Abstracts

Rationale: Genetic variation in the GRIN gene encoding NMDA receptor subunits has emerged as a significant underlying cause of multiple neurological disorders ranging from intractable epilepsy to intellectual disability. We developed a genetic knock-in mouse expressing the GRIN2B p.S810R variant (associated with seizures and early-onset epileptic encephalopathy), which enhances agonist potency, prolongs the deactivation rate following glutamate removal, and increases channel open probability. We have evaluated the behavioral features of the mouse harboring this variant, assessed the effects on circuit function, and explored potential rescue pharmacology.

Methods: The Grin2b-S810R knockin mouse line was generated by the Emory University Mouse Transgenic and Gene Targeting Core Facility (RRID:SCR_023535) using CRISPR/Cas9. Behavioral tests (rotarod, locomotor, and elevated zero maze) were performed on the heterozygous S810R mice and their WT littermates. Golgi staining was performed and the images of hippocampal CA1 pyramidal cells were analyzed using Neurolocida360 to assess the numbers and morphology of the dendrite spine. Schaffer Collateral was stimulated and whole-cell voltage clamp recordings were performed on CA1 pyramidal cells in brain slices to record evoked EPSCs. Seizure susceptibility was evaluated by intraperitoneal (i.p.) injection of pentylenetetrazol (PTZ).


Results: Behavioral tests showed that the S810R variant has no major effect on locomotor activities, movement coordination, and anxiety level. Golgi staining indicates that the S810R variant reduces dendritic spine density (0.54 vs 0.99 per um of WT; p< 0.05, unpaired t-test). CA1 pyramidal cells from the S810R mouse had an enhanced NMDAR EPSC amplitude (121 pA vs 55 pA of WT; p< 0.05), indicating less stimulation is required for S810R to evoke a comparable current response. 3 mM radiprodil (a selective GluN2B inhibitor) can significantly inhibit both S810R and WT NMDAR EPSCs. The adult heterozygous mice showed generalized seizures when the cage lid was lifted. After i.p. injection of PTZ, the S810R mice showed a shorter latency for the first myoclonic jerk (58 sec for S810R vs 115 sec for WT, p=0.0011). All the S810R mice (10/10) showed generalized tonic-clonic seizures (GTCS) with latency of 183 sec, whereas only half of the WT (5/10; no GTCS was observed in the other 5 WT) progressed to GTCS with latency of 309 sec (p=0.011). These data suggest the S810R mice have a higher seizure susceptibility (i.e. a reduced seizure threshold). All 5 S810R mice with vehicle treatment showed GTCS with a latency of 138 sec, while only 6 out of 10 S810R mice with radiprodil treatment showed GTCS with a latency of 242 sec.