Optimizing intact skull intrinsic signal imaging for subsequent targeted electrophysiology across mouse visual cortex

Abstract

Understanding brain function requires repeatable measurements of neural activity across multiple scales and multiple brain areas. In mice, large scale cortical neural activity evokes hemodynamic changes readily observable with intrinsic signal imaging (ISI). Pairing ISI with visual stimulation allows identification of primary visual cortex (V1) and higher visual areas (HVAs), typically through cranial windows that thin or remove the skull. These procedures can diminish long-term mechanical and physiological stability required for delicate electrophysiological measurements made weeks to months after imaging (e.g., in subjects undergoing behavioral training). Here, we optimized and directly validated an intact skull ISI system in mice. We first assessed how imaging quality and duration affect reliability of retinotopic maps in V1 and HVAs. We then verified ISI map retinotopy in V1 and HVAs with targeted, multi-site electrophysiology several weeks after imaging. Reliable ISI maps of V1 and multiple HVAs emerged with ~ 60 trials of imaging (65 ± 6 min), and these showed strong correlation to local field potential (LFP) retinotopy in superficial cortical layers (r2 = 0.74–0.82). This system is thus well-suited for targeted, multi-area electrophysiology weeks to months after imaging. We provide detailed instructions and code for other researchers to implement this system.

Narrowband gamma oscillations propagate and synchronize throughout the mouse thalamocortical visual system

“…Following headplate fixation, a glass coverslip (5 mm diameter, #1 thickness ∼0.15 mm) was centred over the representation of V1 and HVAs (centre of window at ∼ 2.4 mm lateral to midline and ∼ 2.4 mm anterior to lambda) and bonded to the skull using VetBond. Mice were individually housed and monitored for full recovery for at least 3 days before intrinsic signal imaging (ISI) of V1 and HVAs, as detailed in our prior studies (Nsiangani et al, 2022). Briefly, ISI was performed during isoflurane anesthesia with sedation.…”

“…Following ISI, mice were habituated to head fixation for 3 to 5 days before undergoing awake recordings as in prior studies (Speed et al ., 2019). On recording days, small craniotomies (∼100-500 μm) were made in HVAs under isoflurane anesthesia, using the ISI maps and vasculature as references (Nsiangani et al ., 2022). The skull overlying two retinotopically distant sites in V1 (∼ 60 to 90° apart in azimuth) was thinned to facilitate optical stimulation.…”

“…Laser stimulation was only delivered on 25-33% of randomly selected trials with the remaining trials serving as the interleaved control trials. At the conclusion of optogenetic experiments, retinotopy was confirmed at the HVA recording sites by presenting briefly flashed bright and dark bars throughout the visual field (Nsiangani et al ., 2022). Retinotopy of the thinned V1 sites was also subsequently electrophysiologically confirmed the same way.…”

Cranial and Spinal Window Preparation for in vivo Optical Neuroimaging in Rodents and Related Experimental Techniques

“…Typical cortical cranial window size varies from 2 mm up to 8 mm in diameter depending on the region of interest (ROI). Examples include synaptic and dendritic changes in retrosplenial cortex (posterior cortical area) with small-sized CW (~3 mm in diameter) by 2PE imaging [ 79 ]; OIS changes in small targeted region (visual cortex, ~4 mm) using intact skull cranial window model [ 80 ]; cerebral blood flow changes in somatosensory area (~6 mm in diameter) by LSCI and OISI [ 35 ]; large area (7 mm×8 mm) cortical neuronal connectivity analysis with VSDI [ 34 ]; and whole skull optical clearing window (SOWC) for microvessels and synaptic resolution [ 81 ].…”

“…For long-term in vivo imaging, the next generation of cranial and spinal windows covered the brain parenchyma with glass coverslip over the open-skull or thinned skull. However, these window types require further modification, including the retractable type or flexible polymer-based windows, which have been adopted in conjunction with other neuroscience techniques such as brain stimulation or electrophysiological recording [ 30 , 39 , 40 , 77 , 80 , 82 - 84 ]. With advances in MEMS supported by nanotechnology, flexible transparent microelectrode will allow a range of previously unfeasible experiments due to the physical barrier from glass windows.…”