Animal behaviour is controlled by neuronal circuits in the brain and spinal cord. Revealing the circuit structure underlying various animal behaviours remains an immense task for neuroscientists, especially at the whole brain level. We study how the central nervous system of a developing vertebrate, frog tadpoles, controls its swimming and struggling behaviour. The tadpole nervous system is relatively simple, consisting of a limited number and types of neurons. Recording neuronal activity using either electrophysiology or calcium imaging is less a challenge than in mammals. This has allowed the revelation of many common mechanisms controlling vertebrate movements in general (Roberts et al., 2010).
Technically, conventional electrophysiology can only record the activity of one or a few neurons each time. Current calcium imaging using synthetic dyes (e.g. Fluo-4-AM) critically depends on the successful loading of the dye into surgically exposed neurons. The shortcomings of both technologies have limited our recording options to certain areas of the CNS in one experimental setting. We plan to take advantage of the recently developed and improved GCaMP probes (Nakai et al., 2001; Zhao et al., 2011), modify them to get reliable expression in the whole tadpole CNS. This will allow us to monitor the activity of more brain regions simultaneous using Calcium imaging, which will guide and greatly improve the efficiency of the subsequent electrophysiology recordings.
The first step of the project is to design and synthesize a new plasmid to include the calcium sensing and EGFP domains of GCamp7 and the beta-actin promotor for frogs. Secondly, the plasmid will be microinject into the early stages of frog embryonic cells. Thirdly, full brain imaging will be carried out after stimulating the tadpole sensory systems to evoke swimming or struggling. Plasmid injection leads to stochastic expression of GCamp7 and more universal expression can be achieved by injecting GCamp7 cRNAs. One system we know very little is the lateral line system. We plan to use the imaging tool to reveal the neuronal pathway that mediates the escape response when the lateral system is stimulated. The final stage is to use electrophysiology to record the active neurons identified by calcium imaging and analyse their properties and synaptic connections. The prospective student will receive extensive training in molecular biology, microinjections, calcium imaging and electrophysiology.
Robert et al., (2010) ‘How neurons generate behavior in a hatchling amphibian tadpole: An outline’, Frontiers in Behavioral Neuroscience, 4.
Nakai et al., (2001). "A high signal-to-noise Ca(2+) probe composed of a single green fluorescent protein". Nature Biotechnology. 19 (2): 137–41.
Zhao et al., (2011). "An expanded palette of genetically encoded Ca²⁺ indicators". Science. 333 (6051): 1888–91.
To apply for this project, please go to this link.