Figure 1.
Experimental design and ganglion staining.
A. Schematic of STNS showing experimental design for bath application of the dyes and electrophysiological recording of the triphasic pyloric rhythm (LP, PD and PY neurons). The STG was surrounded by a petroleum jelly well holding the VSD. An additional petroleum jelly well was placed around the dvn for extracellular recording. CoG, commissural ganglion; dvn, dorsal ventricular nerve; LP, lateral pyloric neuron; OG, oesophageal ganglion; PD, pyloric dilator neurons; PY, pyloric constrictor neurons; STG, stomatogastric ganglion. B. Similar to A, but after dye wash-out. The STG was continuously superfused with saline. VSDs were excited by green fluorescence light and red emission was recorded. C. Both dyes stained the STG in a time-dependent manner. Photos of the STG during bath-application of dyes at a concentration of 0.2 mM (top: RH795, bottom: Di-4-ANEPPS) at indicated times after dye application. The shutter speed of the camera was 200 msec for RH795 and 100 msec for Di-4-ANEPPS.
Figure 2.
A. Di-4-ANEPPS exhibited higher fluorescence emission than RH795.
Mean grey values of pictures taken from STGs after bath application of 0.05(green) or Di-4-ANEPPS (orange) (mean+SD, N = 3). Photos were taken with a camera exposure time of 200 msec. Dye type and time had significant influences on fluorescence emission (Two-way ANOVA, time: p<0.0001, dye type: p<0.01 for all measurement >10 minutes, Bonferroni multiple comparison test). B. Comparison of staining intensity for both dyes at different concentrations. Photos were taken 60 minutes after dye application.
Figure 3.
Optical recordings exhibited sufficient S/N to identify neuronal activity patterns.
A. Original single-sweep optical recording of three different neurons (LP, PY, PD) after Di-4-ANEPPS application plus extracellular recording of the pyloric rhythm on the dvn (bottom trace). Analyzed areas are marked with circles in the high resolution photo (left). Areas included the cell membranes of the neurons analyzed. Scale bars mark the changes in fluorescence intensity. B. Cycle-triggered average (n = 16 cycles) of neurons shown in A. Averaging multiple cycles of optical imaging data improved S/N. C. The S/N was always higher for Di-4-ANEPPS, but independent of dye concentration. S/N was calculated using averaged data. D. Comparison of single-sweep LP optical recording in Di-4-ANEPPS (taken from inset in A.) and RH795 (different preparation).
Figure 4.
The S/N of RH795 is independent of LED strength.
A. Left: S/N for RH795 did not change with excitation light strength (n = 12 trials in N = 3 animals, p>0.05). Right: S/N for Di-4-ANEPPS depended on excitation light strength (n = 10 trials in N = 3 animals, p<0.05). B. Fluorescence emission (mean grey values) increased with increasing excitation light strength for Di-4-ANEPPS (left; p<0.05; n = 22 trials from N = 4 animals), but not for RH795 (right; p>0.05; n = 23 trials from N = 4 animals).
Figure 5.
Di-4-ANEPPS and RH795 showed bleaching over time.
A. Photos of STGs taken every 10 minutes during 60 minutes of constant illumination. Ganglia had been stained for 24 hours with 0.1(top) or 0.05 mM Di-4-ANEPPS (bottom) prior to light exposure. Camera shutter speed was 200 msec for RH795 and 100 msec for Di-4-ANEPPS. B. Bleaching was time-dependent for both dyes (Two-way ANOVA: p<0.0001; N = 12). Di-4-ANEPPS bleached faster than RH795 (Two-way ANOVA: p<0.05; N = 12). Mean grey values were normalized to the maximum and plotted over the duration of illumination. C. Comparison of the normalized mean grey values after 60 min of illumination, for all concentrations used (green: RH795; orange: Di-4-ANEPPS).
Figure 6.
Both dyes were internalized during constant illumination.
A. Di-4-ANEPPS was bath-applied for 24 hours and then washed out. Photos were taken every 10 minutes for 1 hour. The plot shows the frequency density of pixel brightness of the somata area. While in general the frequency of smaller grey values increased over time (due to bleaching), there were fewer dark pixels in the 20 minutes picture (blue) than in the initial picture (orange; bottom inset) as indicated by the shift of the frequency density to the right. No fluorescence: grey value of 0; highest fluorescence: grey value of 255. Binwidth: 1. For display purposes only bins 1 to 250 are shown. Top inset: Photos showing neuron staining immediately after dye wash-out and after 20 minutes of illumination. Immediately after washout cell bodies are dark while their surrounding is brightly stained. After 20 minutes of illumination the cell cytoplasm shows fluorescence and the cell nucleus is visible (orange arrow). B. Photos were analyzed and internalization was noted if at least the nucleus of one cell was visible. Group 1: Preparations that had been used for 180 minutes staining experiments (including illumination) (RH795: N = 6, Di-4-ANEPPS: N = 4). Photos were inspected for signs of internalization after the 180 minutes of staining experiments, after 24 hours staining and after bleaching. Group 2: Preparations were stained for 24 hours. Internalization was measured after the staining process and then again after 1 hour of bleaching (RH795: N = 4, Di-4-ANEPPS: N = 7). Group 1 Di-4-ANEPPS values after bleaching and group 2 RH795 values after bleaching were included for the sake of completeness (all preparations had shown internalization in the previous experimental step already).
Figure 7.
Dye toxicity and phototoxicity increased over time.
A. Example of an extracellular recording of the triphasic pyloric rhythm on the dvn showing the activity patterns of PD, LP and PY and plot of instantaneous firing frequency (f.f.) of LP. Top: saline control. Bottom: after 180 minutes exposed to Di-4-ANEPPS (0.1 mM). Arrows indicate extra LP spikes. B. The distribution of LP instantaneous spike frequencies changed over time. The frequencies ranges given are 0–2 Hz, 3–9 Hz and 10–55 Hz. The firing frequency distribution changed for RH795, between different conditions (saline control and 0.05 mM, 0.1 mM and 0.2 mM RH795) and for illuminated and non-illuminated preparations (N = 3, measurements every ten minutes for one hour, then at 90 minutes, 120 minutes and 180 min). Each bar includes data from three experiments. C. Similar to B., for Di-4-ANEPPS.
Figure 8.
RH795 had weaker pharmacological and phototoxic effects than Di-4-ANEPPS.
Separate ganglia were used for each dye and concentration. During the experiment the pyloric rhythm was recorded extracellularly from the dvn and the instantaneous firing frequencies of LP were monitored. Frequencies were measured for 60 seconds each, every 10 minutes for one hour, than at 90 minutes, 120 minutes and 180 minutes. Colors code differences from a control measurement before dye application. Cool colors indicate small deviations from control and hot colors represent large deviations. Data from one animal is represented in one line, from left to right. Preparations that experienced the same treatment are highlighted with the same grey boxes (N = 3). The saline control is always represented by the bottom three lines highlighted in white. Firing frequency classes are shown by columns (left to right: 0–2 Hz, 3–9 Hz and 10–55 Hz).
Figure 9.
The pyloric rhythm was unaffected during imaging.
A. There were no short-term influences on the pyloric rhythm during illumination in RH795 stained preparations. Original recordings of the pyloric dilator nerve (pdn) and the dvn before, during and after illumination. Spike activities and phasing of the pyloric neurons were virtually identical in all conditions. B. Original recordings of pdn and dvn in a preparation that had been stained with Di-4-ANEPPS. There was no apparent change in the pyloric rhythm during illumination. C. Phase diagram showing the relative contribution of PD and LP neurons to the pyloric cycle before, during and after illumination in RH795. There were no significant differences (One-way ANOVA, p>0.6 for all comparisons, N = 4). D. Phase diagram for illumination in Di-4-ANEPPS. Illumination did not affect the phasing of LP and PD (One-way ANOVA, p>0.6 for all comparisons, N = 4). E. Comparison of burst duration (left), number of spikes per burst (middle) and intraburst spike frequencies (right) of PD and LP before, during and after illumination in RH795 stained preparations. No significant differences were found (One-way ANOVA, p>0.9 for all comparisons, N = 4). F. Similar comparison in preparations that were stained with Di-4-ANEPPS (One-way ANOVA, p>0.9 for all comparisons, N = 4).