Optical Imaging and Microscopy of Neurovascular Coupling
Neurovascular coupling is the process by which blood flow is modulated in the brain in response to neuronal activity. This blood flow modulation is the basis of the signals measured in functional MRI, but is also an essential component of normal brain health.
Speakers: Dr Elizabeth Hillman (Associate Professor of Biomedical Engineering, Laboratory of Functional Optical Imaging, Columbia University, New York City) and Dr Mark Nelson (Professor of Cardiovascular Medicine, University of Manchester)
During this webinar, Dr Elizabeth Hillman discussed her work which focuses on developing and using novel in-vivo optical imaging and microscopy strategies to better understand the cellular basis of neurovascular coupling. Some of the newest optical techniques include simultaneous wide-field imaging of GCaMP dynamics, blood flow and oxygenation in the “awake” mouse brain, and a new high-speed volumetric microscopy technique based on light-sheet illumination which can image the intact rodent brain as well as freely behaving small organism’s in-vivo.
Dr Hillman also discussed recent discoveries and highlighted how the vascular endothelium of blood vessels in the brain plays a central role in generating functional hyperemia during neuronal activity.
Dr Mark Nelson is currently a University Distinguished Professor and Chair of the Pharmacology Department at the University of Vermont. During the webinar he discussed optical measurements of ion channel function.
The behaviour of single channels has typically been examined by measuring currents in isolated cells using the patch-clamp technique. Dr Nelson discussed how his lab have developed approaches for measuring calcium influx through single TRPV4 (transient receptor potential vanilloid 4) channels in native endothelium of intact arteries, referring to these unitary events as “sparklets”.
The properties of endothelial TRPV4 channels are measured optically by imaging Ca2+ signals in arteries from genetically engineered mice that express the circularly permutated, enhanced green fluorescent protein-based Ca2+ biosensor, GCaMP2, specifically in the endothelium under the control of the connexin 40 promoter (Sonkusare et al., Science, 2012). The GCaMP2 mouse model permits long-term, stable recordings, including in pressurized and slit-open (en face) artery preparations. Images are generally obtained at rates of 15–30/s with a multi-point confocal system and are analyzed using our custom-written software.
The imaging conditions used allow the activity of single TRPV4 channels to be measured with high spatial and temporal resolution under physiological conditions. With the en face preparation, about 14 intact endothelial cells—each with an approximate surface area of 1,700 mm2—are imaged simultaneously. Thus, one imaging experiment is equivalent to hundreds of successful on-cell patch-clamp experiments. Dr Nelson’s lab has successfully extended this approach to the measurement of sparklets mediated by TRPV3 and TRPV1 channels. Unitary calcium influx rate through TRPV channels can be determined by comparing the amplitudes of TRPV sparklets to those of L-type voltage-dependent calcium channels, which conduct primarily calcium ions. The criteria for obtaining unitary measurements in native preparations and insights that can be gained is discussed in detail throughout this webinar.