalamus of the rat. Neuroscience 42:49–59
Somers DL, Beckstead RM (1990) Striatal preprotachykinin and preproenkephalin mRNA levels and the levels of nigral substance P and pallidal Met5-enkephalin depend on corticostriatal axons that use the excitatory amino acid neurotransmitters aspartate and glutamate quantitative radioimmunocytochemical and in situ hybridization evidence. Mol Brain Res 8:143–158
Takagi H, Somogyi P, Somogyi J, Smith AD (1983) Fine structural studies on a type of somatostatin-immunoreactive neuron and its synaptic connections in the rat neostriatum: a correlated light and electron microscopic study. J Comp Neurol 214:1–16
Uehara-Kunugi Y, Terai K, Taniguchi T, Tooyama I, Kimura H (1991) Time course of in vitro expression of NADPH-diaphorase in cultured rat brain neurons: comparison with in vivo expression. Dev Brain Res 59:157–162
Uhl GR, Navia B, Douglas J (1988) Differential expression of preproenkephalin and preprodynorphin mRNAs in striatal neurons: high levels of preproenkephalin expression depend on cerebral cortical afferents. J Neurosci 8:4755–4764
Vincent SR, Johansson O (1983) Striatal neurons containing both somatostatin- and avian pancreatic polypeptide (APP)-like immunoreactivities and NADPH-diaphorase activity: a light and electron microscopic study. J Comp Neurol 217:264–270
Vincent SR, Skirboll L, Hökfelt T, Johansson O, Lundberg JM, Elde RP, Terenius L, Kimmel J (1982) Coexistence of somatostatin- and avian pancreatic polypeptide (APP)-like immunoreactivity in some forebrain neurons. Neuroscience 7:439–446
Vincent SR, Johansson O, Hökfelt T, Skirboll L, Elde RP, Terenius L, Kimmel J, Goldstein M (1983) NADPH-diaphorase: a selective histochemical marker for striatal neurons containing both somatostatin- and avian pancreatic polypeptide (APP)-like immunoreactivities. J Comp Neurol 217:252–263
Vuillet J, Kerkerian L, Kachidian P, Bosler O, Nieoullon A (1989a) Ultrastructural correlates of functional relationships between nigral dopaminergic or cortical afferent fibers and neuropeptide Y-containing neurons in the rat striatum. Neurosci Lett 100:99–104
Vuillet J, Kerkerian L, Salin P, Nieoullon A (1989b) Ultrastructural features of NPY-containing neurons in the rat striatum. Brain Res 477:241–251
Wallace MN, Fredens K (1992) Activated astrocytes of the mouse hippocampus contain high levels of NADPH-diaphorase. NeuroReport 3:953–956
Young WS III, Bonner TI, Brann MR (1986) Mesencephalic dopamine neurons regulate the expression of neuropeptide mRNAs in the rat forebrain. Proc Natl Acad Sci USA 83:9827–9831
Zielasek J, Tausch M, Toyka KV, Hartung H-P (1992) Production of nitrite by neonatal rat microglial cells/brain macrophages. Cell Immunol 141:111–120
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Østergaard, K., Finsen, B. & Zimmer, J. Organotypic slice cultures of the rat striatum: an immunocytochemical, histochemical and in situ hybridization study of somatostatin, neuropeptide Y, nicotinamide adenine dinucleotide phosphate-diaphorase, and enkephalin. Exp Brain Res 103, 70–84 (1995). https://doi.org/10.1007/BF00241966
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DOI: https://doi.org/10.1007/BF00241966