Robust and sensitive GFP-based cGMP reporter for real time imaging of intact neurons and neural circuits
- Author(s): Woldemariam, Sarah
- Advisor(s): L'Etoile, Noelle
- et al.
cGMP is a ubiquitous second messenger implicated in a multitude of neurobiological processes, including sensory transduction, learning and memory. FRET-based and GFP-based cGMP reporters have been developed to complement the genetic and biochemical tools used to probe the role of cGMP in these processes. While FRET-based cGMP sensors have been expressed in C. elegans to explore the spatiotemporal regulation of cGMP in sensory neurons in response to stimuli, their use requires a dual emission system, which limits their ability to be coexpressed with other fluorophores, such as red calcium sensors that can indirectly read out neural activity. This work demonstrates that WincG2, a GFP-based cGMP sensor codon-optimized for use in C. elegans, can report changes in cGMP levels in living, behaving C. elegans. We demonstrate that coexpression of WincG2 and light-activatable guanylyl cyclases in body wall muscle cells results in an increase in WincG2 fluorescence upon light exposure that corresponds with the rate of cGMP production. Furthermore, WincG2 fluorescence changes in the cell bodies of the gustatory neuron ASER and the phasmid neuron PHB in response to [NaCl] step changes and sodium dodecyl sulfate, respectively. This provides the first visual evidence that suggests GMP levels change in these neurons in response to stimuli. Intriguingly, preliminary data suggest that in ASER, cGMP levels decrease linearly in the cell body while increasing transiently in the cilia in response to a [NaCl] downstep, which could have implications for cGMP’s potential role in both sensation and memory in ASER. Finally, we demonstrate that cGMP could act as a neuromodulator in a nociceptive neural circuit. WincG2 fluorescence increases in the nociceptive neuron ASH - which is not known to express guanylyl cyclases - in the absence of food while remaining relatively constant in the presence of LB. These results suggest that cGMP could be flowing from other neurons into ASH to signal food status, resulting in the modulation of ASH activity. Taken together, this work demonstrates that WincG2 could be used the uncover cGMP’s role in diverse neurobiological processes in living, behaving C. elegans.