Noninvasive ultrasound deep brain stimulation of nucleus accumbens induces behavioral avoidance

Lili Niu, Yanchen Guo, Zhengrong Lin, Zhe Shi, Tianyuan Bian, Lin Qi, Long Meng, Anthony A. Grace, Hairong Zheng, Ti-Fei Yuan

Science China Life Sciences 2020, 63, 1328-1336 · 10.1007/s11427-019-1616-6

rodenthealthysubstance use disorderbehaviourhistology molecular

Abstract

Ultrasound stimulation is an emerging noninvasive option in treating neuropsychiatric disorders. The present study investigates the behavioral alterations resulting from ultrasound stimulation on the nucleus accumbens (NAc) in freely moving mice. Our results show that an acute ultrasound stimulation on the NAc, rather than the visual cortex or auditory cortex, led to a pronounced avoidance behavior, while repeated NAc ultrasound stimulation resulted in an obvious conditioned place aversion with changes in synaptic protein (GluA1/2 subunit) expression. Notably, NAc ultrasound stimulation suppressed the morphine-induced conditioned place preference. The results provide evidence that NAc ultrasound stimulation can be applied as a potential noninvasive therapeutic option in treating psychiatric disorders.

Abstract via europepmc.

Speciesmouse (C57BL/6J)

Subjects4, 6, 6, 8, 7, 14, 4swept animals

Sessions per subject5

Randomisedyes

Blindingnot reported

Sham / controlinactive transducer

Auditory controldeafened subjects

Readout timingoffline

Anaesthesiaawake

Readoutsbehaviour, histology molecular — real-time place preference test; conditioned place preference test; morphine-induced conditioned place preference; c-Fos immunostaining; Western blot (GluA1, GluA2, GluA3)

Direction of effectexcitatoryUltrasound stimulation of the NAc shell increased c-Fos expression and GluA1 expression (with decreased GluA2/GluA3), and produced behavioral avoidance/aversion (acute real-time place aversion and conditioned place aversion), and suppressed morphine-induced conditioned place preference.

Adverse eventsnot reportedThe negative peak pressure of ultrasound stimulation is 304 kPa before passing through the skull, substantially below the magnitude required for cavitation; the maximum temperature increase was estimated to be less than 0.003C; both MI (0.16) and Ispta (230 mW/cm2) were substantially below FDA-approved clinical ultrasound imaging thresholds (MI=1.9, Ispta=720 mW/cm2).

Exposures

Exposure 1: Ultrasound stimulation of nucleus accumbens (NAc) shell

Target: nucleus accumbens — “nucleus accumbens (NAc), shell”
Device: custom-built

Pulse timing
Waveform	pulsed
Fundamental frequency (kHz)	3,400	✓✓✓
Pulse duration (ms)	0.05, 0.5swept	✓✓✓⚑
Pulse repetition frequency (Hz)	1,000, 100swept	✓✓✓
Duty cycle (%)	not reported	⚑
Sonication duration (s)	1,800	✓✓✓

Pressure and intensity, by domain
Free-field pressure (kPa)	304	✓✓✓
Free-field Isppa (W/cm²)	not reported
Free-field Ispta (W/cm²)	not reported
In-situ estimate	not reported
In-situ pressure (kPa)	not reported
In-situ Isppa (W/cm²)	not reported
In-situ Ispta (W/cm²)	not reported
Ispta, domain unspecified (W/cm²)	0.23	✓✓✓

Protocol, in the paper’s words

In the real-time place preference test, ultrasound was delivered into the NAc when the mouse entered the ultrasound stimulation side until it returned to the non-stimulation side; two sonication parameter sets were tested: 0.5 ms TBD/100 Hz PRF and 0.05 ms TBD/1,000 Hz PRF (n=6 per group), both at 304 kPa. For the conditioned place preference test, mice received 0.05 ms TBD, 1,000 Hz PRF stimulation for 30 min per day for 5 consecutive days (n=14), while sham mice wore the transducer without stimulation for 30 min per day for 5 days (n=18).

Exposure 2: Ultrasound stimulation of visual cortex (V1), specificity control

Target: primary visual cortex — “visual cortex (V1)”
Device: custom-built

Pulse timing
Waveform	pulsed
Fundamental frequency (kHz)	3,400	✓✓✓
Pulse duration (ms)	0.05	✓✓✓
Pulse repetition frequency (Hz)	1,000	✓✓✓
Duty cycle (%)	not reportedpulse duration × PRF gives 5%
Sonication duration (s)	not reported

Pressure and intensity, by domain
Free-field pressure (kPa)	304	✓✓✓
Free-field Isppa (W/cm²)	not reported
Free-field Ispta (W/cm²)	not reported
In-situ estimate	not reported
In-situ pressure (kPa)	not reported
In-situ Isppa (W/cm²)	not reported
In-situ Ispta (W/cm²)	not reported
Ispta, domain unspecified (W/cm²)	0.23	✓✓✓

Protocol, in the paper’s words

Ultrasound waves with 0.05 ms TBD and 1,000 Hz PRF were delivered to visual cortex (V1) as a target-specificity control (n=8) and did not result in behavioral place-preference changes, unlike NAc stimulation.

Flags from extraction

n_subjects — Multiple, only partly overlapping sub-experiments each report their own group size (c-Fos n=4, RTPP groups n=6/6/8/7, CPP n=14 US/18 sham, morphine-CPP n=4, western blot n=6); the paper never states one overall total exposed to ultrasound, so group sizes are listed rather than summed.
exposures[0].timing.pulse_duration_ms — Figure 1 legend states the tone burst duration as '5 μs' (0.005 ms) at 1,000 Hz PRF, which conflicts with the 0.05 ms TBD stated in the main text and other figure legends for the same 1,000 Hz PRF condition; likely a typo/unit error in the figure legend, both durations are recorded via protocol_description/quotes but pulse_duration_ms uses the main-text value (0.05 ms).
unspecified_domain.ispta_w_cm2 — The paper does not state whether the reported Ispta (230 mW/cm2) and MI (0.16) refer to free-field or in-situ/transcranial values; placed in unspecified_domain.
exposures[0].timing.duty_cycle_pct — Duty cycle is not stated explicitly in the text; it could be computed from pulse duration and PRF but this was not done per instructions to avoid unstated arithmetic.
blinding — c-Fos cell quantification is stated to have been performed 'in a double-blind fashion', but no blinding is described for the behavioral testing itself; overall blinding recorded as not_reported.