Kohtaro Kamino

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.

Optical mapping of the early development of the response pattern to vagal stimulation in embryonic chick brain stem.

1. In both intact and slice preparations of vagus‐brain stem isolated from 3‐ to 8‐day‐old chick embryos, the spatial pattern of neural responses to vagal stimulation and its development were assessed by means of multiple‐site optical recording of electrical activity, using a voltage‐sensitive merocyanine‐rhodanine dye (NK2761) and a 12 x 12‐element photodiode array. 2. The first neural responses, viz. fast optical signals (related to the action potential), were recorded in the 4‐day‐old brain stem preparation, and slow optical signals (related to excitatory postsynaptic potentials) were detected from late 7‐ and 8‐day‐old brain stem preparations. 3. The evoked optical signals appeared to be concentrated longitudinally in the central region of the stimulated side of the intact brain stem preparation and in a limited dorsal area in the slice preparation. The signal size gradually increased and the response area expanded as development proceeded. 4. Based on the above results, we have constructed developmental maps of the spatial patterns of the fast and slow optical responses. In the maps, the positions of the peak‐size regions of the fast and slow signals were assessed and we have found that there were differences in the location of these areas for the fast vs. the slow signals in the late 7‐ and 8‐day‐old embryonic brain stem preparations. 5. In the maps for the late 7‐ and 8‐day‐old embryonic brain stems, the fast signal response area seems to correspond to the dorsal motor nucleus of the vagus nerve and the slow response area to the nucleus tractus solitarii.

Early events in development of electrical activity and contraction in embryonic rat heart assessed by optical recording.

Spontaneous action potential and contraction in the early embryonic heart of the rat have been monitored optically using a voltage‐sensitive merocyanine‐rhodanine dye together with a multiple‐element photodiode matrix array, and the onset of rhythmical action‐potential activity in the early phases of rat cardiogenesis was conclusively determined for the first time. Spontaneous rhythmical action potentials were first generated in the central part of the embryonic heart at the middle period of the 3‐somite stage of development, at 91/2 days after copulation. Subsequently, contractions coupled with the action potential also appeared at the end of the 3‐somite stage. Usually, at the 3‐somite stage, spontaneous action signals were synchronized among the different areas in the heart. From this result, it is evident that the paired right and left cardiac primordia are fused completely at the time of initiation of spontaneous electrical activity. In the 3‐somite embryonic heart, excitatory waves were conducted radially over the heart, at a uniform rate (0.4‐0.8 mm/s), from the pace‐making area. However, the regional priority of pace‐making activity is not rigid but is flexible.

Optical detection of postsynaptic potentials evoked by vagal stimulation in the early embryonic chick brain stem slice.

1. A voltage‐sensitive dye and multiple‐site optical recording of changes in membrane potential were used to reveal the postsynaptic potentials in the early embryonic chick brain stem slice preparation. 2. Vagus‐brain stem preparations were isolated from 8‐day‐old chick embryos and then transverse slice preparations were prepared with both the right and left vagus nerve fibres intact. The slice preparations were stained with a voltage‐sensitive merocyanine‐rhodanine dye (NK2761). 3. Voltage‐related optical (absorbance) changes evoked by vagus nerve stimulation with positive square current pulses using a suction electrode were recorded simultaneously from 127 contiguous loci in the preparation, using a 12 x 12‐element photodiode array. Optical responses appeared in a limited area near the dorsal surface of the stimulated side. 4. When relatively large stimulating currents were applied, optical changes having two (or sometimes three) components were recorded. One component was the fast spike‐like signal and another the delayed, long‐lasting slow signal. 5. The size of the slow signal was decreased by continuous stimulation, reduced by low external calcium ion concentrations and eliminated in the presence of manganese or cadmium ions. 6. The slow signals were eliminated in the presence of kynurenic acid, and they were reduced by 2‐APV (DL‐2‐amino‐5‐phosphono‐valeric acid) and by CNQX (6‐cyano‐7‐nitroquinoxaline‐2,3‐dione). We conclude that the slow signals correspond to excitatory postsynaptic potentials which are glutamate mediated.

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.

Optical indications of electrical activity and excitation-contraction coupling in the early embryonic heart

Complete understanding of the ontogenesis and early development of electrical activity and its related contraction has been hampered by our inability to apply conventional electrophysiological techniques to the early embryonic heart. Direct intracellular measurement of electrical events in the early embryonic heart is impossible because the cells are too small and frail to be impaled with microelectrodes. Optical signals from voltage-sensitive dyes have provided a new and powerful tool for monitoring changes in membrane potential in a wide variety of living preparations. With this technique it is possible to make optical recordings from cells which are inaccessible to microelectrodes. An additional advantage of the optical method for recording membrane potential activity is that electrical activity can be monitored simultaneously from many sites in a preparation. Thus, applying a multiple-site optical recording method with a 100- or 144-element photodiode array and voltage-sensitive dyes, we have been able to monitor for the first time spontaneous electrical activity in pre-fused cardiac primordia in early chick embryos at the 6- and early 7-somite stages of development; we have been able to determine that the time of initiation of the heartbeat is the middle period of the 9-somite stage. In the rat embryonic heart, the onset of spontaneous electrical activity and contraction occurs at the 3-somite stage. This article describes ionic properties of the spontaneous action potential and genesis of excitation-contraction coupling in the early embryonic chick and rat hearts. In addition, an improved view of the ontogenetic sequence of spontaneous electrical activity and its implications for excitation-contraction coupling in the early embryonic heart are proposed and discussed.

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.