NMDA receptors are heteromeric complexes that contribute to excitatory synaptic transmission and plasticity. The presence of specific variants of GluN2 subunits in these complexes enables diversity in NMDA receptor function and regulation. At brain synapses, there is a switch from slow GluN2B-mediated NMDA receptors to faster GluN2Adominated NMDA receptors as well as an increase in the ratio of AMPA to NMDA receptors during early postnatal development. This glutamate receptor switch is observed across brain regions and is critical for synaptic maturation, circuit development, and associative learning. However, whether a similar receptor subunit switch occurs within pain processing neurons in the developing spinal cord remains untested. To investigate this, we performed wholecell patch clamp recordings of excitatory synaptic responses from lamina II dorsal horn neurons of one to three week-old rats. We found that GluN2B and GluN2A both prominently contribute to NMDA receptor responses at neonatal lamina II synapses, with a small contribution from GluN2D as well. Surprisingly, we found that this molecular identity of NMDA receptor responses as well as the relative contribution of AMPA receptors versus NMDA receptors did not change at lamina II synapses across early postnatal development (P7 to P21). The lack of a developmental switch and persistence of slow-decaying GluN2B-and GluN2D-mediated synaptic responses throughout neuronal maturation in the dorsal horn has implications for understanding both the regulation of synaptic glutamatergic receptors as well as spinal mechanisms of pain processing.

NMDA receptors are heteromeric complexes crucial to the regulation of excitatory synaptic transmission, including in the spinal cord. The presence of specific subtypes of GluN2 subunits determines the kinetic properties of receptor activity. The Hildebrand lab has demonstrated that slow-decaying GluN2B and GluN2D dominate NMDAR responses at lamina I adult spinal synapses, which is unlike the fast GluN2A-dominated synapses found throughout most of the mature CNS. The functional contribution of specific GluN2 subunits is less characterized for synaptic NMDAR responses in lamina II neurons. We performed whole-cell patch clamp recordings of mEPSCs in the presence and absence of subtype-specific NMDAR pharmacological blockers. We observed a relatively equal and stable contribution of GluN2A and GluN2B throughout lamina II development, contrasting the shift in contribution from GluN2B to GluN2A commonly observed during postnatal development in the brain. We also identified a slower synaptic NMDAR component that is blocked by a GluN2D antagonist. These findings provide key insights into the differential roles of specific NMDAR subtypes in nociceptive signaling at juvenile spinal synapses.Keywords: NMDA receptor, GluN2A, GluN2B, GluN2D, spinal cord development, lamina II, dorsal horn hyperexcitablity, mEPSCsiii Last but not least, I am grateful for the support of my family and friends during this trying time. This publication is as much yours as it is mine.iv