ASKING BIOLOGICAL QUESTIONS WITH CAGED COMPOUNDS Samuel S.-H. Wang.
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Transcript of ASKING BIOLOGICAL QUESTIONS WITH CAGED COMPOUNDS Samuel S.-H. Wang.
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ASKING BIOLOGICAL QUESTIONS WITH CAGED COMPOUNDS
Samuel S.-H. Wang
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Design principles of caged compounds
H. Lester and J. Nerbonne (1982)Ann. Rev. Biophys. Bioeng. 11:151
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The dark reaction
Decay of the aci-nitro intermediate of NPE-caged ATP
J.W. Walker et al.(1988) JACS 110:7170
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K.R. Delaney and R.S. Zucker (1990) J.Physiol. 426:473
Fast temporal control:caged calcium at the squid giant synapse
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Delays in Ca release after IP3 uncaging
K. Khodakhah and D. Ogden (1993) PNAS 90:4976
Note: 1) [IP3]-dependent delay in Ca rise and IK(Ca); 2) phosphorescence artifact
Temporal dissection of signal kinetics
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Judging a caged compound
• In practice, most caged compounds marketed have pretty fast dark reaction. A more variable quantity is the effectiveness with which caged compounds use light.
• The uncagability index depends on:
• Absorption (Tends to be constant for a given cage group)
• Quantum yield (Varies with modified molecule)
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THE UNCAGABILITY INDEX Extinction coefficient:
where transmission through a thick sample is given by the Beer-Lambert law
where C = concentration of absorber and L is thickness.
For a thin absorber sample [C]L << 1 and so I/I0 2.303 CL.
Quantum yield:
can vary significantly as a function of (308 nm values may be an underestimate). For a good caged compound the uncagability, , should exceed 500 M–1cm–1 (Lester and Nerbonne 1982). The probability that a given molecule will absorb a photon and be photolyzed is 2.303 I0 .
= extinction coefficient of a compound at wavelength .
Units of are M–1cm–1. For cross-sections 26,000 M–1cm–1 = 10–16 cm2.
I/I0 = 10–CL
= quantum yield of a compound (unitless)
= the probability of chemical conversion after a photon is absorbed
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ESTIMATING THE EXPECTED UNCAGING EFFICIENCY FROM POWER MEASUREMENTS Principles:
- Uncaging is proportional to flash energy
- Uncaging is proportional to "uncagability index,"
- Upper limit for efficiency is 1 (total conversion) Formula: where is in M–1cm–1, is in µm, E is in µJ, and A is in µm2. For high E, the uncaging probability is 1 – e–p. Example: Caged ATP on a Xe flash setup (Rapp and Güth, 1988) Measured E/A = 2 mJ/mm2 = 0.002 µJ/µm2 p = 0.84 (420 M–1cm–1)(0.35 µm)(0.002 µJ/µm2) = 0.25 Measured p = 0.20
Probability of uncaging = p = 0.84 ()()(E/A)
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Focal uncaging
Wang and Augustine (1995)
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Two-photon excitation: the third dimension of resolution
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Caged fluorescein dextran
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Svoboda, Tank & Denk (1996)Science 272:716
Uncaging in single dendritic spines
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Furuta et al. (1999) PNAS 96:1193
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Comparison of a new caging group,6-bromo-7-hydroxycoumarin-4-ylmethyl (Bhc),
with previous caged compounds
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Scanning two-photon uncaging of glutamate
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Chemical two-photon uncaging
• Achieving a multiphoton effect by chemical means
• A new design principle: multiple-site caging
• Reduction of effective spontaneous hydrolysis
• Effective cross-section is MUCH larger (109-fold) than true two-photon excitation
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Chemical two-photon uncaging
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Improved axial resolution viachemical two-photon uncaging
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Wang, Khiroug and Augustine (2000)PNAS 97:8635
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Wang, Khiroug and Augustine (2000)PNAS 97:8635
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LTD induction causes a spreading decrease in receptor sensitivity
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PART 2:TECHNICAL PRACTICALITIES
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Handling caged compounds
• Regarding the necessity of keeping the compound in the dark.
• Storage.
• Vendor impurities - aftermarket purification.
• Cost control: recirculating and local perfusion.
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Picking a caged compound
• Caged glutamates: a consumer report
• Fastest: CNB- or desyl-
• Best optical cross-section: Brc-
• Most efficient two-photon effect: bis-CNB-
• Future potential for two-photon uncaging: Corrie’s Magickal Indoline
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CAGED GLUTAMATE COMPOUNDS - AUGUST 2000
Compound
max (nm) (M–1cm–1) ph dark
-O-CNB-glutamate*** 264 180 ( =360 nm) 0.14 25 0.007* 21 µsec 510 (350 nm) 70 0.05* N-Nmoc-glutamate ~265 300 (360 nm) 0.11 30 0.015* 5 msec -O-Desyl-glutamate 150 (360 nm) 0.29 44 0.010* 0.1 µsec Corrie’s indoline ~340 2720 (347 nm) 0.043 120 0.4* <260 µsec (“Ani-glutamate”) -Brc-glutamate 368 17,300 (365 nm) 0.019 330 0.4-1 GM ~10 msec -bis-CNB-glutamate 264 960 (350 nm) 0.14,0.16 140 108 GM** 21, 80 µsec *estimated using the formula ph 2ph (ph
2)(10–15s)(1ph). **chemical two-photon equivalent ph 2ph (ph
2)(10–5s)(1ph2).
***significant spontaneous hydrolysis (F.M. Rossi et al. 1997, J. Biol. Chem. 272:32933).
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Furuta et al. (1999) PNAS 96:1193
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PROPERTIES OF PHOTOLABILE CHELATORS KD(Ca) KD KD(Mg) Quantum Extinction Rate of Rate of Prods Yield Coefficient Photolysis Ca Release nM mM mM M–1cm–1 s–1 s–1 M–1cm–1 NPE 80 1 9 0.20-0.23 975 5x105 6.8x104 200-225 DM-nitrophen 5 3 0.0025 0.18 4,300 8x104 3.8x104 775 (a.k.a. DMNP-EDTA) DMNPE 125 1 10 4,570 BNPE 48 10 1,950 DMNPE-2 313 DMNPE-3 >5000 DMNPE-4 40 1 10 ca.0.7 4,570 ? ca. 3200 nitr-5 145 0.0063 8.5 0.012-0.035 5,500 2,500 ND 30-190 nitr-7 54 0.003 5.4 0.011-0.042 5,500 2,500 ND 55-230 Information courtesy Johann Bollmann, MPIMF Heidelberg.
Nd:YAG 355 nm
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UNCAGING PARAMETERS FOR SOME CAGED COMPOUNDS CNB-caged compounds compound/linkage 350 nm 308nm uncaging index Reference carbachol (N-) ~600M-1cm-1 0.8 480 Milburn et al. (1989)
NMDA (-COO-) 0.43 220* Gee et al. (1995)
kainate (-COO-) 0.34 170*
glutamate ( or -COO-) 500 0.15 75 Wieboldt et al. (1994)
GABA (COO-) 0.15 75* Gee et al. (JACS)
not commercially available:
GABA (N-) 0.06-0.10 30-50* Wieboldt et al.
glutamate (N-) 0.04 20*
*estimated assuming = 500M-1cm-1.
2-nitrobenzyl / l-(2-nitrophenyl)ethyl (NPE) caged compounds compound/linkage 350 nm 308nm uncaging index Reference carbachol (N-) ~100M-1cm-1 0.29* 480 Walker et al. (1986)
ATP 660 0.63† 420
IP3 500 0.65† 325 Walker et al. (1988)
caged phosphates in general 0.49-0.63†
DM-nitrophen 4330‡ 0.18‡ 780 Kaplan & Ellis-Davies
NP-EGTA ~4000 0.35 1400 Ellis-Davies e-mail
fluorescein dextran 4000¶ 0.15 600
*Measured at 0.25 at 347 nm. †Measured at 300-350 nm. ‡Measured at 350 nm. ¶Measured at 338 nm and normalized per mole fluorescein.
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Picking a light source
• If temporal only, light source can be uncollimated
• Flashlamps (Rapp)
• Mercury arc (Denk)
• Nd:YAG laser
• Argon laser
• Ti:S laser
• …see CSHL chapters by Delaney, Kandler
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Achieving lateral resolution
• Full-field epi-illumination (>50 µm)
• Fiber optic directly into the preparation (20 µm)
• Epi-illumination with an aperture (5-50 µm)
• Focal beam direction (2-5 µm) - Ar laser or intense conventional UV source
• Diffraction-limited focus (<1 µm) - Ar or Ti:S laser
• Diffusion: another fundamental limit
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How much light is enough?
• Light density• Focal or subthreshold uncaging: 0.01-0.1 µJ/µm2
• Going through thick tissue may require more• Photostimulation may require more
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Alignment and focusing
• Light metering• General focusing: fluorescence or caged
fluorescein• In epi-illumination mode, strive for parfocality• With a UV objective, direct viewing is sufficient
to achieve parfocality
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Absorption bands imply chromatic aberration
H. Piller, Microscope Photometry (1977)
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REFERENCES General reviews on caged compounds Adams, S.R. and R.Y. Tsien (1993) Controlling cell chemistry with caged compounds. Annu. Rev. Physiol. 55, 755-784. Corrie, J.E.T. and D.R. Trentham (1993) Caged nucleotides and neurotransmitters. pp. 243-305, Bioorganic Photochemistry Volume 2:
Biological applications of photochemical switches, ed. H. Morrison. Marriott, G., ed. (1998) Caged compounds. Methods in Enzymology Vol. 291. Yuste, R., F. Lanni, and A. Konnerth, eds. (2000) Imaging neurons: a laboratory manual. Cold Spring Harbor Laboratory Press. Methods and equipment Denk, W. (1997) Pulsing mercury arc lamps for uncaging and fast imaging. J. Neurosci. Meth. 72, 39-42. Furuta, T., S.S.-H. Wang, J.L. Dantzker, T.M. Dore, W.J. Bybee, E.M. Callaway, W. Denk, and R.Y. Tsien (1999) Brominated 7-
hydroxycoumarin-4-ylmethyls: novel photolabile protecting groups with biologically useful cross-sections for two photon photolysis. Proc. Natl. Acad. Sci., 96(4):1193-1200.
Katz, L.C. and M.B. Dalva (1994) Scanning laser photostimulation: a new approach for analyzing brain circuits. J. Neurosci. Meth. 54, 205-218.
Papageorgiou, G., D.C. Ogden, A. Barth and J.E.T. Corrie (1999) Photorelease of carboxylic acids from 1-acyl-7-nitroindolines in aqueous solution: rapid and efficient photorelease of L-glutamate. J. Am. Chem. Soc. 121, 6503-6504.
Pettit, D.L., S.S.-H. Wang, K.R. Gee and G.J. Augustine (1997) Chemical two-photon uncaging: a novel approach to mapping glutamate receptors. Neuron 19, 465-471.
Rapp, G. and K. Güth (1988) A low cost high intensity flash device for photolysis experiments. Pflügers Archiv. 411, 200-203. Biological results learned using caged compounds Dantzker, J.L. and E.M. Callaway (2000) Laminar sources of synaptic input to cortical inhibitory interneurons and pyramidal neurons. Nat.
Neurosci. 3, 701-707. Delaney, K.R. and R.S. Zucker (1990) Calcium released by photolysis of DM-nitrophen stimulates transmitter release at squid giant
synapse. J. Physiol. 426, 473-498. Dodt, H., M. Eder, A. Frick, W. Zieglgansberger (1999) Precisely localized LTD in the neocortex revealed by infrared-guided laser
stimulation. Science 286, 110-113. Svoboda, K., D.W. Tank and W. Denk (1996) Direct measurement of coupling between dendritic spines and shafts. Science 272, 716-719. Wang, S.S.-H. and G.J. Augustine (1995) Confocal imaging and local photolysis of caged compounds: dual probes of synaptic function.
Neuron 15, 755-760. Wang, S.S.-H., L. Khiroug, and G.J. Augustine (2000) Lateral spread of long-term synaptic depression from active to inactive cerebellar
synapses revealed using chemical two-photon uncaging. Proc. Natl. Acad. Sci. 97, 8635-8640.