Lynn Landmesser

The development of motor projection patterns in the chick hind limb.

1. Retrograde transport of horseradish peroxidase was used to map the initial projection patterns of lumbosacral motoneurones to the embryonic chick hind limb. 2. The stage 28 segmental projection pattern to each of the four primary muscle masses was characteristic and indistinguishable from the stage 36 projection pattern to the sum of the muscles derived from that mass. In addition, the adductor motoneurone pool was found to be similar in position (both rostro‐caudal and mediolateral) at stages 29, 30, 32, 33 1/2 and 36. 3. Therefore axons from lumbosacral motoneurones project for the most part only to appropriate regions from early times shortly after they grow into the limb bud. Furthermore, the attainment of the segmental projection pattern occurs prior to the normal time of, and therefore without the aid of, cell death. This conclusion was supported by electrophysiological recordings made from muscle nerves. 4. A regionalization of the projection patterns within a single muscle mass could be shown both anatomically and physiologically prior to the cleavage of the mass into individual muscles and the projections were in a general way appropriate for the muscles derived from those regions. 5. Therefore the process of muscle cleavage does not in itself create the specific projection patterns observed, and motoneurone axons appear to grow to and to ramify and make synapses only within regions which correspond to their adult muscles. 6. Finally, the termination site of each motoneurone axon in the early limb was found to be tightly correlated in a somatotopic fashion with the position occupied by its soma in the cord. This suggests that some feature of the motoneurone related to its position may be of importance in achieving the specific projection patterns observed.

The distribution of motoneurones supplying chick hind limb muscles.

1. The motor nuclei supplying many of the hind limb muscles were localized in late chick embryos (stage 36‐37; 10‐11 days) by utilizing the technique of retrograde transport of horseradish peroxidase. 2. Each nucleus was found to be localized in a characteristic position in both the rostro‐caudal and transverse plane of the spinal cord with only slight individual variation. 3. Each motor nucleus consisted of an elongate, coherent cluster of labelled cells, with few cells occurring outside the cluster. Thus, there did not appear to be extensive overlap of nuclei nor extensive intermingling of motoneurones projecting to different muscles. 4. The position of a motor nucleus in the transverse plane was not correlated with whether its muscle was used as an extensor or flexor; nor were adjacent nuclei necessarily co‐activated during normal unrestrained walking movements as deduced from e.m.g. recordings. The position of a motor nucleus also was not correlated in a topographical manner with the adult position in the limb of the muscle to which it projected. 5. Further, while no correlation was found between the rostrocaudal position of a motor nucleus and the embryonic muscle mass from which its muscle was derived, such a relationship existed for the medio‐lateral position; all muscles arising from the dorsal muscle mass, regardless of their function or adult position, were innervated by laterally situated motoneurones, all muscles arising from the ventral muscle mass by medially situated motoneurones. 6. It is concluded that motoneurone position is most closely correlated with ontogenetic events presumaeriphery. It can also be inferred that the central connexions onto motoneurones, responsible for their proper activation, cannot be achieved by a simple mechanism based largely on the position of the motoneurone soma.

SYNAPTIC TRANSMISSION AND CELL DEATH DURING NORMAL GANGLIONIC DEVELOPMENT

1. During normal embryonic development of the chick ciliary ganglion, cell death over a 4‐day period (Stages 35–39) reduces the number of ganglion cells by half, from 6500 to 3200. Both ciliary and choroid populations are affected by approximately the same amount.

The onset and development of transmission in the chick ciliary ganglion.

1. The onset and development of transmission has been studied electro‐physiologically in the isolated chick ciliary ganglion from Stage 25 (Hamburger & Hamilton, 1951) until 28 days after hatching. Ultrastructure of the synapses was concomitantly investigated.

Synapse formation during embryogenesis on ganglion cells lacking a periphery

1. The development of transmission was studied in chick ciliary ganglia that had been deprived of their periphery during early embryonic development.

Pathway selection by chick lumbosacral motoneurons during normal development

Pathways taken by motoneuron axons from the lumbosacral lateral motor column to individual hindlimb muscles have been characterized throughout the normal period of outgrowth and the establishment of specific functional connections in the chick embryo. Axon pathways from individual cord segments were identified after injections of horseradish peroxidase (HRP) directly into the cord. Labelled motoneuron axons were then traced through the plexus and major nerve trunks to termination sites within the limb. At stages 23-24 labelled axons within spinal nerves have just reached the base of the limb and have begun to converge and form the crural and the ischiadic plexus. Even at this early stage, before periods of muscle cleavage, motoneuron cell death and muscle nerve formation, axons show no evidence of widespread random distribution within the limb. Rather, they generally maintain their anterior-posterior position as far as the base of the limb. At stages 27-30, although axons to individual muscles were found to course in discrete tracts within the plexus and nerve trunks they also changed their topographical position with respect to other axons. Axon pathways to single muscles were characterized by tracing retrogradely labelled axons back to the cord after injections of HRP into specific muscle nerves. Axons destined for a single muscle are intermingled with other axons in the spinal nerves and proximal plexus but by the distal plexus have converged to form a discrete tract which then diverges as an individual muscle nerve at more distal levels. These observations exclude models for the establishment of specific connections in which there is widespread testing of the environment with removal of projection errors by cell death and/or axon retraction. They also exclude models that require axons to maintain their topographical position with respect to each other throughout their course.

The development of functional innervation in the hind limb of the chick embryo.

1. The development of functional motor innervation was studied in the hind limb of chick embryos from Stages 25 to 43 by observing contraction of individual muscles and by recording the resultant tension when individual spinal nerves were electrically stimulated. 2. At later developmental stages (35–43) a given muscle always received functional innervation from specific spinal nerves. This pattern, with respect to the craniocaudal position of motoneurones, was similar to those described for amphibians and mammals. 3. The observed pattern was similar throughout development from the time that movement could first be elicited at Stages 27–28. There was no indication that motoneurones form initial synapses with inappropriate muscles. 4. Recordings from muscle nerves during excitation of individual spinal nerves gave results similar to the tension recordings, showing that even at early developmental stages muscle nerves did not contain substantial numbers of inappropriate axons. 5. Most limb muscles or primitive muscle masses became functionally innervated at the same time with no clearly defined proximo‐distal sequence of limb innervation. 6. It appears that chick motoneurones are initially specified with respect to their peripheral destination and grow out selectively to synapse with appropriate muscles from the outset.

Pathway selection by embryonic chick motoneurons in an experimentally altered environment

To characterize cues used by motoneuron axons to reach their appropriate targets, connectivity patterns within the embryonic chick hindlimb have been analysed after early experimental manipulations of the limb or spinal cord. The manipulations altered the anterior–posterior (a.–p.) relationship between motoneurons within the lumbosacral motor column and their specific targets in the limb. Primary emphasis was placed on analysing the pathways taken by embryonic motoneuron axons at stages 23–36 which had been orthogradely labelled by horseradish peroxidase (HRP) injection into the motor column. Motoneuron pool topography and functional patterns of connectivity were also identified by retrograde HRP labelling and spinal cord stimulation coupled with electromyographic recording. With small shifts in position, as in two or three segment a.–p. cord reversals or a.–p. limb shifts, motoneuron axons frequently entered the appropriate plexus but in an inappropriate spinal nerve sequence. Despite this, axons altered their course to innervate specifically and consistently their correct target. When motoneuron axons entered an inappropriate plexus as the result of a greater positional shift (i. e. more extensive cord reversal or limb shift) or in experiments where posterior cord segments were replaced with anterior cord segments and supernumerary limbs were added, they behaved in one of two ways.They either formed inappropriate and largely unpatterned or unordered connections or they took totally aberrant paths within the limb to reach their appropriate target. We conclude that axons are capable of responding in a precise and specific manner to environmental cues when displaced up to a certain distance from their target or normal point of entry into the limb. Their failure to form patterned connections at more extreme distances suggests that the cues to which they are responding may be local, or that an axon’s ability to respond to them is restricted to subclasses of the motoneuron population.

Motoneurone projection patterns in the chick hind limb following early partial reversals of the spinal cord.

1. The development of motoneurone projection patterns in the chick hind limb from reversed spinal cord segments was studied from the onset of axonal outgrowth (St. 24) to the establishment of mature connectivity patterns (St. 36). Approximately the first three lumbosacral cord segments were reversed along the anterior‐posterior axis at St. 15‐16. 2. Projection patterns from reversed cord segments were assessed electrophysiologically by direct spinal cord and spinal nerve stimulation and anatomically by retrograde horseradish peroxidase (HRP) labelling of motoneurones in St. 30‐36 embryos. In younger embryos, paths taken by reversed axons were characterized by orthograde HRP labelling of motoneurones in specific reversed cord segments. 3. Lumbosacral motoneurones formed appropriate functional connexions with individual limb muscles in spite of anterior‐posterior shifts in their spinal cord position aned consequent shifts in their spinal nerve entry point into the limb bud. Reversed motoneurones supplying individual hind limb muscles formed discrete nuclei in the transverse plane of the cord. Each nucleus and the lateral motor column as a whole showed reversed topographical characteristics when compared to control embryos. These observations were made before (St. 30) and after (St. 35‐36) the major period of motoneurone cell death. 4. Correct connectivity resulted from specific alterations in axonal pathways within the plexus or major nerve trunks proximal to the branching of individual muscle nerves. Further such directed outgrowth was present from the earliest times that axons could be traced into the limb which is before the onset of motoneurone cell death and muscle cleavage. 5. It is concluded that motoneurones are specified to project to individual muscles or to follow particular pathways prior to motoneurone birthdays and limb bud formation. The establishment of specific motoneurone connectivity can not be accounted for by passive or mechanical guidance models alone. Rather, motoneurones must also actively respond to cues within the limb or interact among themselves on the basis of an early central specification.

Activation patterns of embryonic chick hind limb muscles recorded in ovo and in an isolated spinal cord preparation.

Muscle activation patterns of embryonic chick hind limb muscles were determined from electromyographic (e.m.g.) recordings in an isolated spinal cord/hind limb preparation of stage 34‐36 embryos, and were compared with in ovo e.m.g. activity from similarly staged embryos. Muscle activity in ovo consisted of periodically recurring sequences of bursts during which antagonistic muscles often alternated and synergistic muscles were co‐active, as compatible with their mature function. However, more variable behaviour was also observed. Burst sequences in ovo were often initiated by a short‐duration, high‐amplitude discharge that occurred synchronously in all muscles studied, and which was followed by a period of electrical silence that was longest in the flexor muscles. This type of activity has not been described previously in mature animals. In ovo movement sequences were generally initiated by extensor activity which progressively declined in duration and intensity throughout the sequence, while flexor activity progressively intensified. The onset of activity in extensor muscles was accompanied by an abrupt decrease in flexor activity, whereas the converse was not observed. Spontaneous movement sequences also occurred when the spinal cord and hind limb were isolated and maintained in oxygenated Tyrode solution for several hours. Deafferentation experiments indicated that the motor pattern in this preparation was generated centrally by circuits within the spinal cord. Activity from the isolated cord was less variable than that occurring in ovo, consisting of sequences of highly regular recurring bursts. Each burst began with a brief high‐amplitude discharge that occurred synchronously in all muscles and which was similar to that observed in ovo. This was followed by a silent period, which was longest in the flexors, and then by a more prolonged burst. Although its behaviour differs from that in ovo in some respects, it is concluded that the isolated cord maintained in vitro produces a spontaneous and patterned motor output.