The biophysical properties of glutamatergic synaptic responses in lamina II neurons are conserved across early postnatal spinal cord development
To characterize postsynaptic glutamatergic responses at developing lamina II synapses, we performed whole-cell patch-clamp recordings on lamina II neurons from acute transverse spinal sections of postnatal day 7 (P7) to 21 (P21) male rats. We recorded miniature excitatory postsynaptic currents (mEPSCs) at − 60 mV and + 60 mV to study AMPAR- and NMDAR-mediated postsynaptic responses, respectively [7]. In lamina II neurons held at − 60 mV, we observed inward AMPAR-mediated mEPSCs with a peak amplitude of − 15.9 ± 0.9 pA and a decay constant (τdecay) of 11.1 ± 1.1 ms (n = 40 animals, 62 neurons, Fig. 1a). At a holding potential of + 60 mV, outward mEPSCs were much slower than mEPSCs at − 60 mV for the same neurons (Fig. 1a). To estimate the amplitude of the NMDAR component of mEPSCs at + 60 mV, we measured the mEPSC amplitude at 20 ms from onset, a timepoint of peak NMDAR response where the contribution from fast deactivating AMPARs is minimal [7]. The average amplitude of NMDAR mEPSCs at + 60 mV was 16.3 ± 0.7 pA, which was not significantly different from the absolute peak amplitude of mEPSCs at − 60 mV (n = 62 neurons, p = 0.68). The decay constant for NMDAR mEPSCs at + 60 mV (169 ± 7 ms) was significantly slower than that for AMPAR mEPSCs at − 60 mV (n = 62, p = 1.9e− 31, Fig. 1a). We next compared the biophysical properties of AMPAR and NMDAR mEPSCs at distinct time points in early postnatal development. Our developmental age bins of P7-P11, P12-P16, and P17-P21 correspond to postnatal periods that encompass the complete synaptic switch process across brain regions [11] and also correspond to periods of maximal change in primary afferent-induced postsynaptic responses and plasticity for developing dorsal horn neurons [13]. We found that the ratio of the peak amplitude of AMPAR mEPSCs at − 60 mV compared to the amplitude of NMDAR mEPSCs at + 60 mV (at 20 ms) remained unchanged (p = 0.67) near a value of 1 across the three postnatal time periods (P7-P11, 1.1 ± 0.1, n = 22 neurons; P12-P16, 1.0 ± 0.1, n = 19; P17-P21, 1.1 ± 0.1, n = 21; Fig. 1b). We therefore conclude that AMPARs and NMDARs contribute equally to excitatory synaptic responses in lamina II neurons across early postnatal rodent development.
To further investigate the NMDAR component of lamina II synaptic responses across early development (Fig. 1c), we calculated the charge transfer through mEPSCs at + 60 mV from 20 ms to 500 ms after event onset. Synaptic charge transfer during this time range is exclusively mediated by NMDAR and not AMPAR activity [7]. We found that there was no significant difference (p = 0.42) in the magnitude of NMDAR charge transfer between P7-P11 (n = 22), P12-P16 (n = 19), and P17-P21 (n = 21; Fig. 1d). Similarly, there was no significant change in decay constants for NMDAR mEPSCs at lamina II synapses across early postnatal development (Fig. 1c, e). Taken together, our results suggest that the relative contribution and biophysical properties of synaptic NMDARs do not change during the postnatal maturation of lamina II spinal cord neurons.
Both GluN2B- and GluN2A-containing NMDARs mediate lamina II synaptic responses across early postnatal development
The lack of change in charge transfer and decay constants of NMDAR mEPSCs in developing lamina II neurons suggests that there may not be an early postnatal switch from GluN2B-containing NMDARs to GluN2A-containing NMDARs at these specific central synapses. In fact, a stable decay constant between approximately 160 ms and 180 ms across lamina II development (Fig. 1e) suggests that both GluN2A- and GluN2B-containing NMDARs may mediate synaptic responses throughout the maturation process [4]. We therefore used GluN2 subunit-specific pharmacological antagonists to directly explore the relative contribution of individual GluN2 subtypes to NMDAR-mediated mEPSCs. The contribution of GluN2B was investigated using the GluN2B-specific antagonist, Ro25–6981, at a concentration (1 μM) that inhibits recombinant GluN2B- but not GluN2A-containing diheteromeric NMDARs [14] and that we have shown to selectively inhibit GluN2B-like NMDAR responses at lamina I synapses [7] (see Methods for further discussions on NMDAR antagonists). NMDAR charge transfer was calculated for a 10-min baseline control recording period and compared to a time period where inhibition reached a steady-state level, typically after 40 minutes of drug administration. We found that administration of 1 μM Ro25–6981 resulted in a significant block of NMDAR mEPSCs at P7-P21 lamina II synapses (56.9 ± 7.1%, n = 13, p = 0.00035, Fig. 1f). To test for the contribution of GluN2A to NMDAR-mediated mEPSCs, we utilized the GluN2A-specific antagonist, TCN-201, at a concentration (10 μM) that selectively blocks only GluN2A-containing recombinant NMDARs [15] and that inhibits fast-decaying GluN2A-like but not slower decaying GluN2B-like synaptic NMDAR responses at rat lamina I synapses [7]. In contrast to mature lamina I neurons [7], we found that administration of 10 μM TCN-201 also robustly reduced NMDAR-mediated charge transfer at early postnatal lamina II synapses (41.6 ± 7.2%, n = 14, p = 0.00064, Fig. 1g). There was no significant difference between the baseline NMDAR charge transfers for 1 μM Ro25–6981- and 10 μM TCN-201-treated neurons (p = 0.90), suggesting that NMDAR biophysical properties did not diverge between lamina II neurons from the two treatment groups (Supplementary Fig. 1). To assess the GluN2 subunit-selectivity of the specific pharmacological antagonists, the drug-sensitive mEPSC difference current was graphed and fitted with an exponential decay constant (Fig. 1f, g). The 1 μM Ro25–6981-sensitive difference current displayed a decay constant of 354 ms (Fig. 1f), which falls within the deactivation range for GluN2B-containing diheteromeric NMDARs [4, 7]. The 10 μM TCN-201-sensitive difference current had a decay constant of 158 ms (Fig. 1g), consistent with the faster deactivation rate observed for GluN2A-containing NMDARs [4, 7]. From these findings, we conclude that synaptic NMDAR responses are primarily mediated by a combination of GluN2A- and GluN2B-containing receptors in P7 to P21 lamina II neurons.
We next explored whether the functional contribution of GluN2A versus GluN2B-containing receptors to synaptic responses changes during the postnatal maturation of lamina II neurons. To test this, we analyzed the effects of Ro25–6981 and TCN-201 on NMDAR charge transfer across the three distinct postnatal development periods: P7-P11(nRo = 4, nTCN = 5), P12-P16 (nRo = 5, nTCN = 5), and P17-P21 (nRo = 4, nTCN = 4). Two-way ANOVA analysis revealed no significant difference in the magnitude of NMDAR block for either 1 μM Ro25–6891 (p = 0.24) or 10 μM TCN-201 (p = 0.72) between the three postnatal development periods (Fig. 1h). We therefore conclude that the relative contribution of GluN2A versus GluN2B to synaptic NMDAR responses in lamina II neurons does not change across early postnatal development.
GluN2D has a minor contribution to mEPSCs at early postnatal lamina II synapses
Given that GluN2D-containing receptors prominently contribute to NMDAR responses at lamina I synapses [7], we next explored whether GluN2D NMDARs mediate a component of NMDAR mEPSCs at developing lamina II synapses. We utilized the GluN2D-selective antagonist, DQP-1105, at a concentration (10 μM) that inhibited approximately 85% of GluN2D NMDARs with negligible effects on GluN2A or GluN2B NMDARs [16] and that also inhibited native GluN2D-like synaptic NMDAR responses in lamina I neurons [7]. Treatment with 10 μM DQP-1105 resulted in a 23.7 ± 8.6% reduction in NMDAR charge transfer at P7-P21 lamina II synapses (Control 2.78 ± 0.32 pC, DQP-1105 2.07 ± 0.40 pC, n = 14, p = 0.033, Fig. 1i). The 10 μM DQP-1105-sensitive mEPSC difference current exhibited a decay constant of 596 ms (Fig. 4A), which is consistent with slow deactivating GluN2D-containing NMDARs [4, 7]. We found that the NMDAR blockade by DQP-1105 at lamina II synapses was less than that produced by Ro25–6981 or TCN-201 (Fig. 1j).