אנו מודים ברנרד Foucart לקבלת סיוע טכני. עבודה זו נתמכה על ידי הקרן הבלגית למחקר מדעי (FRS-FNRS) ועל ידי קרן מלכת אליזבט למחקר רפואי. אגנס Villers הוא עמית מחקר בקרן הבלגית למחקר מדעי.

אין ניגודי האינטרסים הכריזו.

פיתחנו במעבדה שלנו וכתוצאה מפרוטוקול השילוב של שיטות שפותחו והופעל על ידי מעבדות אחרות בעלי מומחיות גדולה בLTP הקלטות 11,17. פרוטוקול זה מותאם ליפוקמפוס עכבר המבוגר, וניתן להשתמש בבעלי חיים, בכל גיל ובכל גנוטיפ רקע. זה גם מאפשר ניתוח של LTP בעכברים מהונדסים לפתח מחל?...

כיום, יש הבנה מוגבלת של איך זכרונות מאוחסנים מורכבים ונזכרו ברמת המעגל העצבית. עם זאת, השערה מאחדת של זכרון אחסון זמינה ומקובל באופן כללי: זכרונות מאוחסנים כשינויים בעוצמת קשרים סינפטיים בין תאי עצב במערכת העצבים המרכזית. בכוחות עצמו, מחקר על פלסטיות הסינפטית יש נהנה במידה רבה משתי תגליות פריצת דרך. (1) בניסוי זרע, בליס ו1 Lomo, תוך שימוש בהרדמה הארנב בשלמותה, מצאו כי מסירה של תדר גבוה קצר (1 שניות, 100 הרץ) גירוי לנתיב perforant של ההיפוקמפוס שנגרמה לטווח ארוך (כמה שעות) להגדיל בקשרים סינפטיים בנושא. תופעה מרתקת זו נקראה &quot;הגברה לטווח ארוך&quot; או LTP על ידי דאגלס וגודארד בשנת 1975 2. (2) בהמשך, נמצא כי תופעה דומה יכולה להיות מופעלת בפרוסות מוח (0.4 מ&quot;מ) באופן מלאכותי נשמר בחיים במבחנה </eמ &#39;&gt;. LTP הנחקר ביותר נצפה במבחנה על ידי מתן אחד או כמה tetani לחבילה של אקסונים (ביטחונות שפר כביכול) בעת הקלטת הפוטנציאל עורר בנוירונים פירמידליים של מה שמכונה אזור CA1 הסינפטי מעורר שדה שנוצר. המנגנונים של האינדוקציה LTP שהתגלו במידה רבה. בעיקרון, 2 + Ca זרם דרך קולטני NMDA מפעיל אנזימים עם שתי תוצאות: זירחון של קולטני AMPA (אשר מגדיל את היעילות שלהם) ושילוב של קולטני AMPA נוסף בקרום postsynaptic 3. לעומת זאת, את המנגנונים של שלב התחזוקה של-LTP הם ידועים ברובו, בעיקר משום שהוא בניסוי הרבה יותר קשה לשמור על נתח בריא במשך שעות רבות יותר מאשר ל30 עד 60 דקות. הרבה מחקרים הוקדשו להבנת מנגנוני LTP ותאוריות מעניינות כבר הרחיב במהלך השנים 4-11. אבל האו&quot;םtil עכשיו, את המנגנונים המולקולריים שבבסיס המדויק גידול היציב בחוזק סינפטי לא הובהרו. זה יכול להיות גם בגלל הקושי לשחזר תוצאות קודמות במעבדות שונות תוך שימוש בטכניקות שונות להכנה והתחזוקה של פרוסות בהיפוקמפוס. בנייר המתודולוגיה שלהם, Sajikumar et al. -12 הדגיש את החשיבות של תנאי ניסוי להכנת פרוסות בהיפוקמפוס חולדה וההקלטה של LTP היציב. בסרטון הזה אנו מציגים את כל שלבי אופטימיזציה שפותחו במעבדה שלנו במשך השנים כדי להיות מסוגל להקליט LTP יציב מאוד בפרוסות בהיפוקמפוס עכבר. אופטימיזציה זו נעשתה מפרוטוקולים שפותחו ושימש בהצלחה על ידי מעבדות אחרות שלומדות מנגנוני LTP בעכברי 13 ו 11 חולדות. זה מאפשר לחוקרים מנוסים כדי לגרום ולהקליט LTP טווח ארוך מאוד בעכברים בוגרים עם שיעור גבוה של הצלחה. עמ &#39;בסיס hysiological של LTP המושרה נבדק בקפידה והפגין 14. בנייר מתודולוגיה זו, אנו מראים כי כל שינוי של תנאי ניסוי, כמו טמפרטורה או חמצון יכול להיות השפעה עמוקה על תחזוקת LTP בעוד הליך הנתיחה יכול לשנות רגישות עמוקה פרוסות. כמו כן, יש להדגיש כי השליטה המדויקת של כל הפרמטרים הללו דורשת הכשרה של מספר חודשים לתלמידים מתחילים.

<table border="1">
	<tbody>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Reagent/Material</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma - Aldrich</td>
			<td>S7653</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Sigma - Aldrich</td>
			<td>S8875</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma - Aldrich</td>
			<td>P9333</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>D-glucose</td>
			<td>Sigma - Aldrich</td>
			<td>G7528</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>NaH2PO4</td>
			<td>Sigma - Aldrich</td>
			<td>S9638</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>MgSO4 1M</td>
			<td>Sigma - Aldrich</td>
			<td>63126</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Sigma - Aldrich</td>
			<td>C4901</td>
			<td>&nbsp;</td>
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			<td>Carbogen</td>
			<td>Air Liquide (Belgium)</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
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			<td>Capillaries</td>
			<td>WPI, Inc. (UK)</td>
			<td>TW150-4</td>
			<td>&nbsp;</td>
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		<tr>
			<td>Stimulating Electrodes</td>
			<td>FHC (USA)</td>
			<td>CE2B30</td>
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			<td>Surgical tools</td>
			<td>FST (Germany)</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
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		<tr>
			<td>Filter paper 84 g/m2</td>
			<td>Sartorius</td>
			<td>FT-3-105-110</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Mesh</td>
			<td>Lycra</td>
			<td>15 den</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Glue</td>
			<td>UHU</td>
			<td>plus endfest300</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Instrument</td>
		</tr>
		<tr>
			<td>Amplifier</td>
			<td>WPI, Inc. (UK)</td>
			<td>ISO-80</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Interface recording chamber</td>
			<td>FST (Germany)</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Peristaltic pumps</td>
			<td>Gilson (USA)</td>
			<td>Minipuls 3</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Temperature controller</td>
			<td>University of Edinburgh</td>
			<td><a href="http://www.etcsystem.com" target="_blank">www.etcsystem.com</a></td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Tissue Chopper</td>
			<td>Mcllwain</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Stimulators</td>
			<td>Grass (USA)</td>
			<td>S88X + SIU-V</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Program analysis</td>
			<td>WinLTP</td>
			<td><a href="http://www.winltp.com" target="_blank">www.winltp.com</a></td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Micromanipulators</td>
			<td>Narishige</td>
			<td>MM-3 and MMO-220A</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Surgical microscope</td>
			<td>Leica Microsystem</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>A/D converter</td>
			<td>National Instruments</td>
			<td>NIPCI-6229 M-series</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

improved preparation and preservation of hippocampal mouse slices for a very stable and reproducible recording of long-term potentiation

הגברה לטווח ארוך (LTP) הוא סוג של פלסטיות הסינפטית מתאפיין בעלייה בכח הסינפטי והאמינה להיות מעורבים בקידוד זיכרון. LTP שהושרו באזור CA1 של פרוסות בהיפוקמפוס חריפות, נחקר בהרחבה. עם זאת את המנגנונים המולקולריים שבבסיס שלב התחזוקה של תופעה זו עדיין הם הבינו היטב. זה יכול להיות גם בגלל תנאי הניסוי השונים המשמשים במעבדות שונות. ואכן, שלב התחזוקה של-LTP הוא מאוד תלוי בפרמטרים חיצוניים כמו חמצון, טמפרטורה ולחות. זה גם תלוי בפרמטרים פנימיים, כמו נטייה של מטוס החיתוך וכדאיות פרוסה לאחר נתיחה. אופטימיזציה של כל הפרמטרים הללו מאפשרת אינדוקציה של potentiation מאוד לשחזור ומאוד יציב לטווח ארוך. מתודולוגיה זו מציעה את האפשרות להמשיך לחקור את המנגנונים המולקולריים מעורבים בגידול היציבבחוזק סינפטי בפרוסות בהיפוקמפוס. זה גם מדגיש את החשיבות של תנאי ניסוי במבחנה בחקירה של תופעות neurophysiological.

מתודולוגיה זו נעשתה שימוש כדי לנתח את המאפיינים של הגברה לטווח ארוך לטווח ארוך הנגרם בפרוסות בהיפוקמפוס חריפות מעכברים בוגרים (C57BL/6J Janvier SAS, צרפת) 14. באופן מפתיע, שיפור של תנאי הניסוי הוביל לדרך חדשה להסתכל על LTP. אנחנו הראיתי כי עלייה ארוכת טווח בחוזק סינפטי לא ד?...

Watch this Scientific Journal Video about Improved Preparation and Preservation of Hippocampal Mouse Slices for a Very Stable and Reproducible Recording of Long-term Potentiation at JoVE.com

Improved Preparation and Preservation of Hippocampal Mouse Slices for a Very Stable and Reproducible Recording of Long-term Potentiation

מאמר זה מציג מתודולוגיה שלמה להכין ולשמור<em&gt; במבחנה</em&gt; פרוסות בהיפוקמפוס חריפות מעכברים בוגרים. פרוטוקול זה מאפשר הקלטה של ​​הגברה לטווח ארוך לטווח ארוך מאוד יציב (LTP) ליותר מ 8 שעות עם שיעור הצלחה של 95%.

הכנה משופרת ושימור פרוסות עכבר היפוקמפוס להקלטה יציבה מאוד ושחזור של הגברה לטווח ארוך

Long-term potentiation (LTP) is a type of synaptic plasticity characterized by an increase in synaptic strength and believed to be involved in memory ...

improved-preparation-preservation-hippocampal-mouse-slices-for-very

Research

JoVE Journal

Neuroscience

26.6K Views.  University of Mons. מאמר זה מציג מתודולוגיה שלמה להכין ולשמור במבחנה פרוסות בהיפוקמפוס חריפות מעכברים בוגרים. פרוטוקול זה מאפשר הקלטה של ​​הגברה לטווח ארוך לטווח ארוך מאוד יציב (LTP) ליותר מ 8 שעות עם שיעור הצלחה של 95%.

Video: Improved Preparation and Preservation of Hippocampal Mouse Slices for a Very Stable and Reproducible Recording of Long-term Potentiation

Preparation of Oligomeric &beta;-amyloid1-42 and Induction of Synaptic Plasticity Impairment on Hippocampal Slices

Impairment of synaptic connections is likely to underlie the subtle amnesic changes occurring at the early stages of Alzheimer s Disease (AD). &beta;-amyloid (A&beta;), a peptide produced in high amounts in AD, is known to reduce Long-Term Potentiation (LTP), a cellular correlate of learning and memory. Indeed, LTP impairment caused by A&beta; is a useful experimental paradigm for studying synaptic dysfunctions in AD models and for screening drugs capable of mitigating or reverting such synaptic impairments. Studies have shown that A&beta; produces the LTP disruption preferentially via its oligomeric form. Here we provide a detailed protocol for impairing LTP by perfusion of oligomerized synthetic A&beta;1-42 peptide onto acute hippocampal slices. In this video, we outline a step-by-step procedure for the preparation of oligomeric A&beta;1-42. Then, we follow an individual experiment in which LTP is reduced in hippocampal slices exposed to oligomerized A&beta;1-42 compared to slices in a control experiment where no A&beta;1-42 exposure had occurred.

Preparation of Oligomeric &amp;#946;-amyloid1-42 and Induction of Synaptic Plasticity Impairment on Hippocampal Slices

One feature of Alzheimer's Disease is the elevation of A&beta;1-42 peptide. Here we provide a protocol for preparing synthetic A&beta;1-42 oligomers, which impairs hippocampal Long-Term Potentiation, a cellular correlate of memory. This procedure is useful for investigating mechanisms of A&beta;-induced pathology and drug screening.

הכנת Oligomeric β עמילואיד 1-42 ו אינדוקציה של ירידת ערך הפלסטיות הסינפטית על Slices בהיפוקמפוס

Impairment of synaptic connections is likely to underlie the subtle amnesic changes occurring at the early stages of Alzheimer s Disease (AD). ...

Preparation of Acute Hippocampal Slices from Rats and Transgenic Mice for the Study of Synaptic Alterations during Aging and Amyloid Pathology

The rodent hippocampal slice preparation is perhaps the most broadly used tool for investigating mammalian synaptic function and plasticity. The hippocampus can be extracted quickly and easily from rats and mice and slices remain viable for hours in oxygenated artificial cerebrospinal fluid. Moreover, basic electrophysisologic techniques are easily applied to the investigation of synaptic function in hippocampal slices and have provided some of the best biomarkers for cognitive impairments. The hippocampal slice is especially popular for the study of synaptic plasticity mechanisms involved in learning and memory. Changes in the induction of long-term potentiation and depression (LTP and LTD) of synaptic efficacy in hippocampal slices (or lack thereof) are frequently used to describe the neurologic phenotype of cognitively-impaired animals and/or to evaluate the mechanism of action of nootropic compounds. This article outlines the procedures we use for preparing hippocampal slices from rats and transgenic mice for the study of synaptic alterations associated with brain aging and Alzheimer's disease (AD)1-3. Use of aged rats and AD model mice can present a unique set of challenges to researchers accustomed to using younger rats and/or mice in their research. Aged rats have thicker skulls and tougher connective tissue than younger rats and mice, which can delay brain extraction and/or dissection and consequently negate or exaggerate real age-differences in synaptic function and plasticity. Aging and amyloid pathology may also exacerbate hippocampal damage sustained during the dissection procedure, again complicating any inferences drawn from physiologic assessment. Here, we discuss the steps taken during the dissection procedure to minimize these problems. Examples of synaptic responses acquired in "healthy" and "unhealthy" slices from rats and mice are provided, as well as representative synaptic plasticity experiments. The possible impact of other methodological factors on synaptic function in these animal models (e.g. recording solution components, stimulation parameters) are also discussed. While the focus of this article is on the use of aged rats and transgenic mice, novices to slice physiology should find enough detail here to get started on their own studies, using a variety of rodent models.

This article outlines procedures for preparing hippocampal slices from rats and transgenic mice for the study of synaptic alterations associated with brain aging and age-related neurodegenerative diseases, such as Alzheimer&#x2019;s disease.

הכנת פרוסות בהיפוקמפוס של חולדות חריפה העכברים הטרנסגניים לחקר שינויים Synaptic במהלך ההזדקנות עמילואיד פתולוגיה

The rodent hippocampal slice preparation is perhaps the most broadly used tool for investigating mammalian synaptic function and plasticity. The ...

Reproducible Mouse Sciatic Nerve Crush and Subsequent Assessment of Regeneration by Whole Mount Muscle Analysis

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative response mounted by PNS neurons thereby possibly illuminating the failures of CNS regeneration1. Sciatic nerve crush (axonotmesis) is one of the most common models of peripheral nerve injury in rodents2. Crushing interrupts all axons but Schwann cell basal laminae are preserved so that regeneration is optimal3,4. This allows the investigator to study precisely the ability of a growing axon to interact with both the Schwann cell and basal laminae4. Rats have generally been the preferred animal models for experimental nerve crush. They are widely available and their lesioned sciatic nerve provides a reasonable approximation of human nerve lesions5,4. Though smaller in size than rat nerve, the mouse nerve has many similar qualities. Most importantly though, mouse models are increasingly valuable because of the wide availability of transgenic lines now allows for a detailed dissection of the individual molecules critical for nerve regeneration6, 7. Prior investigators have used multiple methods to produce a nerve crush or injury including simple angled forceps, chilled forceps, hemostatic forceps, vascular clamps, and investigator-designed clamps8,9,10,11,12. Investigators have also used various methods of marking the injury site including suture, carbon particles and fluorescent beads13,14,1. We describe our method to obtain a reproducibly complete sciatic nerve crush with accurate and persistent marking of the crush-site using a fine hemostatic forceps and subsequent carbon crush-site marking. As part of our description of the sciatic nerve crush procedure we have also included a relatively simple method of muscle whole mount we use to subsequently quantify regeneration.

In this report we describe a method to crush mouse sciatic nerve. This method uses readily available hemostatic forceps and easily and reproducibly produces complete sciatic nerve crush. In addition, we describe a method to prepare muscle whole mounts suitable for analysis of nerve regeneration after sciatic nerve crush.

עכבר לשחזור Sciatic עצב קראש והערכה לאחר ההתחדשות של ניתוח הר שלם שריר

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative ...

A Polished and Reinforced Thinned-skull Window for Long-term Imaging of the Mouse Brain

In vivo imaging of cortical function requires optical access to the brain without disruption of the intracranial environment. We present a method to form a polished and reinforced thinned skull (PoRTS) window in the mouse skull that spans several millimeters in diameter and is stable for months. The skull is thinned to 10 to 15 &mu;m in thickness with a hand held drill to achieve optical clarity, and is then overlaid with cyanoacrylate glue and a cover glass to: 1) provide rigidity, 2) inhibit bone regrowth and 3) reduce light scattering from irregularities on the bone surface. Since the skull is not breached, any inflammation that could affect the process being studied is greatly reduced. Imaging depths of up to 250 &mu;m below the cortical surface can be achieved using two-photon laser scanning microscopy. This window is well suited to study cerebral blood flow and cellular function in both anesthetized and awake preparations. It further offers the opportunity to manipulate cell activity using optogenetics or to disrupt blood flow in targeted vessels by irradiation of circulating photosensitizers.

We present a method to form an imaging window in the mouse skull that spans millimeters and is stable for months without inflammation of the brain. This method is well suited for longitudinal studies of blood flow, cellular dynamics, and cell/vascular structure using two-photon microscopy.

מלוטשת מחוזק דליל, הגולגולת חלון הדמיה לטווח ארוך של מוח עכבר

In vivo imaging of cortical function requires optical access to the brain without disruption of the intracranial environment. We present a method to form ...

Preparation of an Awake Mouse for Recording Neural Responses and Injecting Tracers

It is well known that anesthesia alters neural response properties in various regions of the brain.13. In the auditory system, fundamental response properties of brainstem neurons including threshold, frequency specificity, and inhibitory sidebands are altered in significant ways under anesthesia1-2. These observations prompted physiologists to seek ways to record from single neurons without the contaminating effects of anesthesia. One result was a decerebrate preparation, where the brainstem was completely transected at the level of the midbrain4. The drawbacks of this preparation are a formidable surgery, the elimination of descending projections from the forebrain, and an inability to use sensory stimulation to examine structures above the midbrain. A different strategy has been to implant electrode arrays chronically to record from single neurons and multiunit clusters while the animal is awake and/or behaving5,6. These techniques however are not compatible with injecting tracer dyes after first electrophysiologically characterizing a brain structure. To avoid altering neural response properties with anesthetics while recording electrophysiological response properties from single neurons, we have adapted a head restraint technique long used in bats7-9 to mouse10-12. Using this method, we are able to conduct electrophysiological recordings over several days in the unanesthetized mouse. At the end of the recording sessions, we can then inject a dye to reconstruct electrode positions and recording sites or inject a tracer so that pathways to and from the recording loci can be determined. This method allows for well isolated single neuron recordings over multiple days without the use anesthetics.

Electrophysiological characterization of neuronal responses is important for understanding brain function and for guiding the placement of dyes for pathway tracing. However, many studies are performed in anesthetized animals. To understand brain function without anesthetics, we developed a method to record neuronal response properties and inject dyes in awake mouse.

הכנת עכבר ער להקלטת תגובות עצביות והזרקת קליעים נותבים

It is well known that anesthesia alters neural response properties in various regions of the brain.13.  In the auditory system, fundamental response ...

A Molecular Readout of Long-term Olfactory Adaptation in C. elegans

During sustained stimulation most sensory neurons will adapt their response by decreasing their sensitivity to the signal. The adaptation response helps shape attention and also protects cells from over-stimulation. Adaptation within the olfactory circuit of C. elegans was first described by Colbert and Bargmann1,2. Here, the authors defined parameters of the olfactory adaptation paradigm, which they used to design a genetic screen to isolate mutants defective in their ability to adapt to volatile odors sensed by the Amphid Wing cells type C (AWC) sensory neurons. When wildtype C. elegans animals are exposed to an attractive AWC-sensed odor3 for 30 min they will adapt their responsiveness to the odor and will then ignore the adapting odor in a chemotaxis behavioral assay for ~1 hr. When wildtype C. elegans animals are exposed to an attractive AWC-sensed odor for ~1 hr they will then ignore the adapting odor in a chemotaxis behavioral assay for ~3 hr. These two phases of olfactory adaptation in C. elegans were described as short-term olfactory adaptation (induced after 30 min odor exposure), and long-term olfactory adaptation (induced after 60 min odor exposure). Later work from L'Etoile et al.,4 uncovered a Protein Kinase G (PKG) called EGL-4 that is required for both the short-term and long-term olfactory adaptation in AWC neurons. The EGL-4 protein contains a nuclear localization sequence that is necessary for long-term olfactory adaptation responses but dispensable for short-term olfactory adaptation responses in the AWC4. By tagging EGL-4 with a green fluorescent protein, it was possible to visualize the localization of EGL-4 in the AWC during prolonged odor exposure. Using this fully functional GFP-tagged EGL-4 (GFP::EGL-4) molecule we have been able to develop a molecular readout of long-term olfactory adaptation in the AWC5. Using this molecular readout of olfactory adaptation we have been able to perform both forward and reverse genetic screens to identify mutant animals that exhibit defective subcellular localization patterns of GFP::EGL-4 in the AWC6,7. Here we describe: 1) the construction of GFP::EGL-4 expressing animals; 2) the protocol for cultivation of animals for long-term odor-induced nuclear translocation assays; and 3) the scoring of the long-term odor-induced nuclear translocation event and recovery (re-sensitization) from the nuclear GFP::EGL-4 state.

A Molecular Readout of Long-term Olfactory Adaptation in C. elegans

Here we describe a molecular readout of long-term olfactory adaptation in Caenorhabditis elegans. The Protein Kinase G, EGL-4, is necessary for stable adaptation responses in the primary sensory neuron pair called AWC. During prolonged odor exposure EGL-4 translocates from the cytosol to nucleus of the AWC.

Readout מולקולרי של הסתגלות חוש ריח לטווח הארוך ב<em&gt; ג elegans</em

During sustained stimulation most sensory neurons will adapt their response by decreasing their sensitivity to the signal. The adaptation response helps ...

Paired Whole Cell Recordings in Organotypic Hippocampal Slices

Pair recordings involve simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling not only direct electrophysiological characterization of the synaptic connections between individual neurons, but also pharmacological manipulation of either the presynaptic or the postsynaptic neuron. When carried out in organotypic hippocampal slice cultures, the probability that two neurons are synaptically connected is significantly increased. This preparation readily enables identification of cell types, and the neurons maintain their morphology and properties of synaptic function similar to that in native brain tissue. A major advantage of paired whole cell recordings is the highly precise information it can provide on the properties of synaptic transmission and plasticity that are not possible with other more crude techniques utilizing extracellular axonal stimulation. Paired whole cell recordings are often perceived as too challenging to perform. While there are challenging aspects to this technique, paired recordings can be performed by anyone trained in whole cell patch clamping provided specific hardware and methodological criteria are followed. The probability of attaining synaptically connected paired recordings significantly increases with healthy organotypic slices and stable micromanipulation allowing independent attainment of pre- and postsynaptic whole cell recordings. While CA3-CA3 pyramidal cell pairs are most widely used in the organotypic slice hippocampal preparation, this technique has also been successful in CA3-CA1 pairs and can be adapted to any neurons that are synaptically connected in the same slice preparation. In this manuscript we provide the detailed methodology and requirements for establishing this technique in any laboratory equipped for electrophysiology.

Pair recordings are simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling precise electrophysiological and pharmacological characterization of the synapses between individual neurons. Here we describe the detailed methodology and requirements for establishing this technique in organotypic hippocampal slice cultures in any laboratory equipped for electrophysiology.

הקלטות תא כל לזווג בפרוסות בהיפוקמפוס Organotypic

Pair recordings involve simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling not only direct ...

Long-term Time Lapse Imaging of Mouse Cochlear Explants

Here we present a method for long-term time-lapse imaging of live embryonic mouse cochlear explants. The developmental program responsible for building the highly ordered, complex structure of the mammalian cochlea proceeds for around ten days. In order to study changes in gene expression over this period and their response to pharmaceutical or genetic manipulation, long-term imaging is necessary. Previously, live imaging has typically been limited by the viability of explanted tissue in a humidified chamber atop a standard microscope. Difficulty in maintaining optimal conditions for culture growth with regard to humidity and temperature has placed limits on the length of imaging experiments. A microscope integrated into a modified tissue culture incubator provides an excellent environment for long term-live imaging. In this method we demonstrate how to establish embryonic mouse cochlear explants and how to use an incubator microscope to conduct time lapse imaging using both bright field and fluorescent microscopy to examine the behavior of a typical embryonic day (E) 13 cochlear explant and Sox2, a marker of the prosensory cells of the cochlea, over 5 days.

Live imaging of the embryonic mammalian cochlea is challenging because the developmental processes at hand operate on a temporal gradient over ten days. Here we present a method for culturing and then imaging embryonic cochlear explant tissue taken from a fluorescent reporter mouse over five days.

לטווח ארוך זמן פקיעת הדמיה של Explants שבלול העכבר

Here we present a method for long-term time-lapse imaging of live embryonic mouse cochlear explants. The developmental program responsible for building ...

A Simplified Method for Ultra-Low Density, Long-Term Primary Hippocampal Neuron Culture

Culturing primary hippocampal neurons in vitro facilitates mechanistic interrogation of many aspects of neuronal development. Dissociated embryonic hippocampal neurons can often grow successfully on glass coverslips at high density under serum-free conditions, but low density cultures typically require a supply of trophic factors by co-culturing them with a glia feeder layer, preparation of which can be time-consuming and laborious. In addition, the presence of glia may confound interpretation of results and preclude studies on neuron-specific mechanisms. Here, a simplified method is presented for ultra-low density (~2,000 neurons/cm2), long-term (&#62;3 months) primary hippocampal neuron culture that is under serum free conditions and without glia cell support. Low density neurons are grown on poly-D-lysine coated coverslips, and flipped on high density neurons grown in a 24-well plate. Instead of using paraffin dots to create a space between the two neuronal layers, the experimenters can simply etch the plastic bottom of the well, on which the high density neurons reside, to create a microspace conducive to low density neuron growth. The co-culture can be easily maintained for &#62;3 months without significant loss of low density neurons, thus facilitating the morphological and physiological study of these neurons. To illustrate this successful culture condition, data are provided to show profuse synapse formation in low density cells after prolonged culture. This co-culture system also facilitates the survival of sparse individual neurons grown in islands of poly-D-lysine substrates and thus the formation of autaptic connections.

Low density cultures of primary hippocampal neurons usually require glia feeder layer to supply neurotrophic factors and sustain longevity. We describe here a simplified method to culture ultra-low density neurons on glass coverslips in the presence of a high density neuronal feeder layer, which facilitates investigation of specific neuronal-autonomous mechanisms.

שיטה פשוטה עבור צפיפות Ultra-Low, לטווח ארוך ראשית בהיפוקמפוס Neuron תרבות

Culturing primary hippocampal neurons in vitro facilitates mechanistic interrogation of many aspects of neuronal development. Dissociated embryonic ...

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 ...