Determining the spatial limits and strengths of neurovascular coupling across layers of the cerebral cortex by using three-photon imaging of synapses, neurons and blood vessels - PROJECT SUMMARY This proposal introduces advanced techniques like three-photon optical imaging, two-photon optogenetics and two-photon photothrombosis to determine the precision of blood flow control across different layers of the neocortex, both in healthy animals and after inducing single capillary strokes. Our approach is significant for interpreting fMRI data, particularly laminar fMRI which links high-level brain functions to specific cortical layers. An fMRI signal is certainly triggered by neural activity-dependent dilation of arteries which then leads to downstream changes in blood flow in capillaries and veins. A neural trigger of a hemodynamic event does not mean that the full enunciation of the vascular response is going to match the neural event, or that the vascular response could be used to faithfully predict the underlying neural event. Our prevailing hypothesis suggests that if the artery dilation propagates from one functional cortical domain to an adjacent domain, a mismatch between neural and hemodynamic signals will occur. If the neural feature map lacks sufficient clustering, a mismatch will also occur. Tight matching between neural and vascular signals occurs when the stimuli are generic or when only the responses to the preferred stimulus in a functional domain is analyzed. A popular approach in neurovascular coupling research has been to use sparse sampling of neural activity via LFP and single-unit electrode recordings. Relating these electrical signals to vascular responses is controversial because different results are obtained with each new study: spikes match hemodynamics, LFP match vascular responses, or that spikes, LFPs and vascular responses are all tightly coupled. Multi-photon imaging has sub- cellular resolution and eliminates sampling bias. We previously examined some aspects of neurovascular coupling in layer 2/3 of the neocortex. We imaged visually evoked responses in neurons and synapses that surrounded individual blood vessels in a non-rodent model system. We previously ignored neurovascular coupling within cortical layer 1 because it has a low density of neurons. However, layer 1 is well suited to contrast neurovascular coupling (Aim 1) with other cortical layers because there is no recurrent excitation from neighboring neurons in layer 1. New technical advances such as three-photon microscopy now enable us to probe the strength of neurovascular coupling in layer 4 which is very deep in non-rodents, typically 850–1150 μm below the brain surface (Aim 2). New techniques also allow us to test our prevailing hypothesis on the “propagation of dilation” from one functional domain into the immediately adjacent one and whether a single capillary stroke in layer 2/3 can disrupt neural function across cortical columns (Aim 3). Our research provides ground truth and mechanistic data that will specify where and why selective neural signals may be decoded from hemodynamic signals in specific cortical layers. Our preliminary data suggests that there is no “one-size- fits-all” coupling function across cortical layers. In the long-term, our approach could uncover the neurovascular underpinnings of neurological diseases such as vascular cognitive impairment.
