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.
Part 1: Tissue preparation
Part 2: Preparation of tissue samples for optical recording
Part 3: Staining with di-4-ANEPPDHQ
Part 4: Electrical stimulation
Part 5: Perfusion
We use a 2-channel peristaltic pump with one channel for input of oxygenated solutions, and the other for output. To eliminate mechanical disturbances induced by the perfusion, the pump is turned-off during the acquisition of optical data.
Part 6: Apparatus for multiple site optical recording of membrane potential
Part 7: Optical experiments
Part 8: How to achieve technical quality in an optical experiment
The technical success of an optical experiment is measured, ultimately, by the signal-to noise ratio of the recordings. Furthermore, a good signal-to-noise ratio stems not from an epiphany but from a journey. As in an opera performance, the final outcome is determined by a multiplicity of factors that interact synergistically. Thus, to achieve a technically sound optical experiment, it is essential to refine, independently, the optical apparatus and the skills required for each individual step of the protocol. What follows is a list of potential trouble-areas, and some suggestions for avoiding problems not always anticipated 5:
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 model in which to correlate molecular and cellular events with the electrical behavior of the two neuronal plexuses and their biological outputs.
We would like to thank D.A. Coulter (CHOP) for allowing us to use his 2-photon microscope to acquire the 2-photon images shown at the beginning of the video, and H. Takano for sharing with us his technical expertise.
Material Name | Type | Company | Catalogue Number | Comment |
---|---|---|---|---|
Medium 199 | Sigma, St. Louis, MO | M3769 | With Earle’s salts Without L-Glutamine, Phenol Red and Sodium Bicarbonate | |
Sylgard | Dow Corning Corp., Midland, MI | 184 silicone elastomer kit | ||
Insect pins used for dissection | F.S.T., Foster City, CA | 26001-30 | ||
Dumont forceps | F.S.T., Foster City, CA | 11203-23 or 11200-33 | ||
Moria MC19/B Pascheff-Wolff Spring Scissors | F.S.T., Foster City, CA | 15371-92 | ||
Pins to hold the tissue during the experiment | Seirin Corp., Shizuoka, Japan | Seirin Spinex | ||
Collagenase VII | Sigma, St. Louis, MO | C0773 | ||
Protease IX | Sigma, St. Louis, MO | P6141 | ||
Fetal Bovine Serum | Invitrogen, Carlsbad, CA | 10439-016 | ||
PenStrep | GIBCO, BRL | 15140 | ||
L-Glutamine 100X | GIBCO, BRL | 25030 | ||
Nifedipine | Sigma, St. Louis, MO | |||
Di-4-ANEPPDHQ | Molecular Probes, Eugene, OR | D36802 | ||
4-Channel Stimulus Generator | Multi Channel System MCS GmbH, Reutlingen, Germany | 2000 Series | ||
Peristaltic pump | Rainin, Woburn, MA | 2-channel Dinamax RP-1 | ||
High-speed camera | RedShirtImaging, LLC Decatur, GA | NeuroCCD-SM | ||
1:10 demagnifier / relay lens | Optem, Qioptiq Linos, Inc., Rochester, NY | |||
Upright microscope | Olympus Optical Co., Tokyo, Japan | BX61TRF | ||
Gibraltar fixed stage with motorized X-Y translator | Gibraltar, Burleigh Instruments, EXFO, Quebec, Canada | PSSG–BX2 | ||
Linear matrix electrodes | FHC, Inc., Bowdoin, ME | 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 Optical Glass, Duryea, PA | KG-1 | ||
High-Q interference filter | Chroma, Inc., Rockingham, VT | HQ530/50 | ||
Dichroic mirror | Chroma, Inc., Rockingham, VT | 565 nm (50% transmission) | ||
Long-pass filter | Chroma, Inc., Rockingham, VT | HQ572LP | ||
Acoustic curtains | Acoustical Surfaces, Inc. | BSC-25 | ||
Neuroplex | RedShirtImaging, Inc., Decatur, GA | |||
IDL | ITT Visual Information Solutions, Boulder, CO | |||
High-resolution CCD camera, with its own camera controller | Hamamatsu Photonics, KK, Hamamatsu City, Japan | |||
Frame-grabber for high-resolution camera | Data Translation, Marlboro, MA | DT3120K-1 | ||
Imaging software for high-resolution camera | Data Translation, Marlboro, MA | Global Lab Image/2 |