Signal Transduction in the Olfactory Lobe

Signal transduction in the olfactory system. The anatomy and organization of the olfactory system has striking similarities across many different organisms. There are olfactory receptor neurons exposed to the periphery that interact with odor molecules in the environment. These neurons then project to higher order processing centers usually within neuropil structures called glomeruli within the brains of the organisms. We are interested in signal transduction events in both these locations. In the olfactory receptor neuron, how does detection of the odorant become translated into an action potential? How is the sensitivity of the neuron to outside stimuli monitored and modified? In the central brain neuropil, we are interested in how the signals from the periphery get processed and codified for higher order processing. Finally, we are also interested in the signal transduction events that mediate axon pathfinding during the development of these structures.

Model systems. We are studying these processes using two insects as model systems: Manduca sexta and Drosophila melanogaster. Manduca is a large, agriculturally-important insect whose olfactory anatomy, behavior, and physiology have been well studied. Its large size allows for sophisticated biochemical and molecular cloning approaches, and its history allows us to place those results in a relevant anatomical and ethological context. The use of Drosophila allows us to bring its powerful molecular genetic tools to bear to address questions of both function and development.

Regulation of cGMP. One focus of our lab has been on the regulation of the synthesis of the intracellular messenger cGMP through the actions of guanylyl cyclases (GCs). The GCs are usually classified as being either soluble GCs (sGCs) or receptor GCs (rGCs). sGC is an obligate heterodimer comprising an alpha and a beta subunit. Upon heterodimer formation, sGC can be strongly stimulated by the gaseous messenger nitric oxide (NO) to generate cGMP. The rGCs usually act as homodimers and are activated either through ligand binding or interactions with guanylyl cyclase activating proteins (GCAPs).

We have identified eight different GCs expressed in the olfactory system of Manduca: three with sequence similarity to sGCs and five with similarity to rGCs. We have obtained full-length clones of one alpha (MsGCa1) and two beta sGC (MsGCb1 and MsGCb3) subunits. The a1 and b1 isoforms form an NO-sensitive heterodimer while the b3 isoform is active alone and is not strongly stimulated by NO. We have also identified three full-length rGCs including MsGC-II, a GC with similarity to mammalian GC-E, MsGC-III, a GC with similarity to mammalian GC-B, and MsGC-I, a novel form of GC (Nighorn et al., 1998a, Nighorn et al., 1999, Simpson et al., 1999). Because we are interested in the regulation of the activity of these GCs, we have also cloned nitric oxide synthase, the enzyme that generates NO, and two GCAPs.

We are currently examining the function of these molecules in the olfactory system in three ways. First, we are identifying the expression patterns of each of these molecules in the olfactory system. Using Northern blots, in-situ hybridization, and immunocytochemistry we have been able to show that all of these GCs are expressed in the olfactory system. Their expression patterns demonstrate that each GC examined so far is expressed in a particular subset of olfactory neurons suggesting specific functions.

Second, we are using pharmacology to examine the function of these molecules. We are treating the olfactory system with inhibitors of the NO/sGC signaling pathway and measuring their effects on the electrophysiological output of antennal lobe neurons. Finally, we are examining the function of these molecules using Drosophila. We have identified 9 GCs expressed in Drosophila. We will first determine which of these GCs are expressed in the olfactory system and then use the molecular genetics of Drosophila to examine the consequences of manipulation of these GCs on olfactory-mediated behavioral assays.

Receptor tyrosine kinases. RTKs are transmembrane molecules that are activated as homodimers through ligand binding. Their activation sets off a signal transduction cascade that usually leads to changes either in the behavior or gene expression of a particular cell. We have isolated the Manduca homologs of the EPH and RYK families of receptor tyrosine kinases. These rTKs are known to play important roles in the development of the nervous system in mammals. We are now investigating whether these molecules also mediate events in the development of the olfactory system in either Manduca or Drosophila. We are particularly interested to see if these molecules help to guide the developing axons of olfactory receptor neurons as they find their appropriate glomerular targets within the antennal lobe.