Estrogen signaling is necessary for exercise-mediated enhancement of motoneuron participation in axon regeneration after peripheral nerve injury in mice.

Thousands of people each year suffer from peripheral nerve injury. Treatment options are limited, and recovery is often incomplete. Treadmill exercise can enhance nerve regeneration; however, this appears to occur in a sex-dependent manner. Females respond best to short duration, high speed interval training; whereas, males respond best to slower, continuous training. Previous studies have shown a role for testosterone in this process, but the role of estrogen is unknown. To evaluate the role of estrogen signaling in treadmill exercise, we blocked estrogen receptor (ER) signaling during treadmill exercise in males and female wild type mice. The right common fibular (CF) branch of the sciatic nerve was cut and repaired with fibrin glue that contained the ER antagonist ICI 182,780. Estradiol-filled or blank Silastic capsules were implanted subcutaneously at the time of nerve transection. Starting three days post-transection, exercised mice received treadmill training using the paradigm appropriate to their sex 5 days a week for 2 weeks. Fourteen days after the initial nerve transection, motoneurons whose axons had regenerated at least 1.5 mm distal to the original cut sites were labeled with a retrograde tracer. Regeneration was quantified by counting the number of fluorescent labeled motoneurons in the lumbar region of the spinal cord. Both treadmill training and estradiol administration increased the number of motoneurons participating in axon regeneration, but these effects were blocked by ER antagonist treatment. Estrogen signaling is important for the enhancing effects of treadmill exercise on motoneuron participation after peripheral nerve cut. © 2017 Wiley Periodicals, Inc. Develop Neurobiol 77: 1133-1143, 2017.

Optically-Induced Neuronal Activity Is Sufficient to Promote Functional Motor Axon Regeneration In Vivo.

Peripheral nerve injuries are common, and functional recovery is very poor. Beyond surgical repair of the nerve, there are currently no treatment options for these patients. In experimental models of nerve injury, interventions (such as exercise and electrical stimulation) that increase neuronal activity of the injured neurons effectively enhance axon regeneration. Here, we utilized optogenetics to determine whether increased activity alone is sufficient to promote motor axon regeneration. In thy-1-ChR2/YFP transgenic mice in which a subset of motoneurons express the light-sensitive cation channel, channelrhodopsin (ChR2), we activated axons in the sciatic nerve using blue light immediately prior to transection and surgical repair of the sciatic nerve. At four weeks post-injury, direct muscle EMG responses evoked with both optical and electrical stimuli as well as the ratio of these optical/electrical evoked EMG responses were significantly greater in mice that received optical treatment. Thus, significantly more ChR2+ axons successfully re-innervated the gastrocnemius muscle in mice that received optical treatment. Sections of the gastrocnemius muscles were reacted with antibodies to Synaptic Vesicle Protein 2 (SV2) to quantify the number of re-occupied motor endplates. The number of SV2+ endplates was greater in mice that received optical treatment. The number of retrogradely-labeled motoneurons following intramuscular injection of cholera toxin subunit B (conjugated to Alexa Fluor 555) was greater in mice that received optical treatment. Thus, the acute (1 hour), one-time optical treatment resulted in robust, long-lasting effects compared to untreated animals as well as untreated axons (ChR2-). We conclude that neuronal activation is sufficient to promote motor axon regeneration, and this regenerative effect is specific to the activated neurons.

Neuronal BDNF signaling is necessary for the effects of treadmill exercise on synaptic stripping of axotomized motoneurons.

The withdrawal of synaptic inputs from the somata and proximal dendrites of spinal motoneurons following peripheral nerve injury could contribute to poor functional recovery. Decreased availability of neurotrophins to afferent terminals on axotomized motoneurons has been implicated as one cause of the withdrawal. No reduction in contacts made by synaptic inputs immunoreactive to the vesicular glutamate transporter 1 and glutamic acid decarboxylase 67 is noted on axotomized motoneurons if modest treadmill exercise, which stimulates the production of neurotrophins by spinal motoneurons, is applied after nerve injury. In conditional, neuron-specific brain-derived neurotrophic factor (BDNF) knockout mice, a reduction in synaptic contacts onto motoneurons was noted in intact animals which was similar in magnitude to that observed after nerve transection in wild-type controls. No further reduction in coverage was found if nerves were cut in knockout mice. Two weeks of moderate daily treadmill exercise following nerve injury in these BDNF knockout mice did not affect synaptic inputs onto motoneurons. Treadmill exercise has a profound effect on synaptic inputs to motoneurons after peripheral nerve injury which requires BDNF production by those postsynaptic cells.

Exercise, neurotrophins, and axon regeneration in the PNS.

Electrical stimulation and exercise are treatments to enhance recovery from peripheral nerve injuries. Brain-derived neurotrophic factor and androgen receptor signaling are requirements for the effectiveness of these treatments. Increased neuronal activity is adequate to promote regeneration in injured nerves, but the dosing of activity and its relationship to neurotrophins and sex steroid hormones is less clear. Translation of these therapies will require principles associated with their cellular mechanisms.

Cooperative roles of BDNF expression in neurons and Schwann cells are modulated by exercise to facilitate nerve regeneration.

After peripheral nerve injury, neurotrophins play a key role in the regeneration of damaged axons that can be augmented by exercise, although the distinct roles played by neurons and Schwann cells are unclear. In this study, we evaluated the requirement for the neurotrophin, brain-derived neurotrophic factor (BDNF), in neurons and Schwann cells for the regeneration of peripheral axons after injury. Common fibular or tibial nerves in thy-1-YFP-H mice were cut bilaterally and repaired using a graft of the same nerve from transgenic mice lacking BDNF in Schwann cells (BDNF(-/-)) or wild-type mice (WT). Two weeks postrepair, axonal regeneration into BDNF(-/-) grafts was markedly less than WT grafts, emphasizing the importance of Schwann cell BDNF. Nerve regeneration was enhanced by treadmill training posttransection, regardless of the BDNF content of the nerve graft. We further tested the hypothesis that training-induced increases in BDNF in neurons allow regenerating axons to overcome a lack of BDNF expression in cells in the pathway through which they regenerate. Nerves in mice lacking BDNF in YFP(+) neurons (SLICK) were cut and repaired with BDNF(-/-) and WT nerves. SLICK axons lacking BDNF did not regenerate into grafts lacking Schwann cell BDNF. Treadmill training could not rescue the regeneration into BDNF(-/-) grafts if the neurons also lacked BDNF. Both Schwann cell- and neuron-derived BDNF are thus important for axon regeneration in cut peripheral nerves.

Sex differences in the effectiveness of treadmill training in enhancing axon regeneration in injured peripheral nerves.

Exercise in the form of daily treadmill training results in significant enhancement of axon regeneration following peripheral nerve injury. Because androgens are also linked to enhanced axon regeneration, we wanted to investigate whether sex differences in the effect of treadmill training might exist. The common fibular nerves of thy-1-YFP-H mice were cut and repaired with a graft of the same nerve from a strain-matched wild-type donor mouse. Animals were treated with one of two daily treadmill training paradigms: slow continuous walking for 1 h or four higher intensity intervals of 2 min duration separated by 5-min rest periods. Training was begun on the third day following nerve injury and continued 5 days per week for 2 weeks. Effects on regeneration were evaluated by measuring regenerating axon profile lengths in optical sections through the repair sites and grafts at the end of the training period. No sex differences were found in untrained control mice. Continuous training resulted in significant enhancement of axon regeneration only in males. No effect was found in females or in castrated males. Interval training was effective in enhancing axon regeneration only in females and not in intact males or castrated males. Untrained females treated with the aromatase inhibitor, anastrozole, had significant enhancement of axon regeneration without increasing serum testosterone levels. Two different mechanisms exist to promote axon regeneration in a sex-dependent manner. In males treadmill training uses testicular androgens. In females, a different cellular mechanism for the effect of treadmill training must exist.

Enhancing recovery from peripheral nerve injury using treadmill training.

Full functional recovery after traumatic peripheral nerve injury is rare. We postulate three reasons for the poor functional outcome measures observed. Axon regeneration is slow and not all axons participate. Significant misdirection of regenerating axons to reinnervate inappropriate targets occurs. Seemingly permanent changes in neural circuitry in the central nervous system are found to accompany axotomy of peripheral axons. Exercise in the form of modest daily treadmill training impacts all three of these areas. Compared to untrained controls, regenerating axons elongate considerably farther in treadmill trained animals and do so via an autocrine/paracrine neurotrophin signaling pathway. This enhancement of axon regeneration takes place without an increase in the amount of misdirection of regenerating axons found without training. The enhancement also occurs in a sex-dependent manner. Slow continuous training is effective only in males, while more intense interval training is effective only in females. In treadmill trained, but not untrained mice the extent of coverage of axotomized motoneurons is maintained, thus preserving important elements of the spinal circuitry.

Compensatory changes in cellular excitability, not synaptic scaling, contribute to homeostatic recovery of embryonic network activity.

When neuronal activity is reduced over a period of days, compensatory changes in synaptic strength and/or cellular excitability are triggered, which are thought to act in a manner to homeostatically recover normal activity levels. The time course over which changes in homeostatic synaptic strength and cellular excitability occur are not clear. Although many studies show that 1-2 days of activity block are necessary to trigger increases in excitatory quantal strength, few studies have been able to examine whether these mechanisms actually underlie recovery of network activity. Here, we examine the mechanisms underlying recovery of embryonic motor activity following block of either excitatory GABAergic or glutamatergic inputs in vivo. We find that GABA(A) receptor blockade triggers fast changes in cellular excitability that occur during the recovery of activity but before changes in synaptic scaling. This increase in cellular excitability is mediated in part by an increase in sodium currents and a reduction in the fast-inactivating and calcium-activated potassium currents. These findings suggest that compensatory changes in cellular excitability, rather than synaptic scaling, contribute to activity recovery. Further, we find a special role for the GABA(A) receptor in triggering several homeostatic mechanisms after activity perturbations, including changes in cellular excitability and GABAergic and AMPAergic synaptic strength. The temporal difference in expression of homeostatic changes in cellular excitability and synaptic strength suggests that there are multiple mechanisms and pathways engaged to regulate network activity, and that each may have temporally distinct functions.

GABAA transmission is a critical step in the process of triggering homeostatic increases in quantal amplitude.

When activity levels are altered over days, a network of cells is capable of recognizing this perturbation and triggering several distinct compensatory changes that should help to recover and maintain the original activity levels homeostatically. One feature commonly observed after activity blockade has been a compensatory increase in excitatory quantal amplitude. The sensing machinery that detects altered activity levels is a central focus of the field currently, but thus far it has been elusive. The vast majority of studies that reduce network activity also reduce neurotransmission. We address the possibility that reduced neurotransmission can trigger increases in quantal amplitude. In this work, we blocked glutamatergic or GABA(A) transmission in ovo for 2 days while maintaining relatively normal network activity. We found that reducing GABA(A) transmission triggered compensatory increases in both GABA and AMPA quantal amplitude in embryonic spinal motoneurons. Glutamatergic blockade had no effect on quantal amplitude. Therefore, GABA binding to the GABA(A) receptor appears to be a critical step in the sensing machinery for homeostatic synaptic plasticity. The findings suggest that homeostatic increases in quantal amplitude may normally be triggered by reduced levels of activity, which are sensed in the developing spinal cord by GABA, via the GABA(A) receptor. Therefore, GABA appears to be serving as a proxy for activity levels.