Compensatory network changes in the dentate gyrusrestore long-term potentiation following ablation ofneurogenesis in young-adult miceBenjamin H. Singera,b, Amy E. Gamellic, Cynthia L. Fullera, Stephanie J. Temmeb,c, Jack M. Parenta,b,d,1,and Geoffrey G. Murphyb,c,e,1

aDepartment of Neurology, bNeuroscience Graduate Program, cMolecular and Behavioral Neuroscience Institute, and eDepartment of Molecular andIntegrative Physiology, University of Michigan Medical School, Ann Arbor, MI 48109; and dVA Ann Arbor Healthcare System, Ann Arbor, MI 48105

Edited* by Pasko Rakic, Yale University, New Haven, CT, and approved February 15, 2011 (received for review October 13, 2010)

It is now well established that neurogenesis in the rodent sub-granular zone of the hippocampal dentate gyrus continues through-out adulthood. Neuroblasts born in the dentate subgranular zonemigrate into the granule cell layer, where they differentiate intoneurons known as dentate granule cells. Suppression of neuro-genesis by irradiation or genetic ablation has been shown to disruptsynaptic plasticity in the dentate gyrus and impair some forms ofhippocampus-dependent learning and memory. Using a recentlydeveloped transgenic mouse model for suppressing neurogenesis,we sought to determine the long-term impact of ablating neuro-genesis on synaptic plasticity in young-adult mice. Consistent withprevious reports, we found that ablation of neurogenesis resulted insignificant deficits in dentate gyrus long-term potentiation (LTP)when examined at a time proximal to the ablation. However, theobserved deficits in LTP were not permanent. LTP in the dentategyrus was restored within 6 wk and this recovery occurred inthe complete absence of neurogenesis. The recovery in LTP wasaccompanied by prominent changes within the dentate gyrus, in-cluding an increase in the survival rate of newborn cells that wereproliferating just before the ablation and a reduction in inhibitoryinput to the granule cells of the dentate gyrus. These findingssuggest that prolonged suppression of neurogenesis in young-adultmice results in wide-ranging compensatory changes in the structureand dynamics of the dentate gyrus that function to restore plasticity.

adult neurogenesis | thymidine kinase | metaplasticity | miniatureinhibitory postsynaptic currents

Forebrain neurogenesis persists into adulthood in the sub-granular zone (SGZ) of the hippocampal dentate gyrus in

rodents (1–4). Under normal conditions (i.e., in the absence ofovert pathology) neuroblasts that arise in the SGZmigrate a shortdistance into the dentate granule cell layer (GCL) and differen-tiate into dentate granule cells (DGCs), where they subsequentlyreach functional maturity (5, 6). The birth, integration, and sur-vival of DGCs are modulated by environmental enrichment (7),exercise (8), stress (9, 10), hippocampus-dependent learning (11),and direct manipulation of neuronal activity (12, 13). In addition,adult-born DGCs respond preferentially in hippocampus-dependentmemory tasks (14) and display increased synaptic plasticity rela-tive to mature DGCs (15, 16).The correlation of increased DGC neurogenesis with cogni-

tively demanding tasks has led to the hypothesis that adult-bornneurons are integral participants in hippocampus-dependentmemory processing and behavior. The role of adult-born DGCsin hippocampal function has been studied at the behavioral levelin rodents after suppressing neurogenesis with antimitotic agents(17, 18), radiation (19), or genetic targeting (19–22). Thesestudies indicate that adult-born DGCs are necessary for somehippocampus-dependent tasks, although results have been in-consistent and vary by rodent species and strain, behavioralparadigm, and ablation method (reviewed in ref. 23). Deficits infear conditioning (19) and spatial memory (22) correlate in these

models with impaired perforant path (PP)-dentate gyrus long-term potentiation (LTP). Conversely, conditions that stimulateadult neurogenesis in mice also increase LTP (8, 24). ImmatureDGCs generated in the adult exhibit a period of enhanced syn-aptic plasticity (15, 16), suggesting that this small cell cohortcontributes significantly to dentate gyrus function.Two forms of DGC LTP can be induced by PP stimulation (25).

When GABAA receptors are blocked, PP stimulation greatly po-tentiates DGC postsynaptic responses. With GABAA receptor-mediated neurotransmission intact, PP stimulation induces alower-amplitude, NR2B-dependent LTP. This LTP is blocked bymanipulations that acutely suppress adult neurogenesis and isthought to be mediated by young granule neurons that are underless resting inhibitory tone than mature DGCs (19, 25).However, the long-term impact of suppressing neurogenesis,

as it relates to synaptic plasticity, has yet to be investigated. Toaddress this question, we have examined LTP in the nestin-thymidine kinase (nestin-tk)mousemodel, inwhich herpes simplexvirus (HSV) thymidine kinase expression is regulated by the nestinenhancer, enabling the specific and temporally controlled ablationof neurogenesis after the intracerebroventricular administration ofganciclovir (GCV). In this model, treatment with GCV for 28 dresults in nearly complete suppression of neurogenesis in young-adult mice (26). Consistent with previous results, we found thatLTP was significantly disrupted when assessed immediately fol-lowing ablation. This deficit in LTP was in marked contrast to thenormal levels of LTP observed in GCV-treated nestin-tk mice42 d following the ablation. This restoration of LTP occurred inthe complete absence of neurogenesis, which failed to recover.Interestingly, recovery of LTP was associated with an increase insurvival of DGCs that were born immediately before the ablation.Moreover, 42 d following the ablation there was a marked re-duction in expression of the inhibitory synaptic marker vesicularGABA transporter (VGAT) within the dentate gyrus. The de-crease in VGAT staining was concomitant with a reduction inoverall inhibitory tone in the dentate gyrus, as assessed by mea-suring miniature inhibitory postsynaptic currents (mIPSCs) inDGCs. These findings suggest that the ablation of neurogenesis inyoung-adult mice results in wide-ranging compensatory changes tothe structure and dynamics of the dentate gyrus.

Author contributions: B.H.S., A.E.G., C.L.F., S.J.T., J.M.P., and G.G.M. designed research;B.H.S., A.E.G., C.L.F., S.J.T., and G.G.M. performed research; B.H.S., A.E.G., S.J.T., J.M.P.,and G.G.M. analyzed data; and B.H.S., J.P., and G.M. wrote the paper.

The authors declare no conflict of interest.

*This Direct Submission article had a prearranged editor.1To whom correspondence may be addressed. E-mail: murphyg@umich.edu or parent@umich.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1015425108/-/DCSupplemental.

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ResultsSuppressing Neurogenesis in Nestin-tk Mice Disrupts PP-DentateGyrus LTP. We first examined how suppression of neurogenesisfor 4 wk in young-adult nestin-tk mice influenced PP-dentategyrus LTP. Field potential excitatory postsynaptic potentials(fEPSPs) in response to medial PP stimulation were recordedadjacent to the GCL in hippocampal slices from 2- to 4-mo-oldnestin-tk mice (tk+/GCV; 17 slices/nine mice) or wild-type lit-termate controls (WT/GCV; 15 slices/eight mice) after 28 d ofGCV treatment. As shown in Fig. 1A, a tetanus consisting offour bursts (500 ms) of 100-Hz stimulation with a 30-s interburstinterval was used to induce LTP after 10 min of baseline re-cording. A two-way repeated-measures ANOVA revealed thatthe tetanus significantly increased the normalized fEPSP slope[F(69, 1035) = 26.4; P < 0.001]. However, slices prepared fromnestin-tk mice treated with 28 d of GCV exhibited significantlyless LTP than those from wild-type mice [F(1,15) = 5.6; P = 0.014for main effect of genotype]. In fact, comparison of the averagefEPSP slope for the last 10 min (minutes 60–70) of the experi-ment with the average fEPSP slope of the 10 min before thetetanus yielded no significant difference for the GCV-treatednestin-tk group (P = 0.2). This LTP deficit was not a result ofinsertion of the tk transgene or a disruption in basal synaptictransmission at medial PP-DGC synapses (Fig. S1). These resultsdemonstrate that 28 d of GCV treatment is sufficient to disruptLTP in 2- to 4-mo-old nestin-tk mice.Newborn DGCs are minimally affected by feed-forward in-

hibition. Thus, immature DGCs are thought to selectively de-polarize after medial PP stimulation and preferentially expressLTP. In contrast, when inhibition is blocked, mature DGCs aresignificantly depolarized by medial PP stimulation and ablationof newborn granule cells is thought to have minimal impact (19,25). To examine the effects of complete disinhibition, LTP wasinduced using the same stimulation paradigm (four 500-msbursts at 100-Hz stimulation; 30-s interburst interval) in thepresence of artificial cerebrospinal fluid containing the GABAAreceptor antagonist picrotoxin (50 μM). Results are presented inFig. 1B. As in the previous experiment, mice were chronicallytreated with GCV for 28 d and then acute hippocampal sliceswere prepared from nestin-tk (tk+/GCV; six slices/five mice) orwild-type (WT/GCV; six slices/five mice) animals. Tetanic stimu-lation of the medial PP in the presence of picrotoxin substantiallyincreased the normalized fEPSP slope for both groups [F(69, 483) =138.3; P < 0.001 two-way repeated-measures ANOVA], andno significant difference in LTP was observed between groups[F(1,7) = 1.4; P = 0.27 for main effect of genotype]. These resultssuggest that the LTP deficit seen in nestin-tk mice treated withGCV is specific to the loss of newborn DGCs. Recent work sug-gests that 28 d of GCV treatment in nestin-tk mice results in a lossof newborn DGCs that persists for at least 42 d (6 wk) after thediscontinuation of the GCV infusion (26). To determine the im-pact of persistently suppressed neurogenesis on LTP, experimentssimilar to those described above (Fig. 1A) were performed onslices from nestin-tk and wild-type mice treated for 28 d with GCVand then allowed to remain drug free for 42 d. As shown in Fig. 1C,robust LTP was observed in wild-type (WT/GCV; 18 slices/nine mice) and nestin-tk (tk+/GCV; 13 slices/seven mice) groups[F(69, 966) = 138.3; P< 0.001 two-way repeated-measures ANOVA].In contrast to the LTP that was observed immediately following28 d of GCV treatment, there was no significant difference in themagnitude of LTP between groups after 42 d of recovery [F(1,14) =0.175; P= 0.68]. Importantly, the recovery of LTP occurred in theabsence of ongoing neurogenesis as indicated by the loss of im-munostaining for doublecortin after 28 d of treatment or 28 d plus42 d of recovery (Fig. 2). The 28-d GCV infusion significantlyreduced the number of doublecortin-immunoreactive neurons inthe dentate gyrus of nestin-tk mice (Fig. 2 A and B). Consistent

with previous findings (26), very few doublecortin-positive neu-roblasts were observed 42 d after the termination of GCV treat-ment (Fig. 2 C and D). Similar results were obtained when slicesused in the LTP experiments were fixed, resectioned, and immu-

A

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Fig. 1. Suppression of neurogenesis disrupts LTP in the dentate gyrus ofyoung adult mice. (A) After 10 min of baseline recording, a tetanus of fourbouts of 100-Hz stimulation (500 ms) separated by 30 s was delivered(arrows). Following the tetanus, the average fEPSP slope (as a percentage ofbaseline) was significantly enhanced in slices from wild-type mice treated for28 d with GCV (wt/GCV). In contrast, slices from nestin-tk mice treated withGCV (tk+/GCV) exhibited no significant increase in fEPSP slope after thetetanus. (B) LTP in response to a high-frequency tetanus was examined in thepresence of artificial cerebrospinal fluid that contained 50 μM picrotoxin.Slices from wild-type mice (wt/GCV) and nestin-tk mice (tk+/GCV) treatedwith 28 d of GCV both exhibited robust and comparable LTP. All data aremean ± SEM. (C) LTP is restored 42 d after termination of GCV treatment.Wild-type mice and nestin-tk mice were treated with 28 d of GCV (wt/GCVand tk+/GCV, respectively), and hippocampal slices prepared 42 d later. Slicesfrom both groups exhibited robust LTP following the high-frequency teta-nus. All data are mean ± SEM. Insets present representative extracellularfEPSP recordings made before (average of first 10 min) and after (average oflast 10 min) the tetanus. (Scale bars, 0.25 mV/5 ms.)

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nostained (Fig. 2 E–H). It seems unlikely that the deficits in LTPthat we observed immediately following GCV treatment werea result of nonspecific effects, such as inflammation, as immu-nostaining with ED1 (a marker of microglial activation) wasminimal and indistinguishable between groups (Fig. S2). Takencollectively, these findings suggest that LTP of medial PP-DGCsynapses is disrupted by the loss of newborn granule cells, but thatLTP recovers despite the prolonged absence of neurogenesis.

Enhanced Survival of Cells Born Immediately Before SuppressingNeurogenesis. The abolition and restoration of LTP over 10 wkduring and after GCV treatment could also be explained by al-tered survival, maturation, or integration of a cohort of DGCsborn before suppression of neurogenesis. To explore the possi-bility that the survival of newborn DGCs is changed by the ab-sence of continued neurogenesis, we labeled dividing cells byadministering BrdU 5 to 10 d before the initiation of GCV in-fusion (Fig. 3A) such that the cells would be postmitotic at thetime of GCV infusion and therefore not be susceptible to GCV-induced death. BrdU-labeled cells were then examined after 14or 28 d of GCV administration. Basal rates of neurogenesis didnot differ between nestin-tk and wild-type mice (26). After 14 dof GCV infusion, there was no significant difference in BrdU-labeled cell numbers in wild-type and nestin-tk mice (Fig. 3C)(P > 0.3, n = 4 per group). After 28 d of GCV, BrdU+ cells inboth groups (n = 4 mice per group) decreased compared withthe 14-d time point, as expected. The number of surviving BrdU-labeled cells in nestin-tk mice, however, was double the numberseen in controls (Fig. 3 B and C) (P < 0.02). This finding suggeststhat suppression of DGC neurogenesis leads to feedback signalsthat stimulate the survival of previously generated DGCs that arestill developing (i.e., < 5 wk old) at the time of ablation.

Immunoreactivity for VGAT Is Reduced in the Dentate Gyrus FollowingChronic Suppression of Neurogenesis. The restoration of basal LTP(i.e., LTP evoked without GABAA receptor blockade) after 42 d ofrecovery may have also resulted from changes in the mature den-tate gyrus network that normally expresses LTP only in the pres-ence of picrotoxin. Such network changes involving mature DGCscould arise either from disinhibition by decreased GABAergic in-put or increased excitation via excessive glutamatergic input. Toassess these possibilities, we examined immunoreactivity for theGABAergic presynaptic marker VGAT and the glutamatergicpresynaptic marker vesicular glutamate transporter-1 (VGLUT-1)(27, 28) in hippocampal sections prepared fromWT and tk+ mice.In all groups, VGAT immunostaining was most intense (brightest)in the outer GCL and outermolecular layer (OML) of the dentate,with less labeling in the inner and middle molecular layers (IMLand MML, respectively) (Fig. 4A, Top and Middle). There wasa uniform lack of labeling in the white matter tracts of the fornix(Fig. 4A, Bottom). Although VGAT immunoreactivity in the GCLof nestin-tk mice was slightly decreased relative to controls after28 d of GCV treatment, it was profoundly decreased after 70 d(28 d of GCV + 42 d of recovery) (Fig. 4A, Top and Middle).Quantitative immunofluorescence microscopy using the fornixfor normalization showed no significant difference in VGAT la-beling between nesin-tk (six mice) and wild-type (six mice) groupsafter 28 d of GCV (P > 0.1 for all layers). After 42 d of recovery,however, VGAT immunoreactivity in the GCL was 65% less innestin-tk animals (three mice) vs. controls (six mice) (Fig. 4B) (P <0.001). VGAT labeling was also decreased, but less robustly, in theIML/MML and OML (Fig. 4B) (P < 0.05). Note that VGAT la-beling is heterogeneous within dentate gyrus such that the nor-malized optical density is not directly comparable between layers(e.g., the outer GCL is heavily labeled, but the overall opticaldensity of the GCL is lower than the OML because of the absenceof reactivity in the inner GCL).

Fig. 2. GCV treatment persistently disrupts neurogenesis. Immunoreactivityfor doublecortin was examined in both frozen-brain sections (A–D) and indentate gyrus slices used for electrophysiological recordings (E–H). Intra-cerebroventricular infusion of GCV for 28 d dramatically reduces immatureneurons in nestin-tk (tk+) mice, shown here by absence of doublecortin immu-noreactivity (B and F) vs. controls receiving GCV (A and E). No recovery of dou-blecortin expression was seen after 28 d of GCV administration plus a 42-drecovery (D and H) vs. similarly treated controls (C and G). (Scale bar, 100 μm.)

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Fig. 3. Ablation of newborn neurons increases survival of cells born beforestarting the GCV infusion. (A) A cohort of proliferating cells labeled with BrdU5 to 10 d before GCV infusion was examined after 14 or 28 d of GCV infusion.(B) After 28-d GCV infusion, more BrdU-labeled cells were evident in the SGZand GCL of tk+ mice than WT controls (arrowheads). (C) Quantification ofBrdU labeling revealed no difference after 14 d of GCV treatment, but a sig-nificantly greater number of surviving BrdU+ cells in tk+ mice after a 28-d GCVinfusion. Data presented as mean ± SEM. *P < 0.02. (Scale bar, 100 μm.)

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Although normalization of the optical density to the fornixcontrols for nonspecific overlabeling, it is possible that the ob-served differences in VGAT labeling were a result of nonspe-cific globally decreased immunoreactivity because, for example, oftissue-processing artifacts. To control for these artifacts, we ex-amined VGAT labeling in the cingulate cortex, which would notbe expected to be affected by the experimental protocol. Whendentate gyrus VGAT optical density was normalized using non-specific changes in cortical VGAT immunoreactivity among ani-mals, VGAT labeling in nestin-tk mice was specifically decreasedin the GCL (Fig. S3) [F(1,7) = 184.7, P < 0.001] and IML/MML[F(1,7) = 73.6, P < 0.001]. Thus, the difference in VGAT opticaldensity observed in the dentate gyrus was robust regardless ofwhether the measurement was controlled for processing artifactsresulting in over- or underimmunoreactivity.Although decreased inhibitory innervation of mature DGCs

would be most concordant with changes in basal (picrotoxin-independent) LTP, an increased excitatory drive would also be

expected to enhance LTP in mature DGCs. We therefore ex-amined immunolabeling for VGLUT-1 as a marker for gluta-matergic presynaptic terminals in the dentate molecular layer(Fig. S4). VGLUT-1 expression did not differ between nestin-tkand wild-type mice at either the 28-d or 6-wk recovery timepoints (Fig. S4 A and B) (28-d recovery P > 0.80; 6-wk recovery,P > 0.90). The preserved VGLUT-1 immunoreactivity supportsthe specificity of reduced VGAT expression in nestin-tk miceafter 42 d of recovery from GCV treatment.To investigate the functional consequence of the observed

decrease in VGAT staining, we recorded mIPSCs in DGCs inhippocampal slices prepared from wild-type and tk+ mice trea-ted with GCV for 28 d, followed by 42 d of recovery (Fig. 5A).Compared with slices prepared from wild-type mice treated withGCV, slices prepared from tk+ GCV-treated mice exhibited asignificant reduction in mIPSC frequency (indicated by a right-ward shift in the log interevent interval) (Fig. 5 B and C). Con-versely, we did not find any significant differences in the averagemIPSC amplitude (Fig. 5D), area (Fig. 5E), or decay (Fig. 5F)when we compared wild-type GCV-treated mice with tk+-treatedmice. Our observation of a reduction in mIPSC frequency in theabsence of altered mIPSC amplitude would suggest an alterationin presynaptic release (29). Taken together with our results fromthe VGAT immunostaining experiments, these results stronglysuggest that there is a reduction in inhibitory tone in the GCLfollowing prolonged loss of neurogenesis in young-adult mice.

DiscussionOur results indicate that administration of GCV to young-adultnestin-tk mice for 28 d leads to suppression of neurogenesis andthe concomitant loss of picrotoxin-independent PP-dentate gyrusLTP in young-adult mice. This result is consistent with previousreports describing cranial irradiation, genetic ablation (19, 25),or knockdown of growth factor signaling to reduce integration ofnewborn DGCs (30). We have also now investigated the long-term consequences of suppressing neurogenesis in young-adultmice. We found that 42 d after the cessation of GCV adminis-tration (10 wk from starting GCV treatment), LTP recovered tocontrol levels. Importantly, this LTP recovery occurred in thecomplete absence of neurogenesis. Although we previouslyreported that sparse doublecortin-labeled neurons may appear inthe dentate 42 d after the cessation of GCV in this model (26),we did not find any doublecortin-immunoreactive cells in thehippocampal slices used for in vitro recordings in this study.Even if the dentate begins to generate new neurons during therecovery phase after the GCV infusion ended, most would likelybe less than 4 wk old by the end of our experimental recoveryperiod, and thus would have not yet entered the critical period ofenhanced synaptic plasticity (15, 26). Thus, we sought alternativeexplanations for the apparent dissociation between picrotoxin-independent LTP and neurogenesis.When neurogenesis is suppressed by cranial irradiation, neither

neurogenesis nor LTP recover 3 mo later (19). However, irradia-tion alters the neurogenic microenvironment and induces in-flammation in addition to directly affecting dividing cells (31, 32),and may also have off-target effects on the hippocampus. Genet-ically specified ablation of neurogenesis should have fewer off-target effects than radiation, and we found no evidence of micro-glial activation in our model. Thus, it is unlikely that our resultsrepresent a nonspecific suppression of LTP because of transientinflammation.We hypothesize that LTP recovers 42 d after the cessation of

GCV via compensatory changes in dentate gyrus networks in-duced by suppressing neurogenesis. These changes may reflectan altered fate of young DGCs continuing to develop and in-tegrate as successive generations of neurons are ablated. Recentresults suggest that the developmental program of adult-bornDGCs is plastic and may be altered and even reversed by alter-

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Fig. 4. Ablation of neurogenesis in young adult mice decreases inhibitoryinnervation in the dentate gyrus. (A) Staining for VGAT in the dentate gyrus(Top row at 20× and Middle row at 63×) and fornix (Bottom row). The lefttwo columns are immediately after 28-d GCV treatment, and the right twocolumns are 70 d after initiating GCV infusion (28 d + 42 d recovery). Im-munoreactivity for VGAT (lighter staining) is maximal in the outermostportion of the GCL and in the OML, and is significantly decreased in tk+ miceonly after treatment with GCV for 28 d followed by a 42-d recovery (70 d;right column of Top and Middle). The white matter tracts of the fornix(Bottom) show minimal VGAT immunoreactivity. [Scale bars: 100 μm (Topand Bottom), 25 μm (Middle.)] (B) Quantification of the optical densities(normalized to the white matter of the fornix). Although a trend towardlower normalized optical density was evident after 28 d of GCV treatment,VGAT immunoreactivity was significantly decreased after 42 d of recoveryfrom GCV treatment. Data presented as mean ± SEM. *P < 0.05, **P < 0.001.

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ations in neuromodulatory tone (33). Furthermore, survival ofimmature DGCs is dependent on both GABAergic and gluta-matergic input and may be controlled by competition amongDGCs for connections within the dentate network (34, 35). Ourresults indicate that survival of cells born 5 to 10 d before thebeginning of the GCV infusion was twofold greater when suc-cessive generations of neurons were ablated. These cells werenearly 3 mo old at the time LTP recovered, and under normalcircumstances would have passed the window of enhancedplasticity. Given that ablation of neurogenesis so profoundlyalters cell survival in this cohort, however, it is possible that theirmaturation is altered as well, and further studies will be neces-sary to fully elucidate the physiology of these cells. Nevertheless,their altered survival indicates that the dynamics of newbornDGC integration is influenced by the subsequent ablation ofDGC progenitors.Alternatively, the reemergence of LTP 42 d after the cessation

of GCV infusion may not depend on newborn neurons. Instead,“homeostatic-like” changes within mature dentate gyrus networksmay restore the balance of inhibition and excitation in the absence

of immature DCGs. Indeed, the level of neurogenesis correlates in-versely with rates of synaptic turnover under certain conditions (36).Given that picrotoxin-independent PP-DGC LTP is thought to

depend on immature DGCs because they receive less inhibitoryinput than their mature counterparts (19, 25), we hypothesizedthat a reduction in the inhibitory innervation of the GCL as awhole may accompany the recovery of LTP. Indeed, we foundthat VGAT immunoreactivity, a marker of GABAergic synapses,decreased in association with recovery of LTP. In contrast, nosignificant difference in VGAT density between nestin-tk andwild-type mice was present after 28 d of GCV administration,when LTP was absent.This concordance suggests that LTP in the dentate gyrus may

recover as a function of decreased inhibition over time, ashomeostatic-like changes occur within the network. Supportingthe specificity of changes in inhibition, we did not find concurrentchanges in immunoreactivity for VGLUT-1, a marker of gluta-matergic synapses. Furthermore, we observed that the frequencyof DGC mIPSCs was significantly decreased in tk+ GCV-treatedmice at 70 d after the beginning of the GCV infusion, further

Fig. 5. Miniature IPSC frequency is reduced in tk+ mice 42 d after treatment with GCV. (A) Representative whole-cell voltage clamp recordings from wild-type(A1) and tk+ (A2) mice 42 d after termination of GCV treatment. (B) Plot of cumulative fraction of the log10 interevent intervals revealed an increase in timebetween events as evidenced by a significant rightward shift in the tk+ curve (Kolmogorov-Smirnov, P < 0.0001). (C) Histograms of the number of eventsobserved in recordings from slices prepared from wild-type mice (C1) and tk+ mice (C2). Log10 (interval) bins were set to 0.1 ms. Average mIPSC amplitude (D),area (E), and decay (F) were not significantly different 42 d after treatment with GCV (P > 0.5, Student t test). Data in D to F presented as mean ± SEM. (Scalebars, 10 pA/200 ms.)

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suggesting a functional relationship between the loss of VGATstaining and a decrease in inhibitory tone.Our findings demonstrate that widespread dynamic changes

occur in the dentate gyrus following the chronic suppression ofneurogenesis in young-adult mice, and that these changes cor-relate with alterations in network physiology. A number of re-ports suggest that inhibiting neurogenesis has little or no impact onlearning and memory (for review, see ref. 23). It is notable that inmany cases the behavioral assessments were made several weeksafter ablation of neurogenesis. Although it is unknown if the ab-lation methods in these experiments resulted in permanent dis-ruption of neurogenesis (as in our experiments), our data raise theintriguing possibility that the absence of behavioral deficits mayreflect a compensatory rearrangement within the dentate gyrus.Similarly, recent work suggests that adult DGC neurogenesis isrequired for the transition of remote contextual fear memoriesfrom a hippocampus-dependent to a hippocampus-independentform (37). Interestingly, suppression of adult neurogenesis in theseexperiments did not completely inhibit fear memories from beingconverted to a hippocampus-independent form. The authorsspeculate that this may result from an incomplete suppression ofneurogenesis or that perhaps adult neurogenesis plays more of amodulatory role in this process (37). In light of our data, it ispossible that the compensatory shift in plasticity that follows dis-ruption of neurogenesis may be sufficient to promote the extra-hippocampal consolidation of remote memories. Setting thesespeculations aside, our findings suggest that chronic disruption ofneurogenesis in young-adult mice transiently impairs LTP and thatthis transient disruption is reversed by compensatory changes thattake place at the systems or circuit level.

Materials and MethodsDetails of materials and methods are in SI Materials and Methods.

Mice. Themice used in these experiments have been previously described (26).Briefly, the tk gene from HSV-1, with the DNA sequence of the viral genemodified by humanizing the usage codon and eliminating all of the CpGs,was fused downstream of a minimal tk promoter element followed by a 1.8-kb fragment of the second intronic nestin enhancer. Mice were generatedand maintained on a FVB genetic background. All procedures involvinganimals were approved by the University Committee on the Use and Care ofAnimals of the University of Michigan.

Statistical Analysis. All data are presented as mean ± SEM. Data from field-potential recordings were analyzed using one-way or two-way repeated-measures ANOVA, as noted. Inter mIPSC interval data were analyzed bysubjecting the cumulative fraction of the log10 interevent interval to a Kol-mogorov-Smirnov two-sample test. Mean mIPSC amplitude, area, and decaywere averaged across neurons and compared using unpaired Student’s ttests. Cell counts were compared among nestin-tk and wild-type mice usinga Student’s t test. Normalized optical densities of each region of interestwere compared among nestin-tk and wild-type mice using a general linearmodel with fixed effects of genotype and time point and random effect ofsubject, and post hoc comparisons made with Bonferroni-corrected t tests.Statistical analysis was performed in SPSS (SPSS Inc.) or in Statview (SASInstitute Inc.).

ACKNOWLEDGMENTS. This workwas supported in part by National Institutesof Health (NIH) Grants HD044775 (to J.P.) and AG28488 (to G.M.) and NIH/National Institute on Aging Grant T32-AG000114 and NIH/National Instituteof Neurological Disorders and Stroke Grant T32 NS007222-30 (to A.E.G. andB.H.S.). S.J.T. is a National Science Foundation Graduate Research Fellow andwas also supported by a University of Michigan Rackham Merit Fellowship.

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