Nurr1 mRNA expression in neonatal and adult rat brain following kainic acid-induced seizure activity

Nurr1 mRNA expression in neonatal and adult rat brain following kainic acid-induced seizure activity

Molecular Brain Research 59 Ž1998. 178–188 Research report Nurr1 mRNA expression in neonatal and adult rat brain following kainic acid-induced seizu...

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Molecular Brain Research 59 Ž1998. 178–188

Research report

Nurr1 mRNA expression in neonatal and adult rat brain following kainic acid-induced seizure activity Marianna Crispino

a,)

, Georges Tocco a , Jonathan D. Feldman b, Harvey R. Herschman c , Michel Baudry a

a

c

Neuroscience Program, Hedco Neuroscience Building, Room 311, UniÕersity of Southern California, Los Angeles, CA 90089-2520, USA b Department of Pediatrics, UCLA Center for the Health Sciences, Los Angeles, CA 90095-1570, USA Department of Biological Chemistry and Department of Molecular and Medical Pharmacology, and Molecular Biology Institute, UCLA Center for the Health Sciences, Los Angeles, CA 90095-1570, USA Accepted 9 June 1998

Abstract Nurr1 is an immediate early gene encoding a member of the steroid–thyroid hormone receptor family. In PC12 cells, Nurr1 is readily induced by membrane depolarization, but not by growth factors. Nurr1 is predominantly expressed in the brain, and is essential to the differentiation of midbrain dopaminergic neurons. However, Nurr1 is also expressed in brain regions unrelated to dopaminergic neurons, e.g., hippocampus and cerebral cortex, and its immediate induction following seizure activity suggests a potential involvement of this transcription factor in modulating gene expression in the nervous system. To investigate the response of Nurr1 to neuronal activation, we analyzed Nurr1 mRNA expression in neonatal and adult rat brain following kainic acid ŽKA.-induced seizure. In P7 animals, systemic injection of KA increased Nurr1 mRNA levels in a few hilar cells of the dentate gyrus and some pyramidal cells of the CA3 region of the hippocampus. In older animals, Nurr1 induction progressively expanded to all hippocampal regions ŽP14, P21. and eventually to cortical regions Žadult.. The increase was rapid and transient in the dentate gyrus, a structure resistant to the neurotoxic effect of KA, and was more prolonged in other regions more susceptible to KA toxicity. Induction of Nurr1 at early postnatal stages and rapid increase in the dentate gyrus following KA-induced seizure, suggest that Nurr1 expression is modulated by neuronal activity. On the other hand, prolonged Nurr1 induction in regions sensitive to KA toxicity indicates a possible involvement of Nurr1 in selective neuronal vulnerability. q 1998 Elsevier Science B.V. All rights reserved. Keywords: Nurr1; mRNA; Kainic acid; Seizure; Hippocampus; In situ hybridization

1. Introduction The nuclear receptor super family refers to transcription factors that regulate gene expression in response to a variety of chemical signals w7,20,34x. The nuclear receptor super family has been classified into two groups. Ligandactivated members comprise receptors for specific ligands such as retinoic acid, steroid and thyroid hormones w1,7x. The other members of the family are so-called orphan receptors, termed ‘orphan’ because their gene activation mechanism and cognate ligands, if any, are unknown w13,17,20x. Nurr1 belongs to the orphan receptor subfamily. The deduced amino acid sequence of mouse Nurr1 corresponds to a 66 kDa protein containing a DNA-bind) Corresponding author. [email protected]

Fax:

q 1-213-740-5687;

E-mail:

0169-328Xr98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 9 - 3 2 8 X Ž 9 8 . 0 0 1 4 3 - 0

ing domain with zinc finger structural motifs w12,23x. The amino acid sequence of Nurr1 DNA-binding domain exhibits over 92% homology to the same domain of Nur77, a closely related orphan receptor, and this structural homology was the basis for naming Nurr1, i.e., Nur-related factor 1 w12x. Both Nurr1 and Nur77 receptors are encoded by immediate early genes whose expression is readily induced by depolarization in PC12 cells, a rat pheocromocytoma cell line often used as a neuronal model system w10x. However, Nurr1 and Nur77 exhibit different patterns of expression, as Nurr1 gene is not activated by nerve growth factor in PC12 while Nur77 is. Furthermore, whereas Nur77 is widely expressed in most tissues including brain, ovary, testis, muscle and submaxillary gland, Nurr1 is highly expressed in the brain and only moderately in the lung and testis w12,33x. Thus, despite their structural similarity, Nurr1

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and Nur77 may play distinct roles in transcriptional regulation of gene expression in brain. Nurr1 expression was recently found to be essential to the differentiation of midbrain dopaminergic neurons in the developing brain. In adult, Nurr1 helps to maintain the dopaminergic phenotype by regulating the production of an adequate amount of dopamine w36x. On the other hand, Nurr1 mRNA is present in brain regions unrelated to dopaminergic neurons, e.g., the cerebral cortex and the pyramidal cell layers of hippocampus w32,35x, and Nurr1 biological function in these regions is not known. Nurr1 mRNA level in rat brain was recently shown by dot-blot analysis to increase after lindane-induced seizures w19x, and Nurr1 expression was found to be differentially regulated in the dentate gyrus by electroconvulsive seizures and kindling w33x. These observations have led to the suggestion that this orphan receptor may play a role in synaptic plasticity of the nervous system by mediating activity-dependent gene regulation. To further characterize the potential involvement of Nurr1 in synaptic plasticity, we analyzed the regional distribution and time course of Nurr1 expression in developing and adult rat brain following kainic acid ŽKA.-induced seizure activity. Our data demonstrate a transient and region-specific induction of Nurr1 mRNA following seizure activity in adult and neonatal rat brain, suggesting that Nurr1 is involved in regulating gene expression following intense synaptic activation.

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2. Materials and methods 2.1. Kainate treatment and preparation of brain sections Neonatal, juvenile ŽP7, 14, 21., and adult male Sprague–Dawley rats were maintained in a 12 h light–dark cycle and received food and water ad libitum. Systemic administration of KA was performed by subcutaneous injection with doses ranging from 2 to 10 mgrkg b.wt to compensate for age-related alterations in brain uptake w4x. Animals were sacrificed by decapitation following methoxyflurane anesthesia at different time points after seizure onset Ž1, 4, 8, 16, 30 and 120 h.. Brains were dissected and rapidly frozen in y208C isopentane for subsequent cryosectioning Ž10 mm coronal section.. Brain sections were mounted on microscope slides and stored at y708C until used. 2.2. Probe labeling Nurr1 mRNA-specific oligonucleotides corresponding to antisense Ž5X-TTTGTTTTGTAGCTCTTCCACTCTCTTGGGTTCCTTGAG-3X ; nucleotides 1632–1594. and sense sequence Ž5X-CCACCAGCAATAATTGACAAACTTTTCCTGGACACCTTAC-3X ; nucleotides 1792–1831. were selected from the rat Nurr1 cDNA sequence w18x. Oligonucleotide probes were end-labeled using terminal deoxynucleotidyl transferase ŽPromega, Madison, WI. in the presence of a-w35 Sxthio-dATP ŽNEN, Boston, MA;

Fig. 1. Changes in Nurr1 mRNA expression in brain regions during development. Groups of animals were sacrificed at various developmental ages and processed for in situ hybridization. Autoradiograms similar to those shown in Fig. 2 and Fig. 7 were analyzed as described in Section 2. Data were expressed as Relative Optical Densities ŽROD. and represent means" S.E.M. of 3–5 animalsrgroup. )Significantly different from adult group Ž P - 0.05.. CA1: pyramidal cell layer of the CA1 region of the hippocampus; CA3: pyramidal cell layer of the CA3 region of the hippocampus; DG: granule cell layer of the dentate gyrus; CTX: cortical layers overlying the hippocampus; VI: deep cortical layers; PIR: piriform cortex.

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specific activity 1250 Cirmmol. at 378C for 1 h. The incorporation ratio was between 70 and 80%. As a negative control, some slides were hybridized in the presence of 100-fold excess unlabeled probe. 2.3. In situ hybridization Frozen sections were briefly thawed and fixed in 4% Žwrv. paraformaldehyde in phosphate buffered saline ŽPBS. pH 7.4, for 30 min at room temperature ŽRT.. After three washes in PBS Ž10 min each., sections were dehy-

drated in increasing concentrations of ethanol and air-dried. As a negative control, some slides were treated with RNAse A Ž20 mgrml RNAse in 0.5 M NaCl, 10 mM Tris–Cl pH 8, 1 mM EDTA. at 378C for 30 min, prior to hybridization. Probes were diluted to 5000 c.p.m.rml in hybridization buffer Ž50% formamide, 4 = SSC, 5 = Denhardts solution, 1% SDS, 10% dextran sulfate, 0.1 M DTT, 25 mgrml poly ŽA., 25 mgrml poly ŽC. and 0.25 mgrml tRNA.. To each slide, 60 ml of radioactive hybridization solution was applied and covered with a parafilm strip. Hybridization was performed overnight at

Fig. 2. Nurr1 mRNA expression at various times following KA-induced seizure activity in animals of different postnatal ages. Groups of animals at postnatal ages of 7, 14 and 21 days received s.c. injection of kainic acid and were sacrificed 4 and 16 h, and 5 days after seizure onset. Brains were processed for in situ hybridization as described in Section 2. The figure depicts representative autoradiograms of in situ hybridization with w35 Sx-Nurr1 oligonucleotide probe on coronal sections of the brains from untreated animal Žcontrol. and animals sacrificed 4 and 16 h, and 5 days after seizure onset. Top row: brains of animals at postnatal day 7; middle row: brains of animal at postnatal day 14; bottom row: brains of animals at postnatal day 21. Arrowhead: cortical layers overlying hippocampus; arrow: deep cortical layers.

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448C in a humidified chamber. Sections were washed in 4 = SSC at RT for 20 min, 2 = SSC at RT for 3–4 h, and a high stringency wash was carried out in 0.2 = SSC at 558C for 30 min. Sections were dehydrated through graded ethanol concentrations containing 0.3 M ammonium acetate and air-dried before being exposed to Hyperfilm bmax ŽAmersham, Arlington Heights, IL.. After 10 days, films were developed and autoradiograms were analyzed. Specificity of hybridization was demonstrated by the observation that signals were abolished when 100-fold excess of unlabeled probe was added to the hybridization solution, when sections were pretreated with RNAse A, and when the sense sequence oligonucleotide was used as a probe Ždata not shown.. Sections were dipped in photographic emulsion ŽLM-1, Amersham., developed after 30 days, and subsequently counter-stained with hematoxylin–eosin.

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3. Results 3.1. Nurr1 expression during deÕelopment We analyzed changes in Nurr1 mRNA levels in control animals during the postnatal period from P7 to P60 Žadult. using in situ hybridization ŽFig. 1.. In the hippocampal formation ŽCA1, CA3 and DG., Nurr1 mRNA levels remained constant from P7 to P21, but decreased significantly at P28 Ž65% of P7 values. and reached the lowest level Ž50% of P7 values. in the adult. In cortical areas ŽCTX, VI, and PIR., Nurr1 expression gradually decreased throughout development, reaching the lowest level in the adult. 3.2. Effect of KA treatment on Nurr1 expression during deÕelopment

2.4. Quantitation of autoradiograms Autoradiograms were quantitated using a computerized image analysis system ŽBrain software running on DUMAS system from Drexel University.. Relative optical densities ŽROD. were measured for CA1, CA3, dentate gyrus, cortical layers overlying hippocampus, deep cortical layers, and piriform cortex. Analysis of variance ŽANOVA. was performed with STATISTICA software ŽStatsoft, Tulsa OK. with respect to animal groups and brain regions, followed by post-hoc analyses with the LSD test. P values - 0.05 were considered statistically significant.

Animals at different developmental stages ŽP7, P14, and P21. were sacrificed at various time points Ž4, 8, 16, 30 and 120 h. following KA treatment, and changes in Nurr1 mRNA levels were determined in different brain regions by in situ hybridization. At P7, Nurr1 mRNA levels were not changed at early time points but 20–25% decreased, 16 h after seizure onset, in almost all regions analyzed ŽFigs. 2 and 3.. However, the decrease was statistically significant only for CA3. From 30 h to 5 days after KA treatment, Nurr1 expression levels returned to the control levels Ži.e., levels determined at P7. except for piriform cortex,

Fig. 3. Nurr1 mRNA expression at various times following KA-induced seizure activity in 7-day-old rats. Postnatal day seven rats received s.c. injection of kainic acid and were sacrificed 4, 8, 16 and 30 h, and 5 days after seizure onset. Brains were processed for in situ hybridization as described in Section 2. Autoradiograms similar to those shown in Fig. 2 were quantitatively analyzed. Results represent means" S.E.M. of 3–5 animalsrgroup, and are expressed as the ratios of ROD in KA-treated animals to ROD in control animals. )Significantly different from control group Ž P - 0.05.. CA1: pyramidal cell layer of the CA1 region of the hippocampus; CA3: pyramidal cell layer of the CA3 region of the hippocampus; DG: granule cell layer of the dentate gyrus; CTX: cortical layers overlying the hippocampus; VI: deep cortical layers; PIR: piriform cortex.

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where Nurr1 mRNA levels remained decreased. In addition, closer examination of the emulsion-dipped sections revealed a dramatic increase of Nurr1 mRNA levels in a few select pyramidal cells of CA3 and some scattered neurons in the hilus of the dentate gyrus 4 h after seizure onset ŽFig. 4.. This effect was transient and was not observed at any other time-points. At P14, Nurr1 mRNA levels were significantly increased in all hippocampal subfields 4 h after seizure onset ŽFigs. 2 and 5.. By 8 h after seizure onset, Nurr1 expression had returned to basal levels in every structure except for CA3 where it was still elevated. By 16 h, Nurr1 mRNA levels decreased in most brain structures, an effect that reached statistical significance in CA1 and piriform cortex 5 days after KA treatment. At P21, Nurr1 mRNA levels increased significantly in the hippocampus and piriform cortex 4 h after seizure onset ŽFigs. 2 and 6.. In contrast to P7 and P14, Nurr1 expression was still increased at 8 h in both CA1 and CA3.

However, by 16 h after seizure onset, Nurr1 expression returned to the control levels in every structure, and no significant decrease was observed in piriform cortex 5 days after KA treatment. 3.3. Effect of KA treatment on Nurr1 expression in adult rat brain In agreement with previous reports w32,35x, Nurr1 mRNA was widely expressed in hippocampus, piriform cortex, medial and lateral habenular nuclei, as well as in some layers of neocortex in control animals ŽFig. 7.. Nurr1 mRNA levels rapidly increased in all hippocampal fields and piriform cortex of adult rat brain following KA-induced seizure activity ŽFigs. 7 and 8.. The granule cells of the dentate gyrus exhibited the largest extent of induction Žabout 500% of control values. as early as 1 h after seizure onset. The induction was transient as mRNA levels returned to control levels by 16 h. In contrast, pyramidal

Fig. 4. Nurr1 mRNA expression in the hilus and pyramidal cells of CA3 region following KA-induced seizure activity in 7-day-old rats. Postnatal day seven rats received s.c. injection of kainic acid and were sacrificed 4 h after seizure onset. Brains were processed for in situ hybridization as described in Section 2. Emulsion autoradiography of w35 Sx-Nurr1 oligonucleotide hybridization was performed to evaluate cellular levels of Nurr1 mRNA expression. Arrows indicate some of the cells with elevated expression of Nurr1 mRNA. Scale bar: 100 mm.

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Fig. 5. Nurr1 mRNA expression at various times following KA-induced seizure activity in 14-day-old rats. Postnatal day 14 rats received s.c. injection of kainic acid and were sacrificed 4, 8, 16 and 30 h, and 5 days after seizure onset. Brains were processed for in situ hybridization as described in Section 2. Autoradiograms similar to those shown in Fig. 2 were quantitatively analyzed. Results represent means" S.E.M. of 3–5 animalsrgroup, and are expressed as the ratios of ROD in KA-treated animals to ROD in control animals Žcontrol.. )Significantly different from control group Ž P - 0.05.. CA1: pyramidal cell layer of the CA1 region of the hippocampus; CA3: pyramidal cell layer of the CA3 region of the hippocampus; DG: granule cell layer of the dentate gyrus; CTX: cortical layers overlying the hippocampus; VI: deep cortical layers; PIR: piriform cortex.

Fig. 6. Nurr1 mRNA expression at various times following KA-induced seizure activity in 21-day-old rats. Postnatal day 21 rats received s.c. injection of kainic acid and were sacrificed 4, 8, 16 and 30 h, and 5 days after seizure onset. Brains were processed for in situ hybridization as described in Section 2. Autoradiograms similar to those shown in Fig. 2 were quantitatively analyzed. Results represent means" S.E.M. of 3–5 animalsrgroup, and are expressed as the ratios of ROD in KA-treated animals to ROD in control animals Žcontrol.. )Significantly different from control group Ž P - 0.05.. CA1: pyramidal cell layer of the CA1 region of the hippocampus; CA3: pyramidal cell layer of the CA3 region of the hippocampus; DG: granule cell layer of the dentate gyrus; CTX: cortical layers overlying the hippocampus; VI: deep cortical layers; PIR: piriform cortex.

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Fig. 7. Nurr1 mRNA expression at various times following KA-induced seizure activity in adult animals. Groups of adult animals received s.c. injection of kainic acid and were sacrificed 4 and 16 h, and 5 days after seizure onset. Brains were processed for in situ hybridization as described in Section 2. The figure depicts representative autoradiograms of in situ hybridization with w35 Sx-Nurr1 oligonucleotide probe on coronal sections of the brains from untreated animal Žcontrol. and animals sacrificed 4 and 16 h, and 5 days after seizure onset.

cells in CA1 and CA3 as well as neurons in piriform cortex exhibited a more prolonged increase in Nurr1 expression, as Nurr1 mRNA levels were still significantly elevated in these structures 16 h after seizure onset ŽFig. 8B.. In addition, a small increase in Nurr1 expression was observed in deep cortical layers, 1 and 4 h after seizure onset, whereas Nurr1 mRNA levels in the cortex overlying the hippocampus did not significantly change following KA treatment. Interestingly, closer examination of emulsion-dipped sections revealed that pyramidal neurons with eosinophilic cytoplasm and condensed nuclei Ži.e., neurons with evident morphological damage. were still expressing large amounts of Nurr1 mRNA even 5 days after KA treatment ŽFig. 9.. We also verified that Nurr1 induction following KA-induced seizure was not due to the stress caused by the

hypodermic injection per se as animals injected with the vehicle alone did not exhibit any change in Nurr1 mRNA levels Ždata not shown..

4. Discussion 4.1. Nurr1 mRNA expression in the brain during postnatal deÕelopment Nurr1 expression is developmentally regulated in hippocampus and cortex of normal rat brain. Quantitative analysis of in situ hybridization signals in rat brains at different developmental stages indicated that Nurr1 mRNA levels decreased, in a region-specific manner, from early postnatal period to adulthood. In hippocampus, a marked

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Fig. 8. Quantitative analysis of changes in Nurr1 mRNA levels in various brain regions of adult animals following KA-induced seizure activity. Groups of adult rats received s.c. injection of KA and were sacrificed 1, 4, 8, 16 and 30 h, and 5 days after seizure onset. Brains were processed for in situ hybridization as described in Section 2. Autoradiograms similar to those shown in Fig. 7 were quantitatively analyzed. ŽA. Results represent means " S.E.M. of 4–8 animalsrgroup, and are expressed as the ratios of ROD in KA-treated animals to ROD in control animals. )Significantly different from control group Ž P - 0.05.. CA1: pyramidal cell layer of the CA1 region of the hippocampus; CA3: pyramidal cell layer of the CA3 region of the hippocampus; DG: granule cell layer of the dentate gyrus; CTX: cortical layers overlying the hippocampus; VI: deep cortical layers; PIR: piriform cortex. ŽB. Time course of Nurr1 expression following KA-induced seizure in three hippocampal regions of adult rat. Values Žmeans" S.E.M.. are plotted on a semi-logarithmic scale to stress the prolonged expression in CA1 and CA3 as opposed to dentate gyrus.

decrease in Nurr1 expression took place during the period between P21 and P28, whereas in cortical structures the decrease in Nurr1 mRNA occurred at later stages ŽFig. 1.. During postnatal development, neurons acquire mature and differentiated properties through a number of different mechanisms, including activity-dependent processes. High levels of Nurr1 expression during the early postnatal stages might be linked to processes of axonal elongation, synaptic maturation, and synaptic stabilization. The transition period between P21 and P28 corresponds to the weaning of the animals and to a metabolic shift from a mostly anaerobic to aerobic mechanism. Thus, it is conceivable that these events could modify the factorŽs. involved in the regulation of Nurr1 expression. Our results are at variance

with a previous report indicating that Nurr1 expression did not change in the hippocampus and cortical regions during postnatal development w35x. The reason for this discrepancy is not clear, but it could be due to differences in the method used for quantitative analysis. 4.2. Nurr1 mRNA induction in the deÕeloping rat brain following KA-induced seizures Systemic injection of KA leads to different effects in adult and neonatal rat brains. In adult rats, subcutaneous KA injection produces recurrent seizure activity that results in selective neuronal death in the limbic system. In contrast, animals younger than 3 weeks injected with KA

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Fig. 9. Increased Nurr1 mRNA expression in damaged pyramidal neurons 5 days following KA-induced seizure activity in adult rats. Adult rats received s.c. injection of KA and were sacrificed 5 days later. Brains were processed for in situ hybridization as described in Section 2. Emulsion autoradiography of w35 Sx-Nurr1 oligonucleotide hybridization was performed and the sections were counterstained with hematoxylin–eosin as described in Section 2. Section from the CA1 region of the hippocampus. Arrows indicate some of the eosinophilic cells expressing increased levels of Nurr1 mRNA. Scale bar: 20 mm.

undergo intense tonic–clonic seizures, but do not exhibit neuronal cell death w3,16,30x. KA-induced seizure is accompanied by increased neuronal activity in select regions of rat brain, as measured by EEG recording as well as by w14 Cx2-deoxyglucose Ž2DG. uptake autoradiography. In animals before P7, the increased 2DG utilization observed following KA-induced seizure is restricted to the CA3 and hilus regions of the hippocampus. On the other hand, KA-induced seizure activity in animals older than 1 week results in large increase of 2DG labeling in the granule cell layer of dentate gyrus, as well as in pyramidal cell layers of CA1 and CA3 regions w30x. Thus, KA-induced seizures at different developmental stages are characterized by increased neuronal activities in distinct regions of the hippocampal formation. The spatio-temporal induction of Nurr1 elicited by KA treatment in developing rat brain paralleled the changes in neuronal activity represented by 2DG uptake. As with 2DG labeling, after KA-induced seizures, Nurr1 mRNA induction at P7 was restricted to a few pyramidal cells in CA3 and some scattered cells in the hilus, whereas the induction of Nurr1 mRNA at P14 and P21 was much more widespread in the dentate gyrus and CA1 and CA3 subfields of the hippocampus. Moreover, KA treatment pro-

duces neuronal activation of the cortical structures only in animals older than P24 w30x, and we did not observe any significant KA-induced Nurr1 expression in cortical regions until after P21. Although KA-induced seizure activity in neonatal and juvenile rats does not result in neuronal damage, it produced a decrease in Nurr1 expression 5 days after the treatment ŽFigs. 3 and 5.. While the significance of this effect is not clear, it could be related to long-lasting consequences on brain development of seizure activity during the developmental period w11x. In any event, our results strongly suggest that Nurr1 expression in the hippocampus of neonatal brains is directly modulated by neuronal activity. This idea fits well with previous observations indicating that Nurr1 is induced by membrane depolarization in PC12 cells w12x. Screening PC12 cDNA library for genes differentially induced by depolarization and growth factors, a number of immediate early genes such as synaptotagmin IV, and PIM-1, have been identified. We found that these genes are induced by KA treatment in the adult rat brain ww31x, Feldman et al., unpublished observationsx. In contrast to Nurr1, however, synaptotagmin IV mRNA was not inducible by seizure until P14 w29x. Similarly, many other

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immediate early genes such as BDNF, c-fos and SGP-2 are not activated by seizure activity until the third week of postnatal development w6,24,25x. Thus, modulation of Nurr1 expression differs from that of a number of other immediate early genes, suggesting that Nurr1 plays a distinct role in linking neuronal activity to regulation of gene expression. 4.3. Nurr1 mRNA induction in adult rat brain following KA-induced seizures It was previously demonstrated that Nurr1 expression increased in the adult rat brain as early as 15 min after lindane-induced seizures, and remained elevated 4 h after seizure onset, as evidenced by dot-blot analysis w19x. A more recent study also indicated that Nurr1 expression was differentially regulated by electroconvulsive seizures and amygdala-kindled seizures. Whereas electroconvulsive seizures produced a rapid and transient induction in the dentate gyrus, amygdala-kindled seizures did not produce a consistent pattern of expression w33x. In the present study, we performed extensive and quantitative analyses of regional distribution and time course of Nurr1 expression in the adult rat brain following KA-induced seizure activity. Our in situ hybridization data demonstrate that Nurr1 induction takes place specifically in the hippocampal formation and piriform cortex. Nurr1 mRNA levels were elevated in these regions as early as 1 h after seizure onset, and returned to basal values by 30 h ŽFigs. 7 and 8.. These results, observed in autoradiographic films, were also confirmed at the cellular level by examination of emulsion-dipped sections. Interestingly, numerous neurons with obvious morphological damage at 30 h and 5 days after KA treatment, still exhibited elevated Nurr1 mRNA levels as compared to control neurons ŽFig. 9.. The rapid induction of Nurr1 following KA-induced seizure activity indicates that Nurr1 is not directly related to cell death, as neuronal damage takes place several hours or days after seizure onset. In addition, the increase in Nurr1 mRNA, 1 h after seizure onset, was more prevalent in the dentate gyrus than in the pyramidal cell layers of CA3 or CA1. As granule cells of the dentate gyrus are resistant to KA toxicity while pyramidal cells of hippocampal regions CA3 and CA1 are most vulnerable w2,14,15,27x, the early induction of Nurr1 is more likely related to seizure activity rather than to the neurotoxic effect of KA. This interpretation fits also well with the fact that Nurr1 is induced following KA treatment during the postnatal period when this treatment does not induce neuronal damage. However, it is also evident that Nurr1 induction is more prolonged in CA3 and CA1 than in the dentate gyrus and is present in damaged neurons. Similarly, mRNAs for other immediate early genes such as c-fos and synaptotagmin IV showed transient increases in areas resistant to KA toxicity Ži.e., dentate gyrus., and prolonged increases in vulnerable regions ŽCA1, CA3, and

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piriform cortex. w26,29x. It is thus conceivable that prolonged expression of Nurr1 in certain neuronal populations is linked to their vulnerability to insults. Several different groups have demonstrated that limbic seizures are able to modulate the expression of numerous genes in hippocampal neurons. Seizure activity, chemically- or electrically-induced, causes a rapid increase in the mRNAs for preproenkephalin and enkephalin peptide w8x, SGP-2 w25x, as well as some other immediate early genes ŽIEG. such as NGF w9x, BDNF w5x, c-fos w26,28x, NF-kB w21x, synaptotagmin IV w29,31x, and p53 w22x. It is plausible that the combinatorial nature of gene expression in each individual neuron determines the ultimate fate of neurons in the hippocampal formation. In this regard, the balance between induction of apoptotic and anti-apoptotic genes would be the critical determinant of neuronal survival. In conclusion, our results strongly support the hypothesis that the modulation of Nurr1 expression following KA-induced seizures in the neonatal and adult rat brain is due to enhanced neuronal activity rather than to the neurotoxic effect of KA. However, our data do not exclude a possible role for Nurr1 expression in the selective vulnerability of specific neuronal populations. Acknowledgements This research was supported by NINDS grant NS18427 and a grant from Sankyo Phamaceuticals to M.B., grant AR01870 to J.D.F. and grant NS28660 to H.R.H. M.C. was supported in part by a fellowship from University of Naples, Italy: Dottorato di ricerca in Neuroscienze. References w1x M. Beato, Gene regulation by steroid hormones, Cell 56 Ž1989. 335–344. w2x Y. Ben Ari, Limbic seizure and brain damage produced by kainic acid: mechanisms and relevance to human temporal lobe epilepsy, Neuroscience 14 Ž1985. 375–403. w3x M.L. Berger, E. Tremblay, L. Nitecka, Y. Ben Ari, Maturation of kainic acid seizure–brain damage syndrome in the rat: III. Postnatal development of kainic acid binding sites in the limbic system, Neuroscience 13 Ž1984. 1095–1104. w4x M.L. Berger, J.M. Lefauconnier, E. Tremblay, Y. Ben Ari, Y., Limbic seizure induced by systematically applied kainic acid: how much kainic acid reaches the brain, in: R. Schwarz, Y. Ben Ari ŽEds.., Kainic Acid-Induced Seizures, Plenum, New York, 1986, pp. 199–209. w5x M.M. Dugich-Djordjevic, G. Tocco, P.A. Lapchack, G.M. Pasinetti, I. Najm, M. Baudry, F. Hefti, Regionally specific and rapid increases in brain-derived neurotrophic factor messenger RNA in the adult rat brain following seizures induced by systemic administration of kainic acid, Neuroscience 47 Ž1992. 303–315. w6x M.M. Dugich-Djordjevic, G. Tocco, D.A. Willoughbly, I. Najm, G.M. Pasinetti, F.R. Thompson, M. Baudry, P.A. Lapchack, F. Hefti, BDNF mRNA expression in the developing rat brain following kainic acid-induced seizure activity, Neuron 8 Ž1992. 1127–1138. w7x R.M. Evans, The steroid and thyroid hormone receptor superfamily, Science 240 Ž1988. 889–895.

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M. Crispino et al.r Molecular Brain Research 59 (1998) 178–188

w8x C. Gall, J. Lauterborn, P. Isackson, J. White, Seizures, neuropeptide regulation, and mRNA expression in the hippocampus, Prog. Brain Res. 83 Ž1990. 371–390. w9x C. Gall, K. Murray, P.J. Isackson, Kainic acid-induced seizures stimulate increased expression of nerve growth factor mRNA in rat hippocampus, Mol. Brain Res. 9 Ž1991. 113–123. w10x L.A. Greene, A.S. Tischler, Establishment of a noradrenergic clonal line of rat adrenal pheocrhromocytoma cells which respond to nerve growth factor, Proc. Natl. Acad. Sci. U.S.A. 73 Ž1976. 2424–2428. w11x G.L. Holmes, The long-term effects of seizures on the developing brain: clinical and laboratory issues, Brain and Development 13 Ž1991. 393–409. w12x S.W. Law, O.M. Conneely, F.J. DeMayo, B.W. O’Malley, Identification of a new brain-specific transcription factor, Nurr1, Mol. Endocrinol. 6 Ž1992. 2129–2135. w13x S. Lopes da Silva, J.P.H. Burbach, The nuclear hormone-receptor family in the brain: classics and orphans, TINS 18 Ž1995. 542–548. w14x E.W. Lothman, R.C. Collins, Kainic acid induced limbic seizures: metabolic, behavioral, electroencephalographic and neuropathological correlates, Brain Res. 218 Ž1981. 299–318. w15x J.V. Nadler, Kainic acid as a tool for the study of temporal lobe epilepsy, Life Sci. 29 Ž1981. 2031–2042. w16x L. Nitecka, G. Tremblay, G. Charton, P. Bouillot, M.L. Berger, Y. Ben Ari, Maturation of kainic acid seizure–brain damage syndrome in the rat: II. Histopathological sequelae, Neuroscience 13 Ž1984. 1073–1094. w17x B.W. O’Malley, O.M. Conneely, Orphan receptor: in search of a unifying hypothesis for activation, Mol. Endocrinol. 6 Ž1992. 1359– 1361. w18x S. Pena de Ortiz, M. Cannon, G.A. Jamieson, Expression of nuclear hormone receptors within the rat hippocampus: identification of novel orphan receptors, Mol. Brain Res. 23 Ž1994. 278–283. w19x S. Pena de Ortiz, G.A. Jamieson, HZF-3, an immediate-early orphan receptor homologous to NURR1rNOT: induction upon membrane depolarization and seizure, Mol. Brain Res. 38 Ž1996. 1–13. w20x R.F. Power, O.M. Conneely, B.W. O’Malley, New insights into activation of the steroid hormone receptor superfamily, TIPS 13 Ž1992. 318–323. w21x Y. Rong, M. Baudry, Seizure activity results in a rapid induction of nuclear factor-kappaB in adult but not juvenile rat limbic structures, J. Neurochem. 67 Ž1996. 662–668. w22x S. Sakhi, A. Bruce, N. Sun, G. Tocco, M. Baudry, S. Schreiber, p53 induction is associated with neuronal damage in the central nervous system, Proc. Natl. Acad. Sci. U.S.A. 91 Ž1994. 7525–7529. w23x O. Saucedo-Cardenas, R. Kardon, T.R. Edinger, J.P. Lydon, O.M. Conneely, Cloning and structural organization of the gene encoding the murine nuclear receptor transcription factor, Nurr1, Gene 187 Ž1997. 135–139. w24x S.S. Schreiber, G. Tocco, I. Najm, C.E. Finch, S.A. Johnson, M.

w25x

w26x

w27x

w28x

w29x

w30x

w31x

w32x

w33x

w34x w35x

w36x

Baudry, Absence of c-fos induction in neonatal rat brain after seizures, Neurosci. Lett. 136 Ž1992. 31–35. S.S. Schreiber, G. Tocco, I. Najm, M. Baudry, Seizure activity causes a rapid increase in sulfated glycoprotein-2 messenger RNA in the adult but not the neonatal rat brain, Neurosci. Lett. 153 Ž1993. 17–20. S.S. Schreiber, G. Tocco, I. Najm, R.F. Thompson, M. Baudry, Cycloheximide prevents kainate-induced neuronal death and c-fos expression in adult rat brain, J. Mol. Neurosci. 4 Ž1993. 149–159. J.E. Schwob, T. Fuller, J.L. Price, J.W. Olney, Widespread patterns of neuronal damage following systemic or intracerebral injections of kainic acid: a histological study, Neuroscience 5 Ž1980. 991–1041. J.L. Sonnenberg, C. Mitchelmore, P.F. Mac-Gregor-Leon, J. Hempstead, J.I. Morgan, T. Curran, Glutamate receptor agonists increase the expression of Fos, Fra and AP-1 DNA binding activity in the mammalian brain, J. Neurosci. Res. 24 Ž1989. 72–80. G. Tocco, X. Bi, L. Vician, I.K. Lim, H. Hershman, M. Baudry, Two synaptotagmin genes, Syt1 and Syt4, are differentially regulated in adult brain and during postnatal development following kainic acid-induced seizures, Mol. Brain Res. 40 Ž1996. 229–239. E. Tremblay, L. Nitecka, M.L. Berger, Y. Ben Ari, Maturation of kainic acid seizure–brain damage syndrome in the rat: I. Clinical, electrographic and metabolic observations, Neuroscience 13 Ž1984. 1051–1072. L. Vician, I.K. Lim, G. Ferguson, G. Tocco, M. Baudry, H.R. Herschman, Synaptotagmin IV is an immediate early gene induced by depolarization in PC12 cells and in brain, Proc. Natl. Acad. Sci. U.S.A. 92 Ž1995. 2164–2168. Q. Xiao, S.O. Castillo, V.M. Nikodem, Distribution of messenger RNAs for the orphan nuclear receptor Nurr1 and Nur77 ŽNGFI-B. in adult rat brain using in situ hybridization, Neuroscience 75 Ž1996. 221–230. G. Xing, L. Zhang, L. Zhang, T. Heynen, X.L. Li, M.A. Smith, S.R.B. Weiss, A.N. Feldman, S. Detera-Wadleigh, D.-M. Chuang, R.M. Post, Rat nurr1 is prominently expressed in perirhinal cortex, and differentially induced in the hippocampal dentate gyrus by electroconvulsive vs. kindled seizures, Mol. Brain Res. 47 Ž1997. 251–261. K.R. Yamamoto, Steroid receptor regulated transcription of specific genes and gene networks, Annu. Rev. Genet. 19 Ž1985. 209–225. R.H. Zetterstrom, R. Williams, T. Perlmann, L. Olson, Cellular expression of the immediate early transcription factors Nurr1 and NGFI-B suggests a gene regulatory role in several brains regions including the nigrostriatal dopamine system, Mol. Brain Res. 41 Ž1996. 111–120. R.H. Zetterstrom, L. Solomin, L. Jansson, B.J. Hoffer, L. Olson, T. Perlmann, Dopamine neuron agenesis in Nurr1-deficient mice, Science 276 Ž1997. 248–250.