DOI: 10.1126/science.1240405, 1120 (2013);341 Science

et al.Michelle W. AntoineDysfunctionA Causative Link Between Inner Ear Defects and Long-Term Striatal

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Acknowledgments: We thank Rose Phillips, Roger Phillips,and J. Thorpe for technical support; M. Ramaswami for theantibody to Ca-P60A; and F. Casares, M. Baylies, I. Galindo,C. Alonso, and laboratory members for manuscript comments.E.M. was supported by Conacyt, F.P. was supported by aDaphne Jackson Fellowship and the UK Medical ResearchCouncil, and J.N. was supported by a Royal SocietyUniversity Fellowship. Otherwise, this work was funded by aWellcome Trust Fellowship (ref 087516) awarded to J.P.C.The GenBank accession number for Drosophila Scl sequences isNR_001662.

Supplementary Materialswww.sciencemag.org/cgi/content/full/341/6150/1116/DC1Materials and MethodsFigs. S1 to S8Tables S1 to S9References (20–34)Movies S1 and S2Supplementary data file S1

5 April 2013; accepted 7 August 201310.1126/science.1238802

A Causative Link Between InnerEar Defects and Long-TermStriatal DysfunctionMichelle W. Antoine,1 Christian A Hübner,2 Joseph C. Arezzo,1 Jean M. Hébert1,3*

There is a high prevalence of behavioral disorders that feature hyperactivity in individuals withsevere inner ear dysfunction. What remains unknown is whether inner ear dysfunction can alter thebrain to promote pathological behavior. Using molecular and behavioral assessments of micethat carry null or tissue-specific mutations of Slc12a2, we found that inner ear dysfunction causesmotor hyperactivity by increasing in the nucleus accumbens the levels of phosphorylated adenosine3′,5′-monophosphate response element–binding protein (pCREB) and phosphorylated extracellularsignal-regulated kinase (pERK), key mediators of neurotransmitter signaling and plasticity. Hyperactivitywas remedied by local administration of the pERK inhibitor SL327. These findings reveal that asensory impairment, such as inner ear dysfunction, can induce specific molecular changes in thebrain that cause maladaptive behaviors, such as hyperactivity, that have been traditionallyconsidered exclusively of cerebral origin.

The inner ear contains the cochlea, devotedto hearing, and the vestibular end organs,dedicated to balance. In 20 to 95% of chil-

dren with severe hearing loss, auditory and ves-tibular dysfunction occur concurrently (1, 2). Insuch cases, there is a high incidence of behavioral

disorders that feature hyperactivity as a core di-agnostic symptom (3–5). Although socioenviron-mental variables have been proposed as riskfactors (6), it is unclear whether sensory impair-ments, such as inner ear defects, can directlyinduce specific changes in the brain that lead to

maladaptive behavior. In nonhuman vertebrates,including rodents and frogs, surgical or pharma-cological lesions to the vestibulo-auditory systemare also linked to long-term changes in locomotoractivity, although, to date, the associations be-tween ear dysfunction and behavior remain un-explained (7–9). Genetic mouse models of innerear dysfunction can exhibit increased levels oflocomotor hyperactivity (10), but because thegene is mutated in the brain, as well as the innerear, the causal neural underpinnings of this be-havior remain unknown.

Slc12a2 (also known as Nkcc1) is a gene thatencodes a sodium-potassium-chloride cotrans-porter broadly expressed in tissues, includingthe inner ear and central nervous system (CNS)(11, 12). The Slc12a2 mutant mice used in thisstudy arose spontaneously in our mouse colonyand exhibit increased levels of motor hyperac-tivity, including locomotion, circling, and headtossing (Fig. 1, A and B; movie S1; and fig. S1A),

1Department of Neuroscience, Albert Einstein College of Med-icine, Bronx, NY 10461, USA. 2Jena University Hospital, Insti-tute of Human Genetics, Jena 07743, Germany. 3Departmentof Genetics, Albert Einstein College of Medicine, Bronx, NY10461, USA.

*Corresponding author. E-mail: jean.hebert@einstein.yu.edu

Fig. 1. Slc12a2K842*/K842* mutants display adopamine receptor–mediated increase in loco-motor activity that cannot be explained by dis-ruption of Slc12a2 in the brain. (A) Traces and(B) quantification of mouse locomotion in an openfield showing that haloperidol alleviates locomotoractivity and circling in Slc12a2K842*/K842* mice with-out affecting grooming [***P < 0.0001; repeatedmeasures analysis of variance (ANOVA) with Bonferronipost hoc comparison]. (C) Germline recombinationof Slc12a2fx/fx mice recapitulates the increased loco-motion of the Slc12a2K842*/K842*mutant (P = 0.0032,unpaired two-tailed test). Mice lacking Slc12a2 in theneocortex and hippocampus (Emx1Cre/+;Slc12a2fx/rec),striatum (Dlx5/6-Cre;Slc12a2 fx/fx), cerebellum(En1Cre/+;Slc12a2fx/fx), and CNS (Nestin-Cre;Slc12a2fx/fx)display normal levels of motor activity (unpairedtwo-tailed test). (D) Germline recombination ofSlc12a2fx/fx also recapitulates the circling behaviorof the Slc12a2K842*/K842* mutants. n = 4 to 11 miceper genotype. All data are means T SEM.

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as with previously characterized Slc12a2mutants(13–16). The spontaneous mutation was identifiedas an A to T mutation in exon 17 that changescodon 842 of Slc12a2 from encoding a lysine(K) to a stop codon (*) (hereafter referred to asSlc12a2K842*), which results in no detectableSLC12A2 protein, as shown by antibodies raisedto either theN or C terminus (fig. S1, B to E). Theinner ear of Slc12a2K842*/K842* mice revealed acollapse of Reissner’s membrane and the mem-branes of the vestibular compartments (fig. S1F).These morphological defects were associated withprofound deafness and balance deficits (figs. S1Gand S2) (15).

An increase in locomotor activity is not read-ily explained by dysfunction of the inner ear butrather points to a disruption of brain functionsthat regulate movement. Consistent with this no-tion, the dopaminergic antagonist haloperidol,which acts in the brain to alleviate increased mo-tor behavior in humans (17), normalized the open-field locomotor behavior of Slc12a2K842*/K842*

mice (Fig. 1, A and B). The same dose of halo-peridol did not decrease the locomotor activity inlittermate controls, which indicated that the doseused was not sedating. Haloperidol did not affectgrooming in either mutants or controls, nor did itameliorate performance of mutants on a rotating

rod (rotarod) test, which requires an intact innerear, and which therefore indicates a specificity inthe behaviors modified by haloperidol (Fig. 1Band fig. S2).

To test whether loss of Slc12a2 expression inthe brain leads to increased locomotor activity,we deleted a floxed Slc12a2 allele (Slc12a2fx)specifically from individual brain areas that con-trol movement using Emx1Cre for the cortex,Dlx5/6-Cre for the striatum, En1Cre for the cer-ebellum, andNestin-Cre for the entire CNS.Withthese lines, recombination occurs before neuro-genesis and expression of Slc12a2 (18–21). West-ern blot analyses confirmed the loss of Slc12a2specifically in the expected tissues (fig. S3, Aand C). Although mice having a germline dele-tion of the Slc12a2fx allele, which lack expressionin all tissues, are behaviorally indistinguishablefrom Slc12a2K842*/K842* mice, mice with dele-tions in the cortex, striatum, cerebellum, or entireCNS are behaviorally normal (Fig. 1, C and D,and fig. S3B), which indicates that loss of Slc12a2from any single or combination of brain areasdoes not cause the behavior. Note that inner earmorphology and Slc12a2 expression were nor-mal in Nestin-Cre;Slc12a2fx/fxmutants (fig. S3C).

To determine whether loss of Slc12a2 ex-pression in the inner ear causes the behavioral

phenotype, the Foxg1Cre line was used to deleteSlc12a2 from the embryonic precursors of theinner ear (Fig. 2A). However, because Foxg1Cre

also targets the telencephalon (neocortex, hip-pocampus, and striatum) (Fig. 2B), we deletedSlc12a2 from the inner ear using a combinationof Pax2-Cre and Tbx1Cre, which together suffi-ciently deleted Slc12a2 to result in the anatomicalear defects indicative of dysfunction (fig. S4). InPax2-Cre;Tbx1Cre-driven mutants, Slc12a2 dele-tion also occurred in the mid-hindbrain area, butnot the telencephalon. Both the Pax2-Cre;Tbx1Cre

and Foxg1Cre-driven mutants recapitulated allthe hyperactive features of Slc12a2K842*/K842*

mice (Fig. 2C, movie S2, and fig. S4). Togetherwith the absence of the behavioral phenotype,when Slc12a2 is deleted from the entire CNS,these results demonstrate that inner ear dysfunc-tion caused the abnormal behavior of these mice.

Because increased locomotor activity and aresponsiveness to haloperidol are indicative ofbrain, rather than ear, dysfunction, we reasonedthat inner ear defects may cause abnormal func-tioning of the striatum, a central brain area thatregulates motor output levels. Striatal levels of26 candidate proteins involved in neurotransmittersignaling were examined byWestern blot (Fig. 3,A and B, and fig. S5). Initially, Slc12a2K842*/K842*

Fig. 2. Increased locomotor activity of Slc12a2 mutants depends oninner ear dysfunction. (A) The inner ear defects of Foxg1Cre/+;Slc12a2fx/fx

mice are identical to those of the Slc12a2K842*/K842* mice: vestibular (vm) andReissner’s (rm) membrane collapse and degeneration of the vestibular endorgans (sacculae, utricles, and cristae). In Foxg1Cre/+;Slc12a2fx/fx mice, SLC12A2immunostaining (brown) is reduced in the endolymph secreting stria vascularis(sv) of the cochlea and in the vestibular end organs. Purple: Nissl counterstain.(B) Western blot analysis shows loss of SLC12A2 in the neocortex (Ctx),hippocampus (Hipp), and striatum (Str) in Foxg1Cre/+;Slc12a2fx/fx mice, butnot in the thalamus (Thal), midbrain (MB), cerebellum (Cb), or hindbrain(HB). (C) Foxg1Cre/+;Slc12a2fx/fx mice recapitulate the abnormal behavior ofSlc12a2K842*/K842* mice (n = 7 to 9 mice per genotype, ***P < 0.0001,unpaired two-tailed test, means T SEM).

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mutants were examined, and proteins showingsignificant differences in expression levels com-pared with controls were then also examinedin striata from Nestin-Cre;Slc12a2fx/fx andFoxg1Cre/+;Slc12a2fx/fx mice to identify the rele-vant changes that correlate with the presence ofthe ear defects and behavioral phenotype. Amongthe proteins examined, we observed significantincreases only in the levels of phosphorylatedextracellular signal-regulated kinase (pERK), akey component of dopamine and glutamate neu-

rotransmission in the striatum, and its commondownstream target, phosphorylated adenosine3′,5′-monophosphate response element–bindingprotein (pCREB-Ser133) (22–24) (Fig. 3, A andB).Although phosphorylated formswere elevated,total ERK and CREBwere unaffected. IncreasedpERKand pCREBwere specific to the striatum andnot observed in other forebrain regions (fig. S5D).Immunohistochemical analysis of Slc12a2K842*/K842*

mutants revealed that the number of pERK-positive (pERK+) cells was up-regulated specif-

ically in the nucleus accumbens, the ventral partof the striatum (Fig. 3C). All pERK+ cells wereNeuN+, which indicated that they were neurons.Of these, most were DARPP32+ medium-sizedspiny neurons (MSNs) and a few were somato-statin+ interneurons (Fig. 3D) but not calretinin+,parvalbumin+, or choline acetyltransferase (ChAT)+interneurons. The proportions of pERK+ cell typesin the mutants were nevertheless similar to those ofcontrols. In addition, pCREB+ cells were pERK+(Fig. 3D).

Fig. 4. The increased striatal pERK levelsinduced by inner ear defects promote in-creased locomotor activity. (A) Traces and(B) quantification of motor activity in an open-field 1 hour after systemic SL327 administra-tion, which ameliorates both locomotion andcircling in the Slc12a2K842*/K842* mutants whilehaving little effect on grooming (n = 6 miceper genotype, *P = 0.015, ***P = 0.0002). (C)Local SL327 administration to the nucleus ac-cumbens of Slc12a2K842*/K842* mice restoredopen-field activity and circling to control lev-els, which were unaffected by treatment, for2 days postinjection, without affecting groom-ing (n = 5 to 7 mice per genotype and pertreatment condition, ***P = 0.0025, **P =0.0082, *P = 0.018; means T SEM; two-wayrepeated measures ANOVA with Bonferronipost hoc comparison).

Fig. 3. Inner ear dysfunction contributes to the up-regulation of pERK1 in striatal neurons. (A) Westernblot analyses reveal increased (arrowheads) pERK1 (upperband of doublet) and its common target pCREB in micewith inner ear defects and increased motor activity(Slc12a2K842*/K842* and Foxg1Cre/+;Slc12a2fx/fx mice) butnot in phenotypically normal mice. (B) Quantification ofWestern analyses (pERK: P = 0.009 for Slc12a2K842*/K842*;P = 0.006 for Foxg1Cre/+;Slc12a2fx/fx; pCREB: P = 0.028 forSlc12a2K842*/K842*; P = 0.033 for Foxg1Cre/+;Slc12a2fx/fx;n = 3 to 6 mice per genotype; unpaired two-tailed t test).(C) Immunohistochemical analysis showed an increasein the number of pERK+ cells in the ventral striatum ofmutants (n = 4 mice per genotype, **P = 0.0066, ***P =0.00046, unpaired two-tailed t test). (D) In both controlsand mutants, pERK+ cells in the ventral striatum areprimarily DARPP32+, whereas in the dorsal striatumthey are somatostatin+ (SST+) interneurons (n = 3 miceper genotype) (means T SEM).

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Robust increases in pERK occur in MSNsof the nucleus accumbens in response to psy-chostimulants and other drugs of abuse and areconsidered critical for enabling their long-lastingbehavioral changes (22, 25, 26). The induction ofsuch behaviors is inhibited by the local or sys-temic application ofMAPKkinase (MEK) or ERKkinase inhibitors, such as SL327 (22, 27–29). Todetermine whether striatal ERK phosphorylationwas necessary for the abnormal increase in loco-motor activity, Slc12a2K842*/K842*micewere givenan intraperitoneal injection or a local injectionof SL327 to the nucleus accumbens. In both setsof experiments, SL327 administration restoredlocomotor activity to normal levels without af-fecting the levels of activity in controls (Fig. 4).Mutant mice treated with local SL327 returnedto their baseline, presurgery locomotor levels ofactivity by day 3, which suggested that the in-jection did not cause permanent damage. SL327administration did not affect grooming, whichsuggested that increased striatal pERK selec-tively elevates locomotor activity levels and notgeneral activity.

This study demonstrates that inner ear dys-function can induce molecular changes in thestriatum that promote increased motor hyper-activity. The neural circuits linking inner ear defectsto abnormal striatal function are likely transmittedby the normal auditory and vestibular input path-ways, primarily via the thalamus and neocortex(30), but this remains to be demonstrated. Ourresults also suggest that a neurobiological cause,rather than simply socioenvironmental factors,contributes to the high incidence of behavioraldisorders associated with inner ear dysfunction in

children and adolescents. Moreover, disruptionof the ERK pathway in the striatum provides apotential target for intervention. Finally, it is in-triguing to ponder whether sensory impairmentsother than those associated with inner ear defectscould also cause or contribute to psychiatric or mo-tor disorders that have traditionally been consideredexclusively of cerebral origin.

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Acknowledgments: We thank K. Khodakhah and P. Calderonfor support with surgical approaches, rotarod, and earlyinputs to the project; G. Fishell for the Nestin-CreER mice inwhich Slc12a2K842* arose; S. Zukin for Western blotting;C. Heinze for Slc12a2fx genotyping; University of California atDavis and NIH NeuroMab for antibodies; and the DevelopmentalStudies Hybridoma Bank for the T4 antibody developed byLytle and Forbush. This work was supported by the TouretteSyndrome Association (J.M.H. and K. Khodakhah), NIHR21NS073761 and R01MH083804 (J.H.), and DeutscheForschungsgemeinschaft HU 800/5-1 (C.A.H.).

Supplementary Materialswww.sciencemag.org/cgi/content/full/341/6150/1120/DC1Materials and MethodsFigs. S1 to S5Movies S1 and S2References (31–40)

13 May 2013; accepted 8 August 201310.1126/science.1240405

Topographic Representationof Numerosity in the HumanParietal CortexB. M. Harvey,1* B. P. Klein,1 N. Petridou,2 S. O. Dumoulin1

Numerosity, the set size of a group of items, is processed by the association cortex, but certainaspects mirror the properties of primary senses. Sensory cortices contain topographic mapsreflecting the structure of sensory organs. Are the cortical representation and processing ofnumerosity organized topographically, even though no sensory organ has a numerical structure?Using high-field functional magnetic resonance imaging (at a field strength of 7 teslas), wedescribed neural populations tuned to small numerosities in the human parietal cortex. They areorganized topographically, forming a numerosity map that is robust to changes in low-levelstimulus features. The cortical surface area devoted to specific numerosities decreases withincreasing numerosity, and the tuning width increases with preferred numerosity. Theseorganizational properties extend topographic principles to the representation of higher-orderabstract features in the association cortex.

Humans and many other animals use nu-merosity to guide behavior and decisions(1–4). Numerosity perception becomes

less precise as the size of numbers increases (4–8)and is particularly effective for small numbers

(9). Animals, infants, and tribes with no numer-ical language perceive numerosity (1, 10–12),although they cannot count or use symbolic rep-resentations of number. Thus, numerosity pro-cessing is an evolutionarily preserved cognitive

function, distinct from counting and humans’unique symbolic and mathematical abilities (12).Because aspects of numerosity processing mirrorprimary sensory perception, it has been referredto as a “number sense” (2, 3).

The primary sensory and motor cortices inthe brain are organized topographically. Is theneural organization for numerosity similarly topo-graphic? The neural representation of numeros-ity resides in higher-order association cortices,including the posterior parietal cortex. Humanfunctionalmagnetic resonance imaging (fMRI) con-sistently identifies this region as particularly re-sponsive to numerosity manipulations (6, 12–14),and in similar regions, macaque neurophysiologydescribes neurons tuned to visual numerosity(4, 5, 7, 15). Both human fMRI and macaque neu-rophysiological response properties are closelylinked to behavioral numerosity performance (4, 6).

We elicited responses to visual patterns withvarying numerosity in study participants, while

1Department of Experimental Psychology, Helmholtz Institute,Utrecht University, Utrecht, 3584 CS, Netherlands. 2Departmentof Radiology, Rudolf Magnus Institute of Neuroscience, Uni-versity Medical Center Utrecht, Utrecht, 3584 CX, Netherlands.

*Corresponding author. E-mail: b.m.harvey@uu.nl

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