Brain Research, 117 (1976)437--460 © Elsevier/North-Holland Biomedical Press, Amsterdam - Printed in The Netherlands

437

SYNAPTIC T R A N S M I S S I O N BETWEEN R A T SPINAL C O R D EXPLANTS A N D DISSOCIATED SUPERIOE. C E R V I C A L G A N G L I O N N E U R O N S 1N TISSUE C U L T U R E *

CHIEN-PING KO**, HAROLD BURTON*** and RICHARD P. BUNGE Departments of Physiology-Biophysics and Anatomy=Neurobiology, Washington University School of Medicine, St. Louis, Vlo. 63110 (U.S.A.)

(Accepted April 5th, 1976)

SUMMARY Physiological properties of the synapses formed between explants of spinal cord and dissociated autonomic ganglion neurons in tissue culture were studied using intracellular and extracellular stimulation and recording techniques (as well as iontophoresis) with a culture perfusion system allowing continuous microscopic observation during repeated changes of the bathing medium. The principal neurons of the superior cervical ganglion (SCGN) were dissociated from perinatal rats and the spinal cord explants were obtained from 15-day rat fetuses; these were allowed to mature for 3-10 weeks in co-culture. Recordings from over 1000 SCGN established that: (a) spontaneous small depolarizations and action potentials occurred in 20% of the SCGN studied, (b) the EPSPs observed in SCGN after spinal cord stimulation were sensitive to decreased Ca ~+ and increased Mg 2+, as well as to D-tt, bocurare, hexamethonium and mecamylamine, but not to atropine (at 10-6M concentration) or to the aipha-adrenergic blocking agents phentolami~z or phenoxybenzamine; no potentiation of the EPSPs was seen with neostigmine or esedne, (c) acetylchol|ne directly applied to the SCGN was seen to mimic the responses seen after spinal cord stimulation; tetrodotoxin blocked both direct and iontophoretically fired action potentials, with only a suprathreshold acetylcholine potential remaining, l hese synapses were not sensitive to a-bungarotoxin. It is concluded that the synapses formed by spinal cord neurites on principal SCGN in tissue culture are nicotinic cholinergic, and that

* Part of this study published in: C.-P. Ko, H. Burton and R. Bunge, Cholinergic synapses between spinal cord and sympathetic neurons in tissue culture, Neurosci. Abstr., 1 (1975) 816. ** Present address: Department of Anatomy, University of Colorado School of Medicine, Denver, Colo., U.S.A. *** Please address t.eprint requests to: Dr. H. Burton, Department of Anatomy and Neurobiology, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, Mo. 63110, U.S.A.

438 the evoked EPSPs recorded in this study are thus similar to the orthodromic fast EPSPs observed in vivo. No slow synaptic responses were observed and no demonstrable effects were noted that could be attributed to adrenergic transmission.

INTRODUCTION

In previous studies n.es, we demonstrated that nerve fibers from rat thoracic spinal cord explants formed synapses on dissociated cell or organotypic cultures of rat superior cervical ganglia, and that the morphology of these endings resembled the preganglionic innervation of sympathetic ganglia noted in the animalge-2s,59,53,sl. This innervation was noted on the principal cells of the ganglia and consisted of asymmetric contacts where the presynaptic component contained clear round vesicles of 40-50 nm. Preliminary analyses indicated that the small intensely fluorescent cells known to be present in this ganglion in vivo 52'Sa,sl did not survive in the isolated cell cultures and only the principal superior cervical ganglion neuron (SCGN) was available for innervation. The purpose of the present study was to characterize the preganglionic input formed on a homogeneous ganglionic neuronal population of known identity by physiological and pharmacological methods. Previous anatomical studies had indicated that synapse formation in this culture system has some degree of specificity; thus, superior cervical ganglion neurons were shown to be selectively innervated by spinal cord rather than cerebral cortex tissue in culture 05. It was, therefore, anticipated that the synaptic physiology of connections formed would be comparable to the wellcharacterized, preganglionic innervation of these neurons in the animal. This comparison provided a specific reference for analyzing the expression of synaptic properties in culture. In addition to the connections from the spinal cord explants, some or all of the dissociated SCGN in culture may be innervated by nerve fibers from neighboring dissociated sympathetic neurons. The study of these interconnections between dissociated sympathetic neurons is presented in the following paper 4e. I~4ETHODS AND MATERIALS

(I) Culture preparation Experiments were conducted with 72 nerve tissue cultures that consisted of dissociated superior c~rvical ganglion neurons prepared from 19-21-day Holtzman rat fetuses (or l-2-day newborns) and explants from thoracic spinal cord taken from 15.5 day rat fetuses. The SCGN were dis~oc!ated mechanically by the method of Bray v in a modified medium (see Table I) and were seeded onto collagen-coatede dishes molded from Aclar plastic that were carried in altered Falcon petri dishes TM. During the first 24 h the SCGN settled onto the collagen and began to send out processes. At this time two to three 0.5 mm thick transverse sections from embryonic thoracic spinal c~;rd were placed around the center of the dish. The spinal cord tissue had the

439 TABLE I

Component amounts for making 104 ml of media

Leibovitz-15 Eagle's MEM without glutamine Human placental serum Chick ~9-day) embryo extract 20 % glucose 0.15 M KCI 200 mM L-glutamine Units of NGF* FUDR** Uridine Penicillin G (units) Streptomycin ~ulfate

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meninges removed and was opened out in the mid-dorsal line to form a flattened, often heart-shaped explant with the original ventral horns situated at the apex 1i. Because the spinal cord tissue did not thrive in the modified Bray's medium, the cultures were carried for the next 7-10 days in a medium of intermediate 'richness" to foster spinal cord explant development and suppress the outgrowth of non-neuronal cells (medium II, Table I). They then received a 2-day treatment with 10-5 M cytosine arabinoside to further suppress subsequent non-neuronal cell growth. The cultures were thereafter can'ied in a standard medium (see Table I) until the recording experiments when they were bathed with Eagle's minimum essential medium (MEM) with added glucose and antibiotics (Table I). All cultures were refed 3 times weekly and were incubated at 35-36 °C in air (with Bray's medium) or 5 % CO2 (with the other media); the pH of all bathing solutions was maintained at about 7.4. (I1} Maintenance o f cultures during recording experiments In preparation for intracellular recordings, cultures of 3-10 weeks of age were placed on a rigid platform which was mounted over a Zeiss inverted microscope equipped with phase contrast optics. Tracking the alignment of the microelectrodes with respect to particular cells was accomplished by sliding the microscope on a grease plate. The cultures were maintained and the effects of various drugs were studied by using a continuous perfusion system that delivered and withdrew media from the

44O Aclar dish at a rate of approximately 0.4 ml/min from one of 6 difIerent reservoirs. It took about 4 min to fill the 1.5 ml dish, includi~.g dead space in the delivery tubing, and about 20 min after switching to a new solution 99 % of the initial medium on the dish was replaced (as measured by dye dilution). Changes in neuronal activity were generally monitored only after 5-10 min of perfusion with a new drug, 15-20 min were frequently allowed for final effects to occur and, at least 20 min of perfusion with control medium was used to demonstrate recoveries. During the extensive pharmacological analyses 3-4 h of continuous intracellular recordings from a single neuron were often achieved. The cultures and all perfusion media were kept at 34-36 °C by enclosing the entire system in an insulated chamber warmed by infra-red lamps. The perfusion system allowed changes either in the ionic composition or in the drug content of the bathing medium. The effect of raising the Mg2÷/Caz+ ratio was tested on evoked and spontaneous activity. The role of acetylcholine was examined by noting the effects of medium containing one of the standard nicotinic (D-tubocurare, hexamethonium bromide or mecamylamine HCI) or muscarinic (atropine sulfate) blocking agents and by noting the consequences of anticholinesterase agents (eserine or neosfidmine methylsulfate). The role of catecholamines in the recorded synaptic activity was investigated by perfusion with phenoxybenzamine (an alpha-adrenergic blocking agent). The concentrations u~ed for these various drugs are stated in the results.

(!il) Recording methods Intraceilular recordings were made with l M potassium citrate filled electrodes with DC resistances between 70-200 Mf~. These electrodes were connected to W-P Instrument electrometers whose output was recorded on a 502 Tektronix oscilloscope. The noise level of the recording system, including the electrodes, was approximately 0.5 mV and the rise time was at least 25/~sec. All electrodes were filled by the fiber glass technique. Prior to filling, each electrode was bent at an angle of 30-45 ° 4-5 mm from t.he tip in order to facilitate a vertical approach to the neurons. Focal extracellular stimulation was delivered through bipolar glass microelectrodes filled with 4 M NaC! in 0.5 % agar. Tip diameters of these electrodes were 10-20/~m and DC resistances were 5-10 Mf~. Electrical stimulation was localized to a small region of less than 5/~m by independently adjusting the distance between the tips of the stimulating electrodes. In searching for synaptic transmission from the spinal cord tissue to a SCGN, the stimulating electrodes were moved to several loci over the explants or across the neuritic bundles contiguous with the spinal cord tissue. Stimulus parameters varied at each position such that stimulus voltage ranged from l to 100 V, durations were 0.i-I msec and stimulus repetition rate was 0.5-10 Hz. All stimulus parameters were controlled by a multichannel W-P Instrument stimulator (Model 831). Positive iontophoretic currents of 1-100 nA and usually 0. I-2 msec duration were injected through 100-200 M f~ electrodes filled with 1 M ACh. Currents were kept constant by using a 109 f~ resistance in series with the electrode and current levels were monitored through the ground circuit which formed one of the inputs to

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Fig. I. A camera lucida drawing of an isolated SCGN stained with Sudan black and showing the extensive branching of processes emanating from the proximal neurites and soma. Processes that could be traced back to the cell body or proximal neurites are drawn although all of the most distal branches may not be from the same cell since it is difficult to track these fine neurites when they taper to their terminal points. A clearly demarcated axon was not noted in this cell. Original drawing made at × 1200 magnification with phase contrast optics.

442 an operational amplifier. Under visual control, the tips of the iontophoretic electrodes first touched the soma or proximal processes so that current injection directly triggered an AP; the iontophoretic electrode was then withdrawn sufficiently to limit the level of current passing through the membrane. Sensitivity to ACh was measured over several adjacent spots until the region providing the maximum ACh potential was located. RESULTS

(I) The SCGN networks The growth of SCGN under the conditions used in the experiments has been described in considerable detail and these studies have indicated that the surviving SCGN retain the morphological characteristics of the principal sympathetic neuron found in the intact rat superior cervical ganglionV,9,u, St. After individual or small groups of neurons settle onto the collagen surface, fine unmyelinated processes grow out from the soma and proximal dendrites to form a complex network (Fig. 1). The processes from a neuron branch repeatedly as they extend a considerable distance out into the surrounding neuropil. Many of the processes are smaller than 1 pm and these frequently join with the processes from other neurons to form small neuritic fascicles. Consequently, it is impossible to distinguish all the terminal branches for a particular SCGN, although some of the straight fibers can be traced for more than 100 pm while other processes scatter several branches from one locus close to the soma (Fig. 1). No unique axonal branches could be identified although stimulating separate branches from a cell could directly evoke an action potential u. Conduction velocities of approximately 0.5 m/sec were determined for several fibers by noting the latency between activity recorded in the somas and distance of the stimulated locus in the networks. Exact measurements were not always obtainable because the length of the conducting path frequently could not be distinguished. However, stimulating fibers as distant as 4 mm could sometimes evoke a direct response. The pattern of neurite growth from the spinal cord explants is similar to that noted for the SCGN TM. Within 24 h of placing the spinal cord explants onto the collagen, a corona of processes forms and several bundles may be followed as they approach individual SCGN 74. After the spinal cord neurites join the established SCGN network the identity of individual processes can no longer be discerned.

(II) Spontaneous activity In intracellular recordings from over 1000 SCGN spontaneous activity, consisting of small depolarizations and action potentials (Fig. 2), occurred in more than ! 8 of the dissociated neurons. No examples of isolated hyperpolarizations were seen. Most frequently these responses were present in cultures grown with spinal cord tissue but they were also observed after removal of the spinal cord explants or in pure SCGN cultures 6a (WakshuU and Burton, unpublished observations). The pattern of spontaneous activity in half of the cells appeared to be random since sub- and suprathreshold responses happened at any moment and usually at rates below 5/sec (Fig. 2A). In some cells~ responses clustered into bursts consisting of subthreshold (Fig. 2C) or combined sub- and suprathreshold responses (Fig 2B). The activity during a burst

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Fig. 2. Patterns of spontaneous activity noted during recording from SCGN grown with spinal cord explants. A: random activity with supra- and subthreshold potentials. B: irregular bursting activity with various periods of silence. C: regular bursts of subthreshold responses lasting for 5 sec (C1) followed by 20 sec of silence (C~); the responses and silent periods repeat in a regular cycle in this neuron. All records within each frame are continuous. Calibration of 10 mV and 100 msec applies to all records except amplitude calibration of 15 mV, which applies to A and time marker 50 msec applies to B.

could build up to a crescendo of discharges, but the occurrence of the bursts was random (Fig. 2B). Activity repeated at more regular intervals in 5 cases where cycles of short bursts of responses, followed by long silent periods (Fig. 2C), recurred continually but the duration of the cycles was not always constant. The average size of the subthreshold response in two neurons was 3 mV and the te~nporal course of these responses (see Table II) tended to be similar to subsequent observations made on evoked synaptic activity (see below). The observed spontaaeous activity was probably due to chemically mediated synaptic transmission since spontaneous activity was reversibly blocked in several neurons when the perfusing medium contained 15 mM MgCle or 0.05 mM CaCi2 (Fig. 5, and see below). In several additional neurons with spontaneously occurring responses activity also ceased at the same point where a directly evoked AP was blocked by perfusion with 10-~ Mrtetrodotoxin (Fig. 3) indicating that most of the observed

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Fig. 3. Effects of tetrodotoxin (TTX) on anode break excitation of action potentials (upper trace) and spolitaneous potentials (lower trace) from the same SCGN grown with spinal cord. Both spontaneous activityand AP in control solution (A) are blocked within 5 min in TTX 10-?M(B) and these responses recover 19 min after the TTX solution is washed out by control medium (C). Calibration of 20 mV and 20 msec applies to upper trace; 2 mV and 200 msec apply to lower trace. Records of spontaneous activity within each frame are continuous in time. spontaneous subthreshold responses were not due to spontaneous leakage of transmitter. This toxin should differentiate between miniature and transmitted synaptic potentials because it does not block spontaneous leakage of transmitter 44.

(II) Excitatory connections The spontaneous activity just described could partly be due to conducted impulses from the spinal cord explants since synaptic connections could be demonstrated between the spinal cord explants and $ C G N by electrically stimulating one of the explants while recording from a nearby $CGN. Generally neurons within a few hundred micra of the explants were studied. All neurons within this vicinity did not respond and sometimes negative results were obtained from ceils whose somas lay astride the neuritic bundles emanating from the cord tissue. No consistent morphological pattern identified innervated SCGN, and a random search had to be used to find each synaptic potential. As shown by the records in Fig. 4, sub- and suprathreshold excitatory postsynaptic potentials (EPSPs) were encountered that varied in amplitude over successive trials. Multiple innervation was clearly evident for some neurons since two or more distinct EPSPs occurred with consistent latencies even when the first EPSPs fired an AP (Fig. 4B), and repetitive trains of impulses sometimes followed a single stimulus (Fig. 4C). As expected, these evoked responses were sensitive to the Mge+/Ca 2+ ratio in the extracellular fluid. For example, in control bathing medium a complex response was recorded from one SCGN consisting of a short latency EPSP

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Fig. 4. Five consecutive traces of orthodromic responses from 3 different SCGN following constant intensity stimulation of spinal cord explants (A and B) and neuritic bundles coming from the spinal cord (C). A: several EPSPs of varying amplitudes are evoked follow:,g each single stimulus. Summation of some of these multiple synaptic potentials could trigger an AP (second and third tracesA. B: two EPSPs occur with constant delay bu~.with fluctuating size following a single stimulus. The first EPSP could sometimes fire an AP; the res.dual depolarizations (third and fourth traces) are probably caused by additional EPSPs. C: single stimulation could sometimes evoke a subthreshold EPSP (third trace), single AP (fi~'st and fourth trace) or repetitive AP, and EPSPs (second and fifth trace) indicating that polysynaptic pathways might be activated. Calibrations in A apply to all records except amplitude marker of 20 mV which applies to C followed by a series of additional responses that sometimes summated sufficiently to fire an AP (Fig. 5). These late responses and most of the spontaneous activity disappeared within I min after switching to a medium with 15 m M MgCI2; by 15 min the short latency responses had ceased. This blockage of all activity was reversed by returning to normal perfusion medium. A similar reversible sequence occurred after perfusion with medium containing 0.05 m M CaCle (Fig. 5). A characteristic feature of chemical synaptic transmission is a correlation between the amplitude of the EPSP and resting membrane potential. As shown in Fig. 6A, appropriate changes occurred in the amplitude of the EPSPs evoked in cultures during the injection of hyperpolarizing and depolarizing currents. However,

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447 no evidence for a reversal potential was seen probably because the electrode resistances were above 100 MD, or the synaptic site was too distant to be effectively influenced by current injection across the somal membrane. The characteristic amplitude and time course of the synaptic responses from 3 SCGN are listed in Table 1I. The amplitudes of these EPSPs are typical for single, subthreshold responses and ranged from 0.6 to 6.4 mV. Sometimes EPSPs of 12-15 mV appeared in the records from other neurons and these larger responses frequently lead to APs. The rising phases of the subthreshold EPSPs to peak amplitudes were gradual, ranging from 3 to 14 msec and fall times tended to be exponential with decay to half the peak amplitt.de occurring in 4-15 msec. The duration of most EPSPs was under 40 msec; no responses lasted beyond 100 msec even after the presyr.~aptic neuron was stimulated repetitively. No inhibitory postsynaptic potentials were observed although sometimes an extended hyperpolarization followed an evoked EPSP (Fig. 6B). This was probably not due to direct transmitter action since a similar hyperpolarization could be elicited by injecting a depolarizing pulse (Fig. 6C). It is likely that this late phase of hyperpolarization in cultured SCGN is due to a delayed rectification involving increased K + permeability as was previously suggested to explain similar observations from parasympathetic neurons in the frog heart septum ~o.

(IV) Sensitivity to ACh In order to determine whether acetylcholine (ACh) was an appropriate transmitter for cultured SCGN, ACh was iontophoretically applied to the surface of these neurons with positive current. When the ACh electrode was kept at some distance from the somal membrane (as judged by visual control), a gradually graded small depolarization followed injection of a I msec iontophoretic pulse (Fig.7A). Successive increases in iontophoretic current fired an AP (Fig. 7B), or an AP with residual depolarization (Fig. 7C) or evoked repetitive firing (Fig. 7D). Previous analyses at the neuromuscular junction~O, have shown that conductance across the membrane increases during the action of transmitter. Similarly, in culture (see Fig. 8A) a drop in membrane resistance accompanies the application of ACh onto the SCGN somal membrane. In addition, the amount of depolarization caused by a fixed amplitude iontophoretic pulse was directly related to the membrane potential (Fig. 8B). Although a

Fig. 5. Effects of high concentration of MgCI~ (15 mM) and low concentration of CaCI2 (0.05 mM) in Eagle's minimum essential medium (MEM) on evoked orthodromic and spontaneous activity. In control medium, 4 successive traces of potentials show an early EPSP with constant delay followed by ~, ~eries of additional EPSPs and APs plus an occasionai spontaneous barrage (second trace in frame on upper left). Introducing a solution containing 15 mM MgCle blocks, within 1 min both the complex late responses and the spontaneous activity but not the short latency unitary EPSP (middle frame, upper row) in 15 rain all evoked synaptic responses are gone (upper right). These effects are reversible and all activity, including the spontaneous potentials (first trace in frame on lower left) reappear in control MEM. Similar blockage of all synaptic activity occurs in the presence of 0.05 mM CaCle (middle frame, lower row) and these effects are again reversible ,(lower right). All traces within

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Fig. 6. A: varying the membrane potential of an SCGN effects the amplitudes of evoked EPSPs following single stimulation of a spinal cord explant. The size of the EPSP at resting potential (RP) increases with membrane hyperpolarization (Hp) and decreases with membrane depolarization (Dp). The membrane potential is altered by injecting 475 msec pulses of current through the recording electrode. B, C: after-hyperpolarizations recorded from two different SCGN follow (a) stimulation of spinal cord, or (b) intraceUular injection of depolarizing current (C). In B, 3 superimposed traces of evoked responses show one failure and two amplitudes of subthreshold EPSPs followed by hyperpolarizing potentials. The magnitude of these after-hyperpolarizing potentials depends on the size of the EPSPs. In C, injection of a 20 msec depolarizing pulse through the recording electrode causes similar after-hyperpolarizations. 5 mV calibration applies to both B and C.

reversal potential was not observed, extrapolation from the data shown indicated that the reversal potential was probably around --30 mV which is equal to the --30 mV noted by Skok es for orthodromic EPSPs in cat sympathetic ganglia. In extensive studies of ACh receptors, evidence has been presented for decreased synaptic or ACh response shortly after exposing the postsynaptic membrane to ACh 45. This phenomenon of desensitization s~as also demonstrated in these cultures (Fig. 8C) where the response to a 1 msec injection of ACh was reduced for up to 4 sec following a 100 msec conditioning injection of ACh. Acetylcholine probably opens sodium and potassium channels at specialized receptor sites even in cultured neurons, and consequently, tetrodotoxine (TTX) o u g h t to have no direct effect on synaptic c u r r e n t s ao,44,Ss or, as s h o w n in Fig. 9, on

TABLE ii

Synaptic potential characteristics spontaneous EPSPs Cell no.

Size (m V)

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V/sec

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Total duration (msec)

Nmnber of trials

(1) (2)

3.30 2.98

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34.5 94.8

40 25

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21 27.4 36.5

12 42 50

Average values for evoked EPSPs f rmn spinal cord (3) (4) (5)

2.58 2.03 3.25

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the ACh potential. For this example, when the TTX blocked both direct and iontophoretically fired APs, only a suprathreshold ACh potential remained.

(V) Pharmacology of synaptic activity (a) Nicotinic cholinergic blocking agents. Several dru~s (D-tubocurare, hexamethonium and mecamylamine)are noted for their ability to prevent depolarization from occurring during synaptic transmission at cholinergic receptor sites that are quite sensitive to the application of nicotine ag. These drugs effectively and reversibly eliminated evoked syna9tic activity transmitted from the spinal cord explants to the SCGN (Fig. 10A, B). The blockade usually began to appear within 2-5 rain indicating that the effective concentration for these drugs was below the final i0 -4 or l 0 -5 M present in the perfusing medium; recoveries generally occurred within 15-20 rain following subsequent perfusion with control medium. The accepted mechanism of action for these drugs is that they competitively bind to postsynaptic receptors thereby blocking interaction with acetylcholine. Evidence for the postsynaptic action of these drugs in culture is presented in Fig. 10C where only the iontophoretically triggered AP is reversibly blocked by hexamethonium. As shown elsewhere in this paper cholinergic nicotinic receptors exist on S C G N grown with spinal cord tissue. a-Bungarotoxin, which is known to bind irreversibly with nicotinic receptors at the neuromuscular junction xa, had no effect on these syrapathetic neurons (Fig. 10D) even after 10-5 g/ml of this venom in the medium was left on the cells for 30 min. (b) Muscarinic cholinergic blocking agents. The sensitivity of the ~,~pinal cord evoked EPSPs to high and low concentrations ofatropine, a muscarinic blocking drug, was explored since some reports have indicated that muscarinic receptors may also be found in mammalian superior cervical ganglia 25,59. As shown in Fig. 11A, l~w (2.8 × l0 -6 M) concentrations of atropine had no effect whereas higher (10 -4 M) concentrations (Fig. 1I B) reversibly blocked synaptic transmission. The mechanism of the blockade is still unknown although the site of action in culture is probably postsynaptic since similar high concentrations of atropine had the same effect on ACh potentials as on the orthodromic EPSP (Fig. 1 I D). The lower concentration of atropine also caused some (25 %) reduction in the ACh potential (middle column of

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Fig. 8. Properties of ACh potentials recorded from 3 different $CGN grown with spinal cord. A: membrane resistance changes during ACh application in one neuron. A 10 sec pulse ofiontophoretic current (upper trace) produces a massive depolarization and several APs (lower trace). Superimposed on this response is a series of hyperpolarizing pulses of constant strength (0--0.5 nA) and duration (50 msec duration) applied at l/see through the intracellular recording electrode. The magnitude of these electrotonic potentials decreases during the prolonged ACh poteotial indicating a drop in membrane resistance. B: the effect of membrane potential on the amplitude of ACh potentials from another SCGN. The amplitude of the ACh potential increases as the membrane potential is raised by injecting a long (350 msec) pulse of hyperpolarizing current through the intracellular recording electrode. Anomalous rectification is noted since the magnitude of the membrane potential gradually decreases with time when stronger hyperpolarizing currents are injected. C: desensitization. C1: a 1 msec test pulse of iontophoretic current (lower trace) produces approximately a 15 mV depolarization. Cs: a 100 msec pulse of ACh causes a massive depolarization. When this is followed 1 sec later by another msec test pulse, the size of the ACh potential, caused by this test pulse, decreases to less than 10 mV due to desensitization of ACh receptors caused by the previous 100 msec conditioning pulse of ACh. C3:4 sec after the conditioning pulse, the size of the ACh potential produced by the unconditioned 1 msec test pulse nearly returns to the control size.

Fig. 1 I C) a l t h o u g h this effect was n o t detected during recordings of the EPSPs. (c) Anticholinesterase drugs. A t certain cholinergic synapses the anticholinesterase agent (anti-ChE), eserine and neostigmine, potentiate synaptic a n d A C h responses p r o b a b l y by inhibiting the enzymatic activity of cholinesterase tg,4a. In studies on the effects of anti-ChE on the cholinergic synapses between spinal cord and S C G N no potentiation was observed a n d the EPSPs were reversibly blocked within

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Fig. 10. The effect of nicotinic cholinergic blocking agents on evoked orthodromic responses following stimulation of the spinal cord explants (A, B) and ACh potentials in 4 different SCGN. In A, 10 -4 M mecamylamine and in B 10 -4 M hexamethonium (HC-6) block almost all synaptic activity within 5 rain (middle traces). This blockade is reversible (third column of A + B). In C, hexamethonium also reversibly eliminates the ACh potential but the directly evoked AP potential at anode break remains unchanged throughout the pharmacological sequence. In D, a-bungarotoxin is shown to have no effect on the ACh potential ,oted before or after perfusion with control medium. Calib~ ~tions in B and D apply, respectively, to A and C.

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Fig. 11. The effect of atropine on evoked orthodromic responses following spinal cord stimulation (A + B) and ACh potentials (C + D) in 4 different SCGN. In 2.8 × 10-e M atropine little change occurs in synaptic activity (A) but approximately 25 ~ reduction appears in the ACh potentials (C); in 10-4 M atropine all synaptic (B) and iontophoretic (D) responses are reversiblyeliminated. Consecutive traces are shown in each frame in D. Calibration in B also applies to A. 7 rain of switching to a perfusion medium containing 10 -5 g/ml of neostigmine (Fig. 12A) or eserine (Fig. 12B). Since a continuous perfusion was used, any potentiating effect at lower concentration should have been observed before the final concentrations were reached. The mechanism responsible for these effects is probably not the same for the two anti-ChE drugs since neo~tgg__mjne and eserine had different actions on the ACh potentials. For example, neostigmine had no effect on the AP evoked by ACh application (first AP in Fig. 12C) or the second AP triggered by intracellular current injection and only slightly reduced ( 1 5 ~ ) subthreshold ACh potentials (Fig. 12D). In contrast, eserine completely blocked all iontophoretic potentials (Fig. 12E). These results suggest that neostigmine may block t~e release of transmitter from the presynaptic terminal and secondarily may also slightly affect the postsynaptic receptors whereas eserine may directly act on the postsynaptic region.

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Fig. 12. Effects of anticholinesterases on synaptic and iontophoretic responses recorded from 4 different $CGN grown with spinal cord• In A and B 10 -s g/ml of neostigmine or eserine reversibly blocks the EPSP produced by stimulating the spinal cord. Neostigmine has no (C) or only slight effects (15 % reduction in D) on ACh potentials, but eserine almost completely eliminates ACh potentials (E). Recordings in D and E from same cell. Calibration in B and D applies, respectively, to A and E.

(d) Alpha-adrenergicblockingdrugs. The ACh potential and EPSP from spinal cord to SCGN was also studied in the presence of alpha-adrenergic blocking agents, such as phentolamine and phenoxybenzamine, in order to note whether any unsuspected catecholaminergic transmission interacted with the cholinergic effects (see following paper). No changes appeared in the presence of ! 0 -5 M of either alpha-adrenergic blocking agent even after continuous perfusion for more than 30 min.

454 DISCUSSION The present study shows that cholinergic synapses may form de novo in tissue culture between explants of spinal cord tissue and dissociated sympathetic neurons prepared from embryonic and newborn rat neural tissue. These findings are consistent with the suggestion u that the synaptic terminals seen in combined spinal cord and SCGN cultures, containing round clear vesicles of 40-50 nm in diameter, are probably cholinergic endings and that many of these are analogous to the preganglionic connections from spinal cord neurons found in vivo e~,ev,5~,53. The evoked EPSPs in culture were similar to the so-called fast EPSP recorded from mammalian sympathetic ganglia 4,1s,24,29,6s,¢o and frog sympathetic neurons1,49, el. In all of these instances synaptic depolarization rises to some peak value in about 4 msec 24, generally lasts 15-40 msec and decays exponentially in rat or guinea pig superior cervical ganglia with a time constant of 6.7 or 8.5 msec, respectively es. The parameters cited in Table II for EPSPs in culture are comparable to those seen in the animal. The observation that the amplitudes of many apparently unitary EPSPs fluctuated and that several trials showed no responses is evidence that transmitter release in these cultures is quantal. When similar observations have been made on the amplitude fluctuations of synaptic activity in intact sympathetic gangliae-4,e0, vv or in dissociated cell ~ultures prepared from other tissues 34,67, quantal release has also been suggested. Extensive statistical analyses of the distribution of EPSP amplitude were not attempted in this study but in one case a mean quantum content of 2.87 was determined by the method of failures and an average miniature amplitude was calculated to be approximately 1 mV. These values are consiz,tent with observations from intact superior cervical ganglion of guinea pig 77. One of the significant similarities between the SCGN in culture and in the animal is the pharmacological sensitivity of the fast EPSP in the two situations. The cholinergic nature of these responses was nicotinic rather than muscarinic, since hexamethonium, mecamylamine or D-tubocurare blocked activity, whereas only very high concentrations of atropine were effective. As expected from in vivo studies, the fast EPSP was also insensitive to low concentrations of alpha-adrenergic blocking agents. Further evidence that acetylcholine caused the evoked EPSPs was demonstrated by the depolarizing responses to iontophoretic application of this transmitter 63. In our studies, applied ACh appropriately decreased membrane resistance and, as expected, this shift in permeability was insensitive to TTX 5s. Specific desensitization during prolonged exposure to ACh was also observed and this was equivalent to examples of the same phenomenon at other cholinergic junctions20,4~, 63. Since cholinergic transmission characterizes the preganglionic connections to sympathetic neurons in vivo, these results indicate that ACh production and release and ACh postsynaptic receptors survive or are re-established in culture after the denervation and dissociation procedures followed in growing the tissue. The negative findings with a-bungarotoxin are consistent with its previously demonstrated ineffectiveness to bind to cultured rat autonomic neurons 62,63. These observations indicate that innervation from the spinal cord does not alter the sensitiv-

455 ity of the SCGN ACh receptors to a-bungarotoxin. However, a-bungarotoxin will bind to cholinergic nicotinic receptors on the processes and somas of chick sympathetic neurons in dissociated culture as. It would be interesting to know whether these negative findings for rat SCGN in culture indicate that this toxin will not alter synaptic transmission between the spinal cord and SCGN in the animal. Single shocks to the pre3ynaptic inputs frequently resulted in temporally dispersed EPSPs, repetitive APs or evidence of summated EPSPs indicating that multiple innerration developed in vitro. Convergence of several preganglionic fibers has been hypothesized in mammalian sympathetic ganglia on the basis of quantal size and thresholds 4,77 and from evidence of successive increases in the amplitude of evoked synaptic potentials when stimulus strength is increased in several other sympathetic ganglia 4, ls,2a,sv,6s,6a,7s. In culture, the multiple responses following a single stimulus may, thus, be due to activation of different populations of preganglionic fibers that converge onto the recorded SCGN, with differing conduction velocities. Alternatively, temporally dispersed synaptic potentials may be due to activity in excitatory interneurons 2a. Variation in the occurrence of repetitive APs with constant stimulus strength suggested that the late activity in culture could possibly be due to interneurons. Similarly, early blockade by high Mg 2+ of long latency responses but not the initial EPSP (see Fig. 5) indicated that polysynaptic pathways might exist in culture. Although there is no direct evidence of excitatory interneurons in mammalian sympathetic ganglia in the animal 2a, these may be present in amphibian parasympathetic ganglia ~5,76 and in culture. As O'Lague et al. 64 and Ko et al.46 have shown SCGN can receive excitatory synapses from other SCGN46,64; these, in turn, could be activated by synapses from the spinal cord. Another source of interneurons might be spinal cord neurons. The formation of synaptic networks between spinal cord neurons has been observed in explants 1°,1~-17 and in dissociated cultureS5, 67. Kano and Shimada 42, who observed repetitive spikes and EPSPs on dissociated muscle fibers after a single stimulus to spinal cord explants, suggested that these impulses might be due to repetitive firing of neuronal interconnections in the spinal cord explants. For these reasons the synaptic pathways leading to activation of the SCGN in cultures may be more complex than observed in vivo. Although the cholinergic nature of ganglionic synaptic transmission was expressed in culture, no potentiation of the responses was effected by anticholinesterase drugs; on the contrary, a blockade of synaptic transmission was produced by high concentrations of eserine and neostigmine. The observation that the inhibitory effect of high concentrations ofeserine was manifested postsynaptically has also been observed in vivoas, but the presynaptic and partly postsynaptic inhibition demonstrated with neostigmine has not been reported previously. Even endplate potentials, which are potentiated by low concentrations of anti-ChE al, may be depressed by high concentrations of neostigmine 2a and eserine ao indicating that the blockade may be due to some non-specific inhibitory effects of anti-ChE at high concentrations. Evidence for potentiation of synaptic transmission in autonomic ganglia by low concentrations of anti-ChE have been observed by some authors 5,a2,33,66. This potentiation could be due to the conventional effect of anti-ChE in preventing ACh from being

456 hydrolyzed by ChE 31, or to its effect on the input resistance of the cells 54. The complete absence of potentiation in these cultures could be due to lack of functional ChE as has previously been suggested in cultures of dissociated muscle fibers34,42. Alternatively, these results represent a normal phenomenon since anti-ChE has been shown to have no effect or, ganglionic transmission in vivo by some workers 22,37. The mechanism underlying the effects of different concentrations of anti-ChE on synaptic transmission in autonomic ganglia (e.g., blockade, potentiation, no effects or inconclusive) x,20 are still not clear s2. These confusing results have led to the suggestion that the potentiation effect of anti-ChE is not an absolute indicator of cholinergic synaptic transmission 37. No slow potentials were recorded from these cultures that could be considered equivalent to the late phase of hyperpolarization and depolarization recorded from various sympathetic ganglia, including the rat superior cervical ganglion 2x. In the intact ganglion, a postsynaptic inhibition is believed to arise from the release of catecholamines from small, intensely fluorescent (SIF) cells ~5,5°.53,59. No SIF cells survive in these cultures 73. However, even if some of the SIF cells were present, no evidence for changes in activity accompanied perfusion with alpha-adrenergic blocking agents whereas these drugs block the slow hyperpolarization in mammalian ganglia 25. Furthermore, Obata e3 showed that SCGN in culture do not respond to iontophoretic application of catecholamines and we have also seen no striking changes in activity during bath applications of norepinephrine. These observations suggest that in our cultures the slow IPSP would be unlikely to occur. All of the observations strongly indicated that the synaptic potentials recorded in these cultures were conventional fast EPSPs. None of the characteristics of the late depolarization, known as the slow EPSP, were seen during these experiments. For example, no muscarinic cholinergic receptors were noted, the membrane resistance decreased during ACh application and the amplitude of the evoked synaptic potential and ACh potential were directly related to the degree of membrane hyperpolarization 47,49,s°. These negative observations regarding slow potentials in culture are consistent with previous intraceilular studies in the intact superior cervical ganglion of rat and guinea pig6s.69, or parasympathetic ganglion neurons in the frog heart septum 20. In most of the recordings the spinal cord explants were probably the source of the spontaneous activity since frequent examples of complicated spontaneous responses occurred when the explants were present. This is not surprising because several previous studies have shown that related patterns of spontaneous poteatials may be recorded from embryonic spinal cord that is isolated from supraspinal or intersegmental inputs in chick embryos40, 75, in organotypic cultures grown from embryonic central nervous system tissuesX4,16, and also dissociated cell cultures prepared from chick and mouse embryonic spinal c0rd34,35,67. Even in vivo, spontaneous, tonic and random bursts of activity from mammalian sympathetic ganglion neurons have been related to activity in preganglionic, spinal cord neurons 56,57,7°,Tx,Ts. The possible causes of the spontaneous activity in the intact spinal cords are unknown, in culture the spinal cord neurons may have 'fibrillating' membranes due to

457 removal of the suprasegmental inputs in an analogous fashion to the fibrillation seen in denerva~ed muscle fibers ~'z. Alternatively, the spinal cord tissue may be more active due to t~le loss of supraspinal inhibitory influences s. The source of spontaneous synaptic activity observed in S C G N cultures after removal of the spinal cord explants or in pure S C G N networks 64 (Wakshull and Burton, unpublished observations) must be propagated synaptic activiLy from other sympathetic neurons11,46, 64. The origin of this activity may be intrinsic pacemakerlike responses in some S C G N . Direct recording of these intrinsic responses has not been obtained even in reduced Ca 2~ medium, although this possibility still cannot be ruled out. No spontaneous potentials were noted in denervated frog 41 and mamm_,,ian sympathetic ganglia 54,5~ but reinnervation, which occurs in culture, may impart some u n k n o w n instability to these neurons. Alternatively, some changes in the composition or flow of the medium may activate a response that then propagates through the synaptic networks in the culture eventually reaching the recorded neurt~n. This suggestion is tenable provided that the S C G N are synaptically connected in vitro to a great extent 46. ACKNOWLEDGEMENTS We wish to t h a n k Dr. P. Wood for his assistance in preparing the cultures used in these experiments. Supported by N I H Grants NS-09923, NS-09809, NS-11888, GRS-5501 and RR-05398.

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