1) הכנה <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> כדי להתחיל, במושב בנושא על כסא נוח מול מסך מחשב. </li><li style=";text-align:right;direction:rtl"> השתמש סרט מדידה על מנת להבטיח את המרחק בין המסך nasion של הנבדק הוא 60 ס&quot;מ. </li><li style=";text-align:right;direction:rtl"> לבסוף, במקום מסכה חסימת אור על העיניים של הנבדק. </li></ol> 2) קביעת סף Phosphene על v1/v2 <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הידיעה סף phosphene של הנבדק יהיה חשוב כאשר בוחנים התנהגות phosphenic מאוחר יותר בפרוטוקול זה. כדי לקבוע לסף זה, להגדיר תחילה את המכונה TMS לשלטון 70%. </li><li style=";text-align:right;direction:rtl"> החזיקו את הסליל כך את החלק העליון של קו האמצע דמות-8 פנים הגולגולת. </li><li style=";text-align:right;direction:rtl"> בגין גירוי יחיד הדופק בנקודה על 3 ס&quot;מ מעל inion. </li><li style=";text-align:right;direction:rtl"> אחרי הדופק בכל, לשאול את הנושא לדווח על כל ניסיון phosphenic. </li><li style=";text-align:right;direction:rtl"> בגין העברת סליל ברשת חיפוש קטן סביב האזור הזה (v1/v2). </li><li style=";text-align:right;direction:rtl"> קבע את המיקום שבו גירוי מעורר דוחות phosphene עקבי וחד משמעי מהנושא. זה כבר שיערו כי TMS לא יוכלו להפיק phosphenes ב -40% מהאוכלוסייה, כך יש סיכוי הנושא לא ידווח דבר. אם זה המקרה, למרבה הצער, הניסוי הזה לא יעבוד. הנושא צריך להיות מותר לעזוב. </li><li style=";text-align:right;direction:rtl"> לאחר phosphenic &quot;נקודה חמה&quot; כבר נמצא, ולהתאים את תפוקת החשמל TMS למעלה ולמטה עד phosphenes דיווחים נושא בדיוק 3 מתוך 6 פולסים רצופים. רמה זו היא כוח v1/v2 של הנבדק הסף. אם זמין, neuronavigation ניתן להשתמש כדי להשיג דיוק רב יותר מרחבית כאשר איתור מיקוד hotspot v1/v2. </li></ol> 3) קביעת סף Phosphenic על V5/MT + קומפלקס <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> באמצעות ההליך הנ&quot;ל זהה ופרמטרים, כעת אנו קובעים סף phosphene על V5/MT + מורכבת. כדי לאתר את האזור הזה, להתחיל בנקודה 3 ס&quot;מ הגבי ו 5 ס&quot;מ לרוחב inion של הנושא. </li><li style=";text-align:right;direction:rtl"> אחרי הדופק בכל, לשאול את הנושא לדווח על כל ניסיון phosphenic. </li><li style=";text-align:right;direction:rtl"> שוב, באמצעות פולסים אחת, להתחיל דפוס קטן רשת החיפוש עד phosphenes חד משמעית ועקבית הם עוררו. </li><li style=";text-align:right;direction:rtl"> לבסוף, להתאים את תפוקת החשמל TMS למעלה ולמטה עד phosphenes דיווחים נושא בדיוק 3 מתוך 6 פולסים רצופים. רמה זו היא כוח V5/MT של נושא + סף מורכבים. שוב, אם neuronavigation זמין, ניתן להשתמש כדי להשיג דיוק רב יותר מרחבית כאשר איתור מיקוד V5/MT + נקודה חמה. </li></ol> 4) קביעת Phosphene התנהגות Baseline <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לאחר סף phosphene נקבע הן אזורים ויזואליים, אנחנו צריכים למדוד phosphenic התנהגות בסיסית. כדי לעשות זאת, להסיר תחילה את אור חסימה של הנבדק מסכה. </li><li style=";text-align:right;direction:rtl"> לאחר מכן, להורות את הנושא לבהות צלב קיבוע יציב הציגה במרכז מסך המחשב למשך 60 שניות. </li><li style=";text-align:right;direction:rtl"> החלף את המסכה מעל נקודה v1/v2 החם, ליצור רכבת הדופק יחיד עבור 3 שניות ב 120% v1/v2 הסף. </li><li style=";text-align:right;direction:rtl"> המתן חמש שניות אחר התנהלות הרכבת. </li><li style=";text-align:right;direction:rtl"> שוב, לחכות חמש שניות והתנהגות רכבת השלישי. </li><li style=";text-align:right;direction:rtl"> לאחר הרכבת הדופק השלישי יחיד, לשאול את הנושא כדי לתאר את המיקום ואת המניע המאפיינים של כל עורר phosphenes. זה יהיה הבסיס. </li><li style=";text-align:right;direction:rtl"> חזור על נוהל זה זהה על V5/MT + נקודה חמה (זוכרים לאפס את כוח TMS עד 120% הסף V5/MT +). </li></ol> 5) תנאי מספר אחד <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לאחר ההתחלה phophenic נקבע, להסיר אור חוסם את הנושא המסכה שוב. </li><li style=";text-align:right;direction:rtl"> הדריכו את הנושא לבהות צלב קיבוע יציב הציגה במרכז מסך המחשב למשך 60 שניות. הפעם, במקום מסך ריק, נוכל להטביע סדרה של נקודות נעות במקומות שונים ברחבי הצלב. <img src="/files/ftp_upload/2273/2273fig1.jpg" alt="איור 1" /> איור 1 עבור התאמת גירויים בתנאי אחד:. Translational תנועה פשוטה. נקודות כל מהלך קוהרנטי או שמאלה או ימינה. ודא שכל הנקודות נעות באותו כיוון. גירוי זה צריך לשמש כדי עיבוד חזותי לעורר: תופעה שבה שינויים רגישות עצבית כמו הנגרם על ידי חשיפה ממושכת לגירוי משמש הטיה התפיסה של גירויים שהוצגו לאחר מכן. </li><li style=";text-align:right;direction:rtl"> לאחר 60 שניות, להחליף את המסכה מעל נקודה חמה v1/v2, ליצור רכבת הדופק אחד למשך 3 שניות ב 120% v1/v2 הסף. </li><li style=";text-align:right;direction:rtl"> המתן חמש שניות וחזור פעמיים נוספות. </li><li style=";text-align:right;direction:rtl"> אחרי שלוש רכבות, לשאול את נושא הדיווח מקומות ומאפיינים המניע של כל עורר phosphenes. אם זמין, השימוש במערכת עין התנועה מעקב בחדר חשוך יכול לעזור להשיג דיוק רב יותר ניטור כמותי של תנועות עיניים שנעשו על ידי הנבדקים במהלך elicitations phosphene. </li><li style=";text-align:right;direction:rtl"> המשך רצף three-train/report עד phospהתנהגות hene חוזר לפעילות בסיסית. </li><li style=";text-align:right;direction:rtl"> חזור על הליך זה עבור V5/MT + hotspot מורכבים. </li></ol> 6) תנאי מספר שתיים <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> במצב השני, לחזור על אותו הליך מנוצל בתנאי אחד. הפעם, לעומת זאת, במקום להציג את הנושא עם סדרה של נקודות נעות בכיוון יחיד, להציג את הנושא עם סדרה של נקודות כל אחד בכיוון הקרדינל היחסי במרחק נקודה מרכזית - בדומה לדפוס &quot;פרץ כוכבים&quot; . <img src="/files/ftp_upload/2273/2273fig2.jpg" alt="איור 2" /> איור 2 התאמת גירויים עבור מצב שני:. תנועה רדיאלי. נקודות לנוע בכיוונים הקרדינל בהתאמה גם כלפי או הרחק נקודה מרכזית. </li><li style=";text-align:right;direction:rtl"> כמו בעבר, לאחר 60 שניות, להחליף את המסכה, לערוך סדרה של שלושה, רכבות סף 120% מעל v1/v2, לבקש דו&quot;ח בנושא, וממשיכים עד חוזר הבסיס. </li><li style=";text-align:right;direction:rtl"> חזור עבור + V5/MT. </li></ol> 7) תרשים <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> כאופציה, אחרי כל תנאי (או אחרי כל התנאים הושלמו), לשאול את הנושא כדי לצייר את אזורים התנהגות המניע של כל סדרה של phopsphenes על גבי הגרף. זה לא צעד בלתי נפרד אבל יספק לך סדרה נוספת של נתונים לפירוש. </li></ol> 8) נציג תוצאות עיבוד חזותי של חד כיוונית גירויים המניע צריך לעורר תנועה phosphenic זהה על v1/v2. <img src="/files/ftp_upload/2273/2273fig3.jpg" alt="איור 3" /> איור 3. דוגמאות phosphenes הנובעים v1/v2 בחצי הכדור ימינה ושמאלה במהלך בתנאי אחד (מבוסס על הרישומים של נושאים). V5/MT + phosphenes מורכב צריך גם להיות מושפע, כך phosphene כעת יופיע סכום של תנועה לכיוון הגירוי הסתגלות phosphene הבסיס. עם זאת, עיבוד חזותי לדפוס מהספרים צריך לעורר מהספרים זהה phosphenic התנועה מן V5/MT + מורכב, אבל לא צריך לשנות את הבסיס v1/v2. <img src="/files/ftp_upload/2273/2273fig4.jpg" alt="איור 4" /> איור 4. דוגמאות phosphenes הנובעים V5/MT + מורכב בחצי הכדור ימינה ושמאלה במהלך מצב שני (על סמך הרישומים של נושאים).

<ol>
	<li>Pascual-Leone, A., Davey, M., Wassermann, E. M., Rothwell, J., Puri, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Handbook of Transcranial Magnetic Stimulation. , (2002).</li><li>Walsh, V., Pascual-Leone, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Transcranial Magnetic Stimulation: A Neurochronometrics of Mind. , (2005).</li><li>Silvanto, J., Muggleton, N. G., Cowey, A., Walsh, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+adaptation+reveals+state-dependent+effects+of+transcranial+magnetic+stimulation.">Neural adaptation reveals state-dependent effects of transcranial magnetic stimulation.</a> European Journal of Neuroscience. 25, 1874-1881 (2007).</li><li>Silvanto, J., Pascual-Leone, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=State-Dependency+of+Transcranial+Magnetic+Stimulation.">State-Dependency of Transcranial Magnetic Stimulation.</a> Brain Topography. 21, 1-10 (2008).</li><li>Silvanto, J., Cattaneo, Z., Battelli, L., Pascual-Leone, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Baseline+cortical+excitablility+determines+whether+TMS+disrupts+or+facilitates+behavior.">Baseline cortical excitablility determines whether TMS disrupts or facilitates behavior.</a> Journal of Neurophysiology. 99, 2725-2730 (2008).</li><li>Silvanto, J., Muggleton, N. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Testing+the+validity+of+the+TMS+state-dependency+approach:+targeting+functionally+distinct+motion-selective+neural+populations.">Testing the validity of the TMS state-dependency approach: targeting functionally distinct motion-selective neural populations.</a> Neuroimage. 40, 1841-1848 (2008).</li><li>Tootell, R. B., Rappas, J. B., Kwong, K. K., Malach, R., Born, R. T., Brady, T. J., Rosen, B. R., Belliveau, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+analysis+of+human+MT+and+related+visual+cortical+areas+using+magnetic+resonance+imaging.">Functional analysis of human MT and related visual cortical areas using magnetic resonance imaging.</a> Journal of Neuroscience. 15, 3215-3230 (1995).</li><li>Singh, K. D., Smith, A. T., Greenlee, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatiotemporal+frequency+and+direction+sensitivities+of+human+visual+areas+measured+using+fMRI.">Spatiotemporal frequency and direction sensitivities of human visual areas measured using fMRI.</a> Neuroimage. 12, 550-564 (2000).</li><li>Rutchmann, R. M., Schrauf, M., Greenlee, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain+activation+during+dichoptic+presentation+of+optic+flow+stimuli.">Brain activation during dichoptic presentation of optic flow stimuli.</a> Exp Brain Res. 134, 533-537 (2000).</li><li>Morrone, M. C., Tosetti, M., Montanaro, D., Fiorentini, A., Cioni, G., Burr, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+cortical+area+that+responds+specifically+to+optic+flow+revealed+by+fMRI.">A cortical area that responds specifically to optic flow revealed by fMRI.</a> Nat. Neuroscience. 3, 1322-1328 (2000).</li></ol>

אין ניגודי אינטרסים הכריז.

ניסוי זה נעשה בלב תלות המדינה. נוירונים v1/v2 נחשבים להתכתב עם התנועה, פשוט כיוונית 7,8 לכן, הסתגלות חד כיוונית גירויים מניע הגדילו את רגישות של נוירונים רישום תנועה זו -. ככאלה, הם צריכים להיות הראשונים להגיב על TMS הדופק. V5/MT + מורכב מכיל גם נוירונים מכוון תנועה translation...

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state-dependency effects on tms:  a look at motive phosphene behavior

גירוי מגנטי Transcranial (TMS) הוא neurostimulatory פולשני וטכניקה neuromodulatory שיכולים transiently לאורך זמן רב או לווסת את רגישות קליפת המוח (או להגדיל או להקטין אותו) באמצעות יישום מקומי פולסים השדה המגנטי. 1,2 בתחום של TMS, המונח תלות המדינה מתייחס למצב, הבסיס הראשוני של האזור העצבי בפרט ממוקד לגירוי. כפי שניתן להסיק, את ההשפעות של TMS יכולים (ועושים) להשתנות על פי רגישות זו התגובה הראשוני של קליפת המוח באזור ממוקד. 3,4,5 בניסוי זה, נבחן את המושג הזה של תלות המדינה דרך ההתמחרות ועל החוויה הסובייקטיבית של phosphenes מניע. Phosphenes נתפסים חזותית הבזקים של אורות קטנים מופעלת על ידי פולסים אלקטרומגנטיים לקליפת המוח הראייתית. אלה אורות קטנים יכולים להניח מאפיינים שונים תלוי איזה סוג של קליפת המוח החזותית היא להיות מגורה. במחקר זה בפרט, נהיה מיקוד phosphenes המניע כפי שהושרו על ידי גירוי של v1/v2 ואת V5/MT + מורכב אזורים ויזואליים. 6

Watch this Scientific Journal Video about State-Dependency Effects on TMS:  A Look at Motive Phosphene Behavior at JoVE.com

State-Dependency Effects on TMS:  A Look at Motive Phosphene Behavior

במאמר זה, נבחן את ההשפעות של תלות המדינה הרלוונטיים חזותית על TMS המושרה phosphenic מצגות מניע.

המדינה תלות השפעות על TMS: מבט על התנהגות Phosphene מניע

Transcranial magnetic stimulation (TMS) is a non-invasive neurostimulatory and neuromodulatory technique that can transiently or lastingly modulate ...

state-dependency-effects-on-tms-a-look-at-motive-phosphene-behavior

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JoVE Journal

Neuroscience

10.5K Views.  Beth Israel Deaconess Medical Center. במאמר זה, נבחן את ההשפעות של תלות המדינה הרלוונטיים חזותית על TMS המושרה phosphenic מצגות מניע.

Video: State-Dependency Effects on TMS:  A Look at Motive Phosphene Behavior

The NeuroStar TMS Device: Conducting the FDA Approved Protocol for Treatment of Depression

The Neuronetics NeuroStar Transcranial Magnetic Stimulation (TMS) System is a class II medical device that produces brief duration, pulsed magnetic fields. These rapidly alternating fields induce electrical currents within localized, targeted regions of the cortex which are associated with various physiological and functional brain changes.1,2,3
In 2007, O'Reardon et al., utilizing the NeuroStar device, published the results of an industry-sponsored, multisite, randomized, sham-stimulation controlled clinical trial in which 301 patients with major depression, who had previously failed to respond to at least one adequate antidepressant treatment trial, underwent either active or sham TMS over the left dorsolateral prefrontal cortex (DLPFC). The patients, who were medication-free at the time of the study, received TMS five times per week over 4-6 weeks.4
The results demonstrated that a sub-population of patients (those who were relatively less resistant to medication, having failed not more than two good pharmacologic trials) showed a statistically significant improvement on the Montgomery-Asberg Depression Scale (MADRS), the Hamilton Depression Rating Scale (HAMD), and various other outcome measures. In October 2008, supported by these and other similar results5,6,7, Neuronetics obtained the first and only Food and Drug Administration (FDA) approval for the clinical treatment of a specific form of medication-refractory depression using a TMS Therapy device (FDA approval K061053).
In this paper, we will explore the specified FDA approved NeuroStar depression treatment protocol (to be administered only under prescription and by a licensed medical profession in either an in- or outpatient setting).

In this article, we examine the methodology and considerations relevant to the FDA approved depression treatment protocol using the Neuronetics NeuroStar TMS device.

התקן TMS NeuroStar: ניהול ה-FDA פרוטוקול מאושר לטיפול דיכאון

The Neuronetics NeuroStar Transcranial Magnetic Stimulation (TMS) System is a class II medical device that produces brief duration, pulsed magnetic ...

Cryopreservation of Cortical Tissue Blocks for the Generation of Highly Enriched Neuronal Cultures

In this study, we outline a standardized protocol for the successful cryopreservation and thawing of cortical brain tissue blocks to generate highly enriched neuronal cultures. For this protocol the freezing medium used is 10% dimethyl sulfoxide (DMSO) diluted in Hank's Buffered Salt Solution (HBSS). Blocks of cortical tissue are transferred to cryovials containing the freezing medium and slowly frozen at -1&deg;C/min in a rate-controlled freezing container. Post-thaw processing and dissociation of frozen tissue blocks consistently produced neuronal-enriched cultures which exhibited rapid neuritic growth during the first 5 days in culture and significant expansion of the neuronal network within 10 days. Immunocytochemical staining with the astrocytic marker glial fibrillary acidic protein (GFAP) and the neuronal marker beta-tubulin class III, revealed high numbers of neurons and astrocytes in the cultures. Generation of neural precursor cell cultures after tissue block dissociation resulted in rapidly expanding neurospheres, which produced large numbers of neurons and astrocytes under differentiating conditions. This simple cryopreservation protocol allows for the rapid, efficient, and inexpensive preservation of cortical brain tissue blocks, which grants increased flexibility for later generation of neuronal, astrocyte, and neuronal precursor cell cultures.

Here, we describe a method for efficient cryopreservation and thawing of cortical brain tissue blocks to generate highly enriched neuronal cultures. This simple protocol provides flexibility for later generation of neuronal, astrocyte, and neuronal precursor cell cultures.

Cryopreservation של בלוקים רקמות קליפתיים לדור תרבויות העצבית מועשר

In this study, we outline a standardized protocol for the successful cryopreservation and thawing of cortical brain tissue blocks to generate highly ...

Studying the Integration of Adult-born Neurons

Neurogenesis occurs in adult mammalian brains in the sub-ventricular zone (SVZ) of the lateral ventricle and in the sub-granular zone (SGZ) of the hippocampal dentate gyrus throughout life. Previous reports have shown that adult hippocampal neurogenesis is associated with diverse brain disorders, including epilepsy, schizophrenia, depression and anxiety (1). Deciphering the process of normal and aberrant adult-born neuron integration may shed light on the etiology of these diseases and inform the development of new therapies.
SGZ adult neurogenesis mirrors embryonic and post-natal neuronal development, including stages of fate specification, migration, synaptic integration, and maturation. However, full integration occurs over a prolonged, 6-week period. Initial synaptic input to adult-born SGZ dentate granule cells (DGCs) is GABAergic, followed by glutamatergic input at 14 days (2). The specific factors which regulate circuit formation of adult-born neurons in the dentate gyrus are currently unknown.
Our laboratory uses a replication-deficient retroviral vector based on the Moloney murine leukemia virus to deliver fluorescent proteins and hypothesized regulatory genes to these proliferating cells. This viral technique provides high specificity and resolution for analysis of cell birth date, lineage, morphology, and synaptogenesis.
A typical experiment often employs two or three viruses containing unique label, transgene, and promoter elements for single-cell analysis of a desired developmental process in vivo. The following protocol describes a method for analyzing functional newborn neuron integration using a single green (GFP) or red (dTomato) fluorescent protein retrovirus and patch-clamp electrophysiology.

A way to study the integration of newborn dentate granule cells in adult animals is described. This technique uses an engineered retrovirus to label newborn neurons, followed by electrophysiological recordings to determine in vivo functional integration.

שילוב של לימוד למבוגרים, יליד נוירונים

Neurogenesis occurs in adult mammalian brains in the sub-ventricular zone (SVZ) of the lateral ventricle and in the sub-granular zone (SGZ) of the ...

Investigations on Alterations of Hippocampal Circuit Function Following Mild Traumatic Brain Injury

Traumatic Brain Injury (TBI) afflicts more than 1.7 million people in the United States each year and even mild TBI can lead to persistent neurological impairments 1. Two pervasive and disabling symptoms experienced by TBI survivors, memory deficits and a reduction in seizure threshold, are thought to be mediated by TBI-induced hippocampal dysfunction 2,3. In order to demonstrate how altered hippocampal circuit function adversely affects behavior after TBI in mice, we employ lateral fluid percussion injury, a commonly used animal model of TBI that recreates many features of human TBI including neuronal cell loss, gliosis, and ionic perturbation 4-6.
	Here we demonstrate a combinatorial method for investigating TBI-induced hippocampal dysfunction. Our approach incorporates multiple ex vivo physiological techniques together with animal behavior and biochemical analysis, in order to analyze post-TBI changes in the hippocampus. We begin with the experimental injury paradigm along with behavioral analysis to assess cognitive disability following TBI. Next, we feature three distinct ex vivo recording techniques: extracellular field potential recording, visualized whole-cell patch-clamping, and voltage sensitive dye recording. Finally, we demonstrate a method for regionally dissecting subregions of the hippocampus that can be useful for detailed analysis of neurochemical and metabolic alterations post-TBI.
	These methods have been used to examine the alterations in hippocampal circuitry following TBI and to probe the opposing changes in network circuit function that occur in the dentate gyrus and CA1 subregions of the hippocampus (see Figure 1). The ability to analyze the post-TBI changes in each subregion is essential to understanding the underlying mechanisms contributing to TBI-induced behavioral and cognitive deficits. 
	The multi-faceted system outlined here allows investigators to push past characterization of phenomenology induced by a disease state (in this case TBI) and determine the mechanisms responsible for the observed pathology associated with TBI.

A multi-faceted approach to investigating functional changes to hippocampal circuitry is explained. Electrophysiological techniques are described along with the injury protocol, behavioral testing and regional dissection method. The combination of these techniques can be applied in similar fashion for other brain regions and scientific questions.

חקירות על שינויים בתפקוד המעגל היפוקמפוס בעקבות פגיעה מוחית טראומטית קלה

Traumatic Brain Injury (TBI) afflicts more than 1.7 million people in the United States each year and even mild TBI can lead to persistent neurological ...

A Procedure for Implanting a Spinal Chamber for Longitudinal In Vivo Imaging of the Mouse Spinal Cord

Studies in the mammalian neocortex have enabled unprecedented resolution of cortical structure, activity, and response to neurodegenerative insults by repeated, time-lapse in vivo imaging in live rodents. These studies were made possible by straightforward surgical procedures, which enabled optical access for a prolonged period of time without repeat surgical procedures. In contrast, analogous studies of the spinal cord have been previously limited to only a few imaging sessions, each of which required an invasive surgery. As previously described, we have developed a spinal chamber that enables continuous optical access for upwards of 8 weeks, preserves mechanical stability of the spinal column, is easily stabilized externally during imaging, and requires only a single surgery. Here, the design of the spinal chamber with its associated surgical implements is reviewed and the surgical procedure is demonstrated in detail. Briefly, this video will demonstrate the preparation of the surgical area and mouse for surgery, exposure of the spinal vertebra and appropriate tissue debridement, the delivery of the implant and vertebral clamping, the completion of the chamber, the removal of the delivery system, sealing of the skin, and finally, post-operative care. The procedure for chronic in vivo imaging using nonlinear microscopy will also be demonstrated. Finally, outcomes, limitations, typical variability, and a guide for troubleshooting are discussed.

A Procedure for Implanting a Spinal Chamber for Longitudinal In Vivo Imaging of the Mouse Spinal Cord

In this video, we describe a procedure for implanting a chronic optical imaging chamber over the dorsal spinal cord of a live mouse. The chamber, surgical procedure, and chronic imaging are reviewed and demonstrated.

נוהל השתלה קאמרי בעמוד השדרה לאורך<em&gt; In vivo</em&gt; הדמיה של כבל עכבר השדרה

Studies in the mammalian neocortex have enabled unprecedented resolution of cortical structure, activity, and response to neurodegenerative insults by ...

High-Resolution Quantitative Immunogold Analysis of Membrane Receptors at Retinal Ribbon Synapses

Retinal ganglion cells (RGCs) receive excitatory glutamatergic input from bipolar cells. Synaptic excitation of RGCs is mediated postsynaptically by NMDA receptors (NMDARs) and AMPA receptors (AMPARs). Physiological data have indicated that glutamate receptors at RGCs are expressed not only in postsynaptic but also in perisynaptic or extrasynaptic membrane compartments. However, precise anatomical locations for glutamate receptors at RGC synapses have not been determined. Although a high-resolution quantitative analysis of glutamate receptors at central synapses is widely employed, this approach has had only limited success in the retina. We developed a postembedding immunogold method for analysis of membrane receptors, making it possible to estimate the number, density and variability of these receptors at retinal ribbon synapses. Here we describe the tools, reagents, and the practical steps that are needed for: 1) successful preparation of retinal fixation, 2) freeze-substitution, 3) postembedding immunogold electron microscope (EM) immunocytochemistry and, 4) quantitative visualization of glutamate receptors at ribbon synapses.

The postembedding immunogold method is one of the most effective ways to provide high-resolution analyses of the subcellular localization of specific molecules. Here we describe a protocol to quantitatively analyze glutamate receptors at retinal ribbon synapses.

ניתוח ברזולוציה גבוהה כמותי immunogold של קולטני קרום ב סינפסות רצועת הכלים רשתית

Retinal ganglion cells (RGCs) receive excitatory glutamatergic input from bipolar cells. Synaptic excitation of RGCs is mediated postsynaptically by NMDA ...

Using Linear Agarose Channels to Study Drosophila Larval Crawling Behavior

Drosophila larval crawling is emerging as a powerful model to study neural control of sensorimotor behavior. However, larval crawling behavior on flat open surfaces is complex,&#160;including: pausing, turning, and meandering. This complexity in the repertoire of movement hinders detailed analysis of the events occurring during a single crawl stride cycle. To overcome this obstacle, linear agarose channels were made that constrain larval behavior to straight, sustained, rhythmic crawling. In principle, because agarose channels and the Drosophila larval body are both optically clear, the movement of larval structures labeled by genetically-encoded fluorescent probes can be monitored in intact, freely-moving larvae. In the past, larvae were placed in linear channels and crawling at the level of whole organism, segment, and muscle were analyzed1. In the future, larvae crawling in channels can be used for calcium imaging to monitor neuronal activity. Moreover, these methods can be used with larvae of any genotype and with any researcher-designed channel. Thus the protocol presented below is widely applicable for studies using the Drosophila larva as a model to understand motor control.

Using Linear Agarose Channels to Study Drosophila Larval Crawling Behavior

The Drosophila larva is a powerful model system to study neural control of behavior. This publication describes the use of linear agarose channels to elicit sustained bouts of linear crawling and methods to quantify the dynamics of larval structures during repetitive crawling behavior.

שימוש בערוצי Agarose לינארית ללמוד<em&gt; תסיסנית</em&gt; התנהגות זחל זחילה

Drosophila larval crawling is emerging as a powerful model to study neural control of sensorimotor behavior. However, larval crawling behavior on flat ...

Combining Optogenetics with Artificial microRNAs to Characterize the Effects of Gene Knockdown on Presynaptic Function within Intact Neuronal Circuits

The purpose of this protocol is to characterize the effect of gene knockdown on presynaptic function within intact neuronal circuits. We describe a workflow on how to combine artificial microRNA (miR)-mediated RNA interference with optogenetics to achieve selective stimulation of manipulated presynaptic boutons in acute brain slices. The experimental approach involves the use of a single viral construct and a single neuron-specific promoter to drive the expression of both an optogenetic probe and artificial miR(s) against presynaptic gene(s). When stereotactically injected in the brain region of interest, the expressed construct makes it possible to stimulate with light exclusively the neurons with reduced expression of the gene(s) under investigation. This strategy does not require the development and maintenance of genetically modified mouse lines and can in principle be applied to other organisms and to any neuronal gene of choice. We have recently applied it to investigate how the knockdown of alternative splice isoforms of presynaptic P/Q-type voltage-gated calcium channels (VGCCs) regulates short-term synaptic plasticity at CA3 to CA1 excitatory synapses in acute hippocampal slices. A similar approach could also be used to manipulate and probe the neuronal circuitry in vivo.

This protocol provides a workflow on how to combine artificial microRNA-mediated RNA interference with optogenetics to stimulate specifically presynaptic boutons with reduced expression of selective gene(s) within intact neuronal circuits.

שילוב Optogenetics עם מיקרו Rna מלאכותי כדי לאפיין תופעות נוקאאוט גנטי על פונקציה Presynaptic בתוך מעגלים עצביים ללא פגע

The purpose of this protocol is to characterize the effect of gene knockdown on presynaptic function within intact neuronal circuits. We describe a ...

FM Dye Cycling at the Synapse: Comparing High Potassium Depolarization, Electrical and Channelrhodopsin Stimulation

FM dyes are used to study the synaptic vesicle (SV) cycle. These amphipathic probes have a hydrophilic head and hydrophobic tail, making them water-soluble with the ability to reversibly enter and exit membrane lipid bilayers. These styryl dyes are relatively non-fluorescent in aqueous medium, but insertion into the outer leaflet of the plasma membrane causes a &#62;40X increase in fluorescence. In neuronal synapses, FM dyes are internalized during SV endocytosis, trafficked both within and between SV pools, and released with SV exocytosis, providing a powerful tool to visualize presynaptic stages of neurotransmission. A primary genetic model of glutamatergic synapse development and function is the Drosophila neuromuscular junction (NMJ), where FM dye imaging has been used extensively to quantify SV dynamics in a wide range of mutant conditions. The NMJ synaptic terminal is easily accessible, with a beautiful array of large synaptic boutons ideal for imaging applications. Here, we compare and contrast the three ways to stimulate the Drosophila NMJ to drive activity-dependent FM1-43 dye uptake/release: 1) bath application of high [K+] to depolarize neuromuscular tissues, 2) suction electrode motor nerve stimulation to depolarize the presynaptic nerve terminal, and 3) targeted transgenic expression of channelrhodopsin variants for light-stimulated, spatial control of depolarization. Each of these methods has benefits and disadvantages for the study of genetic mutation effects on the SV cycle at the Drosophila NMJ. We will discuss these advantages and disadvantages to assist the selection of the stimulation approach, together with the methodologies specific to each strategy. In addition to fluorescent imaging, FM dyes can be photoconverted to electron-dense signals visualized using transmission electron microscopy (TEM) to study SV cycle mechanisms at an ultrastructural level. We provide the comparisons of confocal and electron microscopy imaging from the different methods of Drosophila NMJ stimulation, to help guide the selection of future experimental paradigms.

Synaptic vesicle (SV) cycling is the core mechanism of intercellular communication at neuronal synapses. FM dye uptake and release are the primary means of quantitatively assaying SV endo- and exocytosis. Here, we compare all the stimulation methods to drive FM1-43 cycling at the Drosophila neuromuscular junction (NMJ) model synapse.

צבען FM רכיבה על הסינפסה: השוואה בין דפולריזציה אשלגן גבוהה, חשמל וגירוי Channelrhodopsin

FM dyes are used to study the synaptic vesicle (SV) cycle. These amphipathic probes have a hydrophilic head and hydrophobic tail, making them ...

Effects of Blast-induced Neurotrauma on Pressurized Rodent Middle Cerebral Arteries

Though there have been studies on the histopathological and behavioral effects of blast exposure, fewer have been dedicated to blast&#39;s cerebral vascular effects. Impact (i.e., non-blast) traumatic brain injury (TBI) is known to decrease pressure autoregulation in the cerebral vasculature in both humans and experimental animals. The hypothesis that blast-induced traumatic brain injury (bTBI), like impact TBI, results in impaired cerebral vascular reactivity was tested by measuring myogenic dilatory responses to reduced intravascular pressure in rodent middle cerebral arterial (MCA) segments from rats subjected to mild bTBI using an Advanced Blast Simulator (ABS) shock tube. Adult, male Sprague-Dawley rats were anesthetized, intubated, ventilated and prepared for Sham bTBI (identical manipulation and anesthesia except for blast injury) or mild bTBI. Rats were randomly assigned to receive Sham bTBI or mild bTBI followed by sacrifice 30 or 60 min post-injury. Immediately after bTBI, righting reflex (RR) suppression times were assessed, euthanasia at the time points post-injury was completed, the brain was harvested and the individual MCA segments were collected, mounted and pressurized. As the intraluminal pressure perfused through the arterial segments was reduced in 20 mmHg increments from 100 to 20 mmHg, MCA diameters were measured and recorded. With decreasing intraluminal pressure, MCA diameters steadily increased significantly above baseline in the Sham bTBI groups while MCA dilator responses were significantly reduced (p &#60; 0.05) in both bTBI groups as evidenced by the impaired, smaller MCA diameters recorded for the bTBI groups. In addition, RR suppression in the bTBI groups was significantly (p &#60; 0.05) higher than in the Sham bTBI groups. MCA&#39;s collected from the Sham bTBI groups exhibited typical vasodilatory properties to decreases in intraluminal pressure while MCA&#39;s collected following bTBI exhibited significantly impaired myogenic vasodilatory responses to reduced pressure that persisted for at least 60 min after bTBI.

Here, we present a protocol to describe methods for ex vivo vascular reactivity determination following a primary blast traumatic brain injury (bTBI) using isolated, pressurized, rodent middle cerebral arterial (MCA) segments. bTBI induction is accomplished using a shock tube, also known as an Advanced Blast Simulator (ABS) device.

השפעות הפיצוץ-induced Neurotrauma על מכרסם מווסת המזרח התיכון העורקים המוחיים

Though there have been studies on the histopathological and behavioral effects of blast exposure, fewer have been dedicated to blast&#39;s cerebral ...