Functional organization of locomotor interneurons in the ventral lumbar spinal cord of the newborn rat

Abstract

Although the mammalian locomotor CPG has been localized to the lumbar spinal cord, the functional-anatomical organization of flexor and extensor interneurons has not been characterized. Here, we tested the hypothesis that flexor and extensor interneuronal networks for walking are physically segregated in the lumbar spinal cord. For this purpose, we performed optical recordings and lesion experiments from a horizontally sectioned lumbar spinal cord isolated from neonate rats. This ventral hemi spinal cord preparation produces well-organized fictive locomotion when superfused with 5-HT/NMDA. The dorsal surface of the preparation was visualized using the Ca(2+) indicator fluo-4 AM, while simultaneously monitoring motor output at ventral roots L2 and L5. Using calcium imaging, we provided a general mapping view of the interneurons that maintained a stable phase relationship with motor output. We showed that the dorsal surface of L1 segment contains a higher density of locomotor rhythmic cells than the other segments. Moreover, L1 segment lesioning induced the most important changes in the locomotor activity in comparison with lesions at the T13 or L2 segments. However, no lesions led to selective disruption of either flexor or extensor output. In addition, this study found no evidence of functional parcellation of locomotor interneurons into flexor and extensor pools at the dorsal-ventral midline of the lumbar spinal cord of the rat.

Figure 1. Experimental setup.

A, Schematic drawing of the in vitro ventral SC preparation with VRs recording electrodes positioned at L2 and L5 (r = right; l = left). B, Transverse section of an intact SC (B1) and of two SC preparations with a horizontal cut at different dorso-ventral levels (B2, B3). Scale bars = 300 µm. The bottom panels show the motor activity recorded in VRs. F = Flexor activity, E = Extensor activity. C, Schematic drawing of transverse sections of the SC illustrating horizontal cuts at different levels (light grey bar and dark grey bar). The histogram plots the data of the mean period ± SEM of the different horizontal cuts.

Figure 2. Ca²⁺ imaging experiments.

A, Photomicrographs showing Fluo-4 AM labeling in the in vitro ventral SC preparation at the T13-L1 level (A1, magnification ×4; scale bar = 200 µm). Right photomicrographs were taken from areas delineated by grey squares 1 and 2 (A2, A3, magnification ×10; Scale bar = 100 µm). B, Extracellular recordings of left L2 VR and its rectified and low-pass filtered trace illustrating a typical rhythmic activity. Grey bars are aligned with r L2 VR bursts and reveal three cells examples tending to have Ca²⁺ peaks in phase (cell1), out of phase (cell2) or mixed (cell3) with lL2 VR activity. C, Circular plots illustrating the Ca²⁺ peak phases (black circles) for each cell in relation to the timing of L2 VRs bursts. The mean VR bursts over a 60 s recording bout range from spans phases 0 to 0.4 in the circular plot and is illustrated in grey. Vectors show the mean phase values and r values. Vector orientation indicates preferred phase of firing; vector length is proportional to coupling strength. D, Dot diagram shows distribution of all cells with phase relationship with motor output from T12 to L5. Cells were illustrated in phase (red dots), out of phase (black dots) and mixed (grey dots). E, Density plot of in phase and out of phase cells from T12 to L5 segments (n = 4 experiments; cc: central canal; scale bar = 100 µm).

Figure 3. Lesion experiments.

A, Photomicrographs of a ventral SC preparation (top) and its corresponding transverse section (bottom) showing an example of electrocoagulation of a restricted region located at the T13 segment level (magnification 4×; scale bar = 300 µm). B, Schematic drawing presenting all microlesions performed from T12 to L2 segments. The thick circles represent the first lesion (n = 21) whereas the thin circles represent the second lesion n = 16/21). The right and left dashed lines delineate the grey and white matter. The lesion size is expressed as a percentage of SC size (central canal set to 0). C, Lesion effects on locomotor activity. (C1), Extracellular recordings of L2 and L5 VRs before and after a lesion at the L1 level. The arrow represents the onset of locomotor recovery. (C2), Time course of the motor period 1 min before (control) and after a lesion at the T13 level (circles), the L1 level (triangles) and L2 level (squares). The onset of recovery is indicated by successive arrows for each lesion. D, Lesion effect on duration of locomotor activity disruption (D1), motor period (D2), and burst area (D3) of microlesions performed at different SC level (T13-L1-L2). The black circles indicate the first microlesions and the grey triangles illustrate the second microlesions.