Figure - uploaded by Edward Parker
Content may be subject to copyright.
![Pattern of GAD67 expression in αPax6-Cre:Gad1lox/lox (mutant) retina. (a) The spatial distribution of Cre recombinase expression across the retina parallels the pattern of GFP expression in amacrine cells. Cre expression is present in all cells of the embryonic retina, but several days after birth becomes restricted to subpopulations of amacrine cells [63]. At P8, GFP is expressed by relatively few cells in central retina (box, b) and a larger number of amacrine cells in the peripheral retina (box, c). Staining for GAD67 follows an inverse pattern in this mutant retina. (b) Numerous GAD67-immunopositive amacrine cells near the center of the mutant retina. hc, horizontal cells; ac, amacrine cells. (c) GAD67 immunoreactivity is largely absent in the retinal periphery of the mutant retina. Immunofluorescence in this image (arrow) marks blood vessels. There is no GAD67 immunoreactivity at retinal depths where amacrine cells (ac) and horizontal cells (hc) are located. (d) In the wild-type region of a P3 mutant retina, GAD67-immunopositive horizontal cells (hc) also contain GABA. In knockout regions of this retina, GAD67 and GABA immunolabeling are absent. Arrow (mutant) indicates immunoreactivity in some blood vessels.](profile/Edward-Parker/publication/44690331/figure/fig3/AS:195397550120962@1423597814207/Pattern-of-GAD67-expression-in-aPax6-CreGad1lox-lox-mutant-retina-a-The-spatial.png)
Pattern of GAD67 expression in αPax6-Cre:Gad1lox/lox (mutant) retina. (a) The spatial distribution of Cre recombinase expression across the retina parallels the pattern of GFP expression in amacrine cells. Cre expression is present in all cells of the embryonic retina, but several days after birth becomes restricted to subpopulations of amacrine cells [63]. At P8, GFP is expressed by relatively few cells in central retina (box, b) and a larger number of amacrine cells in the peripheral retina (box, c). Staining for GAD67 follows an inverse pattern in this mutant retina. (b) Numerous GAD67-immunopositive amacrine cells near the center of the mutant retina. hc, horizontal cells; ac, amacrine cells. (c) GAD67 immunoreactivity is largely absent in the retinal periphery of the mutant retina. Immunofluorescence in this image (arrow) marks blood vessels. There is no GAD67 immunoreactivity at retinal depths where amacrine cells (ac) and horizontal cells (hc) are located. (d) In the wild-type region of a P3 mutant retina, GAD67-immunopositive horizontal cells (hc) also contain GABA. In knockout regions of this retina, GAD67 and GABA immunolabeling are absent. Arrow (mutant) indicates immunoreactivity in some blood vessels.
Source publication
The inhibitory neurotransmitter gamma-amino-butyric acid (GABA) not only modulates excitability in the mature nervous system but also regulates neuronal differentiation and circuit development. Horizontal cells, a subset of interneurons in the outer retina, are transiently GABAergic during the period of cone photoreceptor synaptogenesis. In rodents...
Contexts in source publication
Context 1
... Cre-expressing regions were marked by the presence of GFP expression because the aPax6-Cre construct contains an IRES-GFP reporter cassette [38]. GFP expression in the mutant retina was apparent from birth through juvenile stages in neurons with a spatial organization consistent with that of ama- crine cells (Figure 3a). GFP expression in amacrine cells could thus be used as a reporter of Cre activity within that field of view. ...Context 2
... expression in amacrine cells could thus be used as a reporter of Cre activity within that field of view. Second, loss of GAD67 expression was directly confirmed by immunostaining for GAD67 (Figure 3b, c). Figure 3d demonstrates that a lack of GAD67 is accompanied by a loss of GABA. ...Context 3
... loss of GAD67 expression was directly confirmed by immunostaining for GAD67 (Figure 3b, c). Figure 3d demonstrates that a lack of GAD67 is accompanied by a loss of GABA. We did not observe expression of GAD65 in horizontal cells in any region of the mutant retina at the neonatal ages exam- ined (data not shown). ...Similar publications
The first inhibitory interneurons of the retina, the horizontal cells, stratify within the outer plexiform layer, extending dendritic terminals that connect to the pedicles of cone photoreceptors and an axon terminal system contacting the spherules of rod photoreceptors. How the horizontal cells acquire this morphology is unknown, but instructive i...
Cell transplantation is a potential strategy for treating blindness caused by the loss of photoreceptors. Although transplanted rod-precursor cells are able to migrate into the adult retina and differentiate to acquire the specialized morphological features of mature photoreceptor cells, the fundamental question remains whether transplantation of p...
Citations
... Quantitative analysis showed that the frequency (ciliation) and length of primary cilia displayed no Figures 4(b)-4(c)). To further explore the cilia profile in different subtypes of amacrine cells, we applied immunostaining for GAD67 (glutamate decarboxylase 67), a protein expressed in horizontal and amacrine cells during retinal development [59,60]. Coimmunostaining for GAD67 and AP2α antibodies identified GAD67-positive amacrine cells; their cilia profile was assessed by Arl13b ( Figure 4D ...
Objectives:
Primary cilia are conserved organelles found in polarized mammalian cells that regulate neuronal growth, migration, and differentiation. Proper cilia formation is essential during eye development. Our previous reports found that both amacrine and retinal ganglion cells (RGCs) contain primary cilia in primate and rodent retinas. However, whether primary cilia are present in the inner retina of human retinal organoids remains unknown. The purpose of this study is to characterize the primary cilia distribution in human embryonic stem cell (hESC-derived retinal organoid development.
Materials and methods:
Retinal organoids were differentiated from a hESC line, harvested at various developmental timepoints (day 44-day 266), and immunostained with antibodies for primary cilia, including Arl13b (for the axoneme), AC3, and Centrin3 (for the basal body). AP2α, Prox1, GAD67, Calretinin, GFAP, PKCα, and Chx10 antibodies as well as Brn3b-promoted tdTomato expression were used to visualize retinal cell types.
Results:
A group of ciliated cells were present in the inner aspects of retinal organoids from day 44 to day 266 in culture. Ciliated Chx10-positive retinal progenitor cells, GFAP-positive astrocytes, and PKCα-positive rod-bipolar cells were detected later during development (day 176 to day 266). Ciliation persisted during all stages of retinal developmental in AP2α-positive amacrine cells, but it was decreased in Brn3b-positive retinal ganglion cells (RGCs) at later time points. Additionally, AC3-positive astrocytes significantly decreased during the later stages of organoid formation.
Conclusions:
Amacrine cells in retinal organoids retain cilia throughout development, whereas RGC ciliation gradually and progressively decreases with organoid maturation.
... Alternative approaches to stain whole HCs, such as immunostaining against GFP in transgenic animals or direct injection of fluorophores into single HCs, may result in signals with a better SNR [42]. ...
Since its invention in 1994, super-resolution microscopy has become a popular tool for advanced imaging of biological structures, allowing visualisation of subcellular structures at a spatial scale below the diffraction limit. Thus, it is not surprising that recently, different super-resolution techniques are being applied in neuroscience, e.g. to resolve the clustering of neurotransmitter receptors and protein complex composition in presynaptic terminals. Still, the vast majority of these experiments were carried out either in cell cultures or very thin tissue sections, while there are only a few examples of super-resolution imaging in thick (> ~50 um) biological samples. In that context, the mammalian whole-mount retina has rarely been studied with super-resolution microscopy. Here, we aimed at establishing a stimulated-emission-depletion (STED) microscopy protocol for imaging whole-mount retina. To this end, we developed sample preparation including horizontal slicing of retinal tissue, an immunolabeling protocol with STED-compatible fluorophores and optimised the STED microscope's settings. We labelled subcellular structures in somata, dendrites, and axons of retinal ganglion cells in the inner mouse retina. Under optimal conditions, we achieved a mean lateral spatial resolution of ~120 nm (using the full width of half-maximum as a proxy) for the thinnest filamentous structures in our preparation and a resolution enhancement of two or higher compared to conventional confocal images. When combined with horizontal slicing of the retina, these settings allowed us visualisation of putative GABAergic horizontal cell synapses in the outer retina with a similar resolution. Taken together, we successfully established a STED protocol for reliable super-resolution imaging in the whole-mount mouse retina, which enables investigating, for instance, protein complex composition and cytoskeletal ultrastructure at retinal synapses in health and disease.
... GAD67 expression was first detected in horizontal and amacrine cells at P3 but this changed at P10, when the GAD67 expression was reduced in horizontal cells and remained unchanging only in amacrine cells. Otherwise, the expression of the enzyme was increased at P21 in amacrine cells and down-regulated around P14 in horizontal cells [26]. GAD67 immunoreactivity occurred in the amacrine, ganglion and horizontal cells at the end of the 1st postnatal week; after this, the proportion of GAD67 positive horizontal cells increased until the opening of the eye lid. ...
... Although the expression of GAD67 has already been widely analysed during the developmental process of the retina [25][26][27], the underlying molecular mechanisms involved in the transcriptional regulation during the postnatal development remain unknown. Our approach is innovative in the sense that to our knowledge no one has ever used three methodologies to study a single microRNA (miRNA) in the regulation of transmitter synthesis enzyme. ...
... This co-labelling disappeared in horizontal cell bodies and only the miR-23 labelling remained at P15. Previous studies also mentioned the same disappearance or down-regulation of GAD67 positivity in horizontal cells at the end of the second postnatal week [25,26]. Likewise, GABA expression is also transient in the outer retina of rat during postnatal development and disappears in horizontal cells after P15, while in the inner retinal layers GABA labelling is increasing until adulthood [23]. ...
As neurotransmitter, GABA is fundamental for physiological processes in the developing retina. Its synthesis enzymes are present during retinal development, although the molecular regulatory mechanisms behind the changes in expression are not entirely understood. In this study, we revealed the expression patterns of glutamic acid decarboxylase 67(GAD67) and its coding gene (GAD1) and its potential miRNA-dependent regulation during the first three postnatal weeks in rat retina. To gain insight into the molecular mechanisms, miRNA-sequencing supported by RT-qPCR and in situ hybridization were carried out. GAD1 expression shows an increasing tendency, peaking at P15. From the in silico-predicted GAD1 targeting miRNAs, only miR-23 showed similar expression patterns, which is a known regulator of GAD1 expression. For further investigation, we made an in situ hybridization investigation where both GAD67 and miR-23 also showed lower expression before P7, with the intensity of expression gradually increasing until P21. Horizontal cells at P7, amacrine cells at P15 and P21, and some cells in the ganglion cell layer at several time points were double labelled with miR-23 and GAD67. Our results highlight the complexity of these regulatory networks and the possible role of miR-23 in the regulation of GABA synthesizing enzyme expression during postnatal retina development.
... Previous studies have shown that a loss of photoreceptor proteins that control the release of glutamate leads to a degeneration of photoreceptor ribbon synapses (Dick et al., 2003;Haeseleer et al., 2004;Mansergh et al., 2005;tom Dieck et al., 2012;Michalakis et al., 2013). However, neither the absence of GABA synthesis (Schubert et al., 2010) nor the elimination of the light-dependent modulation of horizontal cell feedback and feedforward (Ströh et al., 2018) alters the ultrastructure of the photoreceptor ribbon synapse, suggesting that the synaptic activity of horizontal cells is not necessary for the assembly and maintenance of photoreceptor ribbon synapses. ...
The first synapse of the visual pathway is formed by photoreceptors, horizontal cells and bipolar cells. While ON bipolar cells invaginate into the photoreceptor terminal and form synaptic triads together with invaginating horizontal cell processes, OFF bipolar cells make flat contacts at the base of the terminal. When horizontal cells are ablated during retina development, no invaginating synapses are formed in rod photoreceptors. However, how cone photoreceptors and their synaptic connections with bipolar cells react to this insult, is unclear so far. To answer this question, we specifically ablated horizontal cells from the developing mouse retina. Following ablation around postnatal day 4 (P4)/P5, cones initially exhibited a normal morphology and formed flat contacts with OFF bipolar cells, but only few invaginating contacts with ON bipolar cells. From P15 on, synaptic remodeling became obvious with clustering of cone terminals and mislocalized cone somata in the OPL. Adult cones (P56) finally displayed highly branched axons with numerous terminals which contained ribbons and vesicular glutamate transporters. Furthermore, type 3a, 3b, and 4 OFF bipolar cell dendrites sprouted into the outer nuclear layer and even expressed glutamate receptors at the base of newly formed cone terminals. These results indicate that cones may be able to form new synapses with OFF bipolar cells in adult mice. In contrast, cone terminals lost their invaginating contacts with ON bipolar cells, highlighting the importance of horizontal cells for synapse maintenance. Taken together, our data demonstrate that early postnatal horizontal cell ablation leads to differential remodeling in the cone pathway: whereas synapses between cones and ON bipolar cells were lost, new putative synapses were established between cones and OFF bipolar cells. These results suggest that synapse formation and maintenance are regulated very differently between flat and invaginating contacts at cone terminals.
... In rabbit retina, GAD65 and GAD67 immunoreactivities were detected in horizontal cells (Johnson and Vardi, 1998). Several studies report GAD67 immunostaining is present at high levels in horizontal cells of the developing and juvenile mouse, rat, and rabbit retina (Schnitzer and Rusoff, 1984;Osborne et al., 1986;Versaux-Botteri et al., 1989;Schubert et al., 2010), but at low or non-detectable levels in adult horizontal cells (Brandon et al., 1979;Schnitzer and Rusoff, 1984;Brandon, 1985;Osborne et al., 1986;Wässle and Chun, 1989;Brecha et al., 1991;Yazulla et al., 1997;Koulen et al., 1998b), including mouse Schubert et al., 2010;Herrmann et al., 2011). GAD65 immunostaining (Figure 2) and mRNA were detected in adult guinea pig horizontal cells . ...
... In rabbit retina, GAD65 and GAD67 immunoreactivities were detected in horizontal cells (Johnson and Vardi, 1998). Several studies report GAD67 immunostaining is present at high levels in horizontal cells of the developing and juvenile mouse, rat, and rabbit retina (Schnitzer and Rusoff, 1984;Osborne et al., 1986;Versaux-Botteri et al., 1989;Schubert et al., 2010), but at low or non-detectable levels in adult horizontal cells (Brandon et al., 1979;Schnitzer and Rusoff, 1984;Brandon, 1985;Osborne et al., 1986;Wässle and Chun, 1989;Brecha et al., 1991;Yazulla et al., 1997;Koulen et al., 1998b), including mouse Schubert et al., 2010;Herrmann et al., 2011). GAD65 immunostaining (Figure 2) and mRNA were detected in adult guinea pig horizontal cells . ...
... The detection of GAD or GABA in the adult retina may be influenced by numerous factors, including the differential expression of GAD isoforms, regulations of levels of Gad transcripts and GAD proteins, and GABA synthesis in horizontal cells, as well as technical issues related to fixation composition, fixation protocols (perfusion or immersion) and antibody specificity Vardi and Auerbach, 1995;Kalloniatis et al., 1996;Johnson and Vardi, 1998;Deniz et al., 2011). Schubert et al. (2010) confirmed expression of GAD67 during neonatal development in mouse, but never detected GAD65 in horizontal cells. Some investigators observed the volatility of GABA immunoreactivity (Kalloniatis et al., 1996;Deniz et al., 2011) and suggested that it may be due to technical issues with harvesting the retina . ...
Feedback inhibition by horizontal cells regulates rod and cone photoreceptor calcium channels that control their release of the neurotransmitter glutamate. This inhibition contributes to synaptic gain control and the formation of the center-surround antagonistic receptive fields passed on to all downstream neurons, which is important for contrast sensitivity and color opponency in vision. In contrast to the plasmalemmal GABA transporter found in non-mammalian horizontal cells, there is evidence that the mechanism by which mammalian horizontal cells inhibit photoreceptors involves the vesicular release of the inhibitory neurotransmitter GABA. Historically, inconsistent findings of GABA and its biosynthetic enzyme, L-glutamate decarboxylase (GAD) in horizontal cells, and the apparent lack of surround response block by GABAergic agents diminished support for GABA's role in feedback inhibition. However, the immunolocalization of the vesicular GABA transporter (VGAT) in the dendritic and axonal endings of horizontal cells that innervate photoreceptor terminals suggested GABA was released via vesicular exocytosis. To test the idea that GABA is released from vesicles, we localized GABA and GAD, multiple SNARE complex proteins, synaptic vesicle proteins, and Cav channels that mediate exocytosis to horizontal cell dendritic tips and axonal terminals. To address the perceived relative paucity of synaptic vesicles in horizontal cell endings, we used conical electron tomography on mouse and guinea pig retinas that revealed small, clear-core vesicles, along with a few clathrin-coated vesicles and endosomes in horizontal cell processes within photoreceptor terminals. Some small-diameter vesicles were adjacent to the plasma membrane and plasma membrane specializations. To assess vesicular release, a functional assay involving incubation of retinal slices in luminal VGAT-C antibodies demonstrated vesicles fused with the membrane in a depolarization- and calcium-dependent manner, and these labeled vesicles can fuse multiple times. Finally, targeted elimination of VGAT in horizontal cells resulted in a loss of tonic, autaptic GABA currents, and of inhibitory feedback modulation of the cone photoreceptor Cai, consistent with the elimination of GABA release from horizontal cell endings. These results in mammalian retina identify the central role of vesicular release of GABA from horizontal cells in the feedback inhibition of photoreceptors.
... In mammalian retinas, compelling evidence suggests that horizontal cells release GABA in a depolarization-dependent, vesicular manner Grove et al., 2019). In mammals, GABA and the GABA synthetic enzyme, L-glutamate decarboxylase (GAD) are localized to horizontal cells (Schnitzer and Rusoff, 1984;Wässle and Chun, 1989;Grünert and Wässle, 1990;Vardi et al., 1994;Guo et al., 2010;Schubert et al., 2010;Deniz et al., 2011) and VGAT, V-ATPase, multiple SNARE, and vesicle proteins, and Ca V channels mediating vesicular release are localized to horizontal cell dendritic tips and axonal terminals (Dowling and Boycott, 1966;Brandon and Lam, 1983;Linberg and Fisher, 1988;Peters et al., 1991;Catsicas et al., 1992;Ueda et al., 1992;Löhrke and Hofmann, 1994;Grabs et al., 1996;Greenlee et al., 2001;Rivera et al., 2001;Cueva et al., 2002;Hirano et al., 2005Hirano et al., , 2007Hirano et al., , 2011Schubert et al., 2006;Lee and Brecha, 2010;Liu et al., 2013). Furthermore, vesicle membrane fusion and recycling in horizontal cells is depolarization-and Ca 2+ -dependent (Takamori et al., 2000;Vuong et al., 2011), and the deletion of VGAT from horizontal cells abolishes horizontal cell inhibitory feedback to photoreceptor Ca V channels . ...
How neurons in the eye feed signals back to photoreceptors to optimize sensitivity to patterns of light appears mediated by one or more unconventional mechanisms. Via these mechanisms, horizontal cells control synaptic gain and enhance key aspects of temporal and spatial light adaptation, including center-surround receptive field antagonism. After the transduction of light energy into an electrical signal in photoreceptors, a major task in visual processing is transmission of an optimized signal to the follower neurons in the retina. For this to happen, the release of the excitatory neurotransmitter glutamate from photoreceptors is carefully regulated via feedback from horizontal cells, which acts like a thermostat to keep synaptic transmission in an optimal range during changes in light patterns and intensities. A recently described model that casts a classical neurotransmitter system together with ion transport mechanisms to adjust the alkaline milieu outside the synapse, is emphasized here. We review the new evidence showing how this novel inter-neuronal messaging system carries the feedback inhibition using two separate, but interwoven, regulated systems. It may be the complex interplay between these two signaling modalities, creating synaptic modulation-at-a-distance, that has made its definition difficult.
The foundations of our understanding of the feedback mechanism from horizontal cells to photoreceptors have been long established: Horizontal cells have broad receptive fields, suitable for providing surround inhibition, their membrane potential regulates inhibition of photoreceptor voltage-gated calcium channels, and strong artificial pH buffering eliminates this action. This report compares and contrasts models of how these foundations are linked, focusing on a recent report of a novel action of GABA in mammals that shows tonic horizontal cell release of GABA activating chloride and bicarbonate permeable GABA autoreceptors. The membrane potential of horizontal cells provides the driving force for GABAR-mediated bicarbonate efflux, alkalinizing the cleft when horizontal cells are hyperpolarized by light or adding to their depolarization cells in darkness by contributing to cleft acidification via NHE-mediated proton efflux. This model reverses interpretations of earlier studies that were considered to rule out a role for GABA in feedback to cones.
... In mammalian retinas, compelling evidence suggests that horizontal cells release GABA in a depolarization-dependent, vesicular manner Grove et al., 2019). In mammals, GABA and the GABA synthetic enzyme, L-glutamate decarboxylase (GAD) are localized to horizontal cells (Schnitzer and Rusoff, 1984;Wässle and Chun, 1989;Grünert and Wässle, 1990;Vardi et al., 1994;Guo et al., 2010;Schubert et al., 2010;Deniz et al., 2011) and VGAT, V-ATPase, multiple SNARE, and vesicle proteins, and Ca V channels mediating vesicular release are localized to horizontal cell dendritic tips and axonal terminals (Dowling and Boycott, 1966;Brandon and Lam, 1983;Linberg and Fisher, 1988;Peters et al., 1991;Catsicas et al., 1992;Ueda et al., 1992;Löhrke and Hofmann, 1994;Grabs et al., 1996;Greenlee et al., 2001;Rivera et al., 2001;Cueva et al., 2002;Hirano et al., 2005Hirano et al., , 2007Hirano et al., , 2011Schubert et al., 2006;Lee and Brecha, 2010;Liu et al., 2013). Furthermore, vesicle membrane fusion and recycling in horizontal cells is depolarization-and Ca 2+ -dependent (Takamori et al., 2000;Vuong et al., 2011), and the deletion of VGAT from horizontal cells abolishes horizontal cell inhibitory feedback to photoreceptor Ca V channels . ...
How neurons in the eye feed signals back to photoreceptors to optimize sensitivity to patterns of light appears to be mediated by one or more unconventional mechanisms. Via these mechanisms, horizontal cells control photoreceptor synaptic gain and enhance key aspects of temporal and spatial center-surround receptive field antagonism. After the transduction of light energy into an electrical signal in photoreceptors, the next key task in visual processing is transmission of an optimized signal to the follower neurons in the retina. For this to happen, the release of the excitatory neurotransmitter glutamate from photoreceptors is carefully regulated via horizontal cell feedback, which acts like a thermostat to keep synaptic transmission in an optimal range during changes to light patterns and intensities. Novel findings of a recently described model that casts a classical neurotransmitter system together with ion transport mechanisms to adjust the alkaline milieu outside the synapse, are reviewed. This novel inter-neuronal messaging system carries the feedback inhibition using two separate, but interwoven, regulated systems. The complex interplay between these two signaling modalities, creating synaptic modulation-at-a-distance, have obscured its being defined.
The foundations of our understanding of the feedback mechanism from horizontal cells to photoreceptors have been long established: Horizontal cells have broad receptive fields, suitable for providing surround inhibition, their membrane potential, a function of stimulus intensity and size, regulates inhibition of photoreceptor voltage-gated Ca2+ channels, and strong artificial pH buffering eliminates this action. This review compares and contrasts models of how these foundations are linked, focusing on a recent report in mammals that shows tonic horizontal cell release of GABA activating Cl− and HCO3− permeable GABA autoreceptors. The membrane potential of horizontal cells provides the driving force for GABAR-mediated HCO3− efflux, alkalinizing the cleft when horizontal cells are hyperpolarized by light or adding to their depolarization in darkness and contributing to cleft acidification via NHE-mediated H+ efflux. This model challenges interpretations of earlier studies that were considered to rule out a role for GABA in feedback to cones.
... In mammalian retinas, compelling evidence suggests that horizontal cells release GABA in a depolarization-dependent, vesicular manner Grove et al., 2019). In mammals, GABA and the GABA synthetic enzyme, L-glutamate decarboxylase (GAD) are localized to horizontal cells (Schnitzer and Rusoff, 1984;Wässle and Chun, 1989;Grünert and Wässle, 1990;Vardi et al., 1994;Guo et al., 2010;Schubert et al., 2010;Deniz et al., 2011) and VGAT, V-ATPase, multiple SNARE, and vesicle proteins, and Ca V channels mediating vesicular release are localized to horizontal cell dendritic tips and axonal terminals (Dowling and Boycott, 1966;Brandon and Lam, 1983;Linberg and Fisher, 1988;Peters et al., 1991;Catsicas et al., 1992;Ueda et al., 1992;Löhrke and Hofmann, 1994;Grabs et al., 1996;Greenlee et al., 2001;Rivera et al., 2001;Cueva et al., 2002;Hirano et al., 2005Hirano et al., , 2007Hirano et al., , 2011Schubert et al., 2006;Lee and Brecha, 2010;Liu et al., 2013). Furthermore, vesicle membrane fusion and recycling in horizontal cells is depolarization-and Ca 2+ -dependent (Takamori et al., 2000;Vuong et al., 2011), and the deletion of VGAT from horizontal cells abolishes horizontal cell inhibitory feedback to photoreceptor Ca V channels . ...
How neurons in the eye feed signals back to photoreceptors to optimize sensitivity to patterns of light appears to be mediated by one or more unconventional mechanisms. Via these mechanisms, horizontal cells control photoreceptor synaptic gain and enhance key aspects of temporal and spatial center-surround receptive field antagonism. After the transduction of light energy into an electrical signal in photoreceptors, the next key task in visual processing is transmission of an optimized signal to the follower neurons in the retina. For this to happen, the release of the excitatory neurotransmitter glutamate from photoreceptors is carefully regulated via horizontal cell feedback, which acts like a thermostat to keep synaptic transmission in an optimal range during changes to light patterns and intensities. Novel findings of a recently described model that casts a classical neurotransmitter system together with ion transport mechanisms to adjust the alkaline milieu outside the synapse, are reviewed. This novel inter-neuronal messaging system carries the feedback inhibition using two separate, but interwoven, regulated systems. The complex interplay between these two signaling modalities, creating synaptic modulation-at-a-distance, have obscured its being defined.
The foundations of our understanding of the feedback mechanism from horizontal cells to photoreceptors have been long established: Horizontal cells have broad receptive fields, suitable for providing surround inhibition, their membrane potential, a function of stimulus intensity and size, regulates inhibition of photoreceptor voltage-gated Ca2+ channels, and strong artificial pH buffering eliminates this action. This review compares and contrasts models of how these foundations are linked, focusing on a recent report in mammals that shows tonic horizontal cell release of GABA activating Cl− and HCO3− permeable GABA autoreceptors. The membrane potential of horizontal cells provides the driving force for GABAR-mediated HCO3− efflux, alkalinizing the cleft when horizontal cells are hyperpolarized by light or adding to their depolarization in darkness and contributing to cleft acidification via NHE-mediated H+ efflux. This model challenges interpretations of earlier studies that were considered to rule out a role for GABA in feedback to cones.
... We thus examined the morphology of these neurons (Figure 3 and Figure 3-figure supplement 1). In control animals, horizontal cells first emerge as radial neurons (Schubert et al., 2010), exhibiting long apical and basal neurites ( Figure 3A). Between P3 and P5, horizontal cells then undergo a process of neurite refinement in which apically and basally targeted neurites become lateralized through largely unknown mechanisms to form a horizontal neural structure (Poché and Reese, 2009). ...
Structural changes in pre and postsynaptic neurons that accompany synapse formation often temporally and spatially overlap. Thus, it has been difficult to resolve which processes drive patterned connectivity. To overcome this, we use the laminated outer murine retina. We identify the serine/threonine kinase LKB1 as a key driver of synapse layer emergence. The absence of LKB1 in the retina caused a marked mislocalization and delay in synapse layer formation. In parallel, LKB1 modulated postsynaptic horizontal cell refinement and presynaptic photoreceptor axon growth. Mislocalized horizontal cell processes contacted aberrant cone axons in LKB1 mutants. These defects coincided with altered synapse protein organization, and horizontal cell neurites were misdirected to ectopic synapse protein regions. Together, these data suggest that LKB1 instructs the timing and location of connectivity in the outer retina via coordinate regulation of pre and postsynaptic neuron structure and the localization of synapse-associated proteins.
... We thus examined the morphology of these neurons (Figure 3 and Figure 3-figure supplement 1). In control animals, horizontal cells first emerge as radial neurons (Schubert et al., 2010), exhibiting long apical and basal neurites ( Figure 3A). Between P3 and P5, horizontal cells then undergo a process of neurite refinement in which apically and basally targeted neurites become lateralized through largely unknown mechanisms to form a horizontal neural structure (Poché and Reese, 2009). ...
Structural changes in pre and postsynaptic neurons that accompany synapse formation often temporally and spatially overlap. Thus, it has been difficult to resolve which processes drive patterned connectivity. To overcome this, we use the laminated outer murine retina. We identify the serine/threonine kinase LKB1 as a key driver of synapse layer emergence. The absence of LKB1 in the retina caused a marked mislocalization and delay in synapse layer formation. In parallel, LKB1 modulated postsynaptic horizontal cell refinement and presynaptic photoreceptor axon growth. Mislocalized horizontal cell processes contacted aberrant cone axons in LKB1 mutants. These defects coincided with altered synapse protein organization, and horizontal cell neurites were misdirected to ectopic synapse protein regions. Together, these data suggest that LKB1 instructs the timing and location of connectivity in the outer retina via coordinate regulation of pre and postsynaptic neuron structure and the localization of synapse-associated proteins.
