Name: quantifying infra-slow dynamics of spectral power and heart rate in sleeping mice
Uploaded: 2017-08-02T14:00:00
Description: Watch this Scientific Journal Video about Quantifying Infra-slow Dynamics of Spectral Power and Heart Rate in Sleeping Mice at JoVE.com

Vi takker alle laboratoriemedlemmer for deres bidrag til at skrive og omhyggeligt læse dette manuskript. Vi er taknemmelige for Paul Franken for at stimulere diskussioner, Dr. Gisèle Ferrand for nyttige kommentarer til den kirurgiske protokol og Dr. Jean-Yves Chatton for at levere de originale Labview-eksekverbare filer til støjeksponeringen. Finansiering blev ydet af det schweiziske nationale videnskabsstiftelse (Grants 31003A_146244 og 31003A_166318) og Etat de Vaud.

Alle forsøgsprocedurer blev udført i overensstemmelse med Lausanne Animal Care Committee og Service de la Consommation et des Affaires Vétérinaires af Canton de Vaud. 1. Kirurgi til EEG / EMG-EKG-optagelser <ol><li> Dyrhus og udvælgelse. <ol><li> Opbevares dyr (C57Bl / 6J, 7 - 9 uger, 25 - 30 g) i en 12: 12-h mørk / lyscyklus, enkeltindrettet og under standardbetingelser (40% fugtighed, 22 ° C) med mad og vand Tilgængelig ad libitum . </li><li> Brug kun ma...

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Her viser vi, hvordan man etablerer en kontinuerlig tidsmæssig profil for ikke-REM-søvn, der integrerer EEG-, EMG- og EKG-variabler. Dette er et første skridt i retning af at udvikle en integreret beskrivelse af musesøvn, som kan hjælpe med at identificere den tidligere uigenkaldelige tidsskala, over hvilken høj og lav modstandsdygtighed over for støj er organiseret under ikke-REM-søvn 1 . En lignende tidsmæssig struktur blev også beskrevet i human ikke-REM søvn gennem en analog analys...

Sygeplejerske søvn er en adfærdsmæssig hvilestilstand og modstandsdygtighed over for miljømæssige stimuli. På trods af denne tilsyneladende ensartethed indikerer polysomnografiske og autonome parametre, at søvn bevæger sig mellem kvalitativt og kvantitativt forskellige neurale og somatiske tilstande på forskellige tidsmæssige og rumlige skalaer 1 . I løbet af minutter til mange minutter, skifter kontakten mellem ikke-REM og REM søvn. Non-REM-søvn ledsages af stor amplitude lavfrekvent aktivitet i EEG med en spektral peak omkring ~ 0,5-4 Hz, mens REM-søvn viser regelmæssig EEG-aktivitet i theta-båndet (6 - 10 Hz) sammen med Muskel atonia 2 . Inden for ikke-REM søvn cykler mennesker gennem lys (S2) og dyb langsom bølgesøvn (SWS). Som deres navngivning angiver, viser disse to faser henholdsvis lavere og højere ophobningstærskler 3 , 4 , og de afviger primært i tætheden af ​​lavfrekvensEncycortical EEG-effekt, kaldet slow-wave-aktivitet (SWA; 0,75-4 Hz). Ikke-ensartethed fortsætter i løbet af individuelle udfald af S2 og SWS i minut- til sub-anden-tidsskalaen, som udførligt dokumenteret af den variable tilstedeværelse af SWA i løbet af en bout 5 , 6 , men også ved EEG og feltpotentiale rytmer ved Højere frekvenser, herunder spindelbølger i sigma båndet (10-15 Hz) og gamma rytmer (80 - 120 Hz) (for en anmeldelse, se 7 , 8 , 9 , 10 ). I stedet for at være subtile skifter disse variationer den sovende kortikale tilstand hos mennesker til ekstremerne af spektret. For ikke-REM-søvn spænder disse fra en overvejelse af SWA til stater, der tilnærmer sig vævlignende aktivitet, fordi de indeholder en betydelig andel af højfrekvente komponenter 11 <suP&gt;, 12 . Hos gnavere og katte, selvom ikke-REM-søvn ikke er opdelt i stadier, fremkommer en kort periode kaldet mellemliggende søvn (IS) før REM-søvnbegyndelsen 13 . Under IS indleder REM søvnfunktioner såsom hippocampal theta aktivitet og ponto-geniculo-occipitale bølger, mens ikke-REM søvn signaturer, såsom spindelbølger og SWA, stadig er til stede, hvilket indikerer en blanding mellem de to søvntilstande 14 , 15 . Ikke desto mindre kan IS være funktionelt særskilt, fordi det moduleres af antidepressiva 16 og gennem ny objektpræsentation under forudgående vævning 17 , og det bidrager til at indstille ophobningstærsklen 18 . Endvidere viser statens rumtegninger af EEG- og EMG-parametre for frit bevægelige rotter en klynge af punkter 14, som er kontinuerlig mellem ikke-REM-søvn, REM-søvn og vækkelse. Der er også sporadiske fald i SWA uden at indtaste vækkelse eller REM søvn, hvilket fører til betydelige udsving i den relative tilstedeværelse af lav- og højfrekvenskomponenterne under en konsolideret ikke-REM-søvn om 14 , 19 , 20 . Endelig forekommer variable variabler af SWA og højere frekvensrytmer under ikke-REM-søvn ikke kun i tid, men viser også regionale forskelle i amplitude og synkronisering mellem kortikale områder 19 . Mammalian non-REM søvn er langt fra ensartet. Hvorvidt en sådan ikke-ensartethed fører til stater, der adskiller sig i funktion og adfærdsmæssige attributter, er ikke klart. I flere typer søvnforstyrrelser er kontinuerlig søvn forstyrret af spontane vækkelser og uhensigtsmæssig motorisk adfærd. Desuden viser spektrale analyser ændringer i den relative tilstedeværelse af højere frekvenser i EEG 21Og i autonome parametre, såsom vejrtrækninger og hjerteslag 22 . Den ordnede sekvens af stabile søvntilstande er således forstyrret, og elementer af kortisk og / eller autonom oprykning trænger på en ukontrolleret måde. Derfor er forståelse af kontinuum for søvntilstanden mulig for sygdommen. Desuden er forstyrrelsen af ​​søvn ved miljøstøj i bymiljøer forbundet med generelle sundhedsrisici, hvilket gør det afgørende at identificere øjeblikke med øget sårbarhed i søvn 23 . Adfærdsmæssige opvågningsforsøg hos sovende mennesker indikerer, at det er sværest at vågne op fra SWA-domineret ikke-REM-søvn (stadium S3), mens lys ikke-REM-søvn (stadium S2) og REM-søvn viser sammenlignelige og lavere ophobningstærskler 4 . Den kortikale behandling af korte lydstimuli varierer væsentligt mellem REM-søvn, S2 og S3 24 ,25 , hvilket indikerer at statsspecifikke kortikale aktivitetsmønstre modulerer de første faser af sensorisk behandling. For ikke-REM søvn hos mennesker varierer tilbøjelighed til at vågne op som følge af støj med tilstedeværelsen af ​​spindelbølger og alfa-rytmer i EEG 26 , 27 , 28 . Thalamocortical rytmicitet under spindler ledsages af øget synaptisk hæmning ved både thalamins og kortikale niveauer, hvilket antages at bidrage til dæmpning af sensorisk behandling 7 . Hvordan er støjbestandige og sårbare søvnperioder organiseret i tide, og hvad er deres determinanter? I både mus og menneske identificerede vi for nylig en infrarad, 0,02-Hz oscillation i neurale rytmer. Afhængig af fasen af ​​denne 0,02 Hz-svingning viste musene variabel reaktivitet over for eksterne stimuli enten ved at vågne op eller sove throuGh støjen. Interessant nok var denne oscillation korreleret med hjerteslagets hastighed, hvilket indikerer, at det autonome nervesystem deltager i moduleringen af ​​søvnens sårbarhed over for eksterne stimuli 1 . Hukommelsesrelaterede hippocampale rytmer blev også organiseret inden for denne rytme, og mest påfaldende var dets styrke korreleret med kvaliteten af ​​hukommelseskonsolidering hos mennesker. 0,02 Hz-svingningen ser således ud til at være et organiserende princip for gnaver og human ikke-REM-søvn, som modulerer både følsomhed over for miljøet og intern hukommelse. Dette fremhæver igen behovet for multiparametriske og kontinuerlige vurderinger af søvntilstande for at genkende deres funktionalitet og for at identificere steder med potentiel sårbarhed. Her præsenterer vi en procedure for at udvinde bølgeformen af ​​disse dynamikker, herunder kirurgisk implantation af mus til kombineret EEG / ECoG og EMG-EKG målinger, eksponering for sensoriske stimuli, enNd analyse rutiner. Denne procedure tilvejebringer et grundlag for visning af søvn som en kontinuerligt varierende, dog højorganiseret årvågenhedstilstand, hvor forskellige grundlæggende søvnfunktioner udføres sekventielt. Mere generelt er proceduren anvendelig for fremgangsmåder, der sigter mod at udtrække de spektrale og autonome træk, der går forud for et adfærdsmæssigt resultat under søvn i både sundhed og sygdomstilstande.

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		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gold wire</td>
			<td>CMSA</td>
			<td>T.69 5gr</td>
			<td>http://www.cmsa.ch/en/</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hemostatic sponge</td>
			<td>Pfizer</td>
			<td>Gelfoam</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">iodine-based disinfectant (Betadine)</td>
			<td>Mundipharma</td>
			<td>standart solution 60mL</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Komet drill steel 1/005PM104</td>
			<td>UNOR AG</td>
			<td>22310</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Matlab Analysis Software</td>
			<td>MathWorks</td>
			<td>R2016b</td>
			<td>https://ch.mathworks.com/products/matlab.html</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microdrill</td>
			<td>Fine Science Tools</td>
			<td>96758</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mouse Gas Anesthesia Head Holder</td>
			<td>Kopf Instruments</td>
			<td>Model 923-B</td>
			<td>http://kopfinstruments.com/product/model-923-b-mouse-gas-anesthesia-head-holder/</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ophtalmic ointment</td>
			<td>Pharmamedica</td>
			<td>VITA-POS</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Paladur (liquid)</td>
			<td>UNOR AG</td>
			<td>2260215</td>
			<td>for dental cement</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Palavit (powder)</td>
			<td>UNOR AG</td>
			<td>5410929</td>
			<td>for dental cement</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Small Animal Stereotaxic Frame</td>
			<td>Kopf Instruments</td>
			<td>Model 930</td>
			<td>http://kopfinstruments.com/product/model-930-small-animal-stereotaxic-frame-assembly/</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Soldering wire</td>
			<td>Stannol</td>
			<td>593072</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Temperature controller - Mini rectal probe</td>
			<td>Phymep</td>
			<td>4090502</td>
			<td>http://www.phymep.com/produit/dc-temperature-controller/</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Temperature controller- heating pad</td>
			<td>Phymep</td>
			<td>4090205</td>
			<td>http://www.phymep.com/produit/dc-temperature-controller/</td>
		</tr>
	</tbody>
</table>

quantifying infra-slow dynamics of spectral power and heart rate in sleeping mice

Tre årvågenhed stater dominerer pattedyrs liv: vågenhed, ikke-hurtig øjenbevægelse (ikke-REM) søvn og REM søvn. Da flere neurale korrelater af adfærd er identificeret i fritflytende dyr, bliver denne tredobbelte underopdeling for forenklet. Under vågenhed definerer ensembler af globale og lokale kortikale aktiviteter sammen med perifere parametre som pupildiameter og sympatovagalbalance forskellige grader af ophidselse. Det er fortsat uklart, i hvilken grad søvn også udgør et kontinuum af hjernestater - inden for hvilken grad af modstandsdygtighed overfor sensoriske stimuli og arousability og måske andre søvnfunktioner varierer gradvist - og hvordan perifere fysiologiske tilstande varierer. Forskning, der fremmer metoderne til overvågning af flere parametre under søvn, samt tilskrivning af konstellationer af disse funktionelle egenskaber, er centralt for at forbedre vores forståelse af søvn som en multifunktionel proces, hvor mange gavnlige virkninger skal være executed. At identificere nye parametre, der karakteriserer søvntilstand, vil åbne muligheder for nye diagnostiske veje i søvnforstyrrelser. Vi præsenterer en procedure for at beskrive dynamiske variationer af mus-ikke-REM søvntilstande via den kombinerede overvågning og analyse af elektroencefalogram (EEG) / elektrokortikogram (ECoG), elektromyogram (EMG) og elektrokardiogram (EKG) signaler ved anvendelse af standardpolysomnografiske optagelsesteknikker. Ved hjælp af denne tilgang fandt vi, at mus-ikke-REM-søvn er organiseret i cykler af koordinerede neurale og hjerteoscillationer, der genererer successive 25-s intervaller med høj og lav sårbarhed overfor eksterne stimuli. Derfor koordineres centrale og autonome nervesystemer for at danne adfærdsmæssigt forskellige søvntilstande under konsolideret ikke-REM-søvn. Vi præsenterer kirurgiske manipulationer for polysomnografisk ( dvs. EEG / EMG kombineret med EKG) overvågning for at spore disse cykler i den frit sovende mus, analysen til quantiFy deres dynamik, og de akustiske stimuleringsprotokoller til at vurdere deres rolle i sandsynligheden for at vågne op. Vores tilgang er allerede blevet udvidet til menneskelig søvn og løfter om at løse fælles organiseringsprincipper for ikke-REM søvntilstande hos pattedyr.

Figur 2 A (øverste panel) viser 100 min. Spontane søvnvågens adfærd registreret ved hjælp af polysomnografiske elektroder implanteret som beskrevet (se figur 1 ). Øgene og faldene i EEG- og EMG-amplitude ved ikke-REM-søvnbetændelse er tydeligt synlige. Intermitterende REM søvn er præget af et fald i EEG amplitude og et yderligere fald i EMG tone, der ikke er synlig på denne komprimerede tidsskala. Zoo...

Watch this Scientific Journal Video about Quantifying Infra-slow Dynamics of Spectral Power and Heart Rate in Sleeping Mice at JoVE.com

Quantifying Infra-slow Dynamics of Spectral Power and Heart Rate in Sleeping Mice

Her præsenterer vi eksperimentelle og analytiske procedurer for at beskrive den tidsmæssige dynamik af de neurale og hjertevariablerne af ikke-REM-søvn hos mus, som modulerer søvnresponsen over for akustiske stimuli.

Kvantificering af infra-langsom dynamik for spektral kraft og hjertefrekvens i sovende mus

Three vigilance states dominate mammalian life: wakefulness, non-rapid eye movement (non-REM) sleep, and REM sleep. As more neural correlates of behavior ...

The overall goal of this procedure, is to describe how neural and cardiac activities are coordinated during mouse non REM sleep. Through monitoring and analyzing electroencephalographic, electromyographic, and electrocardiographic signals. This method can help answer key questions in the sleep field.Such as how sleep reconciles two essential and evolutionarily preserved needs. Which are to be both continuous and fragile. The main advantage of this technique is that it uses standard polysomnography to score non REM sleep in a novel way that is based on the variable behavioral responses to external stimuli.We first had the idea for this method when we observed that sleeping mice showed variable responses to noise such as waking up and sleeping through. Generally individuals new to this method will struggle because it combines surgical skill, bare minimal experimentation, and advancing analysis. Visual demonstration of this method is critical as several essential steps are new to the field.In particular concerning the behavioral design and data analysis. The implications of this technique extend towards diagnosis of sleep disorders in humans. Because it identifies the moments of sleep that are highly fragile.To begin the procedure, prepare to EEG ecogeolectrodes using 0.5 centimeter long pieces of gold wire, each soldered on top of a gold plated steel screw. Then clean them in 70%ethanol. Next prepare two EMG-ECG electrodes with three to four centimeter long gold wires.Bend the wires at a 90 degree angle one centimeter from one end and prepare a coil at the other end. Between the two ends bend the wire to create a small curvature that corresponds to the surface profile of the bone between the cerebellum and lambda. After that prepare a six channel female to male head connector by covering the connector of both female and male pins at the base with tape.Then add to the pins at the corners some tin to facilitate the soldering process. In this step, place an anesthetized animal on the stereotaxic apparatus by positioning the mouth. This will fix the mouse head.Then fix the head by positioning the ear bars at the skull with their blunt reverse ends. Subsequently lift the skin at the center of the skull and gently cut the lifted portion of the skin along the mid line from the top of the neck to the level of the eyes. Then remove the scalp.Ensure that the window is large enough to clearly see the bregma and the lambda fissures of the skull. Fix the skin on both sides with bulldog serafines to ensure access to the bone. Next carefully remove the conjunctive tissue with a scalpel.Clean the area with iodine based disinfectant and dry the skull with an antiseptic swab. Then using a sharp scalpel blade, scratch the skull to obtain a cleaned and mattified bone surface. Using only the scalpel tip, scratch a grid like meshwork of groves with a distance of one to two millimeters between the grooves.Afterward, perform four craniotomies in the skull at specific locations with a microdrill. While drilling, blow away the bone dust using a pasteur pipette and clean any bleeding with antiseptic swabs. If bleeding occurs, ensure that it is stopped completely before resuming the process.Use a hemostatic sponge to accelerate hemostasis. Fix the screw in a hemostatic clamp. On the left hemisphere, screw two gold plated screws through the craniotomies.Hold the clamp vertically above the craniotomy and rotate the screw while not deviating from the vertical position. Gently open the hemostatic clamp to release the screw. On the right hemisphere screw the previously prepared electrodes through the craniotomies.Add a drop of glue on the middle part of the skull to ensure tight adhesion of the left EMG-ECG. Carefully lift the border of the skin from the neck muscles with forceps and insert the EMG-ECG wires with the coiled ends in the muscles. To detect the heart ECG signals during sleep, ensure that the EMG-ECG wire is inserted into the muscle to a depth of about 0.8 to one centimeter.Position the left EMG-ECG next to the posterior left anchoring screw. Repeat the procedure to insert the right EMG-ECG. Position the right EMG-ECG next to the anterior left anchoring screw.Ensure that the loop ends are placed as far as possible from each other. It is important that the EMG-ECG wires are inserted deeply enough into the muscle in order for the heart rate to be detected reliably. Using a spatula apply the two component epoxy glue to the skull between and around the screws.Let it dry in the light but protect the eyes of the animal from excessive lighting. Try to place the connector pins as closely as possible to minimize the height of the implant using a small crocodile lamp. Solder the four pins at the corner of the connector to the four wires.Minimize the time in contact with the soldering tip as this rapidly heats the screws. Next, fill the space between the glue and the connector with dental cement and cover the soldered parts. Create smooth surface and avoid sharp edges that could hurt the animal.In addition, avoid touching the skin as this leads to itching. Now remove the bulldog serafines. Disinfect the wound with iodine based disinfectant.If necessary, close the wound with suture points in front and behind the connector. Afterwards, monitor the animal every day for a week and look for weight loss, reduced or abnormal activity, and signs of infection. Five to six days after surgery, connect the recording cable to the head connector on the animal, leaving it in its home cage.Wait an additional four to five days before the start of recording so that the animal is habituated to the condition and sleeps naturally. With a commercial polysomnographic software, record EEG and EMG-ECG data over 48 hours using typical settings such as 2000 x gain, a 2000 hertz sampling rate at acquisition, down sampled to 200 hertz after acquisition. Apply a 0.7 hertz high pass filter for all the EEG traces and a 10 hertz high pass filter for all the EMG-ECG traces.In this demonstration, the mouse has been sleeping for more than 40 seconds. Here are the characteristic low frequency, high amplitude signals in the EEG and the low muscle tone in the EMG traces. It is important that the mouse sleep stably for at least 40 seconds before the noise is played to ensure a long base line for the analysis of sleep, prior to wake up or sleep through.Now an acoustic stimulus, here a white noise, will be delivered. In this case, the mouse wakes up within 10 seconds. Note that the EEG desynchronizes when the mouse wakes up.In another case, the mouse does not wake up, even though the noise has been delivered for more than 20 seconds. Note that the EEG does not desynchronize. Shown here are the raw traces of the EEG in black and the corresponding EMG-ECG signals in gray, for 40 seconds before the noise onset and during the 20 seconds of noise.This is a sleep through event. And this is an example of the wake up event. The corresponding analyzed data for sigma power dynamics in the 40 seconds window before noise onset are shown here for sleep through and wake up.The key result of this analysis is that the sigma power is at its trough before noise onset for a sleep through event and at its peak for a wake up event. In contrast to the wake up and sleep through events presented before, we discarded the events with an early transition into REM sleep. And we also discarded the events with an early wake up before noise onset.While attempting this procedure, it's important to ensure that EMG signal detects the heart rate throughout non REM sleep. Moreover remember that the analysis procedure needs to be understood and implemented in a software script to extract the infra-slow oscillation. Following this procedure, other methods, such as local electrophysiological recordings, or imaging approaches can be performed to follow activity patterns in defined brain areas.After redevelopment, this technique paves the way for researchers in the field of human sleep to explore how neural and cardiac activities are organized in the different sleep stages in healthy sleepers and in sleep disorder patients. After watching this video, you should have a good understanding of how to score and analyze non REM sleep retrospectively based on the behavioral reactivity of the sleeping mouse to an acoustic stimulus.

Research

JoVE Journal

Neuroscience

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