Fluorescent Voltage Imaging Technique for the Measurement of Molluscan Neural Activities


The electrophysiological methods using microelectrodes are not appropriate for the simultaneous measurement of neural activities of many neurons. To overcome the difficulty, the fluorescent imaging technique using voltage sensitive dyes can be a powerful technique. The voltage sensitive dyes, however, generally exhibit a relatively small change in their fluorescence intensities, resulting in a low S/N ratio. Additionally, they often exhibit photobleaching and phototoxity. We have therefore improved the fluorescent voltage imaging technique by using a LED as the light source and an electron multiplying (EM)-CCD camera as the fluorescence detector. In this study, we applied our imaging system for the measurement of two kind of molluscan neural activities; one of which is involved in the olfactory processing of the land slug Limax valentianus and the other is involved in the feeding rhythm of the pond snail Lymnaea stagnalis. The system enabled us to measure the neural activities for a long time with a high speed and a high S/N ratio, and the obtained results showed some new physiological findings.

Share and Cite:

Y. Hamasaki, M. Hosoi, S. Nakada, T. Shimokawa and M. Saito, "Fluorescent Voltage Imaging Technique for the Measurement of Molluscan Neural Activities," Open Journal of Biophysics, Vol. 3 No. 1A, 2013, pp. 54-58. doi: 10.4236/ojbiphy.2013.31A007.

Conflicts of Interest

The authors declare no conflicts of interest.


[1] R. P. Haugland, “Handbook of Fluorescent Probes and Research Products,” Molecular Probes Inc., Eugene, 2001.
[2] H. S. Orbach, L. B. Cohen and A. Grinvald, “Optical Mapping of Electrical Activity in Rat Somatosensory and Visual Cortex,” Journal of Neuroscience, Vol. 5, No. 7, 1985, pp. 1886-1895.
[3] K. R. Delaney, A. Gelperin, M. S. Fee, J. A. Flores, R. Gervais, D. W. Tank and D. Kleinfeld, “Waves and Stimulus-Modulated Dynamics in an Oscillating Olfactory Network,” Proceedings of the National Academy of Sciences of the United States of America, Vol. 91, No. 2, 1994, pp. 669-673. doi:10.1073/pnas.91.2.669
[4] K. Okada, R. Kanzaki and K. Kawachi, “High-Speed Voltage-Sensitive Dye Imaging of an in Vivo Insect Brain,” Neuroscience Letters, Vol. 209, No. 3, 1996, pp. 197-200. doi:10.1016/0304-3940(96)12646-X
[5] S. Watanabe, S. Shimozono and Y. Kirino, “Optical Recording of Oscillatory Neural Activities in the Molluscan Brain,” Neuroscience Letters, Vol. 359, No. 3, 2004, pp. 147-150. doi:10.1016/j.neulet.2004.01.063
[6] R. Yoshida, A. Iwamoto and T. Nagahama, “Calcium Imaging for Detection and Estimation of Spike Activities in Aplysia Neurons,” Zoological Science, Vol. 18, No. 5, 2001, pp. 631-643. doi:10.2108/zsj.18.631
[7] N. Takahashi, S. Oba, N. Yukinawa, N. Ujita, M. Mizunuma, N. Matsuki, S. Ishii and Y. Ikegaya, “High-Speed Multineuron Calcium Imaging Using Nipkow-Type Confocal Microscopy,” Current Protocols in Neuroscience, Vol. 57, 2011, pp. 2.14.1-2.14.10.
[8] Y. Kudo, T. Nakamura and E. Ito, “A ‘Macro’ Image Analysis of Fura-2 Fluorescence to Visualize the Distribution of Functional Glutamate Receptor Subtypes in Hippocampal Slices,” Neuroscience Research, Vol. 12, No. 3, 1991, pp. 412-420. doi:10.1016/0168-0102(91)90072-7
[9] S. Sato, H. Osanai, T. Monma, T. Harada, A. Hirano, M. Saito and S. Kawato, “Acute Effect of Corticosterone on N-Methyl-D-Aspartate Receptor-Mediated Ca2+ Elevation in Mouse Hippocampal Slices,” Biochemical and Biophysical Research Communications, Vol. 321, No. 2, 2004, pp. 510-513. doi:10.1016/j.bbrc.2004.06.168
[10] H. Osanai, A. Suzuki, Y. Komatsuzaki, H. Mukai, S. Kawato and M. Saito, “The Binding Site for Acute Corticosterone Effects on N-Methyl-D-Aspartate Receptor-Mediated Ca2+ Signals in Mouse Hippocampal Slices,” Journal of Biophysical Chemistry, Vol. 2, No. 4, 2011, pp. 430-433. doi:10.4236/jbpc.2011.24050
[11] A. Gelperin and D. W. Tank, “Odour-Modulated Collective Network Oscillations of Olfactory Interneurons in a Terrestrial Mollusk,” Nature, Vol. 345, No. 6274, 1990, pp. 437-440. doi:10.1038/345437a0
[12] A. Gelperin, L. D. Rhines, J. Flores and D. W. Tank, “Coherent Network Oscillation by Olfactory Interneurons: Modulation by Endogenous Amines,” Journal of Neurophysiology, Vol. 69, No. 6, 1993, pp. 1930-1939.
[13] T. Kimura, S. Toda, T. Sekiguchi and Y. Kirino, “Behavioral Modulation Induced by Food Odor Aversive Conditioning and Its Influence on the Olfactory Responses of an Oscillatory Brain Network in the Slug Limax marginatus,” Learning & Memory, Vol. 4, No. 5, 1998, pp. 365-375. doi:10.1101/lm.4.5.365
[14] H. Nakamura, S. Kojima, S. Kobayashi, I. Ito, Y. Fujito, H. Suzuki and E. Ito, “Physiological Characterization of Lip and Tentacle Nerves in Lymnaea stagnalis,” Neuroscience Research, Vol. 33, No. 4, 1990, pp. 291-298. doi:10.1016/S0168-0102(99)00020-6
[15] M. Saito, Y. Hamasaki, M. Hosoi and S. Nakada, “Various Firing Patterns Found in a Giant Neuron of the Pond Snail Lymnaea stagnalis and Their Dynamics,” Journal of the Physical Society of Japan, in Press.

Copyright © 2022 by authors and Scientific Research Publishing Inc.

Creative Commons License

This work and the related PDF file are licensed under a Creative Commons Attribution 4.0 International License.