Teratogenic effects of pyridoxine on the spinal cord and dorsal root ganglia of embryonic chickens

Abstract

Our understanding of the role of somatosensory feedback in regulating motility during chicken embryogenesis and fetal development in general has been hampered by the lack of an approach to selectively alter specific sensory modalities. In adult mammals, pyridoxine overdose has been shown to cause a peripheral sensory neuropathy characterized by a loss of both muscle and cutaneous afferents, but predominated by a loss of proprioception. We have begun to explore the sensitivity of the nervous system in chicken embryos to the application of pyridoxine on embryonic days 7 and 8, after sensory neurons in the lumbosacral region become post-mitotic. Upon examination of the spinal cord, dorsal root ganglion and peripheral nerves, we find that pyridoxine causes a loss of neurotrophic tyrosine kinase receptor type 3-positive neurons, a decrease in the diameter of the muscle innervating nerve tibialis, and a reduction in the number of large diameter axons in this nerve. However, we found no change in the number of Substance P or calcitonin gene-related peptide-positive neurons, the number of motor neurons or the diameter or axonal composition of the femoral cutaneous nerve. Therefore, pyridoxine causes a peripheral sensory neuropathy in embryonic chickens largely consistent with its effects in adult mammals. However, the lesion may be more restricted to proprioception in the chicken embryo. Therefore, pyridoxine lesion induced during embryogenesis in the chicken embryo can be used to assess how the loss of sensation, largely proprioception, alters spontaneous embryonic motility and subsequent motor development.

Keywords: DRG; TrkC; chicken embryo; motor neurons; proprioception; pyridoxine.

Fig. 1

Effect of pyridoxine treatment on TrkC immuno-reactivity of neurons in the third lumbosacral DRG. Representative sections of the third lumbosacral DRG immuno-stained for TrkC are shown from a control (A-C) and a pyridoxine-treated (D-F) embryo. Sections are from positions 25%, 50% and 75% from the rostral to caudel extent of the DRG. Panel (G) shows a plot of the number (uc) of TrkC positive neurons in each assayed section for each of the animals. The symbol shape denotes the reaction pairs. The total number (uc) of TrkC positive neurons for each animal are shown grouped by pairs as they were reacted (H).

Fig. 2

Effect of pyridoxine treatment on Substance P and CGRP immuno-reactivity in the third lumbosacral DRG. Panels (A-C) show images of the immuno-reactivity for Substance P (green), CGRP (red) and a superposition of the images, respectively, from the medial section of a control DRG. Panels (D-F) show similar images from the medial section of a pyridoxine-treated embryo. All images are maximum projections from optical sections collected using structured illumination. Notice that the treated DRG is smaller than the control DRG. The total number of cells (uc) from all embryos (mean and standard deviation) expressing Substance P, CGRP or both are shown in (G). There was no significant loss of Substance P or CGRP expressing cells.

Fig. 3

Effect of pyridoxine treatment on axon diameter distribution in the tibialis nerve. Representative cross-sections of the tibialis nerve from a control (A) and pyridoxine treated embryo (B). Images of the two nerves are at the same magnification. The average number of axons greater than and less than the median diameter are plotted in (C). Pyridoxine treatment caused a significant decrease in large diameter axons. The error bars represent the standard deviations. The asterisk denotes a statistically different change.

Fig. 4

Effect of pyridoxine treatment on spinal cord morphology in the second lumbosacral segment. Images of representative sections of the lumbosacral spinal cord (LS2) stained with cresyl violet from a control (A) and a pyridoxine treated embryo (B). Images are at the same magnification. Notice the significant reduction in volume caused by pyridoxine. (C) and (D) show enlargements of the motor column from (A) and (B) respectively.