Yoshiaki Nojyo

Three-dimensional analysis of the whole axonal arbors originating from single CA2 pyramidal neurons in the rat hippocampus with the aid of a computer graphic technique

The axonal arborization of single pyramidal neurons in field CA2 and the rostral adjacent area of the rat hippocampus was studied with intracellular staining following the pressure microinjection of horseradish peroxidase (HRP) in combination with the immunoperoxidase technique, and was analyzed three-dimensionally with the aid of a computer system. The axonal arbors were composed of two types of axon branches, which were distinguished as the primary and secondary axon branches on the basis of morphological criteria. The axon branches in the ipsilateral hippocampus exhibited almost the contour of the dorsal hippocampus. The large amount of axon branches labeled with HRP in the stratum (str.) oriens of field CA1 was comparable to that in the str. radiatum of the field. The labeled axon branches in the dorsal hippocampus were not distributed uniformly in terminal regions but were focused on the caudolateral CA1a-b subfields. Most primary axon branches ran to a focus along the alvear fibers. The lamellar organization in the CA2 pyramidal neurons may be composed of axon branches running caudally and terminal branches forming a focus. The dense association fibers along the septotemporal axis may connect the lamellar organized circuits to each other. Axon branches in the septal nuclei of each hemisphere formed a rather flat plane. The commissural fibers of the CA2 pyramidal neurons seemed to form a symmetrical projection field in the contralateral side against the median plane. The axonal arbors and dendritic expansion of the pyramidal neurons shown in this study appeared to reveal the whole image of the single CA2 pyramidal neuron.

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

The ganglionic eminence (GE) supplies neurons containing γ‐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. J. Comp. Neurol. 455:238–248, 2003.

Columnar organization in the subiculum formed by axon branches originating from single CA1 pyramidal neurons in the rat hippocampus

An intracellular horseradish peroxidase study combined with immunoperoxidase techniques was carried out on hippocampal CA1 pyramidal neurons in the rat. Most axon branches originating from a single CA1 pyramidal neuron ran caudally and terminated in the subiculum. The individual axon branches of the single pyramidal neurons bifurcated repeatedly in the subiculum and finally formed a slab-like or columnar terminal arborization (250-300 microns wide, 500-550 microns high and 1.8-2.2 mm long). The present results suggest, in association with other data, that the CA1 pyramidal neurons receive afferent information through lamellar organized connections and they send efferent information to the subiculum through columnar organized connections.

c-Kit-negative fibroblast-like cells express platelet-derived growth factor receptor α 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 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 Wv/Wv 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.

Interstitial cells of Cajal are innervated by nitrergic nerves and express nitric oxide–sensitive guanylate cyclase in the guinea-pig gastrointestinal tract

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.

Intracranial trajectories of sympathetic nerve fibers originating in the superior cervical ganglion in the rat: WGA-HRP anterograde labeling study

Intracranial trajectories of sympathetic nerve fibers originating in the superior cervical ganglion (SCG) in the rat were investigated by means of anterograde labeling following the injection of wheat germ agglutinin-horseradish peroxidase conjugate (WGA-HRP) into the unilateral SCG. The trajectory of the sympathetic fiber innervating the pineal gland and its continuing structures was found advancing along the abducent nerve, through the cavernous plexus, then along the trochlear nerve. Labeled sympathetic fibers showed two patterns of distribution in the blood vessels on the basal surface of the brain. The sympathetic fibers originating in the unilateral SCG were intermingled with those fibers from the contralateral SCG in the pineal gland, its continuing structures and the choroid plexus of the third ventricle as well as in the cerebral blood vessels.

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

γ‐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. J. Comp. Neurol. 502:113–125, 2007.

Expression of Hex mRNA in early murine postimplantation embryo development

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.

A whole image of the hippocampal pyramidal neuron revealed by intracellular pressure-injection of horseradish peroxidase

The intracellular pressure-injection of HRP was applied to the rat hippocampus and has brought an excellent presentation of the Golgi-like image of the pyramidal neuron. Rats were allowed to survive for 3 days and brain sections were treated with the PAP-immunohistochemical technique to enhance the sensitivity of HRP neurohistochemistry. The pyramidal neuron densely developed axon branches in the ipsilateral hippocampus and sent the commissural axon to the contralateral hippocampus. Moreover, short axon branches diverged from the commissural axon to the bilateral septal nuclei.

Muscarinic M2 acetylcholine receptor distribution in the guinea-pig gastrointestinal tract

In the enteric nervous system, acetylcholine is the most common neurotransmitter to induce gastrointestinal smooth muscle contractions. Cholinergic signaling is mediated by muscarinic acetylcholine receptors on the surface of smooth muscle cells. Five different muscarinic receptor subtypes (M(1)-M(5)) have been identified and characterized, all of which belong to the superfamily of the G-protein-coupled receptor. The muscarinic M(2) acetylcholine receptor is the major muscarinic receptor subtype expressed by smooth muscle tissues in the gastrointestinal tract, where it is coexpressed with a smaller population of M(3) receptor. In this study, we examined the immunohistochemical distribution of the M(2) receptor using a specific antibody in the guinea-pig gastrointestinal tract. M(2) receptor-like immunoreactivity was mainly observed as associated with smooth muscle cells in the gastrointestinal tract. M(2) receptor-like immunoreactivity in smooth muscle cells was distributed throughout the cell membrane associated with caveolae. In the proximal colon, M(2) receptor-like immunoreactivity in the smooth muscle cells was weak. In the small intestine, interstitial cells of Cajal that possessed neurokinin 1 receptor-like immunoreactivity had intense M(2) receptor-like immunoreactivity. In the proximal colon, intramuscular and myenteric interstitial cells of Cajal exhibited M(2) receptor-like immunoreactivity. These findings indicate that, in the gastrointestinal musculature, M(2) receptors are distributed both in the smooth muscle cells and interstitial cells of Cajal, suggesting that the M(2) receptor elicits smooth muscle cell contraction and the interstitial cells of Cajal are the sites of innervation by enteric cholinergic neurons.