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

Abstract

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.

Figure 1. FUS-facilitated viral delivery of ChR2 protein and safety evaluation.

(a) Experimental schematic and the targeted brain region was precisely located by identifying the lambda suture through shaved scalp. (b) The timeline for our technique. Subjects first received systematic injection of microbubbles and viral vectors, followed by FUS sonication. The BBB opening was confirmed via MRI and the animals were allowed to survive for 2 weeks. The bioactivity of the expressed ChR2 was evaluated by performing optical stimulation at the targeted area. (c) Signals collected from a passive cavitation detector (PCD): top panel shows the spectra of pre-microbubble injection signals, while ultra-harmonic signals (e.g. black arrow) indicate ongoing cavitation events. (d) BBB opening confirmation with contrast-enhanced MRI in hippocampus (top panel) and cortex (bottom panel) and the corresponding ChR2 expression (red, last column).

Figure 2

(a) Comparison of ChR2 expression using FUS-facilitated viral delivery (top) and the direct infusion technique (bottom). (b-c) Hematoxylin and Eosin (H&E) staining (b) and Nissl staining (c) were used to evaluate potential structural damage (N = 3, right column is FUS sonicated side). No damage was found in any subjects. (d) Microglia staining revealed no notable inflammatory response on the FUS sonicated side (bottom) compared to the contralateral side (top). Scale bar in (b) indicates 200 μm, while in (c,d) represent 100 μm.

Figure 3. The bioactivity of FUS-facilitated viral delivery was tested via optical stimulation.

(a) Four 2 s blue light (470 nm) pulses were given to elicit neuronal response in freely behaving mice. FUS-facilitated viral delivery was carried out in these mice (N = 3) targeting hippocampus. Sorted spike signals (top) and the corresponding number of spikes (bottom, binned into 0.4 s segments) revealed elevated neuronal activity during stimulation (red). (b) Four 2-s pulses were applied to mice (N = 3) that received direct infusion of viral vectors. An increased neuronal activity was observed during stimulation, which is similar to that of FUS-facilitated viral delivery group. (c) Examples of individual spike signals during stimulation from FUS-facilitated delivery group (red) and direct infusion group (blue). The baseline level spike for each group is indicated in black. (d) The firing rate was calculated for each group and significant increases (p < 0.001) in firing rate were observed during stimulation.

Figure 4

(a) Ten-second long pulses were also applied (red). An increased spike amplitude and number of spikes were observed at the onset of stimulation, which was followed by a gradual decrease to baseline level. (b) Neuronal activity (ChR2 is red) was revealed by labeling c-Fos proteins with fluorescent markers (green). (c) An example of a ChR2-expressing neuron that was activated by optical stimulation. Scale bars represent 20 μm and 5 μm in (b,c), respectively.