이 연구는 상하이 기초 연구를 위한 파일럿 프로그램(Shanghai Pilot Program for Basic Research) - 푸단대학교 21TQ1400100(22TQ019), 상하이시 과학기술 주요 프로젝트, 린강 연구소(Lingang Laboratory, 보조금 번호)의 지원을 받습니다. LG-QS-202203-09) 및 중국국가자연과학재단(32371036).

생쥐의 치상동맥 세포에서 뉴런 활성을 위한 실시간 이미징 

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저자는 경쟁하는 재정적 이익이 없음을 선언합니다.

여기서 우리는 마우스의 DG에서 생체 내 칼슘 이미징 절차를 설명했습니다. 우리는 이 프로토콜이 다양한 인지 과정에서 DG 기능을 연구하려는 연구자, 특히 유전적으로 식별된 하위 집단에 관심이 있는 경우에 유용할 것이라고 믿습니다. 여기에서는 수술의 몇 가지 핵심 사항을 강조하여 프로토콜의 장점을 설명하고 이 방법의 한계에 대해 논의합니다.
우리는 사용 가?...

이전 연구에 따르면 해마는 기억을 처리하고 검색하는 데 필수적인 뇌 구조입니다 1,2. 1950년대 이래로 설치류의 해마의 신경회로는 기억 형성, 저장 및 회수를 연구하는 데 초점이 되어 왔다3. 해마 내의 해부학적 구조에는 치상이랑(DG), CA1, CA2, CA3, CA4 및 subiculum4의 하위 영역이 포함됩니다. 이러한 하위 영역들 사이에는 복잡한 양방향 연결이 존재하며, 그 중 DG, CA1 및 CA3는 과립 세포와 피라미드 세포로 구성된 두드러진 삼시냅스 회로를 형성합니다5. 이 회로는 내측 피질(entorhinal cortex, EC)로부터 주요 입력을 받으며 시냅스 가소성(synaptic plasticity)을 연구하기 위한 고전적인 모델이었습니다. 이전의 생체 내 연구는 해마 기능에 대한 연구가 대부분 CA1 6,7에 집중되어 있었는데, 이는 접근이 용이하기 때문입니다. CA1 뉴런은 기억 형성, 통합 및 검색, 특히 공간 기억을 위한 장소 세포에서 중요한 역할을 하지만 해마의 다른 하위 영역도 중요합니다 8,9. 특히 최근 연구에서는 DG가 기억 형성에 미치는 영향을 강조하고 있다. DG의 장소 셀은 CA1(10)의 장소셀보다 더 안정적인 것으로 보고되었으며, 이들의 활동은 상황별 정보(context-specific information)11를 반영한다. 또한, DG 과립 세포의 활동 의존적 표지는 기억 관련 행동을 유도하기 위해 재활성화될 수 있다12. 따라서 DG의 정보 코딩에 대한 더 깊은 이해를 얻으려면 동물이 메모리 의존적 작업을 수행하는 동안 DG 하위 영역의 활동을 조사하는 것이 중요합니다.
당뇨병 활성에 대한 선행 연구는 주로 in vivo electrophysiology를 사용하였다13. 그러나, 이 기술에는 몇 가지 단점이 있다: 첫째, 전기적 기록에서, 신호를 생성하는 다양한 유형의 세포를 직접 식별하기 어려울 수 있다. 기록된 신호는 억제성 세포와 흥분성 세포 모두에서 나옵니다. 따라서 이 두 가지 세포 유형을 분리하기 위해 추가 데이터 처리 방법이 필요합니다. 더욱이, 투영 특이적 소그룹 또는 활동 의존적 라벨링과 같은 다른 세포 유형 정보를 전기 기록과 결합하는 것은 어렵습니다. 또한 DG의 해부학적 형태로 인해 기록 전극이 직교 방향으로 이식되는 경우가 많아 기록할 수 있는 뉴런의 수가 크게 제한됩니다. 따라서, 전기적 기록이 동일한 동물(14)의 DG 구조로부터 수백 개의 개별 뉴런을 모니터링하는 것은 어렵다.
DG에서 뉴런 활동을 기록하는 보완적인 기술은 in vivo calcium imaging15을 사용하는 것이다. 칼슘 이온은 유기체의 세포 신호 전달 과정에 필수적이며, 특히 포유류 신경계 내에서 많은 생리적 기능에서 중요한 역할을 합니다. 뉴런이 활동할 때 세포 내 칼슘 농도가 급격히 증가하여 뉴런 활동과 신호 전달의 역동적인 특성을 반영합니다. 따라서 뉴런의 세포 내 칼슘 농도의 실시간 변화를 기록하면 신경 코딩 메커니즘에 대한 중요한 통찰력을 얻을 수 있습니다.
칼슘 이미징 기술은 특수 형광 염료 또는 유전자 조작 칼슘 지표(GECI)를 사용하여 형광 강도의 변화를 감지하여 뉴런의 칼슘 이온 농도를 모니터링한 다음 현미경 이미징을 통해 캡처할 수 있습니다16. 일반적으로 녹색 형광 단백질(GFP), 칼모듈린 및 M13 폴리펩티드 서열로 구성된 GCaMP 계열의 칼슘 지시자 유전자가 사용됩니다. GCaMP는 칼슘 이온(17)에 결합할 때 녹색 형광을 방출할 수 있으므로 이미징18을 통해 녹색 형광의 변동을 기록할 수 있습니다. 또한 표적 뇌 영역의 선명한 이미지를 얻기 위해 일반적으로 GRIN 렌즈(Gradient Index Lens)를 관심 영역 위에 이식합니다. GRIN 렌즈는 표면에서 직접 접근할 수 없는 뇌 심부 영역을 이미징할 수 있습니다.
이 기술은 다른 세포 유형을 표지하기 위해 다른 유전 도구와 결합하기가 비교적 쉽습니다. 또한, 이미징 평면이 DG의 세포 방향과 평행하기 때문에 성공적인 수술을 받을 때마다 수백 개의 뉴런에 접근할 수 있습니다. 이 연구에서는 생쥐의 치상이랑에서 생체 내 칼슘 이미징을 위한 완전하고 상세한 수술 프로토콜을 제시합니다(그림 1). 이 절차에는 두 가지 주요 작업이 포함됩니다. 첫 번째는 AAV-CaMKIIα-GCaMP6f 바이러스를 DG에 주입하는 것입니다. 두 번째 수술은 바이러스 주입 부위 위에 GRIN 렌즈를 이식하는 것입니다. 이 두 가지 절차는 같은 자리에서 수행됩니다. 이러한 수술에서 회복 후 다음 단계는 소형 현미경 (miniscope)으로 이미징 품질을 확인하는 것입니다. 이미징 필드에 수백 개의 활성 세포가 있는 경우 후속 절차는 치과용 시멘트를 사용하여 미니스코프 베이스 플레이트를 마우스 두개골에 부착하는 것입니다. 그런 다음 마우스를 이미징 실험에 사용할 수 있습니다. 또한 수집된 칼슘 데이터의 분석을 간소화하기 위한 MATLAB 기반 전처리 파이프라인도 제공합니다.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>200 &#956;L universal pipette tips</td>
			<td>Transcat Pipettes</td>
			<td>1030-260-000-9</td>
			<td>For removing the blood and saline</td>
		</tr>
		<tr>
			<td>25 G luer lock blunt needle (Prebent dispensing tips)</td>
			<td>iSmile</td>
			<td>20-0105</td>
			<td>For removing the brain tissue</td>
		</tr>
		<tr>
			<td>3D printed protective cap</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>To protect the GRIN Lens</td>
		</tr>
		<tr>
			<td>75% ethanol</td>
			<td>Shanghai Hushi Laboratory Equipment Co.,Ltd</td>
			<td>bwsj-230219105303</td>
			<td>For disinfection and cleaning the GRIN lens surface</td>
		</tr>
		<tr>
			<td>AAV2/9-CaMKII&#945;-GCaMP6f virus</td>
			<td>Brain Case</td>
			<td>BC-0083</td>
			<td>For viral injection</td>
		</tr>
		<tr>
			<td>Adobe Illustrator</td>
			<td>Adobe</td>
			<td>cc 2018 version 22.1</td>
			<td>To draw figures</td>
		</tr>
		<tr>
			<td>Anesthesia air pump</td>
			<td>RWD Life Science Co.,Ltd</td>
			<td>R510-30</td>
			<td>For anesthesia</td>
		</tr>
		<tr>
			<td>Camera control software</td>
			<td>Daheng Imaging</td>
			<td>Galaxy Windows SDK_CN (V2)</td>
			<td>For recording the behavioral data</td>
		</tr>
		<tr>
			<td>Cannula/Ceramic Ferrule Holders (GRIN lens holder)</td>
			<td>RWD Life Science Co.,Ltd</td>
			<td>68214</td>
			<td>To hold the GRIN lens</td>
		</tr>
		<tr>
			<td>Carprofen</td>
			<td>MedChemExpress</td>
			<td>53716-49-7</td>
			<td>To reduce postoperative pain of the mouse&#160;</td>
		</tr>
		<tr>
			<td>Coax Cable</td>
			<td>Open ephys</td>
			<td>CW8251</td>
			<td>To connect the miniscope and the miniscope DAQ box</td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>Olympus Life Science&#160;</td>
			<td>FV3000</td>
			<td>For observing the brain slices</td>
		</tr>
		<tr>
			<td>Cotton swab</td>
			<td>Nanchang Xiangyi Medical Devices Co.,Ltd</td>
			<td>20202140438</td>
			<td>For disinfection</td>
		</tr>
		<tr>
			<td>Customized headplate</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>For holding the mouse on the running wheel</td>
		</tr>
		<tr>
			<td>Customized headplate holder</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>To hold the headplate of the mouse</td>
		</tr>
		<tr>
			<td>Denture base matierlals (self-curing)</td>
			<td>New Centry Dental</td>
			<td>430205</td>
			<td>For attaching the miniscope</td>
		</tr>
		<tr>
			<td>Depilatory cream</td>
			<td>Veet</td>
			<td>ASIN : B001DUUPQ0</td>
			<td>For removing the hair of the mouse</td>
		</tr>
		<tr>
			<td>Desktop digital stereotaxic in strument, SGL M</td>
			<td>RWD Life Science Co.,Ltd</td>
			<td>68803</td>
			<td>For viral injection and GRIN lens implantation</td>
		</tr>
		<tr>
			<td>Dexamethasone</td>
			<td>Huachu Co., Ltd.</td>
			<td>N/A</td>
			<td>To prevent postoperative inflammation of the mouse</td>
		</tr>
		<tr>
			<td>Dissecting microscope</td>
			<td>RWD Life Science Co., Ltd</td>
			<td>MZ62-WX</td>
			<td>For observing the conditions during surgeries</td>
		</tr>
		<tr>
			<td>Gas filter canister, large, packge of 6</td>
			<td>RWD Life Science Co.,Ltd</td>
			<td>R510-31-6</td>
			<td>For anesthesia</td>
		</tr>
		<tr>
			<td>GRIN lens</td>
			<td>GoFoton</td>
			<td>CLHS100GFT003</td>
			<td>For GRIN lens implantation</td>
		</tr>
		<tr>
			<td>GRIN lens</td>
			<td>InFocus Grin Corp</td>
			<td>SIH-100-043-550-0D0-NC</td>
			<td>For GRIN lens implantation</td>
		</tr>
		<tr>
			<td>Induction chamber-mouse (15 cm x 10 cm x 10 cm)</td>
			<td>RWD Life Science Co.,Ltd</td>
			<td>V100</td>
			<td>For anesthesia</td>
		</tr>
		<tr>
			<td>Industrial camera</td>
			<td>Daheng Imaging</td>
			<td>MER-231-41U3M-L, VS-0618H1</td>
			<td>For acquiring the behavioral data</td>
		</tr>
		<tr>
			<td>Iodophor disinfectant</td>
			<td>Qingdao Hainuo Innovi Disinfection Technology Co.,Ltd</td>
			<td>8861F6DFC92A</td>
			<td>For disinfection</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>RWD Life Science Co.,Ltd</td>
			<td>R510-22-10</td>
			<td>For anesthesia</td>
		</tr>
		<tr>
			<td>Liquid sample collection tube (Glass Capillaries micropipette for Nanoject III)</td>
			<td>Drummond Scientific Company</td>
			<td>3-000-203-G/X</td>
			<td>For viral injection</td>
		</tr>
		<tr>
			<td>MATLAB</td>
			<td>MathWorks</td>
			<td>R2021b</td>
			<td>For analyzing the data</td>
		</tr>
		<tr>
			<td>Microdrill</td>
			<td>RWD Life Science Co.,Ltd</td>
			<td>78001</td>
			<td>For craniotomy</td>
		</tr>
		<tr>
			<td>Micropipette puller</td>
			<td>Narishige International USA</td>
			<td>PC-100</td>
			<td>For pulling the liquid sample collection tube</td>
		</tr>
		<tr>
			<td>Mineral oil</td>
			<td>Sigma-Aldrich</td>
			<td>M8410</td>
			<td>For viral injection</td>
		</tr>
		<tr>
			<td>Miniscope DAQ Software</td>
			<td>Github (Aharoni-Lab/Miniscope-DAQ-QT-Software)</td>
			<td>N/A</td>
			<td>For recording the calcium imaging data</td>
		</tr>
		<tr>
			<td>Miniscope Data Acquisition (DAQ) Box (V3.3)</td>
			<td>Open ephys</td>
			<td>V3.3</td>
			<td>To acquire the calcium imaging data</td>
		</tr>
		<tr>
			<td>Miniscope V4</td>
			<td>Open ephys</td>
			<td>V4</td>
			<td>For in vivo calcium imaging</td>
		</tr>
		<tr>
			<td>Miniscope V4 base plate (Variant 2)</td>
			<td>Open ephys</td>
			<td>Variant 2</td>
			<td>For holding the miniscope</td>
		</tr>
		<tr>
			<td>nanoject III Programmable Nanoliter Injector</td>
			<td>Drummond Scientific Company</td>
			<td>3-000-207</td>
			<td>For viral injection</td>
		</tr>
		<tr>
			<td>Ophthalmic ointment</td>
			<td>Cisen Pharmaceutical Co.,Ltd.</td>
			<td>H37022025</td>
			<td>To keep the eyes moist</td>
		</tr>
		<tr>
			<td>PCR tube</td>
			<td>LabServ</td>
			<td>309101009</td>
			<td>For dilue the virus</td>
		</tr>
		<tr>
			<td>Personal Computer (ThinkPad)</td>
			<td>Lenovo</td>
			<td>20W0-005UCD</td>
			<td>To record the calcium imaging data and behavioral data</td>
		</tr>
		<tr>
			<td>Running wheel</td>
			<td>Shanghai Edai Pet Products Co.,Ltd</td>
			<td>NA-H115</td>
			<td>For holding the mouse when affixing the base plate</td>
		</tr>
		<tr>
			<td>Screwdriver (M1.6 screws)</td>
			<td>Greenery (Yantai Greenery Tools Co.,Ltd)</td>
			<td>60902</td>
			<td>To unscrew the M1.6 screws</td>
		</tr>
		<tr>
			<td>Screwdriver (set screws)</td>
			<td>Greenery (Yantai Greenery Tools Co.,Ltd)</td>
			<td>S2</td>
			<td>For unscrew the set screws</td>
		</tr>
		<tr>
			<td>Set screw</td>
			<td>TBD</td>
			<td>2-56 cone point set screw</td>
			<td>For fasten the miniscope to its base plate</td>
		</tr>
		<tr>
			<td>Small animal anesthesia machine</td>
			<td>RWD Life Science Co.,Ltd</td>
			<td>R500</td>
			<td>For anesthesia</td>
		</tr>
		<tr>
			<td>Sterile syringe</td>
			<td>Jiangsu Great Wall Medical Equipment Co., LTD</td>
			<td>20163140236</td>
			<td>For rinse the blood</td>
		</tr>
		<tr>
			<td>Surgical scissors</td>
			<td>RWD Life Science Co.,Ltd</td>
			<td>S14016-13</td>
			<td>For cutting off the hair and scalp</td>
		</tr>
		<tr>
			<td>ThermoStar temperature controller,69025 pad incl.</td>
			<td>RWD Life Science Co.,Ltd</td>
			<td>69027</td>
			<td>To maintain the animal&#39;s body temperature</td>
		</tr>
		<tr>
			<td>Ultra fine forceps</td>
			<td>RWD Life Science Co.,Ltd</td>
			<td>F11020-11</td>
			<td>For removing the bone debris and dura</td>
		</tr>
		<tr>
			<td>USB 3.0 cable</td>
			<td>Open ephys</td>
			<td>N/A</td>
			<td>To connect the miniscope DAQ box and the computer</td>
		</tr>
		<tr>
			<td>UV light</td>
			<td>Jinshida</td>
			<td>66105854002</td>
			<td>To fix the GRIN lens on the skull</td>
		</tr>
		<tr>
			<td>UV resin (light cure adhesive)</td>
			<td>Loctite</td>
			<td>32268</td>
			<td>To fix the GRIN lens on the skull</td>
		</tr>
		<tr>
			<td>Vacuum pump</td>
			<td>Kylin-Bell</td>
			<td>GL-802B</td>
			<td>To remove the blood, saline and the brain tissue</td>
		</tr>
	</tbody>
</table>

in vivo calcium imaging of granule cells in the dentate gyrus of hippocampus in mice 

실시간 접근 방식은 일반적으로 학습 및 기억 연구에 필요하며, 생체 내 칼슘 이미징은 행동 작업 중 깨어 있는 동물의 신경 활동을 조사할 수 있는 가능성을 제공합니다. 해마는 일화 및 공간 기억과 밀접하게 관련되어 있기 때문에 이 분야의 연구에서 필수적인 뇌 영역이 되었습니다. 최근 연구에서는 생쥐에서 소형 현미경을 사용하여 해마 CA1 영역의 신경 활동을 기록하여 개방 필드 및 선형 트랙을 포함한 행동 작업을 수행하는 방법을 연구했습니다. 치상이랑은 해마의 또 다른 중요한 영역이지만, 깊이가 깊고 이미징이 어렵기 때문에 생체 내 이미징으로 연구된 적은 거의 없습니다. 이 프로토콜에서는 바이러스를 주입하는 방법, GRIN(Gradient-index) 렌즈를 이식하는 방법, 해마의 치상이랑을 이미징하기 위한 베이스 플레이트를 부착하는 방법을 포함한 칼슘 이미징 과정을 자세히 제시합니다. 또한 MATLAB을 사용하여 칼슘 이미징 데이터를 전처리하는 방법을 설명합니다. 또한 이미징이 필요한 다른 뇌 심부 영역에 대한 연구에도 이 방법이 도움이 될 수 있습니다.

Previous studies found that the hippocampus is a brain structure essential for processing and retrieving memories1,2. Since the 1950s, the neural circuits of the hippocampus in rodents have been a focus in studying memory formation, storage, and retrieval3. The anatomical structures within the hippocampus include the subregions of dentate gyrus (DG), CA1, CA2, CA3, CA4, and subiculum4. Complex bidirectional connections exist among these subregions, of which the DG, CA1, and CA3 form a prominent trisynaptic circuit that consists of granule cells and pyramidal cells5. This circuit receives its primary input from the entorhinal cortex (EC)&#160;and has been a classic model for studying synaptic plasticity. Prior in vivo research on the hippocampus function has mostly concentrated on the CA16,7 due to its easier access. While CA1 neurons serve important roles in memory formation, consolidation, and retrieval, particularly in place cells for spatial memory, other subregions of the hippocampus are also vital8,9. In particular, recent studies have highlighted the functions of DG in memory formation. Place cells in DG have been reported to be more stable than those in CA110, and their activities reflect context-specific information11. Further, activity-dependent labeling of DG granule cells can be reactivated to induce memory-related behaviors12. Therefore, to gain a deeper understanding of the information coding in DG, it is crucial to investigate the activities of the DG subregion while the animal is carrying out memory-dependent tasks.
Prior studies of DG activities have mostly used in vivo electrophysiology13. However, this technique has some drawbacks: First, in electrical recordings, it may be difficult to directly identify the various types of cells that are generating the signal. The recorded signals are from both inhibitory and excitatory cells. Therefore, further data processing methods are required to separate these two cell types. Moreover, it is difficult to combine other cell type information, such as projection-specific subgroups or activity-dependent labeling, with electric recordings. In addition, due to the anatomical morphology of DG, the recording electrodes are often implanted in an orthogonal direction, which greatly limits the number of neurons that can be recorded. Thus, it is difficult for electric recordings to achieve monitoring&#160;hundreds of individual neurons from the DG structure in the same animal14.
A complementary technique of recording neuron activities in DG is to use in vivo calcium imaging15. Calcium ions are fundamental to cellular signaling processes in organisms, playing a crucial role in many physiological functions, especially within the mammalian nervous system. When neurons are active, the intracellular calcium concentration increases rapidly, reflecting the dynamic nature of neuronal activity and signal transmission. Therefore, recording the real-time changes in intracellular calcium levels in neurons provides important insights into the neural coding mechanisms.
Calcium imaging technology utilizes specialized fluorescent dyes or genetically engineered calcium indicators (GECIs) to monitor calcium ion concentrations in neurons by detecting changes in fluorescence intensity, which can then be captured through microscopic imaging16. Commonly, the GCaMP family of calcium indicator genes, comprising green fluorescent protein (GFP), calmodulin, and M13 polypeptide sequences, are employed. GCaMP can emit green fluorescence when it binds to calcium ions17, allowing the fluctuations in green fluorescence to be recorded via imaging18. Additionally, to obtain clear images of the target brain region, a Gradient Index Lens (GRIN lens) is typically implanted above the region of interest. The GRIN lens enables imaging of the deep brain region that cannot be accessed directly from the surface.
This technique is relatively easy to combine with other genetic tools to label different cell types. Moreover, as the imaging plane is parallel to the cells&#39; orientation in DG, hundreds of neurons are accessible for imaging with each successful surgery. In this work, we present a complete and detailed surgery protocol for in vivo calcium imaging in the dentate gyrus in mice (Figure 1). The procedure involves two major operations. The first one is to inject the AAV-CaMKII&#945;-GCaMP6f virus into the DG. The second operation is to implant a GRIN lens above the virus injection site. These two procedures are conducted in the same sitting. After recovery from these surgeries, the next step is to check the imaging quality with miniaturized microscopes (miniscopes). If the imaging field has hundreds of active cells, the subsequent procedure is to attach the miniscope base plate onto the mouse skull using dental cement; the mouse can then be used for imaging experiments. We also present a MATLAB-based preprocessing pipeline for streamlining the analysis of the collected calcium data.

저자 스포트라이트: Investigating Neural Activity of Dentate Gyrus Granule Cells with Miniature Microscope(소형 현미경으로 치상골 과립 세포의 신경 활동 조사)

In Vivo: Calcium Imaging of Granule cells in the dentate gyrus of hippocampus in mice(생쥐의 해마 치열계에 있는 과립 세포의 In vivo 칼슘 이미징) 

Well, in this protocol, we use a miniature microscope to investigate the neural activities in the dentate gyrus of the hippocampus. We aim to investigate neuron coding and spatial representations during the different disease conditions. I believe the key challenge of studying dentate gyrus in vivo involves obtaining high-quality calcium imaging data, whether the virus expression is satisfactory, whether the gradient is implanted at the proper location.And the details like these, the entire surgery are of critical importance for the success of the study. In the traditional calcium imaging surgery, the virus injection and the GRIN lens implantation are typically performed as separate operations. In our protocol, we combined these two steps into a single procedure, which not only reduces the waiting time, but also ensures the accurate placement of the GRIN lens.Our lab uses various microscopy techniques to study the neurodynamics underlying cognitive functions. We hope to understand the neural activity patterns that support cognition. And combining theoretical modeling and causal manipulations, we hope to dissect the processes of information flow and neural computation in the brain.

그림 1 은 바이러스 주입, GRIN 렌즈 주입, 베이스 플레이트 부착, 미니스코프를 통한 생체 내 칼슘 이미징 및 데이터 처리를 포함한 실험 절차의 개략도를 보여줍니다. 일반적으로 전체 절차는 1개월이 소요됩니다. 그림 2 는 두개골에 뚫린 구멍의 위치와 GRIN 렌즈 이식 전 뇌 조직의 상태를 포함한 바이러스 주입 절차의 예를 보여줍니다.
<p class...

해마의 치상이랑(dentate gyrus)은 학습과 기억에서 필수적이고 뚜렷한 기능을 수행합니다. 이 프로토콜은 자유롭게 움직이는 마우스의 치상이랑에 있는 과립 세포의 생체 내 칼슘 이미징을 위한 일련의 강력하고 효율적인 절차를 설명합니다.

Real-time approaches are typically needed in studies of learning and memory, and in vivo calcium imaging provides the possibility to investigate neuronal ...

in-vivo-calcium-imaging-granule-cells-dentate-gyrus-hippocampus

Research

JoVE Journal

Neuroscience

In Vivo Calcium Imaging of Granule Cells in the Dentate Gyrus of Hippocampus in Mice 

811 조회수.  Fudan University. 글쎄요, 이 프로토콜에서는 소형 현미경을 사용하여 해마의 치상이랑의 신경 활동을 조사합니다. 우리는 다양한 질병 조건에서 뉴런 코딩 및 공간 표현을 조사하는 것을 목표로 합니다. in vivo dentate gyrus 연구의 주요 과제는 고품질 칼슘 이미징 데이터를 얻는 것, 바이러스 발현이 만족스러운지, 그래디언트가 적절한 위치에 이식되었는지 여부와 관?...

비디오: 저자 스포트라이트: Investigating Neural Activity of Dentate Gyrus Granule Cells with Miniature Microscope(소형 현미경으로 치상골 과립 세포의 신경 활동 조사)

Real-time approaches are typically needed in studies of learning and memory, and in vivo calcium imaging provides the possibility to investigate neuronal activity in awake animals during behavior tasks. Since the hippocampus is closely associated with episodic and spatial memory, it has become an essential brain region in&#160;this field&#39;s research. In recent research, engram cells and place cells were studied by recording the neural activities in the hippocampal CA1 region using the miniature microscope in mice while performing behavioral tasks including open-field and linear track. Although the dentate gyrus is another important region in the hippocampus, it has rarely been studied with in vivo imaging due to its greater depth and difficulty for imaging. In this protocol, we present in detail a calcium imaging process, including how to inject the virus, implant a GRIN (Gradient-index) lens, and attach a base plate for imaging the dentate gyrus of the hippocampus. We further describe how to preprocess the calcium imaging data using MATLAB. Additionally, studies of other deep brain regions that require imaging may benefit from this method.

The dentate gyrus of the hippocampus carries out essential and distinct functions in learning and memory. This protocol describes a set of robust and efficient procedures for in vivo calcium imaging of granule cells in the dentate gyrus in freely moving mice.

연구

신경과학

Optimized Virus Injection and GRIN Lens Implantation in the Dentate Gyrus of Mice for In Vivo Calcium Imaging

To begin, place the anesthetized mouse on a stereotaxic apparatus. Make a continuous incision along the midline of the scalp using surgical scissors and cut off part of the scalp to expose the skull surface. Make the craniotomy in the specified region with the micro drill and set four points as the border.Stop drilling when brain tissue becomes visible. Use the control panel to set the injector to inject 80 nanoliters of the virus. Click the Inject button on the control panel to inject the virus into the dentate gyrus at a flow rate of 1 nanoliter per second.To suction the brain tissue, revolve the tip of the blunt needle closely above the brain tissue, and press gently to facilitate aspiration. Rinse continuously with cold saline during suctioning to maintain a clear view of the brain. Observe the tissue features closely to monitor the depth of aspiration.The cortical tissue is pale pink. The corpus callosum underneath appears as white fibrous tissue. And the hippocampal CA1 layer is dark red and gray.Stop the suction when the surface of the tissue becomes smooth and a large area of CA1 is exposed. Move the holder attached to the grin lens above the drilled hole. When the grin lens contacts the surface of the brain tissue, set the Z axis to zero.Insert the grin lens into the hole at the dorsal ventral axis minus 1.32 millimeters. Allow the holder to stand for 5 to 10 minutes. Then, raise the holder and rinse the hole with saline.Using a needle, apply the ultraviolet resin around the grin lens. Illuminate the area with ultraviolet light for 15 seconds to ensure that the resin becomes solid. Carefully remove the holder from the grin lens.Cover the entire exposed skull with ultraviolet resin. Place the head plate on the skull in the appropriate position. Illuminate the entire skull and head plate with ultraviolet light for 15 seconds.Cover the exposed grin lens with a 3D printed protective cap. Fix the cap to the head plate using M1.6 screws. Turn on the Miniscope control software and click Select Config File.Then, select vscode, followed by User Configuration Files. Now, click Run. Then, slide the LED power button to adjust the brightness.Slide the focus adjustment button to set the value near zero. Attach the base plate to the mini scope and readjust the position of the mini scope to obtain a field of view with good imaging quality. Carefully apply the first layer of denture base material around the mini scope base plate.Once the first layer of the cement becomes hardened, apply a second layer of cement from the base plate to the head plate. After all denture base material hardens, loosen the set screw and detach the mini scope from the base plate. Put the base plate cap into the base plate to protect the exposed grin lens and tighten the set screw.After imaging, convert the imaging data from AVI to HDF5 format. Apply the non-rigid motion correction and downsample the motion corrected movie. Apply extract on the downsample data to identify single cell signals.Then, apply cell check and manually select the potential cells. Click Save Data before closing the cell check window. Finally, save the data files.In unsuccessful in vivo calcium imaging, no active cells were observed in the imaging field of view. Successful in vivo calcium imaging showed fewer than 10 active cells without obvious blood vessels. More than 50 active cells with visible blood vessels.And hundreds of active cells with clear blood vessels. Extracted individual cells from a successful in vivo calcium imaging recording were superimposed on the field of view with representative calcium traces shown. The position of GCaMP6F expression and the track of the grin lens were verified in the mouse brain slice.