A live cell assay of GPCR coupling allows identification of optogenetic tools for controlling Go and Gi signaling.

BACKGROUND: Animal opsins are light-sensitive G-protein-coupled receptors (GPCRs) that enable optogenetic control over the major heterotrimeric G-protein signaling pathways in animal cells. As such, opsins have potential applications in both biomedical research and therapy. Selecting the opsin with the best balance of activity and selectivity for a given application requires knowing their ability to couple to a full range of relevant Gα subunits. We present the GsX assay, a set of tools based on chimeric Gs subunits that transduce coupling of opsins to diverse G proteins into increases in cAMP levels,  measured with a real-time reporter in living cells. We use this assay to compare coupling to Gi/o/t across a panel of natural and chimeric opsins selected for potential application in gene therapy for retinal degeneration. RESULTS: Of the opsins tested, wild-type human rod opsin had the highest activity for chimeric Gs proxies for Gi and Gt (Gsi and Gst) and was matched in Go proxy (Gso) activity only by a human rod opsin/scallop opsin chimera. Rod opsin drove roughly equivalent responses via Gsi, Gso, and Gst, while cone opsins showed much lower activities with Gso than Gsi or Gst, and a human rod opsin/amphioxus opsin chimera demonstrated higher activity with Gso than with Gsi or Gst. We failed to detect activity for opsin chimeras bearing three intracellular fragments of mGluR6, and observed unexpectedly complex response profiles for scallop and amphioxus opsins thought to be specialized for Go. CONCLUSIONS: These results identify rod opsin as the most potent non-selective Gi/o/t-coupled opsin, long-wave sensitive cone opsin as the best for selectively activating Gi/t over Go, and a rod opsin/amphioxus opsin chimera as the best choice for selectively activating Go over Gi/t.

Melanopsin+RGCs Are fully Resistant to NMDA-Induced Excitotoxicity.

We studied short- and long-term effects of intravitreal injection of N-methyl-d-aspartate (NMDA) on melanopsin-containing (m+) and non-melanopsin-containing (Brn3a+) retinal ganglion cells (RGCs). In adult SD-rats, the left eye received a single intravitreal injection of 5µL of 100nM NMDA. At 3 and 15 months, retinal thickness was measured in vivo using Spectral Domain-Optical Coherence Tomography (SD-OCT). Ex vivo analyses were done at 3, 7, or 14 days or 15 months after damage. Whole-mounted retinas were immunolabelled for brain-specific homeobox/POU domain protein 3A (Brn3a) and melanopsin (m), the total number of Brn3a+RGCs and m+RGCs were quantified, and their topography represented. In control retinas, the mean total numbers of Brn3a+RGCs and m+RGCs were 78,903 ± 3572 and 2358 ± 144 (mean ± SD; n = 10), respectively. In the NMDA injected retinas, Brn3a+RGCs numbers diminished to 49%, 28%, 24%, and 19%, at 3, 7, 14 days, and 15 months, respectively. There was no further loss between 7 days and 15 months. The number of immunoidentified m+RGCs decreased significantly at 3 days, recovered between 3 and 7 days, and were back to normal thereafter. OCT measurements revealed a significant thinning of the left retinas at 3 and 15 months. Intravitreal injections of NMDA induced within a week a rapid loss of 72% of Brn3a+RGCs, a transient downregulation of melanopsin expression (but not m+RGC death), and a thinning of the inner retinal layers.

Aberrant light directly impairs mood and learning through melanopsin-expressing neurons.

The daily solar cycle allows organisms to synchronize their circadian rhythms and sleep-wake cycles to the correct temporal niche. Changes in day-length, shift-work, and transmeridian travel lead to mood alterations and cognitive function deficits. Sleep deprivation and circadian disruption underlie mood and cognitive disorders associated with irregular light schedules. Whether irregular light schedules directly affect mood and cognitive functions in the context of normal sleep and circadian rhythms remains unclear. Here we show, using an aberrant light cycle that neither changes the amount and architecture of sleep nor causes changes in the circadian timing system, that light directly regulates mood-related behaviours and cognitive functions in mice. Animals exposed to the aberrant light cycle maintain daily corticosterone rhythms, but the overall levels of corticosterone are increased. Despite normal circadian and sleep structures, these animals show increased depression-like behaviours and impaired hippocampal long-term potentiation and learning. Administration of the antidepressant drugs fluoxetine or desipramine restores learning in mice exposed to the aberrant light cycle, suggesting that the mood deficit precedes the learning impairments. To determine the retinal circuits underlying this impairment of mood and learning, we examined the behavioural consequences of this light cycle in animals that lack intrinsically photosensitive retinal ganglion cells. In these animals, the aberrant light cycle does not impair mood and learning, despite the presence of the conventional retinal ganglion cells and the ability of these animals to detect light for image formation. These findings demonstrate the ability of light to influence cognitive and mood functions directly through intrinsically photosensitive retinal ganglion cells.

Visual opsin expression and morphological characterization of retinal photoreceptors in the pouched lamprey (Geotria australis, Gray).

Lampreys are extant members of the agnathan (jawless) vertebrates that diverged ~500 million years ago, during a critical stage of vertebrate evolution when image-forming eyes first emerged. Among lamprey species assessed thus far, the retina of the southern hemisphere pouched lamprey, Geotria australis, is unique, in that it possesses morphologically distinct photoreceptors and expresses five visual photopigments. This study focused on determining the number of different photoreceptors present in the retina of G. australis and whether each cell type expresses a single opsin class. Five photoreceptor subtypes were identified based on ultrastructure and differential expression of one of each of the five different visual opsin classes (lws, sws1, sws2, rh1, and rh2) known to be expressed in the retina. This suggests, therefore, that the retina of G. australis possesses five spectrally and morphologically distinct photoreceptors, with the potential for complex color vision. Each photoreceptor subtype was shown to have a specific spatial distribution in the retina, which is potentially associated with changes in spectral radiance across different lines of sight. These results suggest that there have been strong selection pressures for G. australis to maintain broad spectral sensitivity for the brightly lit surface waters that this species inhabits during its marine phase. These findings provide important insights into the functional anatomy of the early vertebrate retina and the selection pressures that may have led to the evolution of complex color vision.

Expressions of rod and cone photoreceptor-like proteins in human epidermis.

Previous reports have suggested the existence of photoreceptors for visible radiation at the surface of the human body. Rhodopsin is a well-known photosensitive protein found in the rod cells of the retina and detects light/dark contrast. Cone opsins are also photosensitive receptors in the cone cells of the retina and detect colour. Here, we describe immunochemical studies using anti-rhodopsin and anti-opsin antibodies on human skin. Both mouse retina and human epidermis showed clear immunoreactivity with each antibody. Interestingly, immunoreactivity against longer-wavelength opsin antibody was observed in the basal layer of the epidermis, while immunoreactivity against rhodopsin and shorter-wavelength opsin was observed in the upper layer. PCR analysis confirmed the expression of rhodopsin-like and opsin-like genes in human retina and the skin. These results suggest that a series of proteins, which play a crucial role in visual perception, are expressed in human epidermis.

Pineal opsin: a nonvisual opsin expressed in chick pineal.

Pineal opsin (P-opsin), an opsin from chick that is highly expressed in pineal but is not detectable in retina, was cloned by the polymerase chain reaction. It is likely that the P-opsin lineage diverged from the retinal opsins early in opsin evolution. The amino acid sequence of P-opsin is 42 to 46 percent identical to that of the retinal opsins. P-opsin is a seven-membrane spanning, G protein-linked receptor with a Schiff-base lysine in the seventh membrane span and a Schiff-base counterion in the third membrane span. The primary sequence of P-opsin suggests that it will be maximally sensitive to approximately 500-nanometer light and produce a slow and prolonged phototransduction response consistent with the nonvisual function of pineal photoreception.

The chemistry of John Dalton's color blindness.

John Dalton described his own color blindness in 1794. In common with his brother, he confused scarlet with green and pink with blue. Dalton supposed that his vitreous humor was tinted blue, selectively absorbing longer wavelengths. He instructed that his eyes should be examined after his death, but the examination revealed that the humors were perfectly clear. In experiments presented here, DNA extracted from his preserved eye tissue showed that Dalton was a deuteranope, lacking the middlewave photopigment of the retina. This diagnosis is shown to be compatible with the historical record of his phenotype, although it contradicts Thomas Young's belief that Dalton was a protanope.

Transgenic mice with a rhodopsin mutation (Pro23His): a mouse model of autosomal dominant retinitis pigmentosa.

We inserted into the germline of mice either a mutant or wild-type allele from a patient with retinitis pigmentosa and a missense mutation (P23H) in the rhodopsin gene. All three lines of transgenic mice with the mutant allele developed photoreceptor degeneration; the one with the least severe retinal photoreceptor degeneration had the lowest transgene expression, which was one-sixth the level of endogenous murine rod opsin. Of two lines of mice with the wild-type allele, one expressed approximately equal amounts of transgenic and murine opsin and maintained normal retinal function and structure. The other expressed approximately 5 times more transgenic than murine opsin and developed a retinal degeneration similar to that found in mice carrying a mutant allele, presumably due to the overexpression of this protein. Our findings help to establish the pathogenicity of mutant human P23H rod opsin and suggest that overexpression of wild-type human rod opsin leads to a remarkably similar photoreceptor degeneration.

Expression and comparative characterization of Gq-coupled invertebrate visual pigments and melanopsin.

A non-visual pigment melanopsin, which is localized in photosensitive retinal ganglion cells and is involved in the circadian photoentrainment and pupillary responses in mammals, is phylogenetically close to the visual pigments of invertebrates, such as insects and cephalopods. Recent studies suggested that melanopsin is a bistable pigment and drives a Gq-mediated signal transduction cascade, like the invertebrate visual pigments. Because detailed electrophysiological properties are somewhat different between the visual cells and the photosensitive ganglion cells, we here expressed and purified the invertebrate visual pigment and melanopsin to comparatively investigate their Gq-activation abilities. We successfully expressed and purified UV and blue light-sensitive visual pigments of the honeybee as well as the amphioxus melanopsin. Although the purified UV-sensitive pigment and the melanopsin lost their bistable nature during purification, reconstitution of the pigments in lipid vesicles resulted in return of the bistable nature. The light-dependent Gq-activation abilities among these reconstituted pigments are similar, suggesting that the electrophysiological differences do not depend on the Gq-activation step but rather on the other signal transduction steps and/or on cell properties. Our findings are also important in that this is the first report describes a heterologous large-scale expression of the Gq-coupled invertebrate visual pigments in cultured cells.

Two types of melanopsin retinal ganglion cell differentially innervate the hypothalamic suprachiasmatic nucleus and the olivary pretectal nucleus.

Melanopsin-expressing intrinsically photosensitive retinal ganglion cells (ipRGCs) innervate the hypothalamic suprachiasmatic nucleus (SCN) and the olivary pretectal nucleus (OPN), providing irradiance information for entrainment of circadian rhythms and for stimulating the pupillary light reflex. In this study, mice were used in which the melanopsin gene was replaced with the tau-lacZ gene. Heterozygous (tau-lacZ+/-) mice express both melanopsin and beta-galactosidase. In tau-lacZ+/- mice, only approximately 50% of melanopsin ipRGCs contain beta-galactosidase, and these cells are specifically labeled with a C-terminus melanopsin antibody. Retrograde tracer injection into the SCN labels beta-galactosidase-expressing ipRGCs (termed M1) that comprise approximately 80% of the SCN-projecting ipRGCs. M1 ipRGCs and an additional set of ipRGCs (termed M2) are labeled with a melanopsin antiserum targeted against the N-terminus of the melanopsin protein; M2 ipRGCs do not contain detectable beta-galactosidase, and these cells make up the remainder of the SCN-projecting RGCs. Tracer injection into the OPN labeled non-melanopsin RGCs and both types of melanopsin ipRGC: 45% M1 and 55% M2. Infection of the iris with pseudorabies virus (PRV) results in retrograde transneuronal label of OPN projection neurons that innervate preganglionic parasympathetic neurons of the Edinger-Westphal nucleus; PRV-labeled cells were located almost exclusively within the terminal field of M1 ipRGCs in the periphery (shell) of the OPN. The OPN core receives retinal input, and we hypothesize that the OPN core receives input from the M2 ipRGCs. Two subtypes of melanopsin ipRGCs project differentially to the SCN and OPN; the functional significance of ipRGCs subtypes is currently unknown.

Loss of melanopsin-containing retinal ganglion cells in a rat glaucoma model.

BACKGROUND: Glaucoma can cause progressive damage to retinal ganglion cells. These cells can be classified as cells projecting to the superior colliculus and melanopsin-containing retinal ganglion cells, which project to the suprachiasmatic nucleus. This study was to investigate the effects of chronic intraocular pressure elevation on melanopsin-containing retinal ganglion cells in rats. METHODS: Chronic intraocular pressure elevation was induced in one eye of adult Wistar rats by cauterization of three episcleral veins. Intraocular pressure was measured at different intervals with a rebound tonometer. Superior collicular retinal ganglion cells were retrogradely labeled from the superior colliculus with Fluorogold. Melanopsin-containing retinal ganglion cells were visualized by free-floating immunohistochemistry on whole-mount retinas. The number of labeled superior collicular and melanopsin-containing retinal ganglion cells were counted in the sample areas on flat-mounted retinas. RESULTS: Compared with contralateral control eyes, the numbers of both superior collicular and melanopsin-containing retinal ganglion cells were significantly reduced after 12 weeks of experimental intraocular pressure elevation ((2317.41 +/- 29.96)/mm(2) vs (1815.82 +/- 24.25)/mm(2); (26.20 +/- 2.10)/mm(2) vs (20.62 +/- 1.52)/mm(2), respectively). The extent of cell loss of the two types of retinal ganglion cells was similar. However, no morphologic changes were found in melanopsin-containing retinal ganglion cells. CONCLUSION: Both melanopsin-containing and superior collicular retinal ganglion cells were damaged by chronic ocular hypertension, indicating that glaucomatous neural degeneration involves the non-image-forming visual pathway.

Presence of glutamate, glycine, and gamma-aminobutyric acid in the retina of the larval sea lamprey: comparative immunohistochemical study of classical neurotransmitters in larval and postmetamorphic retinas.

The neurochemistry of the retina of the larval and postmetamorphic sea lamprey was studied via immunocytochemistry using antibodies directed against the major candidate neurotransmitters [glutamate, glycine, gamma-aminobutyric acid (GABA), aspartate, dopamine, serotonin] and the neurotransmitter-synthesizing enzyme tyrosine hydroxylase. Immunoreactivity to rod opsin and calretinin was also used to distinguish some retinal cells. Two retinal regions are present in larvae: the central retina, with opsin-immunoreactive photoreceptors, and the lateral retina, which lacks photoreceptors and is mainly neuroblastic. We observed calretinin-immunostained ganglion cells in both retinal regions; immunolabeled bipolar cells were detected in the central retina only. Glutamate immunoreactivity was present in photoreceptors, ganglion cells, and bipolar cells. Faint to moderate glycine immunostaining was observed in photoreceptors and some cells of the ganglion cell/inner plexiform layer. No GABA-immunolabeled perikarya were observed. GABA-immunoreactive centrifugal fibers were present in the central and lateral retina. These centrifugal fibers contacted glutamate-immunostained ganglion cells. No aspartate, serotonin, dopamine, or TH immunoreactivity was observed in larvae, whereas these molecules, as well as GABA, glycine, and glutamate, were detected in neurons of the retina of recently transformed lamprey. Immunoreactivity to GABA was observed in outer horizontal cells, some bipolar cells, and numerous amacrine cells, whereas immunoreactivity to glycine was found in amacrine cells and interplexiform cells. Dopamine and serotonin immunoreactivity was found in scattered amacrine cells. Amacrine and horizontal cells did not express classical neurotransmitters (with the possible exception of glycine) during larval life, so transmitter-expressing cells of the larval retina appear to participate only in the vertical processing pathway.

Absorption Characteristics of Vertebrate Non-Visual Opsin, Opn3.

Most animals possess multiple opsins which sense light for visual and non-visual functions. Here, we show spectral characteristics of non-visual opsins, vertebrate Opn3s, which are widely distributed among vertebrates. We successfully expressed zebrafish Opn3 in mammalian cultured cells and measured its absorption spectrum spectroscopically. When incubated with 11-cis retinal, zebrafish Opn3 formed a blue-sensitive photopigment with an absorption maximum around 465 nm. The Opn3 converts to an all-trans retinal-bearing photoproduct with an absorption spectrum similar to the dark state following brief blue-light irradiation. The photoproduct experienced a remarkable blue-shift, with changes in position of the isosbestic point, during further irradiation. We then used a cAMP-dependent luciferase reporter assay to investigate light-dependent cAMP responses in cultured cells expressing zebrafish, pufferfish, anole and chicken Opn3. The wild type opsins did not produce responses, but cells expressing chimera mutants (WT Opn3s in which the third intracellular loops were replaced with the third intracellular loop of a Gs-coupled jellyfish opsin) displayed light-dependent changes in cAMP. The results suggest that Opn3 is capable of activating G protein(s) in a light-dependent manner. Finally, we used this assay to measure the relative wavelength-dependent response of cells expressing Opn3 chimeras to multiple quantally-matched stimuli. The inferred spectral sensitivity curve of zebrafish Opn3 accurately matched the measured absorption spectrum. We were unable to estimate the spectral sensitivity curve of mouse or anole Opn3, but, like zebrafish Opn3, the chicken and pufferfish Opn3-JiL3 chimeras also formed blue-sensitive pigments. These findings suggest that vertebrate Opn3s may form blue-sensitive G protein-coupled pigments. Further, we suggest that the method described here, combining a cAMP-dependent luciferase reporter assay with chimeric opsins possessing the third intracellular loop of jellyfish opsin, is a versatile approach for estimating absorption spectra of opsins with unknown signaling cascades or for which absorption spectra are difficult to obtain.

Classical Photoreceptors Are Primarily Responsible for the Pupillary Light Reflex in Mouse.

Pupillary light reflex (PLR) is an important clinical tool to assess the integrity of visual pathways. The available evidence suggests that melanopsin-expressing retinal ganglion cells (mRGCs) mediate PLR-driven by the classical photoreceptors (rods and cones) at low irradiances and by melanopsin activation at high irradiances. However, genetic or pharmacological elimination of melanopsin does not completely abolish PLR at high irradiances, raising the possibility that classical photoreceptors may have a role even at high irradiances. Using an inducible mouse model of photoreceptor degeneration, we asked whether classical photoreceptors are responsible for PLR at all irradiances, and found that the PLR was severely attenuated at all irradiances. Using multiple approaches, we show that the residual PLR at high irradiances in this mouse was primarily from the remnant rods and cones, with a minor contribution from melanopsin activation. In contrast, in rd1 mouse where classical photoreceptor degeneration occurs during development, the PLR was absent at low irradiances but intact at high irradiances, as reported previously. Since mRGCs receive inputs from classical photoreceptors, we also asked whether developmental loss of classical photoreceptors as in rd1 mouse leads to compensatory takeover of the high-irradiance PLR by mRGCs. Specifically, we looked at a distinct subpopulation of mRGCs that express Brn3b transcription factor, which has been shown to mediate PLR. We found that rd1 mouse had a significantly higher proportion of Brn3b-expressing M1 type of mRGCs than in the inducible model. Interestingly, inducing classical photoreceptor degeneration during development also resulted in a higher proportion of Brn3b-expressing M1 cells and partially rescued PLR at high irradiances. These results suggest that classical photoreceptors are primarily responsible for PLR at all irradiances, while melanopsin activation makes a minor contribution at very high irradiances.

Encephalopsin: a novel mammalian extraretinal opsin discretely localized in the brain.

We have identified a mammalian opsin, encephalopsin, that shows strong and specific expression in the brain. Encephalopsin defines a new family of opsins and shows highest homology to vertebrate retinal and pineal opsins. Encephalopsin is highly expressed in the preoptic area and paraventricular nucleus of the hypothalamus, both regions implicated in encephalic photoreception in nonmammalian vertebrates. In addition, encephalopsin shows highly patterned expression in other regions of the brain, being enriched in selected regions of the cerebral cortex, cerebellar Purkinje cells, a subset of striatal neurons, selected thalamic nuclei, and a subset of interneurons in the ventral horn of the spinal cord. Rostrocaudal gradients of encephalopsin expression are present in the cortex, cerebellum, and striatum. Radial stripes of encephalopsin expression are seen in the cerebellum. In the cortex and cerebellum, encephalopsin expression is considerably higher and more highly patterned in the adult than in the neonate. Encephalopsin is the first putative extraocular opsin identified in mammals and may play a role in encephalic photoreception.

Direct innervation of GnRH neurons by encephalic photoreceptors in birds.

In nonmammalian vertebrates, photic cues that regulate the timing of seasonal reproductive cyclicity are detected by nonretinal, nonpineal deep brain photoreceptors. It has long been assumed that the underlying mechanism involves the transmission of photic information from the photoreceptor to a circadian system, and thence to the reproductive axis. An alternative hypothesis is that there is direct communication between the brain photoreceptor and the reproductive axis. In the present study, light and confocal microscopy reveal that gonadotropin releasing hormone (GnRH) neurons and processes are scattered among photoreceptor cells (identified by their opsin-immunoreactivity) in the lateral septum (SL). In the median eminence (ME), opsin and GnRH immunoreactive fibers overlap extensively. Single and double label ultrastructural immunocytochemistry indicate that in the SL and preoptic area (POA), opsin positive terminals form axo-dendritic synapses onto GnRH dendrites. In the ME, opsin and GnRH terminals lie adjacent to each other, make contact with tanycytes, or terminate on the hypophyseal portal capillaries. These results reveal thatbrain photoreceptors communicate directly with GnRH-neurons; this represents a means by which photoperiodic information reaches the reproductive axis.

Role of the pineal organ in the photoregulated hatching of the Atlantic halibut.

The timing of hatching in the Atlantic halibut (Hippoglossus hippoglossus) has been suggested to be regulated by environmental light conditions. However, the photosensory organ that perceives the triggering light has not been identified. In the present study, we investigated the morphogenesis of the pineal organ and the neurochemical differentiation of photoreceptors in the pineal organ and the retina of the Atlantic halibut during embryonic development. Immunocytochemical techniques were used for detection of integral protein components of the phototransduction process: opsins, arrestin (S-antigen) and alpha-transducin. We also studied the expression of serotonin (5-HT), a precursor of the neurohormone melatonin known to be synthesized by pineal photoreceptors. In the pineal anlage, opsin immunoreactive (ir) cells appear at 11 days post-fertilization (pf), arrestin, alpha-transducin and serotonin ir cells appear at 14 days pf; hatching took place 15 days pf. The retina contained no immunoreactive cells in embryos or in newly hatched larva. During this period, the pineal anlage is morphologically discernible only as a wedge-shaped region in the diencephalic roof, where elongated cells are aligned with their long axes converging toward a centrally located presumptive pineal lumen. The results show that the pineal photoreceptors contain serotonin and molecules involved in the phototransduction cascade before hatching. We suggest that the pineal organ has the capacity to perceive and mediate photic information before hatching in halibut embryos, and may thereby influence the timing of hatching.

Impaired circadian photosensitivity in mice lacking glutamate transmission from retinal melanopsin cells.

Intrinsically photoreceptive retinal ganglion cells (ipRGCs) contain the photopigment melanopsin and convey retinal light inputs to the circadian system via the retinohypothalamic tract (RHT) projection to the suprachiasmatic nucleus (SCN). The principal neurotransmitter of this projection is glutamate, and ipRGCs use the vesicular glutamate transporter 2 (VGLUT2) to package glutamate into synaptic vesicles. However, these neurons contain other potential neurotransmitters, such as pituitary adenylate cyclase activating polypeptide (PACAP). To test the role of glutamate in mediating ipRGC light inputs into the SCN, we crossed mice in which Cre-recombinase expression is driven by the melanopsin promotor (Opn4(Cre/+)) with mice in which the second exon of VGLUT2 is flanked by loxP sites (VGLUT2(fl/fl)), producing ipRGCs that are unable to package glutamate into synaptic vesicles. Such mice had free-running circadian rhythms that did not entrain to a 12:12 light-dark (12:12 LD) cycle, nor did they show a phase delay after a 45-min light pulse administered at circadian time (CT) 14. A small subset of the mice did appear to entrain to the 12:12 LD cycle with a positive phase angle to lights-off; a similar entrainment pattern could be achieved in free-running mice if they were exposed to a 12:12 LD cycle with light of a greater intensity. Glutamate transmission from the ipRGCs is necessary for normal light entrainment of the SCN at moderate (0.35 W/m(2)) light levels, but residual transmission (possibly by PACAP in ipRGCs or by other RGCs) can weakly entrain animals, particularly at very high (6.53 W/m(2)) light levels, although it may be less effective at suppressing locomotor activity (light masking).

Differential expression of retinal proteins in a pineal parenchymal tumor.

The pineal gland and retina share histogenetic features that reflect a similar neurosensory/photosensory ontogeny. Pineal parenchymal tumors demonstrate a highly variable and incomplete photosensory differentiation evidenced by specific cytoarchitectural features and the expression of photosensory retinal S-Antigen (S-Ag). Despite these neuro-ontogenetic parallels, pineal parenchymal tumors have not been well studied for the neuroretinal phenotypes that accompany normal neuroretinal development. The investigation of photoreceptor gene expression may provide an important insight into the histogenesis of pineal parenchymal neoplasms. In this study, a pineal parenchymal tumor of the "mixed pineoblastoma/pineocytoma" type was examined for the expression of several photoreceptor, glial and neuronal proteins including: interphotoreceptor retinoid-binding protein (IRBP), rod opsin, cone opsin, S-Ag and cellular retinaldehyde-binding protein (CRA1BP). The detection of IRBP and its mRNA, the earliest photoreceptor-associated protein expressed during retinal development, corroborated the rudimentary photosensory differentiation of this tumor which had limited cytoarchitectural evidence for pineal differentiation. The analysis of IRBP expression may facilitate the diagnostic recognition of primitive pineal neoplasms and further define the neuroretinal differentiation which occurs in pineal parenchymal tumors.

Retinoblastoma-like phenotype expressed in medulloblastomas.

The previous demonstration of rod-opsin and S-antigen (S-Ag), a protein which arrests visual phototransduction, in retinoblastomas and in a subgroup of medulloblastomas has suggested a relationship between these tumors. We examined 17 medulloblastomas for the presence of a retinoblastoma-like phenotype. Overall 41% of the tumors were immunoreactive for S-Ag. Two tumors with well-differentiated Flexner-Wintersteiner rosettes were also immunoreactive for S-Ag, but not for epithelial membrane antigen (EMA). In contrast, most ependymal rosettes in two ependymomas stained positive for EMA along the luminal surface, consistent with a previous study, and were negative for S-Ag. Because calcification in areas of necrosis is a near constant finding in retinoblastomas, the medulloblastomas were evaluated for the presence of calcification, using Von Kossa staining. Forty-one percent showed calcification in areas of necrosis and 29% were positive for both calcification and S-Ag immunoreactivity. There was a statistically significant concordance between calcification and S-Ag immunoreactivity in the medulloblastomas (p < 0.05). Despite similar phenotypic features, a shared mechanism of tumori-genesis for retinoblastomas and the subgroup of medulloblastomas with photoreceptor differentiation could not be identified since all 17 medulloblastomas were found to express functional Rb protein, as indicated by positive nuclear immunoreactivity.