Investigating Neuromodulatory Mechanisms of Cotransmitter Proctolin in Drosophila Neuromuscular Junction

Abstract

Proctolin is a neuropeptide that induces or increases muscle contraction in nearly all arthropods. In the fruit fly, Drosophila melanogaster, proctolin is released onto skeletal muscles as a cotransmitter with the excitatory neurotransmitter, L-glutamate (Glu). This thesis examines mechanisms of action through which proctolin modulates contractions of Drosophila muscles. Chapter 1 includes a literature review of neuromodulation, cotransmitters, neurohormones and effects of proctolin, and it gives a rationale for the scientific questions addressed in subsequent chapters. Previous work showed that increasing neural activity reduces the threshold and EC50 values for proctolin’s ability to increase nerve-evoked contractions. Chapter 2 provides evidence that this activity-dependence cannot be attributed solely to an increase in Glu release with increasing neural activity. Proctolin potentiates contractions elicited by directly applying Glu to larval muscles but does not consistently shift EC50 values. Effects of proctolin and Glu on contraction are supra-additive but do not appear to be synergistic. Chapter 3 demonstrates that proctolin increases calcium-induced fluorescence in muscle cytoplasm by itself and enhances calcium signals elicited by Glu. The effect of proctolin is blocked by nifedipine, an inhibitor of voltage-gated, L-type calcium channels in the plasma membrane, suggesting a role for calcium influx. Proctolin failed to increase calcium-induced fluorescence changes induced by caffeine in calcium-free saline, suggesting that proctolin may not act directly on ryanodine receptors (RyRs) that release calcium from the sarcoplasmic reticulum (SR) into the cytosol. Chapter 4 examines a hypothesis in which activating the proctolin receptor inhibits calmodulin, since calcium-calmodulin complexes are reported to inhibit RyRs at high calcium concentrations, limiting calcium release from the SR. A calmodulin inhibitor, W7, mimicks some but not all effects of proctolin, suggesting that although calmodulin inhibition may contribute to proctolin’s effects, other mechanisms must be involved. Data are presented implicating the possible involvement of protein kinase C (PKC), the cAMP-dependent protein kinase (PKA) and the plasma membrane calcium ATPase (PMCA), which normally removes calcium from the cytosol. This thesis adds to our understanding of how cotransmission of proctolin with Glu modulates muscle performance in Drosophila, and it suggests an experimental framework for investigating effects of cotransmitters generally.