Current Application and Technology of Functional Multineuron Calcium Imaging

Abstract

Here we describe recent applications and technical advancements of functional multineuron calcium imaging (fMCI), which monitors the firing activity of more than a thousand neurons through their somatic Ca 2؉ signals. fMCI is used for analysis of various neural circuits under normal and pathological conditions. In vitro fMCI is made more sophisticated by using multipoint illumination and scanning technology with spinning-disk and low-laser-intensity imaging, electron-multiplying charge-coupled device cameras, etc. In vivo fMCI is still developing. We review optical technologies for fast scanning imaging, deep tissue imaging, and recording from moving animals.Key words calcium imaging; functional multineuron calcium imaging; large-scale recording; scanning; spinning disk Review January 2009 1 Biol. Pharm. Bull. 32(1) 1-9 (2009) © 2009 Pharmaceutical Society of Japan * To whom correspondence should be addressed. e-mail: ikegaya@mol.f.u-tokyo.ac.jp tation is removed with a small aperture called a pinhole which functions as a spatial filter 31) ; that is, light emitted from the focal spot efficiently passes through the pinhole, while the vast majority of emission light arising from the outside area is physically blocked. To acquire a two-dimensional or three-dimensional image of a specimen, the focal spot is moved with servomotor-controlled mirrors called Galvano-mirrors, servomotor-controlled stages, etc. Scanned emission light is detected by a photomultiplier tube (PMT). PMT detects a photon via a photoelectric effect and amplifies the signal 10 5 -10 8 times through multiple charged cathodes, in which an incoming photoelectron produces secondary electrons. The amplified electrons are converted into electric currents by an anode.Nipkow Spinning Disk Confocal Microscopy: Spinning Disk with Multi-Pinholes The second implementation is the use of multi-point illumination with many pinholes on a spinning plate termed a Nipkow disk. A rotating spinning disk has multiple pinholes through which laser lines embody multisite focal illumination and detection. The commercially available apparatus, Yokogawa's spinning disk, is further sophisticated. The system uses an additional spinning disk that contains a ca. 20000 microlens array for light convergence on the corresponding pinholes on a Nipkow disk.32) The light focusing also helps reduce scattering light, improving the signal-to-noise ratio. In addition, pinholes are strategically aligned in space to achieve uniform illumination over the field of view. 33,34) The latest version of Yokogawa's spinning disk, CSU-X1, is more improved in terms of the light use efficiency, about two-fold higher than the previous version, CSU-10. In CSU-X1, reduction of mechanical noise from its disk-driving part allows realization of 10000 revolutions per minute. Because the helical pinhole distribution covers the whole field of view every 30°of disk rotation, CSU-X1 enables the maximum sampling rate of 2000 Hz.Charge-Coupled Device Technology Spinning disk confocal microscopy simultaneously...

Facilitation of axon outgrowth via a Wnt5a-CaMKK-CaMKIα pathway during neuronal polarization

“…Then, each mouse brain was immersed in this ice-cold solution bubbled with a gas mixture of 95 % O 2 and 5 % CO 2 , and 300 μm-thick coronal slices were prepared using a vibratome (VT1000, Leica). After preparation, the slices were recovered for over 30 min at room temperature in an artificial cerebrospinal fluid (aCSF) consisting of: (in mM) 127 NaCl, 26 NaHCO 3 , 1.5 KCl, 1.24 KH 2 PO 4 , 1.4 MgSO 4 , 2.4 CaCl 2 , and 10 glucose [ 45 ].…”

“…After Fluo-4 AM loading, cortical slices were transferred to a perfusion chamber (RC-22, Harvard Apparatus) attached to a LSM510META confocal laser microscopy system (Carl Zeiss) and perfused with physiological aCSF containing: (in mM) 127 NaCl, 26 NaHCO 3 , 3.3 KCl, 1.24 KH 2 PO 4 , 1.0 MgSO 4 , 1.0 CaCl 2 , and 10 glucose [ 44 , 45 ] bubbled with 95 % O 2 and 5 % CO 2 at 37C°. Time-lapse Fluo-4 and TagRFP images were acquired sequentially using an oil-immersion objective (Plan-Neofluar 40×/1.30NA, Carl Zeiss) and Multi-track mode of data collection software (Zen, Carl Zeiss) at 0.5 fps with 512 × 512 pixels.…”

Heterogeneous effects of antiepileptic drugs in an in vitro epilepsy model – a functional multineuron calcium imaging study

“…It has also been reported that GABA‐mediated excitation in the developing hippocampus contributes to the initiation of ictal epileptiform activity (Dzhala & Staley, ). On the other hand, fMCI is usually performed on juvenile tissue, because of the difficulty of loading adult tissue with Ca 2+ dyes (Namiki & Ikegaya, ); we used postnatal day 6–8 animals to overcome this problem. GABA seemed to at least have an inhibitory effect in this study, because the giant depolarizing potential and epileptiform activity described above were inhibited by the GABA A antagonist bicuculline, whereas synchronous activity in this study was caused by bicuculline.…”

Using light to probe neuronal function

“…Depending on the scan rate, raster scanning could take longer than wide-field imaging CCD cameras but they have higher SNR. Another alternative is to use a spinning-disk confocal microscope with multiple pinholes [24]. For in vivo experiments, however, using a 2P microscope for Ca 2+ imaging allows for deeper imaging of neuronal populations compared to 1P confocal microscopy (see fig.…”