Fiber photometry is increasingly utilized to monitor fluorescent sensors of neural activity in the brain. However, most implementations are based on flat-cleaved optical fibers that can only interface with shallow tissue volumes adjacent to the fiber. We exploit modal properties of tapered optical fibers (TFs) to enable light collection over an extent of up to 2 mm of tissue and multisite photometry along the taper. Using a single TF, we simultaneously observed distinct dopamine transients in dorsal and ventral striatum in freely moving mice performing a simple, operant conditioning task. Collection volumes from TFs can also be engineered in both shape and size by microstructuring the nonplanar surface of the taper, to optically target multiple sites not only in the deep brain but, in general, in any biological system or organ in which light collection is beneficial but challenging because of light scattering and absorption. Fiber photometry with tapered fibers allows monitoring of neural activity in larger volumes than with flat-cleaved fibers. In addition, signals from different depths can be resolved with the same tapered fiber.

Depth-resolved fiber photometry with a single tapered optical fiber implant

Cracking the Function of Layers in the Sensory Cortex

An important technological revolution is underway in the field of neuroscience as we begin the 21st century. The combination of optical methods with genetically encoded photosensitive tools (optogenetics) offers the opportunity to quickly modulate and monitor a large number of neuronal events and the ability to recreate the physiological, spatial, and temporal patterns of brain activity. The use of light instead of electrical stimulation is less invasive, and permits superior spatial and temporal specificity and flexibility. This ongoing revolution has motivated the development of new optical methods for light stimulation. They can be grouped in two main categories: scanning and parallel photostimulation techniques, each with its advantages and limitations. In scanning approaches, a small light spot is displaced in targeted regions of interest (ROIs), using galvanometric mirrors or acousto-optic deflectors, whereas in parallel approaches, the light beam can be spatially shaped to simultaneously cover all ROIs by modulating either the light intensity or the phase of the illumination beam. With amplitude modulation, light patterns are created by selectively blocking light rays that illuminate regions of no interest, while with phase modulation, the wavefront of the light beam is locally modified so that light rays are directed onto the target, thus allowing for higher intensity efficiency. In this review, we will describe the principle of each of these photostimulation techniques and review the use of these approaches in optogenetics experiments by presenting their advantages and drawbacks. Finally, we will review the challenges that need to be faced when photostimulation methods are combined with two-photon imaging approaches to reach an all-optical brain control through optogenetics and functional reporters (Ca and voltage indicators). Journal of Optics J. Opt. 19 (2017) 113001 (27pp) https://doi.org/10.1088/2040-8986/aa8299 Original content from this work may be used under the terms of the Creative Commons Attribution 3.0 licence. Any further distribution of this work must maintain attribution to the author(s) and the title of the work, journal citation and DOI. 2040-8978/17/113001+27$33.00 © 2017 IOP Publishing Ltd Printed in the UK 1

https://iopscience.iop.org/article/10.1088/2040-8986/aa8299/pdf

Recent advances in patterned photostimulation for optogenetics

Characterization of the diffusional and electrotonic coupling of spines to the dendritic shaft is crucial to understanding neuronal integration and synaptic plasticity. Two-photon photobleaching and photorelease of fluorescein dextran were used to generate concentration gradients between spines and shafts in rat CA1 pyramidal neurons. Diffusional reequilibration was monitored with two-photon fluorescence imaging. The time course of reequilibration was exponential, with time constants in the range of 20 to 100 milliseconds, demonstrating chemical compartmentalization on such time scales. These values imply that electrical spine neck resistances are unlikely to exceed 150 megohms and more likely range from 4 to 50 megohms.

Direct measurement of coupling between dendritic spines and shafts

Photoactivated genetically encoded voltage indicators (GEVIs) have the potential to enable optically sectioned voltage imaging at the intersection of a photoactivation beam and an imaging beam. We developed a pooled high-throughput screen to identify archaerhodopsin mutants with enhanced photoactivation. After screening ~105 cells, we identified a novel GEVI, NovArch, whose one-photon near-infrared fluorescence is reversibly enhanced by weak one-photon blue or two-photon near-infrared excitation. Because the photoactivation leads to fluorescent signals catalytically rather than stoichiometrically, high fluorescence signals, optical sectioning, and high time resolution are achieved simultaneously at modest blue or two-photon laser power. We demonstrate applications of the combined molecular and optical tools to optical mapping of membrane voltage in distal dendrites in acute mouse brain slices and in spontaneously active neurons in vivo.

Photoactivated voltage imaging in tissue with an archaerhodopsin-derived reporter.

Electrical properties of neuronal processes are extraordinarily complex, dynamic, and, in the general case, impossible to predict in the absence of detailed measurements. To obtain such a measurement one would, ideally, like to be able to monitor electrical subthreshold events as they travel from synapses on distal dendrites and summate at particular locations to initiate action potentials. It is now possible to carry out these measurements at the scale of individual dendritic spines using voltage imaging. In these measurements, the voltage-sensitive probes can be thought of as transmembrane voltmeters with a linear scale, which directly monitor electrical signals. Grinvald et al. were important early contributors to the methodology of voltage imaging, and they pioneered some of its significant results. We combined voltage imaging and glutamate uncaging using computer-generated holography. The results demonstrated that patterned illumination, by reducing the surface area of illuminated membrane, reduces photodynamic damage. Additionally, region-specific illumination practically eliminated the contamination of optical signals from individual spines by the scattered light from the parent dendrite. Finally, patterned illumination allowed one-photon uncaging of glutamate on multiple spines to be carried out in parallel with voltage imaging from the parent dendrite and neighboring spines.

https://www.spiedigitallibrary.org/journals/Neurophotonics/volume-4/issue-3/031211/Imaging-membrane-potential-changes-from-dendritic-spines-using-computer-generated/10.1117/1.NPh.4.3.031211.pdf

Imaging membrane potential changes from dendritic spines using computer-generated holography.

The physiology of individual synapses on dendritic spines is important because isolated and widely distributed spines are active during sensory information processing in vivo. We used acute cortical brain slices from the rat to investigate synaptic signal integration at the level of individual synapses on dendritic spines in layer 5 pyramidal neurons. We monitored subthreshold synaptic signals using a combination of voltage-sensitive dye recordings, patch-electrode somatic recordings, and 2-photon glutamate uncaging with holographic illumination. We describe the capabilities and limitations of this approach and demonstrate that imaging with an organic voltage-sensitive dye is presently a unique way to monitor temporal summation of uncaging evoked quantal excitatory synaptic potentials locally, at the site of origin on thin basal dendrites. The results indicated that the ability of repetitive activation of individual excitatory synapses on spines to influence the electrical signaling in individual neurons is strictly limited to subthreshold responses. We show that the underlying mechanisms, which control the temporal summation of excitatory synaptic potential in single synapses, can now be investigated with described methodology.

Ju-Yun Weng

Papers

Imaging membrane potential changes from dendritic spines using computer-generated holography.

Using holographic illumination to study synaptic signal integration at individual dendritic spines