Peripheral nerve injury results in persistent motor deficits, even after the nerve regenerates and muscles are reinnervated. This lack of functional recovery is partly explained by brain and spinal cord circuit alterations triggered by the injury, but the mechanisms are generally unknown. One example of this plasticity is the die-back in the spinal cord ventral horn of the projections of proprioceptive axons mediating the stretch reflex (Ia afferents). Consequently, Ia information about muscle length and dynamics is lost from ventral spinal circuits, degrading motor performance after nerve regeneration. Simultaneously, there is activation of microglia around the central projections of peripherally injured Ia afferents, suggesting a possible causal relationship between neuroinflammation and Ia axon removal. Therefore, we used mice (both sexes) that allow visualization of microglia (CX3CR1-GFP) and infiltrating peripheral myeloid cells (CCR2-RFP) and related changes in these cells to Ia synaptic losses (identified by VGLUT1 content) on retrogradely labeled motoneurons. Microgliosis around axotomized motoneurons starts and peaks within 2 weeks after nerve transection. Thereafter, this region becomes infiltrated by CCR2 cells, and VGLUT1 synapses are lost in parallel. Immunohistochemistry, flow cytometry, and genetic lineage tracing showed that infiltrating CCR2 cells include T cells, dendritic cells, and monocytes, the latter differentiating into tissue macrophages. VGLUT1 synapses were rescued after attenuating the ventral microglial reaction by removal of colony stimulating factor 1 from motoneurons or in CCR2 global KOs. Thus, both activation of ventral microglia and a CCR2-dependent mechanism are necessary for removal of VGLUT1 synapses and alterations in Ia-circuit function following nerve injuries. ) for the donation of the csf1-flox mouse line; and Myriam Alvarez for quantification of microglia in CSF1 motoneuron KOs.Synaptic plasticity and reorganization of essential motor circuits after a peripheral nerve injury can result in permanent motor deficits due to the removal of sensory Ia afferent synapses from the spinal cord ventral horn. Our data link this major circuit change with the neuroinflammatory reaction that occurs inside the spinal cord following injury to peripheral nerves. We describe that both activation of microglia and recruitment into the spinal cord of blood-derived myeloid cells are necessary for motor circuit synaptic plasticity. This study sheds new light into mechanisms that trigger major network plasticity in CNS regions removed from injury sites and that might prevent full recovery of function, even after successful regeneration.

Motoneurons axotomized by peripheral nerve injuries experience profound changes in their synaptic inputs that are associated with a neuroinflammatory response that includes local microglia and astrocytes. This reaction is conserved across different types of motoneurons, injuries, and species, but also displays many unique features in each particular case. These reactions have been amply studied, but there is still a lack of knowledge on their functional significance and mechanisms. In this review article, we compiled data from many different fields to generate a comprehensive conceptual framework to best interpret past data and spawn new hypotheses and research. We propose that synaptic plasticity around axotomized motoneurons should be divided into two distinct processes. First, a rapid cell-autonomous, microglia-independent shedding of synapses from motoneuron cell bodies and proximal dendrites that is reversible after muscle reinnervation. Second, a slower mechanism that is microgliadependent and permanently alters spinal cord circuitry by fully eliminating from the ventral horn the axon collaterals of peripherally injured and regenerating sensory Ia afferent proprioceptors. This removes this input from cell bodies and throughout the dendritic tree of axotomized motoneurons as well as from many other spinal neurons, thus reconfiguring ventral horn motor circuitries to function after regeneration without direct sensory feedback from muscle. This process is modulated by injury severity, suggesting a correlation with poor regeneration specificity due to sensory and motor axons targeting errors in the periphery that likely render Ia afferent connectivity in the ventral horn nonadaptive. In contrast, reversible synaptic changes on the cell bodies occur only while motoneurons are regenerating. This cell-autonomous process displays unique features according to motoneuron type and modulation by local microglia and astrocytes and generally results in a transient reduction of fast synaptic activity that is probably replaced by embryonic-like slow GABA depolarizations, proposed to relate to regenerative mechanisms.

The potassium-chloride cotransporter (KCC2) maintains the low intracellular chloride found in mature central neurons and controls the strength and direction of GABA/glycine synapses. We found that following axotomy as a consequence of peripheral nerve injuries (PNIs), KCC2 protein is lost throughout the somatodendritic membrane of axotomized spinal cord motoneurons after downregulation of kcc2 mRNA expression. This large loss likely depolarizes the reversal potential of GABA/glycine synapses, resulting in GABAergic-driven spontaneous activity in spinal motoneurons similar to previous reports in brainstem motoneurons. We hypothesized that the mechanism inducing KCC2 downregulation in spinal motoneurons following peripheral axotomy might be mediated by microglia or motoneuron release of BDNF and TrkB activation as has been reported on spinal cord dorsal horn neurons after nerve injury, motoneurons after spinal cord injury (SCI), and in many other central neurons throughout development or a variety of pathologies. To test this hypothesis, we used genetic approaches to interfere with microglia activation or delete bdnf from specifically microglia or motoneurons, as well as pharmacology (ANA-12) and pharmacogenetics (F616A mice) to block TrkB activation. We show that KCC2 dysregulation in axotomized motoneurons is independent of microglia, BDNF, and TrkB. KCC2 is instead dependent on neuromuscular innervation; KCC2 levels are restored only when motoneurons reinnervate muscle. Thus, downregulation of KCC2 occurs specifically while injured motoneurons are regenerating and might be controlled by target-derived signals. GABAergic and glycinergic synapses might therefore depolarize motoneurons disconnected from their targets and contribute to augment motoneuron activity known to promote motor axon regeneration. Significance StatementThe neuronal potassium-chloride cotransporter KCC2 is dysregulated after numerous types of neuronal injuries and has been related to neuronal dysfunction, hyperalgesia and spasticity. In this study, we investigated KCC2 regulation on spinal motoneurons with axons injured during peripheral nerve transections. We illustrate that KCC2 loss on axotomized motoneurons occurs at the transcriptional level. This loss differs in time course, completeness and signaling mechanisms from KCC2 dysregulation in other neurons after various pathologies or in motoneurons after spinal cord injury (SCI). In contrast, KCC2 loss on axotomized motoneurons relates to muscle innervation, suggesting a dependence on target-derived signals. We argue that KCC2 loss in axotomized motoneurons may be part of the response that facilitates regeneration of motor axons in the periphery.

Peripheral nerve injury (PNI) is an excellent model for studying neural responses to injury and elucidating the mechanisms that can facilitate axon regeneration. As such, several animal models have been employed to study regenerative mechanisms after PNI, including Aplysia, zebrafish, rabbits, cats and rodents. This protocol describes how to perform a sciatic nerve injury and repair in mice, one of the most frequently used models to study mechanisms that facilitate recovery after PNI, and that takes advantage of the availability of many genetic models. In this protocol, we describe a method for using fibrin glue to secure the proximal and distal stumps of an injured nerve in close alignment. This method facilitates the alignment of nerve stumps, which aids in regeneration of both sensory and motor axons and allows successful reconnection with peripheral targets.

The snapping shrimp, Alpheus angulosus McClure, 2002, is a small crustacean with bilaterally asymmetric claws that serve distinct behavioural and sensory functions. If the large claw is lost, the organism switches handedness, transforming its small pincer claw into a large snapping claw while simultaneously developing a small claw on the contralateral side. To better understand the mechanisms required to adapt to this radical change in body composition, we examined developmental plasticity by tracing changes in sensory setae distribution on the claws throughout transformation. We observed only two broad types of setae, simple and plumose. Quantitative analysis across molt stages revealed significant alterations in setae composition and numbers that occurred primarily on the edge of the propodus, where the most drastic morphological changes also occur. These results suggest that previous developmental mechanisms are re-engaged to support the proliferation and differentiation of new setae during transformation.