Retrograde Labeling Of Motoneurons Research Articles

Synaptic stripping and MHC class I expression in the facial motor nucleus of ALS mice

Pathogenetic mechanisms involved in the fatal, still incurable neurodegenerative disease amyotrophic lateral sclerosis (ALS), characterized by progressive motoneuron death, await full clarification, important for the development of new therapeutic approaches. In the ALS murine model provided by mutant SOD1(G93A) mice, we here investigated the presynaptic wiring of facial motoneurons in basal conditions and after facial nerve transection (a classical paradigm to examine the retrograde motoneuron response to injury), and major histocompatibility (MHC) class I antigen expression after axotomy. The study was based on fluorescent retrograde labeling of motoneurons, synaptophysin and MHC class I antigen immunostaining, electron microscopy. A significant decrease of excitatory axosomatic boutons was found in presymptomatic ALS mice compared to the wild-type (Wt) counterpart, indicating the occurrence of excitatory synapse detachment (presynaptic stripping) in mutant motoneurons. Synaptic stripping, which seems to represent a protective mechanism preserving the inhibitory input, became more marked in facial motoneurons of symptomatic ALS mice. After axotomy, synaptic stripping was consistently enhanced in ALS mice. In the axotomized facial motoneurons of Wt mice synaptic stripping was accompanied by induction of MHC class I antigens, immune molecules implicated in activity-dependent changes in synaptic connectivity and regeneration after injury. MHC class I antigen induction was instead decreased in the axotomized facial nucleus of presymptomatic ALS mice, and was very low, occurring only in glial cells, in symptomatic ALS mice. The findings demonstrate enhanced loss of excitatory presynaptic terminals, as well as a dissociation between this process and MHC class I antigen expression after injury, in motoneurons which carry a mutation committing them to death. The findings also implicate MHC class I antigen induction in glial cells surrounding ALS motoneurons in this intercellular crosstalk.

BDNF/TrkB signaling regulates HNK-1 carbohydrate expression in regenerating motor nerves and promotes functional recovery after peripheral nerve repair

Functional recovery after peripheral nerve injury is often poor despite high regenerative capacity of peripheral neurons. In search for novel treatments, brief electrical stimulation of the acutely lesioned nerve has recently been identified as a clinically feasible approach increasing precision of axonal regrowth. The effects of this stimulation appear to be mediated by BDNF and its receptor, TrkB, but the down-stream effectors are unknown. A potential candidate is the HNK-1 carbohydrate known to be selectively reexpressed in motor but not sensory nerve branches of the mouse femoral nerve and to enhance growth of motor but not sensory axons in vitro. Here, we show that short-term low-frequency electrical stimulation (1 h, 20 Hz) of the lesioned and surgically repaired femoral nerve in wild-type mice causes a motor nerve-specific enhancement of HNK-1 expression correlating with previously reported acceleration of muscle reinnervation. Such enhanced HNK-1 expression was not observed after electrical stimulation in heterozygous BDNF or TrkB-deficient mice. Accordingly, the degree of proper reinnervation of the quadriceps muscle, as indicated by retrograde labeling of motoneurons, was reduced in TrkB+/− mice compared to wild-type littermates. Also, recovery of quadriceps muscle function, evaluated by a novel single-frame motion analysis approach, and axonal regrowth into the distal nerve stump, assessed morphologically, were considerably delayed in TrkB+/− mice. These findings indicate that BDNF/TrkB signaling is important for functional recovery after nerve repair and suggest that up-regulation of the HNK-1 glycan is linked to this phenomenon.

C fragment of tetanus toxin hybrid proteins evaluated for muscle-specific transsynaptic mapping of spinal motor circuitry in the newborn mouse

We investigated whether the non-toxic C fragment of tetanus toxin (TTC) fused to either beta-galactosidase or green fluorescent protein could be utilized to transsynaptically trace muscle-specific spinal circuitry in the neonatal mouse after i.m. injection into a single hindlimb muscle. We found that even with careful low volume injection (0.2–1.0 μl) into a single muscle (medial gastrocnemius), the TTC hybrid proteins spread rapidly to many other hindlimb muscles and to trunk musculature such that retrograde labeling of motoneurons could not be constrained to a single motoneuron pool. Retrogradely labeled motoneurons in the lower lumbar segments harboring the medial gastrocnemius motoneuron pool were first observed two hours after the medial gastrocnemius injection. Within the next 10 h, additional lumbar and lower thoracic motoneurons became labeled, and punctate labeling in the neuropil surrounding the motoneurons appeared. Many of the TTC hybrid protein-labeled puncta in the neuropil co-localized synaptotagmin, indicating that they represent presynaptic axon terminals onto motoneurons. Although this is consistent with retrograde transsynaptic passage, we found no evidence that the TTC hybrid proteins were transported further along premotor axons to label interneuron somata. The i.m. TTC injection procedure described here therefore provides an important tool for the study of presynaptic terminals onto motoneurons. However, additional technical modifications will be required to utilize TTC tracers for transsynaptic mapping of muscle-specific spinal motor circuitry in the neonatal mouse. We provide here a set of criteria for assessing the i.m. delivery of TTC tracers as a basis for future improvements in this technique.

The course of aberrant reinnervation following nerve repair with fresh or denatured muscle autografts

Denatured muscle grafts obtained by freeze thawing have been proposed to replace losses in the peripheral nerves. In the present report, we compare the performance of such grafts with fresh grafts in the rat median nerve. A long-term effect of muscle interposition on reinnervation was studied by behavioral assessment, muscle ATPase histochemistry, and retrograde labeling of motoneurons. There was no difference in grasping strength recovery between fresh and denatured 10-mm-long muscle grafts. Recovery was delayed and incomplete. Twelve months after surgery, only 50% of the normal grasping strength was attained. Grasping recovery was not observed in the 20-mm-long graft groups. Pathway reinnervation was non-specific with a huge amount of motor fiber misdirection. A decrease in the number of misdirected motor fibers occurred with time and activity recovery. Muscle reinnervation was not specific with disturbance of the mosaic pattern and type-grouping formation. Preference of type I axons for reinnervating deeper zones was observed. Type I aberrant reinnervation was demonstrated in the muscle periphery. The mosaic distribution of type I and II muscle fibers was not stable, and readjustments were observed with time, correlating with grasping improvement. During grasping strength recovery, there was a decrease in the number of type I fibers peripherally located and an increase of those deeply disposed. A time- and activity-related recovery was associated with readjustment in the pathways and muscle fiber rearrangement. We suggest that muscle activity generates specificity.

Opposite impacts of tenascin‐C and tenascin‐R deficiency in mice on the functional outcome of facial nerve repair

The glycoproteins tenascin-C (TNC) and tenascin-R (TNR) are extracellular matrix proteins involved in the development, plasticity and repair of the nervous system. Altered expression patterns after nerve lesions in adult animals have suggested that these molecules influence axonal regeneration. To test this hypothesis, we investigated adult mice constitutively deficient in the expression of TNC, TNR or both, using the facial nerve injury paradigm. Quantitative analysis of vibrissal movements prior to nerve transection and repair (facial-facial anastomosis) did not reveal genotype-specific differences, and thus impacts of the mutations on motor function in intact animals. Two months after nerve repair, recovery of vibrissal whisking was poor in wild-type mice, a typical finding after facial-facial anastomosis in rodents. Differential effects of the mutations on whisking were found: recovery of function was worse in TNC-deficient and better in TNR null mice compared with wild-type littermates. In double-knockout animals, vibrissal performance was insufficient, but to a lesser extent compared with TNC null mutant mice. Retrograde labelling of motoneurons in the same animals showed that similar numbers of motoneurons had reinnervated the whisker pads in all experimental groups precluding varying extents of motoneuron death and/or axon regeneration failures as causes for the different outcomes of nerve repair. Our results provide strong evidence that TNC promotes and TNR impedes recovery after nerve lesion. These findings are of particular interest with regard to the scanty knowledge about factors determining success of regeneration in the peripheral nervous system of mammals.

Retrograde response of the rat facial motor nucleus to muscle inflammation elicited by phytohaemagglutinin.

To investigate whether motoneurons react to signals deriving from target inflammation, we studied the facial motor nucleus after injections of phytohaemagglutinin in the snout of adult rats. This plant lectin is a tool widely used to induce proliferation and activation of T lymphocytes, and we observed marked lymphocyte infiltration in the injected facial muscles. Retrograde labelling of motoneurons was not detected after peripheral injections of fluorochrome-conjugated phytohaemagglutinin. Nitric oxide synthase, revealed by NADPH-diaphorase histochemistry, OX-42-immunoreactive microglia, and expression of the cell death repressor gene bcl-2, investigated with nonradioactive in situ hybridization and immunohistochemistry, were evaluated in the facial nucleus. Daily phytohaemagglutinin injections for 4 days, mimicking repeated muscle exposure to inflammatory stimuli, resulted after 2-day survival in NADPH-diaphorase induction in motoneurons and marked activation of the surrounding microglia. Quantitative image analysis of NADPH-diaphorase staining, and OX-42 immunoreactivity and microglial cell counts indicated highly significant increases with respect to saline-injected control cases. The occurrence of a neuroprotective retrograde response was evaluated monitoring bcl-2 expression. Following single phytohaemagglutinin administration, bcl-2 mRNA was significantly upregulated at 6 h in facial motoneurons and returned to basal levels at 24 h. Bcl-2 immunoreactivity was markedly upregulated at 24 h and was still significantly higher than in controls at 7 days, when concomitant NADPH-diaphorase induction in motoneurons and microglia activation was also observed. No degenerative features were observed in motoneurons after phytohaemagglutinin injections at the examined time-points. The data point out that local muscle inflammation retrogradely elicits gene activation in motoneurons and their microenvironment.

Phosphatidylinositol 3-kinase activity in murine motoneuron disease: the progressive motor neuropathy mouse.

A murine model of motoneuron disease, the pmn/pmn mouse, shows a reduction in the retrograde transport of fluorescent probes applied directly onto the cut end of sciatic nerve. Brain-derived neurotrophic factor (BDNF), when co-applied with fluorescent tracers, increases the number of retrograde labelled motoneurons. We demonstrate here that spinal cord tissue from pmn/pmn mice had significantly reduced phosphatidylinositol 3-kinase activity and expression in the particulate fraction compared to controls, without changes in the activities or expression of the downstream kinases, protein kinase B/Akt or Erk1. Systemic administration of BDNF augmented phosphatidylinositol 3-kinase specific activity in spinal cord tissue from pmn/pmn and control mice, with a greater elevation in the particulate fractions of pmn/pmn mice than in controls. We examined the effect of inhibitors of phosphatidylinositol 3-kinase and mitogen-activated protein kinase kinase on the retrograde labelling of motoneurons, 24h following the direct application of inhibitors and Fluorogold to the cut end of sciatic nerve in control and pmn/pmn mice (labelling index). The mitogen-activated protein kinase kinase inhibitor PD 98059 had no effect on the labelling index in control or pmn/pmn mice. In the absence of exogenous BDNF, phosphatidylinositol 3-kinase inhibitors reduced the number of labelled motoneurons in control mice, without changing the labelling index in pmn/pmn. Co-application of phosphatidylinositol 3-kinase inhibitors with BDNF to the cut end of sciatic nerve blocked the action of BDNF on retrograde labelling in pmn/pmn mice. These results indicate that the retrograde labelling of motoneurons is mediated by phosphatidylinositol 3-kinase-dependent and -independent pathways. In pmn/pmn mice, phosphatidylinositol 3-kinase activity in spinal neurons is below the level required for optimal retrograde labelling of motoneurons and labelling can be augmented by the administration of growth factors stimulating phosphatidylinositol 3-kinase activity. The data indicate that phosphatidylinositol 3-kinase activity is important in the uptake and/or retrograde transport of substances by motoneurons and is altered in this model of motoneuron diseases.

Topographic mapping of motor pools onto skeletal muscles

We have studied the segmental innervation of 2 rat skeletal muscles, the diaphragm and the serratus anterior. Both muscles are thin, flat, and composed of several sectors that form a clear rostrocaudal progression. Each is innervated through a single nerve, which is in turn supplied by motor neurons from several cervical spinal segments. Using intracellular recording, we found that in both cases, the rostrocaudal axis of the motor pool is systematically mapped onto the rostrocaudal axis of the muscle's surface. For the diaphragm, electrophysiological results were confirmed by immunohistochemical identification of denervated fibers following section of single ventral roots and by retrograde labeling of motoneurons following localized application of fluorescent dyes. In addition, an immunohistochemical method was used to study the arrangement of motor axons in the phrenic nerve, which supplies the diaphragm, and to show that contributions from individual ventral roots are compartmentalized within this nerve. We suggest that segmental ordering of axons in the nerve, axonal guidance at branch points in the nerve, and positional labels within the muscle may all contribute to the rostrocaudal mapping of motor pools onto muscle.

Acrylamide alters oxidative enzyme activity in rat motoneurons

Acrylamide (ACR) produces a central-peripheral distal axonopathy, via an unknown mechanism. We have investigated the effects of ACR on the activity of enzymes responsible for the oxidation of NADH (NADH-tetrazolium reductase activity, NADH-TR) with quantitative histochemical techniques. Chronic (5- or 10-day) injection of ACR (50 mg/kg/day) resulted in a significant decrease in enzyme activity in soleus motoneurons, which normally have high NADH-TR activity. The NADH-TR activity in motoneurons with low oxidative metabolism was not significantly affected. Retrograde labeling of motoneurons with horseradish peroxidase was diminished by the acrylamide treatment. These data demonstrate an acrylamide-induced change in the oxidative metabolism of certain motoneurons; further study will determine whether oxidative metabolism is the primary site of action of ACR in producing the distal axonopathy.

Staining of the dorsal root primary afferent fibers by anterograde movement of horseradish peroxidase and retrograde labelling of motoneurons and preganglionic autonomic cells in the turtle spinal cord

The enzyme horseradish peroxidase (HRP) has been applied to the proximal stumps of severed thoracic and lumbar dorsal roots in the turtles Pseudemys scripta elegans and Testudo hermanni. The results obtained confirm the distribution pattern observed with anterograde degeneration techniques. Primary afferent terminal structures, however, could be demonstrated in more detail. Commonly, ventral roots were also more or less damaged which resulted in the presence of retrogradely labeled motoneurons and small cells in area X. This group of retrogradely labeled small neurons probably represents cells of origin of preganglionic autonomic fibers.