ASKING BIOLOGICAL QUESTIONS WITH CAGED COMPOUNDS Samuel S.-H. Wang

ASKING BIOLOGICAL QUESTIONS WITH CAGED COMPOUNDS

Samuel S.-H. Wang

Design principles of caged compounds

H. Lester and J. Nerbonne (1982)Ann. Rev. Biophys. Bioeng. 11:151

The dark reaction

Decay of the aci-nitro intermediate of NPE-caged ATP

J.W. Walker et al.(1988) JACS 110:7170

K.R. Delaney and R.S. Zucker (1990) J.Physiol. 426:473

Fast temporal control:caged calcium at the squid giant synapse

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

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)

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

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)

Focal uncaging

Wang and Augustine (1995)

Two-photon excitation: the third dimension of resolution

Caged fluorescein dextran

Svoboda, Tank & Denk (1996)Science 272:716

Uncaging in single dendritic spines

Furuta et al. (1999) PNAS 96:1193

Comparison of a new caging group,6-bromo-7-hydroxycoumarin-4-ylmethyl (Bhc),

with previous caged compounds

Scanning two-photon uncaging of glutamate

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

Chemical two-photon uncaging

Improved axial resolution viachemical two-photon uncaging

Wang, Khiroug and Augustine (2000)PNAS 97:8635

Wang, Khiroug and Augustine (2000)PNAS 97:8635

LTD induction causes a spreading decrease in receptor sensitivity

PART 2:TECHNICAL PRACTICALITIES

Handling caged compounds

• Regarding the necessity of keeping the compound in the dark.

• Storage.

• Vendor impurities - aftermarket purification.

• Cost control: recirculating and local perfusion.

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

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).

Furuta et al. (1999) PNAS 96:1193

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

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.

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

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

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

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

Absorption bands imply chromatic aberration

H. Piller, Microscope Photometry (1977)

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.