Development and maintenance of vision's first synapse

Abstract

Synapses in the outer retina are the first information relay points in vision. Here, photoreceptors form synapses onto two types of interneurons, bipolar cells and horizontal cells. Because outer retina synapses are particularly large and highly ordered, they have been a useful system for the discovery of mechanisms underlying synapse specificity and maintenance. Understanding these processes is critical to efforts aimed at restoring visual function through repairing or replacing neurons and promoting their connectivity. We review outer retina neuron synapse architecture, neural migration modes, and the cellular and molecular pathways that play key roles in the development and maintenance of these connections. We further discuss how these mechanisms may impact connectivity in the retina.

Keywords: Bipolar cell; Horizontal cell; Outer plexiform layer; Photoreceptor; Retina; Ribbon; Synapse.

Figure 1.. Structure of the retina.

A. Schematic of outer retina neuron organization in adults. The outer nuclear layer (ONL) contains rods (purple) and cones (blue) that form connections in the outer plexiform layer (OPL) with rod bipolar cells (RBP, green), cone bipolar cells (CBP, yellow), and horizontal cells (HC, magenta) found in the inner nuclear layer (INL). Also present in the INL are amacrine cells (AC, dark purple) that form connections in the inner plexiform layer (IPL) together with bipolar cell axons onto retinal ganglion cells (RGC, red) whose cell bodies reside in the ganglion cell layer (GCL). Müller glia (MG, dark blue) span the length of the outer retina. Three intraretina vascular layers also interdigitate the GCL, IPL, and OPL and are termed the superficial, intermediate, and deep vasculature layers, respectively. B. The OPL contains two sublamina. Rod spherules form the apical sublamina (1), while cone pedicles sit just beneath rod spherules and form the basal sublamina (2). C. Axons from ganglion cells form the optic nerve, which projects to multiple (~50) retinorecipient areas in the brain.

Figure 2.. Axon terminal architecture of photoreceptors.

A. Rod (purple) and cone (blue) photoreceptors are comprised of four general regions: the outer segment, inner segment, soma, and axon terminal. The terminal region is termed the spherule in rods and pedicle in cones. Photoreceptors contain ribbon synapses which hold vesicles close to the active zone for rapid neurotransmitter release. The mature rod synapse contains one ribbon and a corresponding postsynaptic invagination by two lateral horizontal cell (HC; pink) neurites that are regulated by ionotropic AMPA receptors and two or more rod bipolar cells (RBP; green) that contain metabotropic mGluR6 receptors. OFF-cone bipolar cells can also form contacts on the rod spherule. Cone pedicles contain between 20 and 50 sites of invagination. Each invagination contains two lateral horizontal cell neurites that are regulated by ionotropic AMPA receptors and one or more central metabotropic mGluR6 ON-cone bipolar cell (ON BP; yellow) dendrites. Cones also receive flat contacts on the base of the pedicle made by OFF-cone bipolar cells (OFF BP; orange) that are regulated by ionotropic AMPA or Kainate glutamate receptors. Rod bipolar cells can also form contacts on the cone pedicle. B. Electrical signals can also propagate through photoreceptors via gap junctions formed by connexin 36 in mice. These contact sites occur on the tips of telondendria that connect cones to cones and cones to rods. The connexin that electrically couples rods to rods is unknown. Gap junction contacts are pictured in cross section (left panel) and en face (right panel).

Figure 3.. Proteins found at the photoreceptor synapse.

A. Schematic of proteins associated with rod photoreceptor synaptic ribbons and the active zone. RIBEYE is the primary protein of the ribbon, and Kif3a, RIM1, and piccolo also associate with this structure. Bassoon and CAST are localized beneath the ribbon at the active zone and are scaffolding proteins that interact with RIM2, which enhances calcium channel activity. RIM weakly interacts with Rab3a, a vesicle-associated GTPase that regulates vesicle exocytosis, and Munc13. B. Schematic of proteins associated with synaptic vesicles. SNARE proteins are present, including synaptobrevin (V-Snare) and syntaxin3 (T-Snare), while SNAP25 has also been variously reported. Complexin 3 and 4 bind to SNARE complexes to promote vesicle docking. VGLUT1 is a vesicular glutamate transporter, SV2 regulates neurotransmitter release at terminals, and synaptotagmin1 is predicted to be a calcium sensor. C. Schematic of proteins that interact with the extracellular matrix (ECM) and those found at postsynaptic sites. Dystrophin is a cytoskeletal protein that interacts with actin and the dystroglycan beta subunit, while the dystroglycan alpha subunit binds laminin and the retina-specific protein pikachurin. Laminin is a glycoprotein found in the ECM that interacts with the retina specific protein retinoschisin, which in turn regulates calcium channels. Pikachurin is required to connect photoreceptors to ON-bipolar cells via binding to GPR179 on the postsynaptic membrane. GPR179 interacts with regulator of G-protein signaling (RGS) 7 and RGS9. MPP4 is also present in presynaptic photoreceptors and recruits PSD95 and Veli3 to the photoreceptor synapse. In turn, these proteins interact with the calcium-dependent chloride channel TMEM16b and plasma membrane calcium ATPase PMCA. mGluR6 is present on the postsynaptic membrane. Binding of glutamate to mGluR6 activates the G protein G_o, which leads to closure of the constitutively active nonselective cation channel transient receptor potential melastatin 1 (TRPM1). Nyctalopin is required for TRPM1, mGluR6, and GPR179 localization by binding to LRIT3 on the presynaptic membrane. Asterisk (*) denotes protein localization at conventional ribbon synapses.

Figure 4.. Migration of outer retina neurons.

A. Schematic of retinal progenitor cells show that they undergo interkinetic nuclear migration (INM). In this form of movement, nuclei move in apical and basal directions in phase with the cell cycle. B-E. Schematic of fate committed retina neuron subtypes show that retinal neurons exhibit unique migration modes to reach their final laminar position. Horizontal cells (magenta) undergo a bipolar migration phase beginning in the outer neuroblast layer (ONBL), bypassing their final position and descending further into the inner neuroblast layer (INBL), where they switch to a multipolar migration phase (B). They then migrate apically to reach the outer retina where they then fine-tune their final position at the apical side of the future INL. It is presumed that rods (purple) undergo two migration phases (C). From P5 to P8, rods are present at the apical surface of the INL and are thought to migrate apically. During this time, the ONL increases in thickness as the INL decreases in thickness. It is also likely that rods apically and basally migrate within the ONL, as knockout and overexpression studies of CasZ1 results in preferential bias of rods to the apical or basal surface of the ONL. However, when this migration occurs is unknown. Finally, cones (blue) undergo nuclear translocation and move in apical and basal directions from P4 to P12 (D). Together, these neuron subtype specific movements result in proper nuclear lamination in adult retina (E).

Figure 5.. Ultrastructural and anatomical events that underlie outer retina synaptogenesis.

A. Schematic of OPL development at P3. The outer retina contains developing cones (blue) whose axons form contacts with horizontal cell neurites (magenta) at P3 forming synaptic patches (red circles). B. Schematic of OPL development from P5 to P7. Synaptogenesis between cones and horizontal cells begins at P5 forming the nascent OPL (red box). At this time, horizontal cells form a monad connection with cones. At P6, horizontal cells form a dyad connection with cones, while cones and OFF-cone bipolar cells (orange) form flat contacts at the base of the pedicle. At P7, cones and ON-cone bipolar cells (yellow) form triad connections and mGluR6 expression becomes apparent (Sarin et al., 2018). C. Schematic of OPL development from P8 to P10. Rods (purple) undergo synaptogenesis with horizontal cells forming a monad contact. At P9, horizontal cells form a dyad connection with rods, followed by triad connections between rods and rod bipolar cells (green) at P10. mGluR6 is not observed in rod terminals until P13 (Sarin et al., 2018). D. Schematic of OPL development at P13 and beyond. Ribbon synapse formation is complete, and sublaminar patterning of the OPL continues until P21.

Figure 6.. Genes involved in neurite targeting.

A. The OPL of wildtype retina is highly ordered, with neurites from pre and postsynaptic cells precisely targeted to the terminals of rods and cones. Rod spherules contain two lateral horizontal cell axons and two or more central bipolar cell dendrites. Each cone pedicle invagination contains two lateral horizontal cell dendrites, one or more centrally localized ON-cone bipolar cell contact, and flat contacts with OFF-cone bipolar cells. B. Genes involved in bipolar cell targeting to photoreceptors. ELFN1, pikachurin, and dystroglycan are required for rod bipolar cell invagination into rod terminals, while LRIT3, pikachurin, and dystroglycan are required for cone bipolar cell invagination into cone terminals. Defects are denoted by arrows, while cells reported to be normal in the mutant lines are shown in grey. C. Genes involved in horizontal cell neurite targeting. Loss of Sema6a, PlexinA4, or NGL2 results in horizontal cell neurite sprouting into the outer retina and reduced invagination into rod terminals (arrows). NGL2 mutants also exhibit an increase in spherical and club-shaped ribbons (asterisk). Cells reported to be normal in the mutant lines are shown in grey.

Figure 7.. Genes involved in OPL maturation and maintenance.

Schematic of genes involved in OPL maturation and maintenance. Wildtype outer retina synapses maintain their precise connectivity over time. Postsynaptic horizontal cell (magenta), rod bipolar cell (green) and cone bipolar cell (yellow) neurites remain confined to the OPL where they exactly oppose presynaptic cone (blue) and rod (purple) terminals in this region. Bassoon, Cacna1f, Cacna2d4, and RIBEYE are required for proper ribbon synaptic structure in both rod and cone terminals, and interneuron neurite sprouting and rod terminal retraction are observed in mutant lines. Complexin 3 and 4, piccolo isoform piccolino, and SynCAM1 are also required for proper rod ribbon structure, while the ultrastructure of cones was not reported (grey). No optokinetic response (nrc) mutants appear similar to Bassoon mutants but affects cone terminals. Rod terminals have not been examined in this line (grey). Deletion of 4.1G and CAST does not affect ribbon ultrastructure, but remodeling of pre and postsynaptic cells is observed. Similar remodeling of rod terminals occurs with the loss of LKB1, AMPK, or retinoschisin. Cone pedicles were not examined in these lines (grey). Neurite remodeling is denoted by arrowheads, while changes to the ribbon are denoted by a black asterisk. A red asterisk denotes perturbations that occur after P13.