The tetrodotoxin-resistant sodium channel SNS has a specialized function in pain pathways

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1 RESULTS Voltge-gted sodium chnnel suunits contin S4-domin voltge sensors tht re essentil for ctivity 18,19. We therefore generted trgeting construct y sustituting for exons 4 nd 5 of sns, which include the S4 voltge sensor of domin I (Fig. 1), PGK-neo cssette terminting with stop codons in ll three frmes 20,21. Mice heterozygous for the trgeted llele were intercrossed to derive vile homozygotes tht were repetedly ckcrossed onto C57/Bl6 mice to produce congenic lines 22. The expression of voltge-gted sodium chnnel -suunit mrnas ws exmined in null mutnts. We used northern lots (Fig. 1c) nd PCR (Fig. 1d) to exmine whether the deletion of functionl SNS ltered the expression levels of TTX-sensitive suunits 20. Both SNS nd the TTX-sensitive chnnel PN-1 re prerticles The tetrodotoxin-resistnt sodium chnnel SNS hs specilized function in pin pthwys Armen N. Akopin 1, Veronik Souslov 1, Steven Englnd 1,2, Kenji Okuse 1, Noukuni Ogt 3, Jn Ure 4, Andrew Smith 4, Brdley J. Kerr 5, Steven B. McMhon 5, Sue Boyce 6, Ry Hill 6, Louise C. Stnf 7, Anthony H. Dickenson 7 nd John N. Wood 1 1 Moleculr Nociception Group, Deprtment of Biology, Medwr Building, University College, London WC1E 6BT, UK 2 Present ddress: Pfizer Centrl Reserch, Sndwich, Kent, CT13 9NJ, UK 3 Second Deprtment of Physiology, Fculty of Medicine, Hiroshim University, Hiroshim 734, Jpn 4 Centre for Genome Reserch, West Mins Rod, Edinurgh EH9 3JQ, UK 5 Deprtment of Physiology, UMDS, St. Thoms Hospitl Medicl School, Lmeth Rod, London SE1 7EH, UK 6 Merck Shrp nd Dohme Reserch Ls, Terlings Prk, Hrlow, Essex CM20 2QR, UK 7 Deprtment of Phrmcology, University College, London WC1E 6BT, UK Correspondence should e ddressed to J.N.W. (j.wood@ucl.c.uk) Mny dmge-sensing neurons express tetrodotoxin (TTX)-resistnt voltge-gted sodium chnnels. Here we exmined the role of the sensory-neuron-specific (SNS) TTX-resistnt sodium chnnel suunit in nociception nd pin y constructing sns-null mutnt mice. These mice expressed only TTXsensitive sodium currents on step depolriztions from norml resting potentils, showing tht ll slow TTX-resistnt currents re encoded y the sns gene. Null mutnts were vile, fertile nd pprently norml, lthough lowered thresholds of electricl ctivtion of C-fiers nd incresed current densities of TTX-sensitive chnnels demonstrted compenstory upregultion of TTX-sensitive currents in sensory neurons. Behviorl studies demonstrted pronounced nlgesi to noxious mechnicl stimuli, smll deficits in noxious thermoreception nd delyed development of inflmmtory hyperlgesi. These dt show tht SNS is involved in pin pthwys nd suggest tht lockde of SNS expression or function my produce nlgesi without side effects. Severl kineticlly nd phrmcologiclly distinct voltge-gted sodium chnnels re found in dorsl root gnglion (DRG) neurons 1 6. The TTX-resistnt current is insensitive to micromolr concentrtions of tetrodotoxin, with low single-chnnel conductnce, slow ctivtion nd inctivtion kinetics nd more depolrized ctivtion threshold thn other chnnels 5,6. At lest three types of TTX-resistnt sodium currents in DRG hve een distinguished electrophysiologiclly 7,8, nd some evidence suggests tht these chnnels re importnt in the trnsmission of nociceptive informtion to the spinl cord. Both rdykinindependent relese of clcitonin gene-relted peptide nd the depolriztion of dorsl horn neurons elicited through C-fier ctivtion re insensitive to peripherlly pplied TTX 9. In ddition, TTX-resistnt ction potentils hve een detected in C- fiers from humn surl nerve iopsies 10. A role for TTX-resistnt chnnels in nociception is supported y the demonstrtion tht some smll-dimeter sensory neurons (predominntly nociceptors) in culture express only TTX-resistnt sodium currents 11. At lest eight sodium chnnel suunits occur in DRG tht my ccount for sodium currents detected electrophysiologiclly 12. Three TTX-resistnt suunits re known; one chnnel is expressed selectively in hert muscle 13, wheres SNS (or PN3) is selectively expressed in suset of smll-dimeter sensory neurons 14,15. A trnscript encoding low-threshold, rpidly inctivting, TTX-resistnt sodium chnnel nmed NN or SNS-2 hs lso een identified in sensory neurons 16,17. To test the hypothesis tht TTX-resistnt chnnels hve specilized role in dmge sensing, nd to exmine the role of SNS in nociception, we generted null-mutnt mouse for the TTX-resistnt SNS sodium chnnel. We found tht such mice re norml, prt from prtil deficits in perception of noxious therml, mechnicl nd inflmmtory stimuli. nture neuroscience volume 2 no 6 june

2 rticles c Fig. 1. Trgeting constructs nd sodium chnnel expression in sns null mutnts. () Structure of the trgeting construct pxxsns, its reltionship to the wild-type sns locus, nd the resulting trgeted locus, together with the loction of the 5 nd 3 proes used to nlyze integrtion events, nd the distinct sizes of restriction frgments produced y EcoRI digests of wild-type (7.8 k) nd null-mutnt (8.8 k) DNA proed with the 3 ApI EcoRI proe descried in the methods section. () Genomic Southern lotting of til DNA from wildtype, heterozygous nd null-mutnt sns mice proed with the ApI EcoRI frgment. (c) Northern lots of RNA extrcted from dult nd neontl rts, nd dult wild-type nd null-mutnt mice exmined with sutype-specific, voltge-gted sodium chnnel rioproes for SNS, PN-1, NCh6 nd type II sodium chnnels. Some rt tissues re included to show tht the proes re working efficiently. An ethidium romide stin ws used to compre RNA loding on the gel, nd the gel ws susequently proed for the expression of cyclophilin mrna to quntify chnges in suunit expression. (d) RT-PCR nlysis of sodium chnnel trnscripts in null-mutnt nd wild-type mice. Controls were done without reverse trnscriptse nd with primers for the riosoml protein L27. PCR cycles were 35 for voltge-gted sodium chnnel, 25 for L27 nd cyclophilin, 28 for NN/SNS2 nd 30 for SNS. sent t high levels in peripherl ut not centrl neurons. An SNS trnscript running slightly hed of the wild-type nd ws detected on northern lots. The levels of PN-1 trnscript were upregulted 1.53-fold (s.e. 0.08, n = 4, p < 0.05) in null-mutnt DRG compred to control mice, when trnscript levels were normlized ginst cyclophilin housekeeping trnscript. Using PCR to exmine the levels of other less-undnt sodium chnnel trnscripts, we filed to detect the presence of type III or crdic TTX-resistnt trnscripts in wild-type or null-mutnt DRG. Type-I, NN/SNS-2, NCh6 nd type II trnscripts, in descending order of undnce, were present t roughly equl levels s judged y PCR in wild-type nd nullmutnt DRG, lthough up to twofold chnges in the levels of trnscripts would e difficult to detect relily using PCR. Using the sme primers for SNS tht generted the proes for northern lots, we otined wek PCR product representing prt of the truncted mrna from null mutnts, wheres roust signl ws pprent in wild-type DRG (Fig. 1d). Sequencing of the null-mutnt PCR frgment showed tht exons three nd six were spliced, cusing frme-shift muttion 20. Thus non-functionl SNS trnscript is mde in null mutnts, ut there is no induction of mrna encoding TTX-resistnt crdic chnnel suunits in these neurons. d Null mutnts were helthy, fertile nd pprently norml. We exmined the sensory neurons in dult L4 DRG y immunocytochemistry. We used ntiodies to peripherin to define the smlldimeter sensory neurons tht express SNS, nti-neurofilment ntiody N-52 to define lrge-dimeter, predominntly nonnociceptive, sensory neurons nd the lectin IB 4 to stin the c-retexpressing, GDNF (glil cell line-derived neurotrophic fctor)-dependent popultion of sensory neurons 23. The percentges of stined neurons (41% peripherin positive, 53% N52 Tle 1. Distriution of TTX-resistnt nd TTX-sensitive currents in DRG neurons in culture. Wild-type Heterozygous SNS null mutnt TTX sensitive Current present 30/38 (78.9%) 36/42 (85.7 %) 29/36 (80.6 %) Current sent 5/38 (13.2%) 3/42 (7.1 %) 2/36 (5.6 %) Current <0.1 na 3/38 (7.9%) 3/42 (7.1%) 5/36 (13.9 %) TTX resistnt Current present 35/38 (92.1%) 39/42 (92.9 %) 0/36 (0%) Current sent 3/38 (7.9%) 3/42 (7.1%) 36/36 (100%) Men cell cpcitnce (pf) 24.7 ± ± ± 1.3 Distriution of sodium current types in DRG neurons from wild-type, heterozygous nd nullmutnt sns mice. Men cell cpcitnce ws not significntly different mong the wild-type, heterozygous nd null-mutnt mice from which cells were prepred. 542 nture neuroscience volume 2 no 6 june 1999

3 rticles c Fig. 2. Functionl TTX-resistnt sodium chnnel expression in sns null mutnts. Sodium currents recorded from wild-type mouse DRG. () A step to 120 mv in the commnd potentil preceded the step to the test potentil. Both fst (TTX-sensitive) nd slow (TTX-resistnt) currents re pprent. () Currents recorded from the sme cell s (), ut with prepulse to 50 mv preceding the step to the test pulse. Only the slow TTX-resistnt currents re recorded, ecuse of the inctivtion of the TTX-sensitive currents. (c, d) The sme voltge-clmp protocols were used with null-mutnt mouse DRG neurons. The currents re rpidly ctivting nd inctivting in (c), wheres with the inctivting prepulse, no current ws evoked (d). The recordings were mde from smll cells of pproximtely equl size. The cpcitnces were 16 pf (, ) nd 18 pf (c, d). positive nd 28% IB 4 positive) were similr in L4 gngli of the null-mutnt nd wild-type mice, s were the totl cell numers determined y counting seril sections through L4 DRG neurons in null mutnts (7168 ± 435, n = 3) nd wild-type nimls (6830 ± 691, n = 3), demonstrting no loss of sensory neurons in the null mutnts. We next exmined TTX-sensitive nd TTX-resistnt voltgegted sodium currents in wild-type, heterozygous nd homozygous null-mutnt littermtes (Tle 1). In wild-type DRG d neurons, two types of sodium currents were evoked y step depolriztions (10 ms durtion, 5 or 10 mv increments) from holding potentil of 120 mv to etween 80 nd +40 mv. At low thresholds, this procedure evoked rpidly ctivting, rpidly inctivting current, which ws sensitive to lock y TTX. At higher thresholds, more slowly ctivting, slowly inctivting TTX-resistnt sodium current ecme pprent, s oserved in mouse C57/Bl6 DRG 24. We focused on smll-dimeter neurons (men cpcitnce 24 pf) tht express most TTX-resistnt sodium current 11. In wild-type DRG neurons (Fig. 2), when 120 mv prepulse ws incorported into the voltge-clmp protocol, oth the fst nd slow sodium currents were pprent (Fig. 2). When prepulse to 50 mv preceded the chnge in commnd potentil or cells were treted with 0.5 mm TTX, the TTX-sensitive currents were inctivted, nd the slow TTX-resistnt currents were recorded in isoltion (Fig. 2). In similr experiments on DRG from null-mutnt mice, only the TTX-sensitive current ws pprent (Fig. 2c). Injection of null-mutnt DRG neurons with vector encoding the rt suunit of SNS resulted in the re-expression of lrge TTX-resistnt sodium currents. The chrcteristics of these exogenously expressed currents (Fig. 3) were indistinguishle from the TTX-resistnt currents recorded from DRG neurons from wild-type or heterozygous mice 24. These experiments confirm tht SNS is responsile for ll the slow TTX-resistnt currents found in sensory neurons. They lso demonstrte tht there is no requirement for SNS splice vrints to rescue the norml TTX-resistnt phenotype. The DRG from wild-type nd heterozygous mice showed mixture of oth TTX-sensitive nd TTX-resistnt sodium currents, wheres smll proportion (8%) of cells in the null mutnt expressed neither TTX-sensitive nor TTX-resistnt sodium currents. The numer of TTX-sensitive cells ws similr in DRG from wild-type, heterozygous nd null-mutnt mice, suggesting tht the deletion of sns did not lter the cell-type pttern of expression of TTX-sensitive chnnels. However, when the sodium current densities of DRG neurons from wild-type, heterozygote nd null mutnts were exmined quntittively, significnt upregultion (out twofold) of TTX-sensitive currents ws pprent in null mutnts, ut not heterozygotes, which were not different from wild-type mice (Fig. 4). The significnce of ltered sodium chnnel expression ws exmined y mesuring the thresholds of ctivtion of spinl cord wide-dynmic-rnge neurons y trnscutneous electricl c Current (na) Commnd potentil (mv) I/I mx Pek inwrd current (na) Prepulse potentil (mv) Fig. 3. Rescue of TTX-resistnt chnnels in null mutnts following nucler injection of vector encoding the suunit of SNS. The recordings were mde in the presence of 500 nm TTX to olish endogenous TTX-sensitive currents. () Currents recorded from null-mutnt DRG neuron two dys fter nucler injection of the construct. The cell ws voltge clmped t 90 mv, prepulsed to 120 mv, nd then stepped to rnge of potentils (detils in text). Slowly ctivting nd inctivting currents were evoked, similr to those in Fig. 2. () Current voltge reltionship for the recordings in (). The threshold for ctivtion of the current ws pproximtely 40 mv, with pek on stepping to 20 mv. (c) Stedy-stte inctivtion curve constructed from currents recorded using the two-pulse protocol detiled in the text. The dt hs een normlized (I/I mx ) with respect to current evoked following non-inctivting prepulse. The dt points were fitted y single Boltzmn function, yielding hlf-point for inctivtion of 29 mv, nd slope fctor of 5.1 mv. nture neuroscience volume 2 no 6 june

4 wves of compound ction potentils recorded from the L4 dorsl roots of wild-type (n = 4) nd null-mutnt mice (n = 4). Although no chnges were oserved in the recruitment of A- fiers (Fig. 4c; p > 0.05, Kolmogorov-Smirnov test), C-fiers in the null mutnt nimls hd lower electricl threshold, nd the recruitment curve ws shifted significntly to the left (Fig. 4d; p < 0.01, Kolmogorov-Smirnov test). With suprmximl electricl stimultion, the ltency, wveform nd mgnitude of A- nd C-fier compound ction potentils were similr, suggesting tht the differences in recruitment did not result from differences in primry fferent neuron numers. Consistent with the lowered threshold for evoking C-fier ltency response in the dorsl horn neurons, the C-fier recruitment curve in the null mutnt nimls showed lowered threshold of ctivtion of these fiers nd significntly higher level of C-fier ctivtion for given stimulus intensity thn in wild-type mice (p < 0.01, Kolmogorov-Smirnov test). The recruitment curves for the ctirticles Fig. 4. TTX-sensitive chnnels re induced in sns null mutnt C-fier-ssocited sensory neurons. () Wholecell sodium currents were recorded s indicted in the text. Pek currents were normlized with respect to the cpcitnces of the individul cells nd the dt expressed s men ± s.e.m. (n > 20). There ws no significnt difference etween the densities of the tetrodotoxin-sensitive (TTX-S) nd tetrodotoxin-resistnt currents (TTX-R) in +/+ nd +/ mice. In the / mice, no TTX-resistnt current ws detected, wheres the density of TTX-sensitive current ws more thn twice tht found in Current density (pa/pf) the two other types of mouse. () Cell cpcitnces were mesured using the cpcitnce compenstion fcility on the mplifier. There ws no significnt difference in the cell cpcitnces cross the three groups of mice. (c, d) Recruitment of A- nd C-fiers in the scitic nerve of wild-type nd null-mutnt nimls. Compound ction potentils were recorded from the L4 dorsl roots of urethne-nesthetized nimls following electricl stimultion of the scitic nerve t mid-thigh level. The verge compound ction potentil re is plotted ginst stimulus strength for A- nd C-fiers in (c) nd (d), respectively (wild type, ; null mutnt, ; n = 4 for oth). There is no significnt difference in the recruitment curve for A-fiers (p > 0.05, Kolmogorov-Smirnov test), ut C-fiers in the null mutnt nimls re ctivted with significntly less current (p < 0.01, Kolmogorov- Smirnov test). (e, f) Thresholds of electricl ctivtion of dorsl horn neurons vi C-fiers ut not A-fiers re lowered in sns null mutnts. Men (± s.e.m.) threshold current of trnscutneous electricl stimultion of the hindpw receptive field required to evoke A-fier-medited responses (e) or C-fier-medited responses (f) of dorsl horn neurons recorded in wild-type (+/+; n = 21) or null-mutnt ( / ; n = 26) mice. The C-fier threshold of electricl excittion (f) is lowered in null-mutnt mice (p < 0.05), wheres A-fier thresholds (e) re identicl in the two groups. stimultion. We chrcterized the responses of 20 wild-type mouse dorsl horn neurons nd 26 null-mutnt mouse neurons evoked y electricl stimultion of their peripherl receptive fields 25. In null-mutnt mice, the threshold current required to evoke long-ltency (presumly C-fier medited) response (Fig. 4f) ws significntly lower (0.62 ± 0.09 ma, n = 26) thn in wild-type mice (1.10 ± 0.19 ma, n = 20; Mnn-Whitney test, p = 0.021). The poststimulus ltency of the long-ltency evoked response did not differ etween the groups (null mutnts, ± 5.0 ms fter stimulus; wild type, ± 5.9 ms) suggesting no chnge in conduction velocity. In contrst, the threshold current required to evoke short-ltency (presumly A-fier medited) response (Fig. 4e) ws the sme in oth null-mutnt (0.060 ± ma) nd wild-type mice (0.063 ± ma). To confirm tht these chnges in threshold were due to lowered threshold for ctivtion of primry fferent C-fiers, recruitment curves were constructed from the res under the C- nd A-fier pf c d Percent of mximum Percent of mximum pa pa e ma f ma Locomotor ctivity Rotrod Blncing ems Bem reks / 5 min Time spent on rotrod (s) Time le to sty on ems (s) Time (min) Fig. 5. Norml motor ehvior nd spinl reflexes in sns null mutnts. Mice were ssessed for sensorimotor coordintion using rnge of tests. All null mutnt nd ll control mice (n = 11) showed norml plcing nd righting reflexes nd hindlim reflex extension nd hd no difficulty in mintining their lnce on em or on revolving rotrod. Spontneous locomotor ctivity ws not different in sns mutnt nd control mice nture neuroscience volume 2 no 6 june 1999

5 rticles Pressure score Ltency (s) Ltency (s) Pw flick Til pressure Hot plte test Percent response Til flick Von Frey thresholds Force (g) +/+ / Fig. 6. The sns null mutnt mice show prtil nlgesi to noxious therml nd mechnicl stimultion. Both pw-flick nd til-flick ltencies to noxious irrdition were significntly lengthened in null mutnts (n = 11, p > 0.05), lthough no differences in hot-plte ltencies could e detected t rnge of tempertures. Noxious mechnicl stimultion provided y Rndel-Selitto stimultion showed complete nlgesi in null mutnt, which ll filed to respond up to the cutoff point of the experiment (n = 11, p > 0.005). In contrst, thresholds to stimultion with Von-Frey hirs were the sme in wild-type or null-mutnt mice. vtion of A-fiers did not differ etween wild-type nd null mutnt mice (p > 0.05, Kolmogorov-Smirnov test). These dt (Fig. 4c nd d) suggest tht the C-fier-medited, long-ltency input into the dorsl horn of null mutnts is more esily stimulted electriclly thn tht of wild-type nimls, wheres A-fier input is identicl in wild-type nd null-mutnt mice. The higher level of expression of TTX-sensitive chnnels in the somt of DRG null-mutnt neurons proly reflects similr ltered levels of TTX-sensitive ctivity in xons nd provides possile mechnism for this ltertion in electricl excitility. Finlly, ecuse of the selective expression of SNS in nociceptive sensory neurons, we exmined the effect of deleting the SNS chnnel on mouse ehvior, focusing on pin responses. We ckcrossed the sns null-mutnt mice with C57/Bl6 mles nd found tht the ehvior of null mutnts on different genetic ckgrounds from F1 to F4 ws identicl 22. Null-mutnt mice were indistinguishle in their ppernce, spontneous ehvior, ody weight nd ody temperture from ge- nd sex-mtched, wild-type control nimls. All null-mutnt nd wild-type mice exhiited norml plcing nd righting reflexes nd hindlim reflex extension, nd they hd no difficulty in mintining their lnce on ems for the full 120-second tril durtion (Fig. 5). Similrly, null-mutnt mice were le to mintin their lnce on revolving rotorod for the sme durtion s wild-type mice 26. Spontneous locomotor ctivity ws not different in sns nullmutnt nd wild-type mice (Fig. 5). The responses of null-mutnt nd wild-type littermtes to noxious chemicl nd therml stimultion were next exmined. The pw-withdrwl nd til-flick ltencies following exposure to noxious therml (rdint het) stimulus were significntly incresed, leit to smll extent, in null mutnts compred with wild-type mice (Fig. 6). In contrst, there ws no difference in the ltency on the hot plte of null-mutnt nd wild-type mice cross rnge of tempertures (45 60 o C; Fig. 6). Pw-withdrwl responses elicited y grded Von Frey hirs did not differ etween null-mutnt nd wild-type mice (Fig. 6). In contrst, pin thresholds to noxious mechnicl stimuli pplied to the til were mrkedly incresed in the null-mutnt mice (Fig. 6), which ll went to the cutoff point of the test, demonstrting pronounced mechnicl nlgesi. TTX-resistnt sodium chnnels my e involved in persistent inflmmtory pin sttes In wild-type mice, intrplntr injection of crrgeenn induced therml hyperlgesi. Hyperlgesi ws pprent t one hour nd ws mximl for severl hours following injection. Therml hyperlgesi developed more slowly in null-mutnt mice, with significnt hyperlgesi eing oserved t two nd three hours. However, the mximum level of hyperlgesi ws not significntly different in wild-type nd null-mutnt mice (Fig. 7 nd ). Similrly, there ws no difference in pw edem in wild-type nd null-mutnt mice when mesured 3.5 hours fter the injection of crrgeenn (wild-type, 42.1 ± 9.0 mg, n = 6; mutnt, 37.0 ± 7.7 mg, n = 6). The incresed levels of TTX-sensitive currents nd lower thresholds of electricl ctivtion of C-fiers could prtilly compenste for the loss of SNS in null mutnts. There re no specific lockers for TTX-sensitive currents, ut the locl nesthetic lidocine is reltively selective locker of TTX-sensitive currents compred to TTX-resistnt sodium currents (IC 50 of 50 mm for TTX sensitive 4,8,30, 200 mm 1mM for TTX resistnt 4,6,31 ). Using systemic dose of lidocine tht hd no effect on motor function (25 mg per kg, equivlent to clculted systemic concentrtion of pproximtely 90 mm), we compred the ehvior of wild-type nd null-mutnt littermtes. Null-mutnt nimls showed enhnced therml hypolgesi fter lidocine tretment (Fig. 8; p < 0.01, pired t-test), wheres wild-type nimls were unffected (p > 0.2, pired t-test). Lidocine lso ltered crrgeenn-induced hyperlgesi. Three hours fter intrplntr crrgeenn, when stle level of hyperlgesi hd developed, systemic ppliction of lidocine produced prtil reversl of the hyperlgesi in wild-type nimls, ut complete reversl in null mutnts (Fig. 8; p < 0.05, unpired t-test). These dt demonstrte role for SNS in setting pin thresholds nd suggest tht TTX-sensitive chnnel upregultion my compenste for this loss of SNS function in the null mutnt. DISCUSSION Smll-dimeter sensory neurons express unusul TTX-resistnt sodium chnnels 1 11, ut the sence of sutype-specific sodium chnnel lockers hs precluded study of the role of individul chnnel sutypes in nociception. Using gene-trgeting technology to delete the TTX-resistnt sodium chnnel sns, we find evidence to suggest tht there is selective role for this chnnel in dmge sensing. The norml ehvior exhiited y sns null nture neuroscience volume 2 no 6 june

6 rticles Rection time (test/seline) Rection time (test/seline) Hours Minutes Fig. 7. Full inflmmtory hyperlgesi is delyed in the sns null mutnt. Intrplntr injection of crrgeenn induced therml hyperlgesi t 1 h, nd the mximl level of hyperlgesi lsted over 24 h. Therml hyperlgesi ws delyed in null-mutnt mice, with significnt hyperlgesi (p > 0.05) oserved only fter 90 min. However, the mximum level of hyperlgesi ws similr in wild-type nd null-mutnt mice. Two independent experiments re shown. Hours mutnts (prt from deficits in nociception) suggests tht SNS is not essentil in other spects of nervous system function, consistent with the highly selective pttern of expression of this chnnel in sensory neurons 14. The deletion of sns leds to the loss of ll slow TTX-resistnt currents in sensory neurons clmped t norml resting potentils. However, electrophysiologicl studies hve demonstrted two or more types of TTX-resistnt current in DRG neurons 7,8. These pprently contrdictory oservtions cn e reconciled if distinct ccessory suunits exist tht modify the functionl properties of suunits in mnner nlogous to the previously identified sodium chnnel suunits 32. SNS is expressed in two distinct types of sensory neurons, one ering trka nd p75 nd nother expressing the GDNF receptor c-ret 23. It is possile tht different cell types express different memrne-ssocited proteins tht influence SNS -suunit function Alterntively, different TTX-resistnt currents my represent distinct phosphorylted sttes of the sme SNS chnnel 28,29. These explntions could underlie the oservtion of type-b nd -C TTX-resistnt currents 7. Another TTX-resistnt current ws oserved when smll DRG neurons were depolrized from hyperpolrized potentils of 107 mv 7. The properties of this chnnel re similr to those recently descried for heterologously expressed SNS-2 or NN 17. The physiologicl role of this high threshold current is uncertin, ut it will e interesting to nlyze the ehviorl phenotype of SNS-2/NN null mutnts. The loss of essentilly ll TTX-resistnt currents in the sns null mutnt suggests tht typicl sodium chnnels such s NG 33 re unlikely to encode functionl TTX-resistnt ctivity in DRG neurons 17. Re-injection of constructs encoding SNS into nullmutnt DRG neurons reconstituted the repertoire of slow TTXresistnt currents found in norml smll-dimeter neurons. The sns suunit encodes chnnel with errnt electrophysiologicl properties when expressed in cell lines or Xenopus oocytes 14, rising the possiility tht ccessory fctors tht re sent from non-neuronl cells regulte spects of chnnel function in DRG neurons. Concomitnt with the loss of SNS, n incresed expression of TTX-sensitive currents ws oserved in the null mutnt. The moleculr mechnism tht links expression of SNS nd other sodium chnnels is unknown 34. Using the semi-quntittive method of PCR, we could not detect ovious chnges in TTX-sensitive - suunit trnscripts. However, northern lots of the undnt PN- 1 chnnel trnscript did show n increse in expression of more thn 50%. The higher density of TTX-sensitive chnnels tht hve lower threshold of ctivtion thn SNS provides n explntion for the diminished threshold of electricl ctivtion of C-fiers ut not A-fiers detected oth in compound ction potentil studies of peripherl nerve nd in recordings from dorsl horn neurons ctivted vi peripherl stimultion. Behviorl studies demonstrte cler-cut deficit in oth mechno- nd thermoreception nd temporrily diminished response to inflmmtory pin stimuli in null-mutnt nimls ckcrossed etween one nd four times onto pure C57/Bl6 ckground. Inflmmtory hyperlgesi evoked y prostglndin E 2 is reversily locked y ntisense oligonucleotides directed +/+ / Control Crrgeenn Crrgeenen + Lidocine Fig. 8. Systemic lidocine enhnces the nlgesic phenotype of sns null mutnts. () Bseline thresholds for noxious therml stimultion with Hrgreves pprtus were incresed in null-mutnt nimls fter lidocine tretment (p < 0.01, pired t-test), wheres wild-type nimls were unffected (p > 0.2, pired t-test). () Three hours fter intrplntr crrgeenn injection, oth null-mutnt (lck rs) nd wild-type (open rs) nimls show similr level of therml hyperlgesi. Systemic lidocine produces prtil reversl of the hyperlgesi in wild-type nimls (open rs), ut complete reversl in null mutnts (lck rs). The responses of null mutnts re greter thn those of wild-type mice fter lidocine tretment (p < 0.05, unpired t-test). 546 nture neuroscience volume 2 no 6 june 1999

7 rticles ginst sns 39. The less-pronounced nlgesic phenotype demonstrted in null mutnts my reflect developmentl compenstory mechnisms. Thus the prtil nlgesi to noxious inflmmtory or therml stimuli could e enhnced y systemic ppliction of lidocine in the null mutnt t doses tht do not ffect motor ehvior, suggesting tht the phenotype of the sns null mutnt would e more pronounced except for the incresed expression of TTX-sensitive chnnels. The delyed development of inflmmtory hyperlgesi suggests tht trgets other thn SNS hve lter coopertive role in the development of hyperlgesi. Altered potssium current ctivity is lso likely to e involved in setting nociceptor thresholds 38. The prtil nlgesi shown y sns null mutnts is consistent with oservtions out the role of TTX-resistnt currents in nociception 9,10 ut is more difficult to reconcile with the expression of higher levels of TTX-sensitive sodium chnnels nd lower electricl ctivtion thresholds of C-fiers in the null mutnts. A specilized role for the rpidly repriming TTX-resistnt ctivity encoded y SNS in setting thresholds of depolriztion t C-fier terminls, rther thn propgting ction potentils, provides possile explntion for these two sets of oservtions. In summry, the dt presented here show tht the SNS suunit is responsile for ll slow TTX-resistnt sodium chnnel ctivity in sensory neurons, nd tht this chnnel hs selective role in the response of nociceptors to noxious therml, mechnicl nd inflmmtory stimuli. These oservtions, comined with other indirect evidence tht the regultion of TTXresistnt chnnels is importnt in inflmmtory pin sttes 28,29,39 suggest tht lockers of SNS synthesis or ctivity should e specific nlgesics. METHODS Gene trgeting. 129Sv genomic DNA ws used to construct trgeting vector 20. A 2.5-k HincII EcoRI frgment contining prt of sns exon 4 ws ligted into pbluescript (linerized with SmI nd EcoRI) tht contined PGK-neo cssette etween the EcoRI nd HindIII sites. An ApI frgment contining exons 5 9 ws ligted to the PGK-neo cssette nd thymidine kinse dimer cssette (MC1) to give the trgeting construct (Fig. 1). E14-TG2 suclone IV cells were electroported with 150 mg NotI-linerized vector in 600 ml PBS t 0.8 kv, 3.0 mf. Cells were selected with GM418 nd gncyclovir, nd three correctly trgeted single-copy integrtions were identified. Clones were injected into C57BL/6 lstocysts susequently implnted into CBA C57BL/6 F1 foster mothers. Mle chimers when crossed with C57BL/6 femles gve 100% germline trnsmission of ES cell cot color. Trnsmission of the trgeted llele ws confirmed y Southern lot nlysis (Fig. 1). Chimers were lso susequently crossed with 129/Sv femles. Digests of til DNA with EcoRI were Southern lotted nd proed with rndom-prime-leled ApI EcoRI genomic frgment 20 (Fig. 1). Null-mutnt digests contining the PGK-neo cssette produce nd of 8.8 k, compred to the 7.8- k nd found in wild-type nimls (Fig. 1). Sodium chnnels in null-mutnt DRG. Proes for sodium chnnels were cloned y PCR, or the sme primers were used in RT-PCR experiments: SNS, X92184, 5 -CAGAGATCGAGAAGCAGATCGCTG-3, 5 - AGCTTC- CTCACTGAGTGGATC-3 ; NN, AF059030, 5 -CCCTGCTGCGCTCG- GTGAAGAA-3, 5 -GACAAAGTAGATCCCAGAGAGG-3 ; NCh6, U59966, 5 -GAGAATGAGTTCGCAGACGATG-3, 5 -CTCTTCCAGCTCTTCAC- TAGCGTG-3 ; PN-1, X82835, 5 -AGTGCAGTGGACTGCAATGGAGTCG- 3, 5 -GAGCAAATCTGTACCACCATGGTGGACA-3 ; Type I, X03638, 5 -GGGAAGATGCACAGCACAGTGGATTCC-3, 5 -CTTTTAGC- CAATATGGAGAACAG-3 ; Type II, X03639, 5 -CCGAAAAT- GACTTTGCAGACGATG-3, 5 -CCATCACTACCAGATTGACAACGTG-3 ; Type III, Y00766, 5 -AAGTCGGAATCGGAAGACAGTGT-3, 5 -AGGAT- ACTGGCTATGCTCATGGATC-3 ; mh1, A33996, 5 -TGAGGCG- GACTTCGCAGATGACGAG-3 ; 5 -GAGGACACTGACAGCGCTGAGTGCCCG-3 ; L-27, 5 -ATCGCTC- CTCAAACTTGACC-3, 5 -AAAGCCGTCATCGTAAAGAAC-3 ; cyclophilin 5 - ACCCCACCGTGTTCTTCGAC-3, 5 - CATTTGCCATGGACAAGATG- 3. Rndom-primed cdna synthesized from DNse-I-treted RNA ws mplified (94 o C, 1 min), nneled (58 o C, 1 min) nd extended (72 o C, 1 min) for 25, 30 or 35 cycles. Amplified DNA ws sequenced. [- 32 P]UTPleled rioproes were generted from the linerized plsmid using Sp6 or T7 polymerse. We seprted mg totl RNA from rt nd mouse tissues on 1% grose-formldehyde gels nd lotted it onto Hyond-N. Northern lots were done nd quntified s descried 33. Bnds were normlized ginst cyclophilin. Immunocytochemistry. Seven-micron sections of prformldehydefixed L4 DRG were stined with ntiodies ginst peripherin, nd neurofilments (N52) nd with the FITC-lelled lectin IB 4. Strongly positive cell percentges were estimted y counting the totl numer of neurons. Totl cell counts were done y counting nuclei in seril sections of L-4 DRG s descried 36. Electrophysiology. We recorded from DRG neurons (1 5 dys in culture) two hours fter replting t room temperture. The cdna encoding rt SNS in the expression vector pgw-1 (HindIII KpnI 35 ; 200 mg/ml), together with the GFP expression vector pgw-1-gfp (100 mg/ml) nd 0.5 % FITC-dextrn were injected into neuronl nuclei. The DNA injection uffer contined 118 mm NCl, 3 mm KCl, 5 mm HEPES, 22.2 mm NHCO 3 nd 1.2 mm MgCl 2, ph 7. Recordings were mde 1 2 dys fter injection using the whole-cell, ptch-clmp technique 36. pclmp6 softwre (Axon) ws used to cquire nd nlyze the currents. Pipets hd DC resistnces of 3 MW. The pipet solution contined 130 mm CsCl, 10 mm NCl, 1 mm MgCl 2, 10 mm HEPES nd 5 mm EGTA, ph In some experiments, CsCl ws sustituted with equimolr CsF. The extrcellulr solution contined 105 mm choline Cl, 35 mm NCl, 3 mm CsCl, 1 mm MgCl 2, 0.01 mm CCl 2, 0.05 mm CdCl 2, 11 mm glucose nd 5 mm HEPES, ph 7.4. In some experiments, 500 nm TTX ws included in the extrcellulr solution. The extrcellulr sodium concentrtion ws lowered to 35 mm to reduce the mgnitude of the currents. Clcium chnnel ctivity ws locked y the inclusion of either CsF in the intrcellulr or CdCl 2 in the extrcellulr solution. For electrophysiologicl studies in vivo, mice were nesthetized with urethne (3 g per kg, i.p.) Following lminectomy, single-neuron extrcellulr recordings were mde using prylene-coted tungsten electrode in the dorsl horn ( mm; men depth, 445 ± 28 mm). Electricl stimultion (2 ms pulse) in the peripherl receptive field evoked short-ltency, low-threshold response (< 10 ms fter stimulus) nd longer-ltency, high-threshold response ( ms fter stimulus) in the dorsl horn neuron, which reflect recruitment of A- nd C-fiers. Electricl excitility ws mesured directly y studying compound ction potentils from L4 dorsl roots of mice (n = 4) following electricl stimultion of the scitic nerve t mid-thigh level. Compound ction potentil res were mesured fter squre-wve current pulses of 200 ms durtion nd 0 40 ma mplitude for A-fiers, nd 2 ms durtion nd 0 1 ma mplitude for C-fiers. Behviorl studies. Mice were exmined for spinl reflexes nd motor skills s descried in detil elsewhere 26. Nociceptive thresholds to therml stimuli were determined using hot-plte (45, 50, 55 or 60 o C), pw-flick (method of Hrgreves) or til-flick methods. For the hot plte, the ltency for the mouse to lick its hindpw or jump ws recorded. Pw-flick ltencies were determined for oth hind pws on three occsions. Tilflick ltencies were determined on three occsions. Mechnicl sensitivity ws determined using clirted Von Frey hirs. Hirs were pplied three times ech in scending order of force, through mesh floor, until response ws elicited (pw withdrwl, shking or iting). Percent responses were clculted for ech hir. Nociceptive thresholds to noxious mechnicl stimuli were determined using Ugo Bsile lgesiometer. The til ws grdully compressed until n escpe response ws elicited (iting the pprtus, vocliztion or struggle). Crrgeenn-induced therml hyperlgesi ws ssessed 27 using n intrplntr injection of 20 ml of crrgeenn (0.6 mg) or sline into one hind pw. Pw-flick nture neuroscience volume 2 no 6 june

8 rticles ltencies were mesured, nd mice tht hd received crrgeenn were killed nd oth hind feet weighed to mesure edem. To ssess the effect of lidocine, fter seline therml nd mechnicl mesurements, 25 mg per kg lidocine (Sigm) in PBS (5 mg/ml) ws injected i.p. nd further tests crried out ten minutes lter. These experiments were crried out under Home Office supervision t UMDS nd MSD lortories. ACKNOWLEDGEMENTS This work ws supported y the MRC (V.S., K.O., B.K., S.M., A.H.D., L.C.S.), the Wellcome Trust (A.A., S.E., J.N.W.) nd the Royl Society (N.O.). L.C.S. ws supported y Merck Phrmcology Fellowship. The Centre for Genome Reserch ws supported y the BBSRC. We re grteful to Smnth Rvenll, Mdhu Sukumrn, Oro Rufin, Sturt Stevenson, Richrd Pugh, Jne Hley, Ptrique Delms nd Dvid Brown for technicl dvice nd comments. RECEIVED 13 NOVEMBER 1998; ACCEPTED 16 APRIL Mtsud, Y., Yoshid, S. & Yonezw, T. 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Complex lockde of TTX-resistnt N + currents y lidocine nd upivcine reduce firing frequency in DRG neurons. J. Neurophysiol. 79, (1998). 31. Bru, M. E. & Elliott, J. R. Locl nesthetic effects on TTX-r sodium currents in rt dorsl root gnglion neurones Eur. J. Anesthesiol. 15, (1998). 32. Isom, L. L., De-Jongh, K. S. & Ctterll, W. A. Auxiliry suunits of voltgegted ion chnnels. Neuron 12, (1994). 33. Akopin, A. N., Souslov, V., Sivilotti, L. & Wood, J. N. Structure nd distriution of rodly expressed typicl sodium chnnel. FEBS Lett. 400, (1997). 34. Cummins, T. R. & Wxmn, S. G. Downregultion of tetrodotoxin-resistnt sodium currents nd upregultion of rpidly repriming tetrodotoxinsensitive sodium current in smll spinl sensory neurons fter nerve injury. J. Neurosci. 17, (1997). 35. Connolly, C. N., Krishek, B. J., McDonld, B. J., Smrt, T. G. & Moss, S. J. Assemly nd cell surfce expression of heteromeric nd homomeric gmm-minoutyric cid type A receptors. J. Biol. Chem. 271, (1996). 36. Hmill, O. 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