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><tbody><tr><td>Amplifier</td><td>HEKA</td><td>EPC 10 USB Triple</td><td>with 2 - 3 preamplifiers</td></tr><tr><td>Microscope</td><td>Olympus</td><td>BX51WI</td><td>with 2 camera ports, a 4X objective, and a 40X water-immersion objective</td></tr><tr><td>Camera</td><td>TILL Photonics</td><td>VX55</td><td>infrared CCD camera</td></tr><tr><td>Workstation</td><td>Luigs &amp;amp; Neumann</td><td>Infrapatch 240</td><td>with a motorized x-y stage and a motorized focus axis for the microscope</td></tr><tr><td>Micromanipulator</td><td>Luigs &amp;amp; Neumann</td><td>SM-5</td><td>x-y-z manipulators for 2 - 3 preamplifiers</td></tr><tr><td>Faraday cage</td><td>Luigs &amp;amp; Neumann</td><td></td><td></td></tr><tr><td>Anti-vibration table</td><td>Newport Spectra-Physics</td><td></td><td></td></tr><tr><td>Patchmaster</td><td>HEKA</td><td></td><td></td></tr><tr><td>Microtome</td><td>Microm International</td><td>HM650V</td><td></td></tr><tr><td>Micropipette puller</td><td>HEKA</td><td>Sutter P-97</td><td></td></tr><tr><td>Neurolucida system</td><td>Microbrightfield</td><td></td><td>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

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

17.2K 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

Investigations on Alterations of Hippocampal Circuit Function Following Mild Traumatic Brain Injury

Traumatic Brain Injury (TBI) afflicts more than 1.7 million people in the United States each year and even mild TBI can lead to persistent neurological impairments 1. Two pervasive and disabling symptoms experienced by TBI survivors, memory deficits and a reduction in seizure threshold, are thought to be mediated by TBI-induced hippocampal dysfunction 2,3. In order to demonstrate how altered hippocampal circuit function adversely affects behavior after TBI in mice, we employ lateral fluid percussion injury, a commonly used animal model of TBI that recreates many features of human TBI including neuronal cell loss, gliosis, and ionic perturbation 4-6.
	Here we demonstrate a combinatorial method for investigating TBI-induced hippocampal dysfunction. Our approach incorporates multiple ex vivo physiological techniques together with animal behavior and biochemical analysis, in order to analyze post-TBI changes in the hippocampus. We begin with the experimental injury paradigm along with behavioral analysis to assess cognitive disability following TBI. Next, we feature three distinct ex vivo recording techniques: extracellular field potential recording, visualized whole-cell patch-clamping, and voltage sensitive dye recording. Finally, we demonstrate a method for regionally dissecting subregions of the hippocampus that can be useful for detailed analysis of neurochemical and metabolic alterations post-TBI.
	These methods have been used to examine the alterations in hippocampal circuitry following TBI and to probe the opposing changes in network circuit function that occur in the dentate gyrus and CA1 subregions of the hippocampus (see Figure 1). The ability to analyze the post-TBI changes in each subregion is essential to understanding the underlying mechanisms contributing to TBI-induced behavioral and cognitive deficits. 
	The multi-faceted system outlined here allows investigators to push past characterization of phenomenology induced by a disease state (in this case TBI) and determine the mechanisms responsible for the observed pathology associated with TBI.

A multi-faceted approach to investigating functional changes to hippocampal circuitry is explained. Electrophysiological techniques are described along with the injury protocol, behavioral testing and regional dissection method. The combination of these techniques can be applied in similar fashion for other brain regions and scientific questions.

Traumatic Brain Injury (TBI) afflicts more than 1.7 million people in the United States each year and even mild TBI can lead to persistent neurological ...

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 ...

Slice It Hot: Acute Adult Brain Slicing in Physiological Temperature

Here we present a protocol for preparation of acute brain slices. This procedure is a critical element for electrophysiological patch-clamp experiments that largely determines the quality of results. It has been shown that omitting the cooling step during cutting procedure is beneficial in obtaining healthy slices and cells, especially when dealing with highly myelinated brain structures from mature animals. Even though the precise mechanism whereby elevated temperature supports neural health can only be speculated upon, it stands to reason that, whenever possible, the temperature in which the slicing is performed should be close to physiological conditions to prevent temperature related artifacts. Another important advantage of this method is the simplicity of the procedure and therefore the short preparation time. In the demonstrated method adult mice are used but the same procedure can be applied with younger mice as well as rats. Also, the following patch clamp experiment is performed on horizontal cerebellar slices, but the same procedure can also be used in other planes as well as other posterior areas of the brain.

In this paper we show a method for preparing acute brain slices in physiological temperature, using a conventional physiological solution without special modifications for the cutting (such as adding sucrose) and without intracardial perfusion of the animal before slice preparation.

Here we present a protocol for preparation of acute brain slices. This procedure is a critical element for electrophysiological patch-clamp experiments ...

Real-time Electrophysiology: Using Closed-loop Protocols to Probe Neuronal Dynamics and Beyond

Experimental neuroscience is witnessing an increased interest in the development and application of novel and often complex, closed-loop protocols, where the stimulus applied depends in real-time on the response of the system. Recent applications range from the implementation of virtual reality systems for studying motor responses both in mice1 and in zebrafish2, to control of seizures following cortical stroke using optogenetics3. A key advantage of closed-loop techniques resides in the capability of probing higher dimensional properties that are not directly accessible or that depend on multiple variables, such as neuronal excitability4 and reliability, while at the same time maximizing the experimental throughput. In this contribution and in the context of cellular electrophysiology, we describe how to apply a variety of closed-loop protocols to the study of the response properties of pyramidal cortical neurons, recorded intracellularly with the patch clamp technique in acute brain slices from the somatosensory cortex of juvenile rats. As no commercially available or open source software provides all the features required for efficiently performing the experiments described here, a new software toolbox called LCG5 was developed, whose modular structure maximizes reuse of computer code and facilitates the implementation of novel experimental paradigms. Stimulation waveforms are specified using a compact meta-description and full experimental protocols are described in text-based configuration files. Additionally, LCG has a command-line interface that is suited for repetition of trials and automation of experimental protocols.

Closed-loop protocols are becoming increasingly widespread in modern day electrophysiology. We present a simple, versatile and inexpensive way to perform complex electrophysiological protocols in cortical pyramidal neurons in vitro, using a desktop computer and a digital acquisition board.

Experimental neuroscience is witnessing an increased interest in the development and application of novel and often complex, closed-loop protocols, where ...

Preparation of Acute Brain Slices Using an Optimized N-Methyl-D-glucamine Protective Recovery Method

This protocol is a practical guide to the N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. Numerous recent studies have validated the utility of this method for enhancing neuronal preservation and overall brain slice viability. The implementation of this technique by early adopters has facilitated detailed investigations into brain function using diverse experimental applications and spanning a wide range of animal ages, brain regions, and cell types. Steps are outlined for carrying out the protective recovery brain slice technique using an optimized NMDG artificial cerebrospinal fluid (aCSF) media formulation and enhanced procedure to reliably obtain healthy brain slices for patch clamp electrophysiology. With this updated approach, a substantial improvement is observed in the speed and reliability of gigaohm seal formation during targeted patch clamp recording experiments while maintaining excellent neuronal preservation, thereby facilitating challenging experimental applications. Representative results are provided from multi-neuron patch clamp recording experiments to assay synaptic connectivity in neocortical brain slices prepared from young adult transgenic mice and mature adult human neurosurgical specimens. Furthermore, the optimized NMDG protective recovery method of brain slicing is compatible with both juvenile and adult animals, thus resolving a limitation of the original methodology. In summary, a single media formulation and brain slicing procedure can be implemented across various species and ages to achieve excellent viability and tissue preservation.

Preparation of Acute Brain Slices Using an Optimized N-Methyl-D-glucamine Protective Recovery Method

This protocol demonstrates the implementation of an optimized N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. A single media formulation is used to reliably obtain healthy brain slices from animals of any age and for diverse experimental applications.

This protocol is a practical guide to the N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. Numerous recent studies have ...

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 ...

Slice Patch Clamp Technique for Analyzing Learning-Induced Plasticity

The slice patch clamp technique is a powerful tool for investigating learning-induced neural plasticity in specific brain regions. To analyze motor-learning induced plasticity, we trained rats using an accelerated rotor rod task. Rats performed the task 10 times at 30-s intervals for 1 or 2 days. Performance was significantly improved on the training days compared to the first trial. We then prepared acute brain slices of the primary motor cortex (M1) in untrained and trained rats. Current-clamp analysis showed dynamic changes in resting membrane potential, spike threshold, afterhyperpolarization, and membrane resistance in layer II/III pyramidal neurons. Current injection induced many more spikes in 2-day trained rats than in untrained controls.
To analyze contextual-learning induced plasticity, we trained rats using an inhibitory avoidance (IA) task. After experiencing foot-shock in the dark side of a box, the rats learned to avoid it, staying in the lighted side. We prepared acute hippocampal slices from untrained, IA-trained, unpaired, and walk-through rats. Voltage-clamp analysis was used to sequentially record miniature excitatory and inhibitory postsynaptic currents (mEPSCs and mIPSCs) from the same CA1 neuron. We found different mean mEPSC and mIPSC amplitudes in each CA1 neuron, suggesting that each neuron had different postsynaptic strengths at its excitatory and inhibitory synapses. Moreover, compared with untrained controls, IA-trained rats had higher mEPSC and mIPSC amplitudes, with broad diversity. These results suggested that contextual learning creates postsynaptic diversity in both excitatory and inhibitory synapses at each CA1 neuron.
AMPA or GABAA receptors seemed to mediate the postsynaptic currents, since bath treatment with CNQX or bicuculline blocked the mEPSC or mIPSC events, respectively. This technique can be used to study different types of learning in other regions, such as the sensory cortex and amygdala.

The slice patch clamp technique is an effective method for analyzing learning-induced changes in the intrinsic properties and plasticity of excitatory or inhibitory synapses.

The slice patch clamp technique is a powerful tool for investigating learning-induced neural plasticity in specific brain regions. To analyze ...

Application of Automated Image-guided Patch Clamp for the Study of Neurons in Brain Slices

Whole-cell patch clamp is the gold-standard method to measure the electrical properties of single cells. However, the in vitro patch clamp remains a challenging and low-throughput technique due to its complexity and high reliance on user operation and control. This manuscript demonstrates an image-guided automatic patch clamp system for in vitro whole-cell patch clamp experiments in acute brain slices. Our system implements a computer vision-based algorithm to detect fluorescently labeled cells and to target them for fully automatic patching using a micromanipulator and internal pipette pressure control. The entire process is highly automated, with minimal requirements for human intervention. Real-time experimental information, including electrical resistance and internal pipette pressure, are documented electronically for future analysis and for optimization to different cell types. Although our system is described in the context of acute brain slice recordings, it can also be applied to the automated image-guided patch clamp of dissociated neurons, organotypic slice cultures, and other non-neuronal cell types.

This protocol describes how to conduct automatic image-guided patch-clamp experiments using a system recently developed for standard in vitro electrophysiology equipment.

Whole-cell patch clamp is the gold-standard method to measure the electrical properties of single cells. However, the in vitro patch clamp remains a ...

Preparation of Acute Spinal Cord Slices for Whole-cell Patch-clamp Recording in Substantia Gelatinosa Neurons

Recent whole-cell patch-clamp studies from substantia gelatinosa (SG) neurons have provided a large body of information about the spinal mechanisms underlying sensory transmission, nociceptive regulation, and chronic pain or itch development. Implementations of electrophysiological recordings together with morphological studies based on the utility of acute spinal cord slices have further improved our understanding of neuronal properties and the composition of local circuitry in SG. Here, we present a detailed and practical guide for the preparation of spinal cord slices and show representative whole-cell recording and morphological results. This protocol permits ideal neuronal preservation and can mimic in vivo conditions to a certain extent. In summary, the ability to obtain an in vitro preparation of spinal cord slices enables stable current- and voltage-clamp recordings and could thus facilitate detailed investigations into the intrinsic membrane properties, local circuitry and neuronal structure using diverse experimental approaches.

Here, we describe the essential steps for whole-cell patch-clamp recordings made from substantia gelatinosa (SG) neurons in the in vitro spinal cord slice. This method allows the intrinsic membrane properties, synaptic transmission and morphological characterization of SG neurons to be studied.

Recent whole-cell patch-clamp studies from substantia gelatinosa (SG) neurons have provided a large body of information about the spinal mechanisms ...

Evaluation of Synaptic Multiplicity Using Whole-cell Patch-clamp Electrophysiology

In the central nervous system, a pair of neurons often form&#160;multiple synaptic contacts and/or functional neurotransmitter release sites (synaptic multiplicity). Synaptic multiplicity is plastic and changes throughout development and in different physiological conditions, being an important determinant for the efficacy of synaptic transmission. Here, we outline experiments for estimating the degree of multiplicity of synapses terminating onto a given postsynaptic neuron using whole-cell patch clamp electrophysiology in acute brain slices. Specifically, voltage-clamp recording is used to compare the difference between the amplitude of spontaneous excitatory postsynaptic currents (sEPSCs) and miniature excitatory postsynaptic currents (mEPSCs). The theory behind this method is that afferent inputs that exhibit multiplicity will show large, action potential-dependent sEPSCs due to the synchronous release that occurs at each synaptic contact. In contrast, action potential-independent release (which is asynchronous) will generate smaller amplitude mEPSCs. This article outlines a set of experiments and analyses to characterize the existence of synaptic multiplicity and discusses the requirements and limitations of the technique. This technique can be applied to investigate how different behavioral, pharmacological or environmental interventions in vivo affect the organization of synaptic contacts in different brain areas.

Here, we present a protocol for evaluating the functional synaptic multiplicity using whole-cell patch clamp electrophysiology in acute brain slices.

In the central nervous system, a pair of neurons often form&#160;multiple synaptic contacts and/or functional neurotransmitter release sites (synaptic ...

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

Summary

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Preparation of Acute Brain Slices Using an Optimized N-Methyl-D-glucamine Protective Recovery Method

Whole-cell Patch-clamp Recordings in Brain Slices

Slice Patch Clamp Technique for Analyzing Learning-Induced Plasticity

Application of Automated Image-guided Patch Clamp for the Study of Neurons in Brain Slices

Preparation of Acute Spinal Cord Slices for Whole-cell Patch-clamp Recording in Substantia Gelatinosa Neurons

Evaluation of Synaptic Multiplicity Using Whole-cell Patch-clamp Electrophysiology