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The antagonism of neuronal responses to acetylcholine by atropine: a quantitative study
568
Brah~ Research, 88 11975) 568 571 :f) Elsevier Scientific Publishing C o m p a n y , A m s t e r d a m - Printed in The Netherland~,~
The antagonism of neuronal responses to acetylcholine by atropine: a quantitative study
P. B E V A N , C. M. B R A D S H A W
AND E. S Z A B A D I
Department of Psychiatry, University of Edinburgh, Morningside Park, Edinburgh EHIO 5HF (Great Britain) (Accepted J a n u a r y 27th, 1975)
There have been several reports that atropine can antagonise the excitatory effects of acetylcholine on single cortical neuronesg,6, al. This antagonism appears to be specific, since responses to glutamate H and noradrenaline 6 are not affected. These qualitative observations could be greatly strengthened if the nature of the interaction between atropine and acetylcholine could be evaluated quantitatively. For this purpose it would be necessary to construct dose-response curves and to determine whether the curve is displaced to the right in a parallel or non-parallel fashion. When parallel displacement is observed, it is desirable to obtain as high a dose ratio as possiblel~L Attempts have been made to construct dose-response curves using the intensity of the electrophoretic current as the measure of dose and change in firing rate at equilibrium as the measure of response. The justification for this procedure rests on the assumption of a linear relationship between the intensity of the ejecting current and the steady-state rate of drug release from the micropipette'L This procedure has been used successfully for the construction of dose-response curves for depressant amino acids~, 7. There are, however, no reports about the use of this method for the construction of dose-response curves for excitatory responses. Spontaneously active single neurones were used in the somato-sensory cortices of adult cats anaesthetised with halothane 10.6-1.5 ~i). Our methods for the surgical preparation of the animals, for the manufacture of 6-barrelled micropipettes, for the recording of action potentials, and the electrophoretic application of drugs have been described elsewhere z. All the drugs were applied by microelectrophoresis. The micropipettes used in these experiments contained the following solutions: 4 M NaCI (two barrels: one for recording and the other for current balancing), 0.05 M acetylcholine chloride (pH 3.6) (3 barrels) and 0.01 M atropine sulphate (pH 5.9). The results included in this report were obtained from 22 cortical neurones. On all these cells atropine reversibly antagonised the excitation evoked by acetylcholine. Quantitative drug interaction studies were conducted in the following way (an example is shown in Fig. 1). When a suitable unit had been found, the retaining current was removed from one of the acetylcholine-containing barrels. (If the neurone
569 r e s p o n d e d to the s p o n t a n e o u s release o f acetylcholine, it was n o t used for d o s e response studies.) In c o n s t r u c t i n g d o s e - r e s p o n s e curves acetylcholine was a p p l i e d r e p e a t e d l y f r o m one b a r r e l o f the m i c r o p i p e t t e using successively increasing intensities o f ejecting current. Care was t a k e n to ensure t h a t the firing rate h a d reached a p l a t e a u d u r i n g each d r u g a p p l i c a t i o n (see Fig. 1A, t o p line). W h e n increases in the intensity o f the ejecting c u r r e n t no longer resulted in an increase in the size o f the p l a t e a u response (i.e., when the m a x i m u m response was attained,) a t r o p i n e was a p plied c o n t i n u o u s l y , either by passing a weak ejecting current, or by r e m o v i n g the retaining c u r r e n t a n d allowing a t r o p i n e to leak o u t from the pipette. D u r i n g the a p p l i c a t i o n o f a t r o p i n e , acetylcholine was tested r e p e a t e d l y using a s t a n d a r d intensity o f ejecting c u r r e n t until there a p p e a r e d to be no further change in the size o f the response. T h e n the intensity o f the ejecting c u r r e n t for acetylcholine was increased A CONTROL
l
'tr
, 5hA ATROPINE
10nA
25nA
50 nA RECOVERY
100nA
200nA
i
40hA
.
. 50nA
50nA
y'
C, < O Z ua ac
.~
ib
A_ j - " ~ 16o
260
CURRENT (nA)
Fig. 1. Antagonism by atropine of excitatory responses of a cortical neurone to acetylcholine (ACh). A: excerpts from the ratemeter recording of the firing rate of the neurone (ordinate: spikes/sec; abscissa : rain). Horizontal bars indicate applications of acetylcholine. Upper trace : control responses to acetylcholine applied with increasing intensities of ejecting current. Middle trace: responses to acetylcholine obtained during the continuous spontaneous release of atropine from one barrel of the pipette containing a 0.01 M solution. Lower trace: recovery of the response to acetylcholine (50 nA) 25 rain after the application of atropine had been terminated. B: current-response curves derived from this study. The increase in firing rate at plateau is taken as the measure of response (see text) and this is expressed in the graph as a percentage of the maximum plateau response to acetylcholine. Closed circles: plateau responses to acetylcholine before atropine was applied; closed triangles: plateau responses to acetylcholine in the presence of atropine; open circles: plateau responses to acetylcholine 20 and 25 rain after the application of atropine had been terminated.
570 with successive applications until the maximum response was again attained (sce Fig. I A, middle line). If necessary, increases in the total ejecting current were achieved by applying acetylcholine simultaneously from two or more barrels. Finall), the application of atropine was terminated and the progress of recovery was observed (see Fig. IA, bottom line). The quantitative drug interaction studies showed that in the presence of atropine the current-response curve for acetylcholine was displaced to the right in an approximately parallel fashion. Dose ratios of 12 could be obtained in these experiments (see Fig. I B). Our experiments confirm that atropine can antagonise excitatory responses of cortical neurones to acetylcholinc 6m,ll Moreover, our finding of an approximately parallel shift in the current-response curve suggests that this antagonism might be competitive in nature. The use of the intensity of the ejecting current as the measure of dose rests on the assumption of a linear relationship between the intensity of the ejecting current and the steady-state rate of drug release from the micropipette 1,4. However, the validity of this assumption is confounded by the contribution of diffusionat drug release to the total rate of release during an ejection period ~. The relative contribution of diffusional release can be reduced by using dilute drug solutions 1. However, this procedure severely restricts the current-carrying capacity of the drug solution. This in turn places a limit on the dose ratio which can be obtained in an antagonism study, since high ejecting currents are needed to apply the agonist when the doseresponse curve has been shifted to the right by an antagonist. It is apparent therefore that quantitative studies of agonist-antagonist interaction in microelectrophoresis are hampered by two conflicting requirements: (1) dilute drug solutions must be used; (2) it must be possible to pass ejecting currents of high intensity. The use of two or more barrels each containing a dilute solution of the agonist seems to go some way towards resolving this conflict. Using this technique it was possible to obtain dose ratios as high as 12, even with the use of agonist solutions many times weaker than those used in previous studies (0.2-1.0 M). In previous experiments it has seldom been possible to obtain dose ratios in excess of three or four '~,7,~. One objection to our procedure could be that the simultaneous use of more than one barrel to apply the agonist results in an increased spontaneous drug release during ejection periods. However, the error involved is probably not great since rather dilute solutions were used. Moreover, the relative contribution of spontaneous release is likely to have been small since a second or third barrel was only needed when the total rate of release required was very high. During the preparation of this manuscript, Clarke e t al. 3 reported the use of a different method for the quantitative analysis of excitatory responses. These workers constructed cumulative dose-response curves on the basis of single applications of acetylcholine. It is of interest that these curves were displaced to the right in an approximately parallel fashion in the presence of atropine. The authors wish to acknowledge financial support from the Mental Health Trust and Research Fund and the Scottish Home and Health Department.
571 We are grateful to Mr R. Lamb for his technical assistance.
1 BRADSHAW, C. M., AND SZABADI, E., The measurement of dose in microelectrophoresis experiments, Neuropharmacology, 13 (1974) 407415. 2 BRADSHAW, C. M., SZABADI, E., AND ROBERTS, M. H. T., The reflection of ejecting and retaining currents in the time-course of neuronal responses to microelectrophoretically applied drugs, J. Pharm. Pharmacol., 25 (1973) 513-520. 3 CLARKE, G., FORRESTER, P. A., AND STRAUGHAN, D. W., A quantitative analysis of the excitation of single cortical neurones by acetylcholine and L-glutamic acid applied microiontophoretically, Neuropharmacology, 13 (1974) 1047-1056. 4 CURTIS, D. R., Microelectrophoresis. In W. L. NASTUK (Ed.), Physical Techniques in Biological Research, Vol V, IHectrophysiological Methods, Part A, Academic Press, New York, 1964, pp. 144~190.
5 CURTIS, D. R., DUGGAN, A. W,, AND JOHNSTON, G. A. R., The specificity of strychnine as a glycine antagonist in the mammalian spinal cord, Exp. Brain Res., 12 (1971) 547-565. 6 JOHNSON, E.S., ROBERTS, M. H.T., SOBIESZEK, A., AND STRAUGHAN, D.W., Noradrenaline sensitive cells in cat cerebral cortex, hit. J. Neuropharmacol., 11 (1969) 549-566. 7 JOHNSON, E.S., ROBERTS, M. H. T., AND STRAUGHAN, D . W . , Amino-acid induced depression of cortical neurones, Brit. J. Pharmacol., 38 (1970) 659-666. 8 KELLY, J. S., AND RENAUD, L. P., On the pharmacology of the 7-aminobutyric acid receptors on the cuneo-thalamic relay cells of the cat, Brit. J. Pharmacol., (1973) 369-386. 9 KRNJEVI~, K., AND PHILLIS, J . W . , Pharmacological properties of acetylcholine-sensitive cells in the cerebral cortex, J. Physiol. (Lond.), 166 (1963) 328 350. l0 STEPHENSON, R. P,, A modification of receptor theory, Brit. J. Pharmacol., 11 (1956) 379-393. 11 STONE, T. W., Cholinergic mechanisms in the rat somatosensory cerebral cortex, J. Physiol. (Lond.), 225 (1972) 485499.
Brah~ Research, 88 11975) 568 571 :f) Elsevier Scientific Publishing C o m p a n y , A m s t e r d a m - Printed in The Netherland~,~
The antagonism of neuronal responses to acetylcholine by atropine: a quantitative study
P. B E V A N , C. M. B R A D S H A W
AND E. S Z A B A D I
Department of Psychiatry, University of Edinburgh, Morningside Park, Edinburgh EHIO 5HF (Great Britain) (Accepted J a n u a r y 27th, 1975)
There have been several reports that atropine can antagonise the excitatory effects of acetylcholine on single cortical neuronesg,6, al. This antagonism appears to be specific, since responses to glutamate H and noradrenaline 6 are not affected. These qualitative observations could be greatly strengthened if the nature of the interaction between atropine and acetylcholine could be evaluated quantitatively. For this purpose it would be necessary to construct dose-response curves and to determine whether the curve is displaced to the right in a parallel or non-parallel fashion. When parallel displacement is observed, it is desirable to obtain as high a dose ratio as possiblel~L Attempts have been made to construct dose-response curves using the intensity of the electrophoretic current as the measure of dose and change in firing rate at equilibrium as the measure of response. The justification for this procedure rests on the assumption of a linear relationship between the intensity of the ejecting current and the steady-state rate of drug release from the micropipette'L This procedure has been used successfully for the construction of dose-response curves for depressant amino acids~, 7. There are, however, no reports about the use of this method for the construction of dose-response curves for excitatory responses. Spontaneously active single neurones were used in the somato-sensory cortices of adult cats anaesthetised with halothane 10.6-1.5 ~i). Our methods for the surgical preparation of the animals, for the manufacture of 6-barrelled micropipettes, for the recording of action potentials, and the electrophoretic application of drugs have been described elsewhere z. All the drugs were applied by microelectrophoresis. The micropipettes used in these experiments contained the following solutions: 4 M NaCI (two barrels: one for recording and the other for current balancing), 0.05 M acetylcholine chloride (pH 3.6) (3 barrels) and 0.01 M atropine sulphate (pH 5.9). The results included in this report were obtained from 22 cortical neurones. On all these cells atropine reversibly antagonised the excitation evoked by acetylcholine. Quantitative drug interaction studies were conducted in the following way (an example is shown in Fig. 1). When a suitable unit had been found, the retaining current was removed from one of the acetylcholine-containing barrels. (If the neurone
569 r e s p o n d e d to the s p o n t a n e o u s release o f acetylcholine, it was n o t used for d o s e response studies.) In c o n s t r u c t i n g d o s e - r e s p o n s e curves acetylcholine was a p p l i e d r e p e a t e d l y f r o m one b a r r e l o f the m i c r o p i p e t t e using successively increasing intensities o f ejecting current. Care was t a k e n to ensure t h a t the firing rate h a d reached a p l a t e a u d u r i n g each d r u g a p p l i c a t i o n (see Fig. 1A, t o p line). W h e n increases in the intensity o f the ejecting c u r r e n t no longer resulted in an increase in the size o f the p l a t e a u response (i.e., when the m a x i m u m response was attained,) a t r o p i n e was a p plied c o n t i n u o u s l y , either by passing a weak ejecting current, or by r e m o v i n g the retaining c u r r e n t a n d allowing a t r o p i n e to leak o u t from the pipette. D u r i n g the a p p l i c a t i o n o f a t r o p i n e , acetylcholine was tested r e p e a t e d l y using a s t a n d a r d intensity o f ejecting c u r r e n t until there a p p e a r e d to be no further change in the size o f the response. T h e n the intensity o f the ejecting c u r r e n t for acetylcholine was increased A CONTROL
l
'tr
, 5hA ATROPINE
10nA
25nA
50 nA RECOVERY
100nA
200nA
i
40hA
.
. 50nA
50nA
y'
C, < O Z ua ac
.~
ib
A_ j - " ~ 16o
260
CURRENT (nA)
Fig. 1. Antagonism by atropine of excitatory responses of a cortical neurone to acetylcholine (ACh). A: excerpts from the ratemeter recording of the firing rate of the neurone (ordinate: spikes/sec; abscissa : rain). Horizontal bars indicate applications of acetylcholine. Upper trace : control responses to acetylcholine applied with increasing intensities of ejecting current. Middle trace: responses to acetylcholine obtained during the continuous spontaneous release of atropine from one barrel of the pipette containing a 0.01 M solution. Lower trace: recovery of the response to acetylcholine (50 nA) 25 rain after the application of atropine had been terminated. B: current-response curves derived from this study. The increase in firing rate at plateau is taken as the measure of response (see text) and this is expressed in the graph as a percentage of the maximum plateau response to acetylcholine. Closed circles: plateau responses to acetylcholine before atropine was applied; closed triangles: plateau responses to acetylcholine in the presence of atropine; open circles: plateau responses to acetylcholine 20 and 25 rain after the application of atropine had been terminated.
570 with successive applications until the maximum response was again attained (sce Fig. I A, middle line). If necessary, increases in the total ejecting current were achieved by applying acetylcholine simultaneously from two or more barrels. Finall), the application of atropine was terminated and the progress of recovery was observed (see Fig. IA, bottom line). The quantitative drug interaction studies showed that in the presence of atropine the current-response curve for acetylcholine was displaced to the right in an approximately parallel fashion. Dose ratios of 12 could be obtained in these experiments (see Fig. I B). Our experiments confirm that atropine can antagonise excitatory responses of cortical neurones to acetylcholinc 6m,ll Moreover, our finding of an approximately parallel shift in the current-response curve suggests that this antagonism might be competitive in nature. The use of the intensity of the ejecting current as the measure of dose rests on the assumption of a linear relationship between the intensity of the ejecting current and the steady-state rate of drug release from the micropipette 1,4. However, the validity of this assumption is confounded by the contribution of diffusionat drug release to the total rate of release during an ejection period ~. The relative contribution of diffusional release can be reduced by using dilute drug solutions 1. However, this procedure severely restricts the current-carrying capacity of the drug solution. This in turn places a limit on the dose ratio which can be obtained in an antagonism study, since high ejecting currents are needed to apply the agonist when the doseresponse curve has been shifted to the right by an antagonist. It is apparent therefore that quantitative studies of agonist-antagonist interaction in microelectrophoresis are hampered by two conflicting requirements: (1) dilute drug solutions must be used; (2) it must be possible to pass ejecting currents of high intensity. The use of two or more barrels each containing a dilute solution of the agonist seems to go some way towards resolving this conflict. Using this technique it was possible to obtain dose ratios as high as 12, even with the use of agonist solutions many times weaker than those used in previous studies (0.2-1.0 M). In previous experiments it has seldom been possible to obtain dose ratios in excess of three or four '~,7,~. One objection to our procedure could be that the simultaneous use of more than one barrel to apply the agonist results in an increased spontaneous drug release during ejection periods. However, the error involved is probably not great since rather dilute solutions were used. Moreover, the relative contribution of spontaneous release is likely to have been small since a second or third barrel was only needed when the total rate of release required was very high. During the preparation of this manuscript, Clarke e t al. 3 reported the use of a different method for the quantitative analysis of excitatory responses. These workers constructed cumulative dose-response curves on the basis of single applications of acetylcholine. It is of interest that these curves were displaced to the right in an approximately parallel fashion in the presence of atropine. The authors wish to acknowledge financial support from the Mental Health Trust and Research Fund and the Scottish Home and Health Department.
571 We are grateful to Mr R. Lamb for his technical assistance.
1 BRADSHAW, C. M., AND SZABADI, E., The measurement of dose in microelectrophoresis experiments, Neuropharmacology, 13 (1974) 407415. 2 BRADSHAW, C. M., SZABADI, E., AND ROBERTS, M. H. T., The reflection of ejecting and retaining currents in the time-course of neuronal responses to microelectrophoretically applied drugs, J. Pharm. Pharmacol., 25 (1973) 513-520. 3 CLARKE, G., FORRESTER, P. A., AND STRAUGHAN, D. W., A quantitative analysis of the excitation of single cortical neurones by acetylcholine and L-glutamic acid applied microiontophoretically, Neuropharmacology, 13 (1974) 1047-1056. 4 CURTIS, D. R., Microelectrophoresis. In W. L. NASTUK (Ed.), Physical Techniques in Biological Research, Vol V, IHectrophysiological Methods, Part A, Academic Press, New York, 1964, pp. 144~190.
5 CURTIS, D. R., DUGGAN, A. W,, AND JOHNSTON, G. A. R., The specificity of strychnine as a glycine antagonist in the mammalian spinal cord, Exp. Brain Res., 12 (1971) 547-565. 6 JOHNSON, E.S., ROBERTS, M. H.T., SOBIESZEK, A., AND STRAUGHAN, D.W., Noradrenaline sensitive cells in cat cerebral cortex, hit. J. Neuropharmacol., 11 (1969) 549-566. 7 JOHNSON, E.S., ROBERTS, M. H. T., AND STRAUGHAN, D . W . , Amino-acid induced depression of cortical neurones, Brit. J. Pharmacol., 38 (1970) 659-666. 8 KELLY, J. S., AND RENAUD, L. P., On the pharmacology of the 7-aminobutyric acid receptors on the cuneo-thalamic relay cells of the cat, Brit. J. Pharmacol., (1973) 369-386. 9 KRNJEVI~, K., AND PHILLIS, J . W . , Pharmacological properties of acetylcholine-sensitive cells in the cerebral cortex, J. Physiol. (Lond.), 166 (1963) 328 350. l0 STEPHENSON, R. P,, A modification of receptor theory, Brit. J. Pharmacol., 11 (1956) 379-393. 11 STONE, T. W., Cholinergic mechanisms in the rat somatosensory cerebral cortex, J. Physiol. (Lond.), 225 (1972) 485499.