Visual experience dependent control of NMDAR subunit composition and neuronal gene expression: a critical role for the GluN2A C-terminal domain

Abstract

The N-methyl-D-aspartate (NMDAR) NMDA receptor is a Ca²⁺ -permeant glutamate receptor which plays key roles in health and disease. Canonical NMDARs are heterotetramers that contain two GluN2 subunits, of which GluN2A and GluN2B are predominant in the forebrain. Moreover, the relative contribution of GluN2A vs. GluN2B is controlled both developmentally and in an activity-dependent manner. The GluN2 subtype influences the biophysical properties of the receptor through differences in their N-terminal extracellular domain and transmembrane regions, but they also have large cytoplasmic Carboxyl (C)- terminal domains (CTDs) which have diverged substantially during evolution. While the CTD identity does not influence NMDAR subunit specific channel properties, it determines the nature of CTD-associated signalling molecules and has been implicated in mediating the control of subunit composition (2A vs. 2B) at the synapse. However, the role of CTD identity in mediating activity-dependent changes in NMDAR subunit composition remains unclear. First, I investigate the role of sensory experience to changes in the NMDAR GluN2A:GluN2B composition by using two different dark rearing protocols: 1) Mice were dark reared from birth to assess the contribution of sensory experience to the developmental increase in the ratio of 2A to 2B; 2) Mice raised in standard light conditions were taken during the third postnatal week and placed in the dark for 7-days with or without subsequent re-exposure to light to interrogate subunit specific regulation in response to changes in the level of synaptic activity. Here I show that, while dark rearing from birth has little effect on NMDAR subunit composition, 7 days of dark rearing produces a marked decrease in the ratio of 2A to 2B, which is reversed upon re-exposure to light. Crucially, I demonstrate that changes in the ratio are driven by changes in the level of synaptic GluN2A, and not GluN2B. This suggests that GluN2A is dynamically regulated by activity, and points to a GluN2A dependent mechanism of insertion/removal from the synapse.

Historically, much of the research into the differential function of GluN2 CTDs has been conducted in vitro by over-expressing mutant subunits, but more recently, the generation of knock-in (KI) mouse models have allowed CTD function to be probed in vivo and in ex vivo systems without heterologous expression of GluN2 mutants. Taking advantage of a KI mouse model with the GluN2A CTD (CTD²ᴬ) swapped for that of GluN2B (CTD²ᴮ) (GluN2A²ᴮ⁽ᶜᵀᴿ⁾/²ᴮ⁽ᶜᵀᴿ⁾), I next investigate the role of the CTD²ᴬ in experience-dependent changes in the level of synaptic GluN2A. I demonstrate that the CTD²ᴬ is required for activitydependent synaptic expression of GluN2A. Furthermore, I find that the transcriptomic response to changes in sensory experience is blunted in the absence of the CTD²ᴬ.

Collectively, this work establishes an important role for CTD²ᴬ in driving activity-dependent changes to both NMDAR subunit composition and gene transcription.