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Bone morphogenetic protein-5 (BMP5), a member of the transforming growth factor-β (TGF-β) superfamily, has many effects in several biological events. Although BMP5 expression has been well reported in the early development of the central nervous system (CNS), there is little information about its expression in the adult CNS. Thus, we analyzed BMP5 expression in the adult rat CNS by immunohistochemistry. Abundant BMP5 expression was observed in most neurons, and their dendrites and axons. Furthermore, strong BMP5 expression was also detected in the neuropil of the gray matters with high plasticity, such as the molecular layer of the cerebellum, locus coeruleus, and nucleus of the solitary tract. In addition, we showed BMP5 expression also in astrocytes, ependymal cells and meninges. Our data suggest that BMP5 is widely expressed throughout the adult CNS, and this abundant expression in the adult brain strongly supports the idea that BMP5 plays important roles not only in the developing brain but also in the adult brain.

Bone morphogenetic proteins (BMPs) exert its biological functions by interacting with membrane bound receptors. However, functions of BMPs are also regulated in the extracellular space by secreted antagonistic regulators. Chordin is an extracellular BMP antagonist that binds BMP-2, 4, and 7 with high affinity and thus interferes with binding to BMP receptors. Although chordin expression has been well described in the early development of the CNS, little information is available for its expression in the adult CNS. We, thus, investigated chordin expression in the adult rat CNS using immunohistochemistry. Chordin was intensely expressed in most neurons, and their dendrites and axons. In addition, abundant chordin expression was also observed in the neuropil of the gray matters where high plasticity is reported, such as the molecular layer of the cerebellum and the superficial layer of the superior colliculus. Furthermore, we found that astrocytes and ependymal cells also express chordin protein. These data indicate that chordin is more widely expressed throughout the adult CNS than previously reported, and its continued abundant expression in the adult brain strongly supports the idea that chordin plays pivotal roles also in the adult brain.

Rodents exhibit aversive behavior toward a diet that lacks at least one of the essential amino acids. We sought to determine whether the particular form of anorexia caused by such diets could be ameliorated by the administration of orexigenic peptides while simultaneously analyzing the neural mechanisms underlying anorexia. Rats were fed a valine-deficient diet, which induced severe anorexia (reducing food consumption by 80%). The severe anorexia was associated with a significant decrease in the cerebrospinal fluid valine concentration and hyper-ghrelinemia. Between 6 and 12 days after initiation of the valine-deficient diet, we injected rats twice daily with valine and/or an orexigenic peptide (ghrelin, neuropeptide Y, or agouti-related protein) either i.p. or i.c.v.. We then measured dietary intake. An i.c.v. valine injection allowed earlier food intake compared with an i.p valine injection and increased the density of c-Fos-positive ependymal cells lining the third ventricle. Whereas an i.c.v. injection of ghrelin or neuropeptide Y increased consumption of the valine-deficient diet, i.p injection of ghrelin or i.c.v. injection of agouti-related protein did not. Following i.c.v. administration of either valine or ghrelin, we did not observe complete recovery of consumption of the valine-deficient diet. This may be due to the ineffectiveness of peripheral ghrelin and central agouti-related protein and/or to conditioned aversion to the valine-deficient diet. Since ghrelin is known to be involved in food anticipatory activities, whether the hyper-ghrelinemia observed in valine-deficient rats play role in foraging behavior other than food intake is the future study to be investigated.

A stem cell's microenvironment, or "niche," is a critical regulator of its behaviour. In the adult mammalian spinal cord, central canal ependymal cells possess latent neural stem cell properties, but the ependymal cell niche has not yet been described. Here, we identify important similarities and differences between the central canal ependymal zone and the forebrain subventricular zone (SVZ), a well-characterized niche of neural stem cells. First, direct immunohistochemical comparison of the spinal cord ependymal zone and the forebrain SVZ revealed distinct patterns of neural precursor marker expression. In particular, ependymal cells in the spinal cord were found to be bordered by a previously uncharacterized sub-ependymal layer, which is relatively less elaborate than that of the SVZ and comprised of small numbers of astrocytes, oligodendrocyte progenitors and neurons. Cell proliferation surrounding the central canal occurs in close association with blood vessels, but unlike in the SVZ, involves mainly ependymal rather than sub-ependymal cells. These proliferating ependymal cells typically self-renew rather than produce transit-amplifying progenitors, as they generate doublets of progeny that remain within the ependymal layer and show no evidence of a lineage relationship to sub-ependymal cells. Interestingly, the dorsal pole of the central canal was found to possess a sub-population of tanycyte-like cells that express markers of both ependymal cells and neural precursors, and their presence correlates with higher numbers of dorsally proliferating ependymal cells. Together, these data identify key features of the spinal cord ependymal cell niche, and suggest that dorsal ependymal cells possess the potential for stem cell activity. This work provides a foundation for future studies aimed at understanding ependymal cell regulation under normal and pathological conditions.

Inter-alpha Inhibitor Proteins (IAIPs) are a family of related serine protease inhibitors. IAIPs are important components of the systemic innate immune system. We have identified endogenous IAIPs in the central nervous system (CNS) of sheep during development and shown that treatment with IAIPs reduces neuronal cell death and improves behavioral outcomes in neonatal rats after hypoxic-ischemic brain injury. The presence of IAIPs in CNS along with their exogenous neuroprotective properties suggests that endogenous IAIPs could be part of the innate immune system in CNS. The purpose of this study was to characterize expression and localization of IAIPs in CNS. We examined cellular expressions of IAIPs in vitro in cultured cortical mouse neurons, in cultured rat neurons, microglia, and astrocytes, and in vivo on brain sections by immunohistochemistry from embryonic (E) day-18 mice and postnatal (P) day-10 rats. Cultured cortical mouse neurons expressed the light chain gene Ambp and heavy chain genes Itih-1, 2, 3, 4, and 5 mRNA transcripts and IAIPs proteins. IAIP proteins were detected by immunohistochemistry in cultured cells as well as brain sections from E18 mice and P10 rats. Immunoreactivity was found in neurons, microglia, astrocytes and oligodendroglia in multiple brain regions including cortex and hippocampus, as well as within both the ependyma and choroid plexus. Our findings suggest that IAIPs are endogenous proteins expressed in a wide variety of cell types and regions both in vitro and in vivo in rodent CNS. We speculate that endogenous IAIPs may represent endogenous neuroprotective immunomodulatory proteins within the CNS.

Brain edema and severe alterations of the glial and endothelial cells have recently been demonstrated in the dystrophin-deficient mdx mouse, an experimental model of Duchenne muscular dystrophy, and an increase in microvessel density in patients affected by Duchenne muscular dystrophy has also been shown. In order to further elucidate the mechanisms underlying the angiogenetic processes occurring in Duchenne muscular dystrophy, in this study we analyzed matrix-metalloproteinase-2 and -9 expression in the brain of 20-month-old mdx and control mice by means of immunohistochemistry, in situ hybridization, immunoblotting and gelatin zymography. Moreover, we studied vascular endothelial growth factor expression by means of Western blot and immunohistochemistry, and by dual immunofluorescence using anti-vascular endothelial growth factor and anti matrix-metalloproteinase-2 and-9 antibodies. Ultrastructural features of the brain choroidal plexuses were evaluated by electron microscopy. Spatial relationships between endothelium and astrocyte processes were studied by confocal laser microscopy, using an anti-CD31 antibody as a marker of endothelial cells, and anti-glial fibrillary acidic protein (GFAP) as a marker of glial cells. The results demonstrate that high expression of matrix-metalloproteinase-2 and matrix-metalloproteinase-9 protein content occurs in mdx brain and in choroidal plexuses where, by in situ hybridization, matrix-metalloproteinase-2 and matrix-metalloproteinase-9 mRNA was localized in the epithelial cells. Moreover, matrix-metalloproteinase-2 mRNA was found in both mdx perivascular astrocytes and blood vessels, while matrix-metalloproteinase-9 mRNA was localized in mdx vessels. Through zymography, increased expression of matrix-metalloproteinase-2 and matrix-metalloproteinase-9 was found in mdx brain compared with the controls. These enhanced matrix-metalloproteinase levels in mdx mice were found to be associated with increased vascular endothelial growth factor expression, as determined by immunoblotting and immunocytochemistry and with ultrastructural alterations of the mdx choroidal epithelial cells and brain vessels, as previously reported [Nico B, Frigeri A, Nicchia GP, Corsi P, Ribatti D, Quondamatteo F, Herken R, Girolamo F, Marzullo A, Svelto M, Roncali L (2003) Severe alterations of endothelial and glial cells in the blood-brain barrier of dystrophic mdx mice. Glia 42:235-251]. Indeed, in the mdx epithelial cells of the plexuses, the apical microvilli were located on the lateral membranes, whereas in the controls they were uniformly distributed over the free ventricular surface. Moreover, by dual immunofluorescence, a colocalization of vascular endothelial growth factor and matrix-metalloproteinase-2 and matrix-metalloproteinase-9 was found in the ependymal and epithelial cells of plexuses in mdx mice and, under confocal laser microscopy, mdx CD-31 positive vessels were enveloped by less GFAP-positive astrocyte processes than the controls. Overall, these data point to a specific pathogenetic role of matrix-metalloproteinase-2 and matrix-metalloproteinase-9 in neurological dysfunctions associated with Duchenne muscular dystrophy.

Adducins alpha, beta and gamma are proteins that link spectrin and actin in the regulation of cytoskeletal architecture and are substrates for protein kinase C and other signaling molecules. Previous studies have shown that expressions of phosphorylated adducin (phospho-adducin) and protein kinase C are increased in spinal cord tissue from patients who died with amyotrophic lateral sclerosis, a neurodegenerative disorder of motoneurons and other cells. However, the distribution of phospho-adducin immunoreactivity has not been described in the mammalian spinal cord. We have evaluated the distribution of immunoreactivity to serine/threonine-dependent phospho-adducin at a region corresponding to the myristoylated alanine-rich C kinase substrate-related domain of adducin in spinal cords of mice over-expressing mutant human superoxide dismutase, an animal model of amyotrophic lateral sclerosis, and in control littermates. We find phospho-adducin immunoreactivity in control spinal cord in ependymal cells surrounding the central canal, neurons and astrocytes. Phospho-adducin immunoreactivity is localized to the cell bodies, dendrites and axons of some motoneurons, as well as to astrocytes in the gray and white matter. Spinal cords of mutant human superoxide dismutase mice having motoneuron loss exhibit significantly increased phospho-adducin immunoreactivity in ventral and dorsal horn spinal cord regions, but not in ependyma surrounding the central canal, compared with control animals. Increased phospho-adducin immunoreactivity localizes predominantly to astrocytes and likely increases as a consequence of the astrogliosis that occurs in the mutant human superoxide dismutase mouse with disease progression. These findings demonstrate increased immunoreactivity against phosphorylated adducin at the myristoylated alanine-rich C kinase substrate domain in a murine model of amyotrophic lateral sclerosis. As adducin is a substrate for protein kinase C at the myristoylated alanine-rich C kinase substrate domain, the increased phospho-adducin immunoreactivity is likely a consequence of protein kinase C activation in neurons and astrocytes of the spinal cord and evidence for aberrant phosphorylation events in mutant human superoxide dismutase mice that may affect neuron survival.

Each day, approximately 0.5-0.9 l of water diffuses through (primarily) aquaporin-1 (AQP1) channels in the human choroid plexus, into the cerebrospinal fluid of the brain ventricles and spinal cord central canal, through the ependymal cell lining, and into the parenchyma of the CNS. Additional water is also derived from metabolism of glucose within the CNS parenchyma. To maintain osmotic homeostasis, an equivalent amount of water exits the CNS parenchyma by diffusion into interstitial capillaries and into the subarachnoid space that surrounds the brain and spinal cord. Most of that efflux is through AQP4 water channels concentrated in astrocyte endfeet that surround capillaries and form the glia limitans. This report extends the ultrastructural and immunocytochemical characterizations of the crystalline aggregates of intramembrane proteins that comprise the AQP4 "square arrays" of astrocyte and ependymocyte plasma membranes. We elaborate on recent demonstrations in Chinese hamster ovary cells of the effects on AQP4 array assembly resulting from separate vs. combined expression of M1 and M23 AQP4, which are two alternatively spliced variants of the AQP4 gene. Using improved shadowing methods, we demonstrate sub-molecular cross-bridges that link the constituent intramembrane particles (IMPs) into regular square lattices of AQP4 arrays. We show that the AQP4 core particle is 4.5 nm in diameter, which appears to be too small to accommodate four monomeric proteins in a tetrameric IMP. Several structural models are considered that incorporate freeze-fracture data for submolecular "cross-bridges" linking IMPs into the classical square lattices that characterize, in particular, naturally occurring AQP4.

Despite their diverse histological types, most brain tumours cause brain oedema, which is a significant cause of patient morbidity and mortality. Brain tumour oedema occurs when plasma-like fluid enters the brain extracellular space through impaired capillary endothelial tight junctions in tumours. Under-expression of the tight junction proteins occludin, claudin-1 and claudin-5 are key molecular abnormalities responsible for the increased permeability of tumour endothelial tight junctions. Recent evidence suggests that the membrane water channel protein aquaporin-4 (AQP4) also plays a role in brain tumour oedema. AQP4-deficient mice show remarkably altered brain water balance after various insults, including brain tumour implantation. AQP4 expression is strongly upregulated around malignant human brain tumours in association with reduced extracellular volume, which may restrict the flow of extracellular fluid from the tumour bed into the brain parenchyma. Elimination of excess fluid leaking into brain parenchyma requires passage across three AQP4-rich barriers: a) the glia limitans externa, b) the glia limitans interna/ependyma, and c) the blood-brain barrier. Modulation of the expression and/or function of endothelial tight junction proteins and aquaporins may provide novel therapeutic options for reducing brain tumour oedema.

It has been well documented that in adult rats astrocytes in the subventricular zone and subgranular layer of the dentate gyrus are neural stem cells. Elsewhere in the CNS astrocytes are not generally recognized as stem cells. Here we describe nestin expression in a population of astrocytes in the spinal cord of adult rat following cord injury. In either hemitransectioned or longitudinally cut spinal cord, there was widespread nestin expression in astrocytes of both the gray and white matters. Isolation of the lateral part of the spinal cord from the central canal region, where stem cells may reside, could not block the appearance of nestin-immunoreactive astrocytes in the lateral cord, and none of them showed Fast DiI labeling after the central canal ependyma had been labeled by the dye, indicating that the nestin-immunoreactive astrocytes can evolve locally in the lateral cord. They were found to be undergoing a process of de-differentiation. Culture of the nestin-immunoreactive astrocytes of the lateral cord generated neurospheres, the cells of which had the ability of self-renewal, and were able to differentiate into neurons, astrocytes, or oligodendrocytes. Taken together, the results indicate that the astrocytes in injured adult rat spinal cord may acquire the potential of neural stem cells.

A number of signaling molecules have been implicated in the acute response to hypoxia/ischemia in the adult brain. In contrast, the reaction to chronic hypoxemia is largely unexplored. We used a protocol of chronic hypoxia in rat pups during the first three postnatal weeks, encompassing the period of cellular plasticity in the cerebral cortex. We find that the levels of fibroblast growth factor 1 (FGF1) and FGF2, two members of the FGF family, increase after 2 weeks of chronic hypoxia. In contrast, members of the neurotrophin family are unaffected. FGF2 is normally expressed in the nucleus of mature, glial fibrillary acidic protein (GFAP)-containing astrocytes. Under hypoxia, most FGF2-containing cells do not express detectable levels of GFAP, suggesting that chronic low O(2) induces their transformation into more immature glial phenotypes. Remarkably, hypoxia promotes the appearance of radial glia throughout the sub-ventricular and ependymal zones. Most of these cells express vimentin and brain lipid binding protein. A subset of these radial glial cells expresses FGF receptor 1, and are in close contact with FGF2-positive cells in the sub-ventricular zone. Thus, FGF receptor signaling in radial glia may foster cell genesis after chronic hypoxic damage. From the results of this study we suggest that after the chronic exposure to low levels of oxygen during development, the expression of radial glia increases in the forebrain periventricular region. We envision that astroglia, which are the direct descendants of radial glia, are reverting back to immature glial cells. Alternatively, hypoxia hinders the normal maturation of radial glia into GFAP-expressing astrocytes. Interestingly, hypoxia increases the levels of expression of FGF2, a factor that is essential for neuronal development. Furthermore, chronic hypoxia up-regulated FGF2's major receptor in the periventricular region. Because radial glia have been suggested to play a key role in neurogenesis and cell migration, our data suggests that hypoxia-induced FGF signaling in radial glia may represent part of a conserved program capable of regenerating neurons in the brain after injury.

Initial experiments to evaluate the in vivo fate(s) of constitutively proliferating subependymal cells determined that, following in vivo labeling of this population by infection with a retrovirus containing a β-galactosidase reporter gene, there was a progressive and eventually complete loss of histochemically β-galactosidase-positive cells within the lateral ventricle subependyma with increasing survival times of up to 28 days after retroviral infection. Subsequent experiments were designed to ascertain the potential contributions of: (i) the migration of subependymal cells away from the forebrain lateral ventricles; and (ii) the down-regulation of the retroviral reporter gene expression. Retroviral lineage tracing experiments demonstrate that a major in vivo fate for constitutively proliferating subependymal cells is their rostral migration away from the walls of the lateral ventricle to the olfactory bulb. Although down-regulation of retroviral reporter gene expression does not contribute to the loss of detection of β-galactosidase-labeled cells from the lateral ventricle subependyma, it does result in an underestimation of the absolute number of retrovirally labeled cells in the olfactory bulb at longer survival times. Furthermore, a temporal decrease in the double labeling of β-galactosidase-labeled cells with [ 3 H]thymidine was observed, indicating that only a subpopulation of the migratory subependymal-derived cells continue to actively proliferate en route to the olfactory bulb. These two events may contribute to the lack of a significant increase in the total number of retrovirally labeled subependymal cells during rostral migration. Evidence from separately published studies suggests that cell death is also an important regulator of the size of the constitutively proliferating subependymal population. In summary, in vivo studies utilizing retroviral reporter gene labeling demonstrate that constitutively proliferating subependymal cells born in the lateral ventricle migrate rostrally to the olfactory bulb. Loss of proliferation potential and retroviral reporter gene down-regulation contribute to the lack of any significant increase in the total number of labeled cells recovered in the olfactory bulb.

Melatonin binding in the brain of hydrocephalic H-Tx rats was examined by autoradiography. At the time of birth, hydrocephalic animals showed an abnormality in the distribution of high-affinity melatonin receptors dorsal to the cerebral aqueduct when compared to controls. Whereas newborn rats of the H-Tx strain that were unaffected by hydrocephalus had melatonin receptors in a tectal midsagittal strip overlying the aqueduct and spanning the anterior half of the tectum, hydrocephalic rats lacked melatonin receptors in the most anterior part of this region. In these animals, the length of the aqueduct over which receptors were missing was compressed and was additionally occluded by dystrophic ependyma. The first signs of ventricular expansion characteristic of hydrocephalus were evident.

Rat adrenal chromaffin cells attached to either collagen-coated dextran (Cytodex® 3) or glass bead microcarriers, both of 90–200 μm in diameter, were used as dopamine-secreting implants in the caudate-putamen of rats with 6-hydroxydopamine-induced unilateral lesions of the substantia nigra. As controls, beads without cells and cells in suspension alone were implanted. Chromaffin cells adhered to microcarriers reduced apomorphine-induced rotation by 75% in lesioned animals. Animals that were lesioned but not receiving cell implants or receiving beads alone showed no reduction. Animals implanted with cells not attached to beads also showed a reduction in rotation but this effect lasted less than three months. Microcarrier-attached cells, however, maintained their effect in reducing rotation for at least eight months (rotations were reduced from a control mean of 10.9 ± 1.4 to 3.6 ± 1.1 turns/min) without any “drop-off” of the effect. Histological examination showed that after eight months post-implant the cells pre-adhered to beads were still present and could be stained by anti-tyrosine hydroxylase antibody. Sections stained with hematoxylin–eosin showed no signs of an inflammatory response. In contrast to beads implanted into the striatum, Cytodex® bead implants injected into the lateral ventricle induced a histopathological response appearing to involve the ependyma and choroid plexus. Results suggest that the striatal parenchyma but not the ventricle is amenable to studies using the microcarrier approach to transplantation.

This study reports that lesion of the adult lizard medial cortex (lizard hipocampal fascia dentata) induces a short period of intensive neurogenesis which we have termed reactive neurogenesis; a cell proliferation event that occurs in the subjacent ependyma. Specific lesion of the medial cortex was achieved by intraperitoneal injection of the neurotoxin 3-acetylpyridine and proliferating cells were detected using tritiated thymidine or 5-bromodeoxiuridine pulse labelling. After lesion, granule neurons in the lizard medial cortex cell layer appeared pyknotic and died; they were then removed and progressively replaced by a set of new neurons. These neurons were mostly generated from the second to the seventh day post-lesion. A dramatic temporal increment of labelled ependymal cells was detected when either tritiated thymidine or 5-bromodeoxiuridine pulses were delivered in that period. The maximum of about five thousand labelled cells per hemisphere was reached by the fourth day after the lesion. Beyond the seventh day post-lesion, the numbers of labelled cells returned to a level of about four hundred per hemisphere, similar to that of the control specimens. Electron microscopy revealed that the recently generated cells were neuroblasts or immature neurons with a characteristic pattern of chromatin condensation and a high number of ribonucleic granules.

The highly sialylated isoform of the neural cell adhesion molecule is thought to be expressed predominantly in the developing nervous system, where it is implicated in a variety of dynamic events linked to neural morphogenesis. It has become increasingly evident, however, that this "embryonic" neural cell adhesion molecule isoform continues to be expressed in certain adult neuronal systems, and in particular, in those that can undergo structural plasticity. In the present study, we performed light microscopic immunocytochemistry with an antibody specific for polysialylated neural cell adhesion molecule and confirmed our earlier observations [Bonfanti L. et al. (1992) Neuroscience 49, 419-436] showing polysialylated neural cell adhesion molecule-immunoreactive cells in the subependymal layer of the lateral ventricle of the adult rat, a region where cell proliferation continues into the postnatal period. In addition, we used an antibody raised against the proliferating cell nuclear antigen and found that proliferating cells continue to be visible in this area, even in the adult. Double immunolabeling showed that many of these newly generated cells displayed high polysialylated neural cell adhesion molecule immunoreactivity. Cells from a portion of the subependymal layer migrate to the olfactory bulb and contribute to the continual replacement of its granule neurons [Luskin M. B. (1993) Neuron 11, 173-189]. We found polysialylated neural cell adhesion molecule-immunoreactive cells all along the pathway purported to be followed by the newly generated cells to their final destination and in neurons corresponding to granular and periglomerular cells in the olfactory bulb. Our present observations thus support the contention that polysialylation is a feature of neurons capable of dynamic change and may contribute to the molecular mechanisms permitting cell proliferation and migration not only during development but also in the adult.

During development, recognition mechanisms between neurons and their targets are necessary for the formation of the neuronal network. Neurol connections are synaptic or non-junctional. Both types of communication can be found between neurons and glial elements in the periventricular walls. Serotonergic fibers form synaptic contacts on the specialized ependymocytes of the subcommissural organ, a structure which forms the roof of the third ventricle at its junction with the aqueduct. A network of non-junctional fibers containing both GABA and serotonin spread between the cilia of the classical ependymocytes in the ventricles. These anatomical, morphological and biochemical features suggest a tropism and specific recognition mechanisms between glial elements and serotonergic neurons. This hypothesis can be tested by the study of the innervation of the subcommissural organ and the classical ependyma by grafted embryonic neurons after a chemical destruction of the serotonergic endogenous innervation. Solid implants or cell suspensions prepared from embryonic metencephalon were transplanted to either the third ventricle or the periventricular gray matter in 5,7-dihydroxytryptamine denervated rats. Grafted serotonergic neurons were able to reinnervate the classical ependyma and the subcommissural organ. The fibers forming the supraependymal plexus were non-junctional and contained both serotonin and GABA while those innervating the subcommissural organ formed synaptic contacts and contained only serotonin. The signals capable of inducing the ependymal innervation were specific for serotonergic neurons since catecholaminergic neurons present in the grafts were unable to innervate either classical or specialized ependymocytes. These results demonstrate that glial cells are targets for serotonergic neurons and that the morphological and biochemical characteristics of the serotonergic innervation are closely related to the target cell phenotype.

Brain oedema is a major clinical problem produced by CNS diseases (e.g. stroke, brain tumour, brain abscess) and systemic diseases that secondarily affect the CNS (e.g. hyponatraemia, liver failure). The swollen brain is compressed against the surrounding dura and skull, which causes the intracranial pressure to rise, leading to brain ischaemia, herniation, and ultimately death. A water channel protein, aquaporin-4 (AQP4), is found in astrocyte foot processes (blood–brain border), the glia limitans (subarachnoid cerebrospinal fluid—brain border) and ependyma (ventricular cerebrospinal fluid—brain border). Experiments using mice lacking AQP4 or alpha syntrophin (which secondarily downregulate AQP4) showed that AQP4 facilitates oedema formation in diseases causing cytotoxic (cell swelling) oedema such as cerebral ischaemia, hyponatraemia and meningitis. In contrast, AQP4 facilitates oedema elimination in diseases causing vasogenic (vessel leak) oedema and therefore AQP4 deletion aggravates brain oedema produced by brain tumour and brain abscess. AQP4 is also important in spinal cord oedema. AQP4 deletion was associated with less cord oedema and improved outcome after compression spinal cord injury in mice. Here we consider the possible routes of oedema formation and elimination in the injured cord and speculate about the role of AQP4. Finally we discuss the role of AQP4 in neuromyelitis optica (NMO), an inflammatory demyelinating disease that produces oedema in the spinal cord and optic nerves. NMO patients have circulating AQP4 IgG autoantibody, which is now used for diagnosing NMO. We speculate how NMO-IgG might produce CNS inflammation, demyelination and oedema. Since AQP4 plays a key role in the pathogenesis of CNS oedema, we conclude that AQP4 inhibitors and activators may reduce CNS oedema in many diseases.

Increased angiogenesis and an altered blood-brain barrier have been reported in the brain of dystrophin-deficient mdx mouse, an experimental model of Duchenne muscular dystrophy. To further elucidate the mechanisms underlying angiogenesis in Duchenne muscular dystrophy, in this study we evaluated whether nerve growth factor (NGF) and nerve growth factor receptors (NGFRs) are involved, then correlated NGF-NGFRs expression with vascular endothelial growth factor (VEGF) and its receptor-2 (VEGFR-2) content and matrix metalloproteinases-2 and -9 (MMP-2 and -9) activity, by confocal laser microscopy and immunohistochemistry. Results showed that neurons, astrocytes and ependymal cells were strongly labeled by NGF in mdx brain, expressing NGFRs on glial and endothelial cells. In controls, NGF faintly labeled neurons and astrocytes, whereas endothelial cells were negative for NGFRs. Immunogold electron microscopy demonstrated NGFR gold particles on endothelial cells in mdx brain, while in controls few particles were recognizable only on glial end feet. Western blotting and real time polymerase chain reaction (RT-PCR) demonstrated a higher expression of NGF and NGFR mRNA and protein in mdx brain as compared to controls, and increase of VEGF-VEGFR-2 and active MMP-2 and -9 content. Overall, these data suggest that in the brain of mdx mice, an upregulation of the NGF-NGFRs system might be involved directly, or indirectly through the activation of VEGF-VEGFR-2 and MMP-2 and -9, in the angiogenic response taking place in this pathological condition.

Nutrient transporters and ABC efflux pumps at the blood-brain barrier are major determinants of drug penetration into the brain. Immunohistochemical analysis of transporter subcellular localization is challenging due to the close apposition of the luminal and abluminal microvessel plasma membranes. We employed in vivo perfusion of biotinylation reagent through rat brain microvessels to domain-specifically label proteins exposed on the microvessel luminal surface. Using this approach, we analyzed the luminal/abluminal localization of a number of blood-brain barrier transporters identified by quantitative PCR profiling as being highly expressed and enriched in rat brain endothelial cells compared with whole brain. We also examined the apical/basal-lateral distribution of transporters in the choroid plexus, a secondary site for transport of nutrients between the blood and CNS. We detected P-glycoprotein (Pgp) (Abcb1), ATP-binding cassette (Abc) g2, multidrug resistance protein (Mrp) 4 (Abcc4), glucose transporter 1 (Glut1) (Slc2a1), Lat1 (Slc7a5), and monocarboxylate transporter-1 (Mct1) (Slc16a1) on the luminal surface of rat cerebral microvessels by both immunofluorescence staining and Western blotting of in vivo biotinylated proteins. Mrp1 (Abcc1) appeared primarily abluminal by immunofluorescence staining, and was barely detectable in the biotinylated protein fraction. Organic anion transporter (Oat) 3 (Slc22a8), organic anion transporter polypeptide (Oatp) 2b1 (Slco2b1, Oatpb), and Mrp5 (Abcc5) were not detected on the luminal surface using either method, while Oatp1a4 (Slco1a4, Oatp2) appeared to partially localize to the microvessel lumen by immunofluorescence staining, but was not detected in the biotinylated protein fraction by Western blotting. Lat1, Mrp1 and Mrp4 were detected on the basal-lateral surface of lateral ventricle choroid plexus epithelial cells. Mrp5, Oct3 and Oatp2b1 (Oatpb) were detected in the ependymal cells lining the ventricle. We did not detect Pgp expression in choroid plexus by immunofluorescence staining. In vivo biotinylation provides a method for domain-specific labeling of luminal surface proteins within the capillaries of the blood-brain barrier, allowing for biochemical analysis of protein localization and facilitating optical discrimination of the luminal and abluminal endothelial surfaces.

Spatially and temporally restricted populations of neurogenic astrocytes can generate multipotent neurospheres in vitro . To examine the ability of neurogenic astrocytes to respond to in vivo differentiation cues within a germinal matrix, we provided cultured neonatal cerebellar astrocytes access to the subependymal zone (SEZ) by grafting them directly into the lateral ventricle of adult mice. Here we report three events that follow such transplants. 1) Donor cells attach to periventricular structures, and form “neoplastic-like” spheres that penetrate the ventricular wall. These attached spheres can persist for months, as they give rise to “clones” of cells that infiltrate forebrain parenchyma. 2) Many donor cells enter the rostral migratory stream and migrate into the olfactory bulb where a small percentage differentiates as olfactory interneurons. 3) Finally, within the SEZ, some donor cells formed cell clusters that appear to interact with the SEZ neuronal precursor chains, and some donor cells differentiate into distinctive neurons with extensive, beady projections precisely confined between the ependymal layer and the striatum. Further analysis of normal SEZ anatomy reveals indigenous neurons with identical morphologies—some of which are contacted by 5-HT+ fibers—that we propose represent a heretofore uncharacterized, intrinsic SEZ neuron of unknown function. These results suggest that cultured astrocytes derived from non-SEZ brain regions can respond in different ways to in vivo cues provided by the adult lateral ventricle and SEZ by differentiating into neurons that eventually inhabit both the olfactory bulb and SEZ proper.

Ependymal cells of the adult mammalian spinal cord exhibit stem/progenitor cell properties following injury. In the present study, we utilized intraventricular injection of 1,1'-dioctadecyl-6,6'-di(4-sulfophenyl)-3,3,3',3'-tetramethylindocarbocyanine (DiI) to label the ependyma lining the central canal to allow tracking of the migration of endogenous ependymal cells and their progeny after spinal cord injury (SCI). We developed a minimal injury model that preserved the integrity of the central canal and did not interfere with ependymal cell labeling. Three days following SCI, there was an 8.6-fold increase in the proliferative labeling index of the ependymal cells at the level of the needle track based on bromodeoxyuridine labeling, compared with 1 day post-injury. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) positive cells were not detected in the ependyma or surrounding gray matter, indicating that ependymal cells do not undergo apoptosis in response to minimal injury. Nestin was rapidly induced in the ependyma by 1 day and expression peaked by 7 days post-injury. We quantitated the number and distance of ependymal cell migration following minimal injury. The number of ependymal cells migrating from the region of the central canal increased by 3 days following minimal injury and DiI-labeled glial fibrillary acidic protein expressing cells were detected 14 days post-SCI, most of which migrated within 70 microm of the region of the central canal. These results show that a minimal SCI adjacent to the ependyma is sufficient to induce an endogenous ependymal cell response where ependymal stem/progenitor cells proliferate and migrate from the region of the central canal, differentiating primarily into astrocytes.

Young, adult and presumed old specimens of the tropical lizard Tropidurus hispidus, living in an almost steady warm habitat, have been the subjects of a 5-bromodeoxiuridine immunocytochemical study to label proliferating brain cells. All animals showed abundant 5-bromodeoxiuridine-labeled nuclei in the ependyma of their telencephalic lateral ventricles, with these being especially abundant in the medial cortex ependyma. Surprisingly, adult animals displayed higher numbers of labeled nuclei when compared with those of young specimens. In a second experiment, in order to check the evolution of ependymal-labeled nuclei, adult specimens were allowed 4 h or 2, 4, 7, 15 or 30 days of survival after the 5-bromodeoxiuridine pulse. Most labeled nuclei appeared isolated at short survival times (4 h and 2 days after the 5-bromodeoxiuridine pulse) but from day 4 and beyond, labeled nuclei appeared in couples or groups usually located in the ependyma. Labeled nuclei with vertical fusiform appearance in the inner plexiform layer or even recruited in the medial cortex cell layer were assumed to be migratory. These presumed migratory nuclei were unexpectedly few (less than 30%) when compared with other lizards, and they appeared much later; at 15 and 30 days after the pulse. This situation resembles that of mammals where only a small proportion of postnatally generated neurons can develop and survive.

SNAT2 is a neutral amino acid carrier that belongs to the system A family. Since its function in the nervous system remains unclear, we have analyzed its distribution in the rat CNS using specific antisera. Although SNAT2 is expressed widely in the CNS, it is enriched in the spinal cord and the brainstem nuclei, especially those of the auditory system. At the cellular level, SNAT2 was preferentially located in neuronal cell bodies and processes, although it was also strongly expressed in the meninges and ependyma. In astrocytes, the localization of SNAT2 was more restricted since it was intensely expressed in the perivascular end-feet, glia limitans, cerebellar astrocytes and Bergmann glia, but it was less intense in astrocytes of the cerebral parenchyma. Among neurons, the primary sensory neurons of the mesencephalic trigeminal nucleus appeared to be those that most strongly express SNAT2, but many other neurons, including cortical pyramidal cells and their dendrites were also intensely stained. In several regions the transporter was detected in axons, especially in the brainstem, and its presence in both dendrites and axons was confirmed by confocal microscopy and ultrastructural studies. However, while SNAT2 was observed in the large principal dendrites and the small distal dendrites, it was only found in axonal shafts and was excluded from terminals. Some glutamatergic neurons were among the more intensely labeled cells whereas SNAT2 was not detected on GABAergic neurons. The expression of SNAT2 partially coincides with that reported for SNAT1, especially in glutamatergic neurons. Hence, both proteins could fulfill complementary roles in replenishing glutamate pools and be differentially regulated under different physiological conditions. They also seem to co-localize in non-neuronal cells probably contributing to amino acid fluxes through the blood-brain barrier.

Precursor cells in the ependyma of the lateral ventricles of adult mammalian brain have been reported in brain, and also in the spinal cord. The present study used antibody to the intermediate filament protein (nestin) as an immunohistochemical marker for neural stem cells and precursor cells in a rat model of spinal cord trauma. Male Sprague-Dawley rats (n=25) had a laminectomy at Thll-Thl2, and spinal cord contusion was created by compression with 30 g of force for 10 min. The rats were killed at 24 h, 1 week and 4 weeks after injury, and four levels of the spinal cord were examined: 5 mm and 10 mm, both rostral and caudal region to the injury center. Time- and region-dependent alterations of nestin immunoreactivity were analyzed. Revealed at 24 h post-injury, 5 mm rostral and caudal to the lesions, nestin expression was observed in ependymal cells and around the hemorrhagic and necrotic lesion located in dorsal spinal cord, peaking at 1 week after injury. Moreover, nestin expression was also observed in the white matter of ventral spinal cord, extending into arborizing processes centripetally from the pial surface toward the central canal. At 4 weeks after injury, nestin expression in ependyma decreased 10 mm from the injury site. But nestin expression in white matter increased dramatically with a 100-fold increase in nestin originating from the pial surface, and extension now to all the white matter. The latter was accompanied by glial fibrillary acidic protein positivity into very long arborizing processes, morphologically compatible with radial glia. The findings suggest two possible sources of precursor cells in adult mammalian spinal cord; ependyma of the central canal and subpial astrocytes. Subpial astrocytes may be associated with neural repair and regeneration after spinal cord injury.

Cation-chloride cotransporters have been considered to play pivotal roles in controlling intracellular and extracellular ionic environments of neurons and hence controlling neuronal function. We investigated the total distributions of K-Cl cotransporter 1 (KCC1), KCC2 (KCC2), and Na-K-2Cl cotransporter 1 (NKCC1) messenger RNAs in the adult rat nervous system using in situ hybridization histochemistry. KCC2 messenger RNA was abundantly expressed in most neurons throughout the nervous system. However, we could not detect KCC2 messenger RNA expression in the dorsal root ganglion and mesencephalic trigeminal nucleus, where primary sensory neurons show depolarizing responses to GABA, suggesting that the absence of KCC2 is necessary for this phenomenon. Furthermore, KCC2 messenger RNA was also not detected in the dorsolateral part of the paraventricular nucleus, dorsomedial part of the suprachiasmatic nucleus, and ventromedial part of the supraoptic nucleus where vasopressin neurons exist, and in the reticular thalamic nucleus. As vasopressin neurons in the suprachiasmatic nucleus and neurons in the reticular thalamic nucleus produce their intrinsic rhythmicity, the lack of KCC2 messenger RNA expression in these regions might be involved in the genesis of rhythmicity through the control of intracellular chloride concentration. The expression levels of KCC1 and NKCC1 messenger RNAs were relatively low, however, positive neurons were observed in several regions, including the olfactory bulb, hippocampus, and in the granular layer of the cerebellum. In addition, positive signals were seen in the non-neuronal cells, such as choroid plexus epithelial cells, glial cells, and ependymal cells, suggesting that KCC1 and NKCC1 messenger RNAs were widely expressed in both neuronal and non-neuronal cells in the nervous system. These results clearly indicate a wide area- and cell-specific variation of cation chloride cotransporters, emphasizing the central role of anionic homeostasis in neuronal function and communication.

In order to characterize the localization of the sigma(1) receptor in the adult rat central nervous system, a polyclonal antibody was raised against a 20 amino acid peptide, corresponding to the fragment 143-162 of the cloned sigma(1) receptor protein. Throughout the rostrocaudal regions of the central nervous system extending from the olfactory bulb to the spinal cord, intense to moderate immunostaining was found to be associated with: (i) ependymocytes bordering the entire ventricular system, and (ii) neuron-like structures located within the parenchyma. Double fluorescence studies confirmed that, throughout the parenchyma, sigma(1) receptor-immunostaining was essentially associated with neuronal structures immunostained for the neuronal marker betaIII-tubulin. In all rats examined, high levels of immunostaining were always associated with neurons located within specific regions including the granular layer of the olfactory bulb, various hypothalamic nuclei, the septum, the central gray, motor nuclei of the hindbrain and the dorsal horn of the spinal cord. In contrast, only faint immunostaining was associated with neurons located in the caudate-putamen and the cerebellum. Electron microscope studies indicated that sigma(1) receptor immunostaining was mostly associated with neuronal perikarya and dendrites, where it was localized to the limiting plasma membrane, the membrane of mitochondria and of some cisternae of the endoplasmic reticulum. At the level of synaptic contacts, intense immunostaining was associated with postsynaptic structures including the postsynaptic thickening and some polymorphous vesicles, whereas the presynaptic axons were devoid of immunostaining. These data indicate that the sigma(1) receptor antibody prepared here, represents a promising tool for further investigating the role of sigma(1) receptors.

Bovine subcommissural organ-spondin was shown to be a brain-secreted glycoprotein specifically expressed in the subcommissural organ, an ependymal differentiation located in the roof of the Sylvian aqueduct. Also, subcommissural organ-spondin makes part of Reissner's fiber, a phylogenetically and ontogenetically conserved structure present in the central canal of the spinal cord of chordates. This secretion is a large multidomain protein probably involved in axonal growth and/or guidance. As Reissner's fiber is highly conserved in the chordate central nervous system, we sought genes orthologous to the bovine subcommissural organ-spondin gene by Southern blot analysis in several members of the chordate phylum: urochordates, cephalochordates, cyclostomes, and lower and higher vertebrates, including humans. In addition, conserved glycoproteins present in the subcommissural organ and Reissner's fiber were revealed by immunohistochemistry using antibodies raised against bovine Reissner's fiber. Variation in the sites of Reissner's fiber production according to chordate subphylum, presence of this structure in the spinal cord, and conservation of the subcommissural organ-spondin gene are discussed in the context of chordate central nervous system development. These results indicate that subcommissural organ-spondin is an ancient ependymal secretion, making part of Reissner's fiber, that may have had an important function during the evolution of the central nervous system in chordates, including that of the spinal cord.

During the past two decades several revisions of the concepts underlying interneuronal communication in the central nervous system have been advanced. We propose here to classify communicational phenomena between cells of the central neural tissue under two general frames: "wiring" and "volume" transmission. "Wiring" transmission is defined as intercellular communication occurring through a well-defined connecting structure. Thus, wiring transmission is characterized by the presence of physically identifiable communication channels within the neuronal and/or glial cell network. It includes synaptic transmission but also other types of intercellular communication through a connecting structure (e.g., gap junctions). "Volume" transmission is characterized by signal diffusion in a three-dimensional fashion within the brain extracellular fluid. Thus, multiple, structurally often not well characterized extracellular pathways connect intercommunicating cells. Volume transmission includes short- (but larger than synaptic cleft, i.e. about 20 nm) and long-distance diffusion of signals through the extracellular and cerebrospinal fluid. It must be underlined that the definitions of wiring and volume transmission focus on the modality of transmission and are neutral with respect to the source and target of the transmission, as well as type of informational substance transmitted. Therefore, any cell present in the neural tissue (neurons, astroglia, microglia, ependyma, tanycytes, etc.) can be a source or a target of wiring and volume transmission. In this paper we discuss the basic definitions and some distinctive characteristics of the two types of transmission. In addition, we review the evidence for different types of intercellular communication besides synaptic transmission in the central nervous system during phylogeny, and in vertebrates in physiological and pathological conditions.

The suprachiasmatic nucleus of the mammalian brain is thought to be the anatomical locus of circadian rhythms. To examine the functional organization of the suprachiasmatic nucleus in vitro with intact intercellular connections for a prolonged period, we have established an organotypic slice culture system using a roller-tube technique. Brain slices (400 microns in thickness) containing the bilateral suprachiasmatic nuclei, were obtained from newborn rats at four to seven days old and were maintained in vitro for more than three weeks. During this three-week period, the slices flattened to one to three cell layers and two tightly packed neuronal cell-masses (neuronal zones), with diameters of about 1 mm were formed, which were surrounded by a peripheral glial cell-dispersed zone. In the neuronal cell zones, peptides and their messenger RNAs were found cytochemically with characteristic patterns similar to the suprachiasmatic nucleus in the brain. In situ hybridization and immunocytochemistry showed that vasoactive intestinal peptide messenger RNA expressing and vasoactive intestinal peptide-immunoreactive neurons were detected predominantly in the ventrolateral part of the neuronal zones in the suprachiasmatic nucleus slice culture. Vasopressin messenger RNA-expressing and vasopressin-immunoreactive cells were localized in the dorsomedial neuronal zones near the ependymal cell zone. The distribution of cell bodies and fibers containing these neuropeptides and their messenger RNAs in the neuronal zones of suprachiasmatic nucleus organotypic slice culture were similar to that of the suprachiasmatic nucleus in vivo. This suggests that the suprachiasmatic nucleus in these organotypic slice cultures retains the biological characteristics of these cells in vivo as the cells did develop, form compact neuronal masses and did establish connections. To examine the possibility that suprachiasmatic nucleus neurons in slice cultures show a persistent rhythmic activity, we also measured the amount of vasopressin released into the culture medium. Sampling at 4-h intervals combined with enzyme immunoassay revealed that vasopressin concentration in the medium embracing suprachiasmatic nucleus slice cultures fluctuated with a period of approximately 24 h. The present findings suggest that the intranuclear neuronal networks of the suprachiasmatic nucleus are maintained in vitro for a long duration and that organotypic cultures of the suprachiasmatic nucleus produce and release bioactive substances in an oscillating manner. The suprachiasmatic nucleus in slice cultures may be useful for future analysis of circadian rhythms in vitro.

The central gray region (lamina X) of the lumbar spinal cord in cat was examined by electron microscopy. This region consisted of three morphological zones. Medially, the first zone was comprised of ependyma which surrounded the central canal. The ependyma in the cat spinal cord was similar to most vertebrate spinal ependyma. Secondly, a subependymal zone consisted of glial processes arranged parallel to the long axis of the spinal cord. This glial zone was widest lateral to the central canal and extended approximately 75 microns. The lateral edge of the glial zone intermingled with a neuropil zone, the third zone. The components of the neuropil zone consisted of dendrites, myelinated and unmyelinated axons, synaptic terminals, astrocytes and neurons. The dendrites and neurons generally were oriented parallel with the long axis of the spinal cord. Three synaptic terminal types were categorized according to vesicular morphology, i.e. small round vesicles, flattened vesicles and dense core vesicles. The central gray region has been implicated in nociception and has been shown to receive both primary afferent and supraspinal input. The results from this study are consistent with the central gray region being an area of multiple synaptic inputs which may form the morphological basis of nociceptive processing that ascends to brainstem nuclei.

The origin of the serotoninergic innervation of the rat subcommissural organ was studied using radioautography of tritiated serotonin and biochemical determination of endogenous serotonin content after electrolytic lesions of raphe nuclei. The results suggest that this innervation is mainly derived from nuclei raphe centralis superior and raphe dorsalis, each nucleus contributing about one-third of the input. A possible contribution from nucleus raphe pontis is also suggested. Given the different patterns of innervation revealed by silver staining of nerve fibers and the different patterns of secretory activity observed with histochemical methods after the electrolytic lesions, the following working hypothesis is formulated. Nucleus raphe dorsalis would inhibit the synthesis of secretory material in the rat subcommissural organ via medium-sized serotoninergic fibers restricted to the hypendymal region, whereas nucleus raphe centralis superior might inhibit the release of secretory material via rather thin serotoninergic fibers reaching the nuclear level of the ependyma. This hypothesis is in line with the inhibitory effect postulated for the serotoninergic innervation in the rat subcommissural organ in early investigations using serotonin neurotoxins.

The effect of olfactory placode transplantation on the differentiation of the optic vesicle and stalk has been studied in Xenopus laevis embryos. Host embryos (stages 23-24) received the transplant of two olfactory placodes from same-stage donors in place of a partially or totally removed optic vesicle. All tadpoles were sacrificed at stages 47-50. The host tadpoles were subdivided in three groups according to the amount of optic vesicle removed. In the first group all of the optic vesicle was removed. At sacrifice a lobar mass of nervous tissue, continuous with and protruding from the diencephalic wall, was penetrated by the olfactory nerves from the transplanted placodes and a well-defined glomerular layer was present at the entrance zone of the olfactory nerves. The lobar protrusion contained a normal ventricular cavity, connected by a foramen to the third ventricle. The ventricle was lined by a mitotically active ependymal layer. In the second group the host embryos were deprived of two-thirds of the optic vesicle. In these animals we observed the development of a round mass of nervous tissue connected by a peduncle to the diencephalic wall. A ventricle lined by ependyma was present in the formation; however, the cavity was not continuous with the third ventricle. Olfactory nerves from the transplanted placodes penetrated the rostral portion of the nervous mass and formed a distinct glomerular layer. In the third group of embryos only one-third of the optic vesicle was removed. These animals developed irregularly shaped structures where ocular tissue and nervous tissue coexisted. The termination of the supranumerary olfactory nerves and the glomerular layer were restricted to the non-ocular nervous tissue. The interpretation of the histogenetic phenomena determining the protrusions is difficult and further studies are needed. It seems highly probable, however, that the fate of such determined structures as the optic peduncle and vesicle can be influenced by the olfactory input, supporting the hypothesis that the ectopically directed olfactory fibres interfere with the differentiation of the optic structures.

Previous investigations of cortical organization in the brain of the turtle have revealed many features typical of mammalian neocortex. Recent evidence suggests that many neocortical neurons contain neuroactive peptides. The possibility that one such peptide, somatostatin, is found in the turtle brain was tested using immunocytochemical techniques. Intense somatostatin-like immunoreactivity was observed in many neurons and fibers in turtle cortex, as well as in several forebrain nuclei. Cortical neurons with several different dendritic configurations showed immunoreactive labelling, including bipolar, stellate and pyramidal cell types. In addition, stained cells and processes were observed in close association with the ependyma of the lateral ventricle. Other forebrain regions containing immunoreactive neurons included the dorsal ventricular ridge, the basal telencephalic nuclei and the hypothalamus. These data support the idea that peptidergic neurons existed in the pallium of an ancestor common to modern mammals and reptiles. We speculate that somatostatin plays a similar role in the normal function of all types of cortex and suggest that turtle cortex may provide a useful model for the study of this cortical neuropeptide.

An antibody to a 240,000 dalton microtubule-associated protein, microtubule-associated protein 4, was used to illustrate the distribution of this protein in semi-thin sections of the central nervous system. Immunofluorescence microscopy indicated that microtubule-associated protein 4 was restricted to non-neuronal elements of the brain and spinal cord. Astrocytes, oligodendrocytes and “specialized” glia, including tanycytes, Bergmann glia and Muller cells, contained microtubule-associated protein 4. This distribution of microtubule-associated protein 4 in neural tissue is distinct from that described for the other major brain microtubule-associated proteins, microtubule-associated protein 1 and microtubule-associated protein 2. The reactivity of MAP 4 antibody with these glia demonstrates the antigenic relatedness of these cells and further distinguishes glia from other elements of the nervous system.

Quantitative changes in the amount of ribonucleic acid per cell were determined in rat spinal cord cells by means of ultraviolet and visible two-wavelength cytospectrophotometry, both these methods being shown to give similar results. After 2-h hypobaric hypoxia, the ribonucleic acid content was increased in the spinal motor neurons with no changes in the perineuronal neuroglia and ependymal cells. The posthypoxic period was characterized by a number of changes in ribonucleic acid which were rather different in neurons, neuroglial or ependymal cells. Daily injections of adrenaline to rats for 2 weeks resulted in a decrease in ribonucleic acid content in the motor neurons and led to an increase in two other kinds of spinal cord cells. Four days following adrenalectomy there was a decrease in ribonucleic acid content in spinal motor neurons without any changes in perineuronal neuroglia or in ependymal cells. After cortisol injection to adrenalectomized rats, the decrease in the ribonucleic acid content was not reversed in the motor neurons and there was also a decrease in the ependymal cells. Changes in ribonucleic acid content are complex and not obviously related to single or combined actions of adrenal hormones. Similarities and differences in the metabolic responses of ribonucleic acid to stress are discussed with respect to three main types of nerve tissue cells: neurons, neuroglia and ependyma.

Supraependymal, varicose nerve fibres were observed in human post-mortem brain when ventricle surfaces were examined by transmission and scanning electron microscopy. Transmission electron microscopy revealed the presence of numerous profiles of nerve terminals, containing small electron-lucent vesicles and large dense-cored vesicles, and axons with their characteristic neurotubules. Occasionally, desmosome-like junctions were found between the nerve terminals and the adjacent ependymal cells. Scanning electron microscopy demonstrated the topographical distribution of varicose nerve fibres traversing among clusters of cilia and numerous microvilli on the ventricle surface. These nerve fibres, which were most frequently observed in the interventricular foramen, often coursed in bundles. In one of the five cases examined, a discrete formaldehyde-induced fluorescence typical of 5-hydroxytryptamine (yellow colour and rapid fading upon violet-blue irradiation) was observed on the floor of the fourth ventricle. It is concluded that supraependymal nerve fibres in human brain are probably identical to similar nerves previously characterized as serotoninergic in rat brain and occurring in several other mammalian brains.

The subependymal layer underlying the external wall of the lateral ventricle has been studied with freeze-fracture in 3-week-old rats. Membrane specializations corresponding to gap junction and attachment plates were found on ependymal cells, as well as the characteristic features of cilia and microvilli. Hexagonal arrays of particles previously found in the membrane of astrocytes were also present in ependyma. The membranes of subependymal cells were characterized by a paucity of intramembranous particles, when compared to ependymal and other mature glial cells. Specialized regions corresponding to areas of close membrane apposition seen on the sections were tentatively identified as clustering of particles on the protoplasmic face, corresponding with a cobbly texture on the exoplasmic face. Their possible significance, if they are not artifactual, is discussed. The analysis of replicas obtained from freeze-fractured material provides an additional tool for the study of cell differentiation and maturation, in our case gliogenesis. The comparison of membrane characteristics of neuroepithelial cells—precursors of glia—with those of mature glial cells and intermediate forms may contribute to a better understanding of the mechanism of their maturation.

Publisher Summary The neurohypophysis acts as the central interface for the coordination of the nervous and endocrine systems. The part that is called the median eminence contains the neurohemal contact zone where neurohormones regulating the secretion of anterior pituitary cells are discharged. Neurohormone producing nerve cells of the so-called tuberoinfundibular system project with their axons to the external zone of the median eminence and terminate upon the capillary loops of the primary plexus that drain into the portal vessels of the pituitary. Hormones are assumed to be released from the nerve endings to penetrate the walls of blood vessels that are characterized by a fenestrated endothelium, a perivascular space, and by the absence of a blood–-brain barrier. The portal circulation carries the neural hormones to the secondary capillary plexus of the anterior pituitary. This chapter discusses the structure of the median eminence and the significance of the cerebrospinal fluid and presents the transport hypothesis.

Low, repeated doses of X-rays from a Co 60 source were used to impair the development of the granule cells and their dendritic terminals in the olfactory bulb and the resulting effect was studied under light and electron microscopes at 9 days of age. Irradiation of rats from embryonic day 18 ( in utero ) to postnatal day 5 resulted, among others, in maldevelopment of the (internal) granule cell and external plexiform layers. This was accompanied by a decrease in the number and the density of the granule cells and the remaining granule cells contained less ribosomes, regardless of their position within the layer. This implies that both supposed subtypes of granule cells were effected. In the external plexiform layer, a reduced number of mature dendrodendritic synapses and signs of harmed granule gemmules were observed. The results suggest that intrauterinal plus postnatal irradiation with low, repeated doses of X-rays may be an effective tool impairing the development of prenatally forming neurons.

The cellular localization of the regulatory (RI and RII) and catalytic components of the cyclic AMP-dependent protein kinase in selected areas of the central nervous system was determined using a selective and sensitive immunofluorescent technique. The neuronal and glial distribution was similar for the three subunits, however, each unit exhibited differences in the pattern of subcellular localization. Whereas the catalytic unit was confined to intranuclear loci, RI and RII were identified within both the cytoplasm and nucleus, with RII in addition showing distinct staining of the nuclear membrane. A granular distribution of RI in the cell soma further distinguished the cytological localization of these two regulatory components in this cellular compartment. The contrasting distribution of the subunits suggests possible functional differences between the regulatory and catalytic components of cyclic AMP-dependent protein kinase, which are discussed with regard to the role of this enzyme in mediating the physiological effects of cyclic AMP in the central nervous system.