نود أن نعترف أولغا Garaschuk لإجراء مناقشات مفيدة حول الاستفادة المثلى من تحميل الجزء الأكبر من مؤشرات الكالسيوم.

<ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> إعداد مؤشر الكالسيوم الفلورسنت الحل. نستخدم الأصباغ استر acetoxymethyl ، أو الأصباغ AM قصيرة. هذه الأصباغ التي يمكن اتخاذها من قبل الخلايا عند حقنها خارج الخلية. نستخدم عادة ولاية أوريغون والخضر BAPTA - 1 - Fluo صباحا أو 04:00 في تركيز 1 ملم. ويمكن الحصول على الأصباغ صباحا من Invitrogen 50 ميكروغرام في قارورة. نحن نستعد لأول مرة حل 20 ٪ من Pluronic F - 127 في DMSO. نحن الحار الحل قبل استخدامها كل يوم حتى واضحة لها. نحن بحل مؤشر الكالسيوم F-127/DMSO Pluronic 20 ٪ لمدة 15-20 دقيقة إلى تركيز من 10 ملم. ثم أننا تمييع الحل ل1MM في محلول يحتوي ، في ملي : 150 كلوريد الصوديوم ، 2.5 بوكل ، 10 Hepes ، ودرجة الحموضة 7.4. بالإضافة إلى ذلك ، وعادة ما يتم Sulforhodamine 100 ميكرومتر من 101 إلى النجمية التسمية وتصور ماصة خلال الحقن. نحن تصفية حل قبل الحقن مع فلتر ميكرون 0.49 الطرد المركزي (Stosiek وآخرون ، 2003 ؛. Garaschuk وآخرون ، 2006). </li><li style=";text-align:right;direction:rtl"> زرع نافذة الجمجمة على المنطقة المراد تصويره. لقد كنا <a href="http://jove.com/index/details.stp?ID=680" target="_blank">وصفت في النهج العام إن الرب</a> ، ولكن التعديلات التالية مهمة لحقن الصبغة الكالسيوم : جعل أصغر حج القحف من 2 ملم وقطرها على المنطقة القشرية ذات الاهتمام ، مع الحرص الشديد على عدم الإضرار الجافية الكامنة. تأمين ساترة في المكان خلال حج القحف ، ولكن جزئيا فقط تغطية حج القحف ، مما يترك فجوة صغيرة بين حافة حج القحف وساترة الزجاج للسماح للغرفة ماصة للدخول في القشرة بشكل غير مباشر. قبل الحقن ، وجعل بئر ضحل مع الاسمنت الأسنان حول حج القحف. ينبغي أن تمتد بشكل جيد كما هو الحال في اتجاه منقاري وتكون ضحلة بما يكفي للسماح للmicropipette الإدراج للحقن. </li><li style=";text-align:right;direction:rtl"> نقل الماوس نقله إلى مرحلة المجهر ويستوقف في الرأس ، كما هو مبين من خلال موقعنا <a href="http://jove.com/index/details.stp?ID=678" target="_blank">على تصوير الفيديو تدفق الدم</a> . </li><li style=";text-align:right;direction:rtl"> سحب ماصات microelectrode الزجاج ليبلغ قطرها غيض الغلة 2-4 ميغا أوم في المقاومة ، وذلك باستخدام ماصة مجتذب. تحميل الصبغة مؤشر على مزيج من الكالسيوم والزجاج باستخدام ماصة microsyringe. باستخدام micromanipulator ، وانخفاض micropipette برفق على سطح الدماغ باستخدام 4X الهدف الذي وضعه ناعما ثم تحت الهدف 40X. اختيار موقع مناسب للحقن ، مثل أن هناك عدد قليل أو لا الأوعية الدموية المغطي لحجب التصوير. باستخدام اثنين من الفوتون المجهري ، وغيض من ماصة هو تصور والمتقدمة ببطء داخل القشرة ، إلى عمق 200 ميكرومتر تحت الجافية. ثم ، وضغط حقن صبغة الخليط في 10 PSI لمدة 1 دقيقة باستخدام Picospritzer. نقدم عادة 2-4 حقن الصبغة AM مزيج من الكالسيوم ، وفصلها في الفضاء بنحو 200-300 ميكرون. هذا النهج تحميل الجزء الأكبر من الحقن تداخل قليلا يضمن بشكل كاف الملون مساحة كبيرة مثل 600 × 600 ميكرون للتصوير الكالسيوم. يبدأ التصوير 1 بعد ساعة من الحقن من هذا الخليط صباحا صباغة ، للسماح لصبغ لنشر وسيتم تناولها من خلال الخلايا العصبية. </li><li style=";text-align:right;direction:rtl"> أداء داخل الجسم الحي اثنين الفوتون التصوير مع العرف مجهر ثنائي الفوتون باستخدام TI - الياقوت الحرباء ليزر (ضبطها ل800-880 نانومتر) والمرايا كامبريدج الجلفانومتر التكنولوجيا. علينا الحصول على الصور باستخدام برامج ScanImage ضعت في مختبر للكاريل سفوبودا (Pologruto وآخرون ، 2003). الليزر السلطة هي عادة أقل بكثير من 70 ميغاواط في العينة. يتم الحصول على الصور الحقل بأكمله باستخدام إما 20X (0.95 NA) أو 40X (0.8 NA) أهداف أوليمبوس ، 1،95-15،63 في لقطة في الثانية (512 × 256 بكسل إلى 256 × 64 بكسل). يتم الحصول على خط بفحص 500 هرتز. خلال التصوير ، ويتم الاحتفاظ الحيوانات تحت التخدير isoflurane الخفيفة (0،6-0،9 ٪) ، ويتم الاحتفاظ أيضا عند 37 درجة مئوية درجة الحرارة باستخدام جهاز هارفارد جهاز التحكم. هو الحرص على الحفاظ على معدل التنفس الحيوانات أقرب إلى 100 الأنفاس / دقيقة قدر الإمكان. هذا المستوى من استخدام التخدير هو أدنى مستوى الذي يشل هذا الحيوان ، ولكن لا يزال يسمح النشاط العفوي ليتم تسجيلها. </li></ol>

<ol>
	<li>Garaschuk, O., Milos, R. I., 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=Targeted+bulk-loading+of+fluorescent+indicators+for+two-photon+brain+imaging+in+vivo.">Targeted bulk-loading of fluorescent indicators for two-photon brain imaging in vivo.</a> Nat Protoc. 1, (2006).</li><li>Stosiek, C., Garaschuk, O., Holthoff, K., 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=In-vivo+two-photon+calcium+imaging+of+neuronal+networks.">In-vivo two-photon calcium imaging of neuronal networks.</a> Proc Natl Acad Sci USA. 100, 7319-7324 (2003).</li><li>Pologruto, T. A., Sabatini, B. L., 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=ScanImage:+flexible+software+for+operating+laser+scanning+microscopes.&amp;nbsp;&amp;nbsp;+Biomed+Eng+Online+2:13.">ScanImage: flexible software for operating laser scanning microscopes.&amp;nbsp;&amp;nbsp; Biomed Eng Online 2:13.</a> Biomed Eng Online. 2, (2003).</li></ol>

ميزة هذا الأسلوب أكثر من التسجيلات الكهربية هو أنه أقل الغازية ويسمح للتسجيلات نشاط الخلايا العصبية في شبكات كثيفة العصبية. عندما تفعل هذا الإجراء فإنه من المهم أن نتذكر أن تأخذ الحذر الشديد عند أداء حج القحف لا يلحق الضرر الجافية. حتى كمية صغيرة من نزيف تحت الجافية مع غامضة إلى...

<table><tbody><tr><td>Oregon Green BAPTA1-AM</td><td>Reagent</td><td>Invitrogen</td><td>O-6807</td><td></td></tr><tr><td>Fluo 4- AM</td><td>Reagent</td><td>Invitrogen</td><td>F-14201</td><td></td></tr><tr><td>Sulforhodamine 101</td><td>Reagent</td><td>Invitrogen</td><td>S-359</td><td></td></tr><tr><td>Pluronic F-127 in DMSO</td><td>Reagent</td><td>Invitrogen</td><td>P-3000MP</td><td></td></tr><tr><td>Centrifugal Filter (0.45 micron)</td><td>Reagent</td><td>EMD Millipore</td><td>UFC3 0HV 0S</td><td></td></tr></tbody></table>

in vivo 2-photon calcium imaging in layer 2/3 of mice

لفهم ديناميات شبكة من رقائق في القشرة المخية الحديثة ، فإنه من الضروري في نفس الوقت لتسجيل نشاط عدد كبير من الخلايا العصبية. داخل الجسم الحي اثنين الفوتون التصوير الكالسيوم هو الأسلوب الوحيد الذي يسمح احد لتسجيل نشاط الخلايا العصبية من سكان كثيفة مع وحيدة الخلية القرار. طريقة تتمثل في زرع نافذة التصوير الجمجمة ، حقن صبغة الفلورسنت مؤشر الكالسيوم التي يمكن اتخاذها من قبل أعداد كبيرة من الخلايا العصبية وأخيرا تسجيل نشاط الخلايا العصبية مع مرور الوقت باستخدام التصوير الكالسيوم في الجسم الحي ، two المجهر الفوتون. شارك في حقن الأصباغ نجمية محددة يسمح احد لتمييز الخلايا العصبية من الخلايا النجمية. يمكن تنفيذ هذا الاسلوب في التعبير عن جزيئات الفلورسنت الفئران في مجموعات سكانية فرعية معينة من الخلايا العصبية لفهم أفضل للتفاعلات شبكة من مجموعات مختلفة من الخلايا.

Watch this Scientific Journal Video about In Vivo 2-Photon Calcium Imaging in Layer 2/3 of Mice at JoVE.com

In Vivo 2-Photon Calcium Imaging in Layer 2/3 of Mice

لفهم ديناميات شبكة من رقائق في القشرة المخية الحديثة ، فإنه من الضروري في نفس الوقت لتسجيل نشاط عدد كبير من الخلايا العصبية. داخل الجسم الحي اثنين الفوتون التصوير الكالسيوم هو الأسلوب الوحيد الذي يسمح احد لتسجيل نشاط الخلايا العصبية من سكان كثيفة مع وحيدة الخلية القرار.

في فيفو 2 فوتون التصوير الكالسيوم في طبقة 2 / 3 من الفئران

To understand network dynamics of microcircuits in the neocortex, it is essential to simultaneously record the activity of a large number of neurons .  ...

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Research

JoVE Journal

Biology

28.2K Views.  University of California, Los Angeles. لفهم ديناميات شبكة من رقائق في القشرة المخية الحديثة ، فإنه من الضروري في نفس الوقت لتسجيل نشاط عدد كبير من الخلايا العصبية. داخل الجسم الحي اثنين الفوتون التصوير الكالسيوم هو الأسلوب الوحيد الذي يسمح احد لتسجيل نشاط الخلايا العصبية من سكان كثيفة مع وحيدة الخلي...

Video: In Vivo 2-Photon Calcium Imaging in Layer 2/3 of Mice

Two-photon Calcium Imaging in Mice Navigating a Virtual Reality Environment

In recent years, two-photon imaging has become an invaluable tool in neuroscience, as it allows for chronic measurement of the activity of genetically identified cells during behavior1-6. Here we describe methods to perform two-photon imaging in mouse cortex while the animal navigates a virtual reality environment. We focus on the aspects of the experimental procedures that are key to imaging in a behaving animal in a brightly lit virtual environment. The key problems that arise in this experimental setup that we here address are: minimizing brain motion related artifacts, minimizing light leak from the virtual reality projection system, and minimizing laser induced tissue damage. We also provide sample software to control the virtual reality environment and to do pupil tracking. With these procedures and resources it should be possible to convert a conventional two-photon microscope for use in behaving mice.

Here we describe the experimental procedures involved in two-photon imaging of mouse cortex during behavior in a virtual reality environment.

ثنائي الفوتون التصوير الكالسيوم في الفئران التنقل إلى هذا الشخص واقع البيئة

In recent years, two-photon imaging has become an invaluable tool in neuroscience, as it allows for chronic measurement of the activity of genetically ...

Behavior

Multi-layer Cortical Ca2+ Imaging in Freely Moving Mice with Prism Probes and Miniaturized Fluorescence Microscopy

In vivo circuit and cellular level functional imaging is a critical tool for understanding the brain in action. High resolution imaging of mouse cortical neurons with two-photon microscopy has provided unique insights into cortical structure, function and plasticity. However, these studies are limited to head fixed animals, greatly reducing the behavioral complexity available for study. In this paper, we describe a procedure for performing chronic fluorescence microscopy with cellular-resolution across multiple cortical layers in freely behaving mice. We used an integrated miniaturized fluorescence microscope paired with an implanted prism probe to simultaneously visualize and record the calcium dynamics of hundreds of neurons across multiple layers of the somatosensory cortex as the mouse engaged in a novel object exploration task, over several days. This technique can be adapted to other brain regions in different animal species for other behavioral paradigms.

Multi-layer Cortical Ca2+ Imaging in Freely Moving Mice with Prism Probes and Miniaturized Fluorescence Microscopy

Here, we present a procedure for performing large-scale Ca2+ imaging with cellular-resolution across multiple cortical layers in freely moving mice. Hundreds of active cells can be observed simultaneously using a miniature, head-mounted microscope coupled with an implanted prism probe.

متعدد الطبقات الكالسيوم الكالسيوم<sup&gt; 2+</sup&gt; التصوير في الفئران تتحرك بحرية مع تحقيقات بريزم والمصور المجهري مضان

In vivo circuit and cellular level functional imaging is a critical tool for understanding the brain in action. High resolution imaging of mouse cortical ...

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety of brain disorders1,2. To better understand the mechanisms by which brain circuits react to an animal's experience requires the ability to monitor the experience-dependent molecular changes in a given set of neurons, over a prolonged period of time, in the live animal. While experience and associated neural activity is known to trigger gene expression changes in neurons1,2, most of the methods to detect such changes do not allow repeated observation of the same neurons over multiple days or do not have sufficient resolution to observe individual neurons3,4. Here, we describe a method that combines in vivo two-photon microscopy with a genetically encoded fluorescent reporter to track experience-dependent gene expression changes in individual cortical neurons over the course of day-to-day experience. 
One of the well-established experience-dependent genes is Activity-regulated cytoskeletal associated protein (Arc)5,6. The transcription of Arc is rapidly and highly induced by intensified neuronal activity3, and its protein product regulates the endocytosis of glutamate receptors and long-term synaptic plasticity7. The expression of Arc has been widely used as a molecular marker to map neuronal circuits involved in specific behaviors3. In most of those studies, Arc expression was detected by in situ hybridization or immunohistochemistry in fixed brain sections. Although those methods revealed that the expression of Arc was localized to a subset of excitatory neurons after behavioral experience, how the cellular patterns of Arc expression might change with multiple episodes of repeated or distinctive experiences over days was not investigated. 
In vivo two-photon microscopy offers a powerful way to examine experience-dependent cellular changes in the living brain8,9. To enable the examination of Arc expression in live neurons by two-photon microscopy, we previously generated a knock-in mouse line in which a GFP reporter is placed under the control of the endogenous Arc promoter10. This protocol describes the surgical preparations and imaging procedures for tracking experience-dependent Arc-GFP expression patterns in neuronal ensembles in the live animal. In this method, chronic cranial windows were first implanted in Arc-GFP mice over the cortical regions of interest. Those animals were then repeatedly imaged by two-photon microscopy after desired behavioral paradigms over the course of several days. This method may be generally applicable to animals carrying other fluorescent reporters of experience-dependent molecular changes4.

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

Experience-dependent molecular changes in neurons are essential for the brain's ability to adapt in response to behavioral challenges. An in vivo two-photon imaging method is described here that allows the tracking of such molecular changes in individual cortical neurons through genetically encoded reporters.

في الجسم الحي ثنائي الفوتون التصوير من الخبرة التي تعتمد على التغيرات الجزيئية في الخلايا العصبية القشرية

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety ...

Neuroscience

Longitudinal Two-Photon Imaging of Dorsal Hippocampal CA1 in Live Mice

Two-photon microscopy is a fundamental tool for neuroscience as it permits investigation of the brain of live animals at spatial scales ranging from subcellular to network levels and at temporal scales from milliseconds to weeks. In addition, two-photon imaging can be combined with a variety of behavioral tasks to explore the causal relationships between brain function and behavior. However, in mammals, limited penetration and scattering of light have limited two-photon intravital imaging mostly to superficial brain regions, thus precluding longitudinal investigation of deep-brain areas such as the hippocampus. The hippocampus is involved in spatial navigation and episodic memory and is a long-standing model used to study cellular as well as cognitive processes important for learning and recall, both in health and disease. Here, a preparation that enables chronic optical access to the dorsal hippocampus in living mice is detailed. This preparation can be combined with two-photon optical imaging at cellular and subcellular resolution in head fixed, anesthetized live mice over several weeks. These techniques enable repeated imaging of neuronal structure or activity-evoked plasticity in tens to hundreds of neurons in the dorsal hippocampal CA1. Furthermore, this chronic preparation can be used in combination with other techniques such as micro-endoscopy, head-mounted wide field microscopy or three-photon microscopy, thus greatly expanding the toolbox to study cellular and network processes involved in learning and memory.

This method describes a chronic preparation that allows optical access to the hippocampus of living mice. This preparation can be used to perform longitudinal optical imaging of neuronal structural plasticity and activity-evoked cellular plasticity over a period of several weeks.

التصوير الطولي اثنين من الفوتون من CA1 فرس النهر الأورسيال في الفئران الحية

Two-photon microscopy is a fundamental tool for neuroscience as it permits investigation of the brain of live animals at spatial scales ranging from ...

Two-Photon in vivo Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull Preparation

In the mammalian cortex, neurons form extremely complicated networks and exchange information at synapses. Changes in synaptic strength, as well as addition/removal of synapses, occur in an experience-dependent manner, providing the structural foundation of neuronal plasticity. As postsynaptic components of the most excitatory synapses in the cortex, dendritic spines are considered to be a good proxy of synapses. Taking advantages of mouse genetics and fluorescent labeling techniques, individual neurons and their synaptic structures can be labeled in the intact brain. Here we introduce a transcranial imaging protocol using two-photon laser scanning microscopy to follow fluorescently labeled postsynaptic dendritic spines over time in vivo. This protocol utilizes a thinned-skull preparation, which keeps the skull intact and avoids inflammatory effects caused by exposure of the meninges and the cortex. Therefore, images can be acquired immediately after surgery is performed. The experimental procedure can be performed repetitively over various time intervals ranging from hours to years. The application of this preparation can also be expanded to investigate different cortical regions and layers, as well as other cell types, under physiological and pathological conditions.

Two-Photon in vivo Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull Preparation

Time-lapse imaging in the living animal provides valuable information on structural reorganization in the intact brain. Here, we introduce a thinned-skull preparation that allows transcranial imaging of fluorescently labeled synaptic structures in the living mouse cortex by two-photon microscopy.

ثنائي الفوتون<em&gt; في الجسم الحي</em&gt; التصوير من العمود الفقري شجيري في اللحاء باستخدام الماوس الجمجمة ضعفت إعداد

In the mammalian cortex, neurons form extremely complicated networks and exchange information at synapses. Changes in synaptic strength, as well as ...

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 ...

In Vivo Chronic Two-Photon Imaging of Microglia in the Mouse Hippocampus

Microglia, the only immune cells resident in the brain, actively participate in neural circuit maintenance by modifying synapses and neuronal excitability. Recent studies have revealed the differential gene expression and functional heterogeneity of microglia in different brain regions. The unique functions of the hippocampal neural network in learning and memory may be associated with the active roles of microglia in synapse remodeling. However, inflammatory responses induced by surgical procedures have been problematic in the two-photon microscopic analysis of hippocampal microglia. Here, a method is presented that enables the chronic observation of microglia in all layers of the hippocampal CA1 through an imaging window. This method allows the analysis of morphological changes in microglial processes for more than 1 month. Long-term and high-resolution imaging of the resting microglia requires minimally invasive surgical procedures, appropriate objective lens selection, and optimized imaging techniques. The transient inflammatory response of hippocampal microglia may prevent imaging immediately after surgery, but the microglia restore their quiescent morphology within a few weeks. Furthermore, imaging neurons simultaneously with microglia allows us to analyze the interactions of multiple cell types in the hippocampus. This technique may provide essential information about microglial function in the hippocampus.

In Vivo Chronic Two-Photon Imaging of Microglia in the Mouse Hippocampus

This paper describes a method for chronic in vivo observation of the resting microglia in the mouse hippocampal CA1 using precisely controlled surgery and two-photon microscopy.

في الجسم الحي التصوير المزمن ثنائي الفوتون للخلايا الدبقية الصغيرة في قرن آمون الفأر

Microglia, the only immune cells resident in the brain, actively participate in neural circuit maintenance by modifying synapses and neuronal ...

Simultaneous Imaging of Microglial Dynamics and Neuronal Activity in Awake Mice

Since brain functions are under the continuous influence of the signals derived from peripheral tissues, it is critical to elucidate how glial cells in the brain sense various biological conditions in the periphery and transmit the signals to neurons. Microglia, immune cells in the brain, are involved in synaptic development and plasticity. Therefore, the contribution of microglia to neural circuit construction in response to the internal state of the body should be tested critically by intravital imaging of the relationship between microglial dynamics and neuronal activity.
Here, we describe a technique for the simultaneous imaging of microglial dynamics and neuronal activity in awake mice. Adeno-associated virus encoding R-CaMP, a gene-encoded calcium indicator of red fluorescence protein, was injected into layer 2/3 of the primary visual cortex in CX3CR1-EGFP transgenic mice expressing EGFP in microglia. After viral injection, a cranial window was installed onto the brain surface of the injected region. In vivo two-photon imaging in awake mice 4 weeks after the surgery demonstrated that neural activity and microglial dynamics could be recorded simultaneously at the sub-second temporal resolution. This technique can uncover the coordination between microglial dynamics and neuronal activity, with the former responding to peripheral immunological states and the latter encoding the internal brain states.

Here, we describe a protocol combining adeno-associated virus injection with cranial window implantation for simultaneous imaging of microglial dynamics and neuronal activity in awake mice.

التصوير المتزامن لديناميات الخلايا الدبقية الصغيرة والنشاط العصبي في الفئران المستيقظة

Since brain functions are under the continuous influence of the signals derived from peripheral tissues, it is critical to elucidate how glial cells in ...

In Vivo Wide-Field and Two-Photon Calcium Imaging from a Mouse Using a Large Cranial Window

Wide-field calcium imaging from the mouse&#39;s neocortex allows one to observe cortex-wide neural activity related to various brain functions. On the other hand, two-photon imaging can resolve the activity of local neural circuits at the single-cell level. It is critical to make a large cranial window to perform multiple-scale analysis using both imaging techniques in the same mouse. To achieve this, one must remove a large section of the skull and cover the exposed cortical surface with transparent materials. Previously, glass skulls and polymer-based cranial windows have been developed for this purpose, but these materials are not easily fabricated. The present protocol describes a simple method for making a large cranial window consisting of commercially available polyvinylidene chloride (PVDC) wrapping film, a transparent silicone plug, and a cover glass. For imaging the dorsal surface of an entire hemisphere, the window size was approximately 6 x 3 mm2. Severe brain vibrations were not observed regardless of such a large window. Importantly, the condition of the brain surface did not deteriorate for more than one month. Wide-field imaging of a mouse expressing a genetically-encoded calcium indicator (GECI), GCaMP6f, specifically in astrocytes, revealed synchronized responses in a few millimeters. Two-photon imaging of the same mouse showed prominent calcium responses in individual astrocytes over several seconds. Furthermore, a thin layer of an adeno-associated virus was applied to the PVDC film and successfully expressed GECI in cortical neurons over the cranial window. This technique is reliable and cost-effective for making a large cranial window and facilitates the investigation of the neural and glial dynamics and their interactions during behavior at the macroscopic and microscopic levels.

In Vivo Wide-Field and Two-Photon Calcium Imaging from a Mouse Using a Large Cranial Window

The present protocol describes making a large (6 x 3 mm2) cranial window using food wrap, transparent silicone, and cover glass. This cranial window allows in vivo wide-field and two-photon calcium imaging experiments in the same mouse.

في الجسم الحي تصوير الكالسيوم واسع المجال وثنائي الفوتون من فأر باستخدام نافذة جمجمة كبيرة

Wide-field calcium imaging from the mouse&#39;s neocortex allows one to observe cortex-wide neural activity related to various brain functions. On the ...

في فيفو 2 فوتون التصوير الكالسيوم في طبقة 2 / 3 من الفئران

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