A poster from TRENDS IN NEUROSCIENCE, July 1997



William J. Betz & Joseph K. Angleson, Department of Physiology, University of Colorado Medical School, Denver, CO 80262
The poster comprises 16 panels divided into five sections: Capacitance, Amperometry, Optical imaging, Combined techniques, and New techniques. The individual panels and text are shown below.

Monitoring secretion in single living cells
by capacitance, amperometric, and optical techniques

INTRODUCTION: From the early 1950's until about 1980, secretion from individual living cells was measured primarily by intracellular recordings of synaptic signals at neuronal synapses. Beginning in the early 1980's, three new techniques have been developed. The patch clamp capacitance technique (red traces) provides an electrical measure of the surface area of a cell. Amperometry (black traces) can detect the secretion of certain molecules non-invasively and with exquisite sensitivity. Optical techniques offer opportunities to visualize directly exocytosis and endocytosis.

CAPACITANCE
Panel 1. Capacitance measures cell surface area.
The cartoon shows the equivalent circuit for whole cell patch clamp configuration to measure membrane capacitance (Cm), a technique developed by Erwin Neher and colleagues. The red trace shows the capacitance change of an adrenal chromaffin cell in response to a 200 ms depolarization (dot). Scale = 10 s and 10 fF (= surface area of ~4 granules).

Cartoon from Lindau, M., and Neher, E. (1988) Pflugers Arch 411, 137-146. Trace from Neher, and Marty, A. (1982) Proc. Natl. Acad. Sci. USA 79, 6712-6716.

CAPACITANCE
Panel 2. Behavior of single secretory granules.

Capacitance flicker and fusion pore dilation. The cartoon shows the equivalent circuit of a single granule's capacitance (Cg) and fusion pore conductance (Gp). The traces (40 s duration) show flickering capacitance (Cm; red trace; final change = 220 fF) and fusion pore conductance (Gp; black trace; 500-600 pS) in a beige mouse mast cell during exocytosis of a single giant secretory granule.

Cartoon from Monck, J.R., and Fernandez, J.M. (1992) J.Cell Biol. 119, 1395-1404. Traces from Spruce, A.E., Breckenridge, I.J., Lee, A.K., and Almers, W. (1990) Neuron 4, 643-654.

CAPACITANCE
Panel 3. Capacitance monitoring in a nerve terminal.
Bright field image of an isolated goldfish retinal bipolar cell synaptic terminal with patch clamp electrode. This terminal secretes neurotransmitter from small, clear synaptic vesicles, like most neurons. The red trace shows a capacitance jump in response to a 250 ms depolarization, followed by rapid endocytosis. Scale bar = 10 um, 10 s, and 160 fF.

von Gersdorff, H., and Matthews, G. (1994) Nature 367, 735-739.

AMPEROMETRY
Panel 4. Amperometry measures secretion.
The cartoon depicts exocytosis of a granule and detection of secretory products by their oxidation at a Carbon Fiber Electrode placed nearby, a technique developed by Mark Wightman and colleagues. The black trace shows quantal amperometric current spikes due to exocytosis of catecholamine-containing granules from an isolated adrenal chromaffin cell stimulated by 100 mM nicotine. Scale = 20 pA and 2 s.

Trace from Wightman, R.M., et al., (1991) Proc. Natl. Acad. Sci. USA 88, 10754-10758.

AMPEROMETRY
Panel 5. Secretion from a nerve cell monitored by amperometry.
The bright field image shows an isolated leech neuron (70 um diameter) with a carbon fiber electrode placed near its tip. The traces show two amperometric current spikes, quantal responses due to secretion from a large granule (top trace) and, possibly, from a small synaptic vesicle (bottom trace). Scale = 10 pA and 5 ms.

Contributed by D. Bruns and R. Jahn, Yale University Medical School. See Bruns, D. and Jahn, R. (1995) Nature 377, 62-65.

COMBINED
Panel 6. Capacitance and amperometry combined.
An isolated adrenal chromaffin cell with carbon fiber electrode (upper left) and patch clamp electrode for capacitance monitoring (right) is shown in the bright field image. Scale =10 mM. The traces show amperometric spikes (black trace) and capacitance change (red trace) monitored during secretion from an adrenal chromaffin cell in response to photolytic release of intracellular caged-GTPgS. Scale = 8 min, 210 pA, and 1 pF.

Image from Chow, R.H., and von Rüden, L. (1995). in Single Channel Recording, 2 ed. (Sakmann, B., and Neher, E. eds.) pp 245-275, Plenum. Traces from Oberhauser, A.F., Robinson, I.M., and Fenandez, J.M. (1996) Biophys. J. 71, 1131-1139.

OPTICAL IMAGING
Panel 7. Activity dependent staining with fluorescent dyes.
Activity dependent uptake by garter snake motor nerve terminals of two fluorescent dyes - sulfo-rhodamine (red) and 8-OH pyrene trisulfonic acid (blue), a technique developed by Jeff Lichtman and colleagues. Stimulation of one of two adjacent segmental nerves in the presence of one or the other of the dyes revealed two singly innervated neuromuscular junctions (all red or all blue presynaptic boutons) and one doubly innervated junction (red and blue boutons intermixed; boutons are 2-4 um in diameter).

Contributed by J.W. Lichtman and R.S. Wilkinson, Washington University Medical School. See Lichtman, J.W., Wilkinson, R.S., and Rich, MM. (1985) Nature 314, 357-359.

OPTICAL IMAGING
Panel 8. FM1-43 stains recycled synaptic vesicles.
Frog motor nerve terminals are readily stained and destained in an activity-dependent fashion with the fluorescent styryl dye FM1-43. This image shows a terminal that innervates a single muscle fiber; each dye spot marks a cluster of several hundred stained synaptic vesicles. Scale = 12 um.

Contributed by W.J. Betz and J.A. Angleson, University of Coloradado Medical School. See Betz, W.J., Mao, F, Bewick, G.S. (1992) J. Neurosci. 12, 363-375.

OPTICAL IMAGING
Panel 9. Functional synaptic sites are revealed by FM1-43 in cultured hippocampal neurons.
Fluorescence image of cultured rat hippocampal neurons stained with the styryl dye FM1-43 (in red) superimposed on a DIC image of the neurons. Scale = 10 um.

Contributed by T. A. Ryan, Stanford University Medical School. See Ryan, T.A., et al., (1993) Neuron 11, 713-724.

OPTICAL IMAGING
Panel 10. Presynaptic terminals of retinal neurons stain with FM1-43.
A goldfish retinal bipolar cell shown in bright field (left) and stained with FM1-43 (right). The presynaptic terminal (circular structure at top, about 8 (m in diameter) stains heavily, while the cell body (spindle-shaped structure at bottom) does not take up the dye.

Contributed by L. Lagnado, Cambridge University. See Lagnado, L., Gomis, A., and Job, C., (1996) Neuron 17, 957-967.

OPTICAL IMAGING
Panel 11. Fluorescent antibodies to a synaptic vesicle protein mark exocytic sites in cultured neurons.
Living rat hippocampal neurons were stained with fluorescently tagged antibodies to the lumenal domain of the synaptic vesicle protein synaptotagmin I. Antibodies were taken up during endocytosis of synaptic vesicles, but not released during subsequent exocytosis due to their tight binding. The fluorescent image (orange) is superimposed on a bright field image of a single axon (about 2 um in diameter).

Kraszewski et al., (1995) J. Neurosci 15, 4328-4342.

COMBINED
Panel 12. Combined capacitance and FM1-43 fluorescence measurements.
Top: Five superimposed confocal fluorescence images of an isolated adrenal chromaffin cell (11 um diameter) stained with FM1-43 before (images 1-2), during (images 3-4) and after (image 5) nicotine stimulation. Dye was present in the bathing solution for images 1-4. Middle: Bright field image with a patch clamp electrode for capacitance monitoring. Bottom: Simultaneous measurements of changes (~6 %) in capacitance (red trace) and FM1-43 fluorescence (blue trace) in response to a 1 s depolarization.

Contributed by C.B. Smith, J.K. Angleson, and W.J. Betz, University of Colorado Medical School. See Smith, C.B. and Betz, W.J., (1996) Nature 380, 521-524.

COMBINED
Panel 13. 'Patch amperometry' is combined cell-attached capacitance and amperometry.
The bright field image shows a an adrenal chromaffin cell and patch electrode in cell-attached mode, which increases recording sensitivity. In addition, the carbon fiber electrode for amperometry is positioned inside the patch pipette. Thus, every exocytic event is detected by both techniques; the traces show perfect coincidence of capacitance steps (red trace; each step reflects the exocytosis of a single granule) and amperometric spikes (black trace). Scale = 12 s, 10 fF (cap) and 6.5 uA (amp).

Contributed by M. Lindau, A. Albillos, G. Dernick, and G. Alvarez de Toledo, Max-Planck-Institut für Medizinische Forschung, Heidelberg. See Albillos et al (1997) Biophys. J. 72, A14; Lollike, K., Borregaard, N., and Lindau, M. (1995) J Cell Biol. 129, 99-104.

NEW
Panel 14. New imaging by three photon microscopy.
Fluorescence images of unstained RBL cells grown in culture. White spots mark endogenous fluorescence of serotonin-containing granules, excited by infra-red 'three photon' illumination, rather than the usual ultraviolet illumination. Panel width = 65 um.

Maiti, S. et al., (1997) Science 275, 530-532.

NEW
Panel 15. New imaging by total internal reflection microscopy.
A laser evanescent wave excites only those acridine orange-loaded granules that lie within a few hundred nanameters of the cover slip, providing selective fluorescence imaging of granules ready to undergo exocytosis in an adrenal chromaffin cell. The dark area marks a region where granules exocytosed and disappeared before this image was acquired. Scale = 2 um.

Contributed by J. Steyer and W. Almers, Max-Planck-Institut für Medizinische Forschung, Heidelberg, Germany. See Steyer and Almers (1997) Biophys. J. 72, A227.

NEW
Panel 16. Bright field imaging of single exocytic events.
Cortical granule exocytosis imaged in an intact sea urchin egg by differential interference video contrast microscopy. Granules (each about 1 um in diameter) are visible before secretion (left panel) but not afterwards (right panel) because their contents have a different refractive index than sea water and cytoplasm.

Vogel, S.S., Blank, P.B., and Zimmerberg, J. (1996) J. Cell Biol. 134, 329-338.