José M. García-Fernández

VA opsin-based photoreceptors in the hypothalamus of birds.

Studies in the 1930s demonstrated that birds possess photoreceptors that are located within the hypothalamus and regulate photoperiodic responses to day length. Most recently, photoperiod has been shown to alter the activity of the pars tuberalis to release thyrotrophin, which ultimately drives a reproductive response. Despite these significant findings, the cellular and molecular identity of the hypothalamic photoreceptors has remained a mystery. Action spectra implicated an opsin-based photopigment system, but further identification based on rod- or cone-opsin probes failed, suggesting the utilization of a novel opsin. The vertebrate ancient (VA) opsin photopigments were isolated in 1997 but were thought to have a restricted taxonomic distribution, confined to the agnatha and teleost fish. Here, we report the isolation of VA opsin from chicken and show that the two isoforms spliced from this gene (cVAL and cVA) are capable of forming functional photopigments. Further, we show that VA opsin is expressed within a population of hypothalamic neurons with extensive projections to the median eminence. These results provide the most complete cellular and molecular description of a deep brain photoreceptor in any vertebrate and strongly implicate VA opsin in mediating the avian photoperiodic response.

The spatio-temporal pattern of photoreceptor degeneration in the aged rd/rd mouse retina

Abstract.Photoreceptor degeneration in the retina of the rd/rd (retinal degeneration) mice has been studied using immunocytochemistry with antisera against cone- and rod-opsin. The rd/rd mice exhibited different regional specific rates of degeneration for rods and cones. As early as postnatal day 25, cells labelled with the rod-opsin and cone-opsin antisera disappeared preferently from the central retina. Whereas in the inferior half of the retina, degeneration subsequently proceeded towards the periphery, this did not occur in the dorsal hemisphere. By the age of 100 days, many cells immunoreactive for the cone-opsin antiserum and a few cells immunoreactive for the rod-opsin antiserum were located in an area of the dorsal retina. The ventral retina lacked labelled elements at this age. Finally, rd/rd mice at one year or 600 days of age contained a similar number of cone-opsin immunopositive cells (approximately 2000–2800 cells), occupying almost the same area in the retina as that found at 100 days of age. A photoreceptor candidate for the entrainment of non-visual photoreception probably remains in the cone population in aged rd/rd mice.

An immunocytochemical study of encephalic photoreceptors in three species of lamprey

Abstract.The extraretinal and extrapineal photoreceptors of three species of adult lamprey, sea lamprey (Petromyzon marinus), river lamprey (Lampetra fluviatilis) and silver lamprey (Ichthyomyzon unicuspis) were studied using antibodies raised against photoreceptor rod and cone opsins, α-transducin and arrestin. In all three species cells in the pineal organ (P), parapineal organ (PP), nucleus preopticus (T5), nucleus commissurae postopticae (D8), nucleus ventralis hypothalami (D10) and nucleus dorsalis hypothalami (D11) were labelled by one or more of the anti-opsin antibodies. In addition, anti-arrestin antibodies labelled cells within the D8 and anti-α-transducin antibodies labelled cells within the pineal complex and hypothalamus (primarily D8 and/or D10). A more variable and species dependent pattern of opsin, arrestin and α-transducin labelling was observed within the nucleus commissurae postinfundibularis (D12) in an area comprising the nucleus dorsalis thalami pars subhabenularis (D4sh) and nucleus dorsalis thalami pars caudalis/nucleus commissurae posterioris (D4c/M1), and in the proximity of the second Müller cells in the ventrocaudal diencephalon (2.MZ/M6). The majority of the neurons labelled within the pineal and parapineal organs and hypothalamus were periventricular with clear cerebrospinal fluid contacts (CSF-contacting neurons). Labelled neurons in the epithalamic (D4sh and D4c/M1) and caudal diencephalon (2.MZ/M6) had no obvious ventricular contacts. We speculate that the ”primitive” vertebrate brain of lampreys represents an ancestral condition in which different populations of encephalic photoreceptors are associated with different behavioural and physiological responses. Image-forming vision needs an eye, but irradiance detection does not require a specialised organ. Rather the photoreceptors could be closely associated with their effector systems within the brain.

The persistence of cone photoreceptors within the dorsal retina of aged retinally degenerate mice (rd/rd): implications for circadian organization.

Rod- and cone-opsin specific antibodies were used in an attempt to immunolabel remaining photoreceptor cells in the mutant rd (retinal degeneration) mouse retina. We identified a region-specific distribution in the pattern of photoreceptor degeneration, with the dorsal retina showing markedly less photoreceptor degeneration than the ventral retina. All rod and cone immunoreactive cells disappeared in the ventral retina by 100-120 days of age. By contrast, both cone and a small number of rod immunopositive cells were identified in the dorsal retina at this time. By 200 days all rod immunoreactive cells had disappeared. At 360 days numerous cone immunoreactive cells remained within a restricted region of the dorsal retina. As rd mice show unattenuated circadian responses to light, these remaining photoreceptor cells within the dorsal retina become candidates for the regulation of circadian physiology by light.

Vertebrate ancient opsin photopigment spectra and the avian photoperiodic response

In mammals, photoreception is restricted to cones, rods and a subset of retinal ganglion cells. By contrast, non-mammalian vertebrates possess many extraocular photoreceptors but in many cases the role of these photoreceptors and their underlying photopigments is unknown. In birds, deep brain photoreceptors have been shown to sense photic changes in daylength (photoperiod) and mediate seasonal reproduction. Nonetheless, the specific identity of the opsin photopigment ‘sensor’ involved has remained elusive. Previously, we showed that vertebrate ancient (VA) opsin is expressed in avian hypothalamic neurons and forms a photosensitive molecule. However, a direct functional link between VA opsin and the regulation of seasonal biology was absent. Here, we report the in vivo and in vitro absorption spectra (λmax = ∼490 nm) for chicken VA photopigments. Furthermore, the spectral sensitivity of these photopigments match the peak absorbance of the avian photoperiodic response (λmax = 492 nm) and permits maximum photon capture within the restricted light environment of the hypothalamus. Such a correspondence argues strongly that VA opsin plays a key role in regulating seasonal reproduction in birds.

Immunocytochemical identification of photoreceptor proteins in hypothalamic cerebrospinal fluid-contacting neurons of the larval lamprey (Petromyzon marinus)

Antibodies directed against different visual pigment opsins, and an antibody raised against the C terminal of the α-subunit of retinal G protein (transducin) labelled cerebrospinal fluid-contacting cells located within the hypothalamus (postoptic commissural nucleus and ventral hypothalamic nucleus) of ammocoete lampreys (Petromyzon marinus). These antibodies also labelled photoreceptor cells within the retina and the pineal and parapineal organs, but no other areas of the brain. Despite considerable behavioural and physiological evidence for the existence of deep brain photoreceptors, numerous studies have failed to identify photoreceptor proteins within the basal brain. The results presented in this paper support our recent results in the lizard Anolis carolinensis, suggesting that a group of cerebrospinal fluid-contacting neurons within the vertebrate brain have a photosensory capacity. We speculate that these cells mediate extraocular and extrapineal photoreception in nonmammalian vertebrates.

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.

The eye of the african mole-rat Cryptomys anselli: to see or not to see?

In an attempt to clarify its possible physiological role, we studied the eye of the Zambian mole rat Cryptomys anselli by light, electron and confocal microscopy using conventional staining as well as immunolabelling with rod and cone cell markers. The small eyes of Cryptomys are located superficially and display all features typical of sighted animals: iris, pupil and well‐developed lens, separating the anterior chamber and the vitreous. The retina shows a well stratified organization and the folds described in blind subterranean or nocturnal mammals were not observed. The major population of the photoreceptor cells in the Cryptomys retina consists of rod cells, again with a morphology quite similar to that found in sighted animals. The relatively short outer segments contain numerous well‐stacked disks and show a strong rod‐opsin as well as transducin immunoreaction. Synapses were evident in the spherules, the round basal processes of the rod cell, but they lacked the precise organization reported for sighted mammals. Cone cells were present as well, as indicated by peanut lectin staining, but no immunolabelling with polyclonal M/L‐opsin antisera was detectable. The presence of cone cells was also suggested by some basal processes at the outer plexiform layer which displayed several synaptic active sites and irregular contours. While the other retinal layers also showed an organization typical of sighted mammals, there were signs of less tightly preserved morphology as well. Displaced rods and amacrine and/or ganglion cells were observed, and some sparse rod spherules penetrated into the inner nuclear layer. A major reduction was observed in the number of ganglion cells, estimated from the number of axons in the optic nerve, that was very low (≈1000 per retina on average) relative to sighted mammals. The data we have suggest a slow, ongoing loss of cells with ageing. Apoptotic nuclei, mainly corresponding to photoreceptor cells and ganglion cells, were detected in young individuals, and an overall reduction in the thickness of the retina was observed in older animals. The morphological data presented here allow some first speculations on the physiological role of the Cryptomys eye and will hopefully trigger detailed studies on the chronobiology and the anatomy of the retinal projections and of the visual cortex of this remarkable species.

Daily Rhythm of Melanopsin-Expressing Cells in the Mouse Retina

In addition to some other functions, melanopsin-expressing retinal ganglion cells (RGCs) constitute the principal mediators of the circadian photoentrainment, a process by which the suprachiasmatic nucleus (the central clock of mammals), adjusts daily to the external day/night cycle. In the present study these RGCs were immunohistochemically labelled using a specific polyclonal antiserum raised against mouse melanopsin. A daily oscillation in the number of immunostained cells was detected in mice kept under a light / dark (LD) cycle. One hour before the lights were on (i.e., the end of the night period) the highest number of immunopositive cells was detected while the lowest was seen 4 h later (i.e., within the first hours of the light period). This finding suggests that some of the melanopsin-expressing RGCs “turn on” and “off” during the day/night cycle. We have also detected that these daily variations already occur in the early postnatal development, when the rod/cone photoreceptor system is not yet functional. Two main melanopsin-expressing cell subpopulations could be found within the retina: M1 cells showed robust dendritic arborization within the OFF sublamina of the inner plexiform layer (IPL), whilst M2 cells had fine dendritic processes within the ON sublamina of the IPL. These two cell subpopulations also showed different daily oscillations throughout the LD cycle. In order to find out whether or not the melanopsin rhythm was endogenous, other mice were maintained in constant darkness for 6 days. Under these conditions, no defined rhythm was detected, which suggests that the daily oscillation detected either is light-dependent or is gradually lost under constant conditions. This is the first study to analyze immunohistochemically the daily oscillation of the number of melanopsin-expressing cells in the mouse retina.

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.