The overall objective of this research proposal is to determine if the free radical gas nitric oxide (NO) plays a critical role in synaptic pattern formation in the central nervous system. An emerging hypothesis predicts that NO serves as a retrograde messenger to initiate, strengthen, or stabilize developing synapses on nitric oxide synthase (NOS) containing neurons. The hypothesis predicts that NO release from post-synaptic neurons activates a guanylate cyclase mechanism in presynaptic axons to enhance neurotransmitter release. A further prediction is that NO release is produced by NMDA-receptor (NMDAr) mediated calcium influx which activates NOS. To investigate these hypothesizes the investigator proposed to use the development of the retino-tectal pathway and the patch-cluster system in the rat superior colliculus (SC) as a model system. Anatomical, electrophysiological, and calcium imaging techniques will be used to investigate five specific aims. (1) Determine if there is a temporal correlation between the expression of NOS and the NMDA receptor and the growth and refinement of axons in the SC. This will be tested using antibody immunocytochemistry and anterograde labeling techniques. (2) Determine if inhibition of NOS or blockade of the NMDA receptor alter the growth of these axons. Pharmacological agents will be used to block NOS and NMDAr and anterograde tracers will be used to examine alterations in axon distribution. An nNOS knockout mouse will also be used to test this hypothesis. (3) Determine when the synapses onto the NOS neurons become functional and if NMDA channel currents are involved. The applicant will also determine whether synaptic transmission or the membrane properties of NOS cells are affected by NO production or NOS inhibition. These studies will use electrophysiological techniques in isolated brainstem and brain slice preparations, including tissue from nNOS knockout mouse. (4) Determine if NOS-neurons have NMDAr-mediated [Ca2+]i increases or show elevated [Ca2+]i resting levels during periods of synaptic development. This will be determined using calcium imaging techniques. (5) Determine if NO has a role in modulating the calcium transients induced by NMDA and other agonists. Calcium imaging and pharmacological manipulations of NO will be used in these studies. Collectively, these studies should provide important new information concerning the mechanisms underlying the role of NO in synaptic plasticity and development.
Showing the most recent 10 out of 15 publications
