Parathyroid hormone-related peptide is a potent tumor angiogenic factor.

Rat pituitary malignant tumor cells; mGH3, show hypervascularization in in vivo xenografts and overexpress parathyroid hormone-related peptide (PTHrP) compared to original GH3 cells. To elucidate whether PTHrP is involved in tumor-derived angiogenesis, we examined the effect of PTHrP on vascular endothelial cells both in vitro and in vivo. Results of in vivo diffusion chamber assay showed a clear hypervascularization on the outer surface of diffusion chambers containing mGH3 tumor cell implants but not in those containing GH3 cells. Co-incubation with antisense PTHrP oligonucleotide (10 microM), but not sense or mismatched PTHrP oligonucleotide, suppressed hypervascularization in diffusion chambers. To further examine the role of PTHrP on endothelial cell function, PTHrP(1-34) was added at various concentrations to cultured bovine endothelial cells (BAECs) harvested from the aorta. PTHrP(1-34) did not alter the proliferation or migration of endothelial cells, but rather dose-dependently increased capillary formation by endothelial cells on the collagen gel matrix. Furthermore, 0.1 mM of 8-bromo-cAMP caused a similar increase in tube formation, which was dose-dependently inhibited by H89, a protein kinase A inhibitor. Our results indicate for the first time that PTHrP is a potential paracrine factor acting via the PKA pathway to enhance angiogenesis through capillary tube formation by endothelial cells in malignant pituitary tumors.

Neuronal organization of the optic tectum in the river lamprey, Lampetra japonica: a Golgi study.

The neuronal and laminar organization of the optic tectum (OT) in the river lamprey was studied using the rapid Golgi method. Based primarily on the distribution pattern of the dendrites, the OT neurons were divided into vertical, horizontal and stellate neurons. The river lamprey OT shows a laminar structure consisting of eight concentric strata. The stratum ependymale consists of several rows of ependymal cells. The stratum cellulare periventriculare contains one to two rows of vertical neurons. The stratum fibrosum periventriculare is thin and contains a few vertical neurons. The stratum cellulare et fibrosum internum consists of several alternating cellular and fibrous layers: a large variety of vertical and horizontal neurons are distributed in this stratum. The stratum fibrosum centrale consists of compact horizontal fiber bundles, among which a few horizontal neurons are disseminated. In the stratum cellulare et fibrosum externum, numerous fibers run horizontally in a loosely organized plexus; various types of vertical, horizontal and stellate neurons are distributed among these fibers. The stratum opticum is the main terminal area of the optic nerve, and contains stellate and horizontal neurons. The stratum marginale is a thin layer and consists of sparse populations of vertical and horizontal neurons. Besides the above outer to inner laminar structure, the OT is divided into medial (m-OT) and lateral parts (1-OT), based primarily on the distribution pattern of the dendrites. The dendrites of neurons in the m-OT are distributed almost exclusively within the OT. On the other hand, the dendrites of some neurons in the 1-OT extended into the confines of the torus semicircularis (TS), and conversely, the dendrites of some neurons in the TS are distributed in the 1-OT. These findings are discussed in relation to the neuronal and laminar organization of the OT in other lamprey species and to recent hodological studies.

Neuronal organization of the spinal cord in the red stingray (Dasyatis akajei: chondrichthyes).

The neuronal organization of the spinal cord in red stingray was studied using the rapid Golgi method. The gray matter of the spinal cord was divided into seven laminae: RS-I, RS-II, RS-III, RS-IV, RS-V, RS-VI and RS-VII. RS-I is cell dense lamina which occupies the major part of the dorsal horn and corresponds to laminae I and II of the spinal cord of mammals, birds and reptiles. The neurons of the lamina I are interspersed with those of lamina II, without forming a discrete lamina. RS-II is located at the base of the dorsal horn and is considered to correspond to the nucleus proprius. RS-III and IV form the intermediate zone and are highly reticulated. A few neurons of various shapes and sizes are distributed among the numerous fibers. The nuclei such as the intermediolateral, intermediomedial or Clarke's nucleus cannot be identified in the intermediate zone. RS-V and VI constitute the ventral horn. RS-V occupies the major part of the ventral horn and contains motoneurons which are distributed diffusely, without forming any distinct cell groups. RS-VI is located in the ventromedial part of the ventral horn, contains commissural neurons and correspond to lamina VIII. RS-VII is a small area surrounding the central canal and corresponds to lamina X. Thus, while the major features of the spinal cord of the red stingray can be correlated with those of the spinal cord of mammals, birds and reptiles, the neuronal organization of the spinal cord of the red stingray remains in an undifferentiated state.

Distribution of the parathyroid hormone-related peptide and its receptor in the saccus vasculosus and choroid plexus in the red stingray (Dasyatis akajei: Elasmobranch).

1. The exact role of the parathyroid hormone-related peptide (PTHrP) is not fully understood. We used immunohistochemistry to localize the PTHrP and its receptor in the brain of the red stingray, particularly in the saccus vasculosus (SV) and choroid plexus. 2. Immunoreactive PTHrP and its receptor were detected in the epithelial cells of the SV and the choroid plexus. In addition, the neuronal perikarya in the nucleus of the SV located in the hypothalamus is positive for the PTHrP. 3. No PTHrP-containing neurons were detected in the choroid plexus. Extracts of SV and choroid plexus showed positive reactions against the PTHrP and its receptor antibody in Western blot analysis. 4. High levels of immunoreactive PTHrP were detected in the plasma equivalent to those present in human humoral malignant hypercalcemia. In contrast, the immunoreactive PTHrP concentration in the cerebrospinal fluid was below detectable levels. 5. Our results suggest that the regulation of the PTHrP in the SV differs from that in the choroid plexus in the red stingray.

Neuronal organization of the olfactory bulb in the hagfish, Eptatretus burgeri: a Golgi study.

The neuronal organization of the olfactory bulb (OB) in the hagfish (Eptatretus burgeri) was studied using the rapid Golgi method. Cytologically, two groups of cells, the mitral and stellate cells, were discernible in the OB. Cytoarchitecturally, the OB showed a distinct laminar structure. From the periphery inward, the following four strata were distinguished: the stratum nervosum, stratum glomerulosum, stratum mitrale and stratum stellatum. Olfactory fibers from the olfactory epithelium reach the rostral aspect of the OB and form the stratum nervosum. The olfactory fibers run deeply in the OB, enter the stratum glomerulosum and terminate in the olfactory glomeruli which are arranged in three to four rows. Numerous mitral and a few stellate cells are distributed in periglomerular areas. The dendrites of the mitral cells terminate in one to two glomeruli in tufted terminals, while those of the stellate cells are distributed in periglomerular areas. The stratum mitrale also consists of mitral and stellate cells. The mitral cells in this stratum extend long dendrites to 4-5 widely separated glomeruli and generate axons traveling caudally. The dendrites of the stellate cells are long and are distributed in the stratum glomerulosum and stratum stellatum, as well as within the stratum mitrale. The stratum stellatum occupies a narrow caudal area and consists mainly of stellate cells extending long dendrites to the stratum stellatum and stratum mitrale.

Neuronal organization of the optic tectum in the hagfish, Eptatretus burgeri: a Golgi study.

The neuronal organization of the optic tectum (OT) was studied in the hagfish using the rapid Golgi method. The OT shows laminar structure. Beginning from the ventricular surface, the following four concentric strata are discernible: the stratum ependymale, stratum periventriculare, stratum cellulare et fibrosum, and stratum marginale. The stratum ependymale consists of several rows of ependymal cells and neuroblasts lining the mesencephalic ventricle. The stratum periventriculare contains medium-sized and small neurons whose dendrites extend mainly superficially. The stratum cellulare et fibrosum occupies a wide area and consists of densely packed neurons and fibers. Fibers in this stratum are derived mainly from the bulbar lemniscus and run ventrodorsally in several bundles, among which numerous neurons are embedded. Neurons in the stratum cellulare et fibrosum are divided into large, medium-sized and small neurons whose dendrites are arranged in a network rather than being oriented in any particular direction. Some of these dendrites extend contralaterally through the commissure of the OT. The neurons in the stratum marginale are divided into medium-sized and small neurons whose dendrites extend mainly tangentially. The axons of neurons in the stratum periventriculare and those of a few neurons in the stratum cellulare et fibrosum extend rostromedially and can be traced into the stratum periventriculare. On the other hand, the axons of neurons in the stratum marginale and stratum cellulare et fibrosum run rostrally, turn ventrally and join fiber bundles running dorsoventrally.

A Golgi study on the nucleus sphericus of the striated snake, Elaphe quadrivirgata.

The intrinsic organization of the nucleus sphericus (NS) was studied in the striated snake using the rapid Golgi method. The NS is a large aggregation of cells located in the posterior portion of the telencephalon and is, as a whole, cup-shaped with its hilus oriented in the rostral direction. From the periphery inward, the following three concentric layers were discernible: a marginal layer, mural layer and hilar layer. The marginal layer consists of scattered cells extending dendrites internally toward the hilar layer and externally into the anterior commissure (AC). The mural layer contains densely packed polygonal neurons with dendrites extending internally into the hilar layer and externally toward the marginal layer. The hilar layer consists of scattered cells whose dendrites extend in a transverse direction, distributing mainly in the hilar layer. The axons of the neurons in the marginal and mural layers travel rostromedially, and some axons can be traced into the AC, while those of the neurons in the hilar layer run rostrally, and some are lost among fibers of the accessory olfactory tract (AOT). The afferents to the NS are derived mainly from the AOT and AC. The AOT fibers travel caudally in a thick bundle through the hilus and are distributed totally within the hilar layer, forming a dense fiber plexus. The AC fibers enter the nucleus from the rostromedial aspect and run in an arched course, emitting numerous fine short collaterals.

Terminal patterns of the tegmental afferents in the interpeduncular nucleus: a Golgi study in the mouse.

The intranuclear course, distribution and termination of the tegmental afferents in the interpeduncular nucleus (IP) were studied in the mouse by means of the rapid Golgi method. Primarily on the basis of terminal branching patterns and distribution areas, two types of afferents were distinguished. The type 1 fibers distribute mainly within the rostral half in the form of numerous glomerular endings, the size of which corresponds well with that of the tufted terminal dendrites of the IP neurons. On the other hand, the caudal half of the IP has far fewer fibers than the rostral IP and is innervated by the type 2 fibers, which follow a tortuous course, terminating in dense fiber plexus. Thus, the rostral and caudal IP are innervated in a different fashion by different afferents originating from tegmental regions. These results are discussed in relation to the distribution patterns of another conspicuous afferent system of the IP, the fasciculus retroflexus.

Terminal patterns of the fasciculus retroflexus in the interpeduncular nucleus of the mouse: a Golgi study.

The courses and terminal patterns of the fasciculus retroflexus (FR) in the interpeduncular nucleus (IP) were studied in the mouse, using the rapid Golgi method. Mainly on the basis of the distribution areas and terminal patterns, the FR fibers are divided into two types. The type 1 FR fibers are coarse in contour and take zigzag courses to distribute throughout the entire rostral half and core region of the caudal IP. In contrast, the type 2 fibers are fine, travel caudally along the lateral boundary of the IP and terminate in the lateral regions of the caudal half, forming a dense fiber plexus. The distribution areas of the type 1 and type 2 fibers are clearly differentiated from each other, from the cytoarchitectural as well as the fibroarchitectural viewpoint. Thus, the type 1 and type 2 FR fibers form different fiber systems in the IP. These results are discussed in the light of the known hodological, histochemical and ultrastructural studies.

Differentiation of the brain stem structures in the salamander, Hynobius nebulosus.

Differentiation of the internal structure of the brain stem was analyzed in the salamander with special reference to neurons distributed in the marginal layer. It was found that the salamander brain stem was at first composed exclusively of the mantle layer. The marginal layer later differentiated peripherally. In these developmental stages, the mantle and marginal layers were clearly differentiated: the former was made up exclusively of the somata, while the latter was composed mainly of nerve fibers. As the development proceeded, these organization patterns were modified: a few cells migrated into the marginal layer. Cells migrating into the marginal layer formed various nuclei and layers such as the raphe nuclei, reticular formation and superficial cellular layers of the optic tectum. In later development stages, fibers in the marginal layer were myelinated, and neurons in the marginal layer were observed to become embedded among numerous myelinated fibers. Cytologically, the majority of neurons in early developmental stages were unipolar, extending a process peripherally into the marginal layer. In later developmental stages, neurons in a deep zone of the mantle layer remained unipolar, whereas those in the marginal layer and in the superficial zone of the mantle layer differentiated into multipolar cells. Thus, (1) the marginal layer differentiated peripherally as a cell free region; (2) cells in the mantle layer later migrated into the marginal layer, changing into multipolar neurons; (3) cells in the marginal layer formed reticular formation as well as various nuclei and layers in the peripheral white matter; and (4) as development proceeded, fibers in the marginal layer became myelinated.

A Golgi study on the olfactory bulb in the red stingray, Dasyatis akajei.

The intrinsic organization of the olfactory bulb (OB) was studied in the red stingray using the rapid Golgi method. The OB is horse shoe-shaped, surrounding the equator region of the nasal capsule. As seen in the sagittal sections, the OB is round with the long olfactory peduncle extending from the dorsocaudal region and the olfactory fibers in a thick bundle entering from the rostroventral aspect. Although not so distinct, the following areas are distinguished. A rostroventral ovoid area adjacent to the entrance of the olfactory fibers consists exclusively of the olfactory fibers running in various directions. Dorsocaudal to the olfactory fiber area is a wide crescent region containing thin bundles of olfactory fibers, olfactory glomeruli, mitral cells and a few disseminated granule cells. A narrow crescent area made up of scattered granule cells is located dorsocaudally to the above wide crescent area. The outermost region consists of a fiber layer encapsulating the dorsal to caudal aspect of the OB. Thus, while the major constituents of the vertebrate OB are recognized, the lamination is very obscure.

A Golgi study on the neuronal organization of the habenular ganglion in the red stingray, Dasyatis akajei.

The neuronal organization of the habenular ganglion (HG) was studied in the red stingray using the rapid Golgi method. The HG was made up of the medial (MH) and lateral habenular nucleus (LH), and the former nucleus was further divided into a dorsal, intermediate and ventral subnucleus. Only one type of neurons were observed in the MH, while the LH was composed of two types of neurons. In the left HG cut at the rostrocaudal middle of the ganglion, the LH was located in the dorsolateral region, while the dorsal, intermediate and ventral subnuclei of the MH occupied the dorsomedial, intermediate and ventral portions of the ganglion, respectively. In contrast, the right ganglion seen at this level was composed exclusively of the MH, with the dorsal, intermediate and ventral subnuclei located in the dorsomedial, intermediate and ventral portions, respectively. In the caudal level of the left ganglion, each nucleus was seen almost in the same region as in the level of the rostrocaudal middle, however, three subnuclei of the MH fused with the same subnuclei of the opposite side. In the right ganglion at the caudal level, the LH appeared in the intermediate area. The right LH was far smaller and was located more ventrocaudally than the left LH. On account of the LH, the intermediate subnucleus of the MH was divided into a dorsal and ventral part. The dorsal and ventral subnuclei of the MH remained in the same region as in the rostral level. Thus, the HG of the red stingray exhibited a striking left-right asymmetry, the most remarkable aspect of which was considered to be differences of the size, form and location of the LH between the left and right HG.

A Golgi study on the afferent fibers to the habenular ganglion in the red stingray, Dasyatis akajei.

Afferent fibers to the habenular ganglion (HG) were derived mainly from the stria medullaris thalami (SM), which was roughly divided into a dorsal and ventral bundle. In the left ganglion seen at the level of the rostrocaudal middle, the dorsal bundle gave off collaterals to the lateral habenular nucleus (LH) and dorsal subnucleus of the medial habenular nucleus (MH), while the ventral bundle innervated the intermediate and ventral subnuclei of the MH. On the other hand, in the right ganglion at the level of the rostrocaudal middle, the dorsal subnucleus of the MH was innervated by collaterals from the dorsal bundle of the SM, whereas in the intermediate and ventral subnucleus fibers from the ventral bundle were seen. In the left ganglion at the caudal level, the dorsal and ventral bundle extended medially and joined the same bundle of the opposite side to constitute a dorsal and intermediate component of the habenular commissure, respectively. A third component of the HC, a ventral component, was seen to run between the fasciculus retroflexus of both sides. As in the case of the rostral level, the dorsal bundle of the SM emitted collaterals to the LH and dorsal subnucleus of the MH, while the intermediate and ventral subnuclei of the MH were projected upon by collaterals from the ventral bundle of the SM. At the caudal level of the right ganglion, the dorsal bundle gave off collaterals to the dorsal subnucleus of the MH. In contrast, the LH and the intermediate and ventral subnuclei of the MH were innervated by fibers from the ventral bundle. With regard to terminal patterns of the SM, fibers to the MH gave off many short fine branchlets forming the glomerular structures, whereas those to the LH branched out into numerous terminals to form a dense fiber plexus. Thus, the afferent fibers to the HG in the red stingray exhibited a striking left-right asymmetry.

A Golgi study on the red nucleus in the mouse.

The intrinsic organization of the red nucleus (RN) was studied in the mouse using the rapid Golgi method. Cytoarchitecturally, the RN was divided into the magnocellular (RNmc) and parvocellular parts (RNpc). The former occupied the caudal one-third and the latter formed the rostral two-thirds of the RN. Based primarily on the size of somata, the RN neurons were classified into four types: giant, large, medium-sized and small neurons. Of these, the former two types of neurons were distributed mainly in the RNmc, while the latter two types of neurons were seen mainly in the RNpc. Axons of the RN neurons, at least those of the former three types of neurons, ran medially or caudomedially. Some axons ran across the mesencephalic raphe region to be lost in the medial region of the contralateral tegmentum. Two groups of afferent fibers to the RN were distinguished. Group I afferents were fibers composing the superior cerebellar peduncle. After crossing in the decussation of the superior cerebellar peduncle, these fibers entered the RN from the caudomedial aspect, ran rostrally in the nucleus emitting numerous collaterals. Group II afferents reached the RN from the ventrolateral aspect and traveled mediodorsally to be distributed totally within this nucleus.

Differentiation of the brain stem reticular formation in the triturus, Triturus pyrrhogaster.

The brain stem of the triturus was observed to be initially composed exclusively of the mantle layer. A few days before hatching, a narrow marginal layer differentiated peripherally. At the time of hatching, the marginal layer was clearly visible throughout the brain stem, except for in a medial region of the optic tectum. Approximately one week after hatching, a few cells migrated into the marginal layer, and almost simultaneously, a few fibers in that layer were myelinated. Cells migrating into the marginal layer formed reticular neurons as well as the raphe nuclei and superficial cellular layers of the optic tectum. As the development proceeded, the number of myelinated fibers in the marginal layer increased, and cells in that layer, especially reticular neurons, were seen to be embedded among numerous myelinated fibers, assuming the characteristic features of the reticular formation.

A Golgi study on the accessory olfactory bulb in the snake, Elaphe quadrivirgata.

The intrinsic organization of the accessory olfactory bulb (AOB) in the snake was studied using the rapid Golgi method. A distinct laminar organization was observed in the snake AOB. Beginning with the most superficial surface, the following layers were distinguished: the layer of the vomeronasal fibers, the olfactory glomeruli, the mitral cells, the deep fiber plexus, the granule cells and the ependymal cells. While the general organizational pattern of the snake AOB resembles that of the main olfactory bulb (MOB) and the AOB reported in various vertebrate species, the present study shows that: (1) the external and internal plexiform layers cannot be identified as independent layers and are considered to be included in the mitral cell layer; (2) the afferent and efferent paths, which are disseminated in the granule cell layer in the mammalian MOB, accumulate external to the granule cell layer to form the layer of the deep fiber plexus: and (3) as a result of accumulation of the afferent and efferent paths in the layer of the deep fiber plexus, the granule cell layer is very fiber-sparse. These structural patterns are quite similar to those of the snake MOB.

A Golgi study on the main olfactory bulb in the snake Elaphe quadrivirgata.

The intrinsic organization of the main olfactory bulb in the snake was studied using the rapid Golgi method. A distinct laminar structure was recognized. From the periphery inward, the following layers were distinguished: the layer of the olfactory fibers, the olfactory glomeruli, the mitral cells, the deep fiber plexus, the granule cells and the ependymal cells. Olfactory fibers derived from the nasal cavity reached the entire surface of the bulb, forming a dense fiber plexus, then swung deeply and terminated in the olfactory glomeruli which were arranged in 2-4 rows. The mitral cell layer occupied a wide zone and was composed of scattered mitral cells. The mitral cells had 2-9 primary dendrites proceeding externally to terminate in the olfactory glomeruli and 2-4 secondary dendrites extending tangentially in the mitral cell layer to be distributed therein. The axons of the mitral cells travelled deeply and entered the layer of the deep fiber plexus. The deep fiber plexus was the path for the bulbar efferent and afferent fibers and could be traced caudally as the main olfactory tract, up to the anterior olfactory nucleus and vicinity. The granule cell layer was composed of small cells, the granule cells, packed closely with no special arrangement. The granule cells had long processes which extended superficially to be distributed mainly in the mitral cell layer. The ependymal cells were located at the deepest layer forming the wall of the olfactory ventricle and generated a long process which extended towards the surface to terminate in the peripheral portion of the bulb. In the snake bulb, the well-documented external and internal plexiform layers were considered to be included in the wide mitral cell layer. Thus, while several specific structures were observed, the fundamental organization of the main olfactory bulb in the snake seemed to be identical to that of the main olfactory bulb in various other vertebrate species.

A Golgi study on the inferior olivary nucleus in the red sting ray, Dasyatis akajei.

The intranuclear organization of the inferior olivary nucleus (ION) was studied in the red sting ray, using the rapid Golgi method. The ION neurons had polygonal, triangular or spindle cell bodies which generated 3-5 primary dendrites. These dendrites were relatively straight, sparsely spinous, and distributed mainly within the ION. The axons of the ION neurons extended medially and joined fiber bundles which ran transversely in the ION. Three groups of olivary afferents were distinguished: fibers derived from the tegmental area travelled ventrally and ended totally in the ION, composing a dense fiber plexus; collaterals of fibers which extended in a longitudinal direction in and around the ION distributed mainly in the lateral portion of the ION; and collaterals of fibers which ran transversely in the ION also ended in the ION. Some fibers from these 3 afferent groups converged to form pericellular baskets. Thus, the fundamental organization of the ION in the red sting ray was similar to that of the ION in mammals.

A Golgi study on the olfactory bulb in the lamprey, Lampetra japonica.

The intrinsic organization of the olfactory bulb in the lamprey was studied using the rapid Golgi method. Although not as discrete as in many vertebrates, a laminar organization was recognized. From the periphery inward, the following layers were discernible: the layer of the olfactory fibers, the olfactory glomeruli with the mitral cells, the granule cells, and the ependymal cells. Just beneath the surface of the olfactory bulb, the olfactory fibers extended over the entire bulb forming a dense fiber plexus terminating in the olfactory glomeruli which were arranged in one to two layers internally to the layer of the olfactory fibers. The mitral cells formed no discrete layer and were located mainly around the olfactory glomeruli. The mitral cells in the lamprey were lacking in secondary dendrites, but had two or more primary dendrites which terminated in the olfactory glomeruli. The axons of the mitral cells proceeded inwardly and accumulated diffusely in the granule cell layer which occupied a wide area internally to the layer of the olfactory glomeruli with the mitral cells. The granule cell layer was composed of densely packed small spindle or fusiform axonless cells, the processes of which extended superficially to be distributed in the olfactory glomeruli. At the deepest region of the bulb was a layer of the ependymal cells lining the surface of the olfactory ventricle. The external and internal plexiform layers were not evident. Thus, while the major constituents of the olfactory bulb of the vertebrate could be identified in that of the lamprey, the general laminar organization seemed indiscrete.