אנתוני Renda נתמך על ידי אוניברסיטת פרס ויקטוריה מלגה לתואר שני. מחקר זה נתמך על ידי למדעי הטבע וההנדסה מועצת המחקר של קנדה דיסקברי גרנט, פרס NARSAD לחוקר הצעיר (עד RN), קרן ויקטוריה - Myre ו ויניפרד סים הקרן, הקרן הקנדית חדשנות גרנט, פיתוח קולומביה הבריטית קרן ידע ואת למדעי הטבע וההנדסה מועצת המחקר של כלי קנדה מחקר ו גרנט מכשור. אנו מודים Jillian מקיי, כריסטינה בארנס, אריאל סאליבן, ג&#39;ניפר מקדונלד ודניאל Morgado עבור בעלי העכבר מעולה.

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אין לנו מה למסור.

<p class="jove_content"&gt; השימוש של הקולטן ניאון במודל עקום של העכבר כדי לקבוע כמות ולוקליזציה של ערוץ יון מסוים מספק מספר יתרונות. בניגוד חלבונים כגון אקטין, אשר באה לידי ביטוי בכל מקום בגוף בכל התאים, תעלות יונים קיימות במספרים הרבה פחות והביטוי שלהם משתנה בין נוירונים תת ביצוע ניתוח מדויק באמצעות טכניקות מסור...

<table border="1" style=";text-align:right;direction:rtl"><tr><td> שם מגיב </td><td> חברה </td><td> מספר קטלוגי </td><td> תגובות </td></tr><tr><td> מיני האוסמוטי משאבות </td><td> Alzet </td><td> מודל 2002 </td><td></td></tr><tr><td> מלוח </td><td> Teknova </td><td> S5819 </td><td></td></tr><tr><td> (-), ניקוטין tartrate מימן מלח </td><td> סיגמא </td><td> N5260 </td><td></td></tr><tr><td> טיפות עיניים </td><td> נוברטיס </td><td> רימוני ג&#39;ל </td><td></td></tr><tr><td> Vetbond דבק </td><td> 3M </td><td> 1469SB </td><td></td></tr><tr><td> הפרין מלח </td><td> סיגמא </td><td> H4784 </td><td></td></tr><tr><td> 10x PBS </td><td> Invitrogen </td><td> 70011 </td><td></td></tr><tr><td> קטמין </td><td> Wyetח לבריאות בעלי חיים </td><td> 0856-4403-01 </td><td></td></tr><tr><td> medatomidine hydrochloride </td><td> פייזר </td><td> 1950673 </td><td></td></tr><tr><td> 23G מחט פרפר </td><td> בקטון דיקינסון </td><td> 367253 </td><td></td></tr><tr><td> paraformaldehyde </td><td> מיקרוסקופית אלקטרונים למדעים </td><td> 15710 </td><td></td></tr><tr><td> הטבעה פלסטיק עובש </td><td> VWR </td><td> 18986-1 </td><td></td></tr><tr><td> אוקטובר הרכבה מתחם </td><td> רקמת-Tek </td><td> 4583 </td><td></td></tr><tr><td> Mowiol 4-88 </td><td> EMD-Calbiochem </td><td> 475904 </td><td> ה-pH 8.5 </td></tr></table>

spectral confocal imaging of fluorescently tagged nicotinic receptors in knock-in mice with chronic nicotine administration

ליגנד-מגודרת תעלות יונים במערכת העצבים המרכזית (CNS) הם מעורבים תנאים רבים עם השלכות רפואיות וחברתיות קשות. למשל, התמכרות לניקוטין באמצעות עישון טבק הוא הגורם המוביל למוות בטרם עת ברחבי העולם (ארגון הבריאות העולמי) והיא נגרמת ככל הנראה על ידי שינוי של חלוקת ערוץ יונים במוח 1. חשיפה כרונית לניקוטין בשני מכרסמים ותוצאות בני אדם מספרים מוגברת של קולטני אצטילכולין ניאציניות (nAChRs) ברקמת המוח 1-3. כמו כן, שינויים או את GluN1 GluA1 ערוצי glutamatergic היו מעורבים לעורר רגישות לסמים ממכרים אחרים כגון קוקאין, אמפטמינים ו 4-6 בסמי הרגעה. כתוצאה מכך, היכולת למפות ולכמת הפצה ביטוי דפוסים של תעלות יונים מסוימות חשוב ביותר להבנת המנגנונים של התמכרות. מחקר של אזור ספציפי במוח EFfects של תרופות בודדות קידם כניסתו של טכניקות כגון ligands רדיואקטיביים. עם זאת, ברזולוציה מרחבית נמוכה מחייב ליגנד רדיואקטיבי מונע את היכולת לכמת ליגנד-מגודרת תעלות יונים של תת ספציפיים של נוירונים. כתבים ניאון מקודדים גנטית, כגון חלבון פלואורסצנטי ירוק (GFP) ו גרסאות רבות שלה צבע, חוללו מהפכה בתחום הביולוגיה 7. על ידי תיוג גנטית כתב ניאון לחלבון אנדוגני אפשר לדמיין חלבונים in vivo 7-10. אחד היתרונות של fluorescently תיוג חלבונים עם החללית היא חיסול השימוש נוגדנים, אשר יש בעיות של nonspecificity ונגישות חלבון המטרה. השתמשנו באסטרטגיה זו כדי fluorescently nAChRs תווית, אשר אפשרו את לימוד מכלול הקולטן באמצעות פורסטר תהודה העברת אנרגיה (סריג) בתאים בתרבית transfected 11. לאחרונה, השתמשנו knocK-בגישה לעכברים מהנדס עם חלבון פלואורסצנטי צהוב מתויג α4 יחידות משנה nAChR (α4YFP), המאפשר כימות מדויק של vivo הקולטן לשעבר ברזולוציה submicrometer בנוירונים במערכת העצבים המרכזית באמצעות מיקרוסקופיה confocal רפאים 12. ממוקד ניאון עקום של מוטציה משולב לוקוס אנדוגני תחת שליטה של ​​האמרגן המקורית שלו, לייצר רמות נורמליות של ביטוי וויסות של הקולטן בהשוואה קולטנים לא מתויגות בעכברים wildtype. גישה זו דפיקה-in ניתן להרחיב fluorescently לתייג תעלות יונים אחרים ומציע גישה חזקה של הדמיה לכימות קולטני מערכת העצבים המרכזית. במאמר זה אנו מתארים מתודולוגיה לכמת שינויים בביטוי nAChR בנוירונים מסוימים במערכת העצבים המרכזית לאחר החשיפה לניקוטין כרונית. השיטות שלנו כוללות מיני האוסמוטי השתלת המשאבה, קיבעון זלוף intracardiac, הדמיה וניתוח של rec ניקוטינית מתויג fluorescentlyeptor יחידות משנה של α4YFP עקום בעכברים (איור 1). יש לנו אופטימיזציה הטכניקה קיבעון למזער autofluorescence מ tissue.We המוח קבועה תאור מפורט של המתודולוגיה שלנו דימות באמצעות מיקרוסקופ confocal רפאים יחד עם אלגוריתם unmixing ליניארי רפאים כדי להחסיר אות autofluoresent כדי להשיג דיוק האות הקרינה α4YFP. לבסוף, אנו מראים תוצאות upregulation ניקוטין הנגרמת כרונית של קולטני α4YFP בנתיב perforant המדיאלי של ההיפוקמפוס.

Watch this Scientific Journal Video about Spectral Confocal Imaging of Fluorescently tagged Nicotinic Receptors in Knock-in Mice with Chronic Nicotine Administration at JoVE.com

Spectral Confocal Imaging of Fluorescently tagged Nicotinic Receptors in Knock-in Mice with Chronic Nicotine Administration

פיתחנו שיטה חדשנית של לכימות ניאציניות קולטני אצטילכולין שינויים בתוך אזורים subcellular של תת ספציפיים של נוירונים במערכת העצבים המרכזית כדי להבין טוב יותר את המנגנונים של התמכרות לניקוטין באמצעות שילוב של גישות כולל תיוג חלבון פלואורסצנטי של הקולטן באמצעות עקום בגישה רפאים confocal הדמיה.

דימות ספקטרלי Confocal של קולטנים ניאציניות מתויג fluorescently ב עקום בעכברים עם מינהל ניקוטין כרונית

Ligand-gated ion channels in the central nervous system (CNS) are implicated in numerous conditions with serious medical and social consequences.  For ...

spectral-confocal-imaging-fluorescently-tagged-nicotinic-receptors

Research

JoVE Journal

Neuroscience

13.4K Views.  University of Victoria. פיתחנו שיטה חדשנית של לכימות ניאציניות קולטני אצטילכולין שינויים בתוך אזורים subcellular של תת ספציפיים של נוירונים במערכת העצבים המרכזית כדי להבין טוב יותר את המנגנונים של התמכרות לניקוטין באמצעות שילוב של גישות כולל תיוג חלבון פלואורסצנטי של הקולטן באמצעות עקום ...

Video: Spectral Confocal Imaging of Fluorescently tagged Nicotinic Receptors in Knock-in Mice with Chronic Nicotine Administration

Subcutaneous Administration of Muscarinic Antagonists and Triple-Immunostaining of the Levator Auris Longus Muscle in Mice

Hind limb muscles of rodents, such as gastrocnemius and tibialis anterior, are frequently used for in vivo pharmacological studies of the signals essential for the formation and maintenance of mammalian NMJs. However, drug penetration into these muscles after subcutaneous or intramuscular administration is often incomplete or uneven and many NMJs can remain unaffected. Although systemic administration with devices such as mini-pumps can improve the spatiotemporal effects, the invasive nature of this approach can cause confounding inflammatory responses and/or direct muscle damage. Moreover, complete analysis of the NMJs in a hind limb muscle is challenging because it requires time-consuming serial sectioning and extensive immunostaining. 
The mouse LAL is a thin, flat sheet of muscle located superficially on the dorsum of the neck. It is a fast-twitch muscle that functions to move the pinna. It contains rostral and caudal portions that originate from the midline of the cranium and extend laterally to the cartilaginous portion of each pinna. The muscle is supplied by a branch of the facial nerve that projects caudally as it exits the stylomastoid foramen. We and others have found LAL to be a convenient preparation that offers advantages for the investigation of both short and long-term in vivo effects of drugs on NMJs and muscles. First, its superficial location facilitates multiple local applications of drugs under light anesthesia. Second, its thinness (2-3 layers of muscle fibers) permits visualization and analysis of almost all the NMJs within the muscle. Third, the ease of dissecting it with its nerve intact together with the pattern of its innervation permits supplementary electrophysiological analysis in vitro9,5. Last, and perhaps most importantly, a small applied volume (&tilde;50&mu;l) easily covers the entire muscle surface, provides a uniform and prolonged exposure of all its NMJs to the drug and eliminates the need for a systemic approach1,8.

We describe procedures for repeated administration of inhibitors of muscarinic signaling to the levator auris longus (LAL) muscle of young adult mice and for subsequent immunostaining of its neuromuscular junctions (NMJs) in wholemounts. The LAL muscle has unique advantages for revealing in vivo pharmacological effects on NMJs.

תת עורי מינהל היריבים Muscarinic ו Triple-Immunostaining של השריר Levator Longus Auris של עכברים

Hind limb muscles of rodents, such as gastrocnemius and tibialis anterior, are frequently used for in vivo  pharmacological studies of the signals ...

Imaging Calcium Responses in GFP-tagged Neurons of Hypothalamic Mouse Brain Slices

Despite an enormous increase in our knowledge about the mechanisms underlying the encoding of information in the brain, a central question concerning the precise molecular steps as well as the activity of specific neurons in multi-functional nuclei of brain areas such as the hypothalamus remain. This problem includes identification of the molecular components involved in the regulation of various neurohormone signal transduction cascades. Elevations of intracellular Ca2+ play an important role in regulating the sensitivity of neurons, both at the level of signal transduction and at synaptic sites.
New tools have emerged to help identify neurons in the myriad of brain neurons by expressing green fluorescent protein (GFP) under the control of a particular promoter. To monitor both spatially and temporally stimulus-induced Ca2+ responses in GFP-tagged neurons, a non-green fluorescent Ca2+ indicator dye needs to be used. In addition, confocal microscopy is a favorite method of imaging individual neurons in tissue slices due to its ability to visualize neurons in distinct planes of depth within the tissue and to limit out-of-focus fluorescence. The ratiometric Ca2+ indicator fura-2 has been used in combination with GFP-tagged neurons1. However, the dye is excited by ultraviolet (UV) light. The cost of the laser and the limited optical penetration depth of UV light hindered its use in many laboratories. Moreover, GFP fluorescence may interfere with the fura-2 signals2. Therefore, we decided to use a red fluorescent Ca2+ indicator dye. The huge Stokes shift of fura-red permits multicolor analysis of the red fluorescence in combination with GFP using a single excitation wavelength. We had previously good results using fura-red in combination with GFP-tagged olfactory neurons3. The protocols for olfactory tissue slices seemed to work equally well in hypothalamic neurons4. Fura-red based Ca2+ imaging was also successfully combined with GFP-tagged pancreatic &beta;-cells and GFP-tagged receptors expressed in HEK cells5,6. A little quirk of fura-red is that its fluorescence intensity at 650 nm decreases once the indicator binds calcium7. Therefore, the fluorescence of resting neurons with low Ca2+ concentration has relatively high intensity. It should be noted, that other red Ca2+-indicator dyes exist or are currently being developed, that might give better or improved results in different neurons and brain areas.

In this protocol, we update recent progress in imaging Ca2+ signals of GFP-tagged neurons in brain tissue slices using a red fluorescent Ca2+ indicator dye.

תגובות סידן הדמיה בנוירונים GFP-tagged של פרוסות מוח עכבר ההיפותלמוס

Despite an enormous increase in our knowledge about the mechanisms underlying the encoding of information in the brain, a central question concerning the ...

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

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

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

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

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

High-Resolution Quantitative Immunogold Analysis of Membrane Receptors at Retinal Ribbon Synapses

Retinal ganglion cells (RGCs) receive excitatory glutamatergic input from bipolar cells. Synaptic excitation of RGCs is mediated postsynaptically by NMDA receptors (NMDARs) and AMPA receptors (AMPARs). Physiological data have indicated that glutamate receptors at RGCs are expressed not only in postsynaptic but also in perisynaptic or extrasynaptic membrane compartments. However, precise anatomical locations for glutamate receptors at RGC synapses have not been determined. Although a high-resolution quantitative analysis of glutamate receptors at central synapses is widely employed, this approach has had only limited success in the retina. We developed a postembedding immunogold method for analysis of membrane receptors, making it possible to estimate the number, density and variability of these receptors at retinal ribbon synapses. Here we describe the tools, reagents, and the practical steps that are needed for: 1) successful preparation of retinal fixation, 2) freeze-substitution, 3) postembedding immunogold electron microscope (EM) immunocytochemistry and, 4) quantitative visualization of glutamate receptors at ribbon synapses.

The postembedding immunogold method is one of the most effective ways to provide high-resolution analyses of the subcellular localization of specific molecules. Here we describe a protocol to quantitatively analyze glutamate receptors at retinal ribbon synapses.

ניתוח ברזולוציה גבוהה כמותי immunogold של קולטני קרום ב סינפסות רצועת הכלים רשתית

Retinal ganglion cells (RGCs) receive excitatory glutamatergic input from bipolar cells. Synaptic excitation of RGCs is mediated postsynaptically by NMDA ...

Quantifying Infra-slow Dynamics of Spectral Power and Heart Rate in Sleeping Mice

Three vigilance states dominate mammalian life: wakefulness, non-rapid eye movement (non-REM) sleep, and REM sleep. As more neural correlates of behavior are identified in freely moving animals, this three-fold subdivision becomes too simplistic. During wakefulness, ensembles of global and local cortical activities, together with peripheral parameters such as pupillary diameter and sympathovagal balance, define various degrees of arousal. It remains unclear the extent to which sleep also forms a continuum of brain states-within which the degree of resilience to sensory stimuli and arousability, and perhaps other sleep functions, vary gradually-and how peripheral physiological states co-vary. Research advancing the methods to monitor multiple parameters during sleep, as well as attributing to constellations of these functional attributes, is central to refining our understanding of sleep as a multifunctional process during which many beneficial effects must be executed. Identifying novel parameters characterizing sleep states will open opportunities for novel diagnostic avenues in sleep disorders.
We present a procedure to describe dynamic variations of mouse non-REM sleep states via the combined monitoring and analysis of electroencephalogram (EEG)/electrocorticogram (ECoG), electromyogram (EMG), and electrocardiogram (ECG) signals using standard polysomnographic recording techniques. Using this approach, we found that mouse non-REM sleep is organized into cycles of coordinated neural and cardiac oscillations that generate successive 25-s intervals of high and low fragility to external stimuli. Therefore, central and autonomic nervous systems are coordinated to form behaviorally distinct sleep states during consolidated non-REM sleep. We present surgical manipulations for polysomnographic (i.e., EEG/EMG combined with ECG) monitoring to track these cycles in the freely sleeping mouse, the analysis to quantify their dynamics, and the acoustic stimulation protocols to assess their role in the likelihood of waking up. Our approach has already been extended to human sleep and promises to unravel common organizing principles of non-REM sleep states in mammals.

Here, we present experimental and analytical procedures to describe the temporal dynamics of the neural and cardiac variables of non-REM sleep in mice, which modulate sleep responsiveness to acoustic stimuli.

כימות אינפרה איטית דינמיקה של הספק ספקטרלי ואת קצב הלב בעכברים שינה

Three vigilance states dominate mammalian life: wakefulness, non-rapid eye movement (non-REM) sleep, and REM sleep. As more neural correlates of behavior ...

Automated Gait Analysis in Mice with Chronic Constriction Injury

The von Frey test is a classical method that has been widely used to examine the sensory function of neuropathic pain animals. However, it has some disadvantages such as subjective data and the requirement of a skilled, experienced experimenter. To date, a variety of modifications have improved the von Frey method, but it still has a few limitations. Recent reports have suggested that gait analysis produces more accurate and objective data from the neuropathic animals. This protocol demonstrates how to perform the automated gait analysis to determine the degree of neuropathic pain in mice. After several days of acclimation, the mice were allowed to walk freely on the glass floor to illuminate footprints. Then, quantification of the footprints and gait were performed through video clips with automatic analysis of various walking parameters, such as area of paw print, swing time, angle of paw, etc.
The main purpose of this study is to describe the methodology of automated gait analysis and briefly compare it with data from the classical sensory test using von Frey filament.

The precise assessment of pain response in a neuropathic animal model is critical to investigate the pathophysiology of pain diseases and develop new analgesics. We present a sensitive and objective method to determine the sensory function of the rodent hind paw by an automated gait analysis system.

אוטומטיות והרצתי ניתוח אצל עכברים עם פציעה כרונית הכיווץ

The von Frey test is a classical method that has been widely used to examine the sensory function of neuropathic pain animals. However, it has some ...

An In Vivo Blood-brain Barrier Permeability Assay in Mice Using Fluorescently Labeled Tracers

Blood-brain barrier (BBB) is a specialized barrier that protects the brain microenvironment from toxins and pathogens in the circulation and maintains brain homeostasis. The principal sites of the barrier are endothelial cells of the brain capillaries whose barrier function results from tight intercellular junctions and efflux transporters expressed on the plasma membrane. This function is regulated by pericytes and astrocytes that together form the neurovascular unit (NVU). Several neurological diseases such as stroke, Alzheimer&#39;s disease (AD), brain tumors are associated with an impaired BBB function. Assessment of the BBB permeability is therefore crucial in evaluating the severity of the neurological disease and the success of the treatment strategies employed.
We present here a simple yet robust permeability assay that have been successfully applied to several mouse models both, genetic and experimental. The method is highly quantitative and objective in comparison to the tracer fluorescence analysis by microscopy that is commonly applied. In this method, mice are injected intraperitoneally with a mix of aqueous inert fluorescent tracers followed by anesthetizing the mice. Cardiac perfusion of the animals is performed prior to harvesting brain, kidneys or other organs. Organs are homogenized and centrifuged followed by fluorescence measurement from the supernatant. Blood drawn from the cardiac puncture just before perfusion serves for normalization purpose to the vascular compartment. The tissue fluorescence is normalized to the wet weight and serum fluorescence to obtain a quantitative tracer permeability index. For additional confirmation, the contralateral hemi-brain preserved for immunohistochemistry can be utilized for tracer fluorescence visualization purposes.

An In Vivo Blood-brain Barrier Permeability Assay in Mice Using Fluorescently Labeled Tracers

Here we present a mouse brain vascular permeability assay using intraperitoneal injection of fluorescent tracers followed by perfusion that is applicable to animal models of blood-brain barrier dysfunction. One hemi-brain is used for assessing permeability quantitatively and the other for tracer visualization/immunostaining. The procedure takes 5 - 6 h for 10 mice.

In Vivo מחסום הדם - מוח חדירות Assay בעכברים באמצעות Fluorescently עם התווית המשדרים

Blood-brain barrier (BBB) is a specialized barrier that protects the brain microenvironment from toxins and pathogens in the circulation and maintains ...

Spectral Reflectometric Microscopy on Myelinated Axons In Situ

In a mammalian nervous system, myelin provides an electrical insulation by enwrapping the axon fibers in a multilayered spiral. Inspired by its highly-organized subcellular architecture, we recently developed a new imaging modality, named spectral reflectometry (SpeRe), which enables unprecedented label-free nanoscale imaging of the live myelinated axons in situ. The underlying principle is to obtain nanostructural information by analyzing the reflectance spectrum of the multilayered subcellular structure. In this article, we describe a detailed step-by-step protocol for performing a basic SpeRe imaging of the nervous tissues using a commercial confocal microscopic system, equipped with a white-light laser and a tunable filter. We cover the procedures of sample preparation, acquisition of spectral data, and image processing for obtaining nanostructural information.

Spectral Reflectometric Microscopy on Myelinated Axons In Situ

Here, we present a step-by-step protocol for imaging myelinated axons in a fixed brain slice using a label-free nanoscale imaging technique based on spectral reflectometry.

מיקרוסקופ Reflectometric ספקטרלי ב ו דנדריטים בחיי עיר

In a mammalian nervous system, myelin provides an electrical insulation by enwrapping the axon fibers in a multilayered spiral. Inspired by its ...

Quantitative Cell Biology of Neurodegeneration in Drosophila Through Unbiased Analysis of Fluorescently Tagged Proteins Using ImageJ

With the rising prevalence of neurodegenerative diseases, it is increasingly important to understand the underlying pathophysiology that leads to neuronal dysfunction and loss. Fluorescence-based imaging tools and technologies enable unprecedented analysis of subcellular neurobiological processes, yet there is still a need for unbiased, reproducible, and accessible approaches for extracting quantifiable data from imaging studies. We have developed a simple and adaptable workflow to extract quantitative data from fluorescence-based imaging studies using Drosophila models of neurodegeneration. Specifically, we describe an easy-to-follow, semi-automated approach using Fiji/ImageJ to analyze two cellular processes: first, we quantify protein aggregate content and profile in the Drosophila optic lobe using fluorescent-tagged mutant huntingtin proteins; and second, we assess autophagy-lysosome flux in the Drosophila visual system with ratiometric-based quantification of a tandem fluorescent reporter of autophagy. Importantly, the protocol outlined here includes a semi-automated segmentation step to ensure all fluorescent structures are analyzed to minimize selection bias and to increase resolution of subtle comparisons. This approach can be extended for the analysis of other cell biological structures and processes implicated in neurodegeneration, such as proteinaceous puncta (stress granules and synaptic complexes), as well as membrane-bound compartments (mitochondria and membrane trafficking vesicles). This method provides a standardized, yet adaptable reference point for image analysis and quantification, and could facilitate reliability and reproducibility across the field, and ultimately enhance mechanistic understanding of neurodegeneration.

Quantitative Cell Biology of Neurodegeneration in Drosophila Through Unbiased Analysis of Fluorescently Tagged Proteins Using ImageJ

We have developed a simple and adaptable workflow to extract quantitative data from fluorescence-imaging-based cell biological studies of protein aggregation and autophagic flux in the central nervous system of Drosophila models of neurodegeneration.

ביולוגיה של התא כמותית של הקשורים ניוון מוחיים ב דרוזופילה דרך ניתוח לא משוחדת של חלבונים מתויגות Fluorescently באמצעות ImageJ

With the rising prevalence of neurodegenerative diseases, it is increasingly important to understand the underlying pathophysiology that leads to neuronal ...

Probing Nicotinic Acetylcholine Receptor Function in Mouse Brain Slices via Laser Flash Photolysis of Photoactivatable Nicotine

Acetylcholine (ACh) acts through receptors to modulate a variety of neuronal processes, but it has been challenging to link ACh receptor function with subcellular location within cells where this function is carried out. To study the subcellular location of nicotinic ACh receptors (nAChRs) in native brain tissue, an optical method was developed for precise release of nicotine at discrete locations near neuronal membranes during electrophysiological recordings. Patch-clamped neurons in brain slices are filled with dye to visualize their morphology during 2-photon laser scanning microscopy, and nicotine uncaging is executed with a light flash by focusing a 405 nm laser beam near one or more cellular membranes. Cellular current deflections are measured, and a high-resolution three-dimensional (3D) image of the recorded neuron is made to allow reconciliation of nAChR responses with cellular morphology. This method allows for detailed analysis of nAChR functional distribution in complex tissue preparations, promising to enhance the understanding of cholinergic neurotransmission.

This article presents a method for studying nicotinic acetylcholine receptors (nAChRs) in mouse brain slices by nicotine uncaging. When coupled with simultaneous patch clamp recording and 2-photon laser scanning microscopy, nicotine uncaging connects nicotinic receptor function with cellular morphology, providing a deeper understanding of cholinergic neurobiology.

חקירת פונקציה קולטן אצטילכולין Nicotinic פרוסות המוח העכבר באמצעות לייזר פלאש פוטוליזה של ניקוטין Photoactivatable

Acetylcholine (ACh) acts through receptors to modulate a variety of neuronal processes, but it has been challenging to link ACh receptor function with ...