Voltage imaging of many neurons simultaneously at single-cell resolution is hampered by the difficulty of detecting small voltage signals from overlapping neuronal processes in neural tissue. Recent advances in genetically encoded voltage indicator (GEVI) imaging have shown single-cell resolution optical voltage recordings in intact tissue through imaging naturally sparse cell classes, sparse viral expression, soma restricted expression, advanced optical systems, or a combination of these. Widespread sparse and strong transgenic GEVI expression would enable straightforward optical access to a densely occurring cell type, such as cortical pyramidal cells. Here we demonstrate that a recently described sparse transgenic expression strategy can enable single-cell resolution voltage imaging of cortical pyramidal cells in intact brain tissue without restricting expression to the soma. We also quantify the functional crosstalk in brain tissue and discuss optimal imaging rates to inform future GEVI experimental design.

GEVI signal in a patched neuron GEVI signal in a patched HEK293 cell Good news: GEVIs work in all 3 preparations. GEVI optical signals show reasonable amplitudes, and much faster speeds than genetically encoded calcium indicators (e.g. GCaMP6f). GCaMP6 can only detect firing of action potentials. Unlike GCaMP6, GEVIs can track: (i) subthreshold depolarizations (EPSPs), (ii) hyperpolarizations (IPSPs) and (iii) action potentials. As such, GEVIs may be useful for studying brain circuitry in health and disease. Genetically Encoded Voltage Indicators (GEVIs) are fluorescent proteins, which can be used to track membrane potential changes in living neurons. Several labs around the world generously contributed their GEVI constructs to us. In our lab, all GEVIs were tested using the same equipment. We wanted to know how these indicators compare sideby-side in 3 preparations: Prep. 1 (neuron culture), Prep. 2 (mouse brain slice), & Prep. 3 (HEK cells). 20 µm 20 µm 250 µm sampling sampling GEVI expression in primary neuron culture GEVI expression in HEK293 culture GEVI expression in mouse brain slice GEVI-2 GEVI-1 fluoresence Genetically encoded voltage indicators (GEVIs) could potentially be used for mapping neural circuits at the plane of synaptic potentials and plateau potentials-two blind spots of GCaMP-based imaging. In the last year alone, several laboratories reported significant breakthroughs in the quality of GEVIs and the efficacy of the voltage imaging equipment. One major obstacle of using well performing GEVIs in the pursuit of interesting biological data is the process of transferring GEVIs between laboratories, as their

Subplate (SP) neurons exhibit spontaneous plateau depolarizations mediated by connexin hemichannels. Postnatal (P1-P6) mice show identical voltage pattern and drug-sensitivity as observed in slices from human fetal cortex; indicating that the mouse is a useful model for studying the cellular physiology of the developing neocortex. In mouse SP neurons, spontaneous plateau depolarizations were insensitive to blockers of: synaptic transmission (glutamatergic, GABAergic, or glycinergic), pannexins (probenecid), or calcium channels (mibefradil, verapamil, diltiazem); while highly sensitive to blockers of gap junctions (octanol), hemichannels (La3+, lindane, Gd3+), or glial metabolism (DLFC). Application of La3+ (100 μM) does not exert its effect on electrical activity by blocking calcium channels. Intracellular application of Gd3+ determined that Gd3+-sensitive pores (putative connexin hemichannels) reside on the membrane of SP neurons. Immunostaining of cortical sections (P1-P6) detected connexins 26, and 45 in neurons, but not connexins 32 and 36. Vimentin-positive glial cells were detected in the SP zone suggesting a potential physiological interaction between SP neurons and radial glia. SP spontaneous activity was reduced by blocking glial metabolism with DFLC or by blocking purinergic receptors by PPADS. Connexin hemichannels and ATP release from vimentin-positive glial cells may underlie spontaneous plateau depolarizations in the developing mammalian cortex.

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