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Depression of transmission through the isolated superior cervical ganglion of the rat by physostigmine sulphate
DEPRESSION OF TRANSMISSION THROUGH THE ISOLATED SUPERIOR CERVICAL GANGLION OF THE RAT BY PHYSOSTIGMINE SULPHATE” R. J. MCISAAC and ELIZABETH ALBRECHT Department
of Pharmacology
and Therapeutics, School of Medicine, State University at Buffalo. Buffalo. New York 14214 (Accc,pn,d
of New York
11Srptrmb~r1974)
Summary-
Inhibition of acetylcholinesterase by physostigmine or neostigmine caused a depression of postganglionic action potentials elicited by repetitive stimulation of preganglionic sympathetic nerves at a frequency of IO Hz but not at a frequency of 0.1 Hz. The depression was partially antagonized by atropine and phenoxybenzamine but phentolamine and dihydroergotamine were inactive. Although repetitive stimulation of the preganglionic nerve caused hyperpolarization of control ganglia stimulation in the presence of physostigmine caused a depolarization which increased in magnitude throughout the 10 set of stimulation. The depolarization was completely abolished by hexamethonium. When atropine was added to the physostigmine solution. an initial depolarization developed over the first 4 set similar to that which occurred in physostigmine alone, but the depolarization waned during the remaining 6 set of the volley. The effect of phenoxybenzamine was very similar to atropine and it was suggested that the antagonistic effect of phenoxybenzamine on physostigmine-induced ganglionic depression was due to an atropine-like action. It is concluded that the depression of ganglionic transmission by acetylcholinesterase inhibitors during repetitive preganglionic nerve stimulation is due to a depolarization of the ganglion mediated in part, at least. by muscarinic receptors.
The effects of physostigmine on transmission through sympathetic ganglia are complicated. Both facilitation and inhibition of transmission have been reported and the effect depends on concentration, time of exposure and stimulus frequency. FELDBERG and VARTIAINEN ( 1934) showed that at low frequency submaximal stimulation of the preganglionic nerve. physostigmine facilitated the response of the nictitating membrane. However, a secondary paralyzing action was also observed. MASON (1962) reported that neostigmine, lo- ‘- IO- 4 g/ml, perfused through the superior cervical ganglion reduced the response of the nictitating membrane when the preganglionic nerve was stimulated at frequencies greater than 1 Hz. Diisopropylflurophosphate caused a reduction in postganglionic action potentials elicited by stimulation of the preganglionic nerves at volleys ranging from 3 to 28 Hz in rat superior cervical ganglia in vitro (HOLADAY, KAMIJO and KOELLE, 1954) and cat superior cervical ganglia in vitv~(VOLLE and KOELLE, 1961). More recently. ALKADHI and MCISAAC (1973) reported that after prolonged ganglionic block with chlorisondamine or nicotine, stimulation of the preganglionic nerve resulted in an atropine-sensitive ganglionic transmission through cat superior cervical ganglia and that an intravenous injection of physostigmine caused an inhibition of the atropine-sensitive transmission during the volley. The effect of physostigmine on ganglionic transmission was studied in order to determine the mechanism of inhibition of ganglionic transmission and to determine whether or not a postulated adrenergic inhibitory interneurone was involved (LIBET and TOSAKA, 1969). The results show that the physostigmine-induced inhibition correlates best with a depolarization of the ganglion and that muscarinic receptors are partially involved in the depolarization and depression. METHODS
Male Wistar rats (30&4OOg) were anaesthetized with a mixture of sc-chloralosc and urethane (5 mg and 5 g respectively per rat). The superior cervical ganglion was rapidly * This project Stroke.
was supported
by Grant
NS 06 990 from the National 139
Institute
of Neurological
Diseases
and
removed and placed in chilled physiological saline bubbled with a mixture of 953, oxygen and 57: carbon dioxide. Excess tissue was dissected off and the ganglion was carefully desheathed. The ganglion was mounted as described by DUNANT (I967) in a chamber in which the temperature was maintained at 34’C. The composition of the physiological saline solution (ECCLES,1955) was (mM): NaCl. 137.9; KCI. 4.0; CaCl?. 2.0; MgC12, 05; NaHCO,, 12.0; KH2P04, 1-O;and glucose, 1 1.1. Platinum electrodes were used for stimulating and recording. Postganglionic action potentials were amplified using a Grass P5 or P14 preamplifier and were displayed on a Tektronix 502A oscilloscope. Permanent records of traces were made on film. The preganglionic nerve was stimulated using a Grass S8X stimulator and the stimulus was isolated from ground with a stimulus isolation unit. The nerve was stimulated with square pulses of 0.3 msec duration at supramaximal voltage, i.e. 1W’:, of the voltage which caused a maximum action potential. The experimental protocol was similar in most of the experiments. Transmission was tested every 3Omin after an initial equilibration period of 30min. The test consisted of determining the stimulus voltage-response relationship using a single stimu~Llsof increasing strength applied every 10 sec. The response to a repetitive volley applied to the preganglionic nerve was then determined. Three control stimuli at 0.1 Hz were made and then a volley for 10 set at 10 Hz was applied. Following the repetitive train, a single post-tetanic stimulus was made every 10sec for a period of IOOsec. The mean of the three control stimuli at 0.1 Hz just prior to the train of stimuli was considered as the 1000: response and all other action potentials were calculated as per cent of these control responses. The sequence described above is termed a trial, and two trials were made at 30-min intervals for each treatment except for phenoxybenzamine which was allowed to act for I hr before testing. Ganglionic demarcation potentials were recorded from a chamber in which the capillary tube holding the ganglion had an opening large enough so that the distal pole of the ganglion was drawn partially into the tube. The bath and ganglion capillary electrodes were filled with loi;, agar and chlorided silver wires were inserted into the agar to record the demarcation potential. The potentials were amplified with a Grass P- 16 d.c. amplifer and displayed on one beam of the oscilloscope. Drugs used were physostigmine sulphate. neostigmine sulphate, atropine sulphate, dihydroergotamine rnetllanesLllpho~late. p~lentolamine lnethanesuiphonate, phenoxybenzamine hydrochloride, and hexamethonium chloride. All concentrations are expressed in terms of the salt. RESlJLTS
When volleys at 10 Hz were applied to the preganglionic nerve of a rat superior cervical ganglion immersed in physiological saline, the postganglionic action potential increased in amplitude to 14@150”, of control within l-.2 sec. but then decreased slightly to 1191390/, of control after 10 see of continuous stimulation. Control ganglia that were tested every half hour in physiological saline over a a 3-hr period showed the same type of response. The mean of the action potentials recorded between 9 and 10 set of a IO-set train was 1I9 i: 9 (S.E.M.) in the first hour, 130 & 8 in the second hour, and 139 1. 10 in the third hour of incubation. Addition of atropine, either 1 or 3 pg/ml, had no effect on the response in physiological saline. At higher frequencies of stimulation (I 5 and 20 Hz), the response during stimulation was qualitatively similar to IO Hz. However, at 40 Hz the action potentials declined progressively after an initial transient facilitation. and the mean amplitude was 67”,, of control after 100 pulses and 4.5’:; after 400 pulses. When the ganglia were exposed to physostigmine sulphate (0.25-2 &ml), a marked change in the response occurred during a IO-Hz train. The action potentials initially increased in amplitude and reached a maximum in about 1 sec. Thereafter the action potentials became progressively smaller and reached a constant value between 8 and IO sec. The
Physostigmine
I
on ganglionic
transmission
141
Physostigmine
e
0
Set
Fig. I. The effect of physostigmine and neostigmine on the amplitude of postganglionic action potentials during repetitive stimulation of the preganglionic nerve at 10 Hz, in vitro. The amplitude was measured every set and is expressed as per cent of the mean of three action potentials at 0.1 Hz just prior to the stimulus train. The points are for physiological saline, (0); physostigmine or neostigmine, 0.25 pg/ml, (0); 0.5 pg/ml, (0); I.0 pg/ml, (0); and 2 ng/ml, (A). The data are the means of three to four experiments at each concentration.
mean amplitude of the action potentials was depressed to 89% of control between 9 and 10 set with 0.25 pg/ml and further depressed to between 32-48x by 0.5, 1, and 2 pg/ml. No significant difference in the amplitudes was observed over this four-fold range of concentrations (Fig. 1). Neostigmine sulphate had essentially the same effect as physostigmine on the action potentials during a train at 10 Hz. The mean of the action potentials recorded P-10 set in a train was depressed to 83, 38 and 26”/, of control by 0.25, 0.5 and 1.0 pg/ml respectively. Neither neostigmine nor physostigmine in the dose range used had a significant effect on the action potential amplitudes elicited at a frequency of 0.1 Hz. Physostigmine
on post-tetanic
responses
When the preganglionic nerve was stimulated once every 10sec following the tetanic conditioning train at 10 Hz in physiological saline, a small facilitation (108%) was observed l&20 set following the train. When physostigmine in a concentration of 2pg/ml was added to the bath, the post-tetanic action potential was depressed for periods up to 30 set (Fig. 2). With lower concentrations of physostigmine the depression was not consistent. It occurred in four out of six experiments at 1 pg/ml and in two out of five experiments at 0.5 pg/ml. With physostigmine, 1 ,ug/ml, a late facilitation of the post-tetanic action potentials at 30 set was also observed. The post-tetanic response was also depressed by neostigmine sulphate for 20 set with 1 pg/ml and was enhanced at 30 set by 0.5 pg/ml. Antagonism
qf physostigmine-induced
depression
In order to obtain some information about the mechanism of the depression of ganglionic response, the effects of muscarinic and adrenergic receptor blocking drugs on physostigmine depression were tested. When atropine (1 pg/ml) was added to the physostigmine-containing physiological saline solution, a partial antagonism of the depression occurred (Table 1). Increasing the concentration to 2 ,ug/ml had only a slight additional effect. Atropine also antagonized the post-tetanic depression observed with higher concentrations of physostigmine. The effects of atropine on neostigmine-induced depression of repetitive nerve stimulation was similar. Although neither phentolamine (20 pg/ml), nor dihydroergotamine (l-10 pug/ml) antagonized the physostigmine depression of a repetitive response, phenoxybenzamine (1 ,ug/ ml) did block the depression (Table 1). None of these antagonists, in the concentrations
142
R.J. MCISAAC and ELIZABETHALBRECHT
60 t I
0
I
I
IO
I
20
30
I
I
40
Set after
50
I
6O'kk
train
Fig. 2. The effect of physostigmine on single postganglionic action potentials following a conditioning train of 10 Hz for IO sec. The points plotted are for physiological saline (0); physostigmine, I pg/ml, (0); and physostigmine. 2 pgg/ml, (0). The ordinate is the same as Figure I. The points are the mean, k S.E.M., of three experiments. The asterisk indicates that these points are significantly lower (P < 0.01) than physiological saline control and the + shows the point is significantly larger (P < 0.05) than control.
used, had any consistent effect on transmission in control ganglia immersed in physiological saline. Demarcation
potentials
It was initially thought that with physostigmine treatment, a ganglionic inhibitory system was being activated and the depression of transmission was due to the hyperpolarization of ganglionic cells (LIBET and TOSARA, 1969). Ganglionic demarcation potentials were recorded in order to determine if, in fact, a hyperpolarization of ganglion cells occurred. In untreated ganglia a hyperpolarization of the ganglion by about 245 PV was observed during repetitive stimulation of the preganglionic nerve. However, after physostigmine treatment, a rapid depolarization occurred reaching about 500 PV after 4 set of stimulation. This rapid depolarization was followed by a continued slower depolarization to Table
1. The effect of muscarinic physostigmine-induced
and alpha-adrenergic receptor blocking depression of ganglionic transmission
Amount Experiment Control Physostigmine Physostigmine + Atropine Physostigmine + Atropine Control Physostigmine Physostigmine + Phenoxybenzamine Control Physostigmine Physostigmine + Phentolamine Physostigmine + Atropine
used
(,ugiml)
Number of ganglia 6
I .o I .o
Ia I .o 2.0 0.S 0.5
I .o 0.5 0.5 20.0 0.5 I .o
drugs on
Last IO action potentials of the train 113 * 7* 4x f 3 7x f 4 x2 i_ 3 120* 5 66 ) 2 105 * 5 147 ) 17 50 & x 56 * 4 106 & 3
* The results are expressed in per cent of the mean of three control ganglionic action potentials generated at a frequency of 0.1 Hz prior to the IO set train at IO Hr.
Physostigmine on ganglionic transmission
143
-7oo-
-bOO-
Set
Fig. 3. The change in nV of the ganglion demarcation potential during a IO-set train at a frequency of 10 Hz. The points are the mean (+ S.E.M.) for three experiments; (O), no drug; (0), physostigmine sulphate, 0.5 pg/ml; (A) physostigmine plus atropine sulphate, 1 pg/ml, and (0) physostigmine plus hexamethonium chloride, lOOng/ml. The + indicates points significantly less than (P < 0.05) the demarcation potential in the physostigmine solution.
600 PV at 10 set (Fig. 3). After cessation of the preganglionic nerve stimulation, the ganglionic demarcation potential recovered rapidly and a marked hyperpolarization was observed 10 to 20 set after the train. The change in ganglionic potential after atopine was added to the physostigmine solution was more complex. During the first 4 set of the train, depolarization of the ganglia occurred (489 pV) with a time course similar to physostigmine-induced depolarization. However, after 5 set the ganglion potential started to recover and the mean change in demarcation potential was only 358 PV after 10 set of stimulation. Phenoxybenzamine had an effect on the ganglionic demarcation potential similar to atropine. In this series, the mean depolarization at the end of a 10 Hz train was 563 + 106 PV in physostigmine (0.5 pg/ml) and 308 f 21 in physostigmine plus phenoxybenzamine (1 pg/ml). The effect of hexamethonium (100 pg/ml) was tested in two different series. When hexamethonium was added to the physostigmine solution before atropine, the ganglia hyperpolarized (139 + 97 pV) during repetitive stimulation of preganglionic nerve. However when it was added to a physostigmine solution after the ganglion had been treated with atopine, a small depolarization occurred (23-132 pV) in four ganglia but a small hyperpolarization (28-47 pV) occurred in two other ganglia. DISCUSSION
Treatment of the rat superior cervical ganglion with either physostigmine or neostigmine caused a depression of the postganglionic action potential which was elicited by high frequency preganglionic nerve stimulation, but they did not depress the action potential when the nerve was stimulated at a low frequency, once every 10 sec. The depression of transmission is probably related to inhibition of acetylcholinesterase, since depression of transmission during repetitive stimulation of the preganglionic nerve has been observed with a number of different types of cholinesterase inhibitors; physostigmine, neostigmine, and isoflurophate (ALKADHI and MCISAAC, 1973; HOLADAY et al., 1954; VOLLE and KOELLE, 1961). Furthermore we found that the depression of transmission is partially antagonized by atropine and the depolarization of the ganglia during tetanic volley is blocked completely by hexamethonium and partially by atropine. Finally, depression of transmission did not occur at low frequencies of stimulation. Therefore, it would seem reasonable to conclude, that the depression of transmission is caused by an accumulation of acetyl-
144
R.J. MCISAAC and ELIZABETH
ALBRECW
choline during the tetanic volley because the acetylcholine is released at a faster rate than it can be metabolized or can diffuse away. At least two possible mechanisms for the physostigmine inhibition were considered. The first mechanism was based on the concept of an adrenergic inhibitory interneuronal pathway (ECCLES and LIBET, 1961) in which synaptic transmission between the preganglionic nerve terminal and an interneurone is postulated to involve muscarinic acetylcholinc receptors. The inhibitory transmitter released from the interneurone is suggested to be dopamine (LIRETand TOSAKA. 1970). According to this hypothesis. if physostigmine causes an accumulation of acetylcholine which stimulates muscarinic receptors on the interneurone, hyperpolarization of the ganglion cell would be expected to result. However. the data reported here does not support this hypothesis. The records of the demarcation potentials indicated that ganglia depolarized when they were stimulated in the presence of physostigmine. Furthermore, except for phenoxybenzamine, alpha adrenergic receptor blocking drugs did not antagonize the depressive efTect of physostigmine. Alpha adrenergic receptor blocking drugs reduce the hyperpolarizing positive potential in curarized ganglia (E(‘CLKS and LIRET, 1961) and block the inhibitory effect of adrenergic amines on ~~nglioni~ transmission in uivo (MCISAAC, 1966). The antagonistic effect of phelloxybenzamine on physostigmine-induced depression was most likely due to an atropine-like action. Phenoxybenzamine blocks the effect of acetylcholine on muscarinic receptors in rat atria (REFSCJM and LANDMARK.1973), guinea pig ileum (COOK, 1971; KOCH and MCISAAC,unpublished observations), and on vagal stimulation of the heart (BENFEYand GRILLO, 1963). A second mechanism to account for physostigmine-induced depression of ganglionic transmission, assumes that the inhibition is due to the accumulation of sufficient acetylcholine to cause a prolonged depolarization of ganglionic neurones. The results reported here are more consistent with this hypothesis. Depression occurred only at the higher frequency of stimulation, when acetylcholine was presumably being released at a faster rate than it was being removed. The records of the demarcation potentials showed that depolarization occurred only after physostigmine treatment. The two drugs, atropine and phenoxybenzamine, which partially antagonized the physostigmine depression also partially antagonized the ganglionic depolarization. The depolarization which occurred with onset of stimulation had little depressant effect on the amplitude of action potentials during the first part of the train. Only after 3 4 set of stimulation, when the demarcation potential became larger than 40~5~ /IV. did a dccrease in action potential amplitude occur. Both atropine and phenoxyben~~min~ appeared to exert their main effect on this later depolarization, since they caused a partial recovery of the demarcation potential to a value less than - 400 ,uV during the latter part of the volley. If depolarization is responsible for the depression of transmission, then the major depressant effect is not observed until after an appreciable depolarization has occurred or has lasted for some seconds. The observation of FELDHEKGand V~~TIAI~~~ (1934), that during the physostigmine-indLlced depression of ganglionic transmission the ganglionic responses to potassium as well as to acetylcholine and nicotine were depressed. supports the hypothesis that thi: depression is due to depolarization. If it is assumed that atropine is specific for the muscarinic receptors, then it must be concluded that part of the ganglionic depolarization was mediated via muscarinic receptors. The assumption in this investigation that atropine is a specific antagonist for muscarinic receptors is supported by the fact that under the experimental conditions in which transmission was predominantly nicotinic in nature, i.e. untreated control ganglia, atropine, in a concentration three times that used in these experiments, had no effect on transmission. However, the observation that hexamethonium reversed the physostigmine depolarization to a hyperpolarization may also imply that most, if not all, the receptors involved in the depolarization are nicotinic receptors. The apparent divergent results could be explained by the observation of VOL~Eand HANCOCX(1970) that stimulation of perfused ganglia of the rat by muscarine-like drugs was sensitive to blockade by either hexamethonium or atropine. They suggested that the process of perfusion had induced changes in muscarinic receptors to make them sensitive to hexamethonium. Similar changes may also occur in ganglia studied in r‘itt’o.
Physostigmine
on ganglionic
145
transmission
The reduction in the post-tetanic compound action potential observed with higher concentrations of physostigmine and neostigmine was probably caused by a mechanism different from the depression during the volley. Clear post-tetanic depression was observed only with concentrations of physostigmine greater than threshold concentration required to depress the action potential during a volley. Furthermore, at a concentration of physostigmine of 0.5 mg/ml, the ganglion hyperpolarized immediately after cessation of the volley. Although changes in the demarcation potential were not studied at concentrations of physostigmine of 1 and 2 pg/ml, it is reasonable to assume post-tetanic hyperpolarization would still be observed. The hyperpolarization may reflect the slow positive potential observed by ECCLES and LIBET (1961) after a conditioning volley. These experiments have shown that in untreated rat ganglia, atropine-sensitive receptors have little or no role in transmission of either single electrical events or volleys. Only when the ganglion is sensitized by physostigmine (or other procedures) (HAEFELY, 1974) can an atropine-sensitive component of transmission be demonstrated. The possibility that the atropine-sensitive receptor is altered by artificial perfusing media so that it is also sensitive to hexamethonium should be further investigated. Ackrlo~vl~dy~tnrnts~The
authors
wish to thank
K;~~‘HEKOCH for her valuable
technical
assistance
REFERENCES ALKADHI, K. A. and MCISAAC, R. .I. (1973). Non-nicotinic transmission during ganglionic block with chlorisondamine and nicotine. Eur. J. Phurmuc. 24: 78-85. BENFEY.B. J. and GKILLO, S. A. (1963). Antagonism of acetylcholine by adrenaline antagonists. Br. J. Pharmac. 20: 528-533. COOK. D. A. (1971). Blockade by phenoxybenzamine of the contractor response produced by agonists in the isolated ileum of the guinea-pig. Br. J. Pharnzac. 43: 197-209. DUNANT. Y. (1967). Organisation topographique et fonctionnelle du ganglion cervical superieur chez le rat, J. Ph~SiOl.. Paris 59: 17-38. EC‘CLES, R. M. (1955). Intracellular potentials recorded from a mammalian sympathetic ganglion. J. Phqsiol., Lo,Id. 130: 572-584. EccLt.s. R. M. and LIMIT, B. (1961). Origin and blockade of the synaptic responses of curarized sympathetic ganglia. J. Physiol., Land. 157: 484-503. FELDBERG. W. and VAKTIAIN~N, A. (1934). Further observations on the physiology and pharmacology of a sympathetic ganglion. J. Phmiol.. Land. 83: 103-128. HAPPILY, W. (1974). Muscarinic postsynaptic events in the cat superior cervical ganglion in situ. Naunyn-Schmiedeher(ls Arch. Phurmuc. 281: 119-143. HOLAIIAY. D. A.. KAMIJO, K. and KOELLE, G. B. (1954). Facilitation of ganglionic transmission following inhibition of cholinesterases by DFP. J. Pharmac. cup. T/w. 111: 241-254. Llnr:r, B. and TOSAKA. T. (1969). Slow inhibitory and excitatory postsynaptic responses in single cells of mammalian sympathetic ganglia. J. Nrurophysiol. 32: 43-50. LIBET. B. and TOSAKA. T. (I 970). Dopamine as a synaptic transmitter and modulator in sympathetic ganglia. A different mode of synaptic action. Proc. natn. Acud. Sci. U.S.A. 67: 667-673. MASOX. D. F. J. (1962). A ganglion stimulating action ofneostigmine. Er. /. Phrrrmzc. 18: 7&86. McIsAA(.. R. J. (1966). Ganglionic blocking properties of epinephrine and related amines. Inc. .J. Neuropharmac. 5: 15-26. REFSUM. H. and LANDMARK, K. (1973). Competitive antagonism between phenoxybenzamine and acetylcholine in isolated rat atria. Acfu phurmac. tax. 33: 17-22. VOLL~. R. and HAXU)~K. J. C. (1970). Transmission in sympathetic ganglia. Fedn Proc. Frdn Am Sots e.up. Biol. 29: lY13m 1918. VOLLE. R. and KOELLE. G. (1961). The physiological role of acetylcholinesterase in sympathetic ganglia, J. PharI~LI~.~-XII.Thrr. 133: 233-240.
of Pharmacology
and Therapeutics, School of Medicine, State University at Buffalo. Buffalo. New York 14214 (Accc,pn,d
of New York
11Srptrmb~r1974)
Summary-
Inhibition of acetylcholinesterase by physostigmine or neostigmine caused a depression of postganglionic action potentials elicited by repetitive stimulation of preganglionic sympathetic nerves at a frequency of IO Hz but not at a frequency of 0.1 Hz. The depression was partially antagonized by atropine and phenoxybenzamine but phentolamine and dihydroergotamine were inactive. Although repetitive stimulation of the preganglionic nerve caused hyperpolarization of control ganglia stimulation in the presence of physostigmine caused a depolarization which increased in magnitude throughout the 10 set of stimulation. The depolarization was completely abolished by hexamethonium. When atropine was added to the physostigmine solution. an initial depolarization developed over the first 4 set similar to that which occurred in physostigmine alone, but the depolarization waned during the remaining 6 set of the volley. The effect of phenoxybenzamine was very similar to atropine and it was suggested that the antagonistic effect of phenoxybenzamine on physostigmine-induced ganglionic depression was due to an atropine-like action. It is concluded that the depression of ganglionic transmission by acetylcholinesterase inhibitors during repetitive preganglionic nerve stimulation is due to a depolarization of the ganglion mediated in part, at least. by muscarinic receptors.
The effects of physostigmine on transmission through sympathetic ganglia are complicated. Both facilitation and inhibition of transmission have been reported and the effect depends on concentration, time of exposure and stimulus frequency. FELDBERG and VARTIAINEN ( 1934) showed that at low frequency submaximal stimulation of the preganglionic nerve. physostigmine facilitated the response of the nictitating membrane. However, a secondary paralyzing action was also observed. MASON (1962) reported that neostigmine, lo- ‘- IO- 4 g/ml, perfused through the superior cervical ganglion reduced the response of the nictitating membrane when the preganglionic nerve was stimulated at frequencies greater than 1 Hz. Diisopropylflurophosphate caused a reduction in postganglionic action potentials elicited by stimulation of the preganglionic nerves at volleys ranging from 3 to 28 Hz in rat superior cervical ganglia in vitro (HOLADAY, KAMIJO and KOELLE, 1954) and cat superior cervical ganglia in vitv~(VOLLE and KOELLE, 1961). More recently. ALKADHI and MCISAAC (1973) reported that after prolonged ganglionic block with chlorisondamine or nicotine, stimulation of the preganglionic nerve resulted in an atropine-sensitive ganglionic transmission through cat superior cervical ganglia and that an intravenous injection of physostigmine caused an inhibition of the atropine-sensitive transmission during the volley. The effect of physostigmine on ganglionic transmission was studied in order to determine the mechanism of inhibition of ganglionic transmission and to determine whether or not a postulated adrenergic inhibitory interneurone was involved (LIBET and TOSAKA, 1969). The results show that the physostigmine-induced inhibition correlates best with a depolarization of the ganglion and that muscarinic receptors are partially involved in the depolarization and depression. METHODS
Male Wistar rats (30&4OOg) were anaesthetized with a mixture of sc-chloralosc and urethane (5 mg and 5 g respectively per rat). The superior cervical ganglion was rapidly * This project Stroke.
was supported
by Grant
NS 06 990 from the National 139
Institute
of Neurological
Diseases
and
removed and placed in chilled physiological saline bubbled with a mixture of 953, oxygen and 57: carbon dioxide. Excess tissue was dissected off and the ganglion was carefully desheathed. The ganglion was mounted as described by DUNANT (I967) in a chamber in which the temperature was maintained at 34’C. The composition of the physiological saline solution (ECCLES,1955) was (mM): NaCl. 137.9; KCI. 4.0; CaCl?. 2.0; MgC12, 05; NaHCO,, 12.0; KH2P04, 1-O;and glucose, 1 1.1. Platinum electrodes were used for stimulating and recording. Postganglionic action potentials were amplified using a Grass P5 or P14 preamplifier and were displayed on a Tektronix 502A oscilloscope. Permanent records of traces were made on film. The preganglionic nerve was stimulated using a Grass S8X stimulator and the stimulus was isolated from ground with a stimulus isolation unit. The nerve was stimulated with square pulses of 0.3 msec duration at supramaximal voltage, i.e. 1W’:, of the voltage which caused a maximum action potential. The experimental protocol was similar in most of the experiments. Transmission was tested every 3Omin after an initial equilibration period of 30min. The test consisted of determining the stimulus voltage-response relationship using a single stimu~Llsof increasing strength applied every 10 sec. The response to a repetitive volley applied to the preganglionic nerve was then determined. Three control stimuli at 0.1 Hz were made and then a volley for 10 set at 10 Hz was applied. Following the repetitive train, a single post-tetanic stimulus was made every 10sec for a period of IOOsec. The mean of the three control stimuli at 0.1 Hz just prior to the train of stimuli was considered as the 1000: response and all other action potentials were calculated as per cent of these control responses. The sequence described above is termed a trial, and two trials were made at 30-min intervals for each treatment except for phenoxybenzamine which was allowed to act for I hr before testing. Ganglionic demarcation potentials were recorded from a chamber in which the capillary tube holding the ganglion had an opening large enough so that the distal pole of the ganglion was drawn partially into the tube. The bath and ganglion capillary electrodes were filled with loi;, agar and chlorided silver wires were inserted into the agar to record the demarcation potential. The potentials were amplified with a Grass P- 16 d.c. amplifer and displayed on one beam of the oscilloscope. Drugs used were physostigmine sulphate. neostigmine sulphate, atropine sulphate, dihydroergotamine rnetllanesLllpho~late. p~lentolamine lnethanesuiphonate, phenoxybenzamine hydrochloride, and hexamethonium chloride. All concentrations are expressed in terms of the salt. RESlJLTS
When volleys at 10 Hz were applied to the preganglionic nerve of a rat superior cervical ganglion immersed in physiological saline, the postganglionic action potential increased in amplitude to 14@150”, of control within l-.2 sec. but then decreased slightly to 1191390/, of control after 10 see of continuous stimulation. Control ganglia that were tested every half hour in physiological saline over a a 3-hr period showed the same type of response. The mean of the action potentials recorded between 9 and 10 set of a IO-set train was 1I9 i: 9 (S.E.M.) in the first hour, 130 & 8 in the second hour, and 139 1. 10 in the third hour of incubation. Addition of atropine, either 1 or 3 pg/ml, had no effect on the response in physiological saline. At higher frequencies of stimulation (I 5 and 20 Hz), the response during stimulation was qualitatively similar to IO Hz. However, at 40 Hz the action potentials declined progressively after an initial transient facilitation. and the mean amplitude was 67”,, of control after 100 pulses and 4.5’:; after 400 pulses. When the ganglia were exposed to physostigmine sulphate (0.25-2 &ml), a marked change in the response occurred during a IO-Hz train. The action potentials initially increased in amplitude and reached a maximum in about 1 sec. Thereafter the action potentials became progressively smaller and reached a constant value between 8 and IO sec. The
Physostigmine
I
on ganglionic
transmission
141
Physostigmine
e
0
Set
Fig. I. The effect of physostigmine and neostigmine on the amplitude of postganglionic action potentials during repetitive stimulation of the preganglionic nerve at 10 Hz, in vitro. The amplitude was measured every set and is expressed as per cent of the mean of three action potentials at 0.1 Hz just prior to the stimulus train. The points are for physiological saline, (0); physostigmine or neostigmine, 0.25 pg/ml, (0); 0.5 pg/ml, (0); I.0 pg/ml, (0); and 2 ng/ml, (A). The data are the means of three to four experiments at each concentration.
mean amplitude of the action potentials was depressed to 89% of control between 9 and 10 set with 0.25 pg/ml and further depressed to between 32-48x by 0.5, 1, and 2 pg/ml. No significant difference in the amplitudes was observed over this four-fold range of concentrations (Fig. 1). Neostigmine sulphate had essentially the same effect as physostigmine on the action potentials during a train at 10 Hz. The mean of the action potentials recorded P-10 set in a train was depressed to 83, 38 and 26”/, of control by 0.25, 0.5 and 1.0 pg/ml respectively. Neither neostigmine nor physostigmine in the dose range used had a significant effect on the action potential amplitudes elicited at a frequency of 0.1 Hz. Physostigmine
on post-tetanic
responses
When the preganglionic nerve was stimulated once every 10sec following the tetanic conditioning train at 10 Hz in physiological saline, a small facilitation (108%) was observed l&20 set following the train. When physostigmine in a concentration of 2pg/ml was added to the bath, the post-tetanic action potential was depressed for periods up to 30 set (Fig. 2). With lower concentrations of physostigmine the depression was not consistent. It occurred in four out of six experiments at 1 pg/ml and in two out of five experiments at 0.5 pg/ml. With physostigmine, 1 ,ug/ml, a late facilitation of the post-tetanic action potentials at 30 set was also observed. The post-tetanic response was also depressed by neostigmine sulphate for 20 set with 1 pg/ml and was enhanced at 30 set by 0.5 pg/ml. Antagonism
qf physostigmine-induced
depression
In order to obtain some information about the mechanism of the depression of ganglionic response, the effects of muscarinic and adrenergic receptor blocking drugs on physostigmine depression were tested. When atropine (1 pg/ml) was added to the physostigmine-containing physiological saline solution, a partial antagonism of the depression occurred (Table 1). Increasing the concentration to 2 ,ug/ml had only a slight additional effect. Atropine also antagonized the post-tetanic depression observed with higher concentrations of physostigmine. The effects of atropine on neostigmine-induced depression of repetitive nerve stimulation was similar. Although neither phentolamine (20 pg/ml), nor dihydroergotamine (l-10 pug/ml) antagonized the physostigmine depression of a repetitive response, phenoxybenzamine (1 ,ug/ ml) did block the depression (Table 1). None of these antagonists, in the concentrations
142
R.J. MCISAAC and ELIZABETHALBRECHT
60 t I
0
I
I
IO
I
20
30
I
I
40
Set after
50
I
6O'kk
train
Fig. 2. The effect of physostigmine on single postganglionic action potentials following a conditioning train of 10 Hz for IO sec. The points plotted are for physiological saline (0); physostigmine, I pg/ml, (0); and physostigmine. 2 pgg/ml, (0). The ordinate is the same as Figure I. The points are the mean, k S.E.M., of three experiments. The asterisk indicates that these points are significantly lower (P < 0.01) than physiological saline control and the + shows the point is significantly larger (P < 0.05) than control.
used, had any consistent effect on transmission in control ganglia immersed in physiological saline. Demarcation
potentials
It was initially thought that with physostigmine treatment, a ganglionic inhibitory system was being activated and the depression of transmission was due to the hyperpolarization of ganglionic cells (LIBET and TOSARA, 1969). Ganglionic demarcation potentials were recorded in order to determine if, in fact, a hyperpolarization of ganglion cells occurred. In untreated ganglia a hyperpolarization of the ganglion by about 245 PV was observed during repetitive stimulation of the preganglionic nerve. However, after physostigmine treatment, a rapid depolarization occurred reaching about 500 PV after 4 set of stimulation. This rapid depolarization was followed by a continued slower depolarization to Table
1. The effect of muscarinic physostigmine-induced
and alpha-adrenergic receptor blocking depression of ganglionic transmission
Amount Experiment Control Physostigmine Physostigmine + Atropine Physostigmine + Atropine Control Physostigmine Physostigmine + Phenoxybenzamine Control Physostigmine Physostigmine + Phentolamine Physostigmine + Atropine
used
(,ugiml)
Number of ganglia 6
I .o I .o
Ia I .o 2.0 0.S 0.5
I .o 0.5 0.5 20.0 0.5 I .o
drugs on
Last IO action potentials of the train 113 * 7* 4x f 3 7x f 4 x2 i_ 3 120* 5 66 ) 2 105 * 5 147 ) 17 50 & x 56 * 4 106 & 3
* The results are expressed in per cent of the mean of three control ganglionic action potentials generated at a frequency of 0.1 Hz prior to the IO set train at IO Hr.
Physostigmine on ganglionic transmission
143
-7oo-
-bOO-
Set
Fig. 3. The change in nV of the ganglion demarcation potential during a IO-set train at a frequency of 10 Hz. The points are the mean (+ S.E.M.) for three experiments; (O), no drug; (0), physostigmine sulphate, 0.5 pg/ml; (A) physostigmine plus atropine sulphate, 1 pg/ml, and (0) physostigmine plus hexamethonium chloride, lOOng/ml. The + indicates points significantly less than (P < 0.05) the demarcation potential in the physostigmine solution.
600 PV at 10 set (Fig. 3). After cessation of the preganglionic nerve stimulation, the ganglionic demarcation potential recovered rapidly and a marked hyperpolarization was observed 10 to 20 set after the train. The change in ganglionic potential after atopine was added to the physostigmine solution was more complex. During the first 4 set of the train, depolarization of the ganglia occurred (489 pV) with a time course similar to physostigmine-induced depolarization. However, after 5 set the ganglion potential started to recover and the mean change in demarcation potential was only 358 PV after 10 set of stimulation. Phenoxybenzamine had an effect on the ganglionic demarcation potential similar to atropine. In this series, the mean depolarization at the end of a 10 Hz train was 563 + 106 PV in physostigmine (0.5 pg/ml) and 308 f 21 in physostigmine plus phenoxybenzamine (1 pg/ml). The effect of hexamethonium (100 pg/ml) was tested in two different series. When hexamethonium was added to the physostigmine solution before atropine, the ganglia hyperpolarized (139 + 97 pV) during repetitive stimulation of preganglionic nerve. However when it was added to a physostigmine solution after the ganglion had been treated with atopine, a small depolarization occurred (23-132 pV) in four ganglia but a small hyperpolarization (28-47 pV) occurred in two other ganglia. DISCUSSION
Treatment of the rat superior cervical ganglion with either physostigmine or neostigmine caused a depression of the postganglionic action potential which was elicited by high frequency preganglionic nerve stimulation, but they did not depress the action potential when the nerve was stimulated at a low frequency, once every 10 sec. The depression of transmission is probably related to inhibition of acetylcholinesterase, since depression of transmission during repetitive stimulation of the preganglionic nerve has been observed with a number of different types of cholinesterase inhibitors; physostigmine, neostigmine, and isoflurophate (ALKADHI and MCISAAC, 1973; HOLADAY et al., 1954; VOLLE and KOELLE, 1961). Furthermore we found that the depression of transmission is partially antagonized by atropine and the depolarization of the ganglia during tetanic volley is blocked completely by hexamethonium and partially by atropine. Finally, depression of transmission did not occur at low frequencies of stimulation. Therefore, it would seem reasonable to conclude, that the depression of transmission is caused by an accumulation of acetyl-
144
R.J. MCISAAC and ELIZABETH
ALBRECW
choline during the tetanic volley because the acetylcholine is released at a faster rate than it can be metabolized or can diffuse away. At least two possible mechanisms for the physostigmine inhibition were considered. The first mechanism was based on the concept of an adrenergic inhibitory interneuronal pathway (ECCLES and LIBET, 1961) in which synaptic transmission between the preganglionic nerve terminal and an interneurone is postulated to involve muscarinic acetylcholinc receptors. The inhibitory transmitter released from the interneurone is suggested to be dopamine (LIRETand TOSAKA. 1970). According to this hypothesis. if physostigmine causes an accumulation of acetylcholine which stimulates muscarinic receptors on the interneurone, hyperpolarization of the ganglion cell would be expected to result. However. the data reported here does not support this hypothesis. The records of the demarcation potentials indicated that ganglia depolarized when they were stimulated in the presence of physostigmine. Furthermore, except for phenoxybenzamine, alpha adrenergic receptor blocking drugs did not antagonize the depressive efTect of physostigmine. Alpha adrenergic receptor blocking drugs reduce the hyperpolarizing positive potential in curarized ganglia (E(‘CLKS and LIRET, 1961) and block the inhibitory effect of adrenergic amines on ~~nglioni~ transmission in uivo (MCISAAC, 1966). The antagonistic effect of phelloxybenzamine on physostigmine-induced depression was most likely due to an atropine-like action. Phenoxybenzamine blocks the effect of acetylcholine on muscarinic receptors in rat atria (REFSCJM and LANDMARK.1973), guinea pig ileum (COOK, 1971; KOCH and MCISAAC,unpublished observations), and on vagal stimulation of the heart (BENFEYand GRILLO, 1963). A second mechanism to account for physostigmine-induced depression of ganglionic transmission, assumes that the inhibition is due to the accumulation of sufficient acetylcholine to cause a prolonged depolarization of ganglionic neurones. The results reported here are more consistent with this hypothesis. Depression occurred only at the higher frequency of stimulation, when acetylcholine was presumably being released at a faster rate than it was being removed. The records of the demarcation potentials showed that depolarization occurred only after physostigmine treatment. The two drugs, atropine and phenoxybenzamine, which partially antagonized the physostigmine depression also partially antagonized the ganglionic depolarization. The depolarization which occurred with onset of stimulation had little depressant effect on the amplitude of action potentials during the first part of the train. Only after 3 4 set of stimulation, when the demarcation potential became larger than 40~5~ /IV. did a dccrease in action potential amplitude occur. Both atropine and phenoxyben~~min~ appeared to exert their main effect on this later depolarization, since they caused a partial recovery of the demarcation potential to a value less than - 400 ,uV during the latter part of the volley. If depolarization is responsible for the depression of transmission, then the major depressant effect is not observed until after an appreciable depolarization has occurred or has lasted for some seconds. The observation of FELDHEKGand V~~TIAI~~~ (1934), that during the physostigmine-indLlced depression of ganglionic transmission the ganglionic responses to potassium as well as to acetylcholine and nicotine were depressed. supports the hypothesis that thi: depression is due to depolarization. If it is assumed that atropine is specific for the muscarinic receptors, then it must be concluded that part of the ganglionic depolarization was mediated via muscarinic receptors. The assumption in this investigation that atropine is a specific antagonist for muscarinic receptors is supported by the fact that under the experimental conditions in which transmission was predominantly nicotinic in nature, i.e. untreated control ganglia, atropine, in a concentration three times that used in these experiments, had no effect on transmission. However, the observation that hexamethonium reversed the physostigmine depolarization to a hyperpolarization may also imply that most, if not all, the receptors involved in the depolarization are nicotinic receptors. The apparent divergent results could be explained by the observation of VOL~Eand HANCOCX(1970) that stimulation of perfused ganglia of the rat by muscarine-like drugs was sensitive to blockade by either hexamethonium or atropine. They suggested that the process of perfusion had induced changes in muscarinic receptors to make them sensitive to hexamethonium. Similar changes may also occur in ganglia studied in r‘itt’o.
Physostigmine
on ganglionic
145
transmission
The reduction in the post-tetanic compound action potential observed with higher concentrations of physostigmine and neostigmine was probably caused by a mechanism different from the depression during the volley. Clear post-tetanic depression was observed only with concentrations of physostigmine greater than threshold concentration required to depress the action potential during a volley. Furthermore, at a concentration of physostigmine of 0.5 mg/ml, the ganglion hyperpolarized immediately after cessation of the volley. Although changes in the demarcation potential were not studied at concentrations of physostigmine of 1 and 2 pg/ml, it is reasonable to assume post-tetanic hyperpolarization would still be observed. The hyperpolarization may reflect the slow positive potential observed by ECCLES and LIBET (1961) after a conditioning volley. These experiments have shown that in untreated rat ganglia, atropine-sensitive receptors have little or no role in transmission of either single electrical events or volleys. Only when the ganglion is sensitized by physostigmine (or other procedures) (HAEFELY, 1974) can an atropine-sensitive component of transmission be demonstrated. The possibility that the atropine-sensitive receptor is altered by artificial perfusing media so that it is also sensitive to hexamethonium should be further investigated. Ackrlo~vl~dy~tnrnts~The
authors
wish to thank
K;~~‘HEKOCH for her valuable
technical
assistance
REFERENCES ALKADHI, K. A. and MCISAAC, R. .I. (1973). Non-nicotinic transmission during ganglionic block with chlorisondamine and nicotine. Eur. J. Phurmuc. 24: 78-85. BENFEY.B. J. and GKILLO, S. A. (1963). Antagonism of acetylcholine by adrenaline antagonists. Br. J. Pharmac. 20: 528-533. COOK. D. A. (1971). Blockade by phenoxybenzamine of the contractor response produced by agonists in the isolated ileum of the guinea-pig. Br. J. Pharnzac. 43: 197-209. DUNANT. Y. (1967). Organisation topographique et fonctionnelle du ganglion cervical superieur chez le rat, J. Ph~SiOl.. Paris 59: 17-38. EC‘CLES, R. M. (1955). Intracellular potentials recorded from a mammalian sympathetic ganglion. J. Phqsiol., Lo,Id. 130: 572-584. EccLt.s. R. M. and LIMIT, B. (1961). Origin and blockade of the synaptic responses of curarized sympathetic ganglia. J. Physiol., Land. 157: 484-503. FELDBERG. W. and VAKTIAIN~N, A. (1934). Further observations on the physiology and pharmacology of a sympathetic ganglion. J. Phmiol.. Land. 83: 103-128. HAPPILY, W. (1974). Muscarinic postsynaptic events in the cat superior cervical ganglion in situ. Naunyn-Schmiedeher(ls Arch. Phurmuc. 281: 119-143. HOLAIIAY. D. A.. KAMIJO, K. and KOELLE, G. B. (1954). Facilitation of ganglionic transmission following inhibition of cholinesterases by DFP. J. Pharmac. cup. T/w. 111: 241-254. Llnr:r, B. and TOSAKA. T. (1969). Slow inhibitory and excitatory postsynaptic responses in single cells of mammalian sympathetic ganglia. J. Nrurophysiol. 32: 43-50. LIBET. B. and TOSAKA. T. (I 970). Dopamine as a synaptic transmitter and modulator in sympathetic ganglia. A different mode of synaptic action. Proc. natn. Acud. Sci. U.S.A. 67: 667-673. MASOX. D. F. J. (1962). A ganglion stimulating action ofneostigmine. Er. /. Phrrrmzc. 18: 7&86. McIsAA(.. R. J. (1966). Ganglionic blocking properties of epinephrine and related amines. Inc. .J. Neuropharmac. 5: 15-26. REFSUM. H. and LANDMARK, K. (1973). Competitive antagonism between phenoxybenzamine and acetylcholine in isolated rat atria. Acfu phurmac. tax. 33: 17-22. VOLL~. R. and HAXU)~K. J. C. (1970). Transmission in sympathetic ganglia. Fedn Proc. Frdn Am Sots e.up. Biol. 29: lY13m 1918. VOLLE. R. and KOELLE. G. (1961). The physiological role of acetylcholinesterase in sympathetic ganglia, J. PharI~LI~.~-XII.Thrr. 133: 233-240.