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H Komuro

Publications and source records attributed to H Komuro.

At least 55 records · Page 3Linked to original sources
PubMed1993

Optical assessment of spatial patterning of strength-duration relationship for vagal responses in the early embryonic chick brainstem.

The spatial patterning of strength-duration relationship for neural responses to vagus nerve stimulation in the early embryonic chick brainstem preparation was assessed by means of an optical method for monitoring neural activity using a voltage-sensitive dye. Intact brainstem, and brainstem slice preparations, together with the attached vagus nerve, were dissected from 6- to 8-day (d)-old chick embryos. They were stained with a voltage-sensitive merocyanine-rhodanine dye (NK2761). Application of depolarizing square current pulses to the vagus nerve fibers using a micro-suction electrode evoked voltage-related optical (absorption) responses in the brainstem. The optical responses were recorded simultaneously from 127 contiguous sites in both the intact and slice preparations, using a 12 x 12-element photodiode array. The amplitude of the evoked signals and the extent of the response area depended on the strength and duration of the stimulating current pulse. Thus, we determined the threshold current strength needed to evoke the fast spike-like optical signal, corresponding to the action potential, in response to vagal stimulation at a given duration, and we obtained strength-duration curves for 100-200 contiguous regions in the preparation. The strength-duration curves could be apparently fitted by the empirical formula of Weiss. From these curves we obtained the rheobasic currents as a possible indication of neural responsiveness to peripheral vagus stimulation, and we constructed the map of the spatial distribution of the rheobasic currents. The rheobasic current is smallest in the central region of the response area, and it is distributed in a layered pattern of increasing value. In accordance with such a pattern, the minimum rheobasic current decreased and the response area expanded developmentally. The results suggest that the ordered patterns of spatial distributions of the size of the axon, the specific membrane resistance, sodium channel activity, and other electrophysiological factors underlie the initial process of functional organization of the dorsal motor nucleus within the brainstem during early embryonic development.

Animals↗
PubMed1992

Selective role of N-type calcium channels in neuronal migration.

Analysis of neuronal migration in mouse cerebellar slice preparations by a laser scanning confocal microscope revealed that postmitotic granule cells initiate their migration only after the expression of N-type calcium channels on their plasmalemmal surface. Furthermore, selective blockade of these channels by addition of omega-conotoxin to the incubation medium curtailed cell movement. In contrast, inhibitors of L- and T-type calcium channels, as well as those of sodium and potassium channels, had no effect on the rate of granule cell migration. These results suggest that N-type calcium channels, which have been predominantly associated with neurotransmitter release in adult brain, also play a transient but specific developmental role in directed migration of immature neurons before the establishment of their synaptic circuits.

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PubMed1991

Optical determination of impulse conduction velocity during development of embryonic chick cervical vagus nerve bundles.

1. Employing an optical method for multiple-site simultaneous recording of electrical activity, we have determined the conduction velocity in cervical vagus nerve bundles isolated from 5- to 21-day-old chick embryos, and investigated its developmental changes. 2. The preparations were stained with a voltage-sensitive merocyanine-rhodanine dye (NK2761), and action potential- (impulse-) related optical signals were elicited by brief stimuli applied to the end of the vagus nerve bundle with a suction electrode. Optical signals were recorded simultaneously from many contiguous regions using a 12 x 12-element photodiode array. 3. The optical signals spread with small delay from the site of stimulation. From the relationship between the delay and distance from the current-applying electrode, conduction velocities were estimated in each tested preparation: the conduction velocity was very small and increased monotonically from about 0.1 m s-1 at 5 days embryonic age to about 0.4 m s-1 by hatching. The increase in the conduction velocity was closely related to a developmental increase in the diameter of the vagus nerve bundle. 4. In addition, we have examined the spread of electrotonic potentials. The space constant was very small (200-450 microns) and increased as development proceeded. 5. Compound optical action signals having two distinct components were also recorded. They often appeared to be concentrated in the preparations from 8- to 12-day-old embryos. The conduction velocity of the second component was slower than that of the first. We suggest that appearance of the second component reflects degeneration of a subset of axons resulting from 'neural cell death' during the development of the vagus nerve.

Action Potentials↗
PubMed1991

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.

Action Potentials↗
PubMed1991

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.

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PubMed1990

Optical assessment of spatially ordered patterns of neural response to vagal stimulation in the early embryonic chick brainstem.

Spatial dynamic patternings of electrical responses to vagal nerve stimulations in the embryonic chick brainstem preparation were assessed by means of simultaneous multiple-site optical recordings of electrical activity. The vagus/brainstem preparations were dissected from early 7-day-old chick embryos (165-172 h after incubation), and they were stained with a voltage-sensitive merocyanine-rhodanine dye (NK2761). Application of depolarizing square current pulses to the vagus nerve fibers using a suction electrode evoked voltage-related optical (absorbance) signals that were recorded simultaneously from 127 contiguous sites in the whole brainstem preparation using a 12 x 12-element photodiode array. The optical signals evoked by the vagus nerve stimulation appeared to be concentrated longitudinally in the central region and in the lateral region ipsilateral to the site of brainstem stimulation. These response areas were orderly changed according to changes in the strength and in the duration of the stimulating current: the response area expanded as the strength or the duration of the stimulating current was increased. The size of the evoked optical signals also depended on the strength and on the duration of the stimulating current. We also measured critical (threshold) values of the strength and duration of the stimulating current to produce the optical responses, and we found that there was a regionally ordered distribution in the brainstem. In the experiments on transverse slices of the brainstem, the evoked optical responses were detected from limited areas near the dorsal surface of the stimulated side, and the response pattern depended on the strength and duration of the stimulating current in a manner similar to that in the whole brainstem preparations. On the basis of the data obtained from these experiments, we have constructed 3 kinds of response maps for the embryonic brainstem, and the results suggest the functionally ordered arrangement of the neurons in the vagus-related area in the embryonic brainstem.

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PubMed1989

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.

Animals↗
PubMed1989

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.

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PubMed1988

Functional pacemaking area in the early embryonic chick heart assessed by simultaneous multiple-site optical recording of spontaneous action potentials.

Pacemaking areas in the early embryonic chick hearts were quantitatively assessed using simultaneous multiple-site optical recordings of spontaneous action potentials. The measuring system with a 10- X 10- or a 12 X 12-element photodiode array had a spatial resolution of 15-30 microns. Spontaneous action potential-related optical signals were recorded simultaneously from multiple contiguous regions in the area in which the pacemaker site was located in seven- to nine-somite embryonic hearts stained with a voltage-sensitive merocyanine-rhodanine dye (NK 2761). In the seven- to early eight-somite embryonic hearts, the location of the pacemaking area is not uniquely determined, and as development proceeds to the nine-somite stage, the pacemaking area becomes confined to the left pre-atrial tissue. Analysis of the simultaneous multiple-site optical recordings showed that the pacemaking area was basically circular in shape in the later eight- to nine-somite embryonic hearts. An elliptical shape also was observed at the seven- to early eight-somite stages of development. The size of the pacemaking area was estimated to be approximately 1,200-3,000 micron2. We suggest that the pacemaking area is composed of approximately 60-150 cells, and that the pacemaking area remains at a relatively constant size throughout the seven- to nine-somite stages. It is thus proposed that a population of pacemaking cells, rather than a single cell, serves as a rhythm generator in the embryonic chick heart.

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PubMed1988

Regional gradient of pacemaker activity in the early embryonic chick heart monitored by multisite optical recording.

1. Regional gradient of pacemaker activity in the early embryonic precontractile chick heart was quantitatively assessed by means of simultaneous multiple-site optical recordings of changes in membrane potential, using a measuring system with a 10 X 10-element photodiode array which had a spatial resolution of 30 microns. 2. Absorption changes related to spontaneous electrical activity were recorded simultaneously from many contiguous regions in the area in which the pacemaker site was located in seven- to nine-somite embryonic hearts stained with a voltage-sensitive merocyanine-rhodanine dye (NK 2761). 3. The absorption changes related to slow diastolic depolarization were detected, and they were concentrated in and near the pacemaking area. The area in which the absorption changes related to slow diastolic depolarization were detected increased in size as development proceeded. 4. The slope of the absorption change related to diastolic depolarization was measured as an indicator of the pacemaker activity. It was largest in the pacemaking area, and gradually decreased towards the periphery. 5. The maximum slope of the optical change related to slow diastolic depolarization also increased as development proceeded and was related to early development of the heart rate. Thus, these results suggest that formation of a regional gradient of pacemaker activity results in the functional architecture of the pacemaking area in the early phases of cardiogenesis.

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PubMed1987

Mapping of early development of electrical activity in the embryonic chick heart using multiple-site optical recording.

1. Using a multiple-site optical recording method with a 100-element photodiode array and a voltage-sensitive merocyanine-rhodanine dye, we have been able to monitor, for the first time, spontaneous electrical activity in pre-fused cardiac primordia in the 6- and 7-somite chick embryos. 2. To study the regional development of spontaneous electrical activity in the early embryonic pre-contractile chick heart at the later 7-9-somite stages, we have also recorded optically action potentials simultaneously from the entire heart, and constructed maps of the early development of electrical activity. 3. The data show that during the 6-9-somite stages, the size of the active area gradually increases, and that the development of electrical activity was spatially non-uniform: two peaks of activity were found in the right and left sides of the cono-ventricular region at the 7-8-somite stages. As development proceeded to the 9-somite stage, several peak areas of activity appeared. 4. The results are discussed in relation to the spatial pattern of proliferation of electrically active cells in the early phases of cardiogenesis.

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PubMed1986

Conduction pattern of excitation in the amphibian atrium assessed by multiple-site optical recording of action potentials.

Spontaneous action potentials were monitored from multiple sites in the bullfrog atrium using a voltage-sensitive merocyanine-rhodanine dye together with a 100-element photodiode matrix array, and we have assessed the spread of the excitation from the pacemaker. Isochrone curves of conduction were obtained by timing the initiation of the action potential-related optical signals: we constructed maps of the spread. Excitatory waves appeared to conduct radially from the pacemaking area over the atrium, and the conduction velocity in the left atrium exceeded that in the right atrium.

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PubMed1985

Development of electrical rhythmic activity in early embryonic cultured chick double-heart monitored optically with a voltage-sensitive dye.

Double-hearted embryos were produced by whole-embryo culture of chick embryos which were microsurgically cut through the tissue of the anterior intestinal portal at the 1- to 6-somite developmental stage, at the time when the cardiac primordia have not yet fused in the bulboventricular region. The cultured embryos were removed from an incubator usually at the 7- to 10-somite stages of development, and then spontaneous electrical action potentials and/or contractions were optically recorded simultaneously from both the right and left half-hearts, using a 10 X 10- element photodiode matrix array together with a voltage-sensitive merocyanine-rhodanine dye (NK 2761). At the 7- to 8-somite stages, spontaneous action potentials were detected from bilateral prebeating half-hearts or sometimes from one half-heart. In each half-heart, the first spontaneous beating was often observed in the half-heart of the 9 somite embryos. In the beating half-hearts regular activity was always observed, while in the prebeating half-hearts at the 7- to 8-somite stages, both the regular and irregular rhythms of action potentials were detected, and the incidence of occurrence of regular activity significantly outnumbered that of the irregular rhythm. The heart rate in the left half-heart was faster than that in the right half-heart in the great majority of the prebeating and beating double-hearted embryos.

Action Potentials↗
PubMed1985

Optical recording of membrane potential responses from early embryonic chick ganglia using voltage-sensitive dyes.

Changes in absorbance of voltage-sensitive merocyanine-rhodanine dyes were used to monitor electrical responses in the semilunar ganglion of 4-10-day-old developing chick embryos. The electrical responses were simultaneously recorded from many positions in the ganglion. Stimulation of the afferent nerve fibers (the ophthalmic division of cranial nerve V) with a suction electrode led to changes in light absorption of the stained ganglia. With both the depolarizing and hyperpolarizing pulses, the change was largest at 700 nm and was eliminated at a wavelength of 620 nm where the voltage-dependent absorption change of the dyes disappears. In the 4-10-day-old embryonic ganglia, two types of optical membrane potential responses, 'non-conducted' and 'conducted' responses, were identified. The non-conducted response varied with the intensity of the stimulus and had the nature of an electrotonic spread. Furthermore, this non-conducted response exhibited an 'initial upstroke-response' followed by the steady-state plateau evoked by larger depolarizing pulses. The conducted responses were blocked by tetrodotoxin (TTX) or by high external potassium concentration. The incidence of the conducted responses increased as development proceeded from the 5th to the 10th day of age. Thus, the TTX-sensitive action potential activity is probably generated initially in the semilunar ganglion during the 5-10-day-stage of development. These data represent the first demonstration of membrane potential responses in early embryonic intact nervous system. Furthermore, these studies demonstrate the usefulness of voltage-sensitive dyes in the analysis of the organizing process of embryonic neuronal functions during these early stages of development.

Animals↗
PubMed1985

Effects of calcium on electrical propagation in early embryonic precontractile heart as revealed by multiple-site optical recording of action potentials.

The effects of Ca2+ on electrical propagation in early embryonic precontractile chick hearts were studied optically using a voltage-sensitive merocyanine-rhodanine dye. Spontaneous optical signals, corresponding to action potentials, were recorded simultaneously from 25 separate regions of the eight-to-nine-somite embryonic primitive heart, using a square photodiode array. Electrical propagation was assessed by analyzing the timing of the signals obtained from different regions. Electrical propagation in the heart was suppressed by either lowering or raising extracellular Ca2+. Similar effects were produced by a Ca2+ ionophore (A23187). We have also found that electrical propagation across the primordial fusion line at the midline of the heart was enhanced by increasing, and depressed by lowering, external Ca2+. One possible interpretation is that intercellular communication in the embryonic precontractile heart is regulated by the level of the intracellular Ca2+ concentration, and it is suggested that intercellular communication across the primordial fusion line strongly depends on external Ca2+.

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PubMed1985

Optical studies of excitation-contraction coupling in the early embryonic chick heart.

Excitation-contraction coupling at the onset of beating in the 9-10-somite embryonic chick heart was studied by means of an optical method together with a voltage-sensitive merocyanine-rhodanine dye. Spontaneous optical signals were recorded simultaneously from many areas of the embryonic heart, using a square photodiode matrix array. At time of initiation of the heartbeat, spontaneous optical signals consisting of two components were often detected. The first component (1st signal) is a dye-related absorption change due to the action potential, and the second component (2nd signal) is a light scattering change due to contraction. When Ca2+ in the bathing solution was partly replaced by Mg2+, the peak size of both signals was reduced. The correlation between the 1st and 2nd signals corresponded to the relationship between excitation and contraction. The formation of excitation-contraction coupling exhibited a regional non-uniformity in the developing 9-10-somite embryonic hearts: contraction was first generated in the right ventricular region, and then the contractile area spread widely over the whole of the heart. The curves of the excitation-related 1st signal vs. the contraction-related 2nd signal obtained from different areas were not superimposable. Decoupling of excitation from contraction was produced by raising the Ca2+ concentration in the bathing solution, by lowering the Na2+ concentration or by inclusion of a Ca2+ ionophore (A23187). Replacement of the bathing solution with D2O or hypertonic solution also suppressed excitation-contraction coupling. The results suggest that in the early embryonic initial beating chick heart, the contractile system is activated by Ca2+ influx across the sarcolemma accompanying the action potential, and that a Na+-Ca2+ exchange mechanism participates in the relaxation phase of the heartbeat.

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