Figure 4

Gfrα1-TLZ+/Hb9::GFP- motor neurons display structural and synaptic characteristics of gamma motor neurons. (A) Neurolucida tracings of P20 large Gfrα1-TLZ-/Hb9::GFP+ (black) and small strongly Gfrα1-TLZ+/Hb9::GFP- MNs (gray). (B-D) Quantitative analyses of dendritic arbors. Gfrα1-TLZ-/Hb9::GFP+ MN primary dendrites are more numerous, more highly branched (B) and thicker (C), than those of Gfrα1+/Hb9::GFP- MNs. Sholl analysis (D) of Gfrα1-TLZ-/Hb9::GFP+ (black line) and Gfrα1-TLZ+/Hb9::GFP- (gray line) MNs also reveals differences in the distribution of membrane surface at different distances from soma that are characteristic of α- vs. γ-MNs. (E) VGluT1+ contacts (red) are present on P20 Hb9::GFP+ (green) MNs, but absent on Gfrα1-TLZ+ (blue)/Hb9::GFP- neurons. (F) VAChT+ contacts (red) are present on both Hb9::GFP+ (green) and Gfrα1-TLZ+ (blue)/Hb9::GFP- MNs (E and F, regions in white boxes are magnified in insets). (G) Quantification of dendritic and somatic VGluT1 and VAChT positive contacts on Hb9::GFP+ (black bars) and Gfrα1-TLZ+/Hb9::GFP- (gray bars) MNs (error bars indicate SEMs; triple and double asterisks indicate significance levels of P < 0.001 and P < 0.01 in t-test comparisons, respectively). (H-K) Tibialis anterior muscle in a Hb9::GFP mouse showing Cy5-bungarotoxin (Cy5-Bgtx, blue) labeled intra- and extrafusal neuromuscular junctions (NMJ) and PGP9.5 immunolabeled sensory and motor axons (red). Hb9::GFP+ motor axons are in green (H). Spindle afferent annulospiral endings (dual asterisks; also shown in the inset in a serial section) and intrafusal muscle fibers are labeled with PGP9.5. Extrafusal NMJs are innervated by PGP9.5+ and Hb9::GFP+ motor axons (K, high magnification of boxed area). Most motor end-plates on intrafusal fibers lacked GFP. Intrafusal Cy5-Bgtx NMJs (white arrowheads) are innervated by PGP9.5-IR axons that are HB9::GFP- (I, high magnification of boxed area). Yellow arrowheads indicate a few intrafusal NMJs innervated by PGP9.5+ and Hb9::GFP+ motor axons (example boxed and shown at higher magnification in J). Scale bars: (E,F) 20 μm; (H) 200 μm (100 μm in inset); (I,J) 25 μm.