Optical Recording of Electrical Activity in Guinea-pig Enteric Networks using Voltage-sensitive Dyes

Summary

This protocol illustrates how voltage-sensitive dyes enable optical recording of electrical activity from intact neural networks such as the plexuses of the guinea-pig enteric nervous system, with an adjustable resolution that ranges from single-cells to multi-ganglionic circuitry.

Abstract

The enteric nervous system (ENS) is a self-contained network with identified functions, capable of performing complex behaviors in isolation. Its neurons (10 to 25 μm in diameter) are arranged in plexuses that are confined to distinct planes of the gut wall ¹; the myenteric plexus can be found between the longitudinal and circular muscle layers, and the submucous plexus between the circular muscle layer and the mucosa. Since the effector systems for these plexuses (transporting epithelium, endocrine cells, immune elements, blood vessels and smooth muscle) are also contained within the gut wall, semi-intact preparations can be dissected that preserve individual components of different reflex pathways. The behavior of the effector systems is controlled by the submucous and myenteric plexuses acting in concert. Therefore, detailed knowledge of synaptic interactions within and between ganglia, and of communication between the plexuses, is essential for understanding normal gastrointestinal function. The ENS, as an intact nervous system, is a unique experimental model in which one can correlate molecular and cellular events with the electrical behavior of the neuronal network and its physiological outputs. Because of the quasi-two-dimensional organization of its plexuses, the ENS is particularly well suited for the study of neural networks using multiple site optical recording techniques that employ voltage-sensitive dyes ^2,7,8,9. We will illustrate here the use of a relatively new naphthylstyryl-pyridinium dye (di-4-ANEPPDHQ) ³ that offers multiple advantages over its predecessors, including very low phototoxicity, slow rate of internalization, and remarkable chemical stability. When used in conjunction with a camera that permits sub-millisecond time resolution, this dye allows us to monitor the electrical activity of all the neurons in the field of view with a maximal spatial resolution of ~ 2.5 μm at 100X magnification. At lower magnification (10X or 20X), the sacrifice of single-cell resolution is compensated by a gain in perspective, revealing the intricacies of the inter-ganglionic circuitry.

Protocol

Part 1: Tissue preparation

1. The submucous and myenteric plexus preparations are isolated by sequential dissection from the small intestines of 150-200g (2-3 week old) Hartley guinea pigs that have been anesthetized by isoflurane inhalation and decapitated. After euthanasia, the small intestine is excised and its contents removed by flushing the lumen with a warm, oxygenated solution. We use Medium 199 supplemented with 25 mM HEPES, 5 mM NaHCO₃ and 2 mM Glutamine, and pH adjusted to 7.4 (referred to, from now on, as M199-DM), which has been pre-warmed at 37 °C. All the remaining steps of the protocol, however, will be carried out at room temp....

Discussion

This protocol has been written with two goals in mind. The first one is to persuade other investigators that, thanks to the many technological advances of the last decade, optical recording of electrical activity using voltage-sensitive dyes has become one of the most powerful, reliable and affordable methodologies for studying intact neuronal networks; indeed, it could be easily implemented even in a laboratory having modest resources. The second goal is to promote the enteric nervous system as a unique experimental mod.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Medium 199	Sigma-Aldrich	M3769	With Earle’s salts Without L-Glutamine, Phenol Red and Sodium Bicarbonate
Sylgard	Dow Corning	184 silicone elastomer kit
Insect pins used for dissection	F.S.T.	26001-30
Dumont forceps	F.S.T.	11203-23 or 11200-33
Moria MC19/B Pascheff-Wolff Spring Scissors	F.S.T.	15371-92
Pins to hold the tissue during the experiment	Seirin Corp.	Seirin Spinex
Collagenase VII	Sigma-Aldrich	C0773
Protease IX	Sigma-Aldrich	P6141
Fetal Bovine Serum	Invitrogen	10439-016
PenStrep	GIBCO, by Life Technologies	15140
L-Glutamine 100X	GIBCO, by Life Technologies	25030
Nifedipine	Sigma-Aldrich
Di-4-ANEPPDHQ	Molecular Probes, Life Technologies	D36802
4-Channel Stimulus Generator	Multi Channel System MCS GmbH	2000 Series
Peristaltic pump	Rainin	2-channel Dinamax RP-1
High-speed camera	RedShirtImaging, LLC	NeuroCCD-SM
1:10 demagnifier / relay lens	Optem, Qioptiq Inc.
Upright microscope	Olympus Corporation	BX61TRF
Gibraltar fixed stage with motorized X-Y translator	Gibraltar, Burleigh Instruments, EXFO, Quebec, Canada	PSSG--BX2
Linear matrix electrodes	FHC, Inc.	Custom made
Active vibration-isolation table	Halcyonics,Inc., Menlo Park, CA	Micro 60
Ultra-low-ripple feedback stabilized power supply	Kepco, Flushing, NY	ATE 75-15M
Heat filter	Schott AG	KG-1
High-Q interference filter	Chroma Inc.	HQ530/50
Dichroic mirror	Chroma Inc.	565 nm (50% transmission)
Long-pass filter	Chroma Inc.	HQ572LP
Acoustic curtains	Acoustical Surfaces, Inc.	BSC-25
Neuroplex	RedShirtImaging, LLC
IDL	ITT Visual Information Solutions
High-resolution CCD camera, with its own camera controller	Hamamatsu Corp.
Frame-grabber for high-resolution camera	Data Translation	DT3120K-1
Imaging software for high-resolution camera	Data Translation	Global Lab Image/2