Light controls cerebral blood flow in naive animals

Abstract

Optogenetics is increasingly used to map brain activation using techniques that rely on functional hyperaemia, such as opto-fMRI. Here we test 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 light sensitive proteins. Combining two-photon laser scanning microscopy and ultrafast functional ultrasound imaging, we report that in the naive mouse brain, light per se causes a calcium decrease in arteriolar smooth muscle cells, leading to pronounced vasodilation, without excitation of neurons and astrocytes. This photodilation is reversible, reproducible and energy-dependent, appearing at about 0.5 mJ. 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.

Figure 1. Blue light generates a rapid increase of cerebral blood flow (CBF) in the naive mouse brain.

(a) Schematic, light emitted by the optic fibre diffuses through a PMP chronic window into the brain. (b) A single train of blue light pulses (20 ms, 20 Hz, 5 mW, 2 s) reliably generates a power Doppler signal, detectable at the level of single pixels and indicative of a CBF increase. Top, the activation map is superimposed on a Doppler map. Bottom, single pixel responses vary within the activated region. Three trials are superimposed. (c) Average of CBF responses to light in the cortex of three wild type mice. (d) CBF responses spread with the stimulus intensity. The region activated by a 2 mW light train (top left, 20 ms, 20 Hz, 2 s) and limited to the border of the somatosensory and motor cortices, enlarges progressively with the intensity shone. (e) Averages of CBF responses at different light intensities. Data presented as mean±s.d. All scale bars, 1 mm. fUS in plane resolution =100 × 100 μm².

Figure 2. Light-evoked CBF increase results from artery dilation.

(a) In the olfactory bulb (OB) of the naive mouse, a single train of blue light (20 ms, 20 Hz, 5 mW, 2 s) generates a focal increase of the power Doppler signal. Scale bar, 1 mm. In plane resolution: 100 × 100 μm². (b) Averages of CBF responses to light and odour (ethyl tiglate). (c) Light causes a dilation of large arterioles at the dorsal surface of the OB. Vessels were labelled with Texas red and imaged with a two-photon microscope. Light dilated both A1 and A2 arteries but did not affect the vein V. (d) Quantification of the experiment in (c). (e) Dilation is detectable above a light intensity of 1 mW. Data points correspond to mean value of 3–5 trials±s.d. in individual mice.

Figure 3. Light increases CBF independently of neuronal or astrocyte Ca²⁺ dependent mechanisms.

(a) Odour causes a large calcium increase in the glomerular layer of a mouse expressing GCaMP6f under the Thy1 promoter. Top, Fluorescence increases robustly in the dendritic tufts of mitral cells during odour. Images were selected from a frame scan acquisition. The broken line in white indicates the two segments used in linescan acquisition mode to measure calcium and red blood cell (RBC) velocity in b–f. (b,c) Odour generates a calcium increase in the neuropil that precedes the increase in RBC velocity by more than a second. The calcium raw data shown in (b) corresponds to the acquisition comprised between the two arrows in (c). The RBC raw flow data shown in (b) were selected from baseline and following odour. (d-f) Light increases RBC velocity without activating neurons. All grey areas illustrate the time periods used to quantify the effects of odours and light (see main text). Scale bar in (a,d) is 25 μm. (g) An arteriole whose lumen is labelled with Texas red and that is surrounded by astrocyte end-feet expressing GCaMP6f under the connexin 30 promoter. Dashed lines outline endfoot ROIs plotted in (h). Scale, 5 μm. (h) Light dilates the vessel (right) without affecting the spontaneous calcium signals nor the steady state calcium level in the astrocyte end-feet(left). Grey traces show single trials, black trace shows mean of trials.

Figure 4. Light triggers dilation via a decrease in SMC calcium.

(a,b) A glomerulus layer capillary, whose lumen is labelled with Texas red and contacted by the process of a longitudinal-type pericyte expressing GCaMP6f. A broken line scan acquisition to simultaneously measure pericyte calcium and RBC velocity. Scale bar, 3 μm. (b) Spontaneous pericyte Ca²⁺ transients or steady calcium are not affected by consecutive light trains (5 mW, 20 ms, 20 Hz, 2 s), whereas RBC velocity is consistently increased (right). (c,d) An arteriole in which the smooth muscle cell (SMC) wall shows typical stripe patterns of GCaMP6f expression, and in which the lumen is labelled with Texas red. A transversal linescan acquisition allows simultaneous recording of SMC calcium and vessel diameter. Light lowers calcium in the SMC wall and dilates the vessel. Scale bar, 30 μm (d) Left, the reversible responses to three consecutive trains (5 mW, 20 ms, 20 Hz, 2 s) are superimposed. Right, single 100 ms pulses (5 mW) reveal that the decrease in SMC calcium precedes the arteriole dilation (displayed in (c) on right). (e) Summarized data: Left, SMC Ca²⁺ and arteriole diameter (n=3 arterioles, 3 mice). Right, glomerulus pericyte Ca²⁺ and RBC velocity (n=6 capillaries, 3 mice). Data presented as mean±s.d.

Figure 5. Isoflurane blocks light-triggered dilations.

(a) The same vessel that dilated to light under ketamine-xylazine anaesthesia (top left) no longer dilated to light when the mouse was anesthetized with isoflurane (bottom left). Right, postive interleaved controls show response to odour indicating that neurovascular coupling is maintained, although smaller. Black traces: single trials from the same vessel. Red trace: mean. (b) Summarized data (3 mice). Each point represents mean±s.d. for individual mice.