Transcript supplemental material

To confirm VGLUT1 specificity for afferent synaptic inputs we analyzed VGLUT1-immunoreactivity around calbindin-IR
Renshaw cells in P15 Er81-/- mutants that lack ventral horn sensory afferent projections (Arber et al., 2000) and compared them to
wild-type age-matched littermates. Wild-type (Er81+/+) and mutant (Er81-/-) spinal cords did not differ in overall morphology,
density or distribution of NeuN-immunoreactive neurons (we used a mouse monoclonal antibody, Chemicon, dilution 1:1000) or
calbindin-IR Renshaw cells (panels A-D; Er81+/+: 5.9 ± 0.3 Renshaw cell profiles per ventral horn in 40 µm thick sections
compared to 5.1 ± 0.7 in Er81-/- mutants; p>0.05, t-test). VGLUT1-immunolabeling in Er81-/- mutants was restricted to the dorsal
horn (panel B). Correspondingly, VGLUT1-IR contacts on Renshaw cells dropped to almost zero in Er81-/- mutants (panels E-G).
In addition to proprioceptive primary afferents, corticospinal tract (CST) neurons also express VGLUT1 and Er81 (Fremeau et al.,
2001; Yoneshima et al., 2006). The lumbar ventral horn receives a minor CST projection after P6 (Bareyre et al., 2005). The
stability of CST projections in Er81-/- mutants is unknown, but this sparse projection is clearly outnumbered by the high density of
ventral VGLUT1-IR boutons. Moreover, most VGLUT1-IR varicosities in lumbar 5 and 4 ventral horns distribute within lateral
laminae IX and VII, while the ventral CST projects to medial laminae VII and VIII. Taken together, the data suggest that
VGLUT1-IR contacts on Renshaw cells originate from Er81-dependent (proprioceptive) primary sensory afferents.
Figure legend :
A, B, Low magnification images of P15 spinal cords from wild type (Er81+/+, A) and mutant (Er81-/-, B). Neuronal cell bodies
were immunostained with antibodies against NeuN (Cy3, red) and superimposed on VGLUT1-IR boutons (FITC, green). No
major differences in distribution or densities of spinal neurons were observed, however, VGLUT1-IR boutons were reduced in
Er81-/- ventral horns. C, D, Medium magnification images showing calbindin-IR Renshaw cells (red) and VGLUT1-IR boutons
(green, VF, ventral funiculus). Renshaw cell numbers, distribution and calbindin-immunoreactivities showed no differences.
VGLUT1-IR boutons were depleted from the Renshaw cell area. E, High magnification images of calbindin-IR Renshaw cells
(red) receiving contacts from VGLUT1-IR boutons (green) in the wild-type. F, Almost no contacts were observed in Er81-/- . The
rare contacts identified on the dendrites of Renshaw cells from Er81-/- knockout mice were rather small (arrow in F). G,
VGLUT1-IR contact densities were dramatically reduced on both dendrites and somata in Er81-/- knockout animals (t-test;
p<0,001, n=10 Renshaw cells in wild type and in Er81-/- knockout; error bars indicate SEM).
Scale bars, 200 µm in A (B at same magnification); 100 µm in C,D; 20 µm in E,F.
References:
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