Adult-born neurons expressing ChR2 can be manipulated in slice electrophysiological preparations in order to examine their contribution towards the function of olfactory neural circuits.
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Method Article
Using Affordable LED Arrays for Photo-Stimulation of Neurons
In This Article
Summary
Abstract
Standard slice electrophysiology has allowed researchers to probe individual components of neural circuitry by recording electrical responses of single cells in response to electrical or pharmacological manipulations1,2. With the invention of methods to optically control genetically targeted neurons (optogenetics), researchers now have an unprecedented level of control over specific groups of neurons in the standard slice preparation. In particular, photosensitive channelrhodopsin-2 (ChR2) allows researchers to activate neurons with light3,4. By combining careful calibration of LED-based photostimulation of ChR2 with standard slice electrophysiology, we are able to probe with greater detail the role of adult-born interneurons in the olfactory bulb, the first central relay of the olfactory system. Using viral expression of ChR2-YFP specifically in adult-born neurons, we can selectively control young adult-born neurons in a milieu of older and mature neurons. Our optical control uses a simple and inexpensive LED system, and we show how this system can be calibrated to understand how much light is needed to evoke spiking activity in single neurons. Hence, brief flashes of blue light can remotely control the firing pattern of ChR2-transduced newborn cells.
Protocol
1. Optical Calibration: Measuring LED Power
- Attach a LED array to a heatsink actively cooled by a fan and affix this LED/heatsink aparatus to a collimating lens.
- Replace lamp used in brightfield illumination with LED/heatsink/fan/lens apparatus. This apparatus must be carefully positioned so that the collimated LED beam travels along a straight optical path towards the condenser lens. Make sure the heatsink/fan is properly grounded to the system's common ground.
- Drive the LED array with a power supply that can give fast and square pulses of current. This power supply may be controlled by a 5V TTL pulse originating from a pulse genera....
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Discussion
Recent years have seen an explosion in the popularity of optogenetic tools for neuroscience research6. As a result, it is increasingly important to lower the barrier of entry for labs wishing to begin using these new tools. Here we describe how to conduct a simple and low cost retrofitting and calibration of a conventional patch-clamp rig so that it can do full-field optical stimulation of channelrhodopsin-expressing neurons. In particular, we apply this technique to the study of olfactory bulb adult neuroge.......
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Disclosures
No conflicts of interest declared.
Acknowledgements
This work was supported by the life insurance company "AG2R-La-Mondiale", Ecole des Neurosciences de Paris (ENP), the Agence Nationale de la Recherche "ANR-09-NEUR-004" in the frame of "ERA-NET NEURON" of FP7 program by the European Commission, and the Pasteur Foundation. Sebastien Wagner was supported by the Letten Foundation.
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Materials
| Name | Company | Catalog Number | Comments |
|---|---|---|---|
| Ketamine | Imalgène 1000 | 100 mg/ml | |
| Xylazine | Rompun | 2% | |
| NaCl | Sigma-Aldrich | S5886 | &nbps; |
| KCl | Sigma-Aldrich | P5405 | |
| MgSO4 | Sigma-Aldrich | M1880 | |
| NaHCO3 | Sigma-Aldrich | S5761 | |
| NaHPO4 | Sigma-Aldrich | S5011 | |
| Glucose | Sigma-Aldrich | G7021 | |
| CaCl2 | Sigma-Aldrich | C7902 | |
| Agarose | Sigma-Aldrich | A9539 | |
| Pipette Puller | Sutter Instrument Co. | P-97 | |
| Glass Capillaries | Harvard Apparatus | GC150T-10 | 1.5 mm O.D./1.17 mm I.D. |
| LED array | Bridgelux | BXRA-C2000 | |
| Collimating lens | Thorlabs Inc. | LEDC1 | 40 mm beam diameter |
| Power supply | A1W Electronik | HKO2800 | 2.8 amp |
| Optical power meter | Thorlabs Inc. | PM 100 | |
| Heatsink | Thermaltake | A1838 | Silent Boost K8 |
| Fan | Thermaltake | A1838 | Silent Boost K8 |
| Vibratome | Leica Microsystems | VT1200S |
References
- Nissant, A. Adult neurogenesis promotes synaptic plasticity in the olfactory bulb. Nature Neuroscience. 12, 728-730 (2009).
- Apicella, A. Pyramidal Cells in Piriform Cortex Receive Convergent Input from Distinct Olfactory Bulb Glomer....
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