Bethanechol-induced responses in mudpuppy parasympathetic neurons

0028-3908/92 $5.00 + 0.00

h’europhmwcaiogy Vol. 31, No. 12, pp. 131I-1321,1992 Printed in Great Britain. All rights reserved

Copyright 0 1992 Pergamon Press Ltd

BETHANECHOL-INDUCED RESPONSES IN MUDPUPPY PARASYMPATHETIC NEURONS L. M. KONOPKA* and R. L. PARSONS? Department of Anatomy and Neurobiology, College of Medicine, The University of Vermont, Burlington, VT 05405, U.S.A. (Accepted 9 Aprii 1992)

Summary-The effect of bethanechol on membrane potential and excitability was determined in mudpuppy parasympathetic postganglionic neurons. Bethanechol induced a large amplitude hyperpolarization, which was followed by a smaller amplitude depolarization, in I15 out of 135 cells tested. In approximately 20% of these cells, a brief depolarization preceded the hy~rpola~~tion. During the ~~anechol-indu~ hy~r~lari~tion, the membrane input resistance decreased markedly, whereas the input resistance was increased during the subsequent depolarization. The hyperpolarization and depolarization were blocked by atropine and were unaffected by d-tubocurarine, thus, both appeared to be mediated by muscarinic receptors. The bethanechol-induced hypetpolarization was inhibited by the M, muscarinic receptor antagonist AF-DX 116, whereas the bethanechol-induced depolarization was unaffected. Both a nonselective increase in membrane conductance and a decrease in membrane potassium conductance appeared to be involved in the generation of the ~than~hol-indu~ d~ola~zation. Evidence for the first mechanism was obtained in barium-treated cells in which bethanechol initiated a rapid onset depolarization, which was reversed at membrane potentials near 0 mV. Evidence for the second mechanism was obtained when the hyperpolarization was inhibited by AF-DX 116. In AF-DX 116-treated cells, the membrane input resistance was increased during most of the bethanechol-induced depolarization. Mudpuppy neurons initiate repetitive action potential activity in response to long depolarizing current pulses. Following application of bethanechol, with the hyperpolarization negated electrotonically, the number of action potentials produced by a depolarizing current pulse was greater than that produced prior to application of bethanechol. It is suggested that activation of muscarinic receptors on mudpuppy cardiac neurons influences multiple conductance systems and determines the excitability of these neurons. Key words-autonomic

neurons, bethanechol, cardiac ganglion, hyperpolarization,

Muscarinic agonists, such as bethanechol, hyperpolarize parasympathetic postganglionic neurons in the mudpuppy cardiac ganglion, by activating a membrane potassium conductance (Hartzell, Kuffler, Stickgold and Yoshikami, 1977). In a preliminary study, it was observed that, in addition to being hyperpolarized, approximately 75% of the parasympathetic neurons in the mudpuppy cardiac ganglia were also depolarized by bethanechol (Konopka and Parsons, 1989). Recently, Allen and Bumstock (1990) reported that mu~a~nic agonists could initiate both hyperpolarization and depolarization in cultured cardiac ganglion cells, taken from neonatal guinea pigs. It ‘was suggested that, in these cells, the hyperpolariz(ation was due to an increased membrane potassium conductance through activation of M, receptors, while the depolarization was primarily due to the reduction of a voltage-dependent, time-inde~ndent

*Present address: Section of Biological Psychiatry, 116A7, VA Hines Medical Center, Hines, IL 60141, U.S.A. ?To whom correspondence should be addressed.

depolarization.

potassium conductance, through activation of M, receptors. The present study was done to investigate in more detail the properties of the bethanechol-induced hyperpolarization and depolarization, observed in adult amphibian cardiac neurons. Previously, it was reported that the mudpuppy neurons were also hyperpolarized by the neuropeptide galanin (Konopka, McKeon and Parsons, 1989). Membrane excitability was depressed following application of galanin, even if the hyperpolarization was negated el~trotonically (Konopka et uf., 1989; Parsons and Konopka, 1991). Consequently, experiments were also carried out to determine whether or not a similar change in excitability was produced by bethanechol. Brief accounts of some of these results have appeared previously (Konopka and Parsons, 1989, 1990). METHODS

All experiments were carried out in vitro on parasympathetic postganglionic neurons in the cardiac ganglion of the mudpuppy, Necturus maculosus. The procedures used in the isolation and preparation

1311

1312

L. M. KONOPKAand R. L. PARSONS

Fig. 1. Records obtained from 4 cells, which exhibited both hyperpolarizing and depolarizing responses, following either a brief application of bethanechol [O.Sset in (A) and (C) and 0.9 se.c in (B) indicated by the solid arrowheads] or a 0.2 set application of oxytremorine (onset indicated by the open arrowhead). In the first two examples, the bethanechol-induced hyperpolarization (h) was followed by a period of depolarization (d). The depolarization was large enough to initiate spike activity in example (B). In the third example, a depolarization preceded the hyperpolarization. In the fourth example, the response to oxyt~mo~ne consisted of an initial period of d~Ia~~tion, followed by a much longer hsting hyperpolarization. The hyperpolarization was then followed by another period of depolarization. The resting membrane potential was - 51 mV in (A), - 55 mV in (B) and (C) and - 50 mV in (D). Calibrations:

y-axis equals 20 mV, x-axis 20 sec.

of the cardiac ganglion for el~trophysiological studies were identical with those described previously (Konopka et al., 1989; Parsons, Neel, Konopka and McKeon, 1989; Parsons and Konopka, 1991). The preparations were continuously perfused with a HEPES-buffered solution, containing in mM: NaCl 120, KC1 2.5, CaCl, 3.6, HEPES 1.0; pH 7.3. In some experiments, 2 mM barium was added to the calciumcontaining solution or 3.6 mM calcium was replaced by 2-4 mM barium. Four different muscarinic antagonists were added to the bathing solution: atropine, 0.5-5 PM and pirenzepine, 0.3-200 nM (both from Sigma); AF-DX 116 (l l -[(2[(diethylamino)methyl]-I-pinridinyl)acetyl]S, I 1-dihydro 6H-pyrido[2,3-b][ 1,4]benzodiazepine-6-one), 0.05-I .O,uM; and 4-DAMP (4-diphenylacetoxy-N-methylpiperidine methiodide), l-10 nM. AF-DX and 4-DAMP were the generous gift of Dr John Ellis from the Department of Psychiatry, UVM. Equilibration of solution changes occurred within 2min. Membrane voltage was measured with a single electrode Axociamp 2 amplifier system, in the current clamp mode. All recordings were obtained using microelectrodes filled with 1.5 M potassium citrate. The input resistance was determined by measuring the amplitude of voltage changes produced by constant current hyperpoiarizing pulses. Action potentials were initiated in some experiments by applying long duration depolarizing pulses. Bethanechol (Sigma) was applied to individual ganglion cells by localized pressure ejection (Picospritzer II, General

Valve Corp.). The con~ntration of ~thanechoi in the puffer pipette was 10e3 to lo-* M. A few experiments were done using oxytremorine methiodide (oxy-M) (Research Biochemicais, Inc.). In these experiments, the concentration of oxytremorine in the pipette was 10s3 M. Throughout, the results from different experiments are expressed as the mean value + SEM . RESULTS Bethanechol initiated both hyperpoiarization and depolarization in the majority of mudpuppy neurons

Brief application of ~than~hol initiate membrane hyperpolarization, which developed to a peak amplitude within a few seconds, then decayed gradually. In 115 of 135 ceils studied, bethanechoi also produced depolarization. In the majority of ceils, the depolarization was observed only at the termination of the ~than~hol-indu~d h~er~la~~tion (Figs 1A and B) and was consistently smaller in amplitude than the hyperpoiarization. In 35 cells having a mean resting membrane potential of - 52.0 k 1.4 mV, the amplitudes of the hyperpoiarization and depolarization were 15.0 + 0.9 mV and 2.9 + 0.2 mV, respectively. In many ceils, action potentials occurred during the declaration (Fig. 1B). A very brief bethanechol-induced depolarization preceded the hyperpolarization in some cells (Fig. 1C) and in some instances, this initial depolarization evoked action potential activity, prior to the development of the hyperpoiarization (data not shown). Also, as seen in

Bethanechol-induced

potential changes

(A)

Fig 2. Results demonstrating that the bethanechol-induced depolarization could be initiated without any hyperpolarization (A) and that both the bethanechol-induced hyperpolarization and depolarization could be blocked by atropine (B). The records in (A) show a series of responses produced by pulses of bethanechol, increasing in duration 10.02see in (Al), 0.05 set in fA2), 0.07 set in (A3), 0.15 set in (A4}, 0.25 set in (A5) and OS set in (A(j)]. The resting membrane potential was -50 mV. The records in (3) show the ~thane~hoi-induced response (0.2 set application indicated by solid arrowheads in each record) before (Bl), after a 5 min exposure to 5 PM atropine (B2) and after a 20 min washout of atropine (B3), respectively. The resting membrane potential was -61 mV. Calibration bars: y-axis equals 10 mV in (A) and (B); x-axis equals 20 set in (A) and 40 set in (B). Fig. l(C), in these cells depolarization became evident again at the end of the hype~olarization. A bethanechol-induced depolarization, without any hyperpolarization, was observed in 8 cells. In

these instances, depolarization was produced when a 0.01-0.02 set application of bethanechol was applied. However, when the duration of the application of bethan~hol was increased (with the puffer pressure kept constant), hyperpolarization was also produced (Fig. 2A). As the duration of the application of bethanechol was increased, the amplitude and duration of the hyperpolarization also increased progressively, so that eventually the depolarization was recorded only after the h~e~olarization had ended. In 10 cells, the response produced when bethanechol was applied for up to 16 set was also determined. Aa the duration of the application of bethanechol was increased from a fraction of a second to several seconds, the hyperpolarization continued to increase in duration (Fig. 3). Further, in the example shown in Fig. 3, when the duration of the application of bethanechol was 8 set, the time-course of decay of the hyperpolarization became complex. When this occurred, the hyperpolarization appeared to be composed of two components, an initial rapid onset phase of hyperpolarization being followed by a more slowly

developing hype~ola~zation (Fig. 3). In 4 of these 10 cells, depolarization was evident at the end of the long-lasting hyperpolarization. The bethanechol-induced depolarization did not appear to be due to the activation of nicotinic receptors

In the present study, the concentration of bethanechol in the puffer pipette was kept large, in order to initiate consistent responses from neurons in whole mount preparations. Because bethanechol was ejected from pipettes positioned 25-50 pm from the neurons, the concentration reaching the cells must have been less than that in the pipette but the actual con~ntration was not known. The authors were concerned that with large concentrations of bethanechol, the depolarization might have resulted from the activation of nicotinic receptors, which are also present on these cells (Roper, 1976; Hartzell et al., 1977). The results obtained in three additional series of experiments indicated that this explanation was unlikely. First of all, membrane hyperpolarization and depolarization were produced by brief applications of oxytremorine, another muscarinic agonist which is thought to be very specific for muscarinic receptors (Fig. lD, Bernheim, Beech and Hiile, 1991). In the

1314

L. M.

K~NOPKA

and R. L.

PARSONS

Fig. 3. Results illustrating that the duration of the bethanechol-induced hyperpolarization increased progressively and exhibited a complex time-course, when the duration of the application of agonist (onset indicated by the solid arrowheads) was increased to several seconds [O.Sset in (I), 2.0 set in (2), 4.0 set in (3) and 8.0 set in {4)]. The resting membrane potential in this cell was -48 mV. Calibration: y-axis 20 mV; x-axis 40 sec.

example shown in Fig. l(D), a 0.2 set application of oxytremorine initiated an initial, brief depolarization, followed by a longer lasting hyperpolarization. After the hyperpola~zation ended, an additional period of depola~zation was evident. Spikes occurred during both the initial and subsequent periods of depolarization. Similar results were obtained in 7 additional cells. In other experiments (4 cells), it was found that both the hyperpolarization and depolarization, recorded following the application of bethanechol, were reversibly inhibited by exposure to 0.5-5 PM concentrations of atropine (Fig. 2B). In contrast, in a third series of experiments (5 cells), it was found that exposure to 50-500pM concentrations of dtubocurarine had no effect on the amplitude of either the ~thanechol-induced h~erpolarization or depolarization, suggesting further that activation of nicotinic receptors was not involved in the generation of either component (data not shown). Input resistance changes occurred during the bethanechoI-induced hy~erpo~arizat~on and subsequent depolarization Experiments were undertaken to determine the relationship between the change in input resistance and membrane potential, following a brief application of bethanechol. When cells were hyperpolarized, shortly after the application of ~than~hol, the input resistance decreased as denoted by the decrease in the amplitude of the membrane hyperpolarizations produced by 300 msec duration constant current pulses (Fig. 4A). In the cell shown in Fig. 4(A), the decrease in input resistance was approximately 50% at the peak of the ~thanechol-induced hyperpolarization; it gradually returned towards the control level as the hyperpolarization ended (Fig. 4B). In 23 cells, which had an average resting membrane potential of - 51 .O & 1.1 mV, the maximum hyperpolarization and decrease in input resistance, recorded following a brief application (0.2-1.5 set) of bethanechol, was

23.0 + 1.7mV and 37.0* 3.5%, respectively. The relationship between the maximum bethanecholinduced decrease in input resistance and the peak amplitude of hy~~la~zation in these 23 cells is shown in Fig. 4(C); it appeared that the amplitude of the bethanechol-induced hyperpolarization was directly related to the decrease in membrane input resistance. In 5 cells, the input resistance was measured during bethanechol-induced hyperpolarizations, produced by 4-12 set applications of bethanechol. The input resistance was decreased throughout the duration of these long-lasting hyperpolarizations and returned towards control levels progressively as the hyperpolarization ended. The results shown in Fig. 4(B) also demonstrate that when the hyperpolarization had terminated, a small increase in input resistance (approx 10%) occurred during the subsequent period of depolarization. Although this was a consistent observation, no attempt was made to quantify the increase in resistance during the depolarization, because the change was so small. The decrease in input resistance was also measured when the ~than~hol-induced hype~ola~zation was prevented electrotonically. In 5 cells in which the bethanechol-induced hyperpolarization was negated, the decrease in input resistance was 32.0 f 4.8%. In these cells, the peak amplitude of the hyperpolarization, prior to being negated, was 22.0 k 2.0 mV. The M, receptor antagonist AF-DX I16 compfetely eliminated the bethanechol-induced hyperpolarization but not the bethanechol-induced depolarization In 13 cells, the influence of the cardiac Mz receptor antagonist, AF-DX 116, on the ~than~hol-induct h~~lari~tion and depolarization was tested. The range of concentrations tested was l&l000 nM. The bethanechol-induced hyperpolarization was always rapidly and reversibly inhibited by 1 PM AF-DX 116 (Fig. 5A). Further, when long-duration, multicomponent hy~rpolari~tions were initiated by increasing

Rethanechol-induced

potential changes

1315

2 -

I 10

3

40

SO

Time (see)

(0

2

30

20

0 70

0

60 50

40 30 20 10

I

f 0

0

20

Bethanechol-induced

30 hyperpolarization

40

50 (mV)

Fig. 4. Results showing the change in membrane potential and membrane resistance produced by a 0.3 set application of bethanechol (onset indicated by the solid arrowhead) in a cell with a resting membrane potential of - 52 mV. (A) The amplitudes of transient hy~~la~tions [trace (Al)], produced by 3OOmsec hyperpolarizing constant current pulses lo.5 Hz; trace (A2)] were decreased during the bethanechol-induced hyperpolarization and then returned toward control values as the hyperpolarization ended. Note that immediately after the application of bethanechol, a small amplitude, short duration depolarization preceded the hyperpolarization. (B) A plot illustrating the time-course of the change in membrane resistance, which occurred after the application of bethanechol. (C) Results obtained in 23 cells, showing that the peak amplitude of the bethanechol-induced hyperpolarization was correlated with the initial decrease in membrane input resistance. The R value, determined by linear regression, was 0.85. Calibrations in (A): y-axis 20mV (Al), 0.1 nA (A2); x-axis 4sec.

the duration

of application of bethanechol to many seconds, both the initial and late phase of hyperpolarization was completely eliminated by AF-DX

116 (Fig. 6B4). Although AF-DX 116 completely eliminated the bethanechoi-induced hypcrpolarization, the depolarization was not inhibited (Fig. 5A). In 8 cells, the change in membrane input resistance was measured during the bethanechol-induced depolarization, which was present after the hyperpolarixation was

blocked by 1 pM AF-DX 116. In all cases, it was found that, immediately following the application of bethanechol, there was a decrease in the input resistance as the depolarization developed but then the resistance was increased during the remainder of the depolarization. In the example presented in Fig. 5(B), the amplitude of the first hyperpolarixing pulse, recorded on the rising phase of the depolarization, was approximately 40% smaller than the control h~~la~~ng pulses. In contrast, the amplitudes of

L. M. KONOPKAand R. L. PARSONS

1316

2,___,__

Fig. 5. Results which illustrate that AF-DX 116 selectively and reversibly inhibited the bethanecholinduced hyperpolarization, without a similar action on the depolarization. In (A) record (Al) shows the response to a 0.5 set application of bethanechol (onset indicated by the solid arrowhead in all records), prior to exposure to 1 x 10e6 M AF-DX 116. After 9 min in AF-DX 116, the hyperpolarization was inhibited but the depolarization remained [record (A2)]. Record (A3) demonstrates that the bethanecholinduced hyperpolarization returned after removal of the antagonist. The resting membrane potential recorded in this cell was - 50 mV. Calibration: y-axis 20 mV; x-axis 10 sec. (B) Shows that the membrane input resistance was increased during most of the duration of the bethanechol-induced depolarization, recorded in the presence of 1 x 10e6 M AF-DX 116, with the hyperpolarization being blocked. Trace (Bl) shows membrane potential and amplitude of hyperpolarizing responses, produced by 300 msec constant current pulses delivered at 0.5 Hz. Trace (B2) shows the current applied. Note that the amplitude of the first hyperpolarizing response, recorded just after the 0.9 set application of bethanechol (onset indicated by the solid arrowhead) was less than that of the control responses; whereas, during the remainder of the depolarization, the amplitude of the hyperpolarizing responses was greater than those recorded prior to the application of bethanechol. The resting membrane potential was - 53 mV. Calibration: y-axis 10 mV, 0.1 nA; x-axis 2 sec.

(A)

Fig. 6. Results demonstrating that 4-DAMP and pirenzepine preferentially blocked the initial component of the bethanechol-induced hyperpolarization, produced with long-duration applications of agonist. In (A), records (1), (2) and (3) show the response to an 8 set application of bethanechol, prior to exposure to 5 x 10e9 M 4-DAMP and then after equilibration in CDAMP for 2 and 9 min, respectively. Record (4) shows the bethanechol-induced potential change, elicited 15 min after removal of the antagonist. The resting membrane potential in this cell was -48 mV. In (B) record (1) shows the response in another cell, produced by a 2sec application of bethanechol. Record (2) was obtained after a 33min exposure to 5 x 10m8M pirenzepine. Record (3) was obtained in the continued presence of pirenzepine (5 x 10M8M, 53 min) and 5 x 10e9 M 4-DAMP for 20 min. Note that the bethanechol-induced depolarization was still present and was followed by a period of hyperpolarization. Record (4) shows the response to bethanechol after pirenzepine and 4-DAMP were washed out for 46 min and then the preparation exposed for 6 min to 1 x 10e6 M AF-DX 116. The resting membrane potential was - 50 mV. Calibration bars: y-axis 20 mV; x-axis 40 sec.

Bethanechol-induced

the subsequent hyperpolarizing pulses were approximately 35% greater than the control responses during the remainder of the bethanechol-induced depolarization, Experiments were carried out with 10 cells in an attempt to determine the voltage dependence of the ~thanechol-induced depolarization, present after the hyI~erpolarization had been inhibited by exposure to AF-DX 116. The ~thanechol-induced depolarization did not exhibit a consistent voltage dependence. In 2 cells, the amplitude of the depolarization was largest near the resting membrane potential (i.e. -40 to -60 mV) and decreased when the cells were depolarized or hyperpolarized. In 8 other cells, the am-?litude of the depolarization decreased when the cell was depolarized and increased when the membrane potential was adjusted in the current clamp to more negative values. However, in these 8 cells, even though the amplitude of the bethanechol-induced depola~zation increased with hy~~olari~tion, the duration of the bethan~hol-induced depolarization decreased progressively; when the cells were maintained at approximately -90 mV, the duration of the bethanechol-induced depolarization was decreased by 35.2 k 12.0% from that observed at the resting membrane potential (- 50.0 + 9.2 mV). The effect of two other muscarinic receptor antagonists, 4-DAMP and pirenzepine, which are thought to preferentially block other than M, subtypes of muscarinic receptors were also tested. In these experiments, the effects of the antagonists were determined using both brief (< 1 set) and longer duration (l-8 set) applications of ~thanechol. The M, antagonist, 4-DAMP (I-50 nM), preferentially inhibited the initial component of the bethanechol-induced hyperpolarization, regardless of the duration of the application of bethanechol. For instance, in 2 cells, brief applications (~0.5 set) of bethanechol produced hyperpolarizations of 16 and 12 mV, respectively. After an exposure of 3-8 min to 10 or 50 nM 4-DAMP, these hyperpolarizations were inhibited by 82 and 100%. In 4 other cells, longer duration applications of bethanechol produced hy~~rpola~zations of 23-26mV. In the presence of 5-10 nM concentrations of 4-DAMP, these hyperpolarizations were decreased by 1341%. In all cases, the slow component of the hyperpolarization, which persisted in the presence of 4-DAMP, was readily blocked by AF-DX 116 (Fig. 6B4). The effect of pirenzepine (3 x lo-‘O-2 x lo-‘M) was tested in 14 cells with respect to bethanecholhyperpolarizations. also induced Pirenzepine prefarentially decreased the initial phase of the bethanechol-induced hyperpolarization but the concentration required and the extent of the inhibition varied greatly between cells. In 4 of these experiments, short applications of ~thane~hol (2 see or less) produced 5-30 mV hyperpolari~tions, which were reduced by at least 50% in the presence of 2-3OnM pirenzepine. In the other 10 experiments,

potential changes

1317

longer duration applications of bethanechol were used. In these cells, pirenzepine produced a concentration-dependent decrease in the amplitude of the bethanechol-induced hyperpolarizations (data not shown). However, even at a concentration of 200 nM, the hyperpolarization was only partially inhibited (13-92%). Further, the initial phase of hyperpolarization was reduced while the slow ~omponeni of the ~than~hol-induced hyperpolarization remained present (Fig. 6B2). No consistent effects of I-10 nM concentrations of 4-DAMP or 10-200 nM concentrations of pirenzepine were observed on the bethanechol-induced depolarization, which followed the hyperpolarization. The amplitude of the depolarization was not changed in some cases, was decreased in other instances and was increased in 1 cell. A nonselective increase in membrane conductance might be involved in the generation of the bethone~holinduced depolarization

The observation that, in cells treated with AF-DX 116, an increase in input resistance occurred during most of the bethanechol-induced depolarization, suggested that a decrease in potassium conductance was involved in the generation of the depolarization. However, the inconsistent results, obtained in experiments investigating the voltage dependence of the bethanechol-induced depolarization, suggested that a change in several ionic conductances might be involved. Consequently, the bethan~hol-induced depolarization was studied after exposure to millimol~ concentrations of barium, which should depress all membrane potassium conductances. It was found that bethanechoi initiated depolarizations in preparations maintained either in a calcium-deficient solution containing 2-4mM barium or in the control solution containing 2 mM barium; on the other hand, in the presence of barium, the hyperpolarizations produced by either short- or long-duration applications of bethanechol were inhibited. During exposure to the barium-containing solutions, the cells depolarized gradually, presumably due to a decrease in the resting potassium conductance and began to spike spontaneously. Spike activity could be inhibited by hy~rpola~zing the barium-treated cells electrotonically. The sample records shown in Fig. 7(A) were obtained from a cell exposed to the calcium-deficient solution, containing 4mM barium. The membrane potential was - 51 mV and the peak amplitude of the bethanechol-induced hyperpolarization was 16 mV in the control solution, After exposure to the solution containing barium, the cell depolarized to -42 mV and action potential activity was initiated. After approximately 5 min in the barium-substituted solution, the ~thanechol-induced hy~~ola~zation was completely inhibited. However, with the membrane potential electrotonically held at -56 mV, bethanechol induced a 10 mV depolarization, which

L. M. KONOPKA and R. L. PAWONS

1318 (A)

t

-120

-100

-80

-60 Holding

-40 potential

-20

0

20

(mV)

Fig. 7. Results illustrating that the bethanechol-induced hyperpolarization but not the depolarization, was inhibited by barium. (A) Shows the response to bethanechol (0.3 set application, onset indicated by the solid arrowhead), recorded in the control calcium-containing solution (Al), 3 min after the preparation was exposed to a calcium-deficient, 4 mM barium solution (A2) and following 35 min of recovery in the control calcium-containing solution (A3). The membrane potential was - 5 1 mV when the records in the top trace were obtained. The potential decreased to -42 mV in the presence of barium and returned to -48 mV after 35 min in the control calcium-containing solution. Calibration: y-axis equals 20 mV; x-axis equals 40 set in traces (Al) and (A3) and 10 set in trace (A2). (B) The bethanechol-induced depolarization reversed near 0 mV in the barium-substituted solution. The amplitude of bethanechol-induced depolarization is plotted as a function of the membrane holding potential.

initiated action potential activity (Fig. 7A2). The bethanechol-induced depolarization, seen in the barium-treated preparations, developed quickly after application of the agonist. The inhibition of the bethanechol-induced hyperpolarization by barium reversed slowly. In this example, after 30 min of recovery in the calcium-containing solution, with the membrane potential at -42 mV, the amplitude of the bethanechol-induced hyperpolarization was 14 mV. Similar results were obtained in 7 cells exposed to barium-containing solutions. The voltage-dependence of the bethanecholinduced depolarization was also determined in 3 cells, maintained in the barium-substituted solution. The amplitude of the depolarization increased when the membrane potential was made more negative and decreased as the membrane potential was made more positive (Fig. 7B). In these cells, the bethanecholinduced depolarization reversed at 5.8 + 2.6 mV. Complex changes in excitability occurred after application of bethanechol, when the agonist-induced hyperpolarization was negated electrotonically

When mudpuppy cardiac neurons were exposed to 400-700 msec suprathreshold depolarizing current pulses, a brief train of action potentials was initiated. The number of spikes produced was a function of the amplitude of the depolarizing current pulse. Further, the number of spikes produced occurred consistently with repeated stimuli of the same intensity (Konopka et al., 1989; Parsons and Konopka, 1991). When the membrane input resistance was decreased during the bethanechol-induced hyperpolar-

ization (Fig. 4) depolarizing stimuli were shunted and therefore became less effective. Also, as the membrane potential became more negative, the membrane potential was more distant from the threshold for spike generation, For both reasons, it was more difficult to initiate action potentials during the bethanechol-induced hyperpolarization (Konopka and Parsons, unpublished observations). A decrease in excitability during the bethanechol-induced hyperpolarization was also clearly demonstrated in those cells (approx 10%) which exhibited generation of spontaneous action potentials (Konopka et al., 1989). In spontaneously active cells, generation of action potentials ceased during the bethanechol-induced hyperpolarization and was restored when the hyperpolarization ended (Fig. 8). Previously, it was found that the neuropeptide galanin, which similarly to bethanechol hyperpolarized the mudpuppy neurons, decreased membrane excitability, even when the hyperpolarization was negated electrotonically (Konopka et al., 1989; Parsons and Konopka, 1991). Experiments were completed in this study, to determine whether excitability was depressed when the hyperpolarization following a brief application of bethanechol was negated electrotonically. In these experiments, individual neurons were injected with long depolarizing current pulses prior to and shortly after, the application of bethanechol, with the membrane potential maintained at the pre-bethanechol value. In the initial experiments, the response to subthreshold depolarizing current pulses was tested prior to and after the application of bethanechol. After a 0.5 set

Bethanechol-induced potential changes

(A)

Fig. 8. The decrease in spike activity of 2 spontaneously active cells during the bcthanechol-induced hyperpolarizaticn. The application of bethanechol [I.0 set in (A) and 0.3 set in (B), onset indicated by the solid arrowhead] caused the cell to hyperpolarize and the spontaneous spiking to cease. The resting membrane potential was -47 mV in trace (A) and -44mV in trace (B). Calibration: y-axis equals .2OmV; x-axis equals 10sec in (A) and 4sec in (B).

application of bethanechol and with the hyperpolarization negated, action potentials were produced by depolarizing current pulses, which prior to bethanechol did not elicit action potentials. The effect was

1319

reversible (Fig. 9A). Similar results were obtained in the two other cells tested. In other experiments, suprathreshold depolarizing pulses were used which elicited at least four spikes. In 12 of 16 cells tested it was found that, immediately after the bethanechol-induced hyperpolarization was negated electrotonically, the number of spikes produced was greater than that produced by a similar intensity stimulus, prior to the application of bethanechol (Fig. 9B). In the remaining 4 cells, the change in number of action potentials, after the application of bethanechol was more complicated. In these cases, when the depolarizing current pulse was given immediately after the hyperpolarization had reached its peak value and was then negated electrotonically, there was either no change (1 cell) or a slight decrease (3 cells) in the number of spikes in the burst. However, shortly thereafter, at the time when the cells were still hyperpolarized (i.e. if the membrane potential was not maintained at the pre-bethanechol level), subsequent depolarizing pulses initiated an increased number of spikes in the burst (Fig. 9C). This suggested that, in these cases, excitability was increasing progressively in a time-dependent fashion. DlSCUSSION

The results of the present study demonstrated that bethanechol could initiate both hyperpolarization

Fig. 9. Change in excitability, following a brief application of bethanechol, when the hyperpolarization was negated electrotonically. Solid arrowheads indicate onset of the application of bethanechol in each trace. Record (A) shows that constant current depolarizing pulses, which were subthreshold prior to a 0.5 set application of bethanechol, elicited action potentials after the application of agonist. This effect was quickly reversed. (B) For a brief period, following a 0.2 set application of bethanechol to another cell, the number of spikes initiated by the constant current depolarizing pulses was greater than that produced prior to the application of agonist. (C) In a third cell, excitability initially decreased after a 0.3 set application of bethanechol and then increased, so that the number of spikes initiated by the constant current depolarizing pulses was greater than prior to application of the agonist. The increase in excitability, seen in record (C), lasted longer than the duration of hyperpolarization. The resting membrane potential was - 51 mV in (A), -50 mV in (B) and - 52 mV in (C). Calibration: y-axis equals 20 mV; x-axis equals 400 msec, 40 set (indicated by the solid underlines).

1320

L. M. KONOPKA and R.

and depolarization in mudpuppy cardiac neurons. It has been proposed that bethanechol may activate nicotinic receptors in some ganglion cells (Brown, Fatherazi, Garthwaite and White, 1980). However, both the hyperpolarizing and depolarizing responses were blocked by atropine but not by d-tubocurarine. Further, depolarizing and hyperpolarizing responses were also produced by the selective muscarinic agonist, oxytremorine. Therefore, it is suggested that the bethanechol-induced depolarization resulted from the activation of muscarinic, rather than nicotinic receptors. A bethanechol-induced depolarization followed the hyperpolarization in approximately 80% of the mudpuppy parasympathetic neurons; in about 20% of the cells, a bethanechol-induced depolarization preceded the hyperpolarization. Evidence was obtained which suggests that the hyperpolarization and depolarization overlapped in time. Because of this overlap and because the hyperpolarization was much greater in magnitude than the depolarization, the depolarization was very likely masked to some extent in most cells. Therefore, the percentage of cells exhibiting the bethanechol-induced depolarization and the peak amplitude of the depolarization were very likely underestimated. As the duration of the application of bethanechol was increased from a fraction of a second to many seconds, the hyperpolarization increased in amplitude and duration. A similar, long-duration hyperpolarization followed brief applications of oxytremorine. Evidence was obtained in some experiments that the long duration bethanechol-induced hyperpolarizations were composed of two components (Figs 3 and 6). For instance, in the presence of 4-DAMP and pirenzepine, the rapidly developing phase of the bethanechol-induced hyperpolarization could be markedly reduced, without eliminating the later phase of the hyperpolarization. The long-duration hyperpolarizations were completely inhibited by AFDX 116, indicating that both components resulted from the activation of muscarinic receptors. Some of the mudpuppy cardiac neurons are electrically coupled (Roper, 1976). However, it is not thought that the slower developing phase of hyperpolarization represented electrotonic responses caused by hyperpolarizations generated in adjacent, electricallycoupled cells because all of the cells, given long pulses of bethanechol, exhibited two phases. Further, long pulses of bethanechol also produced two phases of hyperpolarization in enzymatically-dissociated neurons (Konopka and Parsons, unpublished observations). These results suggest that at least two ionic mechanisms are involved in the generation of the bethanechol-induced depolarization in the mudpuppy neurons, as indicated by several findings. For instance, it was noted that, in AF-DX 116-treated preparations, the input resistance was decreased during the onset of the bethanechol-induced depolar-

L. PARXINS

ization and increased during the remainder of the depolarization. Furthermore, while in barium the reversal potential for the bethanechol-induced depolarization was approximately 6 mV, as expected for a depolarization resulting from the activation of nonspecific conductance (Dwyer, Adams and Hille, 1980). A decrease in membrane potassium conductance might also be involved in the generation of the bethanechol-induced depolarization, as indicated by measurements of input resistance, made on untreated cells and cells treated with AF-DX 116. In control cells, the input resistance was increased above the pre-bethanechol value, during the depolarization which followed the hyperpolarization (Fig. 4). An increase in resistance also was noted during most of the depolarization recorded in AF-DX 116-treated preparations (Fig. 5). Moreover, in 8 of 10 AF-DX 116-treated cells, the duration of the bethanecholinduced depolarization decreased when the membrane potential was adjusted to voltages approaching the potassium equilibrium potential. These observations suggest that the latter phase of the depolarization resulted from a decrease in membrane potassium conductance (Brown and Adams, 1980; Kuffler and Sejnowski, 1983; Akasu, Gallagher, Koketsu and Shinnick-Gallagher, 1984; Jones, 1985). It is speculated, therefore, that the muscarinic depolarization, observed in the mudpuppy neurons, may be caused by a combination of a receptor-mediated increase in a nonselective cation conductance and decrease in potassium conductance. A similar conclusion has been suggested for the generation of muscarinic receptor-mediated slow depolarizations in other amphibian autonomic neurons (Brown, 1988). Previously, Allen and Burnstock (1990) concluded that the muscarinic hyperpolarization in the guinea pig cardiac neurons was mediated by an M, type of muscarinic receptor. The antagonist of the cardiac M, receptor, AF-DX 116, was the most consistent inhibitor of the bethanechol-induced hyperpolarization in the mudpuppy neurons. Consequently, it is suggested that, in mudpuppy parasympathetic neurons, the bethanechol-induced hyperpolarization is mediated primarily by a muscarinic M,-like receptor (Micheletti, Montagna and Giachetti, 1987). However, 4-DAMP and pirenzepine attenuated the bethanechol-induced hyperpolarization in several cells. Therefore, it is possible that other subtypes of muscarinic receptor might mediate part of the bethanechol-induced hyperpolarization in mudpuppy cardiac neurons. Concentrations of AF-DX 116, which completely eliminated the bethanechol-induced hyperpolarization, did not affect the depolarization. It is suggested, therefore, that different subtypes of muscarinic receptor mediate the generation of the bethanechol-induced hyperpolarization and depolarization. As pirenzepine and 4-DAMP did not consistently affect the bethanechol-induced depolarization, the results are insufficient to suggest which subtype of

Bethanechol-induced

muscarinic receptor mediates the bethanecholinduced depolarization. During the bethanechol-induced hyperpolarization, the ability to elicit spike activity was decreased, while it was increased when the hyperpolarization was negated electrotonically (Fig. 9). Furthermore, in the latter case, the increase in spike activity occurred at times when depolarizing stimuli would still be shunted by the bethanechol-induced increase in potassium conductance (Fig. 4). This result was opposite to that reported previously for galanin (Konopka et al., 1989; Parsonsand Konopka, 1991). Consequently, although bsoth bethanechol and galanin initiate hyperpolarization by activating a membrane potassium conductance, these two agonists have different effects on other ionic conductances, responsible for spike generation. Allen and Burnstock (1990) also found in guinea pig c.ardiac neurons that muscarinic agonists increased the number of action potentials that were evoked by a given constant current depolarizing pulse. This increase in excitability in guinea pig neurons was attributed to depression of the slow spike afterhyperpolarization and decrease in action potential duration, secondary to muscarine-induced attenuation of calcium current (I,,) (Allen and Burnstock, 1990). Recently, Tse, Clark and Giles (1990) demonstrated that muscat-me reduced the Ica in frog cardiac neurons. In frog and mudpuppy parasympathetic neurons, action potential repolarization is controlled primarily by &a,* so that following a reduction in I,-,, the duration of the action potential is increased (Konopka er al., 1989; Clark, Tse and Giles, 1990; Tse et al., 1!)90). In these mudpuppy neurons, the increase in excitability induced by bethanechol occurred under conditions in which the duration of the action potential would be expected to increase, a situation different from that seen in the guinea pig neurons. In conclusion, the present results demonstrate that bethanechol has multiple actions on membrane ionic conductances in the mudpuppy parasympathetic neurons, these actions determining membrane excitability. Acknowledgements-We

thank Mr Dean Melen for his expert technical assistance during the course of this study and Drs Cynthia Forehand, Jean Hardwick, Gary Mawe and Jerome Fiekers for their critical review of this manuscript. We also thank Dr John Ellis for the generous gift of some of the muscarinic receptor antagonists used in parts of this study and his helpful comments made during the course of this work. This work was supported in part by NI:H grants NS23978 and NS25973. REFERENCES

Akasu T., Gallagher J. P., Koketsu K. and Shinnick*Gallagher P. (1984) Slow excitatory post-synaptic currents ;mbull-frog sympathetic neurons. J. Physiol. 351: 583-593.

potential changes

1321

Allen T. G. J. and Bumstock G. (1990) M, and M, muscarinic receptors mediate excitation and inhibition of guineapig intracardiac neurones in culture. J. Physiol. 422: 463-480.

Bemheim L., Beech D. J. and Hille B. (1991) A diffusible second messenger mediates one of the pathways coupling receptors to calcium channels in rat sympathetic neurons. Neuron 6: 859-867. Brown D. (1988) M-currents: an update. Trends Neurosci. 11: 294-299.

Brown D. A. and Adams P. R. (1980) Muscarinic suppression of a novel voltage-sensitive K+ current in a vertebrate neuron. Nature, Land. 233: 673676. Brown D. A., Fatherazi S., Garthwaite J. and White R. D. (1980) Muscarinic receptors in rat sympathetic ganglia. Br. J. Pharmac. 70: 577-592.

Clark R. B., Tse A. and Giles W. R. (1990) Electrophysiology of parasympathetic neurones isolated from the interatrial septum of bull-frog heart. J. Physiol. 427: 89-125.

Dwyer T. M., Adams D. J. and Hille B. (1980) The permeability of the endplate channel to organic cations in frog muscle. J. gen. Physiol. 75: 469-492. Hartzell H. C., Kuther S. W., Stickgold R. and Yoshikami D. (1977) Synaptic excitation and inhibition resulting from direct action of acetylcholine on two types of chemoreceptors on individual amphibian parasympathetic neurons. J. Physiol. 271: 817-846. Jones S. W. (1985) Muscarinic and peptidergic excitation of bull-frog sympathetic neurons. J. Physiol. 366: 63387.

Konopka L. M. and Parsons R. L. (1989) Activation of muscarinic receptors and galanin-like peptide receptors initiate membrane hyperpolarization in mudpuppy parasympathetic neurons but do not exert similar effects on excitability. PASEE 3: A394. Konopka L. M. and Parsons R. L. (1990) The muscarinic agonist bethanechol initiates hyperpolarization and depolarization of mudpuppy intracardiac neurons. Neurosci. Abstr. 16: 1055. Konopka L. M., McKeon T. W. and Parsons R. L. (1989) Galanin-induced hyperpolarization and decreased membrane excitability of neurons in mudpuppy cardiac ganglia. J. Physiol. 410: 107-122. Kuffler S. W. and Sejnowski T. J. (1983) Peptidergic and muscarinic excitation at amphibian sympathetic synapses. J. Physiol. 341: 257-278. Micheletti R., Montagna E. and Giachetti A. (1987) AF-DX 116, a cardioselective muscarinic antagonist. J. Pharmac. exp. Ther. 241: 628434.

Parsons R. L. and Konopka the decrease in membrane mudpuppy parasympathetic

L. M. (1991) Analysis of excitability by galanin in neurons. Neuroscience 43:

647-660.

Parsons R. L., Neel D. S., Konopka L. M. and McKeon T. W. (1989) The presence and possible role of a galaninlike peptide in the mudpuppy heart. Neuroscience 29: 149-159.

Roper S. (1976) An electrophysiological study of chemical and electrical synapses on neurones in the parasympathetic cardiac ganglion of the mudpuppy, Necrurus maculosus: evidence for intrinsic ganglionic innervation. J. Physiol. 254: 427-454.

Tse A., Clark R. B. and Giles W. R. (1990) Muscarinic modulation of calcium current in neurons from the interatrial septum of bull-frog heart. J. Physiol. 427: 127-149.