Name: electroconvulsive seizures in rats and fractionation of their hippocampi to examine seizure-induced changes in postsynaptic density proteins
Uploaded: 2017-08-15T14:00:00
Description: Watch this Scientific Journal Video about Electroconvulsive Seizures in Rats and Fractionation of Their Hippocampi to Examine Seizure-induced Changes in Postsynaptic Density Proteins at JoVE.com

Forfatterne takke Dr. Eric C. Bolton for at lade os bruge sin centrifuge til fraktionering og Dr. Graham H. Diering i Dr. Richard L. Huganir lab på John's Hopkins University for at give os med de små protokol for PSD fraktionering.

Alle eksperimentelle procedurer herunder animalske emner er blevet godkendt af det institutionelle dyr pleje og brug udvalg på University of Illinois i Urbana-Champaign.
1. opretholde en rotte koloni
<ol><li>Racen Sprague-Dawley rotter (Se Tabel af materialer) og fastholde dem i standardbetingelser med en 12-h lys-mørke cyklus og ad libitum adgang til mad og vand.</li>
	<li>Vænne rotte unger på postnatal dag (P) 28 og hus dem i grupper af 2-4 mand eller kvinde littermate...

Her, beskriver vi en ECS induktion metode i rotter, der fremkalder den globale stimulation af neuronal aktivitet i deres hippocampi. ECS er en dyremodel af elektrochok terapi, som er klinisk anvendes til behandling af narkotika ildfaste depressive lidelser i mennesker1,2,3. Trods brugen af elektrochok terapi til behandling af svær depression, den præcise underliggende mekanisme er fortsat uklart. Fordi ECS inducerer depressant...

Elektrochok terapi har været anvendt til behandling af patienter med større depressive lidelser, herunder svær resistent depression, bipolar depression, Parkinsons sygdomme, og skizofreni1,2. I denne behandling genereres beslaglæggelse af elektriske stimulus leveret til lederen af bedøvede patienter via epicranial elektroder1,2,3. Gentagen administration af ECS har været klinisk gavnlig medicinresistente depressive lidelser1,2,3. Imidlertid har den nøjagtige mekanisme bag den langsigtede effekt af antidepressiv effekt hos mennesker forblev undvigende. ECS er en dyremodel af elektrochok terapi og er almindeligt anvendt til at undersøge sin terapeutiske mekanisme. I gnavere, både akut ECS og kronisk ECS behandling fremme voksen neurogenese i hippocampi og omstrukturere de neurale netværk4,5, som forventes at bidrage til forbedringer i kognitiv fleksibilitet. Derudover ændrer globale udvidelse af hjerneaktivitet af ECS overfloden af udskrifter, såsom en hjerne afledt neurotropic faktor6, og flere proteiner, herunder metabotropic glutamat receptor 17 og N-methyl-D-aspartat (NMDA) type glutamat receptor underenheder7. Disse ændringer er involveret i mægler langsigtet ændring af synapse nummer, struktur og styrke i hippocampus7,8,9.
I ECS modeller, er elektrisk stimulation leveret til gnavere via stereotaxically implanterede elektroder, cornea elektroder eller øre elektroder til at fremmane generaliseret tonisk-kloniske anfald10,11. Stereotaxisk implantation af elektroder indebærer hjernekirurgi og kræver en væsentlig tid til at forbedre den eksperimentatoren kirurgiske færdigheder for at minimere skaden. Mindre invasive hornhinde elektroder kan forårsage hornhinde slid og tørhed og kræver bedøvelse. Brugen af øre-klip elektroder omgår disse begrænsninger, fordi de kan bruges på gnavere uden operation eller anæstesi og minimal skade. Faktisk, vi fandt, at nuværende leveres til vågen rotter via øret-klip elektroder pålideligt inducerer fase 4-5 tonisk-kloniske anfald og ændrer synaptic proteiner i deres hippocampi10.
For at undersøge den ECS-induceret overflod af synaptic proteiner i de specifikke hjerneregioner af gnavere, er det vigtigt at vælge de eksperimentelle metoder, der er mest egnet til deres påvisning og kvantificering. Subcellulært fraktionering af hjernen giver mulighed for den rå isolation af opløselige cytosole proteiner; Membranproteiner; organelle-grænser proteiner; og selv proteiner i særlige subcellulært strukturer, såsom PSD12,13,14. PSD er en tæt og velorganiseret subcellulært domæne i neuroner, synaptic proteiner er meget koncentreret på og i nærheden af postsynaptiske membran12,13,15. Isolering af PSD er nyttig for studiet af synaptic proteiner beriget på PSD, da dynamiske ændringer i overflod og funktion af postsynaptiske glutamat receptorer, stilladser proteiner og signaltransduktion proteiner i PSD12 , 15 , 16 , 17 er korreleret med synaptisk plasticitet og synaptopathy observeret i flere neurologiske17,18. En tidligere subcellulært fraktionering metode bruges til at rense PSD involveret isolation af de detergent-uopløselige fraktion fra rå membran brøkdel af hjernen ved den differentierede centrifugering af saccharose forløb14, 19. den store udfordring med denne traditionelle metode er, at det kræver store mængder af gnaver brains14,19. Forberedelse af 10-20 gnavere at isolere PSD brøkdel pr. behandling kræver omfattende omkostninger og tid investeringer og er ikke praktisk muligt, hvis der er mange behandlinger.
For at overvinde denne udfordring, har vi tilpasset en enklere metode, der direkte isolerer PSD fraktion, uden saccharose gradient centrifugering20,21, og revideret den for at være gældende for PSD isolation fra hippocampi af en enkelt rotte hjernen. Vores små PSD fraktionering metode giver om 30-50 µg af PSD proteiner fra 2 hippocampi, tilstrækkelige til brug i flere biokemiske analyser, herunder immunoprecipitation og Western blotting. Western blotting viser succesen med vores metode til at isolere PSD ved at afsløre berigelse af postsynaptiske tæthed protein 95 (PSD-95) og udelukkelse af præsynaptiske markør synaptophysin og opløselige cytoplasmatiske protein α-tubulin. Vores ECS induktion og små PSD fraktionering metoder er let at tilpasse til andre gnavere hjerneregioner og giver en relativt enkel og pålidelig måde at evaluere virkningerne af ECS på udtryk for PSD proteiner.

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			<td>08-772B</td>
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electroconvulsive seizures in rats and fractionation of their hippocampi to examine seizure-induced changes in postsynaptic density proteins

Elektrochok beslaglæggelse (ECS) er en eksperimenterende dyremodel af elektrochok terapi, den mest effektive behandling for svær depression. ECS inducerer generaliseret tonisk-kloniske anfald med lav dødelighed og neuronal død og er en meget anvendt model til skærmen anti-epileptisk medicin. Her, vi beskriver en ECS induktion metode i som en kort 55-mA strøm er leveret til 0,5 s hen til Handyr rats 200-250 g i vægt via øret-klip elektroder. Sådanne bilaterale stimulation produceret etape 4-5 kloniske anfald, der varede cirka 10 s. Efter ophør af akut eller kronisk ECS, de fleste rotter kommet for at blive behaviorally skelnes fra humbug "ingen beslaglæggelse" rotter. Fordi ECS hæver globalt hjerneaktivitet, er det også blevet brugt til at undersøge aktivitet-afhængige ændringer af synaptic proteiner og deres effekter på synaptisk styrke ved hjælp af flere metoder. Især subcellulært fraktionering af postsynaptiske massefylde (PSD) i kombination med Western blotting giver mulighed for kvantitativ bestemmelse af overfloden af synaptic proteiner på denne specialiserede synaptic struktur. I modsætning til en tidligere fraktionering metode, der kræver store mængder af gnaver hjerner, beskriver vi her en mindre fraktionering metode til at isolere PSD fra hippocampi af en enkelt rotte, uden saccharose gradient centrifugering. Ved hjælp af denne metode, viser vi, at den isolerede PSD fraktion indeholder postsynaptiske Membranproteiner, herunder PSD95, GluN2B og GluA2. Præsynaptiske markør synaptophysin og opløselige cytoplasmatiske protein α-tubulin blev udelukket fra PSD brøkdel, demonstrerer vellykket PSD isolation. Derudover faldt kronisk ECS GluN2B udtryk i PSD, der angiver, at vores små PSD fraktionering metode kan anvendes til at registrere ændringerne i hippocampus PSD proteiner fra en enkelt rotte efter genetiske, farmakologiske eller mekaniske behandlinger .

Ved hjælp af en detaljeret procedure præsenteres her, en elektrisk stød (55 mA, 100 pulser/s for 0,5 s) leveret via øre-klip elektroder induceret ikke-tilbagevendende etape 4-5 tonisk-kloniske anfald hos rotter (figur 1A-B). En samlet 8 af rotter modtaget akut ECS induktion og vises etape 4-5 tonisk-kloniske anfald. Anfaldene varede omkring 10 s, og alle rotter inddrevet inden for 1-2 min af beslaglæggelse ophør. Forlorne "ingen beslagl...

Watch this Scientific Journal Video about Electroconvulsive Seizures in Rats and Fractionation of Their Hippocampi to Examine Seizure-induced Changes in Postsynaptic Density Proteins at JoVE.com

Electroconvulsive Seizures in Rats and Fractionation of Their Hippocampi to Examine Seizure-induced Changes in Postsynaptic Density Proteins

Elektrochok beslaglæggelse (ECS) er en eksperimenterende dyremodel af elektrochok behandling for svær depression. ECS stimulerer globalt aktivitet i hippocampus, fører til synaptogenesis og synaptisk plasticitet. Her, beskriver vi metoder for ECS induktion i rotter og subcellulært fraktionering af deres hippocampi til at undersøge beslaglæggelse-inducerede ændringer i synaptic proteiner.

Elektrochok anfald hos rotter og fraktionering af deres Hippocampi til at undersøge beslaglæggelse-inducerede ændringer i postsynaptiske tæthed proteiner

Electroconvulsive seizure (ECS) is an experimental animal model of electroconvulsive therapy, the most effective treatment for severe depression. ECS ...

The overall goal of the electroconvlusive seizure induction and subsequent smallscale fractionation of hippocampus is to induce global seizure activity in the rat brain, and to examine seizure activity induced alterations of proteins in the postsynaptic density of the hippocampus. This method can help answer key questions in the neural process of the film, such as which snaptic proteins are regulated in specific brain regions upon seizure, and whether such regulation can contribute to neuroplasticity. The main advantage of this electroconvulsive seizure induction protocol is that we can lively induce lo-bal allevation of law-dons brain activity without neuronal death.Demonstrating the procedure will be myself and Han Gil Jeong from Dr.Hee Jung Chung's laboratory. To begin this procedure, place a male rat in a clean, empty cage with a lid. Let the rat habituate for 30 minutes, then, set the pulse generator for ECS induction.Prepare the pulse generator by pushing the reset button and ensure that the ready button is lit. Make sure that the ear clips are not attached to the pulse generator. Next, press the shock button for a few seconds, then, plug the ear clips into the pulse generator.In this step, wet the ear clips with sterile saline and ensure that they are saturated. Next, wet the rat's ears with sterile saline by wrapping them in saline soaked gauze. Once they are wet, remove the gauze.Attach the clips to the ears at one clip per ear and position them beyond the main cartilage band. Press the shock button for a few seconds and observe the seizure. For the sham, no seizure control, treat the rat identically, but do not deliver the current.As clonus begins, disconnect the ear clips and record the seizure behavior according to a revised, Racine scale of one to five. This includes rearing with four limb clonus at stage four, and rearing and falling with four limb clonus at stage five. The seizure should last approximately 10 seconds.Record the seizure duration using a timer. Following seizure termination, return the rat to its home cage. Monitor the rat for another five minutes to make sure of its recovery from the seizures.Keep it singly housed in the cage, and return the cage to the recovery room. In this procedure, place two hippocampi from one rat onto a 30 milometer tissue culture dish and mince them into small pieces using scissors. After that, transfer the minced hippocampi to a manual glass homogenizer and add one milliliter of iced cold homogenization buffer.Next, insert a round pecil into a glass homogenizer. While the glass homogenizer is on ice, gently and steadily stroke up and down on the pecil, 10 to 15 times for one minute until small pieces of hippocampal tissue disappear. Then, transfer the homogenote to a 1.7 milometer microcentrifuge tube using a one milometer pipet and centrifuge the homogenote at 800 times G for 10 minutes at four degrees Celsius to separate the post nuclear supernatant from the pellet containing insoluble tissue and nuclei.Afterward, transfer 50 microliters and 950 microliters of the S1 fraction to two separate new 1.7 milliliter microcentrifuge tubes and store these tubes on ice. Also, save the P1 fraction pellet on ice. Subsequently, centrifuge the 950 microliter S1 fraction for 10 minutes at 13, 800 times G and four degrees Celsius to separate the supernatant enriched with cytosolic soluble proteins and the pellet enriched with membrane bound proteins, including synaptosomal proteins.Transfer the S2 fraction to a new 1.7 milliliter microcentrifuge and store it on ice. Following that, resuspend the P2 fraction pellet in 498 microliters of ice cold purified water. Add two microliters of one molar heeps to achieve a final concentration of four millimolar heeps.Incubate the suspension at four degrees Celsius with agitation for 30 minutes. After 30 minutes, store the resuspended P2 fraction on ice. In this step, centrifuge the P2 fraction for 20 minutes at 25, 000 times G and four degrees Celsius to separate the lysed supernatant and the lysed pellet.Transfer the LS2 fraction to a new 1.7 milliliter microcentrifuge tube and store it on ice. Then, resuspend the LP1 pellet in 250 microliters at 50 millimolar heeps, mixed with 250 microliters of one percent detergent, and 1 X PBS. Incubate the suspension at four degrees Celsius with gentle agitation for 15 minutes.Afterward, centrifuge the resuspended LP1 pellet for 3 hours at 25, 000 times G and four degrees Celsius to separate the non PSD fraction supernatant from the PSD fraction pellet. Remove the supernatant to a 1.7 milliliter microcentrifuge tube and resuspend the PSD pellet in 100 microliters of 50 millimolar heeps. In this figure, the representative western blots show the protein expression of NMDA receptive subunit GluN2B, ampa receptor, GluA2, and STEP 61 in the S2, P2, and PSD fractions from the hippocampi of sham, no seizure rats, and rats that received acute ECS.The cytoplasmic soluble protein alpha-Tubulin is enriched in the S2 fraction. Synaptophysin is a presynaptic vesicle protein and is enriched in the crude membrane P2 fraction but not in the PSD fraction, whereas PSD-95 is enriched in both the P2 and PSD fractions. Shown here, are the quantification of GluN2B and GluA2 in the P2 and PSD fractions at three and 24 hours after acute ECS.GluN2B and GluA2 expression in the P2 fraction was normalized to alpha-Tubulin in the S2 fraction. In GluN2B and GluA2 expression in the PSD fraction, was normalized to PSD 95 in the PSD fraction. Once mastered, this technique can be done in five to six hours if it is performed properly.Following this procedure, auto-masser like mass, spector-metry, based on protein mix can be performed in order to answer additional questions, what other synaptic proteins are altered by ECS. After its development, this technique paved the way for researchers in the field of neurological disease to explore the underlying mechanism of synaptic dysfunction in order for act of law-dons, trans-gen-ing motors of neurological diseases. After watching this video, you should have a good understanding of how to induce the ECS in law-dons, perform homogenization and PSD fractionation from the hippocampi and to examine alterations of synaptic proteins after ECS.

Research

JoVE Journal

Neuroscience

A General Method for Evaluating Incubation of Sucrose Craving in Rats

En generel metode til evaluering Inkubation af Saccharose Savner hos rotter

For someone on a food-restricted diet, food craving in response to food-paired cues may serve as a key behavioral transition point between abstinence and ...

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

Dobbelte Elektrofysiologiske Optagelser af synaptisk-evoked astrogliale og Neuronal respons i Akut hippocampusskiver

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs ...

Preparation of Synaptic Plasma Membrane and Postsynaptic Density Proteins Using a Discontinuous Sucrose Gradient

Fremstilling af Synaptic plasmamembranen og postsynaptiske Density Proteiner hjælp af en Diskontinuerlig sucrosegradient

Neuronal subcellular fractionation techniques allow the quantification of proteins that are trafficked to and from the synapse. As originally described in ...

The Neuromuscular Junction: Measuring Synapse Size, Fragmentation and Changes in Synaptic Protein Density Using Confocal Fluorescence Microscopy

Den neuromuskulære forbindelse: Måling Synapse Størrelse, fragmentering og ændringer i Synaptic Protein Density Brug Konfokal Fluorescens mikroskopi

The neuromuscular junction (NMJ) is the large, cholinergic relay synapse through which mammalian motor neurons control voluntary muscle contraction. ...

Using Fluorescence Activated Cell Sorting to Examine Cell-Type-Specific Gene Expression in Rat Brain Tissue

The brain is comprised of four primary cell types including neurons, astrocytes, microglia and oligodendrocytes. Though they are not the most abundant ...

Non-invasive Assessment of Changes in Corticomotoneuronal Transmission in Humans

Ikke-invasiv vurdering af ændringer i kortikomotoneuronal transmission i mennesker

The corticospinal pathway is the major pathway connecting the brain with the muscles and is therefore highly relevant for movement control and motor ...

Simultaneous Two-photon In Vivo Imaging of Synaptic Inputs and Postsynaptic Targets in the Mouse Retrosplenial Cortex

Simultaneous Two-photon In Vivo Imaging of Synaptic Inputs and Postsynaptic Targets in the Mouse Retrosplenial Cortex

Samtidig To-foton<em&gt; In vivo</em&gt; Imaging af synaptisk indgange og Postsynaptiske Mål i Mouse retrospenialis Cortex

This video shows the craniotomy procedure that allows chronic imaging of neurons in the mouse retrosplenial cortex (RSC) using in vivo two-photon ...

The Optical Fractionator Technique to Estimate Cell Numbers in a Rat Model of Electroconvulsive Therapy

Den optiske fraktioneringsteknik til estimering af celle tal i en rotte model for elektrokonvulsiv terapi

Stereological methods are designed to describe quantitative parameters without making assumptions about size, shape, orientation and distribution of cells ...

Quantifying Acute Changes in Renal Sympathetic Nerve Activity in Response to Central Nervous System Manipulations in Anesthetized Rats

Kvantificere akutte ændringer i Renal sympatiske Nerve aktivitet som svar på centralnervesystemet manipulationer i bedøvede rotter

Renal sympathetic nerve activity (RSNA) and mean arterial pressure are important parameters in cardiovascular and autonomic research; however, there are ...

Quantitative Measurement of Intrathecally Synthesized Proteins in Mice

Kvantitativ måling af Intratalalt syntetiseret protein i mus

Cerebrospinal fluid (CSF), a fluid found in the brain and the spinal cord, is of great importance to both basic and clinical science. The analysis of the ...