A quantitative analysis of the spinal motor pool and its target muscle during growth in the dogfish, Scyliorhinus canicula.

In order to relate the phenomenon of continuous growth in fishes to the development of the neuromuscular system, we established the numbers and sizes of spinal motoneurons and red and white axial muscle fibers in late- and post-embryonic dogfish within the size range 6-71 cm. We found that motoneuron somata, ventral root axons, and red and white muscle fibers increase their size throughout the life of the animal; there is an initial increase in the number of motoneurons that ceases as the fish reaches a length of about 40 cm; white muscle fibers initially decrease in number during post-embryonic life; and red muscle fibers increase in number, but this increase levels off at a fish length of about 40 cm. Spinal motoneurons innervating red myotomal muscle fibers or centrally located white muscle fibers were identified after retrograde labeling with horseradish peroxidase, which was injected in small amounts into the respective muscle areas. The motoneurons supplying the red muscle are smaller and occupy a more lateral position in the ventral horn than the white muscle motoneurons. The number of motoneurons in the ventral horn per unit area increases from medial to lateral and this is associated with a reduction in the sizes of the somata. Values for the ratio of number of muscle fibers to number of supplying motoneurons in the various muscle areas were based on axon counts within the peripheral nerves. This ratio was higher for the centrally located white muscle fibers than for the ventral or dorsal white fibers, but was highest for the red fibers.

Changes in the distribution of synapses during growth: a quantitative morphological study of the neuromuscular system of fishes.

The distribution of synaptic sites on multiply innervated muscle fibres was analysed in four teleost fish species (zebrafish, trout, goldfish and stickleback), using acetylcholinesterase histochemistry. Fishes were chosen for this study rather than other vertebrates because of their long period of growth and continuous increase of muscle fibre size. We found that length and diameter of the fibres increase linearly with fish length but that the distance between synaptic sites increases only as the square root of the fish length and of muscle fibre size. This is explained functionally in connection with the increase of the space constant of a muscle fibre that is expected to accompany the increase of its diameter. We suggest that the change in the synaptic distribution is caused by factors associated with the increasingly wider spread of postsynaptic potentials along the growing fibres, as the intersynaptic distance was found to correlate more strongly with fibre size than with other factors, such as age, speed of growth and genetical background.

The development of the spinal motor column in relation to the myotomal muscle fibers in the zebrafish (Brachydanio rerio). I. Posthatching development.

The neuromuscular system in the trunk of larval and adult zebrafishes was studied by means of light and electronmicroscopical methods. Spinal motoneurons were identified with the horseradish peroxidase retrograde transport method. We correlated the differentiation and growth of the myotomal muscle with the number of motoneurons per spinal cord segment and the size of the motoneuron somata. The adult number of motoneurons is reached in an early larval stage, before the muscle fiber type differentiation in the myotomes is completed. The mean motoneuron size does not bear a clear correlation with the size of the myotomal muscle. In adult zebrafishes we could distinguish the motoneurons which innervate the superficial slow red and the deep fast white muscle fibers on the basis of soma size and position in the motor column. The motoneurons of the red muscle part are small; they are located in the ventrolateral part of the motor column. The motoneurons of the deep fast white fibers are large; they lie near the central canal.

Acetylcholinesterase and acetylcholine receptor histochemistry on end plate regions, myotendinous junctions, and sarcolemma in the axial musculature of three teleost fish species.

In this paper we describe a rapid procedure for the identification of motor end plates in fish. We demonstrated the presence of acetylcholine receptors by means of an immune fluorescence technique with alpha-bungarotoxin. Koelle's thiocholine method was used to localize acetylcholinesterase (AChE) activity. Under carefully controlled conditions the AChE activity and the anti-alpha-bungarotoxin fluorescence showed an equal distribution. This means that in the study of motor innervation in fish the AChE reaction can be used to stain only the motor end plates, leaving the AChE rich preterminal axons unstained. Comparison of the AChE reaction pattern with the distribution of binding sites for antibodies raised against neurofilament protein revealed that in end plate regions high concentrations of AChE are only present in axons and end plates. The myotendinous junctions also possess a high receptor density and enzyme activity. A low enzymatic activity was found at the non-junctional periphery of white muscle fibres. This activity probably resides in the sarcolemma. No non-specific cholinesterase activity was found. From light microscopical analysis it appeared that a single end plate may innervate 2 adjacent muscle fibres. This was affirmed by ultrastructural observations. The dual innervation suggests that, in fish, motor units have a limited distribution through the myotome.

Differentiation of the musculature of the teleost Brachydanio rerio. II. Effects of immobilization on the shape and structure of somites.

The development of the shape and structure of somites in the teleost Brachydanio rerio was studied in embryos under normal conditions and in immobilized embryos. Three different immobilization methods were applied: enclosure in agar, a glass rod in the neural tube and anaesthesia in MS-222. When the performance of the lateral body movements is prevented, the shape development of the somites in embryos and young larvae becomes reversed. When the agar-immobilization is terminated, the larvae resume their normal movements. In about 10 days, the shape of the somites is again as in control larvae. We conclude, that the lateral body movements have both a shape-determining and a shape-stabilizing role during the early stages of somite morphogenesis. It is suggested that in normal embryos differences in shortening between lateral and medial muscle fibres, cause differences in longitudinal growth of the muscle fibres and that the oblique muscle fibre arrangement is a consequence of these differences in growth. In immobilized embryos and larvae, the longitudinal growth of the muscle fibres is decreased. Also the difference in the longitudinal growth rate between lateral and medial muscle fibres diminishes in all somites. We conclude that for the normal morphogenesis of the somites the performance of the specific function, that is to bring about lateral body movements, is required. We suggest, that the impact of the lateral body movements on the development of the structure of the somites is mediated through adaptive growth of the muscle fibres. The suggestion may also apply to the development of the pinnate structure of muscles of higher vertebrates.