我们博士Sanika奇鲁瓦的严格审查本手稿。这项工作是由美国国立卫生研究院（DA026947，DA021471，NS071122）的支持。

1。设备和用品<ol><li>安装一个法拉第笼顶部的抗振动台（TMI），以减少背景噪音。 </li><li>的同时膜片钳安培的记录系统需要一个倒置显微镜具有优良的DIC的光学元件和长工作距离镜头。连接到汽车电池的显微镜灯塔。这个直流系统的光源，用于将进一步降低的电噪声。 </li><li>液压的微操纵（锡斯基尤）进一步降低噪音。在我们的配置中，我们使用的全细胞记录，用右手的机械手和左手为安培。 </li></ol> 2。准备电极记录<ol><li>拉块电极使用石英移液管上的P-2000拉出器（萨特）。我们的拉持续约5秒后，用两个热循环。在我们的全细胞膜片吸液管，这导致加热时间一致电阻（MΩ3-4​​）。 </li><li>填充电极与所述液含2 mM多巴胺，它安装于正确的机械手。保持的吸移管溶液包含DA用铝箔包裹的容器。请在冰上。多巴胺是可氧化。保持溶液在冰上，避光将减少多巴胺的氧化速率。 </li><li> （大矸）一个ProCFE低噪声的碳纤维安培电极，轻轻取下的储物盒，填充与汞，安装到安培适配器（在图1所示），然后安装在右边maniupulator。保护免受损害的碳纤维的前端保持的碳纤维的远端。实验室显微镜检查电极的前端，以确保清洁完整。 </li><li>检查通过把包含外部溶液的玻璃底培养皿中的电极的安培电极的完整性。记录的基准电流，在没有多巴胺。添加10微升的为1mm DA解决方案的菜。一个很好的安培电极重帘线的氧化电流的增加。重复此步骤，在每一个实验的开始和结束，使若干安培电极工作正常。 </li></ol> 3。准备主设计，以表达多巴胺转运体的玻璃底培养皿或细胞的多巴胺神经元的神经细胞培养<ol><li>轻轻洗细胞或多巴胺神经元的3倍，温暖的外部解决方案。 </li><li>安装玻璃底培养皿，在显微镜阶段。 </li></ol> 4。可视化细胞进行实验<ol><li>找到正确的焦点，清晰地可视化的细胞。将贴片电极上的正压力。然后，轻轻地使两个电极下降到溶液中，并靠近细胞。 </li><li>旁边的小区（在左侧），和在右边的贴片电极安培电极的位置。 </li><li>对细胞的修补电极达到一个gigaohm的密封。破裂的密封与吸实现整个单元的配置。 </li><li>允许5-8分钟含多巴胺进入细胞内部溶液用于透析。 </li><li>使用所需电压台阶或斜坡协议。同时采集数据而膜电位的补丁移液管通过控制从两个补丁移液器和安培电极测量全细胞电流和反向多巴胺通过多巴胺转运蛋白运输。 </li></ol>

<ol>
	<li>Michael, A. C., Ikeda, M., Justice, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+contributing+to+the+recovery+of+striatal+releasable+dopamine+following+MFB+stimulation.">Mechanisms contributing to the recovery of striatal releasable dopamine following MFB stimulation.</a> Brain Res. 421, 325-335 (1987).</li><li>Gonon, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prolonged+and+extrasynaptic+excitatory+action+of+dopamine+mediated+by+D1+receptors+in+the+rat+striatum+in+vivo.">Prolonged and extrasynaptic excitatory action of dopamine mediated by D1 receptors in the rat striatum in vivo.</a> J. Neurosci. 17, 5972-5978 (1997).</li><li>Sulzer, D., Pothos, E. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+quantal+size+by+presynaptic+mechanisms.">Regulation of quantal size by presynaptic mechanisms.</a> Rev. Neurosci. 11, 159-212 (2000).</li><li>Napolitano, A., Cesura, A. M., Da Prada, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+monoamine+oxidase+and+catechol+O-methyltransferase+in+dopaminergic+neurotransmission.">The role of monoamine oxidase and catechol O-methyltransferase in dopaminergic neurotransmission.</a> J. Neural. Transm. Suppl. 45, 35-45 (1995).</li><li>Sulzer, D., Maidment, N. T., Rayport, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amphetamine+and+other+weak+bases+act+to+promote+reverse+transport+of+dopamine+in+ventral+midbrain+neurons.">Amphetamine and other weak bases act to promote reverse transport of dopamine in ventral midbrain neurons.</a> J. Neurochem. 60, 527-535 (1993).</li><li>Salahpour, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+amphetamine-induced+hyperactivity+and+reward+in+mice+overexpressing+the+dopamine+transporter.">Increased amphetamine-induced hyperactivity and reward in mice overexpressing the dopamine transporter.</a> Proc. Natl. Acad. Sci. U.S.A. 105, 4405-4410 (2008).</li><li>Khoshbouei, H., Wang, H., Lechleiter, J. D., Javitch, J. A., Galli, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amphetamine-induced+dopamine+efflux.+A+voltage-sensitive+and+intracellular+Na+-dependent+mechanism.">Amphetamine-induced dopamine efflux. A voltage-sensitive and intracellular Na+-dependent mechanism.</a> J. Biol. Chem. 278, 12070-12077 (2003).</li><li>Goodwin, J. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amphetamine+and+methamphetamine+differentially+affect+dopamine+transporters+in+vitro+and+in.">Amphetamine and methamphetamine differentially affect dopamine transporters in vitro and in.</a> , 284-2978 (2009).</li><li>Kahlig, K. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amphetamine+induces+dopamine+efflux+through+a+dopamine+transporter+channel.">Amphetamine induces dopamine efflux through a dopamine transporter channel.</a> Proc. Natl. Acad. Sci. U.S.A. 102, 3495-3500 (2005).</li><li>Swant, J., Chirwa, S., Stanwood, G., Khoshbouei, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methamphetamine+reduces+LTP+and+increases+baseline+synaptic+transmission+in+the+CA1+region+of+mouse+hippocampus.">Methamphetamine reduces LTP and increases baseline synaptic transmission in the CA1 region of mouse hippocampus.</a> PLoS One. 5, e11382 (2010).</li><li>Gnegy, M. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracellular+Ca2++regulates+amphetamine-induced+dopamine+efflux+and+currents+mediated+by+the+human+dopamine+transporter.">Intracellular Ca2+ regulates amphetamine-induced dopamine efflux and currents mediated by the human dopamine transporter.</a> Mol. Pharmacol. 66, 137-143 (2004).</li></ol>

没有利益冲突的声明。

同时电压钳位和​​安培法有以下好处。所有类型的细胞都可以访问的，并且可以被用于记录。录音完成后的细胞或神经元的鉴定是简单明了的。特别是，如果该信元被加入荧光标记的兴趣蛋白质的荧光标记的实验者可以很容易地选择目标细胞或神经元。的实验配置允许的药物制剂的均匀和受控的交付，通过修补程序移液管，或通过加入这些试剂的浴溶液10。在同时膜片钳安培的技术允许...

<table border="1"><tbody><tr><td></td><td></td><td></td><td> 设备 </td></tr><tr><td>抗振动表w /法拉第笼</td><td>技术制造公司</td><td> 63-500系列</td><td>我们使用模型63-543 </td></tr><tr><td>倒置显微镜尼康TE-2000。 </td><td>尼康</td><td>停产</td><td>现在Eclipse钛</td></tr><tr><td>两个低噪声放大器Axopatch 200B中</td><td> Molecular Devices公司</td><td></td><td> 800-635-5577 </td></tr><tr><td> 1-CV 203 BU探头</td><td> Molecular Devices公司</td><td></td><td> 800-635-5578 </td></tr><tr><td> 1-HL-U吸管架</td><td> Molecular Devices公司</td><td></td><td> 800-635-5579 </td></tr><tr><td> Digidata 1440A的A / D转换器</td><td> Molecular Devices公司</td><td></td><td> 800-635-5580 </td></tr><tr><td>两个的操控Siskyou，离开和右撇子</td><td>锡斯基尤</td><td> MX6600R MX6600L </td><td> 877-313-6418 </td></tr><tr><td>激光吸管拉马</td><td>萨特仪器</td><td> P-2000 </td><td> 888-883-0128 </td></tr><tr><td>低噪声的碳纤维安培电极</td><td> ProCFE </td><td></td><td> <a href="http://www.dagan.com" target="_blank">www.dagan.com</a> </td></tr><tr><td>低噪音石英移液管</td><td>萨特仪器</td><td> QF100-70-7.5 </td><td> 888-883-0128 </td></tr><tr><td> 12伏汽车电池</td><td></td><td></td><td>广泛使用</td></tr><tr><td>汽车电池充电器</td><td></td><td></td><td>广泛使用</td></tr><tr><td></td><td></td><td></td><td> 试剂 </td></tr><tr><td>氯化钠（NaCl） </td><td>西格玛</td><td> S7653 </td><td></td></tr><tr><td> HEPES </td><td>西格玛</td><td> H3375 </td><td></td></tr><tr><td>葡萄糖</td><td>西格玛</td><td> G7528 </td><td></td></tr><tr><td>硫酸镁（用MgSO 4） </td><td>西格玛</td><td> M2643 </td><td></td></tr><tr><td>磷酸二氢钾（KH 2 PO 4） </td><td>西格玛</td><td> P5655 </td><td></td></tr><tr><td>氯化钾（KCl） </td><td>西格玛</td><td> P9333 </td><td></td></tr><tr><td>氯化钙二水合物（CaCl 2的 •2H 2 0） </td><td>西格玛</td><td> 223506 </td><td></td></tr><tr><td>六水合氯化镁（MgCl 2的 •6H 2 0） </td><td>西格玛</td><td> M2670 </td><td></td></tr><tr><td> EGTA </td><td>西格玛</td><td> E0396 </td><td></td></tr></tbody></table>

single cell measurement of dopamine release with simultaneous voltage-clamp and amperometry

其释放到突触间隙，多巴胺后发挥其生物学特性，通过其前和突触后的目标1。的多巴胺信号被终止通过扩散2-3，胞外酶4，和膜转运5。位于周边的多巴胺神经元的突触间隙的多巴胺转运蛋白，清除释放的胺通过向内多巴胺通量（吸收）。多巴胺转运蛋白也可以在相反的方向，在这个过程被称为向外输送或流出的多巴胺5从内到外释放胺。 20余年前，苏尔寿等。报道，多巴胺转运可以工作在两种模式的活动：向前（吸收）和反向（流出）5。通过外排通过释放神经递质转运到细胞外空间移动大量的多巴胺，并已被证明发挥重要的调控作用，在细胞外多巴胺Ĥomeostasis 6。在这里，我们描述了如何同时膜片钳和安培法记录膜电位被控制以毫秒的时间分辨率时，通过外排机制，可用于测量释放多巴胺。对于这一点，使用Axopatch 200B中放大器（Molecular Devices公司，用一个低通贝塞尔滤波器组在1,000 Hz的全细胞电流记录），全细胞电流和氧化（电流）信号是同时测量的。对于记录的碳纤维电极的安培法连接到第二放大器（Axopatch 200B中），被置于相邻的质膜，并保持在700毫伏。可以被记录的全细胞和氧化的电流（安培），可以使用一个电压阶跃协议生成的电流 - 电压关系。不同于通常安培校准，需要转换为浓度，电流被直接报告，而不考虑的有效容积7。因此，所得到的数据代表一个下限多巴胺流出，因为有些发射机失去本体溶液。

组合膜片钳与安培法可以测量电压依赖性DAT介导的DA流出。 图2A示出有代表性的实验配置和记录DAT介导的DA流出当细胞内以及独特的膜电位，通过全细胞膜片移液器夹紧。使用这种技术，细胞表达YFP-DAT蛋白的电压钳位与全细胞膜片移液器而安培电极被放置到质膜（ 图2A）。整个电池用电极的填充用移液管溶液含有2mM DA。安培触摸质膜的电极被保持在700毫伏，大于DA的氧化?...

Watch this Scientific Journal Video about Single Cell Measurement of Dopamine Release with Simultaneous Voltage-clamp and Amperometry at JoVE.com

Single Cell Measurement of Dopamine Release with Simultaneous Voltage-clamp and Amperometry

电化学技术措施，通过检测自发多巴胺的氧化产生的氧化电流从一个单细胞多巴胺的释放。同时钳位电压和安培的方法揭示了整体的“活动”，这种活动对多巴胺的反向运输的多巴胺转运体的调节作用机理之间的关系。

单细胞的多巴胺释放，同时电压钳和电流分析法测量

After its release into the synaptic cleft, dopamine exerts its biological properties via its pre- and post-synaptic targets1. The dopamine signal is ...

single-cell-measurement-dopamine-release-with-simultaneous-voltage

Research

JoVE Journal

Neuroscience

13.7K 次观看.  University of Florida. 电化学技术措施，通过检测自发多巴胺的氧化产生的氧化电流从一个单细胞多巴胺的释放。同时钳位电压和安培的方法揭示了整体的“活动”，这种活动对多巴胺的反向运输的多巴胺转运体的调节作用机理之间的关系。

视频: Single Cell Measurement of Dopamine Release with Simultaneous Voltage-clamp and Amperometry

Functional Mapping with Simultaneous MEG and EEG

We use magnetoencephalography (MEG) and electroencephalography (EEG) to locate and determine the temporal evolution in brain areas involved in the processing of simple sensory stimuli. We will use somatosensory stimuli to locate the hand somatosensory areas, auditory stimuli to locate the auditory cortices, visual stimuli in four quadrants of the visual field to locate the early visual areas. These type of experiments are used for functional mapping in epileptic and brain tumor patients to locate eloquent cortices. In basic neuroscience similar experimental protocols are used to study the orchestration of cortical activity. The acquisition protocol includes quality assurance procedures, subject preparation for the combined MEG/EEG study, and acquisition of evoked-response data with somatosensory, auditory, and visual stimuli. We also demonstrate analysis of the data using the equivalent current dipole model and cortically-constrained minimum-norm estimates. Anatomical MRI data are employed in the analysis for visualization and for deriving boundaries of tissue boundaries for forward modeling and cortical location and orientation constraints for the minimum-norm estimates.

We use magnetoencephalography (MEG) and electroencephalography (EEG) to map brain areas involved in the processing of simple sensory stimuli.

同时脑磁图和脑电图的功能映射

We use magnetoencephalography (MEG) and electroencephalography (EEG) to locate and determine the temporal evolution in brain areas involved in the ...

科研

神经科学

Presynaptic Dopamine Dynamics in Striatal Brain Slices with Fast-scan Cyclic Voltammetry

Extensive research has focused on the neurotransmitter dopamine because of its importance in the mechanism of action of drugs of abuse (e.g. cocaine and amphetamine), the role it plays in psychiatric illnesses (e.g. schizophrenia and Attention Deficit Hyperactivity Disorder), and its involvement in degenerative disorders like Parkinson's and Huntington's disease. Under normal physiological conditions, dopamine is known to regulate locomotor activity, cognition, learning, emotional affect, and neuroendocrine hormone secretion. One of the largest densities of dopamine neurons is within the striatum, which can be divided in two distinct neuroanatomical regions known as the nucleus accumbens and the caudate-putamen. The objective is to illustrate a general protocol for slice fast-scan cyclic voltammetry (FSCV) within the mouse striatum. FSCV is a well-defined electrochemical technique providing the opportunity to measure dopamine release and uptake in real time in discrete brain regions. Carbon fiber microelectrodes (diameter of ~ 7 &mu;m) are used in FSCV to detect dopamine oxidation. The analytical advantage of using FSCV to detect dopamine is its enhanced temporal resolution of 100 milliseconds and spatial resolution of less than ten microns, providing complementary information to in vivo microdialysis.

Using fast-scan cyclic voltammetry to measure electrically evoked presynaptic dopamine dynamics in striatal brain slices.

突触前多巴胺纹状体脑片的快速扫描循环伏安动态

Extensive research has focused on the neurotransmitter dopamine because of its importance in the mechanism of action of drugs of abuse (e.g. cocaine and ...

Single-cell Profiling of Developing and Mature Retinal Neurons

Highly specialized, but exceedingly small populations of cells play important roles in many tissues. The identification of cell-type specific markers and gene expression programs for extremely rare cell subsets has been a challenge using standard whole-tissue approaches. Gene expression profiling of individual cells allows for unprecedented access to cell types that comprise only a small percentage of the total tissue1-7. In addition, this technique can be used to examine the gene expression programs that are transiently expressed in small numbers of cells during dynamic developmental transitions8.
This issue of cellular diversity arises repeatedly in the central nervous system (CNS) where neuronal connections can occur between quite diverse cells9. The exact number of distinct cell types is not precisely known, but it has been estimated that there may be as many as 1000 different types in the cortex itself10. The function(s) of complex neural circuits may rely on some of the rare neuronal types and the genes they express. By identifying new markers and helping to molecularly classify different neurons, the single-cell approach is particularly useful in the analysis of cell types in the nervous system. It may also help to elucidate mechanisms of neural development by identifying differentially expressed genes and gene pathways during early stages of neuronal progenitor development.
As a simple, easily accessed tissue with considerable neuronal diversity, the vertebrate retina is an excellent model system for studying the processes of cellular development, neuronal differentiation and neuronal diversification. However, as in other parts of the CNS, this cellular diversity can present a problem for determining the genetic pathways that drive retinal progenitors to adopt a specific cell fate, especially given that rod photoreceptors make up the majority of the total retinal cell population11. Here we report a method for the identification of the transcripts expressed in single retinal cells (Figure 1). The single-cell profiling technique allows for the assessment of the amount of heterogeneity present within different cellular populations of the retina2,4,5,12. In addition, this method has revealed a host of new candidate genes that may play role(s) in the cell fate decision-making processes that occur in subsets of retinal progenitor cells8. With some simple adjustments to the protocol, this technique can be utilized for many different tissues and cell types.

A method for the isolation of single retinal cells and subsequent amplification of their cDNAs is described. Single-cell transcriptomics reveals the degree of cellular heterogeneity present in a tissue and uncovers new marker genes for rare cell populations. The accompanying protocol can be adjusted to suit many different cell types.

视网膜神经细胞发育和成熟的单细胞的剖析

Highly specialized, but exceedingly small populations of cells play important roles in many tissues. The identification of cell-type specific markers and ...

Optical Recording of Suprathreshold Neural Activity with Single-cell and Single-spike Resolution

Signaling of information in the vertebrate central nervous system is often carried by populations of neurons rather than individual neurons. Also propagation of suprathreshold spiking activity involves populations of neurons. Empirical studies addressing cortical function directly thus require recordings from populations of neurons with high resolution. Here we describe an optical method and a deconvolution algorithm to record neural activity from up to 100 neurons with single-cell and single-spike resolution. This method relies on detection of the transient increases in intracellular somatic calcium concentration associated with suprathreshold electrical spikes (action potentials) in cortical neurons. High temporal resolution of the optical recordings is achieved by a fast random-access scanning technique using acousto-optical deflectors (AODs)1. Two-photon excitation of the calcium-sensitive dye results in high spatial resolution in opaque brain tissue2. Reconstruction of spikes from the fluorescence calcium recordings is achieved by a maximum-likelihood method. Simultaneous electrophysiological and optical recordings indicate that our method reliably detects spikes (&gt;97% spike detection efficiency), has a low rate of false positive spike detection (&lt; 0.003 spikes/sec), and a high temporal precision (about 3 msec) 3. This optical method of spike detection can be used to record neural activity in vitro and in anesthetized animals in vivo3,4.

Understanding the function of the vertebrate central nervous system requires recordings from many neurons because cortical function arises on the level of populations of neurons. Here we describe an optical method to record suprathreshold neural activity with single-cell and single-spike resolution, dithered random-access scanning. This method records somatic fluorescence calcium signals from up to 100 neurons with high temporal resolution. A maximum-likelihood algorithm deconvolves the underlying suprathreshold neural activity from the somatic fluorescence calcium signals. This method reliably detects spikes with high detection efficiency and a low rate of false positives and can be used to study neural populations in vitro and in vivo.

阈上的神经活动的单细胞和单穗的分辨率的光学的方法记录

Signaling of information in the vertebrate central nervous system is often carried by populations of neurons rather than individual neurons. Also ...

Simultaneous Electroencephalography, Real-time Measurement of Lactate Concentration and Optogenetic Manipulation of Neuronal Activity in the Rodent Cerebral Cortex

Although the brain represents less than 5% of the body by mass, it utilizes approximately one quarter of the glucose used by the body at rest1. The function of non rapid eye movement sleep (NREMS), the largest portion of sleep by time, is uncertain. However, one salient feature of NREMS is a significant reduction in the rate of cerebral glucose utilization relative to wakefulness2-4. This and other findings have led to the widely held belief that sleep serves a function related to cerebral metabolism. Yet, the mechanisms underlying the reduction in cerebral glucose metabolism during NREMS remain to be elucidated.
	One phenomenon associated with NREMS that might impact cerebral metabolic rate is the occurrence of slow waves, oscillations at frequencies less than 4 Hz, in the electroencephalogram5,6. These slow waves detected at the level of the skull or cerebral cortical surface reflect the oscillations of underlying neurons between a depolarized/up state and a hyperpolarized/down state7. During the down state, cells do not undergo action potentials for intervals of up to several hundred milliseconds. Restoration of ionic concentration gradients subsequent to action potentials represents a significant metabolic load on the cell8; absence of action potentials during down states associated with NREMS may contribute to reduced metabolism relative to wake.
	Two technical challenges had to be addressed in order for this hypothetical relationship to be tested. First, it was necessary to measure cerebral glycolytic metabolism with a temporal resolution reflective of the dynamics of the cerebral EEG (that is, over seconds rather than minutes). To do so, we measured the concentration of lactate, the product of aerobic glycolysis, and therefore a readout of the rate of glucose metabolism in the brains of mice. Lactate was measured using a lactate oxidase based real time sensor embedded in the frontal cortex. The sensing mechanism consists of a platinum-iridium electrode surrounded by a layer of lactate oxidase molecules. Metabolism of lactate by lactate oxidase produces hydrogen peroxide, which produces a current in the platinum-iridium electrode. So a ramping up of cerebral glycolysis provides an increase in the concentration of substrate for lactate oxidase, which then is reflected in increased current at the sensing electrode. It was additionally necessary to measure these variables while manipulating the excitability of the cerebral cortex, in order to isolate this variable from other facets of NREMS.
	We devised an experimental system for simultaneous measurement of neuronal activity via the elecetroencephalogram, measurement of glycolytic flux via a lactate biosensor, and manipulation of cerebral cortical neuronal activity via optogenetic activation of pyramidal neurons. We have utilized this system to document the relationship between sleep-related electroencephalographic waveforms and the moment-to-moment dynamics of lactate concentration in the cerebral cortex. The protocol may be useful for any individual interested in studying, in freely behaving rodents, the relationship between neuronal activity measured at the electroencephalographic level and cellular energetics within the brain.

A procedure is described for manipulating the activity of cerebral cortical pyramidal neurons optogenetically while the electroencephalogram, electromyogram, and cerebral lactate concentration are monitored. Experimental recordings are performed on cable-tethered mice while they undergo spontaneous sleep/wake cycles. Optogenetic equipment is assembled in our laboratory; recording equipment is commercially available.

同时脑电图，实时测量的啮齿动物的大脑皮质中的神经元活动的乳酸浓度与Optogenetic的操作

Although the brain represents less than 5% of the body  by mass, it utilizes approximately one quarter of the glucose used by the body  at rest1. The ...

Deep Brain Stimulation with Simultaneous fMRI in Rodents

In order to visualize the global and downstream neuronal responses to deep brain stimulation (DBS) at various targets, we have developed a protocol for using blood oxygen level dependent (BOLD) functional magnetic resonance imaging (fMRI) to image rodents with simultaneous DBS. DBS fMRI presents a number of technical challenges, including accuracy of electrode implantation, MR artifacts created by the electrode, choice of anesthesia and paralytic to minimize any neuronal effects while simultaneously eliminating animal motion, and maintenance of physiological parameters, deviation from which can confound the BOLD signal.&#160;Our laboratory has developed a set of procedures that are capable of overcoming most of these possible issues. For electrical stimulation, a homemade tungsten bipolar microelectrode is used, inserted stereotactically at the stimulation site in the anesthetized subject. In preparation for imaging, rodents are fixed on a plastic headpiece and transferred to the magnet bore. For sedation and paralysis during scanning, a cocktail of dexmedetomidine and pancuronium is continuously infused, along with a minimal dose of isoflurane; this preparation minimizes the BOLD ceiling effect of volatile anesthetics. In this example experiment, stimulation of the subthalamic nucleus (STN) produces BOLD responses which are observed primarily in ipsilateral cortical regions, centered in motor cortex. Simultaneous DBS and fMRI allows the unambiguous modulation of neural circuits dependent on stimulation location and stimulation parameters, and permits observation of neuronal modulations free of regional bias. This technique may be used to explore the downstream effects of modulating neural circuitry at nearly any brain region, with implications for both experimental and clinical DBS.

This protocol describes a standard method for simultaneous functional magnetic resonance imaging and deep brain stimulation in the rodent. The combined use of these experimental tools allows for the exploration of global downstream activity in response to electrical stimulation at virtually any brain target.

深部脑刺激与同步功能磁共振成像在鼠害

In order to visualize the global and downstream neuronal responses to deep brain stimulation (DBS) at various targets, we have developed a protocol for ...

Preparation of Neuronal Co-cultures with Single Cell Precision

Microfluidic embodiments of the Campenot chamber have attracted great interest from the neuroscience community. These interconnected co-culture platforms can be used to investigate a variety of questions, spanning developmental and functional neurobiology to infection and disease propagation. However, conventional systems require significant cellular inputs (many thousands per compartment), inadequate for studying low abundance cells, such as primary dopaminergic substantia nigra, spiral ganglia, and Drosophilia melanogaster neurons, and impractical for high throughput experimentation. The dense cultures are also highly locally entangled, with few outgrowths (&#60;10%) interconnecting the two cultures. In this paper straightforward microfluidic and patterning protocols are described which address these challenges: (i) a microfluidic single neuron arraying method, and (ii) a water masking method for plasma patterning biomaterial coatings to register neurons and promote outgrowth between compartments. Minimalistic neuronal co-cultures were prepared with high-level (&#62;85%) intercompartment connectivity and can be used for high throughput neurobiology experiments with single cell precision.

Protocols for single neuron microfluidic arraying and water masking for the in-chip plasma patterning of biomaterial coatings are described. Highly interconnected co-cultures can be prepared using minimal cell inputs.

神经元共培养与单细胞精度的制备

Microfluidic embodiments of the Campenot chamber have attracted great interest from the neuroscience community. These interconnected co-culture platforms ...

Simultaneous Detection of c-Fos Activation from Mesolimbic and Mesocortical Dopamine Reward Sites Following Naive Sugar and Fat Ingestion in Rats

This study uses cellular c-fos activation to assess effects of novel ingestion of fat and sugar on brain dopamine (DA) pathways in rats. Intakes of sugars and fats are mediated by their innate attractions as well as learned preferences. Brain dopamine, especially meso-limbic and meso-cortical projections from the ventral tegmental area (VTA), has been implicated in both of these unlearned and learned responses. The concept of distributed brain networks, wherein several sites and transmitter/peptide systems interact, has been proposed to mediate palatable food intake, but there is limited evidence empirically demonstrating such actions. Thus, sugar intake elicits DA release and increases c-fos-like immunoreactivity (FLI) from individual VTA DA projection zones including the nucleus accumbens (NAC), amygdala (AMY) and medial prefrontal cortex (mPFC) as well as the dorsal striatum. Further, central administration of selective DA receptor antagonists into these sites differentially reduce acquisition and expression of conditioned flavor preferences elicited by sugars or fats. One approach by which to determine whether these sites interacted as a distributed brain network in response to sugar or fat intake would be to simultaneous evaluate whether the VTA and its major mesotelencephalic DA projection zones (prelimbic and infralimbic mPFC, core and shell of the NAc, basolateral and central-cortico-medial AMY) as well as the dorsal striatum would display coordinated and simultaneous FLI activation after oral, unconditioned intake of corn oil (3.5%), glucose (8%), fructose (8%) and saccharin (0.2%) solutions. This approach is a successful first step in identifying the feasibility of using cellular c-fos activation simultaneously across relevant brain sites to study reward-related learning in ingestion of palatable food in rodents.

The goal of this study is to identify reward-related distributed brain networks by delineating a reliable immunohistological technique using cellular c-fos activation to measure simultaneous changes in dopamine pathways and terminal sites after novel ingestion of fat and sugar in rats.

从脑边缘和Mesocortical多巴胺奖赏网站继朴素的糖和脂肪摄入大鼠c-fos激活的同时检测

This study uses cellular c-fos activation to assess effects of novel ingestion of fat and sugar on brain dopamine (DA) pathways in rats. Intakes of sugars ...

Electrophysiological Method for Whole-cell Voltage Clamp Recordings from Drosophila Photoreceptors

Whole-cell voltage clamp recordings from Drosophila melanogaster photoreceptors have revolutionized the field of invertebrate visual transduction, enabling the use of D. melanogaster molecular genetics to study inositol-lipid signaling and Transient Receptor Potential (TRP) channels at the single-molecule level. A handful of labs have mastered this powerful technique, which enables the analysis of the physiological responses to light under highly controlled conditions. This technique allows control over the intracellular and extracellular media; the membrane voltage; and the fast application of pharmacological compounds, such as a variety of ionic or pH indicators, to the intra- and extracellular media. With an exceptionally high signal-to-noise ratio, this method enables the measurement of dark spontaneous and light-induced unitary currents (i.e.&#160;spontaneous and quantum bumps) and macroscopic Light-induced Currents (LIC) from single D. melanogaster photoreceptors. This protocol outlines, in great detail, all the key steps necessary to perform this technique, which includes both electrophysiological and optical recordings. The fly retina dissection procedure for the attainment of intact and viable ex vivo isolated ommatidia in the bath chamber is described. The equipment needed to perform whole-cell and fluorescence imaging measurements are also detailed. Finally, the pitfalls in using this delicate preparation during extended experiments are explained.

Electrophysiological Method for Whole-cell Voltage Clamp Recordings from Drosophila Photoreceptors

Whole-cell recordings from Drosophila melanogaster photoreceptors enable the measurement of spontaneous dark bumps, quantum bumps, macroscopic responses to light, and current-voltage relationships under various conditions. In combination with D. melanogaster genetic manipulation tools, this method enables the study of the ubiquitous inositol-lipid signaling pathway and its target, the TRP channel.

全细胞电压钳记录的电生理方法<em&gt;果蝇</em&gt;光感受器

Whole-cell voltage clamp recordings from Drosophila melanogaster photoreceptors have revolutionized the field of invertebrate visual transduction, ...

Levator Auris Longus Preparation for Examination of Mammalian Neuromuscular Transmission Under Voltage Clamp Conditions

This protocol describes a technique to record synaptic transmission from the neuromuscular junction under current-clamp and voltage-clamp conditions. An ex vivo preparation of the levator auris longus (LAL) is used because it is a thin muscle that provides easy visualization of the neuromuscular junction for microelectrode impalement at the motor endplate. This method allows for the recording of spontaneous miniature endplate potentials and currents (mEPPs and mEPCs), nerve-evoked endplate potentials and currents (EPPs and EPCs), as well as the membrane properties of the motor endplate. Results obtained from this method include the quantal content (QC), number of vesicle release sites (n), probability of vesicle release (prel), synaptic facilitation and depression, as well as the muscle membrane time constant (&#964;m) and input resistance. Application of this technique to mouse models of human disease can highlight key pathologies in disease states and help identify novel treatment strategies. By fully voltage-clamping a single synapse, this method provides one of the most detailed analyses of synaptic transmission currently available.

Levator Auris Longus Preparation for Examination of Mammalian Neuromuscular Transmission Under Voltage Clamp Conditions

The protocol described in this paper uses the mouse levator auris longus (LAL) muscle to record spontaneous and nerve-evoked postsynaptic potentials (current-clamp) and currents (voltage-clamp) at the neuromuscular junction. Use of this technique can provide key insights into mechanisms of synaptic transmission under normal and disease conditions.

提耳伸肌压钳条件下哺乳动物神经肌肉传输的检查准备

This protocol describes a technique to record synaptic transmission from the neuromuscular junction under current-clamp and voltage-clamp conditions. An ...