우리는이 원고의 중요한 검토 박사 Sanika Chirwa 감사드립니다. 이 작품은 건강의 국립 연구소 (DA026947, DA021471 및 NS071122)에 의해 지원되었다.

1. 장비 및 용품 <ol><li> 배경 잡음을 줄일 수있는 안티 진동 테이블 (TMI)의 상단에 패러데이 케이지를 탑재합니다. </li><li> 동시 패치 클램프 amperometry 기록 시스템은 우수한 DIC 광학 및 긴 작동 거리 렌즈와 역 현미경이 필요합니다. 자동차 배터리에 등대 현미경을 연결합니다. 시스템이 DC 광원은 또한 전기 노이즈를 감소합니다. </li><li> 유압 마이크로 (Siskiyou) 추가 감소 소음. 우리의 구성에서, 우리는 전체 셀 녹음 오른쪽 손잡이 속이는, 그리고 amperometry에 넘겨 왼쪽을 사용합니다. </li></ol> 2. 녹음을위한 전극을 준비 <ol><li> P-2000 풀러 (셔터)에 석영 피펫을 사용하여 패치 전극을 당겨. 우리 풀다운 두 열 사이클과 함께 약 5 초 정도 소요됩니다. 이 열 시간은 우리의 전체 세포 패치 피펫에 일관성 저항 (MΩ 3-4) 결과입니다. </li><li> 로 전극을 기입2 밀리미터 도파민과 오른쪽 속이는에 마운트를 포함하는 피펫 솔루션입니다. 알루미늄 호일로 DA를 포함하는 피펫 솔루션을 가지고있는 용기를 넣어. 얼음 계속. 도파민은 oxidizable입니다. 빛으로부터 보호 얼음에 솔루션을 유지하는 것은 도파민의 산화 속도를 감소합니다. </li><li> 부드럽게, 저장 상자에서 ProCFE (Dagan) 저소음 탄소 섬유 amperometric 전극을 제거 amperometric 어댑터 (그림 1에 묘사)에 수은, 마운트로 입력하고 다음 오른쪽 maniupulator에 탑재합니다. 탄소 섬유의 맨 끝을 잡고 손상에서 탄소 섬유의 끝을 보호합니다. 끝이 깨끗하고 손상되지 않도록하기 위해 실험실 현미경으로 전극을 검사합니다. </li><li> 외부 솔루션을 포함하는 유리 바닥 배양 접시에 전극을 넣어 amperometric 전극의 무결성을 검사합니다. 도파민의 부재에서 기준 전류를 기록합니다. 요리에 1mM DA 솔루션 10 μl를 추가합니다. 좋은 amperometric 전극 재코드 산화 현재의 증가. 특정 amperometric 전극이 제대로 작동하기 위해 각 실험의 시작과 끝에서이 단계를 반복합니다. </li></ol> 3. 유리 바닥 배양 접시에 도파민 전송을 표현하는 엔지니어링 도파민 뉴런 또는 셀의 기본 Neuronal 문화 준비 <ol><li> 부드럽게 따뜻한 외부 솔루션을 셀이나 도파민 뉴런을 세 번 씻는다. </li><li> 현미경 단계에 유리 바닥 페트리 접시를 탑재합니다. </li></ol> 4. 셀을 시각화 및 실험을 수행 <ol><li> 명확하게 세포를 시각화 할 수있는 올바른 초점을 찾아보세요. 패치 전극에 긍정적 인 압력을 넣습니다. 그런 다음, 부드럽게 솔루션으로 두 전극을 가지고 있으며, 셀을 닫습니다. </li><li> 셀 (왼쪽), 그리고 오른쪽에있는 패치 전극 옆에있는 amperometric 전극을 배치합니다. </li><li> 패치 전극과 같은 감방에 gigaohm 인감을 충족해야합니다. 에 흡입과 파열 인감전체 셀 구성을 얻을 수 있습니다. </li><li> 셀에 도파민을 포함하는 내부 솔루션의 투석을위한 5-8 분 소요됩니다. </li><li> 원하는 전압 단계 또는 램프 프로토콜을 사용합니다. 막 가능성이 패치 피펫을 통해 제어되는 동안 동시에 패치 피펫 및 전체 셀 전류를 측정하고 도파민 수송을 통해 도파민의 전송을 바꿀 수있는 amperometric 전극 모두에서 데이터를 수집. </li></ol>

<ol>
	<li>Michael, A. C., Ikeda, M., Justice, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+contributing+to+the+recovery+of+striatal+releasable+dopamine+following+MFB+stimulation.">Mechanisms contributing to the recovery of striatal releasable dopamine following MFB stimulation.</a> Brain Res. 421, 325-335 (1987).</li><li>Gonon, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prolonged+and+extrasynaptic+excitatory+action+of+dopamine+mediated+by+D1+receptors+in+the+rat+striatum+in+vivo.">Prolonged and extrasynaptic excitatory action of dopamine mediated by D1 receptors in the rat striatum in vivo.</a> J. Neurosci. 17, 5972-5978 (1997).</li><li>Sulzer, D., Pothos, E. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+quantal+size+by+presynaptic+mechanisms.">Regulation of quantal size by presynaptic mechanisms.</a> Rev. Neurosci. 11, 159-212 (2000).</li><li>Napolitano, A., Cesura, A. M., Da Prada, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+monoamine+oxidase+and+catechol+O-methyltransferase+in+dopaminergic+neurotransmission.">The role of monoamine oxidase and catechol O-methyltransferase in dopaminergic neurotransmission.</a> J. Neural. Transm. Suppl. 45, 35-45 (1995).</li><li>Sulzer, D., Maidment, N. T., Rayport, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amphetamine+and+other+weak+bases+act+to+promote+reverse+transport+of+dopamine+in+ventral+midbrain+neurons.">Amphetamine and other weak bases act to promote reverse transport of dopamine in ventral midbrain neurons.</a> J. Neurochem. 60, 527-535 (1993).</li><li>Salahpour, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+amphetamine-induced+hyperactivity+and+reward+in+mice+overexpressing+the+dopamine+transporter.">Increased amphetamine-induced hyperactivity and reward in mice overexpressing the dopamine transporter.</a> Proc. Natl. Acad. Sci. U.S.A. 105, 4405-4410 (2008).</li><li>Khoshbouei, H., Wang, H., Lechleiter, J. D., Javitch, J. A., Galli, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amphetamine-induced+dopamine+efflux.+A+voltage-sensitive+and+intracellular+Na+-dependent+mechanism.">Amphetamine-induced dopamine efflux. A voltage-sensitive and intracellular Na+-dependent mechanism.</a> J. Biol. Chem. 278, 12070-12077 (2003).</li><li>Goodwin, J. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amphetamine+and+methamphetamine+differentially+affect+dopamine+transporters+in+vitro+and+in.">Amphetamine and methamphetamine differentially affect dopamine transporters in vitro and in.</a> , 284-2978 (2009).</li><li>Kahlig, K. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amphetamine+induces+dopamine+efflux+through+a+dopamine+transporter+channel.">Amphetamine induces dopamine efflux through a dopamine transporter channel.</a> Proc. Natl. Acad. Sci. U.S.A. 102, 3495-3500 (2005).</li><li>Swant, J., Chirwa, S., Stanwood, G., Khoshbouei, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methamphetamine+reduces+LTP+and+increases+baseline+synaptic+transmission+in+the+CA1+region+of+mouse+hippocampus.">Methamphetamine reduces LTP and increases baseline synaptic transmission in the CA1 region of mouse hippocampus.</a> PLoS One. 5, e11382 (2010).</li><li>Gnegy, M. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracellular+Ca2++regulates+amphetamine-induced+dopamine+efflux+and+currents+mediated+by+the+human+dopamine+transporter.">Intracellular Ca2+ regulates amphetamine-induced dopamine efflux and currents mediated by the human dopamine transporter.</a> Mol. Pharmacol. 66, 137-143 (2004).</li></ol>

관심 없음 충돌이 선언 없습니다.

동시 전압 클램프와 amperometry은 다음과 같은 이점이 있습니다. 모든 세포 유형은 액세스 할 수 있습니다 및 녹음에 사용할 수 있습니다. 녹음이 완료되어있는 셀이나 신경 세포의 식별은 간단하고 간단합니다. 셀이 휘황 관심 단백질에 형광 태그를 추가하여 표시하는 경우 특히, 실험 쉽게 대상 셀이나 신경을 선택할 수 있습니다. 실험 구성은 패치 피펫을 통해, 또는 목욕 솔루션 10이 요?...

<table border="1"><tbody><tr><td></td><td></td><td></td><td> 장비 </td></tr><tr><td> 방진 테이블 w / 패러데이 케이지 </td><td> 기술 제조 기업 </td><td> 63-500 시리즈 </td><td> 우리는 모델 63-543를 사용 </td></tr><tr><td> 역 현미경 니콘 TE-2000 </td><td> 니콘 </td><td> 중단 </td><td> 지금 이클립스 티 </td></tr><tr><td> 두 로우 노이즈 앰프 axopatch 200b </td><td> 분자 장치 </td><td></td><td> 800-635-5577 </td></tr><tr><td> 1 CV 203 BU의 headstage </td><td> 분자 장치 </td><td></td><td> 800-635-5578 </td></tr><tr><td> 1-HL-U 피펫 홀더 </td><td> 분자 장치 </td><td></td><td> 800-635-5579 </td></tr><tr><td> Digidata 1440A A / D 변환기 </td><td> 분자 장치 </td><td></td><td> 800-635-5580 </td></tr><tr><td> 두 manipulators Siskyou, 왼쪽으로오른쪽 넘겨 </td><td> Siskiyou </td><td> MX6600R MX6600L </td><td> 877-313-6418 </td></tr><tr><td> 레이저 피펫 풀러 </td><td> 셔터 악기 </td><td> P-2000 </td><td> 888-883-0128 </td></tr><tr><td> 낮은 소음 탄소 섬유 amperometric 전극 </td><td> ProCFE </td><td></td><td> <a href="http://www.dagan.com" target="_blank">www.dagan.com</a> </td></tr><tr><td> 낮은 소음 석영 피펫 </td><td> 셔터 악기 </td><td> QF100-70-7.5 </td><td> 888-883-0128 </td></tr><tr><td> 12 볼트 자동차 배터리 </td><td></td><td></td><td> 널리 사용되는 </td></tr><tr><td> 자동차 배터리 충전기 </td><td></td><td></td><td> 널리 사용되는 </td></tr><tr><td></td><td></td><td></td><td> 시약 </td></tr><tr><td> 나트륨 염화물 (NaCl) </td><td> 시그마 </td><td> S7653 </td><td></td></tr><tr><td> HEPES </td><td> 시그마 </td><td> H3375 </td><td></td></tr><tr><td> 우선 당 </td><td> 시그마 </td><td> G7528 </td><td></td></tr><tr><td> 황산 마그네슘 (MgSO 4) </td><td> 시그마 </td><td> M2643 </td><td></td></tr><tr><td> 인산 칼륨 일 염기의 (KH 2 PO 4) </td><td> 시그마 </td><td> P5655 </td><td></td></tr><tr><td> 염화칼륨 (KCl) </td><td> 시그마 </td><td> P9333 </td><td></td></tr><tr><td> 칼슘 염화물 이수화 (CaCl 2 • 2H 2 0) </td><td> 시그마 </td><td> 223506 </td><td></td></tr><tr><td> 마그네슘 염화물 hexahydrate (MgCl 2 • 6H 2 0) </td><td> 시그마 </td><td> M2670 </td><td></td></tr><tr><td> EGTA </td><td> 시그마 </td><td> E0396 </td><td></td></tr></tbody></table>

single cell measurement of dopamine release with simultaneous voltage-clamp and amperometry

시냅스 갈라진 틈, 도파민에 출시 된 이후의 사전 및 사후 신경 목표 1을 통해 자사의 생물학적 특성을 발휘. 도파민 신호는 확산 2-3, 세포 효소 4, 막 수송 (5)에 의해 종료됩니다. 도파민 뉴런의 사망시 신경 분열에 위치한 도파민 수송은 안쪽 도파민 유량 (통풍 관)를 통해 출시 아민을 지 웁니다. 도파민 수송은 바깥쪽으로 교통이나 도파민 5 유출이라는 프로세스에서 내부에서 외부로 아민을 공개 역방향으로 작업 할 수 있습니다. . 앞으로 (통풍 관) 및 역방향 (유출) 5 : 20 년 이상 전 Sulzer 외는 도파민 수송이 두 활동의 모드에서 작동 할 수 있습니다 발표했다. 전송을 통해 유출을 통해 발표 신경 전달 물질은 세포 공간으로 도파민 많은 양의 이동 할 수 있으며, 세포 도파민 H의 주요 규제 역할을 보여왔다omeostasis 6. 여기 동시 패치 클램프 및 amperometry 녹음이 막 잠재력이 통제 밀리 초 시간 해상도 유출 메커니즘을 통해 출시 도파민을 측정하는 데 사용할 수있는 방법에 대해 설명합니다. 이 경우 전체 세포 현재와 산화 (amperometric) 신호는 Axopatch 200B 앰프 (분자 장치, 전체 셀 현재 기록은 1,000 Hz에서 로우 패스 베셀 필터가 설정된)를 사용하여 동시에 측정합니다. 탄소 섬유 전극을 기록 amperometry는 두 번째 앰프 (Axopatch 200B)에 연결되어 있고 플라즈마 막에 인접 배치하고 700 뮤직 비디오에서 개최 때문입니다. 전체 셀과 산화 (amperometric) 전류가 기록 할 수 있으며, 전류 전압 관계는 전압 단계 프로토콜을 사용하여 생성 할 수 있습니다. 농도로 변환을 필요로하는 일반적인 amperometric 보정 달리, 현재는 효과적인 볼륨 7 고려하지 않고 직접보고됩니다. 따라서, 결과 데이터일부 송신기는 대량 솔루션으로 손실되기 때문에 도파민 유출에 하한을 나타냅니다.

amperometry와 함께 패치 클램프 전압에 따라 달라 DAT로 인한 DA 유출. 그림 2A는 세포 내 환경과 막 잠재력이 전체 셀 패치 피펫으로 고정 아르 DAT로 인한 DA 유출의 대표 실험 구성 및 기록을 보여줍니다 측정 할 수 있습니다. 이 기법 사용, YFP - DAT 단백질을 표현 세포는 전압 고정 amperometric 전극이 플라즈마 막 (그림 2A)에 위치하는 동안 전체 세포 패치 피펫으로. 전체 전지 전...

Watch this Scientific Journal Video about Single Cell Measurement of Dopamine Release with Simultaneous Voltage-clamp and Amperometry at JoVE.com

Single Cell Measurement of Dopamine Release with Simultaneous Voltage-clamp and Amperometry

amperometric 기술은 자연 도파민의 산화에 의해 생성 된 산화 전류를 검출하여 하나의 세포에서 도파민 릴리스를 측정합니다. 동시 전압 클램프 및 amperometry 방법론은 도파민 수송과 도파민의 역방향 전송에서이 활동의​​ 규제 역할의 전체 &#39;활동&#39;사이의 기계론의 관계를 알 수있다.

동시 전압 클램프 및 Amperometry과 도파민 릴리스의 단일 세포 측정

After its release into the synaptic cleft, dopamine exerts its biological properties via its pre- and post-synaptic targets1. The dopamine signal is ...

single-cell-measurement-dopamine-release-with-simultaneous-voltage

Research

JoVE Journal

Neuroscience

13.7K 조회수.  University of Florida. amperometric 기술은 자연 도파민의 산화에 의해 생성 된 산화 전류를 검출하여 하나의 세포에서 도파민 릴리스를 측정합니다. 동시 전압 클램프 및 amperometry 방법론은 도파민 수송과 도파민의 역방향 전송에서이 활동의​​ 규제 역할의 전체 '활동'사이의 기계론의 관계를 알 수있다.

비디오: Single Cell Measurement of Dopamine Release with Simultaneous Voltage-clamp and Amperometry

Functional Mapping with Simultaneous MEG and EEG

We use magnetoencephalography (MEG) and electroencephalography (EEG) to locate and determine the temporal evolution in brain areas involved in the processing of simple sensory stimuli. We will use somatosensory stimuli to locate the hand somatosensory areas, auditory stimuli to locate the auditory cortices, visual stimuli in four quadrants of the visual field to locate the early visual areas. These type of experiments are used for functional mapping in epileptic and brain tumor patients to locate eloquent cortices. In basic neuroscience similar experimental protocols are used to study the orchestration of cortical activity. The acquisition protocol includes quality assurance procedures, subject preparation for the combined MEG/EEG study, and acquisition of evoked-response data with somatosensory, auditory, and visual stimuli. We also demonstrate analysis of the data using the equivalent current dipole model and cortically-constrained minimum-norm estimates. Anatomical MRI data are employed in the analysis for visualization and for deriving boundaries of tissue boundaries for forward modeling and cortical location and orientation constraints for the minimum-norm estimates.

We use magnetoencephalography (MEG) and electroencephalography (EEG) to map brain areas involved in the processing of simple sensory stimuli.

동시 MEG와 EEG와 기능 매핑

We use magnetoencephalography (MEG) and electroencephalography (EEG) to locate and determine the temporal evolution in brain areas involved in the ...

연구

신경과학

Presynaptic Dopamine Dynamics in Striatal Brain Slices with Fast-scan Cyclic Voltammetry

Extensive research has focused on the neurotransmitter dopamine because of its importance in the mechanism of action of drugs of abuse (e.g. cocaine and amphetamine), the role it plays in psychiatric illnesses (e.g. schizophrenia and Attention Deficit Hyperactivity Disorder), and its involvement in degenerative disorders like Parkinson's and Huntington's disease. Under normal physiological conditions, dopamine is known to regulate locomotor activity, cognition, learning, emotional affect, and neuroendocrine hormone secretion. One of the largest densities of dopamine neurons is within the striatum, which can be divided in two distinct neuroanatomical regions known as the nucleus accumbens and the caudate-putamen. The objective is to illustrate a general protocol for slice fast-scan cyclic voltammetry (FSCV) within the mouse striatum. FSCV is a well-defined electrochemical technique providing the opportunity to measure dopamine release and uptake in real time in discrete brain regions. Carbon fiber microelectrodes (diameter of ~ 7 &mu;m) are used in FSCV to detect dopamine oxidation. The analytical advantage of using FSCV to detect dopamine is its enhanced temporal resolution of 100 milliseconds and spatial resolution of less than ten microns, providing complementary information to in vivo microdialysis.

Using fast-scan cyclic voltammetry to measure electrically evoked presynaptic dopamine dynamics in striatal brain slices.

빠른 스캔 순환 Voltammetry와 Striatal 뇌 조각의 Presynaptic 도파민 역학

Extensive research has focused on the neurotransmitter dopamine because of its importance in the mechanism of action of drugs of abuse (e.g. cocaine and ...

Single-cell Profiling of Developing and Mature Retinal Neurons

Highly specialized, but exceedingly small populations of cells play important roles in many tissues. The identification of cell-type specific markers and gene expression programs for extremely rare cell subsets has been a challenge using standard whole-tissue approaches. Gene expression profiling of individual cells allows for unprecedented access to cell types that comprise only a small percentage of the total tissue1-7. In addition, this technique can be used to examine the gene expression programs that are transiently expressed in small numbers of cells during dynamic developmental transitions8.
This issue of cellular diversity arises repeatedly in the central nervous system (CNS) where neuronal connections can occur between quite diverse cells9. The exact number of distinct cell types is not precisely known, but it has been estimated that there may be as many as 1000 different types in the cortex itself10. The function(s) of complex neural circuits may rely on some of the rare neuronal types and the genes they express. By identifying new markers and helping to molecularly classify different neurons, the single-cell approach is particularly useful in the analysis of cell types in the nervous system. It may also help to elucidate mechanisms of neural development by identifying differentially expressed genes and gene pathways during early stages of neuronal progenitor development.
As a simple, easily accessed tissue with considerable neuronal diversity, the vertebrate retina is an excellent model system for studying the processes of cellular development, neuronal differentiation and neuronal diversification. However, as in other parts of the CNS, this cellular diversity can present a problem for determining the genetic pathways that drive retinal progenitors to adopt a specific cell fate, especially given that rod photoreceptors make up the majority of the total retinal cell population11. Here we report a method for the identification of the transcripts expressed in single retinal cells (Figure 1). The single-cell profiling technique allows for the assessment of the amount of heterogeneity present within different cellular populations of the retina2,4,5,12. In addition, this method has revealed a host of new candidate genes that may play role(s) in the cell fate decision-making processes that occur in subsets of retinal progenitor cells8. With some simple adjustments to the protocol, this technique can be utilized for many different tissues and cell types.

A method for the isolation of single retinal cells and subsequent amplification of their cDNAs is described. Single-cell transcriptomics reveals the degree of cellular heterogeneity present in a tissue and uncovers new marker genes for rare cell populations. The accompanying protocol can be adjusted to suit many different cell types.

개발하고 성숙한 망막 신경 세포의 단일 세포 프로 파일링

Highly specialized, but exceedingly small populations of cells play important roles in many tissues. The identification of cell-type specific markers and ...

Optical Recording of Suprathreshold Neural Activity with Single-cell and Single-spike Resolution

Signaling of information in the vertebrate central nervous system is often carried by populations of neurons rather than individual neurons. Also propagation of suprathreshold spiking activity involves populations of neurons. Empirical studies addressing cortical function directly thus require recordings from populations of neurons with high resolution. Here we describe an optical method and a deconvolution algorithm to record neural activity from up to 100 neurons with single-cell and single-spike resolution. This method relies on detection of the transient increases in intracellular somatic calcium concentration associated with suprathreshold electrical spikes (action potentials) in cortical neurons. High temporal resolution of the optical recordings is achieved by a fast random-access scanning technique using acousto-optical deflectors (AODs)1. Two-photon excitation of the calcium-sensitive dye results in high spatial resolution in opaque brain tissue2. Reconstruction of spikes from the fluorescence calcium recordings is achieved by a maximum-likelihood method. Simultaneous electrophysiological and optical recordings indicate that our method reliably detects spikes (&gt;97% spike detection efficiency), has a low rate of false positive spike detection (&lt; 0.003 spikes/sec), and a high temporal precision (about 3 msec) 3. This optical method of spike detection can be used to record neural activity in vitro and in anesthetized animals in vivo3,4.

Understanding the function of the vertebrate central nervous system requires recordings from many neurons because cortical function arises on the level of populations of neurons. Here we describe an optical method to record suprathreshold neural activity with single-cell and single-spike resolution, dithered random-access scanning. This method records somatic fluorescence calcium signals from up to 100 neurons with high temporal resolution. A maximum-likelihood algorithm deconvolves the underlying suprathreshold neural activity from the somatic fluorescence calcium signals. This method reliably detects spikes with high detection efficiency and a low rate of false positives and can be used to study neural populations in vitro and in vivo.

단일 셀 및 단일 스파이크 해상도 suprathreshold 신경 활동의 광 기록

Signaling of information in the vertebrate central nervous system is often carried by populations of neurons rather than individual neurons. Also ...

Simultaneous Electroencephalography, Real-time Measurement of Lactate Concentration and Optogenetic Manipulation of Neuronal Activity in the Rodent Cerebral Cortex

Although the brain represents less than 5% of the body by mass, it utilizes approximately one quarter of the glucose used by the body at rest1. The function of non rapid eye movement sleep (NREMS), the largest portion of sleep by time, is uncertain. However, one salient feature of NREMS is a significant reduction in the rate of cerebral glucose utilization relative to wakefulness2-4. This and other findings have led to the widely held belief that sleep serves a function related to cerebral metabolism. Yet, the mechanisms underlying the reduction in cerebral glucose metabolism during NREMS remain to be elucidated.
	One phenomenon associated with NREMS that might impact cerebral metabolic rate is the occurrence of slow waves, oscillations at frequencies less than 4 Hz, in the electroencephalogram5,6. These slow waves detected at the level of the skull or cerebral cortical surface reflect the oscillations of underlying neurons between a depolarized/up state and a hyperpolarized/down state7. During the down state, cells do not undergo action potentials for intervals of up to several hundred milliseconds. Restoration of ionic concentration gradients subsequent to action potentials represents a significant metabolic load on the cell8; absence of action potentials during down states associated with NREMS may contribute to reduced metabolism relative to wake.
	Two technical challenges had to be addressed in order for this hypothetical relationship to be tested. First, it was necessary to measure cerebral glycolytic metabolism with a temporal resolution reflective of the dynamics of the cerebral EEG (that is, over seconds rather than minutes). To do so, we measured the concentration of lactate, the product of aerobic glycolysis, and therefore a readout of the rate of glucose metabolism in the brains of mice. Lactate was measured using a lactate oxidase based real time sensor embedded in the frontal cortex. The sensing mechanism consists of a platinum-iridium electrode surrounded by a layer of lactate oxidase molecules. Metabolism of lactate by lactate oxidase produces hydrogen peroxide, which produces a current in the platinum-iridium electrode. So a ramping up of cerebral glycolysis provides an increase in the concentration of substrate for lactate oxidase, which then is reflected in increased current at the sensing electrode. It was additionally necessary to measure these variables while manipulating the excitability of the cerebral cortex, in order to isolate this variable from other facets of NREMS.
	We devised an experimental system for simultaneous measurement of neuronal activity via the elecetroencephalogram, measurement of glycolytic flux via a lactate biosensor, and manipulation of cerebral cortical neuronal activity via optogenetic activation of pyramidal neurons. We have utilized this system to document the relationship between sleep-related electroencephalographic waveforms and the moment-to-moment dynamics of lactate concentration in the cerebral cortex. The protocol may be useful for any individual interested in studying, in freely behaving rodents, the relationship between neuronal activity measured at the electroencephalographic level and cellular energetics within the brain.

A procedure is described for manipulating the activity of cerebral cortical pyramidal neurons optogenetically while the electroencephalogram, electromyogram, and cerebral lactate concentration are monitored. Experimental recordings are performed on cable-tethered mice while they undergo spontaneous sleep/wake cycles. Optogenetic equipment is assembled in our laboratory; recording equipment is commercially available.

동시 Electroencephalography, 설치류 대뇌 피질의 젖산 농도와 Neuronal 활동 Optogenetic 조작의 실시간 측정

Although the brain represents less than 5% of the body  by mass, it utilizes approximately one quarter of the glucose used by the body  at rest1. The ...

Deep Brain Stimulation with Simultaneous fMRI in Rodents

In order to visualize the global and downstream neuronal responses to deep brain stimulation (DBS) at various targets, we have developed a protocol for using blood oxygen level dependent (BOLD) functional magnetic resonance imaging (fMRI) to image rodents with simultaneous DBS. DBS fMRI presents a number of technical challenges, including accuracy of electrode implantation, MR artifacts created by the electrode, choice of anesthesia and paralytic to minimize any neuronal effects while simultaneously eliminating animal motion, and maintenance of physiological parameters, deviation from which can confound the BOLD signal.&#160;Our laboratory has developed a set of procedures that are capable of overcoming most of these possible issues. For electrical stimulation, a homemade tungsten bipolar microelectrode is used, inserted stereotactically at the stimulation site in the anesthetized subject. In preparation for imaging, rodents are fixed on a plastic headpiece and transferred to the magnet bore. For sedation and paralysis during scanning, a cocktail of dexmedetomidine and pancuronium is continuously infused, along with a minimal dose of isoflurane; this preparation minimizes the BOLD ceiling effect of volatile anesthetics. In this example experiment, stimulation of the subthalamic nucleus (STN) produces BOLD responses which are observed primarily in ipsilateral cortical regions, centered in motor cortex. Simultaneous DBS and fMRI allows the unambiguous modulation of neural circuits dependent on stimulation location and stimulation parameters, and permits observation of neuronal modulations free of regional bias. This technique may be used to explore the downstream effects of modulating neural circuitry at nearly any brain region, with implications for both experimental and clinical DBS.

This protocol describes a standard method for simultaneous functional magnetic resonance imaging and deep brain stimulation in the rodent. The combined use of these experimental tools allows for the exploration of global downstream activity in response to electrical stimulation at virtually any brain target.

설치류에 동시 fMRI를 깊은 뇌 자극

In order to visualize the global and downstream neuronal responses to deep brain stimulation (DBS) at various targets, we have developed a protocol for ...

Preparation of Neuronal Co-cultures with Single Cell Precision

Microfluidic embodiments of the Campenot chamber have attracted great interest from the neuroscience community. These interconnected co-culture platforms can be used to investigate a variety of questions, spanning developmental and functional neurobiology to infection and disease propagation. However, conventional systems require significant cellular inputs (many thousands per compartment), inadequate for studying low abundance cells, such as primary dopaminergic substantia nigra, spiral ganglia, and Drosophilia melanogaster neurons, and impractical for high throughput experimentation. The dense cultures are also highly locally entangled, with few outgrowths (&#60;10%) interconnecting the two cultures. In this paper straightforward microfluidic and patterning protocols are described which address these challenges: (i) a microfluidic single neuron arraying method, and (ii) a water masking method for plasma patterning biomaterial coatings to register neurons and promote outgrowth between compartments. Minimalistic neuronal co-cultures were prepared with high-level (&#62;85%) intercompartment connectivity and can be used for high throughput neurobiology experiments with single cell precision.

Protocols for single neuron microfluidic arraying and water masking for the in-chip plasma patterning of biomaterial coatings are described. Highly interconnected co-cultures can be prepared using minimal cell inputs.

단일 셀의 정밀도와의 연결을 공동 문화의 준비

Microfluidic embodiments of the Campenot chamber have attracted great interest from the neuroscience community. These interconnected co-culture platforms ...

Simultaneous Detection of c-Fos Activation from Mesolimbic and Mesocortical Dopamine Reward Sites Following Naive Sugar and Fat Ingestion in Rats

This study uses cellular c-fos activation to assess effects of novel ingestion of fat and sugar on brain dopamine (DA) pathways in rats. Intakes of sugars and fats are mediated by their innate attractions as well as learned preferences. Brain dopamine, especially meso-limbic and meso-cortical projections from the ventral tegmental area (VTA), has been implicated in both of these unlearned and learned responses. The concept of distributed brain networks, wherein several sites and transmitter/peptide systems interact, has been proposed to mediate palatable food intake, but there is limited evidence empirically demonstrating such actions. Thus, sugar intake elicits DA release and increases c-fos-like immunoreactivity (FLI) from individual VTA DA projection zones including the nucleus accumbens (NAC), amygdala (AMY) and medial prefrontal cortex (mPFC) as well as the dorsal striatum. Further, central administration of selective DA receptor antagonists into these sites differentially reduce acquisition and expression of conditioned flavor preferences elicited by sugars or fats. One approach by which to determine whether these sites interacted as a distributed brain network in response to sugar or fat intake would be to simultaneous evaluate whether the VTA and its major mesotelencephalic DA projection zones (prelimbic and infralimbic mPFC, core and shell of the NAc, basolateral and central-cortico-medial AMY) as well as the dorsal striatum would display coordinated and simultaneous FLI activation after oral, unconditioned intake of corn oil (3.5%), glucose (8%), fructose (8%) and saccharin (0.2%) solutions. This approach is a successful first step in identifying the feasibility of using cellular c-fos activation simultaneously across relevant brain sites to study reward-related learning in ingestion of palatable food in rodents.

The goal of this study is to identify reward-related distributed brain networks by delineating a reliable immunohistological technique using cellular c-fos activation to measure simultaneous changes in dopamine pathways and terminal sites after novel ingestion of fat and sugar in rats.

흰쥐의 나이브 설탕과 지방 섭취에 따라 변연계와 Mesocortical 도파민 보상 사이트에서 C-에 Fos 활성화의 동시 검출

This study uses cellular c-fos activation to assess effects of novel ingestion of fat and sugar on brain dopamine (DA) pathways in rats. Intakes of sugars ...

Electrophysiological Method for Whole-cell Voltage Clamp Recordings from Drosophila Photoreceptors

Whole-cell voltage clamp recordings from Drosophila melanogaster photoreceptors have revolutionized the field of invertebrate visual transduction, enabling the use of D. melanogaster molecular genetics to study inositol-lipid signaling and Transient Receptor Potential (TRP) channels at the single-molecule level. A handful of labs have mastered this powerful technique, which enables the analysis of the physiological responses to light under highly controlled conditions. This technique allows control over the intracellular and extracellular media; the membrane voltage; and the fast application of pharmacological compounds, such as a variety of ionic or pH indicators, to the intra- and extracellular media. With an exceptionally high signal-to-noise ratio, this method enables the measurement of dark spontaneous and light-induced unitary currents (i.e.&#160;spontaneous and quantum bumps) and macroscopic Light-induced Currents (LIC) from single D. melanogaster photoreceptors. This protocol outlines, in great detail, all the key steps necessary to perform this technique, which includes both electrophysiological and optical recordings. The fly retina dissection procedure for the attainment of intact and viable ex vivo isolated ommatidia in the bath chamber is described. The equipment needed to perform whole-cell and fluorescence imaging measurements are also detailed. Finally, the pitfalls in using this delicate preparation during extended experiments are explained.

Electrophysiological Method for Whole-cell Voltage Clamp Recordings from Drosophila Photoreceptors

Whole-cell recordings from Drosophila melanogaster photoreceptors enable the measurement of spontaneous dark bumps, quantum bumps, macroscopic responses to light, and current-voltage relationships under various conditions. In combination with D. melanogaster genetic manipulation tools, this method enables the study of the ubiquitous inositol-lipid signaling pathway and its target, the TRP channel.

전 세포 전압 클램프 기록을위한 전기 생리 학적 방법<em&gt; Drosophila</em&gt; 광 수용체

Whole-cell voltage clamp recordings from Drosophila melanogaster photoreceptors have revolutionized the field of invertebrate visual transduction, ...

Levator Auris Longus Preparation for Examination of Mammalian Neuromuscular Transmission Under Voltage Clamp Conditions

This protocol describes a technique to record synaptic transmission from the neuromuscular junction under current-clamp and voltage-clamp conditions. An ex vivo preparation of the levator auris longus (LAL) is used because it is a thin muscle that provides easy visualization of the neuromuscular junction for microelectrode impalement at the motor endplate. This method allows for the recording of spontaneous miniature endplate potentials and currents (mEPPs and mEPCs), nerve-evoked endplate potentials and currents (EPPs and EPCs), as well as the membrane properties of the motor endplate. Results obtained from this method include the quantal content (QC), number of vesicle release sites (n), probability of vesicle release (prel), synaptic facilitation and depression, as well as the muscle membrane time constant (&#964;m) and input resistance. Application of this technique to mouse models of human disease can highlight key pathologies in disease states and help identify novel treatment strategies. By fully voltage-clamping a single synapse, this method provides one of the most detailed analyses of synaptic transmission currently available.

Levator Auris Longus Preparation for Examination of Mammalian Neuromuscular Transmission Under Voltage Clamp Conditions

The protocol described in this paper uses the mouse levator auris longus (LAL) muscle to record spontaneous and nerve-evoked postsynaptic potentials (current-clamp) and currents (voltage-clamp) at the neuromuscular junction. Use of this technique can provide key insights into mechanisms of synaptic transmission under normal and disease conditions.

Levator Auris Longus 포유류 신경 근육 학 전송 전압 클램프 조건에서의 시험을 위한 준비

This protocol describes a technique to record synaptic transmission from the neuromuscular junction under current-clamp and voltage-clamp conditions. An ...