Biomedical subjects
Publications and source records attributed to A Hirota.
Using a multiple-site optical recording technique employing a fast voltage-sensitive dye, we found a novel type of gamma-aminobutyric acid (GABA) response, which is insensitive to GABAA and GABAB antagonists, but is stimulated by either GABAA or GABAB agonist. This evidence was identified in the early embryonic brainstem slice preparation.
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 recording system. Signal size, signal-to-noise ratio, photobleaching, and phototoxicity were examined. Several promising new merocyanine-rhodanine dyes for embryonic nervous systems were found.
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.
Nonenzymatic glycation of bovine serum albumin (BSA) was inhibited in vitro by some extracts of 34 kinds of spices. The methanol extract of thyme (Thymus vulgaris) had the most potent inhibitory activity among them. Chromatographic purification yielded four flavonoids, quercetin (1), eriodictyol (2), 5,6,4'-trihydroxy-7,8,3'-trimethoxyflavone (3), and cirsilineol (4). These known flavonoids suppressed the levels of advanced glycation end products (AGEs) and fructosamines, shown by the measurement of specific fluorescent groups and the reduction of nitroblue tetrazolium (NBT), respectively. The inhibitory activities were compared with those of other structure-related flavonoids and aminoguanidine.
USF-19A, a soybean lipoxygenase (SBL) and human 5-lipoxygenase (5-LO) inhibitor, was isolated from Streptomyces sp. USF-19 strain. Its chemical structure was determined by spectroscopic evidence to be a new member of the antimycin antibiotic family. The IC50 value of USF-19A against 5-LO was 28.0 microM.
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.
The effects of the intensity, duration and quantity (intensity x duration) of exercise on the reduction of intraocular pressure (IOP) in healthy and physically fit individuals were studied. Five minutes after 15 minutes of exercise at 70%, 55% and 40% of maximum exercise load (%HRmax) the IOP decreased 4.3 +/- 0.7 mmHg, 2.2 +/- 0.7 mmHg and 0.6 +/- 0.5 mmHg, respectively. The magnitude of IOP reduction increased with exercise load. Running for 7.5 minutes at 70%HRmax decreased IOP comparable to 15 minutes of running at the same exercise load (4.4 +/- 0.6 mmHg). Twenty-five minutes of running at 40%HRmax is almost the same quantity of exercise as 15 minutes of running at 70%HRmax. However, the former did not result in IOP reductions to equal the latter (2.3 +/- 0.5 vs 4.4 +/- 0.6 mmHg). The amount of IOP reduction after short-term exercise seems to depend on the intensity of exercise, not on the duration of exercise or the quantity of exercise.
Multiple-site optical recording of transmembrane voltage activity, using a voltage-sensitive dye (NK2761) and a 12 x 12 element photodiode array, was employed to monitor neural responses in the slice preparations that were dissected from 6-9-d-old embryonic brainstems. Transmembrane voltage-related optical responses evoked by a brief square current pulse applied with a microsuction electrode to the vagus nerve were recorded simultaneously from many sites in the preparation. In preparations from 6-d-old embryos, only action potential-related spike-like optical signals were often detected in the normal bathing solution containing 0.5 mM Mg2+. However, in several loci of some preparations, excitatory postsynaptic potential-related slow signals were elicited in a Mg(2+)-free bathing medium, and these elicited slow signals were blocked by DL-2-amino-5-phosphonovaleric acid (2-APV). These results show a possible embryonic expression of NMDA receptor-mediated excitatory postsynaptic potentials. The slow signals were usually detected from tested preparations from 7-9-d-old embryos in normal bathing solution. The later phase of the slow signals was enhanced in a Mg(2+)-free bathing medium, and the enhanced component was also sensitive to 2-APV and insensitive to 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). There were regional differences in the degree of the enhancement of the slow signals in the Mg(2+)-free bathing medium, and of the 2-APV-induced reduction of the slow signals. We have constructed early developmental maps of the spatial patterns of the slow signals related to Mg(2+)- and 2-APV-sensitive postsynaptic potentials. These maps reflect the spatial distribution of functional expression of the NMDA receptors during early development of the chick brainstem. The present findings are new data that have not been reported previously, and they have been obtained for the first time using the multiple-site optical recording technique with a voltage-sensitive dye.
Using a voltage-sensitive merocyanine-rhodamine dye (NK2761) and a 12 x 12-element photodiode matrix array, we recorded optically spontaneous membrane potential changes in a slice preparation from the embryonic chick brain stem during early development. The spontaneous optical signals, related to membrane potential changes, showed a simple monophasic shape with a relatively long duration, and they were synchronized among the different regions in the medulla oblongata. The spontaneous signals were first detected from seven-day-old embryos, and were not present in six-day-old embryos. The spontaneous signals appeared sporadically, and their frequency was very low. Three modes of optical signals termed "singlet-mode", "doublet-mode", and "triplet-mode" were observed. In the doublet- and triplet-modes, the spatial pattern of the first signal was primarily similar to that of the singlet-mode signal, whereas the signal size and spatial extent of the second and third signals appeared to decay.
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.
A basic problem in neurophysiology is concerned with the functional organization/architecture of the central nervous system (CNS). This problem has been studied through a variety of experiments and theoretical approaches. The ontogenetic approach is potentially a very useful strategy for analyzing the complex and subtle functional organization/architecture of the central nervous system. However, for investigations of the early embryonic CNS, conventional electrophysiological recording techniques, including microelectrodes, are extremely difficult or impossible to employ, owing to the small size, fragility and topology of the immature or newly matured neurons. For this reason, investigations of electrophysiology in the vertebrate CNS during the early phases of development have been hampered. Optical techniques using voltage-sensitive dyes have made it possible to monitor electrophysiological events in living systems that are inaccessible to microelectrodes [1, 2]. Furthermore, optical recording methods have been developed into a powerful tool for recording electrical activity simultaneously from many sites in one living preparation [3-5]. Applying these optical techniques, we have overcome traditional obstacles to functional approaches to embryonic heart and CNS during early development [6-8]. We describe here some results obtained with optical techniques in the early embryonic chick brain stem.
The binding of ocular hypotensive drugs to synthetic melanin was studied spectrophotometrically in vitro. The ocular hypotensive effects of the drugs, namely, timolol, befunolol, carteolol, pilocarpine, epinephrine, prostaglandin A2, F2 alpha and E2, also were compared in vivo on eyes of pigmented and albino rabbits. At an initial concentration of 10(-4) M, each of the three beta-blockers exhibited a binding rate of 80-85% as compared to only 40% for pilocarpine and 50% for epinephrine. Almost none of the prostaglandins were found to bind to synthetic melanin. Topically applied, 0.5% timolol and 3% pilocarpine significantly lowered the intraocular pressure in albino but not in pigmented rabbits. Epinephrine (1%) caused a significant reduction in the intraocular pressure both in albino and pigmented rabbits; however, the maximum reduction was greater in albino than in pigmented rabbits. Intraocular pressure was reduced to the same extent and with a similar time-course in both albino and pigmented rabbits by 0.02% prostaglandin A2, F2 alpha and E2. These findings show that several ocular hypotensive drugs bind to melanin and suggest that this process can modify the extent of their pharmacological effects when tested in a single dose, or the time-course of their effects when used to treat chronic conditions.
Ca release from the sarcoplasmic reticulum of frog cut muscle fibers has been estimated with two different methods. The first method is to measure the myoplasmic free [Ca] transient under nonperturbing conditions and then estimate the amounts of Ca that are bound to the myoplasmic buffers troponin and parvalbumin. The Ca released from the sarcoplasmic reticulum is estimated from the sum of the concentrations of free Ca and of Ca bound to the indicator, to troponin, and to parvalbumin. The second method is to estimate the amount of Ca release by capturing most of the Ca with a large concentration of a high affinity Ca buffer. Both methods give similar estimates of the amount of Ca release following a single action potential and of the shape and amplitude of the release waveform.
The present study investigated the effects of meaning information on psychophysiological responses during imagery. Eighteen undergraduates were divided into arousal and relaxation groups, which were then given arousal-meaning- and relaxation-meaning-oriented trainings, respectively. After the training, the subjects were given the imagery test, and inter-heartbeat-interval (IBI), respiration, frontalis EMG, and peripheral temperature during neutral, snake, and social imageries were analyzed. The results showed that there was a significant main effect in IBI for groups: the arousal group demonstrated greater IBI changes than the relaxation group. Regarding EMG, significant increases were present during snake and social imageries in the arousal group, whereas no significant increase was observed in the relaxation group. No significant difference between groups was found in respiration and temperature. The results suggest that there are some situations that accompany their primary meanings, and that meaning-oriented trainings can modify the structure of the response propositions.
I investigated stimulation of cyclic adenosine 3':5'-monophosphate (cAMP) production by isoproterenol and forskolin in cultured chick embryo retinal pigment epithelium. In addition, in order to study the interaction between signal transduction systems, I studied the effects of phorbol 12-myristate, 13-acetate (PMA), a protein kinase C (PKC) activator, on isoproterenol- or forskolin-stimulated cAMP production. After stimulation with 10(-9)-10(-5) M isoproterenol and forskolin, a marked and concentration-dependent increase in cAMP level was observed. However, addition of 10(-10)-10(-5) M PMA had no effect on the cAMP level. Preincubation of isoproterenol and forskolin with 100 nM PMA suppressed the increase of cAMP. The suppressive effect of PMA was stronger on isoproterenol-stimulated production than on forskolin-stimulated production. 4 alpha-phorbol 12, 13, didecanoate, a weak activator of PKC, had a weak suppressive effect on cAMP-production stimulated by isoproterenol. Both staurosporin and H-7, 1-(5-isoquinolinylsulfonyl)-methylpiperazine, inhibitors of PKC, decreased the suppression of cAMP production by PMA. It is speculated that in chick retinal pigment epithelium PKC stimulators, such as PMA, may inhibit the adenylate cyclase pathway.
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.
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.
It was reported that 8-Hydroxycarteolol (8-OH CA), the major metabolite of carteolol hydroxychloride (CA), has a slightly different pharmacological effect from CA. We studied the reduction of intraocular pressure (IOP) on a single eyedrop application of 8-OH CA in albino and pigmented rabbit eyes. To determine the characteristic of 8-OH CA and CA, we investigated the binding ability of these drugs to synthetic melanin. In the present study, topically applied 2% CA did not significantly decrease IOP in albino and pigmented rabbit eyes. Topically applied 0.01, 0.05, 0.1 and 1.0% 8-OH CA significantly decreased the IOP of albino rabbit as did 0.1 and 1.0% 8-OH to pigmented rabbit eyes. The maximum reduction of IOP was 3.5 +/- 0.33 mmHg (mean +/- SEM) in albino rabbit and 3.0 +/- 0.45 mmHg (mean +/- SEM) in pigmented rabbit. Maximum IOP reduction was obtained after 30 min. from topical application in albino rabbit, but in pigmented rabbit after 1 hour or later. Our binding studies to melanin show that the melanin binding ability is less for 8-OH CA than for CA at any concentrations. These results may indicate that lower concentrations of topically applied 8-OH CA profoundly reduce IOP compared to CA, and 8-OH CA has less effect on melanin than CA.