Bu çalışma Samsung Bilim ve Teknoloji Vakfı'nın (Proje Numarası SSTF-BA1801-10) hibeleri ile desteklendi.

<ol>
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Yazarın açıklayacak bir şeyi yok.

Burada anlattığımız gibi amigdala gibi daha derin beyin bölgelerinde kafaya monte minyatür mikroskopi ile in vivo optik kalsiyum görüntülemede başarılı olmak için ustaca cerrahi teknikleri şarttır. Bu nedenle, bu protokol temel ataşman ve GRIN lens implantasyonunun optimize edilmiş cerrahi süreçleri için bir kılavuz sağlasa da, kritik adımlar için ek optimizasyon süreçleri gerekebilir. Protokol bölümünde belirtildiği gibi, cerrahide amigdala koordinatları, taban plakası ek adımında hava ...

Kalsiyum her yerde bulunan ikinci bir habercidir, hemen hemen her hücresel işlevde çok önemli bir rol oynar8. Nöronlarda, eylem potansiyeli ateşleme ve sinaptik giriş hücre içi serbest [Ca2+]9,10'unhızlıdeğişmesineneden olur. Bu nedenle, kalsiyum geçicilerini izlemek nöronal aktiviteyi izlemek için bir fırsat sağlar. GECI'ler, tanımlanmış hücre popülasyonlarında ve hücre içi bölmelerde[Ca 2+] izlenmesine izin veren güçlü araçlardır11,12. Protein bazlı kalsiyum göstergesinin birçok farklı türü arasında, tek bir GFP molekülü13'edayanan bir Ca2+ probu olan GCaMP, en optimize edilmiş ve bu nedenle yaygın olarak kullanılan GECI'dir. Birden fazla mühendislik turu sayesinde, GCaMP'nin bir dizi çeşidigeliştirilmiştir 12,14,15,16. Son geliştirilen GCaMPP'lerden birini kullanıyoruz, GCaMP7b, bu protokolde16. GCaMP sensörleri, geliştirme sırasında Ca2+ geçicilerinin görüntülenmesi, belirli bir kortikal tabakada in vivo görüntüleme18, motor görev öğreniminde devre dinamiklerinin ölçümü19ve hipokampus ve amigdala 20,21'deki ilişkisel korku hafızası ile ilgili hücre topluluğu aktivitesinin görüntülenmesi gibi bir dizi model organizmadanöraldevre fonksiyonlarının incelenmesine büyük katkıda bulunmuştur.
GECI'lerin optik görüntülemesinin çeşitli avantajları vardır22. Genetik kodlama, GECI'lerin genetik profil veya belirli anatomik bağlantı kalıpları ile tanımlanan belirli bir hücre alt kümesinde uzun süreli bir süre boyunca saptanmasını sağlar. Optik görüntüleme, canlı hayvanlarda yüzlerce ila binlerce nöronun in vivo kronik eşzamanlı izlenmesini sağlar. Kafa montajlı minyatür floresan mikroskoplar21, 23,24,25ile serbestçe davranan farelerin beynindeki GECI'lerin in vivo görüntüleme ve analizi için birkaç optik görüntüleme sistemi geliştirilmiştir. GECI'lere dayanan in vivo optik görüntüleme tekniğine, GRIN lense ve kafaya monte minyatür mikroskobun nöral devre aktivitesi ve davranışı arasındaki bağlantıyı incelemek için güçlü bir araç olmasına rağmen, bu teknolojinin amigdalaya uygulanması, GÖRÜNTÜ alma kalitesini ciddi şekilde azaltan hareket yapıtlarına neden olmadan amigdaladaki GECI'leri ifade eden hücrelere GRIN lensin hedef alınmasıyla ilgili çeşitli teknik sorunlar nedeniyle zor olmuştur. Bu protokol, amigdalada başarılı in vivo optik kalsiyum görüntüleme için kritik adımlar olan baseplate eki ve GRIN lens implantasyonunun cerrahi prosedürleri için yararlı bir kılavuz sağlamayı amaçlamaktadır. Bu protokol amigdalayı hedeflemesine rağmen, burada açıklanan çoğu prosedür genellikle diğer derin beyin bölgeleri için geçerlidir. Bu protokol belirli bir sisteme (örneğin Inscopix) dayansa da, aynı amaca diğer alternatif sistemlerle kolayca ulaşılabilir.

<table><tbody><tr><td>26G needle</td><td>BD</td><td>302002</td><td>Surgery</td></tr><tr><td>AAV1-Syn-GCaMP7b-WPRE</td><td>Addgene</td><td>104493-AAV1</td><td>Surgery</td></tr><tr><td>AAV2/1-CaMKii&amp;alpha;-GFP</td><td>custom made</td><td></td><td>Surgery</td></tr><tr><td>Acrylic-Dental cement (Ortho-jet Acrylic Pink)</td><td>Lang</td><td>1334-pink</td><td>Surgery &amp;amp; Baseplate Attachment</td></tr><tr><td>Air flow manipulator</td><td>Neurotar</td><td>NTR000253-04</td><td>Baseplate Attachment</td></tr><tr><td>Amoxicillin</td><td>SIGMA</td><td>A8523-5G</td><td>Surgery</td></tr><tr><td>Baseplate</td><td>INSCOPIX</td><td>1050-002192</td><td>Baseplate Attachment</td></tr><tr><td>Baseplate cover</td><td>INSCOPIX</td><td>1050-002193</td><td>Baseplate Attachment</td></tr><tr><td>Behavioral apparatus (chamber)</td><td>Coulbourn Instrument</td><td>Testcage</td><td>Behavior test</td></tr><tr><td>Behavioral apparatus (software)</td><td>Coulbourn Instrument</td><td>Freeze Frame</td><td>Behavior test</td></tr><tr><td>Carbon cage</td><td>Neurotar</td><td>180mm x 70mm</td><td>Baseplate Attachment</td></tr><tr><td>Carprofen</td><td>SIGMA</td><td>PHR1452-1G</td><td>Surgery</td></tr><tr><td>Data processing software</td><td>INSCOPIX</td><td>INSCOPIX Data Processing Software</td><td>Baseplate Attachment &amp;amp; Behavior test</td></tr><tr><td>Dexamethasone</td><td>SIGMA</td><td>D1756-500MG</td><td>Surgery</td></tr><tr><td>Drill</td><td>Seyang</td><td>marathon-4</td><td>Surgery</td></tr><tr><td>Drill bur</td><td>ELA</td><td>US1/2, Shank104</td><td>Surgery</td></tr><tr><td>Glass needle</td><td>WPI</td><td>PG10165-4</td><td>Surgery</td></tr><tr><td>GRIN lens (INSCOPIX Proview Lens Probe)</td><td>INSCOPIX</td><td>1050-002208</td><td>Surgery</td></tr><tr><td>Hamilton Syringe</td><td>Hamilton</td><td>84875</td><td>Surgery</td></tr><tr><td>Head plate</td><td>Neurotar</td><td>Model 5</td><td>Surgery</td></tr><tr><td>Hex-key</td><td>INSCOPIX</td><td>1050-004195</td><td>Baseplate Attachment</td></tr><tr><td>Laptop computer</td><td>Samsung</td><td>NT950XBV</td><td>Surgery &amp;amp; Baseplate Attachment</td></tr><tr><td>Lens holder, Stereotaxic rod (INSCOPIX proview implant kit)</td><td>INSCOPIX</td><td>1050-004223</td><td>Surgery</td></tr><tr><td>Microscope gripper</td><td>INSCOPIX</td><td>1050-002199</td><td>Baseplate Attachment</td></tr><tr><td>Microscope, DAQ software, hardware</td><td>INSCOPIX</td><td>nVista 3.0</td><td>Baseplate Attachment &amp;amp; Behavior test</td></tr><tr><td>Mobile homecage</td><td>Neurotar</td><td>MHC V5</td><td>Baseplate Attachment</td></tr><tr><td>Moterized arm</td><td>Neurostar</td><td>Customized</td><td>Surgery</td></tr><tr><td>Moterized arm software</td><td>Neurostar</td><td>Customized</td><td>Surgery</td></tr><tr><td>NI board</td><td>National instrument</td><td></td><td>Behavior test</td></tr><tr><td>Removable epoxy bond</td><td>WPI</td><td>Kwik-Cast</td><td>Surgery</td></tr><tr><td>Resin cement (Super-bond)</td><td>Sun medical</td><td>Super bond C&amp;amp;B</td><td>Surgery</td></tr><tr><td>Skull screw</td><td>Stoelting</td><td>51457</td><td>Surgery</td></tr><tr><td>Stereotaxic electrode holder</td><td>ASI</td><td>EH-600</td><td>Surgery</td></tr><tr><td>Stereotaxic frame</td><td>Stoelting</td><td>51600</td><td>Surgery</td></tr><tr><td>Stereotaxic manipulator</td><td>Stoelting</td><td>51600</td><td>Baseplate Attachment</td></tr></tbody></table>

successful in vivo calcium imaging with a head-mount miniaturized microscope in the amygdala of freely behaving mouse

In vivo serbestçe hareket eden hayvanlarda nöronal aktivitelerin gerçek zamanlı izlenmesi, nöronal aktiviteyi davranışa bağlamak için temel yaklaşımlardan biridir. Bu amaçla, genetik olarak kodlanmış kalsiyum göstergeleri (GECI'ler), minyatürleştirilmiş bir floresan mikroskobu ve gradyan refraktif indeks (GRIN) lensi kullanarak nöronlardaki kalsiyum geçicilerini tespit eden bir in vivo görüntüleme tekniği geliştirilmiş ve birçok beyinyapısına 1, 2,3,4,5,6başarıyla uygulanmıştır. Bu görüntüleme tekniği özellikle güçlüdür, çünkü genetik olarak tanımlanmış hücre popülasyonlarının birkaç haftaya kadar uzun süreli kronik eşzamanlı görüntülenmesini sağlar. Yararlı olmasına rağmen, bu görüntüleme tekniği, duygusal işlem ve ilişkilendirilebilir korku hafızası için gerekli bir beyin yapısı olan amigdala gibi beynin derinliklerine yer alan beyin yapılarına kolayca uygulanmamıştır7. Görüntüleme tekniğinin amigdalaya uygulanmasını zorlaştırıcı birkaç faktör vardır. Örneğin, hareket eserleri genellikle daha derin beyin bölgelerinde yapılan görüntüleme sırasında daha sık ortaya çıkar, çünkü beynin derinliklerine yerleştirilen bir kafaya monte mikroskop nispeten kararsızdır. Diğer bir sorun ise lateral ventrikülün implante grin lense yakın konumlandırılması ve solunum sırasındaki hareketinin kolayca düzeltilemeyen son derece düzensiz hareket yapıtlarına neden olmasıdır, bu da kararlı bir görüntüleme görünümü oluşturmayı zorlaştırır. Ayrıca, amigdaladaki hücreler genellikle dinlenme veya uyuşturulma durumunda sessiz olduğundan, daha sonraki görüntüleme için temelleme prosedürü sırasında amigdalada GECI'yi ifade eden hedef hücreleri bulmak ve odaklamak zordur. Bu protokol, bu kadar derin bir beyin bölgesinde başarılı in vivo kalsiyum görüntüleme için kafa montajlı minyatür mikroskop ile amigdaladaki GECI'yi ifade eden hücrelerin nasıl verimli bir şekilde hedeflenerek hedeflendirılacağına dair yararlı bir kılavuz sağlar. Bu protokolün belirli bir sisteme (örneğin, Inscopix) dayandığı, ancak bununla sınırlı olmadığı belirtilmiştir.

GRIN lens implantasyonunun doğrulanması Taban plakasını çimentolama ile beyne kronik olarak takmadan önce, GRIN lens implantasyonunun doğrulanması gerekir. Başarılı lens implantasyonu olan hayvanlarda, hem GCaMP ekspresyasyon hücreleri hem de kan damarları, mikroskopun objektif lensi ile implante grin lens arasındaki mesafe ile belirlenen bir odak düzlemi aralığında açıkça gözlenmiştir (Şekil 2A ve B). Buna karş...

Watch this Scientific Journal Video about Successful In vivo Calcium Imaging with a Head-Mount Miniaturized Microscope in the Amygdala of Freely Behaving Mouse at JoVE.com

Successful In vivo Calcium Imaging with a Head-Mount Miniaturized Microscope in the Amygdala of Freely Behaving Mouse

In vivo mikroendoskopik kalsiyum görüntüleme, özgürce davranan hayvanlarda nöronal aktivitelerin gerçek zamanlı izlenmesini sağlayan paha biçilmez bir araçtır. Ancak bu tekniği amigdalaya uygulamak zor olmuştur. Bu protokol, farelerde minyatürleştirilmiş bir mikroskopla amigdala hücrelerini başarıyla hedeflemek için yararlı bir kılavuz sağlamayı amaçlamaktadır.

Özgürce Davranan Farenin Amigdalasında Kafaya Monte Minyatür Mikroskop ile Başarılı In vivo Kalsiyum Görüntüleme

In vivo real-time monitoring of neuronal activities in freely moving animals is one of key approaches to link neuronal activity to behavior. For this ...

successful-vivo-calcium-imaging-with-head-mount-miniaturized

Research

JoVE Journal

Neuroscience

12.1K Görüntüleme.  Korea Advanced Institute of Science and Technology (KAIST). In vivo mikroendoskopik kalsiyum görüntüleme, özgürce davranan hayvanlarda nöronal aktivitelerin gerçek zamanlı izlenmesini sağlayan paha biçilmez bir araçtır. Ancak bu tekniği amigdalaya uygulamak zor olmuştur. Bu protokol, farelerde minyatürleştirilmiş bir mikroskopla amigdala hücrelerini başarıyla hedeflemek için yararlı bir kılavuz sağlamayı amaçlamaktadır.

Video: Successful In vivo Calcium Imaging with a Head-Mount Miniaturized Microscope in the Amygdala of Freely Behaving Mouse

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.

Sanal Gerçeklik Çevre gezinme Farelerde iki foton Kalsiyum Görüntüleme

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

Davranış

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 Fare Kalitesiz Zeytinde Kalsiyum Görüntüleme

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 ...

Nörobilim

Craniotomy Procedure for Visualizing Neuronal Activities in Hippocampus of Behaving Mice

Imaging neuronal activities at single-cell resolution in awake behaving animals is a very powerful approach for the investigation of neural circuit functions in systems neuroscience. However, high absorbance and scattering of light in mammalian tissue limit intravital imaging mostly to superficial brain regions, leaving deep-brain areas, such as the hippocampus, out of reach for optical microscopy. In this video, we show the preparation and implantation of the custom-made imaging window to enable chronic in vivo imaging of the dorsal hippocampal CA1 region in head-fixed behaving mice. The custom-made window is supplemented with an infusion cannula that allows targeted delivery of viral vectors and drugs to the imaging area. By combining this preparation with wide-field imaging, we performed a long-term recording of neuronal activity using a fluorescent calcium indicator from large subsets of neurons in behaving mice over several weeks. We also demonstrated the applicability of this preparation for voltage imaging with single-spike resolution. High-performance genetically encoded indicators of neuronal activity and scientific CMOS cameras allowed the recurrent visualization of subcellular morphological details of single neurons at high temporal resolution. We also discuss the advantages and potential limitations of the described method and its compatibility with other imaging techniques.

This article demonstrates the preparation of a custom-made imaging window supplemented with infusion cannula and its implantation onto the CA1 region of the hippocampus in mice.

Behaving Farelerinin Hipokampusunda Nöronal Aktiviteleri Görselleştirmek için Kraniyotomi Prosedürü

Imaging neuronal activities at single-cell resolution in awake behaving animals is a very powerful approach for the investigation of neural circuit ...

Implantation of a Cranial Window for Repeated In Vivo Imaging in Awake Mice

To fully understand the cellular physiology of neurons and glia in behaving animals, it is necessary to visualize their morphology and record their activity in vivo in behaving mice. This paper describes a method for the implantation of a chronic cranial window to allow for the longitudinal imaging of brain cells in awake, head-restrained mice. In combination with genetic strategies and viral injections, it is possible to label specific cells and regions of interest with structural or physiological markers. This protocol demonstrates how to combine viral injections to label neurons in the vicinity of GCaMP6-expressing astrocytes in the cortex for simultaneous imaging of both cells through a cranial window. Multiphoton imaging of the same cells can be performed for days, weeks, or months in awake, behaving animals. This approach provides researchers with a method for viewing cellular dynamics in real time and can be applied to answer a number of questions in neuroscience.

Implantation of a Cranial Window for Repeated In Vivo Imaging in Awake Mice

Presented here is a protocol for the implantation of a chronic cranial window for the longitudinal imaging of brain cells in awake, head-restrained mice.

Uyanık Farelerde Tekrarlanan In Vivo Görüntüleme için Kraniyal Pencerenin İmplantasyonu

To fully understand the cellular physiology of neurons and glia in behaving animals, it is necessary to visualize their morphology and record their ...

Stereotaxic Viral Injection and Gradient-Index Lens Implantation for Deep Brain In Vivo Calcium Imaging

A miniature fluorescence microscope (miniscope) is a potent tool for in vivo calcium imaging from freely behaving animals. It offers several advantages over conventional multi-photon calcium imaging systems: (1) compact; (2) light-weighted; (3) affordable; and (4) allows recording from freely behaving animals. This protocol describes brain surgeries for deep brain in vivo calcium imaging using a custom-developed miniscope recording system. The preparation procedure consists of three steps, including (1) stereotaxically injecting the virus at the desired brain region of a mouse brain to label a specific subgroup of neurons with genetically encoded calcium sensor; (2) implantation of gradient-index (GRIN) lens that can relay calcium image from deep brain region to the miniscope system; and (3) affixing the miniscope holder over the mouse skull where miniscope can be attached later. To perform in vivo calcium imaging, the miniscope is fastened onto the holder, and neuronal calcium images are collected along with simultaneous behavior recordings. The present surgery protocol is compatible with any commercial or custom-built single-photon and two-photon imaging systems for deep brain in vivo calcium imaging.

Stereotaxic Viral Injection and Gradient-Index Lens Implantation for Deep Brain In Vivo Calcium Imaging

Miniscope in vivo calcium imaging is a powerful technique to study neuronal dynamics and microcircuits in freely behaving mice. This protocol describes performing brain surgeries to achieve good in vivo calcium imaging using a miniscope.

Derin Beyin In Vivo Kalsiyum Görüntüleme için Stereotaksik Viral Enjeksiyon ve Degrade İndeks Lens İmplantasyonu

A miniature fluorescence microscope (miniscope) is a potent tool for in vivo calcium imaging from freely behaving animals. It offers several advantages ...

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.

Uyanık Farelerde Mikroglial Dinamiğin ve Nöronal Aktivitenin Eşzamanlı Görüntülenmesi

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.

İn Vivo Büyük Bir Kraniyal Pencere Kullanarak Bir Fareden Geniş Alan ve İki Foton Kalsiyum Görüntüleme

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 ...

Subcellular Imaging of Neuronal Calcium Handling In Vivo

Calcium (Ca2+) imaging has been largely used to examine neuronal activity, but it is becoming increasingly clear that subcellular Ca2+ handling is a crucial component of intracellular signaling. The visualization of subcellular Ca2+ dynamics in vivo,&#160;where neurons can be studied in their native, intact circuitry, has proven technically challenging in complex nervous systems. The transparency and relatively simple nervous system of the nematode Caenorhabditis elegans enable the cell-specific expression and in vivo visualization of fluorescent tags and indicators. Among these are fluorescent indicators that have been modified for use in the cytoplasm as well as various subcellular compartments, such as the mitochondria. This protocol enables non-ratiometric Ca2+ imaging in vivo with a subcellular resolution that permits the analysis of Ca2+ dynamics down to the level of individual dendritic spines and mitochondria. Here, two available genetically encoded indicators with different Ca2+ affinities are used to demonstrate the use of this protocol for measuring relative Ca2+ levels within the cytoplasm or mitochondrial matrix in a single pair of excitatory interneurons (AVA). Together with the genetic manipulations and longitudinal observations possible in C. elegans, this imaging protocol may be useful for answering questions regarding how Ca2+ handling regulates neuronal function and plasticity.

Subcellular Imaging of Neuronal Calcium Handling In Vivo

The current methods describe a non-ratiometric approach for high-resolution, sub-compartmental calcium imaging in vivo in Caenorhabditis elegans using readily available genetically encoded calcium indicators.

Nöronal Kalsiyum Kullanımının In Vivo Subsellüler Görüntülemesi

Calcium (Ca2+) imaging has been largely used to examine neuronal activity, but it is becoming increasingly clear that subcellular Ca2+ handling is a ...

Superior Colliculus'ta İki Foton ve Geniş Alan Kalsiyum Görüntüleme

Calcium Imaging in Mouse Superior Colliculus

The superior colliculus (SC), an evolutionarily conserved midbrain structure in all vertebrates, is the most sophisticated visual center before the emergence of the cerebral cortex. It receives direct inputs from ~30 types of retinal ganglion cells (RGCs), with each encoding a specific visual feature. It remains elusive whether the SC simply inherits retinal features or if additional and potentially de novo processing occurs in the SC. To reveal the neural coding of visual information in the SC, we provide here a detailed protocol to optically record visual responses with two complementary methods in awake mice. One method uses two-photon microscopy to image calcium activity at single-cell resolution without ablating the overlaying cortex, while the other uses wide-field microscopy to image the whole SC of a mutant mouse whose cortex is largely undeveloped. This protocol details these two methods, including animal preparation, viral injection, headplate implantation, plug implantation, data acquisition, and data analysis. The representative results show that the two-photon calcium imaging reveals visually evoked neuronal responses at single-cell resolution, and the wide-field calcium imaging reveals&#160;neural activity across the entire SC. By combining these two methods, one can reveal the neural coding in the SC at different scales, and such combination can also be applied to other brain regions.

Yazar Gündemi: Superior Colliculus'ta Nöral Kodlamanın ve Görsel İşleme Mekanizmalarının Ortaya Çıkarılması 

This protocol details the procedure for imaging calcium responses in the superior colliculus (SC) of awake mice, including imaging single-neuron activity with two-photon microscopy while leaving the cortex intact in wild-type mice, and imaging the entire SC with wide-field microscopy in partial-cortex mutant mice.

Fare Superior Colliculus'ta Kalsiyum Görüntüleme

The superior colliculus (SC), an evolutionarily conserved midbrain structure in all vertebrates, is the most sophisticated visual center before the ...

Özgürce Davranan Farenin Amigdalasında Kafaya Monte Minyatür Mikroskop ile Başarılı In vivo Kalsiyum Görüntüleme

Özet

Daha Fazla Video Keşfet

Bu videodaki bölümler

Sanal Gerçeklik Çevre gezinme Farelerde iki foton Kalsiyum Görüntüleme

In vivo Fare Kalitesiz Zeytinde Kalsiyum Görüntüleme

Behaving Farelerinin Hipokampusunda Nöronal Aktiviteleri Görselleştirmek için Kraniyotomi Prosedürü

Uyanık Farelerde Tekrarlanan In Vivo Görüntüleme için Kraniyal Pencerenin İmplantasyonu

Derin Beyin In Vivo Kalsiyum Görüntüleme için Stereotaksik Viral Enjeksiyon ve Degrade İndeks Lens İmplantasyonu

Uyanık Farelerde Mikroglial Dinamiğin ve Nöronal Aktivitenin Eşzamanlı Görüntülenmesi

İn Vivo Büyük Bir Kraniyal Pencere Kullanarak Bir Fareden Geniş Alan ve İki Foton Kalsiyum Görüntüleme

Nöronal Kalsiyum Kullanımının In Vivo Subsellüler Görüntülemesi

Fare Superior Colliculus'ta Kalsiyum Görüntüleme

Fare Superior Colliculus'ta Kalsiyum Görüntüleme