وقد تم تمويل هذا العمل من قبل المعاهد الوطنية للصحة منح المجلس الاعلى للقضاة وEY005102 لEY018640 إلى CPR

<ol>
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I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+coupling+between+retinal+horizontal+cells+by+retinoic+acid+and+endogenous+dopamine.">Modulation of coupling between retinal horizontal cells by retinoic acid and endogenous dopamine.</a> Brain. Res. Brain. Res. Rev. 32, 121-129 (2000).</li><li>Xin, D., Bloomfield, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+nitric+oxide+on+horizontal+cells+in+the+rabbit+retina.">Effects of nitric oxide on horizontal cells in the rabbit retina.</a> Vis. Neurosci. 17, 799-811 (2000).</li><li>Ribelayga, C., Cao, Y., Mangel, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+circadian+clock+in+the+retina+controls+rod-cone+coupling.">The circadian clock in the retina controls rod-cone coupling.</a> Neuron. 59, 790-801 (2008).</li><li>Ribelayga, C., Mangel, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+circadian+clock-controlled+neural+pathway+in+the+rabbit+retina.">Identification of a circadian clock-controlled neural pathway in the rabbit retina.</a> PLoS One. 5, e11020-e11020 (2010).</li><li>Ouyang, X., Winbow, V. M., Patel, L. S., Burr, G. S., Mitchell, C. K., O'Brien, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+Kinase+A+mediates+regulation+of+gap+junctions+containing+connexin35+through+a+complex+pathway.">Protein Kinase A mediates regulation of gap junctions containing connexin35 through a complex pathway.</a> Brain. Res. Mol. Brain. Res. 135, 1-11 (2005).</li><li>Patel, L. S., Mitchell, C. K., Dubinsky, W. P., O'Brien, J. <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+gap+junction+coupling+through+the+neuronal+connexin+Cx35+by+nitric+oxide+and+cGMP.">Regulation of gap junction coupling through the neuronal connexin Cx35 by nitric oxide and cGMP.</a> Cell. Commun. Adhes. 13, 41-54 (2006).</li><li>Peters, J. L., Cassone, V. M., Zoran, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Melatonin+modulates+intercellular+communication+among+cultured+chick+astrocytes.">Melatonin modulates intercellular communication among cultured chick astrocytes.</a> Brain. Res. 1031, 10-19 (2005).</li><li>Cusato, K., Bosco, A., Rozental, R., Guimar&#227;es, C. A., Reese, B. E., Linden, R., Spray, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gap+junctions+mediate+bystander+cell+death+in+developing+retina.">Gap junctions mediate bystander cell death in developing retina.</a> J. Neurosci. 23, 6413-6422 (2003).</li><li>Li, H., Chuang, A. Z., O'Brien, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photoreceptor+coupling+is+controlled+by+connexin+35+phosphorylation+in+zebrafish+retina.">Photoreceptor coupling is controlled by connexin 35 phosphorylation in zebrafish retina.</a> J. Neurosci. 29, 15178-15186 (2009).</li><li>Mills, S. L., Massey, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+kinetics+of+tracer+movement+through+homologous+gap+junctions+in+the+rabbit+retina.">The kinetics of tracer movement through homologous gap junctions in the rabbit retina.</a> Vis. Neurosci. 15, 765-777 (1998).</li><li>Mills, S. L., Massey, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+series+of+biotinylated+tracers+distinguishes+three+types+of+gap+junction+in+retina.">A series of biotinylated tracers distinguishes three types of gap junction in retina.</a> J. Neurosci. 20, 8629-8636 (2000).</li></ol>

الإعلان عن أي تضارب في المصالح.

طريقة قص صفها التحميل هنا هو تقنية مفيدة وصريحة لتحديد مدى اقتران تقاطع الفجوة بين الخلايا العصبية في شبكية العين الكاشفة في ظل ظروف إضاءة مختلفة وفي أوقات مختلفة من النهار والليل. مزايا هذه التقنية تشمل القدرة على قياس مدى تقاطع الفجوة بين الخلايا العصبية اقتران التتبع في أنسجة الشبكية سليمة في ظل مجموعة متنوعة من ظروف الإضاءة خلال النهار والليل والقيام بذلك عن جانب الخلايا العصبية التي somata صغيرة القطر. القيود المفروضة على هذه التقنية في دراسة تقاطعات الفجوة في خريف أنسجة سليمة إلى فئتين عامتين. أولا ، قد نشر من خلال التتبع تقاطعات الفجوة المفتوحة من الصعب نسبيا لمراقبة نظرا لقطرها) صغير من التهمة أو المرتبطة مع القنوات المفتوحة وب) 1،14،15 الكميات النسبية للمقصورات الخلوية بالإضافة. وهذا هو ، نشر التتبع من خلية صغيرة إلى أكبر خلية أو مجموعة من الخلايا مقرونة ، شركاتحمراء مكتوب لنشر التتبع من أكبر أصغر خلية إلى خلية ، قد يكون أكثر صعوبة للكشف بسبب تخفيف التتبع. ثانيا ، في ظل بعض الظروف الفسيولوجية ، قد حد من الانتشار من خلال التتبع تقاطعات الفجوة في أنسجة سليمة لا تعكس بدقة قوة تصرف الفجوة صلي بسبب الاختلافات في تصرف الأيونات الصغيرة التي تحمل الكهربائية الحالية ، مقارنة مع نفاذية التتبع كبيرة نسبيا جزيئات 1،14،15. بشكل عام ، دليل على اقتران التتبع يشير بقوة إلى وجود وأداء فتح قنوات تقاطع الفجوة ، ولكن تحت بعض الشروط الفسيولوجية ، والتتبع نشرها أو قد لا يحدث لاحظ أن على الرغم من التسجيلات الكهربية توحي وجود وأداء تقاطعات الفجوة المفتوحة. يبدو من المرجح أنه يمكن أيضا خفض تقنية التحميل يمكن استخدامها لتحقيق في مدى اقتران التتبع تقاطع الفجوة بين الخلايا العصبية في أنسجة سليمة من مناطق أخرى من أنظمة العصبي المركزيTEM.

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> اسم الكاشف </td><td> شركة </td><td> فهرس العدد </td><td> تعليق (اختياري) </td></tr><tr><td> Tricaine سلفونات الميثان (MS222) </td><td> سيغما الدريخ </td><td> A5040 </td><td> 150 ملغم / لتر من الماء مخزنة الأسماك دبابات </td></tr><tr><td> يوريتان </td><td> سيغما الدريخ </td><td> U2500 </td><td> 2 ز / kgloading جرعة </td></tr><tr><td> أنبوب مزدوج للرؤية الليلية حملق </td><td> الولايات المتحدة الأمريكية ليلة بصريات </td><td> D - 221 </td><td></td></tr><tr><td> مرشح ورقة ، رقم الصف 4 </td><td> Whatman </td><td> 1004-090 </td><td></td></tr><tr><td> غرامة الملقط ، دومون رقم 5 ، البيولوجيا ، و 11 سنتيمترا </td><td> أدوات العلوم غرامة </td><td> 11295-10 </td></tr><tr><td> غرامة مقصات بنابض ، 8 مم شفرة ، على التوالي </td><td> أدوات العلوم غرامة </td><td> 15025-10 </td><td></td></tص&gt; <tr><td> Neurobiotin </td><td> النواقل </td><td> SP - 1120 </td><td> 0.5 ٪ </td></tr><tr><td> Streptavidin مترافق اليكسا - 488 </td><td> Invitrogen </td><td> S11223 </td><td> 2 ٪ </td></tr><tr><td> ديكستران رودامين (عالية (&gt; 10000) ميجاواط) </td><td> Invitrogen </td><td> D1817 </td><td> 0.5 ٪ </td></tr><tr><td> Vectashield تصاعد المتوسطة </td><td> النواقل </td><td> H - 1000 </td><td></td></tr><tr><td> زايس META ليزر متحد البؤر مجهر المسح الضوئي 510 </td><td> كارل زايس ، وشركة </td><td></td><td></td></tr><tr><td> LSM - 5 مستعرض الصور 3،2،0،115 </td><td> كارل زايس ، وشركة </td><td></td><td></td></tr><tr><td> ImageJ البرمجيات </td><td> المعاهد الوطنية للصحة </td><td></td><td></td></tr><tr><td> OriginPro 8.0 </td><td> OriginLab كورب </td><td></td><td></td></tr></tbody></table>

cut-loading: a useful tool for examining the extent of gap junction tracer coupling between retinal neurons

بالإضافة إلى انتقال متشابك الكيميائية ، يمكن أن الخلايا العصبية التي ترتبط بها تقاطعات الفجوة التواصل بسرعة أيضا عن طريق نقل متشابك الكهربائية. أدلة متزايدة تشير إلى أن الفجوة لا تقاطعات تصريح تدفق التيار الكهربائي والنشاط متزامن بين الخلايا المترابطة أو إضافة ، إلا أن قوة أو فعالية الاتصالات بين الخلايا الكهربائية مقرونة يمكن التضمين إلى 1،2 حد كبير. بالإضافة إلى ذلك ، وكبيرة قطرها الداخلي (~ 1،2 نانومتر) من العديد من القنوات تقاطع الفجوة التصاريح ليس فقط تدفق التيار الكهربائي ، ولكن أيضا انتشار الخلايا جزيئات صغيرة إشارات والأيضات بين الخلايا مترابطة ، بحيث تقاطعات الفجوة قد توسط أيضا الأيض الاتصالات والكيميائية . ويمكن دراسة مدى قوة الاتصالات بين الخلايا العصبية الفجوة صلي والتشكيل من خلال الموصلات العصبية وغيرها من العوامل التي كتبها في وقت واحد تسجيل كهربائيا من الخلايا وإلى جانب تحديد رانه مدى انتشار جزيئات التتبع ، والتي هي قابلة للاختراق تقاطع الفجوة ، ولكن ليس نفاذية الغشاء ، وبعد حقن الخلايا في iontophoretic واحد. ومع ذلك ، يمكن لهذه الإجراءات سيكون من الصعب للغاية القيام على الخلايا العصبية مع somata صغيرة في الأنسجة العصبية سليمة. وقد أجريت دراسات عديدة حول المشابك الكهربائية والتشكيل من الاتصالات الكهربائية في شبكية الفقاريات ، حيث تم توصيل بالكهرباء كل من الخلايا العصبية للشبكية خمسة أنواع من قبل تقاطعات الفجوة 3،4. وقد أظهرت أدلة متزايدة على أن الإيقاعية (24 ساعة) على مدار الساعة في الشبكية والتغييرات في ضوء التحفيز تنظيم تقاطع الفجوة اقتران 3-8. على سبيل المثال ، أظهرت مؤخرا أن العمل على مدار الساعة الإيقاعية الشبكية يقلل الفجوة بين مفترق طرق توصيل قضيب وخلايا مستقبلة للضوء مخروط خلال النهار عن طريق زيادة الدوبامين تنشيط مستقبلات D2 ، ويزيد بشكل كبير قضيب مخروط اقتران ليلا عن طريق الحد من تفعيل مستقبلات D2 <suف&gt; 7،8. ومع ذلك ، لا يقتصر على هذه الدراسات من الصعب للغاية تنفيذها على الخلايا العصبية مع somata صغيرة في الأنسجة العصبية للشبكية سليمة ، ولكن يمكن أن يكون من الصعب السيطرة عليها على نحو كاف ظروف الإضاءة أثناء الدراسة الكهربية من الخلايا العصبية للشبكية واحدة لتجنب الضوء الناجم عن التغيرات في مفترق الفجوة تصرف. هنا ، نقدم طريقة بسيطة لتحديد مدى اقتران تقاطع الفجوة بين الخلايا العصبية في شبكية العين الكاشفة في ظل ظروف إضاءة مختلفة وفي أوقات مختلفة من النهار والليل. هذه التقنية خفض التحميل هو تعديل لتحميل كشط 9-12 ، الذي يقوم على تحميل صباغة ونشرها من خلال فتح قنوات تقاطع الفجوة. تحميل كشط يعمل بشكل جيد في الخلايا المستزرعة ، ولكن ليس في شرائح سميكة مثل شبكية العين سليمة. وقد استخدمت هذه التقنية خفض التحميل لدراسة اقتران مبصرة في الأسماك سليمة وشبكية العين الثدييات 7 ، 8،13 ، ويمكن استخدامها لدراسة تقارن بينالخلايا العصبية للشبكية أخرى ، كما هو موضح هنا.

Watch this Scientific Journal Video about Cut-loading: A Useful Tool for Examining the Extent of Gap Junction Tracer Coupling Between Retinal Neurons at JoVE.com

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

ووصف طريقة سهلة ومريحة لتحديد مدى اقتران تقاطع الفجوة بين الخلايا العصبية في شبكية العين الكاشفة. هذه التقنية تمكن واحد لتحقيق وظيفة نقاط الاشتباك العصبي بين الخلايا العصبية الكهربائية في الشبكية سليمة في ظل ظروف إضاءة مختلفة وفي أوقات مختلفة من النهار والليل.

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 ...

cut-loading-useful-tool-for-examining-extent-gap-junction-tracer

Research

JoVE Journal

Neuroscience

15.3K Views.  Ohio State University College of Medicine. ووصف طريقة سهلة ومريحة لتحديد مدى اقتران تقاطع الفجوة بين الخلايا العصبية في شبكية العين الكاشفة. هذه التقنية تمكن واحد لتحقيق وظيفة نقاط الاشتباك العصبي بين الخلايا العصبية الكهربائية في الشبكية سليمة في ظل ظروف إضاءة مختلفة وفي أوقات مختلفة من النهار والليل....

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

Patch Clamp Recordings from Mouse Retinal Neurons in a Dark-adapted Slice Preparation

Our visual experience is initiated when the visual pigment in our retinal photoreceptors absorbs photons of light energy and initiates a cascade of intracellular events that lead to closure of cyclic-nucleotide-gated channels in the cell membrane. The resulting change in membrane potential leads in turn to reductions in the amount of neurotransmitter release from both rod and cone synaptic terminals. To measure how the light-evoked change in photoreceptor membrane potential leads to downstream activity in the retina, scientists have made electrophysiological recordings from retinal slice preparations for decades1,2. In the past these slices have been cut manually with a razor blade on retinal tissue that is attached to filter paper; in recent years another method of slicing has been developed whereby retinal tissue is embedded in low gelling temperature agar and sliced in cool solution with a vibrating microtome3,4. This preparation produces retinal slices with less surface damage and very robust light-evoked responses. Here we document how this procedure can be done under infrared light to avoid bleaching the visual pigment.

Here we describe a procedure for generating dark-adapted slices of the mouse retina for electrophysiological recordings.

تسجيلات المشبك التصحيح من الخلايا العصبية في الشبكية ماوس إعداد غامق تكييفها شريحة

Our visual experience is initiated when the visual pigment in our retinal photoreceptors absorbs photons of light energy and initiates a cascade of ...

Dissection of a Mouse Eye for a Whole Mount of the Retinal Pigment Epithelium

The retinal pigment epithelium (RPE) lies at the back of the mammalian eye, just under the neural retina, which contains the photoreceptors (rods and cones). The RPE is a monolayer of pigmented cuboidal cells and associates closely with the neural retina just above it. This association makes the RPE of great interest to researchers studying retinal diseases. The RPE is also the site of an in vivo assay of homology-directed DNA repair, the pun assay. The mouse eye is particularly difficult to dissect due to its small size (about 3.5mm in diameter) and its spherical shape. This article demonstrates in detail a procedure for dissection of the eye resulting in a whole mount of the RPE. In this procedure, we show how to work with, rather than against, the spherical structure of the eye. Briefly, the connective tissue, muscle, and optic nerve are removed from the back of the eye. Then, the cornea and lens are removed. Next, strategic cuts are made that result in significant flattening of the remaining tissue. Finally, the neural retina is gently lifted off, revealing an intact RPE, which is still attached to the underlying choroid and sclera. This whole mount can be used to perform the pun assay or for immunohistochemistry or immunofluorescent assessment of the RPE tissue.

A formal demonstration of the dissection of a mouse eye, resulting in a whole mount of the retinal pigment epithelium.

تشريح العين ماوس لجبل الجامعة للصبغات الظهارة الشبكية

The retinal pigment epithelium (RPE) lies at the back of the mammalian eye, just under the neural retina, which contains the photoreceptors (rods 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.

وحيدة الخلية التنميط من الخلايا العصبية الشبكية المتطورة والناضجة

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

Antibody Transfection into Neurons as a Tool to Study Disease Pathogenesis

Antibodies provide the ability to gain novel insight into various events taking place in living systems. The ability to produce highly specific antibodies to target proteins has allowed for very precise biological questions to be addressed. Importantly, antibodies have been implicated in the pathogenesis of a number of human diseases including systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), paraneoplastic syndromes, multiple sclerosis (MS) and human T-lymphotropic virus type 1 (HTLV-1) associated myelopathy/tropical spastic paraparesis (HAM/TSP) 1-9. How antibodies cause disease is an area of ongoing investigation, and data suggests that interactions between antibodies and various intracellular molecules results in inflammation, altered cellular messaging, and apoptosis 10. It has been shown that patients with MS and HAM/TSP produce autoantibodies to the intracellular RNA binding protein heterogeneous ribonuclear protein A1 (hnRNP A1) 3, 5-7, 9, 11. Recent data indicate that antibodies to both intra-neuronal and surface antigens are pathogenic 3, 5-9, 11. Thus, a procedure that allows for the study of intracellular antibody:protein interactions would lend great insight into disease pathogenesis.
Genes are commonly transfected into primary cells and cell lines in culture, however transfection of antibodies into cells has been hindered by alteration of antibody structure or poor transfection efficiency 12. Other methods of transfection include antibody transfection based on cationic liposomes (consisting of DOTAP/DOPE) and polyethylenimines (PEI); both of which resulted in a ten-fold decrease in antibody transfection compared to controls 12. The method performed in our study is similar to cationic lipid-mediated methods and uses a lipid-based mechanism to form non-covalent complexes with the antibodies through electrostatic and hydrophobic interactions 13. We utilized Ab-DeliverIN reagent, which is a lipid formulation capable of capturing antibodies through non-covalent electrostatic and hydrophobic interactions and delivering them inside cells. Thus chemical and genetic couplings are not necessary for delivery of functional antibodies into living cells. This method has enabled us to perform various antibody tracing and protein localization experiments, as well as the analyses of the molecular consequences of intracellular antibody:protein interactions 9.
In this protocol, we will show how to transfect antibodies into neurons rapidly, reproducibly and with a high degree of transfection efficiency. As an example, we will use anti-hnRNP A1 and anti-IgG antibodies. For easy quantification of transfection efficiency we used anti-hnRNP A1 antibodies labelled with Atto-550-NHS and FITC-labeled IgG. Atto550 NHS is a new label with high molecular absorbtion and quantum yield. Excitation source and fluorescent filters for Atto550 are similar to Cy3 (Ex. 556 Em. 578). In addition, Atto550 has high photostability. FITC-labeled IgG were used as a control to show that this method is versatile and not dye dependent. This approach and the data that is generated will assist in understanding of the role that antibodies to intracellular target antigens might play in the pathogenesis of human diseases.

A rapid approach to investigate interactions and effects on molecular mechanisms related to the presence of antibodies in an intracellular environment is described. The method involves transfection of antibodies into live cells using a non-covalent complex formation based on a lipid formulation. The technique is adaptable to immortalized cell lines and primary cells.

ترنسفكأيشن الأجسام المضادة إلى الخلايا العصبية كأداة للدراسة إمراض مرض

Antibodies provide the ability to gain novel insight into various events taking place in living systems.  The ability to produce highly specific ...

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

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

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

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 ...

A Dual Tracer PET-MRI Protocol for the Quantitative Measure of Regional Brain Energy Substrates Uptake in the Rat

We present a method for comparing the uptake of the brain&#39;s two key energy substrates: glucose and ketones (acetoacetate [AcAc] in this case) in the rat. The developed method is a small-animal positron emission tomography (PET) protocol, in which&#160;11C-AcAc and 18F-fluorodeoxyglucose (18F-FDG) are injected sequentially in each animal. This dual tracer PET acquisition is possible because of the short half-life of 11C (20.4 min). The rats also undergo a magnetic resonance imaging (MRI) acquisition seven days before the PET protocol. Prior to image analysis, PET and MRI images are coregistered to allow the measurement of regional cerebral uptake (cortex, hippocampus, striatum, and cerebellum). A quantitative measure of 11C-AcAc and 18F-FDG brain uptake (cerebral metabolic rate; &#956;mol/100 g/min) is determined by kinetic modeling using the image-derived input function (IDIF) method. Our new dual tracer PET protocol is robust and flexible; the two tracers used can be replaced by different radiotracers to evaluate other processes in the brain. Moreover, our protocol is applicable to the study of brain fuel supply in multiple conditions such as normal aging and neurodegenerative pathologies such as Alzheimer&#39;s and Parkinson&#39;s diseases.

Small-animal positron emission tomography enables the assessment of the brain&#39;s two main energy substrates:&#160;glucose and ketones. In the present method, 11C-acetoacetate and 18F-fluorodeoxyglucose are injected sequentially in each animal, and their uptake is measured quantitatively in specific brain regions determined from the magnetic resonance images.

We present a method for comparing the uptake of the brain&#39;s two key energy substrates: glucose and ketones (acetoacetate [AcAc] in this case) in the ...

An Engulfment Assay: A Protocol to Assess Interactions Between CNS Phagocytes and Neurons

Phagocytosis is a process in which a cell engulfs material (entire cell, parts of a cell, debris, etc.) in its surrounding extracellular environment and subsequently digests this material, commonly through lysosomal degradation. Microglia are the resident immune cells of the central nervous system (CNS) whose phagocytic function has been described in a broad range of conditions from neurodegenerative disease (e.g., beta-amyloid clearance in Alzheimer&#8217;s disease) to development of the healthy brain (e.g., synaptic pruning)1-6. The following protocol is an engulfment assay developed to visualize and quantify microglia-mediated engulfment of presynaptic inputs in the developing mouse retinogeniculate system7. While this assay was used to assess microglia function in this particular context, a similar approach may be used to assess other phagocytes throughout the brain (e.g., astrocytes) and the rest of the body (e.g., peripheral macrophages) as well as other contexts in which synaptic remodeling occurs (e.g. ,brain injury/disease).

Microglia are the resident immune cells of the central nervous system (CNS) with a high capacity to phagocytose or engulf material in their extracellular environment. Here, a broadly applicable, reliable, and highly quantitative assay for visualizing and measuring microglia-mediated engulfment of synaptic components is described.

على الإحاطة الفحص: بروتوكول لتقييم التفاعلات بين الخلايا العصبية والجهاز العصبي المركزي البالعات

Phagocytosis is a process in which a cell engulfs material (entire cell, parts of a cell, debris, etc.) in its surrounding extracellular environment and ...

Optical Control of a Neuronal Protein Using a Genetically Encoded Unnatural Amino Acid in Neurons

Photostimulation is a noninvasive way to control biological events with excellent spatial and temporal resolution. New methods are desired to photo-regulate endogenous proteins expressed in their native environment. Here, we present an approach to optically control the function of a neuronal protein directly in neurons using a genetically encoded unnatural amino acid (Uaa). By using an orthogonal tRNA/aminoacyl-tRNA synthetase pair to suppress the amber codon, a photo-reactive Uaa 4,5-dimethoxy-2-nitrobenzyl-cysteine (Cmn) is site-specifically incorporated in the pore of a neuronal protein Kir2.1, an inwardly rectifying potassium channel. The bulky Cmn physically blocks the channel pore, rendering Kir2.1 non-conducting. Light illumination instantaneously converts Cmn into a smaller natural amino acid Cys, activating Kir2.1 channel function. We express these photo-inducible inwardly rectifying potassium (PIRK) channels in rat hippocampal primary neurons, and demonstrate that light-activation of PIRK ceases the neuronal firing due to the outflux of K+ current through the activated Kir2.1 channels. Using in utero electroporation, we also express PIRK in the embryonic mouse neocortex in vivo, showing the light-activation of PIRK in neocortical neurons. Genetically encoding Uaa imposes no restrictions on target protein type or cellular location, and a family of photoreactive Uaas is available for modulating different natural amino acid residues. This technique thus has the potential to be generally applied to many neuronal proteins to achieve optical regulation of different processes in brains. The current protocol presents an accessible procedure for intricate Uaa incorporation in neurons in vitro and in vivo to achieve photo control of neuronal protein activity on the molecular level.

Here, a procedure to selectively activate a neuronal protein with a short pulse of light by genetically encoding a photo-reactive unnatural amino acid into a target neuronal protein expressed in neurons in culture or in vivo is presented.

مراقبة البصرية من البروتين العصبية باستخدام حمض أميني مصطنعة مشفرة وراثيا في الخلايا العصبية

Photostimulation is a noninvasive way to control biological events with excellent spatial and temporal resolution. New methods are desired to ...

Preparation of Mouse Retinal Cryo-sections for Immunohistochemistry

Preparation of high-quality mouse eye sections for immunohistochemistry (IHC) is critical for assessing the retinal structure and function and for determining the mechanisms underlying retinal diseases. Maintaining structural integrity throughout the tissue preparation is vital for obtaining reproducible retinal IHC data but can be challenging due to the fragility and complexity of retinal cytoarchitecture. Strong fixatives like 10% formalin or Bouin's solution optimally preserve the retinal structure, they often impede IHC analysis by enhancing the background fluorescence and/or diminishing antibody-epitope interactions, a process known as epitope masking. Milder fixatives, on the other hand, like 4% paraformaldehyde, reduces background fluorescence and epitope-masking, meticulous dissection techniques must be utilized to preserve the retinal structure. In this article, we present a comprehensive method to prepare mouse ocular posterior cups for IHC that is sufficient to preserve most antibody-epitope interactions without loss of retinal structural integrity. We include representative IHC with antibodies to various retinal cell type markers to illustrate tissue preservation and orientation under optimal and sub-optimal conditions. Our goal is to optimize IHC studies of the retina by providing a complete protocol from ocular posterior cup dissection to IHC.

This report describes comprehensive methods for preparing frozen mouse retina sections for immunohistochemistry (IHC). Methods described include dissection of the ocular posterior cup, paraformaldehyde fixation, embedding in Optimal Cutting Temperature (OCT) media and tissue orientation, sectioning and immunostaining.

إعداد المقطعين الماهيينيات في شبكية العين للكيمياء المناعية

Preparation of high-quality mouse eye sections for immunohistochemistry (IHC) is critical for assessing the retinal structure and function and for ...

A Standardized Pipeline for Examining Human Cerebellar Grey Matter Morphometry using Structural Magnetic Resonance Imaging

Multiple lines of research provide compelling evidence for a role of the cerebellum in a wide array of cognitive and affective functions, going far beyond its historical association with motor control. Structural and functional neuroimaging studies have further refined understanding of the functional neuroanatomy of the cerebellum beyond its anatomical divisions, highlighting the need for the examination of individual cerebellar subunits in healthy variability and neurological diseases. This paper presents a standardized pipeline for examining cerebellum grey matter morphometry that combines high-resolution, state-of-the-art approaches for optimized and automated cerebellum parcellation (Automatic Cerebellum Anatomical Parcellation using U-Net Locally Constrained Optimization; ACAPULCO) and voxel-based registration of the cerebellum (Spatially Unbiased Infra-tentorial Template; SUIT) for volumetric quantification. 
The pipeline has broad applicability to a range of neurological diseases and is fully automated, with manual intervention only required for quality control of the outputs. The pipeline is freely available, with substantial accompanying documentation, and can be run on Mac, Windows, and Linux operating systems. The pipeline is applied in a cohort of individuals with Friedreich ataxia (FRDA), and representative results, as well as recommendations on group-level inferential statistical analyses, are provided. This pipeline could facilitate reliability and reproducibility across the field, ultimately providing a powerful methodological approach for characterizing and tracking cerebellar structural changes in neurological diseases.

A standardized pipeline is presented for examining cerebellum grey matter morphometry. The pipeline combines high-resolution, state-of-the-art approaches for optimized and automated cerebellum parcellation and voxel-based registration of the cerebellum for volumetric quantification.

خط أنابيب موحد لفحص مورفومتر المادة الرمادية المخيخية البشرية باستخدام التصوير بالرنين المغناطيسي الهيكلي

Multiple lines of research provide compelling evidence for a role of the cerebellum in a wide array of cognitive and affective functions, going far beyond ...