Name: electrophysiological and morphological characterization of neuronal microcircuits in acute brain slices using paired patch-clamp recordings
Uploaded: 2015-01-10T15:00:00
Description: Watch this Scientific Journal Video about Electrophysiological and Morphological Characterization of Neuronal Microcircuits in Acute Brain Slices Using Paired Patch-Clamp Recordings at JoVE.com

We would like to thank all members of &#8216;Function of Neuronal Microcircuits&#8217; Group at Institute of Neuroscience and Medicine, INM-2, Research Centre J&#252;lich and the &#8216;Function of Cortical Microcircuits&#8217; Group in the Dept. of Psychiatry, Psychotherapy and Psychosomatics, Medical School, JARA, RWTH Aachen University for fruitful discussions. This work was supported by the DFG research group on Barrel Cortex Function (BaCoFun).

All experimental procedures have been carried out in accordance with the EU Directive for the Protection of Animals, the German Animal Welfare Act (Tierschutzgesetz) and the Guidelines of the Federation of European Laboratory Animal Science Association.
1. Set-up for Electrophysiology
Before commencing with paired recording, an electrophysiology set-up has to be built. A brief outline how such a set-up is assembled is given below:
<ol>
	<li>Install an anti-vibrati...

Paired recordings from synaptically coupled excitatory and/or inhibitory neurons are a very versatile approach for the study of neuronal microcircuits. Not only does this approach allow one to estimate synaptic connectivity between neuron types but also allows determining the functional characteristics of the connection and the morphology of pre- and postsynaptic neurons. Furthermore, agonist and/or antagonist can easily be applied to neurons in slice preparations. This allows one to study the effects of neuromodulators ...

Neuronal microcircuits between two synaptically coupled neurons are the building blocks of large-scale networks in the brain and are the fundamental units of synaptic information processing. A prerequisite for the characterization of such neuronal microcircuits is to know the morphology and functional properties of both the pre- and postsynaptic partner neurons, the type of the synaptic connection(s) and its structure and functional mechanism. However, in many studies of synaptic connections at least one of the neurons in a microcircuit is not well characterized. This results from the relatively unspecific stimulation protocols often used in studies of synaptic connectivity. Therefore, the structural and functional properties of the presynaptic neuron are either not identified at all or only to a rather small extent (i.e., the expression of marker proteins etc.). Paired recordings in combination with intracellular staining by markers such as biocytin, neurobiotin or fluorescent dyes are better suited for studying small neuronal microcircuits. This technique allows one to investigate many structural and functional parameters of a morphologically identified synaptic connection at the same time.
So-called &#8216;unitary&#8217; monosynaptic connections between two neurons have been investigated in both cortical and subcortical brain regions1-10 using acute slice preparations. Initially, sharp microelectrodes were used in these experiments; later, patch clamp recording was employed in order to obtain recordings of synaptic signals with a lower noise level and an improved temporal resolution.
A significant technical advance was the use of infrared differential interference contrast (IR-DIC) optics11-14, a microscopic technique that significantly improved the visibility and identification of neurons in the brain slice so that it became possible to obtain recordings from visually identified synaptic connections15-17. In general, paired recordings are done in acute slice preparations; only very few publications are available reporting recordings from synaptically connected neurons in vivo18-20.
The most important advantage of paired recordings is the fact that a functional characterization can be combined with a morphological analysis at both the light and electron microscopic level (see e.g.,7,16,21). After histochemical processing, the dendritic and axonal morphology of the synaptically connected neuron pair is traced. Subsequently, it is possible to quantify morphological features such as length, spatial density, orientation, branching pattern etc. These parameters may then provide a basis for an objective classification of a specific synaptic connection. Furthermore, in contrast to most other techniques used for studying neuronal connectivity, paired recordings also permit the identification of synaptic contacts for unitary synaptic connections. This can be done directly using a combination of light and electron microscopy16,21-27 or using calcium imaging28,29 of dendritic spines. However, with the latter approach only excitatory but not inhibitory connections can be studied as it requires calcium influx via the postsynaptic receptor channels.
In addition to a detailed analysis of synaptic transmission at a defined neuronal microcircuit paired recordings also allow the study of synaptic plasticity rules30,31 or &#8211; in combination with agonist/antagonist application &#8211; the modulation of synaptic transmission by neurotransmitters such as acetylcholine32 and adenosine33.

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			<td height="21" style="height:21px;width:199px;">Name</td>
			<td style="width:199px;">Company</td>
			<td style="width:199px;">Catalog Number</td>
			<td style="width:199px;">Comments</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:199px;">Amplifier</td>
			<td style="width:199px;">HEKA</td>
			<td style="width:199px;">EPC 10 USB Triple</td>
			<td style="width:199px;">with 2-3 preamplifiers</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:199px;">Microscope</td>
			<td style="width:199px;">Olympus</td>
			<td style="width:199px;">BX51WI</td>
			<td style="width:199px;">with 2 camera ports and a 4&#215; objective, a 40&#215; water-immersion objective</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:199px;">Camera</td>
			<td style="width:199px;">TILL Photonics</td>
			<td style="width:199px;">VX55</td>
			<td style="width:199px;">infrared CCD camera</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:199px;">Workstation</td>
			<td style="width:199px;">Luigs &#38; Neumann</td>
			<td style="width:199px;">Infrapatch 240</td>
			<td style="width:199px;">with a motorized x-y stage and a motorized focus axis for the microscope</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:199px;">Micromanipulator</td>
			<td style="width:199px;">Luigs &#38; Neumann</td>
			<td style="width:199px;">SM-5</td>
			<td style="width:199px;">x-y-z manipulators for 2-3 preamplifiers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:199px;">Faraday cage</td>
			<td style="width:199px;">Luigs &#38; Neumann</td>
			<td style="width:199px;"></td>
			<td style="width:199px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:199px;">Anti-vibration table</td>
			<td style="width:199px;">Newport Spectra-Physics</td>
			<td style="width:199px;"></td>
			<td style="width:199px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:199px;">Patchmaster</td>
			<td style="width:199px;">HEKA</td>
			<td style="width:199px;"></td>
			<td style="width:199px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:199px;">Microtome</td>
			<td style="width:199px;">Microm International</td>
			<td style="width:199px;">HM650V</td>
			<td style="width:199px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:199px;">Micropipette puller</td>
			<td style="width:199px;">HEKA</td>
			<td style="width:199px;">Sutter P-97</td>
			<td style="width:199px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:199px;">Neurolucida system</td>
			<td style="width:199px;">Microbrightfield</td>
			<td style="width:199px;"></td>
			<td style="width:199px;">with Neurolucida and Neuroexplorer softwares</td>
		</tr>
	</tbody>
</table>

electrophysiological and morphological characterization of neuronal microcircuits in acute brain slices using paired patch-clamp recordings

The combination of patch clamp recordings from two (or more) synaptically coupled neurons (paired recordings) in acute brain slice preparations with simultaneous intracellular biocytin filling allows a correlated analysis of their structural and functional properties. With this method it is possible to identify and characterize both pre- and postsynaptic neurons by their morphology and electrophysiological response pattern. Paired recordings allow studying the connectivity patterns between these neurons as well as the properties of both chemical and electrical synaptic transmission. Here, we give a step-by-step description of the procedures required to obtain reliable paired recordings together with an optimal recovery of the neuron morphology. We will describe how pairs of neurons connected via chemical synapses or gap junctions are identified in brain slice preparations. We will outline how neurons are reconstructed to obtain their 3D morphology of the dendritic and axonal domain and how synaptic contacts are identified and localized. We will also discuss the caveats and limitations of the paired recording technique, in particular those associated with dendritic and axonal truncations during the preparation of brain slices because these strongly affect connectivity estimates. However, because of the versatility of the paired recording approach it will remain a valuable tool in characterizing different aspects of synaptic transmission at identified neuronal microcircuits in the brain.

Paired recordings are the method of choice for an in-depth characterization of morphologically identified uni- or bidirectional synaptic connections as well as gap junction (electrical) connections (Figure 1). An example of a paired recording in layer 4 of the somatosensory barrel cortex is shown in Figure 1A. Both unidirectional excitatory and inhibitory synaptic connections can be characterized (Figure 1B,C). Furthermore paired recordings allow to record from bidirecti...

Watch this Scientific Journal Video about Electrophysiological and Morphological Characterization of Neuronal Microcircuits in Acute Brain Slices Using Paired Patch-Clamp Recordings at JoVE.com

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

Patch-clamp recordings and simultaneous intracellular biocytin filling of synaptically coupled neurons in acute brain slices allow a correlated analysis of their structural and functional properties. The aim of this protocol is to describe the essential technical steps of electrophysiological recording from neuronal microcircuits and their subsequent morphological analysis.

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

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