InVivoBiosensorForNeurotransmitterReleaseAndInSituReceptorActivity
From LabAutopedia
An In Vivo Biosensor For Neurotransmitter Release and In Situ Receptor Activity
Lee Schroeder (University of California, San Diego)
Quoc Thang-Nguyen (Femtoscience, Inc)
Arnaud Muller (University of California, San Diego)
Clayton Dickson (University of Alberta, Edmonton, CA)
Marco Mank (Max Plank Inst. of Neurobio., Martinsried, Germany)
Oiliver Griesbeck (Max Plank Inst. of Neurobio., Martinsried, Germany)
Palmer Taylor (University of California, San Diego)
David Kleinfeld (University of California, San Diego)
We further develop Cell-based Neurotransmitter Fluorescent Engineered Reporters (CNiFERs) for the detection of released signaling molecules and exogenous receptor transmission in vivo. CNiFERs are cultured cells engineered to express a chosen metabotropic receptor, use the Gq-protein cascade to transform receptor binding into increased cytosolic Ca2+, and report this Ca2+ increase via a genetically-encoded FRET-based Ca2+ sensor, TN-XXL. CNiFERs are acutely or chronically (using cyclosporine for up to at least 6 days) implanted in rodent cortex and FRET is measured with in vivo two-photon microscopy.
M1-CNiFERs, expressing the M1 muscarinic receptor, respond within ~1 s in vitro to a ~2.5 s pulse of 100nM acetylcholine. The half-maximal rise-time is ~2 s, and full width at half maximal amplitude (FWHMA) is ~7 s. Bath application of acetylcholine (1 nM - 1 µM for 8 minutes), results in a dose-dependent response up to 100% ∆R/R with a phasic peak followed by a decreased but stable plateau.
In vivo, M1-CNiFERs are implanted with control mCh-CNiFERs: HEK293 cells expressing TN-XXL and mCherry. M1- but not mCh-CNiFERs respond robustly (up to 40% ∆R/R) to electrical stimulation of Nucleus Basalis Magnocellularis (NBM) with a single peak initiated within ~2 s, a half-maximal rise time of ~1 s and a FWHMA of < 10 s. We observe a strong correlation between M1-CNiFER response and electrocorticogram (ECoG) delta band power after NBM stimulation (r=.81). The cholinergic nature of this response is further verified by modulation with physostigmine and atropine.
As CNiFERs report exogenous receptor activity in vivo, they are well suited to measure the net-effect of drugs on that receptor. We explore the effect of atypical antipsychotics on exogenous muscarinic transmission in vivo. Olanzapine and Clozapine are known to cause a large cortical release of acetylcholine but also antagonize the M1 muscarinic receptor. The net-effect on muscarinic transmission is not known. We show that M1-CNiFERs implanted in cortex in fact do not respond to systemic Olanzapine or Clozapine. This suggests that muscarinic antagonism by the atypical antipsychotics outweighs the evoked cortical acetylcholine release.
In another project, we highlight CNiFER’s temporal resolution by detecting cortical acetylcholine release in a urethane sleep model. Urethane has been shown to induce oscillations between an activated and deactivated ECoG state resembling the oscillations between Rapid Eye Movement (REM) and non-REM sleep. We show that M1- but not mCh-CNiFERs respond throughout the activated state. This further substantiates urethane-induced oscillations as a model of sleep.
