이 사업은 삼성과학기술재단(프로젝트 번호 SSTF-BA1801-10)의 보조금으로 지원되었다.

<ol>
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저자는 공개 할 것이 없습니다

숙련된 수술 기술은 우리가 여기에서 설명한 편도체와 같은 더 깊은 두뇌 지구에 있는 헤드 마운트 소형 현미경 검사법을 가진 생체 내 광학 칼슘 화상 진찰에서 성공을 달성하기 위해 필수적입니다. 따라서 이 프로토콜은 베이스플레이트 부착 및 GRIN 렌즈 이식의 최적화된 수술 과정에 대한 지침을 제공하지만 중요한 단계에 추가 최적화 프로세스가 필요할 수 있습니다. 프로토콜 섹션에서 언급?...

칼슘은 거의 모든 세포 기능8에서중요한 역할을하는 유비쿼터스 두 번째 메신저입니다. 뉴런에서, 작용 잠재력 발사 및 시냅스 입력은 세포내 무료 [Ca2+]9,10의급격한 변화를 일으킨다. 따라서 칼슘 과도를 추적하면 신경 활동을 모니터링 할 수있는 기회를 제공합니다. GECI는 정의된 세포 인구 및 셀룰러 내구획(11,12)에서[Ca2+]모니터링을 허용하는 강력한 도구입니다. 단백질 기반 칼슘 표시기의 많은 다른 모형 중, GCaMP, 단일 GFP 분자13에근거를 둔 Ca2+ 프로브는, 가장 최적화되고 이렇게 널리 사용되는 GECI입니다. 여러 차례의 엔지니어링을 통해 GCaMP의 여러 변형이12,14,15,16을개발했습니다. 우리는 이 프로토콜16에서최근에 개발된 GCaMPPs, GCaMP7b 중 하나를 사용합니다. GCaMP센서는 개발 17 동안 Ca2+ 과도의 이미징, 특정 피질층(18)에서의생체 이미징, 모터 작업 학습19의 회로 역학 측정 및 해마및 대미20의 연관 공포 기억과 관련된 세포 앙상블 활동의이미징과 같은 다수의 모델 유기체에서 신경 회로 기능의 연구에 크게기여했다.
GECIs의 광학 이미징은 몇 가지 장점이있습니다 22. 유전 인코딩을 통해 GECI는 유전자 프로파일 또는 해부학 적 연결의 특정 패턴에 의해 정의되는 세포의 특정 하위 집합에서 장기간 안정적으로 표현 될 수 있습니다. 광학 이미징은 살아있는 동물에서 수백에서 수천 개의 뉴런을 생체 내에서 만성 동시 모니터링할 수 있게 합니다. 몇몇 광학 이미징 시스템은 헤드 마운트 소형형 광소 현미경21,23,24,25로마우스를 자유롭게 행동하는 뇌 내에서 GECIs의 생체 이미징 및 분석을 위해 개발되었다. GECIs, GRIN 렌즈 및 헤드 마운트 소형 현미경을 기반으로 한 생체 내 광학 이미징 기술이 신경 회로 활동과 행동 사이의 연관성을 연구하는 강력한 도구임에도 불구하고, 편도체에 이 기술을 적용하는 것은 GRIN 렌즈를 미막도라에서 GEC를 표현하는 세포에 표적화하는 것과 관련된 몇 가지 기술적 문제로 인해 어려웠으며, 이는 GEC의 이미지를 심각하게 저하시키는 동작 아티팩트를 유발하고 이미지를 저하시키는 데 방해가 되고 있다. 이 프로토콜은 편도체에서 생체 내 광학 칼슘 이미징에서 성공적인 단계를 위한 중요한 단계인 베이스플레이트 부착 및 GRIN 렌즈 이식의 외과 적 절차에 유용한 지침을 제공하는 것을 목표로 합니다. 이 프로토콜은 편도체를 대상으로하지만, 여기에 설명 된 대부분의 절차는 일반적으로 다른 깊은 뇌 영역에 적용됩니다. 이 프로토콜은 특정 시스템(예: Inscopix)을 기반으로 하지만 다른 대체 시스템에서동일한 목적을 쉽게 달성할 수 있습니다.

<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

자유롭게 움직이는 동물에서 신경 활동의 생체 내 실시간 모니터링은 행동에 신경 활동을 연결하는 주요 접근 법 중 하나입니다. 이를 위해, 유전자 인코딩된 칼슘 지표(GECIs), 소형형 형광 현미경, 그라데이션 굴절지수(GRIN) 렌즈를 사용하여 뉴런의 칼슘 과도를 검출하는 생체 내 이미징 기술이 개발되어 많은 뇌 구조1,2,3,4,5,6에성공적으로 적용되었다. 이 화상 진찰 기술은 최대 몇 주까지 장기 기간 동안 유전으로 정의된 세포 집단의 만성 동시 화상 진찰을 가능하게 하기 때문에 특히 강력합니다. 유용하지만, 이 화상 진찰 기술은 편도체, 정서적 처리 및 연관 공포 기억 기억7을위한 필수적인 두뇌 구조물과 같은 두뇌 내깊은 찾아내는 두뇌 구조물에 쉽게 적용되지 않았습니다. 편도체에 이미징 기술을 적용하는 것을 어렵게 만드는 몇몇 요인이 있습니다. 예를 들면, 운동 유물은 일반적으로 두뇌에 있는 깊은 이식된 헤드 마운트 현미경이 상대적으로 불안정하기 때문에 더 깊은 두뇌 지구에서 실시하는 화상 진찰 도중 더 빈번해 깁니다. 또 다른 문제는 측면 심실이 이식된 GRIN 렌즈에 가깝게 위치하고 호흡 도중의 움직임이 쉽게 교정할 수 없는 고불규칙한 동작 아티팩트를 유발할 수 있어 안정적인 이미징 뷰를 형성하기가 어렵다는 것입니다. 더욱이, 편도체내의 세포는 일반적으로 휴식 또는 마취 상태에서 조용하기 때문에, 나중에 이미징을 위한 기질도금 절차 도중 편도체에서 GECI를 발현하는 표적 세포를 찾아 서 집중하기 어렵다. 이 프로토콜은 이러한 깊은 뇌 영역에서 생체 내 칼슘 이미징을 성공적으로 위해 헤드 마운트 소형 현미경으로 편도체에서 GECI를 발현하는 세포를 효율적으로 표적으로 하는 방법에 대한 유용한 지침을 제공합니다. 이 프로토콜은 특정 시스템(예: Inscopix)을 기반으로 하지만 제한되지는 않습니다.

GRIN 렌즈 이식 의 검증 만성적으로 시멘트를 통해 뇌에 베이스 플레이트를 부착하기 전에 GRIN 렌즈 이식을 검증해야 합니다. 렌즈 이식이 성공한 동물의 경우, GCaMP 발현 세포와 혈관 모두 현미경의 객관적인 렌즈와 이식된 GRIN렌즈(그림 2A 및 B)사이의거리에 의해 결정된 초점 평면 범위 내에서 명확하게 관찰되었다. 대조적으로, 오프...

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

생체 내 미세 엔도스코픽 칼슘 이미징은 자유롭게 행동하는 동물의 신경 활동을 실시간으로 모니터링 할 수있는 귀중한 도구입니다. 그러나, 편도체에 이 기술을 적용하는 것은 어려웠습니다. 이 프로토콜은 마우스에 있는 소형화된 현미경으로 편도체 세포를 성공적으로 표적으로 하기 위한 유용한 지침을 제공하는 것을 목표로 합니다.

자유롭게 행동하는 마우스의 편도체에 있는 머리 산 소형 현미경을 가진 성공적인 생체 내 칼슘 화상 진찰

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 조회수.  Korea Advanced Institute of Science and Technology (KAIST). 생체 내 미세 엔도스코픽 칼슘 이미징은 자유롭게 행동하는 동물의 신경 활동을 실시간으로 모니터링 할 수있는 귀중한 도구입니다. 그러나, 편도체에 이 기술을 적용하는 것은 어려웠습니다. 이 프로토콜은 마우스에 있는 소형화된 현미경으로 편도체 세포를 성공적으로 표적으로 하기 위한 유용한 지침을 제공하는 것을 목표로 합니다.

비디오: 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.

가상 현실 환경을 탐색 마우스의 두 광자 칼슘 이미징

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

연구

행동분석학

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.

생체 내 마우스 열등올리브의 칼슘 이미징

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 ...

신경과학

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.

마우스를 면도하는 해마에서 신경 활동을 시각화하기위한 두개골 절제술 절차

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.

깨어있는 마우스 에서 반복되는 생체 내 이미징을위한 두개골 창 이식

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.

입체 택시 바이러스 주입 및 생체 내 심부 뇌를위한 그라디언트 인덱스 렌즈 이식

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.

깨어 있는 생쥐의 소교세포 역학 및 신경 활동의 동시 이미징

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.

생체 내 큰 두개골 창을 사용한 마우스의 광시야 및 2광자 칼슘 이미징

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.

생체 내에서 처리되는 신경 칼슘의 세포 내 이미징

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

Superior Colliculus의 Two-photon 및 Wide-field Calcium 이미징

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.

저자 스포트라이트: Unveiling Neural Coding and Mechanisms of Visual Processing in the Superior Colliculus 

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.

Mouse Superior Colliculus의 칼슘 이미징

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

자유롭게 행동하는 마우스의 편도체에 있는 머리 산 소형 현미경을 가진 성공적인 생체 내 칼슘 화상 진찰

요약

더 많은 비디오 탐색

이 비디오의 챕터

가상 현실 환경을 탐색 마우스의 두 광자 칼슘 이미징

생체 내 마우스 열등올리브의 칼슘 이미징

마우스를 면도하는 해마에서 신경 활동을 시각화하기위한 두개골 절제술 절차

깨어있는 마우스 에서 반복되는 생체 내 이미징을위한 두개골 창 이식

입체 택시 바이러스 주입 및 생체 내 심부 뇌를위한 그라디언트 인덱스 렌즈 이식

깨어 있는 생쥐의 소교세포 역학 및 신경 활동의 동시 이미징

생체 내 큰 두개골 창을 사용한 마우스의 광시야 및 2광자 칼슘 이미징

생체 내에서 처리되는 신경 칼슘의 세포 내 이미징

Mouse Superior Colliculus의 칼슘 이미징

Mouse Superior Colliculus의 칼슘 이미징