Demonstration of a direct projection from the retina to the hypothalamic supraoptic nucleus of the hamster

Neurosctence Letter,s, 139 (1992) 149 152 ,c I992 Elsevier Scmnufic Publishers Ireland Ltd. All rights reserved l)3(14-3940/92,,'S 05 00

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Demonstration of a direct projection from the retina to the hypothalamic supraoptic nucleus of the hamster Jens D. M i k k e l s e n ~' a n d J a c q u e s Servi6re b "LltlxtltUle o/:iledlcal ,4,l~ltom v, Departmenl B. Ulll verstl.V o/('op*,'tlhag~elt. Copcn/la~(c~t ( Dcmmlrk ) am/ bLaboralorw de Ph v vudo:,,w Sen ~orwlh'. In ~tma ,%~ttlonal de la Recherche 4gronomtque. Jot(l" Ol Josas : Frailty')

IRecewed 8 Nove,nber 1991, Revised xer,,lon recel,,ed 14 February. 1992, Accepted 17 February' 1992) Key n'otd~

Retina. Cholera toxin subumt B, Visual. Supraoptac nucleus. Retmohypothalamlc pathway', Hypothalamus. Hamster

Hamsters were inJected m the left eye vdth unconjugated cholera toxm ~ubumt B ICHB) and the tracer was locahzed using mlmunohlstochemlstry A large n u m b e r of immunoreacm.e fibers was found m the suprachlasmatm nuclm and the contralateral lateral hypothalamm area Labeled libers coursed around the supraoptm nucleus and nerve terminals accumulated m a zone dorsally' and laterally' to the nucleus Smgle fibers from th~s plexus penetrated mto the supraoptm nucleus, where few fibers arborized into dehcate ~mmunoreacme profile~ possessmg vancosltmS Labeled fibers were Mentlfied only m the dorsal and lateral parts of the nucleus, and mostly at the caudal level of the optic chmsm These resulls show a direct retinal mnervat~on of the magnocellular neurons of the supraoptlc nucleus, and indicate a d~rect phoI]c mlluence on lhe h)pothalamo-neurohypoph,,',ml system.

The mammalian accessory visual pathways include projections to the hypothalamus: the retinohypothalamic tract. Thib pathway' has been convincingly demonstrated using autoradiographic tracing methods [1, 4, 7], and its existence has been confirmed subsequently, using other anterograde tracing techniques in all mammals studied to date [5, 6, 10, 17]. Most importantly, the retinohypothalamic tract densely innervates the suprachiasmatic nuclei and this projection mediates the photic entrainment of the circadian oscillator (see rel~. 8 and 12). In addition, hypothalamic structures outside the suprachiasmatic nuclei, such as the lateral and anterior hypothalamic areas among others, are innervated from the retina [6, 7, 10]. It is unknown whether these neural pathways are involved in regulation of alternative circadian oscillators or play other roles related to visual perception. in contrast to many other mammalian species, the golden hamster is a long-day seasonal breeder and this behavior is photo-dependent [14, 15]. The retinohypothalamic projections manifest a degree of variability' among mammalian species [16], which may explain why various mammals have a different dependence on the external (;)~re,v~omh'm'c J D. M~kkelsen, Inslltute of Medical Anatomy, Department B, The P a n u m Institute, Blegdamsxel 3, DK-2200 Copenhagen N, Denmark. Fax" (45) 35369612

light dark cycle (see refs. 8 and 14). This resulted in speculation that the hamster possesses a more developed retinohypothalamic projection than other rodents [5]. Tracing with various probes has improved during recent years, and now the most sensitive tracers with regard to uptake and filling of labeled neurons appear in many' cases to be various lectins or bacterial toxins (see refs. 2, 9 and 13). By using immunohistochemical identification of cholera toxin subunit B (CHB), we have analyzed the localization of retinal axons outside the suprachiasmatic nuclei in the golden hamster. Eight golden hamsters (Mesocricetus a u r a t u s ) were anesthetized with 30 mg/kg pentobarbital (Sanofi, France) i.p., and 5/21 0.4% CHB (Sigma, St. Louis) dissolved in 50 inM phosphate-buffered saline (PBS, pH 7.4) was pressure-injected into the vitreous of the left eye within a period of 2 rain. After a survival t\~r 24 h. the animals were fixed by' perfusion with 400 ml 4% parat\)rmaldehyde dissolved in 0.1 M phosphate buffer (pH 7.4) for 15 inin. Brain bections were washed for 3 x 10 rain in PBS and incubated in goat anti-CHB ( L i s t Biological, California) diluted 1:8,000 in PBS containing 0.3% Triton X-100 and 1% BSA l\~r 24 h at 4°C. The sections were then processed for immunohistochemistry using the avidin biotin peroxidase complex method as described previously [9].

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A large n u m b e r o f C H B - i m m u n o r e a c t i v e (CHB-Ir) fibers were located in the optic nerve and were followed t h r o u g h the optic chiasm to the optic tract. A dense ac-

cumulation o f nerve terminals was seen in the primary targets o f retinal efferents such as the lateral geniculate bodies and the suprachiasmatic nuclei IFig. 1). Single

151 Figs. 1 6 A very dense accumulation of CHB-Ir fibers is present in the contralaleral SCN Note that numerous fibers are present be.~ond the boundaries of the SCN, and penetrate mto the adjacent anterior, and perlventrlcular hypothalamlc areas (F~g. I ) Along the dorsal surlltce of the optm

chlasm a number of labeled nerve fibers and terminals are accumulated (alrows) (Fig. 2. I High power photomicrograph demonstrates smooth fibers m the lateral part of the optic tract some of which arborize m an angle of 90 ~' into the h,~pothalamus (arrows) I Fig 3) In the lateral hypothalamlc area (LHA) adjacent to the optic chlasm and the supraoptic nucleus (SON), an accumulation of labeled fibers is observed The arrows sho~ two groups of fibers penetrating the SON fronl the dorsal and lateral sides, rcspectivel.\ (Fig 4}. High magnification of positix c ner,~e terminals (arrov, s) In the SON (Fig 51 Note that single fibers arborlze within the SON (arrow ~ (Fig 6) Bar 2(}0# m (Figs. 1.2 and 4~. [O(I # m (F lg. 3), 25 u m I Flgs. 5 and 6)

fibers in the optic chiasm arborized and sent distinct branches into the ventral part of the suprachiasmatic nuclei and the lateral hypothalamic areas (Figs. 1 and 21. In the optic chiasm and tract distinct axons branched in at1 angle of 90 ° into the lateral hypothalamic area (Figs. 3 and 4) and coursed in a dorsal and lateral direction. At the level of the caudal part of the optic chiasm, these fibers accumulated in a perinuclear zone surrounding the supraoptic nucleus (SON), and from this bundle single fibers passed into the SON (Fig. 4). In addition, fibers from the perinuclear zone could be followed dorsally in the lateral hypothalamic area to the perifornical region, and laterally to the extreme medial part of the amygdaloid complex and piriform cortical region. Of the fibers penetrating into the SON proper, most entered into the dorsal pole, but some also entered into its lateral subpart (Fig. 4). The positive fibers were distinct and resembled either delicate axons with boutonsen-passage or fibers possessing bulbous nerve endings (Figs. 5 and 6) Some fibers arbortzed within the nucleus into fine nerve terminals endowed with round immunoreactive endings (Figs. 5 and 6). Most of the CHB-ir fibers were identified in the contralateral SON. No CH Bir fibers were lkmnd in the part of the SON located rostrally to the susprachlasmatic nuclei, nor in the part caudal to the optic chiasm. This study rex.eals the presence of a direct retinal innervation of contralateral SON in the golden hamster. In agreement with earher observations in all mammalian species examined to date, bilateral projections to the lateral geniculate nuclei, the suprachiasmatic nuclei and the lateral hypothalamic areas were identified. It has been suggested that the retinofugal projections are different a m o n g species [5], and with regard to the retino- supraoptic projection this has been emphasized, because this particular prolection has been demonstrated in some studies [6, 17] but not in others [1, 4, 5, 7, 10]. The identification of the retino-supraoptic pathway is likely to bc explained by the high sensitlvit? of the CHB-tracing technique m combination with immunohistochemical staining [9] and not by species differences. In particular, the CHB-tracing technique appears to be a very sensitive anterograde tracer which fills the retinal neurons corn-

pletely, and when stained immunohistochemically, their distribution can be determined in detail [9]. Apparently, this technique allo~vs to distinguish terrninating fibers from passing fibers. Taken together, the topographical organization of the retinohypothalamic projection should be examined in a number of species by using the same technique, to conclude whether the retinal innervation of the SON is a matter of species differences or not. The present stud} emphasizes the observation by Levine et al. [6] that the retinohypothalamic tract can be separated into a medial component innervating mainl:r the suprachiasmatic nuclei, and a ktteral component, with major afferents to the lateral h.~pothalamic area. Whereas the role of the medial component in entrainment of endogenous circadian rhythms to the external photoperiod is x~.ell established, the functional significance of the lateral component is unknown. It is also unknown whether the two components are mnervated by the same population of retinal ganglion cells, but it is evident that the SON is innervated from the lateral component of the tract. The SON is implicated in regulation of diuresis, vascular resistance, lactation and in endocrinological reproductix e function,~. These functions are mediated by a release of either vasopressin or oxytocin fl'om axonal endings in the neurohj, pophy,~is to the general circulation [3]. The neurochemical nature of the neurons mnerwtted from the retinohypothalannc tract is at prcsent undetermined. Ho~vexer, the SON contains magnocellular neuron,~: the oxytoclnerglc

neuron~

m the dorsal

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and vasopres~inergic neurons in the ventral part, suggestmg that the oxytocmergic neurons recm~e the retinal input. The neurons of the SON that receive retinal input and the nulnber of possible contacts the,,' actually estabhsh have not been determmed. It seems that the mput is rather small and only represented by those axons that actually penetrate the nucleus, ~hich is supported by the fact that the dendrites of the neurons in the SON are mostly oriented xentralh and into the xentral glial lamina [11]. Drs. Philip J. Larsen and Morten Moiler are thanked for their helpful comments and suggestions on the man-

152

uscript. This study was supported by Nordlsk Insulinfond, Dir. Jacob Madsen og hustru Olga Madsens Fond, Hotelejer Carl Larsen og hustru Nicohne Larsens Mindelegat and The Danish Medical Research Council (J. 12-9742: J. 12-0954). J.D.M. is recipient of a HallasMoiler Stipend from the NOVO l-:oundation.

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