Research Article
Gap junctional coupling and connexin immunoreactivity in rabbit retinal glia
- KATHLEEN R. ZAHS, PAUL W. CEELEN
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- Published online by Cambridge University Press:
- 09 March 2006, pp. 1-10
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Gap junctions provide a pathway for the direct intercellular exchange of ions and small signaling molecules. Gap junctional coupling between retinal astrocytes and between astrocytes and Müller cells, the principal glia of vertebrate retinas, has been previously demonstrated by the intercellular transfer of gap-junction permeant tracers. However, functional gap junctions have yet to be demonstrated between mammalian Müller cells. In the present study, when the gap-junction permeant tracers Neurobiotin and Lucifer yellow were injected into a Müller cell via a patch pipette, the tracers transferred to at least one additional cell in more than half of the cases examined. Simultaneous whole-cell recordings from pairs of Müller cells in the isolated rabbit retina revealed electrical coupling between closely neighboring cells, confirming the presence of functional gap junctions between rabbit Müller cells. The limited degree of this coupling suggests that Müller cell–Müller cell gap junctions may coordinate the functions of small ensembles of these glial cells. Immunohistochemistry and immunoblotting were used to identify the connexins in rabbit retinal glia. Connexin30 (Cx30) and connexin43 (Cx43) immunoreactivities were associated with astrocytes in the medullary ray region of the retinas of both pigmented and albino rabbits. Connexin43 was also found in Müller cells, but antibody recognition differed between astrocytic and Müller cell connexin43.
The nob2 mouse, a null mutation in Cacna1f: Anatomical and functional abnormalities in the outer retina and their consequences on ganglion cell visual responses
- BO CHANG, JOHN R. HECKENLIVELY, PHILIPPA R. BAYLEY, NICHOLAS C. BRECHA, MURIEL T. DAVISSON, NORM L. HAWES, ARLENE A. HIRANO, RONALD E. HURD, AKIHIRO IKEDA, BRITT A. JOHNSON, MAUREEN A. MCCALL, CATHERINE W. MORGANS, STEVE NUSINOWITZ, NEAL S. PEACHEY, DENNIS S. RICE, KIRSTAN A. VESSEY, RONALD G. GREGG
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- 09 March 2006, pp. 11-24
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Glutamate release from photoreceptor terminals is controlled by voltage-dependent calcium channels (VDCCs). In humans, mutations in the Cacna1f gene, encoding the α1F subunit of VDCCs, underlie the incomplete form of X-linked congenital stationary night blindness (CSNB2). These mutations impair synaptic transmission from rod and cone photoreceptors to bipolar cells. Here, we report anatomical and functional characterizations of the retina in the nob2 (no b-wave 2) mouse, a naturally occurring mutant caused by a null mutation in Cacna1f. Not surprisingly, the b-waves of both the light- and dark-adapted electroretinogram are abnormal in nob2 mice. The outer plexiform layer (OPL) is disorganized, with extension of ectopic neurites through the outer nuclear layer that originate from rod bipolar and horizontal cells, but not from hyperpolarizing bipolar cells. These ectopic neurites continue to express mGluR6, which is frequently associated with profiles that label with the presynaptic marker Ribeye, indicating potential points of ectopic synapse formation. However, the morphology of the presynaptic Ribeye-positive profiles is abnormal. While cone pedicles are present their morphology also appears compromised. Characterizations of visual responses in retinal ganglion cells in vivo, under photopic conditions, demonstrate that ON-center cells have a reduced dynamic range, although their basic center-surround organization is retained; no alteration in the responses of OFF-center cells was evident. These results indicate that nob2 mice are a valuable model in which to explore the pathophysiological mechanisms associated with Cacna1f mutations causing CSNB2, and the subsequent effects on visual information processing. Further, the nob2 mouse represents a model system in which to define the signals that guide synapse formation and/or maintenance in the OPL.
Photic history modifies susceptibility to retinal damage in albino trout
- W. TED ALLISON, TED E. HALLOWS, TRUDI JOHNSON, CRAIG W. HAWRYSHYN, DONALD M. ALLEN
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- 09 March 2006, pp. 25-34
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Albino vertebrates exposed to intense light typically lose photoreceptors via apoptosis, and thus serve as useful models of retinal degeneration. In contrast, albino rainbow trout exposed to intense light maintain populations of rod and cone nuclei despite substantial damage to rod outer segments (ROS). The aim of this study was to differentiate between two hypotheses that could account for this divergent result: (1) trout rod nuclei remain intact during light damage, or (2) rod nuclei die but are replaced by cell proliferation. A further aim was to examine whether photic history modulates retinal damage, as in rodents. Albino and normally pigmented trout were moved from defined photic regimes into full daylight, while some were not moved to serve as protected controls. ROS were always maintained in pigmented fish and in albinos protected from full daylight. In albinos exposed to full daylight, ROS were removed over most of the central retina, whereas rod nuclei were maintained in the outer nuclear layer over 10 days. Pyknotic and TUNEL-labeled rod nuclei were abundant in affected albinos at all time-points tested. Rod death occurred without a decrease in the number of rod nuclei, confirming that proliferation must be replacing cells. Indeed a transient increase in proliferation was observed in retinal progenitors of albinos receiving 5 days of damaging light. This proliferative response was decreased with further damage. Cones remained intact even in areas where rod nuclei had degenerated. Pretreatment with light of moderate versus low intensity light affected the cell death and proliferative responses, and the ectopic localization of rod opsin. We conclude that apoptotic demise of rods, but not cones, occurred during light damage in retinas of albino trout and proliferative responses have a limited a capacity to replace lost rods.
Natural images and contrast encoding in bipolar cells in the retina of the land- and aquatic-phase tiger salamander
- DWIGHT A. BURKHARDT, PATRICK K. FAHEY, MICHAEL A. SIKORA
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- 09 March 2006, pp. 35-47
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Intracellular recordings were obtained from 57 cone-driven bipolar cells in the light-adapted retina of the land-phase (adult) tiger salamander (Ambystoma tigrinum). Responses to flashes of negative and positive contrast for centered spots of optimum spatial dimensions were analyzed as a function of contrast magnitude. On average, the contrast/response curves of depolarizing and hyperpolarizing bipolar cells in the land-phase animals were remarkably similar to those of aquatic-phase animals. Thus, the primary retinal mechanisms mediating contrast coding in the outer retina are conserved as the salamander evolves from the aquatic to the land phase. To evaluate contrast encoding in the context of natural environments, the distribution of contrasts in natural images was measured for 65 scenes. The results, in general agreement with other reports, show that the vast majority of contrasts in nature are very small. The efficient coding hypothesis of Laughlin was examined by comparing the average contrast/response curves of bipolar cells with the cumulative probability distribution of contrasts in natural images. Efficient coding was found at 20 cd/m2 but at lower levels of light adaptation, the contrast/response curves were much too shallow. Further experiments show that two fundamental physiological factors—light adaptation and the nonlinear transfer across the cone-bipolar synapse are essential for the emergence of efficient contrast coding. For both land- and aquatic-based animals, the extent and symmetry of the dynamic range of the contrast/response curves of both classes of bipolar cells varied greatly from cell to cell. This apparent substrate for distributed encoding is established at the bipolar cell level, since it is not found in cones. As a result, the dynamic range of the bipolar cell population brackets the distribution of contrasts found in natural images.
Radial motion bias in macaque frontal eye field
- QUAN XIAO, ANDREI BARBORICA, VINCENT P. FERRERA
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- 09 March 2006, pp. 49-60
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The visual responsiveness and spatial tuning of frontal eye field (FEF) neurons were determined using a delayed memory saccade task. Neurons with visual responses were then tested for direction selectivity using moving random dot patterns centered in the visual receptive field. The preferred axis of motion showed a significant tendency to be aligned with the receptive-field location so as to favor motion toward or away from the center of gaze. Centrifugal (outward) motion was preferred over centripetal motion. Motion-sensitive neurons in FEF thus appear to have a direction bias at the population level. This bias may facilitate the detection or discrimination of expanding optic flow patterns. The direction bias is similar to that seen in visual area MT and in posterior parietal cortex, from which FEF receives afferent projections. The outward motion bias may explain asymmetries in saccades made to moving targets. A representation of optic flow in FEF might be useful for planning eye movements during navigation.
Retinotopic organization of ferret suprasylvian cortex
- GINA CANTONE, JUN XIAO, JONATHAN B. LEVITT
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- 09 March 2006, pp. 61-77
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The retinotopic organization of striate and several extrastriate areas of ferret cortex has been established. Here we describe the representation of the visual field on the Suprasylvian visual area (Ssy). This cortical region runs mediolaterally along the posterior bank of the suprasylvian sulcus, and is distinct from adjoining areas in anatomical architecture. The Ssy lies immediately rostral to visual area 21, medial to lateral temporal areas, and lateral to posterior parietal areas. In electrophysiological experiments we made extracellular recordings in adult ferrets. We find that single and multiunit receptive fields range in size from 2 deg × 4 deg to 21 deg × 52 deg. The total visual field representation in Ssy spans over 70 deg in azimuth in the contralateral hemifield (with a small incursion into the ipsilateral hemifield), and from +36 deg to −30 deg in elevation. There are often two representations of the horizontal meridian. Furthermore, the location of the transition from upper to lower fields varies among animals. General features of topography are confirmed in anatomical experiments in which we made tracer injections into different locations in Ssy, and determined the location of retrograde label in area 17. Both isoelevation and isoazimuth lines can span substantial rostrocaudal and mediolateral distances in cortex, sometimes forming closed contours. This topography results in cortical magnifications averaging 0.07 mm/deg in elevation and 0.06 mm/deg in azimuth; however, some contours can run in such a way that it is possible to move a large distance on cortex without moving in the visual field. Because of these irregularities, Ssy contains a coarse representation of the contralateral visual field.
Ionotropic glutamate receptors of amacrine cells of the mouse retina
- OLIVIA N. DUMITRESCU, DARIO A. PROTTI, SRIPARNA MAJUMDAR, HANNS ULRICH ZEILHOFER, HEINZ WÄSSLE
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- 09 March 2006, pp. 79-90
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The mammalian retina contains approximately 30 different morphological types of amacrine cells, receiving glutamatergic input from bipolar cells. In this study, we combined electrophysiological and pharmacological techniques in order to study the glutamate receptors expressed by different types of amacrine cells. Whole-cell currents were recorded from amacrine cells in vertical slices of the mouse retina. During the recordings the cells were filled with Lucifer Yellow/Neurobiotin allowing classification as wide-field or narrow-field amacrine cells. Amacrine cell recordings were also carried out in a transgenic mouse line whose glycinergic amacrine cells express enhanced green fluorescent protein (EGFP). Agonist-induced currents were elicited by exogenous application of NMDA, AMPA, and kainate (KA) while holding cells at −75 mV. Using a variety of specific agonists and antagonists (NBQX, AP5, cyclothiazide, GYKI 52466, GYKI 53655, SYM 2081) responses mediated by AMPA, KA, and NMDA receptors could be dissected. All cells (n = 300) showed prominent responses to non-NMDA agonists. Some cells expressed AMPA receptors exclusively and some cells expressed KA receptors exclusively. In the majority of cells both receptor types could be identified. NMDA receptors were observed in about 75% of the wide-field amacrine cells and in less than half of the narrow-field amacrine cells. Our results confirm that different amacrine cell types express distinct sets of ionotropic glutamate receptors, which may be critical in conferring their unique temporal responses to this diverse neuronal class.
Long-term ERG analysis in the partially light-damaged mouse retina reveals regressive and compensatory changes
- ADAM RICHARDS, ALFRED A. EMONDI, BAERBEL ROHRER
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- 09 March 2006, pp. 91-97
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Most of the blinding retinopathies are due to progressive photoreceptor degeneration. Treatment paradigms that are currently being investigated include strategies to either halt or slow down photoreceptor cell loss, or to replace useful vision with retinal prosthesis. However, more information is required on the pathophysiological changes of the diseased retina, in particular the inner retina, that occur as a consequence of photoreceptor cell loss. Here we wished to use light damage as a stoppable insult to determine the structural and functional consequences on inner and outer retina, with the overall goal of determining whether survival of a functional inner retina is possible even if the outer retina is damaged. Mice were exposed to a 20-day light-damage period. Electroretinograms (ERG) and morphology were used to assess subsequent recovery. Outer retina was monitored analyzing a-waves, which represent photoreceptor cell responses, and histology. Integrity of the inner retina was monitored, analyzing b-waves and oscillatory potentials (OP1–OP4) and immunohistochemical markers for known proteins of the inner retina. All six ERG components were significantly suppressed with respect to amplitudes and kinetics, but stabilized in a wave-dependent manner within 40–70 days after the end of light exposure. As expected, damage of the outer retina was permanent. However, function of the inner retina was found to recover significantly. While b-wave amplitudes remained suppressed to 60% of their baseline values, OP amplitudes recovered completely, and implicit times of all components of the inner retina (b-wave and OP1–OP4) recovered to a level close to baseline values. Histological analyses confirmed the lack of permanent damage to the inner retina. In summary, these data suggests that the inner retina has the potential for significant recovery as well as plasticity if treatment is available to stop the deterioration of the outer retina.
Cloning and molecular characterization of cGMP-gated ion channels from rod and cone photoreceptors of striped bass (M. saxatilis) retina
- CHRISTOPHE PAILLART, KAI ZHANG, TATIANA I. REBRIK, WOLFGANG BAEHR, JUAN I. KORENBROT
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- 09 March 2006, pp. 99-113
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Vertebrate photoreceptors respond to light with changes in membrane conductance that reflect the activity of cyclic-nucleotide gated channels (CNG channels). The functional features of these channels differ in rods and cones; to understand the basis of these differences we cloned CNG channels from the retina of striped bass, a fish from which photoreceptors can be isolated and studied electrophysiologically. Through a combination of experimental approaches, we recovered and sequenced three full-length cDNA clones. We made unambiguous assignments of the cellular origin of the clones through single photoreceptor RT-PCR. Synthetic peptides derived from the sequence were used to generate monospecific antibodies which labeled intact, unfixed photoreceptors and confirmed the cellular assignment of the various clones. In rods, we identified the channel α subunit gene product as 2040 bp in length, transcribed into two mRNA 1.8 kb and 2.9 kb in length and translated into a single 96-kDa protein. In cones we identified both α (CNGA3) and β (CNGB3) channel subunits. For α, the gene product is 1956 bp long, the mRNA 3.4 kb, and the protein 74 kDa. For β, the gene product is 2265 bp long and the mRNA 3.3 kb. Based on deduced amino acid sequence, we developed a phylogenetic map of the evolution of vertebrate rod and cone CNG channels. Sequence comparison revealed channels in striped bass, unlike those in mammals, are likely not N-linked-glycosylated as they are transported within the photoreceptor. Also bass cone channels lack certain residues that, in mammals, can be phosphorylated and, thus, affect the cGMP sensitivity of gating. On the other hand, functionally critical residues, such as positively charged amino acids within the fourth transmembrane helix (S4) and the Ca2+-binding glutamate in the pore loop are absolutely the same in mammalian and nonmammalian species.
Receptive-field structure of optic flow responsive Purkinje cells in the vestibulocerebellum of pigeons
- IAN R. WINSHIP, DOUGLAS R.W. WYLIE
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- 09 March 2006, pp. 115-126
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Neurons sensitive to optic flow patterns have been recorded in the the olivo-vestibulocerebellar pathway and extrastriate visual cortical areas in vertebrates, and in the visual neuropile of invertebrates. The complex spike activity (CSA) of Purkinje cells in the vestibulocerebellum (VbC) responds best to patterns of optic flow that result from either self-rotation or self-translation. Previous studies have suggested that these neurons have a receptive-field (RF) structure that “approximates” the preferred optic flowfield with a “bipartite” organization. Contrasting this, studies in invertebrate species indicate that optic flow sensitive neurons are precisely tuned to their preferred flowfield, such that the local motion sensitivities and local preferred directions within their RFs precisely match the local motion in that region of the preferred flowfield. In this study, CSA in the VbC of pigeons was recorded in response to a set of complex computer-generated optic flow stimuli, similar to those used in previous studies of optic flow neurons in primate extrastriate visual cortex, to test whether the receptive field was of a precise or bipartite organization. We found that these RFs were not precisely tuned to optic flow patterns. Rather, we conclude that these neurons have a bipartite RF structure that approximates the preferred optic flowfield by pooling motion subunits of only a few different direction preferences.
Unique functional properties of the APB sensitive and insensitive rod pathways signaling light decrements in mouse retinal ganglion cells
- GUO-YONG WANG
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- 09 March 2006, pp. 127-135
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Light decrements are mediated by two distinct groups of rod pathways in the dark-adapted retina that can be differentiated on the basis of their sensitivity to the glutamate agonist DL-2-amino-phosphonobutyric (APB). By means of the APB sensitive pathway, rods transmit light decrements via rod bipolar cells to AII amacrine cells, then to Off cone bipolar cells, which in turn innervate the dendrites of Off ganglion cells. APB hyperpolarizes rod bipolar cells, thus blocking this rod pathway. With APB insensitive pathways, rods either directly synapse onto Off cone bipolar cells, or rods pass light decrement signal to cones by gap junctions. In the present study, whole-cell patch-clamp recordings were made from ganglion cells in the dark-adapted mouse retina to investigate the functional properties of APB sensitive and insensitive rod pathways. The results revealed several clear-cut differences between the APB sensitive and APB insensitive rod pathways. The latency of Off responses to a flashing spot of light was significantly shorter for the APB insensitive pathways than those for the APB sensitive pathway. Moreover, Off responses of the APB insensitive pathways were found to be capable of following substantially higher stimulus frequencies. Nitric oxide was found to selectively block Off responses in the APB sensitive rod pathway. Collectively, these results provide evidence that the APB sensitive and insensitive rod pathways can convey different types of information signaling light decrements in the dark-adapted retina.
Expansion of visual receptive fields in experimental glaucoma
- WAYNE MICHAEL KING, VIMAL SARUP, YVES SAUVÉ, COLLEEN M. MORELAND, DAVID O. CARPENTER, S.C. SHARMA
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- 09 March 2006, pp. 137-142
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Glaucoma is a major cause of blindness and is characterized by death of retinal ganglion cells. In a rat model of glaucoma in which intraocular pressure is raised by cautery of episcleral veins, the somata and dendritic arbors of surviving retinal ganglion cells expand. To assess physiological consequences of this change, we have measured visual receptive-field size in a primary retinal target, the superior colliculus. Using multiunit recording, receptive-field sizes were measured for glaucomatous eyes and compared to both those measured for contralateral control eyes and to homolateral eyes of unoperated animals. Episcleral vein occlusion increased intraocular pressure. This was accompanied by a significant increase in receptive-field size across the superior colliculus. The expansion of receptive fields was proportional to both degree and duration of the increase of intraocular pressure. We suggest that this increase in the size of receptive fields of glaucomatous eyes may be related to the increase in the size of dendritic arbors of the surviving ganglion cells in retina.