Proc Natl Acad Sci U S A. 2016 Nov 22;113(47):13486-13491.

Quasimodo mediates daily and acute light effects on Drosophila clock neuron excitability.
 

Buhl E1,2, Bradlaugh A3,4, Ogueta M3,5, Chen KF6, Stanewsky R7,5, Hodge JJ8.
1 School of Physiology, Pharmacology and Neuroscience, University of Bristol, Bristol BS8 1TD, United Kingdom; e.buhl@bristol.ac.uk r.stanewsky@ucl.ac.uk.
2 Hatherly Laboratories, University of Exeter Medical School, University of Exeter, Exeter EX4 4PS, United Kingdom.
3 Department of Cell and Developmental Biology, University College London, London WC1E 6DE, United Kingdom.
4 School of Biological and Chemical Sciences, Queen Mary College, London E1 4NS, United Kingdom.
5 Institute for Neuro- and Behavioral Biology, Westfälische Wilhelms University, 48149 Muenster, Germany.
6 Institute of Neurology, University College London, London WC1N 3BG, United Kingdom.
7 Department of Cell and Developmental Biology, University College London, London WC1E 6DE, United Kingdom; e.buhl@bristol.ac.uk r.stanewsky@ucl.ac.uk.
8 School of Physiology, Pharmacology and Neuroscience, University of Bristol, Bristol BS8 1TD, United Kingdom.

 

Abstract 

We have characterized a light-input pathway regulating Drosophila clock neuron excitability. The molecular clock drives rhythmic electrical excitability of clock neurons, and we show that the recently discovered light-input factor Quasimodo (Qsm) regulates this variation, presumably via an Na+, K+, Cl cotransporter (NKCC) and the Shaw K+ channel (dKV3.1). Because of light-dependent degradation of the clock protein Timeless (Tim), constant illumination (LL) leads to a breakdown of molecular and behavioral rhythms. Both overexpression (OX) and knockdown (RNAi) of qsm, NKCC, or Shaw led to robust LL rhythmicity. Whole-cell recordings of the large ventral lateral neurons (l-LNv) showed that altering Qsm levels reduced the daily variation in neuronal activity: qsmOX led to a constitutive less active, night-like state, and qsmRNAi led to a more active, day-like state. Qsm also affected daily changes in K+ currents and the GABA reversal potential, suggesting a role in modifying membrane currents and GABA responses in a daily fashion, potentially modulating light arousal and input to the clock. When directly challenged with blue light, wild-type l-LNvs responded with increased firing at night and no net response during the day, whereas altering Qsm, NKKC, or Shaw levels abolished these day/night differences. Finally, coexpression of ShawOX and NKCCRNAi in a qsm mutant background restored LL-induced behavioral arrhythmicity and wild-type neuronal activity patterns, suggesting that the three genes operate in the same pathway. We propose that Qsm affects both daily and acute light effects in l-LNvs probably acting on Shaw and NKCC.

KEYWORDS: GABA reversal potential; circadian rhythms; light input; membrane excitability; potassium currents

 

Supplement:

All organisms are subject to predictable but drastic daily environmental changes caused by the earth’s rotation around the sun. It is critical for the fitness and wellbeing of an individual to anticipate these changes and this is mediated by circadian clocks (Latin for circa=‘around’, dies=‘day’). These clocks regulate changes in behavior, physiology and metabolism to ensure they occur at certain times during the day thereby allowing the optimal adaptation of the organism to its changing environment. Circadian clocks run at a steady pace (with ~24 h period) and if they go wrong, they can be reset by light, temperature or social cues. As a consequence, circadian rhythms are synchronized with the environment.

 

The underlying molecular clock is well studied and work in Drosophila allowed the identification and cloning of the clock genes and elucidation of the clock mechanism, which later was found to be conserved in mice and humans. In the Drosophila brain the anatomical clock consists of ~150 neurons that drive behavioral rhythms. Each of these clock neurons contains a molecular oscillator and in itself functions as a cell-autonomous clock. In addition to the well-described molecular clock that consists of transcription-translation feedback loops, there are rhythmic changes in the membrane properties of the clock neurons. This ‘membrane clock’ controls the circadian firing and thus the release of neurotransmitters of clock neurons and is a special feature of neuronal clocks (in contrast to non-neuronal clocks, operating in almost every animal cell). Both, mammalian and insect clock neurons are more depolarized and active during the day compared to at night. The membrane clock is important for (i) receiving light input information from the eye and from circadian photo-pigments expressed within the clock neurons, (ii) synchronizing different clock neurons, (iii) reinforcing molecular clock oscillations and (iv) conveying circadian time signals to the nervous system and body. While the molecular clock regulates rhythmic events in the membrane via clock-controlled gene expression of ion channels and transporters, rhythmic membrane events also feed back to the molecular clock via calcium signals to regulate transcriptional activation of the core clock. Similar to the molecular clock being conserved from flies to mammals, components of the membrane clock appear to be conserved involving similar neuropeptide and GPCR receptors [1], GABAAR [2], a leak sodium current (NA/NALCN) [3], Shaw/Kv3 [4] and Slowpoke/BK channels [5].

 

In contrast, how these molecular and electrical processes are translated to temporally coordinate behavior is not well understood. Behavior is produced by the brain networks of interacting neurons that communicate with each other via close connections called ‘synapses’. A neurons’ electrical activity, measured in number and frequency of impulses called ‘spikes’, activates or inhibits other neurons using chemical and electrical synapses. It is this exchange of activity between clock neurons that is important for synchronizing these autonomous clocks and for conveying circadian information to the rest of the brain and body to generate rhythmic behavior.

 

Another mystery is how the clock is synchronized to environmental stimuli, and we recently discovered one mechanism for how daily temperature changes synchronize the clock [6]. However, light is the main Zeitgeber (‘time-giver’) and synchronization is mediated via highly redundant visual and non-visual pathways. Non-visual photoreceptors, e.g., the Melanopsin expressing intrinsically photosensitive Retinal Ganglion Cells (ipRGCs) [7] in mammals, and clock neurons expressing Cryptochrome [8, 9] as well as the novel light input factor Quasimodo [10] in flies, seem to play a particularly important role for clock resetting by light.

 

In this study [11] we used a combination of behavioral analysis and whole-cell recordings from clock neurons. We were utilizing the extensive genetic toolbox of Drosophila to over-express and knock-down Quasimodo as well as interacting ion channels and transporters and measured physiological properties of clock neurons and their response to acute light exposure. We show that the membrane-anchored extracellular protein Quasimodo affects both the daily changes in physiological properties and light resetting of clock neurons, possibly via the potassium channel Shaw and the ion transporter NKCC and by interacting with Cryptochrome. Interestingly, NKCC has recently been shown to regulate the daily, light-dependent GABA switch in the mammalian SCN [12], and since Quasimodo interacts with Shaw and NKCC in flies [11, 13], this raises the exciting possibility that a similar mechanism functions in flies. We show that the light-dependent functions of the three interacting proteins help setting the clock neurons to a day and night state and to modify their light and arousal response. Our findings furthermore provide a link how the membrane clock can feed back to the molecular clock, as Quasimodo triggers light-dependent degradation of Timeless that ultimately resets the clock.

 

Our results are of interest to the wider circadian rhythm and sleep communities, but also to scientists more generally interested in mechanisms of behavior and neuronal excitability and underlying ion channels. Disruption of these intrinsic timekeeping processes negatively affects health and well-being and can shorten lifespan. Furthermore, in our ’24/7 society’, an increasing proportion of the population experiences a de-synchronization of their circadian clock with that of the external world, and this so-called ‘social jetlag’ has lead to an alarming increase in circadian clock-related health problems. Forced clock de-synchronization has been associated with cancer, obesity, depression, addiction, and several sleep syndromes resulting in about a third of people experiencing insomnia [14]. Therefore, and because these ion channels are functionally conserved with their mammalian homologues, we believe that our findings present a good starting point for new and important studies utilizing these novel proteins as targets for curing circadian and sleep disorders. Our work will therefore benefit researchers in the healthcare and pharmaceutical sectors.

 

 

Figure 1: The authors and members of both labs during a fly clock symposium. From left: Maite Ogueta, Phil Smith, Simon Lowe, Amanda Deakin, Adam Bradlaugh, Kiah Tasman, Edgar Buhl, Jack Curran, Ralf Stanewsky. Inserts: James JL Hodge (left) and Kofan Chen (right)

 

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