Research Article
Differential induction of gene expression by basic fibroblast growth factor and neuroD in cultured retinal pigment epithelial cells
- RUN-TAO YAN, SHU-ZHEN WANG
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- 01 March 2000, pp. 157-164
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Embryonic chick retinal pigment epithelial (RPE) cells can undergo transdifferentiation upon appropriate stimulation. For example, basic fibroblast growth factor (bFGF) induces intact RPE tissue younger than embryonic day 4.5 (E4.5) to transdifferentiate into a neural retina. NeuroD, a gene encoding a basic helix-loop–helix transcription factor, triggers de novo production of cells that resemble young photoreceptor cells morphologically and express general neuron markers (HNK-1/N-CAM and MAP2) and a photoreceptor-specific marker (visinin) from cell cultures of dissociated E6 RPE (Yan & Wang, 1998). The present study examined whether bFGF will lead to the same transdifferentiation phenomenon as neuroD when applied to dissociated, cultured E6 RPE cells, and whether interplay exists between the two factors under the culture conditions. Dissociated E6 RPE cells were cultured in the presence or absence of bFGF, and with or without the addition of retrovirus expressing neuroD. Gene expression was analyzed with immunocytochemistry and in situ hybridization. Unlike neuroD, bFGF did not induce the expression of visinin, or HNK-1/N-CAM and MAP2. However, bFGF elicited the expression of RA4 immunogenicity; yet, many of these RA4-positive cells lacked a neuronal morphology. Addition of bFGF to neuroD-expressing cultures did not alter the number of visinin-expressing cells; misexpression of neuroD in bFGF-treated cultures did not change the number of RA4-positive cells, suggesting the absence of interference or synergistic interaction between the two factors. Our data indicated that bFGF and neuroD induced the expression of different genes in cultured RPE cells.
Effects of atropine on refractive development, dopamine release, and slow retinal potentials in the chick
- HARTMUT N. SCHWAHN, HAKAN KAYMAK, FRANK SCHAEFFEL
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- 01 March 2000, pp. 165-176
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Atropine has previously been found to suppress visually induced myopia both in animals and humans. The mechanism of its action is unclear. We have studied its retinal effects in an in vitro preparation, using the retina-pigment epithelium-choroid complex of the chick eye. In vivo, deprivation myopia was induced by translucent goggles. Atropine solution was injected into the vitreous at two-day intervals. Dopamine release from the retina following atropine injection in vivo and from the in vitro retina preparation was quantified by HPLC-EC. In vitro preparations of the isolated chick retina–pigment epithelium–choroid were superfused with atropine. Light-induced potentials (local ERG), slow standing potentials from the retinal pigment epithelium/neural retina, and extracellular potassium concentrations were recorded. In line with previous findings, intravitreal injections of atropine (25 μg, 250 μg) reduced deprivation myopia in a dose-dependent manner. Atropine increased the release of the neurotransmitter dopamine into the superfusate in vitro at 100–500 μM and into the vitreous in vivo at 250 μg. Before an increase was measured in the vitreous, the retinal dopamine content was elevated. In concentrations equivalent to the intravitreal concentration to suppress myopia in vivo (200–800 μM), atropine induced spreading depression (SD) in the in vitro preparation. In contrast, muscarinic agonists, acetylcholine and pilocarpine, did not induce SD. Atropine reduced the ERG b- and d-wave, led to damped oscillations of RPE potentials, and reversed the ERG c-wave. Atropine suppressed myopia only at doses at which severe nonspecific side effects were observed in the retina. Atropine seems to intrude massively into the vital functions of the retina as indicated by the occurrence of SD. We conclude that atropine, by inducing SD, boosts neurotransmitter release from cellular stores, which may cancel out a presumed retinal signal that controls eye growth and through this, myopia.
D2-dopamine receptor blockade impairs motion detection in goldfish
- CARLOS MORA-FERRER, VOLKER GANGLUFF
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- 01 March 2000, pp. 177-186
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Under photopic illumination conditions, motion detection in goldfish is dominated by the long-wavelength-sensitive cone type (L-cone), and under scotopic conditions motion it is determined by rods (Schaerer & Neumeyer, 1996). The switch from rod-dominated to cone-dominated motion detection occurs during light adaptation. It has been suggested that dopamine acts as a neuronal light-adaptative signal. It is known that dopamine affects wavelength discrimination through D1-dopamine receptors (Mora-Ferrer & Neumeyer, 1996), and the dorsal light reflex through D1- and D2-dopamine receptors (Lin & Yazulla, 1994a). The purpose of this study was to determine whether dopamine influenced movement detection by goldfish, and if so, which dopamine receptor was involved. The D2-dopamine receptor antagonist sulpiride reduced the animal's sensitivity to the moving stimulus, whereas SCH 23390, a D1-dopamine receptor antagonist, did not have any effect. The effect of sulpiride is discussed in relation to known sulpiride effects on retinal neurons and the retinal pigment epithelium.
NMDAR-1 staining in the lateral geniculate nucleus of normal and visually deprived cats
- JOKUBAS ZIBURKUS, MARTHA E. BICKFORD, WILLIAM GUIDO
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- 01 March 2000, pp. 187-196
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In normal adult cats, a monoclonal antibody directed toward the NR-1 subunit of the N-methyl-d-aspartate (NMDA) receptor (Pharmingen, clone 54.1) produced dense cellular and neuropil labeling throughout all layers of the lateral geniculate nucleus (LGN) and adjacent thalamic nuclei, including the thalamic reticular, perigeniculate, medial intralaminar, and ventral lateral geniculate nuclei. Cellular staining revealed well-defined somata, and in some cases proximal dendrites. NMDAR-1 cell labeling was also evident in the LGN of early postnatal kittens, suggesting that developing LGN cells possess this receptor subunit at or before eye opening. Within the A-layers of the adult LGN, staining encompassed a wide range of soma sizes. Soma size comparisons of NMDAR-1 stained cells with those stained with an antibody directed toward a nonphosphorylated neurofilament protein (SMI-32), which selectively stains Y-relay cells (Bickford et al., 1998), or an antibody to glutamic acid decarboxylase (GAD), which stains for GABAergic interneurons, suggested that NMDA receptors are utilized by relay cells and interneurons. NMDAR-1 staining was also observed in the LGN of cats with early monocular lid suture. Although labeling was apparent in both deprived and nondeprived A-layers of LGN, the distribution of soma sizes was significantly different. In the deprived A-layers of LGN, staining was limited to small- and medium-sized cells. Cells with relatively large soma were lacking. However, cell density measurements as well as soma size comparisons with cells stained for Nissl substance suggested these differences were due to deprivation-induced cell shrinkage and not to a loss of NMDAR-1 staining in Y-cells. Taken together, these results suggest that NMDA receptors are utilized by both relay cells and interneurons in LGN and that alterations in early visual experience do not necessarily affect the expression of NMDA receptors in the LGN.
Chloride efflux inhibits single calcium channel open probability in vertebrate photoreceptors: Chloride imaging and cell-attached patch-clamp recordings
- WALLACE B. THORESON, RON NITZAN, ROBERT F. MILLER
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- 01 March 2000, pp. 197-206
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The present study uses cell-attached patch-recording techniques to study the single-channel properties of Ca2+ channels in isolated salamander photoreceptors and investigate their sensitivity to reductions in intracellular Cl−. The results show that photoreceptor Ca2+ channels possess properties similar to L-type Ca2+ channels in other preparations, including (1) enhancement of openings by the dihydropyridine agonist, (−)BayK8644; (2) suppression by a dihydropyridine antagonist, nisoldipine; (3) single-channel conductance of 22 pS with 82 mM Ba2+ as the charge carrier; (4) mean open probability of 0.1; (5) open-time distribution fit with a single exponential (τ0 = 1.1 ms) consistent with a single open state; and (6) closed time distribution fit with two exponentials (τc1 = 0.7 ms, τc2 = 25.4 ms) consistent with at least two closed states. Using a Cl−-sensitive dye to measure intracellular [Cl−], it was found that perfusion with gluconate-containing, low Cl− medium depleted intracellular [Cl−]. It was therefore possible to reduce intracellular [Cl−] by perfusion with a low Cl− solution while maintaining the extracellular channel surface in high Cl− pipette solution. Under these conditions, the single-channel conductance was unchanged, but the mean open probability fell to 0.03. This reduction can account for the 66% reduction in whole-cell Ca2+ currents produced by perfusion with low Cl− solutions. Examination of the open and closed time distributions suggests that the reduction in open probability arises from increases in closed-state dwell times. Changes in intracellular [Cl−] may thus modulate photoreceptor Ca2+ channels.
Response variability and information transfer in directional neurons of the mammalian horizontal optokinetic system
- COLIN W.G. CLIFFORD, MICHAEL R. IBBOTSON
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- 01 March 2000, pp. 207-215
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This study is concerned with how information about the direction of visual motion is encoded by motion-sensitive neurons. Motion-sensitive neurons are usually studied using stimuli unchanging in speed and direction over several seconds. Recently, it has been suggested that neuronal responses to more naturalistic stimuli cannot be understood on the basis of experiments with constant-motion stimuli (de Ruyter van Steveninck et al., 1997). We measured the variability and information content of spike trains recorded from directional neurons in the nucleus of the optic tract (NOT) of the wallaby, Macropus eugenii, in response to constant and time-varying motion. While the NOT forms part of the mammalian optokinetic system, we have shown previously that the responses of its directional neurons resemble those of insect H1 in many respects (Ibbotson et al., 1994). We find that directional neurons in the wallaby NOT respond with lower variability and higher rates of information transmission to time-varying stimuli than to constant motion. The difference in response variability is predicted by an inhomogeneous Poisson model of neuronal spiking incorporating an absolute refractory period of 2 ms during which no subsequent spike can be fired. Refractoriness imposes structure on the spike train, reducing variability (de Ruyter van Steveninck & Bialek, 1988; Berry & Meister, 1998). A given refractory period has a greater impact when firing rates are high, as for the responses of NOT neurons to time-varying stimuli. It is in just these cases that variability in experimentally observed spike trains is lowest. Thus, differences in response variability do not necessarily imply that different models are required to predict neuronal responses to constant- and time-varying motion stimuli.
Regulation of arrestin mRNA levels in Limulus lateral eye: Separate and combined influences of circadian efferent input and light
- BARBARA-ANNE BATTELLE, CHRISTOPHER D. WILLIAMS, JERI-LYNN SCHREMSER-BERLIN, CHELSI CACCIATORE
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- 01 March 2000, pp. 217-227
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Most animals experience daily changes in light and darkness. The retinas of many of these animals show concomitant rhythmic changes in the levels of mRNAs that encode proteins involved in the photoresponse. These changes may be circadian and independent of light, independent of circadian clocks and regulated by light, or regulated by a circadian clock and light. We have taken advantage of the organization of the Limulus visual system to examine the separate and combined effects of signals from a circadian clock and light on arrestin mRNA levels in photoreceptors. The clock that regulates photoreceptors in the lateral eye of Limulus is in the brain, and signals from the clock reach the lateral eye via activation of a well-characterized efferent projection in the lateral optic nerve. In the experiments described, clock-driven efferent input to the lateral eye was eliminated by cutting the lateral optic nerve, and light input to the lateral eye was eliminated by placing an opaque patch over the eye. Arrestin mRNA levels were quantified relative to 18s rRNA with a ribonuclease protection assay. We observed the following. In lateral eyes exposed to natural diurnal light and endogenous efferent nerve activity, the level of arrestin mRNA was higher during the day in the light than during the night in the dark. Circadian efferent nerve activity was necessary and sufficient to produce normal daily fluctuations in the level of arrestin mRNA. Light influenced arrestin mRNA levels only in eyes with intact and active efferent projections. We conclude that arrestin mRNA levels in lateral eye photoreceptors are controlled entirely by efferent nerve activity, and that light exerts its effects by modulating this output from the circadian clock. Light-stimulated changes in arrestin mRNA in the vertebrate retina may likewise require interactions between light-driven biochemical cascades and clock output.
Spontaneous activity in developing turtle retinal ganglion cells: Statistical analysis
- NORBERTO M. GRZYWACZ, EVELYNE SERNAGOR
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- 01 March 2000, pp. 229-241
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We report on the temporal properties of the spontaneous bursts of activity in the developing turtle retina. Quantitative statistical criteria were used to detect, cluster, and analyze the temporal properties of the bursts. The interburst interval, duration, firing rate, and number of spikes per burst varied widely among cells and from burst to burst in a single cell. Part of this variability was due to the positive correlation between a burst's duration and the interburst interval preceding that burst. This correlation indicated the influence of a refractory period on the bursts' properties. Further evidence of such a refractoriness came from the bursts' auto-covariance function, which gives the tendency of a spike to occur a given amount of time after another spike. This function showed a positive phase (between ≈10 ms and 10 s) followed by a negative one (between 10 s and more than 100 s), suggestive of burst refractoriness. The bursts seemed to be propagating from cell to cell, because there was a long (symmetrically distributed) delay between the activation of two neighbor cells (median absolute delay = 2.3 s). However, the activity often failed to propagate from one cell to the other (median safety factor = 0.59). The number of spikes per burst in neighbor cells was statistically positively correlated, indicating that the activity in the two cells was driven by the same excitatory process. At least two factors contribute to the excitability during bursts, because the positive phase of the cross-covariance function (similar to auto-covariance but for two cells) had a temporally asymmetric fast component (1–3 ms) followed by a temporally symmetric slow component (1 ms to 10 s).
Inhibition is not required for the production of transient spiking responses from retinal ganglion cells
- MARK C. BIEDA, DAVID R. COPENHAGEN
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- 01 March 2000, pp. 243-254
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Ganglion cells responding only transiently to changes in illumination are found in many different vertebrate retinas. The interactions underlying formation of these transient responses are still poorly understood. Two recently proposed hypotheses are (1) functional inhibitory pathways are necessary for transient response production, and (2) direct inhibition of the ganglion cell has little effect on its output. Here, we examine these conclusions by using cell-attached patch-clamp recordings of spiking, whole-cell recordings of synaptic currents, and computer modeling. We found that picrotoxin (a GABAA and GABAC receptor antagonist), bicuculline (a GABAA receptor antagonist), and strychnine (a glycine receptor antagonist), applied either singly or in combination, always failed to convert transient responses to sustained responses. Application of the GABAB antagonist CGP35348 in the presence of picrotoxin and strychnine also failed to convert transient responses into sustained responses. Whole-cell recordings of synaptic currents at various holding potentials indicated that direct inhibitory inputs to ganglion cells limit the duration of net excitation, implying that direct inhibition does act to truncate the ganglion cell spiking response. Computer simulations using spiking and synaptic data from combined cell-attached and whole-cell recordings supported this interpretation. We conclude that inhibitory pathways are not required for generation of transient responses, but these pathways do serve to modulate transient ganglion cell spiking responses. We find that this modulation occurs, in part, via inhibitory inputs directly to the ganglion cell.
Dye coupling in horizontal cells of developing rabbit retina
- DIANNA A. JOHNSON, STEPHEN L. MILLS, MICHAEL F. HABERECHT, STEPHEN C. MASSEY
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- 01 March 2000, pp. 255-262
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In the mature rabbit retina, two classes of horizontal cells, A type and B type, provide lateral inhibition in the outer plexiform layer (OPL) and spatially modify the activation of bipolar cells by photoreceptors. Gap junctions connecting homologous horizontal cells determine the extent to which this inhibitory activity spreads laterally across the OPL. Little is currently known about the expression of gap junctions in horizontal cells during postnatal development or how cell–cell coupling might contribute to subsequent maturational events. We have examined the morphological attributes and coupling properties of developing A and B type horizontal cells in neonatal rabbit retina using intracellular injections of Lucifer Yellow and Neurobiotin. Prelabeling with DAPI permitted the targeting of horizontal cell bodies for intracellular injection in perfused preparations of isolated retina. A and B type horizontal cells were identifiable at birth although their dendritic field sizes had not reached adult proportions and their synaptic contacts in the OPL were minimal. Both cell types exhibited homologous dye coupling at birth. Similar to that seen in the adult, no heterologous coupling was observed, and homologous coupling among A type cells was stronger than that observed among B type cells. The spread of tracer compounds through gap junctions of morphologically immature horizontal cells suggests that ions and other small, bioactive compounds may likewise spread through coupled, horizontal networks to coordinate the subsequent maturational of emerging outer plexiform layer pathways.
Computation of motion direction by quail retinal ganglion cells that have a nonconcentric receptive field
- HIROYUKI UCHIYAMA, TAKAHIDE KANAYA, SHOICHI SONOHATA
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- 01 March 2000, pp. 263-271
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One type of retinal ganglion cells prefers object motion in a particular direction. Neuronal mechanisms for the computation of motion direction are still unknown. We quantitatively mapped excitatory and inhibitory regions of receptive fields for directionally selective retinal ganglion cells in the Japanese quail, and found that the inhibitory regions are displaced about 1–3 deg toward the side where the null sweep starts, relative to the excitatory regions. Directional selectivity thus results from delayed transient suppression exerted by the nonconcentrically arranged inhibitory regions, and not by local directional inhibition as hypothesized by Barlow and Levick (1965).
Modulation by Zn2+ of GABA responses in bipolar cells of the mouse retina
- M. KANEDA, B. ANDRÁSFALVY, A. KANEKO
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- 01 March 2000, pp. 273-281
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The localization of endogenous Zn2+ in the mouse retina was examined histochemically and the inhibitory action of Zn2+ on GABA-induced responses was studied in bipolar cells isolated from the mouse retina. Accumulation of endogenous Zn2+ was detected in photoreceptors, bipolar, and/or amacrine cells by either the bromopyridylazo-diethylaminophenol method or the dithizone method. Under whole-cell recording conditions, GABA induced a Cl− current in isolated bipolar cells. The current consisted of two components. The first component was inhibited completely by application of 100 μM bicuculline, suggesting that this is a GABAA-receptor mediated current. The second component was inhibited completely by 100 μM 3-aminopropyl-(methyl)-phosphinic acid, suggesting that this is a GABAC-receptor mediated current. GABAC receptors were present at a higher density on the axon terminal than on dendrites. Zn2+ inhibited both GABAA and GABAC receptors. GABAC receptors were more susceptible to Zn2+; the IC50 for the GABAA receptor was 67.4 μM and that for the GABAC receptor was 1.9 μM. These results suggest that Zn2+ modulates the inhibitory interaction between amacrine and bipolar cells, particularly that mediated by the GABAC receptor.
The functional influence of nicotinic cholinergic receptors on the visual responses of neurones in the superficial superior colliculus
- K.E. BINNS, T.E. SALT
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- 01 March 2000, pp. 283-289
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In the rat, the superficial gray layer (SGS) of the superior colliculus receives glutamatergic projections from the contralateral retina and from the visual cortex. A few fibers from the ipsilateral retina also directly innervate the SGS, but most of the ipsilateral visual input is provided by cholinergic afferents from the opposing parabigeminal nucleus (PBG). Thus, visual input carried by cholinergic afferents may have a functional influence on the responses of SGS neurones. When single neuronal extracellular recording and iontophoretic drug application were employed to examine this possibility, cholinergic agonists were found to depress responses to visual stimulation. Lobeline and 1-acetyl-4-methylpiperazine both depressed visually evoked activity and had a tendency to reduce the background firing rate of the neurones. Carbachol depressed the visual responses without any significant effect on the ongoing activity, while the muscarinic receptor selective agonist methacholine increased the background activity of the neurones and reduced their visual responses. Lobeline was chosen for further studies on the role of nicotinic receptors in SGS. Given that nicotinic receptors are associated with retinal terminals in SGS, and that the activation of presynaptic nicotinic receptors normally facilitates transmitter release (in this case glutamate release), the depressant effects of nicotinic agonists are intriguing. However, many retinal afferents contact inhibitory neurones in SGS; thus it is possible that the increase in glutamate release in turn facilitates the liberation of GABA which goes on to inhibit the visual responses. We therefore attempted to reverse the effects of lobeline with GABA receptor antagonists. The depressant effects of lobeline on the visual response could not be reversed by the GABAA antagonist bicuculline, but the GABAB antagonist CGP 35348 reduced the effects of lobeline. We hypothesize that cholinergic drive from the parabigeminal nucleus may activate presynaptic nicotinic receptors on retinal terminals, thereby facilitating the release of glutamate onto inhibitory neurones. Consequently GABA is released, activating GABAB receptors, and thus the ultimate effect of nicotinic receptor activation is to depress visual responses.
The role of NMDA channels in rabbit retinal directional selectivity
- DARREL S. TJEPKES, FRANKLIN R. AMTHOR
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- 01 March 2000, pp. 291-302
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It has been previously demonstrated that the majority of the glutamatergic input to directionally selective (DS) ganglion cells in the rabbit retina is mediated by NMDA receptors. To examine whether NMDA channels have any role in directional selectivity, we eliminated magnesium from the superfusion medium to prevent the magnesium block of the channels at hyperpolarized membrane potentials. During superfusion in magnesium-free media, the response to null-direction motion increased to the level of the response to preferred-direction motion. This effect was specifically mediated by NMDA channels because subsequent blocking of the NMDA channels with AP7 restored directional selectivity. We also tested whether the increase in the null-direction response in magnesium-free medium was due to an increased release of acetylcholine from the cholinergic amacrine cells, rather than an effect on the DS ganglion cells themselves, by blocking acetylcholine transmission with d-tubocurarine during superfusion with the magnesium-free medium. During zero-magnesium superfusion, d-tubocurarine reduced both the preferred- and null-direction responses of DS ganglion cells but did not restore directional selectivity. These findings suggest that null-direction motion normally causes portions of the dendritic membrane of the directionally selective ganglion cell to be maintained at a sufficiently negative potential that the NMDA channels are blocked by magnesium ions. This result is discussed in terms of several models for the mechanisms of directional selectivity.
Cloning and tissue localization of a novel zebrafish RdgB homolog that lacks a phospholipid transfer domain
- VECHESLAV A. ELAGIN, RAYA B. ELAGINA, CHRISTOPHER J. DORO, THOMAS S. VIHTELIC, DAVID R. HYDE
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- 01 March 2000, pp. 303-311
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The retinal degeneration B (RdgB) protein family is characterized by an amino-terminal phosphatidylinositol transfer protein (PITP) domain, several hydrophobic domains, and a highly conserved carboxyl terminus. We identified a zebrafish RdgB homolog (pl-RdgB) that lacks the amino-terminal PITP domain, while retaining over 45% amino acid identity with the two mouse RdgB proteins (M-RdgB1 and M-RdgB2). Unlike the widespread retinal expression observed for other vertebrate RdgB homologs, pl-RdgB is restricted in the retina to the cone cell inner segments. The pl-RdgB protein is also expressed in the brain, although its distribution is different than the other RdgB homologs. Analogous to M-RdgB2, pl-RdgB protein is extracted from a retinal homogenate by guanidine and not by Triton X-100. Thus, pl-RdgB and likely all the identified RdgB homologs are not integral membrane proteins, but may associate with the membrane through protein–protein interactions. While expression of either murine RdgB homolog restored the defective light response and prevented retinal degeneration in rdgB mutant flies, expressing zebrafish pl-RdgB in Drosophila rdgB2 null mutants slowed retinal degeneration without restoring the electrophysiological light response. Thus, pl-RdgB may define a previously unrecognized protein family, which includes the other RdgB homologs, that act through a protein complex to maintain photoreceptor viability.
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Organization of brain stem afferents to the ventral lateral geniculate nucleus of rats
- CHRISTIAN KOLMAC, JOHN MITROFANIS
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- 01 March 2000, pp. 313-318
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We have examined the patterns of projections from different nuclei of the brain stem to the ventral lateral geniculate nucleus (vLGN) of the thalamus. Injections of biotinylated dextran were made into different nuclei of the brainstem (i.e., midbrain reticular nucleus, pontine reticular nucleus, deep layers of superior colliculus, periaqueductal grey matter [ventrolateral, dorsolateral, and lateral columns], pedunculopontine tegmental nucleus, parabrachial nucleus, lateral dorsal tegmental nucleus, substantia nigra [pars reticulata], locus coeruleus, and dorsal raphe) of Sprague-Dawley rats using stereotaxic coordinates. Our results show that all of the abovementioned brain-stem nuclei have overlapping projections to the medial regions of the vLGN, within the parvocellular lamina of the nucleus. This if the first instance of the parvocellular lamina being shown to receive a major set of projections. Very few labelled terminals from the brain stem were ever seen within the larger more lateral magnocellular lamina, which has been shown by previous studies to receive heavy inputs from visually associated structures, such as the retina and occipital cortex. Since many of the brain-stem nuclei injected in this study have little to do with visual processing, our results suggest that one can perhaps package the vLGN into distinct visual (magnocellular) and nonvisual (parvocellular) components.