Reversible modulations of neuronal plasticity by VEGFTamar Lichta,1, Inbal Goshenb,1, Avi Avitalc,d, Tirzah Kreiselb, Salman Zubedatc, Ronen Eavria, Menahem Segald,Raz Yirmiyab, and Eli Kesheta,2

aDeparment of Developmental Biology and Cancer Research, Hebrew University Hadassah Medical School, Jerusalem 91120, Israel; bDepartment ofPsychology, Hebrew University, Jerusalem 91905, Israel; cDepartment of Psychology and Center for Psychobiological Research, Yezreel Valley College,Emek Yezreel 19300, Israel; and dDepartment of Neurobiology, Weizmann Institute of Science, Rehovot 76100, Israel

Edited by Napoleone Ferrara, Genentech, South San Francisco, CA, and approved February 16, 2011 (received for review June 3, 2010)

Neurons, astrocytes, and blood vessels are organized in functional“neurovascular units” in which the vasculature can impact neuro-nal activity and, in turn, dynamically adjust to its change. Herewe explored different mechanisms by which VEGF, a pleiotropicfactor known to possess multiple activities vis-à-vis blood vesselsand neurons, may affect adult neurogenesis and cognition. Condi-tional transgenic systems were used to reversibly overexpressVEGF or block endogenous VEGF in the hippocampus of adultmice. Importantly, this was done in settings that allowed theuncoupling of VEGF-promoted angiogenesis, neurogenesis, andmemory. VEGF overexpression was found to augment all threeprocesses, whereas VEGF blockade impaired memory without re-ducing hippocampal perfusion or neurogenesis. Pertinent to thegeneral debate regarding the relative contribution of adult neuro-genesis to memory, we found that memory gain by VEGF over-expression and memory impairment by VEGF blockade werealready evident at early time points at which newly added neu-rons could not yet have become functional. Surprisingly, VEGF in-duction markedly increased in vivo long-term potentiation (LTP)responses in the dentate gyrus, and VEGF blockade completelyabrogated LTP. Switching off ectopic VEGF production resultedin a return to a normal memory and LTP, indicating that ongoingVEGF is required to maintain increased plasticity. In summary, thestudy not only uncovered a surprising role for VEGF in neuronalplasticity, but also suggests that improved memory by VEGF isprimarily a result of increasing plasticity of mature neurons ratherthan the contribution of newly added hippocampal neurons.

vascular biology | neural stem cells | learning

VEGF is the key factor in promoting and coordinating most ifnot all processes of blood vessel formation in the embryo and

adult. VEGF is also required for the maintenance of vascularhomeostasis, including an indispensible role in adjusting thevasculature to meet dynamic changes in oxygen supply and de-mand and in controlling vascular barrier functions (reviewed inref. (1). An increasing body of evidence implicates VEGF inneuronal processes in the adult mammalian brain, notably inadult neurogenesis. Canonically, neurogenesis in the adult braintakes place in two niches: the subventricular zone, which con-tinuously supplies new interneurons to the olfactory bulb, andthe subgranular zone of the dentate gyrus (DG), which gives riseto granule neurons as well as glial cells throughout adult life. Inboth niches, this process takes place in close proximity to bloodvessels (2, 3), which has prompted the notion of a “vascularniche” of adult neurogenesis. Exogenous VEGF was shown toincrease the basal level of adult neurogenesis in the hippocam-pus (4–6). Conversely, inhibition of VEGF negates the increasedrate of neurogenesis usually observed in mice reared in anenriched environment or under increased exercise (5, 7). Nota-bly, enriched housing, exercise, and training in the Morris watermaze induced an increase in endogenous VEGF expression (5).VEGF was also shown to be capable of enhancing hippocam-

pus-dependent memory (5), but it is unclear whether the effects ofVEGF on neurogenesis and memory are causally related. Thisissue should be considered in the broader context of the ongoingdebate regarding the contribution of neurogenesis to neuronalplasticity and hippocampus-dependent memory in comparisonwith other contributing processes, such as increased synaptic

density and enhanced synaptic strength. Findings that favor a rolefor newly added neurons demonstrate that newly born neuroblastsnot only differentiate and integrate into the existing network asfunctional hippocampal granule neurons, but are more responsivethan older neurons to a spatial memory task and display an in-creased synaptic plasticity (8–10). On the contrary, experimentallydamaging the proliferative capacity of DG cells yielded contra-dicting results concerning an impairment in hippocampus-de-pendentmemory (11–15). (Ref. 16 reviews arguments that supportand refute the notion that hippocampal neurogenesis has a majorcontribution to formation of new memories.)VEGF is ideally poised to mediate a vascular–neuronal cross-

talk, considering that (i) increased neuronal activity induceschanges in blood flow and microvascular density and (ii) VEGFproduction is induced whenever there is increased need for ox-ygen and metabolites. However, delineating the multiple waysby which VEGF may affect neuronal activity has been greatlyhampered by the lack of suitable experimental systems in whichto conditionally manipulate endogenous hippocampal VEGF. Inparticular, it has been impossible to rule out that phenotypes ofVEGF loss of function are not secondary to impaired perfusion.Here, we developed transgenic mice models for gain of cerebralVEGF function (GOF) and loss of cerebral VEGF function(LOF) in a conditional and reversible manner. With the aid ofthese unique mice, we uncovered a surprising role for VEGF inthe adult brain in enhancing neuronal plasticity and memoryfunctioning, independent of its described effects on blood per-fusion and adult neurogenesis.

ResultsVEGF Is Constitutively Expressed in Astrocytes of the Adult Hippo-campus Whereas VEGF TK Receptors Are Predominantly Expressed inNearby Endothelial Cells. Endogenous expression of VEGF in theadult hippocampus was demonstrated by using a LacZ knock-inreporter inserted into the 3′UTR of the endogenous VEGF gene.As shown in Fig. S1A (Left), within the hippocampus, VEGFis expressed in CA1 and DG regions. GFAP staining revealedthat VEGF is produced primarily by astrocytes (Fig. S1A, Right).Nevertheless, because VEGF is a secreted protein, it is likely ac-cessible for all CA1 and DG cells in the adult hippocampus.As a first step to examining a putative role for VEGF in

neuronal cells, we wished to determine which cells within the DGmay potentially respond to the constitutively expressed VEGF byvirtue of expressing VEGF receptors. The pattern of expressionof VEGF-R2 (Flk1) was examined by mRNA in situ hybridiza-tion (ISH) and through the use of an Flk1 promoter-LacZ re-porter mouse (17). Both methods revealed that Flk1 expressionwas readily detected on endothelial cells of the DG but not onneuronal cells or astrocytes (Fig. S2A). Coimmunostaining forastrocytes and Flk1 demonstrated a close proximity of the cells

Author contributions: T.L., M.S., R.Y., and E.K. designed research; T.L., I.G., A.A., T.K., S.Z.,and R.E. performed research; I.G. contributed new reagents/analytic tools; T.L., I.G., A.A.,T.K., S.Z., and R.E. analyzed data; and T.L. and E.K. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.1T.L. and I.G. contributed equally to this work.2To whom correspondence should be addressed. E-mail: keshet@cc.huji.ac.il.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1007640108/-/DCSupplemental.

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naturally producing (and secreting) VEGF and endothelial cellsexpressing cognate receptors, respectively (Fig. S2A′′). A similaranalysis was extended to VEGF-R1 (Flt1), and results showedthat, similarly to Flk1, Flt1 ISH signal could be detected only inendothelial cells of the DG (Fig. S2B). Next, we determinedpatterns of hippocampal expression of neuropilin-1 (Nrp1) andneuropilin-2 (Nrp2). Corroborating previous results (18), DGgranule cells were found to be positive for both neuropilins (Fig.S2 C and D).The fact that VEGF and its receptors are constitutively

expressed in the DG prompted us to explore a role for VEGF inthe adult hippocampus via its conditional manipulations.

Genetic System for Conditional and Reversible Gain or Loss of VEGFFunction in the Adult Brain. A tetracycline-regulated system wasused to manipulate VEGF in the adult brain. Briefly, brain-specific expression was achieved by using a driver transgenic linecomposed of tet-regulated transactivator protein (13) driven bya CamKIIα promoter (19). For VEGF GOF, a tet-responsiveVEGFA164 responder line was generated (20). For VEGF LOF,a transgene responder encoding a chimeric tet-regulated proteincomposed of the five Ig-like loops of the extracellular domain ofFlt1 fused to an IgG1-Fc tail was used. The induced secretedreceptor (I-sVEGF-R1) efficiently binds and sequesters VEGF,thereby precluding its signaling (21). Thus, this transgenic mouse

system allows reversible induction and repression of VEGF sig-naling at will (SI Materials and Methods and Fig. S1B providefurther details on the experimental system).The choice of a CamKIIα-based driver mouse was based on

previous studies showing its widespread expression in the hip-pocampus (19). Accordingly, the system was proven suitable forswitching “on” expression of VEGF or, conversely, of theVEGF-trapping protein in the hippocampus (Figs. S1 C and Dand S3). Note that the system is not leaky and fully reversible asevident from full rerepression of the respective transgene ex-pression following readdition of tetracycline (i.e., “on > off”).It is noteworthy that the induced soluble receptor I-sVEGF-

R1 used in the VEGF LOF experiments sequesters not onlyVEGFA (abbreviated as VEGF) but also its family membersVEGFB and PLGF, the inhibition of which may confound thephenotypes described here and is attributed to VEGF.

VEGF Increases Hippocampal Angiogenesis and Neurogenesis andImproves Hippocampal-Dependent Memory. As expected, trans-genic overexpression of VEGF in the hippocampus resulted inrapid, robust, and progressive addition of new blood vessels, asevidenced by staining with the endothelial marker CD31 (Fig.1A). Many of these endothelial cells were also positive for Ki67,which is indicative of active endothelial proliferation (Fig. 1A,Inset). i.v. injection of FITC-dextran was used to show that

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Fig. 1. VEGF GOF increases hippocampal angiogenesis, augments neurogenesis, and improves memory. (A and B). VEGF was switched-on at postnatal day 40and brains analyzed 7 or 21 d later. (A) Sections were immunostained for endothelial cells (CD31+) and nuclei were highlighted with DAPI. Vascular density wasquantified as the area occupied by capillaries relative to total DG area (including the neuropil; n = 3–5 per group and n = 4–6 sections per animal, representingall DG areas at the rostrocaudal axis; *P < 0.0005). (B) Immunostaining for neuroblasts (DCX+). Images are representatives of a 40-μm-thick Z-projection.Quantification was done by calculating the number of DCX+ cell bodies per a volume unit of DG cell bodies (n = 3–5 per group, n = 4–6 sections per animal; *P <0.0005). (C) BrdU pulse was given 4 wk after the onset of VEGF induction (three injections per day of 50 mg/kg for 2 d), and brains were retrieved for analysis 3wk thereafter. BrdU+/NeuN+ cells labeling newly added neurons that have maturedmeanwhile and survived were counted and normalized to the length of theDG in the image (n = 5 per group, n = 4–6 sections per animal; *P< 0.0005). (D) Control and VEGF-induced animals were subjected to a fear conditioning learningparadigm (Materials and Methods). Note a significantly enhanced contextual memory in VEGF-induced mice, reflected by a twofold increase in freezing timebut no change in auditory-cued fear conditioning (during tone; n = 8 animals in the control group and n = 7 in the VEGF group; #P < 0.005). (Scale bars, 200 μm.)

5082 | www.pnas.org/cgi/doi/10.1073/pnas.1007640108 Licht et al.

VEGF-induced hippocampal vessels are functional and wellperfused (Fig. S4).To detect neurogenic activity of VEGF, proliferating neuro-

blasts were visualized by immunostaining with the early neuronalmarker doublecortin (DCX; Fig. 1B). The number of DCX-positive cells in the subgranular zone was doubled within 1 wkfrom VEGF induction, and was further increased to approxi-mately fourfold after an additional 2 wk. To determine whetherthe fourfold increase in the number of neuroblasts is also reflec-ted in a parallel increase in the number of added mature neurons,a short BrdU pulse was given at 1 wk from the onset of VEGFinduction, and brains were analyzed 3 wk thereafter for cellsdouble-positive for BrdU and the mature neuronal marker NeuN.Results showed that VEGF overexpression increased the numberof BrdU+/NeuN+ cells similarly by a factor of four (Fig. 1C).To determine whether VEGF-induced neurogenesis is ac-

companied by improved cognitive performance, we subjected themice to the fear-conditioning memory task. It is noteworthy thatthe experimental design used is capable of distinguishing hip-pocampus-dependent and -independent memory. As shown inFig. 1D, mice analyzed 4 wk after switching-on of transgenicVEGF expression showed a markedly improved hippocampal-dependent contextual memory whereas their hippocampal in-dependent auditory-cued memory remained unchanged.

VEGF LOF Impairs Memory Without Impairing Vascular Density orDecreasing Neurogenesis. To follow the functional consequencesof inhibiting signaling of the naturally produced hippocampalVEGF (Fig. S1A), I-sVEGF-R1 was induced in the adult brainand maintained in the on mode for various times and for as longas 7 wk after induction. The three processes shown here to beaugmented by VEGF overexpression, namely angiogenesis,neurogenesis, and memory, were then similarly analyzed.

We have previously shown that premature withdrawal ofVEGF may result in regression of newly formed vessels but thatmature vessels are refractory to VEGF inhibition (22). We as-sumed, therefore, that withdrawing VEGF in the adult brain, i.e.,after vessels have already matured, will not result in vascular loss.This was indeed confirmed in the experiment shown in Fig. 2A.Further, VEGF inhibition did not reduce vessel patency as judgedby FITC-dextran perfusion (Fig. S4), did not cause hippocampalhypoxia, and did not inflict any detectable cell death (cleavedcaspase-3–positive cells)within thehippocampus (Fig. S5A andB).To determine the effect of VEGF inhibition on basal neuro-

genesis, hippocampal slices were stained with DCX. Resultsshowed no decrease in the number of neuroblasts (Fig. 2B).Additionally, newly formed neurons (highlighted via DG injec-tions of GFP-encoding retrovirus) developed to have typicaldendritic trees and spines with branching points equal in numberto control (Fig. S5C).To determine whether VEGF sequestration is accompanied by

impairment of cognitive performance, animals were subjected toa memory task as described earlier. Despite the fact that neu-rogenesis was not compromised, animals with VEGF LOF dis-played impaired contextual memory compared with their controllittermates (Fig. 2C). Auditory-cued memory was also moder-ately reduced, but this effect did not reach statistical significance.To provide additional support to the finding that VEGF LOFmay result in memory impairment, animals were subjected toa radial eight-arm maze test that measured hippocampus-dependent spatial learning (SI Materials and Methods). As shownin Fig. S6, VEGF LOF animals completely lost spatial learningskills. VEGF GOF animals, however, failed to further improvespatial learning in this particular test.Taken together, GOF and LOF experiments clearly showed

that VEGF is required for proper memory. Intriguingly, however,the LOF experiments also suggest that the role of VEGF in

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Fig. 2. VEGF LOF in the hippocampus impairs memory without reducing vessel density or neurogenesis. (A and B) I-sVEGF-R1 transgenic mice were switchedat postnatal day 40 for 7, 21, and 49 d. (A) CD31 immunostaining: quantification of the area occupied by vessels (Bottom) reveals no significant differenceamong all time points (P > 0.1; n = 4 per group, n = 4–6 images each). (Scale bar, 200 μm.) (B) DCX immunostaining: quantification shows no significantdecrease in neurogenesis (P > 0.1; n = 4 per group, n = 4–6 images each). (C) Control and VEGF LOF animals were subjected to a fear-conditioning learningparadigm. Note a 2.5-fold reduction in contextual memory. Auditory-cued memory was also reduced but this did not reach statistical significance (n = 6–8 inall groups; *P < 0.01). (Scale bars, 200 μm.)

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the memory process is unrelated to its neurogenic activity. There-fore, we wished to further establish that VEGF-induced neuro-genesis and VEGF-improved memory are causally unrelated.

Uncoupling the Effect of VEGF on Memory from Its Effects onNeurogenesis. To evaluate the relative contribution of newlyadded neurons to memory, we subjected mice to a memory taskas early as 5 d after VEGF induction (or, conversely, after VEGFblockade). This experiment is based on the premise that, at thistime point, newly added neurons may not yet have becomefunctional considering that newly added hippocampal neuronsare known to mature within 3 to 4 wk and project their first axonsand dendrites no earlier than 10 d from their birth (23). Asshown in Fig. 3 A and B, at 5 d after VEGF induction or block-ade, there was already a significant improvement or reduction,respectively, in memory functioning and at a magnitude com-parable to changes observed after 1 mo from onset. These resultssuggest that a mechanism other than neurogenesis is likely toaccount for VEGF-enhanced memory.

VEGF-Mediated Cognitive Gain Is Reversible Whereas Cognitive LossInduced by VEGF Inhibition Is Irreversible. To determine whetherthe cognitive gain induced by VEGF could be reversed onwithdrawal of transgene expression, VEGF was switched off af-ter 1 mo from induction (i.e., on > off), and 1 mo later, animalswere subjected to the fear-conditioning test (Fig. 4). Resultsshowed that enhanced memory induced by VEGF was com-pletely lost after switching off transgenic VEGF and was now

indistinguishable from that of control littermates (Fig. 4A). Torule out that the reversal was because the newly added neuronsdid not survive VEGF withdrawal, a BrdU pulse was givenduring the on period and the number of BrdU+ neurons wasdetermined at the end of the off’ period. Results showed thatthe number of BrdU+ neurons remained significantly higher inVEGF on > off animals than in controls (Fig. 4B). Moreover, thenumber of DCX+ neuroblasts remained significantly higher inVEGF on > off animals (Fig. S7), indicating that neuronal cell

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Fig. 4. Reversibility of phenotypes induced byVEGF GOF and LOF. Transgenic expression ofVEGF (A, B, and D) or I-sVEGF-R1 (C) was switchedon for 4 wk. A 4-d long BrdU pulse (50 mg/kg i.p.twice daily) was given during the second week.Animals were then switched off and subjected toanalysis 4 wk later. (A) VEGF on > off transgenicanimals subjected to fear conditioning. Notea comparable performance to control animals(n = 7 animals per group). (B) Hippocampi fromVEGF on > off transgenic animals immunostainedfor BrdU and NeuN. Note an increase in thenumber of double-positive neurons comparableto the increase observed in VEGF on animalsshown in Fig. 2C (n = 3 animals per group, n = 6images each; *P < 0.0001). (Scale bar, 200 μm.) (C)I-sVEGF-R1 on > off transgenic animals subjectedto fear conditioning. Note that the deficit incontextual memory was not rectified (n = 9–11animals per group; #P < 0.05). (D) VEGF on > offtransgenic animals immunostained for CD31.Note that the vascular gain remained unchangedfollowing VEGF withdrawal (n = 4–5 animals pergroup, n = 3–5 images each; *P < 0.0001). (Scalebar, 200 μm.)

5084 | www.pnas.org/cgi/doi/10.1073/pnas.1007640108 Licht et al.

production did not decrease upon VEGF withdrawal. Theseresults indicate that ongoing VEGF signaling is required tomaintain the cognitive gain and that this cannot be attributed tothe neurogenic activity of VEGF.A similar reversal experiment was performed for VEGF LOF.

Here, the memory deficit induced via VEGF inhibition was notrectified after cessation of VEGF blockade (Fig. 4C). The reasonfor this result is currently not understood given that, under theseconditions, perfusion and neurogenesis were not compromised.

Uncoupling the Effect of VEGF on Memory from Its Effects onPerfusion. A bidirectional link between perfusion and neuronalactivity is well established. Because VEGF-induced angiogenesisfunctions to improve tissue perfusion, we wished to determinewhether improved memory could be attributed to increased mi-crovascular density. VEGF induction indeed led to markedly in-creased microvascular density in the hippocampus (highlighted byCD31 staining), which persisted 1mo afterVEGFwithdrawal (Fig.4D). As enhanced memory was lost despite the fact that addedvessels persisted after VEGFwithdrawal, it could be ruled out thatVEGF-induced hyperperfusion is the cause of improved memory.

VEGF Enhances Long-Term Potentiation (LTP) Whereas I-sVEGF-R1Abrogates LTP. As VEGF-induced memory changes could not beaccounted for by VEGF-enhanced neurogenesis, we examinedother mechanisms of neuronal plasticity such as LTP. Animalswith VEGF GOF or LOF were subjected in vivo to tetanicstimulation of the perforant path (PP) while recording thepostsynaptic field potential of DG granule cells. Importantly,LTP was measured 5 to 15 d after induction of the respectivetransgene to rule out a contribution by newly added neurons.Results showed that VEGF overexpression resulted in a highlysignificant reactivity to the afferent stimulation persisting for atleast 60 min. Conversely, VEGF LOF resulted in complete ab-rogation of the normal LTP response (Fig. 5). To characterizechanges in the dynamic range of DG synapses, input/outputrelations were determined; no significant difference between thegroups was found (Fig. S8A). To determine whether there areapparent differences in short-term synaptic plasticity in thesesynapses, paired-pulse responses were examined (Fig. S8B). Adecreased facilitation in the VEGF LOF animals at the in-terstimulus interval of 60 ms was indeed found, which provideda partial explanation for the LTP loss. Remarkably, augmentation

of the LTP response by ectopic VEGF appeared to be hippo-campus region-specific, taking place at the PP, where recording isdone at the DG, but not in the Schaffer collaterals, where re-cording is done at the CA1. This was evident by a parallel in vivorecording from the CA1 region that failed to show a similar in-crease in LTP response despite a similar level of VEGF inductionin both regions and a comparable angiogenic response (Fig. S9).Prompted by the results described here that have demonstrated

a requirement for ongoing VEGF to maintain cognitive gain, wewished to determine whether switching off the respective trans-gene will similarly lead to reversal of the LTP change. As shown inFig. 5, switching off VEGF indeed resulted in return to an LTPresponse that is indistinguishable from control, and terminatingVEGF inhibition resulted in a partial rescue of LTP. These resultsnot only uncovered a role for VEGF in synaptic modulation, butalso suggest that VEGF-induced synaptic strengthening maycontribute to VEGF-induced memory facilitation.

DiscussionThis study uncovered a surprising role for VEGF in neuronalplasticity, namely modulating plasticity of mature neurons. Thestudy not only extends the list of nonangiogenic functions forVEGF, but may also provide insights on complex neuronal/vas-cular cross-talk in general, and on the question of how com-ponents of the vascular system may modify neuronal activity inparticular.Uncoupling multiple functions of VEGF in the adult brain

has been hampered by the lack of appropriate genetic systemssuitable for conditional VEGF manipulations in a tightly con-trolled spatial and temporal manner. Here, we developed systemsof conditional VEGF GOF or LOF in brain regions implicated inadult neurogenesis and memory. Furthermore, the systems wedeveloped allowed for reversal of the respective VEGF manipu-lation at will, thereby allowing us to determine whether ongoingVEGF signaling is also required tomaintain the respectiveVEGF-induced phenotype. With the aid of these systems, we were able tocorroborate and extend previous studies that used exogenous ad-ministrationofVEGForaVEGFinhibitor todemonstrateVEGF-enhanced hippocampal neurogenesis (4–6) and memory (5).Traditionally, it has been difficult to distinguish neuronal

phenotypes resulting from impaired perfusion, which is an an-ticipated consequence of VEGF LOF, from perfusion-in-dependent effects. The ability to inhibit VEGF signaling at timeswhen hippocampal vessels are no longer dependent on VEGFallowed us to conclude that neuronal phenotypes resulting fromVEGF LOF are indeed not caused by impaired perfusion, whichremained unchanged (Fig. 2 and Fig. S4). Because VEGF isprimarily an angiogenic factor and its overexpression indeed ledto increased vascular density in the hippocampus, we could di-rectly evaluate the effect of hyperperfusion on neurogenesis andmemory. Interestingly, an elevated level of basal neurogenesisaccompanying the increase in vascular density (Fig. 1) persistedeven after the return of VEGF to its normal level (followingswitching off of transgene expression; Fig. 4 and Fig. S7), sug-gesting that expansion of the hippocampal vasculature alone issufficient for a sustained increase in basal neurogenesis withouta need for ongoing VEGF signaling. Intriguingly, under theseconditions of augmented hippocampal perfusion, increasedneurogenesis, and normal VEGF levels, the cognitive gain andenhanced LTP returned to control levels.The latter result has implications beyond VEGF biology by

virtue of addressing the general debate regarding the relativecontribution of newly added neurons to learning and memory.Findings reported here suggest that increased neurogenesis byitself is not sufficient for enhancing memory, at least in the fear-conditioning paradigm, nor enhancing LTP. Two additional linesof evidence argue against a major contribution of newly addedneurons to memory processes: First, VEGF LOF compromisedmemory and LTP without reducing neurogenesis (Figs. 2 and 5).We further reason that, even if newborn neurons that wereadded in the setting of VEGF LOF were functionally impaired,their relatively small population could not have accounted forcomplete abrogation of memory and LTP. Second, the VEGF-induced enhancement of memory and LTP—and, conversely,

Fig. 5. VEGF GOF enhances LTP whereas VEGF LOF ablates LTP in the DG. (A)In vivo LTP in the DG of anesthetized adult animals. VEGF on mice (n = 6)switched for 5 to 15 d before the experiment showed a significantly enhancedLTP. Similar switch of I-sVEGF-R1 (n = 8) demonstrate inability to obtain LTP. Inthe VEGF on> off (n = 7) and I-sVEGF-R1 on> off (n = 7) groups, the respectivetransgene was induced for 4 wk followed by 4 wk in the off mode. Resultsshowed complete or partial reversibility, respectively. Representative tracesare presented for control before high-frequency stimulation (HFS) (a), VEGFafter HFS (b), control after HFS (c), and I-sVEGF-R1 after HFS (d).

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memory and LTP deficits under conditions of VEGF inhibition—were already evident within 5 d from onset, i.e., at an early timewhen newly made neurons could have not possibly becomefunctional (Figs. 3 and 5).Although VEGF manipulations were used in the present study

to uncouple adult neurogenesis and learning, the question re-mains to what extent the relatively minor contribution of neu-rogenesis to learning can be generalized. Previous studies haveshown that newborn neurons are essential for memory and thatthey are more responsive to novel stimuli than existing neurons(8–15). The different conclusions drawn from these studies andthe present study could also reflect methodological differences.Notably, the previous studies used selective ablation of newbornneurons (based on antimitotic agents or ablation of nestin- orGFAP-positive cells), which could have affected other cells aswell, whereas the present study circumvented any cellular dam-age and is mostly based on a kinetic argument.It is not known whether the newly discovered effect of VEGF

on neuronal plasticity is direct, taking place via VEGF binding toneuronally expressed VEGF receptors, or indirect, i.e., via in-ducing endothelial or glial cells to secrete some factor acting onneuronal cells. A precedent for an indirect effect vis-à-vis VEGF-induced neurogenesis is the seasonal addition of new neuronsto the high vocal center (HVC) of male songbirds, in whichtestosterone-induced VEGF was found to stimulate nearby en-dothelial cells to secrete BDNF that, in turn, promotes re-cruitment of neurons from the HVC ventricular zone (24). Thenotion of a direct response of neurons to VEGF is supported byreports on expression of the high-affinity VEGF receptors FLK1and FLT1 and of the auxiliary nonsignaling receptor neuropilin onneuronal cells (6, 25–27). Also consistent with a direct mechanismare findings showing that VEGF can induce neurite growth incultured cortical neurons (28–30). In vivo, however, a rigorousdistinction between direct and indirect mechanisms may necessi-tate neuronal-, glial-, and endothelial-specific ablation (or func-tional knockdown) of each VEGF receptor. Supporting an in-direct mechanism, we detected endothelial-specific expression ofVEGF tyrosine kinase receptors FLK1 and FLT1 in the hippo-campus but could not detect their expression on hippocampal

neurons (Fig. S2). However, we cannot exclude the possibility ofa low level of expression that is below our detection threshold.The requirement for ongoing VEGF signaling for proper

memory may have implications for anti-VEGF–based cancertherapy, as a prolonged, systemic treatment with VEGF-neu-tralizing antibodies may potentially impair cognitive functioning.In conclusion, this study uncovered a function of the highly

pleiotropic factorVEGF, linking the brain vasculature to neuronalfunctioning in a more complex manner than previously thought.

Materials and MethodsAnimals. All animal procedures were approved by the animal care and usecommittee of the Hebrew University. The transgenic CamKIIα-tTa brain-specific mouse driver line was purchased from Jackson Labs (19). pTET-VEGF164 responder line (20) and pTET-I-sVEGF-R1 responder line were de-scribed previously (31). For switching-off of VEGF or I-sVEGF-R1, water wassupplemented by 500 mg/L tetracycline (Tevacycline; Teva) and 3% sucrose.For switching on the transgenes, tetracycline-supplemented water wasreplaced by fresh water for the desired time. SI Materials and Methodsprovides additional details.

Fear-Conditioning Assay. Context and cued conditioning were measuredas described (32). The detailed protocol is provided in SI Materials andMethods.

In Vivo LTP Measurements. PP-evoked responses in the DG were measured invivo as described (32). The detailed protocol is provided in SI Materialsand Methods.

Immunohistochemistry. For immunohistochemistry, we used formalin-fixedparaffin-embedded tissues (dissected to 3 μm) and 4% paraformaldehyde-fixed frozen 50-μm floating sections. SI Materials and Methods includes a listof antibodies and detailed immunohistochemistry protocols, along withadditional details on methods.

ACKNOWLEDGMENTS. We thank Andras Nagy and Janet Rossant for miceand Rinnat Porat, Nicola Maggio, and Ahuva Itin for technical assistance.This work was supported by the Israel Science Foundation and the German–Israeli Foundation.

1. Ferrara N (2004) Vascular endothelial growth factor: Basic science and clinicalprogress. Endocr Rev 25:581–611.

2. Palmer TD, Willhoite AR, Gage FH (2000) Vascular niche for adult hippocampalneurogenesis. J Comp Neurol 425:479–494.

3. Shen Q, et al. (2008) Adult SVZ stem cells lie in a vascular niche: A quantitative analysisof niche cell-cell interactions. Cell Stem Cell 3:289–300.

4. Schänzer A, et al. (2004) Direct stimulation of adult neural stem cells in vitro andneurogenesis in vivo by vascular endothelial growth factor. Brain Pathol 14:237–248.

5. Cao L, et al. (2004) VEGF links hippocampal activity with neurogenesis, learning andmemory. Nat Genet 36:827–835.

6. Jin K, et al. (2002) Vascular endothelial growth factor (VEGF) stimulates neurogenesisin vitro and in vivo. Proc Natl Acad Sci USA 99:11946–11950.

7. Fabel K, et al. (2003) VEGF is necessary for exercise-induced adult hippocampalneurogenesis. Eur J Neurosci 18:2803–2812.

8. Tashiro A, Makino H, Gage FH (2007) Experience-specific functional modification ofthe dentate gyrus through adult neurogenesis: A critical period during an immaturestage. J Neurosci 27:3252–3259.

9. Kee N, Teixeira CM, Wang AH, Frankland PW (2007) Preferential incorporation ofadult-generated granule cells into spatial memory networks in the dentate gyrus. NatNeurosci 10:355–362.

10. Ge S, Yang CH, Hsu KS, Ming GL, Song H (2007) A critical period for enhanced synapticplasticity in newly generated neurons of the adult brain. Neuron 54:559–566.

11. Shors TJ, et al. (2001) Neurogenesis in the adult is involved in the formation of tracememories. Nature 410:372–376.

12. Meshi D, et al. (2006) Hippocampal neurogenesis is not required for behavioral effectsof environmental enrichment. Nat Neurosci 9:729–731.

13. Saxe MD, et al. (2006) Ablation of hippocampal neurogenesis impairs contextual fearconditioning and synaptic plasticity in the dentate gyrus. Proc Natl Acad Sci USA 103:17501–17506.

14. Zhang CL, Zou Y, He W, Gage FH, Evans RM (2008) A role for adult TLX-positive neuralstem cells in learning and behaviour. Nature 451:1004–1007.

15. Imayoshi I, et al. (2008) Roles of continuous neurogenesis in the structural andfunctional integrity of the adult forebrain. Nat Neurosci 11:1153–1161.

16. Leuner B, Gould E, Shors TJ (2006) Is there a link between adult neurogenesis andlearning? Hippocampus 16:216–224.

17. Shalaby F, et al. (1995) Failure of blood-island formation and vasculogenesis in Flk-1-deficient mice. Nature 376:62–66.

18. Sahay A, et al. (2005) Secreted semaphorins modulate synaptic transmission in theadult hippocampus. J Neurosci 25:3613–3620.

19. Mayford M, et al. (1996) Control of memory formation through regulated expressionof a CaMKII transgene. Science 274:1678–1683.

20. Dor Y, et al. (2002) Conditional switching of VEGF provides new insights into adultneovascularization and pro-angiogenic therapy. EMBO J 21:1939–1947.

21. May D, et al. (2008) Transgenic system for conditional induction and rescue of chronicmyocardial hibernation provides insights into genomic programs of hibernation. ProcNatl Acad Sci USA 105:282–287.

22. Benjamin LE, Hemo I, Keshet E (1998) A plasticity window for blood vesselremodelling is defined by pericyte coverage of the preformed endothelial networkand is regulated by PDGF-B and VEGF. Development 125:1591–1598.

23. Zhao C, Teng EM, Summers RG Jr, Ming GL, Gage FH (2006) Distinct morphologicalstages of dentate granule neuron maturation in the adult mouse hippocampus. JNeurosci 26:3–11.

24. Louissaint A, Jr, Rao S, Leventhal C, Goldman SA (2002) Coordinated interaction ofneurogenesis and angiogenesis in the adult songbird brain. Neuron 34:945–960.

25. Mani N, Khaibullina A, Krum JM, Rosenstein JM (2005) Astrocyte growth effects ofvascular endothelial growth factor (VEGF) application to perinatal neocortical explants:receptor mediation and signal transduction pathways. Exp Neurol 192:394–406.

26. Jin KL, Mao XO, Greenberg DA (2000) Vascular endothelial growth factor: Directneuroprotective effect in in vitro ischemia. Proc Natl Acad Sci USA 97:10242–10247.

27. Maurer MH, Tripps WK, Feldmann RE Jr, Kuschinsky W (2003) Expression of vascularendothelial growth factor and its receptors in rat neural stem cells.Neurosci Lett 344:165–168.

28. Rosenstein JM, Mani N, Khaibullina A, Krum JM (2003) Neurotrophic effects ofvascular endothelial growth factor on organotypic cortical explants and primarycortical neurons. J Neurosci 23:11036–11044.

29. Khaibullina AA, Rosenstein JM, Krum JM (2004) Vascular endothelial growth factorpromotes neurite maturation in primary CNS neuronal cultures. Brain Res Dev BrainRes 148:59–68.

30. Jin K, Mao XO, Greenberg DA (2006) Vascular endothelial growth factor stimulatesneurite outgrowth from cerebral cortical neurons via Rho kinase signaling. JNeurobiol 66:236–242.

31. Grunewald M, et al. (2006) VEGF-induced adult neovascularization: Recruitment,retention, and role of accessory cells. Cell 124:175–189.

32. Avital A, et al. (2003) Impaired interleukin-1 signaling is associated with deficits inhippocampal memory processes and neural plasticity. Hippocampus 13:826–834.

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