Voltage imaging with ANNINE dyes and two-photon microscopy of Purkinje dendrites in awake mice

Voltage imaging is the next generation of functional imaging in neuroscience. It promises to resolve neuronal activity 10 to 100-times faster than calcium imaging and to report not only supra but also subthreshold activity on a single cell or even subcellular level. Lately, several different voltage sensors and imaging techniques were published which can achieve this. Here, we focus on a technique based on the synthetic pure electrochromic voltage-sensitive dyes ANNINE-6 and ANNINE-6plus and the excitation of this dye at the red spectral edge of absorption to maximize voltage sensitivity and minimize phototoxicity and bleaching. Importantly, voltage imaging with ANNINE dyes can be done with one and two-photon excitation. Two-photon microscopy allows in vivo, depth resolved imaging and line-scan recordings with sub-millisecond temporal resolution. Interestingly for many future applications, the spectral characteristics of ANNINE dyes allows simultaneous imaging with green indicators, like the genetically encoded calcium indicator GCaMP6. We used this method to study supra and subthreshold dendritic voltage changes in Purkinje neurons of awake mice. Simultaneously, we imaged dendritic calcium and recorded electrical activity from the soma or locally applied drugs to show the full potential of the technique to study dendritic integration in awake animals

Simultaneous dendritic voltage and calcium imaging and somatic recording from Purkinje neurons in awake mice

Spatiotemporal maps of dendritic signalling and their relationship with somatic output is fundamental to neuronal information processing, yet remain unexplored in awake animals. Here, we combine simultaneous sub-millisecond voltage and calcium two-photon imaging from distal spiny dendrites, with somatic electrical recording from spontaneously active cerebellar Purkinje neurons (PN) in awake mice. We detect discrete 1−2 ms suprathreshold voltage spikelets in the distal spiny dendrites during dendritic complex spikes. Spikelets and their calcium correlates are highly heterogeneous in number, timing and spatial distribution within and between complex spikes. Back-propagating simple spikes are highly attenuated. Highly variable 5–10 ms voltage hotspots are localized to fine dendritic processes and are reduced in size and frequency by lidocaine and CNQX. Hotspots correlated with somatic output but also, at high frequency, trigger purely dendritic calcium spikes. Summarizing, spatiotemporal signalling in PNs is far more complex, dynamic, and fine scaled than anticipated, even in resting animals

Dendritic coincidence detection in Purkinje neurons of awake mice

Dendritic coincidence detection is fundamental to neuronal processing yet remains largely unexplored in awake animals. Specifically, the underlying dendritic voltage-calcium relationship has not been directly addressed. Here, using simultaneous voltage and calcium two-photon imaging of Purkinje neuron spiny dendrites, we show how coincident synaptic inputs and resulting dendritic spikes modulate dendritic calcium signaling during sensory stimulation in awake mice. Sensory stimulation increased the rate of postsynaptic potentials and dendritic calcium spikes evoked by climbing fiber and parallel fiber synaptic input. These inputs are integrated in a time-dependent and nonlinear fashion to enhance the sensory-evoked dendritic calcium signal. Intrinsic supralinear dendritic mechanisms, including voltage-gated calcium channels and metabotropic glutamate receptors, are recruited cooperatively to expand the dynamic range of sensory-evoked dendritic calcium signals. This establishes how dendrites can use multiple interplaying mechanisms to perform coincidence detection, as a fundamental and ongoing feature of dendritic integration in behaving animals

Fast variational alignment of non-flat 1D displacements for applications in neuroimaging

Background:In the context of signal analysis and pattern matching, alignment of 1D signals for the comparison of signal morphologies is an important problem. For image processing and computer vision, 2D optical flow (OF) methods find wide application for motion analysis and image registration and variational OF methods have been continuously improved over the past decades.New method:We propose a variational method for the alignment and displacement estimation of 1D signals. We pose the estimation of non-flat displacements as an optimization problem with a similarity and smoothness term similar to variational OF estimation. To this end, we can make use of efficient optimization strategies that allow real-time applications on consumer grade hardware.Results:We apply our method to two applications from functional neuroimaging: The alignment of 2-photon imaging line scan recordings and the denoising of evoked and event-related potentials in single trial matrices. We can report state of the art results in terms of alignment quality and computing speeds.Existing methods:Existing methods for 1D alignment target mostly constant displacements, do not allow native subsample precision or precise control over regularization or are slower than the proposed method.Conclusions:Our method is implemented as a MATLAB toolbox and is online available. It is suitable for 1D alignment problems, where high accuracy and high speed is needed and non-constant displacements occur

Dendritic diameters affect the spatial variability of intracellular calcium dynamics in computer models

There is growing interest in understanding calcium dynamics in dendrites, both experimentally and computationally. Many processes influence these dynamics, but in dendrites there is a strong contribution of morphology because the peak calcium levels are strongly determined by the surface to volume ratio (SVR) of each branch, which is inversely related to branch diameter. In this study we explore the predicted variance of dendritic calcium concentrations due to local changes in dendrite diameter and how this is affected by the modeling approach used. We investigate this in a model of dendritic calcium spiking in different reconstructions of cerebellar Purkinje cells and in morphological analysis of neocortical and hippocampal pyramidal neurons. We report that many published models neglect diameter-dependent effects on calcium concentration and show how to implement this correctly in the NEURON simulator, both for phenomenological pool based models and for implementations using radial 1D diffusion. More detailed modeling requires simulation of 3D diffusion and we demonstrate that this does not dissipate the local concentration variance due to changes of dendritic diameter. In many cases 1D diffusion of models of calcium buffering give a good approximation provided an increased morphological resolution is implemented

Photoswitching mechanism of a fluorescent protein revealed by time-resolved crystallography and transient absorption spectroscopy.

International audienceReversibly switchable fluorescent proteins (RSFPs) serve as markers in advanced fluorescence imaging. Photoswitching from a non-fluorescent off-state to a fluorescent on-state involves trans-to-cis chromophore isomerization and proton transfer. Whereas excited-state events on the ps timescale have been structurally characterized, conformational changes on slower timescales remain elusive. Here we describe the off-to-on photoswitching mechanism in the RSFP rsEGFP2 by using a combination of time-resolved serial crystallography at an X-ray free-electron laser and ns-resolved pump-probe UV-visible spectroscopy. Ten ns after photoexcitation, the crystal structure features a chromophore that isomerized from trans to cis but the surrounding pocket features conformational differences compared to the final on-state. Spectroscopy identifies the chromophore in this ground-state photo-intermediate as being protonated. Deprotonation then occurs on the μs timescale and correlates with a conformational change of the conserved neighbouring histidine. Together with a previous excited-state study, our data allow establishing a detailed mechanism of off-to-on photoswitching in rsEGFP2