מימון: עבודה זו נתמכה על ידי מענק הקרן Bertarelli (Djl), מענק MED-EL (Djl), ומכון לאומי לבריאות מענקי DC01089 (MCB).

הערה: כל הליכי הניסוי מבוצעים בהתאם לועדת הטיפול בבעלי חיים ושימוש בעיני מסצ&#39;וסטס ומרפאת אוזן ובית הספר לרפואה של אוניברסיטת הרווארד, שפועל לפי הנחיות טיפול בבעלי החיים לאומיות, ובכלל זה לבריאות ציבור שירות מדיניות על הומניות טיפול ושימוש בבעלי חיים במעבדה, ILAR מדריך, ובעלי חי חוק צער. הפרוצדורות מפורטות להלן פירוט חשיפה של DCN השמאל. השתמש בכלים סטריליים בעת ביצוע ניתוח הישרדות. 1. היסודי Craniotomy וחשיפה הגבי שבלול Nucleus <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הרדמה <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הרדימי עכבר, בגילי 8-12 שבועות, במשקל 18-24 גרם, עם 20 מ&quot;ג / קילוגרם xylazine וקטמין 100 מ&quot;ג / קילוגרם באמצעות ממשל intraperitoneal. אשר הרדמה תקינה על ידי קצב ניטור לב, קצב נשימה, כמו גם רפלקס נסיגת קמצוץ הבוהן. משחה וטרינר מקום על עיניים כדי למנוע יובש ואילו בהרדמה. ברגע כראוי בהרדמה, לא מעל שיער גילוחהוא הקרקפת כדי לספק גישה ללא הפרעה לאתר כירורגית. </li></ol></li><li style=";text-align:right;direction:rtl"> מיקום כירורגי <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> מניחים את העכבר באופן מאובטח בבעל החיים קטן stereotaxic, שנערך במקום על ידי מהדק חוטם. </li><li style=";text-align:right;direction:rtl"> ודא שמהדקים החוטם הוא רופף מספיק כדי לאפשר לנשימה נאותה אבל חזק מספיק כדי לשתק את הראש של העכבר לחלוטין. אם ראש העכבר הוא רופף, בעל החיים עלולים ליפול מבעל הראש במהלך חלק craniotomy של הליך. </li><li style=";text-align:right;direction:rtl"> מניחים אלקטרודות שתל גזע המוח שמיעתיות באופנה סטנדרטית, המאפשרת לניטור קצב לב. קצב נשימה צריך להיות במעקב על ידי הדמיה. שים לב שקצב לב נורמלי וקצב נשימה משתנים בהתאם לגילו של העכבר. </li><li style=";text-align:right;direction:rtl"> מדחום ממוקם וeuthermia מובטח באמצעות מיקום על שמיכת חימום homeothermic. </li></ol></li><li style=";text-align:right;direction:rtl"> חתך בעור וחשיפה של עצמות interparietal והעורפיות של הגולגולת <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> תחת microscאופ, לעשות חתך אנכי דרך העור מתחיל בקו האמצע, ישירות בין האפרכסת, והארכת לחלק הזנב של העורף. בעקבות חתך בעור קו האמצע, לעקור את העור רוחבי. דמיינו את השרירים המכסים את עזב את החלק האחורי של הגולגולת. </li><li style=";text-align:right;direction:rtl"> הסר שריר שמעל השמאל הקודקודית, interparietal, ועצמות עורפיות של הגולגולת על ידי disarticulation של קבצים מצורפים שרירים לעצמות שלהם עם מספריים או אזמל איריס. שים לב במידה קטנה של דימום לאורך קצות שרירים לחתוך. למזער את זה על ידי לחץ עדין עם המוליך כותנה שקצה במשך 10-15 שניות. </li></ol></li><li style=";text-align:right;direction:rtl"> שמעליה craniotomy השבלול Nucleus <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לזהות קווי תפר רלוונטיים, כולל קווי תפרים sagittal ולמבדה (איור 1, שמאל לוח). </li><li style=";text-align:right;direction:rtl"> שימוש rongeurs, להפוך את פתיחת גולגולת על עצם interparietal, השמאלית של קו האמצע, ~ הזנב 2 מ&quot;מ לקו התפר למבדה. אזור זה overlies DCN. </li><li style=";text-align:right;direction:rtl"> Followincraniotomy g, להתבונן בשכבה דקה של דורה שoverlies המוח הקטן. באמצעות סכין אזמל, להסיר את הדורה. (איור 1, פנל באמצע). הערה: הסרת הדורה עלולה לגרום למידה קטנה של דימום. תהליך זה של שימוש בלהב סכין המנתחים כדי להסיר את הדורה נועד להפחית את כמות איבוד דם במהלך שאיפת המוח הקטן, שברכה על פני השטח של גזע המוח וזיהוי אלמוני של DCN. סה&quot;כ נפח דם של עכבר הוא ~ 1.5 מיליליטר לא יעלה&gt; מ -15%. אם הדימום עולה על סכום זה, העכבר צריך להיות מסופק עם עם תוספת נוזלים. </li><li style=";text-align:right;direction:rtl"> הסר דם קרוש כיסה את המוח הקטן עם המוליך קצה כותנה. לחלופין, בעדינות לטפטף מי מלח באמצעות נקודת שיניים על המוח הקטן כדי לנקות דם משם דם. </li></ol></li><li style=";text-align:right;direction:rtl"> המוח הקטן שאיפה <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> באמצעות 5 יניקה צרפתית, לשאוב את החלק לרוחב-ביותר של המוח הקטן השמאלי מעל DCN עד DCN הוא מולרע&quot;מ (איור 1, ימין לוח). הסר כ 1/4 עד 1/3 של המוח הקטן משמאל. ציון דרך העיקרי בסמוך לDCN הוא ampulla של התעלה חצי עגולה מעולה. הערה: שאיפה של המוח הקטן היא צעד מפתח לפרוטוקול. שאיפת בימוי של המוח הקטן מתבצעת ביותר בהצלחה אם היא מתרחשת בניסיון בודד ולא עם מעברים מרובים של היניקה כמו זה יגרום לדימום. כדי לסייע בשאיפה, להגדיר את מישור המוקד של המיקרוסקופ בעומק הצפוי של CN, שיהיה מעט דיסטלי אל פני השטח של המוח הקטן. הגדרת מישור המוקד לCN תשפר הדמיה ולהבטיח תמונה חדה. </li><li style=";text-align:right;direction:rtl"> השאיפה הבאה, דימום נוסף, נוזל השדרתי (CSF) הצטברות, ועקירת המוח הקטן על DCN צפויים. להחדיר 0.5 סמ&quot;ק של תמיסת מלח סטרילית במהירות לפתיחת הגולגולת כדי למנוע קרישת דם. שילוב של ספיגה עדינה עם נקודה ויניקת שיניים יכול להיות אזנהג לסלק דרך להדמיה ישירה של DCN. ישירות לא לפנות אל פני השטח של DCN. </li></ol></li></ol> Microinjection לחץ 2. להעברת גנים בתיווך וירוס ושחזור כירורגי <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לאחר DCN הוא חופשי של דם וCSF שמעליה ונראים בבירור, להפוך microinjections לחץ לDCN באמצעות מזרק המילטון 10 μl על פני תקופה 2 דקות. </li><li style=";text-align:right;direction:rtl"> להציג את המחט עם micromanipulator עד הקצה הוא כבר לא נראה לעין מתחת לפני השטח של DCN. </li><li style=";text-align:right;direction:rtl"> לקבלת ביצועים מיטביים, השתמש במחט 33 או 34 מד (להשתמש במזרק gastight עם מחט 34 מד) עם שיפוע רדוד, כגון 45 מעלות, כדי למזער את הטראומה קהה ולמקם את עוצמת הזריקה בתוך DCN, שהוא דק ו מבנה גזע המוח רדוד (&lt;300 מיקרומטר עבה). הערה: מכשירי microinjection אחרים עשויים להיות מבוססים מנוצלים. באופן אידיאלי, מכשיר ההזרקה צריך להיות גמיש כדי לאפשר לכמה בן קלד, נחוץ אם לפנות ישירות לקהל DCN. יתר על כן, כפי שהעכבר זז מעט לאורך כל ההליך בשל נשימה, המכשיר צריך להיות חזק מספיק כדי לעמוד בכמויות קטנות של תנועה. </li></ol> שחזור כירורגי 3. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> מייד ההזרקה הבאה, מחדש משוערת העור ולאפשר העכבר כדי להתאושש להליך התאוששות סטנדרטי. רקמת המוח הקטן וצלקת שנותרה למלא את החלל שנגרם על ידי שאיפה של המוח הקטן. כל הזמן לפקח על בעלי חיים עד תודעה מספיק הוא חזר כדי לשמור על כיבה sternal. לעקוב אחר קצב לב, קצב נשימה, כמו גם יכולת להאכיל. הערה: אין חיה שעברה ניתוח חזר לכלוב עם בעלי חיים אחרים, עד שהתאושש לחלוטין. </li><li style=";text-align:right;direction:rtl"> אם עכבר מתחיל ללכת במעגלים לאחר ניתוח (&lt;5% מהמקרים), בדרך כלל 2-4 שעות לאחר סגירת החתך בעור, להקריב מייד העכבר, כפי שהוא עלול להביא להאכלת תקופה קשה. Tתופעת הלוואי שלו היא כנראה עקב שאיפה של המוח הקטן. בעלי החיים צריכים להיות במעקב לכאב לאחר הניתוח, ויש לתת סמים מתאימים כדי להבטיח נוחות בעלי חיים המבוססים על תקנים מוסדיים. אנטיביוטיקה עשויה להיות נחוצה, אם עדות לזיהום. </li></ol> 4. חשיפה Craniotomy והשבלול Nucleus משנית <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לאחר 2-4 שבועות של ריפוי ודגירת העברת גני וירוס בתיווך, מחדש להרדים זמן דגירה אופטימלית הזריק בעבר צעד עכבר וחזור על 1.1-1.5 משתנה בהתאם לסוג של גן הועבר. </li><li style=";text-align:right;direction:rtl"> הסרה של רקמת צלקת על אתר craniotomy על ידי שילוב של פינצטה, אזמל, וrongeurs. </li><li style=";text-align:right;direction:rtl"> לאחר לדמיין את פתיחת הגולגולת, לזהות שילוב של רקמת המוח הקטן ונותר צלקת מעל DCN. השתמש 5 יניקה צרפתית כדי לשאוב המוח הקטן שמעליה / רקמת צלקת (בדומה לשאיפת המוח הקטן הקודמת). </li><li style=";text-align:right;direction:rtl"> בעקבות שאיפה, מצפה דימום נוסף, ב CSFuild-אפ, וחתיכות הנותרות של המוח הקטן. במהירות להציג מלוח לפתיחת הגולגולת כדי למנוע קרישת דם. השתמש בשילוב של ספיגה עדינה שיניים נקודה ויניקה כדי לנקות את הנתיב להדמיה ישירה של DCN. </li><li style=";text-align:right;direction:rtl"> שים לב פני השטח של DCN. לבצע ניסויי פיזיולוגיה מבוסס optogenetics כDCN נגיש כעת. להציג את גירוי אור, למשל עם סיב אופטי (איור 2), לנהוג ניסוי מבוסס optogenetics. </li></ol> 5. היסטולוגיה <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> בעקבות מסקנה של ניסויים, להרדים את העכבר עם מנת יתר של קטמין. Perfuse העכבר עם מלח רגיל ואחריו paraformaldehyde 4%. </li><li style=";text-align:right;direction:rtl"> חלץ את גזע המוח מהגולגולת ופוסט-התיקון לשעה 2. Cryoprotect גזע המוח בסוכרוז 30% במשך 24-48 שעות. סעיף גזע המוח באמצעות cryostat סטנדרטי באמצעות 60 מיקרומטר חלקים. </li></ol>

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H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optogenetic+stimulation+of+the+auditory+pathway.">Optogenetic stimulation of the auditory pathway.</a> J Clin Invest. 124 (3), 1114-1129 (2014).</li><li>Adamantidis, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optogenetic+interrogation+of+dopaminergic+modulation+of+the+multiple+phases+of+reward-seeking+behavior.">Optogenetic interrogation of dopaminergic modulation of the multiple phases of reward-seeking behavior.</a> J Neurosci. 30 (31), 10829-10835 (2011).</li><li>Kim, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optogenetic+mimicry+of+the+transient+activation+of+dopamine+neurons+by+natural+reward+is+sufficient+for+operant+reinforcement.">Optogenetic mimicry of the transient activation of dopamine neurons by natural reward is sufficient for operant reinforcement.</a> PloS One. 7 (4), e33612 (2012).</li><li>Britt, J., Bonci, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optogenetic+interrogations+of+the+neural+circuits+underlying+addiction.">Optogenetic interrogations of the neural circuits underlying addiction.</a> Curr Opin Neurobiol. 23 (4), 539-545 (2013).</li><li>Abbott, S., Coates, M., Stornetta, R., Guyenet, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optogenetic+stimulation+of+c1+and+retrotrapezoid+nucleus+neurons+causes+sleep+state-dependent+cardiorespiratory+stimulation+and+arousal+in+rats.">Optogenetic stimulation of c1 and retrotrapezoid nucleus neurons causes sleep state-dependent cardiorespiratory stimulation and arousal in rats.</a> Hypertension. 61 (4), 835-841 (2013).</li><li>Carter, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tuning+arousal+with+optogenetic+modulation+of+locus+coeruleus+neurons.">Tuning arousal with optogenetic modulation of locus coeruleus neurons.</a> Nat Neurosci. 13 (12), 1526-1533 (2010).</li><li>Darrow, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optogenetic+control+of+central+auditory+neurons.">Optogenetic control of central auditory neurons.</a> Assoc. 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(695), (2012).</li><li>Shimano, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+the+AAV-mediated+expression+of+channelrhodopsin-2+and+halorhodopsin+in+brainstem+neurons+mediating+auditory+signaling.">Assessment of the AAV-mediated expression of channelrhodopsin-2 and halorhodopsin in brainstem neurons mediating auditory signaling.</a> Brain Res. 1511, 138-152 (2013).</li><li>Doucet, J., Ryugo, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Projections+from+the+ventral+cochlear+nucleus+to+the+dorsal+cochlear+nucleus+in+rats.">Projections from the ventral cochlear nucleus to the dorsal cochlear nucleus in rats.</a> J Comp Neurol. 385, 245-264 (1997).</li><li>Brown, M., Drottar, M., Benson, T., Darrow, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Commissural+axons+of+the+mouse+cochlear+nucleus.">Commissural axons of the mouse cochlear nucleus.</a> J Comp Neurol. 521, 1683-1696 (2013).</li><li>Verma, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Auditory+responses+to+electric+and+infrared+neural+stimulation+of+the+rat+cochlear+nucleus.">Auditory responses to electric and infrared neural stimulation of the rat cochlear nucleus.</a> Hear Res. 310, 69-75 (2014).</li><li>Taberner, A. M., Liberman, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Response+properties+of+single+auditory+nerve+fibers+in+the+mouse.">Response properties of single auditory nerve fibers in the mouse.</a> J Neurophysiol. 93 (1), 557-569 (2005).</li><li>Rolls, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optogenetic+disruption+of+sleep+continuity+impairs+memory+consolidation.">Optogenetic disruption of sleep continuity impairs memory consolidation.</a> Proc Natl Acad Sci. 108 (32), 13305-13310 (2011).</li><li>Huff, M., Miller, R., Deisseroth, K., Moorman, D., LaLumiere, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Posttraining+optogenetic+manipulations+of+basolateral+amygdala+activity+modulate+consolidation+of+inhibitory+avoidance+memory+in+rats.">Posttraining optogenetic manipulations of basolateral amygdala activity modulate consolidation of inhibitory avoidance memory in rats.</a> Proc Natl Acad Sci. 110 (9), 3597-3602 (2013).</li><li>Stortkuhl, K., Fiala, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Smell+of+Blue+Light:+A+New+Approach+toward+Understanding+an+Olfactory+Neuronal+Network.">The Smell of Blue Light: A New Approach toward Understanding an Olfactory Neuronal Network.</a> Front Neurosci. 5 (72), (2011).</li><li>Hira, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcranial+optogenetic+stimulation+for+functional+mapping+of+the+motor+cortex.">Transcranial optogenetic stimulation for functional mapping of the motor cortex.</a> J Neurosci Methods. 179 (2), 258-263 (2009).</li><li>Ayling, O., Harrison, T., Boyd, J., Foroshkov, A., Murphy, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+light-based+mapping+of+motor+cortex+by+photoactivation+of+channelrhodopsin-2+transgenic+mice.">Automated light-based mapping of motor cortex by photoactivation of channelrhodopsin-2 transgenic mice.</a> Nat Methods. 6 (3), 219-224 (2009).</li><li>Boyden, E., Zhang, F., Bamberg, E., Nagel, G., Deisseroth, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Millisecond-timescale,+genetically+targeted+optical+control+of+neural+activity.">Millisecond-timescale, genetically targeted optical control of neural activity.</a> Nat. Neurosci. 8, 1263-1268 (2005).</li></ol>

יש לי המחברים אין לחשוף.

מאמר זה מתאר את הטכניקה של הדמיה הישירה של DCN במודל העכברי למניפולציה של מערכת השמיעה המרכזית. הגישה המתוארת של הדמיה ישירה מספקת יתרונות משמעותיים על פני החלופה המרכזית, שהם גישות stereotaxic. בעיקר, להדמיה ישירה של DCN מאפשרת לאישור מיידי של האתר של גזע המוח, ואילו גישות ster...

Virus-mediated gene transfer to reverse hearing loss has largely been focused on the peripheral auditory system.1 Targeting the cochlea, investigators have examined a host of delivery routes, including osmotic minipump infusion2, vector-transgene complex-soaked Gelfoam&#174;2 or gelatin sponge3, direct microinjection4; numerous gene transfer vectors, including adeno-associated viral vectors5,6, lentiviral vectors7, and cationic liposomes2; and the dissemination of gene transfer vectors beyond the target tissue2. Most recently, adeno-associated virus (AAV)-1 has been introduced in the cochlea in order to treat deafness in mice due to loss of vesicular glutamate transporter-3.8 Further, the application of optogenetics in peripheral auditory system has recently been described.9
Few studies, however, have examined gene transfer to the central auditory system. The dorsal cochlear nucleus (DCN) of the brainstem contain second order neurons of the auditory pathway. While gene transfer techniques in the cochlear nucleus (CN) may be utilized for a host of investigations, gene transfer of opsins, light-sensitive proteins, to the DCN may also be utilized to enable optogenetics-based experimental techniques. Following virus-mediated gene transfer delivery of an opsin, such as channelrhodopsin-2 (ChR2), the neurons of the DCN becomes sensitive to light stimuli. Optogenetic gene transfer has been previously attempted in several brainstem regions, including the rat retrotrapezoid nucleus, mouse locus coeruleus, monkey superior colliculus, and mouse ventral tegmental area.10-14
Recently, investigators have examined the use of optogenetics in the DCN.15,16 The DCN is the location of placement of auditory brainstem implants in humans, making it an attractive part of the auditory system to study for translational studies on auditory neuroprostheses. However, given the location of the DCN, surgical exposure is challenging. The technique described herein provides a protocol for maximal exposure of the DCN via posterior fossa approach to enable viral vector gene transfer and optogenetics-based experiments in a murine model. Previous studies used stereotactic microinjection into the DCN with channelrhodopsin-2.16 Stereotaxic injections, however, are potentially less accurate than injections made by direct visualization, especially in a nucleus as small and deep along the brainstem as the DCN. Transgenic mice expressing tissue specific proteins in the CN are also an attractive option and would obviate the need for gene transfer. Our protocol for exposure of the DCN would also work in transgenic mice as the DCN would need to be directly exposed for optical stimulation. This technique for surgical exposure of the DCN is adapted from previous protocols involving recordings from the auditory nerve and cochlear nucleus in mice and rat models.15,17-20
The overall goal of the protocol is to provide direct exposure to the CN to allow for gene transfer techniques. More specifically, the approach is compatible with both acute and survival surgery and the preparation can be repeated in the same animal for subsequent neurophysiological testing. The direct exposure of the DCN protocol has implications for optogenetics- and virus-mediated gene transfer-based experimentation in other nuclei of the brainstem.

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</table>

direct visualization of the murine dorsal cochlear nucleus for optogenetic stimulation of the auditory pathway

חקירת השימוש בהעברת גני וירוס בתיווך לעצור או להפוך אובדן השמיעה במידה רבה נדחקה למערכת השמיעה ההיקפית. מחקרים מעטים בחנו העברת גנים למערכת השמיעה המרכזית. גרעין גב השבלול (DCN) של גזע המוח, המכיל נוירונים מסדר שני של המסלול השמיעתי, הוא אתר פוטנציאל להעברת גנים. בפרוטוקול זה, טכניקה לחשיפה ישירה ומקסימלי של DCN העכברי באמצעות גישה הפוסות אחורי מודגמת. גישה זו מאפשרת לאף אחד מניתוח אקוטי או הישרדות. בעקבות הדמיה ישירה של DCN, מארח של ניסויים אפשריים, כולל הזרקה של opsins לתוך גרעין השבלול ולאחר מכן על ידי הגירוי של סיבים אופטיים מצמידים את אור לייזר כחול. ניסויי נוירופיזיולוגיה אחרים, כגון גירוי חשמלי והעתקי מזרק עצביים הם גם אפשריים. רמת visualization ואת משך הזמן של גירוי השגה להפוך גישה זו ישימה למגוון רחב של ניסויים.

חלקית המוח הקטן שאיפה מדגימה גישה לגרעין השבלול לאחר העור והשרירים שמעל הגולגולת יוסרו, ציוני דרך פני גולגולת, כגון קווי תפר העטרה ומדא, מדגים את הלוקליזציה המשוערת של פתיחת הגולגולת. בעקבות פתיחת גולגולת עם rongeurs, המוח הקטן ?...

Watch this Scientific Journal Video about Direct Visualization of the Murine Dorsal Cochlear Nucleus for Optogenetic Stimulation of the Auditory Pathway at JoVE.com

Direct Visualization of the Murine Dorsal Cochlear Nucleus for Optogenetic Stimulation of the Auditory Pathway

The goal of this protocol is to outline a surgical approach to provide direct access to the dorsal cochlear nucleus in a murine model.

הדמיה ישירה של Murine הגבי שבלול Nucleus לoptogenetic גירוי של הנתיב השמיעתי

Investigation into the use of virus-mediated gene transfer to arrest or reverse hearing loss has largely been relegated to the peripheral auditory system. ...

direct-visualization-murine-dorsal-cochlear-nucleus-for-optogenetic

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9.2K Views.  Harvard Medical School. The goal of this protocol is to outline a surgical approach to provide direct access to the dorsal cochlear nucleus in a murine model. 

Video: Direct Visualization of the Murine Dorsal Cochlear Nucleus for Optogenetic Stimulation of the Auditory Pathway

Selective Viral Transduction of Adult-born Olfactory Neurons for Chronic in vivo Optogenetic Stimulation

Local interneurons are continuously regenerated in the olfactory bulb of adult rodents1-3. In this process, called adult neurogenesis, neural stem cells in the walls of the lateral ventricle give rise to neuroblasts that migrate for several millimeters along the rostral migratory stream (RMS) to reach and incorporate into the olfactory bulb. To study the different steps and the impact of adult-born neuron integration into preexisting olfactory circuits, it is necessary to selectively label and manipulate the activity of this specific population of neurons. The recent development of optogenetic technologies offers the opportunity to use light to precisely activate this specific cohort of neurons without affecting surrounding neurons4,5. Here, we present a series of procedures to virally express Channelrhodopsin2(ChR2)-YFP in a temporally restricted cohort of neuroblasts in the RMS before they reach the olfactory bulb and become adult-born neurons. In addition, we show how to implant and calibrate a miniature LED for chronic in vivo stimulation of ChR2-expressing neurons.

Selective Viral Transduction of Adult-born Olfactory Neurons for Chronic in vivo Optogenetic Stimulation

Adult-born neurons of the olfactory bulb can be optogenetically controlled using Channelrhodopsin2-expressing lentiviral injection in the rostral migratory stream and chronic photostimulation with an implanted miniature LED.

התמרה ויראלי סלקטיבי של נוירונים בוגרים יליד חוש הריח של כרוני In vivo Optogenetic גירוי

Local interneurons are continuously regenerated in the olfactory bulb of adult rodents1-3. In this process, called adult neurogenesis, neural stem cells ...

Visualization of Proprioceptors in Drosophila Larvae and Pupae

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other insects proprioception is provided by specialized sensory organs termed chordotonal organs (ChOs) 2. Like many other organs in Drosophila, ChOs develop twice during the life cycle of the fly. First, the larval ChOs develop during embryogenesis. Then, the adult ChOs start to develop in the larval imaginal discs and continue to differentiate during metamorphosis.
The development of larval ChOs during embryogenesis has been studied extensively 10,11,13,15,16. The centerpiece of each ChO is a sensory unit composed of a neuron and a scolopale cell. The sensory unit is stretched between two types of accessory cells that attach to the cuticle via specialized epidermal attachment cells 1,9,14. When a fly larva moves, the relative displacement of the epidermal attachment cells leads to stretching of the sensory unit and consequent opening of specific transient receptor potential vanilloid (TRPV) channels at the outer segment of the dendrite 8,12. The elicited signal is then transferred to the locomotor central pattern generator circuit in the central nervous system.
Multiple ChOs have been described in the adult fly 7. These are located near the joints of the adult fly appendages (legs, wings and halters) and in the thorax and abdomen. In addition, several hundreds of ChOs collectively form the Johnston's organ in the adult antenna that transduce acoustic to mechanical energy 3,5,17,4.
In contrast to the extensive knowledge about the development of ChOs in embryonic stages, very little is known about the morphology of these organs during larval stages. Moreover, with the exception of femoral ChOs 18 and Johnston's organ, our knowledge about the development and structure of ChOs in the adult fly is very fragmentary.
Here we describe a method for staining and visualizing ChOs in third instar larvae and pupae. This method can be applied together with genetic tools to better characterize the morphology and understand the development of the various ChOs in the fly.

Visualization of Proprioceptors in Drosophila Larvae and Pupae

A method to immunostain and visualize chordotonal organs in larvae and pupae of Drosophila melanogaster is described.

ויזואליזציה של Proprioceptors ב<em&gt; תסיסנית</em&gt; זחלים וגלמים

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other ...

Fiber-optic Implantation for Chronic Optogenetic Stimulation of Brain Tissue

Elucidating patterns of neuronal connectivity has been a challenge for both clinical and basic neuroscience. Electrophysiology has been the gold standard for analyzing patterns of synaptic connectivity, but paired electrophysiological recordings can be both cumbersome and experimentally limiting. The development of optogenetics has introduced an elegant method to stimulate neurons and circuits, both in vitro1 and in vivo2,3. By exploiting cell-type specific promoter activity to drive opsin expression in discrete neuronal populations, one can precisely stimulate genetically defined neuronal subtypes in distinct circuits4-6. Well described methods to stimulate neurons, including electrical stimulation and/or pharmacological manipulations, are often cell-type indiscriminate, invasive, and can damage surrounding tissues. These limitations could alter normal synaptic function and/or circuit behavior. In addition, due to the nature of the manipulation, the current methods are often acute and terminal. Optogenetics affords the ability to stimulate neurons in a relatively innocuous manner, and in genetically targeted neurons. The majority of studies involving in vivo optogenetics currently use a optical fiber guided through an implanted cannula6,7; however, limitations of this method include damaged brain tissue with repeated insertion of an optical fiber, and potential breakage of the fiber inside the cannula. Given the burgeoning field of optogenetics, a more reliable method of chronic stimulation is necessary to facilitate long-term studies with minimal collateral tissue damage. Here we provide our modified protocol as a video article to complement the method effectively and elegantly described in Sparta et al.8 for the fabrication of a fiber optic implant and its permanent fixation onto the cranium of anesthetized mice, as well as the assembly of the fiber optic coupler connecting the implant to a light source. The implant, connected with optical fibers to a solid-state laser, allows for an efficient method to chronically photostimulate functional neuronal circuitry with less tissue damage9 using small, detachable, tethers. Permanent fixation of the fiber optic implants provides consistent, long-term in vivo optogenetic studies of neuronal circuits in awake, behaving mice10 with minimal tissue damage.

The development of optogenetics now provides the means to precisely stimulate genetically defined neurons and circuits, both in vitro and in vivo. Here we describe the assembly and implantation of a fiber optic for chronic photostimulation of brain tissue.

השרשה של סיבים אופטיות לגירוי Optogenetic כרוני של רקמת המוח

Elucidating patterns of neuronal connectivity has been a challenge for both clinical and basic neuroscience. Electrophysiology has been the gold standard ...

Optogenetic Stimulation of Escape Behavior in Drosophila melanogaster

A growing number of genetically encoded tools are becoming available that allow non-invasive manipulation of the neural activity of specific neurons in Drosophila melanogaster1. Chief among these are optogenetic tools, which enable the activation or silencing of specific neurons in the intact and freely moving animal using bright light. Channelrhodopsin (ChR2) is a light-activated cation channel that, when activated by blue light, causes depolarization of neurons that express it. ChR2 has been effective for identifying neurons critical for specific behaviors, such as CO2 avoidance, proboscis extension and giant-fiber mediated startle response2-4. However, as the intense light sources used to stimulate ChR2 also stimulate photoreceptors, these optogenetic techniques have not previously been used in the visual system. Here, we combine an optogenetic approach with a mutation that impairs phototransduction to demonstrate that activation of a cluster of loom-sensitive neurons in the fly's optic lobe, Foma-1 neurons, can drive an escape behavior used to avoid collision. We used a null allele of a critical component of the phototransduction cascade, phospholipase C-&beta;, encoded by the norpA gene, to render the flies blind and also use the Gal4-UAS transcriptional activator system to drive expression of ChR2 in the Foma-1 neurons. Individual flies are placed on a small platform surrounded by blue LEDs. When the LEDs are illuminated, the flies quickly take-off into flight, in a manner similar to visually driven loom-escape behavior. We believe that this technique can be easily adapted to examine other behaviors in freely moving flies.

Optogenetic Stimulation of Escape Behavior in Drosophila melanogaster

Genetically encoded optogenetic tools enable noninvasive manipulation of specific neurons in the Drosophila brain. Such tools can identify neurons whose activation is sufficient to elicit or suppress particular behaviors. Here we present a method for activating Channelrhodopsin2 that is expressed in targeted neurons in freely walking flies.

גירוי Optogenetic של התנהגות בריחה ב<em&gt; דרוזופילה melanogaster</em

A  growing number of genetically encoded tools are becoming available that allow non-invasive  manipulation of the neural activity of specific neurons in ...

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution has been a long sought tool for neuroscientists in the systems-level search for the neural circuitry governing complex behavioral states. Optogenetics is a cutting-edge tool that is revolutionizing the field of neuroscience and represents one of the first systematic approaches to enable causal testing regarding the relation between neural signaling events and behavior. By combining optical and genetic approaches, neural signaling can be bi-directionally controlled through expression of light-sensitive ion channels (opsins) in mammalian cells. The current protocol describes delivery of specific wavelengths of light to opsin-expressing cells in deep brain structures of awake, freely-moving rodents for neural circuit modulation. Theoretical principles of light transmission as an experimental consideration are discussed in the context of performing in vivo optogenetic stimulation. The protocol details the design and construction of both simple and complex laser configurations and describes tethering strategies to permit simultaneous stimulation of multiple animals for high-throughput behavioral testing.

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

Optogenetics has become a powerful tool for use in behavioral neuroscience experiments. This protocol offers a step-by-step guide to the design and set-up of laser systems, and provides a full protocol for carrying out multiple and simultaneous in vivo optogenetic stimulations compatible with most rodent behavioral testing paradigms.

בoptogenetic גירוי vivo של מערכת העצבים המרכזית מכרסם

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution ...

Optogenetic Stimulation of the Auditory Nerve

Direct electrical stimulation of spiral ganglion neurons (SGNs) by cochlear implants (CIs) enables open speech comprehension in the majority of implanted deaf subjects1-6. Nonetheless, sound coding with current CIs has poor frequency and intensity resolution due to broad current spread from each electrode contact activating a large number of SGNs along the tonotopic axis of the cochlea7-9. Optical stimulation is proposed as an alternative to electrical stimulation that promises spatially more confined activation of SGNs and, hence, higher frequency resolution of coding. In recent years, direct infrared illumination of&#160;the cochlea has been used to evoke responses in the auditory nerve10. Nevertheless it requires higher energies than electrical stimulation10,11 and uncertainty remains as to the underlying mechanism12. Here we describe a method based on optogenetics to stimulate SGNs with low intensity blue light, using transgenic mice with neuronal expression of channelrhodopsin 2 (ChR2)13 or virus-mediated expression of the ChR2-variant CatCh14. We used micro-light emitting diodes (&#181;LEDs) and fiber-coupled lasers to stimulate ChR2-expressing SGNs through a small artificial opening (cochleostomy) or the round window. We assayed the responses by scalp recordings of light-evoked potentials (optogenetic auditory brainstem response: oABR) or by microelectrode recordings from the auditory pathway and compared them with acoustic and electrical stimulation.

Cochlear implants (CIs) enable hearing by direct electrical stimulation of the auditory nerve. However, poor frequency and intensity resolution limits the quality of hearing with CIs. Here we describe optogenetic stimulation of the auditory nerve in mice as an alternative strategy for auditory research and developing future CIs.

גירוי optogenetic של עצב השמיעה

Direct electrical stimulation of spiral ganglion neurons (SGNs) by cochlear implants (CIs) enables open speech comprehension in the majority of implanted ...

Analysis of Gene Expression Changes in the Rat Hippocampus After Deep Brain Stimulation of the Anterior Thalamic Nucleus

Deep brain stimulation (DBS) surgery, targeting various regions of the brain such as the basal ganglia, thalamus, and subthalamic regions, is an effective treatment for several movement disorders that have failed to respond to medication. Recent progress in the field of DBS surgery has begun to extend the application of this surgical technique to other conditions as diverse as morbid obesity, depression and obsessive compulsive disorder. Despite these expanding indications, little is known about the underlying physiological mechanisms that facilitate the beneficial effects of DBS surgery. One approach to this question is to perform gene expression analysis in neurons that receive the electrical stimulation. Previous studies have shown that neurogenesis in the rat dentate gyrus is elicited in DBS targeting of the anterior nucleus of the thalamus1. DBS surgery targeting the ATN is used widely for treatment refractory epilepsy. It is thus of much interest for us to explore the transcriptional changes induced by electrically stimulating the ATN. In this manuscript, we describe our methodologies for stereotactically-guided DBS surgery targeting the ATN in adult male Wistar rats. We also discuss the subsequent steps for tissue dissection, RNA isolation, cDNA preparation and quantitative RT-PCR for measuring gene expression changes. This method could be applied and modified for stimulating the basal ganglia and other regions of the brain commonly clinically targeted. The gene expression study described here assumes a candidate target gene approach for discovering molecular players that could be directing the mechanism for DBS.

The mechanism underlying the therapeutic effects of Deep Brain Stimulation (DBS) surgery needs investigation. The methods presented in this manuscript describe an experimental approach to examine the cellular events triggered by DBS by analyzing the gene expression profile of candidate genes that can facilitate neurogenesis post DBS surgery.

ניתוח שינויי ביטוי הגנים בהיפוקמפוס העכברוש לאחר Deep Brain Stimulation של Anterior התלמוס הגרעין

Deep brain stimulation (DBS) surgery, targeting various regions of the brain such as the basal ganglia, thalamus, and subthalamic regions, is an effective ...

In Vitro Modeling of Cancerous Neural Invasion: The Dorsal Root Ganglion Model

One way that solid tumors disseminate is through neural invasion. This route is well-known in cancers of the head and neck, prostate, and pancreas. These neurotropic cancer cells have a unique ability to migrate unidirectionally along nerves towards the central nervous system (CNS). The dorsal root ganglia (DRG)/cancer cell model is a three dimensional (3D) in vitro model frequently used for studying the interaction between neural stroma and cancer cells. In this model, mouse or human cancer cell lines are grown in ECM adjacent to preparations of freshly dissociated cultured DRG. In this article, the DRG isolation protocol from mice, and implantation in petri dishes for co-culturing with pancreatic cancer cells are demonstrated. Five days after implantation, the cancer cells made contact with the DRG neurites. Later, these cells formed bridgeheads to facilitate more extensive polarized, neurotropic migration of cancer cells.

In Vitro Modeling of Cancerous Neural Invasion: The Dorsal Root Ganglion Model

This video article shows the use of the dorsal root ganglia (DRG)/cancer cell model in pancreatic ductal adenocarcinoma.

במבחנת דוגמנות של פלישה העצבית סרטנית: מודל גנגליון השורש הגבה

One way that solid tumors disseminate is through neural invasion. This route is well-known in cancers of the head and neck, prostate, and pancreas. These ...

Microelectrode Guided Implantation of Electrodes into the Subthalamic Nucleus of Rats for Long-term Deep Brain Stimulation

Deep brain stimulation (DBS) is a widely used and effective therapy for several neurologic disorders, such as idiopathic Parkinson&#8217;s disease, dystonia or tremor. DBS is based on the delivery of electrical stimuli to specific deep anatomic structures of the central nervous system. However, the mechanisms underlying the effect of DBS remain enigmatic. This has led to an interest in investigating the impact of DBS in animal models, especially in rats. As DBS is a long-term therapy, research should be focused on molecular-genetic changes of neural circuits that occur several weeks after DBS. Long-term DBS in rats is challenging because the rats move around in their cage, which causes problems in keeping in place the wire leading from the head of the animal to the stimulator. Furthermore, target structures for stimulation in the rat brain are small and therefore electrodes cannot easily be placed at the required position. Thus, a set-up for long-lasting stimulation of rats using platinum/iridium electrodes with an impedance of about 1 M&#937; was developed for this study. An electrode with these specifications allows for not only adequate stimulation but also recording of deep brain structures to identify the target area for DBS. In our set-up, an electrode with a plug for the wire was embedded in dental cement with four anchoring screws secured onto the skull. The wire from the plug to the stimulator was protected by a stainless-steel spring. A swivel was connected to the circuit to prevent the wire from becoming tangled. Overall, this stimulation set-up offers a high degree of free mobility for the rat and enables the head plug, as well as the wire connection between the plug and the stimulator, to retain long-lasting strength.

A method for implanting electrodes into the subthalamic nucleus (STN) of rats is described. Better localization of the STN was achieved by using a microrecording system. Furthermore, a stimulation set-up is presented that is characterized by long-lasting connections between the head of the animal and the stimulator.

Microelectrode מודרך השרשה של אלקטרודות לתוך גרעין Subthalamic של חולדות לטווח ארוך עמוק גירוי המוח

Deep brain stimulation (DBS) is a widely used and effective therapy for several neurologic disorders, such as idiopathic Parkinson&#8217;s disease, ...

Isolation of Adult Spinal Cord Nuclei for Massively Parallel Single-nucleus RNA Sequencing

Probing an individual cell&#39;s gene expression enables the identification of cell type and cell state. Single-cell RNA sequencing has emerged as a powerful tool for studying transcriptional profiles of cells, particularly in heterogeneous tissues such as the central nervous system. However, dissociation methods required for single cell sequencing can lead to experimental changes in the gene expression and cell death. Furthermore, these methods are generally restricted to fresh tissue, thus limiting studies on archival and bio-bank material. Single nucleus RNA sequencing (snRNA-Seq) is an appealing alternative for transcriptional studies, given that it accurately identifies cell types, permits the study of tissue that is frozen or difficult to dissociate, and reduces dissociation-induced transcription. Here, we present a high-throughput protocol for rapid isolation of nuclei for downstream snRNA-Seq. This method enables isolation of nuclei from fresh or frozen spinal cord samples and can be combined with two massively parallel droplet encapsulation platforms.

Here, we present a protocol to rapidly isolate high-quality nuclei from the fresh or frozen tissue for downstream massively parallel RNA sequencing. We include detergent-mechanical and hypotonic-mechanical tissue disruption and cell lysis options, both of which can be used for isolation of nuclei.

בידוד של חוט השדרה למבוגרים גרעינים עבור רצפי RNA חד-גרעין בנפט מקבילים

Probing an individual cell&#39;s gene expression enables the identification of cell type and cell state. Single-cell RNA sequencing has emerged as a ...