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

Animal Ethics Statement:
The protocols described in this manuscript follow the animal care guidelines established by The University of Auckland and Stanford University. P7 rat pups were euthanized by rapid decapitation. Hippocampal dissection is then immediately performed as described below.
1. Organotypic Hippocampal Slice Culture
<ol>
	<li>Preparation
	<ol>
		<li>Prepare dissection Medium (used only for dissecting the brain). Combine 200 ml Minimum Essential Medium, 2 ml penicillin-streptomycin solution (10,000 units of each, in 0.85% NaCl), 5 ml HEPES buffer solution, 2 ml 1 M Tris stock solution (pH 7.2), and filter sterilize with 0.22 &#956;m filter. Carry out hippocampal dissection in ice-cold dissection medium.</li>
		<li>Cool the dissection medium. Place the dissection media in the freezer approximately 1 hr prior to beginning the dissection until the liquid is very cold. Do not allow large ice crystals to form. Store on ice until required.</li>
		<li>Prepare culture medium (used for everything except dissection of hippocampi). Combine 100 ml Minimum Essential Medium, (1x concentration, liquid) w/Hank&#8217;s salts, w/ L-glutamine, 2 ml penicillin-streptomycin solution (liquid, 10,000 units of each, in 0.85% NaCl ), 2.5 ml HEPES 1 M buffer solution, 50 ml Hank&#8217;s Balanced Salt Solution, 50 ml Horse Serum (defined, heat inactivated), and filter sterilize with 0.22 &#956;m filter.</li>
		<li>Prepare the culture dishes. Place 1 ml of culture media per 35 mm culture dish, and add a membrane insert to each dish. Put up to seven of these dishes into one 150 mm Petri dish (referred to hereafter as a &#8216;plate&#8217;). Place the plate in a CO2 incubator for at least an hour before the dissection begins so that the culture medium in the dishes attains the proper temperature and pH.</li>
	</ol>
	</li>
	<li>Dissection of the Hippocampi from Rat Pups at Postnatal Day 7 (P7)
	<ol>
		<li>Pre-sterilize all dissection tools under UV light before the procedure.</li>
		<li>After rapid decapitation, remove the brain and place into chilled medium in one dish, and then remove it to a piece of moistened filter paper for the dissection. Tease the cortex away from the midbrain using blunt smooth plastic-coated miniature spatulas, exposing the hippocampus. Cut the fornix, and then gently work the spatula underneath the hippocampus to flip it out (Figure 1A). 
		NOTE: Successful slice cultures can be prepared using animals up to P10.</li>
		<li>Trim the isolated hippocampus away from the rest of the brain. Transfer the hippocampi into a new dish containing chilled dissection media using a moistened soft paintbrush (e.g., white sable #4).</li>
		<li>Clean the underside (i.e. the flatter side) of the hippocampus of choroid plexus during dissection, as these spongy, meninge-like tissues make it difficult to separate hippocampal slices from one another later on. 
		NOTE: Leave these tissues in place if they cannot be teased away gently.</li>
		<li>Perform the entire dissection as quickly as possible without damaging the hippocampi. 
		NOTE: A careful dissection is more important than a fast one, as long as the experimental conditions are chilled. Slice the hippocampi from three rats simultaneously. The slice health is not affected as long as the total time from start to when the plated slices go into the incubator is under 30 min.</li>
	</ol>
	</li>
	<li>Slicing
	<ol>
		<li>Slice the hippocampi transversely into 400 &#956;m cross-sections using a manual tissue slicer. The method by which the slicing is conducted is not important, so long as it is not overly damaging to the tissue and produces slices of consistent thickness with readily visible laminar architecture (i.e. Ammon&#8217;s Horn is easily seen to be preserved in the tissue).</li>
		<li>Lie the hippocampi on the stage of the tissue chopper on top of a triple thickness of #2 filter paper. The hippocampi are sliced entirely without removing any slices individually (i.e. the entire hippocampus is left on the stage of the chopper until all cuts have been made; rather like loaves of sliced bread at this point; Figure 1B).</li>
		<li>Transfer the hippocampi into dissection medium using a soft paintbrush laid next to each hippocampus (white sable #2). Gently push each hippocampus to the side to break the adhesion between the hippocampus and the underlying filter paper. Roll the brush underneath each hippocampus to pick it up off the stage. Place all hippocampi into the same 35 mm Petri dish of chilled dissection medium. Separate the hippocampi into individual slices by manually agitating the dish in alternating clockwise and counterclockwise motions.</li>
		<li>Inspect the slices under a dissecting microscope and discard any which are damaged, small, or which do not possess clearly visible cell body layers. 
		NOTE: It will often be the case that not all the slices are successfully separated from their siblings in this manner. In this case, they can be separated by turning them on edge and prodding them with the tips of fine forceps. &#8220;Good&#8221; slices are then transferred to another clean Petri dish containing chilled dissection medium using a cut off and fire-polished Pasteur pipette (Figures 1C-E).</li>
	</ol>
	</li>
	<li>Storage
	<ol>
		<li>Place the individual slices onto the membrane filter inserts. Using a Pasteur pipette cut off and fire polished to give a larger bore, individually transfer slices to the culture inserts. 
		NOTE: The size of the bore created when cutting and fire-polishing the pipette is important. Too small and the slices will not easily leave the pipette for the membrane. Too large and excess fluid is placed on the surface of the membrane along with the slice. The best bore diameter is typically about half the diameter of the barrel of the pipette. When cutting, this diameter can be chosen by cutting at the proper place on the taper of the pipette.</li>
		<li>Slice Transfer
		<ol>
			<li>Fill the pipette with medium first, and then suck up a single slice with small suction forces. This keeps the slice near the opening of the pipette and prevents too much medium expulsion into the insert to place the slice.</li>
			<li>Apply a slight pressure on the bulb to form a small hanging droplet, allow the slice to settle into that droplet, and then touch that droplet to the membrane and let the slice &#8220;fall&#8221; onto the membrane. Generally three slices are placed onto each membrane insert. 
			NOTE: If the fluid droplet is too large, the droplets from the three slices tend to merge on the membrane surface, and the slices will thus all gather to the center of the membrane, making it more difficult to separate them for electrophysiological recording later.</li>
		</ol>
		</li>
		<li>Fluid Removal
		<ol>
			<li>Aspirate any excess medium from the top of the culture plate insert for all slices in that plate (3 slices per insert = 21 slices per plate), so that slices are not immersed in or surrounded by a pool of dissection medium.</li>
			<li>Perform this is with an uncut, but fire-polished Pasteur pipette. Allow the slice to settle and adhere to the membrane for a minute before pipetting. Take care not to suck the slice up into the pipette. Carry out fluid removal for the inserts that are plated first, to allow sufficient time for the slices to settle.</li>
		</ol>
		</li>
		<li>Slice Storage
		<ol>
			<li>Store slice cultures in a 5.0% CO2 incubator at 37 &#176;C. Observe the final arrangement of the cultures in Figure 1E. The individual slices rest upon a culture plate insert which has a porous membrane for a bottom, and this insert fits inside a Petri dish filled with a small amount of medium; by this means, the slices are not immersed in the medium, but have access to it only through the membrane at the bottom of the culture plate insert.</li>
			<li>Optionally, remove the slices may for study either by removing the culture plate insert or by cutting out a section of insert membrane containing a slice.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Maintenance
	<ol>
		<li>Exchange the medium in the Petri dishes the day after making cultures. Transfer the culture plate insert to a new petri dish containing 1 ml of fresh culture medium that is equilibrated in the incubator for at least an hour prior to transfer.</li>
		<li>Change the medium again in the same manner on the third day after making cultures. On the third day, transfer the cultures to an incubator set at 34 &#176;C. Change the medium twice each week (every 3-4 days).</li>
		<li>Identify healthy cultures. Select healthy cultures for paired whole cell recordings. 
		NOTE: Healthy cultures have a well-defined edge and a clearly defined pyramidal cell layer. Cultures with dark (likely necrotic) areas present or a vacuolated appearance with flattened borders are rejected. Employing these criteria, usually two-thirds of slice cultures are sufficiently healthy for recording.</li>
		<li>Utilize these cultures within a fairly small window of time. Do not used tissues that are cultured for more than two weeks since the neurons begin to display slightly epileptiform behavior which slowly worsens with time. The exact upper limit of neuronal survival is not known but it is possible to record from functioning pyramidal cells as late as 16&#160;weeks in culture.</li>
	</ol>
	</li>
</ol>
2. Paired Whole Cell Recordings
<ol>
	<li>ACSF and Intracellular Solutions
	<ol>
		<li>Prepare presynaptic electrode solution by mixing (in mM) 120 potassium gluconate, 40 HEPES, 5 MgCl2, 2 NaATP, and 0.3 NaGTP (pH 7.2 with KOH; osmolarity: 290 mOsm). Whilst the same electrode solution is used for postsynaptic neurons, cesium gluconate (120 mM) is typically used as the major salt in the postsynaptic electrode, plus 5 mM QX314. 
		NOTE: This enables stable voltage clamping at positive potentials to record postsynaptic NMDAR-mediated currents8-10,13. It also prevents the potassium-induced hyperexcitability of the presynaptic neuron by the internal solution flowing from the postsynaptic recording electrode before a giga ohm seal is made.</li>
		<li>Prepare postsynaptic electrode solution composing (in mM) 120 cesium gluconate, 40 HEPES, 5 MgCl2, 5 QX314, 2 NaATP, and 0.3 NaGTP (pH 7.2 with CsOH; osmolarity: 290 mOsm). Pull glass microelectrodes at 5-10 M&#937; resistance and fill with filtered internal solution.</li>
		<li>Prepare artificial cerebrospinal fluid (ACSF) composing (in mM) 119 NaCl, 2.4 KCl, 1.3 MgSO4, 2.4 CaCl2, 1 Na2HPO4, 26.2 NaHCO3, 11 glucose, pH 7.4, saturated with 95% O2, 5% CO2. NOTE: If AMPAR-mediated responses need to be blocked for examination of NMDAR EPSCs, include 10 &#956;M CNQX or NBQX in the ACSF.</li>
	</ol>
	</li>
	<li>Obtain Paired Whole Cell Recordings
	<ol>
		<li>To examine synaptic transmission between two individual neurons, designate one neuron as the presynaptic neuron and hold in current clamp to induce action potentials and initiate synaptic transmission.</li>
		<li>Designate the second neuron, as the postsynaptic neuron, and hold it in current or voltage clamp depending on the information required by the researcher. Obtain detailed examination of the glutamatergic currents, with AMPAR-mediated EPSCs examined at -65 mV and NMDAR-mediated EPSCs examined at +30 mV6-16 by holding the postsynaptic neuron in voltage clamp</li>
		<li>Establish a paired recording. To establish a paired recording, the presynaptic whole cell recording is always obtained first, enabling multiple sequential postsynaptic neurons to be obtained until one that is synaptically connected is obtained. If performing synaptic plasticity experiments, it is especially critical to obtain the presynaptic neuron first as the induction of LTP in the postsynaptic neuron must be initiated within 10 min of obtaining the whole cell recordings to prevent washout of cytoplasmic factors required for LTP8,19.</li>
		<li>Avoid movement-induced cell loss. A major challenge when performing paired whole cell recordings is avoiding vibration and movement-induced disruption of the first whole cell recording whilst obtaining the second recording.
		<ol>
			<li>Obtain the first whole cell recording and then move the microscope lens 10-200 &#956;m in the x-axis so that the established recording is located at the edge of the area visible on the monitor (Figure 2A).</li>
			<li>Raise the lens of the microscope ~5-10 mm while ensuring that the ACSF still maintains contact with the lens. Mount the second electrode and guide into the fluid meniscus (Figure 2B).</li>
			<li>Move the electrode in the x-y plane until it is directly under the light path through the lens but still significantly above the established recording electrode. Once the electrode is visible under the microscope, ensure the tip is at the far edge of the monitor away from the first electrode.</li>
			<li>Move the second electrode down in sequence with the microscope focus until both electrodes are in the same focal plane. It is important that the level of positive pressure is strong enough to avoid tip blockage but not too strong so as to disrupt the first whole cell recording. This translates to the positive pressure inducing movement in 2-3 neurons from the electrode when it first enters the slice.</li>
			<li>Locate a postsynaptic partner in a similar focal plane to the first presynaptic recording (Figure 2C). Recording is generally Obtain paired recordings between CA3 pyramidal neurons the second whole cell from a 0-200 mm neuron of the first recording (Figures 2C,D). 
			NOTE: Synaptic transmission between neuronal pairs are stable over time periods of up to 3-4 hr when using these standard whole cell recording techniques.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Synaptic plasticity: Once Upon obtaining a successful synaptically-connected pyramidal cell pair (Figures 3A,B), examine the characteristics of synaptic transmission and plasticity.
	<ol>
		<li>LTP induction.
		<ol>
			<li>Induce LTP by pairing presynaptic action potentials (1 Hz) with postsynaptic depolarization to -10 to 0 mV for 1 min (Figure 3C)7-9,12-14. Initiate pairing within 10 min of break-in to the postsynaptic neuron. 
			NOTE: LTP can also be induced with both neurons held in current clamp by pairing presynaptic and postsynaptic action potentials at 1 Hz for 1 min, with postsynaptic action potentials elicited 10 msec following injection of current into the presynaptic neuron8.</li>
		</ol>
		</li>
		<li>Further induce LTD by low frequency stimulation (LFS) at 1 Hz combined with a slight depolarization of the postsynaptic neuron to -55 mV for 5-10 min (Figure 3C)9.</li>
	</ol>
	</li>
</ol>

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V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Presynaptic+FMR1+genotype+influences+the+degree+of+synaptic+connectivity+in+a+mosaic+mouse+model+of+fragile+X+syndrome.">Presynaptic FMR1 genotype influences the degree of synaptic connectivity in a mosaic mouse model of fragile X syndrome.</a> Journal of Neuroscience. 27 (15), 4014-4018 (2007).</li><li>Hanson, J. E., Blank, M., Valenzuela, R. A., Garner, C. C., Madison, D. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+functional+nature+of+synaptic+circuitry+is+altered+in+area+CA3+of+the+hippocampus+in+a+mouse+model+of+Down's+syndrome.">The functional nature of synaptic circuitry is altered in area CA3 of the hippocampus in a mouse model of Down's syndrome.</a> Journal of Physiology. 579, 53-67 (2007).</li></ol>

The authors have nothing to disclose

Here we have described the requirements for establishing successful paired whole cell recordings in organotypic hippocampal slice cultures. Paired recordings can also be performed in multiple preparations, including acute slices and dissociated culture systems26,27. While the focus here has been on the induction of longer forms of synaptic plasticity (namely LTP and LTD), it is important to highlight that paired whole cell recordings in organotypic, acute slice and dissociated cell preparations have provided i...

Glutamate receptors mediate the majority of excitatory synaptic transmission at central nervous system synapses. The two major subtypes of ionotropic glutamate receptors localized at the spine head of the postsynaptic membrane are N-methyl-D-aspartate (NMDA) and &#945;-amino-3-hydroxy-5-methylisoxazole-4-proprionic acid (AMPA) receptors. At resting membrane potentials, AMPA receptors carry most of the postsynaptic current during synaptic transmission. In the hippocampus, the NMDA receptor plays a key role in triggering changes in the number of AMPA receptors in the postsynaptic membrane: by acting as a &#8221;coincidence detector&#8221;1 to initiate changes in synaptic strength1, the NMDA receptor participates in the synaptic mechanisms that are thought to underpin learning and memory at a subcellular level. In response to depolarization of the postsynaptic neuron in parallel with presynaptic transmitter release, calcium enters via the NMDA receptor to initiate AMPA receptor insertion or removal2. These receptor dynamics underlie synapse plasticity: an increase in synaptic strength is long-term potentiation2,3 (LTP), while a decrease in synaptic strength is long-term depression4 (LTD). Therefore AMPA receptor movement is thought to be responsible for synaptic plasticity expression, while NMDA receptors are thought to control its induction.
Determining the precise mechanisms underlying synaptic transmission and plasticity requires studying small populations of synapses, ideally single synapses. While some synapses are highly suited for study at this level, e.g.,&#160;the Calyx of Held5, for most synaptic populations this is extremely difficult due to the small and diffuse nature of the synaptic connections. Two major electrophysiology techniques have been developed to examine single synaptic connections: The first is minimal stimulation, where one presynaptic fiber is presumed stimulated extracellularly. The second technique is paired recordings, where two simultaneous whole cell recordings from synaptically connected neurons is performed. A major advantage of minimal stimulation is that it is rapid and relatively simple to perform, involving placement of an extracellular stimulating electrode into the axonal tract while simultaneously recording from a postsynaptic neuron. The primary concern when using this technique is that reliable stimulation of a single cell can rarely be guaranteed trial after trial.
Over the past fifteen years we have routinely used paired whole-cell recordings from two synaptically connected pyramidal neurons6-17. The major advantage of this technique is that only one presynaptic neuron is consistently and reliably stimulated. It also allows not only electrophysiological characterization but also pharmacological manipulation of the presynaptic neuron6,18. However, the probability of synaptic connectivity between neurons is low, making connected pairs difficult to obtain19. The use of organotypic brain slice cultures circumvents this obstacle as synaptic connectivity can re-establish in vitro and moreover the nature of the resulting connectivity is similar to that in native brain tissue20. In addition, organotypic cultures express LTP, LTD7-10,12-15,21 and additional forms of short-term synaptic plasticity including paired-pulse facilitation (PPF) and depression (PPD)6,22,23, enabling plasticity mechanisms to be studied in pairs of neurons. Here we describe the detailed methodology involved in successfully attaining paired recordings in this in vitro system. This information can readily be adapted to other experimental systems, including acute slices and other brain regions.

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

paired whole cell recordings in organotypic hippocampal slices

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

Synaptic connectivity is evident by stimulating the presynaptic neuron to fire an action potential by passing a depolarizing current pulse (typically 20-50 pA for 20 msec) via the recording electrode. The postsynaptic current trace is then examined for the presence of a monosynaptic EPSC evoked at short (&#60;5 msec) and consistent latencies after the peak of the presynaptic action potential (Figure 3A). In most experiments multiple postsynaptic neurons are tested before a synaptically-connected pair can...

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

Paired Whole Cell Recordings in Organotypic Hippocampal Slices

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

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

paired-whole-cell-recordings-in-organotypic-hippocampal-slices

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Neuroscience

14.6K visualizações.  University of Auckland. 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.

Vídeo: Paired Whole Cell Recordings in Organotypic Hippocampal Slices

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.  ...

Organotypic Hippocampal Slice Cultures

The hippocampus, a component of the limbic system, plays important roles in long-term memory and spatial navigation 1. Hippocampal neurons can modify the strength of their connections after brief periods of strong activation. This phenomenon, known as long-term potentiation (LTP) can last for hours or days and has become the best candidate mechanism for learning and memory 2. In addition, the well defined anatomy and connectivity of the hippocampus 3 has made it a classical model system to study synaptic transmission and synaptic plasticity4.
As our understanding of the physiology of hippocampal synapses grew and molecular players became identified, a need to manipulate synaptic proteins became imperative. Organotypic hippocampal cultures offer the possibility for easy gene manipulation and precise pharmacological intervention but maintain synaptic organization that is critical to understanding synapse function in a more naturalistic context than routine culture dissociated neurons methods.
Here we present a method to prepare and culture hippocampal slices that can be easily adapted to other brain regions. This method allows easy access to the slices for genetic manipulation using different approaches like viral infection 5,6 or biolistics 7. In addition, slices can be easily recovered for biochemical assays 8, or transferred to microscopes for imaging 9 or electrophysiological experiments 10.

We describe a method to prepare organotypic hippocampal slices that can be easily adapted to other brain regions. Brain slices are laid on porous membranes and culture media is allowed to form an interface. This method preserves the gross architecture of the hippocampus for up to 2 weeks in culture.

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

Whole Cell Recording from an Organotypic Slice Preparation of Neocortex

We have been studying the expression and functional roles of voltage-gated potassium channels in pyramidal neurons from rat neocortex. Because of the lack of specific pharmacological agents for these channels, we have taken a genetic approach to manipulating channel expression. We use an organotypic culture preparation (16) in order to maintain cell morphology and the laminar pattern of cortex. We typically isolate acute neocortical slices at postnatal days 8-10 and maintain the slices in culture for 3-7 days. This allows us to study neurons at a similar age to those in our work with acute slices and minimizes the development of exuberant excitatory connections in the slice. We record from visually-identified pyramidal neurons in layers II/III or V using infrared illumination (IR-) and differential interference contrast microscopy (DIC) with whole cell patch clamp in current- or voltage-clamp. We use biolistic (Gene gun) transfection of wild type or mutant potassium channel DNA to manipulate expression of the channels to study their function. The transfected cells are easily identified by epifluorescence microscopy after co-transfection with cDNA for green fluorescent protein (GFP). We compare recordings of transfected cells to adjacent, untransfected neurons in the same layer from the same slice.

This is a protocol to prepare and maintain a neocortical slice preparation in organotypic culture for the purpose of making electrical recordings from pyramidal neurons.

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

Multi-electrode Array Recordings of Neuronal Avalanches in Organotypic Cultures

The cortex is spontaneously active, even in the absence of any particular input or motor output. During development, this activity is important for the migration and differentiation of cortex cell types and the formation of neuronal connections1. In the mature animal, ongoing activity reflects the past and the present state of an animal into which sensory stimuli are seamlessly integrated to compute future actions. Thus, a clear understanding of the organization of ongoing i.e. spontaneous activity is a prerequisite to understand cortex function. 
Numerous recording techniques revealed that ongoing activity in cortex is comprised of many neurons whose individual activities transiently sum to larger events that can be detected in the local field potential (LFP) with extracellular microelectrodes, or in the electroencephalogram (EEG), the magnetoencephalogram (MEG), and the BOLD signal from functional magnetic resonance imaging (fMRI). The LFP is currently the method of choice when studying neuronal population activity with high temporal and spatial resolution at the mesoscopic scale (several thousands of neurons). At the extracellular microelectrode, locally synchronized activities of spatially neighbored neurons result in rapid deflections in the LFP up to several hundreds of microvolts. When using an array of microelectrodes, the organizations of such deflections can be conveniently monitored in space and time. 
Neuronal avalanches describe the scale-invariant spatiotemporal organization of ongoing neuronal activity in the brain2,3. They are specific to the superficial layers of cortex as established in vitro4,5, in vivo in the anesthetized rat 6, and in the awake monkey7. Importantly, both theoretical and empirical studies2,8-10 suggest that neuronal avalanches indicate an exquisitely balanced critical state dynamics of cortex that optimizes information transfer and information processing.
In order to study the mechanisms of neuronal avalanche development, maintenance, and regulation, in vitro preparations are highly beneficial, as they allow for stable recordings of avalanche activity under precisely controlled conditions. The current protocol describes how to study neuronal avalanches in vitro by taking advantage of superficial layer development in organotypic cortex cultures, i.e. slice cultures, grown on planar, integrated microelectrode arrays (MEA; see also 11-14).

A robust way to study neuronal avalanches, i.e. scale-invariant spatio-temporal activity bursts, indicative of critical state dynamics in cortex. Avalanches emerge spontaneously in developing superficial layers of cultured cortex which allows for long-term measurements of the activity with planar integrated multi-electrode arrays (MEA) under precisely controlled conditions.

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

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

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 ...

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

GABAergic inhibitory interneurons play a central role within neuronal circuits of the brain. Interneurons comprise a small subset of the neuronal population (10-20%), but show a high level of physiological, morphological, and neurochemical heterogeneity, reflecting their diverse functions. Therefore, investigation of interneurons provides important insights into the organization principles and function of neuronal circuits. This, however, requires an integrated physiological and neuroanatomical approach for the selection and identification of individual interneuron types. Whole-cell patch-clamp recording from acute brain slices of transgenic animals, expressing fluorescent proteins under the promoters of interneuron-specific markers, provides an efficient method to target and electrophysiologically characterize intrinsic and synaptic properties of specific interneuron types. Combined with intracellular dye labeling, this approach can be extended with post-hoc morphological and immunocytochemical analysis, enabling systematic identification of recorded neurons. These methods can be tailored to suit a broad range of scientific questions regarding functional properties of diverse types of cortical neurons.

Cortical networks are controlled by a small, but diverse set of inhibitory interneurons. Functional investigation of interneurons therefore requires targeted recording and rigorous identification. Described here is a combined approach involving whole-cell recordings from single or synaptically-coupled pairs of neurons with intracellular labeling, post-hoc morphological and immunocytochemical analysis.

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

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

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.

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 ...

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 ...

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

In organotypic hippocampal slice cultures (OHSC), the morphological and functional characteristics of both neurons and glial cells are well preserved. This model is suitable for addressing different research questions that involve studies on neuroprotection, electrophysiological experiments on neurons, neuronal networks or tumor invasion. The hippocampal architecture and neuronal activity in multisynaptic circuits are well conserved in OHSC, even though the slicing procedure itself initially lesions and leads to formation of a glial scar. The scar formation alters presumably the mechanical properties and diffusive behavior of small molecules, etc. Slices allow the monitoring of time dependent processes after brain injury without animal surgery, and studies on interactions between various brain-derived cell types, namely astrocytes, microglia and neurons under both physiological and pathological conditions. An ambivalent aspect of this model is the absence of blood flow and immune blood cells. During the progression of the neuronal injury, migrating immune cells from the blood play an important role. As those cells are missing in slices, the intrinsic processes in the culture may be observed without external interference. Moreover, in OHSC the composition of the medium-external environment is precisely controlled. A further advantage of this method is the lower number of sacrificed animals compared to standard preparations. Several OHSC can be obtained from one animal making simultaneous studies with multiple treatments in one animal possible. For these reasons, OHSC are well suited to analyze the effects of new protective therapeutics after tissue damage or during tumor invasion. 
The protocol presented here describes a preparation method of OHSC that allows generating highly reproducible, well preserved slices that can be used for a variety of experimental research, like neuroprotection or tumor invasion studies.

Organotypic hippocampal slice cultures (OHSC) represent an in vitro model that simulates the in vivo situation very well. Here we describe a vibratome-based improved slicing protocol to obtain high quality slices for use in assessing the neuroprotective potential of novel substances or the biological behavior of tumor cells.

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

Whole-cell Patch-clamp Recordings for Electrophysiological Determination of Ion Selectivity in Channelrhodopsins

Over the past decade, channelrhodopsins became indispensable in neuroscientific research where they are used as tools to non-invasively manipulate electrical processes in target cells. In this context, ion selectivity of a channelrhodopsin is of particular importance. This article describes the investigation of chloride selectivity for a recently identified anion-conducting channelrhodopsin of Proteomonas sulcata via electrophysiological patch-clamp recordings on HEK293 cells. The experimental procedure for measuring light-gated photocurrents demands a fast switchable &#8211; ideally monochromatic &#8211; light source coupled into the microscope of an otherwise conventional patch-clamp setup. Preparative procedures prior to the experiment are outlined involving preparation of buffered solutions, considerations on liquid junction potentials, seeding and transfection of cells, and pulling of patch pipettes. The actual recording of current-voltage relations to determine the reversal potentials for different chloride concentrations takes place 24 h to 48 h after transfection. Finally, electrophysiological data are analyzed with respect to theoretical considerations of chloride conduction.

This article describes how the ion selectivity of channelrhodopsin is determined with electrophysiological whole-cell patch-clamp recordings using HEK293 cells. Here, the experimental procedure for investigating chloride selectivity of an anion-selective channelrhodopsin is demonstrated. However, the procedure is transferable to other channelrhodopsins of distinct selectivity.