Using Affordable LED Arrays for Photo-Stimulation of Neurons

Summary

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.

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 manipulations^1,2. 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 light^3,4. 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

1. Attach a LED array to a heatsink actively cooled by a fan and affix this LED/heatsink aparatus to a collimating lens.
2. 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.
3. 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....

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.......

Materials

List of materials used in this article

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