Fumi Sato

An immunohistochemical study of Ca2+/calmodulin-dependent protein kinase IV in the rat central nervous system : light and electron microscopic observations

We observed the distribution pattern of Ca2+/calmodulin-dependent protein kinase IV in rat brain and spinal cord using an immunohistochemical method by light and electron microscopy. Particularly strong immunoreactivity was detected in the telencephalic structures such as the olfactory bulb, cerebral cortex, hippocampal formation, caudate-putamen, most nuclei of the dorsal thalamus and the granule cell layer of the cerebellum. Relatively weak staining was observed in the amygdaloid body, some neuron groups of the brainstem reticular formation, the inferior olivary nucleus and the posterior horn of the spinal cord. Immunohistochemical reactivity was not detected in the globus pallidus, substantia nigra, sensory and motor nuclei of the cranial nerves, or in the spinal cord anterior horn. Overall, the distribution of Ca2+/calmodulin-dependent protein kinase IV-like immunoreactivity broadly paralleled the sites of expression of signals for messenger RNA of this enzyme. At the subcellular level, Ca2+/calmodulin-dependent protein kinase IV-like immunoreactivity appeared exclusively in the nuclei of neurons in the various brain regions, and immunopositive reactivity, although less strong, was also observed in dendritic processes, as well as on the granular endoplasmic reticulum in neuronal somata in these areas. Axon terminals, however, did not show immunoreactivity. These studies demonstrate that Ca2+/calmodulin-dependent protein kinase IV-like immunoreactivity is distributed widely in the central nervous system. The significance of the localization of this enzyme in nuclei is discussed in relation to gene expression.

An anatomical study of the human cardiac veins with special reference to the drainage of the great cardiac vein.

Many case reports in which the main aim was to clarify the development of the superior vena cava (SVC) with persistent SVC have so far been published. However, there have only been a few systematic studies of the cardiac veins. Therefore, we macroscopically investigated the cardiac veins using 337 human adult hearts to obtain a detailed understanding of the morphology of the heart. From our study, we obtained the following results. 1. The frequency of persistent left SVC including one case of bilateral SVC was observed to be higher 7 cases (2.1%) than in previous reports. 2. We observed a second case in which the great cardiac vein drained directly into the right SVC (the first case being reported by Bergman et al. 1988). 3. Variations in the drainage of the great cardiac vein (GV) were observed from the right SVC to the left SVC, while the middle cardiac vein (MV) showed less variations of the drainage and course. 4. The drainage boundary between the GV and MV was classified into 3 types: MV-dominant type in 123 cases (36.5%), intermediate type in 182 cases (54.0%), and GV-dominant type in 32 cases (9.5%). From our results, we speculated that the MV did not generate any variations due to its short course before draining into the right atrium, while the GV had many variations due to its long course before draining into the right atrium. From the few GV-dominant types, the halfway boundary between the anterior (GV) and posterior (MV) route for drainage into the right atrium could be considered to be the anterior side rather than the Apex cordis.

Distribution of Ca2+/calmodulin-dependent protein kinase α in the rat central nervous system: an immunohistochemical study

Abstract Ca2+/calmodulin-dependent protein kinase IV (CaM-kinase IV) is activated by Cam-kinase IV kinase. We provided a rabbit antiserum against 20 amino acid residues at the carboxyl-terminal end of CaM-kinase IV kinase, and examined regional and intracellular distribution of CaM-kinase IV kinase immunohistochemically in the central nervous system of the rat by light and electron microscopy. The immunoreactivity was found in cellular nuclei of virtually all neurons. However, the immunoreactivity was weak in the nuclei of the granule cells in the cerebellar cortex, although the nuclei of the granule cells were reported to contain high CaM-kinase IV activity. Thus, it was suggested that other types of CaM-kinase IV kinase might exist in the cerebellum, and the present CaM-kinase IV kinase was named as CaM-kinase kinase α.

Immunohistochemical localization of Ca2+/calmodulin-dependent protein kinase kinase β in the rat central nervous system

We examined regional and intracellular distribution of Ca(2+)/calmodulin-dependent protein kinase kinase beta (CaM-KK beta), which activated Ca(2+)/calmodulin-dependent protein kinase I and IV (CaM-K I and IV) immunohistochemically in the central nervous system of the rat by light and electron microscopy. Although most neurons in the brain and spinal cord exhibited the immunoreactivity, no labeled neurons were observed in the globus pallidus or entopeduncular nucleus, and only a small number of neurons showed weak immunoreactivity in the substantia nigra pars reticulata. In general, the immunoreactivity was observed both in the cytoplasm and cellular nucleus, although the immunoreactivity was not found in the cellular nucleus in some large neurons such as in the mesencephalic trigeminal nucleus, lateral vestibular nucleus or gigant cellular reticular formation. As to motoneurons of the cranial nerve nuclei and the anterior horn of the spinal cord, they revealed the immunoreactivity both in the cytoplasm and nucleus. The reaction product appeared as fine granules in the cytoplasm and nucleus under light microscopy. Electron microscopic observations confirmed that the reaction product was localized mainly on the Golgi apparatus or on the nuclear chromatin. Immunolabeling for antibody against CaM-KK beta was discussed with the distribution of CaM-K I, IV and another CaM-KK, CaM-KK alpha, in the central nervous system.

Serial electron microscopic reconstruction of axon terminals on physiologically identified thalamocortical neurons in the cat ventral lateral nucleus

The distribution of different types of terminals on different portions of single thalamocortical neurons (TCNs) was quantitatively investigated in the cat ventral lateral nucleus (VL) by the application of computer‐assisted three‐dimensional reconstruction from serial ultrathin sections. Single neurons in the VL were intracellularly penetrated with a glass micropipette filled with horseradish peroxidase (HRP), and were electrophysiologically identified as TCNs by their antidromic responses to stimulation of the motor cortex. These TCNs received monosynaptic excitation from the contralateral cerebellum. After electrophysiological identification, they were injected with HRP iontophoretically. The spatial distribution of terminals of different types on two identified TCNs was analyzed on serial ultrathin sections, some of which were stained by a postembedding immunogold technique by using a γ‐aminobutyric acid (GABA) antibody. Terminals that synapsed on the injected cells were categorized as LR terminals (GABA‐negative large axon terminals containing round vesicles), SR terminals (GABA‐negative small axon terminals containing round vesicles), P terminals (GABA‐positive axon terminals of various sizes containing pleomorphic vesicles), or PSDs (presynaptic dendrites). The order of dendritic branches of labeled TCNs was determined by computer‐assisted reconstruction from serial sections. LR terminals made contacts mainly with proximal dendrites of TCNs. SR terminals made contacts predominantly with distal dendrites, and were never found on somata or primary dendrites. P terminals were observed on somata and on every portion of the dendritic trees. Synapses formed by PSDs were concentrated on the proximal dendrites and sometimes formed synaptic triads with LR terminals. Only a few terminals were found on somata, all of which were P type. Therefore, terminals belonging to different classes were not uniformly distributed on the somata and dendrites of single TCNs. These results suggest that terminals originating from different sources may preferentially contact specific regions of TCNs in the VL, and their topographical locations reflect the electrophysiological response properties of the TCNs. J. Comp. Neurol. 388:613–631, 1997.

Immunohistochemical study of the distribution of Ca2+/calmodulin-dependent protein kinase phosphatase in the rat central nervous system

Abstract Distribution of Ca2+/calmodulin-dependent protein kinase phosphatase (CaM-K Pase) which dephosphorylate multifunctional Ca2+/calmodulin-dependent protein kinases (CaM-kinases) in the rat brain and spinal cord were examined immunohistochemically by using an antibody against this enzyme. CaM-K Pase was localized only in the cytoplasm as has been investigated in PC 12 cells, and was never observed in the nucleus. Immunostainability varied from cell group to cell group. Mitral cells in the olfactory bulb, pyramidal neurons in the fifth layer of the cerebral cortex, hippocampal and striatal interneurons, dorsal and ventral pallidal, entopeduncular, and the reticular part of the substantia nigra neurons were intensely immunolabeled. Motoneurons in all the cranial nerve nuclei and the anterior horn of the spinal cord also revealed intense immunolabeling. On the contrary, pyramidal neurons in the Ammon’s horn of the hippocampal formation, granule cells in the olfactory bulb, dentate gyrus and cerebellar cortex, Purkinje cells, neurons in the medial habenular nucleus and the inferior olivary nucleus have not shown immunoreactivity. Axons in the white matter or nerve root of the cranial nerve nuclei were immunolabeled. Glial cells in the white matter also showed immunostaining. Because the substrate of CaM-K Pase is multifunctional CaM-kinase II, I and IV, localization of each CaM-kinase was compared with that of CaM-K Pase. The distribution of CaM-K Pase and these CaM-kinases was found to overlap in various regions in the brain and spinal cord. It was concluded, therefore, that CaM-K Pase could regulate the activity of these CaM-kinases by dephosphorylation, when they existed together in neurons.

Morphological analysis of single astrocytes of the adult cat central nervous system visualized by HRP microinjection

The morphology of individual astrocytes of the adult cat was analyzed by HRP microinjection and light microscopy. The astrocytes had generally two types of processes: (1) thread-like processes of relatively constant width with few ramifications and few lamellar appendages and (2) the sinuous processes with clusters of lamellar appendages. The former processes were morphologically characterized as follows: (1) Those of fibrous astrocytes were frequently remarkably long, ranging from 600 to 1500 microns. They were much longer than any astrocytic processes hitherto reported in the literature. In contrast, those of protoplasmic astrocytes were usually short (30-400 microns), and were sometimes decorated with lamellae. (2) The processes often terminated in endfeet on the subpial surface of the brain tissue or on blood vessel walls. The number of endfeet per cell varied from 1-11 with a tendency to split into two populations close to each extreme number. Another type of endfoot was also found, i.e. swellings along the trunk of the processes which made side-to-side contact with the vessel wall. The sinuous processes rich in lamellae were predominant in protoplasmic astrocytes, and clearly corresponded to the sheet- or veil-like processes of Golgi-impregnated astrocytes. They formed an ellipsoidal field (100 microns for the longer, and 50-60 microns for the shorter, diameter) around the nucleus.

Electron microscopic comparison of the terminals of two electrophysiologically distinct types of primary vestibular afferent fibers in the cat.

Two types of electrophysiologically distinct vestibular primary afferents (regular-type and irregular-type) were injected intraaxonally with HRP and their terminals were compared using the electron microscope. In general, regular-type terminals were small and two or more terminals contacted a single dendrite side by side, whereas irregular-type terminals were large and covered a wide surface of the dendrites. Both types of terminals contained spherical clear vesicles with almost the same diameter. Prominent postsynaptic membrane specializations were observed in both types. Mitochondria in both types had an elliptical profile with the same elongation index, but mitochondria in the regular-type terminals were thicker than those in the irregular-type ones.

Three-dimensional analysis of cerebellar terminals and their postsynaptic components in the ventral lateral nucleus of the cat thalamus

Relationships among cerebellar terminals (CTs), dendrites of thalamocortical projection neurons (TCNs), and dendrites of local circuit neurons in the ventral lateral nucleus of the cat thalamus were analyzed quantitatively by observing several series of serial ultrathin sections and by using a computer‐assisted program for the three‐dimensional reconstruction from serial ultrathin sections. In pentobarbital‐anesthetized cats, CTs were labeled either by injections of wheat germ agglutinin conjugated to horseradish peroxidase (WGA‐HRP) into the cerebellar nuclei or by intra‐axonal injection of HRP after electrophysiological identification. By using two series of 133 and 73 serial sections, mutual relationships between 43 WGA‐HRP‐labeled CTs and their postsynaptic structures were analyzed based on their synaptic specializations and shapes of synaptic vesicles. Thirty‐nine of these CTs formed a synapse with one TCN dendrite, whereas only four CTs formed synapses with two TCN dendrites. These CTs also synapsed on dendrites containing pleomorphic synaptic vesicles (presynaptic dendrites). Single CTs synapsed on 0–6 presynaptic dendrites (2.2 ± 1.5, N = 43) through their whole extents, and about 40% of these presynaptic dendrites that were contacted by CTs established synaptic contacts with the same TCN dendrites on which the CTs synapsed. Thus, a CT, a presynaptic dendrite, and a TCN dendrite formed a triadic arrangement. Triadic arrangements were identified in approximately 60% of these 43 CTs. However, they rarely had a glomerulus‐like appearance, as described previously in the ventral lateral nucleus and other main thalamic relay nuclei. In another series of 83 and 43 serial sections along dendrites of TCNs, observations were focused on the triadic arrangement. Triadic arrangements were located evenly on the primary and secondary dendrites of TCNs. Computer‐assisted three‐dimensional reconstructions were made on one WGA‐HRP‐labeled CT and two intra‐axonally labeled CTs (a bouton en passant and a bouton terminal) with their surrounding neuronal elements, and complex spatial arrangement of neuronal processes became obvious. These results provide the quantitative assessment of synaptic arrangements among CTs, presynaptic dendrites, and TCN dendrites and reveal their spatial interrelations in the cat ventral lateral nucleus.

Single-axon tracing study of neurons of the external segment of the globus pallidus in primate Dr. Fumi Sato was on leave of absence from the Department of Anatomy, School of Medicine, Tokyo Medical and Dental University, Tokyo, Japan.

Axonal projections arising from the external segment of the globus pallidus (GPe) in cynomolgus monkeys (Macaca fascicularis) were mapped after labeling small pools (5-15 cells) of neurons with biotinylated dextran amine. Seventy-six single axons were reconstructed from serial sagittal sections with a camera lucida. The majority of labeled GPe cells displayed long, aspiny, and poorly branched dendrites that arborized mostly along the sagittal plane, whereas others showed dendrites radiating in all directions. Numerous GPe axons emitted short, intranuclear collaterals that arborized close to their parent cell body. Based on their axonal targets, four distinct types of GPe projection neurons have been identified: 1) neurons that project to the internal segment of the globus pallidus (GPi), the subthalamic nucleus (STN), and the substantia nigra, pars reticulata (SNr; 13.2%); 2) neurons that target the GPi and the STN (18.4%); 3) neurons that project to the STN and the SNr (52.6%); and 4) neurons that target the striatum (15.8%). Labeled GPe axons displayed large varicosities that often were closely apposed to the somata and proximal dendrites of STN, GPi, and SNr neurons. At striatal levels, however, GPe axons displayed small axonal varicosities that did not form perineuronal nets. These results suggest that the GPe is an important integrative locus in primate basal ganglia. This nucleus harbors several subtypes of projection neurons that are endowed with a highly patterned set of collaterals. This organization allows single GPe neurons to exert a multifarious effect not only on the STN, which is the claimed GPe target, but also on the two major output structures of the basal ganglia, the SNr and the GPi.