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Thein vivo synthesis and release of tritium labeled acetylcholine by cat cerebral cortex
Int.J. Neuropharmacol., 1968,7,351-358
PergamonPrrss.PrintedIII Ct. Britain.
THE IN VW0 SYNTHESIS LABELED ACETYLCHOLINE
AND RELEASE OF TRITIUM BY CAT CEREBRAL CORTEX*
LAWRENCEW. CHAKRIN~, F. E. SHIDEMAN and AMIZDE~ S. MAKKAZZI Department of Pharmacology, College of Medical Sciences, The University of Minnesota, Minneapolis, Minnesota 55455 (Accepted 20 November 1967) Summary-Experiments were conducted in which cerebral cortical acetylcholine in the cat was labeled by the prior intracortical injection of choline-methyl-Hz. A lucite chamber, which was sealed against the cortical surface at the site into which the tritiated choline had been injected 1 hr previously, formed the walls of a pool into which the labeled ester could be released
from the underlying cortical tissue. In the absence of cortical stimulation, the presence of labeled acetylcholine in the tissue underlying the collection chamber was confirmed by chromatographic analysis. Direct stimulation of the ipsilateral cortex and conducted neuronal impulses initiated by stimulation of the contralateral cortex evoked a significant release of the labeled ester from the underlying cortex into the chamber fluid. These findings provide further evidence in support of a transmitter function for acetylcholine in the central nervous system.
INTRODUCTION h‘ WAS established, some years ago, that acetylcholine appeared in the eflluent when the cerebral ventricles were perfused with a fluid containing an anticholinesterase agent (ADAM
et al., 1938; BHATTACHARYAand FELDBERG, 1958). The greatest contribution to the released acetylcholine was made by the caudate nucleus. It was later observed (FELDBERG and FLEISCHHAUER, 1965) that this cerebral output of acetylcholine was influenced by electrical stimulation and by a number of substances which markedly affected the activity of the central nervous system. Central nervous system depressants, including morphine and chloralose, have been
shown to inhibit nonspecifically the central release of acetylcholine and this inhibition presumably results in an elevation of acetylcholine levels in the brain. In contrast, convulsive drugs such as pentylenetetrazol and strychnine greatly increase the cerebral release of the ester with a concomitant decrease in central acetylcholine stores (FELDBERG and FLEISCHHAUER, 1965; BELESLINand POLAK, 1965; BELESLINet al., 1965). These drugs affect not only the release of acetylcholine from the caudate nucleus, as evidenced by ventricular perfusion experiments, but also from the cerebral cortex. Evidence for the cortical release of the ester was obtained by means of a technique introduced by MACINTOSH and OBORIN (1953).
These authors
demonstrated
that acetylcholine,
as shown by subsequent
bioassay
*This paper represents a portion of the research presented in a thesis submitted to the Graduate School of the University of Minnesota by Lawrence W. Chakrin in partial fulfillment of the requirements for the Ph.D. degree. The investigation was supported in part by a training grant (GM 1117) from the National Institutes of Health, U.S. Public Health Service. A preliminary report appeared in Fedn hoc. Fedn Am. Sots exp. Biol. 26: 295 (1967). t Present address: The Department of Biochemistry, The University of Cambridge, Cambridge, England. 351
352
L. W. CIIAKRIN, F. E. SHIDEMAN and A. S. MARRAZZI
procedures, could be collected from the pial surface by placing in contact with the exposed cerebral cortex of adult cats a small volume of saline solution containing eserine. MITCHELL(1963) studied the release of acetylcholine from the cerebral cortex and concluded that direct electrical stimulation of the cortex or excitation by transcallosal stimulation increased the rate of acetylcholine release. COLLIERand MITCHELL(1966) employed a technique identical with that described by MACINTOCH and OBORIN(1953) and demonstrated an increased release of cerebral acetylcholine during stimulation of the visual pathways in the rabbit. More recently, CELESIAand JASPER(1966) investigated the rate of liberation of acetylcholine from the cerebral cortex of adult cats while recording electrical activity during sleep, arousal, direct and transcallosal electrical stimulation, and epileptiform activation by pentylenetetrazol and strychnine. These authors confirmed the electricall-evoked release of acetylcholine by means of a cerebral collecting cup technique identical with that previously described (CHAKRINand SHIDEMAN,1968). It was of considerable interest, therefore, to study the spontaneous and evoked release of acetylcholine-methyl-H3 from a similar preparation, for as COLLIERand MITCHELL(1966) concluded, “ . . . the demonstration of release from central nervous structures, especially as a result of nerve stimulation is usually regarded as essential before the role of a central transmitter may be assigned to acetylcholine”.
METHODS Yreparution of animals for collection of labeled acetylcholine
Adult cats of either sex, weighing from 2 to 3 kg were anesthetized with pentobarbital sodium (35 mg/kg, ip.) and surgically prepared in the same manner as that previously described (CHAKRINand SHIDEMAN,1968). After a tracheotomy and craniotomy, the dura overlying the cerebral cortex was incised and reflected. Choline-methyl-H3-chloride, 77.4 me/m-mol (New England Nuclear Corp.) was injected intracortically in a dose of 20.0 PC in 200 ~1. of isotonic saline by means of a 50 ~1. Hamilton microsyringe, in five to six divided doses, in a selected region of the suprasylvian gyrus of one hemisphere. A lucite chamber (12 mm dia. and capable of holding 1.5 ml. of fluid) was gently placed on the cortical surface at the site into which the tritiated choline had been injected 1 hr previously. The chamber was firmly fixed in position with the aid of a three plane micromanipulator. No disturbance in pial blood flow to the cortex covered by the cup was observed as a result of this procedure. The lack of fluid leakage between the cup and the surface of the cortex was established by noting, under ultraviolet light, no escape of a solution of fluorescein sodium (2 %) from the cup to the surrounding cortical surface. The chamber was flushed and refilled, through a polyethylene sidearm, with a warm (37°C) isotonic saline solution containing physostigmine sulfate (2.1 x 10-4M) and atropine sulfate (7.2 x lo-‘M). This solution was stored in a glass reservoir, maintained at 37”C, and continuously aerated with a mixture of 95 % O2 and 5 % CO,. The reservoir was connected through a manually driven dual syringe infusion pump to the chamber. The chamber fluid (1.5 ml) was mixed approximately six times during the 30 min collection periods, and was removed subsequently by means of a 2.0 ml syringe connected to the upper polyethylene sidearm. Aliquots of this fluid were immediately chromatographed with authentic, unlabeled choline and acetylcholine in a manner to be described. Between collection periods, the cup was flushed and refilled with fresh solution. Ipsilateral stimulation was carried out by means of bipolar silver electrodes placed on the
The
in viva
synthesis and release of tritium labeled acetylcholine by cat cerebral cortex
353
cortex in juxtaposition to the chamber. Stimulation by conducted neuronal impulses was achieved by placing the electrodes in an identical position on the contralateral cortex. In both cases, continuous stimulation of supramaximal intensity (25 V, 1 msec, 20 pulses/ set, oscilloscopically monitored) was utilized. Chromatographic analysis and radioactivity estimations of chamber fluid
The fluid was removed from the chamber at appropriate 30 min intervals, and was transferred to glass vials cooled to and maintained at 4°C. Aliquots (20 ~1) of these samples were spotted on 1 in. strips of chromatography paper (Whatman No. 1) and chromatographed with authentic, unlabeled choline and acetylcholine for 16 hr at room temperature in a descending paper chromatography system. The solvent consisted of n-butanol: ethanol: acetic acid : water in the proportions 8 : 2: 1: 3 (MORLEYand SCHACHTER, 1963; FRIESENet al., 1964, 1965). The developed chromatograms were dried at room temperature and immediately sprayed with iodoplatinate reagent (WALLACHet al., 1967), Areas from the chromatograms, visualized by the iodoplatinate spray and having Rf values identical with choline and acetylcholine (CHAKRINand SHIDEMAN,1968), as well as the area between these, were cut out, placed in individual radioactivity counting vials, eluted and rendered colorless by the addition of 1.0 ml of dilute (7 %) ammonium hydroxide solution. Fifteen ml of dioxane-phosphor scintillation solution (formula No. 3, HAYES, 1963) was added to each sample, and the vials were allowed to cool to 4°C before estimation of radioactivity by means of a Packard liquid scintillation spectrometer. Internal standardization of the samples with a tritiated toluene standard indicated a mean counting efficiency of 13.5 %. Preparation of tissue samples for chromatographic analysis and radioactivity determinations
Immediately after the collection of chamber fluid, the cup was carefully removed and samples of approximately 1.0 g of the underlying cerebral tissue were removed and homogenized at 4°C in 2 volumes of acidified (HCI, pH4) isotonic saline containing physostigmine sulfate (2.1 x 10-lM). The homogenates were centrifuged at an average force of 1200 x g for 5 min after which 20 ~1 aliquots of the supernatant fluid were subjected to chromatographic analysis and, subsequently, radioactivity estimations as described previously.
RESULTS The chamber fluid which had been in contact for 30 min with the cortex in the region injected with tritiated choline 1 hr previously provided the following data. In a series of experiments with nine animals, in the absence of any electrical stimulation, when the chamber fluid was chromatographed and analyzed for its content of radioactivity, the results presented in Fig. 1 were obtained. Radioactivity equivalent to 6-l fl.2 x lo3 DPM/ml. (mean-j$E) of chamber fluid was found at the area of the chromatogram in which unlabeled, carrier acetylcholine was visualized. Part of this radioactivity may actually have been due to the much reported, spontaneously-released ester (MACINTOCH and OBORIN,1953 ; MITCHELL, 1963; COLLIERand MITCHELL, 1966). In contrast, fluid obtained from experiments on seven animals in which continuous stimulation of the ipsilateral cortex was carried out during the 30 min collection period, exhibited a definite peak of radioactivity at the Rf at which authentic carrier acetylcholine was visualized. The level of radioactivity at this F
354
L. W.
CHAKRIN,
m
130.0
F. E. SHIDEMANand A. S. MARRAZZI
STIMULATED CONTROL
m
90.6
bz I.? ‘n T 50.0
;
25.0
r, c1
20.0
(71
f
TT
0.0
L-
ACH R, 0.49
Level of Chromotogrom
FIG. 1. The evoked cerebral cortical release of labeled acetylcholine by electrical stimulation of the ipsilateral cerebral cortex 1 hr following the intracerebral injection of choline-methylHs-chloride. ACH and CHOL indicate the activities in the areas of the chromatograms associated with the unlabeled carriers acetylcholine (I+ 0.49) and choline (Rf 0.39), respectively, which were visualized with the iodoplatinate spray (see Methods). BTWN represents the activity in the area between ACH and CHOL.
Rf was calculated to be equivalent to 25.5 & 3.9 x 1O3DPM/ml (mean f SE) of the chamber fluid. Immediately after the collection of chamber fluid from animals whose cortex had not been stimulated, samples of the cerebral tissue underlying the chamber were removed and homogenized. The chromatographic analysis and estimation of radioactivity in the various areas of the chromatograms were carried out as described previously, and activities at other areas of the chromatogram were calculated as a percent of the activity found at the area of visualized, carrier choline. It is apparent from Fig. 2, that even though no clear cut evidence for the presence of labeled acetylcholine in the chamber fluid could be obtained in these experiments, a significant peak of radioactivity at the Rf of acetylcholine was observed in extracts of brain tissue from these animals. It must be concluded, therefore, that even though labeled acetylcholine could not be shown to be present in the chamber fluid in the absence of stimulation, it is present in the underlying cerebral tissue. With the evidence that ipsilateral electrical stimulation evoked a significant release of labeled acetylcholine from the cerebral cortex, the experimental protocol was modified to allow each animal to serve as its own control. Three successive 30 min collection periods, beginning at 1 hr after the cerebral injection of choline-methyl-H3, were made, the chamber fluid being collected at the end of each period. The data in Table 1 indicate that electrical
355
The in viva synthesis and release of tritium labeled acetylcholine by cat cerebral cortex
CHAMBER FLUID
I IO
TL%SUE HOMOGENATE
-
Level of Chromatogrom FIG. 2. The chromatographic distribution of tissue and released radioactivity in unstimulated cat cerebral cortex. Levels of chromatograms are designated in the same manner as in Fig. 1.
TABLE I. THE STIMULATIONOF
Animal
1
2 3 4 5 6 mean zt S.E.
EVOKED CEREBRAL CORTICAL RELEASE OF LABELED ACETYLCHOLINE FOLLOWING ELECTRICAL THEIPSILATERAL CEREBRAL CORTEX 1 hr AFTER THE INTRACEREBRALINJECTION OF CHOLINEMETHYL-H'
Control period 1 Activity at level of
Period of stimulation Activity at level of
Control period 2 Activity at level of
ACH
BTWN
CHOL
ACH
BTWN
CHOL
ACH
BTWN
CHOL
10.4 1.3 4.1 5.9 2.7 3.1
8.6 4.0 *:.:.
70.9 37.3 15.0 38.6 13.6 65.9
28.2 8.2 6.8 12.3 7.3 9.6
7.2 1.8 0.9 5.9 0.9 0.9
79.5 82.3 25.0 50.0 25.9 26.8
5.0 0.4 0.9 0.4 1.4 1.8
3.2 0.5 0.9 0.9 1.4 1.4
18.2 20.5 7.8 14.5 5.5 15.0
40.2 k9.8
12.1 zk3.3
2.9 rtl.1
48.3 rtll.1
1.6 *to.7
1.4 kO.4
13*6 f2.3
1i.i.
All periods were of 30 min duration. Levels of chromatograms are designated in the same manner as in Fig. 1. Activities at the various levels in the chromatograms are expressed as DPM x l0-3/ml. of chamber fluid. Difference between the concentrations of acetylcholine in the chamber fluid during control period 1 and period of stimulation is significant (p cO.01).
356
L. W. CHAKRIN,F. E. SHIDEMANand A. S.
MARRAZZI
stimulation of the ipsilateral cortex throughout the second period evoked a significant release of tritiated acetylcholine into the chamber fluid (12.153.3 x lo3 DPM/ml). This significant peak of radioactivity from the area of the chromatogram at which carrier acetylcholine was visualized was found only in samples collected during the period of stimulation, and it did not occur during either the period preceding or the period following stimulation. The activity ascribed to the presence of labeled acetylcholine during the period of stimulation was 2.6 times that observed in the period before stimulation. There was, during the period after cessation of electrical stimulation, the suggestion of a peak of radioactivity at the Rf of acetylcholine. Undoubtedly this reflects a delay in the diffusion of the transmitter released during stimulation into the chamber fluid. Certainly a portion of the activity in the chamber found at the Rfof acetylcholine during both control periods may also reflect the spontaneous release of the ester from the cortex. Studies similar to those just described were performed on another series of animals but the contralateral, rather than the ipsilateral, cerebral cortex was stimulated. The results of these experiments are presented in Table 2. A definite peak of radioactivity at the Rf TABLE 2. THE EVOKED CEREBRAL CORTICAL STIMULATIONOFTHECONTRALATERALCORTEX
Control period 1 Activity at level of
RELEASE OF LABELED ACETYLCHOLINE FOLLOWING ELECTRICAL 1 hr AFTERTHEINTRACEREBRALINJECTION OF CHOLINEMETHYL-H3
Period of stimulation Activity at level of
Control period 2 Activity at level of
Animal 1 2 3 4 5 6 Mean i S.E.
ACH
ACH
BTWN
CHOL
1.9 1.9 1.5 0.8 2.3 4.6
2.3 1.1 2.3 4.2 5.8 8.4
35.0 5.0 19.6 20.4 28.2 33.5
6.2 4.6 3.9 3.9 3,9 4.2
2.2 10.5
4.1 kl.0
24.0 f4.5
4.5 rto.4
BTWN
CHOL
ACH
BTWN
CHOL
1.9 0.8 0.8 0.8 1.5 1.9
13.8 5.4 IO.0 11.7 6.5 10.8
2.3 1.5 1.9 1.1 0.4 1.1
0.8 0.8 1.1 0.0 0.4 0.8
11.1 1.5 5.8 1.9 3.9 5.0
1.2 10.2
10.8 311.9
1.4 ho.3
0.7 ho.1
4.9 Al.4
All periods were of 30 min duration. Levels of chromatograms are designated in the same manner as in Fig. 1. Activities at the various levels are expressed as DPM x 10-3/ml of chamber fluid. Difference between the concentrations of acetylcholine in the chamber during control period 1 and period of stimulation is significant (p
at which authentic acetylcholine was visualized was found in samples of fluid collected during the period of stimulation. This peak of radioactivity (4.5 & 0.4 x lo3 DPM/ ml of chamber fluid) was significantly different from, and twice as great as the comparable radioactivity in samples collected prior to stimulation. As in the case of the ipsilateral stimulation, the suggestion of a peak of radioactivity at the Rf of authentic acetylcholine during the period after stimulation possibly reflects a delay in the diffusion of the labeled ester released during stimulation into the collecting chamber.
DISCUSSION
It has been suggested (CELESIA and JASPER, 1966; KRNJEVIC, 1966) that the cholinergic network may be part of the ascending activating system through which lower centres of the
The in
vivo
synthesis and release of tritium labeled acetylcholine
by cat cerebral cortex
357
brain influence the activity of the cerebral cortex. The actual demonstration of acetylcholine release from the intact cerebral cortex, as a result of nerve stimulation, was provided by ELLIOTTet al. (1950), and MACINTOSHand OBORIN(1953) with the introduction of the cerebral collecting cup technique. These authors indicated that this release of the ester was related to an increase in the electrical activity of the brain, thus supporting the observation that the amount of bound cortical acetylcholine is decreased in cortical activation (PEPEU and MANTEGAZZINI,1964). It was not until a decade later, however, that MITCHELL(1963) conclusively established the value of the collecting cup technique for the study of the cerebral release of acetylcholine. In addition to other studies, this investigator established the origin of the ester recovered from the cup. When he removed the underlying cortical tissue and placed the chamber over the glistening white matter, no release of acetylcholine could be detected over periods as long as 3 hr. COLLIERand MITCHELL(1966) reported that the amount of acetylcholine collected from the surface of the cerebral cortex of anesthetized rabbits was increased some two to five times when atropine sulfate was applied topically to the area of the cortex from which the collections were made. Similar results had been reported previously by MACINTOSH and OBORIN(1953) and MITCHELL(1963). It has been suggested (GIARMANand PEPEU, 1964) that atropine might exert its effects on acetylcholine release by occupying central nervous system receptor sites and so prevent the uptake of released acetylcholine. More recently, CELESIAand JASPER(1966) concluded that in order to explain the marked increase in the rate of liberation of free acetylcholine in the presence of atropine, it must be assumed that atropine blocks the uptake of acetylcholine by binding mechanisms which account to a large extent for its inactivation, independent of its hydrolysis by cholinesterase. This action of atropine has not been investigated but has been assumed, as in the experiments of COLLIERand MITCHELL(1966), to increase the amounts of acetylcholine collected. MITCHELL(1963) established that direct electrical stimulation of the cerebral cortex or excitation by transcallosal stimulation increased the rate of acetylcholine release from the cortex and that the amount released depended on the frequency of the stimulation. COLLIER and MITCHELL(1966) recently observed that direct electrical stimulation, at levels up to 50 V, evoked a large increase in release of cerebra1 acetylcholine (3.4 times the spontaneous release) from the ipsilateral visual cortex, and a smaller increase (1.7 times the spontaneous release) from the contralateral cortex. The evoked release of the ester was smallest at low frequency stimulation and increased to a maximum at a frequency of 20 pulses/set. The detailed nature of the evoked release of the ester from the contralateral cerebral cortex remains to be clarified. Considerable evidence exists to suggest that the transcallosal pathway might have a cholinergic component. MARRAZZI(1953, 1957) has, for example, repeatedly demonstrated since 1953 the acetylcholine induced stimulation of an intercortical (transcallosal) system when potentials were evoked in the optic cortex by submaximal electrical stimulation of a symmetrical point on the contralateral cortex. However, COLLIER and MITCHELL(1966) were able to section the transcallosal fibers without abolishing the evoked release of the ester following strong stimulation of the contralateral cortex. These results may be explained by the studies of RUTLEDGEand KENNEDY(1961) who showed that these homotopic areas are also connected by deeper pathways relaying through the brain stem reticulum. They found such a pathway responsible for a quite late component of the intercortically evoked cortical potential, which appeared with very intense contralateral cortical stimulation. Certainly the demonstration of release of labeled acetylcholine from the intact cerebral
358
L. W. CHAKRIN, F. E. SHIDEMANand A. S. MARRAZZI
cortex after electrical stimulation provides further evidence for the role of the ester as a central neurotransmitter. REFERENCES ADAM, H., MCKAIL, S. OBRADOR, S. and WILSON, W. (1938).
Acetylcholine
in the cerebra-spinal fluid. J.
Physiol., Lond. 93: 45 -46. BELESLIN,D. and POLAK, R. (1965). Depression by morphine and chloralose of acetylcholine release from the cat’s brain. J. Physiol., Lond. 177: 411419. BELESLIN,D., POLAK, R. and SPROULL,D. (1965). The effect of leptazol and strychnine on the acetylcholine release from the cat brain. J. Physiol., Lond. 181: 308-316. BHA~TACHARYA,B. and FELDBERG,W. (1958). Perfusion of cerebral ventricles: Effects of drugs on outflow from the cisterna and the aqueduct. Br. J. Pharmac. Chemother. 13: 156-162. CELESIA,G. and JASPER,H. (1966). Acetylcholine released from cerebral cortex in relation to state of activation. Neurology, Minneap. 16: 1053-1064. CHAKRIN, L. W. and SHIDEMAN,F. E. (1968). Synthesis of acetylcholine from labeled choline by brain.
ht. J. Neuropharmacol. 7: 337-349. COLLIER,B. and MITCHELL,J. (1966). The central release of acetylcholine during stimulation of the visual pathway. J. Physiol., Lond. 184: 239-254. ELLIOTT, K., SWANK, R. and HENDERSON,N. (1950). Effects of anesthetics and convulsants on acetylcholine content of brain. Am. J. Physiol. 162: 469474. FELDBERG,W. and FLEISCHHAUR,K. (1965). A new experimental approach to the physiology and pharmacology of the brain. Br. med. Bull. 21: 36-43. FRIESEN,A., KEMP, J. and WOODBURY, D. (1964). Identification of acetylcholine in sympathetic ganglia by chemical and physical methods. Science, N. Y. 145: 157-158. FRIESEN,A., KEMP, J. and WOODBURY, D. (1965). The chemical and physical identification of acetylcholine obtained from sympathetic ganglia. J. Pharmac. exp. Ther. 148: 312-319. GIARMAN, N. and F%PEIJ, G. (1964). The influence of centrally acting cholinolytic drugs on brain acetylcholine levels. Br. J. Pharmac. Chemother. 23: 123-130. HAYES, F. (1963). Solutes and solvents for liquid scintillation counting. Packard Tech. Bull. No. 1, Packard Inst. Co. Inc. KRNJEVIC,K. (1966). Chemical transmission in the central nervous system. Endeavour 25: 8-12. MACINTOSH,F. C. and OBORIN, P. E. (1953). Release of acetylcholine from intact cerebral cortex. Abstr. XIX Int. Physiol. Congr. 580-581. MARRAZZI, A. S. (1953). Some indications of cerebral humoral mechanisms. Science, N. Y. 118: 367-370. MARRAZZI, A. S. (1957). The effects of drugs on neurons and synapses. In: Brain Mechanisms and Drug Action. pp. 45-66. Thomas, Springfield, Ill. MITCHELL, J. F. (1963). The spontaneous and evoked release of acetylcholine from the cerebral cortex. J. Physiol., Lond. 165: 98-116. MORELY, J. and SCHACTER,M. (1963). Acetylcholine in non-nervous tissues of some Lepidoptera. J. Physiol., Lond. 168: 706715. PEPEU,G. and MANTEGAZZINI,P. (1964). Midbrain hemisection: Effect on cortical acetylcholine in the cat. Science, N. Y. 145: 1069-1070. RUTLEDGE,L. T. and KENNEDY,T. T. (1961). Brain-stem and cortical interactions in the interhemispheric delayed response. ExpI. Neurol. 4: 47w83. WALLACH, M., GOLDBERG,A. and SHIDEMAN,F. (1967). The synthesis of labeled acetylcholine by the isolated cat heart and its release by vagal stimulation. Znt. J. Neuropharmacol. 6: 317-323.
PergamonPrrss.PrintedIII Ct. Britain.
THE IN VW0 SYNTHESIS LABELED ACETYLCHOLINE
AND RELEASE OF TRITIUM BY CAT CEREBRAL CORTEX*
LAWRENCEW. CHAKRIN~, F. E. SHIDEMAN and AMIZDE~ S. MAKKAZZI Department of Pharmacology, College of Medical Sciences, The University of Minnesota, Minneapolis, Minnesota 55455 (Accepted 20 November 1967) Summary-Experiments were conducted in which cerebral cortical acetylcholine in the cat was labeled by the prior intracortical injection of choline-methyl-Hz. A lucite chamber, which was sealed against the cortical surface at the site into which the tritiated choline had been injected 1 hr previously, formed the walls of a pool into which the labeled ester could be released
from the underlying cortical tissue. In the absence of cortical stimulation, the presence of labeled acetylcholine in the tissue underlying the collection chamber was confirmed by chromatographic analysis. Direct stimulation of the ipsilateral cortex and conducted neuronal impulses initiated by stimulation of the contralateral cortex evoked a significant release of the labeled ester from the underlying cortex into the chamber fluid. These findings provide further evidence in support of a transmitter function for acetylcholine in the central nervous system.
INTRODUCTION h‘ WAS established, some years ago, that acetylcholine appeared in the eflluent when the cerebral ventricles were perfused with a fluid containing an anticholinesterase agent (ADAM
et al., 1938; BHATTACHARYAand FELDBERG, 1958). The greatest contribution to the released acetylcholine was made by the caudate nucleus. It was later observed (FELDBERG and FLEISCHHAUER, 1965) that this cerebral output of acetylcholine was influenced by electrical stimulation and by a number of substances which markedly affected the activity of the central nervous system. Central nervous system depressants, including morphine and chloralose, have been
shown to inhibit nonspecifically the central release of acetylcholine and this inhibition presumably results in an elevation of acetylcholine levels in the brain. In contrast, convulsive drugs such as pentylenetetrazol and strychnine greatly increase the cerebral release of the ester with a concomitant decrease in central acetylcholine stores (FELDBERG and FLEISCHHAUER, 1965; BELESLINand POLAK, 1965; BELESLINet al., 1965). These drugs affect not only the release of acetylcholine from the caudate nucleus, as evidenced by ventricular perfusion experiments, but also from the cerebral cortex. Evidence for the cortical release of the ester was obtained by means of a technique introduced by MACINTOSH and OBORIN (1953).
These authors
demonstrated
that acetylcholine,
as shown by subsequent
bioassay
*This paper represents a portion of the research presented in a thesis submitted to the Graduate School of the University of Minnesota by Lawrence W. Chakrin in partial fulfillment of the requirements for the Ph.D. degree. The investigation was supported in part by a training grant (GM 1117) from the National Institutes of Health, U.S. Public Health Service. A preliminary report appeared in Fedn hoc. Fedn Am. Sots exp. Biol. 26: 295 (1967). t Present address: The Department of Biochemistry, The University of Cambridge, Cambridge, England. 351
352
L. W. CIIAKRIN, F. E. SHIDEMAN and A. S. MARRAZZI
procedures, could be collected from the pial surface by placing in contact with the exposed cerebral cortex of adult cats a small volume of saline solution containing eserine. MITCHELL(1963) studied the release of acetylcholine from the cerebral cortex and concluded that direct electrical stimulation of the cortex or excitation by transcallosal stimulation increased the rate of acetylcholine release. COLLIERand MITCHELL(1966) employed a technique identical with that described by MACINTOCH and OBORIN(1953) and demonstrated an increased release of cerebral acetylcholine during stimulation of the visual pathways in the rabbit. More recently, CELESIAand JASPER(1966) investigated the rate of liberation of acetylcholine from the cerebral cortex of adult cats while recording electrical activity during sleep, arousal, direct and transcallosal electrical stimulation, and epileptiform activation by pentylenetetrazol and strychnine. These authors confirmed the electricall-evoked release of acetylcholine by means of a cerebral collecting cup technique identical with that previously described (CHAKRINand SHIDEMAN,1968). It was of considerable interest, therefore, to study the spontaneous and evoked release of acetylcholine-methyl-H3 from a similar preparation, for as COLLIERand MITCHELL(1966) concluded, “ . . . the demonstration of release from central nervous structures, especially as a result of nerve stimulation is usually regarded as essential before the role of a central transmitter may be assigned to acetylcholine”.
METHODS Yreparution of animals for collection of labeled acetylcholine
Adult cats of either sex, weighing from 2 to 3 kg were anesthetized with pentobarbital sodium (35 mg/kg, ip.) and surgically prepared in the same manner as that previously described (CHAKRINand SHIDEMAN,1968). After a tracheotomy and craniotomy, the dura overlying the cerebral cortex was incised and reflected. Choline-methyl-H3-chloride, 77.4 me/m-mol (New England Nuclear Corp.) was injected intracortically in a dose of 20.0 PC in 200 ~1. of isotonic saline by means of a 50 ~1. Hamilton microsyringe, in five to six divided doses, in a selected region of the suprasylvian gyrus of one hemisphere. A lucite chamber (12 mm dia. and capable of holding 1.5 ml. of fluid) was gently placed on the cortical surface at the site into which the tritiated choline had been injected 1 hr previously. The chamber was firmly fixed in position with the aid of a three plane micromanipulator. No disturbance in pial blood flow to the cortex covered by the cup was observed as a result of this procedure. The lack of fluid leakage between the cup and the surface of the cortex was established by noting, under ultraviolet light, no escape of a solution of fluorescein sodium (2 %) from the cup to the surrounding cortical surface. The chamber was flushed and refilled, through a polyethylene sidearm, with a warm (37°C) isotonic saline solution containing physostigmine sulfate (2.1 x 10-4M) and atropine sulfate (7.2 x lo-‘M). This solution was stored in a glass reservoir, maintained at 37”C, and continuously aerated with a mixture of 95 % O2 and 5 % CO,. The reservoir was connected through a manually driven dual syringe infusion pump to the chamber. The chamber fluid (1.5 ml) was mixed approximately six times during the 30 min collection periods, and was removed subsequently by means of a 2.0 ml syringe connected to the upper polyethylene sidearm. Aliquots of this fluid were immediately chromatographed with authentic, unlabeled choline and acetylcholine in a manner to be described. Between collection periods, the cup was flushed and refilled with fresh solution. Ipsilateral stimulation was carried out by means of bipolar silver electrodes placed on the
The
in viva
synthesis and release of tritium labeled acetylcholine by cat cerebral cortex
353
cortex in juxtaposition to the chamber. Stimulation by conducted neuronal impulses was achieved by placing the electrodes in an identical position on the contralateral cortex. In both cases, continuous stimulation of supramaximal intensity (25 V, 1 msec, 20 pulses/ set, oscilloscopically monitored) was utilized. Chromatographic analysis and radioactivity estimations of chamber fluid
The fluid was removed from the chamber at appropriate 30 min intervals, and was transferred to glass vials cooled to and maintained at 4°C. Aliquots (20 ~1) of these samples were spotted on 1 in. strips of chromatography paper (Whatman No. 1) and chromatographed with authentic, unlabeled choline and acetylcholine for 16 hr at room temperature in a descending paper chromatography system. The solvent consisted of n-butanol: ethanol: acetic acid : water in the proportions 8 : 2: 1: 3 (MORLEYand SCHACHTER, 1963; FRIESENet al., 1964, 1965). The developed chromatograms were dried at room temperature and immediately sprayed with iodoplatinate reagent (WALLACHet al., 1967), Areas from the chromatograms, visualized by the iodoplatinate spray and having Rf values identical with choline and acetylcholine (CHAKRINand SHIDEMAN,1968), as well as the area between these, were cut out, placed in individual radioactivity counting vials, eluted and rendered colorless by the addition of 1.0 ml of dilute (7 %) ammonium hydroxide solution. Fifteen ml of dioxane-phosphor scintillation solution (formula No. 3, HAYES, 1963) was added to each sample, and the vials were allowed to cool to 4°C before estimation of radioactivity by means of a Packard liquid scintillation spectrometer. Internal standardization of the samples with a tritiated toluene standard indicated a mean counting efficiency of 13.5 %. Preparation of tissue samples for chromatographic analysis and radioactivity determinations
Immediately after the collection of chamber fluid, the cup was carefully removed and samples of approximately 1.0 g of the underlying cerebral tissue were removed and homogenized at 4°C in 2 volumes of acidified (HCI, pH4) isotonic saline containing physostigmine sulfate (2.1 x 10-lM). The homogenates were centrifuged at an average force of 1200 x g for 5 min after which 20 ~1 aliquots of the supernatant fluid were subjected to chromatographic analysis and, subsequently, radioactivity estimations as described previously.
RESULTS The chamber fluid which had been in contact for 30 min with the cortex in the region injected with tritiated choline 1 hr previously provided the following data. In a series of experiments with nine animals, in the absence of any electrical stimulation, when the chamber fluid was chromatographed and analyzed for its content of radioactivity, the results presented in Fig. 1 were obtained. Radioactivity equivalent to 6-l fl.2 x lo3 DPM/ml. (mean-j$E) of chamber fluid was found at the area of the chromatogram in which unlabeled, carrier acetylcholine was visualized. Part of this radioactivity may actually have been due to the much reported, spontaneously-released ester (MACINTOCH and OBORIN,1953 ; MITCHELL, 1963; COLLIERand MITCHELL, 1966). In contrast, fluid obtained from experiments on seven animals in which continuous stimulation of the ipsilateral cortex was carried out during the 30 min collection period, exhibited a definite peak of radioactivity at the Rf at which authentic carrier acetylcholine was visualized. The level of radioactivity at this F
354
L. W.
CHAKRIN,
m
130.0
F. E. SHIDEMANand A. S. MARRAZZI
STIMULATED CONTROL
m
90.6
bz I.? ‘n T 50.0
;
25.0
r, c1
20.0
(71
f
TT
0.0
L-
ACH R, 0.49
Level of Chromotogrom
FIG. 1. The evoked cerebral cortical release of labeled acetylcholine by electrical stimulation of the ipsilateral cerebral cortex 1 hr following the intracerebral injection of choline-methylHs-chloride. ACH and CHOL indicate the activities in the areas of the chromatograms associated with the unlabeled carriers acetylcholine (I+ 0.49) and choline (Rf 0.39), respectively, which were visualized with the iodoplatinate spray (see Methods). BTWN represents the activity in the area between ACH and CHOL.
Rf was calculated to be equivalent to 25.5 & 3.9 x 1O3DPM/ml (mean f SE) of the chamber fluid. Immediately after the collection of chamber fluid from animals whose cortex had not been stimulated, samples of the cerebral tissue underlying the chamber were removed and homogenized. The chromatographic analysis and estimation of radioactivity in the various areas of the chromatograms were carried out as described previously, and activities at other areas of the chromatogram were calculated as a percent of the activity found at the area of visualized, carrier choline. It is apparent from Fig. 2, that even though no clear cut evidence for the presence of labeled acetylcholine in the chamber fluid could be obtained in these experiments, a significant peak of radioactivity at the Rf of acetylcholine was observed in extracts of brain tissue from these animals. It must be concluded, therefore, that even though labeled acetylcholine could not be shown to be present in the chamber fluid in the absence of stimulation, it is present in the underlying cerebral tissue. With the evidence that ipsilateral electrical stimulation evoked a significant release of labeled acetylcholine from the cerebral cortex, the experimental protocol was modified to allow each animal to serve as its own control. Three successive 30 min collection periods, beginning at 1 hr after the cerebral injection of choline-methyl-H3, were made, the chamber fluid being collected at the end of each period. The data in Table 1 indicate that electrical
355
The in viva synthesis and release of tritium labeled acetylcholine by cat cerebral cortex
CHAMBER FLUID
I IO
TL%SUE HOMOGENATE
-
Level of Chromatogrom FIG. 2. The chromatographic distribution of tissue and released radioactivity in unstimulated cat cerebral cortex. Levels of chromatograms are designated in the same manner as in Fig. 1.
TABLE I. THE STIMULATIONOF
Animal
1
2 3 4 5 6 mean zt S.E.
EVOKED CEREBRAL CORTICAL RELEASE OF LABELED ACETYLCHOLINE FOLLOWING ELECTRICAL THEIPSILATERAL CEREBRAL CORTEX 1 hr AFTER THE INTRACEREBRALINJECTION OF CHOLINEMETHYL-H'
Control period 1 Activity at level of
Period of stimulation Activity at level of
Control period 2 Activity at level of
ACH
BTWN
CHOL
ACH
BTWN
CHOL
ACH
BTWN
CHOL
10.4 1.3 4.1 5.9 2.7 3.1
8.6 4.0 *:.:.
70.9 37.3 15.0 38.6 13.6 65.9
28.2 8.2 6.8 12.3 7.3 9.6
7.2 1.8 0.9 5.9 0.9 0.9
79.5 82.3 25.0 50.0 25.9 26.8
5.0 0.4 0.9 0.4 1.4 1.8
3.2 0.5 0.9 0.9 1.4 1.4
18.2 20.5 7.8 14.5 5.5 15.0
40.2 k9.8
12.1 zk3.3
2.9 rtl.1
48.3 rtll.1
1.6 *to.7
1.4 kO.4
13*6 f2.3
1i.i.
All periods were of 30 min duration. Levels of chromatograms are designated in the same manner as in Fig. 1. Activities at the various levels in the chromatograms are expressed as DPM x l0-3/ml. of chamber fluid. Difference between the concentrations of acetylcholine in the chamber fluid during control period 1 and period of stimulation is significant (p cO.01).
356
L. W. CHAKRIN,F. E. SHIDEMANand A. S.
MARRAZZI
stimulation of the ipsilateral cortex throughout the second period evoked a significant release of tritiated acetylcholine into the chamber fluid (12.153.3 x lo3 DPM/ml). This significant peak of radioactivity from the area of the chromatogram at which carrier acetylcholine was visualized was found only in samples collected during the period of stimulation, and it did not occur during either the period preceding or the period following stimulation. The activity ascribed to the presence of labeled acetylcholine during the period of stimulation was 2.6 times that observed in the period before stimulation. There was, during the period after cessation of electrical stimulation, the suggestion of a peak of radioactivity at the Rf of acetylcholine. Undoubtedly this reflects a delay in the diffusion of the transmitter released during stimulation into the chamber fluid. Certainly a portion of the activity in the chamber found at the Rfof acetylcholine during both control periods may also reflect the spontaneous release of the ester from the cortex. Studies similar to those just described were performed on another series of animals but the contralateral, rather than the ipsilateral, cerebral cortex was stimulated. The results of these experiments are presented in Table 2. A definite peak of radioactivity at the Rf TABLE 2. THE EVOKED CEREBRAL CORTICAL STIMULATIONOFTHECONTRALATERALCORTEX
Control period 1 Activity at level of
RELEASE OF LABELED ACETYLCHOLINE FOLLOWING ELECTRICAL 1 hr AFTERTHEINTRACEREBRALINJECTION OF CHOLINEMETHYL-H3
Period of stimulation Activity at level of
Control period 2 Activity at level of
Animal 1 2 3 4 5 6 Mean i S.E.
ACH
ACH
BTWN
CHOL
1.9 1.9 1.5 0.8 2.3 4.6
2.3 1.1 2.3 4.2 5.8 8.4
35.0 5.0 19.6 20.4 28.2 33.5
6.2 4.6 3.9 3.9 3,9 4.2
2.2 10.5
4.1 kl.0
24.0 f4.5
4.5 rto.4
BTWN
CHOL
ACH
BTWN
CHOL
1.9 0.8 0.8 0.8 1.5 1.9
13.8 5.4 IO.0 11.7 6.5 10.8
2.3 1.5 1.9 1.1 0.4 1.1
0.8 0.8 1.1 0.0 0.4 0.8
11.1 1.5 5.8 1.9 3.9 5.0
1.2 10.2
10.8 311.9
1.4 ho.3
0.7 ho.1
4.9 Al.4
All periods were of 30 min duration. Levels of chromatograms are designated in the same manner as in Fig. 1. Activities at the various levels are expressed as DPM x 10-3/ml of chamber fluid. Difference between the concentrations of acetylcholine in the chamber during control period 1 and period of stimulation is significant (p
at which authentic acetylcholine was visualized was found in samples of fluid collected during the period of stimulation. This peak of radioactivity (4.5 & 0.4 x lo3 DPM/ ml of chamber fluid) was significantly different from, and twice as great as the comparable radioactivity in samples collected prior to stimulation. As in the case of the ipsilateral stimulation, the suggestion of a peak of radioactivity at the Rf of authentic acetylcholine during the period after stimulation possibly reflects a delay in the diffusion of the labeled ester released during stimulation into the collecting chamber.
DISCUSSION
It has been suggested (CELESIA and JASPER, 1966; KRNJEVIC, 1966) that the cholinergic network may be part of the ascending activating system through which lower centres of the
The in
vivo
synthesis and release of tritium labeled acetylcholine
by cat cerebral cortex
357
brain influence the activity of the cerebral cortex. The actual demonstration of acetylcholine release from the intact cerebral cortex, as a result of nerve stimulation, was provided by ELLIOTTet al. (1950), and MACINTOSHand OBORIN(1953) with the introduction of the cerebral collecting cup technique. These authors indicated that this release of the ester was related to an increase in the electrical activity of the brain, thus supporting the observation that the amount of bound cortical acetylcholine is decreased in cortical activation (PEPEU and MANTEGAZZINI,1964). It was not until a decade later, however, that MITCHELL(1963) conclusively established the value of the collecting cup technique for the study of the cerebral release of acetylcholine. In addition to other studies, this investigator established the origin of the ester recovered from the cup. When he removed the underlying cortical tissue and placed the chamber over the glistening white matter, no release of acetylcholine could be detected over periods as long as 3 hr. COLLIERand MITCHELL(1966) reported that the amount of acetylcholine collected from the surface of the cerebral cortex of anesthetized rabbits was increased some two to five times when atropine sulfate was applied topically to the area of the cortex from which the collections were made. Similar results had been reported previously by MACINTOSH and OBORIN(1953) and MITCHELL(1963). It has been suggested (GIARMANand PEPEU, 1964) that atropine might exert its effects on acetylcholine release by occupying central nervous system receptor sites and so prevent the uptake of released acetylcholine. More recently, CELESIAand JASPER(1966) concluded that in order to explain the marked increase in the rate of liberation of free acetylcholine in the presence of atropine, it must be assumed that atropine blocks the uptake of acetylcholine by binding mechanisms which account to a large extent for its inactivation, independent of its hydrolysis by cholinesterase. This action of atropine has not been investigated but has been assumed, as in the experiments of COLLIERand MITCHELL(1966), to increase the amounts of acetylcholine collected. MITCHELL(1963) established that direct electrical stimulation of the cerebral cortex or excitation by transcallosal stimulation increased the rate of acetylcholine release from the cortex and that the amount released depended on the frequency of the stimulation. COLLIER and MITCHELL(1966) recently observed that direct electrical stimulation, at levels up to 50 V, evoked a large increase in release of cerebra1 acetylcholine (3.4 times the spontaneous release) from the ipsilateral visual cortex, and a smaller increase (1.7 times the spontaneous release) from the contralateral cortex. The evoked release of the ester was smallest at low frequency stimulation and increased to a maximum at a frequency of 20 pulses/set. The detailed nature of the evoked release of the ester from the contralateral cerebral cortex remains to be clarified. Considerable evidence exists to suggest that the transcallosal pathway might have a cholinergic component. MARRAZZI(1953, 1957) has, for example, repeatedly demonstrated since 1953 the acetylcholine induced stimulation of an intercortical (transcallosal) system when potentials were evoked in the optic cortex by submaximal electrical stimulation of a symmetrical point on the contralateral cortex. However, COLLIER and MITCHELL(1966) were able to section the transcallosal fibers without abolishing the evoked release of the ester following strong stimulation of the contralateral cortex. These results may be explained by the studies of RUTLEDGEand KENNEDY(1961) who showed that these homotopic areas are also connected by deeper pathways relaying through the brain stem reticulum. They found such a pathway responsible for a quite late component of the intercortically evoked cortical potential, which appeared with very intense contralateral cortical stimulation. Certainly the demonstration of release of labeled acetylcholine from the intact cerebral
358
L. W. CHAKRIN, F. E. SHIDEMANand A. S. MARRAZZI
cortex after electrical stimulation provides further evidence for the role of the ester as a central neurotransmitter. REFERENCES ADAM, H., MCKAIL, S. OBRADOR, S. and WILSON, W. (1938).
Acetylcholine
in the cerebra-spinal fluid. J.
Physiol., Lond. 93: 45 -46. BELESLIN,D. and POLAK, R. (1965). Depression by morphine and chloralose of acetylcholine release from the cat’s brain. J. Physiol., Lond. 177: 411419. BELESLIN,D., POLAK, R. and SPROULL,D. (1965). The effect of leptazol and strychnine on the acetylcholine release from the cat brain. J. Physiol., Lond. 181: 308-316. BHA~TACHARYA,B. and FELDBERG,W. (1958). Perfusion of cerebral ventricles: Effects of drugs on outflow from the cisterna and the aqueduct. Br. J. Pharmac. Chemother. 13: 156-162. CELESIA,G. and JASPER,H. (1966). Acetylcholine released from cerebral cortex in relation to state of activation. Neurology, Minneap. 16: 1053-1064. CHAKRIN, L. W. and SHIDEMAN,F. E. (1968). Synthesis of acetylcholine from labeled choline by brain.
ht. J. Neuropharmacol. 7: 337-349. COLLIER,B. and MITCHELL,J. (1966). The central release of acetylcholine during stimulation of the visual pathway. J. Physiol., Lond. 184: 239-254. ELLIOTT, K., SWANK, R. and HENDERSON,N. (1950). Effects of anesthetics and convulsants on acetylcholine content of brain. Am. J. Physiol. 162: 469474. FELDBERG,W. and FLEISCHHAUR,K. (1965). A new experimental approach to the physiology and pharmacology of the brain. Br. med. Bull. 21: 36-43. FRIESEN,A., KEMP, J. and WOODBURY, D. (1964). Identification of acetylcholine in sympathetic ganglia by chemical and physical methods. Science, N. Y. 145: 157-158. FRIESEN,A., KEMP, J. and WOODBURY, D. (1965). The chemical and physical identification of acetylcholine obtained from sympathetic ganglia. J. Pharmac. exp. Ther. 148: 312-319. GIARMAN, N. and F%PEIJ, G. (1964). The influence of centrally acting cholinolytic drugs on brain acetylcholine levels. Br. J. Pharmac. Chemother. 23: 123-130. HAYES, F. (1963). Solutes and solvents for liquid scintillation counting. Packard Tech. Bull. No. 1, Packard Inst. Co. Inc. KRNJEVIC,K. (1966). Chemical transmission in the central nervous system. Endeavour 25: 8-12. MACINTOSH,F. C. and OBORIN, P. E. (1953). Release of acetylcholine from intact cerebral cortex. Abstr. XIX Int. Physiol. Congr. 580-581. MARRAZZI, A. S. (1953). Some indications of cerebral humoral mechanisms. Science, N. Y. 118: 367-370. MARRAZZI, A. S. (1957). The effects of drugs on neurons and synapses. In: Brain Mechanisms and Drug Action. pp. 45-66. Thomas, Springfield, Ill. MITCHELL, J. F. (1963). The spontaneous and evoked release of acetylcholine from the cerebral cortex. J. Physiol., Lond. 165: 98-116. MORELY, J. and SCHACTER,M. (1963). Acetylcholine in non-nervous tissues of some Lepidoptera. J. Physiol., Lond. 168: 706715. PEPEU,G. and MANTEGAZZINI,P. (1964). Midbrain hemisection: Effect on cortical acetylcholine in the cat. Science, N. Y. 145: 1069-1070. RUTLEDGE,L. T. and KENNEDY,T. T. (1961). Brain-stem and cortical interactions in the interhemispheric delayed response. ExpI. Neurol. 4: 47w83. WALLACH, M., GOLDBERG,A. and SHIDEMAN,F. (1967). The synthesis of labeled acetylcholine by the isolated cat heart and its release by vagal stimulation. Znt. J. Neuropharmacol. 6: 317-323.