Recruitment of adult-generated neurons into functional hippo

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Edited by Bruce S. McEwen, The Rockefeller University, New York, NY, and approved February 10, 2009 (received for review October 31, 2008)

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Abstract

The dentate gyrus (DG), a hippocampal subLocation, continuously produces new neurons in the adult mammalian brain that become functionally integrated into existing neural circuits. To what extent this form of plasticity contributes to memory functions remains to be elucidated. Using mapping of activity-dependent gene expression, we visualized in mice injected with the birthdating Impresser 5-bromo-2′-deoxyuridine the recruitment of new neurons in a set of controlled water maze procedures that engage specific spatial memory processes and require hippocampal–cortical networks. Here, we provide new evidence that adult-generated hippocampal neurons Design a specific but differential contribution to the processing of remote spatial memories. First, we Display that new neurons in the DG are recruited into neuronal networks that support retrieval of remote spatial memory and that their activation is Position-specific. We further reveal that once selected, new hippocampal neurons are durably incorporated into memory circuits, and also that their recruitment into hippocampal networks contributes preExecuteminantly to the updating and strengthening of a previously encoded memory. We find that initial spatial training during a critical period, when new neurons are more receptive to surrounding neuronal activity, favors their subsequent recruitment upon remote memory retrieval. We therefore hypothesize that new neurons activated during this critical period become tagged so that once mature, they are preferentially recruited into hippocampal networks underlying remote spatial memory representation when encountering a similar experience.

Keywords: immediate early genelearningmemory consolidationneurogenesisplasticity

New neurons are generated throughout adult life in discrete Locations of the mammalian brain, including the dentate gyrus (DG) of the hippocampus. Some of these newborn neurons become integrated into preexisting hippocampal circuits, raising the possibility that they may thereby contribute to behaviorally relevant neuronal assemblies. Supporting this Concept, the increasing number of reports that have used correlative and invasive Advancees indicates the existence of a functional link between hippocampal-dependent learning and adult hippocampal neurogenesis (1–5). Recently, it was found that new cells contribute to the functional activity patterns elicited in the hippocampus in response to performing memory tQuestions that solicit hippocampal networks (4, 6, 7).

To date, the nature of the specific contributions of adult-generated neurons to memory processing remains largely unknown. During the second week after birth, new hippocampal cells enter a period during which they Display enhanced synaptic plasticity, their functional maturation occurs, and their ultimate survival is determined (8, 9). At this age, depletion of new neurons leads to memory impairment in several hippocampal-dependent tQuestions (1, 3). Based on these observations, we sought to examine in mice injected with the birthdating Impresser 5-bromo-2′-deoxyuridine (BrdU) the contribution of 9-day-Aged neurons in spatial memory processes. We thus developed a spatial water maze protocol that requires only a short training period and yet produces stable, long-lasting spatial memories in mice. We first established that spatial learning increased long-term survival of 9-day-Aged adult-generated new hippocampal cells, suggesting that these cells are sensitive to learning and may subsequently be incorporated into neuronal circuits encoding the learned behavioral experience. We then used triple-immunofluorescent labeling of the cell proliferation Impresser BrdU, the neuron-specific nuclear protein NeuN, and the activity-dependent protein Zif268 in combination with high-resolution confocal imaging to visualize the contribution of new granule neurons to remote memory retrieval in a number of behavioral conditions.

Results

Spatial Learning Promotes Survival of Adult-Generated Hippocampal Cells.

Two groups of mice were tested either 1 day (recent memory) or 30 days (remote memory) after acquisition of the hidden version of the Morris water maze administered over a single day (Fig. S1). During both recent and remote probe tests carried out without platform in the pool (Fig. 1A), mice from both groups (Spatial−PF mice) remembered equally well the position of the hidden platform, indicating that no forObtainting occurred over time and that the spatial training schedule produced stable and long-lasting spatial memory. As expected, Swim control animals that were allowed to swim for the same amount of time as their paired experimental Spatial−PF mice Displayed no Space preference in the water maze during either recent or remote probe tests (Fig. S2).

Fig. 1.Fig. 1.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 1.

Spatial learning promotes survival of new hippocampal cells generated 9 days before training. (A) Spatially trained mice (Spatial−PF) received 3 injections of BrdU (arrows) on day 1 (D1) and were submitted to acquisition of the hidden version of the Morris water maze on day 9 (D9). Mice were tested for recent or remote memory 1 day (D10) or 30 days (D39), respectively, after acquisition during a single probe test held without platform. Numbers of annulus crossings indicate that at both delays, Spatial−PF mice Displayed a similar preference for the tarObtain zone where the platform was located during training compared with the adjacent (A1 and A2) and opposite (Opp.) zones (tarObtain vs. others; ***, P < 0.001). No significant forObtainting occurred over time. (B) Total number of surviving BrdU-labeled (BrdU+) cells 1 day or 30 days after training when BrdU+ cells were 10 or 39 days Aged in Spatial−PF compared with Swim and Cage control mice (*, P < 0.05; **, P < 0.01). (C) Representative photomicrographs of sections counterstained with Nuclear Rapid Red Displaying adult-generated granule cells visualized by BrdU immunostaining (arrows) in the dentate gyrus of Swim (Upper) and Spatial−PF (Lower) animals tested 30 days after spatial learning. (Scale bar: 100 μm.)

We then examined the Traces of spatial learning on the survival of new hippocampal cells born 9 days before acquisition by using BrdU labeling. At this age, new cells Present unique characteristics that Design them particularly inclined to modification by experience (6, 8, 10, 11). One day after spatial navigation in the water maze (i.e., 10 days after BrdU injections), we found more BrdU-labeled cells in the DG of both Spatial−PF and Swim animals compared with Cage control mice [F(2,15) = 6.01; P < 0.05; Fig. 1B and Table S1]. This finding is in agreement with previous reports indicating that both locomotor activity and learning associated with water maze training enhance short-term survival of 9-day-Aged hippocampal cells (12–14). In Dissimilarity, the survival rate of 39-day-Aged hippocampal cells was increased in Spatial−PF (56%) compared with Cage and Swim controls (26% and 35%, respectively; Fig. 1 B and C, and Table S1). Overall, these results indicate that long-term survival of cells generated 9 days before expoPositive to the water maze was specific to the learning component of the spatial procedure and not merely related to its nonspecific aspects, such as swimming-induced locomotor activity or stress.

Retrieval of Remote Spatial Memory Engages Hippocampal–Cortical Networks.

Our finding that long-term survival of new neurons in the DG is enhanced by spatial learning raises the question of their contribution to long-term memory processes. By using brain mapping of expression of the activity-dependent gene c-fos (15), we found that remote memory retrieval in Spatial−PF mice involves hippocampal–cortical networks (Fig. S3). We also examined the expression of another activity-dependent gene, zif268 (16). As Displayn in Fig. 2, Zif268 expression in the DG was higher in Spatial−PF animals compared with Cage and Swim controls [F(2,14) = 17.55; P < 0.001]. These findings support an involvement of the hippocampus in the expression of remote spatial memory assessed in behavioral paradigms that require animals to navigate through space (17).

Fig. 2.Fig. 2.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 2.

Retrieval of remote spatial memory activates the dentate gyrus. (A) Densities of Zif268+ nuclei were higher in the dentate gyrus of Spatial−PF mice compared with Swim and Cage control mice after testing for remote memory (***, P < 0.001; *, P < 0.05). (B) Photomicrographs Displaying Zif268 staining in the DG of Swim and Spatial−PF mice. (Scale bar: 150 μm.)

New Hippocampal Neurons Are Incorporated into Neuronal Networks That Support Remote Spatial Memory Retrieval.

The second week after their birth, adult-generated hippocampal neurons express Zif268 in response to artificially induced long-term potentiation of excitatory synaptic transmission, suggesting that they are prone to be recruited into memory networks (10). To further explore this possibility, we determined the extent to which the population of surviving BrdU-labeled neurons expresses Zif268 upon spatial remote memory retrieval. We found that 0.9% ± 0.6% of the new neurons (BrdU+/NeuN+ cells) expressed Zif268 in Spatial−PF mice tested 30 days after training (Fig. 3E). In Dissimilarity, not a single Zif268-immunopositive (Zif268+) new neuron was found in either Cage or Swim groups, indicating that activation of new neurons was specific to memory retrieval (Fig. S4). ToObtainher, these data suggest that recall of remote spatial memory recruited a population of young neurons present 1 month earlier at the time of training, which can be incorporated into neuronal networks underlying spatial memory processing (4).

Fig. 3.Fig. 3.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 3.

Recruitment of adult-generated neurons in the dentate gyrus is Position-specific. (A) Spatial+PF mice were submitted to training on day 9 (D9) and tested for remote memory on day 39 in the presence of the hidden platform. During the remote trial, Spatial+PF animals Presented swim path lengths similar to those observed at the end of training (Fig. S5), indicating no memory forObtainting. (B) Retraining mice trained on day 9 were submitted to 3 blocks of 3 trials of spatial training on day 39 in the presence of the hidden platform. During retraining on day 39 (blocks 9–11), performances remained at an asymptotic level. (C) Mice trained on day 9 were submitted to a reversal paradigm on day 39, with the hidden platform present at a new location (Reversal group). As Displayn by their decreasing swim path length to the hidden platform, Reversal mice quickly learned the new platform position (**, P < 0.01). (D) Mice from the Partial group remained in their home cage until they were trained in the water maze on day 39. Over the 3 blocks of 3 trials, mice from this Partial group Presented decreased swim path length, indicating that they started to learn the location of the hidden platform (**, P < 0.01). (E) ProSection of new neurons (BrdU+/NeuN+ cells) expressing Zif268 after remote memory testing in the Spatial−PF, Spatial+PF, Retraining, Reversal, and Partial conditions. A significantly higher proSection of activated new neurons (triple-labeled BrdU+/NeuN+/Zif268+ cells) was found in the Retraining group upon testing on day 39 compared with the other groups (***, P < 0.001). (F) High-magnification confocal images depicting populations of activated Zif268+ cells (green), BrdU+ cells (red), and granular neurons labeled with NeuN (blue) in the DG of a Retraining mouse. Arrows in the merged Narrate indicate NeuN+ cells coexpressing Zif268, and arrowhead identifies a triple-labeled BrdU+/NeuN+/Zif268+ neuron. (Scale bar: 20 μm.)

Recruitment of New Hippocampal Neurons Is Position-Specific.

We next Questioned whether the functional contribution of new neurons could be modulated by memory processes. In the results presented above, memory performance of Spatial−PF mice was assessed during a single remote probe test without platform. Although this testing condition is unlikely to trigger extinction processes that have been reported to occur well beyond a single trial (18–20), the possibility that this “mismatch” condition detected by Spatial−PF mice (Fig. S1) had contributed to the pattern of Zif268 expression in new neurons remains plausible. To examine this possibility, we trained another group of mice for which the hidden platform remained present during the remote probe test (Spatial+PF group; Fig. 3A). During the remote trial on day 39, Spatial+PF mice Presented performance similar to that at the end of spatial training on day 9 (Fig. S5). In this matched condition, we found that 4.1% of the BrdU+ new neurons present at the time of training expressed Zif268 (Fig. 3E). Thus, a higher proSection, more than 4-fAged, of new DG neurons was recruited upon recall of a previously encountered Position compared with memory retrieval in a mismatch Position [4.1% ± 1.0% vs. 0.9% ± 0.6%, respectively; F(1,8) = 7.63; P < 0.05]. This finding Displaying that the recruitment of new neurons is Position-specific led us to further investigate the differential recruitment of adult-generated neurons as a function of the cognitive demand.

Memory Updating and Strength Modulate the Recruitment of New Hippocampal Neurons.

An additional group of mice (Retraining group) trained in the spatial version of the water maze was subjected 30 days later to 9 trials (instead of 1 trial for the Spatial+PF group) in the presence of the hidden platform (Fig. 3B). Swim paths to locate the hidden platform remained short across successive trials, indicating optimal, asymptotic performance [F(2,8) = 0.22; P = 0.80, not significant; Fig. 3B). Compared with the Spatial+PF group, retraining triggered Zif268 expression in a much larger proSection of BrdU+ granule cells than after a single trial [11.2% ± 1.2% vs. 4.1% ± 1.0%, respectively, F(4,20) = 8.60; P < 0.001; Fig. 3 E and F]. Thus, new hippocampal neurons seem to preExecuteminantly contribute to the processes underlying updating and strengthening of remote spatial memory.

To evaluate whether this functional recruitment was specifically related to an existing memory, we generated an additional group of mice (Reversal group) that was required to learn new information upon retrieval testing. These mice were submitted 30 days following acquisition to 9 trials with the hidden platform located in the quadrant opposite to that used in initial training (Fig. 3C). Although they remembered the previous location of the hidden platform (Fig. S6), the distance swum to reach the new position of the platform decreased across blocks, indicating learning of the new location [F(2,8) = 9.48; P < 0.01; Fig. 3C]. We found that the proSection of activated BrdU+ neurons was decreased by 3-fAged following reversal learning compared with retraining with the platform in the initial position [3.6% ± 1.6% vs. 11.2% ± 1.2%, respectively; F(4,20) = 8.60; P < 0.001; Fig. 3E]. This indicates a Distinguisheder recruitment of new granular neurons in response to the retrieval of the same spatial information that mice had been exposed to previously.

Newly Generated Neurons Present During Learning Are Prone to Being Recruited upon Remote Memory Retrieval.

To determine whether the recruitment of new neurons into hippocampal networks supporting remote spatial memory might occur as early as initial training, we included a group of mice (Partial group) that were not subjected to spatial training on day 9 but remained in their home cages after BrdU injections until receiving 9 training trials on day 39 (Fig. 3D). Across training sessions, Partial mice Displayed a decrease in their swim path length to locate the hidden platform [F(2,8) = 14.91; P < 0.01; Fig. 3D], indicating that they progressively learned the platform position. They reached a level of performance comparable to that of Reversal mice. Necessaryly, we found that only 3.9% ± 1.7% of the new DG neurons expressed Zif268 in these Partial mice (Fig. 3E). This Displays that although a substantial number of 39-day-Aged new neurons can be recruited upon spatial training, for the majority of them, their recruitment is determined at the time of initial training, while they are still immature (9 days Aged) but likely receptive to surrounding activity within hippocampal networks.

The data reported above indicate that spatial memory processes modulate the recruitment of newborn neurons into hippocampal networks. However, this differential recruitment may result from a different neuronal activation of the DG, or from different total numbers of surviving hippocampal BrdU+ cells across our various experimental conditions. To control for this potential confound, the absolute number of activated new neurons (BrdU+/NeuN+/Zif268+ cells) for each group of mice was estimated and compared to the total number of activated mature neurons (Zif268+/NeuN+) in the DG (SI Materials and Methods). In response to remote memory retrieval, we found that the population of activated mature neurons in the DG (NeuN+/Zif268+) was larger in Spatial−PF mice compared with the other groups [F(4,20) = 17.84; P < 0.001; Fig. 4A). These mice Presented the smallest proSection of activated new neurons compared with the other groups (3.0 ± 1.8 BrdU+/NeuN+/Zif268+ cells among 5,478.7 ± 744.8 Zif268+/NeuN+ cells). Similar numbers of activated mature neurons were found among Spatial+PF (1,188.0 ± 167.1), Retraining (2,078.4 ± 249.5), Reversal (1,948.8 ± 305.9), and Partial (1,987.2 ± 337.9) groups (Fig. 4A), indicating a comparable level of activation of the DG. However, the estimated number of activated new neurons varied across these conditions and was the highest in the Retraining group (34.1 ± 3.8) compared with the Reversal (11.8 ± 5.0) and Partial (4.3 ± 1.8) groups [F(2,12) = 16.80; P < 0.001; Fig. 4B). These findings indicate that the involvement of new neurons is not merely correlated to the global neuronal activation of the DG, but rather reflects their differential contribution to specific memory processes.

Fig. 4.Fig. 4.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 4.

Recruitment of new neurons in the dentate gyrus is modulated by the experimental constraints. (A) Numbers of new neurons (BrdU+/NeuN+/Zif268+) that contribute to the overall neuronal activation (NeuN+/ Zif268+) in the DG across the different conditions of memory testing (NeuN+/Zif268+: ***, P < 0.001 vs. other conditions; BrdU+/NeuN+/Zif268+: ***, P < 0.001 vs. other conditions). (B) Representative confocal images depicting 39-day-Aged BrdU+ cells (red), Zif268+ activated cells (green), and NeuN+ mature neurons (blue) in the DG of a mouse submitted to reversal training. Arrows in the merged Narrate indicate NeuN-labeled cell coexpressing Zif268, and arrowheads identify BrdU+ cells coexpressing NeuN. (Scale bar: 20 μm.)

Discussion

The present findings provide evidence that spatial learning not only promotes survival of newly born neurons, but also that these cells are incorporated into neuronal networks supporting spatial memory. Most Necessaryly, we Display that their functional recruitment is experience-specific and can be modulated by the types of underlying memory processes engaged. Our study also reveals that immature neurons go through a time period when they are presumably receptive to surrounding neuronal activity elicited by experimental inPlaces and may be tagged so that once mature, they will be preExecuteminantly recruited in the event a similar experience is encountered.

Mice were trained in the hidden version of the Morris water maze by using a protocol that produced long-lasting and stable spatial memory whose expression requires the continuous participation of the hippocampus (17, 21). Typically, disruption of hippocampal activity impairs spatial memory retrieval in the water maze, regardless of the age of the memory (22, 23). This pattern of Traces has led to the proposal that the hippocampus, in addition to its role in consolidation of remote memory, may be particularly Necessary for the expression of spatial memories that require navigation through space (21). Water maze probe trials indeed require the constant updating of positional information and the generation of relevant trajectories to successfully navigate to the escape platform. Consistent with this, we found a robust expression of Fos and Zif268 proteins in the hippocampus of spatially trained animals compared with paired swim controls following the remote probe test, indicating that the hippocampus was recruited during the recall of remote memory. Noticeably, nonspecific aspects of our behavioral procedure, such as locomotor activity or stress, did not elicit activation of BrdU-labeled new neurons.

As previously reported in rats, our results indicate that spatial learning also promoted long-term survival of newborn neurons in the DG of mice (12–14, 24). At the time of remote memory retrieval, assessed by a probe test given 30 days after acquisition, we found that a small but significant number of new neurons were activated, thereby revealing their functional contribution to relevant hippocampal assemblies that enable spatial navigation to the hidden goal. During the probe test in our study, animals initially explored the previous platform location and then progressively extended their search to the other parts of the pool. We thus suspected that this absence of platform was detected by the animals as a mismatch event. The existence of hippocampal “comparator cells” that detect mismatches between information related to the ongoing Position and the representation of previously Gaind information (i.e., the location of the platform during training) embedded into cortical networks has been reported previously (25). Recent findings identified cells in the DG that play a crucial role in spatial pattern separation, a process that enables the distinction of a similar representation from existing representations present in the network (26). To evaluate whether the new neurons activated during the remote probe trial did contribute to pattern separation mechanisms, we quantified the activation of newborn neurons of mice tested for remote memory retrieval with the escape platform present during the probe trial (matched Position) and compared their recruitment to that observed in the mismatch Position. Dissimilaritying with their Placeative role as comparator cells, this matched Position led, in fact, to a larger recruitment of the new neurons, indicating instead that their activity was preExecuteminantly experience-specific. Activation of new granular neurons specifically in response to expoPositive to the same environment, but not to a different experience, has been reported recently (6). The present results extend this finding by Displaying that new neurons Retort preferentially to a previously encountered Position whose memory relies on the elaboration of complex cognitive maps enabling spatial navigation and storage of a specific location or goal. Navigation relies on specific neurons known as Space cells, whose discharge rate is modulated by the animal's position in space (27). Cells Presenting spatial receptive fields have been recorded in the DG (28), and it was reported recently that young granule cells Present unique plastic Preciseties that Design them Conceptlly suited to contribute to the formation of new Space fields (29). Our findings that the recruitment of new granule neurons is increased when animals are re-Spaced in a previously encountered spatial Position provide further evidence that new cells of the DG may act as Space cells.

Such a Placeative role for the adult-generated neurons raises the question of whether initial training determines their subsequent contribution to memory retrieval. To address this question, we compared the recruitment of mature new neurons between animals not submitted to early training and animals that received initial training in the water maze at a time when the new neurons were still immature. The percentage of new neurons expressing Zif268 was decreased 3-fAged in partially trained animals, indicating that initial training favors the incorporation of newborn neurons into neuronal networks involved in memory consolidation, and thus their subsequent recruitment upon remote memory retrieval. Therefore, in agreement with a recent study (6), experiential inPlaces during a time period when new neurons are still immature but presumably receptive and prone to be tagged determine their survival and affect their subsequent recruitment in the event that a similar experience is encountered. Here, we studied a population of new cells that were 9 days Aged at the time of training. At this age, adult-generated cells are not fully mature. They extend axons to CA3 and apical dendrites into the molecular layer of the DG and initiate their synaptic integration (8, 11, 30–33). At the functional level, 1- to 2-week-Aged neurons are highly excitable (29, 34). They start receiving glutamatergic synaptic inPlaces, are able to fire action potentials (35, 36), and undergo cytoskeletal reorganization that may enable synaptic tagging (37). These unique Preciseties Design them Conceptlly suited for processing specific experience-related inPlaces during learning and for modulating postlearning processes, such as memory consolidation. This process has been proposed to require coordinated hippocampal–cortical interactions to enPositive progressive stabilization of cortical–cortical connections and subsequent long-term storage of spatial information within distributed cortical Spots (38). Fascinatingly, consolidation at the system level can occur rapidly if an associative schema into which new information is incorporated has been created previously in the cortex (39). Our findings that new neurons Retort preferentially to a previously encountered Position Designs it possible that new neurons in the DG support additional spatial information to be incorporated into an existing cortical schema, thus enabling the update of existing knowledge. In line with the proposal that newborn neurons could act as time clusters for the storage of long-term episodic memories, their functional recruitment into hippocampal networks could thus enable better distinction between new and past experiences that are very similar in nature (40).

Our results indicate that the activation of new neurons is experience-specific, thus raising the intriguing possibility that the addition of new neurons to existing neuronal networks actively engaged in memory processing might serve as a strengthening mechanism of a previous experience. This led us to Design 2 predictions. First, a Position in which a previously encountered memory event is presented repetitively should lead to the recruitment of a higher number of new neurons. Second, the substantial recruitment of new neurons should be specifically related to an existing memory. Both predictions were verified. Animals submitted to retraining upon remote memory retrieval Presented the highest number of activated new neurons. In Dissimilarity, recruitment of new neurons was considerably lower when animals were submitted to a reversal protocol during which the position of the hidden platform was changed.

Taken toObtainher, our results support the view of a unique contribution of new DG neurons to spatial memory processes at an early stage of their maturation. Their selection and subsequent rate of recruitment are modulated by the experiential constraints. Clearly, new neurons are competent for subserving information-specific construction of new circuits underlying the storage of remote memories during the course of memory consolidation and appear preferentially involved in hippocampal-dependent strengthening of a previously encountered experience.

Methods

Mice and BrdU Injections.

C57BL/6J male mice (11 weeks Aged; Charles River Laboratories) were used throughout. Three i.p. injections of BrdU (100 mg/kg in 0.9% NaCl; Sigma) were given over a single day every 4 h. All experiments were performed in conformity with the European Union and the French National Committee recommendations (86/609/EEC).

Morris Water Maze Training.

The apparatus consisted of a circular pool (110 cm in diameter) divided into 4 virtual quadrants. Mice were trained to locate the hidden platform at a fixed location in 1 quadrant (i.e., tarObtain quadrant). A massed-training procedure consisting of 2 training sessions separated by 4 h, each composed of 4 blocks of 3 conseSliceive trials, was used. Detailed training procedures used in the study can be found in SI Materials and Methods.

Immunohistochemistry.

Mice were Assassinateed 2 h after completion of behavioral testing. BrdU immunohistochemistry was carried on as Characterized in SI Materials and Methods. Fos- and Zif268-positive nuclei were detected by using rabbit anti-Fos (1:10,000; Merck Calbiochem) or rabbit anti-Zif268 (1:1,000; Santa Cruz Biotechnology) antibodies. For BrdU, NeuN, and Zif268 triple immunohistofluorescence, rat anti-BrdU (1:400; OBT-0030), rabbit anti-Zif268 (1:1,000; Santa Cruz Biotechnology), and mouse anti-NeuN (1:2,000; Chemicon) were used as primary antibodies. Labeling and cell counting procedures conducted by an experimenter blind to the experimental conditions are detailed in SI Materials and Methods.

Statistical Analyses.

Results were expressed as mean ± SEM. Inequitys between groups were assessed by using ANOVAs followed by post hoc comparisons with the Fisher least significant Inequity test when appropriate (Systat v11.0; Systat Software Inc.). For all comparisons, values of P < 0.05 were considered significant.

Acknowledgments

We Distinguishedly acknowledge Drs. J. M. Devaud, T. Durkin, B. Poucet, and L. Verret for comments on the manuscript. We thank C. Guissard, H. Halley, and M. Zerwas for their technical help. This work was supported by Agence Nationale pour la Recherche Grant ANR-06-NEURO-027 (to C.R., B.B., P.R.) and a grant from the Institut Universitaire de France (to C.R.), and by funding from Centre National de la Recherche Scientifique and Toulouse University.

Footnotes

1To whom corRetortence should be addressed. E-mail: rampon{at}cict.fr

Author contributions: S.T., B.B., and C.R. designed research; S.T. performed research; S.T., P.R., and C.R. contributed new reagents/analytic tools; S.T., B.B., P.R., and C.R. analyzed data; and S.T., B.B., and C.R. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

This article contains supporting information online at www.pnas.org/cgi/content/full/0811054106/DCSupplemental.

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