prompt_front: Now, answer the following question based on the text below, only give me the answer and do not output any other words.

Question: 对于PD3.0协议，FS312BH支持的最高诱骗电压是多少？

Read the following text and answer briefly.

Context 1: '无锡速芯微电子有限公司是一家集芯片 研发，销售和服务于一体的国家高新技 术企业，为客户提供高性能，高集成 度，极致体验的全协议快充芯片。 无锡速芯微电子有限公司 FastSOC Microelectronics Co ,Ltd  销售联系方式： 联系人：顾先生 手机：1800 185 3071 邮箱：gpp@fastsoc com 网址：www fastsoc com 地址：无锡市新吴区菱湖大道200号中国物联网国际创新园E-503室 顾工微信号 速芯微公众号 免责声明：本文所述方法、方案均供客户参考，用于提示或者展示芯片应用的一种或者多种方式，不作为最终产品的实际方案。文中所描述的功能和性能指标在实 验室环境下测试得到，部分可以提供第三方测试报告，但是不保证客户产品上能获得相同的数据。本文信息只作为芯片使用的指导，不授权用户使用本公司或者其 他公司的知识产权。本文信息只作为芯片使用的指导，不承担因为客户自身应用不当而造成的任何损失。 **文中信息仅供参考，详情请联系我司获取最新资料” 无锡速芯微电子有限公司 FastSOC Microelectronics Co ,Ltd  产品手册 2023年 
新品快览 FS312A：PD3 0 诱骗- FS312A支持PD2 0/PD3 0最高诱骗电压：20V - FS312AE支持PD2 0/PD3 0 最高诱骗电压：20V支持Emarker模拟功能 - 封装：SOT23-5 VBUS CC1 CC2 DM DP 用电电路 4 7K 0 47uF R C C 1 V D D F U N C C C 2F S 3 1 2 B D M D P EP GND 应用图 FS8628：A+C快充协议CC2 CC1 VBUS CC2 CC1 FS312A FUNC GND VDD 4 7K GND R 用电电路 1uF GND 应用图 多口极简方案 FS8611SP*2+CCM-8611SP-A+7533B-T 双C智能降功率方案 FS8611S USB-C AC-DC 双变压器 7533B-T CCM-8611SP-A FS8611S USB-C 采用2颗FS8611SP搭配CCM-8611SP-A （MCU），7533B-T配合工作 - 支持多种协议 - 支持I2C控制 - 任意单 C 的为 35W - 双 插 降 功 率 ， 三 档 功 率 智 能 配 置：27 4W+7 4W；17 4W+17 4W； 27 4W - BOM极简，成本低 FS312B：PD3 1 诱骗FS8611K*2+CCM-8611K-A+7550B-T 双C方案 - FS312BL支持PD2 0/PD3 0/PD3 1/第三方协议最高诱骗电压：20V - FS312BLE支持PD2 0/PD3 0/PD3 1/第三方协议最高诱骗电压：20V支持Emarker模拟功能 - FS312BH支持PD2 0/PD3 0/PD3 1/第三方协议最高诱骗电压：48V - FS312BHE支持PD2 0/PD3 0/PD3 1/第三方协议最高诱骗电压：48V 支持Emarker模拟功能 - 封装：DFN2x2-6L - 兼容兼容BC1 2、Apple2 4A、 QC2 0 Class A、QC3 0 Class A/B、 FCP、SCP、AFC、低压直充等 - 兼容Type-C PD2 0、Type-C PD3 0、 Type-C PD3 0 PPS、QC4 0协议 - 支持两路DP/DM - 支持CV/CC（分段CC）功能 - 支持定制PDO - 支持A+C双口工作，电压自动回5V - 支持FB/OPTO反馈 - 封装：QFN3x3-20L VPWR FB PowerSystem 100K GND R1 GND 19 VIN 17 FB FUNC1 FUNC2 20 15 18 13 PLUGIND VFB FS8628 QFN3x3-20L AGATE 47K 7 5K 47K 7 5K 1 16 8 7 3 4 5 6 10 9 11 CGATE CVBUS CC2 CC1 CDP CDM AVBUS DM DP ISP ISN 12 应用图 2 V3P3 100Ω 1u EP GND GND CVBUS TYPE- C CC2 CC1 CDP CDM CGND TYPE-A AVBUS DM DP 10n 200 AGND 5mΩ GND FS8611K USB-C AC-DC DC-DC 7550B-T CCM-8611K-A FS8611K USB-C 采用2颗FS8611K搭配CCM-8611K-A （MCU）工作，7550B-T配合工作 - 支持PD2 0/PD3 0/QC2 0/AFC/FCP - 支持PDO定制 - 任意单 C 的为 35W(可定制) - 双插18W（可定制15W/20W） - BOM极简，成本低 FS212C+ACM-212C-A+7550B-T 双C方案 FS212C USB-C AC-DC DC-DC 7550B-T ACM-212C-A FS8623B-A+C方案 AC-DC DC-DC FS8623B USB-A USB-C USB-A 采 用 1 颗 F S 2 1 2 C 搭 配 ACM-212C-A 工 作，7550B-T配合工作 - 支持PD2 0/PD3 0 - 支持PDO定制 - 任意单 C 的为20W - 双插7 5W回5V - BOM极简，成本低 采用一颗FS8623B实现A+C方案 - 兼容兼容Apple2 4A/低压直充 QC2 0 Class A/QC3 0 Class A/B/ FCP/SCP等 - 兼 容Type -C PD2 0 / PD3 0 / PD3 0PPS/QC4 0协议 - 支持PDO定制 - 双插回5V 
多口方案选型 产品选型 受电端芯片选型 速芯微现有多种多口的方案选择：A+C，C+C，C+C+A，C+C+C，C+C+A+A等方案。对于 A+C的方案，可使用1颗芯片实现，也可用多颗芯片来实现。 速芯微现有多种受电端诱骗芯片，客户可根据应用需求进行选择。 受电端诱骗芯片应用领域 筋膜枪 无线充 线材 无人机 产品型号 PD2 0 PD3 0 PD3 1 第三方协议 诱骗电压(V) 控制方式 内置Emarker 定制 封装 FS312A √ √ 5/9/12/15/20 电阻阻值 可变电压策略 SOT23-5 FS312AE √ √ 5/9/12/15/20 电阻阻值 √ (公头专用) 可变电压策略 SOT23-5 FS312BL √ √ √ √ 5/9/12/15/20 电阻阻值 可变电压策略 DFN2x2-6 FS312BLE √ √ √ √ 5/9/12/15/20 电阻阻值 √ (公头专用) 可变电压策略 DFN2x2-6 FS312BH √ √ √ √ 5/20/28/36/48 电阻阻值 可变电压策略 DFN2x2-6 FS312BHE √ √ √ √ 5/20/28/36/48 电阻阻值 √ (公头专用) 可变电压策略 DFN2x2-6 FS312LC √ √ √ 5/9/12 电阻阻值 可变第三方 协议 SSOP10 FS312HC √ √ √ 5/9/12/15/20 电阻阻值 可变第三方 协议 SSOP10 FS2711Q √ √ √ 任意设置 I2C √ QFN3x3-16 FS2711P √ √ √ 任意设置 I2C √ QFN3x3-16 FS2711PA √ √ 全协议 任意设置 I2C √ SSOP10 FS2711SW √ √ 全协议 SSOP10 FS512 √ √ 全协议 任意设置 I2C √ SSOP10 方案 类型 产品型号 单C 单A 双插 A+C方案 FS8623 20W（PPS）（可定制） A口全协议18w 5V共享3A FS8623B 20W（PPS）(可定制) A口全协议18w 5V共享3A FS8628 20W（PPS）(可定制) A口全协议18w 5V共享3A FS8611RPC+FS116DB 65W（PPS）(可定制) A口全协议18w A口：5V/2 4A C口：45W FS8628RC+FS116DB 35W(可定制) A口全协议18w A口：5V（BC1 2，Apple 2 4） C口：20W 方案类型 产品型号 单C1 单C2 C1/C2 C+C方案 FS8611RPB*2 30W(可定制) 30W(可定制) C1/C2：5V/3A(或5V/2 4A) FS8611GH*2 35W（可定制） 35W（可定制） C1/C2:18W（可定制） FS8628P*2 35W（可定制） 35W（可定制） C1/C2：17 4W可定制） FS8611KL*2 20W（可定制） 20W（可定制） C1/C2：5V/1 5 A FS8611PC*2 35W 35W C1/C2：18W FS8611BH*2 65W（可定制） 65W（可定制） C1：45W（可定制）C2：20W（可定制） FS8628RPC+FS8611RB 45W（可定制）） 36W （可定制）） C1：30W（可定制）C2：5V/1 5A（可定制） 方案类型 产品型号 单C1 单C2 单A C1+C2 C1/C2+A C1+C2+A C+C+A FS8611S*2+FS116DB 65W（可定制） 65W（ 可定制）） A口全协议18w 智能分配功率 45W+18W C1/C2：智能分配功率 A：18W（或5V1 5A） FS8612C+FS8628P 100W（可定制） 35W （可定制）） 20W C1：65W C2：20W C1+A：65W+20W C2+A：7 5W+7 5W C1：65W C2：7 5W A：7 5W 其他 
Source-TYPE C协议芯片选型 Source-TYPE A协议芯片选型 速芯微现有多种TYPE-C的快充协议芯片，支持多种协议，支持客户定制，多样化，满 足客户对TYPE C的各种快充需求。 速芯微现有多种TYPE A快充协议芯片，支持全协议，支持定制，满足客户对A口协议的各种需 求。速芯微的TYPE-A快充协议芯片的协议丰富，FS112系列拥有多种的型号；FS116D 系列带插入指示，可搭配TYPE-C快充协议芯片，实现A+C，A+C+C，A+A+C+C等多口方 案，协议丰富，其中FS116A一般用于插入指示使用 Source-TYPE A协议芯片引脚封装图 D+ VSS FB 1 2 3 FS112 6 5 4 D- VDD FUNC GATE VIN FUNC FB LED/PLUG_IN 1 2 3 4 5 FS116D 10 DM 9 8 7 6 DP CSP CSN VSS速芯微的各TYPE-C快充协议芯片之间可搭配使用，实现多口方案，更多详情请咨 询我司工作人员。 多口降功率专用快充协议芯片：FS8611RB，FS8611RC，FS8611RPB，FS8611RPC， FS8612CP。 带I2C快充协议芯片：FS8611S，FS8611SP 产品型号 BC1 2 Apple 2 4 QC2 0 QC3 0 AFC FCP SCP HISCP 大电流直充 封装 FS112 √ √ √ √ √ √ √ SOT23-6 FS112H √ √ √ √ √ √ √ √ √ SOT23-6 FS113 √ v √ √ √ √ √ √ √ SOT23-6 FS116DP √ √ √ √ √ √ √ √ SSOP10 FS116DB √ √ √ √ √ √ √ √ SSOP10 FS116E √ √ √ √ √ √ √ √ √ SSOP10 FS116A √ √ SSOP10 其他 可定制 PD2 0 PD3 0 PD3 0 PPS 第三方协议 反馈方式 MOS CV/CC 定制 封装 FS212C √ √ FB √ SOT23-6 FS212CM √ √ FB PMOS(可省) √ SOT23-6 FS212D √ √ √ FB √ SOT23-6 FS212DH √ √ √ FB √ SOT23-6 FS212DP √ √ √ FB PMOS √ SOT23-6 FS212DG √ √ √ FB PMOS √ SOT23-6 FS8611G √ √ FB PMOS(可省) √ SOP-8 FS8611K √ √ QC2 0/AFC/FCP FB PMOS(可省) √ SOP8 FS8611J √ √ √ 全协议 FB PMOS(可省) √ SOP8 FS8611B √ √ √ 全协议 FB PMOS(可省) √ SSOP10 FS8611RB √ √ 全协议 FB PMOS √ SSOP10 FS8611RC √ √ 全协议 FB PMOS √ SSOP10 FS8611S √ √ √ 全协议 FB PMOS √ SSOP10 FS8611PP √ √ √ 全协议 FB PMOS √ SSOP10 FS8611BP √ √ √ 全协议 FB PMOS(可省) √ SSOP10 FS8611RPB √ √ √ 全协议 FB PMOS √ SSOP10 FS8611RPC √ √ √ 全协议 FB PMOS √ SSOP10 FS8611SP √ √ √ 全协议 FB PMOS(可省) SSOP10 FS8612 √ √ √ 全协议 OPTO PMOS √ √ SSOP16 FS8612B √ √ √ 全协议 FB PMOS √ √ SSOP16 FS8612BP √ √ √ 全协议 FB PMOS √ √ SSOP16 FS8612C √ √ √ 全协议 FB/OPTO PMOS √ √ QFN4x4-16 FS8612CP √ √ √ 全协议 FB/OPTO PMOS √ √ QFN4x4-16 
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Context 2: 'Quectel_QuecPython_BC25 开发板使用说明 版本：Quectel_QuecPython_BC25 开发板使用说明_V1 1日期：2021-11-30 状态：临时文件
Quectel_QuecPython_BC25 开发板使用说明一、基本概述BC25_QuecPython_EVB_V1 1 开发板（本文简称“V1 1 开发板”）是专门针对 BC25 制造，是一款小巧便携的“口袋型”开发板。体型虽小，但是功能丰富，拥 有 SIM 卡座、板载天线、磁开关、LED 等元件。开发者仅需一条 USB Type-C 数据线即可轻松玩转开发板。二、开发板资源Quectel 移远 BC25 通信模组NANO SIM 自弹卡座USB Type-C 数据接口开机按键，唤醒按键磁开关单色灯GPIO 排针上海市闵行区田林路 1016 号科技绿洲 3 期（B 区）5 号楼 200233 邮箱: info@quectel com 网址: www quectel com 1 / 6
三、开发板介绍Quectel_QuecPython_BC25 开发板使用说明开发板是为方便开发者使用 QuecPython，而设计的一款基于 BC25 通信模块 的开发板，其上集成了开发常用的配置，可以满足开发者的开发需求。V1 1 开发板正面接口V1 1 开发板配置开发板配备了多种外设。明细如下：序 号名称型号是否支持接口类 型1磁开关KTH1601SL-ST3是GPIO2LED 灯S3528UG6W9TLC2G- 是GPIOTJ- 34微动按键GPIOA5--------是是---------上海市闵行区田林路 1016 号科技绿洲 3 期（B 区）5 号楼 200233 邮箱: info@quectel com 网址: www quectel com 2 / 6
Quectel_QuecPython_BC25 开发板使用说明四、功能详解4 1 磁开关开发板集成了一个磁开关。使用磁铁靠近，可使磁开关输出引脚变为低电平， 默认为高电平。4 2 LED 灯开发板集成了一颗高亮度灯珠，可以用来做显著指示灯。上海市闵行区田林路 1016 号科技绿洲 3 期（B 区）5 号楼 200233 邮箱: info@quectel com 网址: www quectel com 3 / 6
4 3 按键开发板集成了 2 个微动按键，其功能是 S1 为开机键，S2 为睡眠唤醒按键。Quectel_QuecPython_BC25 开发板使用说明五、调试步骤1 拿到开发板 V1 1 先插上 USB 安装串口驱动，在官方 QQ 群文件搜 CP210 或者自 行百度下载 CP210x 的串口芯片驱动进行安装。2 使用串口工具（例如 QCOM_V1 6）连接 BC25 的主串口（硬件 17、18 脚）。V1 1 选择 Enhanced COM 口，波特率选择 9600,打开串口，按下 PWK 键约一秒松开进 行开机，串口工具收到消息则代表开机成 功，然后按下 EINT 键串口显示 +QATWAKEUP 表示模组唤醒了。3 从 https://python quectel com/download 下载 BC25QuecPython 版本固件， 使用 Qflash（群文件下载）选择 BC25 的调试串口（硬件 38、39 脚），波特率选 择 921600，选择 lod 后缀的固件，按下 EINT 键串口工具显示模组已经唤醒串口 工具发 AT+QSCLK=0 可关闭睡眠（不会发 AT 则多按几次 EINT 键），点击 Start 开 始下载固件,下载进度条开始下载，等待下载完成。关闭以上所有工具，并给板 子断电重新上电。4 从 https://python quectel com/download 下载 QPYCOM 工具，直接解压运行 工具，选择主串口（同第 2 步），选择 57600 波特率，打开串口。再按 PWK 按键 进行开机，会看到 QPYCOM 有打印 mount Type "help()" for more information 然后就可以进行 QuecPython 的交互调 试了。上海市闵行区田林路 1016 号科技绿洲 3 期（B 区）5 号楼 200233 邮箱: info@quectel com 网址: www quectel com 4 / 6
六、常见问题解决Quectel_QuecPython_BC25 开发板使用说明Q：模块的固件在哪？A：请登录 QuecPython 网站下载：http://python quectel com/downloadQ：哪里有开发板和其他常用资料？A：请登录 QuecPython 网站下载：http://python quectel com/downloadP S  如果您遇到任何问题，请参照本官网在线文档进行解决或访问 QuecPython 社区进行搜索、交流、提问：QuecPython 社区或者联系我们的在线支持：QQ 群 445121768获取 QuecPython 开发固件及加入官方交流群官网主页：https://python quectel com官网文件下载（各类资料、工具）：https://python quectel com/download官网 wiki（常用于视频教程、手把手教程下载、API 库）：https://python quectel com/wiki/#/官网文档中心(拥有从入门到精通的各种文档介绍、必看)：https://python quectel com/doc/工单系统：https://workorder quectel com/QuecPython 社区：https://forumschinese quectel com/c/function-subjects/quectpython/43QuecPython 官方 QQ 开发交流群：445121768微信公众号：QuecPython移远 OTA 升级平台： https://cloudota quectel com/移远 IoT 管理平台：https://python quectel com/doc/doc/Advanced_development/zh/QuecPython Cloud/QuecCloud html上海市闵行区田林路 1016 号科技绿洲 3 期（B 区）5 号楼 200233 邮箱: info@quectel com 网址: www quectel com 5 / 6
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Context 3: JoVE | Peer Reviewed Scientific Video Journal - Methods and Protocols
A role for thrombospondin-1 deficits in astrocyte-mediated spine and synaptic pathology in Downs syndrome  Octavio Garcia, Maria Torres, Pablo Helguera, Pinar Coskun, Jorge Busciglio 
PUBLISHED: 07-02-2010	Downs syndrome (DS) is the most common genetic cause of mental retardation  Reduced number and aberrant architecture of dendritic spines are common features of DS neuropathology  However, the mechanisms involved in DS spine alterations are not known  In addition to a relevant role in synapse formation and maintenance, astrocytes can regulate spine dynamics by releasing soluble factors or by physical contact with neurons  We have previously shown impaired mitochondrial function in DS astrocytes leading to metabolic alterations in protein processing and secretion  In this study, we investigated whether deficits in astrocyte function contribute to DS spine pathology 
Analysis of Dendritic Spine Morphology in Cultured CNS Neurons Authors: Deepak P  Srivastava, Kevin M  Woolfrey, Peter Penzes  Published: 07-13-2011 JoVE Neuroscience
Dendritic spines are the sites of the majority of excitatory connections within the brain, and form the post-synaptic compartment of synapses  These structures are rich in actin and have been shown to be highly dynamic  In response to classical Hebbian plasticity as well as neuromodulatory signals, dendritic spines can change shape and number, which is thought to be critical for the refinement of neural circuits and the processing and storage of information within the brain  Within dendritic spines, a complex network of proteins link extracellular signals with the actin cyctoskeleton allowing for control of dendritic spine morphology and number  Neuropathological studies have demonstrated that a number of disease states, ranging from schizophrenia to autism spectrum disorders, display abnormal dendritic spine morphology or numbers  Moreover, recent genetic studies have identified mutations in numerous genes that encode synaptic proteins, leading to suggestions that these proteins may contribute to aberrant spine plasticity that, in part, underlie the pathophysiology of these disorders  In order to study the potential role of these proteins in controlling dendritic spine morphologies/number, the use of cultured cortical neurons offers several advantages  Firstly, this system allows for high-resolution imaging of dendritic spines in fixed cells as well as time-lapse imaging of live cells  Secondly, this in vitro system allows for easy manipulation of protein function by expression of mutant proteins, knockdown by shRNA constructs, or pharmacological treatments  These techniques allow researchers to begin to dissect the role of disease-associated proteins and to predict how mutations of these proteins may function in vivo 
Play ButtonIsolation and Culture of Mouse Cortical AstrocytesAuthors: Sebastian Schildge, Christian Bohrer, Kristina Beck, Christian Schachtrup  Institutions: University of Freiburg , University of Freiburg  Astrocytes are an abundant cell type in the mammalian brain, yet much remains to be learned about their molecular and functional characteristics  In vitro astrocyte cell culture systems can be used to study the biological functions of these glial cells in detail  This video protocol shows how to obtain pure astrocytes by isolation and culture of mixed cortical cells of mouse pups  The method is based on the absence of viable neurons and the separation of astrocytes, oligodendrocytes and microglia, the three main glial cell populations of the central nervous system, in culture  Representative images during the first days of culture demonstrate the presence of a mixed cell population and indicate the timepoint, when astrocytes become confluent and should be separated from microglia and oligodendrocytes  Moreover, we demonstrate purity and astrocytic morphology of cultured astrocytes using immunocytochemical stainings for well established and newly described astrocyte markers  This culture system can be easily used to obtain pure mouse astrocytes and astrocyte-conditioned medium for studying various aspects of astrocyte biology Neuroscience, Issue 71, Neurobiology, Cellular Biology, Medicine, Molecular Biology, Anatomy, Physiology, brain, mouse, astrocyte culture, astrocyte, fibroblast, fibrinogen, chondroitin sulfate proteoglycan, neuronal regeneration, cell culture, animal model50079Play ButtonImaging Dendritic Spines of Rat Primary Hippocampal Neurons using Structured Illumination MicroscopyAuthors: Marijn Schouten, Giulia M  R  De Luca, Diana K  Alatriste González, Babette E  de Jong, Wendy Timmermans, Hui Xiong, Harm Krugers, Erik M  M  Manders, Carlos P  Fitzsimons  Institutions: University of Amsterdam, University of Amsterdam 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 Neuroscience, Issue 87, Dendritic Spine, Microscopy, Confocal, Fluorescence, Neurosciences, hippocampus, primary neuron, super resolution microscopy, structured illumination microscopy (SIM), neuroscience, dendrite51276Play ButtonSetting-up an In Vitro Model of Rat Blood-brain Barrier (BBB): A Focus on BBB Impermeability and Receptor-mediated TransportAuthors: Yves Molino, Françoise Jabès, Emmanuelle Lacassagne, Nicolas Gaudin, Michel Khrestchatisky  Institutions: VECT-HORUS SAS, CNRS, NICN UMR 7259 The blood brain barrier (BBB) specifically regulates molecular and cellular flux between the blood and the nervous tissue  Our aim was to develop and characterize a highly reproducible rat syngeneic in vitro model of the BBB using co-cultures of primary rat brain endothelial cells (RBEC) and astrocytes to study receptors involved in transcytosis across the endothelial cell monolayer  Astrocytes were isolated by mechanical dissection following trypsin digestion and were frozen for later co-culture  RBEC were isolated from 5-week-old rat cortices  The brains were cleaned of meninges and white matter, and mechanically dissociated following enzymatic digestion  Thereafter, the tissue homogenate was centrifuged in bovine serum albumin to separate vessel fragments from nervous tissue  The vessel fragments underwent a second enzymatic digestion to free endothelial cells from their extracellular matrix  The remaining contaminating cells such as pericytes were further eliminated by plating the microvessel fragments in puromycin-containing medium  They were then passaged onto filters for co-culture with astrocytes grown on the bottom of the wells  RBEC expressed high levels of tight junction (TJ) proteins such as occludin, claudin-5 and ZO-1 with a typical localization at the cell borders  The transendothelial electrical resistance (TEER) of brain endothelial monolayers, indicating the tightness of TJs reached 300 ohm·cm2 on average  The endothelial permeability coefficients (Pe) for lucifer yellow (LY) was highly reproducible with an average of 0 26 ± 0 11 x 10-3 cm/min  Brain endothelial cells organized in monolayers expressed the efflux transporter P-glycoprotein (P-gp), showed a polarized transport of rhodamine 123, a ligand for P-gp, and showed specific transport of transferrin-Cy3 and DiILDL across the endothelial cell monolayer  In conclusion, we provide a protocol for setting up an in vitro BBB model that is highly reproducible due to the quality assurance methods, and that is suitable for research on BBB transporters and receptors Medicine, Issue 88, rat brain endothelial cells (RBEC), mouse, spinal cord, tight junction (TJ), receptor-mediated transport (RMT), low density lipoprotein (LDL), LDLR, transferrin, TfR, P-glycoprotein (P-gp), transendothelial electrical resistance (TEER),51278Play ButtonInducing Plasticity of Astrocytic Receptors by Manipulation of Neuronal Firing RatesAuthors: Alison X  Xie, Kelli Lauderdale, Thomas Murphy, Timothy L  Myers, Todd A  Fiacco  Institutions: University of California Riverside, University of California Riverside, University of California Riverside Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be stimulated by neuronally-released transmitter  However, the ability of astrocytic receptors to exhibit plasticity in response to changes in neuronal activity has received little attention  Here we describe a model system that can be used to globally scale up or down astrocytic group I metabotropic glutamate receptors (mGluRs) in acute brain slices  Included are methods on how to prepare parasagittal hippocampal slices, construct chambers suitable for long-term slice incubation, bidirectionally manipulate neuronal action potential frequency, load astrocytes and astrocyte processes with fluorescent Ca2+ indicator, and measure changes in astrocytic Gq GPCR activity by recording spontaneous and evoked astrocyte Ca2+ events using confocal microscopy  In essence, a “calcium roadmap” is provided for how to measure plasticity of astrocytic Gq GPCRs  Applications of the technique for study of astrocytes are discussed  Having an understanding of how astrocytic receptor signaling is affected by changes in neuronal activity has important implications for both normal synaptic function as well as processes underlying neurological disorders and neurodegenerative disease Neuroscience, Issue 85, astrocyte, plasticity, mGluRs, neuronal Firing, electrophysiology, Gq GPCRs, Bolus-loading, calcium, microdomains, acute slices, Hippocampus, mouse51458Play ButtonInhibitory Synapse Formation in a Co-culture Model Incorporating GABAergic Medium Spiny Neurons and HEK293 Cells Stably Expressing GABAA ReceptorsAuthors: Laura E  Brown, Celine Fuchs, Martin W  Nicholson, F  Anne Stephenson, Alex M  Thomson, Jasmina N  Jovanovic  Institutions: University College London Inhibitory neurons act in the central nervous system to regulate the dynamics and spatio-temporal co-ordination of neuronal networks  GABA (γ-aminobutyric acid) is the predominant inhibitory neurotransmitter in the brain  It is released from the presynaptic terminals of inhibitory neurons within highly specialized intercellular junctions known as synapses, where it binds to GABAA receptors (GABAARs) present at the plasma membrane of the synapse-receiving, postsynaptic neurons  Activation of these GABA-gated ion channels leads to influx of chloride resulting in postsynaptic potential changes that decrease the probability that these neurons will generate action potentials  During development, diverse types of inhibitory neurons with distinct morphological, electrophysiological and neurochemical characteristics have the ability to recognize their target neurons and form synapses which incorporate specific GABAARs subtypes  This principle of selective innervation of neuronal targets raises the question as to how the appropriate synaptic partners identify each other  To elucidate the underlying molecular mechanisms, a novel in vitro co-culture model system was established, in which medium spiny GABAergic neurons, a highly homogenous population of neurons isolated from the embryonic striatum, were cultured with stably transfected HEK293 cell lines that express different GABAAR subtypes  Synapses form rapidly, efficiently and selectively in this system, and are easily accessible for quantification  Our results indicate that various GABAAR subtypes differ in their ability to promote synapse formation, suggesting that this reduced in vitro model system can be used to reproduce, at least in part, the in vivo conditions required for the recognition of the appropriate synaptic partners and formation of specific synapses  Here the protocols for culturing the medium spiny neurons and generating HEK293 cells lines expressing GABAARs are first described, followed by detailed instructions on how to combine these two cell types in co-culture and analyze the formation of synaptic contacts  Neuroscience, Issue 93, Developmental neuroscience, synaptogenesis, synaptic inhibition, co-culture, stable cell lines, GABAergic, medium spiny neurons, HEK 293 cell line52115Play ButtonTwo-Photon in vivo Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull PreparationAuthors: Xinzhu Yu, Yi Zuo  Institutions: University of California, Santa Cruz In the mammalian cortex, neurons form extremely complicated networks and exchange information at synapses  Changes in synaptic strength, as well as addition/removal of synapses, occur in an experience-dependent manner, providing the structural foundation of neuronal plasticity  As postsynaptic components of the most excitatory synapses in the cortex, dendritic spines are considered to be a good proxy of synapses  Taking advantages of mouse genetics and fluorescent labeling techniques, individual neurons and their synaptic structures can be labeled in the intact brain  Here we introduce a transcranial imaging protocol using two-photon laser scanning microscopy to follow fluorescently labeled postsynaptic dendritic spines over time in vivo  This protocol utilizes a thinned-skull preparation, which keeps the skull intact and avoids inflammatory effects caused by exposure of the meninges and the cortex  Therefore, images can be acquired immediately after surgery is performed  The experimental procedure can be performed repetitively over various time intervals ranging from hours to years  The application of this preparation can also be expanded to investigate different cortical regions and layers, as well as other cell types, under physiological and pathological conditions Neuroscience, Issue 87, dendritic spine, mouse cortex, in vivo, two-photon microscopy, thinned-skull, imaging51520Play ButtonModeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered MiceAuthors: Robert S  McNeill, Ralf S  Schmid, Ryan E  Bash, Mark Vitucci, Kristen K  White, Andrea M  Werneke, Brian H  Constance, Byron Huff, C



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Context 3:  Ryan Miller  Institutions: University of North Carolina School of Medicine, University of North Carolina School of Medicine, University of North Carolina School of Medicine, University of North Carolina School of Medicine, University of North Carolina School of Medicine, Emory University School of Medicine, University of North Carolina School of Medicine Current astrocytoma models are limited in their ability to define the roles of oncogenic mutations in specific brain cell types during disease pathogenesis and their utility for preclinical drug development  In order to design a better model system for these applications, phenotypically wild-type cortical astrocytes and neural stem cells (NSC) from conditional, genetically engineered mice (GEM) that harbor various combinations of floxed oncogenic alleles were harvested and grown in culture  Genetic recombination was induced in vitro using adenoviral Cre-mediated recombination, resulting in expression of mutated oncogenes and deletion of tumor suppressor genes  The phenotypic consequences of these mutations were defined by measuring proliferation, transformation, and drug response in vitro  Orthotopic allograft models, whereby transformed cells are stereotactically injected into the brains of immune-competent, syngeneic littermates, were developed to define the role of oncogenic mutations and cell type on tumorigenesis in vivo  Unlike most established human glioblastoma cell line xenografts, injection of transformed GEM-derived cortical astrocytes into the brains of immune-competent littermates produced astrocytomas, including the most aggressive subtype, glioblastoma, that recapitulated the histopathological hallmarks of human astrocytomas, including diffuse invasion of normal brain parenchyma  Bioluminescence imaging of orthotopic allografts from transformed astrocytes engineered to express luciferase was utilized to monitor in vivo tumor growth over time  Thus, astrocytoma models using astrocytes and NSC harvested from GEM with conditional oncogenic alleles provide an integrated system to study the genetics and cell biology of astrocytoma pathogenesis in vitro and in vivo and may be useful in preclinical drug development for these devastating diseases Neuroscience, Issue 90, astrocytoma, cortical astrocytes, genetically engineered mice, glioblastoma, neural stem cells, orthotopic allograft51763Play ButtonPaired Whole Cell Recordings in Organotypic Hippocampal SlicesAuthors: Chantelle Fourie, Marianna Kiraly, Daniel V  Madison, Johanna M  Montgomery  Institutions: University of Auckland, Stanford University Pair recordings involve simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling not only direct electrophysiological characterization of the synaptic connections between individual neurons, but also pharmacological manipulation of either the presynaptic or the postsynaptic neuron  When carried out in organotypic hippocampal slice cultures, the probability that two neurons are synaptically connected is significantly increased  This preparation readily enables identification of cell types, and the neurons maintain their morphology and properties of synaptic function similar to that in native brain tissue  A major advantage of paired whole cell recordings is the highly precise information it can provide on the properties of synaptic transmission and plasticity that are not possible with other more crude techniques utilizing extracellular axonal stimulation  Paired whole cell recordings are often perceived as too challenging to perform  While there are challenging aspects to this technique, paired recordings can be performed by anyone trained in whole cell patch clamping provided specific hardware and methodological criteria are followed  The probability of attaining synaptically connected paired recordings significantly increases with healthy organotypic slices and stable micromanipulation allowing independent attainment of pre- and postsynaptic whole cell recordings  While CA3-CA3 pyramidal cell pairs are most widely used in the organotypic slice hippocampal preparation, this technique has also been successful in CA3-CA1 pairs and can be adapted to any neurons that are synaptically connected in the same slice preparation  In this manuscript we provide the detailed methodology and requirements for establishing this technique in any laboratory equipped for electrophysiology Neuroscience, Issue 91, hippocampus, paired recording, whole cell recording, organotypic slice, synapse, synaptic transmission, synaptic plasticity51958Play ButtonImaging Intracellular Ca2+ Signals in Striatal Astrocytes from Adult Mice Using Genetically-encoded Calcium IndicatorsAuthors: Ruotian Jiang, Martin D  Haustein, Michael V  Sofroniew, Baljit S  Khakh  Institutions: University of California Los Angeles, University of California Los Angeles Astrocytes display spontaneous intracellular Ca2+ concentration fluctuations ([Ca2+]i) and in several settings respond to neuronal excitation with enhanced [Ca2+]i signals  It has been proposed that astrocytes in turn regulate neurons and blood vessels through calcium-dependent mechanisms, such as the release of signaling molecules  However, [Ca2+]i imaging in entire astrocytes has only recently become feasible with genetically encoded calcium indicators (GECIs) such as the GCaMP series  The use of GECIs in astrocytes now provides opportunities to study astrocyte [Ca2+]i signals in detail within model microcircuits such as the striatum, which is the largest nucleus of the basal ganglia  In the present report, detailed surgical methods to express GECIs in astrocytes in vivo, and confocal imaging approaches to record [Ca2+]i signals in striatal astrocytes in situ, are described  We highlight precautions, necessary controls and tests to determine if GECI expression is selective for astrocytes and to evaluate signs of overt astrocyte reactivity  We also describe brain slice and imaging conditions in detail that permit reliable [Ca2+]i imaging in striatal astrocytes in situ  The use of these approaches revealed the entire territories of single striatal astrocytes and spontaneous [Ca2+]i signals within their somata, branches and branchlets  The further use and expansion of these approaches in the striatum will allow for the detailed study of astrocyte [Ca2+]i signals in the striatal microcircuitry Neuroscience, Issue 93, astrocyte, calcium, striatum, GECI, GCaMP3, AAV2/5, stereotaxic injection, brain slice, imaging51972Play ButtonMethods to Assess Subcellular Compartments of Muscle in C  elegansAuthors: Christopher J  Gaffney, Joseph J  Bass, Thomas F  Barratt, Nathaniel J  Szewczyk  Institutions: University of Nottingham Muscle is a dynamic tissue that responds to changes in nutrition, exercise, and disease state  The loss of muscle mass and function with disease and age are significant public health burdens  We currently understand little about the genetic regulation of muscle health with disease or age  The nematode C  elegans is an established model for understanding the genomic regulation of biological processes of interest  This worm’s body wall muscles display a large degree of homology with the muscles of higher metazoan species  Since C  elegans is a transparent organism, the localization of GFP to mitochondria and sarcomeres allows visualization of these structures in vivo  Similarly, feeding animals cationic dyes, which accumulate based on the existence of a mitochondrial membrane potential, allows the assessment of mitochondrial function in vivo  These methods, as well as assessment of muscle protein homeostasis, are combined with assessment of whole animal muscle function, in the form of movement assays, to allow correlation of sub-cellular defects with functional measures of muscle performance  Thus, C  elegans provides a powerful platform with which to assess the impact of mutations, gene knockdown, and/or chemical compounds upon muscle structure and function  Lastly, as GFP, cationic dyes, and movement assays are assessed non-invasively, prospective studies of muscle structure and function can be conducted across the whole life course and this at present cannot be easily investigated in vivo in any other organism Developmental Biology, Issue 93, Physiology, C  elegans, muscle, mitochondria, sarcomeres, ageing52043Play ButtonImproved Preparation and Preservation of Hippocampal Mouse Slices for a Very Stable and Reproducible Recording of Long-term PotentiationAuthors: Agnès Villers, Laurence Ris  Institutions: University of Mons Long-term potentiation (LTP) is a type of synaptic plasticity characterized by an increase in synaptic strength and believed to be involved in memory encoding  LTP elicited in the CA1 region of acute hippocampal slices has been extensively studied  However the molecular mechanisms underlying the maintenance phase of this phenomenon are still poorly understood  This could be partly due to the various experimental conditions used by different laboratories  Indeed, the maintenance phase of LTP is strongly dependent on external parameters like oxygenation, temperature and humidity  It is also dependent on internal parameters like orientation of the slicing plane and slice viability after dissection 
The optimization of all these parameters enables the induction of a very reproducible and very stable long-term potentiation  This methodology offers the possibility to further explore the molecular mechanisms involved in the stable increase in synaptic strength in hippocampal slices  It also highlights the importance of experimental conditions in in vitro investigation of neurophysiological phenomena Neuroscience, Issue 76, Neurobiology, Anatomy, Physiology, Biomedical Engineering, Surgery, Memory Disorders, Learning, Memory, Neurosciences, Neurophysiology, hippocampus, long-term potentiation, mice, acute slices, synaptic plasticity, in vitro, electrophysiology, animal model50483Play ButtonIn Vivo Modeling of the Morbid Human Genome using Danio rerioAuthors: Adrienne R  Niederriter, Erica E  Davis, Christelle Golzio, Edwin C  Oh, I-Chun Tsai, Nicholas Katsanis  Institutions: Duke University Medical Center, Duke University, Duke University Medical Center Here, we present methods for the development of assays to query potentially clinically significant nonsynonymous changes using in vivo complementation in zebrafish  Zebrafish (Danio rerio) are a useful animal system due to their experimental tractability; embryos are transparent to enable facile viewing, undergo rapid development ex vivo, and can be genetically manipulated 1 These aspects have allowed for significant advances in the analysis of embryogenesis, molecular processes, and morphogenetic signaling  Taken together, the advantages of this vertebrate model make zebrafish highly amenable to modeling the developmental defects in pediatric disease, and in some cases, adult-onset disorders  Because the zebrafish genome is highly conserved with that of humans (~70% orthologous), it is possible to recapitulate human disease states in zebrafish  This is accomplished either through the injection of mutant human mRNA to induce dominant negative or gain of function alleles, or utilization of morpholino (MO) antisense oligonucleotides to suppress genes to mimic loss of function variants  Through complementation of MO-induced phenotypes with capped human mRNA, our approach enables the interpretation of the deleterious effect of mutations on human protein sequence based on the ability of mutant mRNA to rescue a measurable, physiologically relevant phenotype  Modeling of the human disease alleles occurs through microinjection of zebrafish embryos with MO and/or human mRNA at the 1-4 cell stage, and phenotyping up to seven days post fertilization (dpf)  This general strategy can be extended to a wide range of disease phenotypes, as demonstrated in the following protocol  We present our established models for morphogenetic signaling, craniofacial, cardiac, vascular integrity, renal function, and skeletal muscle disorder phenotypes, as well as others  Molecular Biology, Issue 78, Genetics, Biomedical Engineering, Medicine, Developmental Biology, Biochemistry, Anatomy, Physiology, Bioengineering, Genomics, Medical, zebrafish, in vivo, morpholino, human disease modeling, transcription, PCR, mRNA, DNA, Danio rerio, animal model50338Play ButtonDirect Imaging of ER Calcium with Targeted-Esterase Induced Dye Loading (TED)Authors: Samira Samtleben, Juliane Jaepel, Caroline Fecher, Thomas Andreska, Markus Rehberg, Robert Blum  Institutions: University of Wuerzburg, Max Planck Institute of Neurobiology, Martinsried, Ludwig-Maximilians University of Munich Visualization of calcium dynamics is important to understand the role of calcium in cell physiology  To examine calcium dynamics, synthetic fluorescent Ca2+ indictors have become popular  Here we demonstrate TED (= targeted-esterase induced dye loading), a method to improve the release of Ca2+ indicator dyes in the ER lumen of different cell types  To date, TED was used in cell lines, glial cells, and neurons in vitro  TED bases on efficient, recombinant targeting of a high carboxylesterase activity to the ER lumen using vector-constructs that express Carboxylesterases (CES)  The latest TED vectors contain a core element of CES2 fused to a red fluorescent protein, thus enabling simultaneous two-color imaging  The dynamics of free calcium in the ER are imaged in one color, while the corresponding ER structure appears in red  At the beginning of the procedure, cells are transduced with a lentivirus  Subsequently, the infected cells are seeded on coverslips to finally enable live cell imaging  Then, living cells are incubated with the acetoxymethyl ester (AM-ester) form of low-affinity Ca2+ indicators, for instance Fluo5N-AM, Mag-Fluo4-AM, or Mag-Fura2-AM  The esterase activity in the ER cleaves off hydrophobic side chains from the AM form of the Ca2+ indicator and a hydrophilic fluorescent dye/Ca2+ complex is formed and trapped in the ER lumen  After dye loading, the cells are analyzed at an inverted confocal laser scanning microscope  Cells are continuously perfused with Ringer-like solutions and the ER calcium dynamics are directly visualized by time-lapse imaging  Calcium release from the ER is identified by a decrease in fluorescence intensity in regions of interest, whereas the refilling of the ER calcium store produces an increase in fluorescence intensity  Finally, the change in fluorescent intensity over time is determined by calculation of ΔF/F0 Cellular Biology, Issue 75, Neurobiology, Neuroscience, Molecular Biology, Biochemistry, Biomedical Engineering, Bioengineering, Virology, Medicine, Anatomy, Physiology, Surgery, Endoplasmic Reticulum, ER, Calcium Signaling, calcium store, calcium imaging, calcium indicator, metabotropic signaling, Ca2+, neurons, cells, mouse, animal model, cell culture, targeted esterase induced dye loading, imaging50317Play ButtonPreparation of Dissociated Mouse Cortical Neuron CulturesAuthors: Lutz G  W  Hilgenberg, Martin A  Smith  Institutions: University of California, Irvine (UCI) This video will guide you through the process for generating cortical neuronal cultures from late embryo and early postnatal mouse brain  These cultures can be used for a variety of applications including immunocytochemistry, biochemistry, electrophysiology, calcium and sodium imaging, protein and/or RNA isolation  These cultures also provide a platform to study the neuronal development of transgenic animals that carry a late embryonic or postnatal lethal gene mutation  The procedure is relatively straight forward, requires some experience in tissue culture technique and should not take longer than two to three hours if you are properly prepared  Careful separation of the cortical rind from the thalamo-cortical fiber tract will reduce the number of unwanted non-neuronal cells  To increase yields of neuronal cells triturate the pieces of the cortical tissue gently after the enzyme incubation step  This is imperative as it prevents unnecessary injury to cells and premature neuronal cell death  Since these cultures are maintained in the absence of glia feeder cells, they also offer an added advantage of growing cultures enriched in neurons Neuroscience, Issue 10, cellular, molecular, neurobiology, neuron, calcium/sodium imaging, primary cultures, mouse562Play ButtonAnalysis of Schwann-astrocyte Interactions Using In Vitro AssaysAuthors: Fardad T  Afshari, Jessica C  Kwok, James W  Fawcett



ground_truth: Online documentation, QuecPython community, online support: QQ group 445121768.

prompt_front: Now, answer the following question based on the text below, only give me the answer and do not output any other words.

Question: What do dendritic spines contain?

Read the following text and answer briefly.

Context 3:  Institutions: University of Cambridge Schwann cells are one of the commonly used cells in repair strategies following spinal cord injuries  Schwann cells are capable of supporting axonal regeneration and sprouting by secreting growth factors 1,2 and providing growth promoting adhesion molecules 3 and extracellular matrix molecules 4  In addition they myelinate the demyelinated axons at the site of injury 5 
However following transplantation, Schwann cells do not migrate from the site of implant and do not intermingle with the host astrocytes 6,7  This results in formation of a sharp boundary between the Schwann cells and astrocytes, creating an obstacle for growing axons trying to exit the graft back into the host tissue proximally and distally  Astrocytes in contact with Schwann cells also undergo hypertrophy and up-regulate the inhibitory molecules 8-13 
In vitro assays have been used to model Schwann cell-astrocyte interactions and have been important in understanding the mechanism underlying the cellular behaviour 
These in vitro assays include boundary assay, where a co-culture is made using two different cells with each cell type occupying different territories with only a small gap separating the two cell fronts  As the cells divide and migrate, the two cellular fronts get closer to each other and finally collide  This allows the behaviour of the two cellular populations to be analyzed at the boundary  Another variation of the same technique is to mix the two cellular populations in culture and over time the two cell types segregate with Schwann cells clumped together as islands in between astrocytes together creating multiple Schwann-astrocyte boundaries 
The second assay used in studying the interaction of two cell types is the migration assay where cellular movement can be tracked on the surface of the other cell type monolayer 14,15  This assay is commonly known as inverted coverslip assay  Schwann cells are cultured on small glass fragments and they are inverted face down onto the surface of astrocyte monolayers and migration is assessed from the edge of coverslip 
Both assays have been instrumental in studying the underlying mechanisms involved in the cellular exclusion and boundary formation  Some of the molecules identified using these techniques include N-Cadherins 15, Chondroitin Sulphate proteoglycans(CSPGs) 16,17, FGF/Heparin 18, Eph/Ephrins19 
This article intends to describe boundary assay and migration assay in stepwise fashion and elucidate the possible technical problems that might occur Cellular Biology, Issue 47, Schwann cell, astrocyte, boundary, migration, repulsion2214Play ButtonQuantifying Synapses: an Immunocytochemistry-based Assay to Quantify Synapse NumberAuthors: Dominic M  Ippolito, Cagla Eroglu  Institutions: Duke University, Duke University One of the most important goals in neuroscience is to understand the molecular cues that instruct early stages of synapse formation  As such it has become imperative to develop objective approaches to quantify changes in synaptic connectivity  Starting from sample fixation, this protocol details how to quantify synapse number both in dissociated neuronal culture and in brain sections using immunocytochemistry  Using compartment-specific antibodies, we label presynaptic terminals as well as sites of postsynaptic specialization  We define synapses as points of colocalization between the signals generated by these markers  The number of these colocalizations is quantified using a plug in Puncta Analyzer (written by Bary Wark, available upon request, c eroglu@cellbio duke edu) under the ImageJ analysis software platform  The synapse assay described in this protocol can be applied to any neural tissue or culture preparation for which you have selective pre- and postsynaptic markers  This synapse assay is a valuable tool that can be widely utilized in the study of synaptic development Neuroscience, Issue 45, synapse, immunocytochemistry, brain, neuron, astrocyte2270Play ButtonPreparation of Acute Hippocampal Slices from Rats and Transgenic Mice for the Study of Synaptic Alterations during Aging and Amyloid PathologyAuthors: Diana M  Mathis, Jennifer L  Furman, Christopher M  Norris  Institutions: University of Kentucky College of Public Health, University of Kentucky College of Medicine, University of Kentucky College of Medicine The rodent hippocampal slice preparation is perhaps the most broadly used tool for investigating mammalian synaptic function and plasticity  The hippocampus can be extracted quickly and easily from rats and mice and slices remain viable for hours in oxygenated artificial cerebrospinal fluid  Moreover, basic electrophysisologic techniques are easily applied to the investigation of synaptic function in hippocampal slices and have provided some of the best biomarkers for cognitive impairments  The hippocampal slice is especially popular for the study of synaptic plasticity mechanisms involved in learning and memory  Changes in the induction of long-term potentiation and depression (LTP and LTD) of synaptic efficacy in hippocampal slices (or lack thereof) are frequently used to describe the neurologic phenotype of cognitively-impaired animals and/or to evaluate the mechanism of action of nootropic compounds  This article outlines the procedures we use for preparing hippocampal slices from rats and transgenic mice for the study of synaptic alterations associated with brain aging and Alzheimer's disease (AD)1-3  Use of aged rats and AD model mice can present a unique set of challenges to researchers accustomed to using younger rats and/or mice in their research  Aged rats have thicker skulls and tougher connective tissue than younger rats and mice, which can delay brain extraction and/or dissection and consequently negate or exaggerate real age-differences in synaptic function and plasticity  Aging and amyloid pathology may also exacerbate hippocampal damage sustained during the dissection procedure, again complicating any inferences drawn from physiologic assessment  Here, we discuss the steps taken during the dissection procedure to minimize these problems  Examples of synaptic responses acquired in "healthy" and "unhealthy" slices from rats and mice are provided, as well as representative synaptic plasticity experiments  The possible impact of other methodological factors on synaptic function in these animal models (e g  recording solution components, stimulation parameters) are also discussed  While the focus of this article is on the use of aged rats and transgenic mice, novices to slice physiology should find enough detail here to get started on their own studies, using a variety of rodent models Neuroscience, Issue 49, aging, amyloid, hippocampal slice, synaptic plasticity, Ca2+, CA1, electrophysiology2330Play ButtonMesenteric Artery Contraction and Relaxation Studies Using Automated Wire MyographyAuthors: Lakeesha E  Bridges, Cicely L  Williams, Mildred A  Pointer, Emmanuel M  Awumey  Institutions: North Carolina Central University, Durham, North Carolina Central University, Durham, Wake Forest University School of Medicine Proximal resistance vessels, such as the mesenteric arteries, contribute substantially to the peripheral resistance  These small vessels of between 100-400 μm in diameter function primarily in directing blood flow to various organs according to the overall requirements of the body  The rat mesenteric artery has a diameter greater than 100 μm  The myography technique, first described by Mulvay and Halpern1, was based on the method proposed by Bevan and Osher2  The technique provides information about small vessels under isometric conditions, where substantial shortening of the muscle preparation is prevented  Since force production and sensitivity of vessels to different agonists is dependent on the extent of stretch, according to active tension-length relation, it is essential to conduct contraction studies under isometric conditions to prevent compliance of the mounting wires  Stainless steel wires are preferred to tungsten wires because of oxidation of the latter, which affects recorded responses3 The technique allows for the comparison of agonist-induced contractions of mounted vessels to obtain evidence for normal function of vascular smooth muscle cell receptors 
Medicine, Issue 55, cardiovascular, resistant arteries, contraction, relaxation, myography3119Play ButtonVisualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero ElectroporationAuthors: Emilie Pacary, Matilda A  Haas, Hendrik Wildner, Roberta Azzarelli, Donald M  Bell, Djoher Nora Abrous, François Guillemot  Institutions: MRC National Institute for Medical Research, National Institute for Medical Research, Université de Bordeaux In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5  To date this tool has been widely used to study the regulation of cellular proliferation, differentiation and neuronal migration especially in the developing cerebral cortex 6-8  Here we detail our protocol to electroporate in utero the cerebral cortex and the hippocampus and provide evidence that this approach can be used to study dendrites and spines in these two cerebral regions 
Finally, IUE provides a useful tool to identify functional interactions between genes involved in dendrite, spine and/or synapse development  Indeed, in contrast to other gene transfer methods such as virus, it is straightforward to combine multiple RNAi or transgenes in the same population of cells  In summary, IUE is a powerful method that has already contributed to the characterization of molecular mechanisms underlying brain function and disease and it should also be useful in the study of dendrites and spines Neuroscience, Issue 65, Developmental Biology, Molecular Biology, Neuronal development, In utero electroporation, dendrite, spines, hippocampus, cerebral cortex, gain and loss of function4163Play ButtonImaging Analysis of Neuron to Glia Interaction in Microfluidic Culture Platform (MCP)-based Neuronal Axon and Glia Co-culture SystemAuthors: Haruki Higashimori, Yongjie Yang  Institutions: Tufts University, Tufts Sackler School of Graduate Biomedical Sciences Proper neuron to glia interaction is critical to physiological function of the central nervous system (CNS)  This bidirectional communication is sophisticatedly mediated by specific signaling pathways between neuron and glia1,2   Identification and characterization of these signaling pathways is essential to the understanding of how neuron to glia interaction shapes CNS physiology  Previously, neuron and glia mixed cultures have been widely utilized for testing and characterizing signaling pathways between neuron and glia  What we have learned from these preparations and other in vivo tools, however, has suggested that mutual signaling between neuron and glia often occurred in specific compartments within neurons (i e , axon, dendrite, or soma)3  This makes it important to develop a new culture system that allows separation of neuronal compartments and specifically examines the interaction between glia and neuronal axons/dendrites  In addition, the conventional mixed culture system is not capable of differentiating the soluble factors and direct membrane contact signals between neuron and glia  Furthermore, the large quantity of neurons and glial cells in the conventional co-culture system lacks the resolution necessary to observe the interaction between a single axon and a glial cell 
In this study, we describe a novel axon and glia co-culture system with the use of a microfluidic culture platform (MCP)  In this co-culture system, neurons and glial cells are cultured in two separate chambers that are connected through multiple central channels  In this microfluidic culture platform, only neuronal processes (especially axons) can enter the glial side through the central channels  In combination with powerful fluorescent protein labeling, this system allows direct examination of signaling pathways between axonal/dendritic and glial interactions, such as axon-mediated transcriptional regulation in glia, glia-mediated receptor trafficking in neuronal terminals, and glia-mediated axon growth  The narrow diameter of the chamber also significantly prohibits the flow of the neuron-enriched medium into the glial chamber, facilitating probing of the direct membrane-protein interaction between axons/dendrites and glial surfaces Neuroscience, Issue 68, Molecular Biology, Cellular Biology, Biophysics, Microfluidics, Microfluidic culture platform, Compartmented culture, Neuron to glia signaling, neurons, glia, cell culture4448Play ButtonFluorescence Recovery After Photobleaching (FRAP) of Fluorescence Tagged Proteins in Dendritic Spines of Cultured Hippocampal NeuronsAuthors: Chan-Ying Zheng, Ronald S  Petralia, Ya-Xian Wang, Bechara Kachar  Institutions: National Institutes of Health, Bethesda 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 
This FRAP protocol shows how to perform a basic FRAP experiment as well as how to analyze the data Neuroscience, Issue 50, Spine, FRAP, hippocampal neurons, live cell imaging, protein mobility2568Play ButtonPrimary Neuronal Cultures from the Brains of Late Stage Drosophila PupaeAuthors: Beatriz Sicaeros, Jorge M  Campusano, Diane K  O'Dowd  Institutions: University of California, Irvine (UCI) In this video, we demonstrate the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae  The procedure begins with the removal of brains from animals at 70-78 hrs after puparium formation  The isolated brains are shown after brief incubation in papain followed by several washes in serum-free growth medium  The process of mechanical dissociation of each brain in a 5 ul drop of media on a coverslip is illustrated  The axons and dendrites of the post-mitotic neurons are sheered off near the soma during dissociation but the neurons begin to regenerate processes within a few hours of plating  Images show live cultures at 2 days  Neurons continue to elaborate processes during the first week in culture  Specific neuronal populations can be identified in culture using GAL4 lines to drive tissue specific expression of fluorescent markers such as GFP or RFP  Whole cell recordings have demonstrated the cultured neurons form functional, spontaneously active cholinergic and GABAergic synapses  A short video segment illustrates calcium dynamics in the cultured neurons using Fura-2 as a calcium indicator dye to monitor spontaneous calcium transients and nicotine evoked calcium responses in a dish of cultured neurons  These pupal brain cultures are a useful model system in which genetic and pharmacological tools can be used to identify intrinsic and extrinsic factors that influence formation and function of central synapses 



ground_truth: They are rich in actin and have been shown to be highly dynamic.