ON ELECTRICAL ACTIVITY IN SYMPATHETIC GANGLIA OF THE BULLFROG

Transcription

1 Br. J. Phrmc. (1974), 50, EFFECTS OF LITHIUM IONS ON ELECTRICAL ACTIVITY IN SYMPATHETIC GANGLIA OF THE BULLFROG K. KOKETSU & K. YAMAMOTO Deprtment of Physiology, Kurume University School of Medicine, Kurume, Jpn 1 The mode of ction of lithium on electricl ctivity in the sympthetic gngli of the bullfrog hs been studied by recording extrcellulr nd intrcellulr potentil chnges. Chnges in nerve conduction nd vrious types of synptic trnsmission were studied when sodium ions in the externl solution were totlly replced by equimolr concentrtions of lithium ions nd lso when lithium ions were dded to the externl Ringer solution. Nerve conduction nd nicotinic trnsmission in sympthetic gngli were completely blocked in sodium-free sucrose solution, but were restored when the preprtions were trnsferred to sodium-free lithium solution. 3 In the sodium-free lithium solution, the slow excittory postsynptic potentil (e.p.s.p.) nd muscrinic cetylcholine-depolriztion were restored while the slow inhibitory postsynptic potentil (i.p.s.p.) nd the muscrinic cetylcholine-hyperpolriztion were not restored. Furthermore, the erly fter-dischrges were ccelerted nd the inhibition of fter-dischrges ws eliminted. These results support the hypotheticl concept tht the slow i.p.s.p. is generted by n ctivtion of the electrogenic sodium pump. 4 In the sodium-free lithium solution, restortion of nerve conduction nd synptic trnsmission were trnsient phenomen; both conduction nd trnsmission were grdully blocked when preprtions were soked in the solution for long periods. The blockde ppered to be due to membrne depolriztion. 5 When lithium ions (0 mm) were dded to the Ringer solution, nicotinic trnsmission ws depressed. The slow e.p.s.p. ws lso depressed, but less so thn the slow i.p.s.p. The erly fter-dischrge ws, however, ccelerted; presumbly due to the mrked depression of the slow i.p.s.p. in this solution. 6 Chnges in synptic trnsmission in Ringer solution contining lithium ions could be explined by membrne depolriztion, reduction of cetylcholine relese nd depression of the electrogenic sodium pump. 7 All results obtined in the present experiments could be explined by supposing tht lithium ions re ble to substitute for sodium ions in pssive ionic membrne trnsport dependent on electrochemicl energy but not in ctive ionic membrne trnsport dependent on metbolic energy. Introduction Lithium ions re known to be effective substitutes mechnism underlying the ctions of lithium ions for sodium ions in mintining membrne excit- on vrious types of synptic trnsmission in tion (Hodgkin, 1951; Huxley & Stmpfli, 1951). bullfrog sympthetic gngli. Nevertheless, it hs been reported tht lithium ions do not ct s effective substitutes for sodium ions in the membrne excittion processes nd consequently impede synptic trnsmission in tissues, Methods such s sympthetic gngli (Klingmn, 1966; Pppno & Volle, 1966; Pppno & Voile, 1967). Prvertebrl sympthetic chins were crefully The present experiments were designed to re- isolted from bullfrogs (Rn Ctesbin) nd used exmine these phenomen nd to nlyse the exclusively in these experiments.

2 70 K. KOKETSU & K. YAMAMOTO Records of chnges in membrne potentil Extrcellulr recordings. Action potentils in the pregnglionic nerve trunk (which represent nerve conduction) nd those in the postgnglionic nerve trunk (which represent monosynptic nicotinic trnsmission), were recorded extrcellulrly by pplying single stimuli (0.5 ms pulses) to the pregnglionic nerve fibre. The recording method is illustrted schemticlly in Figure l. To record postgnglionic fter-dischrges, the rrngement shown in Fig. lb ws used. Bursts of repetitive stimuli (30 Hz) were pplied to the pregnglionic nerve fibre for periods indicted in the text. The membrne potentil chnges in gnglion cells were lso recorded by use of the sucrose-gp method (Koketsu & Nishi, 1967; Kosterlitz, Lees & Wllis, 1968). The method is shown digrmmticlly in Figure 1 c. Membrne potentil chnges in gnglion cells were induced either by pplying single or repetitive stimuli to the pregnglionic nerve or by dding one drop of cetylcholine (ACh) solution (0.1 M) to the bthing solution, flowing through chnnel (4 x 3 x 50 mm) t the rte of 0. ml/second. Records of chnges in membrne potentil by the sucrose-gp method were mde in Ringer solution, sodium-free lithium solution or Ringer solution contining lithium chloride (up to 0 mm): no potentil records were mde in sodium-free sucrose solution. Single nd repetitive stimuli were pplied t intervls of pproximtely 3 s nd 3 min, respectively, nd ACh ws pplied t n intervl of 5 minutes. Intrcellulr recordings. Glss cpillry microelectrodes filled with 3 M KCI (15-5 MQ) were used for intrcellulr recording nd stimulting electrodes. As shown in Fig. Id, single microelectrode ws used both for recording nd lso for pplying direct stimulus to gnglion cell (Nishi & Koketsu, 1960). In order to study the AChsensitivity of single cell, ACh ws pplied electrophoreticlly by use of glss microelectrode filled with cetylcholine chloride ( M), s shown in Fig. le (Blckmn, Ginsborg & Ry, 1963; Koketsu, Nishi & Soed, 1968). Solutions nd drugs The ionic composition of the solutions used ws s follows: Ringer solution (mm): NCl 11, KCI, CCl 1.8 nd NHCO3.4; sodium-free sucrose solution (mm): sucrose 4, CCl 1.8 nd KHCO3 ; sodium-free lithium solution (mm): LiCl 11, CCl 1.8 nd KHCO3. Drugs used: cetylcholine-chloride (Diichi-seiyku), tropine sulphte (Nkri-Kgku), nicotine sulphte b Sci.N. Ggl. -K- A- C\ c R, Sl T P I3 _ L,3' > d GI Pr. N. Fig. 1 Schemtic drwings of experimentl rrngements for recording electricl ctivity in the sympthetic nervous system of the bullfrog: () nd (b) re for extrcellulr recordings nd (c) is for the sucrose-gp method; (d) nd (e) re for intrcellulr recording. The 9th or 10th gnglion (Ggl.) nd scitic nerve (Sci. N.) re shown in (), (b) nd (c); gnglion cell (Ggl. Cell) nd pregnglionic nerve terminl (Pre. N.) re shown in (d) nd (e). Note the positions of recording nd stimulting electrodes in ech drwing; R, S, T nd P shown in (c) re Ringer, sucrose, test nd prffin solutions, respectively. (Ktym-Kogyo) nd (+)-tubocurrine chloride (Wko-Junyku). Results I. Sodium-free lithium solution 1. Nerve conduction. Two successive ction potentils could be recorded by use of the experimentl rrngement shown in Fig l, when single suprmximl stimulus ws pplied to the pregnglionic B nerve (Koketsu, 1969) fibres (Figure ). The initil nd following diphsic ction potentils were those of pregnglionic nd postgnglionic B nerve fibres, respectively. Chnges in nerve conduction in sodium-free lithium solution were studied by observing the chnges in the initil ction (spike) potentil in this solution. In order to ensure tht extrcellulr sodium ions were removed, preprtions were not trnsferred from Ringer solution directly to sodiumfree lithium solution. Insted, preprtions (six experiments) were first soked in sodium-free sucrose solution for t lest 30 min before being

3 LITHIUM AND SYMPATHETIC GANGLIA 71 b1 c d4r_ d _ 3 4 lms JmV Fig. Restortion of nerve conduction nd nicotinic trnsmission in bullfrog sympthetic nerve-gnglion preprtion: () n ction potentil in the pregnglionic nerve fibre (initil spike potentil) nd subsequent diphsic ction potentil in postgnglionic nerve fibre. These ction potentils were produced by single suprmximl stimulus pplied to pregnglionic B nerves; (b) fter 0 min (1) nd 30 min () in the sodium-free sucrose solution. Note the stimulus rtifct in record, in which no ction potentil ws produced; (c) fter 5min (1), 10min (), 30 min (3) nd 90 min (4) in the sodium-free lithium solution; (d) restortion of ction potentil in Ringer solution. trnsferred to the sodium-free lithium solution. It ws presumed tht extrcellulr sodium ions would be lmost completely wshed out by this procedure. As seen in Fig. b, the initil spike potentil of the preprtions completely disppered within pproximtely 30 min in the sodium-free sucrose solution. When the preprtions were trnsferred to the sodium-free lithium solution, the initil spike potentil ws restored within 5 min nd their mplitudes returned lmost to norml within 10 min (Figure c). The restortion of nerve conduction in the sodium-free lithium solution ws trnsient phenomenon; the initil spikes of these preprtions were grdully depressed until they hd completely disppered within min (Figure c). The spike potentils were completely restored by reimmersion in Ringer solution (Figure d).. Synptic trnsmission. A. Nicotinic trnsmission. ) Fst excittory postsynptic potentils (e.p.s.p.) - Monosynptic nicotinic trnsmission from pre- to postgnglionic neurones is medited by ACh producing the fst e.p.s.p. (Koketsu, 1969; Libet, 1970). The second diphsic spike potentil seen in Fig. is the ction potentil in postgnglionic nerve fibres, being medited by the fst e.p.s.p. Thus, chnges in the fst e.p.s.p. cn be determined by observing chnges in these spike potentils in the sodium-free lithium solution. As seen in Fig. b, the second spike potentils of the preprtions (six experiments) disppered in sodium-free sucrose solution within bout 0 min, which ws quicker thn the disppernce of the initil spikes. When preprtions were trnsferred to the sodium-free lithium solution fter soking in sucrose solution for 30 min, second spikes were restored within 5 min nd their mplitude becme lmost norml within pproximtely 10 min (Fig. c); they then grdully diminished until they completely disppered fter bout 90 minutes. Second spikes disppered quicker thn initil spikes. The filure of nicotinic trnsmission ws reversible in Ringer solution (Figure d). b) Nicotinic cetylcholine-depolriztion - There is possibility tht the filure of nicotinic trnsmission observed in sodium-free sucrose solution is simply due to filure of nerve conduction, prticulrly t pregnglionic nerve terminl brnches. In other words, the restortion of the second spikes in sodium-free lithium solution could be simply due to the restortion of nerve conduction. Thus, it must be clrified whether or not lithium ions re ble to substitute for sodium ions in the production of the fst e.p.s.p. itself. To this end, nicotinic ACh-depolriztion ws produced in the presence of tropine by direct ppliction of ACh to gngli. Chnges in the depolriztion produced in sodium-free lithium solution were studied. The trnsient gnglionic depolriztion (Fig. 3), which cn be recorded by the sucrose-gp method in the presence of tropine (0.14 mm) when drop of ACh solution (0.1 M) is dded to the perfuste, is membrne potentil chnge which corresponds to the fst e.p.s.p. (Koketsu, 1969; Libet, 1970). Whether or not the fst e.p.s.p. itself could be restored in the sodiumfree lithium solution, ws determined by observing chnges in the ACh-depolriztion in this solution. Five preprtions were perfused with sodiumfree lithium solution fter being perfused for pproximtely 30 min with sodium-free sucrose solution. The mplitude of the nicotinic AChdepolriztion of these preprtions ws very smll immeditely fter trnsfer to the sodium-free lithium solution, but grdully incresed during the next 0-40 min (Figure 3b). The nicotinic ACh-depolriztion ws grdully depressed when these preprtions were soked in sodium-free lithium solution for more thn 30-40

4 7 K. KOKETSU & K. YAMAMOTO 1 y LJ0mOmV loms b j(o mv s c 40s 5mV Fig. 3 Restortion of the nicotinic cetylcholine (ACh)-depolriztion recorded by the sucrose-gp method: () in Ringer solution; (b) fter 0 (1), 40 (), 50 (3), 90 (4) nd 100 min (5) in sodium-free lithium solution; the preprtion ws previously soked in sodium-free sucrose solution for 30 minutes. A constnt nodl current ws pplied in records 3 nd 5; (c) restortion in Ringer solution. One drop of ACh (0.1 M) solution ws dded to the perfusion fluid t the rrow in ech record. minutes. This seemed to be due to membrne depolriztion in gnglion cells. Indeed, the depression ws compensted when gnglion cell membrnes were hyperpolrized by pplying constnt rtificil nodl current (see Figure 3b, 3 & 5). This suggested tht the ACh-sensitivity of the gnglion cell ws not depressed even when preprtions were soked for long periods in the sodium-free lithium solution. c) Nicotinic cetylcholine-sensitivity Chnges - in the nicotinic ACh-sensitivity in sodium-free lithium solution were studied by recording the intrcellulr potentil chnges of single gnglion cell. The chnges were recorded by the iontophoretic ppliction of ACh (see methods section). In this experiment (see Fig. 4b), AChdepolriztion ws recorded from the sme cell first in Ringer solution nd then in sodium-free lithium solution. When the perfusion fluid ws chnged to the sodium-free lithium solution, the cell membrne ws grdully depolrized nd the size of nicotinic ACh-depolriztion ws decresed. Reduction of the ACh-depolriztion seemed to be due to the membrne depolriztion. Indeed, if the resting membrne potentil ws mintined t norml level by pplying constnt nodl current, the size of the ACh- Fig. 4 () intrcellulr potentil chnges recorded from preprtion perfused with the sodium-free lithium solution for pproximtely hours. The membrne ws strongly depolrized nd produced no ction potentils when stimulted directly (1); n ction potentil, however, ws produced when the membrne ws hyperpolrized by pplying constnt nodl current to the cell (); (b) nicotinic cetylcholine (ACh)-depolriztions recorded from single cell by iontophoretic ppliction of ACh in the presence of tropine (0.14 mm). The nicotinic AChdepolriztion ws first recorded in Ringer solution (1) nd record ws tken pprox. 90 min fter the perfuste ws chnged to the sodium-free lithium solution; note membrne depolriztion nd depression of the response. Record 3 ws tken immeditely fter record while the membrne ws hyperpolrized by pplying constnt nodl current. depolriztion remined unchnged. These results were confirmed in five different cells. d) Action potentil - Chnges in the resting nd ction potentils of 1 gnglion cells from three gngli in sodium-free lithium solution were studied by the intrcellulr recording method. As before, the gnglion cell membrne ws grdully depolrized nd the resting membrne potentil becme generlly less thn -50 mv within h (the resting membrne potentil in Ringer solution rnged between -60 nd -65 mv). In this condition, no ction potentils were produced by either ntidromic (stimultion of postgnglionic nerve trunk) or orthodromic stimultion. Action potentils, however, could be produced by direct stimultion through n intrcellulr electrode. The pek level of these ction potentils ws low nd the threshold for their initition ws high, by comprison with those produced in Ringer solution. Some cells which were depolrized produced no ction potentils even fter powerful direct stimultion (Figure 4,l). However, these cells could produce ction potentils provided the resting membrne potentil ws mintined t

5 LITHIUM AND SYMPATHETIC GANGLIA 73 norml level by pplying constnt rtificil nodl current (Figure 4,). B. Muscrinic trnsmission. ) Positive potentil (P-potentil) nd lte negtive potentil (LN-potentil) - In preprtions in which nicotinic trnsmission ws completely blocked by the presence of nicotine (0.4 mm), the P-potentil nd LN-potentil (see Fig. 5) could be recorded from gngli when repetitive pregnglionic stimultion ws pplied (Koketsu & Nishi, 1967; Nishi & Koketsu, 1968). Both the P-potentil nd LN-potentil re muscrinic responses which represent the slow inhibitory postsynptic potentil (i.p.s.p.) nd the slow e.p.s.p., respectively (Koketsu & Nishi, 1967; Nishi & Koketsu, 1968). In six nicotinized preprtions perfused with the sodium-free sucrose solution, both the P- nd LN-potentils recorded by the sucrose-gp method were completely blocked within pproximtely 30 min (Figure Sb). When the perfusion fluid ws chnged to sodium-free lithium solution, the LN-potentil ws restored within 5 min nd its mplitude becme lmost norml within 10 min (Figure Sc). The P-potentil, by contrst, ws never restored in this solution (Figure 5c). The restortion of the LN-potentil, like tht of nicotinic trnsmission, ws trnsient phenomenon; its mplitude grdully decresed when preprtions were soked in sodium-free lithium solution for more thn 30 minutes. Both the P- nd LN-potentils were reversible in Ringer solution (Figure 5d). b) Muscrinic cetylcholine-responses - Hyperpolriztion followed by depolriztion cn be recorded by the sucrose-gp method when ACh is directly pplied to nicotinized gngli (Figure 6). The ACh-hyperpolriztion nd ACh-depolriztion re muscrinic responses nd correspond to the P-potentil nd LN-potentil, respectively (Koketsu, 1969). Six nicotinized preprtions were perfused with sodium-free lithium solution fter they hd been perfused with sodium-free sucrose solution for t lest 30 minutes. In the sodium-free lithium solution, the muscrinic AChdepolriztion ws grdully restored wheres the muscrinic ACh-hyperpolriztion ws never restored (Fig. 6b); the restortion of AChdepolriztion ws trnsient phenomenon. The muscrinic ACh-depolriztion could be produced even fter the LN-potentil ws completely blocked in sodium-free lithium solution. This indicted tht the cuse of the disppernce of the LN-potentil ws the depression of ACh ACh-D LN b ACh-H b c 1 LN LN -_ LN d / mv los Fig. 5 Restortion of muscrinic synptic trnsmission: () the positive-potentil (P) nd lte negtive-potentil (LN) recorded by the sucrose-gp method in Ringer solution contining nicotine (0.4 mm); (b) fter 30 min in sodium-free sucrose solution; (c) fter 5 (1), 10 () nd 45 min (3) in the sodium-free lithium solution; (d) gin in Ringer solution contining nicotine. These potentil chnges were produced by pplying repetitive pregnglionic B nerve stimultion (30 Hz for 4 s); the durtion of stimultion is shown by the horizontl line under ech record. C 60s mv Fig. 6 Restortion of the muscrinic cetylcholine (ACh)-depolriztion nd disppernce of the muscrinic ACh-hyperpolriztion in sodium-free lithium solution: () the muscrinic ACh-hyperpolriztion (ACh-H) nd the muscrinic AChdepolriztion (ACh-D) recorded by the sucrose-gp method in Ringer solution contining nicotine (0.4 mm); (b) fter 5 (1), 15 (), 5 (3) nd 45 min (4) in the sodium-free lithium solution, following immersion in sodium-free sucrose solution for pproximtely 30 minutes. Note the grdul restortion of the muscrinic ACh-depolriztion; (c) return to Ringer solution contining nicotine. One drop of ACh (0.1 M) solution ws pplied t the point mrked by n rrow in ech record.

6 74 K. KOKETSU & K. YAMAMOTO 1 _ 1 L 3 4 Y_ 5 1OO1pV ms 1 3 d4iifl, los 30ms Fig. 8 Action potentils of postgnglionic nerve fibres produced by single suprmximl pregnglionic B nerve stimultion recorded in Ringer solution contining lithium ions (0 mm): () nd (b) were in the bsence nd the presence of (+)-tubocurrine (0.007 mm), respectively. In both () nd (b), record 1 ws in Ringer solution contining nicotine (0.4 mm) nd records, 3 nd 4 were 3, 5 nd 10 min fter the ddition of lithium ions (0 mm), respectively. Record 5 ws obtined on returning the preprtion to Ringer solution contining nicotine. Fig. 7 Restortion of the erly fter-dischrge nd depression of the inhibition in the sodium-free lithium solution: () the erly fter-dischrge (1) nd its inhibition () in Ringer solution; repetitive pregnglionic B nerve stimultion (30 Hz) ws pplied for 4 s to initite the erly fter-dischrge nd it ws pplied for s to inhibit it. The durtion of stimultion is shown by horizontl lines under ech record; (b) fter 30 min in the sodium-free sucrose solution; (c) fter 15 (1 nd ) nd 45 min (3) in the sodium-free lithium solution. Note the ugmenttion of dischrges nd disppernce of its inhibition; (c) return to Ringer solution contining nicotine. relese from pregnglionic nerve terminls. Both muscrinic ACh-hyperpolriztion nd AChdepolriztion returned to norml in Ringer solution. c) Erly fter-dischrge nd its inhibition -The erly fter-dischrge cn be recorded from postgnglionic nerves when repetitive stimuli re pplied to pregnglionic B nerve fibres (Nishi & Koketsu, 1 968). The erly fter-dischrge is produced by the slow e.p.s.p. nd cn be inhibited by pplying repetitive pregnglionic stimuli which produce the slow i.p.s.p. (Koketsu & Nishi, 1967) (Figure 7). The erly fter-dischrge recorded in four nicotinized preprtions ws completely eliminted when these preprtions were soked in sodium-free sucrose solution for pproximtely 30 min (Figure 7b). When the preprtions were trnsferred to sodium-free lithium solution, the erly fter-dischrge ws not only restored, but lso mrkedly ugmented (Figure 7c). When the erly fter-dischrge ws ugmented, the inhibitory effect of pregnglionic nerve stimultion hd lmost completely disppered (Figure 7c). These results prllel the observtion tht the slow e.p.s.p. ws restored wheres the slow i.p.s.p. ws not restored in sodium-free lithium solution. The restortion of the erly fter-dischrge ws trnsient phenomenon nd ws reversible in Ringer solution. II. Ringer solution contining lithium 1. Nerve conduction. The effects on nerve conduction of lithium ions dded to the externl Ringer solution, were studied. No detectble chnges were obtined in the presence of lithium ions up to 0 mm; in three experiments the ction potentils of pregnglionic nerve fibres showed no chnges during 10 min in these solutions.. Synptic trnsmission. A. Nicotinic trnsmission. Nicotinic trnsmission ws depressed when lithium ions were dded to the externl Ringer solution. When the concentrtion of lithium ions ws less thn 10 mm, the effect could be observed fter more thn h contct. When lithium ions (0 mm) were pplied (three experiments) slight depression of nicotinic trnsmission ws detected within 10 min by use of the sucrose-gp method (Figure 8). As seen in Fig. 8b, the depression could be demonstrted more clerly by using preprtions (three experiments) in which nicotinic trnsmission ws

7 LITHIUM AND SYMPATHETIC GANGLIA 75 prtilly blocked by (+)-tubocurrine (0.007 mm). The cuse of the depression of nicotinic trnsmission ws studied by recording intrcellulr potentil chnges in single cell in the presence of lithium ions (0 mm). Chnges in the fst e.p.s.p. were investigted by use of preprtion in which the fst e.p.s.p. ws depressed by the presence of tubocurrine (0.007 mm). No detectble depolriztion of the resting membrne ws observed for 10 min fter lithium ws dded to the Ringer solution (five cells). As shown in Fig. 9, however, the mplitude of the fst e.p.s.p. in these cells ws mrkedly decresed nd the ction potentil of the gnglion cell ws finlly blocked within 10 min of the ddition of lithium (0 mm). The depression of fst e.p.s.p. ws quickly restored in Ringer solution (Figure 9c). B. Muscrinic synptic trnsmission. The effects of the ddition of lithium to Ringer solution on the P-potentil, LN-potentil nd muscrinic ACh-responses were investigted. In six experiments, nicotinized preprtions were perfused with Ringer solution contining 0 mm lithium ions nd the P-potentil ws mrkedly depressed in bout 5 min (Figure 1 O). The chnge in LN-potentil ws difficult to determine, since it ws lwys superimposed on the P-potentil. As seen in Fig. 1Ob, the muscrinic ACh-hyperpolriztion ws lso depressed under these experimentl conditions. v The erly fter-dischrge ws significntly ugmented 10 min fter lithium ions (0 mm) were dded to the Ringer solution (Figure 1 lb). In three cses, the inhibitory effect of repetitive pregnglionic nerve stimultion ws significntly depressed (Figure 1 lb). These chnges were reversible (Figure 1 I c). 1 LN 7 PU 1b-v , J5mV 0s jmv 40s Fig. 10 Effects of lithium ions (0 mm) on the positive-potentil (P), the lte negtive-potentil (LN) nd the muscrinic cetylcholine-hyperpolriztion (ACh-H). All these potentil chnges were recorded from single preprtion by the sucrose-gp method in the presence of nicotine (0.4 mm); () in Ringer solution contining nicotine; (b) 5 min fter n ddition of lithium ions (0 mm); note the depression of the P-potentil nd the muscrinic ACh-hyperpolriztion; (c) return to Ringer solution contining nicotine. 1 n / 11 b%7 3 b 1 _ ~ ~ 1I I 5OmV loms - 10s Fig. 9 Intrcellulr potentil chnges in single cell: () the fst excittory postsynptic potentil (e.p.s.p.) nd the ction potentil of the cell-body, produced by single suprmximl pregnglionic B nerve stimultion in Ringer solution contining (+)-tubocurrine (0.007 mm); (b) 3, 5 nd 10 min fter the ddition of lithium ions (0 mm). Note the decrese in size of the fst e.p.s.p. with no detectble membrne depolriztion; (c) fter returning to Ringer solution contining (+)-tubocurrine. Fig. 11 Effects of lithium ions (0 mm) on the erly fter-dischrge (1) nd its inhibition (): () Ringer solution contining nicotine (0.4 mm); (b) 10 min fter the ddition of 0 mm lithium ions nd (c) return to Ringer solution contining nicotine. The erly fter-dischrge nd its inhibition were observed by pplying repetitive pregnglionic B nerve stimultion (30 Hz) for 4 s nd s, respectively; the durtion of stimultion is shown by horizontl-lines under ech record. Note slight increse in the erly fterdischrge nd depression of its inhibition in B.

8 76 K. KOKETSU & K. YAMAMOTO Discussion The results show tht lithium ions re ble to substitute for sodium ions in mintining nerve conduction nd nicotinic trnsmission in the sympthetic gngli of the bullfrog. Thus, the generl concept tht lithium ions re effective substitutes for sodium ions for mintining the membrne excittion (Hodgkin, 1951; Huxley & Stmpfli, 1951) cn be pplied to the present preprtion. It hs been suggested tht lithium ions my not be ble to substitute for sodium ions in mintining synptic trnsmission in sympthetic gngli, becuse of the observtion tht nicotinic trnsmission in this preprtion ws grdully blocked when the externl solution ws chnged from Ringer solution to sodium-free lithium solution (Klingmn, 1966; Pppno & Voile, 1966; Pppno & Volle, 1967). The present results gree with the observtion, provided tht the preprtion ws soked in the sodium-free lithium solution for n extended period of time. However, when the nicotinic trnsmission ws completely blocked in sodium-free sucrose solution it ws restored by trnsferring the preprtion to sodium-free lithium solution. This clerly indictes tht lithium ions re ble to substitute for sodium ions in mintining nicotinic trnsmission. The fct tht nicotinic trnsmission ws eventully blocked in sodium-free lithium solution could be explined by: ) blockde of nerve conduction in pre- or postgnglionic nerve fibres; b) reduction in the relese of trnsmitter (ACh) from pregnglionic nerve terminls; or c) depression of the ACh-sensitivity of the subsynptic membrne of gnglion cells. The second nd third explntions seem likely becuse blockde of nerve conduction ws preceded by tht of nicotinic trnsmission. The present experiments show tht the nicotinic ACh-sensitivity of gnglion cells ws not depressed even when preprtions were soked in sodium-free lithium solution for long periods. This observtion leds to the conclusion tht the cuse of the blockde in nicotinic trnsmission my be reduction in ACh relese from presynptic nerve terminls. The reson for the reduction in ACh relese in the sodium-free lithium solution is not completely explined by the present experiments. It ws observed, however, tht the initil chnge in the membrne potentil in the sodium-free lithium solution ws fll in the resting potentil. The ction potentil ws unffected even when the resting membrne ws depolrized provided the resting membrne potentil ws kept norml by pplying constnt nodl hyperpolrizing current. This suggests tht the mechnism of ACh relese (Ktz, 1969) which is ssocited with the production of n ction potentil, my not be ffected in the sodium-free lithium solution. Thus, the reduction in ACh relese ppers to be due to the depolriztion of the resting membrnes of the presynptic nerve terminls, since the mount of ACh relesed is proportionl to the vlue of the resting membrne potentil (Tkeuchi & Tkeuchi, 196). The present results do not provide ny conclusive explntion of the cuse of membrne depolriztion in the sodium-free lithium solution. It would be expected, however, tht lithium ions would ccumulte in the intrcellulr spce when they replce sodium ions in the extrcellulr spce becuse they re pumped out of the cell with difficulty compred with sodium ions (Mizels, 1954; Zerhn, 1955; Keynes & Swn, 1959). Under such experimentl conditions, the intrcellulr potssium concentrtion would be reduced (Crmeliet, 1964; Arki, Ito, Kostyuk, Oscrsson & Oshim, 1965; see, however, Yonemur & Sto, 1967). Thus, decrese in the intrcellulr potssium concentrtion would led to membrne depolriztion, prticulrly t presynptic nerve terminls where the dimeter of nerve fibres is extremely smll. Action potentils in the sodium-free lithium solution showed smller mplitude nd higher threshold, compred with ction potentils in Ringer solution. This would be explined by the fct tht membrne permebility to lithium ions is smller thn tht to sodium ions (Keynes & Swn, 1959; Armett & Ritchie, 1963). When the membrne ws depolrized in the sodium-free lithium solution, the threshold would become higher becuse of the prtil reduction of permebility. Under these experimentl conditions, nerve conduction, prticulrly t the terminls, would be expected to be blocked (Bjegovic & Rndic, 1971). An interesting finding in the present experiments is the fct tht the slow e.p.s.p. or muscrinic ACh-depolriztion ws completely restored while the slow i.p.s.p. or muscrinic ACh-hyperpolriztion ws not restored when preprtions were trnsferred from the sodiumfree sucrose solution to the sodium-free lithium solution. These results re explined by the hypotheticl concept tht the slow i.p.s.p. is generted by ctivtion of the electrogenic sodium pump (Koketsu & Nishi, 1967; Nishi & Koketsu, 1968b). The sodium efflux my be ccelerted when the extrcellulr sodium ions re totlly replced by lithium ions (Beuge & Sjodin, 1968; Bker, Blustein, Hodgkin & Steinhrdt, 1969). In ny cse, the intrcellulr sodium concentrtion would be reduced in preprtion soked successively in

9 LITHIUM AND SYMPATHETIC GANGLIA 77 sodium-free sucrose nd sodium-free lithium solution. The reduction of the intrcellulr sodium concentrtion would led to depression of the sodium pump, proportionl to the fll in the intrcellulr sodium concentrtion (Bker et l., 1969; Bker, Blustein, Keynes, Mnil, Shw & Steinhrdt, 1969). The mode of ction of lithium ions dded to Ringer solution cn esily be explined by the results obtined in the experiments crried out in sodium-free lithium solution. Indeed, there is no reson to ssume specific ction of smll concentrtions of lithium ions on the membrne excittion process. When lithium ions were dded to extrcellulr fluid, they would penetrte to the intrcellulr spce in proportion to the extrcellulr concentrtion nd the durtion of ppliction. The intrcellulr ccumultion of lithium would led to membrne depolriztion nd depression of the sodium pump. In such conditions, synptic trnsmission s well s nerve conduction would be impeded in proportion to the concentrtion of lithium nd the durtion of its ppliction. The uthors express their thnks to Tisho-Seiyku Inc., Jpn for finncil ssistnce in supporting the present work. References ARAKI, T., ITO, M., KOSTYUK, P.G., OSCARSSON, 0. & OSHIMA, T. (1965). The effects of lkline ctions on the responses of ct spinl motoneurons nd their removl from the cell. Proc. Roy. Soc., B, 16, ARMETT, C.J. & RITCHIE, J.M. (1963). On the permebility of mmmlin non-myelinted fibres to sodium nd lithium ions. J. Physiol., Lond., 165, BAKER, P.F., BLAUSTEIN, M.P., HODGKIN, A.L. & STEINHARDT, R.A. (1969). The influence of clcium on sodium efflux in squid xons. J. Physiol., Lond., 00, BAKER, P.F., BLAUSTEIN, M.P., KEYNES, R.D., MANIL, J., SHAW, T.I. & STEINHARDT, R.A. (1969). The oubin-sensitive fluxes of sodium nd potssium in squid gint xons. J. Physiol., Lond., 00, BEAUGE, L.A. & SJODIN, R.A. (1968). The dul effect of lithium ions on sodium efflux in skeletl muscle. J. gen. Physiol., 5, BJEGOVIC, M. & RANDIC, M. (1971). Effect of lithium ions on the relese of ACh from the cerebrl cortex. Nture, Lond., 30, BLACKMAN, J.G., GINSBORG, B.L. & RAY, C. (1963). Synptic trnsmission in the sympthetic gnglion of the frog. J. Physiol., Lond., 167, CARMELIET, E.E. (1964). Influence of lithium ions on the trnsmembrne potentil nd ction content of crdic cells. J. gen. Physiol., 47, HODGKIN, A.L. (1951). The ionic bsis of electricl ctivity in nerve nd muscle. Biol. Rev., 6, HUXLEY, A.F. & STAMPFLI, R. (1951). Effect of potssium nd sodium on resting nd ction potentils of single myelinted nerve fibres. J. Physiol., Lond., 11, KATZ, B. (1969). The relese of neurl trnsmitter substnces. Liverpool University Press in London. KEYNES, R.D. & SWAN, R.C. (1959). The permebility of frog muscle fibres to lithium ions. J. Physiol., Lond., 147, KLINGMAN, J.D. (1966). Effects of lithium ions on the rt superior cervicl gnglion. Life Sci., 5, KOKETSU, K. (1969). Cholinergic synptic potentils nd the underlying ionic mechnisms. Fed. Proc., 8, KOKETSU, K. & NISHI, S. (1967). Chrcteristics of the slow inhibitory postsynptic potentil of bullfrog sympthetic gnglion cells. Life Sci., 6, KOKETSU, K., NISHI, S. & SOEDA, H. (1968). Acetylcholine-potentil of sympthetic gnglion cell membrne. Life Sci., 7, KOSTERLITZ, H.W., LEES, G.M. & WALLIS, D.I. (1968). Resting nd ction potentils recorded by the sucrose-gp method in the superior cervicl gnglion of the rbbit. J. Physiol., Lond., 195, LIBET, B. (1970). Genertion of slow inhibitory nd excittory postsynptic potentils. Fed. Proc., 9, MAIZELS, E. (1954). Active ction trnsport in erythrocytes. Symp. Soc. Exp. Biol., 8, 0-7. NISHI, S. & KOKETSU, K. (1960). Electricl properties nd ctivities of single sympthetic neurons in frogs. J. cell. comp. PhysioL, 55, NISHI, S. & KOKETSU, K. (1968). Erly nd lte fter-dischrges of mphibin sympthetic gnglion cells. J. Neurophysiol., 31, NISHI, S. & KOKETSU, K. (1968b). Anlysis of slow inhibitory postsynptic potentil of bullfrog sympthetic gnglion. J. Neurophysiol., 31, PAPPANO, A. J. & VOLLE, R.L. (1966). Lithium's filure to replce sodium in mmmlin sympthetic gngli. Science, 15, PAPPANO, A.J. & VOLLE, R.L. (1967). Action of lithium ions in mmmlin sympthetic gngli. J. Phrmc. exp. Ther., 157, TAKEUCHI, A. & TAKEUCHI, N. (196). Electricl chnges in pre- nd postsynptic xons of the gint synpse of Loligo. J. gen. Physiol., 45, YONEMURA, K. & SATO, M. (1967). The resting membrne potentil nd ction movement in frog fibers fter exposure to lithium ions. Jp. J. Physiol., 17, ZERAHN, K. (1955). Studies on the ctive trnsport of lithium in the isolted frog skin. A ct physiol. scnd., 33, (Received August 6, 19 73)