Molecular Brain Research, 13 (1992) 355-357 © 1992 Elsevier Science Publishers B.V. All rights reserved. 0169-328X/92/$05.00
355
BRESM 80123
Basal expression of Fos, Fos-related, Jun, and Krox 24 proteins in rat hippocampus P. Hughes, P. Lawlor and M. Dragunow Department of Pharmacology and Clinical Pharmacology, University of Auckland School of Medicine, Private Bag, Auckland (New Zealand)
(Accepted 17 December 1991) Key words: Transcription factor; Limbic system; Memory
The basal expression of the protein products of the inducible immediate early genes (IEGs), Fos, Jun, and Krox 24, was investigated in rat hippocampus using immunocytochemical visualization methods with antisera specific for Fos only, Fos and the Fos-related antigens (FRAs), the Jun family, and Krox 24 (previously described as TIS 8, egr-1, NGF-IA or zif 268). In the normal adult rat brain basal levels of Jun, Krox 24 and Fos-related antigens but not Fos were seen within the hippocampus. More specificallyvery high basal levels of Jun were seen in the dentate granule cells with high basal Krox 24 levels seen in the CAl-subiculum region of the rat hippocampus. Basal FRAs but not Fos-positive cells were seen at low levels in the dentate granule cells. The implications of these results to the functioning of IEG proteins in hippocampal neurons is discussed. Fos, Jun, and Krox 24 are transcription factors that interact with specific regulatory regions within D N A to modulate the transcriptional activity of various, so-called, 'late-response genes '4'5'7'12'13'18. They are highly inducible genes, being induced rapidly and transiently in a protein synthesis independent manner by a large array of documented agents acting through differential second messenger pathways depending on the cell type and inducing agent used 18. Within the CNS their induction could be linked to events that control long-term change in the CNS. They have thus been implicated in long-term potentiation (LTP) and the changes associated with kindling 5. They may also play a role in CNS pathology as well as plasticity and are induced in a number of models of brain injury 6'8. Fos and Jun and their structurally related family members can form many permutations of leucine zipper-mediated dimers to interact with DNA. The more recently characterized Krox 249 also known as NGFI-A 1, TIS 82°, Egr-119, and zif 2682 interacts with D N A using a highly common and conserved structural motif, the zinc finger binding domain. Here we investigate the basal expression of these inducible immediate early gene (IEG) protein products within the hippocampus of the rat, a structure implicated in learning and memory. Male Wistar rats were deeply anaesthetized with sodium pentobarbital and perfused transcardially with isotonic saline followed by 4% paraformaldehyde in 0.1 M
phosphate buffer (pH 7.4). Brains were removed and left in the fixative for approximately 48 h, and then cut coronally on a vibroslice machine at 70 /~m. Sections were then incubated for 10 min in 1% hydrogen peroxide made up in 100% methanol to remove endogenous peroxidase staining and enhance antibody penetration into the section. Sections were then washed with phosphate-buffered saline (PBS) for a further 10 min before rabbit antiserum to the various IEG products was added to the sections. The antisera used were: (1) PCO5 Fos antibody -- specific for Fos and Fos-related antigens (FRAs) (Oncogene Science); (2) 456 Fos -- specific for Fos only (Medac-Genentechnologie); (3) Krox 24 -(generously provided by R. Bravo); (4) Jun -- specific for the Jun family members c-Jun, Jun B, and Jun D (generously provided by J. Leahl°). The antisera were made up in immunobuffer in the following dilutions 1:500, 1:500, 1:50,000, 1:20,000, respectively. These primary antibodies were allowed to remain on the sections incubated with gentle agitation, at 4°C for 72 h upon which the primary antibody solution was removed and replaced by a biotinylated anti-rabbit secondary antibody which was left overnight. The reaction was visualized using avidin-biotin-horseradish peroxidase and the chromogen diaminobenzidine (DAB), as described previously7. In the rat hippocampus scattered FRA-positive neuronal nuclei were detected in the dentate gyrus in sec-
Correspondence: M. Dragunow, Department of Pharmacologyand Clinical Pharmacology, University of Auckland School of Medicine, Private Bag, Auckland, New Zealand.
356 tions stained with the PCO5 antiserum (Fig. lc). No immunostaining was seen in CA1 or CA3, and since the 456 Fos antibody showed no basal staining within the hippocampus, the low positive staining of certain dentate gyrus granule cell nuclei was probably due to low constitutive expression of Fos-related antigens. Both Junand Krox-24 immunopositive neuronal nuclei were detected in rat hippocampus (Fig. la,b). The Krox 24 basal expression was relatively low in the dentate granule cells, higher in CA3, and very high in CA1 and subiculum (Fig. la). Jun immunoreactivity was high in the nuclei
Fig. 1. Basal expression of Krox 24 (a), Jun (b) and FRAs (c) in rat hippocampus. Bar = 650/~rn.
of dentate granule cells (Fig. lb) but low although detectable in CA3 and CA1. Thus, there is basal expression of the inducible IEGs Jun, Krox 24, and Fos-related antigens but not Fos in the rat hippocampus. FRAs, but not Fos itself, were expressed in a few scattered nuclei in the dentate granule cells; Jun was expressed at high levels in granule cell nuclei, and Krox 24 was expressed at high levels in the nuclei of neurons in CA1 and the subiculum. The immunohistochemical results for Krox 24 are in agreement with previous results of the localization of zif/268 m R N A in rat hippocampus ~6'2z and NGF-I-A protein in rat hippocampus 11. The presence of constitutively expressed I E G proteins (IEGPs) in hippocampal neurons raises a number of important questions. First, what are the inducing stimuli? Second, are the constitutively expressed IEGPs constantly regulating gene expression? Thirdly, what are the target genes that are being constitutively regulated? The differential anatomical expression of the different IEGPs (e.g. Jun high in granule cells, Krox 24 high in CA1 and subiculum) further complicates matters. IEGPs are believed to act as inducible genetic switches, which are not normally expressed constitutively (as are other transcription factors activated post-translationally, e.g. CREB), but which act once their transcription has been induced by the appropriate stimulus. Perhaps constitutively expressed lEGs may be involved in controlling housekeeping genes stimulated by tonic messengers, such as glutamate through N-methylD-aspartate (NMDA) receptors. The genes activated basally may include those genes that sustain neuronal cell functioning and viability, while the novel messengers arriving at the surface of the cell, which activate IEGs rapidly and transiently, work to modulate the transcriptional activity of other genes that change the phenotype of the cell depending on the information carried by the messenger. Alternatively, perhaps l E G proteins present basally are not active, and it may be that activation of basaUy present l E G proteins requires post-translational modifications (phosphorylation, dernethylation, etc.), or that it is not the absolute concentrations of I E G P within cells that is important, but dynamic changes that occur in their levels, that mediate cell surface signal transduction induced changes in m R N A synthesis. Another possibility is that the basal lEGs have no activity until they pair with other induced cellular factors or other IEGs. This may be the case with the basal Jun levels seen in the rat hippocampus. Although Jun is able to bind to D N A as a homodimer, and this may well occur basally, its affinity for the D N A AP-1 binding site would however be significantly increased upon Fos induction within the same cells, because of the greater affinity of the Fos-
357 Jun c o m p a r e d with the Jun-Jun transcription factor complex for this site 14. Within the rat h i p p o c a m p u s , although basal K r o x 24 expression in d e n t a t e granule cells is low, there is strong basal expression of Krox 24 in nuclei of neurons in CA1 and the subiculum. If K r o x 24 plays a role as an inducible transcription factor in granule cells to maintain synaptic plasticity, as suggested by recent studies 3'15 does its constitutive expression in C A 1 , m e a n that in this area Krox 24 is playing a similar role? P r o l o n g e d anaesthesia has been shown to greatly reduce basal K r o x 24 expression in the brain 15. This suggests that turnover of K r o x 24 in regions showing basal expression is still occurring with a similar time course to the turnover of LTP induced expression in granule cells. P e r h a p s K r o x 24 acts as a transcriptionally-activated transcription factor in d e n t a t e gyrus granule cells, but as a basally expressed transcription factor in neurons of CA1 and the subiculum. In these neurons, perhaps post-translational modifications, such as p h o s p h o r y l a t i o n (as for C R E B ) o r d e m e t h y l a t i o n as occurs in some i m m u n e cells 17 are required for Krox 24 activation.
1 Changelian, P.S., Feng, E, King, T.C. and Milbrandt, J., Structure of the NGFI-A gene and detection of upstream sequences responsible for its transcriptional induction by nerve growth factor, Proc. Natl. Acad. Sci. USA, 86 (1989) 377-381. 2 Christy, B.A., Lau, L.E and Nathans, D., A gene activated in mouse 3T3 cells by serum growth factors encodes a protein with 'zinc finger' sequences, Proc. Natl. Acad. Sci. USA, 85 (1988) 7857-7861. 3 Cole, A.J., Saffen, D.W., Baraban, J.M. and Worley, P.E, Rapid increase of an immediate early gene messenger RNA in hippocampal neurons by synaptic NMDA receptor activation, Nature, 340 (1989) 474. 4 Curran, T. and Morgan, J.I., Memories of los, Bioessays, 7 (1987) 255-258. 5 Dragunow, M., Currie, R.W., Faull, R.L.M., Robertson, H.A. and Jansen, K., Immediate-early genes, kindling and long-term potentiation, Neurosci. Biobehav. Rev., 13 (1989) 301-313. 6 Dragunow, M., de Castro, D. and Faull, R.L.M., Induction of Fos in glia-like cells after focal brain injury but not during wallerian degeneration, Brain Res., 527 (1990) 41-54. 7 Dragunow, M. and Robertson, H.A., Localization and induction of c-los protein-like immunoreactive material in the nuclei of adult mammalian neurons, Brain Res., 440 (1988) 252-260. 8 Gunn, A.J., Dragunow, M., Faull, R.L.M. and Gluckman, P.D., Effects of hypoxia-ischemia and seizures on neuronal and glial-like c-los protein levels in the infant rat, Brain Res., 531 (1990) 105-116. 9 Lamaire, P., Revelant, O., Bravo, R. and Charnay, P., Two mouse genes encoding potential transcriptional factors with identical DNA-binding domains are activated by growth factors in cultured cell, Proc. Natl. Acad. Sci. USA, 85 (1988) 46914695. 10 Leah, J.D., Herdegen, T. and Bravo, R., Selective expression of Jun proteins following peripheral axotomy and axonal transport block in the rat: evidence for a role in the regeneration process, Brain Res., 566 (1991) 198-207. 11 Mack, K., Day, M., Milbrandt, J. and Gottlieb, D.I., Localization of the NGFI-A protein in the rat brain, MoL Brain Res., 8 (1990) 177-180.
Finally, since basal expression of the I E G s , Jun and K r o x 24 within areas of the rat h i p p o c a m p u s occurs differentially, significantly Jun in dentate, Krox 24 in CA1 and subiculum, and Fos not at all in any rat hippocampal area, this suggests that either the basal extracellular signal(s) that induces the expression of basal K r o x 24 levels within CA1 and subiculum is different from that found to induce the expression of Jun basal levels in the dentate, or that different induction pathways for Jun and Krox 24 m a y exist in different cells within given regions of the rat hippocampus. Alternatively, p e r h a p s the degree of first and/or second messenger activation determines the transcription factor(s) activated. In conclusion, results demonstrating basal I E G protein expression within the rat h i p p o c a m p u s raise fundamental questions about the regulation and function of constitutively expressed I E G P s in neurons.
We are greateful to R. Bravo for the gift of the Krox 24 antiserum and to J. Leah for the Jun antiserum. This work was supported by grants from the New Zealand Medical Research Council and Lottery Board, and the NZ Neurological Foundation.
12 Morgan, J.I., Cohen, D.R., Hempstead, J.L. and Curran, T., Mapping patterns of c-los expression in the central nervous system after seizure, Science, 237 (1987) 192-197. 13 Morgan, J.I. and Curran, T., Stimulus-transcription coupling in the nervous system: involvement of the inducible proto-oncogenes los and jun, Annu. Rev. Neurosci., 14 (1991) 421-451. 14 Nakabeppu, Y., Ryder, K. and Nathans, D., DNA binding activities of three murine jun proteins: stimulation by fos, Cell, 55 (1988) 907-915. 15 Richardson, C.L., Tate, W.P., Mason, S.E., Lawlor, P.A., Dragunow, M. and Abraham, W.C., Correlation between the induction of an immediate early gene, zif/268, and long-term potentiation in the dentate gyms, Mol. Brain Res., in press. 16 Schlingensiepen, K.-H., Ltino, K. and Brysch, W., High basal expression of the zif/268 immediate early gene in cortical layers IV and VI, in CA1 and in the corpus striatum -- an in situ hybridization study, Neurosci. Lett., 122 (1991) 67-70. 17 Seyfert, V.L., McMahon, S.B., Glenn, W.D., Yellen, A.J., Sukhatme, V.P., Cao, X. and Monroe, J.G., Methylation of an immediate-early inducible gene as a mechanism for B cell tolerance induction, Science, 250 (1990) 797-799. 18 Sheng, M. and Greenberg, M.E., The regulation and function of c-los and other immediate early genes in the nervous system, Neuron, 4 (1990) 477-485. 19 Sukhatme, V.P., Cao, Z., Chang, L.C., Tsai-Morris, C.H., Stamenkovich, D., Ferreira, EC.P., Cohen, D.R., Edwards, S.A., Shows, T.B., Curran, T., Le Beau, M.M. and Adamson, E.D., A zinc finger-encoding gene coregulated with c-fos during growth and differentiation, and after cellular depolarization, Cell, 53 (1988) 37-43. 20 Tippetts, M.T., Varnum, B.C., Lim, R.W. and Herschman, H.R., Tumor promoter-inducible genes are differentially expressed in the developing mouse, Mol. Cell Biol., 8 (1988) 4570-4572. 21 Worley, P.E, Christy, B.A., Nakabeppu, Y., Bhat, R.V., Cole, A.J. and Baraban, J.M., Constitutive expression of zif268 in neocortex is regulated by synaptic activity, Proc. Natl. Acad. Sci. USA, 88 (1991) 5106-5110.
355
BRESM 80123
Basal expression of Fos, Fos-related, Jun, and Krox 24 proteins in rat hippocampus P. Hughes, P. Lawlor and M. Dragunow Department of Pharmacology and Clinical Pharmacology, University of Auckland School of Medicine, Private Bag, Auckland (New Zealand)
(Accepted 17 December 1991) Key words: Transcription factor; Limbic system; Memory
The basal expression of the protein products of the inducible immediate early genes (IEGs), Fos, Jun, and Krox 24, was investigated in rat hippocampus using immunocytochemical visualization methods with antisera specific for Fos only, Fos and the Fos-related antigens (FRAs), the Jun family, and Krox 24 (previously described as TIS 8, egr-1, NGF-IA or zif 268). In the normal adult rat brain basal levels of Jun, Krox 24 and Fos-related antigens but not Fos were seen within the hippocampus. More specificallyvery high basal levels of Jun were seen in the dentate granule cells with high basal Krox 24 levels seen in the CAl-subiculum region of the rat hippocampus. Basal FRAs but not Fos-positive cells were seen at low levels in the dentate granule cells. The implications of these results to the functioning of IEG proteins in hippocampal neurons is discussed. Fos, Jun, and Krox 24 are transcription factors that interact with specific regulatory regions within D N A to modulate the transcriptional activity of various, so-called, 'late-response genes '4'5'7'12'13'18. They are highly inducible genes, being induced rapidly and transiently in a protein synthesis independent manner by a large array of documented agents acting through differential second messenger pathways depending on the cell type and inducing agent used 18. Within the CNS their induction could be linked to events that control long-term change in the CNS. They have thus been implicated in long-term potentiation (LTP) and the changes associated with kindling 5. They may also play a role in CNS pathology as well as plasticity and are induced in a number of models of brain injury 6'8. Fos and Jun and their structurally related family members can form many permutations of leucine zipper-mediated dimers to interact with DNA. The more recently characterized Krox 249 also known as NGFI-A 1, TIS 82°, Egr-119, and zif 2682 interacts with D N A using a highly common and conserved structural motif, the zinc finger binding domain. Here we investigate the basal expression of these inducible immediate early gene (IEG) protein products within the hippocampus of the rat, a structure implicated in learning and memory. Male Wistar rats were deeply anaesthetized with sodium pentobarbital and perfused transcardially with isotonic saline followed by 4% paraformaldehyde in 0.1 M
phosphate buffer (pH 7.4). Brains were removed and left in the fixative for approximately 48 h, and then cut coronally on a vibroslice machine at 70 /~m. Sections were then incubated for 10 min in 1% hydrogen peroxide made up in 100% methanol to remove endogenous peroxidase staining and enhance antibody penetration into the section. Sections were then washed with phosphate-buffered saline (PBS) for a further 10 min before rabbit antiserum to the various IEG products was added to the sections. The antisera used were: (1) PCO5 Fos antibody -- specific for Fos and Fos-related antigens (FRAs) (Oncogene Science); (2) 456 Fos -- specific for Fos only (Medac-Genentechnologie); (3) Krox 24 -(generously provided by R. Bravo); (4) Jun -- specific for the Jun family members c-Jun, Jun B, and Jun D (generously provided by J. Leahl°). The antisera were made up in immunobuffer in the following dilutions 1:500, 1:500, 1:50,000, 1:20,000, respectively. These primary antibodies were allowed to remain on the sections incubated with gentle agitation, at 4°C for 72 h upon which the primary antibody solution was removed and replaced by a biotinylated anti-rabbit secondary antibody which was left overnight. The reaction was visualized using avidin-biotin-horseradish peroxidase and the chromogen diaminobenzidine (DAB), as described previously7. In the rat hippocampus scattered FRA-positive neuronal nuclei were detected in the dentate gyrus in sec-
Correspondence: M. Dragunow, Department of Pharmacologyand Clinical Pharmacology, University of Auckland School of Medicine, Private Bag, Auckland, New Zealand.
356 tions stained with the PCO5 antiserum (Fig. lc). No immunostaining was seen in CA1 or CA3, and since the 456 Fos antibody showed no basal staining within the hippocampus, the low positive staining of certain dentate gyrus granule cell nuclei was probably due to low constitutive expression of Fos-related antigens. Both Junand Krox-24 immunopositive neuronal nuclei were detected in rat hippocampus (Fig. la,b). The Krox 24 basal expression was relatively low in the dentate granule cells, higher in CA3, and very high in CA1 and subiculum (Fig. la). Jun immunoreactivity was high in the nuclei
Fig. 1. Basal expression of Krox 24 (a), Jun (b) and FRAs (c) in rat hippocampus. Bar = 650/~rn.
of dentate granule cells (Fig. lb) but low although detectable in CA3 and CA1. Thus, there is basal expression of the inducible IEGs Jun, Krox 24, and Fos-related antigens but not Fos in the rat hippocampus. FRAs, but not Fos itself, were expressed in a few scattered nuclei in the dentate granule cells; Jun was expressed at high levels in granule cell nuclei, and Krox 24 was expressed at high levels in the nuclei of neurons in CA1 and the subiculum. The immunohistochemical results for Krox 24 are in agreement with previous results of the localization of zif/268 m R N A in rat hippocampus ~6'2z and NGF-I-A protein in rat hippocampus 11. The presence of constitutively expressed I E G proteins (IEGPs) in hippocampal neurons raises a number of important questions. First, what are the inducing stimuli? Second, are the constitutively expressed IEGPs constantly regulating gene expression? Thirdly, what are the target genes that are being constitutively regulated? The differential anatomical expression of the different IEGPs (e.g. Jun high in granule cells, Krox 24 high in CA1 and subiculum) further complicates matters. IEGPs are believed to act as inducible genetic switches, which are not normally expressed constitutively (as are other transcription factors activated post-translationally, e.g. CREB), but which act once their transcription has been induced by the appropriate stimulus. Perhaps constitutively expressed lEGs may be involved in controlling housekeeping genes stimulated by tonic messengers, such as glutamate through N-methylD-aspartate (NMDA) receptors. The genes activated basally may include those genes that sustain neuronal cell functioning and viability, while the novel messengers arriving at the surface of the cell, which activate IEGs rapidly and transiently, work to modulate the transcriptional activity of other genes that change the phenotype of the cell depending on the information carried by the messenger. Alternatively, perhaps l E G proteins present basally are not active, and it may be that activation of basaUy present l E G proteins requires post-translational modifications (phosphorylation, dernethylation, etc.), or that it is not the absolute concentrations of I E G P within cells that is important, but dynamic changes that occur in their levels, that mediate cell surface signal transduction induced changes in m R N A synthesis. Another possibility is that the basal lEGs have no activity until they pair with other induced cellular factors or other IEGs. This may be the case with the basal Jun levels seen in the rat hippocampus. Although Jun is able to bind to D N A as a homodimer, and this may well occur basally, its affinity for the D N A AP-1 binding site would however be significantly increased upon Fos induction within the same cells, because of the greater affinity of the Fos-
357 Jun c o m p a r e d with the Jun-Jun transcription factor complex for this site 14. Within the rat h i p p o c a m p u s , although basal K r o x 24 expression in d e n t a t e granule cells is low, there is strong basal expression of Krox 24 in nuclei of neurons in CA1 and the subiculum. If K r o x 24 plays a role as an inducible transcription factor in granule cells to maintain synaptic plasticity, as suggested by recent studies 3'15 does its constitutive expression in C A 1 , m e a n that in this area Krox 24 is playing a similar role? P r o l o n g e d anaesthesia has been shown to greatly reduce basal K r o x 24 expression in the brain 15. This suggests that turnover of K r o x 24 in regions showing basal expression is still occurring with a similar time course to the turnover of LTP induced expression in granule cells. P e r h a p s K r o x 24 acts as a transcriptionally-activated transcription factor in d e n t a t e gyrus granule cells, but as a basally expressed transcription factor in neurons of CA1 and the subiculum. In these neurons, perhaps post-translational modifications, such as p h o s p h o r y l a t i o n (as for C R E B ) o r d e m e t h y l a t i o n as occurs in some i m m u n e cells 17 are required for Krox 24 activation.
1 Changelian, P.S., Feng, E, King, T.C. and Milbrandt, J., Structure of the NGFI-A gene and detection of upstream sequences responsible for its transcriptional induction by nerve growth factor, Proc. Natl. Acad. Sci. USA, 86 (1989) 377-381. 2 Christy, B.A., Lau, L.E and Nathans, D., A gene activated in mouse 3T3 cells by serum growth factors encodes a protein with 'zinc finger' sequences, Proc. Natl. Acad. Sci. USA, 85 (1988) 7857-7861. 3 Cole, A.J., Saffen, D.W., Baraban, J.M. and Worley, P.E, Rapid increase of an immediate early gene messenger RNA in hippocampal neurons by synaptic NMDA receptor activation, Nature, 340 (1989) 474. 4 Curran, T. and Morgan, J.I., Memories of los, Bioessays, 7 (1987) 255-258. 5 Dragunow, M., Currie, R.W., Faull, R.L.M., Robertson, H.A. and Jansen, K., Immediate-early genes, kindling and long-term potentiation, Neurosci. Biobehav. Rev., 13 (1989) 301-313. 6 Dragunow, M., de Castro, D. and Faull, R.L.M., Induction of Fos in glia-like cells after focal brain injury but not during wallerian degeneration, Brain Res., 527 (1990) 41-54. 7 Dragunow, M. and Robertson, H.A., Localization and induction of c-los protein-like immunoreactive material in the nuclei of adult mammalian neurons, Brain Res., 440 (1988) 252-260. 8 Gunn, A.J., Dragunow, M., Faull, R.L.M. and Gluckman, P.D., Effects of hypoxia-ischemia and seizures on neuronal and glial-like c-los protein levels in the infant rat, Brain Res., 531 (1990) 105-116. 9 Lamaire, P., Revelant, O., Bravo, R. and Charnay, P., Two mouse genes encoding potential transcriptional factors with identical DNA-binding domains are activated by growth factors in cultured cell, Proc. Natl. Acad. Sci. USA, 85 (1988) 46914695. 10 Leah, J.D., Herdegen, T. and Bravo, R., Selective expression of Jun proteins following peripheral axotomy and axonal transport block in the rat: evidence for a role in the regeneration process, Brain Res., 566 (1991) 198-207. 11 Mack, K., Day, M., Milbrandt, J. and Gottlieb, D.I., Localization of the NGFI-A protein in the rat brain, MoL Brain Res., 8 (1990) 177-180.
Finally, since basal expression of the I E G s , Jun and K r o x 24 within areas of the rat h i p p o c a m p u s occurs differentially, significantly Jun in dentate, Krox 24 in CA1 and subiculum, and Fos not at all in any rat hippocampal area, this suggests that either the basal extracellular signal(s) that induces the expression of basal K r o x 24 levels within CA1 and subiculum is different from that found to induce the expression of Jun basal levels in the dentate, or that different induction pathways for Jun and Krox 24 m a y exist in different cells within given regions of the rat hippocampus. Alternatively, p e r h a p s the degree of first and/or second messenger activation determines the transcription factor(s) activated. In conclusion, results demonstrating basal I E G protein expression within the rat h i p p o c a m p u s raise fundamental questions about the regulation and function of constitutively expressed I E G P s in neurons.
We are greateful to R. Bravo for the gift of the Krox 24 antiserum and to J. Leah for the Jun antiserum. This work was supported by grants from the New Zealand Medical Research Council and Lottery Board, and the NZ Neurological Foundation.
12 Morgan, J.I., Cohen, D.R., Hempstead, J.L. and Curran, T., Mapping patterns of c-los expression in the central nervous system after seizure, Science, 237 (1987) 192-197. 13 Morgan, J.I. and Curran, T., Stimulus-transcription coupling in the nervous system: involvement of the inducible proto-oncogenes los and jun, Annu. Rev. Neurosci., 14 (1991) 421-451. 14 Nakabeppu, Y., Ryder, K. and Nathans, D., DNA binding activities of three murine jun proteins: stimulation by fos, Cell, 55 (1988) 907-915. 15 Richardson, C.L., Tate, W.P., Mason, S.E., Lawlor, P.A., Dragunow, M. and Abraham, W.C., Correlation between the induction of an immediate early gene, zif/268, and long-term potentiation in the dentate gyms, Mol. Brain Res., in press. 16 Schlingensiepen, K.-H., Ltino, K. and Brysch, W., High basal expression of the zif/268 immediate early gene in cortical layers IV and VI, in CA1 and in the corpus striatum -- an in situ hybridization study, Neurosci. Lett., 122 (1991) 67-70. 17 Seyfert, V.L., McMahon, S.B., Glenn, W.D., Yellen, A.J., Sukhatme, V.P., Cao, X. and Monroe, J.G., Methylation of an immediate-early inducible gene as a mechanism for B cell tolerance induction, Science, 250 (1990) 797-799. 18 Sheng, M. and Greenberg, M.E., The regulation and function of c-los and other immediate early genes in the nervous system, Neuron, 4 (1990) 477-485. 19 Sukhatme, V.P., Cao, Z., Chang, L.C., Tsai-Morris, C.H., Stamenkovich, D., Ferreira, EC.P., Cohen, D.R., Edwards, S.A., Shows, T.B., Curran, T., Le Beau, M.M. and Adamson, E.D., A zinc finger-encoding gene coregulated with c-fos during growth and differentiation, and after cellular depolarization, Cell, 53 (1988) 37-43. 20 Tippetts, M.T., Varnum, B.C., Lim, R.W. and Herschman, H.R., Tumor promoter-inducible genes are differentially expressed in the developing mouse, Mol. Cell Biol., 8 (1988) 4570-4572. 21 Worley, P.E, Christy, B.A., Nakabeppu, Y., Bhat, R.V., Cole, A.J. and Baraban, J.M., Constitutive expression of zif268 in neocortex is regulated by synaptic activity, Proc. Natl. Acad. Sci. USA, 88 (1991) 5106-5110.
