Exercise-induced motor improvement after complete spinal cord transection and its relation to expression of brain-derived neurotrophic factor and presynaptic markers

<p>Abstract</p> <p>Background</p> <p>It has been postulated that exercise-induced activation of brain-derived neurotrophic factor (BDNF) may account for improvement of stepping ability in animals after complete spinal cord transection. As we have shown previously, treadmill locomotor exercise leads to up-regulation of BDNF protein and mRNA in the entire neuronal network of intact spinal cord. The questions arise: (i) how the treadmill locomotor training, supplemented with tail stimulation, affects the expression of molecular correlates of synaptic plasticity in spinal rats, and (ii) if a response is related to BDNF protein level and distribution.</p> <p>We investigated the effect of training in rats spinalized at low thoracic segments on the level and distribution of BDNF immunoreactivity (IR) in ventral quadrants of the lumbar segments, in conjunction with markers of presynaptic terminals, synaptophysin and synaptic zinc.</p> <p>Results</p> <p>Training improved hindlimb stepping in spinal animals evaluated with modified Basso-Beattie-Bresnahan scale. Grades of spinal trained animals ranged between 5 and 11, whereas those of spinal were between 2 and 4. Functional improvement was associated with changes in presynaptic markers and BDNF distribution. Six weeks after transection, synaptophysin IR was reduced by 18% around the large neurons of lamina IX and training elevated its expression by over 30%. The level of synaptic zinc staining in the ventral horn was unaltered, whereas in ventral funiculi it was decreased by 26% postlesion and tended to normalize after the training. Overall BDNF IR levels in the ventral horn, which were higher by 22% postlesion, were unchanged after the training. However, training modified distribution of BDNF in the processes with its predominance in the longer and thicker ones. It also caused selective up-regulation of BDNF in two classes of cells (soma ranging between 100-400 μm²and over 1000 μm²) of the ventrolateral and laterodorsal motor nuclei.</p> <p>Conclusion</p> <p>Our results show that it is not BDNF deficit that determines lack of functional improvement in spinal animals. They indicate selectivity of up-regulation of BDNF in distinct subpopulations of cells in the motor nuclei which leads to changes of innervation targeting motoneurons, tuned up by locomotor activity as indicated by a region-specific increase of presynaptic markers.</p

Overexpression of BDNF increases excitability of the lumbar spinal network and leads to robust early locomotor recovery in completely spinalized rats.

Strategies to induce recovery from lesions of the spinal cord have not fully resulted in clinical applications. This is a consequence of a number of impediments that axons encounter when trying to regrow beyond the lesion site, and that intraspinal rearrangements are subjected to. In the present study we evaluated (1) the possibility to improve locomotor recovery after complete transection of the spinal cord by means of an adeno-associated (AAV) viral vector expressing the neurotrophin brain-derived neurotrophic factor (BDNF) in lumbar spinal neurons caudal to the lesion site and (2) how the spinal cord transection and BDNF treatment affected neurotransmission in the segments caudal to the lesion site. BDNF overexpression resulted in clear increases in expression levels of molecules involved in glutamatergic (VGluT2) and GABAergic (GABA, GAD65, GAD67) neurotransmission in parallel with a reduction of the potassium-chloride co-transporter (KCC2) which contributes to an inhibitory neurotransmission. BDNF treated animals showed significant improvements in assisted locomotor performance, and performed locomotor movements with body weight support and plantar foot placement on a moving treadmill. These positive effects of BDNF local overexpression were detectable as early as two weeks after spinal cord transection and viral vector application and lasted for at least 7 weeks. Gradually increasing frequencies of clonic movements at the end of the experiment attenuated the quality of treadmill walking. These data indicate that BDNF has the potential to enhance the functionality of isolated lumbar circuits, but also that BDNF levels have to be tightly controlled to prevent hyperexcitability

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

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

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

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

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

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

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