Transient calretinin expression defines early postmitotic step ofneuronal differentiation in adult hippocampal neurogenesis of mice

Moritz D. Brandt,a,1 Sebastian Jessberger,b,1 Barbara Steiner,b Golo Kronenberg,a,c

Katja Reuter,a Anika Bick-Sander,b Wolfger von der Behrens,a and Gerd Kempermanna,b,*a Max Delbruck Center for Molecular Medicine (MDC) Berlin-Buch, Robert-Rossle-Strasse 10, 13125 Berlin, Germany

b VolkswagenStiftung Research Group, Department of Experimental Neurology, Charite University Hospital, Humboldt University,Schumannstrasse 20/21, 10117 Berlin, Germany

c Department of Psychiatry, Freie Universitat Berlin, Eschenallee 3, 14050 Berlin, Germany

Received 10 April 2003; revised 4 June 2003; accepted 6 June 2003

Abstract

We here show that the early postmitotic stage of granule cell development during adult hippocampal neurogenesis is characterized bythe transient expression of calretinin (CR). CR expression was detected as early as 1 day after labeling dividing cells with bromodeoxyuri-dine (BrdU), but not before. Staining for Ki-67 confirmed that no CR-expressing cells were in cell cycle. Early after BrdU, CR colocalizedwith immature neuronal marker doublecortin; and later with persisting neuronal marker NeuN. BrdU/CR-labeled cells were negative forGABA and GABAA1 receptor, but early on expressed granule cell marker Prox-1. After 6 weeks, no new neurons expressed CR, but allcontained calbindin. Stimuli inducing adult neurogenesis have limited (enriched environment), strong (voluntary wheel running), and verystrong effects on cell proliferation (kainate-induced seizures). In these models the induction of cell proliferation was paralleled by anincrease of CR-positive cells, indicating the stimulus-dependent progression from cell division to a postmitotic stage.© 2003 Elsevier Inc. All rights reserved.

Introduction

In most studies to date, adult hippocampal neurogenesishas been investigated by assessing two key events: theproliferation of the progenitor cells in the dentate gyrus andthe survival of the progeny and expression of neuronalmarkers about 4 weeks later (Kuhn et al., 1996). Much lessis known about the period of neuronal development inbetween. It appears, however, that the fate choice decisionof new neurons is made early and that those cells thatexpress neuronal markers such as NeuN are likely to be-come persistently integrated into the granule cell layer(Kempermann et al., 2003). A much larger proportion ofnewborn cells express immature neuronal markers such as

doublecortin (DCX) or �-III-tubulin, and it is suggestivethat this group of cells represents a pool of potential newneurons, from which under conditions that stimulate adultneurogenesis more new cells can be recruited for neuronaldevelopment (Kempermann et al., 1997b; Gould et al.,1999). At least a subset of DCX-expressing cells is highlyproliferative (Filippov et al., 2003), and it is reasonable toassume that this group of cells contains lineage-determinedneuronal progenitor cells. How can the developmentalpoint-of-no-return be defined, at which these cells becomepostmitotic and enter definite neuronal development?

This question is important for a number of reasons: (1)precision about the time point when cells become postmi-totic will allow the fate choice decision in adult hippocam-pal neurogenesis to be pinpointed; (2) better characteriza-tion of early postmitotic neuronal development will allowidentifications of the factors within the cells and in theirlocal environment that control this process; (3) compari-sions as early adult neurogenesis with net adult neurogen-esis quantitatively will allow dissection of the effects of

* Corresponding author. Max Delbruck Center for Molecular Medicine(MDC), Berlin-Buch, Robert-Rossle-Str. 10, 13125 Berlin, Germany. Fax:�49-30-9406-3814.

E-mail address: gerd.kempermann@mdc-berlin.de (G. Kempermann).1 Contributed equally to this work.

Molecular and Cellular Neuroscience 24 (2003) 603–613 www.elsevier.com/locate/ymcne

1044-7431/03/$ – see front matter © 2003 Elsevier Inc. All rights reserved.doi:10.1016/S1044-7431(03)00207-0

manipulations aiming at stimulating neurogenesis; (4) char-acterizations of adult neuronal development in detail andover its time course will lead to a clearer picture of how thenew neurons grow into their functional connections and thushow they might contribute to hippocampal function; andfinally (5) identifications of vulnerable development steps incases of disturbed adult hippocampal neurogenesis, as it isfor example discussed for epilepsy (Parent and Lowenstein,2002) or major depression (Jacobs et al., 2000), will help inunderstanding how failing adult neurogenesis might con-tribute to disease.

A chance observation sparked the series of experimentswe report here. Incidentally we found that in the adultmurine subgranular zone (SGZ) a number of cells expressedcalretinin (CR) and contained proliferation marker bro-modeoxyuridine (BrdU) that had been injected into theanimals 4 weeks earlier. As we will see in the followingparagraphs, our initial hypothesis that these cells might benewly generated interneurons turned out to be wrong. Wetherefore set out to study the role of calretinin-expressingcells within the context of adult hippocampal neurogenesis.It soon became evident that CR expression was linked toearly neuronal development in adult hippocampal neurogen-esis. A report by Liu et al., (1996) has speculated thatCR-containing cells might be immature granule cells, but sofar, this idea has not been proven. The rationale of the studypresented here therefore was to characterize calretinin-ex-pressing cells qualitatively and quantitatively within thecontext of adult hippocampal neurogenesis and relate cal-retinin expression to identifiable milestones of neuronaldevelopment.

Results

Newly generated cells in the adult SGZ express calretinin

Mature granule cells in the adult dentate gyrus are knownto express calbindin (Sloviter, 1989) and many subpopula-tions of interneurons can be categorized by their expressionof other calcium-binding proteins such as parvalbumin orCR (Freund and Buzsaki, 1996). In order to find out whethernewly generated neurons in the adult dentate gyrus wereheterogeneous with regard to their expression of calcium-binding proteins, we examined BrdU-labeled, newly gener-ated cells for their expression of parvalbumin and CR.Nowhere in the SGZ were BrdU-labeled parvalbumin-ex-pressing cells detected (Fig. 1A). However, at 4 weeks afterBrdU approximately 6% of the BrdU-labeled cells ex-pressed CR (Figs. 1B and 2A). We have reported earlier thatat 4 weeks after BrdU, about 60 to 70% of the BrdU-labeledcells express calbindin (Kempermann et al., 1997a).

CR-positive cells were localized in the SGZ of the gran-ule cell layer. There were no distinguishable differences inthe localization of CR- or calbindin-expressing newborncells, and the expression of both CR and calbindin in BrdU-

positive cells colocalized with neuronal marker NeuN (Fig.1B). At this time point, in the SGZ the overlap was 100%:there were no CR-positive cells that were NeuN-negative. Inorder to allow extensive combinations of primary antibodiesfrom different species, three different anti-CR-antibodies(from mouse, goat, and rabbit) were used in the presentstudy: They all yielded identical results.

Thus, with regard to the expression of calretinin or cal-bindin two populations of newly generated neurons could beidentified at 4 weeks after BrdU. The question was whetherthis reflected the existence of two stable independent pop-ulations or snapshots from a dynamic developmental pro-cess.

Calretinin is transiently expressed in maturing newneurons

In order to assess the kinetics of CR expression in newlygenerated cells we determined at which time point CR wasfirst expressed in newly generated neurons. For these timecourse studies animals were examined at 4 h, 1 day, 3 days,7 days, 2.5 weeks, and 4 weeks after a single injection ofBrdU.

CR expression in newborn cells was observed as early as1 day after BrdU, reaching a maximum after one week (76.6� 1.1%; Fig. 2A). Thereafter CR-expressing BrdU-positivecells decreased in number. In absolute terms, the number ofBrdU/CR double-labeled cells peaked at 3 days after BrdUand decreased over the following weeks (Fig. 2B). At 6weeks after BrdU no BrdU/CR double-labeled cells couldbe detected (not shown). This finding suggested that CR isexpressed transiently in BrdU-labeled cells.

Doublecortin is a key molecule in cortical and hippocam-pal development (des Portes et al., 1998; Corbo et al., 2002)and is expressed transiently in adult hippocampal neurogen-esis (Kempermann et al., 2003). At 1 day after BrdU wefound a large percentage of BrdU-positive cells that con-comitantly showed CR and DCX immunoreactivity (Fig.1B). This overlap was not found at 4 weeks after BrdU (Fig.1B).

As evident from the time course in Fig. 2B the increasein the absolute number of newborn CR-positive cells par-alleled the increase in the number of BrdU-marked NeuN-positive cells. The number of BrdU/NeuN-double-labeledcells, however, remained stable after 2.5 weeks, whereasBrdU/CR-double-labeled cells faded out. From the findingthat all CR-positive cells expressed NeuN and the overlapbetween CR and DCX at early time points, we concludedthat CR is transiently expressed in newly generated neuronsat early stages of their development.

Newly generated CR-positive cells do not express GABA,GABAA1 receptor, or reelin

The finding that CR is transiently expressed in immatureneurons of the adult dentate gyrus did not allow determina-

604 M.D. Brandt et al. / Molecular and Cellular Neuroscience 24 (2003) 603–613

tion of whether one or two populations of new neuronswould be generated. We thus intended to find out whetherCR-positive cells represented a so far uncharacterized groupof newborn interneurons or whether maturing granule cellswould switch their expression of calcium-binding proteinsduring the first 4 weeks after the division of the progenitorcell.

Because CR-expressing cells in the hippocampal granulecell layer were considered to be GABA-ergic interneurons(Sloviter et al., 2001), we analyzed newborn CR-positivecells for their coexpression of GABA. However, at no timepoint after BrdU did newly generated CR-positive cellscolabel with GABA (Fig. 1C), although many GABA-positive cells were seen in the same sections. Similarly,there was no overlay between the interneuron-specificGABAA1-receptor and CR (Fig. 1C) (Bouilleret et al.,2000; Redecker et al., 2000). Thus it is unlikely that theCR-labeled new cells represented a separate population ofnew inhibitory interneurons.

Along a similar line we investigated whether CR-ex-pressing cells were Cajal–Retzius neurons. Cajal–Retziusneurons play an important role during the embryonic devel-opment of the dentate gyrus (Gebhardt et al., 2002). Thus itseemed conceivable that the CR-expressing new cells of theadult dentate gyrus were Cajal–Retzius neurons whichmight play a similar role for adult neurogenesis. Cajal–Retzius cells can be identified by their expression of thesecreted glycoprotein reelin (D’Arcangelo et al., 1995;Meyer et al., 2000) that is involved in cell positioning anddifferentiation, and the formation of synaptic connections(Rice and Curran, 2001; Forster et al., 2002).

We did not find any colabeling of CR and reelin at anytime point after BrdU (Fig. 1C). Interestingly, however,there was a characteristically shaped population of reelin-expressing cells in the SGZ. Morphologically, these cellswere identified as basket cells. They were consistentlyBrdU-negative. Taken together, we conclude that newbornCR-expressing cells were neither GABAergic interneuronsnor Cajal–Retzius cells.

Calretinin is expressed in maturing granule cells

Mature granule cells are glutamatergic and thus expressthe excitatory amino acid transporter (EAAT). They are alsodistinguished by the basic homeobox transcription factorProx-1 (Pleasure et al., 2000). To determine whether newCR-positive neurons would already colabel with these pro-teins characteristic of granule cells, we analyzed the expres-sion patterns of EAAT and Prox-1 in BrdU/CR-double-positive cells. We found that at 1 week after BrdU virtuallyall BrdU-labeled CR-expressing cells were positive forProx-1 and also coexpressed EAAT (Fig. 1F/G). Thesefindings indicate that the population of CR-positive cells inthe granule cell layer early possessed granule cell charac-teristics.

Maturation of new granule cells in the adult dentategyrus resembles hippocampal developmentat Postnatal Day 7

We also intended to determine whether the expression ofCR during granule cell maturation would be specific to adultneurogenesis or would mirror the situation during hippo-campal development. At Postnatal Day 7 (P7) we detecteda strong expression of CR colabeling with Prox-1 in theinner zone of the developing dentate gyrus (Fig. 1D). Fur-thermore, these CR-positive cells colabeled with NeuNeven at this early stage of development.

Maturing granule cells in the adult dentate gyrus switchthe expression of calcium-binding protein calretininto calbindin

Mature granule cells express calbindin (Sloviter, 1989)and calbindin expression has been used to identify thenewly generated granule cells of the dentate gyrus. At 4weeks after BrdU, most newborn granule cells have becomecalbindin-positive (Kempermann et al., 1997a). As the log-ical next step we analyzed whether there was an observableswitch from CR expression to calbindin expression duringthe maturation of newborn granule cells. Immunohisto-chemical double-staining against CR and calbindin showedsingle newborn cells that expressed CR and calbindin at thesame time (Fig. 1E). This finding was suggestive of eitheran extremely rapid switch in calcium-binding protein ex-pression or the normal absence of both CR and calbindin inan intermediate state. Irrespective of this consideration,however, our data indicate that newborn granule cellsswitched from CR (Fig. 1E, top right; 1 day after BrdUinjections) to calbindin (Fig. 1E, bottom right; 4 weeks afterBrdU injections).

Calretinin is expressed early in postmitotic granule cells,whereas doublecortin is already expressed on the level ofproliferative precursor cells

It is hypothesized that the generation of new granulecells passes from a dividing stem or progenitor cell in theSGZ over a restricted precursor to a mature neuron. Wetherefore analyzed at which time point in this sequence ofdevelopmental steps CR would become detectable. To iden-tify cells that were in the cell cycle at the time point ofinvestigation, we used antibodies against Ki-67, a cell cy-cle-related antigen. Screening hundreds of Ki-67-positivecells we did not detect any cells with a colabeling for Ki-67and CR (Fig. 1C). From this finding we conclude that CR isexpressed in postmitotic, newly generated granule cells. Inclear contrast to this finding numerous DCX-expressingcells showed coexpression of Ki-67, indicating that thesecells could still divide. Thus among the DCX-expressingcells, CR expression defines the subpopulation that hasexited from the cell cycle and has become postmitotic. In

605M.D. Brandt et al. / Molecular and Cellular Neuroscience 24 (2003) 603–613

Fig. 1. (A) Calcium-binding proteins in the adult dentate gyrus. Top left: parvalbumin-expressing basket cells are primarily located in the hilus (arrow). Theyare larger than granule cells and in our hands have never been found to be labeled with BrdU, indicating that they are not generated in adulthood. Scale bar,50 �m. Bottom left: 4 weeks after BrdU, most BrdU-labeled neurons are calbindin-positive (blue), but � 6% are expressing calretinin (CR, green; arrow).Scale bar, 50 �m. Right: Triple-labeling of BrdU-marked cells (red) with neuronal marker NeuN (green) and CR (blue) indicates that a subpopulation ofnewly generated neurons express calretinin. Three-dimensional reconstruction of a z series through the cell in question along the yz axis (right narrow panel)and xz axis (bottom narrow panel) confirms that all three labels are present in the same cell, which thus appears whitish. Scale bar, 10 �m. (B) Left: At earlytime points (3 days) after BrdU, many CR-labeled cells (green) also express doublecortin (DCX, blue), a marker of immature neurons. Right: At 4 weeksafter BrdU, with few exceptions CR-positive cells have lost their DCX expression. Three-dimensional reconstruction as described for (A). Scale bar for bothpanels, 10 �m. A subpopulation of DCX-expressing cells is proliferative (see (G)). (C) CR-expressing BrdU-positive cells of the SGZ are neither GABAergiccells (and thus putative interneurons; left and right top) nor Cajal–Retzius neurons (right bottom), because they contain neither GABA (green; CR: blue;BrdU; red; left) nor GABAAI receptor (blue; CR: green; BrdU: red; right top) nor reelin (green; CR: blue; BrdU: red; right bottom). Three-dimensionalreconstruction as described in (A). Scale bars, 10 �m. Images from 4 weeks after BrdU. (D) On Postnatal Day 7, there is a large overlap of CR expression(blue) with granule cell marker Prox-1 (green, left panel; see also (F)) and neuronal marker NeuN (green, right panel). Scale bar, 50 �m for both panels. (E)Left: In the adult dentate gyrus very few cells are simultaneously positive for CR (green) and calbindin (blue, see also insert); Scale bar, 10 �m. Right top:7 days after BrdU, the majority of BrdU-labeled cells (red) is CR-positive (blue; calbindin: green; BrdU: red). Right bottom: at 4 weeks BrdU-labeled cells(red) are mostly calbindin-positive (green; calretinin: blue). Scale bar, 50 �m for both right panels. (F) CR-positive cells express the excitatory amino acidtransporter EAAT (green; BrdU: red; CR: blue), supporting the idea that the new cells are developing into excitatory neurons. Three-dimensionalreconstruction (right) panel as described in (A). Scale bar, 10 �m. (G) BrdU-positive (red) CR-expressing (blue) cells are Prox-1-positive (green). Prox-1

606 M.D. Brandt et al. / Molecular and Cellular Neuroscience 24 (2003) 603–613

this context, CR can therefore be considered an early andspecific marker of immature, yet postmitotic adult-bornneurons.

Maturation of new granule cells is influenced by physicalactivity, environmental enrichment, and kainate-inducedseizures

The number of newborn neurons in the adult dentategyrus is regulated by a variety of physiological and patho-logical experimental stimuli. To confirm and extend theabove observations we analyzed the dynamic changes inCR-expressing new cells in paradigms of voluntary physicalactivity (RUN), environmental enrichment (ENR), and kai-nate-induced hippocampal seizures (KA) as known inducersof adult neurogenesis. These three stimuli differ with regardto their impact on cell proliferation in the adult dentategyrus. Environmental enrichment has no or limited effectson cell proliferation, but primarily exerts a survival promot-ing effect on the progeny of dividing cells (Kempermann etal., 1997b). Voluntary wheel running robustly increases cellproliferation (Van Praag et al., 1999), and kainate-inducedseizures belong to the strongest stimulators of cell divisionin the dentate gyrus (Parent et al., 1997).

As a general rule, in all three models at 4 weeks afterBrdU net neurogenesis (as calculated by BrdU-numbers andcolabeling with NeuN) was reflected in the absolute num-bers of CR-expressing cells within the granule cell layer

(Fig. 3). ENR did not show a significant difference in theabsolute number of CR-expressing cells or of BrdU/CR-positive cells compared to CTR. In KA the increase inBrdU/CR-positive cells was almost 15-fold in comparisonto CTR (Fig. 3F). The effect of RUN ranged between. Weinterpret this as a stimulus-dependent progression from celldivision into a postmitotic stage of adult neurogenesis.

Discussion

In this study we show that calretinin is transiently ex-pressed in maturing granule cells of the adult dentate gyrus.Until now the population of CR-expressing cells within thegranule cell layer of the murine dentate gyrus had not beencharacterized in great detail. CR has been considered amarker of three different cell types in the dentate gyrus:hilar mossy cells, interneurons in the SGZ, and Cajal–Retzius cells. In addition, a report by Liu et al. has sug-gested that CR-expressing cells of the adult SGZ might beimmature granule cells, but this assumption was based onlyon the presence of PSA-NCAM in these cells and could stillhave been compatible with the generation of new interneu-rons (Liu et al., 1996).

Mossy cells have a characteristic appearance and aredistinctively located in the hilus. They have never beenfound to be BrdU-positive.

Cajal–Retzius neurons secret reelin and a deletion of

Fig. 2. (A) Percentage of CR-labeled cells among the BrdU-positive cells at different time points after BrdU. ***P � 0.001. (B) Time course of phenotypicmarker expression in BrdU-labeled cells at different time points after BrdU. The number of BrdU-positive cells per granule cell layer (GCL; open bars) peaksat 3 days after BrdU and thereafter decreases, reaching a more or less stable level at about 2.5 weeks. Very early after division, neuronal markers such asNeuN and granule cell marker Prox-1 are expressed in BrdU-positive cells. These two populations are most likely identical and become stable after about2.5 weeks after division. CR as a transient postmitotic marker parallels the first half of this curve. CR expression peaks at 3 days in absolute terms, continuesto increase in relative terms (A) until 1 week after BrdU, and thereafter fades out. Later than 6 weeks after BrdU it is no longer detectable in BrdU-markedcells (not shown).

is a transcription factor, active in granule cells (Pleasure et al., 2000). The frame in the central image relates to the enlargement at the right, in which emissionintensities along the horizontal line drawn across the triple-labeled cell are depicted (bottom right graph). Scale bar in central panel, 50 �m. (H) Left:CR-positive cells are postmitotic. CR-expressing cells (blue), labeled with BrdU (red) 7 days earlier and thus identified as “new” are negative for cell cyclemarker Ki-67 (green; arrow) which identifies cells undergoing division at this moment. The bottom panel shows the three detection channels separately, andthe emission intensities measured along the horizontal line drawn in the large panel demonstrates the presence or absence of colocalization of the three labels.Note that one BrdU-labeled cell, marked by the cross-hair, is also positive for Ki-67, indicating continued division. Scale bar, 10 �m. Right: DCX-positivecells (blue) are actively dividing, as visualized by their immunoreactivity for Ki-67 (green; BrdU: red). Scale bar, 10 �m.

607M.D. Brandt et al. / Molecular and Cellular Neuroscience 24 (2003) 603–613

reelin leads to the phenotype of the reeler mutant(D’Arcangelo et al., 1995) with its characteristic distur-bances of cortical and hippocampal layering (Stanfield andCowan, 1979a, 1979b,). The function of reelin seems to gobeyond the regulation of neuronal migration (Forster et al.,2002) and it has been suggested that the reeler mutant hasdisturbed adult hippocampal neurogenesis (Kim et al.,2002). Cajal–Retzius cells are primarily located in the mo-lecular layer (Del Rio et al., 1996; Forster et al., 2002;Gebhardt et al., 2002) and have not been described for theSGZ. Nevertheless, as most studies on Cajal–Retzius neu-rons had examined the early postnatal brain, it remainedconceivable that a related calretinin-expressing cell popula-

tion of the SGZ might have relevance as “pioneer neurons”for adult hippocampal neurogenesis similar to that of Cajal–Retzius neurons in the marginal zone during development.However, we did not find any colocalization of CR andreelin in the SGZ (although numerous cells were CR/reelin-double-positive in the molecular layer).

For the CR-expressing cells of the granule cell layerproper it had been tacitly assumed that these cells wereinterneurons. This, despite the fact that some putative CR-positive interneurons were found to be devoid of GABAstaining (Freund and Buzsaki, 1996). In our study, CR/BrdU-double-positive cells were consistently negative forGABA and GABAA1-receptor. The colocalization of CRwith Prox-1 and EAAT, and the time course of developmentsuggest that the CR-expressing cells of the dentate gyrus arein fact immature granule cells. Our qualitative data fromearly postnatal development (P7) confirm that maturinggranule cells pass through a phase of CR expression. Studiesof the prenatal and postnatal expression of CR in the hip-pocampus of the rat have demonstrated a transient CRexpression in a large number of migrating pyramidal cells atE20 and at birth (Jiang and Swann, 1997). Such a transi-tional CR expression could indicate that CR is generallyrequired for the maturation of certain neuronal populations.This finding leads to the somewhat surprising conclusionthat granule cells apparently switch their calcium-bindingprotein from calretinin to calbindin. They do so roughlybetween 10 days (when the first new calbindin-positive cellscan be found) and about 6 weeks after BrdU. At 4 weeksafter BrdU, the time point commonly chosen in our andother studies, CR-expressing cells account for about 6% ofthe BrdU-marked cells. In general, the transient expressionof calcium-binding proteins is not uncommon in the CNS,as, for example, a study by Liu and Graybiel (1992) hasshown for calbindin in the ganglionic eminence, the stria-tum, and the neocortex.

The switch from calretinin to calbindin seems to occurvery swiftly, because cells expressing CR and calbindinsimultaneously are rare, but have been described before forother hippocampal regions (Wouterlood et al., 2001). Pre-sumably, this time point marks an important stage in granulecell development. Mature granule cells may need calbindinto control calcium homeostasis and to regulate calciumchannel activity (Lledo et al., 1992). However, knowledgeabout the function of different calcium-binding proteins isscant. Therefore, currently no meaningful hypothesis can beproposed for why this switch might be functionally neces-sary. CR-deficient mice show impaired long term potentia-tion (LTP) only if induced in the dentate gyrus but not inCA1 (Schurmans et al., 1997). That study supported the ideathat the excitatory hilar mossy cells were responsible for theimpaired LTP. However, our data suggest that this LTPimpairment could also be due to a disturbed maturation ofnewborn granule cells. CR, however, does not seem to beindispensable, because in the SGZ of rats CR is expressed inonly a few cells (Liu et al., 1996), despite the fact that both

Fig. 3. Voluntary wheel running (RUN; A/B), environmental enrichment(ENR; C/D), and KA-induced seizures (KA; E/F) all robustly induce adulthippocampal neurogenesis, as indicated by the increased number of BrdU-labeled cells per granule cell layer (GCL) at 4 weeks after BrdU (seeExperimental Methods for details of BrdU application in these paradigms)and the parallel increase in BrdU-labeled NeuN-positive cells. Both thenumbers of BrdU-labeled CR-positive cells (“new CR”; B, D, and F) andthe absolute counts of CR-expressing cells (A, C, and E) reflect thisinduction of adult neurogenesis. The magnitude of the induction variesbetween RUN, ENR, and KA compared to controls (CTR), and CR countsand new CR cells mirror these differences. *P � 0.05; **P � 0.01; ***P� 0.001.

608 M.D. Brandt et al. / Molecular and Cellular Neuroscience 24 (2003) 603–613

learning abilities and adult neurogenesis in rats are verysimilar to those of mice.

Electrophysiological analyses of “interneurons” in theSGZ have revealed a variety of partly unexpected responsepatterns (Mott et al., 1997). An interesting albeit speculativepossibility is that some of this variance could be explainedby the presence of immature granule cells among the cells.It has been shown that with maturation over a period ofseveral weeks or months, new granule cells develop elec-trophysiological properties very similar to those of the oldergranule cells (Van Praag et al., 2002), but nothing is knownabout the changing electrophysiological properties of devel-oping adult-generated granule cells. The present immuno-histochemical identification of definable stages of neuronaldevelopment in the adult hippocampus will have to becomplemented by electrophysiological studies and the char-acterization of these stages on a molecular level. Calretininas marker defining the essential step in neuronal develop-ment when the cells become postmitotic will be a veryuseful tool in this context.

Related to our present finding is the question whetherunder normal conditions, new interneurons other than CR-positive could be generated by adult hippocampal neuro-genesis. This was suggested by a recent study in rats (Liu etal., 2003). In our experiment, extensive screening of BrdU-labeled cells did not reveal any cells colabeled withGABAA1 receptor (Bouilleret et al., 2000), GABA (Fig.1C), or GAD67 (not shown). Our finding of Prox-1 andEAAT in CR-positive cells argues in favor of the idea thatthese new cells are acquiring a granule cell phenotype. It hasalso been shown that new neurons early after division ex-tend their axon to CA3 as do older granule cells (Stanfieldand Trice, 1988; Hastings and Gould 1999; Markakis andGage, 1999). Additionally, the GABAergic nature of CR-positive cells in the dentate gyrus has been debated (Miet-tinen et al., 1992; Soriano and Frotscher, 1993). Togetherthese arguments, however, cannot rule out that developingnew granule cells might go through a stage of interneuron-like functional properties. Nor do they exclude that newinterneurons could be generated in the adult hippocampus.Still, a discrepancy between our findings and the Liu studyis palpable, and further experiments are required to settle thequestion of interneurogenesis in the adult dentate gyrus.

Prox-1 is a transcription factor that is expressed in gran-ule cells (Pleasure et al., 2000). Our data indicate thatProx-1 is expressed early in granule cell development, at astage at which the cells are still proliferative. Thus, dividingProx-1-expressing cells could be classified as committedprecursor cells. In contrast, CR-expressing cells are consis-tently Ki-67-negative. As all BrdU-labeled CR-positivecells were also Prox-1-positive, CR expression character-izes the stage, when the developing new granule cells be-come postmitotic.

This has interesting consequences regarding the homo-geneity of the population of DCX-expressing cells. DCX isconsidered an immature neuronal marker (des Portes et al.,

1998), often related to cell migration (Corbo et al., 2002).However, based on the expression of intermediate filamentnestin and their proliferative activity, DCX-expressing cellsalso show features of progenitor cells in vivo (Filippov etal., 2003). The partial overlap with Prox-1 and CR indicatesthat the population of DCX-expressing cells contains pro-genitor cells committed to a neuronal phenotype, as well aspostmitotic cells further destined to acquire a granule cellphenotype.

The expression of Prox-1 and EAAT in CR-expressingcells is an argument in favor of the idea that these cells arein the process of developing into granule cells. We hypoth-esize that CR is a transient marker during normal develop-ment. Alternatively, might CR be expressed in those granulecells that never reach maturity but are eliminated before?This possibility, however, is unlikely to be true. If CR werea cell death marker, essentially all new granule cells wouldhave to be born later than 1 week after BrdU and 40% ofthem later than 2.5 weeks, because at 1 week all CR-positive cells were NeuN- and Prox-1-positive and at 2.5weeks still 39% of the NeuN-positive cells were CR-posi-tive. Thus all new granule cells would originate from celldivisions later than 1 to 2.5 weeks after the initial divisionwhich started the process of “neurogenesis.” One wouldhave to postulate that adult neurogenesis proceeded in atleast two or three waves with a first set of new neurons thatis entirely eliminated within the first 2.5 weeks. This wouldalso indicate a population of only rarely dividing progenitorcells that pick up their proliferative activity late after aninitial division (during which they incorporated BrdU).Hayes and Nowakowski have estimated that dividing cellswould dilute BrdU below the level of detection as soon asafter the third division (Hayes and Nowakowski, 2002).Because NeuN-positive cells are postmitotic, only the smallfraction of BrdU-positive cells which are CR and NeuN-negative could account for this population. An expansion ofthis population seems generally conceivable, but the hypo-thetical replacement wave would have to be unnoticeable onthe level of total BrdU counts because between 2.5 and 4weeks the numbers of both BrdU-positive cells and BrdU-marked cells expressing Prox-1 and NeuN did not changeany more. Also, the progression from immature to matureneuronal markers in CR-expressing cells argues against theidea that CR was linked to the initiation of cell death.Environmental enrichment has a survival-promoting effecton newborn cells. Young et al., (1999) have demonstratedthat the number of apoptotic cells in the SGZ decreases inenriched living animals. In our experiment, however, ENRincreased the number of CR-positive cells in the SGZ.Taken together, the scenario that CR expression indicatesthe population of eliminated new granule cells seems un-likely.

Based on our data the following time course of granulecell development can be proposed (Fig. 4). Uncommittedprogenitor cells are nestin-positive (Lendahl et al., 1990;Filippov et al., 2003). There are two types of nestin-positive

609M.D. Brandt et al. / Molecular and Cellular Neuroscience 24 (2003) 603–613

proliferating cells, astrocyte-like type-1 cells and type-2cells lacking glial features but expressing DCX (Filippov etal., 2003). Presumably, at this stage a fate choice decisiontoward neuronal development is possible, and the develop-ing cells then express Prox-1 as the earliest persistentmarker of future granule cells (Pleasure et al., 2000). How-ever, at this stage the cells can still divide (Fig. 2B). Thus,at present it cannot be proven that the Prox-1-positive cellsare derived from proliferative cells, which are Prox-1-neg-ative, but DCX- and nestin-positive. Prox-1/DCX-double-positive cells, however, can leave the cell cycle and within3 days after division express CR early on as a postmitoticmarker for developing granule cells (Fig. 1C). CR expres-sion peaks between 3 days and 1 week after division. Mostof the newly generated cells die (Biebl et al., 2000; Kem-permann et al., 2003). Approximately 3 days after divisionthe first cells expressing neuronal marker NeuN appear(although a very low number is found even earlier), reach-ing a stable number at about 2.5 weeks after the BrdUinjection. Under normal conditions this number seems toremain fairly constant, with comparatively few cells dyingoff in this population. Among the new neurons, DCX ex-pression declines as does CR, but CR disappears moreslowly. At about 3 weeks after BrdU, CR is replaced bycalbindin. At 4 weeks after BrdU only about 6% of allBrdU-positive cells remain CR-positive; at 6 weeks afterBrdU (and later) BrdU/CR-double-positive cells are absent(not shown).

Our data also show that manipulations with known ef-fects on net neurogenesis possibly affect granule cell devel-opment at the stage of CR expression. Conditions enhancingcell proliferation also enhanced the number of CR-express-ing, early postmitotic cells in the dentate gyrus. Our finding

supports the conclusion that the reported stimuli truly act onthe process of neurogenesis in the sense of neuronal devel-opment. Increased numbers of BrdU/CR-double-positivecells at 4 weeks after BrdU indicate that a larger number ofimmature yet postmitotic granule cells have been commit-ted.

That the total number of CR-expressing cells was notsignificantly increased in ENR compared to CTR mightsuggest that under this particular condition the decision torecruit a new neuron is made when the cell already hasspecific granule cell characteristics. This mechanism mightset apart the more specific stimulators of hippocampal neu-rogenesis, such as environmental enrichment or learning,which primarily affects cell survival (Kempermann et al.,1997b; 2002; Gould et al., 1999), from more general stim-uli, such as physical activity (Van Praag et al., 1999) orseizures (Bengzon et al., 1997; Parent et al., 1997), whichact on earlier steps of adult neuronal development. If thisspeculation holds, the CR-expressing period might repre-sent a phase during which immature neurons have becomeresponsive to direct functional input that triggers their re-cruitment for hippocampal function.

Experimental methods

Animals, housing conditions, and treatments

To determine the time-dependent expression of CR, a setof 6 week-old female C57BL/6 mice (19–24 g; CharlesRiver) were divided into 6 groups and perfused 4 hours (N� 5), 1 day (N � 5), 3 days (N � 5), 1 week (N � 5), 2.5weeks (N � 4), and 4 weeks (N � 5) after a single BrdUinjection (see below). A second set of four mice was exam-ined on P7 to analyze CR expression during hippocampaldevelopment.

An additional 69 female C57BL/6 mice (same age asabove) were divided into four groups: runner (RUN), en-riched (ENR), seizure (KA), and controls (CTR) (RUN: N� 12; ENR: N � 8; KA: N � 6; runner controls: N � 12;enriched controls: N � 8; seizure controls: N � 6). Forreasons of housing capacity at the MDC animal facility, thedifferent experimental paradigms had to be carried out insuccession, so that there was an individual control group foreach condition.

RUN animals lived in standard size cages with unlimitedaccess to a running wheel, two or three animals per cage.Eight mice were housed together in an enriched environ-ment as described earlier (Van Praag et al., 1999). Seizureanimals received 30 mg/kg kainic acid (KA, Sigma) in 0.1M phosphate-buffered saline (PBS) on the day before BrdUinjections (Day 0; see next paragraph), and only those dis-playing continuous convulsive seizure activity were used inthese experiments. Both KA and BrdU were administeredby intraperitoneal injection.

During the first 7 days all animals received one daily

Fig. 4. Schematic visualization of the hypothesized marker progressionduring granule cell development in the adult hippocampus. See Discussionfor details. The precursor cell nomenclature follows our previous report(Filippov et al., 2003). Type-1 cells are characterized by astrocytic prop-erties (including GFAP expression) and a long process spanning the gran-ule cell layer. Type-2 cells lack glial features but express doublecortin(DCX). The question mark (?) reminds that at present it is not knownwhether there are additional distinguishable cells with progenitor cellproperties. The lineage as depicted in the drawing is in many parts spec-ulative and many questions remain open at present. Data from the presentstudy, however, clearly indicate that the early postmitotic stage of devel-oping new granule cells is characterized by transient calretinin expression.

610 M.D. Brandt et al. / Molecular and Cellular Neuroscience 24 (2003) 603–613

injection of 50 mg/kg BrdU (5-bromo-2-deoxyuridine; Sigma)in sterile 0.9% NaCl solution. One day after BrdU injections 7RUN, 4 KA, and 6 CTR animals were perfused (data notshown). The remaining animals continued to live under theirrespective experimental conditions for 28 more days.

All applicable local and federal regulations of animalwelfare were followed.

Tissue preparation

Mice were killed with an overdose of ketamine andperfused transcardially with 4% paraformaldehyde in cold0.1 M phosphate buffer (pH 7.4). The brains were removedand kept in the fixative overnight and then transferred into30% sucrose. Forty-micrometer coronal sections were cutfrom a dry ice-cooled block on a sliding microtome (SM200R, Leica). The sections were stored at �20°C in cryo-protectant with 25% ethylene glycol, 25% glycerin, and0.05 M phosphate buffer. Sections were stained using freefloating immunohistochemistry and prepared for BrdU de-tection by incubation in 2 N HCl for 30 min at 37°C andwashing in 0.1 M borate buffer (pH 8.5) for 10 min.

Immunohistochemistry

To determine the number of BrdU-labeled and CR-ex-pressing cells we used the peroxidase method (ABC system,Vectastain, Vector Laboratories) with biotinylated donkeyanti-rat and anti-rabbit antibodies (1:500; Dianova) andnickel-intensified diaminobenzidine as chromogen. Immu-nofluorescent triple labeling was done as described earlier(Kempermann et al., 2003).

As primary antibodies we used rat anti-BrdU (1:500;Harlan Seralab), mouse anti-NeuN (1:100; Chemicon),mouse anti-calretinin (1:250; Swant), goat anti-calretinin(1:250; Chemicon), rabbit anti-calretinin (1:250; Swant),mouse anti-calbindin D28K (1:250; Swant), goat anti-dou-blecortin (1:200; Santa Cruz Biotech.), goat anti-EAAT(1:250; Chemicon), rabbit anti-GABA (1:250; Sigma), goatanti-parvalbumin (1:1000; Swant), rabbit anti-Prox-1 (1:5000; a gift from Dr. Samuel Pleasure, San Francisco);mouse anti-reelin (clone E4, 1:1000; a gift from Dr. AndreGoffmet, Bruxelles), guinea pig anti-GABAA1 receptor (1:20,000; a gift from Dr. J.M. Fritschy, Zurich), and rabbitanti-Ki67 (1:500; Novocastra).

The fluorescent secondary antibodies used were anti-ratRhodamine-X, anti-mouse FITC, anti-mouse Cy5, anti-rab-bit Cy5, anti-rabbit FITC, anti-goat Cy5 (all 1:250; JacksonLaboratories, distributor: Dianova). Fluorescent sectionswere mounted in polyvinyl alcohol with diazabicyclo-oc-tane as antifading agent.

Quantification of BrdU and calretinin-labeled cells

From all animals series of every sixth section (240 �mapart) were stained for BrdU and for CR with the peroxidase

method. Positive cells were counted using a 40x objective(Leica) throughout the rostro-caudal extent of the granulecell layer. As described previously, the optical dissectormethod was modified in that cells appearing sharp in theuppermost focal plane were not counted (Kempermann etal., 2003). Resulting numbers were multiplied by 6 to obtainthe estimated total number of BrdU and CR-positive cellsper granule cell layer.

Analysis of newly generated cell phenotypes

One-in-twelve series of sections from animals of eachgroup were triple-labeled for BrdU, NeuN, and CR. Fluo-rescent signals were detected using a spectral confocal mi-croscope (Leica TCS SP2). All analyses were performed insequential scanning mode to rule out cross-bleeding be-tween channels. Double-labeling was confirmed by three-dimensional reconstructions of z series covering the entirenucleus (or cell) in question.

From each animal and for every combination of pheno-types 50 BrdU-positive cells within the granule cell layerwere analyzed at the confocal microscope for coexpressionof the different markers. Relative numbers were related tothe absolute counts of BrdU-positive cells per granule celllayer to yield the absolute numbers of BrdU-marked cellsper phenotype. Analogously, series were stained for com-binations of other markers (see Fig. 1).

Images were processed with Adobe Photoshop 6.0, andonly general contrast enhancements and color level adjust-ments were carried out.

Statistical analyses

All numerical analyses were performed using Statview5.0.1. For all comparisons ANOVA was performed fol-lowed by Fisher’s post hoc test, when appropriate. Differ-ences were considered statistically significant at P � 0.05.

Acknowledgments

We thank Samuel Pleasure for the Prox-1 antibody,Andre Goffinet for the antibody against reelin, and Jean-Marc Fritschy for the GABAA1-receptor antibody. Thisstudy was supported by VolkswagenStiftung and DeutscheForschungsgemeinschaft (DFG). M.D.B. was supported bygrant GRK-238 from DFG graduate college.

References

Bengzon, J., Kokaia, Z., Elmer, E., Nanobashvili, A., Kokaia, M., Lindvall,O., 1997. Apoptosis and proliferation of dentate gyrus neurons aftersingle and intermittent limbic seizures. Proc. Natl. Acad. Sci. USA 94,10432–10437.

611M.D. Brandt et al. / Molecular and Cellular Neuroscience 24 (2003) 603–613

Biebl, M., Cooper, C.M., Winkler, J., Kuhn, H.G., 2000. Analysis ofneurogenesis and programmed cell death reveals a self-renewing ca-pacity in the adult rat brain. Neurosci. Lett. 291, 17–20.

Bouilleret, V., Loup, F., Kiener, T., Marescaux, C., Fritschy, J.M., 2000.Early loss of interneurons and delayed subunit-specific changes inGABA(A)-receptor expression in a mouse model of mesial temporallobe epilepsy. Hippocampus 10, 305–324.

Corbo, J.C., Deuel, T.A., Long, J.M., LaPorte, P., Tsai, E., Wynshaw-Boris, A., Walsh, C.A., 2002. Doublecortin is required in mice forlamination of the hippocampus but not the neocortex. J. Neurosci. 22,7548–7557.

D’Arcangelo, G., Miao, G.G., Chen, S.C., Soares, H.D., Morgan, J.I.,Curran, T., 1995. A protein related to extracellular matrix proteinsdeleted in the mouse mutant reeler. Nature 374, 719–723.

Del Rio, J.A., Heimrich, B., Super, H., Borrell, V., Frotscher, M., Soriano,E., 1996. Differential survival of Cajal-Retzius cells in organotypiccultures of hippocampus and neocortex. J. Neurosci. 16, 6896–6907.

des Portes, V., Pinard, J.M., Billuart, P., Vinet, M.C., Koulakoff, A.,Carrie, A., Gelot, A., Dupuis, E., Motte, J., Berwald-Netter, Y., Catala,M., Kahn, A., Beldjord, C., Chelly, I., 1998. A novel CNS generequired for neuronal migration and involved in X-linked subcorticallaminar heterotopia and lissencephaly syndrome. Cell 92, 51–61.

Filippov, V., Kronenberg, G., Pivneva, T., Reuter, K., Steiner, B., Wang,I.P., Yamaguchi, M., Kettenmann, H., Kempermann, G., 2003. Sub-population of nestin-expressing progenitor cells in the adult murinehippocampus shows electrophysiological and morphological character-istics of astrocytes. Mol. Cell. Neurosci. 23, 373–382.

Forster, E., Tielsch, A., Saum, B., Weiss, K.H., Johanssen, C., Graus-Porta,D., Muller, U., Frotscher, M., 2002. Reelin, Disabled 1, and beta 1integrins are required for the formation of the radial glial scaffold in thehippocampus. Proc. Natl. Acad. Sci. USA 99, 13178–13183.

Freund, T.F., Buzsaki, G., 1996. Interneurons of the hippocampus. Hip-pocampus 6, 347–470.

Gebhardt, C., Del Turco, D., Drakew, A., Tielsch, A., Herz, J., Frotscher,M., Deller, T., 2002. Abnormal positioning of granule cells altersafferent fiber distribution in the mouse fascia dentata: morphologicevidence from reeler, apolipoprotein E receptor 2-, and very lowdensity lipoprotein receptor knockout mice. J. Comp. Neurol. 445,278–292.

Gould, E., Beylin, A., Tanapat, P., Reeves, A., Shors, T.J., 1999. Learningenhances adult neurogenesis in the hippoampal formation. Nat. Neu-rosci. 2, 260–265.

Hastings, N.B., Gould, E., 1999. Rapid extension of axons into the CA3region by adult-generated granule cells. J. Comp. Neurol. 413, 146–154.

Hayes, N.L., Nowakowski, R.S., 2002. Dynamics of cell proliferation inthe adult dentate gyrus of two inbred strains of mice. Brain Res. Dev.Brain Res. 134, 77–85.

Jacobs, B.L., Praag, H., Gage, F.H., 2000. Adult brain neurogenesis andpsychiatry: a novel theory of depression. Mol. Psychiatry 5, 262–269.

Jiang, M., Swann, J.W., 1997. Expression of calretinin in diverse neuronalpopulations during development of rat hippocampus. Neuroscience 81,1137–1154.

Kempermann, G., Gast, D., Gage, F.H., 2002. Neuroplasticity in old age:sustained fivefold induction of hippocampal neurogenesis by long-termenvironmental enrichment. Ann. Neurol. 52, 135–143.

Kempermann, G., Gast, D., Kronenberg, G., Yamaguchi, M., Gage,F.H., 2003. Early determination and long-term persistence of adult-generated new neurons in the hippocampus of mice. Development130, 391–399.

Kempermann, G., Kuhn, H.G., Gage, F.H., 1997a. Genetic influence onneurogenesis in the dentate gyrus of adult mice. Proc. Natl. Acad. Sci.USA 94, 10409–10414.

Kempermann, G., Kuhn, H.G., Gage, F.H., 1997b. More hippocampalneurons in adult mice living in an enriched environment. Nature 386,493–495.

Kim, H.M., Qu, T., Kriho, V., Lacor, P., Smalheiser, N., Pappas, G.D.,Guidotti, A., Costa, E., Sugaya, K., 2002. Reelin function in neuralstem cell biology. Proc. Natl. Acad. Sci. USA 99, 4020–4025.

Kuhn, H.G., Dickinson-Anson, H., Gage, F.H., 1996. Neurogenesis in thedentate gyrus of the adult rat: age-related decrease of neuronal progen-itor proliferation. J. Neurosci. 16, 2027–2033.

Lendahl, U., Zimmerman, L.B., McKay, R.D.G., 1990. CNS stem cellsexpress a new class of intermediate filament protein. Cell 60, 585–595.

Liu, F.C., Graybiel, A.M., 1992. Transient calbindin-D28k-positive sys-tems in the telencephalon: ganglionic eminence, developing striatumand cerebral cortex. J. Neurosci. 12, 674–690.

Liu, S., Wang, J., Zhu, D., Fu, Y., Lukowiak, K., Lu, Y., 2003. Generationof functional inhibitory neurons in the adult rat hippocampus. J. Neu-rosci. 23, 732–736.

Liu, Y., Fujise, N., Kosaka, T., 1996. Distribution of calretinin immuno-reactivity in the mouse dentate gyrus. I. General description. Exp. BrainRes. 108, 389–403.

Lledo, P.M., Somasundaram, B., Morton, A.J., Emson, P.C., Mason, W.T.,1992. Stable transfection of calbindin-D28k into the GH3 cell linealters calcium currents and intracellular calcium homeostasis. Neuron9, 943–954.

Markakis, E., Gage, F.H., 1999. Adult-generated neurons in the dentategyrus send axonal projections to the field CA3 and are surrounded bysynaptic vesicles. J. Comp. Neurol. 406, 449–460.

Meyer, G., Schaaps, J.P., Moreau, L., Goffinet, A.M., 2000. Embryonicand early fetal development of the human neocortex. J. Neurosci. 20,1858–1868.

Miettinen, R., Gulyas, A.I., Baimbridge, K.G., Jacobowitz, D.M., Freund,T.E., 1992. Calretinin is present in non-pyramidal cells of the rathippocampus. II Co-existence with other calcium binding proteins andGABA. Neuroscience 48, 29–43.

Mott, D.D., Turner, D.A., Okazaki, M.M., Lewis, D.V., 1997. Interneuronsof the dentate-hilus border of the rat dentate gyrus: morphological andelectrophysiological heterogeneity. J. Neurosci. 17, 3990–4005.

Parent, J.M., Lowenstein, D.H., 2002. Seizure-induced neurogenesis: aremore new neurons good for an adult brain? Prog. Brain Res. 135,121–131.

Parent, J.M., Yu, T.W., Leibowitz, R.T., Geschwind, D.H., Sloviter, R.S.,Lowenstein, D.H., 1997. Dentate granule cell neurogenesis is increasedby seizures and contributes to aberrant network reorganization in theadult rat hippocampus. J. Neurosci. 17, 3727–3738.

Pleasure, S.J., Collins, A.E., Lowenstein, D.H., 2000. Unique expressionpatterns of cell fate molecules delineate sequential stages of dentategyrus development. J. Neurosci. 20, 6095–6105.

Redecker, C., Luhmann, H.J., Hagemann, G., Fritschy, J.M., Witte, O.W.,2000. Differential downregulation of GABAA receptor subunits inwidespread brain regions in the freeze-lesion model of focal corticalmalformations. J. Neurosci. 20, 5045–5053.

Rice, D.S., Curran, T., 2001. Role of the reelin signaling pathway in centralnervous system development. Annu. Rev. Neurosci. 24, 1005–1039.

Schurmans, S., Schiffmann, S.N., Gurden, H., Lemaire, M., Lipp, H.P.,Schwam, V., Pochet, R., Imperato, A., Bohme, G.A., Parmentier, M.,1997. Impaired long-term potentiation induction in dentate gyrus ofcalretinin-deficient mice. Proc. Natl. Acad. Sci. USA 94, 10415–10420.

Sloviter, R.S., 1989. Calcium-binding protein (calbindin-D28k) and parv-albumin immunocytochemistry: localization in the rat hippocampalwith specific reference to the selective vulnerability of hippocampalneurons to seizure activity. J. Comp. Neurol. 280, 183–196.

Sloviter, R.S., Ali-Akbarian, L., Horvath, K.D., Menkens, K.A., 2001.Substance Preceptor expression by inhibitory interneurons of the rathippocampus: enhanced detection using improved immunocytochemi-cal methods for the preservation and colocalization of GABA and otherneuronal markers. J. Comp. Neurol. 430, 283–305.

612 M.D. Brandt et al. / Molecular and Cellular Neuroscience 24 (2003) 603–613

Soriano, E., Frotscher, M., 1993. GABAergic innervation of the rat fasciadentata: a novel type of interneuron in the granule cell layer with extensiveaxonal arborization in the molecular layer. J. Comp. Neurol. 334, 385–396.

Stanfield, B.B., Cowan, W.M., 1979a. The development of the hippocam-pus and dentate gyrus in normal and reeler mice. J. Comp. Neurol. 185,423–459.

Stanfield, B.B., Cowan, W.M., 1979b. The morphology of the hippocam-pus and dentate gyrus in normal and reeler mice. J. Comp. Neurol. 185,393–422.

Stanfield, B.B., Trice, J.E., 1988. Evidence that granule cells generated inthe dentate gyrus of adult rats extend axonal projections. Exp. BrainRes. 72, 399–406.

Van Praag, H., Kempermann, G., Gage, F.H., 1999. Running increases cellproliferation and neurogenesis in the adult mouse dentate gyrus. Nat.Neurosci. 2, 266–270.

Van Praag, H., Schinder, A.F., Christie, B.R., Toni, N., Palmer, T.D.,Gage, F.H., 2002. Functional neurogenesis in the adult hippocampus.Nature 415, 1030–1034.

Wouterlood, F.G., Grosche, J., Hartig, W., 2001. Co-localization of cal-retinin and calbindin in distinct cells in the hippocampal formation ofthe rat. Brain Res. 922, 310–314.

Young, D., Lawlor, P.A., Leone, P., Dragunow, M., During, M.J., 1999.Environmental enrichment inhibits spontaneous apoptosis, preventsseizures and is neuroprotective. Nat. Med. 5, 448–453.

613M.D. Brandt et al. / Molecular and Cellular Neuroscience 24 (2003) 603–613