“If you find yourself needing to reread this paragraph, perhaps it’s not that well written. Or it may be that you are low on acetylcholine.” Acetylcholine (ACh) is a major modulator of brain activity in vivo and its release strongly influences attention. If we could visualize when and where ACh is released, we could more fully understand the large trial to trial variance found in many in vivo recordings of spike activity, and perhaps correlate that to attentional and behavioral states mediated by ACh transmission.
Back in grad school, when I was desperately trying to figure out what biological question to answer with my GluSnFR glutamate sensor, I ended up in a meeting with Kleinfeld, his grad student Lee Schroder and Palmer Taylor. We plotted a strategy to make a FRET sensor for acetylcholine. Palmer had recently solved crystal structures of an acetylcholine binding protein bound to agonists and antagonists. Snails secrete this binding protein into their ACh synapses to modulate their potency. The structures showed a conformational change upon agonist binding. The hope was that by fusing CFP and YFP to the most translocated bits of the protein, they would be able to see an ACh dependent FRET change. I was skeptical that it would work, as the translocation was much less than with calmodulin-M13 or periplasmic binding proteins used in Cameleon and GluSnFR, but thought was at least worth a shot. FRET efficiency is highly dependent on dipole orientation, not just dipole distance, and you never know how a small conformational change might rearrange the FP dipoles…
Of course, the simple idea didn’t work. Instead of giving up on the first dozen attempts, they kept plugging away at alternative strategies for measuring ACh release, and eventually succeeded. In this Nature Neuroscience report, An in vivo biosensor for neurotransmitter release and in situ receptor activity, Nguyen et al demonstrate a mammalian cell based system for optically measuring ACh levels in an intact brain. They coexpressed M1 muscarinic receptors with the genetically-encoded calcium indicator TN-XXL in HEK293 cells. ACh binding to the M1 receptor induced IP3-mediated calcium influx. This calcium rise was then picked up by the TN-XXL and reported as a change in CFP/YFP fluorescence. The crazy part is that they took this cell culture assay and implanted the cells into the brains of living rats!
In culture, the response was highly sensitive and monotonic (for phasic response section, EC50 of 11 nM, a Hill coefficient of 1.9 and a maximum of ΔR/R = 1.1). In vivo, using two-photon imaging through a cortical window, they were able to see clear ACh responses in frontal cortex from electrical stimulation of the nucleus basalis magnocellularis, typically 200-μs current pulses of 200 μA @ 100Hz for 20-500ms.
This was essentially a in vivo proof of principal experiment, showing that one could image ACh release in spatially and temporally precise regions of the brain. However, the imaging was done under urethane anesthesia, which is a much different brain state than an awake, behaving animal. Are CNiFERs sensitive, powerful and stable enough to determine behavioral states via imaging in an awake animal? Would expressing GCaMP3 (an indicator with greater fluorescence dynamic range) improve the performance of the CNiFER system? We used a very similar assay with ACh applied to HEK cells during the initial screens for better GCaMPs. Or, is the performance more limited by the properties of the M1 receptor and the adapting nature of IP3-mediated calcium dynamics? CNiFERS provide an interesting platform for looking at ACh and potentially other G-protein mediated signaling, but it remains to be seen if labs that aren’t as technically proficient with two-photon rig will find it more useful than cyclic voltammetry for measuring acetylcholine levels.
Nature Neuroscience, 13 (1), 127-132 DOI: 10.1038/nn.2469
Nguyen, Q., Schroeder, L., Mank, M., Muller, A., Taylor, P., Griesbeck, O., & Kleinfeld, D. (2009). An in vivo biosensor for neurotransmitter release and in situ receptor activity