Additional Supporting Information may be found in the online version of this article.

CNE_22717_sm_suppfig1.tif4223KSupporting Figure 1 Time course of the infection of the lumbar spinal network following RV inoculation in TS muscle of mouse neonate. (magenta-green version of Figure 2 from the article for the assistance of color-blind readers.) After RV inoculation in the right TS, infected neurons are detected by immunoperoxidase on transverse sections through the L4 segment of the spinal cord. On the low magnification images, the side of the spinal cord ipsilateral to the infected motoneuronal pool is indicated by a star. (A) At 24h post inoculation (p.i.), only MNs are infected by the virus. (B) High magnification of the RV-infected MN in A. Note the strong labeling of the soma and proximal dendrites. (C) The first RV-infected loINs are observed at 30h p.i., both on the ipsi- and contralateral sides of the spinal cord as shown by high magnification images of the ipsilateral laminae V-VI (D), the contralateral ventral grey matter (E) and the ipsilateral ventral horn (F). Note the weak RV-labeling limited to the soma and the proximal dendrites of the infected loINs (blue arrows), indicating their low level of infection compared to MNs. (G) Extent of RV infection within INs in L4 segment at 36h p.i.. At this time, the dendrites of the INs are labeled on long distances, resulting in a fuzzy aspect of the labeling at low magnification. (H) High magnification of the inset in G showing RV-infected loINs on the contralateral side. Note the different intensities of labeling which reflect the relatively high (arrows) and low (arrowheads) levels of infection of these INs. (I) At 32h p.i., within a motoneuronal pool in L4 segment (identified by the ChAT labeling in magenta), one MN with a large cell body and arborized dendrites, is RV-infected (green labeling, designated by a white arrow). At the periphery, faintly labeled loINs are observed (green arrowheads). (J) Kinetics of the propagation of RV infection in the lumbar network. At sequential times following RV inoculation in the right TS muscles, infected MNs (magenta squares) and INs (green circles) were counted in alternate serial sections along the lumbar spinal cord. The number of animals analyzed at each time point is indicated above the squares. The first labeled MNs were observed at 18h p.i. and their number then remained stable. The first labeled INs were detected from 30h p.i. and their number significantly increased 6h later. The pink window indicates the duration of one infection cycle (12h) corresponding to the interval between the detection of the first RV-labeled MNs and the first RV-labeled loINs. Bars: 200 μm (A,C,G); 20 μm (B,D,E,F,H); 50 μm (I).
CNE_22717_sm_suppfig2.tif1911KSupporting Figure 2 Specific retrograde transneuronal transfer of RV via the motor route in the lumbar spinal network. (magenta-green version of Figure 3 from the article for the assistance of color-blind readers.) A: At 42h p.i., numerous RV-infected INs were observed in the L4 segment. Their counting was difficult due to the strong labeling of dendritic arborizations. Even at this advanced infection time, no RV-immunolabeling was detected in the dorsal root (DR) in which the sensory afferents to the TS MNs are running, as shown in B (enlargement of the upper boxed area in A). Note that some autofluorescent pericytes are visible. In contrast, the ventral root (VR) contained bundles of motor axons strongly RV-immunoreactive (C: enlargement of the lower boxed area in A). D: At 48h p.i., in L2 segment of a Hb9:GFP neonate, many RV-infected INs (magenta labeling) were detected in the ventral horn. However, the IML neurons were devoid of RV infection, as demonstrated in the enlargement of the boxed area in which large sympathetic preganglionic neurons are GFP-labeled (green labeling within the dotted circle in E) but do not exhibit RV immunolabeling (magenta labeling within the dotted circle in F). Bars: 200 μm (A); 100 μm (D); 50 μm (B,C,E,F).
CNE_22717_sm_suppfig3.tif1427KSupporting Figure 3 Identification of glycinergic loINs within the lumbar spinal network. (magenta-green version of Figure 5 from the article for the assistance of color-blind readers.) (A) Dual immunodetection of glycine (green labeling) and RV (magenta labeling) in a mouse neonate, at the L4 level of the spinal cord, 32h after inoculation of RV in the TS muscles. Glycinergic loINs (white arrowheads) are found predominantly in the ipsilateral ventral horn, among non glycinergic loINs (magenta arrowheads). The boxed area contains several of these glycinergic loINs identified by their double RV (enlarged in B) and glycine (enlarged in C) labeling. The midline (ML) is represented by a dotted line. The injected side is indicated by a star. Bars: A = 100 μm; B,C = 50 μm. (D-I) Distribution of glycinergic loINs along the spinal lumbar segments. In each segment, the labeled loINs are plotted from 6 sections (10 μm thick), at 150 μm intervals. On the side of injection, glycinergic RV-labeled loINs are found predominantly in lamina VII and to a lesser extent in laminae VI. On the contralateral side, a few glycinergic loINs are observed mainly in lamina VIII. Dotted lines delineate the Rexed's laminae. Abbreviations: L1-L6: segments of the spinal cord. I-X: Rexed's laminae. CC: Clarke columns. IML: intermediolateral column. (J-M) Diagrams showing the rostro-caudal distribution (L1-L6) of contralateral and ipsilateral glycinergic (yellow bars) and non glycinergic (red bars) loINs at 32h p.i.. Neurons are counted in each treated sections. On the ipsilateral side, the glycinergic and non glycinergic loINs have similar distribution along the different segments. Contralaterally, the glycinergic loINs are not numerous (only 5% of the total number of double labeled neurons) and they are observed more frequently in sections containing MNs (indicated by a blue bar) within L4 segment.
CNE_22717_sm_suppfig4.tif2600KSupporting Figure 4 Identification of Renshaw Cells within the lumbar locomotor network. (magenta-green version of Figure 6 from the article for the assistance of color-blind readers.) (A-F) Distribution of calbindin expressing neurons along the lumbar spinal cord of a mouse neonate. The most ventral groups of neurons in segments L3-L5 (arrows in panels C-E) correspond to RCs according to earlier description (Carr et al. 1998). (G) Calbinding-positive neurons are located on the floor of the ventral spinal cord at the L4 level (green arrows). (H) These cells are also RV-labeled (magenta arrows). (I) The co-localization of calbindin and RV labelings (white arrows) in these RCs demonstrates their monosynaptic connection with MNs (dotted white arrows). The antibodies used are indicated on the lower left corner of each panel. The white lines delineate the spinal cord borders. Bars: 500 μm (applied to A-F), 100 μm (G-I).
CNE_22717_sm_suppfig5.tif3657KSupporting Figure 5 Medial cholinergic INs are connected monosynaptically to TS MNs. (magenta-green version of Figure 7 from the article for the assistance of color-blind readers.) (A) At the L2 level, at a time when RV infection (green labeling) is restricted to MNs and loINs, the cholinergic INs detected centrally (magenta labeling) are not infected by RV, despite the presence of loINs in layer VII (asterisks). Note the presence of ChAT immunoreactive neurons in the IML (delineated by a dotted ellipse) confirming the localization of the section in a rostral lumbar segment. (B,C) Enlargement of the boxed area in C showing cholinergic INs devoid of RV infection. (D) Within L3, cholinergic INs are detected near the central canal and some of them are identified as loINs due to their RV labeling. (E,F) Closer view of the region boxed in D showing 2 loINs with a cholinergic phenotype. (G) In the rostral part of L4, the MNs pools are visualized in the ventral horn by their cholinergic phenotype (magenta labeling). Among them, MNs innervating TS are identified by ChAT and RV expression (double magenta and green labelings, indicated by white arrowheads). Most of the loINs have no ChAT phenotype (green labeling only) and they are observed in layer VII (asterisks) and IX (circled asterisk). (H,I) Enlargement of the area boxed in G showing a group of cholinergic INs (magenta labeling in H) including a loIN (green labeling in I). Bars: A,D,G = 100 μm; B,C,E,F,H,I = 20 μm.

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