ARTICLE |
Correspondence to: Nathalie Janel, EA3508 Université Paris 7, Denis Diderot, Case 7104, 2 place Jussieu, 75251 Paris Cedex 05, France. E-mail: janel@paris7.jussieu.fr
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Hyperhomocysteinemia, caused by a lack of cystathionine ß synthase (CBS), leads to elevated plasma concentrations of homocysteine. This is a common risk factor for atherosclerosis, stroke, and possibly neurodegenerative diseases. However, the mechanisms that link hyperhomocysteinemia due to CBS deficiency to these diseases are still unknown. Early biochemical studies describe developmental and adult patterns of transsulfuration and CBS expression in a variety of species. However, there is incomplete knowledge about the regional and cellular expression pattern of CBS, notably in the brain. To complete the previous data, we used in situ hybridization and Northern blotting to characterize the spatial and temporal patterns of Cbs gene expression during mouse development. In the early stages of development, the Cbs gene was expressed only in the liver and in the skeletal, cardiac, and nervous systems. The expression declined in the nervous system in the late embryonic stages, whereas it increased in the brain after birth, peaking during cerebellar development. In the adult brain, expression was strongest in the Purkinje cell layer and in the hippocampus. Immunohistochemical analyses showed that the CBS protein was localized in most areas of the brain but predominantly in the cell bodies and neuronal processes of Purkinje cells and Ammon's horn neurons. (J Histochem Cytochem 51:363371, 2003)
Key Words: CBS, homocysteine, development, Purkinje cells, hippocampus, skeletal system
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
HOMOCYSTEINE, a sulfur-containing amino acid intermediate, occupies a major regulatory branch point of the methionine metabolism. It is either remethylated to methionine or catabolized to form cysteine through the transsulfuration pathway. In the transsulfuration pathway, homocysteine first condenses with serine to form cystathionine in a rate-limiting reaction catalyzed by the B6-dependent enzyme cystathionine ß synthase (CBS). In the methylation cycle, precursors to homocysteine, such as S-adenosyl methionine, S-adenosyl homocysteine, or 5'-methyltetrahydrofolate, regulate the relative activity of the methylation and transsulfuration pathways in a complex interrelationship. A lack of CBS activity causes homocysteine accumulation as well as export of homocysteine from the cell, leading to hyperhomocysteinemia, which may be toxic to cells. Moreover, it perturbs the methylation cycle, such as intracellular accumulation of S-adenosyl homocysteine, which has consequences for cell metabolism.
In humans, pathological concentrations of homocysteine are associated with hyperhomocysteinemia, an autosomal recessive disease. In hyperhomocysteinemic patients, several factors lead to pathological concentrations of homocysteine: mutations in the CBS gene, mutations in the methylenetetrahydrofolate reductase (MTHFR) gene that encodes an enzyme of the remethylation pathway, and deficiencies in vitamins B6, B12, and folate. The major clinical features of hyperhomocysteinemic patients are premature atherosclerosis and thromboembolism, lens dislocation, osteoporosis, major skeletal development abnormalities, and mental retardation with brain damage and a predisposition to schizophrenia and epilepsy (
A murine model of hyperhomocysteinemia has been generated by Cbs gene targeting (
Although central nervous system CBS expression is well documented, the available data are largely based on early biochemical studies by investigators who used enzyme and cell fractionation methods to describe developmental and adult patterns of transsulfuration and CBS expression in liver and brain in a variety of species (
To complete the previous data, we used in situ hybridization (ISH) and Northern blotting to characterize the spatial and temporal patterns of Cbs gene expression during mouse development. To provide additional information on the expression of Cbs during the late developmental stages until adulthood, we characterized the expression pattern in the nervous system. We also characterized the expression pattern in the skeletal system, which has been shown to be affected in a murine model of hyperhomocysteinemia (
In the early stages of development, Cbs is ubiquitously expressed. At later stages, expression becomes more specific in the tissues that are mainly affected in hyperhomocysteinemia patients [liver, skeletal system, cardiac and nervous systems (
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Animals
All procedures were carried out in accordance with internal guidelines for animal handling. Mice were housed in a controlled environment with access to unlimited food and water. We made every effort possible to minimize suffering and the number of mice used. Heterozygous CBS(+/-) mutant mice were generously donated by Maeda Nobuyo (Department of Pathology, University of North Carolina, Chapel Hill, NC). The day of conception was designated as embryonic day (E) and the day of birth was designated as postnatal day (P). Animals were considered adult when they were older than 3 months. The presence of a vaginal plug on the next morning was defined as 0.5 day postconception. Pregnant mice were killed on days 9.5, 10.5, 12.5, 13.5, or 15.5 postconception. The legends are in accordance with the Atlas of Mouse Development (
In Situ Hybridization
ISH of Cbs mRNA in Mouse Sections.
[35S]-UTP-labeled single-stranded RNA was synthesized from the NotI fragment of pBluescript, which contains the rat Cbs cDNA fragment (nucleotides 652945), using T3 RNA polymerase as an antisense riboprobe, and from the EcoRI fragment of pBluescript using T7 RNA polymerase as a sense riboprobe. A conventional protocol was used for ISH (
ISH of Cbs mRNA in Whole-mount Embryos.
Briefly, single-stranded RNA probes containing digoxigenin were synthesized from previously described linearized template exactly as directed by the manufacturer (Roche Diagnostics; Maylan, France). E9.5, 10.5, 12.5, and 13.5 FVB/N embryos were fixed for 3 hr in 4% paraformaldehyde. The 12.5 and 13.5 FVB/N embryos were then cut in half mid-sagittally using a mouse brain matrix (Fracalosso, France) and stored immediately at -20C in 100% methanol (
Northern Blotting Analyses
Liver and brain tissues were obtained from 3-month-old homozygous (-/-) and heterozygous (+/-) CBS-deficient mice and from wild-type (+/+) control mice from the same litter and were stored at -80C. Total RNA was prepared from two mixtures of three different tissues by the guanidinium thiocyanate procedure (-32P]-dCTP (NEN Life Science Products; Boston, MA). We ensured that loading was equal and that the RNA from the liver samples had not been degraded by hybridization with a probe specific for mouse Cu/Zn superoxide dismutase 1 (SOD1). This gene was expressed at equal levels in the three groups of mice. We ensured that the RNA from the brain samples had not been degraded by hybridization with a probe specific for mouse cytochrome C oxidase subunit I (Cyt C Ox). Hybridization was performed at 68C overnight. The blot was then washed and exposed to X-ray film at -80C.
Western Blotting Analyses
The brains from 3-month-old homozygous (-/-) and heterozygous (+/-) CBS-deficient mice and from wild-type (+/+) control mice were quickly removed and dropped in Laemmli solubilizing buffer (50 mM Tris-HCl, pH 6.8, 100 mM DTT, 2% SDS, 10% glycerol) and boiled for 10 min. Then 60 µg of each protein sample was subjected to SDS electrophoresis on 512.5% gels and transfered to nitrocellulose membranes (Immuno-blot PVDF Membrane; Bio-Rad Laboratories, Hercules, CA). The membrane was incubated in a blocking buffer consisting of 5% (w/v) nonfat dried milk in Tris-buffered saline (1.5 mM Tris base, pH 8, 5 mM NaCl, 0.1% Tween-20) for 1 hr at room temperature (RT). The membranes were then incubated in the presence of the anti-CBS antibody (
Immunohistochemistry
Brains were removed from the mice and sagittal cryosections (8 µm) were fixed in 4% paraformaldehyde for 10 min. Brain cryosections from both homozygous (-/-) CBS-deficient mice and wild-type (+/+) mice from the same litter were loaded on each slide. Because a second unspecific band was present on the Western blot, the anti-CBS antibody (
After several washes in TBS containing 0.1% Triton X-100, the sections were treated with freshly made 1% H2O2 for 30 min. The nonspecific binding sites were blocked with 3% BSA in TBS/0.1% Triton X-100 for 1 hr. The sections were then incubated with the anti-CBS antibody (1:1000 dilution) at 4C overnight. Biotinylated goat anti-mouse IgG (Amersham; 1:400 dilution) used as secondary antibody was incubated for 1 hr at RT and detected with streptavidinperoxidase complex. The immunoreaction was visualized by treating the sections with DAB+ substrate chromatogen reagent (DAKO; Glostrup, Denmark) for 2 min at RT. Tissues were then counterstained with hematoxylin, dehydrated through graded concentrations of alcohol, cleared in xylene, and coverslipped.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
ISH Analyses
A 350-bp cDNA fragment containing exons 58 of the Cbs gene was isolated from the rat Cbs orthologue (
|
Spatial Distribution of the Cbs mRNA During Development
Whole-mount hybridization revealed that Cbs was ubiquitously expressed between E9.5 and E10.5. However, the strongest expression was observed in limb buds, tail, and brain vesicles (Fig 2A and Fig 2B). In Fig 2A the purple spot at the base of the neck was an artifact. Between E12.5 and E13.5, Cbs was expressed in developing organs, including the liver, cartilage primordium, cardiac system, and nervous system (Fig 2C and Fig 2D). At these stages, Cbs transcripts were still detected in embryonic limb buds and in the tail. To determine the cellular expression pattern, we performed ISH analyses on embryo sections (Fig 2E). In the digestive system at E12.5, high levels of Cbs mRNA were first detected in certain developing mesoderm regions, including the liver (Fig 2E). Closer analysis showed that primitive hepatocytes were labeled at this stage. In later embryonic stages and in the adult mouse, strong expression was observed in hepatocytes (data not shown). In the cardiovascular system, expression was observed from E12.5 (Fig 2C). At this stage, Cbs transcripts were detected in endocardial cells (data not shown). Cbs mRNA was also detected in the aorta at later stages of development (data not shown).
|
In the skeletal system, Cbs mRNA was also detected in somitic derivatives and in tissues originating from the mesoblast at E12.5. The developing vertebral bodies and arches were labeled, whereas the developing intervertebral disks were not (Figures 3.1A and 3.1B). The original core of each disk, which is composed of cells originating from the notochord, was not labeled. Cbs mRNA was also detected in chondroblasts of the developing bones of the head, pharyngolaryngeal cartilage, ribs, vertebrae, and in areas at which bone ossification is believed to be initiated (Fig 2E). This dense labeling persisted during later stages of development in the perichondrium and the chondroblasts of ribs at P0 (Figure 3.1C) and vertebrae at P7 (Figure 3.1D). In adult mice (data not shown), the signal persists in the perichondrium in cartilage and in periosteum in bone.
Spatial distribution of Cbs mRNA in the Mouse Nervous System
The Cbs gene was expressed in the CNS from an early stage. At E9.5, the entire neural tube was labeled (Fig 2A), with the strongest labeling in the neural tube closure region (Figure 3.2A). This expression changed during neural tube formation and appeared to be more caudal at E10.5 (Figure 3.2B). At E10.5, the telencephalon was strongly labeled.
Between E12.5 and E15.5, strong signal was observed in most proliferating zones of the nervous system. The strongest labeling was observed in the ventricular zone of many structures, such as the neopallial cortex, the striatum and the fourth ventricle, and the medulla oblongata. Weak labeling was observed in the neopallial cortex, the midbrain, the medulla oblongata and the spinal cord (Fig 2E, Fig 4A, and Fig 4B). Although the strong expression in the ventricular zone of brain persisted at E15.5, expression in the nervous system declined in intensity.
|
|
From P0, the expression was uniform and very weak (Fig 4C). At P2, the expression became stronger and more limited to specific regions of the brain, such as the cerebellum and the olfactory bulb (Fig 4D). This expression changed during the later stages of cerebellar development (Fig 4E). We also used Northern blotting to study the expression of Cbs in the nervous system between P0 and adulthood. Northern blotting was performed with mRNA extracted from the brain and cerebellum of P0, P2, P7, P10, and P15 mice and of 3-month-old CBS(+/+) mice (Fig 5). With both brain and cerebellar extracts, a single 2.4-kb band was observed. This band was weak in P0 mice. The intensity then increased from P2 to P10, and decreased from P15 to 3 months. In the adult mice, weak signal was present in the whole brain, whereas strong signal appeared to be restricted to the Purkinje cell layer and to the hippocampus, including Ammon's horn and the dentate gyrus (Fig 4F).
|
Thus, during the earlier stages of brain development, Cbs is highly expressed in neuroblast cells. In the later stages of development (from birth to adulthood), the Cbs expression pattern changes as cells differentiate. This suggests that CBS is involved in neuronal development and function in some neurons.
Spatial Localization of CBS Protein in Mouse Nervous System
Because Cbs expression might be specific to some neurons, we used IHC methods to localize the protein in the adult brain. We carried out Western blotting experiments using an anti-rat CBS antibody (
|
The protein profile closely mirrored the RNA profile. A weak signal was present in the whole brain. Despite the high level of general background staining in the section, we observed uniform and weak expression in the neurons of the cerebral cortex, the striatum, the thalamus, and the spinal cord, whereas no significant expression was detected in the non-neuronal cells of these brain regions. The strongest expression was observed in the hippocampus and the cerebellum (Fig 6B). In the cerebellum, signal was observed in the cell bodies and neuronal processes of the Purkinje cells (Fig 6C6E). Weak signal was observed in cells of the granular cell layer. In the hippocampus, strong signals were detected in the cellular bodies and the neuronal processes of the CA2 and CA3 neurons (Fig 6F and Fig 6H) and weaker signals were observed in the other regions, such as the CA1 neurons (Fig 6F and Fig 6G) and the dentate gyrus.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We characterized the expression profile of the mouse Cbs gene and demonstrated that its spatial and temporal expression patterns are developmentally regulated. In the early stages of development, Cbs was ubiquitously expressed. It then predominantly accumulated in specific tissues, especially those that are affected in homocysteinemic patients (liver, cardiac, skeletal and nervous systems) and a murine model of hyperhomocysteinemia (
We also showed that the CBS protein is localized in the brain in adult mice. A low level of expression was observed in the whole brain cerebral cortex, the striatum, the thalamus, and the spinal cord, whereas the protein was strongly expressed in the cell bodies and the neuronal processes of the Purkinje cells and hippocampal neurons. This pattern is consistent with the pattern of CBS activity in nine regions of the adult rat brain (
The expression of Cbs therefore appears to be developmentally regulated. A high level of expression was observed in neuroblasts during the early stages of development and in some differentiated neurons in the adult CNS, suggesting that CBS is involved in the growth and maturation of neural networks. This regulated pattern during CNS development should be related to a regulated rate of homocysteine and methionine. In addition, it might explain why high homocysteine levels result in developmental defects of the neural tube in most animal models (
Homocysteine, the concentration of which is regulated by CBS activity, is known to be excitotoxic in vitro, and there is evidence for synthesis of homocysteine and its derivatives, such as homocysteic acid, in the brain (
It has been also suggested that high homocysteine concentrations promote hypersensitivity to excitotoxicity and apoptosis in many cultured neuronal cells, such as rat embryonic neuronal cells, human neuronal cell lines, rat cerebrocortical cells, rat cerebellar granule cells, and rat hippocampal neurons (
It is not clear whether the endogenous concentration of homocysteine is involved in neuronal function or development, but the putative role of homocysteine in synaptic transmission might explain the importance of the regulation of homocysteine metabolism in the CNS through CBS activity. In addition, the observed association of elevated plasma homocysteine with some neurodegenerative diseases makes it more pressing to better understand the role of homocysteine metabolism in normal brain.
![]() |
Acknowledgments |
---|
Supported by a grant from the Fondation Jérôme Lejeune and by an EU grant (BMH4-CT98-3039). KR was supported by the Fondation Jérôme Lejeune. ET was supported by the Fondation pour la Recherche Médicale (FRM).
We thank Dr M. Sakaguchi (Department of Molecular Biology, Graduate School of Medical Science, Kyushu University, Japan) for providing the anti-CBS antibody and Dr M. Nobuyo (Department of Pathology, University of North Carolina, Chapel Hill, NC) for providing heterozygous CBS(+/-) mice. We also thank Nicole Créau and Carmela Lopes for helpful advice and discussions.
Received for publication June 21, 2002; accepted November 5, 2002.
![]() |
Literature Cited |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Chomczynski P, Sacchi N (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162:156-159[Medline]
Conlon RA, Rossant J (1992) Exogenous retinoic acid rapidly induces anterior ectopic expression of murine Hox-2 genes in vivo. Development 116:357-368
Dayal S, Bottiglieri T, Arning E, Maeda N, Malinow MR, Sigmund CD, Heistad DD et al. (2001) Endothelial dysfunction and elevation of S-adenosylhomocysteine in cystathionine beta-synthase-deficient mice. Circ Res 88:1203-1209
D'Emilia DM, Lipton SA (1999) Ratio of S-nitrosohomocyst(e)ine to homocyst(e)ine or other thiols determines neurotoxicity in rat cerebrocortical cultures. Neurosci Lett 265:103-106[Medline]
Eberhardt RT, Forgione MA, Cap A, Leopold JA, Rudd MA, Trolliet M, Heydrick S et al. (2000) Endothelial dysfunction in a murine model of mild hyperhomocyst(e)inemia. J Clin Invest 106:483-491
Eroschenko VP (1996) Atlas of Histology with Functional Correlations. 8th ed Baltimore, Williams & Wilkins
Gaitonde MK, Richter D (1957) The Metabolism of 35S-Methionine in the Brain. In Richter D, ed. The Metabolism of the Nervous System. Oxford, Pergamon Press, 449-458
Grieco AJ (1977) Homocystinuria: pathogenetic mechanisms. Am J Med Sci 273:120-132[Medline]
Harker LA, Slichter SJ, Scott CR, Ross R (1974) Homocystinemia. Vascular injury and arterial thrombosis. N Engl J Med 291:537-543[Medline]
Hope DB (1959) Distribution of cystathionine and cystathionine synthase in rat brain. Fed Proc 18:249-258
Ikonomidou C, Bosch F, Miksa M, Bittigau P, Vockler J, Dikranian K, Tenkova TI et al. (1999) Blockade of NMDA receptors and apoptotic neurodegeneration in the developing brain. Science 283:70-74
Ito S, Provini L, Cherubini E (1991) L-homocysteic acid mediates synaptic excitation at NMDA receptors in the hippocampus. Neurosci Lett 124:157-161[Medline]
Kaufman MH (1995) The Atlas of Mouse Development. New York, Academic Press
Kim WK, Pae YS (1996) Involvement of N-methyl-d-aspartate receptor and free radical in homocysteine-mediated toxicity on rat cerebellar granule cells in culture. Neurosci Lett 216:117-120[Medline]
Kohl RL, Quay WB (1979) Cystathionine synthase in rat brain: regional and time-of-day differences and their metabolic implications. J Neurosci Res 4:189-196[Medline]
Kruman II, Culmsee C, Chan SL, Kruman Y, Guo Z, Penix L, Mattson MP (2000) Homocysteine elicits a DNA damage response in neurons that promotes apoptosis and hypersensitivity to excitotoxicity. J Neurosci 20:6920-6926
Lentz SR, Erger RA, Dayal S, Maeda N, Malinow MR, Heistad DD, Faraci FM (2000) Folate dependence of hyperhomocysteinemia and vascular dysfunction in cystathionine beta-synthase-deficient mice. Am J Physiol Heart 279:H970-975
Lipton SA, Kim WK, Choi YB, Kumar S, D'Emilia DM, Rayudu PV, Arnelle DR et al. (1997) Neurotoxicity associated with dual actions of homocysteine at the N-methyl-D-aspartate receptor. Proc Natl Acad Sci USA 94:5923-5928
Mudd SH (1985) Vascular disease and homocysteine metabolism. N Engl J Med 313:751-753[Medline]
Mudd SH, Finkelstein JD, Irreverre F, Laster L (1965) Transsulfuration in mammals. Microassays and tissue distributions of three enzymes of the pathway. J Biol Chem 240:4382-4392
Omura T, Sadano H, Hasegawa T, Yoshida Y, Kominami S (1984) Hemoprotein H-450 identified as a form of cytochrome P-450 having an endogenous ligand at the 6th coordination position of the heme. J Biochem (Tokyo) 96:1491-1500[Abstract]
Parsons RB, Waring RH, Ramsden DB, Williams AC (1998) In vitro effect of the cysteine metabolites homocysteic acid, homocysteine and cysteic acid upon human neuronal cell lines. Neurotoxicology 19:599-603[Medline]
Peuchmaur M, Emilie D, Crevon MC, SolalCeligny P, Maillot MC, Lemaigre G, Galanaud P (1990) IL-2 mRNA expression in Tac-positive malignant lymphomas. Am J Pathol 136:383-390[Abstract]
Platenik J, Kuramoto N, Yoneda Y (2000) Molecular mechanisms associated with long-term consolidation of the NMDA signals. Life Sci 67:335-364[Medline]
Quere I, Paul V, Rouillac C, Janbon C, London J, Demaille J, Kamoun P et al. (1999) Spatial and temporal expression of the cystathionine beta-synthase gene during early human development. Biochem Biophys Res Commun 254:127-137[Medline]
Rassin DK, Gaull GE (1975) Subcellular distribution of enzymes of transmethylation and transsulphuration in rat brain. J Neurochem 24:969-978[Medline]
Rosenquist TH, Finnell RH (2001) Genes, folate and homocysteine in embryonic development. Proc Nutr Soc 60:53-61[Medline]
Rosenquist TH, Schneider AM, Monogham DT (1999) N-methyl-D-aspartate receptor agonists modulate homocysteine-induced developmental abnormalities. FASEB J 13:1523-1531
Sawada S, Takada S, Yamamoto C (1982) Excitatory actions of homocysteic acid on hippocampal neurons. Brain Res 238:282-285[Medline]
Sturman JA, Gaull GE, Niemann WH (1976) Cystathionine synthesis and degradation in brain, liver and kidney of the developing monkey. J Neurochem 26:457-463[Medline]
Sturman JA, Rassin DK, Gaull GE (1970) Distribution of transsulfuration enzymes in various organs and species. Int J Biochem. I: 251253
Swaroop M, Bradley K, Ohura T, Tahara T, Roper MD, Rosenberg LE, Kraus JP (1992) Rat cystathionine beta-synthase. Gene organization and alternative splicing. J Biol Chem 67:11455-11461
Van den Berg M, van der Knaap MS, Boers GH, Stehouwer CD, Rauwerda JA, Valk J (1995) Hyperhomocysteinaemia; with reference to its neuroradiological aspects. Neuroradiology 37:403-411[Medline]
Vialard F, Toyama K, Vernoux S, Carlson EJ, Epstein CJ, Sinet PM, Rahmani Z (2000) Overexpression of mSim2 gene in the zona limitans of the diencephalon of segmental trisomy 16 Ts1Cje fetuses, a mouse model for trisomy 21: a novel whole-mount based RNA hybridization study. Brain Res Dev Brain Res 121:73-78[Medline]
Vollenweider FX, Cuenod M, Do KQ (1990) Effect of climbing fiber deprivation on release of endogenous aspartate, glutamate, and homocysteate in slices of rat cerebellar hemispheres and vermis. J Neurochem 54:1533-1540[Medline]
Volpe JJ, Laster L (1972) Transsulfuration in fetal and postnatal mammalian liver and brain. Cystathionine synthase, its relation to hormonal influences, and cystathionine. Biol Neonate 20:385-403[Medline]
Watanabe M, Osada J, Aratani Y, Kluckman K, Reddick R, Malinow MR, Maeda N (1995) Mice deficient in cystathionine beta-synthase: animal models for mild and severe homocyst(e)inemia. Proc Natl Acad Sci USA 92:1585-1589[Abstract]
Weiss N, Heydrick S, Zhang YY, Bierl C, Cap A, Loscalzo J (2002) Cellular redox state and endothelial dysfunction in mildly hyperhomocysteinemic cystathionine beta-synthase-deficient mice. Arterioscler Thromb Vasc Biol 22:34-41
Yuzaki M, Connor JA (1999) Characterization of L-homocysteate-induced currents in Purkinje cells from wild-type and NMDA receptor knockout mice. J Neurophysiol 82:2820-2826
Yuzaki M, Forrest D, Verselis LM, Sun SC, Curran T, Connor JA (1996) Functional NMDA receptors are transiently active and support the survival of Purkinje cells in culture. J Neurosci 16:4651-4661