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.

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.

Neuroprotection of Retinal Ganglion Cells with AAV2-BDNF Pretreatment Restoring Normal TrkB Receptor Protein Levels in Glaucoma.

Intravitreal delivery of brain-derived neurotrophic factor (BDNF) by injection of recombinant protein or by gene therapy can alleviate retinal ganglion cell (RGC) loss after optic nerve injury (ONI) or laser-induced ocular hypertension (OHT). In models of glaucoma, BDNF therapy can delay or halt RGCs loss, but this protection is time-limited. The decreased efficacy of BDNF supplementation has been in part attributed to BDNF TrkB receptor downregulation. However, whether BDNF overexpression causes TrkB downregulation, impairing long-term BDNF signaling in the retina, has not been conclusively proven. After ONI or OHT, when increased retinal BDNF was detected, a concomitant increase, no change or a decrease in TrkB was reported. We examined quantitatively the retinal concentrations of the TrkB protein in relation to BDNF, in a course of adeno-associated viral vector gene therapy (AAV2-BDNF), using a microbead trabecular occlusion model of glaucoma. We show that unilateral glaucoma, with intraocular pressure ( IOP) increased for five weeks, leads to a bilateral decrease of BDNF in the retina at six weeks, accompanied by up to four-fold TrkB upregulation, while a moderate BDNF overexpression in a glaucomatous eye triggers changes that restore normal TrkB concentrations, driving signaling towards long-term RGCs neuroprotection. We conclude that for glaucoma therapy, the careful selection of the appropriate BDNF concentration is the main factor securing the long-term responsiveness of RGCs and the maintenance of normal TrkB levels.

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/.

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

The effects of stimulation of low-threshold proprioceptive afferents in the tibial nerve on two types of excitatory inputs to α-motoneurons were tested. The first input is formed by glutamatergic Ia sensory afferents contacting monosynaptically α-motoneurons. The second one is the cholinergic input originating from V0c-interneurons, located in lamina X of the spinal cord, modulating activity of α-motoneurons via C-terminals. Our aim was to clarify whether enhancement of signaling to ankle extensor α-motoneurons, via direct electrical stimulation addressed predominantly to low-threshold proprioceptive fibers in the tibial nerve of awake rats, will affect Ia glutamatergic and cholinergic innervation of α-motoneurons of lateral gastrocnemius (LG). LG motoneurons were identified with True Blue tracer injected intramuscularly. Tibial nerve was stimulated for 7 days with continuous bursts of three pulses applied in four 20 min sessions daily. The Hoffmann reflex and motor responses recorded from the soleus muscle, LG synergist, allowed controlling stimulation. Ia terminals and C-terminals abutting on LG-labeled α-motoneurons were detected by immunofluorescence (IF) using input-specific anti- VGLUT1 and anti-VAChT antibodies, respectively. Quantitative analysis of confocal images revealed that the number of VGLUT1 IF and VAChT IF terminals contacting the soma of LG α-motoneurons increased after stimulation by 35% and by 26%, respectively, comparing to the sham-stimulated side. The aggregate volume of VGLUT1 IF and VAChT IF terminals increased by 35% and by 30%, respectively. Labeling intensity of boutons was also increased, suggesting an increase of signaling to LG α-motoneurons after stimulation. To conclude, one week of continuous burst stimulation of proprioceptive input to LG α-motoneurons is effective in enrichment of their direct glutamatergic but also indirect cholinergic inputs. The effectiveness of such and longer stimulation in models of injury is a prerequisite to propose it as a therapeutic method to improve inputs to selected group of α-motoneurons after damage.

The impact of training and neurotrophins on functional recovery after complete spinal cord transection: cellular and molecular mechanisms contributing to motor improvement.

Beneficial effects of locomotor training on the functional recovery after complete transection of the spinal cord indicate that in chronic spinal animals spontaneous recovery processes are enhanced and shaped by the training. The mechanisms of that use-dependent improvement are still not fully understood. This review tackles three aspects of this issue: (1) neurochemical attributes of functional improvement showing that concentrations of excitatory and inhibitory amino acids in the lumbar spinal segments, which were changed after transection, normalize after the training, or even raise beyond normal. As it does not translate to functional equilibrium between excitatory and inhibitory neurotransmission and may lead to hyperexcitability, the postsynaptic mechanisms which might be responsible for the hyperexcitability are discussed, including (i) dysfunction of K(+)-Cl(-) cotransporter KCC2, which controls the strength and robustness of inhibition, and (ii) altered function of 5-HT2 receptors, which may be targeted to restore KCC2 activity and intrinsic inhibition; (2) morphological changes of lumbar motoneurons and their inputs related to functional improvement of spinal animals, pointing to use-dependent diminution/reversal of the atrophy of the dendritic tree of the hindlimb motoneurons and of their synaptic impoverishment, which in paraplegic animals differs depending on the degree of disuse of the muscles; (3) the role of neurotrophins in motor improvement of spinal animals showing, that increases in neurotrophins due to training or due to efficient viral vector-based transgene expression, that might be responsible for the enrichment of the dendritic tree, elongation of processes and influence neurotransmitter systems in the areas subjected to plastic modifications after injury, correlate with improvement of locomotor functions.

Enhancing proprioceptive input to motoneurons differentially affects expression of neurotrophin 3 and brain-derived neurotrophic factor in rat hoffmann-reflex circuitry.

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.

Different effects of spinalization and locomotor training of spinal animals on cholinergic innervation of the soleus and tibialis anterior motoneurons.

Cholinergic input modulates excitability of motoneurons and plays an important role in the control of locomotion in both intact and spinalized animals. However, spinal cord transection in adult rats affects cholinergic innervation of only some hindlimb motoneurons, suggesting that specificity of this response is related to functional differences between motoneurons. Our aim was therefore to compare cholinergic input to motoneurons innervating the soleus (Sol) and tibialis anterior (TA) motoneurons following spinal cord transection at a low-thoracic level. The second aim was to investigate whether deficits in cholinergic input to these motoneurons could be modified by locomotor training. The Sol and TA motoneurons were identified by retrograde labelling with fluorescent dyes injected intramuscularly. Cholinergic terminals were detected using anti-vesicular acetylcholine transporter (VAChT) antibody. Overall innervation of motoneurons was evaluated with anti-synaptophysin antibody. After spinalization we found a decrease in the number of VAChT-positive boutons apposing perikarya of the Sol (to 49%) but not TA motoneurons. Locomotor training, resulting in moderate functional improvement, partly reduced the deficit in cholinergic innervation of Sol motoneurons by increasing the number of VAChT-positive boutons. However, the optical density of VAChT-positive boutons terminating on various motoneurons, which decreased after spinalization, continued to decrease despite the training, suggesting an impairment of acetylcholine availability in the terminals. Different effects of spinal cord transection on cholinergic innervation of motoneurons controlling the ankle extensor and flexor muscles point to different functional states of these muscles in paraplegia as a possible source of activity-dependent signaling regulating cholinergic input to the motoneurons.