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