Research reportLongitudinal elongation of primary afferent axons in the dorsal funiculus of the chick embryo spinal cord
Introduction
The ingrowth of primary afferent axons (PAAs) into the gray matter of the embryonic spinal cord and the subsequent formation of neural connections between the PAAs and their specific target neurons have been well studied in various species including chick embryos (e.g. [3], [5], [6], [17], [18]). However, little is known concerning the longitudinal development of the PAAs in the dorsal funiculus [5]. The PAAs take a stereotyped path during their longitudinal elongation in the dorsal funiculus and they do not intermingle with adjacent axons in the lateral funiculus from either spinal or supraspinal origin. In the rodent spinal cord, the PAAs and corticospinal axons run within the dorsal funiculus in dorsoventrally segregated bundles. The mechanisms underlying pathway selection of PAAs in their longitudinal growth and their segregation in the dorsal funiculus remain to be elucidated.
It is also important to examine the rostrocaudal growth of the PAAs in the dorsal funiculus in order to understand the development of sensory processing of peripheral information, because the PAAs in the dorsal funiculus play a role in intersegmental integration of peripheral information as well as conveying peripheral information to the dorsal column nuclei in the medulla oblongata. In the present study, we labeled the PAAs by a lipophilic tracer DiI in fixed preparations, and examined the development of longitudinal projections of the PAAs in the dorsal funiculus of chick embryos. In addition, the onset of invasion of PAAs into the spinal cord was immunohistochemically examined using a neuron-specific marker, βIII-tubulin.
Section snippets
Chick embryos
Fertilized white leghorn chicken eggs were obtained commercially and were incubated in the laboratory (37.6°C, 60% humidity) until they reached the desired stages. Chick embryos at stage (st) 19–21 of Hamburger and Hamilton [9] (embryonic day 3–3.5, E3–3.5, n=18), st. 23–24 (E4, n=15), st. 29 (E6–6.5, n=16) and st. 35 (E9, n=15) were used in the present experiments.
DiI labeling
Chick embryos were decapitated and immersion-fixed with 4% paraformaldehyde in 0.1 M phosphate buffer (pH. 7.4, PB). After
Results
At st. 19 (E3–3.5), DRG neurons in brachial segments were bipolar, and extended presumptive primary afferent axons (PAAs) both centrally toward the spinal cord and peripherally toward the limb bud (Fig. 1A,B). DiI labeling showed that growing tips of the central process of the PAAs had a growth cone-like swelling (Fig. 1A). By st. 21 (E3.5), PAAs invaded the dorsolateral spinal cord (the dorsal root entry zone; Fig. 1C).
At st. 23–24 (E4), the dorsal funiculus was small oval-shaped in transverse
Discussion
The ingrowth of PAAs into the spinal gray matter and the subsequent formation of neural connections between the PAAs and their target neurons have been studied extensively in frog [12], chick [3], [6], [17], [18], mouse [20], rat [11], [16], [19], [26], [27], and human [14]. However, little is known about the longitudinal development of PAAs in the dorsal funiculus [5]. The present study revealed the time course of longitudinal elongation of PAAs in the dorsal funiculus of chick embryos, and
Acknowledgements
The authors are grateful to Dr. R.W. Oppenheim for his comments and suggestions on the manuscript. This study is supported by Grant-in-Aid for Scientific Research from the Japanese Ministry of Education, Science, Sports and Culture and a Grant for Special Research Project on Dynamics Brain Function and Amenity for the Mind, University of Tsukuba.
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