במעבדה של בן א בארס (אוניברסיטת סטנפורד).: Puncta Plug-in עבור תמונה J נכתב על ידי בארי ווארק (ייעוץ Physion הכתובת הנוכחית) Analyzer מימון; <ul style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> אלפרד פ &#39;סלואן קרן </li><li style=";text-align:right;direction:rtl"> אסתר א ויוסף Klingenstein קרן, Inc </li><li style=";text-align:right;direction:rtl"> רחבה מחקר ביו קרן </li><li style=";text-align:right;direction:rtl"> תרופה יוזמת מחלת הנטינגטון s </li></ul>

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	<li>Barres, B. A., Silverstein, B. E., Corey, D. P., Chun, L. L. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunological,+morphological,+and+electrophysiological+variation+among+retinal+ganglion+cells+purified+by+panning.">Immunological, morphological, and electrophysiological variation among retinal ganglion cells purified by panning.</a> Neuron. 1, 791-803 (1988).</li><li>Meyer-Franke, A., Kaplan, M. R., Pfrieger, F. W., Barres, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+the+signaling+interactions+that+promote+the+survival+and+growth+of+developing+retinal+ganglion+cells+in+culture.">Characterization of the signaling interactions that promote the survival and growth of developing retinal ganglion cells in culture.</a> Neuron. 15, 805-819 (1995).</li><li>Eroglu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gabapentin+receptor+alpha2delta-1+is+a+neuronal+thrombospondin+receptor+responsible+for+excitatory+CNS+synaptogenesis.">Gabapentin receptor alpha2delta-1 is a neuronal thrombospondin receptor responsible for excitatory CNS synaptogenesis.</a> Cell. 139, 380-392 (2009).</li><li>Pfrieger, F. W., Barres, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+efficacy+enhanced+by+glial+cells+in+vitro.">Synaptic efficacy enhanced by glial cells in vitro.</a> Science. 277, 1684-1687 .</li><li>Ullian, E. M., Sapperstein, S. K., Christopherson, K. S., Barres, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+synapse+number+by+glia.">Control of synapse number by glia.</a> Science. 291, 657-661 (2001).</li><li>Christopherson, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thrombospondins+are+astrocyte-secreted+proteins+that+promote+CNS+synaptogenesis.">Thrombospondins are astrocyte-secreted proteins that promote CNS synaptogenesis.</a> Cell. 120, 421-433 (2005).</li><li>Godement, P., Salaun, J., Imbert, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prenatal+and+postnatal+development+of+retinocollicular+projections+in+the+mouse.">Prenatal and postnatal development of retinocollicular projections in the mouse.</a> J. Comp. Neurol. 230, 552-575 (1985).</li><li>Schmidt, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+of+retinotopic+connections:+selective+stabilization+by+an+activity-dependent+mechanism.">Formation of retinotopic connections: selective stabilization by an activity-dependent mechanism.</a> Cell. Mol. Neurobiol. 5, 65-84 (1985).</li><li>Sachs, G. M., Jacobson, M., Caviness, V. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postnatal+changes+in+arborization+patterns+of+murine+retinocollicular+axons.">Postnatal changes in arborization patterns of murine retinocollicular axons.</a> J. Comp. Neurol. 246, 395-408 (1986).</li><li>Elmariah, S. B., Oh, E. J., Hughes, E. G., Balice-Gordon, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astrocytes+regulate+inhibitory+synapse+formation+via+Trk-mediated+modulation+of+postsynaptic+GABAA+receptors.">Astrocytes regulate inhibitory synapse formation via Trk-mediated modulation of postsynaptic GABAA receptors.</a> J. Neurosci. 25, 3638-3650 (2005).</li><li>Hughes, E. G., Elmariah, S. B., Balice-Gordon, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astrocyte+secreted+proteins+selectively+increase+hippocampal+GABAergic+length,+branching+and+synaptogenesis.">Astrocyte secreted proteins selectively increase hippocampal GABAergic length, branching and synaptogenesis.</a> Moll. Cell. Neurosci. 43, 136-145 (2010).</li></ol>

אין ניגודי אינטרסים הכריז.

Assay סינפסה שתוארו לעיל מבוסס בהקשר של מטרות הניסוי שלנו, שבו אנו מתמקדים בעיקר על תחזיות מעוררות של RGCs, לא מטוהרים תרבות או בחלק המוח. סיפקנו שולחן התייחסות לרישום נוגדנים פועלות היטב עבור תיוג סינפסות מעוררות (טבלה 1). Assay זה סינפס?...

<table border="1" cellpadding="0" cellspacing="0" style=";text-align:right;direction:rtl"><tbody><tr><td></td><td> אנטיגן </td><td> מין </td><td> חד שבטיים / polyclonal </td><td> מוכר </td><td> מספר קטלוגי </td><td> מהילה </td><td> יצירות תרבות </td><td> העבודות בסעיפים </td></tr><tr><td rowspan="10"> Presynaptic </td><td> Synapsin </td><td> ארנב </td><td> Polyclonal </td><td> Synaptic מערכות </td><td> 106004 </td><td> 1:750 </td><td> Y </td><td> ND </td></tr><tr><td> Synapsin </td><td> עכבר </td><td> חד שבטיים </td><td> Synaptic מערכות </td><td> 106001 </td><td> 1:500 </td><td> Y </td><td> Y </td></tr><tr><td> בסון </td><td> עכבר </td><td> חד שבטיים </td><td> Assay עיצובים </td><td> VAM-PS003F </td><td> 1:500 </td><td> Y </td><td> Y </td></tr><tr><td> בסון </td><td> קביה </td><td> Polyclonal </td><td> Synaptic מערכות </td><td> 141004 </td><td> 1:1000 </td><td> Y </td><td> ND </td></tr><tr><td> Synaptotagmin 1 </td><td> ארנב </td><td> Polyclonal </td><td> Synaptic מערכות </td><td> 105002 </td><td> 1:750 </td><td> Y </td><td> N </td></tr><tr><td> Synaptobrevin 2 (C1.69.1) </td><td> עכבר </td><td> חד שבטיים </td><td> Synaptic מערכות </td><td> 104211 </td><td> 1:500 </td><td> Y </td><td> Y </td></tr><tr><td> Synaptophysin (C1.7.2) </td><td> עכבר </td><td> חד שבטיים </td><td> Synaptic מערכות </td><td> 101011 </td><td> 1:500 </td><td> Y </td><td> Y </td></tr><tr><td> VGlut1 </td><td> עכבר </td><td> חד שבטיים </td><td> Millipore </td><td> MAB5502 </td><td> 1:2500 </td><td> N </td><td> Y </td></tr><tr><td> VGlut1 </td><td> קביה </td><td> Polyclonal </td><td> Millipore </td><td> AB5905 </td><td> 1:2500 </td><td> N </td><td> Y </td></tr><tr><td> VGlut2 </td><td> קביה </td><td> Polyclonal </td><td> Millipore </td><td> AB2251 </td><td> 1:2500 </td><td> N </td><td> Y </td></tr><tr><td rowspan="6"> Postsynaptic </td><td> PSD-95 (6G6 1C9-clone) </td><td> עכבר </td><td> חד שבטיים </td><td> זיקה ביו ריאגנטים </td><td> MA1-045 </td><td> 1:750 </td><td> Y </td><td> N </td></tr><tr><td> PSD-95 </td><td> ארנב </td><td> Polyclonal </td><td> Zymed </td><td> 51-6900 </td><td> 1:500 </td><td> N </td><td> Y </td></tr><tr><td> הומר </td><td> עכבר </td><td> חד שבטיים </td><td> Synaptic מערכות </td><td> 160011 </td><td> 1:500 </td><td> Y </td><td> ND </td></tr><tr><td> הומר </td><td> עכברוש </td><td> Polyclonal </td><td> Millipore </td><td> AB5875 </td><td> 1:500 </td><td> Y </td><td> Y </td></tr><tr><td> Gephyrin </td><td> ארנב </td><td> Polyclonal </td><td> Synaptic מערכות </td><td> 147003 </td><td> 1:500 </td><td> Y </td><td> Y </td></tr><tr><td> Gephyrin </td><td> עכבר </td><td> חד שבטיים </td><td> Synaptic מערכות </td><td> 147 </td><td> 1:200 </td><td> Y </td><td> Y </td></tr></tbody></table> טבלה 1: רשימת דוגמאות של סמנים טרום postsynaptic טוב שיש לנו מנוצל בהצלחה assay סינפסה שלנו. שימו לב כי זו אינה רשימה ממצה של כל סמנים זמינים. Y = כן, N = לא, ND = לא נקבע. <table border="1" cellpadding="0" cellspacing="0" style=";text-align:right;direction:rtl"><tbody><tr><td> מגיב </td><td> חברה </td><td> חתול. לא. </td></tr><tr><td> PBS </td><td> Invitrogen </td><td> 20012-027 </td></tr><tr><td> poly-d-ליזין </td><td> סיגמא </td><td> P6407 </td></tr><tr><td> Laminin </td><td> Cultrex </td><td> 3400-010-01 </td></tr><tr><td> טריטון X-100 </td><td> Roche Diagnostics GmbH </td><td> 9002-93-1 </td></tr><tr><td> עיזים רגיל סרום </td><td> Gibco </td><td> 16210 </td></tr><tr><td> VectaShield עם DAPI </td><td> וקטור מעבדות </td><td> H-1200 </td></tr><tr><td> אוקטובר </td><td> רקמות-Tek </td><td> 4583 </td></tr><tr><td> טריס-Base (50 מ&quot;מ) </td><td> דיג </td><td> BP152-5 </td></tr><tr><td> שור סרום אלבומין </td><td> סיגמא </td><td> A2153 </td></tr><tr><td> L-ליזין </td><td> סיגמא </td><td> L-1137 </td></tr><tr><td> 16% PFA פתרון </td><td> מיקרוסקופית אלקטרונים למדעים </td><td> 15711 </td></tr><tr><td> גרעיניים PFA </td><td> מיקרוסקופית אלקטרונים למדעים </td><td> 19210 </td></tr><tr><td> 24 גם תרבות צלחת </td><td> בז </td><td> 35-3047 </td></tr><tr><td> אנטי עכבר עיזים אלקסה נוגדנים מצומדות </td><td> Invitrogen </td><td> --- </td></tr><tr><td> אספקה </td><td> חברה </td><td> חתול. לא. </td></tr><tr><td> 12mm, מס &#39;0 זכוכית coverslips </td><td> קרל הכט GmbH </td><td> 1105209 </td></tr><tr><td> מס &#39;1.5 זכוכית coverslip (עבור פרוסות) </td><td> VWR מדעי </td><td> 48393241 </td></tr><tr><td> שקופיות הזכוכית </td><td> VWR מדעי </td><td> 48311-703 </td></tr></tbody></table>

quantifying synapses: an immunocytochemistry-based assay to quantify synapse number

אחת המטרות החשובות ביותר בחקר המוח היא להבין את הרמזים מולקולרית להורות כי בשלבים המוקדמים של היווצרות הסינפסה. ככזה זה הפך להיות הכרחי לפתח גישות אובייקטיבי לכמת שינויים קישוריות סינפטי. החל מ קיבעון המדגם, זה פרטים פרוטוקול איך לכמת את מספר הסינפסות הן בתרבות העצבית הפרידו בסעיפים המוח באמצעות immunocytochemistry. שימוש ספציפי תא נוגדנים, אנו תווית מסופי presynaptic כמו גם באתרים של התמחות postsynaptic. אנו מגדירים סינפסות כנקודות של colocalization בין האותות שנוצר על ידי סמנים אלה. מספר colocalizations אלה היא לכמת באמצעות תקע Puncta Analyzer (שנכתב על ידי Bary ווארק, זמין על פי דרישה, cellbio.duke.edu c.eroglu @) תחת פלטפורמת תוכנה לניתוח ImageJ. Assay סינפסה המתואר פרוטוקול זה יכול להיות מיושם על כל הכנה או רקמה עצבית תרבות שעבורו יש לך סמנים טרום postsynaptic סלקטיבית. זה assay הסינפסה היא כלי רב ערך אשר יכול להיות מנוצל באופן נרחב במחקר של התפתחות הסינפטי.

Watch this Scientific Journal Video about Quantifying Synapses: an Immunocytochemistry-based Assay to Quantify Synapse Number at JoVE.com

Quantifying Synapses: an Immunocytochemistry-based Assay to Quantify Synapse Number

פרוטוקול זה מפרט כיצד לכמת את מספר הסינפסות הן בתרבות העצבית הפרידו בסעיפים המוח באמצעות immunocytochemistry. שימוש ספציפי תא נוגדנים, אנו תווית מסופי presynaptic כמו גם באתרים של התמחות postsynaptic. אנו מגדירים סינפסות כנקודות של colocalization בין האותות שנוצר על ידי סמנים אלה.

סינפסות כימות: Assay Immunocytochemistry מבוססי לכמת מספר סינפסה

One of the most important goals in neuroscience is to understand the molecular cues that instruct early stages of synapse formation. As such it has become ...

quantifying-synapses-an-immunocytochemistry-based-assay-to-quantify

Research

JoVE Journal

Neuroscience

35.5K Views.  Duke University. פרוטוקול זה מפרט כיצד לכמת את מספר הסינפסות הן בתרבות העצבית הפרידו בסעיפים המוח באמצעות immunocytochemistry. שימוש ספציפי תא נוגדנים, אנו תווית מסופי presynaptic כמו גם באתרים של התמחות postsynaptic. אנו מגדירים סינפסות כנקודות של colocalization בין האותות שנוצר על ידי סמנים אלה.

Video: Quantifying Synapses: an Immunocytochemistry-based Assay to Quantify Synapse Number

Neural-Colony Forming Cell Assay: An Assay To Discriminate Bona Fide Neural Stem Cells from Neural Progenitor Cells

The neurosphere assay (NSA) is one of the most frequently used methods to isolate, expand and also calculate the frequency of neural stem cells (NSCs). Furthermore, this serum-free culture system has also been employed to expand stem cells and determine their frequency from a variety of tumors and normal tissues. It has been shown recently that a one-to-one relationship does not exist between neurosphere formation and NSCs. This suggests that the NSA as currently applied, overestimates the frequency of NSCs in a mixed population of neural precursor cells isolated from both the embryonic and adult mammalian brain. This video practically demonstrates a novel collagen based semi- solid assay, the neural-colony forming cell assay (N-CFCA), which has the ability to discriminate stem from progenitor cells based on their long-term proliferative potential, and thus provides a method to enumerate NSC frequency. In the N-CFCA, colonies &ge;2 mm in diameter are derived from cells that meet all the functional criteria of a NSC, while colonies &lt; 2mm are derived from progenitors. The N-CFCA procedure can be used for cells prepared from different sources including primary and cultured adult or embryonic mouse CNS cells. Here we use cells prepared from passage one neurospheres generated from embryonic day 14 mice brain to perform N-CFCA. The cultures are replenished with proliferation medium every seven days for three weeks to allow the plated cells to exhibit their full proliferative potential and then the frequency of neural progenitor and bona fide neural stem cells is calculated respectively by counting the number of colonies that are &lt; 2mm and the ones that are &ge;2mm in reference to the number of cells that were initially plated.

This video protocol demonstrates how to discriminate and enumerate bona fide neural stem cells in a mixed population of neural precursor cells using the neural colony-forming cell assay.

עצבית, המושבה יצירת Assay נייד: Assay להפלות בונה תאים בתום גזע עצביים מן ובתאים עצבית

The neurosphere assay (NSA) is one of the most frequently used methods to isolate, expand and also calculate the frequency of neural stem cells (NSCs). ...

An Assay for Permeability of the Zebrafish Embryonic Neuroepithelium

The brain ventricular system is conserved among vertebrates and is composed of a series of interconnected cavities called brain ventricles, which form during the earliest stages of brain development and are maintained throughout the animal's life. The brain ventricular system is found in vertebrates, and the ventricles develop after neural tube formation, when the central lumen fills with cerebrospinal fluid (CSF) 1,2. CSF is a protein rich fluid that is essential for normal brain development and function3-6.
In zebrafish, brain ventricle inflation begins at approximately 18 hr post fertilization (hpf), after the neural tube is closed. Multiple processes are associated with brain ventricle formation, including formation of a neuroepithelium, tight junction formation that regulates permeability and CSF production. We showed that the Na,K-ATPase is required for brain ventricle inflation, impacting all these processes 7,8, while claudin 5a is necessary for tight junction formation 9. Additionally, we showed that "relaxation" of the embryonic neuroepithelium, via inhibition of myosin, is associated with brain ventricle inflation.
To investigate the regulation of permeability during zebrafish brain ventricle inflation, we developed a ventricular dye retention assay. This method uses brain ventricle injection in a living zebrafish embryo, a technique previously developed in our lab10, to fluorescently label the cerebrospinal fluid. Embryos are then imaged over time as the fluorescent dye moves through the brain ventricles and neuroepithelium. The distance the dye front moves away from the basal (non-luminal) side of the neuroepithelium over time is quantified and is a measure of neuroepithelial permeability (Figure 1). We observe that dyes 70 kDa and smaller will move through the neuroepithelium and can be detected outside the embryonic zebrafish brain at 24 hpf (Figure 2).
This dye retention assay can be used to analyze neuroepithelial permeability in a variety of different genetic backgrounds, at different times during development, and after environmental perturbations. It may also be useful in examining pathological accumulation of CSF. Overall, this technique allows investigators to analyze the role and regulation of permeability during development and disease.

We describe a live whole animal quantitative measurement for permeability of the embryonic zebrafish brain. The technique analyzes the ability to retain cerebrospinal fluid and molecules of different molecular weights within the neural tube lumen and quantifies their movement out of the ventricles. This method is useful for determining differences in epithelial permeability and maturation during development and disease.

Assay לחדירות של Neuroepithelium עוברי דג הזברה

The brain ventricular system is conserved among vertebrates and is composed of a series of interconnected cavities called brain ventricles, which form ...

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.

Assay היבלעות: פרוטוקול להעריך אינטראקציות בין מערכת העצבים המרכזית וPhagocytes נוירונים

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

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Voltage responses of insect photoreceptors and visual interneurons can be accurately recorded with conventional sharp microelectrodes. The method described here enables the investigator to measure long-lasting (from minutes to hours) high-quality intracellular responses from single Drosophila R1-R6 photoreceptors and Large Monopolar Cells (LMCs) to light stimuli. Because the recording system has low noise, it can be used to study variability among individual cells in the fly eye, and how their outputs reflect the physical properties of the visual environment. We outline all key steps in performing this technique. The basic steps in constructing an appropriate electrophysiology set-up for recording, such as design and selection of the experimental equipment are described. We also explain how to prepare for recording by making appropriate (sharp) recording and (blunt) reference electrodes. Details are given on how to fix an intact fly in a bespoke fly-holder, prepare a small window in its eye and insert a recording electrode through this hole with minimal damage. We explain how to localize the center of a cell's receptive field, dark- or light-adapt the studied cell, and to record its voltage responses to dynamic light stimuli. Finally, we describe the criteria for stable normal recordings, show characteristic high-quality voltage responses of individual cells to different light stimuli, and briefly define how to quantify their signaling performance. Many aspects of the method are technically challenging and require practice and patience to master. But once learned and optimized for the investigator's experimental objectives, it grants outstanding in vivo neurophysiological data.

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Sharp microelectrodes enable accurate electrophysiological characterization of photoreceptor and visual interneuron output in living Drosophila. Here we show how to use this method to record high-quality voltage responses of individual cells to controlled light stimulation. This method is ideal for studying neural information processing in insect compound eyes.

שיטת אלקטרו עבור תגובות מתח תאיים הקלטה של<em&gt; תסיסנית</em&gt; קולטני אור ו interneurons יאירו גירויים<em&gt; In vivo</em

Voltage responses of insect photoreceptors and visual interneurons can be accurately recorded with conventional sharp microelectrodes. The method ...

Mass Histology to Quantify Neurodegeneration in Drosophila

Progressive neurodegenerative diseases like Alzheimer&#39;s disease (AD) or Parkinson&#39;s disease (PD) are an increasing threat to human health worldwide. Although mammalian models have provided important insights into the underlying mechanisms of pathogenicity, the complexity of mammalian systems together with their high costs are limiting their use. Therefore, the simple but well-established Drosophila model-system&#160;provides an alternative for investigating the molecular pathways that are affected in these diseases. Besides behavioral deficits, neurodegenerative diseases are characterized by histological phenotypes such as neuronal death and axonopathy. To quantify neuronal degeneration and to determine how it is affected by genetic and environmental factors, we use a histological approach that is based on measuring the vacuoles in adult fly brains. To minimize the effects of systematic error and to directly compare sections from control and experimental flies in one preparation, we use the &#39;collar&#39; method for paraffin sections. Neurodegeneration is then assessed by measuring the size and/or number of vacuoles that have developed in the fly brain. This can either be done by focusing on a specific region of interest or by analyzing the entire brain by obtaining serial sections that span the complete head. Therefore, this method allows one to measure not only severe degeneration but also relatively mild phenotypes that are only detectable in a few sections, as occurs during normal aging.

Mass Histology to Quantify Neurodegeneration in Drosophila

Drosophila is widely used as a model system to study neurodegeneration. This protocol describes a method by which degeneration, as determined by vacuole formation in the brain, can be quantified. It also minimizes effects due to the experimental procedure by processing and sectioning control and experimental flies as one sample.

Mass היסטולוגיה לכמת מוחיים ב<em&gt; תסיסנית</em

Progressive neurodegenerative diseases like Alzheimer&#39;s disease (AD) or Parkinson&#39;s disease (PD) are an increasing threat to human health ...

Evaluation of Synapse Density in Hippocampal Rodent Brain Slices

In the brain, synapses are specialized junctions between neurons, determining the strength and spread of neuronal signaling. The number of synapses is tightly regulated during development and neuronal maturation. Importantly, deficits in synapse number can lead to cognitive dysfunction. Therefore, the evaluation of synapse number is an integral part of neurobiology. However, as synapses are small and highly compact in the intact brain, the assessment of absolute number is challenging. This protocol describes a method to easily identify and evaluate synapses in hippocampal rodent slices using immunofluorescence microscopy. It includes a three-step procedure to evaluate synapses in high-quality confocal microscopy images by analyzing the co-localization of pre- and postsynaptic proteins in hippocampal slices. It also explains how the analysis is performed and gives representative examples from both excitatory and inhibitory synapses. This protocol provides a solid foundation for the analysis of synapses and can be applied to any research investigating the structure and function of the brain.

A protocol for accurately identifying and analyzing synapses in hippocampal slices using immunofluorescence is outlined in this article.

הערכה של צפיפות סינפסה פרוסות המוח מכרסמים בהיפוקמפוס

In the brain, synapses are specialized junctions between neurons, determining the strength and spread of neuronal signaling. The number of synapses is ...

Assay Development for High Content Quantification of Sod1 Mutant Protein Aggregate Formation in Living Cells

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that can be caused by inherited mutations in the gene encoding copper-zinc superoxide dismutase 1 (SOD1). The structural instability of SOD1 and the detection of SOD1-positive inclusions in familial-ALS patients supports a potential causal role for misfolded and/or aggregated SOD1 in ALS pathology. In this study, we describe the development of a cell-based assay designed to quantify the dynamics of SOD1 aggregation in living cells by high content screening approaches. Using lentiviral vectors, we generated stable cell lines expressing wild-type and mutant A4V SOD1 tagged with yellow fluorescent protein and found that both proteins were expressed in the cytosol without any sign of aggregation. Interestingly, only SOD1 A4V stably expressed in HEK-293, but not in U2OS or SH-SY5Y cell lines, formed aggregates upon proteasome inhibitor treatment. We show that it is possible to quantify aggregation based on dose-response analysis of various proteasome inhibitors, and to track aggregate-formation kinetics by time-lapse microscopy. Our approach introduces the possibility of quantifying the effect of ALS mutations on the role of SOD1 in aggregate formation as well as screening for small molecules that prevent SOD1 A4V aggregation.

We describe a method to quantify the aggregation of misfolded proteins. Our protocol details lentiviral induced stable cell line generation, automated confocal imaging, and image analysis of protein aggregates. As an illustrative application, we studied the effect of small molecules in promoting SOD1 aggregation in a time- and dose-dependent manner.

פיתוח assay על כימות תוכן גבוהה של היווצרות צבירה של מוטציה בחלבון Sod1 בתאים חיים

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that can be caused by inherited mutations in the gene encoding copper-zinc ...

Electrophysiological Investigations of Retinogeniculate and Corticogeniculate Synapse Function

The lateral geniculate nucleus is the first relay station for the visual information. Relay neurons of this thalamic nucleus integrate input from retinal ganglion cells and project it to the visual cortex. In addition, relay neurons receive top-down excitation from the cortex. The two main excitatory inputs to the relay neurons differ in several aspects. Each relay neuron receives input from only a few retinogeniculate synapses, which are large terminals with many release sites. This is reflected by the comparably strong excitation, the relay neurons receive, from retinal ganglion cells. Corticogeniculate synapses, in contrast, are simpler with few release sites and weaker synaptic strength. The two synapses also differ in their synaptic short-term plasticity. Retinogeniculate synapses have a high release probability and consequently display a short-term depression. In contrast, corticogeniculate synapses have a low release probability. Corticogeniculate fibers traverse the reticular thalamic nuclei before entering the lateral geniculate nucleus. The different locations of the reticular thalamic nucleus (rostrally from the lateral geniculate nucleus) and optic tract (ventro-laterally from the lateral geniculate nucleus) allow stimulating corticogeniculate or retinogeniculate synapses separately with extracellular stimulation electrodes. This makes the lateral geniculate nucleus an ideal brain area where two excitatory synapses with very different properties impinging onto the same cell type, can be studied simultaneously. Here, we describe a method to investigate the recording from relay neurons and to perform detailed analysis of the retinogeniculate and corticogeniculate synapse function in acute brain slices. The article contains a step-by-step protocol for the generation of acute brain slices of the lateral geniculate nucleus and steps for recording activity from relay neurons by stimulating the optic tract and corticogeniculate fibers separately.

Here, we present protocols for the preparation of acute brain slices containing the lateral geniculate nucleus and the electrophysiological investigation of retinogeniculate and corticogeniculate synapses function. This protocol provides an efficient way to study synapses with the high-release and low-release probability in the same acute brain slices.

החקירות האלקטאליות של הפונקציה Retinogeniculate ו Corticogeniculate סינפסה

The lateral geniculate nucleus is the first relay station for the visual information. Relay neurons of this thalamic nucleus integrate input from retinal ...

A Preclinical Model to Assess Brain Recovery After Acute Stroke in Rats

The purpose of this study was to establish and validate an animal brain ischemia model in the recovery and sequela stages. A middle cerebral artery occlusion/reperfusion (MCAO/R) model in male Sprague-Dawley rats was chosen. By changing the rat&#39;s weight (260&#8722;330 g), the thread bolt type (2636/2838/3040/3043) and the brain infarct time (2-3 h), a higher Longa&#39;s score, a larger infarct volume and a greater model success ratio were screened using the Longa&#39;s score and TTC staining. The optimum model condition (300 g, 3040 thread bolt, 3 h brain infarct time) was acquired and used in a 1-90 day observation period after reperfusion via assessment of sensorimotor functions and infarct volume. At these conditions, the bilateral asymmetry test had a significant difference from 1 to 90 days, and the grid-walking test had a significant difference from 1 to 60 days; both differences could be a suitable sensorimotor functional test. Thus, the most appropriate condition of a novel rat model in the recovery and sequela stages of brain ischemia was found: 300 g rats that underwent MCAO with a 3040 thread bolt for a 3 h brain infarct and then reperfused. The appropriate sensorimotor functional tests were a bilateral asymmetry test and a grid-walking test.

The&#160;purpose of this study is to establish and validate an animal model for research in the recovery and sequela stages of brain ischemia by testing brain infarction and sensorimotor function after middle cerebral artery occlusion/reperfusion (MCAO/R) after 1-90 days in rats.

מודל טרום קליני להעריך את ההתאוששות המוח לאחר שבץ חריף בחולדות

The purpose of this study was to establish and validate an animal brain ischemia model in the recovery and sequela stages. A middle cerebral artery ...

Utilizing In Vivo Postnatal Electroporation to Study Cerebellar Granule Neuron Morphology and Synapse Development

Neurons undergo dynamic changes in their structure and function during brain development to form appropriate connections with other cells. The rodent cerebellum is an ideal system to track the development and morphogenesis of a single cell type, the cerebellar granule neuron (CGN), across time. Here, in vivo electroporation of granule neuron progenitors in the developing mouse cerebellum was employed to sparsely label cells for subsequent morphological analyses. The efficacy of this technique is demonstrated in its ability to showcase key developmental stages of CGN maturation, with a specific focus on the formation of dendritic claws, which are specialized structures where these cells receive the majority of their synaptic inputs. In addition to providing snapshots of CGN synaptic structures&#160;throughout cerebellar development, this technique can be adapted to genetically manipulate granule neurons in a cell-autonomous manner to study the role of any gene of interest and its effect on CGN morphology, claw development, and synaptogenesis.

Utilizing In Vivo Postnatal Electroporation to Study Cerebellar Granule Neuron Morphology and Synapse Development

Here we describe a method to visualize synaptogenesis of granule neurons in the mouse cerebellum over the time course of postnatal brain development when these cells refine their synaptic structures and form synapses to integrate themselves into the overall brain circuit.

שימוש באלקטרופורציה לאחר הלידה של In Vivo לחקר מורפולוגיה של נוירון גרגיר המוח הקטן והתפתחות סינפסה

Neurons undergo dynamic changes in their structure and function during brain development to form appropriate connections with other cells. The rodent ...