Subependymal Layer Research Articles

Expression of NOS, PSA-N-CAM and S100 protein in the granule cell migration pathway of the adult guinea pig forebrain

To investigate the possible role of nitric oxide (NO) in adult neurogenesis and neuron-glial migration in the rostral migratory stream (RMS), we used a double-labeled immunofluorescence technique together with confocal laser scanning microscopy, and examined the localization of nitric oxide synthase (NOS), the highly polysialylated isoform of neural cell adhesion molecule (PSA-N-CAM), and the astroglial marker in brain, S100 protein (S100), throughout the length of the subependymal layer (SEL) to olfactory bulb (OB) pathway of the adult guinea pig forebrain. Blast-like, beaded, clustered immature cellular elements stained for PSA-N-CAM and those having a typical astrocytic phenotypes positive for S100 protein were densely interlaced throughout the entire length of the SEL. Some S100 positive ependymoglial cells (tanycytes) gave off their basal projections into the closely packed PSA-N-CAM immunopositive clusters in the rostral extension of the subependymal zone (SEZre). The SEL was devoid of NOS immunoreactivity. A dense network of punctate, fenestrated and radially oriented immature cellular elements positive both for NOS and PSA-N-CAM intermingled and overlapped in the inner part of the internal granular layer (IGr), whereas in the outer part, PSA-N-CAM expression gradually diminished and the cells shifted to mature bipolar, spherical or spindle-shaped granule cells with uniform cellular contours, which were exclusively immunopositive for NOS. Radially oriented astroglial phenotypes were intertwined with PSA-N-CAM neuronal clusters in the SEL, and were closely apposed to NOS neuronal elements in the IGr. In summary, these results showed a distinct separation of neurons and glia as revealed by PSA-N-CAM and S100 protein immunostaining, and an inverse spatio-temporal correlation of expression between PSA-N-CAM (immature neuroblasts) and NOS (mature neurons) in the adult guinea pig RMS.

Carnosine-like immunoreactivity in astrocytes of the glial tubes and in newly-generated cells within the tangential part of the rostral migratory stream of rodents

In the nervous system, the aminoacylhistidine dipeptide carnosine ( β-alanyl- l-histidine) has been shown to be expressed in the olfactory receptor neurons and in brain astrocytes. [5]Using immunocytochemical techniques, we report here a dense carnosine-like immunoreactivity in the subependymal layer of the rodent forebrain. Since the subependymal layer involves two distinct compartments (astrocytic cells forming glial tubes and newly-generated cells of the rostral migratory stream, here organized to form chains contained within the glial tubes [Brannon-Thomas L. et al. (1996) Glia 17, 1–14; Jancovski A. and Sotelo C. (1996) J. comp. Neurol. 258, 112–124; Lois C. et al. (1996) Science 271, 978–981; Peretto P. et al. (1997) Brain Res. Bull. 42, 9–21], we investigated in detail the cellular distribution of carnosine-like immunoreactivity in this area. By using double labelling techniques with antisera raised against carnosine and specific markers of glial tubes or chains of migrating cells, we show that carnosine-like immunoreactivity is associated with both the compartments. On the other hand, unlike markers of the rostral migratory stream, carnosine-like immunoreactivity was not observed in isolated, migrating cells located outside the subependymal layer, which spread through the olfactory bulb in a radially-oriented manner. This suggests that carnosine is transiently expressed by cells of the rostral migratory stream when moving in the tangentially-oriented part of the migration route. Moreover, we investigated the distribution of carnosine-like immunoreactivity in the postnatal rat forebrain and found that it is detectable in the subependymal layer only starting from the third postnatal week, although it is well known that the dipeptide is present in the olfactory receptor neurons since the embryonic day 16 [Biffo S. et al. (1992) J. chem. Neuroanat. 5, 51–62]. Taken together, these results show that carnosine, other than abundantly present in astrocytes of the glial tubes, is associated to the tangential part of the rostral migratory stream.

Reduced nicotinamide adenine dinucleotide phosphate diaphorase in the spinal cord of dogs

The distribution of somatic, fibre-like and punctate, non-somatic reduced nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase activity was examined in dog spinal cord using horizontal, sagittal and transverse sections. The morphological features of NADPH diaphorase exhibiting neurons divided into six different neuronal types (N1–N6) were described and their laminar distribution specified. Major cell groups were identified in the superficial dorsal horn and around the central canal at all spinal levels, and in the intermediolateral cell column at thoracic level. NADPH diaphorase exhibiting neurons of the pericentral region were distributed in a thin subependymal cell column containing longitudinally-arranged small bipolar neurons with processes penetrating deeply into the intermediolateral cell column and/or running rostrocaudally in the subependymal layer. The second pericentral cell column located more laterally in lamina X contains large, intensely-stained NADPH diaphorase exhibiting neurons with long dendrites radiating in the transverse plane. Neurons of the sacral parasympathetic nucleus seen in segments S 1–S 3 exhibited prominent NADPH diaphorase activity accompanied by heavily-stained fibres extending from Lissauer's tract through lamina I along the lateral edge of the dorsal horn to lamina V. A massive dorsal gray commissure, with high NADPH diaphorase activity, was found in segments S 1–S 3. At the same segmental level a prominent group of moderately-stained motoneurons was detected in the dorsolateral portion of the anterior horn. Fibre-like NADPH diaphorase activity was found in the superficial dorsal horn and pericentral region in all segments studied. Punctate, non-somatic NADPH diaphorase activity was detected in the superficial dorsal horn, in the pericentral region all along the rostrocaudal axis and in the nucleus phrenicus (segments C 4–C 5), nucleus dorsalis (segments Th 2–L 2), nucleus Y (segments S 1–S 3), and the dorsal part of the dorsal gray commissure (S 1–S 3). A schematic diagram documenting the segmental and laminar distribution of NADPH diaphorase activity is given.

Actual problems of the cerebrospinal fluid-contacting neurons.

Cerebrospinal fluid (CSF)-contacting neurons form a part of the circumventricular organs of the central nervous system. Represented by different cytologic types and located in different regions, they constitute a CSF-contacting neuronal system, the most central periventricular ring of neurons in the brain organized concentrically according to our concept. Because the central nervous system of deuterostomian echinoderm starfishes and the prochordate lancelet is composed mainly of CSF-contacting-like neurons, we hypothesize that this cell type represents ancient cells, or protoneurons, in the vertebrate brain. Neurons may contact the ventricular CSF via their dendrites, axons, or perikarya. Most of the CSF-contacting nerve cells send their dendritic processes into the ventricular cavity, where they form ciliated terminals. These ciliated endings resemble those of known sensory cells. By means of axons, the CSF-contacting neurons also may contact the external CSF space, where the axons form terminals of neurohormonal type similar to those known in the neurohemal areas. The most simple CSF-contacting neurons of vertebrates are present in the terminal filum, spinal cord, and oblongate medulla. The dendritic pole of these medullospinal CSF-contacting neurons terminates with an enlargement bearing many stereocilia in the central canal. These cells are also supplied with a 9 x 2 + 2 kinocilium that may contact Reissner's fiber, the condensed secretory material of the subcommissural organ. The Reissner's fiber floating freely in the CSF leaves the central canal at the caudal open end of the terminal filum in lower vertebrates, and open communication is thus established between internal CSF and the surrounding tissue spaces. Resembling mechanoreceptors cytologically, the spinal CSF-contacting neurons send their axons to the outer surface of the spinal cord to form neurosecretory-type terminals. They also send collaterals to local neurons and to higher spinal segments. In the hypothalamic part of the diencephalon, neurons of two circumventricular organs, the paraventricular organ and the vascular sac, of the magnocellular neurosecretory nuclei and several parvocellular nuclei, form CSF-contacting dendritic terminals. A CSF-contacting neuronal area also was found in the telencephalon. The CSF-contacting dendrites of all these areas bear solitary 9 x 2 + 0 cilia and resemble chemoreceptors and developing photoreceptors cytologically. In electrophysiological experiments, the neurons of the paraventricular organ are highly sensitive to the composition of the ventricular CSF. The axons of the CSF-contacting neurons of the paraventricular organ and hypothalamic nuclei terminate in hypothalamic synaptic zones, and those of magno- and parvocellular neurosecretory nuclei also form neurohormonal terminals in the median eminence and neurohypophysis. The axons of the CSF-contacting neurons of the vascular sac run in the nervus and tractus sacci vasculosi to the nucleus (ganglion) sacci vasculosi. Some hypothalamic CSF-contacting neurons contain immunoreactive opsin and are candidates to represent the "deep encephalic photoreceptors." In the newt, cells derived from the subependymal layer develop photoreceptor outer segments protruding to the lumen of the infundibular lobe under experimental conditions. Retinal and pineal photoreceptors and some of their secondary neurons possess common cytologic features with CSF-contacting neurons. They contact the retinal photoreceptor space and pineal recess, respectively, both cavities being derived from the third ventricle. In addition to ciliated dendritic terminals, there are intraventricular axons and neuronal perikarya contacting the CSF. Part of the CSF-contacting axons are serotoninergic; their perikarya are situated in the raphe nuclei. Intraventricular axons innervate the CSF-contacting dendrites, intraventricular nerve cells, and/or the ventricular surface of the ependyma. (ABSTRACT TRUNCATED)

Serotonergic neurons are present and innervate blood vessels in the olfactory bulb of the laboratory shrew, Suncus murinus

The distribution and characteristics of serotonin-immunoreactivity in the olfactory bulb of the laboratory shrew (Suncus murinus, insectivore) was studied immunohistochemically. Serotonergic neurons were found only in the subependymal layer of the main olfactory bulb. These neurons were 8–12 μm in size and bipolar in shape. These serotonergic neurons had smooth nerve fibers which innervate blood vessels located mainly in the subependymal layer of the main olfactory bulb. On the other hand, other serotonergic nerve fibers with varicosities, which must be extrinsic, were detected in most olfactory layers except the olfactory nerve layer. This result suggests that intrinsic serotonergic neurons may control blood vessels and varicose serotonergic nerve fibers may act to modulate the olfactory transmission.

Heterogeneity of antigen expression and lectin labeling on microglial cells in the olfactory bulb of adult rats

Microglia in different layers of the rat olfactory bulb expressed a variety of membrane antigens except for CD4 (OX-35). Bulb microglial cells bearing complement receptor type 3 (OX-42) were ubiquitous and their immunoreactivity varied considerably in different bulb layers. Although very few in number, labeled microglia in all layers also expressed major histocompatibility complex class I antigen (OX-18), leukocyte common antigen (OX-1) and unknown macrophage antigen (ED-2). The latter was localized in cells distributed almost exclusively in the perivascular spaces. The immunoreactivity of ED-1, an unknown cytoplasmic or lysosomal membrane antigen in macrophages, was localized in labeled microglia which were concentrated mainly in the granule cell layer and periglomerular zone of the bulb. A variable number of microglial cells were stained with OX-6 (major histocompatibility complex class II antigen) and they were located predominantly in the periglomerular zone and at the junction between the granule cell layer and the subependymal layer. Ultrastructural study using GSA I-B4 lectin labeling showed that microglia in different layers of the bulb exhibited similar labeling patterns in their subcellular structures. A remarkable feature was the occurrence of some microglia in the olfactory nerve layer, subependymal layer and granule cell layer adjacent to the subependymal layer in which the cells showed intense lectin labeling at their Golgi apparatus, a feature which was absent in microglia of other bulb layers. Present results showed the regional differences in microglial antigen expressions and lectin labeling within the olfactory bulb. It is therefore suggested that the cells subserve very different specific functions depending on their ambient microenvironments. The heterogeneity of microglial functions in the olfactory bulb may be related to the progressive regeneration and degeneration of its containing neurons.

Nonfebrile seizures.

Nonfebrile seizures.

Expression of NCAM-H and S100 Protein in the Rostral Migratory Stream of the Adult Guinea Pig Forebrain.

We demonstrated a double-labeled immunofluorescent cytochemistry under confocal laser scanning microscope, using antibodies against highly polysialylated isoform of neural cell adhesion molecule (NCAM-H), and Ca++ binding astroglial marker in the brain, S100 protein (S100) in the rostral migratory stream (RMS) of the subependymal layer (SEL) of the adult guinea pig forebrain. In the adult olfactory system, NCAM-H is considered to be associated with the continuous generation and growth of granule cells. In the whole length of germinal SEL, varied populations of blast like, beaded, clustered NCAM-H positive elements were densely intermingled with typical astrocytic subpopulations of S100 protein. Some especialized phenotypes of S100 immunopositive ependymoglial tanycytes gave off their basal processes into NCAM-H immunopositive clusters in the rostral extention of subependymal zone (SEZ-re). Our results evinced the complex morphological multitude of neuro-glial (neuronal and neuroglial) coherence in the course of tangential migration of neuronal progenitors in the young adult guinea pig RMS.

Morphological Interactions between Glial Fibrillary Acidic Protein (GFAP)-like Immunoreactive Elements and Lutenizing Hormone Releasing Hormone (LHRH)-like Immunoreactive Nerve Endings in the Median Eminence of the Rat. Double Labeling Immunoelectron Microscopic Study.

In the present study, interactions between lutenizing hormone releasing hormone (LHRH) -like immunoreactive (LHRH-LI) nerve endings and processes of tanycytes and/or astrocytes in the median eminence (ME) particularly its external layer were investigated by double labeling immunoelectron microscopy employing antibodies against glial fibrillary acidic protein (GFAP) and LHRH. GFAP-like immunoreactivity was observed in some tanycytes throughout the ME and/or astrocytes in the subependymal layer of the ME. In the palisade layer of the ME, GFAP-like immunoreactive (GFAP-LI) elements were frequently seen to make intimate contact with LHRH-LI nerve endings. LHRH-LI nerve endings and processes of the tanycytes and/or astrocytes were also found to direct contact with the basement membrane of the pericapillary space. And they were partially engulfed each other in the vicinity of the perivascular region. The above findings strongly suggest that the processes of tanycytes and/or astrocytes with intimate communication of cerebrospinal fluid in the third ventricle may play a role in regulation of secretion of LHRH into the portal vessels.

Glial tubes in the rostral migratory stream of the adult rat.

In the central nervous system cell migration is usually restricted to developmental periods and occurs mainly radially, following the radial glia. Nevertheless, in the subependymal layer of the adult rodent forebrain tangential migration of newly generated neuronal precursors directed to the olfactory bulb, which follow a well-defined pathway without dispersion, has been recently demonstrated. In the present study, by using light microscopic immunocytochemistry for glia-associated antigens (glial fibrillary acidic protein, S-100 and vimentin), and conventional electron microscopy, we observed a dense mesh-work of astrocytic cells and processes throughout the subependymal layer of the adult rat. These cells were organized to form tangentially oriented glial tubes in the subependymal layer of the lateral ventricle and in its rostral extension to the olfactory bulb. Glial tubes were particularly evident within the rostral extension and were widely intercommunicating. Using markers for the proliferating/ migrating cells of the rostral migratory stream (5-bromo-2'-deoxyuridine, PSA-NCAM, class III beta-tubulin), we provide evidence that long chains of PSA-NCAM/beta-tubulin-positive, newly generated cells are consistently observed inside the glial tubes. These results demonstrate the existence of a peculiar glial organization within the subependymal layer of the adult rat, consisting of long astrocytic tubes that likely represent a new type of glial guidance, accounting for the tangential migration of a high number of cells along their restricted pathway, to the olfactory bulb.

Region-specific DNA synthesis in brains of F344 rats following a six-day bromodeoxyuridine infusion.

Prolonged exposure to certain alkylating chemicals induces glial and meningeal tumours in rats, probably resulting from DNA damage to dividing neural cells. The present work evaluated DNA synthesis in the brains of untreated, young adult male F344 rats in order to define a BrdUrd infusion protocol to more adequately assess proliferation in slowly dividing neural cell populations. BrdUrd (2.5 to 160 mg/ml) was administered for 6 days via subcutaneous osmotic pumps. Clinical toxicity was not observed at any dose. The labelling index (LI; % of cells per brain area that incorporated BrdUrd) and unit length labelling index (ULLI; % of cells per meningeal length that incorporated BrdUrd) were calculated for selected regions by counting labelled neural cells in defined areas of the right hemisphere in coronal brain sections. Intensely stained cells were numerous in the cerebral subependymal layer (LI = 35.8%); scattered in cerebral white matter tracts (e.g. corpus callosum and internal capsule; LI = 6.2%) as well as cerebral (ULLI = 4.2%) and cerebellar (ULLI = 3.6%) meninges; and rare in the hippocampus (LI > 0.1%). Mildy stained cells were dispersed in the pons (LI = 2.1%), deep cerebral (LI = 1.8%) and cerebellar (LI = 1.0%) grey matter, and thalamus (LI = 0.3%). Phenotypically, BrdUrd-positive cells in neuropil were glial cell precursors and their progeny, while those associated with meninges were usually located in the superficial subarachnoid space and appeared to be fibrocytes. Using BrdUrd infusion, LI for glial precursors at these sites ranged from two- to 10-fold higher than those reported previously after a brief parenteral pulse dose. These data indicate that continuous BrdUrd infusion for 6 days by subcutaneous osmotic pump is an efficient means of labelling neural cells throughout the brain.

Presence of macrophage migration inhibitory factor (MIF) in ependyma, astrocytes and neurons in the bovine brain

We investigated the immunohistochemical localization of a cytokine macrophage migration inhibitory factor (MIF) in the bovine brain. MIF was present in the ependymal cell linings of the cerebral ventricles throughout. Double immunostaining of the section with anti-glial fibrillary acidic protein (GFAP) antibody and with anti-MIF antibody showed that the astrocytes present in subependymal layer were immunoreactive for MIF. In the hippocampus, the pyramidal cells in the CA3 and CA4 subfields and the granule cells of the dentate gyrus were immunoreactive. The bundles of mossy fibers were stained along their projections to CA3 and CA4 regions. The nuclei of the subpopulation of these MIF-immunoreactive cells were also immunostained. These results indicated the widespread distribution of a cytokine, MIF, in the bovine brain and suggested the possibility that MIF might play additional roles than a proinflammatory mediator role in the brain.

Presence of macrophage migration inhibitory factor (MIF) in ependyma, astrocytes and neurons in the bovine brain

We investigated the immunohistochemical localization of a cytokine macrophage migration inhibitory factor (MIF) in the bovine brain. MIF was present in the ependymal cell linings of the cerebral ventricles throughout. Double immunostaining of the section with anti-glial fibrillary acidic protein (GFAP) antibody and with anti-MIF antibody showed that the astrocytes present in subependymal layer were immunoreactive for MIF. In the hippocampus, the pyramidal cells in the CA3 and CA4 subfields and the granule cells of the dentate gyrus were immunoreactive. The bundles of mossy fibers were stained along their projections to CA3 and CA4 regions. The nuclei of the subpopulation of these MIF-immunoreactive cells were also immunostained. These results indicated the widespread distribution of a cytokine, MIF, in the bovine brain and suggested the possibility that MIF might play additional roles than a proinflammatory mediator role in the brain.

MCD24 expression in the developing mouse brain and in zones of secondary neurogenesis in the adult.

Interactions mediated by cell surface glycoproteins are considered to be crucial during the formation of the nervous system. Using a monoclonal antibody directed to mCD24, a glycosylphos-phatidylinositol-anchored membrane glycoprotein, we have mapped its distribution throughout the mouse cerebral cortex during development and in young adult. Before birth, mCD24 immunoreactivity was observed in the intermediate zone, the cortical plate and the marginal zone, whereas the ventricular zones were immunonegative. After birth, mCD24 expression declined rapidly in the cortex, except in the corpus callosum (and other commissures in the brain) where immunoreactivity was still found until P20. Furthermore, mCD24 expression was maintained in young adults (until P60, at least) in zones of secondary neurogenesis, such as the granule cells of the dentate gyrus, the subventricular zone lining the anterior part of the lateral ventricles and a zone of cells extending between the striatum and the corpus callosum to the centre of the olfactory bulb. In this area mCD24 and polysialic acid neural cell adhesion molecule stainings were superimposed, and this corresponded to the pathway of migration of the olfactory immature neurons (subependymal layer). A layer of ciliated ependymal cells, lining all the ventricular walls, was also immunoreactive for mCD24. Thus, except for these epithelial-like cells, mCD24 was essentially found associated with differentiating postmitotic neurons. Its spatiotemporal expression, both during development and in the adult, is compatible with a role for this glycoprotein in cell surface recognition and in signalling events occurring during neuronal migration and axonal growth.

Ependymal changes in sudden infant death syndrome.

The ependyma may befall a variety of pathogenic noxae during fetal life, resulting in histological changes which may persist after birth but are without clinical manifestations. Eight of a series of 19 children who died suddenly and unexpectedly, where no explanation as to the cause of death was found at autopsy, were shown to have diverse histological features involving the ependyma. Five micrometer paraffin-embedded brain tissue sections including frontal, temporal, occipital, and ventricular horns as well as the fourth ventricle were stained with hematoxylin-eosin (H&E) and luxol fast blue (LFB). Immunohistochemical stains using antibodies to glial fibrillary acidic protein (GFAP), vimentin and S-100 were also performed. Findings included areas of denuded and/or desquamated ependyma, rosettes in different stages of formation, vacuoles and/or pseudocysts, inflammatory changes consisting in macro- and microglial nodules in the subependymal layer, and gliosis. Chronic brain edema was seen in 4 cases. Our findings indicate that ependymal changes in sudden infant death syndrome (SIDS) cases belong to the prenatal or early postnatal period, thus providing, indirectly, a morphological substrate for the previous existence of a noxa that may also affect other CNS areas, and thus being in the position to produce cardiorespiratory control dysfunction.

Cell proliferation in the subependymal layer of the adult mouse in vivo and in vitro.

Pulse labelling experiments with [3H] thymidine (dT) and double labelling experiments with [3H]dT and bromodeoxyuridine (BrdUrd) were carried out on cells of the subependymal layer in the brain of adult normal mice in vivo, in vivo/in vitro and in vitro. The results should (i) lead to information about cell cycle parameters of these cells in the brain of adult mice, since these cells have been studied mostly in the rat brain up to now and (ii) answer the question whether results concerning cell proliferation obtained in vivo correspond with those from brain slices incubated in vitro with or without prelabelling in vivo. In vivo an LI of 20.2 +/- 2.7% (mean +/- SEM) and Ts = 7.2 +/- 0.7 h were found. Furthermore, grain count halving experiments led to a surprisingly short cycle time (Tc) of 11.2-14.2 h. The longer Tc values (18-20 h) reported in the literature for subependymal cells in the rat brain seem to be due to evaluations of different areas around the lateral ventricle without considering the migrating behaviour of these cells which is quite different regionally. The in vitro studies (with or without prelabelling in vivo) showed a significantly reduced LI due to the fact that about 20% of the S phase cells, possibly lying in the middle of S, stopped further DNA synthesis after transfer to culture. This was shown by comparing the cell fluxes at the G1/S and S/G2 borders of in vivo vs. in vitro studies.

Striatal TGF-α: postnatal developmental expression and evidence for a role in the proliferation of subependymal cells

Transforming growth factor alpha (TGF-α) is expressed in the brain and affects cells by binding to the epidermal growth factor receptor (EGF-R). Using a ribonuclease protection assay, we found that TGF-α steady state mRNA levels in the mouse striatum peak during the first week of postnatal life. Temporally this peak correlates with the height of gliogenesis in the subependymal layer (SEL), which lies along the striatal border of the lateral ventricle. In vivo studies demonstrate that TGF-α can stimulate the proliferation of astrocytes, so glial fibrillary acidic protein (GFAP) mRNA levels were measured as well and it was observed that the peak of GFAP expression followed that of TGF-α by 1 week. Furthermore, in a TGF-α deficient mouse, waved-1 (wa-1), a significant reduction of GFAP mRNA levels and immunostaining for GFAP was found in the striatum. Bromodeoxyuridine labeling combined with immunohistochemistry of normal postnatal day 6 brain showed that the proliferating cells in the SEL are EGF-R immunoreactive. In the waved-1 SEL, there were fewer BrdU positive cells and there was a reduced level of [ 3H]thymidine incorporation. EGF-R immunoreactive cells were found in the SEL of the adult mouse brain. Taken together, our data suggest that the TGF-α/EGF-R signaling pathway is involved in postnatal mitogenic events in the brain.

The Development of the Serotonergic Fiber Network of the Lateral Ventricles of the Rat Brain: A Light and Electron Microscopic Immunocytochemical Analysis

The development of the serotonergic innervation of the lateral ventricles of the rat brain during the first five postnatal weeks was studied with immunocytochemical techniques at the light and electron microscopic levels. In the lateral ventricles of newborn rats serotonergic fibers are only rarely seen. During the first postnatal week the number of serotonergic fibers increases but they are straight and thick, bearing only a few varicosities. By the end of the second postnatal week, however, they become finer, exhibit a large number of varicosities, and form a dense supraependymal network. During the following weeks this network becomes slightly denser but the morphology of fibers as well as their distribution pattern remain unchanged. Examination of sagittal vibratome sections revealed that a group of serotonergic fibers leaves the medial forebrain bundle and turning dorsocaudally between the corpus callosum and the caudate/putamen enter the lateral ventricle from its rostral pole. They then spread to form the supraependymal network of the lateral ventricles and probably of the rest of the ventricular system. Ultrastructural analysis showed that serotonin varicosities are located close to the ventricular surface of the ependymal lining but never make synapses with the ependymal cells. Examination of a large number of labeled fibers and varicosities showed that they are never located between the ependymal cells or in the subependymal layer. This finding was confirmed by examining series of semithin sections. On the basis of these and previous findings we suggest that serotonergic fibers arising in the midbrain raphe nuclei enter the lateral ventricle from its rostral pole, form a dense network within the ventricles, and release their content into the cerebrospinal fluid. This system, as judged with morphological criteria, matures by the end of the second postnatal week.

Microvascularization of the cerebellum in the turtle, Pseudemys scripta elegans (Reptilia). A scanning electron microscope study of microvascular corrosion casts, including stereological measurements.

Cerebellar blood supply and microvascular patterns were studied in 12 freshwater turtles Pseudemys scripta elegans by scanning electron microscopy (SEM) of microvascular corrosion casts and histology. Vascular densities were estimated by point counting methods from casts and thin sections (7 microns). Short A2-arterioles and recurrent branches from A3-arterioles supply the capillary bed of the molecular layer, while V2 and V3 venules drain it. The Purkinje cell layer is supplied by horizontal branches ("parallel arteries") of A4 and A3 arterioles, which capillarize toward the granular and molecular layers. V2, V3 and V4 venules drain the areas supplied by A3 arterioles. The deep granular and subependymal layers are supplied by A4 arterioles, those adjacent to the Purkinje cell layer also by A3 arterioles. The areas supplied by A4 arterioles drain via V4 venules. The granular and Purkinje cell layers taken together have an estimated vascular density of 4.1%, while in the molecular layer this value is 3.3%. Our findings largely confirm published data from Testudo geometrica and Pseudemys scripta elegans with respect to gross supply and drainage. The microvascular patterns are similar to those of the human cerebellar cortex, particularly the basic patterns of intracortical arterioles and venules. The molecular layer, like that in the human brain, has a circulatory bed largely independent of that of the Purkinje cell and granular layers. In contrast to the human brain, a cerebellar pial capillary network is present in the brain of the turtle.

Hexokinase I Messenger RNA in the Rat Central Nervous System

Hexokinase I (ATP:d-hexose 6-phosphotransferase, EC 2.7.1.1) is the first enzyme required in the metabolism of glucose in the central nervous system and plays a major role in regulation of the cerebral glycolytic rate. The distribution of hexokinase I mRNA was examined throughout the central nervous system of the rat by use of oligonucleotide probes and in situ hybridization histochemistry. In the rhinencephalon, strong hexokinase I mRNA labeling was demonstrated in the glomerular, mitral, internal granular, and internal plexiform layers, whereas the olfactory nerve, external plexiform, and subependymal layers and ependyma were devoid of labeling. Within the telencephalon, strong labeling was present in all layers (with the exception of the molecular layer) of the cerebral cortex, in the septum, in CA1-4 and dentate gyrus of the hippocampus, and in several amygdaloid nuclei. There was only weak labeling in the nucleus accumbens and caudate putamen. In the diencephalon, there was in general a strong labeling in the epithalamus, in several thalamic nuclei, including the anteriodorsal, anterioventral, anteriomedial, reticular, paravetricular, intermediodorsal, anteriomedial, interanteriomedial, rhomboid, reuniens, and parafascicular thalamic nuclei. Several hypothalamic regions, including the subfornical organ, the medial preoptic area, the suprachiasmatic, supraoptic, paraventricular, dorsomedial, ventromedial nuclei, and the zona incerta, were strongly labeled. In the mesencephalon, there was particularly strong labeling in the pars compacta and reticulata of the substantia nigra, central gray, and red nucleus, in the Darkschewitsch nucleus, and in the medial accessory oculomotor nucleus. In the rhombencephalon, there was strong hybridization in all raphe nuclei, pontine, tegmental, lateral parabrachial, olivary nuclei, and several cranial motor nuclei. All neurons of the locus ceruleus were heavily labeled. Very strong labeling was present in Purkinje and granular cells of the cerebellar cortex. Neurons of the medulla oblongata area postrema, nucleus tractus solitarius, reticular nucleus, nucleus cuneatus and several motor nuclei were strongly labeled. In the spinal cord, labeled cells were present in all laminae, and also neurons of the dorsal root ganglion were heavily labeled. Hexokinase I mRNA was also demonstrated in the epithelium lining the choroid plexus. In the E15 fetus, very strong labeling was seen in the liver, heart, and trigeminal ganglion, with less intense labeling in the brain and other tissues having more moderate labeling. Administration of 2% saline as drinking water resulted in a marked increase in hexokinase I mRNA in the magnocellular neurons of the supraoptic and paraventricular nuclei. In summary, the results show extensive neuronal distribution of hexokinase I mRNA with regional differences in the expression pattern.