Functional characterization of circadian clock neuronal circuits controlling sleep-wake rhythms in the Madeira cockroach Rhyparobia maderae

FG Tierphysiolgie

Cartoon summary: Beta/gamma, beta, and alpha frequencies prevailed at different Zeitgeber times (ZTs) in long-term recordings of the cockroach clock. In vivo long-term recordings of AME neurons in cockroaches revealed differences in activity patterns linked to certain ZTs. Beta/gamma frequencies (20–40 Hz, cyan) peaked around midday, evening, and early night. They are hypothesized to be related to release of the neuropeptide PDF by different PDF clock cells, playing an important role in the synchronization/ensemble formation of clock networks. Beta frequencies (12–20 Hz, purple) with 6-hr periodicity were suggested to be based upon clock-dependent regularly phased neuropeptide release. Alpha frequencies (~8 Hz, red), which dominated around midday, occurred during the cockroaches’ sleep, possibly due to GABAergic networks. Next, we will examine whether there are causal relationships between prevailing frequencies, neuropeptide/neurotransmitter release, and sleep-wake phases. [Figure reprinted from: Rojas et al. 2019, Network Neuroscience under the terms of the Creative Commons Attribution 4.0 International License]

Project members: Jenny Plath, Pablo Rojas, Martin Garcia, Olaf Stursberg, Werner Seiler, Monika Stengl

The Madeira cockroach Rhyparobia maderae is an established model system in chronobiology, especially suited to cellular, electrophysiological, biochemical and behavioural analysis due to its large size and long lifespan. In the Madeira cockroach the accessory medulla (AME) with adjacent pigment-dispersing factor (PDF)-expressing neurons is the circadian pacemaker centre that controls sleep-wake rhythms. Previous studies found that circadian clock neurons that project to the contralateral circadian clock control locomotor activity rhythms (reviews: Stengl et al. 2015, Current Opinion in Insect Science; Stengl & Arendt 2016, Current Opinion in Neurobiology). Thus, we distinguished contralaterally projecting from ipsilaterally remaining circadian clock neurons via backfills from the contralateral AME (Gestrich et al. 2018, Journal of Biological Rhythms). In subsequent Ca2+-imaging experiments we found that PDF activates ipsilateral while inhibiting contralateral AME clock neurons. We propose that PDF release during the day recruits and activates an ipsilateral clock circuit (= morning = M cells) that controls sleep during the day, while inhibiting a contralateral circadian clock circuit (evening = E cells) that maintains activity at night. In summary, we hypothesize that PDF release during the day keeps M and E cells in antiphase, thereby controlling sleep-wake cycles.

To test this hypothesis, we perform in vivo long-term loose-patch clamp recordings of the AME in constant darkness (DD). In addition, we perform electroretinogram (ERG) recordings of the compound eye and electromyogram (EMG) recordings of leg muscles. With the ERG endogenous rhythmic changes of the sensitivity of the compound eye are revealed, with minima in sensitivity during the resting/sleep phase during the subjective day in DD. Accordingly, EMG recordings express minimal activity of motor neurons during sleep and highest during its active phase during the subjective night. We search for M cell activation in the AME at dawn correlating with minima in the ERG and EMG and E cell activation at dusk and correlating with maximal photic sensitivity and maximal locomotor activity. Then, intracellular recordings and iontophoretic injection of backfilled contralateral neurons together with stimulation with light and PDF will reveal the morphology, light-, and PDF-sensitivity of E cells while physiological analysis of non-backfilled AME neurons will unravel the morphology and physiology of M cells. By microinjection of neuropeptides and other neuroactive substances in control cockroaches versus cockroaches treated with RNAi-knockdowns, we can identify the functional roles of neuropeptides in the circadian clockwork. The resulting complex data are subsequently analyzed using wavelet-transform to detect and describe events spanning over eight orders of magnitude. With this, we can identify ultradian, circadian, and infradian activity patterns generated by the clock network. Implementation of 3D-reconstructed M and E cells into our standard cockroach brain as well as quantitative analysis and modeling will allow us to understand the neuronal network of a circadian clock. [Supported by DFG grants STE531/18-1,2,3 and STE531/26-1 to MS]