Studies on rat sympathetic neurons developing in cell culture

DEVELOPMENTAL

BIOLOGY

(1978)

67,424-443

Studies on Rat Sympathetic

Neurons

III. Cholinergic

PAUL H. O'LAGUE' Department

of Neurobiology, Received

May

Developing

in Cell Culture

Transmission

D. D. POTTER, AND E. J. FURSHPAN Harvard

Medical

15, 1978; accepted

School, in revised

Boston, form

July

Massachusetts

02115

31, 1978

Principal neurons were dissociated from the superior cervical ganglia of newborn rats and grown in culture with several types of non-neuronal cells. As described in the second paper of this series, the neurons in such mixed cultures formed two types of excitatory synapses with each other, electrical and chemical. Evidence is presented here that transmission at the chemical synapses was cholinergic. Four nicotinic ganglionic blocking agents (curare, hexamethonium, tetraethylammonium, and mecamylamine) strongly attenuated or eliminated the excitatory postsynaptic potentials (e.p.s.p.‘s) at moderate concentrations; atropine at relatively high concentrations also blocked transmission. Iontophoretic application of acetylcholine (ACh) to the surface of the neurons gave rise to depolarizations that could be made to resemble the e.p.s.p.‘s in size and time course; the ACh potentials and the e.p.s.p.‘s were then similarly affected by nicotinic blocking agents. The sensitivity to ACh was often distributed nonuniformly on the neuronal surface; it was common to find small, sharply localized regions of high sensitivity. Catecholamines (norepinephrine, epinephrine, and dopamine) had only inhibitory actions; in a few experiments adrenergic blocking agents (phenoxybenzamine, propranolol) were found to have no effect on the e.p.s.p.‘s. These observations leave no doubt that the neurons released ACh and had ganglionic, nicotinic ACh receptors on their surfaces. The significance of the fact that a high proportion of the sympathetic neurons in mixed cultures formed cholinergic synapses is discussed.

(Perri et al., 1970). This resemblance and the likelihood that the cultured neurons possessed the nicotinic cholinergic receptors found at preganglionic synapses in viva raised the possibility that the excitatory chemical synapses were cholinergic. Two obvious tests of this possibility were to see whether the synapses were sensitive to agents which block ganglionic transmission and to see (e.g., curare, hexamethonium) whether the depolarizations produced by acetylcholine (ACh) had characteristics similar to those of the e.p.s.p.‘s. The effect of several adrenergic agonists and antagonists was also examined. Preliminary accounts of these experiments have appeared (O’Lague et al., 1974, 1975). Johnson et al. (1976b) and Ko et al. (1976) have also reported that excitatory synapses formed by dissociated neurons from the rat SCG

INTRODUCTION

In the preceding papers of this series, we reported that sympathetic principal neurons dissociated from the superior cervical ganglia (SCG) of newborn rats display resting and action potentials in culture similar to those of principal neurons in the SCG of adult rats (Part I of this series). Furthermore, when the dissociated neurons were cultured in the presence of several types of non-neuronal cells, a high proportion of the neurons formed excitatory chemical synapses with each other (Part II of this series), The excitatory postsynaptic potentials (e.p.s.p.‘s) at these synapses resembled the fast e.p.s.p.‘s produced in adult rat principal neurons by cholinergic preganglionic axons ’ Present address: Department sity of California, Los Angeles,

of Biology, Calif. 90024.

Univer-

424 0012-X06/78/0672-0424$02.00/O

Copyright All

rights

0 1978 by Academic Press, of reproduction in any form

Inc.

reserved.

O’LAGUE,

POTTER,

AND FURSHPAN

grown under rather similar culture conditions are blocked by cholinergic blocking agents. MATERIALS

AND

METHODS

Cultures of dissociated sympathetic neurons were prepared as described in the first paper of this series (O’Lague et al., 1978a). The neurons were grown either in the presence of a gradually developing layer of ganglionic non-neuronal cells (L-15 CO2 cultures) or on a preestablished layer of primary heart or serially propagated rat-embryo fibroblasts (pL-15 COZ cultures). To prevent overgrowth of fibroblasts, pL-15 CO2 cultures were usually irradiated before, and sometimes after, the neurons were plated. The methods for stimulating and recording from the neurons with intracellular microelectrodes and for locating neurons linked by synapses were the same as those already described (O’Lague et al., 1978a,b). In some experiments the standard perfusion fluid (composition given in O’Lague et al., 1978a) was modified by omission of NaHC03 which was replaced by equimolar NaCl or, to increase the buffering capacity, an equimolar mixture of NaCl and ,&glycerophosphate (Sigma; final concentration, 5 mM). Test solutions were made by dissolving drugs in aliquots of the control perfusion fluid. Catecholamine solutions were freshly made up in perfusion fluid containing 50 to 140 fl sodium ascorbate. The characteristics of the perfusion system were such that, following the switch to a test solution (see “multichannel valve” in Fig. 1 of O’Lague et al., 1978a), there was a delay of 1 to 2 min before the effect of the drug appeared. The stability of the system permitted continuous intracellular recording from a pair of neurons for several hours, during repeated changes in the perfusion fluid. Successive e.p.s.p.‘s evoked at these synapses usually fluctuated in size, sometimes

Choline&c

Synapses in Culture

425

conspicuously (see Fig. 3 of O’Lague et al., 1978b). To determine the effect of a drug on the size of the e.p.s.p.‘s, runs of 25 to 200 successive e.p.s.p.‘s were recorded before, during, and after perfusion with a drugcontaining fluid. E.p.s.p.-amplitude histograms were then plotted or the mean amplitudes determined; the control amplitude was taken as the mean of the amplitudes observed before and after application of a test solution, except in experiments with catecholamines where the effects were not fully reversible. For iontophoretic application of ACh, microelectrodes (resistance 200-300 ma) were filled by capillarity with 3 to 4 M ACh . Cl. Optimum “braking” currents (de1 Castillo and Katz, 1955), usually in the range 0.5 to 1.5 nA, were determined by maximizing the response to a repeated standard test pulse (usually l-3 nA). In some experiments tetrodotoxin (3 I-1M) was included in the perfusion fluid to block “local responses” and action potentials. Drugs were obtained from the following sources: acetylcholine chloride, Sigma; atropine sulfate, Merck; hexamethonium chloride, Pfaltz and Bauer; mecamylamine . HCl, Merck, Sharp and Dohme; d-tubocurarine chloride, Mann Research Laboratories; norepinephrine bitartrate, Sigma; epinephrine bitartrate, Pfaltz and Bauer; dopamine. HCl, Sigma; tetraethylammonium chloride, Pfaltz and Bauer; tetrodotoxin, Sigma; phenoxybenzamine HCl, Smith, Klein and French; or,-propranolol HCl, Sigma. RESULTS

As described in the preceding paper (O’Lague et al., 1978b), randomly chosen pairs of neurons were impaled with microelectrodes; to test for interaction each neuron was stimulated in turn while recording from both. The presence of chemical synapses was revealed when action potentials in one neuron (the “driver”) gave rise, after

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426

a minimum delay of about 1 msec, to characteristic e.p.s.p.‘s in the other neuron (the “follower”). The effects of drugs on the transmission process could then be tested by switching from control perfusion fluid to drug-containing fluids. When short-latency e.p.s.p.‘s and complex waves were both evoked by action potentials in the driver neuron (see O’Lague et al., 1978b), the drug sensitivities of both types of responses were observed. It was discovered at the outset that the chemically mediated responses (simple e.p.s.p.‘s and complex waves) were sensitive to cholinergic nicotinic blocking agents but insensitive to either (Y- or /3-adrenergic blockers. In addition the neurons were depolarized by ACh but not by catecholamines (see Obata, 1974; O’Lague et al., 1974). Therefore, our attention was directed mainly at the nicotinic pharmacology of the synapses, and this will be described first. Nicotinic

Blocking

Agents

Both the short-latency e.p.s.p.‘s and the complex waves were strongly affected by moderate concentrations of four agents that are well known to block transmission through intact sympathetic ganglia: hexamethonium, d-tubocurarine (curare), tetraethylammonium (TEA), and mecamylamine (see Discussion for references). We have used hexamethonium and curare routinely; the other two agents, TEA and mecamylamine, were tested in only a few cases (four for each) to check whether the expected effect was present and to determine approximately the range of effective concentrations. The effects of three of these agents are illustrated in Fig. 1. In the case shown in Fig. la, in the presence of normal perfusion fluid (al, a3, a& a single action potential in the driver neuron (middle traces) gave rise to a short-latency, suprathreshold e.p.s.p. in the follower neuron (upper traces) and also activated a complex wave in both neurons. Perfusion with 100 ).I.M TEA elimi-

VOLUME 67.1978

nated the complex waves and reduced the short-latency e.p.s.p. to a small subthreshold value (i.e., one-to-one transmission was interrupted); the record in Fig. la2 was taken 5 min after the start of perfusion with TEA. The disappearance of the complex waves was presumably due to suppression of one-to-one transmission at many synapses in the culture. Within 5 to 7 min of switching from the TEA solution to standard perfusion fluid, the original pattern of responses was fully restored (Fig. las). In a test with 30 fl TEA on the same driver-follower pair, there was also reversible interruption of one-to-one transmission and elimination of the complex waves (not illustrated); the mean amplitude of the residual short-latency e.p.s.p. was about twice that in 100 @f TEA. In a test with 10 fl TEA on a different driver-follower pair in the same culture, there was no obvious effect except for a slight delay in the time of appearance of the first action potentials in the complex waves. Thus, in this experiment, the minimally effective concentration of TEA was about 10 ,uM, while a marked reduction in e.p.s.p. amplitude (apparently about fivefold) was caused by 100 @. Similar effects of TEA in this concentration range were observed in the other experiments. In the highest concentrations used in this group of experiments (100 m, TEA did not markedly affect the time course of the neuronal action potentials (cf. O’Lague et al., 1978a, Fig. 6). In the continuation of the experiment of Fig. la, on the same driver-follower pair, hexamethonium was found to be somewhat more effective than TEA. While 50 w hexamethonium also eliminated one-to-one transmission and complex waves (Fig. lad), it caused a greater reduction than did 100 fl TEA in the mean amplitude of the short-latency e.p.s.p. (by a factor of ca. 2). The effects of hexamethonium, like those of TEA, were readily reversible (e.g., Fig. la5). In the experiment shown in Fig. 1, con-

OXAGUE,

POTTER,

AND FURSHPAN

Cholinergic

Synapses in Culture

427

T=zEEr~ ----I.---, FIG. 1. The effects of three nicotinic ganglionic blocking agents on synaptic transmission. Each set of records shows the responses from the follower neuron (upper trace), the responses from the driver neuron (middle trace), and the stimulus pulse (bottom trace) applied to the driver neuron. Records aI to as, from a pair of neurons in a pL-15 CO* culture: neurons, 21 days old; background layer of rat-embryo fibroblasts, 29 days old. (ai, a3, a) In control solution; (82) in 100 a&f TEA-chloride; (a4) in 50 @f hexamethonium.chloride; (a& spontaneous complex waves. Records b1 to be, from a pair of neurons in a 63-day-old L-15 CO2 culture; each record consists of three superimposed sweeps. (bl, bar b5) In control solution; (b2) in 5 fl mecamylamine. HCI; (b4) in 2.5 @4 mecamylamine. HCl. In both experiments (a, b) the recordings were made in the sequences shown. Vertical calibration: (al-as) 40 mV (top and middle traces) and 2 nA (bottom traces); (bl-b5) 10 mV (top traces), 20 mV (middle traces), and 2 nA (bottom traces). Horizontal calibration: (a,-&) 50 msec; (bl-b5) 25 msec.

centrations of TEA and hexamethonium much higher than those required to block one-to-one transmission caused severe reduction in the amplitude of the short-latency e.p.s.p. without abolishing it (e.g., Fig. lad). Taken together with the scarcity of electrical synapses in these cultures, such observations provide additional evidence that the short-latency e.p.s.p.‘s arise at a direct, monosynaptic link between the driver and follower neurons (seeO’Lague et al., 1978b). Spontaneous complex waves were a common feature of cultures in which there was a high incidence of driver-follower pairs (O’Lague et al., 1978b). Like the evoked complex waves, the spontaneous ones in-

variably appeared in both of the impaled neurons concurrently; an example is shown in Fig. las. This spontaneous activity was abolished by concentrations of nicotinic blockers that eliminated the evoked complex waves. The effect of mecamylamine hydrochloride is illustrated in Figs. lb1 to bj; in control solution (b,, bS, bs), an action potential in the driver neuron (middle traces) evoked a small, presumably monosynaptic, e.p.s.p. (latency ca. 5.5 msec) in the follower neuron (upper traces). The e.p.s.p. was almost abolished by 5 fl mecamylamine (Fig. lbz) and reduced to about 30% of control amplitude by 2.5 $I4 mecamylamine (Fig. lb4). Similar effects were observed in three other expe-

DEVELOPMENTAL

428

BIOLOGY

riments. In each experiment the block caused by mecamylamine was reversed more slowly than that caused by the other three agents. Following a period of perfusion with mecamylamine (2.5-10 p.M), it was necessary to perfuse with control solution for 15 to 30 min before the amplitude of the e.p.s.p. recovered to control levels. Although the number of caseswas small, mecamylamine seemed to be the most effective of the four nicotinic blockers tested, TEA the least effective. Hexamethonium and curare were effective at intermediate levels of concentration, with hexamethonium slightly more potent than curare. The latter point is illustrated in Fig. 2, which shows dose-response relations for hexamethonium and curare, both determined with the same driver-follower pair. The concentrations which reduced the e.p.s.p. amplitude to one-half were about 2 $l! for hexaBEFORE

lOO--

VOLUME

67,1978

methonium and about 3.5 w for curare. Sample records from this experiment (for 3 fl hexamethonium) are shown above the graph in Fig. 2; the arrows indicate the times of onset and cessation of hyperpolarizing pulses applied to the follower neuron to prevent the e.p.s.p.‘s from exceeding threshold. The concentrations of the four nicotinic blocking agents required to reduce the e.p.s.p.‘s to an undetectable level varied somewhat with the strength of transmission; larger e.p.s.p.‘s generally required a higher drug concentration. The concentrations of the four blocking agents which usually reduced transmission by more than 90% are shown in Table 1. These concentrations are somewhat lower than those generally used to block nicotinic transmission in intact ganglia (see Discussion). The fact that each of these agents eliminated

HEXAMETHONIUM

AFTER

0 HEXAMETHONIUM 0 d-TUBOCURARINE

. . 0 .

EPSP INHIBITION %

-.

.

so--

.

0

0 .

t

I 0.1

1.0 DRUG

CONCENTRATION

10.0

106.0

MM

FIG. 2. The concentration dependence of the synaptic blocking effects of hexamethonium and curare. Recordings from a driver-follower pair in a pL-15 CO2 culture: neurons, 20 days old; background layer of heart cells, 33 days old; the culture was y-irradiated 6 days before and 6 days after the neurons were plated. Upper part: Sample recordings of the e.p.s.p.‘s in the follower neuron (upper four traces in each group) evoked by action potentials in the driver (bottom traces); the arrows indicate the beginning and end of hyperpolarixing pulses applied to the follower neuron to keep the e.p.s.p.‘s from exceeding threshold. Lower part: ordinate, percentage reduction of e.p.s.p. amplitude ( ([&&]/&) ~100, where U, and fit are the mean e.p.s.p. amplitudes in the control and test perfusion solutions); abscissa, the concentration (@J of hexamethonium (0) or dtubocurarine chloride (0).

OILAGUE, POTTER, AND FURSHPAN TABLE

1

CONCENTRATIONSOFCHOLINERGICBLOCKING AGENTSUSUALLYREQUIREDTOPRODUCEGREATER THAN~O%BLOCKOFEXCITATORYCHEMICAL TRANSMISSION Concentration hJ.M Hexamethonium d-Tubocumrine Tetraethylammonium Mecamylamine Atropine sulfate

Cl Cl Cl HCl

30-100 50-150 >lOO 5-10” -60

Trials

45 29 4 4 11

D Each of the trials with mecamylamine was made with a small e.p.s.p. (2-4 mV); this may have exaggerated somewhat the effectiveness of mecamylamine relative to the other agents.

the excitatory transmission completely at moderate concentrations suggests that no transmitter other than ACh contributed significantly to production of the e.p.s.p.‘s. Obata (1974), Nurse and O’Lague (1975), and Nurse (1977) found that a-bungarotoxin had no effect on the excitatory transmission at concentrations (0.6-30 M which block transmission at the mammalian neuromuscular junction. Insensitivity to cu-bungarotoxin has been reported in intact mammalian and avian autonomic ganglia by Bursztajn and Gershon (1977), Brown and Fumagalli (1977), and Carbonetta et al., (1978).

Atropine Choline@ transmission in intact ganglia is sensitive to the muscarinic blocking agent atropine. At low concentrations (0.2-0.5 E.LM),this agent blocks the slow inhibitory and slow excitatory postsynaptic potentials recorded from principal neurons in the rabbit SCG (Eccles and Libet, 1961). At higher concentrations (greater than 10 fl, atropine also diminishes the fast e.p.s.p.‘s of intact ganglia, presumably by combining with the nicotinic ACh receptors of principal neurons (e.g., Eccles and Libet, 1961; Quilliam and Shand, 1964). At similar high concentrations, atropine also blocked the e.p.s.p.‘s in our cultures (Table 1; cf.

Cholinergic

Synapses

in Culture

429

Fig. 4 of O’Lague et al., 1974); presumably, this effect was also exerted on nicotinic receptors. We have seen no sign of slow e.p.s.p.‘s or i.p.s.p.‘s in the cultured neurons. This is consistent with evidence obtained by McAfee and Yarowsky (1977) that these slow responses do not occur in the intact SCG of the rat. We have not tested the effects of muscarinic cholinergic agonists.

Iontophoretic

Application

of ACh

To verify the presence of ACh receptors and to examine their spatial distribution, the iontophoretic drug application technique of Nastuk (1953) was used. Every neuron we have tested, in the several types of cultures, exhibited depolarizing responses to relatively small doses of iontophoretically applied ACh (see also Obata, 1974; O’Lague et al., 1974; Ko et al., 1976). With careful positioning of the ACh pipet (see below) and adjustment of the braking current (see Materials and Methods, and de1 Castillo and Katz, 1955) it was common to find depolarizations greater than 10 mV in response to lo-’ nC of charge passed through the ACh pipet (i.e., >lOOO mV/nC; Miledi, 1960). An example is shown in Fig. 3. The oscilloscope traces show the ACh potentials recorded from the neuronal cell body when the tip of the ACh pipet was at the indicated points. The ACh pulse was kept constant at lo-* nC; the number in each inset gives the ACh sensitivity in millivolts per nanocoulomb. Two points of application, one on the cell body and the other at the base of the upper cell process, had relatively high sensitivities. The two most distal sites of ACh application were on neurites at about 50 and 85 pm from the cell body. The observed sensitivity of the neurites in this and other similar experiments was not due to diffusion of ACh to the cell body region since displacement of the ACh pipet by 5 to 10 pm, transversely to the long axis of the neurite, abolished the response. We do not know whether the neurites that re-

430

DEVELOPMENTALBIOLOGY

VOLUME 67,1978

of a neuron to iontophoretically applied ACh; from a 24-day-old L-15 Air culture. FIG. 3. The sensitivity The insets show the responses (lower traces) recorded in a neuron cell body (furthest to the right in the phasecontrast micrograph) to ACh applied at the corresponding white dots; the iontophoretic pulses (upper traces) were constant at 5 nA and 2 msec (IO-’ nC). The vertical position of the ACh pipet was adjusted at each position to obtain the maximum response. The number in each inset gives the ACh sensitivity in mV/nC. TTX (3 CLM) was present throughout.

sponded to ACh were axons or dendrites, or both. It has been shown by Armett and Ritchie (1960) that unmyelinated axons in the vagus nerve of the adult rabbit are depolarized by ACh; of course, dendrites of sympathetic neurons receive cholinergic synapses and must also be sensitive to ACh. The observed sensitivities at the more distal points on the neurites were relatively low. However, these measurements probably underestimated the true sensitivity because of spatial decrement of the ACh potentials along the neurites; there was also uncertainty whether the ACh pipet was optimally localized on fine processes which

usually ran in bundles with many other processes (personal communications from P. Claude and S. Landis). In the experiment shown in Fig. 3, two other points on the cell body were tested (not shown). In each case the sensitivity was greater than 1000 mV/nC. This neuron had been grown in an L-15 Air culture for 24 days. A consistent feature of neurons grown in L-15 Air cultures was substantial sensitivity to ACh at most points on the cell body. In contrast, cell bodies of neurons grown in L-15 CO2 cultures were characterized by areas of low or undetectable sensitivity, and the patches of high sensitivity

OXAGUE,

POTTER,

AND FURSHPAN

Cholinergic Synapses in Culture

431

often had sharp borders and were some- evoked at 20 points, most of which were 5 times only a few micrometers in diameter. to 10 pm apart, are shown superimposed on This difference between the neurons in the a drawing of the neuron. The bars beneath the records indicate both the duration of two media is being investigated further. In the experiment shown in Fig. 4 a map the ACh pulse (kept constant at lop2 nC) of ACh sensitivity was determined for the and the position of the ACh pipet (with an cell body of a neuron grown 21 days in a uncertainty of ca. 1 pm). At two positions (the uppermost in column D and the lowpL-15 COz culture. The ACh potentials ermost in column A) the ACh potentials D 6 C A exceeded threshold; at these points the sensitivity exceeded 1200 mV/nC. The minimum sensitivity encountered (at the lowermost point in column D) was about 130 mV/nC. The slow rise time of the response at this position implies that the ACh diffused to nearby regions of higher sensitivity (e.g. Kuffler and Yoshikami, 1975); the true sensitivity at this point was therefore probably even lower. Thus the range of sensitivities in this experiment was probably greater than lo-fold. This is similar to the range of sensitivities observed by Harris et al. (1971) for innervated parasympathetic neurons in the frog heart. Another aspect of the nonuniformity of ACh sensitivity was that nearby test points could have quite different values. In the experiment shown in Fig. 4, the lowermost points in column A and B were separated by about 10 pm; their sensitivities differed by a factor of about 6. A similar range of nonuniformity was found in two other experiments in which systematic mapping of ACh sensitivity was made on neurons in FIG. 4. The distribution of ACh sensitivity on the CO2 cultures. In addition it was our expecell body of a neuron. From a pL-15 CO, culture; rience with neurons in CO2 cultures that neurons, 21 days in vitro; background heart-cell layer, very fine movements of the ACh pipet were 32 days in u&o; culture irradiated 9 days after the neurons were plated. ACh was applied iontophoretoften required to localize points of maximal ically to 20 points on the cell body surface; the recordsensitivity. At such points, movements of 3 ing site was also in the cell body. The ACh potentials to 5 pm usually caused a sharp reduction in are shown superimposed on a drawing of the cell body the amplitude of the ACh potential. Nonwith its nucleus and three nucleoli. The bar beneath uniformity of ACh sensitivity like that each record indicates both the position at which ACh was applied and the timing of the ACh pulse which shown in Fig. 4 was probably not due to was kept constant at 5 msec and 2 nA (lo-’ nC). The differences in access of the ACh pipet to ACh potential recorded at the lowermost position in the cell surface, since the surface of the cell column A and one of the two superimposed ACh bodies in these cultures appears in electron potentials recorded at the uppermost position in colmicrographs to be free of close investment umn D exceeded threshold and evoked action potentials (peaks off screen). by non-neuronal cells (Claude, 1973, and in

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preparation; O’Lague et al., 1975; Landis, in preparation). We did not encounter the et al. phenomena described by Harris (1971), a large increase in the amplitude and rate of rise of the ACh potential following a “current response,” and attributed by them to penetration of the satellite cell sheath by the ACh pipet. At each point of application, we routinely adjusted the vertical position of the microelectrode to maximize the amplitude of the ACh potential; however, sudden, discontinuous changes in response amplitude were not observed during this procedure. ACh sensitivity is known to be localized to synapses on skeletal muscle fibers in uiuo and in vitro (for discussion see Kuffler and Yoshikami, 1975; Fischbach and Cohen, 1973) and on amphibian parasympathetic neurons (Harris et al., 1971). It is plausible that the points of high sensitivity on these cultured sympathetic neurons were at or near sites of cholinergic synapses. ACh sensitivities greater than 2500 mV/nC were seen occasionally in such tests of localization, but it is possible that still higher sensitivities would be obtained, if synapses and pipets were visualized with Nomarski interference microscopy (e.g. Harris et al., 1971). Whether the high-sensitivity points correspond to synaptic boutons, and several related questions, are now being studied in this laboratory.

Comparison e.p.s.p.‘s

of

ACh

Potentials

with

Further evidence that ACh was the transmitter at the chemical synapses in culture was obtained by showing that the e.p.s.p.‘s could be readily mimicked in size and time course by iontophoretically applied ACh. An example is shown in Fig. 5. In this experiment, the driver neuron (D in the micrograph) and the follower neuron (F) with were impaled microelectrodes; e.p.s.p.‘s evoked in neuron F by action potentials in neuron D are shown on the righthand side of the two lower oscilloscope

FIG. 5. E.p.s.p.‘s and ACh potentials compared. From a 26-day-old L-15 CO2 culture. Responses were evoked in a follower neuron (F, in the phase-contrast micrograph) in two ways: (1) by applying ACh iontophoretically to a point (not shown) on the surface of ita cell body; (2) by evoking action potentials (not shown) in the driver neuron (D). ACh potentials shown at the left side, e.p.s.p.‘s at the right side, of the two lower traces. The current monitor (upper trace) shows first the ACh-ejection pulse (3 msec, 3nA) and then the stimulating pulse applied to the driver neuron. ACh sensitivity, ca. 1200 mV/nC.

traces. A third microelectrode filled with ACh . chloride was carefully maneuvered over the surface of neuron F until a high-

O’LAGUE,

POTTER,

AND

FURSHPAN

sensitivity point (1200 mV/nC) was encountered. Then the ACh-ejecting pulse was adjusted until the amplitudes of the ACh potentials (left side of the two sweeps) were similar to those of the e.p.s.p.‘s (right side of the two sweeps). It can be seen that the time courses of the two types of responses were very similar; the rise time of the e.p.s.p.‘s was about 2.9 msec, that of the ACh potentials, about 3.5 msec. In such an experiment, one might expect that if ACh was the transmitter responsible for the e.p.s.p., the e.p.s.p. and the ACh potential would be similarly affected by blocking drugs. This test was difficult to perform satisfactorily in practice because changes of a few micrometers in the position of the ACh pipet during application of the blocking agent and return to normal perfusion fluid markedly altered the size of the ACh potential. The experiment shown in Fig. 5 was one of the few in which the position of the ACh pipet was reasonably stable throughout several changes of perfusion fluid; a comparison of the effects on the ACh potential and e.p.s.p. of three concentrations of hexamethonium is shown in Fig. 6. Hexamethonium affected the two potential changes similarly but not identically. Identical effects would have been expected only if the ACh pipet and the driverneuron’s synapses delivered equal concentrations of ACh onto receptors with comparable spatial distribution. In this light, the similarity of sensitivity to hexamethonium of the two potential changes reinforces the other evidence that ACh was the transmitter. Effects

of the Catecholamines

Obata (1974) found that bath-applied norepinephrine (NE; 50-1000 @f) or dopamine (DA; 100-500 +%Q had no effect on the resting or action potentials of the sympathetic neurons cultured in Eagle’s Minimum Essential Medium. We found that these compounds and epinephrine (E), applied in ascorbate-containing perfusion

Cholinergic

Synapses in Culture

HEXA METHONIUM (PM) 0

4

18

36

0

\ EE

FIG. 6. A comparison of the effects of hexamethonium on the ACh potentials and on the e.p.s.p.‘s. From the same driver-follower pair as in the experiment of Fig. 5. The responses of the follower neuron to iontophoretically applied ACh and to an action potential in the driver neuron are shown on the three lower traces in each group of records, taken in each of the several indicated concentrations (a.MJ of hexamethonium; other details as for Fig. 5.

fluid at concentrations in the range 30 to 500 fl, had little effect on the resting potential of neurons cultured in L-15 COZ or pL-15 COZ; sometimes a slight hyperpolarization of slow onset was observed. The three catecholamines did, however, pro-

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duce a clear reduction in the strength of the cholinergic transmission in all trials in this concentration range. In contrast to the effects of the ganglionic blocking agents, the effects of the catecholamines were usually poorly reversible at these concentrations. In four experiments with 0.1 fl NE no effects were observed. An example of the effect of NE in the higher concentration range is shown in Fig. 7. The short-latency e.p.s.p.‘s of this driver-follower pair in a 17-day-old pL-15 COZ culture were variable in size, with a mean value of about 3 mV, in normal perfusion fluid (Fig. 7A). Perfusion with 188 fl NE for 10 min (Fig. 7B) reduced the average size of the e.p.s.p.‘s to about onehalf (ca. 1.4 mV); this effect was still present after about 15 min of washing with normal perfusion fluid (Fig. 7C). In two other experiments with NE (500 ,uM), small e.p.s.p.‘s were almost completely abolished. In the remaining six cases the e.p.s.p.‘s were reduced by 40 to 80% of their initial control amplitudes. In most experiments with NE, control solution was perfused for 15 to 20 min following the test period. In eight of the nine cases, no, or only partial, recovery of the e.p.s.p. occurred during this period; usually about one-half of the reduction in amplitude was restored. In one case recovery was nearly complete. In the case illustrated in Fig. 7, no recovery occurred and in another case in which the e.p.s.p.‘s were observed during 90 min of washing, after exposure to 125 @f NE, only slight recovery occurred. With such slow recovery it is difficult to distinguish persistence of drug effects from adventitious deterioration of synaptic function. It should be noted, however, that in most other experiments the amplitude of the e.p.s.p. was well maintained, often for several hours. The few experiments with DA and E gave results similar to those with NE. In a single experiment, DA (315 m reduced the e.p.s.p. by 46%; after 30 min of washing

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FIG. 7. The effect of NE on the e.p.s.p.‘s. From a pL-15 CO, culture; neurons, 17 days in uitro; background heart-cell layer, 19 days in. uitro. (A-C) e.p.s.p.-amplitude histograms. Evoked e.p.s.p.‘s were recorded in a follower neuron in control perfusion fluid (A), in 188 fl NE (B), and again in control fluid; no recovery had occurred after 15 min of washing with control fluid (C). N,r is the number of e.p.s.p.‘s recorded in each solution; U is the mean (+ SEM) of the e.p.s.p. amplitudes. with control solution, the e.p.s.p. had recovered to 86% of its pretest amplitude. In one experiment with E (180 I-1M) the e.p.s.p.‘s were reduced by 33%. Following a wash period of 20 min, practically no recovery took place (the e.p.s.p.‘s increased by 3% over the value in the test solution). In a second experiment with E (30 $M) there was only a slight reduction in e.p.s.p. amplitude (ca. 10%). Four experiments were made with adre-

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nergic blocking agents, two with the ,f3blocker, propranolol (39 I-1M), and two with the a-blocker, dibenamine (17 and 68 $kf). No effect was seen in any case, either on the membrane potential of the neurons or on the amplitude of the e.p.s.p.‘s. In experiments on microcultures (Furshpan et al., 1976), the excitatory interaction between sympathetic neurons was not affected by 0.6 to 1 @f propranolol, but the excitatory interaction between the neurons and heart myocytes was completely blocked; the latter result indicates that heart cells in our culture conditions retain and/or synthesize antagonist-sensitive, adrenergic receptors. The experiments described above with adrenergic agonists and antagonists indicate that the excitatory chemical transmission between the neurons was not mediated by catecholamines and are consistent with the conclusion that this transmission was mediated by ACh. DISCUSSION

The major purpose of the work described in this paper was to test whether the excitatory chemical transmission between the developing sympathetic neurons was sensitive to ganglionic blocking agents and whether the transmitter could be mimicked by iontophoretically applied ACh. The positive result of both tests suggests very strongly that transmission at these synapses was cholinergic, and that no other transmitter played a significant role (see also O’Lague et al., 1974,1975). This finding is consistent with two other lines of evidence. Patterson and Chun (1974) have shown that under similar culture conditions the neurons as a population synthesize substantial quantities of ACh (as well as catecholamines); Claude (in preparation; see O’Lague et al., 1974, 1975) and Landis (in preparation) have found numerous synaptic profiles of cholinergic appearance in electron micrographs of neurons grown in similar conditions. Burton et al. (1975), Johnson et al. (1976b), and Ko et al. (1976)

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have also reported evidence for formation of cholinergic synapses between developing rat sympathetic neurons cultured under rather similar conditions. The sensitivity of the chemical synapses in culture to ganglionic blocking agents (summarized in Table 1) was similar to, or greater than, that usually reported for the synapses between preganglionic fibers and principal neurons in mammalian SCGs in organ baths. For instance, Quilliam and Shand (1964) reported that the following drug concentrations were required to reduce the externally recorded orthodromic response to 50% in the rat SCG: hexamethonium, 50 r*M; curare, 59 fl; TEA, 485 fl, atropine sulfate, 50 fl. Dunant and Dolivo (1967) illustrated small fast e.p.s.p.‘s in the rat SCG even in the presence of 435 FM curare. Kosterlitz and Waller (1966) reported that depression of the initial component of ganglionic transmission to 11 to 23% in the rabbit SCG required 550 fl hexamethonium. Libet (1967) illustrated virtually complete elimination of the fast e.p.s.p.‘s in the rabbit SCG with 217 @f curare. It would not be surprising if the drug sensitivity of the cultured neurons was somewhat greater than that of neurons in intact ganglia simply because of the ready access of the drugs to the surface of the neurons in culture. It is clear, however, that the intensity of the transmission process is also an important factor in determining drug sensitivity in culture. Higher concentrations of blocking agents were required to eliminate one-to-one, short-latency interaction than to eliminate weak interaction. Moreover, we have found that to eliminate the still more intense transmission at nicotinic choline+ synapses formed by sympathetic neurons in microcultures (Furshpan et al., 1976), about lo-fold higher concentrations of hexamethonium or curare were required than were generally needed in the mass cultures described in this series of papers. Of course, the higher the concen-

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tration of ACh secreted at a synapse, the higher the concentration of a competitive blocking agent needed to produce a particular degree of block. In addition, the number of synapses of a certain efficacy formed by a driver neuron on a follower neuron and the density of postsynaptic receptors at each synapse will also affect the concentration of a drug needed for apparent elimination of transmission, that is, reduction of the e.p.s.p.‘s into the noise. The inhibitory effect of catecholamines on the chemical transmission in culture was expected from the extensive evidence that in the adult mammalian sympathetic nervous system the catecholamines depress secretion of ACh by preganglionic terminals, often hyperpolarize principal-neuron cell bodies and depress secretion of NE by terminals of the principal neurons; all these effects are sensitive to a-receptor blockers, and the order of activity of the three catecholamines in each case has been reported to be epinephrine > norepinephrine > dopamine (for discussion, see Libet, 1970; Christ and Nishi, 1971a,b; Dun and Nishi, 1974; Dun and Karczmar, 1977; Starke, 1977). The hyperpolarization of principal neuron cell bodies by E or NE has generally been found to be weak and variable; for example, Kobayashi and Libet (1970) reported that 150 fl NE produced an average hyperpolarization of rabbit principal neurons of about 4mV, but only in the presence of a monoamine oxidase inhibitor; Watson (1972) reported small hyperpolarizations of the rat SCG (external recording) by E or NE in the range 72 to 126 @. DA at rather high concentrations (50-200 $P0 has been reported consistently to produce small hyperpolarizations in rabbit principal neurons (e.g. Libet, 1970; McAfee and Greengard, 1972; Dun and Nishi, 1974). The sensitivity of cholinergic preganglionic terminals to block by the catecholamines is apparently somewhat higher; rabbit preganglionic terminals are consistently depressed by 10 fl E or NE and by 100 fl

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DA (Christ and Nishi, 1971b; Dun and Nishi, 1974). The sensitivity of adrenergic postganglionic terminals to block by catecholamines is apparently higher still; consistent effects have been reported with concentrations in the range 0.1 to 10 fi (for references seeStarke, 1977). It is not known whether the inhibitory effect of the catecholamines on cholinergic transmission in culture is the result of pre- or postsynaptic actions, or both. As in intact ganglia, the catecholamines produced weak, inconsistent effects on the membrane potential of the cultured neurons but consistently depressed cholinergic transmission in a concentration range 50 to 500 p&i. The size of the effect on cultured neurons was similar to that reported in vivo, but we used somewhat higher concentrations than are needed in vivo to depress the transmission from preganglionic cholinergic terminals or from postganglionic adrenergic terminals. We found that NE at 0.1 fl was ineffective but did not test any of the catecholamines in the range 0.1 to 30 PJJ One of the most interesting findings in the experiments reported here and in the preceding paper was that a substantial fraction of the developing principal neurons formed cholinergic synapses with each other when grown in permissive media (see also O’Lague et al., 1974, 1975; Burton et al., 1975; MacLeish, 1976, and in preparation; Ko et al., 1976). Two aspects of this finding have clear precedents in vivo and will be discussed in turn. (i) The expression of cholinergic functions by postganglionic sympathetic neurons. It has been known for many years that there are cholinergic principal neurons in certain mammalian sympathetic ganglia (e.g. Dale and Feldberg, 1934; Sjoqvist, 1963a,b); there may be a minority of cholinergic neurons in the SCG of the adult rat, for about 5% of the neurons are rich in acetylcholinesterase and lack catecholamine fluorescence (Yamauchi et al., 1973).

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Moreover, Crain and Peterson (1974) and Purves et al. (1974) reported that explants of developing mammalian sympathetic ganglia form cholinergic junctions with skeletal, cardiac, and smooth muscle in culture. Thus, an important and long-standing question about sympathetic principal neurons is how the choice between adrenergic and cholinergic transmission is controlled during development; this question is made the more interesting by the fact that the two transmitters have opposite effects on certain sympathetic target tissues. (ii) The formation of synapses by one sympathetic principal neuron on another. There is considerable evidence, from light and electron microscopy, for contacts between sympathetic principal neurons in uivo (e.g. de Castro, 1965; Grillo, 1966; Taxi et al., 1969; Jacobowitz, 1970; Elfvin, 1971; Job et al., 1971; Tamarind and Quilliam, 1971; Yokota and Yamauchi, 1974; see also Raisman et al., 1974; ijstberg et al., 1976; Purves, 1976). In the last three studies, done on decentralized rat and guinea-pig SCG, many of the “intrinsic” synapses on principal neurons appeared to be cholinergic. While the number of “intrinsic” synapses in small compared to that of the synapses formed by preganglionic axons (ijstberg et al., 1976), it is apparently not negligible. Given the extensive evidence that decentralized adult principal neurons accept cholinergic synapses from a variety of sources (for discussion see Dale, 1935; McLachlan, 1974; ijstberg et al., 1976; Purves, 1976) and given the evidence just mentioned that decentralized adult ganglia contain synapses of cholinergic appearance, one would expect to find functional cholinergic synapses between decentralized principal neurons in viuo, as in vitro; evidence for such synapses was recently obtained in the guinea-pig SCG by Purves (1976). While it is not yet known whether rat sympathetic principal neurons form cholinergic synapses with each other at developmental ages comparable to those of the cultured

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neurons, either in normal or in decentralized ganglia, there seems no present reason to consider this behavior abnormal. However, several other aspects of the observations reported here and several other recent findings on these neurons in culture have little or no precedent in uivo and were therefore surprising: (i) The influence of non-neuronal cells. A conspicuous feature of the CO* cultures, with their relatively high incidence of cholinergic driver neurons, was the presence of a large number of non-neuronal cells. Similarly, an obvious aspect of the pL-15 CO2 cultures in which there was early onset and consistent formation of cholinergic synapses was the continuous presence of a layer of non-neuronal cells from the time the neurons were plated (MacLeish, 1976, and confirmed here). MacLeish (1976) also found that the proportion of cholinergic driver neurons in pL-15 CO2 cultures rose as the density of cardiac non-neuronal cells in the preplated layer was increased. These observations on cholinergic synapse formation correlate well with evidence that a variety of non-neuronal cells favor the development of cholinergic metabolism in the neurons (Patterson and Chun, 1974; MacLeish, 1976; Ross and Bunge, 1976). Patterson et al. (1975) and Patterson and Chun (1977a) have found that this influence is exerted, at least in large measure, via the medium (“conditioned medium”), and efforts are now being made to identify the active agent or agents. The effect is graded, in that cultures grown in a higher proportion of conditioned medium exhibit greater enhancement of cholinergic metabolism (Patterson and Chun, 1977a). Landis et al. (1976) have shown in addition that the formation of synapses of cholinergic appearance (lacking small granular vesicles after KMn04 fixation) is enhanced in a graded way by conditioned medium. Cholinergic synapses and metabolism can be detected, although at low levels, in several culture regimes which nominally

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lack non-neuronal cells or medium conditioned by non-neuronal cells (e.g. L-15 Air cultures or CO2 cultures treated to eliminate almost all non-neuronal cells); this suggests that other features of the culture environment also influence transmitter metabolism (Patterson et al., 1975; Patterson and Chun, 1977a; Landis et al., 1976; Ko et al., 1976; Ross and Bunge, 1976; O’Lague et al., 1978b). A clear demonstration that chronic depolarization or activity diminishes responsiveness to conditioned medium was recently reported by Walicke et al. (1977). The control of transmitter functions by the environment of developing sympathetic neurons has been reviewed by Patterson (1978). (ii) Evidence that many of the cultured

neurons are transiently plastic with respect to transmitter choice. Several lines of evidence, taken together, leave little doubt that a majority of the neurons, although probably not all, can be induced to alter their transmitter from NE to ACh and that this plasticity is transient. (a) At least a majority of the neurons express several important adrenergic properties during the first week in culture. Johnson et al. (1976a,b) reported that during the first week, in a culture regime similar to ours, all the synapses made by the neurons on each other possessed a high percentage of small, granular vesicles after exposure to NE; this is evidence for the uptake and storage of NE. Landis has found that during the first week under our culture conditions (L-15 Air or L-15 CO2 plus conditionedmedium) all the synapses and varicosities, even without exposure to exogenous catecholamine, have a high percentage of small, granular vesicles (personal communication) and that all the growth cones examined also have these vesicles (Landis, 1978); this is evidence for synthesis and storage of NE. These observations lead to the conclusion that during the first week in culture all the neurons which grow processes and form synapses and varicosities synthesize, take

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up, and store NE. (b) When the neurons are grown for several weeks in the presence of large numbers of heart cells, a substantial proportion (more than 50% in some cultures) form cholinergic synapses with each other (MacLeish, 1976; this paper). Nurse (1977) found that at least 76% of neurons cocultured with skeletal myotubes were functionally cholinergic. It is also known that in cultures fed medium conditioned by nonneuronal cells, the expression of adrenergic functions falls as the expression of cholinergic functions rises; Landis et al. (1976) found a fall in the proportion of small, dense-cored vesicles in synapses and varicosities, and Patterson and Chun (1977a,b) found a fall in synthesis of catecholamines. These results demonstrate that non-neuronal cells produce a clear change in transmitter expression. A point of particular interest is that under certain culture conditions, all the neurons which grow and form synapses or varicosities display adrenergic properties at short times, and yet a majority of the neurons are functionally cholinergic at longer times (see also Johnson et al., 1976b). One possible explanation of points (a) and (b) is that the original cell suspension contained an adrenergic population of neurons and a second, potentially cholinergic population which is dependent in some way on non-neuronal cells or medium conditioned by such cells; in the absence of this influence the second population either fails to survive or grow or it fails to express transmitter functions (“silent” neurons). However, Patterson and Chun (1977a) have shown that conditioned medium does not affect either survival or growth of the neurons, and Reichardt and Patterson (1977) found no evidence for a population of “silent” neurons under “adrenergic” conditions. An alternative explanation of points (a) and (b) is that the original cell suspension contained a substantial population of neu-

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rons which express adrenergic properties at the outset but which shift to cholinergic function under the influence of non-neuronal cells. If this is the case, during the transition from adrenergic to cholinergic function, both properties might be expressed simultaneously, as adrenergic functions are phased out and cholinergic functions phased in. Such dual-function neurons have been found in microcultures 13 to 18 days old (Furshpan et al., 1976; Landis, 1976). This result is clearly consistent with transmitter plasticity. Two other results which are readily explained by assuming plasticity are that when mass cultures of neurons (about 5000 neurons per dish) are fed a medium containing a high concentration of conditioned medium, adrenergic metabolism declines as cholinergic metabolism rises (Patterson and Chun, 1977a,b) and the proportion of synapses of adrenergic appearance falls as the proportion of synapses of cholinergic appearance rises (Landis et al., 1976; see also Johnson et al., 1976b); evidently, there is a competition between expression of the two sets of functions. (c) Biochemical assays of single neurons grown in microcultures for 4 to 5 weeks indicate that the neurons eventually become either adrenergic or cholinergic; no clear evidence has been obtained for the presence of bifunctional or “silent” neurons at this more advanced culture age (Reichardt and Patterson, 1977). This suggests that the control of transmitter choice operates in a flip-flop manner (transition time unknown). (d) Sensitivity to conditioned medium is progressively reduced and becomes very low at 40 to 50 days in culture (Patterson and Chun, 1977b). Ross et al. (1977) and Hill and Hendry (1977) have provided evidence that a similar loss of plasticity occurs as the neurons mature in uiuo. All the present evidence is consistent with transient plasticity with respect to transmitter mechanisms, but the evidence is indirect; a direct demonstration might be

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obtained by following a single neuron with time to see if the.expected transition from adrenergic function to dual function to cholinergic function occurs. Such experiments are now in progress. It is naturally of interest to know whether adult neurons display plasticity with respect to transmitter choice; experiments which bear on this question have been done for many years. In an influential review, Dale (1935) summarized extensive crossunion experiments in the peripheral nervous system by saying “the results seem to tit well with our classification [of neurons] in terms of functional chemistry. They can be simply summarized by stating that any cholinergic fibre will functionally replace any other cholinergic tibre and that any adrenergic fibre will replace any other adrenergic fibre, but that neither can assume the function of the other”. This implies that when there is a mismatch between an adult axon and a target cell, neither can impose an appropriate transmitter function on the other; that is, transmitter plasticity is not displayed at these adult synapses. Many further cross-union experiments have been done on adult mammals since 1935, and they have generally been consistent with Dale’s summary. The few experiments known to us which suggest plasticity are the following (all done on cats). The possibility that adult adrenergic axons can form cholinergic synapses was raised by Koslow et al. (1971) and Kemplay and Garrett (1976). The possibility that adult cholinergic neurons can form adrenergic synapses was raised by Ceccarelli et al. (1972). The possibility that nominally noncholinergic adult sensory neurons can make choline@ synapses was raised by Fujiwara and colleagues (see Fujiwara and Kurahashi, 1976, for references) and by Vera and Luco (1967). Each of these experiments is flawed, either by the possibility that the new input included axons of the original transmitter class or by the possibility that there are cholinergic afferent neurons in the vagus

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nerve (cholinergic sensory neurons are apparently present at lower levels of the mammalian gut; e.g., Crowcroft et al., 1971; Purinton et al., 1971). Our conclusion is that there is no convincing demonstration of transmitter plasticity in the adult mammalian peripheral nervous system and that the question is still open. The possibility of transmitter plasticity during early embryonic development has been investigated by LeDouarin and her colleagues who have reported that, in the embryonic bird, neural-crest precursors of adrenergic and cholinergic autonomic neurons can be influenced with respect to transmitter as they migrate or after they reach their final ganglionic sites (LeDouarin and Teillet, 1974; LeDouarin et al., 1975, 1977). These experiments provide clear evidence for environmental control of transmitter expression in developing autonomic neurons but leave open the possibility that the local environment selects between two (or more) populations of neuroblasts or neurons, each already irreversibly committed to a single transmitter. This discussion can be summarized by saying that the dissociated sympathetic principal neurons in culture develop important physiological, morphological, and biochemical properties similar to those of adult principal neurons in vivo, in spite of the conspicuous alterations in the cellular and fluid environment of the cultured neurons. The properties displayed by the cultured neurons which are least precedented are the transient plasticity with respect to choice of transmitter, the susceptibility to control of transmitter choice by non-neuronal cells, and the ability to express both adrenergic and cholinergic functions simultaneously; it remains to be seen whether principal neurons in vivo also express these properties. An obviously abnormal behavior of the dissociated cultured neurons is their failure to establish the close and regular relationships with non-neuronal cells seen in vivo; it is not yet known whether this property is related to the fact that a

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majority of these sympathetic become cholinergic.

neurons

can

Dr. K. Obata participated in the early experiments on the effects of cholinergic agents (see O’Lague et al., 1974). We were dependent on the expert assistance of Delores Cox, William Dragun, Karen Fischer, Joseph Gagliardi, James LaFratta, Michael LaFratta, and Doreen McDowell, Much helpful advice was received throughout from P. Claude and P. Patterson. We thank R. E. Mains and Linda Chun for providing NGF and Dr. J. B. Little of the Laboratory of Radiobiology of the Harvard School of Public Health for use of the mCo source. Dr. D. McAfee kindly supplied us with unpublished manuscripts. This work was supported by NIH Research Grants NS-03273, NS-02253, and NS11576. P.H.O. was supported in part by Training Grant NS-05731. REFERENCES ARMETT, C. J., and RITCHIE, J. M. (1960). The action of acetylcholine on conduction in mammalian nonmyelinated fibres and its prevention by an anticholinesterase. J. Physiol. 152, 141-158. BROWN, D. A., and FUMAGALLI, L. (1977). Dissociation of a-bungarotoxin binding and receptor block in the rat superior cervical ganglion. Brain Res. 129, 165-168. BURSZTAJN, S., and GERSHON, M. D. (1977). Discrimination between nicotinic receptors in vertebrate ganglia and skeletal muscle by alpha-bungarotoxin and cobra venoms. J. Physiol. 269,17-31. BURTON, H., Ko, C.-P., and BUNGE, R. (1975). Cholinergic synapses between sympathetic neurons in tissue culture. Fifth Annu. Meet. Sot. Neurosci., Abstract 1251. CARBONETTO, S. T., FAMBROUGH, D. M., and MULLER, K. J. (1978). Nonequivalence of a-bungarotoxin receptors and acetylcholine receptors in chick sympathetic neurons. Proc. Nat. Acad. Sci. USA 75, 1016-1020. CECCARELLI, B., CLEMENTI, F., and MANTEGAZZA, P. (1971). Synaptic transmission in the superior cervical ganglion of the cat after reinnervation by vagus fibres. J. Physiol. 216, 87-98. CHRIST, D. D., and NISHI, S. (1971a). Site of adrenaline blockade in the superior cervical ganglion of the rabbit. J. Physiol. 213, 107-117. CHRIST, D. D., and NISHI, S. (1971b). Effects of adrenaline on nerve terminals in the superior cervical ganglion of the rabbit. Brit. J. Pharmacol. 41, 331-338. CLAUDE, P. (1973). Electron microscopy of dissociated rat sympathetic neurons in vitro. J. Cell Biol. 59, 57a. CRAIN, S. M., and PETERSON, E. R. (1974). Development of neural connections in culture. Ann. N.Y. Acad. Sci. 228, 6-34.

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CROWCROFT,P. J., HOLMAN, M. E., and SZURSZEWSKI, J. H. (1971). Excitatory input from the distal colon to the inferior mesenteric ganglion in the guineapig. J. Physiol. 219, 443-461. DALE, H. (1935). Pharmacology and nerve-endings. Proc.

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H. H., and FELDBERG, W. (1934). The chemical transmission of secretory impulses to the sweat glands of the cat. J. Physiol. 82, 121-128. DE CASTRO, F. (1965). Sympathetic ganglia, normal and pathological. In “Cytology and Cellular Pathology of the Nervous System” (W. Penfield, ed.), Facsimile of the 1932 ed. Hafner, New York. DEL CASTILLO, J., and KATZ, B. (1955). On the localization of acetylcholine receptors. J. Physiol. 128, 157-181. DUN, N., and KARCZMAR, A. G. (1977). The presynaptic site of action of norepinephrine in the superior cervical ganglion of guinea pig. J. Pharmacol. Exp. DALE,

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DUN, N., and NISHI, S. (1974). Effects of dopamine on the superior cervical ganglion of the rabbit. J. Physiol. 239, 155-164. DUNANT, Y., and DOLIVO, M. (1967). Relations entre les potentiels synaptiques lents et l’exitabihte du ganglion sympathique chez le rat. J. Physiol. (Paris) 59, 281-294.

ECCLES, R. M., and LIBET, B. (1961). Origin and blockade of the synaptic responses of curarized sympathetic ganglia. J. Physiol. 157, 464-503. ELFVIN, L.-G. (1971). Ultrastructural studies on the synaptology of the inferior mesenteric ganglion of the cat. III. The structure and distribution of the axodendritic and dendrodendritic contacts. J. Ultrastruct.

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FISCHBACH, G. D., and COHEN, S. A. (1973). The distribution of acetylcholine sensitivity over uninnervated and innervated muscle fibers grown in cell culture. Develop. Biol. 31, 147-162. FUJIWARA, M., and KURAHASHI, K. (1976). Cholinergic nature of the primary afferent vagus synapsed in cross anastomosed superior cervical ganglia. Life Sci. 19, 1175-1180. FURSHPAN, E. J., MACLEISH, P. R., O’LAGUE, P. H., and POTTER, D. D. (1976). Chemical transmission between rat sympathetic neurons and cardiac myocytes developing in microcultures: Evidence for cholinergic, adrenergic and dual-function neurons. Proc.

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M. A. (1966). Electron microscopy of sympathetic tissues. Pharmacol. Rev. 18, 387-399. HARRIS, A. J., KUFFLER, S. W., and DENNIS, M. J. (1971). Differential chemosensitivity of synaptic and extrasynaptic areas on the neuronal surface membrane in parasympathetic neurons of the frog, tested by microapplication of acetylcholine. Proc. Roy. Sot. London B 177,541-553. HILL, C. E., and HENDRY, I. A. (1977). Development

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of neurons synthesizing noradrenaline and acetylcholine in the superior cervical ganglion of the rat in vivo and in vitro. Neuroscience 2, 741-749. JACOBOWITZ, D. (1970). Catecholamine fluorescence studies of adrenergic neurons and chromaffin cells in sympathetic ganglia. Fed. Proc. 29, 1929-1944. JOHNSON, M., Ross, D., MEYERS, M., and BUNGE, R. (1976a). Changing synaptic vesicle cytochemistry in cultured sympathetic neurons. Sixth Annu. Meet. Sot. Neurosci., Abstract 1091. JOHNSON, M., Ross, D., MEYERS, M., REES, R., BUNGE, R,., WAKSHULL, E., and BURTON, H. (197613). Synaptic vesicle cytochemistry changes when cultured sympathetic neurones develop cholinergic interactions. Nature (London) 262, 308-310. Job, F., LEVER, J. D., IVENS, C., MOTTRAM, D. R., and PRESLEY, R. (1971). A fine structural and electron histochemical study of axon terminals in the rat superior cervical ganglion after acute and chronic preganglionic denervation. J. Anat. 110, 181-189. KEMPLAY, S. K., and GARRETT, J. R. (1976). Effects of heterologous cross-suture between the postganglionic sympathetic and the preganglionic parasympathetic nerve trunks of submandibular glands in cats. Cell Tissue Res. 167, 197-210. Ko, C.-P., BURTON, H., JOHNSON, M. I., and BUNGE, R. P. (1976). Synaptic transmission between rat superior cervical ganglion neurons in dissociated cell cultures. Bruin Res. 117,461-485. KOBAYASHI, H., and LIBET, B. (1970). Actions of noradrenaline and acetylcholine on sympathetic ganglion cells. J. Physiol. 208, 353-372. KOSLOW, S. H., STEPITA-KLAUCO, M., OLSON, L., and GIACOBINI, E. (1971). Functional reinnervation of cat sympathetic ganglia with splenic nerve homografts. Experientia 27, 799-801. KOSTERLITZ, H. W., and WALLIS, D. I. (1966). The effects of hexamethonium and morphine on transmission in the superior cervical ganglion of the rabbit. Brit. J. Pharmacol. 26, 334-344. KUFFLER, S. W., and YOSHIKAMI, D. (1975). The distribution of acetylcholine sensitivity at the postsynaptic membrane of vertebrate skeletal twitch muscles: Iontophoretic mapping in the micron range. J. Physiol. 244,703-730. LANDIS, S. C. (1976). Rat sympathetic neurons and cardiac myocytes developing in microcultures: Correlation of the fine structure of endings with neurotransmitter function in single neurons. Proc. Nat. Acad.

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LANDIS, S. C. (1978). Growth cones of cultured sympathetic neurons contain adrenergic vesicles. J. Cell Biol. 78, R8-14. LANDIS, S. C., MACLEISH, P. R., POTTER, D. D., FURSHPAN, E. J., and PATTERSON, P. H. (1976). Synapses formed between dissociated sympathetic neurons: The influence of conditioned medium.

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