Differential organization of gamma-aminobutyric acid type A and glycine receptors in the somatic and dendritic compartments of rat abducens motoneurons.

Premotor inhibitory neurons responsible for the decrease in the firing discharge during fast or slow eye movements selectively target the cell bodies and the dendrites of abducens motoneurons. Gamma-aminobutyric acid (GABA) and glycine, the main inhibitory synaptic neurotransmitters in the central nervous system, act via glycine and GABAA receptors, assembled from various types of subunits, which determine the kinetics of the currents mediated. Therefore, our hypothesis was that the expression of the inhibitory receptors on the somatic and the dendritic compartments, involved in different functions, may differ. In this study, we compared the subcellular patterns of expression of the main GABAA receptor subunits (GABAARalpha1, alpha2, alpha3, alpha5), glycine receptors (GlyRalpha1), and gephyrin in the somatic and dendritic compartments of rat abducens motoneurons, using double or triple immunocytochemical experiments with confocal microscopy. Significant differences exist in the patterns of organization and the synaptic expression of the GlyR and GABAAR subunits in the cell bodies and dendrites of abducens motoneurons. In the somata, only the GABAARalpha1 subunit was expressed, whereas both GABAARalpha1 and GABAARalpha3 were present in the dendrites. The GlyRalpha1 to GABAARalpha1 density ratio was reversed in the somatic and dendritic compartments (0.9 vs. 2.3). A quantitative electron microscopy study showed that the modes whereby gephyrin reaches its postsynaptic inhibitory synaptic target differ between the somata and the dendrites. Therefore, our results support the idea that a structure-function adaptation occurs at the single-neuron level.

Differential expression of GABAA and glycine receptors in ALS-resistant vs. ALS-vulnerable motoneurons: possible implications for selective vulnerability of motoneurons.

Summary Amyotrophic lateral sclerosis (ALS) is a devastating motoneuronal degenerative disease, which is inevitably fatal in adults. ALS is characterized by an extensive loss of motoneurons in the cerebrospinal axis, except for those motoneurons that control eye movements and bladder contraction. The reason for this selectivity is not known. Systematic differences have been found in the organization of excitatory synaptic transmission in ALS-resistant vs. ALS-susceptible motor nuclei. However, although motoneurons express high levels of glycine receptors (GlyR) and GABA(A) receptors (GABA(A)R), no such studies have been carried out yet for inhibitory synaptic transmission. In this study, we compared the subunit composition, patterns of expression, density and synaptic localization of inhibitory synaptic receptors in ALS-resistant (oculomotor, trochlear and abducens) and ALS-vulnerable motoneurons (trigeminal, facial and hypoglossi). Triple immunofluorescent stainings of the major GABA(A)R subunits (alpha1, alpha2, alpha3, and alpha5), the GlyR alpha1 subunit and gephyrin, were visualized by confocal microscopy and analysed quantitatively. A strong correlation was observed between the vulnerability of motoneurons and the subunit composition of GABA(A)R, the GlyR/GABA(A)R density ratios and the incidence of synaptic vs. extrasynaptic GABA(A)R. These differences contrast strikingly with the uniform gephyrin cluster density and synaptic GlyR levels recorded in all motor nuclei examined. These results suggest that the specific patterns of inhibitory receptor organization observed might reflect functional differences that are relevant to the physiopathology of ALS.

Mapping and quantitative analysis of gephyrin cytoplasmic trafficking pathways in motoneurons, using an optimized Transmission Electron Microscopy Color Imaging (TEMCI) procedure.

In the present study, an optimized Transmission Electron Microscopy Color Imaging (TEMCI) procedure was used to map and quantify the pathways involved in the trafficking and subcellular targeting of gephyrin in identified abducens motoneurons. Gephyrin is a scaffolding protein, which plays a crucial role in the clustering of the GABA(A) and glycine receptors to the cytoskeleton. TEMCI associated several accurate tools: (i) nanogold immunodetection of gephyrin in motoneurons identified on the basis of their immunoreactivity to Choline Acetyl Transferase, (ii) low magnification color scale coding of gephyrin densities on series of ultrathin sections of motoneurons, which gave a map of the cytoplasmic distribution of the protein, (iii) statistical analysis of the subcellular distribution of the immunolabeling. The color map of gephyrin densities in the cell bodies reflected the distribution of inhibitory synapses over the membrane. The TEMCI analysis of motoneurons with various patterns of synaptic covering made it possible to visualize for the first time the cytoplasmic transport pathway of gephyrin towards its target at synaptic contact. A high magnification quantitative analysis, including the study of 109 inhibitory synapses, showed that most gephyrin-associated immunogold particles (67%) were located in the subsynaptic regions facing the active zones, and the second most densely occupied regions were the perisynaptic regions (19.5% of immunogold particles). A consistent proportion of the gephyrin (11.5%), significantly higher than densities present in the rest of the cytoplasm (2%), was detected in the extrasynaptic submembrane region.

Heterogeneous synaptic covering and differential charge transfer sensitivity among the dendrites of a reconstructed abducens motor neurone: correlations between electron microscopic and computer simulation data.

Ultrastructural studies on the synaptology of dendritic arborizations of motoneurones have been problematic because dendrites are very thin in relation to their great length, and most of the studies on this topic have therefore dealt with only small parts of the dendritic tree. Here we compared the ultrastructural characteristics of the axon terminals distributed along the various dendrites of a single motoneurone. For this purpose, the light microscopic 3D reconstruction of the dendritic arborization of an intracellularly labelled abducens motoneurone was combined with an electron microscopic analysis of its synaptic contacts. Dendritic profiles were randomly sampled along the various dendrites and the axon terminals they received were classified on the basis of their ultrastructural features and their GABA-immunoreactivity. It emerged that the various dendrites differed according to the type and local arrangement of their synaptic inputs. Our second aim was to incorporate the morphological data obtained into a model giving the charge transfer effectiveness T(x) of the dendritic sites. The sensitivity S(x) of T(x) to changes in the membrane resistivity (Rm) simulating various levels of tonic synaptic activity was calculated. It turned out that both the proximal and distal regions of the dendritic arborization have a dense synaptic covering and a weak sensitivity to changes in the Rm, whereas the intermediate dendrites have a sparse synaptic covering and a high sensitivity to changes in tonic synaptic activity. This pattern of organisation might mediate the "gating" of a population of synapses covering some dendritic regions in a state-dependent fashion.