This work was supported by Ministerio de Econom&#236;a y competitividad (BFU2012-37146) and Junta de Castilla y Le&#242;n (BIO103/VA45/11, VA145U13 and BIO/VA33/13), Spain. MCR was supported by Junta de Castilla y Le&#242;n (Spain) and the European Social Fund. We thank the late Dr. Philippe Br&#251;let (1947-2013) for the mitochondrial GFP Aequorin plasmid.

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None of the authors have competing interests or conflicting interests.

שיפוץ של Ca 2 + הומאוסטזיס תאיים במוח המזדקן כבר קשור לאובדן הקוגניטיבי, הגדיל את הרגישות לנזק איסכמי, הפעלת יתר רעילה וניוון של מערכת עצבים. כדי לחקור את ההשערה הזאת במבחנה, נהלי ההדמיה Ca 2 + זמינים. למרבה הצער, תרבויות קיימא של נוירונים בהיפוקמפוס ישנים...

הפעלת יתר רעילה היא אחד המנגנונים החשובים ביותר שתרמו לנזק עצבי ומוות של תאים בעלבונות נוירולוגיות כגון איסכמיה, ובחלק ממחלות ניווניות כגון מחלת אלצהיימר 1. סוג זה של רעילויות בעיקר בתיווכו של משחק גלוטמט על Ca 2 +, קולטני -permeable ionotropic NMDA (NMDAR) 2. חשיפה של נוירונים בתרבית לגלוטמט יכולה להוביל להפעלת יתר רעילה 3, מה שגורם לאפופטוזיס העצבי 4. אנו ואחרים דיווחו בעבר כי פגיעות עצביות לאפופטוזיס מושרה NMDA יכולות להשתנות עם התפתחות במבחנה והזדקנות 5-8. זה מקובל שעלייה בריכוז Ca ללא cytosolic 2 + (CYT [Ca 2 +]) מובילה להפעלת תאים. עם זאת, אם עלייה זו היא גבוהה מדי ו / או ספג מספיק, זה יכול לגרום לתא מוות 9. יתר על כן, זה כבר הציעהפעלת יתר רעילה שדורשת Ca 2 + 10 המיטוכונדריה ספיגה, מאז טיפול נוירונים עם נוירונים uncoupler המיטוכונדריה מוגנים מפני מוות של תאים הנגרם גלוטמט 11. אם מיטוכונדריה תופסת הרבה יותר מדי Ca 2 +, הפתיחה הנקבובית מעבר חדירות המיטוכונדריה עלולה להתרחש, שהובילה לשחרורו של ציטוכרום C וגורמים פרו-אפופטוטיים אחרים, ואפופטוזיס התרמה. לאחרונה הראה כי ספיגת Ca 2 + המיטוכונדריה זה קשור באופן ישיר לרגישות תלוית גיל להפעלת יתר רעיל, על ידי מדידה ישירה Ca 2 + ספיגה מושרה NMDA המיטוכונדריה בתאי עצב בהיפוקמפוס יחיד 5, שיטה שבה מדווחת במאמר זה. ההיפוקמפוס, מעורב בתהליכים פיסיולוגיים כגון למידה, זיכרון ותהליכים קוגניטיביים אחרים 12, הוא פגיע מאוד להזדקנות והפרעות ניווניות 13. זה כבר הציע, לאחר מספר שבועות במבחנה, בתרביתנוירונים בהיפוקמפוס להראות מספר המאפיינים הטיפוסיים של נוירונים בגיל 14. בהתאם לכך, תאי עצב בהיפוקמפוס תרבותיים ארוך טווח עשוי לספק מודל מקיף כדי לחקור Ca 2 + מנגנוני תיווך של הפעלת יתר רעילה משופר בהזדקנות. המטרה הכוללת של השיטה שהוצגה היא, אפוא, לחקור שינויים מהותיים בCa 2 + הומאוסטזיס תאיים או שיפוץ Ca 2 + במוח המזדקן כולל תגובות ההפרש Ca 2 + שהושרו על ידי אגוניסטים הקולטן NMDA בנוירונים בהיפוקמפוס תרבותיים לטווח ארוך . השיטה כוללת תיאור מפורט של התרבות של תאי עצב בהיפוקמפוס חולדה והניטור של 2 + ריכוזי Ca cytosolic והמיטוכונדריה על ידי הקרינה והדמיה פליטת אור בנוירונים בודדים, בהתאמה. הדמיה הקרינה של 2 + Ca cytosolic בנוירונים בתרבית היא הליך סטנדרטי. עם זאת, שיטה זו היא פחות אמינה למשנההסלולרי Ca 2 + מדידות כוללים Ca 2 + המיטוכונדריה. סיבות לכך כוללות חוסר המיקוד הנכון של בדיקות סינתטיות וזיקה הולמת לCa 2 + ריכוזים שעשויות לשנות במיטוכונדריה מרמת מיקרומטר הנמוכה אפילו לרמת מ&quot;מ. השימוש בבדיקות 2 + Ca מבוססים על חלבונים כלaequorin למשל, אפשרו מיקוד לאברונים subcellular ושימוש בנגזרים שונים Ca 2 + זיקות באמצעות coelenterazines שונה או בדיקות שעברו מוטציה חסרת Ca 2 + אתרי קישור ספציפי 15. בדרך זו, הדמיה פליטת אור של תאים לבטא aequorin-ממוקד מיטוכונדריה עשויה לאפשר הניטור של Ca 2 + ריכוזים של המיטוכונדריה בתאי עצב בודד. עם זאת, הליך זה עשוי לדרוש שימוש במצלמות ספירת פוטון או מצלמות CCD רגישות להדמיה פליטת אור 16-18. שיטה זו עשויה להניב תוצאות רומן שצריך להיות מאושרת יותר establמודלים ished הזדקנות המוח כמו, למשל, פרוסות מוח מבעלי חיים ישנים.

<table>
	<tbody>
		<tr>
			<td>Neurobasal Culture Medium</td>
			<td>Gibco</td>
			<td>21103-049</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS medium</td>
			<td>Gibco</td>
			<td>14170-088</td>
			<td></td>
		</tr>
		<tr>
			<td>Ham&#39;s F-12 medium</td>
			<td>Gibco</td>
			<td>31330-038</td>
			<td></td>
		</tr>
		<tr>
			<td>DNase I (from bovine pancreas)</td>
			<td>Sigma</td>
			<td>D5025-15KU</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bobine Serum</td>
			<td>Lonza</td>
			<td>DE14-801E</td>
			<td></td>
		</tr>
		<tr>
			<td>B27</td>
			<td>Gibco</td>
			<td>17504-044</td>
			<td></td>
		</tr>
		<tr>
			<td>Gentamicin</td>
			<td>Gibco</td>
			<td>15750</td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Gibco</td>
			<td>25030-032</td>
			<td></td>
		</tr>
		<tr>
			<td>12 mm glass coverslips</td>
			<td>Labolan</td>
			<td>0111520/20012</td>
			<td></td>
		</tr>
		<tr>
			<td>Papain</td>
			<td>Worthington</td>
			<td>LS003127</td>
			<td></td>
		</tr>
		<tr>
			<td>4-well multidish plaques</td>
			<td>Nunc</td>
			<td>176740</td>
			<td></td>
		</tr>
		<tr>
			<td>Petry dishes</td>
			<td>JD Catalan s.l.</td>
			<td>2120044T</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile pipettes</td>
			<td>Fisher Scientific</td>
			<td>431030</td>
			<td></td>
		</tr>
		<tr>
			<td>Fura2-AM</td>
			<td>Life Technologies</td>
			<td>F1201</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipofectamine2000</td>
			<td>Invitrogen</td>
			<td>11668-027</td>
			<td></td>
		</tr>
		<tr>
			<td>Coelenterazine n</td>
			<td>Biotium</td>
			<td>BT-10115-2250 uG</td>
			<td></td>
		</tr>
		<tr>
			<td>Digitonin</td>
			<td>Sigma</td>
			<td>D5628</td>
			<td></td>
		</tr>
		<tr>
			<td>NMDA</td>
			<td>Sigma&#160;</td>
			<td>M3262</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Sigma</td>
			<td>50046</td>
			<td></td>
		</tr>
		<tr>
			<td>Zeiss Axiovert S100 TV microscope</td>
			<td>Carl Zeiss Inc.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Xcite ilumination system</td>
			<td>EXFO</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ORCA ER fluorescence camera</td>
			<td>Hamamatsu</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>VIM photon counting CCD camera</td>
			<td>Hamamatsu</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>VC-8 valve controller</td>
			<td>Warner Instruments</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SH-27B heating system&#160;</td>
			<td>Warner Instruments</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Aquacosmos Software</td>
			<td>Hamamatsu Photonics</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

fluorescence and bioluminescence imaging of subcellular ca2+ in aged hippocampal neurons

רגישות למוות של תאי עצב הקשורים לניוון של מערכת עצבים ואיסכמיה גדלה מאוד במוח בגיל אבל מנגנונים אחראים ידועים קשה. הפעלת יתר רעילה, תהליך האמין לתרום לניזק הנגרם על ידי תא עצב שני העלבונות, מתווכת על ידי הפעלה של קולטני גלוטמט שמקדם Ca 2 + זרם ועומס Ca 2 + המיטוכונדריה. שינוי מהותי בCa 2 + הומאוסטזיס או שיפוץ תאיים של הומאוסטזיס Ca 2 + תאיים עשוי להעדיף נזק נוירון בנוירונים ישנים. לחקירת שיפוץ Ca 2 + בהזדקנות השתמשנו הדמיה תא חי בתרבויות ארוכת טווח של תאי עצב בהיפוקמפוס העכברוש שדומות בכמה היבטים בגילי נוירונים in vivo. לשם כך, תאים בהיפוקמפוס הם, במקום הראשון, התפזרו טרי מhippocampi החולדה נולדה חדשה ומצופה על coverslips מצופה, זכוכית Poli-D ליזין. אז תרבויות נשמרות בתקשורת מבוקרת במשך כמה ימים או כמה weeks לחקירת נוירונים צעירים וזקנים, בהתאמה. שנית, נוירונים בתרבית נטענים עם fura2 ונתונים למדידות של ריכוז Ca 2 + cytosolic באמצעות הדמיה יחס הקרינה דיגיטלית. שלישית, נוירונים בתרבית הם transfected עם פלסמידים להביע טנדם של aequorin נמוכה הזיקה וGFP הממוקד למיטוכונדריה. לאחר 24 שעות, aequorin בתוך תאים מחדש עם coelenterazine ונוירונים חשופים להדמית פליטת אור לניטור של ריכוז Ca 2 + המיטוכונדריה. הליך בן שלושת שלבים זה מאפשר הניטור של cytosolic ותגובות Ca 2 + המיטוכונדריה לגירויים רלוונטיים כמו למשל אגוניסט קולטן גלוטמט NMDA ולהשוות בין תגובות אלה ואחרים מושפעות הזדקנות. הליך זה עשוי להניב תובנות חדשות באשר לאופן הזדקנות cytosolic השפעה ותגובות המיטוכונדריה Ca 2 + לגירויים שנבחרו, כמו גם הבדיקות של תרופות שנבחרו נועדו למנוע תא עצבמוות במחלות הקשורות לגיל.

כאן אנו מתארים שיטה פשוטה להעריך שיפוץ Ca 2 + ואת ההשפעות של NMDA בcytosolic והמיטוכונדריה [Ca 2 +] בתאי עצב בגיל. איור 1 מציג את סכמטי של הליך הבידוד וculturing תאי עצב בהיפוקמפוס של חולדות בילוד. לפני שמתחיל, אנחנו צריכים להכין coverslips סטרילי, פולי-D ליזין המצופה, ז?...

Watch this Scientific Journal Video about Fluorescence and Bioluminescence Imaging of Subcellular Ca2+ in Aged Hippocampal Neurons at JoVE.com

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 ...

fluorescence-bioluminescence-imaging-subcellular-ca2-aged-hippocampal

Research

JoVE Journal

Neuroscience

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

8.3K Views.  Consejo Superior de Investigaciones Cientìficas. 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. 

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

Nucleofection and Primary Culture of Embryonic Mouse Hippocampal and Cortical Neurons

Hippocampal and cortical neurons have been used extensively to study central nervous system (CNS) neuronal polarization, axon/dendrite outgrowth, and synapse formation and function. An advantage of culturing these neurons is that they readily polarize, forming distinctive axons and dendrites, on a two dimensional substrate at very low densities. This property has made them extremely useful for determining many aspects of neuronal development. Furthermore, by providing glial conditioning for these neurons they will continue to develop, forming functional synaptic connections and surviving for several months in culture. In this protocol we outline a technique to dissect, culture and transfect embryonic mouse hippocampal and cortical neurons. Transfection is accomplished by electroporating DNA into the neurons before plating via nucleofection. This protocol has the advantage of expressing fluorescently-tagged fusion proteins early in development (~4-8hrs after plating) to study the dynamics and function of proteins during polarization, axon outgrowth and branching. We have also discovered that this single transfection before plating maintains fluorescently-tagged fusion protein expression at levels appropriate for imaging throughout the lifetime of the neuron (&gt; 2 months in culture). Thus, this methodology is useful for studying protein localization and function throughout CNS development with little or no disruption of neuronal function.

This protocol outlines the steps required to dissect, transfect via electroporation and culture mouse hippocampal and cortical neurons. Short-term cultures may be used for studies of axon outgrowth and guidance, while long-term cultures can be used for studies of synaptogenesis and dendritic spine analysis.

Nucleofection ותרבות ראשונית של נוירונים בהיפוקמפוס עכבר עובריים קליפתיים

Hippocampal and cortical neurons have been used extensively to study central nervous system (CNS) neuronal polarization, axon/dendrite outgrowth, and ...

Fluorescence Recovery After Photobleaching (FRAP) of Fluorescence Tagged Proteins in Dendritic Spines of Cultured Hippocampal Neurons

FRAP has been used to quantify the mobility of GFP-tagged proteins. Using a strong excitation laser, the fluorescence of a GFP-tagged protein is bleached in the region of interest. The fluorescence of the region recovers when the unbleached GFP-tagged protein from outside of the region diffuses into the region of interest. The mobility of the protein is then analyzed by measuring the fluorescence recovery rate. This technique could be used to characterize protein mobility and turnover rate.
In this study, we express the (enhanced green fluorescent protein) EGFP vector in cultured hippocampal neurons. Using the Zeiss 710 confocal microscope, we photobleach the fluorescence signal of the GFP protein in a single spine, and then take time lapse images to record the fluorescence recovery after photobleaching. Finally, we estimate the percentage of mobile and immobile fractions of the GFP in spines, by analyzing the imaging data using ImageJ and Graphpad softwares.
This FRAP protocol shows how to perform a basic FRAP experiment as well as how to analyze the data.

Fluorescence Recovery After Photobleaching FRAP of Fluorescence Tagged Proteins in Dendritic Spines of Cultured Hippocampal Neurons

FRAP has been used to quantify the mobility of Green Fluorescence Protein (GFP)-tagged proteins in cultured cells. We examined the mobile/immobile fractions of the GFP by analyzing the fluorescence recovery percentage after photobleaching. In this study, FRAP was performed at spines of hippocampal neurons.

שחזור הקרינה לאחר photobleaching (FRAP) של החלבונים המתויגים הקרינה הקוצים הדנדריטים של נוירונים בהיפוקמפוס Cultured

FRAP has been used to quantify the mobility of GFP-tagged proteins. Using a strong excitation laser, the fluorescence of a GFP-tagged protein is bleached ...

Neuromodulation and Mitochondrial Transport: Live Imaging in Hippocampal Neurons over Long Durations

To understand the relationship between mitochondrial transport and neuronal function, it is critical to observe mitochondrial behavior in live cultured neurons for extended durations1-3. This is now possible through the use of vital dyes and fluorescent proteins with which cytoskeletal components, organelles, and other structures in living cells can be labeled and then visualized via dynamic fluorescence microscopy. For example, in embryonic chicken sympathetic neurons, mitochondrial movement was characterized using the vital dye rhodamine 1234. In another study, mitochondria were visualized in rat forebrain neurons by transfection of mitochondrially targeted eYFP5. However, imaging of primary neurons over minutes, hours, or even days presents a number of issues. Foremost among these are: 1) maintenance of culture conditions such as temperature, humidity, and pH during long imaging sessions; 2) a strong, stable fluorescent signal to assure both the quality of acquired images and accurate measurement of signal intensity during image analysis; and 3) limiting exposure times during image acquisition to minimize photobleaching and avoid phototoxicity.
Here, we describe a protocol that permits the observation, visualization, and analysis of mitochondrial movement in cultured hippocampal neurons with high temporal resolution and under optimal life support conditions. We have constructed an affordable stage-top incubator that provides good temperature regulation and atmospheric gas flow, and also limits the degree of media evaporation, assuring stable pH and osmolarity. This incubator is connected, via inlet and outlet hoses, to a standard tissue culture incubator, which provides constant humidity levels and an atmosphere of 5-10% CO2/air. This design offers a cost-effective alternative to significantly more expensive microscope incubators that don't necessarily assure the viability of cells over many hours or even days. To visualize mitochondria, we infect cells with a lentivirus encoding a red fluorescent protein that is targeted to the mitochondrion. This assures a strong and persistent signal, which, in conjunction with the use of a stable xenon light source, allows us to limit exposure times during image acquisition and all but precludes photobleaching and phototoxicity. Two injection ports on the top of the stage-top incubator allow the acute administration of neurotransmitters and other reagents intended to modulate mitochondrial movement. In sum, lentivirus-mediated expression of an organelle-targeted red fluorescent protein and the combination of our stage-top incubator, a conventional inverted fluorescence microscope, CCD camera, and xenon light source allow us to acquire time-lapse images of mitochondrial transport in living neurons over longer durations than those possible in studies deploying conventional vital dyes and off-the-shelf life support systems.

We describe a protocol that allows imaging of mitochondria in living neurons via fluorescence microscopy over long durations. Imaging over extended periods is accomplished through lentivirus-mediated expression of a mitochondrially targeted fluorescent protein and use of an inexpensive stage-top incubator that was designed and built in our laboratory.

Neuromodulation ותחבורה מיטוכונדריאלי: הדמיה חיה נוירונים בהיפוקמפוס פני משכי זמן ארוכים

To understand the relationship between mitochondrial transport and neuronal function, it is critical to observe mitochondrial behavior in live cultured ...

Isolation and Culture of Hippocampal Neurons from Prenatal Mice

Primary cultures of rat and murine hippocampal neurons are widely used to reveal cellular mechanisms in neurobiology. By isolating and growing individual neurons, researchers are able to analyze properties related to cellular trafficking, cellular structure and individual protein localization using a variety of biochemical techniques. Results from such experiments are critical for testing theories addressing the neural basis of memory and learning. However, unambiguous results from these forms of experiments are predicated on the ability to grow neuronal cultures with minimum contamination by other brain cell types. In this protocol, we use specific media designed for neuron growth and careful dissection of embryonic hippocampal tissue to optimize growth of healthy neurons while minimizing contaminating cell types (i.e. astrocytes). Embryonic mouse hippocampal tissue can be more difficult to isolate than similar rodent tissue due to the size of the sample for dissection. We show detailed dissection techniques of hippocampus from embryonic day 19 (E19) mouse pups. Once hippocampal tissue is isolated, gentle dissociation of neuronal cells is achieved with a dilute concentration of trypsin and mechanical disruption designed to separate cells from connective tissue while providing minimum damage to individual cells. A detailed description of how to prepare pipettes to be used in the disruption is included. Optimal plating densities are provided for immuno-fluorescence protocols to maximize successful cell culture. The protocol provides a fast (approximately 2 hr) and efficient technique for the culture of neuronal cells from mouse hippocampal tissue.

We provide a protocol for the culture of highly purified hippocampal neurons from prenatal mouse brains without the use of a feeder glial cell layer.

בידוד והתרבות של נוירונים בהיפוקמפוס מעכברים טרום לידתי

Primary cultures of rat and murine hippocampal neurons are widely used to reveal cellular mechanisms in neurobiology. By isolating and growing individual ...

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 ...

Calcium Phosphate Transfection of Primary Hippocampal Neurons

Calcium phosphate precipitation is a convenient and economical method for transfection of cultured cells. With optimization, it is possible to use this method on hard-to-transfect cells like primary neurons. Here we describe our detailed protocol for calcium phosphate transfection of hippocampal neurons cocultured with astroglial cells.

סידן פוספט Transfection של ראשוני נוירונים בהיפוקמפוס

Calcium phosphate precipitation is a convenient and economical method for transfection of cultured cells. With optimization, it is possible to use this ...

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. ...

In Situ Ca2+ Imaging of the Enteric Nervous System

Reflex behaviors of the intestine are controlled by the enteric nervous system (ENS). The ENS is an integrative network of neurons and glia in two ganglionated plexuses housed in the gut wall. Enteric neurons and enteric glia are the only cell types within the enteric ganglia. The activity of enteric neurons and glia is responsible for coordinating intestinal functions. This protocol describes methods for observing the activity of neurons and glia within the intact ENS by imaging intracellular calcium (Ca2+) transients with fluorescent indicator dyes. Our technical discussion focuses on methods for Ca2+ imaging in whole-mount preparations of the myenteric plexus from the rodent bowel. Bulk loading of ENS whole-mounts with a high-affinity Ca2+ indicator such as Fluo-4 permits measurements of Ca2+ responses in individual neurons or glial cells. These responses can be evoked repeatedly and reliably, which permits quantitative studies using pharmacological tools. Ca2+ responses in cells of the ENS are recorded using a fluorescence microscope equipped with a cooled charge-coupled device (CCD) camera. Fluorescence measurements obtained using Ca2+ imaging in whole-mount preparations offer a straightforward means of characterizing the mechanisms and potential functional consequences of Ca2+ responses in enteric neurons and glial cells.

In Situ Ca2+ Imaging of the Enteric Nervous System

The enteric nervous system (ENS) is a network of neurons and glia located in the gut wall that controls intestinal reflexes. This protocol describes methods for recording the activity of enteric neurons and glia in live preparations of ENS using Ca2+ imaging.

בהדמיה 2 + Ca Situ של מערכת העצבים המעיים

Reflex behaviors of the intestine are controlled by the enteric nervous system (ENS). The ENS is an integrative network of neurons and glia in two ...

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

Dendritic spines are the protuberances from the neuronal dendritic shafts that contain&#160; excitatory synapses. The morphological and branching variations of the neuronal dendrites within the hippocampus are implicated in cognition and memory formation. There are several approaches to Golgi staining, all of which have been useful for determining the morphological characteristics of dendritic arbors and produce a clear background. The present Golgi-Cox method, (a slight variation of the protocol that is provided with a commercial Golgi staining kit), was designed to assess how a relatively low dose of the chemotherapeutic drug 5-flurouracil (5-Fu) would affect dendritic morphology, the number of spines, and the complexity of arborization within the hippocampus. The 5-Fu significantly modulated the dendritic complexity and decreased the spine density throughout the hippocampus in a region-specific manner. The data presented show that the Golgi staining method effectively stained the mature neurons in the CA1, the CA3, and the dentate gyrus (DG) of the hippocampus. This protocol reports the details for each step so that other researchers can reliably stain tissue throughout the brain with high quality results and minimal troubleshooting.

Here we present a Golgi-Cox protocol in extensive detail. This reliable tissue stain method allows for a high-quality assessment of the cytoarchitecture in the hippocampus, and throughout the entire brain, with minimal troubleshooting.

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 ...

Measuring Synaptic Vesicle Endocytosis in Cultured Hippocampal Neurons

During endocytosis, fused synaptic vesicles are retrieved at nerve terminals, allowing for vesicle recycling and thus the maintenance of synaptic transmission during repetitive nerve firing. Impaired endocytosis in pathological conditions leads to decreases in synaptic strength and brain functions. Here, we describe methods used to measure synaptic vesicle endocytosis at the mammalian hippocampal synapse in neuronal culture. We monitored synaptic vesicle protein endocytosis by fusing a synaptic vesicular membrane protein, including synaptophysin and VAMP2/synaptobrevin, at the vesicular lumenal side, with pHluorin, a pH-sensitive green fluorescent protein that increases its fluorescence intensity as the pH increases. During exocytosis, vesicular lumen pH increases, whereas during endocytosis vesicular lumen pH is re-acidified. Thus, an increase of pHluorin fluorescence intensity indicates fusion, whereas a decrease indicates endocytosis of the labelled synaptic vesicle protein. In addition to using the pHluorin imaging method to record endocytosis, we monitored vesicular membrane endocytosis by electron microscopy (EM) measurements of Horseradish peroxidase (HRP) uptake by vesicles. Finally, we monitored the formation of nerve terminal membrane pits at various times after high potassium-induced depolarization. The time course of HRP uptake and membrane pit formation indicates the time course of endocytosis.

Synaptic vesicle endocytosis is detected by light microscopy of pHluorin fused with synaptic vesicle protein and by electron microscopy of vesicle uptake.