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Infrared Excitation Induces Heating and Calcium Microdomain Hyperactivity in Cortical Astrocytes.

ABSTRACT: Unraveling how neural networks process and represent sensory information and how these cellular signals instruct behavioral output is a main goal in neuroscience. Two-photon activation of optogenetic actuators and calcium (Ca²⁺) imaging with genetically encoded indicators allow, respectively, the all-optical stimulation and readout of activity from genetically identified cell populations. However, these techniques locally expose the brain to high near-infrared light doses, raising the concern of light-induced adverse effects on the biology under study. Combining 2P imaging of Ca²⁺ transients in GCaMP6f-expressing cortical astrocytes and unbiased machine-based event detection, we demonstrate the subtle build-up of aberrant microdomain Ca²⁺ transients in the fine astroglial processes that depended on the average rather than peak laser power. Illumination conditions routinely being used in biological 2P microscopy (920-nm excitation, ∼100-fs, and ∼10 mW average power) increased the frequency of microdomain Ca²⁺ events but left their amplitude, area, and duration largely unchanged. Ca²⁺ transients in the otherwise silent soma were secondary to this peripheral hyperactivity that occurred without overt morphological damage. Continuous-wave (nonpulsed) 920-nm illumination at the same average power was as damaging as femtosecond pulses, unraveling the dominance of a heating-mediated damage mechanism. In an astrocyte-specific inositol 3-phosphate receptor type-2 knockout mouse, near-infrared light-induced Ca²⁺ microdomains persisted in the small processes, underpinning their resemblance to physiological inositol 3-phosphate receptor type-2-independent Ca²⁺ signals, whereas somatic hyperactivity was abolished. We conclude that, contrary to what has generally been believed in the field, shorter pulses and lower average power can help to alleviate damage and allow for longer recording windows at 920 nm.

SUBMITTER: Schmidt E

PROVIDER: S-EPMC7732816 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:There is serious concern about arsenic in the natural environment, which exhibits neurotoxicity and increases the risk of neurodevelopmental disorders. Adverse effects of arsenic have been demonstrated in neurons, but it is not fully understood how arsenic affects other cell types in the brain. In the current study, we examined whether sodium arsenite (NaAsO2) affects the cell cycle, viability, and apoptosis of in vitro-cultured astrocytes isolated from the cerebral cortex of mice. Cultured astrocytes from transgenic mice expressing fluorescent ubiquitination-based cell cycle indicator (Fucci) were subjected to live imaging analysis to assess the effects of NaAsO2 (0, 1, 2, and 4 μM) on the cell cycle and number of cells. Fucci was designed to express monomeric Kusabira Orange2 (mKO2) fused with the ubiquitylation domain of hCdt1, a marker of G1 phase, and monomeric Azami Green (mAG) fused with the ubiquitylation domain of hGem, a marker of S, G2, and M phases. NaAsO2 concentration-dependently decreased the peak levels of the mAG/mKO2 emission ratio when the ratio had reached a peak in astrocytes without NaAsO2 exposure, which was due to attenuating the increase in the mAG-expressing cell number. In contrast, the mAG/mKO2 emission ratio and number of mAG-expressing cells were concentration-dependently increased by NaAsO2 before their peak levels, indicating unscheduled S phase entry. We further examined the fate of cells forced to enter S phase by NaAsO2. We found that most of these cells died up to the end of live imaging. In addition, quantification of the copy number of the glial fibrillary acidic protein gene expressed specifically in astrocytes revealed a concentration-dependent decrease caused by NaAsO2. However, NaAsO2 did not increase the amount of nucleosomes generated from DNA fragmentation and failed to alter the gene expression of molecules relevant to unscheduled S phase entry-coupled apoptosis (p21, p53, E2F1, E2F4, and Gm36566). These findings suggest that NaAsO2 adversely affects the cell cycle and viability of astrocytes by inducing unscheduled S phase entry coupled with cell death that may be caused by mechanisms other than apoptosis.

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Infrared Excitation Induces Heating and Calcium Microdomain Hyperactivity in Cortical Astrocytes.

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