This work has been supported by NINDS, RO1NS52699 and MH84874 to SA. 
We would like to thank Dr. Dave Featherstone (Department of Biological Sciences, University for Illinois at Chicago) for providing us with the suture glue used in the immunohistochemistry work. We thank Michael Alpert for his comments and proofreading of the manuscript.

<ol>
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The authors do not have any competing financial interests or other conflicts of interest to disclose.

פרוטוקול הניתוק שלנו הוא משמעותי על ידי מניב האקסונים reticulospinal מבודדים נטולי 2f איור תחזיות postsynaptic, אך בכל זאת לשמור על 4d איור 4C מסופים ואיור presynaptic הפונקציונלי. היעדר תהליכי postsynaptic המנוגדים מסוף presynaptic מאפשר גישה ישירה להקלטת קרום הפנים שחר...

שידור סינפטי הוא תהליך מאוד מהיר ומדויק. פלישת פוטנציאל פעולה של מסוף presynaptic מובילה לפתיחת VGCCs ממוקם בקרום הפנים שחרור, העלייה וכתוצאה מכך Ca presynaptic 2 + מתנהגת כמו ההדק לשחרור איחוי שלפוחית ​​ומוליך עצבי 1. כל הצעדים האלה להתרחש בתוך מאות מיקרו 2, ולכן דורש צימוד המרחבית הדוק של VGCCs למכונות איחוי שלפוחית ​​3. Ca 2 + והנתיבים Presynaptic מתאפיינים בעיקר באמצעות הדמיה גישות באמצעות הצבעים הרגישים Ca 2 + 4. Ca 2 + מאגרי שילוב המווסתים Ca 2 + בתאי עצב presynaptic נעשה שימוש כדי לאפיין את מערכת היחסים בין סידן ועצבי 3 presynaptic בעקיפין. בנוסף, ויסות Ca 2 + הריכוז החופשי presynaptic ידי משחרר רפרוף Ca 2 + 5 או הקלטת macroscopiג Ca 2 + זרמים היו בשימוש בשילוב עם אמצעים של איחוי שלפוחית ​​ו / או שחרור; כגון מדידות קיבול 6 או postsynaptic תגובות 2 להתייחס לאותה השאלה. עם זאת, המאפיין את Ca 2 + זרמים ישירות בפרצוף השחרור, הסעיף מיוחד של קרום presynaptic בי שלילת קוטביות קרום מתורגמת לCa 2 + זרמים מפעילות איחוי שלפוחית ​​הסינפטית ושחרור הנוירוטרנסמיטר, הוא חלק בלתי נפרד מהשגת מידה מדויקת של Ca 2 + דרישה לאיחוי שלפוחית ​​הסינפטית. בנוסף, היכולת לאפיין באופן ישיר 2 + זרמי Ca במסופי presynaptic בודדים, בשילוב עם מדידות בו זמנית מדויקות של איחוי שלפוחית ​​ושחרור מאפשר הבהרה מדויקת של מערכת היחסים בין עיתוי מהלך פוטנציאל פעולת הזמן, Ca 2 + נוכחי presynaptic, איחוי שלפוחית ​​ושחרור. גישה לקרום הפנים שחרוראינו זמין ברוב המכריע של מסופי presynaptic בשל סגירת פרד על ידי דנדריטים postsynaptic. חוסר נגישות זה היה מכשול עיקרי באפיון של VGCCs שכן הוא מונע מדידות ישירות של זרם במסופי presynaptic פרט. אפיון ישיר של 2 + זרמי presynaptic Ca במסופי presynaptic בודדים עד כה לא היה אפשרי בסינפסות מרכזיות והושג רק בשני מסופי presynaptic סוג calyceal; סינפסה גביע הסוג של calyces גנגליון ריסי חומוס 7-10 וחולדת 11,12. בכל מסופי presynaptic האחרים, כולל סינפסה reticulospinal הענק בחוט השדרה צמד ים 13, היעדר הגישה לקרום הפנים שחרור סינפטי יש חייבת השימוש בגישות עקיפות כגון ההדמיה Ca 2 + ללמוד Ca 2 + והנתיבים presynaptic. <img alt="איור 1" fo: תוכן רוחב = src &quot;5in&quot; = &quot;/ קבצים / ftp_upload / 51925 / 51925fig1highres.jpg&quot; width = &quot;500&quot; /&gt; איור 1 צמד ים סינפסה reticulospinal הענק. (א) חתך של חוט השדרה צמד ים המצביע על הנטייה dorso- הגחון. האקסונים Reticulospinal מסומנים בכוכבית ירוקה. (ב) שיקום 3-D של סינפסה reticulospinal בחוט השדרה צמד ים מראה האקסון presynaptic reticulospinal עושה רב en קשר אגב (מסומנים בחצים ירוקים) אל הנוירון postsynaptic 13. מסופי Presynaptic סומנו בתווית עם אלקסה פלואוריד 488 hydrazide phalloidin מצומדות (ירוק), בזמן שהנוירון postsynaptic כבר מלא בhydrazide אלקסה פלואוריד 568 (אדום). האקסונים reticulospinal ענק צמד ים, הממוקמים באזור הגחון של מקביל בחוט השדרה לאיור 1 א ציר מקורי, זנב, מרובה טופס en קשר אגב הסינפטי על תאי עצב שלקרן השדרה הגחון 14 איור 1b 13. כל תא מקרוסקופית זרמי Ca 2 + נרשמו מהאקסונים reticulospinal בחוט השדרה שלם 13,15. עם זאת, ניסיונות עיוורים קודמים במדידה ישירה של Ca 2 + זרמים באקסונים reticulospinal בכבל צמד ים שלם השדרה באמצעות טכניקת מהדק תיקון מצורף תא הוכיחו לא מוצלחים 13 בשל חוסר הגישה לקרום הפנים שחרור סינפטי בשל תהליכי postsynaptic מתנגדים איור 1b. פני שחרור הקרום נעשה בעבר נגיש על ידי ההסרה של הנוירון postsynaptic 11, ההפרעות מכאניות של סינפסה לפני הקלטת 12 או טיפול אנזימטי בשילוב עם ניתוק מכאני 16. בהתחשב בארגון המורכב של חוט השדרה, זה יהיה קשה מאוד להוכיח לזהות נוירון postsynaptic ולחזור בו באופן מכאני או להפריע הסינפסה דואר. לפיכך, החלטנו להשתמש בטיפול אנזימטי 17 אחרי ניתוק מכאני. שימוש בגישה זו, פיתחנו הכנה בחריפות ניתקת של חוט השדרה צמד שמניב האקסונים reticulospinal מבודדים קיימא עם מסופי presynaptic פונקציונליים נטול כל תהליכי postsynaptic, ובכך לספק גישה חופשית למסופי presynaptic פרט. בשיתוף עם מיקרוסקופ רגיל הפוך ודימות פלואורסצנטי, זה מאפשר לנו לזהות ולמקד את מסופי presynaptic מזוהה fluorescently בודדים, עם טפטפת תיקון המכיל פתרון הקלטה שמבודד 4d איור 4C ואיור 2 + זרמי Ca, להקלטה באמצעות תאי ד&#39; טכניקת מתח מהדק מצורף. Ca 2 + זרמים נרשמו ישירות בקרום הפנים שחרור presynaptic של 4F איור מסופי presynaptic פרט. זה significant פריצת הדרך בתחום של שידור סינפטי שכן הוא ההקלטה הראשונה מסוג זה בוצע בסינפסות מרכזיות.

<table border="0" cellpadding="0" cellspacing="0" width="713">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:211px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">0.2 &#181;m syringe filter</td>
			<td style="width:211px;">EMD Millipore</td>
			<td style="width:119px;">SLGV004SL</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">22x60 mm coverslips</td>
			<td style="width:211px;">Fisherbrand</td>
			<td style="width:119px;">12545J</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Advasep-7</td>
			<td style="width:211px;">Cydex Pharmaceuticals</td>
			<td style="width:119px;">ADV7</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Alexa Fluor 488 Phalloidin</td>
			<td style="width:211px;">Invitrogen/Life Technologies</td>
			<td style="width:119px;">A12379</td>
			<td></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:216px;">Alexa-633 conjugated goat anti-rabbit secondary antibody</td>
			<td style="width:211px;">Invitrogen/Life Technologies</td>
			<td style="width:119px;">A21070</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Antifreeze</td>
			<td style="width:211px;">Prestone</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Boric acid</td>
			<td style="width:211px;">Sigma-Aldrich</td>
			<td style="width:119px;">B7660</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Bovine Serum Albumin</td>
			<td style="width:211px;">Sigma-Aldrich</td>
			<td style="width:119px;">A7906</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Bright field light source</td>
			<td style="width:211px;">Dolan-Jenner</td>
			<td style="width:119px;">Fiberlite 180</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Calcium chloride</td>
			<td style="width:211px;">Sigma Aldrich</td>
			<td style="width:119px;">C4901</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Collagenase Type IA from Cloristridium histolyticum</td>
			<td style="width:211px;">Sigma-Aldrich</td>
			<td style="width:119px;">C9891</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Cover slip</td>
			<td style="width:211px;">Fisher-Scientific</td>
			<td style="width:119px;">12-545-J</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Dextrose</td>
			<td style="width:211px;">Sigma-Aldrich</td>
			<td style="width:119px;">D9559</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Digital CCD Camera</td>
			<td style="width:211px;">Hamamatsu</td>
			<td style="width:119px;">C8484-03G01</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Dissection fine forceps</td>
			<td style="width:211px;">Fine Science Tools</td>
			<td style="width:119px;">91150-20</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Dissection forceps</td>
			<td style="width:211px;">Fine Science Tools</td>
			<td style="width:119px;">11251-20</td>
			<td></td>
		</tr>
		<tr height="20">
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acute dissociation of lamprey reticulospinal axons to enable recording from the release face membrane of individual functional presynaptic terminals

שידור סינפטי הוא תהליך מהיר מאוד. פוטנציאל פעולה מונע זרם של Ca 2 + למסוף presynaptic, באמצעות ערוצי מתח מגודרת סידן (VGCCs) ממוקמים בפני קרום שחרור, הוא הטריגר לאיחוי שלפוחית ​​ושחרור הנוירוטרנסמיטר. חיוני למהירות של העברת הסינפטית הוא תיאום המרחב ובזמן בין הגעתו של פוטנציאל הפעולה, VGCCs ומכונות שחרור הנוירוטרנסמיטר. היכולת להקליט ישירות Ca 2 + זרמים מעל פני שחרור הקרום של מסופי presynaptic פרט הכרחית להבנה מדויקת של מערכת היחסים בין Ca presynaptic 2 + ושחרור הנוירוטרנסמיטר. גישה לפרצוף שחרור קרום presynaptic להקלטת אלקטרו אינה זמינה ברוב ההכנות וCa presynaptic 2 + כניסה התאפיינה תוך שימוש בטכניקות הדמיה וmeasureme הנוכחי מקרוסקופיתNTS - טכניקות שאין לי החלטה זמנית מספיק כדי לדמיין כניסת Ca 2 +. האפיון של VGCCs ישירות במסופי presynaptic אחת לא היה אפשרי בסינפסות מרכזיות ועד כה הושג בהצלחה רק בסינפסה גביע הסוג של גנגליון ריסי חומוס ובcalyces חולדה. יש לנו לטפל בהצלחה בבעיה זו בסינפסה reticulospinal הענק של חוט השדרה צמד ים על ידי פיתוח הכנה בחריפות ניתקת של חוט השדרה שמניב האקסונים reticulospinal מבודדים עם מסופי presynaptic פונקציונליים נטולי מבני postsynaptic. אנו fluorescently יכולים לתייג ולזהות מסופי presynaptic פרט ולמקד אותם להקלטה. שימוש בתכשיר זה, יש לנו מאופיין VGCCs ישירות בפרצוף שחרורו של מסופי presynaptic בודדים באמצעות אימונוהיסטוכימיה ואלקטרופיזיולוגיה גישות. Ca 2 + זרמים נרשמו ישירות בo קרום הפנים שחרורמסופי presynaptic פרט f, ההקלטה הראשונה מסוגו שבוצעו בסינפסות מרכזיות.

<html> האקסונים זה תשואות פרוטוקול ניתוק בריאים ופונקציונליים מבודדים reticulospinal נטול 2f איור תחזיות postsynaptic, אך בכל זאת לשמור על מסופי presynaptic פונקציונליים מסוגלים 4c עורר שלפוחית ​​הסינפטית Exo-ואנדוציטוזה איור ו4d איור. סעיפים של האזורים המבודדי?...

Watch this Scientific Journal Video about Acute Dissociation of Lamprey Reticulospinal Axons to Enable Recording from the Release Face Membrane of Individual Functional Presynaptic Terminals at JoVE.c

Acute Dissociation of Lamprey Reticulospinal Axons to Enable Recording from the Release Face Membrane of Individual Functional Presynaptic Terminals

Recording Ca2+ currents at the presynaptic release face membrane is key to a precise understanding of Ca2+ entry and neurotransmitter release. We present an acute dissociation of the lamprey spinal cord that yields functional isolated reticulospinal axons, permitting recording directly from the release face membrane of individual presynaptic terminals.

החריפה דיסוציאציה של האקסונים Reticulospinal צמד ים כדי לאפשר הקלטה משחרור הפנים ממברנה של מסופי Presynaptic פונקציונליים פרט

Synaptic transmission is an extremely rapid process. Action potential driven influx of Ca2+&#160;into the presynaptic terminal, through voltage-gated ...

acute-dissociation-lamprey-reticulospinal-axons-to-enable-recording

Research

JoVE Journal

Neuroscience

8.7K Views.  University of Illinois at Chicago. Recording Ca2+ currents at the presynaptic release face membrane is key to a precise understanding of Ca2+ entry and neurotransmitter release. We present an acute dissociation of the lamprey spinal cord that yields functional isolated reticulospinal axons, permitting recording directly from the release face membrane of individual presynaptic terminals. 

Video: Acute Dissociation of Lamprey Reticulospinal Axons to Enable Recording from the Release Face Membrane of Individual Functional Presynaptic Terminals

The Subventricular Zone En-face: Wholemount Staining and Ependymal Flow

The walls of the lateral ventricles contain the largest germinal region in the adult mammalian brain. The subventricular zone (SVZ) in these walls is an extensively studied model system for understanding the behavior of neural stem cells and the regulation of adult neurogenesis. Traditionally, these studies have relied on classical sectioning techniques for histological analysis. Here we present an alternative approach, the wholemount technique, which provides a comprehensive, en-face view of this germinal region. Compared to sections, wholemounts preserve the complete cytoarchitecture and cellular relationships within the SVZ. This approach has recently revealed that the adult neural stem cells, or type B1 cells, are part of a mixed neuroepithelium with differentiated ependymal cells lining the lateral ventricles. In addition, this approach has been used to study the planar polarization of ependymal cells and the cerebrospinal fluid flow they generate in the ventricle. With recent evidence that adult neural stem cells are a heterogeneous population that is regionally specified, the wholemount approach will likely be an essential tool for understanding the organization and parcellation of this stem cell niche.

The lateral ventricle walls contain the largest germinal region in the adult mammalian brain. Traditionally, studies on neurogenesis in this region have relied on classical sectioning techniques for histological analysis. Here we present an alternative approach, the wholemount technique, which provides a comprehensive, en-face view of this germinal region.

אזור Subventricular En פנים: מכתים Wholemount ושפל Ependymal

The walls of the lateral ventricles contain the largest germinal region in the adult mammalian brain. The subventricular zone (SVZ) in these walls is an ...

Vibrodissociation of Neurons from Rodent Brain Slices to Study Synaptic Transmission and Image Presynaptic Terminals

Mechanical dissociation of neurons from the central nervous system has the advantage that presynaptic boutons remain attached to the isolated neuron of interest. This allows for examination of synaptic transmission under conditions where the extracellular and postsynaptic intracellular environments can be well controlled. A vibration-based technique without the use of proteases, known as vibrodissociation, is the most popular technique for mechanical isolation. A micropipette, with the tip fire-polished to the shape of a small ball, is placed into a brain slice made from a P1-P21 rodent. The micropipette is vibrated parallel to the slice surface and lowered through the slice thickness resulting in the liberation of isolated neurons. The isolated neurons are ready for study within a few minutes of vibrodissociation. This technique has advantages over the use of primary neuronal cultures, brain slices and enzymatically isolated neurons including: rapid production of viable, relatively mature neurons suitable for electrophysiological and imaging studies; superior control of the extracellular environment free from the influence of neighboring cells; suitability for well-controlled pharmacological experiments using rapid drug application and total cell superfusion; and improved space-clamp in whole-cell recordings relative to neurons in slice or cell culture preparations. This preparation can be used to examine synaptic physiology, pharmacology, modulation and plasticity. Real-time imaging of both pre- and postsynaptic elements in the living cells and boutons is also possible using vibrodissociated neurons. Characterization of the molecular constituents of pre- and postsynaptic elements can also be achieved with immunological and imaging-based approaches.

This report demonstrates a technique for mechanical isolation of individual viable neurons retaining attached presynaptic boutons. Vibrodissociated neurons have the advantages of rapid production, excellent pharmacological control and improved space-clamp without influence from neighboring cells. This method can be used for imaging of synaptic elements and patch-clamp recording.

Vibrodissociation של נוירונים בין פרוסות המוח מכרסמים לחקר הילוכים Synaptic ותמונה מסופי Presynaptic

Mechanical dissociation of neurons from the central nervous system has the advantage that presynaptic boutons remain attached to the isolated neuron of ...

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

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

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

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

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

Laser Nanosurgery of Cerebellar Axons In Vivo

Only a few neuronal populations in the central nervous system (CNS) of adult mammals show local regrowth upon dissection of their axon. In order to understand the mechanism that promotes neuronal regeneration, an in-depth analysis of the neuronal types that can remodel after injury is needed. Several studies showed that damaged climbing fibers are capable of regrowing also in adult animals1,2. The investigation of the time-lapse dynamics of degeneration and regeneration of these axons within their complex environment can be performed by time-lapse two-photon fluorescence (TPF) imaging in vivo3,4. This technique is here combined with laser surgery, which proved to be a highly selective tool to disrupt fluorescent structures in the intact mouse cortex5-9.
This protocol describes how to perform TPF time-lapse imaging and laser nanosurgery of single axonal branches in the cerebellum in vivo. Olivocerebellar neurons are labeled by anterograde tracing with a dextran-conjugated dye and then monitored by TPF imaging through a cranial window. The terminal portion of their axons are then dissected by irradiation with a Ti:Sapphire laser at high power. The degeneration and potential regrowth of the damaged neuron are monitored by TPF in vivo imaging during the days following the injury.

Laser Nanosurgery of Cerebellar Axons In Vivo

Two-photon imaging, coupled to laser nanodissection, are useful tools to study degenerative and regenerative processes in the central nervous system with subcellular resolution. This protocol shows how to label, image, and dissect single climbing fibers in the cerebellar cortex in vivo.

Nanosurgery הלייזר של האקסונים Cerebellar<em&gt; בVivo</em

Only a few neuronal populations in the central nervous system (CNS) of adult mammals show local regrowth upon dissection of their axon. In order to ...

Measuring Spinal Presynaptic Inhibition in Mice By Dorsal Root Potential Recording In Vivo

Presynaptic inhibition is one of the most powerful inhibitory mechanisms in the spinal cord. The underlying physiological mechanism is a depolarization of primary afferent fibers mediated by GABAergic axo-axonal synapses (primary afferent depolarization). The strength of primary afferent depolarization can be measured by recording of volume-conducted potentials at the dorsal root (dorsal root potentials, DRP). Pathological changes of presynaptic inhibition are crucial in the abnormal central processing of certain pain conditions and in some disorders of motor hyperexcitability. Here, we describe a method of recording DRP in vivo in mice. The preparation of spinal cord dorsal roots in the anesthetized animal and the recording procedure using suction electrodes are explained. This method allows measuring GABAergic DRP and thereby estimating spinal presynaptic inhibition in the living mouse. In combination with transgenic mouse models, DRP recording may serve as a powerful tool to investigate disease-associated spinal pathophysiology. In vivo recording has several advantages compared to ex vivo isolated spinal cord preparations, e.g. the possibility of simultaneous recording or manipulation of supraspinal networks and induction of DRP by stimulation of peripheral nerves.

Measuring Spinal Presynaptic Inhibition in Mice By Dorsal Root Potential Recording In Vivo

GABAergic presynaptic inhibition is a powerful inhibitory mechanism in the spinal cord important for motor and sensory signal integration in spinal cord networks. Underlying primary afferent depolarization can be measured by recording of dorsal root potentials (DRP). Here we demonstrate a method of in vivo recording of DRP in mice.

מדידת השדרה Presynaptic עיכוב בעכברים על ידי הקלטה פוטנציאלית הגבי רוט<em&gt; בVivo</em

Presynaptic inhibition is one of the most powerful inhibitory mechanisms in the spinal cord. The underlying physiological mechanism is a depolarization of ...

Imaging Ca2+ Dynamics in Cone Photoreceptor Axon Terminals of the Mouse Retina

Retinal cone photoreceptors (cones) serve daylight vision and are the basis of color discrimination. They are subject to degeneration, often leading to blindness in many retinal diseases. Calcium (Ca2+), a key second messenger in photoreceptor signaling and metabolism, has been proposed to be indirectly linked with photoreceptor degeneration in various animal models. Systematically studying these aspects of cone physiology and pathophysiology has been hampered by the difficulties of electrically recording from these small cells, in particular in the mouse where the retina is dominated by rod photoreceptors. To circumvent this issue, we established a two-photon Ca2+ imaging protocol using a transgenic mouse line that expresses the genetically encoded Ca2+ biosensor TN-XL exclusively in cones and can be crossbred with mouse models for photoreceptor degeneration. The protocol described here involves preparing vertical sections (&#8220;slices&#8221;) of retinas from mice and optical imaging of light stimulus-evoked changes in cone Ca2+ level. The protocol also allows &#8220;in-slice measurement&#8221; of absolute Ca2+ concentrations; as the recordings can be followed by calibration. This protocol enables studies into functional cone properties and is expected to contribute to the understanding of cone Ca2+ signaling as well as the potential involvement of Ca2+ in photoreceptor death and retinal degeneration.

Imaging Ca2+ Dynamics in Cone Photoreceptor Axon Terminals of the Mouse Retina

We describe a protocol to monitor Ca2+ dynamics in the axon terminals of cone photoreceptors using an ex-vivo&#160;slice preparation of the mouse retina. This protocol allows comprehensive studies of cone Ca2+ signaling in an important mammalian model system, the mouse.

ההדמיה Ca<sup&gt; 2 +</sup&gt; Dynamics בסופי קון קולטי האור אקסון של רשתית העכבר

Retinal cone photoreceptors (cones) serve daylight vision and are the basis of color discrimination. They are subject to degeneration, often leading to ...

Live Imaging of Nicotine Induced Calcium Signaling and Neurotransmitter Release Along Ventral Hippocampal Axons

Sustained enhancement of axonal signaling and increased neurotransmitter release by the activation of pre-synaptic nicotinic acetylcholine receptors (nAChRs) is an important mechanism for neuromodulation by acetylcholine (ACh). The difficulty with access to probing the signaling mechanisms within intact axons and at nerve terminals both in vitro and in vivo has limited progress in the study of the pre-synaptic components of synaptic plasticity. Here we introduce a gene-chimeric preparation of ventral hippocampal (vHipp)&#8211;accumbens (nAcc) circuit in vitro that allows direct live imaging to analyze both the pre- and post-synaptic components of transmission while selectively varying the genetic profile of the pre- vs post-synaptic neurons. We demonstrate that projections from vHipp microslices, as pre-synaptic axonal input, form multiple, reliable glutamatergic synapses with post-synaptic targets, the dispersed neurons from nAcc. The pre-synaptic localization of various subtypes of nAChRs are detected and the pre-synaptic nicotinic signaling mediated synaptic transmission are monitored by concurrent electrophysiological recording and live cell imaging. This preparation also provides an informative approach to study the pre- and post-synaptic mechanisms of glutamatergic synaptic plasticity in vitro.

We developed a gene-chimeric preparation of ventral hippocampal &#8211; accumbens circuit in vitro that allows direct live imaging to analyze presynaptic mechanisms of nicotinic acetylcholine receptors (nAChRs) mediated synaptic transmission. This preparation also provides an informative approach to study the pre- and post-synaptic mechanisms of synaptic plasticity.

הדמיה חיה של איתות הניקוטין מושרה סידן והשחרור הנוירוטרנסמיטר לאורך האקסונים בהיפוקמפוס הגחון

Sustained enhancement of axonal signaling and increased neurotransmitter release by the activation of pre-synaptic nicotinic acetylcholine receptors ...

Recording Synaptic Plasticity in Acute Hippocampal Slices Maintained in a Small-volume Recycling-, Perfusion-, and Submersion-type Chamber System

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful analysis of synaptic transmission modulation when performing field potential or intracellular recordings. This manuscript describes methodological aspects that might be helpful in improving experimental conditions for the maintenance of acute brain slices and for recording field excitatory postsynaptic potentials in a commercially available submersion chamber with an outflow-carbogenation unit. The outflow-carbogenation helps to stabilize the oxygen level in experiments that rely on the recycling of a small buffer reservoir to enhance the cost-efficiency of drug experiments. In addition, the manuscript presents representative experiments that examine the effects of different carbogenation modes and stimulation paradigms on the activity-dependent synaptic plasticity of synaptic transmission.

This protocol describes the stabilization of the oxygen level in a small volume of recycled buffer and methodological aspects of recording activity-dependent synaptic plasticity in submerged acute hippocampal slices.

הקלטה הפלסטיות הסינפטית בפרוסות בהיפוקמפוס חריפה, בהשתתפות של מיחזור - בנפח מצומצם, זלוף- ומערכת ועלייתו-סוג תא

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful ...

In Vivo Single-Molecule Tracking at the Drosophila Presynaptic Motor Nerve Terminal

An increasing number of super-resolution microscopy techniques are helping to uncover the mechanisms that govern the nanoscale cellular world. Single-molecule imaging is gaining momentum as it provides exceptional access to the visualization of individual molecules in living cells. Here, we describe a technique that we developed to perform single-particle tracking photo-activated localization microscopy (sptPALM) in Drosophila larvae. Synaptic communication relies on key presynaptic proteins that act by docking, priming, and promoting the fusion of neurotransmitter-containing vesicles with the plasma membrane. A range of protein-protein and protein-lipid interactions tightly regulates these processes and the presynaptic proteins therefore exhibit changes in mobility associated with each of these key events. Investigating how mobility of these proteins correlates with their physiological function in an intact live animal is essential to understanding their precise mechanism of action. Extracting protein mobility with high resolution in vivo requires overcoming limitations such as optical transparency, accessibility, and penetration depth. We describe how photoconvertible fluorescent proteins tagged to the presynaptic protein Syntaxin-1A can be visualized via slight oblique illumination and tracked at the motor nerve terminal or along the motor neuron axon of the third instar Drosophila larva.

In Vivo Single-Molecule Tracking at the Drosophila Presynaptic Motor Nerve Terminal

Here we illustrate how single molecule photo-activated localization microscopy can be carried out on the motor nerve terminal of a live Drosophila melanogaster third instar larva.

אין ויוו מעקב אחר בתחנת האוטובוסים Presynaptic עצבים מוטוריים דרוזופילה מולקולה בודדת

An increasing number of super-resolution microscopy techniques are helping to uncover the mechanisms that govern the nanoscale cellular world. ...

Single Synapse Indicators of Glutamate Release and Uptake in Acute Brain Slices from Normal and Huntington Mice

Synapses are highly compartmentalized functional units that operate independently on each other. In Huntington&#39;s disease (HD) and other neurodegenerative disorders, this independence might be compromised due to insufficient glutamate clearance and the resulting spill-in and spill-out effects. Altered astrocytic coverage of the presynaptic terminals and/or dendritic spines as well as a reduced size of glutamate transporter clusters at glutamate release sites have been implicated in the pathogenesis of diseases resulting in symptoms of dys-/hyperkinesia. However, the mechanisms leading to the dysfunction of glutamatergic synapses in HD are not well understood. Improving and applying synapse imaging we have obtained data shedding new light on the mechanisms impeding the initiation of movements. Here, we describe the principle elements of a relatively inexpensive approach to achieve single synapse resolution by using the new genetically encoded ultrafast glutamate sensor iGluu, wide-field optics, a scientific CMOS (sCMOS) camera, a 473 nm laser and a laser positioning system to evaluate the state of corticostriatal synapses in acute slices from age appropriate healthy or diseased mice. Glutamate transients were constructed from single or multiple pixels to obtain estimates of i) glutamate release based on the maximal elevation of the glutamate concentration [Glu] next to the active zone and ii) glutamate uptake as reflected in the time constant of decay (TauD) of the perisynaptic [Glu]. Differences in the resting bouton size and contrasting patterns of short-term plasticity served as criteria for the identification of corticostriatal terminals as belonging to the intratelencephalic (IT) or the pyramidal tract (PT) pathway. Using these methods, we discovered that in symptomatic HD mice ~40% of PT-type corticostriatal synapses exhibited insufficient glutamate clearance, suggesting that these synapses might be at risk to excitotoxic damage. The results underline the usefulness of TauD as a biomarker of dysfunctional synapses in Huntington mice with a hypokinetic phenotype.

We present a protocol to evaluate the balance between glutamate release and clearance at single corticostriatal glutamatergic synapses in acute slices from adult mice. This protocol uses the fluorescent sensor iGluu for glutamate detection, a sCMOS camera for signal acquisition and a device for focal laser illumination.