Enzyme histochemical adaptive responses of the medial pterygoid muscle and two heads of the lateral pterygoid muscle to long-term soft diet feeding in growing rabbits.

The aim of the present study was to investigate the histochemical effects of long-term soft diet in the medial pterygoid muscle as well as the two heads of the lateral pterygoid muscles in growing rabbits. Eleven young rabbits were divided into two groups as solid diet (control group; n = 6) or soft diet (soft-diet group; n = 5) groups. After 6 months, muscle fibers from the medial and the two heads of the lateral pterygoid muscles were histochemically defined. In the medial pterygoid muscle, the percentage of the type 1 fiber cross-sectional area to total area was 10.1 ± 2.4% in the control group and 8.3 ± 3.0% in the soft-diet group, respectively. In the soft-diet group, there was a trend toward an increase in the number of type 2A fibers, and toward a decrease in the numbers of type 2B fibers in comparison with the controls. In the two heads of the lateral pterygoid muscle, the percentage of the type 1 fiber cross-sectional area to total area was 8.4 ± 7.5 and 3.3 ± 2.7%, respectively. Compared to that of the control group, the two heads in the soft-diet group showed a trend toward a decrease in the number of type 2A fibers. In addition, type 2B had a tendency to decrease in the number in the inferior head. In conclusion, this study suggests that long-term soft diet leads to adaptations of the pterygoid muscles. Two heads of the lateral pterygoid muscle revealed different adaptation from jaw-closing muscles under soft-diet conditions.

Participation of ventral and dorsal tail muscles in bending movements of rat tail.

The rat controls the form of its tail, from straight to curved, by contraction and relaxation of its four tail muscles. The tendons of these muscles insert on any of the cranial articular, transverse, and hemal processes of each of 24 coccygeal vertebrae (Co5-Co28). In this study, we isolated for the four coccygeal muscles each muscular fascicle segment inserting on any process of the coccygeal vertebrae. We measured the length and weight of all muscular fascicles and tendons, and then divided all muscular fascicles into four groups based on their insertion: Co5-Co10, Co11-Co16, Co17-Co22, and Co23-Co28. Moreover, we used soft X-ray imaging to investigate the geometrical relationship between neighboring coccygeal vertebrae. Additionally we carried out serial sectioning at the sacral and caudal portions, and traced the course of the tendons of coccygeal muscles from their origin to the Co4 level. We discuss which muscles and tendons play important roles when coccygeal vertebrae bend along and rotate around the longitudinal axis.

Downregulation of msh-like 2 (msx2) and neurotrophic tyrosine kinase receptor type 2 (ntrk2) in the developmental gut of KIT mutant mice.

In the gastrointestinal tract, interstitial cells of Cajal (ICC) are the regulatory cells of gut movement. W/W mutant mice that have receptor tyrosine kinase KIT mutation lack ICC along the myenteric plexus layer of small intestine. The development and maintenance of the ICC phenotype have been related to KIT, but the other genes involved in ICC development during embryogenesis are not clear. Our aim was to identify ICC-specific genes in the embryonic stage. We examined genes that are expressed less in ICC-deficient W/W mice than in wild type (WT) at embryonic day 14 (E14) in order to clarify the genes associated with the ICC development using subtractive hybridization and microarray. Among them, we identified msh-like 2 (msx2) and neurotrophic tyrosine kinase receptor type 2 (ntrk2). Using real-time PCR, msx2 and ntrk2 were found to be expressed at significantly lower levels in W/W than in WT during embryogenesis. Msx2 immunoreactivity was high in the WT small intestine. These data suggest that the gene expressions of ntrk2 and msx2 were significantly suppressed in KIT mutant mouse embryo and neonate and that these genes are likely to regulate ICC development.

Immunohistochemical demonstration of c-Kit-negative fibroblast-like cells in murine gastrointestinal musculature.

In the gastrointestinal musculature, interstitial cells of Cajal (ICC) distribute and regulate the gastrointestinal motility. Another type of mesenchymal cell, known as the fibroblast-like cell (FLC), has also been reported to be juxtaposed to the ICC. In this study, we examined the immunohistochemical properties of FLC in the murine gastrointestinal musculature using antibodies to small conductance Ca(2+)-activated K(+) channel 3 (SK3), platelet-derived growth factor receptor alpha (PDGFRalpha), and CD34. SK3-immunopositive (SK3-ip) cells were observed in the musculature throughout the gastrointestinal tract. These SK3-ip cells were distinct from the ICC that were identified by c-Kit immunoreactivity. In the muscular layers, SK3-ip cells were bipolar in shape and were associated with the intramuscular ICC and nerve fiber bundles. In the myenteric layer multipolar-shaped SK3-ip cells encompassed the myenteric ganglia. SK3-ip cells in the subserosal plane formed a cellular network with their ramified processes. The distribution pattern of the SK3-ip cells in the ICC-deficient W(v)/W(v) mutant mice was similar to that in normal mice. We also demonstrated that SK3-ip cells showed the intense PDGFRalpha immunoreactivity that was previously examined in FLC. However, CD34 immunoreactivity, one of the markers of human FLC, was not observed in SK3-ip cells with the exception of subserosal FLC. Thus, our observations indicate that SK3- and PDGFRalpha-double immunopositive cells are FLC in the murine gastrointestinal musculature and behave as a basic cellular element throughout the gastrointestinal musculature.

The expression of soluble guanylate cyclase in the vasculature of rat skeletal muscle.

Nitric oxide (NO) has various roles in the skeletal musculature in both normal and pathological conditions. NO primarily activates soluble guanylate cyclase (sGC) and mediates subsequent intracellular signaling in target cells. We sought to identify the target cells of NO in the rat skeletal musculature, using subtypes of sGCalpha1 and sGCbeta1 antibodies. Immunohistochemistry revealed that both antibodies stained the same cells with round or oval shapes, having several long processes. The sGC-immunopositive cells co-expressed NG2 chondroitin sulfate proteoglycan, a marker of pericytes. The sGC-immunopositive cells were associated with capillaries and formed cellular networks with elongated cytoplasmic processes. sGCalpha1 and sGCbeta1 were not found in muscle sarcolemma that were stained by anti-dystrophin, or neuromuscular junctions, as detected by anti-synaptophysin. Based on these findings, we concluded that sGC immunoreactivity was specifically distributed in capillary pericytes. Pericytes in the skeletal musculature have been shown to be target cells of NO and are involved in the microvascular blood flow.

W(sh)/W(sh) c-Kit mutant mice possess interstitial cells of Cajal in the deep muscular plexus layer of the small intestine.

The c-Kit receptor tyrosine kinase regulates the development and differentiation of various progenitor cells. W mutant mice with spontaneous mutations in the c-kit gene show various phenotypes such as anemia, infertility, loss of coat color and mast cells. c-Kit also regulates the development of the interstitial cells of Cajal (ICC) that are responsible for the motility regulation of the gastrointestinal musculature. W(sh)/W(sh) mice possess an inversion mutation upstream of the c-kit promoter region; this mutation is responsible for reducing c-Kit activity, leading to a decrease in the number of mast cells, melanocytes, and ICC. We extensively examined the small intestine of W(sh)/W(sh) mice by using immunohistochemistry and electron microscopy. Although the musculature of the W(sh)/W(sh) mice did not show any c-Kit immunoreactivity, there were neurokinin 1 receptor (NK1R)-immunopositive cells that were associated with the nerve fibers in the deep muscular plexus (DMP) region. These NK1R-immunopositive cells showed a bipolar shape with long processes and were identified as ICC in the DMP layer (ICC-DMP). Electron microscopic analysis revealed that ICC-DMP had numerous mitochondria, caveolae, and gap junctions and were closely associated with nerve terminals. In contrast, ICC were not observed at the myenteric layer. In the small intestine of the W(sh)/W(sh) mice, we detected ICC-DMP that showed NK1R immunoreactivity and ultrastructural characters. This type of ICC may develop and maturate structurally without c-Kit expression and regulate gastrointestinal motility.

c-Kit-negative fibroblast-like cells express platelet-derived growth factor receptor alpha in the murine gastrointestinal musculature.

Platelet-derived growth factor receptors (PDGFRs) belong to the same kinase group as c-Kit receptor tyrosine kinase that is specifically expressed in the interstitial cells of Cajal (ICC) in the gastrointestinal tract. In this study, we examined PDGFRalpha immunoreactivity in the murine gastrointestinal tract. PDGFRalpha-immunopositive (PDGFRalpha-ip) cells were observed in the musculature in all parts of the gastrointestinal tract. Although PDGFRalpha-ip cells were distinct from ICC and neurons, these cells were closely associated with intramuscular ICC and enteric nerve fibers. In the myenteric layer, PDGFRalpha-ip cells formed a cellular network with their ramified processes and encompassed myenteric ganglia. Numerous PDGFRalpha-ip cells were observed in the subserosal plane and showed a multipolar shape. The distribution pattern of the PDGFRalpha-ip cells in the ICC-deficient W(v)/W(v) mutant mice was the same as that in normal mice. PDGFRalpha-ip cells that showed intense immunoreactivity of SK3 potassium channel were considered to correspond to fibroblast-like cells or non-Cajal interstitial cells. Our observations suggest that PDGFRalpha-ip cells are basic cellular elements throughout the gastrointestinal musculature and are involved in the gastrointestinal functions.

Some gamma-motoneurons contain gamma-aminobutyric acid in the rat cervical spinal cord.

Gamma-aminobutyric acid (GABA) is utilized in the peripheral as well as central nervous system. In this study, fibers immunoreactive for 67 kDa isoform of glutamic acid decarboxylase (GAD67), an enzyme which synthesizes GABA, were found to terminate in the intercapsular region of muscle spindles of the upper limb. GABA-containing fibers were also found in the ventral roots of C5 to T5 spinal segments, brachial plexus, and radial nerve. These fibers were thin and immunoreactive for choline-acetyl transferase (ChAT). After transection of the brachial plexus, GABA immunoreactivity disappeared completely in the ipsilateral triceps brachii muscle (TBM). After the injection of fluorogold into the TBM, some retrogradely labeled medium-sized neurons were positive for GAD67, but not VGAT mRNA. All these observations clearly indicate that GABA-containing gamma-motoneurons in the lower cervical spinal cord send their fibers to muscle spindles in the upper extremities. Since we detected neither GABAA nor GABAB receptors in the TBM by RT-PCR, the function of the GABA-containing gamma-motoneurons remains unclear.

Interstitial cells of Cajal in the gastrointestinal musculature of W mutant mice.

Interstitial cells of Cajal (ICC) are important regulatory cells generating electrical rhythmicity and transducing neural signals in the gastrointestinal musculature. ICC express the proto-oncogene c-kit, a receptor tyrosine kinase, and can be examined morphologically using the c-Kit antibody. The c-kit gene is allelic with the murine white-spotting locus W, and the c-kit mutation (W mutation) affects various aspects of hematopoietic cells, germ cells, melanocytes, mast cells, and ICC. Heterozygous W/W( v) mutant mice lack a specific type of ICC and have been used to reveal its function. To search for a new model that lacks a specific type of ICC, we examined homozygous W( v)/W( v) black-eyed-white mice that are viable with anemia. Results showed the principal patterns of ICC deficiency were the same between the W/W( v) and W( v)/W( v) mutants. In the stomach of both mice, intramuscular ICC (ICC-IM) were missing and myenteric ICC (ICC-MY) were reduced in number. In the small intestine, the number of ICC-MY was severely reduced in spite of a normal distribution of deep muscular plexus ICC (ICC-DMP). The cecum also exhibited fewer reduced. ICC-IM in the colon were almost entirely missing, whereas ICC-MY were reduced only in the distal colon. In the small intestine and colon, the number of remaining ICC-MY in W( v)/W( v) mice was greater than that in W/W( v) mice. The enteric nervous system of the two mutant mice showed normal characteristics. From these findings, we conclude that W( v)/W( v) mice represent a new genotype that lacks a part of the ICC in its gastrointestinal musculature.

Gamma-aminobutyric acid-containing sympathetic preganglionic neurons in rat thoracic spinal cord send their axons to the superior cervical ganglion.

Gamma-aminobutyric acid (GABA)-containing fibers have been observed in the rat superior cervical ganglion (SCG) and, to a lesser extent, in the stellate ganglion (STG). The aim of present study is to clarify the source of these fibers. No cell body showed mRNAs for glutamic acid decarboxylases (GADs) or immunoreactivity for GAD of 67 kDa (GAD67) in the cervical sympathetic chain. Thus, GABA-containing fibers in the ganglia are suggested to be of extraganglionic origin. GAD67-immunoreactive fibers were found not in the dorsal roots or ganglia, but in the ventral roots, so GABA-containing fibers in the sympathetic ganglia were considered to originate from the spinal cord. Furthermore, almost all GAD67-immunoreactive fibers in the sympathetic ganglia showed immunoreactivity for vesicular acetylcholine transporter, suggesting that GABA was utilized by some cholinergic preganglionic neurons. This was confirmed by the following results. 1) After injection of Sindbis/palGFP virus into the intermediolateral nucleus, some anterogradely labeled fibers in the SCG were immunopositive for GAD67. 2) After injection of fluorogold into the SCG, some retrogradely labeled neurons in the thoracic spinal cord were positive for GAD67 mRNA. 3) When the ventral roots of the eighth cervical to the fourth thoracic segments were cut, almost all GAD67- and GABA-immunoreactive fibers disappeared from the ipsilateral SCG and STG, suggesting that the vast majority of GABA-containing fibers in those ganglia were of spinal origin. Thus, the present findings strongly indicate that some sympathetic preganglionic neurons are not only cholinergic but also GABAegic.

A part of cholinergic fibers in mouse superior cervical ganglia contain GABA or glutamate.

The localizations and functions of glutamate and GABA, the major amino acid neurotransmitters in the central nervous system, are still unclear in the peripheral nervous system. We immunohistochemically double-stained mouse superior cervical ganglia with antibodies for the vesicular acetylcholine transporter (VAchT), GAD65, the vesicular glutamate transporters 1-3 (VGluTs1-3), the marker of the sympathetic preganglionic neuron (SPN), GABAergic, and glutamatergic terminals, respectively. All GAD65-positive terminals showed VAchT immunoreactivity, indicating that GABAergic fibers originate from SPNs. VGluT2-immunoreactive terminals showing colocalization with VAchT were observed, but VGluT1 and 3 immunoreactive terminals were not. Colocalization of GAD65 and VGluT2 was rarely found. All VGluT2-immunopositive terminals were also immunopositive for neuronal nitric oxide synthase (nNOS), a marker for the subpopulation of the SPNs, while about half of the GABA-immunopositive fibers were immunopositive for nNOS. The origin of these fibers was discussed.

Cochlear nerve demyelination causes prolongation of wave I latency in ABR of the myelin deficient (md) rat.

In this study, we examined the auditory brainstem responses (ABRs), distortion product of otoacoustic emissions (DPOAEs) and cochlear morphology of the myelin deficient (md) rat, which completely lacks central myelin but not peripheral myelin. ABRs showed a marked prolongation not only wave II-IV latencies but also wave I latency. Cochlear nerve fibers near the modiolus lost their myelin halfway into the internal auditory canal. DPOAEs also decreased at a lower frequency of the combined tone. Since nerve fibers ending at the apical turn of the cochlea passed through central portion of the cochlear nerve, wave I prolongation of ABRs and decrease of DPOAEs at a lower frequency might originate mainly from the demyelinated CNS part of the cochlear nerve and efferent olivocochlear bundle in the internal auditory canal.

Motor, sensory and autonomic nerve terminals containing NAP-22 immunoreactivity in the rat muscle.

Neuron-enriched acidic protein having a molecular mass of 22 kDa, NAP-22, is a Ca(2+)-dependent calmodulin-binding protein and is phosphorylated with protein kinase C (PKC). This protein is localized to the biological membrane via myristoylation and found in the membrane fraction of the brain and in the synaptic vesicle fraction. Recent studies showed that NAP-22 is localized in the membrane raft domain in a cholesterol-dependent manner and suggest a role for NAP-22 in maturation and/or maintenance of nerve terminals by controlling cholesterol-dependent membrane dynamics. The present study revealed the immunohistochemical distribution of NAP-22 in the peripheral nerves in rat muscles. In all examined muscles, nerve terminals in the motor endplates showed NAP-22 immunoreactivity associated with the membranes of synaptic vesicles and nerve terminals. In the muscle spindles, annulospiral endings, which made spirals around the intrafusal muscles, showed intense NAP-22 immunoreactivity. Autonomic nerve fibers around the intramuscular blood vessels also showed the immunoreactivity for NAP-22. NAP-22 immunoreactivity in these peripheral nerves was observed from birth to adulthood (100 days after birth). Though growth-associated protein-43 (GAP-43) immunoreactivity in these nerves was observed from birth, this immunoreactivity decreased from 20 days after birth. These findings suggest that NAP-22 is distributed and regulates functions in the motor, sensory and autonomic nerve terminals in the peripheral nervous system.

Evidence that Sema3A and Sema3F regulate the migration of GABAergic neurons in the developing neocortex.

The ganglionic eminence (GE) supplies neurons containing gamma-aminobutyric acid (GABA) to the pallium of the telencephalon. We investigated the molecular guidance mechanisms of GE cell migration in the neocortex and found neuropilin-1 (Npn-1) or neuropilin-2 (Npn-2) on the GE cells. Ectopic Sema3A or -3F expression by COS1 cell clusters placed on embryo neocortical slices reduced the cell migration but did not block it completely. However, the cell migration was almost completely blocked by COS1 cell clusters expressing both Sema3A and -3F. The direction of cell migration could be reversed by placing Sema3A- and -3F-coexpressing COS1 cell clusters at the distal cut end of the neocortical slices. Further slice experiments revealed that migration of half of the GE cells in the neocortex was regulated by Sema3A and that migration of the other half of the GE cells in the neocortex was regulated by Sema3F. When the cells responding to Sema3A were diverted by ectopic Sema3A expression in vivo, Dlx2-positive cells were found predominantly in the lower intermediate zone (IZ). When the cells responding to Sema3F were diverted by ectopic Sema3F expression in vivo, Dlx2-positive cells were found predominantly in the upper IZ. It was speculated that the semaphorin-neuropilin interactions distribute the GABAergic GE cells evenly in the neocortex as well as guide the GE cells from the GE to the neocortex. The Sema3A expression site under the subplate extended dorsally as the embryo developed. The Sema3A expression seemed to block the Npn-1-positive GE cells in the neocortex from entering the cortical plate (CP) and guide them to the dorsal cortex and the hippocampus. Sema3F expression in the CP continued through the embryonic stages. The expression seemed to block Npn-2-positive GE cells in the neocortex from entering the CP and make them migrate into the lower IZ. Finally, the semaphorin-neuropilin interactions sorted GABAergic inteneurons into the CP and white matter neurons into the IZ.

Differences in the density of sympathetic nerve fibers in the arteriolar walls of the rat extensor digitorum longus muscle.

In this study, we electron-microscopically investigated a number of sympathetic axons in the arteriolar walls of the extensor digitorum longus muscles of the rat rear leg. Arterioles in the muscle were divided into two groups: (i) one group consisted of arterioles with accompanying muscle spindles, and (ii) the other consisted of arterioles without accompanying muscle spindles. The number of sympathetic axons present in the arteriolar walls and the ratios to the total number of sympathetic and non-sympathetic axons were compared between the groups. For electron-microscopic identification of sympathetic axons, 5-hydroxydopamine, a pseudotransmitter agent, was used. The number and ratio of sympathetic axons were significantly higher in arterioles with accompanying muscle spindles than arterioles possibly unrelated to muscle spindles. Additionally, amine- and immunohistochemistry were used to confirm the above observation.

Regulation of Cell Number in Formation of the Dorsal Root Ganglion Revealed by Transplantation of Quail Neural Crest Cells into Chick Embryos: (dorsal root ganglia/cell number/chick-quail chimera/neural crest/regulation).

Neural crest cells appear transiently in early embryogenesis on the dorsal surface of the neural tube and subsequently migrate along specific pathways. Some migrate to between the neural tube and somites, aggregating to form the rudiments of dorsal root ganglia (DRG). The size of DRG at a given somite level is almost constant in all chick embryos. To determine the mechanisms controlling the size of DRG, we transplanted neural crest cells of 2.5-day-old quail embryos into 2.5-day-old chick embryos between the neural tube and the somites, and examined the size of DRG in these chimeric embryos with extra neural crest cells 2 days after the operation, when natural cell death in DRG had not yet occurred. The DRG on the operated side were composed of both chick and quail cells in various proportions. The cell numbers of these chimeric DRG were almost the same as those of the normal DRG on the opposite side. That is, there were significantly fewer chick cells in chimeric DRG than in DRG composed of only chick cells on the opposite unoperated side. This finding indicates that the size of DRG is not determined in migrating neural crest cells but is regulated by the circumstances.

Do Peanut Agglutinin Receptors on Somites Control the Behavior of Neural Cells?: (PNA receptors/somite/resegmentation/rostro-caudal axis/gangliogenesis).

Peanut agglutinin (PNA) receptors are expressed in the caudal halves of sclerotomes in chick embryos after 3 days of incubation (stages 19-20 of Hamburger & Hamilton). The neural crest cells forming dorsal root ganglia (DRG) and motor nerves appear to avoid PNA positive regions and concentrate into rostral halves of sclerotomes. To investigate the role of PNA receptors in gangliogenesis and nerve growth, we examined PNA binding ability in quail sclerotomes and in chick-quail chimeric embryos made by transplanting quail somites to chick embryos, comparing the development of DRG, motor nerves and sclerotomes. PNA did not bind to any part of the somites of 4.5-day quail embryos, although dorsal root ganglia and motor nerves appeared only in the rostral halves of sclerotomes as in chick embryos. Moreover, in spite of no PNA binding ability of the transplanted quail somite in 4.5-day chick-quail chimeric embryos, DRG and motor nerves derived from chick tissues appeared only in the rostral halves of the sclerotomes derived from these somites. Thus, both quail and chick neural crest cells and motor nerves recognized the difference between the rostral and caudal halves of sclerotomes of quail embryos in the absence of PNA binding ability, indicating that PNA binding site on somite cells does not support the selective neural crest migration and nerve growth.