Name: evaluation of synaptic multiplicity using whole-cell patch-clamp electrophysiology
Uploaded: 2019-04-23T14:30:00
Description: Watch this Scientific Journal Video about Evaluation of Synaptic Multiplicity Using Whole-cell Patch-clamp Electrophysiology at JoVE.com

J.S. received Ontario Graduate Scholarship. W.I. received a New Investigator Fellowship from Mental Health Research Canada. This work is supported by operating grants to W.I from the Natural Sciences and Engineering Research Council of Canada (06106-2015 RGPIN) and the Canadian Institute for Health Research (PJT 148707).

All animal experiments are approved by the Animal Care Committee of The University of Western Ontario in accordance with the Canadian Council on Animal Care Guidelines (AUP#2014-031).
1. Solutions
<ol>
	<li>Slicing solution 
	<ol>
		<li>Refer to Table 1 for the composition of the slicing solution.</li>
		<li>Prepare a 20x stock solution in advance and store it at 4 &#176;C for up to 1 month.</li>
		<li>For 1x slicing solution, dissolve NaHCO3, glu...

<ol>
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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+binomial+parameters+of+quantal+release+at+crustacean+motor+axon+terminals+during+presynaptic+inhibition.">Changes in binomial parameters of quantal release at crustacean motor axon terminals during presynaptic inhibition.</a> The Journal of physiology. 402, 177-193 (1988).</li><li>Li, G. L., Keen, E., Andor-Ard&#243;, D., Hudspeth, A. J., von Gersdorff, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+unitary+event+underlying+multiquantal+EPSCs+at+a+hair+cell&#8217;s+ribbon+synapse.">The unitary event underlying multiquantal EPSCs at a hair cell&#8217;s ribbon synapse.</a> The Journal of neuroscience the official journal of the Society for Neuroscience. 29 (23), 7558-7568 (2009).</li><li>Wadiche, J. I., Jahr, C. 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B., Messer, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+composition+of+experimental+solutions+on+neuronal+survival+during+rat+brain+slicing.">Effect of composition of experimental solutions on neuronal survival during rat brain slicing.</a> Experimental neurology. 131 (1), 133-143 (1995).</li><li>Tanaka, Y., Tanaka, Y., Furuta, T., Yanagawa, Y., Kaneko, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+cutting+solutions+on+the+viability+of+GABAergic+interneurons+in+cerebral+cortical+slices+of+adult+mice.">The effects of cutting solutions on the viability of GABAergic interneurons in cerebral cortical slices of adult mice.</a> Journal of neuroscience methods. 171 (1), 118-125 (2008).</li><li>Ye, J. H., Zhang, J., Xiao, C., Kong, J. 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One important requirement for a successful patch clamp electrophysiology experiment is obtaining healthy slices/cells. Our described protocol is optimized for hypothalamic slices that contain PVN neurons. Other brain areas may require modified solutions and slicing methods21,22,23,24. For the recording, it is critical to only accept stable recordings by constantly monitoring cell properties suc...

Synaptic transmission is a fundamental mechanism for communication between neurons, and hence, brain function. Synaptic transmission is also labile and can change its efficacy in an activity-dependent manner as well as in response to modulatory signals1. Thus, examining&#160;synaptic function has been a key focus of neuroscience research. Whole-cell patch clamp electrophysiology is a versatile technique that enables us to understand, by devising experimental designs and data analyses, in-depth biophysical and molecular mechanisms of synaptic transmission. A commonly used approach, perhaps owing to the simplicity of the technique and concept, is the measurement of miniature excitatory/inhibitory postsynaptic currents (mE/IPSCs) under the voltage clamp configuration2,3,4,5,6. Individual mPSCs represent the flow of ions through postsynaptic ionotropic receptors (e.g. AMPA and GABAA receptors) in response to the binding of their respective neurotransmitters released from the presynaptic terminal 7. Because the recording is obtained in the presence of the voltage-gated Na+ channel blocker tetrodotoxin (TTX), the release is action potential-independent and normally involves a single synaptic vesicle that contains neurotransmitter. Based on this assumption, the average amplitude of mPSCs is widely used as a crude estimate for the quantal size, which represents the number and functionality of postsynaptic receptors opposing a single release site. On the other hand, the frequency of mPSCs is considered to represent a combination of the total number of synapses terminating onto the postsynaptic cell and their average release probability. However, these parameters do not measure another variable-multiplicativity of synapses, or synaptic multiplicity &#8212; which is important for the efficacy of synaptic transmission.
Based on the quantal theory of synaptic transmission7,8,9, the strength of a given connection between a pair of neurons is dependent on three factors: the number of functional synapses (N), the postsynaptic response to the release of a single synaptic vesicle (quantal size; Q) and the probability of neurotransmitter release (Pr). Synaptic multiplicity is equivalent to N. The development of synaptic multiplicity or the pruning of multiplicative synapses is plastic throughout development and in different disease states3,4,6,10. For this reason, characterizing synaptic multiplicity has important implications for understanding the efficacy of synaptic transmission in health and disease. Techniques, such as electron microscopy can identify structural evidence of synaptic multiplicity by detecting multiple synaptic contacts originating from the same axon onto the same postsynaptic neuron11,12,13,14. However, these structurally identified multisynapses can be functionally silent15,16. Precise functional examination of N requires technically challenging electrophysiological approaches, such as paired whole-cell recordings that can identify whether a given connection has multiple functional release sites and minimal stimulation approaches that aim to recruit a single putative axon.
In this protocol, we describe a simple method for estimating synaptic multiplicity by adopting a method originally developed by Hsia et al2. This technique involves the measurement of spontaneous PSCs (sPSCs) and mPSCs using whole-cell patch clamp electrophysiology, which allows us to estimate the degree of synaptic multiplicity across all inputs to a given neuron.&#160; As previously defined, synaptic multiplicity reflects the number of synapses between a given pre- and postsynaptic neuron. If multiple synapses are recruited in synchrony by an action potential, there will be a high probability of temporal summation of individual (i.e. quantal) PSCs, generating a greater amplitude PSC.&#160; In mPSC recordings (in which action potentials are blocked by TTX), the probability of temporal summation of individual (non-synchronous) mPSCs is low. Using this rationale, synaptic multiplicity can be estimated by comparing the sPSC amplitude (with action potential-dependent release) to the mPSC amplitude.
To examine the existence of multiplicity we describe four experiments and their analyses using glutamatergic EPSCs as an example. However, the same approach can be used for the fast GABAergic/glycinergic transmission (IPSCs). A brief rationale for each experiment is described below. First, as explained above, synaptic multiplicity can be estimated by comparing the amplitude of sEPSCs to mEPSCs. There are two requirements for this approach; 1) presynaptic axons must fire a sufficient number of action potentials during recording, and 2) Pr must be high so that multiple synapses release neurotransmitter upon the arrival of an action potential. In order to meet these requirements, sEPSCs are first recorded in low Ca2+ artificial cerebrospinal fluid (aCSF), and then recorded in the presence of a low concentration of the K+ channel antagonist, 4-Aminopyridine (4-AP) to increase action potential firing and Pr. Then action potential firing is blocked by TTX and Pr decreased by a voltage-gated Ca2+ channel blocker Cd2+. The amplitude of sEPSCs (with 4AP) is compared to that of mEPSC (with 4AP, TTX, and Cd2+). In the second experiment, Ca2+ is replaced by equimolar Sr2+ in the aCSF to desynchronize vesicle release. As Ca2+ is required for the synchronous release of vesicles, replacement with Sr2+ should eliminate the large amplitude sEPSCs that are indicative of multiplicity. Third, mechanistically, multiplicity can result from either multiple synaptic contacts to the same postsynaptic neuron or multivesicular release (i.e. multiple vesicles released within a single synaptic contact)17,18. To differentiate between the two types of multiplicity, the third experiment uses a low affinity, fast dissociating competitive antagonist of AMPA receptors, &#947;-D-glutamylglycine (&#947;-DGG)17,18 to determine whether large sEPSC are the result of the temporal summation of independent synapses or multivesicular release acting on an overlapping population of postsynaptic receptors. If the large amplitude events arise from multivesicular release, &#947;-DGG will be less effective at inhibiting larger compared to smaller sEPSCs, whereas large sEPSCs that arise from the temporal summation of multiple synaptic contacts will be similarly affected by &#947;-DGG. In the fourth experiment, a more physiological method is used to enhance action potential firing, namely afferent synaptic stimulation. Bursts of synaptic activity can transiently increase/facilitate the spontaneous action potential firing and release probability of the stimulated afferents. Therefore, this approach allows multiplicity to manifest in a more physiological manner.
The following protocol describes the method for conducting these experiments in mouse hypothalamic tissue. Specifically, corticotropin releasing hormone (CRH) neurons of the paraventricular nucleus of the hypothalamus (PVN) are used. We describe the procedures for conducting whole-cell patch clamp electrophysiology and explain the specific experiments to test for synaptic multiplicity.

<table>
	<tbody>
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			<td>1 ml syringe</td>
			<td>BD</td>
			<td>309659</td>
			<td></td>
		</tr>
		<tr>
			<td>10 blade</td>
			<td>Fisher Scientific/others</td>
			<td>35698</td>
			<td></td>
		</tr>
		<tr>
			<td>22 blade</td>
			<td>VWR/others</td>
			<td>21909-626</td>
			<td></td>
		</tr>
		<tr>
			<td>22 uM syringe filters</td>
			<td>Milipore</td>
			<td>09-719-000</td>
			<td></td>
		</tr>
		<tr>
			<td>Adson foreceps</td>
			<td>Harvard Instruments</td>
			<td>72-8547</td>
			<td></td>
		</tr>
		<tr>
			<td>Angled sharp scissors</td>
			<td>Harvard Instruments</td>
			<td>72-8437</td>
			<td></td>
		</tr>
		<tr>
			<td>Clampex</td>
			<td>Molecular Devices</td>
			<td>pClamp 10</td>
			<td></td>
		</tr>
		<tr>
			<td>Double edge blade</td>
			<td>VWR</td>
			<td>74-0002</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter paper</td>
			<td>Sigma/others</td>
			<td>1001090</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine paintbrush</td>
			<td>Fisher/various</td>
			<td>15-183-35/various</td>
			<td></td>
		</tr>
		<tr>
			<td>Gas Dispersion Tube</td>
			<td>VWR</td>
			<td>LG-8680-120</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Fresenius Kabi/others</td>
			<td>M60303</td>
			<td></td>
		</tr>
		<tr>
			<td>Krazy glue</td>
			<td>various</td>
			<td>various</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini analysis</td>
			<td>Synaptosoft</td>
			<td>MiniAnalysis 6</td>
			<td></td>
		</tr>
		<tr>
			<td>Osmomoter</td>
			<td>Wescor Inc</td>
			<td>Model 5600</td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>Sigma</td>
			<td>PM-996</td>
			<td></td>
		</tr>
		<tr>
			<td>Pasteur pipette</td>
			<td>VWR</td>
			<td>14672-200</td>
			<td></td>
		</tr>
		<tr>
			<td>ph meter</td>
			<td>Mettler Toledo</td>
			<td>FE20-ATC</td>
			<td></td>
		</tr>
		<tr>
			<td>Rubber bulb</td>
			<td>VWR</td>
			<td>82024-550</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel handle No. 3</td>
			<td>Harvard Instruments</td>
			<td>72-8350</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel handle No. 4</td>
			<td>Harvard Instruments</td>
			<td>72-8356</td>
			<td></td>
		</tr>
		<tr>
			<td>Single edge blade</td>
			<td>VWR</td>
			<td>55411-050</td>
			<td></td>
		</tr>
		<tr>
			<td>Vibratome slicer</td>
			<td>Leica</td>
			<td>VT1200S</td>
			<td></td>
		</tr>
		<tr>
			<td>Water Purification System</td>
			<td>Millipore</td>
			<td>Milli-Q Academic A10</td>
			<td></td>
		</tr>
		<tr>
			<td>Well plate lid</td>
			<td>Fisher/various</td>
			<td>07-201-590/various</td>
			<td></td>
		</tr>
		<tr>
			<td>Chemicals/reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>4-AP</td>
			<td>Sigma</td>
			<td>275875</td>
			<td></td>
		</tr>
		<tr>
			<td>BAPTA</td>
			<td>molecular probes</td>
			<td>B1204</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2*2H2O</td>
			<td>Sigma</td>
			<td>C7902</td>
			<td></td>
		</tr>
		<tr>
			<td>CdCl2</td>
			<td>sigma</td>
			<td>202908</td>
			<td></td>
		</tr>
		<tr>
			<td>DNQX</td>
			<td>Tocris</td>
			<td>189</td>
			<td></td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Sigma</td>
			<td>E3889</td>
			<td></td>
		</tr>
		<tr>
			<td>glucose</td>
			<td>Sigma</td>
			<td>G5767</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma</td>
			<td>H3375</td>
			<td></td>
		</tr>
		<tr>
			<td>K2-ATP</td>
			<td>Sigma</td>
			<td>A8937</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma</td>
			<td>P9333</td>
			<td></td>
		</tr>
		<tr>
			<td>K-gluconate</td>
			<td>Sigma</td>
			<td>G4500</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2*6H2O</td>
			<td>Sigma</td>
			<td>M2670</td>
			<td></td>
		</tr>
		<tr>
			<td>Molecular biology grade water</td>
			<td>Sigma</td>
			<td>W4502-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>Na3GTP</td>
			<td>Sigma</td>
			<td>G8877</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Bioshop</td>
			<td>SOD001.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Na-gluconate</td>
			<td>Sigma</td>
			<td>S2054</td>
			<td></td>
		</tr>
		<tr>
			<td>NaH2PO4</td>
			<td>Sigma</td>
			<td>71504</td>
			<td></td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Sigma</td>
			<td>S6014</td>
			<td></td>
		</tr>
		<tr>
			<td>Picrotoxin</td>
			<td>sigma</td>
			<td>P1675</td>
			<td></td>
		</tr>
		<tr>
			<td>SrCl</td>
			<td>Sigma</td>
			<td>255521</td>
			<td></td>
		</tr>
		<tr>
			<td>sucrose</td>
			<td>Bioshop</td>
			<td>SUC507.1</td>
			<td></td>
		</tr>
		<tr>
			<td>TTX</td>
			<td>Alamone Labs</td>
			<td>T-550</td>
			<td></td>
		</tr>
		<tr>
			<td>yDGG</td>
			<td>Tocris</td>
			<td>6729-55-1</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

The above protocol describes a method for using whole-cell patch clamp electrophysiology to examine the degree of synaptic multiplicity, using mouse hypothalamic neurons as an example. This slice preparation technique should yield healthy viable cells that do not have a swollen membrane or nucleus (Figure 1). Each step in the protocol is important for the health of the tissue and quality of the recordings.
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Watch this Scientific Journal Video about Evaluation of Synaptic Multiplicity Using Whole-cell Patch-clamp Electrophysiology at JoVE.com

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

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 ...

Research

JoVE Journal

Neuroscience

Culturing and Electrophysiology of Cells on NRCC Patch-clamp Chips

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

Whole Cell Patch Clamp for Investigating the Mechanisms of Infrared Neural Stimulation

It has been  demonstrated in recent years that pulsed, infrared laser light can be used to  elicit electrical responses in neural tissue, independent of ...

A Computer-assisted Multi-electrode Patch-clamp System

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

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 ...

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 ...

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 ...

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 ...

Auditory Brainstem Response and Outer Hair Cell Whole-cell Patch Clamp Recording in Postnatal Rats

The outer hair cell is one of the two types of sensory hair cells in the mammalian cochlea. They alter their cell length with the receptor potential to ...

Preparations and Protocols for Whole Cell Patch Clamp Recording of Xenopus laevis Tectal Neurons

Preparations and Protocols for Whole Cell Patch Clamp Recording of Xenopus laevis Tectal Neurons

The Xenopus tadpole retinotectal circuit, comprised of the retinal ganglion cells (RGCs) in the eye which form synapses directly onto neurons in the optic ...

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 ...