Modulation of muscle spindle innervation by neurotrophin-3 following nerve injury

Abstract

Muscle spindles monitor changes in muscle length and are innervated by groups Ia and II sensory axons as well as gamma motor axons. Ia sensory axons respond to neurotrophin-3 (NT-3), which plays an important role in sculpting proprioceptive development. Previously, transgenic mice were generated that overexpress NT-3 in muscle (mlc/NT-3 mice). These mice have alterations in proprioceptive elements due to the developmental actions of NT-3 and neuroprotective effects on Ia axons following nerve injury (Taylor, M.D., Vancura, R., Williams, J.M., Riekhof, J.T., Taylor, B.K., Wright, D.E., 2001. Overexpression of neurotrophin-3 in skeletal muscle alters normal and injury-induced limb control. Somatosens. Motor Res. 18 (4), 286–294.) Here, we investigated the actions of NT-3 on each class of injured axons innervating spindles and explored the mechanisms by which NT-3 acts. Immunohistochemical assessment of muscle spindle innervation following crush revealed that the degeneration of Ia axons innervating spindles in mlc/NT-3 mice was substantially reduced, and overall spindle innervation by group II and gamma fibers was greatly improved at later stages. Mlc/NT-3 mice also displayed a significant reduction in the expression of the injury-induced transcription factor ATF3 by retrogradely labeled muscle afferent neurons. The effects of transgenic NT-3 overexpression on spindle innervation could be mimicked if wild-type mice were treated intramuscularly with recombinant NT-3 prior to but not following injury, suggesting that NT-3's actions were due to preexposure to NT-3. This view was supported by in vitro experiments in which large DRG neurons from mlc/NT-3 mice grew significantly longer neurites than wild-type neurons. The results reveal that improved Ia–spindle interactions after injury may enhance spindle innervation by group II and gamma fibers. Finally, exposure of muscle afferent fibers to NT-3 prior to injury alters axonal responses both in vitro and in vivo.

Introduction

Communication between sensory neurons and sensory receptors is critical for appropriate signaling to the nervous system. Not surprisingly, interactions between sensory axons and their specialized receptors are tightly coupled during embryonic and postnatal development. Many sensory receptors are induced during development by contact from sensory axons, and the survival of both is dramatically altered if either the axon or receptor is damaged (reviewed in Zelená, 1994). Muscle spindles in skeletal muscle are rapidly adapting receptors sensitive to muscle movement. Spindles are comprised of bag and chain intrafusal fibers innervated by three axon subtypes: Ia and II sensory axons, as well as gamma motor axons. Typical spindles in mice are comprised of two-bag and two-chain fibers, and the number of spindles per muscle is consistent within individual muscles (reviewed in Wright et al., 1997, Zelená, 1994).

Neurotrophin-3 (NT-3) is a member of the nerve growth factor (NGF) family of neurotrophins and is critical to the development of spindles (Airaksinen and Meyer, 1996, Ernfors et al., 1994, Farinas et al., 1994). NT-3 is expressed in developing muscle and acts as a target-derived growth factor dictating proprioceptive neuron survival (reviewed in Davies, 1996, Chen and Frank, 1999, Snider, 1994). Contact of specialized myotubes by Ia axons initiates spindle development, allowing for subsequent intrafusal development and sensorimotor innervation (Kucera et al., 1989). Both positive and negative manipulations of NT-3 levels in muscle substantially alter proprioceptive system development (Ernfors et al., 1994, Farinas et al., 1994, Klein et al., 1994, Kucera et al., 1995, Oakley et al., 1995, Oakley et al., 1997, Taylor et al., 2001a, Tessarollo et al., 1994, Wright et al., 1997, Zhang et al., 1994). In adulthood, NT-3 expression becomes restricted to intrafusal fibers in spindles, placing it in an excellent position to continue to regulate proprioceptive signaling (Copray and Brouwer, 1994).

Recently, several transcriptional elements have been identified that are important in proprioceptive axon/spindle development. EGR3 is a transcription factor expressed by intrafusal fibers and is required for spindle formation (Tourtellotte et al., 2001). ER81 and PEA3 are transcription factors expressed by proprioceptive sensory and motor neurons and play a role in specifying target innervation (Arber et al., 2000, Hippenmeyer et al., 2002, Kucera et al., 2002, Patel et al., 2003). The identification of these molecules is providing a much clearer understanding of the steps necessary for spindle formation and innervation (Patel et al., 2003).

Despite these advances, molecular regulators of proprioception in adulthood are poorly understood. Similar to all peripheral nerves, proprioceptive axons are vulnerable to genetic and metabolic diseases, axon injury, and aging. The consequences of disease or injury are poor or reduced sensory feedback and ataxia (Scott, 1996, Taylor et al., 2001b, Van Deursen and Simoneau, 1999). Here, we examined how NT-3 alters the interactions between proprioceptive axons in muscle spindles following peripheral nerve injury. The experiments utilized transgenic mice overexpressing NT-3 in skeletal muscle (mlc/NT-3 mice), a model that provides sensory axons elevated levels of NT-3 in their target during development and adulthood (Taylor et al., 2001a). Our results show that following nerve injury, NT-3 plays an important role during the early stages of spindle denervation that ultimately effects reinnervation by group II and gamma fibers. Moreover, NT-3 reduces the expression of injury-induced gene expression, and exogenous NT-3 can mimic the effects seen in mlc/NT-3 mice if delivered prior to injury. These approaches will be important in identifying molecular components critical for the responses of peripheral axons to injury and the ability to maintain innervation of peripheral targets.