A model of plasma current through a hole of Rogowski probe including sheath effects.

In TST-2 Ohmic discharges, local current is measured using a Rogowski probe by changing the angle between the local magnetic field and the direction of the hole of the Rogowski probe. The angular dependence shows a peak when the direction of the hole is almost parallel to the local magnetic field. The obtained width of the peak was broader than that of the theoretical curve expected from the probe geometry. In order to explain this disagreement, we consider the effect of sheath in the vicinity of the Rogowski probe. A sheath model was constructed and electron orbits were numerically calculated. From the calculation, it was found that the electron orbit is affected by E × B drift due to the sheath electric field. Such orbit causes the broadening of the peak in the angular dependence and the dependence agrees with the experimental results. The dependence of the broadening on various plasma parameters was studied numerically and explained qualitatively by a simplified analytical model.

Colocalization of prostaglandin F(2alpha) receptor FP and prostaglandin F synthase-I in the spinal cord.

Prostaglandin F(2alpha) is synthesized by prostaglandin F synthase, which exists in two types, prostaglandin F synthase I (PGFS I) and prostaglandin F synthase II (PGFS II). Prostaglandin F(2alpha) binds to its specific receptor, FP. Our previous immunohistochemical study showed the distinct localization of prostaglandin F synthases in rat spinal cord. PGFS I exists in neuronal somata and dendrites in the gray substance, and PGFS II exists in ependymal cells and tanycytes surrounding the central canal. Both enzymes are also present in endothelial cells of blood vessels in the white and gray substances of the spinal cord. In this study, we found that FP localizes in neuronal somata and dendrites but not in ependymal cells, tanycytes, or endothelial cells. Immunohistochemical analysis of serial sections showed the colocalization of FP and PGFS I. FP immunoreactivity was intense in spinal laminae I and II of the dorsal horn, a connection site of pain transmission, and was similar to that of PGFS I in neuronal elements. These findings suggest that prostaglandin F(2alpha) synthesized in the neuronal somata and dendrites exert an autocrine action there.

Vesicular acetylcholine transporter-immunoreactive axon terminals enriched in the pontine nuclei of the mouse.

Information to the cerebellum enters via many afferent sources collectively known as precerebellar nuclei. We investigated the distribution of cholinergic terminal-like structures in the mouse precerebellar nuclei by immunohistochemistry for vesicular acetylcholine transporter (VAChT). VAChT is involved in acetylcholine transport into synaptic vesicles and is regarded as a reliable marker for cholinergic terminals and preterminal axons. In adult male mice, brains were perfusion-fixed. Polyclonal antibodies for VAChT, immunoglobulin G-peroxidase and diaminobenzidine were used for immunostaining. In the mouse brain, immunoreactivity was seen in almost all major cholinergic cell groups including brainstem motoneurons. In precerebellar nuclei, the signal could be detected as diffusely beaded terminal-like structures. It was seen heaviest in the pontine nuclei and moderate in the pontine reticulotegmental nucleus; however, it was seen less in the medial solitary nucleus, red nucleus, lateral reticular nucleus, inferior olivary nucleus, external cuneate nucleus and vestibular nuclear complex. In particular, VAChT-immunoreactive varicose fibers were so dense in the pontine nuclei that detailed distribution was studied using three-dimensional reconstruction of the pontine nuclei. VAChT-like immunoreactivity clustered predominantly in the medial and ventral regions suggesting a unique regional difference of the cholinergic input. Electron microscopic observation in the pontine nuclei disclosed ultrastructural features of VAChT-immunoreactive varicosities. The labeled bouton makes a symmetrical synapse with unlabeled dendrites and contains pleomorphic synaptic vesicles. To clarify the neurons of origin of VAChT-immunoreactive terminals, VAChT immunostaining combined with wheat germ agglutinin-conjugated horseradish peroxidase retrograde labeling was conducted by injecting a retrograde tracer into the right pontine nuclei. Double-labeled neurons were seen bilaterally in the laterodorsal tegmental nucleus and pedunculopontine tegmental nucleus. It is assumed that mesopontine cholinergic neurons negatively regulate neocortico-ponto-cerebellar projections at the level of pontine nuclei.

Immunocytochemical localization of prostaglandin F synthase II in the rat spinal cord.

Prostaglandin F synthase has at least two isozymes, i.e. prostaglandin F synthase I and II. Recently, we demonstrated immunocytochemically that prostaglandin F synthase I was localized in neuronal dendrites and somata, and in endothelial cells of blood vessels in the whole area of rat spinal cord. In the present study, we immunocytochemically localized prostaglandin F synthase II in ependymal cells and tanycytes surrounding the central canal and in endothelial cells of blood vessels, but not in any neuronal elements at all segmental levels of the rat spinal cord. Immunoelectron microscopy and confocal laser scanning microscopy confirmed these findings and further revealed that strong immunoreactivity was found in the basal processes of the tanycytes. Our present and recent studies using antibodies against the two isozymes of prostaglandin F synthase clearly indicated that they were localized differentially in ependymal (prostaglandin F synthase II) and neuronal elements (prostaglandin F synthase I), but were co-localized in blood vessels in the rat spinal cord. The distinct localization of the two isozymes suggests that prostaglandin F(2) has different transcellular biological actions via different cell groups.

Structure of intraglomerular dendritic tufts of mitral cells and their contacts with olfactory nerve terminals and calbindin-immunoreactive type 2 periglomerular neurons.

Intraglomerular dendritic tufts of Golgi-impregnated and biotinylated dextran amine (BDA)-labeled mitral cells in the rat main olfactory bulb were analyzed in detail. In particular, the relationships of BDA-labeled tufts with olfactory nerve (ON) terminals and processes of calbindin D-28K-immunoreactive (CB-IR) cells were investigated with confocal laser-scanning light microscopic (CLSM) and electron microscopic (EM) analyses. CB-IR cells were type 2 periglomerular cells that restricted their processes in the ON-free (non-ON) zone of the glomerulus and received few synapses from ON terminals. The mitral tufts varied in complexity, but individual branches were rather simple, smooth processes that bore some branchlets and spines and extended more or less in a straight line or a gentle curve rather than winding tortuously within glomeruli as though they did not consider the compartmental organization, which consisted of ON and non-ON zones that interdigitated in a complex manner with one another. Conventional EM analysis revealed that both thin and thick, presumed proximal branches of mitral/tufted cell dendritic tufts received asymmetrical synapses from ON terminals. Correlated CLSM-EM analysis confirmed direct contacts between the BDA- and CB-labeled processes detected in the CLSM examinations, and synapses were recognized at some of those sites. Furthermore, ON terminals and CB-IR processes were distributed on both proximal and distal dendritic branches in a more or less mosaic pattern. These findings revealed that, on the mitral dendritic tufts, ON terminals and processes of type 2 periglomerular neurons were not clearly segregated proximodistally but, rather, were arranged in a mosaic pattern, which may be important in fine tuning the output from individual glomeruli.

Chemically defined neuron groups and their subpopulations in the glomerular layer of the rat main olfactory bulb--IV. Intraglomerular synapses of tyrosine hydroxylase-immunoreactive neurons.

Synapses of intraglomerular processes of tyrosine hydroxylase-immunoreactive neurons in the rat main olfactory bulb were examined by electron microscopic immunocytochemistry. Prominent characteristics of intraglomerular synapses of tyrosine hydroxylase-immunoreactive elements were that the vast majority (about 80%) of their synaptic inputs were asymmetrical synapses from olfactory nerve terminals and, though far smaller in proportion, one half of the remaining were asymmetrical synapses from mitral/tufted cell dendrites and the other half were symmetrical synapses from gamma-aminobutyric acid-like immunoreactive elements. So far, we have observed no typical reciprocal synapses between tyrosine hydroxylase-immunoreactive processes and mitral/tufted dendrites; however, we have often identified serial synapses; that is, asymmetrical synapses from olfactory nerve terminals or mitral/tufted cell dendrites to tyrosine hydroxylase-immunoreactive processes, and then symmetrical synapses from the latter to different mitral/tufted cell dendrites. These synaptic connections of tyrosine hydroxylase-immunoreactive neurons were very different from those of Calbindin-D(28k)-immunoreactive neurons, which received no synaptic contact directly from olfactory nerve terminals but formed reciprocal synapses with mitral/tufted cells as we analysed previously.Thus, our present and previous electron microscopic studies combined with confocal laser scanning light microscopy clearly indicated for the first time the heterogeneity of periglomerular neurons, not only in their chemical and morphological features, but also in their synaptic organization in the olfactory glomerulus.

Immunocytochemical localization of lung-type prostaglandin F synthase in the rat spinal cord.

Prostaglandin F synthase, producing prostaglandin F(2 alpha) and 9 alpha,11 beta-prostaglandin F(2), has at least two isozymes, lung-type and liver-type ones. The present study including double immunolabelling with microtubule-associated protein 2 indicated that the lung-type isozyme was present in neuronal dendrites and somata of gray matter (relatively intense in lamina I and II in dorsal horn, and IX in ventral horn) and vascular endothelial cells in the rat spinal cord at all segmental levels.

How simple is the organization of the olfactory glomerulus?: the heterogeneity of so-called periglomerular cells.

Recent progress in the studies of the olfactory system, especially in the molecular biological studies, makes it one of the useful sensory model systems for understanding neural mechanisms for the information processing. In the olfactory bulb, the primary center of the olfactory system, glomeruli are regarded as important functional units in the transmission of odorant signals and in processing the olfactory information, but have been believed to be composed by only a small number of neuronal types and thus to be simple in their neuronal and synaptic organization. However, accumulating morphological data reveal that each type of neurons might further consist of several different subpopulations, indicating that the organization of glomeruli might not be so simple as it was believed. Here we describe an aspect of the structural organization of glomeruli, focusing on the heterogeneities of periglomerular neurons in mammalian main olfactory bulb.

Chemically defined neuron groups and their subpopulations in the glomerular layer of the rat main olfactory bulb: III. Structural features of calbindin D28K-immunoreactive neurons.

The present study analyzed three-dimensional structural features and synaptic contacts of morphologically and chemically identified calbindin D28K-immunoreactive neurons in the glomerular layer of the rat main olfactory bulb by means of combined confocal laser scanning light microscopy, high-voltage electron microscopy and electron microscopic serial section/three-dimensional reconstruction. Most of calbindin D28K-immunoreactive neurons were identified as the periglomerular cell type by combined high-voltage electron microscopic and confocal laser scanning light microscopic observations, and the minority were the short-axon cell type and others. The combined confocal laser scanning light microscopic and electron microscopic study revealed that the calbindin D28K-immunoreactive neurons exhibited unique synaptic contact patterns; they received asymmetrical synapses from presumed mitral/tufted dendrites and made conversely symmetrical synapses with them. About 30% of asymmetrical postsynaptic sites and about 40% of symmetrical presynaptic sites formed reciprocal pairs of synapses. Calbindin D28K-immunoreactive dendrites and somata also received synapses from GABA-like-immunoreactive profiles containing numerous pleomorphic, and a few dense-cored, vesicles. On the other hand, surprisingly, calbindin D28K-immunoreactive neurons had almost no synaptic contacts from olfactory nerve terminals. The present study clearly revealed that calbindin D28K-immunoreactive neurons are a type of periglomerular cell involving unique synaptic contacts that have not been reported so far, and thus indicated that so-called periglomerular cells should be heterogeneous in their synaptic connections as well as in their chemical and structural features.

Differentiation of chemically defined neuronal populations in the transplanted olfactory bulb without olfactory receptor innervation.

Olfactory bulbs (OBs) from embryonic day 15 and 17 and postnatal day 1 mice were transplanted into the lateral ventricle of juvenile host mice without bulbectomy, and fine structural and chemical features of neurons and glia in the OB transplants were investigated immunocytochemically and electron microscopically. In the OB transplants there were neither clearly defined glomeruli nor layers, nor olfactory marker protein immunoreactive elements. However, chemically defined neuronal populations resembling those in the normal OBs such as those immunoreactive for gamma-aminobutyric acid (GABA), tyrosine hydroxylase and Ca(2+)-binding proteins (calbindin-D28K, calretinin, parvalbumin) were observed. Electron microscopically, dendrodendritic and somatodendritic reciprocal synapses, that is, synapses characteristic of the OB, were occasionally observed in the OB transplants. These results indicated that at least some embryonic or newborn mouse OB neurons and/or precursor cells could exhibit chemical properties and form typical synaptic contacts observed in normal OB, even when they received no inputs from olfactory receptor cells.

Chemically defined neuron groups and their subpopulations in the glomerular layer of the rat main olfactory bulb--II. Prominent differences in the intraglomerular dendritic arborization and their relationship to olfactory nerve terminals.

In the glomerular layer of the rat main olfactory bulb, we previously reported three chemically defined interneuron groups: GABA-like immunoreactive, calretinin-immunoreactive and Calbindin-D28k-immunoreactive groups [Kosaka K. et al. (1995) Neurosci. Res. 23, 73-88]. In the present study, we analysed the structural features of these three neuron groups using confocal laser scanning light microscopy, focusing on their dendritic arborization pattern, especially on their close apposition to olfactory receptor terminals labeled by olfactory marker protein. Each glomerulus consisted of two zones, the olfactory nerve zone and the non-olfactory nerve zone. The former was mainly occupied by olfactory nerve preterminals and terminals as well as their targets, postsynaptic fine dendritic portions of intrinsic neurons. The latter non-olfactory nerve zone was occupied mainly by olfactory marker protein-negative profiles. Processes of GABAergic neurons and those of one of their subpopulations, tyrosine hydroxylase-immunoreactive neurons, were numerous both in the olfactory nerve and non-olfactory nerve zones, resulting in their frequent close apposition to olfactory marker protein-immunoreactive elements. Combined confocal laser scanning light microscopic electron microscopic examination revealed synaptic contacts from olfactory nerve terminals on tyrosine hydroxylase-immunoreactive processes at these sites of close apposition. In contrast, calretinin-immunoreactive and Calbindin-D28k-immunoreactive processes, particularly Calbindin-D28k-immunoreactive ones, were distributed almost exclusively in the non-olfactory nerve zone, as if they avoided the olfactory nerve zone, showing a net or honeycomb pattern. Thus, calretinin-immunoreactive and Calbindin-D28k-immunoreactive processes were not or very rarely closely apposed to olfactory nerve terminals. These findings suggested that there might be some differences among chemically defined interneuronal groups in their synaptic contacts from olfactory nerves. Further quantitative image analysis clearly exhibited the prominent differences among these neuron groups in their intraglomerular dendritic arborization in relation with the olfactory nerve zone, i.e. the percentages of the area in the olfactory nerve zone occupied by GABAergic and tyrosine hydroxylase-immunoreactive processes were about 10%, respectively, whereas those of calretinin-immunoreactive and Calbindin-D28k-immunoreactive processes were only about 1% and 0.3%, respectively. These findings suggested that so-called periglomerular cells in glomeruli might be heterogeneous not only in their chemical nature, but also in their dendritic arborization pattern and synaptic contacts from olfactory nerve terminals.

Electron microscopic serial-sectioning/reconstruction study of parvalbumin-containing neurons in the external plexiform layer of the rat olfactory bulb.

Neurons containing a calcium-binding protein parvalbumin in the external plexiform layer of the rat olfactory bulb were identified light microscopically with the pre-embedding immunocytochemistry and were subsequently analysed with the electron microscopic serial-sectioning and three-dimensional reconstructions. In the present study we chose several different types of parvalbumin-immunoreactive neurons identified light microscopically as Van Gehuchten cell type, superficial short-axon cell type and multipolar cell type. Parvalbumin-immunoreactive somata were similar to one another in their ultrastructural characteristics, showing nuclear indentations, moderately developed Golgi apparatus and abundant mitochondria; these structural features appeared to resemble those of the short axon cells around the glomeruli and in the granule cell layer reported in previous electron microscopic studies. All neurons analysed in the present study made symmetrical synapses on to dendrites and somata of presumed mitral/tufted cells and received asymmetrical synapses from them, and occasionally formed reciprocal synapses with them. On the parvalbumin-immunoreactive processes, the asymmetrical synapses nearly equalled the symmetrical ones in number and about 30-50% of them were identified as reciprocal pairs. In contrast, no presynaptic sites were observed on parvalbumin-immunoreactive somata, and thick portions (more than approximately 2 microns in diameter) of the proximal dendrites, where they were occasionally postsynaptic in some asymmetrical and symmetrical synapses from parvalbumin-immunonegative profiles. Characteristically, parvalbumin-immunoreactive process frequently make direct contacts with one another; processes regarded light microscopically as arising from a soma or a dendrite or parvalbumin-immunoreactive neurons were sometimes revealed to be separate but directly contacting processes with electron microscopic examinations. Although puncta adherentia were occasionally observed between these contact sites, so far neither gap junctions nor chemical synapses were observed. Until now, it has been believed that in the external plexiform layer only granule cells form reciprocal synapses with mitral/tufted cells. However, the present study clearly demonstrates that interneurons different from granule cells, namely GABAergic neurons containing a calcium-binding protein parvalbumin, also make reciprocal synapses with mitral/tufted cells in the external plexiform layer. Therefore, neuronal processes making reciprocal synapses with mitral/tufted cells in the external plexiform layer cannot be determined a priori as granule cell processes.

Chemically defined neuron groups and their subpopulations in the glomerular layer of the rat main olfactory bulb.

Chemically-defined neuron groups and their subpopulations in the glomerular layer of the rat main olfactory bulb were revealed immunocytochemically using antibodies against gamma-amino butyric acid (GABA), tyrosine hydroxylase (TH), methionin-enkephalin-Arg6-Gly7-Leu8 (ENK), calretinin (CR), calbindin-D28K (calbindin) and thyrotropin-releasing hormone (TRH). GABA-like immunoreactive (GABA-LIR) neurons and CR immunoreactive (CR-IR) neurons were most numerous; they were about 1.5-3 times more numerous than calbindin immunoreactive (calbindin-IR), TH immunoreactive (TH-IR), ENK-like immunoreactive (ENK-LIR) and THR-like immunoreactive (TRH-LIR) neurons. We identified at least three distinct chemically-defined neuron groups, GABA-LIR neurons, CR containing neurons and calbindin containing neurons, since these three neuron groups were almost separate from one another. On the other hand, TH-IR and ENK-LIR neurons were nearly included in and thus considered to be subpopulations of GABA-LIR and CR-IR neurons, respectively, for about 80% of these two neuron groups contained GABA-L and CR immunoreactivities, respectively. TRH-LIR neurons appeared to be divided into two subpopulations, one containing the GABA-L immunoreactivity and the other containing the CR immunoreactivity. Thus in the glomerular layer of the rat olfactory bulb, GABA-LIR, CR-IR and calbindin-IR cells could be considered to be three distinct chemically-defined neuron groups, whereas TH-IR, TRH-LIR and ENK-LIR neurons were regarded as their subpopulations. Furthermore, some neurons groups, whereas TH-IR, TRH-LIR and ENK-LIR neurons were regarded as their subpopulations. Furthermore, some neurons are supposed to contain three substances (e.g. GABA + TH + TRH, GABA + TRH + EnK, CR + TRH + ENK, GABA + TRH + CR) or a few might even contain four substances (e.g. GABA + TRH + CR + ENK). Preliminary quantitative analysis using the optical disector method showed percentages of these three main neuron groups to total cells in the glomerular layer; that is, neuron groups containing GABA, CR and calbindin were about 20%, 20% and 10%, respectively.

Synaptic contacts between mitral/tufted cells and GABAergic neurons containing calcium-binding protein parvalbumin in the rat olfactory bulb, with special reference to reciprocal synapses between them.

It has been believed so far that in the mammalian olfactory bulb, only granule cells form reciprocal synapses with mitral/tufted cells in the external plexiform layer (EPL). However, the present electron microscopic study demonstrated for the first time that another group of immunocytochemically defined interneurons different from granule cells, viz., neurons containing a specific calcium-binding protein parvalbumin, also made reciprocal synapses with mitral/tufted cells in the EPL.

Observation of Merkel cells with scanning electron microscopy.

This paper first elucidated the overall morphology of Merkel cells in the rat touch dome with scanning electron microscopy (SEM). Quinacrine-fluorescent Merkel cells in the touch dome were exposed by enzymatic treatment following application of dithiothreitol, photographed and then fixed. By referring to the photograph, the same fluorescent cells were easily identified under the SEM. Enzymatically isolated Merkel cells were also examined with SEM. Unlike quinacrine negative, ordinary epidermal cells, the Merkel cells had numerous finger-like processes, ranging from 0.1 to 0.25 micron in diameter and attaining to 2.5 microns in length.

The tunica vasculosa lentis; an expedient system for studying vascular formation and regression.

We examined, with scanning electron microscopy, the tunica vasculosa lentis of the postnatal rat. It consisted of an extensive two-dimensional capillary network, spreading over the posterior and lateral surfaces of the lens. During the postnatal days, capillaries were associated with incipient pericytes and frequently had sprouts with a lamellipodial extension. Stellate angioblastic cells occurred in the meshes of the vasculature, which attached to the pre-existing capillaries with their processes. Both capillary sprouts and angioblastic cells may be involved in vasculogenesis. At the eighteenth postnatal day, the tunica underwent remarkable regressive changes: many capillaries degenerated or were totally eliminated, leaving faint traces on the lens surface. What appeared to be macrophages adhered to degenerating capillaries. With its rapid developmental sequence reported previously and the findings obtained herein, the vasculature provides an expedient system to study the cytological mechanism involved in vascular formation and regression.