Intrinsically photosensitive retinal ganglion cells

A new mammalian photoreceptor was recently discovered to reside in the ganglion cell layer of the inner retina.These intrinsically photosensitive retinal ganglion cells(ipRGCs) express a photopigment,melanopsin,that confers upon them the ability to respond to light in the absence of all rod and cone photoreceptor input.Although relatively few in number,ipRGCs extend their dendrites across large expanses of the retina making them ideally suited to function as irradiance detectors to assess changes in ambient...     

Contribution of human melanopsin retinal ganglion cells to steady-state pupil responses

The recent discovery of melanopsin-containing retinal ganglion cells (mRGCs) has led to a fundamental reassessment of non-image forming processing, such as circadian photoentrainment and the pupillary light reflex. In the conventional view of retinal physiology, rods and cones were assumed to be the only photoreceptors in the eye and were, therefore, considered responsible for non-image processing. However, signals from mRGCs contribute to this non-image forming processing along with cone-mediated luminance signals; although both signals contribute, it is unclear how these signals are summed. We designed and built a novel multi-primary stimulation system to stimulate mRGCs independently of other photoreceptors using a silent-substitution technique within a bright steady background. The system allows direct measurements of pupillary functions for mRGCs and cones. We observed a significant change in steady-state pupil diameter when we varied the excitation of mRGC alone, with no change in luminance and colour. Furthermore, the change in pupil diameter induced by mRGCs was larger than that induced by a variation in luminance alone: that is, for a bright steady background, the mRGC signals contribute to the pupillary pathway by a factor of three times more than the L- and M-cone signals.     

The development of intrinsic excitability in mouse retinal ganglion cells

Activity-dependent refinement of synaptic connections occurs throughout the developing nervous system, including the visual system. Retinal ganglion cells (RGCs) overproduce synapses then refine them in an activity-dependent manner that segregates RGC connections into multicellular patterns, such as eye-specific regions and retinotopic maps. Ferrets additionally segregate ON and OFF retinogeniculate pathways in an activity-dependent manner. It was unknown whether differences in ON versus OFF intrinsic and spontaneous activity occur in postnatal mouse. The work reported here measured the intrinsic properties and spontaneous activity of morphologically identified postnatal mouse RGCs, and tested the hypothesis that mouse ON and OFF RGCs develop differences in spontaneous activity. We found developmental changes in resting potential, action potential threshold, depolarization to threshold, action potential width, action potential patterns, and maximal firing rates. These results are consistent with the maturation of the intrinsic properties of RGCs extending through the first three postnatal weeks. However, there were no differences among mouse ON, OFF, and multistratified RGCs in intrinsic excitability, spontaneous synaptic drive or spontaneous action potential patterns. The absence of differences between ON and OFF activity patterns is unlike the differences that arise in ferrets. In contrast to the ferret, the ON and OFF target neurons in the mouse are organized in a random pattern, not layers. This supports the hypothesis that the absence of systematic differences in activity results in the nonlayered distribution of retinogeniculate connections.     

Molecular classification of zebrafish retinal ganglion cells links genes to cell types to behavior

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EGFR-dependent suppression of synaptic autophagy is required for neuronal circuit development

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Changing dendritic field size of mouse retinal ganglion cells in early postnatal development

During early postnatal development, dendrites of retinal ganglion cells (RGCs) extend and branch in the inner plexiform layer to establish the adult level of stratification, pattern of branching, and coverage. Many studies have described the branching patterns, transient features, and regulatory factors of stratification of the RGCs. The rate of RGC dendritic field (DF) expansion relative to the growing retina has not been systematically investigated. In this study, we used two methods to examine the relative expansion of RGC DFs. First, we measured the size of RGC DFs and the diameters of the eyeballs at several postnatal stages. We compared the measurements with the RGC DF sizes calculated from difference of the eyeball sizes based on a linear expansion assumption. Second, we used the number of cholinergic amacrine cells (SACs) circumscribed by the DFs of RGCs at corresponding time points as an internal ruler to assess the size of DFs. We found most RGCs exhibit a phase of faster expansion relative to the retina between postnatal day 8 (P8) and P13, followed by a phase of retraction between P13 and adulthood. The morphological α cells showed the faster growing phase but not the retraction phase, whereas the morphological ON–OFF direction selective ganglion cells expanded in the same pace as the growing retina. These findings indicate different RGCs show different modes of growth, whereas most subtypes exhibit a fast expansion followed by a retraction phase to reach the adult size. © 2009 Wiley Periodicals, Inc. Develop Neurobiol 70: 397–407, 2010     

Competitive interactions between retinal ganglion cells during prenatal development

In the mammalian visual system, retinal ganglion cell axons terminate within the LGN in a series of alternating eye-specific layers. These layers are not present initially during development. In the cat they emerge secondarily following a prenatal period in which originally intermixed inputs from the two eyes gradually segregate from each other to give rise to the characteristic set of layers by birth. Many lines of evidence suggest that activity-dependent competitive interactions between ganglion cell axons from the two eyes for LGN neurons play an important role in the final patterning of retinogeniculate connections. Studies of the branching patterns of individual ganglion cell axons suggest that during the period when inputs from the two eyes are intermixed, axons from one eye send side branches into territory later occupied exclusively by axons from the other eye. Ultrastructural studies indicate that these branches in fact are sites of synaptic contacts, which are later eliminated since the side branches disappear as axons form their mature terminal arbors in appropriate territory. In vitro microelectrode recordings from LGN neurons indicate that they can receive convergent synaptic excitation from electrical stimulation of the optic nerves before but not after the eye-specific layers form, suggesting that at least some of the synaptic contacts seen at the ultrastructural level are functonal. Finally, experiments in which tetrodotoxin was infused intracranially during the two week period during which the eye-specific layers normally form demonstrate that it is possible to prevent, or at least delay, the formation of the layers. Accordingly, individual axons fail to develop their restricted terminal arbor branching pattern and instead branch widely throughout the LGN. These results indicate that all of the machinery necessary for synaptic function and competition is present during fetal life. Moreover, it is highly likely that neuronal activity is required for the formation of the eye-specific layers. If so, then activity would have to be present in the form of spontaneously generated action potentials, since vision is not possible at these early ages. Thus, the functioning of the retinogeniculate system many weeks before it is put to the use for which it is ultimately designed may contribute to the final patterning of connections present in the adult.     

Physiologic diversity and development of intrinsically photosensitive retinal ganglion cells

Intrinsically photosensitive retinal ganglion cells (ipRGCs) mediate numerous nonvisual phenomena, including entrainment of the circadian clock to light-dark cycles, pupillary light responsiveness, and light-regulated hormone release. We have applied multielectrode array recording to characterize murine ipRGCs. We find that all ipRGC photosensitivity is melanopsin dependent. At least three populations of ipRGCs are present in the postnatal day 8 (P8) murine retina: slow onset, sensitive, fast off (type I); slow onset, insensitive, slow off (type II); and rapid onset, sensitive, very slow off (type III). Recordings from adult rd/rd retinas reveal cells comparable to postnatal types II and III. Recordings from early postnatal retinas demonstrate intrinsic light responses from P0. Early light responses are transient and insensitive but by P6 show increased photosensitivity and persistence. These results demonstrate that ipRGCs are the first light-sensitive cells in the retina and suggest previously unappreciated diversity in this cell population.     

The development of retinal ganglion cells deprived of their targets

The influence of central targets on the morphological differentiation of retinal ganglion cells was investigated in Xenopus laevis. Since the ganglion cells mature into distinct morphological subtypes after their axons have reached their central targets, it is possible that the target tissues may influence or specify this aspect of neuronal cell development. To test this idea, Xenopus eyebuds were target-deprived by transplantation to the flank region of host embryos where they developed ectopically. The grafted eyes grew at normal rates, but could not make any projections into the central nervous system. To examine the morphological differentiation of the retinal ganglion cells their structures were revealed using an in vitro retinal preparation and intracellular injections of the dye Lucifer yellow. The elaboration and maturation of ganglion cell dendrites were found to be indistinguishable between control and transplanted eyes throughout development. Thus, the development of retinal ganglion cells into distinct morphological classes can occur even when their axons do not interact with the appropriate central targets.     

Properties of mouse retinal ganglion cell dendritic growth during postnatal development

The property of dendritic growth dynamics during development is a subject of intense interest. Here, we investigated the dendritic motility of retinal ganglion cells (RGCs) during different developmental stages, using ex vivo mouse retina explant culture, Semliki Forest Virus transfection and time-lapse observations. The results illustrated that during development, the dendritic motility underwent a change from rapid growth to a relatively stable state, i.e., at P0 (day of birth), RGC dendrites were in a highly active state, whereas at postnatal 13 (P13) they were more stable, and at P3 and P8, the RGCs were in an intermediate state. At any given developmental stage, RGCs of different types displayed the same dendritic growth rate and extent. Since the mouse is the most popular mammalian model for genetic manipulation, this study provided a methodological foundation for further exploring the regulatory mechanisms of dendritic development.     

Cell-type specific dendritic contacts between retinal ganglion cells during development.

The extent of a neuron's dendritic field defines the region within which information is processed. The dendritic fields of functionally distinct ON and OFF center retinal ganglion cells (RGCs) form separate mosaics across the retina. Within each mosaic, neighboring dendritic fields overlap by a constant amount, sampling the visual field with the appropriate coverage. Contact-mediated lateral inhibition between neighboring RGCs has long been thought to regulate both the extent and overlap of dendritic fields during development. Here we show that dendro-dendritic contact exists between developing RGCs and occurs in a manner that would regulate the formation of ON and OFF mosaics separately. Dye-filled neighboring ON and OFF ferret alpha RGCs were reconstructed using multiphoton microscopy. At all neonatal ages examined, we observed dendro-dendritic contacts between RGCs of the same sign (ON/ON; OFF/OFF), but never between cells of opposite signs (ON/OFF). Terminal dendrites of one cell often touched a dendrite of its neighbor as they intersected. In some instances, the distal dendrite of one cell formed a fascicle with the proximal process of its neighbor. Alpha cells did not form contacts with neighboring beta cells of the same sign. Together, these observations suggest that dendro-dendritic contact between RGCs is cell-type specific. Dendritic contacts were observed even before the alpha cell arbors were completely stratified, suggesting that cell-cell recognition may take place early in their development. For each cell type, the relative overlap of dendritic fields was constant with age, despite a two-fold increase in field area. We suggest that dendro-dendritic contacts may be sites of intercellular signaling that could regulate local extension of dendrites to maintain the relative overlap of RGCs within a mosaic during development.     

Ultrastructural changes during early development of retinal ganglion cells in Xenopus

Summary All cells in the optic vesicle of Xenopus embryos from stages 27 to 31 have the same ultrastructure. They are elongated and appear to extend from the internal to the external surfaces of the optic vesicle. They are bound together by terminal bars at the internal (lumen) margin, have microvilli and a cilium on the internal margin, and are covered with a basement membrane on the external margin. Their cytoplasm contains abundant free ribosomes, polysomes, mitochondria, yolk and lipid inclusions, and sparse endoplasmic reticulum.Although other studies have shown that retinal ganglion cells originate at stages 29–30 and have their central connections determined before stage 31, these events could not be correlated with any ultrastructural changes. The first sign of differentiation in retinal cells was an increase in endoplasmic reticulum and Golgi apparatus at stage 32. Microtubules and microfilaments appeared at stage 33 in association with the first axonal outgrowth from retinal ganglion cells. Cytodifferentiation proceeded gradually until large areas of Nissl substance had developed by stage 35. At larval stage 48 the ganglion cells resembled those in the adult.The authors wish to thank Marija Duda for her excellent technical assistance during this investigation.Supported by Public Health Service Predoctoral Fellowship No. 5 FO 1 GM37746-02 and Postdoctoral Fellowship 1 F2 NB37,746-01.Supported by Grant GB8315 from the National Science Foundation.     

Transient expression of adheron molecules during chick retinal development

Neuritogenesis and synapse formation are transient phenomena mediated in part by filopodial attachments (Tsui, Lankford, and Klein, Proc. Natl. Acad. Sci. 82:8256–8260 1985). These attachments can be labeled by antisera against adherons, adhesive microparticles isolated from cell culture media (Tsui, Schubert, and Klein, J. Cell Biol. 106:2095–2108 1988). Here, two monoclonal antibodies raised against adherons have been found to recognize transiently expressed membrane antigens of developing avian retina. Early in development, monoclonal antibody (mAb) AD1 stained antigens that spanned the entire tissue. With time, immunoreactivity became restricted to optic fiber, ganglion cell, and inner plexiform layers. Immunoblots of embryonic day (E) 13 retina showed a broad band at 66–72 kD for particulate fractions and a fine band at 70 kD for suluble fractions. The particulate forms disappeared as retinas matured, but the soluble form did not. mAb AD2 initially labeled retina antigens of optic fiber, ganglion cell, and inner plexiform layers (IPL). Labeling in the plexiform layer showed discrete lamina. Immunoreactivity first appeared at E9, peaked at E15, and then disappeared shortly after hatching. In isolated cells, AD2 labeled small cell surface aggregates. Cytoarchitectural studies, using whole mount transmission electron microscopy, showed AD2 antigen in cell surface microfilaments, including some that joined filopodia together. The adheron antigens recognized by mAbs AD1 and AD2 thus were (1) topographically restricted; (2) associated with cell surfaces; and (3) developmentally down-regulated. This pattern suggests a role in developmentally transient cell surface phenomena, such as neurite extension or junction biogenesis. © 1992 John Wiley & Sons, Inc.     

Stimulation of brain development in chick embryo by elevated temperature

Summary Experimental chick embryos were incubated at 37.5°C till day 7 and after day 10, and at 40.5°C on days 7–10; their optic lobes and cerebral hemispheres at day 10 and at hatching were compared with controls incubated at 37.5°C only. Cell numbers at day 10 were directly counted by a new method involving formalin fixation and cell disaggregation by gentle sonication. At hatching, body weights, organ weights and organ DNA (cell numbers) were the same in experimentals and in controls, for both optic lobes and cerebral hemispheres, though the protein contents were significantly higher in experimentals. However, at 10 days (end of neuron proliferation) the weights and the cell numbers in experimentals were significantly higher. Two possible explanations have been offered: 1. Elevated neuron population in experimental animals at day 10 is followed by their elevated death rate, or 2. The increment in neuron number is permanent but at hatching it is overshadowed by the population of other cells.An abstract of this work has been presented (Zamenhof, 1975)     

Electrical activity of rat retinal ganglion cells

The electrical activity of rat retinal ganglion cells is described. It was found that most such cells generate tonic discharges, while cells that demonstrate a phasic type of activity are less numerous. Neirofiziologiya/Neurophysiology, Vol. 39, Nos. 4/5, pp. 382–384, July–October, 2007.     

In utero exposure to microwave radiation and rat brain development

Timed-pregnancy rats were exposed in a circular waveguide system starting on day 2 of gestation. The system operated at 2,450 MHz (pulsed waves; 8 microseconds PW; 830 pps). Specific absorption rate (SAR) was maintained at 0.4 W/kg by increasing the input power as the animals grew in size. On day 18 of gestation the dams were removed from the waveguide cages and euthanized; the fetuses were removed and weighed. Fetal brains were excised and weighed, and brain RNA, DNA and protein were determined. Values for measured parameters of the radiated fetuses did not differ significantly from those of sham-exposed fetuses. A regression of brain weight on body weight showed no micrencephalous fetuses in the radiation group when using as a criterion a regression line based on two standard errors of the estimate of the sham-exposed group. In addition, metrics derived from brain DNA (ie, cell number and cell size) showed no significant differences when radiation was compared to sham exposure. We conclude that 2,450-MHz microwave radiation, at an SAR of 0.4 W/kg, did not produce significant alterations in brain organogenesis.     

The distribution of melanopsin (OPN4) protein in the human brain

Until now, melanopsin (OPN4) – a specialized photopigment being responsive especially to blue light wavelengths – has not been found in the human brain at protein level outside the retina. More specifically, OPN4 has only been found in about 2% of retinal ganglion cells (i.e. in intrinsically photosensitive retinal ganglion cells), and in a subtype of retinal cone-cells. Given that Allen Institute for Brain Science has described a wide distribution of OPN4 mRNA in two human brains, we aimed to investigate whether OPN4 is present in the human brain also at protein level. Western blotting and immunohistochemistry, as well as immunoelectron microscopy, were used to analyse the existence and distribution of OPN4 protein in 18 investigated areas of the human brain in samples obtained in forensic autopsies from 10 male subjects (54 ± 3.5 years). OPN4 protein expression was found in all subjects, and, furthermore, in 5 out of 10 subjects in all investigated brain areas localized in membranous compartments and cytoplasmic vesicles of neurons. To our opinion, the wide distribution of OPN4 in central areas of the human brain evokes a question whether ambient light has important straight targets in the human brain outside the retinohypothalamic tract (RHT). Further studies are, however, needed to investigate the putative physiological phototransductive actions of inborn OPN4 protein outside the RHT in the human brain.