Axonal regeneration through the fibrous scar in lesioned goldfish spinal cord.

Spontaneous nerve regeneration beyond the scar frequently occurs in fish after spinal cord lesions, in contrast to mammals. Here we examined the spatiotemporal relationship between the fibrous scar and axonal regeneration in the goldfish. Within 1 week after hemisection of the spinal cord, the open wound was closed by a fibrous scar that was demarcated from the surrounding nervous tissue by the glia limitans, which was immunoreactive for laminin. Within 1 week after hemisection, regenerating axons entered the fibrous scar, and were surrounded by laminin-coated tubular structures continuous with the glia limitans. Regenerating axons that initially entered the fibrous scar were usually accompanied by glial processes. Within 2-3 weeks after hemisection, the tubular structures became enlarged, and the regenerating axons increased in number, fasciculating in the tubules. Glial processes immunoreactive for glial fibrillary acid protein and 5-hydroxytryptamine neurons then entered the tubular structures to associate with the regenerating axons. The tubular structures developed further, creating tunnels that penetrated the fibrous scar, through which the regenerating axons passed. At 6-12 weeks after hemisection, the fibrous scar was smaller and the enlarged tunnels contained many glial processes and several axons. The findings of present study demonstrated that, following spinal lesions in goldfish, regenerating axons enter and pass the scar tissue. The regenerating axons first enter the fibrous scar with glial elements and then grow through laminin-coated tubular structures within the fibrous scar. Invasion by glial processes and neuronal elements into the tubular structures reduces the fibrous scar area and allows for more regenerating axons to pass beyond the fibrous scar.

Increased migration of IgA lymphocytes to VIP nerve fibers after DSS-induced colitis.

Immunoglobulin-positive lymphocytes are present close to vasoactive intestinal polypeptide-positive (VIP(+)) nerve fibers in the lamina propria of the intestinal tract, and have an important role in mucosal defense. The number of immunoglobulin A-positive (IgA(+)) cells close to the epithelial basement membrane and nerve fibers is increased by the administration of lipopolysaccharides, which induce IgA secretion into the intestinal lumen. The relationship between immunoglobulin-positive lymphocytes and the VIP(+) nerve fibers during inflammation, such as in inflammatory bowel disease, however, is not well known. The morphological relationship between immunoglobulin-positive cells and the basement membrane or the VIP(+) nerve fibers in the colon was examined using double immunofluorescent labeling in an inflammatory bowel disease mouse model created by oral administration of dextran sodium sulfate (DSS). DSS administration induced goblet cell loss, crypt loss, intestinal epithelium deformation and infiltration of inflammatory cells in the mucosa. In the colon, the number and percentage of IgA(+) lymphocytes close to the basement membrane and the VIP(+) nerve fibers in the lamina propria increased after DSS administration, in parallel with the pathologic progress in the inflamed tissue. On the other hand, the percentage of immunoglobulin G-positive (IgG(+)) lymphocytes close to the basement membrane and the VIP(+) nerve fibers decreased, although the total number of IgG(+) lymphocytes in the lamina propria increased. We suggest that the immunoglobulin-producing lymphocytes and enteric nerve fibers in the colon normally have a close morphological relationship, and that this relationship is reinforced in a cell-specific manner during inflammation.

Adult neurogenesis with 5-HT expression in lesioned goldfish spinal cord.

In contrast to mammals, spontaneous nerve regeneration occurs in the teleost spinal cord. In the present study, we examined whether neurogenesis is involved in posttraumatic regeneration in the goldfish spinal cord. In intact fish, many spinal cells positive for both a monoclonal neuronal marker (Hu) and bromodeoxyuridine (BrdU) were observed 24 h after i.p. injection of BrdU, suggesting that constant neurogenesis occurs in the goldfish spinal cord. After hemisection of the spinal cord, the number of spinal cells positive for Hu and BrdU was significantly increased around the lesion site. The number of Hu- and BrdU-positive cells reached the maximum level 7 days after hemisection. In intact fish, spinal cells positive for both Hu and BrdU were also observed 5 weeks after BrdU injection, suggesting that newborn neurons survive for a long time. Six weeks after hemisection, the number of surviving Hu- and BrdU-positive cells at the lesion site was significantly increased as compared with that in intact fish, and some of them were also positive for 5-HT. A retrograde tract tracing study showed that the 5-HT+ neurons were close to the regenerated axons passing through the lesion site. These results suggest that adult neurogenesis occurs in the goldfish spinal cord, and that neurogenesis is activated by spinal cord lesion. The newly produced neurons survive a long time at the lesion site, and might participate in the repair of injured tissue and in the regeneration of descending long axons beyond the lesion site.

Selective projections of cholecystokinin-8 immunoreactive fibers to galanin immunoreactive sympathetic preganglionic neurons in a teleost, Stephanolepis cirrhifer.

In the cellular column of sympathetic preganglionic neurons (SPNs) of the filefish Stephanolepis cirrhifer, neurons containing galanin (GAL) form a distinct population projecting specifically to non-adrenergic postganglionic neurons in the celiac and cranial sympathetic ganglia. The present study showed that virtually all of the GAL-immunopositive SPNs made contact with many nerve terminals immunopositive for cholecystokinin octapeptide (CCK-8). GAL-negative preganglionic neurons made contact with only 26% of this type of nerve terminal; CCK-8-immunopositive nerve fibers appeared to project selectively to GAL-immunopositive SPNs with projections to specific targets. The CCK-8-positive nerve fibers might be of primary sensory origin, and participate in the visceral reflexes.

Ultrastructure of the capillary pericytes and the expression of smooth muscle alpha-actin and desmin in the snake infrared sensory organs.

The infrared sensory membranes of pit organs of pit vipers have an extremely rich capillary vasculature that forms many vascular loops, each serving a small number of infrared nerve terminals. We clarified the ultrastructure of capillary pericytes in the pit membranes by scanning and transmission electron microscopy, and examined the immunoreactivity in their cytoplasm to two contractile proteins: smooth muscle alpha-actin (SM alpha-actin) and desmin. The capillary pericytes had two major cytoplasmic processes: thickened primary processes that radiate to embrace the endothelial tube and flattened secondary processes that are distributed widely on the endothelium. Coexpression of SM alpha-actin and desmin was observed in the pericytes of entire capillary segments, and SM alpha-actin was characterized by prominent filament bundles directed mainly at right angles to the capillary long axis. This expression pattern was different from that of capillary pericytes of the scales, where SM alpha-actin was expressed diffusely in the cytoplasm. In a series of electron microscopic sections, we often observed the pericyte processes depressing the endothelial wall. We also observed a close relationship of the pericytes with inter-endothelial cell junctions, and pericyte processes connected with the endothelial cells via gap junctions. From these findings, we surmised that capillary pericytes in the pit membrane have a close functional relationship with the endothelium, and through their contractile and relaxing activity regulate capillary bloodflow to stabilize production of infrared nerve impulses.

Differential distribution of nerve terminals immunoreactive for substance P and cholecystokinin in the sympathetic preganglionic cell column of the filefish Stephanolepis cirrhifer.

Immunoreactivity for substance P and cholecystokinin-8 was examined in the nerve fibers in the central autonomic nucleus, a cell column for sympathetic preganglionic neurons, in the filefish Stephanolepis cirrhifer. Substance P-immunoreactive fibers were distributed throughout the entire rostrocaudal extent, but were more abundant in the caudal part of the column, where substance P-immunoreactive varicosities sometimes made contacts with the sympathetic preganglionic neurons. Cholecystokinin-8-immunoreactive fibers were found almost entirely in the rostral part of the column, where a dense network of varicosities was in close apposition to a considerable number of the sympathetic preganglionic neurons. Double labeling immunohistochemistry showed that substance P fibers and cholecystokin-8 fibers were entirely different, and distinct from serotonin-immunoreactive fibers. By using immunoelectron microscopy, synaptic specialization was sometimes observed between the dendrites of preganglionic neurons and varicosities immunoreactive for substance P and cholecystokinin-8. Substance P- and cholecystokinin-8 fibers were seen from the descending trigeminal tract, through the dorsolateral funiculus and the ventral portion of the dorsal horn, to the central autonomic nucleus. After colchicine treatment, substance P-immunoreactive perikarya were found in the cranial and spinal sensory ganglia. These results suggest that the sympathetic preganglionic neurons of the filefish receive innervation by substance P fibers and cholecystokinin fibers, and that the former might be of primary sensory origin. Topographical distribution of cholecystokinin-8-immunoreactive terminals in the central autonomic nucleus along the rostrocaudal extent might underlie the differential regulation of sympathetic activity via a distinct population of sympathetic preganglionic neurons.

Differential innervation of the goldfish tonic red muscles and twitch white muscles by neuropeptide-immunoreactive motoneurons.

Neuropeptides in the motor nerves innervating the red and white muscles of the goldfish Carassius auratus were examined. In the tonic red muscles, varicose nerve endings immunoreactive for both calcitonin gene-related peptide and substance P were found spread over the surface of the muscle fibers, but in the twitch white muscles only scattered nerve endings immunoreactive for calcitonin gene-related peptide were found. At the electron microscopic observation, dense electron products immunoreactive for calcitonin gene-related peptide and for substance P (SP) were detected in the motor nerve endings making synapses on the muscle fibers of the red muscles. In the spinal cord, all of the motor neurons showed immunoreactivity to calcitonin gene-related peptide, but the motor neurons immunoreactive for substance P were restricted to the ventrolateral group that has been shown to project predominantly to the red muscles. These results suggest that the motor neurons innervating the red and white muscles of the goldfish are distinct in their neuropeptide content. The present study also raises the possibility that SP might be related to the unique physiological properties of the tonic type red muscles, probably by direct binding to the acetylcholine receptors.

Distinct localization and target specificity of galanin-immunoreactive sympathetic preganglionic neurons of a teleost, the filefish Stephanolepis cirrhifer.

Immunoreactivity for galanin was examined in the sympathetic preganglionic neurons in the spinal cord, adrenal glands, sympathetic ganglia, and some sensory ganglia of the filefish Stephanolepis cirrhifer. Galanin-immunoreactive neurons were found only in the rostral part, but not in the caudal part of the central autonomic nucleus (a column of sympathetic preganglionic neurons of teleosts). Many galanin-immunoreactive nerve terminals were found in contact with neurons in the celiac ganglia and the cranial sympathetic ganglia on both sides of the body. Most neurons encircled by galanin-immunoreactive nerve fibers were negative for tyrosine hydroxylase. Galanin-immunoreactive nerve fibers were very sparse in the spinal sympathetic paravertebral ganglia. No galanin-immunoreactive nerve fibers were found in the adrenal glands. No sensory neurons of the trigeminal, vagal, or spinal dorsal root ganglia were positive for galanin-immunoreactivity. These results suggest that galanin-immunoreactive sympathetic preganglionic neurons have distinct segmental localization and might project specifically to a population of non-adrenergic sympathetic postganglionic neurons in the celiac and cranial sympathetic ganglia.

Serotonin-immunoreactive axons in the cell column of sympathetic preganglionic neurons in the spinal cord of the filefish Stephanolepis cirrhifer.

Serotonin-immunoreactive axonal components were observed in the central autonomic nucleus (CAN), a cell column of sympathetic preganglionic neurons in the rostral spinal cord of the filefish Stephanolepis cirrhifer. Serotonin-positive axonal varicosities were seen around neuronal perikarya through the whole rostrocaudal extent of the CAN, although their distribution pattern in the rostral CAN was different from that in the caudal CAN. Electron microscopically, serotonin-positive axonal varicosities were found to make axodendritic and axosomatic synapses on CAN neurons. Many serotonin-positive neuronal cell bodies were seen in the raphe nuclei in the lower brainstem, whereas only a few were found in the spinal cord. Thus most of serotoninergic axons within the CAN were considered to originate from the raphe nuclei in the lower brainstem.

Nitric oxide synthase in the glossopharyngeal and vagal afferent pathway of a teleost, Takifugu niphobles. The branchial vascular innervation.

To examine the presence of nitric oxide synthase (NOS) in the sensory system of the glossopharyngeal and vagus nerves of teleosts, nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd) activity and immunoreactivity for NOS were examined in the puffer fish Takifugu niphobles. The nitrergic sensory neurons were located in the ganglia of both the glossopharyngeal and the vagal nerves. In the vagal ganglion, positive neurons were found in the subpopulations for the branchial rami and the coelomic visceral ramus, but not for the posterior ramus or the lateral line ramus. In the medulla, nitrergic afferent terminals were found in the glossopharyngeal lobe, the vagal lobe, and the commissural nucleus. In the gill structure, the nitrergic nerve fibers were seen in the nerve bundles running along the efferent branchial artery of all three gill arches. These fibers appeared to terminate in the proximal portion of the efferent filament arteries of three gill arches. On the other hand, autonomic neurons innervating the gill arches were unstained. These results suggest that nitrergic sensory neurons in the glossopharyngeal and vagal ganglia project their peripheral processes through the branchial rami to a specific portion of the branchial arteries, and they might play a role in baroreception of this fish. A possible role for nitric oxide (NO) in baroreception is also discussed.

Microvasculature of crotaline snake pit organs: possible function as a heat exchange mechanism.

The infrared sensory membranes of the pit organs of pit vipers have an extremely rich capillary vasculature, which has been noted passim in the literature, but never illustrated or studied in detail. We rendered the pit vasculature visible in various ways, namely, by microinjection of India ink, by a combination of ink and succinate dehydrogenase staining, and by making resin casts for scanning electron microscope study. We also used transmission electron microscopy for identifying the types (arterioles, venules, capillaries) of blood vessels. Then we compared the pit vasculature with that of the retina and the dermis. Good visualization of the vasculature was obtained with both ink and resin injection. Arterioles, venules, and capillaries could be distinguished with all methods used. The monolayer vasculature was denser in the pit membrane than in the retina or skin. Each loop of the network enclosed a small number of infrared receptors so that all receptors were in contact with a capillary on at least one side. The forward-looking areas of the pit had a denser network than side-looking areas. Since infrared rays cause nerve impulses by raising the temperature of individual receptors, the capillary network functions not only as a supplier of energy but also as a cooling mechanism to reduce afterimages. Thus the denser network in the forward-looking areas causes these areas to be more sensitive and have better image resolution than the rest of the membrane.

Gastrin/CCK-ergic innervation of cutaneous mucous gland by the supramedullary cells of the puffer fish Takifugu niphobles.

The supramedullary cells (SMCs) are spinal neurons lying at the dorsal surface of teleosts. In the present study, we examined whether the SMCs of the puffer fish (Takifugu niphobles) might express gastrin/cholecystokinin-immunoreactivity, as observed in some other teleosts. All the SMCs were immunoreactive for gastrin/cholecystokinin. On the other hand, many immunoreactive varicose nerve fibers were also found terminating in the mucous glands in the skin. In addition, immunoreactive fibers were sparsely distributed in the epidermal layer. No neuronal cells other than the SMCs showed gastrin/cholecystokinin-immunoreactivity centrally or peripherally. The results suggest that gastrin/cholecystokinin-immunoreactive axons in the cutaneous mucous glands and epidermal layer are axons of the SMCs. In view of the present findings, the possible nature of SMCs was discussed.

NADPH-diaphorase activity in the vagal afferent pathway of the dogfish, Triakis scyllia.

Nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase activity was examined in the cranial sensory ganglia and brainstem of the banded dogfish, Triakis scyllia. Positive neurons were found in the vagal sensory ganglion projecting to the coelomic organs, but not in those projecting to the gills or the lateral line organs. Nerve terminals in the vagal lobe were also positive. No positive neurons were found in the glossopharyngeal, facial, or trigeminal sensory ganglia. These results suggest that use of nitric oxide in the vagal sensory transmission from the coelomic organs may have been maintained in the evolutionary process from fish to mammals.

Ultrastructure of the crotaline snake infrared pit receptors: SEM confirmation of TEM findings.

BACKGROUND: Crotaline snakes possess a pair of infrared-sensing pit organs that aid the eyes in the detection and apprehension of prey. The morphology of the receptors in the pit organs has been studied by light and transmission electron microscopy, and the ultrastructure of the receptors has been inferred from the results of this work. But this theoretical reconstruction has never been confirmed by any kind of three-dimensional imaging. METHODS: We treated the receptor-containing membrane of the pit organs with potassium hydroxide to remove collagen and expose the receptors, which we then viewed by scanning electron microscopy. RESULTS: We were able to obtain three-dimensional views of all structures previously reported to exist within the receptor-containing membrane: terminal nerve masses formed from free nerve endings, supporting Schwann cells within the nerve masses, unmyelinated and myelinated nerve fibers, a capillary bed, and vacuole cells. CONCLUSIONS: By providing the first three-dimensional views of the infrared receptors, we have confirmed that previous theoretical reconstructions of the receptors were substantially correct and have provided new evidence of the spatial arrangement of the receptors in a monolayer array.

Calcitonin gene-related peptide immunoreactivity in the trigeminal ganglion of Trimeresurus flavoviridis.

Crotaline snakes, which have infrared-sensitive pit organs, provide a good model for linking neuron morphology with sensory modality. In the trigeminal ganglion of the habu, Trimeresurus flavoviridis, cells positive for calcitonin gene-related peptide-like (CGRP) immunoreactivity were found to be of two types, darkly stained and lightly stained. They were pseudo-unipolar, having an axon divided into stem, peripheral branch, and central branch, all of which were 1 micron or less in diameter. Other, CGRP-negative cells in the ganglion were also pseudo-unipolar, but much larger. In configuration, some of the positive cells were similar to the neurons with A-delta fibers, and others to the neurons with C fibers that have been reported by other workers. On the basis of their distribution and density, and physiological studies by other workers, the CGRP-positive cells were judged to be not part of the infrared-receptive system, but to be involved in the transmission of nociception in small fibers.

Somatosensory and visual correlation in the optic tectum of a python, Python regius: a horseradish peroxidase and Golgi study.

In snakes with infrared receptors the optic tectum receives infrared input in addition to visual and general somatosensory inputs. In order to observe their tectal termination patterns in ball pythons, Python regius, we injected horseradish peroxidase (HRP) into the nucleus of the lateral descending trigeminal tract (LTTD) which mediates infrared information, the optic nerve, and the nucleus of the trigeminal descending tract (TTD) which relays general somatosensory information. Fibers from LTTD were found in layers 5-13 of the contralateral optic tectum, and were especially dense in layers 7a-8. Optic nerve fibers terminated in layers 7a-13 of the contralateral tectum, and mainly in layers 12-13. TTD fibers were few, and could be seen in only the rostral half of the contralateral tectum. These fibers were found in layers 5-7b, but mainly in layers 6-7a. Among various types of neurons stained by the Golgi-Cox method, we focused on six types of neurons whose dendritic arborization overlapped with the distribution of the terminals of these sensory afferents described above. It is possible that these different sensory modalities converge on a single neuron of the various types.

Nerve fibers immunoreactive for substance P and calcitonin gene-related peptide in the cervical spinal ventral roots of the mouse.

We demonstrate the existence of nerve fibers possessing substance P (SP) and calcitonin gene-related peptide (CGRP) immunoreactivity in the mouse cervical ventral roots. The distribution of the SP and CGRP fibers was similar, but CGRP fibers were generally more numerous. Both types entered the ventral pia mater or formed hairpin loops, but they did not enter the spinal cord directly through these roots. SP and CGRP fibers in the ventral roots were thin and had many varicosities. We suggest that these SP and CGRP fibers are involved not only in a sensory mechanism, but also in other functions, via the release of SP and CGRP from varicosities in the ventral roots.

Use of a cortico-cancellous bone graft in the repair of a cleft palate in a dog.

We report the successful use of a cortico-cancellous bone graft to repair a cleft of the secondary palate in a 6-month-old Akita dog. The cleft extended from the incisal papilla to the posterior border of the soft palate. It was 10 mm wide in the hard palate and 16 mm in the soft palate. The cleft was repaired using the "push-back" method with single-pedicled mucoperiosteal flaps to close the cleft in the soft tissues, and a free bone graft from the tibia (cortex with cancellous bone attached) to fill the defect in the hard palate. Two and one-half years after the operation the graft was indistinguishable from surrounding bone on radiographs. Maxillary growth appeared to have progressed normally, resulting in normal occlusion.

Visual and infrared input to the same dendrite in the tectum opticum of the python, Python regius: electron-microscopic evidence.

In snakes with infrared receptors, the optic tectum receives input from both the visual and the infrared senses. We investigated the infrared and optic fiber terminations in the tectum with a combination of horseradish peroxidase and degeneration labeling. In addition to synapses by visual and infrared fibers onto individual neurons, we were able to observe for the first time visual and infrared synapses on one and the same dendrite.

Afferent and efferent projections of the glossopharyngeal-vagal nerve in the hagfish.

Anterograde and retrograde transport of horseradish peroxidase was used to examine the afferent and efferent projections of the glossopharyngeal-vagal nerve in the hagfish Eptatretus burgeri. Anterogradely labeled ganglion cells are scattered in the glossopharyngeal-vagal nerve trunk, in the saccular ganglion, and in the brainstem. Afferent fibers of the glossopharyngeal-vagal nerve terminate in both the vagal lobe and the fasciculus communis. Close observation showed no morphological differentiation between these two structures, indicating that they are not separate entities, but a single, continuous structure that is homologous with the nucleus and tractus solitarius of other vertebrates. The median part of this structure (the commissura infima) is displaced more rostrally than the same part of the solitary nucleus in many other vertebrates. Some of the afferent fibers invade the ventral portion of the trigeminal sensory nucleus, which receives the maxillo-mandibular nerve fibers, and terminate there. Our study showed that the hagfish has only one nucleus in the vagal motor system, i.e., the vagal motor nucleus, which contains both parasympathetic and branchiomotor neurons. The dendrites of the vagal motor neurons in the hagfish are more highly developed than those in other vertebrates. This suggests that the motor reflex arc of the glossopharyngeal-vagal nerve in hagfishes may be simpler than in other vertebrates.