Evoked Cortical Depolarizations Before and After the Amyloid Plaque Accumulation: Voltage Imaging Study.

BACKGROUND: In Alzheimer's disease (AD), synaptic dysfunction is thought to occur many years before the onset of cognitive decline. OBJECTIVE: Detecting synaptic dysfunctions at the earliest stage of AD would be desirable in both clinic and research settings. METHODS: Population voltage imaging allows monitoring of synaptic depolarizations, to which calcium imaging is relatively blind. We developed an AD mouse model (APPswe/PS1dE9 background) expressing a genetically-encoded voltage indicator (GEVI) in the neocortex. GEVI was restricted to the excitatory pyramidal neurons (unlike the voltage-sensitive dyes). RESULTS: Expression of GEVI did not disrupt AD model formation of amyloid plaques. GEVI expression was stable in both AD model mice and Control (healthy) littermates (CTRL) over 247 days postnatal. Brain slices were stimulated in layer 2/3. From the evoked voltage waveforms, we extracted several parameters for comparison AD versus CTRL. Some parameters (e.g., temporal summation, refractoriness, and peak latency) were weak predictors, while other parameters (e.g., signal amplitude, attenuation with distance, and duration (half-width) of the evoked transients) were stronger predictors of the AD condition. Around postnatal age 150 days (P150) and especially at P200, synaptically-evoked voltage signals in brain slices were weaker in the AD groups versus the age- and sex-matched CTRL groups, suggesting an AD-mediated synaptic weakening that coincides with the accumulation of plaques. However, at the youngest ages examined, P40 and P80, the AD groups showed differentially stronger signals, suggesting "hyperexcitability" prior to the formation of plaques. CONCLUSION: Our results indicate bidirectional alterations in cortical physiology in AD model mice; occurring both prior (P40-80), and after (P150-200) the amyloid deposition.

Population imaging discrepancies between a genetically-encoded calcium indicator (GECI) versus a genetically-encoded voltage indicator (GEVI).

Genetically-encoded calcium indicators (GECIs) are essential for studying brain function, while voltage indicators (GEVIs) are slowly permeating neuroscience. Fundamentally, GECI and GEVI measure different things, but both are advertised as reporters of "neuronal activity". We quantified the similarities and differences between calcium and voltage imaging modalities, in the context of population activity (without single-cell resolution) in brain slices. GECI optical signals showed 8-20 times better SNR than GEVI signals, but GECI signals attenuated more with distance from the stimulation site. We show the exact temporal discrepancy between calcium and voltage imaging modalities, and discuss the misleading aspects of GECI imaging. For example, population voltage signals already repolarized to the baseline (~ disappeared), while the GECI signals were still near maximum. The region-to-region propagation latencies, easily captured by GEVI imaging, are blurred in GECI imaging. Temporal summation of GECI signals is highly exaggerated, causing uniform voltage events produced by neuronal populations to appear with highly variable amplitudes in GECI population traces. Relative signal amplitudes in GECI recordings are thus misleading. In simultaneous recordings from multiple sites, the compound EPSP signals in cortical neuropil (population signals) are less distorted by GEVIs than by GECIs.

In Vitro Testing of Voltage Indicators: Archon1, ArcLightD, ASAP1, ASAP2s, ASAP3b, Bongwoori-Pos6, BeRST1, FlicR1, and Chi-VSFP-Butterfly.

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 reported qualities (e.g., membrane targeting, brightness, sensitivity, optical signal quality) are often difficult to reproduce outside of the laboratory of the GEVI origin. We have tested eight available GEVIs (Archon1, ArcLightD, ASAP1, ASAP2s, ASAP3b, Bongwoori-Pos6, FlicR1, and chi-VSFP-Butterfly) and two voltage-sensitive dyes (BeRST1 and di-4-ANEPPS). We used the same microscope, lens, and optical detector, while the light sources were interchanged. GEVI voltage imaging was attempted in the following three preparations: (1) cultured neurons, (2) HEK293 cells, and (3) mouse brain slices. Systematic measurements were successful only in HEK293 cells and brain slices. Despite the significant differences in brightness and dynamic response (ON rate), all tested indicators produced reasonable optical signals in brain slices and solid in vitro quality properties, in the range initially reported by the creator laboratories. Side-by-side comparisons between GEVIs and organic dyes obtained in HEK293 cells and brain slices by a "third party" (current data) will be useful for determining the right voltage indicator for a given research application.

Cadmium versus Lanthanum Effects on Spontaneous Electrical Activity and Expression of Connexin Isoforms Cx26, Cx36, and Cx45 in the Human Fetal Cortex.

Electrical activity is important for brain development. In brain slices, human subplate neurons exhibit spontaneous electrical activity that is highly sensitive to lanthanum. Based on the results of pharmacological experiments in human fetal tissue, we hypothesized that hemichannel-forming connexin (Cx) isoforms 26, 36, and 45 would be expressed on neurons in the subplate (SP) zone. RNA sequencing of dissected human cortical mantles at ages of 17-23 gestational weeks revealed that Cx45 has the highest expression, followed by Cx36 and Cx26. The levels of Cx and pannexin expression between male and female fetal cortices were not significantly different. Immunohistochemical analysis detected Cx45- and Cx26-expressing neurons in the upper segment of the SP zone. Cx45 was present on the cell bodies of human SP neurons, while Cx26 was found on both cell bodies and dendrites. Cx45, Cx36, and Cx26 were strongly expressed in the cortical plate, where newborn migrating neurons line up to form cortical layers. New information about the expression of 3 "neuronal" Cx isoforms in each cortical layer/zone (e.g., SP, cortical plate) and pharmacological data with cadmium and lanthanum may improve our understanding of the cellular mechanisms underlying neuronal development in human fetuses and potential vulnerabilities.

Corrigendum: Single-Neuron Level One-Photon Voltage Imaging With Sparsely Targeted Genetically Encoded Voltage Indicators.

[This corrects the article DOI: 10.3389/fncel.2019.00039.].

Single-Neuron Level One-Photon Voltage Imaging With Sparsely Targeted Genetically Encoded Voltage Indicators.

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.

Mechanisms of Spontaneous Electrical Activity in the Developing Cerebral Cortex-Mouse Subplate Zone.

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.