同時電気生理学的記録および視交叉上核ニューロンのカルシウムイメージング
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 translation1,2. 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 cell patch clamping and quantitative imaging of fluorescent calcium probes3-5, allowing the relationship to be studied in individual neurons. The single cell recording technique allows the recording of the activity of individual neurons in identifiable portions of the brain. The whole cell recording technique allows the membrane voltage or current to be controlled allowing for experimental manipulation of specific ion channel currents. Using micropipettes filled with fluorescent calcium probes also ensures that the neuron is well filled with calcium probe. This technique has a clear advantage when working with brain slice preparations from adult slice preparations, since these neurons are particularly difficult to load using the more common cell permeant probes and reduces potential background fluorescence issues6-8.
Light is the principal way mammals adjust their circadian clock, which is located in the hypothalamic suprachiasmatic nucleus (SCN). Light information transduced in the retina is transmitted 9-11 via the retinohypothalamic tract (RHT) where glutamate is released in the SCN12,13. Glutamate opens NMDA and AMPA ionotropic receptors located on SCN neurons producing an influx of calcium and sodium, and initiating an intracellular signaling cascade that ultimately leads to altering the expression of a family of clock genes14-17 and shifts in phase of the circadian clock18-20. However, calcium can enter neurons either directly through ionotropic glutamate receptors or through membrane depolarization and activation of voltage-dependent calcium channels (VDCC)21. We therefore developed an experimental protocol to investigate the relationship between the intracellular calcium concentration and membrane electrical activity in SCN neurons, such as occurs with action potential firing and from synaptic input22.
Protocol
Representative Results
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 investigators6,23.
A number of items must be taken into …
Declarações
The authors have nothing to disclose.
Acknowledgements
The work was funded by a grant from the National Institute of General Medical Sciences (GM096972).
Materials
| 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 |
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