Mapping of neurovascular and neurometabolic couplings by multimodal optical imaging

Neurovascular coupling links calcium (Ca2+)-dependent neuronal activity to cerebral blood volume changes, whereas neurometabolic coupling describes alterations of neuronal activity and glucose uptake. While mesoscale optical imaging of neurovascular coupling is prevalent, neurometabolic coupling has been explored much less. We describe a multiplexed optical system with a closed cranial window setup for longitudinal studies in Thy1-jRGECO1a mice where neuronal activity is measured with Ca2+-dependent red fluorescence, glucose uptake with bolus injections of 2NBDG with green fluorescence, and cerebral blood volume (CBV) with near-infrared spectroscopy (NIRS). Genetically encoded calcium indicators (GECIs) provide strong fluorescent signals for assessing Ca2+-dependent neuronal activity. Thy1-jRGECO1a, a novel GECI with red fluorescence emission that penetrates deeper into tissue, allows for simultaneous imaging of metabolic activity using a green-fluorescent glucose analog, 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2NBDG), which is taken up like glucose and then phosphorylated. Dual-fluorescent (red, green) and NIRS recordings confirm strong neurovascular coupling during hindpaw stimuli (Ca2+-CBV; P = 0.0033, r2 = 0.91), whereas neurometabolic coupling (Ca2+-2NBDG; P < 0.001) was three times stronger during stimulation (r2 = 0.75; slope = 0.6) compared to rest (r2 = 0.49; slope = 0.23). In summary, multiplexed optical imaging can be used to reveal mechanisms of neurovascular and neurometabolic (un)couplings during ischemia, traumatic brain injury, aging, and Alzheimer's disease.