COMMENTARY THE MEASUREMENT OF ACETYLCHOLINE RATE IN BRAIN STRUCTURES*
TURNOVER
G. RACAGNI, D. L. CHENEY, G. ZSILLAand E. COSTA Laboratory of Preclinical Pharmacology, National Institute of Mental Health. Saint Elizabeths Hospital. Washington D.C. 20032
INTRODUCTION The interest of pharmacologists to develop methods that estimate brain a~tylcholine (ACh) turnover rate in L+TO,stems from the belief that ACh turnover rate increases when the activity of cholinergic neurones is enhanced. Historically, this belief developed as a result of the experiments on cat superior cervical sympathetic ganglia performed by MACINTOSH (1963). These experiments established that at rest, the fractional rate constant for the renovation of acetylcholine is about 6 hr...‘. It was also shown that in ganglia the resting ACh turnover rate never exceeded ZOO/, of the maximum rate that could be attained during electrical stimulation of preganglionic nerves. Since 90’:); or more of the 3.5 nmol of ACh stored in cat sympathetic ganglia resides in terminal axons, the ganglion was an almost ideal model to correlate ACh turnover rate with nerve activity. Unfortunately, the whole brain is not such an ideal model to study this correlation. The ubiquity and intricacy of the cholinergic network plays havoc with accurate measurements of ACh turnover rate in terminals. Moreover. it is very unlikely that drugs simultaneously change the activity of every cholinergic neurone in the brain in the same direction. A possible exception might be the drugs which block either cholinesterase or choline a~tyltransferas~. More specifically, the questions that the neuropharmacologist might wish to answer with ACh turnover rate studies may concern whether or not cholinergic mechanisms participate in the action of drugs that modify transmission mediated by serotonin, dopamine, y-aminobutyric acid (GABA), substance P, etc. In fact, an assessment of drug actions on the integ~tion of different synaptic mechanisms may give a new dimension to the profile of centrally acting drugs. Such information may provide a guide in the synthesis of new antipsychotics with a low potential liability to elicit tardive dyskenesia (ZIVKOvie. GUI~)OTTI. RI-‘VUELTAand COSTA (1975). * This paper was presented at the Round Table entitled “Recent Concepts and Methods to Measure in Go the Turnover Rate of Neurotransmitters”, which was part of the official programme of the 5th International Meeting of the International Society of Neurochemistry, held in Barcelona. Spain. September 1975.
CONSIDERATIONS ON VARIOUS APPROACHES TO MEASURE ACh TURNOVER RATE A major stimulus for the development of methods to measure ACh turnover rate it2 uivo has been the possibility that drug actions on central cholinergic mechanisms can be described in terms of their capacity to change the ACh turnover rate. The report of MACINTOSH(1963) not only supported this contention but also suggested that by perfusing brain tissue and measuring the ACh content of the perfusate one might measure the rate of ACh release. By measuring the release of ACh elicited by electrical stimulation of brain slices in the presence or in the absence of eserine and/or atropine, the rate constant of ACh release was measured (SZERB, 1975). However, the physiological significance of these in citro experiments is open to question. A more reliable estimation of the dynamic state of ACh stores can be obtained by in oiro perfusion of limited areas of brain cortex using perfusion cups (BEANI and BIANCHI,1970; COLLIER and MITCHELL, 1967: PEPEU, 1973). In these experiments ACh e@lux rates are obtained which must be corrected for changes in steady-state in order to obtain a measurement of ACh turnover rate. A case in point illustrating the necessity of such a correction is the action of atropine on ACh stores in brain: the ACh efflux rates are increased by atropine but this drug also lowers brain ACh content (HOLMST~~T. LUNDGREN and SUNI>WALL. 1963). When the ACh efflux rates are corrected for the change in ACh steady-state, one often finds that the turnover rate is not increased although atropine increases ACh efflux rates in superfusion experiments (DUDAK and SZEKK, 1969). More recently, push-pull cannulae have been used with profit in studying the action of drugs on cholinergic mechanisms. This approach will not be discussed in the present paper and we refer to a recent critical evaluation made by BARTHOLINI, STADLER,GAD~A-CIKIA and LI.OYI, (1976).
Decline gf brair~ AC% content &er blockade oj synthesis to rneusure ACh turnover rate In brain, the turnover rate of a transmitter can be measured from the slope of the decline of transmitter
124
G. RACAGNI,D. L. CHENEY.G. ZSILI.Aand E.
COSI.A
content, elicited by the blockade of its synthesis Isotopic methods to ~musuw AC/7 t~tntorw txfc Although these methods minimiTe some of the (COSTA and NEFF, 1966). Hemicholinium-3 (HC-3) problems we have discussed as inherent to the nonisoblocks ACh synthesis (GARDINER, 1961) and intraventopic methods, they include a number of ~~ssurnptiolls tricular injection of He-3 was used to measure brain which cannot be fully substantiated by experimental ACh turnover rate (DOMINO and WILSON, 1972). The evidence. Hence, these methods arc not reliable to assumption implicit in this measurement is that at measure absolute values of ACh turnover rate; neversteady-state, the rate of synthesis and degradation of theless, they are adequate for comparative neuropharACh are in equilibrium. The method therefore utilizes macological studies of ACR turnover rate. principles of steady-state kinetics and applies them ~~lili~?u~f~~~~ ~~thoi~s. Radioactive methods utilize to the decrease of ACh content caused by the instanthe measurement of the specific radioactivity of ACh taneous blockade of ACh synthesis. By defining the and that of its precursor choline at various times after steady-state concentration of brain ACh as a dynamic equilibrium between synthesis and metabolism, when the injection of choline or of a radiol;~bcllcd precursor the ACh synthesis is blocked completely, the slope of choline (DROSS and K~w~rz. 1972; SCHU~I:K~FI, whereby brain ACh content declines with time can SPARF and SUNI)WALL, 1969; HANIY. CHIMY. TKAbe used to calculate the rate constant of ACh ef%x. NUCCIII, M~~~~~1.1~1. WAY bc a precursor of tion of its synthesis requires that the efflux rates of brain choline (ANSI-:LLand SPANSI:K. 1971). RadioACh be calculated from the initial rates in the decline active phos~~liorylcholine mivcs rapidly with the of brain ACh content. In almost every pharmacologiendogenous pool of phosphorylcl~oline and yields cal experiment known to us, calculations of ACh radioactive choline which mixes quite readily with turnover rate were made from a single time point taken at a fixed interval after HC-3 injection (DOMINO the endogenous pool of choline (HANIL cl c/l., 197.7). By injecting phosphorylcholine. WC have obtained a and WILSON, 1972). Since the ground rules for the high specific activity of brain choline (RX’Ac;NI. TKAapplication of principles of steady state kinetics 1975). Moreover, using Crfn&l:v. ULJfCH1 and require assessment of initial rates of ACh decrease. [ 1‘C~phospllorylch~~line iiljected cithcr by pulse injecat least three time points must be used to calcution or by constant rate infusion we have mcasurcd late ACh efflux. In addition, since the assumption relabelled ACh in samples of brain tissue weighing quires that the ACh is stored in a single metabolic about I Gt?-300 mg without changing the steady-state compartment open at both ends, one must demonof brain choline or AC% (CHI:U~:Y. TKAI%~,(‘(‘HI. strate that ACh decline follows first order kineRACAC;XIand Cosrh. iY?4). tics.
725
ACh turnover rate Stable isotope methods. When ACh turnover rate measurements are performed in brain nuclei, mass fragmentography must be used because it possesses the necessary sensitivity for these measurements (KosLOW, RACAGNI and COSTA, 1974). This necessity also imposes that labelling be performed with stable isotopes Pulse injections of deuterated phosphorylcholine cannot be used to label substantially the choline and ACh contained in single brain nuclei (tissue weight 1 mg or less) without changing the steadystate. However. a constant rate infusion with deuterated phosphorylcholine can label the choline and ACh content, thus allowing the measurement of ACh turnover rate (COSTA. CHENEY, RACAGNI and ZSILLA, 1975). The necessity to use constant rate infusion of the label derives from the fact that the detection of stable isotopes by mass fragmentography requires that at least loo/, of the pool be labelled. Considering that a brain nucleus usually contains a few picomoles of ACh. the detection of the label at 10:; implies a measurement in the range of 20&300 femtomoles. This is the limit of mass fragmentographic detection of ACh (KOSLOW et al.. 1974).
MEASUREMENT OF
ACh TURNOVER
VARIOUS STRUCTURES CONSTANT
RATE IN
OF RAT BRAIN USING A
RATE INFUSION
OF PHOSPHORYL-
[Me-“CICHOLINE
Kinetic
corwidrrafions
m brain which proceeds at rates of 20nmol/g tissue per min (DROSS and KEWITZ, 1972). Since even the immersion of the rats in liquid nitrogen freezes the brain in not less than 20sec, the errors in the brain choline measurements using immersion in liquid nitrogen can be conspicuous. Only after microwave radiation, can brain dissection be carried out without changing the steady-state content of ACh and choline (GUIDOTTI et al.. 1974). Using constant rate infusion of radioactive phosphorylcholine in combination with microwave radiation (RA~AGNI et al., 1974) is it possible to overcome two of the problems which, in the past. have hampered the measurement of ACh turnover rate. (1) The postmortem changes in ACh and choline due to residual enzyme activity; (2) the distortion in the precursor product relationship between choline and ACh which is due to a feedback labelling caused by the reupt’ake of labelled choline originating from the reuptake of radioactive ACh released by nerve impulses, Normally. 80”. of the choline that is generated from ACh released extraneuronally by nerve impulses is taken back into the nerve endings, where is becomes a substrate for choline acetyltransferase and recycles to form endogenous ACh (HEBB, 1972). This conspicuous participation in the recycling of the choline generated from ACh is due, in part, to a preferential utilization of newly taken up choline for ACh synthesis (GLYEMT. LEFRESNE. BEAUJOVAN and GLOWINSKI, 1975). When [‘4C]phosphorylcholine is infused at constant rate for 10-I 2 min the specific activities of ACh and choline in brain parts increase slowly but continuously (Fig. I). Since during short term constant rate infusion of [r4C]phosphorylcholine, the specific activity of choline is always higher than that of ACh in all structures studied, distortions due to the perferential utilization of the newly taken up choline for ACh synthesis are not apparent (Fig. 1).
Phosphoryl [Me-‘4C]choline (sp. act. 49 &i/pmol) was usually infused intravenously through the tail vein at a constant rate (25 &i/min per kg). At the end of the infusion or at various times thereafter, the rats were killed by microwave radiation (2.0 kW; 2.45 GHz, 75 W/cm’) focussed on the head. A beam of microwave radiation. focussed on the skull of 12&15Og rats for 2 set, kills the rats instantaneously and inactivates brain enzymes promptly and irreversibly (GUIDOTTI, CHENEY, TRARUCCHI, Muthematical unalysis qf thr dtrta: calculation qf’ ACh DOTEUCHI. WANG and HAWKINS, 1974; STAVINOHA. turnoorr rate A basic tenet for the calculation of ACh turnover WEINTRAUB and MODAK, 1973). This technique prevents the postmortem accumulation of free choline rate from the change with time in isotope enrichment of 1000
20 Striatum
t
I/:
Brainstem
min
Fig. 1. Specific activity stem after different
of [“C]ACh (solid line) and [‘T]choline (dashed line) in striatum and braintimes of infusion at constant rates of phosphoryl [Me-‘4C]choline (25 &3/kg per min). Infusion was initiated at time 0.
G. RACAGM,D. L. CHENEY,G. ZSILLA and E. COSTA
726
choline and ACh after labelling with phosphorylcholine is that the compartmentation and steady-state eq~~ilibri~~m among the various pools of ACh and choline do not change during the infusion of the label. Thus: the ACh synthesis rate and not the rate of change of choline specific activity is the factor which determines the change with time of ACh specific activity. This point is documented by the data graphed in Figure I. In the various brain structures. the rate of ACh specific activity increase is clearly independent from the rate of change of choline specific activity. For instance, in Figure 1~ the rate of change of choline specific activity is faster in brainstem than in striatum and yet the increase in ACh specific activity is faster in striatum than in brainstem. Hence, it can be inferred that an endogenous process at different rates in different brain parts regulates the formation of labelled ACh from labelled choline. We infer that this process reflects the rate of endogenous metabolism of ACh. The data of Figure 1 show that Iabelled phosphoryleholine is rapidly transfor~ned into free brain choline. However. it should he apparent that the rate of change in choline specific activity is different in different brain parts. Hence, we might infer that the various metabolic pools of brain choline are enriched with labelled congencr at a rate proportional to their intrinsic metabolic rates of chotine. During the infusion of radioactive pllosphory~choli~l~. some labet may enter the brain as the ester, and phosphorylcholine may bc dephosphorylated irl silzc to generate choline. Moreover. the present understanding of choline metabolism in brain suggests that phosphorylcholine may also be converted into phosphatidylcholine (KEU’ITZ. PLEUL. DROSS and S~~~~AR~.~~~~~, 1975). Therefore, the difference in the rate of radioactive choline accumulation in various brain parts during constant rate infusion of phosphorylcholine (Fig. 1) reflects diRerent rates of phosphorylcholine metabolism in addition to differences in the metabolism of endogenous choline. Possibly, during constant rate infusion phosphoryl~hoiine, conversion in choline may interfere with the measurement of ACh turnover rate. However, when ACh synthesis was measured in whole mouse brain by labeliing with radioactive choline Table
(SPARF, 1974) or phosphorylcholine (HANIN et al., 1973) the rates calculated from data obtained with the two methods were similar (Table 1). The mathematical procedure for the calculation of ACh turnover rate was derived from the following model (RACACN rf ul., 1974).
where k,, + kAZ = k,, and kR1 + kB2 = k8 and A is the compartment of free choline and B is that of ACh in any given brain structure. The rate of entrance of labelled choline into compartment A is indicated by J, while kA and kB are the fractional rate constants for the efRux of products from compartments A and B, respectively. The change of choline specific activity (S,,) and ACh specific activity (SACb) with time is the function of J which can be controlled.
kAe-Q + k,-k,
can be experimentally %I, and SAC,, k,, can be calculated
Injected ___[‘H]Cholim$ Phosphoryl [?ifc-‘“C]choline:
SC&i) m=SACho. Since m can be calculated from the experimental resufts kg and kB can be determined. When k, is known then AC% turnover rate can be calculated and equals k,, (ACh). Since the kA of each brain area is reproducible under identical experimental conditions if drugs fail to change k,, it is then possible to calculate ACh turnover rate after drug treatment from a following pulse intravenous injection by the finite difference method*
AC11 (nmolig) __..-
k,,.,** (min- ‘)
Turnover rate of ACh*** (nmol/g per min)
151-
0.24 * 0.048 0.25 ) 0.012
3.6 4.5
I8 & 0.72
k, and
where
1. Tutnoter rate of acetylcholine in whole mouse brain of [.~H~lcholinc or phosph~~ryl ~~~~-‘4C]choline calculated -
Compound
measured
mathematically:
* NEW (‘I ‘il. ( i 97 1). ** Yractionai rate constant for ACh (k,,.,,) was calculated from 1 min time intervals abstracted from smooth curves of choline and ACh. Specific radioactivities between 1 and 20min after injection of radiolahcl. *** Turnover rate of ACh obtained by multiplying kAch by steady-state concentration of ACh. 4 [3H]-Choline (160 &i/mouse) injected intravenously (SPARF,1974). t Average ACh concentration abstracted from literature (SPARF,1974). $ Phosph(3ryl [Mc-‘JC]choline (250 itCi!kg) injected ~ntraveno~lsiy (HAWN & uf., 1973).
121
ACh turnover rate Table 2. Kinetic parameters and ACh turnover rate in rat brain areas* (h? ‘) Striatum
Occipital cortex Limbic cortex Brainstem Hippocampus
17 47 16 41 17
ACh turnover rate (pmol/g tier hr)
(h%) 25.0 f 13.0 * 8.8 i 2.5 f
1.2 0.91 1.6 0.20
1x + 0.83
1.3 0.20 0.20 0.092 0.52
& 0.063 f 0.010 f 0.035 f 0.0074 ) 0.034
Choline
ACh (nmolig)
(nmol;g)
51 + 5.0 15 * 1.1 24 + 2.2 38 * 2.5 29 i_ 4.2
46 40 44 44 38
k * * i_ +
3.0 3.4 3.0 3.0 3.6
*Each value is the mean k S.E.M. of 10 determinations. 1\&is the fractional rate constant for the efflux of radioactive choline and li, is the correspondent parameter for ACh calculated according to RACAGNI r/ nl. (1974) from the change with infusion time of choline specific activit) and .ACh specific acti\ 11y. Infusion phosphoryl [Me-14C]choline (25 PCiimin per kg) (sp. act. 49 LtCi ‘/tmol).
single data point during the infusion of phosphoryl[Me-14C]choline. Designating the fractional rate constant of choline in the case of drug treated animals as ka it can be expressed by the following relationship: k, = k,(l A
- eekk’l)
~____
R,(l - emkA’l)’
where R, is equal to the ratio of SCh in treated control animals
and
R = SO,(treated) ’ So(control)’ Since R, can be measured, k, can be calculated and therefore also the correspondent ka, can be determined (CHENEY et a/., 1974). The ACh turnover rare of curious brain structures Table 2 shows the values of kA. kR and ACh turnover rate in various areas of rat brain as they are calculated during constant rate of phosphoryl[Me-‘4C]choline infusion from the change with time of choline specific activity and ACh specific activity. The highest value of ACh turnover rate was in striaturn and the lowest value in brainstem where it was less than one tenth of the value found in striatum. The turnover rate of ACh was unrelated to the steady-state concentration of the neurotransmitter and to the value of kA. Since the concentration of choline was similar in the various structures (Table 2), considering the value of k,, and assuming a uniform distribution of choline in the cells of various brain parts, in striatum the turnover time of choline was almost entirely accounted for by the value of the turnover time of ACh. In contrast, in brainstem and cortex, the turnover time of choline was several times faster than that of ACh (Table 2). From these data, one might infer that cholinergic neurones are more uniformly distributed in striatum than in brainstem; perhaps, in this structure there is a high concentration of ACh in a small portion of the tissue sample. When this possibility was investigated it was found that in this tissue, most of the ACh is located in the nucleus interpeduncularis (CHENEY, LE FEVRE and RACAGNI, 1975b) which represents about l/80 in weight of the tissue sample which was termed brainstem in Table 2.
The anatomical substrate. In mammalian brain the striatum is a structure that traditionally has been recognized as a prominent site for the mediation of synaptic transmission by ACh release. This telencephalic nucleus contains a high concentration of ACh which is located in relatively small neurones with short bifurcate axons (LYNCH, LUCASand DEAL>WYLER. 1972; BUTCHER and BUCHER, 1974). These cholinergic neurones are intrinsic to striatum and, therefore, do not participate in the efferent output from this nucleus (BUTCHER and BUTCHER, 1974). Since GABA-minergic neurones contribute greatly to the output from striatum. the role of the intrinsic cholinergic neurones in striatal physiology might be viewed as one of regulation in the filtering of afferent information originating from thalamus and various cortical areas (KEMP and Powell, 1971). The information that the striatum has filtered is conveyed to neurones in the globus pallidus and other structures. Dopaminergic innervation impinging on numerous cholinergic interneurones participates in this filtering by inhibiting the activity of most neurones it innervates (BLOOM and HOFFER, 1973). Available evidence suggests that in tGo the rate of dopamine (DA) release from nerve endings in striatum regulates the cholinergic neurones by inhibiting the efflux of ACh (STADLER, LLOYD and GADEA-CIRIA, 1973). Moreover. evidence has been obtained of dopaminergic innervation of cholinergic neurones (MCGEER, PATRICK. MCGEER, DARSHAN, GREWAAL and SINC;H, 1975). Thus, by studying how the ACh turnover rate in striatum has been modified by drugs which increase or decrease DA synaptic function, we have learned that striatal ACh turnover rate can be increased during antipsychotic-induced catalepsy (TIUHU~~HI. CHI;NEY, RACAGNI and COSTA. 1974) and decreased (TRABUCCHI, CHENEY, RACACNI and COSTA. 1975) concurrently with stereotypic behaviour. Since other data show that the catalepsy elicited by morphine in the striatum is not associated with an increase in ACh turnover rate, a change in striatal ACh turnover rate may not be associated with drug-induced catalepsy. This is not surprising because the output of striatum is not cholinergic. Perhaps GABA and other transmit-
728
G. RATAGNI.D. L. CHENEY,G. ZSILLAand E. COSTA
ters, in addition to ACh. modulate the neuronal output from this nucleus. The nucleus accumbens septi also contains intrinsic cholinergic neurones and important dopaminergic afferents (UNGERSTEDT, 1971). Preliminary experiments show that there are differences in the way DA neurones modulate the ACh turnover rate in striatum and nucleus accumbens septi (COSTA c’t uI.. 1975). An important control in the activity of DA neurones is the feedback loop of GABA-minergic neurones described by MCGEER, FIBIGER, HATTORI, SINGH, MCGE~K and MOLER (1974) and FONNUM, GROFOVA. RINVIK, STORM-MATHISEN and WALBERG (1974). The striatum contains GABAminergic interneurones but also contains GABA neurones which send their axons to the globus pallidus. It is not known whether the same axon bifurcates and sends branches to both pallidum and substantia nigra or if there is a specialized population of striatal GABA-minergic neurones in charge of the connections between striatum and substantia nigra. Howeve’r, it is known that in the pars reticulate of the nigra there are synapses which contain glutamic acid decarboxylase (ROBERTS 1976). Recently BJBRKLUND, LINDVALL and NOBIN (1975) have shown that the dopaminergic neurones of substantia nigra possess elongated dendrites protruding in pars reticulate which contains dopamine. Actually, there is evidence suggesting that dopaminergic dendrites form reciprocal synapses with axon terminals that contain glutamic acid decarboxylase (ROBERTS.1976) and substance P (H~KFELT, 1976). Among the striatal projections to the globus pallidus, two main pathways can be distinguished which project to the external and internal segments (KEMP and POWELL, 1971). In turn. the external segment of the globus pallidus sends fibres to the subthalamic nucleus and the internal segment to the centromedian nucleus of the thalamus and to the midbrain tegmentum. The centromedian nucleus of the thalamus sends important connections to the motor cortex and receives important afferents from it. Moreover. this thalamic nucleus is a centre where pallidal and cerebullar afferents overlap (KEMP and POWELL, 1971). The striatum is strategically placed to integrate the entire cerebral cortex activity with that of the intralaminar nuclei of the thalamus and the midbrain tegmentum and to influence the motor area of the cortex through the intralaminar system. Although we have not yet reached a complete understanding of how the striatum functions, several lines of electrophysiological investigations indicate that the striatum operates as a filter of the cortical efferent that controls motility. The operation of this filter integrates with that of cerebellum and feeds back into the cortex. The biochemical operation The efferent fibres from the striatum are inhibitory in nature. small in number in comparison to the aRerent and probably operate through a release of GABA. This inhibitory transmitter participates at two levels in the biochemical operation of striatum: GABA is released by small
interneurones for local control of excitability (MCGEER and MCGEER, 1975a) and it carries messages initiated in striatum to the globus pallidus. thalamus and substantia nigra (TRUEX and CARENTER. 1969; MCGEER and MCGEER, 1975b: FONNUM et al., 1974). We do not know whether all these fibres are GABA-minergic only. It is probable that they include a number of other transmitters as well. The first transmitter that comes to mind is ACh because choline acetyltransferase (ChA) and ACh are very high in striatum. However, lesions of known afferents and efferents to the neostriatum fail to change ChA levels. indicating that ChA in striatum is not primarily associated with neurones that have their cell bodies outside the ncostriatum (BUTCHER and BUTCHER, 1974). Confirmatory evidence for the existence of cholinergic interneurones has recently been obtained (SINGH, MCGEER and MCGEER. 1975). The cell bodies that generate efferent axons from the striatum arc large or medium sized and so far there is no reliable evidence that ChA is localized in large and medium sized neurones. but the participation of ACh in one of the pathways efferent from the striatum cannot be categorically excluded on the basis of the available evidence. The localization of glutamic acid decarboxylasecontaining cell bodies in the neostriatum is less certain than that of ChA-containing cell bodies (MCGEER and MCGEEK. 1975a). However, uptake studies of [‘HIGABA show that the label can be localized in cell bodies and nerve terminals of striaturn (HATTORI, MCGEER, FIBIGER and McGeer, 1973). These data suggests that in the rat striatum there is a substantial number of GABA-minergic interneurones (MCGEER and MCGEER, 1975a; FONNUM et ul., 1974). KIM, BAK, HASSLERand IKADA (1971) reported a decrease of glutamic acid decarboxylase in the substantia nigra following caudate lesions, suggesting that the primary source of glutamic acid decarboxylase and GABA in substantia nigra is the well known striatum nigra pathway. Other data by MCGEER, MCGUR. WADA and JUNC; (1971) would suggest that there are glutamic acid decarboxylase neurones in globus pallidus with projections to the substantia nigra. The possibility that there is a striato pallidal GABA-minergic pathway is in keeping with lesion studies showing that a lesion between the striatum and globus pallidus reduces the glutamic acid decarboxylase in globus pallidus by 23”,, and fails to reduce the glutamic acid decarboxylase in substantia nigra (HATTORI et (II., 1973). An important inhibitory afferent to ihe striatum is the dopaminergic nigra striatum pathway. When striatal dopamine receptors are stimulated, motility is increased and stereotyped behaviour may follow; when these receptors are blocked motor activity is reduced and muscle tone increases. Recent evidence suggests that dopaminergic terminals innervate small cholinergic interneurones (MCGEER and MCGEER. 1975a).
ACh turnover Table 3. Turnover
129
rate
rate of ACh in striatum of rats after injection tropine and apomorphine
Treatment (Acmolikg i.p.)
ACh (nmolig) 50 46 33 55
Saline Haloperidol (IO) Bcnrtropine (I 2) Apormorphine (I I) Haloperidol (10) + BenTtropine (12) Halopcridol (IO) + Apomorphine ( 11)
k k * +
3.2 4.1 ?.I* 4.0
of haloperidol,
Turnover (h? 22 39 33 12
+ f * +
‘)
(pmol/g
1.8 2.0* 1.9* 1.7*
1.1 1.8 1.09 0.7
+ k * k
benzo-
rate of per hr) 0.088 0.051* 0.12 0.025*
38 k 3.5*
29 * 2.1*
1.09 + 0.073
54 + 3.4
18 k 2.4
0.98 + 0.041
* P < 0.05. Rats were killed by a focussed microwave beam 40 min after the injection of haloperidol and benrtropine and 20 min after apomorphine. I,,, = Fractional rate constant of ACh etllux. The time and rate of radioactlve phosphorylcholine infusion as in Table 2. Each value is the means k S.E.M. of at least six rats. Data reported here taken from
RXAGNI (jr rrl. (1976) T/W cation c?/’~wrious drqs.
To gain
to antagonize stereotypies (RACAGNI, CHENEY, TRABUCCHI and COSTA, 1976). Some differences in the neurones is regulated we measured the steady-state pharmacological profile of clozapine and haloperidol and turnover rate of striatal ACh when the function were found when we compared their action on striatal of dopamininergic neurones was pharmacologically ACh turnover rate. Table 4 shows that clozapine, unperturbed. The ACh turnover rate in striatum of rats like haloperidol, did not change striatal ACh turnover injected with haloperidol, which blocks dopaminergic rate and antagonized the decrease in the turnover rate receptors. is shown in Table 3. Haloperidol increased of ACh elicited by oxotremorine, a muscarinic recepACh turnover rate and this increase was blocked tor agonist. In addition, clozapine given together with either by apomorphine. a direct stimulant of dopahaloperidol prevented the increase in ACh turnover minergic receptors, or by the injection of benztropine, rate caused by the butyrophenone derivative. Haloa muscarinic receptor blocker (Table 3). Apormorperidol and clozapine block DA stimulation of striaphine decreased (TKAHIJC(.HI ef al.. 1975) ACh turntal adenyl cyclase, but only haloperidol induces cataover rate. whereas benztropinc failed to change the tonia, antagonized stereotypies in rats and causes striatal ACh turnover rate but it decreased the ACh extrapyramidal side effects in man. content (Table 3). Studies in vitro (MILLER and HILEY, 1974; SNYDER, Clozapine is a neuroleptic that is devoid of extraGREENBERGand YAMAMURA,1974) and in vivo (STILLE pyramidal side effects in man (DEMAIO, 1972; STILLE. er ul., 1971) have demonstrated the anticholinergic LAVENEKand EITHENBERGER,1971) but it blocks DA properties of clozapine. Our previous work (Table 4) stimulation of adenylcyclasc in striatal homogenates has shown that clozapine, similarly to many antimus(CARENZI. CHKWL., COSTA. GUIDOTTI and RACAGNI, carinic agents, decreases the striatal ACh content 1975). In rats this drug fails to elicit catatonia and without changing ACh turnover rate (RACAGNI er al., standing
on
how
the
Table
activity
some
of striatal
4. Interaction
between
under-
cholinergic
clozapine, oxotremorine rate ACh in rat striatum
Treatment (/lmoljkg i.p.) Saline Clozapine (30) Clozapine (60) Oxotremorine (9) Haloperldol (10) Clorapine (30) (9) + Oxotremorine Clorapine (60) + Haloperidol (IO) * P < 0.05. Rats were killed 40min after morinc injection. Time course 2. Data reported here are taken S.E.M. of at least six rats. kR =
ACh (nmol&) 52 51 35 82 46
k * + + k
2.8 3.3 2.5* 5.0* 4.1
and
(h;’ 1, 20 20 27 8 37
+ + k k *
1.8 1.3 l.l* l.O* 2.0*
haloperidol
on turnover
Turnover rate of ACh (@mol/g per hr) 1.09 1.07 0.95 0.64 1.7
i. + * k k
0.092 0.053 0.10 0.071* 0.081*
57 + 4.8
16 + 2.1
0.92 + 0.074
38 f 23*
28 * 1.3*
1.06 + 0.078
haloperidol and 30min after clozapine and oxotreand rate of phosphorylcholine infusion as in Table from RACACNI ef al. (1976). Each value is the mean k fractional rate constant of ACh efflux.
G. RACA~NI.D. L. CHENEY.G. ZSILLAand E. C~STA
730
1976). Perhaps, the association of anticholinergic and antidopaminergic properties in the molecule of clozapine may be a clue to explain the lack of extrapyramida1 side effects observed with clozapine. However, the pharmacological profile of this drug should be studied more thoroughly with regard to its effects on GABA and substance P. In fact, these two transmitters (ROBERTS, 1976; H?XFEI.T, 1976) may participate in the regulation of doparminergic neurones in the substantia nigra. Several lines of independent investigation indicates tha: the action of several antipsychotics is related to their capacity to block DA receptors (CARLSSON, 1974), whereas some extrapyramidal side effects which they elicit can be prevented by ACh receptor blockers. Other lines of investigations have suggested that an activation of DA receptors is associated with an exacerbation of schizophrenic symptoms (DAVIS, 1974). Since DA terminals innervate ACh neurones in striaturn (MCGEER and MCGEER, 1975b) it is possible that some schizophrenic symptoms are also related to changes in cholinergic function. In Figure 2 we have attempted to analyze on the basis of the present understanding of the synaptic mechanisms that regulate striatal function, why the extrapyramidal side effects of antipsychotics can be reduced by blockers of cholinergic receptors. It appears that in striatum the dopaminergic receptors reside on cholinergic cells (MCGEER and MCGEER, 1975a. b). These interneurones can exert either a stimulatory or an inhibitory influence on the GABA-minergic neuronal loop that links striatum with substantia nigra. Since DA exerts inhibitory influences on striatal cells (BLOOM, COSTA and SALMOIRAGHI,196.5). the blockade of DA receptors may facilitate the activity of cholinergic interneurones. This possibility is documented indirectly by the increase in ACh turnover rate elicited by DA receptor blockers (Table 3). From electrophysiological experiments (AGHAJANIAN and BUNNEY, 1973), we
POSSIBLE MODELS OF NEURAL BETWEEN
INTERACTION
STRIATUM AND SUBSTANTIA NIGRA
RS ..
REClPROCAL . . ..(. GABA-mlnergic
SYNAPSE
.’ B
TURNOVER
G. RACAGNI, D. L. CHENEY, G. ZSILLAand E. COSTA Laboratory of Preclinical Pharmacology, National Institute of Mental Health. Saint Elizabeths Hospital. Washington D.C. 20032
INTRODUCTION The interest of pharmacologists to develop methods that estimate brain a~tylcholine (ACh) turnover rate in L+TO,stems from the belief that ACh turnover rate increases when the activity of cholinergic neurones is enhanced. Historically, this belief developed as a result of the experiments on cat superior cervical sympathetic ganglia performed by MACINTOSH (1963). These experiments established that at rest, the fractional rate constant for the renovation of acetylcholine is about 6 hr...‘. It was also shown that in ganglia the resting ACh turnover rate never exceeded ZOO/, of the maximum rate that could be attained during electrical stimulation of preganglionic nerves. Since 90’:); or more of the 3.5 nmol of ACh stored in cat sympathetic ganglia resides in terminal axons, the ganglion was an almost ideal model to correlate ACh turnover rate with nerve activity. Unfortunately, the whole brain is not such an ideal model to study this correlation. The ubiquity and intricacy of the cholinergic network plays havoc with accurate measurements of ACh turnover rate in terminals. Moreover. it is very unlikely that drugs simultaneously change the activity of every cholinergic neurone in the brain in the same direction. A possible exception might be the drugs which block either cholinesterase or choline a~tyltransferas~. More specifically, the questions that the neuropharmacologist might wish to answer with ACh turnover rate studies may concern whether or not cholinergic mechanisms participate in the action of drugs that modify transmission mediated by serotonin, dopamine, y-aminobutyric acid (GABA), substance P, etc. In fact, an assessment of drug actions on the integ~tion of different synaptic mechanisms may give a new dimension to the profile of centrally acting drugs. Such information may provide a guide in the synthesis of new antipsychotics with a low potential liability to elicit tardive dyskenesia (ZIVKOvie. GUI~)OTTI. RI-‘VUELTAand COSTA (1975). * This paper was presented at the Round Table entitled “Recent Concepts and Methods to Measure in Go the Turnover Rate of Neurotransmitters”, which was part of the official programme of the 5th International Meeting of the International Society of Neurochemistry, held in Barcelona. Spain. September 1975.
CONSIDERATIONS ON VARIOUS APPROACHES TO MEASURE ACh TURNOVER RATE A major stimulus for the development of methods to measure ACh turnover rate it2 uivo has been the possibility that drug actions on central cholinergic mechanisms can be described in terms of their capacity to change the ACh turnover rate. The report of MACINTOSH(1963) not only supported this contention but also suggested that by perfusing brain tissue and measuring the ACh content of the perfusate one might measure the rate of ACh release. By measuring the release of ACh elicited by electrical stimulation of brain slices in the presence or in the absence of eserine and/or atropine, the rate constant of ACh release was measured (SZERB, 1975). However, the physiological significance of these in citro experiments is open to question. A more reliable estimation of the dynamic state of ACh stores can be obtained by in oiro perfusion of limited areas of brain cortex using perfusion cups (BEANI and BIANCHI,1970; COLLIER and MITCHELL, 1967: PEPEU, 1973). In these experiments ACh e@lux rates are obtained which must be corrected for changes in steady-state in order to obtain a measurement of ACh turnover rate. A case in point illustrating the necessity of such a correction is the action of atropine on ACh stores in brain: the ACh efflux rates are increased by atropine but this drug also lowers brain ACh content (HOLMST~~T. LUNDGREN and SUNI>WALL. 1963). When the ACh efflux rates are corrected for the change in ACh steady-state, one often finds that the turnover rate is not increased although atropine increases ACh efflux rates in superfusion experiments (DUDAK and SZEKK, 1969). More recently, push-pull cannulae have been used with profit in studying the action of drugs on cholinergic mechanisms. This approach will not be discussed in the present paper and we refer to a recent critical evaluation made by BARTHOLINI, STADLER,GAD~A-CIKIA and LI.OYI, (1976).
Decline gf brair~ AC% content &er blockade oj synthesis to rneusure ACh turnover rate In brain, the turnover rate of a transmitter can be measured from the slope of the decline of transmitter
124
G. RACAGNI,D. L. CHENEY.G. ZSILI.Aand E.
COSI.A
content, elicited by the blockade of its synthesis Isotopic methods to ~musuw AC/7 t~tntorw txfc Although these methods minimiTe some of the (COSTA and NEFF, 1966). Hemicholinium-3 (HC-3) problems we have discussed as inherent to the nonisoblocks ACh synthesis (GARDINER, 1961) and intraventopic methods, they include a number of ~~ssurnptiolls tricular injection of He-3 was used to measure brain which cannot be fully substantiated by experimental ACh turnover rate (DOMINO and WILSON, 1972). The evidence. Hence, these methods arc not reliable to assumption implicit in this measurement is that at measure absolute values of ACh turnover rate; neversteady-state, the rate of synthesis and degradation of theless, they are adequate for comparative neuropharACh are in equilibrium. The method therefore utilizes macological studies of ACR turnover rate. principles of steady-state kinetics and applies them ~~lili~?u~f~~~~ ~~thoi~s. Radioactive methods utilize to the decrease of ACh content caused by the instanthe measurement of the specific radioactivity of ACh taneous blockade of ACh synthesis. By defining the and that of its precursor choline at various times after steady-state concentration of brain ACh as a dynamic equilibrium between synthesis and metabolism, when the injection of choline or of a radiol;~bcllcd precursor the ACh synthesis is blocked completely, the slope of choline (DROSS and K~w~rz. 1972; SCHU~I:K~FI, whereby brain ACh content declines with time can SPARF and SUNI)WALL, 1969; HANIY. CHIMY. TKAbe used to calculate the rate constant of ACh ef%x. NUCCIII, M~~~~~1.1~1. WAY bc a precursor of tion of its synthesis requires that the efflux rates of brain choline (ANSI-:LLand SPANSI:K. 1971). RadioACh be calculated from the initial rates in the decline active phos~~liorylcholine mivcs rapidly with the of brain ACh content. In almost every pharmacologiendogenous pool of phosphorylcl~oline and yields cal experiment known to us, calculations of ACh radioactive choline which mixes quite readily with turnover rate were made from a single time point taken at a fixed interval after HC-3 injection (DOMINO the endogenous pool of choline (HANIL cl c/l., 197.7). By injecting phosphorylcholine. WC have obtained a and WILSON, 1972). Since the ground rules for the high specific activity of brain choline (RX’Ac;NI. TKAapplication of principles of steady state kinetics 1975). Moreover, using Crfn&l:v. ULJfCH1 and require assessment of initial rates of ACh decrease. [ 1‘C~phospllorylch~~line iiljected cithcr by pulse injecat least three time points must be used to calcution or by constant rate infusion we have mcasurcd late ACh efflux. In addition, since the assumption relabelled ACh in samples of brain tissue weighing quires that the ACh is stored in a single metabolic about I Gt?-300 mg without changing the steady-state compartment open at both ends, one must demonof brain choline or AC% (CHI:U~:Y. TKAI%~,(‘(‘HI. strate that ACh decline follows first order kineRACAC;XIand Cosrh. iY?4). tics.
725
ACh turnover rate Stable isotope methods. When ACh turnover rate measurements are performed in brain nuclei, mass fragmentography must be used because it possesses the necessary sensitivity for these measurements (KosLOW, RACAGNI and COSTA, 1974). This necessity also imposes that labelling be performed with stable isotopes Pulse injections of deuterated phosphorylcholine cannot be used to label substantially the choline and ACh contained in single brain nuclei (tissue weight 1 mg or less) without changing the steadystate. However. a constant rate infusion with deuterated phosphorylcholine can label the choline and ACh content, thus allowing the measurement of ACh turnover rate (COSTA. CHENEY, RACAGNI and ZSILLA, 1975). The necessity to use constant rate infusion of the label derives from the fact that the detection of stable isotopes by mass fragmentography requires that at least loo/, of the pool be labelled. Considering that a brain nucleus usually contains a few picomoles of ACh. the detection of the label at 10:; implies a measurement in the range of 20&300 femtomoles. This is the limit of mass fragmentographic detection of ACh (KOSLOW et al.. 1974).
MEASUREMENT OF
ACh TURNOVER
VARIOUS STRUCTURES CONSTANT
RATE IN
OF RAT BRAIN USING A
RATE INFUSION
OF PHOSPHORYL-
[Me-“CICHOLINE
Kinetic
corwidrrafions
m brain which proceeds at rates of 20nmol/g tissue per min (DROSS and KEWITZ, 1972). Since even the immersion of the rats in liquid nitrogen freezes the brain in not less than 20sec, the errors in the brain choline measurements using immersion in liquid nitrogen can be conspicuous. Only after microwave radiation, can brain dissection be carried out without changing the steady-state content of ACh and choline (GUIDOTTI et al.. 1974). Using constant rate infusion of radioactive phosphorylcholine in combination with microwave radiation (RA~AGNI et al., 1974) is it possible to overcome two of the problems which, in the past. have hampered the measurement of ACh turnover rate. (1) The postmortem changes in ACh and choline due to residual enzyme activity; (2) the distortion in the precursor product relationship between choline and ACh which is due to a feedback labelling caused by the reupt’ake of labelled choline originating from the reuptake of radioactive ACh released by nerve impulses, Normally. 80”. of the choline that is generated from ACh released extraneuronally by nerve impulses is taken back into the nerve endings, where is becomes a substrate for choline acetyltransferase and recycles to form endogenous ACh (HEBB, 1972). This conspicuous participation in the recycling of the choline generated from ACh is due, in part, to a preferential utilization of newly taken up choline for ACh synthesis (GLYEMT. LEFRESNE. BEAUJOVAN and GLOWINSKI, 1975). When [‘4C]phosphorylcholine is infused at constant rate for 10-I 2 min the specific activities of ACh and choline in brain parts increase slowly but continuously (Fig. I). Since during short term constant rate infusion of [r4C]phosphorylcholine, the specific activity of choline is always higher than that of ACh in all structures studied, distortions due to the perferential utilization of the newly taken up choline for ACh synthesis are not apparent (Fig. 1).
Phosphoryl [Me-‘4C]choline (sp. act. 49 &i/pmol) was usually infused intravenously through the tail vein at a constant rate (25 &i/min per kg). At the end of the infusion or at various times thereafter, the rats were killed by microwave radiation (2.0 kW; 2.45 GHz, 75 W/cm’) focussed on the head. A beam of microwave radiation. focussed on the skull of 12&15Og rats for 2 set, kills the rats instantaneously and inactivates brain enzymes promptly and irreversibly (GUIDOTTI, CHENEY, TRARUCCHI, Muthematical unalysis qf thr dtrta: calculation qf’ ACh DOTEUCHI. WANG and HAWKINS, 1974; STAVINOHA. turnoorr rate A basic tenet for the calculation of ACh turnover WEINTRAUB and MODAK, 1973). This technique prevents the postmortem accumulation of free choline rate from the change with time in isotope enrichment of 1000
20 Striatum
t
I/:
Brainstem
min
Fig. 1. Specific activity stem after different
of [“C]ACh (solid line) and [‘T]choline (dashed line) in striatum and braintimes of infusion at constant rates of phosphoryl [Me-‘4C]choline (25 &3/kg per min). Infusion was initiated at time 0.
G. RACAGM,D. L. CHENEY,G. ZSILLA and E. COSTA
726
choline and ACh after labelling with phosphorylcholine is that the compartmentation and steady-state eq~~ilibri~~m among the various pools of ACh and choline do not change during the infusion of the label. Thus: the ACh synthesis rate and not the rate of change of choline specific activity is the factor which determines the change with time of ACh specific activity. This point is documented by the data graphed in Figure I. In the various brain structures. the rate of ACh specific activity increase is clearly independent from the rate of change of choline specific activity. For instance, in Figure 1~ the rate of change of choline specific activity is faster in brainstem than in striatum and yet the increase in ACh specific activity is faster in striatum than in brainstem. Hence, it can be inferred that an endogenous process at different rates in different brain parts regulates the formation of labelled ACh from labelled choline. We infer that this process reflects the rate of endogenous metabolism of ACh. The data of Figure 1 show that Iabelled phosphoryleholine is rapidly transfor~ned into free brain choline. However. it should he apparent that the rate of change in choline specific activity is different in different brain parts. Hence, we might infer that the various metabolic pools of brain choline are enriched with labelled congencr at a rate proportional to their intrinsic metabolic rates of chotine. During the infusion of radioactive pllosphory~choli~l~. some labet may enter the brain as the ester, and phosphorylcholine may bc dephosphorylated irl silzc to generate choline. Moreover. the present understanding of choline metabolism in brain suggests that phosphorylcholine may also be converted into phosphatidylcholine (KEU’ITZ. PLEUL. DROSS and S~~~~AR~.~~~~~, 1975). Therefore, the difference in the rate of radioactive choline accumulation in various brain parts during constant rate infusion of phosphorylcholine (Fig. 1) reflects diRerent rates of phosphorylcholine metabolism in addition to differences in the metabolism of endogenous choline. Possibly, during constant rate infusion phosphoryl~hoiine, conversion in choline may interfere with the measurement of ACh turnover rate. However, when ACh synthesis was measured in whole mouse brain by labeliing with radioactive choline Table
(SPARF, 1974) or phosphorylcholine (HANIN et al., 1973) the rates calculated from data obtained with the two methods were similar (Table 1). The mathematical procedure for the calculation of ACh turnover rate was derived from the following model (RACACN rf ul., 1974).
where k,, + kAZ = k,, and kR1 + kB2 = k8 and A is the compartment of free choline and B is that of ACh in any given brain structure. The rate of entrance of labelled choline into compartment A is indicated by J, while kA and kB are the fractional rate constants for the efRux of products from compartments A and B, respectively. The change of choline specific activity (S,,) and ACh specific activity (SACb) with time is the function of J which can be controlled.
kAe-Q + k,-k,
can be experimentally %I, and SAC,, k,, can be calculated
Injected ___[‘H]Cholim$ Phosphoryl [?ifc-‘“C]choline:
SC&i) m=SACho. Since m can be calculated from the experimental resufts kg and kB can be determined. When k, is known then AC% turnover rate can be calculated and equals k,, (ACh). Since the kA of each brain area is reproducible under identical experimental conditions if drugs fail to change k,, it is then possible to calculate ACh turnover rate after drug treatment from a following pulse intravenous injection by the finite difference method*
AC11 (nmolig) __..-
k,,.,** (min- ‘)
Turnover rate of ACh*** (nmol/g per min)
151-
0.24 * 0.048 0.25 ) 0.012
3.6 4.5
I8 & 0.72
k, and
where
1. Tutnoter rate of acetylcholine in whole mouse brain of [.~H~lcholinc or phosph~~ryl ~~~~-‘4C]choline calculated -
Compound
measured
mathematically:
* NEW (‘I ‘il. ( i 97 1). ** Yractionai rate constant for ACh (k,,.,,) was calculated from 1 min time intervals abstracted from smooth curves of choline and ACh. Specific radioactivities between 1 and 20min after injection of radiolahcl. *** Turnover rate of ACh obtained by multiplying kAch by steady-state concentration of ACh. 4 [3H]-Choline (160 &i/mouse) injected intravenously (SPARF,1974). t Average ACh concentration abstracted from literature (SPARF,1974). $ Phosph(3ryl [Mc-‘JC]choline (250 itCi!kg) injected ~ntraveno~lsiy (HAWN & uf., 1973).
121
ACh turnover rate Table 2. Kinetic parameters and ACh turnover rate in rat brain areas* (h? ‘) Striatum
Occipital cortex Limbic cortex Brainstem Hippocampus
17 47 16 41 17
ACh turnover rate (pmol/g tier hr)
(h%) 25.0 f 13.0 * 8.8 i 2.5 f
1.2 0.91 1.6 0.20
1x + 0.83
1.3 0.20 0.20 0.092 0.52
& 0.063 f 0.010 f 0.035 f 0.0074 ) 0.034
Choline
ACh (nmolig)
(nmol;g)
51 + 5.0 15 * 1.1 24 + 2.2 38 * 2.5 29 i_ 4.2
46 40 44 44 38
k * * i_ +
3.0 3.4 3.0 3.0 3.6
*Each value is the mean k S.E.M. of 10 determinations. 1\&is the fractional rate constant for the efflux of radioactive choline and li, is the correspondent parameter for ACh calculated according to RACAGNI r/ nl. (1974) from the change with infusion time of choline specific activit) and .ACh specific acti\ 11y. Infusion phosphoryl [Me-14C]choline (25 PCiimin per kg) (sp. act. 49 LtCi ‘/tmol).
single data point during the infusion of phosphoryl[Me-14C]choline. Designating the fractional rate constant of choline in the case of drug treated animals as ka it can be expressed by the following relationship: k, = k,(l A
- eekk’l)
~____
R,(l - emkA’l)’
where R, is equal to the ratio of SCh in treated control animals
and
R = SO,(treated) ’ So(control)’ Since R, can be measured, k, can be calculated and therefore also the correspondent ka, can be determined (CHENEY et a/., 1974). The ACh turnover rare of curious brain structures Table 2 shows the values of kA. kR and ACh turnover rate in various areas of rat brain as they are calculated during constant rate of phosphoryl[Me-‘4C]choline infusion from the change with time of choline specific activity and ACh specific activity. The highest value of ACh turnover rate was in striaturn and the lowest value in brainstem where it was less than one tenth of the value found in striatum. The turnover rate of ACh was unrelated to the steady-state concentration of the neurotransmitter and to the value of kA. Since the concentration of choline was similar in the various structures (Table 2), considering the value of k,, and assuming a uniform distribution of choline in the cells of various brain parts, in striatum the turnover time of choline was almost entirely accounted for by the value of the turnover time of ACh. In contrast, in brainstem and cortex, the turnover time of choline was several times faster than that of ACh (Table 2). From these data, one might infer that cholinergic neurones are more uniformly distributed in striatum than in brainstem; perhaps, in this structure there is a high concentration of ACh in a small portion of the tissue sample. When this possibility was investigated it was found that in this tissue, most of the ACh is located in the nucleus interpeduncularis (CHENEY, LE FEVRE and RACAGNI, 1975b) which represents about l/80 in weight of the tissue sample which was termed brainstem in Table 2.
The anatomical substrate. In mammalian brain the striatum is a structure that traditionally has been recognized as a prominent site for the mediation of synaptic transmission by ACh release. This telencephalic nucleus contains a high concentration of ACh which is located in relatively small neurones with short bifurcate axons (LYNCH, LUCASand DEAL>WYLER. 1972; BUTCHER and BUCHER, 1974). These cholinergic neurones are intrinsic to striatum and, therefore, do not participate in the efferent output from this nucleus (BUTCHER and BUTCHER, 1974). Since GABA-minergic neurones contribute greatly to the output from striatum. the role of the intrinsic cholinergic neurones in striatal physiology might be viewed as one of regulation in the filtering of afferent information originating from thalamus and various cortical areas (KEMP and Powell, 1971). The information that the striatum has filtered is conveyed to neurones in the globus pallidus and other structures. Dopaminergic innervation impinging on numerous cholinergic interneurones participates in this filtering by inhibiting the activity of most neurones it innervates (BLOOM and HOFFER, 1973). Available evidence suggests that in tGo the rate of dopamine (DA) release from nerve endings in striatum regulates the cholinergic neurones by inhibiting the efflux of ACh (STADLER, LLOYD and GADEA-CIRIA, 1973). Moreover. evidence has been obtained of dopaminergic innervation of cholinergic neurones (MCGEER, PATRICK. MCGEER, DARSHAN, GREWAAL and SINC;H, 1975). Thus, by studying how the ACh turnover rate in striatum has been modified by drugs which increase or decrease DA synaptic function, we have learned that striatal ACh turnover rate can be increased during antipsychotic-induced catalepsy (TIUHU~~HI. CHI;NEY, RACAGNI and COSTA. 1974) and decreased (TRABUCCHI, CHENEY, RACACNI and COSTA. 1975) concurrently with stereotypic behaviour. Since other data show that the catalepsy elicited by morphine in the striatum is not associated with an increase in ACh turnover rate, a change in striatal ACh turnover rate may not be associated with drug-induced catalepsy. This is not surprising because the output of striatum is not cholinergic. Perhaps GABA and other transmit-
728
G. RATAGNI.D. L. CHENEY,G. ZSILLAand E. COSTA
ters, in addition to ACh. modulate the neuronal output from this nucleus. The nucleus accumbens septi also contains intrinsic cholinergic neurones and important dopaminergic afferents (UNGERSTEDT, 1971). Preliminary experiments show that there are differences in the way DA neurones modulate the ACh turnover rate in striatum and nucleus accumbens septi (COSTA c’t uI.. 1975). An important control in the activity of DA neurones is the feedback loop of GABA-minergic neurones described by MCGEER, FIBIGER, HATTORI, SINGH, MCGE~K and MOLER (1974) and FONNUM, GROFOVA. RINVIK, STORM-MATHISEN and WALBERG (1974). The striatum contains GABAminergic interneurones but also contains GABA neurones which send their axons to the globus pallidus. It is not known whether the same axon bifurcates and sends branches to both pallidum and substantia nigra or if there is a specialized population of striatal GABA-minergic neurones in charge of the connections between striatum and substantia nigra. Howeve’r, it is known that in the pars reticulate of the nigra there are synapses which contain glutamic acid decarboxylase (ROBERTS 1976). Recently BJBRKLUND, LINDVALL and NOBIN (1975) have shown that the dopaminergic neurones of substantia nigra possess elongated dendrites protruding in pars reticulate which contains dopamine. Actually, there is evidence suggesting that dopaminergic dendrites form reciprocal synapses with axon terminals that contain glutamic acid decarboxylase (ROBERTS.1976) and substance P (H~KFELT, 1976). Among the striatal projections to the globus pallidus, two main pathways can be distinguished which project to the external and internal segments (KEMP and POWELL, 1971). In turn. the external segment of the globus pallidus sends fibres to the subthalamic nucleus and the internal segment to the centromedian nucleus of the thalamus and to the midbrain tegmentum. The centromedian nucleus of the thalamus sends important connections to the motor cortex and receives important afferents from it. Moreover. this thalamic nucleus is a centre where pallidal and cerebullar afferents overlap (KEMP and POWELL, 1971). The striatum is strategically placed to integrate the entire cerebral cortex activity with that of the intralaminar nuclei of the thalamus and the midbrain tegmentum and to influence the motor area of the cortex through the intralaminar system. Although we have not yet reached a complete understanding of how the striatum functions, several lines of electrophysiological investigations indicate that the striatum operates as a filter of the cortical efferent that controls motility. The operation of this filter integrates with that of cerebellum and feeds back into the cortex. The biochemical operation The efferent fibres from the striatum are inhibitory in nature. small in number in comparison to the aRerent and probably operate through a release of GABA. This inhibitory transmitter participates at two levels in the biochemical operation of striatum: GABA is released by small
interneurones for local control of excitability (MCGEER and MCGEER, 1975a) and it carries messages initiated in striatum to the globus pallidus. thalamus and substantia nigra (TRUEX and CARENTER. 1969; MCGEER and MCGEER, 1975b: FONNUM et al., 1974). We do not know whether all these fibres are GABA-minergic only. It is probable that they include a number of other transmitters as well. The first transmitter that comes to mind is ACh because choline acetyltransferase (ChA) and ACh are very high in striatum. However, lesions of known afferents and efferents to the neostriatum fail to change ChA levels. indicating that ChA in striatum is not primarily associated with neurones that have their cell bodies outside the ncostriatum (BUTCHER and BUTCHER, 1974). Confirmatory evidence for the existence of cholinergic interneurones has recently been obtained (SINGH, MCGEER and MCGEER. 1975). The cell bodies that generate efferent axons from the striatum arc large or medium sized and so far there is no reliable evidence that ChA is localized in large and medium sized neurones. but the participation of ACh in one of the pathways efferent from the striatum cannot be categorically excluded on the basis of the available evidence. The localization of glutamic acid decarboxylasecontaining cell bodies in the neostriatum is less certain than that of ChA-containing cell bodies (MCGEER and MCGEEK. 1975a). However, uptake studies of [‘HIGABA show that the label can be localized in cell bodies and nerve terminals of striaturn (HATTORI, MCGEER, FIBIGER and McGeer, 1973). These data suggests that in the rat striatum there is a substantial number of GABA-minergic interneurones (MCGEER and MCGEER, 1975a; FONNUM et ul., 1974). KIM, BAK, HASSLERand IKADA (1971) reported a decrease of glutamic acid decarboxylase in the substantia nigra following caudate lesions, suggesting that the primary source of glutamic acid decarboxylase and GABA in substantia nigra is the well known striatum nigra pathway. Other data by MCGEER, MCGUR. WADA and JUNC; (1971) would suggest that there are glutamic acid decarboxylase neurones in globus pallidus with projections to the substantia nigra. The possibility that there is a striato pallidal GABA-minergic pathway is in keeping with lesion studies showing that a lesion between the striatum and globus pallidus reduces the glutamic acid decarboxylase in globus pallidus by 23”,, and fails to reduce the glutamic acid decarboxylase in substantia nigra (HATTORI et (II., 1973). An important inhibitory afferent to ihe striatum is the dopaminergic nigra striatum pathway. When striatal dopamine receptors are stimulated, motility is increased and stereotyped behaviour may follow; when these receptors are blocked motor activity is reduced and muscle tone increases. Recent evidence suggests that dopaminergic terminals innervate small cholinergic interneurones (MCGEER and MCGEER. 1975a).
ACh turnover Table 3. Turnover
129
rate
rate of ACh in striatum of rats after injection tropine and apomorphine
Treatment (Acmolikg i.p.)
ACh (nmolig) 50 46 33 55
Saline Haloperidol (IO) Bcnrtropine (I 2) Apormorphine (I I) Haloperidol (10) + BenTtropine (12) Halopcridol (IO) + Apomorphine ( 11)
k k * +
3.2 4.1 ?.I* 4.0
of haloperidol,
Turnover (h? 22 39 33 12
+ f * +
‘)
(pmol/g
1.8 2.0* 1.9* 1.7*
1.1 1.8 1.09 0.7
+ k * k
benzo-
rate of per hr) 0.088 0.051* 0.12 0.025*
38 k 3.5*
29 * 2.1*
1.09 + 0.073
54 + 3.4
18 k 2.4
0.98 + 0.041
* P < 0.05. Rats were killed by a focussed microwave beam 40 min after the injection of haloperidol and benrtropine and 20 min after apomorphine. I,,, = Fractional rate constant of ACh etllux. The time and rate of radioactlve phosphorylcholine infusion as in Table 2. Each value is the means k S.E.M. of at least six rats. Data reported here taken from
RXAGNI (jr rrl. (1976) T/W cation c?/’~wrious drqs.
To gain
to antagonize stereotypies (RACAGNI, CHENEY, TRABUCCHI and COSTA, 1976). Some differences in the neurones is regulated we measured the steady-state pharmacological profile of clozapine and haloperidol and turnover rate of striatal ACh when the function were found when we compared their action on striatal of dopamininergic neurones was pharmacologically ACh turnover rate. Table 4 shows that clozapine, unperturbed. The ACh turnover rate in striatum of rats like haloperidol, did not change striatal ACh turnover injected with haloperidol, which blocks dopaminergic rate and antagonized the decrease in the turnover rate receptors. is shown in Table 3. Haloperidol increased of ACh elicited by oxotremorine, a muscarinic recepACh turnover rate and this increase was blocked tor agonist. In addition, clozapine given together with either by apomorphine. a direct stimulant of dopahaloperidol prevented the increase in ACh turnover minergic receptors, or by the injection of benztropine, rate caused by the butyrophenone derivative. Haloa muscarinic receptor blocker (Table 3). Apormorperidol and clozapine block DA stimulation of striaphine decreased (TKAHIJC(.HI ef al.. 1975) ACh turntal adenyl cyclase, but only haloperidol induces cataover rate. whereas benztropinc failed to change the tonia, antagonized stereotypies in rats and causes striatal ACh turnover rate but it decreased the ACh extrapyramidal side effects in man. content (Table 3). Studies in vitro (MILLER and HILEY, 1974; SNYDER, Clozapine is a neuroleptic that is devoid of extraGREENBERGand YAMAMURA,1974) and in vivo (STILLE pyramidal side effects in man (DEMAIO, 1972; STILLE. er ul., 1971) have demonstrated the anticholinergic LAVENEKand EITHENBERGER,1971) but it blocks DA properties of clozapine. Our previous work (Table 4) stimulation of adenylcyclasc in striatal homogenates has shown that clozapine, similarly to many antimus(CARENZI. CHKWL., COSTA. GUIDOTTI and RACAGNI, carinic agents, decreases the striatal ACh content 1975). In rats this drug fails to elicit catatonia and without changing ACh turnover rate (RACAGNI er al., standing
on
how
the
Table
activity
some
of striatal
4. Interaction
between
under-
cholinergic
clozapine, oxotremorine rate ACh in rat striatum
Treatment (/lmoljkg i.p.) Saline Clozapine (30) Clozapine (60) Oxotremorine (9) Haloperldol (10) Clorapine (30) (9) + Oxotremorine Clorapine (60) + Haloperidol (IO) * P < 0.05. Rats were killed 40min after morinc injection. Time course 2. Data reported here are taken S.E.M. of at least six rats. kR =
ACh (nmol&) 52 51 35 82 46
k * + + k
2.8 3.3 2.5* 5.0* 4.1
and
(h;’ 1, 20 20 27 8 37
+ + k k *
1.8 1.3 l.l* l.O* 2.0*
haloperidol
on turnover
Turnover rate of ACh (@mol/g per hr) 1.09 1.07 0.95 0.64 1.7
i. + * k k
0.092 0.053 0.10 0.071* 0.081*
57 + 4.8
16 + 2.1
0.92 + 0.074
38 f 23*
28 * 1.3*
1.06 + 0.078
haloperidol and 30min after clozapine and oxotreand rate of phosphorylcholine infusion as in Table from RACACNI ef al. (1976). Each value is the mean k fractional rate constant of ACh efflux.
G. RACA~NI.D. L. CHENEY.G. ZSILLAand E. C~STA
730
1976). Perhaps, the association of anticholinergic and antidopaminergic properties in the molecule of clozapine may be a clue to explain the lack of extrapyramida1 side effects observed with clozapine. However, the pharmacological profile of this drug should be studied more thoroughly with regard to its effects on GABA and substance P. In fact, these two transmitters (ROBERTS, 1976; H?XFEI.T, 1976) may participate in the regulation of doparminergic neurones in the substantia nigra. Several lines of independent investigation indicates tha: the action of several antipsychotics is related to their capacity to block DA receptors (CARLSSON, 1974), whereas some extrapyramidal side effects which they elicit can be prevented by ACh receptor blockers. Other lines of investigations have suggested that an activation of DA receptors is associated with an exacerbation of schizophrenic symptoms (DAVIS, 1974). Since DA terminals innervate ACh neurones in striaturn (MCGEER and MCGEER, 1975b) it is possible that some schizophrenic symptoms are also related to changes in cholinergic function. In Figure 2 we have attempted to analyze on the basis of the present understanding of the synaptic mechanisms that regulate striatal function, why the extrapyramidal side effects of antipsychotics can be reduced by blockers of cholinergic receptors. It appears that in striatum the dopaminergic receptors reside on cholinergic cells (MCGEER and MCGEER, 1975a. b). These interneurones can exert either a stimulatory or an inhibitory influence on the GABA-minergic neuronal loop that links striatum with substantia nigra. Since DA exerts inhibitory influences on striatal cells (BLOOM, COSTA and SALMOIRAGHI,196.5). the blockade of DA receptors may facilitate the activity of cholinergic interneurones. This possibility is documented indirectly by the increase in ACh turnover rate elicited by DA receptor blockers (Table 3). From electrophysiological experiments (AGHAJANIAN and BUNNEY, 1973), we
POSSIBLE MODELS OF NEURAL BETWEEN
INTERACTION
STRIATUM AND SUBSTANTIA NIGRA
RS ..
REClPROCAL . . ..(. GABA-mlnergic
SYNAPSE
.’ B
