This work is supported by a Canadian Health Research Institute (CIHR) Team Grant to Dr. Bolduc (co-PI) and Dr. Guy Rouleau (PI). We would like to thank Dr. Oksana Suchowersky, Dr. Kathryn Todd, the members of the University of Alberta fly club and Dr. Clayton Dickson for help in development of the method and statistical analysis.

La ricerca sulle Drosophila melanogaster era conforme con l&#39;Università di linee di ricerca di Alberta. 1. Fly Collection <ol><li> Raccogliere 20 mosche con CO 2 (g), anestesia e posto in una fiala di raccolta 25 millimetri x 95 millimetri contenente cibo. </li><li> Fiale magazzini contenenti mosche orizzontalmente per evitare di intrappolare le mosche in liquidi che possono accumularsi sul fondo della fiala. </li><li> Incubare mosche per almeno 21 ore a 22 ° C a 45% di umidità in un incubatore per circa per 15 h. Impostare l&#39;incubatore con una luce 12 ore: ciclo scuro. </li></ol> 2. Arrampicata Assay <ol><li> La mattina seguente, il trasferimento di 20 mosche da una singola fiala in un bicchiere da 250 ml cilindro graduato. Segnare la posizione del cilindro per mantenere costante quotidiana. Utilizzare un cilindro di vetro per il genotipo per evitare la contaminazione incrociata tra i genotipi. Lavarsi alla fine di ogni esperimento e roli tate tra genotipi. <ol><li> Condurre gli esperimenti in luce ambiente (o la luce rossa se esiste un potenziale difetto visivo) rispettivamente a temperatura e umidità di 22 ° C e 40%. Per evitare di confondere circadiano ritmo, sempre esperimenti alla stessa ora del giorno. </li></ol></li><li> Chiudere la parte superiore del cilindro con un film barriera (strato di cera) per evitare fuoriuscite di linea (Figura 2). </li><li> Impostare la videocamera su un treppiede. Fotocamera Focus sulla linea di 190 ml dei 250 ml cilindro graduato (17.5 cm). </li><li> Contare il numero di mosche morte sul fondo del cilindro e nelle fiale alimentari. Registrare questo numero come la mortalità. </li><li> Molto battere leggermente il cilindro contro un tappetino in gomma a cellule chiuse ripetutamente con forza sufficiente a spostare le mosche per la superficie inferiore interna. Toccare 5-10 volte, mentre con l&#39;altra mano per premere registrare sulla fotocamera. </li><li> Premere il pulsante &quot;Record&quot; sulla fotocamera. </li><li> Avviare il video registrazione della videocamera e toccare il cilindro sei volte in un modello non ritmico distinti. </li><li> Condurre ogni prova per 2 minuti dal momento in cui le mosche sono ultimo sfruttato e registrare il numero di mosche che attraversano l&#39;altezza di 17,5 cm (190 ml) per ogni punto temporale prescelto (quantificare ogni 10 sec). Nota: La marcatura sul cilindro ml varierà da un cilindro modello all&#39;altro a seconda del diametro. Per evitare errori, misurare l&#39;altezza di ogni cilindro utilizzato. </li><li> Una volta che il processo è terminato, disporre di mosche in etanolo al 95%. </li><li> Ripetere i passaggi 2,1-2,9 fino a quando tutte le repliche sono stati testati con mosche freschi ogni volta. Nota: Anche se 5 repliche possono essere sufficiente con una mutazione che ha un forte effetto sulla locomozione, 10 replicati biologici di 20 mosche (200 mosche) si consiglia di rilevare le differenze più piccole. </li><li> Al termine dell&#39;esperimento, lavare i cilindri in lavastoviglie laboratorio e O secco / N per essere riutilizzato. </li></ol> 3. Analisi <ol><li> Analyvideo ze di ogni volano prova. Ogni 10 secondi, registrare il numero totale di mosche che passano la linea di tiro. <ol><li> Se una mosca si arrampica indietro o scende, record che volano come -1 e contare il prossimo volo per attraversare la linea di destinazione come lo stesso numero del volo che è risalito verso il basso o è caduto. Ad esempio, se il 15 fly scende al di sotto della linea di tiro, il prossimo volo per attraversare la linea (16 th volano) è considerato il 15 ° volo e non 16 °. </li></ol></li><li> Sottrarre mortalità dal numero totale di linea (20) per ottenere il numero di mosche che rimangono nel processo. Ad ogni punto di tempo, ottenere la frazione di mosche sopra la linea di destinazione. </li><li> Tracciare ogni percentuale in ogni momento (vedi figura 3). </li><li> Analizzare le prestazioni al punto dati 120 secondi ed eseguire studente t-test quando sono presenti 2 gruppi o ANOVA e post-test per confronti multipli (con modifica Bonferroni per pianificati e non pianificati Tukey per comparisons). Il Kolmogorov-Smirnov test 17 ​​viene eseguita per l&#39;accertamento normalità e varianza uguale ma anche per confrontare le distribuzioni del gruppo mutante al controllo. </li><li> Per presentare i dati su invecchiamento, tracciare la percentuale di mosche arrampicata a 120 secondi con le mosche di età diverse (2 giorni, 1 settimana, 2 settimane) per vedere se c&#39;è un deficit progressivo (Figura 4). </li></ol>

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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+iterative+negative+geotaxis+(RING):+a+new+method+for+assessing+age-related+locomotor+decline+in+Drosophila.">Rapid iterative negative geotaxis (RING): a new method for assessing age-related locomotor decline in Drosophila.</a> Experimental gerontology. 40, 386-395 (2005).</li><li>Botella, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Drosophila+carbonyl+reductase+sniffer+prevents+oxidative+stress-induced+neurodegeneration.">The Drosophila carbonyl reductase sniffer prevents oxidative stress-induced neurodegeneration.</a> Current biology : CB. 14, 782-786 (2004).</li><li>Feany, M. B., Bender, W. 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The authors have nothing to disclose.

Drosophila ha già dimostrato di essere un ottimo modello di malattia 14 e altre malattie neurodegenerative 1,2 di Parkinson. Oltre agli strumenti genetici disponibili in Drosophila, suo genoma è altamente conservata per geni coinvolti nei disordini neurologici 19. L&#39;avvento di ampi metodi di screening genetico genoma (compresi sequenziamento dell&#39;intero esoma) è probabile che continuerà a fornire una più grande lista di geni candidati associati con disturbi...

Malattie neurodegenerative devastanti come il morbo di Parkinson, la sclerosi laterale amiotrofica, e paraplegia spastica ereditaria sono sempre più riconosciuti. Sfortunatamente, la maggior parte di questi disturbi neurodegenerativi sono ancora senza trattamenti. L&#39;uso clinico diffuso di test genetici, imparziali genoma come il sequenziamento dell&#39;esoma ha portato ad un numero sempre maggiore di geni di essere coinvolto in disturbi locomotiva umani. Nonostante questi progressi, la progressione patologica dalla mattina presto alla fase avanzata, rimane inafferrabile in questi disturbi. Drosophila fornisce uno con gli strumenti genetici per lo studio requisito gene in modo spaziale e temporale controllato. Inoltre, Drosophila è dimostrato utile nello screening farmaci per malattie neurologiche come il Parkinson 1, il morbo di Alzheimer 2, ritardo mentale ed epilessia 3,4 5,6 tra gli altri. Il nostro obiettivo era quello di sviluppare un costo effettivoe il dosaggio affidabile che permetta di alta analisi rendimento che sarebbe ancora abbastanza sensibile per rilevare piccole variazioni nelle prestazioni del motore. Ci sono diversi test utilizzati per quantificare gli effetti di mutazione genetica e / o le condizioni ambientali sul comportamento arrampicata Drosophila. La maggior parte dei saggi capitalizzare la naturale tendenza di mosche da scalare, noti come geotassi negativi, o il saggio arrampicata. Benzer 7 suggerito nel 1967 che l&#39;apparato controcorrente utilizzato per lo studio di fototassi potrebbe anche essere usato per studiare gravitaxis. Da allora, Ganetsky 8 e molti altri 9 -12 hanno costruito sul dosaggio iniziale. Il principio è quello di mettere un numero noto di mosche in una fiala e toccare la fiala fortemente contro una superficie dura, causando linea a cadere sul fondo della fiala. Dato che è un comportamento innato, linea tenteranno di salire fino alla cima del flaconcino, al contrario di gravità. Questo test è quantitativa e measures quanti mosche sono saliti oltre un marcatore sul flacone nel corso di un periodo di tempo assegnato. Misurazione della velocità invece del numero totale di mosche arrampicata è diventata un parametro affidabile e mostrato difetti nei casi in cui il numero dei criteri mosche non era significativo 13. Il dosaggio arrampicata è dimostrata utile nello studio di molte malattie neurodegenerative come il morbo di Parkinson 14. Tuttavia, abbiamo notato che i difetti locomotiva non deve essere rilevabile al momento in cui la neurodegenerazione è già visto in studi patologici 14. Pertanto, l&#39;uso del test tradizionale può limitare la capacità di studiare le prime fasi della patogenesi della malattia. La comparsa di difetti locomotiva durante le fasi successive di patologia può riflettere una malattia la cui progressione è troppo avanzato per la completa salvataggio. Ciò solleva un potenziale problema con la sensibilità del tradizionale test arrampicata. Il potenziale incapacità del tradizionale arrampicata test di rilevare difetti locomotiva lievi può essere attribuito alla quota alla quale linea devono salire. Le tradizionali misure assay 15,16 il numero di mosche a salire con successo su un&#39;altezza di 2 a 5 cm di 10 a 20 sec.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Drosophila stocks</td>
			<td></td>
			<td></td>
			<td>The stocks are selected depending on the experiments. The temperature and humidity in the room and in the incubator must be controled and consistent to avoid flies being too staticky or too wet.</td>
		</tr>
		<tr>
			<td>Video camera</td>
			<td></td>
			<td></td>
			<td>Any digital camcorder will do. Make sure they can focus on close object.</td>
		</tr>
		<tr>
			<td>Graduated cylinder</td>
			<td>Kimble</td>
			<td>20028W</td>
			<td>Different models of graduated cylinder may have different diameter. It is therefore imporant to measure the height.</td>
		</tr>
		<tr>
			<td>computer</td>
			<td></td>
			<td></td>
			<td>Any model will do. We used the computer to monitor the climbing of the flies and record the number of flies at each time point</td>
		</tr>
	</tbody>
</table>

quantitative analysis of climbing defects in a drosophila model of neurodegenerative disorders

Difetti Locomotive derivanti da malattie neurodegenerative possono essere un sintomo di esordio tardivo della malattia, dopo anni di degenerazione subclinica, e quindi attuali strategie di trattamento terapeutico non sono curativi. Attraverso l&#39;uso di sequenziamento exome, un numero crescente di geni sono stati identificati per giocare un ruolo nella locomozione umana. Nonostante l&#39;identificazione di questi geni, non è noto come questi geni sono essenziali al funzionamento normale locomotiva. Pertanto, un test affidabile, che utilizza organismi modello per chiarire il ruolo di questi geni per identificare nuovi bersagli di interesse terapeutico, è necessaria più che mai. Abbiamo progettato una versione sensibilizzata del test geotassi negativi che consente il rilevamento di difetti lievi anteriore e ha la capacità di valutare questi difetti nel tempo. Il saggio viene eseguito in un cilindro graduato in vetro, che è sigillato con un film barriera di cera. Aumentando la distanza limite ad essere scalata a 17,5 cm ed aumentando la durata dell&#39;esperimento per 2 min abbiamo osservato una maggiore sensibilità nella rilevazione disfunzioni mobilità lievi. Il test è conveniente e non richiede una formazione completa per ottenere risultati altamente riproducibili. Questo lo rende una tecnica eccellente per lo screening candidati farmaci in mutanti di Drosophila con difetti di locomozione.

L&#39;arrampicata è un comportamento forte e riproducibile. Infatti, un giorno di vita in linea wild-type di raggiungere le prestazioni arrampicata distanza del bersaglio in rapido (25 - 30 sec). Mosche mutanti presentano una serie di prestazioni da lieve (o ritardato) per completare l&#39;incapacità di salire al bersaglio. Illustriamo questo qui con due diversi alleli mutanti. Il primo è una grave allele del gene spastin causata da una delezione completa del gene spastin (terme 5,75) 18. In questa linea (...

Watch this Scientific Journal Video about Quantitative Analysis of Climbing Defects in a Drosophila Model of Neurodegenerative Disorders at JoVE.com

Quantitative Analysis of Climbing Defects in a Drosophila Model of Neurodegenerative Disorders

We present an optimized inexpensive and reliable negative geotaxis assay in Drosophila melanogaster as a model for neurodegenerative disorders. Being more sensitive to mild locomotor defects, this assay will help screen for potential genetic interactions and drug targets.

Analisi quantitativa dei difetti arrampicata in un modello Drosophila di malattie neurodegenerative

Locomotive defects resulting from neurodegenerative disorders can be a late onset symptom of disease, following years of subclinical degeneration, and ...

quantitative-analysis-climbing-defects-drosophila-model

Research

JoVE Journal

Neuroscience

18.5K Views.  University of Alberta. We present an optimized inexpensive and reliable negative geotaxis assay in Drosophila melanogaster as a model for neurodegenerative disorders. Being more sensitive to mild locomotor defects, this assay will help screen for potential genetic interactions and drug targets. 

Video: Quantitative Analysis of Climbing Defects in a Drosophila Model of Neurodegenerative Disorders

Quantitative Analysis of Synaptic Vesicle Pool Replenishment in Cultured Cerebellar Granule Neurons using FM Dyes

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis. Retrieved SVs are then refilled with neurotransmitter and rejoin the recycling pool, defined as SVs that are available for exocytosis1,2. The recycling pool can generally be subdivided into two distinct pools - the readily releasable pool (RRP) and the reserve pool (RP). As their names imply, the RRP consists of SVs that are immediately available for fusion while RP SVs are released only during intense stimulation1,2. It is important to have a reliable assay that reports the differential replenishment of these SV pools in order to understand 1) how SVs traffic after different modes of endocytosis (such as clathrin-dependent endocytosis and activity-dependent bulk endocytosis) and 2) the mechanisms controlling the mobilisation of both the RRP and RP in response to different stimuli.
FM dyes are routinely employed to quantitatively report SV turnover in central nerve terminals3-8. They have a hydrophobic hydrocarbon tail that allows reversible partitioning in the lipid bilayer, and a hydrophilic head group that blocks passage across membranes. The dyes have little fluorescence in aqueous solution, but their quantum yield increases dramatically when partitioned in membrane9. Thus FM dyes are ideal fluorescent probes for tracking actively recycling SVs. The standard protocol for use of FM dye is as follows. First they are applied to neurons and are taken up during endocytosis (Figure 1). After non-internalised dye is washed away from the plasma membrane, recycled SVs redistribute within the recycling pool. These SVs are then depleted using unloading stimuli (Figure 1). Since FM dye labelling of SVs is quantal10, the resulting fluorescence drop is proportional to the amount of vesicles released. Thus, the recycling and fusion of SVs generated from the previous round of endocytosis can be reliably quantified.
Here, we present a protocol that has been modified to obtain two additional elements of information. Firstly, sequential unloading stimuli are used to differentially unload the RRP and the RP, to allow quantification of the replenishment of specific SV pools. Secondly, each nerve terminal undergoes the protocol twice. Thus, the response of the same nerve terminal at S1 can be compared against the presence of a test substance at phase S2 (Figure 2), providing an internal control. This is important, since the extent of SV recycling across different nerve terminals is highly variable11.
Any adherent primary neuronal cultures may be used for this protocol, however the plating density, solutions and stimulation conditions are optimised for cerebellar granule neurons (CGNs)12,13.

A live fluorescence imaging technique to quantify the replenishment and mobilisation of specific synaptic vesicle (SV) pools in central nerve terminals is described. Two rounds of SV recycling are monitored in the same nerve terminals providing an internal control.

Analisi quantitativa di Replenishment pool delle vescicole sinaptiche in colture di neuroni granuli cerebellari utilizzando coloranti FM

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis.  Retrieved SVs are then refilled with ...

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Neuroscienze

Ex vivo Culturing of Whole, Developing Drosophila Brains

We describe a method for ex vivo culturing of whole Drosophila brains. This can be used as a counterpoint to chronic genetic manipulations for investigating the cell biology and development of central brain structures by allowing acute pharmacological interventions and live imaging of cellular processes. As an example of the technique, prior work from our lab1 has shown that a previously unrecognized subcellular compartment lies between the axonal and somatodendritic compartments of axons of the Drosophila central brain. The development of this compartment, referred to as the axon initial segment (AIS)2, was shown genetically to depend on the neuron-specific cyclin-dependent kinase, Cdk5. We show here that ex vivo treatment of wild-type Drosophila larval brains with the Cdk5-specific pharmacological inhibitors roscovitine and olomoucine3 causes acute changes in actin organization, and in localization of the cell-surface protein Fasciclin 2, that mimic the changes seen in mutants that lack Cdk5 activity genetically.
A second example of the ex vivo culture technique is provided for remodeling of the connections of embryonic mushroom body (MB) gamma neurons during metamorphosis from larva to adult. The mushroom body is the center of olfactory learning and memory in the fly4, and these gamma neurons prune their axonal and dendritic branches during pupal development and then re-extend branches at a later timepoint to establish the adult innervation pattern5. Pruning of these neurons of the MB has been shown to occur via local degeneration of neurite branches6, by a mechanism that is triggered by ecdysone, a steroid hormone, acting at the ecdysone receptor B17, and that is dependent on the activity of the ubiquitin-proteasome system6. Our method of ex vivo culturing can be used to interrogate further the mechanism of developmental remodeling. We found that in the ex vivo culture setting, gamma neurons of the MB recapitulated the process of developmental pruning with a time course similar to that in vivo. It was essential, however, to wait until 1.5 hours after puparium formation before explanting the tissue in order for the cells to commit irreversibly to metamorphosis; dissection of animals at the onset of pupariation led to little or no metamorphosis in culture. Thus, with appropriate modification, the ex vivo culture approach can be applied to study dynamic as well as steady state aspects of central brain biology.

Ex vivo Culturing of Whole, Developing Drosophila Brains

This article describes a method by which one can mimic in vivo development of the Drosophila mushroom body in an ex vivo culture system.

Ex coltura vivo di Whole, in via di sviluppo Brains Drosophila

We describe a method for ex vivo culturing of whole Drosophila brains. This can be used as a counterpoint to chronic genetic manipulations for ...

Assessing Neurodegenerative Phenotypes in Drosophila Dopaminergic Neurons by Climbing Assays and Whole Brain Immunostaining

Drosophila melanogaster is a valuable model organism to study aging and pathological degenerative processes in the nervous system. The advantages of the fly as an experimental system include its genetic tractability, short life span and the possibility to observe and quantitatively analyze complex behaviors. The expression of disease-linked genes in specific neuronal populations of the Drosophila brain, can be used to model human neurodegenerative diseases such as Parkinson's and Alzheimer's 5.
Dopaminergic (DA) neurons are among the most vulnerable neuronal populations in the aging human brain. In Parkinson's disease (PD), the most common neurodegenerative movement disorder, the accelerated loss of DA neurons leads to a progressive and irreversible decline in locomotor function. In addition to age and exposure to environmental toxins, loss of DA neurons is exacerbated by specific mutations in the coding or promoter regions of several genes. The identification of such PD-associated alleles provides the experimental basis for the use of Drosophila as a model to study neurodegeneration of DA neurons in vivo. For example, the expression of the PD-linked human &alpha;-synuclein gene in Drosophila DA neurons recapitulates some features of the human disease, e.g. progressive loss of DA neurons and declining locomotor function 2. Accordingly, this model has been successfully used to identify potential therapeutic targets in PD 8.
Here we describe two assays that have commonly been used to study age-dependent neurodegeneration of DA neurons in Drosophila: a climbing assay based on the startle-induced negative geotaxis response and tyrosine hydroxylase immunostaining of whole adult brain mounts to monitor the number of DA neurons at different ages. In both cases, in vivo expression of UAS transgenes specifically in DA neurons can be achieved by using a tyrosine hydroxylase (TH) promoter-Gal4 driver line 3, 10.

Assessing Neurodegenerative Phenotypes in Drosophila Dopaminergic Neurons by Climbing Assays and Whole Brain Immunostaining

Here we describe two assays that have been established to study age-dependent neurodegeneration of dopaminergic (DA) neurons in Drosophila: the climbing/startle-induced negative geotaxis assay which allows to study the functional effects of DA neurons degeneration and the tyrosine hydroxylase immunostaining which is used to identify and count DA neurons in whole brain mounts.

Valutare fenotipi neurodegenerative<em&gt; Drosophila</em&gt; Neuroni dopaminergici mediante saggi arrampicata e Whole Brain Immunostaining

Drosophila melanogaster is a valuable model organism to study aging and pathological degenerative processes in the nervous system. The advantages of 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.

Veloce micro-ionoforesi di glutammato e GABA: un utile strumento per indagare l&#39;integrazione Synaptic

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

Deriving the Time Course of Glutamate Clearance with a Deconvolution Analysis of Astrocytic Transporter Currents

The highest density of glutamate transporters in the brain is found in astrocytes. Glutamate transporters couple the movement of glutamate across the membrane with the co-transport of 3 Na+ and 1 H+ and the counter-transport of 1 K+. The stoichiometric current generated by the transport process can be monitored with whole-cell patch-clamp recordings from astrocytes. The time course of the recorded current is shaped by the time course of the glutamate concentration profile to which astrocytes are exposed, the kinetics of glutamate transporters, and the passive electrotonic properties of astrocytic membranes. Here we describe the experimental and analytical methods that can be used to record glutamate transporter currents in astrocytes and isolate the time course of glutamate clearance from all other factors that shape the waveform of astrocytic transporter currents. The methods described here can be used to estimate the lifetime of flash-uncaged and synaptically-released glutamate at astrocytic membranes in any region of the central nervous system during health and disease.

We describe an analytical method to estimate the lifetime of glutamate at astrocytic membranes from electrophysiological recordings of glutamate transporter currents in astrocytes.

Derivando La durata di Liquidazione glutammato con una analisi Deconvoluzione delle Correnti Transporter astrociti

The highest density of glutamate transporters in the brain is found in astrocytes. Glutamate transporters couple the movement of glutamate across the ...

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

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

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

Risoluzione alta analisi quantitativa Immunogold di Recettori di membrana a retina nastro Sinapsi

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

Analyzing Synaptic Modulation of Drosophila melanogaster Photoreceptors after Exposure to Prolonged Light

The nervous system has the remarkable ability to adapt and respond to various stimuli. This neural adjustment is largely achieved through plasticity at the synaptic level. The Active Zone (AZ) is the region at the presynaptic membrane that mediates neurotransmitter release and is composed of a dense collection of scaffold proteins. AZs of Drosophila melanogaster (Drosophila) photoreceptors undergo molecular remodeling after prolonged exposure to natural ambient light. Thus the level of neuronal activity can rearrange the molecular composition of the AZ and contribute to the regulation of the functional output.
Starting from the light exposure set-up preparation to the immunohistochemistry, this protocol details how to quantify the number, the spatial distribution, and the delocalization level of synaptic molecules at AZs in Drosophila photoreceptors. Using image analysis software, clusters of the GFP-fused AZ component Bruchpilot were identified for each R8 photoreceptor (R8) axon terminal. Detected Bruchpilot spots were automatically assigned to individual R8 axons. To calculate the distribution of spot frequency along the axon, we implemented a customized software plugin. Each axon&#39;s start-point and end-point were manually defined and the position of each Bruchpilot spot was projected onto the connecting line between start and end-point. Besides the number of Bruchpilot clusters, we also quantified the delocalization level of Bruchpilot-GFP within the clusters. These measurements reflect in detail the spatially resolved synaptic dynamics in a single neuron under different environmental conditions to stimuli.

Analyzing Synaptic Modulation of Drosophila melanogaster Photoreceptors after Exposure to Prolonged Light

Here we show how to quantify the number and spatial distribution of synaptic active zones in Drosophila melanogaster&#160;photoreceptors, highlighted with a genetically encoded molecular marker, and their modulation after prolonged exposure to light.

Analizzando Synaptic Modulazione di<em&gt; Drosophila melanogaster</em&gt; Fotorecettori dopo l&#39;esposizione alla luce prolungata

The nervous system has the remarkable ability to adapt and respond to various stimuli. This neural adjustment is largely achieved through plasticity at ...

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

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

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

L'uso di traccia Eyeblink condizionamento classico per valutare la disfunzione Hippocampal in un modello del ratto dei disturbi dello spettro fetale alcol

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

Assessment of Blood-brain Barrier Permeability by Intravenous Infusion of FITC-labeled Albumin in a Mouse Model of Neurodegenerative Disease

Disruption of blood-brain barrier (BBB) integrity is a common feature for different neurological and neurodegenerative diseases. Although the interplay between perturbed BBB homeostasis and the pathogenesis of brain disorders needs further investigation, the development and validation of a reliable procedure to accurately detect BBB alterations may be crucial and represent a useful tool for potentially predicting disease progression and developing targeted therapeutic strategies.
Here, we present an easy and efficient procedure for evaluating BBB leakage in a neurodegenerative condition like that occurring in a preclinical mouse model of Huntington disease, in which defects in the permeability of BBB are clearly detectable precociously in the disease. Specifically, the high molecular weight fluorescein isothiocyanate labelled (FITC)-albumin, which is able to cross the BBB only when the latter is impaired, is acutely infused into a mouse jugular vein and its distribution in the vascular or parenchymal districts is then determined by fluorescence microscopy.
Accumulation of green fluorescent-albumin in the brain parenchyma functions as an index of aberrant BBB permeability and, when quantitated by using Image J processing software, is reported as Green Fluorescence Intensity.

In this study, we present an easy and efficient procedure for evaluating the disruption of the blood-brain barrier under neurodegenerative conditions. To achieve our objective, we infused high molecular weight fluorescein isothiocyanate labelled (FITC)-albumin into mouse jugular vein and evaluated its leakage into the brain parenchyma by fluorescence microscopy.

Valutazione della permeabilità della barriera emato - encefalica dall'infusione endovenosa di albumina FITC-labeled in un modello murino di malattia di Neurodegenerative

Disruption of blood-brain barrier (BBB) integrity is a common feature for different neurological and neurodegenerative diseases. Although the interplay ...

Quantitative Analysis of Neuronal Dendritic Arborization Complexity in Drosophila

Dendrites are the branched projections of a neuron, and dendritic morphology reflects synaptic organization during the development of the nervous system. Drosophila larval neuronal dendritic arborization (da) is an ideal model for studying morphogenesis of neural dendrites and gene function in the development of nervous system. There are four classes of da neurons. Class IV is the most complex with a branching pattern that covers almost the entire area of the larval body wall. We have previously characterized the effect of silencing the Drosophila ortholog of SOX5 on class IV neuronal dendritic arborization complexity (NDAC) using four parameters: the length of dendrites, the surface area of dendrite coverage, the total number of branches, and the branching structure. This protocol presents the workflow of NDAC quantitative analysis, consisting of larval dissection, confocal microscopy, and image analysis procedures using ImageJ software. Further insight into da neuronal development and its underlying mechanisms will improve the understanding of neuronal function and provide clues about the fundamental causes of neurological and neurodevelopmental disorders.

Quantitative Analysis of Neuronal Dendritic Arborization Complexity in Drosophila

This protocol focuses on quantitative analysis of neuronal dendritic arborization complexity (NDAC) in Drosophila, which can be used for studies of dendritic morphogenesis.

Analisi quantitativa di un neurone Arborization dentritico complessità in Drosophila

Dendrites are the branched projections of a neuron, and dendritic morphology reflects synaptic organization during the development of the nervous system. ...