Abstracts

Rationale: N-methyl-D-aspartate receptors (NMDARs) are a family of ionotropic ligand-gated glutamate receptors that mediate a slow, Ca²⁺-permeable component of fast excitatory neurotransmission. NMDAR activation is critical for proper central nervous system functioning, with mutations in GRIN genes displaying a range of neuropathological diseases. Among the four GluN2-subunits (GluN2A-D), variants within the GluN2A subunit are most commonly associated with epilepsy.  Particularly interesting are the GRIN2A loss-of-function (LOF) mutations, which make up 67% (14/21) of currently identified GRIN2A variants and present an epileptic phenotype.  This suggests that loss of activity from GluN2A-containing NMDARs results in compensatory mechanism(s) that promote hyperexcitability.  In this study, we used a Grin2a-knockout (KO) mouse model to begin to understand how these LOF mutations promote an epileptic phenotype.  Methods: Mouse hippocampal brain slices from either sex were studied using whole-cell patch clamp and extracellular field potential recordings to examine how the loss of GluN2A-containing NMDARs impacts synaptic neurotransmission.  Slices were exposed to elevated levels of extracellular potassium that can generate spontaneous epileptiform activity to examine changes in network excitability.  To determine if changes in inhibitory tone may have promoted this hyperexcitable phenotype, spontaneous inhibitory postsynaptic currents (sIPSCs) onto CA1 pyramidal cells were measured. Finally, to elucidate the effects of Grin2A LOF variants on epilepsy-associated behavior, mice were given systemic injections of lipopolysaccharide (LPS) and exposed to hyperthermia to provoke a generalized tonic-clonic (GTC) seizure. Results: All Grin2a-KO brain slices (N = 3 animals/5 slices) exposed to elevated levels of extracellular potassium (7.0 mM) showed spontaneous interictal field bursts originating in the CA3 region and propagating to the CA1 region of the hippocampus.  By contrast, no wild-type (WT) slices showed epileptiform activity under these same conditions (N = 3 animals/3 slices).  Additionally, Grin2A-KO mice displayed an increased inhibitory tone, measured by an increased frequency of spontaneous IPSCs and a large gabazine-sensitive current, onto CA1 pyramidal cells when compared to WT.  Consistent with our electrophysiological data, 100% (11/11) of Grin2a-KO mice exposed to LPS and hyperthermia exhibited GTC seizures, whereas only 42% (5/12) of WT mice exhibited seizures.  Conclusions: These data strongly indicate that mutations resulting in a loss or decrement of function of GluN2A-containing NMDARs is sufficient to drive a hyperexcitable state. Additionally, this data suggests that loss of GluN2A activity promotes changes in inhibitory tone, possibly as a compensatory mechanism. A better mechanistic understanding of how the loss of GluN2A activity impacts neuronal development and causes these compensatory changes may allow novel therapeutics to be identified that can be used to treat patients with LOF GRIN2A mutations. Funding: This work was supported by NS036654 and NS092989 (SFT), and an Emory University Synergy grant (STF and SK).