עבודה זו נתמכה על ידי מענק פיילוט תחת NIH P20 GM109005 (ARA) ועל ידי המרכז לתרגום Neuroscience IDeA תוכנית הפרס P30 GM110702.

<ol>
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Vibroslice NVSL &amp; Vibroslice NVSLM123 Available from: <a target="_blank" href="https://www.wpiinc.com/clientuploads/pdf/NVSL_NVSLM1_IM.pdf">https://www.wpiinc.com/clientuploads/pdf/NVSL_NVSLM1_IM.pdf</a> (2000)</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Neurolucida 11.03. , (2017).</li><li>Sholl, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendritic+organization+in+the+neurons+of+the+visual+and+motor+cortices+of+the+cat.">Dendritic organization in the neurons of the visual and motor cortices of the cat.</a> J Anat. 87 (4), 387-406 (1953).</li><li>Pillai, A. 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המחברים מצהירים כי אין להם אינטרסים פיננסיים מתחרים.

לעומת טכניקות מודרניות יותר, שיטת Golgi-Cox יש כמה יתרונות שהופכים אותו לשיטה המועדפת לבדיקת מורפולוגיה עמוד השדרה: 1) מכתים ניתן להשתמש עבור כל רקמה, 2) אור בסיסי מיקרוסקופ ההתקנה היא כל מה שצריך לרכוש תמונות מבוסס Golgi, 3) ההדמיה Golgi-Cox הוא מהיר יותר מאשר הדמיה confocal, ו 4) חלקים ...

<html> הדנדריטים הם החלק הגדול ביותר של נוירונים המקבלים ומעבדים קלט presynaptic 1 . לתהליכים הדנדריטים שלהם יש גיאומטריה מורכבת, שבה לענפים הפרוקסימליים יש קוטר גדול יותר מאשר הענפים הדיסטליים. כמו דנדריטים לפתח, הם יוצרים מספר קשרים עם נוירונים אחרים בתהליך המכונה arborization דנדריטים. היקף ודפוס של הסתעפות זו קובעת את כמות התשומות הסינפטי כי דנדריט יכול לעבד כראוי 2 . דבורריזם arborization הוא תהליך הכרחי עבור פעילות גמישות הפלסטיות ופיתוח תקין של מעגלים נוירונים. הארכה, חזרה, הסתעפות וסינפוגנזה הם תהליכים מורכבים הכוללים תוכניות גנטיות פנימיות והשפעות מגורמים חיצוניים. ההבדלים המורפולוגיים וההסתעפויות של הדנדריטים העצביים בתוך ההיפוקמפוס מעורבים בהבנה קוגניטיבית וזיכרוןF &quot;&gt; 3 , 4. השינויים במורכבות הדנדריטים קשורים לשינויים פתופיזיולוגיים והתנהגותיים 5. הפרעות קשורות למספר מצבי מחלה, כולל תסמונת X שבירה ותסמונת דאון 6 . הקוצים הדנדריטים הם תאים תת תאיים מיוחדים של דבורות arbors לקבל קלט מעורר בתוך מערכת העצבים המרכזית. ישנם שלושה סוגים מורפולוגיים של קוצים דנדריטים, עם שם של כל שיעור על פי גודלם וצורתם: 1) קוצים פטריות, אשר יש צפיפויות מורכבות postsynaptic עם קולטני גלוטמט יותר מאשר קוצים אחרים 7 ; 2) קוצים קטנטנים, שאין להם גזע; ו 3) קוצים דקים, המורכבת גזע מתמשך, צר ראש גלובלית 8 . נפח עמוד השדרה הדנדריטי משמש בחלקו להגדרתם, עם קוצים דקים בדרך כלל קטנים יותר (0.01 מיקרומטר 3 </sup&gt;) לעומת קוצים פטריות (0.8 מיקרומטר 3 ) 9 , 10 . הקוצים מתייצבים עם ההתבגרות. לדוגמה, קוצים דקים או נסוג לאחר כמה ימים או להתפתח פטיש spines. לחלופין, קוצים הפטרייה יציבים יחסית יכול לשרוד במשך תקופה ארוכה.החוזק של קשרים נוירונים הוא חשב להיות מבוסס על מספר הקוצים ו / או נפח שלהם 11 , 12 , 13 . שיטת הקולי הקלסית הקלאסית והווריאציות המודרניות יותר שלה היו מועילות לבחינת המורפולוגיה והצפיפות של עמוד השדרה הדנדריטים. היבט ייחודי אחד של מכתים Golgi היא כי כתמי אקראי על 5% מכלל הנוירונים, המאפשר מעקב אחר נוירונים בודדים 14 , 15 . אמנם את המנגנון המדויק שבו Golgi מתכתמי אוד נוירונים בודדים עדיין לא ידוע, העיקרון של השיטה מבוססת על התגבשות של כרומט כסף (Ag 2 CrO 4 ) 16 , 17 . ישנם שלושה סוגים עיקריים של שיטת Golgi: Golgi מהירה, Golgi-Cox, ואת Golgi-Kopsch 18 , 19 . כל שלוש שיטות להתחיל עם שלב הדגירה הראשונית של מלחי כרום במשך מספר ימים עד חודשים, אבל יש הבדלים מפתח מסוימים ביניהם. Golgi מהירה משתמשת tetroxide אוסמיום בשלב הראשון, ואילו Golgi-Kopsch כולל paraformaldehyde. מכתים הן מהיר Golgi ו Golgi-Kopsch ואחריו הדגירה של פתרון חנקתי 1-2% כסף במשך כ -7 ימים. שיטת Golgi-Cox משתמשת כלוריד כספית אשלגן dichromate במקום חנקתי כסף יש זמן הספגה של 2-4 שבועות. הרקמות נחתכות וממוקמות במהירות באמוניה מדוללתפתרון, ואחריו מסדר צילום כדי להסיר מלחים. מבין שלושת הסוגים, שיטת Golgy-Cox נחשבת הטובה ביותר להכתים את הנמלים הדנדריטים ללא הפרעות רקע רבות, בין היתר, משום שמוצרי הבדולח אינם מופיעים על פני הרקמה (בניגוד לשיטת Golgi המהירה) 17 , 20 , 21 . השיטה הנוכחית היא וריאציה קלה של הפרוטוקול שסופק עם ערכת מכתים Golgi מסחרי, והוא נועד להעריך כיצד מינון נמוך יחסית של 5-פו ישפיע על המאפיינים המורפולוגיים הדנדריטים וצפיפות עמוד השדרה. כל הנתונים שנרכשו יכולים לספק תובנה נוספת כיצד טיפול כימותרפי משפיע על המעגל העצבית.

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			<td height="21" style="height:21px;width:216px;">superGolgi Kit&#160;</td>
			<td style="width:207px;">Bioenno Lifesciences</td>
			<td style="width:119px;">30100</td>
			<td style="width:216px;">&#160;Contains hazardous materials.&#160;</td>
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			<td height="21" style="height:21px;width:216px;">PBS 10X powder concentrate</td>
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			<td style="width:119px;">BP665-1</td>
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			<td height="21" style="height:21px;width:216px;">Triton X-100</td>
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			<td height="21" style="height:21px;width:216px;">Permount&#160;</td>
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			<td height="21" style="height:21px;width:216px;">Slide cover&#160;</td>
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			<td height="21" style="height:21px;width:216px;">7mL Transfer pipette&#160;</td>
			<td style="width:207px;">Globe Scientific&#160;</td>
			<td style="width:119px;">135030</td>
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			<td height="21" style="height:21px;width:216px;">10 mL Falcon tubes&#160;</td>
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			<td height="21" style="height:21px;width:216px;">12-well plate&#160;</td>
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			<td height="21" style="height:21px;width:216px;">200 proof Ethanol&#160;</td>
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			<td height="21" style="height:21px;width:216px;">Permabond 200</td>
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			<td height="21" style="height:21px;width:216px;">25 mL serological pipette</td>
			<td style="width:207px;">Sigma</td>
			<td style="width:119px;">SIAL1489</td>
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			<td height="21" style="height:21px;width:216px;">Parafilm</td>
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			<td height="21" style="height:21px;width:216px;">C57Bl/6 Male Mice&#160;</td>
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assessment of hippocampal dendritic complexity in aged mice using the golgi-cox method

קוצים דנדריטים הם הבולטים של פירים דנדריטים נוירונים המכילים סינפסות מעוררות. ההבדלים המורפולוגיים וההסתעפויות של הדנדריטים העצביים בתוך ההיפוקמפוס מעורבים בהבנה קוגניטיבית וזיכרון. ישנן מספר גישות מכתים Golgi, אשר כולם היו שימושיים לקביעת המאפיינים המורפולוגיים של דנדריטים סוכות לייצר רקע ברור. השיטה הנוכחית Golgi-Cox, (וריאציה קלה של הפרוטוקול מסופק עם ערכת מכתים Golgi מסחרי), נועד להעריך כיצד מינון נמוך יחסית של התרופה chemotherapeutic 5-flurouracil (5-Fu) ישפיע על המורפולוגיה הדנדריטים , את מספר הקוצים, ואת המורכבות של arborization בתוך ההיפוקמפוס. 5-Fu מאופיינת באופן משמעותי את המורכבות הדנדריטים והורידה את צפיפות עמוד השדרה לאורך ההיפוקמפוס באופן ספציפי לאזור. הנתונים המוצגים מראים כי שיטת מכתים Golgi efמוכתמים בצורה נוקשה את הנוירונים הבוגרים ב- CA1, CA3, ואת gyrus משוננת (DG) של ההיפוקמפוס. פרוטוקול זה מדווח על הפרטים עבור כל צעד, כך שחוקרים אחרים יכולים לרקום כתם אמין ברחבי המוח עם תוצאות באיכות גבוהה ופתרון בעיות מינימלי.

ההשפעות של טיפול 5-Fu על arborization המורכבות הדנדריטים ואת ההיפוקמפוס של קטעי מוח מוכתמים Golgi היו לכמת ו traced באמצעות תוכנת הדמיה זמין מסחרית. לאחר המעקב, ניתחו את ההסתגלות הדנדריטית, את צפיפות עמוד השדרה ואת מורפולוגיה עמוד השדרה באמצעות ניתוח Sholl ומדד המ?...

Watch this Scientific Journal Video about Assessment of Hippocampal Dendritic Complexity in Aged Mice Using the Golgi-Cox Method at JoVE.com

Assessment of Hippocampal Dendritic Complexity in Aged Mice Using the Golgi-Cox Method

כאן אנו מציגים פרוטוקול Golgi-Cox בפירוט רב. זו שיטה אמינה רקמת כתם מאפשר הערכה איכותית גבוהה של cytoarchitecture בהיפוקמפוס, ובכל רחבי המוח כולו, עם פתרון מינימלי.

הערכת המורכבות בהיפוקמפוס דנדריטי בעכברים בגילאים בשיטת Golgi-Cox

Dendritic spines are the protuberances from the neuronal dendritic shafts that contain&#160; excitatory synapses. The morphological and branching ...

assessment-hippocampal-dendritic-complexity-aged-mice-using-golgi-cox

Research

JoVE Journal

Neuroscience

14.9K Views.  University of Arkansas for Medical Sciences.  כאן אנו מציגים פרוטוקול Golgi-Cox בפירוט רב. זו שיטה אמינה רקמת כתם מאפשר הערכה איכותית גבוהה של cytoarchitecture בהיפוקמפוס, ובכל רחבי המוח כולו, עם פתרון מינימלי. 

Video: Assessment of Hippocampal Dendritic Complexity in Aged Mice Using the Golgi-Cox Method

Assessment of Motor Balance and Coordination in Mice using the Balance Beam

Brain injury, genetic manipulations, and pharmacological treatments can result in alterations of motor skills in mice. Fine motor coordination and balance can be assessed by the beam walking assay. The goal of this test is for the mouse to stay upright and walk across an elevated narrow beam to a safe platform. This test takes place over 3 consecutive days: 2 days of training and 1 day of testing. Performance on the beam is quantified by measuring the time it takes for the mouse to traverse the beam and the number of paw slips that occur in the process. Here we report the protocol used in our laboratory, and representative results from a cohort of C57BL/6 mice. This task is particularly useful for detecting subtle deficits in motor skills and balance that may not be detected by other motor tests, such as the Rotarod.

Deficits in fine motor coordination can be assessed with the balance beam test. Performance on the beam is quantified by the speed at which the beam is traversed and the number of times the mouse slips on the beam.

הערכת מאזן מוטורי ותיאום עכברים באמצעות קרן איזון

Brain injury, genetic manipulations, and pharmacological treatments can result in alterations of motor skills in mice.  Fine motor coordination and ...

Assaying DNA Damage in Hippocampal Neurons Using the Comet Assay

A number of drugs target the DNA repair pathways and induce cell kill by creating DNA damage. Thus, processes to directly measure DNA damage have been extensively evaluated. Traditional methods are time consuming, expensive, resource intensive and require replicating cells. In contrast, the comet assay, a single cell gel electrophoresis assay, is a faster, non-invasive, inexpensive, direct and sensitive measure of DNA damage and repair. All forms of DNA damage as well as DNA repair can be visualized at the single cell level using this powerful technique.
	The principle underlying the comet assay is that intact DNA is highly ordered whereas DNA damage disrupts this organization. The damaged DNA seeps into the agarose matrix and when subjected to an electric field, the negatively charged DNA migrates towards the cathode which is positively charged. The large undamaged DNA strands are not able to migrate far from the nucleus. DNA damage creates smaller DNA fragments which travel farther than the intact DNA. Comet Assay, an image analysis software, measures and compares the overall fluorescent intensity of the DNA in the nucleus with DNA that has migrated out of the nucleus. Fluorescent signal from the migrated DNA is proportional to DNA damage. Longer brighter DNA tail signifies increased DNA damage. Some of the parameters that are measured are tail moment which is a measure of both the amount of DNA and distribution of DNA in the tail, tail length and percentage of DNA in the tail. This assay allows to measure DNA repair as well since resolution of DNA damage signifies repair has taken place. The limit of sensitivity is approximately 50 strand breaks per diploid mammalian cell 1,2. Cells treated with any DNA damaging agents, such as etoposide, may be used as a positive control. Thus the comet assay is a quick and effective procedure to measure DNA damage.

The comet assay is an efficient way of detecting single- and double-strand breaks, including alkali-labile sites and DNA-DNA/DNA-protein cross-links on the DNA in all cells including hippocampal neurons. The method takes advantage of the differential migration of DNA in an electric field due to differences in amount of DNA damage.

Assaying ניזק לדנ&quot;א בנוירונים ביפוקמפוס שימוש Assay השביט

A number of drugs target the DNA repair pathways and induce cell kill by creating DNA damage. Thus, processes to directly measure DNA damage have been ...

Optogenetic Perturbation of Neural Activity with Laser Illumination in Semi-intact Drosophila Larvae in Motion

Drosophila larval locomotion is a splendid model system in developmental and physiological neuroscience, by virtue of the genetic accessibility of the underlying neuronal components in the circuits1-6. Application of optogenetics7,8 in the larval neural circuit allows us to manipulate neuronal activity in spatially and temporally patterned ways9-13. Typically, specimens are broadly illuminated with a mercury lamp or LED, so specificity of the target neurons is controlled by binary gene expression systems such as the Gal4-UAS system14,15. In this work, to improve the spatial resolution to "sub-genetic resolution", we locally illuminated a subset of neurons in the ventral nerve cord using lasers implemented in a conventional confocal microscope. While monitoring the motion of the body wall of the semi-intact larvae, we interactively activated or inhibited neural activity with channelrhodopsin16,17 or halorhodopsin18-20, respectively. By spatially and temporally restricted illumination of the neural tissue, we can manipulate the activity of specific neurons in the circuit at a specific phase of behavior. This method is useful for studying the relationship between the activities of a local neural assembly in the ventral nerve cord and the spatiotemporal pattern of motor output.

Optogenetic Perturbation of Neural Activity with Laser Illumination in Semi-intact Drosophila Larvae in Motion

Here we describe a protocol for optogenetic manipulation of motoneuronal activity while monitoring changes in motor output (muscle contraction) in semi-intact Drosophila larvae using lasers within a conventional confocal microscope. This technique enables researchers to achieve local perturbation of neural activity in a few neuromeres to elucidate the dynamics of motor circuits.

הפרעות optogenetic של פעילות עצבית עם תאורת לייזר בחצי ללא פגע<em&gt; דרוזופילה</em&gt; זחלים בתנועה

Drosophila larval locomotion is a splendid model system in developmental and physiological neuroscience, by virtue of the genetic accessibility of the ...

Visualizing the Effects of a Positive Early Experience, Tactile Stimulation, on Dendritic Morphology and Synaptic Connectivity with Golgi-Cox Staining

To generate longer-term changes in behavior, experiences must be producing stable changes in neuronal morphology and synaptic connectivity. Tactile stimulation is a positive early experience that mimics maternal licking and grooming in the rat. Exposing rat pups to this positive experience can be completed easily and cost-effectively by using highly accessible materials such as a household duster. Using a cross-litter design, pups are either stroked or left undisturbed, for 15 min, three times per day throughout the perinatal period. To measure the neuroplastic changes related to this positive early experience, Golgi-Cox staining of brain tissue is utilized. Owing to the fact that Golgi-Cox impregnation stains a discrete number of neurons rather than all of the cells, staining of the rodent brain with Golgi-Cox solution permits the visualization of entire neuronal elements, including the cell body, dendrites, axons, and dendritic spines. The staining procedure is carried out over several days and requires that the researcher pay close attention to detail. However, once staining is completed, the entire brain has been impregnated and can be preserved indefinitely for ongoing analysis. Therefore, Golgi-Cox staining is a valuable resource for studying experience-dependent plasticity.

This paper describes the procedures for tactile stimulation of rat pups and subsequent Golgi-Cox staining of neuronal morphology. Tactile stimulation is a positive experience that is administered in the perinatal period by stroking pups with a household duster. Golgi-cox staining is a reliable procedure permitting the visualization of entire neurons.

לדמיין את ההשפעות של ניסיון מוקדם חיובי, מישוש גירוי, על דנדריטים מורפולוגיה וSynaptic קישוריות עם Golgi-קוקס מכתים

To generate longer-term changes in behavior, experiences must be producing stable changes in neuronal morphology and synaptic connectivity. Tactile ...

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.

מיקרו-Iontophoresis מהיר של גלוטמט וגאבא: כלי שימושי לחקירת האינטגרציה סינפטית

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

Imaging Dendritic Spines of Rat Primary Hippocampal Neurons using Structured Illumination Microscopy

Dendritic spines are protrusions emerging from the dendrite of a neuron and represent the primary postsynaptic targets of excitatory inputs in the brain. Technological advances have identified these structures as key elements in neuron connectivity and synaptic plasticity. The quantitative analysis of spine morphology using light microscopy remains an essential problem due to technical limitations associated with light's intrinsic refraction limit. Dendritic spines can be readily identified by confocal laser-scanning fluorescence microscopy. However, measuring subtle changes in the shape and size of spines is difficult because spine dimensions other than length are usually smaller than conventional optical resolution fixed by light microscopy's theoretical resolution limit of 200 nm.
Several recently developed super resolution techniques have been used to image cellular structures smaller than the 200 nm, including dendritic spines. These techniques are based on classical far-field operations and therefore allow the use of existing sample preparation methods and to image beyond the surface of a specimen. Described here is a working protocol to apply super resolution structured illumination microscopy (SIM) to the imaging of dendritic spines in primary hippocampal neuron cultures. Possible applications of SIM overlap with those of confocal microscopy. However, the two techniques present different applicability. SIM offers higher effective lateral resolution, while confocal microscopy, due to the usage of a physical pinhole, achieves resolution improvement at the expense of removal of out of focus light. In this protocol, primary neurons are cultured on glass coverslips using a standard protocol, transfected with DNA plasmids encoding fluorescent proteins and imaged using SIM. The whole protocol described herein takes approximately 2 weeks, because dendritic spines are imaged after 16-17 days in vitro, when dendritic development is optimal. After completion of the protocol, dendritic spines can be reconstructed in 3D from series of SIM image stacks using specialized software.

This article describes a working protocol to image dendritic spines from hippocampal neurons in vitro using Structured Illumination Microscopy (SIM). Super-resolution microscopy using SIM provides image resolution significantly beyond the light diffraction limit in all three spatial dimensions, allowing the imaging of individual dendritic spines with improved detail.

הדמיה קוצים הדנדריטים של העכברוש הראשי נוירונים בהיפוקמפוס באמצעות מובנה תאורה מיקרוסקופית

Dendritic spines are protrusions emerging from the dendrite of a neuron and represent the primary postsynaptic targets of excitatory inputs in the brain. ...

Assessment of Vascular Regeneration in the CNS Using the Mouse Retina

The rodent retina is perhaps the most accessible mammalian system in which to investigate neurovascular interplay within the central nervous system (CNS). It is increasingly being recognized that several neurodegenerative diseases such as Alzheimer&#8217;s, multiple sclerosis, and amyotrophic lateral sclerosis present elements of vascular compromise. In addition, the most prominent causes of blindness in pediatric and working age populations (retinopathy of prematurity and diabetic retinopathy, respectively) are characterized by vascular degeneration and failure of physiological vascular regrowth. The aim of this technical paper is to provide a detailed protocol to study CNS vascular regeneration in the retina. The method can be employed to elucidate molecular mechanisms that lead to failure of vascular growth after ischemic injury. In addition, potential therapeutic modalities to accelerate and restore healthy vascular plexuses can be explored. Findings obtained using the described approach may provide therapeutic avenues for ischemic retinopathies such as that of diabetes or prematurity and possibly benefit other vascular disorders of the CNS.

The rodent retina has long been recognized as an accessible window to the brain. In this technical paper we provide a protocol that employs the mouse model of oxygen-induced retinopathy to study the mechanisms that lead to failure of vascular regeneration within the central nervous system after ischemic injury. The described system can also be harnessed to explore strategies to promote regrowth of functional blood vessels within the retina and CNS.

הערכה של כלי דם התחדשות במערכת העצבים המרכזית באמצעות רשתית העכבר

The rodent retina is perhaps the most accessible mammalian system in which to investigate neurovascular interplay within the central nervous system (CNS). ...

Assessment of Dendritic Arborization in the Dentate Gyrus of the Hippocampal Region in Mice

Dendritic arborization has been shown to be a reliable marker for examination of structural and functional integrity of neurons. Indeed, the complexity and extent of dendritic arborization correlates well with the synaptic plasticity in these cells. A reliable method for assessment of dendritic arborization is needed to characterize the deleterious effects of neurological disorders on these structures and to determine the effects of therapeutic interventions. However, quantification of these structures has proven to be a formidable task given their complex and dynamic nature. Fortunately, sophisticated imaging techniques can be paired with conventional staining methods to assess the state of dendritic arborization, providing a more reliable and expeditious means of assessment. Below is an example of how these imaging techniques were paired with staining methods to characterize the dendritic arborization in wild type mice. These complementary imaging methods can be used to qualitatively and quantitatively assess dendritic arborization that span a rather wide area within the hippocampal region.

We describe two methods for visualization and quantification of dendritic arborization in the hippocampus of mouse models: real-time and extended depth of field imaging. While the former method allows sophisticated topographical tracing and quantification of the extent of branching, the latter allows speedy visualization of the dendritic tree.

הערכה של דנדריטים arborization ברכס המשונן של האזור בהיפוקמפוס של עכברים

Dendritic arborization has been shown to be a reliable marker for examination of structural and functional integrity of neurons. Indeed, the complexity ...

A Simple and Inexpensive Method for Determining Cold Sensitivity and Adaptation in Mice

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life. The cold plantar assay allows the objective and inexpensive assessment of cold sensitivity in mice, and can quantify both analgesia and hypersensitivity. Mice are acclimated on a glass plate, and a compressed dry ice pellet is held against the glass surface underneath the hindpaw. The latency to withdrawal from the cooling glass is used as a measure of cold sensitivity.
Cold sensation is also important for survival in regions with seasonal temperature shifts, and in order to maintain sensitivity animals must be able to adjust their thermal response thresholds to match the ambient temperature. The Cold Plantar Assay (CPA) also allows the study of adaptation to changes in ambient temperature by testing the cold sensitivity of mice at temperatures ranging from 30 &#176;C to 5 &#176;C. Mice are acclimated as described above, but the glass plate is cooled to the desired starting temperature using aluminum boxes (or aluminum foil packets) filled with hot water, wet ice, or dry ice. The temperature of the plate is measured at the center using a filament T-type thermocouple probe. Once the plate has reached the desired starting temperature, the animals are tested as described above.
This assay allows testing of mice at temperatures ranging from innocuous to noxious. The CPA yields unambiguous and consistent behavioral responses in uninjured mice and can be used to quantify both hypersensitivity and analgesia. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

The Cold Plantar Assay (CPA) measures cold responsiveness between 30 &#176;C and 5 &#176;C, and can also measure cold adaptation. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

שיטה פשוטה וזולה לקביעה קרה רגישות והסתגלות בעכברים

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life.  The cold ...

Fluorescence and Bioluminescence Imaging of Subcellular Ca2+ in Aged Hippocampal Neurons

Susceptibility to neuron cell death associated to neurodegeneration and ischemia are exceedingly increased in the aged brain but mechanisms responsible are badly known. Excitotoxicity, a process believed to contribute to neuron damage induced by both insults, is mediated by activation of glutamate receptors that promotes Ca2+ influx and mitochondrial Ca2+ overload. A substantial change in intracellular Ca2+ homeostasis or remodeling of intracellular Ca2+ homeostasis may favor neuron damage in old neurons. For investigating Ca2+ remodeling in aging we have used live cell imaging in long-term cultures of rat hippocampal neurons that resemble in some aspects aged neurons in vivo. For this end, hippocampal cells are, in first place, freshly dispersed from new born rat hippocampi and plated on poli-D-lysine coated, glass coverslips. Then cultures are kept in controlled media for several days or several weeks for investigating young and old neurons, respectively. Second, cultured neurons are loaded with fura2 and subjected to measurements of cytosolic Ca2+ concentration using digital fluorescence ratio imaging. Third, cultured neurons are transfected with plasmids expressing a tandem of low-affinity aequorin and GFP targeted to mitochondria. After 24 hr, aequorin inside cells is reconstituted with coelenterazine and neurons are subjected to bioluminescence imaging for monitoring of mitochondrial Ca2+ concentration. This three-step procedure allows the monitoring of cytosolic and mitochondrial Ca2+ responses to relevant stimuli as for example the glutamate receptor agonist NMDA and compare whether these and other responses are influenced by aging. This procedure may yield new insights as to how aging influence cytosolic and mitochondrial Ca2+ responses to selected stimuli as well as the testing of selected drugs aimed at preventing neuron cell death in age-related diseases.

Fluorescence and Bioluminescence Imaging of Subcellular Ca2+ in Aged Hippocampal Neurons

Intracellular Ca2+ remodeling in aging may contribute to excitotoxicity and neuron damage, processes mediated by Ca2+ overload. We aimed at investigating Ca2+ remodeling in the aging brain using fluorescence and bioluminescence imaging of cytosolic and mitochondrial Ca2+ in long-term cultures of rat hippocampal neurons, a model of neuronal aging.

הקרינה ופליטת אור הדמיה של Ca subcellular<sup&gt; 2 +</sup&gt; בבני נוירונים בהיפוקמפוס

Susceptibility to neuron cell death associated to neurodegeneration and ischemia are exceedingly increased in the aged brain but mechanisms responsible ...

הערכת המורכבות בהיפוקמפוס דנדריטי בעכברים בגילאים בשיטת Golgi-Cox

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