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[Molecular mechanisms of receptor-potential generation by the photoreceptor. I. Photic induction of a change in the permeability of artificial lipid membranes modified by photoreceptor membrane fragments].

Artificial lipid membranes modified by ultrasonic fragments of rod outer segments increase their conduction in response to illumination. Conduction increase is followed by the spontaneous foll in the dark to the unitial or somewhat higher level. The time constant of conduction increase was about 30 msec at room temperature, the constant of its following drop was about 300 msec; the activation energy of the last process was 19 +/- 3 kcal/mole.

Light

The lateral photoreceptor of the barnacle, Balanus eburneus: quantitative morphology and fine structure.

The lateral eye of the barnacle, Balanus eburneus, fixed in highly concentrated osmium is a lens-shaped body of approximately 250 mum in diameter and about 75 mum thick. It contains three photoreceptor cells which occupy about 42% of its volume. The photoreceptor cells are irregularly shaped and extend countless dendritic processes which bear rhabdomeres at their ends. Individual rhabdomeres come into contact with rhabdomeres originating from dendrites of the same or of one of the other visual cells. Thirteen per cent of the volume of the photoreceptor cells is taken up by the rhabdomeres. The membranes of the rhabdomeric microvilli contain globular subunits which suggest a 70 A spacing of rhodopsin molecules. There are two kinds of glial cells. One kind, type A glial cells, makes contact with the fibrous capsule of the photoreceptor. The other kind, type B glial cells, is associated with the photoreceptor cells and extends countless tiny cytoplasmic extensions which interdigitate with similar extensions of the receptor cells. There are approximately 95 type B glial cells and 130 type A glial cells in the receptor. The cytoplasm of the photoreceptor cells contains countless small Golgi fields, mitochondria, microtubules, multivesicular and multilamellar bodies. The extracellular space of the photoreceptor is less than 0.1% of its total volume.

Animals

Ultrastructure of photoreceptors in the eye of Hermissenda labelled with intracellular injections of horseradish peroxidase.

The terminal processes of single and of pairs of identified photoreceptors in the eyes of the nudibranch mollusc Hermissenda crassicornis were studied by light and transmission electron microscopy after their somata were labelled by intracellular iontophoresis of horseradish peroxidase (HRP). The HRP spread from the somata into the axons and fine terminal processes within the neuropil of the cerebropleural ganglia. The photoreceptors ended in extensive secondary branches in the neuropil where previous electrophysiological studies had indicated the probable site of synaptic interactions between photoreceptors. Clear round vesicles (54-126 nm diameter) within labelled processes were similar to the vesicles found in the somata and axon hillocks. The terminal processes of pairs of type B photoreceptors contained different intensities of the HRP label. Uniform intensities of the HRP label were found in the terminal processes of single type B photoreceptors. These differences in intensity suggested that the terminal processes were from different type B photoreceptors. This finding suggests that the connections between type B photoreceptors are probably monosynaptic.

Animals

The formation of photoreceptor synapses in the retina of larval Xenopus.

The first appearance and early development of synapses between photoreceptors, bipolar cells and horizontal cells were studied in the retina of larval Xenopus, between stages 37/8 and 46. Synapse morphology was reconstructed from examination of serial sections. Additional data were obtained from E-PTA stained tissue, Photoreceptors initially contacted horizontal cell dendrites via ribbon synapses at the photoreceptor basal surface (stages 37/8--39). Later, horizontal cell dendrites penetrated the receptor as digitiform processes (stages 39--40) which subsequently expanded extensively within the invagination (stages 40--44). At stages 41--46, a photoreceptor ribbon generally was associated with two horizontal processes in a synaptic dyad complex; each ribbon participated in 1 or 2 such complexes. Bipolar cell dendrites made contact with receptors via superficial or invaginating basal-type junctions. Such junctions were observed first at stage 39, but increased greatly in number beginning at stage 42. Basal junctions were characterized by marked staining of the paramembranous receptor cell cytoplasm and a 9--13 nm wide cleft containing an electron-dense material. No association of bipolar cell dendrites with photoreceptor ribbons was noted. Junctional complexes presumed to be gap junctions first were observed at stage 39 between photoreceptor bases and basal processes emitted by neighbouring photoreceptors.

Animals

Cholinergic features of photoreceptor synapses in Hermissenda.

1. A number of observations, as listed below, suggested a cholinergic basis for inhibitory interactions between photoreceptors of the eye in the nudibranch mollusk Hermissenda crassicornis. 2. The isolated eyes synthesized and accumulated acetylcholine but not other putative neurotransmitter substances. Synthesis and accumulation were determined by electrophoretic separation of products that incorporated radioactive label. Electron microscopic visualization of clear round vesicles within the photoreceptors' somata and axon hillocks was consistent with synthesis and storage of acetylcholine within these cells. 3. Pharmacologic experiments indicated the presence of cholinergic receptors on the terminal branches of the photoreceptors, which are pre- and postsynaptic to each other. Carbachol or nicotine produced hyperpolarization of the photoreceptors' membrane accompanied by a reduction of the input resistance. The reversal potential of carbachol-induced hyperpolarization coincided with the reversal potentials of the IPSPs that followed, one for one, impulses of neighboring photoreceptors. Eserine often caused blockade of the IPSPs. This blockade was associated with substantial membrane hyperpolarization and reduction of membrane resistance. 4. Neuronal endings within the optic tract in the area of the photoreceptor's terminal branches stained for acetylcholinesterase. 5. The results of these different experiments, especially when considered together, strongly suggest, although by no means unequivocally demonstrate, that the neurotransmitter of the photoreceptors is acetylcholine.

Acetylcholine

Calcium and the control of discrete wave latency in the ventral photoreceptor of Limulus.

1. Discrete, transient depolarization (discrete waves) of the ventral photoreceptor of the horseshoe crab, Limulus, occur spontaneously in the dark adapted photoreceptor and are also evoked by light. They form the basic events which comprise the receptor potential. A brief, low energy flash of light evokes variable numbers of discrete waves which have variable latencies. Evidence suggesting that discrete wave latency reflects the kinetics of the chemical reactions of phototransduction is reviewed. 2. The concentration of extracellular Ca influences both the average discrete wave latency and its variability. Lowering extracellular Ca prolongs the latency and increases its variability. Increasing extracellular Ca has the opposite effect. 3. Changes in discrete wave latency caused by changes in extracellular Ca require 10--15 min to become fully manifest, whereas when the concentration of extracellular K is increased the photoreceptor achieves a steady-state depolarization in 10-15 sec. 4. Iontophoresis of the Ca-chelating agent EGTA into the photoreceptor increases both the average discrete wave latency and its variability. Iontophoresis of Ca-EGTA mixtures may either increase or decrease discrete wave latency and its variability depending upon the proportion of Ca mixed with EGTA. 5. It is suggested that the concentration of intracellular rather than extracellular ionized Ca is the prime factor indicating discrete wave latency. The effects of changing extracellular Ca can be explained if the photoreceptor is permeable to Ca in the dark and if it maintains a low intracellular Ca concentration by virtue of active metabolic processes (a pump-leak system). 6. Lowering the temperature of the photoreceptor also has the dual effect of increasing discrete wave latency and its variability. However, effects of lowering temperature and Ca simultaneously are greater than the sum of the two effects in individually. This suggests that Ca may be a reactant in the chemical process of phototransduction. 7. Changing the concentration of extracellular Ca does not change the quantum efficiency of discrete wave production. A previous study showed that quantum efficiency is not changed by temperature. Thus, once initiated by the absorption of light, the reactions that subserve phototransduction may be forced to completion. Ca probably exerts its influence by changing one or more rate constants in the reaction sequence.

Action Potentials

Detection of light-induced changes of intracellular ionized calcium concentration in Limulus ventral photoreceptors using arsenazo III.

1. The metallochromic indicator dye, arsenazo III, was injected intracellularly into Limulus ventral photoreceptor cells to concentrations greater than 1 mM.2. The absorption spectrum (450-750 nm) of the dye in single dark-adapted cells was measured by a scanning microspectrophotometer. When a cell was light-adapted, the absorption of the dye changed; the difference spectrum had two maxima at about 610 and 660 nm, a broad minimum at about 540 nm and an isosbestic point at about 585 nm.3. When intracellular calcium concentration was raised in dark-adapted cells previously injected with arsenazo III, the difference spectum had two maxima at about 610 and 660 nm, a broad minimum at about 530 nm and an isosbestic point at about 585 nm. The injection of Mg(2+) into dark-adapted cells previously injected with the dye induced a difference spectrum that had a single maximum at about 620 nm. Also, decreasing the intracellular pH of cells previously injected with the dye induced a difference spectrum that had a minimum at about 620 nm. The evidence suggests that there is a rise of intracellular ionized calcium when a Limulus ventral photoreceptor is light-adapted.4. The intracellular calcium concentration, [Ca(2+)](1), in light-adapted photoreceptors was estimated to reach at least 10(-4)M by compaing the light-induced difference spectra measured in ventral photoreceptors with a standard curve determined in microcuvettes containing 2mM arsenazo III in 400 mM-KCl, 1 mM-MgCl(2) and 25 mM MOPS at pH 7.0.5. In cells injected to less than 3 mM arsenazo III, light induced a transient decrease in optical transmission at 660 nm (T(660)). This decrease in T(660) indicates that illumination of a ventral photoreceptor normally causes a transient increase of [Ca(2+)](1).6. Arsenazo III was found to be sensitive, selective and rapid enough to measure light-induced changes of intracellular ionized calcium in Limulus ventral photoreceptor cells.

Animals

Novel photoreceptor-specific promoters for gene therapy in mid- to late-stage retinal degeneration.

Inherited retinal degenerations (IRDs) cause progressive photoreceptor loss, leading to vision impairment. Gene therapy using adeno-associated viral (AAV) vectors holds immense promise for treating these conditions. However, achieving optimal gene expression at mid to late stages of retinal degeneration remains challenging due to scarcity of efficient photoreceptor-specific promoters expressed at these disease stages. This study aimed to identify and validate novel promoters capable of robust and specific transgene expression when ≥50% of photoreceptors are lost. Analysis of transcriptomic data from two naturally occurring canine IRD models, laser capture microdissection of retinal cryosections followed by qPCR, and RNA in situ hybridization identified six promising genes with sustained or upregulated expression in photoreceptors in late-stage disease. Upstream cis-regulatory elements of both canine and human orthologs were identified and characterized using in silico analyses and dual-luciferase assays. Short promoters (≤840 base pairs) derived from GNGT2, IMPG2, and PDE6H genes exhibited robust reporter gene expression in photoreceptors when delivered via AAV to the subretinal space of two non-allelic canine IRD models at mid and late disease stages. These findings provide a strategy to enhance AAV-mediated gene therapy by enabling sustained transgene expression in degenerating retinas, improving treatment outcomes for patients with progressive vision loss.

Retinal Degeneration

Renewal of normal and degenerating photoreceptor outer segments in the Ozark cave salamander.

To determine whether photoreceptor degeneration in the Ozark cave salamander is associated with cessation or changes in the kinetics of outer segment (OS) renewal, an autoradiographic study of 3H-leucine incorporation in photoreceptors was carried out. Six days after isotope injection rods and cones showed labeling in both inner and outer segments. Cone OS were diffusely labeled whereas rods contained a band of radioactivity at the base of the OS. At 13 and 21 days the radioactive band in rods was located progressively nearer the distal tip of the OS. The rate of rod OS renewal ranged from 0.30 to 0.38 mu of OS length per day at 18 degrees C. L-thyroxin induced metamorphosis and light increased the renewal rate compared to larvae in darkness, and adults with photoreceptors in an early stage of degeneration had a slightly higher renewal rate than larvae. Light and electron microscope autoradiographs of degenerate photoreceptors revealed that even in the final stages of degeneration when OS are reduced to small, irregular whorls of membrane, 3H-leucine labeling was present in inner segments and OS membranes. These observations demonstrate that OS renewal occurs in both larvae and adults, and suggest that photoreceptor degeneration may be due to disruption of some aspect of the OS disposal process.

Animals

Morphology and responses to light of the somata, axons, and terminal regions of individual photoreceptors of the giant barnacle.

1. The median eye of the giant barnacle, B. nubilus, comprises four large photoreceptor neurones which are visible under the dissecting microscope for almost their entire length. We have studied the structure of, and the responses to light recorded in, the somata, axons, and terminal regions of these neurones.2. The photoreceptor somata, each 40-70 mum in diameter, extend numerous light-sensitive dendritic processes whose membranes form rhabdomeric microvilli. Recordings from the soma show that dim light evokes a steady, noisy depolarization; brighter light elicits a transient depolarization which decays to a maintained plateau, followed by a hyperpolarization when the light is turned off.3. Light-induced voltage changes spread decrementally along the photoreceptor axons, which average 10 mm in length and 25 mum in diameter. In distal parts of the axon, near the presynaptic terminals, depolarizations and hyperpolarizations can be as large as 50% or more of their values in the soma.4. There is no demonstrable electrical coupling between photoreceptor neurones as shown by simultaneous recordings from two receptor somata or axons.5. Each photoreceptor axon enters the mid line commissure of the supraoesophageal ganglion, bifurcates, and arborizes in a restricted zone of neuropil in each hemiganglion. The large size of the terminal processes of these neurones and their characteristic cytoplasmic inclusions enable one to trace them with the electron microscope as they branch in the neuropil.6. The terminal processes subdivide and end in 1-3 mum diameter branches which are the sites of apparently chemical synapses. Vesicle-containing, presynaptic loci on these processes of the receptor cell are invariably apposed to two post-synaptic processes from cells as yet unidentified.

Animals

[Electron microscopic study of presumptive photoreceptor cells in the aboral organ of the ctenophore, Beroë cucumis].

Presumptive photoreceptor cells are located in the receptor epithelium of the aboral organ and exhibit some common morphological characters with flagellar photoreceptors of some invertebrates, as well as with photoreceptors of vertebrates. These cells are specifically distinguished by the presence of a special membrane structure in their epinuclear zone, this structure being the derivative of a cilium. Presumptive photoreceptor cells which have a short central projection and form in their basal part the synaptic contacts ("triads") with the adjacent nervous endings, may be classified as pseudosecondary receptor cells. Acetylcholinesterase was found in the synaptic regions. Cytoplasm of the presumptive photoreceptor cells is rich in granular vesicles which contain biogenic amines.

Animals

Enzymic basis for cyclic GMP accumulation in degenerative photoreceptor cells of mouse retina.

The activities of guanylate cyclase, guanosine 3', 5'-monophosphate (cyclic GMP) phosphodiesterase and 5'-nucleotidase were measured during postnatal development in retinas of control and C3H/HeJ mice. In control retina, each of these enzyme activities increases in conjunction with photoreceptor cell differentiation and maturation. In C3H retina, guanylate cyclase and 5-nucleotidase activities increase with photoreceptor cell development and decrease with photoreceptor cell death. However, the activity of a class of cyclic GMP phosphodiesterase which distinguishes the photoreceptor cells of control mice and those of several other species is not demonstrable in retina of C3H mice at any age. It is suggested that the deficiency in cyclic GMP phosphodiesterase activity may account for the accumulation of cyclic GMP which has been shown to occur in the C3H photoreceptor cells before they degenerate.

Aging

Developmental analysis of the cone photoreceptor-less little skate retina reveals distinct Onecut1 isoforms.

The retinal development of elasmobranchs, the subclass comprising sharks, skates, and rays, remains poorly understood. This group is diverse in retinal phenotype, with many sharks and rays possessing rods together with one or more cone types. In contrast, the little skate (Leucoraja erinacea) has only a single rod photoreceptor type, which has been reported to exhibit some physiological and anatomical properties associated with cones. To investigate how this unusual photoreceptor system develops, we first identified an embryonic stage of early photoreceptor formation based on otx2 expression. We then developed a retinal electroporation approach to test whether a onecut1-dependent cone-associated reporter could be activated in the embryonic skate retina. Activation of this reporter was not detected, indicating that the corresponding enhancer is not robustly active under the conditions tested. To assess developmental changes in gene expression, we generated bulk RNA-seq datasets from embryonic, hatchling, and adult retinas. These analyses showed strong embryonic expression of onecut1, increasing expression of rod-associated genes through development, and pseudogenization or loss of multiple cone-enriched genes. We further identified a developmentally regulated onecut1 splice isoform containing an additional 48 amino acid sequence between the CUT and homeodomain DNA-binding domains. This spacer-containing isoform, termed LSOC1X2, was most abundant in the embryonic retina. To test whether LSOC1X2 retained regulatory activity, we assayed it in a mouse retinal reporter system. Both skate Onecut1 isoforms activated the ThrbCRM1 reporter in this heterologous context. Together, these findings identify a novel, developmentally regulated retinal onecut1 isoform in the little skate and establish it as a candidate regulator for future studies of photoreceptor development in this species and its elasmobranch relatives.

Animals

Scanning electron microscopy of developing photoreceptors in the chick retina.

Photoreceptor morphogenesis in the sensory retina of chicks of 2 to 20 days incubation age was studied by scanning electron microscopy (SEM) and correlative transmission electron microscopy (TEM). At 9.5 days spherical inner segments extend into the subretinal space (optic ventricle). They are randomly arranged with chiefly smooth surfaces and contain centrioles, polyribosomes and microtubules. Microvilli project primarily from Müller cells. By the twelfth day immature ellipsoid and myoid regions have formed. Microvilli are abundant on the lateral surfaces of inner segments and extend over the entire spherical surface by the fifteenth day. Occasional cilia with surrounding depressions at their bases were also observed. Inner segments are more symmetrically arranged due to close proximity of photoreceptor cells. Inner segments elongate during the sixteenth day; many display a transitional ovoid form. Microvilli become less numerous but some persist as calycal processes. By the eighteenth day, conical shaped outer segments appear. Thereafter, all photoreceptor cells are comparable to those in the mature retina. Abundant microvilli on the external surface of the sensory retina suggest a supportive role in supplying adequate nutrition to the sensory retina during morphogenesis. The establishment and continual development of the ellipsoid and myoid appear to be primarily responsible for the elongation of photoreceptor cells.

Animals

Differentiation of photoreceptors and horizontal cells in the embryonic mouse retina: an electron microscopic, serial section analysis.

The early differentiation of photoreceptors and horizontal cells in the mouse retina has been studied with serial thin sections and reconstructions in embryos on the fifteenth and seventeenth days of gestation (E15 and E17). The following developmental sequences have been inferred. At E15 photoreceptors develop from ventricular cells when a long vitreal process fails to develop following mitosis, and the end of the ventricular process forms a bulbous enlargement (the future inner segment) which contains a pair of centrioles and a cilium and extends into the optic ventricle. This future inner segment is considerably larger at E17, but otherwise the photoreceptors resemble those seen at E15. At E15 horizontal cells develop from ventricular cells when a long vitreal process fails to develop following mitosis, and the end of the ventricular process detaches from the junctional complex at the ventricular surface. By E17 future horizontal cells are located in the middle of the ventricular layer (neuroblastic layer) and have developed from bipolar shaped cells into cells with multiple branching processes, predominantly radially arranged but rarely with a more tangential orientation. These relatively advanced cells at E17 resemble closely the earliest stage of horizontal cell formation described previously in silver studies by Cajal. A scheme is proposed which explains the initial differentiation of several of the major cell types in the retina in terms of two key features: whether or not the call remains attached to the junctional complex and whether or not a vitreal process grows into the ganglion cell layer. By independent variations in these two features, four classes of cells are produced that, by virtue of their differing environments, differentiate into four cell types: ganglion (and amacrine) cells, horizontal cells, photoreceptors, and Müller cells.

Animals

Neurotransmitter synthesis in Limulus ventral nerve photoreceptors.

Radiochemical precursor compounds for neurotransmitters were incubated with Limulus ventral nerve photoreceptor preparations. Octopamine was preferentially synthesized by a photoreceptor rich fraction of the nerve, acetylcholine was made by a photoreceptor poor fraction, and gamma-aminobutyric acid was made about equally well in both fractions. The possibility that the ventral nerve photoreceptor cells serve a neurosecretory function in the adult Limulus is discussed.

Acetylcholine

A quantitative comparison of the effects of intracellular calcium injection and light adaptation on the photoresponse of Limulus ventral photoreceptors.

Calcium ions were iontophoretically injected into ventral photoreceptors of Limulus by passing current between two intracellular pipettes. Changes in sensitivity and photoresponse time course were measured for both light adaptation and Ca++ injection. We found for some photoreceptors that there was no significant difference in the photoresponse time course for desensitization produced by light adaptation or by Ca++ injection. In other photoreceptors, the time delay of photoresponse for Ca++ injection was slightly longer than for light adaptation. The variability of threshold response amplitude and time delay decreases when the photoreceptor is desensitized by either light adaptation or Ca++ injection. The peak amplitude versus log stimulus intensity relationships for controls, light adaptation, and Ca++ injection all could be described very closely by a single template curve shifted along the log intensity axis. A 40- to 50-fold change in sensitivity is associated with a 2-fold change in photoresponse time delay for both light adaptation and Ca++ injection.

Adaptation, Ocular

Visual pigment and photoreceptor sensitivity in the isolated skate retina.

Photoreceptor potentials were recorded extracellularly from the aspartate-treated, isolated retina of the skate (Raja oscellata and R. erinacea), and the effects of externally applied retinal were studied both electrophysiologically and spectrophotometrically. In the absence of applied retinal, strong light adaptation leads to an irreversible depletion of rhodopsin and a sustained elevation of receptor threshold. For example, after the bleaching of 60% of the rhodopsin initially present in dark-adapted receptors, the threshold of the receptor response stabilizes at a level about 3 log units above the dark-adapted value. The application of 11-cis retinal to strongly light-adapted photoreceptors induces both a rapid, substantial lowering of receptor threshold and a shift of the entire intensity-response curve toward greater sensitivity. Exogenous 11-cis retinal also promotes the formation of rhodopsin in bleached photoreceptors with a time-course similar to that of the sensitization measured electrophysiologically. All-trans and 13-cis retinal, when applied to strongly light-adapted receptors, fail to promote either an increase in receptor sensitivity or the formation of significant amounts of light-sensitive pigment within the receptors. However, 9-cis retinal isin. These findings provide strong evidence that the regeneration of visual pigment in the photoreceptors directly regulates the process of photochemical dark adaptation.

Adaptation, Physiological