Figure 4. FEVIR: fast random-access two-photon imaging of GEVI responses in organotypic hippocampal slice cultures.

(A) Schematic of our random-access multi-photon (RAMP) imaging system. (B) Representative two-photon single-plane image of ASAP2s-expressing neurons in an organotypic hippocampal slice culture. Left, two neurons can be seen (asterisks), together with processes belonging to ASAP2s-expressing neurons in different planes (arrows). Right, overlay of ASAP2s and AlexaFluor594 fluorescence identifies the neuron recorded in whole-cell configuration. Inset shows how 20 individual voxels (squares) can be selected along the plasma membrane when imaging at 925 Hz. Scale bar, 20 μm. (C) Mean number of photons emitted per voxel during a 50 μs exposure (n = 23 neurons for ASAP1, n = 40 for ASAP2s). Data obtained at a holding potential of –70 mV. Individual neurons are shown as gray squares. Black horizontal bars are the means, and error bars are the SEM (p>0.05, Mann-Whitney U-test). (D) Representative ASAP2s and ASAP1 responses to a single current-evoked AP (black trace). Optical recordings were acquired at 925 Hz with 20 voxels per neuron. Traces are the average of 10 trials. (E) Peak response amplitudes at the soma induced by single current-triggered APs were significantly larger with ASAP2s (n = 17 neurons for ASAP1, n = 23 for ASAP2s, ***p<0.001, t-test). Each data point (gray squares) corresponds to the mean response amplitude to 10 APs per neuron. Black horizontal bars are the means, and error bars are the SEM. (F) Representative single-trial ASAP2s response to spontaneous APs. The voltage trace (bottom) was obtained by simultaneous patch clamping.

DOI: http://dx.doi.org/10.7554/eLife.25690.016

Figure 4—figure supplement 1. Plasma membrane excitability of the ASAP indicators.

(A–C) Box and whisker plots of the resting membrane potential (A), membrane capacitance (B), and input resistance (C) of neurons expressing ASAP1 and ASAP2s in organotypic hippocampal slice cultures. All values are statistically indistinguishable from those of untransfected neurons (p>0.05, Mann-Whitney U-test with Bonferroni correction for multiple comparisons). The bottom and top of each box correspond to the first and third quartile, respectively. When the number of data points (neurons) was odd, quartiles were calculated excluding the median value. The midline inside each box is the median. Circles are outliers, defined as datapoints greater than 1.5-fold the interquartile range less than the first quartile or greater than the third quartile. Error bars (‘whiskers’) correspond to the range of data in the first and last quartile, excluding outliers. n = 23 neurons for ASAP1, 56 for ASAP2s, 24 for ASAP2f, and 16 untransfected cells.

DOI: http://dx.doi.org/10.7554/eLife.25690.017

Figure 4—figure supplement 2. Detecting spontaneous APs using ASAP2s.

Representative single-trial ASAP2s responses to spontaneous APs inorganotypic hippocampal slice cultures imaged at 925 Hz by RAMP microscopy. Twenty voxels were imaged per neuron. Voltage traces were simultaneously obtained by patch clamping. Each example is from a different cell.

DOI: http://dx.doi.org/10.7554/eLife.25690.018

Figure 4—figure supplement 3. Detecting evoked APs using ASAP2f.

(A) Representative ASAP1 and ASAP2f responses to a single current-evoked AP (black trace). Optical recordings were acquired at 925 Hz with 20 voxels per neuron. Traces are the average of 10 trials. (B) Mean number of photons emitted per voxel during a 50 μs exposure (n = 23 neurons for ASAP1 and n = 10 for ASAP2f). Data obtained at a holding potential of –70 mV. Individual neurons are shown as gray squares. Black horizontal bars are the means, and error bars are the SEM. (C) Peak response amplitudes to single current-triggered APs. Each data point (gray squares) corresponds to the mean response amplitude to 10 APs per neuron. n = 23 neurons for ASAP1 and n = 10 for ASAP2f. Black horizontal bars are the means, and error bars are the SEM. Data shown for ASAP1 in B and C is the same data shown in Figure 4C and E, respectively.

DOI: http://dx.doi.org/10.7554/eLife.25690.019

Figure 4—figure supplement 4. Plasma membrane localization and RAMP imaging of Ace2N-4AA-mNeon in organotypic hippocampal slice cultures.

(A) Two-photon images of two representative neurons expressing Ace2N-4AA-mNeon, displayed as maximal intensity Z-projections. Scale bar, 20 μm. (B) Representative two-photon single-plane image of a neuron expressing Ace2N-4AA-mNeon (top row, left). Overlay image of Ace2N-4AA-mNeon and AlexaFluor594 fluorescence are also shown (top row, right; magnified image: bottom row, left). The bottom right image shows the 20 voxels selected for RAMP imaging experiments with this neuron. Scale bar, 20 μm. (C) Fluorescence responses from a representative neuron to a 100 mV step depolarization. The data corresponds to the neuron shown in panel B. The trace was averaged over all 20 voxels and over 10 trials. (D) Steady-state fluorescence responses to a 100 mV step depolarization from n = 8 neurons expressing Ace2N-4AA-mNeon. The arrow points to the data collected from the neuron imaged in panels B and C. Black horizontal bars are the means, and error bars are the SEM.

DOI: http://dx.doi.org/10.7554/eLife.25690.020