New spines formed following midazolam treatment are functional.
Figures are generally photos, graphs and static images that would be represented in traditional pdf publications.
(a) 3D volume rendering of images of an mRFP transfected and Fluo-4 AM loaded cell showing newly formed spines detected 24 h after midazolam application (arrow). The dashed lines and the plain line indicate where line scan analysis was performed at 24 h for the 2 new spines (square and diamond) and a pre-existing spine (star), respectively. (b) Top: line scans of the spines with the corresponding symbols in (a), the red channel showing mRFP fluorescence, and the green channel showing Fluo-4 AM fluorescence. Bottom: Fluo-4 AM corresponding signals expressed as ΔF/F0, illustrating the intracellular calcium response obtained upon electrical stimulation of Schaffer collaterals (arrowhead). (c) Percentage of spines that responded to stimulation by an elevation of intracellular free calcium concentration for new (grey) and pre-existing spines (white). (d) Mean amplitude of calcium responses (ΔF/F0 measured at the peak of the response triggered by electrical stimulation of Schaffer collaterals) for spines present at the beginning of the experiment (pre-existing spines, n = 15 spines in 2 neurons) compared to spines that appeared during of after midazolam treatment (new spines, n = 43 in 4 neurons). P = 0.65, unpaired t test. N-resp.: calcium signal in non responding spines (n = 23 in 4 neurons). (e) Calcium signal of newly formed functional spines before (Functional) and after (Blocked) a half an hour bath application of AP5 (50 µM) and NBQX (20 µM) (n = 8 new spines in 2 cells). Compare to the mean signal recorded for non responding spines in (d). F corresponds to the mean amplitude of the Fluo-4 fluorescence measured during the 20 ms consecutive to the stimulation pulse. Scale bars: (a): 1 µm. (b): 2 s.