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 Med...

<ol>
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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...

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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 ...

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14.5K 次观看.  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.