Katsushige Sato

EVALUATION OF OPTIMAL VOLTAGE-SENSITIVE DYES FOR OPTICAL MONITORING OF EMBRYONIC NEURAL ACTIVITY

To evaluate the suitability of a variety of fast voltage-sensitive dyes for optical recording of rapid transmembrane potential activity in the embryonic nervous system, we screened over twenty dyes, including several newly synthesized probes, in three different embryonic neural preparations: cervical vagus nerve bundle, nodose ganglion, and brainstem from 7-day old chick embryos. Measurements of voltage-related optical signals were made using a multiple-site optical recordingsystem. Signal size, signal-to-noise ratio, photobleaching, and phototoxicity were examined. Several promising new merocyanine-rhodanine dyes for embryonic nervous systems were found.

Optical approaches to embryonic development of neural functions in the brainstem

The ontogenetic approach to physiological events is a useful strategy for understanding the functional organization/architecture of the vertebrate brainstem. However, conventional electrophysiological techniques are difficult or impossible to employ in the early embryonic central nervous system. Optical techniques using voltage-sensitive dyes have made it possible to monitor neural activities from multiple regions of living systems, and have proven to be a useful tool for analyzing the embryogenetic expression of brainstem neural function. This review describes recent progress in optical studies made on embryonic chick and rat brainstems. Several technical issues concerning optical recording from the embryonic brainstem preparations are discussed, and characteristics of the optical signals evoked by cranial nerve stimulation or occurring spontaneously are described. Special attention is paid to the chronological analyses of embryogenetic expression of brainstem function and to the spatial patterning of the functional organization/architecture of the brainstem nuclei. In addition, optical analyses of glutamate, GABA, and glycine receptor functions during embryogenesis are described in detail for the chick nucleus tractus solitarius. This review also discusses intrinsic optical signals associated with neuronal depolarization. Some emphases are also placed on the physiological properties of embryonic brainstem neurons, which may be of interest from the viewpoint of developmental neurobiology.

A new simultaneous 1020-site optical recording system for monitoring neural activity using voltage-sensitive dyes

We have constructed a new 1020-site optical system for simultaneous recording of transmembrane electrical activity, using a 34 x 34-element photodiode array. This new apparatus permits analyses of the spatio-temporal pattern of neural activity, such as action potentials and postsynaptic potentials, in the central nervous system, at higher spatial and temporal resolutions.

Evaluation of voltage-sensitive dyes for long-term recording of neural activity in the hippocampus.

Abstract. We searched for an optimal voltage-sensitive dye for optical measurements of neural activity in the hippocampal slice by evaluating several merocyanine-rhodanine and oxonol dyes. The wavelength dependence (action spectra), pharmacological effects of staining, signal size, signal-to-noise ratio, and the utility of the dyes for long-term continuous recording were examined for four merocyanine-rhodanine dyes (NK2761, NK2776, NK3224 and NK3225), which had been reported to be optimal in embryonic nervous systems, and for two oxonol dyes (NK3630 (RH482) and NK3041 (RH155)), which have been among the most popular potentiometric probes for the hippocampal slice preparation. NK2761, NK3224 and NK3225 provided large signal-to-noise ratios, and proved to be useful for optical recordings lasting several hours. NK3630 was most suitable for long-term recording, although the signal-to-noise ratio was slightly inferior to that of the merocyanine-rhodanines. Using NK3630 (RH482) on the hippocampal slice preparation, we demonstrate here that long-term potentiation can be monitored stably for more than 8 hr.

Multiple-site optical monitoring of neural activity evoked by vagus nerve stimulation in the embryonic chick brain stem.

1. Electrical activity in the embryonic chick brain stem has been monitored optically. The vagus‐brain stem preparations isolated from 7‐day‐old chick embryos were stained with voltage‐sensitive merocyanine‐rhodanine dyes. 2. Voltage‐related optical absorption signals evoked by vagus nerve stimulation with depolarizing and hyperpolarizing pulses using a suction electrode were recorded simultaneously from 127 adjacent loci in the brain stem using a 12 x 12‐element photodiode array. 3. The optical signals evoked by the stimulation appeared to be concentrated longitudinally in the central region and in the lateral region, both on the stimulated side of the brain stem, and they did not spread to the opposite side. In addition, the evoked optical responses were detected from small areas on the dorsal surface of the stimulated side, in experiments using transverse slices of brain stem. 4. The optical action potential signals evoked by the brief depolarizing stimulus were conducted slowly and were blocked completely by tetrodotoxin. With relatively long‐duration depolarizing and hyperpolarizing stimulations, electrotonic responses were recorded. 5. When 2 microA/2 ms hyperpolarizing pulse stimulations were applied, anode‐break excitation signals were detected, and these signals were also blocked by tetrodotoxin. 6. On the basis of the data obtained from these experiments, we constructed maps of the electrical response area and demonstrated the spatial pattern of the vagus dorsal nucleus in the 7‐day‐old embryonic chick brain stem.

Responses to glossopharyngeal stimulus in the early embryonic chick brainstem: spatiotemporal patterns in three dimensions from repeated multiple-site optical recording of electrical activity

In an effort to assess the spatial patterning of glossopharyngeal responses in the early embryonic chick brainstem, we used a multiple- site optical recording system with a 12 x 12 element photodiode array and a voltage-sensitive merocyanine-rhodanine dye (NK2761) to monitor neural transmembrane voltage activities. Seven and 8 d old embryonic chick brainstems were sliced into 1400–1600 microns thick sections with the glossopharyngeal and vagal nerves attached, and then stained with the dye. Neural voltage-related optical signals were evoked by a positive brief (depolarizing) square current pulse applied to the glossopharyngeal nerve with a microsuction electrode, and then recorded simultaneously from many loci in the objective two-dimensional image plane of a compound microscope. In addition to the multiple-site optical recording technique, we tried to introduce an optical sectioning method by changing the focal plane of the microscope to obtain three-dimensional information. Thus, we have been able to assess semiquantitatively the three-dimensional profiles of two glossopharyngeal response areas corresponding to the nucleus of the glossopharyngeal nerve (nucleus nervi glossopharyngei) and the nucleus of the tractus solitarius. Furthermore, glutaminergic excitatory postsynaptic potentials were determined within the response area corresponding to the nucleus of the tractus solitarius. In addition, we also compared the glossopharyngeal and vagal response areas and found that the cores of the related nuclei are separated in three dimensions.

Optical Imaging of Intrinsic Signals Induced by Peripheral Nerve Stimulation in the in Vivo Rat Spinal Cord

We examined neural response patterns evoked by peripheral nerve stimulation in in vivo rat spinal cords using an intrinsic optical imaging technique to monitor neural activity. Adult rats were anesthetized by urethane, and laminectomy was performed between C5 and Th1 to expose the dorsal surface of the cervical spinal cord. The median, ulnar, and radial nerves were dissected, and bipolar electrodes were implanted in the forelimb. Changes in optical reflectance were recorded from the dorsal cervical spinal cord in response to simultaneous stimulation of the median and ulnar nerves using a differential video acquisition system. In the region of the cervical spinal cord, intrinsic optical signals were detected between C5 and Th1 at wavelengths of 605, 630, 730, 750, and 850 nm: the image with the largest signal intensity and highest contrast was obtained at 605 nm. The signal intensity and response area expanded with an increase in the stimulation intensity and varied with the depth of the focal plane of the macroscope. The intrinsic optical response was mostly eliminated by Cd(2+), suggesting that the detected signals were mainly mediated by postsynaptic mechanisms activated by sensory nerve fibers. Furthermore, we succeeded in imaging neural activity evoked by individual peripheral nerve stimulation. We found that the response areas related to each peripheral nerve exhibited different spatial distribution patterns and that there were animal-to-animal variations in the evoked neural responses in the spinal cord. The results obtained in this study confirmed that intrinsic optical imaging is a very useful technique for acquiring fine functional maps of the in vivo spinal cord.

Spontaneous depolarization waves of multiple origins in the embryonic rat CNS.

During development, correlated neuronal activity plays an important role in the establishment of the central nervous system (CNS). We have previously reported that a widely propagating correlated neuronal activity, termed the depolarization wave, is evoked by various sensory inputs. A remarkable feature of the depolarization wave is that it spreads broadly through the brain and spinal cord. In the present study, we examined whether the depolarization wave occurs spontaneously in the embryonic rat CNS and, if so, where it originates. In E15–16 rat embryos, spontaneous optically‐revealed signals appeared in association with the rhythmic discharges of cranial motoneurons and propagated widely with similar characteristics to the evoked depolarization wave. At E15, the spontaneous wave mostly originated in the cervical to upper lumbar cords. At E16, the wave was predominantly generated in the lumbosacral cord although a wave associated with the second oscillatory burst was initiated in the rostral cord. At E16, a few waves also originated in the rostral ventrolateral medulla and the dorsomedial pons. When the influence of the caudal cord was removed by transecting the spinal cord, the contribution of the medulla and pons became more significant. These results show that the depolarization wave can be triggered by the spontaneous activity of multiple neuronal populations which are distributed widely from the pons to the lumbosacral cord, although the spinal cord usually plays a predominant role. This network possibly works as a self‐distributing system that maintains the incidence and complicated patterns of the correlated activity in the developing CNS.

Functional representation of the finger and face in the human somatosensory cortex: intraoperative intrinsic optical imaging.

We applied the intrinsic optical imaging technique to the human primary somatosensory cortex during brain tumor/epilepsy surgery for nine patients. The cortical surface was illuminated with a Xenon light through an operating microscope, and the reflected light, which passed through a 605 nm bandpass filter, was detected by a CCD camera-based optical imaging system. Individual electrical stimulation of five digits induced changes in the reflected light intensities. Visualizing the intrinsic optical responses, we constructed maps of finger representation in Brodmanns area 1. In the maps, response areas of Digits I to V were sequentially aligned along the central sulcus in the crown of the postcentral gyrus from the latero-inferior region (Digit I) to the medio-superior region (Digit V). The neighboring response areas partially overlapped each other, as previously described in the monkey somatosensory cortex. Similar results were obtained in the face region with stimulation of the three branches of the trigeminal nerve. These results suggest that the overlap of the response areas is a common feature in the somatosensory cortex not only in monkeys, but also in humans.

Optical illustration of glutamate-induced cell swelling coupled with membrane depolarization in embryonic brain stem slices

USING intrinsicand voltage-sensitive dye optical recordings, we have elucidated coupling of glutamateinduced depolarization and neuronal swelling in early embryonic chick brain stem slices. Twenty-four slices were prepared from 8-day old chick embryos, and stained with a voltage-sensitive merocyanine-rhodanine dye (NK2761). The pressure ejection of glutamate to one site within the preparation evoked changes in transmitted light intensity. With 700 nm incident light, three components were identified in glutamate-induced optical changes. The first component was wavelength dependent, while the second and third components were independent of the wavelength. With reference to the action spectrum of the merocyanine-rhodanine dye and osmotic changes in optical properties, we concluded that the first component reflects glutamate-induced depolarization of the membrane, and that the second component is an intrinsic light-scattering change resulting from neural cell swelling coupled with the membrane depolarization.