In situ hybridization using a probe against full length survivin allowed assessment of the spatiotemporal expression of survivin during embryonic development. Survivin mRNA was detected in the neurogenic areas of the dorsal and ventral telencephalon surrounding the ventricles (neocortex, medial and lateral ganglionic eminences (MGE and LGE, respectively)) (Figure 1A). At E12.5, expression of survivin overlapped with Dlx1, a marker for mitotic cells in the MGE and LGE 24 (Figure 1A, D) and neurogenin 2 (Ngn2) 25, a marker for dividing precursors in the neocortex (Figure 1A, C). There was minimal overlap with Dlx5 (Figure 1B), which is primarily expressed by postmitotic cells in the mantle zone of the MGE and LGE, less so in mitotic cells in the SVZ, and almost absent in the ventricular zone 26. By E17.5, survivin was additionally expressed in the rostral migratory stream (RMS) and at the center of the olfactory bulb (OB) (Figure 1G, I, K). Survivin mRNA was also present in the retina and lens of the developing eye (not shown).

Postnatally, survivin expression was restricted largely to 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) (Figure 2A, C, D, E). Within the SVZ, survivin was not expressed in the ependymal cells immediately adjacent to the lateral ventricle (Figure 2C, D). During the first two postnatal weeks, survivin mRNA was also detected in granule cell precursors in the external germinal layer (EGL) of the developing cerebellum (Figure 2A). In the adult brain, survivin expression remained restricted to proliferating 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 were positive for the proliferation marker PCNA, while at both sites, 25-50% of PCNA positive cells expressed survivin. Thus, survivin expressing cells represent a subpopulation of the mitotically active cells. Doublecortin (DCX), present in immature, migrating neuroblasts 28 also overlapped with survivin in the SVZ, the RMS and the SGZ. However, similar to PCNA, not all DCX positive cells expressed survivin, indicating that survivin is restricted to a subpopulation of cells in the SVZ-RMS (not shown). Mature neuronal marker NeuN 29 immunoreactivity did not overlap with survivin, consistent with the lack of survivin expression by mature neurons (Figure 2C-E).

Overall, survivin expression is restricted during fetal development to NPCs in neurogenic regions of the telencephalon, of which a fraction populates the major neurogenic regions of the postnatal brain, i.e. the SVZ and the SGZ. Within these neurogenic regions, a subpopulation of NPCs continues to express survivin, which is downregulated once the cells differentiate into neurons. In both the embryo or adult, survivin is not detected in neurons in the cortex, OB, or hippocampus.

To study the in vivo role of NPC survivin, we generated mice in which the survivin gene is inactivated in the neurogenic regions of the brain prenatally. Mice expressing cre recombinase driven by the CamKIIα promoter 3031 were bred with mice in which the entire survivin gene is flanked by loxP sites 32. The resultant CamKIIα-cre:survivin^lox/lox (referred to as Survivin^Camcre) mice were born in the expected Mendelian distribution, i.e. there was no evidence of embryonic lethality.

We confirmed previous reports of cre recombinase activity in the CAMKIIα-cre embryos and mice 3031 by breeding the CAMKIIα-cre mice with the ROSA26 reporter mice, followed by immunohistochemical detection of GFP. Cre recombinase activity at E12.5 was detected prominently in the ventral telencephalon (ganglionic eminences), but less in the dorsal telencephalon, and not in the eye (Additional file 1: Supplemental Figure S1). Postnatally, it was restricted to postmitotic NeuN positive neurons in the hippocampus and cortex, with lower levels in the striatum, thalamus, hypothalamus and amygdala 30. Postnatal cre expression was absent in NPCs in the SVZ and SGZ (Additional file 1: Supplemental Figure S2). Thus, cross-breeding with the Survivin^lox/lox mice resulted in deletion of both survivin alleles in the neurogenic regions in the prenatal period (ganglionic eminences and neocortex).

Cre excision of survivin was assessed by in situ hybridization in Survivin^Camcre and corresponding control Survivin^lox/lox embryos. At E14.5 and E17.5, survivin expression in the Survivin^Camcre embyros was markedly reduced in the ganglionic eminences surrounding the lateral ventricles where mitotically active NPCs normally reside, in the RMS and in the OB (Figure 1F, H, J, L). This was associated with increased tunel staining, most evident in the ganglionic eminences, and minimally in the dorsal telencephalon (Figure 3A-D). BrdU labeling studies revealed decreased NPC proliferation in the SVZ, RMS and OB of E17.5 Survivin^Camcre embryos (Figure 3E-J). Thus, lack of NPC survivin results in increased embryonic NPC death and decreased NPC proliferation in specific embryonic neurogenic regions.

Body weights of Survivin^Camcre and control mice were not significantly different at birth. However, during the first month after birth, Survivin^Camcre mice had a significantly higher mortality rate of 28% (n = 27/95) compared to 0% (n = 0/97) in control mice. Adult Survivin^Camcre mice also had significantly smaller brains, and the OBs were strikingly hypoplastic (Figure 4A, B) (for brains: 421 ± 11 gm versus 329 ± 7 gm, for controls versus Survivin^Camcre, respectively, p < 0.001; for OBs: 19.7 ± 0.9 gm versus 3.5 ± 0.2 gm for controls versus Survivin^Camcre, p < 0.001; n = 6 mice per group). In situ hybridization confirmed loss of survivin expression in the SVZ and RMS (Figure 2A, B, F, G). Surprisingly, expression of survivin in the SGZ was not decreased postnatally in Survivin^Camcre mice (Figure 2I). Nissl stained brain sections from Survivin^Camcre mice revealed loss of the RMS, decreased cortical thickness (average reduction to 80% of control at bregma levels -1.34/-1.70/-2.46/-2.80 mm; n = 4 mice per group, p < 0.01 at each bregma level), enlarged ventricles, yet no morphological changes in the hippocampus (Figure 4C, D). The latter was confirmed by quantifying the volumes corresponding to the GCL and the hilus of the DG (GCL: 0.11 ± 0.01 mm³ versus 0.12 ± 0.01 mm³ for control and Survivin^Camcre mice, respectively; hilus: 0.11 ± 0.01 mm³ 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 with reduced expression of NPC survivin in the Survivin^Camcre mice might be caused by decreased NPC proliferation, increased cell death, and/or deficits in migration. Accumulation of cells in the anterior SVZ of the Survivin^Camcre mice was not observed, mitigating against a predominant migration defect. NPC proliferation, assessed 1 hr after a single dose of BrdU, revealed a significant reduction in BrdU labeled cells in the SVZ of the Survivin^Camcre mice (132 ± 16 cells versus 57 ± 8 cells for control and Survivin^Camcre mice, respectively, n = 4-5 mice per group, p = 0.002) (Figure 4E, F, M). Furthermore, both BrdU and DCX labeled cells were almost completely absent in the RMS, in striking contrast to the controls (Figure 4E-H). Cell death in the SVZ of the Survivin^Camcre mice was also significantly increased (Figure 4K, L, N), as quantified by the number of tunel+ cells in the anterior SVZ (1.5 + 0.21/100 nucleated cells versus 3.5 + 0.46 tunel+ cells/100 nucleated cells, for control and Survivin^Camcre mice, respectively, n = 4-5 mice per group, p = 0.004) (Figure 4N). In the RMS and OB, there were some tunel+ cells identified in the brains of the control mice, but not in Survivin^Camcre mice, the latter likely due to the lack of precursor cells in this region.

As noted above, survivin expression in the SGZ of the DG was not appreciably diminished after cre excision. There was also no alteration in the number of proliferating or immature neurons in the SGZ, as quantified by BrdU labeling (1010 + 88 versus 905 + 75 cells in controls and Survivin^Camcre mice, respectively, n = 4-5 mice per group, p = 0.39) (Figure 4I, J, O) or DCX immunoreactivity (not shown). Nor could we detect changes in tunel+ staining (2.3 + 0.18 versus 1.6 + 0.63 tunel+ cells/section in controls and Survivin^Camcre mice, respectively, n = 3-4 mice per group, p = 0.30) (Figure 4P).

In summary, striking survivin-dependent defects in neurogenesis are evident postnatally in the RMS and OB of Survivin^Camcre mice, due to a combination of increased SVZ NPC apoptosis and diminished cellular proliferation. Despite the fetal abnormalities, and in striking contrast to the RMS-OB, there were no obvious structural defects or alterations in hippocampal neurogenesis in the Survivin^Camcre mice that had no appreciable reduction in survivin expression within the hippocampus.

In pilot studies, we assessed whether prenatal administration of survivin to increase expression in NPCs could promote neurogenesis. E12.5 control Survivin^lox/lox and Survivin^Camcre embryos received an intracerebroventricular injection in utero with the lentiviral vector pCHMWS-eGFP-T2A-SRV140 (survivin vector) or pCHMWS-eGFP-T2A-Fluc (control vector). At P21, immunohistochemical analysis of the control Survivin^lox/lox mice that received the survivin vector revealed an increased number of embryonic precursor cell-derived cells in the OB as compared with the control vector (Additional file 1: Supplemental Figures S3A, B). With Survivin^Camcre embryos, the survivin vector did not apparently reverse the OB hypoplasia when examined at P21, but there were notably more embryonic precursor cell-derived cells in the OB of 3 out of 3 Survivin^Camcre mice that received the survivin vector, as compared with the 2 Survivin^Camcre mice that received the control vector (Additional file 1: Supplemental Figures S3C, D). Overall, these preliminary findings suggest that enhanced expression of NPC survivin may increase neurogenesis.

Although the average cortical thickness in the Survivin^Camcre mice was reduced, in situ hybridization with cortical markers (Cux2 for layers 2-4 33, Badlamp for layers 2/3/5 34, and ER81 for layer 5 35 confirmed the correct orientation and presence of all the cortical layers (not shown). Moreover, and in line with limited expression of cre recombinase in the dorsal telencephalon of CamKIIα-cre embryos, the density of cortical vGLUT1+ glutamatergic cells in the Survivin^Camcre mice was not altered (2282 + 157 cells/mm² versus 2480 + 71 cells/mm² in controls and Survivin^Camcre mice, respectivley, n = 4-5 mice per group, p = 0.30) (Figure 5A-C). In contrast, and consistent with prominent apoptosis and diminished BrdU labeling in the ganglionic eminences, prenatal depletion of survivin in the NPCs of Survivin^Camcre mice resulted in a significant reduction in the density of GAD65/67+ GABAergic interneurons in the postnatal adult cortex (493 + 12 cells/mm² versus 417 + 6 cells/mm² in controls and Survivin^Camcre mice, respectively, n = 5-6 mice per group, p < 0.001) (Figure 5D-F). This occurred in the absence of any changes in interneurons in the hippocampus (hilus + granular cell layer: 213 ± 8 cells/mm² versus 209 ± 17 cells/mm² in controls and Survivin^Camcre mice, respectively, n = 4 mice per group, p = 0.82). These data indicate an alteration in the excitatory/inhibitory balance in the brain of Survivin^Camcre mice that might be associated with postnatal alterations in cognition and behavior, and a lower seizure threshold.

Indeed, during routine handling, 2.5% (n = 9/352) of the Survivin^Camcre mice were recorded to have spontaneous tonic-clonic, generalized motor seizures starting from 2 weeks of age (no seizures observed in controls). To investigate seizure susceptibility, the response of control and Survivin^Camcre mice to kainic acid (KA) was assessed over a period of 2 hours. Control, saline-treated animals (n = 9 per group) showed no signs of epileptic activity. However, in response to KA, Survivin^Camcre mice exhibited a lower threshold for seizures that were more severe. Thus, at a subconvulsive dose of 20 mg/kg, KA induced limbic motor convulsions in 15% (n = 2/13) of control mice and 81% (n = 13/16) Survivin^Camcre mice (Figure 6A). The maximum seizure score was significantly higher in the Survivin^Camcre mice (p < 0.001). Seizure severity over the 2 hr observation period was also significantly greater in the Survivin^Camcre mice (p < 0.001) (Figure 6B). At a KA dose of 30 mg/kg, the mean latency to the first seizure was also shorter in the Survivin^Camcre mice compared to control mice (8.75 ± 2.75 versus 30 ± 7.64 min, respectively, n = 4 mice per group, p = 0.031). At that dose, all Survivin^Camcre mice rapidly developed status epilepticus (stage 5-6) and died of severe generalized convulsions, while all control animals survived the KA treatment (Figure 6A). Thus, Survivin^Camcre mice exhibit enhanced susceptibility to KA seizures.

Neuropeptide Y (NPY) is a multifunctional peptide that is expressed in GABAergic interneurons, regulates pre-synaptic excitatory transmission in the DG, and has anti-epileptic properties 36. Hilar NPY interneuron degeneration, ectopic expression of NPY in mossy fibers, and axonal sprouting are common features of limbic hyperexcitability 37. Due to the increased seizure activity in the Survivin^Camcre mice, we examined NPY expression under both basal conditions (saline treatment) and following induction of seizures (KA 20 mg/kg i.p.). The number of hilar NPY+ interneurons was not different between saline-treated control and Survivin^Camcre mice (490 ± 17 versus 398 ± 53 cells/mm², for control and Survivin^Camcre mice, respectively; n = 4-5 mice per group, p = 0.11). However, two weeks after 20 mg/kg KA treatment, there were significantly fewer NPY+ cells in the hilus of the Survivin^Camcre mice (446 ± 22 versus 306 ± 65 cells/mm² for control and Survivin^Camcre mice, respectively; n = 6-9 mice per group, p = 0.032). Moreover, ectopic NPY expression in mossy fibers was readily detected in 3 out of 4 saline-treated Survivin^Camcre mice but not in any of the corresponding controls (n = 5) (Figure 6C, D). This effect became more prominent after KA (5/6 Survivin^Camcre mice as compared to 0/9 controls). In 2 of these Survivin^Camcre mice, we furthermore observed ectopic NPY immunoreactivity in the supragranular layer, likely reflecting sprouting of mossy fibers (not shown).

Since seizure activity modulates hippocampal neurogenesis, we also evaluated the seizure-induced neurogenic response of the control and Survivin^Camcre mice. The volumes of the GCL and the hilus were not different between control and Survivin^Camcre mice (see above), and KA had no effect on that relationship (data not shown). To assess cell proliferation, BrdU was injected 3 days after KA or saline injection, and mice were sacrificed 1 day later. After saline injection, the total number of SGZ BrdU+ cells was not different in control and Survivin^Camcre mice (648 ± 58 cells versus 705 ± 99 cells in controls and Survivin^Camcre mice, respectively, n = 4 mice per group, p = 0.64) (Figure 6E). Compared to saline treated controls, KA treated mice exhibited an increase in the number of BrdU+ cells in the SGZ after KA injection. However, the neurogenic response was significantly dampened in the Survivin^Camcre KA treated mice compared to control KA treated mice (3578 ± 392 cells versus 1955 ± 233 cells for controls and Survivin^Camcre mice, respectively, n = 5 mice per group, p < 0.001) (Figure 6E). Numbers of BrdU+ cells remained reduced 2 weeks following KA in the Survivin^Camcre mice as compared to controls (data not shown). Thus, despite the higher seizure scores following KA in the Survivin^Camcre mice, there was less seizure-induced neurogenesis in the Survivin^Camcre versus the control mice.

Adult Survivin^Camcre mice exhibited several defects that may contribute to disorders in behavior and cognition, including reduced SVZ-RMS-OB neurogenesis 3839, OB hypoplasia 40, diminution of cortical GABAergic neurons, and seizures. We therefore evaluated the effects of depleting NPCs of survivin by subjecting the Survivin^Camcre and matched controls to a range of behavioral studies.

There was no difference in body weight between the Survivin^Camcre and control mice at the start of behavioral testing (21.6 ± 0.6 gm versus 20.1 ± 0.6 gm for the Survivin^Camcre and controls, respectively, p = 0.081), and the Survivin^Camcre mice had normal visual and auditory skills, grip strength, rotarod performance, pain response and cage activity (Additional file 1: Supplemental Figures S4, S5; Additional file 2: Supplemental Methods and Results).

In the open field test, the Survivin^Camcre mice displayed significant disturbances in exploratory behavior, including delayed first entry to the center, fewer center entries, more corner crossings, and less time in the center (Figure 7A). This type of behavioral outcome is often suggestive of increased anxiety, however the Survivin^Camcre mice performed normally in the elevated plus maze test for anxiety. On the maze, there was no difference between control and Survivin^Camcre mice in the total number of beam crossings (148 ± 5 versus 155 ± 9 for control and Survivin^Camcre mice respectively; n = 12 mice per group, p = 0.48), percent time spent in the open arms (33 ± 2 versus 36 ± 4 for control and Survivin^Camcre mice respectively; n = 12, p = 0.76), nor percentage of entries into the open arms (26 ± 2 versus 23 ± 3 for control and Survivin^Camcre mice respectively; n = 12, p = 0.07). The findings suggest that the poor performance of the Survivin^Camcre mice in the open field test may not be due to increased anxiety, but rather due to a distinct defect in exploratory behavior.

The Survivin^Camcre mice exhibited a significant impairment in passive avoidance learning. During the initial training trial, there was no difference in step-through latencies (12.5 ± 4.5 versus 13.9 ± 1.8 sec, Survivin^Camcre and control mice respectively, p = 0.783). However, during testing, the Survivin^Camcre mice demonstrated significantly shorter latency to enter the dark compartment than the controls (235 ± 27 versus 71 ± 24 sec for control and Survivin^Camcre mice, n = 12, p < 0.001) (Additional file 1: Supplemental Figure S6), consistent with poor associative memory of aversive stimuli.

We examined auditory and contextual fear memory using an auditory cue as the conditioned stimulus (CS), and a foot shock as an aversive stimulus. Freezing times in baseline, pre-US, post-US, and pre-CS trials were not different between the groups (Figure 7B). However, Survivin^Camcre mice exhibited significantly less freezing response in the context and auditory cue (CS) trials compared to control mice (context: 25.7 ± 5.5 versus 68.2 ± 4.5; auditory: 34.3 ± 9.0 and 84.4 ± 7.8, respectively for Survivin^Camcre and control mice, n = 12 mice per group, p < 0.001) (Figure 7B).

Lastly, we assessed hippocampus-dependent spatial learning and long-term memory in the Survivin^Camcre mice using the Morris water maze. Swimming velocity was not different between the controls and the Survivin^Camcre mice, excluding defects in motor ability. During acquisition training, the Survivin^Camcre mice showed training-dependent reduction in escape latency and path length. However, the improvements were minimal as compared with controls, and the escape latency and path length were significantly increased in the Survivin^Camcre mice (p < 0.001, n = 12; by two way repeated measures ANOVA) (Figure 8A, B). Notably, lengths of the escape paths were not different between the Survivin^Camcre mice and control mice during 4 visible-platform training days (803 + 88 cm versus 1076 + 110 cm for control, n = 10, and Survivin^Camcre mice, n = 12, respectively; p = 0.075). These results indicate that the Survivin^Camcre mice were fully capable and motivated to choose the shortest pathway towards the platform in the cued, non-spatial condition of the task.

In the first probe trial performed after 5 days of training, the control mice already had a preference for the target quadrant compared to adjacent 1 (p = 0.002) and opposite (p = 0.02) quadrants, whereas the Survivin^Camcre mice had no preference at all (p = 0.46) (Figure 8C). In the second probe trial after 10 days of training, the control mice continued to show a strong preference for the target quadrant compared to all other quadrants (p < 0.001), whereas the Survivin^Camcre mice equally favoured 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 might alter neural cell proliferation, we also quantified the number of Ki67+ cells in the dentate gyrus of the Survivin^Camcre mice (n = 4) and the corresponding littermate controls (n = 4) after the probe trials. There was no significant difference in the number of Ki67+ cells between the two groups (534 + 49 cells versus 480 + 28 cells in controls versus Survivin^Camcre mice, respectively, p = 0.378).

Overall, the Survivin^Camcre mice exhibited exploratory behavioral abnormalities, with global deficits in various forms of learning and memory.

Mice that express Cre recombinase driven by the promoter of the gene for calmodulin-dependent protein kinase IIα (CamKIIα) 3031 (gift of Dr. G. Schütz, Heidelberg, Germany) were bred with mice in which the survivin gene is flanked by loxP sites 32. The resulting offspring that were heterozygous for Cre and homozygous for floxed survivin (Survivin^lox/lox) (hereafter referred to as Survivin^Camcre mice) were compared to littermate control mice which did not express Cre and were Survivin^lox/lox. Survivin^lox/lox embryos and adults were not different from Survivin^lox/wt, Survivin^wt/wt or CamKIIα-cre:survivin^lox/wt mice. Mice were maintained on a C57B/6:Swiss:129svj 75:12.5:12.5 background. Genotyping of tail DNA was performed by PCR as previously reported 3032. Mice were group-housed in standard mouse cages in a room with a 12 h light-dark cycle and ad libitum access to food and water and all animal experiments were approved by the ethics committee of the University of Leuven.

Adult mice and pregnant females were injected intraperitoneally (ip) with 5-bromo-2-deoxyuridine (BrdU, Sigma Aldrich, Bornem, Belgium) at a concentration of 50 mg per kg body weight. For the analysis of the embryos, 1 hour after injection of BrdU, pregnant females were killed by cervical dislocation, after which the embryos were harvested, placed in ice-cold PBS, and then fixed in 4% paraformaldehyde (Para) for cutting 20 μm cryo sections using a microtome/cryostat (HM550, Microm, Walldorf, Germany). For analysis of adults, mice were anesthetized with sodium pentobarbital at 1 hour (unless stated otherwise) after BrdU and perfused transcardially with 0.9% NaCl, followed by fixation with 4% paraformaldehyde. Brains were dissected and post-fixed overnight at 4°C and 40 μm tissue sections were prepared 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 the optical fractionator method 55 with Stereo Investigator software (MicroBrightField, Colchester, VT, USA). Cells were counted with a 40× objective on every sixth section through the entire rostrocaudal extension of one half of the DG, restricted to the subgranular zone (SGZ) 56. The number of BrdU+ cells in the SVZ of one lateral ventricle was counted with a 40× objective on 1 coronal section (bregma level + 0.14 mm) per animal.

Immunostaining protocols were optimized for the different tissue preparations and antibodies. In general, tissue sections were treated with 1% H₂0₂ in PBS/methanol for 15 min, incubated in 5% serum for 30 min, and incubated overnight at 4°C in following primary antibodies: rabbit anti-neuropeptide Y (NPY) antibody (1:5000, Bachem, UK); mouse anti-NeuN (1:500, Chemicon, Hofheim, Germany); mouse anti-PCNA (1:1000, Chemicon, Hofheim, Germany); rabbit anti-DCX (1:500, Cell Signaling, MA, USA); rat anti-BrdU (1:500, Immunologicals Direct, 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). After washes, the corresponding biotinylated secondary antibody was added for 1 hour and the signal was amplified using 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 staining, Alexa-conjugated secondary antibodies (Molecular Probes, Leiden, The Netherlands) were used. BrdU staining was performed as reported previously 57.

Digoxygenin (DIG)-labeled RNA probes for Dlx1 58, Dlx5 24, Ngn2 (gift from Dr. A. Simeone), GAD65 59, GAD67AE 60 and vGLUT1 (Allen Institute for Brain Science) were generated using the DIG RNA Labeling Kit (Roche Diagnostics, Basel, Switzerland), according to the manufacturer's instructions. GAD65 and GAD67AE probes were mixed to detect the total number of GABAergic interneurons. For survivin riboprobes, full-length murine survivin cDNA was cloned into the pcDNA3 plasmid vector (Invitrogen, CA, USA) 61, and linearized for generation of antisense and sense probes using Sp6 RNA polymerase or T7 polymerase, respectively. In situ hybridization and combined immunohistochemistry protocols were adapted from those reported 3362 and completed on 20 μm cryostat or 40 μm vibratome sections.

Coronal sections through the DG were stained with cresyl violet. Pictures were taken at 4× magnification, and the area of the GCL and the hilus was determined off line using Metamorph software (Molecular Devices, Sunnyvale, CA). Volumes were calculated and expressed in mm³.

Detection of cellular apoptosis in 10 μm coronal paraffin sections, prepared using a HM360 microtome (Microm, Walldorf, Germany), was accomplished using the ApopTag Peroxidase In Situ Apoptosis Detection Kit (Chemicon, Hofheim, Germany). The number of tunel+ cells was counted with a 40× objective. The anterior subventricular zone (SVZa) was analyzed at level bregma +0.98 mm 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 of tunel+ cells per section.

Numbers of neurons and interneurons were quantified hemilaterally on coronal vibratome sections at 4 bregma levels (-1.34/-1.70/-2.46/-2.8 mm). GAD65/67+ cells were counted with a 10× objective in the hilus plus the granule cell layer, and in the parieto/temporal cortex, in a 1.4 mm wide band from the white matter to the pial surface. vGLUT1+ cells in the parieto/temporal cortex were counted with a 20× objective in a 0.7 mm wide band. NPY+ cells in the hilus were counted with a 20× objective on every sixth section (40 μm thick). Results are presented as the number of cells per mm².

Ki67 positive cells in the SGZ were counted hemilaterally with a 40× objective on every third 40 μm section between bregma levels -1.34 and -2.8 mm. The number of counted cells was multiplied by 3 to obtain the total number of Ki67 positive cells.

Lentiviral vectors were prepared, encoding enhanced green fluorescent protein (eGFP) and survivin separated by a T2A sequence starting from pCHMWS-eGFP-T2A-Fluc (gift from Dr. V. Baekelandt, KULeuven). The Fluc fragment was removed from pCHMWS-eGFP-T2A-Fluc using BamHI and MluI and replaced by the cDNA encoding full-length murine survivin. Survivin expression from this vector was confirmed by Western blot analysis of lysates from transfected COS cells. Human immunodeficiency virus type 1 (HIV-1)-derived lentiviral vectors were produced by a standard protocol. The viral vector was mixed with Fast Green dye (0.005% final concentration, Sigma-Aldrich, Bornem, Belgium), which allowed visualization of the distribution of the viral vector in the cerebral ventricles after injection. Pregnant mice (stage E12.5) were anesthetized with 50 mg/ml ketamine, 2% xylazine in saline and placed supine on a heating pad. A 2-cm midline incision was made through the skin and the abdominal wall. The uterine horn was drawn out through the hole onto gauze, and with the uterus transilluminated, a 35 gauge needle (beveled NanoFil needle, World Precision Instruments, FL, USA) was inserted into the ventricle, and 1 μl viral vector solution was injected at a speed of 406 nanoliters per second using a Mycro4® MicroSyringe Pump Controller (World Precision Instruments, FL, USA).

Seizures in adult male mice were evoked by ip administration of kainic acid (KA) (Sigma, MO, USA). KA was dissolved in saline and injected at 20 or 30 mg/kg body weight. Saline-injected animals were used as controls. Seizure severity was quantified by an observer blind to the mouse genotype using the following scale 4863: stage 0, normal behavior; stage 1, immobility; stage 2, forelimb and/or tail extension, rigid posture; stage 3, repetitive movements, head bobbing; stage 4, rearing and falling; stage 5, continuous rearing and falling; stage 6, severe whole-body convulsions; and stage 7, death. For each animal, seizure severity was scored every 10 min over a period of 2 hours after KA administration. The maximum score reached by each animal over the entire observation period was used to calculate the maximum seizure score for each treatment group. Seizure severity over the 2 hour observation period was calculated for each mouse as the area under the seizure score versus time curve (AUC), and the average AUC was calculated for each treatment group.

Behavioral tests were initiated when the mice were 3-4 months of age, n = 12-25 per group. Neuromotor, exploration, and learning tests were performed in the following sequence: cage activity, grip strength, rotarod, open field, elevated plus maze, Morris water maze, passive avoidance. Contextual fear conditioning was performed on a separate group of mice. Animals were tested during the light phase of the light-dark cycle. All studies were performed by observers who were blinded to the genotype of the mice.

Open field exploratory activity was assessed in a 50 cm × 50 cm arena using EthoVision video tracking and software (Noldus, Wageningen, The Netherlands). Mice were individually placed in a specific corner of the open field, and were allowed a 1 min adaptation period. The path was recorded for 10 min to measure dwells and entries in different parts of the field. Measures included total path length, percentage path length in the center circle (diameter 30 cm), entries into the four corner squares, entries into the center, time spent in the center versus periphery, latency of first center approach, and frequency of rearing.

The elevated plus maze 6465, to evaluate anxiety-like behavior, had two open arms (21 cm × 5 cm) and two closed arms of the same size, with high side walls, and was raised 30 cm above the table. Each mouse was placed in the central square of the maze, facing one of the closed arms. After 1 min, exploratory behavior was recorded automatically during a 10 min period using five infrared beams, connected to an activity logger. For each mouse, the number of arm entries, percentage of open arm entries, and percentage time spent in the open arms was assessed.

Passive avoidance (aversive) learning 66 was tested in a two-compartment step-through box. Animals were adapted to the dark for 30 min, and then placed into a small illuminated compartment. After 5 s, a sliding door leading to the large dark compartment was opened. Upon entry, the door was closed and the animal received an electric foot shock (0.3 mA, 1s). Twenty-four hours later, the animals were placed again in the light compartment and the latency to enter the dark compartment was measured up to 300 s, to evaluate memory of the foot shock.

Contextual and auditory-cued fear conditioning 6768 was tested in a Plexiglas chamber with a grid floor through which a foot shock could be administered. Mice were trained and tested on 3 consecutive days: On the day 1, the mice were individually placed in the testing chamber and allowed to adapt for 5 min. On the day 2, the animals were allowed to explore the testing chamber for 2 min, after which an auditory cue (conditioned stimulus, CS) was presented for 28 s, followed by a foot shock (0.3 mA, 2 s; unconditioned stimulus, US). The time (%) spent freezing during the first 2 min and 28 s is the pre-US score. The mice were then allowed to explore again for 1 min, and the auditory cue and shock were again presented, followed by another 2 min exploration (post-US score). On day 3 (24 hours after training), mice were returned to the same context in which training occurred, and freezing behavior was recorded for 5 min (context test). Ninety min later, freezing was recorded in a novel environment (the grid floor was hidden and a scent of peppermint was added) for 3 min without the auditory cue stimulus (pre-CS test). Finally, the auditory cue was turned on, and the time spent freezing was recorded over the following 3 min (cue CS test).

Spatial learning and memory were examined in a Morris water maze 6970, which consisted of a circular tank (32.5 cm high × 150 cm diameter), filled with water (up to 16 cm deep), maintained at 26°C, and made opaque with nontoxic white paint. A circular platform (15 cm high × 15 cm diameter) remained hidden 1 cm below the water surface at a fixed position. The room housing the tank had a permanent display of distal extra-maze cues. The swim paths of the mice were recorded using computerized EthoVision video tracking 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 the pool facing the wall at one of four starting positions. If the animal did not find the platform after 120 s, it was guided there by the experimenter. Mice were allowed to rest 15 s on the platform before being removed from the pool. Latency to reach the platform, path length, and average swim speed were recorded. After five training days, there were two days of rest, followed by another five days of training and two days of rest. Probe trials were performed on days 8 and 15. During probe trials, the platform was removed and each animal was monitored once for 100 s, recording the percentage time in each quadrant. Over all the trials, one Survivin^Camcre mouse floated with a speed of < 5 cm/s, and this mouse was therefore excluded from the study.

Data are presented as the mean ± SEM. Data were analyzed with a two tailed t-test, Mann-Whitney Rank Sum Test, one way ANOVA, or two way repeated measures ANOVA. All statistical tests were performed at a significance level of 0.05.