1SISSA, International School for Advanced Studies, 2Istituto di Biofisica, Consiglio Nazionale delle Ricerche, 3SISSA Unit, Italian Institute of Technology
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Boccaccio, A., Sagheddu, C., Menini, A. Flash Photolysis of Caged Compounds in the Cilia of Olfactory Sensory Neurons. J. Vis. Exp. (55), e3195, doi:10.3791/3195 (2011).
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 lamp6,7 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 configuration8-11.
2. Preparing solutions
Patch-clamp recording solutions
Always prepare and use caged compound solutions in dim light to avoid degradation of caged compounds from ambient light. Protect containers from light using aluminum foil.
We prepare intracellular solutions containing 3 mM DMNP-EDTA5 50% loaded with 1.5 mM Ca.
Notes: During experiments, protect caged compound solutions from light using aluminum foil and keep them on ice. Sterile filter the intracellular solution.
3. Dissociation of mouse olfactory sensory neurons
Animals were handled in accordance with the Italian Guidelines for the Use of Laboratory Animals (Decreto Legislativo 27/01/1992, no. 116) and European Union guidelines on animal research (No. 86/609/EEC).
5. Representative results:
You should be able to produce local uncaging of caged cAMP or of caged Ca in the ciliary region of an isolated olfactory sensory neuron and record the current response in the whole-cell voltage-clamp configuration.
Figure 4 shows a typical inward current elicited by a UV flash producing photolysis of caged cAMP, recorded at a voltage of -60 mV in the presence of an extracellular low Ca Ringer’s solution. In this condition the inward current is due to Na entry through CNG channels. The rising phase of the current was fast and was fitted by a single exponential function with a time constant of 3.4 ms.
Figure 5A-B show the responses of another olfactory sensory neuron in low Ca and in Ringer’s solution with 1mM Ca. The rising phase of the current at -60 mV became much slower and multiphasic (Figure 5 A-B). This is due to the action of Ca entering the cilia through CNG channels and activating a secondary Cl current10. The earlier cationic current component, due to activation of CNG, is smaller in 1mM Ca Ringer solution than in low Ca solution because of the block due to the permeating Ca ions that reduce the overall current.
Another way to reduce the increase of Ca in the cilia is to clamp the neuron at +60 mV (Figure 5 C-D). The rising phase of the response due to cAMP uncaging at +60 mV was well described by a single exponential with a time constant of 6.7 ms, indicating the presence of only one current component.
By photoreleasing Ca inside the cilia of an olfactory sensory neuron you should be able to measure a rapidly rising current. This current is carried by Cl ions. Figure 6 A shows inward currents at -50 mV induced by photorelease of caged Ca in response to UV flashes of different intensities. The rising phase of the Ca-activated Cl currents was well described by a single exponential with time constants varying between 3.8 to 5 ms (Figure 6 B).
Figure 1. Olfactory transduction in the cilia of olfactory sensory neurons. Odorant molecules bind to odorant receptors (OR) activating a G protein that in turns activates adenylyl cyclase (ACIII) producing an intracellular increase in cAMP. cAMP opens cyclic nucleotide-gated (CNG) channels allowing the entry of Na and Ca ions. The intracellular Ca increase activates Ca-activated Cl channels. Caged cAMP or caged Ca can be introduced in the cilia diffusing through a patch pipette. A flash of UV light produces photolysis of the caged compound (Modified, with permission, from Pifferi et al. 20062).
Figure 2. The patch-clamp recording and flash photolysis system. The set-up components include a patch-clamp amplifier, a computer, a digitizer, an epifluorescence microscope, a Xenon flash lamp, a CCD camera and a monitor. Blue and violet lines indicate respectively the visible and UV light path.
Figure 3. Xenon flash lamp. (A) Light source used for flash photolysis of caged compounds. (B) Photodiode module used to evaluate the intensity of the light flash. (C) The light guide from the flash lamp was connected to the input of the photodiode and the output was visualized onto an oscilloscope. One of the three available capacitance values (C1, C2 or C3) was selected on the front panel switch of the flash lamp and the voltage was changed turning the knob on the front panel. The output voltage from the photodiode in response to different flash intensities was plotted versus the applied voltage for each capacitance value: C1 = 1000 μF, C2 = 2000 μF, or C3 = 3000 μF. A 600 μm diameter light guide was used.
Figure 4. Patch-clamp recording in response to photolysis of caged cAMP in low extracellular Ca solution. (A) Whole-cell current response induced in an isolated olfactory sensory neuron by photolysis of caged cAMP localized to the cilia. A UV flash was released at the time indicated by the arrow. The holding potential was -60 mV. (B) The current rising phase was well fitted with a single exponential function (dotted line) with a time constant of 3.4 ms.
Figure 5. Current responses induced by photolysis of caged cAMP in low Ca and in Ringer solutions. (A) An olfactory sensory neuron was bathed in Ringer solution containing 1 mM Ca or in low Ca solution at the holding potential of -60 mV. A UV flash was released at the time indicated by the arrow. (B) Current responses plotted on an expanded timescale showed a multiphasic rising phase in Ringer, while the rising phase was well fitted with a single exponential function (dotted line) with a time constant of 3.5 ms for the response recorded in low Ca solution. (C) Currents responses from the same neuron shown in (A) bathed in Ringer’s solution at the holding potential of -60 and +60 mV. (D) Current responses plotted on an expanded timescale displayed a multiphasic rising phase at -60 mV, whereas at +60 mV the rising phase was well fitted by a single exponential with a time constant of 6.7 ms (dotted line).
Figure 6. Responses to photolysis of caged Ca. (A) Whole-cell currents induced by photolysis of caged Ca at -50 mV. UV flashes were released at the time indicated by the arrow. Flash intensities were varied with neutral density filters. (B) Expanded timescale shows the rapid increase in the current after Ca photorelease. Currents were well fitted by a single exponential function (dotted lines), with time constants of 5, 4.8, 3.8 ms. (Reproduced, with permission, from Boccaccio & Menini, 200710).
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 of caged compounds requires pulses of high intensity of near UV light to uncage a sufficient amount of molecules in a short time. Various light sources can be used: a continuously operated mercury or xenon arc lamp controlled by a shutter and coupled to the epifluorescent port of the microscope, a Xenon flash lamp, a UV laser, and the recently developed high power UV light emitting diode (LED). Each type of light source has advantages and disadvantages according to the specific application and to the cost of the apparatus. Compared to a flash lamp, the continuously operated lamps have a lower light intensity and therefore the duration of light pulses controlled by a shutter needs to be increased up to several hundreds of ms to obtain a sufficient amount of uncaged molecules. UV lasers are very expensive. High power UV LEDs14 for flash photolysis are recently commercially available and could provide a good alternative to other methods. However, an advantage of flash lamps is that they have a broader emission spectrum than UV LEDs, allowing the use of several types of caged compounds with different spectral characteristics The main advantages to use a Xenon flash lamp for uncaging in our application are: a good time resolution, indeed the duration of the light pulse is about 1 ms; a broad UV spectrum that is suitable for photolysis of molecules with different photochemical properties; the possibility to choose the dimension of the light spot to illuminate the ciliary region; the possibility to easily select various light intensities6. Additionally, the Xenon flash-lamp has a reasonable cost, it is easily implemented in an electrophysiological set-up, and does not require a special maintenance.
No conflicts of interest declared.
|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|
|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|
|Bovine serum albumin (BSA)||Sigma-Aldrich||A8806|
|CaCl2 standard solution 0.1 M||Fluka||21059|
|Caged Ca: DMNP-EDTA||Invitrogen||D6814|
|Concanavalin A type V (ConA)||Sigma-Aldrich||C7275|