نشكر هانس رينر أرض مستصلحة من البحر، مارتن فورمان ووكر جاكسون لقراءة المخطوطة بعناية. تلقى الكتاب التمويل الذي تم توفيره من قبل وزارة البحث MIWF الدولة Northrhine-WESTFALIA (SR)، وBMBF-Projekträger DLR التعاون بين الولايات المتحدة والألمانية في علم الأعصاب الحسابية (CRCNS؛ SR)، مراكز التميز في أمراض الاعصاب (COEN؛ SR)، وجامعة بون برنامج تمويل جماعية (BONFOR؛ SR).

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يعلن الكتاب أنه ليس لديهم مصالح مالية المتنافسة.

هنا نجد تفسيرا لكيفية تطبيق سريع الصغرى الرحلان الشاردي من الناقلات العصبية للتحقيق التكامل متشابك على التشعبات. وقد استخدمت هذه التقنية بنجاح لتحقيق الجلوتاميرجي وGABAergic انتقال متشابك في مناطق الدماغ المختلفة في المختبر والمجراة 9،20-22. وقد استخدم الصغرى ...

الخلايا العصبية في الجهاز العصبي المركزي الحصول على مجموعة متنوعة من المدخلات متشابك على عملياتها شجيري رقيقة ومتشعبة 1. هناك، يتم بوساطة الغالبية العظمى من المدخلات شجيري مثير عن طريق نقاط الاشتباك العصبي الجلوتاميرجي. يمكن تنشيط هذه نقاط الاشتباك العصبي بطريقة توزيعها مكانيا، مما أدى إلى التكامل الخطي بعد المشبكي من إمكانات بعد المشبكي مثير (EPSPs). إذا تم تنشيط نقاط الاشتباك العصبي في وقت واحد وعلى مقربة المكاني على التغصنات، هذه المدخلات مثير يمكن أن تكون متكاملة فوق خطيا وتوليد المسامير شجيري 2-5. وعلاوة على ذلك، ودمج المدخلات مثير يعتمد على موقع للمدخلات على الشجرة شجيري. الإشارات التي تصل في المنطقة خصل البعيدة هي أكثر من ذلك بكثير الموهن من المدخلات الداني بسبب كابل تصفية 6. في قرن آمون، يتم إنشاء مدخلات بعيدة إلى التشعبات قمية خصل من منطقة في الدماغ تختلف عن تلك في dendri الدانيTES 7. بالتالي مسألة إثارة هو، كيف تتم معالجة المدخلات متشابك من قبل مقصورات شجيري مختلفة، وإذا تكامل شجيري ينظم تأثير هذه المدخلات الطبقات على إطلاق الخلايا العصبية بطرق مختلفة. ليس فقط الخصائص الفنية والسمات الشكلية للالتغصنات، ومكان وتجميع المدخلات التي تؤثر على التكامل شجيري من المدخلات مثير، أيضا المدخلات المثبطة إضافية من محطات GABAergic حاسم تحديد مدى فعالية من نقاط الاشتباك العصبي الجلوتاميرجي 8،9. هذه الجوانب المختلفة من التكامل متشابك يمكن التحقيق بشكل مثالي باستخدام ناقل عصبي الصغرى الرحلان الشاردي، والذي يسمح تطبيق تعريف مكانيا من الناقلات العصبية المختلفة إلى مجالات شجيري. علينا أن نظهر هنا كيفية تأسيس-الرحلان الشاردي الصغيرة من الغلوتامات وGABA بنجاح إلى تحقيق التكامل إشارة في الخلايا العصبية. لهذا التطبيق، ودفع غرامة ذات الرؤوسوتستخدم ماصات مقاومة عالية مليئة حلول العصبي المركزة. يتم وضع هذه الماصات بالقرب من الغشاء الخارجي للخلية، حيث توجد مستقبلات الناقل العصبي. مطلوب التصور الجيد للفروع شجيري. ويتحقق ذلك باستخدام أفضل الأصباغ الفلورية، والتي يتم تقديمها من خلال ماصة التصحيح. ثم (&lt;1 ميللي ثانية) نبض الحالية قصيرة جدا (في نطاق 10-100 غ) يستخدم لإخراج جزيئات الناقل العصبي مشحونة. مع هذه النبضات القصيرة والتعويض السعة فعالة وإمكانات بعد المشبكي أو التيارات يمكن أثار مع ارتفاع الزمانية والمكانية الدقة، مما يعني أن الدولة من المدخلات مثير هو معروف على وجه التحديد. الغلوتامات الصغرى الرحلان الشاردي يمكن تنشيط نقاط الاشتباك العصبي في دائرة نصف قطرها محدد، وهو أصغر من 6 ميكرون كما هو موضح هنا (الشكل 1 9)، ولكن من الممكن أيضا للوصول إلى قرار المشبك واحدة 10-12. هاينه، M.، وآخرون </eوأظهرت M&gt; أن القرار المكاني للسريع الصغرى الرحلان الشاردي حتى يمكن تعديلها على الفور أحجام أقل من 0.5 ميكرون، والتي هي أصغر من حجم بقعة يتحقق بانتظام مع uncaging اثنين من الفوتون من الغلوتامات 13. مع السرعة في الدقيقة الرحلان الشاردي فمن الممكن بسهولة لاستخدام اثنين أو أكثر الماصات iontophoretic ووضعها في اماكن مختلفة، وحتى البعيدة على الشجرة شجيري. في هذه الطريقة، وإدماج الأحداث مثير، بما في ذلك تلك من مسارات مختلفة، ويمكن التحقيق فيها. ومن الممكن أيضا استخدام الغلوتامات وGABA ماصة iontophoretic مليئة في نفس الوقت. وبهذه الطريقة يمكن دراسة تأثير تثبيط GABAergic في مواقع مختلفة نسبة إلى المدخلات مثير (على الطريق، وتثبيط خارج المسار). أيضا، وتأثير تثبيط من قبل interneurons استهداف المجالات العصبية محددة، مثل التشعبات البعيدة، سوما أو محاور 14، يمكن التحقيق باستخدام الرحلان الشاردي GABA. في الخلايا العصبية مثقف، وسرعة متناهية الصغر الرحلان الشاردي يتيح الفرصة لإنفيstigate توزيع المشبك واحد والجوانب الابتدائية الاتصالات متشابك في الخلايا العصبية في أكثر بكثير من التفصيل 10،11. في هذه المقالة ونحن لشرح بالتفصيل كيفية تأسيس الرحلان الشاردي الغلوتامات وGABA للاستخدام في شرائح الدماغ الحاد، والذي يسمح بالتحقيق التكامل متشابك من المدخلات مثير والمثبطة في الاعتماد من الموقع المدخلات، ونقاط القوة المدخلات، وتوقيت، وحده أو في التفاعل. ونشير الى المزايا والقيود المفروضة على هذه التقنية وكيفية استكشاف بنجاح.

<table border="1">
	<tbody>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Material</td>
		</tr>
		<tr>
			<td>Two-photon laser scanning microscope (TRIM Scope II), and Ultima IV, Prairie Technologies, Middleton, Wisconsin)</td>
			<td>LaVision Biotec, Bielefeld, Germany</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Two-photon laser scanning microscope Ultima IV</td>
			<td>Prairie Technologies, Middleton, Wisconsin, USA</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Ti:Sapphire ultrafast-pulsed laser</td>
			<td>Chameleon Ultra II, Coherent</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>60X Objective, NA 0.9</td>
			<td>Olympus</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Zeiss Axioskop 2 FS upright microscope</td>
			<td>TILLPhotonics, Gr&auml;felfing, Germany</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Monochromator</td>
			<td>TILLPhotonics, Gr&auml;felfing, Germany</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Micro-iontophoresis system MVCS-02</td>
			<td>NPI Electronics, Tamm, Germany</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sutter puller P-97</td>
			<td>Sutter Instrument Company, Novato, CA</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Glass filaments (150 GB F 8P)</td>
			<td>Science Products, Hofheim, Germany</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Reagent</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 hydrazide</td>
			<td>Molecular Probes life technologies</td>
			<td>A-10436</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Alexa Fluor 594</td>
			<td>Molecular Probes life technologies</td>
			<td>A-10438</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma Aldrich</td>
			<td>S7653</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma Aldrich</td>
			<td>P9333</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>NaH2PO4</td>
			<td>Sigma Aldrich</td>
			<td>S8282</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Sigma Aldrich</td>
			<td>S6297</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma Aldrich</td>
			<td>S7903</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Sigma Aldrich</td>
			<td>C5080</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma Aldrich</td>
			<td>M2670</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma Aldrich</td>
			<td>G7528</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>K-Gluconate</td>
			<td>Sigma Aldrich</td>
			<td>G4500</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>HEPES-acid</td>
			<td>Sigma Aldrich</td>
			<td>H4034</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Phosphocreatin</td>
			<td>Sigma Aldrich</td>
			<td>P7936</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Sigma Aldrich</td>
			<td>E3889</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Glutamic acid</td>
			<td>Sigma Aldrich</td>
			<td>G8415</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>GABA</td>
			<td>Sigma Aldrich</td>
			<td>A5835</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>Merck</td>
			<td>1.09137.1000</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>HCl</td>
			<td>Merck</td>
			<td>1.09108.1000</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

fast micro-iontophoresis of glutamate and gaba: a useful tool to investigate synaptic integration

واحدة من الاهتمامات الأساسية في علم الأعصاب هو فهم التكامل من المدخلات مثير والمثبطة على طول بنية معقدة جدا من شجرة شجيري، الأمر الذي يؤدي في نهاية المطاف إلى إنتاج الخلايا العصبية من إمكانات العمل في محور عصبي. تأثير العوامل المكانية والزمانية متنوعة من المدخلات متشابك محددة بشأن الانتاج العصبية هي حاليا قيد التحقيق، على سبيل المثال التوهين التي تعتمد على المسافة من المدخلات شجيري، والتفاعل الموقع التي تعتمد على مدخلات من مكانيا منفصلة، ​​وتأثير GABAergig تثبيط على التكامل مثير، وخطي وسائط التكامل غير الخطية، وغيرها الكثير. مع السرعة في الدقيقة الرحلان الشاردي من الغلوتامات وGABA فمن الممكن للتحقيق بدقة التكامل المكاني والزماني للإثارة وتثبيط مستقبلات GABAergic. المتطلبات الفنية الحرجة هي إما مصباح الفلورسنت أثار، ديود باعث للضوء (LED)، أو هيئة السلع التموينية اثنين من الفوتونnning المجهر لتصور فروع شجيري دون إدخال هامة الصور ضرر في الأنسجة. وعلاوة على ذلك، فمن المهم جدا أن يكون مكبر للصوت الرحلان الشاردي الصغيرة التي تسمح للحصول على تعويض السعة بسرعة من ماصات مقاومة عالية. نقطة أخرى مهمة هي أن لا الارسال يتم تحريرها كرها بواسطة ماصة أثناء التجربة. وبمجرد إنشاء، وهذه تقنية تعطي إشارات موثوقة وقابلة للتكرار مع ناقل عصبي عالية وخصوصية المكان. مقارنة الغلوتامات وGABA uncaging، الرحلان الشاردي سريع يسمح باستخدام كل أجهزة الإرسال في نفس الوقت ولكن في أماكن بعيدة جدا دون تحديد لمجال الرؤية. وهناك أيضا مزايا مقارنة التحفيز الكهربائي التنسيق من المحاور: مع الدقيقة الرحلان الشاردي الموقع من موقع الإدخال هو معروف بالتأكيد، وأنه على يقين من أن فقط الناقل العصبي من الفائدة يتم تحريرها. ومع ذلك فإنه لابد من النظر أنه مع الدقيقة الرحلان الشاردي فقطيتم تنشيط وpostsynapse لم يتم حل الجوانب قبل المشبكي من الافراج عن العصبي. في هذه المقالة ونحن لشرح كيفية إعداد الصغرى الرحلان الشاردي في التجارب شريحة الدماغ.

مقاربة بسيطة لتحديد مدى انتشار المكاني للالرحلان الشاردي هو التراجع عن ماصة iontophoretic متدرج من التغصنات، مع الحفاظ على الغلوتامات طرد مستمر. وجدنا أن مدى المكاني للتحفيز-iontophoretic الصغرى كان يبلغ قطرها حوالي 12 ميكرون (الشكل 1 يبين دائرة نصف قطرها). مدى عمق في أنس?...

Watch this Scientific Journal Video about Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration at JoVE.com

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

في هذه المقالة ونحن نقدم سريع الصغرى الرحلان الشاردي من الناقلات العصبية كأسلوب للتحقيق التكامل من إشارات بعد المشبكي مع عالية الدقة المكانية والزمانية.

سريع الصغرى، الرحلان الشاردي من الغلوتامات وGABA: أداة مفيدة لبحث تكامل متشابك

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

fast-micro-iontophoresis-glutamate-gaba-useful-tool-to-investigate

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Neuroscience

19.4K Views.  University of Bonn. في هذه المقالة ونحن نقدم سريع الصغرى الرحلان الشاردي من الناقلات العصبية كأسلوب للتحقيق التكامل من إشارات بعد المشبكي مع عالية الدقة المكانية والزمانية.

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

Cut-loading: A Useful Tool for Examining the Extent of Gap Junction Tracer Coupling Between Retinal Neurons

In addition to chemical synaptic transmission, neurons that are connected by gap junctions can also communicate rapidly via electrical synaptic transmission. Increasing evidence indicates that gap junctions not only permit electrical current flow and synchronous activity between interconnected or coupled cells, but that the strength or effectiveness of electrical communication between coupled cells can be modulated to a great extent1,2. In addition, the large internal diameter (~1.2 nm) of many gap junction channels permits not only electric current flow, but also the diffusion of intracellular signaling molecules and small metabolites between interconnected cells, so that gap junctions may also mediate metabolic and chemical communication. The strength of gap junctional communication between neurons and its modulation by neurotransmitters and other factors can be studied by simultaneously electrically recording from coupled cells and by determining the extent of diffusion of tracer molecules, which are gap junction permeable, but not membrane permeable, following iontophoretic injection into single cells. However, these procedures can be extremely difficult to perform on neurons with small somata in intact neural tissue.
Numerous studies on electrical synapses and the modulation of electrical communication have been conducted in the vertebrate retina, since each of the five retinal neuron types is electrically connected by gap junctions3,4. Increasing evidence has shown that the circadian (24-hour) clock in the retina and changes in light stimulation regulate gap junction coupling3-8. For example, recent work has demonstrated that the retinal circadian clock decreases gap junction coupling between rod and cone photoreceptor cells during the day by increasing dopamine D2 receptor activation, and dramatically increases rod-cone coupling at night by reducing D2 receptor activation7,8. However, not only are these studies extremely difficult to perform on neurons with small somata in intact neural retinal tissue, but it can be difficult to adequately control the illumination conditions during the electrophysiological study of single retinal neurons to avoid light-induced changes in gap junction conductance.
Here, we present a straightforward method of determining the extent of gap junction tracer coupling between retinal neurons under different illumination conditions and at different times of the day and night. This cut-loading technique is a modification of scrape loading9-12, which is based on dye loading and diffusion through open gap junction channels. Scrape loading works well in cultured cells, but not in thick slices such as intact retinas. The cut-loading technique has been used to study photoreceptor coupling in intact fish and mammalian retinas7, 8,13, and can be used to study coupling between other retinal neurons, as described here.

An easy and convenient method to determine the extent of gap junction tracer coupling between retinal neurons is described. This technique enables one to investigate the function of the electrical synapses between neurons in the intact retina under different illumination conditions and at different times of the day and night.

خفض التحميل : أداة مفيدة لفحص مدى تقاطع الفجوة بين الخلايا العصبية توصيلات الراسم الشبكية

In addition to chemical synaptic transmission, neurons that are connected by gap junctions can also communicate rapidly via electrical synaptic ...

MPI CyberMotion Simulator: Implementation of a Novel Motion Simulator to Investigate Multisensory Path Integration in Three Dimensions

Path integration is a process in which self-motion is integrated over time to obtain an estimate of one's current position relative to a starting point 1. Humans can do path integration based exclusively on visual 2-3, auditory 4, or inertial cues 5. However, with multiple cues present, inertial cues - particularly kinaesthetic - seem to dominate 6-7. In the absence of vision, humans tend to overestimate short distances (&lt;5 m) and turning angles (&lt;30&deg;), but underestimate longer ones 5. Movement through physical space therefore does not seem to be accurately represented by the brain.
Extensive work has been done on evaluating path integration in the horizontal plane, but little is known about vertical movement (see 3 for virtual movement from vision alone). One reason for this is that traditional motion simulators have a small range of motion restricted mainly to the horizontal plane. Here we take advantage of a motion simulator 8-9 with a large range of motion to assess whether path integration is similar between horizontal and vertical planes. The relative contributions of inertial and visual cues for path navigation were also assessed.
16 observers sat upright in a seat mounted to the flange of a modified KUKA anthropomorphic robot arm. Sensory information was manipulated by providing visual (optic flow, limited lifetime star field), vestibular-kinaesthetic (passive self motion with eyes closed), or visual and vestibular-kinaesthetic motion cues. Movement trajectories in the horizontal, sagittal and frontal planes consisted of two segment lengths (1st: 0.4 m, 2nd: 1 m; &plusmn;0.24 m/s2 peak acceleration). The angle of the two segments was either 45&deg; or 90&deg;. Observers pointed back to their origin by moving an arrow that was superimposed on an avatar presented on the screen.
Observers were more likely to underestimate angle size for movement in the horizontal plane compared to the vertical planes. In the frontal plane observers were more likely to overestimate angle size while there was no such bias in the sagittal plane. Finally, observers responded slower when answering based on vestibular-kinaesthetic information alone. Human path integration based on vestibular-kinaesthetic information alone thus takes longer than when visual information is present. That pointing is consistent with underestimating and overestimating the angle one has moved through in the horizontal and vertical planes respectively, suggests that the neural representation of self-motion through space is non-symmetrical which may relate to the fact that humans experience movement mostly within the horizontal plane.

An efficient way to gain insight into how humans navigate themselves in three dimensions is described. The method takes advantage of a motion simulator capable of moving observers in ways unattainable by traditional simulators. Results confirm that movement in the horizontal plane is underestimated, while vertical movement is overestimated.

MPI سيبرموتيون محاكي: تنفيذ جهاز محاكاة الحركة الرواية المعنية بالتحقيق في مسار التكامل متعدد الحواس في ثلاثة أبعاد

Path integration is a process in which self-motion is integrated over time to obtain an estimate of one's current position relative to a starting point 1. ...

Automated Quantification of Synaptic Fluorescence in C. elegans

Synapse strength refers to the amplitude of postsynaptic responses to presynaptic neurotransmitter release events, and has a major impact on overall neural circuit function. Synapse strength critically depends on the abundance of neurotransmitter receptors clustered at synaptic sites on the postsynaptic membrane. Receptor levels are established developmentally, and can be altered by receptor trafficking between surface-localized, subsynaptic, and intracellular pools, representing important mechanisms of synaptic plasticity and neuromodulation. Rigorous methods to quantify synaptically-localized neurotransmitter receptor abundance are essential to study synaptic development and plasticity. Fluorescence microscopy is an optimal approach because it preserves spatial information, distinguishing synaptic from non-synaptic pools, and discriminating among receptor populations localized to different types of synapses. The genetic model organism Caenorhabditis elegans is particularly well suited for these studies due to the small size and relative simplicity of its nervous system, its transparency, and the availability of powerful genetic techniques, allowing examination of native synapses in intact animals.
Here we present a method for quantifying fluorescently-labeled synaptic neurotransmitter receptors in C. elegans. Its key feature is the automated identification and analysis of individual synapses in three dimensions in multi-plane confocal microscope output files, tabulating position, volume, fluorescence intensity, and total fluorescence for each synapse. This approach has two principal advantages over manual analysis of z-plane projections of confocal data. First, because every plane of the confocal data set is included, no data are lost through z-plane projection, typically based on pixel intensity averages or maxima. Second, identification of synapses is automated, but can be inspected by the experimenter as the data analysis proceeds, allowing fast and accurate extraction of data from large numbers of synapses. Hundreds to thousands of synapses per sample can easily be obtained, producing large data sets to maximize statistical power. Considerations for preparing C. elegans for analysis, and performing confocal imaging to minimize variability between animals within treatment groups are also discussed. Although developed to analyze C. elegans postsynaptic receptors, this method is generally useful for any type of synaptically-localized protein, or indeed, any fluorescence signal that is localized to discrete clusters, puncta, or organelles.
The procedure is performed in three steps: 1) preparation of samples, 2) confocal imaging, and 3) image analysis. Steps 1 and 2 are specific to C. elegans, while step 3 is generally applicable to any punctate fluorescence signal in confocal micrographs.

Automated Quantification of Synaptic Fluorescence in C. elegans

The abundance of neurotransmitter receptors clustered at synapses strongly influences synaptic strength. This method quantifies fluorescently-labeled neurotransmitter receptors in three dimensions with single-synapse resolution in C. elegans, allowing hundreds of synapses to be rapidly characterized within a single sample without distortions introduced by z-plane projection.

الآلي الكمي من الإسفار متشابك في<em&gt; C. ايليجانس</em

Synapse strength refers to the amplitude of postsynaptic responses to presynaptic neurotransmitter release events, and has a major impact on overall ...

Deriving the Time Course of Glutamate Clearance with a Deconvolution Analysis of Astrocytic Transporter Currents

The highest density of glutamate transporters in the brain is found in astrocytes. Glutamate transporters couple the movement of glutamate across the membrane with the co-transport of 3 Na+ and 1 H+ and the counter-transport of 1 K+. The stoichiometric current generated by the transport process can be monitored with whole-cell patch-clamp recordings from astrocytes. The time course of the recorded current is shaped by the time course of the glutamate concentration profile to which astrocytes are exposed, the kinetics of glutamate transporters, and the passive electrotonic properties of astrocytic membranes. Here we describe the experimental and analytical methods that can be used to record glutamate transporter currents in astrocytes and isolate the time course of glutamate clearance from all other factors that shape the waveform of astrocytic transporter currents. The methods described here can be used to estimate the lifetime of flash-uncaged and synaptically-released glutamate at astrocytic membranes in any region of the central nervous system during health and disease.

We describe an analytical method to estimate the lifetime of glutamate at astrocytic membranes from electrophysiological recordings of glutamate transporter currents in astrocytes.

اشتقاق مدار الساعة من تخليص الغلوتامات مع تحليل Deconvolution من التيارات الناقل نجمي

The highest density of glutamate transporters in the brain is found in astrocytes. Glutamate transporters couple the movement of glutamate across the ...

Using an &#945;-Bungarotoxin Binding Site Tag to Study GABA A Receptor Membrane Localization and Trafficking

It is increasingly evident that neurotransmitter receptors, including ionotropic GABA A receptors (GABAAR), exhibit highly dynamic trafficking and cell surface mobility1-7. To study receptor cell surface localization and endocytosis, the technique described here combines the use of fluorescent &#945;-bungarotoxin with cells expressing constructs containing an &#945;-bungarotoxin (Bgt) binding site (BBS). The BBS (WRYYESSLEPYPD) is based on the &#945; subunit of the muscle nicotinic acetylcholine receptor, which binds Bgt with high affinity8,9. Incorporation of the BBS site allows surface localization and measurements of receptor insertion or removal with application of exogenous fluorescent Bgt, as previously described in the tracking of GABAA and metabotropic GABAB receptors2,10. In addition to the BBS site, we inserted a pH-sensitive GFP (pHGFP11) between amino acids 4 and 5 of the mature GABAAR subunit by standard molecular biology and PCR cloning strategies (see Figure 1)12. The BBS is 3&#39; of the pH-sensitive GFP reporter, separated by a 13-amino acid alanine/proline linker. For trafficking studies described in this publication that are based on fixed samples, the pHGFP serves as a reporter of total tagged GABAAR subunit protein levels, allowing normalization of the Bgt labeled receptor population to total receptor population. This minimizes cell to cell Bgt staining signal variability resulting from higher or lower baseline expression of the tagged GABAAR subunits. Furthermore the pHGFP tag enables easy identification of construct expressing cells for live or fixed imaging experiments.

Using an &amp;#945;-Bungarotoxin Binding Site Tag to Study GABA A Receptor Membrane Localization and Trafficking

Here we demonstrate the use of fluorescent Alexa dye coupled to &#945;-bungarotoxin to measure GABA A receptor surface localization and endocytosis in hippocampal neurons. Through the use of constructs bearing a short extracellular tag that binds &#945;-bungarotoxin, analysis of plasma membrane protein endocytic trafficking can be achieved.

باستخدام α-بنغاروتوكسين تجليد الوسم الموقع لدراسة مستقبلات GABA A غشاء توطين والاتجار

It is increasingly evident that neurotransmitter receptors, including ionotropic GABA A receptors (GABAAR), exhibit highly dynamic trafficking and cell ...

Induction of an Isoelectric Brain State to Investigate the Impact of Endogenous Synaptic Activity on Neuronal Excitability In Vivo

The way neurons process information depends both on their intrinsic membrane properties and on the dynamics of the afferent synaptic network. In particular, endogenously-generated network activity, which strongly varies as a function of the state of vigilance, significantly modulates neuronal computation. To investigate how different spontaneous cerebral dynamics impact single neurons&#39; integrative properties, we developed a new experimental strategy in the rat consisting in suppressing in vivo all cerebral activity by means of a systemic injection of a high dose of sodium pentobarbital. Cortical activities, continuously monitored by combined electrocorticogram (ECoG) and intracellular recordings are progressively slowed down, leading to a steady isoelectric profile. This extreme brain state, putting the rat into a deep comatose, was carefully monitored by measuring the physiological constants of the animal throughout the experiments. Intracellular recordings allowed us to characterize and compare the integrative properties of the same neuron embedded into physiologically relevant cortical dynamics, such as those encountered in the sleep-wake cycle, and when the brain was fully silent.

Induction of an Isoelectric Brain State to Investigate the Impact of Endogenous Synaptic Activity on Neuronal Excitability In Vivo

This procedure performs long-lasting in vivo intracellular recordings from single neurons during physiologically relevant cerebral states and after complete abolition of ongoing electrical activities, resulting in an isoelectric brain state. The physiological constants of the animal are carefully monitored during the transition to the artificial comatose condition.

تحريض دولة ISOELECTRIC الدماغ المعنية بالتحقيق في أثر الذاتية متشابك آخر على العصبية استثارة<em&gt; في فيفو</em

The way neurons process information depends both on their intrinsic membrane properties and on the dynamics of the afferent synaptic network. In ...

Using Enzyme-based Biosensors to Measure Tonic and Phasic Glutamate in Alzheimer's Mouse Models

Neurotransmitter disruption is often a key component of diseases of the central nervous system (CNS), playing a role in the pathology underlying Alzheimer's disease, Parkinson's disease, depression, and anxiety. Traditionally, microdialysis has been the most common (lauded) technique to examine neurotransmitter changes that occur in these disorders. But because microdialysis has the ability to measure slow 1-20 minute changes across large areas of tissue, it has the disadvantage of invasiveness, potentially destroying intrinsic connections within the brain and a slow sampling capability. A relatively newer technique, the microelectrode array (MEA), has numerous advantages for measuring specific neurotransmitter changes within discrete brain regions as they occur, making for a spatially and temporally precise approach. In addition, using MEAs is minimally invasive, allowing for measurement of neurotransmitter alterations in vivo. In our laboratory, we have been specifically interested in changes in the neurotransmitter, glutamate, related to Alzheimer's disease pathology. As such, the method described here has been used to assess potential hippocampal disruptions in glutamate in a transgenic mouse model of Alzheimer's disease. Briefly, the method used involves coating a multi-site microelectrode with an enzyme very selective for the neurotransmitter of interest and using self-referencing sites to subtract out background noise and interferents. After plating and calibration, the MEA can be constructed with a micropipette and lowered into the brain region of interest using a stereotaxic device. Here, the method described involves anesthetizing rTg(TauP301L)4510 mice and using a stereotaxic device to precisely target sub-regions (DG, CA1, and CA3) of the hippocampus.

Using Enzyme-based Biosensors to Measure Tonic and Phasic Glutamate in Alzheimer&#039;s Mouse Models

Here, we describe the setup, software navigation, and data analysis for a spatially and temporally precise method of measuring tonic and phasic extracellular glutamate changes in vivo using enzyme-linked microelectrode arrays (MEA).

باستخدام أجهزة الاستشعار مقرها انزيم لقياس تونيك وطوري الغلوتامات في نماذج الماوس الزهايمر

Neurotransmitter disruption is often a key component of diseases of the central nervous system (CNS), playing a role in the pathology underlying ...

Culturing In Vivo-like Murine Astrocytes Using the Fast, Simple, and Inexpensive AWESAM Protocol

The AWESAM (a low-cost easy stellate astrocyte method) protocol entails a fast, simple, and inexpensive way to generate large quantities of in vivo-like mouse and rat astrocyte monocultures: Brain cells can be isolated from different brain regions, and after a week of cell culture, non-astrocytic cells are shaken off by placing the culture dishes on a shaker for 6 h in the incubator. The remaining astrocytes are then passaged into new plates with an astrocyte-specific medium (termed NB+H). NB+H contains low concentrations of heparin-binding EGF-like growth factor (HBEGF), which is used in place of serum in medium. After growing in NB+H, AWESAM astrocytes have a stellate morphology and feature fine processes. Moreover, these astrocytes have more in vivo-like gene expression than astrocytes generated by previously published methods. Ca2+ imaging, vesicle dynamics, and other events close to the membrane can thus be studied in the fine astrocytic processes in vitro, e.g., using live cell confocal or TIRF microscopy. Notably, AWESAM astrocytes also exhibit spontaneous Ca2+ signaling similar to astrocytes in vivo.

Culturing In Vivo-like Murine Astrocytes Using the Fast, Simple, and Inexpensive AWESAM Protocol

The AWESAM protocol described here is optimal for culturing murine astrocytes in isolation from other brain cells in a fast, simple, and inexpensive manner. AWESAM astrocytes exhibit spontaneous Ca2+ signaling, morphology, and gene expression profiles similar to astrocytes in vivo.

استزراع في فيفو--مثل مورين Astrocytes استخدام بروتوكول أوسام سريعة وبسيطة وغير مكلفة

The AWESAM (a low-cost easy stellate astrocyte method) protocol entails a fast, simple, and inexpensive way to generate large quantities of in vivo-like ...

Combining Optogenetics with Artificial microRNAs to Characterize the Effects of Gene Knockdown on Presynaptic Function within Intact Neuronal Circuits

The purpose of this protocol is to characterize the effect of gene knockdown on presynaptic function within intact neuronal circuits. We describe a workflow on how to combine artificial microRNA (miR)-mediated RNA interference with optogenetics to achieve selective stimulation of manipulated presynaptic boutons in acute brain slices. The experimental approach involves the use of a single viral construct and a single neuron-specific promoter to drive the expression of both an optogenetic probe and artificial miR(s) against presynaptic gene(s). When stereotactically injected in the brain region of interest, the expressed construct makes it possible to stimulate with light exclusively the neurons with reduced expression of the gene(s) under investigation. This strategy does not require the development and maintenance of genetically modified mouse lines and can in principle be applied to other organisms and to any neuronal gene of choice. We have recently applied it to investigate how the knockdown of alternative splice isoforms of presynaptic P/Q-type voltage-gated calcium channels (VGCCs) regulates short-term synaptic plasticity at CA3 to CA1 excitatory synapses in acute hippocampal slices. A similar approach could also be used to manipulate and probe the neuronal circuitry in vivo.

This protocol provides a workflow on how to combine artificial microRNA-mediated RNA interference with optogenetics to stimulate specifically presynaptic boutons with reduced expression of selective gene(s) within intact neuronal circuits.

الجمع بين أوبتوجينيتيكس ميكرورناس الاصطناعية لتوصيف "تأثيرات الجينات ضربة قاضية" في وظيفة Presynaptic داخل "الدوائر العصبية سليمة"

The purpose of this protocol is to characterize the effect of gene knockdown on presynaptic function within intact neuronal circuits. We describe a ...

A High-throughput Calcium-flux Assay to Study NMDA-receptors with Sensitivity to Glycine/D-serine and Glutamate

N-methyl-D-aspartate (NMDA) receptors (NMDAR) are classified as ionotropic glutamate receptors and have critical roles in learning and memory. NMDAR malfunction, expressed as either over- or under-activity caused by mutations, altered expression, trafficking, or localization, can contribute to numerous diseases, especially in the central nervous system. Therefore, understanding the receptor&#39;s biology as well as facilitating the discovery of compounds and small molecules is crucial in ongoing efforts to combat neurological diseases. Current approaches to studying the receptor have limitations including low throughput, high cost, and the inability to study its functional abilities due to the necessary presence of channel blockers to prevent NMDAR-mediated excitotoxicity. Additionally, the existing assay systems are sensitive to stimulation by glutamate only and lack sensitivity to stimulation by glycine, the other co-ligand of the NMDAR. Here, we present the first plate-based assay with high-throughput power to study an NMDA receptor with sensitivity to both co-ligands, glutamate and D-serine/glycine. This approach allows the study of different NMDAR subunit compositions and allows functional studies of the receptor in glycine- and/or glutamate-sensitive modes. Additionally, the method does not require the presence of inhibitors during measurements. The effects of positive and negative allosteric modulators can be detected with this assay and the known pharmacology of NMDAR has been replicated in our system. This technique overcomes the limitations of existing methods and is cost-effective. We believe that this novel technique will accelerate the discovery of therapies for NMDAR-mediated pathologies.

The goal of this protocol is to facilitate the study of NMDA-receptors (NMDAR) at a larger scale and allow the examination of modulatory effects of small molecules and their therapeutic applications.

مقايسة الفائق كالسيوم-التمويه لدراسة مستقبلات NMDA مع حساسية جليكاين/د-سيرين وغلوتامات

N-methyl-D-aspartate (NMDA) receptors (NMDAR) are classified as ionotropic glutamate receptors and have critical roles in learning and memory. NMDAR ...