Vagus nerve interfacing is of interest due to its central role in parasympathetic regulation of the visceral organs, as well as its modulatory effects on the brain, which have been shown to influence epilepsy, depression and migraines. Electrical vagus nerve stimulation (VNS) has shown therapeutic effect in humans, yet it lacks the specificity for controlling and studying targeted pathways. In contrast, optical techniques may enable axon-specific neuromodulation using genetically targeted opsin expression and spatial patterning of the photo-stimulus. In addition to light-activated stimulation, calcium-sensitive fluorescent reporters such as GCaMP6 present a pathway for axon-specific optical recording of activity. We demonstrate in vivo photo-stimulation and two-photon GCaMP6 fluorescence imaging in the vagus nerve using a novel GRIN lens-coupled nerve cuff in the anesthetized mouse. A pulsed near-IR laser (1040 nm, 300 fs) was modified by a spatial light modulator (SLM) in the Fourier plane and focused by the microscope objective through a GRIN relay lens to the cervical vagus nerve. By actuating the SLM, spatially selected regions of axons could be differentially stimulated within the nerve. Mouse vitals were monitored with a MouseOx suite and used to detect physiological changes in response to photo-stimulation. We were able to induce differential modulations in heart rate, respiratory rate, and blood-oxygen saturation upon photo-stimulation of selective spatial regions of the nerve. Additionally, we recorded two-photon GCaMP6 Ca2+ transients in vagal axons in response to both photo-stimulation and electrical stimulation.
Neural-machine interfaces using optogenetics are of interest due to their minimal invasiveness and potential for parallel read in and read out of activity. One possible biological target for such an interface is the peripheral nerve, where axonlevel imaging or stimulation could greatly improve interfacing with artificial limbs or enable neuron/fascicle level neuromodulation in the vagus nerve. Two-photon imaging has been successful in imaging brain activity using genetically encoded calcium or voltage indicators, but in the peripheral nerve, this is severely limited by scattering and aberrations from myelin. We employ a Shack-Hartman wavefront sensor and two-photon excitation guidestar to quantify optical scattering and aberrations in peripheral nerves and cortex. The sciatic and vagus nerves, and cortex from a ChAT-Cre ChR-eYFP transgenic mouse were excised and imaged directly. In peripheral nerves, defocus was the strongest aberration followed by astigmatism and coma. Peripheral nerve had orders of magnitude higher aberration compared with cortex. These results point to the potential of adaptive optics for increasing the depth of two-photon access into peripheral nerves.
Two-photon microscopy is a powerful tool of current scientific research, allowing optical visualization of structures below the surface of tissues. This is of particular value in neuroscience, where optically accessing regions within the brain is critical for the continued advancement in understanding of neural circuits. However, two-photon imaging at significant depths have typically used Ti:Sapphire based amplifiers that are prohibitively expensive and bulky. In this study, we demonstrate deep tissue two-photon imaging using a compact, inexpensive, turnkey operated Ytterbium fiber laser (Y-Fi, KM Labs). The laser is based on all-normal dispersion (ANDi) that provides short pulse durations and high pulse energies. Depth measurements obtained in ex vivo mouse cortex exceed those obtainable with standard two-photon microscopes using Ti:Sapphire lasers. In addition to demonstrating the capability of deep-tissue imaging in the brain, we investigated imaging depth in highly-scattering white matter with measurements in sciatic nerve showing limited optical penetration of heavily myelinated nerve tissue relative to grey matter.
Access to the requested content is limited to institutions that have purchased or subscribe to SPIE eBooks.
You are receiving this notice because your organization may not have SPIE eBooks access.*
*Shibboleth/Open Athens users─please
sign in
to access your institution's subscriptions.
To obtain this item, you may purchase the complete book in print or electronic format on
SPIE.org.
INSTITUTIONAL Select your institution to access the SPIE Digital Library.
PERSONAL Sign in with your SPIE account to access your personal subscriptions or to use specific features such as save to my library, sign up for alerts, save searches, etc.