This project was supported by grant MOP62827 from the Canadian Institutes of Health Research.

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The authors have nothing to disclose.

פרמטרים חשובים לקחת בחשבון בעת ​​התאמת טכניקה זו כוללים את האוכלוסייה העצבית שתילמד, מצע, גודל נקבובית של המסנן ועל פני שטח. כל הפרמטרים הללו ישפיעו על הספציפיות, איכות וכמות של הכנת neurite שהושגה, ויש לשקול בזהירות על ידי משתמש הקצה. בדוגמאות שהוצגו בפרוטוקול זה, השימ?...

תא אקסון של נוירונים מראה מידה רבה של עצמאות תפקודית מתא somato-הדנדריטים. בנוירונים הקרנה, אקסונים מכילים את רוב 1,2 החלבון העצבי. חקר הפיסיולוגיה והפתופיזיולוגיה של אקסונים צבר תאוצה בקהילת מדעי המוח, כי מחקרים שנעשה לאחרונה הראו כי תפקוד לקוי של אקסון המוקדם נראה כי תכונה משותפת של מחלות ניווניות והפרעות נוירופסיכיאטריות 3. סביר להניח כי הבנה טובה יותר של מנגנוני אקסון בלעדי בתנאים נורמלים ופתולוגית שתשפוך אור על האירועים המוקדמים המניע את חוסר תפקוד. הפרוטוקול הנוכחי מתאר כיצד להשתמש בתרבות מוסיף נושאות קרום נקבובי (מסנן) כדי להשיג כמויות גדולות של חומר אקסון טהור כי הוא מתאים ביוכימי וimmunocytochemical מנתח. השימוש במסנן מוסיף ללמוד הביולוגיה אקסון יושמה לראשונה על ידי דייל ועמיתים 4 ופותחו עוד יותר על ידי Twiss ועמיתים 5 לתצורתו הנוכחית. הטכניקה היא כעת בשימוש שוטף על ידי קבוצות לימוד היבטים שונים של אקסון והביולוגיה דנדריט, עם שינויים קלים בכל מקרה כדי להתאים לאוכלוסייה של נוירונים למדו, הטיפולים שבוצעו ואת הסוג של ניתוחים ביוכימיים המשמשים 6-9. הפרוטוקול הנוכחי אינו מתכוון להכיל את כל החלופות למגוון כה רחב של יישומים, אלא מספק גישה פשוטה, שמתאימה למרבית היישומים. בפרט, הפרוטוקול המתואר כאן משתמש בציפוי משולש למיקסום תשואת אקסון עבור ניתוחים ביוכימיים והוא מתאים ביותר ללמוד אקסון ציפויי ניוון-אחר המתוארים בספרות המיוצר פחות אקסונים שיחזרו ולהידרדר מהר יותר כאשר נשללו מגורמים תזונתיים 9. בהליך זה, גופי תא עצביים נותרו מבודדים על גבי עלי לעשות סינוןאקסונים ile לעבור הדרך הנקבוביות ולגדול לאורך המשטח התחתון שלה. אקסונים טהורים (ללא גליה ומזהמים גוף תא עצביים) הגדלים על המשטח התחתון של המסנן יכולים להיות שנאספו עבור ניתוחים ביוכימיים או יכולים להיות קבוע באתר ונבחנו על ידי טכניקות immunocytochemical 9. ההליך מסתמך על השימוש בתאי עצב תחושתי עובריים מהגרעינים הגבי שורש (DRG). DRGs עוברי נמצא בשימוש נרחב ללמוד ביולוגיה אקסון בגלל neurites מהתאים אלה עוברות צמיחה מהירה וחזקה במבחנה, כאשר נשמר בגורם גדילה עצבי (NGF), וכי הם במהירות לעבור ניוון כאשר נשללו של גורם זה. כמו כן, הנוירונים DRG חסרי דנדריטים, כך שכל neurites שנאסף על ידי בשיטה זו הן אך ורק אקסונים. מוסיף מסנן מייצג יתרון גדול בהשוואה לגישות אחרות המשמשות ללמוד אקסונים. מחקרים להסתמך על השימוש במיקרוסקופ לבדל תהליכים המתרחשים בתא סומה מאלוxons לספק מידע ביוכימי מוגבל. כדוגמא נוספת, מערכות ממודרות תרבות (למשל, תאי Campenot 10 או מכשירי microfluidic 11) שימושיות לגישות הדמיה ולטיפול בהפרש של תאי תאים אלא לספק רק כמויות קטנות של חומר באקסון המונעות את השימוש בטכניקות אלה לניתוחים ביוכימיים אשר דורשים כמויות גדולות היחסי של מדגם. יתר על כן, השימוש בם דורש הכשרה משמעותית, כמו גם התקנים מיוחדים, והם גוזלים זמן. גישה אחרת המשמשת לעתים קרובות לבודד את האקסונים מ explants היא להסיר באופן ידני מרכז explant (המכיל גופי תא עצביים) לפני איסוף דגימת אקסון. למרות שזה יכול לייצר כמויות גדולות של תכשירים מועשר האקסון, ניתן עטפו אקסונים בתכשיר זה בגליה ומהירות הסרת הגופים של 20 explant ברציפות מ -6 בארות (כדוגמא לניסוי מינימאלי) היא זמן רב ועל גבול כדאיות. לעומת זאת, בשיטה המוצגת כאן מייצרת הכנות אקסון שופעים וטהורות, כי אז יכול להיות מנותחת על ידי כמעט כל טכניקה ביוכימיים המשמש בדרך כלל כדי לנתח lysates כל התא, כמו כתם מערבי, immunoprecipitation 6, כתם צפוני 5 ספקטרומטריית מסה 6, טיהור RNA 7,12,13, בין יתר. יתר על כן, הפרוטוקול יכול להיות מיושם על Immunofluorescence טכניקות (IF), כפי שאפשר לתקן אקסונים גדל בצד התחתון של המסנן כדי לבחון אותם עוד יותר על ידי אם. גישה זו מאוד מקלה על הניתוח של תהליכי אקסון ספציפי מכיוון שאין גופי תא בהכנה הסופית. זהו יתרון משמעותי שכן אות גבוהה immunofluorescent מגופי תא לעתים קרובות פוגעת בבחינה וניתוח של אותות חלשים שמקורם במבנים דקים כגון אקסונים. שני יישומים של שיטת התרבות ללמוד ar ניוון אקסוןדואר תאר; מודל של גיזום התפתחותיים ומודל של ניוון הנגרם פגיעה (המכונות ניוון Wallerian כ). המודלים של גיזום התפתחותי מבוסס על העובדה שתאי עצב תחושתי in vivo להתחרות על הגבלת סכומי NGF-derived יעד ואלו שלא הצליחו לקבל מספיק מנוון תמיכת נוירוטרופי 14. תופעה זו יכולה להיות חיקה בתרבויות DRG העובריים על ידי נסיגת NGF מהתקשורת והתרבות, אשר, כפי שקורה בvivo, יוזם ניוון אקסון אחריו הרס גוף תא. למודל ניוון Wallerian, הצד העליון של המסנן עם גופי התא שרוט משם. אקסונים שצמחו על הצד התחתון של המסנן להיות מופרד פיזי מהגופים התא ולאחר מכן לעבור את התהליך המהיר וסטריאוטיפי ניווניות המכונה ניוון Wallerian 15, על שמו של א &#39;וולר שדיווח על התופעה ראשונה בשנת 1850 16.

<table border="0" cellpadding="0" cellspacing="0" width="972">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:297px;">CD-1 mouse, E13 timed pregnancy</td>
			<td style="width:140px;">Charles River</td>
			<td style="width:119px;"></td>
			<td style="width:416px;">In house timed pregnancies can be done by mating mice and checking for vaginal plugs (Day E0)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Falcon Cell Culture Inserts</td>
			<td>Corning</td>
			<td>353102</td>
			<td>1 &#181;m pore size, fits 6-well receiving plate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Falcon Cell Culture Companion Plates</td>
			<td>Corning</td>
			<td>353502</td>
			<td>Receiving plates for inserts</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Poly-D-Lysine</td>
			<td>Sigma-Aldrich</td>
			<td>P6407</td>
			<td>Used at 1 mg/ml in water. Can re-use once.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Laminin</td>
			<td>Sigma-Aldrich</td>
			<td>L2020</td>
			<td style="width:416px;">Used at 10 ug/ml in water. Take 100X stock from -80 &#176;C and thaw overnight at 4 &#176;C&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PureCol Bovine Collagen Solution</td>
			<td>Advanced Biomatrix</td>
			<td>5005-B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Leibovitz&#39;s L-15 Medium</td>
			<td>Invitrogen</td>
			<td>11415-064</td>
			<td>Can be replaced by DMEM.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Neurobasal Medium</td>
			<td>Invitrogen</td>
			<td>21103-049</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">B-27 Serum-Free Supplement</td>
			<td>Invitrogen</td>
			<td>17504-044</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">5-Fluoro-2&#8242;-deoxyuridine (FDU)</td>
			<td>Sigma-Aldrich</td>
			<td>F0503</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NGF (2.5S, beta subunit)</td>
			<td>Cedarlane</td>
			<td>CLMCNET-001</td>
			<td></td>
		</tr>
		<tr height="88">
			<td height="109" rowspan="2" style="height:109px;width:297px;">Anti-NGF Antibody</td>
			<td style="width:140px;">Prepared in-house</td>
			<td>-</td>
			<td style="width:416px;">As described in: Acheson, A., Barker, P.A., Alderson, R.F., Miller, F.D. &#38; Murphy, R.A. Detection of brain-derived neurotrophic factor-like activity in fibroblasts and Schwann cells: inhibition by antibodies to NGF. Neuron.7, 265-75, (1991)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alomone Labs</td>
			<td style="width:119px;">AN-240</td>
			<td>Commercial option</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-Tubulin Antibody</td>
			<td>Millipore</td>
			<td>MAB5564</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa Fluor 488 Goat Anti-Mouse Antibody</td>
			<td>Invitrogen</td>
			<td>A-11029</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DRG culture medium</td>
		</tr>
		<tr height="126">
			<td height="126" style="height:126px;width:297px;">Neurobasal Medium 
			2 % B27 Neurobasal Supplement 
			2 mM l-Glutamine 
			1 % Penicillin-Streptomysin 
			10 &#181;M FDU 
			15 ng/ml NGF (bottom compartment only)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1X Laemmli Sample Buffer</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:297px;">10 % SDS 
			0.5 M DTT 
			0.5 M Tris (pH 6.8) 
			5 % Glycerol 
			0.01 % (w/v) Bromophenol blue</td>
		</tr>
	</tbody>
</table>

production and isolation of axons from sensory neurons for biochemical analysis using porous filters

Neuronal axons use specific mechanisms to mediate extension, maintain integrity, and induce degeneration. An appropriate balance of these events is required to shape functional neuronal circuits. The protocol described here explains how to use cell culture inserts bearing a porous membrane (filter) to obtain large amounts of pure axonal preparations suitable for examination by conventional biochemical or immunocytochemical techniques. The functionality of these filter inserts will be demonstrated with models of developmental pruning and Wallerian degeneration, using explants of embryonic dorsal root ganglion. Axonal integrity and function is compromised in a wide variety of neurodegenerative pathologies. Indeed, it is now clear that axonal dysfunction appears much earlier in the course of the disease than neuronal soma loss in several neurodegenerative diseases, indicating that axonal-specific processes are primarily targeted in these disorders. By obtaining pure axonal samples for analysis by molecular and biochemical techniques, this technique has the potential to shed new light into mechanisms regulating the physiology and pathophysiology of axons. This in turn will have an impact in our understanding of the processes that drive degenerative diseases of the nervous system.

explants גנגליון שורש הגבי מעכברי E13.5 לגדול בהרחבה על מסננים ברצף מצופה פולי-D-ליזין, laminin וקולגן (איור 1 ב, משמאל), והגיע עד 2 מ&quot;מ בתוך 2 ימים בתרבות. שילוב מצע זה מניב צמיחה במיוחד שופעת; שילובים אחרים יניבו אקסונים שהם הרבה יותר קצרים (איור 1 ב, מימין). יתר ...

Watch this Scientific Journal Video about Production and Isolation of Axons from Sensory Neurons for Biochemical Analysis Using Porous Filters at JoVE.com

Production and Isolation of Axons from Sensory Neurons for Biochemical Analysis Using Porous Filters

This article describes a neuronal culture system that can be used to obtain large quantities of pure axonal samples for biochemical and immunocytochemical analyses. This technique will allow an improved understanding of normal axonal physiology and signaling pathways leading to neurodegeneration.

הפקה ובידוד של אקסונים מסנסורי ביוכימית ניתוח באמצעות מסננים נקבוביים

Neuronal axons use specific mechanisms to mediate extension, maintain integrity, and induce degeneration. An appropriate balance of these events is ...

production-isolation-axons-from-sensory-neurons-for-biochemical

Research

JoVE Journal

Neuroscience

14.2K Views.  Montreal Neurological Institute, McGill University. This article describes a neuronal culture system that can be used to obtain large quantities of pure axonal samples for biochemical and immunocytochemical analyses. This technique will allow an improved understanding of normal axonal physiology and signaling pathways leading to neurodegeneration. 

Video: Production and Isolation of Axons from Sensory Neurons for Biochemical Analysis Using Porous Filters

Morphological Analysis of Drosophila Larval Peripheral Sensory Neuron Dendrites and Axons Using Genetic Mosaics

Nervous system development requires the correct specification of neuron position and identity, followed by accurate neuron class-specific dendritic development and axonal wiring. Recently the dendritic arborization (DA) sensory neurons of the Drosophila larval peripheral nervous system (PNS) have become powerful genetic models in which to elucidate both general and class-specific mechanisms of neuron differentiation.
There are four main DA neuron classes (I-IV)1. They are named in order of increasing dendrite arbor complexity, and have class-specific differences in the genetic control of their differentiation2-10. The DA sensory system is a practical model to investigate the molecular mechanisms behind the control of dendritic morphology11-13 because: 1) it can take advantage of the powerful genetic tools available in the fruit fly, 2) the DA neuron dendrite arbor spreads out in only 2 dimensions beneath an optically clear larval cuticle making it easy to visualize with high resolution in vivo, 3) the class-specific diversity in dendritic morphology facilitates a comparative analysis to find key elements controlling the formation of simple vs. highly branched dendritic trees, and 4) dendritic arbor stereotypical shapes of different DA neurons facilitate morphometric statistical analyses.
DA neuron activity modifies the output of a larval locomotion central pattern generator14-16. The different DA neuron classes have distinct sensory modalities, and their activation elicits different behavioral responses14,16-20. Furthermore different classes send axonal projections stereotypically into the Drosophila larval central nervous system in the ventral nerve cord (VNC)21. These projections terminate with topographic representations of both DA neuron sensory modality and the position in the body wall of the dendritic field7,22,23. Hence examination of DA axonal projections can be used to elucidate mechanisms underlying topographic mapping7,22,23, as well as the wiring of a simple circuit modulating larval locomotion14-17.
We present here a practical guide to generate and analyze genetic mosaics24 marking DA neurons via MARCM (Mosaic Analysis with a Repressible Cell Marker)1,10,25 and Flp-out22,26,27 techniques (summarized in Fig. 1).

Morphological Analysis of Drosophila Larval Peripheral Sensory Neuron Dendrites and Axons Using Genetic Mosaics

The dendritic arborization sensory neurons of the Drosophila larval peripheral nervous system are useful models to elucidate both general and neuron class-specific mechanisms of neuron differentiation. We present a practical guide to generate and analyze dendritic arborization neuron genetic mosaics.

ניתוח מורפולוגי של תסיסנית הזחל היקפיים Neuron דנדריטים חושי האקסונים באמצעות פסיפסים גנטי

Nervous system development requires the correct specification of neuron position and identity, followed by accurate neuron class-specific dendritic ...

Flash Photolysis of Caged Compounds in the Cilia of Olfactory Sensory  Neurons

Photolysis of caged compounds allows the production of rapid and localized increases in the concentration of various physiologically active compounds1. Caged compounds are molecules made physiologically inactive by a chemical cage that can be broken by a flash of ultraviolet light. Here, we show how to obtain patch-clamp recordings combined with photolysis of caged compounds for the study of olfactory transduction in dissociated mouse olfactory sensory neurons. The process of olfactory transduction (Figure 1) takes place in the cilia of olfactory sensory neurons, where odorant binding to receptors leads to the increase of cAMP that opens cyclic nucleotide-gated (CNG) channels2. Ca entry through CNG channels activates Ca-activated Cl channels. We show how to dissociate neurons from the mouse olfactory epithelium3 and how to activate CNG channels or Ca-activated Cl channels by photolysis of caged cAMP4 or caged Ca5. We use a flash lamp6,7 to apply ultraviolet flashes to the ciliary region to uncage cAMP or Ca while patch-clamp recordings are taken to measure the current in the whole-cell voltage-clamp configuration8-11.

Photolysis of caged compounds allows the production of rapid and localized increases in the concentration of various physiologically active compounds. Here, we show how to obtain patch-clamp recordings combined with photolysis of caged cAMP or caged Ca for the study of olfactory transduction in dissociated mouse olfactory sensory neurons.

פלאש Photolysis של תרכובות כלואה צילה של עצב סנסורי חוש הריח

Photolysis of caged compounds allows the production of rapid and localized increases in the concentration of various physiologically active compounds1. ...

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 ...

Voltage-sensitive Dye Recording from Axons, Dendrites and Dendritic Spines of Individual Neurons in Brain Slices

Understanding the biophysical properties and functional organization of single neurons and how they process information is fundamental for understanding how the brain works. The primary function of any nerve cell is to process electrical signals, usually from multiple sources. Electrical properties of neuronal processes are extraordinarily complex, dynamic, and, in the general case, impossible to predict in the absence of detailed measurements. To obtain such a measurement one would, ideally, like to be able to monitor, at multiple sites, subthreshold events as they travel from the sites of origin on neuronal processes and summate at particular locations to influence action potential initiation. This goal has not been achieved in any neuron due to technical limitations of measurements that employ electrodes. To overcome this drawback, it is highly desirable to complement the patch-electrode approach with imaging techniques that permit extensive parallel recordings from all parts of a neuron. Here, we describe such a technique - optical recording of membrane potential transients with organic voltage-sensitive dyes (Vm-imaging) - characterized by sub-millisecond and sub-micrometer resolution. Our method is based on pioneering work on voltage-sensitive molecular probes 2. Many aspects of the initial technology have been continuously improved over several decades 3, 5, 11. Additionally, previous work documented two essential characteristics of Vm-imaging. Firstly, fluorescence signals are linearly proportional to membrane potential over the entire physiological range (-100 mV to +100 mV; 10, 14, 16). Secondly, loading neurons with the voltage-sensitive dye used here (JPW 3028) does not have detectable pharmacological effects. The recorded broadening of the spike during dye loading is completely reversible 4, 7. Additionally, experimental evidence shows that it is possible to obtain a significant number (up to hundreds) of recordings prior to any detectable phototoxic effects 4, 6, 12, 13. At present, we take advantage of the superb brightness and stability of a laser light source at near-optimal wavelength to maximize the sensitivity of the Vm-imaging technique. The current sensitivity permits multiple site optical recordings of Vm transients from all parts of a neuron, including axons and axon collaterals, terminal dendritic branches, and individual dendritic spines. The acquired information on signal interactions can be analyzed quantitatively as well as directly visualized in the form of a movie.

An imaging technique for monitoring of membrane potential changes with sub-micrometer spatial and sub-millisecond temporal resolution is described. The technique, based on laser excitation of voltage-sensitive dyes, allows measurements of signals in axons and axon collaterals, terminal dendritic branches, and individual dendritic spines.

הקלטת דיי מתח רגישה מהאקסונים, הדנדריטים וקוצים הדנדריטים של נוירונים בודדים בפרוסות מוח

Understanding the biophysical properties and functional organization of single neurons and how they process information is fundamental for understanding ...

Isolation of Sensory Neurons of Aplysia californica for Patch Clamp Recordings of Glutamatergic Currents

The marine gastropod mollusk Aplysia californica has a venerable history as a model of nervous system function, with particular significance in studies of learning and memory. The typical preparations for such studies are ones in which the sensory and motoneurons are left intact in a minimally dissected animal, or a technically elaborate neuronal co-culture of individual sensory and motoneurons. Less common is the isolated neuronal preparation in which small clusters of nominally homogeneous neurons are dissociated into single cells in short term culture. Such isolated cells are useful for the biophysical characterization of ion currents using patch clamp techniques, and targeted modulation of these conductances. A protocol for preparing such cultures is described. The protocol takes advantage of the easily identifiable glutamatergic sensory neurons of the pleural and buccal ganglia, and describes their dissociation and minimal maintenance in culture for several days without serum.

Isolation of Sensory Neurons of Aplysia californica for Patch Clamp Recordings of Glutamatergic Currents

We describe the dissection of the nervous system of the marine sea hare Aplysia after anesthesia, the isolation of neurons for short term-tissue culture, and recordings of single cell ion currents via the patch clamp technique.

בידוד של נוירונים חושיים של<em&gt; Aplysia californica</em&gt; להקלטות מהדק תיקון של זרמי glutamatergic

The marine gastropod mollusk Aplysia californica has a venerable  history as a model of nervous system function, with particular significance in  studies ...

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this information to the brain. As such, determining how olfactory information is transferred to the brain, modifying both perception and behavior, merits investigation. Interestingly, there is emerging evidence that cellular transduction and transcriptional factors are involved in the diversification of olfactory receptor neuron. Here we provide a robust whole mount immunological labeling method to assay in vivo olfactory receptor neuron organization. Using this method, we identified all olfactory receptor neurons with anti-ELAV antibody, a known pan-neural marker and Or49a-mCD8::GFP, an olfactory receptor neuron specifically expressed in Nba neuron using anti-GFP antibody.

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Herein we describe the process of whole mount immunostaining of Drosophila antennae, which enables us to better understand the molecular mechanisms involved in the diversification of olfactory receptor neurons (ORN)s.

הר immunolabeling השלם של חוש הריח קולטן נוירונים<em&gt; דרוזופילה</em&gt; אנטנה

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this ...

Isolation and Culture of Dissociated Sensory Neurons From Chick Embryos

Neurons are multifaceted cells that carry information essential for a variety of functions including sensation, motor movement, learning, and memory. Studying neurons in vivo can be challenging due to their complexity, their varied and dynamic environments, and technical limitations. For these reasons, studying neurons in vitro can prove beneficial to unravel the complex mysteries of neurons. The well-defined nature of cell culture models provides detailed control over environmental conditions and variables. Here we describe how to isolate, dissociate, and culture primary neurons from chick embryos. This technique is rapid, inexpensive, and generates robustly growing sensory neurons. The procedure consistently produces cultures that are highly enriched for neurons and has very few non-neuronal cells (less than 5%). Primary neurons do not adhere well to untreated glass or tissue culture plastic, therefore detailed procedures to create two distinct, well-defined laminin-containing substrata for neuronal plating are described. Cultured neurons are highly amenable to multiple cellular and molecular techniques, including co-immunoprecipitation, live cell imagining, RNAi, and immunocytochemistry. Procedures for double immunocytochemistry on these cultured neurons have been optimized and described here.

Cell culture models provide detailed control over environmental conditions and thus provide a powerful platform to elucidate numerous aspects of neuronal cell biology. We describe a rapid, inexpensive, and reliable method to isolate, dissociate, and culture sensory neurons from chick embryos. Details of substrata preparation and immunocytochemistry are also provided.

בידוד והתרבות של ניתק עצב סנסורי מאפרוח עוברים

Neurons are multifaceted cells that carry information essential for a variety of functions including sensation, motor movement, learning, and memory. ...

Removal of Drosophila Muscle Tissue from Larval Fillets for Immunofluorescence Analysis of Sensory Neurons and Epidermal Cells

Drosophila larval dendritic arborization (da) neurons are a popular model for investigating mechanisms of neuronal morphogenesis. Da neurons develop in communication with the epidermal cells they innervate and thus their analysis benefits from in situ visualization of both neuronally and epidermally expressed proteins by immunofluorescence. Traditional methods of preparing larval fillets for immunofluorescence experiments leave intact the muscle tissue that covers most of the body wall, presenting several challenges to imaging neuronal and epidermal proteins. Here we describe a method for removing muscle tissue from Drosophila larval fillets. This protocol enables imaging of proteins that are otherwise obscured by muscle tissue, improves signal to noise ratio, and facilitates the use of super-resolution microscopy to study da neuron development.

Removal of Drosophila Muscle Tissue from Larval Fillets for Immunofluorescence Analysis of Sensory Neurons and Epidermal Cells

Studies of neuronal morphogenesis using Drosophila larval dendritic arborization (da) neurons benefit from in situ visualization of neuronal and epidermal proteins by immunofluorescence. We describe a procedure that improves immunofluorescence analysis of da neurons and surrounding epidermal cells by removing muscle tissue from the larval body wall.

הסרה של<em&gt; תסיסנית</em&gt; רקמת שריר מן הזחל פילה עבור ניתוח Immunofluorescence של עצב סנסורי ותאי עוריות

Drosophila larval dendritic arborization (da) neurons are a popular model for investigating mechanisms of neuronal morphogenesis. Da neurons develop in ...

Zebrafish In Situ Spinal Cord Preparation for Electrophysiological Recordings from Spinal Sensory and Motor Neurons

Zebrafish, first introduced as a developmental model, have gained popularity in many other fields. The ease of rearing large numbers of rapidly developing organisms, combined with the embryonic optical clarity, served as initial compelling attributes of this model. Over the past two decades, the success of this model has been further propelled by its amenability to large-scale mutagenesis screens and by the ease of transgenesis. More recently, gene-editing approaches have extended the power of the model.
For neurodevelopmental studies, the zebrafish embryo and larva provide a model to which multiple methods can be applied. Here, we focus on methods that allow the study of an essential property of neurons, electrical excitability. Our preparation for the electrophysiological study of zebrafish spinal neurons involves the use of veterinarian suture glue to secure the preparation to a recording chamber. Alternative methods for recording from zebrafish embryos and larvae involve the attachment of the preparation to the chamber using a fine tungsten pin1,2,3,4,5. A tungsten pin is most often used to mount the preparation in a lateral orientation, although it has been used to mount larvae dorsal-side up4. The suture glue has been used to mount embryos and larvae in both orientations. Using the glue, a minimal dissection can be performed, allowing access to spinal neurons without the use of an enzymatic treatment, thereby avoiding any resultant damage. However, for larvae, it is necessary to apply a brief enzyme treatment to remove the muscle tissue surrounding the spinal cord. The methods described here have been used to study the intrinsic electrical properties of motor neurons, interneurons, and sensory neurons at several developmental stages6,7,8,9.

Zebrafish In Situ Spinal Cord Preparation for Electrophysiological Recordings from Spinal Sensory and Motor Neurons

This manuscript describes methods for electrophysiological recordings from spinal neurons of zebrafish embryos and larvae. The preparation maintains neurons in situ and often involves minimum dissection. These methods allow for the electrophysiological study of a variety of spinal neurons, from the initial electrical excitability acquisition through the early larval stages.

זברה<em&gt; באתרו</em&gt; הכנת בחוט השדרה עבור הקלטות אלקטרו מתוך השדרה חושית הנוירונים המוטוריים

Zebrafish, first introduced as a developmental model, have gained popularity in many other fields. The ease of rearing large numbers of rapidly developing ...

Spinal Cord Neurons Isolation and Culture from Neonatal Mice

We present a protocol for the isolation and culture of spinal cord neurons. The neurons are obtained from neonatal C57BL/6 mice and are isolated on postnatal day 1-3. A mouse litter, usually 4-10 pups born from one breeding pair, is gathered for one experiment, and spinal cords are collected individually from each mouse after euthanasia with isoflurane. The spinal column is dissected out and then the spinal cord is released from the column. The spinal cords are then minced to increase the surface area of delivery for an enzymatic protease that allows for the neurons and other cells to be released from the tissue. Trituration is then used to release the cells into solution. This solution is subsequently fractionated in a density gradient to separate the various cells in solution, allowing for neurons to be isolated. Approximately 1-2.5 x 106 neurons can be isolated from one litter group. The neurons are then seeded onto wells coated with adhesive factors that allow for proper growth and maturation. The neurons take approximately 7 days to reach maturity in the growth and culture medium and can be used thereafter for treatment and analysis.

This study presents a technique for the isolation of neurons from WT neonatal mice. It requires the careful dissection of the spinal cord from the neonatal mouse, followed by the separation of neurons from the spinal cord tissue through mechanical and enzymatic cleavage.

נוירונים חוט השדרה בידוד ותרבות מ עכברים ניאונטליים

We present a protocol for the isolation and culture of spinal cord neurons. The neurons are obtained from neonatal C57BL/6 mice and are isolated on ...