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

Summary

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

Abstract

In the central nervous system, a pair of neurons often form 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.

Introduction

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 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....

Protocol

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

1. Slicing solution
   1. Refer to Table 1 for the composition of the slicing solution.
   2. Prepare a 20x stock solution in advance and store it at 4 °C for up to 1 month.
   3. For 1x slicing solution, dissolve NaHCO₃, glucose, and sucrose in ddH₂O, and add the 20x stock. Ensure the osmolarity is between 315-320 mOsm and store the solution for no more than 1 ....

Results

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.

Discussion

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 methods^21,22,23,24. For the recording, it is critical to only accept stable recordings by constantly monitoring cell properties suc.......

Materials

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