Extrinsic Fibers Research Articles

The origin of extrinsic nitrergic axons supplying the human eye.

Nitrergic nerve fibres of intrinsic and extrinsic origin constitute an important component of the autonomic innervation in the human eye. The intrinsic source of nitrergic nerves are the ganglion cells in choroid and ciliary muscle. In order to obtain more information on the origin of extrinsic nitrergic nerves in the human eye, we obtained superior cervical, ciliary, pterygopalatine and trigeminal ganglia from six human donors, and stained them for neuronal nitric oxide synthase (nNOS) and nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-D). In the superior cervical ganglia, nNOS/NADPH-D-positive varicose axons were observed whereas perikarya were consistently negative. Fewer than 1% of perikarya in the ciliary ganglia were labelled for nNOS/NADPH-D. The diameter of nNOS/NADPH-D-positive ciliary perikarya was between 8 and 10 microm, which was markedly smaller than the diameter of the vast majority of negative perikarya in the ciliary ganglion. More than 70% of perikarya in the pterygopalatine ganglia were intensely labelled for both nNOS and NADPH-D. In trigeminal ganglia, 18% of perikarya were nNOS/NADPH-D-positive. The average diameter of trigeminal nNOS/NADPH-D perikarya was between 25 and 45 microm. Pterygopalatine and trigeminal ganglia are the most likely sources for extrinsic nerve fibres to the human eye.

Effects of enamel matrix proteins on tissue formation along the roots of human teeth

Enamel matrix-derived proteins (EMD) are thought to trigger the formation of acellular extrinsic fibre cementum (AEFC), while other reports indicate that EMD may have osteogenic potential. The aim of the present study was to characterize the tissues developing on the root surface following application of EMD. Twelve human periodontitis-affected teeth, scheduled for extraction, were treated with EMD. Two to 6 weeks later, the teeth were extracted, demineralized and processed for embedding in acrylic and epoxy resins. New tissue formation was analysed by light and transmission electron microscopy. New tissue formation on the root was observed in the notch and on both scaled and unscaled root surfaces distant of the notch area in six defects. The newly formed tissues on the root were thick, collagenous, devoid of extrinsic fibres, and had an irregular surface contour. The presence of electron-dense, organic material in the collagenous matrix indicated at least partial mineralization. Embedded cells were numerous and the cells on the matrix surface were very large in size. Abundant rough endoplasmic reticulum and a prominent Golgi complex were evident. The presence of a split between the treated root surfaces and the newly formed tissue was a common observation, as was the presence of bacteria and host cells in the interfacial gap. Following treatment with EMD, a bone-like tissue resembling cellular intrinsic fibre cementum may develop on the root surfaces, instead of AEFC. Furthermore, EMD may both induce de novo formation of a mineralized connective tissue on scaled root surfaces and stimulate matrix deposition on old native cementum. Interfacial bonding appeared to be weak after 6 weeks of healing.

Maladie de Hirschsprung : attitude pratique

Hirschsprung's disease (1/5000 live births) is defined by the congenital absence of neuronal cells in the nervous plexuses in the distal part of the digestive tract. The disease affects the rectum and sigmoid colon in 80% of cases, or is more extensive. Hirschsprung's disease is suspected in cases of low gastrointestinal obstruction in the neonatal period, or in cases of chronic severe constipation in childhood. It is diagnosed by pathological examination of rectal biopsies that include the submucosa. After standard staining, multiple sections are scrutinized for neuronal cells. Acetylcholinesterase staining is performed on a frozen fragment to demonstrate the hyperplasia of cholinergic fibers that is very suggestive of Hirschsprung's disease. This hyperplasia decreases from the rectum to the splenic flexure of the colon. Hyperplasia of extrinsic nerve fibers and rarefaction of neuromuscular junctions in Hirschsprung's disease may be demonstrated immunohistochemically. Differential diagnosis includes chronic intestinal pseudo-obstructions. The treatment for Hirschsprung's disease is, most often, anastomosis of the normally innervated gut to the anal canal. Peri- or pre-operative biopsies assist surgery, but their interpretation is difficult in the transitional zone. The examination of the surgical specimen allows measurement of the aganglionic segment and transitional zone. Different genes (RET, most often) may be involved in sporadic or familial Hirschsprung's disease. Hirschsprung's disease is associated with other digestive or extra-digestive abnormalities in 5 to 30% of patients. Associated abnormalities may delay the diagnosis and treatment of Hirschsprung's disease.

Localization of neurokinin 1 receptor (NK1R) immunoreactivity in rat esophagus.

The aim of the present immunohistochemical study was to investigate the localization of neurokinin 1 receptor (NK1R) in rat esophagus and examine the relationship between NK1Rs and intrinsic cholinergic, nitrergic, or substance P (SP) neurons. NK1R immunoreactivity (IR) was observed on the nerve cell bodies in the myenteric ganglia throughout the esophagus, but not on striated muscles and smooth muscle cells of the muscularis mucosae. The frequency of occurrence of NK1R neurons was highest in the cervical esophagus and lowest in the lower thoracic esophagus. Considerable immunoreactivity was seen on the nerve cell surfaces and was also present in the cytoplasm of cell somas and in the initial part of the axons, but not in any other nerve fibers or terminals. Dogiel type I-like morphology was observed in some of the NK1R neurons; however, the majority exhibited polymorphic morphology. Double immunolabeling indicated that a majority (77%) of the NK1R neurons were immunoreactive for choline acetyltransferase (ChAT), while a minority (23%) were immunoreactive for nitric oxide synthase (NOS)-IR. Most of the NK1R neurons (92%) were innervated by the SP nerve fibers. Triple immunolabeling indicated that 70% of the NK1R neurons were associated with intrinsic SP nerve fibers (without CGRP-IR), 59% were associated with extrinsic SP nerve fibers (with CGRP-IR), and 35% were associated with both intrinsic and extrinsic SP nerve fibers. These results suggest that SP/tachykinin released from the SP nerve fibers of intrinsic and/or extrinsic origin activates the predominantly intrinsic cholinergic neurons via NK1Rs to influence neuronal transmission or motility in rat esophagus.

Immunolocation of proteoglycans and bone-related noncollagenous glycoproteins in developing acellular cementum of rat molars.

To elucidate the roles of proteoglycans of (PGs), bone sialoprotein (BSP), and osteopontin (OPN) in cementogenesis, their distribution was investigated in developing and established acellular cementum of rat molars by an immunoperoxidase method. To characterize PGs, antibodies against five species of glycosaminoglycans (GAGS), chondroitin-4-sulfate (C4S), chondroitin-6-sulfate (C6S), unsulfated chondroitin (C0S), dermatan sulfate (DS), and keratan sulfate (KS) were used. Routine histological staining was also applied. With onset of dentin mineralization, the initial cementum appeared on the dentin surface as a hematoxylin-stained fibril-poor layer. Subsequently, primitive principal fibers attached to the initial cementum. As the acellular cementum containing extrinsic fibers covered the initial cementum, the intal cementum formed the cemento-dentinal junction. Following immunohistochemistry at the earliest time of cementogenesis, the initial cementum was intensely immunoreactive for C4S, C6S, C0S, BSP, and OPN. After the initial cementum was embedded, neither the cemento-dentinal junction nor the cementum was immunoreactive for any GAG species. However, the cementum was immunoreactive for any GAG species. However, the cementum and cemento-dentinal were consistently immunoreactive for BSP. Although the cemento-dentinal junction was consistently immunoreactive for OPN, the remaining cementum showed no significant immunoreactivity. Thus, initial acellular cementogenesis requires a dense accumulation of PGs, BSP, and OPN, which may be associated with the mineralization process independently of collagen fibrils and initial principal fiber attachment.

Universal distribution of c- kit-positive cells in different types of Hirschsprung’s disease : T. Taguchi, S. Suita, K. Masumoto, et al. Pediatr Surg Int 19:273–279, (June), 2003

The deficiency of inhibitory innervation by nonadrenergic, noncholinergic nerves (NANC) seems to be the pathophysiologic background of colonic aperistalsis in Hirschsprung’s disease (HD). Independent of that, the disruption or absence of c-kit–positive cell networks, consisting of the so-called pacemaker “interstitial cells of Cajal” (ICC), should negatively affect colonic function. ICCs mediate gastrointestinal motility by a cell network in the inner border of circular muscle layer and in the intermuscular zone, interposed between nerve endings and smooth muscle cells. The study investigated the distribution of ICCs in long and short segment aganglionosis and analyzed the correlation between ICCs and enteric nerves. Colonic specimens from 9 HD patients were obtained. Six children had short segment aganglionosis, 3 had long segment disease. The control consisted of 3 children with anorectal malformation biopsied during closure of colostomy. c-kit protein was stained for ICC imaging, PGP 9.5 for ganglia and NADPH activity for visualization of neurons. In normoganglionic segments of HD patients, distribution of ICCs and neurons were the same as in the controls. In the oligoganglionic segments, ICCs were decreased within the muscle layer, and the network around myenteric nerve plexus was incomplete. At the inner border of circular muscle, ICCs were present in the short segment cases but not in the long segment cases. In the (distal) aganglionic segments, where numerous nerve bundles and fibers of extrinsic origin could be identified, c-kit–positive cells were decreased slightly in the muscle layers and myenteric plexus, but were even present at the inner border of circular muscle. In the proximal aganglionic segments of long segmental aganglionosis, where no extrinsic innervation was present, even ICCs were absent at the inner border of the muscle layer but present in the intermuscular space. A decrease of c-kit–positive cells in oligo- and aganglionic segments was shown in former studies. In the current examination, ICCs were lacking only at the inner border of circular muscle in long oligo- and proximal aganglionic segments. Here in the vicinity of the submucous plexus space, even NADPH activity was not observed. Probably NANC ganglia and neurons induce c-kit–positive precursor cells to become ICCs. But nerves are not always required for the development of ICCs because c-kit–positive cells were found with reduced density even in the muscle layer of proximal aganglionic segments without any extrinsic nerve fibers. ICCs are not of neurogenic orgin but are influenced by neurons. Neurons are necessary for the development of myenteric ICCs and mature ICC network. The relationship between the presence of ICC in aganglionic segments and aperistalsis is still difficult to explain.

Observation on the Ultrastructure of the Cementurn in Caiman crocodilus

Many reports have regarded Caiman as a very important topic in the study of comparative anatomy, because this creature is a very attractive and significant animal, especially in oral science. It has been made clear by analyzing the structures of Caiman's tooth enamel and dentine that Caiman is in a transitional stage in evolution from amphibia to mammalia, morphologically. However, neither the structure nor the development of its periodontal tissue has been clarified so far.In this investigation, the fine structure of Caiman crocodilus tooth cementum was observed by using light and electron microscopy, in order to clarify the functions of human periodontal tissue, morphologically.The findings indicated that there are at least two layers in Caiman crocodilus cementum, which are named the inner layer of cementum (ILC) and the outer layer of cementum (OLC) in this paper. The ILC is much more mineralized than the OLC and the extrinsic fibers which mainly comprise the ground substance in both layers were revealed. Moreover, there are many cells in the cementum, which are confirmed cementocytes derived from mesenchyme and the epithelial rests of Malassez. Caiman crocodilus tooth cementum still has many primitive features, but has the fundamental structure to play a functional role as human tooth cementum, morphologically.

ワニＣａｉｍａｎ ｃｒｏｃｏｄｉｌｕｓのセメント質の微細構造に関する観察

Many reports have regarded Caiman as a very important topic in the study of comparative anatomy, because this creature is a very attractive and significant animal, especially in oral science. It has been made clear by analyzing the structures of Caiman's tooth enamel and dentine that Caiman is in a transitional stage in evolution from amphibia to mammalia, morphologically. However, neither the structure nor the development of its periodontal tissue has been clarified so far.In this investigation, the fine structure of Caiman crocodilus tooth cementum was observed by using light and electron microscopy, in order to clarify the functions of human periodontal tissue, morphologically.The findings indicated that there are at least two layers in Caiman crocodilus cementum, which are named the inner layer of cementum (ILC) and the outer layer of cementum (OLC) in this paper. The ILC is much more mineralized than the OLC and the extrinsic fibers which mainly comprise the ground substance in both layers were revealed. Moreover, there are many cells in the cementum, which are confirmed cementocytes derived from mesenchyme and the epithelial rests of Malassez. Caiman crocodilus tooth cementum still has many primitive features, but has the fundamental structure to play a functional role as human tooth cementum, morphologically.

Mechanically evoked reflex electrogenic chloride secretion in rat distal colon is triggered by endogenous nucleotides acting at P2Y1, P2Y2, and P2Y4 receptors.

Mechanical activation of the mucosal lining of the colon by brush stroking elicits an intestinal neural reflex and an increase in short circuit current (Isc) indicative of electrogenic chloride ion transport. We tested whether endogenous nucleotides are physiologic regulators of mucosal reflexes that control ion transport. The brush stroking-evoked Isc response in mucosa and submucosa preparations (M-SMP) of rat colon was reduced by the P2Y1 receptor (R) antagonist 2'deoxy-N6-methyl adenosine 3',5'-diphosphate diammonium salt (MRS 2179) and further blocked by tetrodotoxin (TTX). M-SMP Isc responses to serosal application of the P2Y1 R agonist 2-methylthioadenosine-diphosphate (2MeSADP) or the P2Y2/P2Y4 R agonist 5'uridine-triphosphate (UTP) were reduced but not abolished by TTX. The potency profile of nucleotides for increasing Isc was 5'adenosine-triphosphate (ATP; effective concentration at half maximal response [EC50] 0.65 x 10(4) M) congruent with UTP (EC50 1.0 x 10(-4) M) congruent with 2MeSADP (EC50 = 1.60 x 10(-4) M). Mucosal touch and distention-induced Ca2+ transients in submucous neurons were reduced by apyrase and prevented by blocking the P2Y1 R with MRS 2179 and TTX; denervation of the mucosa. It did not occur by touching a ganglion directly. 2MeSADP Ca2+ responses occurred in subsets of neurons with or without substance P (SP) responses. The potency profile of nucleotides on the neural Ca2+ response was 2MeSADP (5 x 10(-7) M) > UTP (6 x 10(-6) M) > ATP (9 x 10(-5) M). The expression of P2Y R immunoreactivity (ir) in nerve cell bodies was in the order of P2Y1 R > P2Y4 R >> P2Y2 R. P2Y1R ir occurred in the cell somas of more than 90% of neuronal nitric oxide synthase, vasoactive intestinal peptide (VIP), calretinin, or neuropeptide Y (NPY)-ir neurons, 78% of somatostatin neurons, but not in calbindin or SP neurons. P2Y2 R ir was expressed in a minority of SP, VIP, NPY, vesicular acetylcholine transporter, and calcitonin gene-related peptide-ir varicose fibers (5-20%) and those surrounding calbindin (5-20%) neurons. P2Y4 ir occurred mainly in the cell somas of 93% of NPY neurons. Reverse transcriptase polymerase chain reaction of the submucosa demonstrated mRNA for P2Y1R, P2Y2, P2Y4, P2Y6, and P2Y12 Rs. Expression of P2Y1, P2Y2, and P2Y4 protein was confirmed by western blots. In conclusion, endogenous nucleotides acting at P2YRs transduce mechanically evoked reflex chloride ion transport in rat distal colon. Nucleotides evoke reflexes by acting primarily at postsynaptic P2Y1 Rs and P2Y4 R on VIP+/NPY+ secretomotor neurons, at P2Y2 Rs on no more than 2% of VIP+ secretomotor neurons, and 2Y2 Rs mainly of extrinsic varicose fibers surrounding putative intrinsic primary afferent and secretomotor neurons. During mucosal mechanical reflexes, it is postulated that P2Y1 R, P2Y2 R, and P2Y4 R are activated by endogenous ATP, UTP, and 5'uridine-diphosphate.

Intrinsic choroidal neurons in the human eye: projections, targets, and basic electrophysiological data.

The chemical coding of intrinsic choroidal neurons (ICNs) has features in common with extrinsic fibers (e.g., from the pterygopalatine ganglion) making it impossible to assess whether a neuronal nitric oxide synthase (nNOS)/vasoactive intestinal polypeptide (VIP)-immunoreactive nerve fiber is of intrinsic or extrinsic origin. Neurobiotin injections into single neurons allow the visualization of projections of these cells and the determination of the origin of target innervation. Thus, this technique was used in the present study to help characterize the organization of the ICN in the human eye. ICNs were visualized with the fluorescent vital dye 4-Di-2-ASP. Electrophysiological properties were determined by means of intracellular recordings. The impaled neurons were iontophoretically filled with neurobiotin. After fixation, immunohistochemistry for neuronal nitric oxide synthase (nNOS), alpha-smooth muscle actin, and calcitonin gene-related peptide (CGRP) was conducted. ICN processes were traced over distances of up to 2.612 micro m. They were found in the immediate vicinity of other nNOS-positive or -negative ICNs and were also found apposed to smooth muscle fibers (vascular and stromal nonvascular). CGRP-positive fibers forming boutons were observed closely associated with ICNs. Electrophysiological recording showed phasic firing without slow afterhyperpolarization, no spontaneous activity, an input resistance of 136 +/-73 MOmega, and a membrane time constant of 7 +/- 1 ms. Apart from the first functional characterization of ICNs, this study provided more precise evidence of reciprocal ICN-to-ICN contacts and innervation of both choroidal nonvascular and vascular smooth muscle. The presented technique offers promising perspectives to further investigate the function of ICNs in ocular homeostasis.

Histological study of deposited cementum in human deciduous teeth with pathological root resorption

Physiological root resorption is a characteristic feature of human deciduous teeth. Pathological root resorption due to apical periodontitis, dental trauma or excessive orthodontic force is also observed in deciduous roots. The root resorption is not continuous, and has resting periods. In the resting period, cementum deposits in resorbed root surface. The deposited cementum in permanent teeth has been reported in detail. However, the deposited cementum in deciduous teeth is unclear. The present study examined apices of roots of human deciduous incisors with apical periodontitis and roots of sound deciduous incisors by light and transmission electron microscopy. Root dentin and original cementum had a severe irregular caved surface. Cementum was partially deposited on the caved root surfaces. The deposited cementum had made the caved root surface relatively flat. The cementum was lax and had some defects. The deposited cementum was belt-like in shape and had a stratified structure. Each layer had various structures consisting of abundant microfibrils and fine granular materials, microfibrils, granular materials, and collagen fibrils, a few fibrils and granular materials and a relatively homogeneous structure. The original cementum had many collagen fibrils, such as intrinsic and extrinsic fibers, and no granular materials or homogeneous structure. Therefore, structure of the deposited cementum was very different from that of original cementum in deciduous teeth and from that of deposited cementum in permanent teeth.

Neuroepithelial cells and associated innervation of the zebrafish gill: a confocal immunofluorescence study.

Peripheral chemoreceptors responsive to hypoxia have been well characterized in air-breathing vertebrates, but poorly in water-breathers. The present study examined the distribution of five populations of neuroepithelial cells (NECs), putative O(2) chemoreceptors, and innervation patterns in the zebrafish gill using whole-mounts and confocal immunofluorescence. Nerve bundles and fibers of the gill were labeled with zn-12 (a zebrafish-specific neuronal marker) and SV2 antisera and NECs were characterized by serotonin (5-HT) immunoreactivity (IR), SV2-IR and the purinoceptor P2X(3)-IR. A zn-12-IR nerve bundle extended the length of the gill filament and gave rise to a nerve plexus surrounding the efferent filament artery (eFA) and a rich network of fibers that innervated both serotonergic and nonserotonergic NECs of the filament and lamellar epithelium. Three populations of serotonergic, SV2-IR neurons intrinsic to the gill filaments are described, one of which provided innervation to NECs of the filament epithelium. Degeneration of nerve fibers in gill arches maintained in explant culture for 2 days revealed the extrinsic origin of nerve fibers of the plexus and lamellae and the innervation of filament NECs by both intrinsic and extrinsic fibers. Intrinsic innervation surrounding the eFA survived in explant cultures, suggesting a mechanism of local vascular control within the gill. In addition, NECs survived in explants after degeneration of extrinsic nerve fibers. Thus, NECs of the zebrafish gill are organized in a manner reminiscent of O(2) chemoreceptors of mammalian vertebrates, suggesting a role in respiratory regulation.

Distribution of vasoactive intestinal polypeptide in the rat heart: effect of guanethidine and capsaicin

Vasoactive intestinal polypeptide (VIP) is believed to coexist with acetylcholine in postganglionic parasympathetic neurones. However, the presence of VIP in extrinsic nerves and/or other types of intrinsic cardiac neurones has not been excluded. The aim of our study was to examine the distribution and origin of VIP-ergic innervation in the rat heart atria using immunocytochemistry and radioimmunoassay (RIA) combined with two types of denervation: sympathectomy, which was produced by guanethidine treatment and sensory denervation achieved by capsaicin administration. In whole-mount preparations of the intact atria, VIP-immunoreactive (IR) nerve fibres and ganglionic cells were found, the latter being much more numerous in the left atria (LA) than in the right ones. Some of VIP-IR nerve fibres forming bundles appeared to be extrinsic in origin. VIP-IR concentrations determined by RIA in the intact rats were significantly higher in the LA than in the right ones (p < 0.01). However, no changes in VIP-IR levels were found in either atrium after both guanethidine and capsaicin treatment protocols, thus indicating that VIP-immunoreactivity is not associated with either sympathetic or sensory innervation. In conclusion, the ganglionated plexus of the rat atria may comprise at least 3 different neuronal populations expressing VIP-positivity: 1. extrinsic preganglionic parasympathetic fibres, 2. intrinsic postganglionic parasympathetic neurones and 3. intrinsic local circuit neurones that do not express a cholinergic phenotype.

Neuronal location of 5-hydroxytryptamine 3 receptor-like immunoreactivity in the rat colon

The presence of 5-hydroxytryptamine 3 receptors on enteric neurons is known from pharmacological data that date back more than 40 years. However, an adequate account of which neurons bear these receptors has not been made because suitable antisera have not been available. We have found that the majority of antisera that have been raised against sequences from the 5-hydroxytryptamine 3 receptor also recognize pre-prosomatostatin. We report that this source of false labeling can be eliminated by pre-incubating the antisera with a peptide designed for this purpose. We have used the pre-absorbed antiserum to localize 5-hydroxytryptamine 3 receptors in the rat colon. Immunoreactive nerve cell bodies occurred in the myenteric and submucosal ganglia. The majority had smooth cell bodies and long, smooth processes, that is, Dogiel type II morphology. The initial segments of the long processes of the Dogiel type II neurons were strongly immunoreactive. About 12% of immunoreactive myenteric nerve cells were of the same or smaller size, and had multiple short filamentous processes. Some of the immunoreactive Dogiel type II neurons were also immunoreactive for calretinin in both plexuses, and the majority were immunoreactive for calbindin in submucosal ganglia. Specific immunoreactivity occurred in non-varicose, but not in varicose, fibers in the myenteric and submucosal ganglia, and in fiber bundles that traversed the longitudinal and circular muscle layers. Immunoreactive varicose fibers were observed only in the mucosa. It is concluded that 5-hydroxytryptamine 3 receptors occur on intrinsic sensory neurons in the rat colon, and on extrinsic sensory nerve fibers that innervate the colon.

Appearance of neurons and glia with respect to the wavefront during colonization of the avian gut by neural crest cells.

The enteric nervous system is formed by neural crest cells that migrate, proliferate, and differentiate into neurons and glia distributed in ganglia along the gastrointestinal tract. In the developing embryo some enteric crest cells cease their caudal movements, whereas others continue to migrate. Subsequently, the enteric neurons form a reticular network of ganglia interconnected by axonal projections. We studied the developing avian gut to characterize the pattern of migration of the crest cells, and the relationship between migration and differentiation. Crest cells at the leading edge of the migratory front appear as strands of cells; isolated individual crest cells are rarely seen. In the foregut and midgut, these strands are located immediately beneath the serosa. In contrast, crest cells entering the colon appear first in the deeper submucosal mesenchyme and later beneath the serosa. As the neural crest wavefront passes caudally, the crest cell cords become highly branched, forming a reticular lattice that presages the mature organization of the enteric nervous system. Neurons and glia first appear within the strands at the advancing wavefront. Later neurons are consistently located at the nodes where branches of the lattice intersect. In the most rostral foregut and in the colon, some neurons initially appear in close association with extrinsic nerve fibers from the vagus and Remak's nerve, respectively. We conclude that crest cells colonize the gut as chains of cells and that, within these chains, both neurons and glia appear close to the wavefront.

Functional GABA B receptors are present in guinea pig nodose ganglion cell bodies but not in peripheral mechanosensitive endings

The effects of the GABA B-selective agonist baclofen were studied on guinea pig nodose ganglion neurones using grease gap and intracellular recording techniques, and on peripheral mechanosensitive endings in the guinea pig oesophagus and stomach with extracellular recordings. GABA dose-dependently reduced the amplitude of the compound action potential of C-type neurones (C spikes, EC 50=30.9 μM), which was prevented by the GABA A antagonist bicuculline (10 μM). The GABA B agonist baclofen (1–300 μM) did not produce any significant effect on the amplitude of C spikes. In microelectrode studies, baclofen (100 μM) evoked hyperpolarisation (by 2.53±0.51 mV, n=6, N=5) in a subset of nodose neurones (6 out of 26, N=18). In seven out of eight neurones ( N=8) with a slow after-hyperpolarisation following action potentials, baclofen significantly inhibited its amplitude by 19±4% ( n=7, p<0.05). GABA (100 μM) evoked a depolarisation of 9.3±2.4 mV (10 nodose neurones, N=9, p<0.05) associated with a decrease in input impedance of 49±12% ( N=4, p<0.05). Baclofen (100–200 μM) did not affect either spontaneous or stretch-evoked firing of distension-sensitive vagal mechanoreceptors of the guinea pig oesophagus and stomach but did inhibit mechanoreceptors in the ferret oesophagus. Antibodies to GABA B receptor 1a splice variants labelled most of the neurones and numerous fibres in the guinea pig nodose ganglion while antibodies to GABA B receptor 1b splice variants stained only nerve cell bodies. There were numerous nerve fibres showing GABA B receptor 1a- and 1b-like immunoreactivity in the myenteric plexus in the guinea pig oesophagus and stomach but not in anterogradely labelled extrinsic vagal nerve fibres. The result indicates that most guinea pig C-type nodose ganglion neurones have GABA B receptors on their cell bodies but their density on distension-sensitive peripheral endings is too low to allow modulation of mechanotransduction. There is a significant species-dependent difference in the expression of GABA B receptors on peripheral vagal mechanosensitive endings.

Neuronal nitric oxide synthase expression in the mouse vomeronasal organ during prenatal development.

The presence and distribution of immunoreactivity for nitric oxide synthase type I and a panel of regulatory neuropeptides was investigated in the vomeronasal organ (VNO) of mouse embryos. Results show that nitric oxide synthase type I is first expressed in putative extrinsic nerve fibers reaching areas of vascular development at embryonic day 16 and in the vomeronasal nerve at embryonic day 15. Immunoreactivity for vasoactive intestinal peptide appears around developing vessels of the VNO during embryonic day 18. No immunoreactivity for atrial natriuretic peptide, substance P and calcitonin gene-related peptide is present in the VNO. It is concluded that, in the mouse, nitric oxide synthesis is a precocious event in the development of peripheral and central neural vomeronasal structures, representing a very early step in the neurochemical maturation of the VNO.

Collagen fiber orientation affects osteoclastic resorption.

The bone surrounding the teeth is constantly remodeled throughout life, particularly during tooth movement and following tooth loss. Alveolar bone proper contains many highly oriented extrinsic fibers, but whether their orientation affects resorption is unknown. To determine if it does, we selected sperm whale cementum to provide large anisotropic substrates resembling extrinsic fiber bone. Slices cut either perpendicular or parallel to the extrinsic fiber axis were cultured for 24 hours with pre-hatched chick osteoclasts. Resorption pit areas and volumes were measured by video-rate reflection confocal microscopic mapping. Pits in the perpendicular group were smaller in area and were deeper than those in the parallel group (median values, 402 micron2 and 572 micron2, n > 950, p < 0.0001; volume/area 5.37 microm and 4.58 microm); volume differences only just reached significance (2,137 micron3 and 2,554 micron3, p < 0.05). These results show that the fiber orientation within a substrate can profoundly influence the shape of resorption lacunae. This pattern of resorption may help in the maintenance of an optimally functioning attachment of periodontal ligament fibers to alveolar bone.

Immunohistochemical and electron microscopic study of extrinsic hepatic reinnervation following orthotopic liver transplantation in rats.

Because little has been known about the morphological and functional consequences of liver transplantation on hepatic autonomic nerves, we examined the time-course of extrinsic hepatic innervation at the level of the porta hepatis of liver allografts. Orthotopic liver transplantation was performed using male Lewis rats. Crosscut tissue specimens were obtained postoperatively for up to 6 months from the porta hepatis of transplanted livers, and processed for immunohistochemical staining for protein gene product 9.5 (PGP 9.5) and growth-associated protein 43 (GAP-43), and for transmission electron microscopy (TEM). Extrinsic nerve fibers at the porta hepatis stained positively for PGP 9.5 throughout the entire study period. In contrast, the immunoreactivity of GAP-43 was negative at postoperative day (POD) 1 and 2. GAP-43-positive nerves were first observed to appear in the porta hepatis at POD 3. The immunoreactivity of GAP-43 remained positive thereafter until 3 months post-OLT, and became negative in all the specimens at 4 months post-OLT. Transmission electron microscopy demonstrated a small number of regenerating axons existing among many degenerating axons at POD 3. At 3 months post-OLT, most regenerating axons had been fully ensheathed by the cytoplasm of Schwann cells, although their density remained at a lower level compared with normal. The results of this study suggest that liver allografts become extrinsically reinnervated, with the regenerating axons reaching the hepatic hilus 3 days after transplantation. The process of extrinsic hepatic reinnervation is considered to almost terminate 4 months after transplantation in rats.

Tachykinins mediate slow excitatory postsynaptic transmission in guinea pig sphincter of Oddi ganglia.

Intracellular recording techniques were used to test whether tachykinins could be mediators of slow excitatory postsynaptic potentials (EPSPs) in guinea pig sphincter of Oddi (SO) ganglia. Application of the tachykinin substance P (SP) onto SO neurons caused a prolonged membrane depolarization that was reminiscent of the slow EPSP in these cells. Pressure ejection of the neurokinin 3 (NK3) receptor-specific agonist senktide caused a similar depolarization; however, no responses were detected on application of NK1 or NK2 receptor agonists. The NK3 receptor antagonist SR-142801 (100 nM) significantly inhibited both SP-induced depolarization and the stimulation-evoked slow EPSP, as did NK3 receptor desensitization with senktide. Capsaicin, which causes the release of SP from small-diameter afferent fibers, induced a depolarization that was similar to the evoked slow EPSP in both amplitude and duration. The capsaicin-induced depolarization was significantly attenuated in the presence of SR-142801. These data indicate that tachykinins, released from extrinsic afferent fibers, act via NK3 receptors to provide slow excitatory synaptic input to SO neurons.