Voltage compartmentalization in dendritic spines in vivo

Abstract

Dendritic spines mediate most excitatory neurotransmission in the nervous system, so their function must be critical for the brain. Spines are biochemical compartments but might also electrically modify synaptic potentials. Using two-photon microscopy and a genetically encoded voltage indicator, we measured membrane potentials in spines and dendrites from pyramidal neurons in the somatosensory cortex of mice during spontaneous activity and sensory stimulation. Spines and dendrites were depolarized together during action potentials, but, during subthreshold and resting potentials, spines often experienced different voltages than parent dendrites, even activating independently. Spine voltages remained compartmentalized after two-photon optogenetic activation of individual spine heads. We conclude that spines are elementary voltage compartments. The regulation of voltage compartmentalization could be important for synaptic function and plasticity, dendritic integration, and disease states.

Fig. 1.. Characterization of postASAP.

(A) Construction of postASAP. D, Asp; G, Gly; H, His; L, Leu; N, Asn; R, Arg; S, Ser; T, Thr. (B) Two-photon imaging of neurons expressing RFP-p2a-postASAP. Somatic expression (scale bar, 15 μm) is shown on the left. Dendritic expression (scale bar, 3 μm) is shown on the right. (C) Sensitivity of postASAP in ND7/23 cells to 500-ms voltage steps (mean ± SD; n =5 cells). The red area corresponds to a linear range: y = 0.6x, coefficient of determination (R²) = 0.9973, and p = 0.0014. (D) Experimental design. (E) On the left, a patched neuron expressing postASAP is shown. The red lines show the pipette outline, and the white dotted square shows the region of interest (ROI) for fluorescence measurement (scale bar, 5 μm). On the right, a representative optical trace of postASAP (light green, raw fluorescence; black, 10-Hz low-pass filtered) and electrical recording (red) are shown. (F) Average somatic electrical subthreshold signals (red, six events) and simultaneous fluorescence changes (black and gray, mean ± SD). (G) Correlation of peak postASAP fluorescent changes and subthreshold electrical amplitude [mean ± SD; n = 317 subthreshold events, 14 cells, and 8 animals; linear regression (red): y = 0.58x, confidence interval = 0.55 to 0.61, R² = 0.7156, and p = 0.0003].

Fig. 2.. Spine and dendritic voltage dynamics in vivo during spontaneous activity.

(A) In vivo two-photon imaging and somatic whole-cell recording of a neuron expressing postASAP is shown at the top (red lines, pipette outline). Imaged dendrites (43 μm from center of the image to cell body) of a patched cell are shown at the bottom (scale bars, 5 μm). (B) A somatic electrical recording of the neuron in (A) is shown at the top. AP, train of APs; Sub, subthreshold depolarization; RMP, resting membrane potential. Simultaneous fluorescence changes of numbered spines and adjacent dendrites in (A) are shown at the bottom. (C) Representative image with peak fluorescence changes in dendrites and spines during three conditions in (B). (D) Depolarization during APs, generated by three 100-ms current pulses (300 pA). Somatic imaging and electrophysiological recording are shown at the top (scale bar, 5 μm). Representative fluorescence changes in a dendrite are shown at the bottom [average three trials; spine at 48 μm from cell body; scale bar, 5 μm; color scale same as (B)]. (E) Peak spine and dendrite fluorescence changes during AP trains (n = 125 spines, 37 dendrite segments, 5 cells, and 4 animals; linear regression: y = 0.93x, R² = 0.823, and p < 0.0001). Box and whiskers represent median (line), 25th to 75th percentiles (box), range (whiskers), and mean as a “+.” (F) Examples of Dendrite+Spines patterns are shown on the left (average 10 events; scale bar, 5 μm). Peak fluorescence changes in spine heads and adjacent dendrites are shown on the right (n = 221 spines, 90 dendritic segments, 13 cells, and 7 animals). n.s., not significant. (G) Same as (F) for Spine-only pattern (n = 116 spines, 90 dendritic segments, 13 cells, and 7 animals; scale bar, 5 μm). ****p < 0.0001.

Fig. 3.. Spine and dendritic voltage dynamics in vivo after sensory stimulation.

(A) Experimental design. (B) Representative image of dendrites and spines expressing postASAP (scale bar, 5 μm). (C) Simultaneous fluorescence changes of numbered spines and adjacent dendrites in (B). (D) Example image with peak fluorescence changes in dendrites and spines in the time point indicated in (C) is shown on the left. Peak fluorescence changes in spine heads and adjacent dendrites for the Dendrite+Spines pattern are shown on the right (n = 255 spines, 49 dendrite segments, and 5 animals). (E) Same as (D) for Spine-only events (n = 181 spines, 49 dendrites, and 5 animals). ****p < 0.0001.

Fig. 4.. Two-photon optogenetics and voltage imaging in vivo.

(A) Experimental design. (B) Construct and representative postASAP fluorescence changes in soma (light green, raw fluorescence; black, 10-Hz low-pass filtered) during 500-ms stimulation trials (red, 100 mW power). (C) Representative soma (top) and peak fluorescence response (bottom) during stimulation trials (×10, 500 ms, 100 mW). The dotted circle shows the stimulation area (scale bar, 10 μm). (D) Representative in vivo voltage-clamp recordings during optogenetic stimulation of proximal dendrites (100 mW, 100 ms) are shown on the left. Peak currents are shown on the right; −22.7 ± 11.3 pA (mean ± SD), 10 trials (n = 7 cells and 4 animals). (E) Representative peak fluorescence changes during optogenetic activation of dendritic shafts are shown on the left [stimulation ROIs are indicated by white dotted circles; 10 trials, 100 ms, 100 mW; color bar same as (C); scale bar, 5 μm]. Peak fluorescence changes in stimulated dendritic shaft (dendrite stim), adjacent dendritic spine (spine), and unstimulated dendritic shaft (dendrite no stim) are shown on the right (n = 34 dendrites and 9 animals). (F) Same as (E) during optogenetic activation of spines (n = 35 spines and 12 animals; scale bar, 5 μm). ****p < 0.0001. (G) Simplified electrical model. (H) Resistance ratio (R_n to R_den) of Spine-only events during spontaneous (Spon, n = 116), sensory stimuli (Sen, n = 181), and optogenetic spine stimulation (Opto, n = 35). (I) Values of spine neck resistance for stimulated spines; median = 213.7 megohms (n = 35 spines). In (D), (H), and (I), boxes and whiskers represent median (line), 25th to 75th percentiles (box), range (whiskers), and mean as a “+.”