{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,5,4]],"date-time":"2026-05-04T02:24:31Z","timestamp":1777861471523,"version":"3.51.4"},"reference-count":35,"publisher":"MDPI AG","issue":"3","license":[{"start":{"date-parts":[[2026,4,25]],"date-time":"2026-04-25T00:00:00Z","timestamp":1777075200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100000065","name":"National Institute of Neurological Disorders","doi-asserted-by":"crossref","id":[{"id":"10.13039\/100000065","id-type":"DOI","asserted-by":"crossref"}]},{"name":"National Institute of Aging of the National Institutes of Health","award":["R01NS126920"],"award-info":[{"award-number":["R01NS126920"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["MPs"],"abstract":"<jats:p>Neuronal activity and cerebral blood flow are tightly coupled to support the high metabolic demands of the brain. Disruption of neurovascular coupling is a defining feature of many neurodegenerative disorders such as Alzheimer\u2019s disease, stroke, small vessel disease, Parkinson\u2019s disease, and aging. Progress in understanding the mechanisms underlying neurovascular coupling requires experimental approaches that can simultaneously measure neuronal activity and vascular dynamics with high spatial and temporal resolution, while also enabling targeted perturbations of the system. Here, we present a methodological framework that combines chronic electrophysiological recordings with two-photon imaging of cerebral blood flow and optogenetic manipulation of the vasculature in vivo. Using a chronically implanted flexible electrode array, we obtain measurements of the single- and multi-unit spiking activity, as well as local field potentials. Concurrently, two-photon microscopy enables high-resolution measurements of vessel diameter and blood flow within individual vascular segments. In addition, optogenetic control of vascular smooth muscle cells allows for rapid and reversible manipulation of the vessel diameter through the same cranial window while simultaneously recording the neural and vascular activity. We provide detailed protocols for surgical implantation, data acquisition, and analysis, and discuss experimental considerations and limitations. This combined platform offers a powerful tool for mechanistic studies of neurovascular coupling and its dysfunction in disease models.<\/jats:p>","DOI":"10.3390\/mps9030068","type":"journal-article","created":{"date-parts":[[2026,4,29]],"date-time":"2026-04-29T10:34:42Z","timestamp":1777458882000},"page":"68","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Simultaneous In Vivo Electrophysiology, Two-Photon Imaging, and Optogenetics for Probing Neurovascular Coupling"],"prefix":"10.3390","volume":"9","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-9847-3329","authenticated-orcid":false,"given":"Dalchand","family":"Ahirwar","sequence":"first","affiliation":[{"name":"Department of Physiology, Augusta University, Augusta, GA 30912, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Kun","family":"Xie","sequence":"additional","affiliation":[{"name":"Department of Physiology, Augusta University, Augusta, GA 30912, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8137-9432","authenticated-orcid":false,"given":"Philip","family":"O\u2019Herron","sequence":"additional","affiliation":[{"name":"Department of Physiology, Augusta University, Augusta, GA 30912, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2026,4,25]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"232","DOI":"10.1038\/nature09613","article-title":"Glial and neuronal control of brain blood flow","volume":"468","author":"Attwell","year":"2010","journal-title":"Nature"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"416","DOI":"10.1038\/s41583-020-0322-2","article-title":"Neuronal regulation of the blood-brain barrier and neurovascular coupling","volume":"21","author":"Kaplan","year":"2020","journal-title":"Nat. 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