Gephyrin accumulates at specific plasmalemma loci during neuronal maturation in vitro

Abstract

The distribution of glycine receptor (GlyR)-associated gephyrin has been investigated in rat spinal cord neurons maintained in vitro by means of immunocytochemical techniques. Gephyrin, which is crucial for the stabilization of postsynaptic GlyR microdomains, is present in mature neurons at postsynaptic differentiations. With immunofluorescence, discontinuous patches of gephyrin were detected within the neuronal soma of spinal cord neurons on the 1st day after plating. Subsequently, gephyrin was present at membrane areas that correspond to points of contact between cells or with the culture dish. By the 5th day, gephrin was mostly associated with the MAP2-positive somatodendritic compartment. With immunoelectron microscopy, gephyrin blobs detected at the earliest stages (1-3 days after plating) were found within the cytoplasm or associated with the plasma membrane. Asymmetrically immunostained intercellular contacts were only detected after 5 days, and gephyrin was found in association with clearly differentiated postsynaptic membranes at 7 days. At later stages, we observed gephyrin immunoreactivity only at some synapses. Our results suggest that gephyrin accumulates initially at the locus of cell-to-cell contacts involved in adhesion processes. These localizations may define hot spots for later accumulation of the GlyR and possibly other receptors.

GSK3 and KIF5 regulate activity-dependent sorting of gephyrin between axons and dendrites

“…In cultured neurons, endogenous gephyrin is mostly associated with the MAP2-positive somato-dendritic compartment from day 5 in vitro (DIV5) onward (Colin et al, 1996) and is mainly localized at subsynaptic and perisynaptic regions (Lorenzo et al, 2004). To study a potential role of neuronal activity and Ca 2+ increase in the sorting of gephyrin between axons and dendrites, we aimed to use neuronal cultures at later stages.…”

“…In young cultured neurons, gephyrin has been detected in axons (Craig et al, 1996), however at later stages, it is mainly restricted to the somatodendritic compartment (Colin et al, 1996). Application of nocodazole or microinjection of KIF5-specific antibodies interfered with the post-Golgi delivery of gephyrin into dendrites of mature cultured hippocampal neurons (Maas et al, 2009), indicating that gephyrin uses microtubule-dependent kinesin transport to reach the cell periphery.…”

Activity-Dependent Movements of Postsynaptic Scaffolds at Inhibitory Synapses

“…This apparent contradiction resulted from the presence of an intracellular pool of gephyrin detected with the VeGe chimera, but which was not well detected with immunofluorescence in nontransfected cells with the mAb7a antibody used in these experiments (Dumoulin et al, 1999;Danglot et al, 2003;Hanus et al, 2004). However, this pool can be visualized unambiguously in nontransfected cells under the electron microscope (Seitanidou et al, 1992;Colin et al, 1996Colin et al, , 1998 or using another commercially available mAb (BD Biosciences, San Diego, CA) (data not shown).…”

Cellular transport and membrane dynamics of the glycine receptor

“…Originally, GlyRs (Bechade et al, 1996) and the GlyR-interacting protein gephyrin (Seitanidou et al, 1992; Colin et al, 1996) were identified at intracellular sites in neurons, and depolymerization of microtubules dispersed the subcellular accumulation of both proteins (Kirsch and Betz, 1995). A direct association of gephyrin with the light chains Dlc-1/Dlc-2, components of the microtubule-dependent dynein motor, suggested that microtubule transport might be involved in the subcellular localization of these factors (Fuhrmann et al, 2002) (Figure 1).…”