Afferent Projections to the of Tsai and lnterfascicular : A Horseradish Peroxidase Study in the Rat

0. T. PHILLIPSON ' Department of Anatomy, Karolinska Institutet, Solnaucigen I, S-I0401 Stockholm, 60, Sweden

ABSTRACT Using the retrograde transport of horseradish peroxidase (HRP), a study has been made of projections to the ventral tegmental area of Tsai (VTA) and related cell groups (A 10). In order to minimise the possibility of damage to fibres of passage, a technique was evolved for the microiontophoresis of HRP such that minimal current strengths and durations were applied. In addition to a sham injection, control injections were also made to the , , deep tegmental , mesen- cephalic and brachium conjunctivum. Following HRP injections confined to the areas of the VTA containing the cell groups, labelled appeared in , dorsal bank of rhinal sulcus, , bed nucleus of , , , , magnocellular , medial and lateral preoptic areas, anterior, lateral and postero-dorsal , later- al habenular, nucleus parafascicular nucleus of , , nucleus raphe dorsalis, nucleus raphe magnus and pontis, dorsal and ventral , and . Regions con- taining groups A 1, A 5, A 6, A 7, A 9, A 13 and the group B 7 corresponded to the topography of labeled cell groups. Injections of HRP to the interfascicular nucleus resulted in labeling predominantly confined to the medial habenular and median . The results are discussed in relation to the known connections of these regions. Other regions of the labelled by VTA injections are assessed in relation to control injections and the limitations of the HRP technique. A review of the organisation of some of these afferents in relation to the known cortical-subcortical-mesencephalic projection systems, suggests that the VTA is in a position to receive information from a massively convergent system derived ultimately from the entire archi-, paleo-, and neo-cerebral cortices. In addition A 10 dopaminergic neurons are known to project to restricted regions of both pre-frontal and entorhinal cortices, which themselves also receive massively convergent association cortico-cortical connections. It would appear reasonable to propose that these neurons perform a correspondingly important integrative function.

A considerable body of evidence exists to known as the A 10 group (Dahlstrom and support the hypothesis that dopaminergic sys- Fuxe, '64) and in the rat project to many "lim- tems in the central provide a bic'' sites as well as to the frontal site of action for the antipsychotic actions cortex. (Ungerstedt, '71; Berger et al., '74; of different chemical classes of neuroleptic Fuxe et al., '74; Lindvall et al., '74a; Lindvall, (Burt et al., '75;Seeman et al., '75;Iver- '75; Lindvall et al., '78). In addition, recent sen, '75). The dopaminergic neurons of the ventral tegmental area of Tsai (VTA) are i Visiting Scientist, Wellcome European Travelling Fellow

J. COMP. NEUR. (1979) 187: 117-144. 117 118 0. T. PHILLIPSON autoradiographic studies have demonstrated HRP were placed in different regions of the descending projections from VTA to parame- ventral mesencephalon in 16 female adult dian structures (Domesick et al., Sprague-Dawley rats (145-180 g) by the tech- '76). Findings obtained with pathway tracing nique of microelectrophoresis (Graybiel and methods have given some information about Devor, '74). Glass micropipettes with bevelled the of their afferent supply. Antero- tips (external diameters 20-40 pm) were pre- grade degeneration experiments have sug- pared and filled with a 40% solution of HRP gested that these pathways arise in septa1 (Sigma, type VI) in 0.1 M NaOH. Rats were area (Nauta, '561, lateral hypothalamic region anaesthetised with chloral hydrate (400 (Guillery, '57; Wolf and Sutin, '66); lateral mg/kg) and positioned in a Kopf stereotaxic preoptic area (Nauta, '581, and lateral haben- frame. Electrodes were lowered with a retain- ula (Agaki and Powell, '68). More recent auto- ing current to target sites determined from radiographic evidence has confirmed these Konig and Klippel's ('63) stereotaxic atlas. projections (Swanson, '76; Arbuthnott et al., Particular care was taken to avoid as far as '76; Herkenham and Nauta, '77a) and pro- possible the major neighbouring nuclei and vided additional evidence of afferents to VTA fibre tracts during penetration. HRP was from substantia innominata (Swanson, '76; ejected with a constant positive current of 1 Meibach and Siegel, '771, diagonal band, bed pA for 5 to 17 minutes (microiontophoresis nucleus of stria terminalis, medial preoptic programmer, model 160, W-P instruments, area, anterior hypothalamus (Swanson, '76; Inc. U.S.A.). In some cases intermittent cur- Conrad and Pfaff, '76a,b; Meibach and Siegel, rent was applied, with one minute on and one '77) and (Conrad et al., minute off. '74; Bobillier et al., '75; Taber-Pierce et A retaining current was reapplied during al., '76). withdrawal. About 24 hours later, rats were More comprehensive information about the deeply anaesthetised and perfused through afferent supply to VTA neurons must be ob- the heart with 100 ml saline followed by 500 tained, however, in order to understand how ml 1% paraformaldehyde and 1.25%glutaral- changes in activity in this predominantly do- dehyde in 0.05 M cacodylate buffer pH 7.2-7.4 paminergic group can arise. This in turn with 5%sucrose. were removed imme- should help to clarify how the connections of diately and postfixed for one to two hours in neuronal systems, which are suspected as the the same fixative in 30% sucrose, and finally targets for antipsychotic action, relate to rinsed overnight in 0.05 M cacodylate buffer in the various clinical aspects of the psychotic 30%sucrose at 4°C. Frozen sections were cut syndrome. at 40 pm in frontal plane and processed imme- The present study by the method of retro- diately according to the highly sensitive grade labelling with horseradish method of Hardy and Heimer ('77) using the peroxidase (HRP) (Kristesson and Olsson, '71; non-carcinogenic substrate tetramethylben- La Vail and La Vail, '72; La Vail et al., '73) zidine (TMB). Sections were counterstained in was undertaken in order to obtain such infor- neutral red and examined for HRP positive mation, and in addition to gain an accurate neurones under bright field conditions. Con- identification of the site of origin of the affer- trol injections were made in addition into me- ents. This is difficult to obtain with antero- dial lemniscus, red nucleus, tegmental de- grade techniques because the cyto-architec- cussations, midbrain reticular formation, and ture of many of the sources of afferents, such superior cerebellar decussations and also by a as the area, is heterogeneous sham injection in which the electrode was and diffuse. In addition to confirming previ- lowered into VTA but no HRP ejected. ously described afferents, the results of the In order to minimise damage at the injec- present study suggest so far unknown systems tion site and to limit the spread of HRP projecting to the VTA from cerebral cortex, around the needle tip preliminary experi- amygdala, hypothalamus, thalamus, tectum, ments were carried out in seven animals to and medulla. assess the minimum positive current strength A preliminary report of some of the present and duration which would eject sufficient findings has been published previously (Phil- HRP for retrograde transport. Additional ex- lipson, '78). periments were also carried out in order to assess the degree of local spread of HRP at the MATERIALS AND METHODS injection site at short survival times. A total Small unilateral and midline injections of of ten animals were thus sacrificed at 0.25, AFFERENTS TO VENTRAL TEGMENTAL AREA 119

2,3, 6, 14 and 24 hours after HRP injection was “squeezed” between the local tissue land and the degree of spread of HRP around marks e.g., the medial lemniscus, interpedun- the pipette tip assessed in non counterstained cular nucleus and ventral border of the brain. sections. Nevertheless, the approximate diameter at 24 Neurons labelled by retrograde transport in hours was about one-third of that seen at four the main series of experiments were counted hours; the point of maximal spread seen. This and categorised into cytoarchitectonic groups. rapidly reduced by about six hours to a diam- Examples of sections bearing labelled neurons eter only slightly greater than that measured were then plotted for visual inspection at rep- at 24 hours. Neurons within the margin of the resentative levels of the neuraxis. expanded HRP stain showed an even faint blue staining. Such neurones were observed in RESULTS red nucleus, medial and a Preliminary experiments very few neurones in the lateral rim of the in- Different current parameters were applied terpeduncular nucleus, at one-fourth and four to a 40% HRP solution to determine the hours after a VTA injection. Dark granular minimum strengths and durations necessary reaction product was seen within neurons in to elicit measureable retrograde transport the same sites (but not the interpeduncular from VTA to basal forebrain areas. Since re- nucleus) at the 14 and 24 hours survival producible retrograde transport in this path- times, although in very much smaller num- way was not obtained below 1,000 nA for any bers. At the one-fourth hour survival time, length of time of application, this was the cur- marked light evenly blue axonal labelling was rent selected for routine use. Even when the seen in the fibres of the oculomotor total current x duration product was the which ran through the injection site, while same for very low current strengths (e.g., 100 uptake to a lesser extent was also observed in nA for 100 minutes), as that used for higher medial lemniscus, , mam- currents (e.g., 1,000 nA for 10 minutes), and millary peduncle, ventral tegmental decussa- presumably the same total quantity of HRP tions and reticular formation around the nee- ejected from the needle tip, adequate retro- dle track. It was noticed that although the grade transport with low currents was not ob- oculomotor fibres were heavily labelled in tained. In such cases labelling was confined to these experiments, only occasionally did la- local glial cells, vascular endothelium and belled neurons appear in the oculomotor nu- some nerve fibre uptake. Thus demonstrable clei. This indicated that although retrograde retrograde transport may only occur when transport occurred from fibres of passage la- HRP is ejected faster into the extracellular belled by this procedure, it was uncommon rel- space than the capacity of local uptake proc- ative to the number of fibres taking up the en- esses in vascular endothelium, glial cells and zyme. This conclusion, however, could only be to remove the HRP, and this rate is only applied to undamaged axons of passage; later achieved at current strenghts greater than 1 experiments showed clear evidence of uptake PA. Naturally, such low current strengths re- and retrograde transport from damaged fibres sulted in only low numbers of labelled neu- of the medial lemniscus. At 24 hours after rons. This disadvantage, however, was bal- injection, glial and vascular endothelial cells anced by a higher accuracy in interpretation, also accumulated HRP and this occurred resulting from minimum tissue damage. mainly in the territory which had been oc- The spread of HRP into the surrounding tis- cupied by the expanded HRP stain, at early sues at a delivery current of 1 pA for seven survival times. minutes was then assessed after injection of the VTA. The main finding of these experi- Injection sites ments was that there was rapid expansion and Figure 1 shows an example of the appear- contraction of the injection spot soon after ance of the injection site after 25 hours sur- current application. The spread at short inter- vival in case 15. The reaction product is large- vals after injection was much greater than ly confined to the VTA and its spread has been that suspected from examining tissue fixed 24 limited laterally by the medial lemniscus and hours after operation. also apparently ventro-medially since it did Exact determination of the diameters of not extend (except at its lowest margins) into spread was complicated because (1) the reac- the . Outside the tion product was rather granular and the margin of the main HRP injection, accumula- margin not well defined; and (2) the staining tion of reaction product can be seen in 120 0. T. PHILLIPSON perivascular and glial cells and in the pia on concomitant labelling of other VTA afferents the ventral surface of the brain. was to be expected (table 1). Figure 2 shows diagrammatically the injec- tion sites of all cases in which successful injec- (2) Injections to the red nucleus. (Case 26) tions were made into dopaminergic cell groups Large numbers of neurons appeared in dor- (cases 16, 10, 11, 32, 15, 21, 12, 31, 29) and the solateral cerebral cortex, but none in supra- control injection sites (cases 25, 17,26, 22, 28, rhinal, cingulate or prefrontal cortex. Heavy 14 - sham injection control is not shown). labelling of (several hundreds of Control injections. (Tables 1 and 2, neurons) and some labelling of the entopedun- right block) cular nucleus were observed. Many neurons were also labelled in the oculomotor nuclei, su- (1) Injections into medial lemniscus perior colliculus and pontine reticular forma- In case 25, a fairly large injection was made tion, while many neurons were also seen in the into medial substantia nigra compacta and principal nucleus of V. The lateral VTA. The needle track, however, in- was heavily labelled (116 neurons). In case 11, stead of avoiding medial lemniscus as in other where much of the HRP injection spread also cases, was deliberately aimed through the me- to red nucleus, 67 neurones were observed in dial edge of the lemniscus, (fig. 2). In this case, the dentate nucleus. In other cases where unlike injections to VTA alone, very large VTA alone was injected, only a few labelled numbers of labelled neurons appeared in the neurons were observed in the dentate nucleus. principle nucleus of V. (264 neurones, com- A considerable number of labelled neurons ap- pared to a total of 16 for all cases of HRP peared in the after injection injection to VTA alone). In case 17, where a of the red nucleus. rather small HRP deposit was made directly into the medial edge of medial lemniscus, (3) Mesencephalic reticular formation. again a large number of labelled neurones ap- (Case 22) peared in the principal nucleus of V (101 neu- The injection was centered on the reticular Po). Since dopamine neurons of the VTA ex- formation dorsal to medial lemniscus and lat- tend into the medial edge of the lemniscus, eral to the red nucleus. Labelled cells were

A bbreuiations aac, , anterior limb MR, ac, anterior commissure mt, mammillo-thalamic tract ABL, basal amygdaloid nucleus, pars lateralis NA, nucleus accumbens AC, central amygdaloid nucleus ntV, nucleus spinal tract of ALA, lateral amygdaloid nucleus, pars anterior och. optic AM, medial amygdaloid nucleus ON, olivary nuclei AVT, ventral tegmental area of Tsai ot, BST, bed nucleus of stria terminalis OT, cc, crus cerebri OTpo, olfactory tubercle, polymorphic layer DBB, diagonal band of Broca OTpy, olfactory tubercle, pyramidal layer EP, entopeduncular nucleus p, f, poma, mammillary peduncle fr, fasciculus retroflexus pcs, superior cerebellar peduncle GP, PN, paraventricular nucleus of hypothalamus IP, interpeduncular nucleus R, red nucleus LC, nucleus linearis raphe pars caudalis SI, substantia innominata LH, SNC, substantia nigra LHa, lateral SO, LPO, lateral preoptic area sm, stria medullaris ml, medial lemniscus ZI, Zona incerta MaPO, magnocellular preoptic area 111, third cranial nerve MPO, medial preoptic area VII, seventh cranial nerve

Fig. 1 Microphotographs (a) and (b) illustrate the centre of the injection site in the same section in caae 15. (a) shows the full extent of spread of HRP reaction product (compare to diagram A1610, fig. 2). (b) shows the same section at a higher magnification and a different exposure to illustrate the extent of the densest central HRP deposit. The dot- ted line indicates the dorsal border of interpeduncular nucleus. Note how the HRP does not spread ventrally to the in- terpeduncular nucleus and is limited laterally in its spread by the medial lemniscus. Note also how perivascular cells outside the margin of the injection site accumulate HRP. In this case an ejection current of 1 pA was applied for seven minutes and the animal sacrificed at 25 hours after injection. Bar, 200 gm. AFFERENTS TO VENTRAL TEGMENTAL AREA 121

Figure I 122 0.T. PHILLIPSON

I0 Fig. 2 Drawings of the central part of the injection site in different regions of the mesencephalon. The thick line represents pipette track. The area of densest extracellular HRP deposit is indicated by the hatched area and a more lightly stained zone by the clear surround. The present results suggest that only tissue very close t.o the point of ejection is taken up and transported in sufficient amounts to be detected by the present method (see technical discussion). No indication, thus, is given of the antero-posterior extent of the HRP deposit. Coordinates are according to Konig and Klip- pel ('63). concentrated in the superior colliculus and pipette was lowered into the VTA but no HRP substantia nigra , while only ejected by current, no labelled neurons could scattered labelling was seen elsewhere. be observed in any part of the brain. This ex- cludes the possibility in the brain that endoge- (4) Deep tegmental decussations. (Case 28) nous peroxidase activity, as recently shown Here the injection site centered on an area for monkey brain, could account for labelled dorsal and medial to the red nucleus and dor- neurons (Wong-Riley, '76). sal to the main body of dopaminergic neurons Sites of afferents detected after injections of the VTA. Both dentate nucleus and red nu- confined to VTA cleus were heavily labelled in this case, while the parabrachial nuclei were also labelled, but (1) Afferents from cortical regions to much lesser extent. Taking all cases of VTA injection together, labelled neurons appeared most frequently in (5) Sham injection the prefrontal cortex (deep layers) and to a In one case where the HRP containing lesser extent in the dorsal bank of rhinal AFFERENTS TO VENTRAL TEGMENTAL AREA 123

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Fig. 3 Positions of labelled neurones in the frontal and in three cases. Each dot represents the posi- tion of a single neuron in a representative section. Number in the left hand margin indicates the experimental animal. sulcus. Prefrontal cortex described here corre- lar insular area and less commonly in ven- sponds to two cytoarchitecturally distinct re- trolateral orbital area (Kerttek and Price, gions (1) the infralimbic area of Rose and '77). Their distribution corresponded closely Woolsey ('48) (equivalent to area 25 of Brod- with the sites of dopaminergic terminal inner- mann, '09) and (2) the prelimbic area of Kret- vation in those regions (Lindvall et al., '78). tek and Price ('77) (equivalent to area 32 of Occasionally, labelled cells also appeared in Brodmann). Dorsal bank of rhinal sulcus con- the dorsal division of the anterior cingulate tained labelled neurons in the ventral agranu- area (part of area 24 of Brodmann), (table 1, AFFERENTS TO VENTRAL TEGMENTAL AREA 125 fig. 3). Neurons only appeared in dorsolateral and may correspond to of parts of cortex when the injection site en- (Nauta and Haymaker, ’69). This croached on the red nucleus, while injections nucleus also contained prominently labelled confined entirely to the red nucleus resulted large neurons in most cases (fig. 9). When in a label only in dorsolateral cortex and none taken together, labelled neurons of both limbs in pre-frontal cortex or dorsal bank of rhinal of diagonal band, substantia innominata and sulcus (case 11 and 26, table 1). No cortical magnocellular preoptic area, make up a prom- neurons were labelled after injections of the inent component of the afferent systems to interfascicular group in the mid-line. VTA as a whole. Neurons labelled in these structures were usually large but medium (2) Afferents from basal forebrain regions. sized neurons also occurred. In primates (Fig. 4) (Jones et al., ’76) such neurons may comprise a The terminology used in the description of single complex stretching from vertical limb this section is based on that of Swanson (‘76). of diagonal band anteriorly to nucleus basalis Labelled neurons were found in the medial posteriorly, and in rats a similar chain of segment of nucleus accumbens anteriorly (fig. “magnocellular nuclei of the basal forebrain” 81, the central segment of accumbens merging has been described (Divac, ’75). with the subcommissural portion of the bed (3) Afferents from hypothalamic areas. nucleus of stria terminalis more posteriorly, (Fig. 5) and the posterior segment of the bed nucleus caudal to the anterior commissure. Neurons of The lateral hypothalamus was labelled in the accumbens and rostra1 bed nucleus were all cases, and appeared to provide one of the small and round and in the caudal bed nucleus principal sites of afferents to the VTA. The la- medium sized and stellate or spindle shaped. belled neurons were usually spindle shaped Although neurons in the bed nucleus were la- and oriented in a dorsoventral axis slanted belled after injections of red nucleus (table 11, medially. Neurons were surrounded by many no excess labelling was observed over that labelled axons travelling towards forebrain seen in many cases of VTA injections, and la- sites in the . In the belling was less than that seen in some VTA minority of cases, neurons were labelled in the injections. Many small to medium sized neu- anterior hypothalamus and posterior and dor- rons were found to be labelled in lateral preop- sal regions of the hypothalamus ventral and tic area (fig. 41, and in smaller numbers also in medial to the mammillo-thalamic bundle (fig. the medial preoptic area (fig. 9). Labelled neu- 5, case 16). More posteriorly labelled cells rons were observed anteriorly in the vertical were observed amongst the assembling fibres limb of the diagonal band, and further poste- of the . Neurons were riorly in larger numbers in the horizontal limb labelled in the itself only where they merged with the large and me- when the injection site encroached upon the dium sized cells of the substantia innominata. mammillo-tegmental fibres, which runs im- Labelled neurons were observed in substantia mediately ventral to the VTA. innominata particularly at the level of the an- (4) Afferents from . terior commissure, and in the polymorphic (Fig. 5) layer of olfactory tubercle. These smaller cells of olfactory tubercle were much less numerous An important source of fibres to VTA was than the large cells of the overlying substan- from the lateral habenula. This nucleus was tia innominata diagonal band and magnocel- labelled in every case. Neurons were only la- lular preoptic area. The most dorsal compo- belled in the medial habenula following injec- nent of substantia innominata identified by tions confined to the mid-line interfascicular Heimer and Wilson (’75) as “ventral palli- group (table 1, cases 31, 29). In no case of dum” also contained some labelled neurons injection of VTA alone was label observed in (fig. 9). More posteriorly and laterally the the medial habenula indicating that although cells of the substantia innominata appear to demonstrable HRP activity was almost cer- aggregate into the magnocellular preoptic tainly present in the interpeduncular nucleus area (Swanson, ’76). This nucleus has also shortly after injection (Preliminary experi- been called the nucleus of the horizontal limb ments), that this was insufficient to elicit of the diagonal band (Price and Powell, ’70) detectable retrograde transport. 126 0. T. PHILLIPSON

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Fig. 6 Cytoarchitectonic position of labelled neurons in the amygdala.

(5) Afferents from parafascicular nucleus zona incerta and both regions of Forel’s fields. A few labelled neurones were observed in Since however there was a marked increase in this area in every case of VTA injection except labelled neurones in both regions when the one. Earlier experiments not reported here needle tract passed through medial lemniscus showed that after the ejection of large quan- or the injeciton was in the lemniscus itself tities of HRP to the VTA, this nucleus was these results must be treated with caution fairly heavily labelled. (table 1). Injections of red nucleus, also led to large number of neurons appearing in zona in- (6) Afferents from amygdala. (Fig. 6) certa. Thus it seems likely that after VTA Labelled neurones were found in the amyg- injections, the labelled neurons in these re- dala in a minority of cases. In the case result- gions resulted from labelling of damaged ing in most labelled neurones, the injection fibres of passage in the medial lemniscus or site was in the anterior portion of VTA. Neu- of labelling of synaptic terminals in the red rons were observed in anterolateral, basolat- nucleus. eral, medial and central divisions. The number of labelled neurons was small. (8) Afferents from midbrain regions (7) Afferents from zona incerta and The superior colliculus was labelled in all Fields of Fore1 cases of VTA injections. The neurons labelled always appeared in the deeper layers (album Most cases contained labelled neurones in profundum, album mediale griseum profun- 0 +++ 0 +++ + +++ 0 0 + + + + +++ ++ + sna~anuaqsquaa urnlla9aJa3 '111 w4ooooo+ ooooo++o+ sysia381 ap~na!qaisnapnN 3 0 0 + t++ 0 + ooooootoo Fapnu ~~qnq!qsa~ O+O++t ooooo++++ snalniaoa sn3g el 0 + + ++t + ++ 00+00+t0+ sI$uod sp3[n3gai snapnN 2 0 +++ + + ++ +++ 0 0 ++ 0 + + +++ + ++ s!pqasJqsisd snapn~ ooo+o+ 0 0 + 0 + 0 + ++++ snqsauna snapnN B 0 0 0 +++ +++ +++ 0 0 0 0 o+++ 0 0 AUsnapnu p?d!auIrd ~ooo+o+00000++t+ s!yod aqdsi snapnN r3 0 0 o++++ ooooo++++ snuFlsm aqdwi snapnN cl 000000 +++ + + + 0 0 +++ 0 + snus!paur aqdw snapnN 4 + ++ 0 0 ++ ++ + + +++ + ++ + +++ ++ +++ s![ss.~opaqdsi snapnN ~+oooooo+o+ooo++ S~BJ~SOJsyvaug snaIanN z1 Wll?lpJW SUOd '11 0 0 I++ 0 0 ++ 0 0 + 0 + O+++++ sfi!u s!$us'+sqnS g +++ +++ + + + 0 +++ 0 0 +++++ 0 snapnu pag +oo++o +o+ooo+++ xqdmoa Jolourolnao r" + 0 + 0 + +++ +++O+O+O+ XaQ 1squa3 z; + + ++ +++ + ++ + 0 + + + + ++ ++ ++ sn1na!1Ioa Jo!iadnS p: uolwyda3uasaH '1 f2I% ?.I 82 zz 9Z Ll sz 6Z I€ ZI IZ '21 Z€ 11 01 91 4 waw suo!iaaru! p~iu~ ednoa8 [laa +3aau!msdop 07 suog3aruI 130 0. T. PHILLIPSON AFFERENTS TO VENTRAL TEGMENTAL AREA 13 1

Fig. 8 Cytoarchitecture of region8 containing labelled neuron8 in case 16. a. Anterior septa1 reigon and nucleus accumbens. Arrows indicate position of labelled neurons in the accumbens; b. magnification of area in a (box) to show large and small (arrow) labelled neuron ventral to nucleus accumbens and dorsal to the olfactory tubercle; c. lateral hy- pothalamic area; d. magnification of box in c. durn, according to Konig and Klippel, '63). tion to VTA is open to question. The few la- Injection of the red nucleus and other control belled neurons appearing in the oculomotor areas also gave rise to considerable labelling nuclei were probably due to HRP entering of the superior colliculus (table 2). Since also damaged or undamaged oculomotor rootlets as the needle track inevitably traversed many they course through the ventral tegmental re- tectotegmental fibres the specificity of projec- gion, (see discussion in technical section). 132 0. T. PHILLIPSON Similar caution relates to neurons labelled in sionally labelled neurons were seen in or near the red nucleus. In cases of injection to the an- the lateral segment of nucleus reticularis lat- terior VTA, heavily labelled neurons were ob- eralis. Their position was equivalent to the re- served in substantia nigra compacta. Since ef- gion containing the A 1 catecholamine group ferent axons of compacta neurons assemble in (Dahlstrom and Fuxe, '64; Palkovits and the medial edge of substantia nigra compacta Jacobowitz, '74). Furthermore their cell size (Lindvall and Bjorklund, '74) some of this la- and dendritic morphology were distinct from bel could represent uptake and retrograde nearby neurons which were occasionally la- transport of HRP in damaged axons. belled in the principal nucleus of fifth nerve laterally and dorsally. This nucleus was heavi- (9) Afferents from pons and medulla ly labelled after injections of red nucleus or Neurons in the dorsal raphe nucleus were medial lemniscus (table 2) indicating that prominently labelled in all cases. Of all the light labelling after VTA injections was prob- nuclei throughout the brain, this nucleus was ably not significant. (Control injections). the most heavily labelled. More caudally, la- belled neurons occasionally appeared in nu- (10) Other afferents cleus raphe magnus and pontis. Large injec- Neurons in the deep cerebellar nuclei were tions of the VTA which involved the midline frequently labelled by injections confined to interfascicular catecholamine group gave rise the VTA. Heavy labelling of the dentate nu- to labelled neurons in the median raphe nu- cleus was obtained after injections of red nu- cleus particularly its dorsal segment (fig. 7, cleus and light labelling obtained after injec- case 11). Smaller laterally placed VTA injec- tions confined to the anterior VTA (table 2, tions did not label this nucleus (fig. 7, case 161, case 16). Most prominent dentate labelling in whereas injections confined to the inter- VTA injections was obtained when the injec- fascicular nucleus gave rise to labelling of tion area overlapped the borders of the red nu- many median raphe neurons (table 2, cases cleus e.g., case 10. In every case of VTA injec- 31, 29). tion the pipette track inevitably traversed the In more lateral positions neurons were la- dense cerebellar-rubral projections either at belled in caudal portions of nucleus cuneatus the tegmental decussations or the cerebellar amongst fibres of the cerebellar peduncle in peduncles more posteriorly. Vestibular neu- an area containing the A8 catecholamine rons were occasionally labelled but much less group (Palkovits and Jacobowitz, '74). Fur- frequently than control injections to red nu- ther posteriorly, many neurons were also la- cleus or medial lemniscus. belled in both dorsal and ventral divisions of the nucleus parabrachialis. In most cases the (11) Contralateral projections ventral division contained more labelled neu- In case 12 where HRP was deposited in the rons than the dorsal. Many of these ventrally most caudal and midline dopamine group, the located parabrachial neurons were located nucleus linearis raphe caudalis, afferents within an area containing the A 7 catechol- were distributed about equally to both sides of amine group (nucleus subcoeruleus). As the the brain. In cases 29 and 31, where HRP was ventral parabrachial group was followed pos- deposited in the midline interfascicular nu- teriorly and medially, labelled neurones ap- cleus, again the main afferent source, from peared in positions immediately ventral to the the medial habenular nucleus, was repre- locus coeruleus and also, though only in small sented bilaterally. numbers, in the locus coeruleus itself (A 6)in In all other cases of unilateral VTA injec- the periaqueductal grey. tions, only occasionally labelled neurones The reticular nuclei of the pons contained were observed contralateral to the injection occasionally labelled neurones, but much site, with the exception of the deep cerebellar larger numbers were observed after red nu- nuclei and oculomotor nuclei which were cleus injections (table 2). In some cases la- Fig. 9 a. Cytoarchitecture of basal forebrain at level of belled neurons were also observed around the anterior cornmisswe. Dots represent position of neurons roots of the VII nerve towards the ventral sur- labelled and their relative density in this and other simi- face of the brain in the region where neurons lar sections. b. Illustration of a large labelled neuron in of the A 5 catecholamine group have been de- horizontal limb of diagonal band and smaller neurons in lateral preoptic area; c. Large labelled neuron in mag- scribed (Dahlstrom and Fuxe, '64; Palkovits nocellular preoptic area clcae to polymorphic layer of the and Jacobowitz, '74). Further caudal, occa- olfactory tubercle. AFFERENTS TO VENTRAL TEGMENTAL AREA 133

Figure 9 134 0. T. PHILLIPSON usually labelled contralateral to the injection, bodies at 24 hours survival. Similarly, after and superior colliculus which was frequently injections of the VTA which avoid direct dam- labelled contralateral to the side of injection. age to the medial lemniscus, few or no labelled DISCUSSION neurons were seen after 24 hours survival in the principal nucleus of the fifth nerve, whose (1) Technical ~UC~OFS efferent fibres are well known to travel in the It has recently been clearly demonstrated medial edge of the medial lemniscus (Walker, that the technique of microiontophoresis of '39).However, after direct damage to the lem- HRP in the will not niscus, large numbers of labelled neurons ap- only label neuron cell bodies as a result of peared in this nucleus. This effect is very simi- uptake and retrograde transport of enzyme lar to the result obtained in the peripheral from terminals, but will also label neu- nervous system by Oldfield and McLachlan rons whose axons pass through the site of ('77) using the diamino-benzidine substrate. injection and are damaged by the injection Thus axons will take up HRP even if not dam- procedure (Herkenham and Nauta, '77b). aged. However, retrograde transport will not Thus although there are advantages in using occur, except to a minor extent, unless axons the microiontophoretic technique, in that ex- are cut or damaged directly. tremely small deposits of HRP can be made, In summary, differentiation between neu- there is still a significant risk of labelling neu- rons labelled by terminal uptake and those rons by "injecting" damaged fibres of passage, from damaged axons of passage is not possible as had been earlier noticed in the case of pe- with the present technique alone. Only ultra- ripheral whose axons had been delib- structural or physiological evidence can defi- erately transected or damaged (Kristensson nitely confirm the presence of a pathway. and Olsson, '74; Halperin and La Vail, '75; Since the VTA is an area densely traversed by Furstman et al., '75; Kuypers and Maisky, '75; many fibre tracts of different origin, this fac- Kristensson and Olsson, '76; Oldfield and tor must strongly influence the interpretation McLachlan, '77). It is possible that the type of of the present results. reaction product seen within the neuron can Finally, it is not certain to what extent HRP give an idea of whether or not the cells are la- which spreads from the pipette tip to synaptic belled from injured axons; evenly filled non- regions outside VTA will be taken up and granular reaction product indicating filling of transported to cell bodies. This factor might be a damaged fibre and granular reaction prod- of importance because the expanded HRP uct indicating axon terminal uptake (Jones injection spot observed at short intervals after and Leavitt, '74; Turner and Harris, '74; Hal- injection invaded many neighbouring struc- perin and La Vail, '75; Colman et al., '76; tures, even at such low ejection currents as 1 Parent, '76). Unfortunately, however, neurons PA. Our preliminary experiments showed ear- labelled from cut or damaged nerves do not ly staining of red nucleus, medial substantia always show even staining (Furstman et al., nigra, and slightly, of lateral interpeduncular '75); while with the highly sensitive TMB nucleus after test VTA injections. However, method used in this study the increased in animals killed 24 hours later no labelled amount of reaction product in neurons well la- neurons were seen in regions projecting to belled by retrograde transport may give a those nuclei e.g., dorsolateral cerebral cortex; rather solidly stained profile (Phillipson, '78). or medial habenula respec- The appearances of the reaction product is tively. Neurons labelled in the deep cerebellar thus of no value in distinguishing those neu- nuclei (a site of afferents to red nucleus) are rons labelled from damaged fibres of passage. discussed below (DISCUSSION: Control injec- A related technical problem is the degree to tions). Thus synaptic sites invaded by the pe- which surrounding undamaged fibres will riphery of the HRP spread, did not result in take up HRP ejected from the pipette and sub- detectable retrograde labelling. The area of ef- sequently transport it to the neuronal soma. fective uptake appears to be restricted to tis- The present results from VTA injections show sues very close to the pipette tip. This result that although fibres of passage in the oculo- agrees with that obtained with the diamino- are prominently labelled soon benzidine substrate by Vanegas et al. ('78) after injection, there is only a very small who found similar rapid expansion and con- degree of resultant labelling of neuronal cell traction of the HRP spot following hydraulic AFFERENTS TO VENTRAL TEGMENTAL AREA 135 injections to the and also that (3) Afferents from cortical regions uptake and transport occurred only from sites The present results showing labelling of close to the needle tip. Neurons may also become labelled by a di- prefrontal cortex after HRP injection to the VTA are compatible with earlier degeneration rect route by means of vesicular uptake of ex- studies in which lesions of large areas of fron- tracellular HRP by dendrites or neuronal peri- tal cortex in the monkey gave rise to de- karya (Turner and Harris, '74). This may generation in or near the VTA (De Vito and account for occasionally labelled neurons seen Smith, '64; Nauta, '64). More restricted le- in red nucleus, substantia nigra and lateral sions confined to medial frontal cortex in the margin of interpeduncular nucleus in the re- monkey, however, gave rise to preterminal de- gion of early expansion on the HRP spot. La- generation only in substantia nigra, (Leich- belled neurons observed close to the injection netz and Astruc, '76). Comparisons of results site should thus be treated with caution al- obtained from rat and monkey, however, are though real short projections cannot be dis- hazardous because of the difficulty of accu- counted by these methods. rately defining homologous cortical regions. (2) Control injections Moreover, negative results obtained with silver techniques used by these authors does It is clear from the results obtained in cases not exclude the presence of a pathway. 25 and 17, that the principal nucleus of V did not provide a projection system to the VTA, Efferent dopaminergic fibres from VTA in- and that any labelled neurons seen in this nu- nervate exactly those deep cortical regions cleus were due to labelling of fibres travelling shown here to contain HRP labelled neurons '78). Thus it seems probable to the thalamus in the medial edge of the me- (Lindvall et al., dial lemniscus. These results are in agreement that in the rat there is a reciprocal loop be- with the arrangement of projection pathways tween VTA and prefrontal cortex, and to a lesser extent also the dorsal bank of rhinal of the V nerve nucleus found by Walker ('39) in the monkey. A similar conclusion can be sulcus and the cingulate cortex. There is evi- made about neurons labelled in the vestibular dence that this prefrontal VTA loop may have nuclei, , zona incerta, oculomo- an important role to play in motor behaviour tor complex, and pontine reticular formation. (Tassin et al., '78). The superior colliculus is discussed below. In (4) Afferentsfrom subcortical dopamine the case of the red nucleus, it would seem rich areas simplest to assume that labelled neurons ap- pearing in the deep cerebellar nuclei resulted Afferents to VTA from nucleus accumbens from labelling of damaged fibres of passage in described here confirm with a retrograde tech- the cerebello-rubral pathway running in the nique previous reports in the rat in which an- tegmental decussations, or labelling due to terograde autoradiographic methods have spread of HRP into the synaptic terminal area been used (Swanson and Cowan, '75; Conrad of this pathway in the red nucleus itself. How- and Pfaff, '76c; Nauta et al., '78). The results ever, recent investigation of the cerebellar- of these workers, however, show that the mesencephalic projections with the Fink- accumbens-VTA projection is a minor one Heimer technique has suggested the presence compared to the accumbens-substantia nigra of a pathway from deep cerebellar nuclei to (pars compacta) projection. This may account ventral mesencephalic dopaminergic neurons for the failure of other studies to identify the (Snider et al., '76). The present VTA HRP VTA input from accumbens (Powell and Le- injections did not label the well documented man, '76; Williams et al., '77). The present re- cortico-rubral tracts, and were mostly made sults, in agreement with the autoradiographic anterior to the cerebello-rubral fibres crossing findings of Nauta et al. ('781, and Troiano and in the main in the brachium conjunctivum. Siege1 ('78) show that the main input to VTA Therefore, labelling of deep cerebellar nuclei is from the medial segment of nucleus accum- may indicate, in agreement with Snider et al., bens, as well as from the more ventral and pos- a cerebello-tegmental pathway innervating terior accumbens as it merges with bed nu- VTA neurons. Clearly further details of such a cleus of stria terminalis. It is interesting, also, pathway at electron microscopic levels, or that the most caudal and ventral neurons la- physiological investigations, are necessary. belled in frontal cortex occurred very close to 136 0. T. PHILLIPSON the labelled neurons in the anterior and medi- were not labelled by injections to the VTA oc- al border of accumbens and in some sections cur in globus pallidus. These neurons may pro- the borders between these two structures were ject to pars compacta of substantia nigra hard to define. (Hattori et al., ’75; Carter and Fibiger, ’78). Judged by the numbers of neurons labelled in all cases, however, the accumbens is not a (6) Afferents from preoptic regions major source of afferents to VTA. This is in The present results showing afferent projec- sharp contrast to the striato-nigral system tions to VTA from both medial and lateral where an extremely heavy projection exists preoptic areas agree well with autoradi- (Bunney and Aghajanian, ’76) accompanied ographic data of Swanson (‘76) and Conrad by marked development of the pars reticulata. and Pfaff (‘76a). Furthermore, like Swanson, It is interesting that there is no well developed the presents results suggest the projection analog to pars reticulata in the VTA. from the lateral preoptic area is more promin- Olfactory tubercle also provides some affer- ent than that of medial preoptic area. These ent fibres and the neurons of origin are usual- VTA projections seem to arise from neurons ly situated in the deep polymorphic layers. which are cytologically and cytoarchitectural- This pathway has not so far been described. ly distinct from those of the magnocellular Bed nucleus of the stria terminalis, both basal forebrain system. dorsal and ventral divisions provided afferent fibres, a finding in agreement with the autora- (7) Afferents from hypothalamic regions diographic data of Swanson (‘761, Conrad and The lateral hypothalamic region was the Pfaff (’76a) and Meibach and Seigel (‘77). most consistently and heavily labelled hypo- thalamic group. These results are in agree- (5) Afferents from other basal ment with the results of fibre degeneration forebrain regions (Guillery, ’75; Nauta, ’58; Wolf and Sutin, ’66) Fibres from the vertical and horizontal and autoradiographis data (Arbuthnott et al., limbs of the nucleus of the diagonal band have ’76; Nauta et al., ’78) in the rat which show been described with anterograde methods by prominent fibre bundles projecting to VTA as Meibach and Seigel (‘77) and Conrad and Pfaff well as to substantia nigra. (‘76a) to project the VTA. The present experi- The anterior hypothalamus also contained ments, confirm those results with a retrograde labelled neurons, though their numbers were method and show that such fibres originate small. Autoradiographic and degeneration from, large neurons near the apex of the verti- evidence has been obtained for such a path- cal limb, and large and medium sized neurons way in rat (Conrad and Pfaff, ’76b). in the middle and horizontal limbs of diagonal The posterior and dorsal regions of hypo- band. Large labelled neurons were also seen in thalamus were labelled to a minor extent and the horizontal limb to merge laterally and pos- extend the suggestion from fibre degeneration teriorly with large labelled neurons of sub- studies (Guillery, ’57) of such a projection. stantia innominata and magnocellular preop- The connections of this region are poorly un- tic area. These results are in agreement with derstood. Sakai et al. (’77a) noticed after HRP autoradiographic data (Swanson, ’76). The injections of the locus coeruleus in the distribution of large labelled neurons in medi- that labelled neurons appeared in the same re- al septum (vertical limb of diagonal band), gion, while similar results were obtained after horizontal limb of diagonal band, magno-cel- HRP injection of nucleus raphe dorsalis in the lular preoptic area and substantia innominata cat. (Sakai et al., ’77b). Thus it may be that agrees well with the distribution of mag- neurons of this region send information to sev- nocellular basal forebrain neurons described eral midbrain and in addition by Divac (‘75). Neurons were also labelled in to the VTA. It is interesting to note also that the “ventral pallidurn” i.e., that region ven- this region corresponds closely with the posi- tral to the region of the anterior commissure tion of the A 13 catecholamine group (Lind- which receives afferents from the dopamine vall et al., ’74). rich olfactory tubercle and nucleus accum- bens (Heimer and Wilson, ’75; Nauta et al., (8) Afferents from habenuta ’78). Other neurons which are part of the mag- The lateral habenular nucleus was a promi- nocellular basal forebrain “system” but which nent source of fibres to the VTA. These results AFFERENTS TO VENTRAL TEGMENTAL AREA 137 confirm the results of degeneration (Akagi jection (Gloor, '55)although these results sug- and Powell, '68) and autoradiographic studies gest multisynaptic rather than direct influ- (Herkenham and Nauta, '77a). Neurons in ence on the midbrain. Autoradiographic trac- both medial and lateral divisions of the lateral ing of amygdalo-tegmental connections has nucleus were labelled. The lateral habenula recently confirmed that amygdala projects to appears to provide a site of convergence for af- VTA in the cat (Krettek and Price, '78). How- ferents from basal forebrain structures; nu- ever, this was only noted after injections to cleus of diagonal band, substantia innomi- the anterior and basomedial nuclei. In other nata, lateral preoptic area and magno-cellular cases of injection to central nucleus in rat, la- preoptic area; as well as the more caudal lat- bel was transported to lateral substantia eral hypothalamic and entopeduncular nuclei. nigra. Thus it is possible that label in central It is thus in a position to integrate infor- amygdala seen in the present experiments was mation from both "limbic" and "caudatoputa- due to damage and transport of HRP in fibres men" systems (GENERALDISCUSSION). of passage to substantia nigra. Further work The medial habenula was only labelled after is necessary to resolve this question, and the HRP injections confined to the interfascicular status of pathways from anterolateral baso- nucleus. This suggests that the interfascicu- lateral and medial nuclei. Only a minority of lar nucleus is related functionally to the inter- cases showed such label and the number of peduncular nucleus, since the bulk of the ef- neurons labelled was small. In any case, it ferent projections of the medial habenula are seems that the amygdala does project to the confined almost entirely to the interpeduncu- mesencephalic area, and the present results, lar nucleus (Herkenham and Nauta, '77a). It in conjunction with those of others, suggest should be emphasized that no evidence of a that both A 9 and A 10 dopaminergic systems projection from medial habenula to VTA was may be one such input area. obtained, and this agrees with anterograde la- belling studies (Akagi and Powell, '68; Her- (11) Afferentsfrom midbrain areas kenham and Nauta, '77a). It is very likely that neurons labelled in oculomotor complex, red nucleus and perhaps (9) Afferentsfromparafascicular area substantia nigra compacta represent labelling The number of neurons labelled in this re- of damaged andlor undamaged axons of pas- gion was small. Nevertheless the labelling was sage. Some label in red nucleus and substantia seen in most cases. It is interesting that the nigra may also have resulted from direct peri- ascending projections of this nucleus to the karya uptake from the HRP injections which pre-frontal cortex, and caudate nucleus-areas had spread from the nearby pipette tip. Such containing dopamine terminals- (Divac et al., spread to red nucleus was deduced in some '76; Powell and Cowan, '54) complement the cases because of concomitant uptake and descending projection shown here to VTA. transport of HRP to the dentate nuclei contra- Some labelled neurons of the parafascicular lateral to the injection site. area also occurred near the region containing More interesting, however, was labelling the A 11 dopamine group of the posterior thal- more distant from the electrode tip, in the amus (Jacobowitz and Palkovits, '74). deeper layers of the superior colliculus. Of course it is possible that damaged tectospinal (10) Afferents from amygdala or tectotegmental fibres may be responsible Neurons were unexpectedly labelled in for this labelling. Furthermore, both degen- amygdala. Anatomical studies usually state eration and autoradiographic studies of effer- that efferent fibres of the amygdala cannot be ents from the superior colliculi fail to demon- traced further caudally than posterior hypo- strate colliculo-VTA fibres (Kawamura et al., thalamus (Nauta, '61; Cowan et al., '65; Hall, '74; Graham, '77). Nevertheless, recent ana- '63; Leonard and Scott, "71). Recently, how- tomical and electrophysiological demonstra- ever, some evidence of amygdalotegmental, tions of nigro-tectal pathways arising from (Hopkins, '75) and amygdalo-nigral pathways pars reticulata of substantia nigra and pro- (Bunney and Aghajanian, '76) has been ob- jecting to deep layers of the superior colliculus tained with the horseradish peroxidase tech- (Deniau et al., '77; Graybiel, '78) suggest that nique. In addition there is electrophysiological a return collicular-VTA connection should not evidence of an amygdalo-mesencephalic pro- be ruled out on negative grounds alone. Fur- 138 0. T. PHILLIPSON ther evidence for this pathway is obviously by other workers in the monkey brain (Nauta necessary. and Mehler, '66; Kim et al., '76). This area ap- pears to receive the most caudal fibres from (12) Afferentsfrompons and medulla the medial segment of globus pallidus in mon- The efferent projections of the dorsal raphe key (Nauta and Mehler, '66) and possibly also nucleus have been repeatedly studied with an- from the analogous structure, the entopedun- terograde methods and all are in agreement cular nucleus, in the rat (Carter and Fibiger, that the VTA receives fibres from this nucleus '78). Furthermore, since the pedunculopontine (Conrad et al., '74; Bobillier et al., '75; Taber- region also receives fibres from the lateral hy- Pierce, '76; Ruda, '76; Graybiel, '77). The pres- pothalamus, (Arbuthnott et al., '76) and sub- ent results confirm this pathway with a retro- stantia innominata (Swanson, '76) it may rep- grade method. Biochemical evidence suggests resent, like the lateral habenula, another that the transmitter used by this pathway important point of convergence of efferents may be 5HT (Saavedra et al., '74a; Brownstein from caudatoputamen and "limbic" systems. et al., '751, a suggestion consistent with the (GENERAL DISCUSSION). earlier histochemical evidence (Fuxe, '65) As the ventral parabrachial neurons were showing 5HT containing cell bodies in dorsal followed posteriorly they merged with neu- raphe nucleus and 5HT varicosities in VTA. rons labelled immediately ventral to the locus Definite proof that synaptic terminals to the coeruleus and some of these at least may well VTA are derived from this pathway, however, have been catecholamine neurons (Palkovits is still lacking and this will require ultra- and Jacobowitz, '74). The number of neurons structural and electrophysiological evidence. labelled within the locus coeruleus itself was Neurons in median raphe nucleus were oc- small and in animals where the posterior A 10 casionally labelled by VTA injections. This oc- group were injected no neurons were found in curred only when the injection involved the this nucleus at all. Degeneration silver meth- midline of the VTA. Whether such labelling ods have failed to demonstrate a pathway results from uptake from fibres of passage from locus coeruleus to VTA (Shimizu et al., from the habenula to median raphe (Pasquier '74). Swanson and Hartman ('751, however, et al., '76) remains to be seen. However, after showed fibres containing adrenaline or nor- labelling restricted to the midline anterior adrenaline in the ventral tegmental area, VTA group, the interfascicular nucleus, many with the more sensitive immunohistochemical neurones were labelled in this nucleus, par- method. Scattered labelling was found in some ticularly its dorsal segment. Since the inter- animals in regions containing the A 5 and A 1 fascicular nucleus also receives afferents from catecholamine groups. The former group the medial habenula (see above) it seems that seems to be noradrenergic in nature (Swanson afferent relations of the interfascicular group and Hartman, '75) while the latter probably differ fundamentally from the rest of the uses adrenaline as its (Hok- VTA. Thus it receives afferents from medial felt et al., '74; Koslow and Schlumpf, '74; habenula and median raphe, whereas the rest Saavedra et al., '74b; Moore and Phillipson, of VTA receives afferents from lateral haben- "75). Of course, in none of these cases where la- ula and dorsal raphe. belled neurons were observed in regions con- Neurons of the raphe magnus and pontis taining catecholamine cell bodies, is it possi- were sometimes labelled in these experiments. ble, except in the case probably of principal However, since labelling was also obtained locus coeruleus and substantia nigra compac- after HRP injections of the red nucleus, these ta, to say whether the afferents observed are results should be treated with caution. aminergic or not. Double labelling procedures The most consistently and heavily labelled will be necessary to prove this point. Never- group in caudal pons were neurons lying theless, the present results suggest that there amongst the decussated fibres of the bra- may be an extensive interconnective network chium conjuntivum in the ventral parabra- between the different aminergic systems of chial nucleus, but also to a lesser extend in the the such that A 10 neurons receive dorsal nucleus. This region also includes the fibres from A 7, A 6, A 5, A 2 and A 1catechol- A 7 catecholamine group (Palkovits and amine neurons (possibly also A 9 and A 8) as Jacobowitz, "74), Labelled neurons lay in an well as the B 7 serotoninergic group in the dor- area which appears to correspond to the nu- sal raphe nucleus. In the case of A 6, A 7, B 7 cleus tegmenti pedunculopontinus described groups this network may involve reciprocal AFFERENTS TO VENTRAL TEGMENTAL AREA 139 connections with other amine systems (Sa- i.e., the midbrain region providing the dopa- kai et al., ’77a,b; Cedarbaum and Aghajanian, minergic innervation of the “ventral stri- ’78). atum.” In this respect the analogy to the neocortico-strio-pallidal system is complete (13) Negatiue findings for in that case some neurons in the globus No retrogradely labelled neurons were ob- pallidus (“dorsal pallidum”) are known to pro- served in any case in or lat- ject to pars compacta of substantia nigra eral septum, in spite of careful search of these (Nauta and Mehler, ’66; Hattori et al., ’75; regions. These are the only forebrain areas Carter and Fibiger, ’78). Thus, for both neocor- which receive dopamine terminals from VTA tical and allocortical systems the “striatal,” neurons but which do not contain labelled as well as “pallidal” stages project back to the cells. Of course negative results obtained with midbrain dopamine nuclei. As might be ex- the HRP method do not exclude the presence pected from the size of the caudate nucleus, of a pathway, and it may well be that larger the volume of afferents to substantia nigra is HRP injections would reveal such projections. much greater than the volume of fibres from Further evidence is clearly required about the smaller accumbens and olfactory tubercle these exceptions. to VTA and this may explain the lack of a well developed analog in VTA to pars reticulata of GENERAL DISCUSSION substantia nigra - the recipient of striatal An attempt to place the findings of the pres- fibres. ent study within the context of a more general The “ventral pallidal” system so defined neuroanatomical framework must start with here, only occupies a portion of substantia in- a brief summary of cortico-subcortical path- nominata. The definition of substantia in- ways and their relation to dopaminergic sys- nominata is variable (for discussion see Hei- tems. These have been reviewed recently by mer and Wilson, ’75; Divac, ’75; Jones et al., Heimer and Wilson (’75). The major conclu- ’76; Swanson, ’76). However, it is interesting sion can be drawn that, in parallel with the that the distribution of neurons included in well-known neocortico-strio-pallidal pathway the term “magnocellular nuclei of the basal in which the A 9 dopamine system innervates forebrain” (MNBF) (Divac, ’75) corresponds the striatal stage (caudate nucleus); there ap- in general very well with the sites of labelled pears to be an analogous allocortico-strio- neurons seen in the present study. These occur pallidal pathway in which the striatal stage is in vertical and horizontal limbs of diagonal innervated by the A 10 dopamine system. In band, substantia innominata (as defined by the second system, according to Heimer and Swanson, ’76, in the rat), magnocellular pre- Wilson, the cortical component is represented optic area and appear to project to cortex. by and pyriform cortex, and the These MNBF neurons may correspond to the striatal component by nucleus accumbens and chain of “aggregated and unaggregated large olfactory tubercle respectively (“ventral stri- cells that lie in the substantia innominata” atum”). The main feature of the system for described by Jones et al. (’76) in the monkey. the present discussion is that the pallidal com- Since the present results show that they ap- ponent of the second pathway is represented pear also to project back to mesencephalic do- by a region which, although quite discrete as paminergic neurons, this provides additional determined in their lesion studies, is poorly evidence that neurons in these regions may defined in currently available rat brain at- have some functional equivalence. There is lases. According to the cytoarchitectural some electrophysiological evidence of connec- study of Swanson (’76) it corresponds to the tion between neurons of the vertical limb of dorsal area of substantia innominata at the the diagonal band and VTA. Thus, macroelec- level of the anterior commissure. The cytology trode recordings from the VTA will register and ultrastructure of this region support the rhythmic potential fluctuations (Trembly and evidence on connectional grounds, that this Sutin, ’62; Le Moal and Cardo, ’75) which ap- area should be considered “pallidal.” An pear to be related to hippocampal theta and important gap in the scheme which remained are driven from the medial septa1 area (Le at that stage was the question of the efferent Moal and Cardo, ’75). relationship of this “ventral pallidum.” The In addition to these important afferent sys- present study clearly indicates that neurons tems from neurons of diagonal band, substan- of the “ventral pallidum” project to the VTA tia innominata and magnocellular preoptic 140 0. T. PHILLIPSON

area to VTA, there appears to be a series of torhinal cortex (Fallon et al., ’78). Since these afferents from a different type of neuron in cortical regions are themselves at the highest the lateral preoptic/lateral hypothalamic con- levels of convergence of association fibres tinuum or “path neurons” of the medial fore- from many sensory cortical areas (Jacobson brain bundle (Millhouse,’69). The anatomical and Trojanowski, ’77; Van Hoesen et al., ‘72), relationship between these two apparently taken together the evidence indicates that distinct systems and their relation in turn to both axon terminals and dendrites of VTA the cortico-strio-pallidal systems of Heimer dopaminergic neurons lie in regions of the and Wilson should provide important clues to nervous system characterised by truly mas- the function of the VTA as a target for affer- sive convergence. It would appear to be rea- ents from both MNBF and “path” neurons. Be- sonable to propose a correspondingly impor- havioural experiments indicate that the re- tant integrative or coordinating function for sponses of some neurons in the lateral hypo- these neurons. thalamus and substantia innominata are modulated by during discrimination ACKNOWLEDGMENTS and extinction tests (Mora et al., ’76). This The author was holder of a Wellcome Euro- suggests that neurons in the VTA, by virtue of pean Travelling Research Fellowship. This their connection with these regions, may play study was also supported by the Swedish Med- a role in learning mechanisms. ical Research Council, Project No. 553, to Doc- The lateral habenula has been shown to be a tor Gunnar Grant. Many thanks are due to site where information from both “limbic” Doctor Gunnar Grant at Department of Anat- and “caudatoputamen” systems can collabor- omy, Stockholm, for generous provision of ate (Herkenham and Nauta, ’77b). Since the facilities, for many helpful disucssions and for present results repeatedly demonstrate affer- reading the manuscript. The author is in- ent fibres to VTA from this nucleus, the VTA debted to Brita Robertson, Britt Meijer and is in a position tv sample information inte- Elisabeth Johansson at Department of Anat- grated not only from both the limbic “ventral” omy, Stockholm, for excellent technical help. strio-pallidal system via substantia innomi- nata but also from the extrapyramidal “dor- LITERATURE CITED sal” neocortico-strio-pallidalpathway via the entopeduncular nucleus. It is interesting to Akagi, K., and E. W. Powell 1968 Differential projections note that the medial segment of lateral haben- of habenular nuclei. J. Comp. Neur., 132: 263-274. Arbuthnott, G. W., M. J. Mitchell, I. F. Tulloch and A. K. ula is supplied with dopaminergic fibres Wright 1976 Efferent pathways from lateral hypotha- (Lindvall et al., ’74) which appear to be de- lamic neurones. J. 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