Honeycomb blocks composed of carbonate apatite, ß-tricalcium phosphate, and hydroxyapatite for bone regeneration: effects of composition on biological responses.

Synthetic scaffolds exhibiting bone repair ability equal to that of autogenous bone are required in the fields of orthopedics and dentistry. A suitable synthetic bone graft substitute should induce osteogenic differentiation of mesenchymal stem cells, osteogenesis, and angiogenesis. In this study, three types of honeycomb blocks (HCBs), composed of hydroxyapatite (HAp), ß-tricalcium phosphate (TCP), and carbonate apatite (CO3Ap), were fabricated, and the effects of HCB composition on bone formation and maturation were investigated. The HC structure was selected to promote cell penetration and tissue ingrowth. HAp and ß-TCP HCBs were fabricated by extrusion molding followed by sintering. The CO3Ap HCBs were fabricated by extrusion molding followed by sintering and dissolution-precipitation reactions. These HCBs had similar macroporous structures: all harbored uniformly distributed macropores (∼160 â€‹µm) that were regularly arrayed and penetrated the blocks unidirectionally. Moreover, the volumes of macropores were nearly equal (∼0.15 â€‹cm3/g). The compressive strengths of CO3Ap, HAp, and ß-TCP HCBs were 22.8 â€‹± â€‹3.5, 34.2 â€‹± â€‹3.3, and 24.4 â€‹± â€‹2.4 â€‹MPa, respectively. Owing to the honeycomb-type macroporous structure, the compressive strengths of these HCBs were higher than those of commercial scaffolds with intricate three-dimensional or unidirectional macroporous structure. Notably, bone maturation was markedly faster in CO3Ap HCB grafting than in ß-TCP and HAp HCB grafting, and the mature bone area percentages for CO3Ap HCBs at postsurgery weeks 4 and 12 were 14.3- and 4.3-fold higher and 7.5- and 1.4-fold higher than those for HAp and ß-TCP HCBs, respectively. The differences in bone maturation and formation were probably caused by the disparity in concentrations of calcium ions surrounding the HCBs, which were dictated by the inherent material resorption behavior and mechanism; generally, CO3Ap is resorbed only by osteoclastic resorption, HAp is not resorbed, and ß-TCP is rapidly dissolved even in the absence of osteoclasts. Besides the composition, the microporous structure of HC struts, inevitably generated during the formation of HCBs of various compositions, may contribute to the differences in bone maturation and formation.

In vitro maturation of porcine oocytes using a defined medium and developmental capacity after intracytoplasmic sperm injection.

To establish a defined in vitro maturation culture system for porcine oocytes, we examined the effects of adding cysteine (Cys) and epidermal growth factor (EGF) to the maturation medium. Furthermore, to evaluate cytoplasmic maturation, we investigated GSH concentrations and embryo development after intracytoplasmic sperm injection (ICSI). The basic media for IVM were modified TCM199 containing 10% newborn calf serum (NBCS) or 0.1% polyvinyl alcohol (PVA), supplemented with amino acids. Adding EGF (10 ng/ml) or EGF + Cys (0.57 mM) to the defined medium (0.1% PVA + amino acids) increased (P < 0.05) the rate of nuclear maturation relative to the defined medium (without these additives). After ICSI, oocytes matured in a medium supplemented with NBCS, Cys and EGF had a higher (P < 0.05) rate of pronuclear formation rate than oocytes matured in the defined IVM medium. Although there was no significant difference in cleavage rates between NBCS- and PVA-containing media supplemented with both Cys and EGF, the rate of blastocyst development was lower (P < 0.05) in the defined medium than in the NBCS-containing medium. Intracellular GSH concentrations of oocytes matured in the NBCS- and PVA-containing media supplemented with both Cys and EGF were higher (P < 0.05) than in oocytes matured in PVA alone or in oocytes before maturation. Adding Cys and EGF to a defined medium for porcine IVM improved rates of nuclear maturation and cleaved oocytes following ICSI, probably due to increased GSH concentrations. Also, embryos derived from oocytes matured in the defined medium (with the addition of Cys and EGF) developed into blastocysts after ICSI.

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.

Nervous control of blood flow microkinetics in the infrared organs of pit vipers.

The pit organ of pit vipers contains a membrane which serves as an infrared retina, processing infrared information by the degree to which the temperature of trigeminal nerve receptors (terminal nerve masses) is raised. The receptors are arranged in a monolayer array within the pit membrane and irrigated by a capillary network which both supplies energy to the terminal nerve masses and serves as a heat exchange mechanism. This mechanism maintains the receptors at a stable temperature level to increase or decrease their sensitivity and to reduce to a minimum the afterimage effect of a moving stimulus. We used a Doppler laser blood flow meter to measure the local changes in blood flow in response to a point heat source (a small soldering iron) and to direct stimuli (red and infrared lasers). Resection of any one of the trigeminal A-delta fiber trunks innervating the pit membrane abolished blood flow response in the area innervated, but resection of the main trunk between the primary neurons and the medulla left the response intact. In addition to the A-delta fibers the pit membrane contains autonomic and sensory C-fiber innervation, but preganglionic resection of parasympathetic neurons, and chemical blocking of postganglionic fibers with atropine and capsaicin had no influence on the blood flow changes. Therefore, on the basis of the rapid response time and the similarity of the blood flow curves to electrophysiological recordings from the receptors, we surmised that all blood flow changes were due to a vasomotor reaction, modulated by the terminal nerve masses directly, resulting in a change in local heat capacity that cools the stimulated receptors back to a basal temperature.

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.

Expression of neuronal type nitric oxide synthase and renin in the juxtaglomerular apparatus of angiotensin type-1a receptor gene-knockout mice.

Angiotensin type-1a (AT1a) receptor gene-knockout (AT1a-/-) mice exhibit chronic hypotension and renin overproduction. In the kidneys of AT1a-/- mice, the activity of neuronal type nitric oxide synthase (N-NOS) was histochemically detected by nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase (NADPHd) reaction combined with N-NOS immunohistochemistry. The localization of renin was detected by immunohistochemistry and the results were analyzed morphometrically. The levels of N-NOS and renin mRNA in the renal cortical tissue were determined by reverse transcription-PCR and Northern blot analysis, respectively. In the renal sections from wild-type mice, NADPHd activity and N-NOS immunoreactivity were localized to the discrete region of the macula densa in contact with the parent glomerulus. In contrast, N-NOS-positive macula densa cells were distributed beyond the original location of the macula densa, occasionally extending to the opposite side of the distal tubules. The mean number of N-NOS positive macula densa cells was significantly increased in AT1a-/- mice (186 per 100 glomeruli) compared with wild-type mice (65 per 100 glomeruli). AT1a-/- mice showed 1.4-times higher N-NOS mRNA levels in the renal cortical tissues than wild-type mice. The plasma renin activity was significantly higher in AT1a-/- mice (205.5 +/- 26.1 ng/ml/hr) than in wild-type mice (8.0 +/- 0.2 ng/ml/hr). The renin-positive areas per glomerulus and renal renin gene expression were 12-times and 2.6-times higher in AT1a-/- mice than in wild-type mice, respectively. These abnormalities, however, were less remarkable in AT1a-/- mice compared with angiotensinogen-knockout mice. When AT1a-/- mice were fed a high-salt diet, the signal intensity of the NADPHd reaction and the number of positively-stained macula densa cells were significantly decreased. The levels of renal cortical N-NOS mRNA were also suppressed by the treatment. Dietary salt loading produced a parallel decrease in plasma renin activity, renal renin-immunoreactive areas, and the levels of renin mRNA without affecting systemic blood pressure. These results provide evidence for the possible involvement of N-NOS at the macula densa in the increased renin production in AT1a-/- mice.

Dietary salt loading decreases the expressions of neuronal-type nitric oxide synthase and renin in the juxtaglomerular apparatus of angiotensinogen gene-knockout mice.

The present study investigates whether neuronal type nitric oxide synthase (N-NOS) in the macula densa participates in the regulation of renal renin expression during altered dietary salt intake in angiotensinogen gene-knockout (Atg-/-) mice. Wild-type (Atg+/+) and Atg+/+ mice were fed a low-salt (0.04% NaCl), normal-salt (0.3% NaCl), or high-salt (4% NaCl) diet for 2 wk. Histochemical staining for NADPH diaphorase (NADPHd) and renin were analyzed morphometrically. Levels of N-NOS and renin mRNA in renal cortical tissues were determined by reverse transcription-PCR and Northern blot analysis, respectively. In animals fed a normal-salt diet, the renal expressions of N-NOS and renin were markedly increased in Atg-/- mice compared with Atg+/+ mice. When mutant mice were fed a high-salt diet, the signal intensity of the NADPHd reaction and the number of positively stained macula densa cells were significantly decreased. The levels of renal cortical N-NOS mRNA were also suppressed by the treatment. These changes were paralleled by decreases in renal renin-immunoreactive areas and the levels of renin mRNA. On the other hand, salt restriction did not produce further significant increases in the renal N-NOS and renin expressions in mutant mice, whereas a parallel inverse relationship was observed between these enzyme expressions and the levels of salt intake in wild-type mice. These results suggest that the N-NOS expression in the macula densa is inversely regulated by salt intake and that the enzyme activity is functionally linked to renal renin production. Salt-modulated renal N-NOS and renin expressions are independent on angiotensin formation in Atg-/- mice.

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.

Contralateral projections by preganglionic neurons to the sympathetic trunk of the puffer fish, Takifugu niphobles.

Previous studies have shown that the sympathetic preganglionic neurons of teleosts send axons to the sympathetic trunk on the contralateral side. After severing the spinal nerve roots at a level proximal to the sympathetic ganglia (i.e., nerve roots containing the preganglionic axons) on one side of puffer fish, Takifugu niphobles, horseradish peroxidase was applied to the other side of the sympathetic trunk. Retrogradely labeled sympathetic preganglionic neurons were found bilaterally in the central autonomic nucleus (a distinct cell column in the rostral part of the spinal cord). The contralaterally labeled neurons were located almost exclusively in the caudal part of the nucleus. These results suggest that some sympathetic preganglionic neurons in teleosts, unlike those in other vertebrates, send their axons across the midline to the contralateral nerve roots.

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.

Spinal and vagal projections to the sympathetic trunk of the wrasse, Halichoeres poecilopterus.

The sympathetic preganglionic neurons of the teleost, Halichoeres poecilopterus, were identified by retrograde axonal tracing. After horseradish peroxidase was applied to the sympathetic trunk, labeled neurons were found at the caudalmost level of the medulla, in the spinal cord near the fourth spinal nerve root (rostral spinal group), and in the spinal cord from rostral to the sixth spinal nerve root to caudal to the tenth spinal nerve root (caudal spinal group). The rostral spinal group has three cell columns segregated mediolaterally from the central gray zone to the lateral funiculus. Labeled neurons were found predominantly on the side ipsilateral to the application. In the caudal spinal group, labeled neurons were found bilaterally in the central gray zone. This condition is different from that previously reported in the puffer fish and filefish. The labeling in the medulla suggests that the preganglionic neurons in the brainstem may send fibers to the sympathetic trunk of this fish, although their peripheral targets are unknown.

Direct projections from the spinal cord to the trigeminal sympathetic ganglion of the puffer fish, Takifugu niphobles.

The sympathetic trunk of teleosts extends into the cranial levels, forming the cranial sympathetic ganglia. When horseradish peroxidase was applied to the trigeminal sympathetic ganglion (a sympathetic ganglion at the level of the trigeminal nerve) of the puffer fish, Takifugu niphobles, retrogradely labeled neurons were found in the central autonomic nucleus (a distinct cell column in the rostral part of the spinal cord). The central autonomic nucleus has been known to contain preganglionic neurons projecting to the sympathetic ganglia at the spinal levels. Thus, the present results indicate that the central autonomic nucleus in the spinal cord of teleosts contains not only preganglionic neurons projecting to the sympathetic ganglia at the spinal levels, but also neurons projecting to the sympathetic ganglia at the cranial levels.

The spinal sympathetic preganglionic cell column in the puffer fish, Takifugu niphobles.

Little is known about the spinal sympathetic organization in teleosts. We examined the location of the sympathetic preganglionic neurons with horseradish peroxidase (HRP) labeling. After HRP application to the sympathetic trunk or celiac ganglion, labeled neurons were found just dorsal - dorsolateral to the central canal. They form a cell column (central autonomic nucleus) at the level of the posterior rootlet of the first spinal nerve to the third spinal nerve. HRP application to the sympathetic trunk produced labeling in almost the entire central autonomic nucleus, but HRP application to the celiac ganglion produced labeling in only the rostral half of the central autonomic nucleus. These results suggest that there is some topographical arrangement in the rostrocaudal part of the central autonomic nucleus. On the other hand, the fact that the sympathetic preganglionic neurons are within a single cell column and have no mediolateral segregation means that the target-related or function-associated mediolateral arrangement found in tetrapods is lacking in this species. We also found some labeling in the central autonomic nucleus after HRP application to the cranial nerves. This may indicate that the preganglionic neurons project to the cranial nerves.

Trigeminal, vagal, and spinal projections of supramedullary cells in the puffer fish, Takifugu niphobles.

The supramedullary cells (SMCs) of teleosts have been studied for nearly 100 years, but their peripheral connections have remained obscure. We examined the supramedullary cells of the puffer fish, Takifugu niphobles, using horseradish peroxidase transport. Horseradish peroxidase labeling was found bilaterally after application to the trigeminal, the posterior branch of the vagal, and the spinal nerves. No labeled neurons were found after application to the anterior or visceral branches of the vagal nerve. Thus, labeled SMCs were found only after application to the nerves containing cutaneous branches. Some rostrocaudal topographical labeling was found after selective application to each of the four branches of the trigeminal nerve. Labeled neurons were more common in the rostral than in the central or caudal part of the SMC region. Some topographical labeling was also found after application to the first, second, and third spinal nerves, but the topography was not very clear, and there was considerable overlap in the distribution of labeled cells. The sum total of labeled SMCs after unilateral horseradish peroxidase application to each peripheral nerve was more than three times the total number of ipsilateral SMCs, indicating that a single SMC projects several peripheral processes into different nerves. From these results, and taking previous studies into consideration, we propose that supramedullary neurons have a phylogenetic relationship with the spinal dorsal cells of the lamprey and with the extramedullary cells of the amphibian embryo.