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Acetylcholinesterase and the basal ganglia : from cytology to function Lehmann, John 1980

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ACETYLCHOLINESTERASE AND THE BASAL GANGLIA -FROM CYTOLOGY TO FUNCTION by JOHN LEHMANN B.Sc, C a l i f o r n i a I n s t i t u t e of Technology, 1974 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in... THE FACULTY OF GRADUATE STUDIES ( I n t e r d i s c i p l i n a r y Studies) We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA May 1980 John Lehmann, 1980 In presenting th is thes is in p a r t i a l fu l f i lment of the requirements for an advanced degree at the Un ivers i ty of B r i t i s h Columbia, I agree that the L ibrary shal l make it f ree ly ava i l ab le for reference and study. I fur ther agree that permission for extensive copying of th is thes is for scho la r ly purposes may be granted by the Head of my Department or by his representat ives . It is understood that copying or pub l ica t ion of th is thes is for f inanc ia l gain sha l l not be allowed without my wri t ten permission. Department of The Univers i ty of B r i t i s h Columbia 2075 Wesbrook P l a c e V a n c o u v e r , Canada V6T 1W5 6 i i ABSTRACT Biochemical, anatomical, and histochemical studies were performed i n the basal ganglia with an emphasis on the l o c a l i z a t i o n of the enzyme acetylcholinesterase (AChE). The existence of the enzyme i n dopaminergic n i g r o - s t r i a t a l neurons was demonstrated. Descending s t r i a t o - n i g r a l and p a l l i d o - n i g r a l axons did not contain detectable amounts of AChE. A c e l l group c a l l e d the nucleus b a s a l i s magno-c e l l u l a r i s , intimately associated with the globus p a l l i d u s , was found to contain high l e v e l s of AChE; furthermore, these neurons were shown to be the source of a ch o l i n e r g i c p r o j e c t i o n to the neocortex. In the striatum, large neurons containing high l e v e l s of AChE were found to be l i k e l y candidates as..the c h o l i n e r g i c neuron of the striatum. Cholinergic perikarya were found to be absent i n the neocortex; nor were perikarya synthesizing large amounts of AChE found i n the neocortex. An empirical hypothesis was formulated on the basis of these and other findings regarding c h o l i n e r g i c neurons: High l e v e l s of AChE are a necessary but not s u f f i c i e n t c r i t e r i o n for i d e n t i f y i n g c h o l i n e r g i c perikarya. i i i TABLE OF CONTENTS Abstract i i Table of Contents i i i L i s t of f i g u r e s i v Acknowledgements v GENERAL INTRODUCTION 1 a) H i s t o r i c a l overview of AChE 1 b) The basal ganglia and c h o l i n e r g i c function 8 c) B r i e f d e s c r i p t i o n of research and 10 r a t i o n a l e GENERAL DISCUSSION 13 a) Review of new contributions reported 13 i n t h i s thesis b) The r o l e of the striatum i n motor and 15 psychological function c) Psychopharmacology of the striatum 17 1) .Psychopharmacology of dopamine i n 17 the striatum 2) Psychopharmacology of ac e t y l c h o l i n e 18 i n the striatum d) Interactions between dopamine and a c e t y l - 19 choline 1) Psychopharmacological studies 19 2) Measurement of biochemical parameters 20 following pharmacological manipulations e) The c y t o l o g i c a l r e l a t i o n s h i p between the 21 dopaminergic and the c h o l i n e r g i c neuron REFERENCES 26 APPENDIX 35 i v LIST OF FIGURES Figure 1. T r a d i t i o n a l cytological model explaining dopamine:.- ac e t y l c h o l i n e i n t e r a c t i o n s 24 Figure 2. A l t e r n a t i v e c y t o l o g i c a l model proposed to explain the same dopamine - acetylcholine i n t e r a c t i o n s 25 V ACKNOWLEDGEMENTS My f i r s t thanks go to a l l the members, past and present, of the D i v i s i o n of Neurological Sciences, of which i t was an honour to be a part. As a cooperative u n i t with the goal of advancing s c i e n t i f i c knowledge, a l l the members of the D i v i s i o n set examples and gave me help and encouragement. Special thanks go to my supervisor, Dr. Chris F i b i g e r , f o r h i s uncanny a b i l i t y to motivate and d i r e c t my work. The actin g Department head, Dr. Edith McGeer, was i n s p i r a t i o n a l through giving me a sense of perspective i n neuroscience, and as an inexhaust-i b l e source of new ideas. Our groups' t e c h n i c a l s t a f f , S t e l l a Atmadja, Amelia Wong, and Betty Richter, not only taught me a great deal of p r a c t i c a l use, but we worked together so that at times the s c i e n t i f i c goal was the secondary reward. The other graduate students of the D i v i s i o n , always ready for discussion or debate, supplied the greatest amount of academic stimulation. F i n a l l y , my thanks go out to a l l the members of my committee and teachers of courses. S p e c i f i c acknowledgements f or each of the p u b l i c a t i o n s l i s t e d i n the appendix of t h i s thesis are as follows: P u b l i c a t i o n A): S. Atmadja for l e s i o n s , h i s t o l o g y , photomicroscopy; H.C. F i b i g e r f o r d i s s e c t i o n s , photomicroscopy. P u b l i c a t i o n B): S. Atmadja f or l e s i o n s ; H.C. F i b i g e r for d i s s e c t i o n s ; L.L. Butcher f o r hi s t o l o g y and photomicroscopy. P u b l i c a t i o n C): J . I . Nagy for le s i o n s and l e s i o n diagrams; S. Atmadja fo r photomicroscopy, HRP-AChE combined histol o g y , layout; H.C. F i b i g e r f o r d i s s e c t i o n , photomicroscopy. P u b l i c a t i o n D) : S. Atmadja for l e s i o n s , h i s t o l o g y . 1 GENERAL INTRODUCTION The general strategy adopted i n t h i s thesis was to investigate c h o l i n e r g i c systems of the extrapyramidal system, e s p e c i a l l y by studying the enzyme acetylcholinesterase (AChE). There i s a s p e c i a l advantage to studying AChE - i t may be assayed biochemically and also l o c a l i z e d histochemically, with excellent r e s o l u t i o n and contrast. Moreover, by pretreating animals with an i r r e v e r s i b l e AChE i n h i b i t o r , commonly diisopropylphosphorofluoridate (DEP), some time preceding s a c r i f i c e (Lynch et a l . , 1972), further advantages may be r e a l i z e d : Neurons which synthesize the enzyme at d i f f e r e n t rates may be d i s t i n -guished. By the use of DFP, the subce l l u l a r d i s t r i b u t i o n of the enzyme may be resolved with the l i g h t microscope, within l i m i t a t i o n s . The transport of AChE along axonal projections may be determined. For these reasons and others to be elaborated below, the study of AChE has been an i n t e g r a l part of each of the investigations described i n t h i s t h e s i s . a) H i s t o r i c a l overview of AChE Since much of the work comprising t h i s thesis i s concerned with AChE, what i s known and what has been speculated about AChE over the past decades w i l l be reviewed i n some d e t a i l . The major early advances i n understanding p o t e n t i a l f u n c t i o n a l r o l e s of AChE were made by Koelle (1962). He noted three major generalizations concern-ing AChE: F i r s t , i t s highest concentrations appeared to be on presynaptic c h o l i n e r g i c terminals, with the possible exception of the neuromuscular junction. Second, the enzyme was found i n many neurons not thought to be c h o l i n e r g i c , although i n dramatically lower 2 l e v e l s than i n neurons thought to ;be c h o l i n e r g i c . F i n a l l y , there did appear to be n i c o t i n i c receptors on the terminals of c h o l i n e r g i c neurons, s p e c i f i c a l l y motoneuron terminals, which could give r i s e to antidromic a c t i o n p o t e n t i a l s recorded i n the alpha-motoneuron. This l a s t observation would of course give AChE a raison d'etre f o r being present p r e s y n a p t i c a l l y . Subsequent research has borne out the f i r s t two of these hypotheses. With regard to the t h i r d , today the existence of the n i c o t i n i c receptors on motoneuron terminals appears to be v a l i d , although i t i s dubious that they are of p h y s i o l o g i c a l s i g n i f i c a n c e (Miyamoto,. 1978). Thev, f i r s t hypothesis, that high AChE l e v e l s appear to be a un i v e r s a l c h a r a c t e r i s t i c of cho l i n e r g i c neurons, appears to be true at le a s t as f a r as the perikaryon i s concerned. Unfortunately, t h i s hypothesis has been received poorly by the neuro-c.:.-science community, since Shute and Lewis (1961, 1965) modified the hypothesis to state that a l l neurons which contained any l e v e l of AChE i n t h e i r axons were c h o l i n e r g i c . This modification was accepted without further examination by a number of other p r o l i f i c i n v e s t i g a t o r s (e.g., Krnjevic and S i l v e r , 1965, 1966), despite e a r l i e r data published by Koelle (1954)^which c l e a r l y i n v a l i d a t e d such;a.hypothesis. The postulate that the post-synaptic occurence of AChE implies cholinoception remains unproven. I t i s c e t a i n l y p l a u s i b l e i n the peripheral nervious system (Koelle, 1962), but there e x i s t s at le a s t one major exception i n the c e n t r a l nervous system: The dentate gyrus of the hippocampal formation receives a massive c h o l i n e r g i c input (Lewis, Shute, and S i l v e r , 1967), but none of the neurons of the e n t i r e hippocampal formation contain appreciable amounts of AChE (Lewis, Shute, and S i l v e r , 1967; Lehmann, unpublished observations). 3 Some attempts have been made to support the hypothesis that low to intermediate_levels of AChE i n ^ c e r t a i n neurons of the c e n t r a l nervous system are i n d i c a t i v e of cholinoception (Butcher and Talbot, 1978a,b), but the majority of the evidence for these s p e c i a l cases i s negative (Lehmann and F i b i g e r , 1979). Two hypotheses were proposed decades ago i n an attempt to explain completely AChE i n a l l i t s l o c a t i o n s . Nachmansohn (1959) believed that a c e t y l c h o l i n e was released and hydrolyzed along the e n t i r e extent of axons i n general, and that t h i s process was e s s e n t i a l to nerve impulse conduction. Burn and Rand (1959) believed that ac e t y l c h o l i n e was contained i n sympathetic (noradrenergic) terminals and that the release of acetylcholine, e l i c i t e d by the a c t i o n p o t e n t i a l , mediated the subsequent release of noradrenaline, by stimulating a pre-junctional, e x t r a c e l l u l a r c h o l i n e r g i c receptor. While there were perhaps data to support these hypotheses at one time, abundant data garnered over the decades have refuted both..hypotheses ( S i l v e r , 1974). Champions of the Nachmansohn (1959) and Burn and Rand (1959) hypotheses are s t i l l to be found, however (Butcher et a l . , 1975). I t remains l i k e l y , however, that c h o l i n e r g i c receptors e x i s t on noradrenergic neurons i n the periphery (Sharma and Banerjee, 1978), and they may e x i s t on catecholamine terminals i n the c e n t r a l nervous system. The l a t t e r case i s of course much more d i f f i c u l t to resolve experimentally (for review of l i t e r a t u r e , see Butcher and Talbot, 1978a,b). Thus exogenous ac e t y l c h o l i n e may have a d i r e c t a c t i o n on catecholamine terminals. Recently, i t has been proposed that butyrylcholinesterase serves as a precursor for AChE, on the basis of.some rather equivocal 4 pharmacological data (Koelle et a l . , 1976). In studies from t h i s laboratory to be reported below, a s e l e c t i v e l e s i o n of dopaminergic neurons i n the substantia nigra resulted i n a s u b s t a n t i a l depletion of AChE with no depletion i n butyrylcholinesterase, suggesting that at l e a s t i n these neurons butyrylcholinesterase cannot serve as the precursor for AChE. I t has since been shown that AChE and b u t y r y l -cholinesterase show large differences i n binding properties, i n immunological c r o s s - r e a c t i v i t y , and, i n human plasma, have separate gene l o c i (Silman et a l . , 1979), making i t d i f f i c u l t indeed to support t h i s l a t e s t hypothesis of Koelle et a l . (1976). A great deal of research has been done on the d i f f e r e n t molecular forms of AChE, resolved by a number of biochemical techniques. Early studies were at disagreement with respect to the molecular weights and numbers of isoenzymes of AChE. This was apparently due to d i f f e r i n g degrees of aggregation and s o l u b i l i z a t i o n occurring due to the d i f f e r e n t procedures used i n the d i f f e r e n t l a b o r a t o r i e s . Vigny et a l . (1976) succeeded i n s o l u b i l i z i n g the enzyme thoroughly and reproducibly by using the very high sodium chl o r i d e concentration of 1 M. They found three c h a r a c t e r i s t i c forms of AChE i n muscle: 4s, 10s, and 16s, following u l t r a c e n t r i f u g a t i o n . I t appeared that the 16s form was s p e c i f i c a l l y associated with muscle end-plate. Subsequently, the same group found that r a t b r a i n contained only the 4s and 10s forms (Rieger and Vigny, 1976). Furthermore, as r a t b r a i n developed, a predominance of the 4s form gave way to an almost exclusive occurrence of.the"10s'form. These data were very suggestive of a synaptic function of an oligomeric form of AChE. In superior c e r v i c a l ganglion, 4s, 6.5s, 10s, and 16s forms 5 were found, although a l l forms but the 16s were found i n preganglionic and postganglionic nerves (Gisiger et a l . , 1978). The 16s form disappeared from the superior c e r v i c a l ganglion following denervation. However, other workers found small amounts of 16s AChE i n pe r i p h e r a l nerves which apparently was transported with unusually high v e l o c i t y (DiGiamberardino and Courard, 1978; Fernandez et a l . , 1979). The^ f u n c t i o n a l implications of these findings for AChE i n synaptic function await further study. I t i s also noteworthy that AChE which i s bound to the external surface of axons (representing about 84% of the t o t a l i n the nerve section) apparently i s not transported at appreciable v e l o c i t i e s i n either the anterograde nor retrograde d i r e c t i o n . Instead, the minority of AChE contained within the axon undergoes rapid axoplasmic transport (Brimijoin et a l . , 1978). In the nerve terminal, AChE appears to have an e x c l u s i v e l y external, membrane-bound l o c a l i z a t i o n , although sometimes electron microscopic studies report r e a c t i o n product within mitochondria or the Golgi apparatus, which was not eliminated by the appropriate histochemical controls, i . e . , i t does not represent true AChE (Er'anko et a l . , 1967; Bridges et a l . , 1973; Koelle et a l . , 1974; Kuhar and Rommelspacher, 1974). This i s i n agreement with s u b c e l l u l a r f r a c t i o n a t i o n studies (Toschi, 1959; DeRobertis et a l . , 1963; Whittaker et a l . , 1964). A l l these observations emphasize the importance of presynaptic AChE, p a r t i c u l a r l y that which i s bound on the external surface of the c h o l i n e r g i c terminal, i n the c e n t r a l nervous system. Obviously, AChE has a d i f f e r e n t r o l e i n regulating a c e t y l c h o l i n e transmission than monoamine oxidase has i n regulating catecholamine transmission (Geffen and L i v e t t , 1971), monoamine oxidase having an i n t r a t e r m i n a l , 6 pr i m a r i l y mitochondrial l o c a l i z a t i o n . A very i n t r i g u i n g possible mechanism for regulating c h o l i n e r g i c transmission v i a AChE has been raised i n recent years, namely the secretion of AChE either by c h o l i n e r g i c neurons or by the neurons or organs which receive c h o l i n e r g i c innervation. The hypothesis that AChE was secreted was f i r s t suggested by electron microscopic histochemical studies (Flumerfelt et a l . , 1973; Kreutzberg and T6th, 1974; Kreutzberg and Schubert, 1975). In a rigorous e l e c t r o n microscopic histochemical study i n the motoneuron, the possible c y t o l o g i c a l mechanism for secretion was proposed (Kreutzberg et a l . , 1975): Following synthesis i n the rough endoplasmic reticulum, AChE apparently passes through^the Golgi apparatus and becomes incorporated into the smooth endoplasmic reticulum. Since there i s d i r e c t c ontinuity between the smooth endoplasmic reticulum and the plasma membrane, th i s suggested a general c y t o l o g i c a l mechanism for AChE secretion. The observation that, r a r e l y , synaptic v e s i c l e s appear to contain AChE (Bodian 1970), suggested that secreted AChE may not only be r e s t r i c t e d to e x t r a c e l l u l a r l o c a t i o n s , but may play some r o l e i n i n t e r c e l l u l a r communication as w e l l . I t i s important to note that most ind i c a t i o n s suggest that i f AChE indeed enters synaptic v e s i c l e s , i t i s by endocytosis. AChE has subsequently been detected i n cerebrospinal f l u i d (Chubb et a l . , 1976; Bareggi and Giacobini, 1978; S c a r s e l l a et a l . , 1979; Greenfield et a l . , 1979). Non-denaturing g e l electrophoresis studies suggested that one isoenzyme was secreted (Chubb et a l . , 1976; S c a r s e l l a et a l . , 1979). Administration of chlorpromazine increases the amount of AChE secreted (Bareggi and Giacobini, 1978; 7 Greenfield et a l . , 1979). The release i s much more pronounced i n c i s t e r n a l than i n v e n t r i c u l a r cerebrospinal f l u i d (Bareggi and Giacobini, 1978; Greenfield et a l . , 1979). The source of the released AChE i s unknown; however, i t i s higher i n a c t i v i t y than plasma AChE, and there i s no concomitant increase i n l a c t a t e dehydroen-ase, a conventional marker f o r c e l l d i s r u p t i o n (Greenfield et a l . , 1979) . This phenomenon i s of obvious importance i n understanding control mechanisms governing c h o l i n e r g i c tone i n the c e n t r a l nervous system. In the cases of the superior c e r v i c a l ganglion (Gisiger and Vigny, 1977) and cat geniohyoid muscle (Inestrosa et a l . , 1977), afferent c h o l i n e r g i c neurons are not necessary to support the release of AChE. In the i s o l a t e d rat hemidiaphragm, however, the r a t i o of 10s and 4s AChE released by e l e c t r i c a l d e p o l a r i z a t i o n match the_r.atio of these forms i n the phrenic nerve, and not muscle (Skau and Brimijoin, 1978) . The l a t t e r authors suggested synaptic, exocytotic release of AChE as_theusource oftextraeellular^AChEi.and as a means of regulating or perhaps supplying postsynaptic AChE. While i t remains questionable whether cho l i n e r g i c axons can secrete AChE, i t c e r t a i n l y i s true that organs which normally receive c h o l i n e r g i c innervation can secrete AChE, following chronic denervation and i n non-depolarizing medium (Gisiger and Vigny, 1977; Inestrosa et a l . , 1977). AChE release from whole b r a i n synaptosomal f r a c t i o n i n non-depolarizing medium has been demonstrated (Burgun et a l . , 1977). What r o l e exactly AChE release plays i n the c e n t r a l nervous system i s more d i f f i c u l t to answer than i t i s i n the perip h e r a l nervous system. I t i s not known, for instance, whether such released AChE reaches 8 cho l i n e r g i c synapses, but i t s presence has not been reported i n e x t r a c e l l u l a r spacelin^the./brain^except.Jforccerebrospinal:fluid. b) The basal ganglia and ch o l i n e r g i c function The postulate that AChE i s concerned i n some way with the function of acetylcholine remains such an a t t r a c t i v e concept that the study of th i s enzyme was the major emphasis of the projects comprising t h i s t h e s i s . That the striatum .is among the highest i n biochemical markers for c h o l i n e r g i c transmission i n the e n t i r e b r a i n (Hoover et a l . , 1978; Kobayashi et a l . , 1978) makes t h i s an a t t r a c t i v e nucleus i n which to study the c h o l i n e r g i c neuron. Many of the psychopharmacological e f f e c t s produced by drugs acting on ch o l i n e r g i c receptors i n the ce n t r a l nervous system, such as tremor and catalepsy (Karczmar,11975) may be mediated i n the striatum. In the early part of the 1970's i t became c l e a r that the markers for_cho.linergic_rieurons, namely choline acetyltransferase and acetylcholine, were completely i n t r i n s i c to the striatum (McGeer et a l . , 1971; Butcher and Butcher, 1974). Lesions of the b r a i n surrounding the striatum had no e f f e c t on either of.these biochemical markers for c h o l i n e r g i c neurons. This led to the suggestion that the. u ... ch o l i n e r g i c neuron was an interneuron (McGeer et a l . , 1971). At about the same time, s i x d i f f e r e n t morphological types of neurons were ident-i f i e d i n the straitum (Kemp and Powell, 1971). One goal of the neuro-s c i e n t i s t i s to construct a wiring diagram of the brain, as i f i t were a computer, and then go on to explain i t s function, and i n cases of disease dysfunction, i n terms of that wiring diagram. The f i r s t step, of course, i s to i d e n t i f y the components. In the case of the basal ganglia, the primary s p e c i f i c goal as 9 f a r as acetylcholine i s concerned, was to i d e n t i f y the ch o l i n e r g i c neuron morphologically. This was f i r s t accomplished by the technique of immunohistochemistry, employing antibodies r a i s e d against p u r i f i e d choline acetyltransferase (Hattori et a l . , 1976). The most common type of neuron i n the striatum, the medium spiny neurons, were i d e n t i f i e d as the putative c h o l i n e r g i c neurons. Biochemico-pharmacological data were used to support the i d e n t i f i c a t i o n of th i s as the ch o l i n e r g i c neuron, which received a d i r e c t dopaminergic input (Hattori et a l . , 1976). When the experiments reported i n t h i s thesis were begun, the study of AChE i n the striatum appeared to be as complex an approach as any other f o r studying c h o l i n e r g i c function. As noted above, AChE has complex forms and c e l l u l a r l o c a l i z a t i o n s , not only within and on the plasma membranes of neurons i n varying l e v e l s of a c t i v i t y , but also quite possibly i n i n t e r c e l l u l a r space. Histochemically, AChE a c t i v i t y i n the striatum appears dense and uniform (Koelle, 1954; McGeer et a l . , 1971). Fortunately, a technique was developed which allowed the r e s t r i c t i o n of histochemical product formed by AChE to the neurons which synthesize the enzyme most r a p i d l y (Lynch et a l . , 1972; Butcher et a l . , 1975): Administration of the i r r e v e r s i b l e i n h i b -i t o r of AChE, diisopropylphosphorofluoridate (DFP), i n h i b i t s the AChE (as well as other enzymes) .everywhere i n the animal. If the animal i s then s a c r i f i c e d four to twelve hours a f t e r administration of DFP, only the enzyme which has been newly synthesized during that time i s v i s u a l i z e d histochemically. Thus, i t became possible to study the i n t r i n s i c neurons of the striatum which synthesize AChE, a study which i s s t i l l being pursued. Preliminary studies (Butcher 10 et a l . , 1975) showed that only a minority of neurons (less than 5%) synthesized the enzyme at appreciable l e v e l s , and of these s t i l l fewer synthesized very large amounts of the enzyme. The papers which follow are e n t i r e l y of an anatomical and biochemical nature. The f u n c t i o n a l implications, whereby some speculations concerning the r o l e of the s t r i a t a l c h o l i n e r g i c neurons i n psychomotor function may be entertained, w i l l be dealt with i n the discussion. In the publications which follow, as the main body of t h i s thesis, a broad data base was sought to substantiate hypotheses which were^and perhaps s t i l l are,considered unorthodox. The candidate i s aware that i n science, as i n a l l "endeavors, today's conclusions have a way of becoming tomorrow's f a l l a c i e s . c) B r i e f d e s c r i p t i o n of research and r a t i o n a l e Butcher et a l . (1975) reported that the dopaminergic neurons of the substantia nigra contained AChE. This conclusion was based mainly on the s i m i l a r topographic d i s t r i b u t i o n of AChE-containing neurons and dopamine-containing neurons, the l a t t e r having been l o c a l i z e d by catecholamine histofluorescence studies (Dahlstr'om and Fuxe, 1964; Ungerstedt, 1971). This was not, however, a compelling argument_to support the conclusion. For t h i s reason, the f i r s t i n v e s t i g a t i o n of t h i s thesis was to test rigorously the hypothesis that dopamine-containing neurons of the substantia n i g r a contained AChE. Select i v e , i f not s p e c i f i c , destruction of dopamine-containing neurons can be achieved by the administration of the neurotoxin 6-hydroxydopamine by either of two routes. The toxin mayche in j e c t e d d i r e c t l y into the ascending axons of the dopaminergic n i g r o s t r i a t a l 11 tract.n.'.Retrograde degeneration of the dopaminergic neurons then ensues. A l t e r n a t i v e l y , the toxin may be inj e c t e d into the l a t e r a l v e n t r i c l e . Desmethylimipramine, which blocks uptake of the toxin into noradrenergic neurons, must be administered also i f noradrenergic f i b r e s and perikarya are to be spared. Bothsof :r: thesecroutes of administration were employed i n the f i r s t i n v e s t i g a t i o n (A) to obtain a complete and s p e c i f i c l e s i o n of the n i g r o s t r i a t a l dopaminergic projection. The extent of the lesions was determined by measuring the enzyme tyrosine hydroxylase, which i s thought to be a s p e c i f i c marker for catecholamine neurons. Choline acetyltransferase was measured to assess the s p e c i f i c i t y of the l e s i o n . F i n a l l y , AChE was both .measured by radioenzymatic assay and examined histochemically. The biochemical c h a r a c t e r i s t i c s of AChE, an; enzyme which does not follow Michaelis-Menten k i n e t i c s , were also studied. The histochemical demonstration of an AChE-containing s t r i a t o -n i g r a l p r o j e c t i o n had been proposed e a r l i e r ( O l i v i e r et a l . , 1970). The second i n v e s t i g a t i o n of t h i s thesis set out to support t h i s hypothesis also. The ..original i n v e s t i g a t i o n ( O l i v i e r et a l . , 1970) had employed e l e c t r o l y t i c l e s i o n s , which destroy axons-passing through the lesioned area, i n ad d i t i o n to neurons. Since that i n v e s t i g a t i o n , a neurotoxin, k a i n i c acid, has been found which spares axons of passage but destroys i n t r i n s i c neurons of the striatum. This second i n v e s t i g a t i o n (B) u t i l i z e d k a i n i c acid i n order to obtain the more s e l e c t i v e s t r i a t a l l e s i o n . Information on the l o c a l i z a t i o n and sources of AChE i n the striatum were also obtained. Histochemical experiments were performed to determine i f blockade of transport 12 along the s t r i a t o n i g r a l axons caused an accumulation of AChE wit h i n them. A group of neurons, the nucleus b a s a l i s magnocellularis, which may belong to the extrapyramidal system (Divac, 1975) or to the r e t i c u l a r group of neurons (Das and Kreutzberg, 1968), became the focus of the next i n v e s t i g a t i o n (C). These neurons contain very high l e v e l s of AChE, and i t had been speculated that they were the source of a c h o l i n e r g i c p r o j e c t i o n to the neocortex (Divac, 1975). A l l the most rigorous means of t e s t i n g t h i s hypothesis, by u t i l i z i n g d i f f e r e n t sorts of l e s i o n s , neuroanatomical tr a c i n g techniques, and AChE histochemistry, were employed to test t h i s hypothesis. There has been a great deal of confusion and numerous hypotheses concerning the i n t e r p r e t a t i o n s which may be drawn from the presence of AChE a c t i v i t y i n d i f f e r e n t neurons. The investigations reported i n t h i s thesis have some bearing on these i n t e r p r e t a t i o n s . A short review on the topic was therefore written (D). This review contains some o r i g i n a l data, the most s t r i k i n g of which r e l a t e to the morphological i d e n t i f i c a t i o n of the s t r i a t a l c h o l i n e r g i c neuron. Campochiara and Coyle (1978) had developed a s e l e c t i v e l e s i o n of the c h o l i n e r g i c neuron. This turned out to be the same morphologically described neuron previously speculated to be the c h o l i n e r g i c neuron on 'j.'.. the basis of anatomical and histochemical arguments set f o r t h i n p u b l i c a t i o n B. 13 GENERAL DISCUSSION a) Preview of new contributions reported i n t h i s t hesis From the preceding papers, three major findings have emerged: 1) A hypothesis for i d e n t i f y i n g p o t e n t i a l c h o l i n e r g i c neurons on the basis of t h e i r high AChE a c t i v i t y has.been proposed. 2) The cho l i n e r g i c neuron of the striatum has been morphologically i d e n t i f i e d . 3) The o r i g i n of a major ch o l i n e r g i c p r o j e c t i o n to the cortex has been i d e n t i f i e d . In the f i r s t i n v e s t i g a t i o n (A), the hypothesis proposed by Butcher et a l . (1975) was supported: S e l e t i v e l e s i o n of the dopaminergic neurons resulted i n .a 30-40% depletion of AChE i n the substantia nigra, and about a 12% depletion of AChE i n the striatum. These r e s u l t s suggest that both the perikarya and axons of dopaminergic n i g r o s t r i a t a l neurons contain AChE. In t h i s i n -ve s t i g a t i o n , the source of a ch o l i n e r g i c input to the substantia nigra could not be found. In the second i n v e s t i g a t i o n (B), the hypothesis of O l i v i e r et a l . (1970) was refuted. A massive k a i n i c acid l e s i o n of s t r i a t a l neurons did not r e s u l t i n a detectable depletion of AChE i n the substantia n i g r a . Furthermore, c o l c h i c i n e i n j e c t i o n s into the axons of the s t r i a t o n i g r a l pathway, which i n general w i l l cause proximal accumulatiori._of AChE i n axons which transport the enzyme, did not cause such an accumulation. While a major portion, i f not a l l , of the choline acetyltransferase a c t i v i t y of the striatum originates from neurons whose perikarya reside within the striatum, about 50% of the AChE a c t i v i t y i s derived from an external source. The t h i r d i n v e s t i g a t i o n provided compelling evidence that the 14 nucleus b a s a l i s magnocellularis i s the source of a c h o l i n e r g i c pr o j e c t i o n to the cortex (C). The efferent neurons were mapped (C, Figs. 1 and 2). I t was not possible to assign t h i s group of neurons to either the r e t i c u l a r formation or the extrapyramidal system, on the basis of currently a v a i l a b l e evidence. The review (D), which evaluates the int e r p r e t a t i o n s which can be made from the l o c a l i z a t i o n of AChE i n various neurons, a r r i v e d at two basic general and empirical conclusions: 1) Very high l e v e l s of AChE four to twelve hours following DFP administration can be taken as a necessary but not s u f f i c i e n t c r i t e r i o n f o r a neuron to be choliner-g i c . This i s a valuable r u l e , since i t would eliminate 99% of the neurons i n the brain as candidates for ch o l i n e r g i c neurons, since they contain but low to intermediate l e v e l s of AChE. For instance, the cerebral cortex i s devoid of intensely AChE-reactive neurons, and thus, one would pr e d i c t , does not contain c h o l i n e r g i c perikarya. This example was experimentally v e r i f i e d (D). 2) At t h i s point i n time, AChE a c t i v i t y contained i n a neuron cannot be taken as evidence that such a neuron i s cholinoceptive. In t h i s i n v e s t i g a t i o n , evidence was also gathered to support the hypothesis that the intensely AChE-reactive neuron was the c h o l i n e r g i c interneuron of the striatum. When r a t pups ten days of age received k a i n i c acid i n j e c t i o n s , choline acetyltransferase a c t i v i t y and AChE a c t i v t y were s e l e c t i v e l y depleted. While h i s t o l o g i c a l l y no other neuron los s was detectable, the intensely AChE-reactive neuron was destroyed. The putative c h o l i n e r g i c neuron makes up only about 1% of the t o t a l s t r i a t a l neuron population. These fundamental studies of the putative c h o l i n e r g i c neuron 15 of the striatum are e s s e n t i a l as a basis for a more comprehensive understanding of that neuronls more complex functions. It would appear to play an important part i n the function of the striatum. For instance, of a l l s t r i a t a l neurons, the large neurons (including the putative c h o l i n e r g i c neuron) undergo t h e i r f i n a l mitosis e a r l i e r than the r e s t of the s t r i a t a l neurons (Brand and Rakic, 1979). They appear to develop s e n s i t i v i t y to k a i n i c a c i d l s neurotoxic e f f e c t s e a r l i e r than the r e s t of the s t r i a t a l neurons. These r e s u l t s r a i s e the p o s s i b i l i t y that they form the primary neural foundation upon which subsequent s t r i a t a l neural networks are b u i l t . A postnatal develop-, mental study of the putative c h o l i n e r g i c neuron-is currently i n progress. b) The r o l e of the striatum i n motor and psychological function The t i t l e of the thesis promises some consideration of the function of the s t r i a t a l c h o l i n e r g i c neuron. This i s , a f t e r a l l , the ultimate i n t r i g u e of brain research. The current concepts concerning the general psychological function of the; striatum w i l l now be reviewed very b r i e f l y . F i r s t , i t must be considered that the striatum does not play an e s s e n t i a l r o l e i n the control of basic metabolic function, elementary sensorimotor functions, or elementary cognitive functions ( V i l l a b l a n c a et a l . , 1976). However, d e f i c i t s i n response i n i t i a t i o n to a conditioned avoidance response occur following l e s i o n of the dopaminergic n i g r o s t r i a t a l p r o j e c t i o n (Fibiger et a l . , 1974). Such d e f i c i t s can be reversed by a n t i c h o l i n e r g i c drugs, such as scopol-amine and atropine (Fibiger et a l . , 1975). This i s one example of an antagonism which appears very frequently between dopaminergic 16 and c h o l i n e r g i c transmission i n the striatum. The striatum appears to play i t s major r o l e as one of the higher feedback systems c o n t r o l l i n g movement. Although t r a d i t i o n a l l y i t has been considered a more p r i m i t i v e motor output system which operates i n -dependently of the pyramidal ( c o r t i c o - s p i n a l ) motor system, d i r e c t motor outputs have been found to be at most a minor portion of efferents from the basal ganglia. On the contrary, c i r c u i t r y which l i n k s the striatum with thalamic pathways which feed back onto the cerebral cortex i s the predominant neuroanatomical c h a r a c t e r i s t i c of the system (Carpenter, 1975). There appear to be rather d i r e c t routes f or v i s u a l input to the striatum v i a the cortex, demonstrable both anatomically (K'unzle and Akert, 1977) and by recording responses to v i s u a l s t i m u l i (Pouderoux and Freton, 1979). C o r t i c a l afferents, not only from motor areas (Kunzle, 1975) but also from a l l sensory and a s s o c i a t i o n a l areas, innervate the striatum (Goldman and Nauta, 1977; Jones et..al., 1977). Somatic and v i s u a l sensory neglect can be induced by les i o n s of the extrapyramidal motor system (Feeney and Wier, 1979). In normal animals, s i n g l e - c e l l recording i n the striatum during v i s u a l l y and somatically guided hand movements ind i c a t e that the the t y p i c a l l y "quiet" striatum f i r e s during correction of these movements (Anderson.et a l . , 1979). A d i r e c t r o l e of the c h o l i n e r g i c system of the striatum i n modulating c o r t i c a l processing of sensory information has been demonstrated by Roemer et a l . (1978): Intracaudate i n j e c t i o n of carbachol has d i r e c t e f f e c t s on somatosensory-evoked c o r t i c a l p o t e n t i a l s . Cryogenic les i o n s of the striatum produce pursuit-tracking d e f i c i t s (Bowen, 1969). 17 c) Psychopharmacology of the striatum Anthropomorphism i s considered to be a p i t f a l l i n science gener-a l l y and i n neuroscience i n p a r t i c u l a r . However, since some drugs used i n the studies to be discussed can induce a syndrome that c l o s e l y resem-bles paranoid schizophrenia (e.g., amphetamine), and others are used therapeutically i n schizophrenics (Snyder, 1974), some of the animal behaviors e l i c i t e d by these drugs may be considered analogous to human psychological disorders (Mattyse, 1974). Thus the reader i s encouraged to consider the following psychopharmacological discussion i n terms of what roles i n thought processes the transmitters dopamine and a c e t y l -choline may play. I t should be noted that while there i s considerable evidence to indic a t e that s i t e of actions of drugs described below i s the striatum, t h i s point is'-actually r a r e l y adequately demonstrated. 1) Psychopharmacology of dopamine i n the striatum 6-Hydroxydopamine lesions of the n i g r o s t r i a t a l p r o j e c t i o n r e s u l t i n decreased spontaneous locomotor a c t i v i t y (Creese and Iversen, 1973; Roberts et a l . , 1975), and an attenuation i n the normal locomotor stimu-l a t i o n induced by amphetamine. Apparently the nucleus accumbens i s more involved i n the locomotor response to amphetamine, while the stereotypy response to higher doses of amphetamine i s l o c a l i z e d to the caudate-putamen (Creese and Iversen,1975) . Neuroleptics, which are thought to block dopamine receptors, cause catalepsy i n high doses (Asper et a l . , (1973) . E l e c t r o p h y s i o l o g i c a l ^ , i t i s thought by some that dopamine i s mono-sy n a p t i c a l l y excitatory, although at longer lat e n c i e s i t s o v e r a l l e f f e c t i s i n h i b i t o r y , an e f f e c t probably mediated by at l e a s t one interneuron ( K i t a i et a l . , 1976; Richardson et a l . , 1977). Dopamine stimulates adenylate cyclase i n the striatum (McGeer et a l . , 1976; Kebabian, 1978), 18 making i t a candidate as a slower acting, humoral agent which.may not function e x a c t l y l i k e a c l a s s i c a l synaptic transmitter. 2) Psychopharmacology of acetylcholine i n the striatum Because cholinergic systems are found i n so many parts of the ce n t r a l and peripheral nervous systems, the only d i r e c t assessment of the behavioral r o l e of acetyl c h o l i n e i n the striatum i s obtained by i i n j e c t i o n of acetylcholine agonists and antagonists d i r e c t l y into the striatum. I n j e c t i o n of muscarinic agonists or AChE i n h i b i t o r s i n t o the jstriatum r e s u l t s i n tremor, limb dystonia, r i g i d i t y , and akinesia - a l l c h a r a c t e r i s t i c s of Parkinson's disease (Connor et a l . , 1966; Goldman and Lehr, 1976; Matthews and Chiou, 1978, 1979). A l l these e f f e c t s are blocked by muscarinic antagonists. I n t r a s t r i a t a l i n j e c t i o n of atropine, on the other hand, e l i c i t s stereo-typy - an e f f e c t which may also be e l i c i t e d by increasing dopaminergic tone (Zambo et a l . , 1979). E l e c t r o p h y s i o l o g i c a l ^ , there i s less agreement about the ef f e c t s of ac e t y l c h o l i n e i n the striatum. Some in v e s t i g a t o r s found predominantly i n h i b i t i o n of iontophoresed a c e t y l c h o l i n e (Bloom et a l . , 1965; McLennan and York, 1966) which was blocked by muscarinic anta-gonists (McLennan and York, 1966). Systemically administered c h o l i n e r g i c agonists and antagonists led others to believe that c h o l i n e r g i c receptors i n the caudate nucleus were i n h i b i t o r y and muscarinic (Roller and Berry, 1976). However, using s t r i a t a l s l i c e s , l o c a l stimulation evokes e x c i t a t i o n which can be blocked by high concentrations of curare or enhanced by physostigmine, while muscarinic antagonists were without e f f e c t (Misgeld and Bak, 1979). In contrast, another study reports both short and long latency 19 e x c i t a t i o n by l o c a l stimulation of s t r i a t a l s l i c e s , the f i r s t being blocked by n i c o t i n i c antagonists and the l a t t e r being blocked by muscarinic antagonists (Weiler et a l . , 1979). d) Interactions between dopamine and acetylcholine On the basis of pharmacological studies alone, i t i s not s u r p r i s i n g to f i n d that a large number of antagonistic e f f e c t s e x i s t between dopaminergic and c h o l i n e r g i c agonists, and between dopaminergic and c h o l i n e r g i c antagonists. While generally Barbeau (1962) i s credited with creating the concept of a dopamine-acetylcholine balance i n the striatum, for h i s t o r i c a l accuracy he c r e d i t s the concept's inceptiontto McGeer et a l . (1961). Both groups of workers drew t h e i r conclusions from the e f f e c t s of drugs on victims of Park-inson's disease, i n whom dopamineragonists or c h o l i n e r g i c antagonists ameliorated, but dopamine antagonists or c h o l i n e r g i c agonists exacerbated the symptoms. The c h o l i n o l y t i c drugs which are success-f u l i n ameliorating the symptoms of Parkinson's disease are muscarinic (Duvoisin, 1967). Huntington's disease i s e s s e n t i a l l y the inverse of Parkinson's disease - i n these patients dopaminergic afferents to the striatum are i n t a c t , but the neurons of the striatum, including the c h o l i n e r g i c neurons, have undergone massive atrophy. The dopamine-acetylcholine balance model gained further support when the -inverse pharmacological r e l a t i o n s h i p held true once more - dopamine antagonists and^cholinergic agonists ameliorate the symptoms of the disease (Aquilonius and Sjostrom, 1971). 1) Psychopharmacological studies In psychopharmacological studies i n experimental animals, examples of the inverse actions of c h o l i n e r g i c and dopaminergic 20 drugs are innumerable. Just a few pertinent examples w i l l be c i t e d here. Methylphenidate induces stereotypy by increasing dopaminergic transmission; i t s e f f e c t s are reversed by administration of the muscarinic agonist oxotermorine (Davis et a l . , 1978). The muscarinic agonist, p i l o c a r p i n e , decreases amphetamine-induced increases i n locomotor a c t i v i t y , while scopolamine, a muscarinic antagonist, potentiates the locomotor e x c i t a t i o n produced by amphetamine (Fibiger et.-_al., 1970). In mice with u n i l a t e r a l 6-hydroxydopamine lesions of the n i g r o s t r i a t a l projection, scopolamine produces c i r c l i n g towards the side of the l e s i o n , i n d i c a t i n g potentiation of dopaminergic tone on the i n t a c t side (Pycock et a l . , 1978). This e f f e c t i s blocked by administration of tyrosine hydroxylase i n h i b i t o r s (which prevent the synthesis of dopamine). S i g n i f i c a n t l y , d i r e c t or i n d i r e c t c h o l i n e r g i c agonists depressed the rates of c i r c l i n g caused by amphetamine or apomorphine, suggesting antagonism of dopaminergic transmission on the i n t a c t side. 2) Measurement of biochemical parameters following pharmacological  manipulation When instead of behavioral measurements, biochemical parameters i n d i c a t i n g rate of transmitter turnover are measured i n response to pharmacological manipulation, data consistent with the dopamine-acetylcholine balance are obtained. Cholinergic agonists increase dopamine turnover i n the striatum, while muscarinic antagonists decrease dopamine turnover i n the striatum, presumably as some sort of compensatory mechanism, as reviewed by B a r t h o l i n ! et a l . (1975). Systemic administration of neuroleptics (dopamine antagonists) causes a marked increase i n the amount of acetylcholine which can 21 b e ^ c o l l e c t d from cat caudate nucleus by push-pull cannula (Stadler et a l . , 1973; B a r t h o l i n i et a l . , 1975). More commonly, the l e v e l of a transmitter i s measured following the pharmacological manipulation; the l e v e l i s considered to be inversely related to the turnover rate of the transmitter. Thus, e l e c t r o l y t i c l e s i o n of the substantia nigra causes a transient decrease i n s t r i a t a l l e v e l s of ace t y l c h o l i n e , while amphetamine increases s t r i a t a l a c e t y l c h o l i n e l e v e l s , but requires an in t a c t n i g r o s t r i a t a l p r o j e c t i o n i n order to do so (Agid e t . a l . , 1975). Neuroleptics such as h a l o p e r i d o l , . s p i r o p e r i d o l , chlorpromazine, and pimozide, and the depleter of biogenic amines, reserpine, cause de^ creases i n s t r i a t a l a cetylcholine l e v e l s (Cohsolo et a l . , 1975; G l i c k et a l . , 1976; Marco et a l . , 1976; Consolo et a l . , 1977;. Bianchi et a l . , 1979) . I t may be noted that such r e s u l t s have not co n s i s t e n t l y been obtained i n " o l f a c t o - s t r i a t a l " regions, namely the nucleus accumbens and o l f a c t o r y tubercle (Consolo et a l . , 1977). e) The c y t o l o g i c a l r e l a t i o n s h i p between the dopaminergic and c h o l i n - ergic neuron The very orderly way i n which the pharmacological data have sug-gested a constant and consistent antagonistic e f f e c t of dopaminergic i n h i b i t o r y influence on the ch o l i n e r g i c neuron, together with immuno-histochemical evidence, make the model of a d i r e c t synaptic contact from the dopamine neuron onto the c h o l i n e r g i c neuron very appealing (Hattori et a l . , 1976), and th i s model has been popular for a number of years (see F i g . 1). However, some of :.the points r a i s e d by the research contained i n t h i s thesis and to be found i n the l i t e r a t u r e make th i s model appear l e s s a t t r a c t i v e . F i r s t , e l ectron microscopic data suggest that dopaminergic neurons 22 account for approximately 15% of the terminals of the striatum (Hattori et a l . , 1973; Hokfelt and Ungerstedt, 1973; A r l u i s o n et a l . , 1978a,b), and these terminals synapse almost e x c l u s i v e l y on spines, or according to others, the d e n d r i t i c shafts of spiny neurons (for review, see Pasik et a l . , 1979). I t i s quite d i f f i c u l t to argue for the existence of d e n d r i t i c spines on the putative c h o l i n e r g i c neuron. Spines are not apparent i n DFP-pretreated material stained for AChE; i f they do e x i s t one must postulate that they f o r some reason do not contain AChE, as does the r e s t of the dendrite at the same post-DFP s u r v i v a l time. Furthermore, since the putative c h o l i n e r g i c neuron only constitutes about 1% of the t o t a l neuron population, i t must have very extensive d e n d r i t i c a r b o r i z a t i o n i n order to receive a large proportion of 15% of the t o t a l s t r i a t a l terminals. The p o s s i b i l i t y i s thus raised that, psychopharmacologically, a neuron other than the c h o l i n e r g i c neuron receives the major portion of dopaminergic input. In the f i n a l a n a l y s i s , i t i s the psychopharmacological experiment which w i l l make or break a hypothesis of neuronal connections. In an early study (Fibiger et a l . , 1970), i t was shown that r a t s older than 25 days modulated t h e i r locomotor stimulation by amphetamine i n r e -sponse to p i l o c a r p i n e (decreasing the stimulation) and scopolamine (increasing the stimulation). However, at 15 days of age, when the amphetamine stimulation could be demonstrated quite e a s i l y , the cho l i n e r g i c agents were without e f f e c t . I f the dopamine neuron were to transmit i t s information e x c l u s i v e l y through the c h o l i n e r g i c neuron, as diagrammed i n Figure 1, then these r e s u l t s would be impossible to obtain: With no c h o l i n e r g i c receptor to a f f e c t the amphetamine r e -sponse, the c h o l i n e r g i c neuron i s , at day 15, mute. The dopaminergic neuron must transmit i t s information through some other neuron, whose 23 post-synaptic receptor has matured by t h i s age. The model of the dopamine neuron synapsing d i r e c t l y onto the ch o l i n e r g i c neuron was the appropriate model for the data a v a i l a b l e at the time,- since i t was the simplest model. Instead of a " s e r i e s " c i r -c u i t , i n which the c h o l i n e r g i c neuron acts as the e s s e n t i a l l i n k through which dopamine must act, we must now consider other models. The next simplest model may involve a dopaminergic and c h o l i n e r g i c neuron termin-ating i n p a r a l l e l on the same neuron, where they may exert t h e i r opposite e f f e c t s , as diagrammed i n Figure 2. With s i x types of neurons (at least) and at l e a s t four types of afferents to the striatum, the model i s bound to become more complex as studies progress. Experiments are currently i n progress to t e s t the hypothesis proposed above, namely that the c h o l i n e r g i c neuron does not receive a large pro-portion of the dopaminergic input to the striatum. More rigorous b i o -r chemical and histochemical studies are being performed to see i f "the s e l e c t i v e c h o l i n e r g i c neuron l e s i o n produced by s t r i a t a l i n j e c t s of k a i n i c acid i n neonatal rats i s as s e l e c t i v e as i t appears. Binding studies, employing neuroleptics as ligands as well as dopamine agonists, should indicate no decrease i n dopamine receptors following l e s i o n of the ch o l i n e r g i c neurons i f the hypothesis proposed here i s c o r r e c t . Likewise, dopamine-stimulated adenylate cyclase should not be a l t e r e d by the s e l e c t i v e c h o l i n e r g i c neuron l e s i o n . Rotation ( i . e . , psychopharmaco-l o g i c a l ) studies are planned to see i f there i s a functional assymmetry in receptors i n the animal. Studies such as these w i l l eventually y i e l d the necessary information to construct a "road map" of the striatum. In time, we may be able to comprehend the " t r a f f i c patterns" that constitute nothing l e s s than thoughts. 24 s u b s t a n t i a n i g r a Figure 1. Pharmacological and immunohistochemical data were a l l neatly explained by t h i s simple model, i n which the c h o l i n e r g i c neuron i s part of a " s e r i e s " c i r c u i t . The dopamine neuron from the substantia n i g r a synapses d i r e c t l y onto the s t r i a t a l c h o l i n e r g i c neuron, which then contacts other neurons of the striatum. In t h i s model, the c h o l i n e r g i c neuron i s an e s s e n t i a l l i n k i n dopaminergic neurotransmission. The expression of dopaminergic function thus requires: 1) A dopamine receptor on the c h o l i n e r g i c neuron; 2) Functional a c e t y l c h o l i n e release; 3) A f u n c t i o n a l c h o l i n e r g i c receptor on other s t r i a t a l neurons. 25 substantia nigra Figure 2. 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Zambo K., Decsi L., and Nagy J . (1979) Stereotypy a f t e r intracaudate i n j e c t i o n of atropine i n the r a t . Neuropharm. 18:727-730. 35 APPENDIX L i s t of Publications A) Lehmann J . and F i b i g e r H.C. (1978) Acetylcholinesterase 36 i n the substantia nigra and caudate-putamen of the r a t : properties and l o c a l i z a t i o n i n dopaminergic neurons. J . Neurochem. 30:615-624. B) Lehmann J . , F i b i g e r H.C, and Butcher L.L. (1979) 45 The ^ l o c a l i z a t i o n of acetylcholinesterase i n the corpus striatum following k a i n i c acid l e s i o n of the corpus striatum: A biochemical and histochemical study. Neurosci. 4_:217-225. C) Lehmann J . , Nagy J . I . , Atmadja S., and Fib i g e r H.C. 53 (1980) The nucleus b a s a l i s magnocellularis: The u... o r i g i n of a c h o l i n e r g i c p r o j e c t i o n to the neocortex of the r a t . Neurosci, i n press.!. D) Lehmann J . and F i b i g e r H.C. (1979) Acetylcholinesterase 87 and the c h o l i n e r g i c neuron. L i f e S c i . 25:1939-1947. 36 Journal (>/ \<'l/^l^('/n'(lN^F^•. |«»78. Vol. 30. pp. 615-624. Pergamon Press. Primed in Great Britain. ACETYLCHOLINESTERASE IN THE SUBSTANTIA NIGRA AND CAUDATE-PUTAMEN OF THE RAT: PROPERTIES AND LOCALIZATION IN DOPAMINERGIC NEURONS JOHN LEHMANN and H . C . FIBIGER Division of Neurological Sciences, Department or Psychiatry, University of British Columbia, Vancouver, B.C., V6T 1W5, Canada (Received 20 June 1977. Accepted 11 August 1977) Abstract—In order to examine the hypothesis that acetylcholinesterase (AChE) is contained within dopaminergic neurons of the nigro-striatal projection, the effects of selective destruction of these neurons by 6-hydroxydopamine (6-OHDA) on cholinesterase, tyrosine hydroxylase, and choline acetyltransferase in substantia nigra (SN) and caudate-putamen (CP) were studied in the rat. Four to five weeks after intraventricular or intracerebral 6-OHDA injections tyrosine hydroxylase in these structures was reduced by 90% or more. Choline acetyltransferase was not affected in the SN or CP, but cholinesterase was reduced by about 40% in the SN and by 12% in the CP. To determine that the observed decreases in cholinesterase activity reflected true AChE and not butyrylcholinesterase (BChE). further experiments were conducted on tissues from animals with intracerebral 6-OHDA lesions. (1) Substrate specificity. Acetylcholine (ACh) was replaced by either acetyl-£-methyl-choline (Ac/?MeCh) or butyrylcholine (BCh) in the cholinesterase assay. SN and CP from 6-OHDA lesioned rats showed 54% and 92% of control tissue cholinesterase activity respectively with Ac/?MeCh as substrate, in good agreement with values found using ACh. No decrease in activity toward BCh was observed. (2) Kinetics. The decrease in cholinesterase activities at different concentrations of ACh was determined. Analysis of the data revealed that cholinesterase in dopaminergic neurons was inhibited by high ACh concentrations, a characteristic property of AChE but not BChE. (3) Selective inhibitors. In the SN, cholinesterase in dopaminergic neurons was inhibited by the selective AChE inhibitors BW284C51 and ambenonium with a dose-res-ponse curve similar to erythrocyte AChE but different from serum BChE. The selective BChE inhibitor, tetraisopropylpyrophosphoramide. inhibited the enzyme in dopaminergic neurons only at concen-trations which inhibited erythrocyte AChE, concentrations somewhat higher than those which inhibited serum BChE. These results support recent histochemical observations indicating that AChE is contained in dopaminergic neurons of the SN. Moreover, these experiments represent the first characterization of AChE from a homogeneous population of non-cholinergic neurons in mammalian CNS. ALTHOUGH it has been known for some years that the zona compacta of the substantia nigra contains high levels of A C h E (EC 3.1.1.7), only recently has evidence been provided for the existence of this enzyme within the dopaminergic neurons of this nu-cleus (BUTCHER et ai, 1975). At present, however, there is no information regarding the biochemical properties of the cholinesterase in these neurons, and it is to this question that the present experiments were addressed. Two techniques were viewed as providing the means for studying A C h E in D A cells of the S N as well as their terminals in the caudate-putamen. When used appropriately. 6 - O H D A is a selective Abbreviations used: 6-OHDA. 6-hydroxydopamine; Ac/?-MeCh, acetyl-/?-methylcholine; BCh, butyrylcholine; BChE. butyrylcholinesterase (EC 3.1.1.8); CAT. choline acetyltransferase (EC2.3.1.6); CP, caudate-putamen; DA. dopamine; DMPH 4 . 6.7-dimethyl-5,6.7,8-tetrahydropteri-dine; isoOMPA. tetraisopropylpyrophosphoramide; NSB. nigrostriatal bundle; SN. substantia nigra; TH. tyrosine hydroxylase (EC 1.14.16.2). neurotoxin for catecholaminergic neurons (JAVOY et ai. 1976). For example, intraventricular injection of 6 - O H D A can produce widespread destruction of cate-cholaminergic neurons without having significant effects upon neurons which contain other neurotrans-mitters (URETSKY & IVERSEN, 1970; M C G E E R et ai, 1973). Furthermore, it has recently been demonstrated that stereotaxic injection of 6 - O H D A into the axons of the nigrostriatal bundle results in both anterograde degeneration of D A terminals in the C P and retro-grade degeneration of D A perikarya in the pars com-pacta of the S N (CLAVIER & FIBIGER. 1977). Thus, by comparing the properties of A C h E in the S N and C P of control and 6 - O H D A lesioned rats, it is poss-ible to deduce some of the characteristics of this enzyme in the D A neurons. Cholinesterase .was characterized by three criteria used to distinguish true A C h E from B C h E (EC3.1.1.8). These criteria were: (1) Kinetics. Under certain conditions. A C h E demonstrates inhibition by high concentrations of substrate, while B C h E shows a monotonically increasing velocity with increasing 615 37 JOHN LEHMANN and H. C. FIBIGER 616 acetylcholine concentrations. (2) Substrate selectivity. AChE and BChE hydrolyze different substrates with different velocities. For AChE, the hydrolysis rates for different substrates are ACh > Ac/?MeCh > B C h while for BChE, B C h > ACh > Ac/3MeCh (ADAMS, 1949). (3) Selective inhibitors. Several selective inhibi-tors of AChE and BChE exist (AUSTIN & BERRY, 1953; DUBOIS et ai, 1950; ALDRIDGE, 1953; LANDS et al., 1955), but these inhibitors have not been tested in the brain using radioenzymatic assay techniques or on specific nuclei within the extrapyramidal system. M E T H O D S Male Wistar rats were obtained from Woodlyn Labora-tories, Guelph, Ontario. 6-OHDA (250 ng, dissolved in 25 y\ of 0.9% saline, 0.1% ascorbic acid) was injected into the left lateral ventricle under light ether anesthesia, 1 h following pretreatment with pargyline (50 mg/kg). This procedure has been shown to produce extensive damage to both dopaminergic and noradrenergic neurons (BREESE & TRAYLOR, 1971). Controls received vehicle injections. Ex-perimental animals were aphagic for an average of 3 days following injection, and were fed iniragastrically to main-tain body weight. Control and experimental animals were killed 4-5 weeks following injection. Another group of rats received 4 /ig of 6-OHDA dis-solved in 2 ftl of the same vehicle, stereotaxically injected into the left NSB at a rate of 0.2 /d/min under pentobarbi-tal anesthesia. These animals received intraperitoneal injec-tions of desipramine H Q (25 mg/kg) 30 min before the 6-OHDA injection to prevent concomitant damage to nor-adrenergic neurons (ROBERTS et ai, 1975). These animals were also allowed to survive 4-5 weeks following surgery. Animals were killed by cervical fracture. Brains were removed rapidly and the CP was dissected on ice. These tissues included globus pallidus and nucleus accumbens, and averaged 45 mg wet tissue weight. The mesencephalon was sectioned on a freezing microtome and the SN care-fully dissected from these sections on ice. Tissues included A9 and A10 areas and pars reticulata, and averaged 8 mg wet tissue weight The right (i.e. contralateral) SN and CP served as control for all enzyme assays in the unilateral NSB lesioned group. Homogenization was in 10 vol of lOmM-sodium phos-phate buffer (pH7.4) containing 0.25% Triton X-100. TH (EC 1.14.16.2) was assayed immediately by a modification of the method of COVLE (1972). Final incubation volume was 30/A with final concentrations: D M P H 4 , 1.11 mM; 2-mercaptoethanol, 111 mM; catalase, 13.9 units; FeS0 4 , 6mM; NaAc, l l l m M (pH 5.80); tyrosine, 50^M. Incuba-tions were for 20 min at 30°C. Enzyme activity was cor-rected for recovery from alumina (60%) and expressed as K^,. Homogenates were frozen and aliquots were taken as needed for subsequent assays. CAT was assayed in the incubation mixture described by MCCAMAN & DEWHURST (1970) in a final volume of 50 ft\. Incubations were for 30 min at 37°C. The product was extracted into 200 /il of 1.5% sodium tetraphenylboron in 3-heptanone. as described by FONNUM (1969). Since most batches of 3-heptanone acidify the aqueous phase, the sol-vent was previously washed in a separatory funnel with 0.5 M-NaOH. followed by six washes with distilled water. This procedure reduces the blank and results in consislenl 100% efficiency for ACh extraction. Following vigorous agitation and centrifugation, 100 /il of the supernatant was added to scintillation vials. Omission of choline or physo-stigmine virtually eliminated measured CAT activity. Cholinesterase activity, using ACh, BCh, or Ac/?MeCh, was assayed in a final incubation volume of 50 jil, with final concentrations of 15mM-sodium phosphate buffer (pH 7.00) and 5 mM-substrate, except where noted. Tissues were diluted in water by at least a factor of 500, so that endogenous substrates and ions known to affect AChE (e.g. K + , C a 2 * , ACh) had insignificant final concentrations. In-cubations were at i l " C for 30 min, and in no case was more than 10% of the substrate consumed. The reaction was linear with lime. Extraction with tetraphenylboron (FONNUM, 1969) was used in this assay to remove the labeled substrate: 200 jil of 1.5% tetraphenylboron in washed 3-heptanone was added to stop the incubation, and agitated vigorously. Following centrifugation, the superna-tant was aspirated, and the extraction procedure repeated. The pH of the aqueous phase remained at 7.0. Following the second aspiration, 25 /il of the aqueous phase was added to scintillation- vials containing 0.5 ml of 0.1 M-NaOH. Ten millilitres of Bray's solution was added to scintilla-tion vials for the T H assay, and 6 ml of Aquasol was added to scintillation vials for CAT arid cholinesterase assays. Internal standards were used to determine specific activity. Histochemical staining was performed by the method of KARNOVSKY & ROOTS (1954). Butyrylthiocholine was used as substrate in control sections. The following chemicals were obtained from the sources listed: Catalase, Boehringer-Mannheim; ACh bromide, B D H chemicals; tyrosine, BCh, Ac/JMeCh, sodium tetra-phenylboron, bovine erthyrocyte AChE (Type I), and horse serum BChE (Type X), Sigma; acetylcoenzyme A, D M P H 4 , Calbiochem; 3-heptanone, Eastman: [acetyl-l-3H]choline. [acetyl-l-^CJcholine, [butyry]-!-" ,C]choline. [acetyl-l- M C]/?MeCh, [U-MC]tyrosine. New England Nuclear; tetraisopropyl pyrophosphoramide (isoOMPA) K & K Laboratories; 1.5-bis-(4 allyldimethylammoniumphenyl) pentan-3-one dibromide (BW284C51). Burroughs Well-come Co., North Carolina; and ambenonium chloride. Sterling-Winthrop. Rensselaer, New York. RESULTS Four to five weeks following intraventricular injec-tion of 6-OHDA. T H in the CP and SN were reduced by 90%, while CAT activity in these areas showed no significant change (Table 1). Cholinesterase ac-tivity decreased by 31% in the SN and by 12% in the CP, both of which results were statistically signifi-cant (P < 0.001, Student's two-tailed t test). Four to five weeks following unilateral 6-OHDA injection into the NSB, similar decreases in TH were obtained in the ipsilateral CP and SN without alter-ing CAT activity in these structures (Table 2). Cho-linesterase activity was decreased in the SN by 43% (P < 0.001) and by 12% in the CP (P < 0.01). AChE histochemistry by the method of KAR-NOVSKY & ROOTS (1954) in a NSB-6-OHDA lesioned animal is shown in Fig. 1. The lesioned side (left) shows reduction in staining in the pars compacta and the ventral tegmental area [the A10 group of dopa-minergic cells according to UNGERSTEDT (1971)]. This 617 38 FIG. 1. Section of mesencephalon has been stained for AChE. The pars compacta of the substantia nigra (SNC) and the A10 area stain quite densely for AChE. This staining is decreased on the left half of the section, where dopaminergic cells have been selectively lesioned with an injection of 6-OHDA placed in their ascending axons (the NSB). 39 AChE in dopaminergic neurons 619 TABLE 1. THE ACTIVITIES OF THREE ENZYMES FROM SN AND CP OF RATS INJECTED INTRA-VENTR1CULARLY WITH 6-OHT \ EXPRESSED AS PER CENT OF CONTROL ± S.E.M. TH CAT AChE SN 10.3% ±1.7%* 88.0% ± 7 . 1 % 69.0% ± 1.4%* CP 9.3% ± 1.7%* 93.6% ± 2.6% 87.6% ± 1.8%* Control values for SN were: T H . 1.14nmol/mg tissue/h; CAT. 2.54nmol/mg tis-sue/h; AChE. 659nmol/mg tissue/h. Control values for CP were: T H . 1.36nmol/mg tissue/h; CAT. 18.8nmol/mg tissue/h: AChE. 2.12/jmol/mg tissue/h. n = 6. * P < 0.001. TABLE 2. THE ACTIVITIES OF ENZYMES FROM SN AND CP OF RATS INJECTED WITH 6-OHDA IN THE NSB EXPRESSED AS PER CENT OF CONTROL + S.E.M. Cholinesterase with different substrates TH CAT ACh Ac/3MeCh BCh SN CP 9.9% + 1.4%*** 3.4% ± 0.9%*** 99.6% + 6.0% 106.0% ± 4.3% 56.7% + 1.6%*** 53.7% ± 3.1%*** 96.4% ± 6.0% 87.9% ± 1.7%** 92.0% ± 3.5%**- 110.7% ± 6.5%* Control values for SN were: T H . 1.21 nmol/mg tissue/h: CAT. 2.37 nmol/mg tissue/h. Control values for CP were: T H . 1.33 nmol/mg tissue/h; CAT. 18.8 nmol/mg tissue/h. Cholinesterase values for control tissues are listed in Table 3. n = 6. * P < 0.05. ** P < 0.01. *** P < 0.001. observation confirms the extensive cell loss in the pars compacta of the S N and ventral tegmental area caused by retrograde degeneration after N S B lesions (CLAVIER et al, 1977). In discussing the results of this and subsequent ex-periments, S N A C h E will be defined as the cholines-terase activity of control substantia nigra tissue; C P A C h E refers to the cholinesterase activity of control caudate-putamen tissue; and A A C h E refers to the dif-ference between S N A C h E and cholinesterase from the SN of 6 - O H D A treated animals (i.e. the A C h E which is probably contained within the dopaminergic neurons). These three categories of cholinesterase were compared with commercially available purified bovine erythrocyte A C h E and horse serum B C h E , which served as standard enzymes. The first criterion employed to characterize these enzymes was the use of selective substrates (Table 2 and 3). Erythrocyte A C h E hydrolyzcd BCh at 1.6% of the rate that it hydrolyzed A C h , while B C h E hyd-rolyzed BCh at 254% of the rate that it hydrolyzed A C h . Erythrocyte A C h E hydrolyzed Ac/?MeCh at 11% of the rate that it hydrolyzed A C h , while B C h E hydrolyzed Ac/?MeCh at only 0.4% of the rate that it hydrolyzed A C h . Activities reported here were not corrected for the racemic nature of Ac/JMeCh, only one enantiomer of which is reportedly hydrolyzable (HOSKIN. 1963). As shown in Table 3, the 6 - O H D A lesions did not affect the already low rate of B C h hydrolysis in the S N . while there was a slight increase in BCh hy-drolysis in the C P , which proved statistically signifi-cant (P < 0.05). In contrast, there was a marked de-crease in the rate of Ac/?MeCh hydrolysis in the S N (46.3%, P < 0.001) and an 8% decrease in the C P (P < 0.001). This decrease in Ac/?MeCh hydrolysis correlates quite well with the decrease in A C h hy-drolysis in both S N and C P after 6 - O H D A lesions. Also, the relative rates of Ac/JMeCh hydrolysis to A C h hydrolysis in S N and C P compared closely to the ratio observed for erythrocyte A C h E (12-14%). For the comparison of the kinetic properties of the enzymes each point was graphed as per cent of ac-tivity at 1 m M - A C h in order to superimpose the TABLE 3. SPECIFIC ACTIVITIES OF CHOLINESTERASES FROM FOUR DIFFERENT SOURCES USING THE THREE SUB-STRATES LISTED Specific activities of cholinesterases with different substrates (nmol/mg/h) ACh Ac/?MeCh BCh SN 684.3 82.8 70.1 CP 1992 278 70.5 Erythrocyte AChE 44.300 4940 725 Serum BChE 583.000 2500 1.480.000 All substrates are 5 mM. n = 6 for brain tissues, n = 2 for commercially supplied erythrocyte AChE and serum BChE. 40 620 JOHN LEHMANN and H. C. FIBIGER I i i i I J 1 1 3.00 2.60 2.30 2.00 1.60 1.30 -log [ACh] FIG. 2. Kinetics of cholinesterase from various sources expressed as per cent of velocity at 1 mM-ACh. O SN: • C P : O Erythrocyte AChE; • Serum BChE (insei). curves (Fig. 2). SN AChE and CP AChE apparently differ slightly in the substrate concentration giving highest activity, and erythrocyte AChE demonstrated a higher and sharper peak activity; all three enzymes, however, are inhibited by high concentrations of ACh, a property characteristic of AChE, but not BChE (in-set, Fig. 2). In order to quantitate the cholinesterase which is found in dopaminergic neurons, the difference between control and 6-OHDA animals at a single concentration of ACh was measured. That difference (AAChE) can be taken as a measure of the AChE activity found in the dopaminergic neuron, assuming that there was negligible plasticity in the other cho-linesterase-containing neurons in the region. In order to characterize the kinetics of the dopaminergic cho-linesterase, velocities at different concentrations of ACh were measured in SN from control and unila-teral NSB 6-OHDA lesioned animals, and AAChE values were calculated and plotted in Fig. 3. AAChE showed the same typical kinetics as the control SN AChE, indicating that it is true AChE. Unfortunately, the low magnitude of AChE decrease in the CP (as well "as the slight increase in BChE) prevented the same analysis from being performed reliably in that region. Figure 4 shows the effect of increasing concen-trations of isoOMPA on cholinesterase activity from the two brain areas, the two standard enzymes, and AAChE, calculated as described. isoOMPA selectively inhibited BChE only at concentrations around 10"5M. Figures 5 and 6 show that BW284C51 and ambenonium were potent inhibitors of AChE, although there was some inhibition of BChE at higher concentrations. In Figs. 4-6 it is evident that SN AChE and AAChE resemble erythrocyte AChE more closely than they resemble BChE on these graphs, but CP AChE resembles erythrocyte AChE more faithfully than the enzymes from SN. The only case where AAChE differs markedly from SN AChE is in its dose-response to ambenonium: the Kj for AAChE is an order of magnitude lower than for SN AChE, and AAChE is more completely inhibited by 10 _ 4M-ambenonium as well (Fig. 6). DISCUSSION The decreases in AChE activity in SN and CP that were observed following 6-OHDA lesions of the dopaminergic nigrostriatal projection by two different methods confirm recent histochemical studies which have suggested that AChE is synthesized by the dopa-3.00 2.60 2.30 2.00 1.60 1.30 -log[ACh] FIG. 3. Kinetics of AChE from SN of animals lesioned unilaterally by 6-OHDA injections into the nigrostriatal bundle. O S N . control side; • S N , lesioned side; A differ-ence calculated between SN control and SN lesioned. 41 AChE in dopaminergic neurons O-i o Jo _c 6? 50 H 100 Con. 9 T 8 7 6 -log fisoOMPA] F I G . 4. Per cent inhibition of cholinesterase by isoOMPA, 1 x 10~9 M to 1 x 1 0"*M. O S N (control); A ASN (SN control-SN lesioned); • CP (control); O Erythrocyte AChE; + Serum BChE. -log fisoOMPA 100 Con. 9 8 7 6 -log [BW 284C51J F I G . 5. Per cent inhibition of cholinesterases by BW 284C51. 1 x 1 0 ~ 9 M t o l x 10"* M . O SN (control); A ASN (SN control-SN lesioned); • CP (control: O Erythrocyte AChE: • S e r u m BChE. -log jambenonium j - log Jotnbenonium] F I G . 6. Per cent inhibition of cholinesterases by ambenonium, 1 x 10 9 M to 1 x 10 4 M . O S N (con-trol); A ASN (SN control-SN lesioned) Cl C P (control); O Erythrocyte A C h E ; • Serum BChE. 622 JOHN LEHMANN ; minergic neurons of the SN (BUTCHER et ai, 1975; BUTCHER & BILEZIKJIAN, 1975; BUTCHER & HODGE, 1976). Both AChE levels measured by radioenzymatic assay and AChE histochemical staining were observed to decrease following treatment with 6-OHDA (Fig. 1 and Tables 1 and 2 ) Some trans-synaptic plasticity of AChE in nondopaminergic cells in the SN and CP following lesion of DA neurons cannot be ruled out However, in a histochemical study BUTCHER et ai (1975) examined the synthesis of AChE in the SN following irreversible inhibition of cholinesterase with diisopropylfluorophosphate and traced the de novo synthesis of AChE within the DA cell bodies. Thus, transsynaptic plasticity can be ruled out as the sole factor mediating the decrease in AChE in the SN following 6-OHDA. This is not a surprising result, since it has been established that all known catecholamine neurons stain for AChE (JACOBOWITZ & PALKOVITS, 1974; PALKOVITS & JACO-BOWITZ, 1974) and that total brain AChE decreases by 13% following intraventricular 6-OHDA injection in mice (BENTON et ai, 1975). The histochemical regimen may not be as reliable as biochemical methods with respect to differentiating between BChE and AChE (CONTESTABILE, 1976; BRIDGES et ai, 1973). In the case of the dopaminergic neurons of the nigrostriatal system, however, the use of several substrates, selective inhibitors, and different concentrations of ACh have confirmed the suggestion that dopaminergic neurons contain true AChE (BUTCHER et ai, 1975). AAChE, the AChE which is depleted from the SN by 6-OHDA lesions, was char-acterized by kinetic properties, substrate specificity, and response to selective inhibitors as true AChE. It is likely that AAChE is AChE contained in dopa-minergic neurons of the SN. The failure to observe a decrease in BCh hydrolysis after 6-OHDA treat-ments indicates that BChE is not contained in these neurons. The difference between AChE and BChE, using the three biochemical criteria, were confirmed with com-mercial preparations from erythrocytes and serum, re-spectively. The cholinesterases from the CP and SN fulfill all the criteria for true AChE: (1) SN AChE and CP AChE are inhibited by high concentrations of ACh. (2) The relative rates of ACh to Ac^MeCh hydrolysis by SN AChE and CP AChE compared quite closely to the rates observed in this study for erythrocyte AChE. (3) The responses of AChE from these three sources to three selective inhibitors were essentially similar and were distinct from the response of BChE. Some qualitative differences between eryth-rocyte, SN and CP AChE were observed Specifically, SN AChE showed some differences from CP AChE with respect to kinetics (Fig. 2) and effects of selective inhibitors (Figs. 4-6). Second, the effects of selective inhibitors on CP AChE paralleled the effects of these inhibitors on erythrocyte AChE more closely than did SN AChE. Previous workers have also found differ-ences in a kinetic parameter of AChE, the Michaelis 4 Z d H. C. FIBIGER constant, in tissue obtained from different regions of the nervous system (TUNNICLIFF et ai, 1976). The qualitative differences of AChE with respect to kinetic properties and response to inhibitors may be related to the heterogeneous molecular properties of this enzyme (CHAN et ai, 1972; V U A Y A N & BOWNSON, 1974; MCINTOSH & PLUMMER, 1976; RIEGER & VIGNY, 1976; SOMOGYI & CHUBB, 1976; G U R D , 1976). There are hazards, of course, with imputing a different mol-ecular structure of AChEs from different sources on the basis of assays performed on a crude homogenate. Although the tissues were diluted at least 500 fold, some very potent component in the tissue may have exerted a modulatory effect on the enzyme's proper-ties. Likewise, the properties of the purified erythro-cyte AChE may be due to an artifact of purification, e.g. a partial proteolysis at some stage of the pro-cedure. The presence of AChE on noncholinergjc neurons has been suggested by some to indicate that those neurons receive a cholinergic input (PARENT & BUTCHER, 1976). The present data and other consider-ations suggest however that AChE cannot be con-sidered a reliable marker for cholinoception. Primar-ily, this is because there is a dramatic lack of correla-tion between CAT and AChE in some regions of the nervous system. For example, the cerebellum of various species contains high levels of AChE, but low levels of CAT, and there is no correlation between the two enzyme levels in the different strata of cere-bellar cortex (GOLDBERG & M C C A M A N , 1967). In addition, rabbit dorsal root ganglion contains high levels of AChE, but insignificant levels of CAT ( M C C A M A N & HUNT, 1965). AChE appears therefore to be playing an unknown role in these _reas where ACh is not being synthesized in significant amounts. It is generally agreed that compared to the striatum the SN also contains a relatively low CAT to AChE ratio (MCGEER et ai, 1973; F O N N U M et ai, 1974) and this raises the question as to the function, if any, of AChE in the dopaminergic neurons of the nigro-stria-tal projection. At present a cholinergic afferent to the SN has not been demonstrated. Hemitransections anterior or posterior to this nucleus have no effects upon nigral CAT activities (MCGEER et ai, 1973; un-published observations). Furthermore, the failure of kainic acid injections into the SN to affect CAT ac-tivity in this nucleus suggests that CAT is not con-tained within perikarya in the SN which might synapse with DA neurons (Nagy, Vincent, Lehmann, Fibiger & McGeer, in preparation). At present there-fore the localization of CAT within the SN is un-known and .there is no firm evidence to indicate that the dopaminergic perikarya or dendrites receive cho-linergic innervation. If AChE in the DA perikarya does not serve to hydrolyze ACh released upon them, this raises the possibility that the enzyme is synthe-sized in the SN but that its function with respect to cholinergic transmission occurs in the axon terminals of the NSB in the striatum. Specifically in view of 43 AChE in dopaminergic neurons 623 the growing evidence supporting dendro-axonic transmission (LLINAS& HESS, 1976), it is possible that dopaminergic neurons synthesize AChE to inactivate ACh released by cholinergic dendrites upon which the dopaminergic terminals are thought to synapse (HAT-TORI et ai, 1976; GIORGUIEFF et ai, 1976). Alterna-tively, cholinergic axons in the striatum may synapse • on dopaminergic axons or boutons. According to conventional criteria, however, there is no ultrastruc-tural evidence for the presence of axo-axonic synapses in the striatum (KEMP & POWELL, 1971; HATTORI, personal communication). As a rule, cholinoceptive cells may be incapable of localizing AChE to the area of cholinoception. In addition to the above considerations, further support for this hypothesis is found in the peripheral sympath-etic system, where AChE has been identified in norad-renergic terminals in the pineal gland (ERANKO et ai, 1970; RODRIGUEZ DE LORES ARNAIZ & PELLIGRINO DE IRALDI, 1972), and where C A T is virtually negli-gible (Lehmann, unpublished observations). In view of the well-established cholinergic input to the norad-renergic cell bodies in the superior cervical ganglion which give rise to the noradrenergic terminals in the pineal gland, the presence of AChE in those terminals may be due to a transport process incapable of specifically localizing AChE to the area of cholinocep-tion. Therefore, although AChE may prove useful as a marker for cells which are cholinoceptive at some locus of the cell, it clearly cannot be utilized to iden-tify the point of cholinergic contact. To the extent that the decrease in AChE in the CP can be attributed entirely to AChE present in dopaminergic axons and terminals, the cell bodies in the SN appear to transport a greater amount of the enzyme to their terminals than resides in the cell bodies: in terms of absolute enzyme activities, a de-crease in the CP of 10,000 nmol/h/CP (i.e. 240nmol/mg tissue/h x 45 mg tissue/CP) correlates with a decrease in the SN of 2400 nmol/h/SN (i.e. 300nmol/mg tissue/h x 8mg tissue/SN). Approxi-mately four times as much AChE is exported to axons and terminals as is retained in the dopaminergic cell bodies and dentrites of the SN—roughly the same ratio as is observed for T H in this system. The presence of AChE in dopaminergic neurons of the SN has been confirmed biochemically, and the transport of AChE by nigrostriatal axons is suggested The results presented here shed no light on the ques-tion of cholinoception by these dopaminergic neurons. 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Vol. 4. pp. 217-225. Pcrgamon Press Lid, 1974. Primed in Great Britain. O IBRO 45 0306-4522/79)0201 -0217S02.0O/O THE LOCALIZATION OF ACETYLCHOLINESTERASE IN THE CORPUS STRIATUM AND SUBSTANTIA NIGRA OF THE RAT FOLLOWING KAINIC ACID LESION OF THE CORPUS STRIATUM: A BIOCHEMICAL AND HISTOCHEMICAL STUDY J. LEHMANN and H . C. FIBIGER Division of Neurological Sciences, Department of Psychiatry, University of British Columbia, Vancouver. B.C. V6T 1W5, Canada and L. L. BUTCHER Department of Psychology and Brain Research Institute. University of California. Los Angeles. California. U.S.A. Abstract—The distribution ofacetylcholinestera.se in the corpus striatum and substantia nigra was examined with the use of kainic acid lesions of the corpus striatum and pharmacohistochemical experi-ments. Histochemically identified acetylcholinesterase-comaining neurons in the striatum were among those which were destroyed by kainic acid. Complementary biochemical studies demonstrated that approximately 50";, of the total acetylcholinesterase activity in the striatum was localized in these acetylcholineslerase-containing neurons. Intrastriatal injections of kainic acid produced a substantial decrease in the activity of the glutamic acid decarboxylase in the substantia nigra, thus demonstrating that neurons contributing to the striato- and/or pallidonigral pathways had been lesioned. However, nigral acetylcholinesterase activity was not significantly reduced by the striatal kainic acid injections. Furthermore, stereotaxic injections of colchicine along the course of the striatonigral projection failed to produce an accumulation of acetylcholinesterase in these fibers proximal to the injection. In contrast, injections of colchicine into the nigrostriatal projection led to a proximal accumulation of acetyl-cholinesterase in the fibers of this system, thus confirming the presence of acetylcholinesterase in the ascending dopaminergic neurons. It is concluded that the striato- and pallidonigral projections in the rat do not contain significant levels of acetylcholinesterase. Furthermore, acetylcholinesterase-containing neurons in the striatum appear to be interneurons rather than the source of striatal efferents. It is suggested that some of these acetylcholinesterase-containing neurons may be striatal cholinergic interneurons. CERTAIN nuclei within the extrapyramidal system such as the caudate, putamen and substantia nigra, pars compacta. contain among the highest levels of acetylcholinesterase (acetylcholine hydrolase, E C 3.1.1.7; AChE) in the central nervous system (SILVER, ' 1974). Recent studies from this laboratory suggest that approximately 40% of the A C h E activity in the substantia nigra of the rat is contained within axons ' or terminals afferent to this nucleus (NAGY, VINCENT, LEHMANN. FIBIGER & M C G E E R , 1978). Consistent with this finding are the results of earlier lesion studies which led POIRER & co-workers (OLIVIER. PARENT. SIMARD & POIRIER. 1970) to propose that in the cat the projection from the striatum to the substantia nigra contains A C h E . Furthermore, it has been sug-gested that this projection may arise from aspiny neurons in the striatum (POIRIER. PARENT, M A R -Abhreviaiions: AChE, acetylcholinesterase: ChAT. cho-line acetyltransferase: DFP. diisopropylphosphorofluori-date: G A D . glutamic acid decarboxylase. CHAND & BUTCHER. 1977). which have been shown in pharmacohistochemical experiments to stain inten-sely for A C h E (BUTCHER, TALBOT & BILEZIKJIAN, 1975a). In the present experiments, we sought to evaluate further the existence and source of A C h E in the striatonigral and pallidonigral projections. In order to destroy these systems selectively without pro-ducing concomitant damage to the dopaminergic nigrostriatal projection, which is also known to con-tain A C h E (BUTCHER ci ai. 1975a; BUTCHER, TALBOT & BILEZIKJIAN, 1975/?; BUTCHER, 1977; LEHMANN & FIBIGER, 1978). kainic acid, a neurotoxin which is thought to destroy perikarya selectively, leaving affer-ent axons and terminals intact (COYLE & SCHWARCZ, 1976: M C G E E R & M C G E E R , 1976), was injected intra-striatally and the effects on A C h E and other neuronal enzyme markers in the corpus striatum were deter-mined. Furthermore, the brains of rats lesioned with kainic acid were examined by A C h E histochemistry to determine if cells which stained for A C h E were destroyed by kainic acid. 17 218 46 J. LEHMANN, H. C. FIBIGER and L. L. BUTCHER E X P E R I M E N T A L P R O C E D U R E S Male Wistar rats (300-325 g) were obtained from Woodlyn Laboratories, Guelph, Ontario. Ten nmol kainic acid in 1 //I 10 m,M sodium phosphate p H 6.5, 0.9% in N a C l was injected over 5 min unilaterally into the caudate-putamen of rats anesthetized with pentobarbital. F rom stereotaxic zeros, coordinates were: anterior 9 .6mm: lateral 2.8 mm: dorsal 4.5 mm. Two weeks later the rats were killed by cervical fracture and the corpora striata dissected on ice. These tissues included the caudate-putamen, the globus pallidus, and the nucleus accumbens, and averaged 45 mg wet tissue weight. The mesencephalon was sectioned on a freezing microtome and the substantia nigra was carefully dissected from these sections on ice. Tissues included A9 and A10 areas and pars reticulata, averaging 8 mg wet tissue weight. Homogenization and assay of tyrosine hydroxylase (L-tyrosine, tetrahydropteri-dine: oxygen oxidoreductase (3-hydroxylating) E C 1.14.6.2). choline acetyltransferase (Acetyl-CoA: choline-O-acetyltransferase E C 2.3.1.6; C h A T ) and A C h E were as previously described (LEHMANN & FIBIGER, 1978). Gluta-mic acid decarboxylase (L-glutamate 1-carboxylase E C 4.1.1.15: G A D ) was assayed by the method of MCGEER, MCGEER & WADA (1971a). Protein was assayed by the method of LOWRY, ROSEBROUGH, FARR & RANDALL (1951). Histochemical staining for A C h E was performed by the method of KARNOVSKY & ROOTS (1964) at 24-48 h follow-ing pretreatment with diisopropylphosphorofluoridate (DFP) . The irreversible inhibitfon of A C h E by the non-spe-cific phosphorylating agent D F P allows A C h E to be visua-lized at various stages of regeneration, i.e. following de novo synthesis and subsequent transport to distal cell pro-cesses. Combined with other experimental manipulations as outlined in the figure captions, this technique permits the identification of AChE-posi t ive neurons simultaneously with anatomical characterization. R E S U L T S Biochemistry A s seen in Tab le 1, the marked fall in the activities of G A D and C h A T , enzymes which are contained in neurons whose per ikarya are located in the corpus s t r ia tum ( M C G E E R , M C G E E R , FIBIGER & WICKSON, 19716; M C G E E R & M C G E E R , 1975 ; HATTORI, SINGH, M C G E E R & M C G E E R , 1976 ; RIBAK, 1978), indicated that the ka in i c acid injections destroyed more t han 7 0 % of the - / -aminobutyrate-containing and cho l ine r -gic cell bodies. T h e act ivi ty o f A C h E was reduced by 4 0 % in the same tissues whereas there was some increase in str iatal tyrosine hydroxylase. In contrast , nigral tyrosine hydroxylase and C h A T activities were not significantly affected. M o s t important in terms o f the possible presence o f A C h E in the s t r ia tonigra l project ion was the lack o f a significant decrease i n the activity of nigral A C h E despite the extensive damage to the corpus s t r ia tum (Table 1 and F i g . 1) and the extensive damage to the s t r ia tonigral and /o r pa l l idon ig ra l projections as shown by the decrease i n nigral G A D act ivi ty. It shou ld be noted that h i s to lo -gical examina t ion of the extent of the k a i n i c ac id lesion indicated that it inc luded the globus pa l l idus as wel l as the s t r ia tum. N o significant cell des t ruct ion was observed outside the corpus str iatum. Histochemistry D F P pretreatment suppresses "background' A C h E s ta ining (which is presumably contained in axons, ter-minals and dendr i t ic processes of neurons) and a l lows the v i sua l iza t ion of discrete per ikarya . A x o n a l l y trans-ported A C h E is also seen more clearly at longer sur-v iva l times. F o r these reasons, D F P pretreatment was employed for a l l A C h E his tochemical experiments. Intrastriatal injections o f ka in i c acid greatly reduced the amount of s tr iatal A C h E revealed by his-tochemistry (F ig . 1A). Fur the rmore , these injections e l iminated the A C h E - p o s i t i v e neuronal pe r ika rya visual ized by means o f D F P pretreatment. wh ich were seen in the unlesioned s t r ia tum of the same rat ( com-pare F i g . I B and C ) . In the uninjected caudate-putamen, stained per ika rya appeared to have aspiny processes, at least at the light mic roscop ic level (F ig . IB). Injection of co lch ic ine at various ros t ro-cau-da l points between the s t r ia tum and substantia n ig ra TABLE I. NEUROTRANSMITTER-RELATED ENZYMES IN CORPUS STRIATUM AND SUBSTANTIA NIGRA 2 WEEKS AFTER INJECTION OF KAINIC ACID (10 nmol) IN THE CAUDATE-PUTAMEN % Control Control value + S.E.M. Corpus striatum acetylcholinesterase choline acetyltransferase glutamic acid decarboxylase tyrosine hydroxylase 62.5% + 6.0%* 27.5% ± 7.3%* 23.3% ± 3.4%* 126.8% ± 5.5%* 43.4 + 2.07 / imol/mg protein/h 109.9 ± 4.01 nmol/mg protein/h 103.7 + 3.71 nmol/mg protein/h 7.88 ± 0 . 3 1 9 nmol/mg protein/h Substantia nigra acetylcholinesterase choline acetyltransferase glutamic acid decarboxylase tyrosine hydroxylase 92.0% ± 4.8%, 104.0% ± 9.8% 51.1% ± 3.8%* 93.9% ± 4.9% 10.9 + 0.132 / imol/mg protein/h 16.7 + 0.96 nmol/mg protein/h 265.0 ± 8.75 nmol/mg protein/h 5.59 + 0.404 nmol/mg protein/h M = 12. *P < 0.001, Student's two-tailed test. 219 47 A CX FIG. 1. Loss of acetylcholinesterase-containing neuronal somata after infusion of I0nmol/1 / i l kainic acid into the right striatum (A). Non-infused side is shown on left side of A and in B: arrows point to individual cell bodies. Dashed lines in A delimit the area displaying loss of acetylcholinesterase activity, shown in detail in C. Acetylcholinesterase method as described previously (BUTCHER el al.. 1975a). 1.5 mg/kg D F P was injected intramuscularly 24 h prior to death, cx, cerebral cortex; fb, fiber bundle perforating the striatum. Scale in A is 4 mm; scale in C is 400//m and this magnification applies also to B. FIG. 2. Partial trajcclories of ihe nigrostriatal pathways (arrows, frames A and B) visualized by aceul-cholinesterasc histochemistry following unilateral intracerebral infusion of colchicine (0.5 /<g in I ; i l 0.9"„ saline: rate = 0.25 /il/min) into the left globus pallidus and adjacent regions (frame A) or into the left medial forebrain bundle regions and contiguous areas (frame Bl . Rats were killed 48 h after treatment with 1.5 mg/kg D F P and 72 h after infusion of colchicine. In frame C is depicled the striato-nigral pathway visualized according to I h e horseradish peroxidase procedure of DE OLMOS (1977): ( s e e also BUTCHER & GIKSLER. 1977): 0.5 /<] of a 40",, horseradish peroxidase solution was unilaterally infused into the caudate-putamen over a 5-min period: rats were killed 48 h after the injection. Antero-grade transport of the enzyme reveals I h e partial trajectory of the striatonigral projection (frame C). which bears considerable resemblance lo the striatonigral palhwa> as demonstrated by protein-incorpo-ration autoradiography with [ 3 H]pro l inc (cf. BUTCHER. 19781. Hor izonla l sections are shown. Dashed lines in frame A bracket the area in which portions of I h e striatonigral pathway are contained. Scale = 4 mm. P C . substantia nigra, pars compacla: PR. substantia nigra, pars reticulata: IC. internal capsule: ct. cannula tract. The PLLLIGRINO & CUSHMAN (I967| coordinates were: striatum: A P = 2.0. Lai = 3.0. vertical from conical surface = 5.0: globus pallidus: A P — 0.8. Lat. 3.5. vertical = 6.5. Medial forebrain bundle and adjacent regions: A P = 1.8. Lat = 1.5. vertical = 8.2. 221 49 • • FIG. 3. Accumulation of acetylcholinesterase in fibers of ascending dopaminergic pathways from the substantia nigra and probably also the ventromedial mesencephalic tegmentum (bracketed by arrows in F and H) following unilateral infusion of 0.5 ug 1 /J1 colchicine into the ventral thalamus (coordinates according to PELLEGRINO & CuSHMAN {1967): AP = 0.0. Lat = 1.5. vertical from cortical surface = 8.0). The non-infused side of the brain is shown at various levels in A, C, E and G corresponding to the same levels from the same brain sections on the infused side (B. D, F and H). Observe the absence of accumulation of acetylcholinesterase in processes of neuronal somata in the caudate-putamen complex on the infused side of the brain (B. D: compare with A and C. respectively). C and D are higher power depictions of A and B. respectively. E and F show the ventral thalamus and adjacent dorsolateral hypothalamus. G and H depict the substantia nigra and adjacent ventromedial mesence-phalic tegmentum. Rats were killed 24 h after intramuscular injection of 1.5 mg/kg DFP and 72 h after intracerebral colchicine infusion. Scale in B is 500nm and applies to A-B and E-H; scale in D is 300 / i m and applies also to C. fb. fiber bundle perforating CP: PC. substantia nigra, pars compacta; PR. substantia nigra, pars reticulata. 50 Acetylcholinesterase in corpus striatum and substantia nigra reliably resulted in accumulation of A C h E in axons of the nigrostriatal projection proximal to the injec-tion site (Fig. 2A and B, Fig. 3E-H). In contrast, ac-cumulation of A C h E was never observed in the proxi-mal segments of axons of the striatonigral or palli-donigral systems after colchicine injections in the vicinity of these projections (Fig. 2A and B, Fig. 3A-D) . Finally, unlike the accumulation and in-creased staining for A C h E in the processes of the cells of the zona compacta of the substantia nigra after these colchicine injections, such a phenomenon was never seen in the striatum (compare Fig. 3E-H with 3A-D). DISCUSSION The present results failed to provide evidence for the presence of A C h E in the striatonigral or pallido-nigral projections in the rat. Thus, although the kainic acid lesions extensively destroyed perikarya in the striatum and globus pallidus, including those which stain heavily for A C h E , no statistically significant loss of this enzyme could be detected biochemically in the substantia nigra ipsilateral to the lesion. Furthermore, in agreement with conclusions drawn by SHUTE & LEWIS (1967), colchicine injections in the vicinity of nigrostriatal and striatonigral fibers produced A C h E accumulation caudal but not rostral to the injection. The factors underlying the apparent discrepancy between these results and those of previous investiga-tors (OLIVIER et ai, 1970) are presently not clear but could conceivably be related to species differences. However, inasmuch as OLIVIER et al. (1970) utilized electrolytic lesions and long survival times it is also possible that retrograde degeneration of the A C h E -containing nigrostriatal projection may have contri-buted to their findings. This possibility has also recently been put forward by POIRIER et al. (1977). Nigral G A D activity was significantly decreased by these lesions, thus confirming previous suggestions that some of the striatonigral and/or pallidonigral fibers contain G A D (HATTORI, MCGEER, FIBIGER & MCGEER , 1973; FONNUM, GROFOVA, RINVIK, STORM-MATHISEN & WALBERG, 1974; GALE, HONG & G u i -DOTTI, 1977). The decrease in nigral G A D activity fol-lowing striatal kainic acid lesions is complementary to the decrease in nigral G A D activity following in-tranigral kainic acid lesions (NAGY et ai, 1978), sug-gesting that nigral G A D is partially derived from in-trinsic nigral neurons and partially derived from stria-tonigral and/or pallidonigral afferents. The modest decrease in striatal A C h E observed after kainic acid lesions indicates that approximately 50% of the A C h E activity in this structure is con-tained within neurons which are intrinsic to the corpus striatum. The figure of 50% is obtained by extrapolating to a 100% lesion of the C h A T and G A D markers. We have recently shown that approximately 12% of the total A C h E activity in the striatum is con-tained within the axons and terminals of the dopa-223 minergic nigro-striatal projection (LEHMANN & FIBIGEE. 1978). Conceivably, cortical, thalamic and raphe afferents could make up the balance. However, two other possibilities must also be considered. First, some A C h E may be contained within non-neuronal elements of the striatum (but cf. BUTCHER et ai, 1975a). Second, denervation plasticity of the sort observed with A C h E in the superior cervical ganglion (SOMOGYI & CHUBB, 1976; GISIGER, VIGNY, GAUTRON & RIEGER, 1978) must be considered as a contributing factor to biochemical changes which result from lesions. Our observation that kainic acid lesions, which produced extensive damage to the AChE-containing neurons in the striatum, did not result in a biochemi-cally detectable change in A C h E activity in the sub-stantia nigra suggests that these AChE-positive cells do not project to the substantia nigra. A C h E reactive neurons are few in number and appear aspiny at the light-microscopic level (BUTCHER et ai. 1975a; POIR-IER et ai, 1977). Furthermore, in the monkey (POIRIER et ai, 1977) A C h E reactive neurons are larger (> 25 /<m) and fewer in number than the medium spiny neuron (12-18 nm, KEMP & POWELL, 1971:13-20 / tm, GROFQVA, 1975). These observations suggest that neurons with high A C h E activity are not medium spiny neurons, and that medium spiny neurons have low, if any, A C h E activity. Although earlier work suggested that the large aspiny neurons were the source of striatal efferents to the globus pallidus and substantia nigra (Fox, RAFOLS & COWAN , 1975), more recent studies have demonstrated that the striatal efferents originate predominantly, if not exclusively, from the medium-sized, spiny neurons of KEMP & POWELL (1971) (GRO-FOVA, 1975; BUNNEY & AGHAJANIAN, 1976: Kocsis, PRESTON & KITAI. 1976: S. T. KITAI, personal com-munication). Lack of A C h E staining in these numerous medium-sized, spiny efferent cells is consis-tent with the absence of a detectable change in A C h E activity in the substantia nigra after the kainic acid lesions. Inasmuch as the aspiny AChE-containing neurons appear not to project to the substantia nigra, this raises the speculation that some of the AChE-reactive cells may be the cholinergic neurons which are also thought to be intrinsic to the corpus striatum (MCGEER et ai, 1971b). Arguing against this specula-tion is the immunohistochemical observation by HAT-TORI et al. (1976) that some dendritic spines in the striatum contain C h A T , and that medium spiny neurons, as characterized by an unindented nucleus (KEMP & POWELL, 1971). contain C h A T (T. HATTORI, personal communication). If this is the case, then on the basis of presently available evidence it would have to be concluded that these cholinergic, spiny neurons do not contain A C h E . Such a situation would be un-precedented for cholinergic neurons which typically contain very high levels of A C h E activity. Clearly, additional work is required to identify the nature of AChE-containing neurons in the corpus striatum. 224 51 J. LEHMANN, H. C. FIBIGER and L. L. BUTCHER Acknowledgements—Supported by the Medical Research Council (J.L. and H.C.F.) and by USPHS grant NS-10928 (L.L.B.). The excellent technical assistance of S. ATMADJA is gratefully acknowledged. Mr. KEN HIRABAYASHI (U.C.L.A.) is thanked for performing the histochemical ex-perirr.f-'s. The authors thank T. HATTORI for stimulating discussions and reviewing the manuscript. REFERENCES BUNNEY B. S. & AGHAJANIAN G. K. (1976) The precise localization of nigral afferents in the rat as determined by a retrograde tracing technique. Brain Res. 117, 423-435. BUTCHER L. L. (1977) Nature and mechanism of cholinergic-monoaminergic interactions in the brain. Life Sci. 21, 1207-1226. BUTCHER L. L. 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(1971a) Glutamic acid decarboxylase in Parkinson's disease and epilepsy. Neurology, Minneap. 21, 1000-1007. NAGY J. I., VINCENT S. R.. LEHMANN J., FIBIGER H. C. & MCGEER E. G. (1978) The use of kainic acid in the localization of enzymes in the substantia nigra. Brain Res. 149, 431-441. OLIVIER A., PARENT A., SIMARD H. & POIRIER L. J. (1970) Cholinesterasic striatopallidal and striatonigral efferents in the cat and the monkey. Brain Res. 18, 273-382. PELLEGRINO L. J. & CUSHMAN A. J. (1967) A Stereotaxic Atlas of the Rat Brain. Appleton-Century-Crofts, New York. POIRIER L. J., PARENT A., MARCHAND R. & BUTCHER L. L. (1977) Morphological characteristics of the acetylcholinester-ase-containing neurons in the CNS of DFP-treated monkeys. J. neurol. Sci. 31, 181-198. Acetylcholinesterase in corpus striatum and substantia nigra 225 RIBAK C. E. (1978) Immunocytochemical localization of glutamic acid decarboxylase (GAD) in the rat corpus striatum. Anal. Rec. 190, 521. SHUTE C. C. D. & LEWIS P. R. (1967) The ascending cholinergic reticular system: cortical, olfactory and subcortical projections. Brain 90, 497-520. SILVER A. (1974) The Biology of Cholinesteruses. Elsevier. New York. SOMOGYI P. & CHUBB 1. W. (1976) The recovery of acetylcholinesterase activity in the superior cervical ganglion of the rat following its inhibition by diisopropylphosphorofluoridate: a biochemical and cytochemical study. Neuroscience 1, 413-421. . • 53 THE NUCLEUS BASALIS MAGNOCELLULARIS: THE ORIGIN OF A CHOLINERGIC PROJECTION TO THE NEOCORTEX OF THE RAT by John Lehmann, J . I . Nagy, S. Atmadja and H.C. F i b i g e r D i v i s i o n of Neurological Sciences Department of Psychiatry University of B r i t i s h Columbia Vancouver, B.C., Canada V6T 1W5 Please send correspondence to: Dr. H.C. Fibiger D i v i s i o n of Neurological Sciences Department of Psychiatry University of B r i t i s h Columbia Vancouver, B.C., Canada V6T 1W5 54 Abbreviations AChE, a c e t y l c h o l i n e s t e r a s e ; CAT, c h o l i n e a c e t y l t r a n s f e r a s e ; DFP, d i i s o -propylphosphorofluoridate; GAD, glutamic a c i d decarboxylase; HRP, horse r a d i s h peroxidase. 55 4 ABSTRACT The c e l l s of o r i g i n of a n e o c o r t i c a l c h o l i n e r g i c a f f e r e n t p r o j e c t i o n have been i d e n t i f i e d by anterograde and retrograde methods i n the r a t . H o r s e r a d i s h ~ peroxidase i n j e c t e d i n t o neocortex lab e l e d l a r g e , a c e t y l c h o l i n e s t e r a s e (AChE)-intense neurons i n the ventromedial extremity of the globus p a l l i d u s . This same group of neurons underwent retrograde degeneration f o l l o w i n g c o r t i c a l a b l a t i o n s . . The region i n which c e l l depletion occurred a l s o showed s i g n i f i c a n t decreases i n the a c t i v i t i e s of c h o l i n e a c e t y l t r a n s f e r a s e and AChE. D i s c r e t e e l e c t r o l y t i c and k a i n i c a c i d l e s i o n s r e s t r i c t e d to the medial "part o f the globus p a l l i d u s each r e s u l t e d i n s i g n i f i c a n t depletions of n e o c o r t i c a l c h o l i n e a c e t y l -t r a n s f e r a s e and AChE. Hemitransections caudal to t h i s c e l l group d i d not r e s u l t i n such d e p l e t i o n s . Taken together these observations suggest t h a t the AChE-intense neurons l y i n g i n the ventromedial extremity of the globus p a l l i d u s , as mapped i n t h i s study, c o n s t i t u t e the o r i g i n of a major s u b c o r t i c a l c h o l i n e r g i c p r o j e c t i o n to the neocortex.' The u t i l i t y of AChE h i s t o c h e m i s t r y i n DFP-pre-treated animals i n i d e n t i f y i n g c h o l i n e r g i c neurons i s d i s c u s s e d i n the l i g h t of t h i s example. S p e c i f i c a l l y , i t i s proposed that high AChE a c t i v i t y 4-8 hrs a f t e r DFP pretreatment i s a necessary, but not s u f f i c i e n t , c r i t e r i o n f o r the i d e n t i f i c a t i o n of c h o l i n e r g i c p e r i k a r y a . The neurons i n question appear to be homologous to the n u c l e u s b a s a l i s of the substantia innominata of primates, and are thus termed "nucleus b a s a l i s mag-n o c e l l u l a r i s " (nBM) i n the r a t . No evidence was obtained to support the hypo-th e s i s that nucleus of the diagonal band p r o j e c t s to neocortex. However, s t r i k i n g s i m i l a r i t i e s i n s i z e and AChE a c t i v i t y were observed among the p u t a t i v e c h o l i n -e r g i c p e r i k a r y a of the nBM, the nucleus of the diagonal band, and the medial s e p t a l nucleus. K a i n i c a c i d l e s i o n s of the neocortex produced uniform and complete p e r i k a r y a l d e s t r u c t i o n . These l e s i o n s decreased n e o c o r t i c a l glutamic a c i d decarboxylase a c t i v i t y , suggesting that there are GABAergic perikarya i n the neocortex- How ever, the same l e s i o n s d i d not a f f e c t n e o c o r t i c a l choline a c e t y l t r a n s f e r a s e . This observation suggests that there are no c h o l i n e r g i c p e r i k a r y a i n the neo-cortex, a conclusion that i s c o n s i s t e n t with the absence of i n t e n s e l y AChE-r e a c t i v e neurons i n neocortex. 57 INTRODUCTION Acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7; AChE) per se has not proven to be a reliab l e marker for cholinergic neurons and the i r pro-jections i n the central nervous system (PHILLIS, 1976). For example, i t i s now known that high levels of AChE are contained in certain dopaminergic and noradrenergic neurons (SILVER, 1974; JACOBOWITZ & PALKOVTTS, 1974; BUTCHER, TALBOT & BILEZIKJIAN, 1975; LEHMANN & FIBIGER-, 1978). Despite this lack of s p e c i f i c i t y for cholinergic neurons, histochemical studies of AChE have proven to be of value in suggesting potential cholinergic projections because a l l neurons that have been unequivocally characterized as cholinergic appear to contain very high levels of AChE (e.g., the septo-hippo-campal projection: LEWIS, SHUTE, & SILVER, 1967; MESULAM, VAN HOESEN & ROSENE, 1977; LYNCH, ROSE & GALL, 1978; the motoneuron, KREUTZBERG, TOTH & KAIYA, 1975; vis c e r a l efferent neurons of the intermediolateral spinal cord, BUTCHER, MARCHAND, PARENT & POIRIER, 1977). On the basis of currently available inform-ation i t can therefore be postulated that a strong histochemical reaction for AChE i s a necessary (but not sufficient) c r i t e r i o n for the i d e n t i f i c a t i o n of cholinergic neurons. KODAMA (1929) f i r s t provided evidence for a projection from the magno-c e l l u l a r neurons of the basal forebrain to the neocortex and this observation has been confirmed and extended by others (DAS, 1971; KIEVET & KUYPERS, 1975; DIVAC, 1975; JONES, BURTON, SAPER & SWANSON, 1976). MESULAM & VAN HOESEN (1976) reported that neurons in the basal forebrain of the rhesus monkey that stain intensely for AChE are also labeled with HRP after injections of the l a t t e r enzyme into the neocortex. On the basis of this evidence these authors sup-ported e a r l i e r suggestions (SHUTE 6. LEWIS, 1967; DIVAC, 1975) that there i s a cholinergic projection from the basal forebrain, mostly from the nucleus basalis of the substantia innominata, which innervates the precentral neocortex. Re-58 cently, KELLY & MOORE (1978) have published results that appear to be consistent with this hypothesis. They found that e l e c t r o l y t i c lesions in the v i c i n i t y of the globus pallidus of the rat produced significant decreases.in c o r t i c a l choline acetyltransferase a c t i v i t y . EMSON & LINDVALL (1979) have also recently com-mented on various magnocellular forebrain nuclei as possible origins of the cholinergic innervation of the neocortex. The purpose of the present experiments was to provide further information concerning the origin and l o c a l i z a t i o n of cholinergic neuronal elements in the neocortex. METHODS Male Wis tar rats weighing approximately 300 gm at the time of surgery were used in a l l experiments. A l l surgery was conducted while animals were under pentobarbital anesthesia. Horseradish peroxidase (HRP; .Sigma type VI) was injected into various c o r t i c a l areas in 0.1 yl volumes at a concentration of 30% in 0.9% saline. The histological protocol of MESULAM & VAN HOESEN (1976); MESULAM (1976a,b) ; MESULAM (.personal communication) was followed for v i s u a l i z a t i o n of HRP with benzidene dihydrochloride: alternate sections were stained for both HRP and AChE, according to the. same procedure. Twenty-four to thirty hours f o l -lowing HRP injections, the animals were perfused under deep pentobarbital anes-thesia with 50 ml 0.9% saline at room temperature; this was followed by a per-fusion of 400 ml cold fixa t i v e (1% paraformaldehyde and 1.25% glutaraldehyde, 10% sucrose, 0.1 M sodium phosphate buffer, pH 7.4). The brain was stored i n fixative for 2-3 hrs, then transferred into 5% sucrose- 0.1 M sodium phosphate buffer (pH 7.4) and kept overnight at 4°C. The HRP reaction was carried out essentially as previously described.. (MESULAM 1976a; MESULAM, personal communication). Free floating sections were rinsed in d i s t i l l e d water for 1 min. They were then incubated for one hour in incubation medium containing 4 mM acetylthiocholine,. 10 mM glycine, 2 mM copper sulfate, and 50 mM sodium acetate. The pH of this incubation medium was main-59 tained at 5.5. Sections were then r i n s e d i n s i x changes of d i s t i l l e d water, 30 sec each. Then they were t r a n s f e r r e d to 50% benzidine d i h y d r o c h l o r i d e (Sigma), 0.1% n i t r o f e r r i c y a n i d e , 0.01 M sodium acetate b u f f e r pH 5.0, 10% i n e t h a n o l , f o r 10 min. The tray was l i f t e d out of t h i s l a s t medium b r i e f l y , w hile 5 ml f r e s h l y prepared 0.3% H 2 O 2 was added to an incubation volume of 100 ml. The t i s s u e s were r e i n s e r t e d i n t o t h i s incubation mixture and shaken g e n t l y f o r 4 to 4.5 min. The HRP-reaction was stopped by t r a n s f e r r i n g the s e c t i o n s i n t o 9% sodium n i t r o f e r r i c y a n i d e , 50% ethanol i n 40 mM sodium acetate b u f f e r „(pH 5.0), which was f r e s h l y prepared and kept at 0-4°C. Following 20 minutes i n t h i s medium, se c t i o n s were r i n s e d i n s i x changes of d i s t i l l e d water. In order to v i s u a l i z e the AChE r e a c t i o n product, the sections were immersed i n 10% potassium f e r r i -cyanide for 15 min. This was followed by extensive r i n s i n g i n s i x changes of d i s t i l l e d water. Subsequently, sections were mounted from water onto g l a s s s l i d e s , and a i r - d r i e d . In some cases the s e c t i o n s were c o u n t e r s t a i n e d with c r e s y l v i o l e t . F i v e types of b r a i n l e s i o n s were u t i l i z e d ; (1) Extensive u n i l a t e r a l c o r t i c a l l e s i o n s were made i n seven animals by s u c t i o n . Care was taken not to damage any s u b c o r t i c a l s t r u c t u r e s such as the hippocampus, septum and s t r i a t u m . The l e s i o n s included f r o n t a l , d o r s a l and l a t e r a l cortex a n t e r i o r to the l e v e l of (T) bregma. Gelfoam was a p p l i e d to f i l l the v o i d . (2) A second group of n i n e r a t s received u n i l a t e r a l e l e c t r o l y t i c l e s i o n s of the nucleus b a s a l i s magnocel-l u l a r i s (nBM). The l e s i o n s were made by ap p l y i n g a current of 2 mA f o r 20 sec at the coordinates AP + 8.1 mm, ML + 2.6 mm and DV + 3.6 mm from s t e r e o t a x i c "•' j . zero. The i n c i s o r bar was set at -4.2 mm. (3) Eleven animals were i n j e c t e d with two nmoles of k a i n i c a c i d d i s s o l v e d i n NaPOi, buffered (7.0) s t e r i l e s a l i n e i n a volume of 0.2 y l over a period of seven minutes a t the same coor-in a t e s used for the e l e c t r o l y t i c nBM l e s i o n s . (4) Another n i n e aminals r e -ceived u n i l a t e r a l i n j e c t i o n s of k a i n i c a c i d (10 nmol i n 2 y l of the same v e h i c l e 60 9 over f i v e minutes) i n t o the f r o n t a l cortex at the coordinates AP +10.8 mm, ML + 2.0 mm, and DV -2.2 mm from s t e r e o t a x i c zero ( i n c i s o r "bar set a t + 5.0 mm). (5) Hemitransections of the b r a i n at a l e v e l j u s t p o s t e r i o r to the entopeduncular nucleus (AP + 5.9 mm from s t e r o t a x i c zero, i n c i s o r bar at - 4.2 mm) were made i n seven animals according to the method of McGEER, FIBIGER, McGEER & BROOKE (1973). A l l animals that had received u n i l a t e r a l c o r t i c a l l e s i o n s by s u c t i o n were used a f t e r a s u r v i v a l time of s i x months. Animals that had r e c e i v e d e l e c t r o l y t i c l e s i o n s , k a i n i c a c i d l e s i o n s of nBM, and hemitransections were s a c r i f i c e d two weeks p o s t o p e r a t i v e l y . Animals which received k a i n i c a c i d l e s i o n s of the cortex were s a c r i f i c e d one week p o s t o p e r a t i v e l y . Some brains were s e c t i o n e d c o r o n a l l y on a f r e e z i n g microtome. The sections were kept co l d on i c e and from these sections the nBM, which included the medial and c e n t r a l globus p a l l i d u s , was d i s -sected from each s i d e of the b r a i n (see F i g . 1). The f r o n t a l c o r t i c a l p o l e was. obtained from unfrozen brains by making a coronal cut j u s t a n t e r i o r to the head of the st r i a t u m . Care was taken to remove the o l f a c t o r y bulbs from the v e n t r a l surface of the f r o n t a l cortex. The a c t i v i t i e s of ch o l i n e a c e t y l t r a n s f e r a s e (Acetyl-CoA: c h o l i n e - O - a c e t y l -t r a n s f e r a s e , EC 2.3.1.6; CAT) and AChE were measured accordi n g to m o d i f i c a t i o n s (LEHMANN & FIBIGER, 1978) of the method of FONNUM (1969) . Glutamic a c i d decar- • boxylase (L-glutamate 1-carboxylase, EC 4.1.1.15; GAD) was assayed by the method of CHALMERS, McGEER, WICKSON & McGEER (1970). P r o t e i n was measured ac c o r d i n g to LOWRY, ROSENSROUGH, FARR & RANDALL, (1951). For h i s t o l o g i c a l v e r i f i c a t i o n of the k a i n i c acid-induced l e s i o n s of the f r o n t a l cortex and nBM, and e l e c t r o l y t i c l e s i o n s of the nBM, the animals were deeply anesthetized and then perfused with 10% Formalin - 0.9% s a l i n e . The brains' were removed and placed i n 10% Formalin - 0.9% s a l i n e f o r two weeks, s e c t i o n e d at 50 ym at the l e s i o n s i t e s on a f r e e z i n g microtome and s t a i n e d w i t h c r e s y l v i o l e t . Where p o s s i b l e these h i s t o l o g i c a l v e r i f i c a t i o n s were conducted on the 61 same brains that were analyzed f o r the various enzymes. For h i s t o c h e m i c a l studies of AChE, animals were injected intramuscularly with d i i s o p r o p y l p h o s -phorofluoridate (DFP, 1.5 mg/kg, Sigma, dissolved i n peanut o i l ) and s a c r i f i c e d at various times l a t e r . Histochemical s t a i n i n g for AChE was performed according to KARNOVSKY & ROOTS (1964) on 25 um, f r e e - f l o a t i n g sections. P e r i k a r y a l dimensions were estimated both photographically and with the a i d of a measuring eyepiece, both of which were cal i b r a t e d with a stage micrometer. 62 11 RESULTS TOPOGRAPHY OF INTENSELY AChE-REACTIVE NEURONS IN THE BASAL FOREBRAIN Nucleus of the diagonal band and medial septal nucleus The o r g a n i z a t i o n of i n t e n s e l y AChE-reactive neurons i s given i n F i g s . 1 and 2. At the most r o s t r a l l e v e l ( F i g . 1A-B), numerous i n t e n s e l y A ChE-reactive neurons i n the medial s e p t a l nucleus and v e r t i c a l limb of the nucleus of the diagonal band (nD3) can be i d e n t i f i e d . Moving caudally ( F i g . 1C-D), the nDB becomes more l a t e r a l l y and v e n t r a l l y located, and i s c a l l e d the h o r i z o n t a l limb of the nDB (PRICE & POWELL, 1970). At more caudal levels" ( F i g . 2A-E), t h i s c e l l group becomes much l e s s compact; now scattered, magnocellular i n -tensely AChE-reactive neurons occupy the regions termed magnocellular p r e o p t i c nucleus and l a t e r a l hypothalamic area (WYSS, SWANSON & COWAN, 1979). A l l o f these i n t e n s e l y AChE-reactive neurons are more or l e s s contiguous at some p o i n t with one another, although as noted there are marked d i f f e r e n c e s i n the popula-t i o n d e n s i t i e s of the neurons. V e n t r a l pallidum A few s c a t t e r e d , magnocellular, i n t e n s e l y AChE-reactive neurons can be seen i n the region termed " v e n t r a l p a llidum" by HEIMER & WILSON (1975) ( F i g . 1B-E). I t i s important to note that the term v e n t r a l p a l l i d u m r e f e r s to the r a t ' s p u t a t i v e homologue of substantia innominata of primates, and does not r e f e r to the v e n t r a l part of the globus p a l l i d u s (HEIMER & WILSON, 1975) . Along the medial border of the globus p a l l i d u s i s a group of i n t e n s e l y AChE-reactive neurons which may be continuous with the nDB at the more d o r s a l and. caudal aspects of the nDB. However, these neurons, which are b e l i e v e d to be the r a t ' s homologue of the primate nucleus b a s a l i s of the substantia innominata (see d i s -cussion) , have a c h a r a c t e r i s t i c morphology and topographical d i s t r i b u t i o n . D i s t r i b u t i o n of the nucleus b a s a l i s m agnocellularis The r o s t r a l pole of the nucleus b a s a l i s m a g n o c e l l u l a r i s (nBM) i s l o c a t e d 63 approximately just caudal to the decussation of the anterior commissure. At this point i t i s situated along the ventral and medial boundaries of the globus pallidus (Fig. IE) u n t i l the t a i l of the globus pallidus i s reached (Fig. 2D). It should be noted, however, that a few scattered c h a r a c t e r i s t i c a l l y large, intensely AChE-reactive neurons typical of nBM are found deep within the core of the globus pallidus at a l l rostrocaudal levels. Furthermore, at i t s most caudal extent,the nBM extends ventrolaterally into" the medial half of the t a i l of the globus pallidus (Fig. 2E). At this point and further caudally (Fig. 2F), many of the intensely AChE-reactive neurons are located i n t e r s t i t i a l l y (DAS & KREUTZBERG,1968), that i s , their perikarya and dendrites are situated between the fibre bundles of the internal capsule. At the most caudal levels i n v e s t i -gated in this study (Fig. 2F) , these large intensely AChE-reactive neurons are located in the ventral edge of the internal capsule, predominantly l a t e r a l to the entopeduncular nucleus, which would appear i n sections s l i g h t l y more caudal than F i g . 2F. Injections of HRP into the frontal and antero-dorsal neocortex resulted in numerous HRP-labeled neurons in nBM (Fig. 3A). With the r o s t r a l l y located injections used in these experiments, the most anteriorly labeled c e l l s were observed just ventral to the GP i n nBM. In those cases in which there was d i f -fusion of HRP from the injection site in the neocortex to subcortical regions, a few labeled c e l l s were occasionally observed in the nDB. However, when the HRP diffusion remained within the confines of the neocortex, no labeling of nDB was observed. The majority of neurons labeled by KRP in the present experiments were found in the group of large, multipolar neurons located in the ventral and medial regions of the posterior half of the GP. Tnese corresponded precisely to the location of the AChE-intense neurons seen in Figs. IF and 2A. With the ros t r a l c o r t i c a l HRP injections employed in this study, the caudal extent of 64 labeled c e l l s in nBM corresponded to the AChE-intense neurons in F i g . 2B. In subsequent preliminary experiments i t has been found that more posterior c o r t i c a l HRP injections (e.g., o c c i p i t a l cortex) label magnocellular neurons at more posterior regions of the nBM, i.e., at the level of the entopeduncular nucleus. In order to determine i f the c o r t i c a l HRP injections did i n fact l a b e l these intensely AChE-reactive neurons of the nBM, the method of MESULAM & VAN HOESEN (1976) was u t i l i z e d to demonstrate HRP and AChE in the same section. Although AChE staining was somewhat reduced by this procedure, i t nevertheless revealed that some c e l l s in the nBM with a high AChE content also contained HRP reaction product. MORPHOLOGICAL OBSERVATIONS OF THE INTENSELY AChE-REACTIVE NEURONS IN THE BASAL FOREBRAIN Nucleus of the diagonal band, medial septal nucleus, and caudate-putamen . The AChE-intense neurons of the medial septal nucleus and nDB have similar morphological features, with the size of the major axis of the perikarya ranging from 19-42 ym, and averaging 29 ym. AChE-intense aspiny neurons of the caudate-putamen have similar dimensions (Fig. 3C, range, 23-47 ym; average, 34 ym) . Since intensely AChE-reactive neurons in the striatum are less densely packed than the intensely AChE-reactive neurons of the medial septal nucleus and nDB, their dendrites are more easily visualized, although for a l l three of these nuclei, the appreciable "background" staining for AChE li m i t s the distance from the soma at which the dendrite may be seen clearly. Nucleus basalis magnocellularis While the perikarya of the nBM have similar dimensions to those neurons described above (major axis ranging from 25 to 45 ym, averaging 35 ym), their dendrites are easily seen against the background of white matter in which they are usually found. Thus the neuropil that these neurons occupy distinguishes them anatomically from the other intensely AChE-reactive neurons of the basal 65 f o r e b r a i n . The most s t r i k i n g morphological feature of the neurons of nBM i s t h e i r " i s o d e n d r i t i c " nature (RAMON-MOLINER & NAUTA,1966; DAS & KREUTZBERG,1968) : t h e i r dendrites taper o f f very gradually from the p e r i k a r y o n , indeed making i t d i f f i c u l t to delineate e x a c t l y where the soma ends and the d e n d r i t e begins ( F i g . 3B) . This morphological feature i n a l l p r o b a b i l i t y accounts f o r t h e ^ d i s -crepancy between the absolute dimensions reported here and those r e p o r t e d by PARENT, GRAVEL & OLIVIER (1979). The dendrites p r o j e c t d i r e c t l y somatofugally, with minimal branching or d e v i a t i o n i n course. Often these c e l l s a r e arranged i n t i g h t l y packed c l u s t e r s , so that the morphology of i n d i v i d u a l neurons cannot e a s i l y be asc e r t a i n e d . These c l u s t e r s are quite o f t e n so densely packed t h a t at low m a g n i f i c a t i o n they appear to form a giant neuron. F u r t h e r , the morphology of these l a r g e , i n t e r s t i t i a l neurons i s v a r i a b l e and i r r e g u l a r compared to those i n the medial s e p t a l nucleus and nDB. LESION STUDIES Retrograde degeneration of nucleus b a s a l i s m a g n o c e l l u l a r i s Extensive l e s i o n s of the neocortex a n t e r i o r to bregma r e s u l t e d i n a marked l o s s of l a r g e , i n t e n s e l y AChE-reactive neurons that were l o c a t e d i n the medial and v e n t r a l aspects of the GP ( F i g . 2A-F) , i n agreement with data presented by DAS (1971) f o r the r a b b i t . Of the few AChE-intense neurons t h a t remained i n t h i s region on the les i o n e d s i d e of the b r a i n , the majo r i t y appeared to be somewhat shrunken and pyknotic ( F i g . A). In sections that were c o u n t e r s t a i n e d with c r e s y l v i o l e t , there was no apparent decrease i n the p o p u l a t i o n o f the s m a l l e r diameter neurons that d i d not s t a i n i n t e n s e l y f o r AChE and that were l o c a t e d i n the r e g i o n of the v e n t r a l and medial GP. The i n t e n s e l y AChE-reactive neurons of the medial s e p t a l nucleus and nDB d i d not appear to undergo ret r o g r a d e changes or degener-a t i o n a f t e r the c o r t i c a l l e s i o n s . On the c o r t i c a l l y - l e s i o n e d s i d e c h o l i n e a c e t y l t r a n s f e r a s e (CAT) and AChE a c t i v i t y were decreased i n the r e g i o n of nBM, while glutamic a c i d decarboxylase (GAD) a c t i v i t y was normal (Table I ) . 66 1 5 Anterograde degeneration f o l l o w i n g l e s i o n s of nucleus b a s a l i s E l e c t r o l y t i c and k a i n i c a c i d l e s i o n s of the nBM r e s u l t e d i n s i m i l a r and p a r a l l e l depletions of CAT and AChE a c t i v i t i e s i n the i p s i l a t e r a l f r o n t a l c o r t e x (Table I I ) . F i g . 5 shows, diagrammatically, the extent of e l e c t r o l y t i c and k a i n i c a c i d l e s i o n s . Comparison of the areas encompassed by the l e s i o n s w i t h the d i s t r i b u t i o n of the l a r g e , i n t e n s e l y AChE-reactive, i n t e r s t i t i a l neurons i n the medial and v e n t r a l globus p a l l i d u s i n d i c a t e d that the l e s i o n s damaged a s i g -n i f i c a n t number of these neurons. Hemitransections j u s t caudal to the entopedun-cular nucleus did not r e s u l t i n s i g n i f i c a n t depletions of e i t h e r CAT or AChE (Table I I ) . C o r t i c a l k a i n i c a c i d l e s i o n s K a i n i c a c i d i n j e c t i o n s i n the f r o n t a l cortex of the r a t r e s u l t e d i n uniform neuronal d e s t r u c t i o n i n a l l l a y e r s of cortex as assessed by c r e s y l v i o l e t h i s t o -logy. Tne a f f e c t e d areas composed roughly 50% of the c o r t i c a l t i s s u e sample d i s -sected and assayed f o r CAT, AChE and GAD. These t i s s u e s showed no d e p l e t i o n i n CAT, a minor d e p l e t i o n i n AChE, and a major d e p l e t i o n . i n GAD (Table I I I ) . T h e ka i n i c a c i d l e s i o n e d t i s s u e s d i d not shrink, since there was no d i f f e r e n c e between weights of le s i o n e d , c o n t r a l a t e r a l , and c o n t r o l t i s s u e s . H i s t o l o g i c a l examination a l s o d i d not i n d i c a t e that any shrinkage had occurred one week a f t e r the k a i n i c a c i d l e s i o n . DISCUSSION The nucleus b a s a l i s - sub s t a n t i a innominata complex In primates, the d e l i n e a t i o n of the substantia innominata i s r e l a t i v e l y s t r a i g h t f o r w a r d , and li k e w i s e the c l u s t e r s of magnocellular, AChE-intense neu-rons found wit h i n the s u b s t a n t i a innominata i s c l e a r ; hence t h i s c e l l group i s a p p r o p r i a t e l y termed "nucleus b a s a l i s of the s u b s t a n t i a innominata" i n primates (KIEVET & KUYPERS, 1975; JONES et a l . , 1976; MESULAM & VAN HOESEN, 1976). How-ever, the topography of these n u c l e i i s not as c l e a r i n cat and r a t (present observations and PARENT, personal communication). HEIMER & WILSON (1975) have 67 attempted to c l a r i f y the current understanding of the topography of the rat's homologue to the substantia innominata called the "ventral pallidum" i n the r a t , describing i t as an area which 1) receives an input from nucleus accumbens and/ or olfactory tubercle, 2) l i e s adjacent to s t r i a t a l structures, and 3) has neuropil identical to the globus pallidus. According to descriptions of the nucleus basalis i n various species, these neurons are characteristically intensely AChE-reactive, large (25-45 ym),and pro-ject to the neocortex (DAS & KREUTZBERG,1968; DAS,1971; DIVAC,1975; JONES et a l . , 1976; MESULAM & VAN HOESEN,1976 ; PARENT et al.,1979). These characteristics have been used as operational c r i t e r i a for mapping nBM in Figs. 1 & 2. I t i s clear that in the rat, the neuropil surrounding these neurons does not always resemble that of the globus pallidus; neither does the d i s t r i b u t i o n of these neurons always follow the region of the ventral pallidum outlined by REIMER & WILSON (1975) and NAUTA, SMITH, "FAULL & DOMESICK (1978), especially i n the more caudal sections (Figs. 1E-2F). This leads us to question the a p p l i c a b i l i t y of the term "nucleus basalis of the substantia innominata" for the rat. Hence, we have adopted the more parsimonious nomenclature, "nucleus basalis magnocel-l u l a r i s , " and i t i s suggested that i n the rat this c e l l group i s homologous to the nucleus basalis of the substantia innominata in primates. The homology of the nBM i n rat and nucleus basalis of the substantia innominata i n primates i s supported by their common characteristic c o r t i c a l projections, morphology, and intense AChE a c t i v i t y . Organization of intensely AChE-reactive neurons in the basal forebrain The s i m i l a r i t i e s and apparent continuity of large, intensely AChE-reactive neurons of the rat forebrain has already been noted (DIVAC ,1975). On the basis of differences in projection areas of these neurons (EMSON & LINDVALL ,1979) , the intensely AChE-reactive neurons in the basal forebrain of DFP-pretreated r a t can be divided into at least three main groups: (a) the medial septal nucleus, 68 x/ (b) the nucleus of the diagonal band of Broca, and (c) the nucleus basalis magnocellularis (nBM). The intensely AChE-reactive neurons of the medial septum are probably the origin of the v e i l known cholinergic septo-hippocampal pro-jection (see LYNCH et al.,1978). The intensely AChE-reactive neurons of the medial septum are continuous with those of the ro s t r a l portion of the nDB (Fig. 1A,B). Furthermore, l i k e the medial septal nucleus, some neurons i n the nDB also project to the hippocampus (CONRAD & PFAFF, 1976; MEIBACH & SIEGEL, 1977). This also suggests that at least part of the nDB can be viewed as a caudal extension of the medial septal nucleus. The other areas to which the nDB projects include the habenula, the anteromedial nucleus of the thalamus, the interpeduncular nucleus and the mammillary nucleus (CONRAD & PFAFF,1976; MEIBACH & SIEGEL, 1977; KERKENKAM & NAUTA.1977). It may be noted that the caudal border of nDB i s i n d i s -tinct from that of the magnocellular nucleus of the preoptic area. E a r l i e r studies (JACOBOWITZ & PALKOVITS,1974) have suggested that the t i g h t l y packed, intensely AChE-reactive c e l l group of nDB continues into a region not recognized as nDB but generally termed l a t e r a l preoptic area (EMSON, PAXINOS, LE GAL LA SALLE, . BZN-ARI & SILVER,1979). It is apparent from Figs. 1 & 2 that the population density of the magnocellular, intensely AChE-reactive neurons decreases i n the lateral preoptic area (Fig. 2A-C) and l a t e r a l hypothalamic area (Fig. 2D). Des-pite the present d i f f i c u l t i e s i n defining the boundary, biochemical evidence sup-ports the concept that d i s t i n c t differences between nDB and l a t e r a l preoptic area do exist. Specifically, nDB has much higher choline acetyltransferase activity than the la t e r a l preoptic area (HOOVER et al.,1978). Further studies, based on retrograde neuroanatomical techniques combined with histochemical and morphological id e n t i f i c a t i o n are required to c l a r i f y the di s t i n c t i o n between the intensely AChE-reactive neurons in the nDB and those in the magnocellular nucleus of the preoptic area (WYSS et a l . , 1979). ' , In a similar vein, the pro-jections of the intensely AChE-reactive neurons in the magnocellular nucleus of 69 the preoptic area and i n the l a t e r a l hypothalamic area are not pr e s e n t l y known _. and require investigation._ The present r e s u l t s suggest that the nDB does not project to neocortex. Thus, HRP i n j e c t i o n s confined to the neocortex did not r e s u l t i n l a b e l e d c e l l s e i t her i n the medial septum or the nDB, i n agreement with JONES et a l . (1976). However, i t remains possible, of course, that the nDB may pr o j e c t to n e o c o r t i c a l areas that were not investigated i n the present experiments. DIVAC (1975) found some labeled c e l l s i n the medial septum and nDB a f t e r c o r t i c a l HRP i n j e c t i o n s but according to h i s F i g . 3, i t appears p o s s i b l e that t h i s was due to d i f f u s i o n of the injected HRP to the hippocampus. Consistent with our f a i l u r e to l a b e l c e l l s i n medial septal nucleus and nDB a f t e r c o r t i c a l HRP i n j e c t i o n s i s the fi n d i n g that the magnocellular, intensely AChE-reactive neurons that characterize these n u c l e i did not appear to undergo retrograde degeneration or loss a f t e r the extensive c o r t i c a l a b l a t i o n s . Characterization of the nBM ' • In constrast to the lack of l a b e l i n g of the medial septum and nDB, c o r t i c a l i n j e c t i o n s of HRP labeled many neurons i n nBM ( F i g . 3A). These r e s u l t s confirm the findings of DIVAC (1975). The l o c a t i o n of these labeled neurons correspond to the d i s t r i b u t i o n of the magnocellular, intensely AChE-reactive neurons which were found to undergo extensive retrograde atrophy or l o s s a f t e r c o r t i c a l a b l a -t i o n . Furthermore, i n one series of animals i n which the ti s s u e s were processed for both HRP and AChE histochemistry, i t was found, that a l l the c e l l s i n the nBM that contained HRP reaction product also stained i n t e n s e l y f o r AChE. These r e s u l t s are i n s u b s t a n t i a l agreement with MESULAM & VAN HOESEN'S (1976) obser-vations i n the monkey. I t should be noted, however, that these l a t t e r authors found a few HRP-labeled, AChE-reactive neurons i n nDB a f t e r c o r t i c a l HRP i n -j e c t i o n s . Since nD3 l a b e l i n g was not observed i n the present experiments, i t i s not known whether t h i s discrepancy i s due to species differences o r to other 70 f a c t o r s such as those discussed above. In any event, on the b a s i s o f the p r e -sent observations and i n agreement v i t h previous f i n d i n g s (DIVAC 11975) , i t can be concluded that the nBM pr o j e c t s widely upon the neocortex of the r a t and that the o r i g i n of t h i s p r o j e c t i o n i s the group of magnocellular, l a r g e l y i n t e r s t i t i a l , i n t e n s e l y AChE-reactive neurons i d e n t i f i e d i n F i g s . 1 & 2. The l e s i o n experiments i n d i c a t e that'the nucleus b a s a l i s - n e o c o r t i c a l p r o -j e c t i o n s i s c h o l i n e r g i c . Thus, e l e c t r o l y t i c l e s i o n s of t h i s r e g i o n r e s u l t e d i n s i g n i f i c a n t reductions i n the n e o c o r t i c a l a c t i v i t y of a r e l i a b l e enzyme marker for c h o l i n e r g i c neurons, c h o l i n e a c e t y l t r a n s f e r a s e . Furthermore, l e s i o n s with k a i n i c a c i d , a neurotoxin which destroys neuronal p e r i k a r y a but g e n e r a l l y leaves f i b r e s of passage i n t a c t (MASON & FIBIGER.1979) y i e l d e d the same decreases i n n e o c o r t i c a l c h o l i n e a c e t y l t r a n s f e r a s e a c t i v i t y . This suggests that the d e s t r u c -t i o n of neuronal perikarya i n the region of the e l e c t r o l y t i c l e s i o n s , - a n d not damage to f i b r e s of passage, was respon s i b l e for the decrease i n c o r t i c a l c h o l i n e a c e t y l t r a n s f e r a s e caused by the e l e c t r o l y t i c l e s i o n s . T h e observ a t i o n s that hemitransections j u s t caudal to the entopeduncular nucleus d i d not a f f e c t c h o l i n e -acetyltransferase a c t i v i t y i n the f r o n t a l cortex supports t h i s c o n c l u s i o n and i n d i c a t e s that neurons caudal to t h i s l e v e l do not c o n t r i b u t e s i g n i f i c a n t l y to the c h o l i n e r g i c i n n e r v a t i o n of t h i s part of the neocortex. The hypothesis that nBM i s a source of a c o r t i c a l c h o l i n e r g i c p r o j e c t i o n i s f u r t h e r supported by the s e l e c t i v e decrease i n choline a c e t y l t r a n s f e r a s e i n nBM caused by retr o g r a d e de-generation f o l l o w i n g c o r t i c a l l e s i o n s (Table I ) . Whither the balance of c o r t i c a l c h o l i n e a c e t y l t r a n s f e r a s e ? While l e s i o n s of the nBM r e s u l t e d i n s i g n i f i c a n t decreases i n n e o c o r t i c a l c h o l i n e a c e t y l t r a n s f e r a s e a c t i v i t y , i n no instance was t h i s l o s s complete. The present l e s i o n s were smaller than those employed by KELLY & M00PJ£ (1978) ; thus i t i s not s u r p r i s i n g that smaller c h o l i n e a c e t y l t r a n s f e r a s e d e p l e t i o n s r e s u l t e d . ' C o r t i c a l i s o l a t i o n a l s o produces much la r g e r depletions i n c o r t i c a l c h o l i n e 71 a c e t y l t r a n s f e r a s e (GREEN, HALPERN & VAN NIEL,1970). These r e s u l t s have been reproduced i n t h i s l a b o r a t o r y (LEHMANN, ATMADJA & FIBIGER, i n p r e p a r a t i o n ) . The f a i l u r e of the present l e s i o n s to produce a complete d e p l e t i o n of c h o l i n e a c e t y l -transferase i n the cortex could therefore be explained by 1) incomplete l e s i o n of the nBM; 2) the existence of other s u b c o r t i c a l neurons which are c h o l i n e r g i c and p r o j e c t to the neocortex, or 3) the existence of c h o l i n e r g i c p e r i k a r y a in the neocortex i t s e l f . Several l i n e s of evidence argue against t h i s l a s t p o s s i -b i l i t y . F i r s t , k a i n i c a c i d i n j e c t i o n s i n t o the f r o n t a l cortex d i d not s i g n i f i -c a n t l y a f f e c t c h o l i n e a c e t y l t r a n s f e r a s e a c t i v i t y i n the c o r t i c a l r e g i o n damaged by the k a i n i c a c i d . These c o r t i c a l i n j e c t i o n s d i d , however, produce h i s t o l o g i c a l evidence of massive neuronal l o s s i n the f r o n t a l cortex and t h i s was c o r r o b o r a t e d by the s i g n i f i c a n t decrease i n c o r t i c a l glutamic acid.decarboxylase a c t i v i t y . This l a t t e r observation suggests that there are GABAer.gic p e r i k a r y a in the neo-cortex. This i s c o n s i s t e n t with the i d e n t i f i c a t i o n of GABAergic p e r i k a r y a by autoradiography of l a b e l e d GABA uptake i n p a r i e t a l cortex (HOKFELT & LJ7JNGDAKL, ~ 1972) and immunohistochemical i d e n t i f i c a t i o n of GABAergic p e r i k a r y a in v i s u a l cortex (RIBAK ,1978). - Second, there are no i n t e n s e l y A C h E - r e a c t i v e - p e r i k a r y a in the neocortex of c o n t r o l or DFP-pretreated r a t s . Inasmuch as a l l i d e n t i f i e d c h o l i n e r g i c perikarya s t a i n s trongly for AChE, t h i s absence i n the n e o c o r t e x is c o n s i s t e n t with a completely e x t r i n s i c source of c h o l i n e r g i c i n n e r v a t i o n of the neocortex of the r a t . This c o n c l u s i o n i s at variance with i n i t i a l immunohisto-chemical observations (McGEER, McGEER, SINGH & CHASE, 1974). The p o s s i b l e b a s i s of t h i s discrepancy has been discussed r e c e n t l y by EMSON & LINDVALL (1979) . The small d e p l e t i o n of AChE that occurred i n the k a i n i c a c i d l e s i o n e d c o r t i c a l t i s s u e s i s c o n s i s t e n t with both our and previous (KRNJEVIC & SILVER, 1965) observations that weakly AChE-reactive neurons e x i s t i n the c o r t e x . These may emit commissural f i b r e s which co n t a i n AChE (KRNJEVIC & SILVER, 1965) . 72 Fun c t i o n a l considerations The r e l a t i o n s h i p between nBM and the v e n t r a l pallidum, the l a t t e r apparently being the r a t ' s homologue f o r the primate substantia innominata, has important i m p l i c a t i o n s f o r a f u n c t i o n a l understanding of these neurons. The s u b s t a n t i a innominata appears to be a part of the system which i s intermediate between extrapyramidal and l i m b i c , termed " o l f a c t o - s t r i a t a l , " and'elegantly d i s c u s s e d by HEIMER & WILSON (1975) . Since the nucleus b a s a l i s i s found w i t h i n the sub-s t a n t i a innominata i n primates, i t may be f u n c t i o n a l l y i n t e g r a t e d w i t h the o l -f a c t o - s t r i a t a l system. Whether t h i s a s s o c i a t i o n occurs i n cat and r a t i s l e s s c l e a r . On the other hand, some evidence has been presented by DAS & KREUTZBERG (1968) that these AChE-rich neurons may be a r o s t r a l extension of the r e t i c u l a r formation. A d d i t i o n a l observations r e l a t i n g to t h i s question are found i n a recent comparative study of nucleus b a s a l i s i n r a t , cat, and monkey (PARENT et a l . , 1979). Because of the very d i f f e r e n t functions these systems are thought to subserve, assignment of nBM to e i t h e r the descending o l f a c t o - s t r i a t a l system or the r e t i c u l a r system w i l l be of considerable value i n a s s i g n i n g ..functions to t h i s c o r t i c a l c h o l i n e r g i c p r o j e c t i o n . The present r e s u l t s r a i s e the p o s s i b i l i t y that the dramatic d e p l e t i o n i n c o r t i c a l c h o l i n e a c e t y l t r a n s f e r -ase and AChE i n c e r e b r a l cortex of v i c t i m s of Alzheimer's disease (DAVIES,1979), may be due to a l e s i o n of these s u b c o r t i c a l neurons. 73 ACKNOWLEDGEMENTS The authors express deep a p p r e c i a t i o n to Dr. Andre" Parent f o r making f r e e l y a v a i l a b l e to us h i s data and expe r t i s e on the nucleus b a s a l i s -su b s t a n t i a innominata complex, which proved i n v a l u a b l e i n the course of these s t u d i e s . Supported by the Medical Research C o u n c i l . 74 REFERENCES BUTCHER L.L., MARCHAND R. , PARENT A. & POIRIER L.J. (1977) Morphological characteristics of acetylcholinesterase-containing neurons in t h e CNS of DFP-treated monkeys. Part 3: Brain stem and spinal cord. J_. neurol. Sci. 32, 169-185. BUTCHER.L.L. , TALBOT K. cV BILEZIKJIAN V. 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P h i l l i s , J.W. (1976) Acetylcholine and synaptic transmission i n the central nervous system. In Chemical Transmission in the Mammalian Central Nervous  System. (ed. Hockman CH. and Bieger D.) pp. 159-213. University Park Press, Baltimore. PRICE J.L. & POWELL T.P.S. (1970). An experimental study of the o r i g i n and the course of centrifugal fibres to the olfactory bulb i n the r a t . J . Anat. 107,215-237. RAMON-MOLINER E. & NAUTA W.J.H. (1966).The isodendritic core of the brain stem. J_. comp. Neur. 126, 311-336. RIBAK C.E. (1978) Aspinous and sparsely-spinous s t e l l a t e neurons i n the visual cortex of rats contain glutamic acid decarboxylase. J_. Neurocytol. 7_, 461-478. SILVER A (1974) The Biology of Cholinesterases. Elsevier, New York. WALAAS I. & FONNUM F. (1979) The distribution and origin of glutamate decarboxylase and choline acetyltransferase in ventral pallidum and other basal forebrain regions. Brain Res. 177, 325-336. WYSS J.M., SWANSON L.W. h COWAN W.M. (1979) A study of subcortical afferents to the hippocampal formation in the rat. Neurosci. 4_, 463-476. 79 Figure 1: Distribution of AChE-reactibe perikarya in rat forebrain AChE stain according to KARNOVSKY & ROOTS (1964) in animals pretreated with 1.5 mg/kg DTP i.m. 5 hours preceding s a c r i f i c e . A few scattered intensely AChE-reactive neurons can be seen in the regions described by HEIKER & WILSON (1975) and NAUTA et a l . (1978) as ventral pallidum (Fig. 1B-E). The d i s t r i -bution of the intensely AChE-reactive neurons operationally defined as nBM (see discussion) d i f f e r s from the topography of the ventral pallidum, which i s thought to represent the rat's homologue to primate substantia innominata (HEIMER & WILSON,1975; WALAAS & FONNUM,1979). Although many groups o f intensely AChE-reactive neurons are contiguous, i f not continuous, the histochemical regimen employed here i s not sufficient by i t s e l f to c l a r i f y the topographical boundaries, for instance, of the nDB which merges with medial septal nucleus along i t s v e r t i c a l limb (Fig. IB) and with the magnocellular preoptic nucleus • at caudal levels (Fig. 2A and further caudal). Tne organization o f AChE-reactive neurons i n the thalamus and hypothalamus of the rat has been previously des-cribed (PARENT & BUTCHER, 1976) . Abbreviations: AC, anterior commissure; CP, caudate-putamen; F, fornix; GP, globus pallidus; IC, internal capsule;- LHA, l a t e r a l hypothalamic area; LPO, l a t e r a l preoptic area; MaPO, magnocellular preoptic nucleus; MS, medial septal nucleus; nBM, nucleus basalis magnocellularis; nDB, nucleus of the diagonal band; OT, olfactory tubercle; SM, s t r i a medullaris; TAD, antero-dorsal thalamic nucleus; TAV, anteroventral thalamic nucleus; TR, r e t i c u l a r thalamic nucleus; TV, ventral thalamus; VP, ventral pallidum, diagrammed according to HEIMER & WILSON (1975). Calibration bar: 1mm. 80 Figure 2: D i s t r i b u t i o n of AChE-reactive perikarya i n r a t f o r e b r a i n (cont.) At more caudal l e v e l s , p e r i k a r y a of nBM become more numerous. I n each s e c t i o n , neurons morphologically t y p i c a l of nBM are o c c a s i o n a l l y found w i t h i n the core of the GP ( F i g . 2A, B) while the population of neurons comprising the bulk of nBM increases g r e a t l y between GP and the i n t e r n a l c a p s u l e , forming a network of perikarya and processes as described by PARENT et a l . (1979) (F i g . 2B-D) . The i n t e r s t i t i a l character of nBM i s seen most d r a m a t i c a l l y near the t a i l of the GP ( r i g . 2E) . The population of nBM p e r i k a r y a d i m i n i s h e s as the l e v e l of the entopeduncular nucleus i s approached ( F i g . 2F). Note the very low "background" AChE s t a i n i n g i n the region of the nBM compared t o a l l other regions described which contain i n t e n s e l y AChE-reactive neurons. Ex-perimental conditions as i n F i g . 1. Abbreviations are found i n legend to F i g . 1. MaPO, LPO, and LHA have been located according to the d e s c r i p t i o n of. WYSS, SWANSON & COWAN (1979). C a l i b r a t i o n bar: 1 mm 81 Figure 3: HRP-labeled (A) and AChE-stained (B) neurons of nucleus b a s a l i s  magnocellularis HRP i n j e c t e d i n t o f r o n t a l cortex labeled neurons i n nBM with morphology i d e n t i c a l to those of neurons i n nBM when stained f o r AChE (B) 5 hours f o l -lowing DFP pretreatment. The-HRP labeled s e c t i o n was counter-stained w i t h c r e s y l v i o l e t (A), while the AChE-stained s e c t i o n was not c o u n t e r s t a i n e d (B) . In some experiments, sections were stained for both AChE and HRP f o l l o w i n g DFP pretreatment. Neurons which contained the blue HRP product always stained with the c h a r a c t e r i s t i c Hatchett's brown for AChE. No other neurons i n the areas lesioned (see F i g . 5) contained HR? product. These micrographs were taken at the l e v e l of F i g . IF. The topography of HRP-labeled neurons f o l l o w i n g c o r t i c a l i n j e c t i o n s followed the topography of i n t e n s e l y AChE-r e a c t i v e neurons l a b e l e d nBM i n F i g s . 1 and 2. C a l i b r a t i o n bar: 50 ym 82 Figure 4: Retrograde degeneration of nucleus b a s a l i s m a g n o c e l l u l a r i s  produced by c o r t i c a l l e s i o n s A,C - lesioned side; B,D - c o n t r o l side. In low power micrographs (A,B), depletion and shrinkage of nBM p e r i k a r y a are evident (A). The smaller, weakly s t a i n i n g neurons do not appear t o be adversely a f f e c t e d . The micrograph i s taken from a s e c t i o n at the l e v e l of F i g . 2B. The animal was s a c r i f i c e d 6 months a f t e r the c o r t i c a l l e s i o n s and 12 hours a f t e r 1.5 mg/kg DFP. AChE s t a i n i n g was performed according t o KARNOVSKY & ROOTS (1964). C a l i b r a t i o n bar: 200 ym Under higher power (C,D), these micrographs r e v e a l the somal shrinkage and apparent d e n d r i t i c atrophy sustained by s u r v i v i n g nBM neurons (C) . C a l i b r a t i o n bar: 50 ym 83 Figure 5: The extent of damage caused by e l e c t r o l y t i c (A) and k a i n i c a c i d (B) l e s i o n , as assessed by c r e s y l v i o l e t h i s t o l o g y , i s i n d i c a t e d d i a g r a m a t i c a l l y . Abbreviations: AC, a n t e r i o r commissure; EP, entopeduncular n u c l e u s ; F, f o r n i x ; GP, globus p a l l i d u s ; IC, i n t e r n a l capsule; SM, s t r i a m e d u l l a r i s ; St, caudate-putamen. Table I. Neurotransmltter-related enzymes i n the region of the nucleus b a s a l i s magnocellularis s i x months a f t e r extensive u n i l a t e r a l c o r t i c a l l e s i o n s % Control c h o l i n e a c e t y l t r a n s f e r a s e a c e t y l c h o l i n e s t e r a s e glutamic a c i d decarboxylase 64.1 ± 6.0% 80.6 ± 3Ja 107.0 ± 9.3% Control a c t i v i t y ± S.E.M. 63.9 ± 2.9 nmol/mg protein/h 19.5 + 1.6 umol/mg protein/h 296 J: 31 nmol/mg protein/h n=4 *P< .02; **P< .001, Student's two-tailed t e s t . Table I I . Choline a c e t y l t r a n s f e r a s e and a c e t y l c h o l i n e s t e r a s e a c t i v i t i e s i n the f r o n t a l cortex a f t e r l e s i o n s i n the region of nucleus b a s a l i s magnocellularis Kainic a c i d l e s i o n s (n=ll) c h o l i n e a c e t y l t r a n s f e r a s e a c e t y l c h o l i n e s t e r a s e E l e c t r o l y t i c l e s i o n s (n=9) choline a c e t y l t r a n s f e r a s e a c e t y l c h o l i n e s t e r a s e Hemitransections - caudal to nBM (n=7) choline a c e t y l t r a n s f e r a s e a c e t y l c h o l i n e s t e r a s e % Control 78.0% ± 2.2%*** 79.3% ± 4.0%*** 76.2% ± 4.9%* 74.2% ± 3.9%** 95.7% ± 5.3% 96.1% ± 7.5% Control a c t i v i t y ± S.E.M. 30.5 ± 0.70 nmol/mg protein/h 5.51 ± 0.10 umol/mg protein/h 27.8 ± 1.94 nmol/mg protein/h 5.85 ± 0.36 umol/mg protein/h 26.9 ± 1.63 nmol/mg protein/h 5.20 ± 0.45 umol/mg protein/h *P <.02; **P. <.0l; ***P <-001, Student's two-tailed test, Table I I I . Neurotransmitter-related enzymes i n the f r o n t a l cortex a f t e r l o c a l k a i n i c acid i n j e c t i o n s choline a c e t y l t r a n s f e r a s e a c e t y l c h o l i n e s terase glutamic a c i d decarboxylase Lesioned side, % Control 92.2% ± 4.9% 83.4% ± 5.3%* 59.3% ± 5.0%** C o n t r a l a t e r a l , % Control 96.4% + 5.5% 86.6% ± 4.6% 100.4% + 4.1% Control (unoperated) a c t i v i t y , + S.E.M. 29.1 ± 2.1 nmol/mg protein/h 3.59 ± 0.22umol/mg protein/h 190 ± 7 . 3 nmol/mg protein/h n=6 *P< .05; **P <.001, Student's two-tailed t e s t . F i g . 1 88 F i g . 2 89 F i g . 3 F i g . 4 91 F i g : 5A F i g . 5B f 93 The following two f i g u r e s are appended to the manuscript for the copies included i n the thesis only. F i g . 6 depicts the area of c o r t i c a l a b l a t i o n which res u l t e d i n retrograde degeneration of the nBM. F i g . 7 depicts the HRP i n j e c t i o n s i t e at i t s greatest two-dimensional s i z e . 94 / F i g . 6 95 F i g . 7 96 Life Sciences, Vol. 25, pp. 1939-1947 Pergamon Press Printed in the U.S.A. MINIREVIEW ACETYLCHOLINESTERASE AND THE CHOLINERGIC NEURON John Lehmann and H.C. Fibiger Division of Neurological Sciences University of Brit ish Columbia Vancouver, Brit ish Columbia, V6T 1W5 Canada The study of acetylcholinesterase (AChE) dates as far back as the dis-covery of acetylcholine (1,2). It has widespread distribution and very high act iv i ty , generally two orders of magnitude higher than choline acetyl-transferase (3). It is stable, and there are many simple assay and histo-chemical techniques for measuring its act ivi ty. For these reasons, AChE has been the subject of a vast amount of research. Yet today no teleolog-ical model has been found to explain the distribution of AChE in the central nervous system: The existence of AChE on a given neuron is not sufficient information to predict that neuron's relationship with acetylcholine. In general there is an excellent correlation between AChE and choline acetyltransferase activity in the rat forebrain on a regional basis (3). It has long been recognized, however, that AChE is radically disproportion-ate with acetylcholine and choline acetyltransferase in some brain regions (4). For instance, the cerebellum is high in AChE compared to its content of choline acetyltransferase (5), while the inverse holds true for the med-ial habenula (3) and median eminence (6). On a cel lular level , AChE is found in fa i r ly high activity on some neurons which are known not to be cholinergic and furthermore are not thought to be cholinoceptive. Two salient examples are the dopaminergic neurons of the substantia nigra (7-9) and the noradrenergic neurons of the locus coeruleus (10). It is clear from just these two examples, as Koelle pointed out in 1955 (11), that the presence of AChE in a given neuron is not sufficient evidence to indicate that such a neuron is cholinergic. Nonetheless, on occasion AChE has proven worthy of study in the pur-suit which may be called "biochemical neuroanatomy", i . e . , the identi f ica-tion of neurons 1) morphologically, 2) by afferent and efferent con-nections, and 3) by the transmitter(s) used. The study of AChE is a use-ful adjunct to more specific enzyme markers and conventional neuroanatomi-cal techniques as an arbitrary and characteristic marker of certain classes of neurons. For example, AChE histochemistry formed the link between the biochemical and anatomical characterization of the non-homogeneous organi-zation of the striatum (12,13). It has also proven useful in the identi-fication of similar classes of neurons within the CNS (14) and across spe-cies (15). Furthermore, when an irreversible inhibitor such as di iso-propylphosphorofluoridate (DFP) is administered in vivo some time preceding sacr i f ice , the morphological features of neurons that contain AChE are revealed with detail exceeded only by the Golgi method. This powerful modi-0024-3205/79/231939-09$02.00/0 Copyright (c) 1979 Pergamon Press Ltd 97 1940 AChE and the Cholinergic Neuron Vol. 25, No. 23, 1979 fication of the AChE histochemical technique was introduced by Lynch and coworkers (16) and has been applied extensively with technical improvements by Butcher and collaborators, particularly in the striatum (7). Beyond these pragmatic applications, we have lately re-examined the validity of an hypothesis conceived as long ago as 1954 by Koelle (17). Having divided nuclei of the brain into four categories according to the intensity of AChE staining, Koelle generalized from the single example of the motoneuron ". . . i t may be postulated that neurons in the intensely and moderately stained categories are likewise cholinergic." In the light of our understanding 25 years later, i t is striking to note how many correct examples of cholinergic neurons are l isted in the "intensely stained" cate-gory and how few correct examples are l isted in the "moderately stained" category. Today, comparison of the density of AChE-staining in various perikarya, although s t i l l qualitative, is facil i tated by advances in histo-chemical technique. Thus, well-character!zed cholinergic projections o r i g i -nating in the central nervous system have been shown to arise from somata which synthesize large amounts of AChE following OFP-pretreatment. Examples of such cholinergic neurons include the septo-hippocampal projection (18,19) and the motoneuron (20,21). Most importantly, there is no known example of a cholinergic neuron that does not have high levels of AChE. We may therefore form an empirical generalization based on cases in the central and peripheral nervous systems which adhere to the rule: high AChE activity is a necessary but not su f f i - cient characteristic for identifying cholinergic neurons. If indeed this rule holds true, the easily determined distribution of AChE will greatly accelerate the elucidation of cholinergic neuroanatomy. The value of this rule depends upon i ts validity in each circumstance; the discovery of one cholinergic neuron without high levels of AChE will destroy the rule's u t i l i t y . This laboratory has made use of the rule in three cases in which i t seemed most l ikely to f a i l . Here we summarize the course of those investigations in three areas of the brain: the striatum, the cerebral cortex, and the globus pallidus. The Striatum There are six types of neurons described in the striatum of the cat by Kemp & Powell (22): the large, so-called "aspiny" neuron (22-30 um, mean of major and minor axes), comprising less than 1% of the total neuron popula-tion; the medium spiny (12-18- um), comprising 96% of the population; three other medium-sized neurons (16-18 pm, 16-18 um, and 12-14 um), together comprising 3% of the population; and the small neuron (5-9 um) f i l l i n g out the last 1%. Traditionally, the large aspiny neuron was considered to be the sole source of the descending projections from the striatum (23,24). This concept has been revised in the light of data gathered in the last decade; now at least 50% of the medium spiny neurons are known to have des-cending projections (25,26), while the large aspiny neuron is thought to be an interneuron (27). In the striatum from DFP-pretreated animals, the large aspiny neuron is unique in that i t stains intensely for AChE; the small neuron stains l ight ly , and the medium cells are generally judged not to stain at al l (7). However, immunohistochemical evidence,obtained with antibodies directed against puri-fied choline acetyltransferase, previously suggested that medium spiny neu-rons were cholinergic (28,29). Similar results were recently obtained by Kaiya et a l . (30) employing a conventional histochemical reaction for local -izing choline acetyltransferase ultrastructurally. These observations are 98 Vol. 25, No. 23, 1979 AChE and the Cholinergic Neuron 1941 in disagreement with the hypothesis that high levels of AChE are necessari-ly contained in cholinergic neurons. This empirical rule led us to suspect that the large aspiny neurons, rather than the medium-sized neurons, were the elusive cholinergic interneurons of the striatum (27), the existence of which was originally proposed by McGeer et a l . (31). Data reported by Campochiara & Coyle (32) has indicated that kainic acid injected into the striatum of 10-21 day old rats preferentially deple-tes choline acetyltransferase, compared to the GABAergic marker glutamic acid decarboxylase. Kainic acid is a neurotoxin which in general destroys neuronal perikarya while leaving afferent axons and terminals intact (33,34). However, striatal neurons appear to require a functional glutamateric inner-vation in order to be susceptible to kainic acid's neurotoxic action, as f i r s t demonstrated by McGeer et a l . (35,36). The simplest hypothesis to explain the preferential depletion of choline acetyltransferase at early postnatal times is that the glutamatergic corticostriatal projection (37,38) establishes a functional synaptic contact with cholinergic neurons sl ightly earl ier than i t does with the other neurons of the striatum. The data re-ported by Campochiaro & Coyle (32) thus presented an opportunity to test a prediction of the hypothesis that the cholinergic neuron of the striatum was the AChE-intense large aspiny neuron. TABLE I The selective depletion of striatal choline acetyltransferase and ace-tylcholinesterase by intrastriatal injections of kainic acid neonatally % of Control Velocity, contralateral striatum ± S.E.M. CAT 74.7 ± 2.0** 153 ± 4.1 nmol/mg protein/h AChE 78.4 ± 2.8* 48.5 ± 2.8 umol/mg protein/h GAD 96.5 ± 4.6 102 ± 5.3 nmol/mg protein/h n = 5 *P < .002; **p < .001, Student' s two tailed test. 10 nmol kainic acid in 0.5 ul sodium phosphate buffered (pH 7.4) isosmolar Ringer solution was injected unilaterally into the corpus striatum of rats 10 days post partum. 14 days after surgery, choline acetyltransferase (CAT) and acetylcholinesterase (AChE) were significantly reduced, while glutamic acid decarboxy-lase (GAD) activity was unaffected. This laboratory reproduced the biochemical data of Campochiara & Coyle (32) in the ten-day old neonate (see Table I) and performed histochemical studies in paral le l . The kainic acid injections resulted in a striatum with large, irregularly shaped areas entirely void of the AChE-intense, large aspiny neuron, when visualized by AChE histochemistry following DFP-pretreatment. In contrast, cresyl-violet stained neurons (being composed 96% of medium spiny neurons) and small, weakly AChE-reactive neuron popula-tions were unaltered (Table II). These data argue strongly that neither the medium spiny nor the small neuron of the striatum is cholinergic. It is possible that one of the three minority medium-sized neurons identified by Kemp & Powell (22) may be cholinergic, but by far the favored candidate is the large, AChE-intense aspiny neuron. Furthermore, in recent developmental studies of the striatum pursued in this laboratory, the latero-medial pro-gression of the postnatal development of large, AChE-intense neurons is 99 •1942 AChE and Cholinergic Neuron Vol. 25, No. 23, 1979 paralleled exactly by the regional development of choline acetyltransferase activity (Lehmann & Fibiger, in preparation). Finally, i t should be noted that in more recent immunohistochemical experiments aimed at the neuronal localization of choline acetyltransferase, preliminary evidence has been obtained that implicates the large aspiny neuron of the striatum as a cho-linergic neuron (39). These observations demonstrate that the criterion requiring high AChE activity as a necessary but not sufficient characteris-t ic of cholinergic neurons was capable of predicting the morphology of the cholinergic neuron in the striatum. TABLE II Quantitation of morphologically identifiable neurons in regions made void of putative cholinergic neuron by neonatal kainic acid lesions Type of neuron % of Control Density, contralateral s t r i -atal ± S.E.M. Cresyl-violet stained 106.5 ± 8.1 n.s. 1574 ± 5 7 . 4 neurons/mm2 Sml weakly AChE-reactive 124.4 ± 10.6 n.s. 17.2 ± 1.39 neurons/mm2 Large AChE-intense Zero 12.2 ± 0.60 neurons/mm2 Identically lesioned rats from the same group on which biochemical assays were performed (Table I) were injected intramuscularly with 2.0 mg/kg DFP. 6 hr later, they were perfused and the brains pro-cessed for AChE histochemical staining. Alternate sections were cresyl-violet counterstained. At least 22 sample areas entirely void of large AChE-intense neurons were counted from 3 rats; the corresponding striatal region contralaterally served as control. There were no significant changes in the density of either of the other morphologically identifiable neurons. Note that each of the AChE-reactive neurons represents roughly 1% of the total neuronal population as estimated by cresyl-violet staining. The Cerebral Cortex There are three major lines of evidence that have argued for the exis-tence of cholinergic perikarya in the neocortex: 1) Chronic isolation of cortical slabs results in a large (65% - 80%) but not complete depletion of choline acetyltransferase (40-42). Other workers have reported no de-creases in cortical choline acetyltransferase activity following similar operations (38,43). 2) Local electrical stimulation of a chronically-iso-lated cortical slab results in a long-lasting inhibition of glutamate-in-duced f ir ing which is blocked by atropine and mimicked by acetylcholine (44,45). 3) Antibodies directed against choline acetyltransferase stain large numbers of neurons in the cortex (46). Further support for the existence of cholinergic perikarya in the cortex has derived from the argu-ments that the decrease in choline acetyltransferase following cortical iso-lation may be due to retrograde degeneration of cholinergic perikarya (46) or a "secondary effect of denervation" (38), and the observation that the cortical inhibition evoked by surface stimulation of the cortex in the slab is identical to that found in intact cortex (44). Arguing against this hypothesis is the observation that there are no intensely AChE-reactive perikarya in the cerebral cortex (17,47,48). This observation becomes more striking in rat cerebral neocortex 5 hours follow-100 Vol. 25, No. 23, 1979 AChE and the Cholinergic Neuron 1943 ing DFP-pretreatment (Lehmann, Atmadja & Fibiger, in preparation), a condi-tion which causes known cholinergic neurons to stain intensely for AChE. The "necessary but not sufficient" rule therefore predicts that there are no cholinergic perikarya in the neocortex. Again, the opportunity to test the validity of the "necessary but not sufficient" criterion presented i tse l f . Kainic acid was employed by this laboratory to effect a complete and uniform neuronal lesion in frontal cortex of rat, as assessed by cresyl-violet histology. The volume of complete perikaryal depletion comprised approximately 50% of the assayed tissue. In this experiment i t was found that glutamic acid decarboxylase activity and high aff ini ty glutamate up-take were decreased in the lesioned tissue by approximately 50 percent. However, choline acetyltransferase activity did not change (Table III). In order to escape the conclusion that there are no cholinergic neurons in the cortex, i t would be necessary to invoke the condition that putative cholin-ergic interneurons of the cortex project heavily for distances exceeding one centimeter, rather than terminating local ly . This condition is in dis-agreement with arguments 1) and 2) cited above as support for the existence of cholinergic neurons in the cortex. Again, pending agreement, of future data, the necessary but not sufficient rule for AChE activity in choliner-gic neurons predicted successfully the absence of cholinergic perikarya in the cerebral cortex suggested by the results of the kainic acid lesion experiment (Table III). It should be noted that an explanation for both residual choline acetyltransferase activity and stimulus-evoked release of acetylcholine following cortical isolation is s t i l l lacking. Similar large-ly unexplained failures to observe complete depletions of either choline acetyltransferase or acetylcholine release have been observed distal to peripheral cholinergic nerves following positively complete transection in several species (49-56). It is d i f f icu l t of course to extrapolate across species and from peripheral to central nervous system in order to suggest that an analogous phenomenon does or does not occur in the neocortex of the rat. To perform such a comparison on a percentage basis (57) is al l the more hazardous in view of the very low specif ic activity of choline acetyl-transferase in the neocortex compared to the peripheral nerves studied. Globus Pallidus On the basis that AChE-rich axons projecting to the neocortex appeared to originate from the pallidum, Shute & Lewis (58) originally proposed the existence of a cholinergic pal 1ido-neocortical projection. This suggestion was subject to question, however, since i t was well-known at that time that AChE content was not sufficient to characterize a projection as cholinergic (11). Furthermore, there was no known projection from the globus pallidus to the cortex; and the globus pallidus was known to have extremely low levels of AChE and choline acetyltransferase (59). Yet in 1976, Kelly & Moore (60) found that pallidal lesions did indeed result in substantial decreases of choline acetyltransferase in large areas of neocortex. Reporting retrograde transport of HRP injected into the cortex, Divac (61) speculated that what Shute & Lewis (58) had identified as neurons in the globus pallidus were actually the rat's homologue of the primate nucleus basalis of the substantia innominata, which also projects to neocortex (62-64). Mesulam & van Hoesen (63) demonstrated that in the primate, HRP was transported to AChE-rich neurons of the nucleus basalis of the substantia innominata, and joined Divac (61) in speculating that these were the source of a cholinergic projection to the neocortex. Ensuing experiments designed to prove that these AChE-rich neurons were the source of the cholinergic projection from the pallidal region in the rat became obvious. 101 1944 AChE and the Cholinergic Neuron Vol. 25, No. 23, 1979 TABLE III Neurotransmitter-related enzymes in the frontal cortex after local kainic acid injections Lesioned side, % of Control Control (unoperated) velocity, + S.E.M. CAT 92.2 ± 4.9 29.1 ± 2.1 nmol/mg protein/h AChE 83.4 ± 5.3* 3.59 ± 0.22 umol/mg protein/h GAD 59.3 ± 5.0** 190 ± 7.3 nmol/mg protein/h Glu-up 56.6 ± 4.5** 1.23 ± 0.04 umol/mg protein/h n = 6 *P < .05; **P < .001, Student's two-tailed test. One week following injection of 10 nmol kainic acid in 2 ul sodium phosphate buffered (pH 7.4) saline into frontal cortex of rat, choline acetyltransferase (CAT) was not signif icantly decreased. Acetylcholinesterase (AChE) activity was sl ightly decreased, while major decreases in glutamic acid decarboxylase (GAD) and high-aff ini ty glutamate uptake (Glu-up) were observed. Introducing the minor refinement of suppressing non-perikaryal AChE staining by DFP-pretreatment to Mesulam & van Hoesen's protocol for simul-taneously visualizing HRP and AChE, this laboratory replicated Mesulam & van Hoesen's (63) findings in the rat; HRP injected in the neocortex labelled only neurons of nucleus basalis magnocellularis (nBM) which stained heavily for AChE. Discrete lesions of nBM produced either e lectrolyt ical ly or with kainic acid produced identical and parallel depletions in choline acetyltransferase and AChE in frontal cortex, while hemitransections sl ight-ly caudal to nBM did not affect these enzymes (Lehmann, Nagy, Atmadja & Fibiger, submitted). Six months following cortical ablations, retrograde degeneration in nBM visualized by AChE-histochemistry following DFP pre-treatment was paralleled by choline acetyltransferase and AChE depletions in nBM (Table IV). What had been speculation in 1967 became an obvious con-clusion by the close of the seventies: the source of the cholinergic inner-vation of the neocortex arising from the pall idal region is the group of intensely AChE-reactive neurons, the nBM. Looking Forward The above have been but three examples where the predictive u t i l i t y of high AChE levels in identified neurons can be demonstrated. . Several other examples have been cited above where the necessary but not sufficient rule also applies, and there is no clear exception. It must be conceded, however, that relatively few cholinergic neurons in the CNS have been unequivocally characterized. In searching for an exception to the rule that al l choliner-gic neurons contain high levels of AChE, the habenular complex particularly presents a challenge. The tightly-packed cluster of neurons comprising the medial habenula stains very weakly for AChE following DFP pretreatment, while the lateral habenula contains only moderately-staining neurons (65; Lehmann & Fibiger, unpublished observations). Although at one time the medial habenula was thought to be the sole source of a massive cholinergic projection to the interpeduncular nucleus (66-71), is is now believed that 102 Vol. 25, No. 23, 1979 AChE and the Cholinergic Neuron 1945 the cholinergic input to the interpeduncular nucleus derives at lease 50% from the nucleus of the diagonal band (72), which does contain large, intensely AChE-reactive neurons (73) and probably projects along stria med-ul lar is (72i74), although others believe that the source is entirely from cholinergic perikarya residing in the lateral habenula (75,76). Kainic acid injections in the habenular region led to approximately 50% depletion of choline acetyltransferase in the interpeduncular nucleus (77), supporting the notion that the habenular complex was not the sole source of choline acetyltransferase in the interpeduncular nucleus. However, the necessary but not sufficient criterion of high AChE levels indicates that no choline acetyltransferase may originate from perikarya in the habenular complex. It may be possible to account for the data obtained with kainic acid by McGeer et a l . (77) on the basis of partial lesion of fibers of passage, which has been demonstrated in a similar dense fiber bundle, namely the dor-sal noradrenergic bundle (78). Apart from the nucleus of the diagonal band, at present we are not able to suggest another candidate as an origin for the cholinergic innervation-of the AChE-rich interpeduncular nucleus. However, i t should be noted that the "simple" neuroanatomy of this complex remains to be c la r i f i ed , and that the habenular complex represents the most striking anomaly in the central nervous system with regard to disproportionate AChE and choline acetyltransferase activit ies (3). TABLE IV Neurotransmitter-related enzymes in the region of nBM six months after decortication % of Control Control velocity ± S.E.M. CAT 64.1 ± 6.0** 63.9 ± 2.9 nmol/mg protein/h AChE 80.6 + 3.4* 19.5 ± 1.6 pmol/mg protein/h GAD 107.0 ± 9.3 296 ± 31 nmol/mg protein/h n = 4 *P < .02; **P < .001, Student's two-tai.led test. The retrograde degeneration of AChE-intense neurons identified as nucleus basalis magnocellularis (nBM) was paralleled by decreases in choline acetyltransferase (CAT) and acetylcholinesterase (AChE), but not glutamic acid decarboxylase (GAD). Data from Lehmann, Nagy, Atmadja and Fibiger, submitted. Summary While the distribution of AChE in the central nervous system remains largely unexplained, neurons with very high levels of AChE are frequently identified as cholinergic, and cholinergic neurons always have high levels of AChE. 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