After spinal cord transection (SCT) the interaction between motoneurons (MNs) and muscle is impaired, due to reorganization of the spinal network after a loss of supraspinal inputs. Rats subjected to SCT, treated with intraspinal injection of a AAV-BDNF (brain-derived neurotrophic factor) construct, partially regained the ability to walk. The central effects of this treatment have been identified, but its impact at the neuromuscular junction (NMJ) has not been characterized. Here, we compared the ability of NMJ pre- and postsynaptic machinery in the ankle extensor (Sol) and flexor (TA) muscles to respond to intraspinal AAV-BDNF after SCT. The gene expression of cholinergic molecules (VAChT, ChAT, AChE, nAChR, mAChR) was investigated in tracer-identified, microdissected MN perikarya, and in muscle fibers with the use of qPCR. In the NMJs, a distribution of VAChT, nAChR and Schwann cells was studied by immunofluorescence, and of synaptic vesicles and membrane active zones by electron microscopy. We showed partial protection of the Sol NMJs from disintegration, and upregulation of the VAChT and AChE transcripts in the Sol, but not the TA MNs after spinal enrichment with BDNF. We propose that the observed discrepancy in response to BDNF treatment is an effect of difference in the TrkB expression setting BDNF responsiveness, and of BDNF demands in Sol and TA muscles.

Complete thoracic spinal cord transection (SCT) impairs excitatory cholinergic inputs to ankle extensor (soleus; Sol) but not to flexor (tibialis anterior; TA) α-motoneurons (MNs) modifiable by locomotor training applied post-transection. The purpose of this study was to investigate whether Sol and TA MNs adapt to changes in cholinergic environment by differential regulation of their muscarinic receptors M2 (M2R). We examined Chrm2 (M2R gene) transcript level, high-affinity 3-quinuclidinyl benzilate- H ([ H]QNB) ligand binding, distribution and density of M2R immunolabeling in lumbar (L) segments in intact and SCT rats, with or without inclusion of 5-week treadmill locomotor training. We show that at the second week after SCT the levels of Chrm2 transcript are reduced in the L3-6 segments, with [ H]QNB binding decreased selectively in the L5-6 segments, where ankle extensor MNs are predominantly located. At 5 weeks after SCT, [ H]QNB binding differences between the L3-4 and L5-6 segments are maintained, accompanied by higher density of M2R immunolabeling in the plasma membrane and cytoplasm of TA than Sol MNs and by enriched synaptic versus extrasynaptic M2R pools (52% TA vs. 25% Sol MNs). Training normalized M2R in TA MNs, improved locomotion, and reduced frequency of clonic episodes. Our findings indicate higher sensitivity of TA than Sol MNs to cholinergic signaling after SCT, which might shorten flexor twitches duration and contribute to generation of clonic movements. Synaptic enrichment in M2R density may reflect a compensatory mechanism activated in TA and Sol MNs to different extent in response to reduced strength of cholinergic signaling to each MN pool. Open Practices Open Science: This manuscript was awarded with the Open Materials Badge. For more information see: https://cos.io/our-services/open-science-badges/.

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Alpha-motoneurons (MNs) innervating ankle extensor muscles show reduced peripheral inputs from Ia proprioceptive afferents and cholinergic afferents after chronic spinalization (SCT). That phenomenon is not observed on ankle flexor MNs, indicating a smaller vulnerability of the latter MNs circuit to SCT. Locomotor training of spinal rats which partially restored those inputs to extensor MNs tended to hyper innervate flexor MNs, disclosing a need for selective approaches. In rats with intact spinal cord 7-days of low-threshold proprioceptive stimulation of the tibial nerve enriched glutamatergic Ia and cholinergic innervation of lateral gastrocnemius (LG) MNs, suggesting usefulness of selective stimulation for restoration of inputs to extensor MNs after SCT. Accordingly, to examine its effectiveness after SCT, tibial nerves and soleus muscles were implanted bilaterally, and for MN identification fluorescence tracers to LG and tibialis anterior (TA) muscles were injected two weeks prior to spinalization. Stimulation of tibial nerve, controlled by H-reflex recorded in the soleus muscle, started on the third post-SCT day and continued for 7 days. Nine days post-SCT the number and volume of glutamatergic Ia and of cholinergic C-boutons on LG MNs was decreased, but stimulation affected neither of them. Postsynaptically, a threefold decrease of NMDAR NR1 subunit and thirtyfold decrease of M2 muscarinic receptor transcripts caused by SCT were not counteracted by stimulation, whereas a threefold decrease of AMPAR GluR2 subunit tended to deepen after stimulation. We conclude that LG MNs, supported with proprioceptive stimuli after SCT, do not transcribe the perceived cues into compensatory response at the transcriptional level in the early post-SCT period.