אנו מודים לקרן פנימית מאוניברסיטת אוקלהומה (ל BZ) וכן מענק NSF (IOS-1025556, ל RAB ו BZ) לתמיכה TRF, ראב, XP, AAO, MLW, CAS, וזה מחקר. מענק NIH (RO1 NS060878, כדי BZ) נתמך בחלקו ראב. RAB מודה NRSA NIH (T32 NS07292) אימון מענק הוענק אוניברסיטת ברנדייס, וכן מענק NIH (RO1 GM21473) הוענק ג&#39;פרי הול באוניברסיטת ברנדייס, לתמיכה בשלבים המוקדמים של המחקר הזה. אנו מודים ג&#39;פרי הול עידוד מתמשך שלו עבור פרויקט זה. אנו מודים לד&quot;ר קריסטין סקוט לספק לנו באמבטיה P&gt; Gal80 זן&gt;.

<ol>
	<li>Bohm, R. A., Welch, W. P., Goodnight, L. K., Cox, L. W., Henry, L. G., Gunter, T. C., Bao, H., Zhang, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+genetic+mosaic+approach+for+neural+circuit+mapping+in+Drosophila.">A genetic mosaic approach for neural circuit mapping in Drosophila.</a> Proc Natl Acad Sci U S A. 107, 16378-16383 (2010).</li><li>Brand, A. H., Perrimon, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+gene+expression+as+a+means+of+altering+cell+fates+and+generating+dominant+phenotypes.">Targeted gene expression as a means of altering cell fates and generating dominant phenotypes.</a> Development. 118, 401-415 (1993).</li><li>Bruno, B., Holbro, N., Reichert, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polycomb+group+genes+are+required+for+neural+stem+cell+survival+in+postembryonic+neurogenesis+of+Drosophila.">Polycomb group genes are required for neural stem cell survival in postembryonic neurogenesis of Drosophila.</a> Development. 134, 1091-1099 (2007).</li><li>Duffy, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GAL4+system+in+Drosophila:+a+fly+geneticist's+Swiss+army+knife.">GAL4 system in Drosophila: a fly geneticist's Swiss army knife.</a> Genesis. , 34-341 (2002).</li><li>Gordon, M. D., Scott, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Motor+control+in+a+Drosophila+taste+circuit.">Motor control in a Drosophila taste circuit.</a> Neuron. 61, 373-384 (2009).</li><li>Lee, T., Luo, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mosaic+analysis+with+a+repressible+cell+marker+for+studies+of+gene+function+in+neuronal+morphogenesis.">Mosaic analysis with a repressible cell marker for studies of gene function in neuronal morphogenesis.</a> Neuron. 22, 451-461 (1999).</li><li>McGuire, S. E., Le, P. T., Osborn, A. J., Matsumoto, K., Davis, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatiotemporal+rescue+of+memory+dysfunction+in+Drosophila.">Spatiotemporal rescue of memory dysfunction in Drosophila.</a> Science. 302, 1765-1768 (2003).</li><li>Osterwalder, T., Yoon, K. S., White, B. H., Keshishian, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+conditional+tissue+specific+transgene+expression+system+using+inducible+GAL4.">A conditional tissue specific transgene expression system using inducible GAL4.</a> Proc Natl Acad Sci USA. 98, 12596-12601 (2001).</li><li>Pignoni, F., Zipursky, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+Drosophila+eye+development+by+decapentaplegic.">Induction of Drosophila eye development by decapentaplegic.</a> Development. 124, 271-278 (1997).</li><li>Roman, G., Endo, K., Zong, L., Davis, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=P[Switch],+a+system+for+spatial+and+temporal+control+of+gene+expression+in+Drosophila+melanogaster.">P[Switch], a system for spatial and temporal control of gene expression in Drosophila melanogaster.</a> Proc Natl Acad Sci U S A. 98, 12602-12607 (2001).</li><li>Xu, T., Rubin, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+genetic+mosaics+in+developing+and+adult+Drosophila+tissues.">Analysis of genetic mosaics in developing and adult Drosophila tissues.</a> Development. 117, 1223-1237 (1993).</li></ol>

אין ניגודי אינטרסים הכריז.

המערכת Gal4/UAS כבר בשימוש נרחב בהצלחה על ידי Drosophilists ללימודים ביולוגיים שונים (Brand &amp; Perrimon, 1993; דאפי, 2002). עד כה, הקהילה לטוס יצרה אלפי האמרגן מונע משפר-מלכודת Gal4 הקווים. הכוח העיקרי של השיטה FINGR היא שזה תואמת למערכת Gal4/UAS, מה שהופך אותו ניתן להרחיב באופן משמעותי את כוחו של Gal4 ב תסיסנית מחקרים. אחד היישומים החשובים של השיטה FINGR היא לכוונן מעגלים עצביים הבסיסית התנהגות דרך צומת Gal4/Gal80. התנהגויות אלה עשויות לכלול, אך אינם מוגבלים, למידה וזיכרון, חיזור, שינה, קצב היממה. השיטה FINGR יכול לשמש גם כדי למפות את הנוירונים קריטי נוטים ניוון מוחיים בדוגמנות מחלות ניווניות אנושיות כגון אלצהיימר, טרשת לרוחב amyotrophic (ALS), מחלות פרקינסון. כמו כן, מוקדים של נוירונים או מעגלים עצביים חיוני קוקאין מתווך, אלכוהול, התמכרות חומר אחר יכול להיות ממופה בשיטת FINGR. יצוין כי Gal4s מותנה (Osterwalder et al, 2001;.. הרומית et al, 2001) ניתן להשתמש בשיטת FINGR על מנת להשיג שליטה הזמני של מעגלים עצביים והתנהגויות. שינוי של Gal80-המרת כלים באמצעות טמפרטורה רגיש Gal80 (Gal80 ts, מקגווייר et al., 2003) יכול לייצר גיגית P&gt; לעצור&gt; Gal80 ts או באמבטיה P&gt; Gal80 ts&gt; והוסיף הגמישות הזמני נוספת מניפולציה מעגל. מעבר למערכת העצבים, שיטת FINGR ישים באותה מידה הגבלת Gal4 ביטוי תת קבוצות של תאים אם היא הזרוע או הרגל. ET-FLPx2 הקווים יהיה משאב יקר ערך עבור כל ביולוגים תסיסנית מעוניין בניתוח FRT / FLP מבוססי המשובטים (שו &amp; Rubin, 1993), כולל MARCM (Lee &amp; Luo, 1999). צפוי גם כי חלק FLPx2-ET קווי עשוי להחליף heatshock-FLP לייצור שיבוטים לשעתקו עבור ניתוחים מורפולוגיים והתנהגותיים חוזרות ונשנות. כמו משפר-מלכודת Gal4 קווים, רוב ET-FLPx2 קווי צפויים לבוא לידי ביטוי באופן ספציפי בתאי עניין אחד. רוב קווי יביע FLP בתאים של ריבית בתאים או ברקמות אחרות. עם זאת, היעדר &quot;סגוליות רקמה&quot; של שניהם Gal4 ו ET-FLPx2 לא תהיה הדאגה העיקרית לשיטת FINGR עוד באזור שלהם חופפים רצוי ניסוי מסוים. FINGR נועד להפוך את שתי רחבה &#39;מערכות לאחת מעודן. שיטות מאויר בפרוטוקולים אלה הם סטנדרטיים למדי, צריך להיות לשחזור. השלבים העיקריים אזהרה המחייבים תשומת לב של אדם הם הבחירה והשימוש מקבע. Fixatives אחר מאשר PFA יש להימנע כי הם להרוות את אות ה-GFP. רשמנו לפנינו גם את המנה לנתיחה לא אמור לשמש עבור קיבעון עם PFA, כדי למנוע את האפשרות כי הביטוי FLP לא יהיה דיווח מהימן על ידי ה-GFP.

mapping and application of enhancer-trap flippase expression in larval and adult drosophila cns

השיטה Gal4 / כטב&quot;מ בינארית היא חזקה עבור גנים מניפולציה המעגלים העצביים ב תסיסנית. עבור מחקרים הנוירוביולוגי ביותר, לעומת זאת, Gal4 ביטוי רק לעתים נדירות רקמות ספציפיות מספיק כדי לאפשר התאמה מדויקת של המעגל עם readouts התנהגותיות. כדי להתגבר על משוכה גדולה, אנחנו לאחרונה פיתח את שיטת FINGR להשיג מגבילים יותר Gal4 ביטוי ברקמות של עניין. השיטה FINGR יש שלושה מרכיבים: 1) מערכת Gal4/UAS המסורתית; 2) קבוצה של FLP / FRT בתיווך Gal80 כלים להמרת: 3), משפר מלכודת FLP (ET-FLP). Gal4 משמש כדי להגדיר את המעגלים העצביים העיקריים של עניין. קילוף Gal4 עם כטב&quot;מ, מפעיל, כגון כטב&quot;מ-MJD78Q או כטב&quot;מ ש.י. ts, מווסת את הפעילות העצבית, אשר בתורו לידי ביטוי על ידי שינויים בהתנהגות לעוף. עם תוספת כטב&quot;מ-הכתב כמו כטב&quot;מ-GFP, המעגל העצבי מעורב בהתנהגות ספציפית ניתן למפות במקביל לניתוח מורפולוגי. עבור Gal4 שורות עם ביטוי רחב, Gal4 הביטוי ניתן להגביל על ידי שימוש בשני משלים Gal80-המרת כלים: גיגית P&gt; Gal80&gt; (&quot;להעיף החוצה&quot;) ו גיגית P&gt; לעצור&gt; Gal80 (&quot;להעיף ב &#39;). לבסוף, החוקרים יכולים לפנות Gal80 או לבטל, בהתאמה, עם עזרה של רקמות ספציפיות ET-FLP. ב להעיף במצב, Gal80 יהיה להדחיק Gal4 ביטוי בכל מקום Gal4 ו ET-FLP מצטלבים. במצב להעיף החוצה, Gal80 ישחרר Gal4 דיכוי התאים בהם חפיפה Gal4 ו FLP. שתי הגישות לאפשר הגבלה של מספר תאים המעגלים Gal4 מוגדרים, אלא דפוס הפוך. השיטה FINGR תואם את האוסף העצום של Gal4 קווי בקהילה לעוף תכליתי מאוד לניתוח המשובטים המסורתי למיפוי מעגלים עצביים. בפרוטוקול זה, אנו מדגימים את מיפוי דפוסי FLP ביטוי לבחור ET-FLPx2 קווים את האפקטיביות של השיטה FINGR בתאים photoreceptor. העיקרון של השיטה FINGR צריך גם לחול על אורגניזמים אחרים מודל גנטי בו Gal4/UAS, Gal80, FLP ו / FRT משמשים.

Watch this Scientific Journal Video about Mapping and Application of Enhancer-trap Flippase Expression in Larval and Adult Drosophila CNS at JoVE.com

Mapping and Application of Enhancer-trap Flippase Expression in Larval and Adult Drosophila CNS

אנו מתארים Flippase-Induced intersectional Gal80/Gal4 (FINGR) שיטת הדיכוי, המאפשר רקמות ספציפיות FLP לקבוע Gal80 דפוסי הביטוי. בכל מקום Gal4 חפיפה FLP, Gal4 הביטוי מופעלת (Gal80 התהפך החוצה) או לבטל (Gal80 התהפך ב). השיטה FINGR הוא תכליתי לניתוח המשובטים ומיפוי במעגל העצבי.

מיפוי יישומים הביטוי Flippase Enhancer-מלכודת הזחל וגם למבוגרים תסיסנית CNS

The Gal4/ UAS binary method is powerful for gene and neural circuitry manipulation in Drosophila. For most neurobiological studies, however, Gal4 ...

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Neuroscience

Mapping and Application of Enhancer-trap Flippase Expression in Larval and Adult Drosophila CNS

16.4K Views.  University of Oklahoma - Norman. אנו מתארים Flippase-Induced intersectional Gal80/Gal4 (FINGR) שיטת הדיכוי, המאפשר רקמות ספציפיות FLP לקבוע Gal80 דפוסי הביטוי. בכל מקום Gal4 חפיפה FLP, Gal4 הביטוי מופעלת (Gal80 התהפך החוצה) או לבטל (Gal80 התהפך ב). השיטה FINGR הוא תכליתי לניתוח המשובטים ומיפוי במעגל העצבי.

Video: Mapping and Application of Enhancer-trap Flippase Expression in Larval and Adult Drosophila CNS

Visualization of Larval Segmental Nerves in 3rd Instar Drosophila Larval Preparations

Drosophila melanogaster is emerging as a powerful model system for studying the development and function of the nervous system, particularly because of its convenient genetics and fully sequenced genome. Additionally, the larval nervous system is an ideal model system to study mechanisms of axonal transport as the larval segmental nerves contain bundles of axons with their cell bodies located within the brain and their nerve terminals ending along the length of the body. Here we describe the procedure for visualization of synaptic vesicle proteins within larval segmental nerves. If done correctly, all components of the nervous system, along with associated tissues such as muscles and NMJs, remain intact, undamaged, and ready to be visualized. 3rd instar larvae carrying various mutations are dissected, fixed, incubated with synaptic vesicle antibodies, visualized and compared to wild type larvae. This procedure can be adapted for several different synaptic or neuronal antibodies and changes in the distribution of a variety of proteins can be easily observed within larval segmental nerves.

Visualization of Larval Segmental Nerves in 3rd Instar Drosophila Larval Preparations

Drosophila melanogaster larvae provide an ideal model system to investigate the mechanisms of axonal transport within larval segmental nerves. Using this procedure, 3rd instar larvae carrying various mutations can be compared to wild type larvae.

ויזואליזציה של עצבים המגזרי הזחל ב 3 Rd Instar תסיסנית הכנות הזחל

Drosophila melanogaster is emerging as a powerful model system for studying the development and function of the nervous system, particularly because of ...

Targeting Olfactory Bulb Neurons Using Combined In Vivo Electroporation and Gal4-Based Enhancer Trap Zebrafish Lines

In vivo electroporation is a powerful method for delivering DNA expression plasmids, RNAi reagents, and morpholino anti-sense oligonucleotides to specific regions of developing embryos, including those of C. elegans, chick, Xenopus, zebrafish, and mouse 1. In zebrafish, in vivo electroporation has been shown to have excellent spatial and temporal resolution for the delivery of these reagents 2-7. The temporal resolution of this method is important because it allows for incorporation of these reagents at specific stages in development. Furthermore, because expression from electroporated vectors occurs within 6 hours 7, this method is more timely than transgenic approaches. While the spatial resolution can be extremely precise when targeting a single cell 2, 6, it is often preferable to incorporate reagents into a specific cell population within a tissue or structure. When targeting multiple cells, in vivo electroporation is efficient for delivery to a specific region of the embryo; however, particularly within the developing nervous system, it is difficult to target specific cell types solely through spatially discrete electroporation. Alternatively, enhancer trap transgenic lines offer excellent cell type-specific expression of transgenes 8. Here we describe an approach that combines transgenic Gal4-based enhancer trap lines 8 with spatially discrete in vivo electroporation 7, 9 to specifically target developing neurons of the zebrafish olfactory bulb. The Et(zic4:Gal4TA4,UAS:mCherry)hzm5 (formerly GA80_9) enhancer trap line previously described 8, displays targeted transgenic expression of mCherry mediated by a zebrafish optimized Gal4 (KalTA4) transcriptional activator in multiple regions of the developing brain including hindbrain, cerebellum, forebrain, and the olfactory bulb. To target GFP expression specifically to the olfactory bulb, a plasmid with the coding sequence of GFP under control of multiple Gal4 binding sites (UAS) was electroporated into the anterior end of the forebrain at 24-28 hours post-fertilization (hpf). Although this method incorporates plasmid DNA into multiple regions of the forebrain, GFP expression is only induced in cells transgenically expressing the KalTA4 transcription factor. Thus, by using the GA080_9 transgenic line, this approach led to GFP expression exclusively in the developing olfactory bulb. GFP expressing cells targeted through this approach showed typical axonal projections, as previously described for mitral cells of the olfactory bulb 10. This method could also be used for targeted delivery of other reagents including short-hairpin RNA interference expression plasmids, which would provide a method for spatially and temporally discrete loss-of-function analysis.

Targeting Olfactory Bulb Neurons Using Combined In Vivo Electroporation and Gal4-Based Enhancer Trap Zebrafish Lines

The temporal and spatial resolution of genetic manipulations determines the spectrum of biological phenomena that they can perturb. Here we use temporally and spatially discrete in vivo electroporation, combined with transgenic lines of zebrafish, to induce expression of a GFP transgene specifically in neurons of the developing olfactory bulb.

מיקוד Bulb נוירונים חוש הריח שימוש משולב In vivo Electroporation ו Gal4 מבוסס Enhancer מלכודת קווי דג הזברה

In vivo electroporation is a powerful method for delivering DNA expression plasmids, RNAi reagents, and morpholino anti-sense oligonucleotides to specific ...

Dissection and Immunohistochemistry of Larval, Pupal and Adult Drosophila Retinas

The compound eye of Drosophila melanogaster consists of about 750 ommatidia (unit eyes). Each ommatidium is composed of about 20 cells, including lens-secreting cone cells, pigment cells, a bristle cell and eight photoreceptors (PRs) R1-R8 2. The PRs have specialized microvillar structures, the rhabdomeres, which contain light-sensitive pigments, the Rhodopsins (Rhs). The rhabdomeres of six PRs (R1-R6) form a trapezoid and contain Rh1 3 4. The rhabdomeres of R7 and R8 are positioned in tandem in the center of the trapezoid and share the same path of light. R7 and R8 PRs stochastically express different combinations of Rhs in two main subtypes5: In the 'p' subtype, Rh3 in pR7s is coupled with Rh5 in pR8s, whereas in the 'y' subtype, Rh4 in yR7s is associated with Rh6 in yR8s 6 7 8.
Early specification of PRs and development of ommatidia begins in the larval eye-antennal imaginal disc, a monolayer of epithelial cells. A wave of differentiation sweeps across the disc9 and initiates the assembly of undifferentiated cells into ommatidia10-11. The 'founder cell' R8 is specified first and recruits R1-6 and then R7 12-14. Subsequently, during pupal development, PR differentiation leads to extensive morphological changes 15, including rhabdomere formation, synaptogenesis and eventually rh expression.
In this protocol, we describe methods for retinal dissections and immunohistochemistry at three defined periods of retina development, which can be applied to address a variety of questions concerning retinal formation and developmental pathways. Here, we use these methods to visualize the stepwise PR differentiation at the single-cell level in whole mount larval, midpupal and adult retinas (Figure 1).

Dissection and Immunohistochemistry of Larval, Pupal and Adult Drosophila Retinas

The Drosophila retina is a crystal-like lattice composed of a small number of cell types that are generated in a stereotyped manner 1. Its amenability to sophisticated genetic analysis allows the study of complex developmental programs. This protocol describes dissections and immunohistochemistry of retinas at three discrete developmental stages, with a focus on photoreceptor differentiation.

נתיחה ואימונוהיסטוכימיה של זחל, גלמים ומבוגרים<em&gt; דרוזופילה</em&gt; רשתיות

The compound eye of Drosophila melanogaster consists of about 750 ommatidia (unit eyes). Each ommatidium is composed of about 20 cells, including ...

Drosophila Adult Olfactory Shock Learning

Drosophila have been used in classical conditioning experiments for over 40 years, thus greatly facilitating our understanding of memory, including the elucidation of the molecular mechanisms involved in cognitive diseases1-7. Learning and memory can be assayed in larvae to study the effect of neurodevelopmental genes8-10 and in flies to measure the contribution of adult plasticity genes1-7. Furthermore, the short lifespan of Drosophila facilitates the analysis of genes mediating age-related memory impairment5,11-13. The availability of many inducible promoters that subdivide the Drosophila nervous system makes it possible to determine when and where a gene of interest is required for normal memory as well as relay of different aspects of the reinforcement signal3,4,14,16.
Studying memory in adult Drosophila allows for a detailed analysis of the behavior and circuitry involved and a measurement of long-term memory15-17. The length of the adult stage accommodates longer-term genetic, behavioral, dietary and pharmacological manipulations of memory, in addition to determining the effect of aging and neurodegenerative disease on memory3-6,11-13,15-21.
Classical conditioning is induced by the simultaneous presentation of a neutral odor cue (conditioned stimulus, CS+) and a reinforcement stimulus, e.g., an electric shock or sucrose, (unconditioned stimulus, US), that become associated with one another by the animal1,16. A second conditioned stimulus (CS-) is subsequently presented without the US. During the testing phase, Drosophila are simultaneously presented with CS+ and CS- odors. After the Drosophila are provided time to choose between the odors, the distribution of the animals is recorded. This procedure allows associative aversive or appetitive conditioning to be reliably measured without a bias introduced by the innate preference for either of the conditioned stimuli. Various control experiments are also performed to test whether all genotypes respond normally to odor and reinforcement alone.

Drosophila Adult Olfactory Shock Learning

The method to measure adult Drosophila associative memory is described. The assay is based on the ability of the fly to associate an odor presented with a negative reinforcer (electric shock) and then recall this information at a later time, allowing memory to be measured.

דרוזופילה מבוגרים חוש הריח הלם למידה

Drosophila have been used in classical conditioning experiments for over 40 years, thus greatly facilitating our understanding of memory, including the ...

Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of the dendritic tree, which eventually leads to neuronal output of action potentials at the axon. The influence of diverse spatial and temporal parameters of specific synaptic input on neuronal output is currently under investigation, e.g. the distance-dependent attenuation of dendritic inputs, the location-dependent interaction of spatially segregated inputs, the influence of GABAergig inhibition on excitatory integration, linear and non-linear integration modes, and many more.
With fast micro-iontophoresis of glutamate and GABA it is possible to precisely investigate the spatial and temporal integration of glutamatergic excitation and GABAergic inhibition. Critical technical requirements are either a triggered fluorescent lamp, light-emitting diode (LED), or a two-photon scanning microscope to visualize dendritic branches without introducing significant photo-damage of the tissue. Furthermore, it is very important to have a micro-iontophoresis amplifier that allows for fast capacitance compensation of high resistance pipettes. Another crucial point is that no transmitter is involuntarily released by the pipette during the experiment.
Once established, this technique will give reliable and reproducible signals with a high neurotransmitter and location specificity. Compared to glutamate and GABA uncaging, fast iontophoresis allows using both transmitters at the same time but at very distant locations without limitation to the field of view. There are also advantages compared to focal electrical stimulation of axons: with micro-iontophoresis the location of the input site is definitely known and it is sure that only the neurotransmitter of interest is released. However it has to be considered that with micro-iontophoresis only the postsynapse is activated and presynaptic aspects of neurotransmitter release are not resolved. In this article we demonstrate how to set up micro-iontophoresis in brain slice experiments.

In this article we introduce fast micro-iontophoresis of neurotransmitters as a technique to investigate integration of postsynaptic signals with high spatial and temporal precision.

מיקרו-Iontophoresis מהיר של גלוטמט וגאבא: כלי שימושי לחקירת האינטגרציה סינפטית

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of ...

Functional Imaging of Auditory Cortex in Adult Cats using High-field fMRI

Current knowledge of sensory processing in the mammalian auditory system is mainly derived from electrophysiological studies in a variety of animal models, including monkeys, ferrets, bats, rodents, and cats. In order to draw suitable parallels between human and animal models of auditory function, it is important to establish a bridge between human functional imaging studies and animal electrophysiological studies. Functional magnetic resonance imaging (fMRI) is an established, minimally invasive method of measuring broad patterns of hemodynamic activity across different regions of the cerebral cortex. This technique is widely used to probe sensory function in the human brain, is a useful tool in linking studies of auditory processing in both humans and animals and has been successfully used to investigate auditory function in monkeys and rodents. The following protocol describes an experimental procedure for investigating auditory function in anesthetized adult cats by measuring stimulus-evoked hemodynamic changes in auditory cortex using fMRI. This method facilitates comparison of the hemodynamic responses across different models of auditory function thus leading to a better understanding of species-independent features of the mammalian auditory cortex.

Functional studies of the auditory system in mammals have traditionally been conducted using spatially-focused techniques such as electrophysiological recordings. The following protocol describes a method of visualizing large-scale patterns of evoked hemodynamic activity in the cat auditory cortex using functional magnetic resonance imaging.

הדמיה תפקודית של השמיעתי Cortex בחתולים בוגרים באמצעות גבוהה שדה fMRI

Current knowledge of sensory processing in the mammalian auditory system is mainly derived from electrophysiological studies in a variety of animal ...

Functional Analysis of the Larval Feeding Circuit in Drosophila

The serotonergic feeding circuit in Drosophila melanogaster larvae can be used to investigate neuronal substrates of critical importance during the development of the circuit. Using the functional output of the circuit, feeding, changes in the neuronal architecture of the stomatogastric system can be visualized. Feeding behavior can be recorded by observing the rate of retraction of the mouth hooks, which receive innervation from the brain. Locomotor behavior is used as a physiological control for feeding, since larvae use their mouth hooks to traverse across an agar substrate. Changes in feeding behavior can be correlated with the axonal architecture of the neurites innervating the gut. Using immunohistochemistry it is possible to visualize and quantitate these changes. Improper handling of the larvae during behavior paradigms can alter data as they are very sensitive to manipulations. Proper imaging of the neurite architecture innervating the gut is critical for precise quantitation of number and size of varicosities as well as the extent of branch nodes. Analysis of most circuits allow only for visualization of neurite architecture or behavioral effects; however, this model allows one to correlate the functional output of the circuit with the impairments in neuronal architecture.

Functional Analysis of the Larval Feeding Circuit in Drosophila

The feeding circuit in Drosophila melanogaster larvae serves a simple yet powerful model that allows changes in feeding rate to be correlated with alterations in the stomatogastric neural circuitry. This circuit is composed of central serotonergic neurons that send projections to the mouth hooks as well as the foregut.

ניתוח פונקציונלי של מעגל האכלת הזחל ב דרוזופילה</i

The serotonergic  feeding circuit in Drosophila  melanogaster larvae can be used to investigate neuronal substrates of  critical importance during the ...

Live Imaging of Drosophila Larval Neuroblasts

Stem cells divide asymmetrically to generate two progeny cells with unequal fate potential: a self-renewing stem cell and a differentiating cell. Given their relevance to development and disease, understanding the mechanisms that govern asymmetric stem cell division has been a robust area of study. Because they are genetically tractable and undergo successive rounds of cell division about once every hour, the stem cells of the Drosophila central nervous system, or neuroblasts, are indispensable models for the study of stem cell division. About 100 neural stem cells are located near the surface of each of the two larval brain lobes, making this model system particularly useful for live imaging microscopy studies. In this work, we review several approaches widely used to visualize stem cell divisions, and we address the relative advantages and disadvantages of those techniques that employ dissociated versus intact brain tissues. We also detail our simplified protocol used to explant whole brains from third instar larvae for live cell imaging and fixed analysis applications.

Live Imaging of Drosophila Larval Neuroblasts

This protocol details a streamlined method used to conduct live cell imaging in the context of an intact larval brain. Live cell imaging approaches are invaluable for the study of asymmetric neural stem cell divisions as well as other neurogenic and developmental processes, consistently uncovering mechanisms that were previously overlooked.

הדמיה חיה של<em&gt; דרוזופילה</em&gt; הזחל Neuroblasts

Stem cells divide asymmetrically to generate two progeny cells with unequal fate potential: a self-renewing stem cell and a differentiating cell. Given ...

Acquisition of High-Quality Digital Video of Drosophila Larval and Adult Behaviors from a Lateral Perspective

Drosophila melanogaster is a powerful experimental model system for studying the function of the nervous system. Gene mutations that cause dysfunction of the nervous system often produce viable larvae and adults that have locomotion defective phenotypes that are difficult to adequately describe with text or completely represent with a single photographic image. Current modes of scientific publishing, however, support the submission of digital video media as supplemental material to accompany a manuscript. Here we describe a simple and widely accessible microscopy technique for acquiring high-quality digital video of both Drosophila larval and adult phenotypes from a lateral perspective. Video of larval and adult locomotion from a side-view is advantageous because it allows the observation and analysis of subtle distinctions and variations in aberrant locomotive behaviors. We have successfully used the technique to visualize and quantify aberrant crawling behaviors in third instar larvae, in addition to adult mutant phenotypes and behaviors including grooming.

Acquisition of High-Quality Digital Video of Drosophila Larval and Adult Behaviors from a Lateral Perspective

Here we describe a simple and widely accessible microscopy technique to acquire high-quality digital video of Drosophila adult and larval mutant phenotypes from a lateral perspective.

רכישת באיכות גבוהה וידאו דיגיטלי של<em&gt; דרוזופילה</em&gt; Behaviors הזחל ומבוגרים מנקודת מבט רוחבי

Drosophila melanogaster is a powerful experimental model system for studying the function of the nervous system. Gene mutations that cause dysfunction of ...

Using Linear Agarose Channels to Study Drosophila Larval Crawling Behavior

Drosophila larval crawling is emerging as a powerful model to study neural control of sensorimotor behavior. However, larval crawling behavior on flat open surfaces is complex,&#160;including: pausing, turning, and meandering. This complexity in the repertoire of movement hinders detailed analysis of the events occurring during a single crawl stride cycle. To overcome this obstacle, linear agarose channels were made that constrain larval behavior to straight, sustained, rhythmic crawling. In principle, because agarose channels and the Drosophila larval body are both optically clear, the movement of larval structures labeled by genetically-encoded fluorescent probes can be monitored in intact, freely-moving larvae. In the past, larvae were placed in linear channels and crawling at the level of whole organism, segment, and muscle were analyzed1. In the future, larvae crawling in channels can be used for calcium imaging to monitor neuronal activity. Moreover, these methods can be used with larvae of any genotype and with any researcher-designed channel. Thus the protocol presented below is widely applicable for studies using the Drosophila larva as a model to understand motor control.

Using Linear Agarose Channels to Study Drosophila Larval Crawling Behavior

The Drosophila larva is a powerful model system to study neural control of behavior. This publication describes the use of linear agarose channels to elicit sustained bouts of linear crawling and methods to quantify the dynamics of larval structures during repetitive crawling behavior.

שימוש בערוצי Agarose לינארית ללמוד<em&gt; תסיסנית</em&gt; התנהגות זחל זחילה

Drosophila larval crawling is emerging as a powerful model to study neural control of sensorimotor behavior. However, larval crawling behavior on flat ...