Ultrasound-Induced Membrane Hyperpolarization in Motor Axons and Muscle Fibers of the Crayfish Neuromuscular Junction
Feiyuan Yu, Wolfgang S. Müller, Gösta Ehnholm, Yoshio Okada, Jen-Wei Lin
Ultrasound in Medicine & Biology 2023, 49, 2527-2536 · 10.1016/j.ultrasmedbio.2023.08.016
Abstract
Objective Focused ultrasound (FUS) can modulate neuronal activity by depolarization or hyperpolarization. Although FUS-evoked depolarization has been studied extensively, the mechanisms underlying FUS-evoked hyperpolarization (FUSH) have received little attention. In the study described here, we developed a procedure using FUS to selectively hyperpolarize motor axons in crayfish. As a previous study had reported that these axons express mechano- and thermosensitive two-pore domain potassium (K2P) channels, we tested the hypothesis that K2P channels underlie FUSH. Methods Intracellular recordings from a motor axon and a muscle fiber were obtained simultaneously from the crayfish opener neuromuscular preparation. FUSH was examined while K2P channel activities were modulated by varying temperature or by K2P channel blockers. Results FUSH in the axons did not exhibit a coherent temperature dependence, consistent with predicted K2P channel behavior, although changes in the resting membrane potential of the same axons indicated well-behaved K2P channel temperature dependence. The same conclusion was supported by pharmacological data; namely, FUSH was not suppressed by K2P channel blockers. Comparison between the FUS-evoked responses recorded in motor axons and muscle fibers revealed that the latter exhibited very little FUSH, indicating that the FUSH was specific to the axons. Conclusion It is not likely that K2P channels are the underlying mechanism for FUSH in motor axons. Alternative mechanisms such as sonophore and axon-specific potassium channels were considered. Although the sonophore hypothesis could account for electrophysiological features of axonal recordings, it is not consistent with the lack of FUSH in muscle fibers. An axon-specific and mechanosensitive potassium channel is also a possible explanation.
Abstract via europepmc.
Exposures
Exposure 1: Temperature-manipulation experiments (motor axon / muscle fiber)
Target: neuromuscular junction, crayfish neurons — “crayfish opener neuromuscular junction (motor axon and muscle fiber)”
Device: custom-built
| Waveform | continuous | |
|---|---|---|
| Fundamental frequency (kHz) | 2,100 | ✓✓✓ |
| Pulse duration (ms) | not applicable | |
| Pulse repetition frequency (Hz) | not applicable | |
| Duty cycle (%) | not applicable | |
| Sonication duration (s) | 0.08 | ✓✓✓ |
| Free-field pressure (kPa) | not reported | |
|---|---|---|
| Free-field Isppa (W/cm²) | not reported | |
| Free-field Ispta (W/cm²) | not reported | |
| In-situ estimate | not applicable | |
| In-situ pressure (kPa) | not applicable | |
| In-situ Isppa (W/cm²) | not applicable | |
| In-situ Ispta (W/cm²) | not applicable | |
| Pressure, domain unspecified (kPa) | 300, 600swept | ✓?⚑ |
| Ispta, domain unspecified (W/cm²) | 0.00094 | ✓?⚑ |
An 80 ms burst of continuous sinusoidal waves at 2.1 MHz was used in temperature experiments. Optimal intensity per preparation (typically 0.3-0.6 MPa) was the level at which FUS-evoked depolarization occurred in <5% of trials; this was then used for the remainder of the experiment. A typical cycle included three trials (FUS alone, an AP train alone, FUS and an AP train simultaneously) with a 10 s rest between trials.
Exposure 2: Pharmacological (K2P channel blocker) experiments (motor axon)
Target: neuromuscular junction, crayfish neurons — “crayfish opener neuromuscular junction (motor axon)”
Device: custom-built
| Waveform | pulsed | |
|---|---|---|
| Fundamental frequency (kHz) | 2,000 | ✓✓✓ |
| Pulse duration (ms) | 0.5 | ✓✓✓ |
| Pulse repetition frequency (Hz) | 1,000 | ✓✓✓ |
| Duty cycle (%) | 50pulse duration × PRF gives 50% | ✓✓✓ |
| Sonication duration (s) | not reported | ⚑ |
| Free-field pressure (kPa) | not reported | |
|---|---|---|
| Free-field Isppa (W/cm²) | not reported | |
| Free-field Ispta (W/cm²) | not reported | |
| In-situ estimate | not applicable | |
| In-situ pressure (kPa) | not applicable | |
| In-situ Isppa (W/cm²) | not applicable | |
| In-situ Ispta (W/cm²) | not applicable | |
| Pressure, domain unspecified (kPa) | 300, 600swept | ✓?⚑ |
| Ispta, domain unspecified (W/cm²) | 0.00094 | ✓?⚑ |
Thirty FUS tone bursts at 1 kHz with 50% duty cycle were delivered to trigger FUSH, chosen to visualize hyperpolarization evoked by individual bursts; each tone burst lasted 500 microseconds. A typical cycle contained eight current steps (four hyperpolarizing, four depolarizing) with a 2 s rest between steps, repeated with and without FUS, in the presence of fluoxetine, norfluoxetine, or Ba2+ to test K2P channel involvement.
Flags from extraction
n_subjects— Paper reports preparation counts per sub-experiment (6 for cooling, 7 for heating, 6 for pharmacological blockade) with unclear overlap between them; a total distinct-animal count is not stated.exposures[0].unspecified_domain.pressure_kpa— Paper does not state whether the acoustic pressure is a free-field or in-situ (target) value; recorded as unspecified domain.exposures[1].unspecified_domain.pressure_kpa— Paper does not state whether the acoustic pressure is a free-field or in-situ (target) value; recorded as unspecified domain.exposures[0].unspecified_domain.ispta_w_cm2— Value is a calculated/theoretical estimate ('should be 0.94 mW/cm2'), not a directly measured quantity.exposures[1].unspecified_domain.ispta_w_cm2— Value is a calculated/theoretical estimate ('should be 0.94 mW/cm2'), not a directly measured quantity.exposures[1].timing.sonication_duration_s— Total duration of the 30-pulse train (30 ms at 1 kHz) is not explicitly stated by the paper; left not_reported rather than computed.