Name: recording gamma band oscillations in pedunculopontine nucleus neurons
Uploaded: 2016-09-14T15:00:00
Description: Watch this Scientific Journal Video about Recording Gamma Band Oscillations in Pedunculopontine Nucleus Neurons at JoVE.com

This work was supported by core facilities of the Center for Translational Neuroscience supported by NIH award P20 GM103425 and P30 GM110702 to Dr. Garcia-Rill. This work was also supported by grants from FONCYT-Agencia Nacional de Promoci&#242;n Cient&#236;fica y Tecnol&#242;gica; BID 1728 OC.AR. PICT-2012-1769 and UBACYT 2014-2017 #20120130101305BA (to Dr. Urbano).

Alle forsøksprotokoller ble godkjent av Institutional Animal Care og bruk komité ved University of Arkansas for Medical Sciences (Protocol nummer # 3593) og var i samsvar med National Institutes of Health retningslinjer for omsorg og bruk av forsøksdyr. 1. Utarbeidelse av Standard-kunstig cerebrospinalvæsken (aCSF) <ol><li> Utarbeidelse av Stock Solution A <ol><li> Legg 700 ml destillert vann til en ren 1 L beger før tilsetning av kjemikalier. </li><li> Under kontinuerlig omrøring et vol...

<ol>
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PPN nevroner har iboende egenskaper som tillater dem å fyre aksjonspotensialer på beta / gamma band frekvenser under in vivo-opptak fra dyr som er våken eller under REM-søvn, men ikke under langsom bølge søvn 2,3,5,13-17. Andre forfattere har vist at hjernestammen transections på flere fremre nivåer enn PPN redusert gamma frekvenser under EEG opptak. Men når hjernestammen lesjoner posterior til hvor denne kjernen ligger, direkte stimulering av PPN tillatt manifestasjon av kortikale gamma akt...

PPN kjernen er anatomisk inkludert i hale mesencefalisk tegmentum. Den PPN er en viktig del av RAS en. Den PPN deltar i opprettholdelsen av atferds aktiverte tilstander (dvs., våkner, REM-søvn) 2. Elektrisk stimulering av den PPN in vivo indusert rask svingning (20 - 40 Hz) i det kortikale EEG 3, mens bilaterale PPN lesjoner i rotte reduseres eller elimineres REM-søvn 4. Mens et flertall av PPN nevroner brann aksjonspotensialer på beta / gamma-band frekvens (20-80 Hz), noen nevroner present lave priser av spontan tenning (&lt;10 Hz) 5. Videre synes PPN å være involvert i andre aspekter av atferd som motivasjon og oppmerksomhet 6. Direkte høy frekvens (40 - 60 Hz) 7 elektrisk stimulering av PPN kjernen i decerebrate dyr kan fremme bevegelse. I de senere år har dype hjernestimulasjon (DBS) av PPN blitt anvendt for å behandle pasienter som lider from lidelser som involverer ganglag underskudd som Parkinsons sykdom (PD) 8. Tidligere rapporter har vist at nesten alle PPN nevroner kan skyte aksjonspotensialer ved gamma band frekvens når depolarized bruker firkant strømpulser 9. På grunn av den drastiske aktivering av spenningsstyrte kaliumkanaler under firkant pulser depolarisering opp eller under -25 mV. Som en konsekvens av dette ble det ikke robuste gamma oscillasjoner observert etter blokkering aksjonspotensialer generering ved hjelp av tetrodotoksin 10. I et forsøk på å omgå et slikt problem, en - ble to sekunder lang depolariserende dagens ramper brukt. Ramper gradvis depolarized membranpotensialet fra hviler verdier opp til 0 mV, mens delvis inaktivere spenningsstyrte kaliumkanaler. Klare gamma bandet membran svingninger var tydelig innenfor spennings avhengighet vinduet av høy terskel kalsiumkanaler (dvs. mellom -25 mV og -0 mV) 10. I konklusjonen, gamma bandet aktivitety ble observert i PPN neuroner 9, og både P / Q-og N-type spenningsstyrte kalsiumkanaler må aktiveres for å generere gammabånd svingninger i PPN 10. En rekke studier bestemmes av plasseringen av høy terskelkalsiumkanaler i PPN neuroner. Injisering kombinasjonen av fargestoffer, viste proporsjonal fluorescens bildebehandling kalsium transienter gjennom spenningsstyrte kalsiumkanaler som aktiveres i forskjellige dendritter når depolarized med dagens ramper 11. Iboende egenskaper PPN nevroner har vært foreslått å tillate samtidig aktivering av disse cellene under våkne og REM søvn, og dermed indusere høyfrekvente oscillerende neuronal aktivitet mellom RAS og thalamocortical sløyfer. Slike langrekkende interaksjon anses å støtte en hjerne tilstand som kan sikkert vurdere verden rundt oss på en kontinuerlig basis 12. Her beskriver vi eksperimentetal vilkår som er nødvendige for å generere og opprettholde gamma båndet oscillasjon i PPN celler in vitro. Denne protokollen er ikke blitt beskrevet tidligere, og vil bidra til en rekke grupper for å studere indre membranegenskaper som medierer gamma bånd aktivitet ved andre hjerneområder. Videre kan eksisterende trinn fører til den feilaktige konklusjon at gamma båndet aktivitet ikke kan genereres i disse celler.

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		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Microscope</td>
			<td>Nikon</td>
			<td>Eclipse E600FN</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Micromanipulator</td>
			<td>Sutter Instruments</td>
			<td>ROE-200</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Micromanipulator</td>
			<td>Sutter Instruments</td>
			<td>MPC-200</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Amplifier</td>
			<td>Molecular Devices</td>
			<td>Multiclamp 700B</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">A/D converter</td>
			<td>Molecular Devices</td>
			<td>Digidata 1440A</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Heater</td>
			<td>Warner Instruments</td>
			<td>TC-324B</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Pump</td>
			<td>Cole-Parmer</td>
			<td>Masterflex L/S 7519-20</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Pump cartridge</td>
			<td>Cole-Parmer</td>
			<td>Masterflex 7519-85</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Pipette puller</td>
			<td>Sutter Instruments</td>
			<td>P-97</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Camera</td>
			<td>Q-Imaging</td>
			<td>RET-200R-F-M-12-C</td>
			<td></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;">Vibratome</td>
			<td>Leica Biosystems</td>
			<td>&#160;Leica VT1200 S</td>
			<td></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;">Refrigeration system</td>
			<td>Vibratome Instruments</td>
			<td>900R</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">microscope</td>
			<td>Nikon</td>
			<td>Eclipse E600FN</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">micromanipulator</td>
			<td>Sutter Instruments</td>
			<td>ROE-200</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">micromanipulator</td>
			<td>Sutter Instruments</td>
			<td>MPC-200</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">amplifier</td>
			<td>Molecular Devices</td>
			<td>Multiclamp 700B</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">A/D converter</td>
			<td>Molecular Devices</td>
			<td>Digidata 1440A</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">heater</td>
			<td>Warner Instruments</td>
			<td>TC-324B</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">pump</td>
			<td>Cole-Parmer</td>
			<td>Masterflex L/S 7519-20</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">pump cartridge</td>
			<td>Cole-Parmer</td>
			<td>Masterflex 7519-85</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">pipette puller</td>
			<td>Sutter Instruments</td>
			<td>P-97</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">camera</td>
			<td>Q-Imaging</td>
			<td>RET-200R-F-M-12-C</td>
			<td></td>
		</tr>
	</tbody>
</table>

recording gamma band oscillations in pedunculopontine nucleus neurons

Synaptiske efferents fra PPN er kjent for å modulere neuronal aktivitet av flere intralaminar thalamic regioner (for eksempel den centrolateral / parafascicular; Cl / Pf kjernen). Aktiveringen av enten PPN eller Cl / Pf kjerner in vivo har blitt beskrevet for å indusere opphisselse av dyret, og en økning i gamma båndet aktivitet i det kortikale elektroencefalogram (EEG). De cellulære mekanismer for generering av gammabånd svingninger i retikulære aktivere systemet (RAS) nevronene er de samme som de som finnes for å generere gammabånd svingninger i andre hjerner kjerner. I løpet av strøm-klemme opptak av PPN nevroner (fra parasagittal skiver fra 9-25 dager gamle rotter), bruk av depolariserende firkant trinn aktiveres hurtig spenningsavhengige kaliumkanaler som hindret PPN neuroner fra å bli depolarisert utover -25 mV. Injisering 1-2 sek lang depolariserende dagens ramper gradvis depolarized PPN membran potensielle resting verdier mot 0 mV. Imidlertid injisere depolariserende firkantede pulser generert gamma-bånd svingninger av membranpotensialet som viste å være mindre i amplitude sammenlignet med de svingninger som genereres av ramper. Alle forsøkene ble utført i nærvær av spenningsstyrte natriumkanaler og raske synaptiske reseptorer blokkere. Det har vist seg at aktiveringen av høy terskelspenningsavhengige kalsiumkanaler som ligger til grunn gamma-bånd oscillerende aktivitet i PPN neuroner. Spesifikke metodiske og farmakologiske tiltak er beskrevet her, gi de nødvendige verktøy for å indusere og opprettholde PPN subthreshold gamma bandet pendling in vitro.

I første omgang ble gamma svingninger fremkalt ved hjelp av firkant strømpulser. Nåværende klemme opptak av PPN neuroner i nærvær av synaptiske blokkere og TTX ble kontinuerlig overvåket for å sikre at hvilende membranpotensial ble holdt stabil ved ~ -50 mV (figur 1A). To andre langfirkant strømpulser ble injisert intracellulært av plasteret klemmen forsterkeren gjennom innspillings Pipette, øker deres amplitude fra 200 til 600 pA pA (figur 1A).</stron...

Watch this Scientific Journal Video about Recording Gamma Band Oscillations in Pedunculopontine Nucleus Neurons at JoVE.com

Recording Gamma Band Oscillations in Pedunculopontine Nucleus Neurons

Den pedunculopontine nucleus (PPN) ligger i hjernestammen og dets nerveceller er maksimalt aktivert under waking og Rapid Eye Movement (REM) søvn hjernetilstander. Dette arbeidet beskriver eksperimentell tilnærming til å registrere in vitro gamma bandet subthreshold membran svingning i PPN nevroner.

Opptak Gamma Band Svingninger i Pedunculopontine Nucleus Nerveceller

Synaptic efferents from the PPN are known to modulate the neuronal activity of several intralaminar thalamic regions (e.g., the ...

The overall goal of this demonstration is to describe a method for detecting high threshold and subthreshold oscillations in pedunculopontine neurons. This method can help answer questions about, for example, how cells maintain their firing frequency. How do populations maintain their firing frequency?We know that circuits cannot maintain gamma band or 40 to 60 per second oscillations. Therefore, you require intrinsic membrane properties to maintain that kind of firing frequency. Demonstrating the procedure will be Susan Mahaffey and Dr.Stasia D'Onofrio.To prepare standard aCSF, first prepare stock solutions A and B and keep them refrigerated at four degrees Celsius for up to two weeks. Before each experiment, mix 50 milliliters of stock A with 50 milliliters of stock B.Then bring the mixture to one liter with distilled water and oxygenate it with carbogen at room temperature for at least 30 minutes. Transfer to a beaker for stirring and oxygenate.After bubbling, add the calcium chloride. Next, prepare sucrose aCSF by preparing stock solution C.Before each experiment, mix 300 milliliters of stock C with 600 milliliters of distilled water and oxygenate it with carbogen at room temperature for at least 30 minutes. After that, transfer 100 milliliters of sucrose aCSF solution to a clean beaker, place it on ice, and keep it oxygenated with carbogen.Now, fill up a vibratome cutting chamber with sucrose aCSF and keep it oxygenated. Turn on the glycerol-based refrigerating system coupled to the cutting chamber and wait 15 minutes to allow it to cool down to about four degrees Celsius. Next, remove the pup's brain by placing a spatula under the olfactory bulb and gently pushing it out from the most rostral area towards the most caudal area.Subsequently, place the brain into the ice-cold oxygenated sucrose aCSF. Make a parasagittal cut on the right hemisphere to remove approximately 1/3 of the hemisphere. Then, glue the trimmed side of the brain onto the metal disk.Subsequently, fix the metal disk to the vibratome cutting chamber and slice 400 micron sagittal sections containing the PPN. Keep the PPN slices at room temperature for 45 minutes prior to whole-cell patch clamp recording. In this procedure, transfer a slice to the submersion chamber, place a screen on top of it to hold the slice down, and perfuse it at 1.5 milliliters per minute with oxygenated aCSF containing the selected receptor antagonist.In order to isolate intrinsic membrane properties, what you need to do is block the fast synaptic transmission around the cell and also block the ability to generate action potentials, so you have to use synaptic blockers and tetrodotoxin, or TTX, in order to block action potentials. That way, the cell that you are patching is the one that generates those properties, and no synaptic input is responsible for those, and that's what we're studying here, intrinsic membrane property. After that, fill the recording patch pipette with intracellular high potassium solution.Insert the pipette in the headstage holder. Next, apply a small positive pressure using a one milliliter syringe connected to the pipette holder. Using a 4x objective together with the near infrared differential interference contrast optics, locate the PPN nucleus, which is dorsal to the superior cerebellar peduncle.Slowly lower the recording pipette to the PPN nucleus using a mechanical micromanipulater. Then position the recording pipette in the PPN pars compacta, which is located immediately dorsal to the posterior end of the peduncle. Using a 40x water immersion lens, bring the recording pipette in contact with the PPN neuron, and rapidly apply negative suction to form a seal with the cell.Use voltage clamp seal software to monitor the pipette resistance during negative suction. Then slowly increase the negative pressure. When the resistance value of the pipette reaches 80 to 100 megaohms, rapidly change the holding potential to 50 millivolts and release the negative pressure.Apply negative pressure continuously until the cell membrane is ruptured and electrical access is achieved in the wholesale configuration. Before suction, the resistance is low, in this case, 2.5 megaohms. As suction is applied, step amplitude decreases towards holding.When the seal is formed, note the resistance increases to 167 megaohms. With additional suction, a gigaseal is formed. Resistance briefly increases to 1.2 gigaohms before acquiring wholesale access.Continuously monitor the resting membrane potential of the PPN neuron being recording. If the resting membrane potential shifts towards depolarizing or hyperpolarizing values, apply a small amount of direct current to keep the optimal potential at 50 millivolts. The reason why we use current ramps is so that we can slowly depolarize the membrane without activating potassium channels that keep you from depolarizing the membrane sufficiently high to reach the threshold of these high-threshold calcium channels that we're studying.Shown here are the representative membrane potentials responding to the depolarizing two-second-long square steps of increasing current injected intracellularly through the recording pipette in the presence of synaptic blockers and TTX. This figure shows a power spectrum of the membrane oscillations induced by the indicated current steps in three recordings. The frequencies induced were all low in power and low in frequency.This figure shows the current ramps of 200, 400, and 700 picoamps and the membrane responses. Note that the membrane is sufficiently depolarized to reach 30 millivolts, at which high-threshold calcium channels begin to open, as in the blue recording, and the 10 millivolt level at which their oscillation amplitude is maximal, as in the red recording. This graph shows a power spectrum of the membrane oscillations induced by the current ramps in the corresponding three recordings.The frequencies induced by the 400 picoamp ramp were in the beta range, and by the 700 picoamp ramp were in the gamma range. Once mastered, this technique can be done in about an hour or less. When you're doing your recording, it's really important to make sure that you have a tight seal and that you have capacity compensation and series resistance well-monitored to make sure that there are no major changes during the recording procedure.Once you've isolated these oscillations, what you can do is test to determine if they're really mediated by N-type or P/Q-type calcium channels. You can use conotoxin to block N-type channel oscillations and agatoxin to block P/Q-type channel oscillations. As we developed this technique, it became much easier for you to demonstrate that indeed, high threshold calcium channels were dependent for intrinsic membrane oscillations, and it became a breakthrough in the field of sleep research to determine that the reticular activating system could actually generate gamma band oscillations by itself.

Research

JoVE Journal

Neuroscience

Slice Preparation, Organotypic Tissue Culturing and Luciferase Recording of Clock Gene Activity in the Suprachiasmatic Nucleus

A central circadian (~24 hr) clock coordinating daily rhythms in physiology and behavior resides in the suprachiasmatic nucleus (SCN) located in the ...

Large-scale Recording of Neurons by Movable Silicon Probes in Behaving Rodents

Storskala opptak av nevroner med bevegelige Silicon Sonder i oppfører Gnagere

A major challenge in neuroscience is linking behavior to the collective activity of neural assemblies. Understanding of input-output relationships of ...

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

Retrograde Fluorescent Merking Tillater målrettet Ekstracellulær Single-enhet Opptak fra Identifiserte Nerveceller<em&gt; In vivo</em

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from ...

Recording Electrical Activity from Identified Neurons in the Intact Brain of Transgenic Fish

Innspilling elektriske aktiviteten fra Identifiserte Nerveceller i Intakt Norgesmester Transgenic Fish

Understanding the cell physiology of neural circuits that regulate complex behaviors is greatly enhanced by using model systems in which this work can be ...

Intracellular Recording, Sensory Field Mapping, and Culturing Identified Neurons in the Leech, Hirudo medicinalis

Intracellular Recording, Sensory Field Mapping, and Culturing Identified Neurons in the Leech, Hirudo medicinalis

Intracellulær opptak, Sensory felttilordningen, og Dyrknings Identifiserte Nerveceller i Leech, Hirudo medicin</i

The freshwater leech, Hirudo medicinalis, is a versatile model organism that has been used to address scientific questions in the fields of ...

Simultaneous Electrophysiological Recording and Calcium Imaging of Suprachiasmatic Nucleus Neurons

Simultaneous electrophysiological and fluorescent imaging recording methods were used to study the role of changes of membrane potential or current in ...

Perforated Patch-clamp Recording of Mouse Olfactory Sensory Neurons in Intact Neuroepithelium: Functional Analysis of Neurons Expressing an Identified Odorant Receptor

Perforert Patch-clamp Opptak av Mouse olfactory sensoriske nevroner i Intakt neuroepithelium: Funksjonell analyse av nerveceller uttrykker en Identifiserte odoranten Receptor

Analyzing the physiological responses of olfactory sensory neurons (OSN) when stimulated with specific ligands is critical to understand the basis of ...

Tuning in the Hippocampal Theta Band In Vitro: Methodologies for Recording from the Isolated Rodent Septohippocampal Circuit

Tuning in the Hippocampal Theta Band In Vitro: Methodologies for Recording from the Isolated Rodent Septohippocampal Circuit

Tuning i Hippocampal Theta Band<em&gt; I Vitro</em&gt;: Metoder for opptak fra isolert gnagere Septohippocampal Circuit

This protocol outlines the procedures for preparing and recording from the isolated whole hippocampus, of WT and transgenic mice, along with recent ...

Optogenetic Entrainment of Hippocampal Theta Oscillations in Behaving Mice

Optogenetic Entrainment av hippocampus Theta svingninger i oppfører seg mus

Extensive data on relationships of neural network oscillations to behavior and organization of neuronal discharge across brain regions call for new tools ...

Preparations and Protocols for Whole Cell Patch Clamp Recording of Xenopus laevis Tectal Neurons

Preparations and Protocols for Whole Cell Patch Clamp Recording of Xenopus laevis Tectal Neurons

Forberedelser og protokoller for hele cellen Patch klemme opptak i Xenopus laevis Tectal nerveceller

The Xenopus tadpole retinotectal circuit, comprised of the retinal ganglion cells (RGCs) in the eye which form synapses directly onto neurons in the optic ...