Figure 1.
Schematic overview of the stomatogastric nervous system (STNS) of the crab, Cancer borealis.
The pink circle marks the application site for the VSD RH795. The black circles illustrate two different extracellular recording sites and the corresponding neuronal signals. The three main neuron types (PD, LP, PY) participating in the pyloric rhythm can be monitored on the lvn. The pdn selectively shows the activity of the two PD neurons. Grey cell bodies illustrate neurons in the stomatogastric ganglion (STG), the oesophageal ganglion (OG) and the commissural ganglia (CoG). Neurons: PD, pyloric dilator neuron; LP, lateral pyloric neuron; PY, pyloric constrictor neuron. Nerves: ivn, inferior ventricular nerve; ion, inferior esophageal nerve; son, superior esophageal nerve; dpon, dorsal posterior oesophageal nerve; stn, stomatogastric nerve; aln, anterior lateral nerve; mvn, median ventricular nerve; dgn, dorsal gastric nerve; dvn, dorsal ventricular nerve; lvn, lateral ventricular nerve; gpn, gastro pyloric nerve; lgn, lateral gastric nerve; pdn, pyloric dilator nerve.
Figure 2.
RH795 stains through the ganglion sheath and allows the visualization of cell body location.
A: Metathoracic ganglion of the cricket G. sigillatus before (white light) and after staining (fluorescence light). Scale bar is 100 µm. (i–iii) Higher magnification of the dotted area in the picture above. Changing the focal plane from dorsal (i) to ventral (iii) revealed multiple layers of distinct neurons (see arrows). Scale bar is 50 µm. B: Subpharyngeal ganglion of the earthworm E. hortensis before and after staining with RH795. Large neuronal somata were visible after staining (see arrows). Scale bar is 100 µm.
Figure 3.
RH795 staining in the stomatogastric nervous system of the crab, Cancer borealis.
A: Cell visibility before (i) and after desheathing (ii) the STG (note that the ganglion was not stained here). Pictures show the same ganglion, illuminated with a dark-field condenser and taken using the same light intensity, magnification, and camera settings. n, neuropil; m, motor neurons; * minuten pin. Scale bar is 100 µm. B: RH795 stains STG neurons through the ganglion sheath and causes a clear and long-lasting staining of neural structures. Comparison of visibility of the non-desheathed ganglion during 180 min of washout of the dye (left 5 pictures) and after desheathing (right). Immediately after removing the dye, the sheath still contained a lot of dye (1 min), but contrast and visibility improved quickly. The dye caused a long-lasting staining through the ganglion sheath without excessive residual staining of the sheath. Visibility did not further improve after desheathing the ganglion. Scale bar is 50 µm. C: RH795 revealed fine neuronal structures like neurites, axons and varicosities. (i) Anterior part of the STG showing individual axons entering the ganglion via the stn (see arrows). Distinct neurites in the STG neuropil could be identified, which is impossible with regular light microscopy. (ii) Middle part of the non-desheathed stn. Individual axons and varicosities (arrows) were visible after staining. (iii) Staining of the ivn, (iv) the pdn and (v) the gpn. Arrow indicates the location of the soma of the gastro-pyloric receptor neuron GPR. Scale bar is 100 µm. D: The AGR soma shown here stained with RH795 is anatomically isolated from other STG neurons, located more posteriorly than the somata of STG motor neurons. Scale bar is 50 µm.
Figure 4.
Desheathing causes subtle changes in neuronal activity.
A: Rhythmic changes in AGR inst. ff. disappear after desheathing. During a gastric mill rhythm, AGR inst. ff. (top trace) changed rhythmically. Bottom traces: extracellular recordings of mvn, dgn and lgn showing the timing of the gastric mill rhythm. Left: before desheathing; right: after desheathing the STG. Raw data are included in Raw Data.xlsx S1. B: Phase diagram of normalized averaged AGR inst. ff. and the gastric mill motor neurons LG, GM and DG before (left) and after (right) desheathing, from the same animal. AGR inst. ff. was binned (bin width 0.1). The grey surface shows the standard deviation. ***significant difference with p<0.001. C: Rhythmic changes in AGR inst. ff. were not affected by RH795. Left: non-desheathed STG, before staining, right: non-desheathed STG after 1 h staining and 20 minutes washout. D: Phase diagram of the same animal before (left) and after (right) staining with RH795. For detail see (B). Phases 0.3 and 0.7 were significantly different. ***p<0.001 before staining and **p<0.01 after staining. There was no significant difference between the mean values of all phases before and after staining (see text for details).
Figure 5.
Optical imaging through the ganglion sheath.
A: Left, Single-sweep simultaneous optical recording of IC and the two PD neurons, along with an extracellular recording of the pdn. In this particular recording the pdn also shows the action potentials of LP. Top: Photo showing the three somata selected for recording. Scale bar is 50 µm; n, neuropil. PDs and IC showed rhythmic and alternating changes in their fluorescence. Vertical scale bars are 0.04%. Right: spectrogram showing the major frequency components of optical and extracellular recordings. Warmer colors indicate higher power. Top traces, correlation of frequency distribution (1–10 Hz) for each time point for PD and pdn, as well as for PD and IC. There was a high correlation of the frequency components at all points in time. Note the different time scale (slightly compressed to fit page). B: Cycle-triggered average of PD and IC, plus pdn, showing the phase dependence of the optical signal. The first PD spike in each cycle was used for triggering. C: LP spike-triggered average of lvn and PD optical recording showing LP-timed synaptic inhibition in PD and its temporal dynamics.
Figure 6.
Repetitive staining permits long-term experiments.
Photos with regions of interest and simultaneous single-sweep recording of two pyloric neurons, plus pdn. Left: first staining; right: second staining. Recordings were taken roughly 5 hours apart from one another. Staining duration was 1 hour in each case. Scale bars: 100 µm (photo) and 0.1% (optical recordings).
Figure 7.
RH795 allows the simultaneous recording of gastric mill neurons through the ganglion sheath.
Left: photo of STG with 5 regions of interest representing the locations of the 4 GM neurons and Int1. Actual optical recording region indicated by dotted-white lines. Scale bar is 100 µm. Right: simultaneous optical recording of GMs and Int1. Scale bars are 0.4%. Bottom traces: extracellular recordings of lgn and dgn, showing the burst activities of LG, DG and GMs. Note that GM spikes (blue) are small, preventing their individual identification on the extracellular recording. Bottom: Waveform correlation of GMs and Int1, showing high synchrony of the 4 GMs and antiphasic correlation of GMs and Int1. Colors code correlation factor (1: strong correlation; 0: no correlation; −1: strong antiphasic correlation). GM1 was used as a reference for all correlations, with the top trace showing the GM1 autocorrelation.