由美国国立卫生研究院授予AG027297，从肯塔基脊髓和颅脑损伤的研究信托奖，并从Kleberg基金会的礼物支持的工作。

<ol>
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生产这种视频文章是徕卡Microsystems公司赞助。

在本议定书中概述的步骤将有助于确保至少为迅速有效地开展脑解剖年龄在年轻成年大鼠，。我们还提供足够的细节，对于初学者LTP和LTD成立自己的切片研究。如果老化和突触功能和可塑性AD变化的进一步探索，是您的目标之一，有至少有两个其他的方法问题，上面提到的，值得进一步考虑。首先，一些实验室已经表明的Ca 2 +:Mg2 +的比例在录音学联可以在2,10,24,25海马诱导突触可塑性的一个显着的效果。在哺乳动物脑脊液中的 Ca 2 +:Mg2 +的比例大约是（如见 26） 。然而，学联的Ca 2 + Mg2 +的比例接近2片突触功能和可塑性研究。在早期的研究中，这种做法很可能是调整，优化诱导的LTP，随后成为日常所有的可塑性研究。然而，这种做法可以在老化和AD的研究问题，因为在神经元的Ca 2 +调节的特点差异。具体来说，CA 2 +内流和/或Ca 2 +诱导的Ca 2 +释放是升高的老年大鼠和/或在AD模型小鼠神经元激活3,27-31。有限公司感应是在学联的Ca 2 +水平的微妙的变化特别敏感。我们的协议，采用2mm的钙2 +和2 mM的镁，在公司通常结果年龄，但不年轻的成年动物2，同时研究利用一个钙2 +:镁+比例接近两个，已观察到的成年人强大的公司在该没有年龄差异2,10结合减少有限公司或在32岁的大鼠。这些意见强调需要仔细考虑学联钙2 +和Mg2 +水平进行比较时，在中青年成年动物的Ca 2 +依赖可塑性。 第二个方法问题， 涉及 33突触后膜的去极化和突触强度的差异可能老化/基因型的LTP的强烈依赖。在一个典型的LTP的实验，基线和LTP的刺激强度是一般调整，以生产半最大（或三季度最大）EPSP的幅度。潜在的问题是，通常表明，老年大鼠和APP/PS1小鼠突触强度降低相对年轻和/或野生型对口，这意味着，基线EPSP值也将被更小，老年大鼠和APP/PS1小鼠。较小的EPSPS可能转化为在LTP的刺激少的去极化，导致减少的概率为诱导LTP的33。由于这种潜在的混淆，这是很难以确定是否这些动物表现出吞吐量赤字，可塑性赤字或两者。也就是说，年龄和/或APP/PS1小鼠LTP的诱导机制可能是功能完好（无可塑性赤字），但在这些条件下不够刺激（吞吐量赤字）。这个区别是至关重要的，吞吐量和可塑性机制机制的反应可能非常不同的一个特定的药物治疗。我们尽量减少吞吐量降低的LTP诱导的影响，通过规范的所有片LTP的刺激前EPSP的幅度相同的水平（如为1 mV）。其他策略可能会有效，以及（如电压或电流钳的使用，以平衡各群体之间在膜电位的LTP的刺激），并调查在这些动物模型的LTP时，应考虑。

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td>氯化钠</td><td>费舍尔</td><td> BP358 - 1 </td></tr><tr><td>氯化钾</td><td>费舍尔</td><td> BP366 - 500 </td></tr><tr><td> KH 2 PO 4（一元） </td><td>西格玛</td><td> P5379 - 100G </td></tr><tr><td> 硫酸镁 </td><td>西格玛</td><td> M2643 - 500G </td></tr><tr><td> 氯化钙 （二水） </td><td>西格玛</td><td> C3306 - 250G </td></tr><tr><td>碳酸氢钠3 </td><td>费舍尔</td><td> S233 - 500 </td></tr><tr><td> C 6 H 12 O 6（葡萄糖） </td><td>费舍尔</td><td> BP350 - 1 </td></tr></tbody></table>试剂要求见表1。 <table border="1"><tbody><tr><td> 设备名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td>锥形瓶</td><td>费舍尔</td><td> FB - 500 - 2000 FB - 500 - 1000 </td><td></td></tr><tr><td>水族馆鼓泡</td><td></td><td></td><td>用于充氧媒体。可在大多数宠物商店</td></tr><tr><td> 50毫升的玻璃烧杯</td><td>费舍尔</td><td> 02，约440克</td><td>对于脑学联storarge </td></tr><tr><td>封口膜</td><td>费舍尔</td><td> 13-374-10 </td><td></td></tr><tr><td>小动物断头台</td><td>世界精密仪器（WPI） </td><td> DCAP - M </td><td></td></tr><tr><td>平纸巾</td><td></td><td></td><td></td></tr><tr><td> ＃11羽手术刀片</td><td>费舍尔</td><td> 08 - 916 - 5B </td><td></td></tr><tr><td> Beebee骨剪刀</td><td>精细科学的工具（FST）的</td><td> 16044-10 </td><td></td></tr><tr><td> Lempert Rongeurs </td><td> Roboz </td><td> RS - 8321 </td><td>使用老鼠</td></tr><tr><td>弗里德曼 - 皮尔森Rongeurs </td><td>福斯特</td><td> 16020-14 </td><td>使用小鼠或大鼠</td></tr><tr><td>海马工具</td><td>福斯特</td><td> 10099-15 </td><td></td></tr><tr><td>勺子</td><td></td><td></td><td>一个塑料小勺将尽</td></tr><tr><td>抹刀</td><td>费舍尔</td><td> 21 - 401 - 25A </td><td>抹刀</td></tr><tr><td> 0虹膜剪刀</td><td>福斯特</td><td> 14058-09 </td><td></td></tr><tr><td>塑料转移吸管</td><td>费舍尔</td><td> 13-711-43 </td><td></td></tr><tr><td> 110毫米的Whatman滤纸</td><td>费舍尔</td><td> 09 - 805E </td><td>滤纸猫。 1001-110 </td></tr><tr><td>玻璃培养皿</td><td>费舍尔</td><td></td><td></td></tr><tr><td>徕卡VT1000P手册振动切片机</td><td> Vibratome </td><td></td><td></td></tr><tr><td> 0.1毫米FA - 10羽毛小号刀片</td><td>特德佩拉</td><td> 121-9 </td><td> 0.1毫米FA - 10羽毛小号刀片</td></tr><tr><td>硼硅玻璃巴斯德吸取（橡胶球） </td><td>费舍尔</td><td> 13 - 678 - 20A </td><td>对于转让切片提示折断更大的开放和热抛光</td></tr><tr><td> 35毫米Polysterine的培养皿</td><td>康宁</td><td> 430588 </td><td>用于收集解剖后切片</td></tr></tbody></table> 表2工具和材料解剖<table border="1"><tbody><tr><td> 设备/材料 </td><td> 公司 </td><td> 评论（可选） </td></tr><tr><td>控股室</td><td>客户定制</td><td></td></tr><tr><td> P - 97水平移液器拖轮</td><td>萨特仪器有限公司</td><td></td></tr><tr><td>振动隔离表</td><td>技术制造公司（TMC） </td><td></td></tr><tr><td>法拉第笼</td><td>自定义内置</td><td></td></tr><tr><td>耐热吸气瓶底部手枪（产品编号1220 - 1L） </td><td>康宁</td><td></td></tr><tr><td>重力contolled四集稳压器（产品编号2C8891） </td><td>巴克斯特</td><td></td></tr><tr><td>中央真空系统</td><td></td><td>可在最现代化的实验室</td></tr><tr><td> 95％的O 2 / 5％的CO 2气体混合</td><td>苏格兰人T -总值有限公司</td><td></td></tr><tr><td> TygonTM实验室管 为O 2 / CO 2交付</td><td> Fisher Scientific则</td><td>无毒，非氧化性，有各种尺寸的。 </td></tr><tr><td> Eclipse的E600FN显微镜</td><td>尼康</td><td> 10倍和40倍的目标近红外线过滤器，和GFP，DS - red2的过滤器， </td></tr><tr><td>酷捕捉ES数码相机</td><td> Photometrics </td><td>酷捕捉ES数码相机</td></tr><tr><td>的X - Cite荧光照明灯</td><td> EXFO </td><td>的X - Cite荧光照明灯</td></tr><tr><td>显微镜平台</td><td>锡斯基尤</td><td>自定义组装</td></tr><tr><td> RC - 22潜水录音室（产品编号64-0228） </td><td>华纳仪器（威斯康星州） </td><td>需要P - 1型平台和舞台适配器（产品从华纳＃64-0277） </td></tr><tr><td> TC2BIP 2/3Ch温度控制器 </td><td>细胞Microcontrols </td><td> TC2BIP 2/3Ch温度控制器 </td></tr><tr><td> 4轴手动微型机械臂</td><td>锡斯基尤</td><td></td></tr><tr><td>铂铱丝（0.002） （＃PTT0203项目） </td><td> WPI </td><td></td></tr><tr><td> A365刺激隔离器</td><td> WPI </td><td> A365刺激隔离器</td></tr><tr><td> Multiclamp 700B放大器</td><td> Axon仪器</td><td></td></tr><tr><td> Digidata 1322A的A / D转换转换器</td><td> Axon仪器</td><td></td></tr><tr><td> PClamp软件</td><td> Axon仪器</td><td></td></tr><tr><td>个人电脑（奔腾4） </td><td>戴尔</td><td></td></tr></tbody></table> 表3。电设备和材料

preparation of acute hippocampal slices from rats and transgenic mice for the study of synaptic alterations during aging and amyloid pathology

啮齿类动物的海马脑片制备也许是最广泛使用的工具为调查哺乳动物突触功能和可塑性。海马可快速，轻松地从大鼠和小鼠中提取和切片仍然可行含氧人工脑脊液小时。此外，基本electrophysisologic的技术很容易应用到海马突触功能的调查，并提供了一​​些认知障碍的最好的生物标志物。海马脑片是在学习和记忆有关的突触可塑性机制的研究，特别是流行的。诱导长时程增强和突触效能的抑郁症（LTP和LTD）在海马（或缺乏）的变化是经常被用来描述神经表型的认知受损的动物和/或评估行动的机制益智化合物。本文概述了我们准备从老鼠和转基因小鼠海马突触改变与大脑衰老和阿尔茨海默氏病（AD）1-3相关的研究中使用的程序。使用的老年大鼠和AD模型小鼠可呈现出一套独特的挑战，习惯于在他们的研究中使用年轻的大鼠和/或小鼠的研究人员。老年大鼠有较厚的比年轻大鼠和小鼠的头骨和更严厉的结缔组织，可延缓脑提取和/或夹层，并因此否定或夸大真正的突触功能和可塑性的年龄差异。老龄化和淀粉样蛋白的病理还可能加剧在解剖过程持续的海马损伤，再复杂的生理评估得出的任何推论。在这里，我们讨论了在解剖过程中采取的步骤，以尽量减少这些问题。在"健康"和"不健康"的片从大鼠和小鼠获得的突触反应的例子，以及有代表性的突触可塑性实验。在这些动物模型（如录音解决方案组件，刺激参数）的突触功能的其他方法因素可能产生的影响进行了讨论。虽然本文的重点是对老年大鼠和转基因小鼠，新手使用切片生理，要找到足够的细节，在这里开始自己的研究，使用各种啮齿类动物模型。

Watch this Scientific Journal Video about Preparation of Acute Hippocampal Slices from Rats and Transgenic Mice for the Study of Synaptic Alterations during Aging and Amyloid Pathology at JoVE.com

Preparation of Acute Hippocampal Slices from Rats and Transgenic Mice for the Study of Synaptic Alterations during Aging and Amyloid Pathology

本文概述了程序，准备从老鼠和转基因小鼠海马突触改变与大脑衰老和与年龄有关的神经退行性疾病如阿尔茨海默氏症，相关的研究。

突触改变老化过程中淀粉样蛋白病理研究从大鼠和转基因小鼠海马急性片的制备

The rodent hippocampal slice preparation is perhaps the most broadly used tool for investigating mammalian synaptic function and plasticity. The ...

preparation-acute-hippocampal-slices-from-rats-transgenic-mice-for

Research

JoVE Journal

Neuroscience

93.9K 次观看.  University of Kentucky College of Public Health. 本文概述了程序，准备从老鼠和转基因小鼠海马突触改变与大脑衰老和与年龄有关的神经退行性疾病如阿尔茨海默氏症，相关的研究。

视频: Preparation of Acute Hippocampal Slices from Rats and Transgenic Mice for the Study of Synaptic Alterations during Aging and Amyloid Pathology

Preparation of Oligomeric &beta;-amyloid1-42 and Induction of Synaptic Plasticity Impairment on Hippocampal Slices

Impairment of synaptic connections is likely to underlie the subtle amnesic changes occurring at the early stages of Alzheimer s Disease (AD). &beta;-amyloid (A&beta;), a peptide produced in high amounts in AD, is known to reduce Long-Term Potentiation (LTP), a cellular correlate of learning and memory. Indeed, LTP impairment caused by A&beta; is a useful experimental paradigm for studying synaptic dysfunctions in AD models and for screening drugs capable of mitigating or reverting such synaptic impairments. Studies have shown that A&beta; produces the LTP disruption preferentially via its oligomeric form. Here we provide a detailed protocol for impairing LTP by perfusion of oligomerized synthetic A&beta;1-42 peptide onto acute hippocampal slices. In this video, we outline a step-by-step procedure for the preparation of oligomeric A&beta;1-42. Then, we follow an individual experiment in which LTP is reduced in hippocampal slices exposed to oligomerized A&beta;1-42 compared to slices in a control experiment where no A&beta;1-42 exposure had occurred.

Preparation of Oligomeric &#946;-amyloid1-42 and Induction of Synaptic Plasticity Impairment on Hippocampal Slices

One feature of Alzheimer's Disease is the elevation of A&beta;1-42 peptide. Here we provide a protocol for preparing synthetic A&beta;1-42 oligomers, which impairs hippocampal Long-Term Potentiation, a cellular correlate of memory. This procedure is useful for investigating mechanisms of A&beta;-induced pathology and drug screening.

低聚β-淀粉样蛋白的制备 1-42和突触可塑性损伤诱导海马脑片

Impairment of synaptic connections is likely to underlie the subtle amnesic changes occurring at the early stages of Alzheimer s Disease (AD). ...

科研

神经科学

Vibrodissociation of Neurons from Rodent Brain Slices to Study Synaptic Transmission and Image Presynaptic Terminals

Mechanical dissociation of neurons from the central nervous system has the advantage that presynaptic boutons remain attached to the isolated neuron of interest. This allows for examination of synaptic transmission under conditions where the extracellular and postsynaptic intracellular environments can be well controlled. A vibration-based technique without the use of proteases, known as vibrodissociation, is the most popular technique for mechanical isolation. A micropipette, with the tip fire-polished to the shape of a small ball, is placed into a brain slice made from a P1-P21 rodent. The micropipette is vibrated parallel to the slice surface and lowered through the slice thickness resulting in the liberation of isolated neurons. The isolated neurons are ready for study within a few minutes of vibrodissociation. This technique has advantages over the use of primary neuronal cultures, brain slices and enzymatically isolated neurons including: rapid production of viable, relatively mature neurons suitable for electrophysiological and imaging studies; superior control of the extracellular environment free from the influence of neighboring cells; suitability for well-controlled pharmacological experiments using rapid drug application and total cell superfusion; and improved space-clamp in whole-cell recordings relative to neurons in slice or cell culture preparations. This preparation can be used to examine synaptic physiology, pharmacology, modulation and plasticity. Real-time imaging of both pre- and postsynaptic elements in the living cells and boutons is also possible using vibrodissociated neurons. Characterization of the molecular constituents of pre- and postsynaptic elements can also be achieved with immunological and imaging-based approaches.

This report demonstrates a technique for mechanical isolation of individual viable neurons retaining attached presynaptic boutons. Vibrodissociated neurons have the advantages of rapid production, excellent pharmacological control and improved space-clamp without influence from neighboring cells. This method can be used for imaging of synaptic elements and patch-clamp recording.

Vibrodissociation从啮齿动物脑切片的神经元，研究突触传递和图像突触前的码头

Mechanical dissociation of neurons from the central nervous system has the advantage that presynaptic boutons remain attached to the isolated neuron of ...

Preparation of Acute Subventricular Zone Slices for Calcium Imaging

The subventricular zone (SVZ) is one of the two neurogenic zones in the postnatal brain. The SVZ contains densely packed cells, including neural progenitor cells with astrocytic features (called SVZ astrocytes), neuroblasts, and intermediate progenitor cells. Neuroblasts born in the SVZ tangentially migrate a great distance to the olfactory bulb, where they differentiate into interneurons. Intercellular signaling through adhesion molecules and diffusible signals play important roles in controlling neurogenesis. Many of these signals trigger intercellular calcium activity that transmits information inside and between cells. Calcium activity is thus reflective of the activity of extracellular signals and is an optimal way to understand functional intercellular signaling among SVZ cells.
Calcium activity has been studied in many other regions and cell types, including mature astrocytes and neurons. However, the traditional method to load cells with calcium indicator dye (i.e. bath loading) was not efficient at loading all SVZ cell types. Indeed, the cellular density in the SVZ precludes dye diffusion inside the tissue. In addition, preparing sagittal slices will better preserve the three-dimensional arrangement of SVZ cells, particularly the stream of neuroblast migration on the rostral-caudal axis.
Here, we describe methods to prepare sagittal sections containing the SVZ, the loading of SVZ cells with calcium indicator dye, and the acquisition of calcium activity with time-lapse movies. We used Fluo-4 AM dye for loading SVZ astrocytes using pressure application inside the tissue. Calcium activity was recorded using a scanning confocal microscope allowing a precise resolution for distinguishing individual cells. Our approach is applicable to other neurogenic zones including the adult hippocampal subgranular zone and embryonic neurogenic zones. In addition, other types of dyes can be applied using the described method.

A method to load subventricular zone (SVZ) cells with calcium indicator dyes for recording calcium activity is described. The postnatal SVZ contains tightly packed cells including neural progenitor cells and neuroblasts. Rather than using bath loading we injected the dye by pressure inside the tissue allowing better dye diffusion.

钙成像的急性下区片的制备

The subventricular zone (SVZ) is one of the two neurogenic zones in the postnatal brain. The SVZ contains densely packed cells, including neural ...

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs through activation of their ion channels and neurotransmitter receptors, and process information in part through activity-dependent release of gliotransmitters. Furthermore, astrocytes constitute the main uptake system for glutamate, contribute to potassium spatial buffering, as well as to GABA clearance. These cells therefore constantly monitor synaptic activity, and are thereby sensitive indicators for alterations in synaptically-released glutamate, GABA and extracellular potassium levels. Additionally, alterations in astroglial uptake activity or buffering capacity can have severe effects on neuronal functions, and might be overlooked when characterizing physiopathological situations or knockout mice. Dual recording of neuronal and astroglial activities is therefore an important method to study alterations in synaptic strength associated to concomitant changes in astroglial uptake and buffering capacities. Here we describe how to prepare hippocampal slices, how to identify stratum radiatum astrocytes, and how to record simultaneously neuronal and astroglial electrophysiological responses. Furthermore, we describe how to isolate pharmacologically the synaptically-evoked astroglial currents.

The preparation of acute brain slices from isolated hippocampi, as well as the simultaneous electrophysiological recordings of astrocytes and neurons in stratum radiatum during stimulation of schaffer collaterals is described. The pharmacological isolation of astroglial potassium and glutamate transporter currents is demonstrated.

双突触诱发的星形胶质细胞和神经元的反应，在急性海马脑片电生理记录

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs ...

Improved Preparation and Preservation of Hippocampal Mouse Slices for a Very Stable and Reproducible Recording of Long-term Potentiation

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.

This paper presents a complete methodology to prepare and preserve in vitro acute hippocampal slices from adult mice. This protocol allows the recording of very stable long-lasting long-term potentiation (LTP) for more than 8 hours with a success rate of 95%.

长时程增强一个非常稳定的和可重复的记录和保存海马鼠标片制备工艺的改进

Long-term potentiation (LTP) is a type of synaptic plasticity characterized by an increase in synaptic strength and believed to be involved in memory ...

Stereotaxic Infusion of Oligomeric Amyloid-beta into the Mouse Hippocampus

Alzheimer&#8217;s disease is a neurodegenerative disease affecting the aging population. A key neuropathological feature of the disease is the over-production of amyloid-beta and the deposition of amyloid-beta plaques in brain regions of the afflicted individuals. Throughout the years scientists have generated numerous Alzheimer&#8217;s disease mouse models that attempt to replicate the amyloid-beta pathology. Unfortunately, the mouse models only selectively mimic the disease features. Neuronal death, a prominent effect in the brains of Alzheimer&#8217;s disease patients, is noticeably lacking in these mice. Hence, we and others have employed a method of directly infusing soluble oligomeric species of amyloid-beta - forms of amyloid-beta that have been proven to be most toxic to neurons - stereotaxically into the brain. In this report we utilize male C57BL/6J mice to document this surgical technique of increasing amyloid-beta levels in a select brain region. The infusion target is the dentate gyrus of the hippocampus because this brain structure, along with the basal forebrain that is connected by the cholinergic circuit, represents one of the areas of degeneration in the disease. The results of elevating amyloid-beta in the dentate gyrus via stereotaxic infusion reveal increases in neuron loss in the dentate gyrus within 1 week, while there is a concomitant increase in cell death and cholinergic neuron loss in the vertical limb of the diagonal band of Broca of the basal forebrain. These effects are observed up to 2 weeks. Our data suggests that the current amyloid-beta infusion model provides an alternative mouse model to address region specific neuron death in a short-term basis. The advantage of this model is that amyloid-beta can be elevated in a spatial and temporal manner.

Here, we present a protocol for direct stereotaxic brain infusion of amyloid-beta. This methodology provides an alternative in vivo mouse model to address the short-term effects of amyloid-beta on brain neurons.

低聚淀粉样蛋白-β的立体灌注到小鼠海马

Alzheimer&#8217;s disease is a neurodegenerative disease affecting the aging population. A key neuropathological feature of the disease is the ...

Investigation of Synaptic Tagging/Capture and Cross-capture using Acute Hippocampal Slices from Rodents

Synaptic tagging and capture (STC) and cross-tagging are two important mechanisms at cellular level that explain how synapse-specificity and associativity is achieved in neurons within a specific time frame. These long-term plasticity-related processes are the leading candidate models to study the basis of memory formation and persistence at the cellular level. Both STC and cross-tagging involve two serial processes: (1) setting of the synaptic tag as triggered by a specific pattern of stimulation, and (2) synaptic capture, whereby the synaptic tag interacts with newly synthesized plasticity-related proteins (PRPs). Much of the understanding about the concepts of STC and cross-tagging arises from the studies done in CA1 region of the hippocampus and because of the technical complexity many of the laboratories are still unable to study these processes. Experimental conditions for the preparation of hippocampal slices and the recording of stable late-LTP/LTD are extremely important to study synaptic tagging/cross-tagging. This video article describes the experimental procedures to study long-term plasticity processes such as STC and cross-tagging in the CA1 pyramidal neurons using stable, long-term field-potential recordings from acute hippocampal slices of rats.

This video article describes experimental procedures to study long-term plasticity and its associative processes such as synaptic tagging, capture and cross-tagging in the CA1 pyramidal neurons using acute hippocampal slices from rodents.

使用急性海马脑片啮齿动物突触标记/捕获和交叉捕获的研究

Synaptic tagging and capture (STC) and cross-tagging are two important mechanisms at cellular level that explain how synapse-specificity and associativity ...

Preparation of Acute Brain Slices Using an Optimized N-Methyl-D-glucamine Protective Recovery Method

This protocol is a practical guide to the N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. Numerous recent studies have validated the utility of this method for enhancing neuronal preservation and overall brain slice viability. The implementation of this technique by early adopters has facilitated detailed investigations into brain function using diverse experimental applications and spanning a wide range of animal ages, brain regions, and cell types. Steps are outlined for carrying out the protective recovery brain slice technique using an optimized NMDG artificial cerebrospinal fluid (aCSF) media formulation and enhanced procedure to reliably obtain healthy brain slices for patch clamp electrophysiology. With this updated approach, a substantial improvement is observed in the speed and reliability of gigaohm seal formation during targeted patch clamp recording experiments while maintaining excellent neuronal preservation, thereby facilitating challenging experimental applications. Representative results are provided from multi-neuron patch clamp recording experiments to assay synaptic connectivity in neocortical brain slices prepared from young adult transgenic mice and mature adult human neurosurgical specimens. Furthermore, the optimized NMDG protective recovery method of brain slicing is compatible with both juvenile and adult animals, thus resolving a limitation of the original methodology. In summary, a single media formulation and brain slicing procedure can be implemented across various species and ages to achieve excellent viability and tissue preservation.

Preparation of Acute Brain Slices Using an Optimized N-Methyl-D-glucamine Protective Recovery Method

This protocol demonstrates the implementation of an optimized N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. A single media formulation is used to reliably obtain healthy brain slices from animals of any age and for diverse experimental applications.

用优化的n-甲基 d-葡乙胺保护恢复法制备急性脑切片

This protocol is a practical guide to the N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. Numerous recent studies have ...

Recording Synaptic Plasticity in Acute Hippocampal Slices Maintained in a Small-volume Recycling-, Perfusion-, and Submersion-type Chamber System

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful analysis of synaptic transmission modulation when performing field potential or intracellular recordings. This manuscript describes methodological aspects that might be helpful in improving experimental conditions for the maintenance of acute brain slices and for recording field excitatory postsynaptic potentials in a commercially available submersion chamber with an outflow-carbogenation unit. The outflow-carbogenation helps to stabilize the oxygen level in experiments that rely on the recycling of a small buffer reservoir to enhance the cost-efficiency of drug experiments. In addition, the manuscript presents representative experiments that examine the effects of different carbogenation modes and stimulation paradigms on the activity-dependent synaptic plasticity of synaptic transmission.

This protocol describes the stabilization of the oxygen level in a small volume of recycled buffer and methodological aspects of recording activity-dependent synaptic plasticity in submerged acute hippocampal slices.

小容积回收-灌注-浸没式室系统中维持的急性海马切片的突触可塑性记录

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful ...

Single Synapse Indicators of Glutamate Release and Uptake in Acute Brain Slices from Normal and Huntington Mice

Synapses are highly compartmentalized functional units that operate independently on each other. In Huntington&#39;s disease (HD) and other neurodegenerative disorders, this independence might be compromised due to insufficient glutamate clearance and the resulting spill-in and spill-out effects. Altered astrocytic coverage of the presynaptic terminals and/or dendritic spines as well as a reduced size of glutamate transporter clusters at glutamate release sites have been implicated in the pathogenesis of diseases resulting in symptoms of dys-/hyperkinesia. However, the mechanisms leading to the dysfunction of glutamatergic synapses in HD are not well understood. Improving and applying synapse imaging we have obtained data shedding new light on the mechanisms impeding the initiation of movements. Here, we describe the principle elements of a relatively inexpensive approach to achieve single synapse resolution by using the new genetically encoded ultrafast glutamate sensor iGluu, wide-field optics, a scientific CMOS (sCMOS) camera, a 473 nm laser and a laser positioning system to evaluate the state of corticostriatal synapses in acute slices from age appropriate healthy or diseased mice. Glutamate transients were constructed from single or multiple pixels to obtain estimates of i) glutamate release based on the maximal elevation of the glutamate concentration [Glu] next to the active zone and ii) glutamate uptake as reflected in the time constant of decay (TauD) of the perisynaptic [Glu]. Differences in the resting bouton size and contrasting patterns of short-term plasticity served as criteria for the identification of corticostriatal terminals as belonging to the intratelencephalic (IT) or the pyramidal tract (PT) pathway. Using these methods, we discovered that in symptomatic HD mice ~40% of PT-type corticostriatal synapses exhibited insufficient glutamate clearance, suggesting that these synapses might be at risk to excitotoxic damage. The results underline the usefulness of TauD as a biomarker of dysfunctional synapses in Huntington mice with a hypokinetic phenotype.

We present a protocol to evaluate the balance between glutamate release and clearance at single corticostriatal glutamatergic synapses in acute slices from adult mice. This protocol uses the fluorescent sensor iGluu for glutamate detection, a sCMOS camera for signal acquisition and a device for focal laser illumination.