Physiological and Morphological Studies on Developing Sympathetic Neurons in Dissociated Cell Culture

Transcription

1 Physiological and Morphological Studies on Developing Sympathetic Neurons in Dissociated Cell Culture P. H. O'LAGuE, P. i%. MAcLEIsH, C. A. NURSE, P. CLAUDE, E. J. FURSHPAN AI~D I). ]). POTTER Department of Neurobiology, Harvard Medical School, Boston, Massachusetts During the past several years, coordinated studies have been made in this laboratory of the physiology, biochemistry and anatomy of isolated sympathetic principal neurons developing in cell culture (Bray 1970, 1973; Mains and Patterson 1973a,b,c; Claude 1973; Bunge 1973; Obata 1974; O'Lague et al. 1974; Patterson and Chun 1974; Nurse and O'Lague 1975; Patterson et al., this volume). The neurons are dissociated from the superior cervical ganglia of newborn rats and grown in quasi-monolayer cultures, either alone or in the presence of nonneuronal cells. Although grown for days or weeks in fluid and cellular environments rather different from those in vivo, the neurons in culture are similar to their counterparts in intact animals with respect to their resting and action potentials, their high sensitivity to acetylcholine, their general fine structure, the differentiation of catecholamine synthesis, and the uptake, degradation, storage and release of norepinephrine. Unexpectedly, however, a large fraction of these postganglionic sympathetic neurons can, under certain culture conditions, synthesize substantial amounts of acetylcholine and form cholinergic synapses with one another and with skeletal myotubes. Patterson et al. (this volume) report that acetylcholine synthesis is strongly affected by nonneuronal cells, being negligible in the absence of these cells and substantial in their presence. This influence of nonneuronal cells is exerted, at least in part, by way of the bulk phase of the medium. Several types of nonneuronal cells are effective. In this paper, we describe certain aspects of these cholinergic synapses and some of the conditions that favor their formation. Among the principal points are the following: (1) The incidence of cholinergic synapses is usually high in the presence of nonneuronal cells and very low "in their absence, consistent with the effects on acetylcholine synthesis. However, in these experiments, we also made a number of changes in the medium, and therefore there is uncertainty about the relative contributions of the medium and the nonneuronal cells. (2) Under favorable conditions, the proportion of neurons making cholinergic synapses exceeds 50% and, given the sampling procedure used, is probably much greater. In many cultures, stimulation of a single neuron can give rise, after a delay, to a multiple synaptic volley in all of the other neurons in the same microscopic field; these "complex waves" indicate a high degree of synaptic connectivity among the neurons. (3) Although it has been reported that adult, postganglionic sympathetic neurons do not form effective junctions with denervated, adult skeletal muscle fibers (Langley and Anderson 1904a,b; Mendez et al. 1970), the developing cultured principal neurons form effective cholinergic junctions with skeletal myotubes. METHODS The culture conditions used in these experiments were in general similar to those reported earlier (Mains and Patterson 1973a; O'Lague et al. 1974). In brief, neurons obtained by mechanical dissociation of superior cervical ganglia of newborn rats were grown in one of three conditions: (1) in the virtual absence of nonneuronal cells, achieved by using "L-15 Air" medium in which the ganglionic nonneuronal cells fail to proliferate; (2) in the presence of gradually increasing numbers of ganglionic nonneuronal cells, as occurs in "L-15 CO2" medium; and (3) on a preexisting layer of heart cells, usually in a modified L-15 COs medium. This last condition was adopted following the observation (P. R. MacLeish, unpubl.) that the neurons grow more reliably and the neuron-neuron synapses form sooner on background monolayers of such cells. In cultures with preplated heart-cell layers, the L-15 COs medium was usually modified by the omission of methocel and bovine serum albumin and the substitution of fetal calf serum (10%) for rat serum. After formation of a monolayer, further proliferation of the preplated heart cells was usually suppressed by irradiation (5000 rad) from a 6~ In addition, the cultures were sometimes irradiated several days after the neurons were plated, in order to inhibit growth of the accompanying ganglionic nonneuronal cells. We saw no evidence that the neuronal function under study was affected by the irradiation. In some experiments, the sympathetic neurons were plated onto skeletal myotubes prepared by dissociating pectoral muscles of newborn rats with collagenase, preplating to reduce the number of fibroblasts, and allowing the myoblasts to divide and fuse for about 1 week; these cultures were irradiated shortly before the neurons were plated. The methods for making these cultures are described in detail elsewhere (Nurse and O'Lague 1975). 399

2 400 O'LAGUE ET AL. The electrophysiological and electron microscopic techniques were similar to those described in O'Lague et al. (1974). The visibility of the cultured sympathetic neurons, the absence of a nonneuronal cell covering from their upper surface, and the ready control of their fluid environment make it easier to study the electrical behavior of these cells as opposed to that of their counterparts in intact sympathetic ganglia. Synaptic interaction between the cultured neurons can readily be tested by impaling two cells simultaneously. The stability of the recording system permits continuous intracellular recording for many hours from a cell, or a pair of cells, during continuous perfusion. RESULTS The appearance of developing cultures in L-15 Air and in L-15 CO2 has been described previously (Mains and Patterson 1973a; O'Lague et al. 1974). Under the influence of nerve growth factor (NGF), the neurons grow long branching processes of rather uniform diameter (0.5 ~m or smaller). These processes form a network that increases in complexity with the age of the culture (in the present experiments, up to 6 weeks) and that brings each neuron into contact with many others. In L-15 CO2 cultures not irradiated after plating the neurons, the growth of the ganglionic nonneuronal cells produces a monolayer during the second week and thereafter a thickening carpet that progressively encloses and obscures the network of neurites. The phase microscopic appearance of such cultures during the second and third weeks is shown in Figures 2 and 6. The fine structure of the neurons (Claude 1973 and in prep.) is generally similar to that in developing rat superior cervical ganglia reported by Er/ink6 (1972) and in rather similar cultures of these principal neurons reported by Bunge et al. (1974) and Rees and Bunge (1974). A structural anomaly of L-15 CO2 cultures is that the neuronal cell bodies and neurites, although in contact with nonneuronal cells, do not acquire the close and regular investments by these cells characteristic of mature neurons in vivo. Consequently, much of the surface of the cultured neuron is exposed to the culture medium within the loose cellular network. The resting and action potentials routinely recorded from the cultured neurons in all three growth conditions were similar to those reported for intact rat superior cervical ganglia by Perri et al. (1970a). In the experiments reported here, the resting potentials were mv. Overshooting action potentials, mV in size, with a maximum rate of rise of V/s and a duration at half-amplitude of ms, were usually recorded. Synapses between Neurons Synaptic interaction was tested by impaling pairs of neuronal cell bodies with microelectrodes and then stimulating each cell in turn while recording from both. Functional neuron-neuron synapses were prevalent in cultures where nonneuronal cells were present but rare in cultures where nonneuronal cells were virtually absent (see Methods). Both chemical and electrical synapses were observed, but the electrical synapses were relatively rare, comprising a few percent of the total. Whether electrically or chemically mediated, the interaction was always excitatory. The frequency of occurrence of synapses and the timing of their formation are discussed in a later section. Chemical synaptic interaction was characterized by (1) a minimal synaptic delay of about 1ms between the peak of the presynaptic action potential and the foot of the excitatory postsynaptic potential (e.p.s.p.); (2) variability in the size of successive e.p.s.p.'s; the variation often appeared to be stepwise, suggesting quantal secretion of transmitter; (3) changes in the amplitude of the e.p.s.p, in the direction expected for chemical transmission following alterations in extracellular [Ca ++] and [Mg (4) lack of detectable transfer of maintained currents; and (5) sensitivity to conventional nicotinic ganglionic blocking agents "such as curare and hexamethonium. In contrast, electrical interaction was characterized by (1) no detectable transmission delay, the e.p.s.p, beginning to rise before the peak of the presynaptic action potential; (2) transfer of maintained potential changes of both polarities; and (3) insensitivity, in a few tests, to raised Mg + or to ganglionic blocking agents in the perfusion fluid. An example of electrical transmission is shown in Figure 1. Simultaneous intracellular recordings were made from neurons 1 and 2. When an action potential was directly evoked in neuron 1 (bottom trace, Fig. la), an e.p.s.p., about 3 mv in amplitude, began to rise nearly simultaneously in neuron 2; transmission also occurred in the opposite direction (i.e., stimulating neuron 2; Fig. lc). Transmission in either direction was unaffected when 180~M hexamethonium was supplied to the perfusion fluid (Fig. lb, d; cf. chemical synapses described below). Examples of chemical transmission are shown in Figure 2. When neuron 1 was stimulated with depolarizing current to produce an action potential, an e.p.s.p. (8 mv) was evoked in neuron 2 (records at upper left of picture) after a delay of 3.5 ms. In contrast, action potentials in neuron 2 did not give rise to detectable potential changes in neuron 1. The microelectrode was then withdrawn from neuron 2 and placed in neuron 3. When neuron 1 was again stimulated, activity appeared in neuron 3 on a one-to-one basis (records at lower right; delay ca. 6 ms). In this case, reciprocal excitatory chemical synapses were present (records at left center; delay, 5 ms). Evidence that transmission at the neuron-neuron synapses is mediated by acetylcholine acting on nicotinic receptors was obtained by applying a variety of ganglionic blocking agents and by applying acetylcholine electrophoretically to the neuronal cell bodies. It was consistently found that traditional nicotinic

3 Downloaded from symposium.cshlp.org on September 18, Published by Cold Spring Harbor Laboratory SYMPATHETIC NEURONS IN C U L T U R E ,.2na lores d " 2--I Figure 1. Electrical transmission between two neurons. Culture grown in L-15 CO2 (no heart cells present), irradiated (5000 tad) 15 days after plating, and recorded from on day 28. Two neurons (1 and 2) were impaled with microelectrodes, and recordings were made from both while stimulating either neuron 1 (a) or neuron 2 (c). In each set of records, the bottom trace shows the action potential of the directly stimulated neuron; the upper trace, the stimulating current supplied to that neuron; and the middle trace, the response in the other neuron. Note that before the peak of the action potential in the directly stimulated neuron, an e.p.s.p, began to arise in the other neuron. (b, d) As in a and c, but in the presence of 180 blm hexamethonium chloride. b l o c k i n g agents affected the e.p.s.p.'s at m o d e r a t e c o n c e n t r a t i o n s. T a b l e 1 gives the c o n c e n t r a t i o n s of several such agents r e q u i r e d for at least 9 0 % r e d u c tion in e.p.s.p, a m p l i t u d e ; t h e s e drug sensitivities are similar to those o b s e r v e d in intact ganglia (see O ' L a g u e et al. 1974). N o t e the a p p r o x i m a t e l y equal sensitivities to curare a n d h e x a m e t h o n i u m, in c o n t r a s t to the situation at t h e n e u r o m u s c u l a r j u n c t i o n (see below), a n d the relatively low sensitivity to a t r o p i n e Figure 2. Transmission at chemical synapses between neurons. Culture grown in modified L-15 CO2 (see Methods) on a background layer of irradiated heart cells; recordings made 15 days after plating neurons. In each pair of intracellular recordings, the action potential of the directly stimulated neuron is shown in the lower trace and the response of the other neuron in the upper trace. Note that neurons 1 and 3 each made synapses with the other, and that transmission from 1 to 3 was one-to-one. The evidence that synapses of this type were chemical and cholinergic is discussed in the text. c o m p a r e d to t h a t of muscarinic r e c e p t o r s in vivo. T r a n s m i s s i o n at t h e n e u r o n - n e u r o n synapses was completely e l i m i n a t e d by m o d e r a t e c o n c e n t r a t i o n s of the nicotinic blocking agents, a result suggesting n o t only t h a t t r a n s m i s s i o n is cholinergic, b u t t h a t n o o t h e r t r a n s m i t t e r m a k e s a n i m p o r t a n t c o n t r i b u t i o n to the g e n e r a t i o n of the e.p.s.p.'s. F u r t h e r e v i d e n c e consistent with cholinergic t r a n s m i s s i o n was o b t a i n e d by showing t h a t e l e c t r o p h o r e t i c a l l y applied acetylcholine p r o d u c e d d e p o l a r i z a t i o n s t h a t c a n b e m a d e to mimic closely the e.p.s.p.'s in size a n d time course. T h e sensitivity to acetylcholine applied i o n t o p h o r e t i c a l l y

4 402 O'LAGUE ET AL. Table 1. Effective Concentrations of Ganglionic Blocking Agents at Neuron-Neuron Chemical Synapses No. trials Conc. (txm) a d-tubocurarine 9 CI Hexamethonium 9 C Mecamylamine. HCI Tetraethylammonium 9 C Atropine sulfate b A compilation of results reported in O'Lague et al. (1974) plus observations from more recent experiments. a Concentrations are those required to reduce the e.p.s.p, amplitude by more than 90%. b TWO atropine moieties per molecule. was usually in the range mv/nc and was often sharply localized on the cell body surface in cultures exhibiting cholinergic interactions. The concentrations of curare and hexamethonium needed to block the acetylcholine potentials and the e.p.s.p.'s were roughly comparable. These observations leave little doubt that at the neuron-neuron synapses, the cultured principal cells secrete acetylcholine; however, they leave open the question whether catecholamines are also secreted. Patterson et al. (this volume) report that neurons in L-15 Air cultures (lacking nonneuronal cells and accumulating negligible acetylcholine) release norepinephrine, apparently by exocytosis, when depolarized by raised extracellular [K +] or veratridine. High concentrations of catecholamines produce inhibitory effects on principal neurons in vivo (see, e.g., Kobayashi and Libet 1970) and in our cultures (unpubl. prelim, exps.). It is plausible that neurons in L-15 CO2 cultures (possessing nonneuronal cells and, as a group, storing both norepinephrine and acetylcholine) also release norepinephrine when depolarized by action potentials, and that the failure to see any inhibitory postsynaptic effect was due to the low sensitivity of the cultured neurons to catecholamines (unpubl. exps.). When cultures exhibiting cholinergic interaction were examined in the electron microscope, synaptic endings were frequently observed on the neuronal cell bodies. In semiserial sections of one neuron, 24 endings were seen. In vivo, peripheral adrenergic junctions are known to differ in fine structure from cholinergic junctions in that they contain small, densecored vesicles (see e.g., Wolfe et al. 1962; Richardson 1966). Tranzer and Thoenen (1967) showed that this difference is enhanced by 5-hydroxydopamine (5-OH- DA), which increases the proportion of vesicles showing dense cores in adrenergic endings. When interacting cultures were fixed in aldehydes, followed by OsO,, the synaptic endings contained irregularly shaped small vesicles (30-70nm in diameter) which only very rarely had dense cores (cf. Rees and Bunge 1974). When one such culture was exposed to 5-OH- DA (60 O.M for 1 hr) before fixation, the synaptic endings on cell bodies contained some small vesicles with dense cores (example shown in Fig. 3), but the proportion of such labeled vesicles was low (10% of Figure 3. Electron micrograph of a nerve ending in contact with the cell body of an electrophysiologically identified "follower" neuron. The cells were grown in L-15 CO2 culture for 21 days, shown to interact physiologically, and perfused for 1 hr with 60 p,m 5-OH-DA. Arrows indicate some of the heterogeneously shaped small vesicles that contain a dense core as a result of the 5-OH-DA treatment. Magnification, 57, vesicles in 39 synaptic endings). Most endings contained some labeled small vesicles (82% of those endings containing more than ten vesicles total). These observations indicate that a high proportion of synaptic endings in interacting cultures can take up 5-OH- DA, a characteristic which they share with adrenergic terminals in vivo. A similar result was recently reported for cultures grown in a modified L-15 Air medium (i.e., in the absence of nonneuronal cells) by Rees and Bunge (1974). Complex Waves In addition to the short-latency, direct e.p.s.p.'s (Fig. 2), a more complex interaction was frequently observed (Fig. 4a). This "complex wave" differed from the simple e.p.s.p, in having a much greater latency (usually 20-50ms) and a much more variable waveform. It consisted of a volley of e.p.s.p.'s, which usually evoked one or more action potentials. A striking aspect of the complex wave was that although it was initiated by an action potential in a single cell, it came to involve a large population of neurons. In the example shown in Figure 4a, stimulation of one neuron of the recorded pair (lower trace) gave rise to similar, but not identical, complex waves in both cells. In several hundred cases of simultaneous recordings from two neurons in the same microscopic field (ca. 0.3 mm in diam.), a complex wave in one of the neurons was always accompanied by a similar wave in the other. In the case illustrated in Figure 4, the initial action potential in the follower cell (upper trace) happened to be important for the further spread of the complex wave. When this action potential was blocked by an appropriately timed hyperpolarization (during bar marked H, Fig. 4b), the rest of the complex wave

5 Figure 4. Complex synaptic interaction. Culture grown in modified L-15 CO2 (see Methods) on a background layer of heart cells. Intracellular recordings from two neurons (not shown) 41 days after plating. The "complex waves" in both neurons (shown in a) were evoked by a single, suprathreshold stimulus to one of them; time of application of current to the stimulated neuron (lower traces) indicated by broad arrows. When the initial action potential in the unstimulated neuron (upper trace) was blocked by a hyperpolarizing current (time of application marked by bar H in b), the rest of the complex wave failed to appear. Directly evoked action potentials in the stimulated neuron were retouched. SYMPATHETIC NEURONS IN CULTURE 403 either failed to appear or was delayed a further ms (not illustrated). The duration of complex waves was usually ms, and they often appeared spontaneously. The waves could only rarely be evoked at frequencies higher than 2-5 Hz. The low maximal frequency of the waves and their rather abrupt onset and termination suggest that their production depended on synapses that transmitted one-to-one (Fig. 4); such synapses usually failed at frequencies higher than 2-5 Hz. In all tests of cholinergic blocking agents, the waves were eliminated, demonstrating that they depended on cholinergic transmission within the network of neurites. These and the preceding observations imply a high degree of connectivity between the neurons. the same microscopic field). Each neuron of a pair was tested for its ability to evoke a response in the other (a short-latency e.p.s.p, or a complex wave), giving a total of 40 trials. Each point in Figure 5 gives the percentage of positive trials for a single culture of the indicated age; different symbols represent different platings. It can be seen that some interaction between the neurons was already present after a week in culture. The incidence of interaction remained low (10% or less) for about another week and then increased sharply. The mean incidence for the three cultures examined on day 18 was 35%, the highest value being 45%. In other experiments, the highest incidence observed was also about 50%. This is likely to be a serious underestimate of the proportion of cholinergic Incidence of Cholinergic Interaction The probability that two neurons impaled at random were linked by chemical synapses generally increased with age in culture. In L-15 Air cultures, which contained few, if any, nonneuronal cells, cholinergic interaction was seen only in a few cultures, all older than 5 weeks, a finding consistent with the demonstration by Mains and Patterson (1973a) and Patterson and Chun (1974) (see also Patterson et al., this volume) that acetylcholine synthesis and accumulation occurs only in older cultures and in small amounts (unless the L-15 Air medium is conditioned by exposure to nonneuronal cells). In L-15 CO2 cultures without preplated monolayers of heart cells, cholinergic e.p.s.p.'s were rarely seen before 21 days and usually only after 3-4 weeks. In L-15 CO2 cultures with preplated monolayers of nonneuronal cells, interaction was seen as early as 4 days after plating. The increase with time of the probability of cholinergic interaction in cultures containing monolayers of cardiac cells is illustrated in Figure 5. In this series (three platings of five cultures each), the heart cells were grown for 6-8 days, to form a complete monolayer, and then irradiated to suppress further proliferation (see Methods). Neurons were then added (day 0) and, starting one week later, they were tested for the presence of synaptic interaction with the following sampling procedure: In each culture, 20 pairs of neurons were chosen at random (except that both neurons of a pair were always within neuron-neuron interaction % ~ 20. I I age of neurons in culture-days Figure 5. The development of synaptic interaction between neurons. Cultures grown in modified L-15 CO2 medium (see Methods); neurons plated on background layers of heart cells. Three platings of five cultures each. Twenty pairs of neurons in each culture were selected at random, except that both neurons of each pair were in the same microscopic field (diam. 0.3 mm). Each point gives the percentage of neurons evoking a response (short-latency e.p.s.p, or complex wave) in the other neuron of the pair; both neurons of each pair were tested. Recordings were made on the days indicated after plating neurons; different symbols indicate different platings. t

6 Downloaded from symposium.cshlp.org on September 18, Published by Cold Spring Harbor Laboratory 404 O ' L A G U E ET AL. "driver" neurons since failure to interact with.a single nearby neuron did not exclude interaction with the many (usually several thousand) other neurons in the culture. The inference is that well over half the neurons in such cultures were capable of secreting acetylcholine. As noted in the preceding section, complex waves, once initiated, were seen in every impaled neuron, implying that all received cholinergic synapses. Formation of Cholinergic Junctions with Skeletal Myotubes When it was found that the cultured sympathetic principal neurons were capable of forming nicotiniccholinergic synapses with each other, it was natural to wonder whether they would do so with skeletal muscle cells, which also normally receive a nicotiniccholinergic innervation, but from a different anatomical source. In cultures prepared as described in Methods, it was found that the cultured neurons did in fact readily form effective junctions with these developing skeletal myotubes (Nurse and O'Lague 1975) (see example in Fig. 6). First, a neuron labeled D ("driver") and a myotube (M) were each impaled. Stimulation of the neuron evoked excitatory junction potentials (e.j.p.'s) in the myotube (top inset). The delay between the peak of the neuronal action potential (time of occurrence indicated by the arrowhead) and the e.j.p.'s was about 3 ms. According to the criteria discussed above for the neuron-neuron synapses, the interaction between neurons and myotubes was chemically mediated and, like the neuron-neuron chemical synapses, was cholinergic. However, the sensitivities of the two types of junctions to receptor blocking agents were quite different. At the neuron-neuron synapses, curare and hexamethonium were about equally effective; either one abolished the e.p.s.p, at about 50/~M. At the n e u r o n myotube junctions, the e.j.p, was abolished by 1-2 /xm curare but only reduced to about one-half amplitude by 100/.LM hexamethonium. A striking difference in sensitivity to a-bungarotoxin was also observed: the e.j.p.'s were completely blocked by about 0.2/xM, whereas the e.p.s.p.'s at the neuron-neuron synapses were entirely unaffected by three times that concentration (see also Obata 1974). The neuron (D) in Figure 6 was one of several that have been observed to interact, after a short latency, with both a myotube and a neuron; in the bottom inset, the responses of the "follower" neuron (F) (upper three traces) to action potentials in neuron D (lower trace; three action potentials superimposed) are shown. These e.p.s.p.'s were almost completely blocked by 37 ~xm hexamethonium; however, the e.j.p.'s in the myotube were unaffected by this concentration, although they were abolished by 1.4 ~M curare. The persistence of the e.j.p.'s in 37 /XM hexamethonium is consistent with a monosynaptic connection between D and M; cholinergic interneuronal Figure 6. Synaptic transmission from a common presynaptic sympathetic neuron to a myotube and another sympathetic neuron. The phase contrast micrograph (a montage) shows the "driver" neuron (D) within a small clump of neurons, a striated myotube (M) and another sympathetic neuron (F). Culture grown in modified L-15 CO: medium. Neurons plated 7 days after myoblasts and recordings made 9 days later. (Top inset) Excitatory junction potentials (e.j.p.'s) recorded in myotube M. Arrowhead indicates time of peak of action potential in D. Oscilloscope beam displaced between sweeps. (Bottom inset) Top three traces (beam displaced between sweeps) show e.p.s.p.'s recorded in neuron F due to three consecutive action potentials (bottom sweep, three traces superimposed) in D. Black bar indicates duration of depolarizing current pulse in D.

7 SYMPATHETIC NEURONS IN CULTURE 405 synapses presumably would have been blocked by this treatment. This argument does not rule out an interneuron activated one-to-one by electrical transmission. The delay in the neuron-neuron interaction (bottom inset, Fig. 6) was 2.6 ms. The time for impulse propagation in the neurite of cell D probably accounted for a major fraction of this delay, consistent with a monosynaptic link in this pathway as well. The acetylcholine receptors of the neurons and myotubes in culture appear to have pharmacological properties similar to those reported for their adult counterparts in vivo. The characteristic differences between the neuron and myotube receptors are present, even though both cells share the same environment and appear to have the same type of neurons (and probably in some cases the same individual neurons) as presynaptic elements. It seems, not surprisingly, that the postsynaptic elements have a major role in determining the characteristics of their own receptors. DISCUSSION The findings on cultured sympathetic neurons reported in this paper and in the accompanying paper by Patterson et al. (this volume) are complementary as they relate to acetylcholine. Under appropriate conditions (e.g., the presence of nonneuronal cells), acetylcholine synthesis and accumulation is substantial, and the formation of chotinergic junctions is prevalent. A rough correspondence of biochemical and physiological findings is not surprising, but it should be emphasized that it is not yet known whether acetylcholine synthesis and formation of cholinergic synapses are tightly linked or dissociable functions in these neurons. The apparent role of nonneuronal cells in accelerating the formation and increasing the incidence of cholinergic neuron-neuron synapses requires additional comment. The most striking difference in the incidence of synapses was between cultures with virtually no nonneuronal cells (grown in L-15 Air) and those with either ganglionic or heart cells present (grown in L-15 CO2). In the biochemical experiments (Patterson et al., this volume), controls were made for the change in the composition of the medium. For example, it was shown that acetylcholine synthesis was enhanced by adding nonneuronal cells to neurons growing in L-15 Air medium as well as in L-15 CO2. Comparable controls have not yet been made in the physiological experiments, so that uncertainty remains about the relative importance of the medium and the nonneuronal cells in enhancing synapse formation. In addition, preliminary experiments suggest that the choice of the serum added to the medium also influences synaptogenesis. The findings with respect to catecholamines are not yet complementary. Although it is clear from biochemical studies on L-15 Air cultures (Patterson et al., this volume) that catecholamines are released in response to depolarization by raised [K+], no physiological evidence has been obtained for catecholamine secretion at junctions formed by the neurons with each other or with skeletal myotubes. Both types of target cell are quite insensitive to catecholamines, and it may be necessary to culture the sympathetic neurons with a more sensitive target cell in order to observe secretion of catecholamines physiologically. Such cultures are now being made. One of the more striking findings in this study was that in favorable culture conditions, the proportion of cholinergic "driver" neurons was greater than 50%. Although there is evidence for the presence of conventional cholinergic principal neurons in certain sympathetic ganglia, the histochemical evidence so far available suggests that the proportion of those neurons in the superior cervical ganglion of the adult rat is only about 5% (Yamauchi et al. 1973). The high proportion of acetylcholine-secreting neurons in culture might be (1) an expression of a normal property of sympathetic principal cells at a comparable stage of development; (2) the result of selection in culture favoring cholinergic neurons present in our original cell suspension; (3) an uninteresting artifact of culture; or (4) the result of conversion of developing neurons, which would otherwise be adrenergic, into two populations (adrenergic and cholinergic) or into a single population of mixed transmitter function, as might occur if normal developmental cues were absent. These possibilities are discussed by Patterson et al. (this volume). In this context, it may be said that none of the techniques we have so far used reveals the presence of two classes of neurons, but neither is any of these techniques decisive. Several methods are now being used to investigate this matter further. One of them is to identify cholinergic "driver" neurons with electrophysiological methods and then to use the Falck- Hillarp histochemical technique to test for the presence of catecholamines in the same neurons. So far, the results have been ambiguous because in L-15 CO2 cultures, the specific formaldehyde-induced fluorescence is often not much greater than the native fluorescence seen in the absence of formaldehyde treatment. Variations in this technique are now being tried. Whatever the origin of the substantial cholinergic activity of the cultured neurons, the fact that they form effective synapses with each other and with skeletal myotubes invites further comment. Neuron-Neuron Synapses There are a number of light and electron microscopic reports that postganglionic sympathetic neurons are in contact with each other in normal and in decentralized ganglia, including the superior cervical ganglion of the rat, and that some of these contacts are clearly synaptic (for a review of light microscopic findings, see Jacobowitz 1970; for a review of electron microscopic evidence, see Matthews 1974).

8 406 O'LAGUE ET AL. Although such contacts have not yet been demonstrated in developing ganglia at postnatal ages similar to those of the cultured neurons, there does not appear at present to be any reason to consider abnormal the existence of synapses between the principal neurons in culture. What is less expected from microscopy of intact ganglia is that the synapses in culture are cholinergic; the contacts and synapses in vivo have generally been interpreted as adrenergic because they contain small, dense-cored vesicles. Rees and Bunge (1974) similarly interpreted synapses seen in electron micrographs of rat sympathetic neurons cultured in a modified L-15 Air medium. However, there is no unambiguous physiological evidence concerning the function of the neuron-neuron synapses in adult or developing rat ganglia (i.e., whether they are adrenergic, cholinergic, or both) and thus no satisfactory basis at present for deciding whether cholinergic neuronneuron synapses in culture are unusual (see, however, Perri et al. 1970b). Whether sympathetic principal neurons normally form cholinergic synapses with each other or not, it is noteworthy that adult sympathetic neurons deprived of their preganglionic input do accept cholinergic axons from anatomically novel sources (for a review of the extensive literature, see McLachlan 1974)- cultured neurons also might be expected to do the same. Neuron-Skeletal Myotube Junctions The formation of these junctions may seem surprising because adult skeletal fibers are not a normal target for postganglionic sympathetic axons, nor have direct attempts to reinnervate denervated adult skeletal muscle fibers with such axons been successful (the literature is discussed by Nurse and O'Lague 1975). However, developing sympathetic neurons and skeletal muscle fibers may display properties different from those of adult tissues, and the behavior of these cells at postnatal ages comparable to those of the cultured neurons has not yet been investigated in vivo. It seems of interest that the formation of neuronneuron and neuron-myotube junctions in culture is at least superficially consistent with an inference drawn by Dale (1935). On the basis of the extensive experiments of Langley and Anderson (1904a,b) on reinnervation of denervated peripheral tissues, Dale concluded: "...the results seem to fit well with our classification [of neurons] in terms of functional chemistry. They can be simply summarized by stating that any cholinergic fibre will replace any other cholinergic fibre, and that any adrenergic fibre will replace any other adrenergic fibre, but that neither can assume the function of the other." One aspect of Dale's inference is self-evident; if a neuron and a target cell are to form an effective junction after they have come in contact, there must be a match between the transmitter of the neuron and the receptors of the target cell. Our observations on cells in culture are consistent with this suggestion. In the L-15 Air cultures, the neurons express a variety of adrenergic functions, possess acetylcholine receptors, but make little acetylcholine (Patterson et al., this volume); in this condition, the ability of the neurons to form junctions with one another appears to be slight. In contrast in L-15 CO2 cultures (nonneuronal cells present), the neurons synthesize appreciable acetylcholine, have acetylcholine receptors, and, in this condition, readily form cholinergic synapses with one another. Similarly under culture conditions that favor acetylcholine synthesis, these neurons form cholinergic junctions with skeletal myotubes, although adult postganglionic sympathetic neurons (mainly adrenergic) are reported as not forming effective junctions with adult skeletal muscle fibers. Another aspect of Dale's suggestion seems more interesting; when there is an initial mismatch, failure to form a junction indicates that neither cell can impose a more appropriate function on the other. Neurons grown in L-15 Air (very few nonneuronal cells present) were unable to induce acetylcholine synthesis and cholinergic synapse formation in one another. It will be interesting to see how widely Dale's simple rule applies during development. If the factors governing synapse formation are so simple after contact has occurred between a neuron and a target cell, the specificity of adult neuronal connections must depend on other mechanisms during the process of innervation. Acknowledgments Expert assistance was provided in various aspects of this work by Delores Cox, William Dragun, Karen Fischer, Joseph Gagliardi, James La Fratta, Michael La Fratta and Doreen McDowell. We thank R. E. Mains and Linda Chun for providing nerve growth factor. This work was supported by National Institutes of Health Research Grants: NS 03273, NS and NS In addition, P.H.O. was partially supported by Training Grant NS05,731 and P.C. by Research Fellowship NS2612. 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10 Physiological and Morphological Studies on Developing Sympathetic Neurons in Dissociated Cell Culture P. H. O'Lague, P. R. MacLeish, C. A. Nurse, et al. Cold Spring Harb Symp Quant Biol : Access the most recent version at doi: /sqb References This article cites 27 articles, 11 of which can be accessed free at: Creative Commons License Alerting Service Receive free alerts when new articles cite this article - sign up in the box at the top right corner of the article or click here. To subscribe to Cold Spring Harbor Symposia on Quantitative Biology go to: