Differential NR2B subunit expression at dorsal root and ventrolateral funiculus synapses on lumbar motoneurons of neonatal rat

Abstract

Synapse specific differences in NR2 subunit expression exist in several systems within the mammalian CNS. Here we have studied such differences on motoneurons in the neonatal rat cord using ifenprodil known to inhibit voltage-, use- and glycine-independent responses mediated by NR2B-containing N-methyl-d-aspartate receptors (NMDARs) with high specificity. In neonatal rats (P1-P9), the synapses made by the dorsal root (DR) fibres were more sensitive to ifenprodil than ventrolateral funiculus (VLF) connections on the same motoneuron. DR connections exhibited very little additional blockade to bath-applied MK-801 whereas VLF connections displayed a further decrease in amplitude. This suggests that at this immediate postnatal age, DR synapses on motoneurons contain a higher proportion of ifenprodil-sensitive diheteromeric NR1/NR2B receptors than VLF synapses. Since DR synapses have been shown in other studies to be less mature than VLF synapses on the same motoneuron at this developmental stage, these data are interpreted as indicating that less mature NMDA receptors feature a higher proportion of NR2B subunits which declines as the synapse matures. This novel finding of staggered development of NMDA receptors from different synaptic inputs on the same motoneuron is discussed in the context of its developmental and functional implications.

Figure 1. Action of Ifenprodil on DR and VLF synaptic EPSP

Illustration of the procedures carried out on an individual motoneuron from a P2 rat. Data from a single motoneuron obtained over a 3 hour and 30 min recording period. V_m was about −70 mV throughout. Stimulation rate 1/40s. Top Row: Ten consecutive NMDA-mediated responses to DR and to VLF obtained after blockade of all non NMDA receptors with CNQX, bicuculline, strychnine and CGP 35348 (CNQX Cocktail) administered to the bath (not shown). Note the reliable responses to DR and the irregular responses to VLF. Insets are averaged monosynaptic responses at higher gain and faster sweep speed. Bottom row: The NMDAR- mediated response was then blocked with ifenprodil (3μM). Alternate stimulation of DR and VLF (a total of 40 stimuli to each input at 0.025 Hz). Trial 1 was obtained immediately after introducing ifenprodil into the bath and the decline of monosynaptic response occurred progressively. The stimulation was stopped when steady state was reached. Single traces (inset) display the initial response (black) and the last response (red) in ifenprodil.

Figure 2. EPSPs produced by DR stimulation are more sensitive to ifenprodil inhibition than those produced by VLF stimulation

Percent blockade of DR and VLF with ifenprodil. See text for definition of % values. Lines connect data from same motoneuron. Note the negative slope for 13 of 14 cells indicating that the blockade of DR was greater than blockade of response to VLF. Further details in text.

Figure 3

Decline of NMDA-mediated responses from DR and VLF in the same motoneuron during 45 minute application of ifenprodil (3 μM). The lines represent the averaged data normalized to 100% at the beginning of the ifenprodil application and smoothed by a process of running averages (Sigmaplot 11; 2-D smoothing; running average; sampling proportion =0.3). Individual DR and VLF EPSPs displayed on the same scale. Note that the reduction in the responses to both inputs began at about the same time, that the decline for both inputs occurred in parallel both reaching a steady state, and that the DR response declined more than the VLF response.

Figure 4. Action of MK-801 on the ifenprodil-insensitive component of DR and VLF EPSP

Illustration of the procedures carried out on an individual motoneuron from a P2 rat. Data from a single motoneuron obtained over a 4 hour and 30 min recording period. V_m was about −70 mV throughout. Stimulation rate 1/40s. Top Row: After blockade of all non NMDA receptors with the CNQX cocktail administered to the bath (not shown), the NMDAR- mediated response was then blocked with ifenprodil. Bottom Row: After maximal blockade with ifenprodil was achieved, the ifenprodil-insensitive response that remained was then blocked with MK-801, stimulating only DR (60 trials at 0.025 Hz). Trial 1 was obtained immediately after introducing MK-801 (10 μM) into the bath (ifenprodil application was discontinued) and the decline of the late response took place progressively as stimulation was continued. Stimulation was stopped when steady state was reached. VLF was then stimulated (n=20) until its response declined to steady state. Single traces (inset) display the initial response (black) and the last response (red) in MK-801.

Figure 5. Comparison of decline of DR and VLF NMDA receptor- mediated EPSPs in ifenprodil and MK- 801

Percent residual (see text for definition) of DR (white) and VLF (grey) responses after maximal inhibition in ifenprodil (clear) and subsequent treatment with MK-801 (diagonals) in P1–P4 neonatal rats. Note that the DR monosynaptic response that remains after ifenprodil treatment does not change in MK-801 indicating a similar level of blockade. A greater percentage of the VLF response remains after ifenprodil treatment and is blocked to a greater extent by subsequent treatment with MK-801.