Signaling of Pigment-Dispersing Factor (PDF) in the Madeira Cockroach Rhyparobia maderae

Abstract

The insect neuropeptide pigment-dispersing factor (PDF) is a functional ortholog of vasoactive intestinal polypeptide, the coupling factor of the mammalian circadian pacemaker. Despite of PDF's importance for synchronized circadian locomotor activity rhythms its signaling is not well understood. We studied PDF signaling in primary cell cultures of the accessory medulla, the circadian pacemaker of the Madeira cockroach. In Ca2+ imaging studies four types of PDF-responses were distinguished. In regularly bursting type 1 pacemakers PDF application resulted in dose-dependent long-lasting increases in Ca2+ baseline concentration and frequency of oscillating Ca2+ transients. Adenylyl cyclase antagonists prevented PDF-responses in type 1 cells, indicating that PDF signaled via elevation of intracellular cAMP levels. In contrast, in type 2 pacemakers PDF transiently raised intracellular Ca2+ levels even after blocking adenylyl cyclase activity. In patch clamp experiments the previously characterized types 1–4 could not be identified. Instead, PDF-responses were categorized according to ion channels affected. Application of PDF inhibited outward potassium or inward sodium currents, sometimes in the same neuron. In a comparison of Ca2+ imaging and patch clamp experiments we hypothesized that in type 1 cells PDF-dependent rises in cAMP concentrations block primarily outward K+ currents. Possibly, this PDF-dependent depolarization underlies PDF-dependent phase advances of pacemakers. Finally, we propose that PDF-dependent concomitant modulation of K+ and Na+ channels in coupled pacemakers causes ultradian membrane potential oscillations as prerequisite to efficient synchronization via resonance.

No Evidence for Ionotropic Pheromone Transduction in the Hawkmoth Manduca sexta

“…The experiments were performed according to published protocols [34]. Briefly, primary cell cultures of ORNs, which reached a mature, pheromone-sensitive developmental stage [32, 35], were loaded with 1 μM Ca 2+ indicator Fura-2 acetoxymethyl ester (Fura-2 AM, Molecular Probes, Eugene, OR, USA) for 40 min at room temperature.…”

“…Excitation wavelengths for Fura-2 were 340 nm (exposure time, 100 ms) and 380 nm (exposure time 50 ms) with 600 ms intervals. The dissociation constant for Fura-2 was calculated with calibration solutions and linear regression analyses (Origin 6.0) [34]. VUAA1 was applied at different concentrations via perfusion (REGLO Digital MS-2/6, Ismatec, IDEX Health & Science, Germany) at 1ml/min flow rate.…”

Molecular and circuit mechanisms mediating circadian clock output in the Drosophila brain

“…In fact, PDF can apparently increase or decrease Ca 2+ levels in different neurons within the DN1p cluster (Chatterjee et al., ). Opposing effects of PDF are also seen in the cockroach Rhyparobia maderae , such that responses to it vary across circadian clock neurons (Gestrich et al., ; Wei et al., ). Neuropeptides have complex roles in the clock network, as they synchronize the phases of molecular clocks and Ca 2+ rhythms; in addition, acute and long‐term effects of neuropeptides on target neurons may be different.…”

Flies as models for circadian clock adaptation to environmental challenges

“…In D. melanogaster, the central clock consists of dorsal and lateral neurons that express the core clock genes and form an extensive neuropeptidergic network in the brain (Figure a; reviewed in Helfrich‐Förster et al., ; Hermann‐Luibl & Helfrich‐Förster, ; Helfrich‐Förster, ; Schubert, Hagedorn, Yoshii, Helfrich‐Förster, & Rieger, ). One of the best conserved and most important neuropeptide in the insect circadian clock is the pigment‐dispersing factor (PDF) (e.g., Beer et al., ; Helfrich‐Förster et al., ; Helfrich‐Förster, ; Ikeno, Numata, Goto, & Shiga, ; Renn, Park, Rosbash, Hall, & Taghert, ; Shafer & Yao, ; Wei et al., ). In D. melanogaster , PDF is expressed in four small ventro‐lateral neurons (s‐LN v ) and in four large ventro‐lateral neurons (l‐LN v ) (Figure a), which have different roles in the clock network.…”