International Journal of Developmental Neuroscience

Gene transfer is an exciting new tool in medical therapy and scientific investigation, but only very recently has it begun to be developed in the auditory system. This paper describes in vivo and ex vivo experiments using an adenoviral vector (Ad. RSVntlacZ), which is a replication‐deficient virus based on a human adenoviral (serotype 5) genomic backbone. The in vivo experiments demonstrate successful gene transfer into multiple types of cochlear cells. We observed a relatively efficient transduction, several weeks of sustained transgene expression and an absence of major lethal cytotoxicity in spiral ganglion and epithelial cells of the cochlea in adult animals. The ex vivo experiments were performed using fibroblasts transduced in vitro with Ad. RSVntlacZ. Two weeks after inoculation of the fibroblasts into the perilymph, we observed transplanted fibroblasts, which were adherent to the lining of the perilymphatic spaces, and were expressing the lacZ transgene. We speculate that, as the genetic basis of degenerative cochlear diseases is characterized on a mutational level, transgene expression will allow us to test hypotheses regarding the effects of specific genes on cochlear cell biology. Gene transfer will not only increase our understanding of the pathophysiology of hearing loss, but also may provide gene therapy for disease.

Hypothyroidism induced by perinatal administration of propylthiouracil (PTU) had profound effects on growth of the heart, with major organ weight deficits persisting well beyond the termination of drug treatment. These effects were preceded by disruption of the developmental patterns of cardiac ornithine decarboxylase (ODC) and the polyamines, which are thought to be intracellular modulators of cellular maturation. Activity of cardiac ODC was depressed in the PTU‐treated group and putrescine and spermidine levels were markedly subnormal. PTU administration also affected brain growth, but much less so than in the heart. The disruption of the brain ODC/polyamine system was also less pronounced, with relatively small degrees of spermidine depletion and a slight elevation of ODC. For both tissues, the biochemical effects of perinatal hypothyroidism were opposite to those found previously for administration of exogenous thyroid hormones. These results support the views that: (1) endogenous thyroid hormones contribute to the maintenance of normal developmental patterns of ODC and the polyamines, and (2) the ODC/polyamine system participates in events modulating subsequent tissue growth.

Glutamate plays an important role in brain development, physiological function, and neurodegeneration. Astrocytes control synaptic concentration of glutamate via the high affinity glutamate transporters, GLT‐1 and GLAST, and the glutamate catabolizing enzyme, glutamine synthetase. In this study we show that astrocytes cultured from rat brain in various stages of development including embryonic (E18), postnatal (P1–P21) and mature (P50), show distinct patterns of GLT‐1 and GLAST expression, glutamine synthetase activity, and phenotypic changes induced by dibutyryl‐cyclic adenosine monophosphate. The transcripts for GLT‐1 message were detectable in embryonic astrocytes only, whereas the GLAST message was highly expressed in E18 and P1–P4 astrocyte cultures, declined in P10–P21, and was undetectable in P50 astrocytes. Uptake of³H‐glutamate correlated well with GLAST expression in astrocyte cultures of all developmental stages. Glutamine synthetase activity significantly declined from high embryonic levels in P4 astrocytes and remained low throughout postnatal maturation. Exposure of astrocyte cultures to the differentiating agent, db‐cAMP (250–500 μM; 6 days), resulted in a pronounced stellation, up‐regulation of GLT‐1 and GLAST in E18, and GLAST in P4 cultures, while it was ineffective in P10 astrocytes. By contrast, db‐cAMP induced a more pronounced stimulation of glutamine synthetase activity (up to 10‐fold above basal) in P10 than in E18 cultures (up to 2 times above basal). The differences in expression/inducibility of glutamate transporters and glutamine synthetase observed in astrocyte cultures derived from various stages of fetal and postnatal development suggest that astrocytes in vivo might also respond differently to environmental or injurious stimuli during development and maturation.

Cultures of astroglia from C₃H/HeJ mice, which are resistant to bacterial cell wall polysaccharide (LPS), initiated from embryos of Theiler stage 14 (9 days of gestation) up to Theiler stage 25 (17 days of gestation) as well as newborn animals, when subjected to nutritional deprivation, i.e. non‐feeding of cultures, form large numbers of macrophage‐like cells. These cells express Mac‐1, Mac‐3, F4/80 and Fc antigens. The cells are negative for GFAP, positive for vimentin, express Ia antigen and take up DiL‐Ac‐LDL. They are positive to non‐specific esterase, secrete lysozyme and are phagocytic. Their morphology and ultrastructure closely resemble those of macrophages. Cultures initiated from neuroepithelium of Theiler stage 13 (8.5 days of gestation), before vascularization, when subjected to nutritional deprivation, also produce macrophage‐like cells. Using spleen colony assay and methyl cellulose cultures, we were unable to detect the presence of hemopoietic (macrophage) precursor cells in astroglia cultures. This supports the hypothesis that the macrophage‐like cells are of neuroectodermal origin and probably correspond to resident microglia of the CNS. Using nutritionally deprived astroglia cultures, a procedure was developed for isolation of macrophage‐like cells and production of highly enriched macrophage‐like (microglia) cultures.

We have investigated the effect of PS‐D induced in gestating rats by treatment with clomipramine or with the platform technique on the process of DNA synthesis taking place in fetal organs. This parameter was taken as a biochemical index of ongoing cellular proliferation. In brain and, to a minor extent, in liver and kidney the rate of fetal DNA synthesis was markedly increased in both experimental groups. The effect was more prominent in the clomipramine group. PS‐D treatment of gestating rats, notably by the platform technique, left long‐lasting effects in the offspring with regard to organ weight and DNA concentration as well as to learning capacity. It is concluded that the occurrence of PS in gestating rats may exert a significant influence on fetal development.