Rod sensitivity and visual pigment concentration in Xenopus.

Xenopus larvae were raised on a vitamin A-free diet under constant illumination until their visual pigment content had decreased to between 8% of normal and an undetectably low level. After the intramuscular injection of 2.1 X 10(13-2.1 X 10(16) molecules of [3H]vitamin A, ocular tissue showed a rapid rate of uptake of label which reached a maximum level of incorporation by 48 h. Light-microscopic autoradiography revealed that the retinal uptake of label was concentrated within the receptor outer segments. Spectral transmissivity measurements at various times after injection were made upon intact retinas and upon digitonin extracts. They showed that visual pigment with a lambdamax of 504 nm was formed in the retina and that the amount formed was a function of incubation time and the magnitude of the dose administered. Electrophysiological measures of photoreceptor light responses were obtained from the PIII component of the electroretinogram, isolated with aspartate. The quantal flux required to elicit a criterion response was determined and related to the fraction of visual pigment present. The results showed that rod sensitivity varied linearly with the probability of quantal absorption.

Formation, conversion, and utilization of isorhodopsin, rhodopsin, and porphyropsin by rod photoreceptors in the Xenopus retina.

The visual pigment content of rod photoreceptors in Xenopus larvae was reduced greater than 90% through a combination of vitamin A-deficient diet and constant light. Thereafter, a dose of either all-trans-retinol or 9-cis-retinal was injected intramuscularly, leading to the formation of a rhodopsin (lambdamax 504 nm) or isorhodopsin (lambdamax 487-493 nm) pigment, respectively. Electrophysiological measurements were made of the threshold and spectral sensitivity of the aspartate-isolated PIII (photoreceptoral) component of the electroretinogram. These measures established that either rhodopsin or isorhodopsin subserved visual transduction with the same efficiency as the 519 nm porphyropsin pigment encountered normally. When animals with rhodopsin or isorhodopsin were kept in darkness or placed on a cyclical lighting regimen for 8 days, retinal densitometry showed that either pigment was being converted to porphyropsin; significantly more porphyropsin was formed as a result of cyclical lighting than after complete darkness.

Glial cells of the parietal eye: structural and biochemical similarities to retinal Müller cells.

The parietal eye of lizards is a relatively simple yet highly structured visual organ. Cone-like photoreceptors synapse directly onto ganglion cells that project to the brain. Golgi staining confirmed the existence of glial cells spanning the sensory epithelium in a manner analogous to the Müller cells of the vertebrate lateral eye retina. These parietal eye glial cells bear expanded end feet at the basal border of the eye. Electron microscopic examination revealed some ultrastructural similarity to Müller cells. Mitochondria, osmiophilic and transparent vesicles, and the Golgi apparatus are found in the apical end of the parietal eye glial cell. Junctional complexes join adjacent parietal eye glial cells and their neighboring photoreceptor cells. Bundles of filaments are found in the basal end of the cell and the plasma membranes of adjacent cells often intertwine tightly in this region. The parietal eye glial cells are immunoreactive to antibodies against glutamine synthetase, also characteristic of Müller cells. Some differences between parietal eye glial cells and Müller cells also are evident. The parietal eye glial cells are not immunoreactive to antibodies against another Müller cell marker, carbonic anhydrase II, and many of them contain melanin granules, while Müller cells are not pigmented. In addition, we have found that the lens cells of the parietal eye and the Müller cells of the lateral eye retina of lizards are immunoreactive to antibodies against both glutamine synthetase and carbonic anhydrase II.

Serotonin and dopamine in the retina of a lizard.

The objective of this paper is to report the presence and localization of serotonin and dopamine in the retina of the lizard Uta stansburiana. High performance liquid chromatography and electrochemical detection were used to identify and quantitate the two amines. Both compounds are present as endogenous molecules in this retina and are found in concentrations similar to those reported in other non-mammalian retinas. The same methods were employed to confirm, in the isolated retina, the synthesis of serotonin from precursor, tryptophan. Immunocytochemical methods were used to localize, in the neural retina, serotonin and the rate-limiting enzyme of dopamine synthesis, tyrosine hydroxylase. Serotonin immunoreactivity was observed in bistratified amacrine cells (ca. 7 micron dia.) with processes ramifying in sublayers 1, 4, and 5 of the inner plexiform layer. Immunoreactivity to tyrosine hydroxylase was observed in a different population of bistratified amacrine cells (ca. 11 micron dia.) that had processes ramifying in sublayers 1 and 5 (and perhaps 3) of the inner plexiform layer. The enzymes for further metabolism of dopamine were not found in the retina of this lizard by immunocytochemical methods. The results of this research suggest that only single classes of serotoninergic and dopaminergic neurons are present in the retina of U. stansburiana. This retina might, therefore be an appropriate place in which to investigate the functioning of these amines in visual information processing.

Neuropeptide immunoreactivity in Limulus. I. Substance P-like immunoreactivity in the lateral eye and photocerebrum.

Substance P-like immunoreactivity was examined in the Limulus lateral eye and photocerebrum by the unlabeled antibody peroxidase-antiperoxidase method. Small-diameter immunoreactive fibers innervate the corneal epidermis of the lateral eye and appear to be part of a generalized epidermal innervation. No immunoreactive neurons or fibers were found in the ventral, median, or lateral optic nerves nor in the lamina or chiasma. The optic medulla contains neurons with immunoreactive perinuclear caps in the ganglion cell layer and fibers of undetermined origin in the posterolateral regions of the neuropil. The central body contains many immunoreactive fibers in its neuropil, some or all of which arise from neurons adjacent to its anteromedial tips. Immunoreactive neurons within the curve of the central body give rise to processes which join the central neuropil of the photocerebrum. The immunoreactive innervation of the corpora pedunculata includes processes which terminate amont Kenyon cell somata, and processes in the peduncular neuropil, all of which arise from a bilateral cluster of neurons along the midline. Attempts to investigate the source of the immunoreactive fibers using cobalt impregnation of the severed circumesophageal connective have revealed two additional innervations of the corpora pedunculata, one from raphe neurons along the midline and one from lateral portions of the midline ganglion adjacent to but separate from that portion containing the immunoreactive neurons. The results demonstrate that immunocytochemical methods are another powerful tool for the study of neuronal pathways in invertebrates.

Habenular asymmetry and the central connections of the parietal eye of the lizard.

Histochemical and autoradiographic analyses of the axonal transport of horseradish peroxidase and tritiated amino acids were employed to study the central connectivity of the lizard parietal eye. Somata and processes of centrifugal fibers to the parietal eye were localized in tissue of the dorsal sac and in the leptomeningeal sheath of the pineal gland. Analyses of series of transverse sections of the brain showed the left medial habenular nucleus to be subdivided into pars dorsolateralis and pars ventromedialis, and the right medial habenular nucleus not to be so subdivided. Centripetal fibers of parietal eye ganglion cells project to only the pars dorsolateralis of the left medial habenular nucleus and terminate there in two distinct fields. The asymmetry of the lizard habenula may be a specialization associated with the unilateral projection from the parietal eye.

GABA as a potential transmitter in lizard photoreceptors: immunocytochemical and biochemical evidence.

The retina of the desert night lizard, Xantusia vigilis, was examined for immunoreactivity to antibodies against gamma-aminobutyric acid and L-glutamate decarboxylase. At the electron microscopic level it was found that a distinct population of the photoreceptor cells was immunoreactive to both antibodies. Computer-assisted reconstruction of serial sections positively identified the immunoreactive receptors as cones. These cones constituted 15% of the photoreceptors in the retinal sections, and they were morphologically distinct. The mean diameter of the labeled cone synaptic pedicles was 5.8 micron whereas that of the unlabeled pedicles was 7.9 micron, a statistically significant difference. L-glutamate decarboxylase was extracted from the lizard brain, positively identified radiometrically, and shown by immunodiffusion to crossreact with the antibody used for localization. The authors suggest that the immunoreactive cones synthesize and accumulate gamma-aminobutyric acid. Whether or not it is used by those cones as a neurotransmitter should be tested directly.

Identification of putative neurotransmitters in the lizard parietal eye.

The retina of the parietal eye of lizards is a simple vertebrate retina that may be useful in studies of basic principles of information processing in visual systems. The chemical substances which mediate the cell-to-cell communication of this eye are not known. As a step in attempting to understand the cellular basis for visual information processing the authors have studied the ability of the parietal eye to synthesize conventional neurotransmitter substances. The eyes were incubated in radiolabeled precursors, then an extract of the tissue was subjected to high-voltage paper electrophoresis. The results of this study indicate that gamma-amino butyric acid (GABA) was synthesized from glutamic acid, acetylcholine (ACh) was synthesized from choline and serotonin (5-HT) was synthesized from tryptophan. Endogenous 5-HT was localized at sites outside the sensory epithelium by immunohistochemical means. Autoradiography following an incubation with 3H-GABA revealed uptake by lens cells and probably both glia and neurons. The authors conclude that GABA and ACh may be involved in the processing of visual information within the parietal eye but that 5-HT probably is not.

Disk shedding in the absence of a pigment epithelium in the lizard parietal eye.

The photoreceptors and pigment epithelia of vertebrate retinas rhythmically synthesize and degrade photosensitive membrane, but the origin of the signal to shed the outer segment tips remains unknown. The parietal eye of lizards contains cone-like photoreceptors but no pigment epithelium. Parietal eye photoreceptors synthesize new disk membrane in a manner similar to rods and cones and also shed their tips rhythmically. The shed material is then engulfed by lumenal macrophages. The signal to shed must originate in, or be transduced by, the photoreceptor.

Hypoglycemia leads to age-related loss of vision.

The retina is among the most metabolically active tissues in the body, requiring a constant supply of blood glucose to sustain function. We assessed the impact of low blood glucose on the vision of C57BL/6J mice rendered hypoglycemic by a null mutation of the glucagon receptor gene, Gcgr. Metabolic stress from moderate hypoglycemia led to late-onset loss of retinal function in Gcgr(-/-) mice, loss of visual acuity, and eventual death of retinal cells. Retinal function measured by the electroretinogram b-wave threshold declined >100-fold from age 9 to 13 months, whereas decreases in photoreceptor function measured by the ERG a-wave were delayed by 3 months. At 10 months of age Gcgr(-/-) mice began to lose visual acuity and exhibit changes in retinal anatomy, including an increase in cell death that was initially more pronounced in the inner retina. Decreases in retinal function and visual acuity correlated directly with the degree of hypoglycemia. This work demonstrates a metabolic-stress-induced loss of vision in mammals, which has not been described previously. Linkage between low blood glucose and loss of vision in mice may highlight the importance for glycemic control in diabetics and retinal diseases related to metabolic stress as macular degeneration.

Parietalectomy and thermal selection in the lizard Sceloporus magister.

Lizards were acclimatized to various regimes of temperature (15 degrees, 35 degrees C) and photoperiod (LD 08:16, 16:8). Parietalectomized, sham operated, and control animals were placed in thermal gradients and their body temperatures monitored for periods of up to 15 days. Daily rhythms of thermal selection were evident with higher temperatures selected in late photophase and lower temperatures selected in scotophase. Thermal selection was more variable in scotophase than in photophase. Parietalectomized lizards chose significantly higher body temperatures than did shams or controls. Acclimatization to temperature and photoperiod had little or no effect on the thermal preferendum. The preferred body temperature of controls was 33.2 degrees C +/- 3.59. Length of time in the gradient tended to change thermal preference, especially in lizards acclimated to 15 degrees C. Through interactions with the pineal gland the parietal eye is probably important in synchronizing many bodily functions with photoperiod.

Parietal eye of the lizard: neuronal photoresponses and feedback from the pineal gland.

The parietal eye of the lizard responds to illumination by sending afferent impulses to the pineal gland during daylight, the photophase. The pineal gland has efferently conducting neurons which are especially sensitive to norepinephrine and whose feedback to the parietal eye enhances its photo responsiveness. During the scotophase, at night, the eye generates afferent impulses to the cessation of light and the pineal efferents are most sensitive to serotonin. Thus, the photo-and chemoresponses of this system of interacting neurons are nearly reversed during the two phases of the daily photoperiod of the lizard.

Electroretinogram of the parietal eye of lizards: photoreceptor, glial, and lens cell contributions.

Local electroretinograms (ERGs) were recorded in the parietal eye of Xantusia vigilis. The responses to monochromatic light under dark- and light-adapted conditions were studied. We found that two antagonistic chromatic mechanisms dominate the overall response. With the electrode tip in the lumen of the eye, light stimulation under dark-adapted conditions evoked responses of negative polarity with maximum sensitivity to green light. Intense green background illumination saturated the green-sensitive mechanism, and superposition of a blue stimulus then elicited responses of opposite polarity, driving the potentials back toward the dark resting level. The spectral sensitivities of the two chromatic mechanisms were determined using chromatic adaptation. The lower threshold, green-sensitive mechanism has a maximum sensitivity at 495 nm while the antagonistic mechanism, with its maximal spectral sensitivity at 430 nm, is at least 2 log units less sensitive. The polarity of the ERG recording inverts as the electrode traverses the photoreceptor layer, suggesting that the photoreceptors are the major source of the ERG. This result was confirmed with intracellular recordings from photoreceptors, glial, and lens cells. The glial and lens cells of the parietal eye respond to local changes in [K+]o. Intracellular recordings of the responses of these cells to light stimuli follow time courses similar to changes in extracellular potassium concentrations measured with K+ -specific electrodes. These results suggest that the glial and lens cell membranes are highly permeable to potassium and, therefore, the electrical responses of these cells are evoked by changes in [K+]o elicited by light stimulation of the photoreceptors. Nevertheless, the major component of the parietal eye ERG is the photoreceptor signal. A circuit model of the ERG sources is presented.

Neuronal structure of the lacertilian parietal eye, I: A retrograde label and electron-microscopic study of the ganglion cells in the photoreceptor layer.

The cellular connectivity of the lacertilian parietal eye is not well understood. Because the intercellular connections establish the foundation for information processing, we have investigated cellular connectivity of one cell type in this simple vertebrate retina. We also developed an in vitro preparation to study the anatomy of the parietal eye visual system. Horseradish peroxidase transport in the in vitro preparation revealed a class of displaced ganglion cells occupying positions among the photoreceptors, in a location where the presence of interneurons had been suggested. Three-dimensional reconstruction at the electron-microscopic level showed that the morphology and synaptic input of these displaced ganglion cells is different from that of the previously known ganglion cells. The displaced ganglion cells receive an average of about 13 ribbon synapses from photoreceptors. The ribbon input is equally distributed between the soma and dendritic arbor. Junctional membrane measurement and ethanolic phosphotungstic acid-staining provided evidence for the existence of non-ribbon synaptic contacts (synaptoid junctions). Displaced ganglion cells make about 20 synaptoid junctions, 65% of which are on the dendritic arbor. The morphology of the displaced ganglion cell is such that a significant measure of synaptic input to the dendritic arbor will be transmitted to the soma.

Antagonistic chromatic mechanisms in photoreceptors of the parietal eye of lizards.

Photoreceptors are the first in the chain of neurons that process visual information. In lateral eyes of vertebrates, light hyperpolarizes rod and cone photoreceptors that synapse onto bipolar and horizontal cells in the first synaptic layer of the retina. The sign of the photoreceptor signal is either conserved or inverted in bipolar cells, resulting in chromatically dependent depolarizing and hyperpolarizing responses to visual stimuli. Visual information is then conveyed to the second synaptic layer for encoding and transmission to the brain by ganglion cells. The parietal (third) eye of lizards does not contain bipolar cells or other interneurons. Photoreceptors synapse directly onto ganglion cells and yet, even in the absence of interneurons, antagonistic chromatic mechanisms modulate the ganglion cell responses. We report here that chromatic antagonism in the third eye originates in the chromatically dependent hyperpolarizing and depolarizing response of the photoreceptors to light. We also suggest that the antagonistic nature of these photoresponses may provide lizards with a mechanism for the enhanced detection of dawn and dusk.

Substance P-like immunoreactivity in the parietal eye visual system of the lizard Uta stansburiana.

We examined the parietal eye visual system of the iguanid lizard Uta stansburiana for the presence of substance P-like immunoreactivity by use of both immunofluorescence and peroxidase-antiperoxidase techniques. In the parietal eye no substance P-containing somata were found; however, its plexiform layer contained small (ca. 1 micron diam) immunoreactive fibers. These fibers apparently originate outside the parietal eye. Immunoreactive fibers also were found in the parietal nerve, the dorsal sac, and the leptomeninx of the pineal gland. No labeled somata were observed in any of these regions in either normal or colchicine treated animals. Previously we demonstrated that a system of centrifugal fibers to the parietal eye originates from neurons in the dorsal sac (Engbretson et al. 1981). The apparent absence of substance P-containing neurons in the dorsal sac suggests that the substance P-containing fibers in the parietal eye are not the previously observed centrifugal fibers. The source of the substance P-containing fibers in the parietal eye is unknown. The pars dorsolateralis of the left medial habenular nucleus receives a dense substance P-positive projection. No such projection was seen in the right habenula. Simultaneous visualization of the terminals of ganglion cells of the parietal eye (labeled with orthograde intraaxonally transported horseradish peroxidase) and substance P-like immunofluorescence showed that the locus of habenular immunoreactivity is distinct from the projection field of the parietal eye. Thus the substance P-positive terminals in the habenula do not originate in the parietal eye. Transection of the parietal nerve confirmed this conclusion.