Name: using enzyme-based biosensors to measure tonic and phasic glutamate in alzheimer's mouse models
Uploaded: 2017-05-03T13:00:00
Description: Watch this Scientific Journal Video about Using Enzyme-based Biosensors to Measure Tonic and Phasic Glutamate in Alzheimer's Mouse Models at JoVE.com

Dette arbeidet ble støttet av National Institute of General Medical Sciences (MNR, U54GM104942), NIA (MNR, R15AG045812), Alzheimers Association (MNR, NIRG-12-242187), WVU fakultet Forskning Senatet Grant (MNR), og WVU PSCOR Grant (MNR).

1. Coating microelectrode Array med enzymer eller Matrix Layer <ol><li> Klargjøring proteinmatriksen løsningen <ol><li> Vei opp 10 mg bovint serumalbumin (BSA) og overføre til 1,5 ml mikrosentrifugerør. </li><li> Legg 985 mL DI vann til mikrosentrifugerør inneholdende BSA. Bland løsningen ved manuell røring (gjen pipettering ved anvendelse av 1,000 ul pipette ~ 3 ganger, inntil de BSA oppløst). MERK: Ikke bruk vortex for å blande løsningen da dette kan introdusere luft inn i løsninge...

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MEA teknikk tillater måling av raske kinetikk for nevrotransmitter-frigjøring og opptak in vitro og in vivo. Derfor frembringer den teknologi en rekke datautgangs inkludert tonisk nevrotransmitter nivåer fremkalt frigjøring av nevrotransmittere og nevrotransmitter klaring. Men fordi bruk av MEAs er en relativt komplisert prosedyre, er det mange faktorer som kan trenge å være optimalisert for vellykket bruk. For eksempel, i løpet av kalibreringen, kan man være oppmerksom på at det ikke er noen ...

Måling av nevrotransmitter endringer i hjernen er et viktig verktøy for å studere nevro sykdommer i sentralnervesystemet (CNS), som kjennetegnes ofte av nevrotransmitter dysregulering. Selv om mikrodialyse i kombinasjon med høytrykks-væskekromatografi (HPLC / EC) har vært den mest anvendte metoden for å måle endringer i ekstracellulære nevrotransmitter nivåer 1, 2, 3, 4, den romlige og tidsmessige bestemte oppløsningen av mikrodialyse-probene kan ikke være ideell for nevrotransmittere , slik som glutamat, som er tett regulert i det ekstracellulære rom 5, 6. På grunn av den nylige fremskritt innen genetikk og billeddannelse, er det flere metoder som kan brukes til å kartlegge glutamat in vivo. Ved hjelp av genetisk kodede glutamat fluorescerende rapportører (iGluSnFR) etd to-foton avbildning, forskere er i stand til å visualisere glutamatfrigiving av neuroner og astrocytter både in vitro og in vivo 7, 8, 9. Spesielt, tillater dette tas opp fra et større synsfelt og ikke forstyrrer ikke den iboende forbindelsene til hjernen. Selv om disse nye optiske teknikker tillate visualisering av glutamatkinetikk og måling av sensoriske fremkalte responser og neuronal aktivitet, de mangler evnen til å kvantifisere mengden av glutamat i det ekstracellulære rom i diskrete hjerneområder. En alternativ metode er den enzymbundne mikroelektrode array (MEA) som selektivt kan måle ekstracellulære nevrotransmitter nivåer, som for eksempel glutamat, ved bruk av en selv referert opptak ordningen. MEA teknikken har blitt brukt til å studere forandringer i ekstracellulær glutamat etter traumatisk hjerneskadeskade 10, 11, 12, aldring 13, 14, stresset 15, 16, epilepsi 17, 18, Alzheimers sykdom 19, 20, og injeksjon av en viral etterligne 21 og representerer en forbedring i forhold til de rommessige og tidsmessige begrensninger som ligger i mikrodialyse. Mens mikrodialyse begrenser muligheten for å måle nær synapsen 22, 23, MEAs har en høy romlig oppløsning som gjør det mulig for selektive målinger av ekstracellulær glutamat overløp nær synapser 24, 25. For det andre, den lave tidsoppløsningen av mikrodialyse - begrenser (1 20 min) evnen til å undersøkeraske dynamikken i glutamatfrigjøring og klaringen som forekommer i millisekund til andre område 26. Fordi forskjeller i frigjørings eller klaring av glutamat kan ikke være innlysende på målinger av tonic, hviler glutamat nivå, kan det være nødvendig at glutamatfrigjøring og klaringen kan måles direkte. MEAs tillate for slike tiltak på grunn av deres høye tidsmessig oppløsning (2 Hz) og lave påvisningsgrenser (&lt;1 um). For det tredje, MEAs muliggjøre undersøkelse av subregional variasjoner i nevrotransmittere i en bestemt hjerneområde, slik som rotte eller mus hippocampus. For eksempel, ved anvendelse av MEAs vi kan hver for målrette dentate gyrus (DG), ove ammonis 3 (CA3) og ove ammonis 1 (CA1) av hippocampus, som er forbundet via et trisynaptic krets 27, for å undersøke subregional forskjeller i ekstracellulær glutamat. På grunn av størrelsen på mikrodialyseprober (til 1 - 4 mm lengde), og skader forårsaket ved implantasjon 28 &lt;/ sup&gt;, 29, subregionale forskjellene er vanskelig å ta opp. Videre er de optiske systemer bare tillater stimulering gjennom eksterne stimuli, slik som en whisker stimulering eller lys flimmer, som ikke tillater subregional stimulering 7. En endelig fordel med MEAs enn andre metoder er evnen til å studere disse subregioner in vivo uten å forstyrre deres ytre og indre forbindelser. Her beskriver vi hvordan et opptakssystem (f.eks FAST16mkIII) i kombinasjon med MEAs, som består av et keramikk-baserte multisite mikroelektrode, kan differensielt belegges på opptakssider for å gi rom for forstyrrende stoffer som skal detekteres og fjernes fra analytten signal. Vi viser også disse matriser kan benyttes for amperometry baserte studier av in vivo-glutamat regulering innenfor DG, CA3, og CA1 hippocampus-subregioner av bedøvet RTG (TauP301L) 4510 mus, en vanlig brukt mouse modell for Alzheimers sykdom. I tillegg gir vi bekreftelse av følsomheten av MEA-systemet til den raske dynamikken i glutamatfrigjøring og klaring ved behandling av musene med riluzol, til et medikament in vitro vist å redusere glutamatfrigjøring og øke glutamat opptak 30, 31, 32, 33, og demonstrerer disse respektive endringer in vivo i TauP301L musemodellen.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
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		<tr>
			<td>FAST-16mkIII-8 channel</td>
			<td>Quanteon</td>
			<td>16mkIII</td>
			<td></td>
		</tr>
		<tr>
			<td>Microelectrode arrays</td>
			<td>CenMet</td>
			<td>W4 or 8-TRK</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>Sigma-Aldrich</td>
			<td>A-3059</td>
			<td>10 g (expires after 1 month)</td>
		</tr>
		<tr>
			<td>Glutaraldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>G-6257</td>
			<td>100 mL (expires after 6 months)</td>
		</tr>
		<tr>
			<td>Glutamate Oxidase</td>
			<td>US Biological or Sigma Aldrich</td>
			<td>G4001-01 or 100646</td>
			<td>50 UI (expires after 6 months)</td>
		</tr>
		<tr>
			<td>Hamilton Syringes</td>
			<td>Hamilton</td>
			<td>#701</td>
			<td>2 syringes</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>BDH</td>
			<td>UN1230</td>
			<td>4 L</td>
		</tr>
		<tr>
			<td>m-Phenylenediamine dihydrochloride (mPD)</td>
			<td>ACROS Organics</td>
			<td>1330560250</td>
			<td>25 g</td>
		</tr>
		<tr>
			<td>Reference Electrodes (RE-5B)</td>
			<td>BAS</td>
			<td>MF-2079</td>
			<td>3 electrodes</td>
		</tr>
		<tr>
			<td>Magnetic stir plate</td>
			<td>Cole-parmer</td>
			<td>EW-04804-01</td>
			<td>Can purchase from different supplier</td>
		</tr>
		<tr>
			<td>Glutamate</td>
			<td>Sigma-Aldrich</td>
			<td>G-1626</td>
			<td>100 g</td>
		</tr>
		<tr>
			<td>Ascorbic Acid</td>
			<td>TCI</td>
			<td>50-81-7</td>
			<td>500 g</td>
		</tr>
		<tr>
			<td>Dopamine Hydrochloride</td>
			<td>Alfa Aesar</td>
			<td>62-31-7</td>
			<td>5 g</td>
		</tr>
		<tr>
			<td>Perchloric acid</td>
			<td>VWR</td>
			<td>UN2920</td>
			<td>500 mL</td>
		</tr>
		<tr>
			<td>Postassium chloride</td>
			<td>VWR</td>
			<td>7447-40-7</td>
			<td>1 kg</td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>VWR</td>
			<td>7647-40-7</td>
			<td>1 kg</td>
		</tr>
		<tr>
			<td>Calcium Chloride</td>
			<td>MP</td>
			<td>153502</td>
			<td>100 g</td>
		</tr>
		<tr>
			<td>Sodium Hydroxide</td>
			<td>BDH</td>
			<td>1310732</td>
			<td>500 g</td>
		</tr>
		<tr>
			<td>Glass pressure ejection pipettes</td>
			<td>CenMet</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sticky wax</td>
			<td>Kerrlab</td>
			<td>625</td>
			<td>Can purchase from different supplier</td>
		</tr>
		<tr>
			<td>Microsyringe</td>
			<td>World Precision Instruments</td>
			<td>MF28G-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Modeling clay</td>
			<td>WalMart</td>
			<td></td>
			<td>Can purchase from different supplier</td>
		</tr>
		<tr>
			<td>Picospritzer III</td>
			<td>Parker</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Silver wire</td>
			<td>AM systems</td>
			<td>782000</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric acid</td>
			<td>BDH</td>
			<td>7647010</td>
			<td>2.5 L</td>
		</tr>
		<tr>
			<td>Platinum wire</td>
			<td>AM Systems</td>
			<td>778000</td>
			<td></td>
		</tr>
		<tr>
			<td>Solder gun</td>
			<td>Lowes or Home Depot</td>
			<td></td>
			<td>Can purchase from different supplier</td>
		</tr>
		<tr>
			<td>Multimeter</td>
			<td>WalMart</td>
			<td></td>
			<td>Can purchase from different supplier</td>
		</tr>
		<tr>
			<td>PhysioSuite</td>
			<td>Kent Scientific</td>
			<td></td>
			<td>Can purchase from different supplier</td>
		</tr>
		<tr>
			<td>SomnoSuite</td>
			<td>Kent Scientific</td>
			<td></td>
			<td>Can purchase from different supplier</td>
		</tr>
		<tr>
			<td>Stereotaxic device</td>
			<td>Stoelting</td>
			<td></td>
			<td>Can purchase from different supplier</td>
		</tr>
		<tr>
			<td>Digital Lab Standard</td>
			<td>Stoelting</td>
			<td></td>
			<td>Can purchase from different supplier</td>
		</tr>
		<tr>
			<td>Meiji EMZ microscope</td>
			<td>Meiji</td>
			<td>EMZ-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Drill</td>
			<td>Dremel</td>
			<td>Micro</td>
			<td></td>
		</tr>
		<tr>
			<td>Metricide</td>
			<td>Metrex</td>
			<td>102800</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel</td>
			<td>VWR</td>
			<td></td>
			<td>Can purchase from different supplier</td>
		</tr>
		<tr>
			<td>Surgery scissors</td>
			<td>VWR</td>
			<td></td>
			<td>Can purchase from different supplier</td>
		</tr>
		<tr>
			<td>Sterile cotton swabs</td>
			<td>Puritan</td>
			<td>25806</td>
			<td>Can purchase from different supplier</td>
		</tr>
		<tr>
			<td>Eye ointment</td>
			<td>Puralube Vet Ointment</td>
			<td></td>
			<td>Obtain from the vet</td>
		</tr>
		<tr>
			<td>Iodine swabs</td>
			<td>VWR</td>
			<td>S48050</td>
			<td>Can purchase from different supplier</td>
		</tr>
		<tr>
			<td>Alcohol swabs</td>
			<td>Local drug store</td>
			<td></td>
			<td>Can purchase from different supplier</td>
		</tr>
		<tr>
			<td>Sterile surgery drape</td>
			<td>Dynarex</td>
			<td>4410</td>
			<td>Can purchase from different supplier</td>
		</tr>
		<tr>
			<td>Sterile saline</td>
			<td>Teknova</td>
			<td>S5815</td>
			<td>Can make own soltuion using filters</td>
		</tr>
		<tr>
			<td>Hydrogen Peroxide (3%)</td>
			<td>Local drug store</td>
			<td></td>
			<td>Can purchase from different supplier</td>
		</tr>
		<tr>
			<td>Heating Pad</td>
			<td>WalMart</td>
			<td></td>
			<td>Can purchase from different supplier</td>
		</tr>
	</tbody>
</table>

using enzyme-based biosensors to measure tonic and phasic glutamate in alzheimer's mouse models

Nevrotransmitter forstyrrelser er ofte en viktig del av sykdommer i sentralnervesystemet (CNS), som spiller en rolle i patologien underliggende Alzheimers sykdom, Parkinsons sykdom, depresjon og angst. Tradisjonelt har mikrodialyse har vært den mest vanlige (hyllet) teknikk for å undersøke nevrotransmitter endringer som oppstår i disse forstyrrelsene. Men fordi mikrodialyse har evnen til å måle langsomme 1-20 minutt endringer over store deler av vev, har den ulempen med invasivitet, potensielt ødelegge iboende forbindelser i hjernen og en langsom prøvetaking evne. En forholdsvis nyere teknikk, mikroelektroden array (MEA), har tallrike fordeler for måling av spesifikke nevrotransmitter endringer i diskrete hjerneområder etter hvert som de forekommer, noe som gir en romlig og tidsmessig nøyaktig tilnærming. I tillegg, ved bruk MEAs er minimal invasiv, slik at for måling av nevrotransmitteren endringer in vivo. I vårt laboratorium, vi hahar vært spesielt interessert i endringer i neurotransmitter, glutamat, relatert til patologien ved Alzheimers sykdom. Som sådan, er den fremgangsmåten som beskrives her er brukt til å vurdere potensielle hippokampale forstyrrelser i glutamat i en transgen musemodell for Alzheimers sykdom. I korthet, den metode som brukes omfatter belegning av et multi-site mikroelektrode med et enzym som er meget selektiv for den nevrotransmitter av interesse, og ved anvendelse av selvreferansesider for å trekke fra bakgrunnsstøy og interferenter. Etter utsåing og kalibrering, kan det MEA være konstruert med en mikropipette og senkes ned i hjerneområdet av interesse ved hjelp av en stereotaktisk anordning. Her er fremgangsmåten som er beskrevet innebærer bedøvelse RTG (TauP301L) 4510 og mus ved hjelp av en stereotaksisk anordning til nøyaktig å målrette subregioner (DG, CA1, og CA3) av hippocampus.

Selv om denne teknologien kan brukes til å måle endringer i glutamatergic signalanlegg i mange typer dyremodeller, for eksempel traumatisk hjerneskade, aldring, stress, og epilepsi, her viser vi hvordan MEA teknologien kan brukes til å undersøke glutamat endringer i transgen musemodell av menneskelig tauopati 19, 20. Den RTG (TauP301L) 4510 mus uttrykker P301L mutasjon i tau forbundet med frontotemporal demens og Parkinsons s...

Watch this Scientific Journal Video about Using Enzyme-based Biosensors to Measure Tonic and Phasic Glutamate in Alzheimer's Mouse Models at JoVE.com

Using Enzyme-based Biosensors to Measure Tonic and Phasic Glutamate in Alzheimer's Mouse Models

Her beskriver vi oppsettet, programvare navigasjon, og dataanalyse for et romlig og tidsmessig nøyaktig metode for å måle tonic og fase-ekstracellulær glutamat endringer in vivo ved hjelp av enzym-bundet mikroelektrode arrays (MEA).

Ved hjelp av enzymbaserte Biosensorer å måle Tonic og phasic Glutamat i Alzheimers musemodeller

Neurotransmitter disruption is often a key component of diseases of the central nervous system (CNS), playing a role in the pathology underlying ...

Research

JoVE Journal

Neuroscience

Mouse Models of Periventricular Leukomalacia

We describe a protocol for establishing mouse models of periventricular leukomalacia (PVL). PVL is the predominant form of brain injury in premature ...

Hyponeophagia: A Measure of Anxiety in the Mouse

Before the present day, when fast-acting and potent rodenticides such as alpha-chloralose were not yet in use, the work of pest controllers was often ...

Detection of Neuritic Plaques in Alzheimer's Disease Mouse Model

Alzheimer's disease (AD) is the most common neurodegenerative disorder leading to dementia. Neuritic plaque formation is one of the pathological hallmarks ...

GABA-activated Single-channel and Tonic Currents in Rat Brain Slices

The GABAA channels are present in all neurons and are located both at synapses and outside of synapses where they generate phasic and tonic currents, ...

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

Fast Micro-iontoforese av glutamat og GABA: Et nyttig verktøy for å etterforske 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 ...

Stereotaxic Infusion of Oligomeric Amyloid-beta into the Mouse Hippocampus

Stereotaksisk Infusjon av Oligomeric amyloid-beta i musen Hippocampus

Alzheimer&#8217;s disease is a neurodegenerative disease affecting the aging population. A key neuropathological feature of the disease is the ...

Combined Optogenetic and Freeze-fracture Replica Immunolabeling to Examine Input-specific Arrangement of Glutamate Receptors in the Mouse Amygdala

Kombinert optogenetic og Freeze-brudd Replica Immunolabeling å undersøke Input-spesifikke Tilrettelegging av glutamatreseptorer i Mouse Amygdala

Freeze-fracture electron microscopy has been a major technique in ultrastructural research for over 40 years. However, the lack of effective means to ...

A Simple Way to Measure Alterations in Reward-seeking Behavior Using Drosophila melanogaster

A Simple Way to Measure Alterations in Reward-seeking Behavior Using Drosophila melanogaster

En enkel måte å måle endringer i Reward-søkende atferd med<em&gt; Drosophila melanogaster</em

We describe a protocol for measuring ethanol self-administration in fruit flies (Drosophila melanogaster) as a proxy for changes in reward states. We ...

A High-throughput Calcium-flux Assay to Study NMDA-receptors with Sensitivity to Glycine/D-serine and Glutamate

En høy gjennomstrømming kalsium-flux analysen å studere NMDA-reseptorer med følsomhet glysin/D-serine og glutamat

N-methyl-D-aspartate (NMDA) receptors (NMDAR) are classified as ionotropic glutamate receptors and have critical roles in learning and memory. NMDAR ...

Using Near-infrared Fluorescence and High-resolution Scanning to Measure Protein Expression in the Rodent Brain

Bruke nær-infrarød fluorescens og høyoppløselig skanning å måle protein uttrykk i gnagere Brain

Neuroscience is the study of how cells in the brain mediate various functions. Measuring protein expression in neurons and glia is critical for the study ...