Proc. Natl. Acad. Sci. USA Vol. 75, No. 8, pp. 4029-4032, August 1978 Neurobiology Involvement of an interneuron in the generation of the slow inhibitory postsynaptic potential in mammalian sympathetic ganglia (disynaptic event/catecholamine hyperpolarization/small intensely fluorescent cells) N. J. DUN AND A. G. KARCZMAR Department of Pharmacology, Loyola University, Stritch School of Medicine, Maywood, Illinois 60153 Communicated by George B. Koelle, May 19,1978 ABSTRACT Acetylchoine (AcCho) was applied electro- causes a hyperpolarizing response resembling the slow ipsp and phoretically to cells of isolated rabbit superior cervical ganglia, that this response is disynaptic in nature. and the response was recorded by means of intracellular re- cording techniques. In the presence of d-tubocurarine (5 M&M), MATERIALS AND METHODS AcCho applied by tetanic current pulses elicited three distinct membrane potential changes: a slow depolarization, a slow Young white rabbits of either sex, weighing 1.5-2.0 kg, were hyperpolarization, and a biphasic response consisting of an used throughout this study. The superior cervical ganglia were initial hyperpolarization followed by a depolarization. Atropine rapidly excised from the rabbits after they were killed by air (I gM) abolished all the membrane potential changes elicited by AcCho. On the other hand, superfusion with a low-Ca/ embolism. Each ganglion was transferred to the recording high-Mg solution, tetrodotoxin (0.1 &M), or haloperidol (0.1 MM) chamber and superfused continuously with Krebs solution (8). selectively and reversibly blocked AcCho-induced hyperpolar- The intracellular recording and iontophoretic techniques used ization without appreciably affecting the depolarization. The have been described (8). Glass microelectrodes filled with 3 M membrane resistance remained relatively constant during the KC1 (tip resistance, 30-50 MO) were used for intracellular re- course of hyperpolarization. Application of steady depolarizing cording and stimulation. The micropipettes used for ionto- and hyperpolarizing currents decreased and increased, re- spectively, the amplitude of hyperpolarization. These results phoretic application of AcChoCl (2 M) had tip resistances of demonstrate that the hyperpolarization elicited by AcCho is about 75-100 MQ; braking currents of about 3-5 nA were used. electrophysiologically and pharmacologically similar to the slow In experiments concerning the effects of tetrodotoxin upon the inhibitory postsynaptic potential induced by nerve stimulation; response to antidromic stimulation, the postginglionic fibers furthermore, they support the view that the slow inhibitory were drawn into a small capillary; repetitive stimuli at 0.3 Hz postsynaptic potential elicited by presynaptic stimulation is a were applied via a pair of platinum electrodes. disynaptic phenomenon involving the release of a second transmitter, possibly dopamine, from an interneuron. RESULTS In the autonomic ganglia, the stimulation of preganglionic Characteristics of Various Potentials Induced by AcCho. nerve elicits, in addition to the familiar fast excitatory postsy- After a ganglion cell was satisfactorily impaled by a micro- naptic potential, slow excitatory and inhibitory postsynaptic electrode, the AcCho-filled .pipette was manipulated into close potentials (1-3). The genesis of the slow excitatory postsynaptic proximity to the ganglion cell. Under these circumstances, potential (slow epsp) is reasonably certain; most likely it can be AcCho discharged by a brief current pulse (<10 msec) caused accounted for in terms of direct activation of muscarinic re- a fast membrane depolarization with a rise time of <30 msec ceptors located on the principal ganglion cells by presynapti- (8). Superfusion of the ganglion with Krebs solution containing cally released acetylcholine (AcCho) (1-3). The generation of d-tubocurarine (5 AM) completely and reversibly blocked the the slow inhibitory postsynaptic potential (slow ipsp), on the fast AcCho potential in <5 min (9). The resting membrane other hand, remains to be established; it may involve either a potential and other membrane electrical properties were not direct muscarinic action (ref. 4; however, see ref. 5) or a cate- significantly affected by d-tubocurarine (10); the average cholamine released from an adrenergic interneuron (1, 6). In resting potential was found to be about -55 mV. The results the superior cervical ganglion of the rabbit, small intensely described hereafter were obtained in the presence of 5 AM fluorescent (SIF) cells with distinct catecholamine fluorescence d-tubocurarine. are located among the principal ganglion cells (7). These small When applied by tetanic current pulses (10-30 Hz, 10 msec neurons are innervated by the cholinergic preganglionic fibers per pulse for 1-2 sec), AcCho elicited three different types of and, in turn, make synaptic contacts with principal ganglion membrane potential change, depending on the neuron. Of the cells (7). Thus, the SIF cells appear morphologically to be likely 94 cells that exhibited satisfactory resting and action potentials, candidates for the status of an adrenergic interneuron, situated the majority of cells, 48, showed slow membrane depolarization in the synaptic pathway and mediating the slow ipsp. Recently, in response to AcCho iontophoresis. The amplitude of the de- we reported (8) that, when dopamine is applied iontophoreti- polarization ranged from 1.2 to 6.8 mV (mean + SD, 3.7 + 1.7 cally to rabbit sympathetic neurons, it elicits a membrane hy- mV) and total duration ranged from 12 to 33 sec (mean, 28 sec). perpolarization that exhibits electrophysiological features Twenty-one cells exhibited a response of another type, con- similar to those of the slow ipsp. To support the hypothesis that sisting of a slow membrane hyperpolarization. The amplitude an adrenergic interneuron mediates the slow ipsp, it should be of hyperpolarization varied from 1.5 to 7.4 mV (mean, 4.9 mV), shown that AcCho applied to the vicinity of the ganglion cells and the duration ranged from 7.2 to 24 sec (mean, 14.4 sec). Both the depolarizing and hyperpolarizing responses showed The costs of publication of this article were defrayed in part by the payment of page charges This article must therefore be hereby marked Abbreviations: AcCho, acetylcholine; SIF, small intensely fluorescent "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate cells; slow ipsp, slow inhibitory postsynaptic potential; slow epsp, slow this fact. excitatory postsynaptic potential. 4029 Downloaded by guest on September 26, 2021 4030 Neurobiology: Dun and Karczmar Proc. Natl. Acad. Sci. USA 75 (1978) A D present study, the effect of haloperidol (0.1 MM) was examined on eight cells that exhibited either hyperpolarizing or biphasic response to AcCho. Haloperidol depolarized the cell membrane by about 2-3 mV and rapidly blocked the AcCho-induced E hyperpolarization (in <2 min). In the case illustrated in Fig. 1, H AcCho elicited a biphasic response; haloperidol selectively blocked the hyperpolarization and affected the depolarizing response only slightly (Fig. 1 B and C). The selective inhibitory C- F I_ action of haloperidol on the hyperpolarizing response was also demonstrated in experiments on cells that exhibited only de- polarizing response to AcCho. As illustrated in Fig. 2B, halop- eridol (0.1 uM) did not appreciably attenuate the depolarization FIG. 1. Effects of superfusing the sympathetic ganglion cell with after 5 min of superfusion. haloperidol, atropine, or low-Ca/high-Mg solution on AcCho hyper- Effects of Low-Ca/High-Mg Solution. The resting mem- polarization and depolarization. The upper tracing ofeach pair rep- brane potential was slightly altered in a low-Ca (0.25 mM)/ resents the current pulses (10 Hz, 10 msec per pulse for 2 sec) used <5 mV. to elicit the AcCho responses. The ganglion was continuously super- high-Mg (12 mM) solution, the change being always fused with Krebs solution containing 5MM d-tubocurarine. (A, D, and Superfusing the ganglion with the low-Ca/high-Mg solution G) Control responses; (B and C) 1 and 2 min after superfusion with rapidly diminished the amplitude of AcCho hyperpolarization haloperidol (0.1 MM), respectively; (E) 4 min after low-Ca (0.25 and abolished it in <5 min (Fig. 1E); this effect was consistently InM)/high-Mg (12 mM) superfusion; (H) 2 min after atropine (1 MAM); observed in all 14 cells examined. Whenever necessary, the (F and I) 10 min after wash with Krebs solution. Records A-F and change in the resting membrane potential due to thesuperfu- G-I were taken from two different ganglion cells. Vertical calibration sion of low-Ca/high-Mg solution was rectified by passing an mark, 10 mV and 1 nA; horizontal calibration mark, 4 sec for records appropriate steady current through the recording microelec- A-F, and 2 sec for records G-I. trode; this maneuver did not unmask AcCho hyperpolarization. a delay of about 100-400 msec from the start of the current The hyperpolarizing response to AcCho returned after the pulses to the initiation of responses. Still another type of response low-Ca/high-Mg solution was replaced with Krebs solution was 9bserved in 16 cells; this response consisted of biphasic (Fig. iF). This clear-cut effect of the low-Ca/high-Mg solution membrane potential change, initial hyperpolarization being on the hyperpolarizing response to AcCho differed dramatically followed by depolarization (Fig. 1A). The amplitude of the from its lack of action on AcCho depolarization. Indeed, the hyperpolarization was invariably larger than that of the de- depolarizing potential was not significantly affected by the polarization; quite often, the latter was barely distinguishable Ca-deficient solution in 10 cells tested (Figs. LE and 2C). from the electrical noise. Finally, no detectable change in Actions of Tetrodotoxin. These experiments were carried membrane potential was noticed in nine cells. out to determine whether the conduction block due to tetro- Effects of Blocking Agents. Superfusing the ganglion with dotoxin would affect AcCho hyperpolarization; antidromically a low concentration of atropine (1 WM) rapidly abolished both evoked action potentials were used to monitor the action of the slow hyperpolarization (Fig. 1H) and the slow depolariza- tetrodotoxin. Tetrodotoxin (0.1 MM) did not noticeably affect tion (Fig. 2D); the resting membrane potential was not affected. the resting membrane potential but it blocked completely the There was no apparent difference in the susceptibility of these antidromic spikes (Fig. 3F). At the same time, tetrodotoxin first two responses to atropine; both were blocked in less than 3 min. The recovery took much longer; frequently, 10-15 min was necessary for the response to return to near control level (Figs. A 11 and 2E). E As described earlier, the hyperpolarization induced by ion- - tophoresis of dopamine is blocked by haloperidol (8). In the B A F D- C B --

__ _ G E

C I FIG. 3. Effects oftetrodotoxin on AcCho hyperpolarization and FIG. 2. Effects of haloperidol, atropine, and low-Ca/high-Mg antidromic spikes. The ganglion was continuously superfused with solution on slow AcCho depolarization. The ganglion was continuously Krebs solution containing 5MIM d-tubocurarine. The upper tracings superfused with Krebs solution containing 5 AM d-tubocurarine. The of records A-D represent the current pulses (10 Hz, 10 msec per pulse upper tracing of each pair, represents the current pulses (30 Hz, 10 for 2 sec) used to elicit the AcCho hyperpolarization. (A and E) msec per pulse for 2 sec) used to discharge AcCho from the pipette. Control responses; (B and C) 1 and 2 min after superfusion with te- (A) Control; (B) 5 min after superfusion with haloperidol (0.1 AM); trodotoxin (0.1 AM), respectively; (D and G) 10 min after wash with (C) 3 min after superfusion with low-Ca/high-Mg solution; (D) 2 min Krebs solution; (F) 1 min after tetrodotoxin. Records were taken from after superfusion with atropine (1 AM); (E) 12 min after wash with the same ganglion cell. Vertical calibration mark, 10 mV and 10 nA atropine-free Krebs solution. Records were taken from the same for A-D and 20 mV for E-G; horizontal calibration mark, 4 sec for ganglion cell. Calibration: 10 mV, 10 nA, and 4 sec. A-D and 10 msec for E-G. Downloaded by guest on September 26, 2021 Neurobiology: Dun and Karczmar Proc. Natl. Acad. Sci. USA 75 (1978) 4031 constant the AcCho- - -20 resistance remained relatively during indhiwd hyperpolarization; similarly, there is no detectable -30 change in membrane resistance during either the slow ipsp (1, .40 mV 2, 12) or the membrane hyperpolarization induced by appli- _50 cation of dopamine (13). Another distinct feature that charac- terizes and clearly separates the slow ipsp from other synaptic -60 potentials is its unusual response to shifts of initial membrane -Nft*mpmo. - -70 potential (1, 12); the amplitude of the slow ipsp is decreased but O^ftooomm -80 not reversed by decreasing the resting potential, whereas in- - .90 creasing the resting membrane potential first enhances and then L I depresses the slow ipsp (1,2, 12). Similar results were obtained with respect to AcCho-induced hyperpolarization; altogether, FIG. 4. Effects of shifting membrane potential on the amplitude of AcCho hyperpolarization. The ganglion was continuously super- the slow AcCho hyperpolarization appears to be indistin- fused with Krebs solution containing 5MgM d-tubocurarine. A steady guishable from the slow ipsp. depolarizing or hyperpolarizing current was passed through the re- The question that remains to be answered is whether cording microelectrode to shift the initial membrane potential; the AcCho-induced hyperpolarization is a mono- or disynaptic resting membrane potential of this cell was -52 mV. Calibration, 10 phenomenon. The AcCho-hyperpolarization was blocked by mV and 4 sec. both atropine and haloperidol; furthermore, haloperidol abolished differentially the AcCho-induced hyperpolarization attenuated and then completely abolished the AcCho hyper- without affecting the depolarizing response to AcCho. These polarization; this effect was achieved in <2 min (Fig. 3C). The data indicate that postsynaptic cholinoceptive and postsynaptic effect of tetrodotoxin was fully reversible; generally, a 10-min dopaminergic sites are involved in AcCho hyperpolarization; period was necessary (Fig. 3 D andG). the best explanation of these findings is to assume that a di- Membrane Resistance and Shifts in Initial Membrane synaptic mechanism is involved. Potential. The amplitude of hyperpolarizing potentials induced The most convincing evidence in support of this concept is by passing constant hyperpolarizing current pulses of relatively provided by experiments involving the block of transmitter long duration (100-200 msec) through the recording micro- release either by low-Ca/high-Mg solution or tetrodotoxin. electrode was used to measure the membrane resistance. In 12 Superfusing the ganglia with a low-Ca/high-Mg solution ef- cells studied, the amplitude of the hyperpolarizing potentials fectively and reversibly abolished the AcCho hyperpolarization was not significantly affected by AcCho-induced hyperpolar- in all the cells that exhibited this response; on the other hand, ization; the mean (± SD) membrane resistance was 34.6 + 4.7 this Ca-deficient solution did not affect the AcCho depolar- MO and 32.3 ± 3.5 MO, respectively, before and at the peak of ization because the latter is due to a direct muscarinic action membrane hyperpolarization; the difference was not statisti- on the ganglion cells (1,2). It cannot be argued that the alter- cally significant. ation of the resting membrane potential by low-Ca/high-Mg The application of a steady hyperpolarizing or depolarizing solution was responsible for abolishing the AcCho hyperpo- current through the recording microelectrodewas used to shift larization, because the block was still present when the mem- the resting membrane potential to a desired level. In five cells, brane potential was returned to the control level. Because the depolarization of the resting membrane potential by 10-20 low-Ca/high-Mg solution impairs the release of transmitter mV attenuated the AcCho-induced hyperpolarization, whereas from virtually all chemically mediated synapses (1, 14), our the amplitude of the hyperpolarization consistently augmented results clearly indicate that the synaptic release of a second when the initial membrane potential was increased by 20-30 transmitter is a necessary step in the generation of the AcCho- mV. In the case of the experiment illustrated in Fig. 4, the hy- induced hyperpolarization. perpolarization was nearly nullified at about -42 mV; however, The results of experiments carried out with tetrodotoxin bear further depolarization could not produce a reversal potential. a similar interpretation. Tetrodotoxin selectively inhibits the The amplitude of hyperpolarization was progressively en- transient sodium channels and thereby blocks the initiation of hanced when the membrane potential was increased to about regenerative action potentials (15). Thus, the finding with te- -80 mV; further displacing the membrane potential to -90 mV trodotoxin suggests the following scheme: AcCho released from reduced the amplitude. the pipette must diffuse to and cause a discharge of a nearby interneuron, and the propagated action potentials in turn lead DISCUSSION to liberation of the hyperpolarizing transmitter. It must be noted The important finding of this study is that AcCho applied that tetrodotoxin was ineffective in the sucrose gap procedure electrophoretically to the vicinity of mammalian sympathetic in blocking the hyperpolarization induced by bethanechol, a neurons in the presence of d-tubocurarine evokes a slow de- specific muscarinic agonist; the author of this report (16) sug- polarization, a slow hyperpolarization, or a combination of gested that, under sucrose gap conditions, bethanechol could both. release dopamine by acting directly on the nerve terminals. The slow depolarization elicited by AcCho has electrophys- In addition, morphological and neurochemical findings iological and pharmacological features similar to those of the support the concept of the involvement of a disynaptic mech- slow epsp evoked by nerve stimulation (1-3, 11). The amplitude anism in the generation of the slow ipsp in rabbit sympathetic and time course of the AcCho-induced hyperpolarization ganglia (7,16-18). Two pertinent points should be raised in this showed marked resemblance to those of the slow ipsp (1-3). context. First, the slow ipsp seems to be present only in those Finally, the biphasic response mimicked the slow biphasic sympathetic ganglia in which the SIF cells synapse with the postsynaptic potentials elicited by nerve stimulation (1, 2). The postganglionic cells. Indeed, in the case of the guinea pig gan- membrane characteristics recorded during the AcCho-induced glion, where the SIF cells are present but do not appear to form potential changes provide the identification of the potentials synapses with the postganglionic neurons, the slow ipsp is absent in question with respect to the postsynaptic potentials evoked (19, 20). Second, there may be an analogy between the chro- by presynaptic stimulation. In the present study, the membrane maffin cells of the adrenal medulla and of the sympathetic Downloaded by guest on September 26, 2021 4032 Neurobiology: Dun and Karczmar Proc. Natl. Acad. Sci. USA 75 (1978) ganglion. Iontophoretically applied AcCho depolarizes the 5. Libet, B. & Kobayashi, H. (1974) J. Neurophysiol. 37, 805- chromaffin cells of the rat adrenal medulla, and this effect is 814. atropine sensitive; furthermore, tetrodotoxin blocks the action 6. Eccles, R. M. & Libet, B. (1961) J. Physiol. (London) 157, potentials of the adrenal medulla chromaffin cells (21). Perhaps 484-503. 7. Libet, B. & Owman, C. (1974) J. Physiol. (London) 237, 635- the chromaffin cells of the adrenal medulla may serve as a 663. model for the study of the synaptic mechanisms of SIF cells, 8. Dun, N. J., Kaibara, K. & Karczmar, A. G. (1977) Science 197, because it has not yet been possible to impale the SIF cells of 778-780. the sympathetic ganglion with a microelectrode. 9. Dun, N. J., Kaibara, K. & Karczmar, A. G. (1977) Neurophar- It should be pointed out that the SIF cells in the sympathetic macology 16, 715-717. ganglion constitute the only known mechanism in the periph- 10. Dun, N., Nishi, S. & Karczmar, A. G. (1976) Neuropharmacology, eral nervous system whereby an excitatory input is converted 15, 211-218. 11. Koketsu, K. (1969) Fed. Proc. Fed. Am. Soc. Exp. Btol. 28, into an inhibitory output; this type of mechanism is reminiscent 101-112. of that involved in the Renshaw cell circuit in the spinal cord 12. Kobayashi, H. & Libet, B. (1968) Proc. Natl. Acad. Scl. USA 60, (22). It must be emphasized, however, that this disynaptic 1304-1311. mechanism is by no means the only mode of action by which 13. Dun, N. & Nishi, S. (1974), J. Physiol. (London) 239, 155- AcCho may produce an inhibitory potential in an autonomic 164. ganglion, because conclusive evidence indicates that AcCho 14. Rubin, R. P. (1970) Pharmacol. Rev. 22,389-428. hyperpolarizes directly the cardiac parasympathetic neurons 15. Narahashi, T. (1972) Fed. Proc. Fed. Am. Soc. Exp. Biol. 31, 1124-1132. (23). 16. Libet, B. (1976) in SIF Cells: Structure and Function ofthe Small In summary, our results provide positive evidence at the Intensely Fluorescent Sympathetic Cells Fogarty International single-cell level for the hypothesis that the slow ipsp is mediated Center Proceedings No. 30, ed. Eranko, 0. (U. S. Government by a cholinoceptive adrenergic interneuron interposed between Printing Office, Washington, DC), pp. 163-177. the preganglionic fibers and principal ganglion cells; when 17. Erinko, 0. (1976) in SIF Cells: Structure and Function of the activated muscarinically by cholinergic fibers, the interneuron Small Intensely Fluorescent Sympathetic Cells Fogarty Inter- liberates dopamine which then acts postsynaptically in the national Center, Proceedings No. 30, ed. Eranko,O. (U. S. Gov- ernment Printing Office, Washington, DC), pp. 1-7. production of slow ipsp. 18. Bjorklund, A., Cegrell, L., Falck, B., Ritzen, M. & Rosengren, E. in Institutes of Health Grant (1970) Acta Physiol. Scand. 78,334-38. This study is supported part by National 19. Elfvin, L. G., Hokfelt, T. & Goldstein, M. (1975) J. Ultrastruct. NS-6455 and by the American Parkinson Disease Foundation. Res. 51,377-396. 1. Libet, B. (1970) Fed. Proc. Fed. Am. Soc. Exp. Biol. 29, 1945- 20. Dun, N. & Karczmar, A. G. (1977) J. Pharmacol. Exp. Ther. 200, 1956. 328-5. 2. Nishi, S. (1974) in The Peripheral Nenvus System, ed. Hubbard, 21. Brandt, B. L., Hagiwara, S., Kidokoro, Y. & Miyazaki, S. (1976) J. L. (Plenum, New York), pp. 225-255. J. Physiol. (London) 263,417-439. 3. Volle, R. L. (1975) in Cellular Pharmacology of Excitable 22. Eccles, J. C., Fatt, P. & Koketsu, K. (1954) J. Phystol. (London) Tissues, ed. Narahashi, T. (Thomas, Springfield, IL) pp. 89- 126,524-562. 140. 23. Hartzell, H. C., Kuffler, S. W., Stickgold, R. & Yoshikami, D. 4. Weight, F. F. & Padjen, A. (1973) Brain Res. 55,225-228. (1977) J. Physiol. (London) 271, 817-846. Downloaded by guest on September 26, 2021