Abstract
Minimally invasive measurements of neuronal activity are essential for understanding how signal processing is performed by neuronal networks. While optical strategies for making such measurements hold great promise, optical sensors generally lack the speed and sensitivity necessary to record neuronal activity on a single-trial, single-neuron basis. Here we present additional biophysical characterization and practical improvements of a two-component optical voltage sensor (2cVoS), comprised of the neuronal tracer dye, DiO, and dipicrylamine (DiO/DPA). Using laser spot illumination we demonstrate that membrane potential-dependent fluorescence changes can be obtained in a wide variety of cell types within brain slices. We show a correlation between membrane labeling and the sensitivity of the magnitude of fluorescence signal, such that neurons with the brightest membrane labeling yield the largest ΔF/F values per action potential (AP; ∼40%). By substituting a blue-shifted donor for DiO we confirm that DiO/DPA works, at least in part, via a Förster resonance energy transfer (FRET) mechanism. We also describe a straightforward iontophoretic method for labeling multiple neurons with DiO and show that DiO/DPA is compatible with two-photon (2P) imaging. Finally, exploiting the high sensitivity of DiO/DPA, we demonstrate AP-induced fluorescence transients (fAPs) recorded from single spines of hippocampal pyramidal neurons and single-trial measurements of subthreshold synaptic inputs to granule cell dendrites. Our findings suggest that the 2cVoS, DiO/DPA, enables optical measurements of trial-to-trial voltage fluctuations with very high spatial and temporal resolution, properties well suited for monitoring electrical signals from multiple neurons within intact neuronal networks.
Highlights
A long-standing goal in neuroscience is to record all neural activity within particular circuits
In Purkinje cell somata, action potential (AP)-induced DiO/DPA fluorescence transients markedly decremented with distance into the dendrites as expected based on the lack of voltage-gated sodium conductance in Purkinje cell dendrites (Figure 1D)
These data demonstrate a more straightforward whole-cell approach which labels proximal membrane compartments quickly and effectively. In many circumstances such an approach will yield excellent results; the more laborious two-pipette labeling method will typically result in a higher ratio of plasma membrane to internal staining
Summary
A long-standing goal in neuroscience is to record all neural activity within particular circuits This benchmark requires methodology to detect APs in parallel from individual neurons with sufficiently high signal-to-noise ratio (SNR) such that in single trials every AP can be recorded. At present this is not possible, but it is widely recognized that optical approaches provide a viable solution to this significant technical challenge [1]. We recently introduced a 2cVoS pair consisting of DiO, a neuronal tracer dye as a FRET donor and DPA as a non-fluorescent acceptor [14] This 2cVoS exhibits one of the largest relative changes per millivolt of membrane potential change (i.e., sensitivity) among rapid (submillisecond) voltage indicators. Genetically encoded indicators [15] and photo-induced electron transfer-based
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