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.

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.

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.

Effects of reinnervation with normal and tetrodotoxin-inactive nerves on resting membrane potential of rat skeletal muscle.

Resting membrane potentials (RMPs) have been recorded in vitro near the end-plate region of rat soleus muscles reinnervated with tetrodotoxin-inactive nerves and compared with those of denervated muscles whose reinnervation had been prevented. The two muscle groups exhibited the same low values of RMP typical of denervated muscles. In control muscles of rats in which impulse conduction was left unimpaired, reinnervation induced the expected increase in RMP values towards normal. It is suggested that, at least for this property, reinnervation restores to normal the muscle fibre membrane essentially through the return of activity.

Interaction of inactivity and nerve breakdown products in the origin of acute denervation changes in rat skeletal muscle.

The action of nerve breakdown products on innervated fibres of soleus and extensor digitorum longus muscles was investigated with the following procedures: partial denervation, sensory or sympathetic denervation, section of a previously transplanted foreign nerve. Each procedure was performed either in isolation or combined with chronic muscle inactivity obtained by blocking impulse conduction along the sciatic nerve. Silastic cuffs containing tetrodotoxin (TTX) and sodium chloride were utilized for the block. Partial denervation induced extrajunctional sensitivity to acetylcholine (ACh) and resistance to tetrodotoxin not only in the denervated but also in the innervated fibres. The effects in the innervated fibres were equal in magnitude to those in the denervated fibres, provided they were paralysed. The onset of the membrane changes was synchronous in the two classes of fibres and their amount correlated with the extent of partial denervation. If the innervated fibres were normally active, the membrane changes were still detectable, but considerably smaller than in the denervated fibres. Sensory denervation (removal of dorsal root ganglia L4 and L5) was followed by the development of moderate ACh supersensitivity and TTX resistance in chronically paralysed muscles. Furthermore, section of radicular nerves (total denervation, i.e. efferent plus afferent) induced larger membrane changes than those observed following section of ventral roots alone (efferent denervation). Sympathetic denervation was ineffective even when associated with chronic muscle paralysis. Section of a previously transplanted mixed nerve (superficial fibular) was ineffective if the soleus muscle was normally active, while it induced marked extrajunctional ACh sensitivity and TTX resistance when combined with chronic paralysis of the muscle. Section of a transplanted sensory nerve (sural) also induced extrajunctional membrane changes in paralysed soleus muscles, but their magnitude was much smaller than after section of mixed nerves. We conclude that products of nerve destruction, especially those of motor axons, induce membrane changes of striking magnitude when potentiated by muscle inactivity. Such an action may also explain the greater efficacy of denervation vs. pure inactivity, at least at early times after their onset.

Effects of reinnervation upon electrical membrane properties of normal and paralyzed muscles.

Resting membrane potentials (RMP) and resistance to tetrodotoxin (TTX) have been compared in denervated rat soleus muscles and muscles reinnervated with tetrodotoxin-inactive nerves for periods of 15-18 days. RMP's of the two muscle groups exhibited the same low values typical of denervated muscles. Similarly, comparable values of TTX-resistance were found in the two muscle groups, although exceptions with slightly lower values in the innervated-paralyzed muscles were noted. It is concluded that muscle reinnervation restores to normal the membrane properties altered by denervation essentially through the return of muscle activity.

The sodium current underlying the responses of toad rods to light.

1. Intracellular responses were recorded from single rods in the retina of the toads Bufo bufo and Bufo marinus during exposure to solutions in which sodium was replaced by equimolar amounts of choline. 2. Upon moderate reduction (80 and 50 mM) of the external sodium the size of responses to bright flashes decreased as a consequence of both an increase in the resting potential and a decrease of the membrane potential at the peak, while the level of the plateau remained fairly constant. 3. Upon reduction of the external sodium to 22 mM or less, rods hyperpolarized to about the plateau level and failed to respond to illumination. Under these circumstances, membrane depolarization induced by an increased external potassium did not restore the cell responsiveness. Addition of 2-5 mM caesium hyperpolarized the membrane and partially restored the photoresponse. 4. Complete replacements of external sodium with potassium depolarized the rod by 40 +/- 10 mV, and no voltage responses to light could be detected. 5. In the presence of caesium, a nearly complete blockage of the photoresponses was obtained when the external sodium was 5 mM or less. Further reductions of the external sodium did not invert the photoresponses. Application of caesium when the external sodium was nominally zero induced a transient hyperpolarization followed by a slow decay. 6. During exposure to steady illumination, the dependence of the plateau level on the external sodium slowly increased. 7. These results indicate that the ionic current which is directly modulated by the light depends primarily on the external sodium. They suggest also that the current associated with the voltage- and time-dependent process responsible for the sag from peak to plateau of the response to a bright flash of light may have multiple components.

Rod photoresponses in the absence of external sodium in retinae treated with phosphodiesterase inhibitors.

Exposure of isolated toad retinae to phosphodiesterase inhibitors, induced changes in the ionic permeability of rod cells. Under similar conditions intracellularly recorded light responses were observed also in the absence of external Na+. Hyperpolarizing photoresponses in Na+-free media required the presence of divalent cations among which Mg2+, Mn2+ and Ba2+ were the most effective.

Electrical responses of rods in the retina of Bufo marinus.

1. Intracellular responses to flashes and steps of light have been recorded from the outer segment and the cell body of rods in the retina of the Bufo marinus. The identification of the origin of recorded responses has been confirmed by intracellular marking.2. Responses to flashes delivered in darkness or superimposed on a background were analysed. Responses recorded from outer segments conform to the principle of ;spectral univariance'. The shape of the response is not affected by enlarging the spot diameter from 150 to 1000 mum.3. The membrane potential measured in darkness at the outer segments varied from -15 to -25 mV. Injection of steady hyperpolarizing currents increases the size of the response to light; depolarizing currents reduce the response. The mean value of the input resistance is 97 +/- 30 MOmega in darkness and increases by 20-30% during illumination.4. The responses obtained from the cell body of rods have the same shape, time course and spectral sensitivity of those recorded at the outer segment. Injection of steady current at the cell body produces different effects than at the outer segment: hyperpolarizing currents reduce the amplitude of the response to light; depolarizing currents increase the response.5. The experimental data are fitted according to a model similar to that used to describe the responses of turtle cones (Baylor & Hodgkin, 1974; Baylor, Hodgkin & Lamb, 1974a, b).6. The model reproduces the electrical responses of the rod outer segment to a variety of stimuli: (a) brief flashes and steps of light in dark adapted conditions; (b) bright flashes superimposed on background illuminations; (c) pairs of flashes delivered at different time intervals. Responses to hyperpolarizing steps of current are also reproduced by the model.