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.     

Retinohypothalamic pathway in the duck (Anas platyrhynchos)

Summary Retinohypothalamic connections were studied in the duck after unilateral optic nerve transection using both light and electron microscopic techniques. Degenerated endings of optic fibers were found only in a circumscribed part of the anterior hypothalamic area, i.e. the ventral region of the contralateral suprachiasmatic nucleus. Images of degenerating boutons were observed in frozen sections (method according to Johnstone-Bowsher), and their presence confirmed by electron microscopic examination. These degenerating boutons make synaptic contacts with dendrites or dendritic spines of neurons of the suprachiasmatic nucleus.In the same material, the decussation of the optic chiasma was studied with the light microscope. Uncrossed retinal fibers were found in the marginal optic tract, the basal optic root and occasionally also in the isthmo-optic tract.Dedicated to Professor Dr. W. Bargmann on the occasion of his 70th birthdaySupported by the DGRST and European Training Program Brain and Behaviour ResearchI wish to express my gratitude to Professor Andreas Oksche, who repeatedly offered me the scientific facilities at the Department of Anatomy of the University of Giessen, and who provided me with valuable neuroanatomical suggestions throughout the progress of these studies.     

Spatio-temporal evolution of optic tract regeneration in Rutilus rutilus

Regeneration of the different components of the optic tract of Rutilus takes place at a variable rate and follows a relatively precise pattern. The first optic centres to be reinnervated belong to the lateral thalamo-pretectal group (5 weeks at 14 degrees C after section of the optic nerve), followed by the anterior optic tectum and lateral geniculate nucleus (8 weeks after section), the central regions of the tectum, the suprachiasmatic nucleus and the nucleus of the basal optic root (10-15 weeks after section), and finally the medial thalamo-pretectal nuclei and the caudal regions of the optic tectum (16-25 weeks after section).     

An autoradiographic study of the retinal projections in the Formosan monkey

This study investigated the retinal projections of the adult Formosan rock monkey by monocular injection of radioactive proline and fucose. We found that the retinofugal fibers terminated bilaterally in the suprachiasmatic, pregeniculate, lateral geniculate, pretectal complex, pulvinar nucleus, superior colliculus, dorsal and lateral terminal nuclei of the accessory optic system. More crossed retinal terminations were observed, with the exception that the suprachiasmatic nucleus received almost equally of both retinal projections. The existence of the retinal projection to the medial terminal nucleus of the accessory nucleus was in doubt. In the geniculate nucleus, the retinal fibers terminated contralaterally in layers 1, 4 and 6; and ipsilaterally in 2, 3 and 5. In the superior colliculus, most retinal fibers were aggregated superficially in a band located in the contralateral striatum griseum superficialis of the superior colliculus, and had few gaps on the ipsilateral one. The present investigation shows that the Formosan rock monkey has a similar pattern of optic fiber distribution to that of other macaques.     

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.     

Catecholaminergic innervation of the suprachiasmatic nucleus in the adult rat: ultrastructural relationships with neurons containing vasoactive intestinal peptide or vasopressin

Catecholaminergic fibers in the suprachiasmatic nucleus of adult rats were investigated by use of light- and electron-microscopic immunocytochemistry. The suprachiasmatic nucleus receives a modest density of tyrosine hydroxylase-containing axons, homogeneously distributed in the nucleus and forming varicosities throughout its entire rostro-caudal extension. Immunolabeling with antibodies against dopamine showed that this catecholamine input comprises a dopaminergic component. Many tyrosine hydroxylase-positive cells were localized at the immediate periphery of the suprachiasmatic nucleus. With electron-microscopic examination, dendrites of these neurons were found within the limits of the nucleus as well as at a border zone between the suprachiasmatic nucleus proper and the optic tract where they received unlabeled synapses, providing a morphological support for a possible role of dopaminergic neurons in the integration and/or transfer of light-related signals. More than 91% of catecholaminergic axonal varicosities were found to establish morphologically defined synapses with dendrites. To investigate whether these synapses might be shared with neurons of one or both of the two main peptidergic populations of the nucleus, namely vasoactive intestinal peptide- and vasopressin-containing neurons, we carried out doublelabeling experments combining immunoperoxidase and immunogold-silver labeling. Results showed only a few cases of direct association of the catecholaminergic terminals with these peptidergic categories. In both types of dually stained sections, catecholaminergic synapses were preferentially made with unlabeled dendrites. The homogeneous distribution of tyrosine hydroxylase-immunoreactive fibers in the suprachiasmatic nucleus could therefore reflect a lack of significant catecholaminergic innervation of both vasoactive intestinal peptide- and vasopressin-synthesizing neurons.     

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     

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.     

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.     

Distribution and ultrastructure of pyramidal tract endings in the cat rhombencephalon

The distribution and ultrastructure of terminals of corticofugal fibers in the cat rhombencephalon were investigated under the optical and electron microscopes at different periods (2–6 days) of experimental degeneration evoked by destruction of the sensomotor cortex. It was shown by the Fink–Heimer method that most degenerating fibers are distributed in the reticular nuclei of the pons and medulla. Massive degeneration of corticofugal fibers also was observed in the nuclei of the dorsal columns (nuclei of Goll and Burdach). Most of the degenerating (the "pale" type of degeneration) axo-dendritic and axo-somatic synapses in the gigantocellular reticular nucleus and the nucleus of Goll retained spherical vesicles. Small endings were found on the branches of the dendrites in which degenerative changes were of the "dark" type. The topography of the degenerating elements and axo-axonal synapses was studied in large areas of sections by the coordinate grid method. The dimensions of most degenerating axons in the gigantocellular reticular nucleus were greater (1.5 µ) than those of the degenerating axons (0.5 µ) in the nucleus of Goll. Most endings of pyramidal fibers and axo-axonal synapses are located in the central part of the nucleus of Goll at a depth of 0.5–1.2 mm from the brain surface. The results are discussed in connection with electrophysiological studies of the mechanisms of cortical control over unit activity of the reticular formation of the brain stem and nuclei of the dorsal columns.     

Visualization of the retino-hypothalamic projection in the rat by cobalt precipitation

Summary The technique of cobalt sulfide precipitation combined with Timm's sulfide-silver method for intensification of heavy metals was used to delineate the retino-hypothalamic projection of the rat. Freshly isolated rat brains were dissected and a solution of cobaltous chloride was applied to one of the cut optic nerves. Sixteen hours later, after cobalt ions had passed into the brain along the entire length of the optic fibers, the preparation was treated with ammonium sulfide to precipitate the cobalt as cobalt sulfide. In thick light microscopic sections, cobalt-filled axons were visualized as black fibers against a light gold background. Such fibers were observed to leave the posterior medial portion of the optic chiasm and, after arching dorsally, to project into the posterior fifth of the suprachiasmatic nucleus (SCN), as well as into the rostral part of the arcuate nucleus. Neither bifurcation of these axons nor looping of the axons back to the chiasm was seen. Most fibers projected to the SCN contralateral to the filled nerve, but the projection represented less than 0.1 % of the total number of fibers in one optic nerve. These observations are considered to be graphic evidence of a retino-hypothalamic projection. The interpretation of the cobalt method is discussed, as are the functions of the connections that have been observed.This work was supported by the Nuffield FoundationWe are grateful to Mr. Clifford Jeal of the Department of Pathology for excellent advice on photomicrography     

The source of transitory innervation of suprachiasmatic nucleus by tyrosine hydroxylase-immunoreactive fibers during the postnatal period in rats

Suprachiasmatic nucleus in the rats during early postnatal development is transitorily innervated by tyrosine hydroxylase-immunoreactive fibers that are neither catecholamine- nor serotoninergic. The goal of this immunocytochemical investigation was to find out if tyrosine hydroxylase-immunoreactive neurons of anterior hypothalamus could be the source of this innervation. According to the obtained immunocytochemical data, multiple multipolar tyrosine hydroxylase-immunoreactive neurons are localized around the suprachiasmatic nucleus in the rats at days 2 and 10 of postnatal development. Most of them were observed ventrally and laterally to the nucleus. The axons of the neurons are oriented towards the suprachiasmatic nucleus. Further investigation demonstrated considerably decreased number of tyrosine hydroxylase-immunoreactive neurons surrounding the suprachiasmatic nucleus in the adult animals as compared to early postnatal period, which correlates to the number of tyrosine hydroxylase-immunoreactive fibers in this nucleus. Hence, tyrosine hydroxylase-immunoreactive neurons in the ventral region of anterior hypothalamus can be considered as a potential source of transitory innervation of suprachiasmatic nucleus by tyrosine hydroxylase-immunoreactive fibers during early postnatal development.     

Topography of retinal axons in the diencephalon of goldfish

Summary The projections of horseradish peroxidase-filled axons from each quadrant of the retina were studied to determine whether retinal projections of goldfish are topographically organized in diencephalic target nuclei. A distinct topography of the dorsal, nasal, ventral and temporal retina exists in the lateral geniculate nucleus and in the dorsolateral optic nucleus of the thalamus. The projections of retinal quadrants show minimal spatial overlap in each of these nuclei. The suprachiasmatic nucleus of the hypothalamus is extensively innervated by ventral retinal fibers, whereas the nucleus is sparsely innervated by fibers from the other three retinal quadrants. A rudimentary topography also exists in the pretectum where the dorsal pretectal area receives projections primarily from the ventral retina and the ventral pretectal area receives projections mostly from the dorsal retina. These data show that retinal projections to some diencephalic nuclei are topographically organized.This work was supported by Research Grant EY-01426 to S.C.S.     

Cholinergic and catecholaminergic neurons relay striatal information to the optic tectum in amphibians.

In the amphibians Rana perezi and Xenopus laevis, the involvement of cholinergic and catecholaminergic neurons in the relay of basal ganglia inputs to the tectum was investigated. Tract-tracing experiments, in which anterograde tracers were applied to the basal ganglia and retrograde tracers to the optic tectum, were combined with immunohistochemistry for choline acetyltransferase and tyrosine hydroxylase. The results of these experiments suggest that dopaminergic neurons of the suprachiasmatic nucleus and pretectal region, noradrenergic cells of the locus coeruleus and the cholinergic neurons of the pedunculopontine and laterodorsal tegmental nuclei mediate at least part of the basal ganglia input to the tectum in anurans.     

Retinohypothalamic projections and immunocytochemical analysis of the suprachiasmatic region of the desert iguana Dipsosaurus dorsalis

Two separate and distinct retinal projections to the hypothalamus in the iguanid lizard Dipsosaurus dorsalis were described using horseradish peroxidase and cobalt-filling techniques. Both of the projections were unilateral and completely crossed; one terminated in the supraoptic nucleus and the other in the suprachiasmatic nucleus. Immunocytochemical analysis showed that the supraoptic nucleus contained cell bodies and fibers that cross-react with antibodies raised against arginine vasopressin, while the suprachiasmatic nucleus contained arginine vasopressin-like immunoreactive fibers emanating from cells in the nearby paraventricular nucleus. The suprachasmatic nucleus contained a dense plexus of fibers that cross-reacted with neuropeptide-Y antibody. Antiserum against vasoactive intestinal polypeptide showed no reactivity in any part of the forebrain, while antiserum against serotonin showed sparse and uniform reactivity throughout the forebrain, including the suprachiasmatic nucleus. These results, together with other data, indicate that the suprachiasmatic nucleus of D. dorsalis is homologous to the suprachiasmatic nuclei of rodents, structures known to contain circadian pacemakers. We suggest that the suprachiasmatic nucleus may play a similar role in the circadian system of D. dorsalis.     

The Source of Transitory Innervation of Suprachiasmatic Nucleus by Tyrosine Hydroxylase-Immunoreactive Fibers during Postnatal Period in Rats

Suprachiasmatic nucleus in the rats during early postnatal development is transitorily innervated by tyrosine hydroxylase-immunoreactive fibers that are neither catecholamine- nor serotoninergic. The goal of this immunocytochemical investigation was to find out if tyrosine hydroxylase-immunoreactive neurons of anterior hypothalamus could be the source of this innervation. According to the obtained immunocytochemical data, multiple multipolar tyrosine hydroxylase-immunoreactive neurons are localized around the suprachiasmatic nucleus in the rats at days 2 and 10 of postnatal development. Most of them were observed ventrally and laterally to the nucleus. The axons of the neurons are oriented towards the suprachiasmatic nucleus. Further investigation demonstrated considerably decreased number of tyrosine hydroxylase-immunoreactive neurons surrounding the suprachiasmatic nucleus in the adult animals as compared to early postnatal period, which correlates to the number of tyrosine hydroxylase-immunoreactive fibers in this nucleus. Hence, tyrosine hydroxylase-immunoreactive neurons in the ventral region of anterior hypothalamus can be considered as a potential source of transitory innervation of suprachiasmatic nucleus by tyrosine hydroxylase-immunoreactive fibers during early postnatal development.     

Synapses of optic nerve afferents in the rat suprachiasmatic nucleus

Summary The identification of optic synapses in the rat suprachiasmatic nucleus (Güldner, 1978) has made it possible to study them morphometrically. The measurements followed the check-list introduced by Palay and Chan-Palay (1976). There are several items which could usefully be added to this list. The structure of essential synaptic components varies considerably in what is apparently one synaptic population based on morphological criteria. The possible reasons for the variable sizes of the optic boutons containing different amounts of clear and dense core vesicles are discussed in terms of different activities or metabolic states of the individual boutons and/or different metabolic states of neuronal and glial elements in their vicinity. The active zones of optic synapses are also extremely variable. One optic bouton can form several active zones of very different sizes, or form Gray-type-I (asymmetric), Gray-type-II (symmetric) and intermediate contacts at the same time. The function and/or functional efficiency of a single optic bouton therefore could then be quite different with respect to its various postsynaptic elements. The different appearance of the active zones is discussed mainly in terms of possible regulative influences from neighboring synapses via the postsynaptic neuron.The author wishes to thank Mrs. Bassirat Pirouzmandi for her excellent technical assistance     

The serotonin-immunoreactive system of the suprachiasmatic nucleus in the hibernating ground squirrel,Spermophilus richardsonii

Summary In the suprachiasmatic nucleus (NSC) of hibernating and non-hibernating ground squirrels, the distribution of serotonin-immunoreactive (5HT-IR) fibers was studied by the use of the peroxidase-antiperoxidase technique. The cytology of perikarya giving rise to these suprachiasmatic 5HT-IR fibers was investigated in the anterior raphe nuclei. Differences in the immunoreactivity of suprachiasmatic fibers between hibernating and non-hibernating ground squirrels were determined by digital image analysis. The cellular activity was determined densitometrically after RNA-staining in anterior raphe neurons and suprachiasmatic perikarya. Abundant 5HT-IR fibers were observed in the medial and ventromedial portions of the NSC. Frequently, the fibers were found in close contact with perikarya of suprachiasmatic neurons. The central portion of the nucleus and the surrounding hypothalamic areas contained only a few scattered 5HT-IR fibers. Inside the raphe nuclei, 5HT-IR fibers and perikarya formed a dense network. In hibernating ground squirrels, the immunoreactivity to serotonin was approximately 45% higher than in non-hibernating controls. This difference is in accordance with signs of higher neuronal activity (40% higher RNA-content, 20% larger cell nuclei) in 5HT-IR perikarya of the raphe nucleus and the persisting activity of the NSC during hibernation; the activity of other brain regions dropped conspicuously in torpid animals.Supported by the Deutsche Forschungsgemeinschaft (Nu 36/2-1)     

Terminal distribution of retinal fibers in the tegu lizard (Tupinambis nigropunctatus)

Summary The retinal projections in the tegu lizard were traced using degeneration-silver methods. Bilateral projections were found to the dorsolateral geniculate and the posterodorsal nuclei. Unilateral, crossed projections were traced to the suprachiasmatic nucleus, the ventrolateral geniculate nucleus, the mesencephalic lentiform nucleus, nucleus geniculatus praetectalis, the ectomammillary nucleus, and the optic tectum. Some of these connections are distinctly different from those reported in other reptiles and suggest that important interspecific variations occur among reptiles.     

Projections of the optic tectum and the mesencephalic nucleus of the trigeminal nerve in the tegu lizard (Tupinambis nigropunctatus)

Summary Fibers undergoing Wallerian degeneration following tectal lesions were demonstrated with the Nauta and Fink-Heimer methods and traced to their termination. Four of the five distinct fiber paths originating in the optic tectum appear related to vision, while one is related to the mesencephalic nucleus of the trigeminus. The latter component of the tectal efferents distributes fibers to 1) the main sensory nucleus of the trigeminus, 2) the motor nucleus of the trigeminus, 3) the nucleus of tractus solitarius, and 4) the intermediate gray of the cervical spinal cord.The principal ascending bundle projects to the nucleus rotundus, three components of the ventral geniculate nucleus and the nucleus ventromedialis anterior ipsilaterally, before it crosses in the supraoptic commissure and terminates in the contralateral nucleus rotundus, ventral geniculate nucleus and a hitherto unnamed region dorsal to the nucleus of the posterior accessory optic tract.Fibers leaving the tectum dorso-medially terminate in the posterodorsal nucleus ipsilaterally and the stratum griseum periventriculare of the contralateral tectum. The descending fiber paths terminate in medial reticular cell groups and the rostral spinal cord contralaterally and in the torus and the lateral reticular regions ipsilaterally. The ipsilateral fascicle also issues fibers to the magnocellular nucleus isthmi.