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Infants who are passively exposed to morphine or heroin through their addicted mothers usually develop neurobiological changes. The postsynaptic density 95 (PSD-95) protein, a submembranous cytoskeletal specialization, is dynamically linked with N-methyl-d-aspartate receptors (NMDARs) to form a synaptic complex in postsynaptic neurons. This complex serves important neurobiological functions, including mammalian learning and memory. However, the effects of prenatal morphine exposure on this synaptic complex are not well understood. In this study, we determined whether prenatal morphine exposure altered the synaptic complex association between PSD-95 and three major NMDAR subunits (NR1, NR2A, and NR2B), at the mRNA and protein levels, within the hippocampal CA1 subregion (an important integration area for mammalian learning and memory) of rat offspring along with the performance of long-term cognitive functions. Sprague-Dawley rat offspring from morphine-addicted mothers were studied at a younger age (postnatal day 14; P14) and at an older age (P45). Subsequently, an eight-arm radial maze task was applied to analyze the working and cued reference memory in such offspring (P45). The real-time polymerase chain reaction results showed that prenatal morphine exposure caused significant decreases in mRNA levels of the PSD-95 and three NMDAR subunits (NR1, NR2A, and NR2B) in offspring (P14 and P45). Similarly, at the protein level, immunoblotting showed that decreased whole levels of PSD-95 and NMDAR subunits were seen in offspring subjected with prenatal morphine. Furthermore, the protein interaction of the synaptic complex between the PSD-95 and NMDAR subunit, as indicated by coimmunoprecipitation, was less in prenatal morphine samples than in vehicle controls (P14 and P45). The prenatal morphine group also showed poorer performance for an eight-arm radial maze task than the vehicle-control group. These results are particularly important for a better understanding of certain opioid-mediated neurobehavioral cognitive changes in offspring associated with altered protein interaction between PSD-95 and NMDAR subunits within the developing brain.

In this study, the mechanism of transferrin-free iron uptake by brain neuronal cells was investigated using the cultured cerebellar granule cells. Effects of incubation time, iron concentration, temperature and other divalent metals on the cellular uptake were determined. After five days of plating, the cells were incubated with different concentrations of transferrin-free iron in isotonic sucrose solution at different temperatures for a certain time. The cellular transferrin-free iron uptake was analysed by measuring the cellular radioactivity with a gamma-counter. The result showed that the cultured cerebellar granule cells had the capacity to acquire transferrin-free iron at pH 6.5, at which it was demonstrated that transferrin binds iron very poorly and only very little transferrin can be internalized by reticulocytes and HeLa cells. The iron uptake by cells increased with incubation time in a linear manner at a rate of 0.1076 pmol/microg protein/min within the first 10 min. The uptake was time- and temperature-dependent, iron concentration saturable, and inhibited by several divalent metal ions, such as Co2+, Zn2+, Mn2+ and Ni2+. These characteristics of transferrin-free iron uptake by the cultured cerebellar granule cells observed in this study, similar to those obtained from cells outside of the brain, implied that a carrier-mediated iron transport system might be present on the membrane of this type of brain neuronal cells. In addition, no significant difference in malondialdehyde measurement was found when the cells were incubated without or with the lower concentrations of iron (< 4 microM) for 20 min at 37 degrees C, demonstrating that this system was valid for studying membrane iron transport in this type of brain neuronal cell.