PYRIDOXINE EFFECT ON NERVE CONDUCTION AND CELL DEATH IN EMBRYONIC CHICK

Author

Yuri Fedorovich, Southern Illinois University CarbondaleFollow

Date of Award

12-1-2013

Degree Name

Master of Science

Department

Molecular Cellular and Systemic Physiology

First Advisor

Sharp, Andrew

Abstract

The role that sensory feedback plays in the proper assembly of the sensorimotor system during early stages of development is not fully understood. Fortunately, vitamin B6 (pyridoxine) has been shown to elicit detrimental effects that are specific to only large diameter sensory neurons in adult humans, dogs, cats (Pearson et al. 2003), rats (Helgren et al. 1997), guinea pigs (Xu et al. 1989), and developing chicken embryos when administered at high dosages (Sharp & Bekoff 2001; Fedorovich & Sharp 2010). The specificity of vitamin B6 was examined in this study as a potential tool for eliminating a specific type of sensory neuron during the development of the sensorimotor system. I used electrophysiological stimulation of the proximal end of N. Ischiadicus while recording from either the distal end of N. Ischiadicus or the short branch of Ramus medialis in eleven and thirteen day old (E11 and E13, respectively) chicken embryo to test the hypothesis that pyridoxine lesion impairs signal propagation in the large diameter sensory axons. Additionally, I used a histological technique to test if pyridoxine specifically eliminated the affected neurons via an apoptotic pathway. Recordings from the short branch of Ramus medialis were first characterized to determine the optimal parameters for nerve stimulations. We found that a one millisecond stimulus duration with a twenty seconds inter-stimulus interval and a current intensity smaller then 300 µA was sufficient to examine the changes in the compound action potential (CAP) after the lesion. Subsequent to pyridoxine overdose on E7 and E8, examination of CAPs recorded from the N. Ischiadicus has shown a significant decrease in the amplitude of trough one when data were normalized to peak one in E11 embryos. In contrast, there was a significant decrease in the amplitude of trough one and a decreasing trend in the amplitude of peak one when the data were normalized to peak two in the E13 embryos. These findings suggest that pyridoxine overdose at E7 and E8 impairs the propagation of action potentials in large diameter axons in the developing embryo by at least E11. In addition, further examination of CAPs has shown a significant increase in peak one velocity and the peak one onset velocity (t-test, p<0.05) when comparing between E11 and E13 control groups. Since myelination greatly contributes to the velocity of potential propagation in the axon, these data suggest that myelination is continuing during this time period of development and likely contributes to the changes seen when examining the effects of pyridoxine in E11 and E13 embryos. Histological examination of the third lumbosacral dorsal root ganglion in nine day old chicken embryo using TUNEL revealed a significant decrease in the number of apoptotic cells between control and treated groups, suggesting that pyridoxine has either eliminated type Ia sensory neurons, including those normally undergoing programmed cell death at E9, by 48 hours of the initial treatment or that pyridoxine has delayed the normal process of programmed cell death to a later stage of development.