Research report Anterograde tracing of retinal … tracing of retinal afferents to the tree shrew...

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Research report

Anterograde tracing of retinal afferents to the tree shrew hypothalamusand raphe

a,b , a,b*Stefan Reuss , Eberhard FuchsaDepartment of Anatomy, School of Medicine, Johannes Gutenberg-University, Saarstr. 19-21, D-55099 Mainz, Germany

b ¨Department of Neurobiology, German Primate Center, D-37077 Gottingen, Germany

Accepted 13 June 2000

Abstract

The anterograde neuronal transport of Cholera toxin B subunit (CTB) was used in this study to label the termination of retinal afferentsin the hypothalamus of the tree shrew Tupaia belangeri. Upon pressure-injection of the substance into the vitrous body of one eye, amajor projection of the retinohypothalamic tract (RHT) was found to the hypothalamic suprachiasmatic nuclei (SCN). Although theinnervation pattern was bilateral, the ipsilateral SCN received a somewhat stronger projection. Labeling was also found in the supraopticnucleus and its perinuclear zone, respectively, mainly ipsilaterally as well as in the bilateral para- and periventricular hypothalamicregions without lateral predominance. In the raphe region, scattered fibers and terminals were seen in the dorsal and median raphe nuclei.CTB-immunoreactive structures were observed neither in the locus ceruleus nor in vagal nuclei. Our results, partly in contradiction toearlier studies using different tracing techniques in another tree shrew species (Tupaia glis), reveal that hypothalamic nuclei, in particularthe SCN, are contacted by retino-afferent fibers which are thought to mediate the effects of light to the endogenous ‘clock’ and to parts ofthe neuroendocrine system. 2000 Elsevier Science B.V. All rights reserved.

Keywords: Anterograde neuronal tracing; Cholera toxin B subunit; Hypothalamus; Paraventricular nucleus; Retina; Suprachiasmatic nucleus; Supraopticnucleus, Locus ceruleus, Tupaia belangeri, Vagal nuclei

1. Introduction neuroanatomical investigations of the circadian system.Many studies, however, have been conducted in rats and in

The hypothalamic suprachiasmatic nucleus (SCN) is hamsters. To extend our respective knowledge of non-regarded as the major pacemaker for circadian rhythms of rodent mammals, the present study was designed tomammalian body functions [27,38]. The nucleus itself is investigate the retinal projection pattern to the hypo-influenced by internal and external factors. The most thalamus in the tree shrew (Tupaia belangeri). Phylo-important of the latter is light which is transmitted from genetically, tree shrews are considered as intermediatesthe retina to the hypothalamus via retino-afferent fibers, between insectivores and primates and serve as a suitablethe so-called retinohypothalamic tract (RHT). The RHT animal model for the study of bio-behavioral consequencesconveys photoperiodic information to the SCN (the endog- of psychosocial stress (cf. [14]).enous ‘clock’ in mammals) and thus mediates the entrain- Although the visual system of tree shrews is relativelyment of circadian and seasonal rhythms (cf. [21,33] for well-investigated [3], the existence and termination of thereview). RHT in Tupaia is a matter of controversy since some

The RHT, first observed in the last century (cf. [33]), authors did not find evidence for such a projection inwas characterized in 1972 by autoradiographic techniques Tupaia glis [1,18,43], while others identified degenerated[15,28] and since then was investigated in a variety of fibers of passage in the contralateral ventral hypothalamusmammalian species. The topography of its termination upon unilateral eye enucleation [2]. A predominantlypattern in the hypothalamus is a major research item of contralateral projection pattern was also found in a horse-

radish peroxidase (HRP) tracing study [30]. However,autoradiography studies revealed the bilateral innervation*Corresponding author. Tel.: 149-6131-392-3207; fax: 149-6131-392-of the SCN [4,5,26].3719.

E-mail address: [email protected] (S. Reuss). We therefore decided to study the projection pattern of

0006-8993/00/$ – see front matter 2000 Elsevier Science B.V. All rights reserved.PI I : S0006-8993( 00 )02578-6

S. Reuss, E. Fuchs / Brain Research 874 (2000) 66 –74 67

retinal afferents in Tupaia belangeri by using Cholera biotinylated mouse anti-goat IgG (1:100 in PBS; Dianova,toxin B subunit (CTB) as an anterograde neuronal tracer. Hamburg, Germany) for 90 min at RT, rinsed three times,CTB was shown in previous studies to exhibit the best and incubated in streptavidin (SA) coupled to Cy3 (1:100results in RHT tracing compared to Fast blue, Phaseolus in PBS; Amersham, Braunschweig, Germany). Sectionsvulgaris-Leucoagglutinin, biocytin or Fluoro-Gold [34,35]. were mounted onto gelatin-coated slides, briefly dehy-We concentrated on the analysis of the hypothalamus but drated, coverslipped with Merckoglas (Merck, Darmstadt,also studied sections of further brain regions known to Germany) and analyzed using a Leitz Orthoplan micro-receive retinal afferents or to be involved in mediation of scope with a Ploemopak epifluorescence unit through filterstress responses, i.e. the raphe nuclei, locus ceruleus and set N2. The retinae were excised, sectioned in the sagittalthe vagal nuclei [9,10]. plane and treated in the same way as the brain sections.

In control experiments in which CTB was applied to theextrabulbar space, we observed retrograde neuronal tracing

2. Materials and methods of the nucleus of the optic tract but no retino-afferentfibers. Specificity studies were carried out by omitting

2.1. Animals and treatment primary or secondary antisera. The specificity of the CTBantiserum was demonstrated by the absence of staining in

The experiments were performed on adult tree shrews animals that did not receive injections.(Tupaia belangeri) of either sex with body weights of After taking microphotographs of selected regions, the400–500 g. The animals stem from the breeding colony of coverslips were removed and the sections stained with

¨the German Primate Center, Gottingen, Germany, and Cresyl violet.were held under long-day conditions (light:dark 16:8 h)with food and water ad libitum (for details see Ref. [13]).All animal experimentation was conducted in accordance 3. Resultswith the European Communities Council Directive of Nov.24, 1986 (86/EEC) and was approved by the Government In frontal brain sections containing the hypothalamus,of Lower Saxony, Germany. retino-afferent fibers and terminals were seen upon antero-

Animals were anesthetized with tribromoethanol (0.3 grade neuronal transport of CTB injected into one eye. Theg/kg b.wt., i.p.) and received two pressure-injection of 0.5 major hypothalamic projection pattern was found to theml each of the anterograde tracer substance Cholera toxin bilateral SCN (Fig. 1). The comparison of both sidesB subunit (CTB) into one eye. The tracer was applied to showed that the nucleus ipsilateral to the injection sitethe vitrous body at the lateral and dorsal pole of the eye by receives a somewhat stronger innervation. In the anteriorusing a glass micropipette connected to a Hamilton parts of the SCN (Fig. 1A,B), retinal innervation wassyringe. CTB (List Biological, Campbell, CA, USA) was concentrated in ventral aspects of the nuclei. In sections ofdiluted in phosphate-buffered physiological saline (PBS) to the medial SCN (Fig. 1C,D), labeling was distributed overa final concentration of 0.5%. the dorso-ventral extension of both nuclei but was concen-

After 1 week, the animals received an overdose of the trated in the ventral two-thirds. In higher magnificationanesthetic i.p. and were perfused transcardially with 250 (Fig. 1E,F), it is seen that labeled terminals are concen-ml of phosphate-buffered 0.9% saline (PBS), to which trated over, or surrounding, SCN perikarya. The innerva-15,000 IU heparin / l were added, at room temperature (RT) tion pattern of the SCN was found to be dense in particularfollowed by 500 ml of ice-cold fixative (4% paraformal- in its central portion (Fig. 1D). The fibers, however, weredehyde, 1.37% L-lysine, 0.21% sodium-periodate in phos- of fine caliber and exhibited small varicosities (Fig. 1F).phate buffer; according to [22] with a constant rate of 20 Immunoreactive structures were further observed in theml /min. The right atrium was opened to enable venous region of the supraoptic nuclei (SON), paraventricularoutflow. All animals were killed at the middle of the light nuclei (PVN), and periventricular nuclei (PeVN). In theperiod. The eyes and the brain were removed, postfixed for SON (Fig. 2), fibers and terminals were seen mainly in2 h, and stored at 48C in phosphate-buffered 30% sucrose. sections of the ipsilateral anterior (Fig. 2B) and middleThe brains were marked on one side and cut at 40 mm part of the nucleus (Fig. 2E), dorsally and laterally to thethickness on a freezing microtome in the frontal plane. optic chiasm. Although some immunoreactive structures

were observed within the borders of the SON, they were2.2. Section treatment predominantly found in the perinuclear region. Fibers in

the peri-SON were of fine- to medium-sized caliber andFor CTB immunohistochemistry, sections were incu- the varicosities mainly were larger compared to those of

bated free-floating for 24 h at RT in goat CTB-antiserum the SCN. In the higher magnifications from both regions,(List Biological), diluted 1:1000 in PBS to which 1% immunoreactive fibers and terminals were seen at sitesnormal swine serum and 0.3% Triton-X 100 were added. where, in the Nissl-stain, neuronal cell bodies were ob-After three rinses in PBS, the sections were incubated in served (Fig. 2C,D and F,G).

68 S. Reuss, E. Fuchs / Brain Research 874 (2000) 66 –74

Fig. 1. Frontal sections of the tree shrew hypothalamus. The left panels depict the Cresyl violet-stain and the right panels show CTB-immunoreactivityupon injection of the tracer into one eye and anterograde neuronal transport. Each pair of panels was taken from the same section. The right sides of thesections are ipsilateral to the injection site. The termination of the retinohypothalamic tract in the anterior SCN of a female tree shrew is demonstrated in(A,B). A section through the mid-SCN at the level where the major projection pattern was found is shown in (C,D). A region from the left ventral SCN isshown in higher magnification in (E,F). Dense accumulations of labeled fibers are seen in close vicinity of small SCN neurons. The asterisks mark thesame region in both panels. Abbreviations: oc5optic chiasm, V5third ventricle. Magnification bars5100 mm (A–D); 20 mm (E,F).


Fig. 2. Frontal section of the tree shrew hypothalamus showing the region of the ipsilateral supraoptic nucleus (SON) in the Cresyl violet-stain (A,C,G)and CTB-immunoreactivity in the same sections (B,D,E,F) upon injection of the tracer into the ipsilateral eye. A relatively dense retinal innervation of theSON and its perinuclear regions is seen at the level of the anterior SON (A,B). The higher magnifications (C,D) demonstrate that CTB-immunoreactivefibers and terminals are seen in close vicinity to neuronal perikarya. In the peri-SON (E), many fibers are seen. The comparison of CTB-immunolabelingand Cresyl violet-stain in higher magnification (F,G) reveals that fibers are present at neuronal cell bodies. Abbreviations: l5lateral, m5medial, ot5optictract. Bars550 mm (A,B,E); 20 mm (C,D,F,G).


In the PVN, we observed immunoreactive fibers mainly Our study, using the reliable anterograde neuronalin its medial subdivision, predominantly in the dorsal parts tracing properties of CTB following injection of the(Fig. 3A–C). The paraventricular innervation was found to substance into one eye, now confirms the presence ofbe bilateral with no apparent dominance. Fiber caliber and retino-afferent projections to hypothalamic nuclei in treevaricosity size appeared larger than those seen in the shrews and provides a detailed description of the dis-peri-SON. In the bilateral periventricular nuclei (PeVN), tribution of RHT termination of the tree shrew Tupaiascattered fibers and terminals were seen in the ventral (Fig. belangeri. The SCN receives the strongest innervation.3D–F) and dorsal (Fig. 3G–I) aspects. In PVN, PeVN and Although the projection pattern is bilateral, it shows a lightperi-SON, the amount of fibers and terminals traced was ipsilateral predominance which was previously describedrelatively large. to be the case in primates including man [7,12,39], in

In sections of the raphe nuclei (Fig. 4A), we observed contrast to the greater contralateral projection as found infew fibers and scattered terminals in the dorsal raphe many rodents [29,42]. It is generally agreed that the RHTnuclei (Fig. 4C), in its ventral part (between the bilateral is the morphological substrate for the regulatory effect ofmedial longitudinal fasciculus; Fig. 4B) and in the median light on the SCN and thus on the circadian timing systemraphe nucleus (Fig. 4 D). (cf. Refs. [29,33]). The widespread distribution of labeled

Immunoreactive fibers and terminals were also seen, fibers and terminals reveals that several neuronal subpopu-predominantly contralateral, in the lateral geniculate nu- lations of the Tupaia SCN (not yet identified) are con-cleus (LGN), mainly in its dorsal subdivision but also in tacted by retinal afferents. The fibers and varicosities in thethe ventral part, and in the intergeniculate leaflet. Scattered SCN were of relatively fine diameter compared to thosefibers were also observed in the anterior central gray, observed in the PVN (see below). Similar results weremainly dorsally to the aquaeduct without preference to previously described in a detailed report on target-specificeither side, and in the accessory optic nuclei. In the differences of hamster retinal projections [20].superior colliculus, intensive labelling was observed pre- Of further interest is the relatively dense innervation ofdominantly contralateral to the injection site, in particular neuroendocrine nuclei, in particular of the supraopticin its laminated (input) structure (data not shown). nucleus and the peri-SON region. This projection may be

In the locus ceruleus and in vagal nuclei (dorsal nuclei involved in the photic effects on mammalian physiologyof vagus nerve, nuclei of solitary tract and nucleus that are not mediated by the circadian timing system.ambiguus), no CTB-immunoreactivity was observed. Degenerating fibers in the contralateral SON region were

The examination of retinal sections showed that gang- found previously upon unilateral eye enucleation in twolion cells exhibited CTB-immunoreactivity. Signs of ne- studies [2,18] but not by other groups investigating treecrosis were not seen (data not shown). shrews. Our data reveal that the SON and its perinuclear

region receives a relatively dense bilateral innervationfrom the RHT; however, the projection to the ipsilateral

4. Discussion SON area was seen to be far greater than that to thecontralateral nucleus. Retinal projections to the ventrome-

The visual system of tree shrews is well-investigated dial SON are present in humans [7], and studies in rats[3], however, comparatively little knowledge is available have previously shown that a retina-supraoptic projectionsas yet that deals with the RHT of these small diurnal exists [19,24]. Electrical recordings from SON neuronsmammals which are considered as intermediates between while stimulating the optic nerve also revealed that a directinsectivores and primates. Early reports contradict one monosynaptic pathway between the RHT and the SON oranother with regard to the existence of the tree shrew RHT. its perinuclear zone, respectively, exists [6]. It is thusLaemle [18] and Abplanalp [1] did not comment upon a conceivable that the retinal input to the SON area isretinal projection to the Tupaia glis hypothalamus, while responsible for daily rhythms of oxytocin and vasopressinTigges [43] mentioned that no evidence was found for a release as found in rats [31,44].retinohypothalamic projection in the same species upon A retino-afferent innervation of the bilateral para- andunilateral eye enucleation and application of the Nauta- periventricular nuclei was observed in the present study. InGygax technique to identify degenerated fibers. In contrast, the dorsal aspects of the PVN, in particular, many labeledenucleation was described to result in degenerating fibers fibers were seen. Scattered fibers and terminals were alsoappearing as fibers of passage in the contralateral ventral present in the ventral PVN and in the PeVN at the lateralTupaia glis hypothalamus [2]. An HRP tracing study in the borders of the third ventricle. The presence of a bilateralsame species pointed to a predominantly contralateral PVN innervation was previously observed in dogs andprojection to the SCN [30]. In addition, retinal projections rabbits [17], hamsters [45] and humans [40]. Since there istraced with radioactive amino acids and/or fucose and experimental evidence that the PVN is a functional com-visualized by autoradiography revealed that labeled fibers ponent of the retina-pineal gland pathway [32,36,37], itenter both the ipsi- and contralateral SCN in Tupaia glis seems possible that photic information is transmitted to the[4,5,26]. pineal also bypassing the SCN and the sympathetic system.


Fig. 3. Frontal section of the tree shrew hypothalamus showing the contralateral paraventricular and periventricular regions in the Cresyl violet-stain(A,C,F,I) and CTB-immunoreactivity in the same sections (B,D,E,G,H) upon injection of the tracer into one eye. Fibers are seen in higher magnification in(B), while (C) shows the Cresyl violet-stain of the same region. The ventral periventricular region (vPeVN) is shown in (D), in higher magnification in (E)and the same region in the Cresyl violet-stain (F). Panels (G–I) depict scattered CTB-immunoreactivity in the dorsal periventricular area (dPeVN), in (H)in a higher magnification from (G), and in (I) as the Cresyl violet-stain of the region shown in (H). Abbreviations: l5lateral, m5medial. Bars5300 mm(A); 50 mm (D,G); 20 mm (B,C,E,F,H,I).


Fig. 4. Frontal sections of the tree shrew raphe regions. Panel (A) depicts the Cresyl violet-stain. The boxed regions are shown in higher magnification in(B–D). The right sides of the sections are ipsilateral to the injection site. (B) demonstrates CTB-immunoreactive structures in the ventral part, (C) in thedorsal part of the dorsal raphe nuclei (DR). (C) was taken from the median raphe nuclei. Abbreviation: Aq5aquaeduct. Bars5300 mm (A); 20 mm (B–D,same magnification).

We also observed CTB labeling of retinal afferents in dorsal and ventral parts of the DRN, and in the medianthe LGN, IGL, SC and in some other retinorecipient raphe nuclei. These data are in accordance with previousnuclei, in accord to previous observations on the tree shrew and recent reports of direct retina-raphe projections in cat,visual pathways (cf. [3]). It should be noted that re- rat and gerbil [8,11,16,41]. It is conceivable that thistrogradely stained perikarya were observed neither in the connection provides an indirect visual pathway to thehypothalamus nor in other brain regions known to receive circadian pacemaker since the dorsal and median rapheretinal afferents. Retinopetal projections in mammals are nuclei innervate the SCN [16,25] and both are capable ofstill a matter of controversy (cf. [29] for review). We have, modulating the phase of the circadian rhythm [23].so far, only occasionally seen retrogradely labeled neuro- In conclusion, the present study using anterogradenal perikarya in the basal hypothalamus, but not within the tracing of retinal afferents by CTB reveals that theborders of the SCN, upon Fluorogold injection into the eye retinohypothalamic tract in Tupaia belangeri provides aof the dwarf hamster [34]. strong innervation of the bilateral SCN, and weaker

The present study has also demonstrated a retinal projections to the area of the ipsilateral SON, the bilateralprojection to the dorsal raphe nuclei (DRN). Fibers and PVN and PeVN regions, and the raphe nuclei. Theterminals containing CTB were detected in the anterior discrepancies found between the present and previous


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