Research Article: New Research Sensory and Motor Systems Massive Loss of Proprioceptive Ia Synapses in Rat Spinal Motoneurons after Nerve Crush Injuries in the Postnatal Period Ariadna Arbat-Plana,1,2,3,p Sara Bolívar,1,2,3,p Xavier Navarro,1,2 Esther Udina,1,2,† and Francisco J. Alvarez3,† https://doi.org/10.1523/ENEURO.0436-22.2023 1 Department of Cell Biology, Physiology and Immunology, Institute of Neurosciences, Universitat Autònoma de Barcelona, 08028 Barcelona, Spain, 2Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), 08193 Bellaterra, Spain, and 3Department of Physiology, Emory University, Atlanta, Georgia 30322 Visual Abstract Peripheral nerve injuries (PNIs) induce the retraction from the ventral horn of the synaptic collaterals of Ia affer- ents injured in the nerve, effectively removing Ia synapses from a-motoneurons. The loss of Ia input impairs functional recovery and could explain, in part, better recovery after PNIs with better Ia synaptic preservation. Synaptic losses correlate with injury severity, speed, and efficiency of muscle reinnervation and requires ven- tral microglia activation. It is unknown whether this plasticity is age dependent. In neonates, axotomized moto- neurons and sensory neurons undergo apoptosis, but after postnatal day 10 most survive. The goal of this study was to analyze vesicular glutamate transporter 1 (VGluT1)-labeled Ia synapses (which also include II af- ferents) after nerve crush in 10 day old rats, a PNI causing little Ia/II synapse loss in adult rats. We confirmed Significance Statement Synaptic plasticity in spinal motor circuits induced by nerve injuries can be rather permanent and hinder motor and sensory function recovery, even when experimental manipulations are designed for fast and effi- cient regeneration and target innervation. One input particularly affected is the Ia afferent synapse on a-mo- toneurons. The degree of loss of this input depends on injury severity and correlates with varying efficiencies while reinnervating peripheral targets. Here we demonstrate that the loss is also age dependent, being higher in the young. The enhanced synaptic pruning capabilities of microglia during critical windows of high synaptic plasticity at an early age might contribute to augment synaptic circuit reorganizations. February 2023, 10(2) ENEURO.0436-22.2023 1–19 Research Article: New Research 2 of 19 fast and efficient reinnervation of leg muscles; however, a massive number of VGluT1/Ia/II synapses were per- manently lost. This synapse loss was similar to that after more severe nerve injuries involving full transection in adults. In adults, disappearance of ventrally directed Ia/II collaterals targeting a-motoneurons was associated with a prolonged microglia reaction and a CCR2 mechanism that included infiltration of CCR2 blood immune cells. By contrast, microgliosis after P10 injuries was fast, resolved in about a week, and there was no evi- dence of peripheral immune cell infiltration. We conclude that VGluT1/Ia/II synapse loss in young animals dif- fers in mechanism, perhaps associated with higher microglia synaptic pruning activity at this age and results in larger losses after milder nerve injuries. Key words: Ia afferent; microglia; motoneuron; neonates; nerve injury; plasticity Introduction long-term motor function: muscle tension recovers to half Outcomes after nerve injury depend on age and injury se- of normal, with fast muscles more affected than slow verity. In adults, injured sensory and motor axons regenerate muscles (Lowrie et al., 1987). Reduced functional recovery and reinnervate their targets (Gordon, 2016), but while func- in the young despite efficient and precise muscle reinnerva- tional recovery after nerve crush is normal, recovery is poor tion (Nguyen et al., 2002) needs an explanation. The second after nerve transection (Navarro et al., 1994; Bodine-Fowler postnatal week in the rat coincides with a critical window of et al., 1997; Lundborg, 2000; Valero-Cabré and Navarro, synaptic plasticity underlying the maturation of weight-bear- 2001; Bontioti et al., 2003; Gordon, 2020). This is partly ex- ing motor function (Altman and Sudarshan, 1975; Westerga plained by connectivity changes after regeneration following and Gramsbergen, 1990; Greensmith and Vrbová, 1992) nerve transections. Peripherally, axon misdirection de- and corresponds to the development of posture and limb grades muscle specificity after nerve transection, but not coordination in 8- to 15-month-old human infants (Altman after crush (Brushart and Mesulam, 1980; Molander and and Bayer, 2001). One possibility is that the developmental Aldskogius, 1992; Madison et al., 1996; Valero-Cabré et al., synaptic plasticity occurring during this period is disrupted 2004; Gordon et al., 2009; Madison and Robinson, 2014; by nerve crush inducing circuit alterations that do not occur Bolívar et al., 2020). Additionally, nerve transections induce in more mature synaptic circuits. long-term changes in central synaptic circuits that contrib- One input that undergoes significant changes after ute to dysfunction (Navarro et al., 2007; Alvarez et al., 2020). nerve injury in adults is that from Ia proprioceptors injured Outcomes are different in young animals. Nerve injuries in the nerve (Alvarez et al., 2010, 2011; Bullinger et al., of any type induce loss of motoneurons (MNs) and sen- 2011; Rotterman et al., 2014). Ia afferents innervate mus- sory neurons in rodents of postnatal day 1 (P1) to P5 cle spindles and inform about muscle length dynamics (Lowrie and Vrbová, 1992; Hart et al., 2008). Motoneuron (Matthews, 1981). Ia afferents synapse on a-motoneur- loss is, however, absent when injuries occur after P7, ons, mediating the stretch reflex, avoiding g -motoneur- although possible sensory neuron loss still occurs up to ons (Shneider et al., 2009). Irreversible loss of Ia afferent the second postnatal week (Kemp et al., 2015). Despite synaptic collaterals in the ventral horn after adult nerve trans- no or little cell loss, nerve crush at P11 or P12 impairs ections abolishes the reflex and contributes to permanent motor dysfunction because diminished feedback information about muscles reaches a-motoneurons and spinal circuits Received October 25, 2022; accepted January 26, 2023; First published coordinating motor function (Cope and Clark, 1993; Cope et February 2, 2023. The authors declare no competing financial interests. al., 1994; Abelew et al., 2000; Haftel et al., 2005; Maas et al., Author contributions: A.-A.P., S.B., E.U., and F.J.A. designed research; 2007; Bullinger et al., 2011; Sabatier et al., 2011a,b; A.-A.P., S.B., X.N., E.U., and F.J.A. performed research; A.-A.P., S.B., X.N., Horstman et al., 2019). In adults, Ia synapse losses are E.U., and F.J.A. analyzed data; A.-A.P., S.B., E.U., and F.J.A. wrote the paper. graded to injury severity with nerve transections caus- This research was supported by National Institutes of Health/National Institute of Neurological Disorders and Stroke Grant R01-NS111969 to F.J.A.; Ministerio ing large losses (Alvarez et al., 2011; Rotterman et al., de Ciencia, Innovación y Universidades of Spain Grants RTI2018-096386-B-I00 2014) and crush injuries no loss (Schultz et al., 2017), although and SAF2017-84464-R to E.U.; Fondo Personal Universitarion Spain Fellowship Ia afferents are similarly axotomized. Correspondingly, Ia mod- FPU17/03657 to S.B.; and Centro de Investigación Biomédica en Red sobre ulation of a-motoneurons and the stretch reflex are preserved Enfermedades Neurodegenerativas Grant CB06/05/1105 and The Cell Therapy Network Grant RD16/0011/0014 from the Instituto de Salud Carlos III of Spain, after nerve crush in adults (Barker and Young, 1947; Prather et cofunded by the European Union (European Regional Development Fund/ al., 2011). The fate of Ia synapses after nerve crush in younger European Social Fund, “Investing in your future”) to E.U. and X.N. animals is unknown. Acknowledgment: We thank Alicia Lane, Dr. Travis Rotterman, and Indeara Loss of Ia synapses after nerve transection depends on a Cogdell for technical help during the performance of these studies. spinal cord neuroinflammatory reaction that varies with in- *A.A.-P. and S.B. contributed equally to this work. † E.U. and F.J.A. are co-senior authors. jury severity (Rotterman et al., 2019). Nerve injuries causing Correspondence should be addressed to Francisco J. Alvarez at Francisco. maximal Ia afferent synapse plasticity associate with longer j.alvarez@emory.edu. microglia activation, CCR2 activation, and infiltration of https://doi.org/10.1523/ENEURO.0436-22.2023 CCR2-expressing immune cells. Adult microglia are ac- Copyright © 2023 Arbat-Plana et al. tivated from a basal state devoted to synapse and neu- This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license, which permits unrestricted use, ron maintenance; however, postnatal microglia already distribution and reproduction in any medium provided that the original work is actively express genes associated with synapse and properly attributed. axon pruning (Matcovitch-Natan et al., 2016; Hammond February 2023, 10(2) ENEURO.0436-22.2023 eNeuro.org Research Article: New Research 3 of 19 et al., 2019, 2021). Thus, nerve injury might influence Table 1: Experimental design: groups and follow-up time microglia differently, depending on age. Indeed, dorsal Study Follow-up horn microglia activation after “spared nerve injuries” lead- Group 1. Electrophysiology/analysis of end 7 dpi (–/n = 3) ing to hyperalgesia, differs among neonatal, young, and plates and microglia in the spinal cord in 14 dpi (n = 18/3) adult rats (Vega-Avelaira et al., 2007). Similar age-related injured animals 21 dpi (n = 12/3) differences in ventral microglia could translate into more 60 dpi (n = 5/2) severe loss of Ia synapses after crush injuries in infants, a Group 2. VGluT1 along cell body and den- 7 dpi (n = 4/4) period of significant Ia afferent synapse pruning by micro- drite analyses (injured/naive) 14 dpi (n = 4/4) glia using complement mechanisms (Vukojicic et al., 2019). 60 dpi (n = 5/5) Group 3. Percentage of proprioceptive neu- 14 dpi (n = 10) We therefore analyzed Ia–motoneuron synapses and mi- rons in L4 DRG in injured animals croglia after nerve crush at P10 in rats. The results show Group 4. Analysis of microglia in the spinal 3 dpi (n = 3) larger synaptic losses than in adults and extensive micro- cord in injured animals 5 dpi (n = 3) glia activation, but no infiltration of blood-derived immune Animals were divided into four main experimental groups. In all of them except cells. Ia synapse permanent loss after nerve crush in young the naive group, the sciatic nerve was crushed. Group 1: electrophysiology rats might thus result from augmented microglia develop- study. A group of animals was followed up for 60 dpi to study CMAPs. To evalu- mental synaptic pruning mechanisms. ate end plate reinnervation, 3 animals were killed for each time point. Group 2: analyses of VGluT1 distribution along dendrites animals were divided based on follow-up times of 7, 14, and 60 dpi. A subgroup of animals was used as a naive Materials and Methods group (see text in results (n = 3)). Group 3: analysis of the percentage of proprio- ceptive neurons in L4 DRGs 2 weeks after injury. Contralateral DRGs were used as controls. Group 4: analysis of microglia reactivity in the spinal cord. Animals Experimental animals were divided based on follow-up times: 3, 5, 7, 14, and 21 dpi. For follow-ups at All experiments were conducted in postnatal Sprague 7, 14, and 21 dpi, spinal cords from animals of group 1 were used. Dawley rats of both sexes (n = 30) housed with their moth- ers until 21 d old, with free access to food and water at were studied at 3, 5, 7, 14, 21, and 60 d postsurgery, de- room temperature (22 6 2°C) under a 12 h light/dark pending on experiments (Table 1). cycle. Tracer injections, when necessary, were performed before P10, and all nerve surgeries were performed at P10 (animal weight, 40–55 g). Recordings and tissue har- Electrophysiology tests vesting were performing at different times depending on Motor reinnervation was assessed by measuring maxi- the experimental groups described in Table 1. All proce- mal compound muscle action potentials (CMAPs) at 14, dures were approved by the local institutional ethics com- 21, and 60 d postinjury (dpi). Electrophysiological evalua- mittee at the University of Barcelona and followed the tion was performed under anesthesia (ketamine, 0.9 ml/ guidelines of the European Commission on Animal Care kg; xylazine, 0.5 ml/kg) and keeping body temperature (EU Directive 2010/63/EU) and the National Institutes of constant between 34°C and 36°C by means of a thermostat- Health in the United States, and adhere to Society for controlled flat coil. The sciatic nerve was stimulated percuta- Neuroscience policies. neously with two needle electrodes inserted at the sciatic notch. We applied single rectangular pulses of 0.1 ms dura- tion and increasing intensity until the voltage required to Retrograde labeling obtain a maximal response. CMAPs were recorded from To identify the gastrocnemius (G) motor pool on both the tibialis anterior (TA) and G muscles with microneedle sides of the spinal cord, the G muscles were bilaterally in- electrodes and an electromyography system (Synergy jected with retrograde tracers 2–3 d before P10. Depending Medelec, Viasys HealthCare). All potentials were ampli- on survival times, we used a short-term tracer [cholera sub- fied, and the amplitude from baseline to maximal nega- unit b coupled to Alexa Fluor 555; CTb-555 (CTb), Thermo tive peak of the direct M wave was recorded for each Fisher Scientific] or a long-term tracer [Fast Blue (FB), animal. Measurements were done bilaterally to compare Polysciences]. Two 1 ml injections of 1% CTb-555 diluted in the injured side to the control side. sterile saline or 2.5% FB diluted in sterile water were injected at different locations into the body of the muscle with glass micropipettes (Sigma-Aldrich). CTb-555-injected animals Tissue harvesting for Histological processing and were analyzed 1 and 2 weeks postinjury, and FB-injected immunohistochemistry animals were used for the 60 d survival time point. At the end of survival periods the animals were deeply anesthetized with intraperitoneal pentobarbital (Dolethal; Nerve surgeries 30 mg/kg) and transcardially perfused with vascular rinse P10 rats were anesthetized by administration of keta- followed by 4% paraformaldehyde in 0.01 M PBS 0.9%. mine (0.3 ml/kg, i.p.; Imalgene 2000, Boehringer Ingelheim) The L4 dorsal root ganglia (DRGs), L3 to L6 spinal cord and xylazine (0.17 ml/kg; Rompun 2%), and prepared for segments, and selected muscles were removed and post- aseptic surgery. The sciatic nerve was exposed at mid- fixed overnight. Tissue samples were cryoprotected and thigh and crushed during 30 s in three different orientations stored in 30% sucrose in PBS until used. with a fine forceps. Afterward, the skin was closed, su- tured, and iodine povidone applied to the wound. Pups re- End plate reinnervation of the G and TA muscles covered in a warm environment under close observation G and TA muscles were quickly frozen in Tissue-Tek and were returned to the cage with the mother. Animals OCT (optimal cutting temperature compound; Sakura), February 2023, 10(2) ENEURO.0436-22.2023 eNeuro.org Research Article: New Research 4 of 19 and cryostat sections (20 mm thick) were collected on Retrogradely labeled motoneurons (CTb-555 or FB) were Fisherbrand Superfrost Plus glass slides (Thermo Fisher localized, and images were captured with a confocal mi- Scientific). Slides were kept at 20°C until used. Labeling croscope (model FV1000, Olympus) using a 10 objective was performed on the slides after bringing them to room for neuron localization and a 60 objective (numerical temperature and washing the OCT with multiple changes aperture 1.35, oil-immersion) to image VGluT1 contacts in PBS. Thereafter, the sections were blocked with 10% along the somatodendritic surface. normal donkey serum diluted in PBS with 0.3% Triton X- Confocal images z-stacks were uploaded to Neurolucida 100 (PBST) for 1 h and incubated overnight at room tem- (version 10.0; MBF Bioscience) to trace the cell bodies and perature in Alexa Fluor 647-conjugated a-bungarotoxin dendrites to their ends within the 50 mm section (most den- (a-btx; 1:100; Thermo Fisher Scientific) and a mixture of drites are cut in these preparations, but they permit enough primary antibodies that included guinea pig anti-vesicular sampling of the proximal dendritic arbor where most acetylcholine transporter (VAChT; 1:200; EMD Millipore; VGluT1 contacts are located). The cell body was re- RRID:AB_11214110) and phosphorylated chicken anti- constructed through a series of contours traced in each neurofilament heavy chain (NFH; 1:200; Aves Labs; RRID: optical plane. Dendrites were traced in 3D. Synaptic lo- AB_2313552). The following day, the slides were washed cations were marked in the traced neurons and VGluT1 in PBS and incubated for 2 h at room temperature with synaptic densities analyzed using NeuroExplorer (ver- donkey anti-guinea pig Ig coupled to Cy3 and donkey anti- sion 10.0, MBF Bioscience). Morphological features in- chicken IgY coupled to fluorescein isothiocyanate (FITC; cluding cell body surface, volume, cell body diameters, 1:200; Jackson ImmunoResearch). After final washes in number of primary dendrites, and total length of individual PBS, the sections were coverslipped with Vectashield and all dendritic segments contained within the section (Vector Laboratories). were measured. Only the most relevant measurements are Sections were imaged in a confocal microscope (model reported. Motoneurons with cross-sectional cell body av- FV1000, Olympus) at high magnification (60; z-step size, erage diameter of ,25 mm were not included in the analy- 1 mm), capturing overlapping image tiles to sample a ses because they could belong to g -motoneurons, which large proportion of the muscles neuromuscular junction lack VGluT1 synapses (Shneider et al., 2009). To obtain (NMJ) fields. Reinnervation of a-btx-labeled neuromus- VGluT1 densities, the number of VGluT1 contacts was di- cular junctions was analyzed in three animals per time vided by estimates of cell body surface, total dendrite sur- point. For each muscle, at least 50 end plates per animal face, total dendrite length, or the surface of individual were imaged at 7, 14, and 21 dpi. At 60 dpi, we captured dendrite segments at different locations from the cell body. 15 a-btx-labeled neuromuscular junctions per animal. The densities of synapses at different distances from the We considered a neuromuscular junction reinnervated cell body were estimated using path distance measure- when VAChT immunofluorescence was present. Full rein- ments or Sholl analyses. The results were similar. We con- nervation was defined as more than 50% a-btx-labeled neu- sidered that path–distance analyses were more rigorous romuscular junction covered by motor end plate VAChT than Sholl analyses because they take into account dendrite immunoreactivity, and partial reinnervation if VAChT immu- tortuosity; thus, the confirmatory Sholl analyses are not in- noreactivity only covered some spots of the a-btx field. The cluded. The traced dendritic arbor was divided into seg- percentage of a-btx neuromuscular junctions partially or ments at path distances 0–50 , 50–100, or 100–150 mm from fully reinnervated were estimated for each muscle and the cell body. We did not analyze segments beyond 150 mm time point. because they had little representation in our sample. This is explained by the limitations of retrograde labeling and den- Analyses of vesicular glutamate transporter 1 drites cut at the plane of section. We analyzed 4–6 a-moto- contacts on retrogradely labeled a-motoneurons neurons (average 6 SD, 5.00 6 0.57) per animal and side Vesicular glutamate transporter 1 (VGluT1) contacts on (contralateral or ipsilateral to the injury) for a total of 130 re- retrogradely labeled G motoneurons were analyzed at 7, constructed a-motoneurons from 13 animals. 15, and 60 dpi in, respectively, four, four, and five animals (Table 1). The contralateral uninjured side was used as a Quantification of proprioceptive sensory neurons in control. To validate the uninjured side as control, it was dorsal root ganglia first compared with motoneurons in three naive animals. In To confirm cellular preservation of proprioceptive sensory all cases, transverse spinal cords were obtained at 50 mm neurons, we counted parvalbumin (PV)-immunoreactive (IR) thickness in a sliding freezing microtome and processed cellular profiles in dorsal root ganglia (DRGs) ipsilateral and for VGluT1 immunofluorescence. The sections were col- contralateral to the nerve crush. We analyzed L4 DRGs from lected free floating, and, after blocking with 10% normal 10 animals with a crush injury of the right sciatic nerve 14 d donkey serum for 1 h, they were incubated overnight in a before. The DRGs were embedded in Tissue-Tek OCT, seri- solution containing rabbit anti-VGluT1 (1:1000; Synaptic ally cut (15 mm thickness) with a cryostat, and collected onto Systems; RRID:AB_887877) diluted in PBST. The following gelatin-coated glass slides. All sections were first blocked day, sections were washed in PBS and incubated for 2 h in with 10% normal bovine serum for 1 h, followed by overnight donkey anti-rabbit IgG antibodies conjugated to FITC incubation at 4°C with primary antibodies, as follows: rabbit (1:100; Jackson ImmunoResearch) in PBST. Slides were anti-parvalbumin (1:1000; catalog #PV28, Swant; RRID:AB_ then washed in PBS, mounted serially on glass slides, 2315235); and mouse biotinylated anti-NeuN (1:200; catalog and coverslipped with Vectashield (Vector Laboratories). #MAB377B, Millipore; RRID:AB_177621). After washes, February 2023, 10(2) ENEURO.0436-22.2023 eNeuro.org Research Article: New Research 5 of 19 donkey anti-rabbit IgG Alexa Fluor 488 (1:200; catalog microscope (model FV1000, Olympus) using 1 mm z-steps #A-21206, Thermo Fisher Scientific; RRID:AB_2535792) and a 20 objective. Overlapping image tiles were cap- and Alexa Fluor 488-streptavidin (1:200; catalog #S32356, tured to sample the whole spinal cord. At least three sec- Thermo Fisher Scientific) were used to reveal IR sites. After tions were imaged for each animal. We found no CD451 2 h of incubation at room temperature, the sections were cells, and therefore quantitative analyses focused on Iba11 thoroughly washed, mounted on slides, and coverslipped microglia. The ventral horn of each hemisection was man- with Fluoromount-G (SouthernBiotech). DRG sections were ually selected by marking a region of interest (ROIs) limited visualized with a fluorescence microscope (model BX51, dorsally by a straight horizontal line above the central canal Olympus); at least four sections per DRG were captured and in all other directions by the border between gray at 40 with a digital camera (model DP50, Olympus) and and white matter. All Iba11 cells inside these ROIs were CellSens Digital Imaging software (version 1.9; Olympus), counted using ImageJ software. Because the spinal cord and were merged using ImageJ software. The number of changes in size with maturation, we calculated cell den- positive parvalbumin and all NeuN neurons were manually sities, as follows: the number of Iba11 microglia divided counted. NeuN was used to ensure that all cellular profiles by the ventral horn volume calculated from the ROI area counted were from mid-cell cross sections that included multiplied by section thickness (50 mm). Densities ratios the nucleus. We then estimated the proportion of NeuN1 were obtained for each section between the side ipsilat- neurons that were parvalbumin1 in DRGs ipsilateral and eral to the injury and the contralateral control side. contralateral to the nerve crush. This procedure normal- ized differences in cell numbers depending on the level Statistical analysis and orientation of L4 DRG section cut. We analyzed four Normality was assessed with the Shapiro–Wilk test, and to five sections per DRG in six control L4 DRGs with an usually the data were compared using one-way or two- average (6SD) of 1560.0 6 398.9 neurons sampled per way ANOVA to test for significant differences in time after ganglia and eight injured L4 DRGs with an average of injury and lesion versus contralateral control side when 1533.6 6 282.4 neurons per ganglia. Ganglia in which appropriate, and any possible interactions. Post hoc pair- sectioning and immunocytochemistry processing did not wise comparisons were performed using Bonferroni-cor- allow recovery of four to five sections with .200 NeuN rected t tests. If normality was not satisfied, we used an neurons per section were not included in the quantitative ANOVA on ranks (with some exceptions indicated in the analysis to avoid errors in percentage estimates because text) and post hoc Dunn’s tests. DRG numbers in control of uneven clustering of parvalbumin-IR neurons in DRG versus injured sides at a single time point were compared or other possible biases in sections with small numbers using a t test. Statistical analyses were performed in of neurons. SigmaPlot (version 12) or Prism (version 9). Correlations between functional (CMAPs) and anatomical reinnervation Quantification of neuroimmune cells were fitted in SPSS 20.0. A value of p , 0.05 was consid- Loss of Ia/VGluT1 synapses requires a microglia reaction ered significant. The power of performed tests was always (Iba11, CD11b1 cells) and activation of CCR2, which is also .0.8 for a = 0.05, unless indicated in text. When power associated with infiltration of CD451 CCR21 CD11b1 periph- was not reached because effect sizes were too small, we eral macrophages and CD451 CCR21 CD3e1 T cells (see performed estimation statistics and a permutation t test to Introduction). The microglia reaction was analyzed by report effect sizes when the null hypothesis of zero differ- counting all Iba11 cells in the ventral horn of the spinal ence is rejected. For each permutation p value, we per- cord at 3 d (n = 3), 5 d (n = 3), 7 d (n = 4), 14 d (n = 4), 21 d formed 5000 bootstrap reshuffles of the control and injured (n = 4), and 60 d (n = 2) after nerve crush injury at P10. To sides, and the data are represented with a Gardner–Altman test for the presence of peripheral immune cells we estimation plot (Ho et al., 2019). Small skews in the distri- tested for Iba11, CD11b1, and/or CD451 cells at 7, 14, bution of mean differences were overcome using a bias- 21, and 60 d after the injury. Transverse sections (50 mm corrected and accelerated bootstrap method. thick) of L4-5 spinal cord were obtained in a sliding freez- ing microtome, collected free floating, blocked for 1 h Results with 10% normal donkey serum diluted in PBST, and in- cubated overnight at room temperature in a mixture con- Functional muscle motor reinnervation after nerve taining a goat polyclonal antibody against Iba1 (1:500; crush injury at P10 Novus Biologicals; RRID:AB_521594) and a rat monoclo- Electromyographic recordings of the G and TA nal antibody against CD11b (1:50; OX-43; catalog #M1/ muscles were performed in the injured and control 70.15.11.5.2-s, Developmental Hybridoma Bank) or a sides of rats with a unilateral sciatic nerve crush at rabbit polyclonal antibody against CD45 (1:50; Abcam; P10. To evaluate the progression of motor reinnerva- RRID:AB_442810). Sections were washed in PBS and in- tion, we examined animals at 14 dpi (n = 18 animals), cubated for 2 h in donkey anti-goat IgG antibody conju- 21 dpi (n = 12), and 60 dpi (n = 5; Fig. 1, Extended Data gated to FITC and donkey anti-rat or anti-rabbit IgG Fig. 1-1). The progressive decrease in sample size with conjugated to Cy3 (all secondary antibodies diluted 1:200 in increased time is explained by a proportion of animals PBST; Jackson ImmunoResearch). The sections were being killed at each time point for tissue harvesting to mounted on slides, coverslipped with Vectashield (Vector examine NMJ reinnervation and/or microglia in the spi- Laboratories), and imaged throughout with a confocal nal cord. No analyses were performed at 7 dpi, since at February 2023, 10(2) ENEURO.0436-22.2023 eNeuro.org Research Article: New Research 6 of 19 Figure 1. Electromyographic testing of muscle responses before and after nerve injury. Results of CMAPs recorded in G and TA muscles at 14, 21, and 60 dpi in the control and injured side. Controls are in light gray and injured animals in medium gray. Data are expressed in ab- solute values as the mean 6 SEM. Each dot represents the average CMAP value in a different animal. The number of animals tested is at the bottom of the bar. A two-way ANOVA for injured versus control and time after injury showed significant differences according to both variables and their interactions (all p , 0.001). Post hoc Bonferroni corrected t tests showed significant differences with development and injury as indicated in the figure: **p , 0.01, ***p , 0.001. n.s., Nonsignificant. Full statistics are shown in Extended Data Figure 1-1. this time no evoked responses were recorded and anatomi- Motor axon reinnervation of neuromuscular junctions cally there was almost no NMJ reinnervation (see below). Functional reinnervation was compared with anatomical The maximal CMAP (or muscle, M response) recorded in reinnervation of G and TA NMJs labeled with Alexa Fluor the G muscle of the intact hindlimb after stimulation of the 555-conjugated a-btx. Motor axons were labeled with anti- ipsilateral uninjured sciatic nerve had mean 6 SEM ampli- bodies against phosphorylated NFH and motor end plates tudes of 40.0 6 6.7 and 42.3 6 3.1 mV at, respectively, P24 with VAChT antibodies (Fig. 2A). Muscles were analyzed at and P31 in age (corresponding to 14 and 21 dpi of the con- 7, 14, 21, and 60 dpi, respectively, in three, three, three, and tralateral injured sciatic nerve), whereas at P70 (60 dpi) it two animals. The time course of partial or full reinnervation was higher (62.0 6 8.1 mV). In the intact TA muscles, the in- was faster in G compared with TA (Fig. 2B). At 7 dpi, some crease in CMAP amplitude was more gradual (from NFH-stained axons were seen close to denervated NMJs, 38.6 6 6.7 mV at P24 to 47.4 6 1.4 mV at P70). CMAPs in but none of the TA NMJs were reinnervated by VAChT (total, the injured side were always lower compared with the con- 230 NMJs) and only 9.8 6 5.4% (6SEM) of NMJs in the G tralateral control side at any time in both muscles. Two-way (total, 144 NMJs) showed some signs of reinnervation where ANOVAs for injury and age showed significant differences VAChT immunofluorescence partially covered the NMJ. according to both variables and their interaction (p , 0.001) Denervated NMJs at this time had ovoid shapes, quite differ- in both G and TA (although normality failed in the Shapiro– ent from the typical “pretzel-like” appearance of intact NMJs Wilk test, we chose to do parametric tests since zeros at or NMJs denervated in mature adult rats. This suggests a the early reinnervation dates lead to the loss of normality in faster structural disorganization of the postsynaptic acetyl- the distribution). Post hoc pairwise Bonferroni’s t tests choline receptor field after denervation at this early age. The (Extended Data Fig. 1-1) showed that at P70, CMAPs in the presence of some VAChT in motor end plates opposite to intact hindlimb G were significantly higher than at P24 or a-btx-labeled NMJs increased significantly in both muscles P31 (p , 0.001). In contrast, CMAP amplitude in the intact at 14 dpi (G: 85.1 6 5.6%, n = 167; TA: 51.6 6 5.3%, n = TA was significantly higher comparing P70 to P24 (p = 0.004), 202), reaching a plateau at 21 dpi (G: 94.9 6 3.5%, n = 157; but not comparing P31 with either. This suggests that G TA: 93.1 6 2.0%, n = 162) and 60 dpi (G: 96.7 6 3.3%, showed a faster developmental increase of CMAP ampli- n = 35; TA: 91.0 6 2.8%, n = 33). “Full” reinnervation of NMJs tudes during the first month compared with TA. was evaluated when VAChT immunofluorescence covered CMAPs were always significantly lower in the injured more than half of the NMJ-labeled receptor field. Fully inner- side compared with age-matched controls in both G and vated end plates increased in both muscles at 14 dpi (G, TA (p , 0.001). In G, CMAP mean amplitude at 14 dpi 45.3 6 14.5%; TA, 19.1 6 7.8%) and 21 dpi (G, 71.3 6 5.3%; (P24) was 10% of control, increasing to 40% of control at TA, 71.3 6 7.0%), and was quite complete at 60 dpi (G, 21 dpi (P31) and 68% at 60 dpi (P70; Extended Data Fig. 93.3 6 3.3%; TA, 91.0 6 2.8%). 1-1). Similar patterns were observed in TA: limited recov- Full NMJ reinnervation at each postinjury date was stat- ery detected at 14 dpi, averaging 6.5% of control ampli- istically compared using one-way ANOVA with average re- tude, increased at 21 dpi to 32% and to 73% at 60 dpi. innervation estimates for each animal. Percentage values Thus, G and TA recovered functional innervation in paral- in animal averages closely parallel those calculated when lel, despite a faster developmental increase in CMAP am- pooling all NMJs from different animals together. As ex- plitudes in G compared with TA. pected, the increase in the proportion of fully reinnervated February 2023, 10(2) ENEURO.0436-22.2023 eNeuro.org Research Article: New Research 7 of 19 Figure 2. Anatomical reinnervation of the end plates. A, Representative images of sections of a G muscle 7, 14, 21, and 60 dpi, immu- nostained against NFH, a-btx, and VAChT, respectively, to label axons, neuromuscular junctions, and the synaptic vesicle-filled motor end plate. Larger numbers of reinnervated NMJs were observed with increased time after injury, with almost complete innervation at 60 dpi. At 7 dpi, there is little innervation by motor end plates of vacated NMJs, although some axons reach some a-btx-labeled NMJs. The nicotinic receptor field expands after early denervation. B, Percentages of reinnervated NMJs with time after injury in TA and G muscles. Average 6 SEM of estimates of NMJ coverage in each animal (for both muscles: n = 3 at 7, 14, and 21 dpi; n = 2 at 60 dpi). Reinnervation indicates NMJs in which NFH axons with or without some VAChT superimposed onto the NMJ are present. Full reinnervation indicates when most of the NMJ area is covered by VAChT immunoreactivity. Statistics were performed on fully reinner- vated percentages averaged from 3 animals at each age and 2 animals at 60 dpi. Error bars indicate the SEM. A one-way ANOVA for time after injury showed a significant change in reinnervation in both TA and G with time after injury (TA, p , 0.0001; G, p = 0.0008; Extended Data Fig. 2-1). Post hoc pairwise comparisons showed significant changes among all ages in TA, except for 7 versus 14 dpi (slow initial reinnervation) and 21 versus 60 dpi (reinnervation plateau). The number of reinnervated NMJs in G was significantly differ- ent at all ages compared with 7 dpi (faster reinnervation), and then it was significant in alternate dates (14 vs 60 dpi) but not consecu- tive dates (suggesting progressive reinnervation). All comparisons are shown in Extended Data Figure 2-1. Asterisks were omitted from the figure for clarity. C, Correlation between the percentage of the muscle (M) response (CMAPs from Fig. 1) and the percentage of NMJs “fully” reinnervated fitted by a simple exponential regression with R2 = 0.843 and was significant at p , 0.001. M response re- covery was delayed with respect to the initiation of anatomic reinnervation of motor end plates. NMJs in TA and G with time after injury was significant (TA, To establish the status of Ia afferent synapses on regener- p , 0.001; G, p , 0.001; Extended Data Fig. 2-1). Post hoc ating a-motoneurons, we analyzed VGluT1 synaptic con- Bonferroni’s t tests demonstrated significant differences in tacts on 3D Neurolucida reconstructions of the cell body alternate dates and sometimes in consecutive dates, sug- and dendrites of lateral G (LG) a-motoneurons retro- gesting progressive increases in innervation (Extended gradely labeled with cholera toxin subunit b coupled to Data Fig. 2-1). Importantly, NMJ reinnervation and CMAP Alexa Fluor 555 (CTb-MNs) or with FB-MNs at 7, 14, and amplitudes were correlated in those animals in which both 60 dpi (Fig. 3A). Synaptic densities were compared with parameters were simultaneously measured (p , 0.001, R2 similarly retrogradely labeled a-motoneurons on the con- = 0.843); however, the relationship was nonlinear (Fig. 2C). tralateral side and used as controls. To validate contralat- Functional recovery was delayed with respect to anatomi- eral a-motoneurons as reliable controls, VGluT1 contacts cal reinnervation and thereafter grew exponentially. At the on the cell body of LG CTb-MNs were compared between study endpoint (P70 at 60 dpi), CMAP amplitudes slightly naive rats at P27 to P17 and P25 LG CTb-MNs contralat- lagged behind toward full recovery despite .90% anatom- eral to nerve injuries (respectively, 7 and 15 dpi). VGluT1 ical reinnervation. density on the cell body of CTb-MNs in P27 naive animals (n = 3 animals) was 0.47 1/– 0.38 VGluT1 contacts per 100 mm2 (6SD; n = 18 CTb-MNs), and on the control side VGluT1 contacts on a-motoneuron cell bodies and of injured animals at P17 (n = 20 CTb-MNs) and P25 dendrites (n = 19 CTb-MNs) was respectively 0.57 6 0.28 and The above studies confirmed efficient muscle reinner- 0.49 6 0.22. Differences were not significant (p = 0.336; vation 2 months after a sciatic nerve crush injury at P10. ANOVA on-ranks, H = 2.184 with 2 df). February 2023, 10(2) ENEURO.0436-22.2023 eNeuro.org Research Article: New Research 8 of 19 Figure 3. VGluT1 synapses on the cell bodies of injured a-motoneurons are significantly reduced compared with controls and are not recovered after muscle reinnervation. A, Representative 2D projection image of a confocal stack (left) and Neurolucida recon- struction (right) of an Alexa Fluor 555 CTb-labeled motoneuron (white in confocal image) and VGluT1 boutons (green in confocal image). In the Neurolucida reconstruction, each dendrite and each optical cell body plane are in a different color. Yellow triangles in- dicate VGluT1 contacts on the cell body. Orange squares are VGluT1 contacts on dendrites. B, Cell body surfaces of all recon- structed a-motoneurons at different ages and different times postinjury. Boxes represent data between the 25th and 75th percentiles with a continuous line within the box marking the median and a dashed line marking the average. Whiskers above and February 2023, 10(2) ENEURO.0436-22.2023 eNeuro.org Research Article: New Research 9 of 19 continued below the box indicate the 90th and 10th percentiles. Dots represent motoneuron surface estimates beyond these distribution boundaries. In brackets is the total sample of motoneurons reconstructed pooled from 4 animals at 7 and 14 dpi and from 5 animals at 60 dpi (4–6 motoneurons reconstructed per animal). Two-way ANOVA for differences according to injury and date showed signifi- cant differences only according to date and no interaction; pairwise comparisons comparing the three dates within controls or in- jured motoneurons showed statistically significant larger cell bodies at P70. ***p , 0.001 from post hoc Bonferroni’s t tests (Extended Data Fig. 3-1). C, VGluT1 contacts per a-motoneuron cell body, by pooling all motoneurons (left) or calculated by animal averages (right). D, VGluT1 density on cell bodies pooling all motoneurons (left) or by animal averages (right). Box plots in C and D represent data the same as in B and show the sample distributions. Per animal data are represented as the mean 6 SEM, with each dot representing the average of each animal (number of animals in each condition/date are indicated in brackets). Two-way ANOVA revealed significant differences in the number of VGluT1 contacts according to time points (age/dpi, p = 0.0012), injury (p , 0.0001), or interaction between time point and injury (p = 0.0318). Post hoc Bonferroni’s corrected t tests for pairwise comparisons are indi- cated in the figure: *p , 0.05, **p , 0.01, ***p , 0.001. They show a significant increase in VGluT1 contact number at P70 in controls, but not injured motoneurons and significant decreases comparing injury and control at each time point (Extended Data Fig. 3-2). However, two-way ANOVA of VGluT1 densities resulted in a significant difference according to injury (p , 0.0001), but not time point (p = 0.792), and no interaction between the two (p = 171). Bonferroni’s corrected t tests of control versus injury at each day postinjury showed significant differences, as represented in the graph, but no difference in density across different time points for either control or injured data (Extended Data Fig. 3-2). The time of the analyses corresponds with a period of between P17 and P24 (Fig. 3C, Extended Data Fig. 3-2). neuronal growth in the spinal cord. To estimate develop- Interestingly, the age increase in VGluT1 contacts matched mental changes in neuronal structure that occur inde- the increase in available cell body surface at P70. Thus, pendent of the injury, we analyzed cell body sizes. Cell two-way ANOVA of VGluT1 densities (number of VGluT1 body total surface estimates of CTb-MNs and FB-MNs contacts per 100 mm2) showed significant differences with (n = 130 motoneurons) did not change from P17 (7 dpi) to injury (p , 0.001), but not with age (p = 0.792), and differen- P25 (15 dpi), but increased in size (48% change) by P70 ces found with injury were not significantly affected by age (60 dpi; Fig. 3B; mean 6 SEM at P70 = 4410.1 6 107.9 (p = 0.171). The percentage decreases in VGluT1 density mm2; n = 52 FB-MNs). We did not detect significant differ- (7 dpi, 72%; 14 dpi, 45%; 60 dpi, 62%) were similar to the ences in cell body size between control and age-matched percentage loss calculated for VGluT1 contacts per cell injured CTb-MNs or FB-MNs at any age. Two-way ANOVA body and post hoc Bonferroni t tests on the averages of for age and injury demonstrated significant differences ac- the animal showed that differences in VGluT1 densities cording to age (p , 0.001), but not according to injury were all consistently significant (at 7 and 60 dpi, p , 0.001; (p = 0.947) or the interaction between age and injury 15 dpi, p = 0.032). The results suggest that VGluT1 synap- (p = 0.761; Extended Data Fig. 3-1). Post hoc Bonferroni’s t ses are permanently removed from the cell bodies of tests showed significant differences at P70 with P17 and a-motoneurons axotomized after crush at P10 and that the P25 (p , 0.001), but not between P17 and P25 (Fig. 3B, normal developmental addition of VGluT1 synapses to Extended Data Fig. 3-1). While control CTb-MNs showed a match the growth in available surface does not occur after range of 2.234 and 1.679 mm2 in surface at P17 and P25, crush. this increased to 3.708 mm2 at P70 in FB-MNs; however, However, the majority of VGluT1 synapses target a-mo- the coefficient of variation remained relatively constant toneuron dendrites, in particular, the proximal dendritic (P17, 5.9; P25, 5.5; P70, 4.9). In conclusion, developmental arbor (Rotterman et al., 2014). VGluT1 depletions on den- cell body growth includes proportional spreading of size drites usually differ in magnitude compared with changes distributions with age (and thus available surface for syn- on cell bodies (Alvarez et al., 2011; Rotterman et al., 2014; aptic contacts), regardless of injury status. Schultz et al., 2017; Rotterman et al., 2019). Thus, we an- The number of VGluT1 contacts was significantly de- alyzed VGluT1 contacts along the retrogradely labeled creased on the cell bodies of injured a-motoneurons dendritic arbors (Fig. 4A). Retrograde labeling preferen- compared with controls at all postinjury ages when pool- tially labels the proximal dendritic tree, and no differences ing a-motoneurons from all animals (Fig. 3C, left). At 7, were found in the longest dendrite path distance to cell 15, and 60 dpi, we found a decrease in VGluT1 contacts body or surface area measured in injured a-motoneurons on the cell body of, respectively, 71%, 42%, and 61%. compared with controls or at different ages (Fig. 4B,C). Similar results were obtained when comparing average The known growth of the dendritic arbor from P15 to P70 densities obtained per animal (Fig. 3C, right, Extended (Kalb, 1994) affects more distal regions, and no differen- Data Fig. 3-2). A two-way ANOVA on animal averages indi- ces were detected in our samples because the limited cated significant differences according to age (p = 0.0012), spread of the retrograde tracer to more proximal dendritic injury (p , 0.001), and their interaction (p = 0.032). Post hoc regions. Since all P70 a-motoneurons were traced with Bonferroni’s t tests on animal averages showed that at FB and all P17 and P25 a-motoneurons with CTb, the every age the decrease in VGluT1 contacts was significant tracer variable was embedded with age. Two-way ANOVA (at 7 and 60 dpi, p , 0.0001; at 25 dpi, p = 0.022), In addi- showed no significant differences according to age/tracer tion, the number of VGluT1 contacts increased with age in (p = 0.756, F(2,129) = 0.281) or injury (p = 0.117, F(2,129) = controls, and this was significant for P70 (60 dpi) compared 2.183). Similarly, there were no statistically significant dif- with younger ages (p , 0.001), but was not significant ferences on the total dendritic surface analyzed in the February 2023, 10(2) ENEURO.0436-22.2023 eNeuro.org Research Article: New Research 10 of 19 Figure 4. Sample properties of dendrite reconstructions. A, Representative 3D Neurolucida reconstructions of control and injured a-motoneurons at each time point. Each dendrite and each optical cell body plane are in a different color. Yellow triangles indicate VGluT1 contacts on the cell body. Orange squares are VGluT1 contacts on dendrites. B, Box plots representing the longest path distance of dendrite from the cell body analyzed for each a-motoneuron. Box: 25th and 75th percentiles with a continuous line with- in the box marking the median and a dashed line marking the average. Whiskers above and below the box indicate the 90th and 10th percentiles. Dots represent data points beyond these distribution boundaries. Light gray boxes are control a-motoneurons, darker gray boxes are injured a-motoneurons. Superimposed white dots represent individual a-motoneuron data. The large majority of motoneurons did not have dendritic segments farther than 200 mm, and within individual motoneurons many dendrites were traced for ,150 mm path distance from the cell body. Two-way ANOVA showed no significant differences according to age/tracer (p = 0.756, F(2,129) = 0.281) or injury (p = 0.117, F(2,129) = 2.183). C, Box plots representing the dendritic surface analyzed for each motoneuron. Despite a few outliers, our a-motoneuron sample did not significantly differ in the amount of dendritic surface analyzed in the different experimental groups (p = 0.169, F(5,129) = 1.584). different experimental groups (p = 0.169, F(5,129) = 1.584). related differences on average surface analyzed in differ- Thus, both retrograde tracers labeled similar proximal re- ent dendritic samples. gions of dendrite independent of age or injury, and by dif- We decided to focus the study on the VGluT1 input to ference in analyses on the cell body, there were no age- the first 150 mm of path distance from the cell body. This February 2023, 10(2) ENEURO.0436-22.2023 eNeuro.org Research Article: New Research 11 of 19 Figure 5. VGluT1 synapses on the dendrites of injured and control a-motoneurons. A–C, Changes in VGluT1 linear density (number of VGluT1 contacts per 100 mm of dendritic length) according to injury, time point, and dendrite path distance to the cell body, either per a-motoneuron analyzed (A), animal averages (B), or percentage change comparing injured motoneurons to the average control (C). Box plots in A are as in previous figures: box represent the 25th to 75th percentile, line represents the median, whiskers repre- sent the 10th to 90th percentiles. Bars in B represent the average VGluT1 linear density 6 SEM; n = number animals. Dots represent each individual animal. In C, the percentage change of VGluT1 linear density shows control versus injured motoneurons. In both A and B, two-way ANOVAs showed statistically significant differences by injury/age and distance, but no interaction between the two February 2023, 10(2) ENEURO.0436-22.2023 eNeuro.org Research Article: New Research 12 of 19 continued (Extended Data Figs. 5-1, 5-2). In all graphs, statistical significance from pairwise post hoc Bonferroni’s t tests is expressed as follows: *p , 0.05, **p , 0.01, ***p , 0.001. Post hoc Bonferroni-corrected tests indicate significant depletions of VGluT1 contacts at each dendritic bin at all time points after injury. There is also a progressive and significant reduction in VGluT1 synapse numbers with distance to the cell body in controls. Finally, there is a significant increase in the number of contacts in most dendritic bins of control and injured a-motoneur- ons at P70 [two-way ANOVAs for dendrite compartment and age in either control or injured motoneurons; significance in changes with age at each dendritic bin is not depicted in graphs for clarity, but is fully described in Results and in Extended Data Figures 5-5 (control) and 5–6 (injured)]. D, E, Same representations as in A and B, but for VGluT1 densities (number of VGluT1 contacts per 100 mm2 of dendrite surface). Similar two-way ANOVAs followed by post hoc Bonferroni’s tests demonstrate that VGluT1 densities are significantly reduced in each den- dritic bin and in all ages, but VGluT1 density does not show significant differences with distance to the cell body (Extended Data Figs. 5-3, 5-4). The same trend is observed when considering individual motoneurons data (D) or animal averages (E), but significance in proximal dendritic compartments between control and injured a-motoneurons at P17 (7 dpi) was lost when pooling data in animal averages. F, Similar to C (on VGluT1 numbers), the percentage decrease in VGluT1 density on all dendrite regions is always ;50%. cutoff distance was chosen because it was reasonably 0–50 mm distance bin at P17 and P25 and in the 100-150 represented in all our a-motoneuron samples; 85% of the distance bin at P17 (Fig. 5E). These bins showed clear re- sample included dendritic points farther than 100 mm from ductions but did not reach significance in Bonferroni-cor- the cell body, but only 39% farther than 150 mm (n = 130 rected t tests of animal averages, although they did when motoneurons in the total sample). For each motoneuron, all a-motoneurons for each experimental condition (age/ we calculated VGluT1 contact numbers and surface den- injury status) were pooled together (Fig. 5D). These bins sities in the following three distance segments: 0–50, 50– also showed significant differences when analyzed as lin- 100, and 100–150 mm, with 0 being defined as the dendritic ear densities whether using animal averages or pooling all origin from the cell body (point of larger convexity between a-motoneurons together. The discrepancy is best inter- cell body and dendrite membranes). The distribution of preted as a loss of statistical power with smaller differen- VGluT1 synapses and their changes after injury in these ces detected when estimating surface densities versus distance bins were analyzed by measuring path distances number of contacts combined with the smaller sample (the distance to the cell body along the center of the traced size when considering n as the number of animals. dendrite) or by Sholl analyses (estimating the length or sur- We also found a significant addition of synapses with face of the dendrite contained with concentric spheres at age in the 0–50 and 50–100 mm distance compartments in different distances from the cell body). Similar results were controls (Fig. 5A, Extended Data Fig. 5-5; post hoc obtained with either method. This should be expected be- Bonferroni’s tests: p , 0.001 for P17 vs P70 and P25 vs cause a-motoneuron dendrites fan out in a radial manner P70 in both compartments) and injured a-motoneurons and therefore there should be little difference between the (Fig. 5A, Extended Data Fig. 5-6; post hoc Bonferroni’s methods. We report here only on path distance results that tests: p , 0.001, P17 vs P70 in both dendritic compart- we consider more rigorous than Sholl analyses, since it ments, and P25 vs P70 in the more proximal compartment; also considers any tortuosity in dendrite trajectories. p = 0.0174, P25 vs P70 in the second compartments). This VGluT1 input was calculated as linear density (number suggests that VGluT1 synapses are added in both dendrite of contacts per 100 mm of dendrite length; Fig. 5A–C) or regions throughout this postnatal period, but the develop- as surface density (number of contacts per 100 mm2 of mental synaptic additions are smaller for injured a-moto- dendrite surface; Fig. 5D–F). In control MNs, VGluT1 linear neurons. In controls, the estimated difference between P25 densities progressively decreased with distance to the and P70 in the number of VGluT1 contacts per 100 mm of cell body (Fig. 5A,B); however, VGluT1 surface densities dendrite was 4.71 6 1.21 contacts 6 SEM at 0–50 mm dis- were unchanged (Fig. 5D,E). This suggests that the de- tance and 4.05 6 1.10 contacts at 50–100 mm distance. In crease in VGluT1 contacts with distance matches the re- contrast, in injured a-motoneurons the estimated differen- duction in surface resulting from dendritic tapering further ces were 3.40 6 0.70 at 0–50 mm and 2.00 6 0.70 at 50– from the cell body. Two-way ANOVA on VGluT1 linear or 100 mm. In conclusion, a-motoneuron dendrites showed surface densities for time point in control and injured after nerve crush at P10 a significant loss of synapses and a-motoneurons versus dendritic distance bins (0–50, 50– fewer synaptic additions with age. As a result, linear and 100, or 100–150 mm) showed significant differences with surface densities were similarly reduced by approximately time point/injury and dendrite compartment (time point/in- 50% in all dendrite distance bins of injured a-motoneur- jury, p , 0.0001; dendrite compartment: linear density, ons compared with controls (Fig. 5C,F). Since by P70 there p , 0.001; surface density, p = 0.0067), but no interaction is ample motoneuron regeneration, these data suggest between the two (linear, p = 0.085; surface, 0.986). Post that injury-induced reduction in VGluT1 synapses is unre- hoc pairwise comparisons showed significant differences coverable after muscle reinnervation. between control and injured a-motoneurons in VGluT1 lin- ear or surface density at each dendritic distance (Fig. 5A, D, Extended Data Figs. 5-1, 5-3), and this conclusion Proprioceptive neurons in L4 DRG 2 weeks after crush was, typically, replicated when animal averages were injury used (Fig. 5B,E, Extended Data Figs. 5-2, 5-4). The ex- One possible explanation for the differences between ception was for surface density differences between con- crush injuries at P10 compared with the adult would be a trol and injury calculated from animal averages in the higher incidence of cell death among proprioceptors in February 2023, 10(2) ENEURO.0436-22.2023 eNeuro.org Research Article: New Research 13 of 19 Figure 6. Lack of loss of proprioceptive neurons in DRG 60 d after crush injury. A, Representative L4 DRG images of control (contra- lateral side) and injured (ipsilateral side) DRG neurons. PV immunofluorescence in green (FITC) and NeuN in red (Cy3). B, Number of PV cells estimated by counting all parvalbumin in serial sections of L4 DRG and obtaining estimates per ganglia (each dot repre- sents one DRG). Box plots represent the 25th to 75th percentile; the continuous line represents the median; the dashed line repre- sents the average; n indicates the number of DRGs sampled. Two-tailed t tests indicate that differences are not statistically significant (p = 0.262; Extended Data Fig. 6-1). Right, Gardner–Altman estimation plot showing the mean difference ( 17.5) depicted as a dot, and the 95% confidence interval (CI; 39.4, 12.4) indicated by the ends of the vertical confidence interval (CI) of the differ- ence between the means from 5000 bootstrap samples taken. The p value of the two-sided permutation t test is 0.265. C, No differen- ces were found between the percentage of PV neurons to all NeuN comparing ipsilateral and contralateral to the injury. Box plots and data points are represented as in B with 95% CI of mean differences represented to the right with an average value of 5.4% and 95% CI between 9.8 and 0.7). Two-tailed t tests indicate that differences are not statistically significant (p = 0.129; Extended Data Fig. 6-1; p = 0.131 for the two-sided permutation t test estimating mean differences values). Both tests in B and C were underpowered, and therefore the lack of statistical significance needs to be interpreted cautiously. However, any possible effect size is small and therefore does not explain the central loss of VGluT1 synapses. younger animals. To evaluate whether crush injury at P10 axotomy is 17.5 (95.0% CI, 39.4, 12.4) for cell numbers affects the survival of proprioceptive neurons, we calcu- and 5.35 (95.0% CI, 9.79, 0.68) for PV percentage ver- lated the number of PV1 neurons, a marker of propriocep- sus NeuN with, respectively, p = 0.265 and p = 0.131 for the tive neurons (de Nooij et al., 2013; Oliver et al., 2021), and two-sided permutation t test (Fig. 6B,C). Therefore, conclu- their percentage to all NeuN neurons in the right (injured sions derived from parametric mean testing and effect side) and left (control) L4 DRGs 60 d after injury. We size statistics are in good agreement. Given the small sam- counted four to five sections per DRG and analyzed eight ple size, a possible small significant effect in cell survival DRGs ipsilateral to the injury and six on the contralateral could still be detected by stronger powered statistics; how- side. In the control side, we estimated 85.6 6 22.4 (6SD) ever, the estimated effect size is too small to explain the PV neurons per DRG 15-mm-thick section, and on the in- large changes (depletions, 70–40%) in VGluT1 synaptic jured side, 68.2 6 30.7. Similarly, we counted 299.7 6 54.8 coverage on MNs. Injury-induced cell death of PV1 DRG NeuN cells in control and 311.4 6 68.9 in the injured side. neurons thus cannot explain deficits of VGluT1 contacts on The high variability is because of differences in the number a-motoneurons after nerve crush injury at P10. of cells contained within each section depending on sec- tion area and cellular content. To avoid biases introduced by section level, we included in the quantitative analyses The central neuroinflammation reaction and microglia only sections with .200 NeuN neurons. The differences in reactivity PV cell numbers were not statistically significant (t tests; The microglial reaction in the lumbar spinal cord was PV cells: p = 0.263, t(12) = 1.175; NeuN cells: p = 0.738, analyzed at 3, 5, 7, 14, and 21 dpi after a P10 injury in t(12) = 0.343). The percentage of PV cells to all NeuN cells sections immunostained for Iba1 and CD11b (OX-42 anti- was 25.7 6 2.4% in control DRGs and 20.3 6 7.2% in in- body; Fig. 7A,B). These markers are also shared by infil- jured DRG sections. This percentage is similar to previous trating immune cells and therefore serial sections were estimates reported in the literature (Patel et al., 2003), and labeled with CD45 antibodies to detect any CD45high the differences in DRGs ipsilateral and contralateral to the blood-derived inflammatory cells. We could not detect any injury were not significant (t test: p = 0.129, t(12) = 1.628). CD45high cells in our samples at any age, and therefore our There was, however, large variability from DRG to DRG, quantitative analyses focused on Iba1/CD11b cells likely and the powers of these tests for a = 0.05 were 0.296 (for derived from resident microglia. We counted microglia in PV cell numbers) and 0.323 (for PV percentage of NeuN the ipsilateral (injured) and contralateral (control) gray mat- cells), which are below the desired 0.8. However, after ter of the ventral horn and normalized the counts to the 5000 bootstrap sample permutations, the estimated effect available volume (area of the ventral horn  section thick- size for the unpaired mean difference between control and ness). We used three animals per time point from 5 to February 2023, 10(2) ENEURO.0436-22.2023 eNeuro.org Research Article: New Research 14 of 19 Figure 7. Microgliosis after sciatic nerve crush injury at P10. A, B, Representative images of Iba1-microglia in the spinal cords of an- imals at 7 dpi (A) and 21 dpi (B) after a unilateral nerve injury at P10. C, D, High-magnification images of ipsilateral (C) and contralat- eral (D) ventral horn Iba1 microglia (green) showing also immunoreactivity for C11b (blue). E, F, High-magnification images of Iba1- labeleld microglia (green) on the ipsilateral side (E) and the contralateral side (F) showing increased CD11b immunoreactivity (blue). G, Iba1 microglia density in control and injured side. Asterisks show significance with respect to 3 d (*p , 0.05, **p , 0.01, ***p , 0.001; post hoc Bonferroni’s tests after two-way ANOVA for postnatal date and injury. Extended Data Figure 7-1; n = 3 ani- mals at all ages except for 60 dpi in which n = 2). Density diminished in both the control and injured side with age because of spinal cord expansion. Density diminished faster and from a higher peak ipsilateral to the injury because of the additional induction and re- version of microgliosis. H, Progressive growth of the area occupied by the ventral horn at different postnatal days (correspondence with days after injury are noted on the x-axis). Asterisks indicate significance, as above (post hoc Bonferroni’s tests after one-way February 2023, 10(2) ENEURO.0436-22.2023 eNeuro.org Research Article: New Research 15 of 19 continued ANOVA for postnatal date; Extended Data Fig. 7-2). I, Quantitative comparison of microglia cell numbers ipsilateral versus contralat- eral to the injury at each time point. Asterisks indicate significance as in H (post hoc Bonferroni’s tests after the same two-way ANOVA as in H; Extended Data Fig. 7-1). J, Microglia density ratios comparing the side ipsilateral to the injury to the contralateral side (ratio, average 6 SEM). The largest increase in microgliosis, considering the effect of changing spinal cord sizes, occurs at 7 d and then rapidly decreases. 21 dpi and two animals at 3 dpi. We imaged three sections postinjury, despite almost complete muscle reinnervation per animal comparing both sides of the cord to obtain one at this time. These VGluT1 synapses on cell bodies and estimate per animal (Fig. 7C,D). In the side ipsilateral to the proximal dendrites of LG a-motoneurons are interpreted injury not only microglia number increased, but also Iba1 as being derived from muscle spindle proprioceptors and and CD11b immunoreactivities (Fig. 7C–F). The density of not corticospinal axons (another source of VGluT1 synap- Iba1 microglia in the injured ventral horn was significantly ses in the lumbar ventral horn; Alvarez et al., 2004; Persson higher than in the control side at all postinjury ages, but in et al., 2006; Du Beau et al., 2012) because the corticospinal addition there were also developmental changes in cell tract does not directly innervate motoneurons in the lateral density with postnatal time because of the progressive motor columns of lumbar segments in normal mice and increase in the size of the ventral horn (Fig. 7G). A two- any cortico–motoneuronal connections formed during way ANOVA for injury and time after injury (also including early development are efficiently pruned later (Bareyre et developmental time) showed significant differences ac- al., 2005; Du Beau et al., 2012; Gu et al., 2017; Moreno- cording to both variables and a significant interaction (all Lopez et al., 2021). Within sensory afferent VGluT1 synap- p , 0.0001; Extended Data Fig. 7-1). The density of mi- ses, those in synaptic contact with a-motoneurons could croglia peaked at 5 dpi, corresponding to P15, but while belong to either type Ia or type II spindle afferents, but not density was significantly increased at 5 dpi compared with to Golgi tendon organ Ib afferents or cutaneous mechanor- 3 dpi in the injured side (p , 0.05, post hoc Bonferroni eceptors that do not enter the ventral horn (Alvarez et al., tests; Fig. 7C), the differences were not significant in the 2011; Vincent et al., 2017). Ia and II ventrally synaptic col- control side (p . 0.999). Thereafter, density was signifi- laterals reach Lamina IX to innervate a-motoneurons in the cantly reduced in both sides, most likely because of pro- rodent spinal cord (Vincent et al., 2017), but the relative gressive significant increases in ventral horn size with age abundance of synapses from each type of sensory axon (Fig. 7H). Nevertheless, the differences in cell density be- suggest a strong bias toward Ia identity. tween injured and uninjured sides remained significant at The large loss of VGluT1/Ia/II synapses on spinal a-mo- all time points analyzed (Fig. 7I). When microglia density on toneurons by 60 d postinjury is clearly different from the the injured side was calculated as a ratio of the density on response to nerve crush in adult rats, in which there is a the control side (normalizing for changes in density be- significant 35–38% decrease in VGluT1 synaptic density cause of development), maximal microgliosis was detected on the cell bodies but no significant decrease on the den- at 7 dpi. At 3 dpi, there was already microgliosis (ratio, drites by 21 d or 3 months after nerve injuries (Schultz et 2.01 6 0.06), increased at 5 dpi (2.11 6 0.06), peaked at al., 2017). In contrast, we detected a larger depletion of 7 dpi (2.32 6 0.11), and decreased afterward (14 dpi, 1.82 6 VGluT1 synaptic densities on cell bodies (.60%) and 0.1; 21 dpi, 1.46 6 0.01; Fig. 7J). Microglia density returned dendrites (;50% at all distance bins) at 60 d postinjury toward control values slowly, and there were still significantly when the nerve crush occurs at P10. This loss was com- more microglia ipsilateral to the injury at 21 d postinjury. Two parable to that detected in adult rats after nerve transec- animals were further processed at 60 dpi, and in these ani- tions (Alvarez et al., 2011). We targeted the sciatic nerve mals Ib1a/CD11b microglia appearance and density were al- at mid-thigh position for neonatal nerve injuries and for most identical on both sides. the tibial nerve after the sciatic nerve trifurcation for adult Thus, activation of microglia after P10 nerve crush injury injuries. We believe that results among these different in- included rapid proliferation and upregulation of comple- juries are comparable since analyses are focused on LG ment receptor components, followed by fast downregula- MNs retrogradely labeled before the injury. The LG MN tion in the second week after injury and no infiltration of pool receives monosynaptic inputs from Ia afferents, and peripheral immune cells. This time course is faster than to a lesser extent II afferents, innervating all triceps surae previously shown in mouse after nerve transection in the muscles, and these travel in both the sciatic and tibial adult and suggests that the permanent deletion of VGluT1 nerves. We have shown previously that the permanent re- synapses after P10 nerve crush involves a microglia moval of VGluT1/Ia/II afferent synapses from MNs is de- mechanism during the first week after injury by difference pendent on injury in the peripheral nerve to the Ia afferent, to the neuroinflammation mechanisms described previ- and not to the MN (Alvarez et al., 2011; Rotterman et al., ously in the second week after adult nerve transection 2019; Alvarez et al., 2020). In other words, Ia/II afferents (Rotterman et al., 2019). retract from ventral horn targets, including interneurons, injured MNs, and uninjured MNs, independent of the in- Discussion jury status of the postsynaptic neuron. Ia/II afferents pre- In this article, we show that a crush nerve injury in synaptic to LG MNs are similarly injured by nerve crush at the early postnatal period (P10) induces a massive loss the sciatic or tibial nerve level, and their response is ex- of VGluT1 synapses on spinal a-motoneurons by 60 d pected to be independent of the size of the injured MN February 2023, 10(2) ENEURO.0436-22.2023 eNeuro.org Research Article: New Research 16 of 19 cohort. Thus, the difference between the present results ;70% of contralateral values 2 months after the injury. In and those previously reported in the adult rat after nerve agreement, histological analysis indicated almost full rein- crush injuries (Schultz et al., 2017) requires an alternative nervation of neuromuscular junctions at that time point. explanation. These data suggest that regeneration after P10 sciatic One major difference is developmental stage. It is well crush results in muscle innervation close to normal. This known that during the first 2 weeks of postnatal life in ro- is expected because, in the absence of motoneuron loss, dents, VGluT1/Ia/II synaptic densities progressively in- surviving axons in the proximal stump after nerve crush crease on their neuronal targets in the ventral horn (Mentis injuries are effectively guided through endoneurial tubes et al., 2006; Siembab et al., 2010; Mentis et al., 2011). In that remain connected to the original peripheral targets particular, VGluT1/Ia/II synapses on the cell body and den- facilitating reinnervation speed and specificity (Nguyen et drites of mouse MNs increase almost four times from P4 to al., 2002; Valero-Cabré et al., 2004). P13 (Mentis et al., 2011). Here we found continued addition Motoneuron sizes were different at P70 compared with of synapses from P17 to P70, in part to match the increase previous ages. This could be because of the selective loss in available surface with late postnatal development neuro- of smaller a-motoneurons or alternatively by biases intro- nal growth. In a detailed previous study (Kalb, 1994), it was duced by the different tracers used in short-term (CTb-555) shown that during the third postnatal week rat MNs exhibit or long-term (Fast Blue) experiments. We believe, however, significant growth in cell body size (confirmed in our study) that the increase in cell body size is a normal developmental and also dendritic length and bifurcations. The effects of sci- feature and cannot be explained by either of these alterna- atic nerve crush were detectable in both a loss of synapses tive explanations. First, the coefficient of variation in size re- and a slowing of synaptic accumulations during this period mained constant, suggesting spread in size range parallel of MN growth, perhaps suggesting that remaining additions to the mean increase. This is what is expected by the mat- occur in inputs of decreased strength with less proliferative uration of different sizes motor units and is similar to the capacity. Together, they result in a large reduction in synap- increase in cell sizes of lumbar a-motoneurons that we re- tic inputs from VGluT1/Ia/II afferents on the mature MN after ported before in mice (Shneider et al., 2009) and others in it has regenerated and reinnervated muscle. the rat (Westerga and Gramsbergen, 1992; Ishihara et al., One possible explanation for the above results is that 1994). If the type of tracer or cell death had introduced a either MNs or Ia/II afferents are more susceptible to dying bias toward certain cell sizes, we should observe a scaling after injury to their axons in young animals. Alternatively, factor for the whole-cell size distribution, not an increase in we previously demonstrated that the loss of VGluT1 Ia/II range. Moreover, the increase in VGluT1 numbers seems to synapses in the ventral horn is dependent on a microglia match the cell size increase, maintaining a constant synaptic mechanism and the ventral horn neuroinflammation in re- density. Finally, motoneuron cell death after certain injuries sponse to the injury in the peripheral nerve (Rotterman et in the mouse are accompanied by specific local microglia al., 2019). Given that the neuroinflammatory reaction in reactions merging in tight macroclusters around the degen- the dorsal horn in response to nerve injuries is different in erating motoneuron (Rotterman and Alvarez, 2020). These neonates, young rats, and adult rats (Vega-Avelaira et al., were never observed in our preparations. 2007), we sought to compare the time course and proper- Alternatively, a proportion of Ia/II afferent sensory neu- ties of the microglia reaction in the ventral horn of P10 in- rons might die when disconnected from muscle at this de- jured animals with previous observations after similar velopmental time point. Approximately 50% of sensory injuries in adult. We hypothesized that a biased microglia neurons die within 2–3 d after axotomy because of sciatic phenotype at this age toward synaptic pruning might in- nerve transection or crush in P0 to P1 rat pups (Yip et al., duce a differential effect on Ia/II synapse preservation 1984; Whiteside et al., 1998). In contrast, sciatic nerve when combined with further activation because of periph- crush in 5-week-old rats results in limited and protracted eral nerve injuries. This possibility is further supported by (occurring for several days) cell death of sensory neurons a recent report demonstrating microglia synaptic pruning (Ekstrom, 1995). A recent study suggested that DRG neu- of Ia afferent synapses in the neonate via a canonical C1q ron cell loss can occur after injuries during the juvenile age complement mechanism (Vukojicic et al., 2019). (Kemp et al., 2015). To address the possibility of some cell loss from proprioceptors after axotomy following the crush Cell death cannot explain the massive loss of VGluT1 nerve injury at P10, we evaluated the percentage of PV1 synapses neurons in DRGs 2 weeks after the injury. Parvalbumin is a We applied nerve crush injuries in P10 rats because well known marker of proprioceptive afferents (de Nooij et motoneuron and sensory DRG cell death is expected to al., 2013; Oliver et al., 2021). We found a very small and be limited at this age (Lowrie and Vrbová, 1992; Kemp et nonsignificant reduction observed in the total number of al., 2015). A direct comparison of MN and DRG cell sur- PV1 cells or the ratio of PV cells to all NeuN DRG neurons. vival after sciatic nerve crush in P3, P5, P7, P30, and adult If the small change were to become significant by increas- rats showed that, while injury at P3 resulted in loss of ing sample size (number of animals), the estimated effect MNs and DRGs in .50% of cells, these losses were mini- size would remain too small to explain the large loss of mal (,20%) when the injury occurred at P7, and limited VGluT1/Ia/II synapses on MNs. Previous studies already cell loss was only observed in the DRGs when injuries suggested that large-diameter cutaneous and muscle affer- were conducted at P30 (Kemp et al., 2015). In our study, ents are more resistant to axotomy than small cutaneous we estimated that CMAP amplitudes would recover to afferents (Tandrup et al., 2000; Hu and McLachlan, 2003). February 2023, 10(2) ENEURO.0436-22.2023 eNeuro.org Research Article: New Research 17 of 19 Neuroinflammation after neonatal nerve crush differs the microglia reaction in the ventral horn distributes along from crush in the adult the regions targeted by synapses from injured Ia/II pro- Comparison of microglia in nerve cut versus crush in prioceptors, from medial lamina V to lateral lamina IX, the adult rat showed that microglia activation is similar crossing through LVII in the ventrolateral direction (Fig. during the first 2 weeks postinjury but differs at later time 7A). Previously, we showed that after adult injuries in the points because of a prolongation of microglia activation mouse the microglia reaction in the ventral horn was criti- after nerve cut (He et al., 1996; Campos et al., 2021). We cal, while dorsal horn microglia activation (induced by sig- obtained similar results in adult mice (Rotterman et al., nals from sensory afferents) was dispensable (Rotterman 2019), and indeed the prolonged microglia activation cor- et al., 2019). In the adult rat injury to one muscle nerve related with infiltration of blood-borne CCR2 cells after [e.g., the medial G (MG)] deletes Ia inputs from injured MG nerve transection, but not after nerve crush, and the time a-motoneurons, but also affects reciprocal inhibition of course of immune cell infiltration correlated with the dis- uninjured synergists (Horstman et al., 2019). This agrees appearance of VGluT1 synapses from the ventral horn with the die-back of ventrally directed Ia synaptic collater- (Rotterman et al., 2019). Moreover, VGluT1/Ia/II synapses als after nerve injury (Alvarez et al., 2011), suggesting re- were partially preserved in the cell bodies and fully pre- traction from not only axotomized a-motoneurons, but served on the dendrites of MNs that regenerate and rein- also other ventral horn targets, including uninjured syner- nervate muscle after axotomy in CCR2 knock-out animals gists and ventral proprioceptive interneurons. Whether a (Rotterman et al., 2019). These results lead to the conclu- similar die-back occurs after nerve crush in young animals sion that a different neuroinflammatory environment after will need to be assessed in the future by labeling individual each type of injury, particularly at later time points after in- regenerated Ia afferents, as before (Alvarez et al., 2011), jury (.15 d), contributed to the permanent deletion of and using more focalized nerve injuries to study homony- VGluT1/Ia/II afferent synapses. The time course of micro- mous and heteronymous connections. glia activation following nerve crush injuries in the neonate has not been analyzed in detail before. Surprisingly, mi- Functional implications crogliosis declined quite fast after the first week of acti- Deleterious effects on motor function after injuries in vation and despite a massive loss of VGluT1 synapses. the postnatal period have been described even when Moreover, CD45 immunoreaction could not demonstrate there is no motoneuron death (Lowrie et al., 1982; Lowrie any infiltration of peripheral immune cells at early or late and Vrbová, 1984; Lowrie et al., 1987). For example, postnatal ages. We conclude that the microglia reaction muscle tension recovers to only half of normal following after crush injury at P10 differs from those in transection muscle reinnervation after nerve crush injuries at P11 to injuries in the adult and that the lack of CD45 cell infiltra- P12 (Lowrie et al., 1987). Nerve crush injuries inflicted up tion suggests limited activation of CCR2 signaling. We to P30 caused permanent muscle impairments and did therefore propose that the mechanism of loss of VGluT1/Ia/II not recover full function (Kemp et al., 2015). These stud- synapses after nerve crush injury at P10 likely differs from ies suggest that factors unrelated to motoneuron death mechanisms driving the loss of VGUT1/Ia/II synapses in lead to poor motor function recovery after crush injuries the adult after nerve transection injuries. in the postnatal period. Ia afferent input to the ventral Recent single-cell RNA sequencing analyses showed horn is very significant for motor function, from maintain- that microglia during early postnatal development display ing muscle tone to regulating motor unit recruitment phenotypes highly active in synaptic and axon pruning during motor activity and serving for rapid online mus- (Matcovitch-Natan et al., 2016; Hammond et al., 2019). It cle adjustments during performance. Therefore, it is is also known that Ia afferent synapses are actively quite possible that the massive loss of VGluT1 synap- pruned through C1q complement mechanisms during a ses we report here could impair the recovery of motor critical window of synaptic selection and strengthening function when nerve crush occurs in the neonate. during the first 2 postnatal weeks (Vukojicic et al., 2019). In conclusion, after crush injury in the postnatal stage, Indeed, we found a large increase in CD11b expression in there is a major loss of Ia synapses on a-motoneurons. activated microglia. CD11b and CD18 to form CR3, a Further research should focus on identifying the mecha- phagocytic complement receptor that activates down- nisms behind this permanent synaptic loss to improve the stream of C1q and contributes to synaptic plasticity during functional recovery after postnatal nerve injuries. developmental synaptic pruning (Stephan et al., 2012). 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