Neuregulin 1 Regulates Excitability of Fast-Spiking Neurons Through Kv1.1

© 2011 Nature America, Inc. All rights reserved. X.-M.L. ([email protected]). X.-M.L. China. Zhejiang, Hangzhou, China. Zhejiang, Hangzhou, Medicine, of School University 1 cellular major a are interneurons parvalbumin that suggest studies brain adult in (LTP) activ potentiation neuronal long-term and ity pyramidal of regulation NRG1 for interneu critical parvalbumin is rons in ErbB4 that demonstrate studies recent cells to confined classes of specific parvalbumin interneurons, particularly interneurons are clustered (refs. ErbB4 including receptors, of ErbB kinase tyrosine the by activating activity. their by increasing inhibition of loss the to counteract our efforts has directed and seizures inhibition interneuron–mediated fast-spiking reduced between link causal the epilepsy of pathogenesis the in implicated is interneurons neurons pyramidal of output the regulating of neurons, region target axons onto perisomatic the their project They preferentially system in dominant neocortex. inhibitory parvalbumin, comprising 40–50% of GABAergic interneurons, are the treatments. safe and effective finding for urgent and necessary is seizures of occurrence the underlying pathology the of causes the for a minority. Understanding only suitable is focus leptic treatment anticonvulsant cological appropriate pharma despite seizures to continue have breakthrough individuals of affected 30% About ages. of all population general the of 1% about affects that disorder neurological a disabling is Epilepsy drugs anticonvulsant in epilepsy. signaling contributes to human epilepsy through regulating the excitability of FS-PV ErbB4 interneurons. may be a new target for of ErbB4, but not ErbB2, was in downregulated human tissue. epileptogenic Together, our findings suggest that NRG1–ErbB4 models of epilepsy. Exogenous NRG1 delayed the onset of seizures and decreased their incidence and stage. Moreover, expression specific deletion of ErbB4 in parvalbumin interneurons were more susceptible to and pentylenetetrazole- pilocarpine-induced decreasing the voltage threshold for action potentials through Kv1.1, a potassium voltage-gated channel. Furthermore, mice with the intrinsic excitability of FS-PV This interneurons. effect was mediated by increasing the near-threshold and responsiveness ErbB4 signaling in FS-PV interneurons is involved in epilepsy. We found that NRG1, through its receptor ErbB4, increased interneurons are a major target of NRG1-ErbB4 signaling in adult brain. Thus, we hypothesized that of downregulation NRG1- key Neuregulin 1 (NRG1) receptor, is mainly expressed in this type of and interneurons, recent studies suggest that parvalbumin Dysfunction of (FS-PV) interneurons is parvalbumin-positive fast-spiking, implicated in the pathogenesis of epilepsy. ErbB4, a Xiao-Juan Chen Ke-Xin Li neurons through Kv1.1 and acts in epilepsy Neuregulin 1 regulates excitability of fast-spiking nature nature Received 13 May; accepted 7 November; published online 11 December 2011; Department of Neurobiology, Key Laboratory of Medical Neurobiology of Ministry of Health of China, Zhejiang Province Key Laboratory of Neurobiology, Zhejiang Zhejiang Neurobiology, of Laboratory Key Province Zhejiang China, of Health of Ministry of Neurobiology Medical of Laboratory Key Neurobiology, of Department NRG1 is a member of a family of neurotrophic factors that acts acts that factors neurotrophic of family a of member a is NRG1 protein calcium-binding the expressing interneurons Fast-spiking 9– 5–7). 5–7). In adult brains, 1 3 . NRG1 regulates GABAergic transmission GABAergic regulates NRG1 . NEUR 1 , , Ying-Mei Lu OSCI EN 1 , , Zhong Chen C E

3 Soft Matter Research Center, Zhejiang University, Hangzhou, Zhejiang, China. Correspondence should be addressed to to addressed be should Correspondence China. Zhejiang, Hangzhou, University, Zhejiang Center, Research Matter Soft 8 advance online publication online advance Erbb4 . . Furthermore, ErbB4 expression is largely 1 , , Zheng-Hao Xu mRNA is in enriched regions where 2 . Dysfunction of the fast-spiking fast-spiking the of Dysfunction . 1 , and surgical removal of epi removal the , and surgical 1 , , Jian-Hong Luo 2 1 Department of Neurosurgery, Second Affiliated Hospital, Zhejiang University School of Medicine, Medicine, of School University Zhejiang Hospital, Affiliated Second Neurosurgery, of Department , , Jing Zhang 1 14– 3 . In addition, addition, In . 1 7 . All these these All .

1 3 , , 4 3 . Thus, Thus, . , , Shumin Duan 1 doi:10.1038/nn.300 - - - -

, , Jun-Ming Zhu brane properties and firing patterns was based upon their distinctive distinctive upon their was based patterns and firing brane properties mem their measuring by cells fast-spiking of identification Further characterized well is protein lines in these Expression interneurons. in most parvalbumin (EGFP) fluorescent green enhanced expressing G42 and B13 lines mouse transgenic the from slices cortical acute in recordings clamp current- whole-cell performed first we interneurons, FS-PV of func tion the regulates signaling NRG1-ErbB4 whether investigate To NRG1 increases the excitability of FS-PV interneurons RESULTS drugs. anticonvulsant of class new a of development the for target human epileptogenic tissue. Our findings suggest that ErbB4 may be a to animal models of epilepsy. Expression of ErbB4 was also reduced in deletion of ErbB4 in parvalbumin interneurons were more susceptible the voltage through Kv1.1.threshold mice Furthermore, with specific ability of excit FS-PV interneurons. This was effect the mediated by decreasing increased ErbB4, receptor Weits through brain. NRG1, adult that found in epilepsy and signaling NRG1-ErbB4 regulated down between link a is there whether determined we Furthermore, mechanisms. underlying the and interneurons FS-PV of excitability is and epilepsy. in NRG1-ErbB4 involved of inhibition dysfunction that hypothesize to us led interneuron–mediated seizures FS-PV reduced between link causal the and brain, adult in signaling NRG1-ErbB4 of target Here we investigated whether NRG1-ErbB4 directly regulates the the regulates directly NRG1-ErbB4 whether investigated we Here 1 , 3 & Li Xiao-Ming 6 2 , , Jian-Ming Zhang 2 , 18– 2 0 as effectively targeting fast-spiking cells. cells. fast-spiking targeting effectively as 1 , 3 2 , , Shu-Xia Cao t r a

C I 1 ,

e l

s - - - - © 2011 Nature America, Inc. All rights reserved. current injection, there was a reduction of action potential numbers of numbers potential ms action of reduction a was 500 there injection, Within current neurons. pyramidal 2/3 layer of excitability the ( adaptive firing and dendrites apical prominent their by indentified were neurons Pyramidal neurons. pyramidal 2/3 layer from recorded we affects the intrinsic properties of FS-PV interneurons. ( remained ISI on ecto-ErbB4 antagonistpicrotoxin (100 20 (DNQX; dinitroquinoxaline-2,3-dione ( acid with the NMDA receptor antagonist discriminate between these two possibilities, or we cells blocked synaptic fast-spiking inputs onto (ii) input direct modulation inhibitory of the or intrinsic excitability excitatory of the fast-spiking (i) cells. Toby caused excitabilitybe could cell onoffast-spiking NRG1 effect the so interneurons. FS-PV neocortical in results with consistent ( treatment ecto-ErbB4 after increased but treatment NRG1 recordings on FS-PV cells in mouse hippocampus. ISI after decreased same the performed we regions, brain other in interneurons spiking ( ecto-ErbB4 or NRG1 of inactivation heat by abolished was it because specific seemed effect ( frequency firing the or by decreasing ISI (142.8 by prolonging interneurons fast-spiking of excitability the dampened substantially (2 Ecto-ErbB4 peptide. NRG1-neutralizing a ecto-ErbB4, we then interfered with the of activity NRG1 endogenous by applying interneurons, fast-spiking of excitability the regulates NRG1 enous glia and neurons by released and 1a Fig. Fig. (82.9 (ISI) interval interspike average recordings showed that bath application of 10 nM NRG1 decreased clamp Patch the injections. current 500-ms-step of series a by measured characteristics ecto-ErbB4). + (drugs ecto-ErbB4 (50 drugs of presence *** 2 or NRG1 nM 10 of application bath after and before interneurons FS-PV hippocampal *** ecto-ErbB4. denatured ecto-ErbB4, 10 nM denatured NRG1 or 2 2 NRG1, nM 10 of application bath after and before interneurons FS-PV 2/3 ( NRG1. of application bath after acceleration frequency spike illustrating ISI), the of inverse the as (IFF, quantified frequency firing instantaneous ( NRG1 of application bath after and before ISI of ( gray). dark (bottom, ecto-ErbB4 2 or gray) light (top, NRG1 nM 10 of application bath after and (black) before 2/3 layers cortical in interneurons FS-PV ( interneurons. FS-PV hippocampal and cortical of excitability the increase NRG1 1 Figure t r a 14.83 from a

) µ To determine whether NRG1 specifically acts on fast-spiking cells, cells, on acts fast-spiking ToNRG1 specifically whether determine Some studies have shown that NRG1 regulates synaptic currents fast- of excitability the regulates also NRG1 whether Toexamine

g ml g Representative action potentials of two two of potentials action Representative P P 1 < 0.001. ( 0.001. < ( 0.001. < ) and increased the firing frequency ( dl ). It has been reported that NRG1 is endogenously expressed expressed endogenously is NRG1 that reported been Ithas ). −1 Supplementary Table 1 Table Supplementary C I

-AP5; 50 50 -AP5; Both exogenous and endogenous endogenous and exogenous Both ecto-ErbB4. ecto-ErbB4. b e ) or ecto-ErbB4 ( ecto-ErbB4 or ) ) Normalized ISI of cortical layer layer cortical of ISI Normalized ) e l ± 1.42 to 11.83 11.83 to 1.42 1 g f 8 ) Normalized ISI of of ISI Normalized ) ) Normalized ISI in the the in ISI Normalized ) ( s Supplementary TableSupplementary 1 µ M), AMPA and kainate receptor antagonist 6,7- antagonist receptor kainate and AMPA M), µ n n M M = 6 or 7; * 7; or 6 = = 7 or 8; ** 8; or 7 = ± DL Fig. 1 Fig. 7.2% of the control; of control; the 7.2% c µ -AP5, 20 20 -AP5, ). ( ). M) and found that the effect of NRG1 orofNRG1 effect andfound M) thatthe Fig. 1 Fig. d ± e b ) Plot of of Plot ) ). ). Treatment with NRG1 decreased decreased NRG1 with Treatment ). 1.30 after treatment with NRG1 NRG1 with treatment after 1.30 n , µ 13 c P = 7; ** 7; = g ml g µ ) Plots Plots ) , < 0.05, 0.05, < P g 2 g ml g dl 1 < 0.01, 0.01, < ), suggesting that NRG1 directly directly NRG1 that suggesting ), µ . To determine whether endog whether To. determine µ ± M DNQX and 100 100 and DNQX M −1 Supplementary Fig. 1b Fig. Supplementary

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700 120 500 800 NRG1 Control 600 ished in parvalbumin interneurons, but not in parvalbumin-negative butinterneurons, not parvalbumin-negative in parvalbumin in ished cre;Erbb4 ( parvalbumin for positive not were that neurons in and interneurons In control littermates, ErbB4 was detectable in almost all parvalbumin we stained cortical sections with antibodies to parvalbumin and ErbB4. the extent determine of interneurons, ErbB4 in deletion parvalbumin by crossing interneurons parvalbumin in specifically expression ErbB4 ablate to cells. the fast-spiking of excitability maintain intrinsic to necessary be may activity NRG1 endogenous that (137 pressed the excitability of interneurons fast-spiking by prolonging ISI sup substantially alone AG1478 NRG1. by excitability interneuron fast-spiking of regulation the in ErbB4 of role a suggests result This signaling ( ISI the in decrease ErbB4 NRG1-induced the prevented AG1478 prevents specifically which AG1478, antagonist ErbB4 the with by treatment approach cological FS-PV interneurons through ErbB4 receptors, adult we first used a pharma of interneurons parvalbumin brain in expressed highly is ErbB4 excitability FS-PV on effect NRG1’s mediates ErbB4 interneurons. FS-PV of excitability intrinsic the regulates directly NRG1 that indicated findings our together, Taken mission. trans GABAergic activity-dependent through neurons of pyramidal excitability the regulates indirectly NRG1 that indicate results These ( (mIPSCs) IPSCs miniature on effect no ( IPSCs spontaneous of frequency and amplitude the both increased NRG1 of application Bath neurons. pyramidal 2/3 layer from recorded were (IPSCs) rents cur postsynaptic inhibitory neurons, pyramidal onto transmission Tothe GABAergic NRG1 regulates whether transmission. determine by GABAergic is mediated neurons of on NRG1 pyramidal effect the GABA study ( i. 2 Fig. Supplementary Fig. 2a Fig. Supplementary 700 160 Then, Then, to confirm this effect, we used the Cre- 800 ± 8 1 12.7% of control, , 4 A 9 c . . However, by was abolished picrotoxin (100 effect this . To determine whether NRG1 regulates the excitability of of excitability the regulates NRG1 whether determine To . receptor antagonist ( , n gemn wt peiu studies previous with agreement in ), e −/− advance online publication online advance Normalized ISI 0.5 1.0 1.5 2.0 10 ms 20 mV Erbb4 sections, by was sections, contrast, abol immunoreactivity ErbB4 Control 0

DenaturedDenatured Ecto-ErbB4ecto-ErbB NRG1 NRG1 b loxP/loxP ISI (ms) ** 10 12 14 Cortex 4 6 8 *** 400 Supplementary Fig. 2d Fig. Supplementary P mice , < 0.01; 500 b Current (pA)

). This is in agreement with a previous previous a with agreement in is This ). 4 Supplementary Supplementary Fig. 2c 2 600 7 with f Fig. Fig. 2 Normalized ISI 700 1.0 1.5 2.0 0.5 NRG1 Control 0

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Pvalb- b M), M), a EN ). ). To ** 800 a C ). ). ). ). E - - - - - © 2011 Nature America, Inc. All rights reserved. shape shape parameters, (−21.8 including afterhyperpolarization spike other in change significant no found we However, treatment. (−40.9 after NRG1 treatment, whereas they shifted to more depolarized levels larized levels (−40.2 age threshold for action potential generation shifted to more hyperpo rate of change d ( effect opposite the ecto- had ErbB whereas initiation, of slope the increased NRG1 parameters. FS-PV interneuron of excitability, regulation NRG1 we furtherthe analyzed underlying the action mechanisms potential the Toinvestigate potentials action of threshold voltage the decreases NRG1 tion of FS-PV interneuronal excitability by NRG1. interneurons. These results indicate that ErbB4 is critical for the regula ment of endogenous NRG1 in regulating the excitability of fast-spiking ( ErbB4 of inhibition 11.7 (8.9 ISI prolonged a by shown as interneurons, spiking cre;Erbb4 in applied NRG1 by ISI 7.4 to ms 0.7 abbreviation of ISI by NRG1 treatment in the to contrast In mice. knockout ErbB4 parvalbumin-specific from slices in NRG1 of effect the tested we Then labeled. fluorescently are G42, in which the parvalbumin-positive subset of GABAergic neurons with from FS-PV interneurons, in interneurons.parvalbumin To assist electrophysiological recording neurons ( treatment. (−) ACSF control or (+) NRG1 from slices in ISI of acceleration NRG1-induced of histogram ( treatment. gray) (light NRG1 nM 10 after and (black) before potentials action of Superimposition mice. from slices in 2/3 layers cortical in interneurons FS-PV bar, 10 Scale neurons. parvalbumin-negative arrowheads, neurons; parvalbumin Arrows, nuclei. indicate to DAPI with stained also were Slices antibody. parvalbumin and anti-ErbB4 with from slices Cortical neurons. parvalbumin in ErbB4 of ablation The allele. The pairs. primers n ( 2 Figure nature nature regulation of action potential generation of FS-PV interneurons. of FS-PV generation potential of action regulation from those than mV) 1.1 depolarized voltage threshold for action potential generation (−34.4 from slices in interneurons slices from in interneurons FS-PV in potentials action of generation the for old P mV; 1.64 −22.1 to a P a = 6, ** = 6, = 0.25) after NRG1 treatment. We also checked the voltage thresh voltage Wethe treatment. checked NRG1 also after = 0.25) ) Histogram of ISI of action potentials in slices with 5 with slices in potentials action of ISI of ) Histogram < 0.01), providing providing 0.01), < Normalized ISI 0.5 1.0 1.5 2.0 0

± Control G42;Erbb4 0.7 ms; ms; 0.7 ± AG1478

1.09 mV to −35.8 P NEUR Ablation of ErbB4 in parvalbumin neurons prevents NRG1 from increasing excitability of FS-PV interneurons. interneurons. FS-PV of excitability increasing from NRG1 prevents neurons parvalbumin in ErbB4 of Ablation ± Fig. 2 −/− < 0.01. ( < 0.01. P Pvalb-cre **

1.92 mV; 1.92 AG1478 G42;Erbb4 = 0.44) and half-width (0.34 (0.34 half-width and 0.44) = ie lo a a eue itisc xiaiiy f fast- of excitability intrinsic reduced a had also mice NRG1 ± OSCI c

P + 0.6 ms, ms, 0.6 V loxP/loxP ). These results demonstrated the specific loss of ErbB4 < 0.05; 0.05; < /d Erbb4 b primers generated a 250-bp product and and product a 250-bp generated primers 400 250 363 500 b t EN ) Genotyping of of ) Genotyping ± versus membrane potential showed that the volt P in vivo in loxP/loxP 1.47 mV to −43.6 Fig. C = 0.45), amplitude (68.5 (68.5 amplitude 0.45), = mice, where where mice, primers generated a 363-bp product in the wild-type or a 500-bp product in the the in product a 500-bp or wild-type the in product a 363-bp generated primers E P

Fig. 2d Fig.

< 0.01; 0.01; < ± Pvalb-cre;Erbb4 2

G42;Pvalb-cre;Erbb4 G42;Pvalb-cre advance online publication online advance G42;Erbb4 1.54 mV, a G42;Pvalb-cre;Erbb4 evidence that ErbB4 is important in the the in important is ErbB4 that evidence and ) and in further support of the involve the of support further in and ) G42;Pvalb-cre;Erbb4 Fig. 3 Fig. , e G42;Pvalb-cre;Erbb4

), consistent with pharmacological pharmacological with consistent ), G42;Pvalb-cre;Erbb4 Fig. 2d Fig. G42;Pvalb-cre;Erbb4 P a G42 Pvalb-cre;Erbb4 loxP/loxP < 0.01; ). Plots of membrane potential potential membrane of Plots ). n = 7, * = 7, ± G42;Erbb4 represents ± loxP/− −/− 1.26 mV, G42;Erbb4 , e 0.01 ms to 0.35 0.35 to ms 0.01 ), there was no change in in change no was there ), mice were further crossed Erbb4 −/− mice (−40.0 (−40.0 mice Fig. 3

P −/− loxP/loxP

< 0.05, ** < 0.05, loxP/loxP −/− ± −/− 0.94 mV to 67.6 67.6 to mV 0.94 mice. mice. P c loxP/loxP

G42;Erbb4 G42; mice had a more more a had mice ) after ecto-ErbB4 c < 0.01; µ Gad1-EGFP

M AG1478 treatment for 15 min before the addition of NRG1. NRG1. of addition the before min 15 for treatment M AG1478 G42; −/− Pvalb-cre;

− −/− loxP/loxP

/ Erbb4 Erbb4 − mice. FS-PV mice. DNA was isolated for PCR with three indicated indicated three with PCR for isolated was DNA mice. ± P G42;Erbb4 mice (8.9 G42;Pvalb- G42 G42;Pvalb-cre;Erbb4 < 0.01. Error bars represent s.e.m. represent bars Error < 0.01. Parvalbumi 0.5 ms to ms 0.5 ± ± ±

Fig. Fig. 3b 0.01 ms; ms; 0.01 1.65 mV 1.1 mV, 1.1 loxP/loxP primers generated a 400-bp product. ( product. a 400-bp generated primers µ m. ( m. line line , n c d loxP/loxP ± ± ± (top) and and (top) - - - - - ) ) Representative action potentials of two two of potentials action ) Representative

resistance, resting membrane potential, membrane time constant constant time membrane potential, membrane resting resistance, activity of activity We NRG1 endogenous with ecto-ErbB4. applied a of series K the overall mine whether deter to recordings voltage-clamp out carried first we further, nels channels. potassium DTx-K-sensitive by mediated is NRG1 by excitability interneuronal ( frequency firing the and current decreasing threshold the from increasing ecto-ErbB4 5 Fig. Supplementary Supplementary ( Fig. 5 generation potential action for current threshold the DTx-K blocker Kv1.1-specific the that showed data our approaches.and ical biochemical In agreement with a previous study, excitability by NRG1. To test this hypothesis, we usedinterneuronal electrophysiologFS-PV of modulation the for targets are Kv1 channels operate atnels potentials near-threshold that the Considering Kv1 subfamily of voltage-gated chanpotassium signaling NRG1-ErbB4 of target a molecular is Kv1.1 threshold. potential action and initiation) spike of slope (the ability of FS-PV cells by the regulating responsiveness near-threshold 4a Fig. ( ecto-ErbB4 of application the for described that to ecto-ErbB4. The effect of NRG1 on of the thresholdremoval current the was oppositeafter min 15 about disappeared and min 15 at mum a maxi reached application, 5 min of within ecto-ErbB4 was evident a ( firing frequency decreased spiking interneurons in the absence of exogenous NRG1, as shown by ( current threshold discharges. high-frequency Ecto-ErbB4 alone reversibly increased the steps With current showed of all interneurons FS-PV current, the increase injected small with probing by generation potential action for current threshold the measured we directly generation, potential neurons. these of that NRG1 treatment does not affect the passive membrane properties NRG1 treatment ( with difference and found no and capacitance, significant membrane and and ErbB4 Next we measured passive membrane properties, including input input including properties, membrane passive measured we Next To investigate the involvement of DTx-K-sensitive potassium chan action for threshold voltage the on NRG1 of effect Tothe confirm G42;Pvalb-cre;Erbb4 −/− G42;Pvalb-cre;Erbb4 , b and and ). These results indicated that NRG1 influences the excit the influences NRG1 that indicated results These ). G42;Erbb4 Merge Fig. 4d Fig. Supplementary Fig. Supplementary 3a Fig. 4a Fig. ) and increased the firing frequency ( ). Moreover, treatment with DTx-K prevented DTx-K Moreover, with ). treatment loxP/loxP e − , e ) Summary ) Summary c / − loxP − , ), suggesting that the regulation of FS-PV FS-PV of regulation the that suggesting ), / ) Specific ) Specific b + (bottom) (bottom) − current was changed by neutralizing the was current changed by neutralizing mice with with mice ) and suppressed the excitability of fast- of excitability the suppressed and ) DAPI P stained stained -flanked -flanked < 0.001;

Fig. Fig. 4

18 G42; d –

, G42; 32 Pvalb-cre; d −/− loxP/loxP ). ). These results indicated , Erbb4 Erbb4 3 c 3 ). ). The inhibitory effect , we hypothesized that we hypothesized , e

NRG1 ISI (ms) G42;Erbb4 12 15 0 3 6 9 t r a

Supplementary Supplementary G42;Pvalb-cre;Erbb4 loxP/loxP + – Fig. 4 Fig. ** Fig. 4 3 C I 4 reduced reduced + – * e l d 20 mV 10 ms e –/– and and and 1 s 8 ------. © 2011 Nature America, Inc. All rights reserved. ished ( ished K the DTx-K with treatment ( subsequent by inhibited completely were K outward the upregulated application Ecto-ErbB4 ( component current ( DTx-K of absence the in those ( ( a K revealed ecto-ErbB4 of absence the in those from presence the in or current ecto-ErbB4-regulated components. Subtraction of currents Kv1.1 channels. the from contribution maximal a with voltage clamp pulses from −80 mV to −20 mV to generate a K threshold. voltage on effects NRG1’s d as defined was threshold Voltage NRG1. of ( plot plane phase Right, application. NRG1 gray) (light after and (black) before conditions. indicated under initiation of slope normalized of plot summary Right, discharge. sustained during gray) (light washout then application, gray) (dark ecto-ErbB4 2 and after (black) before (bottom) interneuron FS-PV and another from 10 gray) and after nM (light NRG1 (black) before (top) interneuron FS-PV 3 Figure t r a sustained discharge. discharge. sustained ecto-ErbB4. time. over 2 after and n ( 270 top, to bottom ( 360 340, top, to bottom from ( generation spike for current threshold (the 230 top, to bottom from of, injections current to cortex mouse of 2/3 layer in interneuron FS-PV a representative of 4 Figure a a Fig. Fig. 5a Fig. 5 Fig. = 7, *** = 7,

Current subtraction analysis was used to separate the DTx-K-sensitive Control NRG1 + Control Fig. 5b Fig. a urns peuae b et-rB wr ttly abol totally were ecto-ErbB4 by upregulated currents 230 pA 400 pA

300 pA C I , 600 pA c , bottom). Subtraction of currents in the presence from from presence the in currents of Subtraction bottom). , Enhancement of initiation of action potential in FS-PV interneurons by NRG1. ( NRG1. by interneurons FS-PV in potential action of initiation of Enhancement Reversible increase of threshold current for action potential generation by ecto-ErbB4 and inhibition by DTx-K. ( DTx-K. by inhibition and ecto-ErbB4 by generation potential action for current threshold of increase Reversible P ). ). Moreover, when brain were slices with DTx-K, pretreated n < 0.001; < 0.001; µ = 8, ** = 8, I TH n g g ml e l + = 7, ** = 7, current component that was regulated by ecto-ErbB4 ecto-ErbB4 by regulated was that component current )), 300, 400 and 600 pA. Middle, same interneuron as in left panel after bath application of 2 of application bath after panel left in as interneuron same Middle, pA. 600 and 400 300, )), Washout Control , d −1 s ). These results indicated that the whole current current whole the that indicated results These ). Ecto-ErbB4 ecto-ErbB4 (dark gray) application, then washout (light gray) during sustained discharge. Right, summary plot of firing frequency frequency firing of plot summary Right, discharge. sustained during gray) (light washout then application, gray) (dark ecto-ErbB4 P 2 ms I < 0.01, *** < 0.01, TH n n P ### 20 mV = 6, ** = 6, = 7, ** = 7, < 0.01. ( < 0.01. ), 300, 400 and 600 pA. ( pA. 600 and 400 300, ), Fig. 5 Fig. P < 0.001 versus ecto-ErbB4. ( ecto-ErbB4. versus < 0.001 b P P I , bottom) that was sensitive to DTx-K. DTx-K. to sensitive was that bottom) , < 0.01, *** < 0.01, TH < 0.01, *** < 0.01, Ecto-ErbB4 Normalized slope of e P 400 pA 340 pA 360 pA ) As in in ) As ), 400 and 600 pA. Right, same interneuron as in middle after washout of ecto-ErbB4. Current injections, from from injections, Current ecto-ErbB4. of washout after middle in as interneuron same Right, pA. 600 and 400 ),

600 pA initiation < 0.001. ( < 0.001. 1.5 0.5 1.0 Fig. 5 Fig. 0 n Control = 8, ** = 8, c

, before (black) and after DTx-K (light gray) application and further ecto-ErbB4 treatment (dark gray) during during gray) (dark treatment ecto-ErbB4 further and application gray) (light DTx-K after and (black) , before NRG1 b Ecto-ErbB4 P P V ) revealed a Kv1.1 channel channel Kv1.1 a revealed ) ** < 0.001. Error bars represent s.e.m. represent bars Error < 0.001. /d d < 0.001; < 0.001; P Ecto-ErbB4 ) Summary histogram of threshold current before and after DTx-K application and further treatment with with treatment further and application DTx-K after and before current threshold of histogram ) Summary < 0.01. Error bars represent s.e.m. represent bars Error < 0.01. t *** = 10 mV ms mV = 10

b washout ) Summary histogram of threshold current before and after ecto-ErbB4 application and then washout. washout. then and application ecto-ErbB4 after and before current threshold of histogram ) Summary ### + + ### c currents, which which currents, Washout ) Left, superimposed action potentials from a representative FS-PV interneuron before (black) (black) before interneuron FS-PV a representative from potentials action superimposed ) Left, 270 pA 300 pA 400 pA 600 pA P < 0.001 versus ecto-ErbB4. ( ecto-ErbB4. versus < 0.001 −1 Control b . The region around threshold has been magnified for clarity. ( clarity. for magnified been has threshold around region . The + Fig. 5 Fig. current NRG1 20 mV 100 ms a 20 mV - ) 5 ms

d b Threshold current (pA) Threshold current (pA) treatment ( treatment ecto-ErbB4 after amplitude the in doubling a about found and tion ( with ponent com DTx-K-sensitive of the compared dependence voltage and we amplitude the Next sensitive. DTx-K was ecto-ErbB4 by regulated treatment ( treatment which revealed no significant change in the levels of Kv1.1 Kv1.1 after NRG1 with blots western performed we treatment, NRG1 without or with Kv1.1 of expression the test to the on channels of membrane, and number the other is regulatingthe channel phosphorylation. First, changing is one NRG1-ErbB4: by Kv1.1 the on effect current. channel inhibitory powerful a has NRG1 endogenous 200 400 600 200 400 V 0 There are two possible means for the regulation of Kv1.1 channels of Kv1.1 channels regulation the for means possible two are There –1 Control Control m dV/dt (V s ) versus d versus

Ecto-ErbB4 200 400 600 0

DTx- –50

Washout *** K ** a advance online publication online advance ecto-ErbB4DTx-K ) Left, superimposed action potentials from a representative a representative from potentials action superimposed ) Left, Fig. 5 Fig. Membrane potential(mV) Fig. 5 Fig. b ### V –40 ** ) Left, superimposed truncated action potentials recorded recorded potentials action truncated superimposed ) Left, NRG1 Control + /d Fig. 5 Fig. t ) for action potentials before and after bath application application bath after and before potentials action ) for f e and and –30 e c ). Together, the above results demonstrated that that demonstrated results above the Together, ). a Control DTx- ) and without ( without and ) Control Supplementary Fig. 6 Fig. Supplementary –20 K Ecto-ErbB4 Ecto-ErbB4 Washout –40 µ –20 g g ml 0 600 2 ms −1 20 20 mV ecto-ErbB4. Current injections, injections, Current ecto-ErbB4. α Fig. 5 Fig. subunit-specific ant subunit-specific

Firing frequency (Hz) c a Firing frequency (Hz) 100 175 125 150 100 150 ) Left, voltage response response voltage ) Left, Threshold (mV) 50

–50 –45 –40 –35 –30 c nature nature b ) Summary histograms of histograms ) Summary 1 5 1 5 ). ) ecto-ErbB4 applica ecto-ErbB4 ) DTx-

Ecto-ErbB4 Control 5 5 ** K Time (min) Time (min) NEUR ** ** NRG1 *** *** 25 25 ***

*** Control OSCI ** Ecto-ErbB4 Washout i 35 35 µ bodies, bodies, Ecto-ErbB4 g g ml EN 45 45 C −1 E - -

© 2011 Nature America, Inc. All rights reserved. pilocarpine and quantified the percentage of mice that developed developed that mice of percentage the quantified and pilocarpine to (22.2% 75%; 4.4 Pvalb-cre;Erbb4 The antagonist. receptor GABA a be to considered is which (PTZ), Erbb4 in susceptibility seizure measured we interneurons, FS-PV in function dys of NRG1-ErbB4 consequences To pathophysiological the assess in susceptibility seizure Increased interneurons. parvalbumin in signaling NRG1-ErbB4 5 Fig. control in than the in lower was Kv1.1 of phosphorylation the in Kv1.1 of phosphorylation sine Kv1.1 channel in interneurons, parvalbumin we investigated the tyro signaling. by NRG1 regulated was protein Kv1.1 channel the of tyrosine-phosphorylation of level the that demonstrated data These ( extracts membrane cortical mouse from Kv1.1 immunoprecipitated the in anti-phosphoserine with detected control; of progressively over time after NRG1 treatment (maximum about 500% that coimmunoprecipitated with Kv1.1 from the membranes increased or phosphoserine in membrane phosphotyrosine extracts and of mouse Kv1.1 brain. Phosphotyrosine of coimmunoprecipitation assayed Erbb4 n and 10 nM NRG1-treated slices at indicated times. Cav-1, caveolin-1 loading control. ( (IB) of (IP) Kv1.1 of (top) and Kv1.1 (pY;coimmunoprecipitation and phosphotyrosine bottom) in cortical membrane extracts prepared from control (0 min) *** ### K of outward are percentage Results conditions. ( of 2 addition further and application nM DTx-K 100 (middle) after and (left) before top, (left) and after (middle) 2 Figure 5 nature nature the of half than More epilepticus. status c a b = 4, ** ) To further confirm a modulation by NRG1-ErbB4 signaling of the signaling by NRG1-ErbB4 a modulation To confirm further we Kv1.1, phosphorylate can NRG1 whether explore to Second,

P P Summary histogram of overall outward K outward of overall histogram Summary Pvalb-cre;Erbb4 ± < 0.001 versus ecto-ErbB4. ( ecto-ErbB4. versus < 0.001 < 0.001. ( 0.38; 0.38; loxp/loxp −/− h Subtraction Subtraction ). This result suggest that the Kv1.1 channel is regulated by regulated is channel Kv1.1 the that suggest result This ).

Control Control P NRG1-ErbB4 signaling regulates Kv1.1 channels. ( NEUR mice the common convulsant agent pentylenetetrazole pentylenetetrazole agent convulsant common the mice < 0.01; *** P Fig. 5f Fig. and < 0.05) and a greater percentage of generalized seizures seizures generalized of percentage greater a and 0.05) < e OSCI l ) Plot of the voltage dependence and peak amplitude of the DTx-K-sensitive outward currents. P ecto-ErbB4 Erbb4 −/− Pvalb-cre;Erbb4 l control < 0.05) than the , g mice developed higher seizure stages (2.7 (2.7 stages seizure higher developed mice −/− EN ). No significant change of immunoreaction was was immunoreaction of change significant No ). – P l DTx-K loxP/loxP – < 0.001. ( C mice. We gave gave We mice. l control µ E

g ml Ecto-ErbB advance online publication online advance DTx-K −1 mice (67.7 (67.7 mice ecto-ErbB4 application and further addition of 100 nM DTx-K (right); bottom, current subtraction analysis. (

l − ecto-ErbB4 h d / l − DTx- ) Immunoblot ) of Immunoblot Kv1.1 (top) and co-IP of Kv1.1 and pY (middle) in cortical membrane extracts prepared from ) Summary histogram of outward K outward of histogram ) Summary Erbb4 mice. Error bars blots s.e.m. represent Full-length are in presented 4 K+ ecto-ErbB4 – Pvalb-cre;Erbb4 Pvalb-cre;Erbb4 l ecto-ErbB loxp/loxp Erbb4 + ± current activated at depolarizing voltage of −20 mV under control, ecto-ErbB4 and ecto-ErbB4 + DTx-K and ecto-ErbB4 of ecto-ErbB4 −20 voltage mV control, under at depolarizing activated current 5.6% of control; of control; 5.6% DTx-K +ecto-ErbB4 E + – Pvalb-cre;Erbb4 Supplementary Fig. 7 Fig. Supplementary Pvalb-cre;Erbb4 current amplitude before drug administration normalized to 100%. normalized drug administration before amplitude current cto-ErbB4 +DTx-K 4+ l DTx-K loxp/loxp mice. We also injected DTx-K 200 pA 200 pA and and 1 1 a s −/− s ) Top, current evoked by voltage clamping an FS-PV interneuron from −80 to −20 mV before −/− 1 nA 1 nA mice. The The mice. Pvalb-cre;

P mice −/− < 0.001, < 0.001, −/− ± 0.4 to mice mice mice mice c f + + IB IB current under control, DTx-K and DTx-K + ecto-ErbB4 conditions. conditions. + ecto-ErbB4 DTx-K and DTx-K control, under current Normalized K current (%) ). 100 120 140 - - 60 80

NRG Cav- Kv1. Kv1. Control Ecto-ErbB4 pY IP cation cation altered the expression of ErbB4. We previously investigated the 2 years of medical therapy, we wondered whether anticonvulsant medi in group control ( TLE the with subjects in that from different not was ErbB2 of sion control the in group ( that of 60% about only was samples TLE in protein ErbB4 of level The individuals. control from samples in and (TLE) epilepsy lobe temporal with subjects in foci epileptogenic the from to of the expression assess in immunoblotting ErbB4 membranes cell To demonstrate further the ErbB4 link and between epilepsy, we used tissue epileptogenic human in expression ErbB4 Decreased (33.5 of onset seizures 40%; to (89% epilepticus status developed that mice of percentage the reduced greatly treatment NRG1 group, (3.0 vehicle given mice 5.0 to 0.2 (5.6 stages seizure lower showed NRG1 given mice weight); the body however, kilogram per mg (60 PTZ administrating of min 30 within seizures generalized developed mice All application. carpine ventricular delivery of recombinant NRG1 20 min before PTZ or pilo in NRG1 extracellular increased ErbB4 axis should contribute to anticonvulsant action. To test this, we susceptibility. seizure increased parvalbumin interneurons in ErbB4 of loss chemoconvulsants, both with Thus, 56%; to (10% epilepticus status from suffer to likely less the whereas epilepticus, status developed 1 1 1 1

: Ecto-ErbB4 Considering that the individuals with TLE had undergone more than Our findings predict that pharmacological stimulation of the NRG1- 5 0 *** Kv1.1 g

DTx-K * ) Summary histogram of immunoblot analysis of membrane Kv1.1 pY. 10 ### Fig. Fig. 6a + µ 20 ± g g ml 0.2; 0.2; (min) d −1 , +

b Normalized K current (%) P ecto-ErbB4; bottom, current subtraction analysis. analysis. subtraction current bottom, ecto-ErbB4; and 100 120 60 80 < 0.05) and less frequent generalized seizures than than seizures generalized frequent less and 0.05) < Control g Fig. 6a Fig. Ratios of intensities NRG1 Supplementary Fig. 6 Supplementary (pY-Kv1.1 / Kv1.1) DTx-K ± Supplementary Figure 6 Supplementary ± 0 1 2 3 4 5 6 7 *** 2.1 min to 54.5 to 54.5 min 2.1

0.3 to 0.3 1.5 ecto-ErbB4DTX-K , 0 b n = 8, * ). + ** 1 5 Erbb4 ** 2 0 e P n ± < 0.05, **

*** DTx-K–sensitive = 8, * 0.3; 0.3; + 0 K current (pA) Loxp/Loxp (min) 10 20 30 40 50 ± P 0 0 0 0 0 0 7.7 min; min; 7.7 P < 0.01). In the pilocarpine In < pilocarpine the 0.01). P < 0.05; *** –80 Erbb4 ); ); by the expres contrast, < 0.05) and delayed the the delayed and 0.05) < . h P Membrane potential(mV) mice by mice intracerebro –70 < 0.01. ( IB IB Control Ecto-ErbB Loxp/Loxp –6 Cav-1 t r a Kv1. Kv1. P – 0 < 0.05). Erbb4 pY IP –50 1 1 : P Pvalb-cre;Erbb4 loxP/loxP < 0.001; f 4 ) n b

= 8, Immunoblot ) As in mice were were mice 40 C I Kv1. P < 0.05). 0.05). < –3 1 * – 0

–/– e l a

, ** 20

s ± - - -

© 2011 Nature America, Inc. All rights reserved. on Kv1.1 channel current. It has been shown that tyrosine phosphor on ItKv1.1 that tyrosine shown current. has been channel is further supported the conclusion powerful inhibitory This effect of endogenous NRG1 signaling. NRG1-ErbB4 of target molecular a is that the Kv1.1 suggesting DTx-K, channel blocker potassium specific the and potential, action was this effect byblocked the Kv1.1 subunit- regulating the near-threshold responsiveness and voltage threshold of results showed that NRG1 influenced the excitability of FS-PV cells by lation of voltage threshold and near-threshold responsiveness powerfully influence the excitability of fast-spiking cells through regu cortex in interneurons of segment initial axon to the localized are channels Kv1.1-containing specifically potassium does not directly regulate the activity of pyramidal neurons. to suppress pyramidal neuronal activity, suggesting that NRG1-ErbB4 firmed because in the presence of picrotoxin NRG1 was no longer able ing the excitability of interneurons by NRG1. In our study, this wasby conGABAergic release neurons perisomatic region of target neurons, regulating the output the ontoaxons their preferentiallyproject They of neocortex. in systempyramidal pattern firing fast-spiking a have picrotoxin,of cation appli the by blocked not were NRG1 of effects the because inputs, more, that this effect does not occur through the regulation of synaptic directly regulates FS-PV interneuronal intrinsic excitability and, signaling. In further the present study, we provide evidence that NRG1-ErbB4 that GABAergic interneurons are a major cellular target ofLTP NRG1-ErbB4 by means of GABAergic transmission for epilepsy. factor is a risk signaling of NRG1-ErbB4-Kv1.1 tion of interneuronal excitability. These findings suggest that the level Kv1.1 as a downstream target of in signaling NRG1-ErbB4 the regula In our identified study sum, reduced. was TLE with from individuals showed that of foci the expression protein ErbB4 in the epileptogenic models of epilepsy, and seizures were ameliorated by NRG1. pilocarpine-induced Finally,and PTZ- to we susceptible more much were mice Fourth, channels. potassium Kv1.1voltage-gated found that the shift of voltage threshold was mediated by inhibition of threshold responsiveness increased after NRG1 treatment. Notably,near- the we and levels hyperpolarized more toward shifted generation potential action for threshold voltage the Third, ErbB4. receptor its on dependent is NRG1 of effect this that suggesting NRG1, by tion potentia firing the blocked interneurons parvalbumin in ErbB4 of or blockade ablation Second, ecto-ErbB4. peptide by the neutralizing increased the excitability of FS-PV interneurons, but this was inhibited The main of findings this study are as follows. First, exogenous NRG1 DISCUSSION ( vehicle with treated from those differ significantly not did phenytoin sodium or acid valproic with treated clinically used anticonvulsant drugs phenytoin (10 mg kg effects of chronic treatment with valproic acid (260 mg kg in presented are blots Full-length groups. TLE and control ( loading. protein equal shows antibody (cav-1) caveolin-1 with blotting Western TLE. with individuals or individuals control from cortex of ( 6 Figure t r a ylation suppresses the whole-cell outward current of this channel b a

) Immunoblotting with ErbB4 and ErbB2 in the membrane fraction fraction membrane the in ErbB2 and ErbB4 with ) Immunoblotting ) Quantification of ErbB4 (180 kDa) and ErbB2 (180 kDa) proteins in proteins kDa) (180 ErbB2 and kDa) (180 ErbB4 of ) Quantification Electron microscopy and immunofluorescence studies show that that show studies immunofluorescence and microscopy Electron It has been shown that NRG1 regulates pyramidal neuron activity and

C I 2 Decreased ErbB4 expression in human epileptogenic tissue. tissue. epileptogenic human in expression ErbB4 Decreased . The NRG1 regulation of pyramidal neuron activity and LTP e l s −1 dl 14 ) on the expression of ErbB4 in mice n , 1 -AP5 and DNQX. Parvalbumin interneurons Parvalbumin DNQX.and -AP5 = 4, *** = 4, 5 may therefore occur downstream of regulat Supplementary Figure 6 Figure Supplementary P 2 < 0.001. Error bars represent s.e.m. s.e.m. represent bars Error < 0.001. , 3 8 36 and are the dominantinhibitory the are and , 3 7 Supplementary Fig. 8 Fig. Supplementary . ErbB4 expression in the mice 14 , 1 5 . All these studies suggest 32 , 3 Pvalb-cre;Erbb4 9 . These channels channels These . . −1 ) or sodium 3 5 . Both are 18 ). , 4 0 41– . Our −/− 4 3 ------.

ingKv3.2) regulating excitability in fast-spiking interneurons,interneurons.FS-PV reportedthatgenesdeletionsomeItof been has such as ing contributes to human epilepsy through decreasing the excitability of tissue. Our findings suggest that downregulationepileptogenichuman in decreased ofwas ErbB2, notbut NRG1-ErbB4 ErbB4, of sion signal interneurons led to a high seizure sensitivity in mice. parvalbumin Moreover,in ErbB4 of deletionexpres signaling, NRG1-ErbB4 by ability mechanisms. molecular underlying the investigate of receptor. to the ErbB4 kinase Future tyrosine are studies necessary sible involvement of of tyrosine phosphorylation Kv1.1 protein by the of Kv1.1 in membrane. These results suggest for the first time the pos phosphorylation the tyrosine we Indeed, found that NRG1 increased Methods and any associated references are available in the online online the at http://www.nature.com/natureneuroscience/. paper of the in version available are references associated any and Methods M epilepsies. the in effects anticonvulsant attain to targeted be might tion in the pathology of epilepsy. Our findings also suggest that ErbB4 may func signaling that NRG1-ErbB4-Kv1.1 and reveals excitability interneuronal FS-PV of regulation the in signaling NRG1-ErbB4 of interneurons. in deficits developmental to due not be may mice nal excitability and increased sensitivity to epilepsy in pyramidal in mIPSCs neurons of frequency reduced a have mice 13.5, day bination from Pvalb-cre;Erbb4 in unchanged are mIPSCs example, For deletion. gene early require 13. This late-onset deletion may not cause developmental deficits that knockout mice instead of rons, by using respectively required to investigate Kv1.1 effects in interneuronsunclear.is remainsFuture work pathology and this of pyramidal basis cellular exact neu knockout mice might be mainly due to pyramidal cell Kv1.1dysfunction. in phenotype The observed the that possibility the raises therefore increased susceptibility tothe seizures to in contribute mice lackingmay Kv1.1ErbB4 by proteins. excitability This interneuronal FS-PV of mice in epilepsy syn epilepsy human in dromes described been have proteins associated ErbB4 may be a new target for anticonvulsant effects in the epilepsies. genes. In addition, our result that NRG1 reduces seizures suggests that epilepsysusceptibilityarealso they worthwhile determinewhetherto ERBB4 sharebiologicalgenesandphenotypes relation between schizophrenia and epilepsy, and the two diseases may is involved in the etiopathogenesis of seizures all support the idea that dysfunctional FS-PV interneuronal inhibition et Consistent with the powerful inhibition of FS-PV interneuronal excit Overall, Overall, our work identifies Kv1.1 channel as a new molecular target in Cre expression Point mutations Kv1.1 the in gene ErbB2 ErbB4 a Cav-1 h ods are known to be schizophrenia susceptibility genes, it would be 45– 9 . Therefore, the NRG1-ErbB4 regulation of FS-PV interneuro advance online publication online advance 4 3 Control 8 and . Ablation of Kv1.1 in the hippocampus produces profound Dlx5/6-cre −/− Scn1a 4 9 . Our results imply that suppressing the inhibition the suppressing that imply results Our . TLE hippocampus Pvalb-cre;Erbb4 (ref. , which begins to , at begins be which expressed embryonic Pvalb Kcna1 4),leads to epilepsy inmice. Those studies b - Kcna1 −/− Ratio of intensities 1

4 (ErbB4/cav-1) (Kv1.1 null) knockout mice. . . When ErbB4 is ablated by recom 0.2 0.4 0.6 0.8 1.0 1.2 1.4 −/− 0 KCNA1 and 4 4 mice occurs on postnatal day on postnatal occurs mice . Consideringthat. Control

Camk2a 3 nature nature and mutations Kv1.1- in , 4 . There is a bidirectional - ** Kcna1 TLE Pvalb-cre;Erb4 * NEUR Kcnc2 conditional NRG1 OSCI ErbB2 ErbB4 (encod EN and −/− C E ------

© 2011 Nature America, Inc. All rights reserved. 17. 16. 15. 14. 13. 12. 11. 10. 9. 8. 7. 6. 5. 4. 3. 2. 1. reprints/index.html. http://www.nature.com/ at online available is information permissions and Reprints Published online at http://www.nature.com/natureneuroscience/. The authors declare no competing interests.financial all phases suggestions; of X.-M.L. supervised the project and wrote the manuscript. X.-J.C. the purified compounds; Z.C., S.D. and J.-H.L. contributed experimental S.-X.C. conducted part of the electrophysiological recording and gene identification; J.-M. Zhang performed surgery, provided the human tissues and analyzed data; analyzed data; J.Z. performed the immunostaining experiments; J.-M. Zhu and manuscript; Z.-H.X. performed the studies on the mouse model of epilepsy and the manuscript; Y.-M.L. conducted the western blot analyses and wrote the K.-X.L. conducted the electrophysiological studies, analyzed data and wrote Innovation Team (2010R50049). for the Central Universities (2011XZZX002) and Zhejiang Province Key Technology Provincial Qianjiang Talent Plan (2010R10057), the Fundamental Research Funds (200937), the Science Foundation of Chinese Universities (JD09023), the Zhejiang Foundation for the Author of National Excellent Doctoral Dissertation of China the Zhejiang Provincial Natural Science Foundation of China (Z2090127), the Ministry of Science and Technology of China (2010CB912004, 2010CB912002), (30970916, 31070926 and 30725047), the Major Research Program from the state was supported by grants from the National Natural Science Foundation of China reading of this manuscript, and Q.L. Miao for technical assistance. This work families for their participation. We also thank T.M. Gao and I.C. Bruce for critical providing reagents and/or mice. We express our gratitude to the subjects and their (Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences) for X.H. Zhang (Institute of Neuroscience, Chinese Academy of Sciences) and L. Bao to T.M. Gao (Southern Medical University), Z.J. Huang (Cold Spring Harbor), We thank L. Mei (Georgia Health Sciences University) for advice. We are grateful Note: Supplementary information is available on the nature nature COM AUTH Acknowledgmen

ice, G.M. Pitcher, Neuregulin- A. Buonanno, & D.A. Hoffman, D., Vullhorst, M., Longart, O.B., Kwon, Chen, Y.J. L. Wen, R.S. Woo, Vullhorst,D. neuregulin the of development Neural F.C. Liu, & C. Lai, H.F., Wang, H.J., Yau, Y.Z. Huang, P.Fazzari, Lai, C. & Lemke, G. An extended family of protein-tyrosine kinase genes differentially Corfas, G., Roy, K. & Buxbaum, J.D. Neuregulin 1-erbB signaling and the molecular/ and plasticity synaptic development, neural in 1 W.C.Neuregulin Xiong, & L. Mei, to genes from schizophrenia: and pathway signaling neuregulin The A. Buonanno, I. Ogiwara, D. Lau, Y.Kawaguchi, Y.Kubota, & connections synaptic their and subtypes cell GABAergic the drugs: antiepileptic new V.Tomson,versus T.Alexandre, Jr.& Old E., Perucca, suppresses Src upregulation of NMDA receptors. NMDA of upregulation Src suppresses (2005). 9378–9383 synapses. hippocampal CA1 at potentiation long-term reverses 1 potentiation. long-term (2010). 21818–21823 of regulation 1 (2010). interneurons. parvalbumin-positive Neuron hippocampus. rodent the of cells, 13 by interneurons tangentially migrating from the ganglionic eminences. expression preferential hippocampus: the and cortex cerebral the in ErbB4 receptor synapses. CNS at ErbB4 signalling. system. nervous vertebrate the in expressed schizophrenia. of basis cellular schizophrenia. circuits. neural and synapses mutation. gene Scn1a an carrying mice in seizures epileptic for basis circuit a interneurons: 20 proteins. channel K+ Kv3.2 lacking mice in seizures to susceptibility cortex. frontal rat in study. SANAD P , 252–264 (2003). 252–264 , (2000). 9071–9085 , O E TI R R NEUR

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Subcellular domain-restricted GABAergic innervation in primary in innervation GABAergic domain-restricted Subcellular Neuregulin-1 protects ventricular myocytes from anthracycline- from myocytes ventricular protects Neuregulin-1 542 t al. et

eesv smtmtc oa eies ad uat contactin- mutant and epilepsy focal symptomatic Recessive

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© 2011 Nature America, Inc. All rights reserved. current needed to elicit at least one action potential.Membraneactionone leastelicitat constant to time currentneeded ( generation spike for current Threshold lastfiveISIs evoked bysuprathreshold current train.ofbyfirstintervalthe the amplitude. Firing adaptation (ISI voltage after the action potential peak. Spike width was measured at zationhalf thewas spikemeasured as the difference between the spike threshold betweenand theminimum peak and the threshold of the action potential. Fast afterhyperpolariaction potential initiation. Spike amplitude was measured troughas voltagethe voltageof action differencepotential repolarization to the subsequent voltage befored potentials evoked by 500-ms current injection. Voltage threshold was defined as ability of FS-PV interneurons. The shape parameters were measured from action 500-ms suprathreshold current of 400 to 800 pA, was used to quantify the excit intervals between sequential action potentials in a train elicited in response to a and0.2 mM EGTA, pH 7.2 with KOH, 288 mOsm. ISI, calculated for averaged phosphocreatine disodium mM Na-GTP,Mg-ATP,10 mM mM 0.3 4 buffer, a solution containing 130 mM potassium gluconate, 20 mM KCl, 10 mM HEPES Devices). For action potential recording, glass pipettes (3–4 M converter)to-digitalsoftware(Axon pClampInstruments/Molecular10.2and using whole-cell techniques (MultiClamp 700B Amplifier,camera with a ×40 water-immersion Digidata lens (Nikon, ECLIPSE FN1) and1440A recorded analog- E solutions were saturated with 95% O the recording chamber and superfused (3 ml min ACSF at 33 °C was followed by ~60 min at 22 °C, when slices were transferred to 25 mM NaHCO NaH mM 1.25 KCl, mM 3 NaCl, mM 125 cerebrospinalice-cold1000S)in artificial VT (ACSF),fluid consistedwhich of mice about 4 weeks old. Slices (300 preparation.Slice ioral experiments, only male mice were used. Laboratory Animals. In total, about 287of Usemice wereand Careused in the this forwork. Guidelines ForHealth behavof InstitutesNational US the and AdvisoryCommitteeAnimalguidelines theof at Zhejiangthe with University had access to food and water G42;Erbb4 valbumin interneurons, the conditional mutant mice were further crossed with Erbb4 a carrying a bygenerated were mice knockout conditional ErbB4 cells. fast-spiking targeting effectively for protein (EGFP) in parvalbumin interneurons, have been well characterized (Sigma). DAPI was used to stain nuclei. anti– monoclonal mouse and Technology) Signaling (Cell caveolin-1 Cruz Biotechnology), rabbit anti-ErbB2 (Santa Cruz Biotechnology), rabbit anti- mouse monoclonal anti-parvalbumin (PV235, Swant), rabbit anti-ErbB4 (Santa monoclonal anti- phosphoserine (p-Ser (1C8), Santa Cruz Biotechnology, Inc.), monoclonal anti-phosphotyrosine (P-Tyr-102, Cell Signaling Technology), mouse applied to brain slices. sulfoxide(DMSO, Sigma); concentrationthe final of DMSO was <0.01% when dimethyl in dissolved were chemicalsnecessary, When Sigma-Aldrich. from were chemicals Other Lisheng. Tianjing fromphenytoin sodium Aventisand Alomone Labs. PTZ was from Research Biochemicals, valproic acid from Sanofi picrotoxinandTocris purchasedfromwerefrom DTx-K andBioscience TTX column (Amersham). HiTrapa by purified was ErbB4ex/Fc medium. conditioned of collection for medium low–immunoglobulinG in cultured and generatedwere ecto-ErbB4 expressing stably cells HEK-293 pErbB4ex/Fc. generate to pC4DNA/Fc into forecto-domainthe of ErbB4(amino acids1–659, ecto-ErbB4) wassubcloned the entire EGF domain of Reagentsmice. and ONLINE MET nature nature ( τ V lectrophysiology. ) was measured using a single exponential fit of the voltage deflection produced Transgenic mice lines B13 and G42, expressing enhanced green fluorescentexpressingTransgenicgreenG42,enhanced and B13 lines mice Primary antibodies used were rat monoclonal anti-Kv1.1 (NeuroMab), mouse /d t loxP/loxP = 10 mV ms NEUR loxP/loxP loxP mice as a control.a asmice To assist electrophysiological recording ofpar -flanked -flanked 3 OSCI H and 10 mM glucose. After recovery for ~60 min, incubation in mice. All mice were housed under a 12-h light/dark cycle and −1 We coronalmade slicescortexoftheor hippocampus from ODS Neurons were visualized with an infrared-sensitive CCD CCD infrared-sensitive an with visualized were Neurons The NRG1 used is a recombinanta is used NRG1The polypeptide containing . The slope of initiation was measured as the slope from the loxP dl EN -2-Amino-5-phosphonovaleric acid ( Erbb4 -Cre strategy.-Cre C β E -type Neuregulin 1 (Prospec). The coding sequence ad libitum gene to generate to gene 1 /ISI µ 2 m) were prepared with a Vibroslice (Leica , 5% CO n ) was calculated by dividing the averaged . The use and care of the mice complied Pvalb-cre I TH 2 PO ) was the minimum depolarizing minimum the was ) 2 . 4 , 2 mM MgSO mM 2 , −1 Pvalb-cre;Erbb4 mice were crossed with mice with crossed were mice ) with ACSF at 32–33 °C. All Ω dl ) were filled with 4 , 2 mM CaCl mM 2 , -AP5), DNQX, −/− mice, with mice, β -actin -actin 2 , 19 , 2 2 0 - - - - - ,

pH 7.4. Brain tissues were then postfixed at 4 °C for 24 h, and coronal slices coronal and h, 24 for °C 4 at postfixed then were tissues Brain 7.4. pH PBS, in ml) 100 per g (4 sucrose 4% and ml) 100 per g (4 paraformaldehyde previously described Immunocytochemistry. and potassium current recordings. kHz for action potentials and filtered at 3 kHz and sampled at 10 kHz for IPSCs carefully adjusted. Recordings were Bessel-filtered at 10 kHz and sampled compensationatwere whole-cell 100and balance Bridge analysis. for used were ing with series resistance below 20 M Neurons70%. to up bycompensated was resistance accessrecording, During holdingpotentialrecordingspotentialactionIPSCsandformV. both −70 was recorded in the presence of 50 50 containedACSF the IPSCs, tine and 0.5 mM EGTA, pH 7.2 with KOH, 288 mOsm. To isolate spontaneous 2 mM MgCl for recording IPSCs: 120 mM KCl, 20 mM potassium gluconate, 10used was mM mM) HEPES,(in solution internal chloride symmetric recordings,a current postsynaptic GABAergicFor currents. large with associated errors resistance This enabled us to investigate low-threshold currents without introducing series atedanoutward current. Currents were evoked by voltage steps up tomV.−20 gener and s 3 for mV −20 to mV −80 from pulses voltageof series a applied (3–4 M analysis was performed using customized routines in Matlab. by small hyperpolarizing current injection from resting membrane potential. All PTZ doses totesthypothesesdosesPTZ specific and decreaseoverallthe number of mice different Wechose seizures. 5 and 4 stage by identified were which seizures, seizuresorWe5 deathmin.within 30 scoredincidence thealso ofgeneralized clonic-tonic convulsions and loss ofgeneralized postural(5) side; control; the onto turning (6) and convulsionsseveral clonic episodesgeneralized (4) of stage and facial twitching; (2) myoclonic body jerks; (3) clonic forelimb convulsions; response;ear no(1) following(0) theusing scale: min for 30 observed wasior were performed in the laboratory between 12:00 and 14:00. The seizure behav experimentsthe All min. 30 totransparentforup observed andcagePlexiglas a in placed was mouse the injection, After dose. indicated the at peritoneally effects of ERB4 on seizure severity, PTZ was dissolved in PBS and injected intra Behavioral assays. Institutes of Health Image program. NationalUS the usingmeasured wereimages digital and scanner flatbed a on digitized were immunoblotsquantification, densitometric For film. X-ray on 1:2,000). Signals were detected with enhanced chemiluminescence and(polyclonalErbB2antibody, 1:500), anddeveloped1:500) 1:1,000), caveolin-1 (polyclonal antibody, 1:1,000), ErbB4 (polyclonal antibody, antibody,(monoclonalphosphoserineantibody, (monoclonal 1:1,000),rosine Membranes with proteins were immunoblotted using antibodies to phosphoty electrophoresis sample buffer. in Bound proteins pellet were loaded the on 10% acrylamide SDS-PAGE resuspended and gels. supernatant the aspirated we statin A, 1 1 mM Na containing 50 mM Tris-HCl, pH 7.5, 0.5 mM bufferNaCl, 4 with mM EDTA, times three 4 mM washed EGTA, then centrifugation, by collected were plexes (Upstate Biotechnology) suspension (50%, vol/vol). Immunoprecipitated com body to Kv1.1, which was captured by adding Protein A–Sepharose CL-4B beads 60 carried out using membrane extracts of mouse brain. Briefly, previouslyextracts containingdescribed as performed was NRG1 (10 nM) for 5, 10 or 20 min, and membrane fractionationImmunoprecipitation from and immunoblotting.brain slices anti–mouse IgG (1:400). Immunoreactivity was imaged with Alexa 488– and Alexa 594–conjugated goat (1:400) and mouse monoclonal anti-parvalbumin (1:1,000) at 4 °C for 36–48 h. X-100 for 1 h. Then Tritonthe brain0.5% containingsections PBS were in incubated serum withgoat ErbB4normal (vol/vol)antibody 3% 0618 with treated (40 To measure the potassium channel currents in voltage clamp, the patch pipette µ µ g proteing were incubated overnight 10with at°C 4 m) were prepared using a Vibroslice (VT 1000S). Brain sections were sections Brain 1000S). (VT Vibroslice a using prepared were m) Ω ) was filled with the same solution as for current-clamp recording. We 3 VO µ g ml 2 , 4 mM Mg-ATP, 0.3 mM Na-GTP 10 mM disodium phosphocrea 4 , 50 mM NaF, 1 mM dithiothreitol, 1 mM PMSF, 2 −1 leupeptin and 100 nmol l For seizure induction with PTZ or pilocarpine, to assess the 1 4 . Briefly, mice were perfused transcardially with 4% 4% with transcardially perfused were mice Briefly, . Immunofluorescence staining was carried out as as out carried was staining Immunofluorescence µ µ M Ω M dl and changing <20% throughout the record dl -AP5, 20 -AP5 and 20 and -AP5 5 0 . Immunoprecipitation of Kv1.1was Immunoprecipitationof . −1 calyculin A. After the final wash, Brain slices were incubated with µ β M DNQX and 1 -actin (monoclonalantibody,-actin µ M DNQX. mIPSCs were DNQX.mIPSCs M µ l ofmonoclonall anti doi:10.1038/nn.3006 µ µ M TTX. MThe TTX. g ml −1 pep ------

© 2011 Nature America, Inc. All rights reserved. criteria: (i) no history of central nervous system disease; and (ii) no structural no (ii)and disease; systemnervouscentral of history no (i)criteria: discharges in preoperative evaluation and operative indications. Control group lepsy and no potential inducing causes; and (v) localization-related epileptiform nance imaging examination; (iv) no other nervous system disease other than epiprogressive(iii)no foundfoci aftercomputed tomography andmagnetic reso syndromes fitting the 1981 International Anti-epilepsy Federation classification; antiepileptic first-line drugs with effective blood drug concentrations; (ii) seizure sisting despite more than 2 years of medical therapy with three or more kinds of (i) typical epilepsy symptoms and electroencephalographic features, seizures intractablewith epilepsyorbrainper traumaconforming followingthe to criteria: Human material. behavior was scored as above. 15 min recovery, mice were given PTZ or pilocarpine intraperitoneally once and from bregma; lateral, −1 mm; ventral, −2.2 mm) over the course of 5 min. After 3 in mmol (6 NRG1 andStoelting) apparatusstereotaxic (512600, a in (2%) sia scored seizure severity. the effect of NRG1 treatment. A trained observer, who was unaware of genotype, ticus in 200 mg kg gated the incidence of status epilepticus. As with PTZ, pilocarpine was given at investiwepilocarpine, to response in severity seizure on ErbB4 of effects the kg efficientlytest anticonvulsantthe ofeffects NRG1,(60mg PTZdose a we used to And mice.control the ofpercentage small a in seizures generalized evokes have more severe seizures than controls, we used a PTZ dose (40 mg kg the thathypothesis the test example,to Forrequired. doi:10.1038/nn.3006 µ −1 For NRG1 administration, mice were mounted under isoflurane anesthe isoflurane under mounted were mice administration, NRG1 For l) was injected into the right lateral ventricle (anteroposterior, −0.5 mm mm −0.5 (anteroposterior, ventricle lateral right the into injected was l) ) that evoked a severe seizure score in the ACSF control group. Togroup.scorecontrol ACSF the in score seizure severe a evoked that ) Erbb4 −1 for comparing the percentage of mice that developed status epilep loxP/loxP We used cortical tissue from the temporal lobes of five people and Pvalb-cre;Erbb4 −/− mice and at 250 mg kg Pvalb-cre;Erbb4 −1 for testing −/− −1 mice ) that ------

calculated by two-tailed Student’s Student’s ual samples). For comparison of means from the same group of cells, Statisticala two-tailedanalysis. were quantified using the US National Institutes of Health Image program. quantification, immunoblots were digitized on a flatbed scanner and the images enhancedchemiluminescence developedandForX-rayondensitometricfilm. ted using antibodies to ErbB4, ErbB2 and caveolin-1. Signals were detected with isolated the membrane fraction as described above.tical tissueMembranes was dissectedwere fromimmunoblot fresh frozen brainHuman sections tissue maintainedpreparation andat western−80 °C.blot We analysis. foci. The resected brain tissues were obtained for study. raphy examination after resection of foci and abnormal discharge tissue near the waves disappeared or were markedly reduced in the immediate electrocorticog operation was monitored by an intraoperative neocortical electrode. Abnormal gram monitoring and magnetic resonance imaging. Epileptic focus location and clinical manifestations, sphenoidal electrode recording, video electroencephalo of Zhejiang University School of Medicine (No. 1-009). guardians.legal researchThe approvedwas Medicalbythe Ethical Committee and subjects by signed and subjects control and epilepsy both from obtained statisticaldifferencegroups.the age betweenin Written informed consent was and functional injury that can induce epilepsy other than trauma. There was no 50. Brain stereotactic identification was carried out by comprehensive analysis of

Ostrom, R.S. coupling efficiency to adenylyl cyclase. adenylyl (2001). to efficiency coupling t -test was used. For two-group comparisons, statistical differences were et al. Data are presented as mean Receptor number and caveolar co-localization determine receptor t -test or by Pearson’s chi-squared test. . il Chem. Biol. J. ± s.e.m. ( nature nature One gram of temporal cor n = number of individ

276 NEUR 42063–42069 , OSCI EN C E - - - - -