All-optical bidirectional neural interfacing using hybrid multiphoton holographic optogenetic stimulation.

Our understanding of neural information processing could potentially be advanced by combining flexible three-dimensional (3-D) neuroimaging and stimulation. Recent developments in optogenetics suggest that neurophotonic approaches are in principle highly suited for noncontact stimulation of network activity patterns. In particular, two-photon holographic optical neural stimulation (2P-HONS) has emerged as a leading approach for multisite 3-D excitation, and combining it with temporal focusing (TF) further enables axially confined yet spatially extended light patterns. Here, we study key steps toward bidirectional cell-targeted 3-D interfacing by introducing and testing a hybrid new 2P-TF-HONS stimulation path for accurate parallel optogenetic excitation into a recently developed hybrid multiphoton 3-D imaging system. The system is shown to allow targeted all-optical probing of in vitro cortical networks expressing channelrhodopsin-2 using a regeneratively amplified femtosecond laser source tuned to 905 nm. These developments further advance a prospective new tool for studying and achieving distributed control over 3-D neuronal circuits both in vitro and in vivo.

Holographic optogenetic stimulation of patterned neuronal activity for vision restoration.

When natural photoreception is disrupted, as in outer-retinal degenerative diseases, artificial stimulation of surviving nerve cells offers a potential strategy for bypassing compromised neural circuits. Recently, light-sensitive proteins that photosensitize quiescent neurons have generated unprecedented opportunities for optogenetic neuronal control, inspiring early development of optical retinal prostheses. Selectively exciting large neural populations are essential for eliciting meaningful perceptions in the brain. Here we provide the first demonstration of holographic photo-stimulation strategies for bionic vision restoration. In blind retinas, we demonstrate reliable holographically patterned optogenetic stimulation of retinal ganglion cells with millisecond temporal precision and cellular resolution. Holographic excitation strategies could enable flexible control over distributed neuronal circuits, potentially paving the way towards high-acuity vision restoration devices and additional medical and scientific neuro-photonics applications.

Cellular Resolution Panretinal Imaging of Optogenetic Probes Using a Simple Funduscope.

PURPOSE: To acquire and characterize cellular-resolved in vivo fluorescence images of optogenetic probes expressed in rodent retinal ganglion cells, by adapting a low-cost and simple fundus system based on a topical endoscope. METHODS: A custom endoscope-based fundus system was constructed (adapted from the design of Paques and colleagues). Bright field and fluorescence images were acquired from head-fixed transgenic mice expressing Channelrhodopsin2-eYFP, and Sprague Dawley rats virally transfected with the optogenetic probe GCaMP3. Images were compared to in vitro images of the same structures and were analyzed. RESULTS: The fundus system provides high-quality, high-resolution fluorescence images of the eye fundus that span the whole retina. The images allow resolving individual cells and axon bundles in the Channelrhodopsin2-eYFP mice and cellular-scale structures in the GCaMP3 expressing rats. The resolution in mouse eyes was estimated to be better than 20 µm (full width at half maximum) and is only marginally dependent on movement-related blurring. CONCLUSIONS: The fluorescence-endoscopy fundus system provides a powerful yet simple and widely accessible tool for obtaining cellular resolved fluorescent images of optogenetic and other fluorescent probes. TRANSLATIONAL RELEVANCE: The new system could prove to be a basic tool for non-invasive in vivo small animal retinal imaging in a wide array of translational vision applications, including the tracking of fluorescently tagged cells and the expression of gene-therapy and optogenetic vectors.