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

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

Boletín Oficial de la Provincia de Oviedo: Número 15 - 1932 enero 19

The cerebellum has often been referred to as the "neuronal machine", so called for the elegant architecture of neuronal circuitry and necessarily clockwork precision of signal transmission therein (Eccles 1967; Ito 2006). To maintain such precision, many synapses within the cerebellar micro-circuitry are highly plastic and frequency dependant. In particular, short-term synaptic plasticity (STP) at an important excitatory synaptic pathway between cerebellar granule cells (CGCs) and Purkinje neurons (PN), called the "parallel fibre to Purkinje neuron synapse" (PF-PN), is an integral feature of the synapse and for cerebellar function as a whole (Dittman, Kreitzer et al. 2000; Boyden, Katoh et al. 2004). During transmission at PF-PN synapses, the dynamics of elevated pre-synaptic calcium (or residual calcium) dramatically impact the STP exhibited by the synapse (Zucker and Regehr 2002). Multiple cellular mechanisms function cooperatively to carefully control residual calcium dynamics and two principle pre-synaptic mechanisms expressed in CGCs include the sodium calcium exchanger (NCX) and plasma membrane calcium ATPase (PMCA) (Blaustein, Juhaszova et al. 2002; Ivannikov, Sugimori et al. 2010). Both PMCA and NCX proteins function to remove elevated intracellular calcium and their cooperative activity is thought to be critical for maintaining PF-PN synaptic behaviour (Regehr 1997; Empson, Garside et al. 2007). However, characterisation of NCX activity in pre-synaptic calcium control, its influence on synaptic transmission, and how it might interplay with PMCA activity at PF-PN synapses remains to be established. This research aimed to understand the cooperative activity of NCX and PMCA2 and its functional impact on PF-PN synaptic behaviour. To do this, calcium fluorescent imaging and electrophysiological recordings were made from the mouse cerebellum in vitro, whilst PMCA2 and NCX were sequentially removed by either pharmacological or genetic manipulation to assess their individual and combined activities. Two fluorescent calcium imaging techniques using a genetically encoded calcium indicator, GCaMP2 and a conventional calcium indicator, Calcium Green-1 Dextran, were utilised to determine the influence that cooperative NCX and PMCA2 activity has on PF pre-synaptic calcium dynamics. The impact that cooperative NCX and PMCA2 activity had on PF-PN synaptic behaviour was addressed using patch clamp electrophysiology to record PF-PN post-synaptic currents and assess STP of the synapse. To supplement experimental studies, a computational modelling approach was used throughout to aid interpretation of calcium fluorescent imaging experiments. The model simulated pre-synaptic calcium dynamics to provide a theoretical basis for how calcium efflux characteristics exhibited by NCX and PMCA2 dictate their cooperative interplay for pre-synaptic calcium control. This investigation has provided strong evidence for an important cooperative interplay of pre-synaptic NCX and PMCA2 activity, capable of influencing PF-PN synapse behaviour. The extent of cooperation between NCX and PMCA2 activity depends upon on pre-synaptic calcium load (hence pre-synaptic activity) and this influences the behaviour of the PF-PN synapse. It is proposed that the kinetic balance for this cooperative activity within the PF pre-synaptic terminal is predominantly governed by the kinetic properties that each mechanism exhibits, including their calcium affinity for activation and maximal efflux capacities

Chronic cranial window with access port for repeated cellular manipulations, drug application, and electrophysiology

Chronic cranial windows have been instrumental in advancing optical studies in vivo, permitting long-term, high-resolution imaging in various brain regions. However, once a window is attached it is difficult to regain access to the brain under the window for cellular manipulations. Here we describe a simple device that combines long term in vivo optical imaging with direct brain access via glass or quartz pipettes and metal, glass, or quartz electrodes for cellular manipulations like dye or drug injections and electrophysiological stimulations or recordings while keeping the craniotomy sterile. Our device comprises a regular cranial window glass coverslip with a drilled access hole later sealed with biocompatible silicone. This chronic cranial window with access port is cheap, easy to manufacture, can be mounted just as the regular chronic cranial window, and is self-sealing after retraction of the pipette or electrode. We demonstrate that multiple injections can be performed through the silicone port by repetitively bolus loading calcium sensitive dye into mouse barrel cortex and recording spontaneous cellular activity over a period of weeks. As an example to the extent of its utility for electrophysiological recording, we describe how simple removal of the silicone seal can permit patch pipette access for whole-cell patch clamp recordings in vivo. During these chronic experiments we do not observe any infections under the window or impairment of animal health

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

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