Bilateral tectal projection of single nigrostriatal dopamine cells in the rat

0306-4522/89$3.00+ 0.00 Pergamon Press plc 0 1989IBRO

NeuroscienceVol. 33, No. 2, pp. 311-321, 1989 Printed in Great Britain

BILATERAL TECTAL PROJECTION OF SINGLE NIGROSTRIATAL DOPAMINE CELLS IN THE RAT K. J. CAMPBELL* and M. TAXADA? Department of Anatomy, University of Toronto, Medical Sciences Building, Toronto, Ontario, Canada M5S IA8 Abstract-Employing fluorescent retrograde double/triple labeling techniques, we found that a substantial population of substantia nigra pars reticulata cells send divergent axon collaterals to koth the ipsilateral striatum and bilateral superior colliculi in the rat. These multi-collateralized neurons were localized predominantly in the ventrolateral portion of the substantia nigra pars reticulata at its rostra1 level. Furthermore, tyrosine hydroxylase immunofluorescence histochemistry combined with fluorescent retrograde tracing techniques showed that the vast majority (more than 85%) of such specifically branched cells are dopaminergic. This novel nigral cell population seems to be in a strategic position to evoke dopamine-mediated motor impairments (i.e. abnormal saccadic eye movements in Parkinsonism) and/or behavioral syndromes (i.e. compulsive turning behavior) through the GABA-containing nigrotectal pathway.

In view of the existence of massive efferents projecting outside the basal ganglia, the substantia nigra [especially the pars reticulata (SNr)], in conjunction with the pallidum, has been recognized as an output source of the basal ganglia.1’~s2~53The superior colliculus (SC)6,7,‘3,20,26,30-33.38.~.48,58 is one of two major target sites of the SNr [the other is the ventromedial

thalamic nucleus 01M)6,‘Z.‘6*26,30,32,34.57], The fiber projection from the SNr to the SC is relatively well-organized in a topographic manner; most of projection neurons originate in the ventral portion of the SNr through its rostra1 two-thirds extent, and terminate in the intermediate/deep layers of the SC through its caudal two-thirds extent. This nigrotectal pathway is predominantly ipsilateral and to a lesser degree contralateral.7~2’~22~30~3*@‘~4E~s8 The pathway has also been implicated in the initiation of saccadic eye movements.‘3~3’~59~62~80~8’ Indeed, a subpopulation of SNr cells attenuate their activity prior to saccadic eye movements and exert a tonic, GABAergic inhibition on saccade-related cells in the SC.35m37 Many clinical reports have shown that individuals suffering from Parkinsonism often exhibit abnormal saccadic eye movements.‘“~‘8~‘9~4’~50~60~6’~70~77 In this respect, we have recently suggested in the rat that a specific population *To whom correspondence should be addressed. tPresent address:- Department of Anatomy and Neurobioloev. The Universitv of Tennessee Memphis, 875 Monroe Avenue, Memphis, TN 38163, U.S.A. Abbreviations: DY, Diamidino Yellow; FITC, fluorescein isothiocyanate; MFB, medial forebrain bundle; MPTP, I-methyl-4-phenyl-1,2,3,6_tetrahydropyridine; PI, propidium iodide; SC, superior colliculus; SNc, substantia nigra pars compacta; SNl, substantia nigra pars lateralis; SNr, substantia nigra pars reticulata; TB, True Blue; TH, tyrosine hydroxylase; VM, ventromedial thalamic nucleus; VTA, ventral tegmental area; WGAHRP, wheat germ agglutinin conjugated horseradish peroxidase. 311

of SNr cells may provide a critical neuronal substrate for such clinical findings.68,69 Thus, this cell group, which is localized primarily in the ventrolateral portion of the SNr at its rostra1 level, projects to both the striatum and SC by way of axon collaterals.68 At least part of these bifurcating projection neurons are dopaminergic and can be eliminated by injecting a Parkinsonism-inducing drug, 1-methyl-4-phenyl1,2,3,6-tetrahydropyridine (MPTP) into the medial forebrain bundle (MFB, nigrostriatal dopamine fiber tract).69 In the present report, we have investigated the possible existence of a specific nigral dopamine cell population which simultaneously innervates both the ipsilateral striatum and bilateral SC in the rat. EXPERIMENTAL PROCEDURES Eighteen adult male albino rats (Wistar, 280-320 g body weight) were used for this study. Animals were anesthetized i.p. with sodium pentobarbital (60 mg/kg body weight) and were each injected into the bilateral SC with the following two different fluorescent retrograde tracers: True Blue (TB, 5% aqueous suspension) and Diamidino Yellow (DY, 3% aqueous suspension). TB injections were made stereotaxically into the SC on one side of the brain over two needle penetrations, while DY injections were made into the SC on the opposite side of the brain over two needle penetrations. A total volume of 0.2-0.4~1 of each tracer was symmetrically deposited centered on the intermediate/deep layers of the SC at its middle level (12 rats) or at its caudal level (six rats), because nigrotectal fibers terminate mainly within the intermediate/deep layers of the nucleus over its caudal two-thirds extent.6,30,3’.”Of 12 rats injected bilaterally with TB and DY at the middle level of the SC, six rats also received unilateral injections of a third fluorescent retrograde tracer, propidium iodide (PI, 10% aqueous suspension), into the striatum over two needle penetrations. A total of 0.6 pl of PI was usually deposited ipsilaterally to the tectal DY injections. The tracers were slowly delivered through a l-p1 Hamilton microsyringe for 20 min to avoid unnecessary diffusion to neighboring regions (especially mutual tracer contamination from the bilateral SC injec-

tions). The iniection needle was kept in place for an additional 20 min to minimize the leakage-to overlying structures along the needle track. The animals were allowed to survive for two to four days, deeply reanesthetized with an overdose of sodium pentobarbital, and fixed by transcardial perfusion with 30Oml of 10% ice-cold formaline dissolved in 0.1 M phosphate buffer (pH 7.4). The brains were immediately removed. immersed in the same buffer containing 25% sucrose at 4 C, and then cut serially into coronal sections of 30-pm thickness on a cryostat. The sections through the midbrain (including the SC injection sites and substantia nigra) obtained from the

brains with the bilateral SC injections. were mounted onto clean slides and observed with a Leitz epifluorescence microscope to examine and quantify the distribution of SNr cells labeled with TB and/or DY. The nigral sections from the brains which received the unilateral striatal injections combined with the bilateral SC injections, were divided into two groups. The first group was mounted onto slides and

observed under the same microscope to examine and quantify the distribution of SNr cells labeled with TB, DY and/or PI. The second group was subsequently processed

for the indirect immunotluorescence technique of Coons.” Briefly, the sections were incubated in rabbit antisera against TH (Eugene) for 48 h at 4°C. followed bv, ourified eoat I

anti-rabbit igG conjugated to tluorescein isothiocyanate (FITC, Cappel, 1 : 50 dilution) for 1h at room temperature, and were then observed under three different filters: a UV filter providing excitation light of approximately 360 nm wavelength to view the blue-emitting TB (labeling only cytoplasm)-containing and yellow-emitting DY (labeling only nuclei)-containing cells, a green filter providing excitation light of approximately 550 nm wavelength to view the red-emitting PI-containing cells, and a blue filter providing excitation light of approximately 440 nm wavelength to view the green-emitting tyrosine hydroxylase (TH)-immunopositive cells. The alternate use of these three Biters on the same section greatly aided identification of single SNr ceiis quadruple-labeled with the four different markers. RESULTS

Bilateral

tectal injections

injections of TB and DY almost symmetrically involved the intermediate/deep layers of the middle third or caudal third of the SC on each side (Fig. 1). Indeed, the tracer deposits infringed dorsally on the major portion of the superficial layers of’ the nucleus and to some extent the overlying occipital cortex. In some cases, the tracers aiso slightly diffused ventrally into the adjacent dorsolateral central gray and mesencephalic reticular formation. Furthermore, the injections which encompassed the middle third or caudal third of the SC, sometimes spread rostrocaudally as far as the rostra1 third of the SC and the rostral-most part of the inferior colliculus, respectively. However, there was no discernible encroachment of either tracer on the opposite side of the SC across the midline. These two types of bilateral SC injections (middle third and caudal third) resulted in markedly different distribution patterns of SNr cells retrogradely labeled with TB and/or DY. Counts of labeled cells were carried out on the side ipsilateral to DY injections. Following bilateral injections over the middle third portion of the SC, a large number of retrogradely labeled neurons were observed in the SNr throughout Bilateral

rts rostra1 two-thirds (to three-uuarters) extent with ;r rustrocaudal gradient (Fig. IA). These labeled cells

were found predominantly ipsilateral and to a lesser degree (32.3% of total labeled and 37.8% of iphi.laterally labeled cells) contralateral to each tracer injection (Table 1). Thus, the SNr ipsilateral to the DY injection site contained a multitude of DYpositive and a smaller number of TB-positive cells. and vice versa. The neuronal labeling from the ipsilateral and contralateral SC occurred largely in the ventral half and far less frequently in the dorsal half ofthe SNr (Fig. IA). Moreover, along with nun~erous single-labeled cells, bilateral SC injections of this type produced a substantial number (17.9% of the total) of cells double-labeled with both TB and DY in the SNr (Fig. 2). The number of these double-labeled SNr cells also amounted to 55.4% of the oontralaterally labeled or 2 1.0% of the jpsilaterally labeled cell population (Table 1). As might be predicted from each distribution pattern of single-labeled ceils, the SNr cells containing both tracers were concentrated in the ventral half of the nucleus, and more significantly, they were most often seen in the ventrolateral portion at the level of the more rostra1 SNr (in the rostra1 third) (Fig. 1A). Additionally, retrogradety labeled neurons were also found in the substantia nigra pars lateralis (SNl) ipsilateral to each tracer injection. However, the SNl was almost devoid of cell labeling from the contralateral and therefore bilateral SC. More caudal injections (involving the caudal third} of TB and DY into the bilateral SC, on the other hand, showed a striking difference in the retrograde cell labeling pattern in the SNr. Although SNr cells were, in fact, single- and double-labeled with either or both of the tracers with a rostrocaudal gradient, each number of these three types of (ipsilaterally. contralaterally and bitaterally) labeled cells noticeably went down. In particular, the double-labeled cell population only made up 6.2% of total labeled. 30.6% of contralaterally labeled and 7.2% of ipsilaterally labeled cells (Table 1). Furthermore. the SNr cell labeling as a whole seemed to be restricted more ventrally with a slight rostrocaudal gradient. However, double-labeled cells were not localized specifically to the more lateral portion of the SNr, as compared to the case of the more rostra1 SC injections described above, but distributed scattered over the mediolateral plane (Fig. IB). Additionally. fewer neurons labeled only ipsilaterally with either of the tracers were detected in the SNl. Striatal

combined with bilateral tectal injections

In a second series of experiments, the possible existence of SNr cells simultaneously innervating both the ipsilateral stratum and bilateral SC. was examined on the basis of our recente and present data. Given a similar distribution pattern between nigrotectal cells of origin sending axon collaterals to the ipsilateral striatumhX and the contralateral

Multi-ColIateralization of the rat substantia nigra

313

Fig. 1. (Al, Bl) Line drawings of representative coronal sections through the sites of bilateral injections of DY (filled circles) and TB (open circles) into the SC at the middle (Al) and caudal (Bl) levels. (A2, B2) Projection drawings made from eight rostrocaudally (a-h) arranged coronal sections through the substantia nigra following the rostra1 (A2) and caudal (B2) SC injections. The drawings are of the rostra1 three-quarters of the substantia nigra ipsilateral to the DY injection site. DY-single-labeled SNr (ipsilaterally projecting nigrotectal) cells are represented with filled circles, TB-single-labeled SNr ~contralaterally projecting n&rote&al) cells with open circles, and double-ladle SNr (bilaterally projecting nigrotectal) cells with filled triangles. CP, cerebral peduncle; MTN, medial terminal nucleus of

the accessory optic tract; SNc, substantia nigra pars compacta; iii n, root fibers of the oculomotor nerve.

SC (especially in the case of the more rostra1 SC injections as documented above), unilateral striatal injections of PI were designed to be combined with bilateral injections of TB and DY into the middle third of the SC. Striatal PI was usually deposited on the side ipsilateral to DY injections. All cell counts were therefore performed on this side.

‘T’Band DY injections into the middle third of the bilateral SC and the resultant retrograde labeling pattern of SNr ceils were almost identical to those mentioned above (see Fig. 1A). The cell population containing both tracers appeared mostly in the rostral, ventrolateral portion of the SNr and again with similar frequency (16.1% of total, 54.6% of

314 Table I. Distribution Injection

TB (X)

DY (%)

14.4 14.0

67.7 79.8

Middle SC Caudal SC % % % %

Total Total TB + TB +

TB TB DY DY

of nuorescent-positive suh.\Lantia nigra pars reticulata cells following the bilateral tectal injections*

of of of of

total total total total

TB + DY (X) No. of cells (No. of sections)?

TB and DY DY TB DY

17.9 b.2 Middle SC 32.3 37.8 55.4 21.0

874(15) 448 (IO) Caudal SC 20.2 23.4 30.6 7.2

*TB and DY were injected into the bilateral SC through the middle third or caudal third extent. The SNr ipsilateral to DY injections was examined here. tEach number represents the mean of cells counted and sections examined (bracket) for three animals. contralateral and 18.6% of ipsilateral nigrotectal cells) (compare Table 2 with Table 1). PI injections

into the striatum involved the major portion of the nucleus with some diffusion to the overlying frontal cortex. Dense cell labeling with PI was evident in the substantia nigra pars compacta @NC) throughout its entire extent. These labeled SNc cells were found bilaterally with an ipsilateral predominance. A considerable number of PI-positive cells were also seen exclusively ipsilaterally in the SNr. These SNr cells labeled from the striatum were located primarily in its ventral portion (ventral third) and to a lesser degree in its more dorsal part. At the more rostra1 level (in the rostra1 third), the ventrally situated PI-labeled neurons were observed more laterally. As the SNr level went more caudally, they became more numerous and were aggregated more medially (see Fig. 1 in our recent papeF). Together with more massive single labeling, triple tracer injections of this type often yielded the appearance of SNr cells labeled with more than one tracer.

Thus, not only the above-documented double labeling with both TB and DY from the bilateral SC, but three other conceivable combinations of multiple labeling concurred as shown in Table 2. A sibilant number of SNr cells were double-labeled with both TB and DY from the bilateral SC (but not with PI from the striatum) (5.9% of the total), or with both PI and DY from the striatum and ipsilateral SC (but not with TB from the contralateral SC) (4.9% of the total). On the other hand, only occasional cells (0.4% of total) were encountered double-labeled with both PI and TB from the striatum and contralateral SC (but not with DY from the ipsilateral SC). Furthermore, 5.8% of total labeled SNr cells contained all three tracers. These triple-labeled cells also constituted 27.1% of the contr~a~ral, 9.2% of the ipsilateral and as many as 49.6% of the bilateral nigrotectal cell population. Interestingly, all these combinations of multiple labelings were again present primarily in the rostral, ventrolaterai SNr (Fig. 3). In addition to cells single-labeled ipsilateraliy with

Fig. 2. Photomicrograph of fluorescent-positive SNr cells following the bilateral SC injections of DY (ipsilateral) and TB (contralateral) through the middle third extent as depicted in Fig. l(A1). The rostral, ventrolateral SNr ipsilateral to DY injections. Arrowheads or filled arrows indicate cells single-labeled with DY and TB, respectively, while open arrows point to cells double-labeled with both tracers. CP, cerebral peduncle. Magnification: x 200.

315

Multi-collaterali~tion of the rat substantia nigra Table 2. Distribution of fluorescent-positive substantia nigra pars reticulata cells following the ipsilateral striatal combined with bilateral tectal injections* TB

DY

PI

% Total TB of total TB and DY % Total TB of total DY % Total TB + DY of total TB and DY % Total TB + DY of total TB % Total TB + DY of total DY % TB + DY + PI of total TB % TB+DY+PI of total DY % TB $ DY + PI of total TB+DY

TB+DY

TB+PI

DYtPI

TB+DY+PI

16.1 54.6 18.6 27.1 9.2 49.6

*TB and DY were deposited into the bitateral SC through the middle third extent, while PI was deposited into the striatum ipsilateral to the DY injection site. The SNr ipsilateral to DY (PI) injections was examined here. The mean of cells counted or sections examined for three animals was 764 and 10, respectively. one of the three tracers, neuronal cell bodies in the SNI were relatively frequently double-labeled with both PI and DY ipsilateral to each injection site. A number of PI-positive neurons were seen bilaterally in the ventral tegmental area (VTA) with an ipsilateral predominance. Tyrosine hydroxyiase immunohistochemistry combined with trip/e tracer injections

In a third series of experiments, the possible existence of dopaminergic SNr cells giving off axon collaterals to both the ipsilateral striatum and bilateral SC, was examined on the basis of our recent work6’ showing the dopaminergic nigrotectal projection. Following triple tracer injections into the bilateral SC (TB and DY) and unilateral (ipsilateral to DY) striatum (PI) as mentioned above, nigral sections were incubated in TH antisera. All cell counts were done again on the side ipsilateral to DY (PI) injections. After immunofluorescence procedure using FITC, cell bodies in the SNc and VTA were extensively double-labeled with both TH antisera and PI from the striatum. A considerable number of SNr cells also exhibited the characteristic green fluorescence induced by FITC. The site of these TH-immunopositive cells corresponded well with that of PI-positive cells in the SNr (see section on striatal combined with bilateral SC injections). Indeed, almost all the PI-labeled cells were double-labeled with TH antisera. In the rostral, ventrolateral SNr region, these double-labeled cells also often contained TB (from the contralateral SC) and/or DY (from the ipsilateral SC). Thus, at least 75% of cells containing PI and either or both of TB and DY were TH-immunoreactive. Interestingly, the vast majority (more than 85%) of SNr cells triple-labeled with all the three

tracers were imrnunostained (quadruple-labeled) with TH antisera as well (Fig. 3). However, SNr cells confining either or both of TB and DY only (but not PI) failed to display TH immunoreactivity. Additionally, in many instances neurons labeled with Pi only, or both PI and DY, were TH-immu~o~sitive in the SNl. DISCUSSlON

Technical considerations

Before opening the general discussion of the present data, there exists a methodologi~l matter to be precluded from emerging. To our knowledge, only two previous studies using the anterograde axonal transport of radioactive proteinsPO or wheat germ agglutinin conjugated horseradish peroxidase (WGAprojecting HRP),j” reported that contralaterally nigrotectal fibers first penetrate the ipsilateral SC and then cross the SC commissure to terminate in the contralateral counterpart. Supposing our tracer injected on one side of the SC can easily be taken up by fibers-of-passage heading towards the opposite side of the SC, then the resultant retrograde cell labeling in the SNr ipsilateral to the injection, comes to represent not only ispilateral but contralateral nigrotectal cells. Hence, it may be suggested that our double labeling experiments executed may yield an over-estimate of SNr cells giving rise to collateral projections to the bilateral SC, or may discredit even the existence of such collateralization. However, three pieces of evidence strongly argue against the matter. First of ail, it should be emphasized here that DY injected on the side identical to the SNr examined, is the very tracer that might be excluded from the possible uptake by fibers-of-passage. Our recent experiment has shown that DY infused into the MFB which

Fig. 3. Photomicrographs of TH-immunor~act~vc SNr cell< following PI injections into the ipsilatcral striatum combined with DY (ipsilateral) and TB (contralateral) injections into the bilateral SC through the middle third extent. The rostral. bentrolateral SNr ipsilateral to DY (PI) injections. (a) DY- and or TB-positive (unilaterally or bilaterally projecting nigrotectal) cells. (c) PI-positive (ipsilaterally projectmg nigrostriatal) cells in the same section as (a). (e) TH-immunoredctive (dopaminergic) cells in the same section as (a) and (c). 1. 2, 4. 5 and 7. cells quadruple-labeled with the four markers; 3, cell triple-labeled with TB, PI and TH antisera; 6. cell single-labeled with TB: 8, cell single-labeled with DY: 9 and IO. cells double-labeled with PI and TH antisera. CP. cerebral pedunclc A cluster of quadruple-labeled cells in (a). (c) and (e) are shown with higher magnification in (b), (d) and (f), respectively. The same blood ve~el is represented with V. Magnifications: (a, c. e) x200; (b. d, f) x 400.

nigrostriatal fibers travel through, fails to label any nigral cells (unpublished observation). Two supplementary findings in the present study (especially in the case of the bilateral middle third SC injections) further augment the fact. Firstly, on the side ipsilateral to DY injections, the ratio of double labeling to contralateral labeling of SNr cells is maintained (at

approximately 559’0) among experiments. Secondly, the double-labeled cell population occurs in a fairly restricted region of the nucleus (the rostral, ventrolateral SNr) in contrast to the diffuse distribution or cells labeled only contralaterally. Thus, these facts taken together make our double labeling results tenable in an> respect. Conversely. it appears

Multi-collateralization of the rat substantia nigra

317

imperative from the above-mentioned viewpoint to constitutes almost half of bilaterally projecting re_examine many previous papers5*‘,8,26.30,‘3.3**~.3*,58 n~~ote~tal ~41s discussed above. These SNr-derived concerning the dist~bution of cells of origin of the nigrostriatal fibers have recently been reported to nigrotectal projection with retrograde axonal tracing terminate preferentially in the lateral patchy comtechniques. partments of the striatum.2q The overall results from our triple tracer labeling experiments indicate that a Bilateral nigrotectal projections substantial number of cells in the specific (rostral, Our present double labeling study demonstrates ventrolateral) SNr region innervate simultaneously that the rostra1 (middle third) versus caudal (caudal more than one structure of the striatum and bilateral third) SC injections produce a great diversity in SC. In contrast, it might be of some importance to note the paucity of the branching pattern to both the the dist~bution pattern of double-ladle SNr cells, resulting in a topography that the majority of striatum and contralateral SC only (but not to the bilaterally projecting nigrotectal cells arise from the ipsilateral SC). In view of the fact that no available rostra], ventrolateral SNr and terminate in the SC at evidence has so far supported the existence of either the more rostra1 levels. These bifurcating cells are collateral SNr projections to both the striatum and equivalent to more than one-sixth of the total, half of thalamus,8.55~6aor crossed SNr projections to the the contralateral and one-fifth of the ipsilateral nigrostriatum,67 the possibility that the specifically coltectal cell population. It should be added here that lateralized cells under study issue additional branches crossed nigrotectal fiber te~ination (especially in the to the thalamus and/or ~ontralateral striatum, would rat30) is confined to the lateral portion of the deep not be respected. layers of the middle third SC. It has previously been suggested that when a projection to a region is Dopaminergic traits of multi-collateralized nigral cells bilateral, individual cells may send axon collaterals Furthermore, our TH immunofluorescence histoto both sides of the brain, as shown by the existence chemistry combined with the triple tracer labeling of bilateral VTA projections to the locus coeruleus show that the majority (more than 75%) of SNr cells (15% of the tota1).‘j3 However, most (if not a1163,74) with collaterals to both the striatum (ipsilaterally) bilateral projections of dien~phali~/mesencephalic and SC (unilaterally or bilaterally), express do(the enzyme structures situated in close proximity to, or even on, pamine. Since dopamine-~-hydroxylase the midline, including the midline thalamic nuclei,65 required to convert dopamine to noradrenaline) is mammillary body,74 SNC/VTA,~,~‘@ and dorsal not detected in any cell bodies in the ventral mesencells in this brain raphe,47,73are strictly unilateral (either ipsilateral or cephalon,64 TH-immunoreactive area can be considered to be dopaminergic. Above contralateral but not bilateral) at the single neuron all, the dopamine-containing population of cells level, regardless of the laterality of their cell bodies. Therefore, it seems extraordinary that individual SNr giving rise to multi-collateral projections to both the cells remote from the midline are highly collateralized striatum and bilateral SC, remarkably exceeds 85%. to the bilateral SC. Of interest is to ask about the As discussed in our recent paper>’ it is unequivocal developmental mechanism(s) that induce the specific that dopamine in the SC subserves an independent bilateral organization of nigrotectal projection cells. transmission role in the nigrotectal system as well as An abundance of bilateral innervation may allow for a precursor role in the noradrenergic pathway derived an information processing capacity which would be from the locus coeruleus.46@ A recent pharmacoimpossible in the case of overwhelming unilateral logical work’* has shown that noradrenaline release innervation. Another major target site of the SNr is in the SC is modulated specifically by dopamine the VM.6.‘2,‘6.26,30,32,34,57 Given that this nigrothalamic D,-receptor-selective drugs, thus indicative of the pathway is bilateral with an ipsilateral predomipresence of sparse25 but by no means functionally nance 7b~12~‘6~22.30.34 and that both the nigrotectal and trivial amounts of dopamine receptors in the SC. It nigrothalamic projections, albeit partly, take their should also be emphasized here that even in the origin from single SNr neurons,3-5,8.22,55it is also rostral, ventrolateral SNr neurons projecting to the intriguing to investigate the relationships between SC (unilaterally or bilaterally) never displayed these two bilateral projections at the individual cell dopaminergic traits, if they lacked axon collaterals level. innervating the striatum. This finding suggests that dopamine expression in SNr cells may closely be Bilateral nigrotectaI cell branching to the striatum associated with the outgrowth of their axons towards Our recent studies6x,69have elucidated the fact that the striatum or the accomplishment of their consingle dopaminergic neurons in the rostral, ventronection with the striatum. However, the causal lateral SNr send axon collaterals ipsilaterally to both relation between these two events remains to be the striatum and SC. The present data confirm and established definitively. It is generally accepted that extend these reports with the demonstration that as the nigrotectal pathway exerts GABAergic inhibitory many as more than half of such bifurcating SNr cells transmission.‘4~24~75Accordingly, the existence of also issue third collateral branches to the contradopaminergic nigrotectal projection cells recently6’ lateral SC. This multi-collateralized cell population and presently identified, favors the possible co-

localization of dopamine and GABA in these specific SNr cells. However, two previous immunohistochemical studiesM.s4have reported to the contrary; the co-existence of dopamine and GABA does not occur in cells in the rat substantia nigra. It should be considered in this respect that the authors might have missed the specific (rostral, ventrolateral) SNr region under study. Meticulous re-examination of the nigrai localization of dopamine and GABA would be warranted.

a.c,d:DA+GABA? b: GABA

/r .-.---STRIATUM

j

only

rvl

Functional implications

s

-

As described in the introduction, the nigrotectal pathway has been suggested to play a crucial role in the onset of saccadic responses by cells in the deep layers of the SC.‘“~3’~59~62~so~8’ in fact, Hikosaka and Wurtz have clearly shown in the monkey that a certain population of SNr ceils exert a tonic, GABAergic inhibition on saccade-related cells in the SC, and that these saccadic eye movements are severely disrupted by muscimol infused into the SNr as well as into the SC.35-37In many clinical cases, abnormalities in saccadic eye movements are manifested by parkinsonian patients. 10,18,19~41~50~60~61~70,77 This oculomotor disorder has been proposed to result from imbalances of GABAergic transmission to the SC caused by serial damage to putative indirect the nigro-striato-nigro-tectal and/or pathways, visual cortico/frontal eye field-striato-nigro-tectal links.9~27~28~45~4*~49.59.77 However, our SNr dopamine cell population simultaneously innervating both the striatum (ipsilaterally) and SC (unilaterally or bilaterally), might constitute an alternative and presumably more prominent neuronal basis for such a clinical phenomenon. Indeed, we have supported the notion of its vulnerability in Parkinsonism, in view of the fact that MPTP treatment of the MFB induces dopaminergic nigrotectal cell degeneration.69 Moreover, our specifically collateralized dopamine cells may also have an implication in the elicitation of motor and behavioral syndromes such as compulsive turning. Considerable e~dence23,39.~~.43.56 has previously documented that the nigrotectal pathway is involved in the execution of dopamine-mediated turning behaviour. For example, 6-hydroxydopamine-induced nigrostriatal lesions markedly attenuate the effect of intra-SNr muscimol subject to contraversive turning,“’ thus indicative of there being dopaminergic SNr cells (with axon collaterals to both the striatum and SC) responsible for this event. Conceivably, the manifestation of turning behavior may be cooperated with eye movements in higher mammals (especially in primates37). Given that rodents’ eyes have no clear fovea” and consequently do not require precise eye movements,37 turning

~

SC

Fig. 4. Schematic diagram showing four different patterns of collateral projections from the rostral, ventrolateral (rvl) SNr to the ipsilateral striatum and bilateral SC. Cell (a) is far less frequently observed than cells (b), (c) and (d). Cells (a), (c) and (d) possibly contain both dopamine and GARA, while cell (b) contains GABA only. See the text and Table 2 for details.

behavior in rodents might be initiated not by eye movements, but by head/neck movements via the nigro-tecto-spinal link. 1,15,51,72,76,79 1n any case, phar_ macological manipulation of this multi-collateralized dopaminergic nigrotectal system would be expedient to evaluate the viability of our hypotheses. CONCLUSION

The present study provides evidence that individual dopamine (possibly GABA as well) cells in the rostral, ventrolateral SNr give off various patterns of divergent axon collaterals to the ipsilateral striatum and bilateral SC in the rat (Fig. 4). This specific cell population seems to be in a strategic position to exercise a pronounced dopaminergic control over the SC-related somatic motor functions and hence express motor impairments (i.e. abnormal saccadic eye movements in Parkinsonism) and/or behavioral syndromes (i.e. compulsive turning behavior) affected by dopamine. It is extremely attractive to pursue the development of the SNr cell population studied in terms of its novel multi-collateralization and transmitter phenotype. ~ek~ow~edg~~rs-Th;s work was supported by grants from the Medical Research Council of Canada to Prof. T. Hattori. We wish to thank him for giving us invahrable advice and encouragement. M. Takada and K. J. Campbell are recipients of an MRC Fellowship and Studentship+ respectively.

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