Gli autori desiderano ringraziare Ikiel Ptito per il suo continuo supporto tecnico. NSERC concedere a MP.

Parte 1: Pre-trattamento dei tessuti <ol><li> Tessuto deve essere ben perfusi con paraformaldeide, glutaraldeide, o formalina. Questo può essere raggiunto attraverso standard di perfusione transcardial tipicamente usato per raccogliere altri organi. Nel presente studio il soggetto era profondamente sedato con cloridrato di ketamina (10 mg / kg, im), eutanasia con una dose eccessiva di sodio pentobarbital (25 mg / kg, iv) e perfusi transcardially con 0,1 M PBS fino a completo exsanguinated.This è seguito da una soluzione di paraformaldeide al 4% in PBS per 5 min (~ 1 litro). </li></ol> Parte 2: Stereotassica blocco <ol><li> Prima di mettere la testa nel frame stereotassico le coordinate dello zero Horsley-Clarke/interaural aereo deve essere preso. Questo è il punto medio teorico tra le orecchie. Per misurare questo piano, le barre orecchio devono essere ugualmente montato l&#39;apparecchio, poi posto una lama di bisturi nel braccio manipolatore stereotassica e misurare il punto medio tra le sbarre orecchio. Questo è importante per determinare dove per bloccare il tessuto in base alle esigenze di ricerca. Una volta che questo è completato il cranio deve essere preparato per la fissazione nell&#39;apparecchio stereotassica. </li><li> Al fine di posizionare la testa nel frame stereotassico la mascella inferiore dovrà essere rimosso con Rongeurs osso e un bisturi. Inoltre la pelle, muscoli e tessuto connettivo deve essere rimosso al fine di esporre il cranio. Una volta che il tessuto connettivo viene rimosso dal cranio, esporre il cervello intaccando il calvaria (la parte tabellare dell&#39;osso occipitale e parietale, le ossa ad frontale). Nella parte presente esperimento della calvaria è tutto pronto stato rimosso. Fare attenzione a non rimuovere completamente le ossa temporali, perché il canale uditivo dovrà essere intatto per posizionare la testa nel telaio stereotassico. La questione restante durata dovrebbe essere rimosso dal cervello esposto. Il cranio è ora pronto per essere inserito nel telaio stereotassico. </li><li> Nello stesso modo in cui si effettuerebbe un intervento di chirurgia stereotassica, regolare l&#39;occhio, il palato, e bar orecchio in modo che la testa è saldamente fissato l&#39;apparato stereotassico. Posizionare il manipolatore stereotassica nelle coordinate predeterminato anteriore / posteriore (A / P) e spostare il manipolatore alla faccia laterale del cervello. Abbassare lentamente la lama nel cervello, completamente sollevare la lama dal cervello, quindi spostare medialmente e abbassare la lama di nuovo. Ripetere questi due passi fino a quando la lama ha raggiunto la superficie laterale dell&#39;emisfero opposto. Questo completa il primo blocco coronale. Per i successivi blocchi coronale spostare i 1cm manipolatore in asse A / P e ripetere fino a tutto il cervello è stato bloccato. </li></ol> Parte 3: Rimozione del cervello dal cranio <ol><li> Togliere la testa dal telaio stereotassico e tenere il cervello esposti nel palmo della vostra mano. Provate a fissare il cervello con una leggera ponendo vezzeggiare il cervello nel palmo della tua mano e il dito mignolo tra i lobi frontali, questo movimento minimizza del cervello nel cranio. Al fine di evitare l&#39;essiccamento della superficie piale del cervello, posto un pezzo di garza imbevuto PBS in tutto il cervello. Tenere la testa saldamente dal cranio e sgretolare le ossa rimanenti occipitale e temporale insieme alla colonna vertebrale. Questo espone gli aspetti di base e laterale del cervello. Infine togliere qualunque osso residuo frontale e l&#39;osso nasale, che permetterà l&#39;accesso ai bulbi olfattivi. E &#39;importante rimuovere l&#39;osso frontale ultima perché, come si rimuove la base del cranio, il cervello e si muove leggermente i lobi frontali possono essere danneggiati ai bordi frastagliati delle ossa fontale. Tagliare e rimuovere i restanti materia dura. Sollevare con cautela la parte anteriore del cervello, far scorrere il bisturi sotto il cervello e liberare il cervello dal cranio. </li></ol> Parte 4: Misure <ol><li> Ci sono una serie di misure utili che possono essere fatte prima del congelamento. Ad esempio, l&#39;asse A / P del cervello con una pinza. Inoltre la densità specifica può essere misurata dal peso del cervello, misurazione del volume dallo spostamento d&#39;acqua in un cilindro graduato poi dividendo il peso per il dislocamento del volume (Tabella 1). </li></ol> Parte 5: Fine bloccando il cervello. <ol><li> In genere quando il bisturi viene inserito nel cervello non è abbastanza a lungo per penetrare completamente l&#39;intero dorso-ventrale misura del cervello. Una volta che il cervello viene rimosso e le misurazioni sono ottenute lordo, prendere un tessuto-slicing lama e finire bloccando il cervello attraverso il lato ventrale del cervello (Figura 2). </li><li> Prima del congelamento dei blocchi, è necessario cryoprotect il tessuto in PBS graduato-buffered soluzioni di saccarosio (10, 20, e 30%) hanno mantenuto a 4 ˚ C fino a quando il cervello affonda. Ci vuole in genere una notte di incubazione a 10%, 2-3 giorni nel 20% e ulteriori 3-5 giorni nel 30% per i blocchi a depositarsi sul fondo del contenitore. Un cambio giornaliero di secolosoluzione di saccarosio e 30% è raccomandato. </li></ol> Parte 6: Risultati Rappresentante Ci sono una serie di gravi misure morfologiche che possono essere fatte una volta che il cervello è stato rimosso dal cranio. Questi includono un rapporto lunghezza / P, il peso e la densità specifica (Tabella 1). In genere bloccare il cervello in 6-7 blocchi di misura 1cm (Figura 1). Ogni pezzo viene poi fotografato (Figura 2) e può essere ulteriormente sezionato a seconda delle esigenze di ricerca o pronti per essere congelati in soluzioni di saccarosio classificato. <table border="1"><tbody><tr><td> Soggetto </td><td> A Lunghezza / P (Mm) </td><td> Peso (Grammi) </td><td> Acqua Cilindrata (Ml) </td><td> Densità specifica (G / ml) </td></tr><tr><td> O2303-2-1-1 </td><td> 64,3 </td><td> 28,1 </td><td> 24 </td><td> 1,171 </td></tr><tr><td> O5180-1 </td><td> 71,3 </td><td> 38,7 </td><td> 34 </td><td> 1,138 </td></tr><tr><td> O2708-3-1 </td><td> 62,8 </td><td> 28,7 </td><td> 26 </td><td> 1,104 </td></tr><tr><td> O9184-4-2 </td><td> 65,3 </td><td> 29,5 </td><td> 24 </td><td> 1,229 </td></tr><tr><td> N459-1-14-2 </td><td> 68,2 </td><td> 31,6 </td><td> 26 </td><td> 1,215 </td></tr><tr><td> MEDIA </td><td> 66,38 </td><td> 31,32 </td><td> 26,8 </td><td> 1,171 </td></tr><tr><td> STD DEV </td><td> 3,38 </td><td> 4,33 </td><td> 4,17 </td><td> 0,052 </td></tr></tbody></table> Tabella 1. Misure lordo morfologica della Right Hemisphere su 5 cercopitechi mesi <img src="/files/ftp_upload/1259/1259fig1.jpg" alt="figura 1" /> <img src="/files/ftp_upload/1259/1259fig2.jpg" alt="figura 1.5" /> Figura 1. Schemi per i piani coronali utilizzato per bloccare il cervello. Questo è un esempio di un cervello esteriorizzato vervet adulto. Esempio della procedura di blocco. Le linee verticali qui sono distanziati di 1 cm, di solito produce 7 blocchi coronale da ciascun emisfero. <img src="/files/ftp_upload/1259/1259fig3.jpg" alt="figura 2" /> Figura 2. Blocchi di tessuto cerebrale nello spazio stereotassico. Ciascun blocco produrrà circa 200 sezioni prese a 50 micron. Con questo schema di campionamento oltre 1200 sezioni attraverso la corteccia sarà presa e un ulteriore 400-500 dal cervelletto al taglio nel piano coronale.

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	<li>Gallagher, M., Rapp, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+animal+models+to+study+the+effects+of+aging+on+cognition.+Annu+Rev+Psychol+48.">The use of animal models to study the effects of aging on cognition. Annu Rev Psychol 48.</a> , 339-370 (1997).</li><li>Clancy, B., Darlington, R., Finlay, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translating+developmental+time+across+mammalian+species.">Translating developmental time across mammalian species.</a> Neuroscience. 105, 7-17 (2001).</li><li>Nowakowski, R. 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N., Guzman-Flores, C., Juarez, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Voluntary+consumption+of+beverage+alcohol+by+vervet+monkeys:+population+screening,+descriptive+behavior+and+biochemical+measures.">Voluntary consumption of beverage alcohol by vervet monkeys: population screening, descriptive behavior and biochemical measures.</a> Pharmacol Biochem Behav. 36, 367-373 (1990).</li><li>Palmour, R. M., Mulligan, J., Howbert, J. 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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+neural+stem+cells+migrate+along+the+nigrostriatal+pathway+in+a+primate+model+of+Parkinson's+disease.">Human neural stem cells migrate along the nigrostriatal pathway in a primate model of Parkinson's disease.</a> Exp Neurol. 211, 362-369 (2008).</li><li>Lemere, C. A., Beierschmitt, A., Iglesias, M., Spooner, E. T., Bloom, J. K., Leverone, J. F., Zheng, J. B., Seabrook, T. J., Louard, D., Li, D., Selkoe, D. J., Palmour, R. M., Ervin, F. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alzheimer's+disease+abeta+vaccine+reduces+central+nervous+system+abeta+levels+in+a+non-human+primate,+the+Caribbean+vervet.">Alzheimer's disease abeta vaccine reduces central nervous system abeta levels in a non-human primate, the Caribbean vervet.</a> Am J Pathol. 165, 283-297 (2004).</li><li>Mash, D. C., Staley, J. K., Doepel, F. M., Young, S. N., Ervin, F. R., Palmour, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altered+dopamine+transporter+densities+in+alcohol-preferring+vervet+monkeys.">Altered dopamine transporter densities in alcohol-preferring vervet monkeys.</a> Neuroreport. 7, 457-462 (1996).</li><li>Burke, M. W., Palmour, R. M., Ervin, F. 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The authors have nothing to disclose.

Il St. Kitts cercopiteco (Chlorocebus aethiops sabeus) è un primate del Vecchio Mondo, con modelli simili e tassi di sviluppo del cervello corticali e sottocorticali a quella degli esseri umani. Questa specie è stata usata per modellare complessi disturbi del comportamento umano come comportamento ansioso, ipertensione 8, emisferectomia 9, 10 morbo di Parkinson, il morbo di Alzhemier il 11 e l&#39;abuso di alcool 12. Più recentemente, questa specie è stata utilizz...

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dissecting the non-human primate brain in stereotaxic space

L&#39;utilizzo di primati non umani fornisce un modello eccellente traslazionale per la nostra comprensione dei processi di sviluppo e di invecchiamento negli esseri umani<sup&gt; 1-6</sup&gt;. Inoltre, l&#39;uso di primati non umani ha recentemente dato l&#39;opportunità di modellare naturalmente complessi disturbi psichiatrici, come l&#39;abuso di alcool<sup&gt; 7</sup&gt;. Qui si descrive una tecnica per bloccare il cervello nel piano coronale della scimmia cercopiteco (<em&gt; Chlorocebus aethiops sabeus</em&gt;) Nel cranio intatto nello spazio stereotassico. Il metodo descritto fornisce: ecco un piano standard di sezione tra blocchi e soggetti e minimizza le sezioni parziali tra i blocchi. Sezionando un blocco di tessuto sul piano coronale facilita anche la definizione di una zona di interesse. Questo metodo fornisce blocchi di dimensioni gestibili in quanto un singolo emisfero della scimmia cercopiteco produce più di 1200 parti quando taglio a 50 micron. Inoltre, bloccando il cervello in blocchi 1 cm, che facilita la penetrazione di saccarosio per cyroprotection e permette il blocco da affettare su un criostato standard.

Watch this Scientific Journal Video about Dissecting the Non-human Primate Brain in Stereotaxic Space at JoVE.com

Dissecting the Non-human Primate Brain in Stereotaxic Space

Il primate non umano è una specie importante traslazionale per la nostra comprensione della normale elaborazione del cervello. L&#39;organizzazione anatomica del cervello dei primati in grado di fornire spunti importanti in condizioni normali e patologiche nell&#39;uomo.

Dissezione di non-umano cervello dei primati nello spazio Stereotassica

The use of non-human primates provides an excellent translational model for our understanding of developmental and aging processes in humans1-6. In ...

dissecting-the-non-human-primate-brain-in-stereotaxic-space

Research

JoVE Journal

Biology

10.0K Views.  University of Montreal. Il primate non umano è una specie importante traslazionale per la nostra comprensione della normale elaborazione del cervello. L'organizzazione anatomica del cervello dei primati in grado di fornire spunti importanti in condizioni normali e patologiche nell'uomo.

Video: Dissecting the Non-human Primate Brain in Stereotaxic Space

Survivable Stereotaxic Surgery in Rodents 

The ability to measure extracellular basal levels of neurotransmitters in the brain of awake animals allows for the determination of effects of different systemic challenges (pharmacological or physiological) to the CNS. For example, one can directly measure how the animal's midbrain dopamine projections respond to dopamine-releasing drugs like d-amphetamine or natural stimuli like food. In this video, we show you how to implant guide cannulas targeting specific sites in the rat brain, how to insert and implant a microdialysis probe and how to use high performance liquid chromatography coupled with electrochemical detection (HPLC-EC) to measure extracellular levels of oxidizable neurotransmitters and metabolites. Local precise introduction of drugs through the microdialysis probe allows for refined work on site specificity in a compound s mechanism of action. This technique has excellent anatomical and chemical resolution but only modest time resolution as microdialysis samples are usually processed every 20-30 minutes to ensure detectable neurotransmitter levels. Complementary ex vivo tools (i.e., slice and cell culture electrophysiology) can assist with monitoring real-time neurotransmission.

Survivable Stereotaxic Surgery in Rodents

The monitoring of extracellular neurotransmitter levels in distinct brain regions of freely moving animals offers insights on the link between neurotransmitter release and behavior. In vivo microdialysis coupled with electrochemical detection provides excellent anatomical and chemical resolution; and information on how basal neurotransmission is altered by pharmacological or physiological manipulations.

Survivable Chirurgia Stereotassica in roditori

The ability to measure extracellular basal levels of neurotransmitters in the brain of awake animals allows for the determination of effects of different ...

Ricerca

Biologia

Dissecting and Recording from The C. Elegans Neuromuscular Junction

Neurotransmission is the process by which neurons transfer information via chemical signals to their post-synaptic targets, on a rapid time scale. This complex process requires the coordinated activity of many pre- and post-synaptic proteins to ensure appropriate synaptic connectivity, conduction of electrical signals, targeting and priming of secretory vesicles, calcium sensing, vesicle fusion, localization and function of postsynaptic receptors and finally, recycling mechanisms. As neuroscientists it is our goal to elucidate which proteins function in each of these steps and understand their mechanisms of action. Electrophysiological recordings from synapses provide a quantifiable read out of the underlying electrical events that occur during synaptic transmission. By combining this technique with the powerful array of molecular and genetic tools available to manipulate synaptic proteins in C. elegans, we can analyze the resulting functional changes in synaptic transmission. 
The C. elegans NMJs formed between motor neurons and body wall muscles control locomotion, therefore, mutants with uncoordinated locomotory phenotypes (known as unc s) often perturb synaptic transmission at these synapses 1. Since unc mutants are maintained on a rich supply of a bacterial food source, they remain viable as long as they retain some pharyngeal pumping ability to ingest food. This, together with the fact that C. elegans exist as hermaphrodites, allows them to pass on mutant progeny without the need for elaborate mating behaviors. These attributes, coupled with our recent ability to record from the worms NMJs 2,3,7 make this an excellent model organism in which to address precisely how unc mutants impact neurotransmission. 
The dissection method involves immobilizing adult worms using a cyanoacrylic glue in order to make an incision in the worm cuticle exposing the NMJs. Since C. elegans adults are only 1 mm in length the dissection is performed with the use of a dissecting microscope and requires excellent hand-eye coordination. NMJ recordings are made by whole-cell voltage clamping individual body wall muscle cells and neurotransmitter release can be evoked using a variety of stimulation protocols including electrical stimulation, light-activated channel-rhodopsin-mediated depolarization 4 and hyperosmotic saline, all of which will be briefly described.

Application of electrophysiology to accessible synapses provides a quantifiable measure of synaptic activity, useful in analyzing synaptic mutants. This article describes a dissection method used to expose the neuromuscular junctions (NMJ) of Caenorhabditis elegans (C. elegans) and briefly discusses some of the uses to which this preparation can be applied.

Dissezione e registrazione dal bivio C. Elegans neuromuscolare

Neurotransmission is the process by which neurons transfer information via chemical signals to their post-synaptic targets, on a rapid time scale.   This ...

The Gateway to the Brain: Dissecting the Primate Eye

The visual system in humans is considered the gateway to the world and plays a principal role in the plethora of sensory, perceptual and cognitive processes. It is therefore not surprising that quality of vision is tied to quality of life . Despite widespread clinical and basic research surrounding the causes of visual disorders, many forms of visual impairments, such as retinitis pigmentosa and macular degeneration, lack effective treatments. Non-human primates have the closest general features of eye development to that of humans. Not only do they have a similar vascular anatomy, but amongst other mammals, primates have the unique characteristic of having a region in the temporal retina specialized for high visual acuity, the fovea1. Here we describe a general technique for dissecting the primate retina to provide tissue for retinal histology, immunohistochemistry, laser capture microdissection, as well as light and electron microscopy. With the extended use of the non-human primate as a translational model, our hope is that improved understanding of the retina will provide insights into effective approaches towards attenuating or reversing the negative impact of visual disorders on the quality of life of affected individuals.

The non-human primate is an important translational species for our understanding of development and aging. The anatomical organization of the primate retina may provide important insights into normal and pathological conditions in humans.

La Porta del Cervello: Dissezione l&#39;Occhio Primate

The visual system in humans is considered the  gateway  to the world and plays a principal role in the plethora of sensory, perceptual and cognitive ...

Knowing What Counts: Unbiased Stereology in the Non-human Primate Brain

The non-human primate is an important translational species for understanding the normal function and disease processes of the human brain. Unbiased stereology, the method accepted as state-of-the-art for quantification of biological objects in tissue sections2, generates reliable structural data for biological features in the mammalian brain3. The key components of the approach are unbiased (systematic-random) sampling of anatomically defined structures (reference spaces), combined with quantification of cell numbers and size, fiber and capillary lengths, surface areas, regional volumes and spatial distributions of biological objects within the reference space4. Among the advantages of these stereological approaches over previous methods is the avoidance of all known sources of systematic (non-random) error arising from faulty assumptions and non-verifiable models. This study documents a biological application of computerized stereology to estimate the total neuronal population in the frontal cortex of the vervet monkey brain (Chlorocebus aethiops sabeus), with assistance from two commercially available stereology programs, BioQuant Life Sciences and Stereologer (Figure 1). In addition to contrast and comparison of results from both the BioQuant and Stereologer systems, this study provides a detailed protocol for the Stereologer system.

The anatomical organization of the primate brain can provide important insights into normal and pathological conditions in humans. Unbiased stereology is a method for accurately and efficiently estimating the total neuron number (or other cell type) in a given reference space1.

Conoscere ciò che conta: stereologia Tutti nel settore non umano cervello dei primati

The non-human primate is an important translational species for understanding the normal function and disease processes of the human brain. Unbiased ...

Trajectory Data Analyses for Pedestrian Space-time Activity Study

It is well recognized that human movement in the spatial and temporal dimensions has direct influence on disease transmission1-3. An infectious disease typically spreads via contact between infected and susceptible individuals in their overlapped activity spaces. Therefore, daily mobility-activity information can be used as an indicator to measure exposures to risk factors of infection. However, a major difficulty and thus the reason for paucity of studies of infectious disease transmission at the micro scale arise from the lack of detailed individual mobility data. Previously in transportation and tourism research detailed space-time activity data often relied on the time-space diary technique, which requires subjects to actively record their activities in time and space. This is highly demanding for the participants and collaboration from the participants greatly affects the quality of data4.
Modern technologies such as GPS and mobile communications have made possible the automatic collection of trajectory data. The data collected, however, is not ideal for modeling human space-time activities, limited by the accuracies of existing devices. There is also no readily available tool for efficient processing of the data for human behavior study. We present here a suite of methods and an integrated ArcGIS desktop-based visual interface for the pre-processing and spatiotemporal analyses of trajectory data. We provide examples of how such processing may be used to model human space-time activities, especially with error-rich pedestrian trajectory data, that could be useful in public health studies such as infectious disease transmission modeling.
The procedure presented includes pre-processing, trajectory segmentation, activity space characterization, density estimation and visualization, and a few other exploratory analysis methods. Pre-processing is the cleaning of noisy raw trajectory data. We introduce an interactive visual pre-processing interface as well as an automatic module. Trajectory segmentation5 involves the identification of indoor and outdoor parts from pre-processed space-time tracks. Again, both interactive visual segmentation and automatic segmentation are supported. Segmented space-time tracks are then analyzed to derive characteristics of one's activity space such as activity radius etc. Density estimation and visualization are used to examine large amount of trajectory data to model hot spots and interactions. We demonstrate both density surface mapping6 and density volume rendering7. We also include a couple of other exploratory data analyses (EDA) and visualizations tools, such as Google Earth animation support and connection analysis. The suite of analytical as well as visual methods presented in this paper may be applied to any trajectory data for space-time activity studies.

A suite of spatiotemporal processing methods are presented to analyze human trajectory data, such as that collected using a GPS device, for the purpose of modeling pedestrian space-time activities.

Le analisi per i dati di traiettoria pedonale Lo spazio-tempo di attività di studio

It is well recognized that human movement in the  spatial and temporal dimensions has direct influence on disease transmission1-3.  An infectious disease ...

Sampling Blood from the Lateral Tail Vein of the Rat

Blood samples are commonly obtained in many experimental contexts to measure targets of interest, including hormones, immune factors, growth factors, proteins, and glucose, yet the composition of the blood is dynamically regulated and easily perturbed. One factor that can change the blood composition is the stress response triggered by the sampling procedure, which can contribute to variability in the measures of interest. Here we describe a procedure for blood sampling from the lateral tail vein in the rat. This procedure offers significant advantages over other more commonly used techniques. It permits rapid sampling with minimal pain or invasiveness, without anesthesia or analgesia. Additionally, it can be used to obtain large volume samples (upwards of 1 ml in some rats), and it may be used repeatedly across experimental days. By minimizing the stress response and pain resulting from blood sampling, measures can more accurately reflect the true basal state of the animal, with minimal influence from the sampling procedure itself.

Blood samples are useful for assessing biomarkers of physiological states or disease in vivo. Here we describe the methodology to sample blood from the lateral tail vein in the rat. This method provides rapid samples with minimal pain and invasiveness.

Prelievo di sangue dalla vena della coda laterale del Topo

Blood samples are commonly obtained in many experimental contexts to measure targets of interest, including hormones, immune factors, growth factors, ...

Micropuncture of Bowman's Space in Mice Facilitated by 2 Photon Microscopy

Renal micropuncture and renal 2-photon imaging are seminal techniques in renal physiology. However, micropuncture is limited by dependence on conventional microscopy to surface nephron features, and 2-photon studies are limited in that interventions can only be assessed at the organ, rather than the nephron level. In particular, micropuncture studies of the glomeruli of mice have been challenged by the paucity of surface glomeruli in mice. To address this limitation in order to pursue studies of aspirate from Bowman&#39;s space in mouse physiologic models, we developed 2-photon glomerular micropuncture. We present a novel surgical preparation that allows lateral access to the kidney while preserving the required vertical imaging column for 2-photon microscopy. Administration of high molecular weight fluorescein isothiocyanate (FITC)-dextran is used to render the renal vasculature and therefore glomeruli visible for 2-photon imaging. A quantum dot-coated pipette is then introduced under stereotactic guidance to a glomerulus selected from the several to many which may be visualized within the imaging window. In this protocol, we provide details of the preparation, materials, and methods necessary to carry out the procedure. This technique facilitates previously-impossible physiologic study of the kidney, including recovery of filtrate from Bowman&#39;s space and all segments of the nephron within the imaging depth limit, about 100 &#181;m below the renal capsule. Pressure, charge and flow may all be measured using the introduced pipette. Here, we provide representative data from liquid chromatography/mass spectrometry performed on aspirate from Bowman&#39;s space. We expect this technique to have wide applicability in renal physiologic investigation.

Micropuncture of Bowman&#039;s Space in Mice Facilitated by 2 Photon Microscopy

We present use of 2-photon microscopy to place a micropipette within Bowman&#39;s urinary space in mice, combining 2 foundational techniques of renal physiology. Use of 2-photon microscopy overcomes critical limitations of conventional microscopy for micropuncture renal physiology studies.

Micropuntura dello spazio di Bowman in topi nei paraggi 2 Photon microscopia

Renal micropuncture and renal 2-photon imaging are seminal techniques in renal physiology. However, micropuncture is limited by dependence on conventional ...

Reverse Dissection and DiceCT Reveal Otherwise Hidden Data in the Evolution of the Primate Face

Facial expressions, or facial displays, of social or emotional intent are produced by many mammalian taxa as a means of visually communicating with conspecifics at a close range. These displays are achieved by contraction of the mimetic muscles, which are skeletal muscle attached to the dermis of the face. Reverse dissection, removing the full facial mask from the skull and approaching mimetic muscles in reverse, is an effective but destructive way of revealing the morphology of mimetic muscles but it is destructive. DiceCT is a novel mechanism for visualizing skeletal muscles, including mimetic muscles, and isolating individual muscle fascicles for quantitative measurement. Additionally, DiceCT provides a non-destructive mechanism for visualizing muscles. The combined techniques of reverse dissection and DiceCT can be used to assess the evolutionary morphology of mimetic musculature as well as potential contraction strength and velocity in these muscles. This study further demonstrates that DiceCT can be used to accurately and reliably visualize mimetic muscles as well as reverse dissection and provide a non-destructive method for sampling mimetic muscles.

Facial expressions are a mode of visual communication produced by mimetic muscles. Here, we present protocols for the novel techniques of reverse dissection and DiceCT to fully visualize and assess mimetic muscles. These combined techniques can examine both morphological and physiological aspects of mimetic musculature to determine functional aspects.

Invertire la dissezione e dati DiceCT Reveal altrimenti nascosti nell'evoluzione del Primate viso

Facial expressions, or facial displays, of social or emotional intent are produced by many mammalian taxa as a means of visually communicating with ...

Probing mRNA Kinetics in Space and Time in Escherichia coli using Two-Color Single-Molecule Fluorescence In Situ Hybridization

Single-molecule fluorescence in situ hybridization (smFISH) allows for counting the absolute number of mRNAs in individual cells. Here, we describe an application of smFISH to measure the rates of transcription and mRNA degradation in Escherichia coli. As smFISH is based on fixed cells, we perform smFISH at multiple time points during a time-course experiment, i.e., when cells are undergoing synchronized changes upon induction or repression of gene expression. At each time point, sub-regions of an mRNA are spectrally distinguished to probe transcription elongation and premature termination. The outcome of this protocol also allows for analyzing intracellular localization of mRNAs and heterogeneity in mRNA copy numbers among cells. Using this protocol many samples (~50) can be processed within 8 h, like the amount of time needed for just a few samples. We discuss how to apply this protocol to study the transcription and degradation kinetics of different mRNAs in bacterial cells.

Probing mRNA Kinetics in Space and Time in Escherichia coli using Two-Color Single-Molecule Fluorescence In Situ Hybridization

This protocol describes an application of single-molecule fluorescence in situ hybridization (smFISH) to measure the in vivo kinetics of mRNA synthesis and degradation.

Sondare la cinetica dell'mRNA nello spazio e nel tempo in Escherichia coli utilizzando la fluorescenza a singola molecola a due colori in ibridazione situ

Single-molecule fluorescence in situ hybridization (smFISH) allows for counting the absolute number of mRNAs in individual cells. Here, we describe an ...

Dissecting Mechanoenzymatic Properties of Processive Myosins with Ultrafast Force-Clamp Spectroscopy

Ultrafast force-clamp spectroscopy (UFFCS) is a single molecule technique based on laser tweezers that allows the investigation of the chemomechanics of both conventional and unconventional myosins under load with unprecedented time resolution. In particular, the possibility to probe myosin motors under constant force right after the actin-myosin bond formation, together with the high rate of the force feedback (200 kHz), has shown UFFCS to be a valuable tool to study the load dependence of fast dynamics such as the myosin working stroke. Moreover, UFFCS enables the study of how processive and non-processive myosin-actin interactions are influenced by the intensity and direction of the applied force.
By following this protocol, it will be possible to perform ultrafast force-clamp experiments on processive myosin-5 motors and on a variety of unconventional myosins. By some adjustments, the protocol could also be easily extended to the study of other classes of processive motors such as kinesins and dyneins. The protocol includes all the necessary steps, from the setup of the experimental apparatus to sample preparation, calibration procedures, data acquisition and analysis.

Presented here is a comprehensive protocol to perform ultrafast force-clamp experiments on processive myosin-5 motors, which could be easily extended to the study of other classes of processive motors. The protocol details all the necessary steps, from the setup of the experimental apparatus to sample preparation, data acquisition and analysis.

Dissezione delle proprietà meccanoenzimatiche delle miosine processive con spettroscopia ultraveloce a force-clamp

Ultrafast force-clamp spectroscopy (UFFCS) is a single molecule technique based on laser tweezers that allows the investigation of the chemomechanics of ...