The long-term goal of the proposed research is to understand how functionally appropriate synapses are established during development. The aspects of synapse formation to be examined are how neurons acquire their repertoire of neurotransmitters and how target tissues acquire their complement of transmitter receptors and effector proteins. These questions will be addressed in the sympathetic nervous system because (1) the transmitters and receptors are well characterized and tools exist to study them, (2) culture techniques permit dissection of mechanism in a reduced system and (3) development which occurs postnatally is accessible to manipulation in vivo. Previous studies revealed that during normal development, a population of sympathetic neurons changes their transmitter phenotype, from noradrenergic to cholinergic. This switch is retrogradely specified by a soluble factor(s) produced by their target tissue, sweat glands. While innervation is not required for sweat glands to express muscarinic receptors, the appearance of cholinergic function in sweat gland neurons is required for the glands to develop secretory responsiveness. We now plan to determine whether sympathetic neurons, in addition to those that innervate sweat glands, undergo a transmitter switch and if so whether the target induces it. The role of target tissues in determining neuronal phenotype will be examined in several other paradigms. Tabby mutant mice lacking sweat glands will be used to determine if target tissues are required to guide sympathetic axons. Mice in which the CDF/LIF gene has been "knocked out" will be studied to establish whether CDF/LIF plays a role in the transmitter switch of sweat gland neurons. Transplantation experiments will be used to determine whether sympathetic target tissues transneuronally specify the neuropeptide content of their preganglionic innervation. Retrograde tracing will be combined with axotomy to assess the stability of target-specified transmitter properties in sweat gland neurons. We will also determine which aspects of target tissue development are regulated by innervation. Preliminary evidence suggests that innervation regulates cholinergic differentiation factor production by sweat glands. We will ascertain whether catecholamines are responsible. Other receptors in sweat glands will be examined to determine if their expression is controlled by innervation. The expression of candidate effector proteins required for responsiveness and regulated by cholinergic innervation will be studied. Finally, we will determine whether a critical period exists for target-induced changes in transmitter properties. These studies will elucidate the cellular and molecular mechanisms responsible for normal synaptogenesis and provide insight into where this process may be disrupted during abnormal development.
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