nor-1 Regulates Hippocampal Axon Guidance, Pyramidal Cell Survival, and Seizure Susceptibility

The nuclear receptor transcription factor, nor-1, is expressedduring mammalian development predominantly in the nervous systemand is induced in a cell-specific manner in nonneuronal cellsin response to a variety of extracellular stimuli. To elucidatethe essential developmental functions of this transcriptionfactor, we have analyzed the consequences of its eliminationon central nervous system development in mice. Here we showthat null mutant mice lacking nor-1 respond with increased limbicseizure activity to the excitotoxic glutamate receptor agonistkainic acid. We demonstrate that these abnormalities are associatedwith defective postnatal hippocampal development exemplifiedby abnormal axonal guidance of dentate gyrus granule and mossycells, disorganization of the pyramidal cell layer, and earlypostnatal death of pyramidal neurons in the CA1 field of thehippocampus. Our data indicate that nor-1 plays a critical rolein neuronal survival and axonal guidance in the developing murinehippocampus and that hippocampal dysgenesis in nor-1^–/–mice may be an underlying cause of seizure susceptibility.

INTRODUCTION

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Introduction

nor-1 (NR4A3) is a member of the nuclear receptor family oftranscription factors, whose developmental and physiologicalfunctions are poorly understood. nor-1 (NR4A3) and two highlyhomologous transcription factors, nurr1 (NR4A2) and nur77 (NR4A1),constitute the NR4A subfamily of nuclear receptors (2). Unlikemost nuclear receptors, the regulator function of NR4A receptorsis constitutively active and does not require ligand modulation(17, 40, 43). All three proteins are products of immediate-earlygenes whose transcription is rapidly induced in response toa variety of extracellular stimuli, including mitogens, apoptoticsignals, and neurotransmitters (reviewed in reference 21). Theinduced nuclear receptors undergo site-specific phosphorylationto modulate their transcriptional activity (8, 14, 27) and bindto specific cis-acting DNA elements containing the sequencemotif AAAGGTCA to regulate the expression of defined sets oftarget genes in a stimulus- and cell-specific manner (17, 26,43).

Previous studies have suggested that members of the NR4A subfamilyof nuclear receptors, including nor-1, play key roles in mediatingneuronal differentiation during central nervous system (CNS)development and in activity-dependent maintenance of neuronalplasticity in the adult CNS. For example, expression of nurr1in the ventral midbrain is essential for embryonic specificationof dopaminergic neurons (5, 33, 46). All three NR4A receptorsare expressed in the limbic system, including hippocampal pyramidalneurons of the adult CNS (32, 47), suggesting a potential involvementin the regulation of long-term changes in gene expression associatedwith learning and memory. In addition, their expression is rapidlyand transiently induced in both pyramidal neurons and dentategranule cells of the hippocampus in experimental animal modelsof temporal lobe epilepsy (TLE), including chemically and electricallyinduced limbic seizure activity (7, 28, 42, 44).

Pathological seizure activity in both human TLE and experimentalrodent models of TLE is associated with region-specific celldeath and lasting synaptic reorganization of hippocampal circuitryof the mature CNS (3, 4, 39). These changes require new proteinsynthesis and are associated with a rapid upregulation of immediate-earlygene products, among which are several classes of transcriptionfactors whose activity is thought to mediate the long-term changesin synaptic plasticity associated with pathological seizureactivity (22). Consistent with this hypothesis, several membersof the AP-1 class of transcription factors, including c-junand c-fos, are induced by seizures, and inhibition of c-fos(41) or the c-jun-activating kinase jnk3 (45) results in a reductionin seizure activity and associated pathological synaptic plasticity.The rapid seizure-dependent upregulation of NR4A receptors inthe hippocampus predicts that this subfamily of nuclear receptortranscription factors may also contribute to the regulationof specific gene programs whose function is to modulate activity-dependentlong-term synaptic plasticity. However, elucidation of the specificroles of these receptors in hippocampal plasticity is complicatedby their overlapping spatiotemporal expression and activity-dependentinduction patterns in the adult hippocampus and by their abilityto regulate overlapping genes through interaction with commoncis-acting DNA elements. The potential for functional redundancyamong NR4A nuclear receptors in mediating epileptogenesis isalso underscored by the observation that the development ofelectrically induced seizure activity (kindling) and its associatedsynaptic reorganization are unaffected in mice lacking nur77(48).

To further explore the role of NR4A nuclear receptors in thehippocampus, we have analyzed the selective contribution ofnor-1 to hippocampal development and seizure-associated synapticplasticity by examination of the consequences of its eliminationin mice. Previously, our laboratory demonstrated that null mutationof the nor-1 gene in mice (nor-1^–/–) did not affectpostnatal viability but resulted in abnormal vestibular functiondue to defective outgrowth of the semicircular canals of theinner ear (30). In the present study, we demonstrate that inhibitionof nor-1 expression also results in defective axonal growthand region-specific cell death in the hippocampus during theearly postnatal stages of development. The developmental defectspersist to adulthood and are associated with lasting changesin hippocampal excitability and increased susceptibility ofnor-1^–/– mice to chemically induced seizures.

MATERIALS AND METHODS

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Materials and Methods

Animals. The generation and genotyping of mice lacking a functional nor-1gene (nor-1^–/–) has been previously described (30).The mice contained the lacZ reporter gene inserted into thenor-1 locus. Mixed-strain C57BL/6/129Sv mice were used in thesestudies from two independent embryonic stem cell clone lines.Only heterozygous animals (interbred >8 generations) wereused in breeding, and nor-1^–/– offspring and theirwild-type littermates were used in the experiments.

All protocols involving the use of animals were in compliancewith the National Research Council's Guide for the Care andUse of Laboratory Animals (23a) and the Baylor College of MedicineInstitutional Animal Care and Use Committee. The tests wereperformed with the experimenter being blind with respect tothe genotype.

Induction of limbic seizures. Kainic acid (KA; Sigma) was administered intraperitoneally insaline at 15, 20, or 25 mg/kg of body weight. After KA injection,seizure activity was observed for 45 min and scored and stagedby a blinded observer as previously described (23) accordingto the following stages: stage 0, no effect; 1, motionless;2, rigid posture with forelimb and/or tail extension; 3, myoclonicjerks of head and neck; 4, forelimb clonus and partial rearing;5, forelimb clonus, rearing, and falling; 6, generalized tonic-clonicconvulsions with loss of postural tone.

Terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) assay for apoptosis. The brains were fixed overnight in 10% formalin, processed toparaffin, and cut to 6 µm. For staining, the sectionswere rehydrated and digested with proteinase K (40 ng/ml) for7 min. After washes with phosphate-buffered saline, the sectionswere blocked with TdT buffer (30 mM Tris [pH 7.4], 140 mM Na-cacodylate,1 mM CaCl₂) and incubated with TdT-dUTP mix (8 µl of UTPand 6 µl of TdT [Roche] in 1,200 µl of TdT buffer)for 1 h at 37°C. The signal was detected by using VectastainElite and peroxidase substrate kits (Vector). Methyl green wasused as a counterstain.

Immunohistochemistry. Specimens were fixed in Bouin's fixative overnight, washed with70% ethanol, processed for paraffin sectioning, and cut to 6µm. Staining was done in accordance with standard avidin-biotinimmunohistochemical procedures (Vector). Rabbit polyclonal immunoglobulinGs against calretinin and calbindin (1:500 dilution) were fromCalbiochem.

ß-Galactosidase staining. Brains were embedded in Tissue-Tec OCT compound and cut to 20µm. The sections were fixed in 2% formaldehyde-0.2% glutaraldehydefor 15 min and rinsed with phosphate-buffered saline. The stainingsolution consisted of 1.3 mM MgCl₂, 15 mM NaCl, 44 mM HEPES(pH 7.9), 3 mM potassium ferro- and ferricyanides, and 0.5 mgof 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside/mlin H₂O.

Silver staining. Sevier-Munger silver staining (Polyscientific) was performedaccording to the manufacturer's recommendation on Bouin's fixed6-µm sections.

In situ hybridization. Twenty-micrometer frozen coronal sections were cut and processedusing nurr1 and nur77 probes as described elsewhere (32), withthe exception of using digoxigenin-labeled probes (10 ng/slide).For signal development, a substrate solution (BCIP/NBT kit IV;Vector) was applied and incubated in a moist chamber in thedark.

Cell counts and statistical analysis. For cell counting in the CA1 hippocampal field, x400 imageswere captured from coronal brain sections (nine images fromeach animal) at the level of the arch of the dorsal hippocampus,and all cells in the image were counted using the Imagetoolsprogram. Clearly visible split nuclei were included in the count.TUNEL-positive cells were counted from the whole dorsal CA1area (nine sections from each animal). Data collected from eachexperimental group are expressed with the standard error ofmean. Student's t test was used for statistical analysis (n= 6 except for 12-month-old animals, for which n = 4).

RESULTS

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Results

nor-1^–/– mice have increased susceptibility to KA-induced limbic seizures. Observation of adult nor-1^–/– mice revealed thata small percentage exhibited brief freezing spells with tonicposturing that were exacerbated with handling. To elucidatethe neurological basis of this behavior, we examined the seizuresusceptibility of nor-1^–/– mice to a convulsantstimulus. Adult wild-type (nor-1^+/+) and nor-1^–/–mice were injected intraperitoneally with KA (25 mg/kg), whichelicits limbic seizures by stimulating glutamate receptors andby increasing the release of excitatory amino acids (20). Thedevelopment of behavioral seizures was scored as previouslydescribed (23), and the latency of progression to a stage 6seizure was used to assess seizure susceptibility. While bothnor-1^+/+ and nor-1^–/– mice exhibited freezing spellsthat progressed to head bobbing, forepaw tremor, and loss ofpostural control, nor-1^–/– mice had a significantlyshorter latency to continued convulsions than nor-1^+/+ mice(Fig. 1A). In addition, they developed several maximal seizuresduring the experiment, while most of the nor-1^+/+ mice developedonly one stage 6 tonic-clonic convulsion (Fig. 1B). nor-1^–/–mice also exhibited enhanced sensitivity to KA relative to nor-1^+/+mice; four out of seven nor-1^–/– mice died aftercontinuous tonic-clonic convulsions, whereas only one out ofeight nor-1^+/+ mice died. In addition, when injected with alower dosage of KA (15 mg/kg), six out of eight nor-1^–/–mice and only two out of nine nor-1^+/+ mice developed seizures(data not shown). These results demonstrate an essential rolefor nor-1 in modulating the severity of convulsant stimulus-inducedlimbic seizure activity.

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FIG. 1. Increased susceptibility of nor-1^–/– mice to KA-induced seizures. The latency to a stage 6 behavioral score was used to assess seizure susceptibility. (A and B) nor-1^–/– mice showed a heightened susceptibility to KA-induced seizures by reaching stage 6 tonic-clonic convulsion faster than the nor-1^+/+ mice (A) and developed several seizures during the time observed (45 min) (B). Error bars represent the means ± standard errors of the mean. *, P < 0.05 (Student's t test). (C to F) nor-1 expression in the developing hippocampus. Sagittal (C) and coronal (D to F) sections of heterozygous nor-1^lacZ brains were processed for ß-galactosidase staining to establish the expression pattern for nor-1 in the hippocampus. At embryonic day 14.5, nor-1 was expressed in the primitive plexiform layer of the hippocampus (Hc) and the outermost lamina in the neocortex (Cx). At P0, all the principal hippocampal cell layers had formed, and nor-1 expression was evident in the pyramidal cell layer of the subiculum (S) and CA1-CA3. In the dentate gyrus, nor-1 was expressed in the granule cell layer (Gcl) and in the hilar (H) mossy cells. nor-1 was retained in all these areas in the adult hippocampus, shown at the dorsal (E) and ventral (F) levels. Bar, 500 µm (C) and 250 µm (D to F).

To determine whether nor-1 is expressed during development ofthe limbic system, we examined its expression during the embryonicand postnatal stages of hippocampal development. For these analyses,we used ß-galactosidase as a reporter for nor-1 promoteractivity in heterozygous nor-1 mutant mice carrying the lacZreporter gene inserted into the nor-1 locus (30). Expressionof ß-galactosidase at embryonic day 14.5 was observedin the primitive plexiform layer of the hippocampus, which isa continuation of the outermost lamina in the neocortex (Fig.1C). By postnatal day 0 (P0), after the granule cell layersof the dentate gyrus begin to develop at late gestation, nor-1expression was observed in both the pyramidal and granule celllayers (Fig. 1D). The majority of granule cells develop postnatallyduring the first 3 weeks of life, and nor-1 expression persistedin these cells in the adult hippocampus after their postnatalmaturation (Fig. 1E). In addition, nor-1 expression was alsoobserved in the hilar mossy cells of the dentate gyrus, whichform a second major synaptic relay system within this structure(Fig. 1F).

Disorganization and postnatal neuronal cell loss in the pyramidal layer of the hippocampus in nor-1^–/– mice. To examine the structural integrity of the hippocampus, coronalsections of the hippocampus of adult nor-1^–/– micewere compared with those of nor-1^+/+ littermates. Analysis ofthese sections indicated that both the pyramidal and granulecell layers were formed in nor-1^–/– mice (Fig. 2B);however, the pyramidal cell layer appeared less dense than thatobserved in nor-1^+/+ littermates (Fig. 2A). No overt abnormalitiesin either organization or cell number were observed in the granulecell layer of the dentate gyrus (Fig. 2C and D). In contrast,further examination of the hippocampal subfields at higher magnificationrevealed that a compact pyramidal cell layer of Ammon's hornwas not formed in any of the nor-1^–/– mice analyzed(n = 15), as shown at the levels of CA3 (Fig. 2F) and CA1 (Fig.2H) when compared with nor-1^+/+ littermates (Fig. 2E and G).Further, pyramidal cell disorganization was accompanied by anapparent decrease in the total number of cells in the CA1 regionof nor-1^–/– mice (Fig. 2H) relative to that in nor-1^+/+mice (Fig. 2G), but not in the CA3 region (Fig. 2F versus E).

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FIG. 2. Abnormal pyramidal cell layer in nor-1^–/– mice, as shown in H&E-stained coronal sections of nor-1^+/+ hippocampus (A, C, E, and G) and nor-1^–/– hippocampus (B, D, F, and H). Both the pyramidal (CA1 to CA3) and granule cell (Gcl) layers were formed in nor-1^–/– mice (B). The granule cell layer of the hippocampus appeared normal in the nor-1^–/– mice (D); however, the pyramidal cell layer appeared disorganized both at the level of CA3 (F) and CA1 (H). Bars, 100 µm (A and B) and 25 µm (C to H).

To confirm the apparent decrease in pyramidal cell numbers inthe CA1 field and to establish the developmental cause of thesedefects, we quantitated the numbers of pyramidal neurons inthe CA1 region in nor-1^+/+ and nor1^–/– mice (Fig.3) during early postnatal development. Examination of coronalsections of newborn animals (P0) showed that the nor-1^–/–CA1 pyramidal cell layer (Fig. 3E) was comparable to that innor-1^+/+ mice (Fig. 3A), with similar cell numbers observedin both genotypes (Fig. 3K). By P7, however, the disorganizedphenotype of the CA1 pyramidal cell layer became evident innor-1^–/– animals (Fig. 3F). The cellular loss wasmaximal at the second (Fig. 3G) and third (Fig. 3H) postnatalweeks and persisted to adulthood (Fig. 3K). Interestingly, thisloss of the pyramidal CA1 neurons in the nor-1^–/–mice was restricted to the early postnatal stages of developmentand did not progress with age (Fig. 3K).

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FIG. 3. Cell loss due to increased apoptosis during development of the CA1 pyramidal cells. The CA1 pyramidal layer appeared normal in newborn nor-1^–/– mice (E) compared to nor-1^+/+ mice (A). However, disorganization of the cell layer that was observed at P7 (F) became more severe by P14 (G) and P21 (H). In addition, a decrease in cell number was significant at P14 and persisted in the adult nor-1^–/– mice (K). *, P < 0.05 (Student's t test). TUNEL labeling analysis of nor-1^+/+ and nor-1^–/– CA1 regions showed a small but significant increase in the number of apoptotic cells at P0, and cell death was highly increased at P5 in the nor-1^–/– mice (J and L). Inset in J is a 1,250x magnification of the TUNEL-positive cells. By P7, apoptosis in the nor-1^–/– CA1 pyramidal cell layer was decreased to the level of the nor-1^+/+ mice (L). Bars, 25 µm (A to H) and 50 µm (I and J).

To investigate whether the loss of CA1 pyramidal cells in thenor-1^–/– mice was a result of postnatal cell death,we next performed TUNEL staining to monitor the degree of apoptosisoccurring in CA1 pyramidal neurons at the early postnatal stagesbefore the cell loss in these mice became evident. The results(Fig. 3I, J, and L) indicated a significant increase in apoptoticcells in the CA1 region of nor-1^–/– mice relativeto that in nor-1^+/+ mice as early as P0 (Fig. 3L). The increasesin apoptosis in nor-1^–/– sections were maximal atP5 (Fig. 3L) and, from P7 onwards, only occasional TUNEL-positivecells were found in sections from both genotypes. Thus, thedecrease in pyramidal neurons observed in the CA1 region ofnor-1^–/– mice appears to be the result of enhancedcell death that is restricted to the early postnatal stagesof development.

Finally, in contrast to the CA1 field, similar analysis of theCA3 region of nor-1^–/– mice indicated that despitea clear disorganization of pyramidal cells, no cellular losswas observed in this region (data not shown), indicating a specificrole for nor-1 in the survival of CA1 pyramidal cells.

Defective axonal guidance in the dentate gyrus of nor-1^–/– mice. Because both mossy and granule cells of the dentate gyrus playa critical role in regulation of hippocampal circuits associatedwith epileptogenesis, we next examined the integrity of bothpopulations of neurons and their axonal projections. Mossy fibersrepresent the axonal projections of the dentate granule cellsthat develop postnatally and form synapses with apical dendritesof CA3 pyramidal cells and with excitatory mossy cells in thedentate hilus (1). To examine the integrity of this system,we used calbindin immunostaining to label granule cells andtheir axonal and dendrite projections (36). These analyses (Fig.4A to F) revealed normal immunostaining patterns in the granulecell somata and of their dendrites in the molecular layer inboth neonatal (P7) (Fig. 4A and B) and P14 (Fig. 4C and D) andadult (Fig. 4E and F) nor-1^+/+ and nor-1^–/– mice.However, a significant decrease in the density of immunostainingin mossy fiber projections to the CA3 area was observed in thenor-1^–/– mice at P7 and persisted through adulthood(Fig. 4E and F). This reduction in mossy fiber projections tothe CA3 pyramidal neurons in the nor-1^–/– mice wasconfirmed by neuronal process-specific Sevier-Munger silverstaining, which showed a marked decrease in staining of axonalprojections in nor-1^–/– mice (Fig. 4H) relativeto that in nor-1^+/+ mice (Fig. 4G).

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FIG. 4. Reduced outgrowth of granule cell mossy fibers (arrows in E and F) into CA3 stratum lucidum. Whereas calbindin staining in the granule cell bodies (Gcl) and molecular layer (Ml) was normal, the granule cell mossy fibers (Mf) extending toward CA3 pyramidal cell layer were reduced at P7 (B), P14 (D), and in the adult (F) nor-1^–/– mice. This reduction in the outgrowth of mossy fibers in the nor-1^–/– mice was confirmed by Sevier-Munger silver staining (H). nor-1^+/+ controls are shown in panels A, C, E, and G. Bars, 100 µm.

In order to assess the morphology of the second major excitatorypathway in the dentate gyrus, the hilar mossy cell system, weused immunostaining for calretinin, whose expression in theadult hippocampus is localized to hilar mossy cells and widelydistributed subsets of GABAergic interneurons (18). Glutamatergicmossy cells in the dentate hilus give rise to the commissural-associationalsystem that sends axonal projections to both the contralateraland ipsilateral dentate gyrus, where they form synaptic connectionswith both excitatory granule cells and inhibitory interneuronslocated in the inner molecular layer of the dentate gyrus (15).At P14, calretinin staining was observed in hilar mossy cellsoma, newly generated immature granule cells located in thesubgranular zone at the hilar border (18), and in mossy cellaxonal projections located in the inner molecular layer (IML)of the dentate gyrus of wild-type mice (Fig. 5A). This stainingpattern was similar to that in nor-1^–/– mice (Fig.5B), with the exception of the mossy cell axon belt, which showedmarkedly reduced staining relative to that in nor-1^+/+ mice.As development proceeded to adulthood, calretinin immunostainingwas extinguished in mature granule cells but was increased inthe mossy cell axon belt that comprises the supra- and infrapyramidalblades of the dentate gyrus of nor-1^+/+ mice (Fig. 5C and E).In contrast, calretinin staining was almost completely lostin the suprapyramidal blade (Fig. 5D and F) and was decreasedin the inferior pyramidal blade (Fig. 5D) of the nor-1^–/–dentate gyrus.

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FIG. 5. Reduced outgrowth of mossy cell axons. By P14, calretinin-positive mossy cell axons had reached the inner molecular layer (Iml) of the granule cell layer (Gcl) in the nor-1^+/+ dentate gyrus (A), whereas only a weak calretinin staining could be seen in the nor-1^–/– mice (B). An even more obvious decrease in these calretinin-positive mossy cell axons was seen in the adult nor-1^–/– dentate gyrus (D). (F) Higher magnification of the suprapyramidal blade (Spb), which was almost devoid of calretinin-positive axons in the nor-1^–/– dentate gyrus. No difference was found between nor-1^+/+ and nor-1^–/– mice in the number of calretinin-positive mossy cell bodies in the hilus of the ventral hippocampus, where the majority of these cells are located (G and H), or in the commissural fibers that cross the midline at the level of the hippocampal commissure (I and J), indicating a specific reduction in mossy cell associational fibers in the nor-1^–/– mice. Bars, 100 µm (A to D and G to J) and 25 µm (E and F).

To determine whether the loss of calretinin staining in themossy cell axons was due to reduced differentiation of mossycells, we also examined the distribution of calretinin-positivecells in sections obtained from the ventral hippocampus, wherethe majority of these neurons reside. In contrast to the reducedcalretinin immunoreactivity observed in the mossy cell axonsinnervating the IML, there was no reduction in the number ofmossy cell soma in the nor-1^–/– (Fig. 5H) relativeto that in the nor-1^+/+ (Fig. 5G) ventral hippocampus. Thesedata indicate that in the absence of nor-1, mossy cells appearto migrate to their appropriate positions but are defectivein axonal outgrowth. Finally, to determine whether the commissuralaxons from mossy cells were capable of crossing the midlineto the contralateral hippocampus, we visualized these projectionsby calretinin staining at the level of the hippocampal commissure.Comparison of nor-1^+/+ (Fig. 5I) and nor-1^–/– (Fig.5J) mice confirmed that these axons crossed the midline, indicatingthat the defect in mossy cell axon outgrowth appears to specificallydisrupt mossy fiber innervation of the suprapyramidal bladeof the IML of the dentate gyrus (11, 24, 25).

Since we have used mixed-background 129Sv/C57BL/6 mice in ourstudies, we next asked whether the major defects we observedin nor-1^–/– mice relative to wild-type littermatesmay have been due in part to strain variations between the parentalstrains. In these experiments, we compared hippocampal regionsrelating to each of the defects in adult parental C57BL/6 and129Sv mouse strains. No differences in the pyramidal CA1 regionbetween strains were observed by using hematoxylin and eosin(H&E) staining (Fig. 6A and B). Secondly, immunohistochemicalanalysis with calretinin (Fig. 6C and D) and calbindin (Fig.6E and F) showed no overt differences in the mossy cell axonbundle in the suprapyramidal blade of the dentate gyrus or inthe suprapyramidal mossy fiber pathway to the CA3 region, respectively.

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FIG. 6. Analysis of the parental C57BL/6 (A, C, and E) and 129Sv (B, D, and F) strain hippocampus. (A and B) No overt differences were found between these two strains in the CA1 field using H&E staining. (F) In addition, immunohistochemical analysis using calretinin (C and D) and calbindin (E and F) showed no overt differences in the mossy cell axons in the suprapyramidal blade of the dentate gyrus or in the suprapyramidal mossy fiber pathway to the CA3 region, respectively.

Nonredundancy of function of NR4A receptors during hippocampal development. To determine whether nor-1 plays a distinct functional rolein hippocampal development relative to its closely related subfamilymembers, nurr1 and nur77, we used in situ hybridization to examinethe comparative spatiotemporal expression of the two NR4A receptorsin the hippocampus at P5, when the neurodevelopmental defectsbecome apparent in nor-1^–/– mice. Our results showedthat while nur77 was not expressed in the hippocampus at thisdevelopmental stage (data not shown), nurr1 expression couldbe observed in the CA1 field of the pyramidal cell layer (Fig.7A) of nor-1^+/+ mice and was retained in this region in nor-1^–/–mice (Fig. 7B). This early postnatal pattern of expression ofnurr1 in CA1 pyramidal neurons was consistent with that previouslyreported by others (47) but was unable to compensate for theloss of nor-1 in preventing cell death of CA1 pyramidal neurons.The expression of nurr1 persisted in pyramidal neurons of theadult hippocampus (Fig. 7C), and the expression of nur77 overlappedwith nurr1 in this region (Fig. 7D). In contrast, the levelof both receptors in the adult dentate gyrus was extremely lowto undetectable, consistent with the lack of expression of eitherreceptor observed in this region during early postnatal development.Treatment with KA resulted in robust induction of both nurr1(Fig. 7E) and nur77 (Fig. 7G) in the dentate gyrus of nor-1^+/+mice as has been reported previously (7, 16). Significantly,however, this induction was unaffected in nor-1^–/–mice (Fig. 7F and H), indicating that nurr1 and nur77 inductionin the adult hippocampus is unable to compensate for earlierdevelopmental defects that contribute to enhanced seizure susceptibilityin nor-1^–/– mice.

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FIG. 7. nurr1 and nur77 expression in the hippocampus. nurr1 was expressed in the pyramidal cell layer at P5 (A), and its expression was unchanged in nor-1^–/– mice (B). nurr1 (C) and nur77 (D) are expressed in adult hippocampus. Both nurr1 and nur77 were induced by KA injection (20 mg/kg) in the dentate gyrus in adult nor-1^+/+ mice (E and G) and to the same extent in the nor-1^–/– mice (F and H). Bars, 50 µm (A and B), 150 µm (C and D), and 75 µm (E and H).

We conclude that the specific requirement for nor-1 in earlypostnatal hippocampal development appears to be due in partto its selective expression relative to the additional NR4Areceptors in the dentate gyrus and also to its distinct functionalactivity relative to nurr1 in CA1 pyramidal neurons.

DISCUSSION

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Discussion

We have demonstrated that inhibition of nor-1 expression inmice results in defective hippocampal axonal growth and postnatalneuronal cell death. These abnormalities arise during earlypostnatal hippocampal development and result in lasting changesin neuronal excitability in the adult hippocampus. Specifically,we have shown that mice lacking nor-1 display abnormal developmentof axonal projections of granule and mossy cells of the dentategyrus and early postnatal death of pyramidal neurons in theCA1 field of Ammon's horn. The defects in hippocampal axonalgrowth and neuron survival are associated with a lowered seizurethreshold of nor-1^–/– mice relative to nor-1^+/+mice when challenged with the glutamatergic excitotoxin KA.

Guidance of outgrowing axons toward and within the hippocampusis directed by the combined action of long- and short-rangerepellant and attractive signals (37), which recognize specificreceptor proteins at the growing tip of developing axons. Whilethe signaling mechanisms that mediate outgrowth of hilar mossycell axons of the dentate gyrus are poorly understood, severalextracellular molecules known to mediate mossy fiber outgrowthfrom granule cells have recently been identified. These includethe secreted semaphorin Sema3F and its receptor, neuropilin-2,limbic system-associated protein (29, 37), and polysialylatedneural cell adhesion molecule (35). Repellant guidance by thesefactors is required for prevention of inappropriate layer terminationof invading axons, and their absence results in ectopic layertermination of mossy fibers within the hippocampus. However,despite the availability of a growing body of information onthe identity of these extracellular guidance cues, little informationis known regarding the specific intracellular signaling pathwaysthat mediate guided outgrowth and target selection by growingaxons or the gene regulatory mechanisms that may control thesepathways. Despite their decreased density, mossy fibers in nor-1^–/–mice appear to respect their appropriate lamina-specific terminationborder with regard to the SL of CA3 and the CA3-CA1 border,suggesting that nor-1 may be responsible for regulating theexpression of CA3 target-derived attractive guidance cues and/orintracellular signaling pathways within the growing axons ofgranule cells that promote neurite outgrowth and target selection.

We have also demonstrated that elimination of nor-1 resultsin selective loss of mossy cell axons that project to the suprapyramidalblade of the IML. However, the commissural mossy cell axonsappear to cross the midline of the hippocampal commissure andappropriately terminate in the infrapyramidal layer of the IML.These findings suggest that nor-1 expression is not requiredfor the elaboration of long-range guidance cues required forthe guidance of extrinsic hippocampal commissural axons butplays an essential role in regulation of intrinsic signals responsiblefor guidance and target selection by commissural-associationalfibers within the dentate gyrus. The molecular signals thatmediate differential guidance and branching of fibers from mossycells to the suprapyramidal and infrapyramidal blades of theIML remain elusive. Guidance of mossy cell axons is thoughtto be strongly influenced by as-yet-unidentified target-derivedsignals emanating from granule cells (9, 10, 12). Although thegranule cell layer of the dentate gyrus was previously thoughtto be relatively homogeneous, emerging evidence supports theconclusion that the suprapyramidal and infrapyramidal layersexhibit significant structural and functional differences, includingmarked variation in dendritic architecture (6), lack of uniformityin the inhibitory circuitry, and greater contribution of theinfrapyramidal blade to seizure activity (34). The identificationof signaling pathways regulated by nor-1 should provide importantnew insights into the mechanisms underlying the structural andfunctional asymmetry of fiber pathways innervating the suprapyramidaland infrapyramidal layers of the dentate gyrus as well as thecontribution of granule and mossy cell signaling to these pathways.

The developmental abnormalities observed in nor-1^–/–mice show significant similarities to some aspects of hippocampalpathologies observed in human patients with TLE and experimentalanimal models of epilepsy. Histopathologically, human TLE associatedwith hippocampal sclerosis is characterized by neuronal deathwhich is most severe in the CA1 region and in the hilus of thedentate gyrus (13, 38). Stimulation of rodents with KA or pilocarpineresults in the development of acute severe seizures and hippocampalabnormalities that overlap with those observed in human TLE.These abnormalities include death of pyramidal neurons in theCA1 and/or CA3 fields of Ammon's horn, aberrant sprouting ofmossy fiber axons of dentate granule cells to the inner molecularlayer of the dentate gyrus, and retraction of axons from hilarmossy cells to the IML in the absence or presence of death ofmossy cell soma (3, 19, 31). Although the hippocampal pathologiesobserved in the experimental animal models of epilepsy significantlyoverlap with those observed in human TLE, it remains unclearwhether they are the cause or result of limbic seizure activity.The defects in axonal outgrowth from both mossy and granulecells and decreased survival of CA1 pyramidal cells observedduring early postnatal development of nor-1^–/– micesupport the conclusion that underlying developmental defectsin the hippocampus in these animals may contribute to laterdevelopment of seizure susceptibility. The nor-1^–/–mouse thus provides an excellent model system to dissect thegene regulatory mechanisms underlying axonal path finding inthe dentate gyrus and pyramidal cell survival during postnatalneurogenesis whose aberrant activity may contribute activity-dependentsynaptic plasticity associated with pathological seizure development.

The ability of NR4A nuclear receptors to interact with overlappingcis-acting DNA elements together with their overlapping expressionpatterns in the hippocampus and induction by seizure stimulipredict that members of this subfamily of nuclear receptorsare likely to function redundantly to regulate activity-dependentchanges in gene expression in the hippocampus. However, comparativeanalysis of the developmental expression patterns of the threenuclear receptors suggests that despite their structural relatednessand potential for interaction with overlapping target genes,nor-1 and nurr1 have distinct functions in the hippocampus duringearly postnatal development. Our observations that both nurr1and nur77 show a similar induction pattern by KA in nor-1^–/–mice and their inability to compensate for nor-1 loss in nor-1^–/–mice add further support to the conclusion that the early developmentaldefects in nor-1^–/– mice contribute to the lowerthreshold of seizure induction in nor-1-deficient mice.

The identification of nor-1 as an essential regulator of developmentalpathways that ultimately control seizure susceptibility suggeststhat modulation of nor-1 activity may provide a novel therapeuticpoint of intervention to control epileptogenesis. In addition,the identification of nor-1-dependent target genes whose expressionis disrupted in the hippocampus of nor-1^–/– miceshould facilitate the identification of novel molecular signalingpathways that mediate the establishment of hippocampal circuitsassociated with the development of epilepsy as well as uncovernew approaches to clinical intervention.

ACKNOWLEDGMENTS

We thank Jeffry Brace for technical assistance.

We acknowledge the Mental Retardation Research Center (HD 24064)behavioral core and NIH grant DK57743 to O.M.C.

FOOTNOTES

* Corresponding author. Mailing address: Department of Molecular and Cellular Biology, Baylor College of Medicine, Houson, TX 77030. Phone: (713) 798-6233. Fax: (713) 798-7583. E-mail: orlac{at}bcm.tmc.edu.

REFERENCES

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