Hyperpolarizing potentials induced by Ca-mediated K-conductance increase in hamster submandibular ganglion cells

Hyperpolarizing Potentials Induced by Ca- Mediated K-Conductance Increase in Hamster

Submandibular Ganglion Cells

TAKASHI SUZUKI* and KIYOSHI KUSANO?

Neurophysiology Laboratory, Department of Biology, Illinois Institute of Technolog Chicago, Illinois 60616

Received February 14,1978; revised May 1,1978

SUMMARY

The mechanisms of three types of hyperpolarizing electrogenesis in hamster submandibular ganglion cells were analyzed with intracellular microelectrodes. These included (1) spike-induced hyperpolarizing afterpotential (S-HAP), (2) spontaneous transient hyperpolarizing potential (HP), and (3) the hyperpolarizing (H) phase of postsynaptic potential (PSP). Most of these hyperpolarizing potentials were due to conductance increases and reversed polarity at membraie potential (Em) between -70 and -85 mV, which was close to the K-equilibrium potential. The average resting potential of ganglion cells was -53 mlr. Action potential overshoot increased slightly in high [Ca2+]o and decreased in low [Ca2+]o. In most neurons action potentials were completely suppressed by lov7- M tetrodotoxin (TTX). The S-HAP has an initial component due to delayed rectification and a late component. The late component is enhanced by increasing [Ca2+]o, or by applying Ca-ionophore (A23187), TEA, caffeine, or dibutyryl cyclic (DBc-) AMP; it is suppressed by decreasing [Ca2+]o, or by applying Mn2+. Perfusion with C1--free saline reduced membrane potential slightly but did not modify the S-HAP. Depolarizing pulses also induced hyperpolarizing afterpotential (D-HAP), similar to the S-HAP. Spontaneous transient HPs occurred in some neurons at irregular intervals. HPs were insensitive to TTX but were suppressed by Mn2+. Caffeine induced low frequency rhythmic HPs in many neurons, often alternating with periods of repetitive spiking. The PSP was a monophasic depolarizing (D-) potential in some neurons, but in others the D-phase was followed by a small H-phase. Perfusion with A23187, caffeine or DBc-AMP increased the H-phase of the PSP. Perfusion with K+-free saline or treatment with 10-5M ouabain did not abolish the H-phase of PSPs. These membrane potential-dependent phenomena appear to be induced mainly by Ca-mediated K-conductance increases. This mechanism contributes to the regulation of low-frequency repetitive firing in submandibular ganglion cells.

* Present Address: Department of Oral Physiology, Matsumoto Dental College, Shiojiri, 399-07,

+ To whom reprint requests should be addressed. Japan.

Journal of Neurobiology, Vol. 9, No. 5, pp. 367-392 (1978) 0 1978 John Wiley & Sons, Inc. 0022-3034/78/0009-0392$01.00

368 SUZUKI AND KUSANO

INTRODUCTION

It is generally accepted that both sympathetic and parasympathetic nerves supply the salivary glands with secretory function (Langley, 1980; Emmelin, 1967), but the role of the sympathetic innervation is not well understood. The parasympathetic nerve controls the release and flow rate of saliva. Both monosynaptic and multisynaptic convergence types of contact have been found in the rat submandibular ganglion (Suzuki and Sakada, 1972). It is also known that some of the preganglionic fibers diverge to more than one postganglionic neuron (Lichtman, 1977). At the grandular cell level the postganglionic parasympathetic nerve fibers are known to converge (Lundberg, 1958; Garrett, 1966).

Modulation of repetitive impulse discharge pattern set by the CNS occurs at the submandibular ganglion level; however, an understanding of this mechanism in terms of the membrane properties of postganglionic parasympathetic neurons has scarcely been attempted. In this paper, we analyze the mechanisms of three types of hyperpolarizing electrogeneses which modulate the pattern of impulse transmission at the hamster submandibular ganglion under both in situ and in uitro conditions: (1) The spike-induced hyperpolarizing afterpotential (S-HAP), (2) the spontaneous hyperpolarizing potential (HP), and (3) the hyperpolarizing (H) phase of postsynaptic potential (PSP). We observed that most of these potentials had a reversal potential near the K-equilibrium potential, E K , and that they were very sensitive to extracellular Ca2+ concentration, [Ca2+]o and to agents which modify the Ca-conductance of the membrane. We assume these potentials are induced by a Ca-mediated K-conductance increase (Meech, 1972). The mechanism described contributes to modulation of the postganglionic impulse pattern, especially to the reduction of repetitive firing frequency. A brief preliminary report of this work has appeared elsewhere (Suzuki and Kusano, 1977).

METHODS

I n s i tu preparation

Young hamsters weighing about 100 g were used for both in situ and in uitro experiments. Animals were anesthetized by intraperitoneal injection of a chloralose (50 mgkg) and urethane (500 mg/kg) mixture dissolved in Krehs saline. After tracheotomy, the left submandibular and sublingual glands with secretory ducts, blood vessels, and nerves were freed from surrounding connective tissue, up to the posterior region of m. digastricus anterior belly. The animal was placed on its left side on a plexiglass plate and the head was fixed. The plate consisted of a small shallow partition chamber (2 cc volume) which was made of plexiglass and dental wax. The left jaw was placed near the chamber and both glands were stretched horizontally by means of pins onto the bottom of this chamber. The chamber was filled with Krebs saline which was saturated with 95% 0 2 and 5% COz. The blood vessels of the scblingual gland were ligated and removed. The clustered submandibular ganglia (usually 3 or 4) could then be seen near the proximal outlet of the glandular duct with the associated pre- and postganglionic nerve trunks running along the duct. The size of the largest ganglion which we customarily used was about 1 mm long and 500 pm thick. Since mammalian ganglia are covered by a thick layer of Connective tissue making microelectrode penetration difficult, as much as possible of the connective tissue on the surface of the ganglion was removed with needles and forceps avoiding damage to the blood supply to the ganglion. Both pre- and postganglionic nerve trunks were sepa- rated from the neighboring arteries and cut 3-4 mm away from the ganglion. The diameter of the

HPs IN PARASYMPATHETIC NEURONS 369

preganglionic nerve trunk was about 200-300 fim and that of the postganglionic nerve trunks (3-5) were less than 100 gm each. Each cut end of the nerve trunk was picked up by a suction electrode in order to apply orthodromic or antidromic stimulation. Since ganglion cells were small (about 25 fim in diameter), clear visualization of individual cells was not achieved under the dissection mi- croscope (80x) with transillumination. Insertion of microelectrodes was begun after an operated animal showed a slight recovery from anesthesia judged by response to touch stimuli applied to the facial area. When the animal recovered from initial anesthesia, 0.05 ml nembutal was injected in- traperitoneally. The Krebs saline in the recording chamber was continuously renewed

Isolated preparation Animals were anesthetized with intraperitoneal injection of 0.1 ml nembutal. Preliminary pro-

cedures were similar to those for in situ preparations. Both submandibular and sublingual glands, together with associated ducts and blood vessels, were cut out a t the level of posterior region of rn. digastricus anterior belly. The isolated tissue was stretched on the plexiglass plate and covered with Krebs saline. All glandular tissues, blood vessels, and ducts were dissected away leaving the ganglia and the nerve trunks exposed. The ganglionic preparation was then transferred into the recording chamber. The recording chamber was made of dental wax on the plexiglass plate with slightly elevated bottom in the center of the chamber. The preparation was fixed on the bottom of the chamber by stretching out surrounding connective tissue by means of pins. The connective tissue covering the surface of the ganglion was removed as was done for in si tu preparation. The pre- and postganglionic nerve trunks were picked up with suction electrodes for stimulation. Re- cordings were begun after perfusing with Krebs saline for a t least 30 min.

Electrical recordings

Glass microelectrodes were filled with 3 M KC1 and their dc resistance ranged between 40 and 60 Ma. A single microelectrode was inserted into the cell and used for both recording and for current application. Signals were led through a Bak dc amplifier (ELSA-4) and a dc differential amplifier to a two beam Tektronix oscilloscope. Stimulators used were Tektronix 160 series and intracellular currents were applied through a Wheatstone bridge circuit having lo+$ Q limiting resistance in series with the microelectrode. A voltage, time calibrator, and a current to voltage converter were inserted in series between the preparation and ground. For antidromic and orthodromic stimulation, pre- and postganglionic nerve strands were stimulated via suction electrodes. A saline agar-3 M KCI bridge was used as an indifferent electrode as needed. Oscilloscope traces were recorded by means of a bromide paper Kymograph camera (Nihon Koden).

Since the bridge circuit was balanced either hy adjusting the threshold of the directly evoked spike with a short pulse [e.g., Fig. 1 (Al)] or by comparing the amplitude of a directly evoked spike with that of an antidromic or orthodromic spike [e.g., Fig. 6 (Al ) and (A2)], measurements of the displaced membrane potential and the reversal potertial of membrane electrical responses contained certain errors. In addition, possible non-isopotential neuron geometry was another source of erroneous measurements.

Perfusion salines and drugs

Ionic composition of the control saline (Krebs saline) was as follows: 136.9 mM NaCl, 5.0 mM KCl, 2.0 mM CaC12,0.5 mM MgC12,12.0 mM NaHC03,l mM NaH2P04, and 11 mM glucose. For making K+-free saline equiosmolar Na was added. For making Ca2+-free saline, Mg2+ was substituted for Ca2+. Ca2+-free, Mn2+ (BmM)-containing saline was prepared by removing NaHCOB, NaH2P04 and CaClz from the Krebs saline and adjusting to pH 7.4 with Tris. For Cl--free saline, C1- in the Krebs saline was replaced with equiosmolar amount of acetate. The following drugs were used: caffeine (Sigma), tetraethylammonium (TEA) chloride (Sigma), dibutyryl cyclic 3Wadenosine monophosphate (DBc-AMP) (Sigma), tetrodotoxin (TTX) (Sankyo), ouabain (Sigma), and Ca- ionophore (A231871) (Eli Lilly). These were added to the control saline or to the K+-free saline. Ca-ionophore was initially dissolved in dimethyl sulfoxide (DMSO) and then diluted with control saline. Final concentration of Ca-ionophore was 2.5 pg/rnl and that of DMSO was 0.1%. DMSO at this concentration was found to be without effect. All perfusion salines were saturated with 95% 0 2 and 5% COZ gas, and all experiments were carried out a t 3Oo-32OC.


RESULTS

Passive and active membrane properties of submandibular ganglion cells

In situ analysis. Passive membrane properties. The data on the resting E , was taken from cells in which the recording electrode could be maintained for more than 30 min for analysis. The mean resting Em in situ was -53 f 8.0 mV (mean f S.D., n = 54). The current-voltage (I vs V), relation was tested by delivering current of varying intensity as pulses of 100 msec duration. An example is shown in Figure 1. The I vs V relation near the resting E , was ohmic. The input resistance of submandibular ganglion cells ranged from 10 M Q to 90 h4Q (40.4 f 26.2 M Q , n = 16). The membrane time constant was approximated by applying hyperpolarizing current pulses and taking the time required for the membrane potential to rise to 63% of its maximal value, without correcting for the dendritic component. The value ranged from 5 to 10 msec.

Active membrane properties. The E , of some neurons fluctuated spontaneously, thereby inducing a transient hyperpolarizing potential. These properties will be described in a later section. Most neurons responded to a long lasting depolarizing current pulse by a single spike but in some neurons a few repetitive spikes occurred [Fig. 1 (A4)]. An example of membrane response to outward and inward current pulses is shown in Figure l(Al)-l(B4). The critical depolarization for spike firing was 14.4 f 5.4 mV ( n = 36). In general the amplitudes of directly evoked spikes in response to intracellularly injected current (69 f 9.8 mV, n = 36) and those of the antidromically stimulated spikes (71.5 f 12.4 mV, n = 21) were not significantly different. Both directly evoked and antidromically elicited spikes were followed by a transient hyperpolarizing afterpotential of similar amplitude. The average amplitude of the S-HAP was about 13 mV and about 250 msec duration at the level of the average E,. However, as shown in Figures 2 and 3, the amplitude of S-HAP was E , -dependent. As the Em became more negative, the amplitude of the S-HAP decreased. An example is shown in Figure 2, where 2(A1) and 2(A2) were obtained at Em = -72 mV. There was a very small (3 mV) S-HAP. On the other hand, as the E , became less negative, both amplitude and duration of S-HAP were increased. In Figure 2(B1) and 2(B2) the resting E , was -53 mV. Figure 2(Cl) shows an antidromic spike obtained at Em = -43 mV. There was a large S-HAP of 28 mV in amplitude and 160 msec in duration. In Figure 2(C2) and 2(C3) a directly evoked spike was followed by an antidromically elicited spike at different intervals. The amplitudes of HAP induced by a directly evoked spike and that of antidromic spike are about the same. Since the peak of the HAP is close to its equilibrium potential and the membrane resistance falls during the HAP, further development of HAP due to subsequent spike is small or negligible. The threshold for the antidromic spike increased during HAP induced by the previous spike. The relationship among amplitudes of the resting E,, the directly or antidromically evoked spike and those S-HAPS obtained from individual neurons are shown in Figure 3(A) and 3(B). The most negative resting E , which nullified the S-HAP was about -75 mV. Spike induced depolarizing afterpotentials were not observed.


-2 I IO-"A

-30 mV t Fig. 1. Current-voltage relation of a submandibular ganglion cell in control saline (in situ re-

cordings). (Al-4). Membrane response to various intensities of outward current pulse. Bridge circuit was balanced by adjusting the threshold voltage of a directly evoked spike to a short depolarizing pulse as shown in (Al). (Bl-4) Membrane response to various intensities of inward current pulse. Resting Em = -72 mV. A calibration pulse of 20 mV, 10 msec preceded each response and is also seen in Figures 2,4-6. Potential recordings of (Bl-4) are approximately half-gain of that used for (Al-4). Upper traces monitor currents and lower traces monitor membrane potential changes. The current calibration shown in (B4) is 1 X lo-$ A and applies to (A) and (B). Graph I vs V-relation from above data.

The amplitude of directly evoked S-HAP decreased as the Em was increased by injecting various intensities of inward current. With hyperpolarization to about the -75 mV level, the S-HAP became about zero. However, in some cells, the apparent reversal potential of an antidromic S-HAP was about -100 mV [e.g., Fig. 2(Dl)-2(D3)], which was much more negative than a graphical estimation made from Figure 3(A) and 3(B). Since the threshold voltage for the antidromic spike increased during application of hyperpolarizing currents, the action potential which was recorded during application of a large hyperpolarization may be elicited remote from the site of recording, and consequently the apparent reversal potential obtained was in error due to the cable loss of applied current. The average reversal potential of the antidromic S-HAP was -84.6 f 15.9 mV ( n = 8). The membrane resistance decreased from one fourth to two thirds of the resting resistance at the peak of S-HAP [Fig. 2(E1) and 2(E2)].


*l i LJL c1

81 82 A2 I

c2 c3

Fig. 2. Action potentials recorded from five neurons which showed different membrane potentials (in situ recordings). The resting Em of cell-1 [(Al) and (A2)] = -72 mV. (Al) shows an antidromic spike, (A21 an orthodromic spike. The resting Em of cell-2 [(Bl) and (B2)] = -53 mV. (Bl) shows an antidromic spike, (B2) an orthodromic spike. The resting Em of cell-3 [(Cl)-(C3)] = -43 mV. (Cl) shows an antidromic spike. In (C2) and (C3) a directly evoked spike preceded an antidromically elicited spike at different intervals. The resting Em of cell-4 [(Dl)-(D3)] = -40 mV. Antidromic spike (Dl) was hyperpolarized by applying conditioning inward current pulse [(D2) and (D3)]. Note the changes in amplitude of spike after potential. The resting Em of cell-5 [(El) and (E2)] = -40 mV. Small repetitive outward current pulses of 20 msec in duration a t 25 Hz were applied during antidromic S-HAP (El) and at resting Em level (E2) to test for membrane conductance change. For further explanation see text. Voltage calibration shown in each record except (El) and (E2) is 20 mV, 10 msec. Current calibration for (C) and (D) shown in (D3) is 1 X A. Voltage and time calibrations for (E l ) and (E2) are 20 mV, 200 msec.

Stimulation of the preganglionic nerve induced an orthodromic spike. The amplitude of the synaptic potential was large [e.g., Fig. Z(A2) and 2(B2)] and it was difficult to separate it from the spike by applying postsynaptic hyperpolarization. An orthodromic spike also induced an HAP. In vitro analysis. Similar analyses were made on the preparations in uitro.

The average resting Em was -53 f 7.2 mV ( n = 63). Passive membrane con- stants and active responses were similar to in situ values. The relationships among resting Em, both directly and antidromically evoked spike amplitudes, and the S-HAPS are shown in Figure 3(C) and 3(D). The average reversal potential of S-HAPS was -81.2 f 4.3 mV (n = 15).

The minimum conduction velocities of both pre- and postganglionic fibers were estimated from the latencies of orthodromic and antidromic intracellular spikes. Since there appeared to be no difference in velocity between under in situ and in uitro measurements, the data obtained from two conditions were pooled. For the preganglionic fibers, the synaptic delay of 0.5 msec was sub- tracted from each measurement. The conduction velocity of some preganglionic fibers was slightly faster than 1 mhec (n = 8), but the mean velocity of the fibers


B 0 0 90

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-80mV -600 -40 -80mV -60 -40 I I I I I I

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Fig. 3. Relationships among the amplitudes of resting membrane potential (abscissa), spike height (from the resting Em to peak of the spike, positive ordinates) and spike afterpotential (negative ordinates). Data were accumulated from many preparations. (A) Antidromic spikes in situ. (B) Directly evoked spike in situ. (C) Antidromic spikes in uitro. (D) Directly evoked spikes in uitro. 0 = spike amplitude, 0 = S-HAP.

was 24.8 f 17.2 cmlsec (n = 21). The postganglionic fibers had a conduction velocity of 24.0 f 11.7 cm/sec (n = 21). One reason for our observation of relatively slow conduction velocities of preganglionic fibers might be their short length in this preparation, as the impulse conduction velocity is expected to decrease near the axon terminal.

It appeared that there was no characteristic difference in passive and active membrane properties of hamster submandibular ganglion cells under in situ and in uitro conditions. The experiments discussed subsequently were carried out in uitro.


Ef fec ts of ions and drugs o n the hyperpolarizing afterpotentials induced by spikes and depolarizing pulses

Effects of K+. The Em vs [K+]o-relation was examined using cells with both high Em (e.g., -70 mV) and intermediate Em (e.g., -45 mV). The former group showed a Nernst relationship between 15 and 100 mM of [K+]o of about 56 mV per ten fold [K+]o change, but the latter group showed a smaller slope. The E m of both groups approached zero as [K+]o was increased to about 130 mM. As- suming pure K-electrode behavior, the E K is estimated as -84 mV. The Em of the intermediate cell group presumably involves the participation of other ion permeabilities. The S-HAP was also sensitive to changing [K+]o. The S-HAP disappeared when the perfusion saiine contained more than 15 mM K+. Re- moval of K+ control saline induced one of the following three results: In the first type, there was no obvious change in Em, amplitude and duration of S-HAP as shown in Figure 4(A1) and 4(A2). In the second type, the Em decreased gradually, and consequently induced a repetitive spike firing with periods of subthreshold membrane potential fluctuations. The maximum frequency of the reptitive spiking was 13 Hz. The peak amplitude of the S-HAP, however, increased. In the third type, the resting E , became more negative (e.g., by 6 mV from -45 mV) and both amplitude of action potential and S-HAP increased. The resting membrane resistance of above three types increased in general. The reversal potential for the S-HAP shifted slightly in the negative direction in K+-free saline compared to the value in 5 mM [K+]o-saline.

Effect of tetrodotoxin (TTX). Application of 10-7M TTX abolished directly evoked action potentials in many meurons, but some were very resistant to TTX, even at concentrations as high as 10-6M. The HAPS were also induced by depolarizing pulses without spike [e.g., (Bl)-(C4) in Fig. 41. The amplitude of the depolarizing pulse induced HAP (D-HAP) increased by increasing the amplitude of depolarizing pulse to some extent but saturated at a similar value for the S-HAP. The D-HAP behaved similarly as the S-HAP did in K+-free saline. An example is shown in Figure 4(Cl) and 4(C2).

Effects of tetraethylammonium (TEA) ions. Application of TEA (2.5-5 mM) depolarized neurons up to about 10 mV, but in some neurons a few millivolts of hyperpolarization was noticed. The synaptic potential was increased in amplitude and duration. The duration of the depolarizing phase of the action potential was prolonged from about 5 msec in control to about 9 msec in TEA. Further prolongation of the action potential duration was not observed. In one preparation, TEA induced spontaneous repetitive PSPs and spikes (max 2 Hz). Amplitudes of both S-HAP (not shown in Fig. 4) and D-HAP were increased by TEA [compare Fig. 4(B1) and 4(BZ)].

Effects of Cl--free saline. Substitution of total [Cl-], with acetate for 20 min caused the resting Em to decrease by several millivolts. The S-HAP [compare (A) and (B) in Fig. 51 and the input resistance of the neuron were not altered. The reversal potentials of both S-HAP and D-HAP were also unchanged. Continued perfusion with acetate saline beyond 20 min reduced the resting Em further and induced repetitive spiking (max 2 Hz) [Fig. 5(C)], with reduction of spike amplitude and of S-HAP. These effects were reversible in control saline [(D)].


A1 A 2 01 8 2 I L -’

Fig. 4. Effects of K+-free saline, TEA and caffeine on the hyperpolarizing afterpotentials induced by directly evoked spikes [(Al) and (A2)] and by depolarizing pulses in three different neurons in (A), (B), and (C), respectively. (Al) Control response, and (A2) response in K+-free saline. Both resting E,, = -48 mV. (Bl) Control response in TTX-containing saline, resting Em = -49 mV. Recording of the current pulse was accidentally lost; a slightly larger pulse than that shown in (B2) was applied. (B2) Response in 5 mM TEA with TTX-containing saline, resting Em = -53 mV. (Cl) Control response in TTX-containing saline, Em = -46 mV. (C2) Response in TTX-containing K+-free saline, resting Em = -48 mV. (C3) Response in 2.5 mM caffeine with TTX in K+-free saline, resting En, = -49 mV. (C4) Response in 2.5 mM caffeine-containing control saline, resting Em = -39 mV. Lower Em value in (C4) might be due to prolonged exposure to caffeine. Voltage calibration bars are 40 mV each and the time scale shown in (C4) is 200 msec and applies to all records.

Effects of Ca2+-free saline. The orthodromic response was easily blocked by perfusing the preparation with Ca2+-free, Mg2+-substituted saline. In this saline the resting Em was not stable (see p. 378). The amplitudes of both directly evoked and antidromic spikes decreased to about 90% of the control. The amplitude of S-HAP decreased and its duration was reduced [compare Figs. 6(A1) and 6(B)]. Record B was taken immediately after orthodromic response had been blocked by Ca2+-free saline perfusion. The amplitude of the directly evoked S-HAP decreased to one third of the control. A t this time, changes in reversal potentials for both S-HAP and D-HAP were, however, insignificant. When the Ca2+-free, Mn2+-substituted saline was used the resting Em was slightly hyperpolarized (by about 2 mV). The amplitude of the action po-

A B q--, t _ D *

C

2 - Fig. 5. Effects of ClF-free saline. (A) Directly evoked spike in control saline. (B) directly evoked

spike in Cl--free, acetate substituted saline 20 min after perfusion. The resting Em in control was -44 mV and that in acetate saline was -40 mV. (C) Repetitive spiking occurred in acetate saline due to membrane depolarization. (D) Action potential elicited as an off-response of the hyperpolarizing pulse, 20 min after reperfusing with control saline. Calibration bars: 40 mV, 200 msec for (A), (B), and (D); 40 mV, 1 sec for (C).


tential decreased to nearly half of that observed in the control. Both S-HAP (not shown in Fig. 6) and D-HAP [compare Fig. 6(E) and 6(F)] were reduced more than in the Ca2+-free, Mg2+-substituted saline. These effects, induced by Ca2+ substitution were reversible on reperfusion of the preparation with control saline.

Effects of caffeine, Ca-ionophore (A23187) and DBc-AMP. Effects of 2.5 mM caffeine on the resting Em, amplitudes of spike height and S-HAP were Em-dependent. When the resting Em was very negative (e.g., -70 mV) caffeine effects on these parameters are not detected. However, when the resting Em was less negative in control saline, one of the following three changes was induced: a small increase in Em negativity, an induction of periodic hyperpolarizing potentials, or a sudden increase in Em negativity (max 30 mV). These changes are time dependent and longer exposures exhibiting gradual depolarization of Em. The amplitude and duration of the D-HAP, and those for the S-HAP, together with the spike amplitude were considerably increased [compare Fig. 4(C2) and 4(C3); Fig. 6(C) and 6(D)]. Effects of 2.5 pgIml Ca-ionophore were examined in three preparations. Results were similar to the caffeine effects. Addition of 2 mM DBc-AMP in control saline increased the amplitude of S-HAP without changing the resting Em or the membrane resistance.

Periodic transient membrane hyperpolarizations

Spontaneous fluctuation in membrane potential. Spontaneous, periodic, transient membrane hyperpolarizations were observed in some neurons in both in situ and in vitro conditions. The intervals, amplitude, and time course of

A1 A2 B

C I DI E F

- Fig. 6. Effects of Ca2+-free saline [(Al), (A2), and (B)], 2.5 mM caffeine [(C) and (D)] and of 2

mM Mn2+ in Caz+-free saline [(E) and (F)] on the hyperpolarizing afterpotentials. Data shown were obtained from three neurons [(Al), (A2) and (B); (C) and (D); (E) and (F)]. (Al) Control spike by a direct stimulation. (A2) Control spike by orthodromic stimulation. (B) Directly evoked spike in Ca"+-free, Mg2+-substituted saline. The resting Em in the control saline was -44 mV and that in Ca2+-free saline was -46 mV. (C) Directly evoked spike in control saline. (D) Directly evoked spike in 2.5 mM caffeine containing saline. The resting Em was -42 mV in the control and was not changed by caffeine. Both spike height and hyperpolarizing afterpotentials were increased in amplitude. The duration of the hyperpolarizing afterpotential was narkedly prolonged. (E) D-HAP in Ca2+-free, Mg2+-substituted saline (an unusually large amplitude was selected). (F) The D-HAP was reduced by applying 2 mM Mn2+ in Ca2+-free saline. Calibration bars: 40 mV for records (Al), (A2), (B), (E), and (F); 20 mV for (C) and (D). The 200 msec [in (F)] applies to all records.


such hyperpolarizing potentials (HPs) varied considerably among neurons and even in the same neuron. An example of such parameters were as follows: at the resting Em = -50 mV, the peak amplitude of the HPs was 16 mV, with a rise time of 1125 msec, a half-decay time of 1160 msec and total duration of about 3500 msec. The interval between HPs was irregular and the frequency was low [see Table I(A) and Fig. 7(A)]. In a few neurons, such HPs were evoked by the spike.

Effects of caffeine and DBc-AMP. Since the spontaneous fluctuation of membrane potential appeared to be similar to that of caffeine-induced HPs seen in frog sympathetic neurons (Kuba and Nishi, 1976)) we studied the effects of caffeine on the present system. Application of 2.5 mM caffeine in control saline induced HPs, which were larger and more regular than the spontaneous ones. The resting Em was gradually reduced and repetitive spiking was induced, initially as an off-response of the HPs. Such repetitive spiking ( 2 4 Hz) continued for more than 15 min (Fig. 7; also see Fig. 9). When the frequency of repetitive spiking is low, the peak of the spike undershoot and that of the HPs were at about the same Em level [Fig. 7(B) and 7(C)]. Application of TTX M ) abolished the repetitive spiking without affecting the HPs [Fig. 7(D)].

The caffeine induced HPs were Em-dependent. In general the amplitude of HPs was the largest in the Em range between -43 and -55 mV. When the resting Em was less negative than -40 mV, the HPs were reduced to base line level. I t was also not seen in neurons with highly negative resting Em (e.g., >-65 mV). The amplitude of the HP was reduced by applying conditioning hyperpolarization and nullified in the Em range between -70 and -80 mV; its polarity was reversed by increasing the conditioning hyperpolarization further. Appli-

C

I D

Fig. 7. Spontaneous HPs and effects of 2.5 mM caffeine on membrane potential. This preparation showed spontaneous periodic HPs of various amplitudes, durations at irregular intervals in control saline (A). Application of 2.5 mM caffeine induced more regular HPs (B). Amplitude of HPs increased and, spikes were initiated at the ends of HPs as an off-response. Record (B) was obtained approximately 10 min after caffeine had been applied. Record (C) was taken 5 min after that. The repetitive spiking began as an off-response of the HPs. When M TTX was applied, the repetitive spiking ceased but rhythmic HPs continued (D). The resting Em was about -50 mV. Calibrations: 40 mV, 2 sec.


cation of conditioning depolarization also reduced the amplitude of HPs, although frequency was increased. An example of the relationship between the amplitude of the H P and En, is shown in Figure 8.

Addition of 2 mM DBc-AMP in control saline induced rhythmic HPs in one neuron which did not show any demonstrable HPs in control saline alone. No effects were noted in any other neuron.

Effects of Ca2+-free saline and of caffeine in Ca2+-free saline. In neurons which did not show any detectable HPs in control saline large HPs were observed in Ca2+-free, Mg2+-substituted saline [e.g., Fig. 9(A) and Table I(B)]. Addition of 2.5 mM caffeine in Ca2+-free saline increased both amplitude and frequency

-47mV

- 7 2

-93 h -I-

Fig. 8. Reversal potential of HPs induced by 2.5 mM caffeine. Left hand numbers indicate Em levels which were held by injecting various intensities of inward or outward currents. Relation between the amplitude of HPs (ordinate) and various En, levels (abscissa) is shown in the graph. The HP observed a t -47 mV was induced as an off-response of injected outward current. The reversal potential of caffeine-induced HP approximated by linear interpolation of values between -57 and -93 mV is -79 mV. The resting E,,, was -54 mV. Calibrations: 50 mV, 2 sec.


D E

Fig. 9. Effects of Ca2+-free saline and of 2.5 mM caffeine in Ca2+-free saline on HPs. This preparation did not show spontaneous HPs in control saline. The resting Em = -43 mV. Appli- cation of Ca2+-free, Mg2+-substituted saline induced HPs as shown in (A). In (B)-(E), 2.5 mM caffeine was applied in Ca2+-free condition. Two minutes after caffeine application the amplitude of HP increased and became very rhythmic (B). Five minutes later, the repetitive spiking occurred as an off-resonse of each HP (C). Thereafter, the membrane depolarized gradually. Injection of short inward current pulses demonstrated a greater conductance at the peak of HP [(D) and (E)] than during the resting state. In (D) pulses were applied at 1 sec intervals as indicated by arrow signs. 40 mV, 2 sec calibration shown in A applies to records (B)-(D). Calibrations in (E) are 20 mV, 400 msec.

of HPs further [Fig. 9(B)]. Gradual decrease in E , following each HP and increase in HP frequency induced repetitive rhythmic spiking as shown in Figure 9(C). In 9(D) and 9(E) short pulses were injected during HPs to examine possible changes in membrane resistance. A t the peak of the H P the membrane resistance decreased from one half to two thirds of the resting value. The resistance recovered gradually, following the recovery time course of HP and then slightly increased immediately before the spike firing. Therefore, not only recovery from decreased resistance state (K-activation) but also K-inactivation might be oc- curring in some degree at this time. When Mg2+-substituted Ca2+-free saline was switched to Mn2+-substituted Ca2+-free saline, the amplitude of the HPs increased initially with gradual increase of their intervals, and then within a few minutes the HPs were completely suppressed.

As mentioned previously in 2.5 mM caffeine in control saline the peak Em of the S-HAP and those of the HPs were about the same (Fig. 7). In 2.5 mM caffeine-containing, Ca2+-free medium, the situation was different. The amplitude of the S-HAP was markedly reduced by Ca2+-free saline and this was not restored by the addition of 2.5 mM caffeine. The peak of the HPs induced by Ca2+-free caffeine was much more negative than that of the S-HAP, even at lower frequency of spike firing and at relatively negative resting Em level [e.g., Fig. 9(C)] This might indicate that caffeine increases intracellular Ca2+ activity, by releasing Ca2+ from an intracellular reservoir and that this increases the K-conductance of the membrane; however, the action potential itself cannot increase the Ca2+ activity by mediating an influx of Ca2+ since this ion is absent in the external

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medium, and consequently the late K-conductance cannot be activated even in the presence of caffeine. These phenomena, therefore, further suggest that the processes involved in Ca-mediated K-conductance increase by spike and oc- curring spontaneously are not the same.

Hyperpolarizing phase of the synaptic potential

General property of the synaptic potentials. Characteristics of the PSPs were analyzed in neurons which did not generate spikes in response to orthodromic stimulation. Both monosynaptic and multisynaptic (up to 3) contacts were observed, but the former type was the majority. The PSPs were monophasic depolarizing potentials in some meurons, but in others the D-hpase was followed by a small H-phase. Both types of PSPs were observed in those neurons which showed a low resting Em. However, when the resting Em was highly negative (e.g., -70 mV), biphasic PSPs were rarely observed. Application of repetitive preganglionic stimulation at 50 Hz caused summation in both types of PSP. In general, a slight facilitation was observed at the beginning of stimulation and then the depression followed for both D-phases of PSPs. The amplitude and duration of the H-phase were increased by repetitive preganglionic stimulation. Consequently the peak Em level of the D-phase of the PSP elicited during the H-phase of PSPs was reduced. Thus, the H-phase of PSPs behaved as an inhibitory postsynaptic potential [e.g., Fig. l l (Al)] . An example of a monophasic depolarizing PSP is shown in Figure lO(A1). The subthreshold amplitude of depolarizing synaptic potential obtained from twenty-one neurons showed the following characteristics: 13 f 5.6 mV in amplitude; rise time of 3.9 f 1.6 msec, and decay half-time of 11.8 f 4.6 msec. The reversal potential of +1.4 f 4.9 mV for D-phase of the PSP was obtained only from five neurons. The reversal potential estimated by an extrapolation technique in other neurons was -2.2 f 10.7 mV ( n = 12) [Fig. lO(Al)-lO(A4)]. An example of a single biphasic PSP is shown in Figure 12(A). The amplitude of the H-phase was 7.2 mV and the duration was 140 msec at -35 mV resting Em level. An increase of the postsynaptic conductance was obtained during the H-phase of the PSPs in such neurons [e.g., Fig. ll(Bl)-ll(B3)] and the average reversal potential was -77.7 f 8.4 mV (n = 6). However, in some neurons postsynaptic conductance changes were not clearly demonstrated during the H-phase of PSPs [e.g., Fig. ll(A1)- ll(A3)l.

Effects of caffeine, K+-free saline, and ouabain on PSP. Perfusion of the preparation with saline containing 2.5 mM caffeine induced an increase in both D- and H-phases of PSP in most neurons within a few minutes. The effect was more pronounced in the case of the H-phase [Fig. 10(B1)-10(B4) and lO(C1)-lO(C4)l. Caffeine, during the period of measurement, did not change the membrane resistance nor the resting Em. When the resting Em in control saline was very negative (e.g., -70 mV), caffeine did not induce an H-phase in the PSP. The reversal potential of the D-phase of the PSP was shifted a few millivolts in the positive direction by caffeine (Fig. 10, crosses on the uninterrupted line). However, this small potential shift may not be significant, as the


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Fig. 10. Effects of 2.5 mM caffeine on the synaptic potentials. All records were made on a single neuron. The synaptic potential of this neuron in the control saline was a monophasic depolarizing potential a t the resting Em of -40 mV. Amplitude of the depolarizing synaptic potential was increased by the application of conditioning postsynaptic hyperpolarization [(Al)-(A4)]. The relationship between Em (abscissa) and the amplitude of depolarizing PSP (positive ordinate) was linear (open circles in the graph) and the expected reversal potential for this PSP was -23 mV. Application of 2.5 mM caffeine increased the amplitude of the PSP, and the PSP developed a small H-phase (€31). The resting Em increased to -45 mV. Application of conditioning postsynaptic hyperpolarizations increased the amplitude of the D-phase of PSP [(B2)-(B4)], hut the amplitude of H-phase of PSP (negative ordinate) was reduced and became indistinguishable from the base line at -70 mV (cross, dotted line in the graph). The expected reversal potential for the D-phase of PSP did not change significantly from the control value (crosses, uninterrupted line in the graph). The summated PSPs in response to five presynaptic stimuli a t 20 msec intervals induced a 16 mV H-phase as shown in C1. Application of moderate postsynaptic hyperpolarization increased the H-phase slightly, but further hyperpolarization reduced the H-phase and then reversed the polarity at about -110 mV, as shown in the series of records ((C2)-(C4)]. This relationship is shown by triangles and the interrupted line in the graph. In (C4), the resting Em was displaced to about -120 mV. Calibrations: 40 mV, 40 msec [(Al)-(A4) and (Rl)-(B4)] and 40 mV, 200 msec [(Cl)-(C4)].

new value was still within the range of the control values. The amplitude of the H-phase of PSP decreased as the postsynaptic conditioning hyperpolarization was applied and it was nullified at about -72 mV (crosses on dotted line). However, when five consecutive preganglionic stimuli at 20 msec intervals were applied, the amplitude of the H-phase of five summated PSPs increased [compare Fig. lO(B1) and lO(Cl)]. This potential was, however, slightly enhanced


A1 A 2 A 3

81 8 2 83

Fig. 11. Postsynaptic membrane conductance change during the H-phase of the PSPs. Data obtained from two preparations [(Al)-(A3); (Bl)-(B3)] are shown. The resting Em of the first neuron [(Al)-(A3)] was -38 mV and it was pretreated with 10-3M hexamethonium for short periods in order to reduce synaptic transmission. Amplitude of the D-phase of a single PSP was 17 mV and that of the H-phase of the PSP (not shown) was 4 mV. Five repetitive preganglionic stimuli a t 50 Hz increased the H-phase of the PSPs to about 10 mV (Al). Small hyperpolarizing current pulses (4 msec in duration a t 50 Hz) were injected postsynaptically during orthodromic response in order to examine the membrane conductance change (A2) and compared with that obtained in the non- stimulated condition (A3). The second neuron [(Bl)-(B3)] was not treated with hexamethonium. The resting Em was -46 mV. Five repetitive preganglionic stimuli as above increased the H-phase of the PSPs to about 11 mV (Bl). A small conductance increase was noticed during the H-phase of PSPs in this neuron (B2) more clearly than the previous neuron (A2). Calibration bars: 10 mV, 200 msec for (Al)-(A3) and 20 mV, 200 msec for (Bl)-(B3). For further explanation see text.

[Fig. 1O(C2)] or unchanged by the application of moderate postsynaptic hyperpolarization. Application of further hyperpolarization reduced the amplitude of the H-phase and it was then reversed at a more negative Em level than the estimated value of E K (see triangles on the interrupted line). In this particular neuron t,he apparent reversal potential for the H-phase of PSPs was -110 mV. Such large discrepancy between the E K and the reversal potential of the H-phase of summated PSPs was observed at least in several neurons. This problem will be discussed later.

As described previously, perfusion with K+-free saline induced several different effects on the Em and S-HAP. In some neurons the amplitude of the H-phase of PSPs increased but in some neurons there were no clear changes. In the example shown in Figure 12, the resting Em did not change on introduction of K+-free saline. The amplitude of the D-phase of PSPs increased to some extent [compare Fig. 12(C) and 12(D)]. The reversal potential of the D-phase of PSPs shifted from -21 mV to -15 mV; but that of the H-phase, which was -68 mV in control did not change (compare with the relationship between filled circles and crosses of the hyperpolarizing potential side of the graph).

Effects of caffeine on PSPs were also examined in K+-free saline. Addition of 2.5 mM caffeine increased both D- and H-phases of PSPs [Fig. 12(E)]. The reversal potential of the D-phase shifted about 5 mV in the positive direction and that of the H-phase shifted to -78 mV. Caffeine effects on PSPs were reversible. When the caffeine treated preparation in K+-free saline was reperfused with control saline, the resting Em decreased slightly and the postsynaptic response recovered to the control value [Fig. 12(F)].

Effects of 10-5M ouabain on the H-phase of PSPs were examined on three


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Effects of DBc-AMP. Figure 13 shows the effects of DBc-AMP on PSPs.. Application of 1 aM DBc-AMP caused a small increase in both D- and H-phases of PSPs [compare Fig. 13(A) and 13(B); Table II(Cl)]. Amplitudes of both D- and H-phases of the PSPs were further increased by the application of 2 mM DBc-AMP for 20 min; the effect was most pronounced in the case of the H-phase

-mV

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and in 2.5 mM caffeine-containing, K+-free saline. The resting Em was -35 mV throughout the experiment. (A) A single biphasic PSP in control saline. (B) The H-phase was augmented by five consecutive PSPs (20 msec intervals) in control saline. (C)-(F) Low gain recordings of consecutive PSPs in control saline (C), in K+-free saline (D), in 2.5 mM caffeine-containing, K+-saline (E), and the recovered response after washout with control saline (F). Calibrations: 20 mV, 80 msec for (A); 20 mV, 200 msec for (B); 40 mV, 200 msec for (C)-(F), Graph shows the relationship among Em (abscissa), D-phase (positive ordinate of the graph), and H-phase (negative ordinate of the graph) of PSPs when the postsynaptic membrane potential was displaced by injecting various intensities of inward current. The relationships shown by filled circles ( - - - - -), crosses (- - - - -), and triangles (---) were obtained in control, in K+-free, and 2.5 m M caffeine in K+-free salines respectively. Each line was drawn by linear regression analysis, except that one triangle at the extreme left for the D-phase of PSP was excluded from the calculation.


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Effects of dibutyryl cyclic AMP on synaptic potential. Five presynaptic stimuli were applied at 20 msec intervals except in (D). (A) Response in control saline. (B) Response obtained 10 min later in 1 mM DBc-AMP in saline. (C) Response obtained 20 min later in 2 mM DBc-AMP in saline. (D) Response obtained 20 min after the drug was washed out with control saline. In (D), the sixth presynaptic stimulus was applied during hyperpolarizing phase of previous PSPs. Cali- bration: 20 mV, 200 msec. The graph shows the changes in amplitude of H-phase of PSPs (ordinate) as the postsynaptic membrane was hyperpolarized (abscissa) by injected inward current. Filled circles were obtained in the control saline. Crosses were obtained in 2 mM DBc-AMP containing saline. The dotted line is the regression line for crosses and extrapolated intercept of the line with Em was at -78 mV.

Fig. 13.

[Fig. 13(C); Table II(C)]. However, the reversal potential of the H-phase of the PSPs was approximately at the -78 mV Em level, which was about the same as the average value obtained in the control saline (crosses in the graph). Effects of DBc-AMP were not easily reversible. Even 20 min after the drug had been washed out with control saline, and enhanced synaptic response persisted [Fig. 13(D) and also see Table II(Cl)]. In this particular neuron, the amplitude of H-phase induced by five consecutive PSPs was about 8 mV at the resting Em of -51 mV. Changes in membrane conductance were not clearly observed. In Figure 13(D), the sixth orthodromic stimulus was applied during the H-phase of PSPs. It was observed that the D-phase of the sixth PSP was the largest at the peak of the conditioning H-phase. Some effects of caffeine, K+-free and DBc-AMP on the D- and H-phases of PSPs were summarized in Table 11, which should be read, however, with caution, because both parameters are Em -dependent.

DISCUSSION

The resting Em of the submandibular ganglion cells of the hamster ranged between -40 and -70 mV under both i n vivo and i n vitro conditions. The membranes of those cells with highly negative Em values (e.g., -70 mV) behaved as K-electrodes, while the membranes of intermediate Em cells (e.g., -40 mV) did not behave as K-electrodes. The estimated EK from E , vs [K+]o-relations obtained from both highly negative and less negative Em cells was about -84 mV. The low resting Em values appeared not to be due to injury caused by electrode penetration, since such low Em cells showed a relatively large amplitude of spike overshoot, S-HAP, and spontaneous or drug induced HPs. It was also

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observed that Em of such cells exhibited increased negativity on the application of caffeine and Ca-ionophore. These observations suggest that low resting Em in such cells are due to some degree of K-inactivation, rather than other factors, such as lower [K+];, relatively high Na-permeability a t the resting state, or decreased Na-pump activity.

The reversal potentials of the HAPS induced by both directly and antidromically evoked spikes and those of the D-HAP were about the same, being about -75 to -85 mV Em level. Caffeine induced HPs and the H-phase of the PSPs observed in some neurons also showed a similar reversal potential. Analysis of the ionic mechanism involved in these reversal potentials would suggest that K + is critically involved. The ionic channels which are involved in the present K+ system are different in several respects from the well-known depolarizing K- activation system, which introduces a delayed rectification (Barrett and Barrett, 1976). For example, TEA suppresses the latter system but enhances the former system. The present system is enhanced by increasing [Ca2+]o or by Ca ionophore (A23187), while, lowering the [Ca2+]o or application of Mn2+, reduces it. The delayed rectification system on the other hand is not affected by these ma- nipulations. The kinetics of the activation time course of this system is much slower than the latter (Connor and Stevens, 1971; Neher and Lux, 1972). Various means that activate Ca-conductance of the neuronal membrane or increase [Ca’+]i would appear to trigger an increase of the late K-conductance of the membrane. Such Ca-mediated K-conductance increases were first recognized in the red cell membrane (Gardos, 1958; Whittam, 1968; Lew, 1970) and then in several excitable cells: Alplysia neuron (Meech, 19721, leech neuron (Jansen and Nicholls, 19731, electroreceptors of skate (Clusin e t al., 1975), frog sympathetic neuron (Minota, 1974; Busis and Weight, 1976; Kuba and Nishi, 1976), frog spinal motoneuron (Barrett and Barrett, 19761, cat myenteric plexus neuron (North, 1973), cat spinal motoneuron (Krnjevic and Lisiewicz, 1972; Krnjevic e t al., 1975), rat superior cervical ganglion (Yarowsky and McAfee, 1977), and cardiac Purkinje fibers of the sheep (Isenberg, 1975). How increased [Ca2+]i suhsequent,ly increases the K-conductance of the cell memhraiie is still un- clear.

Ca-activation may occur during various depolarizing electrogeneses. First. of all, it will occur during the action potential. No systematic study of the effect of [Ca‘+lo on the amplitude of the action potential was made, but it was reduced in low Ca”‘-media, and increased slightly in higher Ca”-media. Moreover, the requirement of a relatively high concentration (10+jM) of T T X to abolish the action potential and the reduction of the spike amplitude by Mn’+, both suggest that Ca“+, as well as Na+, enters the cell during the action potential, as in the case in amphibian (Koketsu and Nishi, 1969) and mammalian sympathetic neurons (Yarowsky and McAfee, 1977). There is also supporting evidence in other preparations that the postsynaptic membrane Ca-conductance increases during depolarizing synaptic electrogenesis (Takeuchi, 1963; Kusano et al., 1975; Kusano and Janiszewski, 1976). I t is also well-known that application of depolarizing pulses alone triggers an increase in Ca-conductance in some nerve membranes (Raker e t al., 1971). In skeletal muscle fibers, depolarizing pulses


are known to release internal stores of Ca2+ (Winegrad, 1968). In this connection, the development of low-frequency HPs in Ca2+-free saline found in this study invites a comment. Kuba and Nishi (1976) did not observe such responses in frog sympathetic neurons under similar experimental conditions. It is generally observed that when [Ca2+]o is reduced [Ca2+], decreases (Baker et al., 1971; Kusano et al., 1975). However, a sporadic increase in local [Ca2+], during perfusion with Ca2+-free saline may possibly occur due to release of Ca2+ from Ca- reservoirs in the cell. Evidence which supports such a suggestion has been observed in the salivary gland cells of an insect (Rose and Lowenstein, 1976), employing the aequorin technique. Suppression of HPs in Ca2+-free saline by extracellularly applied Mn2+ suggests that this Ca-reservoir is accessible from outside the cell.

Caffeine is known to mobilize intracellular Ca2+ (Weber, 1968). It is also known to potentiate the metabolism-stimulating action of c-AMP in many tissues by inhibiting phosphodiesterases (Robinson et al., 1971). Twitch potentiation in muscle fibers by caffeine (Weber, 1968) and augmentation of PSPs by caffeine (Elmqvist and Feldman, 1965; Hofmann, 1969) have been assumed to be due to Ca-activation by caffeine. The caffeine induced rhythmic HPs are, therefore, assumed to occur through a Ca-mediated K-conductance increase (Kuba and Nishi, 1976).

Although a large amount of data on the relationship between c-AMP and [Ca2+], has been accumulated from various points of interest using a variety of tissues, observations have been to some extent inconsistent. A number o i publications suggest that c-AMP increases [Ca2+], or Ca-conductance of cell membranes (Goldberg and Singer, 1969; Greengard and Kebabian, 1974; Krnjevic and Van Meter, 1976; Reuter, 1974). There is a report which suggests c-AMP decreases [Ca2+], in some cells (e.g., Lichtenstein and Margolis, 1968). There are also some reports which suggest that c-AMP does not stimulate Ca2+ release from mitochondria in contradiction to earlier observations (e.g., Scarpa et al., 1976). In the present preparation, we observed enhancement of S-HAP and both phases of PSP by c-AMP. In one neuron, c-AMP induced HPs but not in any others. Whether c-AMP increases [Ca2+], by releasing Ca2+ from an accessible calcium reservoir (e.g., endoplasmic reticulum and mitochondria) has to be de- termined by more direct methods. It would be interesting to investigate how the time course and the frequency of HPs correlate with changes in [Ca2+], and [ C-AMP], .

The first intracellular recording of biphasic synaptic potentials was obtained from amphibian sympathetic neurons (Tosaka and Libet, 1965). It was then found in molluscan neurons (Kehoe, 1967; Wachtel and Kandel, 1967) and amphibian parasympathetic neurons (Dennis et al., 1971). Cultured rat spinal-cord neurons are also known to respond to ionophoretically applied glutamate with biphasic potentials (Ranson et al., 1975). The observation of biphasic synaptic potential in mammalian parasympathetic neurons reported here adds another example. The mechanism involved in the hyperpolarizing phase of PSP varies among preparations. A prolonged or delayed K-activation has been suggested as the underlying mechanism for the H-phase of PSP in the frog cardiac para-


sympathetic neurons (Dennis et al., 1971). On the other hand several mechanisms haJe been suggested for the slow inhibitory postsynaptic potential (slow-IPSP) in the vertebrate sympathetic neurons such as Na-inactivation (Weight and Padjen, 1973), activation of electrogenic Na-pump (Nishi and Koketsu, 1967; Volle, 1969) and additional transmitter action due to the presence of interneurons (Eccles and Libet, 1961; Tosaka et al., 1968). Although we have not yet completed a detailed analysis of the mechanism involved in this potential, an unequivocal increase in membrane conductance during the H-phase of the PSP was not observed in all neurons examined. In some neurons no clear change in postsynaptic conductance was observed. However, even in such neurons the amplitude of the H-phase of the PSP was reduced by the application of postsynaptic hyperpolarization and it appeared to be nullified at an Em level of around -90 to -110 mV. This Em level was much more negative than the estimated EK. Unfortunately, we have not yet succeeded in comparing the reversal potential of the S-HAP with that of the H-phase of PSPs in the same neuron. We found, however, that the S-HAP reversal potential was also very negative in several neurons [e.g., Fig. 2(D1)-2(D3)]. Although there was some uncertainty about the location of the H-phase generation and the precise amount of the hyperpolarizing effect with the intracellular electrode, the discrepancy between the E K and the reversal potential of the H-phase of PSPs resembles the problem encountered in the slow IPSPs of both amphibian and mammalian sympathetic ganglia (Nishi and Koketsu, 1968; Kobayashi and Libet, 1968). In the present experiment, [K+]o was lowered to inhibit the Na+-K+ exchange pump (Livengood and Kusano, 1972). The facts that the H-phase of the PSP was not depressed by ouabain treatment, that it was rather slightly enhanced in K+-free saline, and that the H-phase has a reversal potential would suggest that the transmitter activated Na-pump mechanism is insufficient as an explanation for this phe- nomenon, as for slow IPSP in the bullfrog sympathetic ganglion (Smith and Weight, 1977). Experiments for evaluating a contribution of transmitter induced Na-conductance decrease was difficult to perform under present experimental conditions. However, if the relative K+ permeability is increased by the decrease in Na+ permeability during the H-phase of PSPs and the S-HAP, the reversal potential of both H-phase of PSPs and that of the S-HAP may not be different from the E K and the change in membrane conductance may be small. In fact we observed no clear resistance change during the H-phase of PSPs in some neurons. It is conceivable that in addition to Ca-mediated K-conductance increase, some degree of Na-inactivation have occurred in such case. Morpho- logical correlation between pre- and postganglionic fibers in the hamster submandibular ganglion is also not well-understood and a possible interneuronal activity, that might release unidentified transmitter and consequently induce the H-phase of the PSP in the postganglionic neurons, cannot be ruled out. The enhancement of the amplitude of PSPs by extracellularly applied caffeine, DBc-AMP and Ca-ionophore, may be due to both pre- and postsynaptic effects. The presented data does not clarify this problem; however, we have recently observed that an intracellular injection of Ca2+ into the postsynaptic cells induced an increase in the H-phase amplitude of PSPs and a slow transient membrane hyperpolarization (unpublished).


Although our pharmacological studies on this preparation have been very preliminary, we have observed that bath applied carbamylcholine and 1,l dimethyl-4-phenylpiperazinium in the concentration range between 5 X 10-4M and 5 X 10-6M induced long-lasting biphasic membrane potential changes. This response consisted of a depolarizing phase followed by a hyperpolarizing phase. A clear change in membrane conductance during drug induced hyperpolarizing potentials was not observed. It appears that the hyperpolarizing potentials produced in the hamster submandibular ganglion are manifested by more than one mechanism, and require further analysis.

Whatever the underlying mechanisms for these hyperpolarizing potentials are, the potentials undoubtedly contribute to the reduction of the frequency of the repetitive spike firing of the neuron. Hyperpolarization might occur trans-synaptically or in the postganglion neuron per se. It has been observed that neurons of the rat submandibular ganglion respond with low frequency repetitive spiking (max 13 Hz) to taste stimuli applied on the tongue (Suzuki, unpublished); and the secretory activity of salivary glands of cat reaches a maximum when the preganglionic nerve is stimulated at 10-20 Hz (Emmelin, 1967). It appears important to maintain repetitive spiking at this low frequency in the parasympathetic nerve to maintain salivary secretion. The role of th.e Ca-mediated K-conductance increase in modulating the low-frequency repetitive spiking has also been implicated in several other preparations (Connor and Stevens, 1971; Barrett and Barrett, 1976).

This work was supported in part by USPHS Grant (NS-12275). We are indebted to Dr. D. C. Koblick and Dr. F. F. Weight for helpful discussion and criticism.

REFERENCES

BAKER, P. F., HODGKIN, A. L., and RIDGWAY, E. B. (1971). Depolarization and calcium entry in squid giant axons. J. Physiol. (London) 218: 709-755.

BARRETT, E. F. and BARRETT, J. N. (1976). Separation of two voltage-sensitive potassium currents, and demonstration of a tetrodotoxin-resistant calcium current in frog motoneurones. J. Physiol. (London) 255: 737-774.

BUTCHER, R. W. and SUTHERLAND, E. W. (1962). Adenosine 3’,5’-phosphate in biological materials. J . Riol. Chem. 237: 1244-1250.

BUSIS, N. A. and WEIGHT, F. F. (1976). Spike after-hyperpolarization of a sympathetic neurone is calcium sensitive and is potentiated by theophylline. Nature 263: 434-436.

CLUSIN, W., SPRAY, D. C., and BENNETT, M. V. L. (1975). Activation of a voltage-insensitive conductance by inward calcium current. Nature 256: 425-427.

CONNOR, J. A. and STEVENS, C. F. (1971). Prediction of repetitive firing behavior from voltage clamp data on an isolated neurone soma. J. Physiol. (London) 213: 31-53.

DENNIS, M. J., HARRIS, A. J., and KUFFLER, S. W. (1971). Synaptic transmission and its duplication by focally applied acetylcholine in parasympathetic neurons in the heart of the frog. Proc. R. SOC. London, Ser. B. 177: 509-539.

ECCLES, R. M. and LIBET, B. (1961). Origin and blockade of the synaptic responses ofcurarized sympathetic ganglia. J. Physiol. (London) 157: 484-503.

ELMQUIST, D. and FELDMAN, D. S. (1965). Calcium dependance of spontaneous acetylcholine release a t mammalian motor nerve terminals. J. Physiol. (London) 181: 487-497.

EMMELIN, N. (1967). Nervous control of salivary glands. Handb. Physiol. 2 (6), 595-632. GARDAS, G. (1958). The function of calcium in the potassium permeability of human erythrocytes.

GARRETT, J. R. (1966). The innervation of salivary glands. J. R. Microsc. Soc. 85: 149-162. Biochem. Biophys. Acta 3 0 653-654.

HPs IN PARASYMPATHETIC NEUKONS 3 Y l

GOLDBERG, A. L. and SINGER, J. J. (1969). Evidence for a role of cyclic AMP in neuromuscular transmiss,ion. Proc. Natl. Acad. Sci. 64: 134-141.

GREENGAR~D, P. and KEBABIAN, J. W. (1974). Role of cyclic AMP in synaptic transmission in the mammalian peripheral nervous system. Fed. Proc. 33: 1059-1067.

HOFMANN, W. W. (1969). Caffeine effects on transmitter depletion and mobilization at motor nerve terminals. Am. J . Physiol. 216: 621-629.

ISENBERG, G. (1975). Is potassium conductance of cardiac Purkinje fibers controlled by [Ca],? Nature :!53: 273-274.

JANSEN, J. K. S. and NICHOLLS, J. G. (1973). Conductance changes, an electrogenic pump and the hyperpolarization of leech neurones following impulses. J. Physiol. (London) 2 2 9 635-655.

KEHOE, J. (1 967). Pharmacological characteristics and ionic basis of two component post-synaptic inhibition. Nature 215: 1503-1505.

KOBAYASHI, H. and LIBET, B. (1968). Generation of slow post-synaptic potentials without increase in ionic conductance. Proc. Natl. Acad. Sci. 6 0 1304-1311.

KOKETSU, K. and NISHI, S. (1969). Calcium and action potentials of bullfrog sympathetic ganglion cells. J. Gen. Physiol. 5 3 608-623.

KRNJEVIC, K. and LISIEWICZ, A. (1972). Injection of calcium ions into spinal motoneurones. J. Physiol'. (London) 225: 363-390.

KRNJEVIC, K., PUIL, E., and WERMAN, R. (1975). Evidence for Ca2+-activated K+ conductance in cat spinal motoneurons from intracellular EGTA injection. Can. J . Physiol. 53: 1214-1218.

KRNJEVIC, K. and VAN METER, W. G. (1976). Cyclic nucleotides in spinal cells. Can. J . Physiol. Pharmacol. 54: 416-421.

KUBA, K.. and NISHI, S. (1976). Rhythmic hyperpolarizations and depolarization of sympathetic ganglion cells induced by caffeine. J . Neurophysiol. 3 9 547-563.

KUSANCI, K. and JANISZEWSKI, L. (1976). Neuromuscular transmission in mealworm larvae (Tenebrio molitor). In: Electrobiology of Nerue, Synapse, and Muscle, J. P. Reuben, D. P. Purpura, M. V. L. Bennett, and E. R. Kandel, Eds., Raven Press, New York, pp. 93-103.

KUSANO, K., MILEDI, R., and SITRNAKRE, J. (1975). Post-synaptic entry of calcium induced by transmitter action. Proc. R. Soc. London, Ser. B 189: 49-56.

LANGLIEY, J. N. (1890). In the physiology of the salivary secretion. Part VI. Chiefly upon the connections of peripheral nerve cells with the nerve fibers which run to the sublingual and sub- maxillary glands. J . Physiol. (London) 11: 123-158.

LEW, V. Lo. (1970). Effect of intracellular calcium on the potassium permeability of human red cells. J. Physiol. (London) 206: 35-36P.

LIBET, B. and TOSAKA, T. (1969). Slow inhibitory and excitatory postsynaptic responses in single cells of mammalian sympathetic ganglia. J . Neurophysiol. 32: 43-50.

LICHTENSTEIN, L. M. and MARGOLIS, S. (1968). Histamine release in vitro: inhibition by cate- cholamines and methylxanthines. Science 161: 902-903.

LICHTMAN, J. W. (1977). The reorganization of synaptic connections in the rat submandibular ganglion during post-natal development. J. Physiol. (London) 273: 155-177.

LIVENGOOD, D. R. and KUSANO, K. (1972). Evidence for an electrogenic sodium pump in follower cells of the lobster cardiac ganglion. J . Neurophysiol. 35: 170-186.

LUNDRERG, A. (1958). Electrophysiology of salivary glands. Phyisol. Reu. 38: 21-40. MEECH, R. W. (1972). Intracellular calcium injection causes increased potassium conductance in

Aplysia nerve cells. Conp . Biochem. Physiol. 4 2 A 493-499. MINOTA, S. (1974). Calcium ions and the posttetanic hyperpolarization of bullfrog sympathetic

ganglion cells. Jpn. J . Physiol. 24: 501-512. NEHER, E. and LUX, H. D. (1972). Differential actions of TEA on two K+ current components of

a molluscan neurone. Pflugers Arch. Ges. Physiol. 3 3 6 87-100. NISHI, S. and KOKETSU, K. (1967). Origin of ganglionic inhibitory postsynaptic potential. Life

Scz. 6: 2049-2055. NISHI, S. and KOKETSU, K. (1968). Analysis of slow inhibitory postsynaptic potential of bullfrog

sympathetic ganglion. J. Neurophysiol. 31: 717-728. NoRrfr, R. A. (1973). The calcium-dependent slow after-hyperpolarization in myenteric plexus

neurons with tetrodotoxin-resistant action potentials. Br. J . Pharmacol. 49: 709-711.

SUZUKI AND KUSANO

RANSON, B. R., BAKER, J. L., and NELSON, P. G. (1975). Two mechanisms for poststimulus hyperpolarizations in cultured mammalian neurons. Nature 256: 424-425.

REUTER, H. (1974). Localization of beta adrenergic receptors, and effects of noradrenaline and cyclic nucleotides on action potentials, ionic currents and tension in mammalian cardiac muscle. J. Physiol. (London) 242: 429-451.

ROBINSON, G. A., BUTCHER, R. W., and SUTHERLAND, E. W. (1971). Cyclic AMP, Academic Press, New York.

ROSE, B. and LOEWENSTEIN, W. R. (1976). Permeability of a cell junction and the local cytoplasmic free ionized calcium concentrations: A study with aequorin. J. Membr. Biol. 28: 87-119.

SCARPA, A., MALMSTORM, K., CHIESI, M., and CARAFOLI, E. (1976). On the problem of the release of mitochondria1 calcium by Cyclic AMP. J . Membr. B i d . 29: 205-207.

SMITH, P. A. and WEIGHT, F. F. (1977). Role of electrogenic sodium pump in slow synaptic inhibition is reevaluated. Nature 267: 68-70.

SUZUKI, T. and KUSANO, K. (1977). Hyperpolarizing potential induced by Ca-mediated K-conductance increase in hamster parasympathetic neurons. Soc. Neurosci., Abstr. 3: 95.

SUZUKI, T. and SAKADA, S. (1972). Synaptic transmission in the submandibular ganglion of the rat. Bull. Tokyo Dental College 13: 145-164.

TAKEUCHI, N. (1963). Effects of calcium on the conductance change of the end-plate membrane during the action of transmitter. J . Physiol. (London) 45: 243-265.

TOSAKA, T., CHICHIBU, S., and LIBET, B. (1968). Intracellular analysis of slow inhibitory and excitatory postsynaptic potentials in sympathetic ganglia of the frog. J. Neurophysiol. 31: 396-409.

TOSAKA, T. and LIBET, B. (1965). Slow postsynaptic potentials recorded intracellularly in sympathetic ganglia of frog. Int . Congr. Physiol. Sci., 23rd, Tokyo, Abstr., p. 386.

VOLLE, R. L. (1969). Ganglionic transmission. Anna. Reu. Pharm. 9: 135-146. WACHTEL, H. and KANDEL, E. R. (1967). A direct synaptic connection mediating both excitation

and inhibition. Science 158: 1206-1207. WEBER, A. (1969). The mechanism of the action of caffeine on sarcoplasmic reticulum. J . Gen.

Physiol. 5 2 760-722. WEIGHT, F. F. and PADJEN, A. (1973). Slow synaptic inhibition: evidence for synaptic inactivation

of sodium conductance in sympathetic ganglion cells. Brain Res. 5 5 219-224. WHITTAM, R. (1968). Control of membrane permeability to potassium in red blood cells. Nature

219: 610. WINEGRAD, S. (1968). Intracellular calcium movements of frog skeletal muscle during recovery

from tetanus. J . Gen. Physiol. 51: 65-83. YAROWSKY, P. and MCAFEE, D. A. (1977). Calcium dependent potentials in the rat superior cervical

ganglion. SOC. Neurosci. Abstr., 3 25.

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Hyperpolarizing potentials induced by Ca-mediated K-conductance increase in hamster submandibular ganglion cells