Differential plasticity of the GABAergic and glycinergic synaptic transmission to rat lumbar motoneurons after spinal cord injury

Abstract

Maturation of inhibitory postsynaptic transmission onto motoneurons in the rat occurs during the perinatal period, a time window during which pathways arising from the brainstem reach the lumbar enlargement of the spinal cord. There is a developmental switch in miniature IPSCs (mIPSCs) from predominantly long-duration GABAergic to short-duration glycinergic events. We investigated the effects of a complete neonatal [postnatal day 0 (P0)] spinal cord transection (SCT) on the expression of Glycine and GABA(A) receptor subunits (GlyR and GABA(A)R subunits) in lumbar motoneurons. In control rats, the density of GlyR increased from P1 to P7 to reach a plateau, whereas that of GABA(A)R subunits dropped during the same period. In P7 animals with neonatal SCT (SCT-P7), the GlyR densities were unchanged compared with controls of the same age, while the developmental downregulation of GABA(A)R was prevented. Whole-cell patch-clamp recordings of mIPSCs performed in lumbar motoneurons at P7 revealed that the decay time constant of miniature IPSCs and the proportion of GABAergic events significantly increased after SCT. After daily injections of the 5-HT(2)R agonist DOI, GABA(A)R immunolabeling on SCT-P7 motoneurons dropped down to values reported in control-P7, while GlyR labeling remained stable. A SCT made at P5 significantly upregulated the expression of GABA(A)R 1 week later with little, if any, influence on GlyR. We conclude that the plasticity of GlyR is independent of supraspinal influences whereas that of GABA(A)R is markedly influenced by descending pathways, in particular serotoninergic projections.

Figures

Figure 1.

The developmental upregulation of GlyR is not affected by a neonatal SCT. Dual or triple labeling of GlyRα1 with GlyT2 and gephyrin on fast blue (FB) retrogradely identified TS motoneurons. The experiments were performed on control rats at P1 (control-P1), P7 (control-P7), and 7 d after neonatal SCT (SCT-P7). Each panel corresponds to a single optical section. In each horizontal series, the antigens detected (and their color of detection) are indicated in the lower part of the left panels. In control-P1 (A1), the density of GlyRα1 reached 29.5 clusters per 100 μm on the cell bodies, 45 on the dendrites (Whiskers Box in D). At this stage, 60% of the clusters of GlyRα1 faced GlyT2-immunoreactive axon terminals on the cell bodies, 75.1% on the dendrites (A2, E), and >75% of the GlyRα1 clusters were colocalized with gephyrin on the cell bodies, 94.8% on the dendrites (A3, F). On motoneurons of control-P7 (B1), the rate of expression of the GlyRα1 significantly increased compared with control-P1 and their density reached 48.9 on the cell bodies, 62.9 on the dendrites (D; Kruskal–Wallis nonparametric test, P1 vs P7, *p < 0.05). At this stage, the association of GlyRα1 with GlyT2 reached 69.4% on the soma, 75.6% on the dendrites (B2, E), while the colocalization of GlyRα1 with gephyrin was almost total on both soma (91.8%) and dendrites (91.7%; B3, F). Seven days after neonatal SCT (SCT-P7), the density of GlyRα1-immunopositive clusters remained remarkably similar (C1) to those quantified on motoneurons of age matched-controls, on both cell bodies and dendrites (D; Control-P7 vs SCT-P7, p = 0.99 for both soma and dendrites). The neonatal SCT did not significantly affect the rate of association of GlyRα1 with GlyT2 on both cell bodies (68.1%) and dendrites (80.3%) compared with P7-control animals (C2, E; Control-P7 vs SCT-P7; p = 0.11 for soma and 0.8 for dendrites). Similarly, on SCT-P7 rats, the rate of colocalization of GlyRα1 with gephyrin (C3) did not significantly change compared with P7 animals on both soma (98.05%) and dendrites (91.04%; F; Control-P7 vs SCT-P7, p = 0.6 for soma and 0.88 for dendrites).

Figure 2.

Lack of a developmental downregulation of the expression of GABAARα2 and GABAARα3 after neonatal SCT. Triple fluorescent labeling of GlyRα1 and GABAARα2 or GABAARα3 on FB retrogradely identified TS motoneurons. Images are single optical plan sections. In each horizontal series, the antigens detected (and their color of detection) are indicated in the lower part of the left panels. In control-P1 pups, immunopositive clusters of GABAARα2 were expressed at the membrane of the motoneurons (A1) with densities of 15 clusters per 100 μm on the soma, 28 on the dendrites (C1, histograms). In control-P7, only a few GABAARα2 clusters were still expressed on the motoneuronal membrane (A2), and their density significantly dropped down on both cell bodies and dendrites (C1; 4 and 0.1 clusters per 100 μm, respectively; Kruskal–Wallis nonparametric test, control-P1 vs control-P7; *p < 0.05 in both soma and dendrites). After neonatal SCT, the expression of the GABAARα2 did follow on a normal developmental decrease. Indeed, densities were significantly higher than in control-P7 (18.5 on the somata, 35 on the dendrites; control-P7 vs SCT-P7: p < 0.05 for both compartments) and very similar to the values measured in control-P1 (A3, C2; P1 vs SCT-P7; p > 0.05, on both somata and dendrites). A similar scenario was observed concerning GABAARα3: In control-P1 the median density of GABAARα3 reached 8 on the soma, 22 on the dendrites (B1, C2). In control-P7, GABAARα3 densities significantly decreased (B2) to 4 ± 17 on the soma, and 0.5 ± 4 on the dendrites (p < 0.05). After neonatal SCT the expression of the GABAARα3 did not follow a normal developmental decrease (B3) and their densities (12 on soma, 27 on the dendrites) were significantly higher than in control-P7 (C2; control-P7 vs SCT-P7, p < 0.05, on both soma and dendrites). Whiskers above and below the box indicate the 90th and 10th percentiles; the number of motoneurons analyzed is indicated in each box.

Figure 3.

Deactivation decay time of global mIPSCs increase after SCT. A1, Example of mIPSCs from a motoneuron of control-P7 with glycine events (filled star), GABAA events (open star), and mixed GABA/glycine events (open circle). A2, Example of mIPSCs from SCT-P7 motoneuron. B1, Normalized mean of mIPSCs from the control-P7 motoneuron illustrated in A1 (light gray trace, mean of 124 events) and from the SCT-P7 motoneuron illustrated in A2 (dark gray trace, mean of 114 events). B2, Mean of mIPSCs from the control-P7 motoneuron illustrated in A1 and from the SCT-P7 motoneuron illustrated in A2. B3, B4, Cumulative probability function of decay times and amplitudes for the individual events from control-P7 group (light gray traces; n = 2051 events from 8 motoneurons) and from SCT-P7 group (dark gray traces; n = 2099 events from 9 motoneurons). The mean decay time increased significantly after SCT [10.16 ± 0.86 ms in control group (n = 8) and 15.61 ± 1.25 ms in SCT group (n = 9); p < 0.01], whereas a nonsignificant trend toward a reduction of the amplitude of mIPSCs was observed after SCT (36.33 ± 5.38 pA vs 25.03 ± 3.94 pA in control and SCT group, respectively; p > 0.05).

Figure 4.

Increase of the proportion of GABAergic but not glycinergic or mixed GABA/Gly events after SCT. A1, Miniature glycinergic events were recorded after 20 min of bicuculline methiodide superfusion (20 μm). A2, GABAergic events were recorded after superfusion of strychnine (1 μm) during 15 min. A3, All mIPSCs were blocked by cocktails of strychnine and bicuculline. B1, Means of glycine events from control-P7 (light gray trace, 134 events) and SCT-P7 (dark gray trace, 112 events) motoneurons were superimposed and normalized. B2, Means of GABA events from control-P7 (light gray trace, 53 events) and SCT-P7 (dark gray trace, 39 events) were superimposed and normalized. B3, Cloud point representation of GABA (red triangles) and glycine (green dots) events after application of strychnine or bicuculline as a function of the amplitude and decay time of each event from control-P7 and SCT-P7 (517 glycine events in control-P7 (n = 5) and 646 glycine events in SCT-P7 (n = 6); 308 GABA events in control-P7 (n = 5) and 329 in SCT-P7 (n = 4)). C1, Histogram showing the percentages of pharmacologically isolated glycine and GABA events, relative to the total number of mIPSCs recorded before drug application in the same motoneurons, in control-P7 (light gray bars) and in SCT-P7 (dark gray bars). C2, Histogram of percentage of glycine, GABA and mixed GABA/glycine events as function of kinetics, in control-P7 (light gray bars) and after SCT-P7 (dark gray bars).

Figure 5.

Effects of an SCT made on P5, on the expression of GABAAR and GlyR. To differentiate maturational and reactional postlesional effects, SCT was made at P5, when the levels of GABAARs are already low in the lumbar cord. The effects of the lesion on the motoneuronal immunoexpression of GlyRα1 (green), GABAARα2, and GABAARα3 (red) were compared on fast blue-labeled motoneurons of control-P12 and on transected SCT-P12 animals. For each column, the combination of antibodies used is mentioned in the lower part of the upper picture. A1, A2, The membrane densities of GlyRα1s observed on TS motoneurons of SCT-P12 animals remained unchanged compared with control-P12 rats (D: control-P12 vs SCT-P12: p = 1 and p = 0.9 for soma and dendrites, respectively). B1, B2, On SCT-P12 rats, the cluster densities of GABAARα2 significantly increased compared with control-P12. C1, C2, Similarly, the GABAARα3 densities were significantly higher on motoneurons of SCT-P12 rats than in control-P12 (D: control-P12 vs SCT-P12: p < 0.05 for both somata and dendrites). Whiskers above and below the box indicate the 90th and 10th percentiles; the number of motoneurons analyzed is indicated in each box. (Kruskal–Wallis nonparametric test, *p < 0.05; ns, nonsignificant).

Figure 6.

Effects of the activation of 5-HT2Rs on the maturational expression of GABAAR subunits and GlyR. Dual immunofluorescent labeling of GABAARβ2,3 subunits (left, Alexa 488, green) and GlyRα1 (right, Cy3, red) on FB retrogradely labeled TS motoneurons of control-P7 rats (A1, B1), transected SCT-P7 rats treated with NaCl (A2, B2, SCT-P7 + NaCl), and SCT-P7 rats treated with DOI (A3, B3, SCT-P7 + DOI). Images are single optical plan sections. The antigens labeled are indicated in the lower part of the top. The average membrane fluorescence intensity of GABAARβ2,3, which increased on motoneurons of SCT-P7-NaCl rats compared with control-P7, significantly dropped down on motoneurons of SCT-P7 + DOI animals (see histograms in A4; p < 0.05) to reach values observed in control-P7. In contrast, the average membrane fluorescence intensity of GlyRα1, which did not significantly change after SCT (compare histograms for control-P7 vs SCT-P7 rats treated with NaCl in B4) was not affected by the treatment with DOI (histograms in B4; p = 0.35). Whiskers above and below the box indicate the 90th and 10th percentiles; the number of motoneurons analyzed is indicated in each box. (Kruskal–Wallis nonparametric test, *p < 0.05; ns, nonsignificant).

Figure 7.

Summary of the analysis of cluster densities of GlyRα1 (green), GABAARα2 (orange), and GABAARα3 (red) on the membrane of the cell bodies of TS motoneurons. Circles indicate membrane densities quantified in control rats at P1, P7, and P12, whereas triangles show the densities observed 7 d after a neonatal SCT or a SCT performed at P5. The glycinergic system appears to be relatively insensitive to SCT. In contrast, a neonatal SCT prevents the downregulation of GABAA receptors that normally occurs during the first postnatal week and a SCT performed at a later stage (P5), when the expression of these receptors is low, upregulates this system.