1) 준비 <ol><li> 컴퓨터 화면의 전면에 편안한 의자에 앉아 주제를 시작합니다. </li><li> 화면과 피사체의 nasion는 60cm입니다 사이의 거리를 보장하기 위해 줄자를 사용하십시오. </li><li> 마지막으로, 피사체의 눈을 통해 빛이 차단 마스크를 놓으십시오. </li></ol> 2) V1/V2 이상 Phosphene 임계값 결정 <ol><li> 나중에이 프로토콜의 phosphenic 동작을 탐험 때 피사체의 phosphene 한계를 아는 것이 중요합니다. 이 임계값을 결정하려면 먼저 70 %의 전력으로 TMS 시스템을 설정합니다. </li><li> 코일을 잡고 있도록 그림 - 8 얼굴 두개골 정중선의 상단. </li><li> inion 위 3cm에 대한 시점에 단일 펄스 자극을 시작합니다. </li><li> 각 펄스 후에는, phosphenic 경험을보고 제목을 부탁드립니다. </li><li> 이 지역 주변에 작은 검색 그리드 (V1/V2)에 코일을 이동 시작. </li><li> 자극이 주제에서 일관성 있고 모호하지 phosphene 보고서를 elicits 위치를 결정합니다. 그것은 TMS는 인구의 40 %에 phosphenes을 이끌어 수 없게됩니다하고 있다고 가정되어 있으므로, 피사체가 아무것도보고하지 않습니다 가능성이 있습니다. 이 경우, 불행히도,이 실험이 작동하지 않습니다. 주제는 떠날 수 있어야합니다. </li><li> phosphenic &quot;핫스팟&quot;이있는되면 정확히 3 6의 연속 펄스에 대한 주제 리포트 phosphenes까지 TMS 전원 출력을 조정 아래. 이 전력 수준은 주제의 V1/V2 한계입니다. 가능한 경우, neuronavigation가 위치하고 V1/V2 핫스팟을 대상으로 할 때 큰 공간 정밀도를 달성하는 데 사용할 수 있습니다. </li></ol> 3) V5/MT + 콤플렉스 이상 Phosphenic 임계값 결정 <ol><li> 동일한 상기 절차와 매개 변수를 사용하여, 우리는 지금 V5/MT 이상 phosphene 임계값 + 복잡한를 결정합니다. 이 지역을 찾으려면 피사체의 inion에 3cm와 5cm 등의 측면 지점에서 시작합니다. </li><li> 각 펄스 후에는, phosphenic 경험을보고 제목을 부탁드립니다. </li><li> 모호하지하고 일관된 phosphenes이 elicited 때까지 다시 단일 펄스를 사용, 작은 검색 격자 패턴을 시작합니다. </li><li> 마지막으로, 정확히 3 6의 연속 펄스에 대한 주제 리포트 phosphenes까지 TMS 전원 출력을 조정 아래. 이 전력 수준은 주제의 V5/MT + 복잡한 한계입니다. 다시가 없습니다 neuronavigation가 위치하고 V5/MT + 핫스팟을 대상으로 할 때 큰 공간 정밀도를 달성하는 데 사용할 수있는 경우. </li></ol> 4)베이스 라인 Phosphene 동작을 결정 <ol><li> phosphene 임계값이 두 시각 영역에 대한 결정되면, 우리는베이스 라인 phosphenic 동작을 측정해야합니다. 이렇게하려면 먼저 피사체의 빛을 차단 마스크를 제거합니다. </li><li> 다음 60 초 동안 컴퓨터 화면의 중앙에 표시 안정적인 고정 크로스에 응시하기 위해 주제를 지시합니다. </li><li> 마스크를 교체하고, V1/V2 핫 스팟 이상, 120 %를 3 초 동안 단일 펄스 트레인 V1/V2 한계를 생성합니다. </li><li> 다섯 초 기다려 다른 열차를 실시하고 있습니다. </li><li> 다시 다섯 초 정도 기다린 세 번째 기차를 실시하고 있습니다. </li><li> 세 번째 싱글 펄스 트레인 후 어떤의 위치와 동기 특성 phosphenes을 elicited 설명하는 제목을 부탁드립니다. 이것은 기준 것입니다. </li><li> V5/MT를 통해이 같은 절차를 반복 + 뜨거운 자리를 (V5/MT + 임계값 120 %로 TMS 전원을 재설정 기억). </li></ol> 5) 조건 넘버 원 <ol><li> phophenic 기준이 결정되면 다시 한번 주제의 빛을 차단 마스크를 제거합니다. </li><li> 60 초 동안 컴퓨터 화면의 중앙에 표시 안정적인 고정 크로스 쳐다보지 수있는 주제를 지시한다. 이번에는 오히려 빈 화면보다, 우리는 십자가 주위를 여러 위치에서 이동하는 점들의 시리즈를 포함합니다. <img src="/files/ftp_upload/2273/2273fig1.jpg" alt="그림 1" /> 그림 1 조건 하나에 자극들을 수정 :. 간단한 translational 운동. 모든 점들은 왼쪽 또는 오른쪽으로 중 coherently 이동합니다. 확실히 모든 점들이 동일한 방향으로 이동합니다. 자극에 장기간 노출에 의해 유도로 신경 흥분의 변화는 편견에 제출 후 자극의 인식을 제공하고있는 현상이 자극이 생기다 시각 적응하는 역할을한다. </li><li> 60초 후, 마스크를 교체하고, V1/V2의 핫스팟을 통해 120 %로 3 초간 단일 펄스 트레인 V1/V2 한계를 생성합니다. </li><li> 다섯 초 후에 두 번 더 반복합니다. </li><li> 세 기차 후, 위치 및 어떤의 동기 특성이 phosphenes을 elicited보고 제목을 부탁드립니다. 가능한 경우, 어두운 방에서 눈을 운동 추적 시스템의 사용은 더 정교하고 phosphene의 elicitations 동안 과목 의한 안구 운동의 양적 모니터링을 달성할 수 있습니다. </li><li> 이 three-train/report 시퀀스를 계속 phosp까지hene 동작은 기본 활동으로 돌아갑니다. </li><li> V5/MT + 복잡한 핫스팟에 대해이 절차를 반복합니다. </li></ol> 6) 조건 넘버 투 <ol><li> 이 두 번째 조건에서 조건 하나를 활용하여 동일한 절차를 반복하십시오. &#39;스타 버스트&#39;패턴과 유사한 -이 시간은, 그러나, 오히려 단수 방향으로 이동하는 점들의 일련의 주제를 제시하는 것보다 각 거리 중앙 지점으로부터 상대적으로 추기경의 방향으로 움직이는 점들의 일련의 주제를 제시 . <img src="/files/ftp_upload/2273/2273fig2.jpg" alt="그림 2" /> 그림 2 상태 두 자극들을 수정 :. 레이디얼 운동. 점들은 어느 방향이나 떨어진 중앙 지점에서 각각의 추기경 방향으로 이동합니다. </li><li> 이전처럼, 60 초 후, V1/V2 세, 120 %의 임계값 열차 일련의 실시, 마스크를 대체, 주제 보고서에 대한 요청 및 기준 돌아올때까지 계속합니다. </li><li> V5/MT +를위한 반복합니다. </li></ol> 7) 다이어그램 <ol><li> 옵션으로 각 조건 이후에 (또는 모든 조건이 완료된 후), 그래프에 지역 및 phopsphenes 각 시리즈의 동기 행동을 그릴 수있는 주제를 부탁드립니다. 이것은 필수적인 단계는 아니지만 interpretable 데이터의 다른 세트를 제공합니다. </li></ol> 8) 대표 결과 유니 방향 동기 자극에 시각적인 적응은 V1/V2 이상의 동일한 phosphenic 움직임을 이끌어내는 것입니다. <img src="/files/ftp_upload/2273/2273fig3.jpg" alt="그림 3" /> 그림 3. 조건 하나 (과목의 도면에 따라)에 좌우 반구의 V1/V2에서 유도된 phosphenes의 예. V5/MT + 복잡한 phosphenes도, 영향을 줄 수 있어야 phosphene 지금 채택 자극과 기준 phosphene의 운동 방향의 합계로 표시됩니다 이러한. 그러나, 스타 버스트 패턴 시각 적응은 V5/MT + 복잡한에서 동일한 스타 버스트 phosphenic 운동을 이끌어해야하지만 V1/V2 기준을 변경해서는 안됩니다. <img src="/files/ftp_upload/2273/2273fig4.jpg" alt="그림 4" /> 그림 4. V5/MT에서 유도된 phosphenes의 예 + 2 급 (과목의 도면에 따라)에 좌우 반구에서 복잡한.

<ol>
	<li>Pascual-Leone, A., Davey, M., Wassermann, E. M., Rothwell, J., Puri, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Handbook of Transcranial Magnetic Stimulation. , (2002).</li><li>Walsh, V., Pascual-Leone, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Transcranial Magnetic Stimulation: A Neurochronometrics of Mind. , (2005).</li><li>Silvanto, J., Muggleton, N. G., Cowey, A., Walsh, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+adaptation+reveals+state-dependent+effects+of+transcranial+magnetic+stimulation.">Neural adaptation reveals state-dependent effects of transcranial magnetic stimulation.</a> European Journal of Neuroscience. 25, 1874-1881 (2007).</li><li>Silvanto, J., Pascual-Leone, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=State-Dependency+of+Transcranial+Magnetic+Stimulation.">State-Dependency of Transcranial Magnetic Stimulation.</a> Brain Topography. 21, 1-10 (2008).</li><li>Silvanto, J., Cattaneo, Z., Battelli, L., Pascual-Leone, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Baseline+cortical+excitablility+determines+whether+TMS+disrupts+or+facilitates+behavior.">Baseline cortical excitablility determines whether TMS disrupts or facilitates behavior.</a> Journal of Neurophysiology. 99, 2725-2730 (2008).</li><li>Silvanto, J., Muggleton, N. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Testing+the+validity+of+the+TMS+state-dependency+approach:+targeting+functionally+distinct+motion-selective+neural+populations.">Testing the validity of the TMS state-dependency approach: targeting functionally distinct motion-selective neural populations.</a> Neuroimage. 40, 1841-1848 (2008).</li><li>Tootell, R. B., Rappas, J. B., Kwong, K. K., Malach, R., Born, R. T., Brady, T. J., Rosen, B. R., Belliveau, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+analysis+of+human+MT+and+related+visual+cortical+areas+using+magnetic+resonance+imaging.">Functional analysis of human MT and related visual cortical areas using magnetic resonance imaging.</a> Journal of Neuroscience. 15, 3215-3230 (1995).</li><li>Singh, K. D., Smith, A. T., Greenlee, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatiotemporal+frequency+and+direction+sensitivities+of+human+visual+areas+measured+using+fMRI.">Spatiotemporal frequency and direction sensitivities of human visual areas measured using fMRI.</a> Neuroimage. 12, 550-564 (2000).</li><li>Rutchmann, R. M., Schrauf, M., Greenlee, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain+activation+during+dichoptic+presentation+of+optic+flow+stimuli.">Brain activation during dichoptic presentation of optic flow stimuli.</a> Exp Brain Res. 134, 533-537 (2000).</li><li>Morrone, M. C., Tosetti, M., Montanaro, D., Fiorentini, A., Cioni, G., Burr, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+cortical+area+that+responds+specifically+to+optic+flow+revealed+by+fMRI.">A cortical area that responds specifically to optic flow revealed by fMRI.</a> Nat. Neuroscience. 3, 1322-1328 (2000).</li></ol>

관심 없음 충돌 선언하지 않습니다.

이 실험은 상태 의존성의 중심에 도착. V1/V2의 뉴런이 간단한 방향 운동과 대응 것으로 생각되고 있습니다 따라서 7,8, 단일 방향 동기의 자극에 적응이 움직임을 등록 뉴런의 흥분 증가 -. 같은를 그들은 TMS에 반응을 가장 먼저해야 펄스. V5/MT + 복합도 따라서이 지역에서 유도 phosphenes도 채택 자극에 의​​해 영향을해야, 간단한 translational 모션으로 조정 뉴런이 포함되어 있습니다. 그러나...

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<td>Light Blocking Eye Mask</td>
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<td>Ear Plugs</td>
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<td>Swim Cap</td>
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<td>Tape Measure</td>
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<td>Any Single Pulse Capable TMS Device</td>
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<td>Any Figure-of-Eight Coil</td>
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state-dependency effects on tms:  a look at motive phosphene behavior

Transcranial 자석 자극 (TMS)는 transiently 또는 lastingly화된 자기장 펄스의 응용을 통해 대뇌 피질의 흥분을 (중 증가 또는 감소) 변조 수있는 비침습 neurostimulatory 및 neuromodulatory 기술이다. TMS 분야의 용어 내에서 1,2가 상태 의존성은 자극에 대한 타겟 특정 신경 지역의 초기, 기본 상태를 말합니다. 유추 할 수 있듯이, TMS의 효과 (그리고 할) 대상 피질 영역의 주요 자화율 및 응답에 따라 달라질 수 있습니다. 3,4,5 본 실험에서, 우리는 이끌어 냄과 동기 phosphenes의 주관적 경험을 통해 상태 의존성이 개념을 검토합니다. Phosphenes는 시각 시각 피질에 전자기 펄스에 의해 트리거 작은 불빛의 깜박 인식하고 있습니다. 이 작은 불빛은 시각 피질의 종류가 자극되고있는에 따라 다양한 특성을 추측할 수있다. 이 특정 연구에서는, 우리는 동기의 phosphenes을 대상으로 될 것이로 V1/V2의 자극과 V5/MT + 복잡한 시각 영역을 통해 elicited 6.

Watch this Scientific Journal Video about State-Dependency Effects on TMS:  A Look at Motive Phosphene Behavior at JoVE.com

State-Dependency Effects on TMS:  A Look at Motive Phosphene Behavior

이 문서에서는, 우리는 동기 phosphenic 프레 젠 테이션을 유도 TMS에 대한 시각 관련 국가 종속성의 효과를 검사합니다.

TMS의 국가 종속성 효과 : 동기 Phosphene 동작 좀보세요

Transcranial magnetic stimulation (TMS) is a non-invasive neurostimulatory and neuromodulatory technique that can transiently or lastingly modulate ...

state-dependency-effects-on-tms-a-look-at-motive-phosphene-behavior

Research

JoVE Journal

Neuroscience

10.5K 조회수.  Beth Israel Deaconess Medical Center. 이 문서에서는, 우리는 동기 phosphenic 프레 젠 테이션을 유도 TMS에 대한 시각 관련 국가 종속성의 효과를 검사합니다.

비디오: State-Dependency Effects on TMS:  A Look at Motive Phosphene Behavior

The NeuroStar TMS Device: Conducting the FDA Approved Protocol for Treatment of Depression

The Neuronetics NeuroStar Transcranial Magnetic Stimulation (TMS) System is a class II medical device that produces brief duration, pulsed magnetic fields. These rapidly alternating fields induce electrical currents within localized, targeted regions of the cortex which are associated with various physiological and functional brain changes.1,2,3
In 2007, O'Reardon et al., utilizing the NeuroStar device, published the results of an industry-sponsored, multisite, randomized, sham-stimulation controlled clinical trial in which 301 patients with major depression, who had previously failed to respond to at least one adequate antidepressant treatment trial, underwent either active or sham TMS over the left dorsolateral prefrontal cortex (DLPFC). The patients, who were medication-free at the time of the study, received TMS five times per week over 4-6 weeks.4
The results demonstrated that a sub-population of patients (those who were relatively less resistant to medication, having failed not more than two good pharmacologic trials) showed a statistically significant improvement on the Montgomery-Asberg Depression Scale (MADRS), the Hamilton Depression Rating Scale (HAMD), and various other outcome measures. In October 2008, supported by these and other similar results5,6,7, Neuronetics obtained the first and only Food and Drug Administration (FDA) approval for the clinical treatment of a specific form of medication-refractory depression using a TMS Therapy device (FDA approval K061053).
In this paper, we will explore the specified FDA approved NeuroStar depression treatment protocol (to be administered only under prescription and by a licensed medical profession in either an in- or outpatient setting).

In this article, we examine the methodology and considerations relevant to the FDA approved depression treatment protocol using the Neuronetics NeuroStar TMS device.

NeuroStar TMS 장치 : FDA를 실시는 우울증 치료 프로토콜을 승인

The Neuronetics NeuroStar Transcranial Magnetic Stimulation (TMS) System is a class II medical device that produces brief duration, pulsed magnetic ...

연구

신경과학

Cryopreservation of Cortical Tissue Blocks for the Generation of Highly Enriched Neuronal Cultures

In this study, we outline a standardized protocol for the successful cryopreservation and thawing of cortical brain tissue blocks to generate highly enriched neuronal cultures. For this protocol the freezing medium used is 10% dimethyl sulfoxide (DMSO) diluted in Hank's Buffered Salt Solution (HBSS). Blocks of cortical tissue are transferred to cryovials containing the freezing medium and slowly frozen at -1&deg;C/min in a rate-controlled freezing container. Post-thaw processing and dissociation of frozen tissue blocks consistently produced neuronal-enriched cultures which exhibited rapid neuritic growth during the first 5 days in culture and significant expansion of the neuronal network within 10 days. Immunocytochemical staining with the astrocytic marker glial fibrillary acidic protein (GFAP) and the neuronal marker beta-tubulin class III, revealed high numbers of neurons and astrocytes in the cultures. Generation of neural precursor cell cultures after tissue block dissociation resulted in rapidly expanding neurospheres, which produced large numbers of neurons and astrocytes under differentiating conditions. This simple cryopreservation protocol allows for the rapid, efficient, and inexpensive preservation of cortical brain tissue blocks, which grants increased flexibility for later generation of neuronal, astrocyte, and neuronal precursor cell cultures.

Here, we describe a method for efficient cryopreservation and thawing of cortical brain tissue blocks to generate highly enriched neuronal cultures. This simple protocol provides flexibility for later generation of neuronal, astrocyte, and neuronal precursor cell cultures.

고도의 연결을 강화 문화의 생성을위한 두피 조직 블록의 Cryopreservation

In this study, we outline a standardized protocol for the successful cryopreservation and thawing of cortical brain tissue blocks to generate highly ...

Studying the Integration of Adult-born Neurons

Neurogenesis occurs in adult mammalian brains in the sub-ventricular zone (SVZ) of the lateral ventricle and in the sub-granular zone (SGZ) of the hippocampal dentate gyrus throughout life. Previous reports have shown that adult hippocampal neurogenesis is associated with diverse brain disorders, including epilepsy, schizophrenia, depression and anxiety (1). Deciphering the process of normal and aberrant adult-born neuron integration may shed light on the etiology of these diseases and inform the development of new therapies.
SGZ adult neurogenesis mirrors embryonic and post-natal neuronal development, including stages of fate specification, migration, synaptic integration, and maturation. However, full integration occurs over a prolonged, 6-week period. Initial synaptic input to adult-born SGZ dentate granule cells (DGCs) is GABAergic, followed by glutamatergic input at 14 days (2). The specific factors which regulate circuit formation of adult-born neurons in the dentate gyrus are currently unknown.
Our laboratory uses a replication-deficient retroviral vector based on the Moloney murine leukemia virus to deliver fluorescent proteins and hypothesized regulatory genes to these proliferating cells. This viral technique provides high specificity and resolution for analysis of cell birth date, lineage, morphology, and synaptogenesis.
A typical experiment often employs two or three viruses containing unique label, transgene, and promoter elements for single-cell analysis of a desired developmental process in vivo. The following protocol describes a method for analyzing functional newborn neuron integration using a single green (GFP) or red (dTomato) fluorescent protein retrovirus and patch-clamp electrophysiology.

A way to study the integration of newborn dentate granule cells in adult animals is described. This technique uses an engineered retrovirus to label newborn neurons, followed by electrophysiological recordings to determine in vivo functional integration.

성인에서 태어난 뉴런의 통합을 공부

Neurogenesis occurs in adult mammalian brains in the sub-ventricular zone (SVZ) of the lateral ventricle and in the sub-granular zone (SGZ) of the ...

Investigations on Alterations of Hippocampal Circuit Function Following Mild Traumatic Brain Injury

Traumatic Brain Injury (TBI) afflicts more than 1.7 million people in the United States each year and even mild TBI can lead to persistent neurological impairments 1. Two pervasive and disabling symptoms experienced by TBI survivors, memory deficits and a reduction in seizure threshold, are thought to be mediated by TBI-induced hippocampal dysfunction 2,3. In order to demonstrate how altered hippocampal circuit function adversely affects behavior after TBI in mice, we employ lateral fluid percussion injury, a commonly used animal model of TBI that recreates many features of human TBI including neuronal cell loss, gliosis, and ionic perturbation 4-6.
	Here we demonstrate a combinatorial method for investigating TBI-induced hippocampal dysfunction. Our approach incorporates multiple ex vivo physiological techniques together with animal behavior and biochemical analysis, in order to analyze post-TBI changes in the hippocampus. We begin with the experimental injury paradigm along with behavioral analysis to assess cognitive disability following TBI. Next, we feature three distinct ex vivo recording techniques: extracellular field potential recording, visualized whole-cell patch-clamping, and voltage sensitive dye recording. Finally, we demonstrate a method for regionally dissecting subregions of the hippocampus that can be useful for detailed analysis of neurochemical and metabolic alterations post-TBI.
	These methods have been used to examine the alterations in hippocampal circuitry following TBI and to probe the opposing changes in network circuit function that occur in the dentate gyrus and CA1 subregions of the hippocampus (see Figure 1). The ability to analyze the post-TBI changes in each subregion is essential to understanding the underlying mechanisms contributing to TBI-induced behavioral and cognitive deficits. 
	The multi-faceted system outlined here allows investigators to push past characterization of phenomenology induced by a disease state (in this case TBI) and determine the mechanisms responsible for the observed pathology associated with TBI.

A multi-faceted approach to investigating functional changes to hippocampal circuitry is explained. Electrophysiological techniques are described along with the injury protocol, behavioral testing and regional dissection method. The combination of these techniques can be applied in similar fashion for other brain regions and scientific questions.

가벼운 외상성 뇌 손상 후 Hippocampal 회로 기능의 수선에 관한 조사

Traumatic Brain Injury (TBI) afflicts more than 1.7 million people in the United States each year and even mild TBI can lead to persistent neurological ...

A Procedure for Implanting a Spinal Chamber for Longitudinal In Vivo Imaging of the Mouse Spinal Cord

Studies in the mammalian neocortex have enabled unprecedented resolution of cortical structure, activity, and response to neurodegenerative insults by repeated, time-lapse in vivo imaging in live rodents. These studies were made possible by straightforward surgical procedures, which enabled optical access for a prolonged period of time without repeat surgical procedures. In contrast, analogous studies of the spinal cord have been previously limited to only a few imaging sessions, each of which required an invasive surgery. As previously described, we have developed a spinal chamber that enables continuous optical access for upwards of 8 weeks, preserves mechanical stability of the spinal column, is easily stabilized externally during imaging, and requires only a single surgery. Here, the design of the spinal chamber with its associated surgical implements is reviewed and the surgical procedure is demonstrated in detail. Briefly, this video will demonstrate the preparation of the surgical area and mouse for surgery, exposure of the spinal vertebra and appropriate tissue debridement, the delivery of the implant and vertebral clamping, the completion of the chamber, the removal of the delivery system, sealing of the skin, and finally, post-operative care. The procedure for chronic in vivo imaging using nonlinear microscopy will also be demonstrated. Finally, outcomes, limitations, typical variability, and a guide for troubleshooting are discussed.

A Procedure for Implanting a Spinal Chamber for Longitudinal In Vivo Imaging of the Mouse Spinal Cord

In this video, we describe a procedure for implanting a chronic optical imaging chamber over the dorsal spinal cord of a live mouse. The chamber, surgical procedure, and chronic imaging are reviewed and demonstrated.

종의 척추 상공 회의소를 주입하는 절차<em&gt; 생체</em&gt; 마우스 척수의 영상

Studies in the mammalian neocortex have enabled unprecedented resolution of cortical structure, activity, and response to neurodegenerative insults by ...

Live Imaging of Nicotine Induced Calcium Signaling and Neurotransmitter Release Along Ventral Hippocampal Axons

Sustained enhancement of axonal signaling and increased neurotransmitter release by the activation of pre-synaptic nicotinic acetylcholine receptors (nAChRs) is an important mechanism for neuromodulation by acetylcholine (ACh). The difficulty with access to probing the signaling mechanisms within intact axons and at nerve terminals both in vitro and in vivo has limited progress in the study of the pre-synaptic components of synaptic plasticity. Here we introduce a gene-chimeric preparation of ventral hippocampal (vHipp)&#8211;accumbens (nAcc) circuit in vitro that allows direct live imaging to analyze both the pre- and post-synaptic components of transmission while selectively varying the genetic profile of the pre- vs post-synaptic neurons. We demonstrate that projections from vHipp microslices, as pre-synaptic axonal input, form multiple, reliable glutamatergic synapses with post-synaptic targets, the dispersed neurons from nAcc. The pre-synaptic localization of various subtypes of nAChRs are detected and the pre-synaptic nicotinic signaling mediated synaptic transmission are monitored by concurrent electrophysiological recording and live cell imaging. This preparation also provides an informative approach to study the pre- and post-synaptic mechanisms of glutamatergic synaptic plasticity in vitro.

We developed a gene-chimeric preparation of ventral hippocampal &#8211; accumbens circuit in vitro that allows direct live imaging to analyze presynaptic mechanisms of nicotinic acetylcholine receptors (nAChRs) mediated synaptic transmission. This preparation also provides an informative approach to study the pre- and post-synaptic mechanisms of synaptic plasticity.

니코틴 유도 칼슘 신호 전달 및 복부 해마 축삭을 따라 신경 전달 물질 방출의 라이브 영상

Sustained enhancement of axonal signaling and increased neurotransmitter release by the activation of pre-synaptic nicotinic acetylcholine receptors ...

High-Resolution Quantitative Immunogold Analysis of Membrane Receptors at Retinal Ribbon Synapses

Retinal ganglion cells (RGCs) receive excitatory glutamatergic input from bipolar cells. Synaptic excitation of RGCs is mediated postsynaptically by NMDA receptors (NMDARs) and AMPA receptors (AMPARs). Physiological data have indicated that glutamate receptors at RGCs are expressed not only in postsynaptic but also in perisynaptic or extrasynaptic membrane compartments. However, precise anatomical locations for glutamate receptors at RGC synapses have not been determined. Although a high-resolution quantitative analysis of glutamate receptors at central synapses is widely employed, this approach has had only limited success in the retina. We developed a postembedding immunogold method for analysis of membrane receptors, making it possible to estimate the number, density and variability of these receptors at retinal ribbon synapses. Here we describe the tools, reagents, and the practical steps that are needed for: 1) successful preparation of retinal fixation, 2) freeze-substitution, 3) postembedding immunogold electron microscope (EM) immunocytochemistry and, 4) quantitative visualization of glutamate receptors at ribbon synapses.

The postembedding immunogold method is one of the most effective ways to provide high-resolution analyses of the subcellular localization of specific molecules. Here we describe a protocol to quantitatively analyze glutamate receptors at retinal ribbon synapses.

망막 리본 시냅스에서 막 수용체의 고해상도 양이 Immunogold 분석

Retinal ganglion cells (RGCs) receive excitatory glutamatergic input from bipolar cells. Synaptic excitation of RGCs is mediated postsynaptically by NMDA ...

Using Linear Agarose Channels to Study Drosophila Larval Crawling Behavior

Drosophila larval crawling is emerging as a powerful model to study neural control of sensorimotor behavior. However, larval crawling behavior on flat open surfaces is complex,&#160;including: pausing, turning, and meandering. This complexity in the repertoire of movement hinders detailed analysis of the events occurring during a single crawl stride cycle. To overcome this obstacle, linear agarose channels were made that constrain larval behavior to straight, sustained, rhythmic crawling. In principle, because agarose channels and the Drosophila larval body are both optically clear, the movement of larval structures labeled by genetically-encoded fluorescent probes can be monitored in intact, freely-moving larvae. In the past, larvae were placed in linear channels and crawling at the level of whole organism, segment, and muscle were analyzed1. In the future, larvae crawling in channels can be used for calcium imaging to monitor neuronal activity. Moreover, these methods can be used with larvae of any genotype and with any researcher-designed channel. Thus the protocol presented below is widely applicable for studies using the Drosophila larva as a model to understand motor control.

Using Linear Agarose Channels to Study Drosophila Larval Crawling Behavior

The Drosophila larva is a powerful model system to study neural control of behavior. This publication describes the use of linear agarose channels to elicit sustained bouts of linear crawling and methods to quantify the dynamics of larval structures during repetitive crawling behavior.

선형 아가로 오스 채널을 사용하여 공부하기<em&gt; 초파리</em&gt; 애벌레 크롤링 동작

Drosophila larval crawling is emerging as a powerful model to study neural control of sensorimotor behavior. However, larval crawling behavior on flat ...

Combining Optogenetics with Artificial microRNAs to Characterize the Effects of Gene Knockdown on Presynaptic Function within Intact Neuronal Circuits

The purpose of this protocol is to characterize the effect of gene knockdown on presynaptic function within intact neuronal circuits. We describe a workflow on how to combine artificial microRNA (miR)-mediated RNA interference with optogenetics to achieve selective stimulation of manipulated presynaptic boutons in acute brain slices. The experimental approach involves the use of a single viral construct and a single neuron-specific promoter to drive the expression of both an optogenetic probe and artificial miR(s) against presynaptic gene(s). When stereotactically injected in the brain region of interest, the expressed construct makes it possible to stimulate with light exclusively the neurons with reduced expression of the gene(s) under investigation. This strategy does not require the development and maintenance of genetically modified mouse lines and can in principle be applied to other organisms and to any neuronal gene of choice. We have recently applied it to investigate how the knockdown of alternative splice isoforms of presynaptic P/Q-type voltage-gated calcium channels (VGCCs) regulates short-term synaptic plasticity at CA3 to CA1 excitatory synapses in acute hippocampal slices. A similar approach could also be used to manipulate and probe the neuronal circuitry in vivo.

This protocol provides a workflow on how to combine artificial microRNA-mediated RNA interference with optogenetics to stimulate specifically presynaptic boutons with reduced expression of selective gene(s) within intact neuronal circuits.

연 접 기능 그대로 신경 회로 내에 유전자 최저 효과 특성 인공 microRNAs Optogenetics 결합

The purpose of this protocol is to characterize the effect of gene knockdown on presynaptic function within intact neuronal circuits. We describe a ...

FM Dye Cycling at the Synapse: Comparing High Potassium Depolarization, Electrical and Channelrhodopsin Stimulation

FM dyes are used to study the synaptic vesicle (SV) cycle. These amphipathic probes have a hydrophilic head and hydrophobic tail, making them water-soluble with the ability to reversibly enter and exit membrane lipid bilayers. These styryl dyes are relatively non-fluorescent in aqueous medium, but insertion into the outer leaflet of the plasma membrane causes a &#62;40X increase in fluorescence. In neuronal synapses, FM dyes are internalized during SV endocytosis, trafficked both within and between SV pools, and released with SV exocytosis, providing a powerful tool to visualize presynaptic stages of neurotransmission. A primary genetic model of glutamatergic synapse development and function is the Drosophila neuromuscular junction (NMJ), where FM dye imaging has been used extensively to quantify SV dynamics in a wide range of mutant conditions. The NMJ synaptic terminal is easily accessible, with a beautiful array of large synaptic boutons ideal for imaging applications. Here, we compare and contrast the three ways to stimulate the Drosophila NMJ to drive activity-dependent FM1-43 dye uptake/release: 1) bath application of high [K+] to depolarize neuromuscular tissues, 2) suction electrode motor nerve stimulation to depolarize the presynaptic nerve terminal, and 3) targeted transgenic expression of channelrhodopsin variants for light-stimulated, spatial control of depolarization. Each of these methods has benefits and disadvantages for the study of genetic mutation effects on the SV cycle at the Drosophila NMJ. We will discuss these advantages and disadvantages to assist the selection of the stimulation approach, together with the methodologies specific to each strategy. In addition to fluorescent imaging, FM dyes can be photoconverted to electron-dense signals visualized using transmission electron microscopy (TEM) to study SV cycle mechanisms at an ultrastructural level. We provide the comparisons of confocal and electron microscopy imaging from the different methods of Drosophila NMJ stimulation, to help guide the selection of future experimental paradigms.

Synaptic vesicle (SV) cycling is the core mechanism of intercellular communication at neuronal synapses. FM dye uptake and release are the primary means of quantitatively assaying SV endo- and exocytosis. Here, we compare all the stimulation methods to drive FM1-43 cycling at the Drosophila neuromuscular junction (NMJ) model synapse.