이 작품은 &quot;ERA - NET 신경 세포의 프레임에있는 생명 보험 회사&quot;AG2R 라 Mondiale &quot;가르슈 DES Neurosciences 드 파리 (ENP), 회사 직원 Nationale 드 라 공들인&quot;ANR - 09 - NEUR - 004 &quot;에 의해 지원되었다 &quot;유럽위원회, 그리고 파스퇴르 재단 FP7 프로그램의. ㅁ 와그너는 Letten 재단에 의해 지원되었다.

1. 광학 보정 : 측정 LED 전원 <ol><li> 적극적으로 collimating 렌즈에 팬 부착이 LED / 방열판의 aparatus에 의해 냉각 방열판에 LED 배열을 연결합니다. </li><li> LED / 히트싱크 / 팬 / 렌즈 장치와 brightfield 조명에 사용되는 램프를 교체하십시오. 이 장치는 조심스럽게 collimated LED 광선은 콘덴서 렌즈를 향해 직선 광 경로를 따라 여행 있도록 배치해야합니다. 히트싱크 / 팬 제대로 시스템의 공통 접지에 접지되어 있는지 확인하십시오. </li><li> 현재의 신속하고 사각형 펄스를 줄 수있는 전원 공급 장치와 LED 배열을 드라이브. 이 전원 공급 장치는 펄스 발생기에서 발생하는 5V TTL 펄스에 의해 제어 수 있습니다. </li><li> 필드 다이아 프램과 condensor 렌즈 사이에 정의된 빛의 경로를 따라 센터는 collimated 빔. 이상적으로, LED 광선은 약간 완전히 오픈 필드 다이어프램을 너무 많이 넣다 것입니다. 일반적으로, 더 단단히 collimated LED 빔 덜이 조리개를 채울 것이며, mor을 생산합니다균일의 비용으로 전자 파워. 우리의 설정에 우리는 consenser 다이어프램에의 활용 비행기에서 LED 배열의 약간 확장된 이미지를 투영 collimating 렌즈를 선택하여 빛을 균일 증가했습니다. </li><li> 필드 다이아 프램 (광원에 가장 가까운 다이아 프램)의 이미지가 슬라이스 챔버 (그림 1)에 초점을 맞추고 있습니다 있도록 콘덴서를 집중하여 쾰러 조명을 얻을 수 있습니다. 렌즈 종이의 얇은 조직은 다른 깊이에있는 필드 다이아 프램의 초점을 맞춘 이미지를 시각화하기 위해 프로젝션 스크린의 역할을 수행할 수 있습니다. </li><li> 불투명 재료로 알려진 직경 pinholes 일련의 드릴. 광 파워 미터의 센서를 통해 이러한 작은 pinholes 중 하나를 놓으십시오. 그것이 최대 읽기를 제공하기 전까지 단순히 전원 측정기를 이동하여 필드 다이아 프램의 초점을 맞춘 이미지 위에 표본 무대와 센터의 전원 측정기 전원 측정기를 놓습니다. 이 위치에 접사 전원 측정기. </li><li> 완벽하게 모든 apertures (aperature 다이어프램과를 엽니다분야 다이어프램). 체계적으로 광학 lightpath에 상대적으로 첨부된 슬라이스 챔버 / 파워 미터를 이동하고 조명 지역 광 전력의 균일을 계산합니다. 시스템의 균일 계획을 작성할 수 있습니다. 현미경이 제대로 목표의 초점을 중심 쾰러 조명과 함께 설치하는 경우, 최대 전력 직접 목표 아래 있어야하고,이 초점 이외의 지역은 지금 균일 계획에 따라 전력 알려진 금액을 받으실 수 있습니다. </li><li> 핀홀의 각 크기, 광학 전력 대 핀홀 면적의 표준 곡선을 빌드합니다. LED 배열 전류 입력을 조정하여 여러 전력 레벨에서이 곡선을 생산하고 각 곡선에서 mm2 당 전력을 계산합니다. LED 배열 패치 광학 사용하는 경우, 빛이 패치 조명 아래에서 전염이 얼마나 알고 패치 (콘덴서, pinholes 및 필터)에 필요한 광학 요소를 소개했는지 확인하십시오. </li><li> 의 전원 측정기를 내리고수은 램프가 켜져 때 목표를 얼굴, 470nm에서 조명의 강도를 계산합니다. </li><li> 실로 살아있는 슬라이스를 추가하고, 산란 뇌 조직을 통해 전달 빛을 전력을 결정하는 표준 곡선을 재건. </li></ol> 2. 깔끔히 절차 및 전기 생리학 부품 A : 슬라이스 준비 <ol><li> 마우스를 마취 (60 MG / kg 케타민을 및 2mg/kg Xylazine)와 목을 벨. ;의 혼합물과 함께 부풀어 오른 ~ 310mOsm, 산도 7.4 124 NaCl, 3 KCl, 1.3 MgSO 4, 26 NaHCO 3, 1.25 NaHPO 4, 20 포도당, 2 CaCl 2 : 인공 대뇌 척수에서 두뇌 (ACSF, MM의 해부하다 95 % O2와 5 %는 후각 전구를 손상하지 않도록 돌봐 2 5,1)를 CO. 하나도 에지 (가로 섹션)와 복부 표면 한천에있는 두 개의 반구과 장소를 구분합니다. </li><li> 접착제 vibr에 한천 각 피질 반구의 표면 지느러미atome 척, 천천히 얼음처럼 차가운 ACSF와 함께 목욕을 입력합니다. 300 μm의 섹션에서 복부 표면에서 슬라이스, 예열 (34-36 ° C)에 각 섹션을 전송하는 산소 ACSF, 그들 30~45분에 대한 복구하는 수. </li></ol> 파트 B : 스파이크에 대한 임계값의 루스 패치 측정 <ol start="3"><li> 30 분 동안 실내 온도에 조각을 가져갈 후, 부드럽게 산소 ACSF의 지속적인 재관류 아래 현미경 챔버의 기록 챔버에서 슬라이스를 배치합니다. 지나치게 ChR2 감염된 신경을 자극 위험, EYFP - ChR2의 존재는 epifluorescence (그림 2A)에서 확인할 수 있습니다 </li><li> 피펫 풀러 (셔터 P - 97)에 유리 전극을 당기세요. ACSF이 전극을 입력합니다. ACSF 목욕에 배치하면 끝 저항은 70-10 MOhms 사이 여야합니다. </li><li> 형광등 조명 아래에서 성숙한 형태로 슬라이스 건강한 ChR2 - EYFP 신경 세포에서 찾습니다. 또한 패치 광학에서이 신경 세포의 소마를 찾습니다. </li><li> 전극 팁 통과 밝은 긍정적인 압력을 통해 확인된 형광 뉴런쪽으로 패치 전극을 낮춥니다. 막 연락이 이루어질 때, 신속하게 긍정적인 압력을 풀어 팁을 통해 흡입의 작고 간단한 금액을 적용합니다. 기가 - 옴 시일이 플라즈마 막과 전극 패치의 벽 사이에하여야한다. </li><li> 기가 - 인감은 측정 지역 현장 잠재력을 생산해야하는 전극이 충분히 형광등 신경 요동 치고 활동에 가까운 경우, 형성했다. 없다고해도 빛의 여러 복용을 깜박하여이 신경 세포에 ChR2을 활성화합니다. 광 선량은 LED 전원 및 기간 모두의 함수이기 때문에, 많은 빛이 여러 권력 및 기간이 있습니다 (그림 2B)에서 실천 가능성을 연상하는 데 필요한 방법을 계산합니다. 급상승은 수은 램프 조명 아래에서 발생하는 얼마나 많은도 관찰합니다. </li></ol> 3. 대표 결과 : 우리의 현미경에 (올림푸스 BX51WI), 우리 LED가 나는 것입니다이 apertures과 콘덴서 렌즈와 n은 라인, 따라서 공장 설치된 아크 램프의 원래 lightpath을 유지. 필드 다이아 프램과 aperature 다이어프램을 모두 닫아, 우리는 패치 클램프 레코딩 (그림의 1B)에 대한 brightfield 대비가 충분 얻을 수 있습니다. 모든 diaphragms과 완벽하게 우리가 channelrhodopsin 활성화 (그림 1A)에 대한 최대의 조명 전원 슬라이스 노출 엽니다. 우리의 현미경에서이 패치 구성 (4.1 μW / mm 2 대 6.88 MW / mm 2) 최대 전체 필드 밀도보다 크기의 약 세 주문 낮은 광 밀도를 생산하고 있습니다. 한 성인 태어나 ChR2 - EYFP은 과립 세포를 표현하는 것을 나타냅니다 우리는 주동이의 철새 스트림 (그림 2A)에서 느슨한 - 패치 녹음에 neuroblasts 마이 그 레이션의 lentiviral 감염 이후 성인 태어난 후각 망울의 과립과 periglomerular의 뉴런 주의 강력한 상표를 참조하십시오 맥심에서 5 MS의 자극음 전력 (6.88 MW / mm 2) (그림 2B) 요동 치고 연상하기에 충분합니다. 표현 수준이 세포 사이에 차이가 있기 때문에, 스파이크에 대한 임계값을 전달 빛의 양은 다를 수 있으며 관심있는 각 세포 유형에 대한 통계 설명한다. <img src="/files/ftp_upload/3379/3379fig1.jpg" alt="그림 1" /> 그림 1. 전체 분야 photostimulation 및 패치 - 클램프 슬라이스 전기 생리학에 대한 배열 설정을 이끌었습니다. channelrhodopsin를 활성화하려면 (ChR2) 우리는 오픈 뒤로 apertures 및 콘덴서 광학 (A)을 통해 collimated 광선을 프로젝트. 이 구성은 완전히 필드 다이어프램을 폐쇄하고 필드 다이아 프램의 modulating 폭 (B)에 의해 높은 대비 패치 광학으로 변경할 수 있습니다. 약어 : A. 팬, B.의 히트싱크, C. LED 배열, D. collimating 렌즈, E. 거울, F. 필드 다이어프램, G. 조리개 다이어프램, H. 콘덴서 렌즈, 난 SAmple 단계, J. 객관적. <img src="/files/ftp_upload/3379/3379fig2.jpg" alt="그림 2" /> 그림 2. 패치 클램프 및 전체 필드 photostimulation에 대한 후각 망울의 300μm 수평 슬라이스 (A)의 이미지. ChR2 - EYFP을 표현 Lentivirally 감염된 성인 출생 과립 세포는 후각 망울의 핵심에서 발산 볼 수 있습니다. 급상승 연상하는 데 필요한 빛을 복용량은 LED 플래시 (B)의 기간을 늘려 찾을 수 있습니다. 이 과립 세포에 대한 임계값은 전체 LED 강도 (2.43mW/mm 2)에서 5ms되었습니다. 규모 (A) = 500μm, (B) = 50ms 규모.

<ol>
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관심 없음 충돌 선언하지 않습니다.

최근 몇 년 동안은 신경 과학 연구 6 optogenetic 도구의 인기가 폭발을 보았다. 결과적으로, 이러한 새 도구를 사용하여 시작하고자하는 실험실에 대한 항목의 장벽을 낮출 점점 더 중요합니다. 여기서 우리는 channelrhodopsin - 표현 뉴런의 전체 필드 광 자극을 할 수 있도록 기존 패치 - 클램프 장비의 간단하고 저렴한 비용 retrofitting 및 교정을 실시하는 방법을 설명합니다. 특히, 우리는 특별히...

<table border="1">
	<tbody>
		<tr>
			<td>Material</td>
			<td>Type</td>
			<td>Company</td>
			<td>Catalogue No.</td>
		</tr>
		<tr>
			<td>Ketamine</td>
			<td>100 mg/ml</td>
			<td>Imalg&egrave;ne 1000</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>2%</td>
			<td>Rompun</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>&nbps;</td>
			<td>Sigma </td>
			<td>S5886</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>&nbsp;</td>
			<td>Sigma </td>
			<td>P5405</td>
		</tr>
		<tr>
			<td>MgSO4</td>
			<td>&nbsp;</td>
			<td>Sigma </td>
			<td>M1880</td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>&nbsp;</td>
			<td>Sigma </td>
			<td>S5761</td>
		</tr>
		<tr>
			<td>NaHPO4</td>
			<td>&nbsp;</td>
			<td>Sigma </td>
			<td>S5011</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>&nbsp;</td>
			<td>Sigma </td>
			<td>G7021</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>&nbsp;</td>
			<td>Sigma </td>
			<td>C7902</td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>&nbsp;</td>
			<td>Sigma </td>
			<td>A9539</td>
		</tr>
		<tr>
			<td>Pipette Puller</td>
			<td>P-97</td>
			<td>Sutter</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Glass Capillaries</td>
			<td>1.5 mm O.D./1.17 mm I.D.</td>
			<td>Harvard Apparatus</td>
			<td>GC150T-10</td>
		</tr>
		<tr>
			<td>LED array</td>
			<td>&nbsp;</td>
			<td>Bridgelux</td>
			<td>BXRA-C2000</td>
		</tr>
		<tr>
			<td>Collimating lens</td>
			<td>40 mm beam diameter</td>
			<td>Thor Labs</td>
			<td>LEDC1</td>
		</tr>
		<tr>
			<td>Power supply</td>
			<td>2.8 amp</td>
			<td>A1W Electronik</td>
			<td>HKO2800</td>
		</tr>
		<tr>
			<td>Optical power meter</td>
			<td>&nbsp;</td>
			<td>Thor Labs</td>
			<td>PM 100</td>
		</tr>
		<tr>
			<td>Heatsink </td>
			<td>Silent Boost K8</td>
			<td>Thermaltake</td>
			<td>A1838</td>
		</tr>
		<tr>
			<td>Fan</td>
			<td>Silent Boost K8</td>
			<td>Thermaltake</td>
			<td>A1838</td>
		</tr>
		<tr>
			<td>Vibratome</td>
			<td>&nbsp;</td>
			<td>Leica</td>
			<td>VT1200S</td>
		</tr>
	</tbody>
</table>

using affordable led arrays for photo-stimulation of neurons

표준 슬라이스 전기 생리학은 연구자가 전기 또는 약리 조작 1,2에 대한 응답으로 단일 세포의 전기적 반응을 기록하여 신경 회로의 개별 구성 요소를 프로브 수 있습니다. 광학 유전자 타겟으로 뉴런을 (optogenetics) 제어 방법의 발명과 함께, 연구자들은 이제 표준 슬라이스 준비 뉴런의 특정 그룹을 제어할의 전례없는 수준을했습니다. 특히, channelrhodopsin - 2 (ChR2) 감광성의 연구자들이 빛을 3,4과 뉴런을 활성화 할 수 있습니다. 표준 슬라이스 전기 생리학과 ChR2의 LED 기반 photostimulation주의 교정을 결합함으로써, 우리는보다 세부와 후각 망울, 후각 시스템의 첫 번째 중앙 릴레이에 성인 태어나 interneurons의 역할을 탐침 수 있습니다. 성인에서 태어난 뉴런에 특별히 ChR2 - YFP의 바이러스 표현을 사용하여, 우리는 선택 이전의 환경에서 젊은 성인에서 태어난 뉴런을 제어할 수 있습니다D 성숙한 뉴런. 우리 광학 제어는 간단하고 저렴한 LED 시스템을 사용하고, 우리는이 시스템이 더 많은 햇빛이 하나의 뉴런의 활동을 급상승 연상하는 데 필요한 방법을 이해 조정 할 수있는 방법을 보여줍니다. 따라서, 파란색 빛을 짧은 깜박 원격 ChR2 - transduced 신생아 세포의 발사 패턴을 제어할 수 있습니다.

Watch this Scientific Journal Video about Using Affordable LED Arrays for Photo-Stimulation of Neurons at JoVE.com

Using Affordable LED Arrays for Photo-Stimulation of Neurons

ChR2을 표현 성인에서 태어난 뉴런은 후각 신경 회로의 기능들에 대한 그들의 기여를 검토하기 위해 슬라이스 electrophysiological 준비로 조작할 수 있습니다.

뉴런의 사진 자극에 저렴한 LED 배열을 사용하여

Standard slice electrophysiology has allowed researchers to probe individual components of neural circuitry by recording electrical responses of single ...

using-affordable-led-arrays-for-photo-stimulation-of-neurons

Research

JoVE Journal

Neuroscience

18.5K 조회수.  Institut Pasteur and Centre National de la Recherche Scientifique (CNRS). ChR2을 표현 성인에서 태어난 뉴런은 후각 신경 회로의 기능들에 대한 그들의 기여를 검토하기 위해 슬라이스 electrophysiological 준비로 조작할 수 있습니다.

비디오: Using Affordable LED Arrays for Photo-Stimulation of Neurons

Scalable Fluidic Injector Arrays for Viral Targeting of Intact 3-D Brain Circuits

Our understanding of neural circuits--how they mediate the computations that subserve sensation, thought, emotion, and action, and how they are corrupted in neurological and psychiatric disorders--would be greatly facilitated by a technology for rapidly targeting genes to complex 3-dimensional neural circuits, enabling fast creation of "circuit-level transgenics." We have recently developed methods in which viruses encoding for light-sensitive proteins can sensitize specific cell types to millisecond-timescale activation and silencing in the intact brain. We here present the design and implementation of an injector array capable of delivering viruses (or other fluids) to dozens of defined points within the 3-dimensional structure of the brain (Figure. 1A, 1B). The injector array comprises one or more displacement pumps that each drive a set of syringes, each of which feeds into a polyimide/fused-silica capillary via a high-pressure-tolerant connector. The capillaries are sized, and then inserted into, desired locations specified by custom-milling a stereotactic positioning board, thus allowing viruses or other reagents to be delivered to the desired set of brain regions. To use the device, the surgeon first fills the fluidic subsystem entirely with oil, backfills the capillaries with the virus, inserts the device into the brain, and infuses reagents slowly (&lt;0.1 microliters/min). The parallel nature of the injector array facilitates rapid, accurate, and robust labeling of entire neural circuits with viral payloads such as optical sensitizers to enable light-activation and silencing of defined brain circuits. Along with other technologies, such as optical fiber arrays for light delivery to desired sets of brain regions, we hope to create a toolbox that enables the systematic probing of causal neural functions in the intact brain. This technology may not only open up such systematic approaches to circuit-focused neuroscience in mammals, and facilitate labeling of brain regions in large animals such as non-human primates, but may also open up a clinical translational path for cell-specific optical control prosthetics, whose precision may enable improved treatment of intractable brain disorders. Finally, such devices as described here may facilitate precisely-timed fluidic delivery of other payloads, such as stem cells and pharmacological agents, to 3-dimensional structures, in an easily user-customizable fashion.

Controlling and analyzing neural circuits in vivo would be facilitated by a technology for delivery of viruses and other reagents to desired 3-dimensional sets of brain regions. We demonstrate customized fluidic injector array fabrication, and delivery of virally-encoded optical sensitizers, enabling optical manipulation of complex brain circuits.

손상 3 - D 뇌 회로의 타겟팅 바이러스성을위한 확장 가능한 유체 인젝터 배열

Our understanding of neural circuits--how they mediate the computations that subserve sensation, thought, emotion, and action, and how they are corrupted ...

연구

신경과학

Quantitative Analysis of Synaptic Vesicle Pool Replenishment in Cultured Cerebellar Granule Neurons using FM Dyes

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis. Retrieved SVs are then refilled with neurotransmitter and rejoin the recycling pool, defined as SVs that are available for exocytosis1,2. The recycling pool can generally be subdivided into two distinct pools - the readily releasable pool (RRP) and the reserve pool (RP). As their names imply, the RRP consists of SVs that are immediately available for fusion while RP SVs are released only during intense stimulation1,2. It is important to have a reliable assay that reports the differential replenishment of these SV pools in order to understand 1) how SVs traffic after different modes of endocytosis (such as clathrin-dependent endocytosis and activity-dependent bulk endocytosis) and 2) the mechanisms controlling the mobilisation of both the RRP and RP in response to different stimuli.
FM dyes are routinely employed to quantitatively report SV turnover in central nerve terminals3-8. They have a hydrophobic hydrocarbon tail that allows reversible partitioning in the lipid bilayer, and a hydrophilic head group that blocks passage across membranes. The dyes have little fluorescence in aqueous solution, but their quantum yield increases dramatically when partitioned in membrane9. Thus FM dyes are ideal fluorescent probes for tracking actively recycling SVs. The standard protocol for use of FM dye is as follows. First they are applied to neurons and are taken up during endocytosis (Figure 1). After non-internalised dye is washed away from the plasma membrane, recycled SVs redistribute within the recycling pool. These SVs are then depleted using unloading stimuli (Figure 1). Since FM dye labelling of SVs is quantal10, the resulting fluorescence drop is proportional to the amount of vesicles released. Thus, the recycling and fusion of SVs generated from the previous round of endocytosis can be reliably quantified.
Here, we present a protocol that has been modified to obtain two additional elements of information. Firstly, sequential unloading stimuli are used to differentially unload the RRP and the RP, to allow quantification of the replenishment of specific SV pools. Secondly, each nerve terminal undergoes the protocol twice. Thus, the response of the same nerve terminal at S1 can be compared against the presence of a test substance at phase S2 (Figure 2), providing an internal control. This is important, since the extent of SV recycling across different nerve terminals is highly variable11.
Any adherent primary neuronal cultures may be used for this protocol, however the plating density, solutions and stimulation conditions are optimised for cerebellar granule neurons (CGNs)12,13.

A live fluorescence imaging technique to quantify the replenishment and mobilisation of specific synaptic vesicle (SV) pools in central nerve terminals is described. Two rounds of SV recycling are monitored in the same nerve terminals providing an internal control.

FM 염료를 사용하여 양식 소뇌 과립 뉴런에서 시냅틱 소포 풀 보충의 정량 분석

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis.  Retrieved SVs are then refilled with ...

Optogenetic Activation of Zebrafish Somatosensory Neurons using ChEF-tdTomato

Larval zebrafish are emerging as a model for describing the development and function of simple neural circuits. Due to their external fertilization, rapid development, and translucency, zebrafish are particularly well suited for optogenetic approaches to investigate neural circuit function. In this approach, light-sensitive ion channels are expressed in specific neurons, enabling the experimenter to activate or inhibit them at will and thus assess their contribution to specific behaviors. Applying these methods in larval zebrafish is conceptually simple but requires the optimization of technical details. Here we demonstrate a procedure for expressing a channelrhodopsin variant in larval zebrafish somatosensory neurons, photo-activating single cells, and recording the resulting behaviors. By introducing a few modifications to previously established methods, this approach could be used to elicit behavioral responses from single neurons activated up to at least 4 days post-fertilization (dpf). Specifically, we created a transgene using a somatosensory neuron enhancer, CREST3, to drive the expression of the tagged channelrhodopsin variant, ChEF-tdTomato. Injecting this transgene into 1-cell stage embryos results in mosaic expression in somatosensory neurons, which can be imaged with confocal microscopy. Illuminating identified cells in these animals with light from a 473 nm DPSS laser, guided through a fiber optic cable, elicits behaviors that can be recorded with a high-speed video camera and analyzed quantitatively. This technique could be adapted to study behaviors elicited by activating any zebrafish neuron. Combining this approach with genetic or pharmacological perturbations will be a powerful way to investigate circuit formation and function.

Optogenetic techniques have made it possible to study the contribution of specific neurons to behavior. We describe a method in larval zebrafish for activating single somatosensory neurons expressing a channelrhodopsin variant (ChEF) with a diode-pumped solid state (DPSS) laser and recording the elicited behaviors with a high-speed video camera.

요리사 tdTomato를 사용하여 Zebrafish Somatosensory의 뉴런의 Optogenetic 활성화

Larval  zebrafish are emerging as a model for describing the development and function  of simple neural circuits. Due to their  external fertilization, ...

Imaging Dendritic Spines of Rat Primary Hippocampal Neurons using Structured Illumination Microscopy

Dendritic spines are protrusions emerging from the dendrite of a neuron and represent the primary postsynaptic targets of excitatory inputs in the brain. Technological advances have identified these structures as key elements in neuron connectivity and synaptic plasticity. The quantitative analysis of spine morphology using light microscopy remains an essential problem due to technical limitations associated with light's intrinsic refraction limit. Dendritic spines can be readily identified by confocal laser-scanning fluorescence microscopy. However, measuring subtle changes in the shape and size of spines is difficult because spine dimensions other than length are usually smaller than conventional optical resolution fixed by light microscopy's theoretical resolution limit of 200 nm.
Several recently developed super resolution techniques have been used to image cellular structures smaller than the 200 nm, including dendritic spines. These techniques are based on classical far-field operations and therefore allow the use of existing sample preparation methods and to image beyond the surface of a specimen. Described here is a working protocol to apply super resolution structured illumination microscopy (SIM) to the imaging of dendritic spines in primary hippocampal neuron cultures. Possible applications of SIM overlap with those of confocal microscopy. However, the two techniques present different applicability. SIM offers higher effective lateral resolution, while confocal microscopy, due to the usage of a physical pinhole, achieves resolution improvement at the expense of removal of out of focus light. In this protocol, primary neurons are cultured on glass coverslips using a standard protocol, transfected with DNA plasmids encoding fluorescent proteins and imaged using SIM. The whole protocol described herein takes approximately 2 weeks, because dendritic spines are imaged after 16-17 days in vitro, when dendritic development is optimal. After completion of the protocol, dendritic spines can be reconstructed in 3D from series of SIM image stacks using specialized software.

This article describes a working protocol to image dendritic spines from hippocampal neurons in vitro using Structured Illumination Microscopy (SIM). Super-resolution microscopy using SIM provides image resolution significantly beyond the light diffraction limit in all three spatial dimensions, allowing the imaging of individual dendritic spines with improved detail.

구조적 조명 현미경을 사용하여 쥐 차 해마 신경 세포의 돌기 쪽 이미징

Dendritic spines are protrusions emerging from the dendrite of a neuron and represent the primary postsynaptic targets of excitatory inputs in the brain. ...

Production and Isolation of Axons from Sensory Neurons for Biochemical Analysis Using Porous Filters

Neuronal axons use specific mechanisms to mediate extension, maintain integrity, and induce degeneration. An appropriate balance of these events is required to shape functional neuronal circuits. The protocol described here explains how to use cell culture inserts bearing a porous membrane (filter) to obtain large amounts of pure axonal preparations suitable for examination by conventional biochemical or immunocytochemical techniques. The functionality of these filter inserts will be demonstrated with models of developmental pruning and Wallerian degeneration, using explants of embryonic dorsal root ganglion. Axonal integrity and function is compromised in a wide variety of neurodegenerative pathologies. Indeed, it is now clear that axonal dysfunction appears much earlier in the course of the disease than neuronal soma loss in several neurodegenerative diseases, indicating that axonal-specific processes are primarily targeted in these disorders. By obtaining pure axonal samples for analysis by molecular and biochemical techniques, this technique has the potential to shed new light into mechanisms regulating the physiology and pathophysiology of axons. This in turn will have an impact in our understanding of the processes that drive degenerative diseases of the nervous system.

This article describes a neuronal culture system that can be used to obtain large quantities of pure axonal samples for biochemical and immunocytochemical analyses. This technique will allow an improved understanding of normal axonal physiology and signaling pathways leading to neurodegeneration.

생화학 적 분석에 대한 감각 뉴런에서 생산 및 축삭의 분리는 다공성 필터를 사용하여

Neuronal axons use specific mechanisms to mediate extension, maintain integrity, and induce degeneration. An appropriate balance of these events is ...

External Excitation of Neurons Using Electric and Magnetic Fields in One- and Two-dimensional Cultures

A neuron will fire an action potential when its membrane potential exceeds a certain threshold. In typical activity of the brain, this occurs as a result of chemical inputs to its synapses. However, neurons can also be excited by an imposed electric field. In particular, recent clinical applications activate neurons by creating an electric field externally. It is therefore of interest to investigate how the neuron responds to the external field and what causes the action potential. Fortunately, precise and controlled application of an external electric field is possible for embryonic neuronal cells that are excised, dissociated and grown in cultures. This allows the investigation of these questions in a highly reproducible system.
In this paper some of the techniques used for controlled application of external electric field on neuronal cultures are reviewed. The networks can be either one dimensional, i.e. patterned in linear forms or allowed to grow on the whole plane of the substrate, and thus two dimensional. Furthermore, the excitation can be created by the direct application of electric field via electrodes immersed in the fluid (bath electrodes) or by inducing the electric field using the remote creation of magnetic pulses.

Neuronal cultures are a good model for studying emerging brain stimulation techniques via their effect on single neurons or a population of neurons. Presented here are different methods for stimulation of patterned neuronal cultures by an electric field produced directly by bath electrodes or induced by a time-varying magnetic field.

전기 단일 및 두 개 차원 문화에 자기장을 사용하여 신경 세포의 외부인가 전압

A neuron will fire an action potential when its membrane potential exceeds a certain threshold. In typical activity of the brain, this occurs as a result ...

Live Images of GLUT4 Protein Trafficking in Mouse Primary Hypothalamic Neurons Using Deconvolution Microscopy

Type 2 diabetes mellitus (T2DM) is a global health crisis which is characterized by insulin signaling impairment and chronic inflammation in peripheral tissues. The hypothalamus in the central nervous system (CNS) is the control center for energy and insulin signal response regulation. Chronic inflammation in peripheral tissues and imbalances of certain chemokines (such as CCL5, TNF&#945;, and IL-6) contribute to diabetes and obesity. However, the functional mechanism(s) connecting chemokines and hypothalamic insulin signal regulation still remain unclear.
In vitro primary neuron culture models are convenient and simple models which can be used to investigate insulin signal regulation in hypothalamic neurons. In this study, we introduced exogeneous GLUT4 protein conjugated with GFP (GFP-GLUT4) into primary hypothalamic neurons to track GLUT4 membrane translocation upon insulin stimulation. Time-lapse images of GFP-GLUT4 protein trafficking were recorded by deconvolution microscopy, which allowed users to generate high-speed, high-resolution images without damaging the neurons significantly while conducting the experiment. The contribution of CCR5 in insulin regulated GLUT4 translocation was observed in CCR5 deficient hypothalamic neurons, which were isolated and cultured from CCR5 knockout mice. Our results demonstrated that the GLUT4 membrane translocation efficiency was reduced in CCR5 deficient hypothalamic neurons after insulin stimulation.

This protocol describes a technique for observation of real-time Green Fluorescence Protein (GFP) tagged Glucose Transporter 4 (GLUT4) protein trafficking upon insulin stimulation and characterization of the biological role of CCR5 in the insulin&#8211;GLUT4 signaling pathway with Deconvolution Microscopy.

마우스 기본 Hypothalamic 뉴런 Deconvolution 현미경을 사용 하 여 매매 GLUT4 단백질의 라이브 이미지

Type 2 diabetes mellitus (T2DM) is a global health crisis which is characterized by insulin signaling impairment and chronic inflammation in peripheral ...

An In Vitro Model for Studying Tau Aggregation Using Lentiviral-mediated Transduction of Human Neurons

Aberrant aggregation of the protein tau is pathogenically involved in a number of neurodegenerative diseases, including Alzheimer&#8217;s disease (AD). Although mouse models of tauopathy have provided a valuable resource for investigating the neurotoxic mechanisms of aggregated tau, it is becoming increasingly apparent that, due to interspecies differences in neurophysiology, the mouse brain is unsuitable for modeling the human condition. Advances in cell culture methods have made human neuronal cultures accessible for experimental use in vitro and have aided in the development of neurotherapeutics. However, despite the adaptation of human neuronal cell cultures, in vitro models of human tauopathy are not yet widely available. This protocol describes a cellular model of tau aggregation in which human neurons are transduced with lentiviral-derived vectors that code for pathogenically mutated tau fused to a yellow fluorescent protein (YFP) reporter. Transduced cultures produce tau aggregates that stain positively for thioflavin and display markers of neurotoxicity, such as decreased axonal length and increased lysosomal volume. This procedure may be a useful and cost-effective model for studying human tauopathies.

This protocol details a procedure in which human neuronal cultures are transduced with lentiviral constructs coding for mutant human tau. Transduced cultures display tau aggregates and associated pathologies.

인간 뉴런의 렌 티 바이러스 매개 된 형질 도입을 이용한 타우 응집 연구를 위한 시험관 내 모델

Aberrant aggregation of the protein tau is pathogenically involved in a number of neurodegenerative diseases, including Alzheimer&#8217;s disease (AD). ...

Chronic Implantation of Multiple Flexible Polymer Electrode Arrays

Simultaneous recordings from large populations of individual neurons across distributed brain regions over months to years will enable new avenues of scientific and clinical development. The use of flexible polymer electrode arrays can support long-lasting recording, but the same mechanical properties that allow for longevity of recording make multiple insertions and integration into a chronic implant a challenge. Here is a methodology by which multiple polymer electrode arrays can be targeted to a relatively spatially unconstrained set of brain areas.
The method utilizes thin-film polymer devices, selected for their biocompatibility and capability to achieve long-term and stable&#160;electrophysiologic recording interfaces. The resultant implant allows accurate and flexible targeting of anatomically distant regions, physical stability for months, and robustness to electrical noise. The methodology supports up to sixteen serially inserted devices across eight different anatomic targets. As previously demonstrated, the methodology is capable of recording from 1024 channels. Of these, the 512 channels in this demonstration used for single neuron recording yielded 375 single units distributed across six recording sites. Importantly, this method also can record single units for at least 160 days.
This implantation strategy, including temporarily bracing each device with a retractable silicon insertion shuttle, involves tethering of devices at their target depths to a skull-adhered plastic base piece that is custom-designed for each set of recording targets, and stabilization/protection of the devices within a silicone-filled, custom-designed plastic case. Also covered is the preparation of devices for implantation, and design principles that should guide adaptation to different combinations of brain areas or array designs.

Described below is a method for implantation of multiple polymer electrode arrays across anatomically distant brain regions for chronic electrophysiological recording in freely moving rats. Preparation and surgical implantation are described in detail, with emphasis on design principles to guide adaptation of these methods for use in other species.

다중 유연한 폴리머 전극 어레이의 만성 이식

Simultaneous recordings from large populations of individual neurons across distributed brain regions over months to years will enable new avenues of ...

Stereotaxic Surgery for Implantation of Microelectrode Arrays in the Common Marmoset (Callithrix jacchus)

Marmosets (Callithrix jacchus) are small non-human primates that are gaining popularity in biomedical and preclinical research, including the neurosciences. Phylogenetically, these animals are much closer to humans than rodents. They also display complex behaviors, including a wide range of vocalizations and social interactions. Here, an effective stereotaxic neurosurgical procedure for implantation of recording electrode arrays in the common marmoset is described. This protocol also details the pre- and postoperative steps of animal care that are required to successfully perform such a surgery. Finally, this protocol shows an example of local field potential and spike activity recordings in a freely behaving marmoset 1 week after the surgical procedure. Overall, this method provides an opportunity to study the brain function in awake and freely behaving marmosets. The same protocol can be readily used by researchers working with other small primates. In addition, it can be easily modified to allow other studies requiring implants, such as stimulating electrodes, microinjections, implantation of optrodes or guide cannulas, or ablation of discrete tissue regions.

Stereotaxic Surgery for Implantation of Microelectrode Arrays in the Common Marmoset Callithrix jacchus

This work presents a protocol to perform a stereotaxic, neurosurgical implantation of&#160;microelectrode arrays in the common marmoset. This method specifically enables electrophysiological recordings in freely behaving animals but can be easily adapted to any other similar neurosurgical intervention in this species (e.g., cannula for drug administration or electrodes for brain stimulation).