In Chinese hamster Don cells, fusion of an interphase cell with a metaphase cell resulted either in prophasing of the interphase nucleus, including loss of the nuclear envelope (NE), or in the formation of a double membrane around the metaphase chromosomes . Only one of these phenomena occurred in a given interphase-metaphase (I-M) binucleate cell . At pH 7 .4, there was about an equal probability that either event could occur amongst the population of I-M cells . The effect of pH changes in the medium containing the fused cells was examined . At pH 6 .6, prophasing was the predominant event ; at pH 8 .0, membrane formation predominated . It was found that the rate of progression of a mononucleate cell from Gs to metaphase was appreciably faster at pH 6 .6 than at pH 8 .0 . Conversely, the progression from metaphase to G, was faster at pH 8 .0 than at pH 6 .6 . These results with the mononucleate cells strengthen the hypothesis that structural changes in I-M cells are reflections of normal mitotic phenomena . Additional evidence for this hypothesis was produced by electron microscope examination after direct fixation in chrom-osmium . The double membrane around the chromosomes of the I-M cell was indistinguishable from the normal NE . The results obtained by varying the pH of the medium containing the fused cells provide an indication that disruption or formation of the NE of Don cells depends on the balance reached between disruptive and formative processes .

Fusing human HeLa metaphase cells with HeLa interphase cells resulted within 30 min in either of two phenomena in the resultant binucleate cell: either prophasing of the interphase nucleus or formation of a normal-appearing nuclear envelope around the metaphase chromosomes. The frequency of either occurrence was strongly dependent on environmental pH. At pH's of 6.6 8.0, prophasing predominated; at pH 8.5 nuclear envelope formation predominated. Additionally, the frequencies of the two events in multinucleate cells depended on the metaphase/interphase ratio. When the ratio was 0.33 nuclear envelope formation predominated; when it was 2.0 prophasing predominated. In their general features, the results with fused HeLa cells resembled those reported earlier with fused Chinese hamster Don cells. However, the results provided an indication that between pH 6.6 and 8.0 the HeLa metaphase cells possessed a much greater capacity than the Don metaphase cells to induce prophasing. Fusion of Don metaphase cells with HeLa interphase cells or of Don interphase cells with HeLa metaphase cells at pH 8.0 resulted in nuclear envelope formation or prophasing in each kind of heterokaryon. As in the homokaryons, the frequencies of the two events in the heterokaryons depended on the metaphase/interphase ratio. The statistics of prophasing and nuclear envelope formation in the homo-and heterokaryon populations were consistent with the notion that disruption or formation of the nuclear envelope depends on the balance attained between disruptive and formative processes.

To ascertain cocaine's effects in the fetus, we developed a nonhuman primate model in which pregnant monkeys were administered cocaine (3 mg/kg im) or saline four times a day from day 20 through days 40-70 of a 165-day gestation. At the time of cesarean section, plasma levels of cocaine in fetal blood were 231 ± 70 ng/ml. Fetal brains were examined using immunocytochemistry, in situ hybridization, receptor autoradiography, and nuclease protection assay analysis. No differences were found in the expression of tyrosine hydroxylase and dopamine receptor mRNAs by days 40-45 of gestation. However, by day 60 the midbrain of monkeys exposed to cocaine had significantly reduced expression of tyrosine hydroxylase, the rate-limiting enzyme in dopamine synthesis. Moreover, dopamine D and D receptor mRNAs were significantly elevated in the rostral forebrain as were D and D receptor binding sites in days 60-70 cocaine-exposed fetuses. Cocaine treatment from day 20 to days 60 and 70 of gestation also significantly increased the mRNA concentrations of dynorphin and enkephalin in the rostral forebrain. These findings suggest that in utero cocaine exposure has profound effects on the developing dopamine neurocircuitry.

Cocaine exposure in utero is known to cause a variety of behavioral and motor deficits that may be attributable to alterations in the dopamine neurocircuitry. To ascertain cocaine effects in the fetus, we developed a nonhuman primate model in which pregnant monkeys were administered cocaine from day 20 through day 60 or 70 of gestation. Fetuses from these pregnancies develop a repertoire of neural deficiencies, including decreased mRNA expression of tyrosine hydroxylase in the midbrain and increased mRNA expression of dopamine receptor subtypes in the rostral forebrain. Presently, we studied the effects of maternal cocaine treatment on the mRNA expression of the endogenous opioids preprodynorphin (PPD) and preproenkephalin (PPE) in fetal monkey brains. Fetuses exposed to saline (0.9%) or cocaine (3 mg/kg) were delivered by Caesarean section, the fetal brains were dissected, and tissue RNA was extracted and quantified using ribonuclease protection assay analysis. The opioid peptides PPD and PPE were expressed in the fetal monkey brain by day 60, and even higher levels were found in day 70 fetuses. Maternal exposure to cocaine increased gene expression of PPD and PPE in the fetus at both day 60 and day 70 of gestation. Dynorphin mRNA levels were significantly elevated in the striatum, whereas enkephalin mRNA was elevated in both the frontal cortex and the striatal area of fetuses whose mothers received cocaine. Changes in the expression of these opioid peptides in presumed dopamine target neurons, which mediate motivation and reward, as well as motor control, provide further evidence for profound consequences of in utero cocaine exposure on the developing dopamine neurocircuitry.