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Pharmacological studies in frog sympathetic ganglion: support for the cholinergic monosynaptic hypothesis for slow IPSP mediation
175
Brain Research, 452(1988) 175-183 Elsevier
BRE 13639
Pharmacological studies in frog sympathetic ganglion" support for the cholinergic monosynaptic hypothesis for slow IPSP mediation Parviz Yavari* and Forrest F. Weight Section on Electrophysiology, Laboratory of Physiologic and Pharmacologic Studies, National Institute on Alcohol Abuse and Alcoholism, Rockville, MD 20852 (U.S.A.)
(Accepted 15 December 1987) Key words: Sympathetic ganglion; Sucrose-gap; Slow inhibitory postsynaptic potential; Adrenergic pharmacology; Bullfrog
The slow inhibitory postsynaptic potential (slow IPSP), the slow excitatory postsynaptic potential (slow EPSP), the late slow excitatory postsynaptic potential (late slow EPSP), and the fast excitatory postsynaptic potential/compound action potential (fast EPSP) were recorded from the 9th or 10th paravertebral sympathetic ganglia of bullfrogs (and some Rana pipiens frogs) by the sucrose-gap technique. The adrenergic antagonists phentolamine, dihydroergotamine and propranolol did not show any antagonistic effect on the slow IPSP when used at concentrations of up to 10, 100 and 10 ~M, respectively. U-0521 (3',4'-dihydroxy-2-methylpropriophenone, 50/~g/ml), a specific inhibitor of catechol-O-methyltransferase, did not show any potentiating effect on the slow IPSP. The cholinesterase inhibitor neostigmine (0.5-1/~M) induced a large increase in the duration and amplitude of slow IPSP. When phentolamine and propranolol at concentrations greater than 10/~M were used the slow IPSP (and all other synaptic potentials) were non-specifically reduced in amplitude by these drugs. The results reported in this paper do not lend any support to the hypothesis that the slow IPSP in frog sympathetic ganglia is mediated by an adrenergic interneuron. The results are consistent with the proposal that the slow IPSP in this ganglion is mediated by a direct action of acetylcholine released from cholinergic preganglionic fibers.
INTRODUCTION The n e u r o t r a n s m i t t e r and pathway m e d i a t i n g the slow inhibitory postsynaptic potential (IPSP) in sympathetic ganglia have been controversial in both m a m m a l i a n and amphibian species (for reviews see refs. 11 and 26). In frog sympathetic ganglia two hypotheses exist for the m e d i a t i o n of this potential. One hypothesis proposes that the slow IPSP is mediated by a direct muscarinic action of acetylcholine ( A C h ) released from cholinergic preganglionic fibers 27. A second hypothesis proposes that the slow IPSP is m e d i a t e d by a catecholamine r e l e a s e d from an adrenergic interneuron (later defined as small intensely fluorescent (SIF) cells) 1s'24. T h e latter hypothesis is based, in part, upon the observations that the a - a d r e n e r g i c antagonists p h e n t o l a m i n e and dih y d r o e r g o t a m i n e in a concentration of 4 0 / ~ M (or greater) partially depress the slow IPSP, and that U-
0521 (50 /~g/ml), a specific inhibitor of catecholO-methyltransferase ( C O M T ) , p o t e n t i a t e s slow IPSP 18. In the p r e s e n t study we carried out a careful re-examination of the adrenergic t h e o r y for slow IPSP m e d i a t i o n in frog ganglia by studying the effects of p h e n t o l a m i n e , d i h y d r o e r g o t a m i n e , p r o p r a n o l o l and U-0521 on this potential using the sucrose-gap method. T h e results showed no specific interaction of these agents with the p r o p o s e d adrenergic system in this ganglion. The results are thus consistent with several recent reports 8'22'23'25'28 presenting evidence for,the 'direct muscarinic '27 m e d i a t i o n of slow IPSP in frog sympathetic ganglia. A n abstract describing some of this w o r k has a p p e a r e d 29. MATERIALS AND METHODS E x p e r i m e n t s were carried out on the isolated p a r a vertebral ganglionic p r e p a r a t i o n of bullfrogs (Rana
* Present address: Department of Physiology and Cellular Biophysics, Columbia University, College of Physicians and Surgeons, 630 West 168th Street, New York, NY 10032, U.S.A. Correspondence: F.F. Weight, Section on Electrophysiology, LPPS, NIAAA, 12501 Washington Avenue, Rockville, MD 20852, U.S.A.
176
catesbeiana), and in a few experiments on large (1>4inch) Rana pipiens frogs. The sucrose-gap method used in the present study has been described in detail elsewhere 23, and is based on that of Nishi and Koketsu 21. Briefly, the ganglionic preparation was mounted in a sucrose-gap chamber (a slab of clear plexiglass with 4 compartments engraved in it from end-to-end) so that the sympathetic chain and the rostral portion of the 8th spinal nerve were stationed in a compartment under mineral oil, the 9th or 10th ganglion was laid in an adjacent compartment through which Ringer solution flowed, the 9th or 10th ganglionic ramus passed through a third compartment through which isotonic sucrose solution flowed, and finally, the distal portion of the ramus (and a portion of attached spinal nerve) was placed in a fourth compartment in isotonic KC1 solution. Grooves between compartments were tightly sealed with a mixture of petroleum jelly and wax. Synaptic potentials were recorded between the ganglion and the distal portion of its ramus in isotonic KC1 solution by use of a pair of isotonic KCl-agar bridge-calomel electrode assemblies (with the ganglion compartment grounded). Synaptic potentials were elicited by stimulation of the sympathetic chain or the 8th spinal nerve with a pair of platinum electrodes under mineral oil. The sympathetic chain between the 6th and 7th ganglia was stimulated with 0.5 ms pulses to activate the preganglionic B fibers. The 8th spinal nerve was stimulated with 1 ms pulses to activate preganglionic C fibers. Supramaximal stimuli were used in both cases. The Ringer solution had the following composition (in mM): NaC1 100, KC12, CaC12 1.8, Tris-HC1 16, glucose 1 g/liter, pH 7.2. Except when the effect of phentolamine on synaptic transmission was studied, the Ringer solution also contained 70~M D-tubocurarine (d-Tc) in order to reduce the amplitude of the fast EPSP and thus prevent action potential generation 26. All experiments were carried out at room temperature. The fast excitatory postsynaptic potential (EPSP)/compound action potential was photographed from the oscilloscope, while the slow potentials were recorded on a Gould rectiliniar penrecorder. Effects of drugs on synaptic potentials were studied by switching the ganglionic superfusion system to Ringer solutions containing known concentrations of these agents (by use of a 3-way tap system). The following chemicals were used: d-Tc chlor-
ide, (_+)-propranolol hydrochloride and neostigmine bromide (Sigma), dihydroergotamine methanesulfonate (gift of Sandoz), U-0521 (gift of Upjohn) and phentolamine hydrochloride (gift of Ciba-Geigy). All chemicals were dissolved directly in Ringer's solution, except for dihydroergotamine which was first dissolved in dimethyl sulfoxide (DMSO) and was then added to Ringer solution (final DMSO concentration was 0.1-0.4% (v/v) which did not have any adverse effects on control responses). RESULTS
Effects of phentolamine This agent, which is an effective blocker of all subtypes of a-adrenergic receptors in other systems 2°, produced little or no reduction in the amplitude of slow IPSPs when used at doses of up to 10/~M. When phentolamine at concentrations greater than 10 ~M was used there was a non-specific reduction in the amplitude of slow IPSP as well as those of other synaptic potentials (Fig. 1). Fig. 1A illustrates the effects of phentolamine on the synaptic responses elicited by stimulation of the 8th spinal nerve. It can be seen in Fig. 1A 1 that 10~M phentolamine leaves the synaptic potentials (fast EPSP, slow IPSP and late slow EPSP) virtually unaffected. However, it is seen in Fig. 1Az-A 4 that when concentrations of phentolamine higher than 10/~M (i.e. 40, 100 and 200 ~M, respectively) are used there is a dose-dependent and nonspecific reduction in the amplitude of fast EPSP, late slow EPSP (a peptide-mediated synaptic potential 16) and the slow IPSP. Note particularly the traces of Fig. 1A 4 where the late slow EPSP has been abolished after superfusion with 200 /~M phentolamine and there is a large antagonism of the fast EPSP, whereas a good-size slow IPSP response could still be elicited after exposure to this very high concentration of phentolamine (Fig. 1A 4, central trace). Similarly the non-specific antagonism of the slow EPSP (a synaptic potential that is known in this ganglion to be mediated by a direct action of ACh in a separate pathway than the slow IPSP 26) by phentolamine at doses of 40, 100 and 200 ~M is illustrated in Fig. lB. Thus, these experiments indicate that the reduction of the slow IPSP amplitude by high doses of phentolamine is very unlikely to be due to a specific blockade of an a-adrenergic receptor in this gan-
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Fig. 1. A: effects of phentolamine on the synaptic responses of bullfrog 9th or 10th sympathetic ganglia elicited by stimulation of the VIII spinal nerve at a frequency of 40 Hz for 1 s (period of stimulation indicated by bar). The response of VIII nerve stimulation consists of the following synaptic potentials26: (i) an initial short duration depolarization (upward deflection) at the beginning of the stimulus - - the nicotinic fast EPSP; (ii) a hyperpolarizing response (downward deflection) following the fast EPSP - - the slow IPSP; and (iii) a long-lasting depolarization subsequent to the slow I P S P - - the peptide-mediated late slow EPSP. In order to show the late slow EPSP on the same tracing as the slow IPSP, the speed of the chart recorder was slowed at the end of the slow IPSP (see arrow in bottom right hand tracing). Consequently, the late slow EPSP has a separate time-base calibration (to the right of arrow in bottom right hand tracing). Left: control responses prior to the administration of phentolaminc. Middle: 60 rain after beginning superfusion with Ringer solution containing phentolamine (at concentrations as indicated for each set of tracings). Right: 4 h after beginning wash-out with phentolamine-free Ringer solution. Each set of tracings are from different experiments. Time and voltage calibrations in A 4 are for all traces. B: effects of phentolamine on the synaptic iesponses in bullfrog 9th or 10th sympathetic ganglia elicited by stimulation of the sympathetic chain between 6th and 7th ganglia at a frequency of 100 Hz for 2 s (period of stimulation indicated by bar). The following synaptic potentials comprise the response to chain stimulation26: (i) an initial short duration depolarization (upward deflection) - - the nicotinic fast EPSP; (ii) a small short duration hyperpolarization following the fast EPSP - - a slow IPSP; and (iii) a long-lasting depolarization following the small slow IPSP - - the muscarinic slow EPSP. Left: control responses prior to the administration of phentolamine. Middle: 60 min after beginning superfusion with Ringer solution containing phentolamine (at concentrations as indicated for each set of tracings). Right: 6 h after beginning wash-out with phentolamine-free Ringer solution. Each set of tracings are from different experiments. Tops of the fast EPSP have been cut away in all traces except the central and right hand trace of B 2. C: graphs of the effects of phentolamine on the amplitude of the slow IPSP (filled circles), slow EPSP (open circles), late slow EPSP (filled triangles), and fast EPSP (open triangles). Percent reduction in the synaptic response amplitude is plotted as a function of antagonist concentration. Response amplitude was measured 60 min after beginning superfusion with phentolamine. The data points for each concentration of phentolamine represent the mean + S.E.M. from 4-10 experiments. These experiments were carried out as described above and in the text.
178 glion. To obtain a further understanding of the mechanism of blockade by high doses of phentolamine on the slow IPSP, we constructed d o s e - r e s p o n s e curves of the effects of this drug (at 1-200 ktM) on the various synaptic potentials in this ganglion (Fig. 1C). It is seen in the curves of Fig. 1C that the high doses of phentolamine (i.e. 40, 100 and 200/~M) reduce the amplitude of synaptic potentials (fast EPSP, slow IPSP, late slow E P S P and slow EPSP) more or less to the same degree. Thus it is very unlikely that phentolamine blockade of these potentials (including the slow IPSP) has a postsynaptic mechanism since these potentials each have a different receptor and/or ionic mechanisms of generation in this ganglion (for example the late slow E P S P involves a peptide transmitter while all other PSPs involve ACh) 26. It is likely, how-
PHENTOLAMINE
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Effects of dihydroergotamine
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ever, that the actions of phentolamine are due to a presynaptic suppressive effect on transmitter release in this ganglion (since phentolamine and other ablockers can block calcium channels 3'12 at high concentrations) or are due to block of nerve conduction. In order to investigate these possibilities further, we studied the effects of 1-200 ktM phentolamine on synaptic transmission of the B and C pathways in this ganglion. Phentolamine (40 ktM, superfused for 30 min) reduced the amplitude of the transmitted B action potential by 13 + 2% (mean + S.E.M., n = 8) and the C spike by 12 + 3% (n = 4). When phentolamine 40/zM was superfused for 60 min, the reductions were 23 _+ 5% (n = 4) for the B spike and 20 + 7% (n = 3) for the C spike. At the concentrations of 100 and 200/~M, phentolamine showed 19 + 9% (n = 3) and 38 + 6% (n -- 3) reductions, respectively, in the height of the B volley when it was superfused for 30 min. Fig. 2 illustrates the antagonistic effect of phentolamine on synaptic transmission in a B fiber pathway in frog ganglion. Since synaptic transmission in bullfrog sympathetic ganglia is mediated by a nicotinic cholinergic mechanism 26, these results indicate that high doses of phentolamine can have a nonspecific depressant effect on synaptic transmission (either due to depression of transmitter release, or by conduction block). Taken together, the experiments with phentolamine indicated that this agent has no specific interaction with slow IPSP in the frog ganglion.
,~
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Fig. 2. Effect of phentolamine on the fast EPSP/compound action potential of bullfrog 9th or 10th sympathetic ganglion elicited by single supramaximal shocks to the sympathetic chain between the 6th and 7th ganglia (at arrows). The action potential (upward deflection) and spike afterhyperpolarization are identified as occurring in the ganglionic B cells since there is only a very short (about 10 ms) interval between shock artifact (seen in majority of traces just before the spike) and spike onset26. Left: control responses prior to the administration of phentolamine. Middle: 30 rain after beginning superfusion with Ringer solution containing phentolamine (at concentrations as indicated for each set of tracings). Right: 45 min after beginning with phentolamine Ringer superfusion. Each set of tracings are from different experiments. Time calibration is for all traces.
As with our previous failure to observe a specific blockade of slow IPSP by low doses of dihydroergotamine in frog ganglion 25, neither could we detect any reduction in the amplitude of the slow IPSP when doses of this drug as high as 100 jzM were used (100 /~M = 5 experiments; 40,uM = 12 experiments; 20 y M = 4 experiments; drug superfusion times of up to 2 h) (Fig. 3). With 100 /~M concentrations of dihydroergotamine there was an increase in the duration of the slow IPSP as recorded in the sucrose-gap (e.g. as in central trace of Fig. 3C). However, since the slow IPSP and the late slow EPSP overlap in time in these sucrose-gap records, it is difficult to pinpoint the exact locus of this enhancing action of dihydroergotamine on the two potentials. Alternatively, the high doses of dihydroergotamine may have an inhibi-
179 CONTROL
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Fig. 3. Effect of dihydroergotamine on the synaptic responses of bullfrog 9th or 10th sympathetic ganglia elicited by stimulating the VIII spinal nerve at a frequency of 40 Hz for 1 s (period of stimulation indicated by bar). See Fig. 1 for description of these responses. Left: control responses prior to the administration of dihydroergotamine. Middle: 60 min after beginning superfusion with Ringer solution containing dihydroergotamine (at concentrations as indicated for each set of tracings). Right: 1 h after beginning wash-out with dihydroergotaminefree Ringer solution. Each set of tracings are from different experiments. Time calibrations in C also apply to traces of A.
tory effect on cholinesterase and thus enhance the duration of the slow IPSP. Doses of dihydroergotamine higher than 100pM were not studied.
Effects of propranolol This agent which is an effective blocker of all subtypes of fl-adrenergic receptors in other systems 1 showed no significant reduction in the amplitude of slow IPSP up to a concentration of 10 pM. When doses of propranolol higher than 10 ,uM were used there was a non-specific reduction in the amplitude of slow IPSP as well as those of the other synaptic potentials. Fig. 4 illustrates these effects of propranolol on synaptic responses elicited by stimulation of the 8th spinal nerve. It can be seen in Fig. 4A 1 that exposure to 10pM propranolol has no significant effect on synaptic potentials (fast EPSP, slow IPSP, and late slow EPSP). However, it is seen in Fig. 4 A z - A 4 that the higher doses of propranolol (i.e. 20, 50 and 100
pM) reduce not only the amplitude of slow IPSP but also those of the other synaptic potentials in a nonspecific and dose-related manner. Note particularly t h e traces of Fig. 4A3 where after superfusion with 50 pM propranolol there is a severe reduction in the amplitude of fast EPSP and late slow EPSP, but that a slow IPSP of appreciable size could still be elicited in this very high dose of propranolol (Fig. 4A 3, central trace). Similarly, the non-specific blockade of slow EPSP by propranolol (at 20, 50 and 100 p M ) is illustrated in Fig. 4B. As in studies with phentolamine (see above), the results with propranolol indicate that the reduction of slow IPSP amplitude by high doses of this agent is unlikely to be due to a specific blockade of an adrenergic receptor (/3) in this ganglion. Again, construction of dose-response curves of the propranolol effect on synaptic responses (Fig. 4C) indicate that the action of these high doses of propranolol is very likely due to either a presynaptic depression of transmitter release or by conduction block by this agent. The conclusion on the transmitter release block by propranolol is substantiated further by findings in other systems that high doses of propranolol can inhibit calcium current and block synaptic transmission 2'15. Taken together, the experiments with propranolol indicated that this agent has no specific interaction with slow IPSP in frog ganglion.
Effects of U-0521 and neostigmine Libet and Kobayashi 18 reported that in bullfrog
(Rana catesbeiana) sympathetic ganglia the drug U0521 (50 pg/ml), a C O M T inhibitor, enhanced the amplitude and duration of slow IPSP both in curarized and nicotinized ganglia. In the present study we re-examined the effect of U-0521 (50pg/ml; from two fresh batches of the drug kindly supplied by the Upjohn Company) on slow IPSP elicited in curarized ganglia by the same parameters of preganglionic stimulation as reported by Libet and Kobayashi 18, but could not see any potentiation of slow IPSP due to U-0521 (total of 21 experiments; see Fig. 5). Indeed, we consistently observed a slight reduction in the amplitude of slow IPSP (and of other synaptic potentials) after U-0521 (50 #g/ml) was superfused for periods of 45-70 min (Fig. 5 A - C and Table I). Thus the reported potentiation by this drug on slow IPSP was not confirmed. Contrary to U-0521, the cholines-
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lqg. 4. A: effects of propranolol on the synaptic responses of bullfrog 9th or 10th sympathetic ganglia elicited by stimulation of the VIII spinal nerve at a frequency of 40 Hz for 1 s (period of stimulation indicated by bar). See Fig. 1A legend for description of these responses. Left: control responses prior to the administration of propranolol. Middle: 60 min after beginning superfusion with Ringer solution containing propranolol (at concentrations indicated). Right: 3.5 h after beginning wash-out with propranolol-free Ringer solution. Each set of tracings are from different experiments. Time calibrations in A 4 are for all traces. B: effects of propranolol on the synaptic responses of bullfrog 9th or t0th sympathetic ganglia elicited by stimulation of sympathetic chain between 6th and 7th ganglia (period of stimulation indicated by bar). See Fig. 1B legend for description of these responses, Left: control responses prior to the administration of propranolol. Middle: 60 rain after beginning superfusion with propranolol (at concentrations as indicated for each set of tracings), Right: 3.5 h after beginning superfusion with propranolol-free Ringer solution. Each set of tracings are from different experiments. Time calibration in B 3 is for all traces. Tops of the fast EPSP have been cut away in all traces except in the central trace of B 3. C: graphs of the effect of propranolol on the amplitude of slow IPSP (filled circles), slow EPSP (open circles), late slow EPSP (closed triangles), and fast EPSP (open triangles). Percent reduction in synaptic response is plotted as a function of antagonist concentration. Response amplitude was measured 60 min after beginning superfusion with propranolol. The data points for each concentration of propranolol represent the mean _+_S.E.M. from 4-8 experiments. Synaptic responses elicited and experiments carried out as described in Fig. 1A,B legends and the text.
terase inhibitor neostigmine (0.5-1~M)
showed con-
H M i n c r e a s e d t h e s l o w I P S P d u r a t i o n b y 44 + 8 % (n
s i s t e n t a n d l a r g e i n c r e a s e s in t h e d u r a t i o n a n d a m p l i -
= 3, m e a n + S . E . M . ) a n d t h e a m p l i t u d e b y 24 + 6 %
t u d e o f slow I P S P in 7 e x p e r i m e n t s . N e o s t i g m i n e 0.5
(n = 3), w h i l e 1 H M n e o s t i g m i n e i n c r e a s e d t h e d u r a -
181 CONTROL
U-0521
WASH
tion by 149 _+ 10% (n = 4) and t h e a m p l i t u d e by 37 + 3 % (n = 4) w h e n it was s u p e r f u s e d for 1 h (see Fig. 5 D ) .
10 Hz
mV
DISCUSSION
.~ Adrenergic antagonists do not specifically block slow IPSP T h e p r e s e n t e x p e r i m e n t s s h o w e d that t h e slow I P S P in frog s y m p a t h e t i c ganglia c a n n o t be b l o c k e d by low d o s e s of a d r e n e r g i c a n t a g o n i s t s w h i c h are eflmV
fective in b l o c k i n g a d r e n e r g i c r e s p o n s e s in o t h e r syst e m s 1'2° (i.e. at 1 ~ M or lower). L i k e w i s e , r e c e n t intracellular
C
showed
studies
in
frog
sympathetic
that the a d r e n e r g i c a n t a g o n i s t s
ganglia (phentol-
a m i n e , p r o p r a n o l o l and h a l o p e r i d o l , at 5 ~tM or lower) w e r e i n e f f e c t i v e in b l o c k i n g slow I P S P s. A l s o , N a k a m u r a 19 s h o w e d
that
the
neuroleptics
chlor-
p r o m a z i n e , h a l o p e r i d o l and p i m o z i d e ( c o m p o u n d s NEOSTIGMINE
which also b l o c k d o p a m i n e r e c e p t o r s ) w e r e e i t h e r ine f f e c t i v e or w e r e n o n - s p e c i f i c b l o c k e r s o f slow I P S P in frog s y m p a t h e t i c g a n g l i o n . T h e s e studies, t h e r e fore, i n d i c a t e that a d r e n e r g i c and d o p a m i n e r g i c receptors are not i n v o l v e d in m e d i a t i o n of the slow 5
see
Fig. 5. Effects of U-0521 50ktg/ml (A-C) and neostigmine 1 pM (D) on the synaptic responses of bullfrog 9th or 10th sympathetic ganglia elicited by stimulation of the VIII spinal nerve at the frequencies as shown (stimulus frequency in D is 40 Hz; stimulation duration is 1 s in A, B and D, and 2 s in C). See Fig. 1 legend for description of these responses. Left: control responses prior to the administration of U-0521 or neostigmine. Middle: 1 h after beginning superfusion with Ringer solution containing these drugs. Right: 1-3 h after beginning wash-out with U-0521- or neostigmine-free Ringer solution. Each set of tracings is from different experiments. Time calibration in D is for all traces.
IPSP in the frog g a n g l i o n . C o n s i s t e n t with o u r conclusion of n o n - i n v o l v e m e n t of c a t e c h o l a m i n e s in the slow I P S P , R a f u s e and S m i t h 22 h a v e r e c e n t l y s h o w n in Rana pipiens ganglia that the slow I P S P is n o t pot e n t i a t e d by the c a t e c h o l a m i n e u p t a k e b l o c k e r desm e t h y l i m i p r a m i n e and that it is n o t b l o c k e d by yoh i m b i n e (an a 2 - a d r e n e r g i c b l o c k e r ) .
U-0521 does not potentiate slow I P S P L i b e t and K o b a y a s h P s r e p o r t e d large i n c r e a s e s in
TABLE I
Effect of COMT-inhibitor U-0521 (50 ~g/ml," superfused for 45-70 min) on synaptic potentials in bullfrog sympathetic ganglia recorded by sucrose-gap technique Values (mean + S.E.M. from number of experiments in parentheses) are percent decrease in the amplitude of control response due to drug.
Synaptic potential
Slow IPSP Slow EPSP Late slow EPSP Fast EPSP
Stimulation frequency and duration* lO Hz, 1 s
40 Hz, l s
50 Hz, 2 s
lOO Hz, 2 s
9 _+ 1 (5) ** 35 + 6 (5)
11 + 3 (5) 13 + 3 (4) 40 + 3 (6)
14 + 3 (4) 19 _+ 5 (4) 34 + 6 (4)
31 _+ 2 (11) -
* See Materials and Methods for further details. ** Data not shown since the late slow EPSP elicited by this stimulation was very small and unreliable to measure in most experiments.
182 the amplitude and the duration of slow IPSP in bullfrog sympathetic ganglia after superfusion of U-0521 50/~g/ml (a COMT inhibitor). Although the primary mechanism for transmitter removal at adrenergic synaptic junctions is uptake (and not enzymatic destruction) 4'13'14, we decided nonetheless to repeat the experiments of Libet and Kobayashi is (see above). In no case, however, were we able to see a potentiation of slow IPSP by U-0521 (Fig. 5 A - C ) . Indeed, this dose of U-0521 showed a consistent non-specific reduction in the amplitude of all synaptic responses (including the slow IPSP, Table I and Fig. 5 A - C ) . Thus the reported potentiation by U-0521 on slow IPSP was not confirmed in the present experiments. The anticholinesterase compound neostigmine, on the other hand, showed a large potentiation in the duration and amplitude of slow IPSP (thus confirming the involvement of a cholinergic step in mediation of this potential; compare Fig. 5 A - C with 5D). Can slow I P S P be mediated in both a direct and an indirect pathway ?
It has been reported recently that a portion of SIF cells in frog sympathetic ganglia are innervated exclusively by preganglionic C fibers and that, upon stimulation of the preganglionic C fibers, give rise to action potentials 9. Since the slow IPSP is generated predominantly by stimulation of preganglionic C fibers and the SIF cells are the proposed interneurons (in the adrenergic hypothesis) for slow IPSP mediation in frog ganglion, the findings by Dunn and Marshall 9 may seem to provide an anatomical substrate whereby the slow IPSP could be mediated in a portion of synaptic pathways using the SIF cells. It has also been suggested that the slow IPSP in frog ganglion may be mediated in both direct (by ACh, monosynaptically) and indirect (via adrenergic interneuronal) pathways 17. However, the following findings strongly argue against the dual mode of slow IPSP mediation in frog: first, no efferent synaptic contact from SIF cells to ganglionic principal cells has been
REFERENCES 1 Ahlquist, R.P., Adrenergic beta-blocking agents, Prog. Drug Res., 20 (1976) 27-43. 2 Akaike, N., Ito, H., Nishi, K. and Oyama, Y., Further analysis of inhibitory effects of propranolol and local anaesthetics on the calcium current in Helix neurones, Br. J.
seen in frog 28, and second, several recent studies (including the present one) have used the sucrose-gap method of recording the slow IPSP 22'23 (which records from the entire population of cells in the ganglion) and thus the results (which support the cholinergic monosynaptic mediation of slow IPSP) are applicable to the entire ganglion. Thus the results of the present report do not lend support to the hypothesis that the slow IPSP in frog ganglion is mediated in a synaptic pathway using an adrenergic interneuron (wholly or partially). The results are consistent with several recent intracellular and extracellular studies in frog ganglion which provide evidence for a cholinergic monosynaptic pathway for this potential in frog ganglion s'22'23'25'28. The adrenergic interneuron hypothesis for slow IPSP in the mammalian sympathetic ganglia m was based partially on the observation that dibenamine blocked this potential. However, dibenamine (and other adrenergic blockers) have consequently been shown to have antagonistic action at muscarinic receptors (as well as having other non-specific actions) 5'12. It has also been suggested that the slow IPSP in mammalian sympathetic ganglia is mediated by a direct action of ACh released from cholinergic preganglionic fibers 6'7. Thus, in view of the present findings with adrenergic antagonists in frog ganglion, it would seem appropriate to carry out similar studies in the mammalian ganglia to resolve the controversy regarding the mediation of the slow IPSP in these ganglia.
ACKNOWLEDGEMENTS We thank Dr. Paul Gallant for reviewing an earlier version of this manuscript, and Jill Lindahl for typing the manuscript. We also thank the following companies for providing the drugs indicated: Sandoz (dihydroergotamine); Upjohn (U-0521); and Ciba-Geigy (phentolamine).
Pharmacol., 76 (1982) 37-43. 3 Atlas, D. and Adler, M., Alpha-adrenergic antagonists as possible calcium channel inhibitors, Proc. Natl. Acad. Sci. U.S.A., 78 (1981) 1237-1241. 4 Axelrod, J., The fate of adrenaline in the sympathetic neurone, Harvey Lect., 67 (1973) 175-197. 5 Beddoe, F., Nicholls, P.J. and Smith, H.J., Inhibition of
183 muscarinic receptor by dibenamine, Biochem. PharmacoL, 20 (1971) 3367-3776. 6 Cole, A.E. and Shinnick-Gallagher, P., Alpha-adrenergic and dopamine receptor antagonists do not block the slow inhibitory postsynaptic potential in sympathetic ganglia, Brain Research, 187 (1980) 226-230. 7 Cole, A.E. and Shinnick-Gallagher, P., Muscarinic inhibitory transmission in mammalian ganglia mediated by increased potassium conductance, Nature (Lond.), 307 (1984) 270-271. 8 Dodd, J. and Horn, J.P., Muscarinic inhibition of sympathetic C neurones in the bullfrog, J. Physiol. (Lond.), 334 (1983) 271-291. 9 Dunn, P.M. and Marshall, L.M., Innervation of small intensely fluorescent cells in frog sympathetic ganglia, Brain Research, 339 (1985) 371-374. 10 Eccles, R.M. and Libet, B., Origin and blockade of the synaptic responses of curarized sympathetic ganglia, J. Physiol. (Lond.), 157 (1961) 484-503. 11 Gallagher, J.P., Shinnick-Gallagher, P., Cole, A.E., Griffith, W.H. and Williams, B.J., Current hypotheses for the slow inhibitory postsynaptic potential in sympathetic ganglia, Fed. Proc., 39 (1980) 3009-3015. 12 Gengo, P.J., Yousif, F., Janis, R.A. and Triggle, D.J., Interaction of phenoxybenzamine with muscarinic receptors and calcium channels, Biochem. Pharmacol., 33 (1984) 3445-3449. 13 Guldberg, H.C. and Marsden, C.A., Catechol-O-methyltransferase: pharmacological aspects and physiological role, Pharmacol. Rev., 27 (1975) 135-206. 14 Hanbauer, I., Johnston, D.G., Silberstein, S.D. and Kopin, I.J., Pharmacological and kinetic properties of uptake of 3H-norepinephrine by superior cervical ganglia in rats in organ culture, Neuropharmacology, 11 (1972) 857-862. 15 Ito, H. and Nishi, K., Frequency-dependent depression of ganglionic transmission by propranolol and diltiazem in the superior cervical ganglion of the guinea-pig, Br. J. Pharmacol., 77 (1982) 359-362. 16 Jan, L.Y. and Jan, Y.N., Peptidergic transmission in sympathetic ganglia of the frog, J. Physiol. (Lond.), 327 (1982) 219-246.
17 Libet, B., Nonclassical synaptic functions of transmitters, Fed. Proc., 45 (1986) 805-814. 18 Libet, B. and Kobayashi, H., Adrenergic mediation of slow inhibitory postsynaptic potential in sympathetic ganglia of the frog, J. Neurophysiol., 37 (1974)805-814. 19 Nakamura, J., The effects of neuroleptics on the dopaminergic and cholinergic systems in sympathetic ganglia, Kurume Med. J., 25 (1978) 241-253. 20 Nickerson, M. and Collier, B., Drugs inhibiting adrenergic nerves and structures by them. In: L.S. Goodman and A. Gilman (Eds.), The Pharmacological Basis of Therapeutics, Macmillan, New York, 1975, pp. 533-564. 21 Nishi, S. and Koketsu, K., Analysis of slow inhibitory postsynaptic potential of bullfrog sympathetic ganglion, J. Neurophysiol., 31 (1968) 717-728. 22 Rafuse, P.E. and Smith, P.A., Alpha2-adrenergic hyperpolarization is not involved in slow synaptic inhibition in amphibian sympathetic ganglia, Br. J. PharmacoL, 87 (1986) 409-416. 23 Smith, P.A. and Weight, F.F., The pathway for the slow inhibitory postsynaptic potential in bullfrog sympathetic ganglia, J. Neurophysiol., 56 (1986) 823-834. 24 Tosaka, T., Chichibu, S. and Libet, B., Intracellular analysis of slow inhibitory and excitatory postsynaptic potentials in sympathetic ganglia of the frog, J. Neurophysiol., 31 (1968) 396-409. 25 Weight, F.F., Slow synaptic inhibition and adrenergic antagonism in sympathetic ganglion, Soc. Neurosci. Abstr., 3 (1973) 312. 26 Weight, F.F., Synaptic mechanisms in amphibian sympathetic ganglia. In L.-G. Elfvin (Ed.), Autonomic Ganglia, Wiley, New York, 1983, pp. 309-344. 27 Weight, F.F. and Padjen, A.L., Acetylcholine and slow synaptic inhibition in frog sympathetic ganglion cells, Brain Research, 55 (1973) 225-228. 28 Weight, F.F. and Weitsen, H.A., Identification of small intensely fluorescent (SIF) cells as chromaffin cells in bullfrog sympathetic ganglia, Brain Research, 128 (1977) 213-226. 29 Yavari, P. and Weight, F.F., Effects of phentolamine on synaptic transmission in bullfrog sympathetic ganglia, Soc. Neurosci. Abstr., 7 (1981) 807.
Brain Research, 452(1988) 175-183 Elsevier
BRE 13639
Pharmacological studies in frog sympathetic ganglion" support for the cholinergic monosynaptic hypothesis for slow IPSP mediation Parviz Yavari* and Forrest F. Weight Section on Electrophysiology, Laboratory of Physiologic and Pharmacologic Studies, National Institute on Alcohol Abuse and Alcoholism, Rockville, MD 20852 (U.S.A.)
(Accepted 15 December 1987) Key words: Sympathetic ganglion; Sucrose-gap; Slow inhibitory postsynaptic potential; Adrenergic pharmacology; Bullfrog
The slow inhibitory postsynaptic potential (slow IPSP), the slow excitatory postsynaptic potential (slow EPSP), the late slow excitatory postsynaptic potential (late slow EPSP), and the fast excitatory postsynaptic potential/compound action potential (fast EPSP) were recorded from the 9th or 10th paravertebral sympathetic ganglia of bullfrogs (and some Rana pipiens frogs) by the sucrose-gap technique. The adrenergic antagonists phentolamine, dihydroergotamine and propranolol did not show any antagonistic effect on the slow IPSP when used at concentrations of up to 10, 100 and 10 ~M, respectively. U-0521 (3',4'-dihydroxy-2-methylpropriophenone, 50/~g/ml), a specific inhibitor of catechol-O-methyltransferase, did not show any potentiating effect on the slow IPSP. The cholinesterase inhibitor neostigmine (0.5-1/~M) induced a large increase in the duration and amplitude of slow IPSP. When phentolamine and propranolol at concentrations greater than 10/~M were used the slow IPSP (and all other synaptic potentials) were non-specifically reduced in amplitude by these drugs. The results reported in this paper do not lend any support to the hypothesis that the slow IPSP in frog sympathetic ganglia is mediated by an adrenergic interneuron. The results are consistent with the proposal that the slow IPSP in this ganglion is mediated by a direct action of acetylcholine released from cholinergic preganglionic fibers.
INTRODUCTION The n e u r o t r a n s m i t t e r and pathway m e d i a t i n g the slow inhibitory postsynaptic potential (IPSP) in sympathetic ganglia have been controversial in both m a m m a l i a n and amphibian species (for reviews see refs. 11 and 26). In frog sympathetic ganglia two hypotheses exist for the m e d i a t i o n of this potential. One hypothesis proposes that the slow IPSP is mediated by a direct muscarinic action of acetylcholine ( A C h ) released from cholinergic preganglionic fibers 27. A second hypothesis proposes that the slow IPSP is m e d i a t e d by a catecholamine r e l e a s e d from an adrenergic interneuron (later defined as small intensely fluorescent (SIF) cells) 1s'24. T h e latter hypothesis is based, in part, upon the observations that the a - a d r e n e r g i c antagonists p h e n t o l a m i n e and dih y d r o e r g o t a m i n e in a concentration of 4 0 / ~ M (or greater) partially depress the slow IPSP, and that U-
0521 (50 /~g/ml), a specific inhibitor of catecholO-methyltransferase ( C O M T ) , p o t e n t i a t e s slow IPSP 18. In the p r e s e n t study we carried out a careful re-examination of the adrenergic t h e o r y for slow IPSP m e d i a t i o n in frog ganglia by studying the effects of p h e n t o l a m i n e , d i h y d r o e r g o t a m i n e , p r o p r a n o l o l and U-0521 on this potential using the sucrose-gap method. T h e results showed no specific interaction of these agents with the p r o p o s e d adrenergic system in this ganglion. The results are thus consistent with several recent reports 8'22'23'25'28 presenting evidence for,the 'direct muscarinic '27 m e d i a t i o n of slow IPSP in frog sympathetic ganglia. A n abstract describing some of this w o r k has a p p e a r e d 29. MATERIALS AND METHODS E x p e r i m e n t s were carried out on the isolated p a r a vertebral ganglionic p r e p a r a t i o n of bullfrogs (Rana
* Present address: Department of Physiology and Cellular Biophysics, Columbia University, College of Physicians and Surgeons, 630 West 168th Street, New York, NY 10032, U.S.A. Correspondence: F.F. Weight, Section on Electrophysiology, LPPS, NIAAA, 12501 Washington Avenue, Rockville, MD 20852, U.S.A.
176
catesbeiana), and in a few experiments on large (1>4inch) Rana pipiens frogs. The sucrose-gap method used in the present study has been described in detail elsewhere 23, and is based on that of Nishi and Koketsu 21. Briefly, the ganglionic preparation was mounted in a sucrose-gap chamber (a slab of clear plexiglass with 4 compartments engraved in it from end-to-end) so that the sympathetic chain and the rostral portion of the 8th spinal nerve were stationed in a compartment under mineral oil, the 9th or 10th ganglion was laid in an adjacent compartment through which Ringer solution flowed, the 9th or 10th ganglionic ramus passed through a third compartment through which isotonic sucrose solution flowed, and finally, the distal portion of the ramus (and a portion of attached spinal nerve) was placed in a fourth compartment in isotonic KC1 solution. Grooves between compartments were tightly sealed with a mixture of petroleum jelly and wax. Synaptic potentials were recorded between the ganglion and the distal portion of its ramus in isotonic KC1 solution by use of a pair of isotonic KCl-agar bridge-calomel electrode assemblies (with the ganglion compartment grounded). Synaptic potentials were elicited by stimulation of the sympathetic chain or the 8th spinal nerve with a pair of platinum electrodes under mineral oil. The sympathetic chain between the 6th and 7th ganglia was stimulated with 0.5 ms pulses to activate the preganglionic B fibers. The 8th spinal nerve was stimulated with 1 ms pulses to activate preganglionic C fibers. Supramaximal stimuli were used in both cases. The Ringer solution had the following composition (in mM): NaC1 100, KC12, CaC12 1.8, Tris-HC1 16, glucose 1 g/liter, pH 7.2. Except when the effect of phentolamine on synaptic transmission was studied, the Ringer solution also contained 70~M D-tubocurarine (d-Tc) in order to reduce the amplitude of the fast EPSP and thus prevent action potential generation 26. All experiments were carried out at room temperature. The fast excitatory postsynaptic potential (EPSP)/compound action potential was photographed from the oscilloscope, while the slow potentials were recorded on a Gould rectiliniar penrecorder. Effects of drugs on synaptic potentials were studied by switching the ganglionic superfusion system to Ringer solutions containing known concentrations of these agents (by use of a 3-way tap system). The following chemicals were used: d-Tc chlor-
ide, (_+)-propranolol hydrochloride and neostigmine bromide (Sigma), dihydroergotamine methanesulfonate (gift of Sandoz), U-0521 (gift of Upjohn) and phentolamine hydrochloride (gift of Ciba-Geigy). All chemicals were dissolved directly in Ringer's solution, except for dihydroergotamine which was first dissolved in dimethyl sulfoxide (DMSO) and was then added to Ringer solution (final DMSO concentration was 0.1-0.4% (v/v) which did not have any adverse effects on control responses). RESULTS
Effects of phentolamine This agent, which is an effective blocker of all subtypes of a-adrenergic receptors in other systems 2°, produced little or no reduction in the amplitude of slow IPSPs when used at doses of up to 10/~M. When phentolamine at concentrations greater than 10 ~M was used there was a non-specific reduction in the amplitude of slow IPSP as well as those of other synaptic potentials (Fig. 1). Fig. 1A illustrates the effects of phentolamine on the synaptic responses elicited by stimulation of the 8th spinal nerve. It can be seen in Fig. 1A 1 that 10~M phentolamine leaves the synaptic potentials (fast EPSP, slow IPSP and late slow EPSP) virtually unaffected. However, it is seen in Fig. 1Az-A 4 that when concentrations of phentolamine higher than 10/~M (i.e. 40, 100 and 200 ~M, respectively) are used there is a dose-dependent and nonspecific reduction in the amplitude of fast EPSP, late slow EPSP (a peptide-mediated synaptic potential 16) and the slow IPSP. Note particularly the traces of Fig. 1A 4 where the late slow EPSP has been abolished after superfusion with 200 /~M phentolamine and there is a large antagonism of the fast EPSP, whereas a good-size slow IPSP response could still be elicited after exposure to this very high concentration of phentolamine (Fig. 1A 4, central trace). Similarly the non-specific antagonism of the slow EPSP (a synaptic potential that is known in this ganglion to be mediated by a direct action of ACh in a separate pathway than the slow IPSP 26) by phentolamine at doses of 40, 100 and 200 ~M is illustrated in Fig. lB. Thus, these experiments indicate that the reduction of the slow IPSP amplitude by high doses of phentolamine is very unlikely to be due to a specific blockade of an a-adrenergic receptor in this gan-
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Fig. 1. A: effects of phentolamine on the synaptic responses of bullfrog 9th or 10th sympathetic ganglia elicited by stimulation of the VIII spinal nerve at a frequency of 40 Hz for 1 s (period of stimulation indicated by bar). The response of VIII nerve stimulation consists of the following synaptic potentials26: (i) an initial short duration depolarization (upward deflection) at the beginning of the stimulus - - the nicotinic fast EPSP; (ii) a hyperpolarizing response (downward deflection) following the fast EPSP - - the slow IPSP; and (iii) a long-lasting depolarization subsequent to the slow I P S P - - the peptide-mediated late slow EPSP. In order to show the late slow EPSP on the same tracing as the slow IPSP, the speed of the chart recorder was slowed at the end of the slow IPSP (see arrow in bottom right hand tracing). Consequently, the late slow EPSP has a separate time-base calibration (to the right of arrow in bottom right hand tracing). Left: control responses prior to the administration of phentolaminc. Middle: 60 rain after beginning superfusion with Ringer solution containing phentolamine (at concentrations as indicated for each set of tracings). Right: 4 h after beginning wash-out with phentolamine-free Ringer solution. Each set of tracings are from different experiments. Time and voltage calibrations in A 4 are for all traces. B: effects of phentolamine on the synaptic iesponses in bullfrog 9th or 10th sympathetic ganglia elicited by stimulation of the sympathetic chain between 6th and 7th ganglia at a frequency of 100 Hz for 2 s (period of stimulation indicated by bar). The following synaptic potentials comprise the response to chain stimulation26: (i) an initial short duration depolarization (upward deflection) - - the nicotinic fast EPSP; (ii) a small short duration hyperpolarization following the fast EPSP - - a slow IPSP; and (iii) a long-lasting depolarization following the small slow IPSP - - the muscarinic slow EPSP. Left: control responses prior to the administration of phentolamine. Middle: 60 min after beginning superfusion with Ringer solution containing phentolamine (at concentrations as indicated for each set of tracings). Right: 6 h after beginning wash-out with phentolamine-free Ringer solution. Each set of tracings are from different experiments. Tops of the fast EPSP have been cut away in all traces except the central and right hand trace of B 2. C: graphs of the effects of phentolamine on the amplitude of the slow IPSP (filled circles), slow EPSP (open circles), late slow EPSP (filled triangles), and fast EPSP (open triangles). Percent reduction in the synaptic response amplitude is plotted as a function of antagonist concentration. Response amplitude was measured 60 min after beginning superfusion with phentolamine. The data points for each concentration of phentolamine represent the mean + S.E.M. from 4-10 experiments. These experiments were carried out as described above and in the text.
178 glion. To obtain a further understanding of the mechanism of blockade by high doses of phentolamine on the slow IPSP, we constructed d o s e - r e s p o n s e curves of the effects of this drug (at 1-200 ktM) on the various synaptic potentials in this ganglion (Fig. 1C). It is seen in the curves of Fig. 1C that the high doses of phentolamine (i.e. 40, 100 and 200/~M) reduce the amplitude of synaptic potentials (fast EPSP, slow IPSP, late slow E P S P and slow EPSP) more or less to the same degree. Thus it is very unlikely that phentolamine blockade of these potentials (including the slow IPSP) has a postsynaptic mechanism since these potentials each have a different receptor and/or ionic mechanisms of generation in this ganglion (for example the late slow E P S P involves a peptide transmitter while all other PSPs involve ACh) 26. It is likely, how-
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ever, that the actions of phentolamine are due to a presynaptic suppressive effect on transmitter release in this ganglion (since phentolamine and other ablockers can block calcium channels 3'12 at high concentrations) or are due to block of nerve conduction. In order to investigate these possibilities further, we studied the effects of 1-200 ktM phentolamine on synaptic transmission of the B and C pathways in this ganglion. Phentolamine (40 ktM, superfused for 30 min) reduced the amplitude of the transmitted B action potential by 13 + 2% (mean + S.E.M., n = 8) and the C spike by 12 + 3% (n = 4). When phentolamine 40/zM was superfused for 60 min, the reductions were 23 _+ 5% (n = 4) for the B spike and 20 + 7% (n = 3) for the C spike. At the concentrations of 100 and 200/~M, phentolamine showed 19 + 9% (n = 3) and 38 + 6% (n -- 3) reductions, respectively, in the height of the B volley when it was superfused for 30 min. Fig. 2 illustrates the antagonistic effect of phentolamine on synaptic transmission in a B fiber pathway in frog ganglion. Since synaptic transmission in bullfrog sympathetic ganglia is mediated by a nicotinic cholinergic mechanism 26, these results indicate that high doses of phentolamine can have a nonspecific depressant effect on synaptic transmission (either due to depression of transmitter release, or by conduction block). Taken together, the experiments with phentolamine indicated that this agent has no specific interaction with slow IPSP in the frog ganglion.
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Fig. 2. Effect of phentolamine on the fast EPSP/compound action potential of bullfrog 9th or 10th sympathetic ganglion elicited by single supramaximal shocks to the sympathetic chain between the 6th and 7th ganglia (at arrows). The action potential (upward deflection) and spike afterhyperpolarization are identified as occurring in the ganglionic B cells since there is only a very short (about 10 ms) interval between shock artifact (seen in majority of traces just before the spike) and spike onset26. Left: control responses prior to the administration of phentolamine. Middle: 30 rain after beginning superfusion with Ringer solution containing phentolamine (at concentrations as indicated for each set of tracings). Right: 45 min after beginning with phentolamine Ringer superfusion. Each set of tracings are from different experiments. Time calibration is for all traces.
As with our previous failure to observe a specific blockade of slow IPSP by low doses of dihydroergotamine in frog ganglion 25, neither could we detect any reduction in the amplitude of the slow IPSP when doses of this drug as high as 100 jzM were used (100 /~M = 5 experiments; 40,uM = 12 experiments; 20 y M = 4 experiments; drug superfusion times of up to 2 h) (Fig. 3). With 100 /~M concentrations of dihydroergotamine there was an increase in the duration of the slow IPSP as recorded in the sucrose-gap (e.g. as in central trace of Fig. 3C). However, since the slow IPSP and the late slow EPSP overlap in time in these sucrose-gap records, it is difficult to pinpoint the exact locus of this enhancing action of dihydroergotamine on the two potentials. Alternatively, the high doses of dihydroergotamine may have an inhibi-
179 CONTROL
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Fig. 3. Effect of dihydroergotamine on the synaptic responses of bullfrog 9th or 10th sympathetic ganglia elicited by stimulating the VIII spinal nerve at a frequency of 40 Hz for 1 s (period of stimulation indicated by bar). See Fig. 1 for description of these responses. Left: control responses prior to the administration of dihydroergotamine. Middle: 60 min after beginning superfusion with Ringer solution containing dihydroergotamine (at concentrations as indicated for each set of tracings). Right: 1 h after beginning wash-out with dihydroergotaminefree Ringer solution. Each set of tracings are from different experiments. Time calibrations in C also apply to traces of A.
tory effect on cholinesterase and thus enhance the duration of the slow IPSP. Doses of dihydroergotamine higher than 100pM were not studied.
Effects of propranolol This agent which is an effective blocker of all subtypes of fl-adrenergic receptors in other systems 1 showed no significant reduction in the amplitude of slow IPSP up to a concentration of 10 pM. When doses of propranolol higher than 10 ,uM were used there was a non-specific reduction in the amplitude of slow IPSP as well as those of the other synaptic potentials. Fig. 4 illustrates these effects of propranolol on synaptic responses elicited by stimulation of the 8th spinal nerve. It can be seen in Fig. 4A 1 that exposure to 10pM propranolol has no significant effect on synaptic potentials (fast EPSP, slow IPSP, and late slow EPSP). However, it is seen in Fig. 4 A z - A 4 that the higher doses of propranolol (i.e. 20, 50 and 100
pM) reduce not only the amplitude of slow IPSP but also those of the other synaptic potentials in a nonspecific and dose-related manner. Note particularly t h e traces of Fig. 4A3 where after superfusion with 50 pM propranolol there is a severe reduction in the amplitude of fast EPSP and late slow EPSP, but that a slow IPSP of appreciable size could still be elicited in this very high dose of propranolol (Fig. 4A 3, central trace). Similarly, the non-specific blockade of slow EPSP by propranolol (at 20, 50 and 100 p M ) is illustrated in Fig. 4B. As in studies with phentolamine (see above), the results with propranolol indicate that the reduction of slow IPSP amplitude by high doses of this agent is unlikely to be due to a specific blockade of an adrenergic receptor (/3) in this ganglion. Again, construction of dose-response curves of the propranolol effect on synaptic responses (Fig. 4C) indicate that the action of these high doses of propranolol is very likely due to either a presynaptic depression of transmitter release or by conduction block by this agent. The conclusion on the transmitter release block by propranolol is substantiated further by findings in other systems that high doses of propranolol can inhibit calcium current and block synaptic transmission 2'15. Taken together, the experiments with propranolol indicated that this agent has no specific interaction with slow IPSP in frog ganglion.
Effects of U-0521 and neostigmine Libet and Kobayashi 18 reported that in bullfrog
(Rana catesbeiana) sympathetic ganglia the drug U0521 (50 pg/ml), a C O M T inhibitor, enhanced the amplitude and duration of slow IPSP both in curarized and nicotinized ganglia. In the present study we re-examined the effect of U-0521 (50pg/ml; from two fresh batches of the drug kindly supplied by the Upjohn Company) on slow IPSP elicited in curarized ganglia by the same parameters of preganglionic stimulation as reported by Libet and Kobayashi 18, but could not see any potentiation of slow IPSP due to U-0521 (total of 21 experiments; see Fig. 5). Indeed, we consistently observed a slight reduction in the amplitude of slow IPSP (and of other synaptic potentials) after U-0521 (50 #g/ml) was superfused for periods of 45-70 min (Fig. 5 A - C and Table I). Thus the reported potentiation by this drug on slow IPSP was not confirmed. Contrary to U-0521, the cholines-
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lqg. 4. A: effects of propranolol on the synaptic responses of bullfrog 9th or 10th sympathetic ganglia elicited by stimulation of the VIII spinal nerve at a frequency of 40 Hz for 1 s (period of stimulation indicated by bar). See Fig. 1A legend for description of these responses. Left: control responses prior to the administration of propranolol. Middle: 60 min after beginning superfusion with Ringer solution containing propranolol (at concentrations indicated). Right: 3.5 h after beginning wash-out with propranolol-free Ringer solution. Each set of tracings are from different experiments. Time calibrations in A 4 are for all traces. B: effects of propranolol on the synaptic responses of bullfrog 9th or t0th sympathetic ganglia elicited by stimulation of sympathetic chain between 6th and 7th ganglia (period of stimulation indicated by bar). See Fig. 1B legend for description of these responses, Left: control responses prior to the administration of propranolol. Middle: 60 rain after beginning superfusion with propranolol (at concentrations as indicated for each set of tracings), Right: 3.5 h after beginning superfusion with propranolol-free Ringer solution. Each set of tracings are from different experiments. Time calibration in B 3 is for all traces. Tops of the fast EPSP have been cut away in all traces except in the central trace of B 3. C: graphs of the effect of propranolol on the amplitude of slow IPSP (filled circles), slow EPSP (open circles), late slow EPSP (closed triangles), and fast EPSP (open triangles). Percent reduction in synaptic response is plotted as a function of antagonist concentration. Response amplitude was measured 60 min after beginning superfusion with propranolol. The data points for each concentration of propranolol represent the mean _+_S.E.M. from 4-8 experiments. Synaptic responses elicited and experiments carried out as described in Fig. 1A,B legends and the text.
terase inhibitor neostigmine (0.5-1~M)
showed con-
H M i n c r e a s e d t h e s l o w I P S P d u r a t i o n b y 44 + 8 % (n
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t u d e o f slow I P S P in 7 e x p e r i m e n t s . N e o s t i g m i n e 0.5
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181 CONTROL
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tion by 149 _+ 10% (n = 4) and t h e a m p l i t u d e by 37 + 3 % (n = 4) w h e n it was s u p e r f u s e d for 1 h (see Fig. 5 D ) .
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mV
DISCUSSION
.~ Adrenergic antagonists do not specifically block slow IPSP T h e p r e s e n t e x p e r i m e n t s s h o w e d that t h e slow I P S P in frog s y m p a t h e t i c ganglia c a n n o t be b l o c k e d by low d o s e s of a d r e n e r g i c a n t a g o n i s t s w h i c h are eflmV
fective in b l o c k i n g a d r e n e r g i c r e s p o n s e s in o t h e r syst e m s 1'2° (i.e. at 1 ~ M or lower). L i k e w i s e , r e c e n t intracellular
C
showed
studies
in
frog
sympathetic
that the a d r e n e r g i c a n t a g o n i s t s
ganglia (phentol-
a m i n e , p r o p r a n o l o l and h a l o p e r i d o l , at 5 ~tM or lower) w e r e i n e f f e c t i v e in b l o c k i n g slow I P S P s. A l s o , N a k a m u r a 19 s h o w e d
that
the
neuroleptics
chlor-
p r o m a z i n e , h a l o p e r i d o l and p i m o z i d e ( c o m p o u n d s NEOSTIGMINE
which also b l o c k d o p a m i n e r e c e p t o r s ) w e r e e i t h e r ine f f e c t i v e or w e r e n o n - s p e c i f i c b l o c k e r s o f slow I P S P in frog s y m p a t h e t i c g a n g l i o n . T h e s e studies, t h e r e fore, i n d i c a t e that a d r e n e r g i c and d o p a m i n e r g i c receptors are not i n v o l v e d in m e d i a t i o n of the slow 5
see
Fig. 5. Effects of U-0521 50ktg/ml (A-C) and neostigmine 1 pM (D) on the synaptic responses of bullfrog 9th or 10th sympathetic ganglia elicited by stimulation of the VIII spinal nerve at the frequencies as shown (stimulus frequency in D is 40 Hz; stimulation duration is 1 s in A, B and D, and 2 s in C). See Fig. 1 legend for description of these responses. Left: control responses prior to the administration of U-0521 or neostigmine. Middle: 1 h after beginning superfusion with Ringer solution containing these drugs. Right: 1-3 h after beginning wash-out with U-0521- or neostigmine-free Ringer solution. Each set of tracings is from different experiments. Time calibration in D is for all traces.
IPSP in the frog g a n g l i o n . C o n s i s t e n t with o u r conclusion of n o n - i n v o l v e m e n t of c a t e c h o l a m i n e s in the slow I P S P , R a f u s e and S m i t h 22 h a v e r e c e n t l y s h o w n in Rana pipiens ganglia that the slow I P S P is n o t pot e n t i a t e d by the c a t e c h o l a m i n e u p t a k e b l o c k e r desm e t h y l i m i p r a m i n e and that it is n o t b l o c k e d by yoh i m b i n e (an a 2 - a d r e n e r g i c b l o c k e r ) .
U-0521 does not potentiate slow I P S P L i b e t and K o b a y a s h P s r e p o r t e d large i n c r e a s e s in
TABLE I
Effect of COMT-inhibitor U-0521 (50 ~g/ml," superfused for 45-70 min) on synaptic potentials in bullfrog sympathetic ganglia recorded by sucrose-gap technique Values (mean + S.E.M. from number of experiments in parentheses) are percent decrease in the amplitude of control response due to drug.
Synaptic potential
Slow IPSP Slow EPSP Late slow EPSP Fast EPSP
Stimulation frequency and duration* lO Hz, 1 s
40 Hz, l s
50 Hz, 2 s
lOO Hz, 2 s
9 _+ 1 (5) ** 35 + 6 (5)
11 + 3 (5) 13 + 3 (4) 40 + 3 (6)
14 + 3 (4) 19 _+ 5 (4) 34 + 6 (4)
31 _+ 2 (11) -
* See Materials and Methods for further details. ** Data not shown since the late slow EPSP elicited by this stimulation was very small and unreliable to measure in most experiments.
182 the amplitude and the duration of slow IPSP in bullfrog sympathetic ganglia after superfusion of U-0521 50/~g/ml (a COMT inhibitor). Although the primary mechanism for transmitter removal at adrenergic synaptic junctions is uptake (and not enzymatic destruction) 4'13'14, we decided nonetheless to repeat the experiments of Libet and Kobayashi is (see above). In no case, however, were we able to see a potentiation of slow IPSP by U-0521 (Fig. 5 A - C ) . Indeed, this dose of U-0521 showed a consistent non-specific reduction in the amplitude of all synaptic responses (including the slow IPSP, Table I and Fig. 5 A - C ) . Thus the reported potentiation by U-0521 on slow IPSP was not confirmed in the present experiments. The anticholinesterase compound neostigmine, on the other hand, showed a large potentiation in the duration and amplitude of slow IPSP (thus confirming the involvement of a cholinergic step in mediation of this potential; compare Fig. 5 A - C with 5D). Can slow I P S P be mediated in both a direct and an indirect pathway ?
It has been reported recently that a portion of SIF cells in frog sympathetic ganglia are innervated exclusively by preganglionic C fibers and that, upon stimulation of the preganglionic C fibers, give rise to action potentials 9. Since the slow IPSP is generated predominantly by stimulation of preganglionic C fibers and the SIF cells are the proposed interneurons (in the adrenergic hypothesis) for slow IPSP mediation in frog ganglion, the findings by Dunn and Marshall 9 may seem to provide an anatomical substrate whereby the slow IPSP could be mediated in a portion of synaptic pathways using the SIF cells. It has also been suggested that the slow IPSP in frog ganglion may be mediated in both direct (by ACh, monosynaptically) and indirect (via adrenergic interneuronal) pathways 17. However, the following findings strongly argue against the dual mode of slow IPSP mediation in frog: first, no efferent synaptic contact from SIF cells to ganglionic principal cells has been
REFERENCES 1 Ahlquist, R.P., Adrenergic beta-blocking agents, Prog. Drug Res., 20 (1976) 27-43. 2 Akaike, N., Ito, H., Nishi, K. and Oyama, Y., Further analysis of inhibitory effects of propranolol and local anaesthetics on the calcium current in Helix neurones, Br. J.
seen in frog 28, and second, several recent studies (including the present one) have used the sucrose-gap method of recording the slow IPSP 22'23 (which records from the entire population of cells in the ganglion) and thus the results (which support the cholinergic monosynaptic mediation of slow IPSP) are applicable to the entire ganglion. Thus the results of the present report do not lend support to the hypothesis that the slow IPSP in frog ganglion is mediated in a synaptic pathway using an adrenergic interneuron (wholly or partially). The results are consistent with several recent intracellular and extracellular studies in frog ganglion which provide evidence for a cholinergic monosynaptic pathway for this potential in frog ganglion s'22'23'25'28. The adrenergic interneuron hypothesis for slow IPSP in the mammalian sympathetic ganglia m was based partially on the observation that dibenamine blocked this potential. However, dibenamine (and other adrenergic blockers) have consequently been shown to have antagonistic action at muscarinic receptors (as well as having other non-specific actions) 5'12. It has also been suggested that the slow IPSP in mammalian sympathetic ganglia is mediated by a direct action of ACh released from cholinergic preganglionic fibers 6'7. Thus, in view of the present findings with adrenergic antagonists in frog ganglion, it would seem appropriate to carry out similar studies in the mammalian ganglia to resolve the controversy regarding the mediation of the slow IPSP in these ganglia.
ACKNOWLEDGEMENTS We thank Dr. Paul Gallant for reviewing an earlier version of this manuscript, and Jill Lindahl for typing the manuscript. We also thank the following companies for providing the drugs indicated: Sandoz (dihydroergotamine); Upjohn (U-0521); and Ciba-Geigy (phentolamine).
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