Flash Photolysis of Caged Compounds in the Cilia of Olfactory Sensory Neurons

Summary

Photolysis of caged compounds allows the production of rapid and localized increases in the concentration of various physiologically active compounds. Here, we show how to obtain patch-clamp recordings combined with photolysis of caged cAMP or caged Ca for the study of olfactory transduction in dissociated mouse olfactory sensory neurons.

Abstract

Photolysis of caged compounds allows the production of rapid and localized increases in the concentration of various physiologically active compounds¹. Caged compounds are molecules made physiologically inactive by a chemical cage that can be broken by a flash of ultraviolet light. Here, we show how to obtain patch-clamp recordings combined with photolysis of caged compounds for the study of olfactory transduction in dissociated mouse olfactory sensory neurons. The process of olfactory transduction (Figure 1) takes place in the cilia of olfactory sensory neurons, where odorant binding to receptors leads to the increase of cAMP that opens cyclic nucleotide-gated (CNG) channels². Ca entry through CNG channels activates Ca-activated Cl channels. We show how to dissociate neurons from the mouse olfactory epithelium³ and how to activate CNG channels or Ca-activated Cl channels by photolysis of caged cAMP⁴ or caged Ca⁵. We use a flash lamp^6,7 to apply ultraviolet flashes to the ciliary region to uncage cAMP or Ca while patch-clamp recordings are taken to measure the current in the whole-cell voltage-clamp configuration^8-11.

Protocol

1. Instrumentation

1. To measure the response of olfactory sensory neurons to photolysis of caged compounds we use a flash lamp in combination with a typical patch-clamp recording system including: a patch-clamp amplifier, a recording electrode and a reference electrode connected to the head-stage of a patch-clamp amplifier, a digitizer, a computer, software for data acquisition, micro-manipulators, an epifluorescence microscope, a perfusion system, an anti-vibration table and a Faraday cage (Figure 2).
2. To generate a high-intensity flash of ultraviolet (UV) light we use a Xenon flash lamp (Rapp OptoElectronic JML-C2 Flash Unit, Figure 3A) that functi....

Discussion

Flash photolysis of caged compounds combined with patch-clamp recordings is a useful technique to obtain rapid and local jumps in the concentration of physiologically active molecules both inside and outside cells. Several types of caged compounds1 have been synthesized, and this technique can be applied to various types of cells, including cultured cells expressing ion channels that can be activated or modulated by photolysis of some of the available caged compounds¹¹.

Photolysis o.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Adapter module flash lamp to microscope	Rapp OptoElectronic	FlashCube 70
Air table	TMC	MICRO-g 63-534
Digitizer	Axon Instruments	Digidata 1322A
Data Acquisition Software	Axon Instruments	pClamp 8
Data Analysis Software	WaveMetrics	Igor
Mirror for adapter module	Rapp OptoElectronic	M70/100
Electrode holder	Axon Instruments	1-HL-U
Faraday’s cage	Custom Made
Filter cube	Olympus Corporation	U-MWU	Excitation filter removed
Flash lamp	Rapp OptoElectronic	JML-C2
Forceps Dumont #55	World Precision Instruments, Inc.	14099
Glass capillaries	World Precision Instruments, Inc.	PG10165-4
Glass bottom dish	World Precision Instruments, Inc.	FD35-100
Illuminator	Olympus Corporation	Highlight 3100
Inverted microscope	Olympus Corporation	IX70
Micromanipulators	Luigs & Neumann	SM I
Micropipette Puller	Narishige International	PP-830
Monitor	HesaVision	MTB-01
Neutral density filters	Omega Optical	varies
Objective 100X	Carl Zeiss, Inc.	Fluar 440285	Either Zeiss or Olympus
Objective 100X	Olympus Corporation	UPLFLN 100XOI2	Either Zeiss or Olympus
Optical UV shortpass filter	Rapp OptoElectronic	SP400
Patch-clamp amplifier	Axon Instruments	Axopatch 200B
Photo Diode Assembly	Rapp OptoElectronic	PDA
Quartz light guide	Rapp OptoElectronic	varies	We use 600 μm diameter
Silver wire	World Precision Instruments, Inc.	AGT1025
Silver ground pellet	Warner Instruments	64-1309
Xenon arc lamp	Rapp OptoElectronic	XBL-JML
Reagent	Company	Catalogue number
BCMCM-caged cAMP	BioLog	B016
Bovine serum albumin (BSA)	Sigma-Aldrich	A8806
CaCl2 standard solution 0.1 M	Fluka	21059
Caged Ca: DMNP-EDTA	Invitrogen	D6814
Cysteine	Sigma-Aldrich	C9768
Concanavalin A type V (ConA)	Sigma-Aldrich	C7275
CsCl	Sigma-Aldrich	C4036
DMSO	Sigma-Aldrich	D8418
DNAse I	Sigma-Aldrich	D4527
EDTA	Sigma-Aldrich	E9884
EGTA	Sigma-Aldrich	E4378
Glucose	Sigma-Aldrich	G5767
HEPES	Sigma-Aldrich	H3375
KCl	Sigma-Aldrich	P3911
KOH	Sigma-Aldrich	P1767
Leupeptin	Sigma-Aldrich	L0649
MgCl2	Fluka	63020
Papain	Sigma-Aldrich	P3125
Poly-L-lysine	Sigma-Aldrich	P1274
NaCl	Sigma-Aldrich	S9888
NaOH	Sigma-Aldrich	S5881
NaPyruvate	Sigma-Aldrich	P2256