The Journal of Membrane Biology

, Volume 246, Issue 9, pp 679–688 | Cite as

Evaluation of Voltage-Sensitive Fluorescence Dyes for Monitoring Neuronal Activity in the Embryonic Central Nervous System

  • Saad Habib-E-Rasul Mullah
  • Ryo Komuro
  • Ping Yan
  • Shihori Hayashi
  • Motoki Inaji
  • Yoko Momose-Sato
  • Leslie M. Loew
  • Katsushige Sato


Using an optical imaging technique with voltage-sensitive dyes (VSDs), we investigated the functional organization and architecture of the central nervous system (CNS) during embryogenesis. In the embryonic nervous system, a merocyanine-rhodanine dye, NK2761, has proved to be the most useful absorption dye for detecting neuronal activity because of its high signal-to-noise ratio (S/N), low toxicity and small dye bleaching. In the present study, we evaluated the suitability of fluorescence VSDs for optical recording in the embryonic CNS. We screened eight styryl (hemicyanine) dyes in isolated brainstem–spinal cord preparations from 7-day-old chick embryos. Measurements of voltage-related optical signals were made using a multiple-site optical recording system. The signal size, S/N, photobleaching, effects of perfusion and recovery of neural responses after staining were compared. We also evaluated optical responses with various magnifications. Although the S/N was lower than with the absorption dye, clear optical responses were detected with several fluorescence dyes, including di-2-ANEPEQ, di-4-ANEPPS, di-3-ANEPPDHQ, di-4-AN(F)EPPTEA, di-2-AN(F)EPPTEA and di-2-ANEPPTEA. Di-2-ANEPEQ showed the largest S/N, whereas its photobleaching was faster and the recovery of neural responses after staining was slower. Di-4-ANEPPS and di-3-ANEPPDHQ also exhibited a large S/N but required a relatively long time for recovery of neural activity. Di-4-AN(F)EPPTEA, di-2-AN(F)EPPTEA and di-2-ANEPPTEA showed smaller S/Ns than di-2-ANEPEQ, di-4-ANEPPS and di-3-ANEPPDHQ; but the recovery of neural responses after staining was faster. This study demonstrates the potential utility of these styryl dyes in optical monitoring of voltage changes in the embryonic CNS.


Optical recording Voltage-sensitive dye Embryo Nervous system Fluorescence Screening 



This research was supported by Grants from the Monbu-Kagaku-sho of Japan, the Human Frontier Science Program (Grant RGP0027/2009) and the US National Institutes of Health (grant R01EB001963). S. H. M. was supported by the Opto-Medical Institute as a postdoctoral fellowship.


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Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  • Saad Habib-E-Rasul Mullah
    • 1
  • Ryo Komuro
    • 2
  • Ping Yan
    • 3
  • Shihori Hayashi
    • 4
  • Motoki Inaji
    • 4
  • Yoko Momose-Sato
    • 2
  • Leslie M. Loew
    • 3
  • Katsushige Sato
    • 1
  1. 1.Department of Health and Nutrition Sciences, Faculty of Human HealthKomazawa Women’s UniversityInagi-shiJapan
  2. 2.Department of Health and Nutrition, College of Human and Environmental StudiesKanto Gakuin UniversityYokohamaJapan
  3. 3.R. D. Berlin Center for Cell Analysis and ModelingUniversity of Connecticut Health CenterFarmingtonUSA
  4. 4.Department of Neurosurgery, Graduate School and Faculty of MedicineTokyo Medical and Dental UniversityTokyoJapan

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