Search

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.

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.

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.

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 PDGFRα immunoreactivity in the murine gastrointestinal tract. PDGFRα-immunopositive (PDGFRα-ip) cells were observed in the musculature in all parts of the gastrointestinal tract. Although PDGFRα-ip cells were distinct from ICC and neurons, these cells were closely associated with intramuscular ICC and enteric nerve fibers. In the myenteric layer, PDGFRα-ip cells formed a cellular network with their ramified processes and encompassed myenteric ganglia. Numerous PDGFRα-ip cells were observed in the subserosal plane and showed a multipolar shape. The distribution pattern of the PDGFRα-ip cells in the ICC-deficient W v /W v mutant mice was the same as that in normal mice. PDGFRα-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 PDGFRα-ip cells are basic cellular elements throughout the gastrointestinal musculature and are involved in the gastrointestinal functions.

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.

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.

Nitric oxide (NO) is a major signaling molecule in the gastrointestinal tract, and released NO inhibits muscular contraction. The actions of NO are mediated by stimulation of soluble guanylate cyclase (sGC, NO-sensitive GC) and a subsequent increase in cGMP concentration. To elucidate NO targets in the gastrointestinal musculature, we investigated the immunohistochemical localization of the beta1 and alpha1 subunits of sGC and the distribution of neuronal NO synthase (nNOS) -containing nerves in the guinea-pig gastrointestinal tract. Distinct immunoreactivity for sGCbeta1 and sGCalpha1 was observed in the interstitial cells of Cajal (ICC), fibroblast-like cells (FLC) and enteric neurons in the musculature. Double immunohistochemistry using anti-c-Kit antibody and anti-sGCbeta1 antibody revealed sGCbeta1 immunoreactivity in almost all intramuscular ICC throughout the entire gastrointestinal tract. Immunoelectron microscopy revealed that sGCbeta1-immunopositive cells possessed some of the criteria for intramuscular ICC: presence of caveolae; frequently associated with nerve bundles; and close contact with smooth muscle cells. sGCbeta1-immunopositive ICC were closely apposed to nNOS-containing nerve fibers in the muscle layers. Immunohistochemical and immunoelectron microscopical observations revealed that FLC in the musculature also showed sGCbeta1 immunoreactivity. FLC were often associated with nNOS-immunopositive nerve fibers. In the myenteric layer, almost all myenteric ganglia contained nNOS-immunopositive nerve cells and were surrounded by myenteric ICC and FLC. Myenteric ICC in the large intestine and FLC in the entire gastrointestinal tract showed sGCbeta1 immunoreactivity in the myenteric layer. Smooth muscle cells in the stomach and colon showed weak sGCbeta1 immunoreactivity, and those in the muscularis mucosae and vasculature also showed evident immunoreactivity. These data suggest that ICC are primary targets for NO released from nNOS-containing enteric neurons, and that some NO signals are received by FLC and smooth muscle cells in the gastrointestinal tract.

γ-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 γ-motoneurons in the lower cervical spinal cord send their fibers to muscle spindles in the upper extremities. Since we detected neither GABA A nor GABA B receptors in the TBM by RT-PCR, the function of the GABA-containing γ-motoneurons remains unclear.

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.

Objective: Recently young people have an increasing tendency to intake an easily chewable diet and spend less time on mastication. The aim of the present study was to investigate the histochemical effects of long-term soft diet on the masseter muscle in growing rabbits. Materials and methods: Twelve young male Japanese white rabbits were divided into two groups (n = 6 each) at weaning (1 month after birth) and fed a solid diet (control group) or a powder diet (soft-diet group). The duration of the experimental period was 6 months. Masseter fibers from the superficial and the deep portions were histochemically defined as type 1, 2A, 2B, or 2C fibers. Results: As compared with that of the control, the deep masseter of the soft-diet group showed a significantly lower ratio of type 1 fiber cross-sectional area to total area (6.3 and 10.1% for the soft-diet and control group, respectively), significantly more type 2A fibers (74.0%vs 50.3%) and significantly fewer type 2B fibers (4.3%vs 12.5%). However, fiber size did not differ between the two groups. NADH-tetrazolium-reductase (NADH-TR) of the masseter was less reactive in the soft-diet group, reflecting a lower oxidative capacity. Conclusions: These findings indicate that the alteration of the functional activities contributed to selective disuse influences on the type 1 and type 2B fibers, and a resultant increase in type 2A fibers. This study suggests that long-term alteration of jaw function induced by a soft diet can lead to adaptations of the masseter muscle.

Maxillofacial growth and histochemical effects of long-term unilateral chewing caused by unilateral facial paralysis on the masseter muscle were studied in growing rabbits. The sample for this study consisted of a total of 20 young male Polish rabbits. The experimental group was comprised of 15 rabbits, which were subjected to dissection of the right facial nerve on the 21st day after birth, while the remaining 5 animals served as a control. The duration of the experimental period was 6 months. In the experimental group, the rabbits with facial abnormality on the paralyzed right side performed unilateral chewing only on the surgically unmanipulated side. Morphometric analysis showed facial asymmetry, with the maxillofacial region severely atrophied on the side that had undergone surgery. Both 4 weeks and 6 months after the operation, type 1 fibers of the masseter muscle on the denervated side, i.e., the non-chewing side, were significantly fewer in number and smaller than those on the unmanipulated side. However, type 2 fibers did not differ in size between sides. NADH-tetrazolium-reductase (NADH-TR) of the masseter was less reactive on the denervated side, reflecting a lower oxidative capacity. These results indicate that the alteration of the functional activities contributed to selective disuse atrophy of the type 1 fibers and a conversion from type 1 to type 2A fibers on the non-chewing side. In other words, the larger diameter and greater proportion of type 1 fibers of the masseter was indicative of work hypertrophy on the unmanipulated, chewing side. This study reveals that facial nerve dissection changes the maxillofacial growth and the histochemical features of the portion of the masseter muscle innervated by the trigeminal nerve, but not by the facial nerve.

Oligodendrocyte precursors are produced in restricted foci of the germinative neuroepithelium in embryo brains and migrate to their sites of function, while astrocytes are produced in a wider area in the neuroepithelium. We investigated the guidance mechanisms of glial precursor (GP) cell migration in the optic nerve. GP cell migration in newborn rat optic nerve was monitored by the UV-thymine-dimer (TD) method. A double labeling study using NG2 and TD revealed that many of these in vivo migrating cells were NG2 positive, while some of them with large TD-positive nuclei were NG2 negative. An in vitro cell migration study using optic nerve with chiasma and/or eyeball tissue revealed that the GP cells migrated under the guidance of repulsive cues secreted from the optic chiasma. We detected the expression of netrin 1 and Sema3a in the optic chiasma, and that of Unc5h1 and neuropilin 1 in the optic nerve. Co-culture experiments of the optic nerve with cell clusters expressing guidance cues revealed that the migrating GP cells in the optic nerve were heterogeneous. Netrin 1 repelled a subtype of NG2-positive and PLP-positive GP cells with small nuclei. Sema3a repelled a subtype of GP cells with large nuclei.

A neuronal tracer, WGA-HRP, was injected into the rat parotid gland (PG), submandibular gland (SMG), and sublingual gland (SLG) to label the superior cervical ganglion (SCG) neurons, and the number, size of soma, dendritic arborization and immunoreactivities (IR) of vasoactive intestinal polypeptide (VIP) and neuropeptide Y (NPY) were investigated. The number and size of neurons projecting into SMG were greater than those of neurons projecting into the PG or SLG. The PG and SLG projecting neurons did not show significant differences in their number and size of soma, even the weight of the SLG was much smaller than the other two glands. The total length of dendrites of the SMG projecting neurons was greater than those of the PG or SLG projecting neurons. PG and SLG projecting neurons showed no significant difference in the total length of their dendrites. PG and SLG projecting neurons with NPY-IR were larger in number than SMG projecting neurons. SLG projecting neurons with VIP-IR were greater in number than neurons projecting into the other two glands. Our results indicate the differences in morphology and neuropeptide immunoreactivities among the SCG neurons which may be due to the differences in the neuronal functions and the participation of neurotrophic factors.

The onset of Hex expression and its role in early murine development was analyzed using in situ hybridization. Hex mRNA was first detected in the chorion of the ectoplacental cavity and weakly at the visceral endoderm of the future yolk sac at embryonic age (E) 7.5. Expression in embryonic tissues was detected exclusively in the hepatic anlage and thyroid primordium at E 9.5. At E 12.5 and E 15.5, Hex expression persisted in the fetal liver and thyroid, and was also detected in the fetal lung. These results suggest that Hex has its role in differentiation and/or organogenesis of several embryonic tissues.

Anterograde labeling technique with Phaseolus Vulgaris leucoagglutinin (PHA-L) was employed to observe how a single preganglionic axon arborizes in the superior cervical ganglion (SCG) and stellate ganglion (STG) of rats. PHA-L was injected into the intermediolateral nucleus of the spinal cord at the middle point between segments T1 and T2, and labeled axons were detected immunohistochemically in serial sections. We traced and drew three preganglionic axons over their full length in the SCG and STG. In SCG, the labeled axons bifurcated repeatedly and extended to a length of 600–700 μ m in the rostrocaudal direction, and about 200 μ m in the transverse direction. These three preganglionic axons made 11, 14 and 11 dense terminal plexus regions along their trajectory. The pattern of the most dense terminal plexus corresponded to the pericellular type dendritic plexus, one of the plexus patterns of dendritic collaterals of SCG neurons. In the STG, the extent of axonal arborization was more variable than that in the SCG, ranging from 400 to 800 μ m in the rostrocaudal direction and about 400 μ m in the transverse direction. The three analyzed axons made 21, 19 and 20 dense terminal plexus regions along their trajectory, with a similar pattern to those in SCG. These results indicated that there might be a columnar or ellipsoidal organization of postganglionic neurons which are innervated by single preganglionic axons.

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.

1. Neurogenic responses to transmural electrical stimulation were examined in endothelium-denuded extrameningeal (vertebral and carotid) and intrameningeal (spinal, basilar and middle cerebral) arteries isolated from dogs. 2. In the extrameningeal arteries, transmural electrical stimulation produced a phasic contraction. This contraction was abolished by tetrodotoxin, prazosin and guanethidine. However, alpha,beta-methylene ATP and NG-nitro-L-arginine (L-NOARG) had no significant effect on the contractile responses. 3. In the intrameningeal arteries, the neurogenic responses to electrical stimulation were composed of a transient contraction and relaxation. The transient contraction was selectively inhibited by guanethidine L-NOARG abolished the relaxation but not the contraction induced by electrical stimulation. Prazosin had no effect on either neurogenic response. 4. Noradrenaline produced a large contraction in the extrameningeal arteries which was selectively inhibited by prazosin. alpha,beta-Methylene ATP produced neither contraction nor inhibition of the response to noradrenaline in the extrameningeal arteries. 5. In the intrameningeal arteries, alpha,beta-methylene ATP produced a greater contraction than noradrenaline. The response to alpha,beta-methylene ATP was selectively abolished by desensitization of P2x-purinoceptors with alpha,beta-methylene ATP itself. The contractile response to noradrenaline was inhibited by rauwolscine but not by prazosin. 6. ATP produced endothelium-dependent relaxations in the extrameningeal and intrameningeal arteries, which were attenuated by endothelium removal. 7. NADPH diaphorase-positive fibres were dense in the middle cerebral and basilar arteries but rare or absent in the spinal artery. In the extrameningeal arteries diaphorase-positive traces were observed in the vasa vasorum. 8. The present findings indicate that the neurogenic responses of intrameningeal arteries of dogs are composed of NO-ergic and sympathetic purinergic components, while the extrameningeal arteries tested produced only sympathetic adrenergic responses, suggesting that regional heterogeneity may be associated with a sudden transition in innervation and receptor expression at the meninx.

In order to examine whether the entorhinal-hippocampal-entorhinal circuit is reciprocal and topographic, the connections between the subiculum, the CA1 field, and the entorhinal cortex were studied with the carbocyanine dye (Dil), which moves in both retrograde and anterograde directions. We investigated the organization of reciprocal connections revealed by injections of Dil in the entorhinal cortex along the rhinal sulcus. Anterograde fluorescent labeling showed the same pattern reported in previous studies of the dorsal hippocampus. When the injection site of DiI extended into the deep layers (IV-VI) of the same cortical column, the anterograde labeling of the perforant path was accompanied by retrograde labeling of the subicular neurons and the CA1 neurons. The distribution of labeled cells overlapped the distribution of labeled fibers, and the distribution of labeled cells paralleled that of the labeled fibers in the CA1 field. DiI injection into the medial entorhinal cortex revealed fewer retrogradely labeled subicular neurons than injection into the lateral entorhinal cortex, whereas the number of labeled CA1 neurons was not dependent on the injection site. The number of labeled CA1 neurons was always several times greater than the number of subicular neurons. Thus, the amount of information conveyed by the CA1 projection might be higher than that conveyed by the subicular projection. These results indicate that the entorhinal cortex, CA1, and the subiculum are connected reciprocally and topographically. We believe that the framework of the major hippocampal circuit proposed in previous studies should be reconsidered. We propose that the CA1 projection, rather than the subicular projection, is the main projection that feeds back information from the hippocampus to the entorhinal cortex.

We evaluated the conditions of fixation for ultrastructurally demonstrating the endogenous peroxidase (PO) activity of macrophages in biopsied human liver. The application of microwaving and immersion fixation with tannic acid and aldehydes allowed excellent visualization of PO activity in the nuclear envelope (NE), rough endoplasmic reticulum (rER), and cytoplasmic granules (CG), with good preservation of cellular ultrastructures. The macrophages with PO activity showed one of the following five patterns of PO localization: positive in both the NE and rER but negative in the CG (type 1); negative in both the NE and rER but positive in the CG (type 2); negative in the NE but positive in both the rER and CG (type 3); positive in all three (type 4); PO negative (type 5). The type 1 cells resembled typical Kupffer cells, type 2 cells monocytes, and type 3 and 4 cells the exudate-resident macrophages considered to be a transitional form between exudate and resident macrophages. Type 5 cells may also be a transitional form between the exudate and resident macrophage, or an end-stage macrophage derived from exudate macrophages which have lost their PO activity. Tannic-acid-aldehyde immersion fixation with microwaving may be a useful method in the study of the PO activities of macrophages in biopsied human liver specimens.

Different patterns of distribution of chemically different nerve fibers in the rat iris were studied by immunoelectron microscopy. The iris was immunostained either singly for tyrosine hydroxylase (TH), or doubly for substance P (SP) and calcitonin gene-related peptide (CGRP), or triply for TH, SP and CGRP. TH-positive fibers were distributed in the stroma near the iris dilator and within the iris sphincter. SP/CGRP-positive fibers were found mainly in the anterior half of the stroma and partly in the stroma close to the sphincter, but were hardly ever seen in the stroma near the dilator or within the sphincter. Fibers neither positive for TH, SP nor CGRP were observed in the stroma near the dilator and within the sphincter.

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.

Nitric oxide (NO) is an inhibitory signalling molecule in the gastrointestinal (GI) tract that is released from neurons and from leucocytes during inflammation. NO stimulates soluble guanylate cyclase (sGC), elevates cyclic guanosine 3',5'-monophospate (cGMP), and subsequently activates cGMP-dependent protein kinase (PKG). Targets for NO in the guinea pig caecum were investigated by characterizing the cellular distribution of sGC, cGMP and PKG. Immunoreactivity for both isoforms of sGC, sGCalpha1 and sGCbeta1, was observed in the interstitial cells of Cajal (ICC) and enteric neurons in the tunica muscularis. Double labelling with anti-Kit and anti-sGC antibodies showed sGCalpha1 and sGCbeta1-like immunoreactivity (LI) in almost all intramuscular (IM) and myenteric ICC. Neuronal processes with neuronal NO synthase were closely apposed to ICC expressing sGC-LI. Cells with sGC-LI possessed ultrastructural features of ICC-IM: caveolae, close association with nerve bundles and contacts with smooth muscle cells (SMC). Sodium nitroprusside, added with the phosphodiesterase inhibitors (3-isobutyl-1-methylxanthine and zaprinast), enhanced cGMP-LI in almost all ICC and in some enteric neurons. Nerve stimulation also increased cGMP-LI in ICC and enteric neurons. In contrast, no resolvable increase in cGMP-LI was observed in any cells when the sGC inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one was present. ICC and SMC also expressed PKG type I-LI. These data show that ICC express the downstream signalling molecules necessary to transduce nitrergic signals and activate inhibitory pathways and thus are primary targets for NO released from neurons and other cells in the GI tract.

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.

The hippocampus is thought to be an area where the neuronal circuits for short-term memory or the cognitive map may reside. In order to advance theoretical studies and neuronal model simulations of such circuits, the projection of the CA1 pyramidal neurons in the rat dorsal hippocampus, especially in the subiculum, was studied by means of intracellular and extracellular HRP injection. The CA1 pyramidal neurons project principally to the subiculum where each forms a slab-like axonal field 2 mm long along the septotemporal axis, which may be regarded as a module for columnar organization, at a specific rostrocaudal level of the subiculum. The modules of the CA1a pyramidal neurons are disposed in the rostral part of the subiculum, those of the CA1c pyramidal neurons in the caudal part, and those of the CA1b pyramidal neurons in the middle part of the subiculum. The CA1 pyramidal neurons also participate in the construction of the lamellar organization in the hippocampus in that their axon branches run rostrocaudally following the stream of the alvear fibers. The CA1 pyramidal neurons in the dorsal rat hippocampus transfer the topographic map from field CA1 to the subiculum with reversed order in the lamellar direction. The topographical relationship is composed of partially shifted, overlapping slab-like modules. As a result, information conveyed through a lamella will diverge into the subiculum approximately 2 mm wide, and information through a group of lamellae 2 mm wide will converge upon single subicular neurons.

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.

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.

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.

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.

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.

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.

The gastroduodenal junction differs in morphology and function from the stomach and the duodenum. We studied the immunohistochemical distribution of the gap junction protein, connexin43, and the nerve terminal proteins, SNAP-25 and synaptotagmin, in the musculature of the guinea pig gastroduodenal junction. Connexin43-immunopositive structures were distributed throughout the circular layer of the gastroduodenal junction, most densely in the duodenal circular layer. The difference in the distribution patterns of these structures between the stomach and the duodenum was readily observed in the gastroduodenal junction. In the inner part of the circular muscle layer of the gastroduodenal junction, the connexin43-immunopositive structures were relatively few or non-existent, whereas the SNAP-25-containing nerve fibers and synaptotagmin-containing nerve terminals, clearly observed, were numerous. These findings show a heterogeneous distribution of the gap junctions and nerves in the gastroduodenal junction. The results suggest that the gastroduodenal junction has heterogeneous electrical connections among smooth muscle cells via gap junctions, and specific nerve innervation, which regulates gastroduodenal motility.

In the telencephalon, the corticospinal (CS), callosal, and hippocampal commissural neurons are the major types of neurons that have axons crossing the midline of the brain. To understand the mechanisms involved in crossing the midline structure and to examine whether the expression patterns of L1 and TAG-1 in the commissural neurons are similar to those in the spinal cord, we investigated L1 and TAG-1 expression in these neurons in rats by using a double-labeling technique involving retrograde labeling and in situ hybridization. Expression of L1 messenger RNA was detected in the retrogradely labeled CS projection neurons by 1,1`-dioctadecyl-3,3,3`,3`-tetramethylindocarbocyanine perchlorate (DiI) injection into the pons at embryonic day (E) 19, but expression of TAG-1 messenger RNA was not detected in these neurons. Also, after their axons crossed the pyramidal decussation, continued expression of L1 but no expression of TAG-1 in the CS projection neurons was shown by an additional double-labeling experiment involving DiI injection into the spinal cord at postnatal day (P) 1. An immunohistochemical study showed that L1 was continuously present in each level of the CS tract at E21 and P3, but TAG-1 immunoreactivity was not found in any level at any stage. Finally, we examined the expression of L1 and TAG-1 messenger RNAs in the callosal and hippocampal commissure neurons after their axons had crossed the midline by using the double-labeling technique. In both cases, hybridization signals of the L1 and TAG-1 messenger RNAs were observed in the retrogradely labeled neurons at P3. These results suggest that the roles of L1 and TAG-1 in the formation of the commissures in the forebrain are different from their roles in the spinal cord. J. Comp. Neurol. 417:275–288, 2000. © 2000 Wiley-Liss, Inc.

To determine the postganglionic targets of NOS-containing preganglionic neurons, we studied the association of NADPH-diaphorase positive preganglionic fibers and retrogradely labeled postganglionic neurons in the superior cervical ganglion (SCG) in rats. Wheat germ agglutinin-horseradish peroxidase solution was applied to the anterior chamber of the eye, middle cerebral artery, subcutaneous layer of the facial skin, or submucosal layer of the inside of the lip. Two days after tracer application, the rats were perfused with fixative solution. Serial sections of the SCG were stained histochemically for NADPH-diaphorase followed by diaminobenzidine reaction. More than 80% of the labeled postganglionic neurons innervating the structures in the subcutaneous or submucosal layer showed close association with NADPH-diaphorase positive preganglionic nerve terminals; approximately one-third of these labeled neurons were encircled by dense baskets of pericellular terminals. On the other hand, most of the postganglionic neurons innervating the iris (69%) or the cerebral artery (90%) did not show a distinct association with NADPH-diaphorase positive terminals. These results suggest that one of the principal roles of the NOS-containing preganglionic neurons may be in controlling the postganglionic neurons which innervate the structures in the subcutaneous or submucosal layer.

Wheat germ agglutinin-horseradish peroxidase conjugate was injected in the unilateral superior cervical ganglion (SCG), and the projection pathways of postganglionic sympathetic nerve fibers innervating the cochlea were traced in the rat. The labeled axons advanced along the internal carotid artery (ICA), and a few advanced caudally in the major petrosal nerve (MPN) and entered the facial nerve, while the majority ran rostral to the pterygopalatine ganglion at the point where they crossed the MPN in the carotid canal. The rest of the labeled fibers remained on the surface of the ICA and advanced to the cranial cavity. Most of the labeled fibers along the facial nerve joined the cochlear nerve and finally reached the osseous spiral lamina through the spiral ganglion. Some of the labeled fibers ran along the anterior inferior cerebellar artery from the basilar artery which was previously thought to have been the only pathway. We could not find any labeled fiber on the modiolar artery from anterior inferior cerebellar artery in the cochlea. These observations are consistent with our hypothesis that the sympathetic fibers innervating the neural tissues or related structures follow nerve fibers and meninges as matrices of projection pathways rather than arteries.

A component of the perforant path, projection of the entorhinal layer II neurons, was investigated by recovering intracellularly labeled layer II neurons from the medial or intermediate entorhinal cortex in rats. The labeled layer II spiny stellate neurons had axon collaterals in layers I, II, and III of the entorhinal cortex as well as some axon collaterals in the subiculum. The stem axons gave rise to terminal axon branches that covered the entire extent (suprapyramidal blade, crest, and infrapyramidal blade) of the dentate gyrus and the CA2-3 fields in the transverse plane, forming a sheet-like formation. The axon arbor in the hippocampal formation spread up to 2 mm wide in a septotemporal direction. The sheet-like formation of the axon arbors was a narrow layer in the suprapyramidal blade and in the stratum lacunosum-moleculare of the CA2-3 fields. The layer became wider in the crest and infrapyramidal blade of the dentate gyrus. This study shows that the entorhinohippocampal circuit is not a simple circle from single cells level.

The ototoxicity of aminoglycoside and anticancer platinum drug was analysed using an organ culture system. The effect of calcium antagonism on aminoglycoside ototoxicity was investigated by the same system. The inner ears of mice, 16-day embryos, were cultured for 5 days with or without gentamicin (GM) or kanamycin (KM) or streptomycin (SM), or ribostamycin (RSM), including 1, 10, 100, or 1000 micrograms/ml respectively. The 21st gestational day-inner ear was cultured in vitro during 4 days with or without Cisplatin (CDDP) or platinum analog DWA2114R (DWA), including 0.1, 1, or 10 micrograms/ml, respectively. The 16th gestational day-inner ear was cultured in vitro for 5 days with 10 micrograms/ml KM, adding 5 mM Ca2+ or 10 mM Ca2+ to the culture medium. The damages of crista ampullaris and macula utriculi of cultured inner ear were estimated according to the ototoxicity score based on morphological changes by a light microscopic observation of serial sections of the materials. We defined four grades for the damages according to the following criteria; grade 1: damage of apical surface of the hair cells, grade 2: the existence of debris in the endolymph space, grade 3: disappearance of the hair cells, grade 4: degeneration of the supporting cells. Using this system following results were obtained: 1) the effect of aminoglycoside was dose dependent, 2) the order of ototoxicity was following; GM greater than KM greater than SM greater than RSM, 3) the drug concentration of 1000 micrograms/ml is sufficient to study its ototoxic potential in this system, 4) the effect of both CDDP and DWA was obvious at a concentration of 0.1 microgram/ml, 5) DWA showed almost the same ototoxicity as CDDP at the same concentration, 6) Adding 5 mM Ca2+ or 10 mM Ca2+ to the culture medium, the ototoxic damage induced by 10 micrograms/ml of KM was not noticed. A protective effect of Ca2+ against KM ototoxicity was observed in vitro. This organ culture and ototoxicity score system can serve as a useful and adequate model system for evaluating the ototoxicity.

The hippocampus is a neural substrate playing a key role in short-term memory. In order to achieve a better understand-ding of how the hippocampus functions in “learning and memory,” we conducted an intracellular horseradish peroxidase (HRP) study of the CA3 pyramidal neurons and the granule cells of the fascia dentata. The axon of the CA3 pyramidal neurons has two components, the longitudinal association system and the Schaffer collateral system. The latter component is organized in a lamellar fashion and follows the alvear fiber stream. An electron microscopic analysis of myelinated fibers suggested that most myelinated fibers in the hippocampus are organized parallel to the alvear fibers. The mossy fibers of the granule cells, however, do not follow the alvear fiber stream. We propose a new model of the organization of the intrinsic excitatory circuitry of the rat hippocampus in which the distinct lamellar organization of the pyramidal and granule cells creates a crossing neural network.