Two-photon imaging induces brain heating and calcium microdomain hyperactivity in cortical astrocytes

ABSTRACT

Unraveling how neural networks process and represent sensory information and how this cellular dynamics instructs behavioral output is a main goal in current neuroscience. Two-photon activation of optogenetic actuators and fluorescence calcium (Ca²⁺) imaging with genetically encoded Ca²⁺ indicators allow, respectively, the all-optical stimulation and readout of activity from genetically identified cell populations. However, these techniques expose the brain to high near-infrared light doses raising the concern of light-induced adverse effects on the biological phenomena being studied. Combing Ca²⁺ imaging of GCaMP6f-expressing cortical astrocytes as a sensitive readout for photodamage and an unbiased machine-based event detection, we demonstrate the subtle build-up of aberrant microdomain Ca²⁺ signals in fine astroglial processes. Illumination conditions routinely being used in biological two-photon microscopy (920-nm excitation, 100-fs regime, ten mW average power) increased the frequency of microdomain Ca²⁺ events, but left their amplitude, area and duration rather unchanged. This increase in local Ca²⁺ activity was followed by Ca²⁺ transients in the otherwise silent soma. Ca²⁺ hyperactivity occurred without overt morphological damage. Surprisingly, at the same average power, continuous-wave 920-nm illumination was as damaging as fs pulses, indicating a linear, heating-mediated (rather than a highly non-linear) damage mechanism. In an astrocyte-specific IP₃-receptor knock-out mouse (IP₃R2-KO), Near-infrared light-induced Ca²⁺ microdomains signals persisted in the small processes, underpinning their resemblance to physiological IP₃R2-independent Ca²⁺ signals, while somatic activity was abolished. Contrary to what has generally been believed in the field, shorter pulses and lower average power are advantageous to alleviate photodamage and allow for longer useful recording windows.