Guidance of axon growth cones to their correct target cells is a fundamental step for establishing neural connections in the developing and regenerating nervous system. Nerve growth cones are guided to their targets by extracellular guidance molecules. Recent studies have shown that most of these guidance molecules have bi-functional roles, serving as either attractants or repellents, that depend on the state of intracellular second messengers (i.e., Ca2+ and cyclic nucleotides) in nerve growth cones. However, the molecular mechanisms by which second messenger signaling is regulated in response to guidance molecules are largely unknown. Our laboratory studies these second messenger signaling mechanisms in axon guidance using developing Xenopus spinal neurons in vitro. By combining approaches of nerve growth cone turning assays, Ca2+ imaging, electrophysiology, and pharmacological and molecular manipulations, we analyze the behavior of growth cone turning and Ca2+ dynamics in response to guidance molecules, such as netrin-1 and Sema 3A. We have shown that direct modulation of Ca2+ channels by cyclic nucleotide signaling in the plasma membrane (PM) and endoplasmic reticulum (ER) determines the polarity of bi-directional turning of a growth cone in response to netrin-1. Currently, we study the detailed molecular mechanisms resulting from Ca2+ entry through channels in the PM and release via internal channels in the ER that underlie Ca2+ dynamics in growth cone response to guidance signals. Using models developed from these studies, our future goal is to develop paradigms to promote post-injury adult nerve regeneration and to explain the limitations involved. For that reason, we are establishing a mature Xenopus spinal neuron culture system and will compare second messenger signaling cascades in the mature neurons in response to guidance signals to those seen in developing neurons. We hope that our studies of axon guidance signaling in developing and mature neurons will lead to a better understanding of the cellular and molecular mechanisms of the normal nervous system and will provide information leading to the design of drugs and treatments for nervous system abnormalities and injury.