Ken Asamoto

Neurons in Golgi-stain-like images revealed by GFP-adenovirus infection in vivo.

Neurons in the adult brain have a very complex morphology with many processes, including tremendously long axons. Since dendrites and axons play key roles in the input and output of neural information, respectively, the visualization of complete images of these processes is necessary to reveal the mechanism of neural information processing. Here we made a recombinant adenovirus vector which encodes green fluorescent protein (GFP) tagged with a palmitoylation site, a membrane-targeting signal, produced specific antibodies to GFP, and used them as probes for staining the nervous system. In the neocortex, after injection of the recombinant virus and immunoperoxidase staining with the antibodies, many different types of cells were labeled in a Golgi stain-like fashion. Although the number of labeled cells varied depending on the amount of virus injected, the recombinant virus was considered to be infectious to cortical neurons of all cell types without selectivity. In contrast, the viral infection in the cerebellar cortex and superior cervical ganglion showed some selectivity toward the cell type. It is expected that this recombinant virus will be a useful tool for the morphological analysis of neuronal connections, especially the analysis of microcircuitry in the cerebral cortex.

The developmental fate of the rostral/caudal half of a somite for vertebra and rib formation: experimental confirmation of the resegmentation theory using chick-quail chimeras.

To determine whether resegmentation of somites forms the axial skeleton, we traced the development of the rostral and the caudal half of a somite during skeletogenesis in chick-quail chimeras by replacing the rostral or caudal half of a newly formed chick somite with that of a quail somite. The rostral half-somite transplant formed the caudal half of the vertebral body, the entire spinous process and the distal rib, while the caudal half-somite transplant formed the rostral half of vertebral body, the rostral half of spinous process, the vertebral arch, the transverse process and the entire rib. These findings confirm the resegmentation theory except the spinous process and the distal rib.

Do peanut agglutinin receptors on somites control the behavior of neural cells

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.

Heterogeneous distribution of a gap junction protein, connexin43, in the gastroduodenal junction of the guinea pig

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.

NOS-positive preganglionic neurons innervate a subpopulation of postganglionic neurons in superior cervical ganglion in rats

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.

ARBORIZATION PATTERN OF SYMPATHETIC PREGANGLIONIC AXONS IN THE RAT SUPERIOR CERVICAL AND STELLATE GANGLIA

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 microns in the rostrocaudal direction, and about 200 microns 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 microns in the rostrocaudal direction and about 400 microns 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.

Regulation of Cell Number in Formation of the Dorsal Root Ganglion Revealed by Transplantation of Quail Neural Crest Cells into Chick Embryos

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.

2409 Morphological analysis of laterality of sympathetic innervation between spinal cord and celiac ganglion and between celiac ganglion and stomach

KEN ASAMOTO, YULIN YAO, YOSHIAKI NOJYO Celiac ganglion (CC) is the center of abdominal sympathetic nervous system and very interested because the right and left ganglia are fused in the medial region, although they are distinguishable by its triangular shape of the right and elongated of the left ganglion. We studied the laterality of relationship between spinal cord, CG and stomach using anterograde and retrograde labeling techniques. After injection of WGA-HRP into anterior wall of stomach, retrogradely labeled cells are distributed almost equally in both left and right CG. Injection of DiI into right or left CG showed ipsilateral relationship between spinal cord and CG, although very few labeled neurons were shown in intermediolateral nucleus of contralateral side of injection. Anterograde labeling study using WGA-HRP also indicated ipsilateral relationship between spinal cord and CG. Labeled axons and their terminals are dominantly distributed in CG of ipsilateral side.

2422 Morphological analysis of innervation pattern of the sympathetic preganglionic neurons projecting to the adrenal medulla of rat

Hiroyuki Fukuda, Tomoshige Koga It is well known that NKl receptor antagonists abolish vomiting mediated by vagal afferents. Capsaicin in the 4th ventricle has been shown to suppress vomiting as well as the response of medial solitary nucleus (mNTS) neurons to vagal stimulation. These results suggest that substance P is the transmitter in the synapse between emetic vagal afferents and mNTS neurons. To evaluate this possibility, the effects of a new NKl receptor antagonist (GR205171, GR) on retching and mNTS neurons were observed in dogs. Retching was induced by stimulation of abdominal vagal afferents. An i.v. injection of GR (>O.O25mg/kg) abolished retching induced by vagal as well as mNTS stimulation, but did not have any effects on the firing of mNTS neurons in response to vagal stimulation. These results suggest that substance P is not the transmitter in the synapse between emetic vagal afferents and mNTS neurons, and that the active site of this antagonist is a more central portion of the vomiting reflex arc.