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

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To confirm VGLUT1 specificity for afferent
synaptic inputs we analyzed VGLUT1-immunoreactivit
y 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 11000)
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 pgt0.05, 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-immunorea
ctivities 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 plt0,001, n10 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 Arber S, Ladle DR, Lin JH, Frank
E, Jessell TM (2000) ETS gene Er81 controls the
formation of functional connections between group
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