Elektrophysiologische Charakterisierung des isolierten circadianen Schrittmachers der Schabe Leucophaea maderae

While considerable information about the molecular machinery of the core circadian pacemaker is available (Honma and Honma 2003; Hardin 2005), little is known about the clock’s physiological properties. It is largely unresolved how circadian coupling in multicellular networks is accomplished and how the neuropeptides act within the clock. The aim of this doctoral thesis was the electrophysiological and pharmacological characterization of the isolated circadian pacemaker of the cockroach Leucophaea maderae, with special focus on the effects of PDF and GABA on the electrical activity of pacemaker neurons. In addition, we addressed the question if the neuronal network of the isolated AMe comprises the capacity to generate a circadian rhythm of electrical activity. Therefore an in vitro essay was established, which enables long-term recordings of multiunit neuronal activity of isolated AMe. For the first time it is possible to investigate and therefore characterize the coordination of electrical activity in the insect circadian neuronal network. Furthermore, it provides insight into the function of neuropeptides and neurotransmitters in the clock`s neuronal network. Here we show that circadian pacemaker candidates of the AMe of the cockroach produce regular interspike-intervals. Therefore, the cells` membrane potential oscillates with ultradian periods. Most or all oscillating cells within the AMe are coupled via synaptic and nonsynaptic mechanisms, forming different assemblies. The cells within an assembly share the same ultradian period (= interspike interval) and the same phase (timing of spikes), while cells between assemblies differ in phase. Apparently the majority of these assemblies are formed by inhibitory GABAergic synaptic interactions. Application of pigment-dispersing factor phase-locked and thereby synchronized different assemblies. These data suggest that phase-control of action potential oscillations in the ultradian range is a main task of the circadian pacemaker network. Interestingly, the recordings revealed that even without synaptic connections all cells remain synchronized and fire with the same frequency at a stable phase relationship. When the extracellular saline is substituted by calcium free saline, which causes disruption of synaptic transmission, more cells become active, apparently due to loss of synaptic inhibitions, but all cells maintain phase coupling and fire very regularly with the same frequency but with a new constant phase difference. After wash out, the cells return to fire with zero phase difference. To determine whether these coupling mechanisms of AMe neurons, which are independent of synaptic release, are based upon electrical synapses between the circadian pacemaker cells, the gap junction blockers halothane, octanol and carbenoxolone were employed in presence and absence of synaptic transmission. We could show that different populations of AMe neurons appear to be coupled via gap junctions to maintain synchrony at a stable phase difference. This synchronization via gap junctions is a prerequisite to phase-locked assembly formation via synaptic interactions and to synchronous gamma-type action potential oscillations within the circadian clock. In long-term recordings we examined the distribution of activity peaks independently of the absolute action potential frequency. We show that electrical activity peaks are predominantly distributed to the mid-subjective night with a minimum at the middle of the day. Additionally, the analysis of electrical activity peak distribution revealed ultradian periods, that are multiples of a fundamental 2 hours period.