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Neostriatal enkephalin-immunoreactive neurones project to the globus pallidus
Brain Research, 231 (1982) 1-17 Elsevier Biomedical Press
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Research Reports NEOSTRIATAL ENKEPHALIN-IMMUNOREACTIVE NEURONES P R O J E C T TO T H E G L O B U S P A L L I D U S
M. DEL F1ACCO*, G. PAXINOS** and A. C. CUELLO*** Departments o['Human Anatomy and Pharmacology, University of Oxford, Oxford (U.K.)
(Accepted June 25th, 1981) Key words: corpus striatum - - striatal pathways - - opiates -- enkephalins --neuropeptide immunocytochemistry
SUMMARY The origin of enkephalin-immunoreactive nerve terminals in the globus pallidus was investigated by combining immunocytochemistry with stereotaxic injection of neurotoxic agents (colchicine and kainic acid) and microknife deafferentations. The intracerebral administration of colchicine, irrespective of whether in the caudate putamen or in globus pallidus, induces the appearance of enkephalin-immunoreactive cell bodies and fibres in the caudate putamen. No immunoreactive cell bodies were depicted in the globus pallidus after this treatment. Kainic acid injections in the caudate putamen produced topographic depletions of enkephalin-immunoreactive terminals in the globus pallidus. The more anterior injections produced medial-anterior depletions, while posterior injections gave lateroposterior depletions. Injections in the globus pallidus produced only a non-specific loss of fluorescence restricted to the tip of the cannula. Coronal microknife cuts produced a combination of build-up and depletion of enkephalin immunofluorescence according to the position of the cut. The build-up of immunoreactive materials was always observed in the caudate-putamen side of the cut while depletions observed in the globus pallidus were related to the extent of deafferentation of this nucleus from the caudate putamen. All these observations confirmed the neostriatal origin (caudate putamen) of the enkephalinergic fibres present in the paleostriatum (globus pallidus).
* Permanent address: Istituto di Anatomia Umana Normale, Universitzi di Cagliari, 09100 Cagliari, Italy. ** Permanent address: Department of Psychology, University of New South Wales, Australia 2033. *** To whom correspondence and reprint requests should be addressed. 0006-8993/82/0000-0000/$02.75 © Elsevier Biomedical Press
INTRODUCTION The endogenous pentapeptides leucine- and methionine-enkephatin IEKs) ~; are present in large amounts in the corpus striatum, seemingly acting as endogenou~ ligands to opiate receptorslAT,Z4, :~5,43,46. Indeed, the highest concentration of enkephalins in the brain are found in the corpus striatum '%'~,:~'5,'4°,5°. Using immunohistochemical techniques, it appears evident that within this nuclear complex the two pentapeptides are largely restricted to the globus pallidus (paleostriatum) where they form an extremely dense mesh of EK-immunoreactive nerve terminals 9,~}.~~,:,s, ~ . ~ . ~ 7 4s. The neostriatum (caudate-putamen) also contains enkephalins. Enkephalin-containing neuronal cell bodies, as well as nerve terminals, have been demonstrated in this subdivision of the corpus striatum z,~:l,''~,''s,:~9,'H Studies with blockers of the axonal transport have revealed immunorcactive cct l bodies to the two pentapeptides in local circuit neurones while very few long enkephalin containing pathways ale described in the literature ~:~,~'~.:: ~=~,~,~:~,~s Experimental data indicate that the enkephalin content of the giobus pallidus depends upon the integrity of its connections with the cuadate-putamen 1~. which strongly suggests the existence of a long EK-containing pathway between these t ~ o ~auclei. To further study this possibility we examined the distribution of the EK-likc in~munoreactive material in the corpus striatum after large and restricted knife lesions and following intracereb~ al microinjections ef the neurotoxic substances~ MATERIALS AND METHODS Male adult Sprague-Dawley rats, weighing 200-230 g and male \Vistar rats (250-450 g) served as subjects for these experiments. Under equithesin anaesthesia, each animal received one stereotaxic unilateral injection of either colchicine or kainic acid, or was subjected to a unilateral knife cut. Colchicine (Sigma) was dissolved in freshly prepared Ringer's solution and 2.50 /~g in a volume of 0.5/hi were injected into the caudate-putamen (coordinates: AP 2.4; k :~ 3.0; V 5.5) or into the globus pallidus (coordinates: AP 1,2: k 2.8: V --6.5). Kainic acid (1.25/zg in 0.5 ~1 of saline) was injected either into the anterior part of the caudate-putamen (coordinates: AP } 3.0; L I 2.5; V - 5.5), the posterior part of the caudate-putamen (coordinates: AP 0.0; L 5:: 4.5" V --4.8) ol into the globus pallidus. Bregma was used as the rostrocaudal and lateral zero reference point and the surface of the dura mater as the vertical zero, according to the stereotaxic atlas of Pellegrino aS. The site of injections are illustrated in Fig. 1. Injections were pert\~rmed through a 30 gauge stainless steel cannula, connected with a polythylene tube to a Hamilton syringe, mounted on a mechanically driven pump. The rate of delivery was 1 #1/3 rain. Animals were sacrificed 48 h after injections. The knife cuts were performed while the animals were kept in the stereotaxic instrument in the flat skull position, that is with bregma and lambda at the same vertical level. The zero reference point was the bregma, the midline and the skull surface for the AP, L and V levels, respectively. Two types of knives were used. They con-
Fig. I. Cresyl violet stained horizontal section of the rat brain. Dashed lines indicate the caudateputamen (neostriatum) and the nucleus accumbens septi. Continuous lines indicate the globus pallidus (paleostriatum). Asterisks mark the approximate location of the injections of kainic acid and colchicine in the corpus striatum. Scale bar 2 ram.
sisted of a tungsten wire of0.13 m m diameter placed inside a 30 gauge guide cannula. In the first type of knife the tungsten wire extended 7.3 m m from the top of the guide c a n n u l a a n d was bent in such a way that the first 3.5 m m were perpendicular to the c a n n u l a a n d the r e m a i n i n g 3.8 m m were parallel to it. This knife when rotated inscribes the top and sides of a cylinder 1~. It was used to separate the globus pallidus from the c a u d a t e - p u t a m e n . Its tip was inserted into the b r a i n at the A P - - 0.0 a n d L ~1.9. The c a n n u l a was lowered until the tip of the knife reached 8.0 m m below the skull surface a n d the t u n g s t e n wire was then rotated 180 °, following the anterior a n d lateral
borders of the globus pallidus. Using the same kind of knife, the caudate-putamen was sepalated from the surrounding cortex. Fer this cut, it was inserted at: AP 2°0 mm: L 1.0 mm and V 6,5 ram, before rotating it 180. in the second type of knife, the 30 gauge cannula was bent at one end in such a way that the tungsten wire, forced through it, would extend in the direction of the curved end. This t~pe of knife was used to make coronal cuts at different A P levels of the corpus striatum. Some of these cuts were aimed at the anterior part o['t he caudateputamen (AP 3.2; 2.8; 2.4; L ~ 1.9), some affected both the neostriatum and the medially located globus pallidus (AP .... 1.8: 1.6; 0.8: L 1.9) and, tinally, coronal cuts were made which only bisected the globus pallidus (AP :- 0.0: I. . . . 2.9). The guide cannula was inserted at the desired AP and L levels. It was then lowmed until the tip of the wiie would, if extended, reach a V 4.0 ram. A 3 mm wire was then forced through the cannula at an angle of 4 5 to the sagittal plane and lowered for 4.2 mm Rats were sacrificed 3-5 days after the knife cuts. The experimental animals we,'e anaesthetized with equithesm and perfuscd intracardiacally with 4(~,i paraformaldehyde in 0.1 M phosphate buffer, pH 7.2 at room temperature. Brains were removed and kept in the same fixative solution, at 4 C , for 2-4 h: then transferred to 0.1 M phosphate buffer, pH 7.2, containing 51!, sucrose, for up to 5 days. Horizontal sections of the brain l0 /~n~ thick were cut on a l)uspiva Deittes cryostat at - 20 C . Series of three consecutive sections at the caudate-putamen globus pallidus level were collected on coated slides. For the determination of the enkephalin-immunoreactive sites the indirect immunofluorescence technique of Coons and co-workers was followed ~. A rabbit Leu-enkephalin antiserum a'' raised and characterised by Dr. R. Miller (Department of Pharmacological and Physiological Sciences, Chicago University, U.S.A.) diluted 1:100 was applied to sections n. 2. Sections n. I were run as control preparations, using the same antiserum previously incubated for 60 min at 37 ~C with the pure antigen. The second antiserum was a FITC-conjugated anti-rabbit serum (Miles-Yeda), diluted 1:6. Sections ¢~ 3 wcrc stained with clesyl violet. The tissue preparations were observed under a Leitz Dialux microscope, equipped with a 50 W high prcssure mercur? lamp and epifluorescencc optics. Photographs were taken with a l,eitz Orthomat camera using a K o d a k 400 ASA Tri-X pan film. RESULTS In the intact side of the brain, the specific EK-like immunoreactivity makes the globus pallidus stand out clearly as a thick interwoven mesh of intensively fluorescent elements, against a dark background. The anteriorly and laterally located caudate-putamen shows only patches of rare, little fluorescent dots and has a much darker appearance (Figs. 2A and 3A). The medially located fibres of the capsula interna appear negative to the immunoreaction. Neither the globus pallidus, nor the caudate-putamen show any fluorescent element in control preparations. This pattern of distribution conforms to previous descriptions by other authors "~,~*,~l,es,a')'4j.
Fig. 2. Enkephalin-immunofluorescence of the rat globus pallidus in a horizontal section, after one unilateral injection of kainic acid into the anterior middle caudate-putamen. A: untreated side. B: injected side. CP, caudate-putamen; GP, globus pallidus. Arrow points to unspecific fluorescence. Scale bar ~- 200 tin1.
K a i n i c acid is an a n a l o g u e o f glutamic acid 37, r e p o r t e d to destroy neurons b e a r i n g g l u t a m a t e receptors in the area o f injection, while sparing a x c n s 'en passage' a n d nerve terminals8, 32. The intracerebral injections o f small a m o u n t s o f kainic acid resulted in a very restricted a r e a o f necrosis with a m o d e r a t e gliosis i m m e d i a t e l y s u r r o u n d i n g the cannula tract which represented the 'unspecific' d a m a g e , accom-
Fig. 3. Enkephalin-immunofluorescence of tile rat globus pallidus in a horizontal section, after one unilateral injection of kainic acid into the posterior caudate-putamen. A: untreated side. B: injected side. CP; caudale-putamen ; GP, globus pallidus. Arrows point to unspecific fluorescence. Scale bar 200/~m.
panicd by a more extended loss o f neuronal elements, which was tile specitic effect or" this neurotoxic c o m p o u n d . Fig. 2 shows the effect o f a unilateral kainic acid injection into thc caudatep u t a m e n on the E K - i m m u n o r e a c t i v i t y of the globus pallidus. This injection caused the d e g e n e r a t i o n o f most o f the neuronal cell bodies l o c a t e d in the a n t e r i o r two-thirds o f the right c a u d a t e - p u t a m e n . C o r r e s p o n d i n g l y , the right globus pallidus showed a r e m a r k a b l e loss in its e u k e p h a l i n i m m u n o r e a c t i v e m a t e r i a l which was however restricted to the a n t e r i o r a n d middle p a r t of the nucleus (Fig. 2B), T h e degree a n d
Fig. 4. Enkephalin-immunofluorescence of the rat globus pallidus in a coronal section, after one unilateral injection of kainic acid into the paliidum itself. A: untreated side. B: injected side. CP, caudate putamen. Scale bar - 100/~m. location of the depletion correlated with the lesion in the caudate-putamen. Alternatively, when the injections of the neurotoxic compound were aimed to the caudal part of the caudate-putamen, a distinct depletion in enkephalin immunoreactive material appeared in the caudal part of the globus pallidus, while the remaining part of this nucleus still appeared very bright (Fig. 3). The area of depletion in both cases was well defined and restricted to a single part of the globus pallidus. Within these depleted areas either very few, or no, immunofluorescent fibres were detected. When the same amount of kainic acid was injected into the globus pallidus, it caused the disappearance of the large multipolar neurones, characteristic of this nucleus, but left the whole caudate-putamen practically unaffected. Fig. 4 illustrates the enkephalin-immunofluorescence of the globus pallidus after such a treatment. In the injected side (Fig. 4B) the tissue appeared damaged in Nissl preparations, but the specific fluorescence is completely unaffected by the neurotoxic compound. Discrete intracerebral injections of small amounts of colchicine were aimed to the caudate-putamen or the globus pallidus, in order to block the axonal flow in the striatal neurones and cause an accumulation of neuroactive substance both in axons and cell somata. Both, injections into the neostriatum and the globus pallidus, ~esulted in the appearance of EK-immunoreactive neuronal cell bodies in the caudate-putamen (Figs. 5 and 6,A, B). These were distributed fairly evenly in the lateral part of the
Fig. 5. E n k e p h a l i n - i m m u n o r e a c t i v i t y of the rat c a u d a t e - p u t a m e n after one unilatcrai injection ,.! colcbicine into the g l o b u s patlidus. A, B: E K - i m m u n o f l u o r e s c e n t neuronal cell bodic>~ and nerxe fibres in the c a u d a t e - p u t a m e n of the injected side. Arrows poinl to the emergence o f a x o n s from thc perikaria. D o u b l e - h e a d e d arrow indicates a bundle oi" positive fibres. C : bundles o f beaded fluorescent fibres in the c a u d a t e - p u t a m e n of the injected side, close to the globus paltidus. D: l)!K-immun~. fluorescence in the c a u d a t e - p u t a m e n of the unireated side. A r r o w h e a d s indicale the bundlea ,~I" unreactive fibres. Scale bar 50 i,m.
Fig. 6. Enkephalin-immunofluorescence of the caudate-putamen after one unilateral injection of colchicine into this nucleus. A, B : EK-immunoreactive cell bodies and bundles of fibres in the injected caudate-putamen. Arrow points to the origin of an axon from an immunoreactive perikaryon. Double-headed arrows indicate bundles of positive fibres. C: bundles of EK-immunofluorescent beaded fibres run in the injected caudate-putamen towards the globus pallidus. D: EK-immunoreactivity in the contralateral untreated caudate-putamen. Arrowheads indicate the bundles of unreactive fibres running towards the globus paIlidus. Scale bar 50 Itm.
10 neostriatum both in the anterior and posterior aspects. However, a much smaller number of enkephatin-positive cell bodies were detectable in the most medial part of the caudate-putamen, surrounding the globus pallidus (Figs. 5 and 6C)., These neurones were usually round or oval in shape and of small to medium size. Often the' origin of the axon could be followed for a certain distance from the perikaryon and many swollen tracts of fibres were detectable after this treatment in the caudateputamen, which gave this nucleus a much brighter overall appearance compared to the untreated contralateral side (Figs. 5 and 6,D). Moreover, again in both cases of colchicine injections into the caudate-putamen or the globus pallidus, bright fluorescent bundles of beaded fibres appeared, running radially between the two n uclei. These were mostly evident in the medial part of the caudate-putamen (Figs. 5 and 6,C) intermingled with negative ones, though they were also detectable in its lateral aspects, among the positive cell bodies (Fig. 6A). ]-he administration of colchicine injections in the globus pallidus did not affect the enkephalin-immunoreactivity in this nucleus and neither positive cell bodies nor bundles of varicose fibres were ever detected here. After colchicine ir~jections in the caudate putamen the specific fluorescence in the globus pallidus appeared a little diminished. In addition, single intensely fluorescent beaded fibres appeared in the ventral capsula interna, running in a lateral to medial and caudal direction. These fibres were not apparent in control material. Knife cuts produced an unspecific restricted area of necrosis along the extent of the knife tract and an anterograde degeneration of the fibres running perpendicula~ ly to the section. Knife cuts which completely isolated the globus pallidus from the ~urt'ounding caudate-putamen produced a viltually total disappearance of the specific pallidal immunofluorescence. A 'build-up' of immunoreactive material was also evident in
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Fig. 7. 'Build-up' of enkephalin-immunoreactive material in tile neostriatal side of a kBile cut, which isolates the globus pallidus from the caudate-putamen. In the insert, the dashed fine indicates the knife cut and the rectangle shows approximately the field of photomontage. CP, caudate-putamen; GP, globus pallidus: NAS, nucleus accumbens septi; a.c., anterior commissure. Scale bar 50 ,,era.
Fig. 8. Enkephalin-immunofluorescence of the rat globus pallidus after a coronal knife cut, which bisects it. CP, caudate-putamen; CI, capsula interna. Arrows point to the extremities of the cut. Note 200 /tm. the permanence of immunofluorescence at both sides of lesion. Scale bar
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Fig. 9. Enkephalin-immunofluorescence of the corpus striatum after a coronal cut placed in the caudate-putamen, anterior to the globus pallidus. Build-up of enkephalin-immunoreac~ive material i>. observed in the anterior side of the cut while a marked loss of specific fluorescet~ce i-; ~¢c~ its. ~hc antero-medial globus pallidus. CP, caudate-putamen; GP, globus pallidus. Dashed line h~dicatcs lhc approximate limits of the globus pallidus. Arrows point to the extremities of the cut. Scale bal 200 ~/Ill.
13 axonal bundles all over the striatal side of the cut (Fig. 7). The accumulation of enkephalin-immunoreactive material, however, never extended so far as to render any cell body detectable by the immunoreaction. The cortical deafferentation of the corpus striatum did not produce any noticeable loss of enkephalin-immunofluorescence in the globus pallidus. Also, coronal cuts which only bisected the globus pallidus, did not produce any relevant depletion in the specific immunoreactivity of this nucleus (Fig. 8). On the contrary, coronal cuts which were placed in the anterior part of the caudate-putamen, produced a combination of partial depletion of the specific immunofluorescence of the globus pallidus arrd of'build-up' of enkepha!in-immunoreactive material on the anterior side of the cut. Fig. 9 illustrates one of these experiments. When caudal to knife cuts, a portion of the caudate-putamen was left in connection with globus pallidus (as in Fig. 9) and a corresponding band of immunofluorescence remained caudal to the depleted area of the globus pallidus. Up to a certain degree, the more laterally the cut extended in the caudate-putamen, the bigger the depleted area of the globus palWidus. More posteriorly placed cuts, which affected both the anteromedial globus pallidus and the adjacent caudate-putamen, produced a similal effect on the enkephalin-immunoreactivity of the corpus striatum. In such cases, however, the enkephalin-specific fluorescence in the portion of the globus pallidus extending rostral to the cut always remained completely unaffected. Progressively more posteriorly located cuts produced smaller areas of depletion in the globus pallidus and a less evident buildup of immunoleactive material in the caudate-putamen. In some cases, a small 'pileup' of enkephalin-immunoreactive material was noticed in the neostriatum in the caudal side of a coronal cut. DISCUSSION The existence of a long enkephalin-containing pathway, projecting from the caudate-putamen to the globus pallidus, previously proposed by Cuello and Paxinos tl is confirmed by the present work. The results obtained suggest that enkephalincontaining neurones located in the caudate-putamen send their axons to the globus pallidus in a radiall2~ oriented fashion, determining a topographical correspondence between these two nuclei. The terminal fields of the enkephalin immunoreactive fibres coming from different sectors of the caudate-putamen show virtually no overlap within the globus pallidus. This arrangement is in agreement with the classical descriptions of the organization of the strio-pallidal prejections4, 24. Small, discrete knife lesions produced a restricted, topographical loss of enkephalin immunoreactive material. This cannot be attributed to non-specific damage of pallidal local circuit neurones 6, as in rat (and man) the globus pallidus is virtually supplied by perforating vessels. This assumption is further stressed by the limited loss of Leu-enkephalin observed following transections of the globus pallidus itself. The depletion of enkephalin-immunoreactive material in the globus pallidus after kainic acid injections is in agreement with the finding of the loss of radioimmunoassayable enkephalin in the corpus striatum caused by the same neurotoxic
14 compound 2a. Also consistent with the presence of this tl io-pallidal "enket:~halinergic pathway is the evidence that enkephalins are produced within the corpu,~ ~iriatmn i~y ribosomal synthesis of larger precursors and successively cleaved as penta,~eptides:~;L The fact that retrogradely transported fluorescent dyes injected into the globus pallidus have been shown in enkephalin-immunoreactive neurones of lh~~ caudat,:putamen but not in the globus pallidus adds further support to this strio-pallidal link:~2 The considerable lack of specific immunofluorescence in the dcpleied areas oi the globus pallidus, after intraneostriatal kainate or knife cuts, indicates li~al most ~q the enkephalin-immunoreactive material of this nucleus comes from the_: caudate-putamen. Conversely, the lack of detectable enkephalin-immunoreacti~c ,~e!t bodic> after intlapallidal injections of colchicine, together with the inabilit? of' i~3trapallidaI kainic acid to modify the enkephalin-immunofluorescence of the gtobu3 pal{idus. demonstrates that the en kephali n-i mmu noreactivity of this nucleus is not a~tri bt|tabt c to short axon interneurones intrinsic to il. Eurthermore, intrastrialai colchicine injections clearly revealed enkephalin-immunofluorescent bundles radiati~3g towards the globus pallidus. Recently, the disappearance of enkephalin-containing neostriatal ncurones and nerve terminals, after chronic kainate lesions, has been brought in support of the view that such cells are small interneurones intrinsic to the caudate-putamen ii~,elf t::'`, -[hc authors, however, do not report any observation on the enkephalin-imm uno~ eactivity of the globus pallidus, after the same treatment. The disappearance of ihc spccilic immunofluorescence from the nerve terminals and cell bodies, intrinsic to the neostriatum following kainate lesion, does not rule out the existence oi ne~striatai enkephalin-containing long-pr(\iecting neurones. The nature of" the enkcphalin-immunoreactive processes in the caudateputamen have been investigated by Pickel et al. ~:). They have found enkephalinimmunoreactive material in axons and nerve terminals, as well as cell bodies and dendrites in the caudate-putamen. These axons and nerve terminals could belong to shorter collaterals of the enkephalin-containing neostriatal neurones. ]-he interruption of these collaterals by the knife cuts may explain the presence of a certain -tmount of 'build-up' in the caudal side of the coronal cuts. The presence o!: enkcphalinimmunoreactive fibres in the capsula interna after intrastriata[ colchicinc can bc interpreted as an indication for an ascending component. The origin and termination of these fibres present in the capsula interna remain obscure. The results presented here do not exclude the possibility of further projections of this strio-pallidal path~'a'~ or even the existence of different minor enkephalinergic pathways terminating in ,,,r passing through the corpus striatum. According to the present results the cell bodies ofenkephalinergic neurons of the corpus striatum are restricted to the caudate-putamen and the bulk of their nerve terminal network is located in the globus pallidus, from where the endogenous opiate would be mainly released, as in vitro experiments also suggest "°,~v. This conflicts with the distribution of opiate receptor, The caudate-putamen seems to have a severat-R~ld higher concentration of opiate receptor than the globus pallidus ~, H. It could simply be postulated that they respond to an unknown opiate ligand present in neostriatal nerve
15 terminals. M o r e simply, that they are a different s u b c a t e g o r y o f opiate receptors. F o r this there is a m p l e evidence o f multiplicity o f opiate receptors in the C N S t5,3°. W h a t e v e r the case, a third o f the neostriatal opiate receptors seems to be located p r e s y n a p t i c a l l y in ascending nigro-striatal d o p a m i n e r g i c fibres 3,4°. The nerve terminal n e t w o r k o f these d o p a m i n e r g i c fibres is in t u r n restricted to the n e o s t f i a t u m ls,22,3l. The potential n u m b e r o f enkephalinergic short axons in the c a u d a t e - p u t a m e n w o u l d not m a t c h the richness o f the d o p a m i n e r g i c network. It is possible that enkephalin released from c a u d a t e - p u t a m e n neurones could explain this interaction ~2. The anatomical basis for these interactions is far from clear. The simultaneous detection at electron microscopical level o f a m i n e a n d e n k e p h a l i n i m m u n o r e a c t i v e sites could c o n t r i b u t e to the clarification o f the synaptic nature o f these interactivities. ACKNOWLEDGEMENTS The a u t h o r s gratefully acknowledge the technical assistance o f Juliette L l o y d a n d T o n y Barclay. The e n k e p h a l i n antiserum was a generous gift from Dr. R. Miller (Chicago University, U.S.A.) T h a n k s are due to Mrs. Ella lies for typing the m a n u s c r i p t a n d for secretarial help. M . D . F . was s u p p o r t e d by a N A T O F e l l o w s h i p f r o m the I t a l i a n Consiglio N a z i o n a l e delle Ricerche and an E M B O s h o r t - t e r m fellowship. G.P. acknowledges grants f r o m A R G C ( A u s t l a l i a ) a n d A.C.C. g i a n t s from the M e d i c a l Research Council, The W e l l c o m e Trust a n d The R o y a l Society (U.K.).
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17 37 Olney, |. W., Rhee, V. and Ho, O. L., Kainic acid as a powerful neurotoxic analogue of glutamate Brain Research, 77 (1974) 507-512. 38 Pellegrino, L. J., Pellegrino, A. S. and Cushman, A. L., A Stereotaxic Atlas o f the Rat Brain, Plenum Press, New York, NY, 1979. 39 Pickel, V. M., Sumal, K. K., Beckley, S. C., Miller, R. J. and Reis, D. J., lmmunocytochemical localization of enkephalin in the neostriatum of the rat brain: a light and electron microscopy study, J. comp. Neurol., 189 (1980) 721 740. 40 Pollard, H., Llorens-Cortes, C. and Schwartz, J. C., Enkephalin receptors on dopaminergic neurones in rat striatum, Nature (Lond.), 268 (1977) 745-747. 41 Sat, M., Stumpf, E., Miller, R. J. and Cuatrecasas, P., lmmunohistochemical localization of enkephalin in rat brain and spinal cord, J. camp. Neurol., 182 (1978) 17-38. 42 Schwarcz, R., Fuxe, K., H6kfelt, T., Terenius, L. and Goldstein, M., Effects of chronic striatal kainate lesions on some dopaminergic parameters and enkephalin immunoreactive neurons in the basal ganglia, J. Neurochem., 34 (1980) 772 778. 43 Simantov, R., Kuhar, M. J., Pasternak, G. W. and Snyder, S. H., The regional distribution of a morphine-like factor enkephalin in monkey brain, Brain Research, 106 (1976) 189 197. 44 Simantov, R., Kuhar, M. J., Uhl, G. R. and Snyder, S. H., Opioid peptide enkephalin : immunohistochemical mapping in the rat central nervous system, Proc. nat. Acad. Sci. U.S.A., 74 (1977) 2167 2171. 45 Simantov, R. and Snyder, S. H., Isolation and identification of a morphine-like peptide enkephalin in bovine brain, L(/? Sci., 18 (1976) 781 788. 46 Terenius, L. and Wahlstrom, A., Search for an endogenous ligand for the opiate receptors, Acta physiol, stand., 94 (1975) 74-81. 47 Uhl, G. R., Goodman, R. R., Kuhar, M. J. and Snyder, S. H., Enkephalin and neurotensin: immunohistochemical localization and identification of an amigdalofugal pathway. In E. Costa and M. Trabucchi (Ed.), Adv. Biochem. Psychopharmacol., 18 (1978) 71-87. 48 Watson, S. J., Akil, H., Sullivan, S. and Barchas, J. D., lmmunocytochemical localization of methionine-enkephalin: preliminary observations, Life Sci., 21 (1977) 733 738. 49 Yang, H. Y., Hong, J.-S. and Costa, E., Regional distribution of Leu- en Met-enkephalin in rat brain, Neuropharn;acology, 16 (1977) 303-307. 50 Yang, H.-Y. T., Hong, J. S., Fratta, W. and Costa, E., Rat brain enkephalins: distribution and biosynthesis, Adv. Biochem. Psychopharmacol., 18 (1978) 149-159.
l
Research Reports NEOSTRIATAL ENKEPHALIN-IMMUNOREACTIVE NEURONES P R O J E C T TO T H E G L O B U S P A L L I D U S
M. DEL F1ACCO*, G. PAXINOS** and A. C. CUELLO*** Departments o['Human Anatomy and Pharmacology, University of Oxford, Oxford (U.K.)
(Accepted June 25th, 1981) Key words: corpus striatum - - striatal pathways - - opiates -- enkephalins --neuropeptide immunocytochemistry
SUMMARY The origin of enkephalin-immunoreactive nerve terminals in the globus pallidus was investigated by combining immunocytochemistry with stereotaxic injection of neurotoxic agents (colchicine and kainic acid) and microknife deafferentations. The intracerebral administration of colchicine, irrespective of whether in the caudate putamen or in globus pallidus, induces the appearance of enkephalin-immunoreactive cell bodies and fibres in the caudate putamen. No immunoreactive cell bodies were depicted in the globus pallidus after this treatment. Kainic acid injections in the caudate putamen produced topographic depletions of enkephalin-immunoreactive terminals in the globus pallidus. The more anterior injections produced medial-anterior depletions, while posterior injections gave lateroposterior depletions. Injections in the globus pallidus produced only a non-specific loss of fluorescence restricted to the tip of the cannula. Coronal microknife cuts produced a combination of build-up and depletion of enkephalin immunofluorescence according to the position of the cut. The build-up of immunoreactive materials was always observed in the caudate-putamen side of the cut while depletions observed in the globus pallidus were related to the extent of deafferentation of this nucleus from the caudate putamen. All these observations confirmed the neostriatal origin (caudate putamen) of the enkephalinergic fibres present in the paleostriatum (globus pallidus).
* Permanent address: Istituto di Anatomia Umana Normale, Universitzi di Cagliari, 09100 Cagliari, Italy. ** Permanent address: Department of Psychology, University of New South Wales, Australia 2033. *** To whom correspondence and reprint requests should be addressed. 0006-8993/82/0000-0000/$02.75 © Elsevier Biomedical Press
INTRODUCTION The endogenous pentapeptides leucine- and methionine-enkephatin IEKs) ~; are present in large amounts in the corpus striatum, seemingly acting as endogenou~ ligands to opiate receptorslAT,Z4, :~5,43,46. Indeed, the highest concentration of enkephalins in the brain are found in the corpus striatum '%'~,:~'5,'4°,5°. Using immunohistochemical techniques, it appears evident that within this nuclear complex the two pentapeptides are largely restricted to the globus pallidus (paleostriatum) where they form an extremely dense mesh of EK-immunoreactive nerve terminals 9,~}.~~,:,s, ~ . ~ . ~ 7 4s. The neostriatum (caudate-putamen) also contains enkephalins. Enkephalin-containing neuronal cell bodies, as well as nerve terminals, have been demonstrated in this subdivision of the corpus striatum z,~:l,''~,''s,:~9,'H Studies with blockers of the axonal transport have revealed immunorcactive cct l bodies to the two pentapeptides in local circuit neurones while very few long enkephalin containing pathways ale described in the literature ~:~,~'~.:: ~=~,~,~:~,~s Experimental data indicate that the enkephalin content of the giobus pallidus depends upon the integrity of its connections with the cuadate-putamen 1~. which strongly suggests the existence of a long EK-containing pathway between these t ~ o ~auclei. To further study this possibility we examined the distribution of the EK-likc in~munoreactive material in the corpus striatum after large and restricted knife lesions and following intracereb~ al microinjections ef the neurotoxic substances~ MATERIALS AND METHODS Male adult Sprague-Dawley rats, weighing 200-230 g and male \Vistar rats (250-450 g) served as subjects for these experiments. Under equithesin anaesthesia, each animal received one stereotaxic unilateral injection of either colchicine or kainic acid, or was subjected to a unilateral knife cut. Colchicine (Sigma) was dissolved in freshly prepared Ringer's solution and 2.50 /~g in a volume of 0.5/hi were injected into the caudate-putamen (coordinates: AP 2.4; k :~ 3.0; V 5.5) or into the globus pallidus (coordinates: AP 1,2: k 2.8: V --6.5). Kainic acid (1.25/zg in 0.5 ~1 of saline) was injected either into the anterior part of the caudate-putamen (coordinates: AP } 3.0; L I 2.5; V - 5.5), the posterior part of the caudate-putamen (coordinates: AP 0.0; L 5:: 4.5" V --4.8) ol into the globus pallidus. Bregma was used as the rostrocaudal and lateral zero reference point and the surface of the dura mater as the vertical zero, according to the stereotaxic atlas of Pellegrino aS. The site of injections are illustrated in Fig. 1. Injections were pert\~rmed through a 30 gauge stainless steel cannula, connected with a polythylene tube to a Hamilton syringe, mounted on a mechanically driven pump. The rate of delivery was 1 #1/3 rain. Animals were sacrificed 48 h after injections. The knife cuts were performed while the animals were kept in the stereotaxic instrument in the flat skull position, that is with bregma and lambda at the same vertical level. The zero reference point was the bregma, the midline and the skull surface for the AP, L and V levels, respectively. Two types of knives were used. They con-
Fig. I. Cresyl violet stained horizontal section of the rat brain. Dashed lines indicate the caudateputamen (neostriatum) and the nucleus accumbens septi. Continuous lines indicate the globus pallidus (paleostriatum). Asterisks mark the approximate location of the injections of kainic acid and colchicine in the corpus striatum. Scale bar 2 ram.
sisted of a tungsten wire of0.13 m m diameter placed inside a 30 gauge guide cannula. In the first type of knife the tungsten wire extended 7.3 m m from the top of the guide c a n n u l a a n d was bent in such a way that the first 3.5 m m were perpendicular to the c a n n u l a a n d the r e m a i n i n g 3.8 m m were parallel to it. This knife when rotated inscribes the top and sides of a cylinder 1~. It was used to separate the globus pallidus from the c a u d a t e - p u t a m e n . Its tip was inserted into the b r a i n at the A P - - 0.0 a n d L ~1.9. The c a n n u l a was lowered until the tip of the knife reached 8.0 m m below the skull surface a n d the t u n g s t e n wire was then rotated 180 °, following the anterior a n d lateral
borders of the globus pallidus. Using the same kind of knife, the caudate-putamen was sepalated from the surrounding cortex. Fer this cut, it was inserted at: AP 2°0 mm: L 1.0 mm and V 6,5 ram, before rotating it 180. in the second type of knife, the 30 gauge cannula was bent at one end in such a way that the tungsten wire, forced through it, would extend in the direction of the curved end. This t~pe of knife was used to make coronal cuts at different A P levels of the corpus striatum. Some of these cuts were aimed at the anterior part o['t he caudateputamen (AP 3.2; 2.8; 2.4; L ~ 1.9), some affected both the neostriatum and the medially located globus pallidus (AP .... 1.8: 1.6; 0.8: L 1.9) and, tinally, coronal cuts were made which only bisected the globus pallidus (AP :- 0.0: I. . . . 2.9). The guide cannula was inserted at the desired AP and L levels. It was then lowmed until the tip of the wiie would, if extended, reach a V 4.0 ram. A 3 mm wire was then forced through the cannula at an angle of 4 5 to the sagittal plane and lowered for 4.2 mm Rats were sacrificed 3-5 days after the knife cuts. The experimental animals we,'e anaesthetized with equithesm and perfuscd intracardiacally with 4(~,i paraformaldehyde in 0.1 M phosphate buffer, pH 7.2 at room temperature. Brains were removed and kept in the same fixative solution, at 4 C , for 2-4 h: then transferred to 0.1 M phosphate buffer, pH 7.2, containing 51!, sucrose, for up to 5 days. Horizontal sections of the brain l0 /~n~ thick were cut on a l)uspiva Deittes cryostat at - 20 C . Series of three consecutive sections at the caudate-putamen globus pallidus level were collected on coated slides. For the determination of the enkephalin-immunoreactive sites the indirect immunofluorescence technique of Coons and co-workers was followed ~. A rabbit Leu-enkephalin antiserum a'' raised and characterised by Dr. R. Miller (Department of Pharmacological and Physiological Sciences, Chicago University, U.S.A.) diluted 1:100 was applied to sections n. 2. Sections n. I were run as control preparations, using the same antiserum previously incubated for 60 min at 37 ~C with the pure antigen. The second antiserum was a FITC-conjugated anti-rabbit serum (Miles-Yeda), diluted 1:6. Sections ¢~ 3 wcrc stained with clesyl violet. The tissue preparations were observed under a Leitz Dialux microscope, equipped with a 50 W high prcssure mercur? lamp and epifluorescencc optics. Photographs were taken with a l,eitz Orthomat camera using a K o d a k 400 ASA Tri-X pan film. RESULTS In the intact side of the brain, the specific EK-like immunoreactivity makes the globus pallidus stand out clearly as a thick interwoven mesh of intensively fluorescent elements, against a dark background. The anteriorly and laterally located caudate-putamen shows only patches of rare, little fluorescent dots and has a much darker appearance (Figs. 2A and 3A). The medially located fibres of the capsula interna appear negative to the immunoreaction. Neither the globus pallidus, nor the caudate-putamen show any fluorescent element in control preparations. This pattern of distribution conforms to previous descriptions by other authors "~,~*,~l,es,a')'4j.
Fig. 2. Enkephalin-immunofluorescence of the rat globus pallidus in a horizontal section, after one unilateral injection of kainic acid into the anterior middle caudate-putamen. A: untreated side. B: injected side. CP, caudate-putamen; GP, globus pallidus. Arrow points to unspecific fluorescence. Scale bar ~- 200 tin1.
K a i n i c acid is an a n a l o g u e o f glutamic acid 37, r e p o r t e d to destroy neurons b e a r i n g g l u t a m a t e receptors in the area o f injection, while sparing a x c n s 'en passage' a n d nerve terminals8, 32. The intracerebral injections o f small a m o u n t s o f kainic acid resulted in a very restricted a r e a o f necrosis with a m o d e r a t e gliosis i m m e d i a t e l y s u r r o u n d i n g the cannula tract which represented the 'unspecific' d a m a g e , accom-
Fig. 3. Enkephalin-immunofluorescence of tile rat globus pallidus in a horizontal section, after one unilateral injection of kainic acid into the posterior caudate-putamen. A: untreated side. B: injected side. CP; caudale-putamen ; GP, globus pallidus. Arrows point to unspecific fluorescence. Scale bar 200/~m.
panicd by a more extended loss o f neuronal elements, which was tile specitic effect or" this neurotoxic c o m p o u n d . Fig. 2 shows the effect o f a unilateral kainic acid injection into thc caudatep u t a m e n on the E K - i m m u n o r e a c t i v i t y of the globus pallidus. This injection caused the d e g e n e r a t i o n o f most o f the neuronal cell bodies l o c a t e d in the a n t e r i o r two-thirds o f the right c a u d a t e - p u t a m e n . C o r r e s p o n d i n g l y , the right globus pallidus showed a r e m a r k a b l e loss in its e u k e p h a l i n i m m u n o r e a c t i v e m a t e r i a l which was however restricted to the a n t e r i o r a n d middle p a r t of the nucleus (Fig. 2B), T h e degree a n d
Fig. 4. Enkephalin-immunofluorescence of the rat globus pallidus in a coronal section, after one unilateral injection of kainic acid into the paliidum itself. A: untreated side. B: injected side. CP, caudate putamen. Scale bar - 100/~m. location of the depletion correlated with the lesion in the caudate-putamen. Alternatively, when the injections of the neurotoxic compound were aimed to the caudal part of the caudate-putamen, a distinct depletion in enkephalin immunoreactive material appeared in the caudal part of the globus pallidus, while the remaining part of this nucleus still appeared very bright (Fig. 3). The area of depletion in both cases was well defined and restricted to a single part of the globus pallidus. Within these depleted areas either very few, or no, immunofluorescent fibres were detected. When the same amount of kainic acid was injected into the globus pallidus, it caused the disappearance of the large multipolar neurones, characteristic of this nucleus, but left the whole caudate-putamen practically unaffected. Fig. 4 illustrates the enkephalin-immunofluorescence of the globus pallidus after such a treatment. In the injected side (Fig. 4B) the tissue appeared damaged in Nissl preparations, but the specific fluorescence is completely unaffected by the neurotoxic compound. Discrete intracerebral injections of small amounts of colchicine were aimed to the caudate-putamen or the globus pallidus, in order to block the axonal flow in the striatal neurones and cause an accumulation of neuroactive substance both in axons and cell somata. Both, injections into the neostriatum and the globus pallidus, ~esulted in the appearance of EK-immunoreactive neuronal cell bodies in the caudate-putamen (Figs. 5 and 6,A, B). These were distributed fairly evenly in the lateral part of the
Fig. 5. E n k e p h a l i n - i m m u n o r e a c t i v i t y of the rat c a u d a t e - p u t a m e n after one unilatcrai injection ,.! colcbicine into the g l o b u s patlidus. A, B: E K - i m m u n o f l u o r e s c e n t neuronal cell bodic>~ and nerxe fibres in the c a u d a t e - p u t a m e n of the injected side. Arrows poinl to the emergence o f a x o n s from thc perikaria. D o u b l e - h e a d e d arrow indicates a bundle oi" positive fibres. C : bundles o f beaded fluorescent fibres in the c a u d a t e - p u t a m e n of the injected side, close to the globus paltidus. D: l)!K-immun~. fluorescence in the c a u d a t e - p u t a m e n of the unireated side. A r r o w h e a d s indicale the bundlea ,~I" unreactive fibres. Scale bar 50 i,m.
Fig. 6. Enkephalin-immunofluorescence of the caudate-putamen after one unilateral injection of colchicine into this nucleus. A, B : EK-immunoreactive cell bodies and bundles of fibres in the injected caudate-putamen. Arrow points to the origin of an axon from an immunoreactive perikaryon. Double-headed arrows indicate bundles of positive fibres. C: bundles of EK-immunofluorescent beaded fibres run in the injected caudate-putamen towards the globus pallidus. D: EK-immunoreactivity in the contralateral untreated caudate-putamen. Arrowheads indicate the bundles of unreactive fibres running towards the globus paIlidus. Scale bar 50 Itm.
10 neostriatum both in the anterior and posterior aspects. However, a much smaller number of enkephatin-positive cell bodies were detectable in the most medial part of the caudate-putamen, surrounding the globus pallidus (Figs. 5 and 6C)., These neurones were usually round or oval in shape and of small to medium size. Often the' origin of the axon could be followed for a certain distance from the perikaryon and many swollen tracts of fibres were detectable after this treatment in the caudateputamen, which gave this nucleus a much brighter overall appearance compared to the untreated contralateral side (Figs. 5 and 6,D). Moreover, again in both cases of colchicine injections into the caudate-putamen or the globus pallidus, bright fluorescent bundles of beaded fibres appeared, running radially between the two n uclei. These were mostly evident in the medial part of the caudate-putamen (Figs. 5 and 6,C) intermingled with negative ones, though they were also detectable in its lateral aspects, among the positive cell bodies (Fig. 6A). ]-he administration of colchicine injections in the globus pallidus did not affect the enkephalin-immunoreactivity in this nucleus and neither positive cell bodies nor bundles of varicose fibres were ever detected here. After colchicine ir~jections in the caudate putamen the specific fluorescence in the globus pallidus appeared a little diminished. In addition, single intensely fluorescent beaded fibres appeared in the ventral capsula interna, running in a lateral to medial and caudal direction. These fibres were not apparent in control material. Knife cuts produced an unspecific restricted area of necrosis along the extent of the knife tract and an anterograde degeneration of the fibres running perpendicula~ ly to the section. Knife cuts which completely isolated the globus pallidus from the ~urt'ounding caudate-putamen produced a viltually total disappearance of the specific pallidal immunofluorescence. A 'build-up' of immunoreactive material was also evident in
j./
•
NAS
"
/
/
/
cp
~.
" a~ "
Fig. 7. 'Build-up' of enkephalin-immunoreactive material in tile neostriatal side of a kBile cut, which isolates the globus pallidus from the caudate-putamen. In the insert, the dashed fine indicates the knife cut and the rectangle shows approximately the field of photomontage. CP, caudate-putamen; GP, globus pallidus: NAS, nucleus accumbens septi; a.c., anterior commissure. Scale bar 50 ,,era.
Fig. 8. Enkephalin-immunofluorescence of the rat globus pallidus after a coronal knife cut, which bisects it. CP, caudate-putamen; CI, capsula interna. Arrows point to the extremities of the cut. Note 200 /tm. the permanence of immunofluorescence at both sides of lesion. Scale bar
12
Fig. 9. Enkephalin-immunofluorescence of the corpus striatum after a coronal cut placed in the caudate-putamen, anterior to the globus pallidus. Build-up of enkephalin-immunoreac~ive material i>. observed in the anterior side of the cut while a marked loss of specific fluorescet~ce i-; ~¢c~ its. ~hc antero-medial globus pallidus. CP, caudate-putamen; GP, globus pallidus. Dashed line h~dicatcs lhc approximate limits of the globus pallidus. Arrows point to the extremities of the cut. Scale bal 200 ~/Ill.
13 axonal bundles all over the striatal side of the cut (Fig. 7). The accumulation of enkephalin-immunoreactive material, however, never extended so far as to render any cell body detectable by the immunoreaction. The cortical deafferentation of the corpus striatum did not produce any noticeable loss of enkephalin-immunofluorescence in the globus pallidus. Also, coronal cuts which only bisected the globus pallidus, did not produce any relevant depletion in the specific immunoreactivity of this nucleus (Fig. 8). On the contrary, coronal cuts which were placed in the anterior part of the caudate-putamen, produced a combination of partial depletion of the specific immunofluorescence of the globus pallidus arrd of'build-up' of enkepha!in-immunoreactive material on the anterior side of the cut. Fig. 9 illustrates one of these experiments. When caudal to knife cuts, a portion of the caudate-putamen was left in connection with globus pallidus (as in Fig. 9) and a corresponding band of immunofluorescence remained caudal to the depleted area of the globus pallidus. Up to a certain degree, the more laterally the cut extended in the caudate-putamen, the bigger the depleted area of the globus palWidus. More posteriorly placed cuts, which affected both the anteromedial globus pallidus and the adjacent caudate-putamen, produced a similal effect on the enkephalin-immunoreactivity of the corpus striatum. In such cases, however, the enkephalin-specific fluorescence in the portion of the globus pallidus extending rostral to the cut always remained completely unaffected. Progressively more posteriorly located cuts produced smaller areas of depletion in the globus pallidus and a less evident buildup of immunoleactive material in the caudate-putamen. In some cases, a small 'pileup' of enkephalin-immunoreactive material was noticed in the neostriatum in the caudal side of a coronal cut. DISCUSSION The existence of a long enkephalin-containing pathway, projecting from the caudate-putamen to the globus pallidus, previously proposed by Cuello and Paxinos tl is confirmed by the present work. The results obtained suggest that enkephalincontaining neurones located in the caudate-putamen send their axons to the globus pallidus in a radiall2~ oriented fashion, determining a topographical correspondence between these two nuclei. The terminal fields of the enkephalin immunoreactive fibres coming from different sectors of the caudate-putamen show virtually no overlap within the globus pallidus. This arrangement is in agreement with the classical descriptions of the organization of the strio-pallidal prejections4, 24. Small, discrete knife lesions produced a restricted, topographical loss of enkephalin immunoreactive material. This cannot be attributed to non-specific damage of pallidal local circuit neurones 6, as in rat (and man) the globus pallidus is virtually supplied by perforating vessels. This assumption is further stressed by the limited loss of Leu-enkephalin observed following transections of the globus pallidus itself. The depletion of enkephalin-immunoreactive material in the globus pallidus after kainic acid injections is in agreement with the finding of the loss of radioimmunoassayable enkephalin in the corpus striatum caused by the same neurotoxic
14 compound 2a. Also consistent with the presence of this tl io-pallidal "enket:~halinergic pathway is the evidence that enkephalins are produced within the corpu,~ ~iriatmn i~y ribosomal synthesis of larger precursors and successively cleaved as penta,~eptides:~;L The fact that retrogradely transported fluorescent dyes injected into the globus pallidus have been shown in enkephalin-immunoreactive neurones of lh~~ caudat,:putamen but not in the globus pallidus adds further support to this strio-pallidal link:~2 The considerable lack of specific immunofluorescence in the dcpleied areas oi the globus pallidus, after intraneostriatal kainate or knife cuts, indicates li~al most ~q the enkephalin-immunoreactive material of this nucleus comes from the_: caudate-putamen. Conversely, the lack of detectable enkephalin-immunoreacti~c ,~e!t bodic> after intlapallidal injections of colchicine, together with the inabilit? of' i~3trapallidaI kainic acid to modify the enkephalin-immunofluorescence of the gtobu3 pal{idus. demonstrates that the en kephali n-i mmu noreactivity of this nucleus is not a~tri bt|tabt c to short axon interneurones intrinsic to il. Eurthermore, intrastrialai colchicine injections clearly revealed enkephalin-immunofluorescent bundles radiati~3g towards the globus pallidus. Recently, the disappearance of enkephalin-containing neostriatal ncurones and nerve terminals, after chronic kainate lesions, has been brought in support of the view that such cells are small interneurones intrinsic to the caudate-putamen ii~,elf t::'`, -[hc authors, however, do not report any observation on the enkephalin-imm uno~ eactivity of the globus pallidus, after the same treatment. The disappearance of ihc spccilic immunofluorescence from the nerve terminals and cell bodies, intrinsic to the neostriatum following kainate lesion, does not rule out the existence oi ne~striatai enkephalin-containing long-pr(\iecting neurones. The nature of" the enkcphalin-immunoreactive processes in the caudateputamen have been investigated by Pickel et al. ~:). They have found enkephalinimmunoreactive material in axons and nerve terminals, as well as cell bodies and dendrites in the caudate-putamen. These axons and nerve terminals could belong to shorter collaterals of the enkephalin-containing neostriatal neurones. ]-he interruption of these collaterals by the knife cuts may explain the presence of a certain -tmount of 'build-up' in the caudal side of the coronal cuts. The presence o!: enkcphalinimmunoreactive fibres in the capsula interna after intrastriata[ colchicinc can bc interpreted as an indication for an ascending component. The origin and termination of these fibres present in the capsula interna remain obscure. The results presented here do not exclude the possibility of further projections of this strio-pallidal path~'a'~ or even the existence of different minor enkephalinergic pathways terminating in ,,,r passing through the corpus striatum. According to the present results the cell bodies ofenkephalinergic neurons of the corpus striatum are restricted to the caudate-putamen and the bulk of their nerve terminal network is located in the globus pallidus, from where the endogenous opiate would be mainly released, as in vitro experiments also suggest "°,~v. This conflicts with the distribution of opiate receptor, The caudate-putamen seems to have a severat-R~ld higher concentration of opiate receptor than the globus pallidus ~, H. It could simply be postulated that they respond to an unknown opiate ligand present in neostriatal nerve
15 terminals. M o r e simply, that they are a different s u b c a t e g o r y o f opiate receptors. F o r this there is a m p l e evidence o f multiplicity o f opiate receptors in the C N S t5,3°. W h a t e v e r the case, a third o f the neostriatal opiate receptors seems to be located p r e s y n a p t i c a l l y in ascending nigro-striatal d o p a m i n e r g i c fibres 3,4°. The nerve terminal n e t w o r k o f these d o p a m i n e r g i c fibres is in t u r n restricted to the n e o s t f i a t u m ls,22,3l. The potential n u m b e r o f enkephalinergic short axons in the c a u d a t e - p u t a m e n w o u l d not m a t c h the richness o f the d o p a m i n e r g i c network. It is possible that enkephalin released from c a u d a t e - p u t a m e n neurones could explain this interaction ~2. The anatomical basis for these interactions is far from clear. The simultaneous detection at electron microscopical level o f a m i n e a n d e n k e p h a l i n i m m u n o r e a c t i v e sites could c o n t r i b u t e to the clarification o f the synaptic nature o f these interactivities. ACKNOWLEDGEMENTS The a u t h o r s gratefully acknowledge the technical assistance o f Juliette L l o y d a n d T o n y Barclay. The e n k e p h a l i n antiserum was a generous gift from Dr. R. Miller (Chicago University, U.S.A.) T h a n k s are due to Mrs. Ella lies for typing the m a n u s c r i p t a n d for secretarial help. M . D . F . was s u p p o r t e d by a N A T O F e l l o w s h i p f r o m the I t a l i a n Consiglio N a z i o n a l e delle Ricerche and an E M B O s h o r t - t e r m fellowship. G.P. acknowledges grants f r o m A R G C ( A u s t l a l i a ) a n d A.C.C. g i a n t s from the M e d i c a l Research Council, The W e l l c o m e Trust a n d The R o y a l Society (U.K.).
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