Activation of N-Methyl-D-aspartate receptor contributed to the ultrasonic modulation of neurons in vitro

Wu Yong, Dongming He, Taiheng Chen, Zeng Rui, Yinchuan Wen, Kin Fung Wong, Lei Sun, Xiaofei Qi, Jinghui Guo

Biochemical and Biophysical Research Communications 2023 · 10.1016/j.bbrc.2023.07.034

in vitro cellhealthycellular imaginghistology molecular

Abstract

Ultrasound stimulation is increasingly used to investigate brain function and treat brain diseases due to its high level of safety and precise spatiotemporal resolution. Therefore, it is crucial to understand the underlying mechanisms involved in ultrasound brain stimulation. In this study, we investigate the role of NMDA receptors in mediating the effects of ultrasound on primary hippocampal neurons in mice. Our results show that ultrasound alone can activate heterologous NMDA receptor subunits, including NR1A, NR2A, and NR2B, in 293T cells, as well as endogenous NMDA receptors in primary neurons. This activation leads to an influx of calcium and an increase in nuclear c-Fos expression in primary neurons that have not been pre-treated with an NMDA receptor inhibitor. In conclusion, our findings demonstrate that NMDA receptors contribute to neuronal activation by ultrasound stimulation in vitro, providing insight into the molecular mechanisms of ultrasound neuromodulation and a new mediator for the sonogenetics technique.

Abstract via europepmc.

Speciesmouse (primary hippocampal neurons, E19); human (HEK293T cells)

Subjectsnot reported cultures

Sessions per subjectnot applicable

Randomisednot applicable

Blindingnot applicable

Sham / controlno treatment control

Auditory controlnot reported

Readout timingboth

Anaesthesianot applicable

Readoutscellular imaging, histology molecular — Fura-2 calcium imaging; c-fos immunostaining; qPCR

Direction of effectexcitatoryUltrasound evoked Ca2+ influx in NMDA-receptor-expressing HEK293T cells and primary hippocampal neurons and raised c-fos; the effects were reduced by AP-5 and ketamine.

Adverse eventsnot applicableATP applied after ultrasound still produced a calcium response, taken to show the cells were not damaged.

Exposures

Exposure 1: Ultrasound to cultured hippocampal neurons and HEK293T cells

Target: cultured neurons — “primary hippocampal neurons and HEK293T cells”
Device: not reported

Pulse timing
Waveform	pulsed
Fundamental frequency (kHz)	500	✓✓✓
Pulse duration (ms)	not reportedimplied by duty cycle ÷ PRF: 0.5 ms (not stated by the paper)
Pulse repetition frequency (Hz)	1,000	✓✓✓
Duty cycle (%)	50	✓?⚑
Sonication duration (s)	0.4	✓✓✓⚑

Pressure and intensity, by domain
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	✓?⚑

Protocol, in the paper’s words

Each stimulus is 400 tone burst pulses at 500 kHz centre frequency and 1 kHz repetition frequency, lasting 400 ms, repeated every 5 s. For c-fos counts neurons received 0.3 MPa ultrasound for 30 min.

Flags from extraction

exposures[0].timing.duty_cycle_pct — Text states 50% in section 3.1 and 33.3% in the following paragraph for the same parameters; 50% recorded.
exposures[0].unspecified_domain.pressure_kpa — Pressures 0.3 and 0.6 MPa described as acoustic pressure mapped with a hydrophone; medium not stated.
exposures[0].timing.sonication_duration_s — 400 ms converted to 0.4 s; stimulus repeated every 5 s, number of repeats not stated.
n_subjects — Number of cultures or replicates not stated as a total; three independent experiments in some assays.