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There is experimental evidence indicating that the non-competitive NMDA receptor antagonist MK-801 impairs cognition and produces a series of schizophrenia-like symptoms in rodents (hypermotility, stereotypies, and ataxia). The present study was designed to investigate the efficacy of the nitric oxide (NO) donor molsidomine in counteracting these MK-801-induced behavioral effects in the rat. In a first study, post-training administration of molsidomine (at 4 but not 2 mg/kg) successfully antagonized MK-801-induced performance deficits in a recognition memory test. In a subsequent study, molsidomine (2 and 4 mg/kg) was shown to be unable to reverse MK-801-induced hypermotility but attenuated stereotypies (continuous movement whole cage, body sway, and head weaving) produced by MK-801. Moreover, at 4 mg/kg this NO donor counteracted MK-801-induced ataxia. Our findings indicate that molsidomine attenuates behavioral effects related to the hypofunction of the NMDA receptor suggesting that NO might be involved in the psychotomimetic effects of non-competitive NMDA receptor antagonists.

Cells from human amniotic fluid derived from the fetus are considered a source of multipotent cells. Their properties have not been fully exploited, partially because unlike other embryonic sources such as embryonic stem (ES) cells, cell lines from amniocentesis samples have not been generated. We have established and characterized the properties of eight individual cell lines. Flow cytometry using several cell surface markers showed that all cell lines generated consisted of homogeneous populations that lack HLAII antigenicity. Using a combination of immunocytochemistry, Western blotting, and RT-PCR, we found weak expression of Oct4 and nestin and strong expression of tubulin-betaIII, MAP2, and tau. Specific markers for cholinergic, (nor)adrenergic, and GABAergic neurons or glia were weakly expressed or absent, whereas expression of factors implicated in early induction of dopaminergic neurons, TGF-beta3 and beta-catenin were present. Further analysis showed strong expression of EN-1, c-RET, PTX3, and NURR1 essential for induction and survival of midbrain dopaminergic neurons, TH, AADC, and VMAT2 components of dopamine synthesis and secretion, and syntaxin1A and SNAP-25 necessary for neurotransmitter exocytosis. This phenotype was retained throughout passages and up to the current passage 36. Expression of neuronal and dopaminergic markers in individual AF cell lines was comparable to expression in neurons induced from ES cells and in IMR-32 and SH-SY5Y neuroblastomas. Our data show that cell lines can be derived from subcultures of amniocentesis, and are primarily composed of a population of progenitors with a phenotype similar to that of committed mesencephalic dopaminergic neurons., (Copyright 2006 Wiley-Liss, Inc.)

Cultures were prepared by dissociating 3-day-old whole chick embryos and plating the dispersed cells on poly-L-lysine-coated dishes in Dulbecco's Modified Eagle's Medium with 10% fetal calf serum. By 48 hr in culture, aggregates and neuritic sprouting were observed. Long neuritic bundles connecting cell aggregates were evident by 4 days in culture. Consistent patterns throughout the lifespan of the cultures were contacts between neurites, and flat isolated cells, presumptively glial, emerged. Throughout the lifespan of the cultures, the cholinergic cell population was characterized histochemically by the method of Karnovsky and Roots and biochemically by assaying choline acetyltransferase. By 4 days in culture, all aggregates showed light cholinesterase-positive staining; however, with days in culture, several aggregates had no staining, and some positive-stained aggregates were interconnected with other aggregates showing only spotted positive staining. Choline acetyltransferase activity showed a developmental profile in agreement with the histological findings. The early presence of choline acetyltransferase activity is taken as indication of the early commitment of cholinergic neurons.

Glial cell cultures derived from newborn and aged (18-month-old) mouse cerebral hemispheres and maintained up to cell passage 11 were characterized immunocytochemically by using glial fibrillary acidic protein (GFA), and biochemically by using glutamine synthetase (GS), for astrocytes, and 2',3' cyclic nucleotide 3' phosphohydrolase (CNP) for oligodendrocytes. We report here the changes occurring during passages 5-11. GS and CNP activities did not significantly change with cell passage in cultures from newborn mouse. In cultures derived from aged mouse, CNP activity did not change significantly whereas GS activity increased severalfold. A characteristic finding in higher cell passages (passage #7) was the loss of GFA-positive stained cells and the appearance of multinucleated cells. We interpret these changes in culture to represent possible signs of cellular senescence.

Changes occurring with days in culture and cell passage in cultured glial cells derived from newborn vs aged (18-mo) mouse cerebral hemispheres were compared. The activities of the enzymes glutamine synthetase (GS), an astrocyte marker, and 2',3'-cyclic nucleotide 3'- phosphohydrolase (CNP), an oligodendrocyte marker, were determined. In addition, glial fibrillary acidic protein (GFA) and glycerol phosphate dehydrogenase (GPDH) immunoreactivity was used to morphologically identify astrocytes and oligodendrocytes, respectively. In cultures derived from newborn mouse cerebral hemispheres, both GS and CNP activity and GFA-positive and GPDH-rhodamine-positive cells were present with cell passage. In general, GS activity did not change in early cell passage in cultures from either newborn or aged mouse; in passage 5, GS was high in both sources of cell populations. CNP activity increased with cell passage in cultures derived from newborn mouse; in cultures derived from aged mouse CNP was low in the primary cultures, increased with cell passages 2 and 3, and declined with passages 4 and 5. The survival of astrocytes as shown by GS and the decline in oligodendrocytes as shown by CNP was also supported by an increase in the proportions of GFA and GPDH immunoreactive cells. We interpret the increase in GS activity to parallel the astrogliosis observed in vivo in the aging brain. Moreover, the decline in oligodendrocytes in culture may represent a shift of balance between glial cell types that appears to be influenced by the age of brain tissue and time in culture.

Glutamine synthetase (GS) activity was used as a marker to examine differences in astrocyte development in mice selectively bred for ethanol sensitivity: long sleep (LS), short sleep (SS), mild ethanol withdrawal (MEW), severe ethanol withdrawal (SEW) and control ethanol withdrawal (CEW). We found that 1) GS activity in MEW and SEW was higher than in LS and SS during the first 2 weeks of postnatal development, in the forebrain but not in the cerebellum; 2) lower GS activity was observed consistently in all areas examined with the SS mice as compared to the LS; 3) glutamine synthetase activity in MEW and SEW differed significantly from their controls (CEW) during the early developmental period regardless of the brain region examined; however, after 30 days of maturation, GS activity in SEW was higher than that in MEW and CEW in the forebrain. Astrocytes are known to contribute in the regulation of the neuronal microenvironment. Therefore, we interpret the differences we found in astrocytic function during early brain development among these lines of mice to account in part for the neuronal predisposition to ethanol sensitivity.