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Dark adaptation of human rod bipolar cells measured from the b-wave of the scotopic electroretinogram.

To examine the dark adaptation of human rod bipolar cells in vivo, we recorded ganzfeld ERGs to (a) a family of flashes of increasing intensity, (b) dim test flashes presented on a range of background intensities, and (c) dim test flashes presented before, and up to 40 min after, exposure to intense illumination eliciting bleaches from a few per cent to near total. The dim flash ERG was characterized by a prominent b-wave response generated principally by rod bipolar cells. In the presence of background illumination the response reached peak earlier and desensitized according to Weber's Law. Following bleaching exposures, the response was initially greatly desensitized, but thereafter recovered slowly with time. For small bleaches, the desensitization was accompanied by acceleration, in much the same way as for real light. Following a near-total bleach, the response was unrecordable for >10 min, but after approximately 23 min half-maximal sensitivity was reached, and full sensitivity was restored between approximately 35 and 40 min. With smaller bleaches, recovery commenced earlier. We converted the post-bleach measurements of desensitization into 'equivalent background intensities' using a Crawford transformation. Across the range of bleaching levels, the results were described by a prominent 'S2' component (0.24 decades min(-1)) together with a smaller and slower 'S3' component (0.06 decades min(-1)), as is found for dark adaptation of the scotopic visual system. We attribute the S2 component to the presence of unregenerated opsin, and we speculate that the S3 component results from ion channel closure by all-trans retinal.

Adaptation, Ocular↗

The fine structure of the rod-bipolar cell synapse in the retina of the albino rat.

The fine structure of the rod-bipolar synapse is described and illustrated. Each rod spherule possesses a large, single, oval or elongate mitochondrion approximately 0.5 x 2.0 microns. Surrounding the mitochondrion are elements of agranular endoplasmic reticulum. The bipolar dendrite projects into the lower pole of the spherule and usually terminates in two lobes separated by a cleft. The plasma membranes appear dense and thicker in the region of the synapse. In the rod spherule cytoplasm, contiguous with the plasma membrane is a dense, slightly concave arciform structure, the rod arciform density, extending from the base of the bipolar bifid process through the cleft to an equivalent point on the opposite side. Also within the spherule, and external (towards the sclera) to the rod arciform density, is a parallel, dense, thin lamella, the rod synaptic lamella. This is approximately 25 mmicro in thickness and 400 mmicro in width at its widest extent. This halfmoon-shaped plate straddles the cleft between the two lobes of the bipolar process. The lamella appears to consist of short regular rodlets or cylinders 5 to 7 mmicro in diameter, oriented with their long axes perpendicular to the plane of the lamella. Minute cytoplasmic vesicles found in the cytoplasm of both the rod spherule and the bipolar terminal are most abundant near the rod synaptic lamella.

Animals↗

Cross-correlation analysis of the maintained discharge of rabbit retinal ganglion cells.

1. Simultaneous recordings were made from pairs of rabbit retinal ganglion cells. Physiological tests were used to classify the receptive field properties of each cell and the receptive field locations were mapped. 2. The statistical dependence between simultaneously recorded retinal ganglion cells was assessed by cross-correlating the maintained discharge of the simultaneously recorded cells. Cross-correlations from cell pairs in which the constituent cells had non-overlapping receptive field centres were statistically flat, reflecting no statistical dependence. 3. Most cell pairs consisting of transient and sustained concentric cells and having overlapping receptive field centres exhibited a correlated maintained discharge indicative of statistical dependence. The strength of the statistical dependence varied approximately inversely with the degree of overlap between the two cells comprising the cell pair. 4. Cell pairs consisting of two ON-centre cells or two OFF-centre cells and having overlapping receptive field centres possessed incremental cross-correlations which were characterized by a peak centred near zero. Cell pairs consisting of an ON-centre cell with an OFF-centre and having overlapping receptive field centres possessed decremental cross-correlations which were characterized by a valley centred near zero. 5. The results are consistent with the hypothesis that a noise source provides shared input to two or more retinal ganglion cells. Bipolar and photoreceptors are the most likely sources of noise responsible for the statistical dependency between retinal ganglion cells.

Action Potentials↗

Vesicle depletion and synaptic depression at a mammalian ribbon synapse.

We estimated the size of the readily releasable pool (RRP) of vesicles at a ribbon synapse in the rat retina by making paired voltage-clamp recordings from presynaptic rod bipolar cells (RBCs) and postsynaptic AII amacrine cells in an in vitro retinal slice preparation. The RRP at each active zone was estimated to constitute seven vesicles, in the range of estimated RRP sizes at conventional synapses. During sustained presynaptic Ca(2+) entry, the RRP could be released with a time constant of about 4 ms. This ribbon synapse exhibited pronounced paired-pulse depression (PPD), which was attributable primarily to vesicle depletion. Recovery from PPD was slow (tau approximately 4 s) but could be accelerated by increasing the duration of the depressing stimulus. The small RRP and very high release probability likely contribute to the transient characteristics of neurotransmission at RBC synapses.

Amacrine Cells↗

Synaptic contacts between an identified type of ON cone bipolar cell and ganglion cells in the mouse retina.

We surveyed the potential contacts between an identified type of bipolar cell and retinal ganglion cells in the mouse. By crossing two existing mouse strains (line 357 and line GFP-M), we created a double transgenic strain in which GFP is expressed by all members of a single type of ON cone bipolar cell and a sparse, mixed population of retinal ganglion cells. The GFP-expressing bipolar cells appear to be those termed CB4a of Pignatelli & Strettoi [(2004) J. Comp. Neurol., 476, 254-266] and type 7 of Ghosh et al. [(2004) J. Comp. Neurol., 469, 70-82 and J. Comp. Neurol., 476, 202-203]. The labelled ganglion cells include examples of most or all types of ganglion cells present in the mouse. By studying the juxtaposition of their processes in three dimensions, we could learn which ganglion cell types are potential synaptic targets of the line 357 bipolar cell. Of 12 ganglion cell types observed, 10 types could be definitively ruled out as major synaptic targets of the line 357 bipolar cells. One type of monostratified ganglion cell and one bistratified cell tightly cofasciculate with axon terminals of the line 357 bipolar cells. Double labelling for kinesin II demonstrates colocalization of bipolar cell ribbons at the sites of contact between these two types of ganglion cell and the line 357 bipolar cells.

Animals↗

Cerebellar Purkinje cell markers are expressed in retinal bipolar neurons.

Previous studies have been directed at the elucidation of neuron-specific gene expression in the mammalian central nervous system. In particular, we have identified a series of marker molecules that are expressed in cerebellar Purkinje cells with varying degrees of specificity. Here, we show by light microscopic immunocytochemistry and Northern transfer and hybridization that two of these markers, namely, L7 and PEP19, are expressed in the retina of mouse and rabbit, while a third marker, cerebellin, is absent. Light and electron microscopic immunocytochemistry proves that L7-like immunoreactivity is restricted to rod bipolar cells, while PEP 19-like immunoreactivity is distributed in both rod and cone bipolars. PEP19 is also expressed by subsets of amacrine and ganglion cells. The density of PEP19-positive bipolar cells is greater than that of L7-positive bipolar cells, although the density of each is approximately equal in central and peripheral portions of the retina. An antiserum to a fourth Purkinje cell marker, vitamin D-dependent calcium-binding protein-28 kD (CaBP), reveals primarily axonless horizontal cells, but also subsets of rod bipolar, amacrine, and, in the mouse but not in the rabbit, ganglion cells. The processes of immunoreactive cell bodies form discrete bands in the internal plexiform layer, and mixtures of the antisera help distinguish their identity. Thus, these Purkinje cell markers can be used at the electron microscopic level to unravel the extremely complex neuropil of this retinal layer. Furthermore, knowledge of the retinal distribution of this panel of molecules is of general value for future studies of retinal neuronal typology and can serve to map the densities of subsets of bipolar cells throughout the retina. The expression of L7 and PEP19 in bipolar cells and in Purkinje cells suggests a biochemical relationship between these two spatially distant neuronal populations.

Animals↗

Tracking mouse visual pathways with WGA transgene.

By use of wheat germ agglutinin (WGA) cDNA as a transgene, we have succeeded in generating a transgenic mouse line in which the visual pathways can be accurately and reproducibly visualized. The WGA transgene was expressed in the retinal rod bipolar cells under the control of mouse L7 promoter. The transgene product, WGA protein, was transferred from the bipolar cells to the amacrine cells and the ganglion cells across synapses in the retinal neural circuitry and further conveyed along the optic nerve to the visual centers such as the suprachiasmatic nucleus, the lateral geniculate nucleus, the pretectal nucleus and the superior colliculus. By crossing the WGA-expressing transgenic mice with the retinal degeneration mutant mice, we analyzed change in the visual pathways by monitoring WGA immunoreactivity and found that the disorganization process of the visual pathways was relatively slow in spite of the rapid degeneration of the photoreceptor cells. Thus, this transgenic mouse line would provide a useful tool for analyzing phenotypic changes in the visual pathways of various mutant mice.

Animals↗

Distribution of phosphorylated protein kinase C alpha in goldfish retinal bipolar synaptic terminals: control by state of adaptation and pharmacological treatment.

Protein kinase C (PKC) is a signalling enzyme critically involved in many aspects of synaptic plasticity. In cyprinid retinae, the PKC alpha isoform is localized in a subpopulation of depolarizing bipolar cells that show adaptation-related morphological changes of their axon terminals. We have studied the subcellular localization of phosphorylated PKC alpha (pPKC alpha) in retinae under various conditions by immunohistochemistry with a phosphospecific antibody. In dark-adapted retinae, pPKC alpha immunoreactivity is weak in the cytoplasm of synaptic terminals, labelling being predominantly associated with the membrane compartment. In light-adapted cells, immunoreactivity is diffusely distributed throughout the terminal. Western blot analysis has revealed a reduction of pPKC alpha immunoreactivity in cytosolic fractions of homogenized dark-adapted retinae compared with light-adapted retinae. Pharmacological experiments with the isoform-specific PKC blocker Goe6976 have shown that inhibition of the enzyme influences immunolabelling for pPKC alpha, mimicking the effects of light on the subcellular distribution of immunoreactivity. Our findings suggest that the state of adaptation modifies the subcellular localization of a signalling molecule (PKC alpha) at the ribbon-type synaptic complex. We propose that changes in the subcellular distribution of PKC alpha immunoreactivity might be one component regulating the strength of the signal transfer of the bipolar cell terminal.

Adaptation, Ocular↗

Hepatic retinopathy: morphological features of retinal glial (Müller) cells accompanying hepatic failure.

More than 80 years ago, Alzheimer described changes in the brains of patients who had suffered hepatic failure. Astrocytes are primarily affected; their nuclei become swollen, their intermediate filament protein composition is altered and their cytoplasm becomes vacuolated. Cells with these features are called Alzheimer type II astrocytes and these changes have been attributed to the toxic effects of elevated ammonia levels. The present study investigates whether the dominant glia of another part of the central nervous system, the Müller cells of the retina, undergo similar changes. Retinae of patients who had died with symptoms of hepatic failure were processed for histology, histochemistry, and immunocytochemistry. Cell nuclei were measured from brain astrocytes (insula cortex), Müller cells, and retinal bipolar neurons. Hepatic failure resulted in the enlargement of nuclei in astrocytes and Müller cells, and the enhanced expression in Müller cells of glial fibrillary acidic protein, cathepsin D, and the beta-subunit of prolyl 4-hydroxylase (glial-p55). In some retinae, signs of gliosis were also observed. We conclude that increased levels of serum ammonia resulting from hepatic insufficiency cause changes in Müller cells that are similar to those seen in brain astrocytes. We term this condition hepatic retinopathy.

Adult↗

Localization of NMDA receptor subunits and mapping NMDA drive within the mammalian retina.

Glutamate is a major neurotransmitter in the retina and other parts of the central nervous system, exerting its influence through ionotropic and metabotropic receptors. One ionotropic receptor, the N-methyl-D-aspartate(NMDA) receptor, is central to neural shaping, but also plays a major role during neuronal development and in disease processes. We studied the distribution pattern of different subunits of the NMDA receptor within the rat retina including quantifying the pattern of labelling for all the NRI splice variants, the NR2A and NR2B subunits. The labelling pattern for the subunits was confined predominantly in the outer two-thirds of the inner plexiform layer. We also wanted to probe NMDA receptor function using an organic cation, agmatine (AGB); a marker for cation channel activity. Although there was an NMDA concentration-dependent increase in AGB labelling of amacrine cells and ganglion cells, we found no evidence of functional NMDA receptors on horizontal cells in the peripheral rabbit retina, nor in the visual streak where the type A horizontal cell was identified by GABA labelling. Basal AGB labelling within depolarizing bipolar cells was also noted. This basal bipolar cell AGB labelling was not modulated by NMDA and was completely abolished by the use of L-2-amino-4-phosphono-butyric acid,which is known to hyperpolarize retinal depolarizing bipolar cells. AGB is therefore not only useful as a probe of ligand-gated drive, but can also identify neurons that have constitutively open cationic channels. In combination,the NMDA receptor subunit distribution pattern and the AGB gating experiments strongly suggests that this ionotropic glutamate receptor is functional in the cone-driven pathway of the inner retina.

Agmatine↗

Morphological impairments in retinal neurons of the scotopic visual pathway in a monkey model of Parkinson's disease.

Physiological abnormalities resulting from death of dopaminergic neurons of the central nervous system in Parkinson's disease also extend to the retina, resulting in impaired visual functions. In both parkinsonian patients and animal models, low levels of dopamine and loss of dopaminergic cells in the retina have been reported. However, the morphology and connectivity of their postsynaptic neurons, the amacrine cells, have not been analyzed. Here we report, with macaques chronically treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) as a model of Parkinson's disease, that morphological impairments in dopaminergic retinal neurons and their plexus in the inner retina are accompanied by an immunoreactivity decrease in gamma-aminobutyric acidergic and glycinergic amacrine cells. Especially deteriorated were AII amacrine cells, the main neuronal subtype postsynaptic to dopaminergic cells, which exhibited a marked loss of lobular appendages and dendritic processes. Concomitantly, electrical synapses among AII cells, as well as chemical synapses between these and rod bipolar cells, were highly deteriorated in parkinsonian monkeys. These results highlight that the scotopic visual pathway is severely impaired in the parkinsonian condition and provide a morphological basis for a number of abnormalities found in electrophysiological and psychophysical trials in Parkinson's disease patients and animal models.

Amacrine Cells↗

Losses of immunoreactive parvalbumin amacrine and immunoreactive alphaprotein kinase C bipolar cells caused by methylmercury chloride intoxication in the retina of the tropical fish Hoplias malabaricus.

To quantify the effects of methylmercury (MeHg) on amacrine and on ON-bipolar cells in the retina, experiments were performed in MeHg-exposed groups of adult trahiras (Hoplias malabaricus) at two dose levels (2 and 6 microg/g, ip). The retinas of test and control groups were processed by mouse anti-parvalbumin and rabbit anti-alphaprotein kinase C (alphaPKC) immunocytochemistry. Morphology and soma location in the inner nuclear layer were used to identify immunoreactive parvalbumin (PV-IR) and alphaPKC (alphaPKC-IR) in wholemount preparations. Cell density, topography and isodensity maps were estimated using confocal images. PV-IR was detected in amacrine cells in the inner nuclear layer and in displaced amacrine cells from the ganglion cell layer, and alphaPKC-IR was detected in ON-bipolar cells. The MeHg-treated group (6 microg/g) showed significant reduction of the ON-bipolar alphaPKC-IR cell density (mean density = 1306 +/- 393 cells/mm2) compared to control (1886 +/- 892 cells/mm2; P < 0.001). The mean densities found for amacrine PV-IR cells in MeHg-treated retinas were 1040 +/- 56 cells/mm2 (2 microg/g) and 845 +/- 82 cells/mm2 (6 microg/g), also lower than control (1312 +/- 31 cells/mm2; P < 0.05), differently from the data observed in displaced PV-IR amacrine cells. These results show that MeHg changed the PV-IR amacrine cell density in a dose-dependent way, and reduced the density of alphaKC-IR bipolar cells at the dose of 6 microg/g. Further studies are needed to identify the physiological impact of these findings on visual function.

Amacrine Cells↗

NADPH-diaphorase positive neurons in the retina of Bufo marinus: selective staining of bipolar and amacrine cells.

Unfixed retinal tissues of adult Bufo marinus were reacted for NADPH-diaphorase histochemistry that resulted in selective staining of populations of bipolar and amacrine cells. The number of stained bipolar cells in the retina was estimated to be 233,600 +/- 38,900 (mean +/- S.D.). These cells were non-uniformly distributed across the retina with the highest density of 4,308 cells/mm2 along the visual streak decreasing to lowest density of 1,378 cells/mm2 at the dorsal and ventral poles of the retina. The stained bipolar cells represented a morphologically uniform population. The number of labelled amacrine cells in the retina amounted to 3,116 +/- 405 including 251 cells that were displaced into the ganglion cell layer. Labelled amacrine cells were also unevenly distributed; with no stained cells in the retinal centre from where the density increased steadily to 72 cells mm2 in the retinal periphery. NADPH-diaphorase positive amacrine cells were classified on the basis of their dendritic arborization pattern into three morphological types: 1) narrow-field cells both at the periphery and the centre of the retina; 2) wide-field amacrine cells, and 3) amacrines with eccentric medium-sized dendritic field. The results of this study provide evidence for the presence of NADPH-diaphorase containing neurons in the anuran retina. Furthermore, this is the first report on the selective staining of bipolar cells for NADPH-diaphorase in the vertebrate retina.

Animals↗

Specific deficit of the ON response in visual transmission by targeted disruption of the mGluR6 gene.

Taking advantage of the restricted expression of metabotropic glutamate receptor subtype 6 (mGluR6) in retinal ON bipolar cells, we generated knockout mice lacking mGluR6 expression. The homozygous mutant mice showed a loss of ON responses but unchanged OFF responses to light. The mutant mice displayed no obvious changes in retinal cell organization nor in the projection of optic fibers to the brain. Furthermore, the mGluR6-deficient mice showed visual behavioral responses to light stimulation as examined by shuttle box avoidance behavior experiments using light exposure as a conditioned stimulus. The results demonstrate that mGluR6 is essential in synaptic transmission to the ON bipolar cell and that the OFF response provides an important means for transmitting visual information.

Animals↗

Interaction of heterotrimeric G protein Galphao with Purkinje cell protein-2. Evidence for a novel nucleotide exchange factor.

The heterotrimeric G protein Galphao is ubiquitously expressed throughout the central nervous system, but many of its functions remain to be defined. To search for novel proteins that interact with Galphao, a mouse brain library was screened using the yeast two-hybrid interaction system. Pcp2 (Purkinje cell protein-2) was identified as a partner for Galphao in this system. Pcp2 is expressed in cerebellar Purkinje cells and retinal bipolar neurons, two locations where Galphao is also expressed. Pcp2 was first identified as a candidate gene to explain Purkinje cell degeneration in pcd mice (Nordquist, D. T., Kozak, C. A., and Orr, H. T. (1988) J. Neurosci. 8, 4780-4789), but its function remains unknown as Pcp2 knockout mice are normal (Mohn, A. R., Feddersen, R. M., Nguyen, M. S., and Koller, B. H. (1997) Mol. Cell. Neurosci. 9, 63-76). Galphao and Pcp2 binding was confirmed in vitro using glutathione S-transferase-Pcp2 fusion proteins and in vitro translated [35S]methionine-labeled Galphao. In addition, when Galphao and Pcp2 were cotransfected into COS cells, Galphao was detected in immunoprecipitates of Pcp2. To determine whether Pcp2 could modulate Galphao function, kinetic constants kcat and koff of bovine brain Galphao were determined in the presence and absence of Pcp2. Pcp2 stimulates GDP release from Galphao more than 5-fold without affecting kcat. These findings define a novel nucleotide exchange function for Pcp2 and suggest that the interaction between Pcp2 and Galphao is important to Purkinje cell function.

Animals↗

G-protein alpha subunit interaction and guanine nucleotide dissociation inhibitor activity of the dual GoLoco motif protein PCP-2 (Purkinje cell protein-2).

Purkinje cell protein-2 (PCP-2; L7/GPSM4) is a GoLoco motif-containing protein that is specifically expressed in Purkinje and retinal ON bipolar cells. An alternative splice variant of PCP-2 has recently been isolated which contains two GoLoco motifs. Although the second GoLoco motif (GL2) of PCP-2 has been reported to interact with Galpha-subunits, a complete biochemical analysis of each individual motif of PCP-2 has not been performed. We demonstrate that the first GoLoco motif (GL1) of PCP-2 is equipotent as a guanine nucleotide dissociation inhibitor (GDI) towards Galphai1 and Galphai2, while it has sevenfold lower GDI activity for Galphai3 and greater than 20-fold lower GDI activity against Galphao. In contrast we found PCP-2 GL2 to be essentially equipotent as a GDI for all Galphai subunits, but it had negligible activity toward Galphao. Using co-immunoprecipitation from COS-7 cells, we found that PCP-2 was only able to interact with Galphai1 but not Galphao nor Galpha-subunits from other families (Galphas, Galphaq, or Galpha12). Mutational analysis of a non-canonical residue (glycine 24) in human PCP-2 GL1 provided evidence for heterogeneity in mechanisms of Galphai interactions with GoLoco motifs. Collectively, the data demonstrate that PCP-2 is a comparatively weak GoLoco motif protein that exhibits highest affinity interactions and GDI activity toward Galphai1, Galphai2, and Galphai3 subunits.

Amino Acid Motifs↗