Simultaneous Electrophysiological Recording and Calcium Imaging of Suprachiasmatic Nucleus Neurons

Summary

Procedures are described to perform simultaneous recordings of membrane potential or current and changes of intracellular calcium concentration. Suprachiasmatic nucleus neurons are filled with the calcium indicator bis-fura-2 using a patch clamp electrode in the whole cell patch clamp configuration.

Abstract

Simultaneous electrophysiological and fluorescent imaging recording methods were used to study the role of changes of membrane potential or current in regulating the intracellular calcium concentration. Changing environmental conditions, such as the light-dark cycle, can modify neuronal and neural network activity and the expression of a family of circadian clock genes within the suprachiasmatic nucleus (SCN), the location of the master circadian clock in the mammalian brain. Excitatory synaptic transmission leads to an increase in the postsynaptic Ca²⁺ concentration that is believed to activate the signaling pathways that shifts the rhythmic expression of circadian clock genes. Hypothalamic slices containing the SCN were patch clamped using microelectrodes filled with an internal solution containing the calcium indicator bis-fura-2. After a seal was formed between the microelectrode and the SCN neuronal membrane, the membrane was ruptured using gentle suction and the calcium probe diffused into the neuron filling both the soma and dendrites. Quantitative ratiometric measurements of the intracellular calcium concentration were recorded simultaneously with membrane potential or current. Using these methods it is possible to study the role of changes of the intracellular calcium concentration produced by synaptic activity and action potential firing of individual neurons. In this presentation we demonstrate the methods to simultaneously record electrophysiological activity along with intracellular calcium from individual SCN neurons maintained in brain slices.

Introduction

Changes in gene expression are known to occur in neurons as a consequence of synaptic signaling. Signaling by the excitatory neurotransmitter glutamate can depolarize the neuronal membrane potential eventually leading to gene transcription and translation^1,2. Activation of ionotropic receptors by glutamate allows extracellular calcium ions to enter the cell, which is thought to play a critical role as a second messenger in activating gene transcription. Evaluating the relationship between membrane electrical activity, such as action potential firing frequency, and changes of intracellular calcium concentration requires the combination of two methods - whole....

Protocol

1. Preparation of Hypothalamic Brain Slices

1. Obtain, in advance, Institutional Animal Care and Use Committee (IACUC) approval for any procedure involving animals. The animal procedures described here are consistent with the AVMA Guidelines for the Euthanasia of Animals 2013 and were approved in advance by the Oregon Health & Science University IACUC.
2. Prepare 500 ml of the slicing buffer without the MgCl₂ and CaCl₂ (Table 1). Bubble the solution with 95% O₂ and 5% CO₂ at room temperature then add the MgCl₂ and CaCl₂ while continuing to aer....

Results

Using hypothalamic brain slices, we simultaneously recorded changes in intracellular calcium in whole cell mode under both voltage and current clamp conditions. The microelectrode shown in Figure 1A is lowered into position under high magnification (40X or 63X UV objectives) with a small amount of applied positive pressure. After touching the neuron, a gigaohm seal is formed with gentle suction. In voltage-clamp mode after setting the cell membrane potential to -60 mV, additional suction breaks the membr.......

Discussion

The methods described above provide a powerful tool to simultaneously record the link between neuron membrane electrical activity and the intracellular calcium concentration. The method has a number of strengths in that it combines two very well characterized methods - whole cell patch clamp recording and measurement of intracellular calcium using fluorescent dyes. Our approach is similar to those described by other investigators^_6,23.

A number of items must be taken into .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
NaCl	Fisher Scientific Co.	S271-3
KCl	Fisher Scientific Co.	P217-500
NaH2PO4•H2O	Sigma Chemical Co.	S-9638
MgCl2•6H2O	Fisher Scientific Co.	M33-500
CaCl2•2H2O	Fisher Scientific Co.	C79-500
D-glucose	Fisher Scientific Co.	D16-500
NaHCO3	Fisher Scientific Co.	S233-500
Sucrose	Fisher Scientific Co.	S5-500
Potassium D-gluconate	Sigma-Aldrich	G4500
HEPES	Sigma-Aldrich	H4034
Adenosine 5′-triphosphate dipotassium salt dihydrate	Sigma-Aldrich	A8937
Guanosine 5′-triphosphate tris salt	Sigma-Aldrich	G9002
Agarose (ultrapure)	Life Technologies	15510-027	Gel pored into sterile Petri dish 6 mm thick layer
KOH	Sigma-Aldrich	P-6310
NaOH	Sigma-Aldrich	S-5881
Bis-fura-2, hexapotassium salt	Invitrogen	B6810	Cell impermeant
Name of Equipment	Company	Catalog Number	Comments
Microtome	Leica	VT1000S	Tissue slicing
Cyanoacrylic glue (Roti-Coll1)	Carl Roth GmbH+Co	Art-Nr. 0258.1
Microelectrode puller	Narshige International USA	PP-83
Microelectrode Capillary Tubes	World Precision Instruments	1B150F-4
Microfil 34 g	World Precision Instruments	MF34G-5
Syringe filter	Corning	#431212
Microscope	Leica	DM LFS	With 4X, 40X UV and 63X UV objectives, and epifluorescence
CCD Camera	Hamamatsu	ORCA ER	12 bit CCD
Fura-2 Filter Cube	Chroma	71500A	Set with UG11 filter
Polychrome IV	Till Photonics GmBH		Monochronometer
Ultraviolet blocking safety glasses	Ultra-Violet Products
EPC-9 amplifier	HEKA Eletronik
Metafluor	Molecular Devices		Imaging Software
Patchmaster	HEKA Eletronik		Data Acquisition Software
Igor version 6	Wavemetrics		Electrophysiology & Ca2+ Data Analysis
VAPRO 5520	Westcor		Vapor pressure osmometer