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Release of acetylcholine from isolated human cortical slices: Inhibitory effect of norepinephrine and phenytoin
EXPERIMENTALNEUROLOGY
73, 144-153 (1981)
Release of Acetylcholine from Isolated Human Cortical Slices: Inhibitory Effect of Norepinephrine and Phenytoin E. S. VIZI AND E. P~ZTOR Institute of Experiment01 Medicine, Hungarian Academy of Sciences, H-1450 Budapest, POB 67. and National Institute of Neurosurgery, Budapest, Hungary Received July 7. 1980: revision received Jonuary 12, 1981 The release of acetylcholine from human isolated brain cortical slices was studied. Both K excess (47.3 mM) and inhibition of Na+-K+-activated ATPase by ouabain (2 X 10e5 M) significantly enhanced the release of acetylcholine. L-Norepinephrine (1 Om6M) and phenytoin (1 Om6M) reduced the release of acetylcholine evoked by ouabain. When the temperature was decreased to 15°C ouabain failed to increase the release of acetylcholine, although the resting release was not affected.
INTRODUCTION Only a limited number of publications (20, 27, 28, 36) have appeared reporting data on the distribution of acetylcholine (ACh) and choline acetylase activity in various parts of the nervous system in postmortem human brain tissue. In those experiments there was a postmortem delay, which is quite likely to produce a partial hydrolysis of ACh and partial or complete loss of enzyme activity. There is no report on the release and synthesis of ACh in human brain tissue removed during surgery. Because EEG seizure activity (11) and electroencephalographic arousal (3, 26) reportedly correlate with the release of ACh from the cerebral cortex, we studied the effect of norepinephrine (NE) and phenytoin on the release of ACh from fresh human brain cortical slices obtained from patients during surgery. From the data obtained it can be concluded that human and rat cerebral cortex may be strictly comparable with regard to the release and synthesis of ACh. Abbreviations: ACh-acetylcholine, tase.
NE-norepinephrine,
144 0014-4886/81/070144-10$02.00/O Copyright Q 1981 by Academic Press. Inc. All rights of reproduction in any form reserved.
ATPase-adenosine
triphospha-
ACh
RELEASE
FROM
HUMAN
CORTEX
145
METHODS Preparation. Human brain cortical samples were taken as follows: five neurosurgical patients presented with deep intracerebral tumors and referred to the National Institute of Neurosurgery were operated under general anesthesia (3:l N20 + 02). The intracerebral lesions did not extend to the surface of the cortex and were accessible for removal only after cortical incision. Slices weighing 10 to 40 mg and less than 0.5 mm thick were dissected from one or the other edge of the cortical incisions, placed immediately in cold Krebs solution, and transferred to the laboratory within 30 min. The collection of human brain specimens was with the written consent and approval of the Medical Ethics Committee of the Institute. The specimens were incubated at 37°C in 2 ml Krebs solution containing eserine sulfate (2 pg/ml) and bubbled with 5% CO, in oxygen. The Krebs solution had the following millimolar composition: NaCl, 113; KCl, 4.7; CaCl,, 2.5; KH2P04, 1.2; MgS04, 1.2; NaHC03, 25.0; and glucose, 11.5. Any changes in the ionic concentrations of the Krebs solution were compensated by equimolar changes in the NaCl content. Estimation of the Acetylcholine Content. To measure the ACh content, the tissue was homogenized in 1 ml 10% trichloroacetic acid. The ACh was extracted and assayed as described earlier (35). The content was expressed as picomoles gram-’ wet weight. In this study immediate homogenization in an ice-cold trichloroacetic acid (10%) solution was also carried out to find out how the ACh content ( 1100 f 2 10 pmol g-r; N = 3) of fresh human tissue changed after removal from the body. In agreement with animal studies [for references, see (20)], it was observed that the ACh content began to decline within minutes. In three experiments there was a 40 -t 8% (SE) reduction in content within 30 min. Assay ofAcetylcholine. Before the collection of the first sample of bathing fluid, the slices were allowed to equilibrate 60 min in the eserinized Krebs solution. Samples of bathing fluid were assayed for ACh at 20-min intervals. Acetylcholine, collected from the slices, was assayed as described by Paton and Vizi ( 16) using guinea pig ileum. The minimum concentration of ACh that could be assayed reproducibly was 0.5 rig/ml. Activity in the samples was antagonized by hyoscine (1O-6 M). Gel filtration was used to identify the substance obtained from human tissue and assayed on guinea pig ileum as acetylcholine. A Sephadex G10 column of 4 ml (20 cm in length and 5 mm in diameter) was prepared and the samples collected or extracted were eluted through the column. The void volume measured with dextran blue was 1.5 ml. When a sample had entered the top of the gel bed, the collection of fractions was begun.
146
VIZ1 AND PASZTOR
The eluate fractions of 0.5 ml were then tested on guinea pig ileum or their radioactivity was measured when [r4C]ACh was eluted and the elution profiles were compared. The elution pattern of “C-labeled ACh was measured by bioassay plus radioassay taking 0.5-ml fractions of eluate. Because the elution of both biological activity and label occurred in the same 2 to 3.5 ml where the endogenous substance appeared, it was concluded that the substance assayed on guinea pig ileum was ACh. Estimation of Acetylcholine Synthesis. The rate of acetylcholine synthesis was measured and calculated as described by Vizi et al. (34). The tissue content of ACh was measured before and after the experiments. The release of ACh between the two estimations was calculated. RESULTS Acetylcholine Content. Thirty minutes after the dissection in six different specimens, the content of ACh in the cerebral cortex was 596.4 f 135.5 pmol g-’ (SE), a value similar to that observed by Tower and Elliott (28). The spasmogenic substance extracted from the tissue and assayed behaved like ACh on a Sephadex G- 10 column, the elution profiles of [ “C]ACh, cold ACh, and the substance extracted from the tissue being identical. Release of Acetylcholine. Under resting conditions and in response to ouabain administration, there was a release of ACh from isolated cerebral cortex slices dissected from man. The gel filtration technique was used to identify the substance released as ACh. Under resting conditions 7.02 f 1.87 pmol g-’ min-’ of ACh was released from the slices. Ouabain (2 X 10m5 M) enhanced the release of ACh to 45.4 + 7.5 pmol g-’ min-‘, which is about sixfold increase. The difference is significant at 0.05 (paired t test; N = 4). A similar increase was obtained in isolated cerebral cortex of the rat (29). When the temperature was decreased to 15°C ouabain failed to increase the release of ACh (Table 1). The resting release, however, was not changed by cooling. Potassium excess greater than 23.5 mM also stimulated the release of ACh (Fig. 1). Tetrodotoxin ( 10m6M) failed to affect the release induced by K excess (47.3 mM), the output being 70.2 f 14.1 pmol g-’ min-’ in the absence and 66.5 f 5.2 pmol g-’ mitt-’ in the presence of tetrodotoxin. The rate of release at rest did not change for 2 h; later it tended to decline. The release induced by K excess proved to be calcium dependent (Figs. 2a and b). Because phenytoin, an anticonvulsant drug, was shown to depress discharges in neural structure (6, 19), its effect was also studied on ACh release. Phenytoin significantly reduced the release of ACh induced in the
ACh RELEASE
FROM HUMAN TABLE
147
CORTEX
1
Effect of Temperature on the Release of Acetylcholine from Isolated Human Cerebral Cortex Slices Acetylcholine release, pmol g-’ mitt-’ Condition
Nontreated
Ouabain (2 X lo-5 M)
37OC
7.02 + 1.87 (3Y 5.88 f 0.33 (3)
45.4 + 7.5 (3) 11.8 + 2.1 (3)
>0.05
1YC PC
(SE)
P”
o. 1
a Paired r test. b Number of experiments. ’ By t test for two means; collection period, 20 min.
presence of ouabain, but failed to affect release during the resting period (Table 2). A similar observation was made with rat cortical slices (Vizi, E. S., unpublished). In our earlier work with rat cortical slices, it was shown that NE reduced the release of ACh evoked by ouabain (29, 31) and the removal of the ceruleus-cortex nonadrenergic pathway resulted in an enhanced release of ACh from the cerebral cortex (31). NE at a concentration of 10m6 M reduced the release in human tissue as in the rat (Table 2). Phentolamine ( 10e6 M), an cY-adrenoceptor inhibitor, prevented
FIG. 1. The release of acetylcholine from isolated human cortical slices as a function of the external potassium concentration. The concentration of NaCl was reduced to maintain a normal isosmolarity when the concentration of KCI was enhanced. For experimental details, see Methods.
148
VIZ1 AND PASZTOR
b
FIG. 2. Ca dependence of K excess-evoked acetylcholine release from isolated human cortical slices. Average of two identical experiments. For experimental details, see Methods. (a) Effect of K excess (47.3 mM) on acetylcholine release in time function. (b) Effect of K excess (47.3 mM) in the absence of CaClz and presence of I mM EGTA. Note that the effect of K excess was markedly reduced when the Ca was removed from the medium.
the effect of NE on ouabain-evoked ACh release. The release was 43.1 + 8.9 pm01 g-’ min-’ in the presence of phentolamine and 20.3 & 1.1 pmol g-’ min-’ (N = 4) in its absence. Synthesis of Acetylcholine. The ACh content of the tissue was measured before and after the experiments. Because the ACh content of the slice did not decline (it might even have increased), the destruction of ACh by TABLE Effect of Norepinephrine
2
and Phenytoin on Acetylcholine Release from Isolated Human Cortical Slices ACh release, pmol g-’
(1) Control (10) (2) Norepinephrine, 10m6M (4) (3) Phenytoin, 10e6 M (4) (4) Ouabain, 2 X IO-’ M (4) (5) Ouabain, 2 X 10e5 M, + norepinephrine, 10e6 M (4) (6) Ouabain, 2 X IO-’ M, + norepinephrine, 1OW6M, + phentolamine, 10m6M (4) (7) Ouabain, 2 X lo-’ M, i- phenytoin, 10m6M (4) a Number of experiments.
min-’
9.4 8.4 9.1 45.4
+ + k k
(SE)
P
1.8 0.6 0.8 7.5
2:1, >O.l 3:1, s-o.1 41,
20.3 2 1.1
5:4, co.01
43.1 + 8.9
6:4, NS
18.5 + 1.4
7:4.
ACh
RELEASE
FROM
HUMAN
149
CORTEX
eserine was prevented, and the synthesis of ACh could he calculated from that in the bathing fluid (34). The synthesis rate was enhanced when the release was elevated: the increase was about fourfold (Table 3). The depletion of ACh stores by K-induced release was also compensated for by an increase in synthesis. DISCUSSION The purpose of this study was to provide data for the comparison of fresh human tissue with fresh animal tissue (Table 4). Human brain cortical slices like those dissected from rats synthesize and release ACh. The inhibition of Na+-K+-activated ATPase [cf. (30)] by ouabain resulted in an increase in ACh release. The release in response to K excess proved to be calcium dependent. The removal of calcium and addition of a calcium chelator, EGTA, prevented the effect of K excess. It is concluded that rat and human cortical tissue are comparable with regard to the release and synthesis of ACh. Membrane Adenosine Triphosphatase Inhibition and Acetylcholine Release. In this study it was shown that ouabain, an inhibitor of membrane
ATPase, is able to enhance the release of ACh from human cerebral cortex. It was reported that ouabain produced convulsions (8). In addition, it was suggested that primary dysfunction of the Na+-K’-activated ATPase plays TABLE Effect
of K Excess on the Synthesis Cerebral
3
Rate of Acetylcholine Cortex Slices’
in Isolated
Human
Acetylcholine Content,
Condition Resting K excess (47.3 rnM)
Before incubation 780.8
k 45.1 (3Y
651.0 (1)
pmol g-’ After incubation 906.3
+ 60.8 (3) 895.8 (1)
Gain
Release, pmol g-’ 2 h-’
Rate of synthesis, pmol g-’ 2 h-’
+125.5
1081.2 (3)
1026.7
+244.1
4640.2 (1)
4664.7
’ The acetylcholine content was measured before and after the experiments. Acetylcholine released in 2 h was collected and measured. Rate of acetylcholine synthesis was calculated as described by Vizi et al. (34). Note that the content of acetylcholine was not decreased after a 2-h incubation, although during that period about six times more acetylcholine was released than the content of the slice. ’ Number of experiments.
150
VIZ1 AND PASZTOR TABLE
4
Comparative Data on the Content, Release, and Synthesis of Acetylcholine in Human and Rat Cerebral Cortex Slices Human Content
0.78
Release Resting K excess (47.3 mM) Ouabain (20 Synthesis
PM)
nmol g-’
7-10 pm01 g-’ mitt-’ Tenfold increase (Ca dependent) Sixfold increase 1000 pmol g-’ 2 h-r (depends on the rate of release)
Rat” 2.39
nmol g-’
8-20 pmol g-’ mitt-’ Eightfold increase (Ca dependent) Sixfold increase 1508 pmol g-’ 2 h-’ (depends on the rate of release)
a Data from Vizi and Palkovits (33) and Vizi er of. (34).
a role in the pathomechanism of epilepsy: epileptogenic focal cortical areas showed abnormally low K uptake (8). In addition, some of the anticonvulsant drugs were shown to stimulate the activity of Na+-K+-activated ATPase (36). NE also stimulated the activity of membrane ATPase of the rat (2, 23, 30) and was able to inhibit the release of ACh from cerebral cortex evoked by ouabain. When the temperature was decreased to 15”C, ouabain failed to enhance the release of ACh, although the rate of resting release was not affected. This observation is in good agreement with the rat, where the Q10 for evoked release proved to be 2.1 but the tetrodotoxin-insensitive resting release was not affected by temperature. Andersen et al. ( 1) observed a reduced synaptic transmission during local cooling (21 “C), although an increased nerve action potential was seen. Sosenkov and Chirkov (25) demonstrated that cortical neurons cease firing when the tissue was exposed to hypothermia. In addition, Pasztor er al. (18) and Rosen (21) provided clear-cut evidence that cooling of rat cortex adjacent to the penicillin spike focus resulted in a decrease in both the frequency and the amplitude of the recorded spike (17). Therefore it is very likely that the reduction in ACh release during cooling might be at least partly responsible for the reduced epileptogenic activity observed by different authors and might be beneficial in the treatment of epilepsy. Inhibitory Effect of Norepinephrine and Phenytoin on Acetylcholine Release. From our data that ouabain-evoked ACh release can be reduced by NE or by phenytoin and since there is a relationship between EEG activity and ACh release ( 1 I), it is suggested that cortical epileptogenic
ACh RELEASE
FROM HUMAN
CORTEX
151
activity could result at least partly from the excessive release of ACh (22). This suggestion is supported by the findings that convulsants are able to decrease ACh concentrations (19) and enhance ACh release (4,5, 10) and that the accumulation of potassium in extracellular space-a condition also known to enhance ACh release-is involved in the generation of paroxysmal discharge (9, 24). Considerable interest has focused on the role of catecholamines in the control of seizures (12-l 5). In rats profound depletion of NE results in an enhanced susceptibility of the central nervous system ( 13,14) to convulsion, an observation which is in line with the clinical data that patients on reserpine treatment-i.e., with partly depleted NE stores-are much more sensitive to electroconvulsive therapy. In experiments with rats we succeeded in showing (3 1) that the reduction in noradrenergic outflow in the cerebral cortex resulted in an enhanced release of ACh. This indicates that the release of ACh due to cortical activity is continuously controlled by NE released from nerve endings derived from ceruleus-cortical neurons. It is suggested therefore, that the nonsynaptic (31, 32) control of ACh release by NE plays a critical role in the clinical epilepsies. It was shown that phenytoin is able to reduce repetitive firing and repetitive discharges of neurons characteristic of seizure states (7). It is likely therefore that the ability of phenytoin to reduce repetitive firing and ACh release contributes to the drug’s anticonvulsant effect. REFERENCES P., L. GJERSTAD, AND E. P~SZTOR. 1972. Effect of cooling on synaptic transmission through the cuneate nucleus. Acta Physiol. &and. 84: 433-447. 2. AD;\M-VIZI, V., M. ~RD&H, I. HORV~TH, J. SOMOGYI, AND E. S. VIZI. 1980. Effect of noradrenaline and vanadium on Na+-KC-activated ATPase in rat cerebral cortex synaptosomal preparation. J. New. Transm. 47: 53-60. 3. BEANI, L., C. BIANCHI, AND A. CASTELLIJCCI. 1974. Correlation of brain catecholamines with cortical acetylcholine outflow, behavior and electrocorticogram. Eur. J. Pharmocol. 1. ANDERSEN,
26: 63-68. 4.
BELESLIN, D. R., L. POLAK, AND D. H. SPROULI. 1965. The effect of leptazol and strychnine on the acetylcholine release from the cat brain. J. PhysioL (London) 181: 308-320.
CELESIA, G. G., AND H. H. JASPER. 1966. Acetylcholine released from cerebral cortex in relation to state of activation. Neurobiology (Minneupolis) 16: 1503-l 510. 6. DELDAGQ J. M. F., AND L. MIHAILKOVIC. 1956. Use of intracerebral electrodes to evaluate drugs that act on the central nervous system. Ann. N.Y. Acad. Sci. 64: 6445.
666.
ESPLIN, D. M. 1957. Effect of diphenylhydantoin on synaptic transmission in cat spinal cord and stellate ganglion. J. Pharmacol. Exp. Ther. 120: 301-323. 8. ESCUETA, A. V. 1974. The freezing lesion. II. Potassium transport within nerve terminals isolated from epileptogenic focus. Brain. Res. 78: 223-237. 7.
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AND
PkSZTOR
9. FERTZIGER, A. P., AND J. B. RANCK. 1970. Potassium accumulation in interstriatal space during epileptiform seizures. Exp. Neural. 26: 571-585. 10. HEMSWORTH, B. A., AND M. J. NEAL. 1968. The effect of central stimulant drugs on acetylcholine release from rat cerebral cortex. Br. J. Phurmacol. 34: 543. 11. GARDNER, C. R., AND R. A. WEBSTER. 1977. Convulsant-anticonvulsant interaction in seizure activity and cortical acetylcholine release. Eur. J. Pharmacol. 42: 247-256. 12. LOWELL, R. 1971. Some neurochemical aspects of convulsion. Pages 63-102 in A. LAJTHA, Ed., Handbook of Neurochemistry, Vol. 6. Plenum Press, New York. 13. MASON, S. T., AND M. E. CORCORAN. 1979. Noradrenaline and seizures. Science 203: 1265. 14. MASON, S. T., AND M. E. CORCORAN. 1979. Catecholamines and convulsion. Brain Res. 170: 497-507.
15. MAYNERT, E. W. 1969. The role of biochemical and neurochemical factors in the laboratory evaluation of antiepileptic drugs. Epilepsia 10: 145-162. 16. PATON, W. D. M., AND E. S. VIZI. 1969. The inhibitory action of noradrenaline and adrenaline on acetylcholine output by guinea-pig ileum longitudinal muscle strip. Br. 1. Pharmacol. 35: l&28. 17. P~SZTOR, E. 1976. Local cooling of the brain. Experimental and clinical studies. Pages 183-233 in K. L&K, Ed., Results in Neurochemistry and Neurobiology. Akademiai Kiadb, Budapest. 18. P&ZZTOR, E., J. TOMKA, AND G. DESK. 1965. The effect of local cerebral hypothermia on the electrocorticogram and on experimental epileptic activity. J. Neurosurg. 23: 4953. 19. PEDATA, F., A. MULAS, I. C. PEPEU, AND G. PEPEU. 1976. Changes in regional brain acetylcholine levels during drug-induced convulsions. Eur. J. Pharmacol. 40: 329-335. 20. RICHTER, J. A., E. K. PERRY, AND B. E. TOMLINSON. 1980. Acetylcholine and choline levels in post-mortem human brain tissue: preliminary observations in Alzheimer’s disease. Life Sci. 26: 1683-1689. 21. ROSEN, A. D. 1979. Cortical modification of an epileptogenic focus. Epilepsio 20: 387593. 22. SPEHLMANN, R., M. B. DANIEL& AND C. M. CHANG. 1971. Acetylcholine and the epileptiform activity of chronically isolated cortex. I. Marcoelectrode studies. Arch. Neural. 24: 401-408. 23. SCHAEFER, A,, A. SEREGI, AND M. K&L&. 1974. Ascorbicacid like effect of the soluble fraction of rat brain on adenosine triphosphates and its relation to catecholamines and chelating agents. Biochem. Pharmacol. 23: 2257-227 1. 24. S&J~N, G. G. 1980. Influence of potassium and neuroglia in the generation of seizures and their treatment. Pages 155-167 in G. H. GLASER, J. K. PENRY, AND D. M. WOODBURY, Eds., Antiepileptic Drugs: Mechanisms of Action. Raven Press, New York. 25. SOSENKOV, V. A., AND V. D. CHIRKOV. 1970. Electrical activity of neurons in the cat cortex during cooling. Neurojziologia 2: 59-63. 26. SZERB, J. C. 1967. Cortical acetylcholine release and electroencephalographic arousal. J. Physiol. [London) 192: 329-341. 27. TOWER, D. B., AND K. A. C. ELLIOTT. 1952. Activity of acetylcholine system in cerebral cortex of various anesthetized mammals. Am. J. Physiol. 168: 747-759. 28. TOWER, D. B., AND K. A. C. ELLIOTT. 1952. Activity of acetylcholine system in human epileptogenic focus. J. Appt. Physiol. 4: 669-676. 29. VIZI, E. S. 1972. Stimulation by inhibition of (Na+-KC-Mg’+)-activated ATPase of
ACh
30. 31. 32. 33. 34. 35. 36.
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CORTEX
153
acetylcholine release in cortical slices from rat brain. J. Physiol. (London) 226: 95117. VIZ], E. S. 1979. Presynaptic modulation of neurochemical transmission. Prog. Neurobiol. 12: 120-259. VIZI, E. S. 1980. Modulation of cortical release of acetylcholine by noradrenaline released from nerves arising from the rat locus coeruleus. Neuroscience 5: 2139-2144. VIZI, E. S. 1980. Non-synaptic modulation of neurochemical transmission: pharmacological implications. TIPS 1: 172-175. VIZI, E. S., AND M. PALKOVITS. 1978. Acetylcholine content in different regions of the rat brain. Brain Res. Bull. 3: 93-96. VIZI, E. S., P. ILL&, A. R&NAI, J. KNOLL. 1972. The effect of lithium on acetylcholine release and synthesis. Neuropharmacology 11: 521-530. QUASTEL, J. A. 1972. Acetylcholine distribution and synthesis in the central nervous system. Pages 431451 in K. A. S. ELLIO-~~, I. A. PAGE, AND J. H. QUASTEL, Eds., Neurochemistry. Charles C Thomas, Springfield, Illinois. WOODBURY, D. M. 1965. Mechanism of action of anticonvulsants Pages 647-651 in H. H. JASPER, A. A. WARD, JR., AND A. POPE, Eds., Basic Mechanisms of the Epilepsies. Little, Brown, Boston.
73, 144-153 (1981)
Release of Acetylcholine from Isolated Human Cortical Slices: Inhibitory Effect of Norepinephrine and Phenytoin E. S. VIZI AND E. P~ZTOR Institute of Experiment01 Medicine, Hungarian Academy of Sciences, H-1450 Budapest, POB 67. and National Institute of Neurosurgery, Budapest, Hungary Received July 7. 1980: revision received Jonuary 12, 1981 The release of acetylcholine from human isolated brain cortical slices was studied. Both K excess (47.3 mM) and inhibition of Na+-K+-activated ATPase by ouabain (2 X 10e5 M) significantly enhanced the release of acetylcholine. L-Norepinephrine (1 Om6M) and phenytoin (1 Om6M) reduced the release of acetylcholine evoked by ouabain. When the temperature was decreased to 15°C ouabain failed to increase the release of acetylcholine, although the resting release was not affected.
INTRODUCTION Only a limited number of publications (20, 27, 28, 36) have appeared reporting data on the distribution of acetylcholine (ACh) and choline acetylase activity in various parts of the nervous system in postmortem human brain tissue. In those experiments there was a postmortem delay, which is quite likely to produce a partial hydrolysis of ACh and partial or complete loss of enzyme activity. There is no report on the release and synthesis of ACh in human brain tissue removed during surgery. Because EEG seizure activity (11) and electroencephalographic arousal (3, 26) reportedly correlate with the release of ACh from the cerebral cortex, we studied the effect of norepinephrine (NE) and phenytoin on the release of ACh from fresh human brain cortical slices obtained from patients during surgery. From the data obtained it can be concluded that human and rat cerebral cortex may be strictly comparable with regard to the release and synthesis of ACh. Abbreviations: ACh-acetylcholine, tase.
NE-norepinephrine,
144 0014-4886/81/070144-10$02.00/O Copyright Q 1981 by Academic Press. Inc. All rights of reproduction in any form reserved.
ATPase-adenosine
triphospha-
ACh
RELEASE
FROM
HUMAN
CORTEX
145
METHODS Preparation. Human brain cortical samples were taken as follows: five neurosurgical patients presented with deep intracerebral tumors and referred to the National Institute of Neurosurgery were operated under general anesthesia (3:l N20 + 02). The intracerebral lesions did not extend to the surface of the cortex and were accessible for removal only after cortical incision. Slices weighing 10 to 40 mg and less than 0.5 mm thick were dissected from one or the other edge of the cortical incisions, placed immediately in cold Krebs solution, and transferred to the laboratory within 30 min. The collection of human brain specimens was with the written consent and approval of the Medical Ethics Committee of the Institute. The specimens were incubated at 37°C in 2 ml Krebs solution containing eserine sulfate (2 pg/ml) and bubbled with 5% CO, in oxygen. The Krebs solution had the following millimolar composition: NaCl, 113; KCl, 4.7; CaCl,, 2.5; KH2P04, 1.2; MgS04, 1.2; NaHC03, 25.0; and glucose, 11.5. Any changes in the ionic concentrations of the Krebs solution were compensated by equimolar changes in the NaCl content. Estimation of the Acetylcholine Content. To measure the ACh content, the tissue was homogenized in 1 ml 10% trichloroacetic acid. The ACh was extracted and assayed as described earlier (35). The content was expressed as picomoles gram-’ wet weight. In this study immediate homogenization in an ice-cold trichloroacetic acid (10%) solution was also carried out to find out how the ACh content ( 1100 f 2 10 pmol g-r; N = 3) of fresh human tissue changed after removal from the body. In agreement with animal studies [for references, see (20)], it was observed that the ACh content began to decline within minutes. In three experiments there was a 40 -t 8% (SE) reduction in content within 30 min. Assay ofAcetylcholine. Before the collection of the first sample of bathing fluid, the slices were allowed to equilibrate 60 min in the eserinized Krebs solution. Samples of bathing fluid were assayed for ACh at 20-min intervals. Acetylcholine, collected from the slices, was assayed as described by Paton and Vizi ( 16) using guinea pig ileum. The minimum concentration of ACh that could be assayed reproducibly was 0.5 rig/ml. Activity in the samples was antagonized by hyoscine (1O-6 M). Gel filtration was used to identify the substance obtained from human tissue and assayed on guinea pig ileum as acetylcholine. A Sephadex G10 column of 4 ml (20 cm in length and 5 mm in diameter) was prepared and the samples collected or extracted were eluted through the column. The void volume measured with dextran blue was 1.5 ml. When a sample had entered the top of the gel bed, the collection of fractions was begun.
146
VIZ1 AND PASZTOR
The eluate fractions of 0.5 ml were then tested on guinea pig ileum or their radioactivity was measured when [r4C]ACh was eluted and the elution profiles were compared. The elution pattern of “C-labeled ACh was measured by bioassay plus radioassay taking 0.5-ml fractions of eluate. Because the elution of both biological activity and label occurred in the same 2 to 3.5 ml where the endogenous substance appeared, it was concluded that the substance assayed on guinea pig ileum was ACh. Estimation of Acetylcholine Synthesis. The rate of acetylcholine synthesis was measured and calculated as described by Vizi et al. (34). The tissue content of ACh was measured before and after the experiments. The release of ACh between the two estimations was calculated. RESULTS Acetylcholine Content. Thirty minutes after the dissection in six different specimens, the content of ACh in the cerebral cortex was 596.4 f 135.5 pmol g-’ (SE), a value similar to that observed by Tower and Elliott (28). The spasmogenic substance extracted from the tissue and assayed behaved like ACh on a Sephadex G- 10 column, the elution profiles of [ “C]ACh, cold ACh, and the substance extracted from the tissue being identical. Release of Acetylcholine. Under resting conditions and in response to ouabain administration, there was a release of ACh from isolated cerebral cortex slices dissected from man. The gel filtration technique was used to identify the substance released as ACh. Under resting conditions 7.02 f 1.87 pmol g-’ min-’ of ACh was released from the slices. Ouabain (2 X 10m5 M) enhanced the release of ACh to 45.4 + 7.5 pmol g-’ min-‘, which is about sixfold increase. The difference is significant at 0.05 (paired t test; N = 4). A similar increase was obtained in isolated cerebral cortex of the rat (29). When the temperature was decreased to 15°C ouabain failed to increase the release of ACh (Table 1). The resting release, however, was not changed by cooling. Potassium excess greater than 23.5 mM also stimulated the release of ACh (Fig. 1). Tetrodotoxin ( 10m6M) failed to affect the release induced by K excess (47.3 mM), the output being 70.2 f 14.1 pmol g-’ min-’ in the absence and 66.5 f 5.2 pmol g-’ mitt-’ in the presence of tetrodotoxin. The rate of release at rest did not change for 2 h; later it tended to decline. The release induced by K excess proved to be calcium dependent (Figs. 2a and b). Because phenytoin, an anticonvulsant drug, was shown to depress discharges in neural structure (6, 19), its effect was also studied on ACh release. Phenytoin significantly reduced the release of ACh induced in the
ACh RELEASE
FROM HUMAN TABLE
147
CORTEX
1
Effect of Temperature on the Release of Acetylcholine from Isolated Human Cerebral Cortex Slices Acetylcholine release, pmol g-’ mitt-’ Condition
Nontreated
Ouabain (2 X lo-5 M)
37OC
7.02 + 1.87 (3Y 5.88 f 0.33 (3)
45.4 + 7.5 (3) 11.8 + 2.1 (3)
>0.05
1YC PC
(SE)
P”
a Paired r test. b Number of experiments. ’ By t test for two means; collection period, 20 min.
presence of ouabain, but failed to affect release during the resting period (Table 2). A similar observation was made with rat cortical slices (Vizi, E. S., unpublished). In our earlier work with rat cortical slices, it was shown that NE reduced the release of ACh evoked by ouabain (29, 31) and the removal of the ceruleus-cortex nonadrenergic pathway resulted in an enhanced release of ACh from the cerebral cortex (31). NE at a concentration of 10m6 M reduced the release in human tissue as in the rat (Table 2). Phentolamine ( 10e6 M), an cY-adrenoceptor inhibitor, prevented
FIG. 1. The release of acetylcholine from isolated human cortical slices as a function of the external potassium concentration. The concentration of NaCl was reduced to maintain a normal isosmolarity when the concentration of KCI was enhanced. For experimental details, see Methods.
148
VIZ1 AND PASZTOR
b
FIG. 2. Ca dependence of K excess-evoked acetylcholine release from isolated human cortical slices. Average of two identical experiments. For experimental details, see Methods. (a) Effect of K excess (47.3 mM) on acetylcholine release in time function. (b) Effect of K excess (47.3 mM) in the absence of CaClz and presence of I mM EGTA. Note that the effect of K excess was markedly reduced when the Ca was removed from the medium.
the effect of NE on ouabain-evoked ACh release. The release was 43.1 + 8.9 pm01 g-’ min-’ in the presence of phentolamine and 20.3 & 1.1 pmol g-’ min-’ (N = 4) in its absence. Synthesis of Acetylcholine. The ACh content of the tissue was measured before and after the experiments. Because the ACh content of the slice did not decline (it might even have increased), the destruction of ACh by TABLE Effect of Norepinephrine
2
and Phenytoin on Acetylcholine Release from Isolated Human Cortical Slices ACh release, pmol g-’
(1) Control (10) (2) Norepinephrine, 10m6M (4) (3) Phenytoin, 10e6 M (4) (4) Ouabain, 2 X IO-’ M (4) (5) Ouabain, 2 X 10e5 M, + norepinephrine, 10e6 M (4) (6) Ouabain, 2 X IO-’ M, + norepinephrine, 1OW6M, + phentolamine, 10m6M (4) (7) Ouabain, 2 X lo-’ M, i- phenytoin, 10m6M (4) a Number of experiments.
min-’
9.4 8.4 9.1 45.4
+ + k k
(SE)
P
1.8 0.6 0.8 7.5
2:1, >O.l 3:1, s-o.1 41,
20.3 2 1.1
5:4, co.01
43.1 + 8.9
6:4, NS
18.5 + 1.4
7:4.
ACh
RELEASE
FROM
HUMAN
149
CORTEX
eserine was prevented, and the synthesis of ACh could he calculated from that in the bathing fluid (34). The synthesis rate was enhanced when the release was elevated: the increase was about fourfold (Table 3). The depletion of ACh stores by K-induced release was also compensated for by an increase in synthesis. DISCUSSION The purpose of this study was to provide data for the comparison of fresh human tissue with fresh animal tissue (Table 4). Human brain cortical slices like those dissected from rats synthesize and release ACh. The inhibition of Na+-K+-activated ATPase [cf. (30)] by ouabain resulted in an increase in ACh release. The release in response to K excess proved to be calcium dependent. The removal of calcium and addition of a calcium chelator, EGTA, prevented the effect of K excess. It is concluded that rat and human cortical tissue are comparable with regard to the release and synthesis of ACh. Membrane Adenosine Triphosphatase Inhibition and Acetylcholine Release. In this study it was shown that ouabain, an inhibitor of membrane
ATPase, is able to enhance the release of ACh from human cerebral cortex. It was reported that ouabain produced convulsions (8). In addition, it was suggested that primary dysfunction of the Na+-K’-activated ATPase plays TABLE Effect
of K Excess on the Synthesis Cerebral
3
Rate of Acetylcholine Cortex Slices’
in Isolated
Human
Acetylcholine Content,
Condition Resting K excess (47.3 rnM)
Before incubation 780.8
k 45.1 (3Y
651.0 (1)
pmol g-’ After incubation 906.3
+ 60.8 (3) 895.8 (1)
Gain
Release, pmol g-’ 2 h-’
Rate of synthesis, pmol g-’ 2 h-’
+125.5
1081.2 (3)
1026.7
+244.1
4640.2 (1)
4664.7
’ The acetylcholine content was measured before and after the experiments. Acetylcholine released in 2 h was collected and measured. Rate of acetylcholine synthesis was calculated as described by Vizi et al. (34). Note that the content of acetylcholine was not decreased after a 2-h incubation, although during that period about six times more acetylcholine was released than the content of the slice. ’ Number of experiments.
150
VIZ1 AND PASZTOR TABLE
4
Comparative Data on the Content, Release, and Synthesis of Acetylcholine in Human and Rat Cerebral Cortex Slices Human Content
0.78
Release Resting K excess (47.3 mM) Ouabain (20 Synthesis
PM)
nmol g-’
7-10 pm01 g-’ mitt-’ Tenfold increase (Ca dependent) Sixfold increase 1000 pmol g-’ 2 h-r (depends on the rate of release)
Rat” 2.39
nmol g-’
8-20 pmol g-’ mitt-’ Eightfold increase (Ca dependent) Sixfold increase 1508 pmol g-’ 2 h-’ (depends on the rate of release)
a Data from Vizi and Palkovits (33) and Vizi er of. (34).
a role in the pathomechanism of epilepsy: epileptogenic focal cortical areas showed abnormally low K uptake (8). In addition, some of the anticonvulsant drugs were shown to stimulate the activity of Na+-K+-activated ATPase (36). NE also stimulated the activity of membrane ATPase of the rat (2, 23, 30) and was able to inhibit the release of ACh from cerebral cortex evoked by ouabain. When the temperature was decreased to 15”C, ouabain failed to enhance the release of ACh, although the rate of resting release was not affected. This observation is in good agreement with the rat, where the Q10 for evoked release proved to be 2.1 but the tetrodotoxin-insensitive resting release was not affected by temperature. Andersen et al. ( 1) observed a reduced synaptic transmission during local cooling (21 “C), although an increased nerve action potential was seen. Sosenkov and Chirkov (25) demonstrated that cortical neurons cease firing when the tissue was exposed to hypothermia. In addition, Pasztor er al. (18) and Rosen (21) provided clear-cut evidence that cooling of rat cortex adjacent to the penicillin spike focus resulted in a decrease in both the frequency and the amplitude of the recorded spike (17). Therefore it is very likely that the reduction in ACh release during cooling might be at least partly responsible for the reduced epileptogenic activity observed by different authors and might be beneficial in the treatment of epilepsy. Inhibitory Effect of Norepinephrine and Phenytoin on Acetylcholine Release. From our data that ouabain-evoked ACh release can be reduced by NE or by phenytoin and since there is a relationship between EEG activity and ACh release ( 1 I), it is suggested that cortical epileptogenic
ACh RELEASE
FROM HUMAN
CORTEX
151
activity could result at least partly from the excessive release of ACh (22). This suggestion is supported by the findings that convulsants are able to decrease ACh concentrations (19) and enhance ACh release (4,5, 10) and that the accumulation of potassium in extracellular space-a condition also known to enhance ACh release-is involved in the generation of paroxysmal discharge (9, 24). Considerable interest has focused on the role of catecholamines in the control of seizures (12-l 5). In rats profound depletion of NE results in an enhanced susceptibility of the central nervous system ( 13,14) to convulsion, an observation which is in line with the clinical data that patients on reserpine treatment-i.e., with partly depleted NE stores-are much more sensitive to electroconvulsive therapy. In experiments with rats we succeeded in showing (3 1) that the reduction in noradrenergic outflow in the cerebral cortex resulted in an enhanced release of ACh. This indicates that the release of ACh due to cortical activity is continuously controlled by NE released from nerve endings derived from ceruleus-cortical neurons. It is suggested therefore, that the nonsynaptic (31, 32) control of ACh release by NE plays a critical role in the clinical epilepsies. It was shown that phenytoin is able to reduce repetitive firing and repetitive discharges of neurons characteristic of seizure states (7). It is likely therefore that the ability of phenytoin to reduce repetitive firing and ACh release contributes to the drug’s anticonvulsant effect. REFERENCES P., L. GJERSTAD, AND E. P~SZTOR. 1972. Effect of cooling on synaptic transmission through the cuneate nucleus. Acta Physiol. &and. 84: 433-447. 2. AD;\M-VIZI, V., M. ~RD&H, I. HORV~TH, J. SOMOGYI, AND E. S. VIZI. 1980. Effect of noradrenaline and vanadium on Na+-KC-activated ATPase in rat cerebral cortex synaptosomal preparation. J. New. Transm. 47: 53-60. 3. BEANI, L., C. BIANCHI, AND A. CASTELLIJCCI. 1974. Correlation of brain catecholamines with cortical acetylcholine outflow, behavior and electrocorticogram. Eur. J. Pharmocol. 1. ANDERSEN,
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15. MAYNERT, E. W. 1969. The role of biochemical and neurochemical factors in the laboratory evaluation of antiepileptic drugs. Epilepsia 10: 145-162. 16. PATON, W. D. M., AND E. S. VIZI. 1969. The inhibitory action of noradrenaline and adrenaline on acetylcholine output by guinea-pig ileum longitudinal muscle strip. Br. 1. Pharmacol. 35: l&28. 17. P~SZTOR, E. 1976. Local cooling of the brain. Experimental and clinical studies. Pages 183-233 in K. L&K, Ed., Results in Neurochemistry and Neurobiology. Akademiai Kiadb, Budapest. 18. P&ZZTOR, E., J. TOMKA, AND G. DESK. 1965. The effect of local cerebral hypothermia on the electrocorticogram and on experimental epileptic activity. J. Neurosurg. 23: 4953. 19. PEDATA, F., A. MULAS, I. C. PEPEU, AND G. PEPEU. 1976. Changes in regional brain acetylcholine levels during drug-induced convulsions. Eur. J. Pharmacol. 40: 329-335. 20. RICHTER, J. A., E. K. PERRY, AND B. E. TOMLINSON. 1980. Acetylcholine and choline levels in post-mortem human brain tissue: preliminary observations in Alzheimer’s disease. Life Sci. 26: 1683-1689. 21. ROSEN, A. D. 1979. Cortical modification of an epileptogenic focus. Epilepsio 20: 387593. 22. SPEHLMANN, R., M. B. DANIEL& AND C. M. CHANG. 1971. Acetylcholine and the epileptiform activity of chronically isolated cortex. I. Marcoelectrode studies. Arch. Neural. 24: 401-408. 23. SCHAEFER, A,, A. SEREGI, AND M. K&L&. 1974. Ascorbicacid like effect of the soluble fraction of rat brain on adenosine triphosphates and its relation to catecholamines and chelating agents. Biochem. Pharmacol. 23: 2257-227 1. 24. S&J~N, G. G. 1980. Influence of potassium and neuroglia in the generation of seizures and their treatment. Pages 155-167 in G. H. GLASER, J. K. PENRY, AND D. M. WOODBURY, Eds., Antiepileptic Drugs: Mechanisms of Action. Raven Press, New York. 25. SOSENKOV, V. A., AND V. D. CHIRKOV. 1970. Electrical activity of neurons in the cat cortex during cooling. Neurojziologia 2: 59-63. 26. SZERB, J. C. 1967. Cortical acetylcholine release and electroencephalographic arousal. J. Physiol. [London) 192: 329-341. 27. TOWER, D. B., AND K. A. C. ELLIOTT. 1952. Activity of acetylcholine system in cerebral cortex of various anesthetized mammals. Am. J. Physiol. 168: 747-759. 28. TOWER, D. B., AND K. A. C. ELLIOTT. 1952. Activity of acetylcholine system in human epileptogenic focus. J. Appt. Physiol. 4: 669-676. 29. VIZI, E. S. 1972. Stimulation by inhibition of (Na+-KC-Mg’+)-activated ATPase of
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153
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