Central and Peripheral Actions of Nitric Oxide

Tufts University, Medford MA

Investigators

Abstract

Nitric oxide (NO) is a small, highly reactive molecule produced in both plants and animals. It is used to protect tissues from infections and acts as a signaling molecule in the vasculature of vertebrates and the central nervous system (CNS) of most species. Because NO can dissolve in both cell membranes and the fluid around cells it is thought to spread widely from where it is produced. However, the range of NO signaling has not been rigorously defined and may vary between tissues and species. In the CNS an effective range of 200 microns would enable NO to control hundreds of neurons, but in peripheral tissues such as muscles this range could limit NO to a very local or even intracellular role. Hence, the immense differences in size, metabolic function and cellular architecture of NO target tissues raise important questions about the mechanisms of NO signaling at different locations. The proposed experiments take advantage of a well-characterized NO signaling system in an insect model system (larval Manduca sexta) to establish the functional roles of NO at central and peripheral sites. A key advantage of studies in this insect is that signaling can be manipulated in the intact, freely moving larva, or in tissues dissected from the insect and maintained alive for several hours under defined conditions. Pharmacological tools are available to block or supplement NO production and to block or increase its effectiveness. It is now also possible to alter the synthesis of key enzymes at the molecular level in intact animals using injections of double-stranded RNA. This treatment interferes with the expression of specific genes in the fully developed animal or in selected parts of the CNS. The results of preliminary physiological experiments, together with the anatomical distribution of enzymes that make or respond to NO (nitric oxide synthase, NOS; and soluble guanylyl cyclase, sGC; respectively), suggest that NO is involved in the control of feeding (chewing movements and foregut activity), the regulation of normal body wall tension and the direct cellular immune response. The neural and muscular activity will be recorded using electrodes and pressure sensors implanted in normal larvae and in larvae that are experimentally deficient in NOS or sGC. These actions of NO on motor patterns and neuromuscular physiology will also be examined pharmacologically in isolated tissues. The role of NO in the insect immune response will be tested by implanting fluorescent micro spheres into the body cavity and assessing the degree of encapsulation in normal and NO-deficient larvae. It is expected that feeding (and growth), gut movements and activity of body wall sensory neurons will all be strongly modulated by NO. The control of body turgor, motor coordination, and abiotic encapsulation are also potentially very important sites of action. The long-term goal of the proposed activity is to better understand how NO carries out its diverse functions. In particular, the results will help to define the specializations and limitations of NO signaling in very different tissue environments. Because NO is involved in the locomotion and feeding behavior of this herbivorous insect, the results could also impact the development of pest-specific antifeedants for crop protection.