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Benin Research Bulletin, VuJ. 40, No, 3, pp. 2 1 9 -2 2 8 , 1996Copyright © 1996 Elsevier Science Inc.

Printed in the USA. All rights reserved0361-9230/96 $15.00 + .00

PII S0361-9230Î96)00048-1

Efferent Projections of the Retrorubral Nucleus to the Substantia nigra and Ventral Tegmental Area in Cats as

Shown by Anterograde TracingM. P. M. ARTS,* H. J. GROENEWEGEN,* J. G. VEENINGt AND A. R. COOLS*1

Departments o f * Psychoneuropharmacology andfAnatomy and Embryology, Graduate School of Pathophysiology of the Nervous System, University of Nijmegen, P.O. Box 9101,

6500HB Nijmegen, The Netherlands and *Graduate School Neurosciences Amsterdam, Research Institute Neurosciences, Vrije Universlteit, Department of Anatomy and Embryology, Amsterdam, The Netherlands

[Received 27 November 1995; Revised 30 January 1996; Accepted 2 February 1996]

ABSTRACT: The aim of the present study was to determine whether the retrorubral nucleus projects to the dopaminergic nuclei in the ventral midbrain of the cat. For this purpose, injec­tions of biotinylated dextran-amine or Phaseolus vulgaris-leu- coagglutinin were placed into the retrorubral nucleus under ste­reotaxic guidance. The tracers were visualized by means of (immune) histochemical procedures. In addition, tyrosine hy­droxylase immunohistochemistry was used to evaluate the lo­cation of the injection sites and the distribution of the antero- gradely labeled fibers. Both tracers reveal the same topography of labeled fibers in the ventral mesencephalon. Labeled fibers with varicosities were found ipsilaterally in the substantia nigra pars compacta, the substantia nigra pars lateralis, the ventral tegmental area and, contralaterally, In the substantia nigra pars compacta* the ventral tegmental area, and the retrorubral nu­cleus, A considerable number of labeled axons with varicosities were observed to be wrapped around the dendrites and peri- karya of tyrosine hydroxylase-positive neurons in these areas. The present results are discussed in view of the possible role of the A8 dopaminergic cell group in the coordination of A9 nigro- striatal and A10 mesolimbic systems, as well as in the progres­sive pathology seen In patients suffering from Parkinson’s dis­ease.

KEY WORDS: A8, A9, A10, Dopamine, Tyrosine hydroxylase im­munohistochemistry, Phaseolus vulgaris-leucoagglutinin, Bioti­nylated dextran-amine.

INTRODUCTION

Parkinson's disease is a progressive neurological disorder, char­acterized by ihe degeneration of dopaminergic neurons in the midbrain. This degeneration of neurons appears to proceed, de­spile the improvement of neurological symptoms following treat­ment with routine anti-Parkinson medication. Why tine dopami­nergic neurons cease to function and eventually die is as yet unknown.

The dopaminergic neurons in the midbrain are distributed over three major cell groups: the substantia nigra pars compacta

(SNC, A9), the ventral tegmental area (VTA, A 10), and the retmrubrai nucleus (RRN, A8). The RRN contains a large num­ber of dopaminergic neurons in the tegmentum, located dorsally and eaudally to the SNC [1,3,11,25,32].

The A8 cell group differs in a number of aspects from the other dopaminergic cell groups. First, the striatal projection areas of A8 cells play a role in the control of facial musculature in cats [421, in this context, it is relevant to note that an early sign in Parkinson’s disease is masking of facial expression [7]. I f the A8 neurons in humans have a function similar to that in cats, this may imply that A8 cells degenerate early in the disease. Second, it has been reported [12] that, in primates, AS dopaminergic neu­rons have a higher vulnerability to the neurotoxin l-methyl-4- phenyM,2,3,6-tetrahydropyridine (MPTP) as compared to the other dopaminergic cell groups [cf, refs. 40,47]. Animals intox­icated with this drug display an unequivocal parkinsonian syn­drome. This, again, suggests an important role for A8 cells in the degeneration seen in Parkinson patients. Third, the three men­tioned cell groups collectively give rise to the mesotelencephalic dopaminergic pathway which displays a broad topographical or­ganization [5,13,26,43,45]. The A9 cell group primarily inner­vates the dorsolateral "motor” part of the caudate nucleus and putamen, whereas the A 10 cell group gives rise to fibers which predominantly terminate in the ventral, ' ‘limbic” striatum, i.e., nucleus aceumbens and olfactory tubercle [15,35], In contrast, the A8 dopaminergic efferents innervate both the “ motor” and the “ limbic’' striatum [6,13,14,17,26,35,44,45]. As such, the A8 cell group can control the activity in these two striatal domains. Fourth, both the projections from the SNC to the dorsal striatum and the projections from the VTA to the ventral striatum are reciprocal [24,27,35], suggesting that the A9 and A 10 nuclei receive direct feedback from their respective target areas in the striatum. In contrast, the dorsal striatal A8-innervated region has no projection back to the A8 cell group, implying that there does not exist such a feedback loop [19,20,27,30,37, see however ref. 39 ]. The ventral striatal target region of the A8 cell group has relatively sparse projections to A8 cells [2,19,22,35]. Finally, it

To whom requests lor reprints should he addressed.

219

220 ARTS ET AL.

has been saggested that A8 cells directly innervate A10 cells, at least in rats [13]. If the latter could be confirmed, it is evident that degeneration of A8 cells will have far-reaching conse­quences for the functioning of A10 cells.

The aim of the present study was to determine whether there exist projections from the RRN to the A9 and A 10 dopaminergic cell groups in the cat. For this purpose, injections of the tracers biotinylated dextran-amine (BDA) and Phaseolus vulgaris-leu- coagglutinin (PHA-L) were placed in the retrorubral nucleus.

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MATERIALS AND METHODS

Thirteen adult male cats (13-16 months old, 4.3-5.2 kg) were used in the present study. Two cats were perfused for ty­rosine hydroxylase (TH)-immunohistochemical analysis of the ventral mesencephalon. Two cats received an injection of the anterograde tracer Phaseolus vw/gam-leucoagglutinin (PHA-L) [18], and nine cats received an injection of the tracer biotinylated dextran-amine (BDA) [46]. In addition, these nine cats were also processed for a TH-immunohistochemical staining.

Surgery

Prior to surgery,_the animals were anaesthetized with a mix­ture of 0 2 and 5% Ethrane® (Abbot BV, The Netherlands) and subsequently intubated. The animals received an injection of Al- bipen® LA (Mycofarm Nederland BV, The Netherlands; 20 mg/ kg SC) to prevent infection and Atropini Sulfas® (Pharmaehemie BV, The Netherlands; 0.5 mg/4,5 kg IM), to prevent mucous secretion^Surgical levels were maintained with a mixture of 0 2, 3-3.5% Ethrane® and N20.

The animals were unilaterally injected with either 0.3 p\ BDA (Brunschwig Chemie, The Netherlands; 1% solution in distilled water) or 0.3 p\ PHA-L (Brunschwig Chemie, The Netherlands; 1% solution in distilled water) aimed at the RRN. Coordinates with respect to the interaural line were: anterior ( + ) 2.8 mm (with an angle of 10°, tip pointing caudal), lateral 2,8 mm, and ventral ( —) 5.7 mm according to the atlas of Snider and Niemer[41]. The tracers were delivered by means of a 1 p\ syringe (Hamilton Bonaduz AG, Switzerland) at a speed of 0.02 jt/l/min. Survival times ranged between 6 and 9 days.

General Fixation Procedure

The cats were anaesthetized with a lethal dose of pentobar­bital, IP (Narcovet®, Apharmo, The Netherlands) and perfused transcardially with 500 ml saline followed by 1 1 lixative, which consisted of 4% paraformaldehyde, 0.05% glutaraldehyde in 0.1 M phosphate buffer (PB, pH 7.4), either with (in case of TH- staining alone) or without (in case of double staining) 0.2% of a saturated picric acid solution. After this fixation procedure, the cats were finally perfused with 500 ml of a 10% sucrose solution in the same fixative. The brains were removed and stored in a 30% sucrose solution in PB for three days at 4°C. Thereafter, transverse sections were cut at either 60 pm (BDA tracer) or 40 /¿m (PHA-L tracer) on a freezing microtome and collected in 8 vials (providing 8 parallel series of sections) containing a 30% sucrose solution in PB to store at -20°C or in PB for further (free floating) processing (modified procedure from Spooren et al.) [42].

Immunohistochemical Staining

Sections from the mesencephalon of the non-operated cats (n = 2) were stained for tyrosine hydroxylase-immunoreactivity (TH) according to Spooren et al. [42] Following repeated rinses in PB and subsequently in Tris-buffered saline (TBS; pH 7.4,

0.05 M), the sections w'ere treated with a \% H A in TBS to reduce endogenous peroxidase. After rinsing (standard proce­dure: 3 X 15 min), the sections were incubated for 18 h in the primary antibody (a monoclonal mouse anti-TH-antiserum, Inc- star Corp. Nijmegen, MN, USA, 1:750), diluted in supermix (0.05 M TBS, 0.5% Trilon-X-100, 0.25% gelatine, pH 7.4).Thereafter, the sections were rinsed with TBS, and incubated for1 h in the secondary antibody (goat anti-mouse IgG, Dakopatts, Denmark; 1:50) diluted in supermix. Subsequently, the sections were again rinsed in TBS and incubated for 1 h in rat peroxidase anti-peroxidase complex (PAP, Nordic Immunology, The Neth­erlands; 1:100 in supermix). After rinsing the sections three times in TBS and lastly in PB, the sections were treated with nickel-enhanced diaminobenzidine (DAB, 12.5 mg in 25 ml 0.1 M PB, pH 7.4 with I ml 2% ammonium-Ni-sulphate and 6.6 fj\ H A ) for 11 min. Finally, the sections were mounted and cov- erslipped with Entellan^ (Mereks, The Netherlands).

BDA and PHA-L Staining

In the tracing experiments, 1 out of 4 sections were stained for either BDA (n = 9; sections 240 pm apart) or PHA-L (/; = 2; sections 160 pm apart). Following a pretreatment with a 1% HiCK solution in TBS for 10 min, the sections containing BDA were incubated using an avidin biotin complex-staining ( ABC, Elite PK-6100, Brunschwig Chemie, The Netherlands) for 1 hour. Prior to the ABC-staining, the sections containing PHA-L tracer were incubated with a biotinylated anti-PHA-L antibody (affinity purified, raised in goat, Brunschwig Chemie, The Neth­erlands; 1:2000 in supermix) at 4°C overnight. Thereafter, the sections were treated with nickel-enhanced DAB to stain the la­beled libers and injection site, resulting in a black reaction prod­uct. These sections were further processed for the double-staining with antibodies against tyrosine hydroxylase. The tyrosine hydroxylase-immunoreactive neurons were visualized as de­scribed above (see Immunohistochemical Staining); however, the end-product was turned brown by means of the DAB reaction(12,5 mg DAB in 20 ml Tris-HCI, 6.6 ¿/I 30% HA»). Through­out the staining procedures, the rinses between the incubations were in TBS.

RESULTS

Tyrosine Hydroxylase-Positive Cell Groups

A brief overview will be presented of the different tyrosine hydroxylase-immunoreactive midbrain areas as identified in this study (Fig. 1A-D).

Retrorubral nucleus. The A8 cell group is located within the RRN as designated by Berman ¡3]. Rostrally, this nucleus is situated medial to the medial lemniscus and ventral to the red nucleus. At this level, the AH cells appear as a eell-bridge joining the TH-positive neurons of the A 10 cell group and the A 9 neu­rons of the SNC (Fig. IB ). More caudally, two subdivisions are distinguished within the RRN [ 16): a dorsal ( A8d) and a ventral part ( A8v; Fig, IC,E). The dorsal subdivision appears as a rather diffuse population of neurons, while the ventral subdivision con­tains more densely packed neurons. The AXv extends more cau­dally than the SNC(dz) (Fig. ID).

Substantia nigra. The substantia nigra is composed of three divisions: pars compacta (SNC), pars lateralis {SNL), and pars reticulata (SNR) 13]. The pars compacta consists of at least two parts [261, a densoeellular zone (SNC(dz)), which is charac­terized by its densely packed neurons and finger-like extensions, and a cell-sparse region (SNC(cs)), which is located rostrally, dorsolateral to the SNC(dz) (Fig. 1 A~C,F).

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222 ARTS ET AL.

The pars lateralis of the substantia nigra (SNL) forms a clus­ter of rather loosely packed cells and is located ventral to the medial lemniscus and dorsolaieral to the SNC (Fig. 1A,B).

The SNR comprises a large area located ventral to the SNC and SNL (Fig. IA,B). This area is almost devoid of TH-stained neurons; however, it contains many TH-positive dendrites and axons, originating from the TH-immunoreactive cells of theSNC,

Ventral tegmental area. The A10 cell group is distributed over five nuclei, collectively referred to as ventral tegmental area (VTA) [23,36]. Three nuclei are arranged along the midline: the nucleus linearis rostralis (LR), the nucleus linearis centralis (LC), and the more ventrally located nucleus interfascicularis (NIF). Two groups are located more laterally: the nucleus par- abrachialis pigmentosus (PB) dorsally and the nucleus parani- gralis ventrally (PN; Fig. 1A-C).

Anterograde Tracing ExperimentsThe location and extent of the injection sites of the BDA {n

= 9; 93-11, 93-30, 93-31, 93-35, 93-36, 93-37, 93-43; controls: 93-14, 93-42) and PHA-L (n = 2; 94-343; control: 93-316) in different parts of the RRN and adjacent structures are schemati­cally represented in Figure 2. The majority of BDA injection sites are ovoid in shape with an intensely stained centre varying be­tween 300 and 600 /im in diameter (Fig. 1G, corresponding to Fig. 3E ). In a few cases in which the centre of the BDA injection site is located in the reticular formation dorsal to the RRN, the peripheral part of the injection site extends medially and ventrally into the RRN (cases 93-36 and 93-43). The BDA injections in the rostrolateral RRN result in labeling of nearby dendrites and perikarya located in the SNC (Fig. 3). The PHA-L injection site in the RRN has a more compact appearance, with a smaller pe­ripheral zone. No labeled perikarya were seen outside the PHA- L injection site.

Following all injections of BDA and PI-IA-L in the RRN, three types of labeled fibers can be distinguished in the ventral mesencephalon. The first type of fibers is smooth and very thin (with a diameter less than 1 ¿¿m; Fig. 4A), The second type of labeled fibers, with approximately the same diameter as the first type, carries numerous varicosities (Fig. 4A). These varicosities have diameters slightly larger than the interconnecting axonal segments and are 1.7-2.1 jj,m long. The distance between the varicosities ranges from 5-20 ¿¿m. The third type of fibers is rather coarse and smooth (Fig. 4B). These fibers are ribbon­shaped (approximately 4 -6 ¡xm in width and 2.5 ¡xm thick). Most of these fibers traverse the mid line and bend ventrally and caudally following crossing,

RRN-Effevents Within the Ventral Mesencephalon

Following the PHA-L injection in the RRN, a pattern of la­beled fibers is found in the mesencephalon that does not differ from the pattern seen following the BDA experiments.

In view of the fact that several of the injection sites primarily located in the RRN also involve parts of the overlying reticular formation, the pattern of labeling following injections in the re­ticular formation just dorsal to the RRN will be briefly described first. Three cats received such an injection in the reticular for­mation, dorsal to the RRN (cats 93-14, 93-42, and 94-316). In cat 93-14, sparse labeling of the fibers with varicosities is seen in the ipsilateral A9 and A10 cell groups, whereas the A8, A9, and A 10 cell groups contralateral to the injection site are devoid of labeled axons. In cats 93-42 and 94-316, no terminal labeling is seen ipsi- or contralaterally in the three TH-positive areas. In these three cases, however, the above-mentioned coarse fibers

are most prominent. These fibers are directed primarily in a cau­dal direction and do not appear to terminate in the SNC. Since the present study focuses on the afferents of the dopaminergic cell groups in the ventral mesencephalon, the following descrip­tion will concentrate on the distribution of the thin fibers with or without varicosities, ignoring the coarse fibers (Fig. 4A,B).

Cat 93-11, with a BDA injection in the most caudolateral part of the RRN, displays a representative liber pattern in the ventral mesencephalon (Fig. 3). From the injection site, fibers run either in a medial direction through the RRN to the VTA, or they traverse the medial lemniscus and extend ventrally into the ipsilateral A9. At caudal levels, thin fibers are present predominantly in the SNC(dz). An example of a labeled fiber with varicosities oriented along the dendrites of a TH-positive neuron located in the most caudal part of the SNC is given in Figure 5 A. More rostrally, libers can be distinguished in the medial and lateral regions of the SNC and in the substantia nigra pars lateralis (SNL; Figs. 4A, 5B). Fiber and terminal labeling display similar patterns in the ipsi- and contralateral A 10 cell groups. In the most caudal part of the A 10 cell group, densest labeling is found in the central linear nucleus and the nucleus paranigralis (Fig. 5C,D). At more rostral levels, fibers are confined to the nucleus paranigralis, parabrachialis pig­mentosus, and nucleus rostralis linearis ( Fig. 5EJF) of the A 10 cell group. Contralateral to the injected side, terminal labeling has the highest density in the eaudomedial SNC. More rostrally, axons with varicosities arc present in the contralateral medial and lateral SNC, but not in the SNL. A few fibers can be traced to the con­tralateral A8 (Fig, 5G,H). Most fibers are concentrated in the ven­tral part, namely A8v.

Following smaller injections in the RRN (e.g., cat 93-43 ), the overall pattern of termination in the ventral mesencephalon is similar to that in cases 93-11 and 94-343. However, the number of labeled fibers is smaller.

Compared with the injections described above, a small injec­tion of BDA (case 93-30), which was placed in the rostral part of the RRN, results relatively in the largest number of labeled fibers. These fibers traverse the medial lemniscus and distribute over the entire ipsilateral SNC, including the SNL (Fig. 4A). Moreover, the dorsolateral part of the A 10 cell group, both ipsi- and contralaterally, is densely labeled, whereas the medial All) contains only sparse labeling. Contralaterally, labeled libers are found in the RRN, both in its caudal and rostral parts. No fibers can be traced to the lateral part of the A9 cell group.

Considering all injections in which the RRN is involved, the total number of anterogradely labeled fibers with varicos­ities is rather small, however, they bear a specific relationship with dendrites and cell bodies of TH-positive neurons. In most cases, these axons appear to have close appositions with the dendrites and cell bodies of the TH-positive neurons. In some cases, the labeled axons have the same orientation as the den­drites (Fig, 5A,B,H). This relationship between the retroru- bral varicose axons with somata and proximal dendrites of TH-positive neurons is most prominent at the ipsilateral side in the SNC(dz) and SNL. At the contralateral side in the A8, similar phenomena are seen ( Fig. 5G,H ). In the ipsi- and con­tralateral VTA, labeled libers with varicosities are present in the neuropil, however less frequently in close apposition with TH-positive neurons (Fig, 5 C -F ) .

RRN-Efferents to the Telencephalon

Several labeled, thin fibers with or without varicosities are found in the lateral part of the caudate nucleus, the nucleus accumbens, olfactory tubercle, hypothalamus, and thalamus (data not shown).

RETRORUBRAL BF FI: RENTS TO THE SUBSTANTIA NIGRA 223

I-'Ki. 2. Schematic drawings of transverse sections to show the location of the injection sites, ordered from rostral (A) to caudal (D).On the left side of each section, the BOA (A ..D) and PHA-L ( B \ C ) injection sites are depicted. On the right side of each section,the different TH-positive cell groups are delineated. The number of each cat is abbreviated in the drawings (316 corresponds with 94- 31h, and 1 I corresponds with 93-11). AS, A8 cell group; ML, medial lemniscus; RN, red nucleus; SNC(cs), cell-sparse zone of the SNC; SNC(dz). densoceliular zone of the SNC; SNL, substantia nigra pars lateralis; SNR, substantia nigra pars reticulata; VTA, ventral tegmenta! area.

DISCUSSION groups within the ventral mesencephalon. One indication thatsuch connections exist in rats is the description by Lind vail and

Until now, little has been reported on the efferent connections Bjorklund [32] of the course of the ascending catecholaminergic of the retro rubral nucleus ( RRN ) to the other dopaminergic cell libers in the mesencephalon. Tegmental radiations from the dor-

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FIG. 3. Schematic representation of the distribution of ante mg rudely labeled thin libers with vari­cosities in the ventral mesencephalon following an injection of BDA in the RRN (cat l)3 - l l ) . illustrating the BDA labeling, seen at six different rostroeaudal levels. (A) rostral section; (F) caudal section. The injection site is also depicted. Dots mark the location o f a presumptively retrogradely labeled neurons outside the injection site. Ill, oculomotor nerve; ML, medial lemniscus; RN, red nucleus.

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RETRORUBRAL EFFERENTS TO THE SUBSTANTIA NIGRA 227

cauda te nucleus. However, it should be noted tha t previous de­scriptions of these projections have been based on different tra­cing techniques, i.e. the anterograde tridated amino acid tracing [26,27 ] or retrograde tracing [ 42]. It is possible that PI-IA-L and BDA used in the present study, are less suitable to visualize small calibre mesostriatal liber systems over such long distances in cats. This implies that the present study does not answer the question whether and how the pattern of labeling in the TH- positive cell groups in the ventral mesencephalon, originating in the RRN, is related to the projections ascending to the forebrain.

F unction ai Considerations

One can only speculate on the function of these RRN efferents to the SNC and VTA. The present data suggest that the RRN can directly influence the activity of the A9 and A 10 cells. This has two far-reaching consequences. First, the RRN may play a crucial role in coordinating the function of the dopaminergic A9 and A10 systems, each of which have their own specific function in reg­ulating cognitive and motor behavior [9,10]. As discussed else­where in detail [ 9 ], there appears to exist a delicate balance and interplay between both systems. The present study implies that the RRN, including the AS cells, may be involved in the modu­lation of this balance and interplay. Second, degeneration of A8 cells may have a strong impact on the functioning of the dopa­minergic cell groups. Once A8 cells start to degenerate, there are no, or only slight, feedback mechanisms from the striatal regions to counteract these disturbances (see Introduction). If this is in­deed true, degeneration of A8 cells may play a key role in the progression of the pathology seen in Parkinson’s disease.

At'KNOWLHDCiMliNTS

The authors wish to thank Dr. W. Spooren for critically reading the manuscript. We arc grateful to T, Peters for excellent help with the an­aesthesia, and to T. Hnfmans and IX de Jong for expert photographic assistance. This study was supported by the Graduate School of Patho­physiology of the Nervous System (Nijmegen, Utrecht, and Wagenin- gen).

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