Neurokinin-1 receptor immunoreactive neuronal elements in the superficial dorsal horn of the chicken spinal cord: with special reference to their relationship with the tachykinin-containing central axon terminals in synaptic glomeruli.

Synaptic glomeruli that involve tachykinin-containing primary afferent central terminals are numerous in lamina II of the chicken spinal cord. Therefore, a certain amount of noxious information is likely to be modulated in these structures in chickens. In this study, we used immunohistochemistry with confocal and electron microscopy to investigate whether neurokinin-1 receptor (NK-1R)-expressing neuronal elements are in contact with the central primary afferent terminals in synaptic glomeruli of the chicken spinal cord. We also investigated which neuronal elements (axon terminals, dendrites, cell bodies) and which neurons in the spinal cord possess NK-1R, and are possibly influenced by tachykinin in the glomeruli. By confocal microscopy, NK-1R immunoreactivities were seen in a variety of neuronal cell bodies, their dendrites and smaller fibers of unknown origin. Some of the NK-1R immunoreactive profiles also expressed GABA immunoreactivities. A close association was observed between the NK-1R-immunoreactive neurons and tachykinin-immunoreactive axonal varicosities. By electron microscopy, NK-1R immunoreactivity was seen in cell bodies, conventional dendrites and vesicle-containing dendrites in laminae I and II. Among these elements, dendrites and vesicle-containing dendrites made contact with tachykinin-containing central terminals in the synaptic glomeruli. These results indicate that tachykinin-containing central terminals in the chicken spinal cord can modulate second-order neuronal elements in the synaptic glomeruli.

Immunohistochemical demonstration of the calcium channel alpha2 subunit in the chicken dorsal root ganglion and spinal cord: a special reference to colocalization with calbindin-D28k in dorsal root ganglion neurons.

We used immunohistochemical methods to examine the distribution of the calcium channel alpha2 (CCalpha2) subunit in the chicken spinal cord and dorsal root ganglion (DRG) neurons and determine its relationship with calbindin-D28k (CB) in the DRG neurons. In the spinal cord, CCalpha2 subunit was detected in nerve terminals, which were observed as dot-like structures, and in laminae I, II, III and Lissauer's tract in the dorsal horn. In the DRG neurons, approximately 65% of the total neurons were CCalpha2 subunit positive, and most (86%) of these neurons were small to medium sized, suggesting that the CCalpha2 subunit and/or a complex of the CCalpha2 and delta subunits is possibly localized in a number of nociceptive neurons. A majority (77%) of the positive neurons showed CB immunoreactivity and most (88%) of these neurons were small to medium sized. This may indicate a close correlation between the CCalpha2 subunit and CB in the nociceptive neurons. Thus, it is postulated that the mode of nociceptive transmission may involve a cellular Ca(2+)-regulating system that consists of both Ca(2+) entry via calcium channels with the alpha2delta subunit and intracellular Ca(2+)-binding activity of CB in the nociceptive neurons of the DRG.

Mast cells appearing in long-term skeletal muscle cell cultures of rat.

Mast cells are known to be involved in type I allergy and to be localized in almost all tissues in the body. However, they have slightly different properties depending on their tissue of residence. Although mast cells are found in skeletal muscle tissue, there have been no reports of their appearance in cultured skeletal muscles. We report here that mast cells appear in long-term cultures of skeletal muscles from neonatal rats and rat fetuses. When muscle cells were disseminated and cultured in minimum essential medium with 10% fetal calf serum and 10% horse serum, oval cells containing large granules started to appear on myotube sheets at 5 days of culture. These oval cells continued to proliferate for 2-3 months, and showed immunoreactivity for histamine, tryptase, Fc(epsilon)RI, and c-kit. They showed metachromatic staining with 0.5% toluidine blue at pH 0.5 and were stained with both Alcian blue and safranin. Biochemically measured histamine content per dish was significantly higher in 2-month than in 5-day culture. From these results, we concluded that these oval cells were mast cells. Because proteases from mast cells have been reported previously to affect myoblast proliferation, the present findings suggest that there may be some interaction between mast cells and muscle cell proliferation or differentiation. The present finding that mast cells are easily obtained from ordinary skeletal muscle cultures provides a useful method for the study of the diverse functions of mast cells.

Cyclo-oxygenase-2-immunoreactive neurons in the lumbar dorsal horn in a chicken acute inflammation model.

Acute and chronic peripheral inflammation is known to induce the expression of cyclo-oxygenase (COX)-2 in spinal cord neurons and increase the synthesis and release of prostaglandins (PG). Although these PG are presumed to cause inflammatory pain or hyperalgesia, the relationship between PG-producing cells in the dorsal horn and substance P (SP)-containing, pain-transmittimg nerve fibers remains unknown. In the present study we investigated immunohistochemically changes in the number of COX-2-containing neurons using the avidin-biotinylated peroxidase complex method in dorsal horn superficial laminae in chicken lumbosacral enlargement (L4, L5) under inflammatory conditions induced by unilateral intraplantar injection of complete Freund's adjuvant. After 12-24 h, a significant increase in the number of small COX-2-containing neurons was observed in lamina II on the injected side compared with the contralateral side. Furthermore, using fluorescent double-labeling for COX-2 and SP, an increase in the number of small COX-2-containing neurons in contact with SP-containing elements was observed ipsilaterally (1.4-1.6-fold compared with the contralateral side) in lamina II. Fluorescence triple-labeling of COX-2, SP and calcitonin gene-related peptide (CGRP) confirmed that the majority of these SP-containing elements coexisted with CGRP, indicating that these elements originated from primary afferent neurons. Using electron microscopy, two types of SP-containing axon terminals were found to form synapses with COX-2-containing neurons in lamina II. These results indicate that the number of COX-2-containing neurons increases concomitantly with an increase in the number of contacts of these neurons with SP-containing primary afferent fibers and suggest that this phenomenon is associated with PG production and the persistence of inflammatory pain.

Subsurface cisterna-lined axonal invaginations and double-walled vesicles at the axonal-myelin sheath interface.

The axonal-myelin sheath interface of vertebrate myelinated axons possesses special structural complexities, and there may be an intercellular macromolecular traffic transversing the periaxonal cleft that spans the internodal axon. By conventional electron microscopy and serial sectioning, we observed a category of double-walled vesicles at the axonal-myelin sheath interface, which often contained ribosome-like particles or endoplasmic reticulum. Some of them were demonstrated to continue with the subjacent axon with a thin stalk. In addition, we described a special category of axonal invaginations, probably mediated by subsurface cisternae. The functional implications of these specialized structures were discussed.

Immunohistochemical study of delta and mu opioid receptors on synaptic glomeruli with substance P-positive central terminals in chicken dorsal horn.

In an attempt to clarify the mechanism underlying the regulation of the release of substance P (SP) from the central axon terminals of the synaptic glomeruli in lamina II of the dorsal horn, we examined the expression patterns of delta and mu opioid receptors (DOR and MOR) in relation to those of enkephalin (ENK) and SP in the synaptic glomeruli. DOR, MOR, ENK and SP immunoreactivities in lamina II of the dorsal horn in the chicken were examined by confocal laser scanning and electron microscopies. DOR immunoreactivity was localized in both SP-positive central terminals and peripheral elements, while MOR immunoreactivity was only localized in the peripheral elements of the synaptic glomeruli. Both of the peripheral DOR- and MOR-immunoreactive elements were shown to be vesicle-containing dendrites by electron microscopy. Dual immunohistochemistry indicated that DOR, MOR and ENK immunoreactivities were located in distinct peripheral elements. On the basis of present results, the possible roles of DOR and MOR in the regulation of the release of SP from the central axon terminals in the synaptic glomeruli are discussed.

Beyond the initial axon segment of the spinal motor axon: fasciculated microtubules and polyribosomal clusters.

Dense undercoating, microtubular fascicles and scattered polyribosomal clusters have until now been considered to be the three structural features of the initial segment, and were thought not to extend beyond the initial segment into the myelinated parts of the axon. The aim of the present study was to make clear whether there is a sudden change in morphology between the unmyelinated and myelinated part. We followed spinal motor axons from the initial segment to the first internode by conventional electron microscopy and serial sectioning, and found that the microtubular fascicles and polyribosomal clusters do exist in the internodal axoplasm. The fasciculated microtubules were observed mainly in the first paranode. The polyribosomal clusters were found along the course of the first internode at a random distance, however, they occurred mainly in the proximal part of the first internode. The proportion of sections in which ribosomes were found, i.e. the incidence of ribosomes, in the first 30-microm-long portion was 71 +/- 24% (mean +/- SD, n = 4), and significantly different from that in the second 30-microm-long portion (3.2 +/- 1.3%) (mean +/- SD, n = 4) (P < 0.005). The more distal part of the first internode was not investigated.

An immunocytochemical study of calbindin-D28K in laminae I and II of the dorsal horn and spinal ganglia in the chicken with special reference to the relation to substance P-containing primary afferent neurons.

The localization of calbindin-D28K (CB) was studied immunocytochemically in laminae I and II of the dorsal horn and in spinal ganglia in the chicken, and compared with the distribution of substance P (SP) using double immunolabeling. At the light microscopic level, CB immunoreactivity was observed most intensely in the lamina II using the avidin-biotinylated peroxidase complex (ABC) and immunofluorescence methods. At the electron microscopic level using the ABC method, CB immunoreactivity was observed in the following three neuronal elements: 1) the scalloped central terminal with many dense-cored vesicles (DCVs) in the synaptic glomerulus; 2) some vesicle-containing dendrites (VCDs) inside or outside the synaptic glomerulus; and 3) some axon terminals outside the synaptic glomerulus. The CB-immunoreactive (IR) VCDs in the synaptic glomerulus often formed reciprocal synapses with the central terminal. Strong immunoreactivity was observed at the postsynaptic membrane of CB-IR elements. Double immunofluorescence and immunolabeling methods at the electron microscopic level showed that CB and SP colocalized in the scalloped central terminal with DCVs of the synaptic glomerulus. Almost all SP-IR neurons in the spinal ganglion revealed the coexistence of CB in serial sections in the chicken. In light of previous biochemical and physiological reports, our findings suggest that CB - coexisting with SP - plays an important role in the control of pain transmission through its strong Ca(2+)-buffering action in the chicken.

Mitochondrial accumulation in the distal part of the initial segment of chicken spinal motoneurons.

The axonal initial segment is the initiation site of action potentials and is characterized morphologically by a dense undercoating and fascicles of microtubules connected by cross-bridges. In order to analyze subcellular structures in the initial segment, we made serial transverse sections of initial segments of identified chicken motoneurons by retrograde transport of horseradish peroxidase (HRP) injected into the muscle. The mean (+/-SD) length of the initial segment was 28.1+/-2.3 microm (n=6). Mitochondria accumulated in the distal part of the initial segment, which was 1.4-6.9 microm in length (5-23% of the total length of the initial segment). In the transverse section of the distal part, mitochondrial density was 15.8+/-6.2% (n=5), while in the middle and proximal parts it was 6.1+/-1.6% and 5.6+/-1.4%, respectively. Mitochondrial accumulation was observed in common in phasic and tonic motoneurons in the chicken, and also observed in the distal part of the initial segment of the large ventral horn neurons of the chicken without HRP injection. These findings suggest that accumulated mitochondria play an important role in maintaining the physiological function of the distal part of the motoneuron initial segment.

Clusters of VIP-36-positive vesicles between endoplasmic reticulum and Golgi apparatus in GH3 cells.

The vesicular integral membrane protein VIP36 belongs to the family of animal lectins and may act as a cargo receptor trafficking certain glycoproteins in the secretory pathway. Immunoelectron microscopy of GH3 cells provided evidence that endogenous VIP36 is localized mainly in 70-100-nm-diameter uncoated transport vesicles between the exit site on the ER and the neighboring cis-Golgi cisterna. The thyrotrophin-releasing hormone (TRH) stimulation and treatment with actin filament-perturbing agents, cytochalasin D or B or latrunculin-B, caused marked aggregation of the VIP36-positive vesicles and the appearance of a VIP36-positive clustering structure located near the cis-Golgi cisterna. The size of this structure, which comprised conspicuous clusters of VIP36, depended on the TRH concentration. Confocal laser scanning microscopy confirmed the electron microscopically demonstrated distribution and redistribution of VIP36 in these cells. Furthermore, VIP36 colocalized with filamentous actin in the paranuclear Golgi area and its vicinity. This is the first study to show the ultrastructural distribution of VIP36 in the early secretory pathway in GH3 cells. It suggests that actin filaments are involved in glycoprotein transport between the ER and cis-Golgi cisterna by using the lectin VIP36.

Localization of VIP36 in the post-Golgi secretory pathway also of rat parotid acinar cells.

VIP36 (36-kD vesicular integral membrane protein), originally purified from Madin-Darby canine kidney (MDCK) epithelial cells, belongs to a family of animal lectins and may act as a cargo receptor. To understand its role in secretory processes, we performed morphological analysis of the rat parotid gland. Immunoelectron microscopy provided evidence that endogenous VIP36 is localized in the trans-Golgi network, on immature granules, and on mature secretory granules in acinar cells. Double-staining immunofluorescence experiments confirmed that VIP36 and amylase co-localized in the apical regions of the acinar cells. This is the first study to demonstrate that endogenous VIP36 is involved in the post-Golgi secretory pathway, suggesting that VIP36 plays a role in trafficking and sorting of secretory and/or membrane proteins during granule formation.

Involvement of VIP36 in intracellular transport and secretion of glycoproteins in polarized Madin-Darby canine kidney (MDCK) cells.

VIP36, an intracellular lectin that recognizes high mannose-type glycans (Hara-Kuge, S., Ohkura, T., Seko, A., and Yamashita, K. (1999) Glycobiology 9, 833-839), was shown to localize not only to the early secretory pathway but also to the plasma membrane of Madin-Darby canine kidney (MDCK) cells. In the plasma membrane, VIP36 exhibited an apical-predominant distribution, the apical/basolateral ratio being approximately 2. Like VIP36, plasma membrane glycoproteins recognized by VIP36 were found in the apical and basolateral membranes in the ratio of approximately 2 to 1. In addition, secretory glycoproteins recognized by VIP36 were secreted approximately 2-fold more efficiently from the apical membrane than from the basolateral membrane. Thus, the apical/basolateral ratio of the transport of VIP36-recognized glycoproteins was correlated with that of VIP36 in MDCK cells. Upon overproduction of VIP36 in MDCK cells, the apical/basolateral ratios of both VIP36 and VIP36-recognized glycoproteins were changed from approximately 2 to approximately 4, and the secretion of VIP36-recognized glycoproteins was greatly stimulated. In contrast to the overproduction of VIP36, that of a mutant version of VIP36, which has no lectin activity, was of no effect on the distribution of glycoproteins to apical and basolateral membranes and inhibited the secretion of VIP36-recognized glycoproteins. Furthermore, the overproduction of VIP36 greatly stimulated the secretion of a major apical secretory glycoprotein of MDCK cells, clusterin, which was found to carry at least one high mannose-type glycan and to be recognized by VIP36. In contrast to the secretion of clusterin, that of a non-glycosylated apical-secretion protein, galectin-3, was not stimulated through the overproduction of VIP36. These results indicated that VIP36 was involved in the transport and sorting of glycoproteins carrying high mannose-type glycan(s).