In vivo widefield calcium imaging of the mouse cortex for analysis of network connectivity in health and brain disease

Abstract

The organization of brain areas in functionally connected networks, their dynamic changes, and perturbations in disease states are subject of extensive investigations. Research on functional networks in humans predominantly uses functional magnetic resonance imaging (fMRI). However, adopting fMRI and other functional imaging methods to mice, the most widely used model to study brain physiology and disease, poses major technical challenges and faces important limitations.Hence, there is great demand for alternative imaging modalities for network characterization. Here, we present a refined protocol for in vivo widefield calcium imaging of both cerebral hemispheres in mice expressing a calcium sensor in excitatory neurons. We implemented a stringent protocol for minimizing anesthesia and excluding movement artifacts which both imposed problems in previous approaches. We further adopted a method for unbiased identification of functional cortical areas using independent component analysis (ICA) on resting-state imaging data. Biological relevance of identified components was confirmed using stimulus-dependent cortical activation. To explore this novel approach in a model of focal brain injury, we induced photothrombotic lesions of the motor cortex, determined changes in inter-and intrahemispheric connectivity at multiple time points up to 56 days post-stroke and correlated them with behavioral deficits. We observed a severe loss in interhemispheric connectivity after stroke, which was partially restored in the chronic phase and associated with corresponding behavioral motor deficits. Taken together, we present an improved widefield calcium imaging tool accounting for anesthesia and movement artifacts, adopting an advanced analysis pipeline based on human fMRI algorithms and with superior sensitivity to recovery mechanisms in mouse models compared to behavioral tests. This tool will enable new studies on interhemispheric connectivity in murine models with comparability to human imaging studies for a wide spectrum of neuroscience applications in health and disease.

Short-Chain Fatty Acids Improve Poststroke Recovery via Immunological Mechanisms

“…These novel findings introduce SCFA as the most likely missing link in the pathophysiological function of the gut-brain axis in stroke and poststroke recovery, for which the microbiotaderived effector molecules were so far unknown (Benakis et al, 2016;Houlden et al, 2016;Singh et al, 2016). Our use of a novel imaging modality, in vivo wide-field calcium imaging, provided a highly sensitive tool for assessing cortical network plasticity after stroke (Cramer et al, 2019). This tool allowed us to perform analyses of network plasticity by comparable analytical approaches to fMRI in patients, with the exception of using a genetically encoded reporter for direct neuronal activation instead of the blood flow surrogate marker (BOLD) used in MRI (Cramer et al, 2019).…”

“…Our use of a novel imaging modality, in vivo wide-field calcium imaging, provided a highly sensitive tool for assessing cortical network plasticity after stroke (Cramer et al, 2019). This tool allowed us to perform analyses of network plasticity by comparable analytical approaches to fMRI in patients, with the exception of using a genetically encoded reporter for direct neuronal activation instead of the blood flow surrogate marker (BOLD) used in MRI (Cramer et al, 2019). The analysis of connectivity within the cortical network provides information about the dynamic changes in defined cortical areas under resting-state conditions, which gave us a unique ability to measure even the more subtle effects of SCFA supplementation on poststroke plasticity.…”

“…Wide-field calcium imaging. In vivo wide-field calcium imaging was performed as previously published (Cramer et al, 2019). Briefly, we used Thy1GCaMP6s heterozygous reporter mice.…”

“…Analogous functional connectivity between seed pairs was calculated as correlation between signal time courses of two seeds within the sensorimotor cortex. Seeds were defined as previously described (Cramer et al, 2019). Mean correlation per group was calculated for each time point.…”

Disrupted population coding in the prefrontal cortex underlies pain aversion

“…Functional connectivity between groups of neurons provides understanding of population-level neural responses to sensory inputs, and it has been used to characterize pain and other neuropsychiatric disorders based on various modalities of neural recording techniques, such as microcircuit-level calcium imaging (Cramer et al, 2019) and brain-wide neuroimaging (Baliki et al, 2012;Kano et al, 2020;Mutso et al, 2012;Spisak et al, 2020). Functional connectivity analyses have successfully identified long-range nociceptive information flow within the brain in chronic pain conditions (Baliki et al, 2012;Vachon-Presseau et al, 2019), thereby having the potential to provide insight into population neural responses within a specific cortical area as well.…”