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Impaired neurogenesis, learning and memory and low seizure threshold associated with loss of neural precursor… Coremans, Vanessa; Ahmed, Tariq; Balschun, Detlef; D'Hooge, Rudi; DeVriese, Astrid; Cremer, Jonathan; Antonucci, Flavia; Moons, Michaël; Baekelandt, Veerle; Reumers, Veerle; Cremer, Harold; Eisch, Amelia; Lagace, Diane; Janssens, Tom; Bozzi, Yuri; Caleo, Matteo; Conway, Edward M Jan 5, 2010

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RESEARCH ARTICLE Open AccessImpaired neurogenesis, learning and memoryand low seizure threshold associated with loss ofneural precursor cell survivinVanessa Coremans1, Tariq Ahmed2, Detlef Balschun2, Rudi D’Hooge2, Astrid DeVriese1, Jonathan Cremer1,Flavia Antonucci3, Michaël Moons1, Veerle Baekelandt5, Veerle Reumers5, Harold Cremer6, Amelia Eisch7,Diane Lagace8, Tom Janssens1, Yuri Bozzi4,9, Matteo Caleo4, Edward M Conway1,10*AbstractBackground: Survivin is a unique member of the inhibitor of apoptosis protein (IAP) family in that it exhibitsantiapoptotic properties and also promotes the cell cycle and mediates mitosis as a chromosome passengerprotein. Survivin is highly expressed in neural precursor cells in the brain, yet its function there has not beenelucidated.Results: To examine the role of neural precursor cell survivin, we first showed that survivin is normally expressedin periventricular neurogenic regions in the embryo, becoming restricted postnatally to proliferating and migratingNPCs in the key neurogenic sites, the subventricular zone (SVZ) and the subgranular zone (SGZ). We then used aconditional gene inactivation strategy to delete the survivin gene prenatally in those neurogenic regions. Lack ofembryonic NPC survivin results in viable, fertile mice (SurvivinCamcre) with reduced numbers of SVZ NPCs, absentrostral migratory stream, and olfactory bulb hypoplasia. The phenotype can be partially rescued, asintracerebroventricular gene delivery of survivin during embryonic development increases olfactory bulbneurogenesis, detected postnatally. SurvivinCamcre brains have fewer cortical inhibitory interneurons, contributing toenhanced sensitivity to seizures, and profound deficits in memory and learning.Conclusions: The findings highlight the critical role that survivin plays during neural development, deficiencies ofwhich dramatically impact on postnatal neural function.BackgroundIn the adult, two major, well-defined neurogenic regionspersist [1]. In the subventricular zone (SVZ), neural pre-cursor cells (NPCs) that arise mostly from the embryo-nic lateral ganglionic eminence (LGE) [2], continuouslyproliferate, and then migrate tangentially along the ros-tral migratory stream (RMS) towards the olfactory bulb(OB) where they differentiate into granular and periglo-merular inhibitory interneurons [3]. In the subgranularzone (SGZ) of the hippocampus, newborn NPCs alsomigrate, but for shorter distances, into the granule celllayer, where they become excitatory granule cells [4].From these neurogenic sites, adult-generated neuronscan migrate to regions of brain injury [5], and establishsynaptic contacts and functional connections [6,7].Decreased neurogenesis induced by prenatal or postnatalstresses is implicated in the development of seizures anddisorders in learning, memory and cognition [8,9]. Thepossibility of preventing onset or progression of theseneural diseases by therapeutically enhancing neurogen-esis [10-15] is prompting efforts to delineate themechanisms and regulatory factors underlying NPC sur-vival, proliferation, differentiation, migration and func-tion. Indeed, numerous neuro-regulatory transcriptionfactors, growth factors and receptors have been identi-fied and characterized (reviewed in [16-19]). However,in spite of advances, effective approaches to prevent andtreat diseases of the central nervous system are lacking,underlining the urgent need to develop better models toelucidate the molecular mechanisms regulating* Correspondence: emconway@interchange.ubc.ca1KU Leuven, VIB Vesalius Research Center (VRC), Herestraat 49, GasthuisbergO&N1, B3000 Leuven, BelgiumCoremans et al. BMC Neuroscience 2010, 11:2http://www.biomedcentral.com/1471-2202/11/2© 2010 Coremans et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the CreativeCommons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, andreproduction in any medium, provided the original work is properly cited.integration of new neurons in the developing and adultbrain. Survivin is a member of the inhibitor of apoptotisprotein (IAP) family, that also promotes the cell cycleand is a chromosome passenger protein [20,21]. Duringembryonic development, it is expressed by several tis-sues, but is particularly prominent in the nervous system[22]. Inactivation of the survivin gene in neuroepithelialcells early in development [23] results in massive apop-tosis throughout the central nervous system, with totaldestruction of the architecture of the brain and lethality.The severity of this phenotype precluded investigatorsfrom delineating the specific role of neural precursorcell survivin on postnatal neural function.Therefore, to elucidate the properties of survivin inneural development and function, we inactivated thesurvivin gene in NPCs in the late-midterm murineembryo and evaluated the effects post-natally. By thisapproach, we generated a unique in vivo mouse modelin which reduced neurogenesis is associated with epi-lepsy and profound deficits in learning and memory.Pilot rescue studies suggest that embryonic administra-tion of survivin may enhance neurogenesis. The findingshighlight the critical role that survivin plays duringneural development, deficiencies of which dramaticallyimpact on postnatal neural function.ResultsSurvivin is expressed in precursor cells in the neurogenicareas of the brainIn situ hybridization using a probe against full lengthsurvivin allowed assessment of the spatiotemporalexpression of survivin during embryonic development.Survivin mRNA was detected in the neurogenic areas ofthe dorsal and ventral telencephalon surrounding theventricles (neocortex, medial and lateral ganglionic emi-nences (MGE and LGE, respectively)) (Figure 1A). AtE12.5, expression of survivin overlapped with Dlx1, amarker for mitotic cells in the MGE and LGE [24] (Fig-ure 1A, D) and neurogenin 2 (Ngn2) [25], a marker fordividing precursors in the neocortex (Figure 1A, C).There was minimal overlap with Dlx5 (Figure 1B),which is primarily expressed by postmitotic cells in themantle zone of the MGE and LGE, less so in mitoticcells in the SVZ, and almost absent in the ventricularzone [26]. By E17.5, survivin was additionally expressedin the rostral migratory stream (RMS) and at the centerof the olfactory bulb (OB) (Figure 1G, I, K). SurvivinmRNA was also present in the retina and lens of thedeveloping eye (not shown).Postnatally, survivin expression was restricted largelyto rapidly proliferating cells [21] and migrating NPCs[27]. At P7, survivin mRNA was detected in the SVZ,the RMS, the OB, and the dentate gyrus (DG) (Figure2A, C, D, E). Within the SVZ, survivin was notexpressed in the ependymal cells immediately adjacentto the lateral ventricle (Figure 2C, D). During the firsttwo postnatal weeks, survivin mRNA was also detectedin granule cell precursors in the external germinal layer(EGL) of the developing cerebellum (Figure 2A). In theadult brain, survivin expression remained restricted toproliferating and migrating precursor cells in the SVZ,the RMS, and the subgranular zone (SGZ) of the DG(Figure 2F, H).In both the SVZ (Figure 2J-L) and the SGZ of the DG(Figure 2M-O), >95% of survivin expressing cells werepositive for the proliferation marker PCNA, while atboth sites, 25-50% of PCNA positive cells expressed sur-vivin. Thus, survivin expressing cells represent a subpo-pulation of the mitotically active cells. Doublecortin(DCX), present in immature, migrating neuroblasts [28]also overlapped with survivin in the SVZ, the RMS andthe SGZ. However, similar to PCNA, not all DCX posi-tive cells expressed survivin, indicating that survivin isrestricted to a subpopulation of cells in the SVZ-RMS(not shown). Mature neuronal marker NeuN [29] immu-noreactivity did not overlap with survivin, consistentwith the lack of survivin expression by mature neurons(Figure 2C-E).Overall, survivin expression is restricted during fetaldevelopment to NPCs in neurogenic regions of the tele-ncephalon, of which a fraction populates the major neu-rogenic regions of the postnatal brain, i.e. the SVZ andthe SGZ. Within these neurogenic regions, a subpopula-tion of NPCs continues to express survivin, which isdownregulated once the cells differentiate into neurons.In both the embryo or adult, survivin is not detected inneurons in the cortex, OB, or hippocampus.In vivo Prenatal Survivin gene inactivationTo study the in vivo role of NPC survivin, we generatedmice in which the survivin gene is inactivated in theneurogenic regions of the brain prenatally. Mice expres-sing cre recombinase driven by the CamKIIa promoter[30,31] were bred with mice in which the entire survivingene is flanked by loxP sites [32]. The resultant Cam-KIIa-cre:survivinlox/lox (referred to as SurvivinCamcre)mice were born in the expected Mendelian distribution,i.e. there was no evidence of embryonic lethality.We confirmed previous reports of cre recombinaseactivity in the CAMKIIa-cre embryos and mice [30,31]by breeding the CAMKIIa-cre mice with the ROSA26reporter mice, followed by immunohistochemical detec-tion of GFP. Cre recombinase activity at E12.5 wasdetected prominently in the ventral telencephalon(ganglionic eminences), but less in the dorsal telence-phalon, and not in the eye (Additional file 1: Supple-mental Figure S1). Postnatally, it was restricted topostmitotic NeuN positive neurons in the hippocampusand cortex, with lower levels in the striatum, thalamus,Coremans et al. BMC Neuroscience 2010, 11:2http://www.biomedcentral.com/1471-2202/11/2Page 2 of 19Figure 1 Expression of survivin mRNA in developing mouse brain. In situ hybridizations were performed on coronal brain sections fromcontrol (wt) (A-D, G, I, K) and SurvivinCamcre (ko) (H, J, L) embryos, and on transverse brain sections from control (E) and SurvivinCamcre (F)embryos. (A-D) mRNA expression of survivin (A), dlx5 (B), ngn2 (C), and dlx1 (D) on adjacent brain sections from control mice illustrates overlapof survivin expression with dlx1 and ngn2. Dashed white line (A, B) indicates minimal overlap of survivin with dlx5. (E-L) Expression of survivinmRNA is reduced in the medial and lateral ganglionic eminences (MGE, LGE) (F, H), the RMS (arrow, J), and the OB (L) in SurvivinCamcre embryosas compared to controls. NCX, neocortex; HP, hippocampus. Scale bars: 500 μmCoremans et al. BMC Neuroscience 2010, 11:2http://www.biomedcentral.com/1471-2202/11/2Page 3 of 19Figure 2 Postnatal expression of survivin mRNA in neural precursor cells. Sagittal sections of brains were used to examine expression ofsurvivin. (A, B) At P7, Survivin mRNA was detected in the DG, SVZ, RMS, OB, and EGL of controls (A), and absent in the SVZ and RMS, but not inthe DG and EGL of SurvivinCamcre mouse brains (B). (C-E) Double labeling to detect survivin mRNA (blue) and NeuN protein (orange) expressionin the SVZ-RMS (C, D) and the DG (E) in P7 control brains, shows that survivin is not expressed in NeuN+ cells. D is a magnified view of thedashed box in C. (F-I) Survivin mRNA expression at P35 is restricted to neural precursor cells in the SVZ, RMS and SGZ in control mice (F, H).Survivin expression is reduced in the SVZ and RMS (G), but not in the SGZ (I) of SurvivinCamcre mice (ko) as compared to controls (wt). (J-O)Sagittal sections of control P7 brains through the SVZ (J-L) and through the SGZ (M-O) were stained for PCNA (red nuclei) (J, M) and survivinmRNA (white cytoplasm) (K, N) and the confocal images were overlaid (L, O). Only a subpopulation of PCNA+ cells express survivin. EGL, externalgerminal layer; OB, olfactory bulb; LV, lateral ventricle. Scale bars: A, B 1000 μm; C, F, G 500 μm; D 50 μm; E 200 μm; H, I 100 μm, J-O 20 μm.Coremans et al. BMC Neuroscience 2010, 11:2http://www.biomedcentral.com/1471-2202/11/2Page 4 of 19hypothalamus and amygdala [30]. Postnatal cre expres-sion was absent in NPCs in the SVZ and SGZ (Addi-tional file 1: Supplemental Figure S2). Thus, cross-breeding with the Survivinlox/lox mice resulted in dele-tion of both survivin alleles in the neurogenic regions inthe prenatal period (ganglionic eminences andneocortex).Neurogenesis defects in SurvivinCamcre embryosCre excision of survivin was assessed by in situ hybridi-zation in SurvivinCamcre and corresponding control Sur-vivinlox/lox embryos. At E14.5 and E17.5, survivinexpression in the SurvivinCamcre embyros was markedlyreduced in the ganglionic eminences surrounding thelateral ventricles where mitotically active NPCs normallyreside, in the RMS and in the OB (Figure 1F, H, J, L).This was associated with increased tunel staining, mostevident in the ganglionic eminences, and minimally inthe dorsal telencephalon (Figure 3A-D). BrdU labelingstudies revealed decreased NPC proliferation in theSVZ, RMS and OB of E17.5 SurvivinCamcre embryos(Figure 3E-J). Thus, lack of NPC survivin results inincreased embryonic NPC death and decreased NPCproliferation in specific embryonic neurogenic regions.Altered postnatal neurogenesis in SurvivinCamcre miceBody weights of SurvivinCamcre and control mice werenot significantly different at birth. However, during thefirst month after birth, SurvivinCamcre mice had a signifi-cantly higher mortality rate of 28% (n = 27/95)compared to 0% (n = 0/97) in control mice. Adult Survi-vinCamcre mice also had significantly smaller brains, andthe OBs were strikingly hypoplastic (Figure 4A, B) (forbrains: 421 ± 11 gm versus 329 ± 7 gm, for controls ver-sus SurvivinCamcre, respectively, p < 0.001; for OBs: 19.7± 0.9 gm versus 3.5 ± 0.2 gm for controls versus Survi-vinCamcre, p < 0.001; n = 6 mice per group). In situhybridization confirmed loss of survivin expression inthe SVZ and RMS (Figure 2A, B, F, G). Surprisingly,expression of survivin in the SGZ was not decreasedpostnatally in SurvivinCamcre mice (Figure 2I). Nisslstained brain sections from SurvivinCamcre mice revealedloss of the RMS, decreased cortical thickness (averagereduction to 80% of control at bregma levels -1.34/-1.70/-2.46/-2.80 mm; n = 4 mice per group, p < 0.01 ateach bregma level), enlarged ventricles, yet no morpho-logical changes in the hippocampus (Figure 4C, D). Thelatter was confirmed by quantifying the volumes corre-sponding to the GCL and the hilus of the DG (GCL:0.11 ± 0.01 mm3 versus 0.12 ± 0.01 mm3 for controland SurvivinCamcre mice, respectively; hilus: 0.11 ± 0.01mm3 versus 0.15 ± 0.02 mm3 for control and Survivin-Camcre mice, respectively; n = 4-5 mice per group, p >0.05).The hypoplastic OB and absent RMS, in concert withreduced expression of NPC survivin in the Survivin-Camcre mice might be caused by decreased NPC prolif-eration, increased cell death, and/or deficits inFigure 3 Neurogenesis defects in SurvivinCamcre embryos. (A-D) Tunel labeling of coronal sections of E14.5 brains illustrates increasednumber of apoptotic cells in the ganglionic eminences (GE) of SurvivinCamcre embryos (arrows). (E-J) BrdU immunostaining of coronal sections ofE17.5 embryos reveals reduced cell proliferation in the SVZ, the RMS (arrow in G, H), and the OB of SurvivinCamcre embryos as compared tocontrols. NCX, neocortex; HP, hippocampus. Scale bars: 500 μmCoremans et al. BMC Neuroscience 2010, 11:2http://www.biomedcentral.com/1471-2202/11/2Page 5 of 19Figure 4 Altered neurogenesis in adult SurvivinCamcre mice. (A, B) Dorsal view of control (wt) and SurvivinCamcre (ko) whole brains at 16weeks, reveals OB hypoplasia in SurvivinCamcre mice. (C, D) Nissl stained sagittal sections of littermates, shows thinner cortex (double arrow) andabsence of the RMS in the SurvivinCamcre mice. (E, F, I, J) BrdU labeling of sagittal sections of brains from 12 week old mice shows proliferationin the SVZ-RMS pathway (E, F) and in the SGZ (I, J), that is reduced only in the SVZ of SurvivinCamcre mice (F) as compared with controls (E). (G,H) DCX immunostaining of sagittal sections through the forebrain of control (G) and SurvivinCamcre (H) mice. (M, O) Quantification of BrdU+ cellsin the SVZ (M) and the SGZ (O) was performed as detailed in Methods. There was a significant reduction in BrdU+ cells in the SVZ, but not inthe SGZ of SurvivinCamcre mice as compared to controls. (K, L) Coronal sections through the anterior SVZ of control (K) and SurvivinCamcre (L) micewere stained for tunel+ apoptotic cells (arrows). (N, P) The number of tunel+ cells in the SVZ (N) and the SGZ (P) of control and SurvivinCamcremice was quantified as described in Methods. There was a significant increase in tunel+ cells in the SVZ but not in the SGZ of SurvivinCamcremice as compared to controls. LV, lateral ventricle. Results in panels M, N, O, P are reflected as means + SEM, n = 4-5 mice per group. *P <0.005. Scale bars: A-D 2 mm; E-H 500 μm; I-L 100 μm. (C-L) 12 weeks.Coremans et al. BMC Neuroscience 2010, 11:2http://www.biomedcentral.com/1471-2202/11/2Page 6 of 19migration. Accumulation of cells in the anterior SVZ ofthe SurvivinCamcre mice was not observed, mitigatingagainst a predominant migration defect. NPC prolifera-tion, assessed 1 hr after a single dose of BrdU, revealeda significant reduction in BrdU labeled cells in the SVZof the SurvivinCamcre mice (132 ± 16 cells versus 57 ± 8cells for control and SurvivinCamcre mice, respectively, n= 4-5 mice per group, p = 0.002) (Figure 4E, F, M).Furthermore, both BrdU and DCX labeled cells werealmost completely absent in the RMS, in striking con-trast to the controls (Figure 4E-H). Cell death in theSVZ of the SurvivinCamcre mice was also significantlyincreased (Figure 4K, L, N), as quantified by the numberof tunel+ cells in the anterior SVZ (1.5 + 0.21/100nucleated cells versus 3.5 + 0.46 tunel+ cells/100nucleated cells, for control and SurvivinCamcre mice,respectively, n = 4-5 mice per group, p = 0.004) (Figure4N). In the RMS and OB, there were some tunel+ cellsidentified in the brains of the control mice, but not inSurvivinCamcre mice, the latter likely due to the lack ofprecursor cells in this region.As noted above, survivin expression in the SGZ of theDG was not appreciably diminished after cre excision.There was also no alteration in the number of prolifer-ating or immature neurons in the SGZ, as quantified byBrdU labeling (1010 + 88 versus 905 + 75 cells in con-trols and SurvivinCamcre mice, respectively, n = 4-5 miceper group, p = 0.39) (Figure 4I, J, O) or DCX immunor-eactivity (not shown). Nor could we detect changes intunel+ staining (2.3 + 0.18 versus 1.6 + 0.63 tunel+cells/section in controls and SurvivinCamcre mice, respec-tively, n = 3-4 mice per group, p = 0.30) (Figure 4P).In summary, striking survivin-dependent defects inneurogenesis are evident postnatally in the RMS and OBof SurvivinCamcre mice, due to a combination ofincreased SVZ NPC apoptosis and diminished cellularproliferation. Despite the fetal abnormalities, and instriking contrast to the RMS-OB, there were no obviousstructural defects or alterations in hippocampal neuro-genesis in the SurvivinCamcre mice that had no appreci-able reduction in survivin expression within thehippocampus.Embryonic survivin administration increases neurogenesisIn pilot studies, we assessed whether prenatal adminis-tration of survivin to increase expression in NPCs couldpromote neurogenesis. E12.5 control Survivinlox/lox andSurvivinCamcre embryos received an intracerebroventricu-lar injection in utero with the lentiviral vectorpCHMWS-eGFP-T2A-SRV140 (survivin vector) orpCHMWS-eGFP-T2A-Fluc (control vector). At P21,immunohistochemical analysis of the control Survivinlox/lox mice that received the survivin vector revealed anincreased number of embryonic precursor cell-derivedcells in the OB as compared with the control vector(Additional file 1: Supplemental Figures S3A, B). WithSurvivinCamcre embryos, the survivin vector did notapparently reverse the OB hypoplasia when examined atP21, but there were notably more embryonic precursorcell-derived cells in the OB of 3 out of 3 SurvivinCamcremice that received the survivin vector, as compared withthe 2 SurvivinCamcre mice that received the control vec-tor (Additional file 1: Supplemental Figures S3C, D).Overall, these preliminary findings suggest thatenhanced expression of NPC survivin may increaseneurogenesis.SurvivinCamcre mice have a thinner cortex with fewerGABAergic interneurons and a lower seizure thresholdAlthough the average cortical thickness in the Survivin-Camcre mice was reduced, in situ hybridization with cor-tical markers (Cux2 for layers 2-4 [33], Badlamp forlayers 2/3/5 [34], and ER81 for layer 5 [35] confirmedthe correct orientation and presence of all the corticallayers (not shown). Moreover, and in line with limitedexpression of cre recombinase in the dorsal telencepha-lon of CamKIIa-cre embryos, the density of corticalvGLUT1+ glutamatergic cells in the SurvivinCamcre micewas not altered (2282 + 157 cells/mm2 versus 2480 + 71cells/mm2 in controls and SurvivinCamcre mice, respec-tivley, n = 4-5 mice per group, p = 0.30) (Figure 5A-C).In contrast, and consistent with prominent apoptosisand diminished BrdU labeling in the ganglionic emi-nences, prenatal depletion of survivin in the NPCs ofSurvivinCamcre mice resulted in a significant reduction inthe density of GAD65/67+ GABAergic interneurons inthe postnatal adult cortex (493 + 12 cells/mm2 versus417 + 6 cells/mm2 in controls and SurvivinCamcre mice,respectively, n = 5-6 mice per group, p < 0.001) (Figure5D-F). This occurred in the absence of any changes ininterneurons in the hippocampus (hilus + granular celllayer: 213 ± 8 cells/mm2 versus 209 ± 17 cells/mm2 incontrols and SurvivinCamcre mice, respectively, n = 4mice per group, p = 0.82). These data indicate an altera-tion in the excitatory/inhibitory balance in the brain ofSurvivinCamcre mice that might be associated with post-natal alterations in cognition and behavior, and a lowerseizure threshold.Indeed, during routine handling, 2.5% (n = 9/352) ofthe SurvivinCamcre mice were recorded to have sponta-neous tonic-clonic, generalized motor seizures startingfrom 2 weeks of age (no seizures observed in controls).To investigate seizure susceptibility, the response ofcontrol and SurvivinCamcre mice to kainic acid (KA) wasassessed over a period of 2 hours. Control, saline-treatedanimals (n = 9 per group) showed no signs of epilepticactivity. However, in response to KA, SurvivinCamcremice exhibited a lower threshold for seizures that weremore severe. Thus, at a subconvulsive dose of 20 mg/kg,KA induced limbic motor convulsions in 15% (n = 2/13)Coremans et al. BMC Neuroscience 2010, 11:2http://www.biomedcentral.com/1471-2202/11/2Page 7 of 19of control mice and 81% (n = 13/16) SurvivinCamcre mice(Figure 6A). The maximum seizure score was signifi-cantly higher in the SurvivinCamcre mice (p < 0.001). Sei-zure severity over the 2 hr observation period was alsosignificantly greater in the SurvivinCamcre mice (p <0.001) (Figure 6B). At a KA dose of 30 mg/kg, the meanlatency to the first seizure was also shorter in the Survi-vinCamcre mice compared to control mice (8.75 ± 2.75versus 30 ± 7.64 min, respectively, n = 4 mice pergroup, p = 0.031). At that dose, all SurvivinCamcre micerapidly developed status epilepticus (stage 5-6) and diedof severe generalized convulsions, while all control ani-mals survived the KA treatment (Figure 6A). Thus, Sur-vivinCamcre mice exhibit enhanced susceptibility to KAseizures.Neuropeptide Y (NPY) is a multifunctional peptidethat is expressed in GABAergic interneurons, regulatespre-synaptic excitatory transmission in the DG, and hasanti-epileptic properties [36]. Hilar NPY interneurondegeneration, ectopic expression of NPY in mossy fibers,and axonal sprouting are common features of limbichyperexcitability [37]. Due to the increased seizure activ-ity in the SurvivinCamcre mice, we examined NPY expres-sion under both basal conditions (saline treatment) andfollowing induction of seizures (KA 20 mg/kg i.p.). Thenumber of hilar NPY+ interneurons was not differentbetween saline-treated control and SurvivinCamcre mice(490 ± 17 versus 398 ± 53 cells/mm2, for control andSurvivinCamcre mice, respectively; n = 4-5 mice pergroup, p = 0.11). However, two weeks after 20 mg/kgKA treatment, there were significantly fewer NPY+ cellsin the hilus of the SurvivinCamcre mice (446 ± 22 versus306 ± 65 cells/mm2 for control and SurvivinCamcre mice,respectively; n = 6-9 mice per group, p = 0.032). More-over, ectopic NPY expression in mossy fibers was readilydetected in 3 out of 4 saline-treated SurvivinCamcre micebut not in any of the corresponding controls (n = 5)(Figure 6C, D). This effect became more prominentafter KA (5/6 SurvivinCamcre mice as compared to 0/9controls). In 2 of these SurvivinCamcre mice, we further-more observed ectopic NPY immunoreactivity in thesupragranular layer, likely reflecting sprouting of mossyfibers (not shown).Since seizure activity modulates hippocampal neuro-genesis, we also evaluated the seizure-induced neuro-genic response of the control and SurvivinCamcre mice.The volumes of the GCL and the hilus were not differ-ent between control and SurvivinCamcre mice (seeabove), and KA had no effect on that relationship (datanot shown). To assess cell proliferation, BrdU wasinjected 3 days after KA or saline injection, and micewere sacrificed 1 day later. After saline injection, thetotal number of SGZ BrdU+ cells was not different incontrol and SurvivinCamcre mice (648 ± 58 cells versus705 ± 99 cells in controls and SurvivinCamcre mice,respectively, n = 4 mice per group, p = 0.64) (FigureFigure 5 GABAergic and glutamatergic inter/neurons. In situ hybridizations of coronal sections were performed to detect and quantifyvGLUT1+ glutamatergic neurons (A-C) and GAD65/67+ GABAergic interneurons (D-F) in adult littermates. (A-C) In spite of SurvivinCamcre micehaving a thinner cortex, the density of vGLUT1+ cells in the cortex was not signficantly different between SurvivinCamcre and control mice. (D-F)The density of GAD65/67+ cells in the cortex was significantly reduced in SurvivinCamcre mice, but not in the hippocampus (see text). Results inpanels B and E are reflected as means + SEM, n = 4-6 mice per group. **P < 0.001 Scale bars: 500 μm.Coremans et al. BMC Neuroscience 2010, 11:2http://www.biomedcentral.com/1471-2202/11/2Page 8 of 196E). Compared to saline treated controls, KA treatedmice exhibited an increase in the number of BrdU+cells in the SGZ after KA injection. However, the neuro-genic response was significantly dampened in the Survi-vinCamcre KA treated mice compared to control KAtreated mice (3578 ± 392 cells versus 1955 ± 233 cellsfor controls and SurvivinCamcre mice, respectively, n = 5mice per group, p < 0.001) (Figure 6E). Numbers ofBrdU+ cells remained reduced 2 weeks following KA inthe SurvivinCamcre mice as compared to controls (datanot shown). Thus, despite the higher seizure scores fol-lowing KA in the SurvivinCamcre mice, there was less sei-zure-induced neurogenesis in the SurvivinCamcre versusthe control mice.SurvivinCamcre mice exhibit learning and memory defectsAdult SurvivinCamcre mice exhibited several defects thatmay contribute to disorders in behavior and cognition,including reduced SVZ-RMS-OB neurogenesis [38,39],OB hypoplasia [40], diminution of cortical GABAergicneurons, and seizures. We therefore evaluated theeffects of depleting NPCs of survivin by subjecting theSurvivinCamcre and matched controls to a range of beha-vioral studies.There was no difference in body weight between theSurvivinCamcre and control mice at the start of beha-vioral testing (21.6 ± 0.6 gm versus 20.1 ± 0.6 gm forthe SurvivinCamcre and controls, respectively, p = 0.081),and the SurvivinCamcre mice had normal visual andFigure 6 SurvivinCamcre mice exhibit increased seizure activity. (A) Scatter plot showing the maximum seizure score assigned to eachexperimental animal during a 2 hr observation period following KA administration. Seizure scores were significantly higher in the SurvivinCamcremice as compared to controls. Horizontal bars indicate the mean for each group. (B) KA (20 mg/kg ip) induced signficantly more severe seizureactivity in SurvivinCamcre mice as compared to controls, P < 0.001, n = 13-16 mice per group. (C, D) Representative NPY-stained coronal sectionsthrough the hippocampus of saline-treated control (wt) (C) and SurvivinCamcre mice (ko) (D) reveals ectopic NPY expression by mossy fibers inSurvivinCamcre mice (arrows in D). (E) Quantification of BrdU+ cells (1 day after BrdU injection) in the SGZ from saline and KA treated control andSurvivinCamcre mice. The neurogenic response to KA was significantly dampened in SurvivinCamcre mice as compared to controls, n = 4-5 mice pergroup. Results in panels B and E are reflected as means + SEM. **P < 0.001. Scale bars: C-D 500 μm.Coremans et al. BMC Neuroscience 2010, 11:2http://www.biomedcentral.com/1471-2202/11/2Page 9 of 19auditory skills, grip strength, rotarod performance, painresponse and cage activity (Additional file 1: Supplemen-tal Figures S4, S5; Additional file 2: Supplemental Meth-ods and Results).In the open field test, the SurvivinCamcre mice dis-played significant disturbances in exploratory behavior,including delayed first entry to the center, fewer centerentries, more corner crossings, and less time in the cen-ter (Figure 7A). This type of behavioral outcome isoften suggestive of increased anxiety, however the Survi-vinCamcre mice performed normally in the elevated plusmaze test for anxiety. On the maze, there was no differ-ence between control and SurvivinCamcre mice in thetotal number of beam crossings (148 ± 5 versus 155 ± 9for control and SurvivinCamcre mice respectively; n = 12mice per group, p = 0.48), percent time spent in theopen arms (33 ± 2 versus 36 ± 4 for control and Survi-vinCamcre mice respectively; n = 12, p = 0.76), nor per-centage of entries into the open arms (26 ± 2 versus 23± 3 for control and SurvivinCamcre mice respectively; n =12, p = 0.07). The findings suggest that the poor perfor-mance of the SurvivinCamcre mice in the open field testmay not be due to increased anxiety, but rather due to adistinct defect in exploratory behavior.The SurvivinCamcre mice exhibited a significant impair-ment in passive avoidance learning. During the initialtraining trial, there was no difference in step-throughlatencies (12.5 ± 4.5 versus 13.9 ± 1.8 sec, SurvivinCamcreand control mice respectively, p = 0.783). However, dur-ing testing, the SurvivinCamcre mice demonstrated signifi-cantly shorter latency to enter the dark compartmentthan the controls (235 ± 27 versus 71 ± 24 sec for con-trol and SurvivinCamcre mice, n = 12, p < 0.001) (Addi-tional file 1: Supplemental Figure S6), consistent withpoor associative memory of aversive stimuli.We examined auditory and contextual fear memoryusing an auditory cue as the conditioned stimulus (CS),and a foot shock as an aversive stimulus. Freezing timesin baseline, pre-US, post-US, and pre-CS trials were notdifferent between the groups (Figure 7B). However, Sur-vivinCamcre mice exhibited significantly less freezingresponse in the context and auditory cue (CS) trialscompared to control mice (context: 25.7 ± 5.5 versus68.2 ± 4.5; auditory: 34.3 ± 9.0 and 84.4 ± 7.8, respec-tively for SurvivinCamcre and control mice, n = 12 miceper group, p < 0.001) (Figure 7B).Lastly, we assessed hippocampus-dependent spatiallearning and long-term memory in the SurvivinCamcremice using the Morris water maze. Swimming velocitywas not different between the controls and the Survivin-Camcre mice, excluding defects in motor ability. Duringacquisition training, the SurvivinCamcre mice showedtraining-dependent reduction in escape latency and pathlength. However, the improvements were minimal ascompared with controls, and the escape latency andpath length were significantly increased in the Survivin-Camcre mice (p < 0.001, n = 12; by two way repeatedmeasures ANOVA) (Figure 8A, B). Notably, lengths ofthe escape paths were not different between the Survi-vinCamcre mice and control mice during 4 visible-plat-form training days (803 + 88 cm versus 1076 + 110 cmfor control, n = 10, and SurvivinCamcre mice, n = 12,respectively; p = 0.075). These results indicate that theSurvivinCamcre mice were fully capable and motivated tochoose the shortest pathway towards the platform in thecued, non-spatial condition of the task.In the first probe trial performed after 5 days of train-ing, the control mice already had a preference for thetarget quadrant compared to adjacent 1 (p = 0.002) andopposite (p = 0.02) quadrants, whereas the Survivin-Camcre mice had no preference at all (p = 0.46) (Figure8C). In the second probe trial after 10 days of training,the control mice continued to show a strong preferencefor the target quadrant compared to all other quadrants(p < 0.001), whereas the SurvivinCamcre mice equallyfavoured the target and adjacent 2 quadrants (p = 0.60)versus the other 2 quadrants (p < 0.05) (Figure 8D, E).Since the Morris water maze test is a stress that mightalter neural cell proliferation, we also quantified thenumber of Ki67+ cells in the dentate gyrus of the Survi-vinCamcre mice (n = 4) and the corresponding littermatecontrols (n = 4) after the probe trials. There was no sig-nificant difference in the number of Ki67+ cells betweenthe two groups (534 + 49 cells versus 480 + 28 cells incontrols versus SurvivinCamcre mice, respectively, p =0.378).Overall, the SurvivinCamcre mice exhibited exploratorybehavioral abnormalities, with global deficits in variousforms of learning and memory.DiscussionIn this report, we show that survivin is prominentlyexpressed in the neurogenic regions of the embryonicmouse brain, and that its expression by a subpopulationof NPCs is maintained postnatally in the two key sitesof adult neurogenesis - the SVZ and the SGZ. Lack ofexpression of survivin in the NPCs during embryonicdevelopment, was associated with profound SVZ-RMS-OB postnatal defects in neurogenesis and loss of inter-neurons, manifest by major deficits in learning andmemory, and heightened sensitivity to seizures. Prenataladministration of survivin in the brain enhanced neuro-genesis in the SVZ-RMS-olfactory system. Our findingsposition survivin as a central player in regulating neuro-genesis during embryonic development, alterations ofwhich impact on postnatal brain function.The embryonic forebrain, the telencephalon, consistsof two parts. The dorsal aspect is the origin ofCoremans et al. BMC Neuroscience 2010, 11:2http://www.biomedcentral.com/1471-2202/11/2Page 10 of 19Figure 7 Open field and fear conditioning defects in SurvivinCamcre mice. (A) Open field test data are provided, with representative paths ofcontrol (wt) and SurvivinCamcre (ko) mice. (B) Contextual and auditory-cued fear conditioning in control and SurvivinCamcre mice. Freezing times inpre-US, post-US and pre-CS trials were not different between control and SurvivinCamcre mice. SurvivinCamcre mice showed significantly lessfreezing responses as compared to controls during both the context and the auditory cue (CS) trials. US: unconditioned stimulus, shock; CS:conditioned stimulus, auditory cue. Results in panel B are reflected as means + SEM, n = 12 mice per group. **P < 0.001.Coremans et al. BMC Neuroscience 2010, 11:2http://www.biomedcentral.com/1471-2202/11/2Page 11 of 19Figure 8 Poor performance of SurvivinCamcre mice in water maze. Morris water maze studies were performed as detailed in Methods. (A)SurvivinCamcre mice (ko) exhibited a significantly longer escape latency as compared to controls (wt) during acquisition of the task (P < 0.001). (B)Representative swim paths of control and SurvivinCamcre mice during acquisition training. (C) The mean percent time spent in each quadrantduring the 1st probe trial is plotted for both genotypes. In the first probe trial performed after 5 days of training, the control mice already had apreference for the target quadrant compared to adjacent 1 and opposite quadrants, whereas the SurvivinCamcre mice had no preference at all. (D,E) In the second probe trial (representative swim path shown in D), control mice spent most of the time in the target quadrant, whileSurvivinCamcre mice spent equal amounts of time in the target and adjacent 2 quadrant. Open triangle and black dot represent location of thestart and the platform, respectively. adj, adjacent 1 or adjacent 2 quadrant; opp, opposite quadrant. Results in panels A, C and E are reflected asmeans + SEM, n = 12 mice per group. *P < 0.05; **P < 0.001.Coremans et al. BMC Neuroscience 2010, 11:2http://www.biomedcentral.com/1471-2202/11/2Page 12 of 19glutamatergic excitatory neurons of the cerebral cortexand hippocampus [41]. The ventral part, comprising theganglionic eminences, gives rise to the basal ganglia.The LGE provides neurons for the striatum [42], inter-neurons of the olfactory bulb (OB) [43], and most adultSVZ NPCs [2]. The MGE is the source of most neocor-tical [42] and hippocampal interneurons [44], as well asstriatal interneurons. At E12.5, survivin is widelyexpressed in the neurogenic region of the ventral anddorsal telencephalon. Since cre recombinase expressionin the CamKIIa-cre mice is low in the dorsal telence-phalon, generation of principal glutamatergic neuronswas largely unaffected in the adult, and the overallintegrity of the hippocampus and cortex was main-tained, albeit the latter was thinner. In contrast, thenumber of cortical GABAergic neurons, which arise pri-marily in the ganglionic eminences and comprise 25-30% of cortical neurons, was significantly reduced in theSurvivinCamcre mice. This reduction may have beenfurther contributed to by the paucity of SVZ NPCs,recently shown to be a continuous postnatal source ofGABAergic interneurons in the cortex [45].Imbalances in inhibitory and excitatory circuits due todecreases in numbers of interneurons, are well knownto be associated with seizures in humans and experi-mental animal models [46,47]. This was clearly evidentin the SurvivinCamcre mice which, even under naïve con-ditions, displayed spontaneous, generalized tonic-clonicmotor seizures, a phenotype that was more dramaticallyrevealed following challenge with KA. Indeed, Survivin-Camcre mice showed a rapid and consistent generalizationof seizures at KA doses that normally result in focal hip-pocampal epileptic activity [48]. Thus, a defect in thecortical inhibitory system may explain the higher sus-ceptibility to generalized convulsions in the Survivin-Camcre mice.Our studies demonstrate that the loss of a subpopula-tion of NPCs in the SVZ of neonatal and adult Survivin-Camcre mice, with resultant near-absence of the RMS andOB, was due to a combination of increased apoptosisand decreased cellular proliferation of NPCs in the cor-responding embryonic neurogenic region (ganglioniceminences). Indeed, this is in line with the fact that sur-vivin is a pro-survival molecule with the capacity toinhibit apoptosis and to promote the cell cycle andmitosis (reviewed in [49]). Somewhat surprisingly, inspite of profound disturbances in neurogenesis in theSVZ, we did not detect baseline changes in neurogenesisin the SGZ of the DG in the SurvivinCamcre mice, or sig-nificant loss of survivin expressing DG NPCs. Althoughthis may be due to cre recombinase inefficiency, thefinding may also be due to the embryonic origin of SGZNPCs being different from the SVZ NPCs, which stillremains to be clarified [50]. There is however, a defectin SGZ neurogenesis in the SurvivinCamcre mice that isonly evident under stress conditions. This may meanthat the baseline source(s) of SGZ NPCs is differentfrom that recruited during stress, an hypothesis thatrequires testing. In the SurvivinCamcre mice, the neuro-genic response was significantly impaired as comparedto the controls after KA-induced seizures. Alterations inGABA signaling in the SurvivinCamcre mice may beimplicated [51], but other factors that are important inmaintaining the function of the neurogenic niche in thehippocampus could also contribute to the dampenedresponse [52]. Further study to identify those that arerelevant is ongoing.Although the integrity of the hippocampus was appar-ently maintained under baseline conditions, upon test-ing, the SurvivinCamcre mice exhibited striking defects inmemory and cognition, that are consistent with hippo-campal dysfunction. In fact, the behavioral abnormalitieswere associated with a significant impairment of long-term potentiation (LTP) in the CA1 region of the hippo-campus (not shown), a finding that frequently is asso-ciated with poor memory, and often with increasedepileptic activity. As with the seizure disorder, a loss ofcortical inhibitory interneurons likely contributed to thebehavioral abnormalities and cognitive defects in theSurvivinCamcre mice. We also cannot exclude a contribu-tion of suboptimal neurogenic responses to the beha-vioral phenotype, as neurogenic defects in both the SGZand SVZ have been implicated in memory, cognition,mood, and hippocampal-dependent learning [53]. More-over, olfactory bulbectomy in rodents impairs neurogen-esis in both the SGZ and the SVZ, disrupts normalhippocampal LTP, and causes significant deficits inlearning and memory. Thus, the OB, which sends pro-jections to the hippocampus [54], also plays a role innormal behavior and cognition [12,13,40]. Indeed, sincethe SurvivinCamcre mice have major defects in neurogen-esis, as well as notable hypoplasia of the OB, all ofwhich are associated with epileptic activity and majoralterations in behavior, it is reasonable to consider thatthe effective lack of an OB exacerbates the loss of SVZand possibly SGZ NPCs, which in turn, contributes tothe behavioral abnormalities and enhanced seizureactivity.ConclusionsWe have established that prenatal expression of survivinin neurogenic regions of the developing brain plays akey role in learning and memory and in determining sei-zure susceptibility. Prenatal stresses are recognized tosuppress postnatal neurogenesis that in turn, inducesbehavioral abnormalities in the neonate and adult [8,9].While the underlying molecular mechanisms have notbeen delineated, it is reasonable to consider thatCoremans et al. BMC Neuroscience 2010, 11:2http://www.biomedcentral.com/1471-2202/11/2Page 13 of 19alterations in embryonic NPC survivin expression mightcontribute to those phenotypic changes. Pilot data indi-cate that prenatal administration of survivin canenhance neurogenesis in the olfactory system. We donot yet know whether the resultant new neurons differ-entiate or integrate, or whether the SGZ is also affected.Nonetheless, the findings are promising, supporting thecritical nature of this molecule, and its potential as atherapeutic target. Our mouse model provides theopportunity to elucidate the relevance of survivin-expressing NPC subpopulations in vivo in response to arange of environmental stresses, and genetic or epige-netic factors.MethodsTransgenic mice and genotypingMice that express Cre recombinase driven by the pro-moter of the gene for calmodulin-dependent proteinkinase IIa (CamKIIa) [30,31] (gift of Dr. G. Schütz, Hei-delberg, Germany) were bred with mice in which thesurvivin gene is flanked by loxP sites [32]. The resultingoffspring that were heterozygous for Cre and homozy-gous for floxed survivin (Survivinlox/lox) (hereafterreferred to as SurvivinCamcre mice) were compared to lit-termate control mice which did not express Cre andwere Survivinlox/lox. Survivinlox/lox embryos and adultswere not different from Survivinlox/wt, Survivinwt/wt orCamKIIa-cre:survivinlox/wt mice. Mice were maintainedon a C57B/6:Swiss:129svj 75:12.5:12.5 background. Gen-otyping of tail DNA was performed by PCR as pre-viously reported [30,32]. Mice were group-housed instandard mouse cages in a room with a 12 h light-darkcycle and ad libitum access to food and water and allanimal experiments were approved by the ethics com-mittee of the University of Leuven.BrdU labeling and quantificationAdult mice and pregnant females were injected intraper-itoneally (ip) with 5-bromo-2-deoxyuridine (BrdU,Sigma Aldrich, Bornem, Belgium) at a concentration of50 mg per kg body weight. For the analysis of theembryos, 1 hour after injection of BrdU, pregnantfemales were killed by cervical dislocation, after whichthe embryos were harvested, placed in ice-cold PBS, andthen fixed in 4% paraformaldehyde (Para) for cutting 20μm cryo sections using a microtome/cryostat (HM550,Microm, Walldorf, Germany). For analysis of adults,mice were anesthetized with sodium pentobarbital at 1hour (unless stated otherwise) after BrdU and perfusedtranscardially with 0.9% NaCl, followed by fixation with4% paraformaldehyde. Brains were dissected and post-fixed overnight at 4°C and 40 μm tissue sections wereprepared using a vibrating microtome (HM650V,Microm, Walldorf, Germany).The number of BrdU+ cells in the adult dentate gyrus(DG) was quantified using a modified version of theoptical fractionator method [55] with Stereo Investigatorsoftware (MicroBrightField, Colchester, VT, USA). Cellswere counted with a 40× objective on every sixth sec-tion through the entire rostrocaudal extension of onehalf of the DG, restricted to the subgranular zone (SGZ)[56]. The number of BrdU+ cells in the SVZ of one lat-eral ventricle was counted with a 40× objective on 1coronal section (bregma level + 0.14 mm) per animal.ImmunohistochemistryImmunostaining protocols were optimized for the differ-ent tissue preparations and antibodies. In general, tissuesections were treated with 1% H202 in PBS/methanol for15 min, incubated in 5% serum for 30 min, and incu-bated overnight at 4°C in following primary antibodies:rabbit anti-neuropeptide Y (NPY) antibody (1:5000,Bachem, UK); mouse anti-NeuN (1:500, Chemicon, Hof-heim, Germany); mouse anti-PCNA (1:1000, Chemicon,Hofheim, Germany); rabbit anti-DCX (1:500, Cell Sig-naling, MA, USA); rat anti-BrdU (1:500, ImmunologicalsDirect, Oxford, UK); chicken anti-GFP (1:3000, Aves,Oregon, USA); and rabbit anti-Cre recombinase (1:3000,gift from Dr. Schütz, Heidelberg, Germany); rabbit anti-Ki67 (1:1000 Monosan, Uden, The Netherlands). Afterwashes, the corresponding biotinylated secondary anti-body was added for 1 hour and the signal was amplifiedusing the Vectastain Elite ABC kit (Vector Laboratories,CA, USA). Peroxidase activity was detected with 3,3’-diaminobenzidine (DAB peroxidase substrate tablet set,Sigma Aldrich, Bornem, Belgium). For fluorescent stain-ing, Alexa-conjugated secondary antibodies (MolecularProbes, Leiden, The Netherlands) were used. BrdUstaining was performed as reported previously [57].In situ hybridizationDigoxygenin (DIG)-labeled RNA probes for Dlx1 [58],Dlx5 [24], Ngn2 (gift from Dr. A. Simeone), GAD65[59], GAD67AE [60] and vGLUT1 (Allen Institute forBrain Science) were generated using the DIG RNALabeling Kit (Roche Diagnostics, Basel, Switzerland),according to the manufacturer’s instructions. GAD65and GAD67AE probes were mixed to detect the totalnumber of GABAergic interneurons. For survivin ribop-robes, full-length murine survivin cDNA was clonedinto the pcDNA3 plasmid vector (Invitrogen, CA, USA)[61], and linearized for generation of antisense andsense probes using Sp6 RNA polymerase or T7 poly-merase, respectively. In situ hybridization and combinedimmunohistochemistry protocols were adapted fromthose reported [33,62] and completed on 20 μm cryostator 40 μm vibratome sections.Coremans et al. BMC Neuroscience 2010, 11:2http://www.biomedcentral.com/1471-2202/11/2Page 14 of 19Measurement of granular cell layer (GCL) volume andhilar volumeCoronal sections through the DG were stained with cre-syl violet. Pictures were taken at 4× magnification, andthe area of the GCL and the hilus was determined offline using Metamorph software (Molecular Devices,Sunnyvale, CA). Volumes were calculated and expressedin mm3.Quantification of apoptosis (tunel+ cells)Detection of cellular apoptosis in 10 μm coronal paraffinsections, prepared using a HM360 microtome (Microm,Walldorf, Germany), was accomplished using the Apop-Tag Peroxidase In Situ Apoptosis Detection Kit (Chemi-con, Hofheim, Germany). The number of tunel+ cellswas counted with a 40× objective. The anterior subven-tricular zone (SVZa) was analyzed at level bregma +0.98mm and the data are presented as the number of tunel+cells per 100 nucleated cells. The subgranular zone(SGZ) was analyzed at bregma levels -1.34/-1.70/-2.46/-2.80 mm and the data are presented as the number oftunel+ cells per section.Quantification of inhibitory and excitatory neuronsNumbers of neurons and interneurons were quantifiedhemilaterally on coronal vibratome sections at 4 bregmalevels (-1.34/-1.70/-2.46/-2.8 mm). GAD65/67+ cellswere counted with a 10× objective in the hilus plus thegranule cell layer, and in the parieto/temporal cortex, ina 1.4 mm wide band from the white matter to the pialsurface. vGLUT1+ cells in the parieto/temporal cortexwere counted with a 20× objective in a 0.7 mm wideband. NPY+ cells in the hilus were counted with a 20×objective on every sixth section (40 μm thick). Resultsare presented as the number of cells per mm2.Quantification of Ki67 positive cellsKi67 positive cells in the SGZ were counted hemilater-ally with a 40× objective on every third 40 μm sectionbetween bregma levels -1.34 and -2.8 mm. The numberof counted cells was multiplied by 3 to obtain the totalnumber of Ki67 positive cells.Intracerebroventricular (ICV) injection of lentiviral vectorin embryosLentiviral vectors were prepared, encoding enhancedgreen fluorescent protein (eGFP) and survivin separatedby a T2A sequence starting from pCHMWS-eGFP-T2A-Fluc (gift from Dr. V. Baekelandt, KULeuven). The Flucfragment was removed from pCHMWS-eGFP-T2A-Flucusing BamHI and MluI and replaced by the cDNAencoding full-length murine survivin. Survivin expres-sion from this vector was confirmed by Western blotanalysis of lysates from transfected COS cells. Humanimmunodeficiency virus type 1 (HIV-1)-derived lentiviralvectors were produced by a standard protocol. The viralvector was mixed with Fast Green dye (0.005% finalconcentration, Sigma-Aldrich, Bornem, Belgium), whichallowed visualization of the distribution of the viral vec-tor in the cerebral ventricles after injection. Pregnantmice (stage E12.5) were anesthetized with 50 mg/mlketamine, 2% xylazine in saline and placed supine on aheating pad. A 2-cm midline incision was made throughthe skin and the abdominal wall. The uterine horn wasdrawn out through the hole onto gauze, and with theuterus transilluminated, a 35 gauge needle (beveledNanoFil needle, World Precision Instruments, FL, USA)was inserted into the ventricle, and 1 μl viral vectorsolution was injected at a speed of 406 nanoliters persecond using a Mycro4® MicroSyringe Pump Controller(World Precision Instruments, FL, USA).Seizure studiesSeizures in adult male mice were evoked by ip adminis-tration of kainic acid (KA) (Sigma, MO, USA). KA wasdissolved in saline and injected at 20 or 30 mg/kg bodyweight. Saline-injected animals were used as controls.Seizure severity was quantified by an observer blind tothe mouse genotype using the following scale [48,63]:stage 0, normal behavior; stage 1, immobility; stage 2,forelimb and/or tail extension, rigid posture; stage 3,repetitive movements, head bobbing; stage 4, rearingand falling; stage 5, continuous rearing and falling; stage6, severe whole-body convulsions; and stage 7, death.For each animal, seizure severity was scored every 10min over a period of 2 hours after KA administration.The maximum score reached by each animal over theentire observation period was used to calculate the max-imum seizure score for each treatment group. Seizureseverity over the 2 hour observation period was calcu-lated for each mouse as the area under the seizure scoreversus time curve (AUC), and the average AUC was cal-culated for each treatment group.Behavioral studiesBehavioral tests were initiated when the mice were 3-4months of age, n = 12-25 per group. Neuromotor,exploration, and learning tests were performed in thefollowing sequence: cage activity, grip strength, rotarod,open field, elevated plus maze, Morris water maze, pas-sive avoidance. Contextual fear conditioning was per-formed on a separate group of mice. Animals weretested during the light phase of the light-dark cycle. Allstudies were performed by observers who were blindedto the genotype of the mice.Open field exploratory activity was assessed in a 50cm × 50 cm arena using EthoVision video tracking andsoftware (Noldus, Wageningen, The Netherlands). Micewere individually placed in a specific corner of the openfield, and were allowed a 1 min adaptation period. Thepath was recorded for 10 min to measure dwells andentries in different parts of the field. Measures includedtotal path length, percentage path length in the centercircle (diameter 30 cm), entries into the four cornerCoremans et al. BMC Neuroscience 2010, 11:2http://www.biomedcentral.com/1471-2202/11/2Page 15 of 19squares, entries into the center, time spent in the centerversus periphery, latency of first center approach, andfrequency of rearing.The elevated plus maze [64,65], to evaluate anxiety-like behavior, had two open arms (21 cm × 5 cm) andtwo closed arms of the same size, with high side walls,and was raised 30 cm above the table. Each mouse wasplaced in the central square of the maze, facing one ofthe closed arms. After 1 min, exploratory behavior wasrecorded automatically during a 10 min period usingfive infrared beams, connected to an activity logger. Foreach mouse, the number of arm entries, percentage ofopen arm entries, and percentage time spent in theopen arms was assessed.Passive avoidance (aversive) learning [66] was tested ina two-compartment step-through box. Animals wereadapted to the dark for 30 min, and then placed into asmall illuminated compartment. After 5 s, a sliding doorleading to the large dark compartment was opened.Upon entry, the door was closed and the animalreceived an electric foot shock (0.3 mA, 1s). Twenty-four hours later, the animals were placed again in thelight compartment and the latency to enter the darkcompartment was measured up to 300 s, to evaluatememory of the foot shock.Contextual and auditory-cued fear conditioning[67,68] was tested in a Plexiglas chamber with a gridfloor through which a foot shock could be administered.Mice were trained and tested on 3 consecutive days: Onthe day 1, the mice were individually placed in the test-ing chamber and allowed to adapt for 5 min. On theday 2, the animals were allowed to explore the testingchamber for 2 min, after which an auditory cue (condi-tioned stimulus, CS) was presented for 28 s, followed bya foot shock (0.3 mA, 2 s; unconditioned stimulus, US).The time (%) spent freezing during the first 2 min and28 s is the pre-US score. The mice were then allowed toexplore again for 1 min, and the auditory cue and shockwere again presented, followed by another 2 minexploration (post-US score). On day 3 (24 hours aftertraining), mice were returned to the same context inwhich training occurred, and freezing behavior wasrecorded for 5 min (context test). Ninety min later,freezing was recorded in a novel environment (the gridfloor was hidden and a scent of peppermint was added)for 3 min without the auditory cue stimulus (pre-CStest). Finally, the auditory cue was turned on, and thetime spent freezing was recorded over the following 3min (cue CS test).Spatial learning and memory were examined in aMorris water maze [69,70], which consisted of a circulartank (32.5 cm high × 150 cm diameter), filled withwater (up to 16 cm deep), maintained at 26°C, andmade opaque with nontoxic white paint. A circularplatform (15 cm high × 15 cm diameter) remained hid-den 1 cm below the water surface at a fixed position.The room housing the tank had a permanent display ofdistal extra-maze cues. The swim paths of the micewere recorded using computerized EthoVision videotracking equipment. During training (acquisition phase),the mice were given four swim trials daily with an inter-trial interval of 15 min. The mice were placed in thepool facing the wall at one of four starting positions. Ifthe animal did not find the platform after 120 s, it wasguided there by the experimenter. Mice were allowed torest 15 s on the platform before being removed fromthe pool. Latency to reach the platform, path length,and average swim speed were recorded. After five train-ing days, there were two days of rest, followed byanother five days of training and two days of rest. Probetrials were performed on days 8 and 15. During probetrials, the platform was removed and each animal wasmonitored once for 100 s, recording the percentagetime in each quadrant. Over all the trials, one Survivin-Camcre mouse floated with a speed of < 5 cm/s, and thismouse was therefore excluded from the study.Statistical analysesData are presented as the mean ± SEM. Data were ana-lyzed with a two tailed t-test, Mann-Whitney Rank SumTest, one way ANOVA, or two way repeated measuresANOVA. All statistical tests were performed at a signifi-cance level of 0.05.Additional file 1: Supplemental Figures S1-S6. Supplemental FigureS1: CAMKIIa-cre activity in neurogenic regions of embryo. CAMKIIa-cre recombinase activity in the embryonic brain was checked bybreeding CAMKIIa-cre mice with ROSA26-stop-YFP reporter mice. GFPstained coronal section through the ganglionic eminence and dorsaltelencephalon of E12.5 CAMKIIa-cre+/-:ROSA26-stop-YFP/wt mice revealsprominent CAM-cre activity in the ganglionic eminences, but less in thedorsal telencephalon. Survivin mRNA expression is shown in adjacentsection. Scale bars 500 μm. HP, hippocampus; GE, ganglionic eminence;NCX, neocortex. Supplemental Figure S2: CAMKIIa-cre is notexpressed in SGZ or SVZ postnatally. Sagittal sections through thedentate gyrus (A, C-E) and lateral ventricle (B, F-H) of CAMKIIa-cre+/-adult mouse brain (6 weeks). (A, B) Staining for cre recombinase (red)and DAPI nuclear staining (blue) shows that cre expression is present inthe dentate granule cell layer (GCL), the striatum (ST) and the cortex(CTX). Lack of red staining of DAPI+ nuclei in the SGZ and SVZ/RMSconfirms that CAMKIIa-cre is not expressed in the SGZ or SVZ NPCspostnatally. Double stainining of the dentate gyrus (C-E) and the SVZ (F-H) for cre recombinase (red) and mature neuronal marker NeuN (green),with overlay of fields (E and H), confirms that CAMKIIa-cre expressioncolocalizes 100% with NeuN and is not present in SGZ or SVZ NPCs. LV,lateral ventricle. Supplemental Figure S3: Exogenous gene delivery ofsurvivin in embryonic NPCs may increase OB neurogenesis. GFPlabeling of sagittal sections through the olfactory bulb (OB) of P21control (A, B) and SurvivinCamcre (ko) (C, D) mice that were injected in thecerebral ventricle at E12.5 with control-GFP (A, C) or survivin-GFP (B, D)lentiviral vector. Injection of survivin results in an increased number ofembryonic NPC-derived cells in the OB. Scale bars: 500 μm.Supplemental Figure S4: Cage activity recordings. Cage activity wasrecorded at 30 min intervals over 23 hours, monitoring the number oflaser beam crossings by each mouse (n = 12 per group). There were nosignificant differences between control (wt) and SurvivinCamcre (ko) miceCoremans et al. BMC Neuroscience 2010, 11:2http://www.biomedcentral.com/1471-2202/11/2Page 16 of 19in the total number of beam crossings (p = 0.40) and no alterations incircadian activity profiles (p = 0.30). Results reflect means + SEM.Supplemental Figure S5: Visual evoked potentials. Visual evokedpotentail (VEP) recordings from control (wt) and SurvivinCamcre (ko) mice,reveal similar peak latency and amplitude for both genotypes.Supplemental Figure S6: Passive avoidance studies. The SurvivinCamcremice (ko) exhibited a significant impairment in passive avoidancelearning, indicated by their shorter latency to enter the darkcompartment than the controls (wt).Click here for file[ http://www.biomedcentral.com/content/supplementary/1471-2202-11-2-S1.PPTX ]Additional file 2: Supplemental Methods and Results. Additionalmethods and resultsClick here for file[ http://www.biomedcentral.com/content/supplementary/1471-2202-11-2-S2.rtf ]AcknowledgementsThis work was supported in part by the Fonds voor WetenschappelijkOnderzoek (FWO), Belgium. YB was supported by grants from ParentsAgainst Childhood Epilepsy (PACE, Inc.), NY, USA, and the Italian NationalResearch Council (CNR - “Ricerche Spontanee a Tema Libero” - RSTLProgram).Author details1KU Leuven, VIB Vesalius Research Center (VRC), Herestraat 49, GasthuisbergON1, B3000 Leuven, Belgium. 2KU Leuven Laboratory of BiologicalPsychology, Tiensestraat 102, B3000 Leuven, Belgium. 3Dept ofPharmacology, University of Milan, via Vanvitelli 32, Milan, Italy. 4Istituto diNeuroscienze, Consiglio Nazionale delle Ricerche, via G. Moruzzi 1, 56100Pisa, Italy. 5KU Leuven, Laboratory for Neurobiology and Gene Therapy,Kapucijnenvoer 33, B3000 Leuven, Belgium. 6Developmental Biology Instituteof Marseille, NMDA CNRS, INSERM, Univ. de Mediterranee, Campus deLuminy, 13288 Marseille, France. 7Department of Psychiatry, University ofTexas Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas, Texas,75390-9070 USA. 8Department of Cellular and Molecular MedicineNeuroscience Program, University of Ottawa, 451 Smyth Road, Ottawa, K1H8M5 Canada. 9Laboratory of Molecular Neuropathology, Centre forIntegrative Biology, University of Trento, via delle Regole 101, 38060 Trento,Italy. 10Center for Blood Research, Faculty of Medicine, University of BritishColumbia, 2350 Health Sciences Mall, Vancouver, V6T 1Z3 Canada.Authors’ contributionsVC was involved in designing and performing all experiments. VC, DL, AE,RD’H, UB, MC, VB and HC helped in drafting the manuscript. VC, VR, AD, JC,MM and TJ prepared riboprobes, did cDNA cloning and sequencing, in situhybridizations, in vivo studies, histologic sectioning, acquisition of data andanalyses. TA, DB, RD’H helped in behavioral studies. FA, YB, MC helped inseizure studies. AE and DL provided continuous intellectual input, evaluationand interpretation of data. EC conceived, designed and co-ordinated theproject, and drafted the manuscript. All authors read and approved the finalmanuscript.Received: 24 July 2009Accepted: 5 January 2010 Published: 5 January 2010References1. Zhao C, Deng W, Gage FH: Mechanisms and functional implications ofadult neurogenesis. Cell 2008, 132:645-660.2. Young KM, Fogarty M, Kessaris N, Richardson WD: Subventricular zonestem cells are heterogeneous with respect to their embryonic originsand neurogenic fates in the adult olfactory bulb. J Neurosci 2007,27:8286-8296.3. 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Brain research 2001, 36:60-90.doi:10.1186/1471-2202-11-2Cite this article as: Coremans et al.: Impaired neurogenesis, learning andmemory and low seizure threshold associated with loss of neuralprecursor cell survivin. BMC Neuroscience 2010 11:2.Publish with BioMed Central   and  every scientist can read your work free of charge"BioMed Central will be the most significant development for disseminating the results of biomedical research in our lifetime."Sir Paul Nurse, Cancer Research UKYour research papers will be:available free of charge to the entire biomedical communitypeer reviewed and published immediately upon acceptancecited in PubMed and archived on PubMed Central yours — you keep the copyrightSubmit your manuscript here:http://www.biomedcentral.com/info/publishing_adv.aspBioMedcentralCoremans et al. BMC Neuroscience 2010, 11:2http://www.biomedcentral.com/1471-2202/11/2Page 19 of 19


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