Saoko Atsumi

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.

Iron supports myogenic cell differentiation to the same degree as does iron-bound transferrin.

T. Hasegawa, K. Saito, I. Kimura, and E. Ozawa (1981, Proc. Jopan Acad. B 57, 206-210) have shown that Fe ion can promote myogenic cell growth as Fe-bound transferrin. In the present work, the effects of these substances in supporting myogenic cell differentiation were examined. The hallmarks of differentiation adopted were appearance of structural and regulatory proteins, myofibrils, sarcoplasmic reticulum, and Ca-activated activities of myosin B and phosphorylase kinase; isoform transition of creatine kinase; and acquisition of cell membrane excitability and contractility following electrical stimulation of myotubes. The degree of differentiation of myotubes cultured in the presence of Fe ion was almost the same as that of myotubes cultured in the presence of Fe-bound transferrin. These facts suggest that transferrin protein molecules do not play a primary role in differentiation. Further, it has also been shown that myotubes acquire excitation-contraction and metabolism coupling qualitatively similar to that of adult muscle fiber.

Synaptology of α-motoneurons in the chicken spinal cord

Abstract The synaptology of α-motoneurons innervating the anterior and posterior latissimus dorsi muscle (ALD and PLD) in the chicken was studied electron microscopically. These motoneurons were identified by means of retrograde transport of horseradish peroxidase injected into each muscle. Presynaptic boutons on their somata and dendrites were classified as S, F, C and M types, fundamentally similar to those previously reported in the monkey, cat and rat. Besides them, the presynaptic terminals which contained dense-cored vesicles, designated as the G type collectively for practical purposes, were newly found on both the somata and dendrites of chicken α-motoneurons and divided into five subtypes characterized by the presence of: (1) elongated-cored vesicles and flattened clear vesicles; (2) small spherical-cored vesicles (the range of diameter, 55–100 nm) and spherical clear vesicles; (3) middle-sized spherical-cored vesicles (60–120 nm) concomitant with spherical clear vesicles; (4) large spherical cored vesicles (85–145 nm) with a few spherical clear vesicles; and (5) cored vesicles of various shapes and sizes intermingled with tubular structures with dense content in them. The frequent occurrence of the G-type boutons on α-motoneurons in the chickens as compared with the rat, cat or monkey may suggest that the somatic motor activity in the chicken is modulated by neuropeptides and/or biogenic amines more than in the mammals.

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‐µm‐long portion was 71 ± 24% (mean ± SD, n = 4), and significantly different from that in the second 30‐µm‐long portion (3.2 ± 1.3%) (mean ± SD, n = 4) (P < 0.005). The more distal part of the first internode was not investigated.

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.

Large dorsal horn neurons which receive inputs from numerous substance P-like immunoreactive axon terminals in the laminae I and II of the chicken spinal cord

Large neurons outlined with numerous substance P (SP)-like immunoreactive (LI) boutons were detected immunocytochemically in the dorsal horn of the chicken spinal cord at the light microscopic level. The cervical enlargement was mainly used for observations. By electron microscopy, asymmetrical synapses were observed between the SP-LI axon terminals and the soma and dendrites of the large neurons. Cell bodies of the large neurons were mostly localized in the lamina I and the region lateral to the lamina I. Some of the cell bodies were also located in the lamina II. Their dendrites extended in the lamina I, in the region lateral to the lamina I, and deeply in the lamina II. In the lamina II, dendrites of these neurons formed synapses with SP-containing central terminals in synaptic glomeruli known to originate from primary afferents. The findings suggest that these large neurons receive nociceptive information directly from primary afferents. In the light of previous investigations, these neurons are considered to be pain-transmitting long ascending tract neurons.

Enkephalin-like immunoreactive axon terminals make synapses with α-motoneurons in the chicken

Abstract Methionine (Met)-enkephalin (Enk)-like immunoreactive axon terminals on HRP-labeled α-motoneurons were studied using the indirect antibody peroxidase-anti-peroxidase technique in the chicken. Electron microscopically, Met-Enk-like immunoreactive axon terminals containing dense-cored vesicles and clear vesicles made axosomatic and axodendritic synapses with PLD α-motoneurons. Dense-cored vesicles were sometimes observed to be adjacent to the presynaptic membrane accompanied by the postsynaptic density. These results suggest morphologically that α-motoneurons may be directly modulated by enkephalins.

Immunohistochemical demonstration of the calcium channel α2 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 Lissauers 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, FcεRI, 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. Anat Rec, 1424‐1430, 2007.

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.