A lie detector test for presynaptic capacitance measurements.

Measurements of membrane capacitance are a mainstay of studies of exocytosis in single cells. However, capacitance reflects both addition and retrieval of membrane, and an increase in capacitance can be taken as an index of the amount of release only if exocytosis and endocytosis do not overlap in time. Because of this potential problem, workers in the field have long appreciated the need for an independent index of the release of secreted substances, in parallel with capacitance measurements. For cells that release oxidizable substances such as norepinephrine, electrochemical detection of the released molecules is commonly used for this purpose (e.g., Albillos et al. 1997xAlbillos, A., Dernick, G., Horstmann, H., Almers, W., Alvarez de Toledo, G., and Lindau, M. Nature. 1997; 389: 509–512Crossref | PubMed | Scopus (378)See all ReferencesAlbillos et al. 1997). For nonoxidizable neurotransmitters such as glutamate, however, some other detection scheme is needed. In a paper published in this issue of Neuron, von Gersdorff and colleagues 1998xvon Gersdorff, H., Sakaba, T., Berglund, K., and Tachibana, M. Neuron. 1998; 21: 1177–1188Abstract | Full Text | Full Text PDF | PubMed | Scopus (126)See all Referencesvon Gersdorff and colleagues 1998 have met this need, by using glutamate receptors of a “postsynaptic” detector cell to determine whether presynaptic capacitance changes tell the truth about glutamate release from the terminal.In a series of elegant and technically demanding experiments, von Gersdorff et al. patch clamped single glutaminergic synaptic terminals of retinal bipolar neurons, a ribbon-type synapse that allows presynaptic capacitance measurements (von Gersdorff and Matthews 1994xvon Gersdorff, H. and Matthews, G. Nature. 1994; 367: 735–739Crossref | PubMed | Scopus (322)See all Referencesvon Gersdorff and Matthews 1994). Simultaneously, they used current through AMPA receptors of a closely apposed retinal horizontal cell to detect glutamate released from the bipolar cell terminal, after treating the AMPA receptors with cyclothiazide to prevent desensitization and ensure temporal fidelity of the glutamate response. They found that over a wide range of conditions, capacitance changes and glutamate release match remarkably well. In keeping with the view developed from previous capacitance work, glutamate release activated rapidly upon opening calcium channels and terminated rapidly upon closing the channels. Thus, exocytosis in bipolar cell terminals does not overlap appreciably with endocytosis, which takes place on a much slower time scale. Depolarization tapped a limited pool of glutamate that was exhausted within a few hundred ms or less, depending on the magnitude of the calcium current. After the pool was depleted, complete recovery of glutamate release required 20–30 s. Again, these conclusions from direct measurement of glutamate release agree quantitatively with prior conclusions based on capacitance measurements.A major advantage of the approach taken by von Gersdorff et al. is that, unlike capacitance measurements, the glutamate-activated current allows a direct view of the kinetics of glutamate release during the presynaptic depolarization. Two components of release were observed. The first component activated in less than 1 ms after onset of depolarization and was exhausted in less than 10 ms. This component matches well with the ultrafast component of exocytosis described in capacitance measurements by Mennerick and Matthews 1996xMennerick, S. and Matthews, G. Neuron. 1996; 17: 1241–1249Abstract | Full Text | Full Text PDF | PubMed | Scopus (178)See all ReferencesMennerick and Matthews 1996, which was interpreted to represent vesicles docked at the plasma membrane. After a delay that depended on the magnitude of the calcium current, the fast component was followed by a second, slower wave of glutamate release. This second component represents recruitment of a reserve pool—possibly the population of vesicles tethered to synaptic ribbons (von Gersdorff et al. 1996xvon Gersdorff, H., Vardi, E., Matthews, G., and Sterling, P. Neuron. 1996; 16: 1221–1227Abstract | Full Text | Full Text PDF | PubMed | Scopus (227)See all Referencesvon Gersdorff et al. 1996). The reserve was depleted within a few hundred milliseconds during weak depolarizations that did not strongly activate calcium channels. With strong depolarization, the two components merged into a single transient burst of glutamate release that decayed to baseline within 100 ms during sustained depolarization, even though presynaptic calcium influx continued unabated.One interesting remaining question is how the results from capacitance measurements—and now from glutamate release—can be reconciled with the very different conclusions reached from studies of uptake of the fluorescent membrane dye FM1-43 in retinal bipolar cells. On the one hand, capacitance and glutamate release measurements demonstrate rapid fusion of a limited pool of vesicles, driven by microdomain levels of calcium (Heidelberger et al. 1994xHeidelberger, R., Heinemann, C., Neher, E., and Matthews, G. Nature. 1994; 371: 513–515Crossref | PubMed | Scopus (546)See all ReferencesHeidelberger et al. 1994). On the other hand, uptake of FM1-43 was interpreted to indicate continuous exocytosis for minutes of a very large population of synaptic vesicles, driven by submicromolar calcium (Lagnado et al. 1996xLagnado, L., Gomis, A., and Job, C. Neuron. 1996; 17: 957–967Abstract | Full Text | Full Text PDF | PubMed | Scopus (145)See all ReferencesLagnado et al. 1996; see also Rouze and Schwartz 1998xRouze, N.C. and Schwartz, E.A. J. Neurosci. 1998; 18: 8614–8624PubMedSee all ReferencesRouze and Schwartz 1998). The experiments of von Gersdorff and colleagues leave little doubt that capacitance measurements indeed give an accurate indication of glutamate release. However, one possibility is that both views are correct, and the much longer time scale of the dye experiments reveals a component of continuous release that would be difficult to detect with glutamate-activated currents. It seems unlikely that the copious release estimated by Lagnado et al. (up to 3800 vesicles per second for periods of minutes) could have been missed, but longer-duration depolarizations need to be tested in the glutamate release experiments. AMPA responses did show some asynchro-nous glutamate release continuing for about 300 ms after termination of strong depolarization, suggesting that even more continuing release might occur after long-duration stimuli that elevate global calcium to sufficiently high levels in the terminal. Recent work on uptake of FM dyes also suggests that both continuous and transient exocytosis coexist in bipolar cell terminals (Rouze and Schwartz 1998xRouze, N.C. and Schwartz, E.A. J. Neurosci. 1998; 18: 8614–8624PubMedSee all ReferencesRouze and Schwartz 1998).Another possible explanation for continuous FM1-43 uptake in bipolar cell terminals is that the dye uptake may be unrelated to neurotransmitter release and perhaps reflects other forms of membrane turnover, such as the remodeling of the terminal that is known to occur during the light/dark cycle in the intact retina (Yazulla and Studholme 1992xYazulla, S. and Studholme, K.M. J. Comp. Neurol. 1992; 320: 521–530Crossref | PubMed | Scopus (31)See all ReferencesYazulla and Studholme 1992). Finally, the possibility should be considered that FM1-43 is somehow able to cross the plasma membrane directly in certain instances, either passively or by means of active cellular mechanisms. Rouze and Schwartz 1998xRouze, N.C. and Schwartz, E.A. J. Neurosci. 1998; 18: 8614–8624PubMedSee all ReferencesRouze and Schwartz 1998 have presented data consistent with such direct entry of FM dyes into bipolar cells. More work is clearly needed to explore these possibilities. For now, though, it is clear from the work of von Gersdorff et al. 1998xvon Gersdorff, H., Sakaba, T., Berglund, K., and Tachibana, M. Neuron. 1998; 21: 1177–1188Abstract | Full Text | Full Text PDF | PubMed | Scopus (126)See all Referencesvon Gersdorff et al. 1998 that presynaptic capacitance measurements have passed the lie detector test.