Input-specific plasticity of N-methyl-D-aspartate receptor-mediated synaptic responses in neonatal rat motoneurons

Abstract

Lumbar motoneurons can be activated monosynaptically by two glutamatergic synaptic inputs: the segmental dorsal root (DR) and the descending ventrolateral funiculus (VLF). To determine whether their N-methyl-d-aspartate (NMDA) receptors are independent, we used (5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine-hydrogen-maleate (MK-801), known to induce a use-dependent irreversible block of NMDA receptors (NMDARs). In the presence of MK-801 (in bath) and non-NMDA antagonists (in bath, to isolate NMDARs pharmacologically), we first stimulated the DR. After MK-801 blockade of DR synaptic input, the VLF was stimulated. Its response was found to be not significantly different from its control value, suggesting that the DR stimulus activated very few, if any, receptors also activated by VLF stimulation. Similar findings were obtained if the stimulation order was reversed. Both inputs also elicited a polysynaptic NMDAR-mediated response. Evoking the DR polysynaptic response in the presence of MK-801 eliminated the corresponding VLF response; the reverse did not occur. Surprisingly, when MK-801 was washed from the bath, both the DR and the VLF responses could recover, although the recovery of the DR monosynaptic and polysynaptic responses was reliably greater than those associated with the VLF. Recovery was prevented if extrasynaptic receptors were activated by bath-applied NMDA in the presence of MK-801, consistent with the possibility that recovery was due to movement of extrasynaptic receptors into parts of the membrane accessible to transmitter released by DR and VLF stimulation. These novel findings suggest that segmental glutamatergic inputs to motoneurons are more susceptible to plastic changes than those from central nervous system white matter inputs at this developmental stage.

Figure 1

Several superimposed responses to DR stimulation in a motoneuron. Note that the early monosynaptic responses are superimposed while the later polysynaptic responses are highly variable although they have a similar latency. Stimulation rate was 1/40s.

Figure 2

Illustration of the procedures carried out on an individual motoneuron and their inputs. Spinal cord obtained from a P2 rat. Data from a single motoneuron obtained over a 4 hour and 30 min recording period. V_m was about −75 mV throughout. Stimulation rate 1/40s. Control: Blockade of all non NMDA receptors with CNQX, bicuculline, strychnine and CGP 35348 (CNQX Cocktail) administered to the bath. Ten consecutive responses to DR and then to VLF are displayed. Note the reliable responses to DR and the irregular responses to VLF. “Insets” placed at top are average monosynaptic responses at higher gain and faster sweep speed. MK-801. The NMDA receptor- mediated response 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 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. Insets display the initial response (black) and the last response (red) in MK-801. MK-801 was then washed from the bath with ACSF for 45 min. in the absence of stimulation while continuing to apply the non NMDA antagonists. Wash. Superimposed responses (n=10 for both DR and VLF) after MK-801 washout are displayed along with the initial response at high gain (insets). Note the recovery of the DR NMDA response followed by further blockade when repetitively stimulated despite absence of MK-801 in the bath. Note also the lesser recovery of VLF NMDA response- the late response never reappeared in contrast to DR. The bottom graphs are average AMPA/kainate responses before CNQX cocktail (black) and at the very end of the experiment after the CXQX cocktail was washed out for about 45 min. (green). Note the similarity in the responses before and after (except for the briefer time course of the DR response which elicited spikes after the wash phase).

Figure 3

Organization and protocol similar to figure 2 except for the order of stimulation. P4 rat. Control: NMDA receptor-mediated response (10 consecutive stimuli for DR and VLF); MK-801: response in MK-801 (VLF: 80 stimuli; DR: 60 stimuli); Wash: response after 45 min. washout of MK-801 in the absence of stimulation (20 stimuli for both VLF and DR). Insets in the bottom row display the last monosynaptic response in MK-801 (black) and first response after washout of MK-801 (red). Note recovery of DR response and lack of recovery of VLF response.

Figure 4

Bar graphs displaying the mean amplitude of the NMDA receptor- mediated monosynaptic EPSPs elicited in the same motoneuron by DR and VLF stimulation. The left group of bars were experiments (n= 20) where DR (blue) was stimulated before VLF (green) during MK-801. The first blue bar is the mean NMDA control response averaged over all cells. The second blue bar (with horizontal lines) is the mean response at the onset of MK-801 and the third blue bar (with crossed diagonals) is mean final response in MK-801. DR and VLF stimuli in control conditions were interdigitated, but in MK-801 the DR was stimulated until steady state was reached after which VLF was stimulated. Note that the initial VLF response in MK-801 was similar to the control response, i.e., stimulating DR in MK-801 did not affect the response to VLF. The same was observed when the experimental protocol was reversed (n=6), i.e., stimulating VLF first in MK-801 did not affect the response to DR. Note also the depression of all response after stimulation in MK-801. Further details in the text.

Figure 5

Percent recovery of DR and VLF from MK-801 block when DR was stimulated before VLF. See text for definition of % values. Line connects data from same motoneurons. Note the negative slope for 9 of 10 cells indicating that recovery of response to DR was greater than recovery of response to VLF. Further detail in text.

Figure 6

Effects of MK-801 in the absence of any stimulation. Control: NMDA receptor mediated EPSPs recorded in CNQX – cocktail in response to 10 consecutive DR stimuli with the initial 5 in red and the final 5 in black. Inset displays average response to the initial 4 VLF stimuli (red) and the last 4 VLF stimuli (black). Stimulation rate 1/40s. Note the uniform amplitude of the monosynaptic component. MK-801 wash: similar records after exposure to MK-801 for 45 min and washout for 45 min., both in the absence of any stimulation throughout this 90 minute period. Red traces are progressively decreasing responses to 1^st 10 stimuli; black traces are the last 10 responses (of 50). Note that the monosynaptic EPSP declined progressively in amplitude as if MK-801 was present at the synapse. Inset displays the response to VLF as in the Control records above. Note that unlike controls, the VLF EPSPs exhibited a decline in amplitude with repetitive stimulation.

Figure 7

Effects of NMDA drop on recovery of synaptic response after MK-801. P2 rat. DR stimulation. A. NMDA receptor- mediated response to DR stimulation. Note the monosynaptic response followed by the late polysynaptic response. B. NMDA response after DR stimulation for 1 h with MK-801 in the bath illustrating the blockade of the synaptic response. C. Response to bath applied 2 NMDA drops in the presence of MK-801. Note the initial depolarizing response (black) despite the presence of MK-801 beginning about 1 h previously. The second drop elicited no response (red). D. Response to DR after immediately after NMDA administration. E. Response to DR after 1 hour MK-801 washout without any stimulation. Note the lack of the usual recovery of the early or late DR- mediated NMDA receptor- mediated response after MK-801 washout when NMDA drop is delivered (compare to recovery in Figs 2 and 3). F. Despite lack of recovery of NMDA response, AMPA response recovered after washout of all non NMDA antagonists. Thus the failure for NMDA recovery was not due to loss of synaptic input.