Article Figures & Data
Figures
- Figure 1.
The medicinal leech and experimental design. A, Diagram of the central nervous system of the leech, characterized by a ventral nerve cord interspersed with 21 segmental ganglia descending from a compound cephalic ganglion. B, Schematic of the placement of neuronal somata on the ventral surface of a single ganglion. The bilateral Retzius cells are colored red and labeled R. C, Neurobiotin fill of a Retzius cell showing its soma, neurites, and axons (a faintly labeled contralateral soma is present because of electrical coupling of the two cells). D, US paradigm demonstrating the positioning of the transducer, intracellular electrode, and ganglion preparation. E, Side view of the electrode displacement paradigm demonstrating the movement of the recording electrode.
- Figure 2.
US parameters. A, In this graph, all the pressures used in this study and their corresponding intensities (spatial peak pulse average) are indicated. Intensities were calculated using the equation shown in A, where Pn = pressure; Þ = density of nerve tissue, estimated to be 1.03 × g/cm3; c = speed of sound in saline medium, estimated to be 1507 m/s. B, US pulse parameters; 960-kHz US was applied for a single tone of 100-ms duration. Tones consisted of 100 pulses of 300 cycles of US (313-μs pulse duration). C, Linearly interpolated pressure distribution maps overlaid with scale preparation, dish, and electrode.
- Figure 3.
Comparison of the effects of US and electrode displacement on the resting membrane potential of Retzius neurons. A, Plots demonstrating changes in mean membrane potential in response to US applied at increasing pressures (upper plot, pink) and electrode displacements of increasing distance (lower plot, green), aggregated across preparations. Error bars denote SEM. B, Intracellular recordings demonstrating effects of US applied at increasing pressures to the same cell (pink, upper); recordings demonstrating effects of electrode displacement at increasing distances on the same cell (green, lower). C, Intracellular recordings demonstrating typical waveforms of depolarizations elicited by US (upper) and electrode displacement (lower). D, Scatter plots comparing time to peak depolarization following start of US (pink) and electrode displacement (green). Horizontal lines denote medians. The difference between the two was significant (Z = 2.6275, *p = 0.0086, Wilcoxon rank-sum test).
- Figure 4.
Comparison of the effects of electrode displacement on the spike frequency and amplitude of Retzius neurons. A, Intracellular recordings demonstrating US (upper, pink) and electrode-displacement (lower, green) associated increase in spike frequency. B, Scatter plots comparing the normalized change in spike frequency, during the period of peak effect, in US (pink) and electrode displacement (green) conditions. Horizontal lines denote medians. The difference between the two did not reach the threshold for significance (Z = 0.1890, p = 0.8501, Wilcoxon rank-sum test). C, Intracellular recordings showing that US (pink) and electrode displacement (green) induce reductions in spike amplitude. Averaged spike waveforms (left) demonstrate reduction in spike amplitude (black waveforms = averaged from the two spikes before stimulus onset, pink and green waveforms = averaged from the two spikes fired during the peak effect period following US application and electrode displacement, respectively). D, Scatter plots comparing normalized change in spike amplitude during peak effect period in US (pink) and electrode displacement (green) conditions. Horizontal lines denote medians. There was no significant (n.s.) difference between the two (t(17.3329) = 0.2777, p = 0.7845, Welch’s t test).
- Figure 5.
US application and electrode displacement yield similar results when a different neuron (N cell) and different pulse parameters are used. A, Schematic of ventral surface of a single leech ganglion with N neurons marked. B, US parameters applied to N cells. We applied one tone (300-ms duration) of continuous (vs pulsed) US per trial. C, Representative intracellular traces of N cell voltage during a trial of US application (upper, pink) and electrode displacement (lower, green). When upper trace is expanded (inset), the waveform closely resembles that observed in the electrode displacement paradigm. The difference in the duration of the US-induced depolarization can be attributed to the difference in stimulus duration.
- Figure 6.
Retzius neuron membrane potential following extended US application is influenced by prior sharp electrode impalement. A, Schematic of extended US application. Pulsed US was applied for a 20-min duration. Tones were delivered the first 10 s of each minute (tone duration = 10 s, tone frequency = 0.167 Hz). Tones consisted of 10,000 pulses of 300 cycles of 960-kHz US (pulse repetition frequency = 1 kHz, pulse duration = 312.5 μs). Pressure applied was 111 kPa in all trials. B, Schematics of trial design for extended application paradigm. Upper, Retzius neuron was impaled (blue), and resting membrane potential was recorded. The recording electrode was then removed (middle cartoon) and US was applied for 20 min. Following US application, the electrode was re-inserted into the same Retzius cell for a second baseline recording. Lower, In a different preparation, the electrode was inserted into the Retzius cell (blue) to record the resting membrane potential. As in the previous experiment, the electrode was removed before 20 min of US application (middle cartoon). After application, the contralateral Retzius cell (orange) was impaled to record baseline activity; this cell was thus not previously impaled. C, Intracellular recordings taken from the same Retzius cell before and after extended application of US demonstrating post-US depolarization of the resting membrane potential. D, Scatter plots comparing differences between pre-US and post-US membrane potential (mV) in the same cell (blue) and contralateral cell (orange). Control paradigms replaced the US application period with a waiting period of equivalent time. Membrane potentials of the US-treated and control groups differed significantly (Wilcoxon rank-sum test, *p = 1.55e-4) when the same Retzius cell was re-impaled. However, the US and control groups showed no significant (n.s.) difference (Wilcoxon rank-sum test, p = 0.1605) when the contralateral cell was recorded.
Tables
Data structure Type of test Result Effect size Power a Non-normal
US condition:
W(11) = 0.7185, p = 0.0018
ED condition:
W(11) = 0.6417, p = 4.38e-04Wilcoxon rank-sum test Z = 2.6275, p = 0.0086 d = 1.3018 0.8438 b Non-normal
US condition:
W(9) = 0.7890, p = 0.0141
ED condition:
W(9) = 0.5623, p = 2.6799e-04Wilcoxon rank-sum test Z = 0.1890, p = 0.8501 d = 0.0135 0.0501 c Normal
US condition:
W(9) = 0.9659 , p = 0.8508
ED condition:
W(9) = 0.9713 p = 0.9027Welch’s t test t(17.3329) = 0.2777,
p = 0.7845d = 0.0343 0.0506 d Non-normal
US condition:
W(7) = 0.8499, p =0.0951
Control condition:
W(7) = 0.9543 p = 0.7547Wilcoxon rank-sum test Z =100, p = 1.554E-4 d = 3.613 0.99 e Non-normal
US condition:
W(7) = 0.8802, p = 0.189
Control condition:
W(7) = 0.8802, p = 0.0274Wilcoxon rank-sum test p = 0.1605 d = 1.3432 0.68 Letters (leftmost column) correspond to p values of statistical tests as reported in Results. The data structure, test type, result, effect size, and statistical power of these tests are described. Results of Shapiro–Wilk test for normality of data in US and electrode displacement (ED) conditions (α = 0.05) are reported under Data structure. Normally distributed data were compared with Welch’s t test, and non-normal data were compared with the nonparametric Wilcoxon rank-sum test. Effect sizes were calculated as Cohen’s d with correction for small sample sizes as described by Durlak (2009).
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