Konrad Schultz

Localization of heterotypic gap junctions composed of connexin45 and connexin36 in the rod pathway of the mouse retina

The primary rod pathway in mammals contains gap junctions between AII amacrine cells and ON cone bipolar cells which relay the rod signal into the cone pathway under scotopic conditions. Two gap junctional proteins, connexin36 (Cx36) and connexin45 (Cx45), appear to play a pivotal role in this pathway because lack of either protein leads to an impairment of visual transmission under scotopic conditions. To investigate whether these connexins form heterotypic gap junctions between ON cone bipolar and AII amacrine cells, we used newly developed Cx45 antibodies and studied the cellular and subcellular distribution of this protein in the mouse retina. Specificity of the Cx45 antibodies was determined, among others, by Western blot and immunostaining of mouse heart, where Cx45 is abundantly expressed. In mouse retina, Cx45 immunosignals were detected in both plexiform layers and the ganglion cell layer. Double staining for Cx45 and Cx36 revealed a partial overlap in the punctate patterns in the ON sublamina of the inner plexiform layer of the retina. We quantified the distributions of these two connexins in the ON sublamina, and detected 30% of the Cx45 signals to be co‐localized with or in close apposition to Cx36 signals. Combining immunostaining and intracellular dye injection revealed an overlap or tight association of Cx36 and Cx45 signals on the terminals of injected AII amacrine and two types of ON cone bipolar cells. Our results provide direct evidence for heterotypic gap junctions composed of Cx36 and Cx45 between AII amacrine and certain types of ON cone bipolar cells.

Horizontal cell receptive fields are reduced in connexin57-deficient mice.

Horizontal cells are coupled by gap junctions; the extensive coupling of the horizontal cells is reflected in their large receptive fields, which extend far beyond the dendritic arbor of the individual cell. In the mouse retina, horizontal cells express connexin57 (Cx57). Tracer coupling of horizontal cells is impaired in Cx57‐deficient mice, which suggests that the receptive fields of Cx57‐deficient horizontal cells might be similarly reduced. To test this hypothesis we measured the receptive fields of horizontal cells from wildtype and Cx57‐deficient mice. First, we examined the synaptic connections between horizontal cells and photoreceptors: no major morphological alterations were found. Moreover, horizontal cell spacing and dendritic field size were unaffected by Cx57 deletion. We used intracellular recordings to characterize horizontal cell receptive fields. Length constants were computed for each cell using the cells responses to concentric light spots of increasing diameter. The length constant was dependent on the intensity of the stimulus: increasing stimulus intensity reduced the length constant. Deletion of Cx57 significantly reduced horizontal cell receptive field size. Dark resting potentials were strongly depolarized and response amplitudes reduced in Cx57‐deficient horizontal cells compared to the wildtype, suggesting an altered input resistance. This was confirmed by patch‐clamp recordings from dissociated horizontal cells; mean input resistance of Cx57‐deficient horizontal cells was 27% lower than that of wildtype cells. These data thus provide the first quantification of mouse horizontal cell receptive field size and confirm the unique role of Cx57 in horizontal cell coupling and physiology.

Effects of Nitric Oxide on the Horizontal Cell Network and Dopamine Release in the Carp Retina

In the teleost retina the intercellular messenger nitric oxide can be synthesized by several cell types including cone photoreceptors and H1 horizontal cells, indicating a modulatory role within the outer plexiform layer, the first stage of the visual information processing. Therefore, the aim of this study was to elucidate the effects of nitric oxide on the physiology of cone horizontal cells in the intact retina. The nitric oxide donor sodium nitroprusside (0.5-2.5 mM) enhanced the light responsiveness of cone horizontal cells and reduced the degree of electrical coupling in the network. Furthermore, the spread of intracellularly injected Lucifer Yellow was restricted. The effects on light responsiveness and electrical coupling were qualitatively mimicked by 8-bromo-cGMP (0.5 mM) and could not be achieved by ferrocyanide (1 mM), the byproduct of nitric oxide liberation from nitroprusside. The effects of NO on the responsiveness of horizontal cells may be due to an action on green- and red-sensitive cones. Nitroprusside (0.1 mM) diminished the K(+)-stimulated release of endogenous dopamine by 50%, whereas the basal dopamine release was not affected, indicating that the effects on electrotonic horizontal cell coupling were not elicited by an NO-induced release of dopamine. With respect to the morphologic plasticity of the cone-horizontal cell synapse the inhibitor of endogenous nitric oxide synthesis L-nitroarginine (0.1 mM) had no influence on the formation or retraction of spinules. These results show that NO affects the responsiveness and coupling of the horizontal cell network in a dopamine-independent way.

Identification and localization of connexin26 within the photoreceptor-horizontal cell synaptic complex.

Connexin26 (Cx26) is a member of the family of integral membrane proteins that normally form intercellular gap junctional channels. We have used Western blotting, immunofluorescence, immunoelectron microscopy, and single-cell reverse-transcriptase polymerase chain reaction amplification (RT-PCR) to analyze the expression and cellular localization of Cx26 in the carp retina. In the outer plexiform layer, strong clustered Cx26 immunolabeling was concentrated at and restricted to the terminal dendrites of horizontal cells. Single-cell RT-PCR confirmed the expression of Cx26 in carp retinal horizontal cells. 248-bp fragments amplified from cDNAs of four different horizontal cells were cloned and each nucleotide sequence encodes a protein fragment (AA 104-185) with highly significant homology to rat and mouse Cx26. Immunoelectron microscopy revealed that only the invaginating dendrites of horizontal cells in intimate lateral association with the presynaptic ribbon complex were labeled. No labeling was found at the photoreceptor membrane and there was no septalaminar structure, indicative of gap junctions, between photoreceptors and horizontal cells. The focal location of Cx26 at the membrane of the dendritic tips of horizontal cells and the lack of gap junctional morphology suggests that Cx26 might form hemichannels.

AMPA-PREFERRING RECEPTORS WITH HIGH CA2+ PERMEABILITY MEDIATE DENDRITIC PLASTICITY OF RETINAL HORIZONTAL CELLS

The synaptic complex formed by the cone photoreceptor pedicles and the dendrites of horizontal cells in the teleost retina undergoes structural changes during light adaptation. Numerous spinules are formed by the terminal dendrites, and they are subsequently retracted during dark adaptation. In a retina kept under continuous illumination, the retraction process can be initiated by analogues of the neurotransmitter glutamate acting at AMPA/kainate receptors. On the other hand, the retraction process depends on calcium influx and the subsequent activation of CaMkII. We show here that the retraction of spinules induced by AMPA or kainate is not impaired in the presence of cobalt, making an involvement of voltage‐gated calcium channels unlikely. Using calcium imaging techniques with isolated horizontal cells, we demonstrate that AMPA and kainate, but not NMDA, increase [Ca2+]i in the presence of nicardipine, caffeine and thapsigargin. The increase of [Ca2+]i under these conditions depends on [Ca2+]o and on the agonist in a dose‐dependent manner, suggesting that the increase of [Ca2+]i is largely due to calcium influx through the agonist‐gated channel. Pharmacological studies were performed to determine whether AMPA‐ and/or kainate‐preferring receptors mediate the calcium influx. The AMPA‐preferring receptor antagonist LY303070 blocked glutamate‐ and kainate‐evoked increases of [Ca2+]i in a concentration‐dependent manner, indicating that kainate‐preferring receptors contributed little or nothing to the observed [Ca2+]i increase. This was supported by experiments where cyclothiazide (which blocks the desensitization of AMPA receptors) and concanavalin A (which potentiates responses mediated by kainate receptors) were applied. In all cases, LY303070 blocked the agonist‐evoked increase of [Ca2+]i. The presence of AMPA‐preferring receptors with high Ca2+ permeability on horizontal cells was also supported by measuring agonist‐induced currents using whole‐cell recording techniques. Furthermore, LY303070 was able to impair the retraction of spinules during dark adaption in the in vivo situation.

Contribution of connexin26 to electrical feedback inhibition in the turtle retina

The first synaptic integration in the neuronal cascade of vision in vertebrates includes a feedback from horizontal cells to cones by a mechanism yet not fully understood. Recent observations in teleosts suggested an electrical feedback mechanism mediated by connexin26 (Cx26) hemichannels at the terminal dendrites of horizontal cells. By using reverse transcription‐polymerase chain reaction and immunoblotting from retinal homogenate, we detected Cx26 mRNA transcripts in the turtle retina and demonstrated that they were translated into protein. Cx26 immunoreactivity was especially prominent in the outer plexiform layer. Subcellularly, immunoreactivity was located mainly between horizontal cell axon terminals and in horizontal cell dendrites forming the lateral elements at the ribbon synaptic complex of the cone pedicle. The label was confined to the horizontal cell membrane flanking the ribbon and was not found on the opposing photoreceptor membrane. No gap junctions at this location are known, so immunosignaling suggested the presence of hemichannels. Their relevance to the feedback mechanism was investigated by intracellular recordings from horizontal cells during application of the hemichannel blocker carbenoxolone. Carbenoxolone hyperpolarized the dark membrane potential by about 25 mV, decreased the amplitudes of responses to full‐field light flashes, and suppressed the feedback‐induced depolarizing inflexion in the response profile. These physiological findings are compatible with the involvement of hemichannels in the feedback between horizontal cells and cones and support the anatomical findings. Together, these data suggest the presence of an electrical feedback mechanism in the turtle retina, which therefore might be a common mechanism at the first visual synapse in vertebrates. J. Comp. Neurol. 466:468–477, 2003.

Connexin57 is expressed in dendro-dendritic and axo-axonal gap junctions of mouse horizontal cells and its distribution is modulated by light

Mouse horizontal cells are coupled by gap junctions composed of connexin57. These gap junctions are regulated by ambient light via multiple neuromodulators including dopamine. In order to analyze the distribution and structure of horizontal cell gap junctions in the mouse retina, and examine the effects of light adaptation on gap junction density, we developed antibodies that detect mouse retinal connexin57. Using immunohistochemistry in retinal slices, flat‐mounted retinas, and dissociated retinal cells, we showed that connexin57 is expressed in the dendrites and axon terminal processes of mouse horizontal cells. No staining was found in retinas of connexin57‐deficient mice. Significantly more connexin57‐positive puncta were found in the distal than in the proximal outer plexiform layer, indicating a higher level of expression in axon terminal processes than in the dendrites. We also examined the gap junctions using immunoelectron microscopy and showed that connexin57 does not form hemichannels in the horizontal cell dendritic tips. Light adaptation resulted in a significant increase in the number of connexin57‐immunoreactive plaques in the outer plexiform layer, consistent with previously reported effects of light adaptation on connexin57 expression in the mouse retina. This study shows for the first time the detailed location of connexin57 expression within mouse horizontal cells, and provides the first ultrastructural data on mouse horizontal cell gap junctions. J. Comp. Neurol. 513:363–374, 2009.

A novel type of interplexiform amacrine cell in the mouse retina

Mammalian retinas comprise an enormous variety of amacrine cells with distinct properties and functions. The present paper describes a new interplexiform amacrine cell type in the mouse retina. A transgenic mouse mutant was used that expressed the gene for the enhanced green fluorescent protein (EGFP) instead of the coding DNA of connexin45 in several retinal cell classes, among which a single amacrine cell population was most prominently labelled. Staining for EGFP and different marker proteins showed that these amacrine cells are interplexiform: they stratify in stratum S4/5 of the inner plexiform layer and send processes to the outer plexiform layer. These cells were termed IPA‐S4/5 cells. They belong to the group of medium‐field amacrine cells and are coupled homologously and heterologously to other amacrine cells by connexin45. Immunostaining revealed that IPA‐S4/5 cells are GABAergic and express GAT‐1, a plasma‐membrane‐bound GABA transporter possibly involved in non‐vesicular GABA release. To characterize the light responses of IPA‐S4/5 cells, patch‐clamp recordings in retinal slices were made. Consistent with their stratification in the ON sublamina of the inner plexiform layer, cells depolarized in response to light ON stimuli and transiently hyperpolarized in response to light OFF. Responses of cells to green (578 nm) and blue (400 nm) light suggest that they receive input from cone bipolar cells contacting both M‐ and S‐cones, possibly with reduced S‐cone input. A new type of interplexiform ON amacrine cell is described, which is strongly coupled and uses GABA but not dopamine as its neurotransmitter.

Differential Regulation of Cone Calcium Signals by Different Horizontal Cell Feedback Mechanisms in the Mouse Retina

Controlling neurotransmitter release by modulating the presynaptic calcium level is a key mechanism to ensure reliable signal transmission from one neuron to the next. In this study, we investigated how the glutamatergic output of cone photoreceptors (cones) in the mouse retina is shaped by different feedback mechanisms from postsynaptic GABAergic horizontal cells (HCs) using a combination of two-photon calcium imaging and pharmacology at the level of individual cone axon terminals. We provide evidence that hemichannel-mediated (putative ephaptic) feedback sets the cone output gain by defining the basal calcium level, a mechanism that may be crucial for adapting cones to the ambient light level. In contrast, pH-mediated feedback did not modulate the cone basal calcium level but affected the size and shape of light-evoked cone calcium signals in a contrast-dependent way: low-contrast light responses were amplified, whereas high-contrast light responses were reduced. Finally, we provide functional evidence that GABA shapes light-evoked calcium signals in cones. Because we could not localize ionotropic GABA receptors on cone axon terminals using electron microscopy, we suggest that GABA may act through GABA autoreceptors on HCs, thereby possibly modulating hemichannel- and/or pH-mediated feedback. Together, our results suggest that at the cone synapse, hemichannel-mediated (ephaptic) and pH-mediated feedback fulfill distinct functions to adjust the output of cones to changing ambient light levels and stimulus contrasts and that the efficacy of these feedback mechanisms is likely modulated by GABA release in the outer retina.

Selective synaptic distribution of AMPA and kainate receptor subunits in the outer plexiform layer of the carp retina

The subunit composition of ionotropic glutamate receptors (GluRs) is extremely diverse and responsible for the diversity of postsynaptic responses to the release of glutamate, which is the major excitatory neurotransmitter in the retina. To understand the functional consequences of this diversity, it is necessary to reveal the synaptic localization and subunit composition of GluRs. We have used immuno light and electron microscopy to localize AMPA and kainate (GluR1, GluR2/3, GluR4, GluR5–7) subunits in identified carp retinal neurons contributing to the outer plexiform layer. GluR1 could not be detected within the outer plexiform layer. Rod and cone horizontal cells all express only GluR2/3 at the tips of their invaginating dendrites. These receptors are also inserted into the membrane of spinules, light‐dependent protrusions of the horizontal cell dendrites, flanking the synaptic ribbon of the cone synapse. Bipolar cells express GluR2/3, GluR4, and GluR5–7 at their terminal dendrites invaginating cone pedicles and rod spherules. Colocalization data suggest that each subunit is expressed by a distinct bipolar cell type. The majority of bipolar cells expressing these receptors seem to be of the functional OFF‐type; however, in a few instances, GluR2/3 could also be detected on dendrites of bipolar cells that, based on their localization within the cone synaptic complex, appeared to be of the functional ON‐type. The spatial arrangement of the different subunits within the cavity of the cone pedicle appeared not to be random: GluR2/3 was found predominantly at the apex of the cavity, GluR4 at its base and GluR5–7 dispersed between the two. J. Comp. Neurol. 435:433–449, 2001.