מימון למחקר זה מסופק על ידי תכנית במדעי המוח בפרס ממדינת מכללת פלורידה לרפואה של האוניברסיטה ותמיכה מהמחלקה למדע ביולוגי בברית המועצות לשעבר. מימון נוסף מסופק על ידי גרנט-in-Aid מקרן המחקר של אלכוהול משקאות היצרן.

<ol>
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החוקרים מצהירים כי אין להם אינטרסים כלכליים מתחרים.

העלויות של שימוש לרעה באלכוהול ואלכוהוליזם לחברה היא אדירות, הן במונחים של עלויות אנושיות 29 וכלכליות 30,31. דרוזופילה כמודל מציעה מערכת מהירה ותכליתית כדי לבחון במהירות את התגובות התנהגותיות של מספר גדול של אנשים ובתור שכזו כבר נעשה שימוש נרחב עבור שנ...

תסיסנית להפגין תגובות התנהגותיות biphasic לאלכוהול 1 שהם מקבילים לתגובות אנושיות לתרופה זו 2,3. לאחר החשיפה ראשונית לריכוזים נמוכים של אלכוהול, זבובי פעילות של תנועה מוגברת תערוכה, הוחלף על ידי חוסר הקואורדינציה מוטורית, אובדן השליטה ביציבה ורפלקסים ליישר (אובדן ליישר רפלקס: LoRR), והרגעה (חוסר הפעילות מוטורית בתגובה מלאה לגירוי מכאני) כחשיפה לאלכוהול מתקדמת 4-9. השעון הביולוגי אנדוגני הוא מאפנן חזק של רגישות אלכוהול ורעילות כפי שנצפה בעכברים 10,11, חולדות 12, ובני האדם 13. ההתקדמות שחלה באחרונה במחקר דרוזופילה הראו שעון היממה מודולציה רגישות אלכוהול חריף אך לא אלכוהול סובלנות 1. גישות רבות עוצמה הגנטית הזמינות בדרוזופילה באמצעות מחקרי מוטציה ומניפולציות מהונדסים של המרחביתוביטוי גנים של זמן מספק מערכת המאפשרת התקדמות מהירה בזיהוי המנגנונים התאיים ומולקולריים שבבסיס להתנהגויות מורכבות. השימוש בדרוזופילה ככלי חקירה התירה התקדמות מהותית בהבנת נוירוביולוגיה אלכוהול שיכול להיות מתורגמת במהירות ליונקים 14-16. על מנת להקל על ההבנה של המנגנונים המולקולריים שבאמצעותו השעון הביולוגי מודולציה רגישות אלכוהול ולמדוד באופן אחיד על פני תגובות התנהגותיות נקודות זמן היממה, פרוטוקול ממשל אלכוהול מתאים לשימוש בתנאי אור אדום עמומים נדרש. לדרוזופילה, אלכוהול יכול להינתן באמצעות תוספי מזון לחשיפה כרונית או באופן אמין דרך בניהול אלכוהול בצורה של אדים לחשיפות אקוטיות. כאן אנו מתארים פרוטוקול ממשל אלכוהול מתאים להערכה של אפנון היממה של רפלקס הפסד של ליישר (LoRR) 1, כמו גםתרופות הרגעה. זבובים נגררו עם שעות 12: 12 מחזורי LD שעות בטמפרטורה קבועה ולאחר מכן הועברו למשטר אור נשלט על 2-5 ימים, תלוי בשאלה הניסיונית. זבובים נחשפים לאדי אתנול במכשיר המכונה FlyBar. במכשיר זה, כמויות מבוקרות של אוויר מבעבעים דרך מים ואלכוהול; לאחר מכן האדים מעורבבים וביימו לדיור בקבוקון הזבובים. כל 5 דקות זבובי כבש עבור המספר שאינו מצליח להציג ליישר רפלקסים או הפכו מסומם. אחוזי LoRR עבור כל נקודת זמן מחושבים והשוואה בין נקודות זמן היממה או בין הזנים של זבובים. הפשטות והאמינות של אספקת אלכוהול באמצעות משלוח אלכוהול FlyBar בשילוב עם אפשרויות ניתוח התנהגות מספקת יתרון משמעותי לניסויים ביולוגיים שנערכו בתנאים חשוכים.

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			<td height="21" style="height:21px;width:285px;">Alcohol 190 proof</td>
			<td style="width:115px;">Various</td>
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			<td height="21" style="height:21px;width:285px;"></td>
			<td style="width:115px;"></td>
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			<td height="21" style="height:21px;width:285px;">Tool clips</td>
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</table>

the flybar: administering alcohol to flies

זבובי פירות (דרוזופילה melanogaster) הם מודל שהוקם עבור שני מחקרי אלכוהול וביולוגיה היממה. לאחרונה, הראה כי השעון הביולוגי מווסת את רגישות לאלכוהול, אך לא היווצרות של סובלנות. כאן אנו מתארים הפרוטוקול שלנו בפירוט. אלכוהול ניתן לזבובים באמצעות FlyBar. בהגדרה זו, אדי אלכוהול רוויים הוא מעורבב עם אוויר humidified בפרופורציות קבועות, וניתן לזבובים בארבע שפופרות בו זמנית. זבובים שגודלו בתנאים אחידים כדי למזער וריאציה בין משכפל. זבובים ישנים שלושה ימים של גנוטיפים או טיפולים שונים המשמשים לניסויים, רצוי על ידי זבובים משתי נקודות זמן שונות (למשל, CT 5 ו-CT 17) מה שהופך את השוואה ישירה אפשרית תואם. במהלך הניסוי, זבובים נחשפים לשעה 1 לשיעור שנקבע מראש של אדי אלכוהול ומספר הזבובים שמפגין אובדן ליישר רפלקס (LoRR) או sedation נספרים כל 5 דקות. ניתן לנתח את הנתונים באמצעות שלוש גישות סטטיסטיות שונות. הראשון הוא לקבוע את המועד שבי 50% מהזבובים איבדו את רפלקס ליישרם ולהשתמש בניתוח השונה (ANOVA) כדי לקבוע אם הבדלים משמעותיים קיימים בין נקודות זמן. השני הוא לקבוע את אחוז הזבובים שמראים LoRR לאחר מספר מסוים של דקות, ואחרי ניתוח ANOVA. השיטה האחרונה היא לנתח את סדרת כולה פעמים באמצעות סטטיסטיקה רב משתני. גם הפרוטוקול יכול לשמש לניסויים או השוואות שאינן היממה בין גנוטיפים.

אפנון היממה של אלכוהול רגישות באמצעות LoRR 50% כסמן. דוגמא מייצגת מראה אפנון היממה ברגישות אלכוהול במהלך היום מוצגת באיור 2. LoRR נמדד בשש נקודות זמן במהלך היום 2 nd של DD בקנטון-S ו50% LoRR נקבעו עבור כל נקודת זמן. ניתוח הראה הש?...

Watch this Scientific Journal Video about The FlyBar: Administering Alcohol to Flies at JoVE.com

The FlyBar: Administering Alcohol to Flies

דרוזופילה התפתחה כמערכת מודל משמעותית עבור לנתח את הבסיס התאי ומולקולרית של תגובות התנהגותיות לאלכוהול. כאן אנו מציגים פרוטוקול לאיסוף נתונים רגישות אלכוהול בהקשר היממה שיכולה להיות מיושם בקלות לניסויים אחרים, והוא מתאים היטב למחקר לתואר ראשון.

FlyBar: מתן אלכוהול לזבובים

Fruit flies (Drosophila melanogaster) are an established model for both alcohol research and circadian biology. Recently, we showed that the circadian ...

the-flybar-administering-alcohol-to-flies

Research

JoVE Journal

Neuroscience

10.6K Views.  Florida State University.  דרוזופילה התפתחה כמערכת מודל משמעותית עבור לנתח את הבסיס התאי ומולקולרית של תגובות התנהגותיות לאלכוהול. כאן אנו מציגים פרוטוקול לאיסוף נתונים רגישות אלכוהול בהקשר היממה שיכולה להיות מיושם בקלות לניסויים אחרים, והוא מתאים היטב למחקר...

Video: The FlyBar: Administering Alcohol to Flies

Experimental Methods for Testing the Effects of Neurotrophic Peptide, ADNF-9, Against Alcohol-induced Apoptosis during Pregnancy in C57BL/6 Mice

Experimental designs for investigating the effects of prenatal alcohol exposure during early embryonic stages in fetal brain growth are challenging. This is mostly due to the difficulty of microdissection of fetal brains and their sectioning for determination of apoptotic cells caused by prenatal exposure to alcohol. The experiments described here provide visualized techniques from mice breeding to the identification of cell death in fetal brain tissue. This study used C57BL/6 mice as the animal model for studying fetal alcohol exposure and the role of trophic peptide against alcohol-induced apoptosis. The breeding consists of a 2-hr matting window to determine the exact stage of embryonic age. An established fetal alcohol exposure model has been used in this study to determine the effects of prenatal alcohol exposure in fetal brains. This involves free access to alcohol or pair-fed liquid diets as the sole source of nutrients for the pregnant mice.
The techniques involving dissection of fetuses and microdissection of fetal brains are described carefully, since the latter can be challenging. Microdissection requires a stereomicroscope and ultra-fine forceps. Step-by-step procedures for dissecting the fetal brains are provided visually. The fetal brains are dissected from the base of the primordium olfactory bulb to the base of the metencephalon.
For investigating apoptosis, fetal brains are first embedded in gelatin using a peel-away mold to facilitate their sectioning with a vibratome apparatus. Fetal brains embedded and fixed in paraformaldehyde are easily sectioned, and the free floating sections can be mounted in superfrost plus slides for determination of apoptosis or cell death.
TUNEL (TdT-mediated dUTP Nick End Labeling; TdT: terminal deoxynucleotidyl transferase) assay has been used to identify cell death or apoptotic cells. It is noteworthy that apoptosis and cell-mediated cytotoxicity are characterized by DNA fragmentation. Thus, the visualized TUNEL-positive cells are indicative of cell death or apoptotic cells.
The experimental designs here provide information about the use of an established liquid diet for studying the effects of alcohol and the role of neurotrophic peptides during pregnancy in fetal brains. This involves breeding and feeding pregnant mice, microdissecting fetal brains, and determining apoptosis. Together, these visual and textual techniques might be a source for investigating prenatal exposure of harmful agents in fetal brains.

The experimental designs proposed here focus on studying the effects of alcohol exposure in apoptosis and the application of neurotrophic peptide during pregnancy in fetal brain. A detailed description from the breeding to the collection of fetal brains is described. Techniques for determination of apoptosis are also described in detail.

שיטות ניסיוניות לבדיקת ההשפעות של הפפטיד neurotrophic, ADNF-9, נגד אפופטוזיס מושרה אלכוהול במהלך הריון בעכברי C57BL / 6

Experimental designs for investigating the effects of prenatal alcohol exposure during early embryonic stages in fetal brain growth are challenging. This ...

Electroporation of the Hindbrain to Trace Axonal Trajectories and Synaptic Targets in the Chick Embryo

Electroporation of the chick embryonic neural tube has many advantages such as being quick and efficient for the expression of foreign genes into neuronal cells. In this manuscript we provide a method that demonstrates uniquely how to electroporate DNA into the avian hindbrain at E2.75 in order to specifically label a subset of neuronal progenitors, and how to follow their axonal projections and synaptic targets at much advanced stages of development, up to E14.5. We have utilized novel genetic tools including specific enhancer elements, Cre/Lox - based plasmids and the PiggyBac-mediated DNA transposition system to drive GFP expression in a subtype of hindbrain cells (the dorsal most subgroup of interneurons, dA1). Axonal trajectories and targets of dA1 axons are followed at early and late embryonic stages at various brainstem regions. This strategy contributes advanced techniques for targeting cells of interest in the embryonic hindbrain and for tracing circuit formation at multiple stages of development.

How neuronal networks are established in the embryonic brain is a fundamental question in developmental neurobiology. Here we combined an electroporation technique with novel genetic tools, such as Cre/Lox&#x2013;plasmids and PiggyBac-mediated DNA transposition system in the avian hindbrain to label dorsal interneurons and track their axonal projections and synaptic targets at various developmental stages.

Electroporation של המוח האחורי כדי לעקוב אחר מסלולים ויעדי axonal Synaptic בעובר האפרוח

Electroporation of  the chick embryonic neural tube has many advantages such as being quick and efficient  for the expression of foreign genes into ...

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety of brain disorders1,2. To better understand the mechanisms by which brain circuits react to an animal's experience requires the ability to monitor the experience-dependent molecular changes in a given set of neurons, over a prolonged period of time, in the live animal. While experience and associated neural activity is known to trigger gene expression changes in neurons1,2, most of the methods to detect such changes do not allow repeated observation of the same neurons over multiple days or do not have sufficient resolution to observe individual neurons3,4. Here, we describe a method that combines in vivo two-photon microscopy with a genetically encoded fluorescent reporter to track experience-dependent gene expression changes in individual cortical neurons over the course of day-to-day experience. 
One of the well-established experience-dependent genes is Activity-regulated cytoskeletal associated protein (Arc)5,6. The transcription of Arc is rapidly and highly induced by intensified neuronal activity3, and its protein product regulates the endocytosis of glutamate receptors and long-term synaptic plasticity7. The expression of Arc has been widely used as a molecular marker to map neuronal circuits involved in specific behaviors3. In most of those studies, Arc expression was detected by in situ hybridization or immunohistochemistry in fixed brain sections. Although those methods revealed that the expression of Arc was localized to a subset of excitatory neurons after behavioral experience, how the cellular patterns of Arc expression might change with multiple episodes of repeated or distinctive experiences over days was not investigated. 
In vivo two-photon microscopy offers a powerful way to examine experience-dependent cellular changes in the living brain8,9. To enable the examination of Arc expression in live neurons by two-photon microscopy, we previously generated a knock-in mouse line in which a GFP reporter is placed under the control of the endogenous Arc promoter10. This protocol describes the surgical preparations and imaging procedures for tracking experience-dependent Arc-GFP expression patterns in neuronal ensembles in the live animal. In this method, chronic cranial windows were first implanted in Arc-GFP mice over the cortical regions of interest. Those animals were then repeatedly imaged by two-photon microscopy after desired behavioral paradigms over the course of several days. This method may be generally applicable to animals carrying other fluorescent reporters of experience-dependent molecular changes4.

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

Experience-dependent molecular changes in neurons are essential for the brain's ability to adapt in response to behavioral challenges. An in vivo two-photon imaging method is described here that allows the tracking of such molecular changes in individual cortical neurons through genetically encoded reporters.

In vivo שני פוטוני הדמיה של שינויים מולקולריים ניסיון תלוי בנוירונים בקליפת מוח

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety ...

A Method to Make a Craniotomy on the Ventral Skull of Neonate Rodents

The use of a craniotomy for in vivo experiments provides an opportunity to investigate the dynamics of diverse cellular processes in the mammalian brain in adulthood and during development. Although most in vivo approaches use a craniotomy to study brain regions located on the dorsal side, brainstem regions such as the pons, located on the ventral side remain relatively understudied. The main goal of this protocol is to facilitate access to ventral brainstem structures so that they can be studied in vivo using electrophysiological and imaging methods. This approach allows study of structural changes in long-range axons, patterns of electrical activity in single and ensembles of cells, and changes in blood brain barrier permeability in neonate animals. Although this protocol has been used mostly to study the auditory brainstem in neonate rats, it can easily be adapted for studies in other rodent species such as neonate mice, adult rodents and other brainstem regions.

A surgical method is described to expose the ventral skull in neonate rats. Using this approach it is possible to open a craniotomy to perform acute electrophysiology and two-photon microscopy experiments in the brainstem of anesthetized pups.

שיטה להרוויח Craniotomy על גולגולת הגחון של מכרסמים ילוד

The use of a craniotomy for in vivo experiments provides an opportunity to investigate the dynamics of diverse cellular processes in the mammalian brain ...

An Inexpensive, Scalable Behavioral Assay for Measuring Ethanol Sedation Sensitivity and Rapid Tolerance in Drosophila

Alcohol use disorder (AUD) is a serious health challenge. Despite a large hereditary component to AUD, few genes have been unambiguously implicated in their etiology. The fruit fly, Drosophila melanogaster, is a powerful model for exploring molecular-genetic mechanisms underlying alcohol-related behaviors and therefore holds great promise for identifying and understanding the function of genes that influence AUD. The use of the Drosophila model for these types of studies depends on the availability of assays that reliably measure behavioral responses to ethanol. This report describes an assay suitable for assessing ethanol sensitivity and rapid tolerance in flies. Ethanol sensitivity measured in this assay is influenced by the volume and concentration of ethanol used, a variety of previously reported genetic manipulations, and also the length of time the flies are housed without food immediately prior to testing. In contrast, ethanol sensitivity measured in this assay is not affected by the vigor of fly handling, sex of the flies, and supplementation of growth medium with antibiotics or live yeast. Three different methods for quantitating ethanol sensitivity are described, all leading to essentially indistinguishable ethanol sensitivity results. The scalable nature of this assay, combined with its overall simplicity to set-up and relatively low expense, make it suitable for small and large scale genetic analysis of ethanol sensitivity and rapid tolerance in Drosophila.

An Inexpensive, Scalable Behavioral Assay for Measuring Ethanol Sedation Sensitivity and Rapid Tolerance in Drosophila

Straightforward assays for measuring ethanol sensitivity and rapid tolerance in Drosophila facilitate the use of this model organism for investigating these important ethanol-related behaviors. Here, a relatively simple, scalable assay for measuring ethanol sensitivity and rapid tolerance in flies is described.

Assay זול, Scalable התנהגות למדידת אתנול הרגעת רגישות וסובלנות מהירה ב<em&gt; דרוזופילה</em

Alcohol use disorder (AUD) is a serious health challenge. Despite a large hereditary component to AUD, few genes have been unambiguously implicated in ...

An Assay for Measuring the Effects of Ethanol on the Locomotion Speed of Caenorhabditis elegans

Alcohol use disorders are a significant public health concern, for which there are few effective treatment strategies. One difficulty that has delayed the development of more effective treatments is the relative lack of understanding of the molecular underpinnings of the effects of ethanol on behavior. The nematode, Caenorhabditis elegans (C.&#160;elegans), provides a useful model in which to generate and test hypotheses about the molecular effects of ethanol. Here, we describe an assay that has been developed and used to examine the roles of particular genes and environmental factors in behavioral responses to ethanol, in which locomotion is the behavioral output. Ethanol dose-dependently causes an acute depression of crawling on an agar surface. The effects are dynamic; animals exposed to a high concentration demonstrate an initial strong depression of crawling, referred to here as initial sensitivity, and then partially recover locomotion speed despite the continued presence of the drug. This ethanol-induced behavioral plasticity is referred to here as the development of acute functional tolerance. This assay has been used to demonstrate that these two phenotypes are distinct and genetically separable. The straightforward locomotion assay described here is suitable for examining the effects of both genetic and environmental manipulations on these acute behavioral responses to ethanol in C.&#160;elegans.

An Assay for Measuring the Effects of Ethanol on the Locomotion Speed of Caenorhabditis elegans

C. elegans is a useful model for studying the effects of ethanol on behavior. We present a behavioral assay that quantifies the effects of ethanol on the locomotion speed of crawling worms; both initial sensitivity and the development of acute functional tolerance to ethanol can be measured with this assay.

Assay למדידת ההשפעות של אתנול במהירות התנועה של<em&gt; Elegans Caenorhabditis</em

Alcohol use disorders are a significant public health concern, for which there are few effective treatment strategies. One difficulty that has delayed the ...

Video Imaging and Spatiotemporal Maps to Analyze Gastrointestinal Motility in Mice

The enteric nervous system (ENS) plays an important role in regulating gastrointestinal (GI) motility and can function independently of the central nervous system. Changes in ENS function are a major cause of GI symptoms and disease and may contribute to GI symptoms reported in neuropsychiatric disorders including autism. It is well established that isolated colon segments generate spontaneous, rhythmic contractions known as Colonic Migrating Motor Complexes (CMMCs). A procedure to analyze the enteric neural regulation of CMMCs in ex vivo preparations of mouse colon is described. The colon is dissected from the animal and flushed to remove fecal content prior to being cannulated in an organ bath. Data is acquired via a video camera positioned above the organ bath and converted to high-resolution spatiotemporal maps via an in-house software package. Using this technique, baseline contractile patterns and pharmacological effects on ENS function in colon segments can be compared over 3-4 hr. In addition, propagation length and speed of CMMCs can be recorded as well as changes in gut diameter and contraction frequency. This technique is useful for characterizing gastrointestinal motility patterns in transgenic mouse models (and in other species including rat and guinea pig). In this way, pharmacologically induced changes in CMMCs are recorded in wild type mice and in the Neuroligin-3R451C mouse model of autism. Furthermore, this technique can be applied to other regions of the GI tract including the duodenum, jejunum and ileum and at different developmental ages in mice.

This article describes a video imaging technique and high-resolution spatiotemporal mapping to identify changes in the neural regulation of colonic motility in adult mice. Subtle effects on gastrointestinal (GI) function can be detected using this approach in isolated tissue preparations to advance our understanding of GI disease.

הדמיה וידאו ומפות Spatiotemporal לנתח תנועתיות מערכת העיכול של עכברים

The enteric nervous system (ENS) plays an important role in regulating gastrointestinal (GI) motility and can function independently of the central ...

A Simple Way to Measure Alterations in Reward-seeking Behavior Using Drosophila melanogaster

We describe a protocol for measuring ethanol self-administration in fruit flies (Drosophila melanogaster) as a proxy for changes in reward states. We demonstrate a simple way to tap into the fly reward system, modify experiences related to natural reward, and use voluntary ethanol consumption as a measure for changes in reward states. The approach serves as a relevant tool to study the neurons and genes that play a role in experience-mediated changes of internal state. The protocol is composed of two discrete parts: exposing the flies to rewarding and nonrewarding experiences, and assaying voluntary ethanol consumption as a measure of the motivation to obtain a drug reward. The two parts can be used independently to induce the modulation of experience as an initial step for further downstream assays or as an independent two-choice feeding assay, respectively. The protocol does not require a complicated setup and can therefore be applied in any laboratory with basic fly culture tools.

A Simple Way to Measure Alterations in Reward-seeking Behavior Using Drosophila melanogaster

We describe a protocol for inducing rewarding and nonrewarding experiences in fruit flies (Drosophila melanogaster) using voluntary ethanol consumption as a measure for changes in reward states.

דרך פשוטה למדוד שינויים בהתנהגות שוחרת זכה שימוש<em&gt; תסיסנית</em

We describe a protocol for measuring ethanol self-administration in fruit flies (Drosophila melanogaster) as a proxy for changes in reward states. We ...

The Use of Trace Eyeblink Classical Conditioning to Assess Hippocampal Dysfunction in a Rat Model of Fetal Alcohol Spectrum Disorders

Neonatal rats were administered a relatively high concentration of ethyl alcohol (11.9% v/v) during postnatal days 4-9, a time when the fetal brain undergoes rapid organizational change and is similar to accelerated brain changes that occur during the third trimester in humans. This model of fetal alcohol spectrum disorders (FASDs) produces severe brain damage, mimicking the amount and pattern of binge-drinking that occurs in some pregnant alcoholic mothers. We describe the use of trace eyeblink classical conditioning (ECC), a higher-order variant of associative learning, to assess long-term hippocampal dysfunction that is typically seen in alcohol-exposed adult offspring. At 90 days of age, rodents were surgically prepared with recording and stimulating electrodes, which measured electromyographic (EMG) blink activity from the left eyelid muscle and delivered mild shock posterior to the left eye, respectively. After a 5 day recovery period, they underwent 6 sessions of trace ECC to determine associative learning differences between alcohol-exposed and control rats. Trace ECC is one of many possible ECC procedures that can be easily modified using the same equipment and software, so that different neural systems can be assessed. ECC procedures in general, can be used as diagnostic tools for detecting neural pathology in different brain systems and different conditions that insult the brain.

Trace eyeblink classical conditioning (ECC) was used to assess hippocampal-dependent associative learning in adult rats that were administered a high concentration (11.9% v/v) of alcohol during early neonatal brain development. In general, ECC procedures are sound diagnostic tools for detecting brain dysfunction across many psychological and biomedical settings.

השימוש של התניה קלאסית Eyeblink מעקב כדי להעריך תפקוד בהיפוקמפוס במודל של עכברים של אלכוהול עוברית ספקטרום הפרעות

Neonatal rats were administered a relatively high concentration of ethyl alcohol (11.9% v/v) during postnatal days 4-9, a time when the fetal brain ...

Assay to Measure Nucleocytoplasmic Transport in Real Time within Motor Neuron-like NSC-34 Cells

Nucleocytoplasmic transport refers to the import and export of large molecules from the cell nucleus. Recently, a number of studies have shown a connection between amyotrophic lateral sclerosis (ALS) and impairments in the nucleocytoplasmic pathway. ALS is a neurodegenerative disease affecting the motor neurons and resulting in paralysis and ultimately in death, within 2-5 years on average. Most cases of ALS are sporadic, lacking any apparent genetic linkage, but 10% are inherited in a dominant manner. Recently, hexanucleotide repeat expansions (HREs) in the chromosome 9 open reading frame 72 (C9orf72) gene were identified as a genetic cause of ALS and frontotemporal dementia (FTD). Importantly, different groups have recently proposed that these mutants affect nucleocytoplasmic transport. These studies have mostly shown the final outcome and manifestations caused by HREs on nucleocytoplasmic transport, but they do not demonstrate nuclear transport dysfunction in real time. As a result, only severe nucleocytoplasmic transport deficiency can be determined, mostly due to high overexpression or exogenous protein insertion. 
This protocol describes a new and very sensitive assay to evaluate and quantify nucleocytoplasmic transport dysfunction in real time. The rate of import of a NLS-NES-GFP protein (shuttle-GFP) can be quantified in real time using fluorescent microscopy. This is performed by using an exportin inhibitor, thus allowing the shuttle GFP only to enter the nucleus. To validate the assay, the C9orf72 HRE translated dipeptide repeats, poly(GR) and poly(PR), which have been previously shown to disrupt nucleocytoplasmic transport, were used. Using the described assay, a 50% decrease in the nuclear import rate was observed compared to the control. Using this system, minute changes in nucleocytoplasmic transport can be examined and the ability of different factors to rescue (even partially) a nucleocytoplasmic transport defect can be determined.

This protocol describes a method to sensitively measure the nucleocytoplasmic transport rate within motor neuron-like NSC-34 cells by quantifying the real-time change in the nuclear import of a NLS-NES-GFP protein.