An Investigative Laboratory Exercise Examining the Cell Signaling and Regulatory Properties of Neurons in the Regenerating Forelimbs of the Axolotl Ambystoma mexicanum

Many students understand the electrical properties of neurons and can adequately describe the creation and transmission of electrical impulses. However, students often have difficulty when it comes to understanding how neurons have an equally important role in cell signaling. This latter function is crucial in the establishment of proper cell fate during regeneration. I have devised a lab that includes in its goals: 1) an investigation into the cell signaling role of neurons, 2) experience with non-lethal invasive surgery, 3) an opportunity for student-collected data, and 4) the chance to work with charismatic animals. In addition, the exercise provides insight into development because development and regeneration follow similar pathways. The lab also introduces the topic of stem cells. Finally, the eventual regeneration of the denervated limb can provide an opportunity to discuss the mechanisms of nerve repair. A critical event in urodele limb regeneration is the formation of a blastema. This event requires an intact nerve supply. Nerves secrete a substance called the neurotrophic growth factor(s) that appear to stimulate the reentry of blastema cells into the cell cycle, through a complex series of signaling events. In this laboratory exercise, students examine this effect by amputating both front limbs, but denervating only one. They then compare limb regeneration under and exempt from nerve control within the same animal. Students control for denervation using a behavioral assay, and monitor limb growth for six weeks. All sixteen of the surgeries were successful, and all showed the expected difference between the denervated and the control limb.