אנו מכירים סילקה Sachse וביאטה Eisermann בפיתוח מתודולוגיה זו, פבלו Traversa לשרטוט תוכניות לחסום הרכבה ודניאלה פלץ לשימוש סכמטית באיור 1. אנו מודים סילקה Sachse, פוואן Ramdya ורפאל Rytz על הערות על כתב היד. ר &#39;בל נתמך על ידי קרן Boehringer Ingelheim PhD מלגה. מחקר במעבדה של ר &#39;בנטון ממומן על ידי המועצה האירופית למחקר החל גרנט חוקר עצמאי וקרן השוויצרית הלאומית למדע.

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החוקרים אין לי מה לחשוף.

הכנה וניתוח השיטות שתוארו כאן ניתן להשתמש כדי לנתח ריח עורר הדים בכל אוכלוסייה של נוירונים באונה מחושים (OSNs, LN ו PNS) אשר transgene הנהג המתאים זמין. בעוד אנו מתארים היישום שלה באמצעות מיקרוסקופ פלואורסצנטי, דימות אופטי של הכנה זו יכולה באותה מידה להתבצע באמצעות מיקרוסקופ confocal או שני פוטונים 22,32. אכן, אלה הכלים האחרונים להקל על בעיית פיזור האור של מיקרוסקופית שדה רחב, אשר יכולה להפחית את הרזולוציה של תמונות, במיוחד כאשר מספר גדול של נוירונים אקספרס הכתב. הכנה זו בדרך כלל מאפשר הקלטות של ריח עוררו תגובות במשך שעה לפחות 1/2, אבל הפעם יכול להיות משמעותי יותר או קצרה יותר בהתאם למספר ואורך של מדידות שהוקלטו, זמן החשיפה לשמש עבור כל מסגרת ואת מרווח הזמן בין הגירוי. יתר על כן, שני הפוטונים עירור יכול להפחית photobleaching של נציג ניאוןorter, כמו גם phototoxicity הסלולר, על מנת לאפשר מדידה על פני תקופות זמן ארוכות יותר 22,32. לא משנה הבחירה של המיקרוסקופ, תנועה של המוח זבוב בתוך הקפסולה הראש הוא הבעיה חוזרת ביותר כאשר הדמיה בבעלי חיים שלמים. תוך תיקון תנועת יכול לתקן בעיות קטנות (שלב 5.1), זה בדרך כלל טוב יותר רק כדי להקליט את ההכנות מאוד יציבה. אסטרטגיות להפחתת חפצים התנועה כוללות: (i) כמו שצריך להגביל את החדק של זבוב עם עמוד השדרה קקטוס, (ii) משתק את הרגליים של הזבוב על ידי לתקן אותם לשלב עם eicosane (לפני שלב 2.6) (eicosane ניתן נמס ב ~ 37 ° C; להשתמש חוטי כסף כדי להאריך ולשפר את קצה ברזל הלחמה למטרה זו), (iii) מוכנים לעצור הוושט (גלוי בין העצבים מחושים) עם פינצטה בסדר. בנוסף G-CaMP1.6 משמש כאן, מספר גרסאות אופטימיזציה של ה-G-קמפ האינדיקטורים סידן אחרים זמינים,אשר נבדלות ביחס שלהן האות לרעש, הקרינה המחקר ולאחר הדינמיקה הזמני 12,28,33. בחירה מדויקת של חיישן צריך לקחת בחשבון את כל המאפיינים האלה, סוג של הנוירונים כדי להיות מנותח השאלה הביולוגית שפונים. חיישנים הנוכחי לדווח שינויים סידן המתואמים די טוב עם פוטנציאל פעולה 12,34, ו שיפור נוסף שלהם, יחד עם מספר גדל והולך של מנהלי התקנים ספציפיים קווי גנטיים 35 תמשיך לשפר את הפוטנציאל של דימות אופטי של פעילות עצבית לטוס ללא פגע.

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> מגיב </td><td> ספק </td><td> מספר קטלוגי </td><td> תגובות </td></tr><tr><td> קקטוס עמוד השדרה </td><td> גן מרכז </td><td></td><td> ~ דקה ארוכה 7mm, לא גמישה מדי </td></tr><tr><td> נטף </td><td> חנות כלי מיתר </td><td></td><td></td></tr><tr><td> דו רכיבי סיליקון </td><td> WPI </td><td> KWIK-SIL </td><td></td></tr><tr><td> מותאמים ללקוח, גוש פרספקס גובר </td><td> הזמינות שרטוטים <a href="http://neuro.uni-konstanz.de/jove_mountingblock_blueprint/" target="_blank">http://neuro.uni-konstanz.de/j ove_mountingblock_blueprint /</a> </td><td></td><td></td></tr><tr><td> נחושת רשתות </td><td> אתנה רשתות </td><td> G220-5 </td><td> בדרך כלל משתמשים עבור דגימות במיקרוסקופ אלקטרונים. </td></tr><tr><td> ברגים </td><td> חומרה החנות </TD&gt; <td></td><td> 2 מ&quot;מ קוטר </td></tr><tr><td> פלסטיק coverslips </td><td> Plano-EM </td><td> L4193 </td><td> 22 X 22 מ&quot;מ </td></tr><tr><td> ברזל טיפים הלחמה </td><td> קונרד אלקטרוניקה </td><td> 830283-62 </td><td></td></tr><tr><td> הכוח הסדיר אספקת </td><td> קונרד אלקטרוניקה </td><td> 511407-62 </td><td></td></tr><tr><td> חוט דק </td><td> Isabellenhütte </td><td> עיסא אוהם </td><td> 0.013 מ&quot;מ </td></tr><tr><td> Eicosane </td><td> סיגמא </td><td> 21,927-4 </td><td></td></tr><tr><td> מיקרוסקופ </td><td> Zeiss </td><td> Axioexaminer D1 </td><td></td></tr><tr><td> מצלמת CCD </td><td> Visitron </td><td> CoolSnap HQ </td><td></td></tr><tr><td> Metafluor </td><td> Visitron </td><td></td><td> רכישת התוכנה </td></tr><tr><td> Monochromator </td><td> Visitron </td><td> Visichrome </td&gt; <td></td></tr><tr><td> שביר להבי </td><td> מדע כלים עדינים </td><td> 10050-00 </td><td></td></tr><tr><td> מחזיק את הלהב </td><td> WPI </td><td> 14134 </td><td></td></tr><tr><td> ספיר להב </td><td> WPI </td><td> 500314 </td><td> פעמיים שולי להב 1mm </td></tr><tr><td> גז משאבות ממברנה </td><td> KNF נויברגר AG </td><td> NMP 830 KVE </td><td></td></tr><tr><td> ספיר בעל להב </td><td> WPI </td><td> 500317 </td><td></td></tr><tr><td> שלוש דרך שסתום </td><td> לי החברה </td><td> LFAA1200118H </td><td> תצורת ה </td></tr><tr><td> Rotameter 500 מ&quot;ל / דקה </td><td> Analyt-MTC </td><td> 112-08SA </td><td></td></tr><tr><td> Rotameter 5 ליטר / דקה </td><td> Analyt-MTC </td><td> 102-08SA </td><td></td></tr><tr><td> דונג </td><td> Siladent Technik </td><td> 209212 </td><td>; </td></tr><tr><td> תאית כרית </td><td> Kettenbach רפואי </td><td> 31003 </td><td></td></tr><tr><td> מלקטת </td><td> Plano-EM </td><td> T5130 </td><td> דומון הביולוגיה # 5 </td></tr><tr><td> מזרקים </td><td> BD </td><td> 300928 </td><td> 2ml, Discardit השנייה, 2pieces </td></tr><tr><td> מחטים </td><td> בראון Sterican </td><td> 4665120 </td><td> OD 1.2 מ&quot;מ </td></tr></tbody></table>

calcium imaging of odor-evoked responses in the drosophila antennal lobe

האונה מחושים היא מרכז ההרחה במוח העיקרי חרקים מייצג המקבילה אנטומיים ותפקודיים של הנורה חוש הריח חוליות 1-5. מידע חוש הריח בעולם החיצוני מועבר באונה מחושים על ידי עצב סנסורי (חוש הריח OSNs), אשר לבודד לאזורים שונים של neuropil בשם glomeruli על פי חוש הריח לקולטן ספציפי שהם מבטאים. כאן, OSN אקסונים סינפסה עם שני interneurons המקומיות (LNs), אשר יכול innervate glomeruli תהליכים שונים, ונוירונים הקרנה (PNS), המעבירים מידע חוש הריח כדי גבוהות יותר אזורים במוח חוש הריח. דימות אופטי של פעילות OSNs, LNs ו PNS באונה מחושים - מסורתי באמצעות אינדיקטורים סידן סינתטיים (כגון סידן ירוק, פורעה-2) או רגישים מתח צבעים (למשל RH414) - כבר זמן רב בטכניקה חשוב להבין כיצד חוש הריח הגירויים מיוצגים תבניות במרחב ובזמןפעילות הסינון של מינים רבים של חרקים 6-10. התפתחות גנטית מקודדים כתבים פעילות עצבית, כגון האינדיקטורים סידן ניאון G מחנות 11,12 ו Cameleon 13, מדד סידן bioluminescent GFP-aequorin 14,15, או כתב של הולכה סינפטית, synapto-pHluorin 16 הפכה את מערכת ההרחה של פריזבוב, תסיסנית melanogaster, נגיש במיוחד הדמיה נוירופיזיולוגי, המשלימה נכסים מקיף, המתוארים שלה מולקולרית, אלקטרו ו neuroanatomical 2,4,17. כתבים אלו יכולים לבוא לידי ביטוי באופן סלקטיבי באמצעות בינאריים מערכות בקרה תעתיק (למשל GAL4/UAS 18, לכסה / LexAop 19,20, ש מערכת 21) בקרב אוכלוסיות מוגדרות של נוירונים בתוך המעגלים חוש הריח לנתח עם רזולוציה גבוהה במרחב ובזמן איך ריח עורר פעילות עצבית מיוצג, מווסתת והפך 22-24 &lt;/ Sup&gt;. כאן אנו מתארים את שיטות הכנה וניתוח למדוד ריח עורר הדים באונה תסיסנית מחושים באמצעות G-מחנה 25-27. הכנת בעלי חיים היא פולשנית והוא יכול להיות מותאם דימות באמצעות מיקרוסקופ שדה רחב הקרינה, confocal ואת שני הפוטונים.

Watch this Scientific Journal Video about Calcium Imaging of Odor-evoked Responses in the Drosophila Antennal Lobe at JoVE.com

Calcium Imaging of Odor-evoked Responses in the Drosophila Antennal Lobe

אנו מתארים טכניקה הוקמה כדי למדוד ולנתח ריח עוררו תגובות סידן באונה מחושים חיים<em&gt; תסיסנית melanogaster</em&gt;.

סידן הדמיה של ריח עוררו התגובות תסיסנית מחושים האונה

The antennal lobe is the primary olfactory center in the insect brain and represents the anatomical and functional equivalent of the vertebrate olfactory ...

calcium-imaging-odor-evoked-responses-drosophila-antennal

Research

JoVE Journal

Neuroscience

Calcium Imaging of Odor-evoked Responses in the Drosophila Antennal Lobe

25.8K Views.  University of Lausanne. אנו מתארים טכניקה הוקמה כדי למדוד ולנתח ריח עוררו תגובות סידן באונה מחושים חיים תסיסנית melanogaster.

Video: Calcium Imaging of Odor-evoked Responses in the Drosophila Antennal Lobe

Imaging Odor-Evoked Activities in the Mouse Olfactory Bulb using Optical Reflectance and Autofluorescence Signals

In the brain, sensory stimulation activates distributed populations of neurons among functional modules which participate to the coding of the stimulus. Functional optical imaging techniques are advantageous to visualize the activation of these modules in sensory cortices with high spatial resolution. In this context, endogenous optical signals that arise from molecular mechanisms linked to neuroenergetics are valuable sources of contrast to record spatial maps of sensory stimuli over wide fields in the rodent brain.
Here, we present two techniques based on changes of endogenous optical properties of the brain tissue during activation. First the intrinsic optical signals (IOS) are produced by a local alteration in red light reflectance due to: (i) absorption by changes in blood oxygenation level and blood volume (ii) photon scattering. The use of in vivo IOS to record spatial maps started in the mid 1980's with the observation of optical maps of whisker barrels in the rat and the orientation columns in the cat visual cortex1. IOS imaging of the surface of the rodent main olfactory bulb (OB) in response to odorants was later demonstrated by Larry Katz's group2. The second approach relies on flavoprotein autofluorescence signals (FAS) due to changes in the redox state of these mitochondrial metabolic intermediates. More precisely, the technique is based on the green fluorescence due to oxidized state of flavoproteins when the tissue is excited with blue light. Although such signals were probably among the first fluorescent molecules recorded for the study of brain activity by the pioneer studies of Britton Chances and colleagues3, it was not until recently that they have been used for mapping of brain activation in vivo. FAS imaging was first applied to the somatosensory cortex in rodents in response to hindpaw stimulation by Katsuei Shibuki's group4.
The olfactory system is of central importance for the survival of the vast majority of living species because it allows efficient detection and identification of chemical substances in the environment (food, predators). The OB is the first relay of olfactory information processing in the brain. It receives afferent projections from the olfactory primary sensory neurons that detect volatile odorant molecules. Each sensory neuron expresses only one type of odorant receptor and neurons carrying the same type of receptor send their nerve processes to the same well-defined microregions of &tilde;100&mu;m3 constituted of discrete neuropil, the olfactory glomerulus (Fig. 1). In the last decade, IOS imaging has fostered the functional exploration of the OB5, 6, 7 which has become one of the most studied sensory structures. The mapping of OB activity with FAS imaging has not been performed yet.
Here, we show the successive steps of an efficient protocol for IOS and FAS imaging to map odor-evoked activities in the mouse OB.

This article presents the protocols of intrinsic optical signals and flavoproteins autofluorescence signals imaging to map odor-evoked activities at the surface of the olfactory bulb in mice.

פעילויות הדמיה ריח ועוררה הנורה חוש הריח באמצעות עכבר אופטי החזר אותות autofluorescence

In the brain, sensory stimulation activates distributed populations of neurons among functional modules which participate to the coding of the stimulus. ...

Imaging Neuronal Responses in Slice Preparations of Vomeronasal Organ Expressing a Genetically Encoded Calcium Sensor

The vomeronasal organ (VNO) detects chemosensory signals that carry information about the social, sexual and reproductive status of the individuals within the same species 1,2. These intraspecies signals, the pheromones, as well as signals from some predators 3, activate the vomeronasal sensory neurons (VSNs) with high levels of specificity and sensitivity 4. At least three distinct families of G-protein coupled receptors, V1R, V2R and FPR 5-14, are expressed in VNO neurons to mediate the detection of the chemosensory cues. To understand how pheromone information is encoded by the VNO, it is critical to analyze the response profiles of individual VSNs to various stimuli and identify the specific receptors that mediate these responses.
The neuroepithelia of VNO are enclosed in a pair of vomer bones. The semi-blind tubular structure of VNO has one open end (the vomeronasal duct) connecting to the nasal cavity. VSNs extend their dendrites to the lumen part of the VNO, where the pheromone cues are in contact with the receptors expressed at the dendritic knobs. The cell bodies of the VSNs form pseudo-stratified layers with V1R and V2R expressed in the apical and basal layers respectively 6-8. Several techniques have been utilized to monitor responses of VSNs to sensory stimuli 4,12,15-19. Among these techniques, acute slice preparation offers several advantages. First, compared to dissociated VSNs 3,17, slice preparations maintain the neurons in their native morphology and the dendrites of the cells stay relatively intact. Second, the cell bodies of the VSNs are easily accessible in coronal slice of the VNO to allow electrophysiology studies and imaging experiments as compared to whole epithelium and whole-mount preparations 12,20. Third, this method can be combined with molecular cloning techniques to allow receptor identification.
Sensory stimulation elicits strong Ca2+ influx in VSNs that is indicative of receptor activation 4,21. We thus develop transgenic mice that express G-CaMP2 in the olfactory sensory neurons, including the VSNs 15,22. The sensitivity and the genetic nature of the probe greatly facilitate Ca2+ imaging experiments. This method has eliminated the dye loading process used in previous studies 4,21. We also employ a ligand delivery system that enables application of various stimuli to the VNO slices. The combination of the two techniques allows us to monitor multiple neurons simultaneously in response to large numbers of stimuli. Finally, we have established a semi-automated analysis pipeline to assist image processing.

The vomeronasal organ (VNO) detects intraspecies chemical signals that convey social and reproductive information. We have performed Ca2+ imaging experiments using transgenic mice expressing G-CaMP2 in VNO tissue. This approach allows us to analyze the complicated response patterns of the vomeronasal neurons to large numbers of pheromone stimuli.

התגובות הדמיה העצבית ההכנות Slice של איברים Vomeronasal הבעת חיישן סידן מקודד גנטית

The vomeronasal organ (VNO) detects chemosensory signals that carry information about the social, sexual and reproductive status of the individuals within ...

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 ...

Identification of Olfactory Volatiles using Gas Chromatography-Multi-unit Recordings (GCMR) in the Insect Antennal Lobe

All organisms inhabit a world full of sensory stimuli that determine their behavioral and physiological response to their environment. Olfaction is especially important in insects, which use their olfactory systems to respond to, and discriminate amongst, complex odor stimuli. These odors elicit behaviors that mediate processes such as reproduction and habitat selection1-3. Additionally, chemical sensing by insects mediates behaviors that are highly significant for agriculture and human health, including pollination4-6, herbivory of food crops7, and transmission of disease8,9. Identification of olfactory signals and their role in insect behavior is thus important for understanding both ecological processes and human food resources and well-being.
	To date, the identification of volatiles that drive insect behavior has been difficult and often tedious. Current techniques include gas chromatography-coupled electroantennogram recording (GC-EAG), and gas chromatography-coupled single sensillum recordings (GC-SSR)10-12. These techniques proved to be vital in the identification of bioactive compounds. We have developed a method that uses gas chromatography coupled to multi-channel electrophysiological recordings (termed 'GCMR') from neurons in the antennal lobe (AL; the insect's primary olfactory center)13,14. This state-of-the-art technique allows us to probe how odor information is represented in the insect brain. Moreover, because neural responses to odors at this level of olfactory processing are highly sensitive owing to the degree of convergence of the antenna's receptor neurons into AL neurons, AL recordings will allow the detection of active constituents of natural odors efficiently and with high sensitivity. Here we describe GCMR and give an example of its use.
	Several general steps are involved in the detection of bioactive volatiles and insect response. Volatiles first need to be collected from sources of interest (in this example we use flowers from the genus Mimulus (Phyrmaceae)) and characterized as needed using standard GC-MS techniques14-16. Insects are prepared for study using minimal dissection, after which a recording electrode is inserted into the antennal lobe and multi-channel neural recording begins. Post-processing of the neural data then reveals which particular odorants cause significant neural responses by the insect nervous system.
	Although the example we present here is specific to pollination studies, GCMR can be expanded to a wide range of study organisms and volatile sources. For instance, this method can be used in the identification of odorants attracting or repelling vector insects and crop pests. Moreover, GCMR can also be used to identify attractants for beneficial insects, such as pollinators. The technique may be expanded to non-insect subjects as well.

Identification of Olfactory Volatiles using Gas Chromatography-Multi-unit Recordings GCMR in the Insect Antennal Lobe

Olfactory cues mediate many different behaviors in insects, and are often complex mixtures comprised of tens to hundreds of volatile compounds. Using gas chromatography with multi-channel recording in the insect antennal lobe, we describe a method for the identification of bioactive compounds.

זיהוי נדיפים חוש ריח באמצעות קלטות כרומטוגרפיה-Multi-יחידת גז (GCMR) באונת מחושי החרקים

All  organisms inhabit a world full of sensory stimuli that determine their  behavioral and physiological response to their environment. Olfaction is ...

Ex Vivo Calcium Imaging for Visualizing Brain Responses to Endocrine Signaling in Drosophila

Organ-to-organ communication by endocrine signaling, for example, from the periphery to the brain, is essential for maintaining homeostasis. As a model animal for endocrine research, Drosophila melanogaster, which has sophisticated genetic tools and genome information, is being increasingly used. This article describes a method for the calcium imaging of Drosophila brain explants. This method enables the detection of the direct signaling of a hormone to the brain. It is well known that many peptide hormones act through G-protein-coupled receptors (GPCRs), whose activation causes an increase in the intracellular Ca2+concentration. Neural activation also elevates intracellular Ca2+ levels, from both Ca2+ influx and the release of Ca2+ stored in the endoplasmic reticulum (ER). A calcium sensor, GCaMP, can monitor these Ca2+ changes. In this method, GCaMP is expressed in the neurons of interest, and the GCaMP-expressing larval brain is dissected and cultured ex vivo. The test peptide is then applied to the brain explant, and the fluorescent changes in GCaMP are detected using a spinning disc confocal microscope equipped with a CCD camera. Using this method, any water-soluble molecule can be tested, and various cellular events associated with neural activation can be imaged using the appropriate fluorescent indicators. Moreover, by modifying the imaging chamber, this method can be used to image other Drosophila organs or the organs of other animals.

Ex Vivo Calcium Imaging for Visualizing Brain Responses to Endocrine Signaling in Drosophila

This paper describes a protocol for ex vivo calcium imaging of the Drosophila brain. In this method, natural or synthetic compounds can be applied to the buffer to test their ability to activate particular neurons in the brain.

לשעבר Vivo הדמיית סידן להמחשת התגובות המוח איתות האנדוקרינית ב דרוזופילה

Organ-to-organ communication by endocrine signaling, for example, from the periphery to the brain, is essential for maintaining homeostasis. As a model ...

In Vivo Optical Calcium Imaging of Learning-Induced Synaptic Plasticity in Drosophila melanogaster

Decades of research in many model organisms have led to the current concept of synaptic plasticity underlying learning and memory formation. Learning-induced changes in synaptic transmission are often distributed across many neurons and levels of processing in the brain. Therefore, methods to visualize learning-dependent synaptic plasticity across neurons are needed. The fruit fly Drosophila melanogaster represents a particularly favorable model organism to study neuronal circuits underlying learning. The protocol presented here demonstrates a way in which the processes underlying the formation of associative olfactory memories, i.e., synaptic activity and their changes, can be monitored in vivo. Using the broad array of genetic tools available in Drosophila, it is possible to specifically express genetically encoded calcium indicators in determined cell populations and even single cells. By fixing a fly in place, and opening the head capsule, it is possible to visualize calcium dynamics in these cells whilst delivering olfactory stimuli. Additionally, we demonstrate a set-up in which the fly can be subjected, simultaneously, to electric shocks to the body. This provides a system in which flies can undergo classical olfactory conditioning - whereby a previously na&#239;ve odor is learned to be associated with electric shock punishment - at the same time as the representation of this odor (and other untrained odors) is observed in the brain via two-photon microscopy. Our lab has previously reported the generation of synaptically localized calcium sensors, which enables one to confine the fluorescent calcium signals to pre- or postsynaptic compartments. Two-photon microscopy provides a way to spatially resolve fine structures. We exemplify this by focusing on neurons integrating information from the mushroom body, a higher-order center of the insect brain. Overall, this protocol provides a method to examine the synaptic connections between neurons whose activity is modulated as a result of olfactory learning.

In Vivo Optical Calcium Imaging of Learning-Induced Synaptic Plasticity in Drosophila melanogaster

Here we present a protocol with which pre- and/or postsynaptic calcium can be visualized in the context of Drosophila learning and memory. In vivo calcium imaging using synaptically localized calcium sensors is combined with a classical olfactory conditioning paradigm such that the synaptic plasticity underlying this type of associative learning may be determined.

ב-Vivo סידן הדמיה אופטית של למידה הנגרמת הפלסטיות הסינפטית בדרוזופילה מלאנוסטר

Decades of research in many model organisms have led to the current concept of synaptic plasticity underlying learning and memory formation. ...

Culture of Brain Capillary Pericytes for Cytosolic Calcium Measurements and Calcium Imaging Studies

Pericytes are associated with endothelial cells and astrocytic endfeet in a structure known as the neurovascular unit (NVU). Brain capillary pericyte function is not fully known. Pericytes have been suggested to be involved in capillary development, regulation of endothelial barrier tightness and trancytosis activity, regulation of capillary tone and to play crucial roles in certain brain pathologies.
Pericytes are challenging to investigate in the intact brain due to the difficulties in visualizing processes in the brain parenchyma, as well as the close proximity to the other cells of the NVU. The present protocol describes a method for isolation and culture of primary bovine brain capillary pericytes and their following usage in calcium imaging studies, where effects of agonists involved in brain signaling and pathologies can be investigated. Cortical capillary fragments are allowed to attach to the bottom of culture flasks and, after 6 days, endothelial cells and pericytes have grown out from the capillary fragments. The endothelial cells are removed by gentle trypsinization and pericytes are cultured for 5 additional days before passaging.
Isolated pericytes are seeded in 96-well culture plates and loaded with the calcium indicator dye (Fura-2 acetoxymethyl (AM)) to allow for measurements of intracellular calcium levels in a plate reader setup. Alternatively, pericytes are seeded on coverslips and mounted in cell chambers. Following loading with the calcium indicator (Cal-520 AM), calcium live-imaging can be performed using confocal microscopy at an excitation wavelength of 488 nm and emission wavelength of 510-520 nm.
The method described here has been used to obtain the first intracellular calcium measurements from primary brain capillary pericytes, demonstrating that pericytes are stimulated via ATP and are able to contract in vitro.

Brain capillary pericytes are essential players in the regulation of blood-brain barrier properties and blood flow. This protocol describes how brain capillary pericytes can be isolated, cultured, characterized with respect to cell type and applied for investigations of intracellular calcium signaling with fluorescent probes.

תרבות של פריציטים נומי מוח למדידות סידן ציטוסולית מחקרי הדמיית סידן

Pericytes are associated with endothelial cells and astrocytic endfeet in a structure known as the neurovascular unit (NVU). Brain capillary pericyte ...

In vivo Calcium Imaging of Mouse Geniculate Ganglion Neuron Responses to Taste Stimuli

Within the last ten years, advances in genetically encoded calcium indicators (GECIs) have promoted a revolution in in vivo functional imaging. Using calcium as a proxy for neuronal activity, these techniques provide a way to monitor the responses of individual cells within large neuronal ensembles to a variety of stimuli in real time. We, and others, have applied these techniques to image the responses of individual geniculate ganglion neurons to taste stimuli applied to the tongues of live anesthetized mice. The geniculate ganglion is comprised of the cell bodies of gustatory neurons innervating the anterior tongue and palate as well as some somatosensory neurons innervating the pinna of the ear. Imaging the taste-evoked responses of individual geniculate ganglion neurons with GCaMP has provided important information about the tuning profiles of these neurons in wild-type mice as well as a way to detect peripheral taste miswiring phenotypes in genetically manipulated mice. Here we demonstrate the surgical procedure to expose the geniculate ganglion, GCaMP fluorescence image acquisition, initial steps for data analysis, and troubleshooting. This technique can be used with transgenically encoded GCaMP, or with AAV-mediated GCaMP expression, and can be modified to image particular genetic subsets of interest (i.e., Cre-mediated GCaMP expression). Overall, in vivo calcium imaging of geniculate ganglion neurons is a powerful technique for monitoring the activity of peripheral gustatory neurons and provides complementary information to more traditional whole-nerve chorda tympani recordings or taste behavior assays.

Here we present how to expose the geniculate ganglion of a live, anesthetized laboratory mouse and how to use calcium imaging to measure the responses of ensembles of these neurons to taste stimuli, allowing for multiple trials with different stimulants. This allows for in depth comparisons of which neurons respond to which tastants.

In vivo סידן הדמיה של עכבר גניציה גנגליון נוירון תגובות לגירויים טעם

Within the last ten years, advances in genetically encoded calcium indicators (GECIs) have promoted a revolution in in vivo functional imaging. Using ...

In vivo Calcium Imaging in Mouse Inferior Olive

Inferior olive (IO), a nucleus in the ventral medulla, is the only source of climbing fibers that form one of the two input pathways entering the cerebellum. IO has long been proposed to be crucial for motor control and its activity is currently considered to be at the center of many hypotheses of both motor and cognitive functions of the cerebellum. While its physiology and function have been relatively well studied on single-cell level in vitro, presently there are no reports on the organization of the IO network activity in living animals. This is largely due to the extremely challenging anatomical location of the IO, making it difficult to subject to conventional fluorescent imaging methods, where an optic path must be created through the entire brain located dorsally to the region of interest.
Here we describe an alternative method for obtaining state-of-the-art -level calcium imaging data from the IO network. The method takes advantage of the extreme ventral location of the IO and involves a surgical procedure for inserting a gradient-refractive index (GRIN) lens through the neck viscera to come into contact with the ventral surface of the calcium sensor GCaMP6s-expressing IO in anesthetized mice. A representative calcium imaging recording is shown to demonstrate the feasibility to record IO neuron activity after the surgery. While this is a non-survival surgery and the recordings must be conducted under anesthesia, it avoids damage to life-critical brainstem nuclei and allows conducting large variety of experiments investigating spatiotemporal activity patterns and input integration in the IO. This procedure with modifications could be used for recordings in other, adjacent regions of the ventral brainstem.

In vivo Calcium Imaging in Mouse Inferior Olive

We present a protocol to expose the brainstem of adult mouse from the ventral side. By using a gradient-refractive index lens with a miniature microscope, calcium imaging can be used to examine the activity of inferior olive neural somata in vivo.

אין ויוו הדמיית סידן בעכבר זית נחות

Inferior olive (IO), a nucleus in the ventral medulla, is the only source of climbing fibers that form one of the two input pathways entering the ...

הערכת פעילות הסידן בשכבת תאי גנגליון ברשתית

Calcium Imaging In Electrically Stimulated Flat-Mounted Retinas

Retinal dystrophies are a leading cause of blindness worldwide. Extensive efforts are underway to develop advanced retinal prostheses that can bypass the impaired light-sensing photoreceptor cells in the degenerated retina, aiming to partially restore vision by inducing visual percepts. One common avenue of investigation involves the design and production of implantable devices with a flexible physical structure, housing a high number of electrodes. This enables the efficient and precise generation of visual percepts. However, with each technological advancement, there arises a need for a reliable and manageable ex vivo method to verify the functionality of the device before progressing to in vivo experiments, where factors beyond the device's performance come into play. This article presents a comprehensive protocol for studying calcium activity in the retinal ganglion cell layer (GCL) following electrical stimulation. Specifically, the following steps are outlined: (1) fluorescently labeling the rat retina using genetically encoded calcium indicators, (2) capturing the fluorescence signal using an inverted fluorescence microscope while applying distinct patterns of electrical stimulation, and (3) extracting and analyzing the calcium traces from individual cells within the GCL. By following this procedure, researchers can efficiently test new stimulation protocols prior to conducting in vivo experiments.

Author Spotlight: גישה חדשנית לגירוי חשמלי עצבי באמצעות הדמיית סידן 

Retinal prostheses have the ability to generate visual perceptions. To advance the development of new prostheses, ex vivo methods are needed to test devices before implantation. This article provides a comprehensive protocol for studying calcium activity in the retinal ganglion cell layer when subjected to electrical stimulation.

הדמיית סידן ברשתית שטוחה עם גירוי חשמלי

Retinal dystrophies are a leading cause of blindness worldwide. Extensive efforts are underway to develop advanced retinal prostheses that can bypass the ...