Darcy S. Peterka

SLM microscopy: scanless two-photon imaging and photostimulation with spatial light modulators

Laser microscopy has generally poor temporal resolution, caused by the serial scanning of each pixel. This is a significant problem for imaging or optically manipulating neural circuits, since neuronal activity is fast. To help surmount this limitation, we have developed a “scanless” microscope that does not contain mechanically moving parts. This microscope uses a diffractive spatial light modulator (SLM) to shape an incoming two-photon laser beam into any arbitrary light pattern. This allows the simultaneous imaging or photostimulation of different regions of a sample with three-dimensional precision. To demonstrate the usefulness of this microscope, we perform two-photon uncaging of glutamate to activate dendritic spines and cortical neurons in brain slices. We also use it to carry out fast (60 Hz) two-photon calcium imaging of action potentials in neuronal populations. Thus, SLM microscopy appears to be a powerful tool for imaging and optically manipulating neurons and neuronal circuits. Moreover, the use of SLMs expands the flexibility of laser microscopy, as it can substitute traditional simple fixed lenses with any calculated lens function.

Nanotools for neuroscience and brain activity mapping.

Neuroscience is at a crossroads. Great effort is being invested into deciphering specific neural interactions and circuits. At the same time, there exist few general theories or principles that explain brain function. We attribute this disparity, in part, to limitations in current methodologies. Traditional neurophysiological approaches record the activities of one neuron or a few neurons at a time. Neurochemical approaches focus on single neurotransmitters. Yet, there is an increasing realization that neural circuits operate at emergent levels, where the interactions between hundreds or thousands of neurons, utilizing multiple chemical transmitters, generate functional states. Brains function at the nanoscale, so tools to study brains must ultimately operate at this scale, as well. Nanoscience and nanotechnology are poised to provide a rich toolkit of novel methods to explore brain function by enabling simultaneous measurement and manipulation of activity of thousands or even millions of neurons. We and others refer to this goal as the Brain Activity Mapping Project. In this Nano Focus, we discuss how recent developments in nanoscale analysis tools and in the design and synthesis of nanomaterials have generated optical, electrical, and chemical methods that can readily be adapted for use in neuroscience. These approaches represent exciting areas of technical development and research. Moreover, unique opportunities exist for nanoscientists, nanotechnologists, and other physical scientists and engineers to contribute to tackling the challenging problems involved in understanding the fundamentals of brain function.

Imaging voltage in neurons

In the last decades, imaging membrane potential has become a fruitful approach to study neural circuits, especially in invertebrate preparations with large, resilient neurons. At the same time, particularly in mammalian preparations, voltage imaging methods suffer from poor signal to noise and secondary side effects, and they fall short of providing single-cell resolution when imaging of the activity of neuronal populations. As an introduction to these techniques, we briefly review different voltage imaging methods (including organic fluorophores, SHG chromophores, genetic indicators, hybrid, nanoparticles, and intrinsic approaches) and illustrate some of their applications to neuronal biophysics and mammalian circuit analysis. We discuss their mechanisms of voltage sensitivity, from reorientation, electrochromic, or electro-optical phenomena to interaction among chromophores or membrane scattering, and highlight their advantages and shortcomings, commenting on the outlook for development of novel voltage imaging methods.

Two-photon optogenetics of dendritic spines and neural circuits

We demonstrate a two-photon optogenetic method that generates action potentials in neurons with single-cell precision, using the red-shifted opsin C1V1T. We applied the method to optically map synaptic circuits in mouse neocortical brain slices and to activate small dendritic regions and individual spines. Using a spatial light modulator, we split the laser beam onto several neurons and performed simultaneous optogenetic activation of selected neurons in three dimensions.

RuBi-Glutamate: Two-Photon and Visible-Light Photoactivation of Neurons and Dendritic spines.

We describe neurobiological applications of RuBi-Glutamate, a novel caged-glutamate compound based on ruthenium photochemistry. RuBi-Glutamate can be excited with visible wavelengths and releases glutamate after one- or two-photon excitation. It has high quantum efficiency and can be used at low concentrations, partly avoiding the blockade of GABAergic transmission present with other caged compounds. Two-photon uncaging of RuBi-Glutamate has a high spatial resolution and generates excitatory responses in individual dendritic spines with physiological kinetics. With laser beam multiplexing, two-photon RuBi-Glutamate uncaging can also be used to depolarize and fire pyramidal neurons with single-cell resolution. RuBi-Glutamate therefore enables the photoactivation of neuronal dendrites and circuits with visible or two-photon light sources, achieving single cell, or even single spine, precision.

Imprinting and recalling cortical ensembles

Building new networks in the brain Donald Hebbs hypothesis that coactivation of neurons leads to the formation of ensembles of neurons has inspired neuroscientists for decades. The experimental creation of such ensembles has been technically challenging. Using two-photon optogenetic stimulation with single-cell resolution, Carrillo-Reid et al. discovered that recurrent activation of a group of neurons creates an ensemble that is imprinted in the brain circuitry. Activation of a single neuron can lead to recall of the entire ensemble in a phenomenon called pattern completion. The artificial ensemble persists over days and can be reactivated at later time points without interfering with endogenous circuitry. Science, this issue p. 691 Hebbian synaptic plasticity can be artificially introduced in the neocortex of awake animals. Neuronal ensembles are coactive groups of neurons that may represent building blocks of cortical circuits. These ensembles could be formed by Hebbian plasticity, whereby synapses between coactive neurons are strengthened. Here we report that repetitive activation with two-photon optogenetics of neuronal populations from ensembles in the visual cortex of awake mice builds neuronal ensembles that recur spontaneously after being imprinted and do not disrupt preexisting ones. Moreover, imprinted ensembles can be recalled by single- cell stimulation and remain coactive on consecutive days. Our results demonstrate the persistent reconfiguration of cortical circuits by two-photon optogenetics into neuronal ensembles that can perform pattern completion.

Simultaneous imaging of neural activity in three dimensions

We introduce a scanless optical method to image neuronal activity in three dimensions simultaneously. Using a spatial light modulator and a custom-designed phase mask, we illuminate and collect light simultaneously from different focal planes and perform calcium imaging of neuronal activity in vitro and in vivo. This method, combining structured illumination with volume projection imaging, could be used as a technological platform for brain activity mapping.

Calcium imaging of neural circuits with extended depth-of-field light-sheet microscopy.

Increasing the volumetric imaging speed of light-sheet microscopy will improve its ability to detect fast changes in neural activity. Here, a system is introduced for brain-wide imaging of neural activity in the larval zebrafish by coupling structured illumination with cubic phase extended depth-of-field (EDoF) pupil encoding. This microscope enables faster light-sheet imaging and facilitates arbitrary plane scanning-removing constraints on acquisition speed, alignment tolerances, and physical motion near the sample. The usefulness of this method is demonstrated by performing multi-plane calcium imaging in the fish brain with a 416×832×160  μm field of view at 33 Hz. The optomotor response behavior of the zebrafish is monitored at high speeds, and time-locked correlations of neuronal activity are resolved across its brain.

Targeted intracellular voltage recordings from dendritic spines using quantum-dot-coated nanopipettes

Dendritic spines are the primary site of excitatory synaptic input onto neurons, and are biochemically isolated from the parent dendritic shaft by their thin neck. However, due to the lack of direct electrical recordings from spines, the influence that the neck resistance has on synaptic transmission, and the extent to which spines compartmentalize voltage, specifically excitatory postsynaptic potentials, albeit critical, remains controversial. Here, we use quantum-dot-coated nanopipette electrodes (tip diameters ∼15-30 nm) to establish the first intracellular recordings from targeted spine heads under two-photon visualization. Using simultaneous somato-spine electrical recordings, we find that back propagating action potentials fully invade spines, that excitatory postsynaptic potentials are large in the spine head (mean 26 mV) but are strongly attenuated at the soma (0.5-1 mV) and that the estimated neck resistance (mean 420 MΩ) is large enough to generate significant voltage compartmentalization. Nanopipettes can thus be used to electrically probe biological nanostructures.

Multi-scale approaches for high-speed imaging and analysis of large neural populations

Progress in modern neuroscience critically depends on our ability to observe the activity of large neuronal populations with cellular spatial and high temporal resolution. However, two bottlenecks constrain efforts towards fast imaging of large populations. First, the resulting large video data is challenging to analyze. Second, there is an explicit tradeoff between imaging speed, signal-to-noise, and field of view: with current recording technology we cannot image very large neuronal populations with simultaneously high spatial and temporal resolution. Here we describe multi-scale approaches for alleviating both of these bottlenecks. First, we show that spatial and temporal decimation techniques based on simple local averaging provide order-of-magnitude speedups in spatiotemporally demixing calcium video data into estimates of single-cell neural activity. Second, once the shapes of individual neurons have been identified at fine scale (e.g., after an initial phase of conventional imaging with standard temporal and spatial resolution), we find that the spatial/temporal resolution tradeoff shifts dramatically: after demixing we can accurately recover denoised fluorescence traces and deconvolved neural activity of each individual neuron from coarse scale data that has been spatially decimated by an order of magnitude. This offers a cheap method for compressing this large video data, and also implies that it is possible to either speed up imaging significantly, or to “zoom out” by a corresponding factor to image order-of-magnitude larger neuronal populations with minimal loss in accuracy or temporal resolution.