Netrin-1-Dependent Spinal Interneuron Subtypes Are Required for the Formation of Left-Right Alternating Locomotor Circuitry

Loss of commissural interneurons in newborn Ntn1^Gt/Gt mice. A , Overview of intrasegmental and intersegmental tracings of CINs in P0 mice. B–G , Photomicrographs of transverse sections of the spinal cord L2 segment from control and Ntn1^Gt/Gt mice. Low power ( B–E ) and higher magnification ( F , G ). Scale bars 200 μm. H , Quantification of aCINs (green), dCINs (red) and adCINs (yellow). Values are given as means ± SEM, count on 5 consecutive sections for n = 6 animals for each group, ***p < 0.0001. The number of commissural neurons is dramatically reduced in P0 Ntn1^Gt/Gt mice. White boxes indicate areas of higher magnification. Error bars indicate SEM.

Ntn1^Gt/Gt mice display synchronous left-right coordination in fictive locomotion. A , B , Recorded activity in ventral roots of left (l) and right (r) lumbar segment (L)2 and L5 from P0 spinal cords of control mice ( A ) and Ntn1^Gt/Gt mice ( B ). Control mice displayed normal left-right alternation (n = 10) whereas Ntn1^Gt/Gt mice displayed left-right synchrony (n = 7). Circular phase diagrams of intersegmental L2/L5 coordination show alternation in both control (n = 8) and Ntn1^Gt/Gt (n = 6) mice. Scale bar 5 s. Each dot represent the mean phase of one animal and stippled circle indicate significant (inner) or highly significant (outer) values. C , Schematic illustration of ventral root recording set-up to measure fictive locomotion.

Remaining commissural interneurons in Ntn1^Gt/Gt mice are predominantly excitatory. A–D , Photomicrographs of in situ staining for Vglut2 or VIAAT mRNA on FDA traced spinal cord sections. Arrowheads show CINs positive for Vglut2 or VIAAT respectively, while arrows indicate single labeled CINs. Scale bar 25 μm. E , Quantification of the neurotransmitter used by traced CINs on lumbar spinal cord sections from control or Ntn1^Gt/Gt mice, counted on 6–10 consecutive sections and n = 3 animals for each group. F , Schematic drawing illustrating glutamatergic (red) or glycinergic/GABAergic (blue) neurotransmitter phenotype of commissural neurons in control and Ntn1^Gt/Gt mice. G , The ratio of excitatory versus inhibitory CINs for control, Ntn1^Gt/Gt mice from data presented in ( E ) showing a shift toward more Vglut2-positive CINs in Ntn1^Gt/Gt mice. Values are given as means ± SEM, ***p < 0.001. H , Circular phase diagrams of analyzed fictive locomotion experiments performed on spinal cords from Ntn1^Gt/Gt mice before and after treatment with 100 μm sarcosine. The arrows represent the grand mean of all animals in each condition. Only animals that subsequently were treated are included in the diagrams (n = 4). Sarcosine treatments left the synchronous left-right coordination unchanged in the Ntn1^Gt/Gt mice.

Loss of Netrin-1 has a more severe effect on dorsal compared with ventral originating CIN subpopulations. A , B , Schematic representation of the E12.5 neuronal tube and the position of traced CINs according to the transverse immunostained section of an FDA-traced control embryo shown in B . C–J , Photomicrographs of control and Ntn1^Gt/Gt animals showing the region of traced CINs according to the black box in A and staining for transcription factors as indicated to the left. White boxes in C–J indicate areas of higher magnification in respective panel to the right ( C′–J′ ). C , D , G–J , Arrows point to Brn3a⁺/Lbx1⁻ ( C′ , D′ ), Evx1⁺ ( G′ , H′ ), Nkx2.2⁺ ( I′ , J′ ), CINs. E , F , Pax2⁺/Lbx1⁻ (arrows) and Lbx1⁺/Pax2⁺ (arrowheads) CINs are found within the ventral group of traced neurons ( E′ ) while the majority of Lbx1⁺/Pax2⁻ CINs (stars) are more dorsally located ( E″ ). Scale bars, 30 μm. K , Schematic drawing of progenitor domains and early neuronal domains labeled by the transcription factors used in this study. L , Quantification of transcription factor phenotype in traced CINs shows significant differences in affected CIN populations in Ntn1^Gt/Gt mice. CIN remaining in Ntn1^Gt/Gt animals colabel predominantly with Evx1 and Nkx2.2. Error bars indicate SEM n = 5 for control and n = 6 for Ntn1^Gt/Gt embryos for all markers analyzed. For Brn3a/Lbx1 measurements in control embryos; T (total count traced neurons) = 724, s (sections) = 19; for Ntn1^Gt/Gt embryos: T = 460, s = 39; ***p < 0.0001. Lbx1/Pax2 measurements in control embryos; T = 555, s = 15; for Ntn1^Gt/Gt embryos: T = 533, s = 46; p < 0.0001. Evx1 measurements in control embryos: T = 541, s = 18; Ntn1^Gt/Gt embryos: T = 569, s = 50; **p = 0.0033. Nkx2.2 in control embryos: T = 332, s = 11; Ntn1^Gt/Gt embryos: T = 586, s = 51; p = 0.9731.

V0_d neurons express Pax2. A–C , Photomicrographs of immunohistochemistry experiments using antibodies against indicated transcription factors on E11.5 ( A , B ) or E12.5 ( C ) spinal cord transverse sections. White box in A indicates areas of higher magnification in B panels. Arrowheads indicate Pax2⁺ cells and arrows indicate Evx1⁺ (V0_v) cells. At E11.5, cells in the ventral V0 domain express Pax2 at clearly distinguishable levels whereas cells in the dorsal V0 domain express low or no Pax2 (see arrows a and b ). Lbx1, expressed in dI4 to dI6 subpopulations (Gross et al., 2002), is here used to delineate the border between the dI6 and V0 subpopulations. C , One day later, a similar situation is observed; V0_v neurons, as labeled by Evx1 (arrows c and d ), express lower or no levels of Pax2 protein while V0_d neurons express high levels of Pax2. Scale bars: A , 100 μm, B , C , 30 μm.