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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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glutamate,+the+dominant+excitatory+transmitter+in+neuroendocrine+regulation.">Glutamate, the dominant excitatory transmitter in neuroendocrine regulation.</a> Science (New York, N.Y). 250 (4985), 1276-1278 (1990).</li><li>Mikl&#243;s, I. H., Kov&#225;cs, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reorganization+of+synaptic+inputs+to+the+hypothalamic+paraventricular+nucleus+during+chronic+psychogenic+stress+in+rats.">Reorganization of synaptic inputs to the hypothalamic paraventricular nucleus during chronic psychogenic stress in rats.</a> Biological Psychiatry. 71 (4), 301-308 (2012).</li><li>Korn, H., Triller, A., Mallet, A., Faber, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluctuating+responses+at+a+central+synapse:+n+of+binomial+fit+predicts+number+of+stained+presynaptic+boutons.">Fluctuating responses at a central synapse: n of binomial fit predicts number of stained presynaptic boutons.</a> Science (New York, N.Y.). 213 (4510), 898-901 (1981).</li><li>Tracey, D. J., Walmsley, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+input+from+identified+muscle+afferents+to+neurones+of+the+dorsal+spinocerebellar+tract+in+the+cat.">Synaptic input from identified muscle afferents to neurones of the dorsal spinocerebellar tract in the cat.</a> The Journal of physiology. 350, 599-614 (1984).</li><li>Lin, J. W., Faber, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+transmission+mediated+by+single+club+endings+on+the+goldfish+Mauthner+cell.+II.+Plasticity+of+excitatory+postsynaptic+potentials.">Synaptic transmission mediated by single club endings on the goldfish Mauthner cell. II. Plasticity of excitatory postsynaptic potentials.</a> The Journal of neuroscience the official journal of the Society for Neuroscience. 8 (4), 1313-1325 (1988).</li><li>Atwood, H. L., Tse, F. 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. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multivesicular+release+at+climbing+fiber-Purkinje+cell+synapses.">Multivesicular release at climbing fiber-Purkinje cell synapses.</a> Neuron. 32 (2), 301-313 (2001).</li><li>Oliet, S. H., Malenka, R. C., Nicoll, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bidirectional+control+of+quantal+size+by+synaptic+activity+in+the+hippocampus.">Bidirectional control of quantal size by synaptic activity in the hippocampus.</a> Science (New York, N.Y.). 271 (5253), 1294-1297 (1996).</li><li>Inoue, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noradrenaline+is+a+stress-associated+metaplastic+signal+at+GABA+synapses.">Noradrenaline is a stress-associated metaplastic signal at GABA synapses.</a> Nature Neuroscience. 16 (5), 605-612 (2013).</li><li>Ting, J. T., Daigle, T. L., Chen, Q., Feng, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+Brain+Slice+Methods+for+Adult+and+Aging+Animals:+Application+of+Targeted+Patch+Clamp+Analysis+and+Optogenetics.">Acute Brain Slice Methods for Adult and Aging Animals: Application of Targeted Patch Clamp Analysis and Optogenetics.</a> Methods in molecular biology (Clifton, N.J.). , 1183-242 (2014).</li><li>Richerson, G. 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. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patch-clamp+studies+in+the+CNS+illustrate+a+simple+new+method+for+obtaining+viable+neurons+in+rat+brain+slices:+glycerol+replacement+of+NaCl+protects+CNS+neurons.">Patch-clamp studies in the CNS illustrate a simple new method for obtaining viable neurons in rat brain slices: glycerol replacement of NaCl protects CNS neurons.</a> Journal of neuroscience methods. 158 (2), 251-259 (2006).</li><li>Gunn, B. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dysfunctional+astrocytic+and+synaptic+regulation+of+hypothalamic+glutamatergic+transmission+in+a+mouse+model+of+early-life+adversity:+relevance+to+neurosteroids+and+programming+of+the+stress+response.">Dysfunctional astrocytic and synaptic regulation of hypothalamic glutamatergic transmission in a mouse model of early-life adversity: relevance to neurosteroids and programming of the stress response.</a> Journal of Neuroscience. 33 (50), 19534-19554 (2013).</li><li>Su, H., Alroy, G., Kirson, E. D., Yaari, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+calcium+modulates+persistent+sodium+current-dependent+burst-firing+in+hippocampal+pyramidal+neurons.">Extracellular calcium modulates persistent sodium current-dependent burst-firing in hippocampal pyramidal neurons.</a> The Journal of neuroscience the official journal of the Society for Neuroscience. 21 (12), 4173-4182 (2001).</li><li>Frankenhaeuser, B., Hodgkin, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+action+of+calcium+on+the+electrical+properties+of+squid+axons.">The action of calcium on the electrical properties of squid axons.</a> The Journal of physiology. 137 (2), 218-244 (1957).</li><li>Xiong, G., Metheny, H., Johnson, B. N., Cohen, A. 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The authors have nothing to disclose

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>
		<tr>
			<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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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 ...

In the brain, a pair of neurons often form multiple synaptic contacts, which is called synaptic multiplicity. However, precise examination of synaptic multiplicity requires technically challenging experiments. This protocol describes a simple method for gross estimation of synaptic multiplicity using whole-cell patch-clamp electrophysiology.This method can be applied to any species and brain area to investigate synaptic multiplicity. This method requires basic skills in whole-cell patch-clamp electrophysiology. Obtaining high-quality recordings with low and stable access resistance is critical for the accurate interpretation of the data.To obtain whole-cell configuration, place the recording pipette just above the slice and offset pipette current in the voltage clamp mode. Apply slight positive pressure to the pipette, and lock the stopcock. Next, select a healthy cell with an intact membrane, and approach the tissue with the pipette.The positive pressure should cause a slight disturbance on the tissue. Slowly bring the pipette closer to the cell in a diagonal motion until a small dimple is formed on the cell surface. Then, release the positive pressure lock.The cell will begin to form a seal, and the resistance will increase above one gigaohm. In voltage clamp, hold the cell at minus 68 millivolts. Subsequently, slightly pull the pipette away from the cell diagonally to remove excess pressure.Compensate for the fast and slow pipette capacitance. Apply a brief suction through the tube connected to the pipette holder to break through the cell and obtain a whole-cell configuration. Then, switch to cell mode on the membrane test window in an electrophysiology data acquisition and analysis software.Maintain the temperature of the recording bath at 27 to 30 degrees Celsius and the flow rate at 1.5 to two milliliters per minute for subsequent experiments. In a multiplicity synapse, an action potential synchronizes neurotransmitter release and generates a larger postsynaptic current. Blocking action potential and calcium-dependent vesicular release with TTX and cadmium prevents the postsynaptic current summation and decreases the amplitude.When there is no multiplicity, blocking action potential will not change the amplitude. In experiment one, to estimate multiplicity, hold the cell at minus 68 millivolts while perfusing it with low-calcium aCSF. Record the spontaneous EPSCs for at least five minutes to ensure a stable baseline.Next, add 30 micromolars 4-AP to the aCSF to increase action potential-dependent events, and record spontaneous EPSCs for at least 10 minutes to obtain the full drug effect. Then, add 0.5 micromolars TTX and 10 micromolars cadmium to the aCSF with 4-AP, and record the miniature EPSCs for at least 10 minutes. For offline analysis, use the last one minute of baseline immediately before the application of 4-AP, the 10th minute of 4-AP application, and the 10th minute of TTX application.In this experiment, extracellular calcium is replaced with strontium to desynchronize the release of synaptic vesicles. Therefore, if multiplicity is present, this should decrease the amplitude of postsynaptic currents. In experiment two, record spontaneous EPSCs for at least five minutes while perfusing the cell with normal calcium aCSF.To desynchronize vesicle release, begin perfusing the cell with strontium aCSF, and record spontaneous EPSCs. For offline analysis, to determine whether the large amplitude spontaneous EPSCs are due to the synchronous release of vesicles, compare the last minute of baseline to the 10th minute of strontium aCSF application. Multiplicity can involve multivesicular release, which causes higher neurotransmitter concentration in the synaptic cleft.Addition of gamma-DGG, a low-affinity AMPA receptor antagonist, leads to less effective inhibition of larger multiquantal compared to smaller uniquantal postsynaptic currents. Without multivesicular release, gamma-DGG will be equally effective on larger and smaller postsynaptic currents. In experiment three, to test for multivesicular release, record spontaneous EPSCs in low-calcium aCSF for at least five minutes.Add 30 micromolars 4-AP to the aCSF through the perfusion system. Record the spontaneous EPSCs for at least 10 minutes. Then, add 200 micromolars gamma-DGG to the aCSF with 4-AP, and record the spontaneous EPSCs for at least 10 minutes.As a control experiment in a separate cell, repeat the procedures, but apply a low concentration of DNQX instead of gamma-DGG. For offline analysis, analyze the last minute of each drug application. Bursts of synaptic activity can transiently increase spontaneous action potential firing and release probability of the stimulated afferents.If neurons exhibit multiplicity, the increase in action potentials should cause a transient increase in the amplitude of postsynaptic currents. In experiment four, record spontaneous EPSCs in normal calcium aCSF. To increase action potential firing, stimulate the afferents using a monopolar glass electrode filled with aCSF at a rate of 20 hertz for two seconds, and repeat 10 times with an inter-burst interval of 20 seconds.For analysis, use 5, 000 milliseconds spontaneous EPSCs before the first stimulus as the baseline and compare to the 10 to 300 milliseconds spontaneous EPSCs after the final stimulus. Then, take the average amplitude and frequency change over 10 trials. Analyze spontaneous EPSCs and miniature EPSCs using a program that detects and analyzes synaptic currents.Use the suggested detection parameters and nonstop analysis function for detecting AMPA receptor-mediated EPSCs. Manually scan each recording to ensure the program is accurately detecting each event. Export the event data by copying it to the clipboard, and paste it into a data management software.Next, calculate the average frequency and amplitude for each drug treatment, and perform the relevant statistical analyses. In an example shown here, 4-AP increases both the amplitude and frequency of spontaneous EPSCs. Subsequent application of TTX and cadmium decreases both the amplitude and frequency.Here is the distribution of spontaneous EPSC amplitude from the recording. In the hypothalamic neurons examined here, the amplitude and frequency of the baseline and TTX conditions are the same, suggesting that the baseline spontaneous EPSCs contain very few action potential-dependent EPSCs. Accordingly, subsequent experiments can compare the difference between baseline and 4-AP to measure multiplicity.The strength of synaptic transmission can be transiently increased by bursts of synaptic activity. To investigate multiplicity under more physiological conditions, afferent stimulation can be used to increase action potential firing and release probability. Here are the summaries of spontaneous EPSC frequency and amplitude changes following synaptic stimulation.Stable recordings are essential for accurate interpretation of the data. Describe the data if the access resistance changes by more than 20%during the recording, as this could confound the analysis. This protocol offers a simple way to estimate synaptic multiplicity, which is a key determinant of synaptic efficacy and its plasticity in different physiological and pathophysiological conditions.

evaluation-synaptic-multiplicity-using-whole-cell-patch-clamp

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JoVE Journal

Neuroscience

12.6K Views.  University of Western Ontario. In the brain, a pair of neurons often form multiple synaptic contacts, which is called synaptic multiplicity. However, precise examination of synaptic multiplicity requires technically challenging experiments. This protocol describes a simple method for gross estimation of synaptic multiplicity using whole-cell patch-clamp electrophysiology.This method can be applied to any species and brain area to investigate synaptic multiplicity. This method requires basic skills in whole-cell patc...