Listening in on neuron conversations: Novel fluorescent cAMP indicator facilitates neuronal live-cell imaging

Cyclic adenosine monophosphate (cAMP) is an intracellular messenger molecule responsible for the functioning of numerous cells, including neurons, where it promotes the growth of axons and maintains neuronal communication. The molecular pathways of cAMP are well studied and it is known to play a major role in regulating synaptic functions; however, indicators that can precisely monitor its intracellular activity are yet to be developed.

Now, in a study published in Proceedings of the National Academy of Sciences, a team of researchers led by Associate Professor Naoto Saitoh from Doshisha University solved this problem through their development of a novel green fluorescent cAMP indicator called gCarvi. Their indicator can be easily expressed using an adeno-associated virus vector and is capable of quantitatively measuring the basal concentration of cAMP (called [cAMP]_i) intracellularly in neurons. “Our new indicator, gCarvi, monitors [cAMP]_i at concentrations ranging from 0.2 to 20 μM with a subsecond time resolution and a high specificity over cGMP. It is bright enough to monitor cAMP kinetics in single cells and even at single presynaptic boutons,” explains Dr. Saitoh, who is the corresponding author of the study. His team included Ms. Seiko Kawata, a PhD student from Doshisha University and the first author of the study, and Professor Tomoyuki Takahashi from Okinawa Institute of Science and Technology. This paper was made available online on July 6, 2022 and was published in Volume 119 Issue 28 of the journal on July 12, 2022.

An ideal cAMP indicator must span the entire intracellular range of cAMP concentration, exhibit high specificity for cAMP over the other nucleotides such as cyclic guanosine monophosphate (cGMP) which is also responsible for synaptic modulation, not interfere with physiological functions of endogenous cAMP pathways, and enable stable intracellular imaging. Most existing cAMP indicators consist of regulatory subunits of the cAMP binding domain (CBD) in mammals. However, these subunits are endogenous molecules and their use could disrupt the physiological functions of cAMP pathways. gCarvi overcomes this problem as it contains the CBD of a bacterial cAMP receptor protein which does not interfere with the cAMP pathways in mammalian cells.

The researchers fused the bacterial CBD to a circular permutated green fluorescent protein (cpGFP), which detects cAMP concentration by causing corresponding changes in the intensity of fluorescent light emitted upon binding to it. Additionally, gCarvi was found to counter another setback of existing cAMP indicators in preventing cGMP detection by exhibiting a high cAMP/cGMP specificity of >100 but a low affinity to cGMP.

The team developed a ratiometric probe by fusing gCarvi with a red fluorescent protein called mCherry to improve its ability to detect various concentrations of cAMP. This probe was expressed in hippocampal neurons to monitor variations in [cAMP]_i as a proof of concept. Similarly, gCarvi was co-expressed with a red fluorescent calcium ion (Ca²⁺) indicator to detect cAMP and Ca²⁺, both which play a role in neuronal regulation.

This simultaneous detection of cAMP/Ca²⁺ in the hippocampal neurons revealed a strong link between cAMP and Ca²⁺ signals. Upon treatment with forskolin, which is a compound that raises cAMP levels, and a phosphodiesterase (PDE) inhibitor, an increase in intracellular cAMP was observed above a certain threshold in these neurons, which resulted in Ca²⁺ elevation. Similarly, in cerebellar presynaptic boutons, forskolin was used to induce irregular cAMP elevations that demonstrated positive increases in the number of synaptic vesicles being recycled. This indicates the presence of presynaptic cAMP domains in individual boutons, which improve synaptic strength by compartmentalizing signaling pathways to avoid cross-talk.

Speaking of future applications of this novel indicator, Dr. Saitoh says “gCarvi can precisely detect the time of degradation of cAMP signaling in the brain, which results in neuro-degenerative conditions like Alzheimer’s disease. gCarvi can be employed as a screening tool in live-neuron imaging to monitor outcomes of drugs targeting such conditions. Additionally, fluorescent probes help detect invisible molecular dynamics in cells; therefore, the gCarvi cAMP indicator presents invaluable knowledge on neuronal talking.”

The future is certainly looking bright for gCarvi!
 

About Associate Professor Naoto Saitoh from Doshisha University, Japan
Dr. Naoto Saitoh is an Associate Professor from the Faculty of Biomedical Sciences at Doshisha University. Dr. Saitoh holds a PhD in Agriculture from the University of Tokyo. He has held many notable research positions prior to joining Doshisha University, from being a CREST researcher at the Department of Functional Biology to being a specially appointed lecturer at the Department of Neurophysiology at the University of Tokyo. Dr. Saitoh has over 20 research publications to his credit with a research focus on Cellular and Molecular Neuroscience.

Funding information
This study was supported by the Core Research for Evolutional Science and Technology of the Japan Science and Technology Agency, Grant-in-Aid for Scientific Research from MEXT Japan, and the Doshisha Harris Grant.

Media contact:
Organization for Research Initiatives & Development
Doshisha University
Kyotanabe, Kyoto 610-0394, JAPAN
E-mail：jt-ura@mail.doshisha.ac.jp