- Email: [email protected]
Intrinsic optical imaging of retinal response to Transcorneal Electrical Stimulation
Abstracts / Neuroscience Research 68S (2010) e223–e334
P2-r18 Fluorescent voltage imaging of Aplysia neuron assisted by potassium channel blocker Yasuo Yoshimi , Kazuto Aoki, Hiriomi Ohnishi, Naoko Matsumoto Dept Appl Chem, Shibaura Inst of Technol, Tokyo Aplysia has been used as an ideal experimental model for studying neuronal networks involved in learning and memory because of the presence of large and identifiable neurons in its central nervous system (CNS). A voltage imaging using a voltage-sensitive dye (VSD) is a potential tool for multiple-site monitoring of neuronal activity. The voltage imaging of CNS of Aplysia would contribute greatly toward basic research of neuronal networks; however, this is yet to be implemented. It is probably due to insufficient sensitivity and frame rate of actual hardware for detecting the action potential. In the present study, we developed a variation of the voltage imaging protocol for Aplysia ganglion neurons using di-4-ANEPPS, which is the most commonly used fluorescent VSD using a potassium channel blocker. A buccal ganglion was extracted from an Aplysia californica. The ganglion is soaked in SL-15 medium which contains 100 mM tetraethylammonium (TEA) chloride for 180 min. The width of the action potential was dramatically prolonged to by the treatment. Then the ganglion was stained with di-4-ANEPPS solution containing 100 mM TEA and washed with SL-15 containing TEA. The voltage image of the stained ganglion was sampled by MiCAM 02 (Brainvison Inc., Tokyo) with stimulating a nerve bundle electrically in SL-15 medium which contains 100 mM. The fluorescent image detects the action potential clearly. And transmission of nerve signal in the ganglion can be tracked by the imaging. As a conclusion, the potassium channel blocker TEA can potentially assist the fluorescent voltage imaging of Aplysia ganglion. doi:10.1016/j.neures.2010.07.1467
P2-r19 Intrinsic optical imaging of retinal response to Transcorneal Electrical Stimulation Tomomitsu Miyoshi 1 , Hiroyuki Kanda 2 , Takeshi Morimoto 3 , Toshiyuki Mihashi 2,4 , Yoko Hirohara 2,4 , Takashi Fujikado 2 1
2
Dept Integrative Physiology, Grad Sch Med, Osaka Univ, Osaka, Japan Dept Applied Visual Science, Grad Sch Med, Osaka Univ, Osaka, Japan 3 Dept Ophthalmol, Grad Sch Med, Osaka Univ, Osaka, Japan 4 Research Institute, Topcon Corp, Tokyo, Japan We previously reported that Transcorneal Electrical Stimulation (TES) had neuroprotective effect for axotomized rat retinal ganglion cells and degenerating photoreceptors of RCS rat (Morimoto et al., Inv Ophtalmol. Vis. Sci., 2005, 2007; Okazaki et al., Neurosci. Res., 2008; Tagami et al., Jpn. J. Ophthalmol., 2009). TES also improved the visual function of the patients with nonarteritic ischemic optic neuropathy or traumatic optic neuropathy. However, the retinal spacial response by TES was not described, except for the subjective human study. Here, we tried to describe the response properties to TES, especially in terms of stimulus frequency, by intrinsic optical imaging. Detailed procedure of intrinsic optical imaging was described previously (Okawa et al., Inv. Ophthalmol. Vis. Sci., 2007). In brief, the reflectance of 820–870 nm was recorded via the fundus camera during 26 s with 40 frames/sec, from adult cat under general anesthesia and paralysis. TES current was applied between the ring electrode sutured on the limbus and the silver plate electrode placed under the occipital skin as reference. The stimulating pulses were anodic-first biphasic ones with 5 ms/phase duration and 0.5 mA intensity. The pulse train was applied at 2 s after the start of recording, and the frequency (5–50 Hz) and the number of pulses were changed such that their product was constant. The change of reflectance started immediately after the onset of stimulation and lasted in pulse number-dependent manner. The reflectance change in the retinal vessels was the main part of TES’ retinal response. The number of pixels that had reflectance change depended on the pulse frequency of TES. doi:10.1016/j.neures.2010.07.1468
P2-r20 A critical view of the dipolar model in the neocortex Jorge Riera 1 , Takakuni Goto 1 , Takeshi Ogawa 1 , Akira Sumiyoshi 1 , Hiroi Nonaka 1 , Akitake Kanno 1 , Kazuyuki Kose 2 , Hiroyoshi Miyakawa 3 , Ryuta Kawashima 1 1
Functional Brain Imaging, The Institute for Development, Aging and Cancer, Tohoku University 2 MEG Gr. Bio Analyitical Center, Yokogawa Electric Corporation, Japan 3 School of Life Sciences, Tokyo University of Pharmacy and Life Sciences, Tokyo, Japan The neuroimaging technique resulting from the electro- (EEG) and magneto(MEG) encephalograms is very useful to understand the time course of
e331
neuronal events. The respective forward problems, which establish an instantaneous relationship between the underlying brain sources and these two observation modalities, were formulated about four decades ago based on the quasi-static approach for electromagnetic fields. The quasi-static approach have two implications: (a) the displacement/circulating-eddies currents are small at a macroscopic scale and (b) the currents sources can be represented by a dipole in the microscopic scale. In this study, we present evidences in which this assumption is violated at the microscopic level in the neocortex, leading to a misrepresentation of the actual current sources. These evidences are from both local field potentials obtained using laminar electrodes arrays implanted in the barrel cortex of rats and concurrent EEG/MEG recordings from epileptic subjects. We proposed a new mesoscopic model for the cortical current sources which comprises: (a) compartmentspecific dynamics of pyramidal cells, the main far open-field neurons in the neocortex, and (b) details about the organization of these cells in the cortical microcolumns. Based on this model, we formulated a generalized elliptic boundary problem and brought about a new type of EEG and MEG dynamic inverse solution. doi:10.1016/j.neures.2010.07.1469
P2-r21 Potentiometric dye imaging for cortical neurons with a novel measurement system using a implantable CMOS imaging device Takuma Kobayashi 1,2,3 , Ayato Tagawa 1 , Toshihiko Noda 1,3 , Kiyotaka Sasagawa 1,3 , Takashi Tokuda 1,3 , Yumiko Hatanaka 1,2,3 , Hideki Tamura 2,3 , Yasuyuki Ishikawa 2,3 , Sadao Shiosaka 2,3 , Jun Ohta 1,3 1 Graduate School of Materials Science, Nara Institute of Science and Technology 2 Graduate School of Biological Science, Nara Institute of Science and Technology 3 CREST, Japan Science and Technology Agency
How are the functional neural networks which regulate the animal behavior organized and represented? To clarify this question, it is necessary to measure the real-time multineural activity in living animals. Here, we present a novel optical imaging technique using the implantable biomedical photonic LSI device which has red absorptive light filter for voltage-sensitive dye imaging (BpLSI-red). The BpLSI-red was developed for sensing fluorescence by the on-chip LSI which was designed by using complementary metal-oxide semiconductor (CMOS) technology, and includes 120 × 268 pixel and its dimensions are 1.0 mm × 3.5 mm. Micro-electro-mechanical systems (MEMS) microfabrication technique was used to post-process the CMOS sensor chip; integration of light emitting diodes (LEDs; Ex = 525 nm) for illumination and formation to be suitable for long-term cell culture. Using the device, we succeeded in visualizing the depolarization of pheochromocytoma cells (PC12 cells) and mouse cerebral cortical neurons in primary culture. After high-K+ stimulation, the fluorescence intensities were rapidly decreased on the almost pixels, indicating that more than several thousands of neurons were activated. Therefore, our measurement application enables to detect multiple neural activities simultaneously as a compact instrument, and this methodology could be applied to analyze the dynamic neural network of living animals. doi:10.1016/j.neures.2010.07.1470
P2-r22 Nano-resolution x-ray tomography for deciphering a wiring diagram of the mouse cerebral cortex Haruo Mizutani 1 , Hiroshi Sagara 2 , Akihisa Takeuchi 3 , Takuji Ohigashi 4 , Wataru Yashiro 5 , Kentaro Uesugi 3 , Yoshio Suzuki 3 , Atsushi Momose 5 , Toshihisa Takagi 1 1
Science Integration Program, University of Tokyo, Chiba, Japan 2 Institute of Medical Science, University of Tokyo, Tokyo, Japan 3 Japan Synchrotron Radiation Research Institute / SPring-8, Hyogo, Japan 4 Research Organization of Science & Engineering, Ritsumeikan University, Shiga, Japan 5 Department of Advanced Materials Science, Graduate School of Frontier Sciences, University of Tokyo, Chiba, Japan Neural circuits in the central nervous system are the substrate of various high-order brain functions. Anatomical and functional graph structures of neural networks with actual connections will provide us with perspectives to elucidate the brain complex system. Here, we aim to develop a three-dimensional mouse brain atlas of neural circuits using high resolution x-ray tomography by synchrotron radiation. It will not only identify a large number of synapses but also clarify the structure of neuronal networks for understanding the most complicated organ in the body. In this study,
P2-r18 Fluorescent voltage imaging of Aplysia neuron assisted by potassium channel blocker Yasuo Yoshimi , Kazuto Aoki, Hiriomi Ohnishi, Naoko Matsumoto Dept Appl Chem, Shibaura Inst of Technol, Tokyo Aplysia has been used as an ideal experimental model for studying neuronal networks involved in learning and memory because of the presence of large and identifiable neurons in its central nervous system (CNS). A voltage imaging using a voltage-sensitive dye (VSD) is a potential tool for multiple-site monitoring of neuronal activity. The voltage imaging of CNS of Aplysia would contribute greatly toward basic research of neuronal networks; however, this is yet to be implemented. It is probably due to insufficient sensitivity and frame rate of actual hardware for detecting the action potential. In the present study, we developed a variation of the voltage imaging protocol for Aplysia ganglion neurons using di-4-ANEPPS, which is the most commonly used fluorescent VSD using a potassium channel blocker. A buccal ganglion was extracted from an Aplysia californica. The ganglion is soaked in SL-15 medium which contains 100 mM tetraethylammonium (TEA) chloride for 180 min. The width of the action potential was dramatically prolonged to by the treatment. Then the ganglion was stained with di-4-ANEPPS solution containing 100 mM TEA and washed with SL-15 containing TEA. The voltage image of the stained ganglion was sampled by MiCAM 02 (Brainvison Inc., Tokyo) with stimulating a nerve bundle electrically in SL-15 medium which contains 100 mM. The fluorescent image detects the action potential clearly. And transmission of nerve signal in the ganglion can be tracked by the imaging. As a conclusion, the potassium channel blocker TEA can potentially assist the fluorescent voltage imaging of Aplysia ganglion. doi:10.1016/j.neures.2010.07.1467
P2-r19 Intrinsic optical imaging of retinal response to Transcorneal Electrical Stimulation Tomomitsu Miyoshi 1 , Hiroyuki Kanda 2 , Takeshi Morimoto 3 , Toshiyuki Mihashi 2,4 , Yoko Hirohara 2,4 , Takashi Fujikado 2 1
2
Dept Integrative Physiology, Grad Sch Med, Osaka Univ, Osaka, Japan Dept Applied Visual Science, Grad Sch Med, Osaka Univ, Osaka, Japan 3 Dept Ophthalmol, Grad Sch Med, Osaka Univ, Osaka, Japan 4 Research Institute, Topcon Corp, Tokyo, Japan We previously reported that Transcorneal Electrical Stimulation (TES) had neuroprotective effect for axotomized rat retinal ganglion cells and degenerating photoreceptors of RCS rat (Morimoto et al., Inv Ophtalmol. Vis. Sci., 2005, 2007; Okazaki et al., Neurosci. Res., 2008; Tagami et al., Jpn. J. Ophthalmol., 2009). TES also improved the visual function of the patients with nonarteritic ischemic optic neuropathy or traumatic optic neuropathy. However, the retinal spacial response by TES was not described, except for the subjective human study. Here, we tried to describe the response properties to TES, especially in terms of stimulus frequency, by intrinsic optical imaging. Detailed procedure of intrinsic optical imaging was described previously (Okawa et al., Inv. Ophthalmol. Vis. Sci., 2007). In brief, the reflectance of 820–870 nm was recorded via the fundus camera during 26 s with 40 frames/sec, from adult cat under general anesthesia and paralysis. TES current was applied between the ring electrode sutured on the limbus and the silver plate electrode placed under the occipital skin as reference. The stimulating pulses were anodic-first biphasic ones with 5 ms/phase duration and 0.5 mA intensity. The pulse train was applied at 2 s after the start of recording, and the frequency (5–50 Hz) and the number of pulses were changed such that their product was constant. The change of reflectance started immediately after the onset of stimulation and lasted in pulse number-dependent manner. The reflectance change in the retinal vessels was the main part of TES’ retinal response. The number of pixels that had reflectance change depended on the pulse frequency of TES. doi:10.1016/j.neures.2010.07.1468
P2-r20 A critical view of the dipolar model in the neocortex Jorge Riera 1 , Takakuni Goto 1 , Takeshi Ogawa 1 , Akira Sumiyoshi 1 , Hiroi Nonaka 1 , Akitake Kanno 1 , Kazuyuki Kose 2 , Hiroyoshi Miyakawa 3 , Ryuta Kawashima 1 1
Functional Brain Imaging, The Institute for Development, Aging and Cancer, Tohoku University 2 MEG Gr. Bio Analyitical Center, Yokogawa Electric Corporation, Japan 3 School of Life Sciences, Tokyo University of Pharmacy and Life Sciences, Tokyo, Japan The neuroimaging technique resulting from the electro- (EEG) and magneto(MEG) encephalograms is very useful to understand the time course of
e331
neuronal events. The respective forward problems, which establish an instantaneous relationship between the underlying brain sources and these two observation modalities, were formulated about four decades ago based on the quasi-static approach for electromagnetic fields. The quasi-static approach have two implications: (a) the displacement/circulating-eddies currents are small at a macroscopic scale and (b) the currents sources can be represented by a dipole in the microscopic scale. In this study, we present evidences in which this assumption is violated at the microscopic level in the neocortex, leading to a misrepresentation of the actual current sources. These evidences are from both local field potentials obtained using laminar electrodes arrays implanted in the barrel cortex of rats and concurrent EEG/MEG recordings from epileptic subjects. We proposed a new mesoscopic model for the cortical current sources which comprises: (a) compartmentspecific dynamics of pyramidal cells, the main far open-field neurons in the neocortex, and (b) details about the organization of these cells in the cortical microcolumns. Based on this model, we formulated a generalized elliptic boundary problem and brought about a new type of EEG and MEG dynamic inverse solution. doi:10.1016/j.neures.2010.07.1469
P2-r21 Potentiometric dye imaging for cortical neurons with a novel measurement system using a implantable CMOS imaging device Takuma Kobayashi 1,2,3 , Ayato Tagawa 1 , Toshihiko Noda 1,3 , Kiyotaka Sasagawa 1,3 , Takashi Tokuda 1,3 , Yumiko Hatanaka 1,2,3 , Hideki Tamura 2,3 , Yasuyuki Ishikawa 2,3 , Sadao Shiosaka 2,3 , Jun Ohta 1,3 1 Graduate School of Materials Science, Nara Institute of Science and Technology 2 Graduate School of Biological Science, Nara Institute of Science and Technology 3 CREST, Japan Science and Technology Agency
How are the functional neural networks which regulate the animal behavior organized and represented? To clarify this question, it is necessary to measure the real-time multineural activity in living animals. Here, we present a novel optical imaging technique using the implantable biomedical photonic LSI device which has red absorptive light filter for voltage-sensitive dye imaging (BpLSI-red). The BpLSI-red was developed for sensing fluorescence by the on-chip LSI which was designed by using complementary metal-oxide semiconductor (CMOS) technology, and includes 120 × 268 pixel and its dimensions are 1.0 mm × 3.5 mm. Micro-electro-mechanical systems (MEMS) microfabrication technique was used to post-process the CMOS sensor chip; integration of light emitting diodes (LEDs; Ex = 525 nm) for illumination and formation to be suitable for long-term cell culture. Using the device, we succeeded in visualizing the depolarization of pheochromocytoma cells (PC12 cells) and mouse cerebral cortical neurons in primary culture. After high-K+ stimulation, the fluorescence intensities were rapidly decreased on the almost pixels, indicating that more than several thousands of neurons were activated. Therefore, our measurement application enables to detect multiple neural activities simultaneously as a compact instrument, and this methodology could be applied to analyze the dynamic neural network of living animals. doi:10.1016/j.neures.2010.07.1470
P2-r22 Nano-resolution x-ray tomography for deciphering a wiring diagram of the mouse cerebral cortex Haruo Mizutani 1 , Hiroshi Sagara 2 , Akihisa Takeuchi 3 , Takuji Ohigashi 4 , Wataru Yashiro 5 , Kentaro Uesugi 3 , Yoshio Suzuki 3 , Atsushi Momose 5 , Toshihisa Takagi 1 1
Science Integration Program, University of Tokyo, Chiba, Japan 2 Institute of Medical Science, University of Tokyo, Tokyo, Japan 3 Japan Synchrotron Radiation Research Institute / SPring-8, Hyogo, Japan 4 Research Organization of Science & Engineering, Ritsumeikan University, Shiga, Japan 5 Department of Advanced Materials Science, Graduate School of Frontier Sciences, University of Tokyo, Chiba, Japan Neural circuits in the central nervous system are the substrate of various high-order brain functions. Anatomical and functional graph structures of neural networks with actual connections will provide us with perspectives to elucidate the brain complex system. Here, we aim to develop a three-dimensional mouse brain atlas of neural circuits using high resolution x-ray tomography by synchrotron radiation. It will not only identify a large number of synapses but also clarify the structure of neuronal networks for understanding the most complicated organ in the body. In this study,