Trabajar en el laboratorio con el apoyo de Nederlandse Organisatie voor Wetenschappelijk Onderzoek (NWO) (917.10.372) para RMM. JD está afiliado a la Unión Europea del 7 º PM BrainTrain programa ( <a href="http://www.brain-train.nl">www.brain-train.nl</a> ). Damos las gracias a Pieter Laurens-Baljon y Sabine Schmitz (tanto CNCR, VU University Amsterdam) para obtener imágenes de formas de onda de FP amplia multielectrodo y culturas neuronal utilizada en la secuencia de vídeo.

<ol>
	<li>Katz, L. C., Shatz, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+activity+and+the+construction+of+cortical+circuits.">Synaptic activity and the construction of cortical circuits.</a> Science. 274, 1133-1138 (1996).</li><li>Khazipov, R., Luhmann, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+patterns+of+electrical+activity+in+the+developing+cerebral+cortex+of+humans+and+rodents.">Early patterns of electrical activity in the developing cerebral cortex of humans and rodents.</a> Trends in Neurosciences. 29, 414-418 (2006).</li><li>Spitzer, N. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrical+activity+in+early+neuronal+development.">Electrical activity in early neuronal development.</a> Nature. 444, 707-712 (2006).</li><li>Adelsberger, H., Garaschuk, O., Konnerth, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cortical+calcium+waves+in+resting+newborn+mice.">Cortical calcium waves in resting newborn mice.</a> Nat. Neurosci. 8, 988-990 (2005).</li><li>Garaschuk, O., Linn, J., Eilers, J., Konnerth, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large-scale+oscillatory+calcium+waves+in+the+immature+cortex.">Large-scale oscillatory calcium waves in the immature cortex.</a> Nat. Neurosci. 3, 452-459 (2000).</li><li>Lamblin, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electroencephalography+of+the+premature+and+term+newborn.+Maturational+aspects+and+glossary.">Electroencephalography of the premature and term newborn. Maturational aspects and glossary.</a> Neurophysiol. Clin. 29, 123-219 (1999).</li><li>Rakic, P., Komuro, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+receptor/channel+activity+in+neuronal+cell+migration.">The role of receptor/channel activity in neuronal cell migration.</a> J. Neurobiol. 26, 299-315 (1995).</li><li>Spitzer, N. C., Root, C. M., Borodinsky, L. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orchestrating+neuronal+differentiation:+patterns+of+Ca2++spikes+specify+transmitter+choice.">Orchestrating neuronal differentiation: patterns of Ca2+ spikes specify transmitter choice.</a> Trends. Neurosci. 27, 415-421 (2004).</li><li>Canto, C. B., Wouterlood, F. G., Witter, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=What+does+the+anatomical+organization+of+the+entorhinal+cortex+tell+us.">What does the anatomical organization of the entorhinal cortex tell us.</a> Neural. Plast. , 381243-381243 (2008).</li><li>Bureau, I., Shepherd, G. M., Svoboda, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Precise+development+of+functional+and+anatomical+columns+in+the+neocortex.">Precise development of functional and anatomical columns in the neocortex.</a> Neuron. 42, 789-801 (2004).</li><li>Ikegaya, Y., Le Bon-Jego, M., Yuste, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large-scale+imaging+of+cortical+network+activity+with+calcium+indicators.">Large-scale imaging of cortical network activity with calcium indicators.</a> Neuroscience research. 52, 132-138 (2005).</li></ol>

LaVision Biotec GmbH (Bielefeld, Alemania), patrocinada por los honorarios de presentación de este artículo manuscrito.

Los métodos que demostrar aquí muestran protocolos adecuados para la imagen de calcio de la dinámica de la red a partir de células de identificación en el desarrollo de redes corticales y del hipocampo en el ratón y también en el cerebro de la rata. Estos métodos proporcionan una resolución espacial óptima para visualizar una red local de somas celulares al mismo tiempo para medir la actividad por encima del umbral. La resolución temporal de la actividad de la red puede variar, dependiendo de la configuración del marco de la adquisición de la cámara CCD, para optimizar la señal: las mediciones de ruido por mucho tiempo los acontecimientos duración por encima del umbral, por lo general se encuentran en el sistema nervioso en desarrollo. Estos protocolos no se limitan a las redes del hipocampo y la corteza, sino también las células de la etiqueta a través del sistema nervioso en desarrollo. La limitación de este método es que la actividad por encima del umbral sólo se pueden grabar.

functional calcium imaging in developing cortical networks

Un patrón distintivo de la actividad en el sistema nervioso en desarrollo es una actividad espontánea, sincronizar la red. Actividad sincronizada se ha observado en intacta la médula espinal, tronco cerebral, la retina, la corteza y disociado preparativos cultivo neuronal. Durante los períodos de actividad espontánea, las neuronas se despolarizan de disparo simple o ráfagas de potenciales de acción, la activación de muchos canales de iones. Despolarización activa canales dependientes de voltaje de calcio en las dendritas y espinas que median el influjo de calcio. Actividad altamente eléctrica sincronizada se ha medido a partir de las redes locales de neuronas con electrodos de campo. Esta técnica permite una alta frecuencia de muestreo temporal pero de menor resolución espacial debido a la gestión integrada de lectura de múltiples neuronas en uno de los electrodos. Resolución única célula de la actividad neuronal es posible con patch-clamp electrofisiología en las neuronas individuales para medir la actividad de disparo. Sin embargo, la capacidad de medir de una red se limita a la cantidad de neuronas parcheado simultaneously, y por lo general es sólo una o dos neuronas. El uso de calcio dependientes de tintes indicadores fluorescentes ha permitido a la medición de la actividad sincronizada a través de una red de células. Esta técnica ofrece una alta resolución espacial y temporal de muestreo suficiente para registrar la actividad espontánea de la red de desarrollo. Una característica clave de la nueva formación de redes corticales y del hipocampo durante el desarrollo postnatal antes y temprano es espontánea, la actividad neuronal sincronizada (Katz y Shatz, 1996; Khaziphov y Luhmann, 2006). Esta actividad de la red correlacionadas se cree que es esencial para la generación de circuitos funcionales en el sistema nervioso en desarrollo (Spitzer, 2006). En tanto los primates y el cerebro de roedores, a principios de electricidad y las ondas de calcio de la red se observan antes y después del nacimiento en vivo e in vitro (Adelsberger et al, 2005;. Garaschuk et al, 2000;.. Lamblin et al, 1999). Estos patrones de las primeras actividades, que se sabe que el control de variasprocesos de desarrollo incluyendo la diferenciación neuronal sinaptogénesis y plasticidad (Rakic ​​y Komuro, 1995;. Spitzer et al, 2004) son de vital importancia para el correcto desarrollo y maduración de los circuitos corticales. En este video JOVE, que demuestran los métodos utilizados para la actividad espontánea de la imagen en el desarrollo de redes corticales. Calcio sensible a los indicadores, como la Fura 2-AM éster difusa a través de la membrana celular donde la actividad de la esterasa intracelular rompe el AM ésteres de salir de la forma de células impermeable de tinte indicador. La forma de impermeant indicador tiene grupos ácido carboxílico que son capaces de detectar y luego se unen los iones de calcio intracelular .. La fluorescencia del colorante sensible al calcio se altera transitoriamente al unirse al calcio. Técnicas de imagen de una o varias de fotones se utilizan para medir el cambio de los fotones emitidos por el medio de contraste, y por lo tanto indicar una alteración en el calcio intracelular. Además, estas calcio-dindicadores ependent se puede combinar con otros marcadores fluorescentes para investigar los tipos de células dentro de la red activa.

Watch this Scientific Journal Video about Functional Calcium Imaging in Developing Cortical Networks at JoVE.com

Functional Calcium Imaging in Developing Cortical Networks

La actividad espontánea de la creación de redes neuronales se puede medir con AM-éster formas de tintes indicadores sensibles de calcio. Cambios en el calcio intracelular, lo que indica la activación neuronal, se detectan los cambios transitorios de la fluorescencia con un indicador o imagen de dos fotones. Este protocolo puede ser adaptado para una serie de redes neuronales del desarrollo que dependen de<em&gt; In vitro</em&gt;.

Imagen de calcio funcionales en el desarrollo de redes corticales

A hallmark pattern of activity in developing nervous systems is spontaneous, synchronized network activity. Synchronized activity has been observed in ...

functional-calcium-imaging-in-developing-cortical-networks

Research

JoVE Journal

Neuroscience

38.8K vistas.  VU University, Amsterdam. La actividad espontánea de la creación de redes neuronales se puede medir con AM-éster formas de tintes indicadores sensibles de calcio. Cambios en el calcio intracelular, lo que indica la activación neuronal, se detectan los cambios transitorios de la fluorescencia con un indicador o imagen de dos fotones. Este protocolo puede ser adaptado para una serie de redes neuronales del desarrollo que dependen de In vitro.

Video: Functional Calcium Imaging in Developing Cortical Networks

Functional Mapping with Simultaneous MEG and EEG

We use magnetoencephalography (MEG) and electroencephalography (EEG) to locate and determine the temporal evolution in brain areas involved in the processing of simple sensory stimuli. We will use somatosensory stimuli to locate the hand somatosensory areas, auditory stimuli to locate the auditory cortices, visual stimuli in four quadrants of the visual field to locate the early visual areas. These type of experiments are used for functional mapping in epileptic and brain tumor patients to locate eloquent cortices. In basic neuroscience similar experimental protocols are used to study the orchestration of cortical activity. The acquisition protocol includes quality assurance procedures, subject preparation for the combined MEG/EEG study, and acquisition of evoked-response data with somatosensory, auditory, and visual stimuli. We also demonstrate analysis of the data using the equivalent current dipole model and cortically-constrained minimum-norm estimates. Anatomical MRI data are employed in the analysis for visualization and for deriving boundaries of tissue boundaries for forward modeling and cortical location and orientation constraints for the minimum-norm estimates.

We use magnetoencephalography (MEG) and electroencephalography (EEG) to map brain areas involved in the processing of simple sensory stimuli.

Mapeo funcional con MEG y EEG simultáneo

We use magnetoencephalography (MEG) and electroencephalography (EEG) to locate and determine the temporal evolution in brain areas involved in the ...

Investigación

Neurociencias

Time-lapse Live Imaging of Clonally Related Neural Progenitor Cells in the Developing Zebrafish Forebrain

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To understand the behavior of neural progenitor cells in the complex in vivo environment, time-lapse live imaging of neural progenitor cells in an intact brain is critically required. In this video, we exploit the unique features of zebrafish embryos to visualize the development of forebrain neural progenitor cells in vivo. We use electroporation to genetically and sparsely label individual neural progenitor cells. Briefly, DNA constructs coding for fluorescent markers were injected into the forebrain ventricle of 22 hours post fertilization (hpf) zebrafish embryos and electric pulses were delivered immediately. Six hours later, the electroporated zebrafish embryos were mounted with low melting point agarose in glass bottom culture dishes. Fluorescently labeled neural progenitor cells were then imaged for 36hours with fixed intervals under a confocal microscope using water dipping objective lens. The present method provides a way to gain insights into the in vivo development of forebrain neural progenitor cells and can be applied to other parts of the central nervous system of the zebrafish embryo.

The present video demonstrates a method which takes advantage of the combination of electroporation and confocal microscopy to perform live imaging on individual neural progenitor cells in the developing zebrafish forebrain. In vivo analysis of the development of forebrain neural progenitor cells at a clonal level can be achieved in this way.

Time-lapse de imágenes en vivo de la relación clonal con células progenitoras neurales en el cerebro anterior El desarrollo de pez cebra

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To ...

In vivo Neuronal Calcium Imaging in C. elegans

The nematode worm C. elegans is an ideal model organism for relatively simple, low cost neuronal imaging in vivo. Its small transparent body and simple, well-characterized nervous system allows identification and fluorescence imaging of any neuron within the intact animal. Simple immobilization techniques with minimal impact on the animal's physiology allow extended time-lapse imaging. The development of genetically-encoded calcium sensitive fluorophores such as cameleon 1 and GCaMP 2 allow in vivo imaging of neuronal calcium relating both cell physiology and neuronal activity. Numerous transgenic strains expressing these fluorophores in specific neurons are readily available or can be constructed using well-established techniques. Here, we describe detailed procedures for measuring calcium dynamics within a single neuron in vivo using both GCaMP and cameleon. We discuss advantages and disadvantages of both as well as various methods of sample preparation (animal immobilization) and image analysis. Finally, we present results from two experiments: 1) Using GCaMP to measure the sensory response of a specific neuron to an external electrical field and 2) Using cameleon to measure the physiological calcium response of a neuron to traumatic laser damage. Calcium imaging techniques such as these are used extensively in C. elegans and have been extended to measurements in freely moving animals, multiple neurons simultaneously and comparison across genetic backgrounds. C. elegans presents a robust and flexible system for in vivo neuronal imaging with advantages over other model systems in technical simplicity and cost.

In vivo Neuronal Calcium Imaging in C. elegans

With its small transparent body, well-documented neuroanatomy and a host of amenable genetic techniques and reagents, C. elegans makes an ideal model organism for in vivo neuronal imaging using relatively simple, low-cost techniques. Here we describe single neuron imaging within intact adult animals using genetically encoded fluorescent calcium indicators.

Formación de imágenes in vivo de calcio neuronal en C. elegans

The nematode worm C. elegans is an ideal model organism  for relatively simple, low cost neuronal imaging in vivo. Its small transparent body and simple, ...

Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of the dendritic tree, which eventually leads to neuronal output of action potentials at the axon. The influence of diverse spatial and temporal parameters of specific synaptic input on neuronal output is currently under investigation, e.g. the distance-dependent attenuation of dendritic inputs, the location-dependent interaction of spatially segregated inputs, the influence of GABAergig inhibition on excitatory integration, linear and non-linear integration modes, and many more.
With fast micro-iontophoresis of glutamate and GABA it is possible to precisely investigate the spatial and temporal integration of glutamatergic excitation and GABAergic inhibition. Critical technical requirements are either a triggered fluorescent lamp, light-emitting diode (LED), or a two-photon scanning microscope to visualize dendritic branches without introducing significant photo-damage of the tissue. Furthermore, it is very important to have a micro-iontophoresis amplifier that allows for fast capacitance compensation of high resistance pipettes. Another crucial point is that no transmitter is involuntarily released by the pipette during the experiment.
Once established, this technique will give reliable and reproducible signals with a high neurotransmitter and location specificity. Compared to glutamate and GABA uncaging, fast iontophoresis allows using both transmitters at the same time but at very distant locations without limitation to the field of view. There are also advantages compared to focal electrical stimulation of axons: with micro-iontophoresis the location of the input site is definitely known and it is sure that only the neurotransmitter of interest is released. However it has to be considered that with micro-iontophoresis only the postsynapse is activated and presynaptic aspects of neurotransmitter release are not resolved. In this article we demonstrate how to set up micro-iontophoresis in brain slice experiments.

In this article we introduce fast micro-iontophoresis of neurotransmitters as a technique to investigate integration of postsynaptic signals with high spatial and temporal precision.

Fast Micro-iontoforesis de glutamato y GABA: una herramienta útil para investigar la integración sináptica

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of ...

Live Imaging of Mitosis in the Developing Mouse Embryonic Cortex

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the developing cerebral cortex, abnormal mitosis of neural progenitors can cause defects in brain size and function. Hence, there is a critical need for tools to understand the mechanisms of neural progenitor mitosis. Cortical development in rodents is an outstanding model for studying this process. Neural progenitor mitosis is commonly examined in fixed brain sections. This protocol will describe in detail an approach for live imaging of mitosis in ex vivo embryonic brain slices. We will describe the critical steps for this procedure, which include: brain extraction, brain embedding, vibratome sectioning of brain slices, staining and culturing of slices, and time-lapse imaging. We will then demonstrate and describe in detail how to perform post-acquisition analysis of mitosis. We include representative results from this assay using the vital dye Syto11, transgenic mice (histone H2B-EGFP and centrin-EGFP), and in utero electroporation (mCherry-&#945;-tubulin). We will discuss how this procedure can be best optimized and how it can be modified for study of genetic regulation of mitosis. Live imaging of mitosis in brain slices is a flexible approach to assess the impact of age, anatomy, and genetic perturbation in a controlled environment, and to generate a large amount of data with high temporal and spatial resolution. Hence this protocol will complement existing tools for analysis of neural progenitor mitosis.

Neural progenitor mitosis is a critical parameter of neurogenesis. Much of our understanding of neural progenitor mitosis is based on analysis of fixed tissue. Live imaging in embryonic brain slices is a versatile technique to assess mitosis with high temporal and spatial resolution in a controlled environment.

Imágenes en vivo de la mitosis en el desarrollo de la corteza de embriones de ratón

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the ...

In Vivo Functional Brain Imaging Approach Based on Bioluminescent Calcium Indicator GFP-aequorin

Functional in vivo imaging has become a powerful approach to study the function and physiology of brain cells and structures of interest. Recently a new method of Ca2+-imaging using the bioluminescent reporter GFP-aequorin (GA) has been developed. This new technique relies on the fusion of the GFP and aequorin genes, producing a molecule capable of binding calcium and&#160;&#8212; with the addition of its cofactor coelenterazine &#8212; emitting bright light that can be monitored through a photon collector. Transgenic lines carrying the GFP-aequorin gene have been generated for both mice and Drosophila. In Drosophila, the GFP-aequorin gene has been placed under the control of the GAL4/UAS binary expression system allowing for targeted expression and imaging within the brain. This method has subsequently been shown to be capable of detecting both inward Ca2+-transients and Ca2+-released from inner stores. Most importantly it allows for a greater duration in continuous recording, imaging at greater depths within the brain, and recording at high temporal resolutions (up to 8.3 msec). Here we present the basic method for using bioluminescent imaging to record and analyze Ca2+-activity within the mushroom bodies, a structure central to learning and memory in the fly brain.

In Vivo Functional Brain Imaging Approach Based on Bioluminescent Calcium Indicator GFP-aequorin

Here we present a novel Ca2+-imaging approach using a bioluminescent reporter. This approach uses a fused construct GFP-aequorin which binds to Ca2+ and emits light, eliminating the need for light excitation. Significantly this method permits long continuous imaging, access to deep brain structures and high temporal resolution.

En Vivo Cerebral Funcional Imaging Enfoque Basado en calcio bioluminiscente Indicador GFP-aequorina

Functional in vivo imaging has become a powerful approach to study the function and physiology of brain cells and structures of interest. Recently a new ...

Simultaneous Recordings of Cortical Local Field Potentials, Electrocardiogram, Electromyogram, and Breathing Rhythm from a Freely Moving Rat

Monitoring the physiological dynamics of the brain and peripheral tissues is necessary for addressing a number of questions about how the brain controls body functions and internal organ rhythms when animals are exposed to emotional challenges and changes in their living environments. In general experiments, signals from different organs, such as the brain and the heart, are recorded by independent recording systems that require multiple recording devices and different procedures for processing the data files. This study describes a new method that can simultaneously monitor electrical biosignals, including tens of local field potentials in multiple brain regions, electrocardiograms that represent the cardiac rhythm, electromyograms that represent awake/sleep-related muscle contraction, and breathing signals, in a freely moving rat. The recording configuration of this method is based on a conventional micro-drive array for cortical local field potential recordings in which tens of electrodes are accommodated, and the signals obtained from these electrodes are integrated into a single electrical board mounted on the animal's head. Here, this recording system was improved so that signals from the peripheral organs are also transferred to an electrical interface board. In a single surgery, electrodes are first separately implanted into the appropriate body parts and the target brain areas. The open ends of all of these electrodes are then soldered to individual channels of the electrical board above the animal's head so that all of the signals can be integrated into the single electrical board. Connecting this board to a recording device allows for the collection of all of the signals into a single device, which reduces experimental costs and simplifies data processing, because all data can be handled in the same data file. This technique will aid the understanding of the neurophysiological correlates of the associations between central and peripheral organs.

This study introduces a method for the simultaneous recording of local field potentials in the brain, electrocardiograms, electromyograms, and breathing signals of a freely moving rat. This technique, which reduces experimental costs and simplifies data analysis, will contribute to the understanding of the interactions between the brain and peripheral organs.

Grabaciones simultáneas de potenciales de campo Local Cortical, Electrocardiograma, electromiograma y respirar a ritmo de una rata libremente móvil

Monitoring the physiological dynamics of the brain and peripheral tissues is necessary for addressing a number of questions about how the brain controls ...

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

Two-photon imaging is a powerful tool for the in vivo analysis of neuronal circuits in the mammalian brain. However, a limited number of in vivo imaging methods exist for examining the brain tissue of live newborn mammals. Herein we summarize a protocol for imaging individual cortical neurons in living neonatal mice. This protocol includes the following two methodologies: (1) the Supernova system for sparse and bright labeling of cortical neurons in the developing brain, and (2) a surgical procedure for the fragile neonatal skull. This protocol allows the observation of temporal changes of individual cortical neurites during neonatal stages with a high signal-to-noise ratio. Labeled cell-specific gene silencing and knockout can also be achieved by combining the Supernova with RNA interference and CRISPR/Cas9 gene editing systems. This protocol can, thus, be used for analyzing the developmental dynamics of cortical neurons, molecular mechanisms that control the neuronal dynamics, and changes in neuronal dynamics in disease models.

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

We present an in vivo two-photon imaging protocol for imaging the cerebral cortex of neonatal mice. This method is suitable for analyzing the developmental dynamics of cortical neurons, the molecular mechanisms that control the neuronal dynamics, and the changes in neuronal dynamics in disease models.

En Vivo Proyección de imagen de dos fotones de neuronas corticales en ratones neonatales

Two-photon imaging is a powerful tool for the in vivo analysis of neuronal circuits in the mammalian brain. However, a limited number of in vivo imaging ...

In vivo Calcium Imaging in Mouse Inferior Olive

Inferior olive (IO), a nucleus in the ventral medulla, is the only source of climbing fibers that form one of the two input pathways entering the cerebellum. IO has long been proposed to be crucial for motor control and its activity is currently considered to be at the center of many hypotheses of both motor and cognitive functions of the cerebellum. While its physiology and function have been relatively well studied on single-cell level in vitro, presently there are no reports on the organization of the IO network activity in living animals. This is largely due to the extremely challenging anatomical location of the IO, making it difficult to subject to conventional fluorescent imaging methods, where an optic path must be created through the entire brain located dorsally to the region of interest.
Here we describe an alternative method for obtaining state-of-the-art -level calcium imaging data from the IO network. The method takes advantage of the extreme ventral location of the IO and involves a surgical procedure for inserting a gradient-refractive index (GRIN) lens through the neck viscera to come into contact with the ventral surface of the calcium sensor GCaMP6s-expressing IO in anesthetized mice. A representative calcium imaging recording is shown to demonstrate the feasibility to record IO neuron activity after the surgery. While this is a non-survival surgery and the recordings must be conducted under anesthesia, it avoids damage to life-critical brainstem nuclei and allows conducting large variety of experiments investigating spatiotemporal activity patterns and input integration in the IO. This procedure with modifications could be used for recordings in other, adjacent regions of the ventral brainstem.

In vivo Calcium Imaging in Mouse Inferior Olive

We present a protocol to expose the brainstem of adult mouse from the ventral side. By using a gradient-refractive index lens with a miniature microscope, calcium imaging can be used to examine the activity of inferior olive neural somata in vivo.

In vivo Imágenes de calcio en la aceituna inferior de ratón

Inferior olive (IO), a nucleus in the ventral medulla, is the only source of climbing fibers that form one of the two input pathways entering the ...

Reliable Acquisition of Electroencephalography Data during Simultaneous Electroencephalography and Functional MRI

Simultaneous electroencephalography (EEG) and functional magnetic resonance imaging (fMRI), EEG-fMRI, combines the complementary properties of scalp EEG (good temporal resolution) and fMRI (good spatial resolution) to measure neuronal activity during an electrographic event, through hemodynamic responses known as blood-oxygen-level-dependent (BOLD) changes. It is a non-invasive research tool that is utilized in neuroscience research and is highly beneficial to the clinical community, especially for the management of neurological diseases, provided that proper equipment and protocols are administered during data acquisition. Although recording EEG-fMRI is apparently straightforward, the correct preparation, especially in placing and securing the electrodes, is not only important for safety but is also critical in ensuring the reliability and analyzability of the EEG data obtained. This is also the most experience-demanding part of the preparation. To address these issues, a straightforward protocol that ensures data quality was developed. This article provides a step-by-step guide for acquiring reliable EEG data during EEG-fMRI using this protocol that utilizes readily available medical products. The presented protocol can be adapted to different applications of EEG-fMRI in research and clinical settings, and may be beneficial to both inexperienced and expert operators.

This article provides a straightforward protocol for acquiring good quality electroencephalography (EEG) data during simultaneous EEG and functional magnetic resonance imaging by utilizing readily available medical products.

Adquisición confiable de datos de electroencefalografía durante la electroencefalografía simultánea y la RMN funcional

Simultaneous electroencephalography (EEG) and functional magnetic resonance imaging (fMRI), EEG-fMRI, combines the complementary properties of scalp EEG ...