Introduction

Previously, an intrinsic astrocytic calcium spike was detected to be negatively correlated to the BOLD fMRI signal¹. By simultaneous calcium with electrophysiology or BOLD fMRI recording, the intrinsic astrocytic Ca²⁺ spikes coincided with suppressed LFP activity, as well as negative BOLD fMRI signal through the entire cortex. Interestingly, the intrinsic astrocytic Ca²⁺ spikes could be initiated in bilateral hemispheres at the same time with sensory stimulation. Upon the appearance the intrinsic astrocytic Ca²⁺ spikes, the whole-brain BOLD fMRI was used to search for the specifically activated brain regions, which may serve as the source to elicit the astrocytic Ca²⁺ spike. Increased BOLD signal was detected at the central and mediodorsal thalamic nuclei specifically, as well as increased LFP activity upon stimulation. This work indicates that the intrinsic astrocytic Ca²⁺ signal could play a crucial role to mediate the brain state changes through the arousal thalamic pathways.

Methods

All images were acquired with a 14.1 T/26cm horizontal bore magnet (Magnex), interfaced to an AVANCE III console (Bruker) and equipped with a 12 cm gradient set, capable of providing 100 G/cm with a rise time of 150 us (Resonance Research). A transreceiver surface coil was used to acquire fMRI images. Electrodes were placed on the forepaw (FP) to deliver a 1.0 mA pulse sequence (4s, 300μs duration repeated at 3Hz). GCaMP6f ² was expressed by AAV5 in the FP somatosensory cortex (FP-S1) with Syn or GFAP promoter. Fiber optic (200um) was inserted into the area which expressed GCaMP for calcium-based fluorescent signal recording.

Results

Intrinsic astrocytic Ca²⁺ spikes were detected in coincidence with the suppressed spontaneous LFP signal, as well as the negative BOLD fMRI signal (Fig 1). This intrinsic astrocytic Ca²⁺ signal could be initiated by sensory stimulation and was detected from multiple cortical areas, e.g. FP-S1, barrel cortex, or cross hemispheres, with negative BOLD signal through the entire cortex (Fig 2). Upon the 30s electrical unilateral sensory stimulation, the intrinsic astrocytic Ca²⁺ spikes were detected in a few epochs at both hemispheres, showing spikes with amplitude 2-3 times higher than the normal evoked astrocytic Ca²⁺ signal (Fig 3). Interestingly, the bilateral intrinsic astrocytic Ca²⁺ spikes showed no onset delay regardless of the evoked cortical side (Fig 3e), indicating the intrinsic astrocytic Ca²⁺ spikes are not propagating from the activated cortical areas. The whole-brain BOLD fMRI was simultaneously acquired with the bi-hemispheric astrocytic Ca²⁺ recording, showing specific thalamic activation upon the appearance of the intrinsic astrocytic Ca²⁺ spikes but not under the normal evoked astrocytic Ca²⁺ events (Fig 4). This result indicates that the thalamic activation may directly mediate the large-scale cortical intrinsic astrocytic Ca²⁺ spike events. Furthermore, simultaneous thalamic LFP recording at the central and mediodorsal region with cortical astrocytic Ca²⁺ recordings showed that the increased LFP power spectrum density in thalamus upon the intrinsic astrocytic Ca²⁺ spikes in comparison to the normal evoked astrocytic Ca²⁺ signal (Fig 5). These results indicates that the intrinsic astrocytic Ca²⁺ spikes may directly get involved in the brain state changes through the arousal thalamic pathways.

Conclusion

The intrinsic astrocytic Ca²⁺ spikes were detected in the cortex with suppressed spontaneous LFP and negative BOLD fMRI signal. These astrocytic Ca²⁺ spike events were different from the normal activity-evoked Ca²⁺ signal and also differentiate themselves from the lesion/stimulation-induced large-scale depolarization or spreading depression given the instantaneous whole-brain pattern and correlation with the thalamic LFP. The intrinsic astrocytic Ca²⁺ spikes may mediate the brain states through the arousal thalamic pathway.

Acknowledgements

This research was supported by the internal funding from Max Planck Society.