J. Phy0iol. (1975), 244, pp. 741-756 With 5 text-ftgure Printed in Great Britain
741
THE EFFECT OF GRADED FORELIMB AFFERENT VOLLEYS ON ACETYLCHOLINE RELEASE FROM CAT SENSORIMOTOR CORTEX
BY W. J. MULLIN AND J. W. PHILLIS From the Department of Physiology, College of Medicine, University of Saskatchewan, Saskatoon, Saskatchewan S 7N OWO, Canada
(Received 17 June 1974) SUMMARY
1. The acetylcholine (ACh)-releasing system in the cerebral cortex of pentobarbital anaesthetized cats was investigated by examining the effect of graded afferent volleys in forelimb nerves on ACh release from the sensorimotor cortices contralateral and ipsilateral to the site of stimulation. 2. Cortical ACh release was determined by bio-assay of neostigminecontaining perfusates which had been in contact with the cortical surfaces for 5-10 min periods. 3. Afferent volleys, generated by stimuli that were effective in activating as many fibres of a fibre group as possible without stimulating fibres in the group with the next highest threshold for activation, were monitored from dorsal roots C7 or C8 before entering the spinal cord. 4. Stimulation of the deep (DR) and superficial (SR) radial nerves and the radial (R) nerve proximal to the junction of the DR and SR were effective in enhancing ACh release only when either group III or groups III and IV fibres were included in the afferent volley. 5. The rates of ACh release from the primary receiving area of the sensorimotor cortex contralateral to the site of stimulation did not differ from those from the same area of the ipsilateral sensorimotor cortex. 6. The pertinence of this data to the various hypotheses concerning the nature of the ACh-releasing pathways to the cerebral cortex is discussed. INTRODUCTION
The release of acetylcholine (ACh) from the cerebral hemispheres of unanaesthetized and anaesthetized cats has been extensively investigated (see review by Pepeu, 1973). In attempts to define the role of ACh as a synaptic transmitter in the cerebral cortex, as well as the pathways in which it may function, many investigators have examined the effects of 29-2
742 W. J. MULLIN AND J. W. PHILLIS stimulation of a variety of afferent pathways on the release of cortical ACh. These studies, however, contain contradictory findings concerning the distribution of the enhancement of release during stimulation of different afferent pathways. Some investigators have described a marked localization of the augmented release to the primary cortical receiving area for the afferent pathway stimulated, while others have observed a diffusely spread increase in release over both hemispheres. Not surprisingly, the conclusions drawn by these investigators have differed. Mitchell and his colleagues (Mitchell, 1963; Collier & Mitchell, 1966, 1967; Hemsworth & Mitchell, 1969) inferred that a greater enhancement of release from the primary receiving area is indicative of a specific thalamo-cortical cholinergic projection, while others (Phillis & Chong, 1965; Phillis, 1968; Bartolini, Weisenthal & Domino, 1972) postulated that the ACh-releasing system projects diffusely to the cortex and may be associated with the system mediating electroencephalographic and behavioural arousal. The latter point of view has found support in the observation that ACh release is increased during states of e.e.g. desynchronization (arousal and rapid eye movement sleep: Jasper & Tessier, 1971, Gadea-Ciria, Stadler, Kenneth & Bartholini, 1973). The present experiments were designed to further investigate the nature of the pathways involved in ACh release from the cerebral cortex by examining the effects of activation of different fibre groups in forelimb nerves on release from both cerebral hemispheres. Since much information is available concerning the nature of the projections of the various groups of forelimb afferents to the cerebral cortex as well as their effects on the e.e.g. and behaviour, we have examined the effect on ACh release from the ipsilateral and contralateral sensorimotor cortices of graded afferent volleys in the superficial (SR) and deep (DR) radial nerves of cats. By monitoring the afferent volley as it entered the spinal cord, the effect of peripheral stimulation on cortical ACh release was related to the fibre group(s) in the SR or DR which had been activated by the stimuli. The possibility of a specific cholinergic thalamo-cortical projection to the contralateral sensorimotor cortex was tested by comparing the ACh release from both contralateral and ipsilateral sensorimotor cortices during stimulation of the SR or DR. METHODS The experiments were performed on nine adult cats of either sex that were anaesthetized with sodium pentobarbitone (Nembutal, Abbott Laboratories Ltd) in an initial dose of 30-35 mgikg (i.v. or I.P.). In all experiments, the trachea was cannulated and a polyethylene cannula was inserted in a forelimb cephalic vein to facilitate supplemental injections of the anaesthetic, when necessary. Body temperature was maintained between 37-38° C by an electric heating pad controlled by a feed-back circuit using a rectal probe.
ACh RELEASE FROM CAT CEREBRAL CORTEX
743
Stimulating and recording The following forelimb nerves were prepared for stimulation: the superficial radial nerve (SR), the deep radial nerve (DR) or alternatively, in four animals, the radial nerve (R) proximal to the junction of the SR and the DR. The nerves were dissected, cut distally and placed on pairs of silver wire electrodes. The electrical stimuli were rectangular pulses (0-1 msec duration) delivered through a stimulus isolation unit at a frequency of I/sec. The dorsal roots of C7 and C8 were exposed by a laminectomy in the cervical region. The afferent input volleys were recorded triphasically by a silver ball electrode in contact with the dorsal root as far distal to its insertion into the spinal cord as possible together with an indifferent electrode placed on a nearby muscle. This location of the electrode on the dorsal root minimized interference with the recording of the peripheral afferent volleys by the cord dorsum potential. The responses from these afferent fibres were amplified and then either displayed on an oscilloscope or averaged 30-60 times by a signal averager-memory control unit (Ortec models 4623 and 4620) and plotted by a X-Y recorder (Model 7035B Hewlett Packard Company). All exposed nervous tissues were kept covered with warmed (36-380 C) paraffin oil in pools that were retained by skin flaps. Heating elements controlled by sensing elements were utilized to maintain the paraffin pool temperatures. The different fibre group components of the compound action potential of the SR, DR or R afferent volley were activated by stimulating these nerves with pulses of different intensities. The fibre groups (nomenclature according to Lloyd, 1943) in the afferent volleys were identified on the basis of the maximum conduction velocity (MCV) of the fibres in that group; the conduction velocity being calculated from the latency of the negative-going deflexion representing that group in the compound action potential and from the conduction distance which was determined at the conclusion of each experiment. The stimulus intensity was adjusted to activate as many fibres of a particular group as possible (as judged from the action potential record displayed on the oscilloscope) without stimulating fibres in the group with the next highest threshold for activation. Wherever possible, stimuli supramaximal for the fibres in each group were used. All stimulus intensities are expressed as multiples of that stimulus intensity which activated the lowest threshold fibres in the nerve (designated at 1-OT; where T denotes 'times threshold'). Group II fibres (MCV = 63-75 mfsec) in the SR were activated at 1-0T and reached maximum at 3-8-4-4T. As the group III (MCV = 13-40 m/sec) threshold occurred at 4-4-9T it was possible to maximally activate group II fibres without apparently including group III fibres in the afferent volleys in three of the four experiments in this series. In the remaining experiment a slightly submaximal stimulus had to be used in order to avoid Group III contamination of the afferent volley. Stimulus intensities of 3-8-4-IT were used, therefore, to activate group II afferent fibres. Although it was sometimes difficult to define full activation of group III fibres, an apparent maximum occurred at 15-19T. Stimuli of twice this intensity were therefore used to activate group III fibres. With the recording conditions used in these experiments, it was not possible to consistently define a group IV component in the afferent volley at high stimulus intensities. From the data of Collins & Randt (1961). it is apparent that this threshold could be at least 250T. In this present study, stimulation of the SR with stimuli of intensity 750-900T was assumed to activate group IV unmyelinated fibres. The lowest threshold group I afferent fibres (MCV = 85-124 m/sec) in the DR were maximally activated at 1-8-2-2T, while the threshold for excitation of group II fibres (MCV = 61-76 m/sec) was 2-1-2-4T. This slight overlap in responses, which is
744
W. J. MULLIN AND J. W. PHILLIS
agreement with the data of Holmqvist,Oscarsson & Uddenberg(1963) and Pompeiano & Swett (1963), necessitated the use of stimuli (2-2-3T) which were just or slightly submaximal for group I fibres in order to avoid contamination maximal of the volley by the activation of the lowest threshold fibres of group II. The threshold for the appearance of group III fibres (MCV = 20-30 m/sec) in theafferent volley occurred at stimulus intensities (5-6-8T) which were greater than those required for apparent maximal activation of group II fibres (4-2-4.7T). This finding is not in agreement with Pompeiano & Swett (1963) who indicated that the group II imum may occur above the threshold for activation of group III fibres (5-7T max in their experiments). In this present study, the stimulus intensities which were used to activate group II fibres (4.7-5.5T) were just below those required for threshold activation of group III afferents and were above the strength required for apparent maximal activation of group II fibres. Stimuli of 1-5-2 times the intensity required for maximal activation of the fibre group (11-15T) were used to include group III fibres in the afferent volley, while group IV fibres were assumed to be activated at 660-830T). When the R was stimulated at a site proximal to the junction of the SR and DR, the afferent volley generated was composed of peaks corresponding to an interof the responses to stimulation of the SR or DR alone described above. mingling Stimulus intensities used to activate the fibre groups in the radial nerve were: group I, 1'7-2-2T; groups I and II, 4'3-5-4T; groupsI+II+III, 22-29T; groups in
I+II+III+IV, 730-830T.
CoUection of actylcholine Both sensorimotor cortices were exposed by craniotomy and of the dura. Perspex cylinders (cups) with an inside diameter of -0 cm were then placed bilaterally over the forelimb receiving areas I. Leakage of fluid from the cups was prevented by a layer of silicone rubber affixed to the surface of the cup which was in contact with the cortical surface. When the cups were in place, the exposed cortical surfaces were covered with a generous amount of 4 % agar in physiological saline or the artificial cerebrospinal fluid of Merlis (1940). solution of The cortical surfaces inside the cups were bathed with a in artificial neostigmine methyl sulphate (Koch-Light Laboratories) 0-05 cerebrospinal fluid. The cortical tissues were exposed to the neostigmine for at least 30 min prior to the collection of the first sample and the solutions used to bathe the cortical tissues over this 30 period were removed and discarded. The cups were rinsed and refilled with 0-6-0'9 ml. of fresh neostigmine containing solution. Thereafter, the contents of the cups were removed and stored in glass tubes after 5 or 10 min of contact with the cortical tissues and the cups were immediately rinsed and refilled with fresh neostigmine-containing solution. The samples collected in this manner were analysed for ACh content in a biological assay on the hearts of the mollusc Mercenaria mercenaria. To ensure that the inhibitory activity of the samples on the molluscs hearts was due to ACh, they were frequently retested at the conclusion of the assay in the presence of the ACh antagonist benzoquinonium chloride (Mytolon). Samples which were not assayed soon after collection were stored frozen until the next day. From these tions of the concentration of ACh in each sample, the rate of liberation of ACh (in cm2 of cortex) from the sensorimotor cortices was determined. The techng/min.for niques the collection and estimation of ACh release from the cerebral cortex have been described in detail in previous publications (Phillis, 1968; Jhamadas, Phillis & Pinsky, 1971).
1
reflexion
warmed
mg/ml.
min
determina-
ACh RELEASE FROM CAT CEREBRAL CORTEX
745
RESULTS
The experiments conducted on the nine animals used in this study differed only in the nerve(s) that were stimulated, the sequence of activation of the fibre groups and, in one instance to be described later, the duration of the sample collection period. The typical effects on ACh release from the contralateral and ipsilateral sensorimotor cortices that were observed upon low frequency stimulation (1 Hz) of the DR and SR with pulses of different intensities are illustrated by the data from one animal presented in Figs. 1 and 2. The columns of the histograms compare the rates of ACh release (in ng/min. cm2 of cortex) over 10 min intervals when the DR or SR were stimulated throughout the collection period with pulses of the intensities indicated below the histograms (hatched columns) or when no stimuli were applied to the forelimb nerves (clear columns). As depicted in Fig. 1, the constant rate of ACh release from the contralateral cortex over the first eight 10 min collection periods indicates that stimulation of the DR with intensities that activated group I (2*OT) or groups I and II (4-7T) fibres had no effect on ACh release. When, however, the stimuli were applied at an intensity which supramaximally activated groups I, II and III afferents (30T), a marked increase in ACh release was observed. The rates of release of ACh over the two subsequent unstimulated collection periods were less than that evoked by stimulation but were greater than those over the two unstimulated collection periods preceding the stimulation. A higher rate of spontaneous release of ACh following a period of stimulation that was effective in enhancing ACh release was consistently observed in these experiments (see also Figs. 2 and 5 and Phillis, 1968). The rate of ACh release from the contralateral cortex that was observed during very high intensity stimulation of the DR (830T), which was assumed to have also activated group IV afferents, was, in terms of absolute rate, greater than that evoked by stimulation of the DR at 30T. With a few exceptions (e.g. the evoked release ofACh from the ipsilateral cortex in this experiment), this was a consistent finding which is further analysed in Table 2. The effects of DR stimulation on ACh release from the ipsilateral cortex were similar to those described for the contralateral cortex. Activation of Group I or Group I and II fibres did not alter release rates. Inclusion of Group III fibres in the afferent volley induced a marked increase in release which subsided slowly after the period of stimulation. The addition of Group IV afferents did not, in this particular experiment, cause any further enhancement of release. Low frequency stimulation of the SR was found to affect the rate of release of ACh from the sensorimotor cortices (Fig. 2) in a manner similar
W. J. MULLIN AND J. W. PHILLIS
746
Deep radial ACh release
3*0
Contralateral cortex
2-0 1.0 0
%_U 0 E
.S
nt
C
310 cpsilateral
cortex 2-0
10 10 Mnin collection periods Stimulus
2.0
4-7
I
I and II
30
830
I, fi
1. II IlIl and IV
T
intensity Activated fibre groups
and IlIl
Fig. 1. Rates of ACh release from the contralateral and ipsilateral sensorimotor cortices of a pentobarbitone anaesthetized cat measured either when the DR nerve was stimulated at 1/sec throughout the entire 10 min collection period (hatched columns) or when no stimuli were applied to the forelimb nerve (open columns). The afferent volleys (shown in the signal averager tracings at the bottom) were recorded from dorsal root C 7 and generated by stimuli of the intensities indicated below the histograms (where T denotes that all stimuli are multiples of that stimulus intensity which activated the very low threshold fibres in the nerve). Activated fibre groups (designated below the histograms) were identified on the basis of the maximum conduction velocity of the fibres in each group. Calibration = 1 msec.
ACh RELEASE FROM CAT CEREBRAL CORTEX
747
Superficial radial ACh release 3-0 Ad Contralateral cortex
2*0 1.0 K x L.
0
0
0 1 *3-0
A.
Ipsilateral cortex
2-0 1.0 0
10 min collection periods
Stimulus intensity Activated fibre groups
38
11
27'5
900
IH and IlIl
II, i
1T
rq i
1!
C-1
and IV
Fig. 2. The influence of afferent volleys in the SR nerve of the same animal used to obtain the data in Fig. 1 on the rates of ACh release from the contralateral and ipsilateral sensorimotor cortices. The afferent volley was recorded from dorsal root C8. For further explanation see Fig. 1.
to that observed following DR stimulation. Stimuli of an intensity (3-8T) that activated only group II fibres in the SR did not affect the rate of ACh release. Both contralateral and ipsilateral cortical ACh release were enhanced when group III (27-5T) or groups III and IV (900T) fibres were included in the afferent volley; the absolute rate of release being considerably greater at the higher intensity of stimulation.
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W. J. MULLIN AND J. W. PHILLIS
748
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741
THE EFFECT OF GRADED FORELIMB AFFERENT VOLLEYS ON ACETYLCHOLINE RELEASE FROM CAT SENSORIMOTOR CORTEX
BY W. J. MULLIN AND J. W. PHILLIS From the Department of Physiology, College of Medicine, University of Saskatchewan, Saskatoon, Saskatchewan S 7N OWO, Canada
(Received 17 June 1974) SUMMARY
1. The acetylcholine (ACh)-releasing system in the cerebral cortex of pentobarbital anaesthetized cats was investigated by examining the effect of graded afferent volleys in forelimb nerves on ACh release from the sensorimotor cortices contralateral and ipsilateral to the site of stimulation. 2. Cortical ACh release was determined by bio-assay of neostigminecontaining perfusates which had been in contact with the cortical surfaces for 5-10 min periods. 3. Afferent volleys, generated by stimuli that were effective in activating as many fibres of a fibre group as possible without stimulating fibres in the group with the next highest threshold for activation, were monitored from dorsal roots C7 or C8 before entering the spinal cord. 4. Stimulation of the deep (DR) and superficial (SR) radial nerves and the radial (R) nerve proximal to the junction of the DR and SR were effective in enhancing ACh release only when either group III or groups III and IV fibres were included in the afferent volley. 5. The rates of ACh release from the primary receiving area of the sensorimotor cortex contralateral to the site of stimulation did not differ from those from the same area of the ipsilateral sensorimotor cortex. 6. The pertinence of this data to the various hypotheses concerning the nature of the ACh-releasing pathways to the cerebral cortex is discussed. INTRODUCTION
The release of acetylcholine (ACh) from the cerebral hemispheres of unanaesthetized and anaesthetized cats has been extensively investigated (see review by Pepeu, 1973). In attempts to define the role of ACh as a synaptic transmitter in the cerebral cortex, as well as the pathways in which it may function, many investigators have examined the effects of 29-2
742 W. J. MULLIN AND J. W. PHILLIS stimulation of a variety of afferent pathways on the release of cortical ACh. These studies, however, contain contradictory findings concerning the distribution of the enhancement of release during stimulation of different afferent pathways. Some investigators have described a marked localization of the augmented release to the primary cortical receiving area for the afferent pathway stimulated, while others have observed a diffusely spread increase in release over both hemispheres. Not surprisingly, the conclusions drawn by these investigators have differed. Mitchell and his colleagues (Mitchell, 1963; Collier & Mitchell, 1966, 1967; Hemsworth & Mitchell, 1969) inferred that a greater enhancement of release from the primary receiving area is indicative of a specific thalamo-cortical cholinergic projection, while others (Phillis & Chong, 1965; Phillis, 1968; Bartolini, Weisenthal & Domino, 1972) postulated that the ACh-releasing system projects diffusely to the cortex and may be associated with the system mediating electroencephalographic and behavioural arousal. The latter point of view has found support in the observation that ACh release is increased during states of e.e.g. desynchronization (arousal and rapid eye movement sleep: Jasper & Tessier, 1971, Gadea-Ciria, Stadler, Kenneth & Bartholini, 1973). The present experiments were designed to further investigate the nature of the pathways involved in ACh release from the cerebral cortex by examining the effects of activation of different fibre groups in forelimb nerves on release from both cerebral hemispheres. Since much information is available concerning the nature of the projections of the various groups of forelimb afferents to the cerebral cortex as well as their effects on the e.e.g. and behaviour, we have examined the effect on ACh release from the ipsilateral and contralateral sensorimotor cortices of graded afferent volleys in the superficial (SR) and deep (DR) radial nerves of cats. By monitoring the afferent volley as it entered the spinal cord, the effect of peripheral stimulation on cortical ACh release was related to the fibre group(s) in the SR or DR which had been activated by the stimuli. The possibility of a specific cholinergic thalamo-cortical projection to the contralateral sensorimotor cortex was tested by comparing the ACh release from both contralateral and ipsilateral sensorimotor cortices during stimulation of the SR or DR. METHODS The experiments were performed on nine adult cats of either sex that were anaesthetized with sodium pentobarbitone (Nembutal, Abbott Laboratories Ltd) in an initial dose of 30-35 mgikg (i.v. or I.P.). In all experiments, the trachea was cannulated and a polyethylene cannula was inserted in a forelimb cephalic vein to facilitate supplemental injections of the anaesthetic, when necessary. Body temperature was maintained between 37-38° C by an electric heating pad controlled by a feed-back circuit using a rectal probe.
ACh RELEASE FROM CAT CEREBRAL CORTEX
743
Stimulating and recording The following forelimb nerves were prepared for stimulation: the superficial radial nerve (SR), the deep radial nerve (DR) or alternatively, in four animals, the radial nerve (R) proximal to the junction of the SR and the DR. The nerves were dissected, cut distally and placed on pairs of silver wire electrodes. The electrical stimuli were rectangular pulses (0-1 msec duration) delivered through a stimulus isolation unit at a frequency of I/sec. The dorsal roots of C7 and C8 were exposed by a laminectomy in the cervical region. The afferent input volleys were recorded triphasically by a silver ball electrode in contact with the dorsal root as far distal to its insertion into the spinal cord as possible together with an indifferent electrode placed on a nearby muscle. This location of the electrode on the dorsal root minimized interference with the recording of the peripheral afferent volleys by the cord dorsum potential. The responses from these afferent fibres were amplified and then either displayed on an oscilloscope or averaged 30-60 times by a signal averager-memory control unit (Ortec models 4623 and 4620) and plotted by a X-Y recorder (Model 7035B Hewlett Packard Company). All exposed nervous tissues were kept covered with warmed (36-380 C) paraffin oil in pools that were retained by skin flaps. Heating elements controlled by sensing elements were utilized to maintain the paraffin pool temperatures. The different fibre group components of the compound action potential of the SR, DR or R afferent volley were activated by stimulating these nerves with pulses of different intensities. The fibre groups (nomenclature according to Lloyd, 1943) in the afferent volleys were identified on the basis of the maximum conduction velocity (MCV) of the fibres in that group; the conduction velocity being calculated from the latency of the negative-going deflexion representing that group in the compound action potential and from the conduction distance which was determined at the conclusion of each experiment. The stimulus intensity was adjusted to activate as many fibres of a particular group as possible (as judged from the action potential record displayed on the oscilloscope) without stimulating fibres in the group with the next highest threshold for activation. Wherever possible, stimuli supramaximal for the fibres in each group were used. All stimulus intensities are expressed as multiples of that stimulus intensity which activated the lowest threshold fibres in the nerve (designated at 1-OT; where T denotes 'times threshold'). Group II fibres (MCV = 63-75 mfsec) in the SR were activated at 1-0T and reached maximum at 3-8-4-4T. As the group III (MCV = 13-40 m/sec) threshold occurred at 4-4-9T it was possible to maximally activate group II fibres without apparently including group III fibres in the afferent volleys in three of the four experiments in this series. In the remaining experiment a slightly submaximal stimulus had to be used in order to avoid Group III contamination of the afferent volley. Stimulus intensities of 3-8-4-IT were used, therefore, to activate group II afferent fibres. Although it was sometimes difficult to define full activation of group III fibres, an apparent maximum occurred at 15-19T. Stimuli of twice this intensity were therefore used to activate group III fibres. With the recording conditions used in these experiments, it was not possible to consistently define a group IV component in the afferent volley at high stimulus intensities. From the data of Collins & Randt (1961). it is apparent that this threshold could be at least 250T. In this present study, stimulation of the SR with stimuli of intensity 750-900T was assumed to activate group IV unmyelinated fibres. The lowest threshold group I afferent fibres (MCV = 85-124 m/sec) in the DR were maximally activated at 1-8-2-2T, while the threshold for excitation of group II fibres (MCV = 61-76 m/sec) was 2-1-2-4T. This slight overlap in responses, which is
744
W. J. MULLIN AND J. W. PHILLIS
agreement with the data of Holmqvist,Oscarsson & Uddenberg(1963) and Pompeiano & Swett (1963), necessitated the use of stimuli (2-2-3T) which were just or slightly submaximal for group I fibres in order to avoid contamination maximal of the volley by the activation of the lowest threshold fibres of group II. The threshold for the appearance of group III fibres (MCV = 20-30 m/sec) in theafferent volley occurred at stimulus intensities (5-6-8T) which were greater than those required for apparent maximal activation of group II fibres (4-2-4.7T). This finding is not in agreement with Pompeiano & Swett (1963) who indicated that the group II imum may occur above the threshold for activation of group III fibres (5-7T max in their experiments). In this present study, the stimulus intensities which were used to activate group II fibres (4.7-5.5T) were just below those required for threshold activation of group III afferents and were above the strength required for apparent maximal activation of group II fibres. Stimuli of 1-5-2 times the intensity required for maximal activation of the fibre group (11-15T) were used to include group III fibres in the afferent volley, while group IV fibres were assumed to be activated at 660-830T). When the R was stimulated at a site proximal to the junction of the SR and DR, the afferent volley generated was composed of peaks corresponding to an interof the responses to stimulation of the SR or DR alone described above. mingling Stimulus intensities used to activate the fibre groups in the radial nerve were: group I, 1'7-2-2T; groups I and II, 4'3-5-4T; groupsI+II+III, 22-29T; groups in
I+II+III+IV, 730-830T.
CoUection of actylcholine Both sensorimotor cortices were exposed by craniotomy and of the dura. Perspex cylinders (cups) with an inside diameter of -0 cm were then placed bilaterally over the forelimb receiving areas I. Leakage of fluid from the cups was prevented by a layer of silicone rubber affixed to the surface of the cup which was in contact with the cortical surface. When the cups were in place, the exposed cortical surfaces were covered with a generous amount of 4 % agar in physiological saline or the artificial cerebrospinal fluid of Merlis (1940). solution of The cortical surfaces inside the cups were bathed with a in artificial neostigmine methyl sulphate (Koch-Light Laboratories) 0-05 cerebrospinal fluid. The cortical tissues were exposed to the neostigmine for at least 30 min prior to the collection of the first sample and the solutions used to bathe the cortical tissues over this 30 period were removed and discarded. The cups were rinsed and refilled with 0-6-0'9 ml. of fresh neostigmine containing solution. Thereafter, the contents of the cups were removed and stored in glass tubes after 5 or 10 min of contact with the cortical tissues and the cups were immediately rinsed and refilled with fresh neostigmine-containing solution. The samples collected in this manner were analysed for ACh content in a biological assay on the hearts of the mollusc Mercenaria mercenaria. To ensure that the inhibitory activity of the samples on the molluscs hearts was due to ACh, they were frequently retested at the conclusion of the assay in the presence of the ACh antagonist benzoquinonium chloride (Mytolon). Samples which were not assayed soon after collection were stored frozen until the next day. From these tions of the concentration of ACh in each sample, the rate of liberation of ACh (in cm2 of cortex) from the sensorimotor cortices was determined. The techng/min.for niques the collection and estimation of ACh release from the cerebral cortex have been described in detail in previous publications (Phillis, 1968; Jhamadas, Phillis & Pinsky, 1971).
1
reflexion
warmed
mg/ml.
min
determina-
ACh RELEASE FROM CAT CEREBRAL CORTEX
745
RESULTS
The experiments conducted on the nine animals used in this study differed only in the nerve(s) that were stimulated, the sequence of activation of the fibre groups and, in one instance to be described later, the duration of the sample collection period. The typical effects on ACh release from the contralateral and ipsilateral sensorimotor cortices that were observed upon low frequency stimulation (1 Hz) of the DR and SR with pulses of different intensities are illustrated by the data from one animal presented in Figs. 1 and 2. The columns of the histograms compare the rates of ACh release (in ng/min. cm2 of cortex) over 10 min intervals when the DR or SR were stimulated throughout the collection period with pulses of the intensities indicated below the histograms (hatched columns) or when no stimuli were applied to the forelimb nerves (clear columns). As depicted in Fig. 1, the constant rate of ACh release from the contralateral cortex over the first eight 10 min collection periods indicates that stimulation of the DR with intensities that activated group I (2*OT) or groups I and II (4-7T) fibres had no effect on ACh release. When, however, the stimuli were applied at an intensity which supramaximally activated groups I, II and III afferents (30T), a marked increase in ACh release was observed. The rates of release of ACh over the two subsequent unstimulated collection periods were less than that evoked by stimulation but were greater than those over the two unstimulated collection periods preceding the stimulation. A higher rate of spontaneous release of ACh following a period of stimulation that was effective in enhancing ACh release was consistently observed in these experiments (see also Figs. 2 and 5 and Phillis, 1968). The rate of ACh release from the contralateral cortex that was observed during very high intensity stimulation of the DR (830T), which was assumed to have also activated group IV afferents, was, in terms of absolute rate, greater than that evoked by stimulation of the DR at 30T. With a few exceptions (e.g. the evoked release ofACh from the ipsilateral cortex in this experiment), this was a consistent finding which is further analysed in Table 2. The effects of DR stimulation on ACh release from the ipsilateral cortex were similar to those described for the contralateral cortex. Activation of Group I or Group I and II fibres did not alter release rates. Inclusion of Group III fibres in the afferent volley induced a marked increase in release which subsided slowly after the period of stimulation. The addition of Group IV afferents did not, in this particular experiment, cause any further enhancement of release. Low frequency stimulation of the SR was found to affect the rate of release of ACh from the sensorimotor cortices (Fig. 2) in a manner similar
W. J. MULLIN AND J. W. PHILLIS
746
Deep radial ACh release
3*0
Contralateral cortex
2-0 1.0 0
%_U 0 E
.S
nt
C
310 cpsilateral
cortex 2-0
10 10 Mnin collection periods Stimulus
2.0
4-7
I
I and II
30
830
I, fi
1. II IlIl and IV
T
intensity Activated fibre groups
and IlIl
Fig. 1. Rates of ACh release from the contralateral and ipsilateral sensorimotor cortices of a pentobarbitone anaesthetized cat measured either when the DR nerve was stimulated at 1/sec throughout the entire 10 min collection period (hatched columns) or when no stimuli were applied to the forelimb nerve (open columns). The afferent volleys (shown in the signal averager tracings at the bottom) were recorded from dorsal root C 7 and generated by stimuli of the intensities indicated below the histograms (where T denotes that all stimuli are multiples of that stimulus intensity which activated the very low threshold fibres in the nerve). Activated fibre groups (designated below the histograms) were identified on the basis of the maximum conduction velocity of the fibres in each group. Calibration = 1 msec.
ACh RELEASE FROM CAT CEREBRAL CORTEX
747
Superficial radial ACh release 3-0 Ad Contralateral cortex
2*0 1.0 K x L.
0
0
0 1 *3-0
A.
Ipsilateral cortex
2-0 1.0 0
10 min collection periods
Stimulus intensity Activated fibre groups
38
11
27'5
900
IH and IlIl
II, i
1T
rq i
1!
C-1
and IV
Fig. 2. The influence of afferent volleys in the SR nerve of the same animal used to obtain the data in Fig. 1 on the rates of ACh release from the contralateral and ipsilateral sensorimotor cortices. The afferent volley was recorded from dorsal root C8. For further explanation see Fig. 1.
to that observed following DR stimulation. Stimuli of an intensity (3-8T) that activated only group II fibres in the SR did not affect the rate of ACh release. Both contralateral and ipsilateral cortical ACh release were enhanced when group III (27-5T) or groups III and IV (900T) fibres were included in the afferent volley; the absolute rate of release being considerably greater at the higher intensity of stimulation.
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