Molecular dynamics of photoreceptor synapse formation in the developing chick retina

Abstract

The cellular and molecular mechanisms underlying photoreceptor synaptogenesis are poorly understood. Furthermore, a detailed picture of the molecular composition of photoreceptor synapses, or their subtypes, is not yet available, nor do we know what differences, if any, exist among those subtypes. To address these questions, we investigated temporal and spatial patterns of expression and assembly of photoreceptor presynaptic components during chick embryo retinal development and early posthatched life by using reverse transcriptase polymerase chain reaction (RT-PCR), dissociated retinal cells, laser-capture microdissection (LCM), immunocytochemistry and confocal microscopy. Immunocytochemistry in tissue sections and dissociated cells showed many similarities and few differences in the synaptic composition of rods and cone subtypes, which, however, were found to project to different strata within the outer plexiform layer. A striking finding was the precise timetable of expression of synaptic genes and proteins during synaptogenesis. Although mRNAs for some synaptic molecules appeared as early as embryonic day (ED) 5-8 (the time of inner retina synaptogenesis), others were undetectable before the time of onset of photoreceptor synaptogenesis on ED13, including CAST, rim2, synapsin-2, syntaxin-3, synaptotagmin, glutamate receptors -1, -4, and -5, homer-1 and -2, and tenascin-R. Most synaptic proteins in photoreceptors followed a similar sequence of expression: they were negative or weakly positive before ED13, appeared in inner segments between ED13 and ED15, became subsequently detectable in perinuclear and axonal regions, and by ED18 were assembled into synaptic terminals and became undetectable in the inner segments. The identity of the signals that regulate the coordinated expression of these synaptic components remains to be investigated. J. Comp. Neurol. 506:822-837, 2008 Indexing terms: synaptogenesis; cone; rod; presynaptic; postsynaptic; ribbonThe neural retina has a laminated structure, in which three nuclear layers (outer nuclear layer, inner nuclear layer, and ganglion cell layer) are separated by two synaptic layers (outer plexiform layer and inner plexiform layer). The outer nuclear layer consists of rod and cone photoreceptors, the inner nuclear layer contains the cell bodies of bipolar, horizontal and amacrine neurons and Mü ller glia, and the ganglion cell layer contains ganglion and displaced amacrine neurons. Synapses between photoreceptors and bipolar and horizontal neurons occur in the outer plexiform layer, and those between bipolar, amacrine, and ganglion neurons occur in the inner plexiform layer. This highly stereotyped laminar organization represents an advantage over other regions of the central nervous system for studies of synapse formation. This paper will focus on the development of the specialized "ribbon" synaptic terminals of photoreceptor cells, known as rod spherules and cone pedicles, and their contacts with postsynaptic processes from bipolar and horizontal neurons. Syn...

Developmental regulation of muscleblind‐like (MBNL) gene expression in the chicken embryo retina

“…7). When that pattern was compared with the distribution of PSD95 and SV2, molecules previously shown to be components of photoreceptor synaptic terminals (Chen et al, 2005;Wahlin et al, 2007), clear colocalization was observed (Fig. 7).…”

“…The isolated photoreceptors were readily identified in the dissociates as rods, double cones, and single cones based on their structure and the shape of their outer segments; single cone subtypes were distinguished based on their characteristic oil droplets (López et al, 2005). These identification criteria have been verified by PCR of individual cells (Wahlin et al, 2004;Huang and Adler, 2005;Wahlin et al, 2007). The segregation of MBNL1 and MBNL2 immunoreactivities was retained in the dissociated cells because, as shown in Figure 8, MBNL1 immunoreactivity was restricted to the synaptic terminal and MBNL2 to inner segments in all photoreceptor subtypes.…”

“…Retinae from 20-day-old chick embryos were dissociated after papain treatment as described by Wahlin et al (2004Wahlin et al ( , 2007. Single rods, green cones, or red cones, identified by morphological criteria corroborated by ICC, were individually captured with a capillary pipette, and cell identity was further verified by PCR amplification of cell type-specific genes Wahlin et al, 2007).…”

“…Single rods, green cones, or red cones, identified by morphological criteria corroborated by ICC, were individually captured with a capillary pipette, and cell identity was further verified by PCR amplification of cell type-specific genes Wahlin et al, 2007). Aliquots of the dissociated cell suspensions were attached to poly-Lornithine-coated Superfrost (ϩ) slides and processed for immunofluorescence as described below.…”

Effects of acoustic trauma on the auditory system of the rat: The role of microglia

“…The SV2 monoclonal antibody was raised against unique components of cholinergic synaptic vesicles from the electric organ of Discopyge ommata and recognizes a band of 75–110 kDa in immunoblots of brain synaptosomes (Wahlin et al, 2008). The antibody has been used as a marker of presynaptic terminals (D’Sa et al, 2007).…”