Voltage sensitive dye experiments and analysis

Voltage sensitive dye (VSD) recording was performed in isolated leech midbody ganglia simultaneously to a double intracellular recording from a P and a T cell. Both mechanosensory cells were stimulated with intracellular current injection, while the activities of all visible cells on the ventral side of the ganglion were monitored through a microscope [Zeiss Examiner.D1, objective plan-apochromat 20 x/1.0 DIC (UV)] with a CCD camera (Photometrics QuantEM:512SC), using bath-applied VF2.1.CL dye (λ_max = 522 nm, λ_em = 535 nm, see Miller et al., 2012). Imaging was performed with a temporal sampling frequency of 94 Hz and a spatial resolution of 64 × 128 pixels. Prior to the recording, a snap shot was taken with the full spatial resolution of the camera, 512 × 512 pixels (Figure ​(Figure2A),2A), based on which regions of interest (ROIs) representing individual cell bodies were selected manually (Fathiazar et al., 2016; see Figure 7B for an example). In this manuscript, data from one representative VSD recording is presented. Similar results were obtained in seven additional preparations.

VSD data analysis method. (A) Snapshot of a ganglion with full resolution. The red circle indicates the cell body of the P cell, which was stimulated by intracellular current injection into the soma during the experiment. (B) Time course of the electrical P cell stimulation, applied through an intracellular electrode (top red trace) and corresponding single VSD response of the P cell (red spikes) in comparison to a single P cell response to control condition (not stimulated, gray) and baseline (averaged P cell responses to 7 traces of control condition, black). The activity map (colored line below) indicates for each recording frame in how many of 7 response traces the activity differed significantly from control condition. (C) Histogram of filtered differences between P cell responses to control condition (7 traces with 110 frames each) and baseline. Black vertical lines indicate thresholds of the p < 0.05 criterion for activity differing significantly from baseline. (D) Histogram of filtered differences between P cell responses to stimulated condition (7 traces with 110 frames each) and baseline. Black vertical lines indicate the significance thresholds determined in (C), showing that the activity differs significantly from baseline more often than in control condition. The activity map in (B) depicts in which frames the significant deviations from baseline occurred, indicating consistent activation during current stimulation.

Electrical stimulation, consisting of 10 ms long pulses of 2 nA (T cell) or 3 nA (P cell), was designed to mimic these cells' spiking patterns in response to tactile stimulation of 70 mN in the ventral midline of the skin in a semi-intact preparation (Pirschel and Kretzberg, 2016). Four different stimulus conditions were compared (see Figure 7): In the PT-stimulated condition, both sensory cells were electrically stimulated in a pattern that reproduces natural responses to tactile stimulation. In the P-stimulated and the T-stimulated condition only one of the cells was stimulated, while the other cell remained unstimulated. In control condition, both cells were not stimulated. In our experiments, responses to 7 repetitions of each condition (trials) were recorded.

For data analysis, 55 ROIs corresponding to visible cell bodies were drawn over the first frame of the VSD recording presented in this manuscript. VSD signals of the cells were extracted by averaging and normalizing the brightness of the pixels in the corresponding ROIs. Movement and bleaching artifacts were corrected as described in Fathiazar and Kretzberg (2015). For each cell, baseline (black line in Figure ​Figure2B)2B) was calculated as an average of the seven trials of control condition. Baseline was subtracted from all VSD signals obtained for all four stimulus conditions. To reduce the noise level, the difference signal was filtered with a moving average filter of three frames window size.

Statistical analysis to identify stimulus-activated cells was performed as described in Fathiazar et al. (2016). In short, the histogram of the filtered VSD difference signals in control conditions was calculated for each cell. Applying a statistical significance level of α = 0.05 on this histogram, we defined the thresholds of activity differing significantly from baseline (black vertical lines in Figure ​Figure2C),2C), indicating very strong de- or hyperpolarization of the cell's membrane potential. These thresholds (quantiles 2.5 and 97.5% of control response distribution) were applied to the filtered VSD difference signals obtained for the three conditions of mechanoreceptor stimulation (Figure ​(Figure2D:2D: P-stimulated condition) to discriminate which individual cells were activated at each time frame (activation map of the P cell in Figure ​Figure2B).2B). The activation maps I(i,j) in Figures 7C–F show the pooled activity for all seven trials of each condition, where I(i,j)ϵ{0, …,7} and iϵ{1, …,55} indicates the cell number (chosen by the sequence of cells' activations after stimulus onset, not corresponding to the cell numbers in the standard ganglion map shown e.g., in Lockery and Kristan, 1990b) and jϵ{1, …,110} is the frame (referring to times 0.16 < t < 1.36 in s). I(i,j) has the value of 0 (shown in dark blue) if the cell i in frame j was not activated in any of the seven trials. If cell i was found to be activated in all the trials in frame j, I(i,j) has the value of seven (shown in yellow). A cell i was classified as a “stimulus-activated” cell for a specific stimulus condition (PT-, P-, or T-stimulated), if at least six of the seven trials revealed significantly increased or decreased activity compared to baseline in at least one time frame in the period 0.53 < t < 0.87 s (from stimulus onset to offset plus five frames).