Recording Synaptic Plasticity in Acute Hippocampal Slices Maintained in a Small-volume Recycling-, Perfusion-, and Submersion-type Chamber System

Summary

This protocol describes the stabilization of the oxygen level in a small volume of recycled buffer and methodological aspects of recording activity-dependent synaptic plasticity in submerged acute hippocampal slices.

Abstract

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful analysis of synaptic transmission modulation when performing field potential or intracellular recordings. This manuscript describes methodological aspects that might be helpful in improving experimental conditions for the maintenance of acute brain slices and for recording field excitatory postsynaptic potentials in a commercially available submersion chamber with an outflow-carbogenation unit. The outflow-carbogenation helps to stabilize the oxygen level in experiments that rely on the recycling of a small buffer reservoir to enhance the cost-efficiency of drug experiments. In addition, the manuscript presents representative experiments that examine the effects of different carbogenation modes and stimulation paradigms on the activity-dependent synaptic plasticity of synaptic transmission.

Introduction

In 1951, the first-reported acute brain slice experiments were conducted¹. In 1971, after successful in vitro recordings from the piriform cortex^2,3 and the discovery that hippocampal neurons are interconnected transversely along the septotemporal axis of the hippocampus⁴, one of the first in vitro recordings of hippocampal neuronal activity was achieved⁵. The similarity of the neurophysiological or neurostructural parameters of neurons under in vivo and in vitro conditions are still the subject of some ....

Protocol

The animals were maintained in accordance with the established standards of animal care and procedures of the Institutes of Brain Science and State Key Laboratory of Medical Neurobiology of Fudan University, Shanghai, China.

1. Solution Preparation

NOTE: See the Table of Materials.

1. Prepare the slicing buffer (modified Gey's solution): 92 mM NaCl, 2.5 mM KCl, 1.25 mM NaH₂PO₄, 30 mM NaHCO₃, 25 mM glucose, 20 mM HEPES, 3 mM Na⁺-pyruvate, 10 mM MgSO₄, and 0.5 mM CaCl₂.
2. Adjust the pH to 7.6 using 1 M NaOH without carbogenation.
3. Store the buff....

Results

In the protocol section, we described the preparation of acute hippocampal slices from the ventral and intermediate part of the hippocampal formation (Figure 1) of male C57BL/6 mice and male Wistar rats (5-8 weeks). The position of the hemispheres on the slicer platform helps to keep them stable and removes the need of stabilization with agar or agarose. The perfusion system itself is based on a peristaltic pump operated on high rotation to give the required .......

Discussion

Although interface slice chambers exhibit more robust synaptic responses^25,26,27,28, submersion chambers provide additional convenience for patch-clamp recording and fluorescence imaging. Thus, we have described several aspects of field potential recordings in acute hippocampal slices using a commercial submersion slice chamber that can easily be extended to the imaging of fluorescence probes i.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Reagents required
NaCl	Sinopharm Chemical Reagent, China	10019318
KCl	Sinopharm Chemical Reagent, China	10016318
KH2PO4	Sinopharm Chemical Reagent, China	10017618
MgCl2·6H2O	Sinopharm Chemical Reagent, China	10012818
CaCl2	Sinopharm Chemical Reagent, China	10005861
NaHCO3	Sinopharm Chemical Reagent, China	10018960
Glucose	Sinopharm Chemical Reagent, China	10010518
NaH2PO4	Sinopharm Chemical Reagent, China	20040718
HEPES	Sigma	H3375
Sodium pyruvate	Sigma	A4043
MgSO4	Sinopharm Chemical Reagent, China	20025118
NaOH	Sinopharm Chemical Reagent, China	10019718
Tools and materials for dissection
Decapitators	Harvard apparatus	55-0012	for rat decapitation
Bandage Scissors	SCHREIBER	12-4227	for mouse decapitation
double-edge blade	Flying Eagle, China	74-C
IRIS Scissors	RWD, China	S12003-09
Bone Rongeurs	RWD, China	S22002-14
Spoon	Hammacher	HSN 152-13
dental cement spatula	Hammacher	HSN 016-15
dental double end excavator	Blacksmith Surgical, USA	BS-415-017
Vibrating Microtome	Leica, Germany	VT1200S
surgical blade	RWD, China	S31023-02
surgical holder	RWD, China	S32007-14
Electrophysiology equipment and materials
Vertical Pipette Puller	Narishige, Japan	PC-10
Vibration isolation table	Meirits, Japan	ADZ-A0806
submerged type recording chamber	Warner Instruments	RC-26GLP
4 Axis Micromanipulator	Sutter, USA	MP-285, MP-225
Platinum Wire	World Precision Instruments	PTP406
Amplifier	Molecular Devices, USA	Multiclamp 700B
Data Acquisition System	Molecular Devices, USA	Digidata 1440A
Anaysis software	Molecular Devices, USA	Clampex 10.2
Fluorescence Microscope	Nikon, Japan	FN1
LED light source	Lumen Dynamics Group, Canada	X-cite 120LED
micropipettes	Harvard apparatus	GC150TF	extracelluar recording
borosilicate micropipettes	Sutter, USA	BF150-86	patch clamp
tungsten electrode	A-M Systems, USA	575500
peristaltic pump	Longer, China	BT00-300T
tubes for peristaltic pump	ISMATEC, Wertheim, Germany	SC0309	1x inflow, ID: 1.02 mm
tubes for peristaltic pump	ISMATEC, Wertheim, Germany	SC0319	2x tubes for outflow, ID: 2.79 mm
CCD camera	PCO, Germany	pco.edge sCMOS
lens cleaning paper	Kodak
50 mL conical centrifuge tube	Thermo scientific	339652
Prechamber	Warner Instruments	BSC-PC
Inline heater	Warner Instruments	SF-28
Temperature Controller	Warner Instruments	TC-324B