סמואל ל Pfaff הוא פרופסור במעבדות ביטוי גנים במכון סאלק למחקרים ביולוגיים החוקר את הווארד יוז רפואי במכון. עבודה זו נתמכה על ידי כריסטופר ודנה ריב קרן. ג&#39;ו Belcovson, קנט Schnoeker ומייק סאליבן ב משאבי מולטימדיה במכון סאלק סיוע בצילום ועריכה.

הקלטות אלקטרו של מיתרי השדרה מבודד יכול לגלות שינויים גנטיים והתפתחותיים על המעגלים העצביים 1. אנחנו קודם לכן הפגינו שיטה לנתח מיתרי עכבר בילוד השדרה 2. כאן אנו מציגים שיטה להכנת אלקטרודות יניקה שימושי הקלטה תנועה פיקטיבי של מיתרי השדרה מבודד 3. קצה אלקטרודה צינורות פלסטיק ניתן להסיק עד קצה דק מאוד ביד באמצעות מנורה בטמפרטורה נמוכה אלכוהול 3-5. צינורות הפלסטיק (PE90, קליי אדמס Intramedic TM) מוחזק על הלהבה כדי לרכך את צינורות לצורה נזיל. כמו צינורות מתחיל להתמוסס והופך שקוף יותר הוא מוסר ממקור החום מסתיים הם משכו בעדינות לגזרים. העיתוי של הוצאת צינור מהאש חשוב להבטיח הפלסטיק לא יקרוס או לפצל בעוד נמשכים. החלק הדק של הצינור הוא חתך בסכין גילוח לפי קוטר פנימי הרצוי, כמו מוכתב על ידי גיל הדגימה או רמת סגמנטלי של חוט. על מנת להקל על ביצוע O-טבעת על צינורות, חתך 45 ° נעשית בסוף עבה של צינורות עם סכין גילוח. טבעת חזוק A ו-O-טבעת מותקנים על קצה עבה לצרף את קצה האלקטרודה על חבית של האלקטרודה. כדי לאפשר אלקטרודות בגודל ספציפי למנוע את הקשיים של יד משיכת טיפים אלקטרודה, אלקטרודות ניתן לבנות באמצעות צינור קטן מוחדר צינור עבה (PE90). אורכי בצינור הן ספציפיות לדרישות של הגדרת הקלטה משתנה. אנו מציגים את המדידות השתמשו בניסויים שלנו 1-3,5. אורך 1cm של צינורות PTFE קנס (זאוס, חלקים קטנים) ו - 10 ס&quot;מ אורך של צינורות PE90 נחתכים בסכין גילוח. קצוות של צינורות קטנים צריכים להיבדק כדי לוודא שהם אינם כתוש או סגור. הם יכולים להיפתח באמצעות סיכה חרקים (כלים המדע פיין). ירידה של דבק (JB וולד) ממוקמת על נקודת האמצע של צינורות PTFE. צינורות PTFE דבק נמשכים לתוך צינורות PE90 באמצעות יניקה הפה על צינורות PE90. לחתוך 45 ° נעשית בסוף עבה של צינורות ועל טבעת חזוק ו O-טבעת מחוברים, כמו הקודם. מחבר BNC ו טבעת חזוק הם פירק מן אלקטרודה יניקה מסחרי. מחבר BNC היא ספוגה קסילן במשך שעה אחת כדי להסיר את חותם השעווה. מחבר היא שטפה אז במים יבשים. נמל התחתון בצד זווית חייבת להתבצע בצד של החבית. מוט מתכת מחומם באמצעות מבער בונזן ולחץ לתוך החור הקיים בצד של הקנה בזווית נמוכה. אורך 15 ס&quot;מ של חוט כסף 0.010 אינץ&#39; (AM Systems) היא לגזור טבול באקונומיקה במשך רבע שעה. זה נעשה כדי ליצור ציפוי כסף כלוריד המסייע הולכה אות 6. חוט היא שטפה עם מים, יבשים, מולחמים אז על המחבר BNC. חוט מחבר BNC וכסף מוכנסים לתוך חבית יניקה אלקטרודה עם מספיק מקום מחבר BNC להישאר בצד של החבית. צינורות יניקה מוכנס לתוך הנמל בצד 2-3 ס&quot;מ. סיליקון איטום מוזרק לתוך החבית כ 2-3 ס&quot;מ. מחבר BNC הוא מושחל לתוך חבית של האלקטרודה דוחפים את איטום קדימה. קצה האלקטרודה הוא מושחל לתוך עכשיו הקצה הקדמי של האלקטרודה. צינורות הפסיכולוג ניתן גם להוסיף ולחזק את קו יניקה מנמל לוואי. אלקטרודות הם רכובים על micromanipulators המצורפים עומד מגנטי, והניח ליד צלחת את ההקלטה. כבלים קואקסיאליים BNC מחוברים headstage שהוא מחובר למגבר. המגבר מחובר למתאם (Polyview) שמחובר אנלוגי לדיגיטלי כרטיס (מכשירים הלאומי) על מחשב PC עם תוכנת רכישת נתונים (Polyview). בעת הקלטת נתונים אלקטרו, יש צורך לסלק את כל התערבות חיצונית. המנה הקלטה, מגבר, בשלב הראש מונחות על שולחן האוויר בתוך כלוב פאראדיי כדי למנוע הפרעה חשמלית מכנית, בהתאמה. נציג תוצאות: <img src="/files/ftp_upload/2580/2580fig1.jpg" alt="איור 1" /> באיור 1. אלקטרודה יניקה זמינים מסחרית ניתן לשנות על ידי הוספת כסף כבד מד תיל טיפ פלסטיק כי הוא נמשך על יד מנורת אלכוהול, או לבנות עם צינורות פלסטיק זמינים מסחרית בגדלים ספציפיים. בנוסף, איטום פרפין מוחלף יותר סיליקון איטום עמיד קולק. <img src="/files/ftp_upload/2580/2580fig2.jpg" alt="איור 2" /> איור 2. סכימטי של האלקטרודה יניקה ביחס שולחן אוויר (עבור בידוד מכני), כלוב פאראדיי (חשמל isolatiב), מגבר ומחשב. יצוין כי קיימת קרקע הפניה אל צלחת הקלטה קרקע השני הוא הפניה אל שולחן אוויר כלוב פאראדיי.

<ol>
	<li>Gallarda, B. W., Sharpee, T. O., Pfaff, S. L., Alaynick, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defining+rhythmic+locomotor+burst+patterns+using+a+continuous+wavelet+transform.">Defining rhythmic locomotor burst patterns using a continuous wavelet transform.</a> Ann N Y Acad Sci. 1198, 133-139 (2010).</li><li>Meyer, A., Gallarda, B. W., Pfaff, S., Alaynick, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spinal+cord+electrophysiology.">Spinal cord electrophysiology.</a> J Vis Exp. , (2010).</li><li>Gallarda, B. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Segregation+of+axial+motor+and+sensory+pathways+via+heterotypic+trans-axonal+signaling.">Segregation of axial motor and sensory pathways via heterotypic trans-axonal signaling.</a> Science. 320, 233-236 (2008).</li><li>Landmesser, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+development+of+motor+projection+patterns+in+the+chick+hind+limb.">The development of motor projection patterns in the chick hind limb.</a> J Physiol. 284, 391-414 (1978).</li><li>Myers, C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cholinergic+input+is+required+during+embryonic+development+to+mediate+proper+assembly+of+spinal+locomotor+circuits.">Cholinergic input is required during embryonic development to mediate proper assembly of spinal locomotor circuits.</a> Neuron. 46, 37-49 (2005).</li><li>Chanin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+determination+of+chloride+by+use+of+the+silver-silver+chloride+electrode.">The determination of chloride by use of the silver-silver chloride electrode.</a> Science. 119, 323-324 (1954).</li><li>Goulding, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circuits+controlling+vertebrate+locomotion:+moving+in+a+new+direction.">Circuits controlling vertebrate locomotion: moving in a new direction.</a> Nat Rev Neurosci. 10, 507-518 (2009).</li></ol>

אין ניגודי אינטרסים הכריז.

התפתחות מערכת העצבים ניתן ללמוד באמצעות מיתרי מכרסם מבודד השדרה. בנוכחות של נוירוטרנסמיטורים, תנועה פיקטיבי יכול להיווצר מתוך חוט השדרה בצורת פעילות בדוגמת חשמל 1,3. התפרצויות אלה קצבית מיוצרים הרץ 0.2-0.5 ו - הם בדוגמת ב החילופים משמאל לימין ו-extensor מכופף. בשלבים הת...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>PTFE Sub Lite Wall Tubing (Small tubing)</td>
<td>&nbsp;</td>
<td>Zeus</td>
<td>36AWG</td>
<td>0.005&#x201D;ID x 0.003&#x201D; Wall (Small Parts) Also available in 0.003&#x201D; to 0.006&#x201D;</td>
</tr>
<tr>
<td>Large tubing (0.86mm (0.34&#x201D;))</td>
<td>&nbsp;</td>
<td>Clay Adams Brand Intramedic Becton Dickinson and Company</td>
<td>427420</td>
<td>0.86mm (0.34&#x201D;) O.D. 1.27mm (.050&#x201D;)</td>
</tr>
<tr>
<td>Electrode Barrel</td>
<td>&nbsp;</td>
<td>A-M Systems</td>
<td>573000</td>
<td>&nbsp;</td>
<tr>
<td>Adhesive</td>
<td>&nbsp;</td>
<td>JB Weld</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Adhesive: Silicone caulk</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Solder and soldering iron</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Bleach</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Xylene</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Silver wire: 0.010&#x201D;</td>
<td>&nbsp;</td>
<td>A-M Systems</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Insect pins: Austerlitz 0.1mm</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>26002-10</td>
<td>&nbsp;</td>
<tr>
<td>Magnetic Stand</td>
<td>&nbsp;</td>
<td>Narishige</td>
<td>GJ-8</td>
<td>&nbsp;</td>
<tr>
<td>Micromanipulator</td>
<td>&nbsp;</td>
<td>Narishige</td>
<td>MN 151</td>
<td>&nbsp;</td>
<tr>
<td>Miniboard (Headstage)</td>
<td>&nbsp;</td>
<td>Grass Industries</td>
<td>F-15EB/B1</td>
<td>&nbsp;</td>
<tr>
<td>Polyview Adaptor Unit</td>
<td>&nbsp;</td>
<td>Grass Industries</td>
<td>PVA 8</td>
<td>&nbsp;</td>
<tr>
<td>Bipolar Portable Physiodata Amplifier System</td>
<td>&nbsp;</td>
<td>Grass Industries</td>
<td>15LT</td>
<td>&nbsp;</td>
<tr>
<td>ANALOG TO DIGITAL CARD</td>
<td>&nbsp;</td>
<td>National Instruments</td>
<td>6035E</td>
<td>&nbsp;</td>
<tr>
<td>Air Table; Vibraplane</td>
<td>&nbsp;</td>
<td>Kinetic Systems</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

spinal cord electrophysiology ii: extracellular suction electrode fabrication

פיתוח circuitries עצבי תנועה ניתן ללמוד באמצעות מכרסם בילוד חוט השדרה המרכזי דפוס גנרטור (CPG) התנהגות. אנו להדגים שיטה לייצר אלקטרודות שאיבה המשמשים לבחינת פעילות CPG, או תנועה פיקטיבי, על מיתרי מכרסם גזור השדרה. מיתרי מכרסם השדרה ממוקמים הנוזל השדרתי מלאכותי לשורשי הגחון נמשכים אל האלקטרודה יניקה. אלקטרודה בנויה על ידי שינוי אלקטרודה יניקה זמינים מסחרית. חוט כסף כבד משמש במקום חוט תקן שניתן על ידי האלקטרודה זמינים מסחרית. קצה הזכוכית על האלקטרודה מסחרי מוחלף עם קצה פלסטיק עמידות מוגברת. אנחנו מכינים אלקטרודות נמשך יד אלקטרודות עשויות בגדלים מסוימים של צינורות, המאפשר שחזור עקביות. הנתונים נאספו באמצעות מגבר ורכישת תוכנה neurogram. הקלטות מבוצעות על שולחן האוויר בתוך כלוב פאראדיי כדי למנוע הפרעות מכניות וחשמליות, בהתאמה.

Watch this Scientific Journal Video about Spinal Cord Electrophysiology II: Extracellular Suction Electrode Fabrication at JoVE.com

Spinal Cord Electrophysiology II: Extracellular Suction Electrode Fabrication

הפגנה של ייצור ושימוש אלקטרודה יניקה תאיים המשמשת למדידת אלקטרו הקלטות של מיתרי מכרסם השדרה בילוד<em&gt; במבחנה</em

חוט השדרה Electrophysiology II: ייצור תאי אלקטרודה יניקה

Development of neural circuitries and locomotion can be studied using neonatal rodent spinal cord central pattern generator (CPG) behavior. We demonstrate ...

spinal-cord-electrophysiology-ii-extracellular-suction-electrode

Research

JoVE Journal

Neuroscience

15.0K Views.  The Salk Institute for Biological Studies. הפגנה של ייצור ושימוש אלקטרודה יניקה תאיים המשמשת למדידת אלקטרו הקלטות של מיתרי מכרסם השדרה בילוד במבחנה

Video: Spinal Cord Electrophysiology II: Extracellular Suction Electrode Fabrication

Chicken Embryo Spinal Cord Slice Culture Protocol

Slice cultures can facilitate the manipulation of embryo development both pharmacologically and through gene manipulations. In this reduced system, potential lethal side effects due to systemic drug applications can be overcome. However, culture conditions must ensure that normal development proceeds within the reduced environment of the slice. We have focused on the development of the spinal cord, particularly that of spinal motor neurons. We systematically varied culture conditions of chicken embryo slices from the point at which most spinal motor neurons had been born. We assayed the number and type of motor neurons that survived during the culture period and the position of those motor neurons compared to that in vivo. We found that serum type and neurotrophic factors were required during the culture period and were able to keep motor neurons alive for at least 24 hr and allow those motor neurons to migrate to appropriate positions in the spinal cord. We present these culture conditions and the methodology of preparing the embryo slice cultures using eviscerated chicken embryos embedded in agarose and sliced using a vibratome.

Slice cultures facilitate the manipulation of embryo development by gene and pharmacological perturbations. However, culture conditions must ensure that normal development can proceed within the reduced environment of the slice. We illustrate a protocol that facilitates normal spinal cord development to proceed for at least 24 hr.

פרוטוקול עוף עובר חוט שדרת Slice תרבות

Slice cultures can facilitate the manipulation of  embryo development both pharmacologically and through gene manipulations. In  this reduced system, ...

Spinal Cord Transection in the Larval Zebrafish

Mammals fail in sensory and motor recovery following spinal cord injury due to lack of axonal regrowth below the level of injury as well as an inability to reinitiate spinal neurogenesis. However, some anamniotes including the zebrafish Danio rerio exhibit both sensory and functional recovery even after complete transection of the spinal cord. The adult zebrafish is an established model organism for studying regeneration following spinal cord injury, with sensory and motor recovery by 6&#160;weeks post-injury. To take advantage of in vivo analysis of the regenerative process available in the transparent larval zebrafish as well as genetic tools not accessible in the adult, we use the larval zebrafish to study regeneration after spinal cord transection. Here we demonstrate a method for reproducibly and verifiably transecting the larval spinal cord. After transection, our data shows sensory recovery beginning at 2&#160;days post-injury (dpi), with the C-bend movement detectable by 3 dpi and resumption of free swimming by 5 dpi. Thus we propose the larval zebrafish as a companion tool to the adult zebrafish for the study of recovery after spinal cord injury.

After spinal transection, adult zebrafish have functional recovery by six weeks post-injury. To take advantage of larval transparency and faster recovery, we present a method for transecting the larval spinal cord. After transection, we observe sensory recovery beginning at 2&#160;days post-injury, and C-bend movement by 3 days post-injury.

חיתוך רוחב חוט השדרה בדג הזברה הזחל

Mammals fail in sensory and motor recovery following spinal cord injury due to lack of axonal regrowth below the level of injury as well as an inability ...

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal degeneration. Lack of growth cone formation, regeneration, and loss of additional myelinated axonal projections within the spinal cord greatly limits neurological recovery following injury. To assess how central myelinated axons of the spinal cord respond to injury, we developed an ex vivo living spinal cord model utilizing transgenic mice that express yellow fluorescent protein in axons and a focal and highly reproducible laser-induced spinal cord injury to document the fate of axons and myelin (lipophilic fluorescent dye Nile Red) over time using two-photon excitation time-lapse microscopy. Dynamic processes such as acute axonal injury, axonal retraction, and myelin degeneration are best studied in real-time. However, the non-focal nature of contusion-based injuries and movement artifacts encountered during in vivo spinal cord imaging make differentiating primary and secondary axonal injury responses using high resolution microscopy challenging. The ex vivo spinal cord model described here mimics several aspects of clinically relevant contusion/compression-induced axonal pathologies including axonal swelling, spheroid formation, axonal transection, and peri-axonal swelling providing a useful model to study these dynamic processes in real-time. Major advantages of this model are excellent spatiotemporal resolution that allows differentiation between the primary insult that directly injures axons and secondary injury mechanisms; controlled infusion of reagents directly to the perfusate bathing the cord; precise alterations of the environmental milieu (e.g., calcium, sodium ions, known contributors to axonal injury, but near impossible to manipulate in vivo); and murine models also offer an advantage as they provide an opportunity to visualize and manipulate genetically identified cell populations and subcellular structures. Here, we describe how to isolate and image the living spinal cord from mice to capture dynamics of acute axonal injury.

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

We present a protocol utilizing two-photon excitation time-lapse microscopy to simultaneously visualize the dynamics of axon and myelin injuries in real time. This proposed protocol permits studies of both intrinsic and extrinsic factors which can influence central myelinated axon fate after injury and contribute to permanent clinical disability.

<em&gt; Ex Vivo</em&gt; חוט השדרה מושרה לייזר הפגיעה דגם להערכת מנגנוני axonal הניוון בזמן אמת

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal ...

Diffusion Imaging in the Rat Cervical Spinal Cord

Magnetic resonance imaging (MRI) is the state of the art approach for assessing the status of the spinal cord noninvasively, and can be used as a diagnostic and prognostic tool in cases of disease or injury. Diffusion weighted imaging (DWI), is sensitive to the thermal motion of water molecules and allows for inferences of tissue microstructure. This report describes a protocol to acquire and analyze DWI of the rat cervical spinal cord on a small-bore animal system. It demonstrates an imaging setup for the live anesthetized animal and recommends a DWI acquisition protocol for high-quality imaging, which includes stabilization of the cord and control of respiratory motion. Measurements with diffusion weighting along different directions and magnitudes (b-values) are used. Finally, several mathematical models of the resulting signal are used to derive maps of the diffusion processes within the spinal cord tissue that provide insight into the normal cord and can be used to monitor injury or disease processes noninvasively.

The goal of this protocol is to obtain high-quality diffusion weighted magnetic resonance imaging (DWI) of the rat spinal cord for noninvasive characterization of tissue microstructure. This protocol describes optimizations of the MRI sequence, radiofrequency coil, and analysis methods to enable DWI images free from artifacts.

ההדמיה דיפוזיה בחוט השדרה עכברוש צוואר הרחם

Magnetic resonance imaging (MRI) is the state of the art approach for assessing the status of the spinal cord noninvasively, and can be used as a ...

Electrophysiology on Isolated Brainstem-spinal Cord Preparations from Newborn Rodents Allows Neural Respiratory Network Output Recording

While it is well known that the central respiratory drive is located in the brainstem, several aspects of its basic function, development, and response to stimuli remain to be fully understood. To overcome the difficulty of accessing the brainstem in the whole animal, isolation of the brainstem and part of the spinal cord is performed. This preparation is maintained in artificial cerebro-spinal fluid where gases, concentrations, and temperature are controlled and monitored. The output signal from the respiratory network is recorded by a suction electrode placed on the fourth ventral root. In this manner, stimuli can be directly applied onto the brainstem, and the effect can be recorded directly. The signal recorded is linked to the inspiratory signal sent to the diaphragm via the phrenic nerve, and can be described as bursts (around 8 bursts per minute). Analysis of these bursts (frequency, amplitude, length, and area under the curve) allows precise characterization of the stimulus effect on the respiratory network. The main limitation of this method is the viability of the preparation beyond the early post-natal stages. Thus, this method greatly focuses on the study of the whole network without the peripheral inputs in the newborn rat.

The central respiratory drive is located in the brainstem. Spontaneous respiratory motor output from an isolated brainstem-spinal cord is recorded by placing an electrode on the fourth ventral root. This experimental approach is valuable for pharmacological investigations or the assessment of respiratory challenges and genetic manipulations on rhythmic motor behavior.

אלקטרופיזיולוגיה בהכנות כבל מבודדות גזע המוח-השדרה מיילוד מכרסמים מאפשרת הקלטת תפוקת רשת הנשימה עצבית

While it is well known that the central respiratory drive is located in the brainstem, several aspects of its basic function, development, and response to ...

Investigating Functional Regeneration in Organotypic Spinal Cord Co-cultures Grown on Multi-electrode Arrays

Adult higher vertebrates have a limited potential to recover from spinal cord injury. Recently, evidence emerged that propriospinal connections are a promising target for intervention to improve functional regeneration. So far, no in vitro model exists that grants the possibility to examine functional recovery of propriospinal fibers. Therefore, a representative model that is based on two organotypic spinal cord sections of embryonic rat, cultured next to each other on multi-electrode arrays (MEAs) was developed. These slices grow and, within a few days in vitro, fuse along the sides facing each other. The design of the used MEAs permits the performance of lesions with a scalpel blade through this fusion site without inflicting damage on the MEAs. The slices show spontaneous activity, usually organized in network activity bursts, and spatial and temporal activity parameters such as the location of burst origins, speed and direction of their propagation and latencies between bursts can be characterized. Using these features, it is also possible to assess functional connection of the slices by calculating the amount of synchronized bursts between the two sides. Furthermore, the slices can be morphologically analyzed by performing immunohistochemical stainings after the recordings. 
Several advantages of the used techniques are combined in this model: the slices largely preserve the original tissue architecture with intact local synaptic circuitry, the tissue is easily and repeatedly accessible and neuronal activity can be detected simultaneously and non-invasively in a large number of spots at high temporal resolution. These features allow the investigation of functional regeneration of intraspinal connections in isolation in vitro in a sophisticated and efficient way.

Here we present a protocol that is based on spinal cord slices cultured on multi-electrode arrays to study functional regeneration of propriospinal connections in vitro.

חוקר התחדשות פונקציונלית בחוט השדרה שיתוף תרבויות Organotypic גדלו על מערכים רב-אלקטרודה

Adult higher vertebrates have a limited potential to recover from spinal cord injury. Recently, evidence emerged that propriospinal connections are a ...

Spiral Ganglion Neuron Explant Culture and Electrophysiology on Multi Electrode Arrays

Spiral ganglion neurons (SGNs) participate in the physiological process of hearing by relaying signals from sensory hair cells to the cochlear nucleus in the brain stem. Loss of hair cells is a major cause of sensory hearing loss. Prosthetic devices such as cochlear implants function by bypassing lost hair cells and directly stimulating SGNs electrically, allowing for restoration of hearing in deaf patients. The performance of these devices depends on the functionality of SGNs, the implantation procedure and on the distance between the electrodes and the auditory neurons. 
We hypothesized, that reducing the distance between the SGNs and the electrode array of the implant would allow for improved stimulation and frequency resolution, with the best results in a gapless position. Currently we lack in vitro culture systems to study, modify and optimize the interaction between auditory neurons and electrode arrays and characterize their electrophysiological response. To address these issues, we developed an in vitro bioassay using SGN cultures on a planar multi electrode array (MEA). With this method we were able to perform extracellular recording of the basal and electrically induced activity of a population of spiral ganglion neurons. We were also able to optimize stimulation protocols and analyze the response to electrical stimuli as a function of the electrode distance. This platform could also be used to optimize electrode features such as surface coatings.

We present a protocol to culture primary murine spiral ganglion neuron explants on multi electrode arrays to study neuronal response profiles and optimize stimulation parameters. Such studies aim to improve the neuron-electrode interface of cochlear implants to benefit hearing in patients as well as the energy consumption of the device.

ספירלת גנגליון Neuron explant תרבות Electrophysiology על מערכי אלקטרודה רבות

Spiral ganglion neurons (SGNs) participate in the physiological process of hearing by relaying signals from sensory hair cells to the cochlear nucleus in ...

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 ...

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in adult mammalian animals remains elusive. In this study, we describe an experimental procedure for measuring calcium dynamics through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy. This method enables recordings and quantifications of neural activity in bilateral mouse brain regions one at a time in the same experiment for a prolonged period in vivo. Key aspects of this method, which can be completed within an hour, include minimally invasive surgery procedures for creating dual optical windows, and the use of 2P imaging. Although we only demonstrate the technique in the S1 area, the method can be applied to other regions of the living brain facilitating the elucidation of structural and functional complexities of brain neural networks.

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

We describe an experimental procedure for measuring neuronal activity through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy in vivo.

הדמיה פעילות עצבית בתוך קליפת המגע הראשי באמצעות Thy1-העכברים הטרנסגניים GCaMP6s

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in ...

Activity-based Training on a Treadmill with Spinal Cord Injured Wistar Rats

Spinal cord injury (SCI) results in lasting deficits that include both mobility and a multitude of autonomic-related dysfunctions. Locomotor training (LT) on a treadmill is widely used as a rehabilitation tool in the SCI population with many benefits and improvements to daily life. We utilize this method of activity-based task-specific training (ABT) in rodents after SCI to both elucidate the mechanisms behind such improvements and to enhance and improve upon existing clinical rehabilitation protocols. Our current goal is to determine the mechanisms underlying ABT-induced improvements in urinary, bowel, and sexual function in SCI rats after a moderate to severe level of contusion. After securing each individual animal in a custom-made adjustable vest, they are secured to a versatile body weight support mechanism, lowered to a modified three-lane treadmill and assisted in step-training for 58 minutes, once a day for 10 weeks. This setup allows for the training of both quadrupedal and forelimb-only animals, alongside two different non-trained groups. Quadrupedal-trained animals with body weight support are aided by a technician present to assist in stepping with proper hind limb placement as necessary, while forelimb-only trained animals are raised at the caudal end to ensure no hind limb contact with the treadmill and no weight-bearing. One non-trained SCI group of animals is placed in a harness and rests next to the treadmill, while the other control SCI group remains in its home cage in the training room nearby. This paradigm allows for the training of multiple SCI animals at once, thus making it more time-efficient in addition to ensuring that our pre-clinical animal model mimics the clinical representation as close as possible, particularly with respect to the body weight support with manual assistance.

This protocol demonstrates our model of activity-based locomotor treadmill training for rats with spinal cord injury (SCI). Included is both quadrupedal and forelimb-only groups, in addition to two distinct types of non-trained control groups. Investigators are able to assess training effects on SCI rats using this protocol.

ההדרכה מבוססי פעילות על הליכון עם חוט השדרה נפגע Wistar חולדות

Spinal cord injury (SCI) results in lasting deficits that include both mobility and a multitude of autonomic-related dysfunctions. Locomotor training (LT) ...