Presynaptic glycine receptors as a potential therapeutic target for hyperekplexia disease

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1 Presynpti glyine reeptors s potentil therpeuti trget for hyperekplexi isese Wei Xiong 1,2, Sho-Rui Chen 3, Liming He 4, Kejun Cheng, Yi-Lin Zho 3, Hong Chen 3, De-Pei Li 3, Gregg E Homnis 6,7, John Peever 8, Kenner C Rie 3, Ling-gng Wu 4, Hui-Lin Pn 3 & Li Zhng 1 Although postsynpti glyine reeptors (GlyRs) s b heteromers ttrt onsierble reserh ttention, little is known bout the role of presynpti GlyRs, likely homomers, in iseses. Here, we emonstrte tht ehyroxylnnbiiol (), nonpsyhotive nnbinoi, n resue GlyR funtionl efiieny n exggerte ousti n ttile strtle responses in mie bering point muttions in 1 GlyRs tht re responsible for hereitry strtle-hyperekplexi isese. The GlyRs expresse s 1 homomers either in HEK-293 ells or t presynpti terminls of the lyel synpses in the uitory brinstem re more vulnerble thn heteromers to hyperekplexi muttion inue impirment. Homomeri mutnts re more sensitive to thn re heteromers, suggesting presynpti GlyRs s primry trget. Consistent with this ie, seletively resues impire presynpti GlyR tivity n iminishe glyine relese in the brinstem n spinl or of hyperekplexi mutnt mie. Thus, presynpti 1 GlyRs emerge s potentil therpeuti trget for ominnt hyperekplexi isese n other iseses with GlyR efiieny. Glyine hs been efine s mjor inhibitory neurotrnsmitter in the spinl or for nerly hlf entury 1. The GlyRs re the lst therpeuti orphn mong members of the ysteine-loop lign-gte ion hnnel superfmily tht inlues GABA A, niotini etylholine (nach) n -HT 3 reeptors. Ntive GlyRs my onsist of either homomers or heteromers. Although ll α subunits (α1, α2, α3 n α4) re pble of forming funtionl homomeri hnnels, the β subunit n form funtionl hnnels only fter ossembly with the α subunits 2. Postsynpti GlyRs hve been well efine s heteromeri α1β subunits beuse the β subunit bins to gephyrin, whih is postsynpti sffoling protein tht is essentil for the lustering n trgeting of GlyRs in the postsynpti membrne 3,4. These reeptors re thought to be the primry trget for severl neurologil iseses, suh s pin, nxiety, rug ition n hyperekplexi isese. Presynpti GlyRs were first esribe in lyel synpses in the meil nuleus of the trpezoi boy (MNTB) in the rt brinstem 6. These presynpti GlyRs were subsequently foun to be expresse in the spinl or n ventrl tegmentl re 7,8. In ontrst to postsynpti heteromeri GlyRs, presynpti GlyRs re likely forme by homomeri α subunits 7,8. A reent stuy hrterize in etil the ntomil segregtion of presynpti n postsynpti GlyRs in lyx of Hel neurons from the uitory brinstem 9. This stuy provie strong eviene to show tht GlyRs t presynpti terminls of lyel synpses re ompose of homomeri α1 subunits. Although presynpti GlyRs hve been propose to moulte neurotrnsmitter relese uner physiologil onitions, little is known bout the roles of presynpti GlyRs in pthologil proesses. Missense point muttions in the humn α1 GlyR subunit gene isrupt GlyR funtion n result in fmilil hyperekplexi-strtle isese 1,11. Although rre, this isese is hrterize by exessive strtle retions to unexpete uitory n ttile stimuli followe by musle stiffness 12. Among ozens of point muttions tht re ssoite with hyperekplexi isese, the most frequently ourring muttion using humn ominnt hyperekplexi isese is the or R271L muttion in the α1 GlyR subunit 13. Mie rrying the muttion exhibit severe hypersensitivity to ttile n ousti stimuli, losely resembling humn strtle isese 14. In ition to the mie rrying, other lines of genetilly engineere mie rrying, or muttions in the α1 subunit lso isply hyperekplexi behviors 1,16. Despite overwhelming eviene for the funtionl efiieny of GlyRs in strtle isese, urrent therpeuti gents o not trget GlyRs 12. In ition, the role of presynpti GlyRs in strtle isese hs been lrgely ignore, s our knowlege bout presynpti GlyRs is very limite. Allosteri positive moultors of GlyRs hve been propose to hve therpeuti potentil in the tretment of iseses with GlyR efiieny 17. This ppers to be the se, s reent stuies hve shown tht hemilly moifie nnbinoi,, n suppress ute n hroni pin by trgeting α3 GlyRs speifilly 18,19. We esigne the present stuy to ress two questions. Cn tret exggerte 1 Lbortory for Integrtive Neurosiene, Ntionl Institute on Alohol Abuse n Aloholism, Ntionl Institutes of Helth, Bethes, Mryln, USA. 2 Shool of Life Sienes, University of Siene n Tehnology of Chin, Hefei, Chin. 3 Center for Neurosiene n Pin Reserh, Deprtment of Anesthesiology n Periopertive Meiine, The University of Texs MD Anerson Cner Center, Houston, Texs, USA. 4 Synpti Trnsmission Setion, Ntionl Institute of Neurologil n Stroke Disorers, Ntionl Institutes of Helth, Bethes, Mryln, USA. Chemil Biology Reserh Brnh, Ntionl Institute on Drug Abuse n Ntionl Institute on Alohol Abuse n Aloholism, Ntionl Institutes of Helth, Bethes, Mryln, USA. 6 Deprtment of Anesthesiology, University of Pittsburgh Shool of Meiine, Pittsburgh, Pennsylvni, USA. 7 Deprtment of Phrmology n Chemil Biology, University of Pittsburgh Shool of Meiine, Pittsburgh, Pennsylvni, USA. 8 Deprtment of Cell n Systems Biology, University of Toronto, Toronto, Ontrio, Cn. Corresponene shoul be resse to L.Z. (lzhng@mil.nih.gov). Reeive April 213; epte 3 Deember 213; publishe online Jnury 214; oi:1.138/nn VOLUME 17 NUMBER 2 FEBRUARY 214 nture NEUROSCIENCE

2 Figure 1 The α1 muttion impirs GlyR funtion n uses exggerte strtle behvior in mie. () Tre reors of I gly in HEK-293 ells expressing wil-type () or α1 mutnt reeptors. (b) Glyine onentrtion-response urves for the wil-type () n α1 mutnt () reeptors. The glyine EC vlues re 6 ± 8 µm (men ± s.e.m.) for the wil-type reeptors n 21, ± 3,21 µm for the α1 mutnt reeptors (P =.6, t(1) = 9.3, unpire t test). () Tre reors of glyinergi (Gly) sipscs in spinl orsl horn neurons isolte from wil-type n α1 mutnt mie. () The verge vlues (±s.e.m.) n t points of glyinergi sipsc frequeny n mplitue (wil type, n = 23 from 6 mie;, n = 11 Frequeny (HZ) Gly 1 mm Gly 1 mm 3 na 3 s Amplitue (pa) from mie; frequeny: wil type ompre to, P =.22, t(32) = 2.4; mplitue: wil type ompre to, P =.31, t(32) = 3.227; unpire t test). (e) The verge vlues of strtle response inue by white noise rnging from 8 to 12 B in wil-type () n α1 () mie. The mximum strtle mplitue (V m ) is shown s funtion of soun intensity. (f) The verge vlues of strtle responses inue by ir puff in wil-type (n = ) n α1 (n = 1) mie. b I/I mx Glyine (mm) e 2, 1, 1, Aousti strtle response (mv) Soun intensity (B) f 1, Ttile strtle response (mv) 1, Gly sipsc 2 s 2 pa Air pressure (PSI) strtle responses by restoring efiieny in GlyR funtion, n wht is the role of presynpti α1 GlyRs in hyperekplexi isese? RESULTS GlyR efiieny n exggerte strtle responses The α1 muttion substntilly impire α1 GlyRs when expresse in HEK-293 ells. Glyine t 1 mm proue the mximl mplitue of I gly (glyine-inue mximl urrent) in HEK-293 ells expressing wil-type α1 GlyRs, wheres glyine t even 1 mm evoke reltively smll urrents in ells expressing the mutnt reeptors (α1 GlyRs) (Fig. 1). The muttion reue the mximl mplitue of I gly (I mx ) by 71% n signifintly inrese the glyine hlf-mximum effetive onentrtion (EC ) vlue (Fig. 1b). In line with previous stuy 14, there ws pronoune reution in both the frequeny n mplitue of glyinergi spontneous inhibitory postsynpti urrents (sip- SCs) in spinl slies from ult mie rrying the α1 missense muttion (Fig. 1,). The mutnt mie only survive s heterozygotes n isplye exggerte strtle response to both ousti (Fig. 1e) n ir-puff (Fig. 1f) stimuli. However, these hyperekplexi mie oul be trine to perform t level similr to their wil-type littermtes in the rotro test when they were hnle gently n refully (Supplementry Fig. 1). restores GlyR efiieny inue behviors is syntheti nnbinoi tht is slightly moifie from CBD, whih is the mjor nonpsyhotive omponent of mrijun (Fig. 2). hs been shown to be more effiious thn CBD in potentiting I gly in HEK-293 ells expressing GlyRs n in suppressing pin hypersensitivity in mie through GlyR-epenent mehnism 18. We pplie ontinuously for t lest min with intermittent pplitions of glyine in our eletrophysiologil experiments. t 1 µm substntilly reovere the glyine I mx in HEK-293 ells expressing α1 mutnt GlyRs (Fig. 2). t 1 µm signifintly reue the glyine EC vlue n inrese the mximl I gly vlue for the α1 GlyRs (Fig. 2b). The resue of α1 mutnt GlyR efiieny ws epenent on onentrtions over rnge of 1 nm to 3 µm (Supplementry Fig. 2). Next we exmine whether n tret exggerte strtle reflexes in mutnt mie. The most typil sign of α1 mutnt mie tht resembles humn hyperekplexi is exggerte strtle reflexes to suen noise (Supplementry Vieo 1). We mesure the strtle responses to ifferent soun stimuli (8 12 B) in α1 mutnt mie n wil-type littermtes. Wil-type mie were nerly insensitive to ll levels of soun stimulus in the rnge 8 12 B (Fig. 2). In ontrst, α1 mutnt mie exhibite exggerte strtle reflexes to the soun stimulus t ll intensity levels (Fig. 2). For instne, the verge vlues of strtle responses to the ousti soun stimulus (12 B) were 13 ± 42 mv (men ± s.e.m.) in the wil-type mie n 1,66 ± 222 mv in the α1 mutnt mie. Intrperitonel injetion of inhibite the exggerte strtle response of α1 mutnt mie in ose-epenent mnner (Fig. 2 n Supplementry Vieo 2). Similrly, α1 mutnt mie isplye exggerte responses to ttile ir-puff stimuli, suggesting tht there is efiieny in sensory-motor reflexes t the spinl level in these mie (Fig. 2). This ttile-inue exggerte reflex ws signifintly suppresse by intrperitonel injetion of t mg per kg boy weight (Fig. 2). Similrly, these mie were supersensitive to touhing n hnling, s they isplye hin feet lenhing behvior n exggerte tremors when pike up by the til (Fig. 2e). Intrperitonel injetion of t 3 mg per kg boy weight mrkely llevite this symptom. Wheres wil-type mie righte themselves in less thn 1 s, α1 mutnt mie neee more thn 2 s to get bk on their feet (Fig. 2f,g n Supplementry Vieo 3). Intrperitonel injetion of reverse the ely of righting reflex in α1 mutnt mie in onentrtionepenent mnner (Fig. 2f,g n Supplementry Vieo 4). restortion: site- n genotype-speifi effet We next onstrute eight itionl point muttions in the α1 GlyR. Eh of these muttions hs been foun to use ominnt fmilil strtle isese in roents or humns 11,14,16. We foun tht ll of these point muttions inrese glyine EC vlues (Fig. 3). signifintly reue glyine EC vlues in seven of nine hyperekplexi mutnt reeptors when expresse in HEK-293 ells (Fig. 3). However, the impt of hyperekplexi muttions on the glyine I mx ppere to be less onsistent n vrie substntilly (Fig. 3b). For instne, the S27T n muttions resulte in n inrese of glyine EC vlues, wheres these muttions i not signifintly lter the glyine I mx when expresse in HEK-293 ells (P S27T =.7, t(14) =.32; P =.81, t(14) =.2; unpire t test). either prtilly or totlly resue the reue glyine nture NEUROSCIENCE VOLUME 17 NUMBER 2 FEBRUARY

3 CBD Gly (mm) 3 na 3 s Gly (mm) + 3 na 3 s I mx of the, n mutnts but i not signifintly ffet those of the other hyperekplexi mutnt reeptors. Thus, ppere to resue the glyine EC in n extent tht ws more onsistent thn its resue of the glyine I mx of hyperekplexi mutnt reeptors. In ition, two mutnt α1 GlyRs, α1 n α1, were ompletely insensitive to, suggesting tht the resue of GlyR ysfuntion by is site speifi. We next ske whether the effiy of the resue of GlyR funtion by is orrelte with its restortion of exggerte strtle behviors. We therefore ollete four iniviul mouse lines tht rry sensitive (α1 n α1 ) or insensitive (α1 n α1 ) point muttions. Consistent with previous observtions 14 16, ll four heterozygous mutnt mouse lines showe signifintly inrese strtle responses to soun stimuli t 12 b (Fig. 3). Intrperitonel injetion of ( mg per kg boy weight intrperitonelly (i.p.)) inhibite the strtle response in both the α1 n α1 mutnt mie (Fig. 3). In ontrst, the α1 n α1 mutnt mie were insensitive to inhibition of the strtle response by. Stryhnine t 1 nm proue rightwr, prllel shift in the glyine onentrtionresponse urve of wil-type α1 GlyRs expresse in HEK-293 ells (Supplementry Fig. 3). In line with previous stuy 2, stryhnine (1. mg per kg boy weight i.p.) inrese the strtle response in wil-type C7BL/6J mie (Supplementry Fig. 3b). i not signifintly lter stryhnine-inue in vitro or in vivo effets. There ws strong orreltion between the effiy of the restortion of GlyR ysfuntion by in vitro n the effiy of the restortion of exggerte strtle behviors by in vivo (Fig. 3; γ 2 =.93, P =., liner regression). b subunit prtilly reues mutnt 1 GlyR efiieny The ition of β subunits resue the α1 muttion inue efiieny in HEK-293 ells oexpressing the β subunit with the α1 b I/I mx Glyine (mm) Figure 2 resues α1 muttion inue GlyR efiieny n hyper-reflexi in mie. () Chemil struture of CBD n. Below re tre reors of I gly without or with (1 µm) in HEK-293 ells expressing α1 GlyRs. (b) Glyine onentrtionresponse urves with or without (1 µm) in HEK-293 ells expressing α1 GlyRs (glyine EC vlues: 21 ± 3.2 mm (men ± s.e.m.) without () n 1.2 ±.1 mm with (n = ); P =.7, t(9) =., unpire t test). () Restortion by of strtle responses inue by ifferent levels of ousti soun in α1 mutnt mie (wil type, ;, ; e f Aousti strtle response (mv) 2, 1, 1, + vehile + 1 mg kg mg kg 1 + mg kg Soun intensity (B) + + subunit (Fig. 4). This resue ws epenent on the rtio of the β n α1 subunits (Fig. 4b). Inresing the rtio of the β subunit reltive to the α1 mutnt subunit trnsfete into ells erese the glyine EC vlue n inrese the glyine I mx. Coexpression of the β subunit with the α1 mutnt subunit in rtio of 3:1 ompletely resue the impire mximl effiy of glyine. Conversely, the β subunit i not lter the expression levels of GlyR proteins t the ell surfe (Supplementry Fig. 4), suggesting tht the resue inue by the β subunit is not ue to ltertion of reeptor trffiking n reeptor protein trnsltion. Similr to its resue of the efiieny in α1 mutnt reeptors, the β subunit lso resue the efiieny in GlyR funtion use by the other hyperekplexi muttions when oexpresse with eh of eight hyperekplexi α1 mutnt reeptors (Fig. 4). Compre to homomeri α1 mutnts, heteromeri α1β mutnt GlyRs were either less sensitive or insensitive to (Fig. 4). Aition of the β subunit lso resue the reue glyine I mx of most, but not ll, mutnt reeptors (Fig. 4e). Conversely, i not signifintly lter the glyine I mx for these heteromeri mutnt α1β GlyRs (Fig. 4f). resues iminishe spinl glyine relese Our bove-esribe t suggest tht GlyR homomers, but not heteromers, re the reeptors tht re most sensitive to nnbinois. This ie fvors hypothesis tht presynpti, but not postsynpti, GlyRs re the primry therpeuti trget for nnbinoi tretment of exggerte strtle isese. To test this hypothesis, we first exmine the effets of on glyinergi trnsmission by reoring glyinergi sipscs in spinl or slies of ult heterozygous α1 mutnt n wil-type mie. Although the frequeny n mplitue of the glyinergi sipscs were profounly reue in the mutnt mie, bth pplition of t 2 µm inrese only the iminishe frequeny but not the mplitue of g Righting reflex time (s) Ttile strtle response (mv) 1, 1, + 1. PSI PSI (mg kg 1 i.p.) +, ; P <., P <.1, F(1,13) =.3, F(1,13) = 14.74, two-wy nlysis of vrine (ANOVA) ompre to the vehile-injete group). () Restortion by ( mg per kg boy weight i.p.) of strtle responses inue by ifferent levels of ttile ir-puff stimuli in α1 mutnt mie (1. PSI: wil type, ;, n = 1; +, ; PSI: wil type, ;, ; +, ; P <., P <.1, F(,31) = 2.6, one-wy ANOVA followe by Tukey s post ho test). (e) Hin feet lenhing behvior in the α1 mutnt mouse n restortion of this behvior by (3 mg per kg boy weight i.p.). (f) Photo imges of the restortion of righting reflex behvior by. (g) Conentrtion epenene of restortion by of prolonge righting reflex time in the α1 mutnt mie (wil type, ; + ( 3 mg per kg boy weight), n = 1; P <.1, P <.1, F(4, 39) = 27.1, one-wy ANOVA followe by Tukey s post ho test). The t in b n g re shown s the men ± s.e.m. 234 VOLUME 17 NUMBER 2 FEBRUARY 214 nture neuroscience

4 Glyine EC (mm) n = V26M Vehile + the glyine sipscs (Fig. ). This suggests tht resues glyinergi efiieny by trgeting presynpti GlyRs. Unlike the postsynpti GlyRs tht typilly hyperpolrize mture neurons fter tivtion, stimultion of presynpti GlyRs les to epolriztion of presynpti terminls n n inrese in glyine n = 1 P2T S27T b I mx (na) 1 1 V26M Vehile + Figure 3 Site-speifi restortion of hyperekplexi GlyR ysfuntion n strtle responses by. () Averge onentrtion-response urves of glyine EC vlues without or with (1 µm) in HEK-293 ells expressing vrious hyperekplexi mutnt α1 subunits (P <., P <.1, P <.1, t(12 18) > 2.2, unpire t test). (b) The verge I mx vlues of glyine onentrtion-response urves without n with (1 µm) for vrious hyperekplexi mutnt α1 subunits (P <., P <.1, t(13 19) > 2.2, unpire t test). () The verge strtle responses to ousti stimuli in wil-type littermtes n α1, α1, α1 n α1 mutnt mie injete with vehile (blue) or with t mg per kg boy weight i.p. (re) (P <., P <.1, t(12 1) > 2.2 between vehile n injetion in mutnt mie, unpire t test). () Correltion nlysis of inue perentge hnges of glyine EC vlues n inue perentge hnges of strtle responses in mie rrying the orresponing mutnt GlyRs (P =., liner regression). The t in re shown s the men ± s.e.m. Glyine EC (mm) Gly 1 mm α1 Gly 1 mm 3 s α1 + β na V26M b Glyine I/I mx Vehile α:β = 1:3 1.2 α:β = 1:3 1. α:β = 1:1.8 α:β = 3:1.6 α n = 12 n = α1β1 GlyR P2T S27T Glyine (mm) e I mx (na) 1 1 n = 1 P2T S27T Perentge hnges in glyine EC inue by Aousti strtle response (mv) 2, 1, 1, r 2 =.93 Stryhnine Perentge hnges in strtle response inue by relese ue to hlorie efflux 6,7. To further ress whether ts on presynpti glyinergi tivity, we exmine glyinergi miniture IPSCs (mipscs) in spinl orsl horn neurons of α1 mie. To reor glyinergi mipscs, we e tetrootoxin (TTX; 1 µm) to the externl solution in ition to glutmte n GABA A Glyine EC (mm) α1 α1 + β1 V26M P2T S27T f I mx (na) V26M α1β1 GlyR α1 α1 + β1 Mutnt Mutnt + P2T S27T n = 1 Vehile + V26M P2T S27T Figure 4 Differentil sensitivity of homomeri n heteromeri GlyRs to hyperekplexi muttions n. () Tre reors of urrent tivte by glyine (1 mm) in HEK-293 ells expressing homomeri α1 or heteromeri α1 β GlyRs. (b) Glyine onentrtion-response urves of wil-type or α1 n β1 GlyR DNAs trnsfete t ifferent rtios (2 µg ml 1 for wil type () or α1 DNA plus 6 (), 2 (),.67 () or (n = ) µg ml 1 β1 DNA) in HEK-293 ells. α inites the α1 subunit. () EC vlues of homomeri n heteromeri hyperekplexi mutnt GlyRs (rtio of mutnt α1 DNA to β1 DNA, 1:3). () Glyine EC vlues of heteromeri mutnt α1β (1:3) subunits without or with (1 µm). (e) Glyine I mx vlues of homomeri n heteromeri hyperekplexi mutnt GlyRs expresse in HEK-293 ells. (f) Glyine I mx vlues of heteromeri mutnt α1β subunits without or with (1 µm). P <., P <.1, P <.1, t(11 1) > 2.4, unpire t test ( f). The t in b f re shown s the men ± s.e.m. nture NEUROSCIENCE VOLUME 17 NUMBER 2 FEBRUARY

5 + Gly sipsc 2 s 2 pa Frequeny (HZ) Amplitue (pa) Figure Resue by of iminishe glyine relese in spinl slies from α1 mutnt mie. () Tre reors of glyinergi sipscs in spinl slies from wil-type n α1 mutnt mie before n fter (2 µm). The br grphs represent the verge frequeny n mplitue of glyinergi sipscs (wil type: vehile, n = 24 ells from 6 mie; Vehile Control + Wsh Gly mipsc () reeptor ntgonists. Similr to our observtion with the glyinergi sipscs, profounly inrese the frequeny, but not the mplitue, of the glyinergi mipscs in spinl slies from mutnt mie (Fig. b). The umultive probbility nlysis of glyinergi mipscs revele tht shifte the istribution pttern of the inter-event intervl, but not the mplitue, of the mipscs to the left (Fig. ). We next teste the effet of on the pirepulse rtio (PPR) of evoke glyinergi IPSCs in α1 spinl slies (Fig. ) n foun tht signifintly inrese the PPR, suggesting mehnism involving presynpti moultion of GlyRs. The restortion of the efiieny in glyinergi trnsmission by ppere to be point-muttion speifi, s i not lter the iminishe frequeny or mplitue of glyinergi sipscs in spinl slies from α1 mutnt mie (Supplementry Fig. ). This observtion is in line with our previous observtion tht potentition of hyperekplexi mutnt GlyRs by is site-speifi effet. This surprising fining revels presynpti mehnism for moultion of GlyRs by. To further test this ie, we pplie low onentrtion of pirotoxin (PTX) in n ttempt to ifferentite the presynpti from the postsynpti effet of on GlyRs. Low onentrtions of PTX hve been shown to preferentilly inhibit homomeri α GlyRs without ltering heteromeri αβ GlyRs tht re expresse in HEK-293 ells 21,22. Similrly, PTX t low oses hs lso been foun to seletively ffet presynpti GlyRs, whih re likely homomers, in the spinl or n brinstem 7,9,23. We first exmine the sensitivity of homomeri n heteromeri α1 mutnt GlyRs to PTX-inue inhibition when expresse in HEK-293 ells. The homomeri α1 GlyRs were substntilly more sensitive thn were heteromeri α1 β reeptors to PTX-inue inhibition (Fig. e). Control + Wsh b Pire pulse () pa 2 ms PPR Con 2 s 2 pa + Wsh Frequeny (HZ) Amplitue (pa) e I PTX /I Con + Wsh α1 α1 + β1 HEK-293 ells Cumultive probbility , PTX (µm) Control Inter-event intervl (s) Amplitue (pa) For exmple, PTX t 3 µm signifintly reue the I gly by % in ells expressing α1 mutnt homomers, (P =.72, t(1) = 3.36, unpire t test) wheres PTX t this ose h no effet on the I gly in ells expressing α1 β1 mutnt heteromers. This fining is in line with the ie tht PTX t low onentrtions (3 µm) preferentilly inhibits homomeri or presynpti GlyR tivity. We next teste whether PTX t 3 µm n blok the effet of on glyinergi sipscs, s similrly potentite the frequenies of both glyinergi sipscs n glyinergi mipscs in spinl slies from α1 mie. In these slies, PTX t 3 µm ompletely eliminte the inue potentiting effet on glyinergi sipsc frequeny (Fig. f). In ontrst, PTX i not signifintly lter glyinergi sipsc mplitue in the α1 spinl slies (P =.91, t(14) =.11, unpire t test; Supplementry Fig. 6). seletively resues presynpti GlyR efiieny The lyx of Hel is one of very few sites in the mmmlin CNS t whih iret reoring of presynpti hnnel onutne n be hieve 24. The lyx of Hel n its postsynpti trget, the MNTB, funtion s ritil rely in the brinstem uitory iruitry. Moreover, these lyel terminls seeme to be n iel preprtion for us to stuy homomeri α1 subunits beuse they lk the β, α2 n α3 GlyR subunits 9. We next reore I gly using whole-ell pth lmps from both presynpti n postsynpti terminls in lyx of Hel neurons from postntl (P) mie (Fig. 6). Applition of 1 mm glyine evoke stryhnine-sensitive n nerly mximl urrents in postsynpti MNTB prinipl neurons fter P12 in wiltype mie (Fig. 6b). Conversely, there ws no etetble urrent when we pplie 3 mm of glyine to lyel terminls from P12 mie. The I gly beme etetble in only 3% of lyel terminls from f sipsc frequeny (Hz) PTX Control spinl slies, n = 12 ells from 4 mie; : vehile, n = 14 ells from mie,, n = 17 ells from mie; P <., P <.1, F(3,6) = 7.2, one-wy ANOVA followe by Tukey s post ho test). (b) Tre reors n verge frequeny n mplitue of glyinergi mipscs before n fter (2 µm). There ws signifint ifferene in the glyinergi mipsc frequeny before n fter (n = 1 ells from 3 mie; P =.3, t(18) = 2.37, unpire t test). Con, ontrol; wsh, ells were wshe with rtifiil erebrospinl flui fter tretment n then mesure gin. () Cumultive plot nlysis of the istribution of the inter-event intervl n mplitue of glyinergi mipscs without or with. () Tre reors n the rtio of pire pulse responses reore in spinl neurons before n fter (2 µm) ( ells from 3 mie; P =.72, t(12) = 3.23, unpire t test). (e) PTX inhibition of I gly in HEK-293 ells expressing humn α1 GlyRs without () or with () β1 subunits. (f) The effet of PTX (3 µm) on glyinergi sipsc frequeny in the bsene (n = 12 ells from 4 mie) or presene (n = 12 ells from 4 mie) of in spinl slies of α1 mutnt mie (P <., P <.1, F(3,41) = 7., one-wy ANOVA followe by Tukey s post ho test). The t in, b n f re shown s the men ± s.e.m VOLUME 17 NUMBER 2 FEBRUARY 214 nture neuroscience

6 Figure 6 Differentil sensitivity of presynpti n postsynpti GlyRs to hyperekplexi muttion n resue by. () Photo imging of lyx ssoite with postsynpti neuron. Sle br, 1 µm. The shemes illustrte the reoring onfigurtions of lyel terminls (re rrows) n n MNTB prinipl neuron (blue str). On the right is the I gly reore from either presynpti terminls (re tre) or the postsynpti membrne of n MNTB prinipl neuron (blue tre). (b) The verge mximl mplitues of I gly reore from presynpti lyel terminls (re; P12, n = 4 ells from 3 mie; P14, ells from 3 mie; P16, n = 4 ells from 3 mie; P18, n = 3 ells from 2 mie) n postsynpti MNTB neurons (blue; P12, ells from 3 mie; P14, n = 4 ells from 2 mie; P16, n = ells from 4 mie; P18, n = ells from 2 mie) uring evelopment from P12 to P18. () Glyine onentrtion-response urves reore from lyel terminls of wil-type littermtes (open squres; ells from 3 mie) n homozygous (homo) mutnt mie (soli squres; ells from 4 mie). () Glyine onentrtion-response urves reore from MNTB prinipl neurons of wil-type littermtes (open squres; ells from 3 mie) n mutnt mie (soli squres; ells from 3 mie). (e) The verge mplitues of I gly (3 µm glyine) from lyel presynpti terminls of wil-type ( ells from 3 mie) n mutnt mie in the bsene ( ells from mie) or presene ( ells from mie) of (P =.23, t(16) = 3.62, unpire t test). (f) The verge mplitues of I gly (1 µm glyine) from MNTB prinipl neurons of wil-type (n = 1 ells from 3 mie) n mutnt mie in the bsene ( ells from 3 mie) or presene ( ells from mie) of (P =.98, t(12) =.31, unpire t test). The t in b f re shown s the men ± s.e.m. P14 mie, n the mplitue of I gly rehe mximum fter P16. This expression pttern is oinient with the timing of when the α1 subunits emerge n reple embryoni-ominnt α2 subunits in the brinstem n spinl or 2. A previous stuy reporte very similr observtion tht presynpti GlyR tivity in the rt lyx epens on evelopmentl stge 2. Among four mutnt mouse lines, only the line n yiel homozygous offspring. These mutnt homozygous mie isplye seizure-like behviors in response to ousti n ttile stimuli fter P14 (Supplementry Fig. 7). This bnorml behvior in the mutnt mie (P16 P2) ws fully reverse shortly fter intrperitonel injetion of ( mg per kg boy weight) (Supplementry Fig. 7b). In lyel neurons isolte from homozygous mie t P16 P18, the muttion substntilly impire the funtion of presynpti GlyRs (Fig. 6). Both the glyine I mx n the pprent glyine ffinity of presynpti GlyRs from the mutnt mie were signifintly reue s ompre with presynpti GlyRs from their wil-type littermtes (P EC =.137, t(11) = 2.93; P Imx =.7, t(11) = 3.31, unpire t test). In ontrst, the point muttion i not signifintly lter the funtionlity of postsynpti GlyRs in prinipl neurons (Fig. 6). t 1 µm signifintly enhne the verge mplitue of the I gly reore in presynpti terminls from 6 ± 1 pa (men ± s.e.m.) to 181 ± 43 pa (Fig. 6e). In ontrst, DH- CBD i not signifintly lter the postsynpti I gly reore in MNTB neurons from mutnt mie (Fig. 6f). This result is onsistent with our observtion tht the homomeri mutnt reeptor, but not its heteromeri ounterprt, is nnbinoi-sensitive reeptor. Thus, nine hyperekplexi mutnt α1 GlyRs n be lssifie s nnbinoi-sensitive n nnbinoi-insensitive reeptors on the bsis of their responses to nnbinoi potentition of I gly n their resue of strtle behvior (Supplementry Fig. 8). Aition of the β subunits lone n substntilly resue the isrupting effet inue by ll strtle ominnt muttions in the α1 subunits (Supplementry Fig. 8b). Cnnbinois pper to restore the glyinergi ysfuntion inue by hyperekplexi point muttions in homomeri α1 GlyRs, whih re most likely lote in presynpti sites (Supplementry Fig. 8). I/I mx e I gly (na) Presynpti Gly Gly s Gly 1 mm Postsynpti Presynpti Wil type homo s Gly 1 mm Glyine (mm) Presynpti.1 na 1 na + bimx (na) I/I mx I gly (na) P12 P14 P16 P18 P12 P14 P16 P18 DISCUSSION Despite bunnt eviene for presynpti GlyRs in the CNS, it is fr from ler to wht extent they hve role in physiologil n pthologil proesses. This stuy highlights the potentil importne of presynpti GlyRs in both the pthophysiologil mehnisms n therpeuti trgets of ominnt hyperekplexi isese. First, hyperekplexi point muttions in the α1 subunits isrupte the funtion of homomers more substntilly thn heteromers when expresse in HEK-293 ells n in segregte presynpti n postsynpti sites of lyel n MNTB synpses. Seon, hyperekplexi mutnt homomers were more sensitive thn mutnt heteromers to DH- CBD inue resue. Thir, potentite only presynpti homomeri α1 GlyRs without signifintly ltering postsynpti GlyR tivity in the lyx of Hel of the uitory brinstem from hyperekplexi mutnt mie. Consistent with this observtion, preferentilly resue the iminishe frequenies without signifintly ffeting the mplitues of glyinergi sipscs n mipscs in spinl or slies. Suh resue by ws bolishe ompletely by PTX t onentrtion tht preferentilly bloke homomeri GlyRs, suggesting tht resue is meite by presynpti GlyRs. Fourth, n inrese in the PPR inue by inites n enhne probbility of neurotrnsmitter relese in spinl or slies of ult hyperekplexi mutnt mie. One oul lso rgue tht extrsynpti GlyRs my be trget of hyperekplexi isese n. However, there is no goo eviene for homomeri extrsynpti α1 subunits. Most extrsynpti GlyRs tht hve been hrterize re homomeri α2 subunits in the brin uring erly evelopmentl stges 2. Conversely, extrsynpti GlyRs in the brinstem re α1β heteromers 9. These reeptors re usully lolize in either postsynpti or presynpti som but not t presynpti terminls 4,9. Our previous stuies hve shown tht the α3 GlyRs re the trgets of nnbinois in the tretment of ute n hroni pin 18,19. This rises the question of whether the α3 subunit is lso involve in the resue by of hyperekplexi f Presynpti Wil type homo Postsynpti Postsynpti Glyine (mm) Postsynpti + nture NEUROSCIENCE VOLUME 17 NUMBER 2 FEBRUARY

7 muttion inue in vivo effets. It is unlikely tht this is the se. Pin sensitivity to therml stimuli is unhnge in mutnt mie 26, suggesting tht there is no α3 GlyR efiieny epenent behviorl hnge in these mie. Although n potentite both wil-type α1 n α3 GlyRs, oes not ffet the bseline of either loomotor tivity or strtle responses in norml mie 18,19. In this regr, the potentition of the α3 GlyRs by probbly oes not ontribute to its resue of exggerte strtle responses in α1 mutnt mie. Consistent with reent stuies 27,28, oexpression of the β subunits prtilly but mrkely restore the funtionl efiieny in some hyperekplexi mutnt GlyRs expresse in HEK-293 ells. Our t lso suggest tht homomeri-like presynpti α1 GlyRs in ntive neurons sustin more hnnel-funtion mge s result of hyperekplexi muttions thn o heteromeri postsynpti GlyRs. In generl, these mutnt homomers were more sensitive to resue by. One hypothesis for this β subunit epenent resue is tht luster of mino is in the trnsmembrne omins of the β subunit my shiel or ompromise the gting impirment from hyperekplexi point muttions in the α1 subunit 27. There is strong eviene to inite reution of GlyR funtion in both hroni inflmmtory n neuropthi pin in nimls 29,3. Yet the role of presynpti GlyRs in the regultion of pin trnsmission is unler. Future stuies shoul be rrie out to etermine whether presynpti GlyRs re potentil trget in hroni pin. We emphsize the presynpti GlyRs in this stuy beuse the effet of on glyinergi input is presynpti. In ition to the efiieny in presynpti GlyR funtion (iminishe glyine relese), we lso observe postsynpti GlyR efiieny (reue mplitue of glyinergi IPSCs) in spinl or slies from mie. Thus, both impire presynpti n postsynpti α1 GlyRs shoul ount for the iminishe glyinergi synpti trnsmission use by the muttion. Hyperekplexi is usully introue s neuromotor isorer. However, there is substntil unerestimtion of the impt of sensory ysfuntion in this isese beuse of loss of glyinergi innervtion n trnsmission either in sensory neurons or from sensory neurons to motor neurons in the spinl or. This unerestimtion oul be present espeilly in ptients with hyperekplexi bering ominnt muttions in the α1 subunits. These ptients usully isply hyper-reflexi n exggerte strtle responses to noise, ttile stimuli (suh s ir), hnling (feeing or touhing the he or nose) n even visul stimuli shortly fter birth It ppers tht n inrese in ousti strtle responses in hyperekplexi mutnt mie oes not lwys exten to more generl motor behviors suh s blne n oorintion in rotro test, s hs been reporte in previous stuies n in our urrent stuy 1. This fining strongly suggests tht isinhibition by iminishe glyinergi input is mjor use of hyperekplexi. Consistent with this notion, hyperekplexi muttions lrgely impire the funtion of α1 GlyRs expresse t the presynpti terminls of uitory brinstem n spinl orsl horn neurons. Conversely, restore glyinergi trnsmission efiieny in both the brinstem n spinl or by trgeting presynpti α1 GlyRs, t lest in prt. There re few hyperekplexi muttions tht hve been etete in postsynpti proteins suh s gephyrin n the GlyR β subunit 11,34. However, bout 3% of ptients with hyperekplexi o not rry muttions in genes enoing postsynpti proteins suh s gephyrin n the GlyR β subunits 13. In ition, most hyperekplexi muttions ourring in the α1 subunits re linke to ominnt fmilil strtle isese, wheres muttions etete in the β subunit n glyine trnsporter genes re inherite priniplly in reessive strtle isese 11,3,36. In ition to, five itionl nnbinoisensitive mutnts teste in this stuy re inherite in humn ominnt hyperekplexi isese 11. The mjority of these mutnts, suh s V26M, P2T, S27T n, were very sensitive to resue by when expresse s homomers but were less sensitive or insensitive when oexpresse with the β subunits s heteromers. In this regr, the notion tht presynpti GlyRs re n importnt therpeuti trget shoul be pplible to ominnt fmilil hyperekplexi. However, this hypothesis shoul be teste further one geneti or phrmologil pproh tht is seletive for the homomeri α1 subunit beomes vilble. Therpeuti mehnisms n pplitions of nonpsyhotive nnbinois hve beome renewe topis of reent reserh 17,37. The results presente here revel new potentil for nonpsyhotive nnbinois in the tretment of severe hereitry neurologil isese. Unlike GABA A reeptor moultors tht re plgue by vrious sie effets 38, oes not proue mjor psyhotive or setive effets even t high onentrtions 19. In ition, lonzepm, ommonly use gent in the tretment of hyperekplexi, is not lwys effetive in the ontrol of some symptoms in this isese 39. More thn 3 hyperekplexi missense, nonsense n frmeshift muttions in the α1 GlyRs hve been foun to be linke to hyperekplexi isese 11. Our t suggest tht trgets nnbinoi-sensitive GlyRs speifilly, whih re likely to be homomers lote t presynpti sites. It is worth noting tht the muttion proue more funtionl efiieny of GlyRs when expresse in lyel neurons thn in HEK-293 ells. This is onsistent with reent stuy tht showe erese glyine-meite urrents in isolte neurons of the brin stem from mutnt mie 4. One possible explntion for this isrepny is tht neuron-speifi post-trnsltionl moifition my lter the sensitivity of GlyRs to hyperekplexi muttions in vivo. This ie is supporte by previous stuy tht showe ifferent funtionl properties of the mutnt α1 GlyR reeptors when expresse in orsl horn neurons n HEK-293 ells 41. Tken together, we provie new eviene showing tht presynpti GlyRs re n emerging trget for the mehnism n therpeutis of hyperekplexi isese. These presynpti-like GlyRs hve been propose to hve role in ethnol tion n rewr mehnisms in the brin 8, Thus, these GlyRs tht resie t presynpti terminls my represent previously unerestimte trget with potentil importne for vrious iseses involving GlyR efiieny or GlyR signling pthwys. Methos Methos n ny ssoite referenes re vilble in the online version of the pper. Note: Any Supplementry Informtion n Soure Dt files re vilble in the online version of the pper. Aknowlegments We thnk A. Hrris n Y. Blenov (University of Texs t Austin) for proviing the α1, α1 n α1 mutnt mie. We thnk D.M. Lovinger for instrumentl support n omments on the mnusript. This work ws supporte by funs from the intrmurl progrms of the Ntionl Institute on Alohol Abuse n Aloholism, Ntionl Institute on Drug Abuse n US Ntionl Institutes of Helth grnts to G.E.H. (AA1422) n from the Ntionl Institute of Neurologil Disorers n Stroke to H.-L.P. (NS462 n NS7393). AUTHOR CONTRIBUTIONS W.X. n L.Z. onute mutgenesis n niml behviorl tests. W.X. onute pth-lmp reorings in HEK-293 ells. S.-R.C., Y.-L.Z., H.C., D.-P.L. n H.-L.P. onute spinl slie reorings. L.H., W.X. n L.W. onute brin stem lyel reoring. K.C. n K.C.R. synthesize. G.E.H. onstrute 238 VOLUME 17 NUMBER 2 FEBRUARY 214 nture neuroscience

8 genetilly engineere mouse lines. J.P. provie the trnsgeni mouse line. W.X. n H.-L.P. prtiipte in the stuy esign n mnusript writing. L.Z. initite, esigne n supervise the projet n wrote the mnusript. COMPETING FINANCIAL INTERESTS The uthors elre no ompeting finnil interests. Reprints n permissions informtion is vilble online t reprints/inex.html. 1. Dvioff, R.A., Shnk, R.P., Grhm, L.T. Jr., Aprison, M.H. & Wermn, R. Assoition of glyine with spinl interneurones. Nture 214, (1967). 2. Betz, H. & Lube, B. Glyine reeptors: reent insights into their struturl orgniztion n funtionl iversity. J. Neurohem. 97, (26). 3. Gruzinsk, J. et l. The β subunit etermines the lign bining properties of synpti glyine reeptors. Neuron 4, (2). 4. Weltzien, F., Puller, C., O Sullivn, G.A., Prmnn, I. & Betz, H. Distribution of the glyine reeptor β-subunit in the mouse CNS s revele by novel monolonl ntiboy. J. Comp. Neurol. 2, (212).. Lynh, J.W. & Cllister, R.J. Glyine reeptors: new therpeuti trget in pin pthwys. Curr. Opin. Investig. Drugs 7, 48 3 (26). 6. Tureek, R. & Trussell, L.O. Presynpti glyine reeptors enhne trnsmitter relese t mmmlin entrl synpse. Nture 411, 87 9 (21). 7. Jeong, H.-J., Jng, I.-S., Moorhouse, A.J. & Akike, N. Ativtion of presynpti glyine reeptors filittes glyine relese from presynpti terminls synpsing onto rt spinl srl orsl ommissurl nuleus neurons. J. Physiol. (Lon.), (23). 8. Ye, J.-H. et l. Presynpti glyine reeptors on GABAergi terminls filitte ishrge of opminergi neurons in ventrl tegmentl re. J. Neurosi. 24, (24). 9. Hruskov, B. et l. Differentil istribution of glyine reeptor subtypes t the rt lyx of hel synpse. J. Neurosi. 32, (212). 1. Shing, R. et l. Muttions in the α1 subunit of the inhibitory glyine reeptor use the ominnt neurologi isorer, hyperekplexi. Nt. Genet., (1993). 11. Hrvey, R.J., Topf, M., Hrvey, K. & Rees, M.I. The genetis of hyperekplexi: more thn strtle!. Trens Genet. 24, (28). 12. Bkker, M.J., vn Dijk, J.G., vn en Mgenberg, A.M. & Tijssen, M.A. Strtle synromes. Lnet Neurol., (26). 13. Dvies, J.S. et l. The glyinergi system in humn strtle isese: geneti sreening pproh. Front. Mol. Neurosi. 3, 8 (21). 14. Beker, L. et l. Disese-speifi humn glyine reeptor α1 subunit uses hyperekplexi phenotype n impire glyine- n GABA(A)-reeptor trnsmission in trnsgeni mie. J. Neurosi. 22, (22). 1. Finly, G.S. et l. Glyine reeptor knok-in mie n hyperekplexi-like phenotypes: omprisons with the null mutnt. J. Neurosi. 23, (23). 16. Blenov, Y.A., Benviez, J.M., Homnis, G.E. & Hrris, R.A. Behviorl hrteriztion of knokin mie with muttions n in the glyine reeptor α1 subunit. J. Phrmol. Exp. Ther. 34, (212). 17. Zhng, L. & Xiong, W. Nonpsyhotive nnbinoi tion on -HT3 n glyine reeptors. in Enonnbinois: Ations t Non-CB1/CB2 Cnnbinoi Reeptors (es. Aboo, M.E., Sorensen, R.G. & Stell, N.) (Springer, 213). 18. Xiong, W. et l. Cnnbinois suppress inflmmtory n neuropthi pin by trgeting α3 glyine reeptors. J. Exp. Me. 29, (212). 19. Xiong, W. et l. Cnnbinoi potentition of glyine reeptors ontributes to nnbis-inue nlgesi. Nt. Chem. Biol. 7, (211). 2. Kehne, J.H., Gllger, D.W. & Dvis, M. Stryhnine: brinstem n spinl meition of exittory effets on ousti strtle. Eur. J. Phrmol. 76, (1981). 21. Pribill, I., Tkgi, T., Lngosh, D., Bormnn, J. & Betz, H. The typil M2 segment of the β subunit onfers pirotoxinin resistne to inhibitory glyine reeptor hnnels. EMBO J. 11, (1992). 22. Yng, Z., Cromer, B.A., Hrvey, R.J., Prker, M.W. & Lynh, J.W. A propose struturl bsis for pirotoxinin n pirotin bining in the glyine reeptor pore. J. Neurohem. 13, 8 89 (27). 23. Deleuze, C. et l. Struturl ifferene between heteromeri somti n homomeri xonl glyine reeptors in the hypothlmo-neurohypophysil system. Neurosiene 13, (2). 24. Shneggenburger, R. & Forsythe, I.D. The lyx of Hel. Cell Tissue Res. 326, (26). 2. Tureek, R. & Trussell, L.O. Reiprol evelopmentl regultion of presynpti ionotropi reeptors. Pro. Ntl. A. Si. USA 99, (22). 26. O She, S.M., Beker, L., Weiher, H., Betz, H. & Lube, B. Propofol restores the funtion of hyperekplexi mutnt glyine reeptors in Xenopus ooytes n mie. J. Neurosi. 24, (24). 27. Shn, Q., Hn, L. & Lynh, J.W. Funtion of hyperekplexi-using α1/l glyine reeptors is restore by shifting the ffete resiue out of the llosteri signlling pthwy. Br. J. Phrmol. 16, (212). 28. Lpe, R., Pleste, A.J., Moroni, M., Colquhoun, D. & Sivilotti, L.G. The α1 strtle isese muttion revels multiple intermeite sttes in the gting of glyine reeptors. J. Neurosi. 32, (212). 29. Hrvey, R.J. et l. GlyR α3: n essentil trget for spinl PGE2-meite inflmmtory pin sensitiztion. Siene 34, (24). 3. Zhou, H.Y. et l. N-methyl--sprtte reeptor- n lpin-meite proteolyti levge of K + -Cl otrnsporter-2 impirs spinl hlorie homeostsis in neuropthi pin. J. Biol. Chem. 287, (212). 31. Anermnn, F., Keene, D.L., Anermnn, E. & Quesney, L.F. Strtle isese or hyperekplexi: further elinetion of the synrome. Brin 13, (198). 32. Zhou, L., Chillg, K.L. & Nigro, M.A. Hyperekplexi: tretble neurogeneti isese. Brin Dev. 24, (22). 33. Prveen, V., Ptole, S.K. & Whitehll, J.S. Hyperekplexi in neontes. Postgr. Me. J. 77, 7 72 (21). 34. Rees, M.I. et l. Hyperekplexi ssoite with ompoun heterozygote muttions in the β-subunit of the humn inhibitory glyine reeptor (GLRB). Hum. Mol. Genet. 11, (22). 3. Chung, S.K. et l. GLRB is the thir mjor gene of effet in hyperekplexi. Hum. Mol. Genet. 22, (213); errtum 22, 22 (213). 36. Rees, M.I. et l. Muttions in the gene enoing GlyT2 (SLC6A) efine presynpti omponent of humn strtle isese. Nt. Genet. 38, (26). 37. Izzo, A.A., Borrelli, F., Cpsso, R., Di Mrzo, V. & Mehoulm, R. Non-psyhotropi plnt nnbinois: new therpeuti opportunities from n nient herb. Trens Phrmol. Si. 3, 1 27 (29). 38. Ashton, H. Guielines for the rtionl use of benzoizepines. When n wht to use. Drugs 48, 2 4 (1994). 39. Tijssen, M.A. et l. The effets of lonzepm n vigbtrin in hyperekplexi. J. Neurol. Si. 149, (1997). 4. Borghese, C.M. et l. Chrteriztion of two muttions, n, in the α1 glyine reeptor subunit tht moify sensitivity to lohols. J. Phrmol. Exp. Ther. 34, (212). 41. Kung, A.Y., Rik, C., O She, S., Hrrison, N.L. & MGehee, D.S. Expression of glyine reeptors in rt sensory neurons vs. HEK293 ells yiels ifferent funtionl properties. Neurosi. Lett. 39, (21). 42. Sebe, J.Y., Eggers, E.D. & Berger, A.J. Differentil effets of ethnol on GABA A n glyine reeptor meite synpti urrents in brin stem motoneurons. J. Neurophysiol. 9, (23). 43. Chu, P., Hoifot-Lio, H., Lof, E., Soerplm, B. & Erison, M. Glyine reeptors in the nuleus umbens involve in the ethnol intke-reuing effet of mproste. Alohol. Clin. Exp. Res. 34, 39 4 (21). 44. Li, J. et l. Miroinjetion of glyine into the ventrl tegmentl re seletively ereses ethnol onsumption. J. Phrmol. Exp. Ther. 341, (212). nture NEUROSCIENCE VOLUME 17 NUMBER 2 FEBRUARY

9 ONLINE METHODS Animls. Unless otherwise inite, mle hyperekplexi GlyR mutnt mie n their wil-type littermtes weighing between 2 n 28 g (7 12 weeks ol) were use in ll experiments. The nimls bre speifilly for this stuy were bkrosse to C7BL/6J mie for t lest six genertions. They were house two mie per ge on 12 h light, 12 h rk yle n were not use in ifferent behviorl tests. All behviorl tests were ouble blin n performe uring the light yle (9:.m. to : p.m.). These mie were pte to the experimentl environment for t lest 2 h before the tests. All niml stuies were rrie out uner protools pprove by the Animl Cre n Use Committees of the University of Pittsburgh, the University of Texs MD Anerson Cner Center n the Ntionl Institute on Alohol Abuse n Aloholism. The α1, α1, α1 n α1 mutnt mie were generte s previously esribe 14,16,4,4. All genetilly engineere mie stuie were heterozygous for the mutnt α1 subunit. Genotyping of the α1 mutnt mie ws one using the following primers: forwr: -GCCTGCTCATCGTCATCCTG-3 ; reverse: -CCAATCTGATCTGTGCAATCCT-3. Genotyping of the α1 mutnt mie ws one using the following primers: forwr: -GCTT TAACTTCTGCCCTATGG-3 ; reverse: -GTTGTTGTTAACTTGTTTAT TG-3. Genotyping of the α1 mutnt mie ws one using the following primers: forwr: -GAATCTTCCAGGCAACATTTCAG-3 ; reverse: -AGTATCCCACCAAGCCAGTCTTT-3. Genotyping of the α1 mutnt mie ws one using the following primers: forwr: -CTCATCTTTGA GTGGCAGGA-3 ; reverse: -GCATCCATGTTGATCCAGAA-3. Wil-type mie n mutnt (α1, α1, α1 n α1 ) heterozygous mie use for the behvior experiments were proue from heterozygous n orresponing wil-type breeing pirs. Site-irete mutgenesis. Point muttions of humn α1 GlyR were introue using QuikChnge Site-Direte Mutgenesis kit (Strtgene). The uthentiity of the DNA sequene through the muttion sites ws onfirme by ouble-strne DNA sequening using CEQ 8 Geneti Anlysis System (Bekmn Coulter, In.). Eletrophysiologil reoring. HEK-293 ell trnsfetion n reoring. HEK-293 ells were ulture s esribe previously 46. Plsmi DNAs oing for wil-type n mutnt GlyR subunits were trnsfete using the SuperFet Trnsfetion kit (Qigen, Hien, CA). Eletrophysiologil reorings were rrie out 2 fter trnsfetion. HEK-293 ells were trete with.2% (wt/vol) trypsin n.3 mm ethyleneiminetetreti i 2 h before reoring. The HEK-293 ells were lifte n ontinuously superfuse with solution ontining 14 mm NCl, mm KCl, 1.8 mm CCl 2, 1.2 mm MgCl 2, mm gluose n 1 mm 4-(2-hyroxyethyl)-1-piperzineethnesulfoni i (HEPES) (ph 7.4 with NOH, ~34 mosm with surose). Pth pipettes (3 MΩ) were fille with intrellulr solution tht ontine 12 mm CsCl, 4 mm MgCl 2, 1 mm ethyleneglyol-bis(2-minoethylether)-n,n,n,n -tetreti i (EGTA), 1 mm HEPES,. mm N-GTP n 2 mm Mg-ATP (ph 7.2 with CsOH, ~28 mosm). Membrne urrents were reore in the whole-ell onfigurtion using n Axopth 2B mplifier (Axon) t 2 22 C. Cells were hel t 6 mv unless otherwise inite. Dt were quire using pclmp 9.2 softwre (Moleulr Devies, Sunnyvle, CA). Dt were filtere t 1 khz n igitize t 2 khz. Bth solutions were pplie through three-brrel squre glss tubing (Wrner Instrument, Hmen, CT) with tip imeter of ~7 µm. Drugs were pplie using Wrner fst-step stepper motor riven system. The solution exhnge time onstnts were ~4 ms for n open pipette tip n 4 12 ms for whole-ell reorings. Spinl or slie preprtion n reoring. Lumbr spinl or slies t the L L6 level were prepre from ult mie s we esribe previously 47,48. The lumbr spinl or ws remove through lminetomy uring isoflurneinue nesthesi n slie (4 µm) using vibrtome. The slies were superfuse ontinuously with rtifiil erebrospinl flui ontining (in mm) 117. NCl, 3.6 KCl, 1.2 MgCl 2, 2. CCl 2, 1.2 NH 2 PO 4, 11. gluose n 2. NHCO 3 (bubble with 9% O 2 n % CO 2 ). Neurons in the lmin II of the spinl or were visulize using fixe-stge mirosope (BXWI, Olympus, Tokyo, Jpn) with ifferentil interferene ontrst n infrre illumintion. We obtine ll whole-ell pth-lmp reorings t 34 C using glss pipettes fille with solution ontining (in mm) 11 Cs 2 SO 4, tetrethylmmonium ion, 2. MgCl 2,. CCl 2,. HEPES,. EGTA,. ATP-Mg,. N-GTP n 1 lioine N-ethyl bromie, juste to ph with 1 M CsOH (29 3 mosm). Spinl slies were reore t holing potentil of mv. DH- CBD ws pplie by puff pplition iretly to the reore neuron using positive pressure system (4 PSI, 1 ms; Toohey Compny, Firfiel, NJ). Glyinergi sipscs were reore in the presene of 1 µm 6-yno-7-nitroquinoxline-2,3- ione (CNQX), 1 µm biuulline n µm AP (( )-2-mino-- phosphonovleri i), whih were bth pplie uring the reoring perio. Glyinergi mipsc were reore in the presene of TTX (1 µm). The input resistne ws monitore ontinuously, n the reoring ws bnone if the resistne hnge more thn 1%. All spinl slie reorings were performe uner ouble-blin onition. Dt quisition n nlysis of postsynpti urrents were one s esribe previously 47,48. Clyel slie preprtion n reoring. Prsgittl brinstem slies (1-µm thik) ontining the MNTB were prepre from 12- to 18-y-ol mie of either sex using vibrtome (7 SMZ, Cmpen Instrument). For the issetion n storge of slies, we use solution ontining (in mm) 9 NCl, 2 NHCO 3, 2 gluose, surose, 2. KCl, 1.2 NH 2 PO 4,.1 CCl 2, 3 MgCl 2,.4 sorbi i, 3 myo-inositol n 2 soium pyruvte (9% O 2 n % CO 2 ). The slies were inubte for 3 min t 37 C n then hel t room temperture (22 24 C). Whole-ell urrent reorings were me from lyx of Hel terminls n prinipl neurons of the MNTB. The smpling intervl n filter setting were 2 µs n 2.9 khz, respetively. Cells were visulize by ifferentil interferene ontrst mirosopy through 4 wter-immersion objetive using n upright Olympus mirosope. All experiments were me with the EPC-1 mplifier. The holing potentil ws mv. The bth solution (22 24 C) ontine (in mm) 12 NCl, 2. KCl, 1 MgCl 2, 2 CCl 2, 2 extrose, 1.2 NH 2 PO 4,.4 sorbi i, 3 myo-inositol, 2 soium pyruvte n 2 NHCO 3, ph 7.4, bubble with 9% O 2 n % CO 2. The presynpti n postsynpti pipette solution ontine (in mm) 12 Cs-gluonte, 2 CsCl, 4 Mg-ATP, 1 N 2 -phosphoretine,.3 GTP, 1 HEPES n. 1,2-bis-(o-minophenoxy)- ethne-n,n,n,n -tetreti i (BAPTA), ph 7.2, juste with CsOH. The BAPTA onentrtion ( µm) mimike the enogenous lium buffer pity t lyes 49. Strtle response mesurement. Aousti strtle response test. Strtle responses were mesure using SR-LAB test sttions n softwre (Sn Diego Instruments, Sn Diego, CA). The metho of strtle response mesurement ws moifie from previous stuy 16. Briefly, test sttions were both stnrize n librte. Iniviul mie were then ple in the Plexigls holing yliner for -min limtion perio. A bkgroun noise level of 7 B ws mintine over the urtion of the test session. The reoring perio of ousti strtle responses onsiste of six bloks. Eh blok onsiste of six trils: one ontrol tril with no stimulus (bseline) n five trils with single 4-ms soun of 8, 9, 1, 11 or 12 B presente in rnom orer with 1- to 2-s intertril intervl. The entire session omprise 36 trils n took ~1 min. The strtle mplitue ws mesure every 1 ms over 6-ms perio beginning t the onset of the strtle stimulus tril. The strtle response ws ientifie s the mximum strtle mplitue (V m ) to given stimulus minus the V m to no stimulus (bseline). Ttile strtle response test. The Sn Diego Instruments, In. (Sn Diego, CA) SR-LAB system ws use to eliver the ttile ir-puff stimulus n mesure the flinh response of the mouse. The mouse ws ple in Plexigls yliner, whih ws lote within ventilte soun-ttenuting hmber to reue noise ontmintion, n left unisturbe for -min limtion perio. Air-puff stimuli of 1. or PSI ontrolle by regultor were elivere to the bks of the mie for urtion of 1 ms. Vibrtions ourring in the Plexigls yliner use by whole-boy strtle responses were reore over 2 ms fter presenttion of the stimulus n were trnsue into nlog signls by piezoeletri unit tthe to the pltform. The entire session omprise six trils with 1- to 2-min intertril intervl. Righting reflex test. Eh mouse ws ple on its sie on flt surfe, n the time it took to turn over to rest in the norml position with ll four feet on the groun ws reore. The utoff time ws set s 3 s. This experiment ws repete three times, n the verge time reore ws ientifie s the righting reflex time. nture NEUROSCIENCE oi:1.138/nn.361