우리는 전자 제품에 대한 지원은 G의 qi5 및 G QS5 cDNA를, A. 슈바이처를 제공 B. 콘 클린 (캘리포니아 대학 샌프란시스코)를 감사, N. 테일러 증거 읽기 클론, 이안 Glaaser 로버트 리프킨의 심사에 대한 지원 및 TN-XXL에 대한 올리비에 Griesbeck. (; DA037170 DA029706), 바이오 메디컬 이미징 및 생물의 국립 연구소 (NIBIB) (EB003832), 호프만 - 라 로슈 (88610A)과 &quot;신경 과학이 작품은 약물 남용에 미국 국립 연구소 (NIDA)를 통해 연구비에 의해 지원되었다 NIDA (DA007315)를 통해 남용 &quot;훈련 보조금의 마약 관련.

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저자는 공개 아무것도 없어.

CNiFERs의 생성은 광학적으로 생체 내에서 뇌의 신경 전달 물질의 방출을 측정하기위한 혁신적이고 독특한 전략을 제공합니다. CNiFERs 이상적으로 신경 전달 물질에 대한 예 extrasynaptic 해제, 볼륨 전도를 측정하기에 적합합니다. 중요한 것은, 각 CNiFER은 뇌의 신경 전달 물질의 농도의 변화 생리 광 측정을 제공하는 천연 GPCR의 특성을 가지고있다. 지금까지 CNiFERs은 아세틸 콜린을 검출 ?...

완전히 신경 뇌에서 통신하는 방법을 이해하기 위해, 생체 내에서 신경 전달 물질의 방출을 측정하는 방법을 갖는 것이 필요하다. 생체 내에서 신경 전달 물질을 측정하기위한 몇 가지 잘 확립 된 기술이있다. 하나의 일반적으로 사용되는 기술은 캐뉼라 뇌에 삽입 뇌척수액 소량의 수집 및 고성능 액체 크로마토 그래피 및 전기 검사 1을 사용하여 분석되는 미세 투석이다. 미세 투석 프로브 몇 직경 정도의 공간 분해능을 가지며, 예를 들어 ~ 200 μm의 마이크로 프로브 직경 0.5 mm. 이 기법의 시간 해상도는, 그러나, 일반적으로 ~ 5 분 1 이상을 지속 샘플링 간격으로 느립니다. 또한, 분석은 실시간으로 제작되지 않습니다. 다른 기술은 뇌에 삽입되는 탄소 섬유 프로브를 사용하여 고속 스캐닝 순환 전압 전류 법 (FSCV)이다. FSCV 우수한 온도가구강 해상도 (초 단위), 고감도 (나노 몰), 30 μm의 5의 프로브 직경 공간 해상도. 그러나 FSCV는 탄소 전위차 프로브 (2)에 전압 특성 산화 및 환원 프로파일을 생성 송신기들로 제한된다. 신경 전달 물질을 측정하는 세 번째 기술은 유 전적으로 인코딩 된 신경 전달 물질 (NT) 바이오 센서 (3)를 통해 직접입니다. 이 방법으로 융합 단백질은 형광 공명 에너지 전달 (FRET) - 기반 형광체 (4)의 쌍 또는 순열 GFP (5)에 결합 된 송신기에 대한 리간드 결합 도메인을 포함하는 생성된다. 앞의 두 방법과 달리,이 바이오 센서는 전적으로 인코딩 된 형질 전환 동물의 생산을 통해 또는 급성 세포 감염 바이러스 제제의 사용과 같은 신경 세포와 같은 숙주 세포의 표면에 발현. 현재까지 유전자 부호화 바이오 센서 만 detectin 개발되어왔다g 글루타메이트와 GABA 3-5. 이러한 기술에 대한 제한은 nM의 범위에서 낮은 감도 및 G 단백질 결합 수용체 (GPCR에)를 통해 신호를 예를 들어 송신기, 고전 신경 전달 물질, 신경 펩티드 및 신경 조절의 다수 검출 확대 없게되었다. 실제로, 인간 게놈에서 거의 800 GPCRs에있다. 이러한 부족을 해결하기 위해, 우리는 GPCR을 통해 신호를 어떤 신경 전달 물질의 광학적 측정 자료에 혁신적인 도구를 개발했습니다. CNiFERs (셀 기반의 신경 전달 물질의 형광 설계 기자)가 자극하면 [Ca를 2+ 즉이 유전자 부호화 FRET 계 칼슘 센서에 의해 검출되어, 세포 내에서의 증가를 유발 특정 GPCR을 발현하도록 유전자 클론 HEK293 세포이다 TN-XXL. 따라서 CNiFERs은 신경 전달 물질 수용체, 형광의 변화에​​ 직접 결합 실시간 광 (R)을 제공하는 변환지역의 신경 전달 물질 활동의 EAD 아웃. 특정 신경 전달 물질에 대한 고유 수용체를 이용하여, CNiFERs 화학 특이성, 친 화성 및 내생 발현 수용체의 시간적 동성을 보유한다. 지금까지, 우리는 CNiFERs 세 가지 유형, 도파민을 검출하는 M1 수용체를 이용하여 아세틸 콜린의 검출을 작성한 D2 수용체를 사용하고 α1a 수용체 6,7을 사용하여 노르 에피네프린을 검출하기위한 하나. CNiFER 기술은 GPCR의 유형은 의무가있어 쉽게 확장과 확장 성이다. 이 조브 기사에서, 우리는 설명하고 모든 응용 프로그램에 대한 생체 CNiFERs의 설계, 실현 방법, 테스트를 보여줍니다.

<table><tbody><tr><td>pCDH-CMV-MCS-EF1-Puro&amp;nbsp;</td><td>System Biosciences</td><td>CD510B-1</td><td>Cloning: for generating lentivirus</td></tr><tr><td>12 x 75 *BD Falcon High Clarity Polypropylene Round Bottom Test Tube</td><td>BD Biosciences</td><td>352063</td><td>FACS</td></tr><tr><td>BD 40 um Falcon cell strainers</td><td>BD Biosciences</td><td>352340</td><td>FACS</td></tr><tr><td>0.05% Trypsin EDTA&amp;nbsp;</td><td>Invitrogen</td><td>25200056</td><td>FACS</td></tr><tr><td>96 Well Plate, flat bottom, clear&amp;nbsp;</td><td>Corning&amp;nbsp;</td><td>3596</td><td>FACS</td></tr><tr><td>96 well cell culture plates&amp;nbsp;</td><td>Corning&amp;nbsp;</td><td>CLS3997</td><td>Flexstation</td></tr><tr><td>Optilux black clear bottom&amp;nbsp;</td><td>Corning&amp;nbsp;</td><td>3603</td><td>Flexstation</td></tr><tr><td>Flexstation pipet tips</td><td>Molecular Devices</td><td>9000-0911</td><td>Flexstation</td></tr><tr><td>Acetylcholine Chloride&amp;nbsp;&amp;nbsp;</td><td>Sigma-Aldrich</td><td>A2661</td><td>Flexstation</td></tr><tr><td>Norepinephrine&amp;nbsp;&amp;nbsp;</td><td>Sigma-Aldrich</td><td>A7256</td><td>Flexstation</td></tr><tr><td>Dopamine Hydrochloride&amp;nbsp;&amp;nbsp;</td><td>Sigma-Aldrich</td><td>PHR1090</td><td>Flexstation</td></tr><tr><td>GABA&amp;nbsp;&amp;nbsp;</td><td>Sigma-Aldrich</td><td>A2129</td><td>Flexstation</td></tr><tr><td>Histamine&amp;nbsp;&amp;nbsp;</td><td>Sigma-Aldrich</td><td>H7125</td><td>Flexstation</td></tr><tr><td>Glutamate&amp;nbsp;&amp;nbsp;</td><td>Sigma-Aldrich</td><td>49621</td><td>Flexstation</td></tr><tr><td>Epinephrine&amp;nbsp;&amp;nbsp;</td><td>Sigma-Aldrich</td><td>E4642</td><td>Flexstation</td></tr><tr><td>Somatostatin&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>Sigma-Aldrich</td><td>S1763</td><td>Flexstation</td></tr><tr><td>5HT&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>Sigma-Aldrich</td><td>H9523</td><td>Flexstation</td></tr><tr><td>VIP&amp;nbsp;</td><td>Alpha Diagnostics Inc.&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>SP-69627</td><td>Flexstation</td></tr><tr><td>Orexin A</td><td>Alpha Diagnostics Inc.&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>12-p-01</td><td>Flexstation</td></tr><tr><td>Substance P&amp;nbsp;&amp;nbsp;</td><td>Sigma-Aldrich</td><td>S6883</td><td>Flexstation</td></tr><tr><td>Adenosine</td><td>Sigma-Aldrich</td><td>A4036</td><td>Flexstation</td></tr><tr><td>Melatonin&amp;nbsp;</td><td>Sigma-Aldrich</td><td>M5250C</td><td>Flexstation</td></tr><tr><td>Fluorescence Plate Reader &amp;amp; software</td><td>Molecular Devices</td><td>Flexstation 3</td><td>Flexstation</td></tr><tr><td>&amp;nbsp;DMEM (high glucose) with Glutamax&amp;nbsp;&amp;nbsp;</td><td>Life Technologies</td><td>10569-010</td><td>Tissue culture</td></tr><tr><td>&amp;nbsp;Fetal bovine serum</td><td>Life Technologies</td><td>10082-139</td><td>Tissue culture</td></tr><tr><td>&amp;nbsp;Pen/Strep&amp;nbsp; antibiotics</td><td>Life Technologies</td><td>15140-122</td><td>Tissue culture</td></tr><tr><td>&amp;nbsp;Puromycin&amp;nbsp;&amp;nbsp;</td><td>InvivoGen</td><td>ant-pr-1</td><td>Tissue culture</td></tr><tr><td>&amp;nbsp;Fibronectin&amp;nbsp;</td><td>Sigma-Aldrich</td><td>F0895</td><td>Tissue culture</td></tr><tr><td>CoolCell LX Alcohol-free controlled-rate cell freezing box</td><td>Bioexpress</td><td>D-3508)</td><td>Tissue culture</td></tr><tr><td>cyanoacrylate glue&amp;nbsp;</td><td>Loctite</td><td>Loctite no. 495</td><td>surgery and stereotaxic injection</td></tr><tr><td>plastic paraffin film&amp;nbsp;</td><td>VWR</td><td>Parafilm&amp;reg;</td><td>surgery and stereotaxic injection</td></tr><tr><td>Nanoinjector</td><td>Drummond</td><td>3-000-204</td><td>surgery and stereotaxic injection</td></tr><tr><td>Glass electrodes</td><td>Drummond</td><td>3-000-203G</td><td>surgery and stereotaxic injection</td></tr><tr><td>hand held drill</td><td>OSADA</td><td>Exl-M40</td><td>surgery and stereotaxic injection</td></tr><tr><td>Burrs for drill</td><td>Fine Scientific</td><td>19007-05; 19007-07)</td><td>surgery and stereotaxic injection</td></tr><tr><td>Sterilizing bath</td><td>FST</td><td>18000-45, Hot Bead Sterilizer</td><td>surgery and stereotaxic injection</td></tr><tr><td>isoflurane chamber/mask</td><td>Highland Medical Equipment</td><td>564-0427, HME 109 Table Top Anesthetic Machine with Isoflurane Vaporizer, O2 Flowmeter, Gang Valve; 564-0852, Induction Chamber 16X7X7.5cm</td><td>surgery and stereotaxic injection</td></tr><tr><td>3D scope with arm</td><td>Zeiss</td><td></td><td>surgery and stereotaxic injection</td></tr><tr><td>fiber optic light</td><td></td><td></td><td>surgery and stereotaxic injection</td></tr><tr><td>Betadine&amp;nbsp;</td><td></td><td></td><td>surgery and stereotaxic injection</td></tr><tr><td>70 % (v/v) isopropyl alcohol</td><td></td><td></td><td>surgery and stereotaxic injection</td></tr><tr><td>Povidone-Iodine Prep Pads</td><td>dynarex</td><td>1108</td><td>surgery and stereotaxic injection</td></tr><tr><td>NaCl 0.9% (injection, USP, 918610)</td><td></td><td></td><td>surgery and stereotaxic injection</td></tr><tr><td>CYCLOSPORINE (INJECTION, USP)</td><td></td><td></td><td>surgery and stereotaxic injection</td></tr><tr><td>Buprenex (injection) buprenorphine (0.03 &amp;mu;g per g rodent)</td><td>Sigma-Aldrich</td><td></td><td>surgery and stereotaxic injection</td></tr><tr><td>Ophthalmic ointment&amp;nbsp;</td><td>Akorn</td><td>NDC 17478-235-35</td><td>surgery and stereotaxic injection</td></tr><tr><td>Surgifoam</td><td>Ethicon</td><td></td><td>surgery and stereotaxic injection</td></tr><tr><td>Grip dental cement</td><td>Dentsply</td><td>#675571, 675572</td><td>surgery and stereotaxic injection</td></tr><tr><td>Instant SuperGlue&amp;nbsp;</td><td>NDindustries</td><td></td><td>surgery and stereotaxic injection</td></tr><tr><td>LOCTITE 4041</td><td></td><td></td><td>surgery and stereotaxic injection</td></tr><tr><td>METABOND</td><td>C&amp;amp;B</td><td></td><td>surgery and stereotaxic injection</td></tr><tr><td>no. 0 cover glass</td><td>Fisher</td><td></td><td>surgery and stereotaxic injection</td></tr><tr><td>stereotaxic frame&amp;nbsp;</td><td>Kopf</td><td></td><td>surgery and stereotaxic injection</td></tr><tr><td>Rectal probe and heating pad</td><td>FHC</td><td>40-90-8D, DC Temperature Controller,40-90-2-06, 6.5X9.5cm Heating Pad40-90-5D-02, Rectal Thermistor Probe</td><td>surgery and stereotaxic injection</td></tr><tr><td>optical breadboard for imaging</td><td>Thorlabs</td><td></td><td>surgery and stereotaxic injection</td></tr><tr><td>Mineral oil</td><td>Fisher</td><td>S55667</td><td>surgery and stereotaxic injection</td></tr><tr><td>Kwik-Cast (Silicone elastomer)</td><td>World Precision Instruments</td><td></td><td>surgery and stereotaxic injection</td></tr><tr><td>Suture&amp;nbsp;&amp;nbsp;</td><td>Ethicon</td><td>18&amp;rsquo;&amp;rsquo;, 1667, 4-0</td><td>surgery and stereotaxic injection</td></tr><tr><td>Scissors</td><td>Fine Scientific Tools</td><td>91500-09, 15018-10</td><td>surgery and stereotaxic injection</td></tr><tr><td>Forcepts</td><td>Fine Scientific Tools</td><td>11252-30; #55, 11295-51; Grafe, 11050-10</td><td>surgery and stereotaxic injection</td></tr><tr><td>Student Halsted-Mosquito Hemostats</td><td>Fine Scientific Tools</td><td>91308-12</td><td>surgery and stereotaxic injection</td></tr><tr><td>Small Vessel Cauterizer Kit</td><td>Fine Scientific Tools</td><td>18000-00</td><td>surgery and stereotaxic injection</td></tr><tr><td>Hot Bead Sterilizers</td><td>Fine Scientific Tools</td><td>18000-45</td><td>surgery and stereotaxic injection</td></tr><tr><td>Instrument Case with Silicone Mat</td><td>Fine Scientific Tools</td><td>20311-21</td><td>surgery and stereotaxic injection</td></tr><tr><td>Plastic Sterilization Containers with Silicone Mat</td><td>Fine Scientific Tools</td><td>20810-01</td><td>surgery and stereotaxic injection</td></tr><tr><td>2P fixed-stage fluorescence scope for &lt;em&gt;in vivo&lt;/em&gt; imaging</td><td>Olympus</td><td>FV1200 MPE</td><td>in vivo imaging</td></tr><tr><td>Multiphoton laser</td><td>SpectraPhysics</td><td>Mai Tai DeepSee</td><td>in vivo imaging</td></tr><tr><td>Green Laser</td><td>Olympus</td><td>473 nm Laser</td><td>in vivo imaging</td></tr><tr><td>xy translational base</td><td>Scientifica</td><td>MMBP</td><td>in vivo imaging</td></tr><tr><td>FRET filter cube for YFP and CFP</td><td>Olympus</td><td></td><td>in vivo imaging</td></tr><tr><td>10x and 40x water immersion objectives</td><td>Olympus</td><td></td><td>in vivo imaging</td></tr><tr><td>air table</td><td>Newport</td><td></td><td>in vivo imaging</td></tr><tr><td>custom built light-tight cage</td><td>Thorlab</td><td></td><td>in vivo imaging</td></tr></tbody></table>

construction of cell-based neurotransmitter fluorescent engineered reporters (cnifers) for optical detection of neurotransmitters in vivo

세포 기반 신경 전달 형광 설계 기자 (CNiFERs)는 신경 과학자 광학적 생체의 뇌에서 신경 전달 물질의 방출을 검출하기위한 새로운 수단을 제공한다. 특정 CNiFER 안정적 특정 G 단백질 결합 수용체, G의 Q에 커플 / 11 G 단백질 및 FRET 기반 칼슘) 검출기 (, TN-XXL를 표현하는 인간 배아 신장 세포에서 생성됩니다. 수용체의 활성화는 FRET 신호의 증가를 이끈다. CNiFER 클론은 도파민에 대한 D2R, 예를 들어, 특정 신경 전달 물질의 고유 수용체를 이용하기 때문에 CNiFERs는 nM의 감도와 초 시간 응답이 있습니다. CNiFERs 직접 뇌에 이식 생체 내에서 볼륨 전송을 측정하기에 이상적 미만 백 ㎛의 공간 해상도와 신경 전달 물질 방출을 감지 할 수 있도록하고 있습니다. CNiFERs 또한 VI 잠재적 교차 - 반응성에 대한 다른 약물을 스크리닝하는데 사용될 수있다VO. 우리는 최근의 GPCR에게 G I / O G 단백질에 그 부부를 포함 CNiFERs의 제품군을 확장했다. CNiFERs은 아세틸 콜린 (ACH), 도파민 (DA)과 노르 에피네프린 (NE)를 검출 할 수 있습니다. 어떤 GPCR이 CNiFER 소설을 생성하는데 사용될 수 있음을 감안할 때 대략 인간 게놈 800 GPCRs에 있다는 것을 우리는 설계, 실현 및 CNiFER 임의의 타입을 테스트하기 위해 여기 일반적인 방법을 설명한다.

특정 G 단백질 결합 수용체 (GPCR)와 유전자 코드 [칼슘 2 +] 센서, TN-XXL하십시오 CNiFER 안정적 적어도 두 개의 단백질을 발현하도록 유전자 조작 된 인간 배아 신장 (HEK293) 세포에서 파생됩니다. TN-XXL은 칼슘 이온 -6,15-의 질문에 답변 시안과 노란색 형광 단백질 각각 eCFP과 황수정, 사이의 형광 공명 에너지 전달 (FRET)을 겪는다. 내생 G q를 G 단?...

Watch this Scientific Journal Video about Construction of Cell-based Neurotransmitter Fluorescent Engineered Reporters CNiFERs for Optical Detection of Neurotransmitters In Vivo at JoVE.com

Construction of Cell-based Neurotransmitter Fluorescent Engineered Reporters CNiFERs for Optical Detection of Neurotransmitters In Vivo

우리는 체적 신경 전달 물질 방출의 광학 검출을위한 셀 기반의 신경 전달 물질의 형광 설계 기자 (CNiFERs)를 만들 수있는 프로토콜을 제시한다.

건설 셀 기반의 신경 전달 물질의 광학 검출을위한 신경 전달 물질 형광 엔지니어링 기자 (CNiFERs)<em&gt; 생체</em

Cell-based neurotransmitter fluorescent engineered reporters (CNiFERs) provide a new tool for neuroscientists to optically detect the release of ...

construction-cell-based-neurotransmitter-fluorescent-engineered

Research

JoVE Journal

Neuroscience

Construction of Cell-based Neurotransmitter Fluorescent Engineered Reporters (CNiFERs) for Optical Detection of Neurotransmitters In Vivo

13.1K 조회수.  Icahn School of Medicine at Mount Sinai.  우리는 체적 신경 전달 물질 방출의 광학 검출을위한 셀 기반의 신경 전달 물질의 형광 설계 기자 (CNiFERs)를 만들 수있는 프로토콜을 제시한다. 

비디오: Construction of Cell-based Neurotransmitter Fluorescent Engineered Reporters CNiFERs for Optical Detection of Neurotransmitters In Vivo

In vitro and in vivo Bioluminescence Reporter Gene Imaging of Human Embryonic Stem Cells

The discovery of human embryonic stem cells (hESCs) has dramatically increased the tools available to medical scientists interested in regenerative medicine. However, direct injection of hESCs, and cells differentiated from hESCs, into living organisms has thus far been hampered by significant cell death, teratoma formation, and host immune rejection. Understanding the in vivo hESC behavior after transplantation requires novel imaging techniques to longitudinally monitor hESC localization, proliferation, and viability. Molecular imaging has given investigators a high-throughput, inexpensive, and sensitive means for tracking in vivo cell proliferation over days, weeks, and even months. This advancement has significantly increased the understanding of the spatio-temporal kinetics of hESC engraftment, proliferation, and teratoma-formation in living subjects.

A major advance in molecular imaging has been the extension of noninvasive reporter gene assays from molecular and cellular biology into in vivo multi-modality imaging platforms. These reporter genes, under control of engineered promoters and enhancers that take advantage of the host cell s transcriptional machinery, are introduced into cells using a variety of vector and non-vector methods. Once in the cell, reporter genes can be transcribed either constitutively or only under specific biological or cellular conditions, depending on the type of promoter used. Transcription and translation of reporter genes into bioactive proteins is then detected with sensitive, noninvasive instrumentation (e.g., CCD cameras) using signal-generating probes such as D-luciferin.

To avoid the need for excitatory light to track stem cells in vivo as is required for fluorescence imaging, bioluminescence reporter gene imaging systems require only an exogenously administered probe to induce light emission. Firefly luciferase, derived from the firefly Photinus pyralis, encodes an enzyme that catalyzes D-luciferin to the optically active metabolite, oxyluciferin. Optical activity can then be monitored with an external CCD camera. Stably transduced cells that carry the reporter construct within their chromosomal DNA will pass the reporter construct DNA to daughter cells, allowing for longitudinal monitoring of hESC survival and proliferation in vivo. Furthermore, because expression of the reporter gene product is required for signal generation, only viable parent and daughter cells will create bioluminescence signal; apoptotic or dead cells will not. 

In this video, the specific materials and methods needed for tracking stem cell proliferation and teratoma formation with bioluminescence imaging will be described.

With the growing interest in stem cell therapies, molecular imaging techniques are ideal for monitoring stem cell behavior after transplantation. Luciferase reporter genes have enabled non-invasive, repetitive assessment of cell survival, location, and proliferation in vivo. This video will demonstrate how to track hESC proliferation in a living mouse.

체외에서 인간 배아 줄기 세포의 생체내의 Bioluminescence 리포터 진 영상에

The discovery of human embryonic stem cells (hESCs) has dramatically increased the tools available to medical scientists interested in regenerative ...

연구

생물학

Spectral Confocal Imaging of Fluorescently tagged Nicotinic Receptors in Knock-in Mice with Chronic Nicotine Administration

Ligand-gated ion channels in the central nervous system (CNS) are implicated in numerous conditions with serious medical and social consequences. For instance, addiction to nicotine via tobacco smoking is a leading cause of premature death worldwide (World Health Organization) and is likely caused by an alteration of ion channel distribution in the brain1. Chronic nicotine exposure in both rodents and humans results in increased numbers of nicotinic acetylcholine receptors (nAChRs) in brain tissue1-3. Similarly, alterations in the glutamatergic GluN1 or GluA1 channels have been implicated in triggering sensitization to other addictive drugs such as cocaine, amphetamines and opiates4-6.

Consequently, the ability to map and quantify distribution and expression patterns of specific ion channels is critically important to understanding the mechanisms of addiction. The study of brain region-specific effects of individual drugs was advanced by the advent of techniques such as radioactive ligands. However, the low spatial resolution of radioactive ligand binding prevents the ability to quantify ligand-gated ion channels in specific subtypes of neurons. 

Genetically encoded fluorescent reporters, such as green fluorescent protein (GFP) and its many color variants, have revolutionized the field of biology7.By genetically tagging a fluorescent reporter to an endogenous protein one can visualize proteins in vivo7-10. One advantage of fluorescently tagging proteins with a probe is the elimination of antibody use, which have issues of nonspecificity and accessibility to the target protein. We have used this strategy to fluorescently label nAChRs, which enabled the study of receptor assembly using F&ouml;rster Resonance Energy Transfer (FRET) in transfected cultured cells11.More recently, we have used the knock-in approach to engineer mice with yellow fluorescent protein tagged &alpha;4 nAChR subunits (&alpha;4YFP), enabling precise quantification of the receptor ex vivo at submicrometer resolution in CNS neurons via spectral confocal microscopy12. The targeted fluorescent knock-in mutation is incorporated in the endogenous locus and under control of its native promoter, producing normal levels of expression and regulation of the receptor when compared to untagged receptors in wildtype mice. This knock-in approach can be extended to fluorescently tag other ion channels and offers a powerful approach of visualizing and quantifying receptors in the CNS.

In this paper we describe a methodology to quantify changes in nAChR expression in specific CNS neurons after exposure to chronic nicotine. Our methods include mini-osmotic pump implantation, intracardiac perfusion fixation, imaging and analysis of fluorescently tagged nicotinic receptor subunits from &alpha;4YFP knock-in mice (Fig. 1). We have optimized the fixation technique to minimize autofluorescence from fixed brain tissue.We describe in detail our imaging methodology using a spectral confocal microscope in conjunction with a linear spectral unmixing algorithm to subtract autofluoresent signal in order to accurately obtain &alpha;4YFP fluorescence signal. Finally, we show results of chronic nicotine-induced upregulation of &alpha;4YFP receptors in the medial perforant path of the hippocampus.

We have developed a novel technique of quantifying nicotinic acetylcholine receptor changes within subcellular regions of specific subtypes of CNS neurons to better understand the mechanisms of nicotine addiction by using a combination of approaches including fluorescent protein tagging of the receptor using the knock-in approach and spectral confocal imaging.

만성 니코틴 관리와 마우스 노크 인의 Fluorescently 태그를 Nicotinic 수용체의 분광 공촛점 이미징

Ligand-gated ion channels in the central nervous system (CNS) are implicated in numerous conditions with serious medical and social consequences.  For ...

신경과학

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from individual neurons are critical for understanding how neural circuits function under natural conditions. Traditionally, these recordings have been performed 'blind', meaning the identity of the recorded cell is unknown at the start of the recording. Cellular identity can be subsequently determined via intracellular1, juxtacellular2 or loose-patch3 iontophoresis of dye, but these recordings cannot be pre-targeted to specific neurons in regions with functionally heterogeneous cell types. Fluorescent proteins can be expressed in a cell-type specific manner permitting visually-guided single-cell electrophysiology4-6. However, there are many model systems for which these genetic tools are not available. Even in genetically accessible model systems, the desired promoter may be unknown or genetically homogenous neurons may have varying projection patterns. Similarly, viral vectors have been used to label specific subgroups of projection neurons7, but use of this method is limited by toxicity and lack of trans-synaptic specificity. Thus, additional techniques that offer specific pre-visualization to record from identified single neurons in vivo are needed. Pre-visualization of the target neuron is particularly useful for challenging recording conditions, for which classical single-cell recordings are often prohibitively difficult8-11. The novel technique described in this paper uses retrograde transport of a fluorescent dye applied using tungsten needles to rapidly and selectively label a specific subset of cells within a particular brain region based on their unique axonal projections, thereby providing a visual cue to obtain targeted electrophysiological recordings from identified neurons in an intact circuit within a vertebrate CNS.
The most significant novel advancement of our method is the use of fluorescent labeling to target specific cell types in a non-genetically accessible model system. Weakly electric fish are an excellent model system for studying neural circuits in awake, behaving animals12. We utilized this technique to study sensory processing by "small cells" in the anterior exterolateral nucleus (ELa) of weakly electric mormyrid fish. "Small cells" are hypothesized to be time comparator neurons important for detecting submillisecond differences in the arrival times of presynaptic spikes13. However, anatomical features such as dense myelin, engulfing synapses, and small cell bodies have made it extremely difficult to record from these cells using traditional methods11, 14. Here we demonstrate that our novel method selectively labels these cells in 28% of preparations, allowing for reliable, robust recordings and characterization of responses to electrosensory stimulation.

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

Retrograde transport of fluorescent dye labels a sub-population of neurons based on anatomical projection. Labeled axons can be visually targeted in vivo, permitting extracellular recording from identified axons. This technique facilitates recording when neurons cannot be labeled through genetic manipulation or are difficult to isolate using 'blind' in vivo approaches.

역행 형광 라벨이 확인 된 신경 세포의 표적 세포 외 단일 단위 기록을 허용<em&gt; 생체 내</em

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from ...

An Enhanced Green Fluorescence Protein-based Assay for Studying Neurite Outgrowth in Primary Neurons

Neurite outgrowth is a fundamental event in the formation of the neural circuits during nervous system development. Severe neurite damage and synaptic dysfunction occur in various neurodegenerative diseases and age-related degeneration. Investigation of the mechanisms that regulate neurite outgrowth would not only shed valuable light on brain developmental processes but also on such neurological disorders. Due to the low transfection efficiency, it is currently challenging to study the effect of a specific protein on neurite outgrowth in primary mammalian neurons. Here, we describe a simple method for the investigation of neurite outgrowth by the co-transfection of primary rat cortical neurons with EGFP and a protein of interest (POI). This method allows the identification of POI transfected neurons through the EGFP signal, and thus the effect of the POI on neurite outgrowth can be determined precisely. This EGFP-based assay provides a convenient approach for the investigation of pathways regulating neurite outgrowth.

In this report, we describe a simple protocol for studying neurite outgrowth in embryonic rat cortical neurons by co-transfecting with EGFP and the protein of interest.

1 차적인 뉴런에 있는 Neurite 파생을 공부를 위한 향상된 녹색 형광 단백질 기지를 둔 분석

Neurite outgrowth is a fundamental event in the formation of the neural circuits during nervous system development. Severe neurite damage and synaptic ...

발생학

TIRFM and pH-sensitive GFP-probes to Evaluate Neurotransmitter Vesicle Dynamics in SH-SY5Y Neuroblastoma Cells: Cell Imaging and Data Analysis

Synaptic vesicles release neurotransmitters at chemical synapses through a dynamic cycle of fusion and retrieval. Monitoring synaptic activity in real time and dissecting the different steps of exo-endocytosis at the single-vesicle level are crucial for understanding synaptic functions in health and disease.
Genetically-encoded pH-sensitive probes directly targeted to synaptic vesicles and Total Internal Reflection Fluorescence Microscopy (TIRFM) provide the spatio-temporal resolution necessary to follow vesicle dynamics. The evanescent field generated by total internal reflection can only excite fluorophores placed in a thin layer (&#60;150 nm) above the glass cover on which cells adhere, exactly where the processes of exo-endocytosis take place. The resulting high-contrast images are ideally suited for vesicles tracking and quantitative analysis of fusion events.
In this protocol, SH-SY5Y human neuroblastoma cells are proposed as a valuable model for studying neurotransmitter release at the single-vesicle level by TIRFM, because of their flat surface and the presence of dispersed vesicles. The methods for growing SH-SY5Y as adherent cells and for transfecting them with synapto-pHluorin are provided, as well as the technique&#160;to perform TIRFM and imaging. Finally, a strategy aiming to select, count, and analyze fusion events at whole-cell and single-vesicle levels is presented.
To validate the imaging procedure and data analysis approach, the dynamics of pHluorin-tagged vesicles are&#160;analyzed under resting and stimulated (depolarizing potassium concentrations) conditions. Membrane depolarization increases the frequency of fusion events and causes a parallel raise of the net fluorescence signal recorded in whole cell. Single-vesicle analysis reveals modifications of fusion-event behavior (increased peak height and width). These data suggest that potassium depolarization not only induces a massive neurotransmitter release but also modifies the mechanism of vesicle fusion and recycling.
With the appropriate fluorescent probe, this technique can be employed in different cellular systems to dissect the mechanisms of constitutive and stimulated secretion.

This paper provides a method for investigating neurotransmitter vesicle dynamics in neuroblastoma cells, using a synaptobrevin2-pHluorin construct and Total Internal Reflection Fluorescence Microscopy. The strategy developed for image processing and data analysis is also reported.

TIRFM과 pH 민감성 GFP - 프로브는 SH-SY5Y 신경 모세포종 세포에서 신경 전달 물질 소포 역학을 평가하기 : 세포 이미징 및 데이터 분석

Synaptic vesicles release neurotransmitters at chemical synapses through a dynamic cycle of fusion and retrieval. Monitoring synaptic activity in real ...

In Vivo Functional Brain Imaging Approach Based on Bioluminescent Calcium Indicator GFP-aequorin

Functional in vivo imaging has become a powerful approach to study the function and physiology of brain cells and structures of interest. Recently a new method of Ca2+-imaging using the bioluminescent reporter GFP-aequorin (GA) has been developed. This new technique relies on the fusion of the GFP and aequorin genes, producing a molecule capable of binding calcium and&#160;&#8212; with the addition of its cofactor coelenterazine &#8212; emitting bright light that can be monitored through a photon collector. Transgenic lines carrying the GFP-aequorin gene have been generated for both mice and Drosophila. In Drosophila, the GFP-aequorin gene has been placed under the control of the GAL4/UAS binary expression system allowing for targeted expression and imaging within the brain. This method has subsequently been shown to be capable of detecting both inward Ca2+-transients and Ca2+-released from inner stores. Most importantly it allows for a greater duration in continuous recording, imaging at greater depths within the brain, and recording at high temporal resolutions (up to 8.3 msec). Here we present the basic method for using bioluminescent imaging to record and analyze Ca2+-activity within the mushroom bodies, a structure central to learning and memory in the fly brain.

In Vivo Functional Brain Imaging Approach Based on Bioluminescent Calcium Indicator GFP-aequorin

Here we present a novel Ca2+-imaging approach using a bioluminescent reporter. This approach uses a fused construct GFP-aequorin which binds to Ca2+ and emits light, eliminating the need for light excitation. Significantly this method permits long continuous imaging, access to deep brain structures and high temporal resolution.

발광 생물 칼슘 표시 GFP - 쿼린을 기반으로 생체 기능성 뇌 영상의 접근 방식

Functional in vivo imaging has become a powerful approach to study the function and physiology of brain cells and structures of interest. Recently a new ...

In Vivo Monitoring of Circadian Clock Gene Expression in the Mouse Suprachiasmatic Nucleus Using Fluorescence Reporters

This technique combines optical fiber mediated fluorescence recordings with the precise delivery of recombinant adeno-associated virus based gene reporters. This new and easy to use in vivo fluorescence monitoring system was developed to record the transcriptional rhythm of the clock gene, Cry1, in the suprachiasmatic nucleus (SCN) of freely moving mice. To do so, a Cry1 transcription fluorescence reporter was designed and packaged into Adeno-associated virus. Purified, concentrated virus was injected into the mouse SCN followed by the insertion of an optic fiber, which was then fixed onto the surface of the brain. The animals were returned to their home cages and allowed a 1-month post-operative recovery period to ensure sufficient reporter expression. Fluorescence was then recorded in freely moving mice via an in vivo monitoring system that was constructed at our institution. For the in vivo recording system, a 488 nm laser was coupled with a 1 &#215; 4 beam-splitter that divided the light into four laser excitation outputs of equal power. This setup enabled us to record from four animals simultaneously. Each of the emitted fluorescence signals was collected via a photomultiplier tube and a data acquisition card. In contrast to the previous bioluminescence in vivo circadian clock recording technique, this fluorescence in vivo recording system allowed the recording of circadian clock gene expression during the light cycle.

In Vivo Monitoring of Circadian Clock Gene Expression in the Mouse Suprachiasmatic Nucleus Using Fluorescence Reporters

This newly developed fluorescence-based technology enables long-term monitoring of the transcription of circadian clock genes in the suprachiasmatic nucleus (SCN) of freely moving mice in real-time and at a high temporal resolution.

Vivo에서 형광 기자를 사용 하 여 마우스 Suprachiasmatic 핵에서 Circadian 시계 유전자 발현의 모니터링

This technique combines optical fiber mediated fluorescence recordings with the precise delivery of recombinant adeno-associated virus based gene ...

Real-Time Fluorescent Measurement of Synaptic Functions in Models of Amyotrophic Lateral Sclerosis

Before neuronal degeneration, the cause of motor and cognitive deficits in patients with amyotrophic lateral sclerosis (ALS) and/or frontotemporal lobe dementia (FTLD) is dysfunction of communication between neurons and motor neurons and muscle. The underlying process of synaptic transmission involves membrane depolarization-dependent synaptic vesicle fusion and the release of neurotransmitters into the synapse. This process occurs through localized calcium influx into the presynaptic terminals where synaptic vesicles reside. Here, the protocol describes fluorescence-based live-imaging methodologies that reliably report depolarization-mediated synaptic vesicle exocytosis and presynaptic terminal calcium influx dynamics in cultured neurons.
Using a styryl dye that is incorporated into synaptic vesicle membranes, the synaptic vesicle release is elucidated. On the other hand, to study calcium entry, Gcamp6m is used, a genetically encoded fluorescent reporter. We employ high potassium chloride-mediated depolarization to mimic neuronal activity. To quantify synaptic vesicle exocytosis unambiguously, we measure the loss of normalized styryl dye fluorescence as a function of time. Under similar stimulation conditions, in the case of calcium influx, Gcamp6m fluorescence increases. Normalization and quantification of this fluorescence change are performed in a similar manner to the styryl dye protocol. These methods can be multiplexed with transfection-based overexpression of fluorescently tagged mutant proteins. These protocols have been extensively used to study synaptic dysfunction in models of FUS-ALS and C9ORF72-ALS, utilizing primary rodent cortical and motor neurons. These protocols easily allow for rapid screening of compounds that may improve neuronal communication. As such, these methods are valuable not only for the study of ALS but for all areas of neurodegenerative and developmental neuroscience research.

Two related methods are described to visualize subcellular events required for synaptic transmission. These protocols enable the real-time monitoring of the dynamics of presynaptic calcium influx and synaptic vesicle membrane fusion using live-cell imaging of in vitro cultured neurons.

근위축성 측삭 경화증 모델에서 시냅스 기능의 실시간 형광 측정

Before neuronal degeneration, the cause of motor and cognitive deficits in patients with amyotrophic lateral sclerosis (ALS) and/or frontotemporal lobe ...

Expanding the Toolkit for In Vivo Imaging of Axonal Transport

Axonal transport maintains neuronal homeostasis by enabling the bidirectional trafficking of diverse organelles and cargoes. Disruptions in axonal transport have devastating consequences for individual neurons and their networks, and contribute to a plethora of neurological disorders. As many of these conditions involve both cell autonomous and non-autonomous mechanisms, and often display a spectrum of pathology across neuronal subtypes, methods to accurately identify and analyze neuronal subsets are imperative.
This paper details protocols to assess in vivo axonal transport of signaling endosomes and mitochondria in sciatic nerves of anesthetized mice. Stepwise instructions are provided to 1) distinguish motor from sensory neurons in vivo, in situ, and ex vivo by using mice that selectively express fluorescent proteins within cholinergic motor neurons; and 2) separately or concurrently assess in vivo axonal transport of signaling endosomes and mitochondria. These complementary intravital approaches facilitate the simultaneous imaging of different cargoes in distinct peripheral nerve axons to quantitatively monitor axonal transport in health and disease.

Expanding the Toolkit for In Vivo Imaging of Axonal Transport

Using transgenic fluorescent mice, detailed protocols are described to assess in vivo axonal transport of signaling endosomes and mitochondria within motor and sensory axons of the intact sciatic nerve in live animals.

축삭 수송의 생체 내 이미징을위한 툴킷 확장

Axonal transport maintains neuronal homeostasis by enabling the bidirectional trafficking of diverse organelles and cargoes. Disruptions in axonal ...

Genetic Barcoding with Fluorescent Proteins for Multiplexed Applications

Fluorescent proteins, fluorescent dyes and fluorophores in general have revolutionized the field of molecular cell biology. In particular, the discovery of fluorescent proteins and their genes have enabled the engineering of protein fusions for localization, the analysis of transcriptional activation and translation of proteins of interest, or the general tracking of individual cells and cell populations. The use of fluorescent protein genes in combination with retroviral technology has further allowed the expression of these proteins in mammalian cells in a stable and reliable manner. Shown here is how one can utilize these genes to give cells within a population of cells their own biosignature. As the biosignature is achieved with retroviral technology, cells are barcoded &#180;indefinitely&#180;. As such, they can be individually tracked within a mixture of barcoded cells and utilized in more complex biological applications. The tracking of distinct populations in a mixture of cells is ideal for multiplexed applications such as discovery of drugs against a multitude of targets or the activation profile of different promoters. The protocol describes how to elegantly develop and amplify barcoded mammalian cells with distinct genetic fluorescent markers, and how to use several markers at once or one marker at different intensities. Finally, the protocol describes how the cells can be further utilized in combination with cell-based assays to increase the power of analysis through multiplexing.

Since the discovery of the green fluorescent protein gene, fluorescent proteins have impacted molecular cell biology. This protocol describes how expression of distinct fluorescent proteins through genetic engineering is used for barcoding individual cells. The procedure enables tracking distinct populations in a cell mixture, which is ideal for multiplexed applications.

멀티 플렉스 애플리케이션을위한 형광 단백질 유전자 바코드

Fluorescent proteins, fluorescent dyes and fluorophores in general have revolutionized the field of molecular cell biology. In particular, the discovery ...

건설 셀 기반의 신경 전달 물질의 광학 검출을위한 신경 전달 물질 형광 엔지니어링 기자 (CNiFERs)

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