Targeted transgene expression in neuronal precursors: watching young neurons in the old brain

Targeted transgene expression in neuronal precursors:watching young neurons in the old brain

Sebastien Couillard-Despres,1,2 Beate Winner,2 Claudia Karl,1,2, Gudrun Lindemann,2 Peter Schmid,2 Robert Aigner,1,2

Joern Laemke,2 Ulrich Bogdahn,2 Juergen Winkler,2 Josef Bischofberger3 and Ludwig Aigner1,2

1Volkswagen-Foundation Junior Group, University of Regensburg, Franz-Josef-Strauss-Allee 11, 93053 Regensburg, Germany2Department of Neurology, University of Regensburg, Universitatsstrasse 84, 93053 Regensburg, Germany3Institute of Physiology, University of Freiburg, Hermann-Herder-Strasse 7, 79104 Freiburg, Germany

Keywords: adult neurogenesis, doublecortin, fluorescent reporter protein, neural stem cell, neuronal migration, transgenic mouse

Abstract

Progress in the field of neurogenesis is limited by the lack of animal models allowing direct detection and analysis of living cellsparticipating in neurogenesis. We engineered a transgenic mouse model that expresses the fluorescent reporter proteins enhancedgreen fluorescent protein or Discoma sp. reef coral red fluorescent protein under the control of the doublecortin (DCX) promoter, agene specifically and transiently active in neuronal precursors and young neurons. The expression of the reporter proteins correlatedwith expression of the endogenous DCX protein, and with developmental and adult neurogenesis. Neurogenesis was unaffected bythe presence of the fluorescent proteins. The transgenic mice allowed direct identification of the very few newly generated neuronspresent in the aged brain. We performed electrophysiological analysis and established that newly generated hippocampal granulecells in aged and young mice shared identical physiological properties. Hence, although the rate of neurogenesis tapers with ageing,a population of highly excitable young neurons indistinguishable to those found in younger animals is continuously generated.Therefore, maintenance of the fundamental properties of neuronal precursors even at advanced age suggests that stimulation ofneurogenesis may constitute a valid strategy to counteract age-related neuronal loss and cognitive declines.

Introduction

Neurogenesis in the mammalian adult CNS is maintained by neuralstem cells located in the subventricular zone of the lateral ventriclesand in the dentate gyrus of the hippocampal formation (Altman & Das,1965; Altman, 1969; Luskin, 1993). These stem cells possess thepotential to proliferate, self-renew and differentiate into neurons andglia. Adult neural stem cells are thought to proliferate slowly (Doetschet al., 1999). However, they can generate fast dividing transit-amplifying progenitors bearing a limited self-renewal capacity (Doe-tsch, 2003). A fraction of the latter is or becomes a pool of neuronalrestricted precursors. The lack of tools for the identification andcharacterization of the subpopulation of cells endowed with neuro-genic capacity impeded advances in the field of adult neurogenesis andstem cell-based CNS regeneration.

The rate of adult neurogenesis is intimately linked to the number ofneuronal precursors, as these cells compose the amplification popu-lation having the potential to generate new neurons. Doublecortin(DCX) is a protein specifically and transiently expressed in neuronalprecursors and young neurons. Detailed analysis demonstrated thatDCX expression was already induced in fast dividing neuronalprecursors, persisted for approximately 30 days and was terminatedthereafter as a consequence of neuronal maturation (Brown et al.,2003). Within this period, the fate of newly generated cells is

determined with respect to migration, survival and elimination(Petreanu & Alvarez-Buylla, 2002). Most importantly, the timing ofDCX expression coincides with the temporal window for synapticintegration of new neurons into the functional network (Carleton et al.,2003; Song et al., 2005).We recently reported that the transient expression pattern of DCX in

neuronal precursors could be used to quantify modulations inneurogenesis levels (Couillard-Despres et al., 2005). Importantly,expression of DCX was observed specifically when new neuronalprecursors were generated and not observed during gliogenesis orneurite regrowth (Couillard-Despres et al., 2005). To take advantageof this specific expression pattern, we isolated a genomic DNAfragment containing regulatory elements of the human DCX gene(hereafter DCX-promoter). In vitro, this human DCX-promoterspecifically drives expression of reporter proteins [e.g. enhancedgreen fluorescent protein (EGFP)] in DCX-expressing neuroblasts andneuronal precursors (Karl et al., 2005).In the present report, we describe the generation of transgenic mice

using the human DCX-promoter to drive expression of the reporterprotein EGFP and Discoma sp. reef coral red fluorescent protein(DsRed2). We documented that the human DCX-promoter was capableof driving expression of transgenes with a pattern similar to the murineendogenous DCX. The detection of living neuroblasts and youngneurons through expression of reporter proteins allowed a direct accessto this intriguing subpopulation, even in aged animals. We report herethat neuronal precursors and young neurons, although getting fewerwith ageing, maintain the same cardinal properties of high excitabilityand plasticity during the whole lifespan of individuals. Hence,

Correspondence: Dr L. Aigner, 1Volkswagen-Foundation Junior Group, as above,

or Dr J. Bischofberger, as above.E-mail: [email protected] [email protected]

Received 7 April 2006, revised 23 June 2006, accepted 3 July 2006

European Journal of Neuroscience, Vol. 24, pp. 1535–1545, 2006 doi:10.1111/j.1460-9568.2006.05039.x

ª The Authors (2006). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing Ltd

stimulation of neurogenesis in ageing constitutes a valid approach tocounteract age-related neuronal deficits.

Materials and methods

Animals and generation of transgenic mouse lines

All experiments were carried out in accordance with the EuropeanCommunities Council Directive of 24 November 1986 (86 ⁄ 609 ⁄ EEC)and were approved by the local governmental commission for animalhealth. For generation of transgenic mouse lines, 100 lg of the vectorphuDCX3509-EGFP or phuDCX3509-DsRed2 (Karl et al., 2005) wasdigested with AflII and XhoI. The encoded EGFP and DsRed2reporter were expressed as cytoplasmic polypeptides, but the latter candistribute within the nucleus of expressing cells. A fragment of 4.5 kbbearing the human DCX regulatory sequences (3.5 kb) and thereporter gene was purified and injected in B6C3F1 mouse embryos at1-cell stage according to standard procedures (Transgenic Mouse Unit,Clinical Research Institute, Montreal, Canada). Transgenic animalswere identified by polymerase chain reaction analysis for the presenceof transgene in their genomic tail DNA using the following primers:EGFPf GCT GAC CCT GAA GTT CAT CTG and EGFPr GGA CTTGAA GAA GTC GTG CTG or DsRed2f CTG TCC CCC CAG TTCCAG T and DsRed2r CGT TGT GGG AGG TGA TGT CCA G. Oneline for each construct was selected for its high levels of transgeneexpression and was further characterized. Similar results wereobtained with the DCX-promoter-EGFP mice and the DCX-promo-ter-DsRed2 mice.

Protein expression analysis

To analyse the expression of DCX and the reporter protein EGFP inthe transgenic mice, tissues were homogenized and processed asdescribed (Brown et al., 2003). The primary antibodies used were:goat anti-doublecortin 1 : 1000 (DCX C-18, Santa Cruz Laboratories,Santa Cruz, USA), goat anti-GFP 1 : 1000 (Rockland, Gilbertsville,USA) and rabbit anti-actin 1 : 5000 (Sigma, Taufkirchen, Germany).Detection of bound primary antibodies was performed using peroxi-dase-conjugated rabbit anti-goat 1 : 50 000 (Sigma) and peroxidase-conjugated donkey anti-rabbit 1 : 50 000 (Jackson ImmunoResearchLaboratories, West Grove, USA), and the ECL Plus chemiluminescentsubstrate according to the manufacturer’s protocol (AmershamBioscience, Buckinghamshire, England). Signals were recorded onHyperfilm ECL chemiluminescence films (Amersham Bioscience).Quantification of protein expression was performed by densitometryon photographic films from two independent Western blots using theImageJ software (available under http://rsb.info.nih.gov/ij/). Levels ofexpression of EGFP and DCX at the various ages were normalized onmeasurements obtained for P0 animals and on the actin detectionsignal.

In vivo analysis of the reporter’s temporal expression pattern innewly generated cells

5-Bromo-2-deoxyuridine (BrdU) labelling was performed as described(Winner et al., 2002) on 2-month-old transgenic and non-transgenicmouse littermates (total n ¼ 110, n ¼ 5 for each time ⁄ genotype). Forthe time points analysed shortly after BrdU injection (i.e. 2 h and 1, 2,4 and 7 days), high temporal resolution concerning the age of newlygenerated cells was achieved using a single BrdU injection. Tocounterbalance the decrement of labelled cells over time (Winner

et al., 2002), higher numbers of labelled cells were generated by dailyinjections of BrdU on Days 1–4 for the medium- and long-termanalysis (7, 10, 14, 21, 30 and 60 days).

Tissue processing, immunohistology and counting procedure

Perfusion of deeply anaesthetized (20.38 mg ⁄ mlketamine, 5.38 mg ⁄ mlxylazine and 0.29 mg ⁄ ml acepromazine) animals, tissue processingand counting were performed as described (Winner et al., 2002).Histological analysis and micrographs were performed using confocalmicroscopy (TCS-NT, Leica Microsystems, Bensheim, Germany). Thefollowing antibodies and final dilutions were used. Primary antibodies:rat anti-BrdU 1 : 250 (Oxford Biotechnology, Oxford, UK); goat anti-doublecortin 1 : 500 (DCX C-18, Santa Cruz Laboratories); mouseanti-NeuN 1 : 500 (Chemicon, Temecula, CA, USA); mouse anti-ratnestin 1 : 500 (Pharmingen International, USA); rabbit anti-glialfibrillary acidic protein (GFAP) 1 : 1000 (Dako, Denmark); guinea piganti-GFAP 1 : 250 (Progen Biotechnik, Heidelberg, Germany); rabbitanti-GFP 1 : 1000 (Molecular Probes, Eugene, OR, USA).

Electrophysiology

Transverse 350-lm-thick hippocampal slices were prepared using acustom-built vibroslicer as described previously (Geiger et al., 2002;Schmidt-Hieber et al., 2004). Briefly, mice were anaesthetized byisoflurane and killed by decapitation, in accordance with institutionalguidelines. Aged mice were kept in a pure oxygen atmosphere for10 min prior to decapitation in order to increase the viability of cellswithin acute slices. For the dissection and storage of the slices, asolution containing (in mm): NaCl, 64; NaHCO3, 25; glucose, 10;sucrose, 120; KCl, 2.5; NaH2PO4, 1.25; CaCl2, 0.5; MgCl2, 7(equilibrated with 95% O2 ⁄ 5% CO2) was used. Slices were incubatedat 35 �C for 30 min and subsequently held at room temperature.Dentate gyrus granule cells were identified by infrared-difference-interference-contrast (IR-DIC) video-microscopy and simultaneousdetection of DsRed2 fluorescence with a back-illuminated cooled-frame-transfer CCD-camera (EBFT 512, Princeton Instruments), asdescribed previously (Normann et al., 2000). The excitation lightsource (Polychrome II with 75 W Xenon lamp, TILL Photonics,Munich, Germany) was coupled to the epifluorescent port of themicroscope (Axioskop FS2, Zeiss; 60 · water immersion objective,Olympus) via a light guide. To minimize bleaching, the intensity of theexcitation light (550 nm) was reduced to 10%. The filter combinationfor excitation and emission comprised a beam splitter (FT580) and anemission filter (LP590) from Zeiss (Jena, Germany). DsRed2-expres-sing newly generated neurons were readily detected in young and agedtransgenic mice. However, as a result of the severe decrease ofneurogenesis in the dentate gyrus of aged mice, some aged animals usedfor this study were provided with a running wheel. This resulted in anincrease in the number of newly generated DsRed2-expressing neurons.For patch-clamp recordings, slices were superfused with a physio-

logical extracellular solution containing (in mm): NaCl, 125; NaH-CO3, 25; glucose, 25; KCl, 2.5; NaH2PO4, 1.25; CaCl2, 2; MgCl2, 1(equilibrated with 95% O2 ⁄ 5% CO2). Voltage signals were measuredwith an Axopatch 200A amplifier (Axon Instruments) in the I-clampfast mode, filtered at 5 kHz, and digitized at 10 or 20 kHz using a1401plus interface (Cambridge Electronic Design). For whole-cellrecordings, patch pipettes were filled with a solution containing (inmm): K-gluconate, 135; KCl, 20; MgCl2, 2; K2ATP, 4; NaGTP, 0.3;HEPES, 10 (pH 7.2). Bridge balance was used to compensate theseries resistance of 10–50 MW. The holding potential was set to

1536 S. Couillard-Despres et al.

ª The Authors (2006). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing LtdEuropean Journal of Neuroscience, 24, 1535–1545

)80 mV. Recordings were performed at 21–23 �C. Traces wereanalysed with IGOR (Wavemetrics). The membrane time constant wasestimated by fitting a monoexponential function to the voltage decayafter a hyperpolarizing current-pulse (1 s). Values of the electrophys-iological analysis indicate mean ± SEM if not stated differently.

Biocytin staining and morphology

Cells were filled with biocytin (1 mg ⁄ mL) during whole-cell record-ing and subsequently fixed in 4% paraformaldehyde overnight. Afterwash, tissue sections were incubated at 4 �C with fluoresceinisothiocyanate (FITC)-conjugated avidin-D (2 lL ⁄ mL, Vector) and0.3% Triton X-100 for 24 h. After wash, slices were embedded inProlong Antifade (Molecular Probes). Fluorescence was analysed witha confocal laser scanning microscope (LSM 510, Zeiss), using anargon laser (488 nm) for the selective excitation of FITC, and a HeNelaser (543 nm) for the excitation of DsRed2. The FITC and DsRed2fluorescence were recorded in 1-lm optical sections using a 40 ·objective (oil, NA 1.4). The morphology of the cells was analysed inmerged images from 50–100 confocal sections of 1 lm thickness.

Statistical analysis

Data are presented as mean values ± SD. Statistical analysis wasperformed using the unpaired, two-sided t-test comparison Student’st-test and linear regression analysis (Prism 4, GraphPad Software).The significance level was assumed at P < 0.05, unless otherwiseindicated.

Results

Reporter gene expression in transgenic mice is co-regulatedwith endogenous DCX

DCX is abundantly expressed in neuronal precursors during embry-onic development. Its presence was shown to be required forappropriate migration and positioning of neuronal precursors intothe developing cortex (des Portes et al., 1998; Gleeson et al., 1998).After birth, levels of DCX protein expression detected in whole-brainhomogenates fall rapidly to the limit of detection. However, highlevels of DCX expression are maintained in regions bearing activeadult neurogenesis, namely the hippocampal dentate gyrus and thesubventricular zone-rostral migratory stream-olfactory bulb axis(Brown et al., 2003).

Expression of the EGFP reporter protein driven by the human DCX-promoter was assessed at various developmental and postnatal ages.Figure 1A shows an example of EGFP and DCX immunodetection inwhole-brain homogenates for the various ages analysed. Relativedensitometric measurements realized on Western blots provided thetemporal patterns of DCX and EGFP expression (Fig. 1B). Thesuperimposition of the two expression curves showed that activity ofthe human DCX-promoter in transgenic mice reflects activity of theendogenous DCX gene. Therefore, it correlates with developmentalneurogenesis and with the age-dependent decrease of neurogenesis.

Expression of DCX is specific for the nervous system. Theexpression pattern of the EGFP reporter protein driven by the humanDCX-promoter was therefore investigated within various organs ofadult (3 months old) transgenic mice. Western blot analysis forexpression of DCX and EGFP confirmed that both proteins wererestricted to the nervous system (Fig. 1C). Absence of EGFPexpression in non-CNS tissue was also confirmed by fluorescencemicroscopy (data not shown).

We recently reported a detailed spatio-temporal analysis of DCXexpression at the cellular level during adult neurogenesis in rats(Brown et al., 2003). DCX expression was induced in fast dividingneuronal precursors, persisted for approximately 4 weeks and wasdownregulated during neuronal maturation (Brown et al., 2003). Here,a temporal expression analysis of the EGFP reporter protein expres-sion driven by the human DCX-promoter was achieved in parallel withthe analysis of endogenous DCX expression in adult transgenic mice.The expression of endogenous DCX in the mouse hippocampus and

in the olfactory bulb followed a temporal pattern similar to the oneobserved in rats (Supplementary material, Fig. S1). Moreover, thetemporal expression patterns of EGFP and of endogenous DCX innewly born cells were nearly identical (Fig. 1D). Figure 1E shows anexample of a BrdU-labelled newly generated neuron expressing theEGFP reporter and DCX. Confocal z-axis projections demonstratethe co-localization of the three signals within the same cell. In theolfactory bulb, already 2 h after labelling, EGFP expression was foundin approximately 10% of the BrdU-positive cells (Fig. 1D). Thehighest frequency (68%) of EGFP expression in BrdU-positive cellsoccurred 10 days after labelling. Thereafter, a constant decline of theco-localization was observed, and 60 days after labelling no signifi-cant expression of EGFP could be found in BrdU-positive cells. Atthis time point, most of the BrdU-positive cells in the granule celllayer of the transgenic mice expressed NeuN (approximately 95%),indicating that these cells became mature neurons (SupplementaryFig. S1). In summary, the temporal expression pattern of the EGFPreporter in newly generated cells strongly correlated with theexpression of endogenous DCX (Fig. 1D). Moreover, the expressionof EGFP in the neuronal precursors in transgenic mice did not impedetheir maturation into NeuN-expressing neurons.To further rule out perturbation of neurogenesis rates resulting from

the transgene insertion site or reporter protein expression, cellproliferation and survival was monitored in transgenic animals andcompared with their non-transgenic littermates. Short- and long-termexaminations of BrdU-labelled cells in the olfactory bulb andhippocampus did not reveal any significant differences in theproliferation and survival of newly generated neurons in the transgenicmice as compared with their non-transgenic littermates (Supplement-ary Fig. S2).

Reporter gene expression in neuronal precursors

At the anatomical level, the expression patterns of the reporter genesEGFP and DsRed2 in the adult brain of transgenic mice were verysimilar. However, fluorescence emission of DsRed2 was brighter andmore resistant to bleaching. Thus, the detailed analysis was restrictedto DCX-promoter-DsRed2 mice. Most importantly, the expressionpattern of the reporter genes was very similar to the one described forDCX. For example, DsRed2 (Fig. 2) and DCX expression werepredominantly present in the neurogenic regions, the hippocampaldentate gyrus and the subventricular zone ⁄ rostral migratorystream ⁄ olfactory bulb. At the cellular level, DsRed2 was expressedin the neurogenic regions in cells with morphologies reminiscent ofneuronal precursors migrating between the subventricular zone and theolfactory bulb (Fig. 2B–D) and in cells located in the lower part of thegranular cell layer of the dentate gyrus (Fig. 2E).Within the neurogenic regions, the hippocampus is of particular

interest, because it is critically important for learning and memory.Neurodegeneration in the hippocampus and functionally relatedregions has devastating consequences for memory function andeveryday life. Therefore, we restricted the more detailed analysis at the

Doublecortin-reporter mice 1537


cellular level to the dentate gyrus of adult (3 months old) DCX-promoter-DsRed2 mice. DsRed2-expressing cells were scrutinized forco-expression with cell type-specific markers: GFAP (mainly astro-cytes, but also neural stem cells), nestin (neural stem cells) and NeuN(mature neurons). Importantly, co-expression of the DsRed2 transgene

and the endogenous DCX within newly generated neuronal precursorswas confirmed, and is illustrated in Fig. 3A–D.Additionally, we examined the expression of NeuN in the DsRed2-

expressing cells, a marker for mature neurons (Mullen et al., 1992).During the maturation process of neuronal precursors, there is a period



during which expressions of DCX and NeuN overlap (Brown et al.,2003). In agreement with the transient co-expression of endogenousDCX together with NeuN, we observed some NeuN expression in theDsRed2-positive cells, especially those bearing a lower content ofreporter proteins (Fig. 3E–H).

Nestin is a marker frequently used to identify the neural stem cellpopulation (Lendahl et al., 1990). Upon neuronal specification,expression of nestin is abruptly terminated and does not overlap withthe expression of DCX (Couillard-Despres et al., 2005). Asdocumented in Fig. 3I–L, no expression of nestin could be detectedin DsRed2-expressing cells. Note that the nestin antibody used isknown to react with other structures, including blood vessels.Similarly, we did not observe a co-localization of GFAP and DsRed2expression in the dentate gyrus (Fig. 3M–P). However, occasionalDsRed2-expressing cells were detected in the molecular layer andwere found to co-express GFAP (asterisks Fig. 3M–P). This ectopicexpression was not observed in transgenic mice expressing thereporter EGFP, and is likely to represent a transgene insertion siteinfluence.

Co-localization analyses were also performed in the subventricularzone ⁄ rostral migratory stream ⁄ olfactory bulb system, and compar-able results were obtained. In summary, in regions bearing activeneurogenesis, expression of transgenic reporters under the control ofthe human DCX-promoter correlates with endogenous DCX andallows direct identification of neuronal precursors and youngneurons.

DsRed2-expressing neurons represent young granule cells withenhanced excitability

Newly generated granule cells of the adult hippocampus werepreviously reported to show enhanced electrical excitability (Sch-midt-Hieber et al., 2004). To investigate the electrophysiologicalproperties of the neurons expressing DsRed2 under the control of theDCX-promoter, whole-cell patch-clamp recordings were performed inacute hippocampal slices from 2- to 3-month-old transgenic mice.Granule cells of the dentate gyrus were visually identified using IR-DIC video microscopy. DsRed2 fluorescence was simultaneouslydetected with a cooled CCD camera.

Figure 4A and B shows an example of a DsRed2-negative granulecell, presenting a typical mature morphology with spiny apicaldendrites and no basal dendrites. Its electrophysiological propertiescorresponded to those repeatedly recorded in mature granule cells, i.e.an input resistance (Rin) of 387 ± 107 MW and a membrane timeconstant of s¼ 48 ± 10 ms (Fig. 4C, n ¼ 4). In contrast, DsRed2-expressing cells in the granule cell layer (Fig. 4D and E) showed atotally different morphology with short and immature dendritic trees,

short basal dendrites and a low number of dendritic spines. Theseproperties are typical for newly generated young granule cells (vanPraag et al., 2002; Schmidt-Hieber et al., 2004). On average, theseyoung neurons had a more than 10 times larger input resistance, ascompared with the mature granule cells (Rin ¼ 4.4 ± 1.1 GW), as wellas a substantially slower membrane time constant of s¼ 104 ± 15 ms(Fig. 4F, n ¼ 5). Note the extremely small excitatory currentamplitude of 7 pA, which was sufficient to reach firing threshold inthe cell shown in Fig. 4D.The electrophysiological properties of DsRed2-positive granule

cells were very similar to the properties previously reported fornewly generated granule cells in the rat hippocampus with an inputresistance of 4.5 ± 1.9 GW (Fig. 4F, insert) and s¼ 123 ± 10 ms(described in more detail in Schmidt-Hieber et al., 2004). Thisindicates that the enhanced electrical excitability is a general featureof newly generated young granule cells, identified according to theirexpression of polysialic acid neural cell adhesion molecule (PSA-NCAM) or DCX. This enhanced excitability might be important forthe formation of new synaptic contacts and the functional integrationof the newly generated neurons into the adult hippocampal circuitry.

Newly generated young neurons in the dentate gyrus have thesame properties in young adult and aged mice

Figure 5A and B shows the presence of newly generated granule cells,identified through the expression of DCX and DsRed2, in thehippocampus of a 21-month-old mouse. This demonstrates that,although the rate of neurogenesis dramatically decreases with age(Seki & Arai, 1995; Kuhn et al., 1996; Kempermann et al., 1998),young granule cells are continuously present throughout the wholelifespan of adult mice (� 2 years).To test the properties of newly generated young granule cells in the

aged hippocampus, we performed whole-cell patch-clamp recordingsin acute slices of animals with an age of 16 or 21 months. The inputresistance of DsRed2-positive granule cells in the aged animals wasnot significantly different from Rin values recorded in 2–3-month-oldanimals (Rin ¼ 2.7 ± 1.5 GW, n ¼ 7, P > 0.1). Linear regressionanalysis (Fig. 5C, red line) revealed no significant change of inputresistance according to the age of the mouse (r2 ¼ 0.04, P > 0.5).Examples of the morphology of recorded granule cells in aged animalsare shown in Fig. 5, with representative DsRed2-positive cells atdifferent stages of cellular development (Fig. 5D–F) and a represen-tative DsRed2-negative cell (Fig. 5G). All the recorded DsRed2-positive cells in the granule cell layer showed a high input resistanceand generated action potentials with small excitatory currents in therange of a few pA, such as presented in Fig. 4F. DsRed2-positive cellswith a more immature morphology (Fig. 5D) had the highest Rin, and

Fig. 1. Expression pattern of doublecortin (DCX) and enhanced green fluorescent protein (EGFP) polypeptides in transgenic mice. EGFP transgene expression,under the control of the human DCX-promoter, was compared with the endogenous DCX expression. (A) Western blots on whole-brain homogenates at differentdevelopmental and postnatal time points. (B) Densitometry on Western blots was performed to measure the relative expression levels of the EGFP reporter proteinvs. the endogenous DCX. Gel loading correction was performed using the actin signal, and densitometry values were expressed in percentages of measurementobtained at P0. The superimposition of the expression curves revealed that endogenous and transgenic promoters activities are comparable during development. Errorbars represent the standard deviation. (C) Protein homogenates from various organs of 3-month-old DCX-promoter-EGFP mice were analysed in parallel to confirmthe specificity of EGFP expression in neural tissues. Detection of DCX and EGFP reporter polypeptides was performed on duplicate Western blots. Loading controlwas performed by Coomassie blue staining on parallel gels (data not shown). (D) Expression kinetic of the EGFP reporter protein driven by the human DCX-promoter was compared with the expression of endogenous DCX in 5-bromo-2-deoxyuridine (BrdU)-labelled cells in the olfactory bulb of 2-month-old transgenicanimals at various time points after BrdU incorporation. The percentage of cells expressing DCX or EGFP within the population of BrdU-positive cells is shown.Note the nearly identical temporal pattern of expression of DCX and EGFP in newly born cells. Error bars represent the standard deviation.(E) Confocal micrograph of the olfactory bulb of a DCX-promo-EGFP transgenic mouse showing a newly generated neuron labelled with BrdU (red, 7 dayspost-BrdU injection) concomitantly expressing EGFP (green) and DCX (blue). Z-axis projection confirmed the co-localization of BrdU, EGFP and DCX within thesame cell. Scale bar, 50 lm.



morphological maturation correlated with a decrease in Rin (Fig. 5Eand F). In summary, these data indicated that although the number ofnewly generated young granule cells in the dentate gyrus of agedanimals was decreased as compared with young adults, the electro-physiological properties were very similar throughout the wholelifespan of an animal.

Discussion

The use of DCX as a marker for newly generated neuronal precursorsbecame widespread over the last years (for example, see Magavi et al.,2000; Jin et al., 2001; Arvidsson et al., 2002; Brown et al., 2003; Rao& Shetty, 2004). Moreover, we recently reported that detection of

Fig. 2. Expression of the DsRed2 reporter gene in migrating neuroblasts and young neurons in adult neurogenic brain regions. (A) A schematic representation ofthe system with the hippocampus (HC), subventricular zone (SVZ), ventricle (V), the rostral migratory stream (RMS) and the olfactory bulb (OB). DsRed2expression in young migrating neurons of a 4-month-old transgenic mice detected in (B) the lateral ventricle wall, (C) RMS, (D) OB, and (E) granule cell layer(GCL) of the dentate gyrus. Note the typical chain-like structure of migratory neuroblasts leaving the SVZ.



DCX could be used as a means to monitor the levels of ongoingneurogenesis (Couillard-Despres et al., 2005), and hence to follow therate of neurogenesis and its modulation without the need to performin vivo labelling such as BrdU incorporation. However, because DCXis a cytoplasmic protein, cells need to be fixed and processed forimmunohistology in order to detect the polypeptide, thus prohibitingthe analysis of living cells. Here, we report the analysis ofneurogenesis and young neurons, based on the transgenic expression

of reporter proteins under the control of the human DCX-promoter.Expression of the reporter transgene was found to be specific forneuronal tissue and appropriately regulated during development.Moreover, high levels of reporter expression were retained in theneurogenic regions of the adult CNS, allowing direct detection andanalysis of living neuronal precursors and young immature neurons.A detailed analysis of the cell types expressing the transgenic

reporter under the control of the human DCX-promoter demonstrated

Fig. 3. Histological characterization of human doublecortin (DCX) promoter activity within the hippocampal dentate gyrus. (A–D) Expression of Discoma sp. reefcoral red fluorescent protein (DsRed2) reporter was observed in DCX-expressing cells with processes parallel to the granular cell layer (arrows), as well as DCX-expressing cells already integrated in the granular cell layer (arrowheads). (E–H) Co-expression of DsRed2 together with NeuN was mostly observed in cells bearinglower levels of reporter expression (arrowheads) rather than in cells with high levels of reporter expression (arrows). (I–L) No expression overlap was observedbetween DsRed2 (arrows) and nestin (arrowheads). (M–P) No expression overlap for DsRed2 and glial fibrillary acidic protein (GFAP) were observed within thegranular cells. However, occasional cells in the molecular layer expressed DsRed2 and were found to be GFAP-positive (asterisks). To-Pro3 was used as a nuclearcounterstain, shown in blue (C, G, K and O). Scale bar, 20 lm (P).



Fig.4.

Excitabilityof

Discomasp.reef

coralredfluo

rescentprotein(D

sRed2)-expressingyo

unggranulecellsin

theadulthipp

ocam

pus.Granule

cellsof

thedentategy

ruschosen

fortheirexpression

,or

lack

ofexpression,of

DsR

ed2reporter

werepatch-clam

pedandconcom

itantlylabelled

withbiocytin.(A

andB)Representativemorphologyof

abiocytin-filled

maturegranulecell(green)no

texpressing

theDsR

ed2

reporter

(red).(C)Current-clamprecordings

ofthecellshow

nin

(A).

Tracesrepresentvoltageresponsesto

current-clam

ppulses

(1s)

witham

plitudes

of)20,0,

80and340pA

.(D

andE)Representative

morph

ologyof

abiocytin-filled

younggranulecell(green)expressing

theDsR

ed2repo

rter

(red)un

dertheDCX-promoter

(overlay

perceivedin

yellow

).(F)Current-clamprecordingof

thecellshow

nin

(D)withcurrentam

plitudes

of)1,

0,2,

4and7pA

.Inset,comparisonof

theinpu

tresistance

ofDsR

ed2-negative

and-positivegranulecellswithPSA-N

CAM-negativeand-positivegranulecells(see

Schmidt-

Hieberet

al.,20

04).Error

bars

representthestandard

errorof

themean.



Fig.5.

Fun

ctionalproperties

ofDsR

ed2-expressing

younggranulecellsin

theaged

hipp

ocam

pus.(A

)DsR

ed2

expression

ingranulecells(red)locatedin

thedentategyrusof

a21-m

onth-old

mouse

correlated

with(B)doublecortin

(DCX)expression

(green)as

confi

rmed

byim

munodetection.(C)The

inputresistancesof

DsR

ed2-positive

(red

squares)andDsR

ed2-negative

(black

diam

onds)granulecellswereplotted

againstthe

ageof

theDCX-promoter-D

sRed2transgenicanim

als.Linearregression

revealed

nosignificant

change

oftheinputresistancein

DCX-positivecellswithanim

alage.(D

–F)Representativemorphologyof

differentmaturationstages

ofbiocytin-filled

(green)yo

unggranulecells(D

sRed2-positive)recorded

inaged

anim

als(16–

21mon

ths)andtherespective

Rin.N

otethecorrelationbetweenim

maturemorphologyand

high

Rin.(G

)Representativemorphologyof

abiocytin-filled

(green)maturegranulecell(D

sRed2-negative),withan

inputresistance

of260M

Wrecorded

inan

aged

anim

al(21mon

ths).



predominant expression in neuronal precursors and young neurons.Expression of DCX and transgenic reporter in newly generatedneuronal precursors and young neurons of the adult mouse CNS wastransient and could be detected for approximately 1 month, similarlyto the expression pattern of DCX described for the adult rat CNS(Brown et al., 2003). Downregulation of DCX and reporter expressioncoincided with the induction of NeuN expression, a marker of matureneurons. Importantly, the expression of the reporter protein did notinfluence the cell proliferation, cell survival and ⁄ or the cell fate of thenewly generated cells. Hence, through the transient fluorescentreporter protein expression, the small population of newly generatedneuronal precursors and young neurons could be directly identifiedwithin the neurogenic brain structures. This is of particular interest forthe aged brain, as hippocampal dysfunction due to reduced neuro-genesis may be an important prerequisite for age-related decline ofcognition and memory.Recently, a transgenic model using the proopiomelanocortin

(POMC) promoter was presented as a hippocampal neurogenesisreporter (Overstreet et al., 2004). In this transgenic mouse model, thePOMC promoter was reported to support the expression of the EGFPreporter gene in newly generated neurons of the dentate gyrus.However, this promoter is not specific for newly generated neurons, asit is also expressed in the arcuate nucleus and the nucleus of the tractussolitarius. Moreover, the reporter is not expressed in newly generatedneurons of the olfactory bulb. Finally, expression of the reporter isinduced some days after the birth of newly generated neuronalprecursors. Therefore, although the POMC transgenic mouse is aninteresting model, it does not fulfil the ultimate needs for a generalin vivo neurogenesis reporter.As a consequence of the low abundance of neuronal precursors and

young neurons in the adult dentate gyrus and the former lack of meansto directly identify them, electrophysiological characterization ofnewly generated young neurons has only been recently reported(Overstreet et al., 2004; Schmidt-Hieber et al., 2004). The directidentification of neuronal precursors and young neurons in the dentategyrus of the DCX-promoter transgenic mouse model, however,permitted to easily and specifically target recording electrodes toneuronal precursors in order to study their electrophysiological profile.In agreement with the first report identifying young neurons aposteriori using immunolabelling for PSA-NCAM (Schmidt-Hieberet al., 2004), the recorded DsRed2-expressing young granule cellspresented a strongly enhanced excitability, as compared with maturegranule cells. As shown previously, the firing of action potentials withsmall excitatory currents measured in young neurons appears to becritically important for associative synaptic plasticity (Doetsch & Hen,2005).Due to the scarce abundance of neuronal precursors in the dentate

gyrus of aged animals, most of the accumulated knowledge aboutadult neurogenesis has been obtained by studies of young adultanimals. Consequently, electrophysiological analysis of neuronalprecursors and young neurons in the aged brain has, to our knowledge,not yet been achieved. Direct detection of DsRed2 expression drivenby the human DCX-promoter allowed us to specifically record fromthe small subpopulation of young neurons in the aged hippocampus.Interestingly, no significant differences were documented between theelectrophysiological properties of neuronal precursors recorded inyoung or aged mice. This finding implies that the newly generatedneuronal precursors, although getting fewer with age, bear the samecharacteristics and capacity during the whole lifespan of the animal.Furthermore, this also substantiates the concept that the source ofneuronal precursors, namely the neural stem cells, is also maintainingits properties intact during the whole lifetime of the individual.

Behavioural studies indicate that cognitive performance and spatiallearning decline with ageing (van Praag et al., 2005). This deficit canbe partially reversed by physical exercise, as for example voluntaryrunning in running-wheels. On the other hand, voluntary runningincreases the rate of neurogenesis that is typically very low in agedbrains, suggesting a link between neurogenesis and spatial learning.Understanding adult neurogenesis and the functional maturation ofyoung neurons during ageing will help to establish a causal linkbetween neurogenesis and learning. Moreover, this might finally helpto identify mechanisms to increase cognitive performance and learningin advanced age (van Praag et al., 2005). In addition, the demonstra-tion that young neurons produced in the aged brain possess that samecapacity as those generated in young individuals strongly supports thehypothesis that the age-dependent reduction of neurogenesis could beclosely linked to cognitive deficits. In addition it suggests that theexercise-induced increase in neurogenesis is indeed the mechanism bywhich voluntary running facilitates learning during ageing.In summary, we report here a new transgenic reporter model, based

on the human DCX-promoter, to visualize neurogenesis in situ, infixed or living specimens. Moreover, using fluorescent proteinreporters, neuronal precursors could be identified in living brain slicesfor electrophysiological analysis. This tool thereby opens new avenuesfor detailed studies of this intricate, but limited, cell subpopulation. Inaddition, future experiments using transgenic mice expressing reporterproteins under different cell type-specific promoters might permit tofollow, step by step, the transition of a non-committed stem cell toneuronal restricted precursors and young neurons, and furthermorefollow their integration into the neuronal network, in young as well asin aged animals. Moreover, monitoring of neurogenesis rates afterapplication of modulating stimuli together with the assessment of theassociated functional consequences will allow to identify mechanismsof brain plasticity in the adult and aged brain.

Supplementary material

The following supplementary material may be found onhttp://www.blackwell-synergy.comFig. S1. Expression pattern of endogenous DCX during adult mouse

neurogenesis.Fig. S2. Survival of newly generated cells in neurogenic regions of

adult DCX-promotor-EGFP transgenic vs. non-transgenic littermates.

Acknowledgements

This work was supported by the Volkswagen-Foundation (Hanover, Germany),Fritz-Thyssen-Foundation (Cologne, Germany), by the German FederalMinistry of Education and Research (BMBF # 01GA0510 and # 0312134),and the Deutsche Forschungsgemeinschaft (SFB505 ⁄ C9) (Bonn, Germany).B.W. was supported by the Hochschul- und Wissenschaftsprogramm of theUniversity of Regensburg.

Abbreviations

BrdU, 5-bromo-2-deoxyuridine; DCX, doublecortin; DsRed2, Discoma sp. reefcoral red fluorescent protein; EGFP, enhanced green fluorescent protein; FITC,fluorescein isothiocyanate; GFAP, glial fibrillary acidic protein; IR-DIC,infrared-difference-interference-contrast; POMC, proopiomelanocortin; PSA-NCAM, polysialic acid neural cell adhesion molecule; Rin, input resistance; s,membrane time constant.

References

Altman, J. (1969) Autoradiographic and histological studies of postnatalneurogenesis. IV. Cell proliferation and migration in the anterior forebrain,with special reference to persisting neurogenesis in the olfactory bulb.J. Comp. Neurol., 137, 433–457.



Altman, J. & Das, G.D. (1965) Autoradiographic and histological evidenceof postnatal hippocampal neurogenesis in rats. J. Comp. Neurol., 124,319–335.

Arvidsson, A., Collin, T., Kirik, D., Kokaia, Z. & Lindvall, O. (2002) Neuronalreplacement from endogenous precursors in the adult brain after stroke. Nat.Med., 8, 963–970.

Brown, J.P., Couillard-Despres, S., Cooper-Kuhn, C.M., Winkler, J., Aigner, L.& Kuhn, H.G. (2003) Transient expression of doublecortin during adultneurogenesis. J. Comp. Neurol., 467, 1–10.

Carleton, A., Petreanu, L.T., Lansford, R., Alvarez-Buylla, A. & Lledo, P.M.(2003) Becoming a new neuron in the adult olfactory bulb. Nat. Neurosci., 6,507–518.

Couillard-Despres, S., Winner, B., Schaubeck, S., Aigner, R., Vroemen, M.,Weidner, N., Bogdahn, U., Winkler, J., Kuhn, H.G. & Aigner, L. (2005)Doublecortin expression levels in adult brain reflect neurogenesis. Eur. J.Neurosci., 21, 1–14.

Doetsch, F. (2003) A niche for adult neural stem cells. Curr. Opin. Genet. Dev.,13, 543–550.

Doetsch, F., Garcia-Verdugo, J.M. & Alvarez-Buylla, A. (1999) Regenerationof a germinal layer in the adult mammalian brain. Proc. Natl Acad. Sci. USA,96, 11619–11624.

Doetsch, F. & Hen, R. (2005) Young and excitable: the function of newneurons in the adult mammalian brain. Curr. Opin. Neurobiol., 15, 121–128.

Geiger, J.R., Bischofberger, J., Vida, I., Frobe, U., Pfitzinger, S., Weber, H.J.,Haverkampf, K. & Jonas, P. (2002) Patch-clamp recording in brain sliceswith improved slicer technology. Pflugers Arch., 443, 491–501.

Gleeson, J.G., Allen, K.M., Fox, J.W., Lamperti, E.D., Berkovic, S., Scheffer,I., Cooper, E.C., Dobyns, W.B., Minnerath, S.R., Ross, M.E. & Walsh, C.A.(1998) Doublecortin, a brain-specific gene mutated in human X-linkedlissencephaly and double cortex syndrome, encodes a putative signalingprotein. Cell, 92, 63–72.

Jin, K., Minami, M., Lan, J.Q., Mao, X.O., Batteur, S., Simon, R.P. &Greenberg, D.A. (2001) Neurogenesis in dentate subgranular zone androstral subventricular zone after focal cerebral ischemia in the rat. Proc. NatlAcad. Sci. USA, 98, 4710–4715.

Karl, C., Couillard-Despres, S., Prang, P., Munding, M., Kilb, W., Brigadski,T., Plotz, S., Mages, W., Luhmann, H., Winkler, J., Bogdahn, U. & Aigner,L. (2005) Neuronal precursor-specific activity of a human doublecortinregulatory sequence. J. Neurochem., 92, 264–282.

Kempermann, G., Kuhn, H.G. & Gage, F.H. (1998) Experience-induced neurogenesis in the senescent dentate gyrus. J. Neurosci., 18,3206–3212.

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

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

Luskin, M.B. (1993) Restricted proliferation and migration of postnatallygenerated neurons derived from the forebrain subventricular zone. Neuron,11, 173–189.

Magavi, S.S., Leavitt, B.R. & Macklis, J.D. (2000) Induction of neurogenesisin the neocortex of adult mice. Nature, 405, 951–955.

Mullen, R.J., Buck, C.R. & Smith, A.M. (1992) NeuN, a neuronal specificnuclear protein in vertebrates. Development, 116, 201–211.

Normann, C., Peckys, D., Schulze, C.H., Walden, J., Jonas, P. &Bischofberger, J. (2000) Associative long-term depression in thehippocampus is dependent on postsynaptic N-type Ca2+ channels.J. Neurosci., 20, 8290–8297.

Overstreet, L.S., Hentges, S.T., Bumaschny, V.F., de Souza, F.S., Smart, J.L.,Santangelo, A.M., Low, M.J., Westbrook, G.L. & Rubinstein, M. (2004) Atransgenic marker for newly born granule cells in dentate gyrus. J. Neurosci.,24, 3251–3259.

Petreanu, L. & Alvarez-Buylla, A. (2002) Maturation and death of adult-bornolfactory bulb granule neurons: role of olfaction. J. Neurosci., 22, 6106–6113.

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, J. (1998) A novel CNS gene required for neuronalmigration and involved in X- linked subcortical laminar heterotopia andlissencephaly syndrome. Cell, 92, 51–61.

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.

van Praag, H., Shubert, T., Zhao, C. & Gage, F.H. (2005) Exercise enhanceslearning and hippocampal neurogenesis in aged mice. J. Neurosci., 25,8680–8685.

Rao, M.S. & Shetty, A.K. (2004) Efficacy of doublecortin as a marker toanalyse the absolute number and dendritic growth of newly generatedneurons in the adult dentate gyrus. Eur. J. Neurosci., 19, 234–246.

Schmidt-Hieber, C., Jonas, P. & Bischofberger, J. (2004) Enhanced synapticplasticity in newly generated granule cells of the adult hippocampus. Nature,429, 184–187.

Seki, T. & Arai, Y. (1995) Age-related production of new granule cells in theadult dentate gyrus. Neuroreport, 6, 2479–2482.

Song, H., Kempermann, G., Wadiche, L.O., Zhao, C., Schinder, A.F. &Bischofberger, J. (2005) New neurons in the adult mammalian brain:synaptogenesis and functional integration. J. Neurosci., 25, 10366–10368.

Winner, B., Cooper-Kuhn, C.M., Aigner, R., Winkler, J. & Kuhn, H.G. (2002)Long-term survival and cell death of newly generated neurons in the adult ratolfactory bulb. Eur. J. Neurosci., 16, 1681–1689.



Documents

Targeted transgene expression in neuronal precursors: watching young neurons in the old brain