Biomedical subjects

C C Naus

Publications and source records attributed to C C Naus.

72 records · Page 4Linked to original sources

PubMed1992

In vivo growth of C6 glioma cells transfected with connexin43 cDNA.

In order to examine the possible role of intercellular communication via gap junctions in the control of tumor growth, we have transfected C6 glioma cells with connexin43 cDNA. We obtained several clones with variable expression of connexin43. The growth rate of these clones in culture was inversely related to the degree of expression of the transfected cDNA. To examine the growth of these transfected cells in vivo, cells were grown in spinner culture flasks to form spheroids 250-300 microns in diameter. Spheroids of nontransfected C6 cells produced large gliomas. Immunohistochemical and in situ hybridization analyses revealed relatively high levels of connexin43 protein and mRNA in the host tissue, while little of this protein was detected in the glioma. In contrast, spheroids of connexin43-transfected cells grew more slowly and exhibited elevated levels of connexin43 protein and mRNA. These findings suggest that the expression of connexin43 may be associated with the control of brain tumor growth in vivo.

Animals↗

PubMed1992

Characteristics of C6 glioma cells overexpressing a gap junction protein.

1. C6 glioma cells transfected with connexin43 cDNA display a dramatic increase in the level of connexin43 mRNA and protein. 2. This overexpression of connexin43 is evident at the cellular level, as revealed with in situ hybridization and immunocytochemistry. Transfection with connexin43 cDNA also induced actin stress fibers in these glioma cells. 3. Although we observed up to a 50-fold increase in the level of connexin43 mRNA following transfection, virtually all of this mRNA was present in the polysomal fraction. 4. Overexpression of connexin43 mRNA did not appear to compete with other cellular mRNAs for access to the translational machinery. 5. It is likely that the reduced proliferation rate of the transfected cells, reported earlier, is due to enhanced connexin43 expression and intercellular coupling.

Animals↗

PubMed1992

Intercellular calcium signaling via gap junctions in glioma cells.

Calcium signaling in C6 glioma cells in culture was examined with digital fluorescence video microscopy. C6 cells express low levels of the gap junction protein connexin43 and have correspondingly weak gap junctional communication as evidenced by dye coupling (Naus, C. C. G., J. F. Bechberger, S. Caveney, and J. X. Wilson. 1991. Neurosci. Lett. 126:33-36). Transfection of C6 cells with the cDNA encoding connexin43 resulted in clones with increased expression of connexin43 mRNA and protein and increased dye coupling, as well as markedly reduced rates of proliferation (Zhu, D., S. Caveney, G. M. Kidder, and C. C. Naus. 1991. Proc. Natl. Acad. Sci. USA. 88:1883-1887; Naus, C. C. G., D. Zhu, S. Todd, and G. M. Kidder. 1992. Cell Mol. Neurobiol. 12:163-175). Mechanical stimulation of a single cell in a culture of non-transfected C6 cells induced a wave of increased intracellular calcium concentration ([Ca2+]i) that showed little or no communication to adjacent cells. By contrast, mechanical stimulation of a single cell in cultures of C6 clones expressing transfected connexin43 cDNA induced a Ca2+ wave that was communicated to multiple surrounding cells, and the extent of communication was proportional to the level of expression of the connexin43 cDNA. These results provide direct evidence that intercellular Ca2+ signaling occurs via gap junctions. Ca2+ signaling through gap junctions may provide a means for the coordinated regulation of cellular function, including cell growth and differentiation.

Calcium↗

PubMed1991

Expression of gap junction genes in astrocytes and C6 glioma cells.

The expression of the gap junction genes coding for the liver-type connexin32 and the heart-type connexin43 was examined in primary cultures of astrocytes and in cultures of C6 glioma cells. In both cell types, only connexin43 mRNA was detectable. However, the level of this mRNA was greatly reduced in C6 glioma cells compared to astrocytes. This was consistent with the further observation that astrocytes in primary culture were extensively dye-coupled, whereas such coupling was very restricted in cultures of C6 glioma cells. Connexin43 was immunocytochemically localized in astrocytes, but was not readily detected in C6 cells.

Animals↗

PubMed1991

Transfection of C6 glioma cells with connexin 43 cDNA: analysis of expression, intercellular coupling, and cell proliferation.

C6 glioma cells express low levels of the gap junction protein connexin 43 and its mRNA and display very weak dye coupling. When implanted into the rat cerebrum, these cells quickly give rise to a large glioma. To investigate the role of gap junctions in the tumor characteristics of these cells, we have used Lipofectin-mediated transfection to introduce a full-length cDNA encoding connexin 43. Several transfected clones were obtained that exhibited various amounts of connexin 43 mRNA transcribed from the inserted cDNA. Immunocytochemical analysis revealed an increase in the amount of connexin 43 immunoreactivity in the transfected cells, being localized at areas of intercellular contact as well as in the cytoplasm. The level of dye coupling was also assessed and found to correlate with the amount of connexin 43 mRNA. When cell proliferation was followed over several days, cells expressing the transfected cDNA grew more slowly than non-transfected cells. These transfected cells will be useful in examining the role of gap junctions in tumorigenesis.

Animals↗

PubMed1991

Effect of prenatal ethanol exposure on postnatal neural gene expression in the rat.

To examine the effects of ethanol exposure on neural development, pregnant rats were fed a liquid diet in which 37.5% of the total caloric content was ethanol-derived. The developmental appearance of the messenger RNAs coding for preprosomatostatin, glial fibrillary acidic protein, and proteolipid protein was examined by Northern blotting of total cellular RNA obtained from forebrain and hindbrain at various times after birth. In general, there was a delay in the developmental pattern of appearance of these mRNAs which was most noticeable at the early postnatal times. These results suggest that the previously reported delay in neural maturation is reflected at the level of the gene expression.

Animals↗

PubMed1991

Expression of gap junction genes during postnatal neural development.

The timing of appearance of mRNAs encoding gap junction proteins was examined during development of the rat and mouse brain. Complementary DNAs (cDNAs) specific for the mRNA for the liver-type gap junction protein, connexin32, and the heart-type gap junction protein, connexin43, were used to probe Northern blots of total RNA isolated from the forebrain and hindbrain of mice and rats at various times before and after birth. Prior to postnatal day 10, connexin32 mRNA is detectable only at low levels. By postnatal days 10 to 16, a sharp increase occurs in the level of this mRNA. This increase is detectable first in the hindbrain, and subsequently in the forebrain. In contrast, connexin43 mRNA is readily detectable at birth, and the level of this mRNA also increases during subsequent development. The developmental appearance of the gap junction proteins, connexin32 and connexin43, was similar to that of their respective mRNAs. These results indicate that the genes encoding connexin32 and connexin43 are differentially expressed during neural development.

Animals↗

PubMed1991

Gap junction gene expression in human seizure disorder.

Increased intercellular coupling has been implicated in contributing to the synchronization of discharges characteristic of epileptic foci. Using RNA blot analysis, the level of mRNA for the heart-type gap junction protein, connexin43, is shown to be elevated in samples of temporal lobe neocortex obtained at the time of surgical resection for intractable seizure disorder. Much lower levels of this mRNA are detectable in peritumoral temporal lobe tissue samples obtained during removal of cerebral tumors. However, in cases where the tumors had induced acute seizures, the level of connexin43 mRNA is higher than that detected in epileptic samples. Similar changes are observed for the mRNA for the liver-type gap junction protein, connexin32, although the observed differences are less dramatic. These findings indicate that there is an increase in the level of connexin mRNA in the temporal cortex of patients exhibiting seizure disorders, suggesting an increase in the synthesis of gap junction protein that may lead to an increase in intercellular coupling.

Adolescent↗

PubMed1990

Regional differences in connexin32 and connexin43 messenger RNAs in rat brain.

The regional distribution of gap junction mRNAs was examined in the adult rat brain. The level of connexin43 mRNA is more abundant than connexin32, being homogeneously distributed throughout different regions of brain. In contrast, there is dramatic heterogeneity in the level of connexin32 mRNA, with the highest level in the hindbrain. These results suggest that the gap junction genes are differentially expressed in regions of the adult rat brain.

Animals↗

PubMed1990

Ontogeny of somatostatin expression in primary cultures of cortical neurons parallels that seen in vivo.

The expression of somatostatin (SS) was followed during the development of rat cortical neurons growing in primary cultures. The appearance of SS mRNA is delayed in cultures of cortical neurons, where it is not readily detectable prior to 5 days in culture. Between 5 and 11 days in vitro, there is a large increase in the level of SS mRNA in these cultures. The analysis of SS immunoreactive neurons indicates that they represent a significant proportion of the total neuronal population in these cultures. The majority of these neurons are bipolar or stellate in morphology, although some pyramidal cells are also immunoreactive for SS. More neurons are immunoreactive for SS28 than for SS28(1-12). Immunoreactivity for SS28(1-12) is primarily localized throughout the entire soma and neurites, while SS28 reactivity is mainly perinuclear in location. These results indicate that the development of SS neurons in primary cortical culture parallels normal cortical SS ontogeny in vivo, suggesting that these cultures provide a suitable model for the study of intrinsic neocortical peptidergic neurons.

Animals↗

PubMed1990

Developmental appearance of somatostatin in the rat cerebellum: in situ hybridization and immunohistochemistry.

The postnatal appearance of the mRNA for preprosomatostatin, as well as the presence of somatostatin 28 and somatostatin 28, was examined in the rat cerebellum. RNA blotting analysis indicates a dramatic decrease in the level of this mRNA during cerebellar maturation. In situ hybridization reveals that many cells contain this mRNA at early postnatal stages, with progressively fewer cells being labeled after postnatal day 20. Immunohistochemical analysis indicates that there is a changing pattern in the distribution of these somatostatin peptides through cerebellar development. Initially, cells in the granule cell layer are stained, and fiber immunoreactivity is localized primarily in this same layer. During the second postnatal week, this granule cell layer staining decreases, and some Purkinje cells display immunoreactivity, particularly in the flocculus and paraflocculus. This Purkinje cell staining decreases by postnatal day 30, but immunoreactive cells are still present in the granule cell layer. In contrast, in the adult cerebellum, very few of these cells remain immunoreactive.

Aging↗

PubMed1989

Development of somatostatin-like immunoreactivity in the hippocampal formation of normal and reeler mice.

Using antisera directed against somatostatin-28 or somatostatin-28(1-12), the development of somatostatin-like immunoreactivity (SS-LI) was examined in the hippocampal formation of normal and reeler mice. As early as postnatal day 5, SS-labeled neurons exhibit the adult pattern of distribution in the normal hippocampal formation, these neurons being situated predominantly in the stratum oriens of the hippocampus and the hilus of the dentate gyrus. In contrast, SS-labeled neurons in the reeler hippocampal formation are dispersed throughout the various layers, reflecting the disrupted laminar organization of the hippocampus in this mutant. In both the normal and reeler hippocampal formation, SS-labeled fibers are most abundant in the stratum lacunosum moleculare. However, in the reeler, there appears to be increase in the density of SS-LI fibers, not only in the stratum lacunosum moleculare but also in the stratum oriens, stratum radiatum and pyramidal cell layer of the hippocampus.

Animals↗

PubMed1988

Immunohistochemical analysis of the development of somatostatin in the reeler neocortex.

The development of somatostatin (SS) neurons and fibers has been examined in the dorsolateral cortex of the mouse mutant reeler. Immunohistochemistry was performed using antisera directed primarily against SS28 or SS28(1-12). In the normal mouse at postnatal day 5 (P5), somatostatin (SS) neurons are concentrated in the ventral half of the cortex, in the developing layers V and VI. In the reeler mutant, SS neurons are scattered throughout the radial extent of the cortex, being concentrated in the dorsal half of the cortex in the polymorphic and large pyramidal cell layers. By P20, when the adult pattern of SS neuron distribution is evident in the normal mouse cortex, the distribution of similar neurons in the reeler appears inverted: immunoreactive neurons are concentrated in the dorsal half of the cortex. Immunoreactive fiber distribution follows a developmental pattern similar to that observed for SS neurons. At P5, SS fibers are most dense in layer I and V-VI of the normal cortex, while in the reeler, fibers are predominant in the polymorphic and large pyramidal cell layers. By P10, many fewer immunoreactive fibers can be detected in either normal or reeler mice than at P5. Nevertheless, while SS fibers in the normal cortex are most dense in layers I and V-VI, the reeler cortex exhibits little laminar heterogeneity in the distribution of these fibers. Thus, the SS fiber distribution appears less organized in the reeler cortex. These results suggest that whatever the nature of the genetic alteration resulting in cortical cellular developmental malposition in the reeler, SS cells and fibers, representing a completely intrinsic neocortical cellular system, behave as do all other cortical elements.

Aging↗

PubMed1988

Development of somatostatin-containing neurons and fibers in the rat hippocampus.

Using a combination of in situ hybridization and immunohistochemistry, the development of somatostatin (SS)-containing neurons and fibers was examined in the rat dorsal hippocampus and dentate gyrus. The major development of this hippocampal peptidergic system occurs postnatally. At postnatal day 1 (P1), neurons containing SS mRNA are evident primarily in the stratum oriens, but also in the hilus of the dentate gyrus. Similar neurons are also immunoreactive for SS28 and SS28(1-12), suggesting a minimal lag in the transcription of SS mRNA and its translation into specific SS peptides. The number of SS neurons increases postnatally to P10, followed by a decrease in number in the adult. This transient change in the number of SS neurons coincides with dramatic changes in SS28(1-12)-immunoreactive fibers, which are initially present in the stratum lacunosum moleculare, with no significant immunoreactivity in the dentate gyrus. By P15, the molecular layer of the dentate gyrus is densely innervated, while similar immunoreactivity in the stratum lacunosum moleculare is greatly reduced. These data are consistent with a transient projection from the stratum oriens to the stratum lacunosum moleculare, which is replaced by a projection from the hilus to the molecular layer of the dentate gyrus as this structure matures.

Animals↗

PubMed1988

Immunohistochemical and in situ hybridization analysis of the development of the rat somatostatin-containing neocortical neuronal system.

The chemical differentiation of somatostatin (SS) neurons in rat neocortex was characterized by molecular biochemical and morphological methods. Northern (RNA) blotting indicates that regional distribution of SS mRNA correlates with the known distribution patterns of SS-containing neurons in the adult, while similar analysis of poly (A)+ RNA isolated from telencephalon at various times postnatally shows an increase between P9 and P15, with a slight decrease in the adult. In situ hybridization with a probe specific to SS mRNA, and immunohistochemistry using antisera specific for the N-terminally extended form of SS, SS28, and SS28(1-12), were used to detect neocortical neurons containing this mRNA or its translation product. The appearance of SS mRNA is coincident with detectable immunoreactivity for SS peptides. The expression of the SS gene by cortical neurons occurs in two waves. From P1 to P11, hybridizing neurons are predominant below the cortical plate in the developing infragranular layers. Immunohistochemical analysis of immunoreactivity to SS28 reveals a significant development of this neocortical system by late gestation (E20). At this point SS28(1-12), the predominant SS form detected, is mainly in neurons of the subplate, with less detectable immunoreactivity in the intermediate zone and cortical plate. By P2, neurons in the subplate exhibit detectable SS28 and SS28(1-12). Although immunoreactive perikarya are no longer detectable at P2 in the cortical plate or marginal zone, a very dense plexus of SS28(1-12) fibers is seen in the subplate, marginal zone, and intermediate zone; relatively few immunoreactive fibers are found in the cortical plate. By P12, a dramatic shift occurs; a large supragranular population of these SS neurons is observed by both mRNA and antibody methods, as is a subsequent decrease in number in the adult. The shift in immunoreactivity occurs with supragranular SS28-containing neurons now prominent, and SS28(1-12)-containing neurons and fibers greatly diminished. The number of neurons containing SS mRNA or SS28 immunoreactivity decreases from P12 to adult, when these neurons exhibit a bilaminar distribution. Neurons immunoreactive for SS28(1-12) are now sparsely distributed throughout the cortex, while SS28(1-12) fibers densely innervate layers I and V/VI.

Age Factors↗

PubMed1987

Isotypes of alpha-tubulin are differentially regulated during neuronal maturation.

The mRNAs for two isotypes of alpha-tubulin, termed T alpha 1 and T26, are known to be expressed in the rat nervous system. We have compared the expression of these two alpha-tubulin mRNAs during neural development, using RNA blotting and in situ hybridization techniques with probes directed against unique sequences of each mRNA. T alpha 1 mRNA is highly enriched in the embryonic nervous system but is markedly less abundant in the adult brain; T26 mRNA is expressed in many embryonic tissues with little change in abundance during development. Within the nervous system, T alpha 1 mRNA is enriched in regions with neurons actively undergoing neurite extension, such as the cortical plate, whereas T26 mRNA is relatively homogeneous in distribution, with some enrichment in proliferative zones. Expression of T alpha 1 mRNA is also increased in PC12 cells induced to differentiate and extend neurite processes by nerve growth factor. Taken together, the data indicate that T alpha 1-tubulin mRNA is expressed at high levels during the extension of neuronal processes. The abundant expression of T alpha 1-tubulin mRNA may therefore reflect either a means to increase the available pool of alpha-tubulin or a specific requirement for the T alpha 1 isotype for neurite extension.

Cell Differentiation↗

PubMed1987

Developmentally regulated rat brain mRNAs: molecular and anatomical characterization.

In order to identify markers for developing neural cell populations and gain molecular insights into the processes of neural development and differentiation, we have selected cDNA clones of rat brain mRNAs that are expressed in brain at embryonic day 16 (E16) with at least 10-fold greater abundance than they are in adult brain. Eleven such clones were obtained from a cDNA library of E16 brain poly(A)+ RNA using a combination of differential and subtractive hybridization screens. The temporal and spatial patterns of expression of the mRNAs corresponding to these clones were characterized by Northern (RNA) blotting and by in situ hybridization. Although all the mRNAs were enriched in embryonic brain, different mRNAs demonstrated maximum abundance at different times in late embryogenesis. The mRNAs can be grouped into 3 classes on the basis of their patterns of spatial expression in the embryo: one cDNA clone from each class and its corresponding mRNAs have been characterized in more detail. Class C represents mRNAs that are highly enriched in the nervous system and may be expressed in newly differentiating neurons; the example chosen was shown by nucleotide sequence analysis to encode the brain alpha 1 isotype of tubulin. Class B mRNAs have a broader distribution in the developing embryo but are expressed predominantly in the ventricular germinal zones of the developing nervous system and may represent molecules involved with neurogenesis. A third class (Class A) includes mRNAs with a more homogeneous distribution within the embryo and developing nervous system, which may encode "housekeeping" molecules. These clones and their encoded products will provide markers for cell populations at particular stages of neural development.

Animals↗

PubMed1986

Contralateral corticorubral fibers induced by neonatal lesions are not collaterals of the normal ipsilateral projection.

Unilateral neonatal cortical ablation induces the development of a bilateral corticorubral projection from the remaining sensorimotor cortex. The retrograde fluorescent tracers Fast blue (FB) and Nuclear yellow (NY) were used to determine if the aberrant contralateral projection arises from axon collaterals of the normal uncrossed projection. Six to 8 weeks after unilateral cortical ablation in neonatal rats, the red nuclei were injected with FB on one side and NY on the other to study the source of the normal and aberrant afferents from the cerebral cortex. In control animals, many neurons in layer V of the sensorimotor cortex were retrogradely labeled with the tracer that had been injected into the ipsilateral red nucleus. In animals with unilateral ablations, many neurons throughout the remaining sensorimotor cortex were retrogradely labeled with FB or NY. No cortical neurons were doubly labeled. In addition to demonstrating the bilaterality of the corticorubral projection in animals which had received neonatal lesions, these results indicate that the aberrant contralateral corticorubral projection does not consist of axon collaterals of the normal ipsilateral fibers.

Animals↗