Drosophila clock network
Dr F Rouyer
Dr E Rosato
Uniwersytet Jagielloñski for 3 months in year 2 to learn specialised neuroanatomical techniques.
Queen Mary University for 1 month in year 3, to learn Ca2+ imaging.
- To demonstrate physical proximity between known clock neurons.
- To demonstrate physical proximity between sensory and clock neurons
- To identify interneurons in physical proximity with known clock or sensory neurons.
- To test the circadian relevance of the newly identified interneurons via behavioural and physiological assays
- To employ GRASP (reconstitution of a GFP moiety when the two halves are in close proximity) to demonstrate physical proximity between known components of the circadian network.
- To combine GRASP with neurotrapping (transposon mobilization to identify neurons within part of a circuit) to recognise novel components of the circadian network.
- To exploit information on anatomical proximity to show functional connectivity, for instance using Ca2+ imaging
The “central” clock is composed of brain interneurons expressing two important molecular components of the clock, PER and TIM, at levels detectable by immunofluorescence. However, per-GAL4 and tim-GAL4 transgenics suggest that the pattern of expression of these two circadian genes might be broader and not always overlapping. Likewise, other circadian proteins are found in cells outside of the canonical clock network. Clock cells also receive information, directly or indirectly, from sensory neurons and contact other inter-neurons that eventually communicate with motor centres. Even assuming that the rhythm generator resides solely in the canonical clock cells, it is feasible that neurons upstream and downstream are also involved in the modulation of the rhythmic signal. It thus follows that a better description of the circadian network is necessary. We will employ GRASP (GFP reconstitution across synaptic partners), a technique based on the reconstitution of a GFP moiety when the two GFP halves are in close proximity, to
a. Demonstrate physical proximity between defined known clock neurons.
b. Map any physical proximity between sensory and clock neurons.
c. Identify cells in physical proximity with relevant sensory neurons
d. Identify candidate network cells potentially up- or downstream of clock neurons
Both c & d will be addressed by neurotrapping, namely transposon mobilization to vary the expression of one of the two halves of GFP to trap neurons. The neurons thus identified will be expressing GAL4 and will become available for further manipulations (eg changing electrical properties or downregulating clock proteins) to test their relevance in the circadian network and in other complex behaviours. In a second phase we will use information on anatomical proximity to show functional connectivity. In the first instance this will be achieved by using a GCaMP reporter, expressed in the postsynaptic cells, to monitor Ca2+ release after activation of the presynaptic cell. We will explore with Oxitec and Biofly the possibility of extending this type of approach to insects of economic and medical relevance.
Results and milestones
- Identify clock neurons (CN)/CN proximity by month 24.
- Identify CN/sensory neurons (SN) proximity by month 30.
- Identify CN/novel interneurons proximity by month 42
Synergies, Risks & Exploitation
The partners overlapping expertise in genetic manipulation and imaging significantly enhance the
approach. Risk, sophisticated project genetically but we have performed similar studies successfully. The fly circadian neuronal network provides the anatomical template for insects of economical and medical relevance thereby allowing future targeting of these regions by our SMEs.
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