Simultaneous Ca²⁺ Imaging and Optogenetic Stimulation of Cortical Astrocytes in Adult Murine Brain Slices

Abstract

Astrocytes are actively involved in a neuroprotective role in the brain, which includes scavenging reactive oxygen species to minimize tissue damage. They also modulate neuroinflammation and reactive gliosis prevalent in several brain disorders like epilepsy, Alzheimer's, and Parkinson's disease. In animal models, targeted manipulation of astrocytic function via modulation of their calcium (Ca2+) oscillations by incorporating light‐sensitive cation channels like Channelrhodopsin‐2 (ChR2) offers a promising avenue in influencing the long‐term progression of these disorders. However, using adult animals for Ca2+ imaging poses major challenges, including accelerated deterioration of in situ slice health and age‐ related changes. Additionally, optogenetic preparations necessitate usage of a red‐shifted Ca2+ indicator like Rhod‐2 AM to avoid overlapping light issues between ChR2 and the Ca2+ indicator during simultaneous optogenetic stimulation and imaging. In this article, we provide an experimental setting that uses live adult murine brain slices (2‐5 months) from a knock‐in model expressing Channelrhodopsin‐2 (ChR2(C128S)) in cortical astrocytes, loaded with Rhod‐2 AM to elicit robust Ca2+ response to light stimulation. We have developed and standardized a protocol for brain extraction, sectioning, Rhod‐2 AM loading, maintenance of slice health, and Ca2+ imaging during light stimulation. This has been successfully applied to optogenetically control adult cortical astrocytes, which exhibit synchronous patterns of Ca2+ activity upon light stimulation, drastically different from resting spontaneous activity. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Experimental preparation, setup, slice preparation and Rhod‐2 AM staining Basic Protocol 2: Image acquisition and analysis

Ion Channels and Electrophysiological Properties of Astrocytes: Implications for Emergent Stimulation Technologies

“…However, ChR2 can also allow the influx of Ca 2+ , which has been shown to cause the release of gliostransmitters like ATP (Gourine et al, 2010 ; Chen et al, 2013 ) and glutamate (Haydon and Carmignoto, 2006 ). ChR2 stimulation in astrocytes has been reliably shown to induce changes in intracellular Ca 2+ (Li D. et al, 2012 ; Figueiredo et al, 2014 ; Perea et al, 2014 ; Mederos et al, 2019 ; Balachandar et al, 2020 ); these changes have been observed to enhance both excitatory and inhibitory synaptic transmission in the primary visual cortex (Perea et al, 2014 ). The use of ChR2 to activate astrocytes has also been shown to induce the release of ATP (Gourine et al, 2010 ; Figueiredo et al, 2014 ).…”

Unraveling ChR2-driven stochastic Ca2+ dynamics in astrocytes: A call for new interventional paradigms

“…Simulation results predict that calcium dynamics in astrocytes, as seen in experimental studies [33,64,65], can be heavily regulated by light-induced activation of ChR2. Consistent in all simulations performed in this study, upon light activation, astrocytes underwent increases in their basal calcium level and exhibited changes in the spiking activity (Figs 2 and 3).…”

“…[30], namely WT 1 and WT 2 . Using the model, we analyze the effect of light stimulation in astrocytes congruent with experimental recording durations (in the order of tens of minutes) [31][32][33], and also gauge the potential effect of long-term optogenetic stimulation (in the order of several hours or beyond) in these cells. Secondly, we quantify the effect of varying stimulation light intensities on a ChR2-incorporated astrocyte with respect to its spiking rate and basal level.…”

Shared peripheral blood biomarkers for Alzheimer’s disease, major depressive disorder, and type 2 diabetes and cognitive risk factor analysis

“…Involved in the internalization and trafficking of activated tyrosine kinase receptors such as PDGFRB. A team designed cellular experiments and found lower Rho-related protein HP1 levels in astrocytes than in normal groups, which may be clinically meaningful for some neurological diseases, including epilepsy and Alzheimer's disease [ 65 ].…”