ANALYTICAL SCIENCES APRIL 2015, VOL. 31
321
2015 © The Japan Society for Analytical Chemistry
An Enzyme-entrapped Agarose Gel for Visualization of Ischemia-induced L-Glutamate Fluxes in Hippocampal Slices in a Flow System Kazuhisa TANAKA,* Atushi SHOJI,** and Masao SUGAWARA*† *Department of Chemistry, College of Humanities and Sciences, Nihon University, Sakurajousui, Setagaya, Tokyo 156–8550, Japan **Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji, Tokyo 192–0329, Japan
An agarose gel slip containing L-glutamate oxidase (GluOx), horseradish peroxidase (HRP) and a dye DA-64 is proposed as a tool for visualizing ischemia-induced L-glutamate release in hippocampal slices in a flow system. The agarose slip with a detection limit of 6.0 ± 0.8 μmol L–1 for L-glutamate enabled us to visualize L-glutamate fluxes in a flow system. The leak of a dye from the agarose gel was negligible and a diffusion blur due to spreading of Bindshedler’s Green (BG) within the gel was suppressed. Monitoring the time-dependent change of ischemia-induced L-glutamate fluxes at neuronal regions CA1, DG and CA3 of hippocampal slices is demonstrated. Keywords Enzyme-entrapped agarose gel, imaging of L-glutamate, hippocampal slice, ischemia (Received October 27, 2014; Accepted January 19, 2015; Published April 10, 2015)
Introduction Monitoring and visualizing the regional distribution of L-glutamate release in brain and brain slices are important for knowing the fundamental role of neuronal activity. The monitoring of extracellular L-glutamate has been performed by using a variety of invasive and non-invasive methods.1–3 Although the methods are invasive, in situ microdialysis4–7 and enzyme based microsensors8–11 are useful for monitoring L-glutamate at a single point with temporal resolution of seconds. On the other hand, physically non-invasive techniques for visualizing the distribution of L-glutamate release with cellular resolution have also been exploited by using glutamate binding and related proteins coupled to a fluorescence molecule12–16 and various glutamate-sensitive techniques.17–23 These probes have successfully been applied to imaging of L-glutamate in brain tissues during physiologically relevant stimulation.3,14,17 We have proposed a visualizing technique with regional resolution based on L-glutamate oxidase (GluOx) immobilized in a polymer matrix24,25 and covalently immobilized on a glass slip.26 The methods enabled us to visualize the regional distribution of L-glutamate fluxes in hippocampal slices under ischemia. In our methods, an acute brain slice is set on a GluOx layer, and hence probes are not necessary to be added. Therefore, the method is practically non-invasive. One of the drawbacks of the above-mentioned visualization methods is that, if the methods are applied to a flow system, a loss of spatial resolution is expected because L-glutamate is released into the extracellular space of neurons and is likely to To whom correspondence should be addressed. E-mail: [email protected]
†
diffuse out of brain slices in a flow system. In the present study, we extend the enzymatic imaging approach to a flow system by trapping the GluOx-based system in agarose gels. Fundamental properties of the glutamate oxidase (GluOx)-based agarose gels are investigated for monitoring the regional distribution of ischemia-induced L-glutamate fluxes in hippocampal slices in a flow system. Under ischemic conditions, the release of L-glutamate occurs slowly and its level is significantly augmented, and hence the enzyme-based agarose gel method will be useful for visualizing the regional distribution of L-glutamate fluxes.
Experimental Reagents Glutamate oxidase (GluOx, recombinant E. Coli) (25 U/mg powder) was obtained from Yamasa Co. (Choshi, Japan). Horseradish peroxidase (HRP), N-(carboxymethylaminocarbonyl)-4,4′-bis(dimethylamino)-diphenylamine sodium salt (DA-64), and L-glutamic acid were obtained from Wako Pure Chemicals Co. (Osaka, Japan). A supply of 2-deoxy-D-glucose was obtained from Tokyo Kasei Co. (Tokyo, Japan). Agarose type VII (low gelling temperature, congealing temp DG. In our previous paper,26 biphasic changes in L-glutamate fluxes were observed at CA1. However, in the present study, the change in the L-glutamate flux was monotonous, probably because temporal resolution was not sufficient, owing to slow penetration of L-glutamate into the gel. It is noted that the L-glutamate flux at each region was observed almost immediately after the onset of ischemia stimulation, because the present system allows the exchange of an ACSF for an ischemia solution in a flow system. In our earlier study,26 a time lag was necessary for injecting a stimulant solution and observing L-glutamate release.
The immobilization of GluOx, HRP and a dye in congealed agarose gel responded to L-glutamate in solution, producing a colored dye (BG) in the gel. The agarose-based visualizing system provides a useful method for knowing L-glutamate fluxes from hippocampal slices under ischemia in a flow system. The gel-based approach can be applied to other enzymatic imaging systems.
Acknowledgements The authors thank Mr. N. Tamura for his experimental help. This work was supported in part by Grants-in-Aid for Scientific Research (No. 21350045) from the Ministry of Education, Culture, Sports, Science and Technology, Japan.
Supporting Information Figure S1 shows R(t) vs. t plots, Fig. S2 illustrates a slope vs. flux plot, Fig. S3 shows the effect of HRP concentration, Fig. S4 shows a time course of BG expansion, Fig. S5 shows photos of BG spreading within a gel. This material is available free of charge at http://www.jsac.or.jp//analsci/.
References 1. A. Hirano and M. Sugawara, Mini-Rev. Med. Chem., 2006, 6, 1091. 2. M. Sugawara, Chem. Rec., 2007, 7, 317. 3. Y. Okubo and M. Iino, J. Physiol., 2011, 589, 481. 4. W. J. Albery, P. N. Barlett, and E. G. Cass, Philos. Trans. R. Soc., B, 1987, 316, 107. 5. W. J. Albery, M. G. Boutelle, and P. T. Galley, J. Chem. Soc., Chem. Commun., 1992, 900. 6. S. Y. Zhou, H. Zuo, J. F. Stobaugh, and E. C. Lunte, Anal. Chem., 1995, 67, 594. 7. M. W. Lada and R. T. Kennedy, Anal. Chem., 1996, 68, 2790. 8. Y. Hu, K. M. Mitchell, F. N. Albahadily, E. K. Michaelis, and G. S. Wilson, Brain Res., 1994, 659, 117. 9. W. H. Oldenziel, M. Zeyden, G. Dijkstra, W. E. J. M. Chijsen, H. Karst, T. I. F. H. Cremers, and B. H. C. Westerink, J. Neurosci. Methods, 2007, 160, 37. 10. B. K. Day, F. Pomerlea, J. J. Burmeister, P. Huettl, and G.
ANALYTICAL SCIENCES APRIL 2015, VOL. 31 A. Gerhard, J. Neurochem., 2006, 96, 1626. 11. K. Nakajima, T. Yanagisawa, A. Hirano, and M. Sugawara, Anal. Sci., 2003, 19, 55. 12. J. Okubo, L. L. Looger, K. D. Michaeva, R. J. Reimer, S. J. Smith, and W. B. Frommer, Proc. Natl. Acad. Sci. U. S. A., 2005, 102, 8740. 13. C. Dulla, H. Tani, S. Okumoto, W. B. Frommer, R. J. Reimer, and J. R. Huguenard, J. Neurosci. Methods, 2008, 168, 306. 14. S. Namiki, H. Sakamoto, S. Iinuma, M. Iino, and K. Hirose, Eur. J. Neurosci., 2007, 25, 2249. 15. S. Okumoto, L. L. Looger, K. D. Micheva, R. J. Reimer, S. J. Smith, and W. B. Frommer, Proc. Natl. Acad. Sci. U. S. A., 2005, 102, 8740. 16. S. A. Hires, Y. Zhu, and R. Y. Tsien, Proc. Natl. Acad. Sci. U. S. A., 2008, 105, 4411. 17. J. Okubo, M. Sekiya, S. Namiki, H. Sakamoto, S. Iinuma, M. Yamasaki, M. Watanabe, K. Hirose, and M. Iino, Proc. Natl. Acad. Sci. U. S. A., 2010, 107, 6526. 18. A. Mitani, F. Kadoya, Y. Nakamura, and E. Kataoka, Neurosci. Lett., 1991, 122, 167.
325 19. G. S. Ayoub and K. Dorst, Vision Res., 1998, 38, 2909. 20. G. S. Ayoub, S. Grutsis, and H. Simko, J. Neurosci. Methods, 1998, 81, 113. 21. C. Tohda and Y. Kuraishi, Neurosci. Res., 1996, 24, 183. 22. B. Innocenti, V. Parpura, and P. G. Haydon, J. Neurosci., 2008, 20, 1800. 23. S. Uchino, T. Nakamura, K. Nakamura, S. Nakajima-Iijima, M. Mishina, S. Kohsaka, and Y. Kudo, Eur. J. Neurosci., 2001, 13, 670. 24. A. Hirano, M. Asakawa, N. Kido, and M. Sugawara, Anal. Sci., 2000, 16, 25. 25. A. Hirano, N. Moridera, M. Akashi, M. Saito, and M. Sugawara, Anal. Chem., 2003, 75, 3775. 26. W. Okumura, N. Moridera, E. Kanazawa, A. Shoji, A. Hirano-Iwata, and M. Sugawara, Anal. Biochem., 2009, 385, 326. 27. N. Nakamura, K. Negishi, A. Hirano, and M. Sugawara, Anal. Bional. Chem., 2005, 330, 660. 28. F. Ka-Ming, K. Moriwaki, and M. Jose Dé Rosa, Methods Mol. Biol., 2013, 979, 65.
321
2015 © The Japan Society for Analytical Chemistry
An Enzyme-entrapped Agarose Gel for Visualization of Ischemia-induced L-Glutamate Fluxes in Hippocampal Slices in a Flow System Kazuhisa TANAKA,* Atushi SHOJI,** and Masao SUGAWARA*† *Department of Chemistry, College of Humanities and Sciences, Nihon University, Sakurajousui, Setagaya, Tokyo 156–8550, Japan **Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji, Tokyo 192–0329, Japan
An agarose gel slip containing L-glutamate oxidase (GluOx), horseradish peroxidase (HRP) and a dye DA-64 is proposed as a tool for visualizing ischemia-induced L-glutamate release in hippocampal slices in a flow system. The agarose slip with a detection limit of 6.0 ± 0.8 μmol L–1 for L-glutamate enabled us to visualize L-glutamate fluxes in a flow system. The leak of a dye from the agarose gel was negligible and a diffusion blur due to spreading of Bindshedler’s Green (BG) within the gel was suppressed. Monitoring the time-dependent change of ischemia-induced L-glutamate fluxes at neuronal regions CA1, DG and CA3 of hippocampal slices is demonstrated. Keywords Enzyme-entrapped agarose gel, imaging of L-glutamate, hippocampal slice, ischemia (Received October 27, 2014; Accepted January 19, 2015; Published April 10, 2015)
Introduction Monitoring and visualizing the regional distribution of L-glutamate release in brain and brain slices are important for knowing the fundamental role of neuronal activity. The monitoring of extracellular L-glutamate has been performed by using a variety of invasive and non-invasive methods.1–3 Although the methods are invasive, in situ microdialysis4–7 and enzyme based microsensors8–11 are useful for monitoring L-glutamate at a single point with temporal resolution of seconds. On the other hand, physically non-invasive techniques for visualizing the distribution of L-glutamate release with cellular resolution have also been exploited by using glutamate binding and related proteins coupled to a fluorescence molecule12–16 and various glutamate-sensitive techniques.17–23 These probes have successfully been applied to imaging of L-glutamate in brain tissues during physiologically relevant stimulation.3,14,17 We have proposed a visualizing technique with regional resolution based on L-glutamate oxidase (GluOx) immobilized in a polymer matrix24,25 and covalently immobilized on a glass slip.26 The methods enabled us to visualize the regional distribution of L-glutamate fluxes in hippocampal slices under ischemia. In our methods, an acute brain slice is set on a GluOx layer, and hence probes are not necessary to be added. Therefore, the method is practically non-invasive. One of the drawbacks of the above-mentioned visualization methods is that, if the methods are applied to a flow system, a loss of spatial resolution is expected because L-glutamate is released into the extracellular space of neurons and is likely to To whom correspondence should be addressed. E-mail: [email protected]
†
diffuse out of brain slices in a flow system. In the present study, we extend the enzymatic imaging approach to a flow system by trapping the GluOx-based system in agarose gels. Fundamental properties of the glutamate oxidase (GluOx)-based agarose gels are investigated for monitoring the regional distribution of ischemia-induced L-glutamate fluxes in hippocampal slices in a flow system. Under ischemic conditions, the release of L-glutamate occurs slowly and its level is significantly augmented, and hence the enzyme-based agarose gel method will be useful for visualizing the regional distribution of L-glutamate fluxes.
Experimental Reagents Glutamate oxidase (GluOx, recombinant E. Coli) (25 U/mg powder) was obtained from Yamasa Co. (Choshi, Japan). Horseradish peroxidase (HRP), N-(carboxymethylaminocarbonyl)-4,4′-bis(dimethylamino)-diphenylamine sodium salt (DA-64), and L-glutamic acid were obtained from Wako Pure Chemicals Co. (Osaka, Japan). A supply of 2-deoxy-D-glucose was obtained from Tokyo Kasei Co. (Tokyo, Japan). Agarose type VII (low gelling temperature, congealing temp DG. In our previous paper,26 biphasic changes in L-glutamate fluxes were observed at CA1. However, in the present study, the change in the L-glutamate flux was monotonous, probably because temporal resolution was not sufficient, owing to slow penetration of L-glutamate into the gel. It is noted that the L-glutamate flux at each region was observed almost immediately after the onset of ischemia stimulation, because the present system allows the exchange of an ACSF for an ischemia solution in a flow system. In our earlier study,26 a time lag was necessary for injecting a stimulant solution and observing L-glutamate release.
The immobilization of GluOx, HRP and a dye in congealed agarose gel responded to L-glutamate in solution, producing a colored dye (BG) in the gel. The agarose-based visualizing system provides a useful method for knowing L-glutamate fluxes from hippocampal slices under ischemia in a flow system. The gel-based approach can be applied to other enzymatic imaging systems.
Acknowledgements The authors thank Mr. N. Tamura for his experimental help. This work was supported in part by Grants-in-Aid for Scientific Research (No. 21350045) from the Ministry of Education, Culture, Sports, Science and Technology, Japan.
Supporting Information Figure S1 shows R(t) vs. t plots, Fig. S2 illustrates a slope vs. flux plot, Fig. S3 shows the effect of HRP concentration, Fig. S4 shows a time course of BG expansion, Fig. S5 shows photos of BG spreading within a gel. This material is available free of charge at http://www.jsac.or.jp//analsci/.
References 1. A. Hirano and M. Sugawara, Mini-Rev. Med. Chem., 2006, 6, 1091. 2. M. Sugawara, Chem. Rec., 2007, 7, 317. 3. Y. Okubo and M. Iino, J. Physiol., 2011, 589, 481. 4. W. J. Albery, P. N. Barlett, and E. G. Cass, Philos. Trans. R. Soc., B, 1987, 316, 107. 5. W. J. Albery, M. G. Boutelle, and P. T. Galley, J. Chem. Soc., Chem. Commun., 1992, 900. 6. S. Y. Zhou, H. Zuo, J. F. Stobaugh, and E. C. Lunte, Anal. Chem., 1995, 67, 594. 7. M. W. Lada and R. T. Kennedy, Anal. Chem., 1996, 68, 2790. 8. Y. Hu, K. M. Mitchell, F. N. Albahadily, E. K. Michaelis, and G. S. Wilson, Brain Res., 1994, 659, 117. 9. W. H. Oldenziel, M. Zeyden, G. Dijkstra, W. E. J. M. Chijsen, H. Karst, T. I. F. H. Cremers, and B. H. C. Westerink, J. Neurosci. Methods, 2007, 160, 37. 10. B. K. Day, F. Pomerlea, J. J. Burmeister, P. Huettl, and G.
ANALYTICAL SCIENCES APRIL 2015, VOL. 31 A. Gerhard, J. Neurochem., 2006, 96, 1626. 11. K. Nakajima, T. Yanagisawa, A. Hirano, and M. Sugawara, Anal. Sci., 2003, 19, 55. 12. J. Okubo, L. L. Looger, K. D. Michaeva, R. J. Reimer, S. J. Smith, and W. B. Frommer, Proc. Natl. Acad. Sci. U. S. A., 2005, 102, 8740. 13. C. Dulla, H. Tani, S. Okumoto, W. B. Frommer, R. J. Reimer, and J. R. Huguenard, J. Neurosci. Methods, 2008, 168, 306. 14. S. Namiki, H. Sakamoto, S. Iinuma, M. Iino, and K. Hirose, Eur. J. Neurosci., 2007, 25, 2249. 15. S. Okumoto, L. L. Looger, K. D. Micheva, R. J. Reimer, S. J. Smith, and W. B. Frommer, Proc. Natl. Acad. Sci. U. S. A., 2005, 102, 8740. 16. S. A. Hires, Y. Zhu, and R. Y. Tsien, Proc. Natl. Acad. Sci. U. S. A., 2008, 105, 4411. 17. J. Okubo, M. Sekiya, S. Namiki, H. Sakamoto, S. Iinuma, M. Yamasaki, M. Watanabe, K. Hirose, and M. Iino, Proc. Natl. Acad. Sci. U. S. A., 2010, 107, 6526. 18. A. Mitani, F. Kadoya, Y. Nakamura, and E. Kataoka, Neurosci. Lett., 1991, 122, 167.
325 19. G. S. Ayoub and K. Dorst, Vision Res., 1998, 38, 2909. 20. G. S. Ayoub, S. Grutsis, and H. Simko, J. Neurosci. Methods, 1998, 81, 113. 21. C. Tohda and Y. Kuraishi, Neurosci. Res., 1996, 24, 183. 22. B. Innocenti, V. Parpura, and P. G. Haydon, J. Neurosci., 2008, 20, 1800. 23. S. Uchino, T. Nakamura, K. Nakamura, S. Nakajima-Iijima, M. Mishina, S. Kohsaka, and Y. Kudo, Eur. J. Neurosci., 2001, 13, 670. 24. A. Hirano, M. Asakawa, N. Kido, and M. Sugawara, Anal. Sci., 2000, 16, 25. 25. A. Hirano, N. Moridera, M. Akashi, M. Saito, and M. Sugawara, Anal. Chem., 2003, 75, 3775. 26. W. Okumura, N. Moridera, E. Kanazawa, A. Shoji, A. Hirano-Iwata, and M. Sugawara, Anal. Biochem., 2009, 385, 326. 27. N. Nakamura, K. Negishi, A. Hirano, and M. Sugawara, Anal. Bional. Chem., 2005, 330, 660. 28. F. Ka-Ming, K. Moriwaki, and M. Jose Dé Rosa, Methods Mol. Biol., 2013, 979, 65.
