BDNF Spinal Overexpression after Spinal Cord Injury Partially Protects Soleus Neuromuscular Junction from Disintegration, Increasing VAChT and AChE Transcripts in Soleus but Not Tibialis Anterior Motoneurons.

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.

Molecular Identification of Pro-Excitogenic Receptor and Channel Phenotypes of the Deafferented Lumbar Motoneurons in the Early Phase after SCT in Rats.

Spasticity impacts the quality of life of patients suffering spinal cord injury and impedes the recovery of locomotion. At the cellular level, spasticity is considered to be primarily caused by the hyperexcitability of spinal α-motoneurons (MNs) within the spinal stretch reflex circuit. Here, we hypothesized that after a complete spinal cord transection in rats, fast adaptive molecular responses of lumbar MNs develop in return for the loss of inputs. We assumed that early loss of glutamatergic afferents changes the expression of glutamatergic AMPA and NMDA receptor subunits, which may be the forerunners of the developing spasticity of hindlimb muscles. To better understand its molecular underpinnings, concomitant expression of GABA and Glycinergic receptors and serotoninergic and noradrenergic receptors, which regulate the persistent inward currents crucial for sustained discharges in MNs, were examined together with voltage-gated ion channels and cation-chloride cotransporters. Using quantitative real-time PCR, we showed in the tracer-identified MNs innervating extensor and flexor muscles of the ankle joint multiple increases in transcripts coding for AMPAR and 5-HTR subunits, along with a profound decrease in GABAAR, GlyR subunits, and KCC2. Our study demonstrated that both MNs groups similarly adapt to a more excitable state, which may increase the occurrence of extensor and flexor muscle spasms.

Regulation of perineuronal net components in the synaptic bouton vicinity on lumbar α-motoneurons in the rat after spinalization and locomotor training: New insights from spatio-temporal changes in gene, protein expression and WFA labeling.

Chondroitin sulfate proteoglycans (CSPGs) consist of core proteins and glycosaminoglycan side chains. Tenascins, and hyaluronan and proteoglycan link protein 1 (HAPLN), link CSPGs with a hyaluronan backbone to constitute perineuronal nets (PNNs), which ensheath preferentially highly active neurons to maintain architecture and stabilize synapses, but restrict repair plasticity. Spinal cord injury increases CSPG core protein levels in the lesion proximity, limiting permissiveness of the extracellular milieu for fiber regrowth, however regulation of PNNs structure in the vicinity of distant α-motoneurons (MNs) in the course of degeneration and reorganization of their inputs requires research. Here, we examined early and late changes in CSPGs, HAPLN1, tenascin-R, and glial activation along the spinal cord in male rats with complete spinal cord transection (Th10), and their impact on PNNs ensheathing lumbar MNs innervating ankle extensor and flexor muscles, which are in different loading states in paraplegic rats. We show that (1) distance from the lesion site and time after injury (2-5 weeks) differentiate degree of changes in transcription rates (measured with RT-qPCR) of PNNs proteins with increased CSPGs and decreased HAPLN1 transcripts, suggesting long-term PNN destabilization in majority of spinal segments, (2) in lumbar segments PNN composition is not MN-class (extensor vs flexor) specific, both showing early decrease and late upregulation of Wisteria floribunda agglutinin (WFA) labeling in vicinity of synaptic boutons on MNs, (3) long-term locomotor training tends to reduce WFA(+) PNNs, but not their protein components (immunofluorescence measurements) around MNs. Our results suggest that training-induced regulation may target glycan structures of CSPGs.

Roles of palmitoylation in structural long-term synaptic plasticity.

Long-term potentiation (LTP) and long-term depression (LTD) are important cellular mechanisms underlying learning and memory processes. N-Methyl-D-aspartate receptor (NMDAR)-dependent LTP and LTD play especially crucial roles in these functions, and their expression depends on changes in the number and single channel conductance of the major ionotropic glutamate receptor α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) located on the postsynaptic membrane. Structural changes in dendritic spines comprise the morphological platform and support for molecular changes in the execution of synaptic plasticity and memory storage. At the molecular level, spine morphology is directly determined by actin cytoskeleton organization within the spine and indirectly stabilized and consolidated by scaffold proteins at the spine head. Palmitoylation, as a uniquely reversible lipid modification with the ability to regulate protein membrane localization and trafficking, plays significant roles in the structural and functional regulation of LTP and LTD. Altered structural plasticity of dendritic spines is also considered a hallmark of neurodevelopmental disorders, while genetic evidence strongly links abnormal brain function to impaired palmitoylation. Numerous studies have indicated that palmitoylation contributes to morphological spine modifications. In this review, we have gathered data showing that the regulatory proteins that modulate the actin network and scaffold proteins related to AMPAR-mediated neurotransmission also undergo palmitoylation and play roles in modifying spine architecture during structural plasticity.

Early pre- and postsynaptic decrease in glutamatergic and cholinergic signaling after spinalization is not modified when stimulating proprioceptive input to the ankle extensor α-motoneurons: Anatomical and neurochemical study.

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.

Spinalization and locomotor training differentially affect muscarinic acetylcholine receptor type 2 abutting on α-motoneurons innervating the ankle extensor and flexor 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-3 H ([3 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 [3 H]QNB binding decreased selectively in the L5-6 segments, where ankle extensor MNs are predominantly located. At 5 weeks after SCT, [3 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/.