Neuroanatomical pathways linking vision and olfaction in mammals

Pergamon

Psychoneuroendocrinology, Vol. 19, Nos. 5-7, pp. 623-639, 1994 Copyright © 1994 Elsevier Science Ltd Printed in the USA. All rights reserved 0306-4530/94 $6.00 + .00

0306-4530(94)E0040-G NEUROANATOMICAL PATHWAYS LINKING VISION AND OLFACTION IN MAMMALS H.M. COOPER, 1 F. PARVOPASSU, 1 M. HERBIN, 1'2 and M. MAGNIN 1 ICerveau et Vision, INSERM U-371, 18 Avenue Doyen L6pine, 69500 Bron, France, and 2Laboratoire d'Anatomie Compar6e, M.N.H.M., Paris, France

(Received 5 October 1993; in final form 20 December 1993)

SUMMARY Retinal projections to several telencephalic structures have been demonstrated in a wide range of mammalian species following intraocular injections of tritiated amino acids and cholera toxin subunit-B conjugated to horseradish peroxidase. Since these regions are also innervated by olfactory fibers, we investigated the distribution of convergent projections using simultaneous injections of different anterograde tracers in the eye and olfactory bulbs. Convergent projections from the retina and from the olfactory bulbs were observed in the piriform cortex, olfactory tubercle, the cortical region of the medial amygdala, lateral hypothalamus, and the bed nucleus of the stria terminalis. A few retinal fibers also invade the nucleus of the lateral olfactory tract, the bed nucleus of the accessory olfactory bulb and the diagonal band of Broca. Injections of retrograde tracers in the medial amygdala, the bed nucleus or the lateral hypothalamus shows that the visuo-olfactory convergence mainly involves projections originating from the accessory olfactory bulb, and to a lesser extent from the ventromedial region of the main olfactory bulb. Fewer than 20 retinotelencephalic ganglion cells were identified in the retina, mainly located contralateral to the injection site. Ganglion cells were medium sized and possessed two long slender opposing dendrites. These retinal and olfactory projections could provide an anatomical substrate for the modulation of gonadotropin hormone levels and the olfactory influence on light mediated rhythms related to reproductive physiology. KeywordsmRetinal projections; Olfactory bulb; Accessory olfactory bulb; Gonadotropin hormones; Basal telencephalon; Hypothalamus.

INTRODUCTION BOTH REPRODUCTIVEBEHAVIORSand neuroendocrine physiology are regulated by visual

and chemosensory stimuli in mammals. Levels of circulating gonadotropin and sexual hormones are subject to variation depending on photoperiodic conditions, blindness, exposure to conspecific odors or interruption of olfactory pathways (Karsch et al., 1989; Meredith, 1991; Signoret, 1991; Urbanski, 1992). Although either light or chemoreception alone is sufficient to influence neuroendocrine responses, these effects are often potentiated or offset when the sensory input of both systems are combined or selectively altered. For example, removal of the olfactory bulbs in rodents can modify the normal circadian Address c o r r e s p o n d e n c e and reprint requests to: H . M . Cooper, C e r v e a u et Vision, I N S E R M U-371, 18 A v e n u e D o y e n L6pine, 69500 Bron, France. 623

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response to light cycles by phase shifting or lengthening the period of activity rhythms (Bittman et al., 1989; Pieper & Lobocki, 1991; Possidente et al., 1990). Similarly, temporal alteration of circannual activity rhythms are observed (Miro et al., 1980; Ruby et al., 1993). Olfactory bulbectomy also disrupts seasonal reproductive responses to light cycles by preventing gonadal regression which normally occurs under the influence of short photoperiods in hamsters (Pieper et al., 1984, 1989, 1990a) and in primates (Perret & Schilling, 1993; Schilling & Perret, 1993). Rats express an opposite effect, since bulbectomy can unmask photoperiodic responsiveness and facilitate testicular regression (Nelson & Zucker, 1981; Pieper et al., 1990b; Reiter et al., 1970). Although the specific cellular mechanisms underlying this neurohumoral integration are largely unknown, the influence of olfaction on gonadotropin hormone effects is assumed to be mediated by pathways involving basal telencephalon and hypothalamus (Pieper et al., 1989). These effects could be the consequence of hormonal or neuroendocrine feedback mechanisms acting through indirect pathways, or by direct integration within neurons receiving fibers of both visual and olfactory origin. Teleosts are known to possess a specific pathway which directly links together the retina and olfactory bulb, involving LHRH containing neurons of the telencephalic nucleus olfactoretinalis and/or terminal nerve (Demski & Northcutt, 1983; Stell et al., 1984; Uchiyama, 1990). However, in mammals such retinopetal neurons are absent, and at primary levels these two sensory systems are distinct. The retina is considered to project uniquely to diencephalic and mesencephalic structures (Campbell, 1972; Ebbesson, 1972), whereas the olfactory bulb projects to telencephalon (Davis et al., 1978; Scalia & Winans, 1975; Skeen & Hall, 1977). Several recent studies, using sensitive anterograde tracing techniques, have provided evidence that the retina also innervates the basal telencephalon (Cooper et al., 1989; Levine et al., 1991; Mick et al., 1993; Pickard & Silverman, 1981; Youngstrom et al., 1991). These regions are also sites of efferent projections from the olfactory bulbs, raising the possibility of convergent visual and olfactory integration within structures linked to reproductive physiology. In this study, we address the following questions: Are retinotelencephalic projections a common mammalian feature? Which brain regions receive convergent projections from the retina and from the olfactory bulb? What is the distribution and morphology of the afferent retinal and olfactory neurons? Could these pathways represent a putative anatomical substrate underlying visuo-olfactory relations in mammals? RETINAL PROJECTIONS TO THE BASAL TELENCEPHALON IN MAMMALS Methods

Retinal projections were studied in more than 50 species from 13 orders of mammals (see Table I), The general procedure for demonstration of retinal projections has been described in detail elsewhere (Cooper et al., 1989; Magnin et al., 1989). Animals received an intravitreal injection of tritiated amino acids (3H-proline and 3H-leucine) or horseradish peroxidase-cholera toxin conjugate (CT-HRP, subunit b, Sigma). The amount of anterograde tracer injected varied from 2 to 1000 ~Ci dissolved in a volume of 2-50 ~l sterile saline, depending on the size of the ocular globe. Survival times were generally 48 h, after which animals were perfused with a warm saline rinse, followed by 4% buffered paraformaldehyde, and in the case of HRP histochemistry, 10% buffered sucrose. For autoradiography (Cowan et al., 1972), the brains were either frozen sectioned or

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TABLE I. LIST OF THE MAMMALIANSPECIES STUDIED IN WHICH RETINAL FIBERS WERE OBSERVED IN THE BASALTELENCEPHALONFOLLOWINGINTRAOCULAR INJECTION AND TRITIATED AMINO ACID AUTORADIOGRAPHYOR HRP TRACING TECHNIQUES

Marsupialla Didelphis opossum Philander opossum Caluromys philander Marmosa murina Chironectes minimus Trichosurus vulpecula Edentata Bradypus pilosa Pholidota Manis tricuspus Insectivora Erinaceus europaeus Scandentia Tupaia glis Dermoptera Cynocephalus variegatus Hyracoidea Dendrohyrax dorsalis

Chiroptera Macroderma gigas Hipposideras gigas Carollia perspicillata Sturnira sp. Artibeus lituratus Rousettus aegyptiacus Pteropus alecto Pteropus scapulatus Eonyterus sp. Primates Nyticebus coucang Perodicticus potto Galago demidovii Galago alleni Euoticus elegantulus Microcebus murinus Callithrix jacchus Saimiri sciureus Miopithecus talapoin Macaca fascicularis Macaca mulatta Hylobates concolor

Carnivora Canis familiaris Felis domesticus Mustela putorius Lagomorpha Oryctolagus cuniculus Rodentia Rattus rattus Mus musculus Mesocricetus auratus Phodopus sungorus Glis glis EUobius talpinus Cryptomys hottentotus Spalax ehrenbergi Spalacopus cyanus Agouti paca Protoxerus strangeri Heliosciurus rufobrachium Petaurista petaurista Ungulata Ovis aries

At least one individual of each species listed was studied. No information on retinal projections is currently available on species from the orders: Monotremata, Sirenia, Cetacea, Tubulidentata, or Proboscidea.

embedded in paraffin and cut in the frontal or horizontal plane. Sections were mounted on gelatinized slides, defatted, coated with emulsion (Ilford L-4) and exposed in the dark for 8-20 weeks. The coated slides were subsequently developed (Ilford PL12), fixed and coverslipped with depex. Every other section was counterstained with cresyl-violet for cytoarchitectural observation. For HRP histochemistry, the brains were sectioned on a freezing microtome in the coronal plane at a thickness of 30-40 /xm. Sections were reacted using TMB as a chromogen according to the method of Mesulam (1978) as modified by Gibson et al. (1984), or with ammonium heptamolybdate (Cooper et al., 1993a; Olucha et al., 1985). Results In all species studied, retinal fibers could be observed in the basal telencephalon. The trajectory of retinal fibers to basal telencephalon was essentially similar. Labeled fibers diverge from the dorsolateral margin of the optic tract and chiasm, pass dorsally around the supraoptic nucleus, penetrate the diencephalic-telencephalicjunction lateral to hypothalamus, and subsequently continue rostrally and laterally within the basal telencephaIon. Although the degree of variation is difficult to appreciate due to different tracing techniques employed, the observed amount of retinotelencephalic fibers appears sparse in

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ROSTRAL FIG. 1: Distribution of labeled retinal fibers in the basal telencephalon of the murine rodent species, EUobius talpinus, following injection of the tracer CT-HRP in the contralateral eye. (A) shows a dark field illustration of labeled fibers in the piriform cortex (small arrows) lateral to the olfactory tract. (B) illustrates a series of line drawings made from serial coronal sections showing the course and distribution of fibers in olfactory structures of basal telencephalon (the second section from the top corresponds to the section shown in (A). Note that retinal fibers remain superficial to the surface of the basal telencephalon. DBh = diagonal band of Broca, horizontal limb; LHA = lateral hypothalamic area; LOT = lateral olfactory tract; MPO = median preoptic nucleus; OC = optic chiasm; OT = optic tract; PIR = piriform cortex; SCN = suprachiasmatic nucleus; SON = supraoptic nucleus; TU = olfactory tubercle; III = third ventricle. Scale in (A) = 500 mm.

most animals, while in species such as rodents and primates, this projection is conspicuous. Since a detailed description of this pathway in all animals studied is beyond the scope of the present report, we will restrict our discussion to some noteworthy or rare specimens. The retinal pathway within the basal telencephalon is extensive in rodents such as the mole-lemming (Ellobius talpinus, Fig. 1) and the syrian hamster (Mesocricetus auratus, Fig. 4). In both species, retinal fibers are present in several telencephalic areas, including the cortical region of the medial amygdala adjacent to the optic tract, the mediocaudal

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region of the olfactory tubercle, and the medial margin of piriform cortex. These labeled fibers form a loose superficial plexus which spreads from the lateral margin of the optic tract to the lateral olfactory tract. This group of retinal fibers appears to be associated with a diffusely distributed network of labeled axons which also invades the lateral and anterior hypothalamic areas (Fig. 4). Labeled fibers extending beyond the lateral olfactory tract into piriform cortex are particularly evident in the mole-lemming (Fig. 1). Within the olfactory tubercle and piriform cortex, retinal fibers remain in the superficial plexiform layer Ia. A few fibers also course within the horizontal limb of the diagonal band of Broca, the nucleus of the lateral olfactory tract and the bed nucleus of the accessory olfactory tract (Fig. 4). Other species of mammals show a similar distribution, although the precise topographical location of retinal fibers varies according to interspecific differences in the morphology of the basal forebrain (Fig. 2). Note, however, the close similarity in spatial distribution of retinal fibers between some unrelated species such as the hyrax and pangolin, rodents and hedgehog, and rabbit and marmoset monkey (Fig. 2). Nonprimate mammals share a number of common features. Retinal fibers in the basal telencephalon are mainly located contralateral to the injected eye, although in all species a few fibers are also seen on the ipsilateral side. As in rodents, retinal fibers form a sparse net extending within the cortical region of medial amygdala to the olfactory tubercle, and occasionally including the piriform cortex. Although fibers are located in the superficial plexiform layer in most species labeled, in the tree sloth, retinal terminals are situated along the inner border of the granular cell layer of olfactory tubercle (Fig. 2C). Due to the general caudorostral direction of retinotelencephalic fibers, this projection often appears scant in coronal sections since the labeled axons are cut perpendicular to their axes. This is clearly illustrated in the case of the rabbit (Figs. 2E and 2F), in which this pathway is more conspicuous in horizontal brain sections sectioned parallel to the orientation of the fibers. This section also depicts the superficial distribution of labeled retinal fibers within the prominent bulge formed by the mediocaudal pole of the olfactory tubercle. In primates (Figs. 2 and 3), retinal label is mainly located ipsilaterally and is restricted to the region between the optic tract and the mediocaudal region of the olfactory tubercle, medial to the Islands of Callega. In most primates, anterograde label extends 2-3 mm rostral to the entrance of the optic nerves into the brain. As mentioned above, this pathway is often more apparent in horizontal brain sections, as shown by the fiber distribution within the protuberance of the olfactory tubercle in the marmoset (Fig. 2G). The retinotelencephalic pathway is particularly evident in the macaque (Fig. 3). As in other species of primates, the olfactory tubercle displays a typical horizontal lamination, consisting of a superficial plexiform layer, an intermediate granular cell layer and a deep polymorphic cell layer (Figs. 3C and 3E). Retinal fibers are distributed along the internal border of the granular cell layer, which in this area forms a distinct crescent like thickening (Fig. 3E). Furthermore, labeled fibers and terminal label are often closely apposed with the perforating branches of the anterior cerebral artery, which enter the forebrain in this region. T O P O G R A P H Y OF CONVERGENT VISUAL AND OLFACTORY PROJECTIONS Methods Since several of the regions described above are also the target of the olfactory bulbs, we used simultaneous injections in the retina and olfactory bulb to assess the

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FIG. 2: Distribution of autoradiographic label in the basal telencephalon of several mammals, following an intraocular injection of tritiated amino acids: (A) Pangolin (Pholidota), (B) Tree hyrax (Hyracoidea), (C) Tree sloth (Edentata), (D) Hedgehog (Insectivora), (E,F) Rabbit (Lagomorpha), (G) Marmoset monkey (Primates). All photographs were made using a 6.3-10× objective under dark field (A, C-G) or light field (B) illumination. Sections (A)-(E) are coronal and (F)-(G) are horizontal. (A)-(F) are contralateral and (G) is ipsilateral to the injected eye. Small arrows, or dashed lines, indicate the location of label in the basal telencephalon in (A)-(E). Note the close similarity between certain species (A and B; F and G). Labeled fibers are often more visible in horizontal sections, such as in rabbit and marmoset (F-G). In these two species the rounded protrusion corresponds to the mediocaudal limit of the olfactory tubercle.

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FIG. 3: Coronal sections in the hypothalamic and basal telencephalic regions of the macaque monkey injected in one eye with radioactive amino acids. Ipsilateral to the injection (right side), labeled retinal fibers emerge from the optic chiasm and course dorsally around the SON (A). At higher magnification, slightly rostral to (A), labeled fibers are seen lateral to the SON and terminal aggregations of silver grains lie within the caudomedial limit of olfactory tubercle (B, dark field; C, light field). Further rostral, terminal label is distributed along the inner border of the granular layer of olfactory tubercle (D, dark field; E, light field). Note the crescent shaped thickening of this layer (E, arrows). CH = optic chiasm; DBh = diagonal band of Broca, horizontal limb; LHA = lateral hypothalamic area; OLT = olfactory tubercle; SON = supraoptic nucleus. Scale in (A) --- 500 mm, and in (E) = 250 mm.

degree of convergence of these sensory systems in two species of rodents, the syrian hamster and the blind mole rat (Spalax ehrenbergi). CT-HRP was used as above for tracing retinal fibers, and the fluorescent anterograde tracer, fluororuby (tetramethylrhodamine dextrane-amine, Molecular Probes), was injected in the main (MOB) and/or accessory (AOB) olfactory bulbs. The fluororuby injection (0.5-1.0 ~1, 10% solution) was made 7 days prior to the C T - H R P injection. Animals were perfused two days later and treated for H R P histochemistry as described above. The distribution of the intense red fluorescence of the dye could be observed on the same sections as the HRP reaction product.

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FIG. 4: Drawings of the distribution of retinal and olfactory fibers in the hypothalamus and telencephalon of the hamster from successive coronal sections. The injection site (not illustrated) was extensive and involved both the MOB and the AOB. Olfactory bulb efferent fibers are shown by shading, and retinal fibers by fine lines. Convergence of these two systems is seen in regions of the basal telencephalon: olfactory tubercle, piriform cortex, the cortical region of the medial amygdala and the lateral hypothalamus; and in the encapsulated region of the bed nucleus of the stria terminalis. See text for further details, ac = anterior commissure; AD = anterodorsal nucleus; AV = anteroventral nucleus; BAOT = bed nucleus of the accessory olfactory tract; BNST = bed nucleus of the stria terminalis; CH = optic chiasm; DBh = diagonal band of Broca, horizontal limb; LHA = lateral hypothalamus; LOT = lateral olfactory tract; ME = medial amygdala; MPO = medial preoptic nucleus; NLOT = nucleus of the lateral olfactory tract; OT = optic tract; PC = piriform cortex; PVA = thalamic paraventricular nucleus; PVN = paraventricular nucleus; SCN = suprachiasmatic nucleus; TU = olfactory tubercle.

Results Figure 4, which shows representative line drawings illustrating the distribution of visual and olfactory fibers, summarizes the results obtained in the hamster. In this case, the injections in the olfactory bulb were extensive and involved both the MOB and the AOB. Retinal fibers and fibers of olfactory origin were observed colocalized within the cortical region of the medial amygdala, the mediocaudal region of the olfactory tubercle, the medial margin of piriform cortex adjacent to the lateral olfactory tract, and in the lateral hypothalamus. In the olfactory tubercle, these fibers were located in the superficial most plexiform layer (Figs. 5A and 5B). In the blind mole rat, convergent projections were restricted to the amygdala region, ventral to the optic tract (Figs. 6A and 6B). Within the telencephalon, the retina was also found to innervate the bed nucleus of the stria terminalis (BNST, Figs. 4-6). This pathway is minute in most mammals, but is particularly evident in rodents, including the hamster (Fig 5), blind mole-rat (Fig. 6), and mole-lemming (Herbin et al., in press). In these rodent species, retinal fibers diverge from the dorsal region of the lateral geniculate nuclei, and coursing rostrally over the thalamus, merge with the stria terminalis. Retinal fibers subsequently penetrate sharply into the bed nucleus in association with an obliquely oriented blood vessel, and distribute within the encapsulated region of the BNST. In Spalax ehrenbergi, this projection constitutes a major target of the retina (17% of all optic fibers) despite the highly atrophied

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FIG. 5: Hamster: Convergent distribution of anterogradely labeled retinal (CT-HRP) and olfactory fibers (fluororuby) in adjacent sections of the basal telencephalon (olfactory tubercle, A, B) and the encapsulated region of the bed nucleus of the stria terminalis (C, D). (A) and (C) are dark field photomicrographs; (B) and (D) are fluorescence photomicrographs. These results are from the same animal illustrated in the line drawings of Figs. 4C and 4D.

eye (Cooper et al., 1993a, 1993b). Olfactory bulb projections form a dense oval-shaped focus of label, which overlaps the region occupied by the optic fibers (Figs. 5C and 5D). In addition to the above regions, injections of fluororuby which included the MOB resulted in extensive anterograde label in the anterior olfactory nucleus, piriform and entorhinal cortices, the ventral hippocampal rudiment, and the nucleus of the lateral olfactory tract. Injections which were restricted to the AOB labeled only the lateral hypothalamus, the cortical region of the medial amygdala, the bed nucleus of the accessory olfactory tract, and the BNST. Since these results suggested that retinal input overlaps with afferents from both the MOB and/or the AOB, we next used retrograde tracing techniques to attempt to identify the origin of these projections. TOPOGRAPHICAL DISTRIBUTION OF AFFERENT RETINAL AND OLFACTORY NEURONS Methods This series of experiments was focused on the hamster. Animals were injected in hypothalamic and telencephalic regions which were previously shown to receive projections from both the retina and the olfactory bulbs. Fluorescent tracers (fast blue, diamidino yellow, flourogold, or rhodamine dextran amine) were injected stereotaxically using

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FIG. 6: Blind mole rat (Spalax): Convergent distribution of anterogradely labeled retinal (CT-HRP) and olfactory fibers (fluororuby) in adjacent sections of the basal telencephalon (cortical region of the medial amygdala, A, B) and the encapsulated region of the bed nucleus of the stria terminalis (C, D). (A) and (C) are dark field photomicrographs; (B) and (D) are fluorescence photomicrographs.

a 30-50/zm tip pipette attached to a picopump which delivered calibrated pulses of air. The quantity of tracer injected varied from 0.02 to 0.1 ~1. Injections were made slowly over 20-min periods. Animals were allowed to survive for 7-10 days, and perfused with a warm saline rinse followed by 1 liter of 8% phosphate buffered paraformaldehyde. Brains were frozen sectioned at 30/zm for localization of injection sites and labeled cells in the olfactory bulbs. The retinas were flat mounted (Stone, 1981) and observed under a fluorescence microscope equipped with a motorized XY stage, for plotting of the locations of retrogradely labeled ganglion cells. Results

Following injections of tracer in the BNST, few (<20) labeled ganglion cells were observed, mainly in the retina located contralateral to the injection site (Fig. 7). This asymmetric distribution corresponded to the ratio of anterograde label in the BNST following an intraocular injection. Ganglion cells were scattered over the retina and showed no particular pattern of spatial distribution. These ganglion cells were medium sized (roughly I0 tzm in diameter) and appeared to possess two or three proximally branching dendrites (Fig. 8D). However, it was obvious that the degree of retrograde labeling was incomplete and offered only rudimentary detail of the cell's dentritic organization. In addition to the retina, retrogradely labeled cells were also seen ipsilaterally in the mitral cell layer of the AOB and bilaterally in the ventromedial region of the MOB.

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FIG. 7: Line drawings illustrating the distribution of retrogradely labeled neurons in the olfactory bulbs (right side) and left and right retinas (lower left side) following an injection of fluorogold in the bed nucleus of the stria terminalis (BNST, coronal section upper left side). Labeled ceils are located in the mitral cell layer of the ipsilateral accessory olfactory bulb (AOB) and bilaterally in the ventromedial part of the main olfactory bulb (MOB). Afferent ganglion cells are mainly distributed in the contralateral retina (D = dorsal; N = nasal; T = temporal; V = ventral). CH = optic chiasm; DBh = diagonal band of Broca, horizontal limb; LOT = lateral olfactory tract; MPO = medial preoptic nucleus; PC = piriform cortex.

Injections which were located rostral or ventral to the B N S T (at the level of the septum) failed to label ganglion cells in the retina. Injections in the lateral hypothalamus, dorsal to the optic tract also yielded less than 20 scattered cells in the contralateral retina and a few cells in the ipsilateral retina. Labeled cells were present in ipsilateral AOB, but were absent from the MOB. Injections that were located further dorsally in the hypothalamus, for example adjacent to the paraventricular nucleus, labeled few retinal ganglion cells (<8) but failed to label any neurons in the olfactory bulbs. In contrast, injection sites that included the optic tract and adjacent basal telencephalon produced large numbers of labeled ganglion cells in both retinas, as well as numerous cells in the ipsilateral AOB and a few scattered cells in the MOB. Injections that were located in the basal telencephalon lateral to the optic tract also revealed a few sparse retinal ganglion cells, but labeled extensive regions of both the MOB and AOB. Since the retrograde tracers used in the above experiments were inadequate for demonstrating the morphology of the retinal ganglion cells, we also used implants of the lipophilic

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Fl6.8: Retrogradely labeled retinal ganglion cells in the hamster following crystal implants of DiI in the optic tract (A) or in the basal telencephalon (B, C); or following injection of fluorogold in the bed nucleus of the stria terlminalis (D, see also Fig. 7). Note the simple morphology of the dendritic trees in (B) and (C). Scale bar in (D) = 20 ram.

carbocyanine dye, DiI, implanted into the basal telencephalon of aldehyde fixed hamster brains. This dye displays the property of passive membrane uptake and transport in postmortem fixed material. The time of transport required to label cells in the retina varied from 7 to 11 mo. In the cases in which the crystal implant included fibers in the optic tract, large numbers of labeled ganglion cells were found throughout both retinas. The morphology of these cells resembled that previously described in rodents (Sefton & Dreher, 1985), with soma diameters ranging from 6 to 22 ~m. The dendritic trees of these cells were extensively labeled, elaborate, highly branched and displayed numerous varicose thickenings (Fig. 8A). In animals in which the implant was situated between the optic tract and the lateral olfactory tract, avoiding fibers of the optic tract, 2-12 ganglion cells were observed in the contralateral retina. In only two cases, 1-2 ganglion cells were observed in the ipsilateral retina. Ganglion cells were randomly distributed in the retina. These cells had medium sized somas (9/zm diameter), were elongated in shape and displayed a simplified, asymmetric, dendritic morphology (Figs. 8B and 8C). Typically, two slender dendritic arbors extended in opposite directions from the soma. These dendrites were poorly branched and displayed few varicosities. Dendrites could be followed for a distance of 100-200/xm from the soma. No labeled cells were seen in cases in which the DiI crystal implant was placed lateral to the olfactory tract, or rostral to the olfactory tubercle.

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DISCUSSION

These results agree with several previous reports describing access of the retina to telencephalic and limbic structures (Cooper et al., 1989; Levine et al., 1991; Martinet et al., 1992; Mick et al., 1993; Pickard & Silverman, 1981; Riley et al., 1981; Youngstrom et al., 1991). The sparseness of this projection, the need for sensitive anterograde tracing techniques, the location of retinal fibers rostral to the optic nerve, combined with the difficulty of observing labeled fibers in coronal section, explains why the existence of this pathway has frequently been overlooked. The quantitative differences observed in the extent of this pathway may be related to both technical limitations and species differences. For example, we observed that in the same species, the CT-HRP method was more sensitive for revealing this pathway than the autoradiographic technique (hamster; see also Herbin et al., in press, for Ellobius). However, the ubiquitous occurrence of retinotelencephalic fibers in many unrelated groups, confirms that this pathway is a consistent feature of the mammalian visual system. Our retrograde tracing studies reveal that this projection originates from an extremely small population of retinal ganglion cells. The limited number and scattered distribution of ganglion cells implies that spatial resolution is practically null, and only crude information concerning global ambient light levels can be conveyed by this system. The function of this pathway might thus be similar to the role of the retinohypothalamic tract in photoperiodic light detection (Groos & Meijer, 1985; Meijer & Rietveld, 1989). Indeed, the simple dendritic morphology of the retinotelencephalic ganglion cells described in this study, resembles that of retinohypothalamic cells which innervate the suprachiasmatic nucleus (Cooper et al., 1993c; Pickard & Friauf, 1990). The retinal fibers projecting to these two regions may thus represent collateral branches originating from a single population of ganglion cells. The sparseness of the retinotelencephalic pathway does not imply a reduced or vestigial function, since the number of pineal or deep brain photic receptors in nonmammalian vertebrates are few in number but sustain an essential role in the regulation of seasonal reproduction and physiology (Collin & Oksche, 1981). The retinal projection to these telencephalic structures overlaps with fibers originating from both the AOB and MOB. Our anterograde and retrograde tracing experiments agree with results from previous studies demonstrating the existence of two parallel chemosensory systems corresponding to their origin in the olfactory mucosa and the vomeronasal organ (Davis et al., 1978; Meredith, 1991; Scalia & Winans, 1975; Skeen & Hall, 1977). Convergence with projections of the MOB occurs in the region of the olfactory tubercle and piriform cortex, while the convergence with AOB fibers is more extensive and includes all target regions of AOB (cortical region of the medial amygdala, the lateral hypothalamus, and the BNST). There is, however, some evidence that both the AOB and MOB project to single neurons in the medial amygdala region (Licht & Meredith, 1987). It is also interesting to note that visuo-olfactive convergence is present in species with relatively reduced visual pathways (blind mole rat) or olfactory system (primates). In the absence of ultrastructural evidence, direct synaptic convergence of olfactory and visual inputs on single neurons cannot be assumed. However, the regions receiving retinal and olfactory fibers are implicated in a diversity of sexual and reproductive behaviors. These telencephalic structures are sexually dimorphic (Allen & Gorski, 1990; Guillamon et al., 1988; Mizukami et al., 1983) and contain high densities of neurons with sex steroid receptors (Bonsall et al., 1986; Commins & Yahr, 1985; Simerly, 1990). The

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medial amygdala and BNST share reciprocal connections and have extensive input to hypothalamic regions, such as the medial preoptic nucleus, all of which are putatively involved in regulating reproductive hormone release (Lehman & Winans, 1983; Simerly & Swanson, 1986; Swanson & Cowan, 1979; Weller & Smith, 1982). Furthermore, lesion (Valcourt & Sachs, 1979) or electrical stimulation (Beltramino & Taleisnik, 1980; Han & Ju, 1990) of these nuclei are susceptible of modifying sexual behavior and gonadotropin levels. The evidence from these studies is thus consistent with the concept of an involvement of these structures in pathways controlling the secretion or sites of action of gonadotropin hormones, and associated olfactory alteration of photoperiodic related responses. The alteration of gonadotropin levels and concomittant physiological and behavioral consequences resulting from olfactory bulbectomy could potentially involve three distinct chemosensory systems, the main and accessory olfactory systems and the terminal nerve (Meredith, 1991). However, input from the MOB and/or AOB to the basal telencephalon has been shown to be specifically involved in the olfactory influence on reproductive neuroendocrine events (Pieper et al., 1989). Transection of the caudal fibers of the lateral olfactory tract prevents testicular regression under short photoperiods, while removal of the vomeronasal organ or lesions of the terminal nerve do not prevent (hamsters: Pieper et al., 1989) or facilitate (rats: Nelson & Zucker, 1981; Sanchez-Barcelo et al., 1985) testicular regression, or the pheromonally mediated testosterone surge (Wirsig-Wiechmann, 1993). However, an involvement of the vomeronasal organ in LH release cannot be ruled out (Coquelin et al., 1984; Wysocki & Lepri, 1991; Meredith, 1991). The caudal LOT contains both the AOB and MOB fibers which project to the olfactory tubercle, the cortical regions of the amygdala, and the BNST. Since bulbectomy results in increased gonadotropin secretion (Clancy et al., 1986; Pieper et al., 1984, 1990a) but does not directly block melatonin secretion by the pineal (Pieper et al., 1988), removal of the olfactory bulbs is assumed to prevent the anti-gonadotropin effects of melatonin. The mechanism through which this influence occurs may thus involve antagonistic effects concerning the sites of action of melatonin related-LHRH release within these regions. The integration of redundant and parallel sensory influences involved in regulating the release and subsequent cascade of effects of gonadotropin hormones is advantageous for adjusting reproductive physiology to exogenous and endogenous conditions. Photoperiodic light variations provide a predictable environmental cue for long term regulation of base line levels of gonadotropin secretion in relation to seasonal breeding. The complementary influence of chemosensory stimuli allows the rapid induction of hormonal responses to appropriate behavioral contexts (Coquelin et al., 1984; Meredith, 1991 ; Signoret, 1991). In contrast to our knowledge of the consequences of sensory stimulations, the precise visual and olfactory pathways responsible for the modulation of gonadotropin secretion remain to be defined. Future efforts should be aimed at determining the specific sites and nature of action of these sensory inputs. Acknowledgments: We would like to thank for their excellent assistance Sandrine Richard, Ghislaine Clain, Jean-Louis Borach and Pascal Giroud. This work was supported by grants to H.M. Cooper from the RhoneAlpes Neuroscience Program, INSERM, and the CNRS and to M. Herbin from the F6deration des Aveugles de France, Cannes Blanches de Lyon and Foundation Fouassier, and from M.N.H.N., B.Q.R. '93.

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