0306-4522/91 %3.oo+a.o0 Pe~monP~pk 0 1991IBRO

Neuroscience Vol.44,No. 2,pp.431447,1991 Printed in Great Britain

EFFERENT CONNECTIONS OF THE STRIATOPALLIDAL AND AMYGDALOID COMPONENTS OF THE SUBSTANTIA INNOMINATA IN THE CAT: PROJECTIONS TO THE NUCLEUS ACCUMBENS AND CAUDATE NUCLEUS W. P. J. M. SPOOREN,*~J. G. VEENING,~ I-I. J. GROENEWEGJIN§ and A. R. COOLS* *Department

of Pharmacology, Psychoneuropharmacological Research Unit, University of Nijmegen, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands SDepartment of Anatomy and Embryology, University of Nijmegen, Nijmegen, The Netherlands §Department of Anatomy, Free University, Amsterdam, The Netherlands

Algae-~nkephalin irnrnunor~~ti~ty is used to divide the feline substantia innominata into circumscript subregions, i.e. the “striatopallidal system” and the “extended amygdala”. In addition, enkephalin immunoreactivity is used to subdivide the striatopallidal system into two distinct areas, i.e. the subcommissural part of the globus pallidus displaying high enkephalin immunoreactivity and the ventral pallidum displaying moderate enkephalin immunoreactivity. The anterograde axonal transport of Phuseolw oulgaris-leucoagglutinin is used to study the efferents of these areas innervating the caudate nucleus and the nucleus accumbens. It is found that the enkephalin-immunoreactive subcommissural part of the globus pallidus as well as the dorsal enkeph~in-immuno~active regions of the extended amygdala project topographically along a rostrocaudal and mediolateral dimension to the nucleus accumbens. The far rostra1 parts of the caudate nucleus are found to be innervated by the subcommissural part of the globus pallidus whereas the extended amygdala has no such connection. This pathway is also found to be to~~aphic~ly organized along a mediolateral dimension. The non~nk~h~in-immunor~ctive area ventral and lateral to the subcommissural part of the globus pallidus is found to have no projections to the nucleus accumbens and caudate nucleus. This region rather innervates the olfactory tubercle. In contrast to the striatopallidal system the sublenticular part of the extended amygdala preferentially projects to the adjoining part of the extended amygdala, i.e. the bed nucleus of the stria terminalis. However, the ventral regions preferentially innervate the medial division of the bed nucleus of the stria terminalis whereas the dorsal regions preferentially innervate the Iateral division of the bed nucleus of the stria terminalis. These data indicate that the differential forebrain systems represented in the feline substantia innominata, i.e. fhe striatopallidal system and extended amygdala have differential output stations. The results are discussed in view of the role of the subcommissural part of the globus pallidus and the nucleus accumbens in orofacial dyskinesia and schizophrenia, respectively.

The substantia ~Rnominata is an ill-defined area within the basal forebrain generally bounded by the nucleus accumbens rostrally, the anterior commissure dorsally, the amygdaloid complex ventrolaterally, the ventral surface of the brain or the olfactory tubercle (TU) ventrally and the septum and preoptic area medialiy.~ It appears as a rather homogeneous area in Cresyl Violet-stained sections, but compartmental-

ization becomes apparent when using immunohisto-

tTo whom correspondence should be addressed. Abbreviations: ACB, nucleus accumbens; ACN, bed nucleus of the anterior commissure; BSA, bovine serum albumin; BST, bed nucleus of the stria terminalis; DAB, 3,3’-diaminobenzidine; ENK, enkephalin; GP, globus pallidus; -IR, immunoreactivity; Leu-ENK, Ileulenkephalin; LV, lateral ventricle; PHA-L, Phaseol& vt&u?&-ieucoa&u-

tinin; r-CRM, anterodorsal caudate nucleus: s&P. subcommissural part of the globus pallidus; SP, sub&nce P, TBS, Tris-btiered saline; TBS-T, Tris-buffered saline with 0.5% Triton X-100; TU, olfactory tubercle; VP, ventral pallidum.

Several components of basal chemistry. 1zi,26-28,46*56 forebrain systems seem to be represented in the substantia innominata (for review see Ref. 1).‘*B*40 As such, it has been proposed that the rostra1 portion of the dorsal substantia innominata is occupied by a rostroventral extension of the pallidal complex, i.e. “the st~atopallidal system”.iJ4,29,M Caudal to the striatopallidal system and ventral to the globus pallidus (GP), i.e. the sublenticular substantia innominata, an area is delineated sharing many characteristics with the central and medial amygdala, i.e. the “extended amygdala”.‘*23,36,40Dispersed within these two systems, islands of cortical projection neurons are found belonging to the “magnocellular corticopetal system”.’ In cats and in primates at least two different areas can be delineated within the striatopallidal system ventral to the anterior commissure on the basis of the differential distribution of enkephalin (ENK) and substance P (SP) immunoreacti~ty (-IR): an area immediately ventral to the anterior commissure dis-

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biotinylated antiserum against PHA-L (biotinylated goat playing high ENK-IR and moderate SP-IR which has anti-PHA-L; Vector Laboratories, Burlingame, [J.&A.: been called the subcommissural part of the globus diluted 1:2000 in TBS-T) at 20°C. pallidus (scGP) and an area ventromedial to the scGP Following three rinses of at least 15 min each in T&S-T, displaying moderate ENK-IR and high SP-IR which the sections were incubated with Vectastaina ABC Kit (Vector Laboratories, Burlingame, U.S.A.; PK-4000 has been called the ventral pallidum (VP).2’~Z5*27*46 dilution I: 8000) in TBS-T in the presence of 0.1% bovine Apart from the differential immunohistochemical characteristics, differences between the scGP and VP serum albumin (BSA) for 90 min at 20°C. Subsequentlv. the sections were rinsed in TBS-T (as described above) ani Tris can also be found in afferent connections, such as buffer (0.05 M Tris, pH 7.6). Following these rinses the from the nucleus accumbens (ACB).2’,25.27”6J7 sections were pre-incubated in 0.05 M Tris buffer (pH 7.6) containing 0.02% 3,3’-dia~no~idine (DAB) tetraFrom a functional point of view our knowledge about the scGP and VP is very limited. Recently we hydrochloride (Sigma) and 0.6% nickel a~onium-sulfate (Fluka; Buch Switzerland) and subseauentlv reacted in the collected evidence that the feline scGP is involved in presence of H,O, (O.OlS%j. The reaction was terminated by funneling striatally induced orofacial dyskinesia, i.e. rinsing several times in TBS. The sections were then a syndrome of involuntary persistent movements of mounted on glass slides and dried. Finally, alternate sections In analogy were directly dehydrated through ethanol and xylene and predominantly the oral region. &10,49,50,cf.34,48 coverslipped with Enteftan (Merck, Darmstadt, F.R.G.), of the striato-nigro-coliicular pathway it has been whereas other sections were first countersink with Cresyl hypothesized that the scGP in its turn funnels the Violet before dehydrating and coverslipping. above-mentioned effects towards lower brain struc[Leulenkephalin immunohistochemistry tures.8~50 The brains of two cats were immunostained for [Leu]In order to study the efferent connections of the enkephalin (Leu-ENK) as described before.*‘~“’ scGP and adjoining areas, we employed the anterograde tracer P~~eolus #uig~~~~-leucoa~iutinin (PHA-L) technique.” In the course of this study an RESULTS extensive ascending projection innervating the striaEnkephalin immunoreactivity turn was noticed. Since this connection, especially in the cat, received little attention up to now, it is The distribution of the ENK-immunoreactive studied in more detail. fibers in the substantia innominata complex and bordering structures, as revealed in the present study, is highly comparable with the results of other EXPERIMENTAL PROCEDURES studies (Figs la-Sa). 3,2i.54 Briefly, the GP is recognized Subjects and surgical procedures in these sections as a structure containing an extremely dense plexus of highly ENK-immunoreactive Male cats (n =9; Animal Laboratory, University of Nijmegen) weighing 3.5-5.2 kg were used. Under deep “woolly fibers”, a term introduced by Haber and anesthesia Ipentobarbital, 40 mg/kg, i.p.; Apharmo, Nauta (Figs 3a-5a). 26 An equally dense plexus Arnhem, The Netherlands) PHA-L (Vector Laboratories, extends rostrally from the GP and ventrally to the B~lingame, U.S.A.; 25 pg/$) dissolved in 50nM Trisanterior commissure, forming the so-called scGP buffered saline (TBS; pH 7.4) was iontophoretically injected into various parts of the basal forebrain. The injections were (Figs la, 2a). made through glass micropipettes (tip diameter, IO-25 pm) Rostrally and medioventrally to the scGP and using a positive pulsed 10-12.5 PA d.c. (7 s on 7 s off) for ventral to the ACB a less dense but still easily 2545 min. recognizable area with moderate ENK-IR can be Fourteen to seventeen days after surgery, the cats were identified, i.e. the so-called VP.2’ The VP and scGP deeply reanesthetized with pentobarbital (60 mg/kg, i.p.) and tran~ardially perfused at a constant hydrostatic pressform part of the so-called striatopallidal system as ure with 500 ml normal saline (20°C pH 7.0), immediately introduced by Alheid and Heimer.’ ENK-IR in the followed by 2000ml of buffered fixative containing 4% form of woolly fibers invades also some parts of the paraformaldehyde, 0.05% glutaraldehyde in 0.1% phosTU. Dorsally, immunoreactive fibers surround the phate buffer, pH 7.4 at 4°C. Following perfusion the animals inferior tip of the lateral ventricle (LV) and extend were tightly packed in ice and were kept in this position for at least 90min after which the brains were removed. Pilot into the bed nucleus of the anterior commissure studies revealed that the latter procedure resulted in a better (ACN) as well as into the bed nucleus of the stria fixation of the brain than in cases in which the brain was terminalis (BST; Figs 3a, 4a).4 More caudally, the removed immediately following the perfusion. moderate ENK fiber plexus extends into the dorsoSubsequently, the brain was postfixed overnight in fixative at 4°C and then stored in a 0.1 M phosphate buffer (PH medial parts of the extended amygdala, i.e. the area 7.4) containing 30% sucrose at 4°C until they sank. Then the ventral to the GP as introduced by Alheid and brains were s&ioned at a thickness of 50 irn on a freezing Heimer, which forms a shell around the GP.’ The microtome. Following sectioning, free-floating sections (two ventral parts of the extended amygdala display no or adjoining sections out of every five or 10) were incubated only very low levels of ENK-IR (Figs la-5a). according to a standard PHA-L immunohistochemi~l procedure described below. Injections of the anterograde tracer PHA-L have been made into the striatopallidal system, the exPhaseolus vulgaris-leucoagglutinin immunohistochemistry tended amygdala and abutting areas in order to Following three rinses in 0.05 M TBS-T pH 7.6 with 0.5% investigate the efferent connections of these strucTriton X-100 (Sigma, St Louis, U.S.A.), the sections were tures. incubated overnight under continuous gentle agitation in a

Fig. 2

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Fig. 5. Figs I-5. The distribution pattern of ENK-immunoreactive fibers in the feline basal forebrain is shown in Figs la-k The different injection sites of the anterograde tracer PHA-L are given in Figs la, b, 2a, b, 3b, 4b and Sb. P, putamen; ic, internal capsule; CN, caudate nucleus; ac, anterior commissure; BST, bed nucleus of the stria terminalis, medial part; BSTm, bed nucleus of the stria terminalis, medial part; BSTl, bed nucleus of the stria terminalis, lateral part; ox, optic chiasm. Anterc )grade tracing experiments

The results of seven injections into the atmvedescril bed regions will be presented: two injections st~atopallidal system into ti le ENK-immunoreactive (one czntromedially and one laterally in the scGP;

Fig. 2b, c), three injections into the extended amygdafa [one dorsolateralfy (a moderate ENK-im munoreactive area), one medially (a low ENK-im muno-

reactive area) and one ventrally (an area with no ENK-IR): respectively, Figs 4b, 3b and 5b]. F‘inally, the results of two injections outside the striator jallidal

Efferent connections of the feline substantia innominata: striatal connections

system and extended amygdala will be presented (one injection ventrolateral to the scGP and one dorsal injection into the ventral putamen). injeclions into the SFriaFo~al~~aIsystem injection into the medioceqtral parl of the subcomrn~~~ral part of the ~~ob~ ~a~~id~. The injection site

of PHA-L in cat 479 is located in the medial and central parts of the dense plexus of high ENKimm~~oreact~ve fibers ~media~ly ventral to the anterior commissure, i.e. the subcommissural part of the GP (scGP; Fig. 2b). The injection track is completely free of labeled cells. The injection itself is large but compact and totally confined to the scGP. Many different types of labeled cell bodies are observed. One frequently observed type of cell is shown in Fig. 1%. Varicose fibers are found around the injection site in the scGP but not in areas ventral or lateral-medial to the SCGP nor in the extended amygdala. A huge bundle of labeled fibers leaves the injection site in a caudal direction innervating lateral hypothalamic, subthalamic and brainstem areas (Spooren ef at., ~publish~ observations). Another bundle of labeled fibers ascends directly into the ACB and/or caudate nucleus. Within the caudal ACB some fibers become varicose whereas others traverse through these areas and become varicose within the rostra1 parts of the ACB (Figs 6a-d lOa, b). These labeled fibers form a diagonal strip within the ACB arising in the far lateral comer just ventral to the anterior commissure and running in a dorsomedial direction towards the inferior tip of the LV (Fig. 6c, d). Characteristically, they almost completely avoid the medial ACB (Figs 6a-d IOa). In continuity with the plexus of tabeled fibers in the ACB labeling is present in the ventral and medial parts of the caudate nucleus. Within the caudate nucleus the fibers form cluster-like patches against a more homogeneous background of labeling. Other parts of the basal forebrain such as the TU, the nucleus of the diagonal band of Broca and BST are not labeled in this case. kijection into the lateral part of the subcomm~s~ra~ part of the globus po~~id~. The injection site of

PHA-L in cat 453 is located laterally in the scGP (Fig. 2a, c). The injection is small and compact and contains diRerent types of IaheIed cells (see also previous case]. In this case, some Iabeled cells are present along the injection track. Varicose fibers are found around the injection site but not ventral and lateral-medial to the scGP nor in the extended amygdala. Like in cat 479, a large bundle of labeled fibers runs in a caudal direction. In the rostra1 direction a bundle enters the ACB directly. The greater part of these fibers become varicose within the far rostra1 areas of this nucleus especially along its lateral parts (Fig. 7a, b). The central and medial parts of the ACB are virtually free of PHA-L-immunoreactive fibers.

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Some smooth non-varicose fibers are seen ascending through the ACB and only become varicose after entering the caudate nucleus, inne~a~ng especially its lateral parts. This lateral zone is labeled all the way up to its most dorsolateral tip. Again, the labeled area within the caudate nucleus is contiguous with the area labeled in the ACB, forming one network of PHA-Limmunoreactive fibers (Fig. 7a, b). A few fibers are also found in the central parts of the caudate nucleus. Although single fibers can be observed within the TU as well as the diagonal band of Broca, the BST and the ACN are not labeled in this case. Injections into the extended amygdala ~or~o~ateral j~jection. The injection of PHA-L in cat 347 is located immediately ventrolateral to the rostra1 parts of the GP (Fig. 4b). This area contains moderate ENK-IR (Fig. 4a). Labeled fibers ate found in the caudal regions of the ACB but even more so in the BST. In the latter nucleus, labeling is present in particular in the lateral division, but some fibers are also present in the medial division. Many fibers are also seen in the ACN (Fig. 8a-c). Only a few single fibers are observed within the rostra1 parts of the ACB. Hardly any fibers are found in the caudate nucleus. Many varicose fibers are found around the injection site as well as more rostrally in the low-moderate INK-immunor~ctive regions lateral to the scGP (Fig. 8a, b). However, the scGP is virtually free of varicose fibers. Furthermore, many fibers are caning in a region ventrally to the s~atopalIida1 system (scGP and VP) and through the nucleus of the diagonal band of Broca and TU. These fibers occasionally show vat&o&ties. iwe& injection. The injection of PHA-L in cat 405 is located ventromedially to the most rostra1 parts of the GP and lateral to the VP in an area with relatively low ENK-IR (Fig. 3a, b). Some fibers are running directly from the injection site towards the ACB where they become varicose within the dorsolateral and ventrolaterai parts (Fig. 9a-c). However, the number of fibers found within the ACB is relatively small compared to the more (rostro)dorsal injections. No fibers are observed within the caudate nucleus but in contrast to all dorsal injections some fibers are found in the TU as well as in the diagonal band of Broca. Like in the previous case, many varicose fibers are found in the BST (the fateral division but not the medial division) and the ACN. ventral injection. The injection of PHA-L in cat 316 is located in the ventral parts of the extended amygdala (Fig. Sb). This area contains no ENK-IR (Fig. Sa). This case is our most caudal injection site and varicose fibers are observed in the TU and the nucleus of the diagonal band of Broca. Varicose fibers are also found in the BST ~a~ic~arly in the medial division but also in the lateral division) and the ACN. The caudate nucleus and the ACB are free of labeled fibers. Finally, varicose fibers are found

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7

6a

I

I

Fig. 5. The distribution pattern of F~A-L-i~rnnnorea~t~~e fibers within the AC3 and caudate nucleus after an injection into the medial scGP (cat 479). F or injection site see Fig. 2b. CN, caudate nucleus: CC, corpus calfosum; ic, internal caps&e; lo, lateraf olfactory tract; P, putamen.

Efferent connections of the feline substantia innominata: striatal connections

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b 2mm

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Fig. 7. The distribution pattern of PHA-L-immunoreactive fibers within the ACB and caudate nucleus after an injection into the lateral s&P (cat 453). For injection site see Fig. 2c. ic, internal capsule; CN,

caudate nucleus; lo, lateral olfactory tract.

rostrally in the non-ENK-immunoreactive regions ventral (and lateral) to the striatopallidal system (VP and scGP). Injections outside the striatopallidal system and extended amygdala Rostr~entra~ injection. The injection site in cat 3 is located in the non-ENK-immunoreactive regions ventrolateral to the scGP and also encompasses dorsolateral parts of the TU (Fig. lc). Many labeled varicose fibers are observed around the injection site, but also caudally in the ventral extended amygdala. In this case scarse fibers are found within the ACB. Very high densities of varicose fibers are found within the TU. Clusters of varicose fibers are also observed in and around the insulae of Calieja. Furthermore, many fibers are found in the diagonal band of Broca (the horizontal part). Within the nucleus of the anterior commissure single fibers are found, whereas the BST is free of fibers. In cats 473 and 2, not described in the present report, the injection sites are smaller and more confined to the ventral regions. In these cases many fibers are found in the TU but not in the ACB and/or caudate nucleus. Rostrodorsal injection. The injection site in cat 492 is located ventrally in the putamen, immediately lateral to the internal capsula and also encompasses

the utmost dorsal regions of the scGP (Fig. lb). Although some PHA-L is found along the injection track, it is free of labeled cell bodies. Varicose fibers are found in the ACB dorsolateral and ventromedial to the anterior commissure whereas other fibers penetrate directly the capsula intema and innervate the extreme lateral parts of the ventral caudate nucleus. Many varicose fibers are also found in the sub- and retrocommissural part of the GP.

DISCUSSION

Enkephalin immunoreactivity The distribution pattern of the ENK-immunoreactive fibers within the basal forebrain, as revealed in the present study, is in good agreement with previous reports dealing with this issue.3g2’q” The peptidergic fibers have a ribbon-like appearance and are named woolly fibersz6 It has been postulated that they represent striatal axons ensheathing dendrites of cell~.‘**~~Furthermore, they form the basic elements of the dense immunoreactive plexus as present in the GP (Figs 3a, 4a, 5a).“*26~2’~” In view of these and comparable findings, it has been postulated that among other characteristics, ENK-IR might be an appropriate marker to label presumptive

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Fig. 8. The distribution pattern of PHA-L-immunoreactive fibers within the caudate ~&us, AC3 and abut areas following a~ injection into the dorsal “extended amygdala” (cat 347). For injection site see Fig. 4b. ic, internal capsule; CN, caudate nucleus; P, putamen; cc, corpus callosum; ST, stria terminalis.

I\

Efferent connections of the feline substantia innominata: striatal connections

n

Fig. 9. The distribution pattern of PHA-L-immunoreactive fibers within the caudate nucleus and ACE following an injection into the medial “extended amygdala” (cat 405). For injection site see Fig. 3b. CN, caudate nucleus; cc, corpus callosum; lo, lateral olfactory tract; ic, internal capsule; P, putamen.

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Efferent connections of the feline substantia innominata: striatal connections p&i&f territory (peripaifidal territory).““‘*& In line with this hypotheses the dense and moderate plexuses present within the dorsal subcommissural substantia innominata of the cat have been referred to as the scGP and the VP, respectively (Fig. 2a).*’ Both areas (scGP and VP) form a part of the striatopallidal system as introduced by Alheid and Heimer.’ Caudal (and ventral) to the striatopallidal system an area is delineated that, as found in the rat, shares many characteristics with the central and medial amygdala and as such is called the extended amygdala.’ This area contains only dorsally some low-moderate ENK-IR whereas ventrally it contains no ENK-ER. Innervation of the caudate nucleus and the nucleus accumbens

The present anterograde tracing study clearly shows that the strong ENK-immunoreactive scGP innervates the ACB and the caudate nucleus. Strikingly, the caudate nucleus seems to be almost exclusively innervated by the scGP and not by areas ventral and lateral to the s&P nor by the extended amygdala. In contrast, the ACB not only receives input from the scGP but also from the dorsal ENKimmunorea~tive extended amygdala. As such the ENK-immunor~ctive dorsal extended amygdaia shares some characteristics with the striatopallidal system. However, it is important to stress that the densest networks of PEA-L-immunoreactive fibers in the ACB were found in cases 453 and 479 in which the injection sites were centered in the scGP proper. In a previous anterograde tracing study of the feline substantia innominata complex the peripaltidal-striatal pathway, as outlined in the present study, has not been described.58 These apparently contradictory results can be explained by the fact that the latter study encompassed no dorsal injections whereas the pe~pallidal-st~atal pathway originates, as outlined above, exclusively from the ENK-immunoreactive scGP and dorsal ENK-immunoreactive extended amygdala. Using the retrograde tracer horseradish peroxidase, Jayaraman clearly showed that the feline caudate nucleus is topographically innervated by peripallidal and pallidal cells.3o Others have shown, using the fluorescent retrograde tracer Nuclear Yellow, that the head of the feline caudate nucleus is innervated by cells positioned within the dorsal substantia inno~~ta complex.‘“i6 Beckstead noticed in his study on the pallidostriatal pathway some HRPpositive neurons within the ventral pallidal territory after injections into the ACB of the cat.2 Finally, in the rat it has been shown that the ventral striatum is innervated by neurons located in the VP (Groenewegen, unpublished observations).22 Taken together, our present findings confirm and extend the a~ve-mentioned retrograde and anterograde tracing studies.

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The present results indicate that the non-ENKimmunoreactive region ventrolateral to the SCGP preferenti~~y innervates the TU rather than the ACB. The ventral parts of the extended amygdala also project to the TU, At least in the rat and the hamster it is known that the rostroventral regions of the substantia innominata are mainly innervated by the TU whereas the dorsal regions are mainly innervated by the ACB.28*3g~64 Although similar data are not available for the cat it can be postulated that the efferent connections of these parts of the basal forebrain reciprocate their input. Clear differences are found in the innervation of the medial and lateral parts of the BST by the dorsal and ventral parts of the extended amygdala. The dorsal regions clearly innervate the lateral division whereas the ventral regions clearly innervate the medial division of the BST. These results are in agreement with findings in the rat and indeed nicely correspond with the input into these divisions by, respectively, the central and medial amygdaloid nuclei as discussed by Grove.23*36,40 Finally, our data suggest that the SCGP proper sends no fibers into the BST. These findings nicely correspond with the pamelation of the substantia innominata introduced by Alheid and Heimer, since the scGP forms part of the striatopallidal system and not of the extended amygdala region.’ Topographic organization of the peripallidal-slriatal pathway

When comparing the results of cats 479 and 453 it is evident that the innervation of the ACB and caudate nucleus by the scGP is topographically organized along a mediolateral dimension. As far as the innervation of the AC3 is concerned, there is also evidence in favor of a topographic organi~tion in the rostrocaudal dimension (cf. cats 479, 405 and 347). The topographic innervation also extends caudally into the ACN and BST. A topographic organization of the innervation of the caudate nucleus along the rostrocaudal dimension is not clear. It is evident that the lateral injection (cat 453) into the scGP resulted in varicose fibers in the extreme rostra1 parts of the caudate nucleus and the ACB. Following the medial injection (cat 479) the fibers were found more caudally in the caudate nucleus and ACB. However, it is unclear to what extent this difference is also related to a more rostral, respectively, more caudal position of the injections into the scGP. It has been shown clearly that the ENK-immunoreactive external segment of the GP innervates the body of the caudate nucleus topographically whereas the SP-immunoreactive entopeduncular nucleus (internal pallidal segment) has no such connection.2*5’*5”@When considering the ENK-immunoreactive s&P as the most rostra1 part of the GP it is

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understandable that the scGP innervates the far rostra1 part of the caudate nucleus. This would suggest that the scGP forms an integral part of the external segment of the GP since the scGP not only resembles the GP in certain immunohistochemical characteristics, but also with respect to its (topographically organized) output to the striaturn. ‘,3,2’.5’,52,54~56,63 Finally, the present results indicate that the media1 part of the ACB is not innervated by the scGP nor by any other part of the basal forebrain investigated in the present study. In line with the above-mentioned mediolateral topography as well as in view of reciprocal connections (see below), it can be postulated that these medial regions will most probably be innervated by more medial regions of the striatopallidal system, i.e. the feline VP.c’2’.46However, this remains to be verified, since we have not placed any injection in this area. Reciprocit?

conclusions can be drawn on the basis of these results. Firstly, the st~atopa~idal system as well as the extended amygdala have strong intrinsic connections and secondly both systems are not interconnected. Comparison with other accumbens-caudate

aferents

The target neurons of the PHA-L-immunoreactive fibers within the ACB and caudate nucleus, labeled after injections into the scGP, are unknown, However, the distribution of fibers from the scGP, in particular to the central part of the ACB continues into the medial caudate nucleus (cat 479), is very comparable to the dist~bution of fibers from the parahippocampal area (perirhinal area), the amygdala (basolateral part) and the (pre- and infra-) limbic cortex.‘8.20.“3~42~43~62 This suggests a convergence of inputs from limbic-related structures (see above) and pallidal-related structures within the ACB-caudate nucleus. As such, this connectivity pattern suggests a strong limbic-related role for the peripallidal-striatal pathway (see below). Furthermore, the difference in the distribution of anterogradely transported PHA-L within the ACB following injections into the Iateral and medial scGP may reflect differences in the connectivity patterns of the medial and lateral scGP. It has been observed in the rat that the caudomedial part of the ACB is especially innervated by the paraventricular thalamic nucleus, ventral subiculum and caudal basolateral amygdaloid nucleus, whereas the rostrolateral part of the ACB is innervated by the dorsal subiculum, the rostra1 basolateral amygdaloid nucleus and centromedial thalamic nuclei.‘” Although similar findings in the cat are not yet available, it can be suggested that subtle differences might also exist in the afferent input from the scGP. However, it is evident that more data are necessary on this point.

It has been shown that the scGP is especially innervated by the central and lateral part of the ACB.*’ Taken together with the present findings, our data suggest that the latter area may be reciprocally connected with the scGP. However, whether the efferents of the scGP exactly innervate the area or the neurons of origin remains to be verified. In the case of the caudate nucleus reciprocity is less clear since the caudate afferents of the scGP have not been clearly defined yet. However, our data taken together with the findings reported in the literature suggest that at least part of the caudate area from where the scGP input originates is in turn innervated by the scGP, especially the medial parts of the far rostra1 caudate nuc1eus.9~Z’.45~s9 This kind of organization would agree with the general finding in the rat that areas innervating the substantia innominata tend to be reciprocally connected with it.ZZ.23However, additional evidence is also necessary on this point. The more so, since no reciprocal connections have been It is important to indicate that the peripalliobserved in the present study between the anterodorsal part of the caudate nucleus and the SCGP.~~” dal-striatal pathway is one of several afferent systems of the striatum and that the functional implications Intrinsic connections may be manifold. However, some interesting suggestions can be made. Remarkable for all cases is the presence of varicose Firstly, the anterodorsal caudate nucleus, i.e. a fibers around the injection site. These results confirm subregion of the caudate nucleus which is delineated earlier findings in the rat. 23The intrinsic relationships, on the basis of its susceptibility to the dopaminergic however, do not only involve the areas in the immedidrugs DPI (~3,~ihydrox~henyl~no)-2-imid~ole) ate vicinity. The data suggest that rostra1 and caudal and ergometrine (r-CRM), is known to innervate the regions are interconn~ted. Furthermore, this inter~xGP.~,‘~ In the present experiments we have not connection seems to be organized in a somewhat obtained any evidence that the r-CRM receives an layered manner: rostroventral to cauduventral (both input from the scGP, which implies a lack of direct non-ENK immunoreactive) and rostrodorsai to caufunctional feedback. Although an indirect pathway dodorsal connections (both ENK immunoreactive; this finding can be of significannot be excluded, 15,37 cf. cat 347 with cat 316). However, injections into the cance for orofacial dyskinesia, i.e. a syndrome selecscGP resulted in the presence of varicose fibers tively elicited from the r-CRM.9,‘0,50Neural systems around the injection site only but not in the extended which receive no or only an indirect functional feedamygdala nor in areas ventrolateral or medial to the scGP. Furthermore, no varicose fibers are found in back might be less stable and are therefore more vulnerable for drugs. As such, a reduced functional the scGP following injections outside the scGP. TWO

Efferent connections of the feline substantia innominata: striatal connections

feedback to sites involved in oral motor control, such as the r-CRM, might provide a neuroanatomical explanation for the L-DOPA and neuroleptic-induced dyskinesias.8*Y~q Secondly, the ACB itself as well as certain of its inputs such as the hippocampus and the dopaminergic afferents, have been implicated in the pathology of schizophrenia.s*6,‘3,3’*35,53,5~ Although especially the latter input is thought to play a crucial role in the pathophysiology of schizophrenia, it can be hypothesized that certain symptoms of schizophrenia are related to a reduced functional feedback. Since pathological processes at this level will only indirectly involve dopaminergic processes, these patients should be difficult to treat with neuroleptic drugs, i.e. dopamine antagonists. Interestingly, a subgroup of schizophrenic patients with predominantly negative symptoms are indeed known to be poor responders to neuroleptic drugs.@ Furthermore, they have been found to have higher cognitive deficits compared to other schizophrenics and they are predisposed to the development of orofacial dyskinesia.32@ The scGP is implicated to play a crucial role in both syndromes: orofacial dyskinesia and cognitive deficits.9*61Therefore, it can be suggested that the presently outlined peripallidal-striatal pathway may be part of the

445

neuroanatomical substrate that links the abovementioned syndromes. Thirdly, in the case where the peripallidal-striatal pathway originates from the islands of cells belonging to the corticopetal magnocellular system, which are embedded into the striatopallidal system and extended amygdala, this pathway is likely to be involved in mnemonic processes. Recently, the ACB has been found to be involved in memory processes, especially processes in which retrieval of external cues is involved.4’,47 Although especially the hippocampus is implicated in these functions, the pallidal-striatal pathway may contribute in these processes. The more so since the scGP has been found to project to the cingulate cortex which in turn is strongly interconnected with the hippocampu~.‘~~~~~~Future combined neuroanatomical and neuropharmacological research is required to investigate the mentioned hypotheses on the functional role of the peripallidal-striatal pathway.

Acknowledgemen&--Drs Tom Roeling, Hans Peters, Theo Hafmans and Henk Joosten are acknowledged for assistance during the initial phase of the experiments. Han Luikens is thanked for his excellent photographical support and Cees Nicolasen for making the illustrations.

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