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המאמר הנוכחי מספק מתודולוגיה להערכת תנועה באמצעות קינמטיקה כמותי רציף התגובה הקרקע כוח ונחישות. חשוב לכל מי שמעוניין וכשהפלגתי מתודולוגיה זו היא רקע חזק ביומכניקה של תנועה, התנהגות בעלי חיים הסנסורית, ונתונים ניהול מניפולציה. למרות kinematic הקרקע כוח ונחישות התגובה דורש זמן מומחיות נוספת, לעומת כמה צורות אחרות של ניתוח של תנועה (אמצעי קצה כגון סולמות דירוג סודר), את הנתונים המתקבלים הוא רגיש, אובייקטיבי כמותי עבור מגוון רחב של 14-22 אורתופדיות ונוירולוגיות 1-3, 23-32 דגמים של המחלה, במגוון רחב של מינים. סיפקנו נתונים המתאר הבדלים תנועה בין הגילאים השונים של זן בהתאמה מידע חולדות שלא ניתן לדלות באמצעות צעדים פשוטים ורגישים פחות. יתר על כן, ניתוח kinematic ו הקינטית של תנועה נעשה שימוש כדי לתאר שינויים של תנועה במגוון רחב של מצבים של מערכת העצבים שבו וצורות אחרות של הערכה יהיה מוצלח. 8, 23-25, 27 השימוש באמצעים רגיש הופך להיות חשוב במיוחד כאשר אנו מעריכים therapeutants פוטנציאל עבור דגמים שונים של המחלה. אם הבדיקה אינו רגיש מספיק כדי להבחין אפקט של therapeutant פוטנציאל הנסיין מסתכן בביצוע שגיאה מסוג II סטטיסטית (כלומר הסיכום אין השפעה של הטיפול, כאשר למעשה לא היתה השפעה). יתר על כן, כי אמצעי קצה ובדיקות סובייקטיבי יותר להעריך תנועה, קיים פוטנציאל של משוא פנים. הערכה kinematic ו הקינטית הוא אובייקטיבי לחלוטין בכך, בתנאי הקריטריון המתאים והכללה / דחייה נעשים מראש, הנסיין פשוט אוספת, בוחן ומחיל הנתונים המתאימים הנתונים (כלומר, אין שום מרכיב סובייקטיבי להגדרה נתונים). ניתוח kinematic ו הקינטית גם מקנה את היכולת לשמש במגוון רחב של מינים. למעשה, קינמטיקה, תגובת הקרקע נחישות כוח, או שניהם היו בשימוש במגוון רחב של מינים כגון 33-35 פילים, בקר 36, 37-40 סוסים, כלבים 4, 41-45, חתולים 21, 46-49, שונים מכרסמים 3, 8, 50, 51, הציפורים 4, 52-55, ודגים 56, 57 (הרשימה הזאת היא בהחלט לא ממצה). הניסיון של המחברים, לעומת זאת, השימוש בעכברים הוא בעייתי בהתחשב בעובדה עכברים אינם קלים בתנאי operantly לנסוע לאורך המסלול. לאור זאת, העכברים לא לנסוע במהירות קבועה יחסית, במקום מהירות מעלה האטה כאשר חוצים את המסלול. התנהגות זו עלולה סביר, בין השאר, ניתן להתגבר על ידי הפעלת עכברים על הליכון ו-Video מקליטה locomoting חיה על ההליכון. 58 אם מנגנון ניסיוני לקביעת התגובה הקרקע בכוח היה להיות שונה עבור השימוש הליכון, תגובת הקרקע נחישות כוח היה סביר רק להשיג בקלות לכוח הקרקע אנכי התגובה כמו את החגורה הליכון יפריע הקדמי הירכתיים מדיו, לרוחב כוח ונחישות. בסך הכל, ניתוח kinematic ו הקינטית של תנועה היא שיטה אמין, רגיש, אובייקטיבי, כי יכול להיות מועסק על מודלים שונים של תנאים אורתופדיות ונוירולוגיות. יתר על כן, כל הציוד הפך לזמין לשימוש מכרסמים, ובכך מבטל כל סיבה הקשורה לאי ביצוע צורה זו של ניתוח התנהגות הסנסורית.

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Low load miniature force platform</td>
<td>&nbsp;</td>
<td>AMTI</td>
<td>HE6X6</td>
<td>http://amti.biz/PDFDownloads/0908HE6x6.pdf</td>
</tr>
<tr>
<td>Vicon Motus</td>
<td>&nbsp;</td>
<td>Vicon</td>
<td>Vicon Motus software</td>
<td>http://www.vicon.com/applications/animal.html 

The force platform can be purchased as an integrated package with the Vicon Motus system.</td>
</tr>
<tr>
<td>Vicon Motus KineCalc</td>
<td>&nbsp;</td>
<td>Vicon</td>
<td>Vicon Motus KineCalc software</td>
<td>http://www.vicon.com/applications/animal.html Proprietary software compatible with Vicon Motus data, thus enabling direct and customizable data calculations.
</td>
</tr>
<tr>
<td>Video Cameras</td>
<td>&nbsp;</td>
<td>Vicon</td>
<td>Varies depending upon needs</td>
<td>http://www.vicon.com/applications/animal.html A minimum of 4 cameras are required for bilateral 2D or 3D imaging of hindlimb kinematics during rat locomotion. Cameras that sample at 60 Hz are sufficient for rats locomoting between 60 and 90 cm/s. High speed cameras will permit identification of very small, yet possibly important, differences in interlimb coordination.</td>
</tr>
<tr>
<td>Calibration volume or frame</td>
<td>&nbsp;</td>
<td>Vicon</td>
<td>Custom</td>
<td>http://www.vicon.com/applications/animal.html An appropriate calibration volume must be purchased and used for calibrating the coordinates prior to performing videography each and every time of new data collection or if any of the cameras are moved. This dimensions of this calibration volume are based upon the field of view being examined.
</td>
</tr>
<tr>
<td>Reflective tape</td>
<td>&nbsp;</td>
<td>3M</td>
<td>NA</td>
<td>Can be purchased bulk through a 3M distributor or on an as-needed basis from VICON
 http://solutions.3m.com/en_US/?WT.mc_id=www.3m.com/us http://www.vicon.com/applications/animal.html</td>
</tr>
<tr>
<td>Cheerios</td>
<td>&nbsp;</td>
<td>General Mills</td>
<td>Cheerios</td>
<td>http://www.cheerios.com/</td>
</tr>
<tr>
<td>Tissue adhesive</td>
<td>&nbsp;</td>
<td>3M</td>
<td>&nbsp;</td>
<td>Can be purchased through a 3M distributor 
http://solutions.3m.com/en_US/?WT.mc_id=www.3m.com/us</td>
</tr>		</tbody>
		</table>
		</div>

kinematics and ground reaction force determination: a demonstration quantifying locomotor abilities of young adult, middle-aged, and geriatric rats

התנהגות, בהגדרה הרחבה ביותר, יכולה להיות מוגדרת כביטוי המנוע של תהליכים פיזיולוגיים. ככזה, התנהגויות כל ביטוי דרך מערכת התנועה. בתחומי הנוירולוגיה אורתופדיה, תנועה היא התנהגות הערכה כלל עבור מגוון רחב של מודלים המחלה. לדוגמה, שחזור של תנועה אחרי פציעה טראומטית במערכת העצבים היא אחת ההתנהגויות העריכו לרוב 1-3. למרות תנועה ניתן להעריך באמצעות מגוון של מדידות נקודת קצה (למשל לקח זמן כדי להשלים משימה של תנועה וכו &#39;), אמצעים kinematic semiquantitative (כגון סולמות דירוג סודר (למשל באסו ביטי ו Bresnahan של תנועה (BBB) ​​סולם דירוג, וכו&#39;)) ו פונדקאית מדדים של התנהגות (כגון כוח שריר, מהירות הולכה עצבי, וכו &#39;), רק קינטיקה (מדידות כוח) ו קינמטיקה (מדידות של מקטעים הגוף בחלל) לספק תיאור מפורט של האסטרטגיה שבה חיה היא מסוגלת locomote 1. אמנם לא מדידות חדשות, kinematic ו הקינטית של מכרסמים locomoting כעת יותר נגיש בשל הזמינות של ציוד זמין מסחרית המיועדת למטרה זו. חשוב לציין, עם זאת, הנסיינים צריכים להיות מאוד מוכרת עם התיאוריה של מנתח biomechanical להבין את היתרונות והמגבלות של צורות אלה של מנתח לפני היציאה מה יהיה מחקר יחסית עתירי עבודה. המאמר הנוכחי נועד לתאר שיטה לאיסוף kinematic הקרקע כוח התגובה הנתונים באמצעות ציוד מסחרי זמין. פרטי ציוד ומכשירים הגדרת, אימון טרום של בעלי חיים, הכללת קריטריונים הרחקה של פועל מקובל, שיטות לאיסוף נתונים מפורטים. אנו מדגימים את השירות של טכניקה זו על ידי ניתוח התנהגותי המתאר את קינמטיקה קינטיקה של מבוגרים זן מתאימים צעירים, בגיל העמידה, וחולדות גריאטריות.

Watch this Scientific Journal Video about Kinematics and Ground Reaction Force Determination: A Demonstration Quantifying Locomotor Abilities of Young Adult, Middle-aged, and Geriatric Rats at JoVE.co

Kinematics and Ground Reaction Force Determination: A Demonstration Quantifying Locomotor Abilities of Young Adult, Middle-aged, and Geriatric Rats

תנועה נבחנת לעיתים קרובות תוצאה התנהגותית במודלים שונים של מחלת בתחומים כמו אורתופדיה הנוירולוגיה. מאמר זה וידאו מתכוון לתאר שיטה לאיסוף התגובה הקרקע כוחות קינמטיקה של חולדות במהלך תנועה חסרת מעצורים.

קינמטיקה ואת תגובת קרקע חיל קביעת: הפגנה כימות של תנועה היכולות של מבוגרים צעירים, בגיל העמידה, ועל החולדות הגריאטרי

Behavior, in its broadest definition, can be defined as the motor manifestation of physiologic processes.  As such, all behaviors manifest through the ...

kinematics-ground-reaction-force-determination-demonstration

Research

JoVE Journal

Neuroscience

19.6K Views.  Riverview, NB. תנועה נבחנת לעיתים קרובות תוצאה התנהגותית במודלים שונים של מחלת בתחומים כמו אורתופדיה הנוירולוגיה. מאמר זה וידאו מתכוון לתאר שיטה לאיסוף התגובה הקרקע כוחות קינמטיקה של חולדות במהלך תנועה חסרת מעצורים.

Video: Kinematics and Ground Reaction Force Determination: A Demonstration Quantifying Locomotor Abilities of Young Adult, Middle-aged, and Geriatric Rats

Growth and Differentiation of Adult Hippocampal Arctic Ground Squirrel Neural Stem Cells

Arctic ground squirrels (Urocitellus parryii, AGS) are unique in their ability to hibernate with a core body temperature near or below freezing 1. These animals also resist ischemic injury to the brain in vivo 2,3 and oxygen-glucose deprivation in vitro 4,5. These unique qualities provided the impetus to isolate AGS neurons to examine inherent neuronal characteristics that could account for the capacity of AGS neurons to resist injury and cell death caused by ischemia and extremely cold temperatures. Identifying proteins or gene targets that allow for the distinctive properties of these cells could aid in the discovery of effective therapies for a number of ischemic indications and for the study of cold tolerance. Adult AGS hippocampus contains neural stem cells that continue to proliferate, allowing for easy expansion of these stem cells in culture. We describe here methods by which researchers can utilize these stem cells and differentiated neurons for any number of purposes. By closely following these steps the AGS neural stem cells can be expanded through two passages or more and then differentiated to a culture high in TUJ1-positive neurons (~50%) without utilizing toxic chemicals to minimize the number of dividing cells. Ischemia induces neurogenesis 6 and neurogenesis which proceeds via MEK/ERK and PI3K/Akt survival signaling pathways contributes to ischemia resistance in vivo7 and in vitro 8 (Kelleher-Anderson, Drew et al., in preparation). Further characterization of these unique neural cells can advance on many fronts, using some or all of these methods.

Neural stem cells were prepared from the hippocampus of adult non-hibernating yearling Arctic ground squirrels (AGS). These neural stem cells can be expanded through numerous passages, differentiated and maintained as a nearly 50:50 neuron to glial culture.

גדילה והתמיינות של מבוגרים בהיפוקמפוס סנאי הארקטי קרקע תאים גזע עצביים

Arctic ground squirrels (Urocitellus parryii, AGS) are unique in their ability to hibernate with a core body temperature near or below freezing 1. These ...

Cerebrovascular Casting of the Adult Mouse for 3D Imaging and Morphological Analysis

Vascular imaging is crucial in the clinical diagnosis and management of cerebrovascular diseases, such as brain arteriovenous malformations (BAVMs). Animal models are necessary for studying the etiopathology and potential therapies of cerebrovascular diseases. Imaging the vasculature in large animals is relatively easy. However, developing vessel imaging methods of murine brain disease models is desirable due to the cost and availability of genetically-modified mouse lines. Imaging the murine cerebral vascular tree is a challenge. In humans and larger animals, the gold standard for assessing the angioarchitecture at the macrovascular (conductance) level is x-ray catheter contrast-based angiography, a method not suited for small rodents.
In this article, we present a method of cerebrovascular casting that produces a durable skeleton of the entire vascular bed, including arteries, veins, and capillaries that may be analyzed using many different modalities. Complete casting of the microvessels of the mouse cerebrovasculature can be difficult; however, these challenges are addressed in this step-by-step protocol. Through intracardial perfusion of the vascular casting material, all vessels of the body are casted. The brain can then be removed and clarified using the organic solvent methyl salicylate. Three dimensional imaging of the brain blood vessels can be visualized simply and inexpensively with any conventional brightfield microscope or dissecting microscope. The casted cerebrovasculature can also be imaged and quantified using micro-computed tomography (micro-CT)1. In addition, after being imaged, the casted brain can be embedded in paraffin for histological analysis.
The benefit of this vascular casting method as compared to other techniques is its broad adaptation to various analytic tools, including brightfield microscopic analysis, CT scanning due to the radiopaque characteristic of the material, as well as histological and immunohistochemical analysis. This efficient use of tissue can save animal usage and reduce costs. We have recently demonstrated application of this method to visualize the irregular blood vessels in a mouse model of adult BAVM at a microscopic level2, and provide additional images of the malformed vessels imaged by micro-CT scan. Although this method has drawbacks and may not be ideal for all types of analyses, it is a simple, practical technique that can be easily learned and widely applied to vascular casting of blood vessels throughout the body.

In this article, we present a simple, practical technique for cerebrovascular casting that is easy to perform and can be utilized to image the vascular tree of the adult mouse brain.

יציקה של כלי דם במוח העכבר למבוגרים הדמיה 3D ו ניתוח מורפולוגי

Vascular imaging is crucial in the clinical diagnosis and management of cerebrovascular diseases, such as brain arteriovenous malformations (BAVMs). ...

Rapid Determination of the Thermal Nociceptive Threshold in Diabetic Rats

Painful diabetic neuropathy (PDN) is characterized by hyperalgesia i.e., increased sensitivity to noxious stimulus, and allodynia i.e., hypersensitivity to normally innocuous stimuli1. Hyperalgesia and allodynia have been studied in many different rodent models of diabetes mellitus2. However, as stated by B&ouml;lcskei et al, determination of "pain" in animal models is challenging due to its subjective nature3. Moreover, the traditional methods used to determine behavioral responses to noxious thermal stimuli usually lack reproducibility and pharmacological sensitivity3. For instance, by using the hot-plate method of Ankier4, flinch, withdrawal and/or licking of either hind- and/or fore-paws is quantified as reflex latencies at constant high thermal stimuli (52-55 &deg;C). However, animals that are hyperalgesic to thermal stimulus do not reproducibly show differences in reflex latencies using those supra-threshold temperatures3,5. As the recently described method of B&ouml;lcskei et al.6, the procedures described here allows for the rapid, sensitive and reproducible determination of thermal nociceptive thresholds (TNTs) in mice and rats. The method uses slowly increasing thermal stimulus applied mostly to the skin of mouse/rat plantar surface. The method is particularly sensitive to study anti-nociception during hyperalgesic states such as PDN. The procedures described bellow are based on the ones published in detail by Alm&aacute;si et al 5 and B&ouml;lcskei et al 3. The procedures described here have been approved the Laboratory Animal Care and Use Committee (LACUC), Wright State University.

Here, we describe a rapid reliable and simple procedure to determine the lowest temperature at which rats or mice show nocifensive behavior, i.e. the thermal nociceptive threshold (TNT). This method applies a slowly increasing thermal stimulus allowing precise and reproducible estimation of TNTs with minimum, if any, stress to the animals.

קביעה מהירה של סף nociceptive תרמי בעכברים סוכרתיים

Painful diabetic neuropathy (PDN) is characterized by hyperalgesia i.e., increased sensitivity to noxious stimulus, and allodynia i.e., hypersensitivity ...

Microdialysis of Ethanol During Operant Ethanol Self-administration and Ethanol Determination by Gas Chromatography

Operant self-administration methods are commonly used to study the behavioral and pharmacological effects of many drugs of abuse, including ethanol. However, ethanol is typically self-administered orally, rather than intravenously like many other drugs of abuse. The pharmacokinetics of orally administered drugs are more complex than intravenously administered drugs. Because understanding the relationship between the pharmacological and behavioral effects of ethanol requires knowledge of the time course of ethanol reaching the brain during and after drinking, we use in vivo microdialysis and gas chromatography with flame ionization detection to monitor brain dialysate ethanol concentrations over time. 
Combined microdialysis-behavioral experiments involve the use of several techniques. In this article, stereotaxic surgery, behavioral training and microdialysis, which can be adapted to test a multitude of self-administration and neurochemical centered hypotheses, are included only to illustrate how they relate to the subsequent phases of sample collection and dialysate ethanol analysis. Dialysate ethanol concentration analysis via gas chromatography with flame-ionization detection, which is specific to ethanol studies, is described in detail. Data produced by these methods reveal the pattern of ethanol reaching the brain during the self-administration procedure, and when paired with neurochemical analysis of the same dialysate samples, allows conclusions to be made regarding the pharmacological and behavioral effects of ethanol.

A method to determine the time course of ethanol concentration in the brains of rats during operant ethanol self-administration is described. Gas chromatography with flame ionization detection is used to quantify ethanol in the dialysate samples, because it has the sensitivity required for the small volumes that are generated.

Microdialysis של אתנול במהלך אתנול נחישות ממשל עצמי ואתנול אופרנטית על ידי גז כרומטוגרפיה

Operant self-administration methods are commonly used to study the behavioral and pharmacological effects of many drugs of abuse, including ethanol.  ...

Simultaneous Intracellular Recording of a Lumbar Motoneuron and the Force Produced by its Motor Unit in the Adult Mouse In vivo

The spinal motoneuron has long been a good model system for studying neural function because it is a neuron of the central nervous system with the unique properties of (1) having readily identifiable targets (the muscle fibers) and therefore having a very well-known function (to control muscle contraction); (2) being the convergent target of many spinal and descending networks, hence the name of "final common pathway"; and (3) having a large soma which makes it possible to penetrate them with sharp intracellular electrodes. Furthermore, when studied in vivo, it is possible to record simultaneously the electrical activity of the motoneurons and the force developed by their muscle targets. Performing intracellular recordings of motoneurons in vivo therefore put the experimentalist in the unique position of being able to study, at the same time, all the compartments of the "motor unit" (the name given to the motoneuron, its axon, and the muscle fibers it innervates1): the inputs impinging on the motoneuron, the electrophysiological properties of the motoneuron, and the impact of these properties on the physiological function of the motoneurons, i.e. the force produced by its motor unit. However, this approach is very challenging because the preparation cannot be paralyzed and thus the mechanical stability for the intracellular recording is reduced. Thus, this kind of experiments has only been achieved in cats and in rats. However, the study of spinal motor systems could make a formidable leap if it was possible to perform similar experiments in normal and genetically modified mice.
For technical reasons, the study of the spinal networks in mice has mostly been limited to neonatal in vitro preparations, where the motoneurons and the spinal networks are immature, the motoneurons are separated from their targets, and when studied in slices, the motoneurons are separated from most of their inputs. Until recently, only a few groups had managed to perform intracellular recordings of motoneurons in vivo2-4 , including our team who published a new preparation which allowed us to obtain very stable recordings of motoneurons in vivo in adult mice5,6. However, these recordings were obtained in paralyzed animals, i.e. without the possibility to record the force output of these motoneurons. Here we present an extension of this original preparation in which we were able to obtain simultaneous recordings of the electrophysiological properties of the motoneurons and of the force developed by their motor unit. This is an important achievement, as it allows us to identify the different types of motoneurons based on their force profile, and thereby revealing their function. Coupled with genetic models disturbing spinal segmental circuitry7-9, or reproducting human disease10,11, we expect this technique to be an essential tool for the study of spinal motor system.

Simultaneous Intracellular Recording of a Lumbar Motoneuron and the Force Produced by its Motor Unit in the Adult Mouse In vivo

This new method permits the simultaneous intracellular recording of a single adult mouse motoneuron and the measurement of the force produced by its muscle fibers. The combined investigation of the electrical and mechanical properties of motor units in normal and genetically modified animals is a breakthrough for the study of the neuromuscular system.

הקלטה תאית סימולטני של Motoneuron מותנה והחיל הופק על ידי יחידת המנוע שלה בעכבר למבוגרים<em&gt; בחי</em

The  spinal motoneuron has long been a good model system for studying neural  function because it is a neuron of the central nervous system with the ...

Determination of the Spontaneous Locomotor Activity in Drosophila melanogaster

Drosophila melanogaster has been used as an excellent model organism to study environmental and genetic manipulations that affect behavior. One such behavior is spontaneous locomotor activity. Here we describe our protocol that utilizes Drosophila population monitors and a tracking system that allows continuous monitoring of the spontaneous locomotor activity of flies for several days at a time. This method is simple, reliable, and objective and can be used to examine the effects of aging, sex, changes in caloric content of food, addition of drugs, or genetic manipulations that mimic human diseases.

Determination of the Spontaneous Locomotor Activity in Drosophila melanogaster

Drosophila melanogaster are useful in studying genetic or environmental manipulations that affect behaviors such as spontaneous locomotor activity.&#160; Here we describe a protocol that utilizes monitors with infrared beams and data analysis software to quantify spontaneous locomotor activity.&#160;

קביעת הפעילות של תנועה הספונטנית ב<em&gt; Melanogaster דרוזופילה</em

Drosophila melanogaster has been used as an excellent model organism to study environmental and genetic manipulations that affect behavior. One such ...

Quantifying Microglia Morphology from Photomicrographs of Immunohistochemistry Prepared Tissue Using ImageJ

Microglia are brain phagocytes that participate in brain homeostasis and continuously survey their environment for dysfunction, injury, and disease. As the first responders, microglia have important functions to mitigate neuron and glia dysfunction, and in this process, they undergo a broad range of morphologic changes. Microglia morphologies can be categorized descriptively or, alternatively, can be quantified as a continuous variable for parameters such as cell ramification, complexity, and shape. While methods for quantifying microglia are applied to single cells, few techniques apply to multiple microglia in an entire photomicrograph. The purpose of this method is to quantify multiple and single cells using readily available ImageJ protocols. This protocol is a summary of the steps and ImageJ plugins recommended to convert fluorescence and bright-field photomicrographs into representative binary and skeletonized images and to analyze them using software plugins AnalyzeSkeleton (2D/3D) and FracLac for morphology data collection. The outputs of these plugins summarize cell morphology in terms of process endpoints, junctions, and length as well as complexity, cell shape, and size descriptors. The skeleton analysis protocol described herein is well suited for a regional analysis of multiple microglia within an entire photomicrograph or region of interest (ROI) whereas FracLac provides a complementary individual cell analysis. Combined, the protocol provides an objective, sensitive, and comprehensive assessment tool that can be used to stratify between diverse microglia morphologies present in the healthy and injured brain.

Microglia are brain immune cells that survey and react to altered brain physiology through morphologic changes which may be evaluated quantitatively. This protocol outlines an ImageJ based analysis protocol to represent microglia morphology as continuous data according to metrics such as cell ramification, complexity, and shape.

לכימות מיקרוגלייה מורפולוגיה של Photomicrographs של אימונוהיסטוכימיה מוכן רקמות באמצעות ImageJ

Microglia are brain phagocytes that participate in brain homeostasis and continuously survey their environment for dysfunction, injury, and disease. As ...

Quantifying Spontaneous Ca2+ Fluxes and their Downstream Effects in Primary Mouse Midbrain Neurons

Parkinson&#8217;s disease (PD) is a devastating neurodegenerative disorder caused by the degeneration of dopaminergic (DA) neurons. Excessive Ca2+ influx due to the abnormal activation of glutamate receptors results in DA excitotoxicity and has been identified as an important mechanism for DA neuron loss. In this study, we isolate, dissociate, and culture midbrain neurons from the mouse ventral mesencephalon (VM) of ED14 mouse embryos. We then infect the long-term primary mouse midbrain cultures with an adeno-associated virus (AAV) expressing a genetically encoded calcium indicator, GCaMP6f under control of the human neuron-specific synapsin promoter, hSyn. Using live confocal imaging, we show that cultured mouse midbrain neurons display spontaneous Ca2+ fluxes detected by AAV-hSyn-GCaMP6f. Bath application of glutamate to midbrain cultures causes abnormal elevations in intracellular Ca2+ within neurons and this is accompanied by caspase-3 activation in DA neurons, as demonstrated by immunostaining. The techniques to identify glutamate-mediated apoptosis in primary mouse DA neurons have important applications for the high content screening of drugs that preserve DA neuron health.

Quantifying Spontaneous Ca2+ Fluxes and their Downstream Effects in Primary Mouse Midbrain Neurons

Here we present a protocol to measure in vitro Ca2+ fluxes in midbrain neurons and their downstream effects on caspase-3 using primary mouse midbrain cultures. This model can be employed to study pathophysiologic changes related to abnormal Ca2+ activity in midbrain neurons, and to screen novel therapeutics for anti-apoptotic properties.

כימות ספונטני Ca2+ שטף ואת ההשפעות במורד הזרם שלהם בעכבר ראשי נוירונים המוח האמצעי

Parkinson&#8217;s disease (PD) is a devastating neurodegenerative disorder caused by the degeneration of dopaminergic (DA) neurons. Excessive Ca2+ influx ...

A Metric Test for Assessing Spatial Working Memory in Adult Rats Following Traumatic Brain Injury

Impairments to sensory, short-term, and long-term memory are common side effects after traumatic brain injury (TBI). Due to the ethical limitations of human studies, animal models provide suitable alternatives to test treatment methods, and to study the mechanisms and related complications of the condition. Experimental rodent models have historically been the most widely used due to their accessibility, low cost, reproducibility, and validated approaches. A metric test, which tests the ability to recall the placement of two objects at various distances and angles from one another, is a technique to study impairment in spatial working memory (SWM) after TBI. The significant advantages of metric tasks include the possibility of dynamic observation, low cost, reproducibility, relative ease of implementation, and low stress environment. Here, we present a metric test protocol to measure impairment of SWM in adult rats after TBI. This test provides a feasible way to evaluate physiology and pathophysiology of brain function more effectively.

Traumatic brain injury (TBI) is commonly associated with memory impairment. Here, we present a protocol to assess spatial working memory after TBI via a metric task. A metric test is a useful tool to study spatial working memory impairment after TBI.

מבחן מטרי להערכת זיכרון עבודה מרחבי בחולדות בוגרות בעקבות פגיעה מוחית טראומטית

Impairments to sensory, short-term, and long-term memory are common side effects after traumatic brain injury (TBI). Due to the ethical limitations of ...

A Scalable Model to Study the Effects of Blunt-Force Injury in Adult Zebrafish

Blunt-force traumatic brain injuries (TBI) are the most common form of head trauma, which spans a range of severities and results in complex and heterogenous secondary effects. While there is no mechanism to replace or regenerate the lost neurons following a TBI in humans, zebrafish possess the ability to regenerate neurons throughout their body, including the brain. To examine the breadth of pathologies exhibited in zebrafish following a blunt-force TBI and to study the mechanisms underlying the subsequent neuronal regenerative response, we modified the commonly used rodent Marmarou weight drop for the use in adult zebrafish. Our simple blunt-force TBI model is scalable, inducing a mild, moderate, or severe TBI, and recapitulates many of the phenotypes observed following human TBI, such as contact- and post-traumatic seizures, edema, subdural and intracerebral hematomas, and cognitive impairments, each displayed in an injury severity-dependent manner. TBI sequelae, which begin to appear within minutes of the injury, subside and return to near undamaged control levels within 7 days post-injury. The regenerative process begins as early as 48 hours post-injury (hpi), with the peak cell proliferation observed by 60 hpi. Thus, our zebrafish blunt-force TBI model produces characteristic primary and secondary injury TBI pathologies similar to human TBI, which allows for investigating disease onset and progression, along with the mechanisms of neuronal regeneration that is unique to zebrafish.

We modified the Marmarou weight drop model for adult zebrafish to examine a breadth of pathologies following blunt-force traumatic brain injury (TBI) and the mechanisms underlying subsequent neuronal regeneration. This blunt-force TBI model is scalable, induces a mild, moderate, or severe TBI, and recapitulates injury heterogeneity observed in human TBI.