ويدعم العمل في المختبر بواسطة Organisatie Nederlandse Onderzoek Wetenschappelijk voor (NWO) (917.10.372) إلى RMM. ويتبع دينار لبرنامج الاتحاد الاوروبي FP7 BrainTrain ( <a href="http://www.brain-train.nl">www.brain - train.nl</a> ). نشكر بيتر لورنس ، وسابين Baljon شميتز (سواء CNCR ، VU جامعة أمستردام) للصور من مجموعة multielectrode FP الطول الموجي والثقافات العصبية المستخدمة في تسلسل الفيديو.

<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>

برعاية LaVision بيوتيك GMBH (بيليفيلد الالماني) ورسوم تقديم هذه المادة المخطوطة.

الأساليب نظهر هنا تظهر بروتوكولات مناسبة لتصوير الكالسيوم ديناميات شبكة من خلايا محددة ضمن تطوير شبكات القشرية وقرن آمون في الماوس ، وكذلك في الدماغ الفئران. هذه الأساليب تقديم القرار المكاني الأمثل لتصور شبكة محلية في وقت واحد من الخلية somas لقياس النشاط suprathreshold. ويمكن تغيير القرار الزمني للنشاط الشبكة ، اعتمادا على الإطار إعدادات اقتناء كاميرا CCD ، لتحسين إشارة : قياسات الضوضاء لفترة طويلة أحداث suprathreshold المدة ، والتي توجد عادة في الجهاز العصبي النامي. لا تقتصر هذه البروتوكولات للشبكات قرن آمون ولكن أيضا الخلايا القشرية التسمية في جميع أنحاء النظام العصبي النامي. الحد من هذه الطريقة هي التي يمكن تسجيلها فقط suprathreshold النشاط.

functional calcium imaging in developing cortical networks

وثمة نمط السمة المميزة لنشاط الجهاز العصبي في وضع غير عفوية ، نشاط الشبكة متزامنة. وقد لوحظ نشاط سليمة متزامنة في الحبل الشوكي ، الدماغ ، اللحاء ، الشبكية وفصلها الاستعدادات ثقافة العصبية. خلال فترات من النشاط العفوي ، يزيل الاستقطاب الخلايا العصبية لاطلاق النار واحد أو رشقات نارية من إمكانات العمل ، وتفعيل قنوات أيونات كثيرة. الاستقطاب تنشيط قنوات الكالسيوم الجهد على بوابات التشعبات والأشواك التي تتوسط تدفق الكالسيوم. وقد تم قياس النشاط الكهربائي المتزامن للغاية من الشبكات العصبية المحلية باستخدام أقطاب الحقل. تمكن هذه التقنية ارتفاع معدلات أخذ العينات الزمانية المكانية ولكن القرار يرجع إلى انخفاض قراءة متكاملة من الخلايا العصبية في واحدة متعددة القطب. قرار وحيد الخلية من نشاط الخلايا العصبية من الممكن استخدام التصحيح ، المشبك على الخلايا العصبية الكهربية واحدة لقياس النشاط اطلاق النار. ومع ذلك ، هو محدود القدرة على قياس من شبكة لعدد الخلايا العصبية ليالي مصححةimultaneously ، وعادة ما يكون احد أو اثنين فقط الخلايا العصبية. وقد مكن استخدام الكالسيوم التي تعتمد على الأصباغ الفلورية مؤشر قياس النشاط متزامنة عبر شبكة من الخلايا. هذا الأسلوب يعطي كلا قرار مكانية عالية وأخذ العينات الزمنية كافية لتسجيل النشاط العفوي للشبكة النامية. ومن السمات الرئيسية لتشكيل شبكات حديثا القشرية والحصين خلال التنمية قبل وبعد الولادة المبكرة هي عفوية ، وتزامن نشاط الخلايا العصبية (كاتس و Shatz ، 1996 ؛ Khaziphov وLuhmann ، 2006). ويعتقد ان هذا النشاط شبكة مترابطة لأنها ضرورية لتوليد الدوائر الوظيفية في الجهاز العصبي النامي (سبيتزر ، 2006). الرئيسيات في كل من الدماغ والقوارض ، ويلاحظ والكهربائية في وقت مبكر وموجات شبكة الكالسيوم قبل وبعد الولادة ، والمجراة في المختبر (Adelsberger وآخرون ، 2005 ؛. Garaschuk وآخرون ، 2000 ؛. Lamblin وآخرون ، 1999). هذه أنماط النشاط في وقت مبكر ، والتي هي معروفة للسيطرة على عدةالعمليات التنموية بما في ذلك الخلايا العصبية synaptogenesis ، والتمايز واللدونة (راكيتش وKomuro ، 1995 ؛. سبيتزر وآخرون ، 2004) هي من الأهمية الحاسمة للتنمية الصحيحة ونضوج الدوائر القشرية. إن الرب في هذا الفيديو ، ونحن لشرح الطرق المستخدمة في النشاط صورة عفوية في تطوير شبكات القشرية. الكالسيوم المؤشرات الحساسة ، مثل Fura استر 02:00 ، منتشر عبر غشاء الخلية داخل الخلايا حيث يشق على النشاط استريز AM استرات لمغادرة شكل خلية impermeant صبغة المؤشر. شكل impermeant مؤشر مجموعات حمض الكربوكسيلية التي هي قادرة على الكشف عن ثم ربط أيونات الكالسيوم intracellularly.. يتم تبديل عابر في مضان من الكالسيوم صبغة حساسة على الربط إلى الكالسيوم. وتستخدم تقنيات التصوير واحد أو متعدد الفوتون لقياس التغيير في الفوتونات المنبعثة من الصبغة ، وتبين بالتالي تغيير في الكالسيوم داخل الخلايا. وعلاوة على ذلك ، فإن هذه الكالسيوم دويمكن الجمع بين المؤشرات ependent مع علامات أخرى للتحقيق الفلورسنت أنواع الخلايا النشطة داخل الشبكة.

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

Functional Calcium Imaging in Developing Cortical Networks

ويمكن قياس النشاط العفوي لتطوير شبكات الخلايا العصبية باستخدام AM - استر أشكال الأصباغ مؤشر الكالسيوم الحساسة. تم الكشف عن التغييرات في الكالسيوم بين الخلايا ، مما يدل تنشيط الخلايا العصبية ، والتغييرات في مضان عابرة مع مؤشر واحد أو اثنين الفوتون التصوير. ويمكن تكييف هذا البروتوكول لمجموعة من الشبكات التي تعتمد على الخلايا العصبية تنمويا<em&gt; في المختبر</em&gt;.

أسلوب التصوير الكالسيوم في تطوير شبكات القشرية

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 Views.  VU University, Amsterdam. ويمكن قياس النشاط العفوي لتطوير شبكات الخلايا العصبية باستخدام AM - استر أشكال الأصباغ مؤشر الكالسيوم الحساسة. تم الكشف عن التغييرات في الكالسيوم بين الخلايا ، مما يدل تنشيط الخلايا العصبية ، والتغييرات في مضان عابرة مع مؤشر واحد أو اثنين الفوتون التصوير. ويمكن ?...

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.

تعيين وظيفي مع MEG الفورية وEEG

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

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.

الوقت الفاصل بين التصوير حية من خلايا ذات صلة Clonally السلف العصبية في الدماغ المقدم اسماك الزرد النامية

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.

الكالسيوم في الجسم الحي التصوير الخلايا العصبية في C. ايليجانس

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.

سريع الصغرى، الرحلان الشاردي من الغلوتامات وGABA: أداة مفيدة لبحث تكامل متشابك

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.

التصوير حية من الانقسام في تطوير الجنينية الماوس اللحاء

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.

في فيفو الدماغ أسلوب التصوير النهج القائم على الكالسيوم إضاءة الحيوية المؤشر 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 ...

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.

التسجيلات المتزامنة الإمكانات الميدانية المحلية القشرية، ورسم القلب، اليكتروميوجرام وإيقاع من الفئران تتحرك بحرية في التنفس

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.

في فيفو اثنين-فوتون تصوير الخلايا العصبية القشرية في الفئران حديثي الولادة

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.

في الجسم الحي تصوير الكالسيوم في الزيتون السفلي الماوس

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.

اكتساب موثوق لبيانات تخطيط كهربية الدماغ أثناء التصوير الكهربائي المتزامن والتصوير بالرنين المغناطيسي الوظيفي

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