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-vibration table on which the microscope, the manipulators and all other equipment will be placed. 
	NOTE: Vibration or any other kind of movement needs to be as small as possible when recording from synaptic connections because this requires a change of pipettes (searching electrode replaced by recording electrode).</li>
	<li>Place a Faraday cage around the anti-vibration table to reduce electrical noise. Connect all equipment inside the Faraday cage with the electrical ground.</li>
	<li>Install a microscope with a motorized focus axis that is based on a motorized x-y table on the anti-vibration table according to the manufacturer&#8217;s guidelines. This allows to reliably move the microscope to neurons that are not in the same field of view as it is the case for translaminar connections in the neocortex.</li>
	<li>Install a video camera on the camera port of the microscope and connect it to analog and/or digital screens to observe the slice preparation and the movement of electrodes. Use a camera, which can be switched between a low- and a high-power magnification to obtain an overview and a close-up image, respectively, of the synaptically coupled neuronal cell pair. This also helps to control the movement of the patch electrodes.</li>
	<li>Install a specimen table containing an inset for the bath chamber (drilled in-house from a Perspex block) for the brain slice preparation. This specimen table must not be connected to the microscope, i.e., must be fixed to the air table and the microscope should be maneuverable under it.</li>
	<li>Mount two (or more if required) high-precision micromanipulators that can be moved in all three dimensions. Install patch-clamp pre-amplifier on the manipulators and connect them to the main amplifier. 
	NOTE: If required additional manipulators can be installed for e.g., more than two pre-amplifiers, extracellular stimulation electrodes, drug-delivery systems, etc.</li>
	<li>Place the bath chamber in the specimen table. Install a solution inlet and outlet and connect them to the perfusion system.</li>
	<li>Use a peristaltic pump to maintain the perfusion of the recording chamber with artificial cerebrospinal fluid (ACSF; Table 1) at a stable flow rate of 4 - 6 ml/min for optimal slice viability. 
	NOTE: Do not exceed this flow rate significantly as it will result in turbulence in the ASCF and hence movement of the slice.</li>
	<li>Install holders for the temperature probe and Ag/AgCl ground electrode onto the specimen table. This is necessary to allow the probe and electrode to sample the bath in the chamber.</li>
	<li>For a good visibility of neurons in the slice preparation use infrared illumination in the microscope. This reduces light diffraction and hence image blur. Ensure that the microscope is equipped with differential interference contrast optics to achieve a &#8216;3D&#8217;-like visualization. This will help patching neurons and allow targeting neurons deep in the slice (60 &#181;m or deeper).</li>
	<li>During the experiment, keep brain slices in an experimental chamber with a glass coverslip at the bottom. Place a &#8216;platinum harp&#8217; on the top of slice to prevent slices from floating. A platinum harp is made from U-shaped, flattened platinum wire with strings of dental floss glued onto it.</li>
	<li>Keep the ACSF perfusing the slice at a temperature of 32 - 35 &#176;C to ensure optimal control of oxygenation and pH. This can be done by heating the solution with a Peltier device installed close to the ACSF inflow into the bath chamber. 
	NOTE: Use ultrathin cover slips so that even microscope condensers with a high numerical aperture and short working distance can be focused on the specimen. This is necessary for an optimal illumination of the slice. 
	For an image and a description of an electrophysiology set-up optimized for paired recordings in the brain slice preparation see Figure&#160;4 in Ref. 34.</li>
</ol>
2. Brain Slice Preparation
<ol>
	<li>Mildly anesthetize the animal from which the brain tissue should be taken with isoflurane (final concentration &#60;0.1%). 
	NOTE: Other anesthetics can also be used. Use of the anesthetic should comply with the respective animal committee&#8217;s recommendations and rules. Always ensure that the animal is not irritated or under stress after adding the anesthetic.</li>
	<li>Decapitate the animal, open the skull and remove the brain as quickly as possible using the procedure described in Refs. 34,35.</li>
	<li>Transfer the brain into cooled (4 &#176;C) artificial cerebrospinal fluid aerated with a mixture of carbogen gas with 95% O2 and 5% CO2.</li>
	<li>Isolate the brain region that should be studied. 
	NOTE: The parameters to obtain optimal preservation and minimal truncation of dendrites and axons of pre- and postsynaptic neurons should be determined in advance for each neuronal connection and developmental stage under investigation. In an optimally dissected brain slice, the axon of the presynaptic neuron is not required to be parallel to the slice surface. Rather, they should point into the slices with a shallow angle. The applies for the apical dendrite of postsynaptic pyramidal neurons; this should be checked under the light microscope. This is particularly important for non-local or translaminar synaptic connections, i.e., where pre- and postsynaptic somata are more than 200 &#181;m apart and the probability of dendritic and axonal truncations is likely to be substantially higher than for local connections.</li>
	<li>Transfer the brain to the microtome chamber. Based on empirical findings, different extracellular slicing solutions are used depending on the age of the animal (see Tables 2 and 3; helpful information can also be found under &#8216;<a href="http://www.brainslicemethods.com/" target="_blank">http://www.brainslicemethods.com/</a>&#8217;).</li>
	<li>Trim the brain until the target region is visible.</li>
	<li>To obtain slices with a good cell visibility, cut 300 - 400 &#181;m thick brain slices if the animal is mature (&#62;3&#160;weeks). If the animal is immature (1st to 2nd postnatal week for mice and rats) slices may be cut up to a thickness of ~600 - 800 &#181;m without substantially impairing cell visibility. 
	NOTE: This will improve the stability of &#8216;immature&#8217; slices and reduce the number of possible truncations of long-range dendritic and axonal collaterals.</li>
	<li>Transfer slices from the microtome chamber to an incubation chamber filled with slicing solution aerated with a mixture of 95% O2 and 5% CO2. Keep the slices in the incubation chamber for at least 30 min to 1 hr either at RT or at ~30 &#176;C, depending on the type of experiment.</li>
</ol>
3. Paired Patch-Clamp Recording and Biocytin Filling
Depending on the type of synaptic connection three different approaches are used to find synaptically coupled neurons. If the connection probability is low (as can be expected for most excitatory connections), proceed as follows:
<ol>
	<li>Neuronal connections with a low connectivity
	<ol>
		<li>Fill the patch pipettes with an internal solution of the composition listed in Table 4. For all recordings in the whole-cell configuration add biocytin at concentrations between 3 - 5 mg/ml to the internal (pipette) solution to obtain good staining results for the pre- and postsynaptic neurons. Let biocytin diffuse into the cell for at least 15 - 30 min (depending on the size of the neuron). 
		NOTE: Do not add biocytin to the solution used in the &#8216;searching&#8217; pipettes mentioned below.</li>
		<li>Patch a putative postsynaptic neuron in the whole-cell mode. Use patch pipettes with a long and slender shank. This facilitates the maneuverability of the pipette under the objective and reduces movement artifacts that may occur when the electrode is introduced in the slice.</li>
		<li>Then, patch a potential presynaptic neuron in a cell-attached configuration using a &#8216;searching&#8217; patch pipette of 8 - 10 M&#8486; resistance and a small tip diameter. With these pipettes establish a &#8216;loose&#8217; cell-attached patch. Fill the &#8216;searching&#8217; pipette with an internal solution in which K+ is replaced by Na+ (Table 5) in order not to depolarize neurons during searching for a presynaptic cell. 
		NOTE: The &#8216;loose&#8217; seal resistance is not in the G&#937; range but typically around 30 - 300 M&#937;.</li>
		<li>Hold the membrane potential under the &#8216;loose&#8217; seal to about -30 to -60 mV in current clamp mode. Then, apply large current pulses (0.2 - 2 nA) to elicit an action potential in the potential presynaptic cell. Observe this action potential as a small spikelet on the voltage response.</li>
		<li>Set the stimulation frequency to 0.1 Hz to prevent run-down of a postsynaptic response. Use lower stimulation frequencies in case the brain tissue is very immature or the synaptic connection not very reliable.</li>
		<li>In case the &#8216;postsynaptic&#8217; neuron does not respond to stimulation of the tested &#8216;presynaptic&#8217; neuron, patch a new neuron in &#8216;loose&#8217; seal mode. Do not replace the &#8216;searching&#8217; patch electrode as long as the seal with a resistance of &#62;30 M&#937; is established. Test up to 30 potentially presynaptic neurons in this way. 
		NOTE: To prevent damaging the neuron during the searching procedure with loose patch clamp, only gently suction should be applied to the searching pipette compared with that applied to the whole-cell patch pipette.</li>
		<li>If stimulation in the &#8216;loose&#8217; seal mode results in an EPSP/IPSP with a latency &#60;5 msec in the postsynaptic neuron remove the &#8216;searching&#8217; pipette from the presynaptic neuron.</li>
		<li>Replace the &#8216;searching&#8217; patch pipette with a patch electrode filled with biocytin-containing, regular internal solution. Ensure this recording patch pipette has a resistance of 4 - 8 M&#937;; the larger the soma size the lower the electrode resistance can be.</li>
		<li>Patch the presynaptic neuron and record in whole-cell, current-clamp mode. Elicit action potentials by current injection in the presynaptic neurons and record the postsynaptic response. Store the data on a computer for later off-line analysis.</li>
	</ol>
	</li>
	<li>Neuronal connections with a high connectivity 
	NOTE: For neuronal microcircuits with a high connectivity ratio (&#62; 30%) use a different procedure to find synaptic connections. This procedure is outlined below and is often used for inhibitory synaptic connection that have on average a higher connectivity ratio than excitatory connections36. It should not be used when the connectivity ratio falls below this value.
	<ol>
		<li>Patch a presynaptic neuron directly in the whole-cell mode. Then, patch a potential postsynaptic neuron, also in whole cell mode. Both cells should be under current clamp control. Elicit an action potential in the presynaptic cell. Monitor the prospective postsynaptic neuron for a postsynaptic potential.</li>
		<li>In case the neuron does not show a response to presynaptic stimulation, patch a new neuron in whole-cell mode using a patch electrode filled with regular internal solution. If a connection is found, do not change the patch pipette but proceed with recording. Then, proceed as in step&#160;3.1.9.</li>
	</ol>
	</li>
	<li>Neuronal connections via gap-junctions 
	NOTE: To search for electrical (gap-junction) connections use the following procedure which is a modified version of that used for low-probability connections.
	<ol>
		<li>Patch a postsynaptic neuron in the whole-cell patch clamp configuration.</li>
		<li>Subsequently, patch a putative presynaptic neuron in the loose-seal configuration (low seal resistance of 30 - 300 M&#8486;) using a &#8216;searching&#8217; patch pipette of 7 - 10 M&#8486; resistance. This pipette should be filled with the modified high Na+ internal solution for loose-seal stimulation.</li>
		<li>Inject 100 - 300 msec long hyperpolarizing current pulses via the &#8216;loose&#8217; seal; the pipette command potential should be set to about -60 to -70 mV. Observe a gap junction connection if this stimulation results in a small hyperpolarizing response in the &#8216;postsynaptic&#8217; neuron.</li>
		<li>Repatch the &#8216;presynaptic&#8217; neuron in the whole-cell mode and proceed as described above. 
		NOTE: To ensure a good staining of the axonal and dendritic processes, use the protocol given below. This protocol is described in brief here, however, a detailed protocol for histochemical processing used in our laboratory is given in Ref. 37.</li>
	</ol>
	</li>
</ol>
4. Histochemical Processing
<ol>
	<li>Transfer the brain slice with the pair of synaptically coupled neurons into a vial containing 4% paraformaldehyde dissolved in 0.1 M phosphate buffer and fixate the slice for 24 hr. For electron microscopy, fixate the slice using 2.5% glutaraldehyde and 1% paraformaldehyde.</li>
	<li>On the next day, transfer the slices into normal phosphate buffer containing no fixative. Wash slices with phosphate buffer for 6 - 8 times for 10 - 15 min each to remove excess fixative.</li>
	<li>Incubate slices for 20 min in a 3% H2O2 solution in 0.1 M phosphate buffer to minimize the endogenous peroxidase reaction. Observe strong bubble formation. Continue the reaction until no further bubbles are produced. Then, rinse the slices in 0.1 M phosphate buffer for 6 - 8 times (for 10 min each) to remove any remaining H2O2.</li>
	<li>Immunocytochemical and chromogenic reactions 
	Visualization of biocytin-filled neurons is based on a streptavidin-biotinylated horseradish peroxidase reaction catalyzed with diaminobenzidine; this produces a dark precipitate that produces a crisp stain with a high spatial resolution. The following procedures are used for the chromogenic reaction:
	<ol>
		<li>Prepare the ABC solution as per the manufacture&#8217;s protocol. Add to 14.55 ml phosphate buffer supplemented with 150 &#181;l 10% Triton X100 (Table 6; only for light microscopy) and keep the solution for about 30 min in the dark. Incubate slices in this solution O/N before use.</li>
		<li>Incubate slices in ABC solution on a shaker for 1 hr at RT and in the dark. Subsequently, keep slices O/N at 4 &#176;C in a fridge. On the next morning, incubate slices at RT for an additional hour.</li>
		<li>After this, rinse slices at least four times for 10 min each in phosphate buffer. Then, incubate on a shaker for about 30 min in the dark in 2 ml of a nickel-intensified 3-3&#8217;-diaminobenzidine solution (Table 7). Be very careful when handling diaminobenzidine and use only under the fume hood; it is extremely carcinogenic even at very low concentrations.</li>
		<li>Add 6.5 &#181;l 3% H2O2 to the diaminobenzidine-containing solution. Monitor the chromogenic reaction under a light microscope until brown-black stained neurons are clearly visible. Then, stop the reaction immediately by washing slices several times in phosphate buffer.</li>
		<li>Mount slices with stained neurons on adhesive, silane-coated Histobond or gelatinized standard object slides.</li>
		<li>Keep the slides in a moist chamber with at least 80% humidity O/N. On the next day, let the slices air-dry for a further 10 min. Prior to embedding, transfer slides in solutions with increasing concentrations of ethanol (20 - 100%) in order to dehydrate them. 
		NOTE: The dehydration process should be slow otherwise distortions in the dendritic and axonal processes are likely to occur37. If an alternative embedding procedure is chosen, e.g., one with the glycerol-based Moviol, a dehydration is not required; however this is likely to result in an inhomogeneous compression of the slice and hence a distorted neuronal morphology.</li>
		<li>Finally, incubate for 10 min in xylol, embed the slices in a hydrophobic embedding medium for light microscopy and place an ultra-thin coverslips on top. Let slides dry for 20 min at RT.</li>
	</ol>
	</li>
</ol>
5. Neuronal Reconstruction and Synaptic Contact Localization
<ol>
	<li>Examine slides with biocytin-labeled neurons under a light microscope equipped with a 60X/100X oil immersion objective and a condenser with a high numerical aperture for optimal spatial resolution. Ensure that axonal boutons and dendritic spines are clearly visible.</li>
	<li>Trace neurons or synaptically coupled neuron pairs using a commercial neuron tracing system (see Table of Specific Materials/Equipment) to obtain 3D neuronal reconstructions. Carry out tracing manually under constant visual inspection to detect even small axonal or dendritic collaterals. For paired recordings from synaptically coupled neurons manual tracing is still the method of choice because the attribution of e.g., an axonal profile to either the pre- or postsynaptic neuron requires substantial reconstruction experience.</li>
	<li>If required, make counts of axonal boutons and/or dendritic spines. Because spines or even spine subtypes and axonal boutons may exhibit a specific distribution and density with respect to e.g., cortical layer or area, this could help to characterize neuronal cell types.</li>
	<li>For synaptically coupled neuron pairs, check the presynaptic axon and postsynaptic dendrites for close appositions at the highest magnification available. Observe an axonal bouton and a postsynaptic dendritic spine or shaft is in the same focal plane can be considered as a putative synaptic contact. Mark this contact in the reconstruction.</li>
	<li>Correct the neuronal reconstructions for shrinkage in all three spatial dimensions in the appropriate section of the tracing program. 
	NOTE: During fixation, histochemical processing and dehydration substantial mechanical deformation and shrinkage occur; this will affect axonal and dendritic length measurements and severely distort their spatial arrangement. Shrinkage correction factors for the embedding medium are ~1.1 for x and y directions and ~2.1 for the z-direction37.</li>
	<li>For a quantitative morphological analysis use a data analysis program for neurophysiology (see Table of Specific Materials/Equipment).</li>
</ol>

The authors declare no conflict of interest.

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.

<table border="0" cellpadding="0" cellspacing="0" width="792">
	<tbody>
		<tr height="21">
			<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>
		</tr>
		<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 ...

electrophysiological-morphological-characterization-neuronal

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JoVE Journal

Neuroscience

17.1K Views.  Research Centre Jülich. 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.

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

Patch Clamp Recordings from Mouse Retinal Neurons in a Dark-adapted Slice Preparation

Our visual experience is initiated when the visual pigment in our retinal photoreceptors absorbs photons of light energy and initiates a cascade of intracellular events that lead to closure of cyclic-nucleotide-gated channels in the cell membrane. The resulting change in membrane potential leads in turn to reductions in the amount of neurotransmitter release from both rod and cone synaptic terminals. To measure how the light-evoked change in photoreceptor membrane potential leads to downstream activity in the retina, scientists have made electrophysiological recordings from retinal slice preparations for decades1,2. In the past these slices have been cut manually with a razor blade on retinal tissue that is attached to filter paper; in recent years another method of slicing has been developed whereby retinal tissue is embedded in low gelling temperature agar and sliced in cool solution with a vibrating microtome3,4. This preparation produces retinal slices with less surface damage and very robust light-evoked responses. Here we document how this procedure can be done under infrared light to avoid bleaching the visual pigment.

Here we describe a procedure for generating dark-adapted slices of the mouse retina for electrophysiological recordings.

Our visual experience is initiated when the visual pigment in our retinal photoreceptors absorbs photons of light energy and initiates a cascade of ...

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

Larval zebrafish represent the first vertebrate model system to allow simultaneous patch clamp recording from a spinal motor-neuron and target muscle. This is a direct consequence of the accessibility to both cell types and ability to visually distinguish the single segmental CaP motor-neuron on the basis of morphology and location. This video demonstrates the microscopic methods used to identify a CaP motor-neuron and target muscle cells as well as the methodologies for recording from each cell type. Identification of the CaP motor-neuron type is confirmed by either dye filling or by the biophysical features such as action potential waveform and cell input resistance. Motor-neuron recordings routinely last for one hour permitting long-term recordings from multiple different target muscle cells. Control over the motor-neuron firing pattern enables measurements of the frequency-dependence of synaptic transmission at the neuromuscular junction. Owing to a large quantal size and the low noise provided by whole cell voltage clamp, all of the unitary events can be resolved in muscle. This feature permits study of basic synaptic properties such as release properties, vesicle recycling, as well as synaptic depression and facilitation. The advantages offered by this in vivo preparation eclipse previous neuromuscular model systems studied wherein the motor-neurons are usually stimulated by extracellular electrodes and the muscles are too large for whole cell patch clamp. The zebrafish preparation is amenable to combining electrophysiological analysis with a wide range of approaches including transgenic lines, morpholino knockdown, pharmacological intervention and in vivo imaging. These approaches, coupled with the growing number of neuromuscular disease models provided by mutant lines of zebrafish, open the door for new understanding of human neuromuscular disorders.

Larval zebrafish represent the first vertebrate model system to allow simultaneous patch clamp recording from a spinal motor-neuron and target skeletal muscle. This video demonstrates the microscopic methods used to identify a segmental CaP motor-neuron and target muscle cells as well as the methodologies for recording from each cell type.

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

Culturing and Electrophysiology of Cells on NRCC Patch-clamp Chips

Due to its exquisite sensitivity and the ability to monitor and control individual cells at the level of ion channels, patch-clamping is the gold standard of electrophysiology applied to disease models and pharmaceutical screens alike 1. The method traditionally involves gently contacting a cell with a glass pipette filled by a physiological solution in order to isolate a patch of the membrane under its apex 2. An electrode inserted in the pipette captures ion-channel activity within the membrane patch or, when ruptured, for the whole cell. In the last decade, patch-clamp chips have been proposed as an alternative 3, 4: a suspended film separates the physiological medium from the culture medium, and an aperture microfabricated in the film replaces the apex of the pipette. Patch-clamp chips have been integrated in automated systems and commercialized for high-throughput screening 5. To increase throughput, they include the fluidic delivery of cells from suspension, their positioning on the aperture by suction, and automated routines to detect cell-to-probe seals and enter into whole cell mode. We have reported on the fabrication of a silicon patch-clamp chip with optimized impedance and orifice shape that permits the high-quality recording of action potentials in cultured snail neurons 6; recently, we have also reported progress towards interrogating mammalian neurons 7. Our patch-clamp chips are fabricated at the Canadian Photonics Fabrication Centre 8, a commercial foundry, and are available in large series. We are eager to engage in collaborations with electrophysiologists to validate the use of the NRCC technology in different models. The chips are used according to the general scheme represented in Figure 1: the silicon chip is at the bottom of a Plexiglas culture vial and the back of the aperture is connected to a subterranean channel fitted with tubes at either end of the package. Cells are cultured in the vial and the cell on top of the probe is monitored by a measuring electrode inserted in the channel.The two outside fluidic ports facilitate solution exchange with minimal disturbance to the cell; this is an advantage compared to glass pipettes for intracellular perfusion.
<img alt="Figure 1" src="/files/ftp_upload/3288/3288fig1.jpg" />
Figure 1. Principle of measurement using the NRCC patch-clamp chip
We detail here the protocols to sterilize and prime the chips, load them with medium, plate them with cells, and finally use them for electrophysiological recordings.

We show how planar patch-clamp chips fabricated at the National Research Council of Canada are sterilized, primed, loaded with medium, plated with cells, and used for electrophysiological recordings.

Due to its exquisite sensitivity and the ability to monitor and control individual cells at the level of ion channels, patch-clamping is the gold standard ...

Patch-clamp Capacitance Measurements and Ca2+ Imaging at Single Nerve Terminals in Retinal Slices

Visual stimuli are detected and conveyed over a wide dynamic range of light intensities and frequency changes by specialized neurons in the vertebrate retina. Two classes of retinal neurons, photoreceptors and bipolar cells, accomplish this by using ribbon-type active zones, which enable sustained and high-throughput neurotransmitter release over long time periods. ON-type mixed bipolar cell (Mb) terminals in the goldfish retina, which depolarize to light stimuli and receive mixed rod and cone photoreceptor input, are suitable for the study of ribbon-type synapses both due to their large size (~10-12 &mu;m diameter) and to their numerous lateral and reciprocal synaptic connections with amacrine cell dendrites. Direct access to Mb bipolar cell terminals in goldfish retinal slices with the patch-clamp technique allows the measurement of presynaptic Ca2+ currents, membrane capacitance changes, and reciprocal synaptic feedback inhibition mediated by GABAA and GABAC receptors expressed on the terminals. Presynaptic membrane capacitance measurements of exocytosis allow one to study the short-term plasticity of excitatory neurotransmitter release 14,15. In addition, short-term and long-term plasticity of inhibitory neurotransmitter release from amacrine cells can also be investigated by recordings of reciprocal feedback inhibition arriving at the Mb terminal 21. Over short periods of time (e.g. ~10 s), GABAergic reciprocal feedback inhibition from amacrine cells undergoes paired-pulse depression via GABA vesicle pool depletion 11. The synaptic dynamics of retinal microcircuits in the inner plexiform layer of the retina can thus be directly studied.
The brain-slice technique was introduced more than 40 years ago but is still very useful for the investigation of the electrical properties of neurons, both at the single cell soma, single dendrite or axon, and microcircuit synaptic level 19. Tissues that are too small to be glued directly onto the slicing chamber are often first embedded in agar (or placed onto a filter paper) and then sliced 20, 23, 18, 9. In this video, we employ the pre-embedding agar technique using goldfish retina. Some of the giant bipolar cell terminals in our slices of goldfish retina are axotomized (axon-cut) during the slicing procedure. This allows us to isolate single presynaptic nerve terminal inputs, because recording from axotomized terminals excludes the signals from the soma-dendritic compartment. Alternatively, one can also record from intact Mb bipolar cells, by recording from terminals attached to axons that have not been cut during the slicing procedure. Overall, use of this experimental protocol will aid in studies of retinal synaptic physiology, microcircuit functional analysis, and synaptic transmission at ribbon synapses.

Patch-clamp Capacitance Measurements and Ca2+ Imaging at Single Nerve Terminals in Retinal Slices

Here we describe a protocol for the preparation of agar-embedded retinal slices that are suitable for electrophysiology and Ca2+ imaging. This method allows one to study ribbon-type synapses in retinal microcircuits using direct patch-clamp recordings of single presynaptic nerve terminals.

Visual stimuli are detected and conveyed over a wide dynamic range of light intensities and frequency changes by specialized neurons in the vertebrate ...

Patch Clamp Recordings in Inner Ear Hair Cells Isolated from Zebrafish

Patch clamp analyses of the voltage-gated channels in sensory hair cells isolated from a variety of species have been described previously1-4 but this video represents the first application of those techniques to hair cells from zebrafish. Here we demonstrate a method to isolate healthy, intact hair cells from all of the inner ear end-organs: saccule, lagena, utricle and semicircular canals. Further, we demonstrate the diversity in hair cell size and morphology and give an example of the kinds of patch clamp recordings that can be obtained. The advantage of the use of this zebrafish model system over others stems from the availability of zebrafish mutants that affect both hearing and balance. In combination with the use of transgenic lines and other techniques that utilize genetic analysis and manipulation, the cell isolation and electrophysiological methods introduced here should facilitate greater insight into the roles hair cells play in mediating these sensory modalities.

The purpose of this video is to demonstrate procedures for obtaining healthy, intact hair cells from the inner ear organs of adult zebrafish and then using them for patch clamp studies aimed at characterizing the biophysical properties of their voltage-gated channels.

Patch  clamp analyses of the voltage-gated channels in sensory hair cells isolated  from a variety of species have been described previously1-4 but this  ...

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

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs through activation of their ion channels and neurotransmitter receptors, and process information in part through activity-dependent release of gliotransmitters. Furthermore, astrocytes constitute the main uptake system for glutamate, contribute to potassium spatial buffering, as well as to GABA clearance. These cells therefore constantly monitor synaptic activity, and are thereby sensitive indicators for alterations in synaptically-released glutamate, GABA and extracellular potassium levels. Additionally, alterations in astroglial uptake activity or buffering capacity can have severe effects on neuronal functions, and might be overlooked when characterizing physiopathological situations or knockout mice. Dual recording of neuronal and astroglial activities is therefore an important method to study alterations in synaptic strength associated to concomitant changes in astroglial uptake and buffering capacities. Here we describe how to prepare hippocampal slices, how to identify stratum radiatum astrocytes, and how to record simultaneously neuronal and astroglial electrophysiological responses. Furthermore, we describe how to isolate pharmacologically the synaptically-evoked astroglial currents.

The preparation of acute brain slices from isolated hippocampi, as well as the simultaneous electrophysiological recordings of astrocytes and neurons in stratum radiatum during stimulation of schaffer collaterals is described. The pharmacological isolation of astroglial potassium and glutamate transporter currents is demonstrated.

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

Patch Clamp Recordings from Embryonic Zebrafish Mauthner Cells

Mauthner cells (M-cells) are large reticulospinal neurons located in the hindbrain of teleost fish. They are key neurons involved in a characteristic behavior known as the C-start or escape response that occurs when the organism perceives a threat. The M-cell has been extensively studied in adult goldfish where it has been shown to receive a wide range of excitatory, inhibitory and neuromodulatory signals1. We have been examining M-cell activity in embryonic zebrafish in order to study aspects of synaptic development in a vertebrate preparation. In the late 1990s Ali and colleagues developed a preparation for patch clamp recording from M-cells in zebrafish embryos, in which the CNS was largely intact2,3,4. The objective at that time was to record synaptic activity from hindbrain neurons, spinal cord neurons and trunk skeletal muscle while maintaining functional synaptic connections within an intact brain-spinal cord preparation. This preparation is still used in our laboratory today. To examine the mechanisms underlying developmental synaptic plasticity, we record excitatory (AMPA and NMDA-mediated)5,6 and inhibitory (GABA and glycine) synaptic currents from developing M-cells. Importantly, this unique preparation allows us to return to the same cell (M-cell) from preparation to preparation to carefully examine synaptic plasticity and neuro-development in an embryonic organism. The benefits provided by this preparation include 1) intact, functional synaptic connections onto the M-cell, 2) relatively inexpensive preparations, 3) a large supply of readily available embryos 4) the ability to return to the same cell type (i.e. M-cell) in every preparation, so that synaptic development at the level of an individual cell can be examined from fish to fish, and 5) imaging of whole preparations due to the transparent nature of the embryos.

We have developed an intact brain-spinal cord preparation to record and monitor electrical activity via patch clamp recording from the Mauthner neurons and other reticulospinal cells in zebrafish embryos. Thus, we are able to record excitatory and inhibitory synaptic currents, voltage-gated channel activity and action potentials from key neurons in a developing embryo.

Mauthner cells (M-cells) are large  reticulospinal neurons located in the hindbrain of teleost fish. They are key  neurons involved in a characteristic ...

A Computer-assisted Multi-electrode Patch-clamp System

The patch-clamp technique is today the most well-established method for recording electrical activity from individual neurons or their subcellular compartments. Nevertheless, achieving stable recordings, even from individual cells, remains a time-consuming procedure of considerable complexity. Automation of many steps in conjunction with efficient information display can greatly assist experimentalists in performing a larger number of recordings with greater reliability and in less time. In order to achieve large-scale recordings we concluded the most efficient approach is not to fully automatize the process but to simplify the experimental steps and reduce the chances of human error while efficiently incorporating the experimenter's experience and visual feedback. With these goals in mind we developed a computer-assisted system which centralizes all the controls necessary for a multi-electrode patch-clamp experiment in a single interface, a commercially available wireless gamepad, while displaying experiment related information and guidance cues on the computer screen. Here we describe the different components of the system which allowed us to reduce the time required for achieving the recording configuration and substantially increase the chances of successfully recording large numbers of neurons simultaneously.

Multi-electrode patch-clamp recordings constitute a complex task. Here we show how, by automating of many of the experimental steps, it is possible to accelerate the process leading to qualitative improvement in performance and number of recordings.

The patch-clamp technique is today the most  well-established method for recording electrical activity from individual  neurons or their subcellular ...

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.

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.

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

Whole-cell Patch-clamp Recordings in Brain Slices

Whole-cell patch-clamp recording is an electrophysiological technique that allows the study of the&#160;electrical properties of a substantial part of the neuron. In this configuration, the micropipette is in tight contact with the cell membrane, which prevents current leakage and thereby provides more accurate ionic current measurements than the previously used intracellular sharp electrode recording method. Classically, whole-cell recording can be performed on neurons in various types of preparations, including cell culture models, dissociated neurons, neurons in brain slices, and in intact anesthetized or awake animals. In summary, this technique has immensely contributed to the understanding of passive and active biophysical properties of excitable cells. A major advantage of this technique is that it provides information on how specific manipulations (e.g., pharmacological, experimenter-induced plasticity) may alter specific neuronal functions or channels in real-time. Additionally, significant opening of the plasma membrane allows the internal pipette solution to freely diffuse into the cytoplasm, providing means for introducing drugs, e.g., agonists or antagonists of specific intracellular proteins, and manipulating these targets without altering their functions in neighboring cells. This article will focus on whole-cell recording performed on neurons in brain slices,&#160;a preparation that has the advantage of recording neurons in relatively well preserved brain circuits, i.e., in a physiologically relevant context. In particular,&#160;when combined with appropriate pharmacology, this technique is a powerful tool allowing identification of specific neuroadaptations that occurred following any type of experiences, such as learning, exposure to drugs of abuse, and stress. In summary, whole-cell patch-clamp recordings in brain slices provide means to measure in ex vivo preparation long-lasting changes in neuronal functions that have developed in intact awake animals.

This protocol describes basic procedural steps for performing whole-cell patch-clamp recordings. This technique allows the study of&#160;the electrical behavior of neurons, and when performed in brain slices, allows the assessment of various neuronal functions from neurons that are still integrated in relatively well preserved brain circuits.

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