Nonvisual photoreceptors of the deep brain, pineal organs and retina.

The role of the nonvisual photoreception is to synchronise periodic functions of living organisms to the environmental light periods in order to help survival of various species in different biotopes. In vertebrates, the so-called deep brain (septal and hypothalamic) photoreceptors, the pineal organs (pineal- and parapineal organs, frontal- and parietal eye) and the retina (of the "lateral" eye) are involved in the light-based entrain of endogenous circadian clocks present in various organs. In humans, photoperiodicity was studied in connection with sleep disturbances in shift work, seasonal depression, and in jet-lag of transmeridional travellers. In the present review, experimental and molecular aspects are discussed, focusing on the histological and histochemical basis of the function of nonvisual photoreceptors. We also offer a view about functional changes of these photoreceptors during pre- and postnatal development as well as about its possible evolution. Our scope in some points is different from the generally accepted views on the nonvisual photoreceptive systems. The deep brain photoreceptors are hypothalamic and septal nuclei of the periventricular cerebrospinal fluid (CSF)-contacting neuronal system. Already present in the lancelet and representing the most ancient type of vertebrate nerve cells ("protoneurons"), CSF-contacting neurons are sensory-type cells sitting in the wall of the brain ventricles that send a ciliated dendritic process into the CSF. Various opsins and other members of the phototransduction cascade have been demonstrated in telencephalic and hypothalamic groups of these neurons. In all species examined so far, deep brain photoreceptors play a role in the circadian and circannual regulation of periodic functions. Mainly called pineal "glands" in the last decades, the pineal organs actually represent a differentiated form of encephalic photoreceptors. Supposed to be intra- and extracranially outgrown groups of deep brain photoreceptors, pineal organs also contain neurons and glial elements. Extracranial pineal organs of submammalians are cone-dominated photoreceptors sensitive to different wavelengths of light, while intracranial pineal organs predominantly contain rod-like photoreceptor cells and thus scotopic light receptors. Vitamin B-based light-sensitive cryptochromes localized immunocytochemically in some pineal cells may take part in both the photoreception and the pacemaker function of the pineal organ. In spite of expressing phototransduction cascade molecules and forming outer segment-like cilia in some species, the mammalian pineal is considered by most of the authors as a light-insensitive organ. Expression of phototransduction cascade molecules, predominantly in young animals, is a photoreceptor-like characteristic of pinealocytes in higher vertebrates that may contribute to a light-percepting task in the perinatal entrainment of rhythmic functions. In adult mammals, adrenergic nerves--mediating daily fluctuation of sympathetic activity rather than retinal light information as generally supposed--may sustain circadian periodicity already entrained by light perinatally. Altogether three phases were supposed to exist in pineal entrainment of internal pacemakers: an embryological synchronization by light and in viviparous vertebrates by maternal effects (1); a light-based, postnatal entrainment (2); and in adults, a maintenance of periodicity by daily sympathetic rhythm of the hypothalamus. In addition to its visual function, the lateral eye retina performs a nonvisual task. Nonvisual retinal light perception primarily entrains genetically-determined periodicity, such as rod-cone dominance, EEG rhythms or retinomotor movements. It also influences the suprachiasmatic nucleus, the primary pacemaker of the brain. As neither rods nor cones seem to represent the nonvisual retinal photoreceptors, the presence of additional photoreceptors has been supposed. Cryptochrome 1, a photosensitive molecule identified in retinal nerve cells and in a subpopulation of retinal photoreceptors, is a good candidate for the nonvisual photoreceptor molecule as well as for a member of pacemaker molecules in the retina. When comparing various visual and nonvisual photoreceptors, transitory, "semi visual" (directional) light-perceptive cells can be detected among them, such as those in the parietal eye of reptiles. Measuring diffuse light intensity of the environment, semivisual photoreceptors also possess some directional light perceptive capacity aided by complementary lens-like structures, and screening pigment cells. Semivisual photoreception in aquatic animals may serve for identifying environmental areas of suitable illumination, or in poikilotermic terrestrial species for measuring direct solar irradiation for thermoregulation. As directional photoreceptors were identified among nonvisual light perceptive cells in the lancelet, but eyes are lacking, an early appearance of semivisual function, prior to a visual one (nonvisual --> semivisual --> visual?) in the vertebrate evolution was supposed.

The system of cerebrospinal fluid-contacting neurons.

Cerebrospinal fluid (CSF)-contacting neurons are located periventricularly or inside the brain ventricles; they contact the CSF via their dendrites, perikarya or axons. Most of the CSF-contacting nerve cells send dendritic processes into the ventricular cavity where they form ciliated terminals. These ciliated dendritic endings resemble those of known sensory cells, yet their role is still unknown. There are two types of CSF-contacting dendritic terminals. One bears solitary 9 X 2 + 0 cilia; it is present in different hypothalamic regions such as the paraventricular organ and the vascular sac. The magnocellular neurosecretory nuclei also contain CSF-contacting neurons, which probably furnish information about the parameters of the CSF for the regulatory function of the hypothalamo-hypophyseal system. CSF-contacting nerve cells of the parvocellular hypothalamic nuclei are suspected to participate in hypothalamo-adenohypophyseal regulation. A second type of CSF-contacting dendritic terminal bears many stereocilia and is found in the central canal of the spinal cord. This type of terminal is also supplied with a 9 X 2 + 2 kinocilium that may contact Reissner's fiber, the secretory material of the subcommissural organ. Resembling mainly mechanoreceptors, these spinal CSF-contacting neurons appear to form axon terminals of the neurosecretory type at the external circumference of the spinal cord. Developing and/or regressing photoreceptor cells of the retina and pineal complex may display a similar dendritic structure characteristic of hypothalamic CSF-contacting neurons. Axons penetrating into the ventricles innervate the apical surface of the ependyma and/or the CSF-contacting dendritic terminals. Some bipolar neurons of the retina form so-called Landolt's clubs; these may be considered as the retinal component of the CSF-contacting neuronal system. Since in the lancelet nearly all nerve cells contact the CSF, the CSF-contacting neurons represent a specialized, but phylogenetically old cell type, a "protoneuron" in the vertebrate brain. They may be derived phylogenetically by inversion of the ciliated neurons found in the plate-like nervous system of more primitive deuterostomians.

Cerebrospinal fluid-contacting neurons, sensory pinealocytes and Landolt's clubs of the retina as revealed by means of an electron-microscopic immunoreaction against opsin.

Opsin-immunoreactive sites of hypothalamic cerebrospinal fluid (CSF)-contacting neurons, pinealocytes and retinal cells were studied in various vertebrates (Carassius auratus, Phoxinus phoxinus, Triturus cristatus, Bombina bombina, Rana esculenta) by means of postembedding immuno-electron microscopy with the use of the protein A-gold labeling method. The retina of the rat served as a general reference tissue for the quality of the immunocytochemical reaction. A strong opsin immunoreaction (rat-antibovine opsin serum) was obtained in the rod-type outer segments of photoreceptors in the retina of all species studied. Cone-type outer segments exhibited only very few antigenic binding sites. In the pineal organ of the goldfish and the frog, outer segments of the photoreceptor cells displayed strong immunoreactivity. No immunoreaction was found in hypothalamic CSF-contacting neurons and Landolt's clubs of nerve cells of the bipolar layer of the retina. The morphological similarity between the ciliated dendritic terminal of the Landolt's club and the intraventricular dendritic ending of the CSF-contacting neurons is emphasized.

Ciliated perikarya, "peptidergic" synapses and supraependymal structures in the guinea pig hypothalamus.

There are numerous nerve cells giving rise to solitary cilia of type 9 x 2 + 0, in the hypothalamic areas studied (medial preoptic nucleus, supraoptic and paraventricular nuclei, anterior periventricular nucleus, wavy paraventricular ependyma, and infundibular wall), further in the precentral gyrus and cerebellar flocculus. In the hypothalamic nuclei, the perikarya contained granular vesicles of varying sizes (800 A to 1800 A in diameter). In the supraoptic nucleus, a second neuron type was described among the lateral optic fibres. These nerve cells differ from the main neurosecretory ones by the size of their granular vesicles and their high number of large axo-somatic synapses formed by myelinated axons. In the paraventricular nucleus, axons may terminate on the basal lamina of vessels. A subependymal neuron group was described near the wavy paraventricular ependyma. The subependymal hypothalamic neuropil is characterized by various kinds of synapses including apparent "peptidergic" ones, and by axo-glial synaptic connections. The latter are also present on the apica surface of the ependyma in various regions of the 3rd ventricle. In addition, intraventricular structures (dendrites, axons, neuronal and non-neuronal perikarya, synapses) were studied by scanning (SEM) and transmission electron microscopy (TEM).

Comparison of the pineal complex, retina and cerebrospinal fluid contacting neurons by immunocytochemical antirhodopsin reaction.

The presence of rhodopsin was investigated by an indirect immunocytochemical method in the pineal complex of various vertebrates (Carassius auratus, Cyprinus carpio, Hypophthalamichthys molitrix, Lucioperca lucioperca, Triturus vulgaris, Bombina bombina, Rana esculenta, Pseudemys scripta elegans, Lacerta agilis et viridis, white leghorn chickens, rat), in the retina of Lebistes reticulatus, Lucioperca lucioperca, Rana esculenta, Lacerta agilis, Pseudemys scripta elegans, the chicken and the rat, and the cerebrospinal (CSF) contacting neurons of Triturus vulgaris. The outer segments of the photoreceptor terminals of the pineal organ, frontal organ, parapineal organ of lower vertebrates and of the retina of the species investigated, were intensely stained with the antirhodopsin reaction. There was no significant positivity in the pineal organ of the reptiles, the chicken and the rat, and the parietal eye of the lizards. We failed to demonstrate any immunoreactive staining in the CSF contacting neurons of various hypothalamic areas and of the spinal cord. The light microscopic immunocytochemical results seem to contradict a photoreceptive role of the CSF contacting neurons and strengthen the view that the receptory cells of the pineal complex of lower vertebrates are involved in light perception by means of the visual pigment rhodopsin.

A comparison of epithalamic, hypothalamic and spinal neurosecretory terminals.

Nerve endings of epithalamic, hypothalamic and spinal neurosecretory areas were studied by light and electron microscopy in various vertebrates (from fishes up to mammals) including the lancelet. Areas investigated were the pineal organ, the pulvinar corporis pinealis, the neurohypophysis, the median eminence, the urophysis, the terminal filum and the medullo-spinal neurosecretory zones. We found that in all these areas the neurosecretory endings have common structures, which we call synaptic hemidesmosomes or neurohormonal terminals. These are characterized by accumulation of vesicles, and dense projections in a terminal on the basal lamina of the surface of the nervous tissue. A critical review of the literature suggests that a considerble neuroendocrine activity is associated with synaptic hemidesmosomes as special neurohormonal effector structures of the nerve cells. The cell-to-cell synapses formed by neurosecretory cells are discussed in connection with the dual capacity of these cells to function as both endocrine and "ordinary# neuronal elements. The importance of the external cerebrospinal fluid (CSF) space for the transport of materials released in the so-called neurohemal areas, is stressed.

Scanning and transmission electron microscopy of intraventricular dendrite terminals of hypothalamic cerebrospinal fluid contacting neurons in Triturus vulgaris.

A scanning (SEM) and transmission electron microscopic (TEM) study of the ventricular wall of the hypothalamus of Triturus vulgaris was performed with special regard to the intraventricular dendrite terminals of the cerebrospinal fluid (CSF) contacting neurons of the preoptic area (magnocellular and parvocellular preoptic nuclei), the infundibular lobe (anterior periventricular nucleus, infundibular nucleus), and the paraventricular organ. In the preoptic area and infundibular lobe, the terminals were knob-like or club-shaped, of various sizes (diameter about 0,5 to 3,0 micrometer) and located immediately above the ependyma. Ultrastructurally, they may contain dense-core vesicles of varying sizes. The CSF contacting dendrite endings of the paraventricular organ built up a supraependymal labyrinthic layer which could be divided into a rostral crest-like part and a caudal flat and broad division. In both parts, three main types of terminals of various size and shape could be distinguished: a) ramifying, b) elongated, and c) bulb-like dendrite endings which also differed by their TEM structure. The bulk-like terminals, first of all the small ones, originated from the distal part of the nucleus of the organ (nucleus organi paraventricularis) while the other two types took their origin from its intra- and subependymal part. In all areas investigated, each intraventricular dendrite ending gave rise to a solitary cilium (type 9 X 2 + 0). It differed from the ependymal kinocilia by both SEM and TEM characteristics. In the paraventricular organ, the neuronal cilia were hidden inside, or below the supraependymal layer of terminals. There were intraventricular axons which formed synapses on CSF contacting dendrite endings of both parts of the paraventricular organ. Free intraventricular neurons, further ependymal areas heavily or scarcely ciliated, were described. The CSF contacting dendrite terminals were predominantly present near ventricular recesses and in regions where the ependyma was scarcely ciliated.

Ciliated neurons and different types of synapses in anterior hypothalamic nuclei of reptiles.

The magnocellular paraventricular and supraoptic nuclei and the parvocellular preoptic and periventricular nuclei have been studied by light and electron microscopy in Emys orbicularis, Lacerta agilis and Elaphe longissima. The ultrastructure of cerebrospinal fluid (CSF)-contacting neurons was described in the preoptic and periventricular nuclei of Emys and Lacerta species. Single 9 X 2 + 0 cilia similar to those of the CSF-contacting dendritic terminals were found on perikarya of non CSF-contacting nerve cells, in all four investigated nuclei. The cilia project from funnel-like invaginations of the perikarya into the intercellular space. In the neurons of the nuclei studied, granular vesicles were found, their size being mainly 1,600 A in the paraventricular nucleus, about 1,800 A in the supraoptic nucleus, 1,100 A in the periventricular nucleus and 800 A, or up to 1,250 A in the preoptic nucleus. In general, the neurons possess synapses of the axo-somatic, axo-somatic spine, axo-dendritic and axo-dendritic spine types. In the supraoptic nucleus, multiple interdigitated synapses were observed. Presynaptically, eif different sizes (600 to 800 A, about 1,100 A, 1250 A, and up to 2,000 A) were found. It is discussed whether the above described 9 X 2 + 0 cilia may represent some kind of hypothalamic sensory structure that earlier physiological studies postulated to exist. The ciliated hypothalamic perikarya are considered by the authors to be a more differentiated form of the CSF-contacting neurons. The different types of synapses indicate multilateral connections of the nerve cells of the nuclei studied.

Cerebrospinal fluid-contacting neurons, ciliated perikarya and "peptidergic" synapses in the magnocellular preoptic nucleus of teleostean fishes.

The magnocellular preoptic nucleus of fishes (Anguilla anguilla, Amiurus nebulosus, Cyprinus carpio, Carassius auratus, Ctenopharyngodon idella, Cichlasoma nigrofasciatum) has been studied by light and electron microscopy. Two kinds of neurons Were found: a) large, electron-dense, Gomori-positive cells with moderate acetylcholinesterase (AChE) positivity which contain granulated vesicles of 1400 to 2200 A (in average 1600 to 1800 A), adn b) small, strongly AChE-positive, electron-lucent neurons containing granulated vesicles of 900 to 1200 A. The nerve cells are supplied with axo-somatic and axo-dendritic synapses. These are formed by axon terminals containing either 1. synaptic vesicles of 500 A, or 2. synaptic vesicles of 500 A and dense-core vesicles of 600 to 800 A, or 3. synaptic vesicles of 600 A and granulated vesicles of up to 1100 A, or 4. synaptic vesicles of about 400 A and granulated vesicles of up to 1800 A. The presence of "peptidergic" and numerous other synapses shows the complexity of the organization and afferentation of the magnocellular preoptic nucleus. In the eel, both types of nerve cells form dendritic terminals within the cerebrospinal fluid (CSF). These CSF contacting dendrites are supplied with 9 x 2 + 0 cilia. In the other species investigated, only some large neurons build up intraventricular endings. The ependymofugal process of the CSF contacting neurons enters the preoptic-neurohypophysial tract. Perikarya of both the large and the small cells may give rise to single, paired or multiple 9 x 2 + 0 cilia extending into the intercellular space. The number of CSF contacting neurons is reciprocal to the number of perikarya with intercellular cilium. These latter cells may represent modified, more differentiated forms of the CSF contacting neurons. We think that atypical cilia protruding into the intercellular space may have the same significance for the intercellular fluid as the cilia of the intraventricular dendrites of the CSF contacting neurons for the CSF.

Comparative ultrastructure of cerebrospinal fluid-contacting neurons and pinealocytes.

The pinealocytes of fishes, amphibians, reptiles, birds and mammals have been compared with cerebrospinal fluid (CSF) contacting neurons. We found that the intraventricular dendrite terminal of the latter resembles the pinealocytic inner segment and that the atypical cilium (9x2+0 tubules) of the CSF contacting neurons is analogous with the outer segment of the pinealocytes, even though the outer segment bears photoreceptor lamellae in lower vertebrates. Regular, but small-sized photoreceptor outer segments were also found on pinealocytes of the chicken. In mammals, too, primitive outer segments are present in the form of 9x2 to cilia similar to those of CSF contacting dendritic terminals. In the Golgi areas of the perikarya of both cell types there are granulated vesicles which may contain transmitter substances and/or neurohormones. The synaptic junctions of the pinealocytes differ from those in the CSF contacting neurons. Many synapses occur on the latter, but they appear only rarely on pinealocytes. The axons of the CSF contacting neurons form synaptic connections with other cells, or terminate as neurohormonal synaptic hemidesmosomes on the basal lamina of the brain surface. The pinealocyte axons give rise to terminals containing synaptic ribbons. Such ribbons do not occur in CSF contacting neurons. In Lacertilians, we found pinealocytic terminals without ribbons on dendrite-like profiles. On the basis of the ultrastructural comparisons, we consider the CSF contacting neurons and pinealocytes to be very similar, but not to represent precisely the same cell type.