Differential regulation of NMDA receptors by d-serine and glycine in mammalian spinal locomotor networks

Abstract

Activation of N-methyl-d-aspartate receptors (NMDARs) requires the binding of a coagonist, either d-serine or glycine, in addition to glutamate. Changes in occupancy of the coagonist binding site are proposed to modulate neural networks including those controlling swimming in frog tadpoles. Here, we characterize regulation of the NMDAR coagonist binding site in mammalian spinal locomotor networks. Blockade of NMDARs by d(-)-2-amino-5-phosphonopentanoic acid (d-APV) or 5,7-dichlorokynurenic acid reduced the frequency and amplitude of pharmacologically induced locomotor-related activity recorded from the ventral roots of spinal-cord preparations from neonatal mice. Furthermore, d-APV abolished synchronous activity induced by blockade of inhibitory transmission. These results demonstrate an important role for NMDARs in murine locomotor networks. Bath-applied d-serine enhanced the frequency of locomotor-related but not disinhibited bursting, indicating that coagonist binding sites are saturated during the latter but not the former mode of activity. Depletion of endogenous d-serine by d-amino acid oxidase or the serine-racemase inhibitor erythro-β-hydroxy-l-aspartic acid (HOAsp) increased the frequency of locomotor-related activity, whereas application of l-serine to enhance endogenous d-serine synthesis reduced burst frequency, suggesting a requirement for d-serine at a subset of synapses onto inhibitory interneurons. Consistent with this, HOAsp was ineffective during disinhibited activity. Bath-applied glycine (1-100 µM) failed to alter locomotor-related activity, whereas ALX 5407, a selective inhibitor of glycine transporter-1 (GlyT1), enhanced burst frequency, supporting a role for GlyT1 in NMDAR regulation. Together these findings indicate activity-dependent and synapse-specific regulation of the coagonist binding site within spinal locomotor networks, illustrating the importance of NMDAR regulation in shaping motor output.NEW & NOTEWORTHY We provide evidence that NMDARs within murine spinal locomotor networks determine the frequency and amplitude of ongoing locomotor-related activity in vitro and that NMDARs are regulated by d-serine and glycine in a synapse-specific and activity-dependent manner. In addition, glycine transporter-1 is shown to be an important regulator of NMDARs during locomotor-related activity. These results show how excitatory transmission can be tuned to diversify the output repertoire of spinal locomotor networks in mammals.

Keywords: central pattern generator; motor control; neuromodulation; spinal cord.

Fig. 1.

NMDARs determine the speed and amplitude of locomotor-related activity in spinal cord preparations from postnatal mice. A: raw (top) and rectified/integrated (bottom) traces recorded from the left and right L2 ventral roots (L2 L; L2 R) and right L5 ventral root (L5 R), showing the effect of the competitive glutamate-binding site antagonist d-APV (50 µM) on locomotor-related activity induced by 5-HT (15 µM) and DA (50 µM). B: left-right phase relationship in control conditions (Bi) and during application of d-APV (Bii). Circular plots represent the onset of locomotor bursts recorded from L2 R ventral roots (gray dots) in relation to the onset of activity recorded from corresponding L2 L roots (assigned a value of 0) in the same cycle. Vector direction indicates mean phase, and vector length corresponds to clustering of data points around the mean. We analyzed >100 burst cycles from 4 preparations for each condition. Ci: locomotor-burst frequency over 5 min during a control period, during a 40-min application of d-APV, and during a 30-min washout (Wash). Individual data points are shown in gray, and means are represented by black lines; n = 6 preparations. Cii: time-course plot of normalized data aggregated into 1-min bins showing a reduction in burst frequency during d-APV application; n = 6. Di: locomotor-burst amplitude over 5 min during a control period, during a 40-min application of d-APV, and during a 30-min washout; n = 6. Dii: time-course plot of normalized data aggregated into 1-min bins showing a reduction in burst amplitude during d-APV application; n = 6. E: raw (top) and rectified/integrated (bottom) traces recorded from L2 L and L2 R showing the effect of the competitive coagonist binding site antagonist DCKA (5 µM) on locomotor-related activity. F: left-right phase relationship in control conditions (Fi) and during application of DCKA (Fii). Circular plots represent the onset of locomotor bursts recorded from L2 R ventral roots (gray dots) in relation to the onset of activity recorded from corresponding L2 L roots (assigned a value of 0) in the same cycle. Vector direction indicates mean phase, and vector length corresponds to clustering of data points around the mean. We analyzed >100 burst cycles from 4 preparations for each condition. Gi: locomotor-burst frequency over 5 min during a control period, during a 30-min application of DCKA, and during a 30-min washout; n = 6. Gii: time-course plot of normalized data aggregated into 1-min bins showing a reduction in burst frequency during DCKA application; n = 6. Hi: locomotor-burst amplitude over 5 min during a control period, during a 30-min application of DCKA, and during a 30-min washout; n = 6. Hii: time-course plot of normalized data aggregated into 1-min bins showing a reduction in burst amplitude during DCKA application; n = 6. Error bars: ± SE. Statistically significant difference from control: *P < 0.05, **P < 0.01. a.u., Arbitrary units.

Fig. 2.

Exogenous d-serine modulates locomotor-related activity. A and B: raw (top) and rectified/integrated (bottom) traces recorded from the left and right L2 ventral roots (L2 L; L2 R) and right L5 ventral root (L5 R) showing the effects of d-serine at 1 µM (A) and 3 µM (B) on locomotor-related activity induced by 5-HT (15 µM) and DA (50 µM). C: percentage change in frequency (Ci) and amplitude (Cii) in response to varying concentrations of d-serine, calculated by comparing a 5-min window during a control period with a 5-min window during a 15-min application of d-serine; n = 5–7 preparations. D: left-right phase relationship in control conditions (Di) and during application of 1 µM d-serine (Dii). Circular plots represent the onset of locomotor bursts recorded from L2 R ventral roots (gray dots) in relation to the onset of activity recorded from corresponding L2 L roots (assigned a value of 0) in the same cycle. Vector direction indicates mean phase, and vector length corresponds to clustering of data points around the mean. We analyzed >100 burst cycles from 6 preparations for each condition. Ei: locomotor-burst frequency over 5 min during a control period, during a 15-min application of d-serine (1 µM), and during a 20-min washout. Individual data points are shown in gray, and means are represented by black lines; n = 7. Eii: time-course plot of normalized data aggregated into 1-min bins showing an increase in burst frequency during d-serine (1 µM) application; n = 7. Fi: locomotor-burst amplitude over 5 min during a control period, during a 15-min application of d-serine (1 µM), and during a 20-min washout; n = 7. Fii: time-course plot of normalized data aggregated into 1-min bins showing a reduction in burst amplitude during d-serine (1 µM) application; n = 7. Error bars: ± SE. Statistically significant difference from control: *P < 0.05, **P < 0.01.

Fig. 3.

Bath-applied d-serine acts at NMDARs to modulate fictive locomotion. A: raw (top) and rectified/integrated (bottom) traces recorded from the left and right L2 ventral roots (L2 L; L2 R) showing the effect of d-serine (10 µM) on locomotor-related activity in the presence of the competitive glutamate-binding site antagonist d-APV (50 µM). Bi: locomotor-burst frequency over 5 min during a control period, during a 30-min application of d-serine (1–10 µM), and during a 20-min washout. d-APV was present throughout; n = 6. Bii: time-course plot of normalized data aggregated into 1-min bins showing no change in burst frequency when d-serine is applied in the presence of d-APV. Ci: locomotor-burst amplitude over 5 min during a control period, during a 30-min application of d-serine (1–10 µM), and during a 20-min washout. d-APV was present throughout. Cii: time-course plot of normalized data aggregated into 1-min bins showing no change in burst amplitude when d-serine is applied in the presence of d-APV; n = 6. Error bars: ± SE.

Fig. 4.

Endogenous d-serine acts via NMDARs to modulate the frequency but not the amplitude of locomotor-related activity. A: raw (top) and rectified/integrated (bottom) traces recorded from the left and right L2 ventral roots (L2 L; L2 R) showing the effect of the d-serine scavenger DAAO (0.29 U/ml) on locomotor-related activity induced by 5-HT (15 µM) and DA (50 µM). Bi: locomotor-burst frequency over 5 min during a control period, during a 40-min application of DAAO, and during a 40-min washout. Individual data points are shown in gray, and means are represented by black lines; n = 8. Bii: time-course plot of normalized data aggregated into 1-min bins showing an increase in burst frequency during DAAO application; n = 8. Ci: locomotor-burst amplitude over 5 min during a control period, during a 40-min application of DAAO, and during a 40-min washout; n = 8. Cii: time-course plot of normalized data aggregated into 1-min bins showing no change in burst amplitude during DAAO application; n = 8. D: raw (top) and rectified/integrated (bottom) traces recorded from L2 L and L2 R showing the effect of the serine-racemase inhibitor HOAsp (400 µM) on locomotor-related activity. Ei: locomotor-burst frequency over 5 min during a control period, during a 40-min application of HOAsp, and during a 40-min washout; n = 6. Eii: time-course plot of normalized data aggregated into 1-min bins showing an increase in burst frequency during HOAsp application; n = 6. Fi: locomotor-burst amplitude over 5 min during a control period, during a 40-min application of HOAsp, and during a 40-min washout; n = 6. Fii: time-course plot of normalized data aggregated into 1-min bins showing no change in burst amplitude during HOAsp application; n = 6. G: raw (top) and rectified/integrated (bottom) traces recorded from L2 L and L2 R showing the effect of HOAsp on locomotor-related activity in the presence of the competitive glutamate-binding site antagonist d-APV (50 µM). H: locomotor-burst frequency over 5 min during a control period, during a 30-min application of HOAsp, and during a 20-min washout. d-APV was present throughout; n = 10. Error bars: ± SE. Statistically significant difference from control: **P < 0.01.

Fig. 5.

Racemization of l-serine within the spinal cord results in a decrease in the frequency of locomotor-related activity. A: raw (top) and rectified/integrated (bottom) traces recorded from the left and right L2 ventral roots (L2 L; L2 R) showing the effect of the d-serine precursor l-serine (50 µM) on locomotor-related activity induced by 5-HT (15 µM) and DA (50 µM). B: locomotor-burst frequency over 5 min during a control period, during a 15-min application of l-serine (40–100 µM), and during a 30-min washout. Individual data points are shown in gray, and means are represented by black lines; n = 9 preparations. C: raw (top) and rectified/integrated (bottom) traces recorded from L2 L and L2 R showing the effect of l-serine (50 µM) on locomotor-related activity in the presence of the serine-racemase inhibitor HOAsp (400 µM). D: locomotor-burst frequency over 5 min during a control period, during a 15-min application of l-serine (50 µM), and during a 30-min washout. HOAsp was present throughout; n = 6. Statistically significant difference from control: **P < 0.01.

Fig. 6.

NMDA receptors are active during disinhibited bursting. A: raw (top) and rectified/integrated (bottom) traces recorded from the left and right L2 ventral roots (L2 L; L2 R) showing the effect of the competitive glutamate-binding site antagonist d-APV (50 µM) applied to preparations in which inhibitory transmission was blocked by the GABA_A-receptor antagonist picrotoxin (60 μM) and the glycine-receptor antagonist strychnine (1 μM). Bi: ventral-root burst frequency over 10 min during a control period, during a 30-min application of d-APV, and during a 40-min washout. Individual data points are shown in gray, and means are represented by black lines; n = 6 preparations. Bii: time-course plot of normalized data aggregated into 1-min bins showing burst frequency during d-APV application; n = 6. Ci: ventral-root amplitude over 10 min during a control period, during a 30-min application of d-APV, and during a 40-min washout; n = 6. Cii: time-course plot of normalized data aggregated into 1-min bins showing burst amplitude during d-APV application; n = 6. D: raw (top) and rectified/integrated (bottom) traces recorded from L2 L and L2 R showing the effect of NMDA (10 µM) applied to preparations in which inhibitory transmission was blocked. Ei: ventral-root burst frequency over 10 min during a control period, during a 20-min application of NMDA, and during a 30-min washout; n = 6. Eii: time-course plot of normalized data aggregated into 1-min bins showing an increase in burst frequency during NMDA application; n = 6. Fi: ventral-root burst amplitude over 10 min during a control period, during a 20-min application of NMDA, and during a 30-min washout; n = 6. Fii: time-course plot of normalized data aggregated into 1-min bins showing a reduction in burst amplitude during NMDA application; n = 6. Error bars: ± SE. Statistically significant difference from control: *P < 0.05, ***P < 0.001.

Fig. 7.

Neither exogenous nor endogenous d-serine modulates disinhibited activity mediated by excitatory components of locomotor networks. A: raw (top) and rectified/integrated (bottom) traces recorded from the left and right L2 ventral roots (L2 L; L2 R) showing the effect of d-serine (10 µM) applied to preparations in which inhibitory transmission was blocked by the GABA_A-receptor antagonist picrotoxin (60 μM) and the glycine-receptor antagonist strychnine (1 μM). Bi: ventral-root burst frequency over 10 min during a control period, during a 30-min application of d-serine (1–10 µM), and during a 40-min washout. Individual data points are shown in gray, and means are represented by black lines; n = 6 preparations. Bii: time-course plot of normalized data aggregated into 1-min bins showing no change in burst frequency during d-serine application; n = 6. Ci: ventral-root burst amplitude over 10 min during a control period, during a 30-min application of d-serine (1–10 µM), and during a 40-min washout; n = 6. Cii: time-course plot of normalized data aggregated into 1-min bins showing no change in burst amplitude during d-serine application; n = 6. D: raw (top) and rectified/integrated (bottom) traces recorded from L2 L and L2 R showing the effect of the serine-racemase inhibitor HOAsp (400 µM) applied to preparations in which inhibitory transmission was blocked. Ei: ventral-root burst frequency over 10 min during a control period, during a 30-min application of HOAsp, and during a 40-min washout; n = 7. Eii: time-course plot of normalized data aggregated into 1-min bins showing no change in burst frequency during HOAsp application; n = 7. Fi: ventral-root burst amplitude over 10 min during a control period, during a 30-min application of HOAsp, and during a 40-min washout; n = 7. Fii: time-course plot of normalized data aggregated into 1-min bins showing no change in burst amplitude during HOAsp application; n = 7. Error bars: ± SE.

Fig. 8.

GlyT1 glycine transporters control the availability of glycine at excitatory synapses. A: raw (top) and rectified/integrated (bottom) traces recorded from the left and right L2 ventral roots (L2 L; L2 R) showing the effect of glycine (100 µM) on locomotor-related activity induced by 5-HT (15 µM) and DA (50 µM). B: left-right phase relationship in control conditions (Bi) and during application of 100 µM glycine 100 µM (Bii). Circular plots represent the onset of locomotor bursts recorded from L2 R ventral roots (gray dots) in relation to the onset of activity recorded from corresponding L2 L roots (assigned a value of 0) in the same cycle. Vector direction indicates mean phase, and vector length corresponds to clustering of data points around the mean. We analyzed >100 burst cycles from 3 preparations for each condition. Ci: locomotor-burst frequency over 5 min during a control period, during a 15-min application of glycine, and during a 40-min washout. Individual data points are shown in gray, and means are represented by black lines; n = 6. Cii: time-course plot of normalized data aggregated into 1-min bins showing no change in burst frequency during glycine application; n = 6. Di: locomotor-burst amplitude over 5 min during a control period, during a 15-min application of glycine, and during a 40-min washout; n = 6. Dii: time-course plot of normalized data aggregated into 1-min bins showing no change in burst amplitude during glycine application; n = 6. E: raw (top) and rectified/integrated (bottom) traces recorded from L2 L and L2 R showing the effect of the GlyT1 inhibitor ALX 5407 (30 µM) on locomotor-related activity. Fi: locomotor-burst frequency over 5 min during a control period, during a 30-min application of ALX 5407, and during a 30-min washout; n = 6. Fii: time-course plot of normalized data aggregated into 1-min bins showing an increase in burst frequency during ALX 5407 application; n = 6. Gi: locomotor-burst amplitude over 5 min during a control period, during a 30-min application of ALX 5407, and during a 30-min washout; n = 6. Gii: time-course plot of normalized data aggregated into 1-min bins showing no change in burst amplitude during ALX 5407 application; n = 6. H: raw (top) and rectified/integrated (bottom) traces recorded from L2 L and L2 R showing the effect of ALX 5407 on locomotor-related activity in the presence of the competitive glutamate-binding site antagonist d-APV (50 µM). I: locomotor-burst frequency over 5 min during a control period, during a 30-min application of HOAsp, and during a 30-min washout. d-APV was present throughout; n = 6. Error bars: ± SE. Statistically significant difference from control: **P < 0.01.