Neurobiology of Learning and Memory 105 (2013) 81–92

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Neurobiology of Learning and Memory

journal homepage: www.elsevier .com/ locate/ynlme

Role of adult neurogenesis in hippocampus-dependent memory,contextual fear extinction and remote contextual memory: New insightsfrom ERK5 MAP kinase

1074-7427/$ - see front matter � 2013 Elsevier Inc. All rights reserved.http://dx.doi.org/10.1016/j.nlm.2013.07.011

⇑ Corresponding author.E-mail addresses: dstorm@uw.edu (D.R. Storm), zxia@uw.edu (Z. Xia).

Yung-Wei Pan a, Daniel R. Storm a,⇑, Zhengui Xia b,⇑a Department of Pharmacology, University of Washington, Seattle, WA 98195, United Statesb Toxicology Program in the Department of Environmental and Occupational Health Sciences, University of Washington, Seattle, WA 98195, United States

a r t i c l e i n f o

Article history:Available online 18 July 2013

Keywords:Adult neurogenesisLearning and memorySignal transductionMAPK

a b s t r a c t

Adult neurogenesis occurs in two discrete regions of the adult mammalian brain, the subgranular zone(SGZ) of the dentate gyrus (DG) and the subventricular zone (SVZ) along the lateral ventricles. Signalingmechanisms regulating adult neurogenesis in the SGZ are currently an active area of investigation. Adult-born neurons in the DG functionally integrate into the hippocampal circuitry and form functional syn-apses, suggesting a role for these neurons in hippocampus-dependent memory formation. Althoughresults from earlier behavioral studies addressing this issue were inconsistent, recent advances in condi-tional gene targeting technology, viral injection and optogenetic approaches have provided convincingevidence supporting a role for adult-born neurons in the more challenging forms of hippocampus-depen-dent learning and memory. Here, we briefly summarize these recent studies with a focus on extra signal-regulated kinase (ERK) 5, a MAP kinase whose expression in the adult brain is restricted to the neurogenicregions including the SGZ and SVZ. We review evidence identifying ERK5 as a novel endogenous signalingpathway that regulates the pro-neural transcription factor Neurogenin 2, is activated by neurotrophinsand is critical for adult neurogenesis. We discuss studies demonstrating that specific deletion of ERK5in the adult neurogenic regions impairs several forms of hippocampus-dependent memory formationin mice. These include contextual fear memory extinction, the establishment and maintenance of remotecontextual fear memory, and several other challenging forms of hippocampus-dependent memory for-mation including 48 h memory for novel object recognition, contextual fear memory established by aweak foot shock, pattern separation, and reversal of spatial learning and memory. We also briefly discusscurrent evidence that increasing adult neurogenesis, by small molecules or genetic manipulation,improves memory formation and long-term memory.

� 2013 Elsevier Inc. All rights reserved.

1. Introduction

Neurogenesis is a life-long developmental process starting fromembryonic development to adult. Under normal physiological con-ditions, neurons are born in the adult brain from multipotent neu-ral stem cells (aNSCs) residing in the subventricular zone (SVZ)along the lateral ventricles and the subgranular zone (SGZ) of thedentate gyrus (Altman & Das, 1965; Bull & Bartlett, 2005; Doetsch,Caille, Lim, Garcia-Verdugo, & Alvarez-Buylla, 1999; Doetsch,Garcia-Verdugo, & Alvarez-Buylla, 1997; Gage, 2000; Gage,Kempermann, Palmer, Peterson, & Ray, 1998; Ming & Song,2005). Recent studies have led to the interesting hypothesis thatadult-born neurons may contribute to some forms of hippocampusdependent learning and memory (Aimone, Deng, & Gage, 2011;

Clelland et al., 2009; Creer, Romberg, Saksida, van Praag, & Bussey,2010; Deng, Aimone, & Gage, 2010; Deng, Saxe, Gallina, & Gage,2009; Dupret et al., 2008; Farioli-Vecchioli et al., 2008; Guoet al., 2011; Imayoshi et al., 2008; Kageyama, Imayoshi, & Sakam-oto, 2012; Kim, Christian, Ming, & Song, 2011; Kitamura et al.,2009; Ming & Song, 2011; Pan, Chan, Kuo, Storm, & Xia, 2012;Pan, Storm, & Xia, 2012; Sahay, Wilson, & Hen, 2011; Saxe et al.,2006; van Praag, Kempermann, & Gage, 1999; van Praag et al.,2002; Zhang, Zou, He, Gage, & Evans, 2008; Zhao, Deng, & Gage,2008). Several conditions, including depression, stress, and agingadversely affect hippocampal adult neurogenesis (Ming & Song,2005; Suh, Deng, & Gage, 2009). In contrast, external stimuliincluding environmental enrichment, voluntary exercise, andtreatments with anti-depressants stimulate adult neurogenesis inthe SGZ (Brown et al., 2003; Creer et al., 2010; David et al., 2010;Eisch et al., 2008; Li et al., 2008; Malberg, Eisch, Nestler, &Duman, 2000; Sairanen, Lucas, Ernfors, Castren, & Castren, 2005;

82 Y.-W. Pan et al. / Neurobiology of Learning and Memory 105 (2013) 81–92

Shirayama, Chen, Nakagawa, Russell, & Duman, 2002; van Praaget al., 1999). Furthermore, increased neurogenesis has been ob-served after brain injuries including seizure, stroke and DNA dam-age, promoting the idea that it may be advantageous to recruitendogenous neural stem cells to treat some types of neurodegener-ative diseases. Thus, it is important to elucidate molecular mecha-nisms regulating adult neurogenesis and to generate definitiveevidence supporting a role for adult neurogenesis in hippocam-pus-dependent memory formation.

Adult neural stem cells undergo proliferation, cell-fate specifi-cation, differentiation, maturation, migration and functional inte-gration (Belvindrah, Lazarini, & Lledo, 2009; Ge, Yang, Hsu, Ming,& Song, 2007; Hsieh, 2012; Kee, Teixeira, Wang, & Frankland,2007; Ma, Kim, Ming, & Song, 2009; Pan, Zou, et al., 2012; Petreanu& Alvarez-Buylla, 2002; Ramirez-Amaya, Marrone, Gage, Worley, &Barnes, 2006; Schmidt-Hieber, Jonas, & Bischofberger, 2004; Song,Stevens, & Gage, 2002; Toni et al., 2007; van Praag et al., 2002;Wang et al., 2013; Whitman & Greer, 2009; Winner, Cooper-Kuhn,Aigner, Winkler, & Kuhn, 2002; Yamaguchi & Mori, 2005; Yokoy-ama et al., 2011). Studies have identified cell-type specific proteinsin the SGZ as they progress from stem cells, expressing glial fibril-lary acidic protein (GFAP), Sox2, and nestin, to transiently-amplify-ing progenitor cells expressing polysialic acid-neural cell adhesionmolecule (PSA-NCAM), doublecortin (DCX), and NeuroD, toimmature neurons expressing NeuroD and Calretinin, and to moremature neurons expressing NeuN and Calbindin (Gage, 2000;Kempermann, 2006; Ming & Song, 2005; Taupin, 2006). Newbornneurons in the adult brain are capable of integrating into the exist-ing neuronal circuitry and have been implicated in hippocampus-dependent learning and memory tasks (Epp, Spritzer, & Galea,2007; Gould, Beylin, Tanapat, Reeves, & Shors, 1999; Kim et al.,2011; Leuner & Gould, 2010; Leuner et al., 2004; Pan, Chan,et al., 2012; Pan, Storm, et al., 2012).

In this review, we briefly discuss several recently identified reg-ulatory mechanisms of adult SGZ neurogenesis and the functionalsignificance of adult neurogenesis for memory formation. Moreextensive general reviews concerning this topic have recently beenpublished (Faigle & Song, 2012; Hsieh, 2012; Mu, Lee, & Gage,2010; Schwarz, Ebert, & Lie, 2012; Suh et al., 2009) (Curlik & Shors,2013; Deng et al., 2010; Glasper, Schoenfeld, & Gould, 2012; Guet al., 2012; Kazanis, 2012; Kim et al., 2011; Lee, Clemenson, &Gage, 2012; Marin-Burgin & Schinder, 2012; Ming & Song, 2011;Song, Christian, Ming, & Song, 2012). This review focuses onERK5, one of the newest signaling pathways implicated in adultneurogenesis, fear extinction and persistence of remote memory(Pan, Chan, et al., 2012; Pan, Storm, et al., 2012; Pan, Zou, et al.,2012). Recent data also suggest that ERK5 activation is sufficientto stimulate adult SGZ neurogenesis and to extend long-term, hip-pocampus-dependent memory (unpublished data).

2. Regulatory mechanisms implicated in hippocampal adultneurogenesis

Several recent reviews have extensively discussed transcriptionfactors and signaling molecules regulating adult SGZ neurogenesis(Faigle & Song, 2012; Hsieh, 2012; Mu et al., 2010; Schwarz et al.,2012; Suh et al., 2009). Gene targeting studies have suggested frag-ile X mental retardation protein (FMRP) and TLX, a cell-autono-mous orphan nuclear receptor as regulators of adult SGZneurogenesis (Guo et al., 2011; Zhang et al., 2008). Using an induc-ible FMRP knockout mouse to delete FMRP in nestin+ neural stemcells in the adult mouse, Guo et al. discovered an overall deficitin the total number of adult-born cells in the SGZ, and increasesin the number of DCX�/Ki67+ progenitor cells and GFAP+/S100b+

astrocytes (Guo et al., 2011). They also found decreases in DCX+/

Ki67+ neuroblasts, DCX+/Ki67� immature neurons, and NeuN+ ma-ture neurons. Additionally, the loss of FMRP in aNSCs resulted insignificantly reduced total dendritic length and the number of den-dritic branching points and ends.

Up until recently, the role of mitogen-activated protein kinases(MAPK) in adult neurogenesis has not received much attention.MAP Kinases are serine/threonine-protein kinases includingERK1/2, ERK5, p38 and JNK and are activated in response to growthfactors or stress. They have regulatory roles in cellular prolifera-tion, differentiation, cell-fate specification, cell-cycle progression,survival, and apoptosis (Kuida & Boucher, 2004; Miloso, Scuteri,Foudah, & Tredici, 2008; Raman, Chen, & Cobb, 2007; Rubinfeld &Seger, 2005; Seger & Krebs, 1995; Xia, Dickens, Raingeaud, Davis,& Greenberg, 1995). MAP Kinases have recently been found to playa role in adult neurogenesis. In one study, adult neural progenitorcells (aNPCs) from the hippocampus were isolated and exposed toHIV-1-associated dementia HIV-envelope glycoprotein gp120.Exposure to gp120 resulted in a significant decrease in the numberof proliferating cells. This effect was attributed to the induction ofp38 MAPK-MAPKAPK2-Cdc25c and an arrest of aNPCs in the G1phase of the cell cycle (Okamoto et al., 2007).

The ERK1/2 signaling pathway is activated in SGZ-derived aNS-Cs in culture in response to neuropeptide Y (NPY) or vascular endo-thelial growth factor (VEGF) (Fournier, Lee, Banasr, Elsayed, &Duman, 2012; Howell et al., 2005). A significant decrease in the to-tal number of BrdU+ cells 5 h after BrdU administration was ob-served along the SGZ of NPY�/� mice compared with littermatecontrols (Howell et al., 2005). The total number of DCX+, tran-siently amplifying progenitors and/or immature neurons, was alsogreatly reduced in the SGZ of NPY�/�mice. However, since NPY canactivate other signaling pathways in addition to ERK1/2, these cor-relative data alone do not specifically implicate ERK1/2 in adultneurogenesis. VEGF activates ERK1/2 in vitro and in vivo and in-creases BrdU+/Sox2+ neural stem cells (Fournier et al., 2012). Theeffect of VEGF was blocked by U0126, an ERK1/2 inhibitor, suggest-ing that the neurogenic effect of VEGF is mediated by the ERK1/2signaling pathway. These results suggest that extracellular growthfactors can stimulate adult SGZ neurogenesis and this effect maybe in part mediated by ERK1/2. Nevertheless, there is no direct evi-dence that ERK1/2 regulates adult neurogenesis under normalphysiological conditions.

ERK5 shares high sequence homology to ERK1/2. However,ERK5 contains a unique loop-12 linker between kinase sub-do-mains VII and VIII and an extended C-terminus making it distinctfrom other members of the MAPK family (Raman et al., 2007).The unique C-terminus contains a nuclear-localization signal(NLS), transcriptional activation domain, and a proline-rich region.ERK5 is normally localized in the cytoplasm, but upon stimulationby extrinsic factors, it translocates to the nucleus via the NLS se-quence (English, Vanderbilt, Xu, Marcus, & Cobb, 1995; Hayashiet al., 2004; Lee, Ulevitch, & Han, 1995; Wang, Su, & Xia, 2006;Zhou, Bao, & Dixon, 1995). Additionally, ERK5 is capable of auto-phosphorylation through its large C-terminus, the region that isalso required for the activation of nuclear substrates (Morimoto,Kondoh, Nishimoto, Terasawa, & Nishida, 2007). ERK5 is activatedspecifically by the upstream kinase, MEK5, through dual phosphor-ylation at ERK5’s TEY motif.

Our laboratory and others have demonstrated that neurotro-phin-induced activation of ERK5 promotes the survival of newbornneurons during embryonic development (Cavanaugh et al., 2001;Finegan, Wang, Lee, Robinson, & Tournier, 2009; Liu et al., 2003;Shalizi et al., 2003; Wang et al., 2006; Watson et al., 2001). ERK5also specifies cortical stem/progenitor cells toward a neuronal line-age during development by phosphorylating and modulating theactivity of Neurogenin 1 (Neurog 1), a proneural, helix-loop-helixtranscription factor (Cundiff et al., 2009; Liu et al., 2006). ERK5

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expression in the brain is high during early embryonic develop-ment but declines as the brain matures (Liu et al., 2003). It wasoriginally thought that ERK5 is not expressed in the adult brain(Di Benedetto et al., 2007). However, recent data demonstrate spe-cific and discrete expression of ERK5 in the adult neurogenic re-gions (Pan, Chan, et al., 2012; Pan, Zou, et al., 2012), implying animportant and novel regulatory role of ERK5 in adult neurogenesis.Indeed, using a tamoxifen-inducible knockout mouse strain to con-ditionally delete Mapk7, the gene encoding ERK5, in nestin-expressing aNSCs in the adult mice, it was demonstrated that inhi-bition of endogenous ERK5 signaling significantly reduces the totalnumber of adult-born neurons (Pan, Zou, et al., 2012). This is mostlikely due to a deficit in neuronal differentiation. Inhibition ofendogenous ERK5 signaling in SGZ-derived aNSCs by RNA interfer-ence significantly increases the pool of Sox2+ and nestin+ neuralstem cells, as well as PCNA+ proliferating cells while concomitantlydecreased the pool of b-III tubulin+ neurons. Furthermore, activat-ing endogenous ERK5 signaling by retroviral infection with a con-stitutive active MEK5 increases the production of adult-bornneurons both in vitro and in vivo. These results identify ERK5MAP kinase as a novel signaling pathway that regulates adult neu-rogenesis in the hippocampus, especially in neuronal differentia-tion. It has also been shown that Neurogenin 2 (Neurog 2) is adownstream target while neurotrophin 3 (NT3) is an upstreamactivator of ERK5 in this process. Neurogenin 2 belongs to the fam-ily of proneural basic helix-loop-helix transcription factors that in-cludes Neurogenin 1. Unlike Neurogenin 1, which is only expressedin the developing brain, Neurogenin 2 is expressed in both embry-onic brains as well as in adult hippocampal neural progenitor cells(Hodge et al., 2008; Ozen et al., 2007).

Mechanistic studies highlighted above provide an overview ofour current understanding of regulatory mechanisms responsiblefor adult SGZ neurogenesis. Early studies also identified signalingmolecules including Sonic Hedgehog, TrkB/C, Notch, and Wnt/b-catenin in the regulation of adult SGZ neurogenesis in the rodentbrain (Breunig, Arellano, Macklis, & Rakic, 2007; Breunig et al.,2008; Han et al., 2008; Li et al., 2008; Lie et al., 2005; Pozniak &Pleasure, 2006; Rolls et al., 2007). Transcription factors Pax6,Sox2, Mash1/Ascl1, NeuroD, Tbr2 and the master regulator REST/NRSF are expressed in the SGZ (Faigle & Song, 2012; Gao et al.,2009, 2011; Hodge & Hevner, 2011; Hodge et al., 2008; Hsieh,2012; Kim, Leung, Reed, & Johnson, 2007; Komitova & Eriksson,2004; Kuwabara et al., 2009; Osumi, Shinohara, Numayama-Tsuru-ta, & Maekawa, 2008; Suh et al., 2007). However, many of theseproteins are expressed outside of adult neurogenic regions in thebrain. Earlier studies using non-inducible knockout mouse modelsmay be confounded by a lack of specificity in targeting aNSCs inadult animals and/or by compensatory mechanisms during devel-opment. Future studies using inducible and conditional knockoutmouse models such as those employed in the FMRP and ERK5 stud-ies (Guo et al., 2011; Pan, Zou, et al., 2012) should clarify some ofthose issues and provide a better understanding of signaling mech-anisms governing adult neurogenesis.

3. Adult neurogenesis and hippocampus-dependent learningand memory

Since many recent reviews have addressed issues concerningthe role of adult neurogenesis in hippocampus-dependent memoryformation (Deng et al., 2010; Kazanis, 2012) (Kim et al., 2011) (Leeet al., 2012) (Marin-Burgin & Schinder, 2012) (Ming & Song, 2011)(Song et al., 2012), we will only attempt to highlight a few of thepublished studies here. In the past decade, several experimentalapproaches have been used to ascertain whether modulating nor-mal levels of adult SGZ neurogenesis affects learning and memory.

However, data from different labs have been contradictory and theimportance of adult neurogenesis for memory formation remainscontroversial. For example, although some studies showed thatadult neurogenesis plays a role in contextual fear memory forma-tion (Guo et al., 2011; Imayoshi et al., 2008; Saxe et al., 2006), con-textual fear memory extinction (Deng et al., 2009), and contextualfear memory transfer out of the hippocampus (Kitamura et al.,2009), several other studies do not support a role for adult neuro-genesis in contextual fear memory formation (Deng et al., 2009;Dupret et al., 2008; Jaholkowski et al., 2009; Shors, Townsend,Zhao, Kozorovitskiy, & Gould, 2002; Zhang et al., 2008). Addition-ally, one study found that x-irradiated mice did not exhibit a deficitin contextual fear conditioning while rats did (Snyder et al., 2009),thereby suggesting a species-specific difference in the requirementof adult-born neurons for contextual learning. Similarly, a numberof studies implicated adult neurogenesis in spatial learning andmemory (Clelland et al., 2009; Creer et al., 2010; Dupret et al.,2008; Farioli-Vecchioli et al., 2008; Garthe, Behr, & Kempermann,2009; Guo et al., 2011; Imayoshi et al., 2008; Jessberger et al.,2009; Zhang et al., 2008), in contrast to others (Denis-Doniniet al., 2008; Jaholkowski et al., 2009; Meshi et al., 2006; Saxeet al., 2006; Shors et al., 2002).

It is not totally clear why different studies have led to contradic-tory conclusions. Some possibilities include differences in the sexand strain of the animals, and details of the experimental designof behavior assays which could influence the level of difficulty ofthe training tasks. Perhaps one of the important factors responsiblefor the inconsistent results from earlier studies is the specificity ofthe methods used to ablate adult neurogenesis. Early studies usedx-irradiation or treatment with anti-mitotic drugs. These treat-ments are effective at reducing adult neurogenesis, but may notnecessarily be specific to aNPCs because these methods may alsoaffect neurons and glia. These treatments may also cause side ef-fects including general health deterioration of the animal and neu-ral inflammation. In other studies, specific genes such as cyclin D2,Wnt, PC3/Tis21, TLX and PI3K were deleted or gene products phar-macologically inhibited (Denis-Donini et al., 2008; Farioli-Vecchio-li et al., 2009; Jaholkowski et al., 2009; Jessberger et al., 2009;Zhang et al., 2008). However, these genes are also expressed out-side of the adult neurogenic regions and the deletions in thesegenes in transgenic mice or pharmacological inhibition of the spe-cific molecules were not restricted to adult neurogenic regions. Thepotential off-target effects associated with these various ap-proaches may account for some of the contradictions in theliterature.

Despite the controversy, these earlier studies are interestingand exciting, and provided valuable information regarding the po-tential function of adult-born neurons on hippocampus-dependentfunction. However, the contradictory results in the literatureunderscored the importance of using more specific genetic ap-proaches with less off-target side effects to manipulate adult neu-rogenesis. More recent studies employed transgenic expression oflethal genes including diphtheria toxin (Imayoshi et al., 2008), thy-midine kinase (Deng et al., 2009; Saxe et al., 2006), or the pro-apoptotic protein Bax (Dupret et al., 2008) to selectively kill new-born cells in the adult brain. These transgenic approaches are morefocused because the effects are specific to adult neural stem cells.Nevertheless, the controversy persisted. For example, thymidinekinase (tk) was expressed under the control of the nestin promoter(Deng et al., 2009) or GFAP promoter (Saxe et al., 2006) of two linesof transgenic mice. Administration of the antiviral prodrug ganci-clovir (GCV) effectively kills dividing, thymidine kinase-expressingneural progenitor/precursor cells. However, these two almost iden-tical approaches for deleting adult born neurons gave oppositeconclusions regarding adult neurogenesis and contextual fear con-ditioning: the GFAP-tk mice were impaired in the contextual fear

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conditioning when tested 24 h after a 0.7 mA foot shock (1 s shock)while the nestin-tk transgenic mice (2 s shock) were not. Severalfactors many have contributed to the inconsistent results: theduration of the foot shock (1 s vs. 2 s) or the different promotersused to direct the tk gene expression. It is also conceivable thatthe degree of GCV-thymidine kinase triggered cell death exceedednormal physiological level, thus causing side effects.

Recent advances in transgenic mouse technology have made itfeasible to conditionally disrupt a gene specifically in adult neuro-genic regions. For example, by crossing transgenic mice containinga floxed allele of a gene of interest with the nestin-cre-ERT2 or nes-tin-cre-ER™ driver mice (Kuo et al., 2006, Imayoshi et al., 2008;Lagace et al., 2007), the gene of interest can be specifically deletedin nestin-expressing adult neural stem cells by cre-recombinasefollowing tamoxifen drug administration in adult mice. Indeed, arecent study used this technology to delete the fmrp gene in aNSCsof nestin-cre-ERT2/FMRPlox/lox mice (Guo et al., 2011). These miceare deficient in pattern separation as well as contextual and tonetrace learning. This technology was also used to delete ERK5 inaNSCs, which will be discussed in more detail in the followingsections.

Recent studies have begun to dissect the specific role of adult-born neurons in different stages of memory formation. Using trans-genic mouse technology to specifically block synaptic output ofolder granular neurons while leaving the immature granular neu-rons intact, Nakashiba et al. discovered unique functions of oldvs. young granular neurons in pattern separation; young neuronsmediate pattern separation while old neurons are required for pat-tern completion (Nakashiba et al., 2012). Using a combination ofretroviral and optogenetic approaches to birthdate and reversiblycontrol a group of adult-born neurons, it was found that reversiblysilencing 4-week-old neurons after training, but not neurons ofother ages, significantly disrupted hippocampal memory retrieval(Gu et al., 2012). These results suggest a specific time windowfor adult-born neurons in hippocampal memory retrieval.

4. The role of ERK5 regulated adult neurogenesis inhippocampus-dependent learning and memory and incontextual fear extinction

We generated the nestin-creER™/ERK5loxP/loxP transgenic mousemodel to inducibly and conditionally knockout Mapk7, the geneencoding ERK5 (ERK5 icKO mice) specifically in adult neurogenicregions upon tamoxifen administration (Pan, Chan, et al., 2012;Pan, Zou, et al., 2012). There was a significant reduction in the num-ber of adult-born neurons in the DG in vivo for at least four months.This provides an extended time (4 months) for behavior tests. TheERK5 icKO mice provide a unique and powerful tool to investigatethe relationship between adult neurogenesis and hippocampus-dependent learning and memory because ERK5 expression in adultbrain is generally absent in the adult brain (Di Benedetto et al.,2007) but specific to adult neurogenic regions (Pan, Chan, et al.,2012). Furthermore, ERK5 deletion in the adult brain was tempo-rally and spatially regulated and specific to neural stem cells inERK5 icKO mice (Pan, Chan, et al., 2012; Pan, Zou, et al., 2012).

To assess the physiological role of ERK5-regulated adult neuro-genesis for hippocampal memory formation, adult male nestin-creER™/ERK5loxP/loxP mice (10–12 weeks old) were treated withtamoxifen to delete ERK5. These mice are designated ERK5 icKO.Several lines of control mice were used for the studies. In one setof experiments, we included only one control group of mice, theadult male nestin-creER™/ERK5loxP/loxP littermates treated withthe vehicle for tamoxifen as a control for the genotype (Pan, Chan,et al., 2012). To address the concern that the behavioral defectsobserved with the ERK5 icKO mice reported in that study might

be due to adverse effect of tamoxifen treatment (Fitzsimons & Luc-assen, 2012), adult male ERK5loxP/loxP littermates treated withtamoxifen were used as a drug control (Figs. 1–7). These new dataconfirm behavioral defects observed with the ERK5 icKO mice (Pan,Chan, et al., 2012) and alleviate the concern regarding tamoxifentoxicity (Fitzsimons & Lucassen, 2012). Our more recent data in-cludes all possible controls for the ERK5 icKO mice (Pan, Storm,et al., 2012). We also confirmed that hippocampus-independentlearning and memory, including cued-fear conditioning, was notaffected in ERK5 icKO mice (Figs. 4 and 5, and (Pan, Chan, et al.,2012; Pan, Storm, et al., 2012)).

There is no statistically significant difference between ERK5icKO and either groups of control mice (genotype control or tamox-ifen drug control) in their open field activities or their ability tohabituate to the open field over three consecutive days (Fig. 1and (Pan, Chan, et al., 2012)). ERK5 icKO mice also do not have de-fects in memory for novel objects measured 1 h or 24 h after train-ing (Fig. 2 and (Pan, Chan, et al., 2012)). These results suggest thatERK5 icKO mice are not deficient in their general mobility, curios-ity to explore a novel environment, or short-term memory. Fur-thermore, ERK5 icKO mice are normal in the visible platform andhidden platform water maze tests (Fig. 3 and (Pan, Chan, et al.,2012)), thus they are not ataxic, or deficient in vision, generalmobility, ability to swim, procedural learning, or motivation to es-cape from water. Although ERK5 icKO mice spent significantly lesstime swimming and searching for the escape platform in quadrant1 compared with drug control animals during the probe test, theirspatial memory for the location of the hidden platform was similar.Finally, ERK5 icKO mice exhibit a normal level of contextual fearmemory when trained with a standard 0.7 mA foot shock andtested 24 h post-training (Fig. 4 and (Pan, Chan, et al., 2012)).

However, unlike control mice, ERK5 icKO animals fail atremembering the novel object 48 h after training (Fig. 2 and(Pan, Chan, et al., 2012)), implicating adult neurogenesis in long-term memory. Several lines of evidence also suggest that ERK5and ERK5-regulated adult neurogenesis are important for the morecomplex and challenging forms of hippocampus-dependent learn-ing and memory tasks, including contextual fear and spatial learn-ing and memory. ERK5 icKO mice show reduced contextual fearmemory when trained with 0.3 mA � 3 foot shocks (Fig. 5 and(Pan, Chan, et al., 2012)). This training paradigm may be more chal-lenging compared to the single 0.7 mA foot shock because wildtype mice have to be shocked three successive times to generatecontextual memory as strong as that generated with a single 2 s,0.7 mA foot shock. The impaired contextual fear memory of ERK5icKO mice after training using the weak foot shock paradigm(0.3 mA) is due to a deficit in consolidation and persistence of con-textual fear memory, but not in memory acquisition or retrieval(Pan, Storm, et al., 2012).

Once trained in the hidden platform test of the Morris watermaze task, ERK5 icKO mice were subjected to reversal training inwhich the hidden platform was moved to the opposite location.ERK5 icKO mice swim longer distances than control mice to findthe new platform during reversal training and they could not dis-tinguish between the old and new target quadrants during thereversal probe test (Fig. 3 and (Pan, Chan, et al., 2012)). Reversaltraining is a complex form of spatial learning and memory becauseanimals need to actively forget the old location of the hidden plat-form and learn to find it in a new location.

Another well accepted spatial learning and memory assay ispattern separation, which specifically requires the function of DG(Clelland et al., 2009; Farioli-Vecchioli et al., 2008; Guo et al.,2011). Pattern separation is also considered a difficult form of hip-pocampal memory formation because it tests the process of trans-forming similar representations of memories into highly dissimilar,non-overlapping representations (Clelland et al., 2009; Guo et al.,

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Fig. 1. Motor behavior and habituation in the open field assay is not affected in ERK5 icKO animals compared to the drug control mice. Adult male nestin-creER™/ERK5loxP/loxP

and ERK5loxP/loxP littermate mice (10–12 weeks old) were treated with tamoxifen as in (Pan et al., 2012) and designated ERK5 icKO or drug control respectively. Mice wereexposed to the open field assay for three consecutive days to examine habituation to a novel environment. (A) Total floor plane activity on day 1 (d1), d2, d3. (B) Total amountof time spent along the margin of the open field chamber. (C) Total distance traveled during each exposure to the open field chamber. (D) Average traveling speed during eachexposure. Data represent mean ± SEM of two independent experiments with n P 10 animals per treatment group per experiment.

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2011). ERK5 icKO animals do not perform as well as control ani-mals in the pattern separation assay (Fig. 6 and (Pan, Chan, et al.,2012)). These data are consistent with other reports implicatingadult neurogenesis in pattern separation (Clelland et al., 2009;Creer et al., 2010; Farioli-Vecchioli et al., 2008).

Finally, unlike control mice, ERK5 icKO animals fail to extin-guish contextual fear memory even after repeated exposure (twicea day for 8 d) to the training context without foot shock (Fig. 4 and(Pan, Chan, et al., 2012)). These data suggest that adult neurogen-esis may regulate fear extinction, a notion supported by anotherstudy (Deng et al., 2009). Contextual fear extinction is a form ofhippocampus-dependent learning and memory, distinct from con-textual fear conditioning (Fischer et al., 2007). It may be moredemanding and complex than contextual fear conditioning be-cause animals learn to actively dissociate the context from the footshock. For example, while it takes only a single 2 s, 0.7 mA footshock to establish a strong, thus relatively easy, form of the initialcontextual fear memory that persists at least 15 weeks (Pan, Storm,et al., 2012), it takes repeated trials of context exposure over thecourse of 8 d without the foot shock for this memory to undergoextinction in control mice (Pan, Chan, et al., 2012)).

It is interesting that ERK5 deletion interferes with contextualfear memory extinction and reversal of hidden platform training.It is possible that adult neurogenesis may be particularly importantfor learning that requires active forgetting of a prior memory.

5. The role of ERK5 regulated adult neurogenesis in theestablishment and maintenance of remote contextual fearmemory

How long-term, or remote memory is formed and maintained isan important question. Recent studies have shed light on mecha-

nisms responsible for the formation of recent hippocampus-depen-dent memories, including a role for adult neurogenesis in thisprocess. However, far less is known how recent memories are trans-formed into remote memories and how remote memories, onceestablished, are maintained (Frankland & Bontempi, 2005). Contex-tual fear memory is initially formed in the hippocampus but isthought to be transferred out to extra-hippocampal regions includ-ing the cortex for long-term storage. Whether the maintenance ofestablished remote contextual memory requires the function ofthe hippocampus is controversial with some evidence supporting(Debiec, LeDoux, & Nader, 2002; Goshen et al., 2011; Sutherland &Lehmann, 2011) as well as against its involvement (Anagnostaras,Maren, & Fanselow, 1999; Frankland & Bontempi, 2005; Kim &Fanselow, 1992; Squire & Alvarez, 1995; Squire & Zola-Morgan,1991). A recent study implicated adult neurogenesis in modulatingthe duration of the hippocampus-dependent period of contextualfear memory; disruption of adult neurogenesis accelerated thetransfer of memory out of the hippocampus but did not affect thestrength of the remote memory per se (Kitamura et al., 2009).

We discovered that ERK5 icKO mice have no remote memory21 days after training with a 2 s, 0.7 mA foot shock in the passiveavoidance assay although their 24 h memory is completely normal(Fig. 7, and (Pan, Chan, et al., 2012)). Similarly, their remote contex-tual fear memory at 5 weeks post-training with the 0.7 mA footshock is greatly attenuated (Pan, Storm, et al., 2012). The deficitsin remote memory are specific to hippocampus-dependent tasksand do not occur with hippocampus-independent, remote (up to16 weeks) cued-fear memory. These data are the first evidence thatadult neurogenesis is required for the establishment of fullstrength remote contextual fear memory.

Interestingly, deletion of ERK5 to attenuate adult neurogenesisat 6 d post-training, a time point when mice had sufficient time

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Fig. 2. Drug control animals retain, while ERK5 icKO mice lack memory in the novelobject recognition assay tested 48 h after training. As described in (Pan et al., 2012),animals were trained for two objects (A and B) during the training session for 5 min(A and B) or 10 min (C–F). Following an interval of 1 h, 24 h, or 48 h, object B wasreplaced with object C and animals were permitted to explore both object A(familiar) and object C (novel) in the testing session. Total exploration time of eachobject was recorded identically during the training (A, C, and E) and testing sessions(B, D, and F). A unique set of objects A–C was used for each time point tested.Memory for the familiar object A is manifested as more time spent in exploring thenovel object C than the familiar object A. (A and B) ERK5 icKO mice have normal 1 hmemory. (C and D) ERK5 icKO mice have normal 24 h memory. (E and F) ERK5 icKOmice have no memory 48 h post-training. Data represent mean explorationtime ± SEM from three independent experiments with n P 8 animals per treatmentgroup per experiment. n.s., not significant; **p < 0.01; ***p < 0.001.

86 Y.-W. Pan et al. / Neurobiology of Learning and Memory 105 (2013) 81–92

to consolidate contextual fear memory but it is still within the win-dow of the hippocampus-dependent period, causes decay of re-mote contextual fear memory but not remote cued-fear memory(Pan, Storm, et al., 2012). Perhaps more profoundly, when tamoxi-fen is administered 5 weeks post-training, a time point when re-mote contextual memory has already been established, ERK5

deletion causes retrograde amnesia of remote contextual fearmemory in ERK5 icKO mice, again without affecting remotecued-fear memory (Pan, Storm, et al., 2012). These data suggestthat ongoing ERK5-regulated adult neurogenesis is required forthe maintenance of remote contextual fear memory, even after thismemory has been established.

Collectively, studies using ERK5 icKO indicate a critical role foradult-born neurons in both the expression and maintenance of re-mote contextual fear memory using three different paradigms,exemplified with observed deficits when ERK5 is conditionally de-leted prior to training, 6 d after training, or 5 weeks after training.These data support the hypothesis that interactions between thehippocampus and neocortex, after the formation of recent mem-ory, is critical for the storage of remote memory regardless of theexact storage location for remote memory (Frankland & Bontempi,2005; Nakashiba, Buhl, McHugh, & Tonegawa, 2009; Nakashiba,Young, McHugh, Buhl, & Tonegawa, 2008). Together with a recentreport using diphtheria toxin-based strategy to ablate mature,adult-generated neurons within the week after training (Arruda-Carvalho, Sakaguchi, Akers, Josselyn, & Frankland, 2011), these datasuggest that interference with adult neurogenesis can cause retro-grade amnesia.

6. Enhancing memory formation by increasing adultneurogenesis

Enriched environment (Birch, McGarry, & Kelly, 2013; Kemper-mann, Kuhn, & Gage, 1997), voluntary running (Creer et al., 2010;van Praag et al., 1999), and chronic antidepressant treatment (Da-vid et al., 2009) increases adult neurogenesis and improves mem-ory formation. Petrik and colleagues recently discovered that Isx-9,a synthetic small molecule, increases hippocampal neurogenesisand the time spent in the target quadrant in the Morris water mazeprobe test (Petrik et al., 2012). Metformin, a clinical drug for type IIdiabetes, also enhances adult hippocampal neurogenesis and spa-tial learning in the reversal Morris water maze test (Wang et al.,2012). These pioneering studies established a strong correlativerelationship and generated much excitement about the possibilitythat enhancing adult neurogenesis may improve cognition (Curlik& Shors, 2013; Glasper et al., 2012).

Utilizing the Nestin-Cre-ERT2:Bax mice to delete the pro-apop-totic gene Bax specifically in adult neural stem cells, Sahay and col-leagues reported that more adult-born neurons survived in thesemice and that the mice show enhanced pattern separation (Sahay,Scobie, et al., 2011). We recently generated a conditional knock-inline of mice in which expression of a constitutive active MEK5 canbe induced, allowing selective and conditional activation of endog-enous ERK5 in adult neurogenic regions (Wenbin Wang, ZhenguiXia et al., unpublished data). These mice show increases in adulthippocampal neurogenesis and most interestingly, extension oflong-term spatial memory. This conditional and inducible genemanipulation allows induction of adult neurogenesis without aug-menting other biochemical or biological pathways. These studiesprovide strong evidence that enhancement of adult neurogenesisalone is sufficient to improve cognition.

7. Potential mechanisms by which adult neurogenesis mayregulate long-term memory

ERK5 conditional knockout studies demonstrated that ongoingadult neurogenesis is necessary and required for memory persis-tence, even after the memory has transferred to extra-hippocampalregions (Pan, Storm, et al., 2012). Conversely, conditional knock-inof activated endogenous ERK5 is sufficient to promote adult neuro-genesis and long-term hippocampus-dependent memory (Wenbin

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Fig. 3. ERK5 icKO animals are deficient in acquiring newer spatial information during reversal training in the Morris water maze. (A) As described in (Pan et al., 2012), micewere subjected to 8 d of training (4 sessions/day, 30 min inter-session interval, from d1 to d8) in the hidden platform Morris water maze. Target (in quadrant #1) acquisition,quantified as swim distance to the hidden platform, was similar in drug control and ERK5 icKO mice, demonstrating similar spatial learning. (B) A probe test was conducted24 h later, which demonstrated that drug control and ERK5 icKO mice both acquired spatial memory for the location of the hidden platform. (C) Mice were then subjected to7 d of reversal training (4 sessions/day, 30 min inter-session interval) where the escape hidden platform was relocated to the opposite quadrant (quadrant #3) of the watermaze. ERK5 icKO mice swam longer distances to find the hidden platform in the newer location, suggesting impaired learning of newer spatial information. (D) ERK5 icKOanimals are unable to differentiate between the new and old locations of the escape platform in the reversal probe test, conducted 24 h after reversal hidden platformtraining. Data represent mean ± SEM from two independent experiments with n P 10 animals per treatment group per experiment. n.s., not significant; *p < 0.05; ***p < 0.001.

Y.-W. Pan et al. / Neurobiology of Learning and Memory 105 (2013) 81–92 87

Wang, Zhengui Xia et al., unpublished data). How do adult-bornneurons in the hippocampus contribute to memory persistence?Memory persistence may require recurrent rounds of consolidationbecause it requires repeated rounds of transcription and proteinsynthesis, for example, for the production of NMDA receptors(Bekinschtein et al., 2007; Cui et al., 2004). The cAMP/ERK1/2MAP kinase/CREB pathway is important for memory consolidationand is activated during REM sleep in the circadian cycle (Eckel-Ma-han et al., 2008; Luo, Phan, Yang, Garelick, & Storm, 2013). Therecurrent activation of this pathway during REM sleep may gener-ate repeated rounds of transcription and translation necessary tosustain memory. Interestingly, the same sequence of synaptic fir-ing that occurs during spatial learning is repeated during sleep(Lee & Wilson, 2002). Perhaps new synapses, including those fromadult-born neurons, are recruited to the circuitry during repeatedsynaptic firing in sleep for continued maintenance of establishedmemories. Immature adult born neurons and mature neurons inthe dentate gyrus have a different activation threshold. Weak affer-ent activity activates a significant proportion of the immatureadult born neurons but not mature neurons in the dentate gyrus(Marin-Burgin, Mongiat, Pardi, & Schinder, 2012). Since mostmemory traces, even long-term memories, eventually decay, it istempting to speculate that the strength of recurrent synaptic firingmay gradually reduce over time, leading to memory decay. Becauseof their higher sensitivity to lower threshold stimulation, imma-ture adult-born neurons may be uniquely suited for new synapse

recruitment during recurrent synaptic firing in sleep. When adultneurogenesis is attenuated, fewer synapses from adult-born neu-rons will respond to the gradually weakening synaptic firing andbe recruited to the circuitry, thus accelerating memory decay.However, increasing adult neurogenesis may increase the pool ofresponding synapses, thus strengthening the circuitry and extend-ing long-term memory.

8. ERK5 regulation of adult neurogenesis in the SVZ and inolfactory memory

ERK5 is also expressed in SVZ (Pan, Kuo, Storm, & Xia, 2012).Deletion of ERK5 reduces adult neurogenesis in the SVZ and olfac-tory bulb (OB) of the ERK5 icKO mice. ERK5 has also been impli-cated in prolactin stimulation of adult neurogenesis in the SVZ/OB (Wang et al., 2013). Conditional deletion of ERK5 in ERK5 icKOmice has no effect on olfactory discrimination among discreteodorants. However, it reduces short-term olfactory memory as wellas detection sensitivity to odorants and pheromones. Furthermore,ERK5 icKO mice show impaired acquisition of odor-cued associa-tive olfactory learning. These data suggest that ERK5 MAP kinaseis critical for the regulation of adult neurogenesis in the SVZ/OB,and provide strong evidence supporting a role for adult neurogen-esis in several distinct complex forms of olfactory behavior.

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Fig. 4. ERK5 icKO mice are deficient in contextual fear memory extinction. As described in (Pan et al., 2012), all animals were trained in the contextual fear conditioning assayusing a single 0.7 mA foot shock lasting 2 s. A 30 s tone was presented prior to foot shock delivery and co-terminated with the foot shock. (A) Drug control and ERK5 icKOanimals have similar levels of contextual fear memory 24 h after training. (B) Fear memory is context specific since neither drug control nor ERK5 icKO mice froze whenplaced in a novel context 24 h after training. (C) Cued-fear response is similar between drug control and ERK5 icKO animals when tested 24 h after training. (D) Fear extinctionis impaired in ERK5 icKO animals compared to drug controls. Day 0: the day of contextual fear testing. Day 1: the first day animals were subjected to fear extinction training.(E) Following fear extinction training, mice were placed into a novel context on Day 9. Both drug control and ERK5 icKO animals froze minimally when placed in the novelcontext, suggesting that the persistent fear memory associated with ERK5 icKO mice is context-specific behavior. Data represent mean freezing behavior ± SEM from twoindependent experiments with n P 8 animals per treatment group per experiment. n.s., not significant; ***p < 0.001.

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Fig. 5. ERK5 icKO animals have reduced contextual fear memory when trained using a more challenging training paradigm. As described in (Pan et al., 2012), animals weretrained using a weaker electric foot shock (0.3 mA) delivered 3 consecutive times. Each foot shock was paired with a tone, which co-terminated with the foot shock. (A)Freezing behavior was monitored in the shocking context 24 h after training. ERK5 icKO mice froze less compared with drug control animals. (B) This deficit was context-specific since both groups of animals did not freeze when exposed to a novel context 24 h after training. (C) Cued-fear conditioning was also not affected in either group ofmice when tested 24 h after training. Data represent mean freezing behavior ± SEM from two independent experiments with n P 6 animals per treatment group perexperiment. ***p < 0.001.

88 Y.-W. Pan et al. / Neurobiology of Learning and Memory 105 (2013) 81–92

9. Summary and conclusions

Adult neurogenesis has received much attention in large partbecause of the therapeutic potential of adult-born neurons. Delin-eating molecular mechanisms involved in the genesis of functional

adult-born neurons will aid in the full understanding of how adultneurogenesis is regulated from stem cell self-renewal, prolifera-tion, cell-fate specification, differentiation, maturation and func-tional integration into the existing neuronal circuitry. To realizethe therapeutic potential of aNSCs, it is important to clarify the

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Fig. 7. Remote memory is impaired in ERK5 icKO mice when tested in the passiveavoidance assay. As described in (Pan et al., 2012), mice were trained in the passiveavoidance assay by placing animals in the light side of a light/dark box. The light/dark chambers are separated by a small trap door. When mice entered the dark sideof the box, the trap door immediately closed and a single 0.7 mA foot shock wasdelivered for 2 s. Following a 1 min memory acquisition period, mice were removedfrom the dark side of the box. Mice were placed back to the light side of the trainingbox 24 h later to assess short-term, 24 h memory for the aversive stimulus,manifested as increased latency to cross over into the dark side. No deficits existbetween drug control and ERK5 icKO animals at 24 h. Mice were then returned totheir home cages. At 21 d post-training, animals were again placed back to the lightside of the box to assess for remote memory. Unlike drug control mice, ERK5 icKOanimals had no remote memory. Data represent mean crossover latency ± SEM withn P 10 per treatment group. ***p < 0.001.

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roles of various intrinsic and extrinsic mechanisms regulating thisprocess.

The functional impact of adult-born neurons in the SGZ for var-ious forms of hippocampus-dependent learning and memory sup-ports the idea that the brain is not static in its adult form and iscapable of continuously generating functional neurons. Studies re-viewed in this manuscript identify the ERK5 MAP kinase as a novelkinase signaling pathway regulating adult neurogenesis in the SGZand in several challenging forms of hippocampus-dependent learn-ing and memory tasks including contextual fear extinction. Fur-thermore, studies with ERK5 conditional knockout mice provide

the first evidence that continual formation of new neurons in theDG is integral for the maintenance of remote contextual fear mem-ory, even after the memory has transferred out of the hippocam-pus. This conclusion supports the general hypothesis that thehippocampus has a long-term role in the continued expression ofcontextual fear memory (Sparks, Lehmann, Hernandez, & Suther-land, 2011; Sutherland, O’Brien, & Lehmann, 2008). The discoverythat memory formation and persistence can be improved by di-rectly stimulating adult neurogenesis has important therapeuticimplications for the treatment of memory disorders. Because ofits selective pattern of expression in neurogenic regions of adultbrain, modulation of the ERK5 signaling pathway may provide aunique pharmacological window of opportunity for stimulationof adult neurogenesis.

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