Gli autori desiderano ringraziare Qiuli Wang, Language Resource Center, Colby College, per l&#39;assistenza tecnica con l&#39;elaborazione video e Eric Thomas, dipartimento di musica, Colby College, per fornire la musica di sottofondo. Questo progetto è stato sostenuto da sovvenzioni dal Centro Nazionale per la Ricerca Risorse, INBRE (P20RR016463-12), l&#39;Istituto Nazionale per la General Medical Sciences (P20 GM103423-12), i cittadini Institutes of Health and Science Division di Grant, Colby College (STA). JL e LWM sono stati supportati da sovvenzioni dal Fondo Estate Scholar, Colby College.

1 Drosophila Startle indotta Assay Locomotion <ol><li> Trattamento di droga <ol><li> Sedare immobilizzare il numero desiderato (circa 8-12) di 1-3 giorno-vecchio maschio vola con CO 2 e trasportarli a fiale contenenti il cibo-farmaco integrato. Nota: Un altro anestetico ad esempio, etere o ghiaccio possono essere usati per sedare le mosche per consentire il conteggio e la manipolazione. </li><li> Consenti mosche per recuperare da sedazione per 20 minuti (o fino al recupero) con la fiala in posizione orizzontale (per evitare di rimanere bloccati mosche sul cibo) e quindi posizionare il flacone in posizione verticale in un buio 12 ore, 12 ore di luce incubatore a 25 ° C per il resto dell&#39;esperimento. </li></ol></li><li> Sperimentale Set Up <ol><li> Dividere questa doppia configurazione fiala in tre sezioni uguali di 6,33 centimetri contrassegnando cerchi intorno le fiale con un pennarello indelebile. </li><li> Dopo 3 giorni di esposizione al farmaco, trasferimento vola senza anestetizzante nel flaconcino inferiore e posizionare rapidamente il flaconcino all&#39;iniziosopra l&#39;apertura. Tape le due fiale con nastro adesivo trasparente. </li><li> Consenti mosche per acclimatarsi al nuovo ambiente per 15 min. </li><li> Luogo fiale su uno sfondo bianco e impostare una fotocamera digitale ad una distanza appropriata dal doppio apparato flaconcino con un timer in vista. Assicurarsi che l&#39;intero apparato è visibile in una sola cornice e che tutte le mosche sono a fuoco. Per mantenere cornici coerenti tra prove, segnare la posizione della telecamera e il flacone. </li></ol></li><li> Mobilità Assay <ol><li> Chiaramente visualizzare il numero di prova, trattamento farmacologico, e timer in vista della telecamera. </li><li> Saldamente toccare il doppio apparato fiala contro il piano di lavoro 3 volte e garantire che tutte le mosche cadono sul fondo della fiala. Contemporaneamente avviare il timer. </li><li> Ogni 5 secondi per 1 minuto, scattare una foto dell&#39;apparato. Nota: In alternativa, un video può essere catturato e messo in pausa ad intervalli appropriati per le misurazioni. </li><li> Consenti mosche di recuperare indisturbati per 1 min. </li><li&gt; Ripetere altre 2 volte con tempo di recupero 1 min tra ogni prova. Nota: Ogni apparecchio dovrebbe prendere 5 minuti per completare la raccolta dei dati. Mantenere forza simile di intercettazioni tra prove. Multipla (almeno 3) apparecchio può essere facilmente gestito contemporaneamente. </li></ol></li><li> Analisi dei dati <ol><li> Rivedere le immagini e registrare il numero di mosche in ogni sezione nel corso del tempo. Calcolare la percentuale di mosche in ogni sezione nel tempo. Note: Ripetere l&#39;intera procedura con le stesse mosche a 2 o 3 punti di tempo di interesse, per esempio, il giorno 3, 5, e 7 se troppe mosche muoiono durante l&#39;esperimento, è possibile scalare il numero di prova iniziale per compensare per la mortalità. Utilizzare l&#39;analisi statistica opportuno confrontare i dati. </li></ol></li></ol> 2 Drosophila spontanea Locomotion Assay <ol><li> Preparazione del cibo <ol><li> Ricostituire 3 g di media istantanea Drosophila con 15 ml di acqua deionizzata e rotenone desiderato(O un altro farmaco di interesse) dosaggio. </li><li> Una volta che il composto cibo è diventato impresa (circa 5 min), caricare con cura il cibo per essere circa 1 cm in alto produttore forniti tubi trasparenti (5 mm x 65 mm). Aggiungere il farmaco infuso cibo ai tubi ponendo attenzione le provette verticalmente nel cibo e torsione fino a che non possono essere rimossi con il cibo all&#39;interno del tubo. Nota: È utile posizionare un dito sull&#39;apertura del tubo per creare il vuoto. Il cibo non deve contenere bolle d&#39;aria o avere una superficie irregolare come le mosche possono diventare bloccato. </li></ol></li><li> Setup sperimentale <ol><li> Inserire un tappo di plastica all&#39;estremità del tubo più vicina del cibo. Spingere il tappo di plastica sul tubo il meno possibile, come si può creare una bolla d&#39;aria nel flacone se spinto a forza. </li><li> Sedare 1 giorno-vecchio maschio vola con CO 2 e inserire con cautela 1 maschio vola in ogni provetta con un pennello. Ripetere a seconda del numero di prove desiderati. </li><li> Inserire ilestremità del tubo più lontano dal cibo con una piccola pallina di cotone, che può essere arrotolato a mano dal deposito ingrandito comprato batuffoli di cotone. </li><li> Consenti mosche di recuperare con i tubi in posizione orizzontale per 15 minuti e assicurarsi che tutte le mosche sono vivi e attivi. Inserire i tubi in DAM e assicurarsi che tutti i tubi sono nella stessa posizione relativa alla diga. Nota: E &#39;possibile posizionarle con la zona di monitoraggio al centro del flacone, o di spingere tutte le fiale di lato, in modo che l&#39;estremità del tubo viene monitorato. Nota: Si veda la discussione di variazioni su questo metodo. </li></ol></li><li> Raccolta dati <ol><li> Inserite il DAM in un buio 12 ore, 12 ore di luce incubatrice impostata a 25 ° C. Collegare il DAM al sistema di raccolta dati. Aprire il software DAM e alle preferenze selezionate lunghezza bin a 10 min. Avviare la raccolta dei dati e consentire al programma di raccolta di dati per 7 giorni. Nota: la lunghezza Bin può essere regolata, se necessario. </li><li> Analisi dei dati Nota: Processoi dati per ottenere conteggi per minuto come misura di locomozione spontanea a lungo termine. <ol><li> Programma e monitorare l&#39;accesso ai dati di scansione dei file DAM Aprire facendo clic su Seleziona dati di input. </li><li> Selezionare appropriata gamma del monitor e selezionare la lunghezza bin a intervalli di 10 min. </li><li> Nel tipo di file di output scegliere i file di canale. Lasciare tutte le altre opzioni come predefinite. </li><li> Fare clic su dati di scansione e salvare in una cartella designata. </li><li> Importare dati in un circadiano software di analisi dei dati per ottenere conteggi per minuto. Nota: Per il software di analisi dei dati Clocklab è comunemente usato. Altre opzioni sono disponibili anche. </li></ol></li></ol></li></ol>

<ol>
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B., 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=Mechanism+of+toxicity+in+rotenone+models+of+Parkinson's+disease.">Mechanism of toxicity in rotenone models of Parkinson's disease.</a> The Journal of neuroscience : the official journal of the Society for Neuroscience. 23, 10756-10764 (2003).</li><li>Munoz-Soriano, V., Paricio, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+models+of+Parkinson's+disease:+discovering+relevant+pathways+and+novel+therapeutic+strategies.">Drosophila models of Parkinson's disease: discovering relevant pathways and novel therapeutic strategies.</a> Parkinson's disease. , 520640 (2011).</li><li>Steffan, J. 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J., 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=Increased+glutathione+S-transferase+activity+rescues+dopaminergic+neuron+loss+in+a+Drosophila+model+of+Parkinson's+disease.">Increased glutathione S-transferase activity rescues dopaminergic neuron loss in a Drosophila model of Parkinson's disease.</a> Proceedings of the National Academy of Sciences of the United States of America. 102, 8024-809 (2005).</li><li>Ahmad, S. T., Steinmetz, S. B., Bussey, H. M., Possidente, B., Seggio, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Larval+ethanol+exposure+alters+free-running+circadian+rhythm+and+per+Locus+transcription+in+adult+D.+melanogaster+period+mutants.">Larval ethanol exposure alters free-running circadian rhythm and per Locus transcription in adult D. melanogaster period mutants.</a> Behavioural brain research. 241, 50-55 (2013).</li><li>Seggio, J. A., Possidente, B., Ahmad, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Larval+ethanol+exposure+alters+adult+circadian+free-running+locomotor+activity+rhythm+in+Drosophila+melanogaster.">Larval ethanol exposure alters adult circadian free-running locomotor activity rhythm in Drosophila melanogaster.</a> Chronobiology international. 29, 75-81 (2012).</li><li>Chaudhuri, 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=Interaction+of+genetic+and+environmental+factors+in+a+Drosophila+parkinsonism+model.">Interaction of genetic and environmental factors in a Drosophila parkinsonism model.</a> The Journal of neuroscience : the official journal of the Society for Neuroscience. 27, 2457-2467 (2007).</li><li>Feany, M. B., Bender, W. 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Gli autori non hanno nulla da rivelare.

In questo studio, descriviamo due procedure di misurazione sia locomozione spontanea a lungo termine e di locomozione startle indotta a breve termine in un rotenone indotta Drosophila modello della malattia di Parkinson. Si può anche misurare queste caratteristiche locomozione in mosche esposti ad altri agenti farmacologici noti per modello di malattia di Parkinson, per esempio paraquat 14, modelli genetici della malattia di Parkinson ad esempio, alfa-sinucleina mutanti 15,</s...

Carenze Locomotion sono un sintomo principale della malattia di Parkinson e sono in gran parte causati dal deterioramento dei neuroni dopaminergici della substantia nigra 1. Rotenone è un insetticida chetonici che è stata ampiamente studiata per modellare deficit motori del Parkinson in Drosophila 2-6. Rotenone provoca danno ossidativo bloccando la via della fosforilazione ossidativa, che provoca in ultima analisi, la morte cellulare 7. Neuroni dopaminergici sono più inclini alla tossicità rotenone, rendendo gli effetti della chimica basata principalmente motore 2,7. Per indurre i sintomi della malattia di Parkinson in mosche, possiamo capire meglio la malattia e porre rimedio suoi sintomi 6,8-11. Drosophila offre un buon modello per lo studio di questo effetto, perché sono geneticamente trattabili, di facile manutenzione, e hanno un ciclo di vita veloce. Diversi studi hanno dimostrato che il rotenone provoca a breve termine startle-indottadifetti di locomozione in mosche Drosophila -quando sono mantenuti sul cibo rotenone-integrato, mostrano una risposta più lenta geotactic negativa dopo startle 2-6. La loro incapacità di salire verso l&#39;alto in un apparato fiala più rapidamente le prove di controllo è indice di difetti di locomozione startle-indotta. L&#39;effetto di rotenone on-lungo termine, movimento spontaneo non è ben descritto. Monitor di attività Drosophila (dighe) sono stati utilizzati con successo per monitorare il movimento in Drosophila ritmo circadiano studia 12,13. Le mosche sono posti in tubi singoli, che vengono caricati nel DAM. Questo apparecchio è dotato di un sensore a infrarossi, che conta il numero di volte che una mosca interrompe il raggio infrarosso. Questi conteggi possono essere usati come misura di locomozione e dell&#39;attività 12,13 indisturbati. Inserendo mosche in una diga, l&#39;effetto di rotenone sul loro locomozione a lungo termine può essere caratterizzato. Questo studio descrive metodi per measlocomozione e locomozione spontanea a lungo termine URE breve termine startle-indotta al fine di comprendere meglio gli effetti del rotenone mediata deficit motori. Caratterizzazione delle carenze di locomozione che mimano la malattia di Parkinson sono importanti perché consentono per lo studio di altri composti che possono invertire questi difetti di locomozione.

<table border="0" cellpadding="0" cellspacing="0" width="628">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Standard narrow vials</td>
			<td style="width:127px;">Genesee Scientific</td>
			<td style="width:119px;">32-120</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Rotenone</td>
			<td style="width:127px;">Sigma</td>
			<td style="width:119px;">R8875</td>
			<td>Store in freezer, make fresh for each experiment</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Dimethyl Sulfoxide (DMSO)</td>
			<td style="width:127px;">Sigma</td>
			<td style="width:119px;">D8418</td>
			<td>Solvent for rotenone</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Instant Drosophila medium</td>
			<td style="width:127px;">Carolina Biological&#160;</td>
			<td style="width:119px;">Formula 4-24</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Drosophila activity monitor (DAM)</td>
			<td style="width:127px;">Trikinetics</td>
			<td style="width:119px;">DAM2</td>
			<td>trikinetics.com</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">DAM tubes</td>
			<td style="width:127px;">Trikinetics</td>
			<td style="width:119px;">Tubes 5X65 mm</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;"></td>
			<td style="width:127px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Recipe for Rotenone +food (125 mM dose)</td>
			<td style="width:127px;"></td>
			<td style="width:119px;"></td>
			<td>Make 62.5 mM rotenone stock solution in DMSO by dissolving 25 mg rotenone in 1 ml DMSO&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:127px;"></td>
			<td style="width:119px;"></td>
			<td>For 125 mM dose, add 10 mM rotenone stock in DMSO to 5 ml water.</td>
		</tr>
	</tbody>
</table>

methods to characterize spontaneous and startle-induced locomotion in a rotenone-induced parkinson's disease model of drosophila

La malattia di Parkinson è una malattia neurodegenerativa che deriva dalla degenerazione dei neuroni dopaminergici nel sistema nervoso centrale, soprattutto nella substantia nigra. La malattia provoca deficit motori, che presentano come rigidità, tremori e demenza negli esseri umani. Rotenone è un insetticida che provoca il danno ossidativo inibendo la funzione della catena di trasporto degli elettroni nei mitocondri. Viene anche usato per modellare la malattia di Parkinson in Drosophila. Le mosche hanno una intrinseca geotactic risposta negativa, che obbliga loro di salire verso l&#39;alto dopo essere stato sorpreso. È stato stabilito che rotenone causa di mortalità e di locomozione difetti iniziali che inficiano la capacità delle mosche &#39;a salire dopo che sono stati sfruttati verso il basso. Tuttavia, l&#39;effetto di rotenone sul movimento spontaneo non è ben documentata. Questo studio delinea due test di throughput sensibili, riproducibili, e alti per caratterizzare carenze rotenone-indottalocomozione e locomozione spontanea-lungo termine a breve termine startle-indotta in Drosophila. Questi test possono essere adattati facilmente per caratterizzare altri modelli di Drosophila di difetti di locomozione e l&#39;efficacia degli agenti terapeutici.

Drosophila Startle indotta Assay Locomotion Di tipo selvatico, Canton-S, mosche mostrato una robusta risposta geotactic negativa con solo circa il 88% e il 5% di mosche nelle sezioni superiore e inferiore, rispettivamente, dell&#39;apparato doppio flaconcino dopo 30 sec (Figura 1). Le mosche esposte a 125 mM e 250 mM rotenone per 3 giorni hanno mostrato una lieve diminuzione del numero di mosche nella sezione superiore e leggero aumento del...

Watch this Scientific Journal Video about Methods to Characterize Spontaneous and Startle-induced Locomotion in a Rotenone-induced Parkinson's Disease Model of Drosophila at JoVE.com

Methods to Characterize Spontaneous and Startle-induced Locomotion in a Rotenone-induced Parkinson&#039;s Disease Model of Drosophila

La malattia di Parkinson è una malattia neurodegenerativa che deriva dalla degenerazione dei neuroni dopaminergici nel sistema nervoso centrale, causando difetti di locomozione. Modelli rotenone malattia di Parkinson in Drosophila. Questo documento delinea due saggi che caratterizzano le carenze locomozione sia spontanee e spaventare-indotte causate da rotenone.

Metodi di caratterizzare spontanea e startle indotta Locomozione in un rotenone-indotta malattia di Parkinson Modello di<em&gt; Drosophila</em

Parkinson&#8217;s disease is a neurodegenerative disorder that results from the degeneration of dopaminergic neurons in the central nervous system, ...

methods-to-characterize-spontaneous-startle-induced-locomotion

Research

JoVE Journal

Neuroscience

Methods to Characterize Spontaneous and Startle-induced Locomotion in a Rotenone-induced Parkinson's Disease Model of Drosophila

10.0K Views.  Colby College.  La malattia di Parkinson è una malattia neurodegenerativa che deriva dalla degenerazione dei neuroni dopaminergici nel sistema nervoso centrale, causando difetti di locomozione. Modelli rotenone malattia di Parkinson in Drosophila. Questo documento delinea due saggi che caratterizzano le carenze locomozione sia spontanee e spaventare-indotte causate da rotenone. 

Video: Methods to Characterize Spontaneous and Startle-induced Locomotion in a Rotenone-induced Parkinson's Disease Model of Drosophila

Autologous Blood Injection to Model Spontaneous Intracerebral Hemorrhage in Mice

Investigation of the pathophysiology of injury after intracerebral
hemorrhage (ICH) requires a reproducible animal model. While ICH
accounts for 10-15% of all strokes, there remains no specific
effective therapy. The autologous blood injection model in mice
involves the stereotaxic injection of arterial blood into the basal
ganglia mimicking a spontaneous hypertensive hemorrhage in man. The
response to hemorrhage can then be studied in vivo and the
neurobehavioral deficits quantified, allowing for description of the
ensuing pathology and the testing of potential therapeutic agents. The
procedure described in this protocol uses the double injection
technique to minimize risk of blood reflux up the needle track, no
anticoagulants in the pumping system, and eliminates all dead space
and expandable tubing in the system.

The autologous blood injection model of intracerebral hemorrhage in mice described in this protocol uses the double injection technique to minimize risk of blood reflux up the needle track, no anticoagulants in the pumping system, and eliminates all dead space and expandable tubing in the system.

Iniezione di sangue autologo Modello spontanea emorragia intracerebrale in topi

Investigation of the pathophysiology of injury after intracerebral
hemorrhage (ICH) requires a reproducible animal model.  While ICH
accounts for 10-15% ...

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Neuroscienze

Methods to Assay Drosophila Behavior

Drosophila melanogaster, the fruit fly, has been used to study molecular mechanisms of a wide range of human diseases such as cancer, cardiovascular disease and various neurological diseases1. We have optimized simple and robust behavioral assays for determining larval locomotion, adult climbing ability (RING assay), and courtship behaviors of Drosophila. These behavioral assays are widely applicable for studying the role of genetic and environmental factors on fly behavior. Larval crawling ability can be reliably used for determining early stage changes in the crawling abilities of Drosophila larvae and also for examining effect of drugs or human disease genes (in transgenic flies) on their locomotion. The larval crawling assay becomes more applicable if expression or abolition of a gene causes lethality in pupal or adult stages, as these flies do not survive to adulthood where they otherwise could be assessed. This basic assay can also be used in conjunction with bright light or stress to examine additional behavioral responses in Drosophila larvae. Courtship behavior has been widely used to investigate genetic basis of sexual behavior, and can also be used to examine activity and coordination, as well as learning and memory. Drosophila courtship behavior involves the exchange of various sensory stimuli including visual, auditory, and chemosensory signals between males and females that lead to a complex series of well characterized motor behaviors culminating in successful copulation. Traditional adult climbing assays (negative geotaxis) are tedious, labor intensive, and time consuming, with significant variation between different trials2-4. The rapid iterative negative geotaxis (RING) assay5 has many advantages over more widely employed protocols, providing a reproducible, sensitive, and high throughput approach to quantify adult locomotor and negative geotaxis behaviors. In the RING assay, several genotypes or drug treatments can be tested simultaneously using large number of animals, with the high-throughput approach making it more amenable for screening experiments.

Methods to Assay Drosophila Behavior

Drosophila melanogaster is a genetically and behaviorally tractable model system that has been used to understand the molecular and cellular basis of many important biological processes for over a century 1. Drosophila has been well exploited to gain insights into the genetic basis of fly behavior.

Metodi per test Drosophila Comportamento

Drosophila melanogaster, the fruit fly, has been used to study molecular mechanisms of a wide range of human diseases such as cancer, cardiovascular ...

Multi-unit Recording Methods to Characterize Neural Activity in the Locust (Schistocerca Americana) Olfactory Circuits

Detection and interpretation of olfactory cues are critical for the survival of many organisms. Remarkably, species across phyla have strikingly similar olfactory systems suggesting that the biological approach to chemical sensing has been optimized over evolutionary time1. In the insect olfactory system, odorants are transduced by olfactory receptor neurons (ORN) in the antenna, which convert chemical stimuli into trains of action potentials. Sensory input from the ORNs is then relayed to the antennal lobe (AL; a structure analogous to the vertebrate olfactory bulb). In the AL, neural representations for odors take the form of spatiotemporal firing patterns distributed across ensembles of principal neurons (PNs; also referred to as projection neurons)2,3. The AL output is subsequently processed by Kenyon cells (KCs) in the downstream mushroom body (MB), a structure associated with olfactory memory and learning4,5. Here, we present electrophysiological recording techniques to monitor odor-evoked neural responses in these olfactory circuits.
First, we present a single sensillum recording method to study odor-evoked responses at the level of populations of ORNs6,7. We discuss the use of saline filled sharpened glass pipettes as electrodes to extracellularly monitor ORN responses. Next, we present a method to extracellularly monitor PN responses using a commercial 16-channel electrode3. A similar approach using a custom-made 8-channel twisted wire tetrode is demonstrated for Kenyon cell recordings8. We provide details of our experimental setup and present representative recording traces for each of these techniques.

Multi-unit Recording Methods to Characterize Neural Activity in the Locust Schistocerca Americana Olfactory Circuits

We demonstrate variations of the extracellular multi-unit recording technique to characterize odor-evoked responses in the first three stages of the invertebrate olfactory pathway. These techniques can easily be adapted to examine ensemble activity in other neural systems as well.

Multi-unit Metodi di registrazione per caratterizzare l&#39;attività neurale nel Locust (<em&gt; Schistocerca Americana</em&gt;) Circuiti olfattive

Detection and interpretation of olfactory cues are critical for the survival  of many organisms. Remarkably, species across phyla have strikingly similar ...

Determination of the Spontaneous Locomotor Activity in Drosophila melanogaster

Drosophila melanogaster has been used as an excellent model organism to study environmental and genetic manipulations that affect behavior. One such behavior is spontaneous locomotor activity. Here we describe our protocol that utilizes Drosophila population monitors and a tracking system that allows continuous monitoring of the spontaneous locomotor activity of flies for several days at a time. This method is simple, reliable, and objective and can be used to examine the effects of aging, sex, changes in caloric content of food, addition of drugs, or genetic manipulations that mimic human diseases.

Determination of the Spontaneous Locomotor Activity in Drosophila melanogaster

Drosophila melanogaster are useful in studying genetic or environmental manipulations that affect behaviors such as spontaneous locomotor activity.&#160; Here we describe a protocol that utilizes monitors with infrared beams and data analysis software to quantify spontaneous locomotor activity.&#160;

Determinazione del locomotore attività spontanea in<em&gt; Drosophila melanogaster</em

Drosophila melanogaster has been used as an excellent model organism to study environmental and genetic manipulations that affect behavior. One such ...

A Novel Method to Model Chronic Traumatic Encephalopathy in Drosophila

Chronic Traumatic Encephalopathy (CTE) is an established neurodegenerative disease that is closely associated with exposure to repetitive mild Traumatic Brain Injury (mTBI). The mechanisms responsible for its complex pathological changes remain largely elusive, despite a recent consensus to define the neuropathological criteria. Here, we describe a novel method to develop a model of CTE in Drosophila melanogaster&#160;(Drosophila&#160;)&#160;in an attempt to identify the key genes and pathways that lead to the characteristic hyperphosphorylated tau accumulation and neuronal death in the brain. Adjustable-strength impacts to inflict mild closed injury are delivered directly to the fly head, subjecting the head to rapid acceleration and deceleration. Our method eliminates the potential problems inherent with other Drosophila mTBI models (e.g.,animal death might be induced by damage to other parts of the body or to internal organs). The less labor- and cost-intensive animal care, short life span, and extensive genetic tools make the fruit fly ideal to study CTE pathogenesis and make it possible to perform large-scale, genome-wide forward genetic and pharmacological screens. We anticipate that the ongoing characterization of the model will generate important mechanistic insights on disease prevention and therapeutic approaches.

A Novel Method to Model Chronic Traumatic Encephalopathy in Drosophila

Here, we describe a new approach to inflict closed-head traumatic brain injury in Drosophila melanogaster. Our method has the advantage of directly delivering repetitive impacts with adjustable strength to the head alone. Further exploration of the invertebrate system will help to illuminate the pathogenesis of chronic traumatic encephalopathy.

Un nuovo metodo per modellare l&#39;encefalopatia traumatica cronica in<em&gt; Drosophila</em

Chronic Traumatic Encephalopathy (CTE) is an established neurodegenerative disease that is closely associated with exposure to repetitive mild Traumatic ...

Quantifying Spontaneous Ca2+ Fluxes and their Downstream Effects in Primary Mouse Midbrain Neurons

Parkinson&#8217;s disease (PD) is a devastating neurodegenerative disorder caused by the degeneration of dopaminergic (DA) neurons. Excessive Ca2+ influx due to the abnormal activation of glutamate receptors results in DA excitotoxicity and has been identified as an important mechanism for DA neuron loss. In this study, we isolate, dissociate, and culture midbrain neurons from the mouse ventral mesencephalon (VM) of ED14 mouse embryos. We then infect the long-term primary mouse midbrain cultures with an adeno-associated virus (AAV) expressing a genetically encoded calcium indicator, GCaMP6f under control of the human neuron-specific synapsin promoter, hSyn. Using live confocal imaging, we show that cultured mouse midbrain neurons display spontaneous Ca2+ fluxes detected by AAV-hSyn-GCaMP6f. Bath application of glutamate to midbrain cultures causes abnormal elevations in intracellular Ca2+ within neurons and this is accompanied by caspase-3 activation in DA neurons, as demonstrated by immunostaining. The techniques to identify glutamate-mediated apoptosis in primary mouse DA neurons have important applications for the high content screening of drugs that preserve DA neuron health.

Quantifying Spontaneous Ca2+ Fluxes and their Downstream Effects in Primary Mouse Midbrain Neurons

Here we present a protocol to measure in vitro Ca2+ fluxes in midbrain neurons and their downstream effects on caspase-3 using primary mouse midbrain cultures. This model can be employed to study pathophysiologic changes related to abnormal Ca2+ activity in midbrain neurons, and to screen novel therapeutics for anti-apoptotic properties.

Quantificare i flussispontanei di Ca 2 e i loro effetti a valle nei neuroni Midbrain del topo primario

Parkinson&#8217;s disease (PD) is a devastating neurodegenerative disorder caused by the degeneration of dopaminergic (DA) neurons. Excessive Ca2+ influx ...

Applying the RatWalker System for Gait Analysis in a Genetic Rat Model of Parkinson's Disease

Parkinson's disease (PD) is a progressive neurodegenerative disorder caused by the loss of dopaminergic (DA) neurons in the substantia nigra pars compacta. Gait abnormalities, including decreased arm swing, slower walking speed, and shorter steps are common in PD patients and appear early in the course of disease. Thus, the quantification of motor patterns in animal models of PD will be important for phenotypic characterization during disease course and upon therapeutic treatment. Most cases of PD are idiopathic; however, the identification of hereditary forms of PD uncovered gene mutations and variants, such as loss-of-function mutations in Pink1 and Parkin, two proteins involved in mitochondrial quality control that could be harnessed to create animal models. While mice are resistant to neurodegeneration upon loss of Pink1 and Parkin (single and combined deletion), in rats, Pink1 but not Parkin deficiency leads to nigral DA neuron loss and motor impairment. Here, we report the utility of FTIR imaging to uncover gait changes in freely walking young (2 months of age) male rats with combined loss of Pink1 and Parkin prior to the development of gross visually apparent motor abnormality as these rats age (observed at 4-6 months), characterized by hindlimb dragging as previously reported in Pink1 knockout (KO) rats.

Applying the RatWalker System for Gait Analysis in a Genetic Rat Model of Parkinson&#039;s Disease

Here we describe the RatWalker system, built by redesigning the MouseWalker apparatus to accommodate for the increased size and weight of rats. This system uses frustrated total internal reflection (FTIR), high-speed video capture, and open-access analysis software to track and quantify gait parameters.

Applicazione del sistema RatWalker per l'analisi dell'andatura in un modello genetico di ratto della malattia di Parkinson

Parkinson's disease (PD) is a progressive neurodegenerative disorder caused by the loss of dopaminergic (DA) neurons in the substantia nigra pars ...

The 6-hydroxydopamine Rat Model of Parkinson's Disease

Motor symptoms of Parkinson's disease (PD)-bradykinesia, akinesia, and tremor at rest-are consequences of the neurodegeneration of dopaminergic neurons in the substantia nigra pars compacta (SNc) and dopaminergic striatal deficit. Animal models have been widely used to simulate human pathology in the laboratory. Rodents are the most used animal models for PD due to their ease of handling and maintenance. Moreover, the anatomy and molecular, cellular, and pharmacological mechanisms of PD are similar in rodents and humans. The infusion of the neurotoxin, 6-hydroxydopamine (6-OHDA), into a medial forebrain bundle (MFB) of rats reproduces the severe destruction of dopaminergic neurons and simulates PD symptoms. This protocol demonstrates how to perform the unilateral microinjection of 6-OHDA in the MFB in a rat model of PD and shows the motor deficits induced by 6-OHDA and predicted dopaminergic lesions through the stepping test. The 6-OHDA causes significant impairment in the number of steps performed with the contralateral forelimb.

The 6-hydroxydopamine Rat Model of Parkinson&#039;s Disease

The 6-hydroxydopamine (6-OHDA) model has been used for decades to advance the understanding of Parkinson's Disease. In this protocol, we demonstrate how to perform unilateral nigrostriatal lesions in the rat by injecting 6-OHDA in the medial forebrain bundle, assess motor deficits, and predict lesions using the stepping test.

Il modello di ratto 6-idrossidopamina della malattia di Parkinson

Motor symptoms of Parkinson's disease (PD)-bradykinesia, akinesia, and tremor at rest-are consequences of the neurodegeneration of dopaminergic neurons in ...

Induction and Assessment of Levodopa-induced Dyskinesias in a Rat Model of Parkinson's Disease

Levodopa (L-DOPA) remains the gold-standard therapy used to treat Parkinson&#39;s disease (PD) motor symptoms. However, unwanted involuntary movements known as L-DOPA-induced dyskinesias (LIDs) develop with prolonged use of this dopamine precursor.&#160;It is estimated that the incidence of LIDs escalates to approximately 90% of individuals with PD within 10&#8211;15 years of treatment. Understanding the mechanisms of this malady and developing both novel and effective anti-dyskinesia treatments requires consistent and accurate modeling for pre-clinical testing of therapeutic interventions. A detailed method for reliable induction and comprehensive rating of LIDs following 6-OHDA-induced nigral lesioning in a rat model of PD is presented here. Dependable LID assessment in rats provides a powerful tool that can be readily utilized across laboratories to test emerging therapies focused on reducing or eliminating this common treatment-induced burden for individuals with PD.

Induction and Assessment of Levodopa-induced Dyskinesias in a Rat Model of Parkinson&#039;s Disease

This article describes methods to induce and evaluate levodopa-induced dyskinesias in a rat model of Parkinson&#39;s disease. The protocol offers detailed information regarding the intensity and frequency of a range of dyskinetic behaviors, both dystonic and hyperkinetic, providing a reliable tool to test treatments targeting this unmet medical need.

Induzione e valutazione delle discinesie indotte da levodopa in un modello di ratto della malattia di Parkinson

Levodopa (L-DOPA) remains the gold-standard therapy used to treat Parkinson&#39;s disease (PD) motor symptoms. However, unwanted involuntary movements ...

Analyzing the Parkinson's Disease Mouse Model Induced by Adeno-associated Viral Vectors Encoding Human &#945;-Synuclein

Parkinson&#39;s disease is a neurodegenerative disorder that involves the death of the dopaminergic neurons of the nigrostriatal pathway and, consequently, the progressive loss of control of voluntary movements. This neurodegenerative process is triggered by the deposition of protein aggregates in the brain, which are mainly constituted of &#945;-synuclein. Several studies have indicated that neuroinflammation is required to develop the neurodegeneration associated with Parkinson&#39;s disease. Notably, the neuroinflammatory process involves microglial activation as well as the infiltration of peripheral T cells into the substantia nigra (SN). This work analyzes a mouse model of Parkinson&#39;s disease that recapitulates microglial activation, T-cell infiltration into the SN, the neurodegeneration of nigral dopaminergic neurons, and motor impairment. This mouse model of Parkinson&#39;s disease is induced by the stereotaxic delivery of adeno-associated viral vectors encoding the human wild-type &#945;-synuclein (AAV-h&#945;Syn) into the SN. The correct delivery of viral vectors into the SN was confirmed using control vectors encoding green fluorescent protein (GFP). Afterward, how the dose of AAV-h&#945;Syn administered in the SN affected the extent of h&#945;Syn expression, the loss of nigral dopaminergic neurons, and motor impairment were evaluated. Moreover, the dynamics of h&#945;Syn expression, microglial activation, and T-cell infiltration were determined throughout the time course of disease development. Thus, this study provides critical time points that may be useful for targeting synuclein pathology and neuroinflammation in this preclinical model of Parkinson&#39;s disease.

Analyzing the Parkinson&#039;s Disease Mouse Model Induced by Adeno-associated Viral Vectors Encoding Human &amp;#945;-Synuclein

This work analyzes the vector dose and exposure time required to induce neuroinflammation, neurodegeneration, and motor impairment in this preclinical model of Parkinson&#39;s disease. These vectors encoding the human &#945;-synuclein are delivered into the substantia nigra&#160;to recapitulate the synuclein pathology associated with Parkinson&#39;s disease.