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Following the microiontophoresis of horseradish peroxidase to the ventral tegmental area of Tsai, neurones labelled by retrograde transport of the enzyme were o bse~ed in a large number of sites.throughcut the brain. In addition to confirming previously described afferents, these studies suggest so far unknown s y ~ s p r o j e c t i n g to the area from cerebral cortex, hypothalamus, amygda]a, thalamus, superior colliculus, substantia nigra, parabrachial nuclei and medulla oblongata.

There is suggestive evidence that dopaminergic systems in the CNS provide a substrate for the specifically antipsychotic action of neuroleptic drugs [4,8, 17]. The ventral tegmental area of Tsai (VTA) and adjacent cell groups contain a population ,jf dopaminergic neurons known as A10 [ 5]. Axons ascending from these neurones have been demonstrated by many histochemical studies to project to forebrain sites including the frontal cortex [3,7,12--14,21] while more recent autoradiogrmphic studies have revealed in addition descending projections to paramedian t~idbrain structures [6]. Relatively little attention, however, has been paid to their afferent supply, although incidental findi n p item degeneration and autoradiographic studies exists for some projections. Here, use has been made of the horseradish peroxidme (HRP) method to carry out a more comprehensive examination of the nature of afferents to the VTA. A full report will appear elsewhere. Female Sprague--Dawley rats (about 150 g) were used for all experiments. Rats were anaesthetised with chloral hydrate (400 mg/kg ip.) and positioned in a Kopfstemotsxic f~mme. Glass micropipettes (tip diameters 20--40 #m) containing 40% H R P (Sigma type VI) in 0.1 M N a O H were aimed at various sites within the V T A and lowered to their tar@ets with a retaining current applied to the H R P solution. Particularcam was taken to avoid as far as possible the

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major neighbouring nuclei and fib~ tracts during the penetration. HRP was ejected microiontophoretically (Model 160, microiontophoresis programmer, W.P instnunents Inc, U.S.A.,) with a constant positive current of !::/zA for 5--17 min. A retaining current was reapp]ied during withdrawal. Approx. 24 h after injection, animals w,,'~e perfused and processed for HRP histochemistry according to the method of Hardy and Heimer [ 9] using the presumed noncarcinogenic substrate tetramethyl benzidine (TMB). This method appears to be considerably more sensitive than conventional procedures. Frozen sections were cut at 40 ~m and after incubation counterstained with neutral red. ~.~ery other section was examined for HRP positive neurones under bright field conditions. A series of 11 injections to different regions of the VTA were fully analysed from a total of 32 injections. Control injections were made in addition into the medial lemniscus, red nucleus, tegmental decussations, midbrain reticular formation, and superior cerebellar decussations~ The present report briefly summarises the main findings of this series as illustrated by one injection which was centred on the anterior VTA. Fig. 1 shows the extent .~f the injection site as seen 24 h after operation. The dimensions of the densest extracellular HRP deposit measured about 600 ~m X 200 ~m X 500 um and was almost entirely confined to an area containing dopaminergic cell bodies and dendrites. A more lightly stained area, containing chiefly intracellular glial, vascular and axonal reaction products, extended to include the medial tip of the medial lemniscus, but did not invade the red nucleus or the interpeduncular nucleus. The pipette track a~roided the red nucleus and medial lemniscus but penetrated the deep tegmental descussations and the roots of the third nerve. Fig. 2 summarises the location of some of the labelled neurones seen throughout this brain. Only neurone-like profiles in which cell body and proximal dendrites were clearly filled with dark blue granular reaction product, were comlted. Care was taken to differentiate these from peroxidase containing glial cells or vascular pericytes. Labelled perikarya appeared in mediofrontal cortex; dorsal bank of rhinal sulcus; nucleus a,.-cumbens (medial segment); diagonal band of Broca; olfactory tubercle (polymorph layers); bed nucleus of stria terminalis; lateral preoptic area; substs~.tia innominata; magnocellular proeoptic area; anterior hypothalamus; amygdala (central, medial, basolateral and asterolateral divisions); posterodorsal regions of the hypothalamus; lateral hypothalamus; H~ and H2 fields of Forel; medial zona incerta; lateral habenula; parafascicular nucleus of thalamus; nucleus .linearis raphe rostralis; red nucleus; oculomotor nuclei; superior colliculus (deep layers); substantia nigra compacta; nucleus linearis raphe caudalis; nucleus cunefformis; nucleus parabrachialis ventralis and dorsalis; nucleus raphe dorsally; locus coeruleus; nucleus reticularis pontis caudalis; nucleus raphe magnus; ventrolateral regions of medulla at the exit of seventh nerve fibres; and a region lateral to the dorsal accessory olivary nucleus. In addition, occasionally labelled neurones, not shown in Fig. 2, were seen in medial preoptic area, periventricular hypothalamic area; central grey; nucleus

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Fig. 1. Drawings of transverse sections .~howing the entire extent of the densest exl~acellular HRP depoMt (solid black). An area of r aore. lightly labelled tissue (clear surround) is also shown. This consists of labelled glial cells and axons, but rather little extracellular HRP reaction product. Sections roatral and (audal to those shown also contain the latter, but not the former type of reaction product. PCMA, mamillary peduncle; FR, fasciculus retroflexus; MB, mammilsry body; LM, medial lenmiscus; cc, cerebral peduncle; RN, red nucleus; IP, interpeduncuhtr nucleus, lIIn, third nerve.

superior centralis; nucleus reticulafis pontis oralis; tegmental nucleus of Gudden; and dentate nuclei. The VTA can be divided on c~ toarchitectouic criteriae into different, regions [5 and O.T. Phillipson, unpubl~hed results). The general pattern of labelling obtained by injection of different regior:s of VTA was in agreement with that reported here. Significant differences, however, were seen when HRP was injected into rnldline cell groups. These will be described in a forthcoming paper. Judged by their numbers in all cases, the densest projection ~o VTA as a whole was from nucleus raphe dorsalis; lateral hypothalarnus; lateral habenula; superior colliculus; substantia innominata (including magnocellulr proeoptic

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Fill. 8. Photomicroffrap]~s of labelled neurones. Bar = 50 ~m for all neurones. E n m p l e s were chosen to show u much as possible of the dendritic tree. Solid Gollli-like staining is some. times seen, (i,k) although in all such ceses the reaction product is in fact coarsely granular. In all areas shown many other neuroztu were also stained with a moze obvious granular appearance, a, dorsal bank rhinal sukus; b~ diallonal band] c, bed nucleus stria terminalis; d, substantia innominata, e, lateral preoptic area. f, amylldsdl, (central nucleus) fl, lateral hypothalamus; h, lateral habenula; i, substantia niflra conipscta; j, superior colliculus; k, raphe dorsalis; I, nucleus parabrachialis.

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injection of the nucleus linearis raphe caudalis (the most caudal A I 0 gr0uP). These studies reveal a rich afferent network supplying the region of A10 neurones. To what extent the results represent labelling of f i b e r : o f passage rather than synaptic terminals is u n c e ~ . Uptake and r e t ~ g ~ d e trahsport of HRP is known to occur from axons which are damaged by the injecti'on procedure [10,16]. Since the ventral t~Imentum is densely traversed by such fibers from many CNS regions, this factor must strongly Lnfluence the interpretation. Based on known connections of variousnuclei and the control ttKJe injections made here, it is quite likely that neurones labelled in certain regions of zona ince~-~_, fields of Forel, oculomotor complex, red nucleus, dentate nucleus and reticular nucleus of ports v,ere due to labelling of fibers of passage, or labelling of n e i g h b o ~ g nuclei. On the other hand the methods of the present study cannot exclude projections from such areas to VTA. In the case of other regions, there is evidence from degeneration and autoradiographic studies that projections from nucleus accumbens [19], diagonal band [ 1 5 ] , bed nucleus of stria terminalis, substantia innominata, medial and lateral preoptic areas [ 18] ,lateral hypothalamus [ 2 ] , lateral habenula [ 1,11 ], and nucleus raphe dorsalia [ 20], do in fact project to and may terminate in VTA. The present results support these findings. Previously undescribed systems projecting through or terminating in VTA revealed by the present study are from medio frontal cortex, dorsal bank of rhinal sulcus, amygdala, posterior and dorsal hypothalamus, parafascicular nucleus of thalamus, nucleus linearis raphe rostralis, superior colliculus, substantia nigra compacta, and parabrachial nuclei. In addition, regions of rae(~ulla and pons containing the A1, AS, A6, A7 and A8 catecholamine groups were also labelled. Further work with anterograde methods will be necessary to assess the validity of the~e results. ACKNOWLEDGEMENTS The author was holder of a European Travelling Fellowship from the Wellcome Trust, and the work was supported by the Swedish Medical Research Council grant No. 12X-553 to Dr. Gunnar Grant. Many thanks are due to Brita Robertson, and Elisabeth Johansson for expert technical help and to Dr. Grant for reading the manuscript. REFERENCES

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