Osmolarity modulates K+ channel function on rat hippocampal

Transcription

1 6145 Journl of Physiology (1997), 498.3, pp Osmolrity modultes K+ chnnel function on rt hippocmpl interneurons ut not CI pyrmidl neurons Scott C. rn *, Mrk C. ellinghm t, lert J. erger t nd Philip. Schwrtzkroin * t Deprtments of *Neurologicl Surgery nd tphysiology/iophysics, University of Wshington, Settle, W 98195, US 1. Whole-cell nd single-chnnel recording methods were used in conjunction with infrred video microscopy techniques to exmine the properties of voltge-ctivted potssium chnnels in hippocmpl neurons during the ppliction of hyposmolr solutions to hippocmpl slices from rts. 2. Hyposmolr externl solutions (osmolrity reduced y 1% to 267 mosmol F-) produced significnt potentition of voltge-ctivted K+ current on lcunosum/moleculre (L/M) hippocmpl interneurons, ut not on Cl nd suiculum pyrmidl neurons. Hyperpolriztion-ctivted (IH) nd lek currents were not ltered during the ppliction of hyposmolr solutions in ll cell types. 3. Men chnnel open time nd the proility of chnnel opening were drmticlly incresed under hyposmolr recording conditions for outside-out ptches from L/M interneurons; no chnges were oserved for ptches from Cl pyrmidl neurons. Men current mplitude nd the threshold for chnnel ctivtion were not ffected y hyposmotic chllenge. 4. Hyposmolr externl solutions produced significnt reduction in the firing frequency of L/M interneurons recorded in current-clmp mode. Hyposmolr solutions hd no effect on resting memrne potentil, ction potentil mplitude or durtion, nd spike fterhyperpolriztion mplitude. 5. These results indicte tht selective modultion of interneuron ion chnnel ctivity my e criticl mechnism y which osmolrity cn regulte excitility in the centrl nervous system. reduction in extrcellulr osmolrity promotes epileptiform ctivity oth cliniclly nd in the hippocmpl slice preprtion (ndrew, 1991; llyk, Quckenush & ndrew, 1991). It hs een proposed tht the reduction in extrcellulr spce (ECS) tht occurs during periods of hyposmotic stress promotes neuronl synchroniztion nd hyperexcitility vi non-synptic mechnisms. For exmple, decresing the ECS my exggerte the effects of fluctutions in extrcellulr ion concentrtions nd increse extrcellulr electricl resistnce (ndrew, 1991; Tru, Dudek, Snow & Knowles, 1985). However, it is not known whether chnge in the extrcellulr environment (e.g. chnge in extrcellulr osmolrity) hs direct effect on ion chnnel function. If such modultion occurred, it would serve s n importnt non-synptic mechnism for regulting neuronl excitility under oth norml nd pthophysiologicl conditions. Cell swelling, which occurs with reduced osmolrity of the extrcellulr medium, hs een shown to ctivte stretchsensitive (or inctivte stretch-inctivted) ion chnnels in cultured or cutely dissocited cell preprtions (Guhry & Schs, 1984; Morris & Sigurdson, 1989; Kim & Fu, 1993; Oliet & ourque, 1996). Lignd-gted N-methyl-D-sprtte chnnels nd voltge-ctivted clcium chnnels in cultured neurons re lso sensitive to chnges in osmolrity (Lngton, 1993; Poletti & scher, 1994). In non-neuronl cells, voltge-ctivted K+ currents re influenced y mechnicl chnges (either directly or indirectly) medited y osmotic stress (Schoenmkers, Vudry & Czin, 1995). It hs een hypothesized tht hyposmolr solutions produce mechnicl stretch of chnnel proteins, opening the pore to permit greter influx of ions. This mechnicl stretch hypothesis (Morris, 199; Schs, 1992; Poletti & scher, 1994) lso predicts tht osmotic stress would increse voltge-ctivted ion currents in neuronlly non-specific mnner, i.e. on ll cell types. Since mesurements of chnnel function in response to osmotic stimultion hve een mde exclusively on cultured or cutely dissocited cells, it is not known whether chnges in extrcellulr osmolrity modulte ion chnnel function on neurons mintined in system tht more closely reflects in vivo conditions (i.e. with 'norml' extrcellulr environment).

2 68 S. C. rn nd others J Phy8iol We investigted this issue in the hippocmpl slice preprtion, where cell-to-cell interctions nd the extrcellulr spce reltionships re generlly mintined. METHODS Hippocmpl slice preprtion Trnsverse hippocmpl slices (3 4um thick) were prepred from 15- to 25-dy-old Sprgue-Dwley rts, s previously descried (rn & Schwrtzkroin, 1996). riefly, rts were decpitted, the rin ws rpidly removed nd chilled riefly in ice-cold, oxygented (95% 2-5% C2) sucrose rtificil cererospinl fluid consisting of (mm): 22 sucrose, 3 KCl, 1P25 NH2P4, 1P2 MgSO4, 26 NHCO3, 2 CCl2 nd 1 dextrose ( mosmol F-'; mesured on n osmometer). The sustitution of sucrose for NCl during preprtion yields slices with greter numers of vile neurons (ghjnin & Rsmussen, 1989; Richerson & Messer, 1995). The rin ws then locked nd glued to the stge of Viroslicer (Frederick Her, runswick, ME, US) nd slices cut from the temporl hlf of the hippocmpus in oxygented sucrose rtificil cererospinl fluid t 4 C. The resulting slices were then trnsferred to holding chmer, where they were sumerged in oxygented norml rtificil cererospinl fluid consisting of (mm): 124 NCI, 3 KCl, 1-25 NH2PO4, 1P2 MgSO4, 26 NHCO3, 2 CCl2 nd 1 dextrose ( mosmol I-). Slices were held t room temperture for t lest 45 min nd experiments performed within 8 h. Slices were perfused with extrcellulr solution t rte of -2 ml min-' ll recordings were mde t room temperture (22-24 OC). Whole-cell recording Tight-sel (4-16 GQ2) whole-cell voltge-clmp recordings were mde with n xoptch-id mplifier (xon Instruments) with pproprite series nd cpcitnce compenstion (Hmill, Mrty, Neher, Skmnn & Sigworth, 1981). Tight-sel whole-cell currentclmp recordings were mde with n xoclmp-2 mplifier (xon Instruments). Ptch pipettes were pulled from 1-5 mm orosilicte filment-contining glss tuing (Wrner Instrument Corp., Hmden, CT, US) using two-stge process, fire-polished nd coted with Sylgrd (Dow Corning). The pipette ws positioned under visul control using Zeiss xioscope nd wter-immersion (x 4) ojective with Nomrski differentil interference contrst optics nd n infrred cmer (Hmmtsu Co., Hmmtsu, Jpn). The commnd protocol to ctivte voltge-dependent K+ currents involved hyperpolrizing prepulse to -115 mv, followed y six depolrizing voltge steps (2 mv increments) from the holding potentil (-6 mv); hyperpolrizing pulse following the commnd potentils ws used to monitor cell cpcitnce nd series resistnce. Currents were lek sutrcted on-line using P/4 protocol. Voltge-clmp commnd potentils nd nlysis of currents were performed using pclmp softwre (xon Instruments). Current records were low-pss filtered t 1-2 khz (-3 d, 8-pole essel), digitized t 4-1 khz using Digidt 12 /D interfce, nd stored on IM-comptile 486 microcomputer. Single-chnnel recording Electrophysiologicl investigtions were performed on excised memrne ptches in outside-out configurtion with n xoptch- 1D mplifier (Hmill et l. 1981). Ptch holding potentil ws -6 mv. To otin ensemle currents the commnd potentil ws stepped to -115 mv (15 ms) followed y depolrizing voltge step to +4 mv. For recording stedy-stte currents, the holding potentils rnged from -6 to +2 mv for durtions of 6-3 s. Single-chnnel recordings were digitlly low-pss filtered (-3 d, 8-pole essel) with cut-off frequency of 5 khz nd smpled t 2 khz. The dt were stored on videotpe nd nlysed off-line using pclmp softwre. The single-chnnel events were mesured y 5% threshold detection nd verified individully in Fetchn (xon Instruments). The current mplitude histogrms were fitted with Gussin functions nd the decy of the open time histogrms with single- or i-exponentil functions. Solutions Normosmolr extrcellulr solution (297 mosmol F') consisted (mm) of either: (i) 124 NCl, 3 KCl, 1-25 NH2PO4, 2 MgSO4, 26 NHCO3, 2 CCl2 nd 1 dextrose; or (ii) 14 NCl, 3 KCI, 1-25 NH2PO4, 2 MgSO4, 26 NHCO3, 2 CCl2 nd 1 dextrose with the ddition of sucrose (-32 g Fl). Hyposmolr solutions (267 mosmol Fl) were mde y the reduction of [NCI] (from 124 to 14 mm); hyperosmolr solutions (327 mosmol F') contined 14 mm NCl plus 65 mm sucrose. ll solutions were djusted to ph 7 4 nd uled with 95% 2-5% CO2. Ptch pipettes were filled with internl recording solution consisting of (mm): 16 KCH3SO4, 1 Hepes, 11 EGT, 1 CCl2, 1 MgCl2, 4 N-TP nd 4 N-GTP (ph ws djusted to 7-25 with 1 M KOH nd osmolrity djusted to mosmol 1- with sucrose). Intrcellulr C2+ ws uffered y 11 mm EGT to reduce the potentil effects of cell swellinginduced increses in intrcellulr free C2+ concentrtion (Wong & Chse, 1986; usch, Vrnum, delmn & North, 1992). The osmolrity of ll solutions ws mesured using Wescor 51G vpour pressure osmometer (Wescor Inc., Logn, UT, US). Pipette solutions were filtered through 2,um filter (Millipore). Solutions with different osmolrities were pplied to the slice or outside-out memrne ptches vi rpid th perfusion (-2 ml min-'). Sttistics Results re presented s men vlues + S.E.M. Dt were nlysed using Student's pired t test on the SigmStt progrm (Jndel Scientific, Sn Rfel, C, US). Significnce level ws tken s P < 5, unless otherwise indicted. RESULTS Phrmcologicl chrcteriztion of whole-cell voltge-ctivted potssium current Infrred video microscopy in conjunction with ptchclmp recording ws used to visulize nd monitor lcunosum/moleculre (L/M) interneurons nd CI pyrmidl neurons in hippocmpl slices. In whole-cell recordings, L/M interneurons nd Cl pyrmidl cells displyed prominent voltge-ctivted outwrd currents during cell depolriztion in normosmolr thing medium (297 mosmol F') contining tetrodotoxin (1 /mm ITX; to lock voltge-dependent Ne chnnels) nd cdmium (1 FM CdCl2; to lock C2+ chnnels). In oth cell types, ppliction of tetrethylmmonium chloride (2-5-2 mm TE) reduced the sustined, delyed rectifier K+ currents (presumly IK) in dose-dependent mnner. TE lockde of IK ws reversile. t concentrtion of 2 mm TE, IK ws reduced y 74% in L/M interneurons (n= 4) nd y 64% in CI neurons (n = 3). In oth types of neurons, prominent fst, trnsient K+ current (presumly I) ws oserved during TE ppliction.

3 J Physiol Osmosensitive potssium chnnels 681 Effects of osmolrity on whole-cell voltge-ctivted potssium current When the slice ws thed in hyposmotic solution (osmolrity reduced y 1% to 267 mosmol F1) we oserved comprle increses in cell volume in oth types of neurons (Fig. 1 C nd D). The som length nd dimeter were mesured from frme-grer imges with the id of digitizing tlet. The som volume ws clculted using the formul for prolte spheroid, nrld2/6, where d is dimeter nd 1 is length (Cmeron, verill & erger, 1983). These mesurements were mde t the cell som nd llow for n pproximtion only of som volume chnges; the chnges in dendritic volumes my e significntly different. Our mesure of cell swelling under hyposmolr conditions indicted 55 % increse in som volume for L/M interneurons (n = 3) nd 43 % increse in CI pyrmidl cells (n = 4). Hyposmolr externl solutions produced significnt potentition of the sustined, delyed rectifier K+ currents recorded from L/M interneurons (n = 1; Fig. I nd ), ut did not potentite voltge-ctivted K+ currents on CI pyrmidl neurons (n = 8; Fig. l-c). Potentition c 6 Hypo < 4 : 2 25 p Time (min) 25 ms c 6 Hypo Cl 4 ) : 2 _,oo C D Time (min) Figure 1. Voltge-ctivted potssium current is sensitive to chnges in extrcellulr osmolrity: whole-cell recordings, whole-cell voltge-clmp recording from n L/M interneuron in normosmolr thing medium (297 mosmol F1-; ) nd following ppliction of hyposmotic thing medium (osmolrity reduced y 1% to 267 mosmol Fl; 1 min; ). Note the osmolrity-medited increse in K+ current. c, plot of the current mplitude mesured t the 45 ms time point of 5 ms depolrizing step to +4 mv. Six mesurements were otined for ech time point (); ppliction of hyposmolr solution (r) incresed IK mplitudes., whole-cell voltge-clmp recording from Cl pyrmidl neuron in normosmolr thing medium (297 mosmol F1; ) nd following ppliction of hyposmotic thing medium (267 mosmol F1-; 1 min; ). c, plot of current mplitude vs. time for the C1 neuron, showing no significnt chnge in current mplitude during hyposmolr chllenge. C, frme-grer imges of n L/M interneuron in normosmolr thing medium (C) nd following ppliction of hyposmolr solution (mesured -2 min following hyposmolr solution ppliction; C). D, frme-grer imges of Ct pyrmidl neuron in normosmolr thing medium (D) nd following ppliction of hyposmolr solution (- 2 min; D ). For oth cell types, the hyposmolr chllenges cused cell swelling tht ws reversile when normosmotic solution ws restored. Scle r (in C), 1 um.

4 . 682 S. C. rn nd others J Phy8iol p p C p 6 %,P%.PW%.f I 6 61 // / 2 - / om 2 2 _ ~ ~~~~ mv mv Figure 2. Current-voltge reltionship for hippocmpl neurons exposed to hyposmolr thing medium, representtive current-voltge plot for n L/M interneuron., representtive plot for Cl pyrmidl neuron. C, representtive plot for suiculum neuron. Normosmolr recording conditions (K; continuous lines); hyposmolr recording conditions (; dshed lines) re shown. Note tht only the L/M interneurons showed chnge in the I-V reltionship following exposure to hyposmolr conditions. of interneuron K+ current ws initilly oserved t 513 p; n = 1; P< 1); no significnt chnge (-6%) 2-4 min following ppliction of hyposmolr solution to the ws oserved in IK mplitude for Cl pyrmidl neurons slice, reched mximum t -15 min, ws sustined for (control, p; hyposmolr, p; recording periods of > 6 min, nd recovered to seline n = 8, P = 46). The fst, trnsient K+ current lso current mplitude during return to normosmolr thing incresed during ppliction of hyposmotic solutions ut medium (Fig. 1 c). We mesured nd compred IK ws not quntified owing to heterogeneity of I cross L/M mplitudes in the whole-cell mode, since this current is interneurons (not shown). This oservtion of I vriility stle over long periods of whole-cell recording. The mong L/M interneurons is consistent with previous reports mplitude of IK, mesured t the 45 ms time point of of L/M heterogeneity reveled y morphologicl nd shrp 5 ms depolrizing commnd, ws significntly incresed intrcellulr current-clmp recordings (Lcille & (+51 %) y hyposmolr thing solutions only in L/M Schwrtzkroin, 1988). lthough Fig. l suggests decrese interneurons (control, p; hyposmolr, in ' on this Cl pyrmidl neuron during the ppliction f-, - homro 4 m%~ VOW%o koo i --M.1 --I j ~~~~~~~~~~- -I _ Il_".mot,S 4= ~~~-= I mv 12 p 2 ms Figure 3. Hyperpolriztion-ctivted currents re not modulted y chnges in extrcellulr osmolrity, whole-cell currents (ottom trces) of n L/M interneuron during series of long hyperpolrizing voltge steps (top trces) under normosmolr recording conditions., sme cell pproximtely 25 min fter ppliction of hyposmolr solution. IH is not lek sutrcted; pek current is determined s the difference etween instntneous () nd stedy-stte currents (). Voltge-ctivted K+ current for this neuron is shown in inset in (scle rs: 25 p, 2 ms) efore (ottom trce) nd fter ppliction of hyposmolr solution (top trce).

5 J Physiol Osmosensitive potssium chnnels 683 of hyposmolr solutions, this effect ws not seen in other CI pyrmidl cells; mesurement of this current reveled non-significnt 1 % decrese in current mplitude mong pyrmidl cells (n = 4; P = 68). Hyperosmotic solutions (osmolrity incresed y 1% to 327 mosmol F') reduced K+ current y 48% in oth L/M interneurons nd CI pyrmidl cells in n irreversile mnner (n = 3), s previously demonstrted in neurolstom x gliom (NG18-15) cells (Schoenmkers et l. 1995) nd cutely dissocited CI pyrmidl neurons (Hung, itken & Somjen, 1995). plot of the stedy-stte current-voltge reltionship (mesured t the 45 ms time point of 5 ms current pulse) ws constructed for three representtive types of hippocmpl neurons: L/M interneuron, CI pyrmidl neuron nd suiculum pyrmidl neuron (Fig. 2). ppliction of hyposmolr solutions produced significnt potentition of voltge-ctivted K+ current on L/M interneurons t depolrizing steps etween +6 nd +1 mv; no chnge in the threshold for outwrd current ctivtion ws oserved. Outwrd current mplitude on Cl opow"llo, ---- M..- c IN- -_ I,, p ^ 1~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 21 p 4 ms C -36 mv -34 mv.*, Time (ms) Time (ms) 4 Figure 4. Voltge-ctivted potssium chnnels re modulted y extrcellulr osmolrity: single-chnnel outside-out ptch recordings Outside-out ptches were held t memrne potentil of -6 mv nd depolrized (following hyperpolrizing prepulse) with protocol designed to mtch tht used in whole-cell experiments; 1-15 current trces were summed to otin n ensemle current., verge ensemle current otined during depolrizing voltge step to +4 mv (following prepulse hyperpolriztion); holding potentil, -6 mv. Ensemle verge from 15 such trils in normosmolr thing medium ( ) nd following ppliction of hyposmolr solution ( ; -1 min). Currents re lek sutrcted., sme protocol for single-chnnel ptch recording from CI pyrmidl neuron in normosmolr thing medium () nd following ppliction of hyposmolr solution (; -1 min). C, voltge-rmp protocol (top; -1 to +2 mv) to ctivte K+ chnnels on n L/M interneuron ptch under normosmolr recording conditions (297 mosmol F'; C) nd during ppliction of hyposmolr solution (267 mosmol F'; 15 min; C). Note the osmolrity-medited increse in chnnel open time in the sence of chnge in threshold for ctivtion.

6 684 S. C. rn nd others J Phy8iol pyrmidl neurons nd suiculum pyrmidl neurons (control, p; hyposmolr, p; n = 3; P = -61) were not ltered y ppliction of hyposmolr solutions (Fig. 2 nd C). To test the selectivity of hyposmotic effects on IK' we lso exmined osmolrity influences on hyperpolriztionctivted current (Fig. 3). Long hyperpolrizing voltge commnds (2 s; Fig. 3, top set of trces) ctivted slowly developing inwrd current (presumly IH) in oth types of neurons. Hyposmolr solutions did not significntly lter the pek mplitude of this current in L/M interneurons (+15%; n = 6; P = O77) or in CI pyrmidl neurons (+4%; n= 3; P = 84). lthough IH ws not ltered during the ppliction of hyposmolr solutions to L/M interneurons, potentition of IK could still e oserved (see 15 p 11. ~ L - I~~~~~~~~7s - E~~~tM_. u_-lf.- r_je ---wo k, c d 1 -o o 8 2o 6 EEe 4 o I & -2 O1. E ) : I Chnnel open time (ms) -L.L..- [Ih Time (ms)..~~~~r-"e mplitude (p) c co 18 E ' E 6._ QM._ d 2 O c E o Chnnel open time (ms) Time (ms) 5 1 mplitude (p) Figure 5. Kinetics of single-chnnel currents from L/M interneurons Stedy-stte single-chnnel recordings from representtive L/M interneuron ptch under normosmolr recording conditions (, open proility -1) nd 15 min following ppliction of hyposmolr = solution (, open proility 113). Currents = were elicited t holding potentil of +2 mv. Note the osmolrity-medited increse in chnnel ctivity. Kinetics of single-chnnel K+ currents were mesured t stedy-stte holding potentil of +2 mv (12 s recording) during seline (297 mosmol F') nd hyposmolr recording conditions (267 mosmol l-1). Histogrms of the chnnel open time were computed for seline ( ) nd hyposmolr recording conditions (). single exponentil function fitted the decy of the histogrm in, ut two exponentils were needed to fit the decy in, indicting significnt difference in chnnel opening etween normosmolr nd hyposmolr sttes. Histogrms of the proility of single-chnnel openings during normosmolr ( c) nd hyposmolr recordings (c) were lso significntly different. However, mplitude histogrms of single-chnnel K+ currents under normosmolr ( d) nd hyposmolr recording conditions (d), fitted with Gussin functions, showed the sme men mplitudes for normo- nd hyposmolr conditions. 15

7 J Phy8iol Osmosen8itive potssium chnnels 685 Tle 1. Single-chnnel potssium currents Normosmolr Hyposmolr Cell type (297 mosmol I1) (267 mosmol F1) L/M interneuron (n = 6) Open time (ms) 7o 1P * (1P8-2-5) (44-79) T * ( ) Open proility '3 * (-1--14) ( 5-242) mplitude (p) ( ) ( ) CI pyrmidl neuron (n =3) Open time (ms) To 1P t19 (-87-1P4) (-96-1P4) Open proility ( ) ( ) mplitude (p) (4--4-3) ( ) Currents were mesured t stedy-stte holding potentil of +2 mv (12 s recording period). The vlues represent mens + S.E.M. Rnge of vlues is represented in prentheses. * Sttisticlly significnt differences from seline vlues using Student's pired t test (P < 5). Fig. 3, inset). Further, we mesured the mplitude of L/M interneuron lek current efore nd fter the ppliction of hyposmolr solutions. Lek current ccounted for < 1% of whole-cell voltge-ctivted current in L/M interneurons nd did not chnge y more thn 1 p during the ppliction of hyposmolr solutions. These experiments demonstrte neuron- nd current-specific modultion of K+ chnnel function in the hippocmpus during periods of osmolrity-induced cell swelling. Effect of osmolrity on single-chnnel voltgectivted potssium current Single-chnnel currents were studied in outside-out ptches pulled from visully identified L/M interneurons nd CI pyrmidl neurons. In these experiments, we mesured nd compred the mplitude of ensemle IK' t the 45 ms time point of 5 ms depolrizing commnd. This current ws significntly potentited y hyposmolr thing solutions in L/M interneurons (6 + 1%; n = 7; P< 5; Fig. 4); no chnge ws oserved in ensemle IK mplitude for CI pyrmidl neurons (1 ± 12%; n = 3; Fig. 4). Slow voltge-rmp protocols (-1 to +2 mv; 35 ms durtion) were used to test whether osmolrity-induced chnges in K+ ion chnnel function could e scried to shift in the threshold for chnnel ctivtion. Hyposmolr-medited chnges in K+ chnnel ctivity were oserved t holding potentils etween -25 nd +4 mv, nd were not ssocited with significnt chnge in the threshold for chnnel ctivtion (Fig. 4C; control, mv; rnge, to -41P2 mv; hyposmolr, mv; rnge, to -41P5 mv; n= 6; P = 98). Single-chnnel voltge-ctivted current ws reduced y 52 % during 2 mm TE ppliction (n = 3), s expected for K+selective ion chnnel (Storm, 199). The pprent difference etween whole-cell nd outside-out ptch sensitivity to TE my simply reflect n uneven distriution of TEselective K+ chnnels in these memrne ptches, especilly since the rnge of sensitivities (38-7%) is consistent with the sensitivity to TE seen in whole-cell recording. seprte protocol ws used to exmine stedy-stte single-chnnel kinetics during chnges in osmolrity. Under normosmolr recording conditions (297 mosmol 1-l with 1 /LM TTX nd 1 /SM cdmium), the chnnel open time, the proility of chnnel opening, nd the mplitude of singlechnnel currents were determined t holding potentil of +2 mv. In L/M interneuron ptches held t +2 mv to ctivte K+ chnnels, significnt increse in K+ chnnel ctivity ws oserved 1-2 min following ppliction of hyposmolr solutions. This increse persisted for recording periods of > 4 min nd chnnel ctivity recovered to seline levels upon return to normosmolr thing medium (Fig. 5 nd ); no chnge ws oserved for ptches from Cl neurons. The men mplitude of single-chnnel currents ws not ffected y hyposmolr solutions nd dditionl chnnel open sttes were not oserved (Tle 1 nd Fig. 5 nd d). However, chnnel open time for ptches from L/M interneurons ws drmticlly incresed during hyposmolr recording conditions (Tle 1). In normosmolr thing medium the men open time ws fitted y single exponentil for ptches from L/M

8 686 S. C. rn nd others J Physiol Tle 2. Current-clmp properties of L/M interneurons Resting ction ction Spike Numer of memrne potentil potentil HP spikes potentil mplitude durtion mplitude per 5 ms (mv) (mv) (ms) (mv) Normosmolr P P (297 mosmol I-) (-54 to -71) (71-13) (3-5) (5-22) (7-22) Hyposmolr P ' * (267 mosmol F-) (-52 to -69) (78-96) (3-6) (6-16) (1-1) The vlues represent mens + S.E.M. (n = 11). Rnge of vlues is represented in prentheses. HP, fterhyperpolriztion. * Sttisticlly significnt difference from seline vlues using Student's pired t test (P< 1). interneurons (time constnt, To = 1P ms); this vlue incresed (To = 4 9 ± 9 ms; P< 5) following ppliction of hyposmolr recording solutions. Under hyposmolr recording conditions chnnel open time histogrms were est fitted y i-exponentil eqution yielding two time constnts, To nd T1 (Tle 1; compre Fig. 5 nd ). The proility of chnnel opening for ptches from L/M interneurons ws lso ltered during ppliction of hyposmolr solutions. These chnges in single-chnnel kinetics were not oserved for ptches from CI pyrmidl neurons (Tle 1). Therefore, chnges in potssium current oserved during conditions of hyposmolr-induced cell swelling re selectively medited y chnges in singlechnnel kinetics. ( E LO 2 mv 1 ms Cz E z C 1 ms 2 mv Figure 6. L/M interneuron firing frequency is modulted y extrcellulr osmolrity, representtive current-clmp recording of n L/M interneuron in normosmolr solution during ppliction of 1 ms depolrizing current injection (ottom trce; 1 n for 2 ms)., plot of the firing frequency for ll eleven L/M interneurons tested, illustrting the difference in dischrge properties under normosmolr conditions (*) nd following ppliction of hyposmolr solutions (-2 min; O). C, representtive current-clmp recording of n L/M interneuron under normosmolr recording conditions (resting memrne potentil, -7 mv; C) nd 2 min fter ppliction of hyposmolr solution (resting memrne potentil, -71 mv; C). Depolrizing current step is 5 n for 5 ms. ction potentil mplitudes clipped y digitizing process.

9 J Physiol Osmosensitive potssium chnnels 687 Effects of osmolrity on the firing properties of L/M interneurons In seprte set of experiments we used current-clmp recordings to determine whether osmolrity-medited chnges in K+ chnnel function were ssocited with chnge in cell firing properties. Eleven visully identified L/M interneurons were recorded using infrred video microscopy in whole-cell current-clmp mode. These cells did not fire ction potentils spontneously nd, when cused to fire y intrcellulr current injection, were chrcterized y lrge spike fter-hyperpolriztion, s reported previously (Lcille & Schwrtzkroin, 1988; Willims, Smulck, eulieu & Lcille, 1994). representtive recording from one such L/M interneuron is shown in Fig. 6. ppliction of hyposmolr solutions did not significntly lter intrinsic cell properties such s resting memrne potentil, ction potentil mplitude nd durtion, nd spike fter-hyperpolriztions (Tle 2), suggesting tht voltge-ctivted sodium nd clcium chnnels re not sensitive to chnges in osmolrity. However, hyposmolr solutions did produce significnt modultion of the firing frequency of L/M interneurons. The numer of ction potentils generted during long depolrizing step (5 ms, 5 n) ws determined for ll neurons efore nd 2 min fter ppliction of hyposmolr solutions (Tle 2; Fig. 6). Hyposmolr solutions drmticlly reduced the firing frequency in these neurons. representtive recording of n L/M interneuron efore nd fter ppliction of hyposmolr solution is shown in Fig. 6C. Prtil recovery of firing frequency ws oserved pproximtely 2 min fter return to normosmolr thing medium. These results suggest tht osmotic stress cn directly modulte neuronl excitility in the hippocmpl slice preprtion. DISCUSSION In these studies, we oserved selective nd neuron-specific modultion of K+ current during modest chnges in osmolrity (3 mosmol F1). Our dt suggest tht (i) osmolrity my ply criticl role in regulting neuronl excittion during oth norml nd pthophysiologicl conditions nd (ii) there my e fundmentl differences in pyrmidl vs. interneuron K+ chnnel structure, which produce differentil sensitivity to chnges in extrcellulr osmolrity. Potentil mechnism of ction for osmosensitive potssium chnnels There re numer of potentil mechnisms tht might contriute to the phenomenon oserved in our studies. First, mechnicl memrne stretch of ion chnnels hs een descried for severl cell types (Sckin, 1989; Morris & Sigurdson, 1989) nd my contriute to the modultion of ion chnnel function oserved in this study. It hs een suggested tht these stretch-sensitive ion chnnels re ctivted (or inctivted) y memrne tension. In previous studies, stretch-sensitive chnnels generlly did not show much voltge dependence nd were difficult to oserve in whole-cell recordings (Sckin, 1989; Poletti & scher, 1994). This mechnism seems unlikely given our findings tht osmosensitive K+ chnnels on L/M interneurons re clerly voltge dependent nd could e oserved in wholecell recordings. Second, it is lso possile tht chnges in extrcellulr N+ concentrtion, with corresponding effects on ion fluxes, could contriute to the modultion of chnnel function oserved in this study. However, we oserved the sme osmoticlly medited potentition of K+ current in experiments where extrcellulr N+ concentrtion did not chnge. Third, tht the oserved effect on L/M interneuron potssium chnnels is osmoticlly rther thn mechniclly medited is suggested y the fct tht it would e difficult to produce significnt mount of mechnicl stretch in outside-out memrne ptches during 1% chnge in osmolrity. In previous studies (Sckin, 1989; ckermn, Wickmn & Clphm, 1994; Rees, Vndenerg, Wright, Yoshid & Powell, 1995; Oliet & ourque, 1996), osmotic chllenges sufficient to increse the lterl tension experienced y ion chnnels emedded in ptch were in the rnge of 5-2 mosmol F-'. The chnge in extrcellulr osmolrity required to produce modultion of K+ current in our study ws only 3 mosmol 1F' nd is well within the physiologicl rnge of cererospinl fluid osmolrity fluctutions (11-5 mosmol I1) mesured in 'norml' ptients (Nishimur, Shimuzi, Imng, Kuo & Yoshid, 1977; Kurokow et l. 199). Furthermore, chnges in extrcellulr osmolrity did not pper to produce non-specific 'mechnicl' stretch of ion chnnels in our experiments. In whole-cell voltge-clmp recordings, lek current nd hyperpolriztion-ctivted current (I.) were not significntly ltered during the ppliction of hyposmolr solutions to L/M interneurons. In whole-cell current-clmp recordings, ction potentil properties nd clcium-dependent spike fter-hyperpolriztions were not ltered during the ppliction of hyposmolr solutions. In ddition, two distinct types of pyrmidl neurons (in Cl nd suiculum) were lso not ffected y chnges in osmolrity in vitro. s such, our dt suggest novel, direct nd neuron-specific effect of hyposmotic (rther thn mechnicl) stress on voltgectivted K+ chnnels. Zimmererg nd collegues recently proposed model for osmosensitive K+ chnnels in the squid gint xon: 'spring-loded' K+ chnnel comprising spring etween voltge-dependent gte nd n osmoticlly sensitive chnnel (Zimmererg & Prsegin, 1986; Zimmererg, eznill & Prsegin, 199). The fetures of this model re consistent with our oservtions on L/M interneurons insmuch s osmotic fctors did not ffect the voltge dependence of gting (i.e. no shift in the threshold for chnnel ctivtion), ut did directly modulte K+ chnnel opening kinetics (i.e. increse in the proility of opening nd chnnel open time). One possile explntion for cell type-specific osmoticlly sensitive ion chnnel could e cell-to-cell

10 688 S. C. rn nd others J Phy8iol differences in moleculr structure. For exmple, single mino cid se pir residue could confer osmosensitivity on L/M interneuron potssium chnnels, in the sme wy tht individul K+ chnnel residues re involved in ph sensitivity, gting kinetics nd ion selectivity (usch, Hurst, North, delmn & Kvnugh, 1991; McCormck, Lin & Sigworth, 1993; Stnfield, Dvies, Shelton, Sutcliffe, rmmr & Conley, 1994; Nvrtnm, Escor, Covrruis & Oerholtzer, 1995). Functionl significnce of n osmosensitive potssium chnnel Interneurons ply centrl role in regulting network excitility in the hippocmpus (Miles & Wong, 1987). In the CI sufield, L/M interneurons medite primrily feedforwrd inhiition to Cl pyrmidl cells (Lcille & Schwrtzkroin, 1988; Willims et l. 1994), re directly ctivted y excittory synptic input to the hippocmpus, nd their influence on pyrmidl cells is medited y synpticlly relesed neurotrnsmitters nd neuromodultors (lger & Nicoll, 1982). n increse in interneuron K+ current during periods of cell swelling, without compenstory chnges in other interneuron currents (or ltertion of pyrmidl cell K+ current) could hve profound effects on network excitility. For exmple, if cell swelling during hypoxic-ischemic or epileptic episodes produced n increse in interneuron IK, the firing frequency of L/M interneurons would decrese (s shown in Results); the resultnt reduction of inhiition to pyrmidl neurons would contriute to the hyperexcitility oserved under these conditions (ndrew, 1991; llyk et t. 1991). Indeed, hyposmolr sttes (e.g. during hypersecretion of ntidiuretic hormone or wter intoxiction) result in cell swelling, hyperexcitility nd n incresed incidence of seizures (ndrew, 1991; Roper, Oenus & Dudek, 1992). Recent work from our lortory hs demonstrted tht furosemide (n gent which reduces cell swelling) cn lock epileptiform ctivity in vriety of in vitro nd in vivo models (Hochmn, rn, Owens & Schwrtzkroin, 1995). In these studies, intrinsic opticl signls otined during stimultion-induced epileptiform fter-dischrge ctivity indicted significnt degree of cell (neuronl or glil) swelling in the strtum lcunosum/strtum moleculre region of the hippocmpus. The cell odies nd dendritic roriztions of L/M interneurons re prominently represented in this hippocmpl region (Kunkel, Lcille & Schwrtzkroin, 1988; Lcille & Schwrtzkroin, 1988; Willims et l. 1994). Following tretment with furosemide, intrinsic opticl signl nd stimultion-induced epileptiform fter-dischrge were olished (Hochmn et l. 1995). Thus, direct non-synptic modultion of L/M interneuron excitility during chnges in cell swelling (coincident with reduction in extrcellulr spce) is one possile mechnism for the nti-epileptiform ctions of furosemide nd other gents which limit or reverse cell swelling. Conclusion We hve demonstrted tht L/M interneurons possess unique ility to respond to chnges in extrcellulr osmolrity in the in vitro hippocmpl slice preprtion. In contrst to the predicted non-specific effects of mechnicl stretch on ion chnnel function (Morris, 199; Poletti & scher, 1994), this effect ws only oserved on L/M interneurons nd ws specific for TE-sensitive voltgectivted potssium chnnels. 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