- Email: [email protected]
The effect of chlorpromazine on the concentration of acetylcholinesterase in the cerebrospinal fluid of rabbits
THE EFFECT OF CHLORPROMAZINE ON THE CONCENTRATION OF ACETYLCHOLINESTERASE IN THE C~REBROSPINAL FLUID OF RABBITS SUSANA.
GREENFIELD*,
I. W. CHt.rBBtand A. D.
University Department of Pharmacology,
SMITH
South Parks Road, Oxford, OX1 3QT, U.K.
(Accepted 2 Augusr 1978)
Summery-~oliowing administration of chlorpromazine (4mg/kg iv.). there was an increase of up to 5-fold in the concentration of acetylcholinesterase in rabbit cerebraspinal fluid sampled from the cisterna magna. but not in cerebrospinal fluid from a fateral ventricle. The pattern of multiple molecular forms and specific activity of acetylcholinesterase in cerebrospinal fluid did not resemble that of blood plasma and so a breakdown of the blood-cerebrospinal fluid barrier can be excluded. The concentration of lactate dehydrogcnase in cisternal cerebrospinal fluid did not change, hence a non-specific increase in permeability of eel1 membranes seems unlikely. Prior administratjon of atropine sulphate (3 mg/kg i.v.), but not the same dose of atropine methylnitrate. prevented the effect of chlorpromazine. The possibility that the rise in the concentration of acetyicholinesterase was the result of increased activity of central neurons containing this enzyme is discussed.
It has been proposed that some of the acetylcholinesterase (AChE) found in cerebrospinal fluid (CSF) is released from neurons as a result of their activity (Chubb, Goodman and Smith, 1976). This hypothesis filllowed from the observation that the concentration cf AChE in rabbit cerebrospinal fluid (CSF) increased after procedures (sensory nerve stimulation) known to evoke the release of a~tylcholine from sufaces of the brain. Furthermore, stimulation via electrodes implanted in the rabbit caudate nucleus also led to an increase in the concentration of AChE in CSF (Greenfield and Smith, 1976). The caudate nucleus has a very high content of AChE (Silver, 1974) and since it borders on the lateral ventricle it is a possible site of origin of AChE in the CSF. The release of acetylcholine from the AChEcontaining cholinergic interneurons in the caudate nucleus is markedly increased following peripheral administration of the antipsychotic phenothiazine, c?lorpromazine (Stadler, Lloyd, Gadea-Ciria and Bartholini, 1973; Trabucchi, Cheney, Racagni and Costa, 1974; McGeer, Grewaal and McGeer, 1974). The purpose of the studies to be described was to see whether AChE is released into the CSF following administration of chlorpromazine.
solution in 0.9% (w/v) NaCI) administered via a marginal ear vein (approximately 6mI/kg). The trachea was cannulated and the head placed in a head holder (C. F. Palmer, Ltd, High Wycombe, Bucks). The dura covering the cisterna magna was then exposed. A hypodermic needle (23 G 0.63 mm dia.) was inserted into the dura (see Fig. 1) and CSF was continuously sampled by slow suction of a peristaltic pump, at a rate between 4 and 6hl/min, which is beIow the normal rate of production in the rabbit (Davson, 1967). The rectal temperature was maintained at 37 k 1°C by means of a warm blanket and lamp. Sanlpling
METHODS S~~rn~~ing of CSF from
from
the lateral
ventricle
the cisterira manna
Zealand White male rabbits (3.0-3.5 kg body weight) were anaesthetized with urethane (25% (w/v) New
*Present address: University Laboratory of Physiology, Oxford, U.K. + Present address: Department of Physiology, Flinders University Medical School, Bedford Park, S. Australia. N.P.182-e
ofCSF
Rabbits were anaesthetized with sodium pentobarbitone (Nembutal) (30 mg/kg i.v.1 and ether. The head was then fixed in a stereotaxic apparatus so that the coronal suture bregma was 1.5 mm higher than lambda. A stainless-steel cannula 35 mm in length was permanently implanted into the ventricle, using dental acrylic, at co-ordinates AP 0, L 3, V 5. The animal was then left to recover for a minimum of 3 days. Rabbits implanted in this way were anaesthetized with urethane. as described above, prior to sampling CSF. CSF was removed at a rate of -11 per min. The sites of impIantation were verified histologically.
Acetylcholinesterase activity was determined using acetylthiocholine as substrate at pH 7.0. The method is a modification (Chubb and Smith, 1975a) of that described by Ellman, Courtney, Andres and Featherstone (1961). One unit (U) of enzyme activity represents the hydrolysis of f pmol substrate per minute at 30°C.
127
SUSAN A. GREENFIELD.I. W. CHUBB and
128
Fig. 1. Position
of rabbit’s head in head-holder. The needle (N) is inserted just above and to a depth such that the entire bevel is covered by the dura.
Lactate dehydrogenase activity was measured by the method of Wroblewski and La Due (195.5) and expressed in international units. Total protein was estimated according to the method of Lowry, Rosebough, Farr and Randall (1951) with bovine serum albumin as standard. Electrophoresis in 6% (w/v) polyacrylamide gels was carried out by the method described by Chubb and Smith (1975a). Drug
administration
Chlorpromazine (Largactil) and promethazine (Phenergan) were used as supplied in injection form. The chlorpromazine vehicle was made up immediately before use, the composition being 0.1% (w/v) Na sulphite, 0.75% (w/v) Na metabisulphite, 0.4% (w/v) NaCl and 0.1% (w/v) Na citrate in distilled HzO. All drugs were administered via a marginal ear vein. RESULTS
Acetylcholinesterase magna
and lateral
A. D. SMITH
in
CSF
sampled
from
cisterna
ventricle
The concentration of AChE in cistemal CSF was often, but not always, high for the first 3G60min.
the Atlas vertebra
following the start of sampling (e.g. Fig. 2a). This has been observed in previous experiments (Chubb et al., 1976) and it was suggested that the initial high values may have been a consequence of activation of sensory nerves during surgery. After this initial period, the concentration of AChE in CSF remained fairly constant and a mean value of 35.0 ) 3.55 (S.E.) mu/ml was obtained (n = 20). In any individual rabbit, the “resting” concentration of AChE varied by no more than 10 mu/ml on either side of the mean concentration for that rabbit. The mean concentration of AChE in CSF withdrawn from the lateral ventricle was 15.5 k 5.0 (SE.) in four rabbits. This is significantly (P < 0.01) lower than the mean concentration in cisternal CSF. EfSect of chlorpromazine cholinesterase
on the concentration
in cerebrospinal
of acetyl-
jluid
chlorpromazine Following administration of (4 mg/kg i.v.) to seven rabbits there was no significant change in the concentration of AChE in CSF withdrawn continuously from the lateral ventricle (Fig. 2b). However, when CSF was withdrawn from the cisterna magna, there was a marked increase in
90r
E i 2
~~_i&Jl_a
u
1
0
I 200 min
-b . 400
Fig, 2. Experiments demonstrating the effect of chlorpromazine (4mg/kg i.v.) on the concentration of AChE in (a) cisternal; (b) ventricular CSF collected from the same rabbit. The vertical axis shows AChE concentration in mu/ml of CSF where a mU is one nanomole of substrate hydrolysed in one minute. The horizontal axis gives the time in minutes: the horizontal bars represent the period of collection of each fraction.
Chlorpromazine
Table 1. Effect of chlorpromazine Dose of CPZ (mg/kg i.v.)
on AChE in cisternal cerebrospinal
Pre-treatment Average AChE Cont. (mu/ml)
Increase “/, after CPZ
30 50 30 20 60 30 20 30
200 200 100 400 100 300 300 300
4 (AM) 4 4 (AM) 4 (AM) 4 4 3 4
AM indicates atropine methylnitrate absolute additional amount of AChE average concentration of AChE is that was calculated for the oeriod during “resting” level.
Efect of atropine on the chlorpromazine-induced crease in AChE concentration
-
in-
of atropine sulphate (4 mg/kg i.v.) no longer
Table 2. Effect of chlorpromazine
Protein @g/ml) 228 552 450
Absolute additional amount “released” (mu)
Latency (min) 0
16 27 15 27 21 13 17 15 18.8 f 1.9 (Mean f S.E.M.)
90 90 20 0 0 75 0
caused a rise in the concentration of AChE in the cisternal CSF in 3 experiments (Fig. 3). The same dose of atropine sulphate given alone did not change the concentration of the enzyme. However, when atropine methylnitrate (3 mg/kg i.v.) was administered instead of atropine sulphate, the chlorpromazine-induced rise in the AChE concentration still occurred (Fig. 3b and Table 1). DISCUSSION
Latency of the rise in concentration
of AChE
A marked increase in the concentration of AChE in cisternal CSF occurred following administration of chlorpromazine. The wide range of latencies in the onset of the effect is, however, puzzling. The drug was always administered, systemically, in the same way. There are, however, some possible variables which could not be controlled. For example, the depth to which the needle was inserted through the dura into the cisterna magna was hard to standardize, beyond the criterion that the whole bevel of the needle should be inserted. It is possible that small differences in the proximity of the sampling needle to the brain caused the variation in the latency of
(4mg/kg i.v.) on AChE and protein in cisternal cerebrospinal
Before chlorpromazine (A pre-treatment sample) AC’hE Cont. (m U/ml) 23.‘) 20.0 40.5
fluid
(3 mg/kg i.v.) given 10 min prior to chlorpromazine (CPZ) (4 mg/kg iv.) The “released” refers only to that in the CSF actually sampled. The pretreatment in the 3 samples prior to the time of injection: the absolute additional amount which the concentration was above the normal variation (+ lOmU/ml) in the
the enzyme concentration following administration of the same dose of chlorpromazine; the result of one such experiment is shown in Fig. 2a. The variation in the magnitude and latency of the effect is shown in Table 1. Changes in total protein concentration in CSF occurred, but these did not correlate with those in the concentration of AChE (Table 2). In all samples of CSF taken, only one multiple molecular form of AChE was detectable after polyacrylamide gel electrophoresis carried out exactly as desc-ibed by Chubb er al. (1976) who found a single fo’-m in CSF but several in soluble extracts of rabbit brain. Lactate dehydrogenase activity, in four experiments, remained constant throughout each experiment. The mean activity was 14mU/ml. Administration of either the non-antipsychotic phenothiazine promethazine (4mg/kg i.v.) or the chlorpromazine vehicle had no effect on the concentrs tion of AChE in the CSF collected from the cisterna magna.
Following administration (3 mg/‘kg i.v.), chlorpromazine
129
and cerebrospinal fluid acetylchohnesterase
AChE specific activity (mU/ng) 0.105 0.04 0.09 0.078 f 0.02 (M k S.E.M.)
fluid
After chlorpromazine (Sample containing maximal AChE activity) AChE specific AChE Cont. Protein activity (mu/ml) (pg/mh (mU/pg) 83.7 90 105
186 150 210
The specific activity is significantly higher. (P < 0.01, Student’s paired f-test) after administration
0.45 0.60 0.50 0.51 f 0.04 (M k S.E.M.) of chlorpromazine.’
130
SUSAN
A.
GREENFIELD.
I. W.CHUBBand A. D.
o/ 60
SMITH
180
80 min
(b)
min
Fig. 3. Experiments showing the effects of chlorpromazine (4mg,kg iv.) on AChE in cisternal CSF, following prior administration of: (a) atropine sulphate (AS) (3 mg/kg iv.); (b) atropine methylnitrate (AM) (3 mgjkg
the onset of the effect because it is. likely that the enzyme would take some time to be distributed homogenously throughout the CSF pool. Another variable was the position of the rabbit’s head. Although we attempted to standardize this by keeping constant the angles (see Methods and Fig. If the variable amount of mandible muscle in different animals makes small variations in the position unavoidable. It is likely that the head position couId influence the supply of CSF from different brain regions to the cisterna magna. Origin of the AChE
Since the specific activity of AChE in the CSF was increased after administration of chlorpromazine and only one of the several molecular forms of the enzyme was ever detected, it is possible to exclude penetration of blood plasma as a cause of the effect; there are several soluble forms of AChE in rabbit blood plasma and specific activity of the enzyme is one twentieth of that in CSF (Chubb et al., 1976). Alternative
i.v.).
explanatjons for the effect could be that chlorpromazine caused gross damage to cells in the brain, or that there was a loss of water from the CSF. Both these can be excluded by the observation that the lactate dehydrogenase (LDH) concentration in the CSF remained constant throughout these experiments. A similar constancy in LDH concentration was observed in earlier experiments where central stimulation (Greenfield and Smith, 1976) and peripheral stimulation. (Chubb et al., 1975) were used to evoke a rise in the AChE concentration. It seems then that the artefacts of cell damage, water loss or contamination with plasma through a change in the permeability of the blood-CSF barrier, could not account for the chlorpromazine-induced increase in AChE concentration. Site of action
of chlorpromazine
The resufts of the experiments with atropine suggest that the increase in AChE concentration in CSF, following chlorpromazine administration, may be due
Chlorpromazine
and cerebrospinal
to increased activity of cholinergic neurons in the central nervous system. There is good evidence that ACh is released from the caudate nucleus of cats following systemic doses of chlorpromazine (Stadler er a[., 1973; Trabucchi et al., 1974: kcGeer et al., 1974). Since a substantial part of the caudate nucleus borders on the lateral ventricle, it could seem reasonable to expect an increase in the AChE concentration in the \.entricular CSF, if the enzyme was being released from the structure; however, none was found. There z.re two alternative explanations for the rise in AChE concentration found in cisternal CSF but not in CSF sampled from the lateral ventricle; firstly, that AChE i:; released from the caudate nucleus, yet instead of crossing the ependyma into the ventricle, it first passes along the nerves (Wagner, Pilgrim and Brandl, 1974) to another region; secondly, that the AChE i:, released from a structure posterior to the caudate. The first suggestion is unlikely since it is known that proteins introduced into the cerebral ventricles can c’oss the ependyma and eventually be taken up into nerve terminals (Brightman, 1968). Furthermore, exogenous AChE, when injected into cerebral ventricles, can be taken up into nerve terminals and. when introd Jced into forebrain parenchyma, can diffuse through the intercellular spaces at a few mm per hour (Kreutzberg
and
Kaija,
1974).
From whereabouts in the brain, posterior to the caudate nucleus, could the additional amount of AChE be released, following administration of chlorpromazine? Although the evidence referred to above suggests that activity of choline@ neurons in the striatum is increased upon administration of chlorpromazine,
this drug
catecholaminergic ccncentration
also
neurons; of
has a pronounced
effect
on
there is an increase in the
catecholamine
metabolites
in
the
striatum following treatment with chlorpromazine (Carlsson and Lindqvist, 1963; Anden, 1974; Laverty and Sharman, 1965; Da Prada and Pletscher, 1966). Carlsson and Lindqvist (1963) suggested that the incrl:ased levels of dopamine metabolites after chlorpromazine treatment reflected an activation of dopaminergic neurons, brought about by a feedback mechanism compensating for the blockade of central dopamine receptors. Recordings of unit activity of cells in the substantia nigra have provided more direct evidence that the activity of the nigro-striatal dopaminergic fibres is incrc ased following the administration of chlorpromazine (Bunney, Walters, Roth and Aghajanian. 1973). It is therefore oertinent that the dooaminergic cell bo*jies in the suktantia nigra of the rat are v&y rich in intracellular AChE (Butcher, Talbot and Bilesikjian.
1975). Could
lead
to the release of AChE from cell bodies or den-
drites ccl
just
increased
as stimulation
(Chubb
of the
in an extracellular and Smith, 1975b;
results
Smith,
activity
of these adrenal
neurones chromaffin
release of the enzyme Somogyi, Chubb and
1975)‘?
The present
finding
that
the increases
in AChE
con-
fluid acetylcholinesterase
131
centration following administration of chlorpromazine were blocked by the prior administration of atropine sulphate could be explained by the fact that dopaminergic cells in the substantia nigra pars compacta show atropine-sensitive excitation by cholinomimetic drugs (Lichensteiner, Felix, Ehart and Hefti, 1976; Dray, Gonye and Oakley, 1976; Wolfarth. Dulska and Lacki, 1974; Javoy, Agid, Bouvet and Glowinski, 1974). Yet the physiological significance of this pharmacological sensitivity is not clear, since there is, so far, no evidence of a cholinergic input to the substantia nigra (Lehmann and Fibiger, 1978). No firm conclusions can, therefore, be drawn about the mechanism by which chlorpromazine evokes a rise in the concentration of AChE in cisternal CSF. Acknowledgements-We would like to thank May & Baker for the gift of promethazine. S.A.G. held an M.R.C. Training Scholarship and was supported by the J. H. Burn Trust. I.W.C. was a Royal Society Horace Le Marquand and Dudley Bigg Research Fellow.
REFERENCES
And&n, N. E. (1974).
Antipsychotic drugs and catecholRes. 11: 97-104. Brightman, M. N. (1968). The intracerebral movement of uroteins iniected into blood and cerebrosoinal fluid mice. Prog.‘Brain Res. 29: 19-37. Bunney, B. S., Walters. J. R.. Roth. R. H. and Aghajanian. K. (1973). Dopaminergic neurons: effect of antipsychotic drugs and amphetamine on single cell activity. J. Pharmat. cup. Ther. 185: 560-571. Butcher. L. L.. Talbot. K. and Bilesikjian, L. (1975). Acetylcholinesterase neurons in dopamine containing regions of the brain. J. Neural Transm. 37: 127~153. Carlsson. A. and Lindqvist, M. (1963). Effect of chlorpromazine or haloperidol on formation of 3-methoxytryramine and normetanephrine in mouse brain. Acta Pharmat. tax. 20: 14&144. Chubb. I. W., and Smith, A. D. (1975a). Isoenzymes of soluble and membrane-bound acetylcholinesterase in bovine splanchnic nerve and adrenal medulla. Proc. R. Sot. Lond. B. 191: 245-26 I. Chubb. I. W. and Smith. A. D. (1975b). Release of acetylcholinesterase into the perfusate from the ox adrenal
amine synapses. J. Psychiatr.
gland. Proc. R. Sot. Lond. B. 191: 263-269. Chubb, I. W., Goodman, S. and Smith, A. D. (1976). Is acetylcholinesterase secreted from central neurons into the cerebrospinal fluid’? Neuroscience. 1: 57-62. Da Prada, M. and Pletscher, A. (1966). Acceleration of the cerebral dopamine turnover by chlorpromazine. Experientia. 22: 465-466. Davson, H. (1967). Physiology of the Cerebrospinal Fluid. J. & S. Churchill Ltd, London. Dray, A., Gonye J. J. and Oakley, N. R. (1976). Caudate stimulation and substantia nigra activity in the rat. J. Physiol., Lond. 259: 825-849. Ellman, G. I.., Courtney, K. D., Andres, V. and Featherstone, K. M. (1961). A new and rapid calorimetric determination of acetvlcholinesterase activitv. Biochem. Pharmat. 7: 88.-95. . Greenfield, S. A. and Smith. A. D. (1976). Changes in acetylcholinesterase concentration in rabbit cerebrospinal fluid following central electrical stimulation. J. Physiol.. Land. 258: lO8-109P. Javoy, F.. Agid. Y.. Bouvet, D. and Glowinski. J. (1974) Changes in neostriatal dopamine metabolism agter car-
132
SUSAN
A.
GREENFIELD,
I. W. CHUBB and A. D.
bachol or atropine micro injection into the substantia nigra. &-air7 Rex 68: 253-260. Kreutzberg, G. W. and Kaija, H. (1974). Exogenous acetylcholinesterase as tracer for extracellular pathways in the brain. Histochentie 42: 233-237. Laverty, R. and Sharman. D. H. (1965). Modification by drugs of the metabolism of 3,4-Dihydroxyphenylethylamine, noradrenaline and 5-hydroxytryptamine in the brain. Br. J. Pharmac. 24: 759-772. Lehmann. J. and Fibiger. H. C. (1978). Acetylcholinesterase in the substantia nigra and caudate putamen of the rat: properties and ‘localization in dopaminergic neurons. J. Nwrochew. 30: 615-624. Lichensteiner. W.. Felix. D.. Ehart. R. and Hefti, F. (1976). A quantitative correlation between single unit activity and fluorescence intensity of dopamine in zona compacta of the substantia nigra as demonstrated under influence of nicotine and physostigmine. Brain Rrs. 117: 85- 103. Lowry, 0. H., Rosebough. N. J., Farr. A. L. and Randall, R. J. (1951). Protein measurement with the Folin phenol reagent. J. hiol. Chem. 193: 265-275. McGeer. P. L., Grewaal, D. S. and McGeer, E. G. (1974). Influence of non-cholinergic drugs on rat striatal acetylcholine levels. Brain Res. 80: 211-217.
SMITH
Silver, A. (1974). The Biology of the Cholinesterases. Nosh Holland, Amsterdam. Somogyi, P., Chubb, I. W. and Smith A. D. (1975). A possible structural basis for the release of acetylcholinesterase. Proc. R. Sot. Lond. B. 191: 272-283. Stadler. H.. Lloyd. K. G., Gadea-Ciria, M. and Bartholoni. G. (1973). Enhanced striatal acetylcholine release by chlorpromazine and its reversal by apomorphine. Brain Res. 55: 476481. Trabucchi, M., Cheney, D. L., Racagni. G. and Costa, E. (1974). Involvement of brain cholinergic mechanisms in the action of chlorpromazine. Nature, Land. 249: 664666. Wagner, H. T., Pilgrim, C. H. and Brandl, J. (1974). Penetration and removal of horseradish peroxidase injected into the CSF: role of cerebral perivascular spaces, endothelium and microglia. Acta Neuropath. 27: 299-315. Wolfarth, S., Dulska, E. and Lacki, M. (1974). Comparison of effects of intranigral injections of cholinomimetics with systemic injections of the dopamine receptor stimulating and blocking agents in the rabbit. Neuropharmacology 13: 867-875. Wroblewski, F. and La Due, J. S. (1955). Lactic dehydrogenase activity in blood. Proc. Sot. Exp. Biol. Med. 20: 21@213.
GREENFIELD*,
I. W. CHt.rBBtand A. D.
University Department of Pharmacology,
SMITH
South Parks Road, Oxford, OX1 3QT, U.K.
(Accepted 2 Augusr 1978)
Summery-~oliowing administration of chlorpromazine (4mg/kg iv.). there was an increase of up to 5-fold in the concentration of acetylcholinesterase in rabbit cerebraspinal fluid sampled from the cisterna magna. but not in cerebrospinal fluid from a fateral ventricle. The pattern of multiple molecular forms and specific activity of acetylcholinesterase in cerebrospinal fluid did not resemble that of blood plasma and so a breakdown of the blood-cerebrospinal fluid barrier can be excluded. The concentration of lactate dehydrogcnase in cisternal cerebrospinal fluid did not change, hence a non-specific increase in permeability of eel1 membranes seems unlikely. Prior administratjon of atropine sulphate (3 mg/kg i.v.), but not the same dose of atropine methylnitrate. prevented the effect of chlorpromazine. The possibility that the rise in the concentration of acetyicholinesterase was the result of increased activity of central neurons containing this enzyme is discussed.
It has been proposed that some of the acetylcholinesterase (AChE) found in cerebrospinal fluid (CSF) is released from neurons as a result of their activity (Chubb, Goodman and Smith, 1976). This hypothesis filllowed from the observation that the concentration cf AChE in rabbit cerebrospinal fluid (CSF) increased after procedures (sensory nerve stimulation) known to evoke the release of a~tylcholine from sufaces of the brain. Furthermore, stimulation via electrodes implanted in the rabbit caudate nucleus also led to an increase in the concentration of AChE in CSF (Greenfield and Smith, 1976). The caudate nucleus has a very high content of AChE (Silver, 1974) and since it borders on the lateral ventricle it is a possible site of origin of AChE in the CSF. The release of acetylcholine from the AChEcontaining cholinergic interneurons in the caudate nucleus is markedly increased following peripheral administration of the antipsychotic phenothiazine, c?lorpromazine (Stadler, Lloyd, Gadea-Ciria and Bartholini, 1973; Trabucchi, Cheney, Racagni and Costa, 1974; McGeer, Grewaal and McGeer, 1974). The purpose of the studies to be described was to see whether AChE is released into the CSF following administration of chlorpromazine.
solution in 0.9% (w/v) NaCI) administered via a marginal ear vein (approximately 6mI/kg). The trachea was cannulated and the head placed in a head holder (C. F. Palmer, Ltd, High Wycombe, Bucks). The dura covering the cisterna magna was then exposed. A hypodermic needle (23 G 0.63 mm dia.) was inserted into the dura (see Fig. 1) and CSF was continuously sampled by slow suction of a peristaltic pump, at a rate between 4 and 6hl/min, which is beIow the normal rate of production in the rabbit (Davson, 1967). The rectal temperature was maintained at 37 k 1°C by means of a warm blanket and lamp. Sanlpling
METHODS S~~rn~~ing of CSF from
from
the lateral
ventricle
the cisterira manna
Zealand White male rabbits (3.0-3.5 kg body weight) were anaesthetized with urethane (25% (w/v) New
*Present address: University Laboratory of Physiology, Oxford, U.K. + Present address: Department of Physiology, Flinders University Medical School, Bedford Park, S. Australia. N.P.182-e
ofCSF
Rabbits were anaesthetized with sodium pentobarbitone (Nembutal) (30 mg/kg i.v.1 and ether. The head was then fixed in a stereotaxic apparatus so that the coronal suture bregma was 1.5 mm higher than lambda. A stainless-steel cannula 35 mm in length was permanently implanted into the ventricle, using dental acrylic, at co-ordinates AP 0, L 3, V 5. The animal was then left to recover for a minimum of 3 days. Rabbits implanted in this way were anaesthetized with urethane. as described above, prior to sampling CSF. CSF was removed at a rate of -11 per min. The sites of impIantation were verified histologically.
Acetylcholinesterase activity was determined using acetylthiocholine as substrate at pH 7.0. The method is a modification (Chubb and Smith, 1975a) of that described by Ellman, Courtney, Andres and Featherstone (1961). One unit (U) of enzyme activity represents the hydrolysis of f pmol substrate per minute at 30°C.
127
SUSAN A. GREENFIELD.I. W. CHUBB and
128
Fig. 1. Position
of rabbit’s head in head-holder. The needle (N) is inserted just above and to a depth such that the entire bevel is covered by the dura.
Lactate dehydrogenase activity was measured by the method of Wroblewski and La Due (195.5) and expressed in international units. Total protein was estimated according to the method of Lowry, Rosebough, Farr and Randall (1951) with bovine serum albumin as standard. Electrophoresis in 6% (w/v) polyacrylamide gels was carried out by the method described by Chubb and Smith (1975a). Drug
administration
Chlorpromazine (Largactil) and promethazine (Phenergan) were used as supplied in injection form. The chlorpromazine vehicle was made up immediately before use, the composition being 0.1% (w/v) Na sulphite, 0.75% (w/v) Na metabisulphite, 0.4% (w/v) NaCl and 0.1% (w/v) Na citrate in distilled HzO. All drugs were administered via a marginal ear vein. RESULTS
Acetylcholinesterase magna
and lateral
A. D. SMITH
in
CSF
sampled
from
cisterna
ventricle
The concentration of AChE in cistemal CSF was often, but not always, high for the first 3G60min.
the Atlas vertebra
following the start of sampling (e.g. Fig. 2a). This has been observed in previous experiments (Chubb et al., 1976) and it was suggested that the initial high values may have been a consequence of activation of sensory nerves during surgery. After this initial period, the concentration of AChE in CSF remained fairly constant and a mean value of 35.0 ) 3.55 (S.E.) mu/ml was obtained (n = 20). In any individual rabbit, the “resting” concentration of AChE varied by no more than 10 mu/ml on either side of the mean concentration for that rabbit. The mean concentration of AChE in CSF withdrawn from the lateral ventricle was 15.5 k 5.0 (SE.) in four rabbits. This is significantly (P < 0.01) lower than the mean concentration in cisternal CSF. EfSect of chlorpromazine cholinesterase
on the concentration
in cerebrospinal
of acetyl-
jluid
chlorpromazine Following administration of (4 mg/kg i.v.) to seven rabbits there was no significant change in the concentration of AChE in CSF withdrawn continuously from the lateral ventricle (Fig. 2b). However, when CSF was withdrawn from the cisterna magna, there was a marked increase in
90r
E i 2
~~_i&Jl_a
u
1
0
I 200 min
-b . 400
Fig, 2. Experiments demonstrating the effect of chlorpromazine (4mg/kg i.v.) on the concentration of AChE in (a) cisternal; (b) ventricular CSF collected from the same rabbit. The vertical axis shows AChE concentration in mu/ml of CSF where a mU is one nanomole of substrate hydrolysed in one minute. The horizontal axis gives the time in minutes: the horizontal bars represent the period of collection of each fraction.
Chlorpromazine
Table 1. Effect of chlorpromazine Dose of CPZ (mg/kg i.v.)
on AChE in cisternal cerebrospinal
Pre-treatment Average AChE Cont. (mu/ml)
Increase “/, after CPZ
30 50 30 20 60 30 20 30
200 200 100 400 100 300 300 300
4 (AM) 4 4 (AM) 4 (AM) 4 4 3 4
AM indicates atropine methylnitrate absolute additional amount of AChE average concentration of AChE is that was calculated for the oeriod during “resting” level.
Efect of atropine on the chlorpromazine-induced crease in AChE concentration
-
in-
of atropine sulphate (4 mg/kg i.v.) no longer
Table 2. Effect of chlorpromazine
Protein @g/ml) 228 552 450
Absolute additional amount “released” (mu)
Latency (min) 0
16 27 15 27 21 13 17 15 18.8 f 1.9 (Mean f S.E.M.)
90 90 20 0 0 75 0
caused a rise in the concentration of AChE in the cisternal CSF in 3 experiments (Fig. 3). The same dose of atropine sulphate given alone did not change the concentration of the enzyme. However, when atropine methylnitrate (3 mg/kg i.v.) was administered instead of atropine sulphate, the chlorpromazine-induced rise in the AChE concentration still occurred (Fig. 3b and Table 1). DISCUSSION
Latency of the rise in concentration
of AChE
A marked increase in the concentration of AChE in cisternal CSF occurred following administration of chlorpromazine. The wide range of latencies in the onset of the effect is, however, puzzling. The drug was always administered, systemically, in the same way. There are, however, some possible variables which could not be controlled. For example, the depth to which the needle was inserted through the dura into the cisterna magna was hard to standardize, beyond the criterion that the whole bevel of the needle should be inserted. It is possible that small differences in the proximity of the sampling needle to the brain caused the variation in the latency of
(4mg/kg i.v.) on AChE and protein in cisternal cerebrospinal
Before chlorpromazine (A pre-treatment sample) AC’hE Cont. (m U/ml) 23.‘) 20.0 40.5
fluid
(3 mg/kg i.v.) given 10 min prior to chlorpromazine (CPZ) (4 mg/kg iv.) The “released” refers only to that in the CSF actually sampled. The pretreatment in the 3 samples prior to the time of injection: the absolute additional amount which the concentration was above the normal variation (+ lOmU/ml) in the
the enzyme concentration following administration of the same dose of chlorpromazine; the result of one such experiment is shown in Fig. 2a. The variation in the magnitude and latency of the effect is shown in Table 1. Changes in total protein concentration in CSF occurred, but these did not correlate with those in the concentration of AChE (Table 2). In all samples of CSF taken, only one multiple molecular form of AChE was detectable after polyacrylamide gel electrophoresis carried out exactly as desc-ibed by Chubb er al. (1976) who found a single fo’-m in CSF but several in soluble extracts of rabbit brain. Lactate dehydrogenase activity, in four experiments, remained constant throughout each experiment. The mean activity was 14mU/ml. Administration of either the non-antipsychotic phenothiazine promethazine (4mg/kg i.v.) or the chlorpromazine vehicle had no effect on the concentrs tion of AChE in the CSF collected from the cisterna magna.
Following administration (3 mg/‘kg i.v.), chlorpromazine
129
and cerebrospinal fluid acetylchohnesterase
AChE specific activity (mU/ng) 0.105 0.04 0.09 0.078 f 0.02 (M k S.E.M.)
fluid
After chlorpromazine (Sample containing maximal AChE activity) AChE specific AChE Cont. Protein activity (mu/ml) (pg/mh (mU/pg) 83.7 90 105
186 150 210
The specific activity is significantly higher. (P < 0.01, Student’s paired f-test) after administration
0.45 0.60 0.50 0.51 f 0.04 (M k S.E.M.) of chlorpromazine.’
130
SUSAN
A.
GREENFIELD.
I. W.CHUBBand A. D.
o/ 60
SMITH
180
80 min
(b)
min
Fig. 3. Experiments showing the effects of chlorpromazine (4mg,kg iv.) on AChE in cisternal CSF, following prior administration of: (a) atropine sulphate (AS) (3 mg/kg iv.); (b) atropine methylnitrate (AM) (3 mgjkg
the onset of the effect because it is. likely that the enzyme would take some time to be distributed homogenously throughout the CSF pool. Another variable was the position of the rabbit’s head. Although we attempted to standardize this by keeping constant the angles (see Methods and Fig. If the variable amount of mandible muscle in different animals makes small variations in the position unavoidable. It is likely that the head position couId influence the supply of CSF from different brain regions to the cisterna magna. Origin of the AChE
Since the specific activity of AChE in the CSF was increased after administration of chlorpromazine and only one of the several molecular forms of the enzyme was ever detected, it is possible to exclude penetration of blood plasma as a cause of the effect; there are several soluble forms of AChE in rabbit blood plasma and specific activity of the enzyme is one twentieth of that in CSF (Chubb et al., 1976). Alternative
i.v.).
explanatjons for the effect could be that chlorpromazine caused gross damage to cells in the brain, or that there was a loss of water from the CSF. Both these can be excluded by the observation that the lactate dehydrogenase (LDH) concentration in the CSF remained constant throughout these experiments. A similar constancy in LDH concentration was observed in earlier experiments where central stimulation (Greenfield and Smith, 1976) and peripheral stimulation. (Chubb et al., 1975) were used to evoke a rise in the AChE concentration. It seems then that the artefacts of cell damage, water loss or contamination with plasma through a change in the permeability of the blood-CSF barrier, could not account for the chlorpromazine-induced increase in AChE concentration. Site of action
of chlorpromazine
The resufts of the experiments with atropine suggest that the increase in AChE concentration in CSF, following chlorpromazine administration, may be due
Chlorpromazine
and cerebrospinal
to increased activity of cholinergic neurons in the central nervous system. There is good evidence that ACh is released from the caudate nucleus of cats following systemic doses of chlorpromazine (Stadler er a[., 1973; Trabucchi et al., 1974: kcGeer et al., 1974). Since a substantial part of the caudate nucleus borders on the lateral ventricle, it could seem reasonable to expect an increase in the AChE concentration in the \.entricular CSF, if the enzyme was being released from the structure; however, none was found. There z.re two alternative explanations for the rise in AChE concentration found in cisternal CSF but not in CSF sampled from the lateral ventricle; firstly, that AChE i:; released from the caudate nucleus, yet instead of crossing the ependyma into the ventricle, it first passes along the nerves (Wagner, Pilgrim and Brandl, 1974) to another region; secondly, that the AChE i:, released from a structure posterior to the caudate. The first suggestion is unlikely since it is known that proteins introduced into the cerebral ventricles can c’oss the ependyma and eventually be taken up into nerve terminals (Brightman, 1968). Furthermore, exogenous AChE, when injected into cerebral ventricles, can be taken up into nerve terminals and. when introd Jced into forebrain parenchyma, can diffuse through the intercellular spaces at a few mm per hour (Kreutzberg
and
Kaija,
1974).
From whereabouts in the brain, posterior to the caudate nucleus, could the additional amount of AChE be released, following administration of chlorpromazine? Although the evidence referred to above suggests that activity of choline@ neurons in the striatum is increased upon administration of chlorpromazine,
this drug
catecholaminergic ccncentration
also
neurons; of
has a pronounced
effect
on
there is an increase in the
catecholamine
metabolites
in
the
striatum following treatment with chlorpromazine (Carlsson and Lindqvist, 1963; Anden, 1974; Laverty and Sharman, 1965; Da Prada and Pletscher, 1966). Carlsson and Lindqvist (1963) suggested that the incrl:ased levels of dopamine metabolites after chlorpromazine treatment reflected an activation of dopaminergic neurons, brought about by a feedback mechanism compensating for the blockade of central dopamine receptors. Recordings of unit activity of cells in the substantia nigra have provided more direct evidence that the activity of the nigro-striatal dopaminergic fibres is incrc ased following the administration of chlorpromazine (Bunney, Walters, Roth and Aghajanian. 1973). It is therefore oertinent that the dooaminergic cell bo*jies in the suktantia nigra of the rat are v&y rich in intracellular AChE (Butcher, Talbot and Bilesikjian.
1975). Could
lead
to the release of AChE from cell bodies or den-
drites ccl
just
increased
as stimulation
(Chubb
of the
in an extracellular and Smith, 1975b;
results
Smith,
activity
of these adrenal
neurones chromaffin
release of the enzyme Somogyi, Chubb and
1975)‘?
The present
finding
that
the increases
in AChE
con-
fluid acetylcholinesterase
131
centration following administration of chlorpromazine were blocked by the prior administration of atropine sulphate could be explained by the fact that dopaminergic cells in the substantia nigra pars compacta show atropine-sensitive excitation by cholinomimetic drugs (Lichensteiner, Felix, Ehart and Hefti, 1976; Dray, Gonye and Oakley, 1976; Wolfarth. Dulska and Lacki, 1974; Javoy, Agid, Bouvet and Glowinski, 1974). Yet the physiological significance of this pharmacological sensitivity is not clear, since there is, so far, no evidence of a cholinergic input to the substantia nigra (Lehmann and Fibiger, 1978). No firm conclusions can, therefore, be drawn about the mechanism by which chlorpromazine evokes a rise in the concentration of AChE in cisternal CSF. Acknowledgements-We would like to thank May & Baker for the gift of promethazine. S.A.G. held an M.R.C. Training Scholarship and was supported by the J. H. Burn Trust. I.W.C. was a Royal Society Horace Le Marquand and Dudley Bigg Research Fellow.
REFERENCES
And&n, N. E. (1974).
Antipsychotic drugs and catecholRes. 11: 97-104. Brightman, M. N. (1968). The intracerebral movement of uroteins iniected into blood and cerebrosoinal fluid mice. Prog.‘Brain Res. 29: 19-37. Bunney, B. S., Walters. J. R.. Roth. R. H. and Aghajanian. K. (1973). Dopaminergic neurons: effect of antipsychotic drugs and amphetamine on single cell activity. J. Pharmat. cup. Ther. 185: 560-571. Butcher. L. L.. Talbot. K. and Bilesikjian, L. (1975). Acetylcholinesterase neurons in dopamine containing regions of the brain. J. Neural Transm. 37: 127~153. Carlsson. A. and Lindqvist, M. (1963). Effect of chlorpromazine or haloperidol on formation of 3-methoxytryramine and normetanephrine in mouse brain. Acta Pharmat. tax. 20: 14&144. Chubb. I. W., and Smith, A. D. (1975a). Isoenzymes of soluble and membrane-bound acetylcholinesterase in bovine splanchnic nerve and adrenal medulla. Proc. R. Sot. Lond. B. 191: 245-26 I. Chubb. I. W. and Smith. A. D. (1975b). Release of acetylcholinesterase into the perfusate from the ox adrenal
amine synapses. J. Psychiatr.
gland. Proc. R. Sot. Lond. B. 191: 263-269. Chubb, I. W., Goodman, S. and Smith, A. D. (1976). Is acetylcholinesterase secreted from central neurons into the cerebrospinal fluid’? Neuroscience. 1: 57-62. Da Prada, M. and Pletscher, A. (1966). Acceleration of the cerebral dopamine turnover by chlorpromazine. Experientia. 22: 465-466. Davson, H. (1967). Physiology of the Cerebrospinal Fluid. J. & S. Churchill Ltd, London. Dray, A., Gonye J. J. and Oakley, N. R. (1976). Caudate stimulation and substantia nigra activity in the rat. J. Physiol., Lond. 259: 825-849. Ellman, G. I.., Courtney, K. D., Andres, V. and Featherstone, K. M. (1961). A new and rapid calorimetric determination of acetvlcholinesterase activitv. Biochem. Pharmat. 7: 88.-95. . Greenfield, S. A. and Smith. A. D. (1976). Changes in acetylcholinesterase concentration in rabbit cerebrospinal fluid following central electrical stimulation. J. Physiol.. Land. 258: lO8-109P. Javoy, F.. Agid. Y.. Bouvet, D. and Glowinski. J. (1974) Changes in neostriatal dopamine metabolism agter car-
132
SUSAN
A.
GREENFIELD,
I. W. CHUBB and A. D.
bachol or atropine micro injection into the substantia nigra. &-air7 Rex 68: 253-260. Kreutzberg, G. W. and Kaija, H. (1974). Exogenous acetylcholinesterase as tracer for extracellular pathways in the brain. Histochentie 42: 233-237. Laverty, R. and Sharman. D. H. (1965). Modification by drugs of the metabolism of 3,4-Dihydroxyphenylethylamine, noradrenaline and 5-hydroxytryptamine in the brain. Br. J. Pharmac. 24: 759-772. Lehmann. J. and Fibiger. H. C. (1978). Acetylcholinesterase in the substantia nigra and caudate putamen of the rat: properties and ‘localization in dopaminergic neurons. J. Nwrochew. 30: 615-624. Lichensteiner. W.. Felix. D.. Ehart. R. and Hefti, F. (1976). A quantitative correlation between single unit activity and fluorescence intensity of dopamine in zona compacta of the substantia nigra as demonstrated under influence of nicotine and physostigmine. Brain Rrs. 117: 85- 103. Lowry, 0. H., Rosebough. N. J., Farr. A. L. and Randall, R. J. (1951). Protein measurement with the Folin phenol reagent. J. hiol. Chem. 193: 265-275. McGeer. P. L., Grewaal, D. S. and McGeer, E. G. (1974). Influence of non-cholinergic drugs on rat striatal acetylcholine levels. Brain Res. 80: 211-217.
SMITH
Silver, A. (1974). The Biology of the Cholinesterases. Nosh Holland, Amsterdam. Somogyi, P., Chubb, I. W. and Smith A. D. (1975). A possible structural basis for the release of acetylcholinesterase. Proc. R. Sot. Lond. B. 191: 272-283. Stadler. H.. Lloyd. K. G., Gadea-Ciria, M. and Bartholoni. G. (1973). Enhanced striatal acetylcholine release by chlorpromazine and its reversal by apomorphine. Brain Res. 55: 476481. Trabucchi, M., Cheney, D. L., Racagni. G. and Costa, E. (1974). Involvement of brain cholinergic mechanisms in the action of chlorpromazine. Nature, Land. 249: 664666. Wagner, H. T., Pilgrim, C. H. and Brandl, J. (1974). Penetration and removal of horseradish peroxidase injected into the CSF: role of cerebral perivascular spaces, endothelium and microglia. Acta Neuropath. 27: 299-315. Wolfarth, S., Dulska, E. and Lacki, M. (1974). Comparison of effects of intranigral injections of cholinomimetics with systemic injections of the dopamine receptor stimulating and blocking agents in the rabbit. Neuropharmacology 13: 867-875. Wroblewski, F. and La Due, J. S. (1955). Lactic dehydrogenase activity in blood. Proc. Sot. Exp. Biol. Med. 20: 21@213.