Retrograde response in axotomized motoneurons: nitric oxide as a key player in triggering reversion toward a dedifferentiated phenotype.

The adult brain retains a considerable capacity to functionally reorganize its circuits, which mainly relies on the prevalence of three basic processes that confer plastic potential: synaptic plasticity, plastic changes in intrinsic excitability and, in certain central nervous system (CNS) regions, also neurogenesis. Experimental models of peripheral nerve injury have provided a useful paradigm for studying injury-induced mechanisms of central plasticity. In particular, axotomy of somatic motoneurons triggers a robust retrograde reaction in the CNS, characterized by the expression of plastic changes affecting motoneurons, their synaptic inputs and surrounding glia. Axotomized motoneurons undergo a reprograming of their gene expression and biosynthetic machineries which produce cell components required for axonal regrowth and lead them to resume a functionally dedifferentiated phenotype characterized by the removal of afferent synaptic contacts, atrophy of dendritic arbors and an enhanced somato-dendritic excitability. Although experimental research has provided valuable clues to unravel many basic aspects of this central response, we are still lacking detailed information on the cellular/molecular mechanisms underlying its expression. It becomes clear, however, that the state-switch must be orchestrated by motoneuron-derived signals produced under the direction of the re-activated growth program. Our group has identified the highly reactive gas nitric oxide (NO) as one of these signals, by providing robust evidence for its key role to induce synapse elimination and increases in intrinsic excitability following motor axon damage. We have elucidated operational principles of the NO-triggered downstream transduction pathways mediating each of these changes. Our findings further demonstrate that de novo NO synthesis is not only "necessary" but also "sufficient" to promote the expression of at least some of the features that reflect reversion toward a dedifferentiated state in axotomized adult motoneurons.

Evidence for a detrimental role of nitric oxide synthesized by endothelial nitric oxide synthase after peripheral nerve injury.

Physical injury to a nerve is the most common cause of acquired peripheral neuropathy. Identification of molecules involved in degenerative and regenerative processes is a key step toward development of therapeutic tools in order to accelerate motor, sensory and/or autonomic function recovery. We have studied the role of nitric oxide (NO) using as a model the severe crushing of a motor nerve in adult rats. This type of injury up-regulates the three isoforms of nitric oxide synthase (NOS) in the affected nerve. Chronic systemic inhibition of NOS accelerated the onset of functional muscle reinnervation evaluated by the recording of compound muscle action potential evoked by electrical stimulation of the injured nerve. Besides, it increased the number of back-labeled motoneurons by application, 2 days after injury, of a retrograde marker 10 mm distal to the crushing site. These effects were mimicked by chronic specific inhibition of the endothelial isoform of nitric oxide synthase (eNOS), but not by specific inhibitors of the neuronal or inducible isoform. Next, we intraneurally injected a replication-deficient adenoviral vector directing the expression of a dominant negative mutant of eNOS (Ad-TeNOS). A single injection of Ad-TeNOS on the day of crushing significantly accelerated functional recovery of neuromuscular junction and increased axonal regeneration. Moreover, Ad-TeNOS did not compromise motoneuron viability or stability of reestablished neuromuscular junctions. Taken together, these results suggest that NO of endothelial origin slows down muscle reinnervation by means of detrimental actions on axonal regeneration after peripheral nerve injury. These experiments identify eNOS as a potential therapeutic target for treatment of traumatic nerve injuries and highlight the potential of gene therapy in treating injuries of this type using viral vectors to suppress the activity of eNOS.

Reversible deafferentation of abducens motoneurons and internuclear neurons with tetanus neurotoxin.

Tetanus neurotoxin (TeNT) is a blocker of synaptic vesicle exocytosis in central synapses with preferential affinity for inhibitory neurotransmission. Following its intramuscular injection, TeNT is retrogradely and trans-synaptically transported towards the premotor terminals. Therefore, we have used TeNT as a tool to study the consequences of functional deafferentation on motoneurons following its peripheral administration. For this, we injected the toxin into the lateral rectus muscle at doses of 5 or 0.5 ng/kg and recorded the discharge activity of abducens motoneurons and internuclear neurons in the alert cat. Our results showed that: (i) TeNT blocked selectively the afferent inhibitory signals on abducens neurons only when used at a low dose, whereas both excitatory and inhibitory synaptic drive was lost after the high dose treatment; (ii) all effects were reversible within one month; and (iii) strikingly, the internuclear neurons of the abducens nucleus showed similar discharge alterations to the motoneurons, suggesting a TeNT action on shared common afferences.