Name: paired whole cell recordings in organotypic hippocampal slices
Uploaded: 2014-09-28T14:00:00
Description: Watch this Scientific Journal Video about Paired Whole Cell Recordings in Organotypic Hippocampal Slices at JoVE.com

We would like to thank the members of the Montgomery and Madison labs for helpful discussion. We acknowledge the funding received from the following sources in this research: NFNZ, AMRF, Marsden Fund, HRC, and NIH.

Déclaration d&#39;éthique animale: Les protocoles décrits dans ce manuscrit suivent les directives de protection des animaux établies par l&#39;Université d&#39;Auckland et de l&#39;Université de Stanford. Les ratons P7 ont été euthanasiés par décapitation rapide. Dissection de l&#39;hippocampe est alors immédiatement effectué comme décrit ci-dessous. 1. organotypique hippocampe Slice Culture <ol><li> Préparation <ol><li> Préparer dissection moye...

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Ici, nous avons décrit les exigences pour établir des enregistrements cellulaires succès paires entières dans les cultures de tranches d&#39;hippocampe organotypiques. Les enregistrements par paires peuvent également être effectuées dans plusieurs préparations, y compris les tranches aiguës et des systèmes de culture 26,27 dissociées. Si l&#39;accent a été mis sur l&#39;induction de formes plus longues de la plasticité synaptique (à savoir LTP et LTD), il est important de souligner que les enre...

Les récepteurs du glutamate médient la majorité de la transmission synaptique excitatrice dans les synapses du système nerveux central. Les deux principaux sous-types de récepteurs ionotropiques du glutamate localisées à la tête de la colonne vertébrale de la membrane post-synaptique sont la N-méthyl-D-aspartate (NMDA) et α-amino-3-hydroxy-5-méthylisoxazole-4-propionique acide (AMPA) des récepteurs. A des potentiels de membrane au repos, les récepteurs AMPA portent plus du courant post-synaptique au cours de la transmission synaptique. Dans l&#39;hippocampe, le récepteur NMDA joue un rôle clé dans le déclenchement de l&#39;évolution du nombre de récepteurs de l&#39;AMPA dans la membrane post-synaptique: en agissant comme un &quot;détecteur de coïncidence&quot; 1 à initier des changements dans la force synaptique 1, le récepteur NMDA participe aux mécanismes synaptiques qui sont pensés pour soutenir l&#39;apprentissage et la mémoire à un niveau subcellulaire. En réponse à la dépolarisation du neurone post-synaptique en parallèle avec la libération du transmetteur présynaptique, le calcium pénètre par le NMDArécepteur pour lancer l&#39;insertion du récepteur AMPA ou d&#39;enlèvement 2. Cette dynamique des récepteurs à la base de plasticité synaptique: une augmentation de la force synaptique est potentialisation à long terme 2,3 (LTP), alors qu&#39;une diminution de la force synaptique est la dépression à long terme 4 (LTD). Par conséquent, le mouvement du récepteur AMPA est considéré comme responsable de l&#39;expression de la plasticité synaptique, tandis que les récepteurs NMDA sont considérées pour contrôler son induction. Déterminer les mécanismes précis qui sous-tendent la transmission synaptique et la plasticité nécessite l&#39;étude de petites populations de synapses, idéalement synapses individuelles. Alors que certaines synapses sont parfaitement adaptés pour l&#39;étude à ce niveau, par exemple, le calice de Held 5, pour les populations les plus synaptiques ce qui est extrêmement difficile en raison de la petite et diffuse la nature des connexions synaptiques. Deux principales techniques d&#39;électrophysiologie ont été développés pour examiner les connexions synaptiques simples: La première est la stimulation minimale, where une fibre présynaptique est présumé stimulée extracellulaire. La deuxième technique est jumelé enregistrements, où deux enregistrements simultanés de cellules entières de neurones reliés par des synapses est effectuée. Un avantage majeur de stimulation minimal est qu&#39;elle est rapide et relativement simple à réaliser, comportant le placement d&#39;une électrode de stimulation extracellulaire dans le tube tout en enregistrant simultanément des axones d&#39;un neurone post-synaptique. La principale préoccupation lors de l&#39;utilisation de cette technique est que la stimulation fiable d&#39;une seule cellule peut rarement être garantie procès après procès. Au cours des quinze dernières années, nous avons utilisé couramment associé enregistrements de cellules entières de deux neurones pyramidaux reliés par des synapses 6-17. L&#39;avantage majeur de cette technique est que seul neurone présynaptique est stimulée cohérente et fiable. Il permet aussi non seulement la caractérisation électrophysiologique, mais aussi la manipulation pharmacologique du neurone présynaptique 6,18 &lt;/ Sup&gt;. Cependant, la probabilité de la connectivité synaptique entre les neurones est faible, ce qui rend difficile les paires connectées à obtenir 19. L&#39;utilisation de cultures de tranches de cerveau organotypiques de contourner cet obstacle comme la connectivité synaptique peut rétablir in vitro et d&#39;ailleurs la nature de la connexion qui en résulte est semblable à celle dans le tissu cérébral 20 native. En outre, les cultures organotypiques expriment LTP, LTD 7-10,12-15,21 et d&#39;autres formes de plasticité synaptique à court terme, y compris la facilitation double choc (PPF) et la dépression (PPD) 6,22,23, permettant mécanismes de plasticité à être étudiée dans des paires de neurones. Nous décrivons ici en détail la méthodologie nécessaire pour atteindre des succès enregistrements appariés dans ce système in vitro. Cette information peut aisément être adapté à d&#39;autres systèmes expérimentaux, y compris les tranches aiguës et d&#39;autres régions du cerveau.

<table border="0" cellpadding="0" cellspacing="0" width="686">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:256px;">Organotypic cultures</td>
			<td style="width:111px;"></td>
			<td style="width:255px;"></td>
			<td style="width:64px;">Paired recordings</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Minimum Essential Medium</td>
			<td></td>
			<td></td>
			<td>Stable motorized micromanipulators&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin-Streptomycin solution&#160;</td>
			<td></td>
			<td></td>
			<td>Shallow tissue bath</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HEPES buffer solution</td>
			<td></td>
			<td></td>
			<td>DIC camera</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1M Tris stock solution&#160;</td>
			<td></td>
			<td></td>
			<td>Amplifier</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hank&#8217;s Balanced Salt Solution</td>
			<td></td>
			<td></td>
			<td>Computer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Horse Serum&#160;</td>
			<td></td>
			<td></td>
			<td>Vibration isolation table</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">plastic-coated miniature spatulas&#160;</td>
			<td></td>
			<td></td>
			<td>Upright microscope</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">soft paintbrush&#160;</td>
			<td></td>
			<td></td>
			<td>Data acquistion and analysis software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">manual tissue chopper</td>
			<td></td>
			<td></td>
			<td>Electrode puller</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">#2 filter paper</td>
			<td></td>
			<td></td>
			<td>Faraday cage</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">#5&#160; forceps</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Membrane inserts</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">CO2 incubator&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dissection hood</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Class II hood</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

paired whole cell recordings in organotypic hippocampal slices

Pair recordings involve simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling not only direct electrophysiological characterization of the synaptic connections between individual neurons, but also pharmacological manipulation of either the presynaptic or the postsynaptic neuron. When carried out in organotypic hippocampal slice cultures, the probability that two neurons are synaptically connected is significantly increased. This preparation readily enables identification of cell types, and the neurons maintain their morphology and properties of synaptic function similar to that in native brain tissue. A major advantage of paired whole cell recordings is the highly precise information it can provide on the properties of synaptic transmission and plasticity that are not possible with other more crude techniques utilizing extracellular axonal stimulation. Paired whole cell recordings are often perceived as too challenging to perform. While there are challenging aspects to this technique, paired recordings can be performed by anyone trained in whole cell patch clamping provided specific hardware and methodological criteria are followed. The probability of attaining synaptically connected paired recordings significantly increases with healthy organotypic slices and stable micromanipulation allowing independent attainment of pre- and postsynaptic whole cell recordings. While CA3-CA3 pyramidal cell pairs are most widely used in the organotypic slice hippocampal preparation, this technique has also been successful in CA3-CA1 pairs and can be adapted to any neurons that are synaptically connected in the same slice preparation. In this manuscript we provide the detailed methodology and requirements for establishing this technique in any laboratory equipped for electrophysiology.

Connectivité synaptique est évident en stimulant le neurone présynaptique de tirer un potentiel d&#39;action en passant une impulsion de courant de dépolarisation (typiquement 20-50 pA pour 20 ms) via l&#39;électrode d&#39;enregistrement. La trace de courant post-synaptique est alors examiné pour rechercher la présence d&#39;un RPEC monosynaptique évoquée à court (&lt;5 ms) et des temps de latence cohérents après le pic du potentiel d&#39;action présynaptique (Figure 3A). Dans la plupart de...

Watch this Scientific Journal Video about Paired Whole Cell Recordings in Organotypic Hippocampal Slices at JoVE.com

Paired Whole Cell Recordings in Organotypic Hippocampal Slices

Pair recordings are simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling precise electrophysiological and pharmacological characterization of the synapses between individual neurons. Here we describe the detailed methodology and requirements for establishing this technique in organotypic hippocampal slice cultures in any laboratory equipped for electrophysiology.

Appariés enregistrements de cellules entières dans organotypiques coupes d&#39;hippocampe

Pair recordings involve simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling not only direct ...

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JoVE Journal

Neuroscience

Paired Patch Clamp Recordings from Motor-neuron and Target Skeletal Muscle in Zebrafish

Jumelés Recordings Patch Clamp du motoneurone et de muscle ciblé squelettiques chez le poisson zèbre

Larval zebrafish represent the first vertebrate model system to allow simultaneous patch clamp recording from a spinal motor-neuron and target muscle.  ...

Recherche

Neurosciences

Organotypic Hippocampal Slice Cultures

Cultures organotypiques d&#39;hippocampe Slice

The hippocampus, a component of the limbic system, plays important roles in long-term memory and spatial navigation 1. Hippocampal neurons can modify the ...

Whole Cell Recording from an Organotypic Slice Preparation of Neocortex

Enregistrement à partir de cellules entières une préparation de tranches organotypiques du néocortex

We have been studying the expression and functional roles of voltage-gated potassium channels in pyramidal neurons from rat neocortex.  Because of the ...

Multi-electrode Array Recordings of Neuronal Avalanches in Organotypic Cultures

Enregistrements tableau multi-électrodes des Avalanches neuronale dans des cultures organotypiques

The cortex is spontaneously active, even in the absence of any particular input or motor output.  During development, this activity is important for the ...

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

Deux enregistrements électrophysiologiques des synapses-réponses évoquées astrogliales et neuronales aiguës dans des coupes d&#39;hippocampe

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

Whole-cell Patch-clamp Recordings from Morphologically- and Neurochemically-identified Hippocampal Interneurons

De cellules entières de patch-clamp enregistrements de Morphologically- et interneurones hippocampiques neurochemically identifiés

GABAergic inhibitory interneurons play a central role within neuronal circuits of the brain. Interneurons comprise a small subset of the neuronal ...

Electrophysiological and Morphological Characterization of Neuronal Microcircuits in Acute Brain Slices Using Paired Patch-Clamp Recordings

Électrophysiologiques et Caractérisation morphologique des neurones Les circuits dans aiguë tranches cerveau en utilisant jumelés patch-clamp enregistrements

The combination of patch clamp recordings from two (or more) synaptically coupled neurons (paired recordings) in acute brain slice preparations with ...

Whole-cell Patch-clamp Recordings in Brain Slices

Recordings cellules entières Patch-clamp en tranches cérébrales

Whole-cell patch-clamp recording is an electrophysiological technique that allows the study of the&#160;electrical properties of a substantial part of the ...

Organotypic Hippocampal Slice Cultures As a Model to Study Neuroprotection and Invasiveness of Tumor Cells

Hippocampe organotypiques comme modèle d’étude Neuroprotection et son caractère invasif des cellules tumorales

In organotypic hippocampal slice cultures (OHSC), the morphological and functional characteristics of both neurons and glial cells are well preserved. ...