TTX Resistance Research Articles

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 administred 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.

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.

Length density and total length of acetylcholinesterase positive nerves related to cystometry and in vitro studies of muscle strips in mini‐pig urinary bladder after chronic outflow obstruction and recovery from obstruction

Chronic partial bladder outlet obstruction was created in mini-pigs by implanting a 6-7 mm ring around the proximal urethra. After a median obstruction period of 63 days, the ring was removed and after a median recovery period of 60 days the animals were sacrificed. At each occasion stepwise cystometry, measurement of residual urine, muscle strips studies with electrical and carbachol stimulation, and stereological estimations of length density and total length of acetylcholinesterase positive nerves were performed. The results can be summarized as follows: (1) unchanged sensitivity of muscle strips to carbachol, but markedly decreased contractility and rate of contraction to carbachol, (2) no evidence of detrusor instability, but severely decompensated bladders in two pigs, (3) a significant increase in residual volume, (4) a pronounced decrease in length density and total length of acetylcholinesterase positive nerves, and (5) at field stimulation strips from some pigs showed increased sensitivity and contractility with high atropine and TTX resistance, while strips from the other pigs revealed decreased sensitivity and markedly decreased contractility to electrical stimulation. In general, most of the changes were markedly, though incompletely, reversed after recovery. Light and electron microscopy of muscle strips showed no histological or ultrastructural changes during the experiments or after storing 1 day at 4 degrees C.

The development of tetrodotoxin-resistant action potentials in long-term organ culture of rat muscle.

The development and long-term maintenance of tetrodotoxin (TTX)-resistant action potentials in culture was examined. The amplitude and maximum rate of rise of the action potential in normal Ringer solution fell during culture. At the same time the amplitude and maximum rate of rise of the TTX-resistant action potential increased, demonstrating that TTX resistance can develop fully in culture. Actinomycin D inhibited the onset of denervation changes in culture and was also able to delay the fall in the maximum rate of rise of the action potential in normal Ringer solution. When applied to muscle after 48 h of culture, it was relatively ineffectual, showing that de novo protein synthesis during the first 48 h of culture result in denervation changes which are not reversed by subsequent exposure to actinomycin D. The onset of TTX resistance occurred more rapidly in dissociated muscle fibres, presumably as a result of the early removal of the nerve stump.

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.

Control of ACh sensitivity in temporarily unconnected (“decentralized”) segments of diaphragm-muscle fibres of the rat

1. After a local lesion of the diaphragm muscle, which produced a segment of innervated muscle fibres connected with an intact nerve-free segment by a region of crushed muscle fibres, the sensitivity to ACh, the presence of tetrodotoxin resistant action potentials (AP) and the transmission of AP along the muscle fibres were studied. 2. Three days after local injury of the diaphragm muscle ACh sensitivity and TTX resistance appeared in the crushed and nerve-free segments between the place of injury and the tendineous attachment. 5-7 days after injury transmission of action potentials through the damaged to the undamaged ("decentralized"), nerve-free part of the fibres is restored. High ACh sensitivity and TTX resistance of the latter segment, however, are completely lost only 20 days after the local injury. During this period contractility of the muscle remains practically unchanged. Enzymatic activities (SDH, ATPase and phosphorylase) of the damaged part were lost 3 days after crushing and recovered slowly between 7-10 days of regeneration of the diaphragm muscle fibres. 3. The experiments suggest that during regeneration of damaged muscle fibres supersensitivity to ACh remains high inspite of normal AP activity and that intracellular mechanisms may be involved in the induction and disappearance of ACh hypersensitivity.

Action potentials in chick atria. Increased susceptibility to blockade by tetrodotoxin during embryonic development.

Tetrodotoxin (TTX) at a concentration of 1.6 x 10 -7 M blocked intracellularly recorded action potentials in 12- and 18-day-old embryonic chick atria. However, TTX, 1 x 10 -5 M, did not abolish action potentials recorded from cells in 6-day-old atria. TTX-sensitive receptor sites were present in 6-day-old atrial membranes because the toxin depressed the early Na + conductance that generated the rapid depolarization phase and the maximum rate of rise of the action potential. TTX at 0.9 x 10 -8 M reduced the maximum rate of rise by 50% in 6-day-old atria, and TTX at 2 x 10 -8 M had the same effect in 12- and 18-day-old preparations. Action potential overshoot changed about 10-20 mv/tenfold variation in external Na + concentration in 6-day-old atria in contrast to the change of about 60 mv/decade observed in 18-day-old atria. Moreover, the TTX-resistant action potentials in 6-day-old atria depended primarily on an inward Ca 2+ current because overshoot changed 24 mv/decade variation in external Ca 2+ concentration. The improvement in sodium-electrode properties that occurred during maturation was accompanied by an increase in the maximum rate of rise of the action potential, which averaged 64 v/sec in 6-day-old atria and 94 v/sec in 18-day-old atria. These findings suggested that the early Na + conductance mechanism became more critical in generating the action potential in older embryonic atria. These data were consistent with the hypothesis that the membrane density of early conductance sites increased with embryonic age. The TTX resistance of 6-day-old atria depended on an inward Ca 2+ current that generated action potentials after the early Na + conductance sites had been blocked by the toxin.