Distribution of the glycine receptor β‐subunit in the mouse CNS as revealed by a novel monoclonal antibody

Abstract

Inhibitory glycine receptors (GlyRs) are composed of homologous α- (α1-4) and β-subunits. The β-subunits (GlyRβ) 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 GlyRβ transcripts throughout the mammalian central nervous system (CNS), whereas GlyRα mRNAs and proteins display more restricted expression patterns. Here we report the generation of a monoclonal antibody that specifically recognizes rodent GlyRβ (mAb-GlyRβ) and does not exhibit crossreactivity with any of the GlyRα1-4 subunits. Immunostaining with this antibody revealed high densities of punctate GlyRβ 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 GlyRβ transcripts in all major regions of the rodent brain and suggests that GlyRβ protein levels are regulated posttranscriptionally. When mAb-GlyRβ was used in double-labeling experiments with GlyRα1-, α2-, α3-, or α4-specific antibodies to examine the colocalization of GlyRβ with these GlyR subunits in the mouse retina, >90% of the GlyRα1-3 clusters detected were found to be GlyRβ-immunoreactive. A subset (about 50%) of the GlyRα4 puncta in the inner plexiform layer, however, was found to lack GlyRβ and gephyrin immunostaining. These GlyRα4-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.

Glycine receptor α3 and α2 subunits mediate tonic and exogenous agonist-induced currents in forebrain

“…In the present study, we first confirmed the presence of specific GlyR-mediated currents in the PFC, striatum, hippocampus, amygdala, and BNST of adolescent mice. Our results are consistent with earlier electrophysiological reports of glycine-activated currents in brain (22,(24)(25)(26)(27)44), as well as studies of GlyR mRNA and protein expression, which demonstrate that four (α1, α2, α3, and β) of the five known GlyR subunits are expressed in the mammalian brain (30,31,36,37,45,46).…”

“…Recent evidence suggests that coassembly of the β subunit, which is required for GlyR synaptic localization, with α subunits occurs to a lesser extent in the forebrain than in spinal cord and hindbrain (31); thus, homomeric GlyRs may be preferentially formed in this brain region. Our demonstration of picrotoxin-sensitive tonic GlyR currents in the NAc is consistent with this view and provides evidence for the existence of functional GlyR α homomers in this region of the forebrain.…”

“…GlyR α subunits exist in many higher brain regions (30) and may include populations of homopentameric GlyRs expressed in the absence of β subunits (31,32).…”

Electrophysiological Signature of Homomeric and Heteromeric Glycine Receptor Channels

“…5A). The finding that mRNA coding for GlyR ␤ was also detected actually also supports recent evidence for the lack of GlyR ␤ protein expression in hippocampal neurons (36). Furthermore, these results indicate that the identified voltage-dependent rectification of homomeric GlyR ␣2/3 can be used as physio-molecular signature to identify these channels at the single cell level.…”

“…It was recently shown that the GlyR ␤ subunit is expressed as mRNA, not as protein, in mouse hippocampal neurons (36). We performed patch clamp experiments on rat primary hippocampal neurons.…”