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Induction of NGFI-A mRNA following middle cerebral artery occlusion in rats: In situ hybridization study
ELSEVIER
Neuroscience Letters 17l (1994) 163 166
NEURO$ClENCi lETTERS
Induction of NGFI-A mRNA following middle cerebral artery occlusion in rats: in situ hybridization study Hiroyuki Kinouchi "'*, Frank R. Sharp u'd, Pak H. Chan b'~, Jari Koistinaho b'd'e, Stephen M. Sagar ba, Takashi Yoshimoto ~ "Division ~[" Neurosurge13', Institute (~/'Brain Diseases, Tohoku University School (~/' Medicine. l-I Seio'o-machi, Aoha-ku, Scmhti 9~'0, Japan D~7~artments (~1 t'Neurology and ' Neurosurgery. University ~[ Cal(/~rnia at San Francisco. ('A, USA JDepartment q[' Neurology, Veterans it[]ilirs Medical Centel: San Francisco, CA. USA 'Department of Biomedical Science. University ~[" Tampere, Tampere, Finhmd Received 21 January 1994; Revised version received 24 February 1994" Accepted 24 February 1994
Abstract
Middle cerebral artery (MCA) occlusion in halothane-anesthetized rats induced the zinc finger gene, NGFI-A, in brain. In situ hybridization studies showed that NGFI-A was induced throughout all of the cortex following MCA occlusion. By 24 h after MCA occlusion there was little expression of NGFI-A mRNA in the core of the MCA infarct, but the mRNA was still induced in all of cortex outside the infarct. MCA occlusion also induced this gene in subcortical structures: ipsilateral medial striatum; most of thalamus including medial and lateral geniculate nuclei; substantia nigra; and hippocampus at 4 h of MCA occlusion which generally disappeared by 24 h of MCA occlusion. Most of these structures, except for the striatum, are not supplied by the MCA. These data show that changes in brain gene expression can occur in many regions remote from an infarction.
Key words'. Immediate early gene; Zinc finger: Central nervous system: Stroke: Ischemia
Following cerebral ischemia, the synthesis of most m R N A species and their respective proteins decrease [7,14,25]. However, several groups of genes are induced by ischemia. These include members of immediate early gene (IEG) families encoding transcription factors [1-3,5,8,9,12,18,19,21]. Among the transcription factors, the fos and jun families have been, widely investigated even in cerebral ischemia. In focal cerebral ischemia, c:[os, c:/un, andjunB IEGs were induced throughout the cortex [3,8,12,21,23] and also in subcortical regions in areas remote from ischemic territory [3,12,23]. Those results suggested that widespread changes in gene expression occur outside regions of infarction following focal cerebral ischemia. However, the expression of another family of transcription factors, the zinc finger genes including N G F I A (:if/218, erg-1, or krox 24), NGFI-B, NGFI-C, and krox 20 has been studied very little following cerebral ischemia [1-3,9]. The present study demonstrates that * Corresponding author: Fax: (81 ) (22) 272-9539. 0304-3940/94/$7.00 ,'~:~1994 Elsevier Science Ireland Ltd. All rights reserved S S D I 0 3 0 4 - 3 9 4 0 ( 9 4 ) 0 0 19 6 - H
the induction pattern of N G F I - A m R N A following middle cerebral artery (MCA) occlusion closely follows that of c-Jbs and junB m R N A reported previously [12]. Focal cerebral ischemia was produced by MCA occlusion using an intraluminal suture without craniectomy. Briefly, male Sprague-Dawley rats (280--300 g) were anesthetized with a 2-0.5% halothane and nitrous oxide/oxygen mixture (70:30). The rectal temperature of the animal was maintained at 37°C with a heating pad. The left common carotid artery was exposed and the external carotid and pterygopalatine arteries were ligated. A 4-0 monofilament nylon suture, blunted at the tip, was threaded into the internal carotid artery through the external carotid artery stump up to the anterior cerebral artery (ACA) [11]. The entire procedure was performed within 10 rain. Three controls underwent identical surgery except that the suture was not inserted. Rats were anesthetized with ketamine (80 mg/kg) and xylazine (20 mg/kg) and decapitated at 1, 4 and 24 h following MCA occlusion (n = 4 for each time). The brains were removed, frozen, embedded and stored at
164
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C
,
I h
]
r
4h
I
24h
i
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.................
Fig. 1. NGFI-A m R N A in situ hybridization autoradiographs o f coronal brain sections from rats that were subjected to sham surgery (C, control) or M C A occlusions 1, 4 or 24 h. Certain amounts of m R N A were hybridized in the control animals. Note induction o f NGFI-A m R N A in ipsilateral cortex, caudate-putamen, thalamus and hippocampus, and some induction in the ipsilateral substantia nigra and contralateral cortex and medial geniculate nucleus, with little expression o f m R N A in the core of the infarct at 4 h.
-70°C. Twenty-micron-thick coronal sections were cut on a cryostat at -20°C, collected on silan-coated slides and processed for in situ hybridization. The brain sections used in this experiment were from the same rats used in the hybridization study of c-los, junB, and c:jun mRNA reported previously [12]. In situ hybridization was performed using oligonucleotides. The probe sequence was (45-mer) complimentary to the base sequence of the mRNA encoding amino acids 2-16 [15]. The oligonucleotide probe was tested on Northern blots of brain poly(A) + RNA and shown to detect specific transcripts of appropriate sizes [10,15]. The oligonucleotide probes were prepared by labeling with [35S]dATP (DuPont) using a 3' labeling system (DuPont/NEN Products). Radiolabeled probes were purified using Nuctrap push column (Stratagene), The slides were hybridized at 42°C for 18 h with 1 x 10 ° cpm of the 35S-labeled probes. After hybridization sections were rinsed in 1 x SSC (150 mM sodium chloride/15 mM sodium citrate, pH 7.4) at 55°C for 60 min with several changes of 1 x SSC, dehydrated, and covered with Kodak XAR-5 film for 3 weeks. In situ hybridization autoradiographs of NGFI-A mRNA following MCA occlusion are shown in Fig. 1. Autoradiographs of brain sections from the sham-operated animals demonstrated a low level of NGFI-A mRNA expression. At 1 h following MCA occlusion, NGFI-A mRNA was induced throughout all of ipsilateral cortex including cingulate, neocortex (frontal, pa-
rietal, temporal and occipital), and piriform cortex. By 4 h there was a decrease of NGFI-A mRNA in the core of the MCA ischemic region but there was a persistent induction in surrounding regions of ipsilateral and contralateral cortex. Although there was no mRNA present in the core of the infarct by 24 h, it persisted in cingulate cortex and regions presumably supplied by the anterior and posterior cerebral artery. There was a slight induction of NGFI-A in the whole striatum at 1 h. However, lateral striatum showed no expression of the mRNA from 4 to 24 h following MCA occlusion and medial striatum and n. accumbens showed a strong induction at 4 h which disappeared by 24 h. NGFI-A was induced in the ipsilateral thalamus (including the medial and lateral geniculates and the anterior thalamic nuclei), CA3 pyramidal neurons in hippocampus, and substantia nigra and in contralateral cortex and medial geniculate nucleus at 4 h, but it had substantially decreased by 24 h. The NGFI-A gene was identified by virtue of its induction by nerve growth factor in PC12 cells [15]. The gene which encodes a zinc finger protein is rapidly activated in response to a variety of stimuli including growth factors [15], excitatory amino acid [22] dopaminergic agents [16], seizure induction [17,20], long term potentiation [6,24], and ischemia/hypoxia [1,3,9]. Abe et al. have shown using Northern blot analysis that NGFI-A mRNA was induced transiently after 30 rain of transient focal cerebral ischemia in rat [l ]. Gubits
H. Kinouchi et al./Neuroscience Letters 171 (1994) 163 166
et al. have also shown the induction of TIS8 (NGFI-A, zif268) following neonatal hypoxia-ischemia by Northern blotting [9]. However, the anatomical pattern of NGFI-A gene has not been elucidated. Only the induction of the zinc finger gene, Krox-20, has been shown using in situ hybridization after transient focal cerebral ischemia [3]. Those authors found that transient focal cerebral ischemia induced Krox-20 mRNA exclusively in the ipsilateral cortex following 30 min of focal ischemia and in ipsilateral cortex and hippocampus following 90 rain of ischemia [3]. The present study shows that NGFI-A mRNA was induced throughout all of cortex bilaterally and ipsilateral medial striatum, most of thalamus including medial (bilateral) and lateral geniculate nucleus, substantia nigra, and hippocampus. The pattern of the NGFI-A induction was similar to that of c-jos and junB in the ipsilateral cortex, striatum, thalamus, and substantia nigra [12]. This suggests that the known calcium/cyclic AMP (CRE) and serum response elements (SRE) in the promoter of the IEG encoding AP-1 binding protein [4,13] might be similar or at least analogous to the regulatory elements in the NGFI-A gene. However, the induction pattern in the hippocampal formation was different for each gene. c-los was induced in ipsilateral hippocampal (CA1-CA3) pyramidal neurons and dentate granule cell neurons, junB was induced in the same cells bilaterally. NGFI-A was induced CA3 pyramidal neurons but not in dentate granule cells. Differential regulation of c-los and zif268 has also been reported in the dentate gyrus of the hippocampus after high-frequency stimulation of the perforant path [6]. However, the underlying mechanism for the differential expression is unknown at present and further investigation is necessary to elucidate. These data on NGFI-A expression confirm our recent studies that c-los and junB IEG can be induced in regions outside of a focal cerebral infarction. A possible mechanism of induction of these genes throughout cortex may be a disinhibition due to spreading depression accounting for NGFI-A induction in cortex supplied by middle, anterior, and posterior cerebral arteries. Induction throughout thalamus likely relates to the spreading depression of cortex. As proposed previously, striatal inl;arction likely decreases inhibitory input to substantia nigra leading to excitation of substantia nigra neurons and induction of NGFI-A in those cells [12]. [1] Abe, K., Kawagoe, J., Sato, S., Sahara, H. and Kogure, K., Induction of the 'zinc finger" gene alter transient focal ischemia in rat cerebral cortex, Neurosci Lett., 123 (1991) 248 250. [2] An, G., Lin, T.-N., Liu, J.-S. and Hsu, C.Y., Induction of Krox20 expression after focal cerebral ischemia, Biochem. Biophys. Res. Commun., 188 (1992) 1104 1110. [3] An, G.. Lin, T.-N., Liu, J,-S., Xue, J.-J., He, Y.-Y. and Hsu, C.Y., Expression of c:/bs and c-jun family genes after ibcal cerebral ischemia. Ann. Neurol., 33 (1993) 457~64.
165
[4] Bading, H., Ginty, D.D. and Greenberg+ M.E., Regulation of gene expression in hippocampal neurons by distinct calcium signaling pathways, Science, 260 (1993)181 186. [5] Blumenfeld, K.S., Welsh, F.A., Harris, V.A. and Pesenson, M.A., Regional expression of c-/bs and heat shock protein-70 mRNA following hypoxia-ischemia in immature rat brain, J. Cerebral Blood Flow Metab., 12 (1992) 987 995. [6] Cole, A.J., Saffen, D.W,, Baraban, J.M. and Worley, P.F., Rapid increase of an immediate early gene messenger RNA in hippocampal neurons by synaptic NMDA receptor activation, Nature, 340 (1989) 474M-76. [7] Dienel, G.A., Pulsinelli, W.A. and Dult}, T.E., Regional protein synthesis in rat brain following acute hemispheric ischemia, J. Neurochem., 35 (1980) 1216 1226. [8] Gass, R, Spranger, T., Herdegen, R., Bravo, R.. Kock, P., Hacke, W. and Kiessling, M., Induction of gos and Jun protein alter focal ischemia in the rat: differential effect of the N-methyl-D- aspartatc receptor antagonist MK-801. Acta Neuropathol., 84 (1992) 545 553. [9] Gubits, R.M., Burke, R.E., Casey-Mclntosh, G., Bandele, A. and Muncll, F.+ Immediate early gene induction after neonatal hypoxia-ischemia, Mol. Brain Res., 18 (1993} 228 238. [10] Hisanaga, K., Sagar, S.M.. Koistinaho, J., Hicks, K,J. and Sharp, F.R., VIP-induced stellation and immediate early gene expression in astrocytes: effects of dexamethasone. Neurosci. Lett., 156 (1993) 57 60. [11] Kinouehi, H., Sharp, F.R., Hill, M.P., Koistinaho, J., Sagar+ S.M. and Chan, RH., Induction of 70 kDa heat shock protein and hsp70 mRNA following transient local cerebral ischemia in the rat, J. Cereb. Blood Flow Metab., 13(1993} 105 115. [12] Kinouchi, H., Sharp, F.R., Chan, P.H., Koistinaho+ J., Sagar, S.M. and Yoshimoto, T., Induction of c:/os,/unB, c:]un and hsp70 mRNA in cortex, thalamus, basal ganglia, and hippocampus t\~llowing middle cerebral artery occlusion, J. Cereb. Blood Flow Metab.. in press. [13] Kitabayashi, 1., Kawakami, Z.. Matsuoka. T., Chiu, R., Gachelin, G+ and Yokoyama, K., Two c/s-regulatory elements that mediate different signaling pathways for serum-dependent activation of the /unB gene, J. Biol. Chem., 268 (1993t 14482 14489. [14] Kleihues, P. and Hossmann, K.-A., Protein synthesis in the cat brain after prolonged cerebral ischemia, Brain Res., 35 (1971) 409 ~-18. [15] Milbrandt, J.+ A nerve growth factor-induced gene encodes a possible transcriptional regulatory lactor, Science, 238 (1987) 797 799. [16] Moratalla, R., Robertson, H.A. and Graybie[, A.M., Dynamic regulation of NGFI-A (zit268, ergl) gene expression in the striatum, J. Neurosci., 12 (1992) 2609 2622. [17] Morgan, J.l., Cohen, D.R., Hempstead, J.k. and Curran, T.+ Mapping patterns ofc-/os expression in the central nervous system after seizure, Science, 237 (19871 192 197. [18] Nowak, T.S., Ikeda, J. and Nakajima, T., 70-kDa heat shock protein and cribs gene expression after transient ischemia, Stroke, 21 (Suppl. Ill) (1990) 107 111. [19] Onodera, H., Kogure, K., Ono, Y., lgarashi, K., Kiyota, Y. and Nagaoka, A., Proto-oncogene c-/bs is transiently induced in the rat cerebral cortex after forebrain ischemia, Neurosci. Lett., 98 (1989) 101 104. [20] Saffen, D.W., Cole, A.J., Worley ,P.F., Christy, B.A., Ryder, K. and Baraban, J.M., Conx ulsant-induccd increase in transcription factor messenger RNAs in rat brain. Proc. Natl. Acad. Sci. USA, 85 (1988) 7795 7799. [21] 13emura Y., Kowall, N.W. and Moskowitz, M.A.+ Focal ischemia in rat causes time-dependent expression of c-los protein immunoreactivity in widespread regions of ipsilateral cortex, Brain Res., 552 (1991} 99 105. [22] Vaccarino, F.M., Hayward, M.D.. Nestler, E.J., Duman, R.S. and
166
tL Kinouchi et al./Neuroscienc~, Letters 171 (1994) 163-166
Tallman, J.F., Differential induction of immediate early genes by excitatory amino acid receptor types in primary cultures of cortical and striatal neurons, Mol. Brain Res., 12 (1992) 233-- 241. [23] Welsh, F.A., Moyer, D.J. and Harris, V.A., Regional expression of heat shock protein-70 mRNA and c-fos mRNA following focal ischemia in rat brain, J. Cereb. Blood Flow Metab., 12 (19921 204-212.
[24] Wisden, W., Errington, M.L., Williams, S., Dunnett, S.13., Waters, C., Hitchcock, D., Evan, G., Bliss, T.V.R and Hunt, S.P., Differential expression of immediate early genes in the hippocampus and spinal cord, Neuron, 4 (1990) 603-614. [25] Yanagihara, T., Experimental stroke in gerbils: effect on translation and transcription, Brain Res., 158 (1978) 435 444.
Neuroscience Letters 17l (1994) 163 166
NEURO$ClENCi lETTERS
Induction of NGFI-A mRNA following middle cerebral artery occlusion in rats: in situ hybridization study Hiroyuki Kinouchi "'*, Frank R. Sharp u'd, Pak H. Chan b'~, Jari Koistinaho b'd'e, Stephen M. Sagar ba, Takashi Yoshimoto ~ "Division ~[" Neurosurge13', Institute (~/'Brain Diseases, Tohoku University School (~/' Medicine. l-I Seio'o-machi, Aoha-ku, Scmhti 9~'0, Japan D~7~artments (~1 t'Neurology and ' Neurosurgery. University ~[ Cal(/~rnia at San Francisco. ('A, USA JDepartment q[' Neurology, Veterans it[]ilirs Medical Centel: San Francisco, CA. USA 'Department of Biomedical Science. University ~[" Tampere, Tampere, Finhmd Received 21 January 1994; Revised version received 24 February 1994" Accepted 24 February 1994
Abstract
Middle cerebral artery (MCA) occlusion in halothane-anesthetized rats induced the zinc finger gene, NGFI-A, in brain. In situ hybridization studies showed that NGFI-A was induced throughout all of the cortex following MCA occlusion. By 24 h after MCA occlusion there was little expression of NGFI-A mRNA in the core of the MCA infarct, but the mRNA was still induced in all of cortex outside the infarct. MCA occlusion also induced this gene in subcortical structures: ipsilateral medial striatum; most of thalamus including medial and lateral geniculate nuclei; substantia nigra; and hippocampus at 4 h of MCA occlusion which generally disappeared by 24 h of MCA occlusion. Most of these structures, except for the striatum, are not supplied by the MCA. These data show that changes in brain gene expression can occur in many regions remote from an infarction.
Key words'. Immediate early gene; Zinc finger: Central nervous system: Stroke: Ischemia
Following cerebral ischemia, the synthesis of most m R N A species and their respective proteins decrease [7,14,25]. However, several groups of genes are induced by ischemia. These include members of immediate early gene (IEG) families encoding transcription factors [1-3,5,8,9,12,18,19,21]. Among the transcription factors, the fos and jun families have been, widely investigated even in cerebral ischemia. In focal cerebral ischemia, c:[os, c:/un, andjunB IEGs were induced throughout the cortex [3,8,12,21,23] and also in subcortical regions in areas remote from ischemic territory [3,12,23]. Those results suggested that widespread changes in gene expression occur outside regions of infarction following focal cerebral ischemia. However, the expression of another family of transcription factors, the zinc finger genes including N G F I A (:if/218, erg-1, or krox 24), NGFI-B, NGFI-C, and krox 20 has been studied very little following cerebral ischemia [1-3,9]. The present study demonstrates that * Corresponding author: Fax: (81 ) (22) 272-9539. 0304-3940/94/$7.00 ,'~:~1994 Elsevier Science Ireland Ltd. All rights reserved S S D I 0 3 0 4 - 3 9 4 0 ( 9 4 ) 0 0 19 6 - H
the induction pattern of N G F I - A m R N A following middle cerebral artery (MCA) occlusion closely follows that of c-Jbs and junB m R N A reported previously [12]. Focal cerebral ischemia was produced by MCA occlusion using an intraluminal suture without craniectomy. Briefly, male Sprague-Dawley rats (280--300 g) were anesthetized with a 2-0.5% halothane and nitrous oxide/oxygen mixture (70:30). The rectal temperature of the animal was maintained at 37°C with a heating pad. The left common carotid artery was exposed and the external carotid and pterygopalatine arteries were ligated. A 4-0 monofilament nylon suture, blunted at the tip, was threaded into the internal carotid artery through the external carotid artery stump up to the anterior cerebral artery (ACA) [11]. The entire procedure was performed within 10 rain. Three controls underwent identical surgery except that the suture was not inserted. Rats were anesthetized with ketamine (80 mg/kg) and xylazine (20 mg/kg) and decapitated at 1, 4 and 24 h following MCA occlusion (n = 4 for each time). The brains were removed, frozen, embedded and stored at
164
H. K i m m c h i c/ , , ' . / , V c t , ' v v c l c m c Lcller,s' /71 ,, / 9 9 4 ) / 6 3 /66
C
,
I h
]
r
4h
I
24h
i
r
~k
.................
Fig. 1. NGFI-A m R N A in situ hybridization autoradiographs o f coronal brain sections from rats that were subjected to sham surgery (C, control) or M C A occlusions 1, 4 or 24 h. Certain amounts of m R N A were hybridized in the control animals. Note induction o f NGFI-A m R N A in ipsilateral cortex, caudate-putamen, thalamus and hippocampus, and some induction in the ipsilateral substantia nigra and contralateral cortex and medial geniculate nucleus, with little expression o f m R N A in the core of the infarct at 4 h.
-70°C. Twenty-micron-thick coronal sections were cut on a cryostat at -20°C, collected on silan-coated slides and processed for in situ hybridization. The brain sections used in this experiment were from the same rats used in the hybridization study of c-los, junB, and c:jun mRNA reported previously [12]. In situ hybridization was performed using oligonucleotides. The probe sequence was (45-mer) complimentary to the base sequence of the mRNA encoding amino acids 2-16 [15]. The oligonucleotide probe was tested on Northern blots of brain poly(A) + RNA and shown to detect specific transcripts of appropriate sizes [10,15]. The oligonucleotide probes were prepared by labeling with [35S]dATP (DuPont) using a 3' labeling system (DuPont/NEN Products). Radiolabeled probes were purified using Nuctrap push column (Stratagene), The slides were hybridized at 42°C for 18 h with 1 x 10 ° cpm of the 35S-labeled probes. After hybridization sections were rinsed in 1 x SSC (150 mM sodium chloride/15 mM sodium citrate, pH 7.4) at 55°C for 60 min with several changes of 1 x SSC, dehydrated, and covered with Kodak XAR-5 film for 3 weeks. In situ hybridization autoradiographs of NGFI-A mRNA following MCA occlusion are shown in Fig. 1. Autoradiographs of brain sections from the sham-operated animals demonstrated a low level of NGFI-A mRNA expression. At 1 h following MCA occlusion, NGFI-A mRNA was induced throughout all of ipsilateral cortex including cingulate, neocortex (frontal, pa-
rietal, temporal and occipital), and piriform cortex. By 4 h there was a decrease of NGFI-A mRNA in the core of the MCA ischemic region but there was a persistent induction in surrounding regions of ipsilateral and contralateral cortex. Although there was no mRNA present in the core of the infarct by 24 h, it persisted in cingulate cortex and regions presumably supplied by the anterior and posterior cerebral artery. There was a slight induction of NGFI-A in the whole striatum at 1 h. However, lateral striatum showed no expression of the mRNA from 4 to 24 h following MCA occlusion and medial striatum and n. accumbens showed a strong induction at 4 h which disappeared by 24 h. NGFI-A was induced in the ipsilateral thalamus (including the medial and lateral geniculates and the anterior thalamic nuclei), CA3 pyramidal neurons in hippocampus, and substantia nigra and in contralateral cortex and medial geniculate nucleus at 4 h, but it had substantially decreased by 24 h. The NGFI-A gene was identified by virtue of its induction by nerve growth factor in PC12 cells [15]. The gene which encodes a zinc finger protein is rapidly activated in response to a variety of stimuli including growth factors [15], excitatory amino acid [22] dopaminergic agents [16], seizure induction [17,20], long term potentiation [6,24], and ischemia/hypoxia [1,3,9]. Abe et al. have shown using Northern blot analysis that NGFI-A mRNA was induced transiently after 30 rain of transient focal cerebral ischemia in rat [l ]. Gubits
H. Kinouchi et al./Neuroscience Letters 171 (1994) 163 166
et al. have also shown the induction of TIS8 (NGFI-A, zif268) following neonatal hypoxia-ischemia by Northern blotting [9]. However, the anatomical pattern of NGFI-A gene has not been elucidated. Only the induction of the zinc finger gene, Krox-20, has been shown using in situ hybridization after transient focal cerebral ischemia [3]. Those authors found that transient focal cerebral ischemia induced Krox-20 mRNA exclusively in the ipsilateral cortex following 30 min of focal ischemia and in ipsilateral cortex and hippocampus following 90 rain of ischemia [3]. The present study shows that NGFI-A mRNA was induced throughout all of cortex bilaterally and ipsilateral medial striatum, most of thalamus including medial (bilateral) and lateral geniculate nucleus, substantia nigra, and hippocampus. The pattern of the NGFI-A induction was similar to that of c-jos and junB in the ipsilateral cortex, striatum, thalamus, and substantia nigra [12]. This suggests that the known calcium/cyclic AMP (CRE) and serum response elements (SRE) in the promoter of the IEG encoding AP-1 binding protein [4,13] might be similar or at least analogous to the regulatory elements in the NGFI-A gene. However, the induction pattern in the hippocampal formation was different for each gene. c-los was induced in ipsilateral hippocampal (CA1-CA3) pyramidal neurons and dentate granule cell neurons, junB was induced in the same cells bilaterally. NGFI-A was induced CA3 pyramidal neurons but not in dentate granule cells. Differential regulation of c-los and zif268 has also been reported in the dentate gyrus of the hippocampus after high-frequency stimulation of the perforant path [6]. However, the underlying mechanism for the differential expression is unknown at present and further investigation is necessary to elucidate. These data on NGFI-A expression confirm our recent studies that c-los and junB IEG can be induced in regions outside of a focal cerebral infarction. A possible mechanism of induction of these genes throughout cortex may be a disinhibition due to spreading depression accounting for NGFI-A induction in cortex supplied by middle, anterior, and posterior cerebral arteries. Induction throughout thalamus likely relates to the spreading depression of cortex. As proposed previously, striatal inl;arction likely decreases inhibitory input to substantia nigra leading to excitation of substantia nigra neurons and induction of NGFI-A in those cells [12]. [1] Abe, K., Kawagoe, J., Sato, S., Sahara, H. and Kogure, K., Induction of the 'zinc finger" gene alter transient focal ischemia in rat cerebral cortex, Neurosci Lett., 123 (1991) 248 250. [2] An, G., Lin, T.-N., Liu, J.-S. and Hsu, C.Y., Induction of Krox20 expression after focal cerebral ischemia, Biochem. Biophys. Res. Commun., 188 (1992) 1104 1110. [3] An, G.. Lin, T.-N., Liu, J,-S., Xue, J.-J., He, Y.-Y. and Hsu, C.Y., Expression of c:/bs and c-jun family genes after ibcal cerebral ischemia. Ann. Neurol., 33 (1993) 457~64.
165
[4] Bading, H., Ginty, D.D. and Greenberg+ M.E., Regulation of gene expression in hippocampal neurons by distinct calcium signaling pathways, Science, 260 (1993)181 186. [5] Blumenfeld, K.S., Welsh, F.A., Harris, V.A. and Pesenson, M.A., Regional expression of c-/bs and heat shock protein-70 mRNA following hypoxia-ischemia in immature rat brain, J. Cerebral Blood Flow Metab., 12 (1992) 987 995. [6] Cole, A.J., Saffen, D.W,, Baraban, J.M. and Worley, P.F., Rapid increase of an immediate early gene messenger RNA in hippocampal neurons by synaptic NMDA receptor activation, Nature, 340 (1989) 474M-76. [7] Dienel, G.A., Pulsinelli, W.A. and Dult}, T.E., Regional protein synthesis in rat brain following acute hemispheric ischemia, J. Neurochem., 35 (1980) 1216 1226. [8] Gass, R, Spranger, T., Herdegen, R., Bravo, R.. Kock, P., Hacke, W. and Kiessling, M., Induction of gos and Jun protein alter focal ischemia in the rat: differential effect of the N-methyl-D- aspartatc receptor antagonist MK-801. Acta Neuropathol., 84 (1992) 545 553. [9] Gubits, R.M., Burke, R.E., Casey-Mclntosh, G., Bandele, A. and Muncll, F.+ Immediate early gene induction after neonatal hypoxia-ischemia, Mol. Brain Res., 18 (1993} 228 238. [10] Hisanaga, K., Sagar, S.M.. Koistinaho, J., Hicks, K,J. and Sharp, F.R., VIP-induced stellation and immediate early gene expression in astrocytes: effects of dexamethasone. Neurosci. Lett., 156 (1993) 57 60. [11] Kinouehi, H., Sharp, F.R., Hill, M.P., Koistinaho, J., Sagar+ S.M. and Chan, RH., Induction of 70 kDa heat shock protein and hsp70 mRNA following transient local cerebral ischemia in the rat, J. Cereb. Blood Flow Metab., 13(1993} 105 115. [12] Kinouchi, H., Sharp, F.R., Chan, P.H., Koistinaho+ J., Sagar, S.M. and Yoshimoto, T., Induction of c:/os,/unB, c:]un and hsp70 mRNA in cortex, thalamus, basal ganglia, and hippocampus t\~llowing middle cerebral artery occlusion, J. Cereb. Blood Flow Metab.. in press. [13] Kitabayashi, 1., Kawakami, Z.. Matsuoka. T., Chiu, R., Gachelin, G+ and Yokoyama, K., Two c/s-regulatory elements that mediate different signaling pathways for serum-dependent activation of the /unB gene, J. Biol. Chem., 268 (1993t 14482 14489. [14] Kleihues, P. and Hossmann, K.-A., Protein synthesis in the cat brain after prolonged cerebral ischemia, Brain Res., 35 (1971) 409 ~-18. [15] Milbrandt, J.+ A nerve growth factor-induced gene encodes a possible transcriptional regulatory lactor, Science, 238 (1987) 797 799. [16] Moratalla, R., Robertson, H.A. and Graybie[, A.M., Dynamic regulation of NGFI-A (zit268, ergl) gene expression in the striatum, J. Neurosci., 12 (1992) 2609 2622. [17] Morgan, J.l., Cohen, D.R., Hempstead, J.k. and Curran, T.+ Mapping patterns ofc-/os expression in the central nervous system after seizure, Science, 237 (19871 192 197. [18] Nowak, T.S., Ikeda, J. and Nakajima, T., 70-kDa heat shock protein and cribs gene expression after transient ischemia, Stroke, 21 (Suppl. Ill) (1990) 107 111. [19] Onodera, H., Kogure, K., Ono, Y., lgarashi, K., Kiyota, Y. and Nagaoka, A., Proto-oncogene c-/bs is transiently induced in the rat cerebral cortex after forebrain ischemia, Neurosci. Lett., 98 (1989) 101 104. [20] Saffen, D.W., Cole, A.J., Worley ,P.F., Christy, B.A., Ryder, K. and Baraban, J.M., Conx ulsant-induccd increase in transcription factor messenger RNAs in rat brain. Proc. Natl. Acad. Sci. USA, 85 (1988) 7795 7799. [21] 13emura Y., Kowall, N.W. and Moskowitz, M.A.+ Focal ischemia in rat causes time-dependent expression of c-los protein immunoreactivity in widespread regions of ipsilateral cortex, Brain Res., 552 (1991} 99 105. [22] Vaccarino, F.M., Hayward, M.D.. Nestler, E.J., Duman, R.S. and
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tL Kinouchi et al./Neuroscienc~, Letters 171 (1994) 163-166
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