Microglia, resident immune cells of the brain, react to the presence of pathogens/danger signals with a large repertoire of functional responses including morphological changes, proliferation, chemotaxis, production/release of cytokines, and phagocytosis. In vitro studies suggest that many of these effector functions are Ca2+-dependent, but our knowledge about in vivo Ca2+ signalling in microglia is rudimentary. This is mostly due to technical reasons, as microglia largely resisted all attempts of in vivo labelling with Ca2+ indicators. Here, we introduce a novel approach, utilizing a microglia-specific microRNA-9-regulated viral vector, enabling the expression of a genetically-encoded ratiometric Ca2+ sensor Twitch-2B in microglia. The Twitch-2B-assisted in vivo imaging enables recording of spontaneous and evoked microglial Ca2+ signals and allows for the first time to monitor the steady state intracellular Ca2+ levels in microglia. Intact in vivo microglia show very homogenous and low steady state intracellular Ca2+ levels. However, the levels increase significantly after acute slice preparation and cell culturing along with an increase in the expression of activation markers CD68 and IL-1β. These data identify the steady state intracellular Ca2+ level as a versatile microglial activation marker, which is highly sensitive to the cell’s environment.

The cerebral cortex is organized in cortical layers that differ in their cellular density, composition, and wiring. Cortical laminar architecture is also readily revealed by staining for cytochrome oxidase—the last enzyme in the respiratory electron transport chain located in the inner mitochondrial membrane. It has been hypothesized that a high-density band of cytochrome oxidase in cortical layer IV reflects higher oxygen consumption under baseline (unstimulated) conditions. Here, we tested the above hypothesis using direct measurements of the partial pressure of O2 (pO2) in cortical tissue by means of 2-photon phosphorescence lifetime microscopy (2PLM). We revisited our previously developed method for extraction of the cerebral metabolic rate of O2 (CMRO2) based on 2-photon pO2 measurements around diving arterioles and applied this method to estimate baseline CMRO2 in awake mice across cortical layers. To our surprise, our results revealed a decrease in baseline CMRO2 from layer I to layer IV. This decrease of CMRO2 with cortical depth was paralleled by an increase in tissue oxygenation. Higher baseline oxygenation and cytochrome density in layer IV may serve as an O2 reserve during surges of neuronal activity or certain metabolically active brain states rather than reflecting baseline energy needs. Our study provides to our knowledge the first quantification of microscopically resolved CMRO2 across cortical layers as a step towards better understanding of brain energy metabolism.

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The rodent olfactory bulb (OB) is continuously supplied with adult-born cells maturing into GABAergic neurons. Using in vivo ratiometric Ca 2+ imaging to readout ongoing and sensory-driven activity, we asked whether mature adult-born cells (mABCs) in the glomerular layer of the bulb become functionally identical to resident GABAergic (Res GABA) neurons. In awake head-restrained mice the two cell populations differed significantly in terms of ongoing spontaneous activity, with 24% of mABCs contributing to a strongly active cell cluster, absent among Res GABA cells. Odor-evoked responses of mABCs were sparse, less reliable, and had smaller amplitudes compared with Res GABA cells. The opposite was seen under anesthesia, with response reliability increasing and response size of mABCs becoming larger than that of Res GABA cells. Furthermore, ongoing activity of mABCs showed increased sensitivity to ketamine/xylazine and was selectively blocked by the antagonist of serotonin receptors methysergide. These functional features of mABCs clearly distinguish them from other OB interneurons.

The cerebral cortex is organized in cortical layers that differ in their cellular density, composition, and wiring. Cortical laminar architecture is also readily revealed by staining for cytochrome oxidase – the last enzyme in the respiratory electron transport chain located in the inner mitochondrial membrane. It has been hypothesized that a high-density band of cytochrome oxidase in cortical layer IV reflects higher oxygen consumption under baseline (unstimulated) conditions. Here, we tested the above hypothesis using direct measurements of the partial pressure of O 2 (pO 2 ) in cortical tissue by means of 2-photon phosphorescence lifetime microscopy (2PLM). We revisited our previously developed method for extraction of the cerebral metabolic rate of O 2 (CMRO 2 ) based on 2-photon pO 2 measurements around diving arterioles and applied this method to estimate baseline CMRO 2 in awake mice across cortical layers. To our surprise, our results revealed a decrease in baseline CMRO 2 from layer I to layer IV . This decrease of CMRO 2 with cortical depth was paralleled by an increase in tissue oxygenation. Higher baseline oxygenation and cytochrome density in layer IV may serve as an O 2 reserve during surges of neuronal activity or certain metabolically active brain states rather than baseline energy needs. Our study provides the first quantification of microscopically resolved CMRO 2 across cortical layers as a step towards informed interpretation and modeling of cortical-layer-specific Blood Oxygen Level Dependent (BOLD) fMRI signals.