21.1 ADULT RESCUE OF NMDA RECEPTORS IMPROVES COGNITION AND ASTROCYTIC GENE EXPRESSION PATTERNS

NMDA receptors are well-known for their role in synaptic plasticity and are implicated in several brain disorders including autism and schizophrenia. We asked whether the developmental consequences of NMDAR hypofunction could be reversed by genetic interventions in adulthood using Cre/lox methodology to reverse a loss-of-function mutation in Grin1. We observed that astrocytic genes showed the greatest fold-change in NMDAR knockdown mice, and these transcripts returned to wildtype levels when NMDAR levels were rescued in adulthood. This observation implicates astrocytes in the pathology caused by NMDAR hypofunction and in the recovery process. A new mouse line was generated to have a hypomorphic insertional mutation in Grin1, creating a knockdown that is reversed by the activation of Cre recombinase. The mutation was reversed with a tamoxifen-inducible Cre (Rosa26-CreERT2) in mice at 10 weeks of age, and mice were studied at 14 weeks of age. Grin1 knockdown and recovery was measured by RNAscope fluorescent in situ hybridization (n=3), western blot (n=6), and immunohistochemistry (n=4). NMDAR levels and function were measured by radioligand binding (n=6) and slice electrophysiology (n=21–24 cells). Changes in gene expression were measured by RNAseq of total RNA from male cortex (n=4) that was analyzed with tuxedo protocol on the Galaxy.org online platform. Behaviours were assessed with digital activity monitors (n=40), sociability test (n=20), puzzle box test (n=20), and pre-pulse inhibition of acoustic startle test (n=20). Insertional mutation of Grin1 caused a knockdown of Grin1 mRNA and GluN1 protein that was 10% of wildtype levels, and Cre-mediated reversal of the mutation led to a substantial increase in Grin1 mRNA and GluN1 protein that was 50% of wildtype levels. Despite the incomplete recovery of protein, the levels of functional NMDAR at the synapse returned to 100% of wildtype levels in the cortex. Cortical behaviours were either substantially or completely normalized in mice with an adult rescue of NMDARs. RNAseq of the cortex indicated that astrocytic transcripts including Gfap, Acan, and Aqp1 were among those with the largest fold-change in both knockdown and rescue animals. While NMDARs play an important role in the developing nervous system, many of the cognitive impairments caused by NMDAR hypofunction can be reversed by interventions that occur in adulthood. Our study of the molecular changes that occur in the process of recovery highlight the role that astrocytes play.

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