Neurotrophin-3 ameliorates sensory-motor deficits in Er81-deficient mice

Abstract

Two factors, the ETS transcription factor ER81 and skeletal muscle-derived neurotrophin-3 (NT3), are essential for the formation of muscle spindles and the function of spindle afferent-motoneuron synapses in the spinal cord. Spindles either degenerate completely or are abnormal, and spindle afferents fail to project to spinal motoneurons in Er81 null mice; however, the interactions between ER81 and NT3 during the processes of afferent neuron and muscle spindle development are poorly understood. To examine if overexpression of NT3 in muscle rescues spindles and afferent-motoneuron connectivity in the absence of ER81, we generated myoNT3;Er81(-/-) double-mutant mice that selectively overexpress NT3 in muscle in the absence of ER81. Spindle reflex arcs in myoNT3;Er81(-/-) mutants differed greatly from Er81 null mice. Muscle spindle densities were greater and more afferents projected into the ventral spinal cord in myoNT3;Er81(-/-) mice. Spindles of myoNT3;Er81(-/-) muscles responded normally to repetitive muscle taps, and the monosynaptic inputs from Ia afferents to motoneurons, grossly reduced in Er81(-/-) mutants, were restored to wild-type levels in myoNT3;Er81(-/-) mice. Thus, an excess of muscle-derived NT3 reverses deficits in spindle numbers and afferent function induced by the absence of ER81. We conclude that muscle-derived NT3 can modulate spindle density and afferent-motoneuron connectivity independently of ER81.

Fig. 1

Morphological features of muscle spindles in wild-type (WT) and mutant mice. The 1-μm transverse sections of the MG muscles stained with toluidine blue (WT, Er81^−/−, myoNT3;Er81^−/−, and myoNT3) or 10-μm sections were processed for β-galactosidase activity (NT3^lacZ/+, NT3^lacZ/; Er81^−/−, and NT3^lacZ/+;myoNT3;Er81^−/−). Note that WT and myoNT3 spindles contain the normal complement of two nuclear bag fibers and two nuclear chain fibers (arrows), whereas spindles in all mutant muscles lacking ER81 contain two nuclear bag fibers only (top row). Spindles in NT3-overexpressing muscles are often clustered (bottom row, left panel). Also note that spindles in ER81-deficient muscles express NT3 similar to WT spindles, as revealed by expression of the lacZ reporter gene (bottom row, right three panels). Magnification is identical for all panels except the one (bottom row, left panel) of which is indicated separately. NT3, neurotrophin-3.

Fig. 2

Histogram of complements of intrafusal muscle fibers in spindles of the gluteus (GL) and medial gastrocnemius (MG) muscle of mutant and wild-type (WT) mice. A,B: Note that most WT spindles contain four intrafusal fibers, whereas most Er81^−/− spindles contain two or three intrafusal fibers in both the GL and MG muscles. Also note that overexpression of NT3 in muscle has little or no effect on numbers of intrafusal fibers in spindles (myoNT3;Er81^−/− compared with Er81^−/−).

Fig. 3

DiI (1,1′, di-octadecyl-3,3,3′,3′,-tetramethylindocarbocyanine perchlorate) labeling of spinal projections of afferents in control and mutant mice at postnatal day (P) 0–P1. A: Extent of the central projections of sensory axons from dorsal spinal roots (DRs) into the spinal cord at different lumbar (L2–L5) segmental levels in Er81^−/− mice. B: Spinal projections of afferents at the L4 level in wild-type (WT), Er81^−/−, myoNT3;Er81^−/−, and myoNT3 mice. Arrows in each panel indicate the normal location of Ia afferents in WT mice, although these projections are largely absent in Er81^−/− mice. Locations of α-motoneurons (MNs) are indicated with asterisks. Some MNs are also DiI (1,1′-dioctadecyl-3,3,3′,3′,-tetramethylindocarbocyanine perchlorate) -labeled in several panels. Note that more afferent projections are present at the L5 than L2 level in Er81^−/− mice and that the density of the projections is higher in myoNT3;Er81^−/− than in Er81^−/− mice. dh, dorsal horn.

Fig. 4

Dextran labeling of spinal projections of afferents in wild-type (WT), Er81^−/−, myoNT3;Er81^−/−, and myoNT3 mice at postnatal day (P) 7. A–D: Extent of the central projections of sensory axons from the lumbar (L4) dorsal spinal root (DR) into the spinal cord in control and mutant mice. A′–D′: Higher magnification of the areas shown above in white rectangles. Arrows indicate the normal ventral projections of Ia sensory afferent collaterals, which are absent in Er81^−/− mice but partially rescued by overexpression of neurotrophin-3 (NT3) in myoNT3;Er81^−/− mice.

Fig. 5

Monosynaptic connections between afferent axons and α-motoneurons (MNs) in wild-type (WT), Er81^−/−, myoNT3;Er81^−/−, and myoNT3 mice. A: Excitatory postsynaptic potentials (EPSPs) were recorded from the lumbar region (L2–L5) ventral spinal roots (VRs) in response to stimulation of the dorsal spinal roots (DRs) of the corresponding segments. Representative traces of EPSPs recorded from the L4 VR are shown. The superimposed red traces show the model EPSPs used to measure monosynaptic amplitudes (see Experimental Procedures section), which are indicated by arrows. B: Average amplitudes of monosynaptic EPSPs in segments L2 to L5 in mutant and control mice. Statistical significance of differences at each segment from WT mice is denoted by asterisks (*P < 0.05, **P < 0.01).

Fig. 6

Intracellular recordings of synaptic potentials evoked by stimulation of Ia muscle afferents and recorded in the corresponding homonymous (projecting to the same muscle) α-motoneurons (MNs). The amplitude of the monosynaptic component of each excitatory postsynaptic potential (EPSP) is indicated with an arrow, as in Figure 5. Note that homonymous Ia–MN EPSPs in rostral lumbar segments (Q MNs at L2) are more severely attenuated in Er81^−/− mice than those in more caudal segments (LG MNs at L4). The remaining connections in caudal segments of Er81^−/− mice are selective; homonymous (LG → LG) Ia EPSPs are larger than those from other muscles (DP → LG), just as in wild-type (WT) mice. Q, quadriceps; LG, lateral gastrocnemius; DP, deep peroneal; L, lumbar.

Fig. 7

Responses of spindles to muscle stretch in wild-type (WT), Er81^−/−, and myoNT3;Er81^−/− mice. A: Representative records of Ia afferent action potentials recorded from the quadriceps (Q) muscle nerve in response to small stretches of the Q muscle at stretch frequencies of 1 Hz and 10 Hz. B: Average responses of spindles in mutant and control mice to varying stretch frequencies. The amplitude of the extracellularly recorded action potential reflects the number of active spindles. The more complex waveforms in myoNT3;Er81^−/− mice indicate a burst of action potentials in response to single stretches. Statistical significance of reductions in amplitude of Er81^−/− compared with WT spindles is denoted by asterisks (**P < 0.01).