The Journal of Physiology

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1 J Physiol (217) pp Heteromeri α/β glyine reeptors regulte exitility in prvlumin-expressing dorsl horn neurons through phsi nd toni glyinergi inhiition M. A. Grdwell 1,2,K.A.Boyle 3,R.J.llister 1,2,D.I.Hughes 3 nd B. A. Grhm 1,2 1 Shool of Biomedil Sienes nd Phrmy, Fulty of Helth, University of Newstle, llghn, NSW, Austrli 2 Hunter Medil Reserh Institute (HMRI), New Lmton Heights, NSW, Austrli 3 Institute of Neurosiene Psyhology, ollege of Medil, Veterinry nd Life Sienes, University of Glsgow, Glsgow, UK The Journl of Physiology Key points Spinl prvlumin-expressing interneurons hve een identified s ritil soure of inhiition to regulte sensory thresholds y gting mehnil inputs in the dorsl horn. This study ssessed the inhiitory regultion of the prvlumin-expressing interneurons, showing tht synpti nd toni glyinergi urrents dominte, loking neuronl or glil glyine trnsporters enhnes toni glyinergi urrents, nd these mnipultions redue exitility. Synptilly relesed glyine lso enhned toni glyinergi urrents nd resulted in deresed prvlumin-expressing interneuron exitility. Anlysis of the glyine reeptor properties mediting inhiition of prvlumin neurons, s well s single hnnel reordings, indites tht heteromeriα/β suunit-ontining reeptors underlie oth synpti nd toni glyinergi urrents. Our findings indite tht glyinergi inhiition provides ritil ontrol of exitility in prvlumin-expressing interneurons in the dorsl horn nd represents phrmologil trget to mnipulte spinl sensory proessing. Astrt The dorsl horn (DH) of the spinl ord is n importnt site for modlity-speifi proessing of sensory informtion nd is essentil for ontextully relevnt sensory experiene. Prvlumin-expressing inhiitory interneurons (PV+ INs) hve funtionl properties nd onnetivity tht enles them to segregte ttile nd noieptive informtion. Here we exmine inhiitory drive to PV+ INs using trgeted pth-lmp reording in spinl ord slies from dult trnsgeni mie tht express enhned green fluoresent protein in PV+ INs. Anlysis of inhiitory synpti urrents showed glyinergi trnsmission is the dominnt form of phsi inhiition to PV+ INs. In ddition, PV+ INs expressed roust glyine-medited toni urrents; however, we found no evidene for toni GABAergi urrents. Mnipultion of extrellulr glyine y loking either, or oth, the glil nd neuronl glyine trnsporters mrkedly deresed PV+ IN exitility, s ssessed y tion potentil dishrge. This deresed exitility ws replited when toni glyinergi urrents were inresed y eletrilly tivting glyinergi synpses. Finlly, we show tht oth phsi nd toni forms of glyinergi inhiition re medited y heteromeri α/β glyine reeptors. This differs from GABA A reeptors in the dorsl horn, where different reeptor stoihiometries underlie phsi nd toni inhiition. Together these dt suggest oth phsi nd toni glyinergi inhiition regulte the output of PV+ INs nd ontriute to the proessing nd segregtion of ttile nd noieptive informtion. The shred stoihiometry for phsi nd toni glyine reeptors suggests phrmology is unlikely to e le to seletively trget eh form of inhiition in PV+ INs. (Resumitted 16 July 217; epted fter revision 1 Septemer 217; first pulished online 14 Septemer 217) orresponding uthor B. A. Grhm: Shool of Biomedil Sienes nd Phrmy, Fulty of Helth, University of Newstle, llghn, NSW 238, Austrli. Emil: rett.grhm@newstle.edu.u This is n open ess rtile under the terms of the retive ommons Attriution Liense, whih permits use, distriution nd reprodution in ny medium, provided the originl work is properly ited. DOI: /JP274926

2 7186 M. A. Grdwell nd others J Physiol Introdution The dorsl horn (DH) of the spinl ord ontins heterogeneous popultion of neurons tht proess informtion relted to noieptive, light touh, ith nd therml modlities (Todd, 21). Integrtion or segregtion of these modlities is onsidered ritil for norml sensory experiene, nd inpproprite mixing of sensory signls in the DH is thought to use errnt senstions suh s llodyni, hyperlgesi, spontneous pin nd ith. A lrge literture, eginning with the gte ontrol theory of pin (Melzk & Wll, 1965), ssigns ruil role for synpti inhiition in mintining ontextully relevnt sensory proessing, employing oth in vivo (Yksh, 1989; Ishikw et l. 2) nd in vitro preprtions (Rusheweyh & Sndkuhler, 25; Tkzw & MDermott, 21). More reently, series of sophistited studies employing neuron-speifi trnsplnttion (Brz et l. 212), pired reordings (Lu & Perl, 23), nd trgeted ltion, silening nd tivtion (Dun et l. 214; Bourne et l. 215; Foster et l. 215; Petitjen et l. 215; ui et l. 216) hve onfirmed the importne of inhiition for norml sensory proessing ndinthedevelopmentofertinpinsttes. We hve hrterized speifi popultion of interneurons tht express the lium inding protein prvlumin (PV+)(Hugheset l. 212) nd re rodly onsidered inhiitory, lthough smll popultion of exittory PV+ INs hs lso een reported (Antl et l. 1991). Despite this, our work in the PV-green fluoresent protein (GFP) trnsgeni mouse showed tht GFP-lelled PV+ INs hd eletrophysiologil, morphologil nd neurohemil properties onsistent with n inhiitory phenotype; nd tht PV-GFP xon vriosities were vesiulr GABA trnsporter (VGAT) positive. This work lso showed tht PV+ INs reeive monosynpti input from myelinted fferents, nd provide soure of xo-xoni input onto the entrl terminls of these fferents (Hughes et l. 212). Suh onnetivity implies feed-forwrd inhiitory iruit ould exist to seletively regulte the effet of innouous ttile input during spinl sensory proessing. It follows tht redution in the inhiition medited y these neurons ould ontriute to development of ttile llodyni. Susequent work hs verified these preditions y showing tht geneti ltion of PV+ INs in the DH redues sensory thresholds s oserved in llodyni (Petitjen et l. 215). Furthermore, this work showed tht inresed tivtion of PV+ INs in neuropthi mie restored norml sensory thresholds nd ttenuted llodyni. Given this role for PV+ INs in modlity-speifi sensory proessing, the mnner in whih their tivtion or exitility is regulted will e importnt for developing strtegies to lter the tivity of this popultion. For exmple, we hve shown tht PV+ INs n support high frequeny tion potentil (AP) dishrge nd express the hyperpolriztion-tivted tion urrent (I h ), whih is ssoited with repetitive AP firing (Hughes et l. 212). Together, these properties mke PV+ INs powerful soure of inhiition in the DH. Prdoxilly, we lso showed tht PV+ INs hve reltively high rheose urrent nd reeive wek synpti exittion, whih imply they my e diffiult to reruit. In order to determine how PV+ INs re reruited during spinl sensory proessing it is importnt to lso fully hrterize the inhiition they reeive. It is well estlished tht oth GABA nd glyine medite fst synpti inhiition in the DH (hery & de Konink, 1999; Grhm et l. 23; Bei & Fitzgerld, 24; Anderson et l. 29; Tkzw & MDermott, 21) nd tht popultions of DH neurons n reeive inhiition dominted y GABA, glyine, or oth. It hs lso een shown tht dorsoventrl grdient exists in the inhiitory trnsmitter phenotype within the DH. GABA is more dominnt in superfiil lyers, wheres glyine domintes in the deep DH nd ventrl horn (ronin et l. 24; Anderson et l. 29). In ddition to their role in fst synpti inhiition, oth GABA nd glyine re known to medite toni inhiitory urrents tht re ple of suppressing AP dishrge (Tkzw & MDermott, 21). Therefore, in this study we ssess the levels of synpti nd toni inhiition medited y GABA nd glyine onto PV+ INs, determine their respetive roles in regulting AP dishrge, nd explore the stoihiometry of synpti nd extrsynpti reeptor popultions. This informtion provides new insights on how ltering GABAor glyinergi trnsmission n ffet spinl inhiition tht is medited y PV+ INs nd how these mehnisms might e trgeted phrmologilly. Methods All experiments were pproved y the University of Newstle (UoN) Animl re nd Ethis ommittee. We used trnsgeni mouse line (oth sexes; ged 29 ± 3 weeks; ody weight g) tht expressed enhned green fluoresent protein (egfp) under the ontrol of the prvlumin promoter (PVeGFP: Meyer et l. 22). The line ws originlly generted y Professor Hn Monyer nd red on the Bl kground with permission t UoN. UV illumintion of the ers of PVeGFP mie ws used to identify egfp-positive nimls: fst twith musle fires lso express prvlumin (nd y ssoition egfp). Aute spinl slie preprtion Spinl ord slies were prepred using previously desried tehniques (Grhm et l. 23; Smith et l. 215). Briefly, nimls (PVeGFP) were nesthetized with ketmine

3 J Physiol Phsi nd toni glyinergi inhiition in the spinl dorsl horn 7187 (1 mg kg 1 I.P.) nd depitted. Using ventrl pproh, the lumosrl enlrgement of the spinl ord ws rpidly removed nd pled in ie-old surose-sustituted rtifiil ererospinl fluid (ASF) ontining (in mm): 25 surose, 25 NHO 2,1gluose, 2.5 Kl, 1 NH 2 PO 4,1Mgl 2 nd 2.5 l 2. Trnsverse or prsgittl slies (from L3 L5 segments ut t 3 nd 2 μm thikness, respetively) were otined using virting mirotome (Lei VT-1S, Heidelerg, Germny). Slies were then trnsferred to n interfe inution hmer ontining oxygented ASF (118 mm Nl sustituted for surose) nd llowed to equilirte for 1 h (t ) prior to reording. Eletrophysiology Slies were trnsferred to reording hmer nd ontinully superperfused (th volume.4 ml; exhnge rte 4 6 th volumes min 1 ) with ASF uled with ronox (95% O 2 nd 5% O 2 )tohievefinlph of Neurons were visulized using ner-infrred differentil interferene ontrst optis. PV+ INs were identified under fluoresene using fluoresein isothioynte (FIT) filter set (488 nm exittion nd 58 nm emission filters) (Hughes et l. 212). Neuroiotin (.2%) ws inluded in internl solutions for post ho onfirmtion of GFP expression in suset of reordings (Vetor Lortories, Peterorough, UK). As previously reported, the mjority of PVeGFP-expressing ells re immunopositive for prvlumin (Hughes et l. 212). Reordings were limited to neurons loted within or lose to the sustnti geltinos. This re is esily identified y its trnsluent pperne in trnsverse nd prsgittl spinl ord slies nd ontins lerly disernle plexus of PV+ INs. All reordings were otined t room temperture (21 24 ). Pth pipettes (4 8 M ) were filled with one of two internl solutions. A esium hloride-sed internl ws used for reording inhiitory urrents. This internl ontined (in mm): 13 sl,1hepes,1egta,1mgl 2,2ATPnd.3GTP (ph djusted to 7.35 with 1 M soh). A potssium gluonte-sed internl solution ws used in experiments where the tion potentil dishrge ws exmined nd nlysed. This internl ontined (in mm): 135 KH 3 SO 4, 6 Nl, 2 Mgl 2, 1 Hepes,.1 EGTA, 2 MgATP,.3 NGTP (ph djusted to 7.3 with KOH). All whole-ell reordings were first estlished in voltge lmp (holding potentil 7 mv). Dt were quired using Multilmp 7B mplifier (Moleulr Devies, Sunnyvle, A, USA), digitized online (smpled t 1 2 khz nd filtered t 5 1 khz) vi n IT-18 omputer interfe (Instruteh, Long Islnd, NY, USA) nd stored on Mintosh omputer using Axogrph Xsoftwre(Kgi,Berkley,A,USA).Afterotining the whole-ell reording onfigurtion, series resistne, neuron input resistne nd memrne pitne were lulted sed on the response to 5 mv hyperpolrizing voltge step (1 ms durtion) from holding potentil of 7 mv. These vlues were monitored t the eginning nd end of eh reording session nd dt were rejeted ifvlueshngedymorethn3%. Miniture inhiitory postsynpti urrents (mipss), whih represent the postsynpti response to spontneous relese of single vesiles of neurotrnsmitter (Ktz & Miledi, 1969; Bekkers & Stevens, 1989; llister & Wlmsley, 1996), were phrmologilly isolted y inluding the sodium hnnel loker tetrodotoxin (TTX; 1 μm) nd the AMPA/kinte reeptor ntgonist 6-yno-7-nitroquinoxline-2,3-dione (NQX; 1 μm) in the th perfuste. The urrents reorded under these onditions in the mouse DH re medited y the tion of GABA, glyine, or oth. In order to isolte glyinergi urrents the GABA A reeptor ntgonist iuulline (1 μm) ws next dded to the th. In suset of experiments NMDA reeptor ntgonist DL-2-mino-5-phosphonovleri id (APV; 5 μm) ws then th pplied to exlude NMDA reeptor tivtion, ut this hd no effet. All remining urrents were olished y th pplition of stryhnine (1 μm). At lest 3 min of dt were quired under oth onditions (pre- nd post-iuulline pplition) for nlysis. In some experiments pirotoxin (1 μm) nd/or lindne (3 μm) ws pplied to test for the presene of homomeri glyine reeptors. Similrly, seletive glyine trnsporter lokers Org (1 μm) nd Org (1μM) were th-pplied to ssess the role of the neuronl nd glil trnsporters (GlyT2 nd GlyT1, respetively) in toni urrent hrteristis. Outside-out memrne pthes (Hmill et l. 1981) were exised t the onlusion of some PV+ IN reordings nd exposed to th pplition of μm glyine to evoke single hnnel glyine reeptor-medited urrents. Single hnnel events were filtered t 1 khz. A seprte series of experiments ssessed PV+ IN AP dishrge in urrent-lmp mode using the potssium gluonte-sed internl solution, nd ridge lne monitored throughout reordings. Neuronl exitility nd AP dishrge were studied y injeting series of depolrizing step urrents (8 ms durtion, 2 pa inrements, delivered every 8 s) into the reorded neuron whenitwsheldtmemrnepotentilof 6 mv. During this protool voltge defletions were limited, to void ell dmge, y terminting the protool if sustined depolriztions exeeded 2 mv (i.e. in prts of the voltge tre not ontining APs). After this initil hrteriztion of exitility glyinergi signlling ws modified y either loking glyine reeptors (GlyRs) or GlyT1/GlyT2 trnsporters with stryhnine or Org nd Org 25543, respetively. Exitility ws ssessed when eitherglyrsorthetrnsporterwereloked.

4 7188 M. A. Grdwell nd others J Physiol To ssess the role of endogenous glyine, eletrilly evoked IPSs (eipss) were eliited using ipolr glss stimulting eletrode (2 μm tip seprtion) positioned 2 8 μm rostrl or udl to the reorded PV+ IN. A numer of stimultion protools were pplied inluding single 1 ms durtion stimulus, 1 stimuli t 1 Hz, 1 stimuli t 2 Hz nd 2 stimuli t 2 Hz vi trnsistor-trnsistor logi (TTL)-driven ISO-Flex stimultor (A.M.P.I., Jeruslem, Isrel). A protool tht delivered three suessive trils of depolrizing step urrents (8 ms durtion, 2 pa inrements, delivered every1s)tothereordedpv+ IN ws used to ssess the effet of stimultion-evoked endogenous glyine on AP dishrge. Toni glyinergi urrents were evoked prior to the seond depolrizing step tril (Fig. 4B) using ipolr stimultion (2 stimuli, t 2 Hz). Importntly, ipolr stimultion ws mintined t 1% elow threshold for N + hnnel tivtion in ll protools nd reordings were mde in the presene of NQX nd iuulline. Dt nlysis Anlysis of mipss ws ompleted using sliding templte method (semi-utomted proedure within Axogrph softwre pkge) to detet nd pture mipss (lements & Bekkers, 1997). All ptured mipss were inspeted individully nd exluded from further nlysis if they ontined overlpping mipss or hd n unstle seline efore the rise or during the dey phse of the mipss. Dt were lso rejeted if signifint trend in either mips mplitude or inter-event intervl ws oserved during the nlysis period. The pek mplitude nd rise time of mipss were mesured for ll epted events (vi semi-utomted proedures in Axogrph) nd instntneous frequeny ws lulted s the reiprol of inter-event intervl. Anlysis of the mips dey time onstnt (lulted over 2 8% of the dey phse) ws performed on verged mipss, generted y ligning the rising phse of ll epted events in reording. Toni urrents were nlysed y lulting the hnge in seline holding urrent nd stndrd devition (seline noise) efore nd fter th pplition of iuulline, stryhnine, APV, pirotoxin, lindne, Org 24598, Org 25543, or oth glyine trnsporter lokers. Single hnnel events reorded in outside-out memrne pthes were ptured from ontinuous reordings using n mplitude threshold detetion nd ll-points histogrms were then onstruted to lulte men single hnnel urrent nd ondutne for eh memrne pth (n = 16). In experiments tht ssessed the reltionship etween evoked endogenous glyine relese nd toni glyinergi urrents we mesured seline urrent nd seline noise over 1 ms epoh (3 4 ms fter the stimulus rteft). Mesurements were mde over this epoh to void ontmintion y evoked synpti urrents. Dt were normlized to pre-stimulus urrent vlues. The time for the evoked urrent to return to seline ws tken s the time from the stimulus rteft until the urrent returned to zero. In experiments involving drug pplition dt were normlized to ontrol responses (no drug) nd tres zeroed to seline. AP dishrge ws lssified ording to previously pulished riteri (Grhm et l. 24, 27). In greement with our previous work, PV+ INs expressed either toni dishrge, hrterized y persistent AP dishrge throughout the depolrizing step, or initil ursting dishrge, hrterized y AP dishrge limited to the eginning of the depolrizing step. The riterion for inlusion of neuron in this nlysis ws resting memrne potentil more negtive thn 5 mv nd series resistne <3 M (filtered t 5 khz). In our nlysis of AP dishrge, individul APs eliited y step-urrent injetion were ptured using derivtive threshold method (dv/dt 15 V s 1 ) with the infletion point during spike initition defined s AP threshold. The differene etween AP threshold nd its mximum positivepekwsdefinedsapmplitude.apsewidth ws mesured t AP threshold. AP fter-hyperpolriztion mplitude ws tken s the differene etween AP threshold nd the mximum negtive pek following the AP. Rheose urrent ws defined s the smllest step urrent tht would eliit t lest one AP. In reordings tht ssessed the impt of ltered glyinergi inhiition on PV+ IN dishrge (i.e. stryhnine or glyine trnsporter loker pplition), the impt of neuron-to-neuron vriility in rheose urrent ws removed y normliztion. This ws hieved y normlizing responses to rheose nd then reporting susequent responses in 2 pa inrements ove rheose. In ddition, the numer of APs eliited nd instntneous frequeny were normlized to vlues otined in the 4 pa urrent step response under ontrol onditions (i.e. no drug). Therefore, AP numer nd instntneous frequeny t this 4 pa step re reported s 1%. Drug-indued differenes in AP numer nd instntneous frequeny re reported s perentge of this vlue. hnges in AP dishrge during endogenously evoked glyine were ssessed y normlizing vlues otined from the seond nd third depolrizing step trils to dt otined from the first tril. Single hnnel events reorded in outside-out memrne pthes were ptured from ontinuous reordings using mplitude threshold detetion nd llpoints histogrms were then onstruted. To lulte men single hnnel urrent nd ondutne, one or two Gussin distriutions were fitted to ll-points histogrms. All drugs were otined from Sigm Aldrih (Sydney, Austrli) unless otherwise stted.

5 J Physiol Phsi nd toni glyinergi inhiition in the spinl dorsl horn 7189 Sttistis Sttistil nlysis ws rried out using SPSS v1 (SPSS In. higo, IL, USA). Student s t tests were used to ompre vriles etween genotypes. Dt tht filed Levene s test for homogeneity of vrine were ompred using the non-prmetri Kruskl Wllis test. Sttistil signifine ws set t P <.5. All vlues re presented s mens ± SEM. Results Glyinergi synpti inhiition domintes in PV+ INs To ssess the reltive ontriution of GABA nd glyine to synpti inhiition in PV+ INs we reorded mixed mipss in the presene of NQX nd TTX (n = 11), nd then ssessed their iuulline sensitivity (Fig. 1A). Any urrents remining fter the ddition of iuulline (i.e. iuulline insensitive) were onsidered glyinergi. This ws onfirmed y th pplition of the glyine reeptor ntgonist stryhnine, whih olished ll remining tivity. In most reordings from PV+ INs omprison of mixed nd glyinergi mipss indited tht there ws little differene etween these onditions (Fig. 1B). Plots showing the hnge in mips frequeny, mplitude, rise time nd dey time onstnt revel tht mixed nd glyinergi mips properties were reltively stle, lthough mips frequeny ws redued in 3 of 11 reordings (Fig. 1). Despite this, the men vlue for frequeny (1.19 ±.26 Hz vs..79 ±.17 Hz), mplitude (19 ± 18 pa vs.99± 17 pa), rise time (.87 ±.7 ms vs..85 ±.9 ms), dey time onstnt (6.67 ±.77 ms vs ±.48 ms) nd goodness of fit of the dey time onstnt (ssessed using the sum of squres error:.67 ±.19 vs..64 ±.14) were similr in mixed nd glyinergi mipss. This nlysis suggests glyinergi trnsmission is the dominnt form of inhiition to PV+ INs with GABAergi synpti trnsmission plying less prominent role. PV+ INs reeive toni glyinergi inhiition A striking feture in the mips reordings mde fter the ddition of stryhnine ws ler shift in the holding urrent nd redution in seline noise (Fig. 2A). This is further emphsized in ll-points histogrm plots for the two epohs: efore nd fter stryhnine ddition. There is ler shift to the left (i.e. redution) in holding urrent s well s nrrowing of the distriution following the ddition of stryhnine. Together, these oservtions indite the presene of toni glyinergi urrent under seline onditions. Group dt omprisons (n = 22) show tht stryhnine signifintly redued the holding urrent in our reordings y 5 pa ( 99 ± 22 pa vs. 49 ± 8 pa efore nd fter stryhnine, P =.12), nd the noise (stndrd devition (SD)) of the seline urrent (9.9 ± 1.59 pa vs. 3.4 ±.46 pa, P <.1). In ontrst, there ws no evidene for the existene of toni GABAergi urrents in PV+ INs fter th pplition of iuulline (n = 11) (Fig. 2B). Group omprisons of holding urrent ( 118 ± 33 pa vs. 12 ± 36 pa, P =.66), nd seline urrent noise (9.7 ± 1.75 pa vs ± 1.96 pa, P =.46) showed no hnge fter iuulline pplition. To exlude possile ontriution of NMDA reeptors to toni urrents suset of experiments ssessed the effet of APV th pplition (n = 6; 2 mie). In no instne did APV use redution in holding urrent ( 14 ± 9pA vs. 111 ± 11 pa, P =.72), or seline urrent noise (6.88 ± 1.21 pa vs ± 1.4 pa, P =.254). In ontrst, stryhnine signifintly deresed holding urrent ( 14 ± 9pAvs. 94 ± 1 pa, P =.11) nd seline urrent noise (6.88 ± 1.21 pa vs ±.27 pa, P =.9) onsistent with our erlier reordings. Thus, PV+ INs in the DH express roust toni urrents medited y glyine, ut we find no evidene for the existene of toni GABAergi or glutmtergi urrents. Glyine trnsporter lokde enhnes toni urrents To further ssess the impt of ltered extrellulr glyine levels on toni inhiition of PV+ INs we undertook series of experiments tht loked the glil (GlyT1, n = 11; 6 mie) nd neuronl (GlyT2, n = 1; 5 mie) glyine trnsporters. Bth ddition of the GlyT1 loker Org inresed the holding urrent nd seline noise. onversely, holding urrent nd noise were redued drmtilly y th pplition of stryhnine (Fig. 3A). These oservtions re further quntified in the ll-points histogrm, whih shows rightwrd shift nd rodening of the urrent distriution fter ddition of Org These effets were olished fter the pplition of stryhnine. Note, the redued mplitude of the toni glyine urrents oserved here, ompred to those oserved in Fig. 2A, emphsizes ell to ell vriility in toni glyine urrent mplitude nd the potentil for rundown ssoited with the longer reordings neessitted y these experiments. Bth ddition of the GlyT2 loker Org produed virtully identil results to GlyT1 lokde. It enhned the toni urrent nd used rightwrd shift nd rodening in the ll-points histogrm (Fig. 3B). These effets were ompletely reversed y th pplition of stryhnine. Group dt omprisons show tht lok of oth glil (GlyT1) nd neuronl (GlyT2) trnsport lokers inresed the holding urrent y pproximtely doule (61 ± 5pAvs. 16 ± 15 pa GlyT1, P =.3; 54 ± 5pAvs. 116 ± 18 pa GlyT2, P =.4) s well s the ssoited seline noise (SD) (5.4 ±.81 pa vs. 9.8 ± 1.1 pa GlyT1, P =.1; 5.56 ±.71 pa vs ± 1.39 pa GlyT2, P =.15). Thus, oth glil nd

6 719 M. A. Grdwell nd others J Physiol neuronl regultion of extrsynpti glyine levels pper to ply n importnt role in mediting toni urrents in PV+ INs. In suset of experiments (n = 8; 4 mie) oth trnsporters were loked (i.e. GlyT1 nd GlyT2) nd, irrespetive of the order of lokde, ddition of the seond trnsporter loker used further inrese in oth holding urrent (67 ± 8pAvs. 324 ± 44 pa, GlyT1 then GlyT2, P =.1) nd seline noise (6.57 ±.91 pa vs.19.6 ± 1.22 pa. GlyT1 then GlyT2, P =.1) (Fig. 3). This effet ws greter thn the summed effet of eh trnsporter lone, suggesting tht under onditions where only one trnsporter is loked some A iuulline stryhnine BF FL 1 pa NB GFP 2 s mixed glyinergi 1 µm B mixed mipss τ = 6.5 ms Frequeny (Hz) Amplitude (pa) mixed glyinergi mixed glyinergi glyinergi mipss 1 pa τ = 6.27 ms Rise time (ms) Dey time onstnt (ms) ms mixed glyinergi mixed glyinergi Figure 1. Synpti inhiition in PV+ INs A, tre showing ontinuous mips reording from PV+ IN. Mixed mipss were reorded in the presene of NQX (1 μm) nd TTX (1 μm). Glyinergi mipss re reveled following th ddition of the GABA A reeptor ntgonist iuulline (1 μm). Bth ddition of the glyine reeptor ntgonist stryhnine (1 μm) olished glyinergi mipss. Inset, neurons were identified in infrred-differentil interferene ontrst (IR-DI) (top left), nd fluoresene (top right) ws susequently used to onfirm the presene of GFP. In some reordings post ho reovery onfirmed neuroiotin-filled ells (ottom left) were GFP+ (ottom right). B, left tres show overlid mixed nd glyinergi mipss ptured from the reording epohs outlined y the light nd drk grey retngles in A. Right tres show verged dey wveforms for mixed nd glyinergi mipss. Red line shows fit for dey time onstnt lultion. They were virtully identil for mixed nd glyinergi mipss., plots ompring men mips properties (frequeny, mplitude, rise time nd dey time onstnt) in PV+ INs under reording onditions tht revel mixed nd glyinergi mipss. The mjority of reordings showed little hnge etween onditions; however, four reordings showed redution in mips frequeny when glyinergi mipss were isolted. Most PV+ INs showed little hnge in mips mplitude, rise time nd dey time onstnt etween onditions. [olour figure n e viewed t wileyonlinelirry.om]

7 J Physiol Phsi nd toni glyinergi inhiition in the spinl dorsl horn 7191 A stryhnine residul pity remins in the other trnsporter system to ler glyine from the extrellulr spe. Blokde of oth trnsporters then uses rpid elevtion in glyine onentrtion, further enhning toni glyine urrents nd the ssoited holding urrent nd seline noise. B ount (x1 3 ) ount (x1 3 ) urrent (pa) iuulline 8 urrent (pa) (pa) Bseline urrent (pa) Bseline urrent 1 pa 3/5 s ns SD (pa) urrent Figure 2. PV+ INs exhiit toni glyine, ut not GABAergi urrents A, tre shows ontinuous glyinergi mips reording (in the presene of TTX, NQX nd iuulline), efore nd fter th ddition of stryhnine. Note tht in ddition to olishing glyinergi mipss, stryhnine uses shift in holding urrent nd redution in seline noise. These fetures re inditive of toni glyine-medited urrent. Insets ( nd ) show expnded epohs highlighting the differene in seline noise efore nd fter stryhnine exposure. An ll-points histogrm (ottom left) from the ove epohs quntify the shift in holding urrent nd redution in seline noise fter stryhnine exposure. Br grphs (ottom right) re group dt showing signifint redution in holding urrent nd seline noise in PV+ INs. Dt for eh neuron used in men lultion for r grphs re shown s filled irles. B, sme nlysis s for A, exept the ontinuous tre shows GABAergi mipss (in the presene of TTX, NQX nd stryhnine) efore nd fter th ddition of iuulline. Note, iuulline olishes ll GABAergi mipss ut does not lter the holding urrent or seline noise. This SD (pa) urrent ns Glyinergi inhiition regultes PV+ IN tion potentil dishrge In order to test the funtionl onsequenes of the dominnt glyinergi inhiitory ontrol of PV+ INs, we ssessed their intrinsi exitility efore nd fter mnipultion of glyinergi input. PV+ IN dishrgews first ssessed y reording the AP dishrge evoked during injetion of series of depolrizing urrent steps. Unlike lssil fst spiking dishrge desried for PV+ INs in higher NS regions (hippompus, ortex) spinl DH PV + IN dishrge ould e lssified s either toni firing, hrterized y sustined dishrge for the durtion of urrent injetion; or initil ursting, where dishrge ws limited to the eginning of urrent injetion; onsistent with our previous report (Hughes et l. 212). Under ontrol onditions, oth the numer of evoked APs nd men instntneous firing frequeny (F) inresed with inresing levels of urrent injetion (from to 1 pa). These dt re omined nd presented s F I plots in Fig. 4 with vlues normlized to the level of dishrge evoked y the 4 pa urrent step (ove rheose) under ontrol onditions. After this initil tril we either loked glyinergi inhiition y th-pplying stryhnine (1 μm), or enhned it y pplying the GlyT1 loker nd/or GlyT2 loker. Ation potentil dishrge ws then ressessed 3 min fter drug pplition from the sme ontrol memrne potentil. In the stryhnine experiments (n = 14; 6 mie) hlf the reordings initilly exhiited toni firing nd hlf showed initil ursting dishrge. omprisons of ontrol nd stryhnine dt showed signifint shift to the left in the F I urve inditing PV+ INs eme more exitle (Fig. 4A). AP dishrge rte more thn douled (216 ± 38%, when normlized to the 4 pa step, P =.9). In ontrst, men instntneous frequeny did not hnge (12 ± 6%, normlized to the 4 pa step, P =.754). In some initil ursting neurons (5/7) ddition of stryhnine hnged or onverted PV+ IN dishrge to the toni firing mode. Likewise, stryhnine ltered the dishrge of toni firing neurons y inresing the numer of APs dishrged per step, without onverting suggests toni GABA urrents re sent in PV+ INs. Insets ( nd ) show no differene in seline noise efore or during iuulline exposure. An ll-points histogrm (ottom left) nd the r grphs (ottom right) show tht holding urrent nd seline noise in PV+ INs do not differ efore or during iuulline exposure.

8 7192 M. A. Grdwell nd others J Physiol A ount (x 1 3 ) B ount (x 1 3 ) ount (x 1 3 ) Org G Org N urrent (pa) urrent (pa) Org N + Org G urrent (pa) Bseline urrent (pa) Bseline urrent (pa) Bseline urrent (pa) Stryhnine 1 pa 6 s Stryhnine Stryhnine urrent SD (pa) urrent SD (pa) urrent SD (pa) pa.5 s 5 Figure 3. PV+ IN toni glyine urrents re enhned y glyine trnsporter lok A, tre shows ontinuous glyinergi mips reording (in the presene of TTX, NQX nd iuulline) during sequentil ddition of the GlyT1 loker Org (Org G) nd stryhnine to the th. Org enhned the toni glyine urrent, s shown y n firing mode. These dt suggest tht under our reording onditions initil ursting neurons re strongly influened y glyinergi inhiition, nd its removl inreses their pity to support sustined AP dishrge. To further explore the lk or smll effet of glyinergi inhiition on toni firing neurons we inresed extrellulr glyine onentrtion y th-pplying Org (n = 11; 3 mie) nd Org (n = 11; 8 mie) either independently or together (n = 9; 5 mie). In these GlyT loker experiments, this mnipultion used rightwrd shift in the F I plots for oth toni firing (n = 16) nd initil ursting (n = 7) neurons (Fig. 4B D). Group dt omprisons show tht pplition of glyine trnsport lokers deresed the numer of tion potentils elow seline vlues (38 ± 9% GlyT1, P =.; 39 ± 14% GlyT2, P =.2; ± % GlyT1 + GlyT2, P =.1, normlized to vlues t 4 pa urrent step) nd lso the instntneous frequeny (73 ± 14% GlyT1, P =.86; 65 ± 16% GlyT2, P =.49; ± % GlyT1 + GlyT2, P =.1). In most ses the toni firing dishrge pttern ws onverted to initil ursting or single spiking (17/21) nd the initil ursting dishrge pttern onverted to single spiking or relutnt firing (6/1) following GlyT lok. Thus, glyine levels nd the resulting inhiition ply n importnt role in shping firing mode in oth toni firing nd initil ursting PV+ INs. inresed holding urrent nd seline noise. Addition of stryhnine olished the toni glyinergi urrent. Insets (, nd ) ove the ontinuous mips tre highlight the enhned seline noise fter the ddition of Org () nd its redution y stryhnine (). An ll-points histogrm (ottom left) from epohs, nd quntifies the holding urrent shift, nd seline noise ltertions during Org nd stryhnine exposure. Br grphs (ottom right) ompre group dt nd show tht Org nd stryhnine shift oth holding urrent nd seline noise in PV+ INs. B, tre shows ontinuous reording of glyinergi mipss during sequentil th pplition of the GlyT2 loker Org nd stryhnine. Org (Org N) lerly enhned the toni glyine urrent. Insets (, nd ) ove the mips tre show Org enhned seline noise wheres stryhnine redued it. An ll-points histogrm (ottom left) from, nd shows the shifts in holding urrent nd seline noise during Org nd stryhnine exposure. Br grphs (ottom right) ompre group dt showing Org nd stryhnine-relted holding urrent nd seline noise shifts in PV+ INs., tre shows ontinuous glyinergi mips reording, with the sequentil th ddition of the GlyT1 loker Org 24598, GlyT2 loker Org 25543, nd then stryhnine. o-exposure to Org nd Org drmtilly enhned the toni glyine urrent. Insets (, nd ) highlight the enhned seline noise during Org Org pplition, nd the stryhnine-medited redution. An ll-points histogrm (ottom left) from these epohs quntifies the holding urrent shift nd seline noise ltertions under Org Org nd stryhnine exposure. Br grphs (ottom right) ompre group dt showing Org Org nd stryhnine-relted holding urrent nd seline noise shifts in PV+ INs.

9 J Physiol Phsi nd toni glyinergi inhiition in the spinl dorsl horn 7193 A Org N+G ontrol Org G ontrol Org N ontrol Stryhnine ontrol B D Spike no. (%) Spike no. (%) Spike no. (%) Stryhnine Rh Org N 16 8 Rh Org G 14 7 Rh 4 3 mv 5 ms Org N+G 16 Spike no. (%) Rh 4 8 Spike frequeny (%) Spike frequeny (%) Spike frequeny (%) Spike frequeny (%) Rh Rh Rh Rh Step (pa) Vm (mv) Vm (mv) Vm (mv) Vm (mv) pa 3 ms Figure 4. PV+ IN exitility is sensitive to levels of glyinergi inhiition A D, left tres show tion potentil (AP) dishrge responses of PV+ INs during depolrizing urrent step injetions, efore nd fter mnipultion of glyinergi inhiition with stryhnine or glyine trnsporter inhiitors. Right plots show group dt summrizing AP dishrge nd frequeny per step ove rheose urrent, s well s suthreshold urrent voltge (I V) reltionships. A, stryhnine ntgonism of glyine reeptors inresed AP dishrge. This inrese is evident in the reltionship etween AP numer versus urrent step (left); however, AP dishrge frequeny (middle) ws not hnged y stryhnine. B, tres show AP dishrge deresed fter GlyT1 loker Org exposure. This derese is ler in the group dt plots of AP dishrge nd frequeny per step ove rheose., tres show AP dishrge deresed fter GlyT2 loker Org exposure. This derese is ler in group dt plots of AP dishrge nd frequeny per step ove rheose. D, tres show omined GlyT1 nd GlyT2 lok with Org nd Org drmtilly deresed AP dishrge. This drmti effet is ler in group dt plots of AP dishrge nd frequeny per step ove rheose. Insets in I V plots show exmple responses to 4 pa depolrizing urrent steps under ontrol onditions nd in the presene of glyine reeptor ntgonist or inhiitors. [olour figure n e viewed t wileyonlinelirry.om]

10 7194 M. A. Grdwell nd others J Physiol In n ttempt to explin the oserved hnges in spike numer nd frequeny when glyine levels re mnipulted we exmined the properties of the APs reorded t spike threshold. omprisons of the prend post-stryhnine dt showed rheose urrent ws unhnged (15 ± 14%, P =.726), wheres pplition of GlyT lokers inresed rheose urrent (136 ± 12% for GlyT1, P =.12; 178 ± 32% GlyT2, P =.39; 328 ± 71% GlyT1 + GlyT2, P =.12). Stryhnine pplition used n inrese in AP height (119 ± 5%, P =.3), ut no hnge in AP hlf-width (14 ± 3%, P =.141), or fter-hyperpolriztion pek (12 ± 9%, P =.835). For the most prt, pplition of GlyT lokers resulted in no hnge to AP pek mplitude (89 ± 3% GlyT1, P =.1; 14 ± 4% GlyT2, P =.334; 97 ± 2% GlyT1 + GlyT2, P =.191), slight inrese in AP hlf-width (115 ± 9% GlyT1, P =.136; 15 ± 4% GlyT2, P =.281; 112 ± 3% GlyT1 + GlyT2, P =.4), nd little hnge in fter-hyperpolriztion pek mplitude (96 ± 1% GlyT1, P =.662; 86 ± 8% GlyT2, P =.13; 76 ± 11% GlyT1 + GlyT2, P =.69). The ler inrese in rheose urrent helps explin the oserved hnges in AP numer nd frequeny. Simply put, enhned glyine reeptor tivtion redues neuronl input resistne (74 ± 6% GlyT1, P =.2; 84 ± 9% GlyT2, P =.9; 71 ± 9% GlyT1 + GlyT2, P =.13) nd neessittes inresed levels of urrent injetion to reh spike threshold. To further exmine this we nlysed the suthreshold voltge defletions during depolrizing urrent injetion steps (Fig. 4). Applition of stryhnine (n = 12) hd no effet on suthreshold voltge responses, wheres GlyT lok redued suthreshold voltge responses (68 ± 11% GlyT1, P =.46; 76 ± 8% GlyT2, P =.6; 62 ± 8% GlyT1 + GlyT2, P =.15 t 8 pa injetion) nd used right-shift in urrent voltge (I V) reltionships. Synptilly evoked toni glyine urrents To exmine whether endogenously relesed glyine ontriuted to toni urrents in PV+ INs we eletrilly evoked glyinergi IPSs using ipolr stimultion in the presene of NQX nd iuulline (n = 1; 2 mie). Eletril stimultion resulted in long lsting enhnement of the toni urrent (sometimes >1 s) seen s inresed holding urrent nd seline noise tht ws sensitive to stimulus durtion (Fig. 5A). From 3 to 4 ms post-stimultion; single eips did not signifintly inrese holding urrent (191 ± 67 pa vs. 28 ± 72 pa, P =.59), seline noise (9.59 ± 3.21 pa vs ± 2.38 pa, P =.628), nd returned to seline in.69 ±.14 s; 1 eipss t 1 Hz inresed holding urrent (141 ± 22 pa vs. 153 ± 23 pa, P =.21), ut not seline noise (6.14 ± 1.29 pa vs ± 1.21 pa, P =.291), nd returned to seline in.85 ±.19 s; 1 eipss t 2 Hz inresed holding urrent (224 ± 86 pa vs. 281 ± 114 pa, P =.32), seline noise (8.26 ± 2.1 pa vs ± 3.68 pa, P =.9), nd returned to seline in 1.14 ±.25 s; 2 eipss t 2 Hz inresed holding urrent (174 ± 37 pa vs. 255 ± 79 pa, P =.11), seline noise (7.29 ± 1.71 pa vs ± 5.91 pa, P =.5), nd returned to seline in 1.23 ±.23 s. Bth ddition of pirotoxin (Fig. 5B; n = 3; 1 mouse) did not ffet the stimulus-evoked hnges to holding urrent (97 ± 3 pa vs.91± 28 pa, P =.713), seline noise (11.72 ± 3.12 pa vs ± 4.6 pa, P =.619), or time to seline (.96 ±.28 s vs..9 ±.25 s, P =.433). In ontrst, these effets were ompletely olished y th pplition of stryhnine (n = 6; 2 mie) holding urrent (97 ± 3 pa vs.4± 2 pa, P <.1), seline noise (11.23 ± 3.3 pa vs ±.17 pa, P =.1), nd redued time to seline (1.5 ±.3 s vs..11 ±.5 s, P <.1). Thus, synpti glyine oming from eipss enhned the toni glyinergi urrent. To ssess the funtionl onsequenes of synptilly enhned toni glyine urrents on PV+ IN AP dishrge, we exmined exitility with (test) nd without (pre-test nd post-test) preonditioning eips input (Fig. 6; n = 13; 4 mie). In these experiments, AP dishrge ws ltered y the preonditioning eipss in 13/34 neurons, supporting toni glyine urrent effet. omprisons etween depolrizing step urrent without (pre-test) nd with (test) preeding eipss (Fig. 6B) showed eipss inresed rheose urrent (55 ± 1 pa for pre-test vs. 71 ± 11 pa for test, P =.35 vs. 55 ± 9 pa for post-test, P =.35), with n ssoited inrese in AP threshold ( ± 1.83 pa for pre-test vs ± 1.97 pa for test, P =.2 vs ± 1.89 pa for post-test, P =.32, not shown). We lso ssessed hnges to AP numer, frequeny nd lteny t rheose + 2 pa (pre-test). Group omprisons show tht preeding eipss redued tion potentil numer y 38 ± 18% (1.3 ± 1.9, pre-test vs. 6.6± 1.8, test, P =.3 vs. 8.9± 1.4, post-test, P =.5) nd inresed lteny to first AP y 193 ± 33% (24.3 ± 3.2 ms, pre-test vs. 5.2 ± 12.4 ms, test, P =.18 vs ± 3.4 ms, post-test, P =.43). In ontrst, preeding eipss did not hnge men AP frequeny (34.6 ± 1.3 Hz, pre-test vs. 3.6 ± 2.6 Hz, test, P =.75 vs ± 1.5 Hz, post-test, P =.222, not shown). Importntly, stryhnine (n = 4; 1 mouse) olished the preonditioning eips effet; rheose (55 ± 9.6 pa, pre-test vs. 6± 11.5 pa, test, P =.391 vs. 65± 15 pa, post-test, P =.391); spike threshold ( 32.8 ± 3.52 pa, pre-test vs ± 3.24 pa, test, P =.95 vs ± 3.51 pa, post-test, P =.321); tion potentil numer (19.3 ± 7.7, pre-test vs ± 5.4, test, P =.96 vs. 12 ± 6.1, post-test, P =.31); men frequeny (4 ± 4.3 Hz, pre-test vs ± 4 Hz, test, P =.77 vs ± 4.7 Hz, post-test, P =.192); lteny to first spike (21.6 ± 5.5 ms, pre-test; 21.5 ± 5.9 ms, test, P =.742

11 J Physiol Phsi nd toni glyinergi inhiition in the spinl dorsl horn 7195 A vs ± 6.7 ms, post-test, P =.68). Thus, endogenously relesed glyine is ple of modulting PV+ IN exitility. Bseline urrent (norm.) B Bseline urrent (norm.) ms 2 ms 1 pa 2 pa urrent SD (norm.) urrent SD (norm.) 1 1 t 1 Hz 2 t 1 Hz 2 t 2 Hz ontrol Piro Stryh Figure 5. Evoked (endogenous) glyine relese enhnes toni glyine urrents A, top urrent tres show the reovery phse reorded following series of glyinergi eipss evoked y stimultion (ipolr eletrode) of glyinergi fferents t the lmin II/III oundry, in the presene of NQX nd iuulline. Four stimulus protools were employed to produe inresing glyine relese: 1 stimulus (lk tre), 1 stimuli t 1 Hz (grey tre), 1 stimuli t 2 Hz (pink tre), 2 stimuli t 2 Hz (red tre). Dt were otined from 1 ms epoh 3 4 ms fter the stimulus rtift (shded re). Note tht inresing levels of stimultion use n inrese in the mplitude nd durtion of the evoked urrent. Br grphs (ottom) show hnges in three urrent properties (seline urrent nd urrent SD) s stimulus intensity is inresed. B, top urrent tres show the Suunit omposition of toni nd synpti glyine reeptors It is well estlished tht differenes in GABA A reeptor suunit omposition determine whether reeptors re lolized to synpti or extrsynpti sites, nd therefore ontriute to phsi or toni inhiition (Brikley & Mody, 212). The sme informtion is not ville for glyine reeptors. Given PV+ INs re under strong synpti nd toni glyinergi inhiition, they represent n idel model to test the reltionship etween glyine reeptor omposition nd synpti/extrsynpti loliztion. For glyine reeptors, it is well estlished tht inorportion of the β suunit in heteromeri glyine reeptors is ritil for synpti stiliztion vi the β suunit s intertions with the ytoskeletl inding protein gephyrin (Geimn et l. 22). onversely, homomeri glyine reeptors, omposed of five α suunits, do not intert with gephyrin nd re therefore more likely to e lolized extrsynptilly. We set out to test if the glyine reeptors mediting toni urrents in PV+ INs hd heteromeri α/β, or homomeri α only suunit omposition. Importntly, heteromeri nd homomeri glyine reeptors n e differentited y their men single hnnel ondutne (heteromeri < homomeri). Non-sttionry flutution nlysis of toni glyinergi urrents during th-pplied stryhnine (Fig. 7A) estimted reltively low men single hnnel ondutne of 28.8 ±.9 ps (n = 19), whih is onsistent with heteromeri glyine reeptors. This finding ws reinfored y the oservtion tht pirotoxin nd lindne, whih show seletivity for homomeri glyine reeptors, hd no effet on toni glyinergi urrent mplitude (pirotoxin: 73.5 ± 14.1 vs ± 14. pa, n = 16, P =.1; lindne: 62.9 ± 7.7 vs ± 8.2 pa, n = 5, P =.4) or noise level (pirotoxin: 7.2 ± 1.1 vs. 7.7 ± 1. pa, n = 16, P =.11; lindne: 6.1 ± 1.4 vs. 6.3 ± 1.3 pa, n = 5, P =.2) (Fig. 7B nd ). These findings suggest tht the toni glyinergi urrents oserved in PV+ INs re not medited y high-ondutne homomeri glyine reeptors. Rther, they re omposed of heteromeri glyine reeptors. reovery phse fter glyinergi eipss (2 stimuli t 2 Hz) under ontrol onditions (lk tre), fter ddition of pirotoxin (grey tre), nd fter ddition of stryhnine (red tre). Note, pirotoxin did not ffet the response wheres stryhnine olished response to eletril stimultion. Br grphs (elow) show group dt onfirming pirotoxin (grey) hs no effet on the eips-relted toni urrent, wheres ddition of stryhnine (pink) olishes the response. [olour figure n e viewed t wileyonlinelirry.om]

12 7196 M. A. Grdwell nd others J Physiol In order to onfirm the synpti glyine reeptors were lso heteromeri, we under took dditionl experiments to determine the men single hnnel ondutne of synptilly loted glyine reeptors nd test the pirotoxin sensitivity of glyinergi mipss. Pek sled non-sttionry noise nlysis on the dey phse of glyinergi mipss estimted reltively low men single hnnel ondutne of ±.78 ps (n = 19). This supports heteromeri α/β suunit omposition t synpses (Fig. 8A). Likewise, pirotoxin hd no effet on A B 1 mv 3 s Rheose urrent (pa) Spike no step e-stim Lteny (ms) + stryhnine Figure 6. Evoked (endogenous) glyine relese lters AP dishrge in PV+ INs A, top tre shows AP dishrge responses to three depolrizing step injetions: () pre-test; step; () test step preeded y rrge of eipss (2 stimuli t 2 Hz vi ipolr eletrode); nd () post-test step. Tres elow show urrent step injetion nd eletril stimultion timing (red). Insets show the onset of AP dishrge on expnded time sle highlighting the dely to AP dishrge nd redued AP numer in the test response (). Right tres shows expnded responses to the sme protool repeted in the presene of stryhnine (1 μm), whih loks glyinergi eipss nd ssoited toni urrent. Stryhnine olishes the delyed nd redued AP dishrge in test step. B, r grphs show group dt ompring rheose urrent, AP numer nd AP lteny etween the pre-test, test nd post-test step responses, onfirming tht eipss nd the resulting toni urrent redued the exitility y modifying AP dishrge properties in PV+ INs. [olour figure n e viewed t wileyonlinelirry.om] 8 4 mips frequeny (.6 ±.1 vs..54 ±.1 Hz, n = 8, P =.39), mplitude ( 54.2 ± 8.3 vs ± 7. pa, n = 8, P =.51), rise time (1.2 ±.1 vs. 1.2±.1 ms, n = 8, P =.89), or dey time onstnt (11.7 ±.8 vs ±.7 ms, n = 8, P =.22) (Fig. 8B nd ). Thus, we onlude tht synpti glyine reeptors in PV+ INs re lso omposed of α/β suunit-ontining A Stryhnine 32 B Bseline urrent (pa) Pirotoxin s ns Stryhnine 1 pa Pirotoxin ns.5 s 4 pa.5 s 1 pa Vrine (pa 2 ) ount (x 1 3 ) urrent SD (pa) I = 2.29 pa g = 33. ps 1 urrent (pa) urrent (pa) (r 2 =.96) Lindne Pirotoxini Lindne Figure 7. Heteromeri glyine reeptors medite toni glyine urrents in PV+ INs A, tres (1 s epohs) extrted from progressive lok of toni glyine urrent vi stryhnine pplition (1 μm). Non-sttionry noise nlysis on the urrent/vrine reltionship during the progressive stryhnine lok (right) ws used to estimte the men single hnnel ondutne of underlying reeptors. B, tre shows ontinuous reording of mipss (in the presene of TTX, NQX nd iuulline), during sequentil th ddition of pirotoxin nd stryhnine (1 μm). Note, pirotoxin does not ffet the holding urrent or seline noise in PV+ INs. In ontrst, stryhnine uses shift in holding urrent nd redues seline noise, thus onfirming the presene of toni glyine urrent. Right insets (, nd ) show no hnge in seline noise fter the ddition of pirotoxin () nd its redution y stryhnine (). An ll-points histogrm (right) from epohs, nd shows tht holding urrent nd seline noise do not differ during pirotoxin exposure, ut re redued following ddition of stryhnine., r plots showing group dt ompre holding urrent mplitude nd seline noise (urrent SD) under ontrol onditions (), following th pplition of pirotoxin or lindne (), nd finlly following stryhnine pplition (). Pirotoxin nd lindne did not lter toni urrent properties suggesting these urrents re medited y heteromeri GlyRs in PV+ INs. 5 ns ns

13 J Physiol Phsi nd toni glyinergi inhiition in the spinl dorsl horn 7197 heteromers. Finlly, the single hnnel ondutne of glyine reeptors ws diretly ssessed in outside-out memrne pthes from PV+ INs during th-pplied glyine (2.5 1μM) (Fig. 9). Under these onditions single hnnel events evoked y glyine pplition hd men ondutne of ± 1.38 ps (n = 16), were pirotoxin insensitive (42.83 ± 1.38 ps vs ± 1.15 ps, n = 6) nd loked y stryhnine (n = 6). These dt re onsistent with the min ondutne stte of previously reorded single hnnel urrents within the spinl ord (Bormnn et l. 1987; Tkhshi et l. 1992) nd provide diret evidene of heteromeri glyine reeptor omposition in PV+ INs (Lynh, 29). A B Vrine (pa 2 ) pa 1 ms ontrol Gly mipss () 15 pa g = 29 ps 3 urrent (pa) 1 ms I = 2. pa n = 3 8 pa 6 Disussion This study hs trgeted PV+ INs in the mouse DH nd shows tht multiple forms of glyinergi inhiition regulte the tivity of this importnt popultion. We show tht synpti inhiition of PV+ INsisdominted y glyinergi soures, nd tht toni glyinergi urrents re lso strongly expressed in this popultion. Given this onfigurtion, we ssessed the stoihiometry of glyine reeptors underlying synpti nd toni urrents nd show tht, surprisingly, heteromeri α/β suunit-ontining reeptors dominte in oth lotions. Funtionlly, our dt emphsize the importne of glyinergi inhiition for shping AP dishrge in PV+ INs, euse enhning or diminishing this inhiition n derese or inrese + Pirotoxin (P) 2 s A 3 glyine B 4 glyine + pirotoxin P ount 15 ount 2 mips frequeny (Hz) ns ns ns ns P mips pek (pa) mips rise (ms) mips dey τ (ms) P P P Figure 8. Heteromeri glyine reeptors medite synpti glyine urrents in PV+ INs A, glyinergi mipss ptured from PV+ INs (overlid tres) were used to undertke pek-sled non-sttionry noise nlysis nd susequently estimte men single hnnel ondutne of synptilly loted glyine reeptors (right plot). PV+ INs exhiited reltively low ondutnes, whih is onsistent with the presene of heteromeri α/β glyine reeptors. B, ontinuous glyinergi mips reordings prior to (ontrol) nd during pirotoxin pplition (left) nd overlid mipss ptured under eh ondition. Inset (ottom right) ompres verged mipss reorded in ontrol nd pirotoxin (tres offset for omprison)., r plots ompre group dt for mips frequeny, mplitude, rise time nd dey time onstnt under ontrol onditions () nd in pirotoxin (P). Vlues for individul neurons re shown s filled irles. Pirotoxin did not lter men mips properties, thus onfirming these urrents re medited y heteromeri α/β GlyRs in PV+ INs. 7 ount urrent (pa) glyine + stryhnine urrent (pa) D urrent (pa) Figure 9. Heteromeri glyine reeptors medite glyinergi urrents in PV+ INs A, plots showing ll-points histogrms generted from single hnnel reordings in outside-out memrne pthes from PV+ INs during th pplition of glyine. Insets re exmple tres from the sme pth, dotted line is 2 pa. A, plot shows single hnnel events fter ddition of glyine. B, plot shows tht ddition of pirotoxin (1 μm) hs no effet on hnnel ondutne., plot shows ddition of stryhnine olishes single hnnel events. D, r plots showing single hnnel ondutne from ll reorded pthes (left r) nd the effet of pirotoxin on single hnnel ondutne (right, ontrol () vs. pirotoxin (P)) in suset of reordings. The reltively low men single hnnel ondutne nd pirotoxin resistne otined from this nlysis supports the presene of heteromeri α/β GlyRs in the pthes. ondutne (ps) ns P