An intracellular study of synaptic transmission and dendritic morphology in sympathetic neurons of the chick embryo

Developmental Brain Research, 22 (1985) 99-111 Elsevier

99

BRD 50264

An Intracellular Study of Synaptic Transmission and Dendritic Morphology in Sympathetic Neurons of the Chick Embryo STUART E. DRYER and VINCENT A. CHIAPPINELLI

Department of Pharmacology, St. Louis University School of Medicine, 1402 South Grand Boulevard, St. Louis, MO 63104 (U.S.A.) (Accepted March 5th, 1985)

Key words: neuronal development - - chick embryo - - slow excitatory postsynaptic potential - - M-current - nicotinic transmission - - substance P - - sympathetic ganglia - - autonomic ganglion pharmacology

The characteristics of synaptic transmission in whole embryonic avian sympathetic ganglia have been examined by intracellular recording. Neurons in lumbar paravertebral ganglia of chick embryos exhibit both fast nicotinic excitatory postsynaptic potentials (EPSPs) and non-cholinergic slow EPSPs. Fast nicotinic transmission is mediated by at least 3-5 convergent preganglionic inputs and can be detected at the earliest embryonic stage examined (Stage 38; 12 days of incubation). Two types of non-cholinergic slow EPSPs have been observed and distinguished by their time course and the resulting changes in input resistance. One of these slow synaptic potentials is mimicked by exogenously applied substance P, but not by exogenous luteinizing hormone-releasing hormone (LH-RH). Muscarinic agonists also evoke slow depolarizations in the ganglia, mediated at least in part by inhibition of the M-current. Intracellular labeling with horseradish peroxidase reveals cells with 5-10 primary dendrites at Stage 42 (16 days of incubation), the earliest stage to exhibit slow EPSPs. The active and passive membrane properties of avian sympathetic neurons, including the presence of the Mcurrent, generally resemble those of adult mammalian and amphibian sympathetic neurons. Functional activity in chick sympathetic neurons is present at a developmental stage where both biochemical and morphological indices of synapse maturation are at low levels. Since this progression has also been observed in the avian ciliary ganglion, it may be of general relevance to neuronal development.

INTRODUCTION A u t o n o m i c ganglia are widely utilized n e u r o n a l systems for the study of synaptic transmission. Vertebrate ganglia are relatively simple in design and highly accessible for in vitro manipulations and therefore provide excellent models for studying n e u r o n a l development. In the avian embryo, synapse formation and maturation have been examined in the parasympathetic ciliary ganglion by morphological, biochemical and electrophysiological means. Extracellular recordings have revealed that ciliary neurons are innervated at an extremely early developmental stage, prior to the period of naturally occurring cell death3S. Few synaptic contacts are visualized by electron microscopy until several days after functional activity is observed37. Moreover, the activities of the neurotransmitter-related enzymes, choline acetyltransfer-

ase and acetylcholinesterase, also increase sharply several days after functional transmission is detected 14. In chick lumbar sympathetic ganglia, the ultrastructure of synapses has been examined at several developmental stages26, 55. Biochemical measures of synaptogenesis, including choline acetyltransferase activity 11,42 and levels of acetylcholine41 have also been determined. Other studies have examined the early formation of the ganglia34.36,40 and the development of preganglionic n e u r o n s in the columns of TernilO,47. Neuronal processes containing substance P-like immunoreactivity have been visualized at early developmental stages 17 and substance P has been identified by H P L C in embryonic sympathetic ganglia25. In contrast, virtually nothing is known about the onset and development of functional synaptic transmission in embryonic chick sympathetic ganglia.

Correspondence: V. A. Chiappinelli, Department of Pharmacology, St. Louis University School of Medicine, 1402 South Grand Boulevard, St. Louis, MO 63104, U.S.A. 0165-3806/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)

100 Such knowledge would enhance the interpretation of previously obtained morphological and biochemical data and would contribute to an understanding of the process of neuronal development and synapse maturation. We have therefore undertaken an intracellular study of fast and slow synaptic transmission in embryonic sympathetic ganglia and have injected horseradish peroxidase (HRP) into sympathetic neurons to determine their dendritic morphology at several developmental stages. We now report that fast nicotinic transmission in embryonic chick sympathetic neurons is mediated by multiple presynaptiE fibers and that nicotinic transmission is observed at the earliest stage examined, prior to the major increases in biochemical indices of synapse formation. Intracellular injections of HRP at later embryonic stages reveal 5-10 primary dendrites per postsynaptic cell. Chick embryo sympathetic neurons contain K+-channels similar to those found in adult amphibian and mammalian sympathetic neurons; these channels are thought to be involved in slow synaptic transmission 5.7. We have observed two types of non-cholinergic slow excitatory postsynaptic potentials (EPSPs); one of these is closely mimicked by substance P but not by luteinizing hormone-releasing hormone (LHRH). These slow potentials appear later in development than the nicotinic EPSPs. The properties of avian sympathetic neurons more closely resemble those of mammalian, rather than amphibian, sympathetic neurons. MATERIALS AND METHODS

lntracellular recording Lumbar paravertebral sympathetic chains were dissected out of White Leghorn (SPAFAS, Peoria, IL) chick embryos (12-21 days of incubation) staged according to Hamburger and Hamilton 22. Connective tissue adhering to the chain was cut away and ganglia L2-L5 (using the numbering system described in ref. 26) were pinned out on the bottom of a plexiglass chamber (1 ml volume) filled with cured Sylgard® resin. The ganglia were superfused with Tyrode's solution12 at 3-5 ml/min and maintained at 37 °C. The interganglionic connective between L1 and L2 and in some cases between L2 and L3, was taken up into a suction electrode. Stimuli were delivered by means of a Grass S-4 stimulator and SIU-5

isolation unit. lntracellular recordings were made with glass microelectrodes filled with 3 M KCI (resistance 60-180 MQ); hyperpolarizing and depolarizing square current pulses were passed through the recording electrode by means of standard bridge balance techniques. Limited manual control of resting membrane potential was achieved by passing DC current through the recording electrode 31. Current and voltage responses were monitored from the balanced bridge outputs of a high impedance electrometer (WPI-M707, WPI, New Haven, CT) and displayed on a storage oscilloscope. Voltage and current responses were photographed off the oscilloscope screen. Upon initial impalement, the membrane potential was usually unstable. Often this situation could be improved by passing a hyperpolarizing DC current through the recording electrode; within a few minutes the cell would recover and the 'clamp' was removed. Cells were used if the resting membrane potential was greater than -40 mV and if application of depolarizing current pulses produced regenerative, overshooting action potentials. Cells were readily impaled, but it was usually not possible to maintain an impalement for longer than 10 rain, although occasionally we maintained contact for up to 1 h. This situation was improved somewhat by using a double-chamber recording bath; in this arrangement, surface turbulence caused by suction was largely isolated from the recording electrode. We have found that embryonic chick sympathetic chains are extremely delicate when compared to several other autonomic ganglion preparations that we have worked with, including the rat superior cervical ganglion, guinea pig inferior mesenteric ganglion, chick ciliary ganglion and frog lumbar sympathetic ganglion; this is particularly true with respect to the tensile strength of the internal connective tissue and the external capsule. The fragility of the preparation seems to be the primary factor limiting the mean duration of impalement and makes pharmacological and ionsubstitution experiments difficult.

Application of drugs Three different methods of drug application were used. Superfusion at a known concentration was performed using separate reservoirs controlled by elec-

101 tric valves. In this case, the dead time was 15-30 s. Drugs were also injected into the flowing bath (3 mm 'upstream' from the ganglion) by means of a small hand-held pipette 15. Finally, using electric valves, drugs were pressure-injected (20 p.s.i, nitrogen gas) from glass micropipettes (broken back under visual control) positioned near the recording electrode. Qualitatively similar results were obtained with agonists by all 3 methods. Agonists appeared to be most potent when applied by superfusion, in agreement with some previous reports 29. Antagonists and high Mg2+low Ca 2+ solutions were always applied by superfusion.

Horseradish peroxidase (HRP) injections Intracellular injections of HRP were performed according to the method of Forehand and Purves Is. Briefly, triangular glass electrodes (Glass Company of America) were filled with a 5% (w/v) solution of HRP (Sigma Type VI) made fresh each day in 0.2 M potassium acetate, 0.05 M Tris, pH 7.6. Electrode resistance was 250-400 Mr2. HRP was ejected by passing depolarizing current pulses (1-3 nA, 50 ms duration) at 5 Hz for 3-5 minutes. The ganglia were processed according to the method of Hanker et al. 23 using reagents obtained from Fluka Chemical Corporation, Hauppauge, New York (gift of Dr. Dale Purves of Washington University, St. Louis, MO). Ganglia were then fixed overnight in 1.25% glutaraldehyde, 0.5% paraformaldehyde in 30 mM HEPES containing 120 mM NaC1 and 3 mM CaC12, pH 7.6. Ganglia were subsequently dehydrated by several passages through ethanol, cleared in xylene and mounted with Permount®. Filled cells were viewed on a Zeiss Universal microscope. A camera lucida device was used to draw the cells, which were characterized by a uniformly dark brown reaction product. Chemicals, substance P, and mammalian LH-RH were obtained from Sigma Chemical, St. Louis, MO. Substance P from Peninsula Labs (Belmont, CA) gave results identical to the Sigma product. RESULTS Intracellular recordings were made from more than 285 cells in 73 preparations of avian lumbar paravertebral sympathetic ganglia. Most of the ganglia studied were from Stage 43-45 embryos (18-21 days

of incubation) and the results reported represent experiments done at these stages, except where otherwise indicated.

Membrane properties Healthy Stage 43-45 sympathetic neurons had resting membrane potentials between -40 and -60 mV (mean + S.E.M. was -50 + 2 mV, n = 13). Application of depolarizing square current pulses evoked overshooting action potentials with a rheobase current of 0.11 ___0.01 nA (mean + S.E.M., n = 8) and chronaxie of 4.3 + 0.2 ms (mean + S.E.M., n = 4). Cell input resistance was 164 + 17 Mr2, the time constant was 10 _+ 1 ms and cell capacitance was 71 + 9 pF (mean + S.E.M., n = 13 cells). Application of small hyperpolarizing square current pulses revealed non-ohmic relaxations in the resulting voltage responses (Fig. 1A). These relaxations disappeared, when larger hyperpolarizing current pulses were applied. The membrane potential at which these relaxations disappeared was equivalent to the K+-equilibrium potential, defined as the membrane potential at which the hyperpolarizing afterpotential reversed, and which was routinely observed between -80 mV and -90 mV (Fig. 1A, see also ref. 31). The relaxations also depended on resting membrane potential, as a 10-20 mV manual hyperpolarization abolished the relaxations (Fig. 1A). This relaxation was also reversibly abolished by superfusion in Tyrode's solution containing 10 mM K +. Under these conditions, the K+-equilibrium potential was shifted to a more depolarized state ( - -45 mV). This procedure caused an 8-13 mV depolarization which was compensated for by passing a hyperpolarizing DC current through the recording electrode (Fig. 1A). Similar results have been reported for adult frog sympathetic ganglia, where this relaxation has been attributed to inactivation of a time- and voltage-sensitive K+-current called the M-current 3. An additional non-ohmic response, observed in about half of the recorded cells, occurred following the break of the hyperpolarizing pulse. This relaxation was characterized by a marked delay in the return to resting potential (Fig. 1B). A relaxation with a superficial resemblance to this response has been seen in mammalian thalamic neurons 28 and in adult rat sympathetic neurons 7. M-current channels are known to be blocked by

102

A. PRESENCE OF M-CURRENT

.~

!

EK ,

~

V

r

J

-

I

_, EK_._.~_.----- ~

Z

Z

i

I

-62

1o mM K° B. ADDITIONAL NON-OHMIC RESPONSE

....

,I,f.-f-

L

C. EFFECTS OF BARIUM CHICK N E U R O N S

CONTROL

3 mM Ba** RAT NEURONS

Fig. 1. Membrane properties of embryonic chick sympathetic neurons. Records in this and subsequent figures are from Stage 43-45 embryos unless otherwise indicated. A: application of small 105-ms hyperpolarizing current pulses (top sweeps) results in a non-ohmic relaxation in the voltage responses (bottom sweeps). Expected ohmic response is indicated by dashed line. Relaxations are not observed (lowest voltage sweep) when current is sufficient to hyperpolarize membrane to E K. In this cell, E K is defined (upper-right panel) as the membrane potential at which the hyperpolarizing afterpotential reverses. In a different cell, manual hyperpolarization to -62 mV (lowerleft panel) abolishes the relaxation observed when resting potential is -45 mV. Superfusion with Tyrode's solution containing 10 mM K + (lower-right panel) abolishes relaxation seen in normal Tyrode's solution containing 3 mM K +. The 10 mM K + solution depolarized the cell and hyperpolarizing DC current was used to compensate for this depolarization (upper sweeps). B: in many cells, at the break of the hyperpolarizing pulse, there is a discernible delay in the return to resting membrane potential (arrow). This is not observed when the membrane is manually hyperpolarized to -66 inV. C: when chick sympathetic neurons were superfused with Tyrode's solution containing 3 m M B a 2+, the action potential duration was increased 100-fold. Rat sympathetic neurons (from superior cervical ganglion) responded to 3 m M B a 2+ with at most a 3-fold increase in action potential duration. Ba 2+ produced a 1020 mV depolarization in neurons from both species. Calibration bars: all voltage sweeps are 20 mV. A: upper left, 0.25 nA, 20 ms; upper right, 0.25 nA, 5 ms; lower left, 1 hA, 20 ms; lower right, 0.5 hA, 20 ms. B: 0.5 nA, 20 ms. C: upper left, 0.25 nA, 10 ms; upper right, 1.0 nA, 10 ms; lower panels, 1 hA, 5 ms.

v e r g e n c e c o m p a r a b l e to adult m a m m a l i a n p a r a v e r t e -

_

bral s y m p a t h e t i c ganglia 6,49 and m u c h g r e a t e r than B n e u r o n s in a m p h i b i a n s y m p a t h e t i c ganglia 46. W e ofCONTROL

3 mM Ba**

ten f o u n d that s i m u l t a n e o u s a c t i v a t i o n of 3 or 4 inputs

e x t e r n a l Ba 2+ (ref. 4). S u p e r f u s i o n with T y r o d e ' s so-

was n e c e s s a r y and sufficient to e v o k e an action po-

lution c o n t a i n i n g 3 m M BaC12 p r o d u c e d d e p o l a r i z a -

tential in the p o s t g a n g l i o n i c cells (Fig. 2 A ) . T h u s , in-

tions of 1 0 - 2 0 m V in a m p l i t u d e , consistent with a

t e g r a t i o n occurs in e m b r y o n i c avian s y m p a t h e t i c gan-

b l o c k a d e of M - c u r r e n t c h a n n e l s . This p r o c e d u r e also

glia.

caused a 5 0 - 1 0 0 - f o l d i n c r e a s e in action p o t e n t i a l duration in chick s y m p a t h e t i c cells (Fig. 1C). This is in

Morphology

m a r k e d contrast to the effects of B a 2+ on adult rat

I n t r a c e l l u l a r i n j e c t i o n s of H R P w e r e used to char-

s y m p a t h e t i c n e u r o n s , w h e r e Ba 2+ caused, at m o s t , a

acterize dendritic m o r p h o l o g y in e m b r y o n i c n e u r o n s .

3-fold increase in action p o t e n t i a l d u r a t i o n (Fig. 1C;

T h e s e injections r e v e a l e d m u l t i p o l a r cells with small, s o m e w h a t triangular, s o m a t a (Fig. 2B). T h e r e w e r e 5

see also ref. 43).

- 1 0 p r i m a r y d e n d r i t e s p e r cell; t h e s e d e n d r i t e s w e r e

Fast synaptic transmission Electrical s t i m u l a t i o n of the rostral i n t e r g a n g l i o n i c c o n n e c t i v e e v o k e d nicotinic fast E P S P s in e m b r y o n i c avian s y m p a t h e t i c g a n g l i o n cells (Fig. 2 A ) . Increasing the stimulus intensity i n c r e a s e d the size of the rec o r d e d E P S P and occasionally e v o k e d a s e c o n d fast E P S P with a l o n g e r latency. As m a n y as 5 g r a d a t i o n s of the E P S P w e r e seen, suggesting a d e g r e e of con-

occasionally b r a n c h e d . In m a n y cases an a x o n c o u l d be identified and f o l l o w e d for a c o n s i d e r a b l e distance. T h e axons a p p e a r e d s m o o t h e r t h a n d e n d r i t e s and w e r e n e v e r b r a n c h e d .

Slow synaptic transmission R e p e t i t i v e stimulation of t h e rostral i n t e r g a n g l i o n ic c o n n e c t i v e r e s u l t e d in slow E P S P s in a b o u t 4 0 % of

103

A°

B.

-j j 20 #M

Fig. 2. Fast synaptic transmission and dendritic morphology. A: nicotinic EPSPs recruited by orthodromic stimulation of the rostral interganglionic connective at increasing stimulus intensities. Evoked EPSPs eventually summate to produce an action potential. When orthodromic stimulation is superimposed upon a hyperpolarizing current pulse (to attenuate regenerative responses) 3-5 gradations of the EPSP are routinely observed. B: camera lucida drawings of sympathetic neurons labeled by intracellular injection of HRP. An identifiable axon (star) is shown. Calibration bars: all voltage sweeps are 20 mV; left panel, 10 ms; middle panel, 0.25 nA, 10 ms; right panel, 1 nA, 20 ms. the recorded cells. These slow EPSPs tended to fall into two groups, although some ambiguity was occasionally encountered. In the first type, repetitive stimulation (10-30 Hz for 2 - 5 s, 0.01-0.5-ms pulse duration) produced a slow depolarization of 3-15 mV in amplitude. This depolarization developed relatively quickly, often being evident by the sixth pulse (Fig. 3A). We have therefore called this the short latency slow EPSP. This response was seen in about 25% of the cells tested. The amplitude and duration of the short-latency slow EPSP could be graded with stimulus intensity (not shown), suggesting that the short latency slow EPSP was due to stimulation of more than one fiber. The short-latency slow EPSP was not associated with any consistent change in input resistance (Fig. 3A) and was occasionally seen in cells where nerve stimulation did not produce a nicotinic fast EPSP (not shown). The short-latency EPSP and fast nicotinic

transmission were both reversibly attenuated by superfusion with Tyrode's solution containing 3.75 mM Mg 2+ and 0.25 mM Ca 2÷ (Fig. 3B). (This solution depolarized sympathetic neurons by 3-15 mV and tended to make the membrane potential somewhat unstable; we compensated for this depolarization by passing small amounts of hyperpolarizing D C current through the recording electrode.) The short-latency slow EPSP did not produce action potentials, even when the depolarizations approached 15 mV in amplitude (Fig. 3B). The short-latency slow EPSP was non-cholinergic, since it could be evoked in the presence of either 1 j~M atropine or 100 ~M (+)-tubocurare (Fig. 3C). A second class of slow EPSPs present in approx. 15% of the cells tested, was also observed following repetitive stimulation of the interganglionic connective (10-30 Hz for 2 - 5 s, 0.01-0.5 ms pulse duration). In this case, there was at least a 500 ms latent

104

A.

B.

C.

CONTROL

HIGH Mg ÷÷

100 I.IM D-TUBOCURARE

RECOVERY

1 IJM ATROPINE

L_

Fig. 3. Short-latency slow synaptic transmission. A: repetitive orthodromic stimulation (13 Hz) of the rostral interganglionic connective evokes a slow EPSP which is apparent by the sixth pulse (arrow) and which is fully developed by the end of the stimulus train. In another cell, application of constant hyperpolarizing pulses (top sweep) reveals, no change in input resistance during a short-latency slow EPSP produced by 30 Hz orthodromic stimulation. B: slow synaptic transmission (30 Hz stimulation, top sweeps) and fast nicotinic transmission (arrow, bottom sweeps) are reversibly blocked by superfusion with Tyrode's solution containing 3.75 mM Mg> and 0.25 mM Ca>. C: non-cholinergic nature of the short-latency slow EPSP is revealed during superfusion with Tyrode's solution containing 100 ¢tM (+)-tubocurare (left panel, drug present for 1.5 h, 25 Hz stimulation) and 1 ¢tM atropine (right panel, drug present longer than 1 h, 30 Hz stimulation). Calibration bars: A: left panel, 10 mV, 1 s; right panel, 0.5 nA, 20 mV, 2 s. B: top sweeps, 20 mV, 2 s; bottom sweeps, 20 mV, 10 ms. C: both panels, 10 mV, 2 s. Resting membrane potentials shown by dashed lines.

period following cessation of stimulation before the cell began to depolarize (Fig. 4A). We have called this the long-latency slow EPSP. The long-latency slow EPSP was associated with an increase in input resistance (Fig. 4A) and had amplitudes ranging from 2 - 2 0 mV. In 12 cells, the long-latency slow EPSP was associated with discharges of action potentials which lasted up to 1 min (Fig. 4B). These action potentials often occurred in cells where the underlying depolarization was small and where repetitive intracellular stimulation had no effect (Fig. 4B). Furthermore, the threshold for intracellular stimulation prior to the slow EPSP was much greater than the depolarizations caused by the slow EPSP (not shown). These spiking responses fatigued rapidly, although the underlying depolarizations were more persistent and could be repeatedly evoked. Spiking responses

have only been seen in cells with resting membrane potentials between - 5 0 and - 4 0 mV. The long-latency slow EPSP was abolished by superfusion with solutions containing 3.75 mM Mg 2+ and 0.25 mM Ca2+ (Fig. 4C) but could be evoked in solutions containing 100 ~M (+)-tubocurare or 1/~M atropine (Fig. 4D); the input resistance increase persisted during manual clamp back to the resting potential (Fig. 4D). In general, unambiguous short-latency slow EPSPs and long-latency slow EPSPS have not been seen in the same cell.

Effects of muscarinic agonists Several autonomic ganglia contain muscarinic receptors which mediate hyperpolarizing2a and depolarizing 2,7,a4 responses. Radioligand binding studies indicate that embryonic chick sympathetic ganglia also

105

A.

iiiiilililili,illi,ifil fflllllll

I__ B. 20 Hz O R T H O D R O M I C rTl

20 Hz I N T R A C E L L U L A R

20 Hz O R T H O D R O M I C

I!

i I

C.

D.

CONTROL

HIGH Mg °°

1 pM A T R O P I N E

III

RECOVERY

100 liM D - T U B O C U R A R E i h l ~ l l l '

/ill.

,"1111tll I , l l " t ' , l n ~ , l '~ i #, , . • --:"., - :~

:i,i'. 'l','' ',

i !

Fig. 4. Long-latency slow synaptic transmission. A: repetitive orthodromic stimulation (30 Hz) evokes a slow EPSP which achieves maximum depolarization following cessation of stimulus train (arrow, left panel). In another cell (right panel), application of constant hyperpolarizing current pulses (current trace not shown) reveals an increase in input resistance during the response (15 Hz stimulation). B: repetitive orthodromic stimulation (20 Hz) evokes a small depolarization and spontaneous action potentials in a previously quiescent cell (left panel). In the same cell, repetitive intracellular stimulation (20 Hz, middle panel) produces no change in membrane potential or spike activity. A second orthodromic stimulus train (20 Hz) again evokes action potentials (right panel). C: in a different cell, repetitive orthodromic stimulation produces a depolarization and spontaneous spiking activity (left panel). Superfusion with Tyrode's solution containing 3.75 mM Mg2÷ and 0.25 mM Ca2÷ (middle panel) blocks this response. Following a recovery period in normal solution, the long-latency slow EPSP is restored, although the spiking response has fatigued (right panel, also see text). Repetitive stimulation is at 30 Hz in all panels. D: non-cholinergic nature of the long-latency slow EPSP is shown by superfusion with 1pM atropine (left panel, drug on for 10 min, 15 Hz stimulation) and 100/~M (+)-tubocurare (right panel, drug on for 30 min, 30 Hz stimulation). Right panel also reveals an increase in input resistance during the slow response which persists during manual clamp back to the resting membrane potential. Calibration bars: A: left panel, 20 mV, 5 s; right panel, 10 mV, 10 s. B: 0.5 nA, 20 mV, 2 s. C: 20 mV, 2 s. D: left panel, 10 mV, 2 s; right panel, 0.5 nA, 20 mV, 2 s. contain muscarinic receptors 35. We have therefore examined the effects of carbachol in the presence and absence of 100 /~M ( + ) - t u b o c u r a r e , a dose which completely blocks nicotinic transmission in avian autonomic ganglia 13A5. Pressure injection of carbachol evoked biphasic depolarizations in all cells tested (Fig. 5A). The early

phase of the carbachol depolarization was associated with a decrease in input resistance and was followed by a prolonged phase during which input resistance was increased. Superfusion with Tyrode's solution containing 100/~M ( + ) - t u b o c u r a r e blocked only the initial decrease in input resistance (Fig. 5B), while the later phase was blocked by 1/~M atropine 13. The

106

rent appeared to be abolished since the voltage relaxations normally caused by small hyperpolarizing current pulses were completely absent (Fig. 5C).

A. I I

........... t;t............. ~

L

..............................................

i ............................

...................................

.......

Effects of peptides Carb

~' Carb

B.

100 pM D-TUBOCURARE

[~

-54

t

Carb c. CONTROL AND CARBACHOL

-.:~

~

C O N T R O L AND R E C O V E R Y

-,3 -60

,---

Fig. 5. Effects of carbachol. A: pressure injection of carbachol (10 4 M in mieropipette, 20 p.s.i, for 5 s at arrow) evokes a biphasic depolarization. The initial phase of the depolarization is associated with a decrease in input resistance while the later phase is associated with an increase in input resistance which persists during manual clamp back to the resting membrane potential. In the same cell (right panel) and during manual hyperpolarization to -70 mV, carbachol again produces a biphasic depolarization. In this case, the initial decrease in input resistance is enhanced while the later decrease in input resistance is attenuated, but detectable. B: pressure injection of carbachol (10 gl of 10-2 M from hand-held pipette) in the presence of 100 ,uM (+)-tubocurare elicits a depolarization associated with only an increase in input resistance. Increase in input resistance persists during manual clamp back to resting membrane potential. Anode break spikes are present in two of the panels. C: blockade of M-current by pressure injection of carbachol (20/A of 10-2 M from hand-held pipette). Application of small hyperpolarizing current pulses (not shown, 120 ms duration) results in non-ohmic relaxation in voltage responses as in Fig. 1A. Left panel shows control response (top sweep) and response 35 s after pressure injection of carbachol (bottom sweep). Membrane potential was manually clamped back to the resting potential during the carbachol exposure. Right panel shows control response and recovery from carbachol (100 s after pressure injection of carbachol). Calibration bars: A: 0.5 nA, 20 mV, 14 s; B: 20mV, 5 s; C: 25 mV, 25 ms.

muscarinic increase in input resistance persisted during manual clamp back to the resting membrane potential (Fig. 5A). When the resting membrane potential was manually clamped down to -70 mV, this increase in input resistance was attenuated but not abolished (Fig, 5A). These results suggest that at least some of the muscarinic depolarization was due to inhibition of a current resembling the M-current. In fact, during the exposure to carbachol, the M-cur-

Both classes of slow EPSPs could be evoked in the presence of high concentrations of cholinergic antagonists. Because peptidergic transmission has been reported in adult amphibian 29,3s and mammalian~S sympathetic ganglia, we have tested the effects of substance P and L H - R H on embryonic chick sympathetic neurons. Superfusion with 1 gM substance P, or pressure injection of substance P by two different methods, produced slow depolarizations in 16 out of 20 cells tested (Fig. 6A, B). The depolarizations were associated with an increase in input resistance and produced A

t Sub P

t

Eq

Sub P

B

1 pM Sub P

1 IJM Sub P

1

G.

--~r]r~!li1~Ilt~lllI"",,., 20 pM LHRH

20 pM LHRH

Fig. 6. Effects of substance P and LH-RH. A: pressure injection of substance P (50~1 of 10-3 M from hand-held pipette, left panel) produces a slow depolarization associated with an increase in input resistance which persists during manual clamp back to resting membrane potential. In another cell, pressure injection of substance P (40 ~M in micropipette, 20 p.s.i, for 5 s at arrow, right panel) evokes a depolarization associated with repetitive spiking and an increase in input resistance. B: bath application of 1 pM substance P (bars) produces effects identical to those seen following pressure injection of substance P. C: bath application of 20 ~M mammalian LH-RH (bars) produces no effects on membrane potential or input resistance. Calibration bars: A: left panel, 0.25 nA, 20 mV, 5 s; right panel, 1 nA, 20 mV, 5 s. B: left panel, 1 nA, 10 mV, 14 s; right panel. 0.5 nA, 20 mV, 14 s. C: left panel, 10 mV, 14 s; right panel, 10 mV, 5 s.

107 spiking in 4 cells, and therefore resembled the longlatency slow EPSP (Fig. 6A, B). The M-current was blocked during the substance P depolarization in much the same way as for the muscarinic depolarization (Fig. 5C; data not shown). In contrast to the results with substance P, superfusion of mammalian L H - R H (1-20 pM) did not produce any effects on membrane potential or input resistance in embryonic chick sympathetic neurons (6 cells tested, Fig. 6C). A.

~

St. 3 9

St. 41

Results from early embryos We have examined the properties of sympathetic neurons in earlier embryos. Successful impalements were obtained from embryos as early as Stage 38 (12 days of incubation, Fig. 7A). Even at this early stage, fast nicotinic EPSPs mediated by multiple inputs were observed following stimulation of the rostral interganglionic connective (Fig. 7B). In contrast, the earliest stage at which we have successfully evoked a slow EPSP was Stage 42 (16 days of incubation), where we have observed slow EPSPs similar to those seen in the Stage 43-45 embryos (Fig. 7C). Intracellular injections of H R P in Stage 42 neurons revealed a dendritic complexity similar to that observed in neurons from Stage 43-45 embryos (Fig. 7D). DISCUSSION

B.

Properties of aviart sympathetic neurons and comparison to amphibian and mammalian sympathetic cells St. 38

St. 39

C.

1_ St. 42

D.

20 pM

St. 42 Fig. 7. Electrophysiological and morphological properties of neurons from earlier embryonic stages. A: action potentials evoked by depolarizing current pulses in Stage 39 and Stage 41 chick sympathetic neurons. B: orthodromic stimulation of the rostral interganglionic connective evokes detectable nicotinic fast EPSPs (arrows) in Stage 38 and Stage 39 neurons. C: shortlatency slow EPSP in Stage 42 neuron is produced by 30-Hz orthodromic stimulation. D: camera lucida drawings of Stage 42 sympathetic neurons labeled by intracellular injection of HRP. Stars indicate identifiable axons. Calibration bars: A: left panel, 0.25 nA, 20 mV, 10 ms; right panel, 0.5 nA, 20 mV, 10 ms. B: left panel, 0.5 nA, 20mV, 10 ms; right panel, 0.25 nA, 20 mV, 10 ms. C: 20 mV, 5 s.

The resting membrane potentials, action potentials and passive membrane properties of Stage 43-45 chick sympathetic neurons resembled those of adult amphibian and mammalian sympathetic neurons 45.49. Moreover, avian sympathetic neurons displayed M-current channels which, as in adult amphibian sympathetic neurons, were inhibited by both substance P and carbachoP. 7.8. A second voltage relaxation resembled a response previously observed in adult rat sympathetic neurons 7. The ionic basis of this response in chick neurons remains to be determined. An unexpected finding was that 3 mM Ba 2÷ produced profound increases in action potential duration in chick sympathetic neurons. In agreement with previous workers 43, we did not see a similar effect in adult rat sympathetic neurons. Voltage-dependent Ca2+-channels are highly permeable to Ba2+ (ref. 21), but Ba 2+ wilt not activate Ca2+-dependent K ÷currents (I~(ca)) l. Thus, it is possible that embryonic avian sympathetic neurons rely primarily on IK(Ca) for repolarization following an action potential. Alternatively, voltage-dependent K+-channels in embryonic chick sympathetic neurons may be unusually susceptible to blockade by Ba 2+. We have not observed anomalous inward rectification in embryonic chick sympathetic neurons, although this response has been observed in cultured mammalian sympathetic neurons 19 and in Stage 43-45 chick parasympathetic

108 neurons 16. We have previously shown that fast synaptic transmission in chick embryo sympathetic ganglia is nicotinic and pharmacologically similar to that in chick ciliary ganglia ~3. In the present study, we were able to demonstrate convergent preganglionic input in most cells. Thus, 3-5 gradations in the fast EPSP were routinely observed. This must be regarded as a minimum estimate of convergence for several reasons. First, it is unlikely that all of the preganglionic fibers course through the rostral interganglionic connective. Indeed, many fine nerves are seen coming out of the sympathetic chain at more caudal levels; at this time, we are unable to stimulate these trunks. Second, because of the delicate nature of embryonic sympathetic chains, it is likely that some damage is sustained during dissection, pinning and application of the suction electrode. Lastly, counting inputs by recruitment relies on differences in stimulus thresholds of the various fibers coursing through the trunk. If two fibers were to have identical thresholds, we would not be able to discriminate them by our techniques. Simultaneous activation of more than one preganglionic fiber is always required to evoke action potentials in postganglionic cells. Thus, integration occurs in embryonic avian sympathetic ganglia and closely resembles that observed in adult mammalian paravertebral sympathetic ganglia 6. In contrast, one class of sympathetic neurons in amphibia (B neurons) are singly innervated 46 and produce all-or-none suprathreshold fast EPSPs. A second class of neurons in the frog (C neurons) do receive convergent preganglionic input. Previous studies of adult avian sympathetic ganglion cells indicated that these cells were multipolar neurons characterized by short unbranched dendrites27, 53. These early studies utilized silver impregnation methods or intra-arterial methylene blue staining. We have used intracellular injections of H R P followed by histochemical processing to obtain a more complete stain. We find that embryonic avian sympathetic neurons are relatively small multipolar cells with 5-10 primary dendrites per cell. These dendrites branch moderately and are somewhat longer than the previous studies indicated. Our results are therefore in substantial agreement (considering the limitations of our recruitment technique) with those

of Purves and Hume52; these workers found a relationship between the number of dendrites and the number of preganglionic inputs in mammalian autonomic neurons. In contrast, the major portion of homologous amphibian sympathetic neurons are known to be large, essentially unipolar cells 27. We have found two classes of non-cholinergic, synaptically-mediated slow EPSPs following stimulation of the rostral interganglionic connective. These slow EPSPs are distinguished on the basis of time course and resulting changes in input resistance. The shortlatency slow EPSP has a relatively rapid onset and is not associated with a discernible change in input resistance. The long-latency slow EPSP has a more delayed onset and is associated with an increase in input resistance. Similarly, B neurons in adult amphibian sympathetic ganglia also display two types of slow EPSPs, associated with either an increase (Type I) or a decrease (Type II) in input resistance 5. In several cells, the long-latency slow EPSP produced spontaneous spiking, thus affecting the integrative activity of the neurons. These spontaneous action potentials may have been due to the increase in input resistance, since spontaneous miniature EPSPs may become suprathreshold under these conditions. Muscarinic agonists produced depolarizations associated with an increase in input resistance and inhibition of the M-current in embryonic chick sympathetic neurons. This finding is in agreement with results obtained with adult mammalian 7,s and amphibian 4 sympathetic neurons. In amphibian ganglia, the early phase of the slow EPSP is muscarinic in nature 30. However, we were unable to detect any muscarinic slow EPSPs in the chick neurons. It is certainly possible that muscarinic potentials do exist within these neurons, but that our recording methods were not able to detect these potentials. Peptidergic synaptic transmission is well-established in adult amphibian and mammalian sympathetic ganglia. In guinea pig prevertebral ganglia, a slow EPSP has been attributed to the release of substance P from sensory nerves 4s, although other peptides may also be involved in this response 50. A plexus of substance P-immunoreactive nerve fibers has been described in embryonic avian sympathetic ganglia 17. Our observations demonstrate that in embryonic chick sympathetic ganglia, substance P mimics the effects of the long-latency slow EPSP. Thus,

109 substance P can be regarded as a putative mediator of the long-latency slow EPSP. Embryonic chick parasympathetic neurons also exhibit a slow EPSP which is mimicked by substance P (Dryer and Chiappinelli, submitted). Thus, choroid neurons in Stage 43-45 ciliary ganglia respond to substance P with a depolarization and an increase in input resistance, although the choroid neurons desensitize to the effects of substance P much more rapidly than do the sympathetic neurons. The choroid neurons are multiply innervated, as are the sympathetic cells. There is also a group of cells in the ciliary ganglion which are singly innervated, namely, the ciliary neurons. These ciliary cells lack both detectable slow EPSPs and any measurable response to applied substance p16. However, the presynaptic terminals synapsing on the ciliary neurons do respond to substance P with a depolarization and an increase in input resistance 16. Substance P, therefore, exhibits both presynaptic and postsynaptic actions in chick autonomic ganglia. A recent study of cultured avian sympathetic and parasympathetic neurons using whole-cell patchclamp methods did not detect depolarizations following application of substance P; instead, high concentrations of substance P (10-20pM) were found to reduce acetylcholine channel open-time 54. It is possible that the differences observed between our results and those obtained in dissociated cell culture are due to developmental factors, as the sympathetic cells were cultured prior to the appearance of significant amounts of substance P-like immunoreactivity25. Additionally, M-currents are not always detectable using whole-cell patch-clamp techniques20 and this could eliminate any depolarizations caused by substance P. Alternatively, some transformations may have occurred following dissociation and plating of the cultured cells. An LH-RH-Iike peptide, possibly teleost-LH-RH, is thought to mediate the late slow EPSP in amphibian sympathetic ganglia 32. In contrast, pressure injection or superfusion of L H - R H had no effect on embryonic avian sympathetic neurons.

Development of synaptic transmission in chick sympathetic ganglia Our results demonstrate that by Stage 43-45, chick sympathetic neurons have many of the properties of homologous adult mammalian neurons, in-

cluding the presence of both fast and slow synaptic potentials and a complex dendritic morphology. We have also used earlier staged embryos to examine the developmental onset of some of these properties. From the earliest age tested (Stage 38, 12 days of incubation), nerve stimulation evoked fast nicotinic EPSPs, often with multiple inputs contributing to the response. It should be noted that at this stage, the ganglia are biochemically and morphologically immature. For example, biochemical indices related to neurotransmission are detected only at low levels at this stage and exhibit rapid rates of increase immediately thereafter11,nl,aL A morphometric analysis using electron microscopy has shown that at 10 days of incubation, fewer than 1% of the adult number of synapses are present26. Thus, our results indicate that nicotinic transmission appears at developmental stages where both biochemical and morphological indicators of synaptogenesis exist at only low levels. This progression has also been observed during the development of the avian ciliary ganglion14,37.38 and may therefore be a general phenomenon. Slow synaptic potentials were not observed until considerably later in development, at Stage 42 (16 days of incubation). This could be due to a later arrival of fibers mediating the slow EPSPs, although substance P-containing fibers can be detected in the ganglia as early as 12 days of incubation 17. However, a large increase in the specific activity of substance P occurs after 16 days of incubation 25, following the appearance of the slow synaptic potentials. Thus, if substance P is indeed the mediator of the long latency slow EPSPs, this progression of large increases in biochemical indices of development following the onset of functional activity would parallel the findings for nicotinic transmission in both sympathetic and ciliary ganglia. When the slow EPSPs first appear, the dendritic morphology of the sympathetic cells is similar to that seen at later stages of development and approximates that of adult mammalian sympathetic neurons 51. Whether or not this dendritic arborization is essential for the inputs responsible for the slow EPSPs remains to be demonstrated. As noted above, chick ciliary neurons, which lack dendrites or have very reduced dendritic processes 9, do not display slow EPSPs. This absence of slow EPSPs in avian autonomic neurons lacking dendrites can be contrasted with observations in frog sympathetic gan-

110 glia, w h e r e n e u r o n s having no d e n d r i t e s exhibit sev-

s y m p a t h e t i c ganglia thus p r o v i d e an e x c e l l e n t m o d e l

eral types of slow E P S P s 5,27,33.

system for the f u r t h e r study of the d e v e l o p m e n t of

A v i a n a u t o n o m i c ganglia offer several distinct ad-

slow and fast synaptic transmission. Studies are cur-

v a n t a g e s for the study of synapse m a t u r a t i o n . T h e

rently u n d e r w a y to d e t e r m i n e the role of synaptic

e m b r y o d e v e l o p s in a s e l f - c o n t a i n e d c h a m b e r free

transmission in the d e v e l o p m e n t of these ganglia.

f r o m m a t e r n a l influences, which facilitates dissection and allows relatively easy access for p h a r m a c o l o g i c a l and surgical m a n i p u l a t i o n s .

ACKNOWLEDGEMENTS

M o r e o v e r , t h e r e is a S u p p o r t e d by N I H G r a n t NS17574 to V . A . C . W e

large b a c k g r o u n d of b i o c h e m i c a l and m o r p h o l o g i c a l of avian

w o u l d like to t h a n k K a t h l e e n W o l f for p r e p a r i n g the

a u t o n o m i c ganglia. In particular, the d e v e l o p m e n t of

figures and M a g g i e K l e v o r n for typing the m a n u -

q u a i l - c h i c k c h i m e r a s has a l l o w e d identification of

script. W e are grateful to Dr. C y n t h i a J. F o r e h a n d

p r o g e n i t o r cells and has p r o v i d e d a m e a n s of d e t e r -

and Dr. Carl M. R o v a i n e n for critical r e a d i n g s of ear-

mining the influences of target tissue on synapse for-

ly drafts of the m a n u s c r i p t , and to Dr. A. R. M a r t i n

m a t i o n in the a u t o n o m i c n e r v o u s system 39. A v i a n

for helpful technical discussions.

information

regarding

the d e v e l o p m e n t

REFERENCES 1 Adams, D. J. and Gage, P. W., Divalent ion currents and the delayed potassium conductance in an Aplysia neurone, J. Physiol. (Lond.), 304 (1980) 297-313. 2 Adams, P. R. and Brown, D. A., Luteinizing hormone-releasing factor and muscarinic agonists act on the same voltage-sensitive K+-current in bullfrog sympathetic neurones, Brit. J. Pharmacol., 68 (1980) 353-355. 3 Adams, P. R., Brown, D. A. and Constanti, A., M-currents and other potassium currents in bullfrog sympathetic neurones, J, Physiol. (Lond.), 330 (1982)537-572. 4 Adams, P. R., Brown, D. A. and Constanti, A., Pharmacological inhibition of the M-current, J. Physiol. (Lond.), 332 (1982) 223- 262. 5 Akasu, T., Gallagher, J. P., Koketsu, K. and Shinnick-Gallagher, P., Slow excitatory postsynaptic currents in bullfrog sympathetic neurones, J. Physiol. (Lond.), 351 (1984) 583-593. 6 Blackman, J. G. and Purves, R., Intracellular recordings from ganglia of the thoracic sympathetic chain of the guinea pig, J. Physiol. (Lond.), 203 (1969) 173-198. 7 Brown, D. A., Adams, P. R. and Constanti, A., Voltagesensitive K-currents in sympathetic neurons and their modulation by neurotransmitters, J, Auton. Nerv. Syst., 6 (1982) 23-35. 8 Brown, D. A. and Constanti, A., Intracellular observations on the effects of muscarinic agonists on rat sympathetic neurones, Brit. J. Pharmacol., 70 (1980) 593-608. 9 Cantino, D. and Mugnaini, E., The structural basis for electrotonic coupling in the avian ciliary ganglion. A study with thin sectioning and freeze-fracturing, J. Neurocytol., 4 (1975) 505-536. 10 Caserta, M. and Ross, L. L., Biochemical and morphological studies of synaptogenesis in the avian sympathetic cell column, Brain Res., 144 (1978) 241-255. 11 Chiappinelli, V. A., Biochemical Development of Cholinergic Synapses in the Peripheral Sympathetic and Parasympathetic Nervous System of the Chick Embryo, Thesis, University of Corinecticut, Storrs, CT, 1977, pp. 1-132. 12 Chiappinelli, V. A., Kappa-bungarotoxin: a probe for the

13

14

15

16

17

18

19

20

21 22 23

24

neuronal nicotinic receptor in the avian ciliary ganglion, Brain Res., 277 (1983) 9-22. Chiappinelli, V. A. and Dryer, S. E., Nicotinic transmission in sympathetic ganglia: blockade by the snake venom neurotoxin kappa-bungarotoxin, Neurosci. Lett., 50 (1984) 239-244. Chiappinelli, V. A., Giacobini, E., Pilar, G. and Uchimura, H., Induction of cholinergic enzymes in chick ciliary ganglion and iris muscle cells during synapse formation, J. Physiol. (Lond.), 257 (1976) 749-766. Dryer, S. E. and Chiappinelli, V. A., Kappa-bungarotoxin: an intracellular study demonstrating blockade of neuronal nicotinic receptors by a snake neurotoxin, Brain Res.. 289 (1983) 317-321. Dryer, S. E. and Chiappinelli, V. A., Substance P depolarizes nerve terminals in an autonomic ganglion. Brain Res.. in press. Fontaine-Perus, J. C., Chanconie, M. and LeDouarin, N. M., Differentiation of peptidergic neurones in quail-chick chimaeric embryos, Cell Diff.. 11 (1982) 183-193. Forehand, C. J. and Purves, D., Regional innervation of rabbit ciliary ganglion cells by the terminals of preganglionic axons, J. Neurosci., 4 (1984) 1-12. Freschi, J. E., Membrane currents of cultured rat sympathetic neurons under voltage clamp, J. Neurophysiol., 50 (1983) 1460-1478. Galvan, M., Satin, L. S. and Adams, P. R., Comparison of conventional microelectrode and whole-cell patch clamp recordings from cultured bullfrog ganglion cells, Soc. Neurosci. Abstr., 10 (1984) 146. Hagiwara, S. and Ohmori, H., Studies of calcium channels in rat clonal pituitary cells With patch electrode voltageclamp, J. Physiol. (Lond.), 331 (1982) 231-252. Hamburger, V. and Hamilton, H. L., A series of normal stages in the development of the chick embryo, J. Morphol., 88 (1951) 49-92. Hanker, J. S., Yates, P. E., Metz, C. B. and Rustioni, A., A new specific, sensitive, and non-carcinogenic reagent for the demonstration of horseradish peroxidase, Histochem. J., 8 (1977) 789-792. Hartzell, H. C., Kuffler, S., Stickgold, R. and Yoshikami,

111 D., Synaptic excitation and inhibition resulting from direct action of acetylcholine on two types of chemoreceptors on individual amphibian parasympathetic neurones, J. Physiol. (Lond.), 271 (1977) 817-846. 25 Hayashi, M., Edgar, D. and Thoenen, H., The development of substance P, somatostatin and vasoactive intestinal peptide in sympathetic and spinal sensory ganglia of the chick embryo, Neuroscience, 10 (1983) 30-39. 26" Hruschak, K., Friedrich, V. L., Jr. and Giacobini, E., Synaptogenesis in chick paravertebral sympathetic ganglia: a morphometric analysis, Develop. Brain Res., 4 (1982) 229-240. 27 Huber, G. C., A contribution on the minute anatomy of the sympathetic ganglia of the different classes of vertebrates, J. Morph., 16 (1900) 27-90. 28 Jahnsen, H. and Llinas, R., Ionic basis for the electro-responsiveness and oscillatory properties of guinea-pig thalamic neurones in vitro, J. Physiol. (Lond.), 349 (1984) 227-247. 29 Jan, L. Y. and Jan, Y. N., Peptidergic transmission in sympathetic ganglia of the frog, J. Physiol. (Lond.), 327 (1982) 219-246. 30 Jan, Y. N. and Jan, L., A LHRH-like peptidergic transmitter capable of action at a distance in autonomic ganglia, Trends Neurosci., 6 (1983) 320-325. 31 Johnson, S. M., Katayama, Y. and North, R. A., Slow synaptic potentials in neurones of the myenteric plexus, J. Physiol. (Lond.), 301 (1980) 505-516. 32 Jones, S. W., Adams, P. R., Brownstein, M. J. and Rivier, J. E., Teleost luteinizing hormone-releasing hormone: action on bullfrog sympathetic ganglia is consistent with role as neurotransmitter, J. Neurosci., 4 (1984) 420-429. 33 Katayama, Y. and Nishi, S., Voltage-clamp analysis of peptidergic slow depolarizations in bullfrog sympathetic ganglion cells, J. Physiol. (Lond.), 333 (1982) 305-313. 34 Kirby, M. L. and Gilmore, S. A., A correlative histofluorescence and light microscopic study of the formation of the sympathetic trunks in chick embryos, Anat. Rec., 186 (1976) 437-449. 35 Kouvelas, E. D. and Greene, L. A., Development of muscarinic cholinergic receptors in chick embryo sympathetic ganglia, Develop. Neurosci., 5 (1982) 375-378. 36 Kuntz, A., The development of the sympathetic nervous system in birds, J. cornp. Neurol., 20 (1910) 283-308. 37 Landmesser, L. and Pilar, G., The onset and development of transmission in the chick ciliary ganglion, J. Physiol. (Lond.), 222 (1972) 691-713. 38 Landmesser, L. and Pilar, G., Synaptic transmission and cell death during normal ganglionic development, J. Physiol. (Lond.), 241 (1974) 737-749. 39 LeDouarin, N. M., The ontogeny of the neural crest in avian embryo chimaeras, Nature (Lond.), 286 (1980) 663-669. 40 Luckenbill-Edds, L. and van Horn, C., Development of chick paravertebral sympathetic ganglia. I. Fine structure

and correlative histofluorescence of catecholaminergic cells, J. comp. Neurol., 191 (1980)65-76. 41 Marchi, M., Giacobini, E. and Hruschak, K., Development and aging of cholinergic synapses. I. Endogenous levels of acetylcholine and choline in developing autonomic ganglia and iris of the chick, Develop. Neurosci., 2 (1979) 201-212. 42 Marchisio, P. C. and Consolo, S., Developmental changes of choline acetyltransferase (ChAc) activity in chick embryo spinal and sympathetic ganglia, J. Neurochem., 15 (1968) 759-764. 43 McLachlan, E. M., The effects of strontium and barium ions at synapses in sympathetic ganglia, J. Physiol. (Lond.), 267 (1977) 497-518. 44 Morita, K., North, R. A. and Tokimasa, T., Muscarinic agonists inactivate potassium conductance of guinea-pig myenteric neurones, J. Physiol. (Lond.), 333 (1982) 125-139. 45 Nishi, S. and Koketsu, K., Electrical properties and activities of single sympathetic neurons in frogs, J. cell. comp. Physiol., 55 (1960) 15-30. 46 Nishi, S., Soeda, H. and Koketsu, K., Studies on sympathetic B and C neurons and patterns of preganglionic innervation, J. cell. comp. Physiol., 66 (1965) 19-32. 47 Oppenheim, R. W., Maderdrut, J. L. and Wells, D. J., Reduction of naturally-occurring cell death in the thoracolumbar preganglionic cell column of the chick embryo by nerve growth factor and hemicholinium-3, Develop. Brain Res., 3 (1982) 134-139. 48 Otsuka, M. and Konishi, S., Substance P - the first peptide neurotransmitters?, Trends Neurosci., 6 (1983) 317-320. 49 Perri, V., Sacchi, O. and Casella, C., Electrical properties and synaptic connections of the sympathetic neurons in the rat and guinea-pig superior cervical ganglion, Pfliigers Arch. ges. Physiol., 314 (1970) 40-54. 50 Peters, S. and Kreulen, D., Vasopressin may be a transmitter of slow potentials in guinea pig inferior mesenteric ganglia, Soc. Neurosci. Abstr., 10 (1984) 1120. 51 Purves, D., Modulation of neuronal competition by postsynaptic geometry in autonomic ganglia, Trends Neurosci., 6 (1983) 10-16. 52 Purves, D. and Hume, R., The relation of postsynaptic geometry to the number of presynaptic axons that innervate autonomic ganglion cells, J. Neurosci., 1 (1981) 441-452. 53 Ramon y Cajal, S., Algunos detalles sobre las cellulas simpaticas, Ergebn. Anat. Entwickl.-Gesch., 2 (1892) 22-51. 54 Role, L. W., Substance P modulation of acetylcholine-induced currents in embryonic chicken sympathetic and ciliary ganglion neurons, Proc. nat. Acad. Sci. U.S.A., 81 (1984) 2924-2928. 55 Wechsler, W. and Schmekel, L., Elektronenmikroskopische Untersuchung der Entwicklung der vegetativen (grenzstrang-) und spinalen Ganglien bei Gallus domesticus, Acta neuroveg., 30 (1967) 427-444.