Acetylcholinesterase-containing neurons in the neostriatum and substantia nigra revealed after punctate intracerebral injection of di-isopropylfluorophosphate

European Journal of Pharmacology, 34 (1975) 115--125

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© North-Holland Publishing Company, Amsterdam -- Printed in The Netherlands

ACETYLCHOLINESTERASE-CONTAINING NEURONS IN THE NEOSTRIATUM AND SUBSTANTIA NIGRA REVEALED AFTER PUNCTATE INTRACEREBRAL INJECTION OF DI-ISOPROPYLFLUOROPHOSPHATE LA RRY L. BUTCHER and LOUISE BILEZIKJIAN

Department of Psychology, University of California, Los Angeles, California 90024, U.S.A. Received 11 March 1975, revised MS received 11 June 1975, accepted 9 July 1975

L.L. BUTCHER and L. BILEZIKJIAN, Acetylcholineslerase-containing neurons in the neostriatum and substantia nigra revealed after punctate intracerebral injection of di-isopropylfluorophosphate, European J. Pharmacol. 34 (1975) 115--125. Infusion of 1 pl arachis oil containing 1.5 pg bis-(1-methylethyl)phosphorofluoridate (di-isopropylfluorophosphate: DFP) into the caudate--putamen nucleus and substantia nigra of rats produced a considerable reduction of histochemical staining for acetylcholinesterase (ACHE) in these two brain regions 30--120 rain after injection. Thereafter, regeneration of AChE occurred within the zone of DFP effect. These new stores of AChE were associated with discrete neuronal perikarya and their processes. Intracerebral DFP administration had little or no histochemically detectable effect on NADH-diaphorase. Thionin staining was similarly unaffected. The results with punctate intracerebral application of DFP were replicated by intramuscular injection of 1.5 mg/kg DFP. Although the significance of dopaminergic--cholinergic interactions in the neostriatum could not be elucidated on the basis of these histochemical data, the thesis was advanced that dopamine neurons in the pars compacta of the substantia nigra also contained ACHE, possibly to inactivate acetylcholine released from cholinergic fibers afferent to this neural structure. Cholinergic--dopaminergic interactions Di-isopropylfluorophosphate

Neostriatum

1. Introduction The increasing interest in interactions among neurons having known neurochemical characteristics is evidenced by augmented efforts to correlate histochemically the topography of brain monoamine neurons with the distribution of cholinergic elements (e.g., see Jacobowitz and Palkovits, 1974; Palkovits and Jacobowitz, 1974). In the present study we address ourselves to this problem by histochemically assaying acetylcholinesterase (ACHE, EC 3.1.1.7) in two regions of the brain, the caud a t e - p u t a m e n nucleus and the substantia nigra, characterized also in terms of their dopamine content (e.g., see Fuxe et al., 1969). Although histochemical methods for choline acetyltransferase (ChAc, EC 2.3.1.6) exist

Substantia nigra

Acetylcholinesterase

(Burt, 1970; McGeer et al., 1974b), only AChE has been used reliably and routinely as a marker for cholinergic systems in the brain. Under usual reaction conditions for ACHE, both the caudate--putamen nucleus and the substantia nigra stain appreciably for the enzyme (e.g., see Shute and Lewis, 1967). It is difficult, however, to discern individual AChE-containing neurons in these two brain regions, presumably because the component cells have intensely staining, overlapping processes which obscure the observation of discrete perikarya, dendrites, and axons (Butcher et al., 1975a). Taking into account the earlier preliminary observations of Lynch et al. (1972), we reasoned that improved morphological detail could be obtained in striatal and nigral neurons containing AChE either by injecting bis-(1-methylethyl)-

116 phosphorofluoridate (di-isopropylfluorophosphate: DFP) directly into these two brain regions or by systemically administering the organophosphorous c o m p o u n d prior to histochemical proc~,ssing. Since DFP inhibits AChE irreversibly recovery of enzyme activity has been attributed primarily to synthesis of new enzyme molecules in the neuronal perikarya followed by their transport to the cellular processes (Blaber and Creasey, 1960; Davis and Agranoff, 1968; Austin and James, 1970; Butcher et al., 1975a). Histochemical staining for these new stores after appropriate survival times following DFP administration should reveal details of AChE-containing neuronal somata and their processes impossible to ascertain in pharmacologically unmanipulated material.

2. Materials and methods

2.1. Experimental animals Female Sprague--Dawley rats weighing 20@-300 g were used. They were obtained from Simonsen Laboratories (Gilroy, California; U.S.A.) and were housed in individual cages under conditions of constant temperature (22°C) and relative humidity (50%).

2.2. Pharmacological, surgical and histochemical procedures DFP (Calbiochem, Inc.; La Jolla, California; U.S.A.) was dissolved in arachis oil in a concentration of 1.5 mg/ml and stored at 3---6°C in a refrigerator until used. After being warmed to room temperature (approximately 2 2 ° C ) t h e drug was administered either (1) directly into the substantia nigra or caudate--putamen nucleus using the stereotaxic procedures described extensively in Butcher et al. (1974) or (2) intramuscularly in a dose of 1.5 mg/kg as detailed in Butcher et al. (1975a). The total amount of intracerebrally administered DFP was 1.5 pg in 1 pl arachis oil. The rate of infusion was 1 pl/min. The rats were sacrificed

L.L. BUTCHER, L. BILEZIKJIAN 30 min--8 hr after intracerebrally infused or systemically injected DFP. Control intracerebral and intramuscular injections of the a~achis oil vehicle were made also. Non-injected, vehicle-injected, or DFPtreated rats were anesthetized with sodium methohexital and sacrificed by subsequent perfusion with 20 ml cold (3---6°C) 0.9% saline followed by 20 ml cold 10% buffered formalin (pH = 7). Immediately after removal of the perfused brain from the cranial cavity, it was post-fixed in cold 10% buffered formalin (pH = 7) for 16----48 hr. The brains were then transferred to cold 30% sucrose for an additional 2 4 - 4 8 hr. After sucrose immersion the brains were blocked in the coronal plane of Pellegrino and Cushman (1967), frozen in 2-methylbutane cooled by solid CO2, mounted on a microtome specimen holder, and sectioned at 20, 40 or 60 pm intervals. 1/3 of the resulting sections was processed for AChE according to the procedure of Karnovsky and Roots (1964), 1/3 for NADH-diaphorase according to the method of Friede (1961), and 1/3 for Nissl substance according to the thionin technique described in Skinner (1971). The present use of the methods of Karnovsky and Roots (1964) and of Friede (1961) has been described extensively in Butcher et al. (1974). To assess the specificity of the histochemical reaction for ACHE, some brain sections were stained according to the Karnovsky and Roots (1964) procedure detailed in Butcher et al. (1974) except that (1) butyrylthiocholine iodide was substituted for acetylthiocholine iodide in the incubation medium or (2) acetylthiocholine iodide was omitted from the reaction mixture. Similarly, the specificity of the NADH-diaphorase reaction was assessed in control brain sections by omitting NADH from the incubation medium. 3. Results

3.1. Histochemical controls Intracerebral or intramuscular injection of the arachis oil vehicle alone had no histochemi-

STRIATAL AND NIGRAL AChE NEURONS

cally detectable effect on ACHE, NADH-diaphorase, or thionin staining. In the brain regions studied by us, omission of acteylthiocholine iodide and NADH from their corresponding incubation media abolished staining for AChE and NADH-diaphorase, respectively, regardless of whether or not the experimental animal was uninjected, given DFP, or administered the arachis oil vehicle alone. In animals systemically administered DFP, substitution of butyrylthiocholine iodide for acetylthiocholine iodide in the reaction mixture failed to produce staining in neuronal elements at the survival times studied in this report; staining in non-neuronal elements was similarly absent or greatly diminished. After intracerebrally infused DFP, butyrylthiocholine iodide incubation produced an absence of cholinesterase staining only in that zone affected by the organophosphorous compound. Brain sections from rats not given DFP showed, after incubation with butyrylthiocholine iodide, a weak reaction which roughly paralleled the sites of cholinesterase activity demonstrated using acetylthiocholine iodide as substrate. Two prominent exceptions were the superior colliculus and interpeduncular nucleus, both of which displayed a strong butyrylcholinesterase reaction (cf. Friede, 1966). In the substantia nigra and caudate---putamen nucleus, however, the staining observed following butyrylthiocholine iodide incubation was

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associated with blood vessels and not neurons, a finding consistent with observations summarized by Friede (1966). We conclude from these data that the hlstochemical reaction we observed in nigral and striatal neurons, using acetylthiocholine iodide as substrate, was selective for ACHE. 3.2. AChE-containing neurons in the caudate-putamen nucleus revealed after DFP administration Intrastriatal infusion of 1.5 pg/1 pl DFP produced a loss of AChE staining 0.4- 0.8 mm from the cavitation caused b y the cannula (fig. 1). The region displaying enzyme inhibition was rather sharply demarcated from adjacent areas showing normal AChE staining (figs. 1 and 3). From 4 to 8 hr after DFP administration, individual AChE-containing neuronal perikarya and their processes could be observed within the zone of AChE diminution (figs. 3 and 4). These cell bodies were not detected consistently at short intervals after DFP infusion but became increasingly more prominent with increasing intervals between application of the organophosphorous compound and euthanasia. The intracerebral DFP injection had little or no effect on NADH~liaphorase activity (fig. 2) or on thionin staining (figs. 5 and 6) in the neostriatum. These results with punctate intra-

Figs. 1--2. Staining for A C h E (fig. 1) a n d N A D H - d i a p h o r a s e (fig. 2) in t h e c a u d a t e - p u t a m e n n u c l e u s a n d cerebral c o r t e x 4 h r a f t e r unilateral i n t r a s t r i a t a l i n f u s i o n of 1.5 pg/l pl DFP. Fig. 1 s h o w s a s e c t i o n f r o m t h e same rat b r a i n as s h o w n in fig. 2. T h e c a l i b r a t i o n b a r in fig. 2 r e p r e s e n t s 5 m m a n d applies also to fig. 1.

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Fig. 3. Higher p o w e r m a g n i f i c a t i o n of fig. 1. A r r o w s p o i n t to A C h E - c o n t a i n i n g n e u r o n a l p e r i k a r y a w i t h i n t h e z o n e of effect of DFP. Asterisk indicates t h e cavit a t i o n p r o d u c e d b y t h e cannula. C a l i b r a t i o n bar for Fig. 3 is t h e s a m e as in fig. 6 a n d r e p r e s e n t s 500 p m .

L.L. B U T C H E R , L. B I L E Z I K J I A N

striatal application of DFP were replicated by intramuscular injection of the organophosphorous c o m p o u n d (see also Butcher et al., 1975a). Most of the neostriatal somata containing AChE were multipolar, although some appeared to be bipolar, and had oval, triangular or fusiform cell bodies (for examples see fig. 4 and Butcher et al., 1975a). On the basis of perikaryal measurements and dendritic topography, the majority of AChE neurons corresponded best to the medium-sized striatal cells described by Kemp and Powell (1971a) and Mensah and Deadwyler (1974). Less frequently, larger AChE-containing neurons were observed having a maximum perikaryon dimension from 30 to 40 pm. Although Kemp and Powell (1971a) report that neurons having morphological features similar to the large AChE neurons we observed constitute less than 1% of the total neuronal population in the cat neostriatum, these cells may be especially important since they probably represent striatal neurons giving rise to axons projecting outside the caudate and putamen nuclei (Kemp and Powell, 1971a). 3. 3. A ChE-cvntaining neurons in the substantia nigra revealed after DFP administration

Fig. 4. A C h E - c o n t a i n i n g cell b o d i e s in t h e c a u d a t e - p u t a m e n n u c l e u s 8 hr a f t e r i n t r a s t r i a t a l i n f u s i o n o f 1.5 pg/l pl DFP. Scale bar is 100 p m .

Pars compacta stained more intensely for AChE than did pars reticulata (figs. 7 and 13).

Figs. 5--6. T h i o n i n s t a i n i n g in t h e c a u d a t e - - p u t a m e n n u c l e u s 4 hr a f t e r i n t r a s t r i a t a l i n f u s i o n of 1.5 pg/1 pl DFP. Figs. 5--6 are f r o m t h e same b r a i n as s h o w n in figs. 1--3. C a l i b r a t i o n bar in fig. 6 is 5 0 0 p m a n d applies also to fig. 5. Asterisk in fig. 5 i n d i c a t e s t h e c a v i t a t i o n p r o d u c e d b y t h e c a n n u l a .

STRIATAL AND NIGRAL AChE NEURONS

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Figs. 7--12. Staining for AChE (figs. 7--8), NADH-diaphorase (figs. 9--10), and thionin (figs. 11--12) in the substantia nigra (SN) after unilateral intranigral infusion of 1.5 pg/1 pl DFP (figs. 8, 10 and 12). All sections are from the same rat which was sacrificed 4 hr after DFP injection. Asterisks in figs. 8, 10 and 12 indicate the cannula tract; the sides opposite the infused side in the same brain section are shown in figs. 7, 9 and 11. Arrows in fig. 7 point to individual AChE-containing neuronal somata. PC = pars compacta, SN; PR = pars reticulata, SN; ML = medial lemniscus. Calibration bar in fig. 9 is 500 g m and applies to figs. 7--12.

I n t r a n i g r a l i n f u s i o n o f 1 . 5 pg/1 pl D F P r e v e a l e d that regeneration of AChE occurred first within n e u r o n a l s o m a t a i n b o t h s u b d i v i s i o n s o f t h e n u c l e u s (fig. 8), a r e s u l t a l s o s e e n a f t e r i n t r a -

muscular injection of the organophosphorous c o m p o u n d (figs. 1 4 a n d 1 5 , see a l s o B u t c h e r e t al., 1 9 7 5 a ) . A l t h o u g h n o t t y p i c a l , i n d i v i d u a l AChE-containing cell bodies could be detected

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Fig. 13. AChE staining in the substantia nigra in pharmacologically unmanipulated material. PC = pars compacta, substantia nigra; PR = pars reticulata, substantia nigra. Scale bar is 300 pm.

L.L. BUTCHER, L. BILEZIKJIAN occasionally on the side o f t h e brain n o t infused with D F P (fig. 7, c o m p a r e with fig. 13). Conceivably, some o f t h e D F P on t h e injected side m a y have been intravascularly incorporated and r e d i s t r i b u t e d to o t h e r parts o f the brain such t h a t a r e d u c e d drug e f f e c t was seen in the non-infused substantia nigra ( c o m p a r e fig. 7 with fig. 13). This particular h y p o t h e s i s is plausible in view o f the fact t h a t individual A C h E - c o n t a i n i n g p e r i k a r y a in t h e substantia nigra were rarely seen in animals n o t given D F P (Fig. 13). A C h E - c o n t a i n i n g nigral n e u r o n s revealed following e n z y m e r e g e n e r a t i o n after D F P either had no cholinesterase-containing processes or were unipolar, bipolar, or m u l t i p o l a r ( f o r examples see figs. 14 and 15 and B u t c h e r et al., 1975a). T h e i r cell bodies were oval, fusif o r m , or, less f r e q u e n t l y , triangular. The A C h E - c o n t a i n i n g n e u r o n s in t h e substantia nigra had a m a x i m u m p e r i k a r y o n d i m e n s i o n f r o m 8 t o 40 p m . On the basis o f s o m a t a size, these A C h E - c o n t a i n i n g n e u r o n s w o u l d be represented within t h e small, m e d i u m and large n e u r o n categories p r o p o s e d b y Gulley and Wood (1971). Finally, some c o m p a c t a AChE n e u r o n s had processes, p r o b a b l y dendrites, which p r o j e c t e d t o w a r d and into pars reticulata (fig. 15). P u n c t a t e intranigral a d m i n i s t r a t i o n o f D F P had no h i s t o c h e m i c a l l y d e t e c t a b l e e f f e c t on N A D H - d i a p h o r a s e activity (figs. 9 and 10) or o n t h i o n i n staining (figs. 11 and 12). Essentially the same h i s t o c h e m i c a l and histological effects were seen a f t e r i n t r a m u s c u l a r D F P injection.

4. Discussion

Fig. 14. AChE staining in the substantia nigra 5 hr after intramuscular injection of 1.5 mg/kg DFP. Scale and abbreviations are the same as in fig. 13.

4.1. D F P as a pharmacological tool in A C h E histochemistry

Fig. 15. AChE staining in the substantia nigra 8 hr after intramuscular injection of 1.5 mg/kg DFP. Arrows point to a process, probably a dendrite, projecting from a pars compacta (PC) neuronal soma into pars reticulata (PR). Calibration bar is 150 pm.

Using p u n c t a t e intracerebral infusion o f DFP, we have shown in the present experim e n t s t h a t t h e AChE in the c a u d a t e - - p u t a m e n nucleus and substantia nigra is associated, at least in major part, with n e u r o n s whose cell

STRIATAL AND NIGRAL AChE NEURONS bodies lie within the two extrapyramidal structures. Discrete neuronal perikarya are not observed reliably or consistently, if at all, in these two brain areas in animals not treated with DFP (e.g., see Butcher et al., 1974, 1975a). The neurons in the neostriatum and substantia nigra have overlapping cellular processes (Kemp and Powell, 1971a; Schwyn and Fox, 1974; Butcher et al., 1975a), and, presumably, enzyme activity in these processes obscures observation of individual cell bodies. Irreversible inhibition of AChE by DFP requires that the neurons synthesize new stores of the enzyme before cholinesterase activity recovers (Blaber and Creasey, 1960; Davis and Agranoff, 1968; Austin and James, 1970; Butcher et al., 1975a,b). Since resynthesis occurs first in the cell b o d y (Austin and James, 1970; Butcher et al., 1975a), histochemical staining for these new stores at appropriate times after DFP enables the visualization of discrete neuronal somata and proximal enzyme-containing processes. Thus, the use of DFP in conjunction with AChE histochemistry emerges as a valuable procedure for morphological studies on the organization of cholinesterase systems in the brain. 4.2. Localization o f AChE in the caudate-putamen nucleus

Since (1) over 96% of all striatal neurons are interneurons (Kemp and Powell, 1971a) and (2) lesioning most known afferents to the caudate--putamen nucleus, as well as ablating those structures known to receive neostriatal efferents, does not produce a histochemically or biochemically detectable loss of striatal AChE (McGeer et al., 1971; Lynch et al., 1972), it must be concluded that the vast majority of AChE in the neostriatum is associated with neurons localized completely within the caudate and putamen nuclei. Some features of the morphology of these ACHEcontaining neurons has been demonstrated in the present study and agrees well with the earlier observations of Lynch et al. (1972). We also observed striatal AChE neurons

121 which had the topographical features characteristic of the large, o u t p u t cells described by Kemp and Powell (1971a). On the basis of known striatal neuroanatomy these AChE neurons should have axons which project to the globus pallidus or substantia nigra (e.g., see Kemp and Powell, 1971b). Some support for this contention derives from the data of Olivier et al. (1971) who reported loss of cholinesterase activity in fibers in the substantia nigra after lesions in the cat caudate nucleus. This diminished staining for nigral AChE was not a strong effect (Olivier et al., 1971), however, and might have been attributable to the fact that only a small proportion of caudate neurons, approximately 3%, have axons projecting outside the neostriatum (Kemp and Powell, 1971a). Lesions confined to the neostriatum also produce retrograde degeneration of ACHEcontaining somata in pars compacta of the substantia nigra (Butcher and Talbot, in preparation; see also Butcher et al., 1975b). Thus, any attempt to ascertain putative striato-nigral projection pa~terns after caudate--putamen lesions, on the basis of diminished AChE staining in the substantia nigra, must take into account not only the cholinesterasic fibers of Olivier et al. (1971) but also retrograde degeneration of AChE-containing nigral perikarya. 4.3. Localization of AChE in the substantia n igra

In a previous paper (Butcher et al., 1975b) we presented evidence that neuronal somata containing dopamine in pars compacta of the substantia nigra also contained ACHE: (1) there was considerable morphological similarity between neurons containing dopamine and those containing ACHE. (2) The proportion of compacta cells containing dopamine, approximately 90% as calculated from data in Gulley and Wood (1971), and the proportion containing ACHE, at least 90%, was too great to permit the simultaneous existence of independent, nonoverlapping sets of neurons (see also Butcher et al., 1975a). (3) Radio-frequency ablations in the ventromedial tegmental area, through

122 which travel axons of the nigro-striatal dopamine pathway (Ungerstedt, 1971), produced retrograde degeneration of AChE-containing neuronal somata in the pars compacta of the substantia nigra (Butcher et al., 1975b). Similarly placed lesions result in loss of nigral perikarya correlated with reductions in neostriatal dopamine (Poirier and Sourkes, 1965). Ablations at other points along the nigro-striatal dopamine projection (i.e., medial forebrain bundle, globus pallidus, caudate--putamen nucleus) also produce degeneration of AChE somata in pars compacta (Butcher and Talbot, in preparation). Although the above results suggest that nigral dopamine neurons contain ACHE, we do not wish to imply that only dopamine neurons in the substantia nigra contain ACHE. Following lesions in the ventromedial tegmental area, the larger, presumably dopaminergic, neurons degenerated. However, a small population of AChE-containing neurons in pars compacta remained after lesioning; their perikarya were small and might not have contained dopamine (Butcher et al., 1975a,b). Recently, F o n n u m et al. (1974) biochemically assayed nigral AChE after lesions in the caudate nucleus (cat) and other subcortical telencephalic nuclei and concluded that the 'observed reduction in A C H E . . . most probably reflects an unspecific localization of the enzyme in non-cholinergic structures in substantia nigra (p. 91)'. Our results agree with this conjecture in suggesting that AChE is localized, at least in major part, within dopamine somata in the pars compacta and in processes of these somata, probably dendrites, projecting into pars reticulata. Similarly, Koelle (1955) suggested that AChE might be localized in adrenergic neurons in the peripheral nervous system. Lesioning the substantia nigra does not produce a biochemically or histochemically detectable loss of AChE in the neostriatum (McGeer et al., 1971; Lynch et al., 1972), and it might be argued that these data invalidate our hypothesis that pars compacta dopamine neurons also contain ACHE. However, the occurrence of a particular molecule in one subcellu-

L.L. BUTCHER, L. BILEZIKJIAN lar compartment of the neuron does not necessarily imply that it is contained in all subcellu. lar constituents. Furthermore, the concentration of the c o m p o u n d may not be the same in the dendrites, soma, and axon. Although AChE may exist in appreciable quantities in the dendrites and somata of compacta dopamine neurons, the concentration in the axons may be zero or too low to detect a loss in the neostriarum after nigral lesions. In addition, since interneurons in the caudate--putamen nucleus contain large amounts of AChE (McGeer et al., 1971; Lynch et al., 1972), enzyme activity in these striatal neurons may obscure detection of AChE contained putatively in axon terminals of the nigro-striatal dopamine pathway. If dopamine neurons in pars compacta of the substantia nigra also contain ACHE, as we believe, then the question can be asked w h y this situation exists. At least two possibilities emerge: in addition to the cholinergic- adrenergic link hypotheses of Koelle (1963) and Burn and Rand (1965), it could be hypothesized that AChE is localized within dopamine neurons in the substantia nigra in order to inactivate a cholinergic input to this neural region. F o n n u m et al. (1974) noted that the 'abnormally high ratio (1000) between AChE and ChAc activities in substantia nigra makes AChE an unreliable marker for cholinergic fibers in this region (p. 90)'. However, AChE in pars compacta of the substantia nigra may be, in part, an excellent marker for dopamine neurons (Butcher et al., 1975a,b). We suggest that neurons can be meaningfully characterized not only in terms of the transmitter used in mediating their o u t p u t effects but also, in some cases, in terms of those post-synaptically localized enzymes necessary to inactivate pre-synaptically released neurotransmitters from other neurons.

4.4. Significance of the present results for dopaminergic--cholinergic interactions in the neostriatum and substantia nigra Since most striatal AChE is associated with interneurons (McGeer et al., 1971) and to the

STRIATAL AND NIGRAL AChE NEURONS extent that AChE is a marker for cholinergic neurons in the caudate--putamen nucleus, it would be tempting to speculate that dopamine nerve terminals deriving from mesencephalic cell b o d y groups synapse on AChE-containing neurons in the neostriatum, particularly since the existence of dopaminergic--cholinergic interactions in the brain has been suggested recently b y abundant pharmacological evidence (Agid et al., 1974; Fibiger and Grewaal, 1974; McGeer et al., 1974a; Sethy and Van Woert, 1974; Stadler et al., 1974; Trabucci et al., 1975). In fact, McGeer et al. (1974a) have hypothesized a direct neuroanatomical linkage between nigro-striatal dopamine terminals and the dendrites of cholinergic interneurons. At the present time, however, there are no morphological data which unequivocably establish such a direct linkage, a fact also acknowledged by McGeer et al. (1974a, p. 211). Although it is clear that drugs influencir~g monoaminergic neurotransmission mechanisms can also alter levels and turnover of brain acetylcholine, data are available suggesting that no simple neuroanatomical relationship exists between nigral dopamine neurons and neostriatal cholinergic or AChE-containing cells: (1) Jones et al. {1973) report that pharmacological manipulation of dopamine systems in the brain has no effect on the release of acetylcholine in the neostriatum. (2) In elegant experiments in which intracellular electrical activity in caudate neurons was recorded after stimulation of the substantia nigra, Buchwald et al. (1973) found that the most frequent response was an initial excitatory post-synaptic potential followed by an inhibitory post-synaptic potential. They concluded that the direct synaptic effects of input fibers to the striatum were all excitatory with the subsequent inhibitory responses being attributable to neuronal stimulation of adjacent inhibitory interneurons -- i.e., 'we view the caudate nucleus as primarily made up of inhibitory interneurons whose extensive extranuclear inputs are all excitatory (Buchwald et al., 1973, p. 321)'. The increases in neostriatal acetylcholine observed by McGeer et al. (1974a) after injection of apomorphine, meth-

123 amphetamine, L-DOPA and amantadine could be explained in terms of these drugs facilitating or mimicing stimulation of inhibitory afferents to cholinergic interneurons in the striatum; presumably, inhibitory input would reduce firing rates of the postulated cholinergic target cells resulting in an intraneuronal accumulation of unreleased acetylcholine. If dopamine is not inhibitory (see Buchwald et al., 1973), however, then in order to explain the data of McGeer et al. (1974a) it must be hypothesized that at least one other neuron is interposed between the neuron upon which dopamine terminals directly synapse and the acetylcholine-containing cell; further, this interposed neuron must be postulated to be inhibitory on the cholinergic neuron. In any case, the data of Buchwald et al. (1973) cast considerable d o u b t on the conjecture of McGeer et al. (1974a) that dopamine released from nigro-striatal axon terminals inhibits striatal cholinergic interneurons directly. Finally (3), reviewing electrophysiological and iontophoretic experiments prior to 1970, Krnjevi6 (1970) concluded that the available evidence did 'not favor the hypothesis of direct cholinergic excitatory or dopaminergic inhibitory projections from the substantia nigra to the striatum (p. 190)'. Iontophoretic application of dopamine to the striatum most often depresses neuronal firing rates (for review, see McLennan, 1970), and it is partly this datum which forms the basis of the conjecture of McGeer et al. (1974a) that a direct inhibitory dopaminergic-~holinergic linkage exists in the neostriatum. However, the problems attendant with the iontophoretic technique are not trivial (for review, see Bloom, 1974), and any hypothesis based on such data should be entertained seriously only after the most careful scrutiny. Despite the ambiguity of the morphological basis for dopaminergic---cholinergic interactions in the caudate--putamen nucleus, we propose, for reasons indicated previously, that a possible neuroanatomical locus for direct interactions between the t w o systems may exist in the substantia nigra (see also Butcher et

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al., 1975a,b). This hypothesis is supported by the following additional evidence: (1)Javoy et al. (1974) found that intranigral infusion of carbachol inhibited dopamine utilization in the neostriatum, whereas atropine increased striatal synthesis and use. (2) Smelik and Ernst (1966) demonstrated that intranigrally applied physostigmine produced stereotyped gnawing, an apparently dopaminergically mediated behavior, which could be blocked by intraperitoneal injection of atropine. Punctate application of physostigmine to the neostriatum and globus pallidus did not elicit gnawing. These authors concluded that 'cholinergic nerve fibers end synaptically on the dopaminergic nigral cells (p. 1487)'. In this and other papers (Butcher et al., 1975a,b) we have presented neuroanatomical and histochemical data which are compatible with the conjecture of Smelik and Ernst (1966). The origin of these postulated cholinergic neurons is currently unknown, but presently available evidence suggests that it is not the neostriatum (McGeer et al., 1971; Kataoka et al., 1974).

Acknowledgement This research was supported in part by NINDS grant NS-10928 from the United States Public Health Service.

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