Focused ultrasound stimulation of an ex-vivo Aplysia abdominal ganglion preparation
Tomas Jordan, James M. Newcomb, Michael B. Hoppa, Geoffrey P. Luke
Journal of Neuroscience Methods 2022, 372, 109536 · 10.1016/j.jneumeth.2022.109536
Abstract
Background A growing body of research demonstrates that focused ultrasound stimulates activity in human and other mammalian nervous systems. However, there is no consensus on which sonication parameters are optimal. Furthermore, the mechanism of action behind ultrasound neurostimulation remains poorly understood. An invertebrate model greatly reduces biological complexity, permitting a systematic evaluation of sonication parameters suitable for ultrasound neurostimulation. New method Here, we describe the use of focused ultrasound stimulation with an ex-vivo abdominal ganglion preparation of the California sea hare, Aplysia californica, a long-standing model system in neurobiology. We developed a system for stimulating an isolated ganglion preparation while obtaining extracellular recordings from nerves. The focused ultrasound stimulation uses one of two single-element transducers, enabling stimulation at four distinct carrier frequencies (0.515 MHz, 1.l MHz, 1.61 MHz, 3.41 MHz). Results Using continuous wave ultrasound, we stimulated the ganglion at all four frequencies, and we present quantitative evaluation of elicited activation at four different sonication durations and three peak pressure levels, eliciting up to a 57-fold increase in spiking frequency. Comparison with electrical stimulation We demonstrated that ultrasound-induced activation is repeatable, and the response consistency is comparable to electrical stimulation. Conclusions Due to the relative ease of long-term recordings for many hours, this ex-vivo ganglion preparation is suitable for investigating sonication parameters and the effects of focused ultrasound stimulation on neurons.
Abstract via europepmc.
Exposures
Exposure 1: 515 kHz continuous-wave FUS to Aplysia abdominal ganglion
Target: aplysia neurons — “abdominal ganglion (Aplysia californica)”
Device: Sonic Concepts · Sonic Concepts · H-204 ✓
| Waveform | continuous | |
|---|---|---|
| Fundamental frequency (kHz) | 515 | ✓✓✓ |
| Pulse duration (ms) | not applicable | |
| Pulse repetition frequency (Hz) | not applicable | |
| Duty cycle (%) | not applicable | |
| Sonication duration (s) | 0.001, 0.01, 0.1, 1swept | ✓✓✓ |
| Free-field pressure (kPa) | 1,110, 2,220, 3,330swept | ✓✓✓ |
|---|---|---|
| Free-field Isppa (W/cm²) | 41.1, 165, 371swept | ✓✓✓ |
| 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 |
For each of four carrier frequencies, continuous-wave FUS was delivered to the isolated abdominal ganglion at four stimulus durations (1, 10, 100 ms, 1 s) and three driving-voltage levels (50, 100, 150 mV rms, corresponding to the peak focal pressures/ISPPA listed); at the 1 s duration only 50 and 100 mV were tested due to concerns about tissue damage at 150 mV. Each duration/amplitude combination was applied twice, with 5 min rest between stimulations, using four animals per frequency.
Exposure 2: 1100 kHz continuous-wave FUS to Aplysia abdominal ganglion
Target: aplysia neurons — “abdominal ganglion (Aplysia californica)”
Device: Sonic Concepts · Sonic Concepts · H-151 ✓
| Waveform | continuous | |
|---|---|---|
| Fundamental frequency (kHz) | 1,100 | ✓✓✓ |
| Pulse duration (ms) | not applicable | |
| Pulse repetition frequency (Hz) | not applicable | |
| Duty cycle (%) | not applicable | |
| Sonication duration (s) | 0.001, 0.01, 0.1, 1swept | ✓✓✓ |
| Free-field pressure (kPa) | 2,610, 5,220, 7,830swept | ✓✓✓ |
|---|---|---|
| Free-field Isppa (W/cm²) | 227, 909, 2,050swept | ✓✓✓ |
| 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 |
For each of four carrier frequencies, continuous-wave FUS was delivered to the isolated abdominal ganglion at four stimulus durations (1, 10, 100 ms, 1 s) and three driving-voltage levels (50, 100, 150 mV rms, corresponding to the peak focal pressures/ISPPA listed); at the 1 s duration only 50 and 100 mV were tested due to concerns about tissue damage at 150 mV. Each duration/amplitude combination was applied twice, with 5 min rest between stimulations, using four animals per frequency.
Exposure 3: 1610 kHz continuous-wave FUS to Aplysia abdominal ganglion
Target: aplysia neurons — “abdominal ganglion (Aplysia californica)”
Device: Sonic Concepts · Sonic Concepts · H-204 ✓
| Waveform | continuous | |
|---|---|---|
| Fundamental frequency (kHz) | 1,610 | ✓✓✓ |
| Pulse duration (ms) | not applicable | |
| Pulse repetition frequency (Hz) | not applicable | |
| Duty cycle (%) | not applicable | |
| Sonication duration (s) | 0.001, 0.01, 0.1, 1swept | ✓✓✓ |
| Free-field pressure (kPa) | 258, 515, 773swept | ✓✓✓ |
|---|---|---|
| Free-field Isppa (W/cm²) | 2.22, 8.85, 20swept | ✓✓✓ |
| 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 |
For each of four carrier frequencies, continuous-wave FUS was delivered to the isolated abdominal ganglion at four stimulus durations (1, 10, 100 ms, 1 s) and three driving-voltage levels (50, 100, 150 mV rms, corresponding to the peak focal pressures/ISPPA listed); at the 1 s duration only 50 and 100 mV were tested due to concerns about tissue damage at 150 mV. Each duration/amplitude combination was applied twice, with 5 min rest between stimulations, using four animals per frequency.
Exposure 4: 3410 kHz continuous-wave FUS to Aplysia abdominal ganglion
Target: aplysia neurons — “abdominal ganglion (Aplysia californica)”
Device: Sonic Concepts · Sonic Concepts · H-151 ✓
| Waveform | continuous | |
|---|---|---|
| Fundamental frequency (kHz) | 3,410 | ✓✓✓ |
| Pulse duration (ms) | not applicable | |
| Pulse repetition frequency (Hz) | not applicable | |
| Duty cycle (%) | not applicable | |
| Sonication duration (s) | 0.001, 0.01, 0.1, 1swept | ✓✓✓ |
| Free-field pressure (kPa) | 506, 1,010, 1,520swept | ✓✓✓ |
|---|---|---|
| Free-field Isppa (W/cm²) | 8.52, 34.1, 76.7swept | ✓✓✓ |
| 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 |
For each of four carrier frequencies, continuous-wave FUS was delivered to the isolated abdominal ganglion at four stimulus durations (1, 10, 100 ms, 1 s) and three driving-voltage levels (50, 100, 150 mV rms, corresponding to the peak focal pressures/ISPPA listed); at the 1 s duration only 50 and 100 mV were tested due to concerns about tissue damage at 150 mV. Each duration/amplitude combination was applied twice, with 5 min rest between stimulations, using four animals per frequency.
Flags from extraction
n_subjects— Two different animal counts are given for different sub-experiments (4 animals per frequency for the main parameter sweep; 9 animals for the separate repeatability experiment); paper does not state a combined total, so both group sizes are listed.exposures[*].in_situ— Pressures/intensities were measured by hydrophone in a water-filled petri dish (free field) prior to placing the ganglion; classified as a bath preparation with no skull/tissue path, so in_situ fields are null rather than not_reported.exposures[*].timing.waveform— Classified stimulus durations (1 ms-1 s) as sonication_duration_s per the single-uninterrupted-burst/continuous-wave rule, even though the paper also calls these 'stimulus durations' resembling pulse-duration language.