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

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

Electrical Stimulation of Low-Threshold Proprioceptive Fibers in the Adult Rat Increases Density of Glutamatergic and Cholinergic Terminals on Ankle Extensor α-Motoneurons - Fig 4

<p><b>Changes in the number (A) and in the aggregate volume (B) of VGLUT1 IF synaptic terminals contacting LG α-MNs after seven days of stimulation of Ia fibers in the tibial nerve.</b> Data are reported as mean +/- SEM. Both the number and aggregate volume of VGLUT1 terminals were increased on the stimulated comparing to sham-stimulated side (*p = 0.03 and *p< 0.05, respectively, <i>Wilcoxon</i> test).</p

Identification of glutamatergic (VGLUT1; indigo) and cholinergic (VAChT; green) terminals abutting on LG α-motoneuron (α-MN) labeled by means of immunofluorescence (IF).

<p>LG MN perikaryon and proximal parts of dendrites were identified with True Blue (turquoise; A, C, E and G) and all synaptic terminals apposing LG α-MN were identified by synaptophysin IF (red). A-C present single optical section (0.21 μm thick); framed areas on C (merge of A-B) are shown with higher magnification in D to demonstrate contiguity of VAChT or VGLUT1 varicosities with the edge of the α-MN perikaryon. E-H are stacks of 20 optical sections of the same α-MN to show 3D reconstruction of the glutamatergic (indigo) and cholinergic (green) terminals among all synaptic terminals (synaptophysin) abutting on LG α- MN. G–merge of E and F. H—glutamatergic (magenta) and cholinergic (yellow) terminals shown to contact α-MN surface were accepted for quantification while the other (white) terminals, which did not fulfill these criteria, were not analyzed.</p

The direct M₁ responses and H₃-reflexes recorded in the rat soleus muscle during stimulation sessions of low-threshold proprioceptive fibers in the tibial nerve.

<p><b>A.</b> Examples of the raw data averaged after 7200 burst repetitions. Arrows indicate stimulus artifacts. <b>B</b>. The mean areas of the H₃-reflexes (blue line) and M₁-responses (green line) during consecutive days of stimulation in six rats. Four 3 min samples daily were taken for the analysis: at the beginning and at the end of the first and fourth stimulation sessions. The data are expressed as a percentage of M_max values for individual animals.</p

The number of TB-identified LG α-MNs subjected to the analysis of VGLUT1- and VAChT IF synaptic terminals apposing their cell bodies.

<p>(N)—the number of animals per group. The numbers of analyzed synaptic terminals are shown in square brackets.</p

Example of typical location of α-motoneurons innervating LG muscle in L5 spinal segment.

<p>The spinal grey matter borderline is marked. Retrogradely labeled motoneurons (framed) are shown enlarged below. Motoneurons were labeled with True Blue fluorescence tracer injected into the LG muscle belly.</p

Enhancing Proprioceptive Input to Motoneurons Differentially Affects Expression of Neurotrophin 3 and Brain-Derived Neurotrophic Factor in Rat Hoffmann-Reflex Circuitry

<div><p>The importance of neurotrophin 3 (NT-3) for motor control prompted us to ask the question whether direct electrical stimulation of low-threshold muscle afferents, strengthening the proprioceptive signaling, could effectively increase the endogenous pool of this neurotrophin and its receptor TrkC in the Hoffmann-reflex (H-reflex) circuitry. The effects were compared with those of brain-derived neurotrophic factor (BDNF) and its TrkB receptor. Continuous bursts of stimuli were delivered unilaterally for seven days, 80 min daily, by means of a cuff-electrode implanted over the tibial nerve in awake rats. The H-reflex was recorded in the soleus muscle to control the strength of stimulation. Stimulation aimed at activation of Ia fibers produced a strong increase of NT-3 protein, measured with ELISA, in the lumbar L3-6 segments of the spinal cord and in the soleus muscle. This stimulation exerted much weaker effect on BDNF protein level which slightly increased only in L3-6 segments of the spinal cord. Increased protein level of NT-3 and BDNF corresponded to the changes of NT-3 mRNA and BDNF mRNA expression in L3-6 segments but not in the soleus muscle. We disclosed tissue-specificity of TrkC mRNA and TrkB mRNA responses. In the spinal cord TrkC and TrkB transcripts tended to decrease, whereas in the soleus muscle TrkB mRNA decreased and TrkC mRNA expression strongly increased, suggesting that stimulation of Ia fibers leads to sensitization of the soleus muscle to NT-3 signaling. The possibility of increasing NT-3/TrkC signaling in the neuromuscular system, with minor effects on BDNF/TrkB signaling, by means of low-threshold electrical stimulation of peripheral nerves, which in humans might be applied in non-invasive way, offers an attractive therapeutic tool.</p></div

NT-3 and BDNF protein level in the tissues involved in the H-reflex circuitry.

<p><b>A.</b> The protein level of NT-3 and BDNF in the intact animals (control group), measured in the lumbar segments (L1-2; L3-6) and the soleus muscle (sol), by means of ELISA. NT-3 level was the highest in the soleus muscle whereas that of BDNF predominated in the spinal cord. <b>B.</b> The changes of NT-3 and BDNF level in the spinal cord segments and soleus muscle after low-threshold, unilateral stimulation of the tibial nerve. Stimulation caused a clear increase of NT-3 protein level in the L3-6 segments of the spinal cord, where the motoneurons innervating the soleus muscles and its synergists acting at the ankle joints are located, and in the soleus muscle. The effect of stimulation on the protein level of BDNF was weaker and detected only in the caudal lumbar segments of the spinal cord. Asterisks indicate statistically significant effects (***P<0.001; **P<0.01; *P<0.05, Mann-Whitney U and Wilcoxon tests).</p