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Scanning Ultrasound (SUS) Causes No Changes to Neuronal Excitability and Prevents Age-Related Reductions in Hippocampal CA1 Dendritic Structure in Wild-Type Mice

Robert John Hatch, Gerhard Leinenga, Jürgen Götz

PLOS ONE 2016 · 10.1371/journal.pone.0164278

rodenthealthyinvasive electrophysiologyhistology molecular

Abstract

Scanning ultrasound (SUS) is a noninvasive approach that has recently been shown to ameliorate histopathological changes and restore memory functions in an Alzheimer's disease mouse model. Although no overt neuronal damage was reported, the short- and long-term effects of SUS on neuronal excitability and dendritic tree morphology had not been investigated. To address this, we performed patch-clamp recordings from hippocampal CA1 pyramidal neurons in wild-type mice 2 and 24 hours after a single SUS treatment, and one week and 3 months after six weekly SUS treatments, including sham treatments as controls. In both treatment regimes, no changes in CA1 neuronal excitability were observed in SUS-treated neurons when compared to sham-treated neurons at any time-point. For the multiple treatment groups, we also determined the dendritic morphology and spine densities of the neurons from which we had recorded. The apical trees of sham-treated neurons were reduced at the 3 month time-point when compared to one week; however, surprisingly, no longitudinal change was detected in the apical dendritic trees of SUS-treated neurons. In contrast, the length and complexity of the basal dendritic trees were not affected by SUS treatment at either time-point. The apical dendritic spine densities were reduced, independent of the treatment group, at 3 months compared to one week. Collectively, these data suggest that ultrasound can be employed to prevent an age-associated loss of dendritic structure without impairing neuronal excitability.

Abstract via europepmc.

Speciesmouse (C57BL/6)
Subjects3, 3, 3, 3swept animals
Sessions per subject1, 6swept
Randomisedyes
Blindingsingle
Sham / controlinactive transducer
Auditory controlnot reported
Readout timingoffline
Anaesthesiaanaesthetised
Readoutsinvasive electrophysiology, histology molecularwhole-cell patch-clamp electrophysiology (AP firing/kinetics, passive properties); Neurolucida dendritic morphometry and confocal spine density imaging
Direction of effectno effectNo change in CA1 neuronal AP firing or kinetics was observed after single or multiple SUS treatments compared to sham at any time-point; however, multiple SUS treatments prevented an age-related reduction in apical dendritic tree length/complexity seen in sham-treated mice at 3 months.
Adverse eventsnone observedDesigned as a safety study; no changes in neuronal excitability, AP kinetics or passive membrane properties were detected after single or repeated SUS/microbubble treatments, and multiple treatments did not damage dendritic structure (indeed preserved apical dendritic tree structure with ageing).

Exposures

Exposure 1: Scanning ultrasound (SUS) treatment of the whole brain with microbubbles

Target: whole brain or unfocused — “the entire brain
Device: Philips Research · Philips Research · Therapy Imaging Probe System (TIPS)

Pulse timing
Waveformpulsed
Fundamental frequency (kHz)1,000✓✓
Pulse duration (ms)10✓✓
Pulse repetition frequency (Hz)10✓✓
Duty cycle (%)10pulse duration × PRF gives 10%✓✓
Sonication duration (s)6✓✓
Pressure and intensity, by domain
Free-field pressure (kPa)700✓✓
Free-field Isppa (W/cm²)not reported
Free-field Ispta (W/cm²)not reported
In-situ estimatenot reported
In-situ pressure (kPa)not reported
In-situ Isppa (W/cm²)not reported
In-situ Ispta (W/cm²)not reported
Protocol, in the paper’s words

Ultrasound was applied sequentially in a scanning mode by applying it for 6 seconds duration per spot, moving the focus 1.5 mm and repeating the application until the entire brain was treated as described previously. The focus of the transducer had a volume of 1.5 mm x 1.5 mm x 12 mm. Mice received either a single SUS treatment (analysed 2 or 24 hours later) or six weekly SUS treatments (analysed one week or three months after the final treatment).

Flags from extraction

  • n_subjectsPaper states total mice (24) split 3 sham/3 SUS per each of 4 protocols; recorded as group sizes for the SUS-exposed arm only (3x4), not summed, and total includes an equal number of sham mice not exposed to ultrasound.
  • exposures[0].free_field.pressure_kpaPaper states 0.7 MPa peak rarefactional pressure 'applied outside the skull' without stating whether this was a water-tank/free-field measurement; recorded as free_field because it is explicitly outside the skull (not in-situ), but the domain wording is not the paper's usual 'measured in water' phrasing.
  • exposures[0].targetUltrasound was scanned over the entire brain (BBB-opening protocol); readouts were specifically from hippocampal CA1 neurons, but the sonication itself was not targeted to the hippocampus.