מימון עבור עבודה זו, וכפי שצוטט 2,10, נקבע, בין השאר, על ידי פרסים מחקר להעניק MF סלווטורה של הפדרציה האמריקאית לחקר הזיקנה, אדוארד פ &#39;סטיילס אמון הקרן ביו קרן המחקר של מערב לואיזיאנה, וכדי BS פרואט מן אחוות אייק Muslow Predoctoral, LSU למדעי הבריאות מרכז, Shreveport.

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אין ניגוד עניינים הצהיר.

כפי שמתואר באיור 1, השיטות שפורטו לעיל צריך להניב readouts רבים של דופאמין ויסות שלה חלבונים ה, DAT, ו VMAT2 ממדגם אחד או SN VTA לקבל חולדה או עכבר. שוב, היתרונות של ביצוע זה הן כי פרוטוקול החוקר יכול להשיג readouts מבצעית המותאמות של כמה דופמין מוסדר in vivo תחת הפרדיגמה כמעט כל ניסי...

HPLC המערכת: המערכת הבסיסית כוללת משאבה LC10-ADvp HPLC Shimadzu, ווטרס 717 Wisp אוטומטית מדגם ההזרקה, 250 X 4.4 מ&quot;מ 5 מיקרון Spherosorb ODS-1 טור C18 הפוך שלבים (ווטרס), מספר מערכות Bioanalytical (BAS) TL12 זכוכית כפולה אלקטרודות פחמן, שני באס LC4B גלאי אלקטרו, וכמה ווטרס העצמת 2 על איסוף מידע מערכת אינטגרציה. עמודה נשמר 30-45 מעלות צלזיוס (BAS דוד LC22A עמודה). בשלב הנייד הוא 0.1 סודיום פוספט M (pH 3.0), 0.1 mM EDTA, 0.2-0.4 mM 1-אוקטן חומצה sulfonic (איסטמן קודאק-), ו אצטוניטריל 0.35% (V / V), מסונן באמצעות מסנן 0.45 מיקרון. קצב הזרימה הוא של 1.2 מ&quot;ל / דקה. ארבע קבוצות ליטר בשלב ניידים מותאמים הפרדות על ידי התאמת ה-pH, חומצה אוקטן sulfonic וטמפרטורה העמודה. בשלב ניידים ממוחזרים, והוא מטוהר ברציפות עם גז הליום להסיר החמצן המומס. מיחזור שלב סלולריהכרחי כמעט כדי לשמור על רזולוציה טובה למשך זמן סביר. בשלב הנייד חיי מדף נשמר באמצעות בורר זרימת (בשליטת אינטגרטור) להסיט לבזבז דקות 1 2-7 של כל סיבוב. האלקטרודות מתוחזקים על פוטנציאל של כ 0.78 ו 0.95V לגבי האלקטרודה Ag/AgC1 התייחסות. האלקטרודה על פוטנציאל גבוה יותר משמש אך ורק לקביעת טריפטופן (ורמת פנימי NMDA). הפוטנציאל 0.78 V מספק אות מעולה יחס רעש כדי לאתר את monoamines ותרכובות, למעט טריפטופן. Chromatograms מאוחסנים על הכונן הקשיח של תחנת העבודה העצמת, ומעובד לאחר מכן והנתונים מועברים ישירות לתוך גיליון אלקטרוני של Excel לצורך חישוב כמויות מטבוליט ו אוסף של נתונים קבוצתיים. משאבת: Shimadzu LC-10AD נייד: זרימה BAS קרוס. הפחמן מזוגגות עובד האלקטרודה על 0.780 ו 0.950 פוטנציאל V. גלאי: BAS LC-4B פעל במצב ערוץ כפול. נתונים ACQ. מערכת: ווטרס העצמת Pro 2. מזרק: Wisp ווטרס 717 טור: ווטרס Spherosorb ODS-1, 5 החלקיקים מיקרומטר, 4.4 מ&quot;מ X 250 מ&quot;מ. <table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> שם מגיב </td><td> חברה </td><td> מספר קטלוגי </td></tr><tr><td> Dodecyl נתרן סולפט (SDS) </td><td> - JT בייקר </td><td> 4095-02 </td></tr><tr><td> מאגר Trizma </td><td> סיגמא </td><td> T1503-1 ק&quot;ג </td></tr><tr><td> Trizma HCl </td><td> סיגמא </td><td> T3253-1 ק&quot;ג </td></tr><tr><td> גליצרול </td><td> סיגמא </td><td> G8773-500 מ&quot;ל </td></tr><tr><td> PVP-40 </td><td> סיגמא </td><td> PVP40-1 ק&quot;ג </td></tr><tr><td> dPBS </td><td> Gibco </td><td> 21600-069 </td></tr><tr><td> Tween20 </td><td> סיגמא </td><td> P1379-500 מ&quot;ל </td></tr><tr><td> גליצין </td><td> סיגמא </td><td> G8898-1 ק&quot;ג</td></tr><tr><td> Ponceau S </td><td> Fluka </td><td> 81460 </td></tr><tr><td> Bromophenol כחול </td><td> סיגמא </td><td> B8026-5G </td></tr><tr><td> Dithiothreitol </td><td> סיגמא </td><td> D-9163 </td></tr><tr><td> חלבון רגיל 2 מ&quot;ג BSA </td><td> סיגמא </td><td> P5619-25VL </td></tr><tr><td> פירס Assay BCA חלבון מגיב </td><td> תרמו פישר סיינטיפיק </td><td> 23223 </td></tr><tr><td> Precision פלוס רגיל חלבון </td><td> ביו רד </td><td> 161-0373 </td></tr><tr><td> [125I], חלבונים, פעילות ספציפית </td><td> פרקין אלמר, </td><td></td></tr></tbody></table> טבלה 2. חומרים כימיים ספציפיים. <table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> ריאגנטים </td><td> נוסחאות </td></tr><tr><td> 10% SDS </td><td> 10 גרם SDS, 100 מ&quot;ל DI H 2 0 </td></tr><tr><td> 1% SDS (pH עד 8.2) </td><td><ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> 10 מ&quot;ל10% SDS </li><li style=";text-align:right;direction:rtl"> 60.5 מ&quot;ג Trizma מאגר </li><li style=";text-align:right;direction:rtl"> 37.22 מ&quot;ג EDTA </li><li style=";text-align:right;direction:rtl"> 90 מ&quot;ל DI H 2 0 </li></ol></td></tr><tr><td> נחושת סולפט II פתרון </td><td><ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> 1 גרם II נחושת גופרתית </li><li style=";text-align:right;direction:rtl"> 25 מ&quot;ל DI H 2 0 </li></ol></td></tr><tr><td> לדוגמה 3X מאגר </td><td><ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> Trizma מאגר 2.27 גרם </li><li style=";text-align:right;direction:rtl"> SDS 6 גרם </li><li style=";text-align:right;direction:rtl"> Dithiothreitol 0.463 גרם </li><li style=";text-align:right;direction:rtl"> גליצרול 30 גרם </li><li style=";text-align:right;direction:rtl"> Bromophenol כחול 10 מ&quot;ג </li><li style=";text-align:right;direction:rtl"> DIH 2 0 (בתחילה להוסיף מעל ריאגנטים ל 40 מ&quot;ל של H 2 0 גליל מדורג, ואז מוסיפים H 2 0 עד נפח מגיע ל 100 מ&quot;ל) </li><li style=";text-align:right;direction:rtl"> HCl (להוסיף לפי הצורך להגיע pH של 6.85) </li><li style=";text-align:right;direction:rtl"> להקפיא את הפתרון ב -50 צינורות 2.0 מ&quot;ל. </li></ol> (עושה 100 מ&quot;ל): נפח מאגר לדוגמה 3X צריך = ½ נפח SDS שימוש במדגם. </td></tr><tr><td> 1X לדוגמא הצפת </td><td> לדלל את מאגר לדוגמה 3X למאגר 1X באמצעות מדגם DI H 2 0 </td></tr><tr><td> 10X<html> הפעלת מאגר (גורם 4 ל &#39;) </td><td><ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> Trizma מאגר 121.1 גרם </li><li style=";text-align:right;direction:rtl"> גליצין 577 גרם </li><li style=";text-align:right;direction:rtl"> SDS 40 גרם </li></ol></td></tr><tr><td> מאגר העברה 10X: (עושה 4 ל &#39;) </td><td><ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> גליצין 360 גרם </li><li style=";text-align:right;direction:rtl"> Trizma בסיס 96 גרם </li></ol></td></tr><tr><td> Ponceau </td><td><ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> Ponceau ס &#39;0.5 גרם </li><li style=";text-align:right;direction:rtl"> חומצה אצטית 5 מ&quot;ל </li><li style=";text-align:right;direction:rtl"> DI H 2 0 95 מ&quot;ל </li></ol></td></tr><tr><td> 0.2% HCl פתרון </td><td> 5.2 מ&quot;ל HCl ב 500 מ&quot;ל של DI H 2 0 </td></tr><tr><td> PVP-T20 חסימת Soln. (גורם 4 ל &#39;) </td><td><ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> PVP-40 40 גרם </li><li style=";text-align:right;direction:rtl"> dPBS 38.2 גרם </li><li style=";text-align:right;direction:rtl"> Tween20 2 גרם </li><li style=";text-align:right;direction:rtl"> Thimerisol 0.4 גרם </li><li style=";text-align:right;direction:rtl"> 1M Tris pH 7.6 (60.6 גרם טריס HCl + 13.9 גרם טריס בסיס 500 מ&quot;ל DI H 2 0) - 50 מ&quot;ל </li></ol></td></tr><tr><td> מאגר כתם 10X (גורם 4 ל &#39;) </td><td><ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> Tween 20 20 גרם </li><li style=";text-align:right;direction:rtl"> טריס בסיס 14 גרם </li><li style=";text-align:right;direction:rtl"> טריס HCl 61 גרם </li></ol></td></tr></tbody></table>&quot;ontent&gt; לוח 3. חלבון Assay והנוסחאות והסתיר המערב. סטנדרטים hydroxylase טירוזין: מכויל על זירחון חלבונים ו ה סטנדרטים המשמשים במעבדה זו נגזרות PC12 תמציות סלולריים, אשר נותחו על תכולת החלבון וה stoichiometries זרחון על תקני ה מכויל בעבר כי בסופו של דבר, שמקורם במעבדה של ד&quot;ר ג&#39;ון הייקוק 11 .

comprehensive profiling of dopamine regulation in substantia nigra and ventral tegmental area

דופמין הוא מוליך עצבי למד במרץ מערכת העצבים המרכזית. אכן, את מעורבותה בפעילות של תנועה ותגמול הקשור להתנהגות טיפחה חמישה עשורים החקירה לעניין הליקויים מולקולריים הקשורים תקנה דופמין. רוב אלה שאלות של רגולציה הדופמין במוח המיקוד על הבסיס המולקולרי של ויסות שלה את שדה באזורים מסוף המסלולים nigrostriatal ו mesoaccumbens, הסטריאטום ו הנסמך הגרעין. יתרה מכך, מחקרים אלה התמקדו בניתוח תוכן רקמות דופמין נורמליזציה עם משקל רק רקמות רטוב. חקירת החלבונים המווסתים דופמין, כגון hydroxylase החלבון טירוזין (ה &#39;), זירחון TH, טרנספורטר דופאמין (DAT), ו טרנספורטר אוקסידאז שלפוחי 2 (VMAT2) חלבון לעתים קרובות אינם כוללים ניתוח של תוכן רקמות הדופמין מדגם זהה. היכולת לנתח את תוכן הן רקמות דופאמין חלבונים המסדירים שלה (כולל פוסט traשינויים nslational) לא רק נותן כוח אינהרנטי לפרש את הקשר של דופמין עם רמת חלבון והתפקוד של TH, DAT, או VMAT2, אלא גם מרחיב את הכלכלה המדגם. זה מיתרגם עלות פחות, ובכל זאת מייצרת תובנות תקנה המולקולרי של הדופמין הפרדיגמה כמעט כל בחירה של החוקרים. אנו מתמקדים בניתוח של המוח התיכון. למרות SN ו VTA מוזנחים בדרך כלל רוב המחקרים תקנה דופמין, גרעינים אלה גזור בקלות עם הפרקטיקה. Readout מקיפה של תוכן רקמות TH דופאמין, DAT, או VMAT2 ניתן לבצע. שם הוא המתפתחת ספרות על ההשפעה של תפקוד הדופאמין SN ו VTA על ההתנהגות, ועל הפרעות וחדירות חיצוניות של חומרים חיצוניים או תהליכי מחלה החלים בהם 1-5. יתר על כן, תרכובות כגון גורמי גדילה יש השפעה עמוקה על חלבונים דופאמין דופאמין מווסת במידה יחסית רבה יותר SN או VTA <sup&gt; 6-8. לכן, שיטה זו מוצג לעיון למעבדות אשר רוצים להרחיב את השאלות שלהם על כמה טיפולים ספציפיים לווסת התנהגות ורגולציה דופמין. הנה, שיטת רב שלבי מוצג על ניתוח תוכן רקמות דופמין, רמות החלבון של TH, DAT, או VMAT2 ו זרחון TH מ nigra substantia ו VTA מן המוח התיכון מכרסם. ניתוח של זירחון TH יכולה להניב תובנות משמעותיות לתוך לא רק כיצד הפעילות TH מוסדר, אלא גם את מפלי איתות מושפע בגרעין somatodendritic פרדיגמה נתונה. אנו מדגימים את הטכניקה לנתיחה כדי לבודד אותם גרעינים 2 ו עיבוד דגימת רקמה גזור שמייצר פרופיל חושף מנגנונים מולקולריים תקנה דופמין in vivo, ספציפיות עבור כל גרעינים (איור 1).

Watch this Scientific Journal Video about Comprehensive Profiling of Dopamine Regulation in Substantia Nigra and Ventral Tegmental Area at JoVE.com

Comprehensive Profiling of Dopamine Regulation in Substantia Nigra and Ventral Tegmental Area

דופאמין הוא מוסדר בבירור בגרעין המוח התיכון, אשר מכילים תאים הגופות דנדריטים של נוירונים דופאמין. כאן נתאר את הגישה לנתיחה ו מדגם הטיפול על מנת למקסם את התוצאות, ולכן מסקנות ותובנות, על ויסות הדופמין בגרעין המוח התיכון של nigra substantia (SN) ואזור tegmental הגחוני (VTA) במכרסמים.

פרופיל מקיף של הסדרת substantia nigra הדופמין ואזור Tegmental הגחון

Dopamine is a vigorously studied neurotransmitter in the CNS. Indeed, its involvement in locomotor activity and reward-related behaviour has fostered five ...

comprehensive-profiling-dopamine-regulation-substantia-nigra-ventral

Research

JoVE Journal

Neuroscience

25.6K Views.  Louisiana State University Health Sciences Center. דופאמין הוא מוסדר בבירור בגרעין המוח התיכון, אשר מכילים תאים הגופות דנדריטים של נוירונים דופאמין. כאן נתאר את הגישה לנתיחה ו מדגם הטיפול על מנת למקסם את התוצאות, ולכן מסקנות ותובנות, על ויסות הדופמין בגרעין המוח התיכון של nigra substantia (SN) ואזור tegmental הגחוני (VTA) במכרסמ...

Video: Comprehensive Profiling of Dopamine Regulation in Substantia Nigra and Ventral Tegmental Area

Single Cell Measurement of Dopamine Release with Simultaneous Voltage-clamp and Amperometry

After its release into the synaptic cleft, dopamine exerts its biological properties via its pre- and post-synaptic targets1. The dopamine signal is terminated by diffusion2-3, extracellular enzymes4, and membrane transporters5. The dopamine transporter, located in the peri-synaptic cleft of dopamine neurons clears the released amines through an inward dopamine flux (uptake). The dopamine transporter can also work in reverse direction to release amines from inside to outside in a process called outward transport or efflux of dopamine5. More than 20 years ago Sulzer et al. reported the dopamine transporter can operate in two modes of activity: forward (uptake) and reverse (efflux)5. The neurotransmitter released via efflux through the transporter can move a large amount of dopamine to the extracellular space, and has been shown to play a major regulatory role in extracellular dopamine homeostasis6. Here we describe how simultaneous patch clamp and amperometry recording can be used to measure released dopamine via the efflux mechanism with millisecond time resolution when the membrane potential is controlled. For this, whole-cell current and oxidative (amperometric) signals are measured simultaneously using an Axopatch 200B amplifier (Molecular Devices, with a low-pass Bessel filter set at 1,000 Hz for whole-cell current recording). For amperometry recording a carbon fiber electrode is connected to a second amplifier (Axopatch 200B) and is placed adjacent to the plasma membrane and held at +700 mV. The whole-cell and oxidative (amperometric) currents can be recorded and the current-voltage relationship can be generated using a voltage step protocol. Unlike the usual amperometric calibration, which requires conversion to concentration, the current is reported directly without considering the effective volume7. Thus, the resulting data represent a lower limit to dopamine efflux because some transmitter is lost to the bulk solution.

The amperometric technique measures dopamine release from a single cell by detecting the oxidative current produced by spontaneous dopamine oxidization. Simultaneous voltage clamp and amperometry methodology reveal the mechanistic relationship between the overall "activity" of dopamine transporter and the regulatory role of this activity on the reverse transport of dopamine.

מדידת תא בודדה של שחרור דופמין במתח-clamp וAmperometry סימולטני

After its release into the synaptic cleft, dopamine exerts its biological properties via its pre- and post-synaptic targets1. The dopamine signal is ...

In ovo Expression of MicroRNA in Ventral Chick Midbrain

Non-coding RNAs are additional players in regulating gene expression. Targeted in ovo electroporation of specific areas provides a unique tool for spatial and temporal control of ectopic microRNA expression. However, ventral brain structures like ventral midbrain are rather difficult to reach for any manipulations. Here, we demonstrate an efficient way to electroporate miRNA into ventral midbrain using thin platinum electrodes. This method offers a reliable way to transfect specific areas of the midbrain and a useful tool for in vivo studies.

In ovo Expression of MicroRNA in Ventral Chick Midbrain

Ectopic expression is one technique to elucidate the microRNAs role in brain development. However, targeting specific areas using in ovo electroporation is challenging. Here, we show an efficient way to selectively electroporate ventral and dorsal midbrain regions.

בביטוי אוב של microRNA בגחון צ&#39;יק מוח התיכון

Non-coding RNAs are additional players in regulating gene expression. Targeted in ovo electroporation of specific areas provides a unique tool for spatial ...

Environmental Modulations of the Number of Midbrain Dopamine Neurons in Adult Mice

Long-lasting changes in the brain or &#8216;brain plasticity&#8217; underlie adaptive behavior and brain repair following disease or injury. Furthermore, interactions with our environment can induce brain plasticity. Increasingly, research is trying to identify which environments stimulate brain plasticity beneficial for treating brain and behavioral disorders. Two environmental manipulations are described which increase or decrease the number of tyrosine hydroxylase immunopositive (TH+, the rate-limiting enzyme in dopamine (DA) synthesis) neurons in the adult mouse midbrain. The first comprises pairing male and female mice together continuously for 1 week, which increases midbrain TH+ neurons by approximately 12% in males, but decreases midbrain TH+ neurons by approximately 12% in females. The second comprises housing mice continuously for 2 weeks in &#8216;enriched environments&#8217; (EE) containing running wheels, toys, ropes, nesting material, etc., which increases midbrain TH+ neurons by approximately 14% in males. Additionally, a protocol is described for concurrently infusing drugs directly into the midbrain during these environmental manipulations to help identify mechanisms underlying environmentally-induced brain plasticity. For example, EE-induction of more midbrain TH+ neurons is abolished by concurrent blockade of synaptic input onto midbrain neurons. Together, these data indicate that information about the environment is relayed via synaptic input to midbrain neurons to switch on or off expression of &#8216;DA&#8217; genes. Thus, appropriate environmental stimulation, or drug targeting of the underlying mechanisms, might be helpful for treating brain and behavioral disorders associated with imbalances in midbrain DA (e.g. Parkinson&#8217;s disease, attention deficit and hyperactivity disorder, schizophrenia, and drug addiction).

This protocol describes two different environmental manipulations and a concurrent brain infusion protocol to study environmentally-induced brain changes underlying adaptive behavior and brain repair in adult mice.

אפנון סביבתי של מספר המוח התיכון דופמין נוירונים בעכברים בוגרים

Long-lasting changes in the brain or &#8216;brain plasticity&#8217; underlie adaptive behavior and brain repair following disease or injury. Furthermore, ...

Laser Capture Microdissection - A Demonstration of the Isolation of Individual Dopamine Neurons and the Entire Ventral Tegmental Area

Laser capture microdissection (LCM) is used to isolate a concentrated population of individual cells or precise anatomical regions of tissue from tissue sections on a microscope slide. When combined with immunohistochemistry, LCM can be used to isolate individual cells types based on a specific protein marker. Here, the LCM technique is described for collecting a specific population of dopamine neurons directly labeled with&#160;tyrosine hydroxylase immunohistochemistry and for isolation of the dopamine neuron containing region of the ventral tegmental area using indirect tyrosine hydroxylase immunohistochemistry on a section adjacent to those used for LCM. An infrared (IR) capture laser is used to both dissect individual neurons as well as the ventral tegmental area off glass slides and onto an LCM cap for analysis. Complete dehydration of the tissue with 100% ethanol and xylene is critical. The combination of the IR capture laser and the ultraviolet (UV) cutting laser is used to isolate individual dopamine neurons or the ventral tegmental area when using PEN membrane slides. A PEN membrane slide has significant advantages over a glass slide as it offers better consistency in capturing and collecting cells, is faster collecting large pieces of tissue, is less reliant on dehydration and results in complete removal of the tissue from the slide. Although removal of large areas of tissue from a glass slide is feasible, it is considerably more time consuming and frequently leaves some residual tissue behind. Data shown here demonstrate that RNA of sufficient quantity and quality can be obtained using these procedures for quantitative PCR measurements. Although RNA and DNA are the most commonly isolated molecules from tissue and cells collected with LCM, isolation and measurement of microRNA, protein and epigenetic changes in DNA can also benefit from the enhanced anatomical and cellular resolution obtained using LCM.

The isolation of individual dopamine neurons or the ventral tegmental area with direct or indirect immunohistochemistry is demonstrated using laser capture microdissection. Parameters for isolation of tissue from a glass slide using an infrared laser and from membrane slides using the combination of an infrared and ultraviolet laser are discussed.

Microdissection לכידת לייזר - הפגנה של הבידוד של פרט דופמין נוירונים ופינת הגחון tegmental השלם

Laser capture microdissection (LCM) is used to isolate a concentrated population of individual cells or precise anatomical regions of tissue from tissue ...

Assessment of Dopaminergic Homeostasis in Mice by Use of High-performance Liquid Chromatography Analysis and Synaptosomal Dopamine Uptake

Dopamine (DA) is a modulatory neurotransmitter controlling motor activity, reward processes and cognitive function. Impairment of dopaminergic (DAergic) neurotransmission is strongly associated with several central nervous system-associated diseases such as Parkinson&#39;s disease, attention-deficit-hyperactivity disorder and drug addiction1,2,3,4. Delineating disease mechanisms involving DA imbalance is critically dependent on animal models to mimic aspects of the diseases, and thus protocols that assess specific parts of the DA homeostasis are important to provide novel insights and possible therapeutic targets for these diseases.
Here, we present two useful experimental protocols that when combined provide a functional read-out of the DAergic system in mice. Biochemical and functional parameters on DA homeostasis are obtained through assessment of DA levels and dopamine transporter (DAT) functionality5. When investigating the DA system, the ability to reliably measure endogenous levels of DA from adult brain is essential. Therefore, we present how to perform high-performance liquid chromatography (HPLC) on brain tissue from mice to determine levels of DA. We perform the experiment on tissue from dorsal striatum (dStr) and nucleus accumbens (NAc), but the method is also suitable for other DA-innervated brain areas.
DAT is essential for reuptake of DA into the presynaptic terminal, thereby controlling the temporal and spatial activity of released DA. Knowing the levels and functionality of DAT in the striatum is of major importance when assessing DA homeostasis. Here, we provide a protocol that allows to simultaneously deduce information on surface levels and function using a synaptosomal6 DA uptake assay.
Current methods combined with standard immunoblotting protocols provide the researcher with relevant tools to characterize the DAergic system.

Synaptosomal dopamine uptake and high-performance liquid chromatography analysis represent experimental tools to investigate dopamine homeostasis in mice by assessing the function of the dopamine transporter and levels of dopamine in striatal tissue, respectively. Here we present protocols to measure dopamine tissue content and assess the functionality of the dopamine transporter.

הערכה של דופאמין הומאוסטזיס בעכברים באמצעות ניתוח ביצועים גבוהים כרומטוגרפיה נוזלית, דופמין Synaptosomal ספיגת

Dopamine (DA) is a modulatory neurotransmitter controlling motor activity, reward processes and cognitive function. Impairment of dopaminergic (DAergic) ...

Stereological Estimation of Dopaminergic Neuron Number in the Mouse Substantia Nigra Using the Optical Fractionator and Standard Microscopy Equipment

In pre-clinical Parkinson&#39;s disease research, analysis of the nigrostriatal tract, including quantification of dopaminergic neuron loss within the substantia nigra, is essential. To estimate the total dopaminergic neuron number, unbiased stereology using the optical fractionator method is currently considered the gold standard. Because the theory behind the optical fractionator method is complex and because stereology is difficult to achieve without specialized equipment, several commercially available complete stereology systems that include the necessary software do exist, purely for cell counting reasons. Since purchasing a specialized stereology setup is not always feasible, for many reasons, this report describes a method for the stereological estimation of dopaminergic neuronal cell counts using standard microscopy equipment, including a light microscope, a motorized object table (x, y, z plane) with imaging software, and a computer for analysis. A step-by-step explanation is given on how to perform stereological quantification using the optical fractionator method, and pre-programmed files for the calculation of estimated cell counts are provided. To assess the accuracy of this method, a comparison to data obtained from a commercially available stereology apparatus was performed. Comparable cell numbers were found using this protocol and the stereology device, thus demonstrating the precision of this protocol for unbiased stereology.

This work presents a step-by-step protocol for the unbiased stereological estimation of dopaminergic neuronal cell numbers in the mouse substantia nigra using standard microscopy equipment (i.e., a light microscope, a motorized object table (x, y, z plane), and public domain software for digital image analysis.

הערכה stereological של דופאמין נוירון במספר העכבר הסובסטנציה ניגרה באמצעות מיקרוסקופ רגיל ציוד אופטי Fractionator

In pre-clinical Parkinson&#39;s disease research, analysis of the nigrostriatal tract, including quantification of dopaminergic neuron loss within the ...

Identification of Dopamine D1-Alpha Receptor Within Rodent Nucleus Accumbens by an Innovative RNA In Situ Detection Technology

In the central nervous system, the D1-alpha subtype receptor (Drd1&#945;) is the most abundant dopamine (DA) receptor, which plays a vital role in regulating neuronal growth and development. However, the mechanisms underlying Drd1&#945; receptor abnormalities mediating behavioral responses and modulating working memory function are still unclear. Using a novel RNA in situ hybridization assay, the current study identified dopamine Drd1&#945; receptor and tyrosine hydroxylase (TH) RNA expression from DA-related circuitry in the nucleus accumbens (NAc) area and substantia nigra region (SNR), respectively. Drd1&#945; expression in the NAc shows a &#34;discrete dot&#34; staining pattern. Clear sex differences in Drd1&#945; expression were observed. In contrast, TH shows a &#34;clustered&#34; staining pattern. Regarding TH expression, female rats displayed a higher signal expression per cell relative to male animals. The methods presented here provide a novel in situ hybridization technique for investigating changes in dopamine system dysfunction during the progression of central nervous system diseases.

Identification of Dopamine D1-Alpha Receptor Within Rodent Nucleus Accumbens by an Innovative RNA In Situ Detection Technology

Identification of dopamine D1-alpha receptor in the nucleus accumbens is critical for clarifying D1 receptor dysfunction during a central nervous system disease. We performed a novel RNA in situ hybridization assay to visualize single RNA molecules in a specific brain area.

זיהוי של דופמין קולטן D1-אלפא בתוך מכרסמים גרעין האקומבנס על ידי טכנולוגיה חדשנית RNA בחיי עיר זיהוי

In the central nervous system, the D1-alpha subtype receptor (Drd1&#945;) is the most abundant dopamine (DA) receptor, which plays a vital role in ...

Preparation of Acute Spinal Cord Slices for Whole-cell Patch-clamp Recording in Substantia Gelatinosa Neurons

Recent whole-cell patch-clamp studies from substantia gelatinosa (SG) neurons have provided a large body of information about the spinal mechanisms underlying sensory transmission, nociceptive regulation, and chronic pain or itch development. Implementations of electrophysiological recordings together with morphological studies based on the utility of acute spinal cord slices have further improved our understanding of neuronal properties and the composition of local circuitry in SG. Here, we present a detailed and practical guide for the preparation of spinal cord slices and show representative whole-cell recording and morphological results. This protocol permits ideal neuronal preservation and can mimic in vivo conditions to a certain extent. In summary, the ability to obtain an in vitro preparation of spinal cord slices enables stable current- and voltage-clamp recordings and could thus facilitate detailed investigations into the intrinsic membrane properties, local circuitry and neuronal structure using diverse experimental approaches.

Here, we describe the essential steps for whole-cell patch-clamp recordings made from substantia gelatinosa (SG) neurons in the in vitro spinal cord slice. This method allows the intrinsic membrane properties, synaptic transmission and morphological characterization of SG neurons to be studied.

הכנה של חוט השדרה חריפה פרוסות תאים כל תיקון-קלאמפ הקלטה בנוירונים הסובסטנציה Gelatinosa

Recent whole-cell patch-clamp studies from substantia gelatinosa (SG) neurons have provided a large body of information about the spinal mechanisms ...

Comprehensive Autopsy Program for Individuals with Multiple Sclerosis

We describe a rapid tissue donation program for individuals with multiple sclerosis (MS) that requires scientists and technicians to be on-call 24/7, 365 days a year. Participants consent to donate their brain and spinal cord. Most patients were followed by neurologists at the Cleveland Clinic Mellen Center for MS Treatment and Research. Their clinical courses and neurological disabilities are well-characterized. Soon after death, the body is transported to the MS Imaging Center, where the brain is scanned in situ by 3 T magnetic resonance imaging (MRI). The body is then transferred to the autopsy room, where the brain and spinal cord are removed. The brain is divided into two hemispheres. One hemisphere is immediately placed in a slicing box and alternate 1 cm-thick slices are either fixed in 4% paraformaldehyde for two days or rapidly frozen in dry ice and 2-methylbutane. The short-fixed brain slices are stored in a cryopreservation solution and used for histological analyses and immunocytochemical detection of sensitive antigens. Frozen slices are stored at -80 &#176;C and used for molecular, immunocytochemical, and in situ hybridization/RNA scope studies. The other hemisphere is placed in 4% paraformaldehyde for several months, placed in the slicing box, re-scanned in the 3 T magnetic resonance (MR) scanner and sliced into centimeter-thick slices. Postmortem in situ MR images (MRIs) are co-registered with 1 cm-thick brain slices to facilitate MRI-pathology correlations. All brain slices are photographed and brain white-matter lesions are identified. The spinal cord is cut into 2 cm segments. Alternate segments are fixed in 4% paraformaldehyde or rapidly frozen. The rapid procurement of postmortem MS tissues allows pathological and molecular analyses of MS brains and spinal cords and pathological correlations of brain MRI abnormalities. The quality of these rapidly-processed postmortem tissues (usually within 6 h of death) is of great value to MS research and has resulted in many high-impact discoveries.

Multiple sclerosis is an inflammatory demyelinating disease with no cure. Analysis of brain tissue provides important clues to understanding the pathogenesis of the disease. Here we discuss the methodology and downstream analysis of MS brain tissue collected through a unique rapid autopsy program in operation at the Cleveland Clinic.

שלאחר המוות תוכנית מקיפה עבור אנשים עם טרשת נפוצה

We describe a rapid tissue donation program for individuals with multiple sclerosis (MS) that requires scientists and technicians to be on-call 24/7, 365 ...

Combined Infusion and Stimulation with Fast-Scan Cyclic Voltammetry (CIS-FSCV) to Assess Ventral Tegmental Area Receptor Regulation of Phasic Dopamine

Phasic dopamine (DA) release from the ventral tegmental area (VTA) to the nucleus accumbens plays a pivotal role in reward processing and reinforcement learning. Understanding how the diverse neuronal inputs into the VTA control phasic DA release can provide a better picture of the circuitry that controls reward processing and reinforcement learning. Here, we describe a method that combines intra-VTA cannula infusions of pharmacological agonists and antagonists with stimulation-evoked phasic DA release (combined infusion and stimulation, or CIS) as measured by in vivo fast-scan cyclic voltammetry (FSCV). Using CIS-FSCV in anesthetized rats, a phasic DA response can be evoked by electrically stimulating the VTA with a bipolar electrode fitted with a cannula while recording in the nucleus accumbens core. Pharmacological agonists or antagonists can be infused directly at the stimulation site to investigate specific VTA receptors&#39; roles in driving phasic DA release. A major benefit of CIS-FSCV is that VTA receptor function can be studied in vivo, building on in vitro studies.

Combined Infusion and Stimulation with Fast-Scan Cyclic Voltammetry CIS-FSCV to Assess Ventral Tegmental Area Receptor Regulation of Phasic Dopamine

The goal of this protocol is to directly manipulate ventral tegmental area receptors to study their contribution to subsecond dopamine release.

עירוי וגירוי משולבים עם וולטמטריה מחזורית סריקה מהירה (CIS-FSCV) כדי להעריך ויסות קולטן אזור טגמנטלי גחוני של דופמין פאזי

Phasic dopamine (DA) release from the ventral tegmental area (VTA) to the nucleus accumbens plays a pivotal role in reward processing and reinforcement ...