Calcium imaging reveals glial involvement in transcranial direct current stimulation-induced plasticity in mouse brain

Transcranical direct current stimulation (tDCS) is a treatment known to ameliorate various neurological conditions and enhance memory and cognition in humans. tDCS has gained traction for its potential therapeutic value; however, little is known about its mechanism of action. Using a transgenic mouse expressing G-CaMP7 in astrocytes and a subpopulation of excitatory neurons, we find that tDCS induces large-amplitude astrocytic Ca2+ surges across the entire cortex with no obvious changes in the local field potential. Moreover, sensory evoked cortical responses are enhanced after tDCS. These enhancements are dependent on the alpha-1 adrenergic receptor and are not observed in IP3R2 (inositol trisphosphate receptor type 2) knockout mice, in which astrocytic Ca2+ surges are absent. Together, we propose that tDCS changes the metaplasticity of the cortex through astrocytic Ca2+/IP3 signalling.


Supplementary Figure 2. Transcranial imaging of the slow oscillations during deep anesthesia in G7NG817
Transcranial imaging on a urethane anesthetized G7NG817 mouse displays large-amplitude and synchronized slow oscillations (A). The normalized fluorescence intensity (ΔF/F) from the visual cortex (A, black square) is plotted in b. The frequency of the oscillations ranges from 0.5 to 2 Hz, consistent with the LFP slow oscillations reported in urethane-anesthetized rodents. The images are taken at a frame rate of 10 Hz. The dotted area in B. is magnified in C. The diamond symbols represent the time points for the images displayed in A.

Supplementary Figure 3.
Visual and tail-pinch responses of the cerebral cortex using G7NG817 A. Visualization of cortical dynamics in response to visual flash stimulation presented to the left eye of an awake mouse. Pseudocoloring is superimposed on the real images. The color range shown is between the mean + 1SD and peak values of the baseline visual evoked response. B. Transcranial imaging of the entire dorsal cortical area to visualize the G-CaMP7 response to tail pinch in anesthetized animals (upper panel). Pseudocoloring is superimposed on the real images. The color range shown is between the mean + 1SD and peak values of the baseline visual evoked response. Note that the response amplitude is tenfold higher than those by flash visual stimulations and the time course is an order of magnitude slower. Two-photon imaging of the layer 2/3 of the sensory cortex during tail-pinch in a different mouse (lower panel). C. Bar graphs for the comparisons of fluorescence signal intensity (left) and time course (right) between tail pinch and visual flash stimulations. D. Astrocytic and neuronal Ca 2+ activities were separated by the SR101 red fluorescent dye that loads selectively into glial cells. SR101-loaded astrocytes (orange) and neurons are marked with squares and circles, respectively (left) and their fluorescent signals are plotted. Red arrowhead indicates the timing of tail pinch. Note that the ΔF/F scale bars are 300 % and 100% for astrocytic and neuronal traces, respectively. The dotted region is magnified on the right panel to show the signal time course of the astrocyte and the neuron. Scale bar: 50 µm. E. Bar graphs show that the tail-pinch induces large and long-lasting G-CaMP7 signal (ΔF/F) in astrocytes. Note that the average Ca 2+ signal amplitude in neurons was also increased by tail pinch, possibly reflecting burst-like firing. pre: 20 s period before tail pinch. post: 20 s period after tail pinch. F. The frequency of the neuronal Ca 2+ events increases in the post-tail pinch period than the pre-tail pinch period. *** p <0.001, ** p < 0.01

Supplementary Figure 4.
Transcranial functional mapping of the cerebral cortex using G7NG817 A. To demonstrate the utility of G7NG817 for functional mapping, individual whiskers were deflected while transcranial imaging was made over the barrel cortex. The barrel area (yellow square) was imaged while individual whiskers were separately deflected at a frequency of 10 Hz for 5 s in an anesthetized G7NG817 mouse. The mean responses for individual whiskers (16 repetitions) are overlaid in the middle panel. The peak response for D1 whisker stimulation (arrow) is ΔF/F: 3.02 %. The stimulated whiskers and the corresponding barrel structure are shown in the right panel. B. Functional mapping of other sensory modalities was also demonstrated in awake and head-restraint conditions. For instance, visual flash stimulation (10 ms) presented to either eye resulted in an activation of the contralateral visual cortex (average of 16 responses). The same mouse as in Supplementary Fig 3A is presented. C. Visualization of cortical dynamics in response to pure tone presentation (5 kHz pure tone for 500 ms) to an anesthetized mouse. The primary auditory cortex is activated (average of 16 trials). Scale bars: a, 1 mm, 250 µm.

Supplementary Figure 5. Determination of the tDCS threshold for cortical Ca 2+ surges
To determine the minimum current necessary to evoke Ca 2+ surges by tDCS, the stimulus intensity was increased in steps. We find that 30-40 µA is sufficient to induce Ca 2+ surges (arrow) in both hemispheres (N = 4 out of 5 mice).

Supplementary Figure 6. Induction of Ca 2+ surges by interhemispheric tDCS
A. To exclude the possibility that peripheral nerve stimulation by the cathode causes the activation of cortex-wide Ca 2+ surges, we placed the anode and cathode on the skull. The interhemispheric tDCS also induced Ca 2+ surges with a current intensity of 102 µA. B. The tDCS current was gradually increased to investigate if the instantaneous current increase is critical in inducing a cortical Ca 2+ surge. In this example, a Ca 2+ surge was induced as the tDCS current exceed 50 µA. Figure 7. tDCS does not induce Ca 2+ surges in IP 3 R2 KO mice. A. Slow oscillations are observed in a urethane-anesthetized IP 3 R2 knockout mouse which carries the G7NG817 transgene (IP 3 R2 -/-;G7NG817 +/-). The G-CaMP7 signal of the area marked with a square is plotted on the right. The amplitude of the slow oscillations is similar between IP 3 R2 -/-;G7NG817 +/and IP 3 R2 +/-;G7NG817 +/-(N = 6 and 3, respectively) B. tDCS is applied to a urethane-anesthetized G7NG817 transgene (IP 3 R2 -/-;G7NG817 +/-). Although the tDCS parameter is the same as in Figure 1D, Ca 2+ surges were not induced in the IP 3 R2 -/-;G7NG817 +/mouse. C. Intracranial two-photon imaging was performed in a urethane-anesthetized IP 3 R2 -/-;G7NG817 +/mice with the glia maker SR101 (red). Cells marked with circles and squares point to neurons and astrocytes, respectively. G-CaMP7 signals of these cells before and during tDCS are plotted on the bottom. Scale bar: 100 µm. D. Data from three urethane-anesthetized IP 3 R2 -/-;G7NG817 +/mice show that tDCS did not induce Ca 2+ surges in astrocytes during tDCS under urethane anesthesia. Figure 8. tDCS alleviates a mouse model of depression by chronic restraint stress A. Schematic diagram of the schedule for the behavioral test. TST: tail suspension test. B. tDCS reduces depression-like behavior after 1 week. TST one week after tDCS (TST6) shows that tDCS group (N = 10) are more active than the group without tDCS treatment (sham; N = 10). This effect is blocked by acute prazosin administration 30 min before tDCS (N = 10) and DSP-4 treatment (N = 9), as well as in IP3R2 KO mice (N = 9). Figure 9. Cortical GFAP and IBA-1 staining patterns at the anodal site three hours after tDCS Five sham-operated adult C57BL/6 mice are compared with five mice that underwent tDCS (0.1 mA, 10 minutes, anodal site: above the primary visual cortex). The mice were sacrificed 3 hours after the offset of tDCS and immunohistochemistry was performed for GFAP and IBA-1 to examine the reactivity of astrocytes and microglia, respectively. A. GFAP immunostaining of the superficial layers of the primary visual cortex in control and tDCS conditions. B. Layer 2/3 IBA-1 immunostaining in the same set of mice as a. The images are the maximum intensity projections of 50 µm stacked images. Neither GFAP nor IBA-1 staining shows obvious inflammatory patterns.