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Keywords:

  • anxiety-related behavior;
  • c-Fos;
  • knockout mouse;
  • neurogenesis;
  • neuronal activity;
  • SPARC

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. References
  7. Acknowledgments
  8. Supporting Information

SPARC (secreted protein acidic and rich in cysteine) is a matricellular protein highly expressed during development, reorganization and tissue repair. In the central nervous system, glial cells express SPARC during development and in neurogenic regions of the adult brain. Astrocytes control the glutamate receptor levels in the developing hippocampus through SPARC secretion. To further characterize the role of SPARC in the brain, we analyzed the hippocampal-dependent adult behavior of SPARC KO mice. We found that SPARC KO mice show increased levels of anxiety-related behaviors and reduced levels of depression-related behaviors. The antidepressant-like phenotype could be rescued by adenoviral vector-mediated expression of SPARC in the adult hippocampus, but anxiety-related behavior persisted in these mice. To identify the cellular mechanisms underlying these behavioral alterations, we analyzed neuronal activity and neurogenesis in the dentate gyrus (DG). SPARC KO mice have increased levels of neuronal activity, evidenced as more neurons that express c-Fos after a footshock. SPARC also affects cell proliferation in the subgranular zone of the DG, although it does not affect maturation and survival of new neurons. SPARC expression in the adult DG does not revert the proliferation phenotype in KO mice, but our results suggest a role of SPARC in limiting the survival of new neurons in the DG. This work suggests that SPARC could affect anxiety-related behavior by modulating neuronal activity, and that depression-related behavior is dependent upon the adult expression of SPARC, which affects adult brain function by mechanisms that need to be elucidated.

SPARC (secreted protein acidic and rich in cysteine, also known as osteonectin) is a matricellular glycoprotein involved in tumor progression and wound healing. SPARC is highly expressed in different tissues during embryogenesis, but in the adult its expression is restricted to tissues undergoing remodeling, repair and tumorigenesis (Bradshaw & Sage 2001, Sage et al. 1989). In the brain, it is expressed both during development and in adulthood. In the embryonic mouse brain, SPARC is expressed by the radial glia in regions undergoing neurogenesis (Vincent et al. 2008). In the postnatal brain, SPARC is still expressed by the radial glia, but it is also present in the rostral migratory stream, surrounding proliferating cells.

In the adult mouse brain, glial cells express SPARC in different regions of the central nervous system, especially in neurogenic regions such as the subventricular zone and the subgranular zone of the dentate gyrus (SGZ-DG) (Vincent et al. 2008). It was also detected in the molecular layers of the CA1 and the cerebellum, linking its expression to regions enriched in synapses (Mendis et al. 1995). Moreover, SPARC expression is upregulated in the hippocampus upon entorhinal deafferentation (Liu et al. 2005) and after epileptic episodes (Ozbas-Gerceker et al. 2006); further supporting that SPARC expression is linked to regions of increased proliferation, axonal sprouting and synaptogenesis.

Recently, it was shown that postnatal astrocytes secrete SPARC and that this protein is essential to the regulation of AMPA receptors at maturing synapses in the hippocampus (Jones et al. 2011). Neurons grown in the presence of SPARC KO astrocytes exhibit increased synaptic strength and a diminished NMDAR/AMPAR ratio, which reduces the capacity of neurons to develop long-term potentiation. Moreover, another recent work shows that SPARC is a negative regulator of synapse formation (Kucukdereli et al. 2011)

We reasoned that the pattern of expression of SPARC and the postnatal role of this protein in determining neuronal activity could reflect a role of this protein in regulating hippocampal-dependent behavior in the postnatal and/or the adult mouse. The aims of this work were then: (1) to determine whether lack of SPARC could result in hippocampal-dependent behavioral abnormalities, (2) to determine the role of adult hippocampal SPARC in these behavioral phenotypes and (3) to identify possible molecular and cellular mechanisms that mediate this effect. The hippocampus is involved both in cognitive function and in mood regulation, modulating anxiety states (Deacon et al. 2002) and depression (Mcewen & Magarinos 2001). We found that SPARC KO mice exhibit anxiety-related behavior and antidepressant-like behavior, and that these mice show a diminished response to spatial novelty. Moreover, we found evidence of increased neuronal activity and reduced cell proliferation in the adult DG of SPARC KO mice. We next found that the re-expression of SPARC in the adult DG of the hippocampus reverts the antidepressant-like behavior in SPARC KO mice.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. References
  7. Acknowledgments
  8. Supporting Information

Animals

B6;129S-Sparctm1Hwe/J mice were obtained from the Jackson Laboratory (Bar Harbor, Maine, USA) and bred in the animal house of the Leloir Institute Foundation. In all experiments, littermates from heterozygous breeding were used. All animals were genotyped by polymerase chain reaction as recommended by the supplier (Jackson Laboratory), using mouse tail genomic DNA as template. Specific primers were as follows: sense, 5′-TTCTTCCTTGCAACCCTCTC-3′; wildtype (WT) reverse, 5′-TGTGGAGCTTCCTCTGTCCT-3′; and KO reverse, 5′-GGGGTTTGCTCGACATTG-3′.

All animals had water and food ad libitum and were housed on a 12:12 light:dark cycle with lights on at 0800 h. Animals were group housed with four to five animals per cage. Surgery, treatments, behavioral studies and euthanasia were performed under conditions that fully comply with National Institute of Health (NIH) guidelines. The experiments were carried out in female mice to acknowledge that affective disorders are more prevalent in women than in men (Gorman 2006) and that there is a need to overcome the sex bias present in neuroscience research (Beery & Zucker 2011). To avoid the confounding effect of previous experience in the analysis of neurogenesis or c-Fos expression, we used independent groups of animals for each experiment. The specific number of animals for each experiment is stated in the figure legends.

Behavior testing

All behavioral testing was performed during the light period (between 0900 and 1600 h). Mice were 8–10 weeks of age at the beginning of testing. The tests were performed in the order listed below, using 1-week intervals to reduce inter-test interactions as previously showed (Lucchina et al. 2010). Animals were habituated to the illumination 30 min before testing. After testing, each mouse was identified and placed in a holding cage until all animals in a cage had been tested. Each apparatus was cleaned with 20% ethanol and dried between sessions. All tests were performed before 4 months of age in order to avoid problems arising from eye degeneration in SPARC KO genotype (Norose et al. 1998). Moreover, at the end of each behavioral test all animals were evaluated for visual placing (Heyser 2004). Two animals were withdrawn from the analysis because they only extended the forearms after vibrissa contact with the surface (<1 cm), which could result from visual problems.

Elevated plus maze

Elevated plus maze (EPM) was performed as previously described (Depino et al. 2008, Lucchina et al. 2010). The maze consisted of two open arms (30 × 5 cm, 0.5 cm black border, 105 lx) and two closed arms (30 × 5 cm, 19 cm high, black walls, 43 lx), with gray polyvinyl chloride (PVC) floor. The apparatus was elevated 50 cm above the floor. Mice were placed into the central platform of the maze facing an open arm and allowed to explore the maze for 5 min. Locomotor behavior was recorded by a video-tracking system (ANY-maze; Stoelting, Wood Dale, IL, USA). Measured locomotor parameters were as follows: total distance, time in the central platform, time in open arms, time in closed arms, % entries into open arms and open arms entries. Ethological parameters were scored manually during each session: time grooming, number of rearings, number of head dippings and number of protected head dippings.

Open field

Open field (OF) was performed as previously described (Depino et al. 2008, Depino et al. 2011, Lucchina et al. 2010). Mice were placed along one side of a 45 × 45 cm arena, with 30-cm high black walls. The center region was defined as the central 23 × 23 cm area. Locomotion data were collected by a video-tracking system (ANY-maze). Measured variables were as follows: total distance, entries to the center, latency to enter the center, time and distance in the center, time and distance in the periphery and percentage of distance in the center. Time grooming and number of rearings were manually scored during the test. Total distance, time grooming, number of rearings, time in the center, latency to enter the center and percentage of distance in the center showed a correlation factor <0.70 and were used in the multiple analysis of variance (manova) analysis. Twenty-four hours after animals performing the test were re-exposed to the same OF and habituation was evaluated.

Light/dark box

Light/dark (LD) test was performed as previously described (Depino et al. 2008, Lucchina et al. 2010). A 45 × 45 cm arena was divided at half with an inverted black box (lit side: 35 lx; dark side: 1 lx). Animals freely moved between the compartments through a 12 × 8 cm hole in the wall. Each mouse was placed under the hole facing the lit side and observed for 5 min. Time spent in the lit compartment was counted using a stopwatch.

Novel object recognition

Novel object recognition (NOR) was performed as previously described (Depino et al. 2008, Depino et al. 2011). An open-field box was conceptually divided into four quadrants, three initially containing objects and one empty. Control objects (object A: 250 ml Erlenmeyer flask, and object C: two 3 × 3 × 3 cm wooden blocks) remained in the same position along the test. The displaced object (object B) was a tennis ball fixed to a plastic petri dish. The novel object (object D) was a plastic, blue pipette tip rack. The mice were tested in six 5-min sessions separated by a 3-min delay, except for between sessions 3 and 4, where there was a 60-min delay. Sessions 1–3 allowed for the habituation to the novel environment and to objects A, B and C (Fig. S1). All animals paid a similar number of visits to objects A–C in session 1, and there was no effect of genotype on the time they spent exploring the objects. However, mice spent less time exploring object A (Fig. S1d). For this reason, object C was used as the control object in the analysis. For the spatial novelty session, object B was moved to the empty quadrant and left there for the rest of the test. For session 6, the novel object session, object D was placed next to object A. Object exploration was scored as the number of visits and the time spent exploring an object (orientation of the animal toward the object with its nose within 1 cm of the object). Results are expressed as the increase in the number of visits or the time exploring the object in the session, with respect to the previous session.

Novel object exploration in cage

Novel object exploration (NOE) in cage was performed as previously described (Depino et al. 2011). Animals were single caged for 24 h and then exposed to a small (3 cm diameter) yellow rubber ball, placed in the corner of the cage. Animals were allowed to explore for 5 min. Total distance was obtained from AnyMaze tracking and NOE (nose less than 2 mm from the ball) was manually measured by the experimenter.

Tail suspension

Tail suspension (TS) test was performed as previously described (Depino et al. 2011). Animals were suspended in the air using an adhesive tape wrapped around the subject's tail and fixed to a horizontal wire at 25 cm of height. Time spent immobile during 5 min was measured with a stopwatch.

Forced swimming test

Porsolt's forced swimming (FS) test was performed as previously described (Depino et al. 2011). Animals were gently placed in a beaker glass (15 cm in diameter and 25 cm in height), filled with 14 cm of water at 25°C. Time spent immobile during a 5-min session was measured with a stopwatch. At the end of the test, animals were dried with a paper towel.

Adenoviral vectors

Adenoviral vectors expressing the nuclearly targeted reporter protein β-galactosidase (Adβgal) or the human SPARC protein (AdSPARC) were generated, controlled and used as previously described (Ferrari et al. 2004, Prada et al. 2007). Stocks were quantified by plaque assay (final titers: Adβgal = 9.66 × 108 pfu/µl, AdSPARC = 1.2 × 109 pfu/µl), and had less than 1 ng/ml of endotoxin (measured with E-TOXATE Reagents; Sigma–Aldrich, St. Louis, MO, USA). Viral stocks were free of autoreplicative particles. Adβgal was kindly provided by Dr. J. Mallet (Hospital Pitie Salpetriere, Paris, France) and AdSPARC was generated and tested in the laboratory (Ferrari et al. 2004, Prada et al. 2007).

Stereotaxic injections

For stereotaxic injections, the animals were anaesthetized with ketamine chlorhydrate (80 mg/kg) and xylazine (8 mg/kg). Animals were 8–9 weeks of age at the time of surgery. The adenoviral vectors were administered with a 50-µm tipped finely drawn glass capillary (to minimize mechanical injury and glial activation). Each animal was injected bilaterally in the DG with 5 × 106 pfu adenoviral particles in 1 µl, infused for 5 min. Preliminary studies showed that this was the highest dose that did not result in extensive inflammation and effectively distributed the vector along the dorsal DG. The stereotaxic coordinates were as follows: bregma, −2.1 mm; lateral, ± 1.3 mm; ventral, −1.3 mm. Animals were kept in a cage placed over a heating pad until they woke up. We previously showed that transgene expression in the DG was high and constant from 7 to 28 days post-injection (Depino et al. 2011), and we confirmed this pattern of expression in preliminary studies here (data not shown). Behavior testing was therefore started 14 days post-injection and performed within 2 weeks.

Analysis of c-Fos expression after footshock

Animals were 8–10 weeks of age at the time of footshock exposure and sacrifice. We used a previously described protocol that elicits mild granule cells activation in the DG of the mouse (Yamasaki et al. 2008). Briefly, we delivered 10 footshocks (0.6 mA, 1 second) to adult mice through the metal grids in the bottom of a fear-conditioning chamber. The interval between shocks was 30 seconds. Two hours after the last footshock, mice were perfused with 4% paraformaldehyde (PFA), brains were post-fixed for 4 h in 4% PFA and then criopreserved in 30% sucrose; 40-µm thick coronal sections were prepared on a cryostat (Leica, Wetzlar, Germany). Every sixth section (a total of eight per animal), spaced by 200 µm and spanning the whole hippocampus from Bregma −1.34 mm to Bregma −3.28 mm (Paxinos & Franklin 2001), was processed for immunohistochemistry. We used the primary antibody rabbit anti-c-Fos (1:5000 in blocking solution; Calbiochem, Darmstadt, Germany), the secondary antibody biotin-SP-conjugated donkey anti-rabbit (Jackson Laboratories) and the ABC kit (Vector Laboratories, Burlingame, CA, USA). Positive cells were counted on × 400 magnification in a light-field microscope (CX31; Olympus, Buenos Aires, Argentina) using a modified version of the optical fractionator (West et al. 1991), as previously reported (Ngwenya et al. 2005). Estimates for the total c-Fos-positive cells (N) for each animal were calculated according to: N = ΣQ × 1/ssf × 1/asf × t/h, where ΣQ is the sum of counted cells, ssf is the section sampling fraction, asf is the area sampling factor, t is the thickness of section and h is the height of the dissector box. We did not use guard zones as typically required for the optical fractionator technique, making h = t. Using full-section thickness may result in undercounting, but we reasoned that this would affect all experimental groups in a similar way and proportionally to the number of positive cells observed, therefore not affecting the reported results. The top plane was used as the exclusion plane. Due to the low numbers of c-Fos-positive cells, the entire granular layer of the DG was used as the counting frame, making asf = 1. The ssf was 1/6. Estimates were based on counting c-Fos-positive nuclei as they came into focus. Total c-Fos-positive cells are presented normalized by the DG granular layer volume for easy visualization. For this, each counted section was photographed under × 40 magnification using a digital camera (Infinity 1; Lumenera Corporation, Ottawa, ON, Canada) attached to the microscope. In each image, the volume of the DG granule cell layer was determined using ImageJ (Rasband 1997–2009) (Fig. S2a): the area in pixels was transformed to mm2, and then multiplied by the thickness of the section (0.04 mm).

Analysis of neurogenesis

In all cases, cells are labeled during the 10th or 11th week of age.

Neural precursors proliferation and differentiation

Animals received daily intraperitoneal (ip) injections of 2-bromo-5-deoxyuridine (BrdU, 50 mg/kg in saline; Sigma) during 7 days, they were sacrificed 24 h after the last injection and their brains processed for immunohistochemistry/immunofluorescence. Animals were 11–12 weeks of age at the time of sacrifice, and their brains were processed as described above.

For total BrdU quantification, every sixth 40-µm thick coronal section was stained using the rat anti-BrdU (1:150; Abcam, Cambridge, MA, USA) and the biotin-SP-conjugated donkey anti-rat antibodies (Jackson Laboratory), and the ABC kit. Total BrdU-positive cells (N) in the SGZ-DG were counted on × 400 magnification in a light-field microscope using the same modified version of the optical fractionator described for c-Fos-positive cells. Positive cells outside of the SGZ were not considered. The number obtained was multiplied by 6. For BrdU + DCX analysis, every 12th 40-µm thick coronal section was processed using the anti-BrdU and the rabbit anti-DCX (Abcam) antibodies, Cy2-conjugated donkey anti-rat (Jackson Laboratory) and Alexa fluor 647-conjugated streptoavidin (Jackson Laboratory). Double-stained cells in the SGZ were quantified using z-scan confocal microscopy (Olympus FV300 confocal-laser scanning microscope equipped with argon and He/Ne laser that emitted at 488 and 633 nm, respectively; Olympus) at × 400 magnification. Each image had a 2-µm thickness in the ‘z’ axis. Images were analyzed using the fv10-asw version 2.0 software (Olympus Fluoview; Fig. S2b). Between 40 and 100 BrdU-positive cells were counted per animal to calculate double-labeled cell percentages. The same images were used to estimate the density of DCX-positive cells, by counting the number of positive cells and dividing it by the volume of the granular cell layer (the area measured with the ImageJ software and multiplied by 0.04 mm).

Neuronal survival and differentiation

Animals received daily ip injections of BrdU (50 mg/kg in saline; Sigma) during 7 days, they were sacrificed 28 days after the last injection and their brains processed. It has been previously shown that this is the time required for neural precursors to fully differentiate into neurons and to integrate to existent hippocampal circuits (Esposito et al. 2005). Animals were 15–16 weeks of age at the time of sacrifice.

We followed the protocol described above to estimate the total number of BrdU cells, but now cells both in the SGZ-DG and in the granular cell layer of the DG were counted. For BrdU + CB analysis, we followed the same protocol used for BrdU + DCX analysis, with the rabbit anti-Calbindin D28k (Swant, Bellinzona, Switzerland) antibody. In each image, we counted the number of BrdU and CB double-labeled cells and divided it by the volume of the granular cell layer as described for DCX.

Statistical analysis

We used StatView version 5.0.1 software (SAS Institute, Cary, NC, USA) or Statistica 7 (StatSoft Inc., Tulsa, OK, USA) for the statistical analyses. Uncorrelated variables (∣r∣ < 0.7) within a test were analyzed by manova. Group comparisons were done using unpaired Student's t-test or analysis of variance (anova) for normally distributed data. The Tukey's multiple comparisons test was used for post hoc comparisons. For all tests, statistical significance was assumed where P < 0.05.

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. References
  7. Acknowledgments
  8. Supporting Information

SPARC KO mice show elevated anxiety and antidepressant-like responses

We first evaluated anxiety-related behavior in WT, heterozygous (Het) and knockout (KO) mice. In the OF test, analysis of the non-correlated variables showed a significant effect of genotype (manova: F12,72 = 2.106, P = 0.027). Knockout mice spent less time in the center (F2,41 = 4.694, P = 0.015; Fig. 1a), walked less in the center (F2,41 = 4.219, P = 0.022; Fig. 1b) and showed reduced vertical exploration (F2,41 = 6.971, P = 0.002; Fig. 1c). But all animals showed similar locomotion (Total distance; F2,41 = 1.529, P = 0.229). Moreover, all genotypes showed habituation to the OF environment 24 h after the first exposure (repeated measures anova: time effect F1,41 = 27.06, P < 0.0001, time × genotype interaction F2,41 = 0.566, P = 0.572). In the LD test, we observed a significant effect of genotype (F2,42 = 4.878, P = 0.012) with Het and KO mice spending less time in the lit side than WT mice (Fig. 1d). No differences among treatments were observed in the EPM and all animals tend to avoid the open arms, spending around 13% of the time in this area (time spent in the open arms, WT, 33.56 ± 6.98 seconds; Het, 36.15 ± 6.12 seconds; KO, 48.54 ± 7.90 seconds; N = 14–15 per group).

image

Figure 1. SPARC KO mice show increased anxiety-related behavior. In the OF, SPARC KO animals spend less time in the center (a), walk less in the center (b) and show reduced vertical exploration (c). SPARC Het mice show an intermediate behavior. In the light/dark test, both SPARC Het and KO mice spend less time in the lit zone (d). Mean ± SEM. N = 14–15 per group. *P < 0.05, **P < 0.01 vs. WT; #P < 0.05 vs. Het, Tukey's Multiple Comparison Test.

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To evaluate the response to spatial novelty and to a novel object, we first performed the NOR test. Across sessions all animals habituated similarly (repeated measures anova: time effect F5,210 = 82.20, P < 0.0001, time × treatment interaction F10,210 = 1.045, P = 0.410; Fig. S1e). During the spatial novelty session, all animals showed a general increase in exploration, although this increase was significatively smaller in KO mice (Fig. S1e). Wildtype and KO mice showed a preference to explore the displaced object than the control object C (paired t-tests: WT, t14 = 16.63, P < 0.0001; Het, t14 = 3.594, P = 0.0029), while KO mice showed no preference (t14 = 1.787, P = 0.0957). Knockout mice showed reduced response to the displaced object, paying less visits to it (F2,42 = 5.23, P = 0.0094; Fig. 2a) and spending less time exploring it (F2,42 = 5.187, P = 0.0097; Fig. 2b). However, exploration of control objects during the displaced object session was similar for all genotypes. During the last session, all animals reacted similarly to a novel object, increasing the number of visits to it (F2,42 = 0.272, P = 0.7632). In line with this, all animals spent the same amount of time exploring a novel object in a familiar cage (F2,42 = 1.977, P = 0.1512; Fig. 2c). However, KO mice showed reduced locomotion (F2,42 = 3.976; P = 0.0262; Fig. 2d), in line with what is reported by the Jackson Laboratories for home cage activity.

image

Figure 2. SPARC KO mice show reduced response to spatial novelty but normal response to a novel object. In the NOR task, SPARC KO mice pay fewer visits to a displaced object (a) and spend less time exploring it (b) that Het and WT mice. In the NOE in cage test, all animals explore the novel object similarly (c), although SPARC KO mice show reduced locomotion (d). Mean ± SEM. N = 15 per group. *P < 0.05 vs. WT; #P < 0.05 vs. Het, Tukey's Multiple Comparison Test.

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Behavioral despair, a depression-related behavior, was evaluated in the TS and FS tests. We observed a significant effect of genotype on both tests (TS: F2,42 = 29.06, P < 00001; FS: F2,42 = 7.12, P = 0.0022), with KO mice spending less time immobile in both tests and Het mice showing an intermediate phenotype in the TS (Fig. 3a) and a KO phenotype in the FS (Fig. 3b).

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Figure 3. SPARC KO and Het mice show reduced depression-related behavior. SPARC KO and Het mice spend less time immobile in both the TS (a) and the FS tests (b). Mean ± SEM. N = 15 per group. *P < 0.05, **P < 0.01, ***P < 0.001 vs. WT; ###P < 0.001 vs. Het, Tukey's Multiple Comparison Test.

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SPARC KO mice show increased neuronal activity in the DG

It was previously shown that SPARC KO mice have increased synaptic strength in postnatal hippocampal neurons (Jones et al. 2011). We therefore evaluated whether this feature can result in increased neuronal activity in adulthood. We first analyzed the expression of c-Fos in the DG of control animals. We could not detect c-Fos expression in the DG, regardless of the genotype of the animals. We reasoned that c-Fos could be not sensitive enough to detect the basal differences in neuronal activity. Therefore, we exposed mice to inescapable footshock, sacrificed the animals 2 h later and analyzed c-Fos expression. We observed a significant effect of genotype (F2,10 = 4.892, P = 0.033), with KO mice having a higher density of c-Fos-positive cells in the DG (Fig. 4). Knockout mice have more c-Fos-positive cells in the DG (WT, 71.7 ± 29.6 cells; Het, 108.4 ± 41.3 cells; KO, 320.1 ± 78.4 cells; F2,12 = 5.38, P = 0.026).

image

Figure 4. SPARC KO mice show increased neuronal activity in the DG after a footshock protocol. Mice received 10 inescapable footshocks and were sacrificed 2 h later. c-Fos expression-positive cells were counted along the DG. Mean ± SEM. N = 4–5 per group. *P < 0.05, Tukey's Multiple Comparison Test.

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SPARC KO mice show reduced proliferation in the subgranular layer of the DG but normal neurogenesis

We analyzed cell proliferation in the subgranular zone of the DG (SGZ-DG; Fig. 5a) and found a significant effect of genotype (F2,16 = 14.64, P = 0.0002). Both Het and KO mice had less BrdU-positive cells in the SGZ-DG (Fig. 5b). However, we observed a similar percentage of BrdU-DCX double-positive cells in all genotypes (Fig. 5c), which could suggest that Het and KO mice have less young neurons. However, all three genotypes showed a similar density of DCX-positive cells in the SGZ-DG (WT, 0.138 ± 0.016; Het, 0.156 ± 0.015; KO, 0.131 ± 0.014 DCX-positive cells/mm3; F2,12 = 0.7780, P = 0.4812).

image

Figure 5. SPARC KO and Het mice have less proliferating cells in the SGL-DG. One day after seven daily BrdU injections (a), KO and Het DG have less BrdU-positive cells in the SGZ-DG (b), but the same percentage of these cells are DCX positive (c). Twenty-eight days after seven daily BrdU injections (d), the same number of BrdU-positive cells is observed in all experimental groups (e) and the density of BrdU − CB double-positive cells is also similar (f). Mean ± SEM. N = 5–7 per group. ***P < 0.001 vs. WT, Tukey's Multiple Comparison Test.

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These results could suggest a delay in the neuronal differentiation of young neurons, so that DCX-positive cells persist for a longer time in Het and KO mice, when compared with WT mice. We reasoned that if that were the case, we should observe less BrdU-CB-positive cells in the KO and Het DG, 28 days after BrdU treatment (Fig. 5d). However, we found no effect of genotype in this analysis (F2,14 = 0.085, P = 0.919; Fig. 5f).

Finally, the total number of BrdU-positive cells in the DG was similar in all genotypes, 28 days after the last BrdU injection (F2,14 = 2.376, P = 0.129; Fig. 5e). The number of BrdU-positive cells at 28 days post-labeling represents 20 and 21% of the values observed 24 h after labeling in Het and KO mice, but only 10% of those estimated for WT mice. These results could be due to a higher probability of survival of proliferating cells in KO and Het animals, aimed to compensate the low levels of proliferation. Alternatively, this could suggest a direct role of SPARC in limiting neuronal survival, resulting in a higher percentage of cell death in WT animals. These hypotheses need to be further studied to clarify the role of SPARC on the survival of neuronal precursors.

Adult hippocampal expression of SPARC rescues SPARC KO antidepressant-like behavior

To evaluate whether adult hippocampal SPARC modulates the behaviors that are altered in SPARC KO mice, we expressed the human SPARC protein in the adult DG using adenoviral vectors. At 2 months of age, SPARC KO mice were injected with either Adβgal or AdSPARC and tested 2 weeks later (Fig. 6a). We focused on behaviors that were altered in KO mice when compared with WT mice: OF, NOR, LD, TS and PS.

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Figure 6. Expression of SPARC in the DG rescues the diminished response to a spatial novelty and the antidepressant-like behavior in SPARC KO mice. Animals were stereotaxically injected with Adβgal or AdSPARC and tested 2 weeks after (a). AdSPARC-injected mice show increased number of visits (b) and exploration (c) of a displaced object. N = 8–9 per group. Animals treated with AdSPARC spend more time immobile in both the TS (d) and the FS (e) tests than Adβgal-injected mice. Mean ± SEM. N = 16–17 per group. Dashed lines represent the values obtained in WT mice. *P < 0.05, **P < 0.01 vs. Adβgal-injected mice, Student's t-test.

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Adult expression of SPARC in the DG did not rescue the anxiety-related phenotype of SPARC KO mice. Animals injected with AdSPARC spent a similar amount of time and a similar percentage of distance in the center of the OF than animals treated with Adβgal, and spent a similar amount of time in the lit zone of the LD (Fig. S3a–d). However, AdSPARC rescued the spatial novelty recognition in the NOR task, showing an increase in the number of visits (t15 = 2.896; Fig. 6b) and in the time exploring the displaced object (t15 = 2.135; Fig. 6c) when compared with Adβgal-injected KO mice. In addition, adult expression of SPARC in the DG rescued the antidepressant-like behavior both in the TS and FS tests (TS, t31 = 3.399, Fig. 6d; FS, t31 = 2.641, Fig. 6e). These results were confirmed in two independent cohorts, and they are presented together because no effect of cohort was observed.

Adult hippocampal expression of SPARC does not rescue the reduction in cell proliferation in the subgranular zone of the DG

To identify a possible mechanism through which adult SPARC expression in the DG reverts the antidepressant-like behavior observed in SPARC KO mice, we characterized the neurogenesis in SPARC KO mice injected with either Adβgal or AdSPARC. Adenoviral vectors were injected bilaterally in the DG, 14 days later all animals received seven daily injections of BrdU and mice were sacrificed 24 h after the last injection (proliferation studies; Fig. 7a) or 28 days later (survival studies; Fig. 7d).

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Figure 7. Expression of SPARC in the DG of SPARC KO mice does not alter neurogenesis. Proliferating cells were labeled between 14 and 20 days after adenoviral vector inoculation. One day after daily BrdU injections (a), SPARC KO animals injected with either adenoviral vector show the same number of BrdU-positive cells (b) and from these cells the same percentage is DCX positive (c). Twenty-eight days after seven daily BrdU injections (d), both experimental groups show a similar number of BrdU-positive cells (e) and a similar density of BrdU + CB double-positive cells (f). Mean ± SEM. N = 5 per group.

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All animals show a similar number of proliferating cells in the SGZ-DG (t8 = 1.126, P = 0.293; Fig. 7b) and the percentage of young neurons among these proliferating cells was equivalent in both experimental groups (t8 = 1.461, P = 0.227; Fig. 7c). Moreover, the density of DCX-positive cells was similar in both treatment groups (Adβgal, 0.050 ± 0.006; AdSPARC, 0.054 ± 0.011 DCX-positive cells/mm3; t8 = 0.339, P = 0.743).

Also a similar amount of proliferating cells differentiated into adult neurons, evidenced as a similar density of CB-positive cells in the granular layer of the DG (t8 = 0.684, P = 0.513; Fig. 7f). When we evaluated the survival of new cells in the DG, we observed no statistically significant effect of treatment on cell survival (t8 = 0.878, P = 0.406; Fig. 7e).

Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. References
  7. Acknowledgments
  8. Supporting Information

We show here for the first time a role of SPARC in anxiety- and depression-related behaviors, and we show that hippocampal SPARC is determining basal levels of behavioral despair in the mouse. Moreover, we show that the increased neuronal activity that was previously reported in the SPARC KO postnatal hippocampus (Jones et al. 2011) is still observable in the adult hippocampus. Finally, we show that SPARC KO and Het mice have less proliferating cells in the SGZ-DG, but that this does not result in reduced neurogenesis.

SPARC KO mice showed increased anxiety-related behavior in the OF and LD tests, but not in the EPM test. Others and we have previously shown that different behavioral tests can reveal diverse coping strategies in mice (Carola et al. 2006, Lucchina et al. 2010) and that, in particular, behavior in the OF does not correlate with EPM in the C57BL/6J strain (Rogers et al. 1999). This study adds to the previous literature and stresses the importance of using multiple behavioral tests to characterize the effects of a particular treatment. Alternatively, time of testing could be affecting our results, preventing us from detecting differences in behavior in the EPM. For practical reasons, we performed all our behavioral experiments during the light phase, i.e. the resting phase of rodents. Previous reports have shown that behavior in the EPM changes along the day (Andrade et al. 2003), and that testing during the dark phase can improve discrimination between experimental groups (Hossain et al. 2004, Huynh et al. 2011). Further studies will help us to determine whether behavior in the EPM is affected in SPARC KO mice in a phase-dependent manner.

SPARC KO mice showed reduced behavioral despair both in the FS and TS tests, a behavior sensitive to antidepressant drugs (Lucki et al. 2001). Whether other symptoms of depression such as anhedonia are also modulated by hippocampal SPARC needs to be further studied.

Considering the high comorbidity between major depression and generalized anxiety disorder (Moller 2002), and the evidence suggesting that these disorders share genetic risk factors (reviewed by Tanti & Belzung 2010), the behavioral phenotype of SPARC KO mice may be unexpected. However, other mouse models have shown these opposing effects on behavior. Heat shock factor 1 KO mice show reduced anxiety-like behavior and increased depression-related behavior (Uchida et al. 2011), while a phenotype of increased anxiety-related behavior and antidepressant-like behavior was found in serotonin-1A receptor (5HT1AR) KO mice (Heisler et al. 1998, Ramboz et al. 1998), GABA(B) KO mice (Mombereau et al. 2004) and corticotropin-releasing factor (CRF)-overexpressing mice (Van Gaalen et al. 2002). Because both 5HT1AR and GABA(B) are localized both pre- and post-synaptically, it has been suggested that different receptor populations could contribute to the expression of anxiety- or depression-related behaviors.

Characterization of the GABAergic or the serotoninergic systems in the SPARC KO hippocampus is still needed. Preliminary evidence from our group shows that mRNA levels of 5HT1AR in SPARC KO mice are half those of WT mice (WT 100 ± 7.37%; KO 46.96 ± 0.28%; t6 = 7.19; P = 0.0004), suggesting that serotonin transmission is indeed affected in SPARC KO mice. In addition, previous work has shown that SPARC levels can determine glutamatergic synaptic strength in the postnatal hippocampus (Jones et al. 2011). We were not able to detect basal neuronal activity in the adult hippocampus by c-Fos immunohistochemistry. This was probably due to the antibody that we used and the protocol that we followed, as other reports have shown basal c-Fos immunoreactivity in this region. Because of this, we could not determine whether the increase in synaptic strength that is observed early in life persists in adult SPARC KO mice. However, our c-Fos analysis is highly reliable for addressing differences in neuronal activation upon different stimuli such as a footshock. We show, indeed, that a mild footshock results in few cells expressing c-Fos in the DG of WT mice. Interestingly, the number of neurons expressing c-Fos upon the footshock protocol is highly increased in SPARC KO animals, supporting the hypothesis that the KO hippocampus has increased neuronal activity also in adulthood.

Our results also show that the antidepressant-like phenotype can be rescued by SPARC re-expression in the adult DG, while anxiety-related behavior in KO mice is not affected by this treatment. This result is also in agreement with a possible mechanism involving 5HT1AR, as it was shown that adult expression of 5HT1AR in the adult forebrain cannot rescue the anxiety-related phenotype observed in 5HT1AR KO mice (Gross et al. 2002). However, we cannot rule out that we failed to rescue this behavior because adult expression was restricted to the DG, and other structures could actually be regulating this behavior through SPARC (e.g. amygdala or cortex). Therefore, we need further evidence to corroborate this hypothesis and to prove that SPARC KO phenotype is dependent upon the downregulation of serotonin transmission in the postnatal hippocampus.

As an alternative cellular mechanism through which SPARC could modulate mood states, we focused on adult neurogenesis. The pattern of expression of SPARC in the normal adult brain – being it particularly high in neurogenic regions such as the SVZ and the SGZ of the DG (Vincent et al. 2008) – pointed us toward studying this plasticity phenomenon. Moreover, a high number of evidence shows that SPARC inhibits cell proliferation in different cell types, and that it also inhibits the proliferative effect of different growth factors (Brekken & Sage 2000). Our behavioral data further pushed us in that direction. The role of neurogenesis in modulating affective states is today a matter of debate. Initially, hippocampal neurogenesis has been linked to depression (Duman 2004). In particular, it was shown that antidepressant treatment increases neurogenesis in the adult hippocampus (Malberg et al. 2000), but also that neurogenesis is necessary for these drugs to have their effects (Santarelli et al. 2003). Therefore, it has been expected that low levels of neurogenesis would be correlated with depression. But studies in which neurogenesis was blocked actually linked neurogenesis to anxiety (reviewed by Petrik et al. 2012): reducing neurogenesis resulted in increased levels of anxiety-like behavior in mice, while depression-related behaviors were not altered (Revest et al. 2009).

We found that SPARC KO and Het mice have reduced numbers of proliferating cells in the SGZ-DG. However, this reduction did not result in diminished levels of neurogenesis, as the density of new young neurons (DCX-positive) and adult neurons (CB-positive) was similar in all genotypes. Interestingly, our results show that the new cells generated in KO and Het mice are more prone to survive for 1 month in the DG than those proliferating in the WT brain. This could be due to either a compensatory effect elicited by the low levels of proliferation observed in KO and Het mice, or to a direct role of SPARC in limiting the number of cells that persist in the DG. In summary, we show that SPARC has a role in promoting cell proliferation in the DG, but we cannot establish whether SPARC also determines the percentage of these cells that will survive or whether there are other mechanisms in the DG that guarantee a certain number of new neurons and that counteract the effect of reduced proliferation in SPARC KO and Het mice. These alternatives need to be further studied.

Adult neurogenesis in the SPARC KO mice was not altered upon SPARC re-expression in the adult hippocampus with adenoviral vectors, a treatment that reverses the antidepressant-like behavior but does not affect the anxiety-related behavior. SPARC KO mice injected with AdSPARC or Adβgal showed similar levels of proliferating cells in the DG, and a similar number of these cells survived after 28 days. Moreover, the proportion of young and adult neurons was similar in both groups. This suggests that the depression-related behavior, i.e. the behavioral despair, is not modulated by neurogenesis in the adult DG. Neurogenesis could underlie the anxiety-related phenotype, as both proliferation and behavior are not rescued upon adult SPARC re-expression. However further studies are needed to confirm this link.

It is also worth to notice that the injection of adenoviral vectors into the DG increased the number of BrdU-positive cells observed 28 days after labeling (Fig. 5e vs. Fig. 7e; t-test with Welch's correction, P = 0.044). We have previously shown that the injection of adenoviral vectors into the DG results in the activation of astrocytes and microglial cells that lasts at least 28 days (Depino et al. 2011). The results presented here suggest that such activation is not relevant in the short term (KO mice in Fig. 5b show similar numbers of BrdU-positive cells when compare with KO mice injected with Adbgal, Fig. 7b) but that this treatment results in more BrdU-positive cells later (Fig. 7e). As the number of new neurons (BrdU + CB-positive cells) is not altered by these treatments, we conclude that SPARC could have a role in the survival of glial cells. Such a role of SPARC on glial cells activation and function needs to be further studied.

In summary, our results prove a role of SPARC in hippocampal function, and suggest that the correct expression of this matricellular protein is essential for the correct establishment of adult behavior and neuronal function. Further studies would contribute to elucidate whether this protein is involved in human disease, e.g. in anxiety and depression, besides the long known role of this protein in cancer and the more recent link of this protein with obesity.

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. References
  7. Acknowledgments
  8. Supporting Information
  • Andrade, M.M., Tome, M.F., Santiago, E.S., Lucia-Santos, A. & de Andrade, T.G. (2003) Longitudinal study of daily variation of rats' behavior in the elevated plus-maze. Physiol Behav 78, 125133.
  • Beery, A.K. & Zucker, I. (2011) Sex bias in neuroscience and biomedical research. Neurosci Biobehav Rev 35, 565572.
  • Bradshaw, A.D. & Sage, E.H. (2001) SPARC, a matricellular protein that functions in cellular differentiation and tissue response to injury. J Clin Invest 107, 10491054.
  • Brekken, R.A. & Sage, E.H. (2000) SPARC, a matricellular protein: at the crossroads of cell-matrix. Matrix Biol 19, 569580.
  • Carola, V., Frazzetto, G. & Gross, C. (2006) Identifying interactions between genes and early environment in the mouse. Genes Brain Behav 5, 189199.
  • Deacon, R.M., Bannerman, D.M. & Rawlins, J.N. (2002) Anxiolytic effects of cytotoxic hippocampal lesions in rats. Behav Neurosci 116, 494497.
  • Depino, A.M., Tsetsenis, T. & Gross, C. (2008) GABA homeostasis contributes to the developmental programming of anxiety-related behavior. Brain Res 1210, 189199.
  • Depino, A.M., Lucchina, L. & Pitossi, F. (2011) Early and adult hippocampal TGF-beta1 overexpression have opposite effects on behavior. Brain Behav Immun 25, 15821591.
  • Duman, R.S. (2004) Depression: a case of neuronal life and death? Biol Psychiatry 56, 140145.
  • Esposito, M.S., Piatti, V.C., Laplagne, D.A., Morgenstern, N.A., Ferrari, C.C., Pitossi, F.J. & Schinder, A.F. (2005) Neuronal differentiation in the adult hippocampus recapitulates embryonic development. J Neurosci 25, 1007410086.
  • Ferrari, C.C., Depino, A.M., Prada, F., Muraro, N., Campbell, S., Podhajcer, O., Perry, V.H., Anthony, D.C. & Pitossi, F.J. (2004) Reversible demyelination, blood-brain barrier breakdown, and pronounced neutrophil recruitment induced by chronic IL-1 expression in the brain. Am J Pathol 165, 18271837.
  • van Gaalen, M.M., Stenzel-Poore, M.P., Holsboer, F. & Steckler, T. (2002) Effects of transgenic overproduction of CRH on anxiety-like behaviour. Eur J Neurosci 15, 20072015.
  • Gorman, J.M. (2006) Gender differences in depression and response to psychotropic medication. Gend Med 3, 93109.
  • Gross, C., Zhuang, X., Stark, K., Ramboz, S., Oosting, R., Kirby, L., Santarelli, L., Beck, S. & Hen, R. (2002) Serotonin1A receptor acts during development to establish normal anxiety-like behaviour in the adult. Nature 416, 396400.
  • Heisler, L.K., Chu, H.M., Brennan, T.J., Danao, J.A., Bajwa, P., Parsons, L.H. & Tecott, L.H. (1998) Elevated anxiety and antidepressant-like responses in serotonin 5-HT1A receptor mutant mice. Proc Natl Acad Sci U S A 95, 1504915054.
  • Heyser, C.J. (2004) Assessment of developmental milestones in rodents. Curr Protoc Neurosci Chapter 8, Unit 8.18.
  • Hossain, S.M., Wong, B.K. & Simpson, E.M. (2004) The dark phase improves genetic discrimination for some high throughput mouse behavioral phenotyping. Genes Brain Behav 3, 167177.
  • Huynh, T.N., Krigbaum, A.M., Hanna, J.J. & Conrad, C.D. (2011) Sex differences and phase of light cycle modify chronic stress effects on anxiety and depressive-like behavior. Behav Brain Res 222, 212222.
  • Jones, E.V., Bernardinelli, Y., Tse, Y.C., Chierzi, S., Wong, T.P. & Murai, K.K. (2011) Astrocytes control glutamate receptor levels at developing synapses through SPARC-beta-integrin interactions. J Neurosci 31, 41544165.
  • Kucukdereli, H., Allen, N.J., Lee, A.T., Feng, A., Ozlu, M.I., Conatser, L.M., Chakraborty, C., Workman, G., Weaver, M., Sage, E.H., Barres, B.A. & Eroglu, C. (2011) Control of excitatory CNS synaptogenesis by astrocyte-secreted proteins Hevin and SPARC. Proc Natl Acad Sci U S A 108, E440E449.
  • Liu, X., Ying, G., Wang, W., Dong, J., Wang, Y., Ni, Z. & Zhou, C. (2005) Entorhinal deafferentation induces upregulation of SPARC in the mouse hippocampus. Brain Res Mol Brain Res 141, 5865.
  • Lucchina, L., Carola, V., Pitossi, F. & Depino, A.M. (2010) Evaluating the interaction between early postnatal inflammation and maternal care in the programming of adult anxiety and depression-related behaviors. Behav Brain Res 213, 5665.
  • Lucki, I., Dalvi, A. & Mayorga, A.J. (2001) Sensitivity to the effects of pharmacologically selective antidepressants in different strains of mice. Psychopharmacology (Berl) 155, 315322.
  • Malberg, J.E., Eisch, A.J., Nestler, E.J. & Duman, R.S. (2000) Chronic antidepressant treatment increases neurogenesis in adult rat hippocampus. J Neurosci 20, 91049110.
  • McEwen, B.S. & Magarinos, A.M. (2001) Stress and hippocampal plasticity: implications for the pathophysiology of affective disorders. Hum Psychopharmacol 16, S7S19.
  • Mendis, D.B., Malaval, L. & Brown, I.R. (1995) SPARC, an extracellular matrix glycoprotein containing the follistatin module, is expressed by astrocytes in synaptic enriched regions of the adult brain. Brain Res 676, 6979.
  • Moller, H.J. (2002) Anxiety associated with comorbid depression. J Clin Psychiatry 63 (Suppl 14), 2226.
  • Mombereau, C., Kaupmann, K., Froestl, W., Sansig, G., van der Putten, H. & Cryan, J.F. (2004) Genetic and pharmacological evidence of a role for GABA(B) receptors in the modulation of anxiety- and antidepressant-like behavior. Neuropsychopharmacology 29, 10501062.
  • Ngwenya, L.B., Peters, A. & Rosene, D.L. (2005) Light and electron microscopic immunohistochemical detection of bromodeoxyuridine-labeled cells in the brain: different fixation and processing protocols. J Histochem Cytochem 53, 821832.
  • Norose, K., Clark, J.I., Syed, N.A., Basu, A., Heber-Katz, E., Sage, E.H. & Howe, C.C. (1998) SPARC deficiency leads to early-onset cataractogenesis. Invest Ophthalmol Vis Sci 39, 26742680.
  • Ozbas-Gerceker, F., Redeker, S., Boer, K., Ozguc, M., Saygi, S., Dalkara, T., Soylemezoglu, F., Akalan, N., Baayen, J.C., Gorter, J.A. & Aronica, E. (2006) Serial analysis of gene expression in the hippocampus of patients with mesial temporal lobe epilepsy. Neuroscience 138, 457474.
  • Paxinos, G. & Franklin, K. (2001). The Mouse Brain in Stereotaxic Coordinates. Academic Press, San Diego, USA.
  • Petrik, D., Lagace, D.C. & Eisch, A.J. (2012) The neurogenesis hypothesis of affective and anxiety disorders: are we mistaking the scaffolding for the building? Neuropharmacology 62, 2134.
  • Prada, F., Benedetti, L.G., Bravo, A.I., Alvarez, M.J., Carbone, C. & Podhajcer, O.L. (2007) SPARC endogenous level, rather than fibroblast-produced SPARC or stroma reorganization induced by SPARC, is responsible for melanoma cell growth. J Invest Dermatol 127, 26182628.
  • Ramboz, S., Oosting, R., Amara, D.A., Kung, H.F., Blier, P., Mendelsohn, M., Mann, J.J., Brunner, D. & Hen, R. (1998) Serotonin receptor 1A knockout: an animal model of anxiety-related disorder. Proc Natl Acad Sci U S A 95, 1447614481.
  • Rasband, W.S. (1997–2009). ImageJ. U.S. National Institutes of Health, Bethesda.
  • Revest, J.M., Dupret, D., Koehl, M., Funk-Reiter, C., Grosjean, N., Piazza, P.V. & Abrous, D.N. (2009) Adult hippocampal neurogenesis is involved in anxiety-related behaviors. Mol Psychiatry 14, 959967.
  • Rogers, D.C., Jones, D.N., Nelson, P.R., Jones, C.M., Quilter, C.A., Robinson, T.L. & Hagan, J.J. (1999) Use of SHIRPA and discriminant analysis to characterise marked differences in the behavioural phenotype of six inbred mouse strains. Behav Brain Res 105, 207217.
  • Sage, H., Vernon, R.B., Decker, J., Funk, S. & Iruela-Arispe, M.L. (1989) Distribution of the calcium-binding protein SPARC in tissues of embryonic and adult mice. J Histochem Cytochem 37, 819829.
  • Santarelli, L., Saxe, M., Gross, C., Surget, A., Battaglia, F., Dulawa, S., Weisstaub, N., Lee, J., Duman, R., Arancio, O., Belzung, C. & Hen, R. (2003) Requirement of hippocampal neurogenesis for the behavioral effects of antidepressants. Science 301, 805809.
  • Tanti, A. & Belzung, C. (2010) Open questions in current models of antidepressant action. Br J Pharmacol 159, 11871200.
  • Uchida, S., Hara, K., Kobayashi, A., Fujimoto, M., Otsuki, K., Yamagata, H., Hobara, T., Abe, N., Higuchi, F., Shibata, T., Hasegawa, S., Kida, S., Nakai, A. & Watanabe, Y. (2011) Impaired hippocampal spinogenesis and neurogenesis and altered affective behavior in mice lacking heat shock factor 1. Proc Natl Acad Sci U S A 108, 16811686.
  • Vincent, A.J., Lau, P.W. & Roskams, A.J. (2008) SPARC is expressed by macroglia and microglia in the developing and mature nervous system. Dev Dyn 237, 14491462.
  • West, M.J., Slomianka, L. & Gundersen, H.J. (1991) Unbiased stereological estimation of the total number of neurons in thesubdivisions of the rat hippocampus using the optical fractionator. Anat Rec 231, 482497.
  • Yamasaki, N., Maekawa, M., Kobayashi, K. et al. (2008) Alpha-CaMKII deficiency causes immature dentate gyrus, a novel candidate endophenotype of psychiatric disorders. Mol Brain 1, 6.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. References
  7. Acknowledgments
  8. Supporting Information

This work was supported by a CONICET Grant PIP2010-2012, a University of Buenos Aires Grant UBACyT GEF2010-2012 and a ANPCyT Grant PICT2010. A.M.D., O.P. and F.P. are members of the Research Career of the National Council of Scientific and Technological Research (CONICET), Argentina. M.C. is fellow of the ANPCyT. We would like to thank Maria Isabel Farias for tissue processing for histology.

Supporting Information

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. References
  7. Acknowledgments
  8. Supporting Information
FilenameFormatSizeDescription
gbb848-sup-0001-FigureS1.epsPS document449K Figure S1: Animals show similar levels of exploration in the NOR test. (a) SPARC genotype did not affect the number of visits to either object in the habituation session 1, and (b) all three objects were visited a similar number of times. (c) SPARC genotype did not affect the amount of time animals spent exploring each object in the habituation session 1, but (d) animals spent less time exploring object A. *P < 0.05 vs. object A. (e) Animals show similar levels of locomotion during the habituation sessions and in the novel object session, but KO mice show reduced locomotion during the spatial novelty session. *P < 0.05 vs. WT. Mean ± SEM. N = 15 per group.
gbb848-sup-0002-FigureS2.epsPS document10494K Figure S2: Representative images for c-Fos and neurogenesis analysis. (a) Representative image of the DG of a KO mouse, processed for immunohistochemistry anti-c-Fos, after a footshock protocol. c-Fos-positive cells in the granular cell layer (arrow head) were counted under × 400 magnification in a light-field microscope. Low magnification images were obtained to determine the area of the granular cell layer (delimited by a yellow line in the figure). ML, molecular layer. Scale bar = 0.25 mm. (b) Representative image obtained after double immunofluorescence anti-BrdU and DCX, for a Het mouse. To determine the percentage of BrdU-positive cells, we obtained × 400 confocal images spanning the ‘z’ axis and we counted the total of BrdU-positive cells and double-labeled cells. Examples of BrdU-positive, DCX-positive and double-labeled cells are shown. GCL, granular cell layer. Scale bar = 50 µm.
gbb848-sup-0003-FigureS3.epsPS document400K Figure S3: Expression of SPARC in the DG does not rescue the increase in anxiety-related behaviors observed in SPARC KO mice. Animals were stereotaxically injected with Adβgal or AdSPARC and tested 2 weeks after (a). AdSPARC- and Adβgal-injected mice spend a similar amount of time (b) and a similar percentage of distance (c) in the center of the open field. Both treatment groups spend the same time in the lit compartment (D) in the light/dark test. Mean ± SEM. N = 10 per group.

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