ORIGINAL RESEARCH published: 26 February 2019 doi: 10.3389/fncel.2019.00065 Role of Noradrenergic Inputs From Locus Coeruleus on Changes Induced on Axotomized Motoneurons by Physical Exercise Ariadna Arbat-Plana, Maria Puigdomenech, Xavier Navarro and Esther Udina* Department of Cell Biology, Physiology and Immunology, Institute of Neurosciences, Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas, Universitat Autònoma de Barcelona, Bellaterra, Spain Physical rehabilitation is one of the cornerstones for the treatment of lesions of the nervous system. After peripheral nerve injuries, activity dependent therapies promote trophic support for the paralyzed muscles, enhance axonal growth and also modulate the maladaptive plastic changes induced by the injury at the spinal level. We have previously demonstrated that an intensive protocol of treadmill running (TR) in rats reduces synaptic stripping on axotomized motoneurons, preserves their perineuronal nets (PNN) and attenuates microglia reactivity. However, it is not clear through which mechanisms exercise is exerting these effects. Here we aimed to evaluate if activation of the locus coeruleus (LC), the noradrenergic center in the brain stem, plays a role in these effects. Since LC is strongly activated during stressful situations, as during Edited by: intensive exercise, we selectively destroyed the LC by administering the neurotoxin Qi Yuan, DPS-4 before injuring the sciatic nerve of adult rats. Animals without LC had increased Memorial University of Newfoundland, microglia reactivity around injured motoneurons. In these animals, an increasing intensity Canada protocol of TR was not able to prevent synaptic stripping on axotomized motoneurons Reviewed by: Elena Vazey, and the reduction in the thickness of their PNN. In contrast, TR was still able to attenuate University of Massachusetts Amherst, microglia reactivity in DSP-4 treated animals, thus indicating that the noradrenergic United States Fani Moreira Neto, projections are important for some but not all the effects that exercise induces on the Universidade do Porto, Portugal spinal cord after peripheral nerve injury. Moreover, animals subjected to treadmill training *Correspondence: showed delayed muscle reinnervation, more evident if treated with DSP-4. However, we Esther Udina did not find differences in treated animals regarding the H/M amplitude ratio, which esther.udina@uab.cat increased during the first stages of regeneration in all injured groups. Received: 29 September 2018 Keywords: peripheral nerve injury, spinal circuitry, physical exercise, motoneuron, noradrenaline, locus coeruleus Accepted: 11 February 2019 Published: 26 February 2019 Citation: INTRODUCTION Arbat-Plana A, Puigdomenech M, Navarro X and Udina E (2019) Role Peripheral nerve injury (PNI) results in a loss of motor, sensory and autonomic function in the of Noradrenergic Inputs From Locus Coeruleus on Changes Induced on denervated territory. Although peripheral axons have the ability to regenerate, this regenerative Axotomized Motoneurons by Physical process is slow and functional recovery is often limited, mainly due to unspecific reinnervation Exercise. Front. Cell. Neurosci. 13:65. of the target organs but also to alterations at the central level. The disconnection between the doi: 10.3389/fncel.2019.00065 neurons and their target organs is accompanied by a disorganization of the central circuitry, in Frontiers in Cellular Neuroscience | www.frontiersin.org 1 February 2019 | Volume 13 | Article 65 Arbat-Plana et al. Training Axotomized Motoneurons Without Locus-Coeruleus part due to the massive stripping of central synapses that N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4), that axotomized motoneurons suffer. The most affected ones are selectively damages noradrenergic projections originating from the proprioceptive afferents from the muscle spindle that never the LC, inducing degeneration of noradrenergic terminals recover basal values (Alvarez et al., 2010). (Dooley et al., 1987; Dudley et al., 1990; Prieto and Giralt, There is extensive literature on the use of activity-dependent 2001; Scullion et al., 2009). Therefore, we evaluated the role of therapies to improve axonal regeneration (van Meeteren et al., the LC on the modulation of physical exercise on the plastic 1997; Al-majed et al., 2000; Sabatier et al., 2008; Asensio-Pinilla changes that motoneurons suffer after axotomy by submitting to et al., 2009), and muscle reinnervation after sciatic nerve injury in TR control rats and rats that previously had suffered chemical rats (Asensio-Pinilla et al., 2009; Udina et al., 2011a). In general, ablation of the LC. when initiated during the denervation phase, moderate exercise training results in accelerated functional recovery (Gutmann and Jokoubek, 1963), whereas forced exercise or long-term MATERIALS AND METHODS hyperactivity tend to have a detrimental effect (Herbison et al., 1974; van Meeteren et al., 1997). Activity can also modulate the Experimental Design plastic changes observed after PNI, as neuropathic pain (Nam Adult female Sprague Dawley rats (8 weeks old, 200–280 g) et al., 2001; Cobianchi et al., 2010) and hyperreflexia (Vivó et al., were housed with free access to food and water at room 2008; Asensio-Pinilla et al., 2009; Udina et al., 2011a). Increased temperature of 22 ± 2◦ C under a 12:12-h light–dark cycle. activity, by reducing excitability of sensory neurons and synaptic All experimental procedures were approved by the ethics stripping of motoneurons, attenuates maladaptive plastic changes committee of Universitat Autònoma de Barcelona and followed in the spinal cord and central pathways (Mòdol et al., 2014; the guidelines of the European Commission on Animal Care Arbat-Plana et al., 2015). (EU Directive 2010/63/EU). For all surgical interventions, rats Although it is well-known that exercise exerts positive effects were anesthetized by intraperitoneal administration of ketamine on the nervous system through modulation of brain derived (90 mg/kg, 0.9 ml/kg; Imalgen 2000) supplemented with xylazine neurotrophic factor (BDNF) (Hutchinson et al., 2004; Molteni (10 mg/kg, 0.5 ml/kg; Rompun 2%). et al., 2004; Gomez-Pinilla et al., 2012; Cobianchi et al., 2013), One group of animals was used as control group; these animals in a recent work we observed that pharmacological activation were only axotomized and did not receive any treatment (AX). of TrkB, the specific receptor of BDNF, did not mimic all Two groups of animals received DSP-4 before the surgery, as the effects of exercise on axotomized spinal motoneurons described below. Animals of one of these groups were subjected (Arbat-Plana et al., 2016). Moreover, we had previously shown to exercise 3 days after the surgery (DSP-4+TR) whereas the that the integrity of sensory inputs from the injured limb is other ones were not trained (DSP-4). A fourth group was important for the maintenance of motoneuron perineuronal subjected to the same protocol of exercise without receiving nets (PNN) induced by treadmill exercise (Arbat-Plana et al., pharmacological treatment (TR). Each group was divided in two 2015). PNN restrict plasticity and stabilize synapses (Kwok subgroups: one subgroup was euthanized 14 days after injury et al., 2011), and thus exercise, by activating proprioceptive and (short-term) to evaluate motoneurons changes and the other was cutaneous receptors, facilitates preservation of central circuits euthanized 75 days after injury (long-term) in order to evaluate after PNI. However, besides peripheral activation of muscles functional recovery (Figure 1 and Table 1). Contralateral side was and sensory receptors, physical exercise is also activating a used as control-intact side. complex central circuitry related with locomotion and stress. Among it, the activation of noradrenergic neurons of the locus coeruleus (LC) may be important. Whereas ascending Retrograde Labeling projections from this brain stem center, as part of the ascending Retrograde tracing [True Blue Chloride (TB, Setareh Biotech) and reticular formation system, regulates arousal and attention Fluorogold (FG, Fluorochrome)] were bilaterally applied 1 week (Berridge and Waterhouse, 2003), descending projections inhibit before intervention to identify motoneuron pools of both tibialis nociceptive transmission (Millan, 2002; Pertovaara, 2006) anterior (TA) and gastrocnemius medialis (GM) muscles. A small and modulate excitability of motoneurons (Heckman et al., incision to the skin was made to expose the muscle, and then two 2003) in the spinal cord. In addition, noradrenaline release injections (2.5 µl/injection) were distributed within the muscle influences microglial function, by suppressing production of with a glass pipette using a Picospritzer (Arbat-Plana et al., 2015). pro-inflammatory cytokines and promoting anti-inflammatory The area of application was rinsed with saline to clean any mediators (Heneka et al., 2010; Jardanhazi-Kurutz et al., 2011). remnants of the tracer and the skin wound was sutured. Interestingly, an intensive protocol of treadmill exercise is able to attenuate the activation of microglia observed in the Surgical Procedure spinal cord after PNI (Cobianchi et al., 2010). This anti- Under anesthesia, the sciatic nerve was exposed at the mid-thigh inflammatory effect may be related to the increased activation and cut by using micro scissors. The proximal and distal stumps of the LC induced by physical activity. However, the role were rejoined with two epineural 10-0 sutures. Afterward, muscle of the LC in the activity-dependent-modulation of the spinal and skin were sutured in layers, iodine povidone was applied changes observed after axotomy has not been addressed yet. to the wound, and the rats were allowed to recover in a warm The LC can be chemically destroyed by using the neurotoxin environment under close observation. Frontiers in Cellular Neuroscience | www.frontiersin.org 2 February 2019 | Volume 13 | Article 65 Arbat-Plana et al. Training Axotomized Motoneurons Without Locus-Coeruleus FIGURE 1 | Diagram of the procedures performed in the study. Intramuscular injection of retrotracers was performed 1 week before the lesion to label motoneurons of both tibialis anterior (TA) and gastrocnemius medialis (GM) muscles. Two days later, DSP-4 was administered in the groups where LC had to be destroyed. Section and direct suture of the sciatic nerve was performed at point 0. Trained animals were exercised from 3 to 14 dpi. Animals were followed up for 14 days for immunohistochemical analysis or for 75 days for long-term electrophysiological assessment. Noradrenergic Depletion Immunohistochemical Analysis of Locus DSP-4 (Sigma Aldrich) was dissolved in sterile NaCl 0.9% saline Coeruleus and Spinal Cord and delivered as a single i.p. dose of 50 mg/kg according to Fourteen days after sciatic nerve injury, deeply anesthetized the work of Lyons et al. (1989) within 10 min of preparation. animals were transcardially perfused with 4% paraformaldehyde DSP-4 was administrated 4 days before the injury to ensure LC in PBS. Brain and L3–L6 spinal cord segment were removed, destruction at the beginning of the treadmill protocol. Control post-fixed for 4 h, cryoprotected in 30% sucrose, and stored animals received a single i.p. injection of saline solution. The at 4◦ C until use. To localize the LC in the brain, we used an doses of DSP-4 used were devoid of adverse effects, and therefore acrylic array of coronal brain matrix (Acrylic brain matrices rats did not require special care during follow up. Alto, for small rat coronal, 175–300 gr). Both brain and spinal cord samples were embedded in Tissue-Tek, serially cut (25 and Treadmill Training Protocol 20 µm thickness, respectively) with a cryostat, and collected To acclimatize the animals subject to training exercise in a motor- onto gelatin-coated glass slides. All sections were first blocked driven rodent treadmill (Treadmill LE 8706, LETICA, Spain), with 10% normal bovine serum for 1 h, followed by overnight they were placed on the treadmill for 60 min twice a week prior incubation at 4◦ C with anti-tyrosine hydroxylase (1:500, Sigma) to surgery. During these training sessions, previous to surgery, for the brain sections or combinations of primary antibodies shock grid intensity was set at 0.4 mA to provide a mild negative for the spinal cord sections: rabbit anti Synaptophysin (1:200, stimulus. The TR protocol was started 3 days after surgery and Covance), guinea pig anti VGlut1 (1:300, Millipore), guinea was carried out during 2 weeks. TR consisted of one session of pig anti VGat (1:200, Synaptic Systems), Lectin from Wisteria TR 5 days/week with duration and intensity being progressively Floribunda (1:100, Sigma), mouse anti GFAP (1:1000, Millipore), increased; running started at a locomotion speed of 10 cm/s that rabbit anti Iba1 (1:500, Wako). After washes, immunoreactive was increased 2 cm/s every 5 min, until a maximum speed of sites were revealed by species-specific secondary antibodies 30 cm/s for 60 min was reached during the final training session conjugated to 488 Alexa Fluor (1:200, Invitrogen), 538 Alexa (Cobianchi et al., 2013). Fluor (1:500, Invitrogen), Cy3 (1:200, Millipore), or Streptavidin 488 Alexa Fluor (1:200, Invitrogen). After 2 h incubation at room temperature, the sections were thoroughly washed, mounted on TABLE 1 | Description of the different experimental groups used in this study, the slides, and coverslipped with Fluoromount-G (SouthernBiotech). type of follow-up and the number of animals used. Back-labeled motoneurons were localized and images captured Group Follow-up Study with a scanning confocal microscope (LSM 700 Axio Observer, Carl Zeiss 40×/1,3 Oil DIC M27). On the brain sections, LC (1) Control group (AX) Short-term Changes around soma (n = 4) was localized and images captured with an epifluorescence Long-term EMG (n = 5) microscope (Nikon eclipse Ni DS-Ri2, 40×). (2) DSP-4 Short-term Changes around soma (n = 4) Image analysis, processing and regression analysis from administration group Long-term EMG (n = 5) (DSP-4) motoneurons labeling quantification were performed by means (3) DSP-4 + Treadmill Short-term Changes around soma (n = 4) of in-house software implemented in MATLAB R2012b (The running (DSP-4+TR) Long-term EMG (n = 5) Mathworks Inc., Natick, MA, United States). Firstly, back-labeled (4) Treadmill running Short-term Changes around soma (n = 4) motoneurons were automatically selected and delineated and a (TR) Long-term EMG (n = 5) constant threshold was used to segment and obtain an estimated Frontiers in Cellular Neuroscience | www.frontiersin.org 3 February 2019 | Volume 13 | Article 65 Arbat-Plana et al. Training Axotomized Motoneurons Without Locus-Coeruleus average density for each labeling. As a control for the background, RESULTS the software checked for each analyzed neuron that the soma had no labeling for the set threshold. Immunoreactivity was evaluated DSP-4 was administrated to destroy the LC and thus, to evaluate in a perimeter of 5 µm thickness surrounding the motoneuron the effects of this center on the spinal changes mediated by soma (Arbat-Plana et al., 2015). Since some variability between TR during 14 days following PNI. Firstly, we explored the state animals, samples and processing can occur, similar amount of of LC after DSP-4 administration using Tyrosine Hydroxylase motoneurons from both sides were analyzed in each slide. For antibody (TH), which is a useful marker for dopaminergic and each animal, 10 to 15 motoneurons of each pool and each noradrenergic neurons. To localize the LC we used sections 9.16 side were analyzed. to 10.04 mm caudal to Bregma, following the Rat Brain Atlas in stereotaxic coordinates (Paxinos and Watson, 1998). In control animals, a group of TH+ neurons organized in Electrophysiology Tests a typical triangular shape was easily localized in each side of For the long term follow-up, motor reinnervation and H the 4th ventricle (Figure 2A). In contrast, in DSP-4 treated reflex were assessed by means of nerve conduction tests, animals, few TH+ neurons were localized (Figure 2B). In some performed at 30, 45, 60, and 75 days after surgery, using animals, isolated brightly stained neurons were observed in an electromyography apparatus (Synergy Medelec, Viasys the region (Figure 2C). HealthCare). Electrophysiological evaluation was performed under ketamine/xylazine anesthesia. During the test, the rat body temperature was kept constant between 34 and 36◦ C Effects of DSP-4 Administration on by means of a thermostated flat coil. The sciatic nerve was Central Changes After Axotomy stimulated by two needle electrodes percutaneously inserted at Synaptophysin (Syn) immunolabeling, a general marker the sciatic notch, applying single rectangular pulses of 0.1 ms for synapses, showed that the number of synaptic coverage duration up to the voltage required to obtain a maximal evoked of motoneurons was reduced after sciatic nerve injury, response. The compound muscle action potentials (CMAP) whereas TR partially preserved that loss (77 ± 3 Syn/µm2 were recorded from the TA, GM and plantar (PL) muscles and 97 ± 3 Syn/µm2 in AX animals vs. 89 ± 2 Syn/µm2 with microneedle electrodes. Onset latency and amplitude from and 104 ± 4 Syn/µm2 in trained animals, in TA and baseline to the maximal negative peak of the direct M wave and GM, respectively, p < 0.01). No significant differences the reflex H wave were measured. The maximal H/M amplitude were found between animals treated with DSP-4 (83 ± 3 ratio was calculated for each muscle tested. The H wave (the Syn/µm2 in both TA and GM) compared to control electrophysiological equivalent of the stretch reflex) suffers a rate injured animals. DSP-4 administration combined with dependent depression (RDD) that is useful to corroborate that TR induced a marked decrease of synapses labeling the recorded wave is truly the H wave. It is important to note on injured motoneurons compared to AX (63 ± 5 that at early stages of regeneration, characterized by polyphasic Syn/µm2 in TA and 71 ± 10 Syn/µm2 in GM, p < 0.05) CMAPs in the reinnervated muscles, the H wave can be masked. (Figures 3A, 4). Thus, to ensure that the selected wave was the H wave, repeat Excitatory synapses were analyzed using VGlut1, a specific stimulation was applied at increasing frequencies (0.3, 1, 3, 5, marker of proprioceptive afferents from the muscle spindle. 10, 15, 20, and 30 pps) and at the threshold intensity for the H Axotomized TA and GM motoneurons had a density of about wave. The ratio between the amplitude of the last and the first 40 ± 2 per µm2 , less than half than in intact motoneurons. After H wave recorded for the different protocols was calculated, in TR, the loss of excitatory synapses was also attenuated (60 ± 4 order to evaluate the RDD. The different tests were performed VGlut1/µm2 in both TA and GM, p < 0.01). Animals treated also in the contralateral non-injured side to obtain control values with DSP-4 did not show significant differences compared to for each animal. injured control animals (42 ± 5 VGlu1/µm2 ). However, animals treated with DSP-4 subjected to TR showed a significant decrease Statistical Analysis of VGlut1 contacts on motoneurons in both TA and GM pools For the immunohistochemical analysis, quantitative variables compared to AX and TR animals (30 ± 3 VGlut1/µm2 , p < 0.01) were normality assessed by Shapiro-Wilk test (Royston, 1993). (Figures 3B, 4). For variables with a normal distribution one-way ANOVA The reduction of excitatory VGlut1 synapses suffered by and post hoc analysis by Bonferroni test were used to test axotomized motoneurons was accompanied by an increase of the significance of the difference between the lesion side and VGat labeled inhibitory synapses (67 ± 1 VGat/µm2 and 100 ± 3 the contralateral side. For non-normal variables such analysis VGat/µm2 in TA and GM, respectively). TR animals, showed was performed by Kruskall-Wallis test. SPSS 20.0 (SPSS Inc., a lower increase in inhibitory synapses after axotomy (49 ± 2 Chicago, IL, United States) was used for statistical analyses. VGat/µm2 and 85 ± 2 VGat/µm2 in TA and GM, p < 0.01) A nested design ANOVA test was used in order to determine than controls. In this case, no significant differences were found if the variability was due to differences between the different between DSP-4 and DSP-4+TR animals compared to control motoneurons or between animals in each group. The H/M animals (Figures 3C, 4). ratio and RDD data were analyzed by two way ANOVA with Wisteria floribunda immunoreactivity was used to analyze Bonferroni post hoc correction. PNN. Axotomized motoneurons had a reduction in the amount Frontiers in Cellular Neuroscience | www.frontiersin.org 4 February 2019 | Volume 13 | Article 65 Arbat-Plana et al. Training Axotomized Motoneurons Without Locus-Coeruleus FIGURE 2 | Representative images from sections of the brain stem regions where LC is localized in animals non-treated (A) or treated with DSP-4 (B,C) and immunostained against TH. The characteristic triangular shape of the LC nucleus (A) is lost 14 days after DSP-4 treatment. Hardly any TH+ neuron can be seen in the region (arrow, B), being some isolated neurons highly stained (arrow, C). FIGURE 3 | Quantitative analysis of synaptic stripping and excitatory/inhibitory synapses after axotomy in the different groups of animals. Evaluation of Synaptophysin (A), VGlut1 (B), VGat (C), GFAP (D), and Iba1 (E) in TA (white bars) and GM (gray bars) 15 days after sciatic nerve cut and suture in sedentary or trained animals, either treated with DSP-4 or untreated. Horizontal dotted lines indicate the average of motoneuron labeling in the non-injured side of both TA and GM motoneurons. Immunoreactivity for GFAP and Iba1 is so low in intact motoneurons that the horizontal dotted lines are missing in D,E graphs. Synaptophysin, Iba1 and GFAP values followed a normal distribution, whereas Vglut1 followed a non-normal one. Data are expressed as mean ± SEM, ∗ p < 0.05, ∗∗ p < 0.01, vs. AX group. of PNN (52 ± 5 PNN/µm2 in AX group compared to vs. AX group). We observed that PNN immunoreactivity in 150 ± 21 PNN/µm2 in intact rats) that was partially prevented injured motoneurons was lower in rats receiving DSP-4 (35 ± 1 if animals were subjected to exercise (75 ± 4 PNN/µm2 ; p < 0.01 PNN/µm2 and 42 ± 2 PNN/µm2 in TA and GM, respectively) Frontiers in Cellular Neuroscience | www.frontiersin.org 5 February 2019 | Volume 13 | Article 65 Arbat-Plana et al. Training Axotomized Motoneurons Without Locus-Coeruleus FIGURE 4 | Quantitative analysis of perineuronal nets (PNN) 15 days after axotomy in animals subjected to treadmill or left untrained, either treated with DSP-4 or untreated, in both experimental side (A) and contralateral side (B). Horizontal dotted lines indicate the mean of motoneuron labeling in the non-injured side of both TA and GM motoneurons. PNN values followed a non-normal distribution. Data are expressed as mean ± SEM, ∗ p < 0.05, ∗∗ p < 0.01, vs. AX group. than in only injured animals (p < 0.01). DSP-4+TR animals Muscle Reinnervation had also a low density of PNN surrounding motoneurons Electrophysiological studies were performed to evaluate motor (35 ± 5 PNN/µm2 in both TA and GM). Interestingly, in DSP- reinnervation during 75 days after sciatic nerve injury. The M 4 group, GM motoneurons at the contralateral side showed a wave values of the muscles at the contralateral side were similar in reduced PNN density (172 ± 8 PNN/µm2 ) compared to those all animals. At the injured side, all the groups showed an increase of AX animals (182 ± 6 PNN/µm2 ), whereas this reduction in the amplitude of the M wave of the muscles tested along time, was not observed in PNN from TA motoneurons (145 ± 18 but with different progression. PNN/µm2 in DSP-4 group and 139 ± 2 PNN/µm2 in AX group) The groups of animals subjected to TR had lower M wave (Figures 4, 5). amplitude of the GM muscle at 45 and 60 days than the untreated Astroglial reactivity, estimated by measuring the density group. However, at the end of follow up, the TR group achieved of GFAP immunolabeling surrounding motoneurons, was similar levels of reinnervation than the control, whereas the significantly increased in animals subjected to TR (261 ± 1 DSP-4+TR group remained with lower levels of reinnervation and 263 ± 1 GFAP/µm2 in TA and GM, respectively) in both GM and TA muscles (Figures 6A,B). GM from DSP-4 compared to the control injured group (5 ± 1 and 6 ± 1 group followed a similar progression of reinnervation than GM GFAP/µm2 , respectively, p < 0.001). Astroglial reactivity was from control group. In contrast, at the end of follow-up, TA also increased in both groups of animals treated with DSP- reinnervation in DSP-4 group was significantly higher than in AX 4 (around 271 ± 3 GFAP/µm2 for both muscles) similarly group. Reinnervation of the PL muscles started in AX and DSP-4 to TR only animals. Thus, administration of DSP-4 did not groups at 45 dpi, and at 60 dpi in TR group. In contrast, in the affect the modulation of astroglial reactivity induced by exercise DSP-4+TR group, we did not record M waves in the PL muscles (Figures 3, 5). at the end of follow up (data not shown), further indicating that Iba1 immunostaining around axotomized motoneurons was motor regeneration was hampered by the combination of both used to evaluate microglial reactivity. Fourteen days after treatments (Figures 6A,B). nerve injury, there was a marked increase of microglia Regarding the recovery of the H wave, there was an increase labeling in control rats (76 ± 4 and 84 ± 5 Iba1/µm2 in of the maximal H/M ratio after sciatic nerve injury, indicative TA and GM, respectively). DSP-4 administration significantly of hyperreflexia, that followed a similar course to attain close to increased microglial reactivity (316 ± 4 and 324 ± 5 normal values at 75 days, without significant differences between Iba1/µm2 in TA and GM, respectively, Figure 3) that also groups (Figures 6C,D). In the injured side, the RDD rate was presented a phagocytic phenotype (Figure 5). Exercise drastically increased at all time points but again no differences between reduced microglia reactivity around injured motoneurons groups were observed. (49 ± 1 and 51 ± 1 Iba1/µm2 in TA and GM, respectively), and similar low activation of microglia was observed in animals treated with DSP-4 and also subjected to exercise DISCUSSION (51 ± 1 Iba1/µm2 for both muscles), thus indicating that the modulation of microglia by exercise was not affected The results of this study corroborate that a high intensity with the loss of LC. protocol of TR applied during 2 weeks after sciatic nerve Frontiers in Cellular Neuroscience | www.frontiersin.org 6 February 2019 | Volume 13 | Article 65 Arbat-Plana et al. Training Axotomized Motoneurons Without Locus-Coeruleus FIGURE 5 | Evaluation of excitatory and inhibitory synapses, PNN, astroglia, and microglia reactivity surrounding axotomized motoneurons. All immunostainings were evaluated in confocal images of spinal cord regions containing back-labeled motoneurons (blue) of TA and GM muscles 15 days after nerve cut repaired with direct suture in the different groups of animals. injury was able to attenuate synaptic stripping and PNN loss intensity of exercise to prevent central changes after PNI in around axotomized motoneurons, as we had shown previously female rats only. (Arbat-Plana et al., 2015). In this work we aimed to analyze The LC is strongly activated in stressful situations and if the noradrenergic neurons of the LC play a role in these besides its key role in arousal (Berridge and Waterhouse, 2003), effects. Since a previous work pointed different responses it also plays an important role in modulating excitability of to exercise in male and female mice (Liu et al., 2015), spinal motoneurons (Heckman et al., 2003) and nociceptive and we had previously evaluated the effects of two protocol transmission (Millan, 2002; Pertovaara, 2006). It has recently of TR in female rats, here we used the more successful demonstrated that contrasting modulation of pain-related Frontiers in Cellular Neuroscience | www.frontiersin.org 7 February 2019 | Volume 13 | Article 65 Arbat-Plana et al. Training Axotomized Motoneurons Without Locus-Coeruleus FIGURE 6 | Plots of the amplitude of the CMAPs (A,B) and of the H/M ratio (ratio between the amplitude of the H reflex wave and the amplitude of the direct M wave after electrical stimulation of the nerve) (C,D) from the electrophysiological tests performed in the different experimental groups. Results recorded in GM (A,C) and TA (B,D) muscles at 30, 45, 60, and 75 dpi. Horizontal dotted lines indicate the average of CMAP or H/M ratio in the non-injured side of both TA and GM muscles. Data are expressed as mean ± SEM, ∗ p < 0.05, ∗∗ p < 0.01, ∗ AX vs. DSP-4+TR; # AX vs. TR; & AX vs. DSP-4. behaviors is mediated with distinct noradrenergic neuronal marked synaptic stripping and larger loss of VGlut1 synaptic populations, being the spinal-projections from the LC the ones contacts than injured control rats. Loss of noradrenergic related with analgesia (Hirschberg et al., 2017). Therefore, we projections from the brain stem also blocked the ability of disrupted the LC by administration of DSP-4 before performing TR exercise to revert the reduction in PNN. These results a sciatic nerve injury in rats. DSP-4 is a neurotoxin that support the hypothesis that the increasing intensity TR protocol quite specifically destroys noradrenergic neurons of the LC, promotes activation of the LC, which play a role on the whereas respects similar neurons of other brain stem regions maintenance of synapses in axotomized motoneurons. As stated (Ross and Stenfors, 2015). before, ascending projections from the LC influence arousal and Destruction of LC did not affect synaptic stripping on attention (Berridge and Waterhouse, 2003) and optimize also axotomized spinal motoneurons. In contrast, when these animals task-performance (Aston-jones and Cohen, 2005). Therefore, were subjected to TR, the preventing effects of exercise on loss of these ascending projections could indirectly affect synaptic stripping were lost. In fact, they even showed more spinal motoneurons. However, since descending neurons project Frontiers in Cellular Neuroscience | www.frontiersin.org 8 February 2019 | Volume 13 | Article 65 Arbat-Plana et al. Training Axotomized Motoneurons Without Locus-Coeruleus directly onto the spinal cord, its logical to assume that are from other sources could compensate the loss of noradrenergic the descending neurons the ones mediating most of the effects inputs from LC and thus, modulate this inflammatory response. we observed on DSP-4 treated animals. In fact, the LC is an Recent works have shown that the inflammatory response important source of descending noradrenergic projections to the is important to switch the peripheral sensory neurons from spinal cord (Grzanna and Fritschy, 1991), and thus its destruction a neurotransmitter state to a pro-regenerative state (Liberto would reduce the noradrenergic inputs that spinal motoneurons et al., 2004; Frey et al., 2008; Niemi et al., 2013). Similarly, receive. Nevertheless, other brainstem noradrenergic nuclei, like reactive microglia might influence the regenerative program in A5 and A7, also send projections to the spinal cord. Since DSP- axotomized motoneurons. Since noradrenaline is a known anti- 4 is a specific neurotoxin for LC neurons (Ross and Stenfors, inflammatory (Heneka et al., 2010), its reduction in the spinal 2015), we did not completely abolish all the NA projections that cord due to LC ablation, by increasing microglia reactivity around motoneurons receive and, therefore, stronger effects could be motoneurons might facilitate faster axonal regeneration and expected if all the spinal sources of NA had been ablated. thus, explain the increased motor reinnervation observed in the It is important to take into account that descending DSP-4 group. On the contrary, TR exercise by itself attenuated noradrenergic projections have a modulatory effect on the the inflammatory response and delayed muscle reinnervation, excitability of motoneurons, facilitating their activation when as suggested by the lower amplitude of CMAPs observed in receiving other synaptic inputs (Heckman et al., 2008). exercised animals during the first 8 weeks. Indeed, the effects of Noradrenaline acts by facilitating persistent inward currents exercise on regeneration and muscle reinnervation are variable, (PICs) in the dendrites (Hultborn et al., 2004; Perrier and depending upon intensity and duration of the protocol, and the Delgado-Lezama, 2005; Heckman et al., 2008, 2003). When period during which it is applied after the injury (Udina et al., activated, PICs amplify ionotropic synaptic inputs and are 2011b; Gordon and English, 2015). It has been hypothesized that essential for normal repetitive firing (Lee and Heckman, 1999; moderate exercise training enhances functional sensorimotor Heckman et al., 2003), and thus for the production and recovery, whereas forced intense exercise, such as the increasing facilitation of movement. Therefore, the loss of noradrenergic intensity TR protocol applied here, may have a detrimental effect. projections can reduce the effects of other synaptic inputs on In our study we also observed that at later stages, several weeks motoneurons and limit the effects of increased activity induced after stopping TR training, reinnervation improved. However, by exercise. It is interesting to note that loss of noradrenergic in exercised animals treated with DSP-4 this late increase of projections also had effects on PNN of uninjured motoneurons, reinnervation was not observed, indicating that LC noradrenergic suggesting a widespread effect. After injury, animals that are pathways are somehow mediating the effects of exercise on not forced to run probably decrease their motor activity and nerve regeneration. thus, the loss of descendent noradrenergic projections does not It is interesting to note that that low intensity TR protocols, potentiate the effects observed after the injury in motoneurons. although being more effective promoting nerve regeneration In contrast, animals forced to run in a treadmill strongly and muscle reinnervation (Udina et al., 2011a), have limited activate these projections. It seems that in these situation, effects on the preservation of synapses and PNN on axotomized the lack of noradrenergic modulatory inputs in axotomized motoneurons (Arbat-Plana et al., 2015), thus indicating that the motoneurons is detrimental on the maintenance of the synaptic protocol that facilitates regeneration differs from the one that arbor and the PNN. favors maintenance of spinal circuitry. As we have already shown (Arbat-Plana et al., 2015), TR also Since the lack of recovery of the functional stretch reflex can affects the contralateral side, increasing the thickness of PNN be due to the permanent loss of proprioceptive afferent/VGlut1 around intact motoneurons, but it is unlikely that this increase synapses onto injured motoneurons even when they reinnervate has any relevant physiological effect in a context where plasticity the muscle (Alvarez et al., 2010; Rotterman et al., 2014) we also is not triggered. In contrast, after a PNI, that lead to changes evaluated the state of the stretch reflex circuit by means of its in the spinal circuitry, preservation of PNN could attenuate electrophysiological equivalent, the H wave. The H/M amplitude maladaptive plasticity and disorganization of the synaptic arbor ratio has been extensively used in the literature to measure spinal of axotomized motoneurons, facilitating functional recovery. excitability. When the spinal reflex response is facilitated, the Besides its ability to increase activity of the injured circuits H/M ratio increases (Burke et al., 1999). At early stages of muscle and to preserve synapses and PNN in spinal motoneurons, TR reinnervation there is an electrophysiological hyperexcitability exercise also modulates the inflammatory response observed in and the H/M ratio increases; as muscle reinnervation is the spinal cord after PNI. Interestingly, noradrenaline has anti- consolidated, it returns to normal values (Valero-Cabré and inflammatory effects in the periphery (Kohm and Sanders, 1999; Navarro, 2001). In our study we observed the same changes along Sanders and Straub, 2002). Loss of the LC increased microglia time, without differences between experimental groups. However, reactivity around axotomized motoneurons in DSP-4 treated it is argued that the electrophysiological recovery of the H reflex animals compared to controls. In contrast, animals forced to run after PNI does not guarantee the functionality of the stretch reflex in the treadmill downregulated microglia reactivity (Cobianchi (Cope et al., 1994; Haftel et al., 2005; Bullinger et al., 2011). et al., 2013). However, this anti-inflammatory effect of exercise In fact, we previously found that a low intensity protocol of at the spinal level seems independent of the LC noradrenergic TR (Vivó et al., 2008; Asensio-Pinilla et al., 2009; Udina et al., system, since similar attenuation of microglia reactivity was 2011a) attenuated such hyperexcitability by means of a decreased observed in exercised animals after its depletion. Of course, NA H/M ratio. However, this protocol was less effective preserving Frontiers in Cellular Neuroscience | www.frontiersin.org 9 February 2019 | Volume 13 | Article 65 Arbat-Plana et al. Training Axotomized Motoneurons Without Locus-Coeruleus proprioceptive synapses on axotomized motoneurons than AUTHOR CONTRIBUTIONS the high intensity TR protocol used here (Arbat-Plana et al., 2015). Therefore, evaluation of the functionality of AA-P contributed to the design of the experiments, performed stretch reflex is needed in order to fully understand the all the experiments, analyzed the data, and wrote the manuscript. functional relevance of proprioceptive synaptic preservation by The work is part of her Ph.D. thesis dissertation. MP performed exercise after PNI. some of the experiments and collaborated in the writing of the In conclusion, LC seems to play a complex role on manuscript. XN contributed to the design of the experiments the effects of exercise on injured motoneurons. This center and the writing of the manuscript. EU designed the experiments, mediates the ability of physical exercise to activate motoneurons, performed some of the experiments, analyzed the data, and and contribute to the maintenance of the normal synaptic wrote the manuscript. inputs but does not participate in the anti-inflammatory effect of TR at the spinal level. Other sources of NA or either other monoamines like serotonin (Lopez-Alvarez et al., FUNDING 2018) can also contribute to the effects of TR on the spinal cord. Interestingly, the sole elimination of LC in injured This study was supported by project grants TV3-201736 animals not subjected to exercise markedly increased microglia from the Fundació la Marató de TV3, PI11/00464, TERCEL reactivity and improved early muscle reinnervation. In contrast, (RD12/0019/0011) and CIBERNED (CB06/05/1105) funds from TR exercise, by reducing microglia reactivity, would delay the Instituto de Salud Carlos III of Spain, the European motor regeneration and muscle reinnervation. However, this Community’s Seventh Framework Programme (FP7-HEALTH- reduced microglia reactivity mediates other positive effects, 2011) under grant agreement n◦ 278612 (BIOHYBRID), and such as attenuation of synaptic stripping and neuropathic FEDER funds. This work is part of the Ph.D. thesis dissertation of pain (Cobianchi et al., 2010; López-Álvarez et al., 2015). AA-P (Arbat-Plana, 2016), who had a four-year fellowship from Therefore, it seems that there is a delicate equilibrium between the Universitat Autonoma de Barcelona as a Ph.D. student. modulation of maladaptive plasticity and enhancement of regeneration. At this stage of research, exercise of moderate intensity seems more adequate than intensive protocols that ACKNOWLEDGMENTS can interfere with axonal regeneration. Use of combined protocols could also compensate the limitations of single We are grateful for the technical help of Jessica Jaramillo and therapeutic approaches. Mónica Espejo. REFERENCES processes. Brain Res. 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Exercise training improves functional recovery and motor nerve Copyright © 2019 Arbat-Plana, Puigdomenech, Navarro and Udina. This is an open- conduction after sciatic nerve crush lesion in tha rat. Arch. Phys. Med. Rehabil. access article distributed under the terms of the Creative Commons Attribution 78, 70–77. doi: 10.1016/S0003-9993(97)90013-7 License (CC BY). The use, distribution or reproduction in other forums is permitted, Vivó, M., Puigdemasa, A., Casals, L., Asensio, E., Udina, E., and Navarro, X. (2008). provided the original author(s) and the copyright owner(s) are credited and that the Immediate electrical stimulation enhances regeneration and reinnervation and original publication in this journal is cited, in accordance with accepted academic modulates spinal plastic changes after sciatic nerve injury and repair. Exp. practice. No use, distribution or reproduction is permitted which does not comply Neurol. 211, 180–93. doi: 10.1016/j.expneurol.2008.01.020 with these terms. Frontiers in Cellular Neuroscience | www.frontiersin.org 12 February 2019 | Volume 13 | Article 65