The role of nicotinic receptor function in the development of synapses and in diabetes-induced dysautonomias

Autonomic circuits depend critically on cholinergic synaptic transmission to develop and function normally, and severe dysautonomias emerge if cholinergic transmission is disrupted. Yet, we do not fully understand how synaptic activity helps cholinergic synapses develop on autonomic neurons nor do we understand whether or not disrupted postsynaptic nAChRs render cholinergic synapses non-functional, resulting in dysautonomias. To study these two issues, I investigated sympathetic neurons from mice with a disruption in the alpha3 nAChR subunit gene (alpha3 KO). I hypothesized that (1) the loss of alpha3 subunits would remove functional nAChRs from cholinergic synapses and abolish synaptic transmission; (2) the loss of postsynaptic activity would prevent cholinergic nerve terminals from developing normally; and (3) functional alpha3 nAChRs are inactivated by diabetes-induced reactive oxygen species (ROS) to render cholinergic synapses non-functional and cause dysautonomias. To test these hypotheses I combined electrophysiological, molecular biology and imaging techniques to examine cholinergic synapses on sympathetic neurons in alpha3 KO mice.;Since the loss of alpha3 subunits in alpha3 KO mice inactivates the sympathetic nervous system and produces severe dysautonomias, I wondered whether dysautonomias that progress during diabetes are produced by disrupted alpha3 nAChRs. To investigate this, I elevated extracellular glucose levels to those seen during diabetes in sympathetic neuron cultures and then simultaneously measured changes in cytosolic ROS levels and whole-cell ACh-evoked currents. I discovered that increased extracellular glucose elevates intracellular ROS and induces a use-dependent, long-lasting rundown of ACh-evoked currents. Using adenoviruses to express mutated alpha3 subunits in alpha3 KO sympathetic neurons, I identified a highly conserved ring of cysteines in the receptor pore that are attacked by ROS to trap nAChRs into a long lasting inactivated state. Finally, using alpha3 KO mice and in vivo gene transfer strategies, I demonstrate that these cysteines are attacked on sympathetic neurons to inactivate nAChRs, depress synaptic transmission and produce cardiovascular and thermoregulatory dysautonomias in diabetic mice.;My results (1) establish that alpha3-containing nAChRs are required for cholinergic synaptic transmission on sympathetic neurons; (2) demonstrate that activity-dependent signals induce and maintain CHT in developing cholinergic nerve terminals; and (3) reveal that diabetes-induced ROS traps nAChRs in an inactivate state, depresses cholinergic transmission and results in dysautonomias. Taken together, my results suggest that diseases that elevate cytosolic ROS would disrupt postsynaptic nAChRs and disrupt activity-dependent retrograde signals that induce and maintain CHT to produce could severe, long lasting dysautonomias.;I found that the loss of alpha3 prevents the appearance of functional nAChRs on sympathetic neurons and abolishes synaptic transmission through sympathetic ganglia. In spite of this, morphologically normal cholinergic synapses form and persist for months on sympathetic neurons from alpha3 KO mice. Surprisingly, in the absence of postsynaptic activity the presynaptic terminals are immature and lack high-affinity choline transporters (CHTs). As a result, they cannot sustain ACh release and become quickly depleted. Moreover, using in vivo gene transfer strategies I demonstrate that CHT expression in nerve terminals is induced and maintained by signals downstream of postsynaptic activity; converting these immature terminals that deplete rapidly to mature terminals capable of sustaining ACh release.