우리는주의 편집을 위해 켈리 존스 감사합니다. 미국 심장 협회 박사 과정 이수 원정대 (DPS),이 작품은 NIH 부여 R01MH 071316, 알츠하이머 협회, 정신 분열증과 우울증 (NARSAD)에 관한 연구에 대한 국가 연합, 그리고 자폐증 연구에 대한 국가 연합 (나르) (PP)에 의해 지원되었다 미국 심장 협회 Predoctoral 원정대 (KMW).

<ol>
	<li>Banker, G., Goslin, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developments+in+neuronal+cell+culture.">Developments in neuronal cell culture.</a> Nature. 336, 185-186 (1988).</li><li>Xie, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kalirin-7+controls+activity-dependent+structural+and+functional+plasticity+of+dendritic+spines.">Kalirin-7 controls activity-dependent structural and functional plasticity of dendritic spines.</a> Neuron. 56, 640-656 (2007).</li><li>Spear, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+adolescent+development+and+alcohol+use+in+animals.">Modeling adolescent development and alcohol use in animals.</a> Alcohol Res Health. 24, 115-123 (2000).</li><li>Srivastava, D. P., Woolfrey, K. M., Liu, F., Brandon, N. J., Penzes, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estrogen+receptor+ss+activity+modulates+synaptic+signaling+and+structure.">Estrogen receptor ss activity modulates synaptic signaling and structure.</a> J Neurosci. 30, 13454-13460 (2010).</li><li>Srivastava, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+enhancement+of+two-step+wiring+plasticity+by+estrogen+and+NMDA+receptor+activity.">Rapid enhancement of two-step wiring plasticity by estrogen and NMDA receptor activity.</a> Proc Natl Acad Sci USA. 105, 14650-14655 (2008).</li><li>Woolfrey, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epac2+induces+synapse+remodeling+and+depression+and+its+disease-associated+forms+alter+spines.">Epac2 induces synapse remodeling and depression and its disease-associated forms alter spines.</a> Nat Neurosci. 12, 1275-1284 (2009).</li><li>Allison, D. W., Gelfand, V. I., Spector, I., Craig, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+actin+in+anchoring+postsynaptic+receptors+in+cultured+hippocampal+neurons:+differential+attachment+of+NMDA+versus+AMPA+receptors.">Role of actin in anchoring postsynaptic receptors in cultured hippocampal neurons: differential attachment of NMDA versus AMPA receptors.</a> J Neurosci. 18, 2423-2436 (1998).</li><li>Harms, K. J., Tovar, K. R., Craig, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synapse-specific+regulation+of+AMPA+receptor+subunit+composition+by+activity.">Synapse-specific regulation of AMPA receptor subunit composition by activity.</a> J Neurosci. 25, 6379-6388 (2005).</li><li>Xie, Z., Huganir, R. L., Penzes, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activity-dependent+dendritic+spine+structural+plasticity+is+regulated+by+small+GTPase+Rap1+and+its+target+AF-6.">Activity-dependent dendritic spine structural plasticity is regulated by small GTPase Rap1 and its target AF-6.</a> Neuron. 48, 605-618 (2005).</li><li>Jones, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+modulation+of+spine+morphology+by+the+5-HT2A+serotonin+receptor+through+kalirin-7+signaling.">Rapid modulation of spine morphology by the 5-HT2A serotonin receptor through kalirin-7 signaling.</a> Proc Natl Acad Sci USA. 106, 19575-19580 (1957).</li><li>Dunaevsky, A., Tashiro, A., Majewska, A., Mason, C., Yuste, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+regulation+of+spine+motility+in+the+mammalian+central+nervous+system.">Developmental regulation of spine motility in the mammalian central nervous system.</a> Proc Natl Acad Sci USA. 96, 13438-13443 (1999).</li><li>Lichtman, J. W., Conchello, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+microscopy.">Fluorescence microscopy.</a> Nat Methods. 2, 910-919 (2005).</li><li>Svoboda, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Do+spines+and+dendrites+distribute+dye+evenly.">Do spines and dendrites distribute dye evenly.</a> Trends Neurosci. 27, 445-446 (2004).</li></ol>

이 작품은 제작이 MetaMorph (분자 디바이스 주식 회사)에 의해 지원되었다.

기술 neuropathologies에 기여할 수있다 포스트 시냅스 메커니즘의 영향을 이해에 초점을 아르 돌기 척추 형태, 중 고정 또는 라이브 주요 대뇌 피질의 뉴런의 선형 밀도와 운동성의 상세한 정량 분석​​을 위해 위에서 설명한. 비슷한 접근 방식은 hippocampal 피라미드, Purkinje, 또는 중간 가시의 뉴런 포함하여 가시의 신경 세포에서 척추의 형태 또는 운동성을 계량하는 데 사용할 수 있습니다. <p clas...

<table border="1"><tbody><tr><th> 시약의 이름 </th><th> 회사 </th><th> 카탈로그 번호 </th><th> 댓글 (옵션) </th></tr><tr><td> 18mm 라운드 커버 유리 번호 1.5 </td><td> 워너 인 스트 루먼트 </td><td> 64-0714 (CS - 18R15) </td><td></td></tr><tr><td> 22mm 사각 커버 글라스 번호 1.5 </td><td> 워너 인 스트 루먼트 </td><td> 64-0721 (CS - 22S15) </td><td></td></tr><tr><td> 폴리 - D - 라이신 </td><td> 시그마 </td><td> P - 0899 </td><td> MW 70 ~ 150 KDA </td></tr><tr><td> Neurobasal 미디어 </td><td> Invitrogen </td><td> 21103049 </td><td></td></tr><tr><td> B27 </td><td> Invitrogen </td><td> 17504044 </td><td></td></tr><tr><td> 글루타민 </td><td> Invitrogen </td><td> 21051024 </td><td></td></tr><tr><td> 페니실린 - 스트렙토 마이신 </td><td> Invitrogen </td><td> 15140148 </td><td></td></tr><tr><td> D, L - APV (AP - 5) </td><td> 아센트 과학 </td><td> ASC - 004 </td><td></td></tr><tr><td> Lipofectamine 2000 </td><td> Invitrogen </td><td> 11668019 </td><td></td></tr><tr><td> DMEM </td><td> Invitrogen </td><td> 11965092 </td><td></td></tr><tr><td> HEPES </td><td> MediaTech Cellgro </td><td> 25-060 - C 1 </td><td> 1M, 산도 7 </td></tr><tr><td> 포름 알데히드 용액 </td><td> EMD 화학 </td><td> FX0415 - 5 </td><td> 36%, 조직학 학년 </td></tr><tr><td> 정상 염소 혈청 </td><td> VWR </td><td> 100188-514 </td><td> 잭슨 Immunoresearch 실험실 </td></tr><tr><td> 트리톤 X - 100 </td><td> 피셔 사이 언티픽 </td><td> AC21568 - 2500 </td><td> Acros의 생명체 </td></tr><tr><td> 알렉사 플루어 높은 488 염소 안티 - 마우스 IgG (H + L) 교차 adsorbed </td><td> Invitrogen </td><td> A - 11029 </td><td></td></tr><tr><td> 알렉사 플루어 488 염소 안티 - 토끼 IgG (H + L) * 높은 상호 adsorbed * </td><td> Invitrogen </td><td> A - 11034 </td><td></td></tr><tr><td> 골드 antifade 시약을 연장 </td><td> Invitrogen </td><td> P36934 </td><td> 특별 포장 </td></tr><tr><td> 동봉된 이미징 단계 챔버 </td><td> 워너 </td><td> RC - 30HV </td><td></td></tr><tr><td> 온도 컨트롤러 유닛 </td><td> 워너 </td><td> TC - 344B </td><td></td></tr><tr><td> MetaMorph </td><td> 범용 이미징 </td><td></td><td></td></tr></tbody></table>

analysis of dendritic spine morphology in cultured cns neurons

돌기 쪽이은 뇌 내에서 흥분성의 연결의 대부분의 사이트이며, 시냅스의 포스트 시냅스 칸막이를 형성하고 있습니다. 이러한 구조는 굴지 풍부하고 매우 동적으로 표시되었습니다. 고전 헵의 소성뿐만 아니라 neuromodulatory 신호에 대응하여, 돌기 쪽이은 모양과 신경 회로의 상세 및 뇌 내의 정보의 처리 및 저장을위한 중요한 것으로 생각됩니다 번호를 변경할 수 있습니다. 돌기 쪽이 내에서 단백질의 복잡한 네트워크 돌기 척추의 형태 및 숫자의 제어를위한 허용 굴지의 cyctoskeleton와 세포외 신호를 연결합니다. Neuropathological 연구는 정신 분열증 자폐증 스펙트럼 장애에 이르기까지 질병 상태의 숫자가, 비정상적인 척추 돌기 형태 또는 숫자를 표시 증명하고있다. 또한, 최근 유전자 연구는 이러한 단백질이 부분이 장애의 pathophysiology을 기초 탈선 척추 소성에 기여할 수있는 제안을 선도, 시냅스 단백질을 인코딩 다수의 유전자에 돌연변이가 확인되었습니다. 돌기 척추 morphologies / 번호를 제어에서이 단백질의 잠재적인 역할을 연구하기 위해, 교양 대뇌 피질의 뉴런의 사용은 몇 가지 장점을 제공합니다. 첫째,이 시스템은 고정된 세포의 돌기 쪽이의 고해상도 영상뿐만 아니라 라이브 세포의 시간 경과 이미징을 허용합니다. 둘째, 체외 시스템에서이 돌연변이 단백질, shRNA 구성 요소로 분해, 또는 약리 치료의 표현에 의해 단백질 기능의 쉬운 조작을 허용합니다. 이러한 기법은 연구자들이 질병 관련 단백질의 역할을 해부하다하고이 단백질의 변이가 생체내에서 작동할 수 방법을 예측하기 시작 수 있습니다.

Watch this Scientific Journal Video about Analysis of Dendritic Spine Morphology in Cultured CNS Neurons at JoVE.com

Analysis of Dendritic Spine Morphology in Cultured CNS Neurons

많은 최근의 연구는 뇌 pathologies와 관련된 시냅스 단백질에 돌연변이가 확인되었습니다. 기본 교양 피질 뉴런이 돌기 척추의 형태와 운동성에서 이러한 질병 관련 단백질의 영향을 조사 큰 유연성을 제공합니다.

교양 CNS의 뉴런의 돌기 스파인 형태론 분석

Dendritic spines are the sites of the majority of excitatory connections within the brain, and form the post-synaptic 
compartment of synapses. These ...

analysis-of-dendritic-spine-morphology-in-cultured-cns-neurons

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34.9K 조회수.  Northwestern University Feinberg School of Medicine. 많은 최근의 연구는 뇌 pathologies와 관련된 시냅스 단백질에 돌연변이가 확인되었습니다. 기본 교양 피질 뉴런이 돌기 척추의 형태와 운동성에서 이러한 질병 관련 단백질의 영향을 조사 큰 유연성을 제공합니다.

비디오: Analysis of Dendritic Spine Morphology in Cultured CNS Neurons

Automated Sholl Analysis of Digitized Neuronal Morphology at Multiple Scales

Neuronal morphology plays a significant role in determining how neurons function and communicate1-3. Specifically, it affects the ability of neurons to receive inputs from other cells2 and contributes to the propagation of action potentials4,5. The morphology of the neurites also affects how information is processed. The diversity of dendrite morphologies facilitate local and long range signaling and allow individual neurons or groups of neurons to carry out specialized functions within the neuronal network6,7. Alterations in dendrite morphology, including fragmentation of dendrites and changes in branching patterns, have been observed in a number of disease states, including Alzheimer's disease8, schizophrenia9,10, and mental retardation11. The ability to both understand the factors that shape dendrite morphologies and to identify changes in dendrite morphologies is essential in the understanding of nervous system function and dysfunction.
Neurite morphology is often analyzed by Sholl analysis and by counting the number of neurites and the number of branch tips. This analysis is generally applied to dendrites, but it can also be applied to axons. Performing this analysis by hand is both time consuming and inevitably introduces variability due to experimenter bias and inconsistency. The Bonfire program is a semi-automated approach to the analysis of dendrite and axon morphology that builds upon available open-source morphological analysis tools. Our program enables the detection of local changes in dendrite and axon branching behaviors by performing Sholl analysis on subregions of the neuritic arbor. For example, Sholl analysis is performed on both the neuron as a whole as well as on each subset of processes (primary, secondary, terminal, root, etc.) Dendrite and axon patterning is influenced by a number of intracellular and extracellular factors, many acting locally. Thus, the resulting arbor morphology is a result of specific processes acting on specific neurites, making it necessary to perform morphological analysis on a smaller scale in order to observe these local variations12.
The Bonfire program requires the use of two open-source analysis tools, the NeuronJ plugin to ImageJ and NeuronStudio. Neurons are traced in ImageJ, and NeuronStudio is used to define the connectivity between neurites. Bonfire contains a number of custom scripts written in MATLAB (MathWorks) that are used to convert the data into the appropriate format for further analysis, check for user errors, and ultimately perform Sholl analysis. Finally, data are exported into Excel for statistical analysis. A flow chart of the Bonfire program is shown in Figure 1.

We have developed a computer program to analyze neuronal morphology. In combination with two existing open source analysis tools, our program performs Sholl analysis and determines the number of neurites, branch points, and neurite tips. The analyses are performed so that local changes in neurite morphology can be observed.

여러 스케일에서 디지털의 연결 형태론의 자동 숄 분석

Neuronal morphology plays a significant role in determining how neurons function and communicate1-3.  Specifically, it affects the ability of neurons to ...

연구

신경과학

Fluorescence Recovery After Photobleaching (FRAP) of Fluorescence Tagged Proteins in Dendritic Spines of Cultured Hippocampal Neurons

FRAP has been used to quantify the mobility of GFP-tagged proteins. Using a strong excitation laser, the fluorescence of a GFP-tagged protein is bleached in the region of interest. The fluorescence of the region recovers when the unbleached GFP-tagged protein from outside of the region diffuses into the region of interest. The mobility of the protein is then analyzed by measuring the fluorescence recovery rate. This technique could be used to characterize protein mobility and turnover rate.
In this study, we express the (enhanced green fluorescent protein) EGFP vector in cultured hippocampal neurons. Using the Zeiss 710 confocal microscope, we photobleach the fluorescence signal of the GFP protein in a single spine, and then take time lapse images to record the fluorescence recovery after photobleaching. Finally, we estimate the percentage of mobile and immobile fractions of the GFP in spines, by analyzing the imaging data using ImageJ and Graphpad softwares.
This FRAP protocol shows how to perform a basic FRAP experiment as well as how to analyze the data.

Fluorescence Recovery After Photobleaching FRAP of Fluorescence Tagged Proteins in Dendritic Spines of Cultured Hippocampal Neurons

FRAP has been used to quantify the mobility of Green Fluorescence Protein (GFP)-tagged proteins in cultured cells. We examined the mobile/immobile fractions of the GFP by analyzing the fluorescence recovery percentage after photobleaching. In this study, FRAP was performed at spines of hippocampal neurons.

교양 Hippocampal 뉴런의 돌기 쪽이에서 형광 단백질의 태그 Photobleaching 후 형광 복구 (FRAP)

FRAP has been used to quantify the mobility of GFP-tagged proteins. Using a strong excitation laser, the fluorescence of a GFP-tagged protein is bleached ...

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 ...

Inducing Dendritic Growth in Cultured Sympathetic Neurons

The shape of the dendritic arbor determines the total synaptic input a neuron can receive 1-3, and influences the types and distribution of these inputs 4-6. Altered patterns of dendritic growth and plasticity are associated with impaired neurobehavioral function in experimental models 7, and are thought to contribute to clinical symptoms observed in both neurodevelopmental disorders 8-10 and neurodegenerative diseases 11-13. Such observations underscore the functional importance of precisely regulating dendritic morphology, and suggest that identifying mechanisms that control dendritic growth will not only advance understanding of how neuronal connectivity is regulated during normal development, but may also provide insight on novel therapeutic strategies for diverse neurological diseases.
Mechanistic studies of dendritic growth would be greatly facilitated by the availability of a model system that allows neurons to be experimentally switched from a state in which they do not extend dendrites to one in which they elaborate a dendritic arbor comparable to that of their in vivo counterparts. Primary cultures of sympathetic neurons dissociated from the superior cervical ganglia (SCG) of perinatal rodents provide such a model. When cultured in defined medium in the absence of serum and ganglionic glial cells, sympathetic neurons extend a single process which is axonal, and this unipolar state persists for weeks to months in culture 14,15. However, the addition of either bone morphogenetic protein-7 (BMP-7) 16,17 or Matrigel 18 to the culture medium triggers these neurons to extend multiple processes that meet the morphologic, biochemical and functional criteria for dendrites. Sympathetic neurons dissociated from the SCG of perinatal rodents and grown under defined conditions are a homogenous population of neurons 19 that respond uniformly to the dendrite-promoting activity of Matrigel, BMP-7 and other BMPs of the decapentaplegic (dpp) and 60A subfamilies 17,18,20,21. Importantly, Matrigel- and BMP-induced dendrite formation occurs in the absence of changes in cell survival or axonal growth 17,18.
Here, we describe how to set up dissociated cultures of sympathetic neurons derived from the SCG of perinatal rats so that they are responsive to the selective dendrite-promoting activity of Matrigel or BMPs.

We describe a protocol for using bone morphogenetic protein-7 (BMP-7) or Matrigel to selectively induce dendritic growth in primary sympathetic neurons dissociated from the superior cervical ganglia (SCG) of perinatal rats.

교양 공감 뉴런의 돌기 성장을 유도

The shape of the dendritic arbor determines the total synaptic input a neuron can receive 1-3, and influences the types and distribution of these inputs ...

The Analysis of Purkinje Cell Dendritic Morphology in Organotypic Slice Cultures

Purkinje cells are an attractive model system for studying dendritic development, because they have an impressive dendritic tree which is strictly oriented in the sagittal plane and develops mostly in the postnatal period in small rodents 3. Furthermore, several antibodies are available which selectively and intensively label Purkinje cells including all processes, with anti-Calbindin D28K being the most widely used. For viewing of dendrites in living cells, mice expressing EGFP selectively in Purkinje cells 11 are available through Jackson labs. Organotypic cerebellar slice cultures cells allow easy experimental manipulation of Purkinje cell dendritic development because most of the dendritic expansion of the Purkinje cell dendritic tree is actually taking place during the culture period 4. We present here a short, reliable and easy protocol for viewing and analyzing the dendritic morphology of Purkinje cells grown in organotypic cerebellar slice cultures. For many purposes, a quantitative evaluation of the Purkinje cell dendritic tree is desirable. We focus here on two parameters, dendritic tree size and branch point numbers, which can be rapidly and easily determined from anti-calbindin stained cerebellar slice cultures. These two parameters yield a reliable and sensitive measure of changes of the Purkinje cell dendritic tree. Using the example of treatments with the protein kinase C (PKC) activator PMA and the metabotropic glutamate receptor 1 (mGluR1) we demonstrate how differences in the dendritic development are visualized and quantitatively assessed. The combination of the presence of an extensive dendritic tree, selective and intense immunostaining methods, organotypic slice cultures which cover the period of dendritic growth and a mouse model with Purkinje cell specific EGFP expression make Purkinje cells a powerful model system for revealing the mechanisms of dendritic development.

We present a protocol that permits to view and to quantitatively asses the morphology of the dendritic tree of individual Purkinje cells grown in organotypic cerebellar slice cultures. This protocol is intended to promote studies on the mechanisms of Purkinje cell dendritic development.

Organotypic 슬라이스 문화의 Purkinje 세포 수지상의 형태학의 분석

Purkinje cells are an attractive model system for studying dendritic development, because they have an impressive dendritic tree which is strictly ...

Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of the dendritic tree, which eventually leads to neuronal output of action potentials at the axon. The influence of diverse spatial and temporal parameters of specific synaptic input on neuronal output is currently under investigation, e.g. the distance-dependent attenuation of dendritic inputs, the location-dependent interaction of spatially segregated inputs, the influence of GABAergig inhibition on excitatory integration, linear and non-linear integration modes, and many more.
With fast micro-iontophoresis of glutamate and GABA it is possible to precisely investigate the spatial and temporal integration of glutamatergic excitation and GABAergic inhibition. Critical technical requirements are either a triggered fluorescent lamp, light-emitting diode (LED), or a two-photon scanning microscope to visualize dendritic branches without introducing significant photo-damage of the tissue. Furthermore, it is very important to have a micro-iontophoresis amplifier that allows for fast capacitance compensation of high resistance pipettes. Another crucial point is that no transmitter is involuntarily released by the pipette during the experiment.
Once established, this technique will give reliable and reproducible signals with a high neurotransmitter and location specificity. Compared to glutamate and GABA uncaging, fast iontophoresis allows using both transmitters at the same time but at very distant locations without limitation to the field of view. There are also advantages compared to focal electrical stimulation of axons: with micro-iontophoresis the location of the input site is definitely known and it is sure that only the neurotransmitter of interest is released. However it has to be considered that with micro-iontophoresis only the postsynapse is activated and presynaptic aspects of neurotransmitter release are not resolved. In this article we demonstrate how to set up micro-iontophoresis in brain slice experiments.

In this article we introduce fast micro-iontophoresis of neurotransmitters as a technique to investigate integration of postsynaptic signals with high spatial and temporal precision.

글루타민산 염과 GABA의 빠른 마이크로 이온 도입 : 시냅틱 통합을 조사하는 유용한 도구

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of ...

Analyzing Dendritic Morphology in Columns and Layers

In many regions of the central nervous systems, such as the fly optic lobes and the vertebrate cortex, synaptic circuits are organized in layers and columns to facilitate brain wiring during development and information processing in developed animals. Postsynaptic neurons elaborate dendrites in type-specific patterns in specific layers to synapse with appropriate presynaptic terminals. The fly medulla neuropil is composed of 10 layers and about 750 columns; each column is innervated by dendrites of over 38 types of medulla neurons, which match with the axonal terminals of some 7 types of afferents in a type-specific fashion. This report details the procedures to image and analyze dendrites of medulla neurons. The workflow includes three sections: (i) the dual-view imaging section combines two confocal image stacks collected at orthogonal orientations into a high-resolution 3D image of dendrites; (ii) the dendrite tracing and registration section traces dendritic arbors in 3D and registers dendritic traces to the reference column array; (iii) the dendritic analysis section analyzes dendritic patterns with respect to columns and layers, including layer-specific termination and planar projection direction of dendritic arbors, and derives estimates of dendritic branching and termination frequencies. The protocols utilize custom plugins built on the open-source MIPAV (Medical Imaging Processing, Analysis, and Visualization) platform and custom toolboxes in the matrix laboratory language. Together, these protocols provide a complete workflow to analyze the dendritic routing of Drosophila medulla neurons in layers and columns, to identify cell types, and to determine defects in mutants.

Here, we show how to analyze dendritic routing of Drosophila medulla neurons in columns and layers. The workflow includes a dual-view imaging technique to improve the image quality and computational tools for tracing, registering dendritic arbors to the reference column array and for analyzing the dendritic structures in 3D space.

열 및 레이어에 수지상 형태학 분석

In many regions of the central nervous systems, such as the fly optic lobes and the vertebrate cortex, synaptic circuits are organized in layers and ...

Quantification of Endosome and Lysosome Motilities in Cultured Neurons Using Fluorescent Probes

In the brain, membrane trafficking systems play important roles in regulating neuronal functions, such as neuronal morphology, synaptic plasticity, survival, and glial communications. To date, numerous studies have reported that defects in these systems cause various neuronal diseases. Thus, understanding the mechanisms underlying vesicle dynamics may provide influential clues that could aid in the treatment of several neuronal disorders. Here, we describe a method for quantifying vesicle motilities, such as motility distance and rate of movement, using a software plug-in for the ImageJ platform. To obtain images for quantification, we labeled neuronal endosome-lysosome structures with EGFP-tagged vesicle marker proteins and observed the movement of vesicles using a time-lapse microscopy. This method is highly useful and simplify measuring vesicle motility in neurites, such as axons and dendrites, as well as in the soma of both neurons and glial cells. Furthermore, this method can be applied to other cell lines, such as fibroblasts and endothelial cells. This approach could provide a valuable advancement of our understanding of membrane trafficking.

The investigation of membrane trafficking is crucial for understanding neuronal functions. Here, we introduce a method for quantifying vesicle motility in neurons. This is a convenient method that can be adapted to the quantification of membrane trafficking in the nervous system.

형광 프로브를 이용한 배양 된 뉴런의 엔도 좀과 리소좀 기능의 정량화

In the brain, membrane trafficking systems play important roles in regulating neuronal functions, such as neuronal morphology, synaptic plasticity, ...

Measuring Synaptic Vesicle Endocytosis in Cultured Hippocampal Neurons

During endocytosis, fused synaptic vesicles are retrieved at nerve terminals, allowing for vesicle recycling and thus the maintenance of synaptic transmission during repetitive nerve firing. Impaired endocytosis in pathological conditions leads to decreases in synaptic strength and brain functions. Here, we describe methods used to measure synaptic vesicle endocytosis at the mammalian hippocampal synapse in neuronal culture. We monitored synaptic vesicle protein endocytosis by fusing a synaptic vesicular membrane protein, including synaptophysin and VAMP2/synaptobrevin, at the vesicular lumenal side, with pHluorin, a pH-sensitive green fluorescent protein that increases its fluorescence intensity as the pH increases. During exocytosis, vesicular lumen pH increases, whereas during endocytosis vesicular lumen pH is re-acidified. Thus, an increase of pHluorin fluorescence intensity indicates fusion, whereas a decrease indicates endocytosis of the labelled synaptic vesicle protein. In addition to using the pHluorin imaging method to record endocytosis, we monitored vesicular membrane endocytosis by electron microscopy (EM) measurements of Horseradish peroxidase (HRP) uptake by vesicles. Finally, we monitored the formation of nerve terminal membrane pits at various times after high potassium-induced depolarization. The time course of HRP uptake and membrane pit formation indicates the time course of endocytosis.

Synaptic vesicle endocytosis is detected by light microscopy of pHluorin fused with synaptic vesicle protein and by electron microscopy of vesicle uptake.

교양된 Hippocampal 신경에 시 냅 스 소포 Endocytosis를 측정

During endocytosis, fused synaptic vesicles are retrieved at nerve terminals, allowing for vesicle recycling and thus the maintenance of synaptic ...

Novel Passive Clearing Methods for the Rapid Production of Optical Transparency in Whole CNS Tissue

Since the development of CLARITY, a bioelectrochemical clearing technique that allows for three-dimensional phenotype mapping within transparent tissues, a multitude of novel clearing methodologies including CUBIC (clear, unobstructed brain imaging cocktails and computational analysis), SWITCH (system-wide control of interaction time and kinetics of chemicals), MAP (magnified analysis of the proteome), and PACT (passive clarity technique), have been established to further expand the existing toolkit for the microscopic analysis of biological tissues. The present study aims to improve upon and optimize the original PACT procedure for an array of intact rodent tissues, including the whole central nervous system (CNS), kidneys, spleen, and whole mouse embryos. Termed psPACT (process-separate PACT) and mPACT (modified PACT), these novel techniques provide highly efficacious means of mapping cell circuitry and visualizing subcellular structures in intact normal and pathological tissues. In the following protocol, we provide a detailed, step-by-step outline on how to achieve maximal tissue clearance with minimal invasion of their structural integrity via psPACT and mPACT.

Here, we present two novel methodologies, psPACT and mPACT, for achieving maximal optical transparency and subsequent microscopic analysis of tissue vasculature in the intact rodent whole CNS.

소설 수동 전체 CNS 조직에 광학 투명도의 급속 한 생산을 위한 방법 삭제

Since the development of CLARITY, a bioelectrochemical clearing technique that allows for three-dimensional phenotype mapping within transparent tissues, ...