Introduction

Over the past decade the use of optogenetics to drive genetically distinct populations of brain cells has profoundly increased our understanding of neural circuitry and brain function in health and disease. Optogenetics is now regularly integrated with functional imaging techniques such as BOLD-fMRI or CBV-fMRI, that rely on neurovascular coupling. However, despite the fact that visible light has been shown to dilate peripheral vessels, the effects of light on cerebral blood flow have not been thoroughly investigated. Here we tested whether light stimulation protocols similar to those commonly used in opto-fMRI or to study neurovascular coupling modulate blood flow in mice that do not express any light sensitive proteins.

Methods

Here we combined two techniques, two-photon laser scanning microscopy and ultrafast functional ultrasound imaging (fUS), approaches that measure blood flow at the microscopic and macroscopic levels. Optical stimulation was performed with lasers (473, 488, 561, 691, 638 nm) connected to a multimode optical fiber (62.5 μm). All the mice received a craniotomy over the Olfactory Bulb or over the neocortex. The exposed tissue was then covered by either an acoustic transparent material or a piece of glass coverslip. A titanium headbar was also placed on the caudal side of the skull. Window and headbar were fixed to the skull with dental cement. Chronic mice were left to recover for > one week before starting with the recordings with either fUS or two-photon laser scanning microscopy. Transgenic mice expressing the fluorescent reporter GCaMP6 under control of different promoters were used to identify activation of the cell types involed: principal neurons (Thy1), mural cells (NG2), astrocytes (Connexin30).

Results

Combining two-photon laser scanning microscopy and ultrafast functional ultrasound imaging (fUS), approaches that measure blood flow at the microscopic and macroscopic levels, we report that light per se causes a pronounced pseudo functional hyperemia, of similar magnitude to a sensory stimulation, in the neocortex and the olfactory bulb. Pulses or trains of light (473 to 638nm), shone on the mouse brain across a chronic cranial window, triggered a reversible and reproducible dilation of arterioles. The effect was energy-dependent appearing at a threshold of ~0.5mW-1mW. Two-photon imaging of GCaMP6f in transgenic mouse lines revealed that light caused a decrease of vascular smooth muscle cell calcium that preceded the onset of dilation and occurred in the absence of neuronal or astrocyte excitation. Furthermore, this photodilation occurred in a peripheral organ (the kidney), indicating that Ca2+ independent mechanisms in neurons or astrocytes were not required.

Discussion

This work shows that light per se, delivered in trains and at intensities commonly used to trigger functional hyperemia and/or fMRI signals in rodents, decreases SMC calcium, either directly or via endothelial cells, leading to dilation of arterioles. The effect of light is in the same range of amplitude as that evoked by sensory stimulation velocity and will thus affect the signal size and threshold detected in opto-fMRI experiments.

Conclusions

These results impose careful consideration on the use of photo-activation in studies involving blood flow regulation, as well as in studies requiring prolonged and repetitive stimulations to correct cellular defects in pathological models. They also suggest that light could be used to locally increase blood flow in a controlled fashion.

Acknowledgements

No acknowledgement found.

References

No reference found.