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The topographic order of inputs to nucleus accumbens in the rat
~euroseieffc@ Vol. 16,No. 2, pp. 275-296, 1985 Printed in Great Britain
0304-4522~85%3.00+ 0.00
Pergamon Press Ltd Q 1985IBRO
THE TOPOGRAPHIC ORDER OF INPUTS TO NUCLEUS ACCUMBENS IN THE RAT 0. T. PHILLIPSON and A. C. GRIFFITHS Department of Anatomy, The Medical School, University Walk, Bristol BS8 IT’D, U.K. A~~act-A~erents to the nucleus accumbens have been studied with the retrograde transport of unconjugat~ wheatgerm agglutinin as detected by immunohist~hemistry using the peroxidaseantiperoxidase method, in order to define precisely afferent topography from the cortex, thalamus, midbrain and amygdala. Cortical afferent topography was extremely precise. The largest number of cells was found following injections to the anterior accumbens. Anteromedial injections labelled a very large extent of the subiculum and part of the entorhinal cortex. Anterolaterai injections produced less subicular and entorhinal label but also labelled the posterior perirhinal cortex. Posteromedial injections labelled only the ventral subiculum and a few cells in the adjacent medial entorhinal cortex. Posterolateral injections labelled few lateral entorhinal neurones but did label a long anteroposterior strip of perirhinal cortex. Prefrontal cortex label was found only after anterior accumbens injections. In the amygdala labelled neurones were found in cortical, central, lateral posterior, anteromedial and basolateral nuclei. Basolateral amygdala projected chiefly to the anteromedial accumbens and central nucleus to anterolateral accumbens. Only a weak amygdala label was found after posterior accumbens injections. In the ventral tegmental area, the midline interfascicular nucleus projected only to medial accumbens. The paranigral ventral tegmentum projected chiefly to the medial accumbens and the parabrachial area chiefly to the lateral aecumbens. In the thalamus, heaviest label was found after anterior accumbens injections. Most cells were found in the paraventricular, reuniens and rhomboid nuclei and at posterior thalamic levels lying medial to the fasciculus retroflexus. There was only restricted topography found from thalamic sites. Retrograde label was also found in the ventral pallidum and lateral hypothalamus. Single small injection sites within accumbens received input from the whole anteroposterior extent of the thalamus and ventral tegmentum. The medial accumbens was found to have a close relationship to habenula, globus pallidus and interfascicular nucleus. It appeared that the heaviest volume of inputs projected to anterom~ial accumbens, where output from hippocampus (CAB, subi~ulum, entorhinal and prefrontal cortices converged with output from amygdala, midline thalamus and ventral tegmentum.
The nucleus accumbens appears to occupy an interface position between the limbic and extrapyramidal motor system. An analysis of its structure, histochemistry and connections has given it a central position in the emerging concept of the ventral striatum.‘4.4’ Afferents to accumbens arise from a wide variety of structures including the thalamus.5,6,7,8.9.24.28.36.40,42 In the course of neurochemical experiments on the influence of thalamic input on dopamine utilization in the caudate-putamen and prefrontal cortex, we have also found changes in the nucleus accumbens.t8.‘9.22.23This raised the question of the full extent of direct thalamic input to accumbens from regions the near mediodorsal and parafascicular-intralaminar nuclei. The existing evidence on thalamic projection to the accumbens using modern techniques shows that the paraventricular thalamus provides input, but there is little information on its full extent or topography in rat,39 although some data has been obtained in the cat and hamster.‘2.29 The present experiments were thus carried out to map more fully the topography of thalamic input to accumbens in the rat using a retrograde CAl, hippocampal subfield CAI; IFN, interfascicular nucleus; VTA, ventral tegmental area; WGA, wheatgerm agglutinin.
Abbreviations:
method with very small injections and a sensitive The immunohistochemical detection technique. topography of inputs to accumbens from cortex, amygdala and midbrain were also assessed.
EXPERIMENTAL PROCEDURES Twenty-one male and female rats of Wistar-derived strain weighing between 180 and 2 IO g were anaesthetized with chloral hydrate (4~mg/kg i.p.) and given injections of unconjugated wheatgerm agglutinin (WGA) to the nucleus accumbens or neighbouring structures by stereotaxic guidance. Wheatgerm agglutinum (Miles-Yeda Ltd, 2.82 pg/pl in 0.05 M Tris-HCl, 0.15 M NaCl buffer pH 8.6) was injected with intermittent ptis of air pressure (about 0.3 kg/cm2) applied, via a reduction valve and metered push-button quick-venting valve mechanism, to a GC2OOF-10 glass micropipette (Clarke Electromedical Instruments) broken to a tip diameter of 20-40pm, filled with WGA and a small quantity of oil. The injected volume was monitored under microscopic control with the aid of a micrometer scale placed against the upper oil meniscus in the pipette. Under these conditions as little as 50 nl could be reliably ejected. In those fully described cases reported in this paper, where regional microinjections to subsectors of nucleus accumbens were achieved, the volumes injected varied between 90 and 280 nl given slowly over several minutes. The pipette was left in place for a further IOmin at the end of the injection. Following recovery from anaesthesia, animals were allowed to survive for 1423 h and then reanaesthetized, 275
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perfused through the ascending aorta with 500ml normal saline, followed by about 500 ml Bouin’s fixative containing 4% paraformaldehyde. Brains were removed and postfixed overnight in the same fixative with 10% sucrose added.
Immunohistochemistry The method for detecting the retrograde transport of lectin is fully described elsewhere.33 Briefly, frozen 40-pm sections were collected into 0.05 M Tris-HCl, pH 7.8, and washed in several changes of this buffer. Floating sections were incubated at room temperature overnight in a l/2000 dilution of rabbit anti-WGA antiserum in 0.05 M Tris-HCI buffer, pH 7.8, containing 0.7% carrageenan and 0.5% Triton X-100. After washing, sections were incubated in the second antibody (sheep anti-rabbit immunoglobulin diluted 1: 15in the Tris/carrageenan/Triton solution) followed by a standard peroxidase-antiperoxidase procedure using freshly prepared 0.05% 3, 3’-diaminobenzidine and H,Oz at a final concentration of 0.03’?. After further washing, sections were mounted on gelatin-coated slides, counterstained with light green, dehydrated and coverslipped.
RESULTS
In the initial stages of this study experiments were carried out with relatively large volumes of tracer injected to nucleus accumbens using WGAhorseradish peroxidase conjugate with tetramethylbenzidine histochemistry. Although useful for summarizing the principal sites of origin of accumbens afferents, the present paper will confine its attention to those brains in which very small deposits of unconjugated WGA were injected since, judging by the injection site, the local spread of unconjugated lectin was considerably less than the horseradish peroxidase conjugate. Furthermore, the precise topography of afferents to accumbens could be more clearly defined with the use of the unconjugated WGA, where it is clear that uptake and transport is confined principally to the area of stained tissue (see discussion of control injections). The main features of afferent topography will be illustrated by four cases in which small deposits of WGA were placed in the anteromedial, the anterolateral, the caudomedial and caudoventrolateral accumbens. Injection sites Anteromedial accumbens (Case 17) (Fig. la). The injected WGA labelled the most rostra1 and medial tip of accumbens but also extended medially to label the taenia tecta. Label in the needle track was seen to stain lightly the deep layers of prefrontal cortex dorsal to the injection site (see results of control injections). Anterolateral accumbens (Case 14) (Fig. lb). In this case the injected WGA surrounded the anterior limb of anterior commissure in the ventroanterolateral accumbens. The centre of the injected WGA lay just ventral and medial to anterior commissure, 1.1 mm lateral to and slightly rostra1 to the centre of injected WGA in Case 17. A narrow tail of label extended dorsally in the pipette track into the ventral part of the head of the caudate-putamen.
A. C.
GRIFFITHS
Caudomedial accumbens (Case 13) (Fig. lc). The centre of this injection lay medial to the ventral tip of the anterior horn of lateral ventricle where it divides the medial caudatoputamen from the lateral septum. The needle track passed through the lateral ventricle and it is probable that some injected WGA leaked into the cerebrospinal fluid since the ependymal lining of the ventricle here was heavily stained. Very light staining was also found in the pipette track as it descended through the cingulate cortex. Caudoventrolateral accumbens (Case 15) (Fig. 1d). Here the centre of the injection site was located ventrolateral to the anterior limb of the anterior commissure, about 700 pm caudal to the centre of Case 14 and about 1 mm lateral and ventral to the centre of Case 13. Light staining was seen in the needle track as it penetrated caudatoputamen. It is of interest to note that this track staining was not uniform but showed patchy extension slightly rostra1 and caudal as well as medial and lateral. The examination of this staining at a high power showed this to be located both in striatal cell bodies and axon terminals in a patch-like arrangement. Midbrain
label (Fig.
2)
Case 17 (anteromedial accumbens). Retrograde label was found in the ventral tegmental area (VTA) throughout its entire anteroposterior extent. Most neurones were found in the paranigral division of VTA and some also in the midline interfascicular nucleus (IFN). In caudal sections cells extended dorsally into nucleus linearis caudalis, whilst rostrally many neurones were also labelled in the supramammillary area. In the posterior half of the VTA a few neurones were found dorsally in VTA in the parabrachial region of VTA. Case 14 (anterolateral accumbens). As in Case 17 the whole anteroposterior extent of VTA was labelled. In contrast to Case 17, however, labelled neurones tended to occupy slightly more lateral and dorsal positions in VTA. Thus more neurones were labelled in the parabrachial and in more lateral divisions of paranigral VTA. Furthermore, unlike Case 17, apart from an occasional neurone, no label was seen in the midline IFN. A few labelled neurones were found in the substantia nigra. Occasionally, neurones were found to be labelled in the contralateral VTA. Case 13 (caudomedial accumbens). As before the whole anteroposterior extent of VTA was labelled. In this case, however, the distribution of midbrain label was different from those cases already described. Here heavy label was found throughout the whole extent of the IFN. Label in the rest of VTA was strictly confined to the paranigral VTA (Fig. 8a). Also, unlike the previous case, substantial numbers of cells were labelled in the medial substantia nigra and small numbers were found labelled also in midsubstantia nigra. Occasionally neurones were found in the contralateral VTA.
Abbreviations used in fisures AHi al am BL BL, CA1 Ce CM ENT fr IFN IP PBP PC PF
amygdalohippocampal transition zone anterolateral accumbens anteromedial accumbens basolateral nucleus of amygdala ventral basolateral nucleus of amygdala CA I subfield of hippocampus central nucleus of amygdala central medial nucleus of thalamus entorhinal cortex fasciculus retroflexus interfascicular nucleus inte~duncuIar nucleus nucleus parabrachialis pigmentosus of VTA paracentral nucleus of thalamus parafascicular nucleus of thalamus
PFC Pl ;: PRC PT PV PV, PV” RE RH SUB SUB, VTA
prefrontal cortex posterolateral accumbens posteromedial accumbens nucleus parani~alis of VTA perirhinal cortex parataenial nucleus of thalamus paraventricular nucleus of thalamus paraventricular nucleus of thalamus (dorsal) paraventricular nucleus of thalamus (ventral) nucleus reuniens of thalamus rhomboid nucleus of thalamus subiculum subiculum (ventral) ventral tegmental area
Fig. I. Injection sites of the cases fully reported in this paper as demonstrated in thionin stained sections through the injection centres. (a) Anteromedial accumbens. x 21. (b) Anterolateral accumbens. x 22. (c) Posteromedial accumbens. x 23. (d) Posterolateral accumbens. x 18. Fig. 8. (a) Retrograde cell labelling in VTA, showing nucleus paranigralis of VTA (PN) and IFN subregions dorsal to the interpeduncular nucleus (IP) x 135. (b) Retrograde cell labelling in the caudal thalamus medial to fasciculus retroflexus (fr) x 145. (c) Cell labelling in ventral paraventricular thalamus. x 300. p. 279. Fig. 9. (a) Large numbers of CAI pyramids labelled following injection of anteromedial accumbens. x 60. (b) Part of the CA1 field shown in (a) at higher power indicating the lightly labelled members of this population. Thus a very large total of labelled cells could be found in CAI. x 200. (c) Retrograde label in central nucleus of amygdala. x 290. (d) Axon labelling in dorsomedial sector of medial habenular nucleus. x 230. p. 28. Fig. 10. (a) Retrograde cell label in medial bank of frontal cortex (prefrontal cortex). Dorsal is at top and midline at left of the micrograph. x 200. (b) Retrograde cell label in nucleus reuniens of thalamus, x 250. (c) Profuse axonal label from WGA injection to posteromedial accumbens in giobus pallidus (position shown also in Fig. 5.) x95. p. 281.
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Case 15 (caudoventrolateral accumbens). Here again the pattern of midbrain label is quite distinct from that described so far for the other three cases. Firstly, there was no label in the IFN. Secondly, the label occupied cells in further lateral and dorsal positions in VTA than in Case 13, i.e. much larger numbers of neurones were found in the parabrachial VTA and very few in the paranigral VTA. The medial substantia nigra was quite markedly labelled throughout its entire anteroposterior length (probably because of WGA in the overlying pipette track) and occasional cells were labelled in the retrorubral (A8) position. Cells were occasionally found in the contralateral VTA. In all cases it was clear that a single small deposit of WGA to the accumbens of diameter about 400 pm labelled a narrow mediolateral extent of VTA only slightly broader than this; while by contrast it labelled the entire anteroposterior extent (about 2000 pm) of the VTA, interfascicular nucleus and nucleus linearis caudalis. Thalamic label (Fig. 3) A notable feature of many cases studied was that a single small injection of WGA labelled the whole anteroposterior extent of the thalamic nuclei described. Thus a length of 3-mm thalamus could be labelled from a deposit of WGA 2400pm in diameter. Case 17 (anteromedial accumbens). The most rostral thalamic label found in this case lay in cells medial to the post-commissural fornix at the point where the base of the posterior septum, the dorsal rostra1 tip of the third and lateral ventricles lie adjacent to one another (Fig. 3a). The label extended caudal from this site to large numbers of cells in the paraventricular thalamus. In its rostra1 portion (i.e. rostra1 to the mediodorsal thalamus) the label extended continuously from the lateral ventricle to the dorsal margin of the third ventricle in nearly all sections. In dorsal positions a negative image of the whole extent of the parataenial nucleus was formed by the surrounding retrograde label. Thus cells were found extending lateral from the paraventricular thalamus to the ventrolateral margin of stria medullaris and this label was continuous with a band of cells found in the rostra1 paracentral thalamus. In ventral positions thalamic label extended in the midline between the anteromedial nuclei in a ventral extension of the paraventricular label to become almost continuous with retrograde label in nucleus reuniens (Fig. 10). A few cells were found in anterodorsal nucleus where they appeared to form a continuous lateral extension of the label found in the paracentral nucleus. At thalamic levels caudal to the parataenial nucleus, the continuous midline dorsoventral strip of label was broken so that the paraventricular and reuniens label was separated by the intervening unlabelled anteromedial and mediodorsal nuclei. Where
the rostra1 tip of mediodorsal and caudal pole of parataenial nuclei overlap, heavy label was found in the “mediodorsal” nucleus in positions suggesting that this label was in fact part of a caudal extension of the lateral paraventricular label already described. Furthermore, and in support of this view, it was continuous with midline paraventricular and paracentral label, as already described at more rostra1 sites. Caudal to this position thalamic label was continuously seen throughout the length of the paraventricular thalamus dorsally and nucleus reuniens ventrally. Smaller numbers of labelled cells were found in some sections in the rhomboid nucleus and a very few in central median and paracentral nuclei. Occasional cells were found in nucleus gelatinosus (gemini). At caudal levels of habenula and at levels posterior to the habenula where fasciculus retroflexus descended through the posterior medial thalamus there was a sharp increase again in the numbers of cells labelled. Here label in the posterior paraventricular nucleus extended laterally under the ventral margin of lateral habenula and the fasciculus retroflexus as it formed at the ventrolateral base of habenula. More scattered label extended ventral in the midline into a poorly defined region of the posterior medial thalamus (central medial) and in positions dorsal to third ventricle, which probably represented a caudal extension of nucleus reuniens, or alternatively nucleus subparafascicularis. Large numbers of neurones were labelled medial to the parafascicular nucleus adjacent to the third ventricle. In summary, the vast majority of label found in this brain lay in the paraventricular nucleus (both stellatocellularis and rotundocellularis divisions were labelled) and in nucleus reuniens throughout their whole extent in the anteroposterior plane. Extentions from this pattern were found into rostra1 mediodorsal and, in small numbers of cells only, into paracentral, anterodorsal, central medial and centrolateral nuclei dorsally and rhomboid, gemini and possibly subparafascicularis ventrally. Case 14 (anterolateral accumbens). The main features of thalamic label found in this brain were similar to that described for Case 17 in that (1) the entire anteroposterior length of thalamus was labelled and (2) the principal nuclei labelled were the paraventricular and reuniens nuclei. The differences between these two brains were as follows. The paraventricular label in the posterior part of this nucleus extended further ventral than in Case 17. The reuniens label in the two cases differed in that heaviest label was found rostrally in Case 17, whilst only light reuniens label was found rostrally in Case 14 and larger numbers of labelled cells were found in mid-reuniens. Furthermore, in Case 14 marked label was found in central medial nucleus (almost unlabelled in Case 17) which extended laterally into paracentral nucleus. A few cells were also labelled in
Afferents to nucleus accumbens dorsal parts of the centrolateral nucleus. In many caudal thalamic sections in this brain continuous label extended from the lateral ventricle in the paraventricular nucleus through to central medial, rhomboid and reuniens nuclei to the third ventricle. This contrasts with Case 17 where rather sparse thalamic label was found at mid to caudal sites. Finally, at the level of the fasciculus retroflexus (Figs 3a and 8b) label in Case 14 lay further lateral than in Case 17; and in some cases neurones were found in parafascicular nucleus itself. Case 13 @osteromedial accumbens). Unlike the two brains so far described, this brain showed thalamic label which was entirely confined to paraventricular label. A few neurones were also labelled in dorsal central lateral nucleus adjacent to the lateral margin of lateral habenula. No label was found in ventral paraventricular nucleus, central medial, paracentral, rhomboid or reuniens nuclei. Case 15 (posterolateral accumbens). The pattern of label in this brain resembled that in Case 14 and 17 in that the greater part of the anteroposterior extent of paraventricular nucleus was labelled. Rostra1 thalamic sections however were unlabelled. In contrast to Cases 13, 14 and 17 paraventricular label was heaviest in its ventral regions, while its dorsal regions were for the most part unlabelled. In some sections label was continuous with labelled neurones found in the central medial and paracentral nuclei. Occasionally labelled neurones were also found in dorsal parts of central lateral nucleus. In posterior thalamic sections the pattern of paraventricular label was similar to that seen in Case 14, although in addition labelled neurones were seen in larger numbers in parafascicular nucleus lateral to the lateral rim of the fasciculus retroflexus. Furthermore, in contrast to Cases 14 and 17 few neurones were labelled in nucleus reuniens and the rhomboid nucleus. The topography of the input to accumbens and its relation to cytoarchitecture is summarized in Fig. 6. Topography is most clearly defined in the posterior thalamus. Cortical label (Fig. 4) Case 17 (anteromedial accumbens). The hippocampal formation and associated cortical areas were extensively labelled in this brain. The most rostra1 extent of this label was found in the ventral CA1 region adjacent to the amygdalohippocampal area, where further retrograde label was found. The CA1 label extended continuously in more posterior sections to include cells in the ventral subiculum and, still further posterior, also in dorsal subiculum, although here the intervening CA1 was unlabelled. At the most posterior cortical levels studied, dorsal subiculum, CA1 and ventral subiculum were continuously and heavily labelled (Fig. 9a and b). Many labelled neurones were also found in the deep layers of entorhinal cortex. The rostra1 margin of this label was continuous with retrograde label
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found in the posterior pole of the basolateral amygdala. Fewer cells were labelled in entorhinal cortex as sections lay further posterior. A few lightly labelled cells were found in the medial edge of entorhinal cortex where it joins subiculum. Scattered label was also found in the deep layers of prefrontal cortex (Fig. 10a). The principal cortical projections, as judged both by numbers of labelled cells and the intensity of staining, was that in CA1 and subiculum. Case 14 (anterolateral accumbens). Fewer cortical neurones were labelled in this brain compared to Case 17. Both CA1 and subiculum (dorsal and ventral) were lightly labelled. The CA1 label did not extend as far anterior or posterior as in Case 17. A few neurones were labelled in the amygdalohippocampal area. Scattered label was also found in the entorhinal cortex and laterally and posteriorly this extended into the most lateral and dorsal entorhinal cortex. Light prefrontal cortical label was also observed. The chief difference from Case 17 was that in addition to the label already described, a long anteroposterior strip of cortex immediately ventral to the rhinal sulcus contained lightly labelled cells in the middle layers. This label extended from the level of anterior thalamus continuously to the most caudal cortical sections examined at the level of posterior subiculum. Case 13 (posteromedial accumbens). In this brain, cortical label was restricted to small numbers of cells in the ventral CAI, adjacent subiculum and amygdalohippocampal area. Case 15 (posterolateral accumbens). The main cortical area labelled in this brain was the perirhinal cortex. This contained a small number of lightly labelled cells in a long anteroposterior strip extending from the level of the anterior commissure to caudal subiculum in every section examined. In anterior sections labelled cells were found both dorsal and ventral to the rhinal sulcus in the middle layers. In posterior sections (caudal to habenula) label was confined ventral to the sulcus and in many sections continued further ventral into the lateral entorhinal cortex. Amygdala (Fig. 4) Case 17 (anteromedial accumbens). In this brain virtually the whole anteroposterior extent of basolateral amygdala was labelled. The posterior extreme of this label seemed to extend continuously into labelling found in deep layers of the entorhinal cortex. Additional label was found in most sections immediately medial to basolateral amygdala in the amygdalohippocampal area and ventral to this site in the posteromedial cortical amygdaloid nucleus. The largest number of labelled neurones was found in the basolateral amygdala. Case 14 (anterolateral accumbens). In anterior amygdala a few cells were found labelled in the
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anterior cortical amygdaloid nucleus. At more caudal levels scattered label also appeared in basolateral, basomedial and medial amygdala. More constantly labelled, however, was the central nucleus in sections taken through the mid-region of amygdala (Fig. SC). At the most caudal levels some cells were labelled in the amygdalohippocampal area and the posteromedial cortical amygdaloid nucleus. Case 13 (posteromedial accumbens). Very few labelled neurones were found in the amygdala of this brain and all of these were confined to the amygdalohippocampal area. Case 15 (posterolateral accumbens). In this brain few amygdalar neurones were labelled and all of these were confined to the ventral part of the basolateral nucleus. Additional
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All sectors of accumbens studied receive inputs from the ventral pallidum. In some cases neurones in
habenula (following posteromedial injection posteromedial injection only). Bar, 500 p.
this area projecting to accumbens lay outside the presently accepted boundaries of ventral pallidum and extended into the subadjacent substantia innominata. Many labelled cell bodies were found in lateral hypothalamus. Eflerent projections from accumbens (Fig. 5)
Although no special attention was paid to axonal labelling, since WGA is not an ideal anterograde marker, a number of incidental observations were made. First, ventral pallidum contained axonal labelling in all cases of accumbens injection. Second, in only one case was axonal label found in globus pallidus, i.e. following injection of the posteromedial accumbens (Figs 5 and 10). Thus the posteromedial accumbens appears unique in sending efferents to both dorsal and ventral pallidurn. Axonal label was also found in the medial segment of lateral habenula following WGA injection of the
Afferents to nucleus accumbens
posteromedial accumbens (Case 13) and in the dorsomedial segment of medial habenula following injection of anterolateral accumbens (Case 14) (Fig. 9d). These are the two habenular regions previously shown to receive dopaminergic innervation from the ventral tegmental area and IFN.34.‘7 Controls Injections of WGA were made into the medial bank of prefrontal cortex, cingulate cortex and lateral septum, to control for possible retrograde label resulting from uptake and transport of WGA from the pipette track overlying the site of accumbens injections. Frontal cortex. Injection of the deep layers resulted in thalamic label in the rhomboid and centrolateral nuclei, IFN and VTA and a few neurones in diagonal band and lateral hypothalamus. Following injections of middle cortical layers many cells were also labelled in the mediodorsal nucleus, mainly its lateral sector and in the basolateral amygdala. Light label was also seen in the ventral subiculum. Elsewhere a similar pattern of label was seen to that found following the deep-layer injection. Cingulate cortex. Thalamic label appeared in anteventromedial, gemini, rhomboid, romedial mediodorsal and centrolateral (adjacent to habenula) nuclei. Claustral, substantia nigra and VTA label were also found. Septum. Thalamic label appeared in rostra1 paraventricular, paracentral nuclei and caudal paraventricular media1 and ventral to fasciculus retroflexus, adjacent to the third ventricle. The hippocampus, VTA, central nucleus of amygdala, lateral hypothalamus and rostra1 periaqueductal grey were also labelled. DISCUSSION
The technique of retrograde transport of WGA is well suited to the analysis of projection topography since very small deposits of tracer can be made. Furthermore, WGA does not apparently diffuse far from the injection site as is the case with horseradish peroxidase injection. 3’ It is apparently superior in sensitivity when compared to methods using WGAhorseradish peroxidase conjugates with tetramethylbenzidine histochemistry; and the transported material, unlike fluorescent tracers, does not diffuse out of the cell body to neighbouring neurones or glia. Ventral tegmen tal area This study shows evidence for a mediolateral topography in the efferent connections of the VTA to the accumbens. Thus cytoarchitectural subgroups already demonstrated in VTA show some evidence of differential projections, since medial accumbens injections label the interfascicular nucleus and paranigral VTA, while lateral accumbens injections label para-
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brachial VTA and a few cells in medial substantia nigra. This result is in agreement with autoradiographic dataI obtained in the rat. Furthermore, a few cells of the A8 group also send fibres to the accumbens, as has been previously described in the rat” and cat12. These findings on topography however, conflict with conclusions from similar experiments carried out in the hamster29 and catI as these authors found no evidence of efferent topography from VTA to accumbens. The reasons for these differences probably lie in the size and location of injection sites employed in the latter studies. The projections of IFN to the medial nucleus accumbens shows that not only does IFN project with dopaminergic fibres to the habenula as has been previously described32.34,37but also sends fibres to further rostra1 sites, presumably by a dorsal route through the stria medullaris.16 This indicates that there is a double ascending pathway from VTA to accumbens, one travelling by the well-known route via media1 forebrain bundle and the lateral hypothalamus and another via a dorsal route through fasciculus retroflexus and the stria medullaris. At least some of these dorsally directed fibres to accumbens are likely to be dopaminergic since in our retrograde-labelling experiments, in some sections, the majority of cells in the IFN were labelled. Some of these IFN cells may therefore project by dopaminergic axon collaterals to both habenula and medial accumbens. Clearly, however, direct evidence on this point is necessary. Autoradiographic data obtained in the rat4 indicate that each small nigral or VTA locus, regardless of its anteroposterior location, sends fibres to terminate throughout the length of the caudate-putamen or accumbens. Furthermore, the present data show that a single small injection of accumbens labels the whole anteroposterior extent of a given register in VTA. The evidence indicates a basic mediolateral efferent topography to the forebrain organized in strips or slabs with broad anteroposterior extent but narrow mediolateral extent.
Thalamus The paraventricular nucleus of thalamus appears to project to all sectors of accumbens. This extends earlier findings with the autoradiographic method.” A degree of topography was discernible and this was most obvious in the posterior midline thalamus (see Fig. 6) on the media1 edge of fasciculus retroflexus. Some evidence for variation of density of accumbens input was obtained, since judging by the numbers of neurones labelled, the anterior accumbens received a heavier thalamic input than posterior accumbens, while the media1 part of the accumbens received heavier input than lateral. This is consistent with autoradiographic evidence.” In addition to paraventricular input, evidence was obtained for rhomboid and reuniens projections to anterior accumbens
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I I Fig. 6. Precise topography of thalamic input to accumbens in relation to cytoarchit~tu~ in rostra1 and caudal thalamus. Stippled area on right of each diagrammatic section indicates ceil position for aceumbens inputs and topography is indicated by conventions of Fig. 2. Cytoarchitecture is indicated on left. In mid-thalamic regions (not shown here) topography is less clear-cut from paraventricular nucleus of the thalamus (PV). However, posteromedial accumbens received mainly dorsal PV input and posterolateral accumbens mainly ventral PV input.
Al&rents to nucleus accumbens
(Fig. 7). This agrees with autoradiographic data obtained in the rat.15 No evidence was found in the present study for suggestions that parataenial nucleus of thalamus (as defined in the atlas of Paxinos and Watson)30 projects to nucleus accumbens in the rat.“,20 Indeed retrograde label from accumbens injections seemed strictly to avoid parataenial nucleus, the labelled cells in the surrounding midline and paracentral intralaminar thalamus providing a “negative image” of the parataenial nucleus (Figs 3a and 6). These findings indicate that the apparent anterograde label in accumbens following parataenial injections, resulted from inadvertent spread of injected tracer to these adjacent neurones. Apparent retrograde label in the parataenial nucleus following accumbens injection in the hamster seems to result from discrepancies in terminology, in the sense that the label in the hamster parataenial nucleus apparently corresponds to caudal paraventricular label reported in the present study. These findings suggest that further work is necessary to support earlier reports that parataenial thalamus in the rabbit and cat may project to nucleus accumbens.6.‘2 The relation between thalamic and ventral tegmental inputs to accumbens shows that in the posterior midline thalamus a mediolateral topography is preserved and if extended laterally, merges with the projection of neurones of the parafascicular nucleus to the caudate-putamen. A similar though less precise picture has been outlined in the cat.12 This orderly topography clearly matches that from midbrain. In those cases where track label in lateral accumbens injections spreads dorsally into caudate-putamen, it was clear that a corresponding extension of thalamic label resulted in the central medial thalamus label extending to paracentral intralaminar label. Thus there appears to be a continuity in the topography of thalamic projections to nucleus accumbens and caudate-putamen from both thalamus and midbrain. An interesting, although minor group of labelled neurones was found in the most dorsal sectors of centrolateral thalamus, immediately adjacent to the lateral border of lateral habenula. This was found in every case except for injections localized to posteromedial accumbens. Their position is similar to that reported for a group of cells in the cat which project to the caudate-putamen Cortex
The main finding from the analysis of cortical input is of an orderly topography of projection from CAl, subiculum, entorhinal cortex and perirhinal cortex to different sectors of accumbens (Fig. 7). Furthermore, prefrontal cortex was found to project only to anterior accumbens. Subicular topography was remarkably precise so that whereas the anterior medial accumbens received widespread subicular label, excluding only the ventral-most tip at the amygdalohippocampal transition zone the posterior
293
medial accumbens received subicular label only from that excluded region and the adjacent amygdalohippocampal transition zone which does not project to anteromedial accumbens. This general picture is in good agreement with cortical afferents found in the cat.13 It differs, however, from the details of the picture found in the hamsteti9 where a detailed topography of projection from entorhinal cortex to accumbens was described but which was not found here. Our evidence suggests instead a more diffuse input principally to the rostra1 accumbens. This appeared to arise mainly from cells in deep layers, as has been found by other workers.13 The most dorsal located cells in cortex were found in area 1325 following posterolateral accumbens injections. The apparently distinctive nature of the cortical inputs to the posteromedial accumbens from ventral subiculum and amygdalohippocampal transition zone agrees with the autoradiographic findings of others.‘3.24.40The output from this sector of accumbens also defines its character, since it is the only region to innervate densely the globus pallidus and medial sector of lateral habenula. Physiological analysis of the projection from the hippocampal formation to the ventral striatum in the cat fully supports the general anatomical picture described above.26 Analysis of the neurotransmitter nature of the pathways suggests that an excitatory amino acid, possibly glutamate or aspartate, may mediate its physiological effects.43,” The demonstration of prefrontal inputs to nucleus accumbens in this study supports the findings in the cat and rat with autoradiographic techniques,2,‘2 emphasizing the widespread nature of frontal cortical influence over basal ganglia and associated circuits. Thus in addition to its projection to accumbens, prefrontal cortex projects bilaterally to widespread areas of caudate-putamen and to both VTA and substantia nigra and nucleus tegmenti pedunFurthermore, these anatomical culopontinus2 findings may be correlated with neurochemical changes found in accumbens following manipulations affecting frontal cortex in the rat.22.35In both cases changes in prefrontal dopamine turnover resulted in changes in nucleus accumbens dopamine turnover. Amygdala
Our main finding is that the basolateral division of amygdala provides the main input to accumbens. Minor inputs are also provided by a restricted region of the central nucleus, basomedial, medial and cortical nuclei. Some of the neurones in the cortical nuclei may have resulted from spread of the injection site to structures medial to the medial border of accumbens. The basolateral projection described fits well with earlier descriptions given for projections from its posterior division to medial border of accumbens and from its anterior division to posterolateral accumbens.‘2.2’.24In confirmation of Krettek and Price24 and contrary to Kelley et aL2’ we find that the
294
0. T.
PROJECTIONS
PHILLIPSON and
TO
GRIFFITHS
ACCUMBENS am
From
A. C.
al
pm
PJ
:
VTA
THALAMUS
CORTEX
AMYGDALA
Fig. 7. Summary of the main topography of projections to accumbens. Thafamic inputs to rostra1 accumbens from paraventricular (PV), rhomboid (RH) and reuniens (RE) nuclei project to both anteromedial (am) and anterolateral (al) subregions, while that from the central medial nucleus (CM) projects only to al. Similarly the prefrontal cortex (PFC) projects to both am and al subregions, while CAI, subiculum (SUB) and enturhinal cortex (EM) send fibres to am and perirhinal cortex (PRC) to al respectively.
Afferents to nucleus accumbens dorsomedial posterior accumbens does receive an amygdalar input and that this arises from cells in the amygdalohippocampal transition zone. Control
injections
Cortical label. Since ventral subiculum was shown to project to the frontal cortex, it seems that either nucleus accumbens and frontal cortex share a common input or that accumbens injections labelled subiculum from track label in the overlying cortex. We favour the former interpretation since results obtained by anterograde techniques demonstrate that both accumbens and cortex receive subicular input.38 Thalamic label. The only thalamic sector apparently providing common input to accumbens and control sites is a projection from rostra1 paracentral thalamus to septum. This arises from a discrete band of cells outlining the ventral border of the parataenial nucleus which was unlabelled. This finding is in contrast to the results of Luiten et a1.27 who found parataenial nucleus labelled following septal injections. Rostra1 paraventricular thalamus, however, seems to provide a dual innervation to accumbens and lateral septum. In caudal diencephalon ventral and medial to the parafascicular nucleus a large number of neurones were labelled adjacent to the third ventricle following septal injection. This appears to be a caudal and ventral extension to the group of posterior paraventricular cells in thalamus innervating the accumbens. Midbrain label. Retrograde label in a few neurones in IFN following prefrontal cortex injections indicate that, although this is a small nucleus, IFN neurones have projections to widely separated brain regions in habenula, nucleus accumbens and prefrontal cortex. Nigral label following cingulate injections may account for the nigral label which was found following WGA injection of the posteromedial accumbens where the pipette track passed through cingulate cortex. General
discussion
A result of some interest arising from this study is the apparently distinctive nature of connections to
295
the medial accumbens particularly its anterior part. Here overlapping cortical afferents from hippocampal formation, prefrontal and entorhinal cortex terminate in territory receiving IFN and paranigral VTA input. This territory of accumbens may be functionally linked to habenular activity because IFN also projects to habenula. 32,34 The suggestion of a link between medial accumbens and habenula is strengthened by evidence that the posteromedial accumbens sends axons to the medial part of lateral habenula. Furthermore, a heavy input to globus pallidus from posteromedial accumbens suggests a strong link to the motor system. Evidence from anterograde-tracing work28 shows that not only does accumbens project to the globus pallidus but heavily to the ventral pallidum and directly to the entire plate of mesencephalic VTA and nigra compacta neurones. The evidence supports the concept of accumbens as an interface between the limbic and extrapyramidal motor system of the basal ganglia and that this interface also connects directly to habenula and the ventral pallidurn. Since ventral pallidum projects further to VTA and mediodorsal nucleus and thence to cortical the nucleus accumbens is also in a regions, 1~“.3’.33 position to exert an influence by indirect routes over many forebrain dopamine functions. The present evidence on topography indicates that selective regions of accumbens may fractionate its relations into more specific components. Not only does our connectional evidence suggest fractionation but studies using a combination of histochemical and pathwaytracing techniques demonstrate a patchy variation in transmitter concentration, receptor density and terminal fields of input projections.” These studies support the view that the posteromedial accumbens has a distinctive character as it receives dorsal periventricular thalamic input to an area rich in opiate receptors and acetylcholinesterase but avoiding cell clusters. The significance of the relationship between these cell clusters, opiate receptors and input-output relations are interesting questions which remain to be investigated. Acknowledgemenfs-We wish to thank the Wellcome Trust for support and Mrs J. Gillard for typing the manuscript.
REFERENCES 1. Beckstead R. M. (1976) Convergent thalamic and mesencephalic projections to the anterior medial cortex in the rat. J. camp. Neural. 166, 403-416. 2. Beckstead R. M. (1979) An autoradiographic examination of cortico-cortical and subcortical projections of the mediodorsal-projection (prefrontal) cortex in the rat. J. camp. Neural. 184. 43-62. 3. Beckstead R.- M. (1984)The thalamostriatal projection in the rat. J. cotnp: Neural. 223, 313-346. 4. Beckstead R. M., Domesick V. 9. and Nauta W. J. H. (1979) Efferent connections of the substantia nigra and ventral tegmental area in the rat. Brain Res. 175, 191-217. 5. Carmen J. B., Cowan W. M. and Powell T. P. S. (1963) The organisation of corticostriate connexions in the rabbit. Brain 86, Z-562. 6. Cowan W. M. and Powell T. P. S. (1955) The projections of the midline and intralaminar nuclei of the thalmus of the rabbit. J. Neural. Neurosurg. Psychiai. 18, 26&279. I. Cragg 9. G. and Hamlyn L. H. (1960) Histological connections and visceral actions of components of the fimbria in the rabbit. Expi Neural. 2, 581-597. 8. Dahlstrom A. and Fuxe K. (1964) Evidence for the existence of monoamine-containing neurons in the central nervous system. I. Demonstration of monoamines in the cell bodies of brain stem neurons. Acra physiol. stand. 62, Suppl. 232, l-55.
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9. De Olmos J. S. and Ingram W. R. (1972) The projection field of the stria terminalis in the rat brain. An experimental study. J. romp. Neural. 146, 303-334. 10. Fallon J. H. and Moore R. Y. (1978). Catecholamine innervation of the basal forebrain. IV. Topography of the dopamine projection to the basal forebrain and neostriatum. J. romp. Neural. 180, 545-580. 11. Groenewegen H. J. and Van Dijk C. A. (1984) Efferent projections of the ventral pallidum in the rat as studied with the anterograde transport of ~~u~e~~~~z~ufgurisleucoagglutinin (PHA-L). Neurasci. Lett. Suppl. 18. S58. 12. Groenewegen H. J., Becker N. E. H. M. and Lohman A. H. M. (1980). Subcortical afferents of the nucleus accumbens septi in the cat, studied with retrograde axonal transport of horseradish peroxidase and bisbenzimid. Neuroscience 5, 1903-1916. 13. Groenewegen H. J., Room P., Witter P. and Lohman A. H. M. (1982) Cortical afferents of the nucleus accumbens in the cat, studied with anterograde and retrograde transport techniques. Neuroscience 7, 977-995. 14. Heimer L. and Wilson R. D. (1975). The subcortical projections of the allocortex: similarities in the neural associations of the hippocampus, the pyriform cortex and the neocortex. In Centennial Symposium, Perspectives in Neuro6joiog~ (ed. Santini M.), pp. 177-193. 15. Herkenham, M. (1978). The connections of the nucleus reuniens thalami: evidence for a direct thalamo-hippocampal pathway in the rat. J. camp. Neurol. 177, 589-610. 16. Herkenham M. and Nauta W. J. H. (1977) Afferent connections of the habenular nuclei in the rat. A horseradish peroxidase study with a note on the fiber-of-passage problem. J. camp. Neural. 173, 123-146. 17. Herkenham M., Moon Edley S., and Stuart J. (1984) Cell clusters in the nucleus accumbens of the rat. and the mosaic relationship of opiate receptors acetylcholinesterase and subcortical afferent terminations. Neuroscience 11, 561-593. 18. Jones M. W., Kilpatrick. I. C. and Phillipson. 0. T. (1985). The parafascicular thalamus and rat forebrain dopamine utilisation. J. Phisiol., Lond. 358, 48P. _ 19. Jones. M. W.. Kiluatrick. I. C. and Phillipson. 0. T. (1985). Dopamine utilisation in the nucleus accumbens is subject to control by the*mediodorsal nucleus 0; the rat thaiam& Nekrosci. Left. Suppf. 21, 258. 20. Keffey A. E. and Stinus L. (1984) Distribution of the projection from the parataenial nucleus of the thalamus to the nucleus accumbens in the rat: an autoradiographic study. Exp. Brain Res. 54, 499-512. 21. Keffey A. E., Domesick B. V. and Nauta W. J. H. (1982) The amygdafostriataf projection in the rat-an anatomical study by anterograde and retrograde tracing methods. Neuroscience 7, 615-630. 22. Kilpatrick 1. C., Phillipson 0. T. and Pycock C. J. (1983) Phasic m~ulation of rat forebrain dopamine utilisation by lesions of the mediodorsal thalamus. J. Physiol.. Land. 334, 130-13 1P. 23. Kifpatrick I. C., Jones M. A., Pycock C. J. and Phillipson 0. T. (1983) Regional telencephalic dopamine utilisation following stimulation and removal of thalamic input. Neurosci. Left. Suppf. 14, Sf96. 24. Krettek J. E. and Price J. L. (1978) Amygdafoid projections to subcortical structures within the basal forebrain and brainstem in the rat and cat. J. camp. Neural. 178, 225-254. 25. Krieg W. J. S. (1946) Connections of the cerebral cortex. 1. The albino rat. (A) Topography of the cortical areas and (B) structure of the cortical areas. J. camp. Neurot. 84, 221-323. 26. Lopes da Sifva F. H., Amofds D. E. A. T. and Neijt H. C. (1984) A functional link between the fimbic cortex and ventral striatum: physiology of the subiculum accumbens pathway. Expf Brain Res. 55, 205-214. 27. Luiten P. G. M., Kuipers F. and Schuitmaker H. (1982) Organisation of diencephalic and brainstem afferent projections to the lateral septum in the rat. Neurosci. Letr. 30, 2ff--216. 28. Nauta W. J. H., Smith G. P., Faulf R. L. M. and Domesick V. B. (1978) Efferent connections and nigral afferents of the nucleus accumbens septi in the rat. Neuroscience 3, 385401. 29. Newman P. and Winans S. S. (1980) An experimental study of the ventral striatum of the golden hamster: I. Neuronal connections of the nucleus accumbens. J. camp. Neural. 191, 167-192. 30. Paxinos G. and Watson C. (1982) The Rat Brain in Steroruxic Coordinaies. Academic Press, New York. 31. Phillipson 0. T. (1979) Afferent projections to the ventral tegmental area of Tsai and interfascicular nucleus: a horseradish peroxidase study in thk rat. J. romp. Neural. 187, 117-144. 32. Phillipson 0. T. and Griffiths A. C. (1980) Neurones of origin for the mesohabenular dopamine pathway. Brain Res. 197, 213-218. 33. Phillipson 0. T. and Griffiths A. C. (1983) Anterograde and retrograde labelling of CNS pathways with unconjugated lectins using the unlabelfed antibody enzyme method. Bruin Res. 265, 199-207. 34. Phillipson 0. T. and Pycock C. J. (1982) Dopamine neurones of the ventral tegmental area project to both medial and lateral habenula. Expl Brain Rex 45, 89-94. 35. Pycock C. J., Carter C. J. and Kerwin R. W. (1980) Effect of 6-hydroxydopamine lesions of the medial prefrontal cortex on neurotransmitter systems in subcortical sites in the rat. J. Neurochem. 34, 91-99. 36. Siegel A. and Tassoni J. P. (1971) Differential efferent projections from the ventral and dorsal hippocampus of the cat. Brain Behav. .!+o/. 4, 185-200.
37. Skagerberg G., Lindvaff 0. and Bjiirklund A. (I 984) Origin, course and termination of the mesohabenular dopamine pathway in the rat. Brain Res. 307, 99-108. 38. Swanson L. W. (198 1) A direct projection from Ammons horn to prefrontal cortex in the rat. Brain Res. 217, 15&l 54. 39. Swanson L. W. and Cowan W. M. (1975) A note on the connections and development of the nucieus accumbens. Brain Res. 92, 324-330. 40. Swanson L. W. and Cowan W. M. (1977) An autoradiographic study of the organisation of the efferent connections of the hippocampaf formation in the rat. J. camp. Neural. 172, 49-84. 41. Switzer R. C., Hill J. and Heimer L. (1982) The globus paffidus and its rostroventral extension into the olfactory tubercle. Neuroscience 7, 1891-1904. 42. Ungerstedt U. (1971) Stereotaxic mapping of the monoamine pathway in the rat brain. Acta physial. stand. 197, Suppf. 367, l-48. 43. Wafaas I. (1981) Biochemical evidence for overlapping neocortical and allocortical glutamate projections to the nucleus accumbens and rostra1 caudatoputamen in the rat brain. Neuroscience 6, 399-405. 44. Walaas I. and Fonnum F. (1979) The effects of surgical and chemical lesions on neurotransmitter candidates in the nucleus accumbens of the rat. Neuroscience 4, 209-216. (Accepted 29 May 198.5)
0304-4522~85%3.00+ 0.00
Pergamon Press Ltd Q 1985IBRO
THE TOPOGRAPHIC ORDER OF INPUTS TO NUCLEUS ACCUMBENS IN THE RAT 0. T. PHILLIPSON and A. C. GRIFFITHS Department of Anatomy, The Medical School, University Walk, Bristol BS8 IT’D, U.K. A~~act-A~erents to the nucleus accumbens have been studied with the retrograde transport of unconjugat~ wheatgerm agglutinin as detected by immunohist~hemistry using the peroxidaseantiperoxidase method, in order to define precisely afferent topography from the cortex, thalamus, midbrain and amygdala. Cortical afferent topography was extremely precise. The largest number of cells was found following injections to the anterior accumbens. Anteromedial injections labelled a very large extent of the subiculum and part of the entorhinal cortex. Anterolaterai injections produced less subicular and entorhinal label but also labelled the posterior perirhinal cortex. Posteromedial injections labelled only the ventral subiculum and a few cells in the adjacent medial entorhinal cortex. Posterolateral injections labelled few lateral entorhinal neurones but did label a long anteroposterior strip of perirhinal cortex. Prefrontal cortex label was found only after anterior accumbens injections. In the amygdala labelled neurones were found in cortical, central, lateral posterior, anteromedial and basolateral nuclei. Basolateral amygdala projected chiefly to the anteromedial accumbens and central nucleus to anterolateral accumbens. Only a weak amygdala label was found after posterior accumbens injections. In the ventral tegmental area, the midline interfascicular nucleus projected only to medial accumbens. The paranigral ventral tegmentum projected chiefly to the medial accumbens and the parabrachial area chiefly to the lateral aecumbens. In the thalamus, heaviest label was found after anterior accumbens injections. Most cells were found in the paraventricular, reuniens and rhomboid nuclei and at posterior thalamic levels lying medial to the fasciculus retroflexus. There was only restricted topography found from thalamic sites. Retrograde label was also found in the ventral pallidum and lateral hypothalamus. Single small injection sites within accumbens received input from the whole anteroposterior extent of the thalamus and ventral tegmentum. The medial accumbens was found to have a close relationship to habenula, globus pallidus and interfascicular nucleus. It appeared that the heaviest volume of inputs projected to anterom~ial accumbens, where output from hippocampus (CAB, subi~ulum, entorhinal and prefrontal cortices converged with output from amygdala, midline thalamus and ventral tegmentum.
The nucleus accumbens appears to occupy an interface position between the limbic and extrapyramidal motor system. An analysis of its structure, histochemistry and connections has given it a central position in the emerging concept of the ventral striatum.‘4.4’ Afferents to accumbens arise from a wide variety of structures including the thalamus.5,6,7,8.9.24.28.36.40,42 In the course of neurochemical experiments on the influence of thalamic input on dopamine utilization in the caudate-putamen and prefrontal cortex, we have also found changes in the nucleus accumbens.t8.‘9.22.23This raised the question of the full extent of direct thalamic input to accumbens from regions the near mediodorsal and parafascicular-intralaminar nuclei. The existing evidence on thalamic projection to the accumbens using modern techniques shows that the paraventricular thalamus provides input, but there is little information on its full extent or topography in rat,39 although some data has been obtained in the cat and hamster.‘2.29 The present experiments were thus carried out to map more fully the topography of thalamic input to accumbens in the rat using a retrograde CAl, hippocampal subfield CAI; IFN, interfascicular nucleus; VTA, ventral tegmental area; WGA, wheatgerm agglutinin.
Abbreviations:
method with very small injections and a sensitive The immunohistochemical detection technique. topography of inputs to accumbens from cortex, amygdala and midbrain were also assessed.
EXPERIMENTAL PROCEDURES Twenty-one male and female rats of Wistar-derived strain weighing between 180 and 2 IO g were anaesthetized with chloral hydrate (4~mg/kg i.p.) and given injections of unconjugated wheatgerm agglutinin (WGA) to the nucleus accumbens or neighbouring structures by stereotaxic guidance. Wheatgerm agglutinum (Miles-Yeda Ltd, 2.82 pg/pl in 0.05 M Tris-HCl, 0.15 M NaCl buffer pH 8.6) was injected with intermittent ptis of air pressure (about 0.3 kg/cm2) applied, via a reduction valve and metered push-button quick-venting valve mechanism, to a GC2OOF-10 glass micropipette (Clarke Electromedical Instruments) broken to a tip diameter of 20-40pm, filled with WGA and a small quantity of oil. The injected volume was monitored under microscopic control with the aid of a micrometer scale placed against the upper oil meniscus in the pipette. Under these conditions as little as 50 nl could be reliably ejected. In those fully described cases reported in this paper, where regional microinjections to subsectors of nucleus accumbens were achieved, the volumes injected varied between 90 and 280 nl given slowly over several minutes. The pipette was left in place for a further IOmin at the end of the injection. Following recovery from anaesthesia, animals were allowed to survive for 1423 h and then reanaesthetized, 275
276
0. T.
PHILLIPSON and
perfused through the ascending aorta with 500ml normal saline, followed by about 500 ml Bouin’s fixative containing 4% paraformaldehyde. Brains were removed and postfixed overnight in the same fixative with 10% sucrose added.
Immunohistochemistry The method for detecting the retrograde transport of lectin is fully described elsewhere.33 Briefly, frozen 40-pm sections were collected into 0.05 M Tris-HCl, pH 7.8, and washed in several changes of this buffer. Floating sections were incubated at room temperature overnight in a l/2000 dilution of rabbit anti-WGA antiserum in 0.05 M Tris-HCI buffer, pH 7.8, containing 0.7% carrageenan and 0.5% Triton X-100. After washing, sections were incubated in the second antibody (sheep anti-rabbit immunoglobulin diluted 1: 15in the Tris/carrageenan/Triton solution) followed by a standard peroxidase-antiperoxidase procedure using freshly prepared 0.05% 3, 3’-diaminobenzidine and H,Oz at a final concentration of 0.03’?. After further washing, sections were mounted on gelatin-coated slides, counterstained with light green, dehydrated and coverslipped.
RESULTS
In the initial stages of this study experiments were carried out with relatively large volumes of tracer injected to nucleus accumbens using WGAhorseradish peroxidase conjugate with tetramethylbenzidine histochemistry. Although useful for summarizing the principal sites of origin of accumbens afferents, the present paper will confine its attention to those brains in which very small deposits of unconjugated WGA were injected since, judging by the injection site, the local spread of unconjugated lectin was considerably less than the horseradish peroxidase conjugate. Furthermore, the precise topography of afferents to accumbens could be more clearly defined with the use of the unconjugated WGA, where it is clear that uptake and transport is confined principally to the area of stained tissue (see discussion of control injections). The main features of afferent topography will be illustrated by four cases in which small deposits of WGA were placed in the anteromedial, the anterolateral, the caudomedial and caudoventrolateral accumbens. Injection sites Anteromedial accumbens (Case 17) (Fig. la). The injected WGA labelled the most rostra1 and medial tip of accumbens but also extended medially to label the taenia tecta. Label in the needle track was seen to stain lightly the deep layers of prefrontal cortex dorsal to the injection site (see results of control injections). Anterolateral accumbens (Case 14) (Fig. lb). In this case the injected WGA surrounded the anterior limb of anterior commissure in the ventroanterolateral accumbens. The centre of the injected WGA lay just ventral and medial to anterior commissure, 1.1 mm lateral to and slightly rostra1 to the centre of injected WGA in Case 17. A narrow tail of label extended dorsally in the pipette track into the ventral part of the head of the caudate-putamen.
A. C.
GRIFFITHS
Caudomedial accumbens (Case 13) (Fig. lc). The centre of this injection lay medial to the ventral tip of the anterior horn of lateral ventricle where it divides the medial caudatoputamen from the lateral septum. The needle track passed through the lateral ventricle and it is probable that some injected WGA leaked into the cerebrospinal fluid since the ependymal lining of the ventricle here was heavily stained. Very light staining was also found in the pipette track as it descended through the cingulate cortex. Caudoventrolateral accumbens (Case 15) (Fig. 1d). Here the centre of the injection site was located ventrolateral to the anterior limb of the anterior commissure, about 700 pm caudal to the centre of Case 14 and about 1 mm lateral and ventral to the centre of Case 13. Light staining was seen in the needle track as it penetrated caudatoputamen. It is of interest to note that this track staining was not uniform but showed patchy extension slightly rostra1 and caudal as well as medial and lateral. The examination of this staining at a high power showed this to be located both in striatal cell bodies and axon terminals in a patch-like arrangement. Midbrain
label (Fig.
2)
Case 17 (anteromedial accumbens). Retrograde label was found in the ventral tegmental area (VTA) throughout its entire anteroposterior extent. Most neurones were found in the paranigral division of VTA and some also in the midline interfascicular nucleus (IFN). In caudal sections cells extended dorsally into nucleus linearis caudalis, whilst rostrally many neurones were also labelled in the supramammillary area. In the posterior half of the VTA a few neurones were found dorsally in VTA in the parabrachial region of VTA. Case 14 (anterolateral accumbens). As in Case 17 the whole anteroposterior extent of VTA was labelled. In contrast to Case 17, however, labelled neurones tended to occupy slightly more lateral and dorsal positions in VTA. Thus more neurones were labelled in the parabrachial and in more lateral divisions of paranigral VTA. Furthermore, unlike Case 17, apart from an occasional neurone, no label was seen in the midline IFN. A few labelled neurones were found in the substantia nigra. Occasionally, neurones were found to be labelled in the contralateral VTA. Case 13 (caudomedial accumbens). As before the whole anteroposterior extent of VTA was labelled. In this case, however, the distribution of midbrain label was different from those cases already described. Here heavy label was found throughout the whole extent of the IFN. Label in the rest of VTA was strictly confined to the paranigral VTA (Fig. 8a). Also, unlike the previous case, substantial numbers of cells were labelled in the medial substantia nigra and small numbers were found labelled also in midsubstantia nigra. Occasionally neurones were found in the contralateral VTA.
Abbreviations used in fisures AHi al am BL BL, CA1 Ce CM ENT fr IFN IP PBP PC PF
amygdalohippocampal transition zone anterolateral accumbens anteromedial accumbens basolateral nucleus of amygdala ventral basolateral nucleus of amygdala CA I subfield of hippocampus central nucleus of amygdala central medial nucleus of thalamus entorhinal cortex fasciculus retroflexus interfascicular nucleus inte~duncuIar nucleus nucleus parabrachialis pigmentosus of VTA paracentral nucleus of thalamus parafascicular nucleus of thalamus
PFC Pl ;: PRC PT PV PV, PV” RE RH SUB SUB, VTA
prefrontal cortex posterolateral accumbens posteromedial accumbens nucleus parani~alis of VTA perirhinal cortex parataenial nucleus of thalamus paraventricular nucleus of thalamus paraventricular nucleus of thalamus (dorsal) paraventricular nucleus of thalamus (ventral) nucleus reuniens of thalamus rhomboid nucleus of thalamus subiculum subiculum (ventral) ventral tegmental area
Fig. I. Injection sites of the cases fully reported in this paper as demonstrated in thionin stained sections through the injection centres. (a) Anteromedial accumbens. x 21. (b) Anterolateral accumbens. x 22. (c) Posteromedial accumbens. x 23. (d) Posterolateral accumbens. x 18. Fig. 8. (a) Retrograde cell labelling in VTA, showing nucleus paranigralis of VTA (PN) and IFN subregions dorsal to the interpeduncular nucleus (IP) x 135. (b) Retrograde cell labelling in the caudal thalamus medial to fasciculus retroflexus (fr) x 145. (c) Cell labelling in ventral paraventricular thalamus. x 300. p. 279. Fig. 9. (a) Large numbers of CAI pyramids labelled following injection of anteromedial accumbens. x 60. (b) Part of the CA1 field shown in (a) at higher power indicating the lightly labelled members of this population. Thus a very large total of labelled cells could be found in CAI. x 200. (c) Retrograde label in central nucleus of amygdala. x 290. (d) Axon labelling in dorsomedial sector of medial habenular nucleus. x 230. p. 28. Fig. 10. (a) Retrograde cell label in medial bank of frontal cortex (prefrontal cortex). Dorsal is at top and midline at left of the micrograph. x 200. (b) Retrograde cell label in nucleus reuniens of thalamus, x 250. (c) Profuse axonal label from WGA injection to posteromedial accumbens in giobus pallidus (position shown also in Fig. 5.) x95. p. 281.
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Case 15 (caudoventrolateral accumbens). Here again the pattern of midbrain label is quite distinct from that described so far for the other three cases. Firstly, there was no label in the IFN. Secondly, the label occupied cells in further lateral and dorsal positions in VTA than in Case 13, i.e. much larger numbers of neurones were found in the parabrachial VTA and very few in the paranigral VTA. The medial substantia nigra was quite markedly labelled throughout its entire anteroposterior length (probably because of WGA in the overlying pipette track) and occasional cells were labelled in the retrorubral (A8) position. Cells were occasionally found in the contralateral VTA. In all cases it was clear that a single small deposit of WGA to the accumbens of diameter about 400 pm labelled a narrow mediolateral extent of VTA only slightly broader than this; while by contrast it labelled the entire anteroposterior extent (about 2000 pm) of the VTA, interfascicular nucleus and nucleus linearis caudalis. Thalamic label (Fig. 3) A notable feature of many cases studied was that a single small injection of WGA labelled the whole anteroposterior extent of the thalamic nuclei described. Thus a length of 3-mm thalamus could be labelled from a deposit of WGA 2400pm in diameter. Case 17 (anteromedial accumbens). The most rostral thalamic label found in this case lay in cells medial to the post-commissural fornix at the point where the base of the posterior septum, the dorsal rostra1 tip of the third and lateral ventricles lie adjacent to one another (Fig. 3a). The label extended caudal from this site to large numbers of cells in the paraventricular thalamus. In its rostra1 portion (i.e. rostra1 to the mediodorsal thalamus) the label extended continuously from the lateral ventricle to the dorsal margin of the third ventricle in nearly all sections. In dorsal positions a negative image of the whole extent of the parataenial nucleus was formed by the surrounding retrograde label. Thus cells were found extending lateral from the paraventricular thalamus to the ventrolateral margin of stria medullaris and this label was continuous with a band of cells found in the rostra1 paracentral thalamus. In ventral positions thalamic label extended in the midline between the anteromedial nuclei in a ventral extension of the paraventricular label to become almost continuous with retrograde label in nucleus reuniens (Fig. 10). A few cells were found in anterodorsal nucleus where they appeared to form a continuous lateral extension of the label found in the paracentral nucleus. At thalamic levels caudal to the parataenial nucleus, the continuous midline dorsoventral strip of label was broken so that the paraventricular and reuniens label was separated by the intervening unlabelled anteromedial and mediodorsal nuclei. Where
the rostra1 tip of mediodorsal and caudal pole of parataenial nuclei overlap, heavy label was found in the “mediodorsal” nucleus in positions suggesting that this label was in fact part of a caudal extension of the lateral paraventricular label already described. Furthermore, and in support of this view, it was continuous with midline paraventricular and paracentral label, as already described at more rostra1 sites. Caudal to this position thalamic label was continuously seen throughout the length of the paraventricular thalamus dorsally and nucleus reuniens ventrally. Smaller numbers of labelled cells were found in some sections in the rhomboid nucleus and a very few in central median and paracentral nuclei. Occasional cells were found in nucleus gelatinosus (gemini). At caudal levels of habenula and at levels posterior to the habenula where fasciculus retroflexus descended through the posterior medial thalamus there was a sharp increase again in the numbers of cells labelled. Here label in the posterior paraventricular nucleus extended laterally under the ventral margin of lateral habenula and the fasciculus retroflexus as it formed at the ventrolateral base of habenula. More scattered label extended ventral in the midline into a poorly defined region of the posterior medial thalamus (central medial) and in positions dorsal to third ventricle, which probably represented a caudal extension of nucleus reuniens, or alternatively nucleus subparafascicularis. Large numbers of neurones were labelled medial to the parafascicular nucleus adjacent to the third ventricle. In summary, the vast majority of label found in this brain lay in the paraventricular nucleus (both stellatocellularis and rotundocellularis divisions were labelled) and in nucleus reuniens throughout their whole extent in the anteroposterior plane. Extentions from this pattern were found into rostra1 mediodorsal and, in small numbers of cells only, into paracentral, anterodorsal, central medial and centrolateral nuclei dorsally and rhomboid, gemini and possibly subparafascicularis ventrally. Case 14 (anterolateral accumbens). The main features of thalamic label found in this brain were similar to that described for Case 17 in that (1) the entire anteroposterior length of thalamus was labelled and (2) the principal nuclei labelled were the paraventricular and reuniens nuclei. The differences between these two brains were as follows. The paraventricular label in the posterior part of this nucleus extended further ventral than in Case 17. The reuniens label in the two cases differed in that heaviest label was found rostrally in Case 17, whilst only light reuniens label was found rostrally in Case 14 and larger numbers of labelled cells were found in mid-reuniens. Furthermore, in Case 14 marked label was found in central medial nucleus (almost unlabelled in Case 17) which extended laterally into paracentral nucleus. A few cells were also labelled in
Afferents to nucleus accumbens dorsal parts of the centrolateral nucleus. In many caudal thalamic sections in this brain continuous label extended from the lateral ventricle in the paraventricular nucleus through to central medial, rhomboid and reuniens nuclei to the third ventricle. This contrasts with Case 17 where rather sparse thalamic label was found at mid to caudal sites. Finally, at the level of the fasciculus retroflexus (Figs 3a and 8b) label in Case 14 lay further lateral than in Case 17; and in some cases neurones were found in parafascicular nucleus itself. Case 13 @osteromedial accumbens). Unlike the two brains so far described, this brain showed thalamic label which was entirely confined to paraventricular label. A few neurones were also labelled in dorsal central lateral nucleus adjacent to the lateral margin of lateral habenula. No label was found in ventral paraventricular nucleus, central medial, paracentral, rhomboid or reuniens nuclei. Case 15 (posterolateral accumbens). The pattern of label in this brain resembled that in Case 14 and 17 in that the greater part of the anteroposterior extent of paraventricular nucleus was labelled. Rostra1 thalamic sections however were unlabelled. In contrast to Cases 13, 14 and 17 paraventricular label was heaviest in its ventral regions, while its dorsal regions were for the most part unlabelled. In some sections label was continuous with labelled neurones found in the central medial and paracentral nuclei. Occasionally labelled neurones were also found in dorsal parts of central lateral nucleus. In posterior thalamic sections the pattern of paraventricular label was similar to that seen in Case 14, although in addition labelled neurones were seen in larger numbers in parafascicular nucleus lateral to the lateral rim of the fasciculus retroflexus. Furthermore, in contrast to Cases 14 and 17 few neurones were labelled in nucleus reuniens and the rhomboid nucleus. The topography of the input to accumbens and its relation to cytoarchitecture is summarized in Fig. 6. Topography is most clearly defined in the posterior thalamus. Cortical label (Fig. 4) Case 17 (anteromedial accumbens). The hippocampal formation and associated cortical areas were extensively labelled in this brain. The most rostra1 extent of this label was found in the ventral CA1 region adjacent to the amygdalohippocampal area, where further retrograde label was found. The CA1 label extended continuously in more posterior sections to include cells in the ventral subiculum and, still further posterior, also in dorsal subiculum, although here the intervening CA1 was unlabelled. At the most posterior cortical levels studied, dorsal subiculum, CA1 and ventral subiculum were continuously and heavily labelled (Fig. 9a and b). Many labelled neurones were also found in the deep layers of entorhinal cortex. The rostra1 margin of this label was continuous with retrograde label
285
found in the posterior pole of the basolateral amygdala. Fewer cells were labelled in entorhinal cortex as sections lay further posterior. A few lightly labelled cells were found in the medial edge of entorhinal cortex where it joins subiculum. Scattered label was also found in the deep layers of prefrontal cortex (Fig. 10a). The principal cortical projections, as judged both by numbers of labelled cells and the intensity of staining, was that in CA1 and subiculum. Case 14 (anterolateral accumbens). Fewer cortical neurones were labelled in this brain compared to Case 17. Both CA1 and subiculum (dorsal and ventral) were lightly labelled. The CA1 label did not extend as far anterior or posterior as in Case 17. A few neurones were labelled in the amygdalohippocampal area. Scattered label was also found in the entorhinal cortex and laterally and posteriorly this extended into the most lateral and dorsal entorhinal cortex. Light prefrontal cortical label was also observed. The chief difference from Case 17 was that in addition to the label already described, a long anteroposterior strip of cortex immediately ventral to the rhinal sulcus contained lightly labelled cells in the middle layers. This label extended from the level of anterior thalamus continuously to the most caudal cortical sections examined at the level of posterior subiculum. Case 13 (posteromedial accumbens). In this brain, cortical label was restricted to small numbers of cells in the ventral CAI, adjacent subiculum and amygdalohippocampal area. Case 15 (posterolateral accumbens). The main cortical area labelled in this brain was the perirhinal cortex. This contained a small number of lightly labelled cells in a long anteroposterior strip extending from the level of the anterior commissure to caudal subiculum in every section examined. In anterior sections labelled cells were found both dorsal and ventral to the rhinal sulcus in the middle layers. In posterior sections (caudal to habenula) label was confined ventral to the sulcus and in many sections continued further ventral into the lateral entorhinal cortex. Amygdala (Fig. 4) Case 17 (anteromedial accumbens). In this brain virtually the whole anteroposterior extent of basolateral amygdala was labelled. The posterior extreme of this label seemed to extend continuously into labelling found in deep layers of the entorhinal cortex. Additional label was found in most sections immediately medial to basolateral amygdala in the amygdalohippocampal area and ventral to this site in the posteromedial cortical amygdaloid nucleus. The largest number of labelled neurones was found in the basolateral amygdala. Case 14 (anterolateral accumbens). In anterior amygdala a few cells were found labelled in the
286
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anterior cortical amygdaloid nucleus. At more caudal levels scattered label also appeared in basolateral, basomedial and medial amygdala. More constantly labelled, however, was the central nucleus in sections taken through the mid-region of amygdala (Fig. SC). At the most caudal levels some cells were labelled in the amygdalohippocampal area and the posteromedial cortical amygdaloid nucleus. Case 13 (posteromedial accumbens). Very few labelled neurones were found in the amygdala of this brain and all of these were confined to the amygdalohippocampal area. Case 15 (posterolateral accumbens). In this brain few amygdalar neurones were labelled and all of these were confined to the ventral part of the basolateral nucleus. Additional
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this area projecting to accumbens lay outside the presently accepted boundaries of ventral pallidum and extended into the subadjacent substantia innominata. Many labelled cell bodies were found in lateral hypothalamus. Eflerent projections from accumbens (Fig. 5)
Although no special attention was paid to axonal labelling, since WGA is not an ideal anterograde marker, a number of incidental observations were made. First, ventral pallidum contained axonal labelling in all cases of accumbens injection. Second, in only one case was axonal label found in globus pallidus, i.e. following injection of the posteromedial accumbens (Figs 5 and 10). Thus the posteromedial accumbens appears unique in sending efferents to both dorsal and ventral pallidurn. Axonal label was also found in the medial segment of lateral habenula following WGA injection of the
Afferents to nucleus accumbens
posteromedial accumbens (Case 13) and in the dorsomedial segment of medial habenula following injection of anterolateral accumbens (Case 14) (Fig. 9d). These are the two habenular regions previously shown to receive dopaminergic innervation from the ventral tegmental area and IFN.34.‘7 Controls Injections of WGA were made into the medial bank of prefrontal cortex, cingulate cortex and lateral septum, to control for possible retrograde label resulting from uptake and transport of WGA from the pipette track overlying the site of accumbens injections. Frontal cortex. Injection of the deep layers resulted in thalamic label in the rhomboid and centrolateral nuclei, IFN and VTA and a few neurones in diagonal band and lateral hypothalamus. Following injections of middle cortical layers many cells were also labelled in the mediodorsal nucleus, mainly its lateral sector and in the basolateral amygdala. Light label was also seen in the ventral subiculum. Elsewhere a similar pattern of label was seen to that found following the deep-layer injection. Cingulate cortex. Thalamic label appeared in anteventromedial, gemini, rhomboid, romedial mediodorsal and centrolateral (adjacent to habenula) nuclei. Claustral, substantia nigra and VTA label were also found. Septum. Thalamic label appeared in rostra1 paraventricular, paracentral nuclei and caudal paraventricular media1 and ventral to fasciculus retroflexus, adjacent to the third ventricle. The hippocampus, VTA, central nucleus of amygdala, lateral hypothalamus and rostra1 periaqueductal grey were also labelled. DISCUSSION
The technique of retrograde transport of WGA is well suited to the analysis of projection topography since very small deposits of tracer can be made. Furthermore, WGA does not apparently diffuse far from the injection site as is the case with horseradish peroxidase injection. 3’ It is apparently superior in sensitivity when compared to methods using WGAhorseradish peroxidase conjugates with tetramethylbenzidine histochemistry; and the transported material, unlike fluorescent tracers, does not diffuse out of the cell body to neighbouring neurones or glia. Ventral tegmen tal area This study shows evidence for a mediolateral topography in the efferent connections of the VTA to the accumbens. Thus cytoarchitectural subgroups already demonstrated in VTA show some evidence of differential projections, since medial accumbens injections label the interfascicular nucleus and paranigral VTA, while lateral accumbens injections label para-
291
brachial VTA and a few cells in medial substantia nigra. This result is in agreement with autoradiographic dataI obtained in the rat. Furthermore, a few cells of the A8 group also send fibres to the accumbens, as has been previously described in the rat” and cat12. These findings on topography however, conflict with conclusions from similar experiments carried out in the hamster29 and catI as these authors found no evidence of efferent topography from VTA to accumbens. The reasons for these differences probably lie in the size and location of injection sites employed in the latter studies. The projections of IFN to the medial nucleus accumbens shows that not only does IFN project with dopaminergic fibres to the habenula as has been previously described32.34,37but also sends fibres to further rostra1 sites, presumably by a dorsal route through the stria medullaris.16 This indicates that there is a double ascending pathway from VTA to accumbens, one travelling by the well-known route via media1 forebrain bundle and the lateral hypothalamus and another via a dorsal route through fasciculus retroflexus and the stria medullaris. At least some of these dorsally directed fibres to accumbens are likely to be dopaminergic since in our retrograde-labelling experiments, in some sections, the majority of cells in the IFN were labelled. Some of these IFN cells may therefore project by dopaminergic axon collaterals to both habenula and medial accumbens. Clearly, however, direct evidence on this point is necessary. Autoradiographic data obtained in the rat4 indicate that each small nigral or VTA locus, regardless of its anteroposterior location, sends fibres to terminate throughout the length of the caudate-putamen or accumbens. Furthermore, the present data show that a single small injection of accumbens labels the whole anteroposterior extent of a given register in VTA. The evidence indicates a basic mediolateral efferent topography to the forebrain organized in strips or slabs with broad anteroposterior extent but narrow mediolateral extent.
Thalamus The paraventricular nucleus of thalamus appears to project to all sectors of accumbens. This extends earlier findings with the autoradiographic method.” A degree of topography was discernible and this was most obvious in the posterior midline thalamus (see Fig. 6) on the media1 edge of fasciculus retroflexus. Some evidence for variation of density of accumbens input was obtained, since judging by the numbers of neurones labelled, the anterior accumbens received a heavier thalamic input than posterior accumbens, while the media1 part of the accumbens received heavier input than lateral. This is consistent with autoradiographic evidence.” In addition to paraventricular input, evidence was obtained for rhomboid and reuniens projections to anterior accumbens
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0. T. PHILLIPSON and A. C. GRWFITHS
I I Fig. 6. Precise topography of thalamic input to accumbens in relation to cytoarchit~tu~ in rostra1 and caudal thalamus. Stippled area on right of each diagrammatic section indicates ceil position for aceumbens inputs and topography is indicated by conventions of Fig. 2. Cytoarchitecture is indicated on left. In mid-thalamic regions (not shown here) topography is less clear-cut from paraventricular nucleus of the thalamus (PV). However, posteromedial accumbens received mainly dorsal PV input and posterolateral accumbens mainly ventral PV input.
Al&rents to nucleus accumbens
(Fig. 7). This agrees with autoradiographic data obtained in the rat.15 No evidence was found in the present study for suggestions that parataenial nucleus of thalamus (as defined in the atlas of Paxinos and Watson)30 projects to nucleus accumbens in the rat.“,20 Indeed retrograde label from accumbens injections seemed strictly to avoid parataenial nucleus, the labelled cells in the surrounding midline and paracentral intralaminar thalamus providing a “negative image” of the parataenial nucleus (Figs 3a and 6). These findings indicate that the apparent anterograde label in accumbens following parataenial injections, resulted from inadvertent spread of injected tracer to these adjacent neurones. Apparent retrograde label in the parataenial nucleus following accumbens injection in the hamster seems to result from discrepancies in terminology, in the sense that the label in the hamster parataenial nucleus apparently corresponds to caudal paraventricular label reported in the present study. These findings suggest that further work is necessary to support earlier reports that parataenial thalamus in the rabbit and cat may project to nucleus accumbens.6.‘2 The relation between thalamic and ventral tegmental inputs to accumbens shows that in the posterior midline thalamus a mediolateral topography is preserved and if extended laterally, merges with the projection of neurones of the parafascicular nucleus to the caudate-putamen. A similar though less precise picture has been outlined in the cat.12 This orderly topography clearly matches that from midbrain. In those cases where track label in lateral accumbens injections spreads dorsally into caudate-putamen, it was clear that a corresponding extension of thalamic label resulted in the central medial thalamus label extending to paracentral intralaminar label. Thus there appears to be a continuity in the topography of thalamic projections to nucleus accumbens and caudate-putamen from both thalamus and midbrain. An interesting, although minor group of labelled neurones was found in the most dorsal sectors of centrolateral thalamus, immediately adjacent to the lateral border of lateral habenula. This was found in every case except for injections localized to posteromedial accumbens. Their position is similar to that reported for a group of cells in the cat which project to the caudate-putamen Cortex
The main finding from the analysis of cortical input is of an orderly topography of projection from CAl, subiculum, entorhinal cortex and perirhinal cortex to different sectors of accumbens (Fig. 7). Furthermore, prefrontal cortex was found to project only to anterior accumbens. Subicular topography was remarkably precise so that whereas the anterior medial accumbens received widespread subicular label, excluding only the ventral-most tip at the amygdalohippocampal transition zone the posterior
293
medial accumbens received subicular label only from that excluded region and the adjacent amygdalohippocampal transition zone which does not project to anteromedial accumbens. This general picture is in good agreement with cortical afferents found in the cat.13 It differs, however, from the details of the picture found in the hamsteti9 where a detailed topography of projection from entorhinal cortex to accumbens was described but which was not found here. Our evidence suggests instead a more diffuse input principally to the rostra1 accumbens. This appeared to arise mainly from cells in deep layers, as has been found by other workers.13 The most dorsal located cells in cortex were found in area 1325 following posterolateral accumbens injections. The apparently distinctive nature of the cortical inputs to the posteromedial accumbens from ventral subiculum and amygdalohippocampal transition zone agrees with the autoradiographic findings of others.‘3.24.40The output from this sector of accumbens also defines its character, since it is the only region to innervate densely the globus pallidus and medial sector of lateral habenula. Physiological analysis of the projection from the hippocampal formation to the ventral striatum in the cat fully supports the general anatomical picture described above.26 Analysis of the neurotransmitter nature of the pathways suggests that an excitatory amino acid, possibly glutamate or aspartate, may mediate its physiological effects.43,” The demonstration of prefrontal inputs to nucleus accumbens in this study supports the findings in the cat and rat with autoradiographic techniques,2,‘2 emphasizing the widespread nature of frontal cortical influence over basal ganglia and associated circuits. Thus in addition to its projection to accumbens, prefrontal cortex projects bilaterally to widespread areas of caudate-putamen and to both VTA and substantia nigra and nucleus tegmenti pedunFurthermore, these anatomical culopontinus2 findings may be correlated with neurochemical changes found in accumbens following manipulations affecting frontal cortex in the rat.22.35In both cases changes in prefrontal dopamine turnover resulted in changes in nucleus accumbens dopamine turnover. Amygdala
Our main finding is that the basolateral division of amygdala provides the main input to accumbens. Minor inputs are also provided by a restricted region of the central nucleus, basomedial, medial and cortical nuclei. Some of the neurones in the cortical nuclei may have resulted from spread of the injection site to structures medial to the medial border of accumbens. The basolateral projection described fits well with earlier descriptions given for projections from its posterior division to medial border of accumbens and from its anterior division to posterolateral accumbens.‘2.2’.24In confirmation of Krettek and Price24 and contrary to Kelley et aL2’ we find that the
294
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PROJECTIONS
PHILLIPSON and
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GRIFFITHS
ACCUMBENS am
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Fig. 7. Summary of the main topography of projections to accumbens. Thafamic inputs to rostra1 accumbens from paraventricular (PV), rhomboid (RH) and reuniens (RE) nuclei project to both anteromedial (am) and anterolateral (al) subregions, while that from the central medial nucleus (CM) projects only to al. Similarly the prefrontal cortex (PFC) projects to both am and al subregions, while CAI, subiculum (SUB) and enturhinal cortex (EM) send fibres to am and perirhinal cortex (PRC) to al respectively.
Afferents to nucleus accumbens dorsomedial posterior accumbens does receive an amygdalar input and that this arises from cells in the amygdalohippocampal transition zone. Control
injections
Cortical label. Since ventral subiculum was shown to project to the frontal cortex, it seems that either nucleus accumbens and frontal cortex share a common input or that accumbens injections labelled subiculum from track label in the overlying cortex. We favour the former interpretation since results obtained by anterograde techniques demonstrate that both accumbens and cortex receive subicular input.38 Thalamic label. The only thalamic sector apparently providing common input to accumbens and control sites is a projection from rostra1 paracentral thalamus to septum. This arises from a discrete band of cells outlining the ventral border of the parataenial nucleus which was unlabelled. This finding is in contrast to the results of Luiten et a1.27 who found parataenial nucleus labelled following septal injections. Rostra1 paraventricular thalamus, however, seems to provide a dual innervation to accumbens and lateral septum. In caudal diencephalon ventral and medial to the parafascicular nucleus a large number of neurones were labelled adjacent to the third ventricle following septal injection. This appears to be a caudal and ventral extension to the group of posterior paraventricular cells in thalamus innervating the accumbens. Midbrain label. Retrograde label in a few neurones in IFN following prefrontal cortex injections indicate that, although this is a small nucleus, IFN neurones have projections to widely separated brain regions in habenula, nucleus accumbens and prefrontal cortex. Nigral label following cingulate injections may account for the nigral label which was found following WGA injection of the posteromedial accumbens where the pipette track passed through cingulate cortex. General
discussion
A result of some interest arising from this study is the apparently distinctive nature of connections to
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the medial accumbens particularly its anterior part. Here overlapping cortical afferents from hippocampal formation, prefrontal and entorhinal cortex terminate in territory receiving IFN and paranigral VTA input. This territory of accumbens may be functionally linked to habenular activity because IFN also projects to habenula. 32,34 The suggestion of a link between medial accumbens and habenula is strengthened by evidence that the posteromedial accumbens sends axons to the medial part of lateral habenula. Furthermore, a heavy input to globus pallidus from posteromedial accumbens suggests a strong link to the motor system. Evidence from anterograde-tracing work28 shows that not only does accumbens project to the globus pallidus but heavily to the ventral pallidum and directly to the entire plate of mesencephalic VTA and nigra compacta neurones. The evidence supports the concept of accumbens as an interface between the limbic and extrapyramidal motor system of the basal ganglia and that this interface also connects directly to habenula and the ventral pallidurn. Since ventral pallidum projects further to VTA and mediodorsal nucleus and thence to cortical the nucleus accumbens is also in a regions, 1~“.3’.33 position to exert an influence by indirect routes over many forebrain dopamine functions. The present evidence on topography indicates that selective regions of accumbens may fractionate its relations into more specific components. Not only does our connectional evidence suggest fractionation but studies using a combination of histochemical and pathwaytracing techniques demonstrate a patchy variation in transmitter concentration, receptor density and terminal fields of input projections.” These studies support the view that the posteromedial accumbens has a distinctive character as it receives dorsal periventricular thalamic input to an area rich in opiate receptors and acetylcholinesterase but avoiding cell clusters. The significance of the relationship between these cell clusters, opiate receptors and input-output relations are interesting questions which remain to be investigated. Acknowledgemenfs-We wish to thank the Wellcome Trust for support and Mrs J. Gillard for typing the manuscript.
REFERENCES 1. Beckstead R. M. (1976) Convergent thalamic and mesencephalic projections to the anterior medial cortex in the rat. J. camp. Neural. 166, 403-416. 2. Beckstead R. M. (1979) An autoradiographic examination of cortico-cortical and subcortical projections of the mediodorsal-projection (prefrontal) cortex in the rat. J. camp. Neural. 184. 43-62. 3. Beckstead R.- M. (1984)The thalamostriatal projection in the rat. J. cotnp: Neural. 223, 313-346. 4. Beckstead R. M., Domesick V. 9. and Nauta W. J. H. (1979) Efferent connections of the substantia nigra and ventral tegmental area in the rat. Brain Res. 175, 191-217. 5. Carmen J. B., Cowan W. M. and Powell T. P. S. (1963) The organisation of corticostriate connexions in the rabbit. Brain 86, Z-562. 6. Cowan W. M. and Powell T. P. S. (1955) The projections of the midline and intralaminar nuclei of the thalmus of the rabbit. J. Neural. Neurosurg. Psychiai. 18, 26&279. I. Cragg 9. G. and Hamlyn L. H. (1960) Histological connections and visceral actions of components of the fimbria in the rabbit. Expi Neural. 2, 581-597. 8. Dahlstrom A. and Fuxe K. (1964) Evidence for the existence of monoamine-containing neurons in the central nervous system. I. Demonstration of monoamines in the cell bodies of brain stem neurons. Acra physiol. stand. 62, Suppl. 232, l-55.
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and A. C. GRIFFITHS
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