because the endings of each neuron were sparsely distributed

Transcription

1 Proc. Natl. Acad. Sci. USA Vol. 73, No. 11, pp , November 1976 Neurobiology Chemical transmission between rat sympathetic neurons and cardiac myocytes developing in microcultures: Evidence for cholinergic, adrenergic, and dual-function neurons (autonomic transmitters/autapses/culture methods/ neuron-heart cell interaction) E. J. FURSHPAN*, P. R. MACLEISH*, P. H. O'LAGUEt, AND D. D. POTTER* * Department of Neurobiology, Harvard Medical School, Boston, Massachusetts 02115; and t Department of Biology, University of California, Los Angeles, Calif Communicated by David H. Hubel, September 7,1976 ABSTRACT Electrophysiological studies were made on microcultures ( Ism in diameter) in which solitary sympathetic principal neurons from newborn rats grew on previously dissociated rat heart cells. Some neurons inhibited, some excited, and others first inhibited and then excited the cardiac myocytes. Application of drugs provided evidence for secretion of acetylcholine by the first group, catecholamines by the second, and both acetylcholine and catecholamines by the third. Solitary neurons which inhibited the myocytes usually excited themselves at nicotinic synapses (autapses). The transmitter functions of rat sympathetic principal neurons developing in culture can be influenced in a striking way by a variety of nonneuronal cells. Neurons cultured in the near absence of nonneuronal cells develop adrenergic functions including synthesis, storage, release, and uptake of norepinephrine (NE); these neurons form synapses of adrenergic appearance (i.e., containing small granular vesicles) on each other (1-4). Adrenergic transmission has not yet been detected at such synapses, apparently because of the insensitivity of the neurons to NE (unpublished; see ref. 5). Moreover, acetylcholine (AcCh) synthesis is usually negligible, and cholinergic transmission is rarely seen (1, 6, 7, 8). In contrast, when the neurons are cultured in the presence of nonneuronal cells (e.g., from ganglia or heart), or in medium conditioned by such cells, the neuronal population synthesizes both AcCh and NE (2, 9), and many neurons form nicotinic cholinergic synapses on each other (6-8, 10, 11). These effects are graded. A higher proportion of conditioned medium or a greater number of nonneuronal cells gives a higher ratio of AcCh synthesis to catecholamine synthesis (12), a higher incidence of cholinergic transmission, and a higher proportion of synapses which lack small granular vesicles (13). This shift to cholinergic functions occurs without appreciable change in the number of neurons per culture (2, 12). This and other evidence indicates that at least a majority of the neurons isolated from ganglia of newborn rats are plastic with respect to transmitter functions; many apparently remain so for at least two weeks in culture (12). It is unclear whether in cultures with both adrenergic and cholinergic functions individual neurons are exclusively adrenergic or cholinergic, or whether they can display, at least temporarily, both functions simultaneously. To investigate this question with biochemical methods, Reichardt, Patterson, and Chun (14) developed techniques for growing single neurons and assayed AcCh and catecholamine synthesis. To investigate this question with electrophysiological methods, MacLeish grew neurons in mass cultures with atrial myocytes (thousands of dissociated neurons and cardiac cells per dish), because each Abbreviations: AcCh, acetylcholine; NE, norepinephrine; e.p.s.p.'s, excitatory postsynaptic potentials myocyte is sensitive to both AcCh and NE (MacLeish, unpublished). He found that the incidence of detectable neuronmyocyte interaction was too low to be useful, perhaps in part because the endings of each neuron were sparsely distributed in the large field of myocytes; the few cases found all appeared to be cholinergic. It was plausible that the incidence of detectable interaction would increase if the innervation field of a given neuron was concentrated on a few cardiac myocytes. Thus, we made microcultures containing a single neuron and a small number of myocytes in an area only a fraction of a millimeter in diameter. In this paper, we report physiological observations on such microcultures; in the accompanying paper, Landis (15) reports electron microscopic observations on the same microcultures. In two previous studies (16, 17), explants of sympathetic ganglia were found to make functional contacts with explants of heart. METHODS Several methods for making microcultures suitable for electrophysiology and microscopy were successful, but none routinely so. The simplest method was, in brief, to apply 25 to 50 equally spaced droplets of dissolved collagen to a nonwetting polystyrene surface. When dried, these produced a grid (ca 50 mm2) of collagen islands, each island Atm in diameter. Cardiac cells (myocytes and fibroblasts) were dissociated from hearts of newborn rats by use of collagenase (EC ) (Worthington Type I; 1 mg/ml), and allowed to settle on the grid for about 2 hr. Almost all cells not adhering to the collagen islands could then be washed away with medium. Proliferation of the cardiac cells was suppressed after 1-2 days by y-irradiation (60Co; 5000 rads in sec, where one rad equals 1 X 10-2 J/kg). One to 5 days later, principal neurons were dissociated from superior cervical ganglia of newborn rats (Charles River CD) as previously described (1, 18), and plated at a density such that many islands received only one or a few neurons. The cultures were grown in L-15 CO2 medium (1) containing 5% adult rat serum or 10% fetal calf serum (Microbiological Associates, ), but lacking bovine serum albumin and Methocel. Six platings (about 30 dishes per plating) were used in experiments reported here. For electrophysiological recording, cultures were placed on a microscope stage and perfused continuously with fluid similar to that used previously (6) but containing 6 mm NaHCO3, 0.14 mm ascorbate and fetal calf serum (1% vol/vol). A change to fluid containing drugs could be made while maintaining impalement of the cells. The culture was kept at Microelectrodes filled with 3 M KCI ( Mohm) were used

2 4226 Neurobiology: Furshpan et al. to pass current and record membrane potential in neurons and myocytes. In some experiments, an optical method was used to record the beating of myocytes (cf. ref. 19). At the end of many experiments, the microculture was fixed for electron microscopic examination as described by Landis (15). RESULTS In microcultures, the neurites grew rapidly, as in mass cultures of dissociated neurons on heart cells (7); two islands containing single neurons (called "solitary neurons" below) are shown in two figures below. A typical island had several clusters of myocytes which usually beat spontaneously and synchronously. The neurites crisscrossed the island and sometimes grew around the perimeter. Occasionally the neurites were seen to extend for short distances from the island to nonneuronal cells or even from island to island. In the cases reported below, no inter-island cellular connections were present. The resting and action potentials recorded from solitary neurons were similar to those of dissociated neurons in mass cultures (6, 7, 10). Stable impalements of neurons for many hours were routine; repeated impalements of myocytes were often necessary. Action potentials recorded in the myocytes were up to 120 mv in size and were preceded by pacemaker potentials. Cholinergic synapses formed by solitary neurons on themselves In mass cultures of dissociated neurons grown on heart cells, the neurons frequently form cholinergic synapses on each other (ref. 7; see also refs. 6, 8, 10, and 11). One question was whether a solitary neuron would form such synapses on itself; many did so. Examples are shown in Fig. lalis. A single action potential in the neuron often evoked a barrage of excitatory postsynaptic potentials (e.p.s.p.'s) (Fig. lal). The initial e.p.s.p. had such short latency that it began during the action potential. The longerlatency e.p.s.p.'s suggested the presence of long conduction pathways. Hexamethonium chloride ( AtM), or d- tubocurarine chloride (350,gM) greatly reduced or abolished the e.p.s.p.'s (Fig. 3a-c), but atropine sulfate had little effect in concentrations which blocked the cholinergic inhibition of the myocytes by these neurons (see below). This leaves little doubt that the e.p.s.p.'s were produced by secretion of AcCh at nicotinic synapses. The useful name autapse was proposed by Van Der Loos and Glaser (20) for a synapse made by a neuron on itself. In the microcultures, almost all neurons that made nicotinic autapses also produced atropine-sensitive inhibition of myocytes (next section). The action of the neurons on the myocytes To test for effects on the myocytes, we stimulated neurons repetitively with one microelectrode while recording myocyte activity with a second electrode. Evidence for Cholinergic Action. Many solitary neurons hyperpolarized the myocytes (Fig. lb1, and cl) and stopped their spontaneous activity (e.g., Fig. lb,). The hyperpolarization of the myocytes always had a longer latency and a much slower time course than did the e.p.s.p.'s on the same neuron. In two cases, hyperpolarization was evoked by a single neuronal impulse; the latency was about 50 msec (cf. ref. 21). An increase in myocyte membrane conductance during the hyperpolarization was demonstrated either with small test current pulses (three cases) or by reversal of the hyperpolarization (one case; Fig. 1CI-c3). In the mammalian heart, hyperpolarization and an increase in K+ permeability are- caused by AcCh acting on atropine- Proc. Natl. Acad. Sci. USA 73 (1976) FIG. 1. Cholinergic transmission at autapses and at neuronmyocyte contacts. (al-a3) autaptic e.p.s.p.'s after evoked spikes in three neurons (stimulating current pulses on lower traces). Culture ages in days: (ai) 17; (a2) 14; (a3) 30. (bl) inhibition of spontaneous impulses in a myocyte (upper trace) evoked by three trains of neuronal stimuli (35 per sec; lower trace). Culture age is 13 days. (b2) perfusion with atropine sulfate (0.1 1AM) blocked the inhibition. (c1-c) reversal of neurally-evoked hyperpolarization of a myocyte when the myocyte resting potential (cl) was shifted to inside-more-negative values (c2, C3) with applied currents. White bar under each trace is the duration of neuronal stimulation. Culture age is 30 days. Scales (y and x axis, respectively) are for (al-a3) 20 mv and 20 msec; (bl) or (b2) 40 mv and 2 sec; (cl-c3) 10 mv and 10 sec. sensitive muscarinic receptors (for references, see 22 and 23). Fig. lb2 shows that atropine (0.1,uM) blocked the inhibitory action of a solitary neuron on the myocytes. This effect of atropine ( ,uM) was present in all cases tested and was readily reversible (not shown). Similar concentrations block vagal and AcCh effects on mammalian hearts (22, 24, 25). AcCh (0.6,uM) in the presence of eserine (1,uM) hyperpolarized the myocytes and stopped their beating (not illustrated; cf. ref.. 5). The inhibition of the myocytes, its time course, and its sensitivity to atropine, taken with the evidence that these solitary neurons simultaneously secreted AcCh at autapses, leave little doubt that the neurons secreted AcCh onto the myocytes. Moreover, there is biochemical evidence that these neurons synthesize substantial amounts of AcCh in mass cultures containing cardiac cells (12), as do single neurons grown in isolation (14). Evidence for Adrenergic Action. Many solitary neurons which did not form cholinergic autapses had an excitatory effect on myocytes. The action potentials of two such neurons are shown in Fig. 2 (inset of phase micrograph and Fig. 2a); no autaptic effect was observed even after an impulse train (not illustrated). Trains of neuronal impulses did, however, produce a depolarization of slow onset and time course in the myocytes and gave rise to a train of cardiac impulses lasting about 20 sec (Fig. 2b). In mammalian heart, these effects are produced by sympathetic stimulation or application of catecholamines and are blocked by propranolol, a f3-blocker, at a concentration of

3 Neurobiology: Furshpan et al. Proc. Natl. Acad. Sci. USA 73 (1976) 4227.ji~~~~~~~~h * 4 V.-. FIG. 2. A microculture containing a solitary neuron; arrow at H indicates a cluster of myocytes. Neuron at 19 days in culture. Inset shows impulse in this neuron (scales are 50 mv, 20 msec for y and x axis, respectively); (d-f) were also from this neuron. (a) impulse in a similar neuron (upper trace), and a slower impulse in a myocyte; duration of stimulus in neuron shown by bar. (b) depolarization and impulses in the myocytes (upper trace) evoked by a train of neuronal impulses (22 per sec at deflection of lower trace). Brief gaps in both traces in this and other records mark start of new sweeps. (c) response of same myocytes to 0.1 IAM NE. Pulse on lower trace marked return to normal fluid. Large gap in record is about 40 sec. Tops of impulses are not shown. (d) three trains of neuronal impulses (33 per sec) excited the myocytes. (e) ten trains had no effect in propranolol (1,M). (f) subsequent recovery in normal fluid. Scales for y and x axis, respectively are (a) 50 mv and 20 msec; (b) 50 mv and 2 sec; (c) 25 mv and 2 sec; (d-f) 50 mv, 5 sec. 1 AM or less (see ref. 22 for references). Fig. 2d-f show that propranolol (1 AM) reversibly blocked the depolarization and initiation of beating; it consistently had this effect in the concentration range AM. Fig. 2c shows that 0.1 MM NE mimicked the depolarization and initiation of beating produced by the solitary neuron; compare Fig. 2b and c. The excitatory neuronal effect on the myocytes, its time course, its sensitivity to block by propranolol, and its similarity to the effect of applied NE all suggest that these solitary neurons secreted catecholamines, presumably including NE, onto the myocytes. Further evidence is provided by Landis' observations on these solitary neurons; varicosities near the myocytes con-

4 4228 Neurobiology: Furshpan et al. Proc. Natl. Acad. Sci. USA 73 (1976) FIG. 3. A microculture containing a dual-function solitary neuron after 13 days in culture. Arrow at Il shows cluster of myocytes. All records were from this neuron. (a-c) neuronal impulse and autaptic e.p.s.p.'s before (a), during (b), and after (c) perfusing with hexamethonium (0.5 mm). (d) a train of neuronal impulses (deflection of lower trace) produced inhibition, and then excitation of spontaneous myocyte activity (upper trace). (e) inhibition was blocked by atropine (0.1 um). In (d) and (e) hexamethonium (0.5 mm) was present. (f) the effect of atropine (0.1 AM; no hexamethonium in (f-i)) at higher sweep speed. (g) block of excitation by propranolol (0.6 MM; atropine still present). (h) about 45 min after removal of propranolol (atropine still present) with excitation restored. (i) the dual effect restored by perfusion with drug-free fluid. Scales 100 mv and 5 sec. are (for y and x axis, respectively) (a-) 50 mv and 30 msec; (d) and (e) 100 mv and 12.5 sec (f-i) tained many small granular vesicles (15). Moreover, when grown on cardiac cells either in mass cultures or in isolation some of the neurons synthesize NE (12, 14), and when grown in the absence (2) or presence (3) of ganglionic nonneuronal cells they release NE by a Ca++-dependent mechanism. Evidence for Dual-Function. In seven microcultures, stimulation of solitary neurons first inhibited and then accelerated the beating of the myocytes, as if both adrenergic and cholinergic transmission were present. One of these ca.ses is illustrated in Fig. 3. Fig. 3d shows the initial hyperpolarization, a pause of about 5 sec in the beating, and the subsequent accelerated beating which lasted about 25 sec. In the presence of atropine (0.1,uM), the hyperpolarization and pause was eliminated, and the excitation enhanced (Fig. 3e). The inhibition was promptly restored after removal of atropine (not shown). This left little doubt that the neuron secreted AcCh onto the myocytes. To see whether the excitation which persisted in the presence of atropine (Fig. 3f; note change in time scale) was

5 Neurobiology: Furshpan et al. sensitive to propranolol, we added this drug at a concentration of 0.6 1sM. The excitation was eliminated (Fig. 3g). It was gradually restored by perfusion with normal solution for about 45 min (Fig. 3h). In other experiments, propranolol alone (0.6 tim) abolished the excitation of the myocytes but left intact the secretion of AcCh at autapses and onto the myocytes; this is consistent with its conventional effect of blocking (-receptors, and rules out a general interference with secretion at nerve endings. In the experiment of Fig. 3 and others, hexamethonium alone had little influence on the action of the neuron on the myocytes, at concentrations which nearly eliminated transmission at cholinergic autapses. The presence of only one neuron in the microculture of Fig. 3 was confirmed by electron microscopic examination (15). Occasional small granular-vesicdes were present in the synaptic endings and varicosities of this neuron; see Landis (15) for discussion. Thus, a single neuron appeared to secrete two transmitters which activated three receptors: nicotinic-cholinergic and fl-adrenergic (excitatory); muscarinic-cholinergic (inhibitory). Burn and Rand (e.g., ref. 26) proposed that adult sympathetic neurons secrete both AcCh and NE, and that the acetylcholine acts on nicotinic receptors to stimulate NE secretion; it should be noted that the apparent secretion of NE by solitary neurons onto myocytes persisted in concentrations of hexamethonium which nearly eliminated cholinergic transmission at nicotinic autapses. There is evidence that certain molluscan neurons synthesize (27, 28) and use (29) more than one transmitter. Multi-Neuron Microcultures. Many islands containing two to six neurons were examined. Further examples of block of neuron-myocyte interactions by atropine and propranolol were obtained. By these criteria, both adrenergic and cholinergic neurons were often present in the same island. In several cases, the cell bodies of two neurons, one apparently adrenergic, the other cholinergic, were in direct contact; we do not yet know whether this difference in transmitter functions arises because of heterogeneity among the neurons at the time of plating or because of environmental differences within a microculture. As they do in mass cultures (6-8, 10, 11), the neurons in these islands often activated each other by cholinergic synapses, and in one case by electrical transmission. Although hexamethonium or curare blocked the cholinergic interaction, the determination of the effects of individual neurons on myocytes remained uncertain because of the possibility of electrical interaction. DISCUSSION We began this work by asking whether individual sympathetic neurons developing in culture express, at any moment, only one set of transmitter functions, adrenergic or cholinergic, or both sets simultaneously. Three lines of evidence (ref. 14, this paper, and ref. 15) demonstrate that some of the developing neurons are at least predominantly adrenergic, and others at least predominantly cholinergic. In preliminary biochemical assays, dual-function neurons were not detected (14); physiological and morphological assays, which may be more sensitive than existing biochemical ones, have now provided preliminary evidence that, at least at shorter times in culture, some neurons use both transmitters (this paper, 15). If further work confirms the existence of such neurons, several questions of interest will be: (i) whether such neurons are capable of synthesizing both transmitters, since it is conceivable that the neurons take up and secrete catecholamines synthesized by adrenergic neurons in nearby islands; (ii) whether two transmitter systems in a single Proc. Natl. Acad. Sci. USA 73 (1976) 4229 neuron can be expressed in any proportion; and (iif) whether a particular proportion is stable. The microcultures have several advantages over mass cultures for investigating these questions. One advantage, which may have wide application, is that confinement of the growing neurites to a small number of target cells increases the probability of synapse formation on these cells and therefore the intensity of synaptic action. This work was done in continuous collaboration with S. Landis (see ref. 15). We thank L. Chun for nerve growth factor, and our colleagues for helpful discussion. Essential help was provided by Delores Cox, William Dragun, Karen Fischer, Joseph Gagliardi, James LaFratta, Michael LaFratta, and Doreen McDowell. We thank Dr. John Little, Harvard School of Public Health for use of the 6OCo-source. Suppoyt was received from National Institutes of Health Research Grants NS02253, NS03273, NS11576, RR-7009, and Training Grant MH Mains, R. E. & Patterson, P. H. (1973) J. Cell. Biol. 59, Patterson, P. H., Reichardt, L. F. & Chun, L. L. Y. (1975) Cold Spring Harbor Symp. Quant. Biol. 40, Burton, H. & Bunge, R. P. (1975) Brain Res. 97, Rees, R. & Bunge, R. P. (1974) J. Comp. Neurol. 157, Obata, K. (1974) Brain Res. 73, O'Lague, P. H., Obata, K., Claude, P., Furshpan, E. J. & Potter, D. D. (1974) Proc. Natl. Acad. Scd. USA 71, O'Lague, P. H., MacLeish, P. R., Nurse, C. A., Claude, P., Furshpan, E. J. & Potter, D. D. (1975) Cold Spring Harbor Symp. Quant. Biol. 40, Johnson, M., Ross, D., Meyers, M., Rees, R., Bunge, R., Wakshull, E. & Burton, H. 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