The primary goal of this project is to elucidate the molecular mechanisms by which thyroid hormones regulate the neuromuscular transmission at the vertebrate neuromuscular junction.

The proposed studies will address a series of fundamental questions regarding the non genomic action of THs in the mammalian NMJ. The pre- and post-synaptic action of THs on the mammalian enplate will be characterized. Specifically, the following 3 aims are proposed:

AIM 1. Non-genomic or non-transcriptional action of THs has been suggested to occur at the earliest step in which the adult NMJ of mammals operate when exposed to THs (Saelim et al. 2004; Scanlan, et al. 2004; Rojas, et al. 2003). Such initial action is evident, for example, in the fast increased spontaneous miniature end-plate currents (MEPCs) frequency observed after the acute application of 3,5,3’ triiodothyronine (T3) occurring inclusive when action potentials are blocked. In specific aim 1 we propose to characterize the acute action of THs on the spontaneous neurotransmitter release from the presynaptic element of the mammalian NMJ. The working hypothesis is that THs control the basal MEPCs frequency. The second hypothesis is that the acute action of THs in mammalian NMJ involves a fast mechanism similar to the one previously characterized in the amphibian NMJ (Rojas et al., 2003). To test these hypotheses we propose to evaluate the synaptic activity of hypo- and hyperthyroid in the mice NMJs using electrophysiological approaches.

AIM 2. Under abnormal levels of THs the functional changes observed in the adult mammal’s muscular activity can be quickly reverted when euthyroid status is restored (Duyff et al. 2000). In specific aim 2 we will study how alterations in THs levels produce phenotype modifications (i.e., in spontaneous locomotion, rearing, etc.) in short-term and long-term hypo- and hyperthyroid mice. We propose that the acute modification of THs levels cause changes in the spontaneous animal locomotion, which can be explained by peripheral THs’ action as has been previously suggested (Ozata et al., 1996). Secondly, we propose that under induced thyroid gland dysfunction, temporal changes in density and distribution of AChR in the skeletal muscle occur. We propose to analyze the distribution of acetylcholine receptors (AChRs) in the post-synaptic membrane during the period of transient hypo- and hyperthyroidism.

AIM 3. During the acute actions of THs in skeletal muscle of adult mouse, the decay time of the MEPCs is increased in the presence of T3. However, it is not clear how the AChRs function is being altered by the THs level. The mechanism by which THs interact with AChRs could be associated with the free membrane diffusion of THs. Consequently, we hypothesize that THs could interact directly with the postsynaptic AChR. To test this hypothesis in Specific Aim 3 we propose to directly assess the activity of the AChR channel in muscle fibers dissociate from hypo and hyperthyroid mice using outside-out patches and fast perfusion methodology.