THE JOURNAL OF COMPARATIVE NEUROLOGY 325~1-21(1992)

Topography and Collateralization of the Dopaminergic Projections to Motor and Lateral Prefrontal Cortex in Owl Monkeys P. GASPAR, I. STEPNIEWSKA, AND J.H. KAAS Department of Psychology, Wilson Hall, Vanderbilt University, Nashville, Tennessee 37240

ABSTRACT The sources and histochemical characteristics of dopaminergic projections to motor and premotor areas of cortex were investigated in owl monkeys in which information from related studies was used to subdivide cortex into motor fields. Brainstem projections to frontal cortex were identified by injections of different fluorescent dyes in the primary motor cortex (Ml) and the supplementary motor area (SMA),first identified by microstimulation. Injections were also placed in dorsal premotor cortex and lateral prefrontal cortex. The distribution of retrogradely labeled neurons was related to the location of tyrosine hydroxylase immunolabeled neurons on the same or alternate brain sections to identify the dopamine (DA) neurons. All DA cortically projecting neurons were located in the AS-A10 complex, largely in its dorsal components, including the parabrachial pigmented n. of the ventral tegmental area (VTA), pars gamma of the substantia nigra compacta, and the dorsal part of the retrorubral area (AS). Fewer cells were in the midline groups of VTA (n. linearis rostralis and caudalis) and in the n. paranigralis. DA neurons projecting to M1, SMA, and prefrontal cortex were largely intermixed, and some of these neurons were double or triple labeled by the fluorescent dyes, indicating collateralization to two or three fields; DA cells projecting to M1 were more numerous than to the other locations. The dorsal components of the AS-A10 complex from which arose the DA mesocortical projection were also characterized by the presence of calbindin-immunoreactive neurons and by a dense neurotensin and noradrenergic terminal innervation. Compared to rodents or felines, the DA neurons projecting to the lateral frontal lobe of primates appear to be shifted dorsally and laterally in the nigral complex. The topographic overlap, partial collateralization, and common histochemical characteristics of the DA mesocortical neurons projecting to different fields of the lateral frontal lobe suggest that some degree of functional unity exists within this projection. T> 1992 Wiley-Liss, Inc. Key words: substantia nigra, VTA, calbindin, neurotensin, noradrenaline

Clinical data from patients with Parkinson's disease as well as studies in experimental animals have shown that dopamine (DA) plays a major role in motor control (Bernheimer et al., '73; Carlsson, '77).At least much ofthe effects of DA seem to occur at the level of the basal ganglia, through the nigrostriatal pathway (Alexander and Crutcher, '90; Delong, '90). However, the primary motor (Ml),supplementary motor (SMA),and premotor areas receive a dense DA input in human (Gaspar et al., '89) and non-human primates (Berger et al., '86, '88; Lewis et al., '87); this suggests a different view, i.e., that DA exerts control at both the levels of the basal ganglia and the cerebral cortex. In favor of this view, DA innervation is depleted in the motor cortex of Parkinsonian patients (Gaspar et al., '91). Presently, little is known about the anatomical organization of DA projections to the motor cortex and, in particular, their origin. Most of the experimental work on the DA

o 1992 WILEY-LISS, INC.

systems has been carried out in rats, which lack a significant DA input to M1 (lateral agranular field) and have only a weak DA projection to the premotor area (medial agranular field) (Berger et al., '85). In rats, the mesocortical DA pathway is primarily directed to the medial prefrontal and anterior cingulate cortices, arising from the ventral tegmental area (VTA) and the medial substantia nigra (SN). In primates, DA fibers reach the entire cortical mantle (Berger et al., '88; Lewis et al., '87; Gaspar et al., '89) and are most abundant in the anterior cingulate and motor cortices. DA terminals are always most abundant in layer I, with laminar-specific patterns in the different cortical areas. DA fibers are distributed to all cortical layers in the motor and Accepted June 30, 1992. P. Gaspar is now at INSERM U106. Bit. de Pediatrie, HBpital Salpetriere, 47, Blvd. de l'HBpita1,75651, Paris cedex 13, France.

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anterior cingulate cortices, while they display a bilaminar World monkeys. Owl monkeys (Aotus trzuirgutus) were pattern (in layers I and V-VI) in granular cortices such as especially useful in these studies because motor and premothe lateral prefrontal cortex. The source of this widespread tor cortical areas have been defined and investigated in a DA cortical input in primates is not clear. In the volumi- series of microstimulation, architectonic, and anatomical nous anatomical literature devoted to motor areas in studies (Gould et al., '86; Preuss et al., '91; Stepniewska et primates, there is hardly any mention of afferents arising al., '90, '91). In the present investigation, multiple injecfrom these areas; only Jurgens ('84), analyzing the affer- tions of different fluorescent tracers enabled us to deterents to the SMA of squirrel monkeys mentions the exist- mine the topography of projections and the extent of their ence of projections from the VTA. However, afferents collateralization to motor and premotor areas. The DAarising from VTA and SN to prefrontal cortex (Porrino and projecting neurons were distinguished by concurrent immuGoldman-Rakic, '82) or to the posterior parietal cortex nocytochemical visualization of tyrosine hydroxylase (TH), (Lewis et al., '88) have been reported, but these studies the rate-limiting enzyme of catecholamine biosynthesis. either lacked a histochemical identification of the retro- Immunolocalization of dopamine-P-hydroxylase (DBH),the gradely labeled cells (Porrino and Goldman-Rakic, '82) or synthetic enzyme of noradrenaline, further allowed us to did not provide a topographical analysis (Lewis et al., '88). distinguish between the DA- and noradrenaline-containing Such information is important for distinguishing the DA neurons, since the 2 types of catecholaminergic cells overlap from other projections arising from the mesencephalon, at caudal midbrain levels (Felten et al., '74; Pearson et al., since non-DA neurons appear to constitute as much as 70% '83). of the projection from the VTA to the cortex in rats (Swanson, '82), and for defining the subsets of DA cells projecting to different areas of cerebral cortex. Indeed, the MATERIALS AND METHODS mesencephalic DA neurons in rodents form very heterogeResults are from 6 adult owl monkeys (Aotus triuirgutus). neous groups with respect to their projections (Lindvall et One monkey was perfused without prior surgery and was al., '78; Fallon et al., '78; Swanson, '82; Fallon and Loughlin, '87; Oades and Halliday, '871, metabolism, firing rates used only for immunocytochemical analysis. Anatomical (Chiodo, "9,and reactivity to stress (Thierry et al., '76; tracing and immunocytochemical methods were combined Zacharco and Anisman, '91). In humans the different DA in the other five. The fluorescent dyes, fluorogold (4%),fast subgroups display different rates of cell death in Parkin- blue (3%),and diamidino yellow (3%),were used as retroson's disease (Hassler, '35; Hirsch et al., '88; German et al., grade neuronal tracers. A different fluorescent tracer was injected in each of the cortical areas studied (Fig. 7). '89) and Alzheimer's disease (Gibb et al., '89). A related issue is collateralization. In rats, the mesocortical neurons have different collateralization patterns, with Surgery SNc neurons being more collateralized than VTA neurons Animals were premedicated with Dexamethasone (5 mg/ (Deniau et al., '80; Fallon and Loughlin, '82; Loughlin and kg, i.m.) and penicillin (30,000 Uikg, s.ic.1and anesthetized Fallon, '84; Takada and Hattori, '87). Since the cortical DA for surgery with Ketamine hydrochloride (25 mgikg i.m., projection fields are more extensive in primates than in rats initial dose) supplemented by xylazine ( 2 mgikg). Surgical (Berger et al., '91), one might expect an increase in the levels of anesthesia were maintained with additional doses proportion of collateralized neurons in primates. (5-10 mg), as needed. Surgery was performed under aseptic In an attempt to answer some of these questions we have conditions while body temperature was maintained around analyzed the midbrain and diencephalic projections to 38°C. Dorsomedial frontal cortex was exposed with a cranelectrophysiologically defined motor areas (Ml, dorsal preiotomy, the dura retracted, and the brain covered with motor, and SMA) and lateral prefrontal cortex in New liquid silicon to prevent dessication. Enlarged photographs of the brain surface were made to mark the location of electrode penetrations and injection sites. Parts of motor and premotor cortex were identified by patterns of moveAbbreuiations ments evoked by a Grass stimulator delivering single or central gray cg multiple trains of cathodal current through low impedence cl central linear n. tungstene microelectrodes, with tips approximately 1.7 mm DH. dorsal raphe beneath the pial surface (see Gould et al., '86; Stepniewska fr fasciculus retroflexus interpeduncular n. iP et al., '90, for details of the intracortical microstimulation if interfascicular n. procedure). The electrophysiological identification of the LC locus coeruleus cortical areas followed previous descriptions by Gould et al. LM lemniscus medialis Im lateral mammillary n. ('861, Stepniewska et al. ('90, '911, and Preuss et al. ('91). In mlf medial longitudinal fasciculus brief, the primary motor area, M1, was recognized as a mm medial mammillary n. large, caudally situated, somatotopically organized, lowMnR median raphe n. threshold area (currents in the 1-40 pA range). The parachialis pigmentosus PbPg supplementary motor area was identified on the dorsal pedunculoponine tegmental n PPtg rl rostral linear n. shoulder of the cortex, rostral to M1, with comparable or red nucleus RN slightly higher movement thresholds than M1 (20-70 PA) RTP n. reticularis tegmenti pontis and with a caudorostrally oriented somatotopic representaSCP superior cerebellar peduncle SNc substantia nigra compacta tion. The dorsal premotor area was recognized as cortex SNR substantia nigra reticulata with higher thresholds for evoked movements just rostral ST subthalamic n. to M1 and lateral to SMA. Other injections were placed in V ventricle prefrontal cortex rostral to the zone where body moveVP ventro-posterior thalamic n ZI zona incerta ments or occulomotor responses could be elicited.

DA AFFERENTS TO MOTOR CORTEX Several closely spaced pressure injections of a given fluorescent dye were placed in each cortical area, with 5 or 10 pl Hamilton syringes attached to a glass micropipette (20 pm tip diameter). A volume of 0.3 to 0.5 p1 of the dye was injected over 3 cortical depths, 1.8, 1.3, and 0.9 mm, to ensure that the injections would involve all the cortical layers. Ten minutes was allowed before removing the pipette. Any overflow of dye was carefully blotted away. Following injections, dura was replaced, the skull opening was closed with dental acrylic, and the skin was sutured. The animals were carefully monitored during recovery from anesthesia. Postoperative care included analgesics (Butorphanol 0.4 mgikg s.c.) and antibiotics (penicilin 20,000 Uiday) during the first 2 days. Histologicalprocessing. After survival times of 11to 16 days, the animals were given a lethal dose of barbiturate and perfused through the heart with saline 0.9% followed by buffered paraformaldehyde (4% in 5 cases, 2% in case 91-24). After extraction, the brain was blocked and postfixed for 6 to 12 hours in 4% paraformaldehyde with 10% sucrose. The blocks were placed in sucrose 15% and then sucrose 30% for 6 to 24 hours and cut on a freezing microtome. The brainstem was serially cut in the coronal plane and 30 pm thick sections were collected in phosphate buffer. At least one section out of five was immediately mounted for examination of the fluorescent dyes, and one section in five was used for combined TH-immunofluorescence; one section out of ten was used for making alternate series of Nissl, acetylcholinesterase, and immunoperoxidase revelation of tyrosine hydroxylase (TH) and calciumbinding protein D28K (CABP). Sections were immunostained with neurotensin and dopamine P-hydroxylase antibodies in three cases. Injection sites and labeling in the cerebral cortex were examined either in the coronal plane or in a tangential plane after flattening of the cortex (91-24) (Stepniewska et al., '91). The series mounted for fluorochromes were rapidly dehydrated and coverslipped with Krystalon (EM-diagnostics), a procedure that helped prevent fading of fluorescence. Immunofluorescence. Floating sections were transferred to phosphate buffered saline (0.02 M, pH = 7.4) to which 0.2% of Triton X-100 and 0.2% gelatin had been added (PBS+).After a 1hour rinse, sections were incubated in the primary antibody (monoclonal antibody to TH from Incstar, diluted 1/1,000 to l/2,000 in PBS+) for 2 to 12 hours at 4°C. The incubation time was 2 hours when DY had been used, as this fluorochrome tended to leak out of neurons with more protracted incubations. The sensitivity of THimmunodetection was similar with the shorter incubation periods, provided the concentration of antibody was high ( l / l , O O O ) . After a 20 minute rinse in PBS+, the secondary antibodies linked to rhodamine (Cappel) diluted l/200 in PBS+ were applied 1-2 hours at room temperature. After a final rinse in 0.05 phosphate buffer, the sections were mounted and coverslipped either with Fluoromount (Polyscience) after a quick drying, or with Krystalon after dehydration. The latter procedure proved more reliable for the optimal preservation of all tracers. The immunoperoxidase procedure followed that previously described (Gaspar et al., '89). Using 48 hours incubations at +4"C, the optimal antisera dilutions were 1/20,000 for TH (Incstar), 1/20,000 for CABP (a monoclonal antibody generously provided by Dr. Heizmann), (Heizmann and Hunziker, '91) l/lO,OOO for Neurotensin (rabbit polyclonal antibody purchased from Incstar), 11500 for dopa-

3 mine beta hydroxylase (polyclonal antibody purchased from Eugene Tech). The streptavidin-biotin-peroxidasecomplex was revealed either with DAB (Sigma) (DAB (0.05% and HzOz 0.005%) or with DAB (O.OJ%)-Nickel Ammonium sulfate (0.6%). Analysis. Sections were plotted on a Leitz-Orthoplan fluoromicroscope equipped with stage transducers. The X-Y coordinates (of contours and labeled cells) were transmitted to a microcomputer to generate 2-D maps (Bioquant). Each type of labeled or double-labeled cell could be plotted with different colors or symbols. DY, FG, FB were examined with filter D, Fluorescein with filter 13, and rhodamine with filter N2.1.

RESULTS The results are presented in two parts. First we present a description of DA cell groups relative to brainstem architecture in owl monkeys. Such a description is the first available for owl monkeys and incorporates several features of previous descriptions of DA cell groups in squirrel monkeys (Felten et al., '74; Arsenault et al., '88), macaques (Garver and Sladek, '75), and humans (Gaspar et al., '83; Pearson et al., '83). DA cell groups are defined according to both the terminology of Dahlstrom and Fuxe (Lindvall and Bjorklund, '78) for catecholaminergic groups, and the more traditional cytoarchitectonic compartments as noted by Olszewski and Baxter ('54), FranGois et al. ('851, and Halliday and Tork ('86). In addition, we discuss the topographic distributions of neurons immunoreactive for CABP and the noradrenergic (DBH+) and neurotensin positive (NT+) innervation on alternate sets of sections as further markers of chemoanatomic compartments. This description of the distribution provides a context for evaluating the second part of the results, where we identify and localize DA and other neurons projecting to different subdivisions of the frontal cortex.

Localization of the DA cell groups in owl monkeys and relationship to chemoanatomic compartments The TH-immunoreactive cell groups are described relative to cytoarchitectonic features in a series of coronal brain sections through the caudal diencephalon and mesencephalon in Figures 1-4. At these levels TH can be considered as marking only DA neurons (Felten et al., '74; Arsenault et al., '88). Adjacent sections show the distributions of CABP, DBH, and NT. Diencephalic cell groups (All-A14). Three distinct cell groups contain DA neurons in the diencephalon: 1) The arcuate (infundibular) group, A12, lines the ventral floor of the 3rd ventricle (level rostra1 to that shown in Fig. 1, not illustrated), and is composed of small neurons that contain no detectable calbindin. A12 was characterized by very dense neurotensin and noradrenergic innervations, and some NT positive neurons. 2) The lateral hypothalamic group A13 is a conspicuous group, located in the posterior hypothalamic area around the thalamo-mammillary tract (Fig. 1).It is formed of large multipolar neurons and overlaps a group of CABP-immunoreactive neurons. Its neurotensin innervation is moderate to low and its DBH+ innervation is low. 3) The periventricular cell group, composed of medium to small cells, is located in the periventricular gray often just below the ependymal lining (Figs. 1-4). It extends from the walls of the 3rd ventricle rostrally, where it has been termed A 14, to the pontine central gray

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4

T H

DBH

\

CABP

1 mm

NT

180

Fig. 1. The topographic distribution of tyrosine hydroxylase (TH) and calcium-binding protein D28K (CABP) positive perikarya (dots) and of dopamine-p-hydroxylase (DBH) and neurotensin (NT) terminal innervations (fine dots and lines) is shown on 4 representative coronal levels through the mesencephalon, from rostra1 (Fig. 1)to caudal (Fig. 4). The catecholaminergic subgroups are indicated with the nomenclature of Dahlstrom and Fuxe on the drawing for TH, and the major

cytoarchitectonic nuclei are indicated on the drawing for CABP. Actual drawings are slightly schematized and reduced from camera lucida drawings of case 90-68. The numbers indicate the section level in a rostrocaudal series cut at 30 km; GOO km separates Figure 1 (level 180) from Figure 2 (level 2001, and Figure 2 (level 200) from Figure 3 (level 220); 1,200 bm separates Figure 3 (level 220) from Figure 4 (level 260).

caudally, where it is referred to as A 11 (Lindvall and Bjorklund, '78). Many CABP-immunoreactive neurons were present in this periaqueductal zone, which received a moderate to dense neurotensin and DBH+ innervation.

Mesencephalic groups (A8-AlO). The substantia nigra pars cornpacta (SNc) or A9 is the largest DA group, extending 3.5 mm rostrocaudally, 3.3 mm mediolaterally, and 0.5 to 1.5 mm dorso-ventrally. While from two to 84

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5

TH

. ....-

CABP

NT Fig. 2

2 00

Same as Figure 1.

different subgroups have been identified in the primate SN (see refs. in Francois et al., '841, Olszewski and Baxter ('54) proposed 3 ventro-dorsal subdivisions of the SNc,a,P,T, in humans. These compartments seemed to best correspond with our histochemical observations (Figs. 1-4, 5). 1) The dorsal part of SNc, pars r, also called pars mixta (Franqois et al., '85) or parabrachialis pigmentosus (Halliday and Tork, '86), contained the most scattered TH+ cells with frequently fusiform morphologies and a horizontal orientation of the main dendrites (Fig. 5c). This zone had the densest NT+ and DBH+ terminal innervation of the entire

SNc and contained a large group of CABP+ neurons, with aspects very similar to the TH+ neurons. Basket-like pericellular arrangements of NT+ fibers and varicosities were observed. 2) The intermediate pars p contained a denser rather homogeneous accumulation of TH + cells with multipolar shapes. This division contained a lower amount of CABP+ neurons and received a weak DBH + and a moderate-to-dense neurotensin-like innervation (Fig. 5b,d). 3) The ventrally located pars 01 contained rounded TH+ neurons grouped in clusters. Their dendrites were often assembled in bundles oriented ventrally into the pars

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6

Fig. 3. Same as Figure 1.

reticulata. No CABP+ neurons were present in this ventral zone of SNc, which was scarcely penetrated by NT+ or DBH+ fibers (Fig. 5 ) . The ventral tegmental area or A10 is distributed over several cytoarchitectonic nuclei where DA and non-DA neurons are intermingled. We have followed the cytoarchitectonic description and nomenclature of Halliday and Tork ('86) to identify 5 subgroups. Three are along the midline: the n. rostralis linearis (rl) (Figs. 2, 31, which lies just below

the I11 cranial nucleus, the n. centralis linearis (cl) ventral and caudal to rl, which merges with the dorsal raphe nucleus (Figs. 2-4), and the n. interfascicularis (if), just above the n. interpeduncularis (Figs. 2 , 4 ) . Two DA groups of the VTA were located more laterally within the nerve rootlets of the IIIrd cranial nerve: the n. paranigralis (pn) ventrally and the n. parabrachialis pigmentosus dorsally (pbpg) (this term refers here only to neurons within or medial to the 3d nerve rootlets contrary to the more

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7

\

f

D BH TH

NT

260 Fig. 4. Same as Figure 1

extensive definition of Halliday and Tork, '86) (Fig. 3). The lateral limits of these cell groups with the SNc are unclear; we have arbitrarily set them as the lateral border of the 3rd cranial nerve. In all VTA cell groups, the CABP+ neurons were abundant and there was a dense NT and D B H innervations. However, the histochemical characteristics of the ventral part of the n. paranigralis were more similar to

those of the a sub-group of the SNc, and may thus be considered as a medial subgroup of SNc a. The retrorubral area or A8 cell group lies in the caudal continuation of the SNc at the level of the decussation of the superior cerebellar peduncle (Fig. 4). Two distinct compartments were distinguished with the present histochemical criteria, a dorsal region, which contained CABP+

Fig. 5. Micrographs of the SNc, showing adjacent sections immunostained with TH (a),DBH (b),CABP (c), and NT (d).The denser ventral part of SNc, pars a,is indicated with arrows; note its scarcity in DBH (b) and NT+ (d)innervations, and the absence of CABP+ neurons at this

level. The dorsal, more diffuse parts and r, contain CABP+ neurons and a dense NT and DBH+ innervation. Medial is to the right. Star points to a landmark vessel visible in all the sections. X 100.

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Fig. 6. Injection sites in case 91-9. A: Fluorogold in primary motor cortex (MU. B: Two closely spaced Fast blue iniections in the supplementary motor area (SMA).The arrow indicates the inferior limit of the _. cortex. Scale bar = 1mm.

neurons and received a dense N T + and DBH+ innervation, and a ventral component which contained no CABPimmunoreactive neurons and received no or a weak NT and DBH input.

Retrograde labeling after cortical fluorochrome injections Injection sites. Fluorochrome injections were used to determine the locations of DA neurons projecting to various subdivisions of the frontal cortex. The injection sites extended throughout the entire cortical depth, being frequently more widespread in the upper

cortical layers (Fig. 6). The injection sites remained limited to the cortical ribbon except in case 91-75 where the diffusion zone of fast blue spread 0.1-0.2 mm into the subcortical white matter of M1. Figure 7 indicates the sites ofthe fluorochrome injections and the zones of diffusion of the tracers in relationship to the cortical areas previously determined in architectonic and electrophysiological studies of cortical organization in owl monkeys (see Gould et al., '86; Preuss et al., '91). In the present cases, microstimulation maps over limited portions of cortex at the time of injections identified the injection fields and some borders between cortical fields. Other relevant borders were deter-

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10 90-75

1

91-51

91-24

L

FLUOROGOLD

FAST BLUE

DlAMlDlNO YELLOW

Fig. 7. Injection sites by 3 different fluorchromes are indicated with stippling or stripes in each of the analyzed cases. The lighter stippling or stripes indicate the diffusion zone around the injection sites. The

borders between the different cortical areas are represented on standardized dorsal views of the owl monkey cortex, as determined after reconstructions with serial coronal sections. Scale bar = 5 mm.

mined from coronal sections stained for Nissl, acetylcholinesterase, or cytochrome oxidase. Reconstructing dorsal views of the brain allowed the localization of borders and injections, which were then transferred to standard dorsal views for comparison across cases (Fig. 7). In case 90-75

injections of fast blue covered a large part of M1 (hindlimb, trunk, and forelimb representations) while injection of fluorogold included much of SMA. I n 2 other cases (91-9, 91-24) the injections included the forelimb representation of M1 and of SMA. I n case 91-9, the fast blue injection site

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Fig. 8. Localization of DA and non-DA cortically projecting neurons in Snc. a: Low power micrograph ( x 1 5 ) through the SNc in case 91-9, at a rostra1 level; medial is to the right. The different symbols (cross, arrowhead, and star) indicate the locations of some retrogradely labeled

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neurons, which are shown at higher power in b,d,f. Labeled neurons in b and d are also TH+ ( c ) and ( e ) .Another FG-labeled neuron (0 lying close to SNc, is not TH+ (g). ~ 3 2 0 .

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involved most of SMA, extending into the neighbouring cortical areas. In case 91-51, the injections were in the forelimb representation of M1 and in the dorsal premotor region. Case 91-76 involved only a limited injection in the forearm representation of the SMA. Prefrontal injections (91-9,91-24,91-51)were located in lateral granular fields of the frontal lobe, rostrally to the motor areas. Localization o f the cortically projecting cells. Cells intensely labeled by the fluorescent dyes were observed in many diencephalic and brainstem structures. The most extensive labeling resulted from the fast blue injections, followed by that resulting from fluorogold and diamidino yellow injections. Thus the type of tracers injected in each area was varied over cases: fast blue was injected in M1 in case 90-75, in SMA in case 91-9, and in lateral prefrontal cortex in cases 91-24 and 91-51. The quality of the fluorogold labeling depended on the time allowed for transport, with optimal labeling after survival times of 2 weeks or more, and fainter labeling after a 10 day survival period (case 91-51). The identification of the retrogradely labeled neurons that were immunoreactive to TH was feasible (Fig. 4) in 3 cases (91-9, 91-51, 91-76). In the 2 other cases, TH-immunofluorescence was either too weak (90-75) or too intense (91-24) (possibly due to differences in the fixation conditions), making the double labeling difficult to interpret. In these cases, consecutive sections with the fluorochromes and with TH-immunolabeling (streptavidin peroxidase) were analyzed to localize regions where cortically projecting neurons were coextensive with DA neurons. Distribution of DA and non-DA cortically projecting cells are illustrated for 2 cases, one (91-9) with predominant injections in the motor areas (M1, SMA and to some extent the dorsal premotor) and a smaller projection in the lateral prefrontal cortex (Fig. 91, and another case (91-51) with a predominant injection in the prefrontal cortex and more limited injections in the motor areas (Fig. 10). In all cases, the DA cortically projecting neurons were found in the substantia nigra pars compacta (SNc) and in the ventral tegmental area (VTA). These neurons were almost always ipsilateral to the cortical injections and were mainly found in the dorsal subgroups of the VTA-SNc complex. A few retrogradely labeled neurons were found contralaterally in VTA but were generally not TH+ and thus appear to be mainly non-DA. The SNc contained 16 to 52% of all the cortically projecting neurons (Table 1); these cells were always in the dorsal SNc zone corresponding to pars r (Fig. 8b,c). The VTA contained 30 to 63% of the DA cortically projecting neurons mainly in the n. parabrachialis pigmentosus (18 to 43%), among the rootlets of the 3rd cranial nerve (Figs. 9-140, 10-2561, fewer neurons being present medially in the rostra1 or central linear groups (6 to 15%),or ventrally in the paranigralis 10 to 10%)and hardly any in the n. interfascicularis (Table 1).Ten to 20% of the DA projecting neurons to either the prefrontal or the motor cortical areas were in the retrorubral group AS, but only in its dorsal component (Figs. 9-262, 10-166). No TH+ retrogradely labeled neurons were observed in the periventricular groups (All-A141 or in the lateral hypothalamic (A13) cell group, although cortically projecting neurons were present in these areas. In the dorsal raphe, at caudal levels of the pons (Fig. 9-2621, a few TH+ neurons were retrogradely labeled. However elution-restainingwith DBH antibodies revealed that these neurons were noradrenergic, and

P. GASPAR ET AL. thus appear to be medially displaced neurons of the locus coeruleus. The DA neurons projecting to M1, SMA, dorsal premotor, and prefrontal cortices overlapped in the same subnuclei of the SNc-VTA complex. The cortically projecting neurons were found throughout the rostro-caudal extent of the SNc-VTA, peaking a t middle levels (Figs. 9, 10) where the DA neurons were most abundant. At these levels, we estimated that the proportion of cortically projecting neurons (in cases 91-9 and 91-51) represented approximately 4% of the total number of ipsilateral TH+ cells. There was no apparent dorsoventral topography among neurons projecting to either prefrontal, M1, or SMA. In the mediolatera1 dimension, the distribution of the TH+ retrogradely labeled cells did not differ significantly between injections placed in lateral M1 and in the lateral prefrontal cortex, with the number of neurons found in the dorsal SNc being roughly equivalent to that found in the VTA (Table 1). However, injections placed more medially, in the SMA (91-9 and 91-24) labeled proportionally more TH+ neurons medially than laterally (Table 1,cases 2 and 3). Some TH+ neurons in the SNc-VTA contained more than one retrogradely transported fluorochrome, indicating that they sent collaterals to two or more cortical sites (Fig. 11).The extent of this collateralization was however difficult to evaluate because of some uncertainties in the recognition of the double-labeled neurons. Double labeling was most easily recognized when the nuclear labeling by DY was associated with the cytoplasmic retrograde labeling by fast blue or fluorogold. Double labeling with fluorogold and fast blue was more difficult to appreciate. With these reservations in mind, the topography of collateralization is shown in one case, 91-24, (Fig. 12) in which the cortical injections were clearly separate from one another, by a few millimeters. In this case, we estimated that 15% of the SNc-VTAneurons projecting to M1 sent also projections to the SMA, and 28% of the neurons projecting to the lateral prefrontal cortex had collaterals to either M1 or SMA. A few neurons (3 out of 111) seemed to contain the 3 fluorochromes (Fig. 12-3211, and could represent neurons collateralizing to the 3 areas. The double-labeled neurons did not appear to have a particular topography relative to singly labeled neurons within the VTA-SNc complex. For the sake of comparison we also examined the extent of collateralization in neighbouring non-DA nuclei which sent projections to the cortical areas studied. In the raphe nuclei (Fig. 8-4011, the proportion of neurons containing two fluorochromes, versus those containing only one, appeared to be similar to that observed in the VTA-SNc, with slightly more double-labeled neurons in the dorsal than in the median raphe. On the other hand, the hypothalamic lateromammillary nucleus (Fig. 12-271) and the locus coeruleus (Fig. 12-451), which contained a fairly large number of retrogradely labeled neurons after each cortical injection, had large numbers of double- and triple-labeled neurons. Non-dopaminergic projections. A number of cortically projecting neurons were non-dopaminergic as assessed by the absence of TH immunolabeling. These neurons were partly intermixed with the DA cell groups. At the level of the diencephalon, retrogradely labeled neurons were located in the latero-mammillary n., and the infundibulotuberal n., which overlapped with the medial substantia nigra (Figs. 9-80, 10-296). In the mesencephalon, the prerubral area always contained retrogradely labeled neu-

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I10

91-9

Fig. 9. Distribution of neurons retrogradely labeled by fluorochromes in case 91-9, on representative coronal sections from hypothalamus to pons (section number is below each drawing: 30 Fm-thick sections). TH+ neurons are indicated with small dots. Circles are for neurons labeled with fluorogold (M1 injection), triangles for fast-bluelabeled neurons (injection in SMA, spreading to dorsal premotor) and

squares for diamidino-yellow-labeledneurons (injection in lateral prefrontal). The solid symbols are for the retrogradely labeled neurons that were also TH positive, the open symbols for those that were TH negative. TH labels both DA (first 5 levels) and noradrenergic (level 262) neurons.

P. GASPAR ET AL.

14

.:. .

91-51

Fig. 10. Same representation as in Figure 9 for case 91-51.Projections to M1 (Fluorogold)are also noted with circles, projections t o lateral prefrontal with squares (Fast blue), and to the dorsal premotor with triangles (diamidinoyellow).

DA AFFERENTS TO MOTOR CORTEX

15

TABLE 1.

Areas SMA-premotor

M1

Cases

Prefrontal

1

2

3

4

1

2

3

4

2

4

N

90

75

166

50

82

31

95

22

42

Gl

SNc PbPg RL+CL pn + if A8 Lm

45 18 15 3 13 3

33 24 17

33 31 11 4 20 1

52 25 22 32 6 18 2 1 1 10 11 8 2

16 29 31 3

25 43 11

50 36 9

52

9

-

16

10 2

-

36 30 21 2 10 9

'z

20

5

6

4

21 6 19 -

'Case 1: 91-75, case 2: 91-24, case 3: 91-9, case 4: 91-51. Neurons retrogradely labeled by the cortical injections in M1, SMA, or premotor, and lateral prefrontal cortices were counted in the substantia nigra compactd (SNc), the different subnuclel of the VTA (phpg, pn, nif, rl, cl), the retrurubral area (A8), and the lateromammillaty area (lm). N 1s the total No. uf cells, and the percentage uf this No. is given for the different nuclei, on semi-serial sections (1/300 kmi throughout the mesencephalon. In cases 1 and 2, all the retrogradely labeled neurons that closely overlapped with TH+ neurons un consecutive sections were counted; in cases 3 and 4, only those retrogradely labeled neurons that were immunolaheled with TH were counted.

ions that could be mistaken for DA neurons as they partly overlapped with the SNC pars gamma neurons (Figs. 8f,g, 10-256).Cortically projecting neurons were also frequently observed in the central gray (Fig. 9-110), intermixed with the DA periventricular cell group ( A l l ) , but were never double-labeled with TH. Some non-DA cortically projecting neurons were also observed in the A9-Al0 group, intermixed with the TH+ retrogradely labeled cells: such neurons were more frequently observed in the VTA cell groups, in particular in the caudal linear nucleus, which merges with the dorsal raphe and contains a number of non-DA, probably serotoninergic neurons (Fig. 10-196,166) that project to the cortex. At the level of the retrorubral area (Figs. 9-202, 10-166) DA and non-DA cortically projecting neurons were intermixed, the latter possibly belonging to the pedunculo-pontine tegmental nucleus, recognizable at this level by the intense acetylcholinesterase positive reaction of its neurons.

DISCUSSION This study demonstrates that the DA projections to the primary motor, supplementary motor, and lateral prefrontal areas of primates originate from a dorsal subgroup of the AB-AlO DA complex, which is further characterized by specific histochemical characteristics. Some of the mesencephalic DA neurons send collaterals to both motor and prefrontal areas. After each of our injections in the frontal lobe, TH positive neurons were retrogradely labeled in the mesencephalic DA complex, indicating that the DA innervation is widespread throughout the lateral frontal lobe of the owl monkey, as shown in prior analyses of dopamine-accumulating terminals (Berger et al., '86, '88) or TH positive axons in monkeys (Lewis et al., '87) and humans (Gaspar et al., '89). In these studies, higher densities of DA fibers were found in the primary motor and supplementary motor areas than in the prefrontal cortical areas. This seems to be also the case in owl monkeys, as injections in M1 labeled more TH positive neurons than injections in the lateral prefrontal cortex. This robust DA projection from the VTA or SNc to all three motor areas (primary, SMA, and dorsal premotor), was not noted in most previous anatomic studies (Kievit and Kuypers, ' 7 5 ; Leichnetz, '86; Markovitsh et al., '87). However, Jurgens '84 did mention afferents from the VTA

Fig. 11. Overlap and collateralization of DA neurons projecting to different cortical areas is shown in this micrograph from case 91-51. a: In the same field of the lateral VTA (n. parabrachialis pigmentosus) a neuron projecting to M 1 (FG indicated by an arrow), to lateral prefrontal (FB indicated by an arrowhead), and to dorsal premotor and M 1 (DY and FG, indicated by the open arrow). b: these 3 neurons contain TH. x 180.

to the SMA. This discrepancy could be due to methodological differences, such as the type of retrograde tracer that was used (peroxidase instead of fluorescent dyes) or the sizes of the injections. However, in other cases, we observed some retrogradely labeled neurons in the VTA-SNc, even after limited WGA-HRP injections in the primary motor area. A more probable reason for this discrepancy is that the mesencephalon was not analyzed in detail in previous studies, because DA afferents to the motor cortex were not suspected from prior studies in the rat. On the other hand, in the prefrontal cortex, or anterior cingulate cortex, where DA innervation had already been shown in the rat (Berger et al., '76; Lindvall et al., ' 7 8 ) , anatomical studies in macaque monkeys using peroxidase, revealed a significant population of neurons labeled in the VTA-SNC (Porrino and Goldman-Rakic, '82).

271

40 I

\

91 2 4

Fig. 12. Selected drawings from 6 coronal levels in case 91-24, to show the topography of neurons containing either one (open symbols), two (solid triangles and circles), or three (stars) retrogradely transported fluorochrome. For singly labeled neurons, circles are for neurons

projecting to M1, triangles to SMA, and squares to the lateral prefrontal cortex. For double-labeled neurons, solid circles are for n. projecting to motor and prefrontal areas, solid triangles for n. projecting to M1 and SMA. Same magnification as in Figure 10.

DA AFFERENTS TO MOTOR CORTEX

Topography of the dopaminergic projection The DA projections to M1, SMA, and dorsal premotor cortex originate from the 3 principal divisions of the mesencephalic DA complex, VTA, SNc, and retrorubral area, throughout their rostrocaudal extent. However, the retrogradely labeled neurons occupied specific positions in the dorsal parts of these divisions. Thus in the VTA, labeled neurons are mainly found in the dorso-lateral group, the n. parabrachialis pigmentosus, and in the medio-dorsal subgroups, then. rostralis linearis and caudalis linearis, whereas the ventral subgroups of VTA, the n. paranigralis and the n. interfascicularis, contained very few retrogradely labeled cells. In the SNc, the labeled neurons are confined to the dorsal diffuse part, pars gamma or pars mixta according to nomenclatures, but span its entire medio-lateral extent. In the retrorubral area, labeled neurons are found only in its dorsal portion. The DA projection to the lateral prefrontal cortex appears to originate from the same subnuclei of the A8-AlO complex. The largest amount of DA neurons projecting to the motor and prefrontal areas were located laterally to the midline, in the n. parabrachialis pigmentosus (a lateral subgroup of the VTA) and in the SNc. The DA neurons projecting to lateral M1 or lateral prefrontal cortices did not have obviously different topographic distributions in the mediolateral plane. However, DA neurons projecting to the SMA were proportionally more numerous medially than laterally, whereas the reverse was true of DA neurons projecting to M1 or lateral prefrontal cortex. Thus, despite some overlap, there could be a crude medio-lateral topography in the meso-cortical DA projections to the frontal lobe. The widespread distribution of the mesencephalic neurons projecting to the frontal lobe in owl monkeys is comparable with that previously reported for macaque monkeys (Porrino and Goldman-Rakic, '82). There are however a few discrepancies in the detail of the topography, which could reflect procedural factors or differences in organization between New and Old World monkeys. Thus, Porrino and Goldman Rakic ('82) described retrograde labeling in the ventral subgroup of VTA, n. paranigralis, a localization that was seldom found in our cases. Furthermore, the reported medio-lateral topography differed from that presently observed in that projections to the medial wall of the hemisphere (to the anterior cingulate cortex), originated more laterally in the SNc than projections to the lateral or orbital prefrontal cortices. Studies on the DA projections to other cortical areas are limited to the posterior parietal cortex in macaque monkeys. Lewis et al. ('88) indicated that TH neurons that projected to these cortical fields are also widespread in the A8-AlO complex, but their precise topography was not shown. A more extensive set of cortical injections should be compared in order to determine whether a topographic organization exists in the DA meso-cortical projection of primates besides the weak mediolateral trend presently observed. The topography of the mesocortical projections has been more extensively studied in rodents and cats. In cats, where diffuse mesocortical projections exist (Schneiber and Tork, '87; Markovitsh et al., '87) part of which may be DA (Tork and Turner '81), the dorsal situation of the mesocortical neurons in the VTA-SNc complex, presently observed in monkeys, was also shown (Schneiber and Tork, '87).However, the nuclear localization of these neurons seemed to

17

differ, being mainly located in the midline cell groups (rostra1 linear) of the VTA, and not in SNc or retrorubral cell groups (Schneiber and Tork, '87). In rats, DA mesocortical neurons are distributed in both the dorsal and ventral subnuclei of the VTA-SNc and appear to be more restricted to the medial DA groups, at least concerning the mesofrontal projection (Lindvall et al., '78; Carter and Fibiger, ' 7 8 ; Swanson, '82; Albanese and Bentivoglio, '82; Loughlin and Fallon, '84). Thus, projections to the prefrontal (infralimbic and prelimbic) and anterior cingulate cortices, which are the main cortical targets of DA input in rodents, are found in the VTA and medial SNc, but not in the lateral part of the SNc or in the retrorubral area (Carter and Fibiger, '77; Lindvall et al., '78; Swanson, '82; Loughlin and Fallon, '84; reviewed in Oades and Halliday, '87). Although some of the observed interspecies differences may reflect differences in methodology or the fact that the examined cortical regions were not homologous, the available data suggest that there is a dorsal and lateral shift of the DA mesocortical neurons in primates relative to rats and cats, which could correspond to a further segregation from the mesostriatal or mesolimbic projection neurons. The larger number of DA meso-cortical neurons in the SNc relative to the VTA in monkeys compared to rats could correspond to the increase of DA innervation to the superficial cortical layers in primates. Rats appear to have two DA mesocortical systems, differing by a number of functional characters; one arising from the VTA, which innervates the deep cortical layers, appears to be rather diffuse, and the other arising from the medial SNc, which innervates the superficial cortical layers, seems relatively restricted in the anterior cingulate cortex (Berger et al., '91). In primates, where DA innervation to layer I is most abundant and topographically widespread, the only indication for the existence of these 2 systems is a differential loss of DA fibers in the superficial or deep cortical layers in the frontal and motor cortices of patients with Parkinson's disease (Gaspar et al., '91). In the present study, where the injections occupied the entire width of the cortex, the question of a different origin of the deep and superficial innervations was not addressed. Separate injections in deep or superficial layers would be necessary to determine the existence of two separate DA systems in primates.

Non-DA projections About half of the cortically projecting neurons in the ventral mesencephalon were non-DA. These non-DA neurons are more frequent in the region of the VTA, and retrorubral area, than in the SNc. In most instances, these non-DA neurons belong to neighbouring nuclei that partly overlap with the DA cell groups. Thus, the large population of cortically projecting neurons lying between the mammillary bodies and the SNc, partly intermixing with the latter, may represent the histaminergic cortically projecting neurons, as described in rats (Wada et al. '91);indeed, histamineimmunoreactive neurons have recently been described in this area in humans, termed the tuberomammilary n. (Airaksinen et al., '91). As indicated by recent observations in the macaque, cholecystokinin could be the neurotransmitter of some non-DA mesocortical neurons in the prerubral area or n. ruber parvocellularis, which intermix with the SNc (Oeth and Lewis, '91). Contrary to previous reports in rats (Yoshida et al., '89; Stratford and Wirtshafter, 'go), we did not observe any DA cortical projection from the dorsal raphe, although DA neurons of the n. centralis (VTA) do

P. GASPAR ET AL.

18

extend rather caudally into the pontine raphe. The few retrogradely labeled neurons that are also TH+ in the dorsal raphe could represent displaced noradrenergic cells (A6-A7 groups) since they also contain dopamine betahydroxylase, the synthetic enzyme to NA. Finally in the retro-rubral area, DA and non-DA neurons projecting to cortex are intermixed; the latter could represent cortical projections either from the midbrain extrapyramidal area (Lee et al., '88) or from the tegmental pedunculopontine cholinergic neurons (Woolf et al., '84).

Collateralization The existence of DA neurons bifurcating to two or three frontal cortical fields was indicated by the presence of 2 or, more rarely, 3 retrogradely transported ffuorochromes in some TH+ neurons. However some caution is needed in evaluating collateralization because of possible interferences between the transport or visibility of the fluorochromes, and since the tracers may be taken up by fibers of passage. In the cases we analyzed for collateralization, there was no overlap in the diffusion zones of the injected sites and the injections remained confined to the cortical grey, thereby reducing the risk of dye uptake by underlying fiber fascicles while some uncertainties remain the present observations are a first step in gaining a better understanding of the organization of the cortical DA system in primates. The collateralization that we observed within the dorso-lateral frontal cortex of owl monkeys did not seem to depend on the interconnections within cortical fields: DA cells sent collaterals to unconnected areas of M1 and lateral prefrontal cortex as frequently as to interconnected zones of M1 and SMA (Stepniewska et al., '91); collaterals were distributed in both the medio-lateral plane (collaterals between lateral M1 and SMA) and rostrocaudal plane (between M1 and lateral prefrontal). Thus individual DA axons may provide terminal arbors at cortical distances of at least 6 mm (maximal distance separating our cortical injections) in either the medio-lateral or rostrocaudal directions. Alternatively, some DA axons may travel intracortically, similarly to the noradrenergic axons (Morrison et al., '82); however such an intracortical route would not appear to be the dominant pathway for the cortical DA axons since fascicles of DA fibers have been observed to course subcortically, with either a tangential orientation in the depth of the sulci, or a radial distribution in the crest of gyri (Levitt et al., '84; Gaspar et al., '89). Loughlin and Fallon ('84; see also Fallon and Loughlin, '82, '87; Takada and Hattori, '86) in rats reported that highly collateralized neurons were more frequent in the medial SNc than in the VTA. In owl monkeys, we observed collateralized DA neurons in all the VTA-SNc subnuclei that sent projections to the cortex. Collateralized neurons were more frequent in the lateral VTA, around the third nerve rootlets (n. parabrachialis pigmentosus), but this may simply be the consequence of this cell group having the highest amount of cortically projecting neurons. Whether some DA mesocortical neurons could also bifurcate to subcortical targets as shown in rats (Deniau et al., '80; Albanese and Bentivoglio, '82; Swanson, '82; Fallon and Loughlin, '82; Takada and Hattori, '87; and see Oades and Halliday, '87 for a review), has not been specifically examined in primates. However, the projections to caudate and putamen arise from ventral locations of the SNc in monkeys (Parent et al., '83; Franqois et al., '84; Hedreen, '91). Thus it seems unlikely that single DA neurons project

to both the striatum and cortex. Meso-cortical neurons might collateralize to other subcortical targets such as the n. accumbens, the bed n. of the stria terminalis, or lateral septum. Although the DA neurons giving rise to these projections have not yet been adequately localized in primates, their topography in rats overlaps with the cortically projecting neurons and neurons bifurcating to the prefrontal cortex and n. accumbens or lateral septum have been frequently reported (Deniau et al., '80; Albanese and Bentivoglio, '82; Fallon and Loughlin, '82).

Chemoanatomic compartments Although distributed over the different parts of the A8-Al0 complex, the DA cortically projecting neurons had common histochemical characteristics that set them apart from other DA groups. Indeed, the different subnuclei of the VTA, and the pars r of the SNc, where the mesocortical neurons were found, were characterized by noradrenergic (NA) and neurotensin (NT) innervations as well as by the presence of neurons immunoreactive to calbindin. The conspicuous NA innervation of all the dorsal components of the VTA, SNc and retrorubral area had not been shown previously in primates. In anterograde tracing studies from the locus coeruleus in macaques, afferents to these areas were not described (Bowden et al., '781, perhaps because of the lower sensitivity of the autoradiographic method. In rats, inputs from the locus coeruleus, or subceoruleus, to the VTA have been demonstrated (Simon et al., '79; Philipson, '79; Herve et al., '82) and a sparse DBH innervation was shown in the VTA by Swanson and Hartman ('75) but this innervation was not shown to extend in the dorsal SNc, or retrorubral area. Whether these differences are species related or linked to an increased sensitivity of the modern methods remains to be answered. The dense neurotensin innervation of the DA mesencephalic neurons of monkeys has also been described in rats (Jennes et al., '82; Woulfe and Beaudet, '89; Bayer et al., '911, cats (Sugimoto and Mizune, '871, and humans (Mai et al., '87).Furthermore, NT receptors have been shown to be localized on the DA neurons (Sadoul et al., '84; Szigethy and Beaudet, '891, and synaptic contacts between NT terminals and DA cells have been visualised (Woulfe and Beaudet, '89; Bayer et al., '91). On the other hand, neurotensin positive neurons in the VTA have been reported only in rodents (Hokfelt et al., '84; Jennes et al., '82) and have not been seen in cats (Sugimoto and Mizune, '871, monkeys (Deutch et al., '88; and present observations), or humans (Mai et al., '87; Gaspar et al., 'go), although negative observations may be compromised by the absence of colchicine pretreatment. The bulk of the nigral NT projection seems to arise from the striatum in cats (Sugimoto and Mizune, '87) but such information is not available in other species. However, the pattern of dense NT innervation in VTA, dorsal SNc, and retrorubral area corresponds to the described pattern of afferents from the n. accumbens (Nauta et al., '78; Haber et al., '901, which contains many NT+ perikarya (Zahm and Heimer, '881, suggesting that the ventral "limbic" component of the striato-nigral pathway may utilize mainly neurotensin, whereas the other striatonigral afferents, which surround the ventral SN components appear to contain mainly enkephalins and substance P in rodents (reviewed in Groenewegen et al. '91) and humans (Gaspar et al., '83; Haber and Groenewegen,

DA AFFERENTS TO MOTOR CORTEX '89). Interestingly these peptides have different effects on the DA mesencephalic neurons (Cador et al., '89). Calbindin D28K, a major calcium-binding protein in the central nervous system, (Heizmann and Hunziker, '91) has been shown to label a subpopulation of DA mesencephalic neurons in rats and monkeys (Gerfen et al., '85, '87). We have extended these observations in monkeys, by the comparative topographic analysis of TH and CABP in the different catecholaminergic cell groups, from diencephalon to pons. In rodents, the neurochemical distinction of the SNc DA cells with respect to their CABP content seemed to coincide with different projection patterns to the striatal matrix (CABP+) or to the striosomes (CABP-) (Gerfen et al., '87). Whether such a pattern exists also in primates is an open question. Our observations of a topographic coincidence of the CABP positive cells and the mesocortically projecting neurons are of interest in this perspective. We have observed that the extent of CABP-TH colocalization varies in the different DA groups of owl monkeys, and that CaBP is indeed present in those DA neurons that project to the cerebral cortex (Gaspar et al., personal observations). The importance of this finding is underscored by the increasing evidence that CABP-containing neurons are more resistant to degenerative (Parkinson's disease) or neurotoxic (MPTP, 6OHDA) cell death (Gerfen et al., '87; Yamada et al., '91; Manaye et al., '91). The coincidence that we observed between the CABP zones of expression and the NT and DBH innervation suggests the testable hypothesis that these innervations could control the expression of this protein.

19

rostrocaudal plane with the caudal A8 group being opposed to the A9 rostra1 group. However, the topographic distribution of the cortically projecting neurons and of the neurochemical markers studied here showed no obvious discontinuities in these planes (between A9 and A10, or AS), whereas there was a clear discontinuity in the dorso-ventral plane. Golgi studies in primate SNc have identified different dendritic orientations between neurons oriented dorsally or ventrally in the SNc (Franqois et al., '87). Similar dorsoventral differences between dorsal and ventral SNc neurons had in fact already been noted in rodents and cats with Golgi, histochemical, and hodological studies (Fallon et al., '78; Fallon and Loughlin, '87, Gerfen et al., '85; JimenezCastellanos and Graybiel, '87; Weiss-Wunder and Chesselet, '91; rev. in Groenewegen et al., '91). The present results are relevant to studies of pathology in humans that correlate cognitive deficits with cell loss in the mesencephalon and DA reductions in the cerebral cortex. Until now, the DA cell groups that have been investigated in such attempts (Javoy-Agid et al., '83; Rinne et al., '89; Torack and Morris, '88) are the n. paranigralis and the n. interfascicularis, the two ventral groups of the VTA that contain few or no cortically projecting neurons in monkeys. Our results in owl monkeys, together with those of others in the macaque, indicate that the dorsal DA group should be investigated more closely, as has been done recently for the melanized neurons (Gibb and Lees, '91) or for the Calbindinpositive neurons (Yamada et al., '91) in the mesencephalon of patients with Parkinson's disease.

Functional implications

ACKNOWLEDGMENTS

The different characteristics of the DA meso-cortical projections, i.e., the topographic overlap of the DA mesocortical neurons projecting to different cortical fields, their partial collateralization, and common histochemical characters, all suggest that there is some functional unity within this pathway, at least within the lateral frontal lobe. In Parkinson's disease, previous findings point in the same direction as DA fibers are similarly reduced (by 70%) in M1, premotor, and prefrontal cortex (Gaspar et al., '91). DA neurons projecting to prefrontal, premotor, and primary motor areas may be subject to similar influences and regulations. The possibility of a simultaneous action of the DA inputs, through collaterals, on different cortical areas is particularly interesting in this perspective: DA could act to synchronize or modulate simultaneously the different cortical centers controlling motor behavior. Physiological studies in the awake monkey indicate that neurons in dorsal SNc are activated by trigger stimuli and reward during the learning phase of a task (Schultz et al., '91). Furthermore the importance of DA in mnemonic processes has been shown at the level of the prefrontal cortex (Sawaguchi and Goldman-Rakic '91). Although DA may influence vast expanses of cortex in primates, similarly to NA, the activation of the DA mesocortical projection has been shown to produce very different effects from that of NA coeruleocortical inputs: DA blocks momentarily the thalamicinduced cortical activation instead of the protracted increase of signal to background caused by NA inputs (Mantz et al., '88). In slice preparations from human cortex, DA was shown to inhibit glutamate-induced volleys whereas NA increased them (Radisavljevic et al., '91). Compartmentations of the DA mesencephalic cell groups have been mainly described in the mediolateral plane, with the A9 group laterally and the A10 group medially, or in the

We thank Todd Preuss and Brigitte Berger for helpful comments on the manuscript, and Dr. Heizmann for providing the antibodies to calbindin D28K. This work would not have been possible without the patience and excellent technical assistance of Judy Ives and Laura Trice. This work was supported by grant NS 16446 (J.H.K.) and Fondation Cino del Ducca (P.G.).

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