Distribution of the Glycine Receptor β-Subunit in the Mouse CNS as Revealed by a Novel Monoclonal Antibody

Inhibitory glycine receptors (GlyRs) are composed of homologous alpha- (alpha 1-4) and beta-subunits. The beta-subunits (GlyR beta) interact via their large cytosolic loops with the postsynaptic scaffolding protein gephyrin and are therefore considered essential for synaptic localization. In situ hybridization studies indicate a widespread distribution of GlyRb transcripts throughout the mammalian central nervous system (CNS), whereas GlyR alpha mRNAs and proteins display more restricted expression patterns. Here we report the generation of a monoclonal antibody that specifically recognizes rodent GlyR beta (mAb-GlyR beta) and does not exhibit crossreactivity with any of the GlyR alpha 1-4 subunits. Immunostaining with this antibody revealed high densities of punctate GlyR beta immunoreactivity at inhibitory synapses in mouse spinal cord, brainstem, midbrain, and olfactory bulb but not in the neocortex, cerebellum, or hippocampus. This contrasts the abundance of GlyR beta transcripts in all major regions of the rodent brain and suggests that GlyR beta protein levels are regulated posttranscriptionally. When mAb-GlyR beta was used in double-labeling experiments with GlyR alpha 1-, alpha 2-, alpha 3-, or alpha 4-specific antibodies to examine the colocalization of GlyR beta with these GlyR subunits in the mouse retina, >90% of the GlyR alpha 1-3 clusters detected were found to be GlyR beta-immunoreactive. A subset (about 50%) of the GlyR alpha 4 puncta in the inner plexiform layer, however, was found to lack GlyR beta and gephyrin immunostaining. These GlyR alpha 4-only clusters were apposed to bassoon immunoreactivity and hence synaptically localized. Their existence points to a gephyrin-independent synaptic localization mechanism for a minor subset of GlyRs. J. Comp. Neurol. 520: 3962-3981, 2012. (C) 2012 Wiley Periodicals, Inc.