No abstract

Velocity sedimentation patterns of acetylcholinesterase (AChE, EC 3.7.1.1) in endplate-free regions of the diaphragm were studied in rats during early postnatal development. A significant amount of 16 S AChE, comprising 20% total activity, was found in endplate-free regions of the diaphragm of 8- and 19-day-old rats. By 32 days after birth, 16 S AChE accounted for less than 5% total AChE activity in endplate-free regions. 16 S AChE is, therefore, not strictly an endplate-specific molecular form. Instead, it becomes restricted to the motor endplate region of the rat diaphragm by the end of the first month of life.

The success of axon regeneration after nerve injury should be judged by the extent to which the target organs regain their function. Recovery of muscle contraction involves axon regeneration, reestablishment of nerve-muscle connections, recovery of transmission, and muscle force. All these processes were investigated under the same experimental conditions and correlated in order to better understand their time-course and interdependence. The sciatic nerve of a rat was crushed in the thigh. The ingrowth of regenerating motor axons into the soleus (SOL) and extensor digitorum longus (EDL) muscles was monitored by measuring the activity of choline acetyltransferase (ChAT), a marker enzyme for cholinergic nerve terminals, in the muscles. The electron microscopic cytochemistry of acetylcholine esterase (AChE) was used to estimate the reestablishment of neuromuscular junctions in these two muscles. The recovery of muscle contraction was followed by measuring the force of isometric contraction in the triceps surae muscle in vivo. The pattern of ChAT recovery during reinnervation was similar in the EDL and SOL. The statistically significant increase of ChAT activity in these muscles, 14 d after the nerve crush, signified the entry of regenerating axons into the calf muscles. Electron microscopic cytochemistry revealed the first small nerve endings in contact with the denervated end plates 12 d after denervation. Subsequently, the number of reinnervated motor end plates and the surface area of the neuromusclar junctions steadily increased. The recovery of muscle force started between d 14 and 21 after the nerve crush. Thirty-five days after denervation, the difference between the muscle force of the reinnervated muscle and the control became statistically insignificant. Morphological normalization of the motor end plates was practically complete 33 d after denervation, concomitant with the normalization of the muscle force. At that time, however, ChAT activity in both muscles was still clearly subnormal (33.5% in EDL and 45% of the control in SOL) and therefore does not reflect the true extent of muscle force recovery. Yet, it seems that in spite of this, the regenerated nerve terminals contained sufficient amounts of acetylcholine (ACh) to trigger normal muscle contractions.

Velocity sedimentation analysis of acetylcholinesterase (AChE) molecular forms was performed separately in endplate-rich and endplate-free regions of the diaphragm muscle of the rat, guinea pig, rabbit, dog, and pig, and in mm. erectores trunci and m. vastus lateralis in man. Several high-ionic-strength media were first tested to achieve better solubilization of AChE from rat muscles than by the usual 1 M NaCl-Triton X-100 medium. Ninety-five percent of the AChE from the motor endplate region of the rat diaphragm was solubilized in a single extraction step by medium containing 1 M lithium chloride instead of NaCl. Homologous molecular forms of AChE were found in all species. The asymmetric forms were invariably present in the endplate regions of muscles but their activity in endplate-free regions was much lower than in endplate regions in all investigated mammals except in man. Essentially the same pattern of AChE molecular forms was present in both regions in human muscles. High extrajunctional activity of the asymmetric forms makes human muscles similar to immature rodent muscles in vivo and in culture. The pattern of AChE molecular forms in the endplate region of the diaphragm in senile 24-month-old rats was not significantly different from that in 3-month-old animals. The persistence of the asymmetric AChE forms in the diaphragm of senile rats suggests that neuromuscular interactions do not become deficient with age in this muscle.

1. Initiation of subsynaptic sarcolemmal specialization and expression of different molecular forms of AChE were studied in fast extensor digitorum longus (EDL) and slow soleus (SOL) muscle of the rat under different experimental conditions in order to understand better the interplay of neural influences with intrinsic regulatory mechanisms of muscle cells. 2. Former junctional sarcolemma still accumulated AChE and continued to differentiate morphologically for at least 3 weeks after early postnatal denervation of EDL and SOL muscles. In noninnervated regenerating muscles, postsynaptic-like sarcolemmal specializations with AChE appeared (a) in the former junctional region, possibly induced by a substance in the former junctional basal lamina, and (b) in circumscribed areas along the whole length of myotubes. Therefore, the muscle cells seem to be able to produce a postsynaptic organization guiding substance, located in the basal lamina. The nerve may enhance the production or accumulation of this substance at the site of the future motor end plate. 3. Significant differences in the patterns of AChE molecular forms in EDL and SOL muscles arise between day 4 and day 10 after birth. The developmental process of downregulation of the asymmetric AChE forms, eliminating them extrajunctionally in the EDL, is less efficient in the SOL. The presence of these AChE forms in the extrajunctional regions of the SOL correlates with the ability to accumulate AChE in myotendinous junctions. The typical distribution of the asymmetric AChE forms in the EDL and SOL is maintained for at least 3 weeks after muscle denervation. 4. Different patterns of AChE molecular forms were observed in noninnervated EDL and SOL muscles regenerating in situ. In innervated regenerates, patterns of AChE molecular forms typical for mature muscles were instituted during the first week after reinnervation. 5. These results are consistent with the hypothesis that intrinsic differences between slow and fast muscle fibers, concerning the response of their AChE regulating mechanism to neural influences, may contribute to different AChE expression in fast and slow muscles, in addition to the influence of different stimulation patterns.