The mPer1 clock gene expression in the rd mouse suprachiasmatic nucleus is affected by the retinal degeneration.

Endogenous rhythms of mammals are controlled by the clock located in the suprachiasmatic nucleus (SCN). The molecular mechanism of a clock involves transcription/translation-based feedback loops in which the expression of the so called "clock genes" is suppressed periodically by their protein products. Previous studies reported influence of the eye itself on the circadian oscillation of the SCN, apart from the well-known photic readjustment of the central clock. With this in mind, we decided to analyze the mPer1 clock gene expression in the retinally degenerate (rd) mouse SCN by means of immunohistochemical techniques. Our objective was to detect possible alterations of the daily endogenous oscillation of PER1 protein in the SCN of these rd mice, as well as to make clear whether or not this protein was involved in the resetting of the central clock in a manner similar to wild-type animals. We found that the endogenous levels of PER1 protein were reduced in the SCN of rd mice throughout the 24-h cycle, which suggests that loss of classic photoreceptors influences somehow the main mechanism of the SCN clock. Light stimulation induced a parallel increase of Per1 expression at the subjective night, but not at the subjective day, in both rd and wild-type mice. Therefore, SCN readjustment by light in the rd mice occurs with a pattern similar to wild-type controls, despite the reduced PER1 protein levels detected. The effect of retinal degeneration on the circadian system and the possible interactions between the retinal and the SCN clocks are discussed.

Darkness during early postnatal development is required for normal circadian patterns in the adult rat.

Early light experience influences the brain during development. Perinatal light exposure has an important effect on the development of the circadian system, although the role of quantity versus quality of light in this process is still unclear. We tested the development of the circadian rhythm of locomotor activity under constant bright light from the day of weaning, of six groups of rats raised under different light conditions during suckling. Results indicated that when rats received daily darkness during suckling (rats reared under constant darkness or light-dark cycles with dim or bright light) became arrhythmic when exposed to continuous bright light after weaning. However, those rats reared in the absence of darkness (constant dim or bright light, or alternating dim and bright light) developed a circadian rhythm, which was stronger and had a shorter period depending on the quantity of light received during suckling. Vasointestinal polypeptide immunoreactivity in the suprachiasmatic nucleus (SCN) was higher in those rats with weaker rhythms. However, no apparent differences among these groups were found in the melanopsin-expressing retinal ganglion cells, which provide the SCN with light input in the photoentrainment process. When bright light was shifted to dim light in three of the groups on day 57 after weaning, all of them generated a circadian rhythm with a longer period in those rats previously arrhythmic. Our results indicate the importance of the amount of light received at the early stages of life in the development of the circadian system and suggest that darkness is needed for the normal development of circadian behaviour.

Cell populations in the pineal gland of the viscacha (Lagostomus maximus). Seasonal variations.

Pineal samples of the viscacha, which were taken in winter and in summer, were analysed using both light and electron microscopy. The differences found between the two seasons were few in number but significant. The parenchyma showed two main cell populations. Type I cells occupied the largest volume of the pineal and showed the characteristics of typical pinealocytes. Many processes, some of which were filled with vesicles, could be seen in intimate contact with the neighbouring cells. The presence in the winter samples of "synaptic" ribbons and spherules, which were almost absent in the summer pineals, suggests a seasonal rhythm. These synaptic-like structures, as well as the abundant subsurface cisterns present in type I cells, appeared as basic differential features which allowed these cells to be distinguished from type II cells. These latter cells, which can be classified as interstitial cells, showed some other distinguishing features, such as irregular-shaped nuclei, abundant deposits of glycogen-like particles and structures of unknown function consisting of concentric cisterns surrounding a dense body. In the summer, interstitial cells displayed numerous large round bodies, which contributed to increase the cellular volume slightly. Regarding other constituents, like glial cell processes, vessels of non-fenestrated endothelium and sympathetic innervation, no qualitative differences were observed between the two seasons studied. We have presented here some morphological evidences of the circannual rhythm of the viscacha pineal, as well as ultrastructural criteria for distinguishing the main cell populations of this organ, which could be useful for studies carried out in other mammals.

Identification of extraretinal photoreceptors in the teleost Phoxinus phoxinus.

The existence of cells capable of detecting changes of the photoperiod within the deep brain, the so-called deep brain photoreceptors, was proposed in the early years of the twentieth century. By using immunocytochemistry with antisera against phototransductory proteins on paraffin and vibratome sections, we have localized several positive areas in the brain of the teleost Phoxinus phoxinus. These areas were restricted to two encephalic regions: the epithalamus and the hypothalamus. Immunopositive (rod-opsin- and a-transducin-like) pinealocytes and parapinealocytes, as well as some sparse neurons in the habenula, were seen in the epithalamus. The immunoreaction of the hypothalamus was represented by a-transducin-like positive (magnocellular and parvicellular) neurons of the Nucleus Preopticus, as well as by a-transducin- and arrestin-like positive fibers corresponding to the hypothalamic-hypophyseal tract and a few fibers running towards the basal telencephalon. These findings corroborate the data published on other teleost fish and fully support the hypothesis of the presence of photosensitive cells in the encephalon of lower vertebrates. The labelling with antisera against different components of the phototransductory cascade also strengthens the idea that such cells employ a biochemical mechanism similar to that in the retinal visual photoreceptor cells, rods and cones. Although the function is still unclear, the detection of the photoperiod seems to be the most likely role for these extraretinal photoreceptors.

Age-related morphometric changes in the pineal gland. A comparative study between C57BL/6J and CBA mice.

Relatively little is known about the effects of melatonin on the aging of the pineal, the organ which is the main place for synthesis of this hormone. Using simple morphometric methods, some parameters of the pineal gland, such as total volume, number of pinealocytes and pinealocyte volume were estimated in two mice strains: normal CBA and melatonin-deficient C57BL/6J. Two age groups, 6 weeks and 10 months, were studied in order to evaluate possible differential age-related changes between both strains. Pineals of both strains have similar morphometric and morphological features at 6 weeks of age. This suggests that pineal development, which has already concluded at 6 weeks of age, is not affected by the absence of melatonin synthesis in the pinealocytes. Later on, CBA pineal showed an increase in size caused by cellular hypertrophy. In contrast, the C57BL/6J pineal volume decreased by loss of pinealocytes in the same period of time. Semithin sections analysed by light microscopy did not show that this cell death was evident in the C57BL/6J strain at any of the ages studied. Thus, a gradual loss of pinealocytes could be hypothesised in these pineals. These results suggest that pineal melatonin could have a role in the maintenance of pinealocyte viability and the increase of pineal size which takes place after development. The abnormal pattern observed in the C57BL/6J pineal should be taken into account in future studies on this gland.

Structural diversity of the ordinary and specialized lateral line organs.

Lateral line organ, a superficial sensory system in amphibia and fish which provides the animal with information about its surrounding environment, is divided classically into two main different types, ordinary and specialized, whose functions are mechanoreceptive and electroreceptive, respectively. Although it has great diversity, the basic sensory unit, which is usually called "neuromast," is composed of sensory cells embedded in accessory cells. The functions of the latter are to support the sensory cells and to secrete the material that covers the organs, forming a cupular structure or filling a canal which enables the organ to communicate with the exterior. Sensory cells of mechanoreceptive neuromasts have a tuft of processes included in the cupular material; these are a kinocilium and a group of stereocilia with a typical staircase arrangement. The displacement of the stereocilia towards or away from the kinocilium produces different stimuli. The electroreceptive organs are more diverse. They include ampullary and tuberous organs. The latter can be subdivided into different types: knollenorgans, mormyromasts, gymnarchomasts, etc. All of these present a great diversity among species, but their morphology is less reported than that of the mechanoreceptive organs. This paper summarizes the structural features of the main different types of lateral line organs, as well as their taxonomic distribution and different patterns of distribution along the surface of the animal.

Spatio-temporal analysis of light-induced Fos expression in the retina of rd mutant mice.

Rd mutant mice are visually blind but they maintain the ability of synchronising their circadian rhythms to the external light-dark cycles. We used immunocytochemical procedures to detect light-induced Fos expression in the rd mice retina. We found that Fos is expressed in the rd retina in an unattenuated pattern through the entire life of the animal. Furthermore, we have found that cells expressing Fos are distributed throughout the whole retina, while opsin expression takes place only in the dorsal half of the retina in the 1-year old rd mice. Finally, we found that light induces Fos expression in the rd retina at the same levels during the subjective day as during the subjective night, whereas in the suprachiasmatic nucleus (SCN), Fos is stimulated by light only during the subjective night. Our results support the hypothesis that new, undiscovered photoreceptors are implicated in light perception for the circadian system.

Ontogeny of a photic response in the retina and suprachiasmatic nucleus in the mouse.

The ontogeny of photic responsiveness in the retina and the suprachiasmatic nucleus (SCN) of C57BL/6J mouse was studied using the enhanced expression of the immediate early gene c-fos as a marker of neuronal activation. c-fos expression was assessed by immunocytochemical localisation of its protein product. Light induction of Fos-like protein in the retina and SCN occurred first at postnatal day four (PD 4). At this stage of development, some cells in the inner part of the neuroblastic layer and in the ganglion cell layer showed positive immunoreaction; the number of Fos-like positive cells increased with age until it reached adult levels by PD 15. Induction of Fos-like expression at PD 4 in the SCN mainly occurred in the ventrolateral region, the region that receives the greatest density of retinal innervation. These results indicate that retinal input can activate cells in the SCN even before eyelids open, and the SCN can be stimulated by photic inputs as early as day 4 after birth.

Fos expression in the retina of rd/rd mice during the light/dark cycle.

An anti-Fos protein antiserum was used to elucidate the diurnal expression of Fos protein in the normal and degenerate rd/rd mice retina. We have found that Fos expression is stimulated in cells of both inner nuclear layer (INL) and ganglion cell layer (GCL) at the onset of light period and reaches its maximum after 2 h. After which, the number of stained nuclei decreases along the light/dark cycle until almost no reaction is observed at the end of dark period. This expression pattern was similar in both normal and rd/rd mice although degenerate retinas showed a much lower number of stained nuclei. Aged rd animals also show Fos expression in GCL and INL in response to light stimuli suggesting that severely degenerate retinas are still able to transduce light stimulus.

Neurotrophin receptors expression in the developing mouse retina: an immunohistochemical study.

Neurotrophins and their receptors (p75 and Trk family of receptors) play an important role in the survival of different populations of neurons in the central and peripheral nervous system. Expression of p75, TrkA, TrkB and TrkC was examined in mouse retinas by means immunohistochemistry in the postnatal development of normal and rd/rd mice (C57BL/6J). The rd/rd mice suffer a degeneration that causes a massive lost of photoreceptor cells. Results showed immunoreactivity to all three Trk proteins in both normal and rd/rd mice during the first 21 postnatal days, but some variations in intensity and localization were found. p75 immunoreaction was only present in rd/rd mice at the end of the degeneration process. These results could indicate a role of neurotrophins and their receptors in both the postnatal development of mouse retina and the degeneration process of rd/rd mice.

Some observations on the fine structure of the sensory cells in the neuromasts (ordinary lateral line) of the teleost Gambusia affinis.

The structural and ultrastructural features of the sensory cells in the neuromasts of G. affinis were studied. These pear-shaped cels present a tuft of sensory hairs rising from the apex and a highly dense cytoplasm with multivesicular bodies, dyctiosomes, granular and smooth endoplasmic reticulum and numerous mitochondria. Large round granular dense bodies of an unknown nature can be observed inside and outside the nucleus. A great number of vesicles are present at the base of the cells, some of them associated with an afferent synapse, which also presents a presynaptic dense body. An efferent synapse with a nerve ending full of vesicles and a postsynaptic subsurface cisterna is also present at the base of these cells.