Abstract
In a typical electrophysiology experiment, synaptic stimulus is delivered in a cortical layer (1–6) and neuronal responses are recorded intracellularly in individual neurons. We recreated this standard electrophysiological paradigm in brain slices of mice expressing genetically encoded voltage indicators (GEVIs). This allowed us to monitor membrane voltages in the target pyramidal neurons (whole-cell), and population voltages in the surrounding neuropil (optical imaging), simultaneously. Pyramidal neurons have complex dendritic trees that span multiple cortical layers. GEVI imaging revealed areas of the brain slice that experienced the strongest depolarization on a specific synaptic stimulus (location and intensity), thus identifying cortical layers that contribute the most afferent activity to the recorded somatic voltage waveform. By combining whole-cell with GEVI imaging, we obtained a crude distribution of activated synaptic afferents in respect to the dendritic tree of a pyramidal cell. Synaptically evoked voltage waves propagating through the cortical neuropil (dendrites and axons) were not static but rather they changed on a millisecond scale. Voltage imaging can identify areas of brain slices in which the neuropil was in a sustained depolarization (plateau), long after the stimulus onset. Upon a barrage of synaptic inputs, a cortical pyramidal neuron experiences: (a) weak temporal summation of evoked voltage transients (EPSPs); and (b) afterhyperpolarization (intracellular recording), which are not represented in the GEVI population imaging signal (optical signal). To explain these findings [(a) and (b)], we used four voltage indicators (ArcLightD, chi-VSFP, Archon1, and di-4-ANEPPS) with different optical sensitivity, optical response speed, labeling strategy, and a target neuron type. All four imaging methods were used in an identical experimental paradigm: layer 1 (L1) synaptic stimulation, to allow direct comparisons. The population voltage signal showed paired-pulse facilitation, caused in part by additional recruitment of new neurons and dendrites. “Synaptic stimulation” delivered in L1 depolarizes almost an entire cortical column to some degree.
Highlights
Modern neuroscience aims to develop a structure–function model of nervous system organization that would allow mechanistic linking of brain and behavior
• All four voltage indicators used in the present study
subtracting exponential fits through data points
Summary
Modern neuroscience aims to develop a structure–function model of nervous system organization that would allow mechanistic linking of brain and behavior. Mapping the connectivity of pairs of neocortical excitatory neurons is limited to the few neurons selected for whole-cell recording (Markram et al, 1997; Holmgren et al, 2003) and cannot address what occurs when a much larger ensemble of neurons is stimulated. Voltage-sensitive dye (VSD) imaging signals can address subthreshold (synaptic) depolarizations in a much larger ensemble of neurons to explore functionally dependent areas, activity in supragranular and infragranular cortical laminas, activity in neighboring cortical columns, the spread of depolarization waves in respect to speed and direction, cortical oscillations, as well as the plasticity of cortical maps induced by alterations in sensory experience (Prechtl et al, 1997; Petersen and Sakmann, 2001; Petersen et al, 2003; Grinvald and Hildesheim, 2004; Huang et al, 2010; Song et al, 2018). GEVIs can be selectively expressed in one neuron cell type [e.g., layer 2/3 (L2/3) neocortical pyramidal neuron], so that the recorded optical signals are not contaminated by activities of other cell types (Empson et al, 2015)
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