Genetic recovery of ErbB4 in adulthood partiallyrestores brain functions in null miceHongsheng Wanga,1, Fang Liub,1, Wenbing Chena,c,1, Xiangdong Sund, Wanpeng Cuia, Zhaoqi Donga, Kai Zhaob,Hongsheng Zhanga, Haiwen Lia, Guanglin Xinga, Erkang Feic, Bing-Xing Panc, Bao-Ming Lic, Wen-Cheng Xionga,e,and Lin Meia,e,2

aDepartment of Neurosciences, School of Medicine, Case Western Reserve University, Cleveland, OH 44106; bDepartment of Neuroscience and RegenerativeMedicine, Medical College of Georgia, Augusta University, Augusta, GA 30912; cInstitute of Life Science, Nanchang University, 330031 Nanchang, China;dSchool of Basic Medical Sciences, The Second Affiliated Hospital of Guangzhou Medical University, 510260 Guangzhou, Guangdong, China; and eLouisStokes Cleveland Veterans Affairs Medical Center, Cleveland, OH 44106

Edited by Richard L. Huganir, The Johns Hopkins University School of Medicine, Baltimore, MD, and approved November 1, 2018 (received for review July1, 2018)

Neurotrophic factor NRG1 and its receptor ErbB4 play a role inGABAergic circuit assembly during development. ErbB4 null micepossess fewer interneurons, have decreased GABA release, and showimpaired behavior in various paradigms. In addition, NRG1 and ErbB4have also been implicated in regulating GABAergic transmission andplasticity in matured brains. However, current ErbB4 mutant strainsare unable to determine whether phenotypes in adult mutant miceresult from abnormal neural development. This important question, aglaring gap in understanding NRG1–ErbB4 function, was addressedby using two strains of mice with temporal control of ErbB4 deletionand expression, respectively. We found that ErbB4 deletion in adultmice impaired behavior and GABA release but had no effect on neu-ron numbers and morphology. On the other hand, some deficits dueto the ErbB4 null mutation during development were alleviated byrestoring ErbB4 expression at the adult stage. Together, our resultsindicate a critical role of NRG1–ErbB4 signaling in GABAergic trans-mission and behavior in adulthood and suggest that restoring NRG1–ErbB4 signaling at the postdevelopmental stage might benefitrelevant brain disorders.

ErbB4 | adulthood | GABA | treatment | schizophrenia

Schizophrenia (SZ) is a disabling mental disorder that affects∼1% of the population worldwide (1). It alters basic brain

processes of perception, emotion, and judgment to cause hallu-cinations, delusions, thought disorder, anhedonia, and cognitivedeficits. Despite extensive efforts to study its pathophysiologicalmechanisms, SZ remains one of the least understood brain dis-orders. Recent identification of SZ susceptibility genes and studiesof their functions have begun to shed light on its pathophysiology.Neuregulin 1 (NRG1) is an EGF-domain–containing trophic fac-

tor that acts by activating ErbB tyrosine kinases including ErbB4.NRG1–ErbB4 signaling is critical for assembling GABAergic cir-cuits. Mutant mice lacking NRG1 or ErbB4 display deficits in inter-neuron migration, axon and dendrite development of interneurons,and synaptogenesis onto and by interneurons (2–10). Both NRG1and ErbB4 are expressed in the adult brain. NRG1 is producedmainly in neurons in an activity-dependent manner (11, 12). On theother hand, 99% of ErbB4-positive neurons in the adult cortex andhippocampus are GABAergic (4, 13–17), the majority of whichexpress parvalbumin (PV) (4, 13, 15, 16). In adult animals, NRG1–ErbB4 signaling could promote GABAergic transmission and thuscontrol pyramidal neuron activity (17–24). It was proposed thatNRG1–ErbB4 signaling serves as a homeostatic mechanism tocontrol the excitation–inhibition (E-I) balance (25, 26).Interestingly, NRG1 and ErbB4 are SZ risk genes in diverse

populations based on family trio studies, case-controlled associ-ation studies, and meta-analysis studies [see review by Mei andNave (25)]. However, their polymorphism did not reach genome-wide significance in a recent genome-wide association study ofSZ (27), perhaps as a result of allelic heterogeneity at NRG1 andErbB4 loci, the existence of haplotypes (28–32), and/or pop-ulation stratification (33–36). The following evidence supports

the notion that NRG1 and ErbB4 alteration contributes topathophysiological mechanisms of SZ. First, NRG1 and ErbB4variants have been shown to be associated with reduced volumeand decreased activation of brain regions and cognitive pheno-types (37–39) and with responses to antipsychotics treatment(40–42). Second, altered NRG1 or ErbB4 levels were detected inpostmortem brain samples and peripheral blood of SZ patients(43–45). Third, mutating NRG1 and ErbB4 or altering theirlevels in mice could recapitulate SZ-related endophenotypes (3,18, 24, 46–48). Finally, recent meta-analyses (49–51), includingone from 2017 on >16,000 schizophrenic patients and >20,000controls (49), identify NRG1 and ErbB4 as risk genes for SZ. In-terestingly, SNP rs7598440 of ErbB4 could predict cortical or ce-rebrospinal fluid GABA concentration in healthy human subjects(52, 53), in agreement with critical roles of ErbB4 in the develop-ment and function of the GABA circuitry from mouse studies.A critical question in understanding how abnormal NRG1–

ErbB4 signaling alters brain functions is whether it also involvesthe E-I imbalance in adulthood. A related question is whetherSZ-associated endophenotypes caused by ErbB4 deficiency inearly development could be diminished by restoring ErbB4 ex-pression in adult animals. These questions require temporalcontrol of ErbB4 mutation and expression. In this paper, wedeveloped genetic approaches; in inducible knockout (iKO)

Significance

NRG1–ErbB4 signaling is implicated in GABAergic circuit as-sembly during development and GABAergic transmission atadulthood. However, it is unclear whether phenotypes in theadult stage in ErbB4 mutant mice result from abnormal neuraldevelopment. By using two strains of mice with temporalcontrol of ErbB4 deletion and expression, we demonstrate thatErbB4 deletion in adult mice impaired behavior and GABA re-lease, whereas deficits due to ErbB4 null mutation during de-velopment were alleviated by restoring ErbB4 expression atthe adult stage. Together, our results indicate that NRG1–ErbB4signaling at adulthood is critical to GABAergic transmission andbehavior and suggest that restoring NRG1–ErbB4 signaling atthe postdevelopmental stage might benefit relevant braindisorders.

Author contributions: H.W., F.L., W. Chen, B.-M.L., W.-C.X., and L.M. designed research; H.W.,F.L., W. Chen, X.S., W. Cui, Z.D., K.Z., H.Z., H.L., G.X., E.F., and B.-X.P. performed research; H.W.,F.L., and W. Chen analyzed data; and H.W., F.L., W. Chen, and L.M. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Published under the PNAS license.1H.W., F.L., and W. Chen contributed equally to this work.2To whom correspondence should be addressed. Email: lin.mei@case.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1811287115/-/DCSupplemental.

Published online November 29, 2018.

www.pnas.org/cgi/doi/10.1073/pnas.1811287115 PNAS | December 18, 2018 | vol. 115 | no. 51 | 13105–13110

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mice, ErbB4 expression is normal until tamoxifen (Tam) treat-ment, whereas in recovery knockout (rKO) mice, ErbB4 is ab-sent during development but can be restored in adulthood uponTam treatment. We studied behaviors and characterizedGABAergic transmission of Tam-treated iKO and rKO mice.ErbB4 deletion in adult mice was sufficient to cause behavioraland synaptic deficits; on the other hand, behavioral and synapticdeficits observed in ErbB4 null mice could be diminished byrestoring ErbB4 expression at the adult stage. These resultsprovide compelling evidence that ErbB4 is critical for synaptictransmission and plasticity after development and suggest thatrestoring ErbB4 signaling could be beneficial to relevant SZ.

ResultsBehavioral Deficits in Mice Lacking ErbB4 in Adulthood. To deleteErbB4 at the adult stage, we generated iKO mice by crossingErbB4f/f mice with CAG::Cre-ER mice (Fig. 1A). CAG::Cre-ERmice express a fusion protein consisting of Cre recombinase and amodified ligand-binding domain of the estrogen receptor (ER) un-der the control of the ubiquitous promoter CAG (CMV enhancer,chicken β-actin promoter, rabbit β-globin polyA). In ErbB4f/f mice,exon 2 is floxed, and exon 2 deletion generates a frame shift todisrupt ErbB4 expression. In the resulting CAG::Cre-ER;ErbB4f/f

(iKO) mice, expression of ErbB4 continues to be controlled by thepromoter of the endogenous ErbB4 gene. iKO mice were injectedwith Tam, whose metabolite binds to ER and activates the Cre.Therefore, ErbB4 is expressed in iKO mice until Tam injection. Weinjected iKO mice with Tam or vehicle (referred to as iKO+Tamand iKO+Veh mice) at 8 wk of age and analyzed ErbB4 expression6 wk after (Fig. 1B). As shown in Fig. 1 C and D, ErbB4 expressionin the cortex and hippocampus was abolished in iKO+Tam micecompared with samples from iKO+Veh mice that were similar towild-type mice. Thus, ErbB4 was expressed at a normal level in iKOmice during development but was ablated upon Tam induction.Next, we determined whether adult ErbB4 deletion alters mousebehavior and focused on the paradigms where ErbB4 null mice werefound to be deficient (Fig. 1 E–M). As shown in Fig. 1 E–M, iKO+Veh mice behaved similarly to WTmice in all paradigms, suggestingno apparent effect of the Cre transgene. In contrast, compared withiKO+Veh mice, iKO+Tam mice were hyperactive in an open-field

test as travel distance was increased (Fig. 1 E–G). They were alsoimpaired in prepulse inhibition (PPI) (Fig. 1 H and I) and socialinteraction (Fig. 1 J and K) as PPI ratio was lower while time spentwith stranger mice was reduced. In addition, freezing time wasreduced in iKO+Tam mice in contextual fear conditioning com-pared with iKO+Veh mice (Fig. 1 L and M). Notice that thefreezing times among groups during training were similar (Fig.1M), suggesting a normal ability to sense or escape from footshock. These results indicate that ErbB4 in adult mice is necessaryfor proper behavior. Interestingly, impairment in iKO+Tam micein open-field (Fig. 1 E–G) and PPI tests (Fig. 1 H and I) was lesssevere than in ErbB4 null mice, but that in social interaction (Fig.1 J and K) and contextual memory (Fig. 1 L and M) was similarbetween the two genotypes. This suggests that the ErbB4 nullmutation was more damaging than adult deletion and ErbB4 inadults is more critical for selective behavioral paradigms.

Decreased Inhibitory Transmission in iKO+Tam Mice. Morphologicalstudies of iKO+Tam mice indicate that adult ErbB4 deletion hadlittle effect on global structures and numbers of interneuronspyramidal neurons of the cortex and hippocampus (SI Appendix,Fig. S1). ErbB4 mutation in excitatory neurons has no effect ondendrites of pyramidal neurons (47). In agreement, iKO+Tammice displayed a similar dendrite length and complexity of CA1pyramidal neurons of the hippocampus (SI Appendix, Fig. S2).ErbB4 null and interneuron-specific mutation caused deficits inspines (3, 47, 55, 56) and excitatory synapses onto interneurons(3, 4, 8). To determine whether similar changes may result frompostdevelopmental ErbB4 deletion, we first crossed iKO micewith Thy1-GFP mice to label spines. Quantification of GFP-labeled spines in the hippocampus CA1 region showed spinedensity and morphology in iKO+Tam mice similar to those iniKO+Veh mice and ErbB4f/f mice (SI Appendix, Fig. S3 A–E). Inagreement, there was no change in miniature excitatory post-synaptic current (mEPSC) frequency or amplitude of CA1 py-ramidal neurons (SI Appendix, Fig. S3 F–I). These resultsdemonstrate that adult ErbB4 has little effect on excitatorysynapses between pyramidal neurons or their function. ErbB4 ispresent at postsynaptic sites of excitatory synapses onto in-hibitory neurons and is implicated in their formation (3, 4, 8).

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13106 | www.pnas.org/cgi/doi/10.1073/pnas.1811287115 Wang et al.

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We investigated whether these synapses are altered by adultErbB4 deletion by using Vglut1 and PV antibodies to label, re-spectively, presynaptic excitatory terminals and postsynapticdendrites of PV+ interneurons, many of which are ErbB4+ (4,13, 15, 16). As shown in SI Appendix, Fig. S3 J and K, thenumbers of Vglut1+ puncta onto PV+ dendrites were similaramong different genotypes, regardless of Tam treatment. Inagreement, there was no difference in mEPSC frequency oramplitude of interneurons in the stratum oriens and stratumpyramidale of the hippocampus CA1 region (SI Appendix, Fig. S3L–O). These results indicate no detectable effect of adult ErbB4deletion on excitatory synapses onto interneurons.Studies of ErbB4 null or PV-specific mutation demonstrated

that ErbB4 is necessary for GABAergic transmission in thehippocampus and cortex (3, 4, 17–24, 55). We next determinedwhether this function requires ErbB4 in adulthood by recordingIPSCs (inhibitory postsynaptic currents) in CA1 pyramidal neu-rons (Fig. 2A). As shown in Fig. 2B, evoked IPSC (eIPSC) am-plitude was reduced in iKO+Tam slices, indicating a necessaryrole of adult ErbB4 in maintaining GABA transmission in thehippocampus. This reduction may be caused by a reduction inGABA release and/or GABA receptor density on postsynapticmembrane. To address this question, we compared miniatureIPSC (mIPSC) frequency and amplitude between iKO+Tam andiKO+Veh mice. There was no change in mIPSC amplitude,suggesting GABA receptor density was not compromised (Fig.2D). However, mIPSC frequency was reduced (Fig. 2E), whichmay suggest fewer numbers of inhibitory synapses or diminishedrelease probability. Next, we quantified the numbers of PV-labeled perisomatic inhibitory synapses onto CA1 pyramidalneurons, which were similar among different genotypes, re-gardless of Tam treatment (Fig. 2 F and G), suggesting that re-duced mIPSC frequency may be due to a problem with releaseprobability. To test this hypothesis, we characterized the paired-pulse ratios (PPRs) by recording eIPSCs in response to tandemstimuli with different intervals. The PPRs were increased iniKO+Tam mice compared with ErbB4f/f and iKO+Veh mice(Fig. 2 H and I), indicating compromised GABA releaseprobability. Besides perisomatic synapses, ErbB4+ neuronsalso form inhibitory synapses onto axon initial segments (AISs) ofpyramidal neurons (3, 4, 16). Adult deletion seemed to have noeffect on the number of these synapses (Fig. 2 J–L). Taken to-gether, these observations suggest that ErbB4 in adult animals isnecessary for GABA activity by maintaining release probability.

Diminished GABA Transmission Deficits by Restoring ErbB4 Expressionin Adult Animals. Next, we determined whether ErbB4 in adult-hood is sufficient for GABA transmission by restoring ErbB4expression in ErbB4 null mice. We generated rKO mice byinserting a loxP-NeotpA-loxP cassette into the first intron (be-tween exons 1 and 2) of the ErbB4 gene to first produce Stop-ErbB4 mice (Fig. 3A). The cassette contained the anti-neomycingene (for embryonic stem cell selection) and a poly-A transcrip-tion stop signal. Homozygous Stop-ErbB4 mice did not expressErbB4 in any tissues or cells and died prematurely due to cardiacdeficits, like ErbB4 null mice. To prevent embryonic lethality,hemizygous Stop-ErbB4 mice were crossed with α-MHC::ErbB4mice, which express ErbB4 specifically in developing heart cells.Finally, Stop-ErbB4;α-MHC::ErbB4 mice were crossed with theaforementioned CAG::Cre-ER mice to generate Stop-ErbB4;α-MHC::ErbB4;CAG::Cre-ER (rKO) mice (Fig. 3B). Therefore,rKO mice do not express ErbB4 in the brain or any other tissueexcept the heart (Fig. 3C). After Tam treatment (Fig. 3 A and B)to release the loxP-NeotpA-loxP cassette, rKO miceexpressed ErbB4 in the brain (Fig. 3 D and E). Notice that al-though ErbB4 recovery is mediated by CAG-CreER, the pro-moter activity of the endogenous ErbB4 gene controls the cells inwhich ErbB4 is expressed. As shown in Fig. 3E, the ErbB4 level inthe brain of rKO mice was restored by Tam treatment to a levelthat was comparable to that of control mice (CAG::Cre-ERmice).ErbB4 null mutation reduces the number of PV+ interneurons

in the cortex (5, 7) and hippocampus (54). In agreement, vehicle-treated rKO mice (that did not express ErbB4, Fig. 3E) displayedfewer PV+ neurons in the cortex (SI Appendix, Fig. S4 A–C) andhippocampus (SI Appendix, Fig. S4 D and E). The reducednumber of PV+ interneurons remained unchanged after Tamtreatment (SI Appendix, Fig. S4 B–E), indicating that interneu-ron migration deficit was not rescued by restoring ErbB4 ex-pression in adult animals. The result is not unexpected becauseinterneuron migration was completed before Tam treatment.GABA transmission in the cortex and hippocampus is reduced

in ErbB4 mutant mice (3, 4, 17–24, 55). We next determinedwhether GABA release deficits due to ErbB4 mutation could berescued by restoring ErbB4 expression in adult animals. Asshown in Fig. 4B, eIPSC amplitude was reduced in hippocampalslices of rKO+Veh mice, in agreement with previous reports (18,19, 21, 25). Remarkably, the reduction was mitigated in rKO+Tam slices (Fig. 4B), indicating that restoring ErbB4 is able toenhance GABAergic transmission. mIPSC frequency was also

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Fig. 2. Decreased inhibitory transmission in Tam-treated iKO mice. (A) Recording diagram. (B) Decreased eIPSC amplitude in iKO+Tam mice. n = 7/8 neuronsfrom three mice. (C) Representative traces of mIPSCs. (Scale bar: 20 pA/s.) (D) No change in mIPSC amplitude. (E) Decreased mIPSC frequency. n = 8/9 neuronsfrom three mice. (F) Representative images of PV+ inhibitory synapses onto NeuN+ postsynaptic somata. (Scale bar: Upper, 5 μm; Lower, 2 μm.) (G) Quan-tification data of F. n = 40/42/42 somata of six mice. (H) Representative IPSC traces induced by paired-pulse stimulation with a 100-ms interval. (Scale bar:250 pA/50 ms.) (I) Increased paired-pulse ratios in iKO+Tam mice. n = 10 neurons from four mice. (J) Diagram of inhibitory synapses onto axon initial segments.(K) Representative images of GAD67+ inhibitory synapses onto AnkG+ axon initial segments. AnkG, Ankyrin G. (Scale bar: 5 μm.) (L) Quantification data of K.n = 42/40/43 neurons of six mice. *P < 0.05, **P < 0.01, compared with ErbB4f/f mice; $P < 0.05, $$P < 0.01, compared with iKO+Veh mice.

Wang et al. PNAS | December 18, 2018 | vol. 115 | no. 51 | 13107

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reduced in rKO+Veh slices (Fig. 4 C–E), in agreement withprevious studies (3, 4, 21, 23). Remarkably, after Tam treatment,the reduction was diminished (i.e., in rKO+Tam slices) (Fig. 4C–E), indicating a rescue effect by adult ErbB4 expression. Todetermine whether this effect resulted from increased numbersof inhibitory synapses, we quantified the number of perisomaticinhibitory synapses onto CA1 pyramidal neurons. As shown inFig. 4 F and G, there was no difference between Tam- and Veh-treated rKO mice, suggesting that the rescue effect on mIPSCfrequency may be mediated by improved release probability. Thisnotion was supported by recovered PPRs (Fig. 4 H and I). Aswith perisomatic synapses, adult recovery of ErbB4 had littleeffect on the number of inhibitory synapses onto AIS of pyra-midal neurons, which remained low in Tam-treated rKO mice(Fig. 4 J–L). mIPSC amplitude was not changed regardless ofgenotypes and treatment (Fig. 4E). Taken together, these ob-servations suggest that restoring ErbB4 in adult animals is able torescue functional deficits of GABA transmission without in-creasing the number of interneurons or inhibitory synapses.

No Effect on Excitatory Synaptic Deficits by Adulthood ErbB4Expression. As stated above, ErbB4 mutation has no effect ondendrites of pyramidal neurons in vivo (47); in agreement, rKO+Veh mice showed a similar dendrite number and complexity (SIAppendix, Fig. S5). To determine whether ErbB4 expression mayrescue deficits of excitatory synapses, we examined their mor-phological and functional properties in Veh- and Tam-treatedrKO mice. As shown in SI Appendix, Fig. S6 A–E, spines of CA1pyramidal neurons, in particular mushroom-shaped spines, were

reduced in Veh-treated rKO mice (that did not express ErbB4), inagreement with previous reports (3, 47, 55, 56), suggesting thatErbB4 is required for spine development. However, the spinedeficits were not diminished by ErbB4 recovery (i.e., in rKO+Tammice). mEPSC frequency of CA1 pyramidal neurons was reducedin rKO+Veh mice compared with control (SI Appendix, Fig. S6 F–I), in agreement with reduced spine number. There was no dif-ference between mEPSC frequencies of Veh- and Tam-treatedrKO mice (SI Appendix, Fig. S6 F–I), indicating that adult ErbB4expression was unable to rescue deficits of excitatory synapses ontopyramidal neurons. The number of excitatory synapses onto in-terneurons is reduced after early ErbB4 mutation (3, 4, 8), asobserved in rKO+Veh mice (SI Appendix, Fig. S6 J and K). Thiswas associated with a reduction in mEPSC frequency of inter-neurons (SI Appendix, Fig. S6 L–O). Both of these deficits (re-duced number of excitatory synapses and mEPSC frequency)remained in rKO+Tam mice (SI Appendix, Fig. S6 J–O), indicatingthat they could not be rescued by adult ErbB4 expression.

Mitigated Behavioral Deficits by Restoring ErbB4 Expression in AdultMice. To determine whether adult ErbB4 expression mitigatesbehavioral deficits caused by ErbB4 null mutation during de-velopment, we characterized behaviors of Veh- and Tam-treatedrKO mice. Compared with control mice, rKO+Veh mice werehyperactive in an open field as travel distance was increased (Fig.5 A–C). They were also impaired in PPI (Fig. 5 D and E) andcontextual fear conditioning (Fig. 5 F and G). Remarkably, thesebehavioral deficits were ameliorated by Tam treatment in rKOmice. Compared with Veh-treated rKO mice, Tam-treated rKO

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Fig. 4. Diminished GABA transmission deficits in Tam-treated rKO mice. (A) Recording diagram. (B) Diminished reduction of eIPSC amplitude in rKO+Tam mice.n = 7/8 neurons from three mice. (C) Representative traces of mIPSCs. (Scale bar: 20 pA/s.) (D) Diminished reduction of mIPSC frequency in rKO+Tam mice. (E) Nodifference in mIPSC amplitude among groups. n = 8/9 neurons from three mice. (F) Representative images of PV+ inhibitory synapses onto NeuN+ postsynapticsomata. (Scale bar:Upper, 5 μm; Lower, 2 μm.) (G) No effect of Tam treatment on decreased number of inhibitory synapses. n = 45/45/47 somata of seven mice. (H)Representative IPSC traces induced by paired-pulse stimulation with a 100-ms interval. (Scale bar: 250 pA/50 ms.) (I) Recovered paired-pulse ratios in Tam-treatedrKO mice. n = 9/10 neurons of four mice. (J) Diagram of inhibitory synapses onto axon initial segments. (K) Representative images of GAD67+ inhibitory synapsesonto AnkG+ postsynaptic axon initial segments. AnkG, ankyrin G. (Scale bar: 5 μm.) (L) No effect of Tam treatment on decreased number of inhibitory synapses.n = 45/44/47 neurons of seven mice. *P < 0.05, **P < 0.01, compared with control, $P < 0.05, $$P < 0.01, compared with rKO+Veh mice.

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mice were less hyperactive in open field (Fig. 5 A–C), displayedbetter PPI (Fig. 5 D and E), and showed increased freezing timein contextual fear conditioning (Fig. 5 F and G), indicating im-proved behavioral scores by restoring ErbB4 in adult mice. No-tice that the recovery from behavioral deficits was incomplete;that is, Tam-treated rKO mice remained deficient in these par-adigms compared with control mice. These results suggest thatrestoring ErbB4 expression in adulthood mitigates but does noteliminate behavioral deficits caused by ErbB4 loss of functionduring development.

DiscussionThis study determined the role of ErbB4 in the adult brain byutilizing mouse lines that enable temporal control of ErbB4deletion or expression. We showed that adult deletion of ErbB4caused behavioral deficits and reduced IPSCs in hippocam-pal slices, indicating compromised GABAergic transmission.However, it had no effect on numbers of interneurons, inhibitorysynapses onto excitatory neurons, and excitatory synapses in thehippocampus. These results suggest that ErbB4 is critical toGABAergic transmission in adult mice. On the other hand, rKOmice did not express ErbB4 during development and displayedbehavioral deficits similar to those of ErbB4 null mutant mice,including hyperactivity in an open field and impaired PPI andcontextual fear memory. Remarkably, these deficits were di-minished after Tam treatment, indicating that deficits caused byErbB4 deletion during development could be mitigated by re-storing ErbB4 expression at the adult stage. Notice that numbersof PV+ interneurons and inhibitory and excitatory synapsesremained depressed after Tam treatment, suggesting that re-storing ErbB4 in adult animals was unable to diminish structuraldeficits caused by ErbB4 loss of function during development.Together, these observations support a working hypothesis thatrestoring ErbB4 at adulthood could improve GABAergic trans-mission even on compromised circuits and thus diminish behav-ioral deficits that are caused by developmental ErbB4 mutation.ErbB4 is expressed in developing and mature interneurons (4,

13–17) and in mature interneurons, is present in somata, den-drites, and, arguably, axons. ErbB4 is necessary for interneuronmigration from ganglion eminences to the cerebral cortex andforming synapses onto and from pyramidal neurons (25, 26, 57).In the adult cortex and hippocampus, ErbB4 is almost exclusivelyexpressed in GAD67+ interneurons, not in pyramidal neurons(4, 13–16), and has been implicated in maintaining GABAergictransmission and synaptic plasticity (3, 4, 17–24, 55). ErbB4 nullmutant mice displayed fewer numbers of PV+ interneurons (5, 7,54) and compromised GABA release (17, 19, 20, 23). Adultablation of ErbB4 impaired GABAergic transmission, in furthersupport of the hypothesis that GABAergic transmission requiresErbB4. However, the number of PV+ neurons was similar iniKO and control mice, suggesting that adult ErbB4 is not re-quired for the migration and survival of PV+ interneurons withinthe experimental period. Similarly, restoring ErbB4 expressionin adults improved GABAergic transmission compared with thatin ErbB4 null mice but was unable to increase the number of PV+

interneurons (because interneuron migration had already finished).Molecular mechanisms by which ErbB4 regulates interneuron migra-tion, synapse formation onto pyramidal neurons and interneurons, andGABA releases are complex. For example, maintaining GABArelease requires kinase activity, whereas interneuron migration andsynapse formation could also be mediated by kinase-independent, cell-adhesion-dependent mechanisms (6, 58). Future studies are warrantedto determine which mechanism is involved in mitigating deficitscaused by ErbB4 null mutation. Evidently, ErbB4 level or promoteractivity is dominantly expressed in interneurons (4, 13–17). However,ErbB4 knockdown was shown to reduce spines in vitro (56), andpyramidal neuron-specific deletion of ErbB4 could reduce thenumber of spines of pyramidal neurons in vivo (47, 59). These resultssuggest a role of ErbB4 in pyramidal neurons. Our data are unableto determine whether phenotypes were due to loss of ErbB4 in in-terneurons and/or pyramidal neurons. For example, loss of spinescould be due to the loss of ErbB4 in pyramidal neurons or a com-pensatory mechanism due to ErbB4 loss in interneurons (3, 55, 60).Earlier studies suggested NRG1–ErbB4 signaling is involved

in controlling E-I balance by maintaining GABAergic trans-mission. First, NRG1 increases mIPSCs and eIPSCs in corticaland hippocampal slices (17, 18, 20, 24). This is likely due to in-creased GABA release probability (17, 23, 24). Second, this effectrequires ErbB4 because it is diminished by pharmacological in-hibition of the kinase activity of ErbB4 or genetic deletion of theErbB4 gene in null or interneuron-specific mutant mice (17, 18,21, 24). Third, treating brain slices with an NRG1-neutralizingpeptide decreases mIPSCs and eIPSCs (17, 18, 20, 21, 23, 24,61). Fourth, NRG1 neutralization and/or ErbB4 mutationimpair behaviors in various paradigms involving prefrontal cor-tex, the hippocampus, and the amygdala (3, 15, 18–24), whereErbB4 is enriched. Results from iKO and rKO mice add to thenotion that ErbB4 is critical to E-I balance in the adult brain.In summary, our study demonstrates a role of ErbB4 signaling at

the adult stage in maintaining normal GABAergic transmission, E-Ibalance, and behaviors. The results suggest that abnormal ErbB4function in adult animals could contribute to the pathophysiologicalmechanism of relevant disorders. A mouse model mimicking highNRG1 levels in patients (32, 62) displayed SZ-related deficits (46).Such deficits could be rescued by reducing the NRG1 level at theadult stage (46). Together, these observations suggest that relevantpatients could benefit from therapeutic approaches of optimizingNRG1–ErbB4 signaling.

Materials and MethodsAnimals, electrophysiology, immunofluorescence, Western blot, Golgistaining, analysis of spine morphology, behavioral tests, and statisticalanalyses are described in SI Appendix. Experimental procedures were ap-proved by the Institutional Animal Care and Use Committees of AugustaUniversity and Case Western Reserve University.

ACKNOWLEDGMENTS. We thank Dr. Cary Lai for ErbB4 antibodies and Dr.Martin Gassmann for MHC-ErbB4 transgenic mice. This work is supported byNIH Grants MH083317, MH109280, NS082007, and NS090083 (to L.M.) andAG051773 and AG045781 (to W.-C.X.).

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Fig. 5. Mitigated behavioral deficits in Tam-treatedrKO mice. (A) Representative travel traces of mice inan open-field test. (B) Mitigation of increased traveldistance (per 5 min) in rKO+Tam mice. (C) Mitigationof increased total travel distance (within 30 min) inrKO+Tam mice. (D) Diagram of the PPI test. PPI (%) =100 × (a − b)/a. (E ) Mitigation of impaired PPI inrKO+Tam mice. (F ) Diagram of contextual fearmemory test. (G) Mitigation of reduced freezing timein rKO+Tam mice. n = 12 mice; *P < 0.05, **P < 0.01,compared with control; $P < 0.05, $$P < 0.01, com-pared with rKO+Veh mice.

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