Retinohypothalamic projection in the mouse: Electron microscopic and iontophoretic investigations of hypothalamic and optic centers

The problem of the direct retinohypothalamic projection in mammals (Moore, 1973) was reinvestigated in the laboratory mouse by electron microscopy and cobalt chloride-iontophoresis. The time-course of the axonal degeneration in the suprachiasmatic nucleus was studied 3, 6 and 12 h, 1, 2, 4, 6, 9 and 12 days after unilateral retinectomy. Specificity of the degenerative changes was controlled by investigation of the superficial layers of the superior colliculus. The ratio of crossed to uncrossed optic fibers could could be determined by counting degenerating structures (axons and terminals) in the optic chiasma and the ipsilateral and contralateral areas of the optic tract, the suprachiasmatic nucleus, and the superior colliculus. The number of degenerating axons in the suprachiasmatic nucleus showed a maximum one day after unilateral retinectomy and was, at all stages studied, two to three times higher in the contralateral than in the ipsilateral nuclear area. In the optic tract and in the superior colliculus the number of degenerating profiles was three times higher in the contralateral than in the ipsilateral area. Retinohypothalamic connections and crossing pattern of retinal fibers were studied light microscopically using impregnation with cobalt sulfide in whole mounts of brains. Most of the optic fibers in the laboratory mouse are crossed crossed (70-80%). A bundle of predominantly crossed optic fibers runs to the suprachiasmatic nucleus.     

Dolphin peripheral visual pathway in chronic unilateral ocular atrophy: Complete decussation apparent

Components of the peripheral visual pathway were examined in two bottlenose dolphins, Tursiops truncatus, each with unilateral ocular degeneration and scarring of 3 or more years' duration. In both animals, the optic nerve associated with the blind eye right eye in Tg419 and left eye in Tt038 had a translucent, gel-like appearance upon gross examination. This translucency was also evident in the optic tract contralateral to the affected eye. In Tg419, myelinated axons of varying diameters were apparent in the left optic nerve, whereas the right optic nerve, serving the blind eye, appeared to be devoid of axons. In Tt038, myelinated axons were associated with the right optic nerve (serving the functional eye) and left optic tract but were essentially absent in the left optic nerve and right optic tract. Examined by light microscopy in serial horizontal sections, the optic chiasm of Tt038 was arranged along its central plane in segregated, alternating pathways for the decussation of right and left optic nerve fibers. Ventral to this plane, the chiasm was comprised of fibers from the left optic nerve, whereas dorsal to the central plane, fibers derived from the right optic nerve. Because of this architectural arrangement, the right and left optic nerves grossly appeared to overlap as they crossed the optic chiasm with the right optic nerve coursing dorsally to the left optic nerve. At the light and electron microscopic levels, the optic nerves and tracts lacking axons were well vascularized and dominated by glial cell bodies and glial processes, an expression of the marked glial scarring associated with postinjury axonal degeneration. The apparent absence of axons in one of the optic tract pairs (right in Tt038 and left in Tg419) supports the concept of complete decussation of right and left optic nerve fibers at the optic chiasm in the bottlenose dolphin. © 1994 Wiley-Liss, Inc.     

Optic nerve regeneration after intravitreal peripheral nerve implants: trajectories of axons regrowing through the optic chiasm into the optic tracts

We have studied axon regeneration through the optic chiasm of adult rats 30 days after prechiasmatic intracranial optic nerve crush and serial intravitreal sciatic nerve grafting on day 0 and 14 post-lesion. The experiments comprised three groups of treated rats and three groups of controls. All treated animals received intravitreal grafts either into the left eye after both left sided (unilateral) and bilateral optic nerve transection, or into both eyes after bilateral optic nerve transection. Control eyes were all sham grafted on day 0 and 14 post-lesion, and the optic nerves either unlesioned, or crushed unilaterally or bilaterally. No regeneration through the chiasm was seen in any of the lesioned control optic nerves. In all experimental groups, large numbers of axons regenerated across the optic nerve lesions ipsilateral to the grafted eyes, traversed the short distal segment of the optic nerve and invaded the chiasm without deflection. Regeneration was correlated with the absence of the mesodermal components in the scar. In all cases, axon regrowth through the chiasm appeared to establish a major crossed and a minor uncrossed projection into both optic tracts, with some aberrant growth into the contralateral optic nerve. Axons preferentially regenerated within the degenerating trajectories from their own eye, through fragmented myelin and axonal debris, and reactive astrocytes, oligodendrocytes, microglia and macrophages. In bilaterally lesioned animals, no regeneration was detected in the optic nerve of the unimplanted eye. Although astrocytes became reactive and their processes proliferated, the architecture of their intrafascicular processes was little perturbed after optic nerve transection within either the distal optic nerve segment or the chiasm. The re-establishment of a comparatively normal pattern of passage through the chiasm by regenerating axons in the adult might therefore be organised by this relatively immutable scaffold of astrocyte processes. Binocular interactions between regenerating axons from both nerves (after bilateral optic nerve transection and intravitreal grafting), and between regenerating axons and the intact transchiasmatic projections from the unlesioned eye (after unilateral optic nerve lesions and after ipsilateral grafting) may not be important in establishing the divergent trajectories, since regenerating axons behave similarly in the presence and absence of an intact projection from the other eye.     

Efferent projections from the lateral geniculate nucleus to the pineal complex of the Mongolian gerbil (Meriones unguiculatus)

Summary The intergeniculate leaflet of the lateral geniculate nucleus is considered to modulate circadian activity rhythms probably mediated by a direct neuronal connection to the suprachiasmatic nucleus. The present study in the gerbil demonstrates, by anterograde tracing with Phaseolus vulgaris-leucoagglutinin (PHA-L), the existence of an additional neuronal projection from a subportion of the lateral geniculate nucleus, involving the intergeniculate leaflet, directly to the pineal gland. PHA-L-immunoreactive nerve fibers originating from perikarya at the injection site were located under the optic tract projecting towards the midsagittal plane. Delicate PHA-L-immunoreactive nerve fibers were observed in the posterior paraventricular thalamic nucleus, precommissural nucleus, olivary pretectal nucleus, anterior and posterior pretectal nuclei, and posterior commissure. Single fibers could be followed from the caudal part of the medial habenular nucleus and the pretectal area into the rostral part of the deep pineal gland. Other fibers continued through the posterior commissure into the contralateral hemisphere to terminate in the same structures as on the ipsilateral side. From the posterior commissure, small bundles of thick fibers entered the deep pineal gland where they arborized among the endocrine cells. A few nerve fibers were observed in the habenular commissure and the pineal stalk, but no fibers were identified in the superficial pineal. This direct geniculo-pineal connection suggests that the pineal gland is directly influenced by the optic system.     

Efferent projections from the lateral septal nucleus to the anterior hypothalamus in the rat: A study combining Phaseolus vulgaris-leucoagglutinin tracing with vasopressin immunocytochemistry

Summary The tracer Phaseolus vulgaris-leucoagglutinin (PHA-L) was injected into the lateral septum of the rat at different rostrocaudal locations to study the efferent septal projections to the anterior hypothalamus. For spatial correlation of these septofugal elements with the vasopressinergic system a dual immunocytochemical technique was used (i) to demonstrate nerve fibers and their corresponding bouton-like structures labeled with the tracer, and (ii) to identify vasopressin in the same section. The hypothalamic paraventricular and supraoptic nuclei, the accessory hypothalamic magnocellular system, and the suprachiasmatic nucleus are recipients of PHA-L-labeled fibers from all parts of the lateral septum. Close appositions between (i) these axons and their varicosities, and (ii) vasopressin-immunoreactive perikarya and their processes, putatively indicating functional interrelationships, were observed in all these nuclear areas, especially in their neuropil formations.Abbreviations F fornix - OC optic chiasm - OT optic tract - PVN paraventricular nucleus - SCN suprachiasmatic nucleus - SON supraoptic nucleus - III third ventricle     

The thalamic paraventricular nucleus relays information from the suprachiasmatic nucleus to the amygdala: A combined anterograde and retrograde tracing study in the rat at the light and electron microscopic levels

The relationship between efferents of the hypothalamic suprachiasmatic nucleus (SCN) and neurons of the thalamic paraventricular nucleus (PVT) projecting to the amygdala was investigated in the rat using tract tracing in light and electron microscopy. Biotinylated dextran amine was used to label anterogradely SCN efferents. These fibers were found to reach the thalamic midline, terminating in PVT, through three pathways: anterodorsally through the preoptic region, dorsally through the periventricular hypothalamus, and through the contralateral medial hypothalamic and preoptic areas after crossing the midline in the optic chiasm. Preterminal and terminal-like elements labeled from the SCN were distributed throughout the rostrocaudal extent of PVT, with an anteroposterior gradient of density. Labeled terminal elements were densest in the dorsal portion of PVT beneath the ependymal lining and some of them entered the ependyma. Anterograde tracing of SCN fibers was combined with injections of retrograde tracers in the amygdala. Numerous retrogradely labeled cell bodies were seen throughout PVT, with a prevalence in its anterodorsal portion. Overlap was detected between puncta labeled from the SCN and retrogradely labeled neurons, especially in the anterodorsal sector of PVT, where numerous puncta were in close apposition to thalamo-amygdaloid cells. Electron microscopy revealed that boutons labeled from the SCN established synaptic contacts with dendritic profiles of PVT neurons labeled from the amygdala. The findings demonstrate that information processed in the biological clock is conveyed to the amygdala through PVT, indicating that this nucleus plays a role in the transfer of circadian timing information to the limbic system.     

Reduced light response of neuronal firing activity in the suprachiasmatic nucleus and optic nerve of cryptochrome-deficient mice

To examine roles of the Cryptochromes (Cry1 and Cry2) in mammalian circadian photoreception, we recorded single-unit neuronal firing activity in the suprachiasmatic nucleus (SCN), a primary circadian oscillator, and optic nerve fibers in vivo after retinal illumination in anesthetized Cry1 and Cry2 double-knockout (Cry-deficient) mice. In wild-type mice, most SCN neurons increased their firing frequency in response to retinal illumination at night, whereas only 17% of SCN neurons responded during the daytime. However, 40% of SCN neurons responded to light during the daytime, and 31% of SCN neurons responded at night in Cry-deficient mice. The magnitude of the photic response in SCN neurons at night was significantly lower (1.3-fold of spontaneous firing) in Cry-deficient mice than in wild-type mice (4.0-fold of spontaneous firing). In the optic nerve near the SCN, no difference in the proportion of light-responsive fibers was observed between daytime and nighttime in both genotypes. However, the response magnitude in the light-activated fibers (ON fibers) was high during the nighttime and low during the daytime in wild-type mice, whereas this day-night difference was not observed in Cry-deficient mice. In addition, we observed day-night differences in the spontaneous firing rates in the SCN in both genotypes and in the fibers of wild-type, but not Cry-deficient mice. We conclude that the low photo response in the SCN of Cry-deficient mice is caused by a circadian gating defect in the retina, suggesting that Cryptochromes are required for appropriate temporal photoreception in mammals.     

Quantitative autoradiographic light- and electron microscopic studies on the retinohypothalamic connections in the rat

Summary Light microscopic autoradiography performed subsequent to intraocular injection of ³H-leucine revealed silver grains (SG) above axons of the optic tract which could be followed into the ventral and caudal portion of the suprachiasmatic nuclei (SCN) and above the contralateral anterior hypothalamic nucleus (AHN). By high resolution photometric measurement and computer processing the labelled areas were analysed, thus yielding statistical data of the relative grain distribution. The highest SG density was found in the ventrolateral part of both SCN (SCvl), confirming earlier reports concerning retinohypothalamic connections. That area exhibiting a cytoarchitecture different from the remaining nucleus was traversed, however, by numerous labelled axons. In the caudal part of both SCN a specific projection field of retinal fibres could be located. Here, almost no traversing fibres contribute to the rather circumscribed marked area. In the ventral part of the contralateral AHN, diffuse labelling well above background levels could be observed. Distinction between bypassing and terminating fibres within the SCvl could not be made using light microscopy. Analysis of SG distribution of the SCvl with electron microscopic autoradiography revealed a specific localization of SG within presynaptic terminals containing clear vesicles and pale mitochondria.     

Anterograde tracing of retinal axons in the avian embryo with low molecular weight derivatives of biotin

Several reactive biotin esters were injected into the eyes of chick and quail embryos at various stages of development. Four of the biotin esters reacted with molecules of the eye tissue and were detected with light and electron microscopy in fluorescein isothiocyanate and peroxidase-avidin incubated sections and whole mounts. Intra and extracellular components of the lens, the vitreous body, and the retina were labeled to different degrees. Three of the biotin esters (biotin-N-hydroxysuccinimidester, biotin-epsilon-aminocaproic acid-N-hydroxysuccinimidester, and desthiobiotin-N-hydroxysuccinimidester) prominently marked the optic fiber layer in the retina and the biotin labels were transported along the optic pathway. The tracers were detected up to the growth cone of axons 24 to 36 hr after injection. Explants from biotin marked retinas were cultured on collagen or basal laminae. During culturing axons grew out from these explants into the substratum showing that labeled tissue and nerve fibers were viable. The development of the optic pathway at the chiasma of quail embryos was studied using the biotin/avidin tracing. The bulk of fibers emerging from the retina crossed as shown by double labeling of both optic nerves in a complex pattern of segregated and interdigitizing axon bundles at the chiasma toward the contralateral side of the brain. From stage 25 onward a minor ipsilateral projection was found. At the same developmental stage a few fibers traveled into the contralateral optic nerve and grew retrogradely toward the contralateral eye. The percentage of specimens having this retino-retinal projection increased during development from 53% (stage 24 to 27; E3.5-E5.5) to 89% (stage 29 to 35; E6-E8) and declined to 40% at late embryogenesis (stage 37 to 41; E9-E12). The fact that all retinal axons were found within predictable pathways with some of them running in the wrong direction suggests that nerve fiber pathways provide accurate positional information, but at best weak directional information for growing nerve fibers.     

Axon routing at the optic chiasm after enzymatic removal of chondroitin sulfate in mouse embryos

The effects of removing chondroitin sulfate from chondroitin sulfate proteoglycan molecules on guidance of retinal ganglion cell axons at the optic chiasm were investigated in a brain slice preparation of mouse embryos of embryonic day 13 to 15. Slices were grown for 5 hours and growth of dye-labeled axons was traced through the chiasm. After continuous enzymatic digestion of the chondroitin sulfate proteoglycans with chondroitinase ABC, which removes the glycosaminoglycan chains, navigation of retinal axons was disrupted. At embryonic day 13, before the uncrossed projection forms in normal development, many axons deviated from their normal course, crossing the midline at aberrant positions and invading the ventral diencephalon. In slices from embryonic day 14 embryos, axons that would normally form the uncrossed projection at this stage failed to turn into the ipsilateral optic tract. In embryonic day 15 slices, enzyme treatment caused a reduction of the uncrossed projection that develops at this stage. Growth cones in enzyme-treated slices showed a significant increase in the size both before and after they crossed the midline. This indicates that responses of retinal axons to guidance signals at the chiasm have changed after removal of the chondroitin sulfate epitope. We concluded that the chondroitin sulfate moieties of the proteoglycans are involved in patterning the early phase of axonal growth across the midline and at a later stage controlling the axon divergence at the chiasm.     

Alterations in the Xenopus retinotectal projection by antibodies to Xenopus N-CAM

The patterned neural projection from the eye to the optic tectum of lower vertebrates (the retinotectal projection) has been proposed to be ordered by interactions between the optic nerve fibers and their surrounding tissues. To investigate the role of one such defined cell interaction, agarose implants containing antibodies to the neural cell adhesion molecule, N-CAM, were inserted into the tectum of the African clawed frog, Xenopus laevis. Both monoclonal and polyclonal antibodies against N-CAM reversibly and specifically distorted the pattern of the retinotectal projection, decreasing the precision of the projection as determined by electrophysiological techniques as well as decreasing the density of retinal innervation of the tectum and the branching of single axons as determined by horseradish peroxidase tracing. The anatomical effects became maximal at 4 to 6 days after implantation and returned to undetectable levels by 2 weeks, whereas the physiological effects became maximal by 8 to 10 days and a normal physiological map was reestablished within 4 weeks. The results are consistent with the hypothesis that anti-N-CAM antibodies perturb the ongoing growth and retraction of the terminal arbors of the optic nerve fibers, such that a region of the tectum becomes largely denuded of fibers. The physiological defects may then be a consequence both of the initial retraction of optic nerve terminals and of the rapid ingrowth of the perturbed and neighboring optic nerve fibers into the denuded region after the antibodies were cleared from the tectum. These results support the concept of a major role for N-CAM-mediated adhesion during map regeneration and maintenance.     

Retinal projections in the chain pickerel (Esox niger Lesueur)

Summary The retinal projections inEsox niger, as determined with the aid of a modified cobalt-lysine method, are considerably more extensive in the diencephalon and pretectum than in other teleost fishes so far examined. Although most retinal axons terminate contralaterally, rare fibers can be traced to the same aggregates ipsilaterally. The retinohypothalamic projection appears larger than hitherto reported in teleosts, and the dorsomedial optic tract issues fibers to a series of cell clusters extending from the rostral thalamus to mid-torus levels. A retinal projection to a presumed ventrolateral optic nucleus (VLO) is described for the first time in a teleost. Other targets of retinal fibers include the nucleus geniculatus lateralis ipse of Meader (GLI), the pretectal nucleus (P), the cortical nucleus and a well-developed ventromedial optic nucleus (VMO). The projection to the optic tectum is principally to the stratum fibrosum et griseum superficiale (SFGS) and stratum marginale (SM), but a considerable number of axons also course through the stratum album centrale (SAC) before terminating there or piercing the stratum griseum centrale (SGC) and terminating in SFGS. Rare terminal arborizations of retinal fibers were also observed in stratum griseum centrale (SGS) and in the stratum griseum periventriculare (SGC) in restricted portions of the tectum. Because of the relatively large size of the visual structures inE. niger it is a potentially useful model for future experimental studies on the visual system.     

Slit1 and Slit2 cooperate to prevent premature midline crossing of retinal axons in the mouse visual system.

During development, retinal ganglion cell (RGC) axons either cross or avoid the midline at the optic chiasm. In Drosophila, the Slit protein regulates midline axon crossing through repulsion. To determine the role of Slit proteins in RGC axon guidance, we disrupted Slit1 and Slit2, two of three known mouse Slit genes. Mice defective in either gene alone exhibited few RGC axon guidance defects, but in double mutant mice a large additional chiasm developed anterior to the true chiasm, many retinal axons projected into the contralateral optic nerve, and some extended ectopically-dorsal and lateral to the chiasm. Our results indicate that Slit proteins repel retinal axons in vivo and cooperate to establish a corridor through which the axons are channeled, thereby helping define the site in the ventral diencephalon where the optic chiasm forms.     

Characterization of the Circadian System in a Brazilian Species of Monkey (Callithrix jacchus): Immunohistochemical Analysis and Retinal Projections

The hypothalamic suprachiasmatic nucleus (SCN) and the thalamic pregeniculate nucleus (PGN), which appears to include the intergeniculate leaflet (IGL), comprise circadian related centers in the primate brain. In this study, these centers were analysed in respect to their cytoarchitecture, retinal afferents and chemical of major cells and axon terminals with tract tracers and immunohistochemical techniques to define cytoarchitecture and connections, in the common marmoset. The SCN was shown to be a triangularly shaped cluster of compact cells just dorsal to the optic chiasm and lateral to the third ventricle. It is innervated in its ventral portion by terminals from the retina, and NPY-ergic fibers. Serotonergic and SP-staining processes are distributed throughout. VIP-neurons form a dorsolateral group of cells and CB-immunoreactive neurons fill much of the nucleus. The PGN was shown to be a wedge-shaped cluster of cells located dorsomedially to the dorsal lateral geniculate nucleus. It appears to comprise a ventral portion which receives a bilateral retinal projection and contains NPY-neurons, suggesting that this portion may correspond to IGL. The PGN also contains CB-neurons, PV-neurons and fibers, and SP- and 5-HT-fibers. These results in marmoset show that, beside a common plan revealed for most mammals, there are significant interspecific variations in the circadian timing system. Future studies are needed in order to elucidate the circadian organization in this primate species.     

Characterization of the Circadian System in a Brazilian Species of Monkey (Callithrix jacchus): Immunohistochemical Analysis and Retinal Projections

The hypothalamic suprachiasmatic nucleus (SCN) and the thalamic pregeniculate nucleus (PGN), which appears to include the intergeniculate leaflet (IGL), comprise circadian related centers in the primate brain. In this study, these centers were analysed in respect to their cytoarchitecture, retinal afferents and chemical of major cells and axon terminals with tract tracers and immunohistochemical techniques to define cytoarchitecture and connections, in the common marmoset. The SCN was shown to be a triangularly shaped cluster of compact cells just dorsal to the optic chiasm and lateral to the third ventricle. It is innervated in its ventral portion by terminals from the retina, and NPY-ergic fibers. Serotonergic and SP-staining processes are distributed throughout. VIP-neurons form a dorsolateral group of cells and CB-immunoreactive neurons fill much of the nucleus. The PGN was shown to be a wedge-shaped cluster of cells located dorsomedially to the dorsal lateral geniculate nucleus. It appears to comprise a ventral portion which receives a bilateral retinal projection and contains NPY-neurons, suggesting that this portion may correspond to IGL. The PGN also contains CB-neurons, PV-neurons and fibers, and SP- and 5-HT-fibers. These results in marmoset show that, beside a common plan revealed for most mammals, there are significant interspecific variations in the circadian timing system. Future studies are needed in order to elucidate the circadian organization in this primate species.     

Retinofugal projections of the big brown bat, Eptesicus fuscus and the neotropical fruit bat, Artibeus jamaicensis

Retinal connections were studied in Eptesicus fuscus and Artibeus jamaicensis using anterograde axonal degeneration and autoradiographic techniques following unilateral enucleations and uniocular injections of radioactive amino acids. Although each retina projected bilaterally to the brainstem, the number of silver grains in the emulsion of autoradiographs indicated that nearly all fibers in the optic nerve entered the contralateral optic tract. Ipsilaterally, a major portion of the projection ended in the suprachiasmatic nucleus; caudal to the suprachiasmatic nucleus, the amount of label was so small that individual silver grains were counted to determine the location and quantity of label in other ipsilateral nuclei. In both species the retinal projection terminated bilaterally in the suprachiasmatic, dorsal lateral geniculate, ventral lateral geniculate, and pretectal olivary nuclei and contralaterally in the posterior pretectal nucleus, superficial gray layers of the superior colliculus, and nuclei of the accessory optic system. In Eptesicus the projection to the nucleus of the optic tract ended contralaterally, and in Artibeus it ended in this nucleus bilaterally. The results of this study revealed a basic theme in the optic projection of the two ecologically different microchiropterans. The results differed, however, in that the projection was larger and visually related nuclei were better developed in Artibeus. Such variations are presumed to relate to eye size and the relative use of vision by the two chiropterans.