Altered GluN2A levels reduce the emergence of seizure-like events in a rat model of GRIN2A haploinsufficiency

Abstract GluN2A-containing NMDA receptors, encoded by the GRIN2A gene, are critical components of excitatory synaptic transmission and are essential for proper brain development and function. Dysregulation of NMDA receptor activity is strongly implicated in the pathophysiology of epilepsy. While excessive GluN2A-mediated signalling can lead to hyperexcitability and seizure generation, loss-of-function mutations in GRIN2A, which result in reduced GluN2A expression, have also been identified in patients with various forms of epilepsy, including focal epilepsies and epileptic encephalopathies. This paradox highlights the complexity of NMDA receptor contributions to network excitability and suggests that both gain- and loss-of-function mutations can be pathogenic. However, the mechanisms through which reduced GluN2A expression influences seizure susceptibility remains unclear. In this study, we examined the impact of reduced and absence of GluN2A on seizure-like activity using in vitro model system. Hippocampal slices were prepared from wild-type littermate controls (Grin2a+/+), heterozygous (Grin2a+/-) and homozygous (Grin2a-/-) transgenic rats and subjected to pro-convulsant conditions to elicit epileptiform-like events. Specifically, we employed three well established in vitro epilepsy models: (i) 4-aminopyridine (4-AP), (ii) zero-magnesium with elevated potassium (0Mg²⁺/5K⁺), and (iii) high-potassium (high-K+). Local field potentials were recorded from the CA1 pyramidal layer to quantify interictal and seizure-like activity, by measuring latency to onset, event frequency, and duration. Spectral analyses were also conducted to assess alterations in network dynamics. Our findings reveal that reduced GluN2A expression alters the susceptibility of hippocampal circuits to seizure-like activity in a model-dependent manner. In the 4-AP model, Grin2a-/- slices exhibited a significantly lower frequency of interictal events and a delayed onset of seizure-like activity relative to both wild-type and heterozygous slices. In the 0Mg²⁺/5K⁺ model, both Grin2a+/- and Grin2a-/- slices displayed reduced seizure susceptibility compared to wild-type. Finally, in the high-K⁺ model, Grin2a-/- slices showed a reduced incidence of seizure-like events compared to wild-type controls when extracellular potassium concentration was increased to 7.5 mM, although this effect was less apparent at higher concentrations (9.5 mM). These results suggest that partial or complete loss of GluN2A-containing NMDA receptors reduces the propensity of hippocampal networks to develop epileptiform activity, potentially by altering intrinsic or synaptic excitability. Our findings provide mechanistic insights into how GRIN2A loss-of-function variants may contribute to epilepsy and highlight the need to consider developmental and circuit-level compensations when interpreting the impact of GluN2A disruption. Competing Interest Statement The authors have declared no competing interest. Footnotes Abbreviations: N-methyl-D-aspartate, (NMDA); artificial cerebral spinal fluid, (ACSF); seizure-like events, (SLE); interictal events, (IIE); analysis of variance, (ANOVA); 4-Aminopyridine, (4-AP).