Non-invasive, Focused Ultrasound-Facilitated Gene Delivery for Optogenetics.

Optogenetics, a widely used technique in neuroscience research, is often limited by its invasive nature of application. Here, we present a noninvasive, ultrasound-based technique to introduce optogenetic channels into the brain by temporarily opening the blood-brain barrier (BBB). We demonstrate the efficiency of the method developed and evaluate the bioactivity of the non-invasively introduced channelrhodopsin channels by performing stimulation in freely behaving mice.

Introduction

Purpose Drug delivery with BBB opening

Study Objective To develop and evaluate a noninvasive ultrasound-based method to deliver channelrhodopsin across the blood–brain barrier and assess its bioactivity via stimulation in freely behaving mice.

Animal model / Human subject Mouse (Mus musculus); strain: not specified; age: not specified; sex: not specified

Disease model Healthy

MRI or image guidance method C-mode ultrasound guidance referenced to the lambda suture (visualized through the shaved scalp using a metallic cross marker) with the FUS transducer positioned on a 3D positioning system to specified stereotactic coordinates (AP/ML/DV); pulse-echo confocal monitoring for cavitation.

Targeted brain region(s) Hippocampus And Motor Cortex

Target coordinates AP +2.6 mm, ML -2 mm, DV +2.5 mm (FUS hippocampus); AP +6.3 mm, ML -1 mm, DV +0.5 mm (FUS motor cortex); AP -2.7 mm, ML -2 mm, DV +1.5 mm (direct infusion hippocampus)

Cargo name and characteristics AAV9-mCherry-ChR2 (adeno-associated virus serotype 9 encoding Channelrhodopsin-2 fused to mCherry); viral vector, titer 2.48 × 10^13 GC/ml, diluted 1:6 in PBS prior to administration; used to express optogenetic ChR2 in mouse brain.

Route of administration Intravenous (tail vein) and direct intracranial stereotaxic infusion (needle)

Outcomes and Safety

Summary of Outcomes Transcranial focused ultrasound (FUS) BBB opening (noninvasive delivery of AAV9-ChR2 with IV microbubbles) produced functional optogenetic expression—blue-light stimulation evoked increased neuronal firing rates and c-Fos activation comparable to direct intracranial infusion, with no histological damage or notable inflammation. Successful FUS parameters reported: 1.5 MHz ultrasound transducer with focal depths ≈2 mm below skull for hippocampus and ≈0.5 mm for cortex, IV AAV9 (~4×10^11 GC/animal) co-administered with lipid-shelled microbubbles, monitored by passive cavitation detection and confirmed by contrast-enhanced MRI.

Duration of biological effect 2 weeks

Safety-related matter Histopathological analysis showed no structural brain damage, and microglia staining showed no significant inflammatory response following FUS BBBO

Brain Region

Ultrasound Parameters

Ultrasound instrument Single-element FUS transducer (Imasonic, France) — focal length 60 mm; radius 30 mm (aperture diameter 60 mm); center frequency 1.5 MHz; −6 dB focus 7.5 × 1 × 1 mm^3.

FUS Frequency 1.5 MHz

FUS Pressure 0.74 MPa

FUS Mode pulsed

Pulse duration 10 ms

Duration of a single FUS session 120 s

Focal Characteristics Focal depth: 2 mm (hippocampus), 0.5 mm (motor cortex); Focal length: 60 mm; Aperture size: radius 60 mm

Treatment frequency Single

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