1. sex-pheromone detection in the hawkmoth Manduca sexta
Insect odor transduction is still under debate and different ionotropic or/and metabotropic cascades are suggested to be employed in different insect species. We focus on the analysis of pheromone transduction in the hawkmoth Manduca sexta. Female moths attract their conspecific mates over long distances of several kilometers by pulsatile release of a sex-pheromone blend from their abdominal glands. Specialized olfactory receptor neurons innervating long trichoid sensilla on the males´ antennae detect these pheromones. One of the two sensory neurons always detects bombykal, the main component of the blend. The detection of the species-specific sex-pheromone blend induces an instantaneous change of behavior: the aroused males start searching for their mates in a zig-zagging upwind flight.
To examine the pheromone transduction cascade of the sensory neurons, primary cell cultures of the olfactory receptor neurons from the antennae of the tobacco hornworm Manduca sexta were developed which can “smell” in vitro. With patch-clamp experiments and pharmacology we examined, which ion channels open after the addition of pheromones and how they are modulated via second messengers. Our current main focus of research is the examination of mechanisms of gain control in the olfactory receptor neurons, depending on time of day and on previous pheromone exposure. In addition, we examine the function of the suggested coreceptor orco in pheromone transduction. We compare our results from in vitro pharmacological studies of primary cell cultures of olfactory receptor neurons with in vivo extracellular recordings of single sensilla on the intact antenna and challenge our hypothesis of pheromone transduction in behavioral assays.
We suggest, that moth olfactory receptor neurons are peripheral circadian pacemakers which generate circadian rhythms of intracellular messengers, thereby modulating olfactory sensitivity during the course of the day. Evidence is accumulating that M. sexta employs a metabotropic pheromone transduction cascade via phospholipase Cβ activation without employment of an orco-based ionotropic mechanism in contrast to odor transduction cascades suggested for the fruit fly Drosophila melanogaster.
2. circadian clock function and the role of neuropeptides in the circadian clock of the Madeira cockroach Rhyparobia maderae
Circadian pacemakers orchestrate physiological processes and behavioral rhythms in probably all organisms on earth. Circadian clocks, with a period length of about 24 hours are the best studied biological oscillators. Thousands of circadian oscillators are located in the brain and body which generate synchronized endogenous rhythms of about 24 hrs. They are entrained via external Zeitgebers such as the light-dark cycle and send outputs to different effectors such as centers of hormone release and control centers of locomotor activity. To obtain a common time scheme in sync with the rhythmic environment, the many clocks per organism are synchronized via coupling pathways and entrained via the light-dark cycle.
The most important circadian coupling signal of insects is the neuropeptide pigment-dispersing factor (PDF) which shares surprising homologies with the most important coupling signal of vertebrate clocks, vasoactive intestinal polypeptide (VIP). The PDF-receptor and the VIP-receptor both signal via adenylyl cyclase activation and show conserved functions. Despite of the importance of VIP and PDF for circadian rhythmicity, our knowledge about their signal transduction cascades and their respective neuropeptide-dependent cellular networks is still very limited. The Madeira cockroach Rhyparobia maderae (Syn.: Leucophaea maderae) is an established circadian model system especially suited for cellular and behavioral analysis because of its large size and longevity. With patch clamp studies, Ca2+ imaging, and FRET on primary cell cultures of R. maderae circadian clock neurons, we examine PDF-signaling. We want to identify PDF-dependent ion channels and PDF-dependent signaling cascades. In addition, with intracellular recordings of circadian clock neurons in intact cockroaches brains, combined with multiple-label immunocytochemistry we decipher the circadian network of the cockroach clock. AMIRA-based 3D-reconstructions of the individually characterized neurons are implemented into a standard cockroach brain to attempt to reconstruct the neuronal circuit of the circadian pacemaker neurons. Furthermore, we analyze neuropeptide contents and neuropeptide sensitivities with MALDI-TOF as well as electrophysiological and imaging techniques. Furthermore, we want to know whether combinations of neuropeptides identified in circadian clock neurons affect circadian locomotor rhythms in behavioral assays. Finally, we employ transcriptome-analysis and realtime PCR to identify the molecular constituents of the circadian clockwork, and use an RNA interference based approach for a functional analysis circadian genes in the cockroach.