Two-photon optogenetics-based assessment of neuronal connectivity in healthy and chronic hypoperfusion mice

Abstract

Significance: Two-photon optogenetics and simultaneous calcium imaging can be used to visualize the response of surrounding neurons with respect to the activity of an optically stimulated target neuron, providing a direct method to assess neuronal connectivity.

Aim: We aim to develop a two-photon optogenetics-based method for evaluating neuronal connectivity, compare it to the existing indirect resting-state synchrony method, and investigate the application of the method to brain pathophysiology.

Approach: C1V1-mScarlet was introduced into GCaMP6s-expressing transgenic mice with an adeno-associated virus. Optical stimulation of a single target neuron and simultaneous calcium imaging of the target and surrounding cells were performed. Neuronal connectivity was evaluated from the correlation between the fluorescence intensity of the target and surrounding cells.

Results: The neuronal connectivity in the living brain was evaluated using two-photon optogenetics. However, resting-state synchrony was not always consistent with two-photon optogenetics-based connectivity. Comparison with neuronal synchrony measured during sensory stimulation suggested that the disagreement was due to external sensory input. Two-photon optogenetics-based connectivity significantly decreased in the common carotid artery occlusion model, whereas there was no significant change in the control group.

Conclusions: We successfully developed a direct method to evaluate neuronal connectivity in the living brain using two-photon optogenetics. The technique was successful in detecting connectivity impairment in hypoperfusion model mice.

Keywords: calcium imaging; chronic cerebral hypoperfusion; neuronal connectivity; two-photon optogenetics.

Fig. 1

Two-photon optogenetics-based neuronal connectivity: (a) a typical two-photon imaging FOV of layer 2/3 in the somatosensory cortex. Neurons co-expressing GCaMP6s (green) and C1V1-mScarlet (red) can be observed with 920 and 1064 nm imaging, respectively. The area surrounded by the white square is shown in panel (b). (b) Neuronal activity induced using two-photon optical stimulation. A neuron expressing C1V1 (ROI 1) responds to optical stimulation, whereas a neuron without C1V1 (ROI 2) does not respond. (c) An example of a correlation coefficient map. The target neuron for optical stimulation is shown in yellow. The correlation coefficient of each cell with respect to the target cell is indicated according to the color scale in the top right of the image. (d) Percent change in GCaMP6s fluorescence ($\mathrm{\Delta }F/F$) of the target cell and the labeled cells (cells 1 to 3) induced after optical stimulation. The graph shows representative data from 10 of the 20 to 25 stimulation periods during the experiments. (e) Scatter plots of ΔActivity for the target cell and the labeled cells (cells 1 to 3). The r-value in the top right corresponds to the correlation coefficient calculated for the scattered data points.

Fig. 2

Comparison of neuronal connectivity evaluated using two-photon optogenetics and resting-state synchrony: (a) a representative FOV where the neuronal activity was assessed. Cells with green fluorescence are GCaMP6s-expressing neurons. (b) Percent changes in GCaMP6s fluorescence ($\mathrm{\Delta }F/F$) during the resting state (upper) and corresponding scatter plots of $\mathrm{\Delta }F/F$ (lower) for the target and labeled cells in panel (a). Correlation coefficients characterizing the connectivity based on resting-state synchrony (r-values) are shown in the top right of the plots. (c) Comparison of correlation coefficient maps based on two-photon optogenetics (left) and resting-state synchrony (middle). The t-values calculated for the two methods are compared in a scatter plot (right). The correlation between these two sets of t-values is $r=0.3445$ ($p<0.05$) in this example. (d) An example where the two sets of t-values are poorly correlated ($r=0.1134$, $p>0.05$). Two target cells were selected for each mouse, making a total of 12 experiments. The distribution of the corresponding r-values is shown in panel (e), and a histogram of the p-values is in panel (f). The black dots in panel (e) correspond to cases where $p<0.05$ in panel (f). A similar relationship exists for the green dots.

Fig. 3

Comparison of two-photon optogenetics-based connectivity and synchrony during sensory stimulation: (a) another representative FOV different from that in Fig. 2 where the neuronal activity was assessed. (b) Percent change in GCaMP6s fluorescence ($\mathrm{\Delta }F/F$) during sensory stimulation (upper) and scatter plots of $\mathrm{\Delta }\mathrm{Activity}$ for the target and labeled cells (lower). The upper part shows representative data from seven out of 20 stimulation periods during the experiments. Correlation coefficients characterizing the connectivity based on synchrony during sensory stimulation (r-values) are shown in the top right of the plots. (c) On the left is a comparison of the correlation coefficient maps obtained with (1) two-photon optogenetics, (2) resting-state synchrony, (3) synchrony during sensory stimulation, and (4) resting-state synchrony after sensory stimulation. To the right of these maps are scatter plots comparing the t-values obtained for two-photon optogenetics with those obtained for resting-state synchrony and synchrony during sensory stimulation. (d) Comparison of the set of r-values obtained for two-photon optogenetics versus resting-state synchrony [same analysis as for Fig. 2(c)], the set of r-values obtained for two-photon optogenetics versus sensory stimulation [shown in panel (c), bottom right], and the set of r-values obtained for two-photon optogenetics versus resting-state after sensory stimulation. The results shown are for the eight experiments where the target neuron did not respond to the external sensory stimulation. The three sets of r-values were compared with the Friedman tests. (e) Comparison of the three sets of p-values accompanying the r-values. The three sets of p-values were also compared with Friedman tests.

Fig. 4

Evaluation of neuronal connectivity in the hypoperfusion model using the two-photon optogenetics-based method: (a) a mouse model of common carotid artery occlusion (CCAO). The left common carotid artery (ipsilateral to the cranial window) was separated from the jugular vein and the vagus nerve (not shown) to be ligated. In this model, there is no significant change in T2WI to indicate cerebral infarction despite reduced blood flow (CBF-ASL). (b) The upper row of images shows a representative FOV before (pre) and 1, 2, and 4 weeks after CCAO. The lower images show corresponding correlation coefficient maps. The target neurons are indicated by the yellow arrows in the upper images and by yellow dots in the lower maps. (c) Mean of the r-values for each animal (dashed lines) and the average over all five animals (solid line) before (pre) and 1, 2, and 4 weeks after CCAO. Differences between the means of the r-values at each of the four timepoints were evaluated with the Friedman test. (d) Mean of the r-values for each animal (dashed lines) and the average (solid line) for five control animals without CCAO measured at the same timepoints as for (c).