Biomedical subjects
Publications and source records attributed to H Tokuno.
Application of a detergent, Triton X-100, dramatically increased the number of impregnated neurons in the Golgi-Kopsch method. This modification is particularly useful for obtaining a panorama of the neuronal field in adult brains.
Using a semihorizontal section plane tangential to the ventral surface of the cerebral peduncle, the authors re-examined cyto-, myelo- and dendroarchitecture, acetylcholinesterase activity, afferent fibers, and efferent projection neurons of the substantia nigra pars reticulata. In the semihorizontal section plane, the substantia nigra pars reticulata was a disc-shaped nucleus and contained two to three myelinated fiber bundles running from anteromedial to posterolateral. Bands of high acetylcholinesterase activity existed parallel to the anteromedial-posterolateral direction. The Golgi silver impregnation study revealed that many nigral neurons extended their varicose dendrites anteromedially and posterolaterally. In cases with injections of wheat germ agglutinated horseradish peroxidase into the neostriatum or injections of tritiated leucine into the subthalamic nucleus, anterogradely labeled afferent fibers and axon terminals in the substantia nigra pars reticulata were organized into bands in the same anteromedial-posterolateral direction. In cases with injections of wheat germ agglutinated horseradish peroxidase into either the superior colliculus, the pedunculopontine tegmental nucleus or the ventromedial nucleus of the thalamus, retrogradely labeled neurons were also clustered along the anteromedial-posterolateral direction with their dendrites extending anteromedially and posterolaterally. The present findings strongly suggest that the substantia nigra pars reticulata has a laminar organization.
1. The effects of W-7, trifluoperazine, and W-5 on Ca2(+)-activated K(+)-channels were investigated with the inside-out patch-clamp method in smooth muscle cells freshly dispersed from pregnant rat myometrium. These drugs are known to have different potencies as calmodulin antagonists. 2. In the presence of 1 microM Ca2+ on the cytoplasmic side ([Ca2+]i), the fraction of time the channel was open (open probability, Po) was about 0.9 and the calmodulin antagonists (1-30 microM) applied to the cytoplasmic face reduced Po to 0.65-0.55 dose-dependently. In the presence of 0.1-0.16 microM Ca2+, when Po was very low (0.02), calmodulin antagonists increased Po. All antagonists used produced almost identical effects at the same concentration. 3. The probability density function of the open time distribution could be described by the sum of two exponentials. W-7 decreased the time constant of slow component of distribution and at 30 microM the slow component disappeared both at 1 and 0.25 microM [Ca2+]i, reflecting the appearance of flickering channel activity. The probability density function of the closed time distribution could be fitted with three exponentials. The time constants of these components were not significantly altered by W-7. 4. Internally applied calmodulin (1-5 microM) did not produce any significant effect on channel activity. 5. The effects of calmodulin antagonists are considered to be due to a direct action of these compounds on the channel, and suggest that channel activation by Ca2+ is not mediated by calmodulin.
1. The action of catecholamines (isoprenaline and noradrenaline) and forskolin on membrane currents was studied in single cells freshly dispersed from the pig coronary artery by use of the whole-cell clamp method, usually with electrodes containing CsCl. 2. In normal Krebs solution, with and without 30 mM tetraethylammonium (TEA) and 0.5 mM 4-aminopyridine, isoprenaline (1-5 microM) clearly increased the inward currents elicited by membrane depolarization, without affecting the holding current at -80 mV. The same effect was observed when the external Cl- was replaced with isethionate. The outward current recorded with K(+)-containing electrodes was not significantly affected by isoprenaline. 3. In the presence of 67 mM Ba2+ and 30 mM TEA, the maximum inward current recorded with CsCl containing electrodes was 119 +/- 7 pA (the mean +/- s.e.mean, n = 90) in cells where the current was larger than 30 pA. The L-type Ca2+ channel was considered to be responsible for these currents, based on the threshold voltage, the slow time course of decay, the large depolarization necessary to produce inactivation, and the high susceptibility to the Ca2+ channel antagonist, nicardipine. 4. Isoprenaline and noradrenaline increased the amplitude of inward currents evoked by depolarizing pulses. The maximum inward current was potentiated by 43 +/- 7% (n = 12) by isoprenaline and 39 +/- 10% by noradrenaline (n = 6) at a concentration of 1 microM. These effects were strongly inhibited by propranolol, but not phentolamine. Forskolin (10 microM) also potentiated the currents to a similar degree. 5. It is suggested that stimulation of beta 3-adrenoceptors increases the amplitude of inward currents through L-type Ca2 + channels in the pig coronary artery and that intracellular cyclic adenosine monophosphate is likely to be inolved in this action.
1. The effects of intracellular pH (pHi) on calcium-activated potassium channels (Ca2(+)-activated K+ channels) were studied in membrane patches of smooth muscle freshly dispersed from the rabbit trachea. Single-channel currents were recorded with an 'inside-out' patch clamp technique, mainly at 0 mV, with the external (electrode) medium containing 130 mM-K+ and the internal (bath) medium 6 mM-K+. 2. With an internal Ca2+ concentration ([Ca2+]i) of 1 microM, the fraction of time during which the channel was in an open state (the open probability, Po) was more than 0.8 at pHi 7.4. The channel activity nearly disappeared at pHi 7.0. The [Ca2+]i-Po relationship was shifted to higher [Ca2+]i by acidosis, the shift being approximately an 8-fold increase for a fall in pHi of 0.5 units. 3. The membrane potential and current intensity (V-I) relationship of single channels between +30 and -50 mV was shifted in a hyperpolarizing direction by intracellular acidosis. The shift was roughly 10 mV for 1 pH unit at 1 microM [Ca2+]i. At pHi 7.4 [Ca2+]i 1 microM, the V-Po relationship was shifted in a depolarizing direction by acidification. When [Ca2+]i was increased to 10 microM, V-Po relationship became less sensitive to V as well as pHi changes. 4. When Po was high, the probability density function of open and closed time distributions could be fitted by two exponentials. When Po was decreased to less than 0.3, either by reducing [Ca2+]i or by lowering pHi, another component having long closed times appeared. At similar Po values, the time constant of open time distribution was smaller with lower pHi. 5. It is concluded that the main effect of an increase in intracellular hydrogen ions is to decrease the open probability of the Ca2(+)-activated K+ channel, by reducing the sensitivity to Ca2+ and also shortening the open state.
Isoprenaline is a beta-adrenergic agonist of clinical importance as a remedy for asthma. In airway smooth muscle its relaxant action is accompanied by hyperpolarization of the membrane and elevation of the level of intracellular cyclic AMP. Hyperpolarization and relaxation are also induced by drugs such as forskolin, theophylline and dibutyryl cAMP, indicating that cAMP-dependent phosphorylation is involved in producing the electrical response. Cyclic AMP-dependent protein kinase (protein kinase A) has been reported to activate Ca2+-dependent K+ channels in cultured aortic smooth muscle cells and snail neurons. The membrane of tracheal smooth-muscle cells is characterized by a dense distribution of Ca2+-dependent K+-channels. We have now examined the effect of isoprenaline and protein kinase A on Ca2+-dependent K+-channels in isolated smooth muscle cells of rabbit trachea, using the patch-clamp technique. Our results show that the open-state probability of Ca2+-dependent K+-channel of tracheal myocytes is reversibly increased by either extracellular application of isoprenaline or intracellar application of protein kinase A. We also show that this effect is significantly enhanced and prolonged in the presence of a potent protein phosphatase inhibitor, okadaic acid.
An electron microscopic study in the cat has suggested that neurons in the substantia nigra pars reticulata (SNr) project to the medial reticular formation of the medulla oblongata (MRF) via the pedunculopontine tegmental nucleus (PPN): PPN neurons, which were labeled with a retrograde tracer (WGA-HRP; horseradish peroxidase conjugated to wheat germ agglutinin) injected into the MRF, were found to be in synaptic contact with axon terminals which were degenerated with neurotoxic agents applied into the SNr.
The cat substantia nigra (SN) pars reticulata (SNR) has been observed electron microscopically after ibotenic acid injections into the caudate nucleus (Cd) and horseradish peroxidase injections into the nucleus tegmenti pedunculopontinus pars compacta (TPC). As a result, degenerating striatal terminals were found to make symmetric synaptic contacts on retrogradely labeled SNR neurons. The present findings, together with our previous study on the tegmentonigrostriatal projection (Tokuno et al., Neurosci. Lett. 85 (1988) 1-4), confirm that the Cd has indirect reciprocal connections with the TPC via the SN.
The neuronal connections of the anterior pretectal nucleus (PTA) were investigated in the cat. For the light microscopy, the retrograde double-labeling technique by means of Fluoro-Gold (FG) and horseradish peroxidase conjugated to wheat germ agglutinin (WGA-HRP) was used. Following injections of one tracer into the central lateral nucleus of the thalamus (CL) and the other into the dorsal accessory olivary nucleus (DAO), distributions of labeled neurons in the PTA were observed. Most of the labeled neurons were single-labeled either with FG or with WGA-HRP. The result indicated that pretecto-thalamic projection neurons were distributed throughout the whole extent of the PTA, whereas pretecto-olivary projection neurons were located in a restricted area of the ventral part of the PTA. Only a very small number of double-labeled neurons were found in the PTA. These two efferent projections thus seemed to be derived from different populations of PTA neurons. For the electron microscopy, a combination of retrograde transport of horseradish peroxidase (HRP) and anterograde degeneration technique was used. After HRP injections into the CL combined with lesions either in the motor cortex (MCx) or in the anterior interpositus nucleus of the cerebellum (Cbl), some degenerating axon terminals originating from the cerebrum or cerebellum were found to synapse with retrogradely labeled pretecto-thalamic projection neurons. We have already observed direct cerebral and cerebellar projections to the pretecto-olivary projection neurons (J. Comp. Neurol., 259 (1987) 348-363). We conclude that the two different populations of PTA neurons comprise two different kinds of neuronal circuitries, i.e. MCx-PTA-CL-MCx and Cbl-PTA-DAO-Cbl, and that these two circuitries might interrelate with each other in the PTA at the cellular level.
The synaptic organization of the feline pedunculopontine tegmental nucleus (PPN) was studied electron microscopically. The bouton covering ratios were calculated in various sizes of PPN neurons, and the ratios of large neurons (56%) were found to be much higher than those of small neurons (16%). The PPN neuron dendrites usually showed some varicosities, and spines were observed on both somatic and dendritic profiles. Among a total of 1021 synapses sampled at random, axosomatic, axodendritic and axospinous synapses comprised 21.7, 61.2 and 14.1%, respectively. On the basis of the postsynaptic junction, these synapses were classified into the symmetric (66.3%) and the asymmetric (33.7%) types. The percentage of symmetric synapses was much higher on the soma (91.0%), and the large (69.4%) and medium-sized (63.2%) dendrite, while that of asymmetric synapses showed a higher value on the small dendrite (55.5%) and the dendritic spine (50.8%). Axoaxonic, dendrodendritic and dendroaxonic synapses, although not so frequent, were, in part, involved in the serial synapse or the synaptic triad. It is concluded that some PPN neurons are spiny, and that axosomatic, axodendritic and axospinous synapses are the main synaptic constituents and besides those synapses a more complex synaptic organization exists in this nucleus.
Effects of cisapride were studied on the whole segments and muscle strips isolated from the guinea pig ascending colon. In whole segment preparations, external application of cisapride (0.4-10 microM) produced relaxation and inhibited spontaneous and evoked intraluminal pressure changes. Intraluminal application (1-10 microM) increased the pressure response. In many circular muscle strips (18 preparations out of 20), cisapride (0.3-1 microM) increased the mechanical activity, particularly when the pH was lowered, associated with an increased spike activity. However, this action was relatively weak, and in some preparations (2 preparations out of 20), almost no effect was observed. At higher concentrations (more than 1-5 microM), cisapride reduced the muscle tone and nerve-mediated contractions. The cisapride effect could be demonstrated in the presence of atropine (0.5 microM) and serotonin (5 microM). It was concluded that cisapride has direct inhibitory as well as excitatory actions. There was a tendency for the excitatory action to appear at a lower concentration than for the inhibitory action.
Convergence of afferent fibers from the entopeduncular nucleus (EP) and the substantia nigra pars reticulata (SNr) onto single neurons in the ventromedial thalamic nucleus (VM) was shown electron microscopically in the cat. Axon terminals from the EP were anterogradely labeled with wheat germ agglutinin-horseradish peroxidase (WGA-HRP) injected into the EP. Axon terminals from the SNr were degenerated by injecting ibotenic acid into the SNr. Both WGA-HRP-labeled axon terminals and degenerating ones were found on single neuronal profiles in the VM.
Pallidal terminals in the cat lateral habenular nucleus and the ventral anterior and ventral lateral nuclear complex of the thalamus have been examined electron microscopically. A significant difference was obtained after a quantitative analysis of size and shape parameters of synaptic vesicles in the identified terminals: synaptic vesicles in the pallidohabenular terminals had smaller mean elongation index and larger mean vesicle profile area than those in the pallidothalamic terminals.
Direct retinothalamic projections in the mole, an animal with a highly reduced visual system, were studied after an intraocular injection of wheat germ-agglutinated horseradish peroxidase (WGA-HRP). Anterogradely labeled axon terminals were found not only in the dorsal and ventral nuclei of the lateral geniculate body but also in the lateroposterior nucleus on the side contralateral to the intraocular WGA-HRP injection.
A retrograde tracing study in the mole using wheat germ-agglutinated horseradish peroxidase (WGA-HRP) indicated that the medial superior olivary nucleus (MSO) projects to the inferior colliculus (IC) bilaterally. Considering the strict ipsilateral projections from the MSO to the IC in all other eutherian species ever reported, the bilateral projection in the mole is quite unique. This may reflect specializations of the peripheral auditory apparatus of the underground dwellers and/or primitiveness of the insectivorous brains.
Electrolytic lesion was placed in the nucleus tegmenti pedunculopontinus pars compacta (TPC) in cats, which were injected simultaneously with horseradish peroxidase (HRP) into the caudate nucleus (Cd). After a survival period of 4 days, the substantia nigra pars compacta (SNC) was examined electron microscopically. Degenerating axon terminals were found to make asymmetric synaptic contacts on dendrites of SNC neurons which were retrogradely labeled with HRP. Thus, TPC neurons were indicated to project monosynaptically to SNC neurons which sent their axons to the Cd.
The authors have operated on and reviewed 38 patients with Chiari malformation. By the introduction of our new operative method, Gardner's operation combined with expansive suboccipital cranioplasty, the operative results were improved.
Projections from the basal ganglia to the nucleus tegmenti pedunculopontinus pars compacta (TPC) were studied by using anterograde and retrograde tracing techniques with horseradish peroxidase conjugated with wheat germ agglutinin (WGA-HRP) in the cat. Following WGA-HRP injections into the medial TPC area, a substantial number of retrogradely labeled cells were seen in the entopeduncular nucleus (EP) and medial half of the substantia nigra pars reticulata (SNr), whereas following WGA-HRP injections into the lateral TPC area, labeled cells were marked in the caudal half of the globus pallidus (GP) and lateral half of the SNr. To confirm the retrograde tracing study, WGA-HRP was injected into the EP or the caudal GP, and anterograde labeling was observed in the TPC areas. Terminal labeling was located in the medial TPC area in the EP injection case, while terminal labeling was observed in the lateral TPC area in the caudal GP injection case. Projections from the striatum to the pallidal complex (the EP and the caudal GP) were also studied autoradiographically by injecting amino acids into various parts of the caudate nucleus and the putamen. Terminal labeling was distributed over the whole extent of the EP and the rostral GP following injections into the rostral striatum (the head of the caudate nucleus or the rostral part of the putamen), while terminal labeling was distributed over the caudal GP following injections into the caudal striatum (the body of the caudate nucleus or the caudal part of the putamen). From these findings, we conclude that there exists a medio-lateral topography in the projection from the basal ganglia to the TPC: The EP receives afferent projections from the rostral striatum and projects to the medial TPC area, whereas the caudal GP receives projections from the caudal striatum and sends fibers to the lateral TPC area.