6. When heptamethonium (30 ,sM)was added to the solution in the

J. Phy8iol. (1987), 388, pp. 141-151 141With 4 texft gurePrinted in Gireat Britain

SINGLE CHANNELS ACTIVATED BY ACETYLCHOLINE IN RATSUPERIOR CERVICAL GANGLION

BY V. A. DERKACH, R. A. NORTH*, A. A. SELYANKO AND V. I. SKOKFrom the Department of Autonomic Nervous Sy8tem Phy8iology,

A. A. Bogomoletz Institute of Physiology, Bogomoletz str. 4,Kiev-24, U.S.S.R.

(Received 3 April 1986)

SUMMARY

1. The elementary currents flowing through single channels opened by acetyl-choline were recorded in rat superior cervical ganglion neurones using patch-clampmethods. Acetylcholine (30 ,zM) was included in the patch electrode (cell-attachedrecordings) or applied by ionophoresis (outside-out configuration). All measure-ments were made at 23-25 °C and mostly at -110 mV.

2. Channel openings appeared both as single events and as bursts of events. Onepopulation of the currents observed had a conductance of 20-0 + 0-2 pS (mean+ s.E.of mean, n = 4). A second population had a conductance of about 50 pS, occurredmore rarely, and was not included in further analysis.

3. Four channel closed time periods and two channel open time periods were foundfrom the distributions of closed and open times. It was found that shorter channelopenings (about 0-2 ms) appeared in isolation, whereas longer openings (duration1-3 + 0-2 ms, n = 4) appeared as bursts of openings separated by the shortest channelclosed time periods (about 0-15 ms). The next shortest closed time (about 2 ms)apparently corresponds to the lifetime of the channel not activated by acetylcholine.The two longer closed times (about 80 ms and 1 s) may reflect desensitization. Themean burst duration was 8-5+ 1-2 ms (n = 4), giving about six openings per burst.

4. Because the time constant of decay of the excitatory post-synaptic current ismore similar to the burst duration than to the duration of individual single openings,it is suggested that acetylcholine released from presynaptic nerves may result in aburst of openings rather than a single opening.

5. On the basis of the above assumption, the rate constants were calculated fora sequential model in which acetylcholine binds to the receptor (forward ratek+1 = 2-3 x 107 M-1 S-1; reverse rate k11 = 1235 s-1) which then undergoes a con-formational change to an open state (forward rate /? =6293 s-1; reverse rate= 894 s-1).6. When heptamethonium (30 ,sM) was added to the solution in the patch electrode,

the burst duration was markedly shortened, but there was no change in the closedtime between two openings within the burst. This effect was voltage-dependent,

* Permanent address: Institute for Advanced Biomedical Research, Oregon Health SciencesUniversity, 3181 SW Sam Jackson Park Road, Portland, OR 97201, U.S.A.

V. A. DERKACH AND OTHERS

which suggests that heptamethonium binds to the channel after it is opened byacetylcholine.

INTRODUCTION

Nicotinic synaptic transmission in sympathetic ganglia has been widely studied byrecording membrane currents from single neurones (Kuba & Nishi, 1979; Selyanko,Derkach & Skok, 1979). It has been suggested that the acetylcholine (ACh) releasedby a single preganglionic impulse opens individual channels for about 4-5 ms; thisestimate is based on measurements of the decay of the excitatory post-synapticcurrent (e.p.s.c.) and the analysis ofACh-induced noise in the rabbit superior cervicalganglion (35 °C, -80 mV) (Derkach, Selyanko & Skok, 1983). This channel lifetimeis considerably longer than that found by the same approaches at the end-plate ofthe frog (1 ms: Magleby & Stevens, 1972; Anderson & Stevens, 1973), rat or mouse(about 0'3 ms: Dreyer, Miller, Peter & Sterz, 1976; Head, 1983).The purpose of the present experiments was to investigate the properties of single

ACh-activated channels in sympathetic neurones by the patch-clamp technique(Neher & Sakmann, 1976; Hamill, Marty, Neher, Sakmann & Sigworth, 1981). A moredirect approach seemed desirable for our understanding of both the physiology ofganglionic transmission and the mechanisms of action of ganglion-blocking drugs.Preliminary reports of this work have appeared (Derkach, North, Selyanko & Skok,1985).

METHODS

The experiments were carried out on neurones of the superior cervical ganglia of rats aged aboutone month. The animals were anaesthetized with ether, and the ganglia were removed and placedin a physiological saline solution. The composition of this solution was (mM): NaCl, 133; KCl, 5 9;MgCl2, 1P2; CaCl2, 2-5; tris (hydroxymethyl)aminomethane hydrochloride, 10; glucose, 11; pH 7-4.The sheath surrounding the ganglion was removed and the tissue was then incubated inphysiological saline which also contained collagenase (0'4 %, Sigma type 1). Incubation was at 33 0Cfor about 1 h. This treatment resulted in some neurones on the surface of the ganglion appearingclean when viewed with differential-interference contrast optics; such cells were chosen forpatch-clamp recordings. All experiments were carried out at 23-25 0C.

Patch-clamp recordings were made both in the cell-attached and outside-out configuration. Forcell-attached recording, the pipette (tip resistance 10-30 MCI) contained the extracellular solutiondescribed above, and ACh (30 uM). In the outside-out mode, the pipettes contained a solution thatresembled the intracellular medium (in mM: potassium acetate, 140; ethyleneglycol-bis(fl-aminoethylether)N,N,N',N'-tetraacetic acid (EGTA) 11; CaCl2, 1; N-2-hydroxyethylpiperazine-N'-2-ethanesulphonic acid (HEPES) 10; pH 74). ACh was applied to outside-out patches bymicroionophoresis from a pipette with its tip situated about 30 ,sm from the detached patch.

Patch currents were recorded on tape with a frequency band width of 0-3-5 kHz, digitized at20 kHz and transferred to an SM-3 computer for further analysis. The amplitude and duration(at half amplitude) of each current pulse, as well as the interpulse intervals, were measured. Pulsesand interpulse intervals of less than 150 4pss were ignored.

RESULTS

The results described were obtained from four cell-attached membrane patches,with the use of ACh only, and from four other cell-attached membrane patches withthe use of ACh and heptamethonium. In control experiments, a number of cell-

142

GANGLIONIC ACh CHANNELS

A

50 msB

180

'o 90.

Ez

00 10

Amplitude (pA)

ACh

Fig. 1. ACh-induced single-channel currents. A, an example of currents recorded from acell-attached patch at -110 mV (calculated membrane potential, assuming that theresting membrane potential was -50 mV). Inward currents are seen as downwarddeflections from the base line. The pipette contained 30 /M-ACh. B, distribution ofamplitudes of single-channel currents, some of which are shown in A. Mean amplitudesof currents were 2-2 pA (low-amplitude mode) and 5-6 pA (high-amplitude currents). C,currents recorded from an outside-out patch at -140 mV (directly measured membranepotential) as produced by ionophoretic application of a threshold dose of ACh (indicatedby bar). Time and current scales in C are the same as in A.

attached membrane patches were examined in the absence ofACh; they did not revealany channel activity, providing evidence that the activity recorded in the presenceofACh is produced by the ACh. In addition, two outside-out membrane patches wereobtained, allowing transient application of ACh. They gave results qualitativelysimilar to those obtained from cell-attached membrane patches.An example of single channel activity in the cell-attached mode is shown in Fig.

1 A. The pipette was polarized by 60 mV with respect to the bath ground (externalsolution); this means that the potential difference across the patch was -110 mVif one assumes a resting potential for the cell of -50 mV. The mean resting potentialobtained in six cells in the whole-cell recording configuration (see Hamill et al. 1981)

143


A

220

a

Qa)CL0,c

.0

Ez

110 -A1

40

CD

CL

a,

4._

cxa)0

Ez

TcI = 0-16 ms

Tc2 = 1-85 ms

UAJs-B

100

C

Q

E

z

50

0

Time (ms)6

80-

C

(DC

a)0,

0

01=0-16ms -

7-2 = 2-97 ms E

z

0

40

20

0

C

Tc3- 148 ms

TC4= 961 ms

Time (s)

Tb, = 0-25 ms

Tb2 = 936 ms

10 0

Time (ms) Time (ms)

Fig. 2. Distributions of single-channel current pulse durations and interpulse intervals ina single cell-attached patch. The pipette contained 30 ,uM-ACh. The calculated membranepotential was -110 mV. A, distribution of interpulse intervals for the initial and laterparts ofthe distribution (note the different ordinate scales). Each distribution was fitted bytwo exponentials (estimated time constants, rc1,2,Trc 3, and rC4, were 0 16, 1P85, 148 and961 ms); fitted numbers of events were 2347, 409, 319 and 259, respectively). B,distribution of current pulse durations fitted to two exponential functions (time constants,701 and rO2, were 0-16 and 2-97 ms; numbers of events were 388 and 575). C, distributionof current pulse durations obtained after short interpulse intervals (0-64 ms = 4 x 0 16 ms)were ignored (see text). The distribution was fitted by the sum of two exponential curveswith time constants equal to 0-25 ms (corresponding to a mean single-pulse duration Tbl)and 9-36 ms (corresponding to a mean burst duration Tb2). Bin width is 50 ss and 20 ms(A), 100l ,s (B) and 150 ,us (C).

was -51 + 2 mV (this and subsequent values are the mean+ s.E. of mean). This issimilar to that found with conventional intracellular electrodes (Selyanko et al. 1979).Fig. 1 A illustrates the two amplitudes of single channel current that were typicallyobserved, and Fig. 1 B shows this in histogram form. The amplitude distribution ofthe currents of lower amplitude was approximately Gaussian, but there was an

additional tail of currents having considerably higher amplitudes (Fig. 1B). Bothcurrents reversed from inward to outward at about 0 mV (assuming a restingpotential of -50 mV) and the mean single channel conductances were 20-0+ 1-2 pS(n = 4) for the smaller currents and about 50 pS for the larger currents.

144

CANGLIONIC ACh CHANNELS

The possibility was considered that the large-amplitude currents were due tooccasional coincidence in the opening oftwo channels of smaller amplitude. However,the higher-amplitude currents were not obvious multiples of the lower-amplitudecurrents and, furthermore, the contributions of the high-amplitude currents to the

tcl tc2, tc3, tC4

Closed

Open

to2 toI, tbI

tb2

Fig. 3. Schematic representation of events comprising single-channel activity. Current-pulse durations (open time, to), interpulse intervals (closed time t,) and durations of burstsof openings (burst time, tb) are indicated by arrows. Mean values of to, t and tb were equaltor0, TC, and Tb, respectively.

over-all number of currents was several times greater than would be predicted fromrandom coincidence of lower-amplitude events. It is not clear whether these twoevents represent different channels or different conductance substates of the samechannel. The over-all activity was predominantly due to low-amplitude events(70-900 of all openings); only the low-amplitude currents were analysed further.An example of such analysis is shown in Fig. 2. The distribution of the time

intervals between openings of the channel (closed time, tc) had at least fourcomponents, each of which could be fitted by an exponential curve (Fig. 2A; notethe different time-scales of the two graphs). The time that the channel spent in theopen state was best described by two exponential distributions (Fig. 2B). Themulti-component distribution of the closed time suggested that the current pulsesappeared in bursts; indeed, bursts ofopenings were readily observed during the courseof the experiments. Mean values (n = 4) of the time constants of the four closedtimes (Tc1, Tc2 TC3' TC4) were 0t15+0-02, 1X78+0X38, 80X2+23X3 and 1083+292 ms,respectively.The burst was defined as the duration of a single event when short closures were

eliminated. To estimate the duration of the burst, closures of up to 4Tc1 were deemedshort (this should include 98% of an exponentially distributed population of eventshaving mean rci). The distribution of burst durations thus calculated had twocomponents, the time constants of which were bi (about 0-32 + 0-06 ms) and 7b2(mean value was 8-54 + 1-22 ms, n = 4). It was thought that the shorter componentmight correspond to the shorter component of the channel open times ro1 describedabove (0'21 +0-04 ms), because a certain number of 'bursts' would presumablycontain only single openings. The second component (Tb2) should therefore representthe true mean burst duration, when a burst contains two or more openings. The T02would thus be a single opening within the burst; its mean duration was 2-6 + 01 ms(n = 4). The mean values of tc, to and tb (Tc' TOr Tb) were estimated from the averagetime constants of the corresponding exponential distributions; they are shownschematically in Fig. 3.

145


It should be pointed out that the value Of T02 estimated from open timedistributions is likely to be too large due to the fact that the fastest interpulseintervals (shorter than 150,ts, see Methods) remained unresolved. The factor ofover-estimation for rol is about two. The corrected value of ro2 can be estimated using

ACh (30 ym)

ACh (30 pM) and heptamethonium (30 MM)

ACh (30 pM) and heptamethonium (100;LM)

5 pA5 ms

Fig. 4. Single-channel currents recorded with a pipette that contained ACh only (30 #M)or ACh and heptamethonium (30 or 100 #M). Records were obtained from three differentpatches.

the approach described by Colquhoun & Sakmann (see Colquhoun & Sakmann, 1985,p. 505). The mean value Of T02 thus obtained was 1-3 + 0-2 ms and it is this value whichwas used in further considerations.

It was possible that the channel openings observed were not causally related tothe presence of ACh in the recording pipette, although activity was never observedat membrane potentials of -110 mV when the patch pipette did not contain ACh.The reversal potential of about 0 mV was appropriate to a nicotinic action of ACh.In addition, in two experiments with outside-out patches, application of ACh bymicroionophoresis caused the appearance ofsingle-channel currents having propertiesvery similar to those described. In this case, the currents of low amplitude appearedin bursts as the very first indication of the presence of ACh. This is consistent withthe observations made at the end-plate (Colquhoun & Sakmann, 1983). As theionophoretic current was increased, the frequency of channel opening increased. Thefinding of discrete bursts (Fig. 1 C) with minimal applications of ACh suggests thatthe bursts might not simply result from desensitization (Sakmann, Patlak & Neher,1980).The effect of heptamethonium was studied in four further patches in the cell-

attached mode. Heptamethonium (30,UM) was present together with ACh in thesolution within the pipette. Heptamethonium was used because of its fast dissociation

146


from the open ACh channel when compared to hexamethonium, and because itschannel-blocking action is more voltage-dependent than that of hexamethonium orother ganglion-blocking drugs (Skok, Selyanko, Derkach, Gmiro & Lukomskaya,1984). Thus, comparison of records at various membrane potentials should allow one

TABLE 1. Effect of heptamethonium on bursts

Pipette solution i (pA) Tel (Ms) T02 (MS) Tb2 (Ms)ACh (30 sM) 2-2+0-01 0415+0-02 1-3+0.2 8-5+1-22

(n = 4)ACh (30 /UM) and 2-0+007 0-12+0-02 04+01 1-5+0-13heptamethonium (30 4uM)(n = 4)

Heptamethonium reduces the mean time spent open within the burst (To2) and the mean durationof the burst (Tb2). There is no change in the time spent closed within the burst (c,) or in the currentamplitude (i). Note that the values of Tr1 and Tb2 were estimated from the interpulse intervals andburst duration distributions, whereas To2 was calculated as described in the text.

more reliably to attribute differences to the presence of the heptamethonium. Burstswere observed whether or not heptamethonium was in the patch pipette; however,both the mean burst duration (rb2) and the duration of openings within the bursts(T02) were significantly shortened in those patches recorded with pipettes containingboth heptamethonium and ACh than in those containing only ACh (Fig. 4; Table 1).The single channel current (at -110 mV apparent) and the time spent closed withinthe burst (xcr) were no different for patches exposed to heptamethonium and AChand patches exposed only to ACh.

DISCUSSION

According to the most generally accepted three-state model (del Castillo & Katz,1957), the interaction between ACh and its receptor might be described by:

k+1 ftACh+R = AChR = AChR*

k-I ac

non-occupied closed open

Tc(l(n+2) 'r2(n+ 1)tc2

(Tb2 + 2Trcl)where AChR is an inactive or closed state and AChR* is an active or open state ofthe receptor (R) and kl1, k+1, a and a are rate constants. If we assume that a singleburst (see Fig. 3) is produced by a single occupancy of the receptor by ACh (seeColquhoun & Hawkes, 1983), then the mean lifetime of AChR* will be To2* If n is thenumber ofinterpulse intervals within a burst (that is, the number of complete channelclosures which separate two openings within the burst), the number of openingswithin the burst will be n+ 1; then a = 1/TO2' because the only way from the openstate is to AChR. Therefore, n = (Tb2 -To2)/(TC + T02)' where Tc. +T2 is the durationof one open-closed cycle. The mean total lifetime of AChR is Tcl (n+ 2), because

147


transition through this state both starts and finishes the period of occupancy of Rby ACh. The rate constant for dissociation of ACh (k-,) is given by (fl/n); this followsbecause the number of times that a channel is open during a single occupancy (n + 1)is determined as (1 +fll/k-). The rate constant for channel opening (fi) is given byn/['rl (n+ 1)], asr,j = 1/(l+k1).

It is possible to estimate the values for these rate constants from the present results.For four patches exposed only to ACh, the mean values (derived from four separateexperiments) were: Lx = 894+ 185 s-51 = 6293+259 s-1 and k1& = 1235+261 s-.These rate constants are considerably slower than those at the end-plate (Colquohoun& Sakmann, 1983). The mean value of n was 5-2+ 0-5, indicating that the averageburst contains 6'2 discrete channel openings.

In the cell-attached configuration, ACh was present in the pipette throughout theexperiment, in a concentration (30 ,SM) that would cause marked desensitization atthe end-plate (Sakmann et al. 1980). Two results suggest that the bursts do not simplyresult from desensitization. First, in outside-out patches bursts of channel openingswere the very first indication of the presence of ACh as the ionophoretic current wasincreased. It is possible that the slower components in the closed time distribution(having means of Tc3 andrC4) reflect the time course of desensitization, as theirnumerical values are quite similar to the time constants of desensitization in otherpreparations (Feltz & Trautmann, 1982; Fenwick, Marty & Neher, 1982). Secondly,heptamethonium shortened the burst duration, and heptamethonium also acceleratesthe decay of the e.p.s.c. in sympathetic ganglion cells (Skok et al. 1984). Presumably,the ACh that is transiently present to bring about the e.p.s.c. does not result insignificant desensitization. The necessity to use rather high concentrations of AChin the patch pipette in order to record channel activity is similar to the finding ofOgden, Gray, Colquhoun & Rang (1984) in their experiments on the chick ciliaryganglion.

It is important to know whether the single-channel activity observed in theseexperiments is relevant to synaptic transmission in the ganglion. In rat lumbarsympathetic ganglia, the time constant of decay of the e.p.s.c. (Td) was 4-2 ms at-60 mV and 37°C; this would correspond to approximately 10 ms at -110 mV and27°C (Hirst & McLachlan, 1984). Rang (1981) found two time constants of decayof the e.p.s.c. in the rat submandibular ganglion at 20°C. These were 5-9 and27-45ms; the faster component showed little voltage dependence between -60 and-100 mV. Derkach et al. (1983) recorded from rabbit superior cervical ganglion cells;at 35°C and -80 mV, Td was 4-5ms. The Qlo of 3-7 indicates a value of about16 ms at 23 'C. The e.p.s.c. in the bull-frog sympathetic ganglion cells decays witha time constant of about 5 ms at -110 mV and 24 'C (Kuba & Nishi, 1979;MacDermott, Connor, Dionne & Parsons, 1980). We recently measured the timeconstant of decay of the e.p.s.c. in rat superior cervical ganglion cells at atemperature (23 'C) and membrane potential (-110 mV) similar to those used in thepresent experiments: the value was 13-9+ 0-6 ms (n = 4) (V. Derkach, A. A. Selyanko& V.I. Skok, unpublished observations). It is obvious that the value determined inthe present experiments for the burst duration rb2 more closely resembles theseestimates of the e.p.s.c. decay than does the estimate of the individual open times(TO2). Thus, at -110 mV and 25 'C the state AChR would last, on average, about

148


9 ms (rb2 +2T) and during this time the channel would close and then reopen anaverage of 6-2 times. A similar burst activity ofACh channels was observed in adrenalchromaffin cells, although the bursts were longer (about 27 ms at -80 mV and20-22 °C; Fenwick et al. 1982). If one assumes Tr3 and Tr4 to reflect desensitization(see above), then it is reasonable to suggest that Tr2 is related to thelifetime of the resting state R. Taking a value of Tr2 of 1-85 ms (for example, theexperiment illustrated in Fig. 2), the rate constant k+1 can be found from (TC2[ACh])-where [ACh] is the ACh concentration. This gives about 2-3 x 107 M-1 s-1.The concentration of ACh in the synaptic cleft following its normal release from

the presynaptic fibres is not known, but let us accept the estimate of Kuffler &Yoshikami (1975) of 300 /M. Since this is ten times higher than that used in thepresent experiments, the average time before a receptor is occupied would be0-1 x or about 0-2 ms. This is within the limits of the time that elapses before theopening of nicotinic receptor channels after the release of ACh as a result of a singlepresynaptic impulse in the sympathetic ganglion (Derkach et al. 1983) and theend-plate (Adams, 1980). On the other hand, it is known that the sensitivity ofganglion cells to ACh may be different at the synaptic and non-synaptic regions (seeHarris, Kuffler & Dennis, 1971).Two single-channel open durations, one long (mean 1-3 ms), the other short (mean

0-2 ms) were found in these experiments. It is possible that they result from thebinding to the receptor of two or one molecules of ACh, respectively (see Colquhoun& Sakmann, 1983). This suggestion is consistent with our observation that thecontribution of short openings was markedly reduced when the concentration ofAChwas raised from 30 to 250 gM.The effect ofheptamethonium on the burst duration can be analysed in the context

of the sequential model (Adams, 1976):fi k+B*[B]

AChR.= AChR*+B = AChR*Ba ~~~k-B*

where AChR*B is an open but blocked state of the open channel AChR* atconcentration [B] ofheptamethonium, and k+B* and klB* are rate constants. The rateconstant k+B* can be estimated as [(r02') - (r02)'1] [B]-1, where T02' in the presenceof the blocking drug corresponds to T02 in the absence of any blocker. This gives a'true' k+B* value of 65 x 106 M-1 s-1. When rb2 and rb2' are used instead of r andT02', the 'apparent' value ofthe rate constant k+B* is 18 x 106 M-1 s-. (b2' is the valuein the presence of heptamethonium that corresponds to 7b2 in the absence of blockingdrug.) These values are not much different from the 'apparent' value of k+B*estimated from the reduction of the e.p.s.c. decay time constant by heptamethoniumin the rabbit sympathetic ganglion (Skok et al. 1984). In that study, the value of k+B*appropriate to a holding potential of -110 mV was 3 x 106 M-1 s-1. These results areconsistent with the notion that heptamethonium blocks the channels opened by ACh(Skok et al. 1984; Skok, 1986).

149


REFERENCES

ADAMS, P. R. (1976). Drug blockade of open end-plate channels. Journal of Physiology 260,531-552.

ADAMS, P. R. (1980). Aspects of synaptic potential generation. In Information Processing in theNervous System, ed. PINKSER, H. M. & WILLIS, W. D., pp. 109-124. New York: Raven.

ANDERSON, C. R. & STEVENS, C. F. (1973). Voltage clamp analysis of produced end-plate currentfluctuations at frog neuromuscular junction. Journal of Physiology 235, 655-691.

COLQUHOUN, D. & HAWKES, A. G. (1983). The principles of the stochastic interpretation ofion-channel mechanisms. In Single-Channel Recording, ed. NEHER, E. & SAKMANN, B., pp.

135-174. New York: Plenum Press.COLQUHOUN, D. & SAKMANN, B. (1983). Bursts of openings in transmitter-activated ion channels.

In Single-Channel Recording, ed. NEHER, E. & SAKMANN, B., pp. 345-364. New York: PlenumPress.

COLQUHOUN, D. & SAKMANN, B. (1985). Fast events in single-channel currents activated byacetylcholine and its analogues at the frogmuscle end-plate. Journal of Physiology 369, 501-557.

DEL CASTILLO, J. & KATZ, B. (1957). Interaction at end-plate receptors between different cholinederivatives. Proceedings of the Royal Society B 146, 369-381.

DERKACH, V., NORTH, R. A., SELYANKO, A. A. & SKOK, V. I. (1985). Activity of single acetylcholine-activated channels in a sympathetic neurone. Reports of the U.S.S.R. Academy of Sciences 284,495-498.

DERKACH, V. A., SELYANKO, A. A. & SKOK, V.I. (1983). Acetylcholine-induced current fluctuationsand fast excitatory post-synaptic currents in rabbit sympathetic neurones. Journal of Physiology336, 511-526.

DREYER, F., MtLLER, K.-D., PETER, K. & STERZ, R. (1976). The m. omohyoideus as a convenientmammalian muscle preparation. A study of junctional and extrajunctional acetylcholinereceptors by noise analysis and cooperativity. Pfluigers Archiv 367, 115-122.

FELTZ, A. & TRAUTMANN, A. (1982). Interaction between nerve-released acteylcholine andbath-applied agonists at the frog end-plate. Journal of Physiology 299, 533-552.

FENWICK, E. M., MARTY, A. & NEHER, E. (1982). A patch-clamp study of bovine chromaffin cellsand their sensitivity to acetylcholine. Journal of Physiology 331, 577-597.

HAMILL, 0. P., MARTY, A., NEHER, E., SAKMANN, B. & SIGWORTH, F. J. (1981). Improvedpatch-clamp techniques for high-resolution current recording from cells and cell-free membranepatches. Pfliuger8 Archiv 391, 85-100.

HARRIS, A. J., KUFFLER, S. W. & DENNIS, M. J. (1971). Differential chemosensitivity of synapticand extrasynaptic areas on the neuronal membrane in parasympathetic neurons of the frog,tested by micro application of acetylcholine. Proceedings of the Royal Society B 177, 541-553.

HEAD, S. D. (1983). Temperature and end-plate currents in rat diaphragm. Journal of Physiology334, 441-459.

HIRST, G. D. S. & McLACHLAN, E. M. (1984). Post-natal development of ganglia in the lower lumbarsympathetic chain of the rat. Journal of Physiology 349, 119-134.

KUBA, K. & NISHI,S. (1979). Characteristics of the fast excitatory postsynaptic current in bullfrogsympathetic ganglion cells. Pfluigers Archiv 378, 205-212.

KUFFLER, S. W. & YOSHIKAMI, D. (1975). The number of transmitter molecules in a quantum: an

estimate from iontophoretic application of acetylcholine at the neuromuscular synapse. Journalof Physiology 251, 465-484.

MAcDERMOTT, A. B., CONNOR, E. A., DIONNE, V. E. & PARSONS, R. L. (1980). Voltage clamp studyof fast excitatory synaptic current in bullfrog sympathetic ganglion cells. Journal of GeneralPhysiology 75, 39-60.

MAGLEBY, K. L. & STEVENS, C. F. (1972). A quantitative description of end-plate currents. Journalof Physiology 223, 173-197.

NEHER, E. & SAKMANN, B. (1976). Single-channel currents recorded from membrane of denervatedfrog muscle fibres. Nature 260, 799-802.

OGDEN, D. C., GRAY P. T. A., COLQUHOUN, D. & RANG, H. P. (1984). Kinetics of acetylcholine-activated ion channels in chick ciliary ganglion neurones grown in tissue culture. Pfluigers Archiv400, 44-50.

150

GANGLIONIC ACh CHANNELS 151

RANG, H. P. (1981). The characteristics of synaptic currents and responses to acetylcholine of ratsubmandibular ganglion cells. Journal of Physiology 311, 23-55.

SAKMANN, B., PATLAK, J. & NEHER, E. (1980). Single acetylcholine-activated channels showburst-kinetics in presence of desensitizing concentrations of agonist. Nature 286, 71-73.

SELYANKO, A. A., DERKACH, V. A. & SKOK, V. I. (1979). Fast excitatory post-synaptic currentsin voltage-clamped mammalian sympathetic ganglion neurones. Journal ofthe Autonomic NervousSystem 1, 127-137.

SKOK, V. I. (1986). Channel-blocking mechanism ensures specific blockade of synaptic transmission.Neuroscience 17, 1-9.

SKOK, V. I., SELYANKO, A. A., DERKACH, V. A., GMIRO, V. E. & LUKOMSKAYA, N. Ya. (1984). Themechanisms of ganglion-blocking action of bis-ammonium compounds. Neurofizologia 16, 46-52.

Documents

6. When heptamethonium (30 ,sM)was added to the solution in the