The timing of impulse activity shapes the process of synaptic competition at the neuromuscular junction.

The development of neuromuscular junctions exhibits profound remodeling that brings from an immature state characterized by multiple motoneuronal inputs per muscle fiber, to a mature mononeuronal innervation. This striking elimination process occurs both perinatally and during adult reinnervation, and is also widely present in the developing CNS. The accelerating influence of the amount of impulse activity on this process, has been shown by various studies, but a more subtle role of the time correlation of action potential firing in the competing inputs, has also been suggested. Here we explore the latter influence using a rat adult model of neuromuscular junction formation, that is reinnervation following a motor nerve crush. This shares all important features with perinatal development, especially the strict juxtaposition of the competing inputs. In fact the regenerating axons converge on a single cluster of postsynaptic receptors, that is the original endplate of each muscle fiber. This focus on the spatial aspect of competition between nerve endings was missing in our previous experiments employing a similar paradigm. We impose a chronic synchronous firing to the competing terminals, by in vivo electrical stimulation of their axons distal to a sciatic nerve conduction block. Control preparations, with similar post-crush reinnervation, are left with their natural impulse activity unperturbed. We find that the experimental muscles display a prolonged duration of polyneuronal innervation with respect to controls, indicating that hebbian mechanisms participate in the synapse elimination process. Another aspect dealt with in our study is the genuine nature of the polyneuronal innervation occurring during adult muscle reinnervation, because it is supported by both confocal microscopy and by appropriate electrophysiological tests that exclude electrical coupling of myofibers by gap junctions.

On the mechanism of action of muscle fibre activity in synapse competition and elimination at the mammalian neuromuscular junction.

Activity-dependent competition plays a crucial role in the refinement of synaptic connections in the peripheral and central nervous system. The reduction in number of axons innervating each neuromuscular junction during development, i.e. synapse elimination, appears to be one such competitive activity-driven event. Recently, we showed that asynchronous firing of competing presynaptic terminals is a key player in synapse elimination. Although some previous studies suggested that activity of the postsynaptic cell may be an intermediary in the disposal of redundant presynaptic inputs, the mechanism involved remains unknown. In the present study, in order to assess the role of evoked muscle activity in this process, we inhibited the generation of postsynaptic action potentials in muscle fibers in vivo, through the overexpression of inwardly rectifying Kir2.1 and Kir2.2 channels, via electroporation of the soleus muscle in the mouse hindlimb. Electrophysiological and morphological data show that overexpression of potassium channels in the endplate region of neonatal muscle fibres induces membrane hyperpolarization and an increase in conductance, inhibition of the action potential mechanism and prolonged persistence of polyneuronal innervation. These changes are not seen in muscle fibres with overexpression of a non-conducting Kir2.1 mutant. Our results are compatible with the interpretation that the block of action potential generation, even in single endplates, can inhibit synapse elimination through local signalling.

Effects of evoked and spontaneous motoneuronal firing on synapse competition and elimination in skeletal muscle.

Synapse elimination is a general feature of the development of neural connections, including the connections of motoneurons to skeletal muscle fibers. Our work addressed two questions: (1) how the action potentials generated in the set of motoneurons innervating an individual muscle ( i.e., in a motor pool) are correlated in time during development in vivo; (2) what influence different firing patterns exert on the processes of polyneuronal innervation and synapse elimination which characterize the establishment of muscle innervation. We recorded the spontaneous electromyographic activity of the tibialis anterior and soleus muscles of late embryonic and neonatal rats, identifying the firing of at least two single motor unit signals in each record. We found that a striking switch occurs a few days after birth from a highly synchronous type of firing to an asynchronous one, the first thus characterizing embryonic while the second one adult motoneurons. We also investigated the effects of an evoked synchronous type of discharge on neuromuscular synapse formation, measuring polyneuronal innervation and synapse elimination. This was done in an adult in vivo model of de novo synapse formation, while a chronic TTX nerve conduction block, placed centrally with respect to the stimulating electrodes, eliminated the natural activity of motoneurons. We found that the imposed synchronous activity greatly inhibits synapse elimination, causing polyneuronal innervation to persist. We conclude that the early synchronous firing, favors the establishment of polyneuronal innervation while the subsequent switch to an asynchronous one promotes synapse elimination.

In vivo acetylcholine receptor expression induced by calcitonin gene-related peptide in rat soleus muscle.

We applied calcitonin gene-related peptide (CGRP) by continuous perfusion of the extrajunctional surface of the adult rat soleus muscle in vivo. We obtained this through a fine polyethylene catheter connected to an Alzet pump implanted in the animal. The perfusion induced a local acetylcholine receptor accumulation in the membrane of the muscle fibres starting with a delay of one to two days, provided a chronic conduction block of soleus innervation was concomitantly present. The effect was prominent, being higher than that following denervation. The lack of acetylcholine receptor accumulation observed in sham perfused animals and the co-administration of CGRP and its competitive antagonist peptide, hCGRP(8-37), eliminates the possibility that the response to CGRP application represents an inflammatory reaction to foreign bodies instead of a specific effect of the peptide. We suggest that CGRP may act on the extrajunctional membrane of muscle fibres to help induce acetylcholine receptor accumulation after appropriate receptors for the peptide are re-expressed due to muscle paralysis. Whilst this is compatible with a role of CGRP in synaptogenesis, a recent study showed that alpha-CGRP(-/-) mutant mice have normal neuromuscular junction development. However, given the redundancy of factors involved in acetylcholine receptor accumulation, further experiments on multiple knock-outs need to be performed before a final conclusion is reached about the physiological significance of CGRP.

The use of in vivo direct drug application to assess neural regulation of muscle properties.

Skeletal muscle is a convenient model system for studying basic questions on the neural regulation of synaptogenesis and of many properties of sarcolemma and contractile apparatus. The study of the neural signals involved in a particular regulation and of the mediating intracellular pathways, requires the chronic application of drugs, second messengers, antibodies, trophic factors and the like. The most common way of application is in vitro treatment of muscle cell lines or primary myotube cultures. As an alternative to tissue culture, we developed a technique for in vivo application of the agents under study directly on skeletal muscle. An initial surgical step secures the tip of a fine polyethylene catheter (

Hebbian mechanisms revealed by electrical stimulation at developing rat neuromuscular junctions.

Synapse competition and elimination are widespread developmental processes, first demonstrated at neonatal neuromuscular junctions. Action potential activity was long shown to exert a powerful influence, but mechanisms and contribution relative to other factors are still not well understood. Here we show that replacement of natural motoneuronal discharge with synchronous activity suppresses elimination of polyneuronal innervation of myofibers. This requires the simultaneous chronic conduction block (tetrodotoxin) and distal electrical stimulation of motor axons during ectopic synaptogenesis in denervated adult soleus muscle. If in fact chronic stimulation is applied without central block of motor axons, the time course of synapse elimination is as fast as in control muscles undergoing natural activity. Our findings follow the prediction of Hebb's postulate and imply that asynchronous activity drives developmental synapse elimination in muscle. They further suggest that motoneurons could become transiently synchronized during development and regeneration, helping to establish the initial polyneuronal innervation.

Studies on anterograde trophic interactions based on general muscle properties.

General properties of rat skeletal muscle (extrajunctional membrane and contractile properties) are subjected to tight physiological neural regulation, as indicated by their striking alterations (up- or down-regulation) following denervation. The main contributions of the literature concerning the nature of the neural signals which mediate this regulation, are reviewed. The physiological regulation of these general properties appears to be operated by the action potential activity evoked by motoneurons in the muscle fibres. No need to postulate the participation of nerve-borne chemical substances, acetylcholine or unidentified "trophic factors", arises from the main experimental evidence. The stronger response to denervation of extrajunctional membrane properties with respect to pure paralysis is best explained by actions of factors released during wallerian degeneration of the transected nerves.

Use of dexamethasone with TTX block of nerve conduction shows that muscle membrane properties are fully controlled by evoked activity.

This paper provides further evidence that motorneurons control extrajunctional properties of skeletal muscles through the activity evoked in the muscle fibres. The experiments compare the amount of action potential resistance to tetrodotoxin (TTX resistance) in denervated soleus muscle with that in soleus whose nerve was crushed and then allowed to regenerate in the presence of a block of the sciatic impulse conduction. Measurements were taken after about 2-3 weeks to allow full reinnervation and recovery of trophic regulation by the nerve. Blocking sciatic impulse conduction with TTX solutions containing low doses of the anti-inflammatory drug dexamethasone induced values of extrajunctional TTX resistance identical to those caused by denervation. In contrast lower levels of TTX resistance were obtained with dexamethasone-free solutions or when the drug was administered through the systemic path rather than topically applied to the nerve. These results indicate that physiological neural regulatory signals other than activity do not participate to the regulation of extrajunctional properties of skeletal muscles. Furthermore the low levels of TTX resistance measured with dexamethasone-free blocks confirm our previous experiments indicating that reported differences between denervation and pure inactivity are attributable to incomplete suppression of nerve impulse conduction.

Paralysis of rat skeletal muscle equally affects contractile properties as does permanent denervation.

The effects of long lasting (4-5 weeks) nerve conduction block and denervation were compared by investigating contractile, morphological and histochemical properties of slow (soleus) and fast (EDL) rat skeletal muscles. The block was based on improved perfusion techniques of the sciatic nerve with a tetrodotoxin (TTX) solution delivered at doses adequate to obtain maximal effects in the muscles. The TTX-inactivated axons retained normal histological and physiological properties such as the ability to evoke full contractile responses, to regenerate, and to completely reinnervate muscle. In spite of their intact innervation or of their full reinnervation, the TTX-paralysed muscles underwent weight loss, fibre atrophy and reduction in force, output quantitatively indistinguishable from those following denervation. The same was true for all other contractile parameters tested, that is, twitch speed, twitch to tetanus ratio, post-tetanic potentiation, endurance, and fibre type composition. The results indicate the fundamental role of activity as a regulatory signal for muscle contractile properties, while they do not support the notion of a participation of chemical, activity-independent factors in this regulation.

Effects of long-term conduction block on membrane properties of reinnervated and normally innervated rat skeletal muscle.

1. Do motoneurons regulate muscle extrajunctional membrane properties through chemical (trophic) factors in addition to evoked activity? We addressed this question by comparing the effects of denervation and nerve conduction block by tetrodotoxin (TTX) on extrajunctional acetylcholine (ACh) sensitivity and action potential resistance to TTX in adult rats. 2. We applied TTX to sciatic or tibial nerves for up to 5 weeks using an improved blocking technique which completely suppresses conduction but avoids nerve damage. 3. Reinnervation by TTX-blocked axons had no effect on the high ACh sensitivity and TTX resistance induced by nerve crush. 4. Long-lasting block of intact nerves (up to 38 days) induced extrajunctional changes as pronounced as after denervation. At shorter times (3 days), however, denervation induced much larger changes than TTX block; such a difference is thus only transiently present in muscle. 5. The effects of long-lasting block were dose dependent. Dose levels (6.6 micrograms day-1) corresponding to those used in the literature to block the rat sciatic nerve induced muscle effects much smaller than those induced by denervation, confirming published data. Our novel finding is that equal effects are obtained using doses substantially higher (up to 10.5 micrograms day-1). For the soleus it was necessary in addition to apply the TTX directly to the smaller tibial nerve. 6. The TTX-blocked nerves were normal in their histological appearance and capacity to transport anterogradely 3H-labelled proteins, to release ACh in quantal and non-quantal form or cluster ACh receptors and induce functional ectopic junctions on denervated soleus muscles. 7. We conclude that muscle evoked activity is the physiological regulator of extrajunctional membrane properties. Chemical factors from the nerve do not appear to participate in this regulation. The stronger response to denervation at short times only is best accounted for by factors produced by degenerating nerves.

An electrophysiological study of calcium entry during normal human T-lymphocyte activation.

Our aim was to observe whether normal human T-cells respond to mitogenic stimulation with large whole-cell inward currents (composed of identifiable single-channel contributions) when [Ca2+]i is not markedly lowered but instead kept normal or moderately low, as has been reported in human leukaemic Jurkat T-cell line and T-cell clones [Kuno et al. (1986) Nature 323, 269-73; Kuno and Gardner (1987) Nature 326, 301-304; Gardner (1990) Annu. Rev. Immunol. 8, 231-252]. Whole-cell patch recordings showed no such currents in cells otherwise normally responding to depolarisation with the macroscopic IK described in T-lymphocytes and thus deemed viable, in agreement with the notion that Ca2+ influx in normal T-cells enterily depends on depletion of internal stores [Putney (1986) Cell Calcium 7, 1-12; Putney (1990) Cell Calcium 11, 611-624].

Nerve stump effects in muscle are independent of synaptic connections and are temporally correlated with nerve degeneration phenomena.

Close or distant denervation of the rat soleus muscle indicated that (1) longer soleus nerve stumps delay the onset of axon terminal degeneration and of muscle membrane changes (spike resistance to TTX) by strictly comparable times, and (2) the stump-induced delay of the muscle effect is independent of synaptic connections, because it is also obtained (RMP fall and TTX-resistance development) when sectioning a foreign nerve previously transplanted on the soleus surface but not making synaptic contacts. Both lines of evidence are consistent with the interpretation that, as far as the extrajunctional membrane properties are concerned, the effect of the length of the nerve stump on muscle is mediated by nerve terminal breakdown.