Methamphetamine use by females of child-bearing age has become a major public health concern in terms of the long-term risk to the exposed fetus. We examined the possibility of enhanced adult neurotoxic potential of the drug in offspring that had been exposed to methamphetamine in utero during gestational days 7 to 18. While basal levels of monoamines were not affected by prenatal exposure to methamphetamine, we observed an enhanced neurotoxicity in adult male offspring following drug challenge with effects localized primarily to the dopaminergic nigrostriatal projection. This was evidenced by greater methamphetamine-induced reductions of dopaminergic markers in the striatum [dopamine (DA), dihydroxyphenylacetic acid, homovanillic acid (HVA), and 3-methoxytyramine (3-MT)] and ventral brainstem (DA) of prenatal methamphetamine-treated males compared with saline-treated animals. Some effects of prenatal methamphetamine exposure were observed in female offspring, but these were limited to striatal levels of 3-MT and HVA. Differential gender sensitivity to the neurotoxic effect of methamphetamine was shown to be correlated with hyperthermic response. Hyperthermic effects, however, do not account for the increased susceptibility of prenatal methamphetamine-treated males to drug-induced striatal DA neurotoxicity since methamphetamine challenge did not evoke a significantly greater hyperthermic response in these animals compared with prenatal saline-treated males. The findings raise the concern that male methamphetamine abusers may be at risk for an enhanced neurotoxic risk if they were exposed to the drug in utero.

1. A mutation in the G-protein-linked, inwardly rectifying K+ channel GIRK2 leads to the loss of cerebellar and dopaminergic mesencephalic neurons in weaver mice. The steps leading to cell death are not well understood but may involve constitutive influx of Na+ and Ca2+ into the neurons. 2. We found that resting [Ca2+]i was dramatically higher in cerebellar neurons from weaver mice compared to wild-type neurons. 3. High-K+ stimuli elicited much smaller changes in [Ca2+]i in weaver cerebellar neurons compared to wild-type neurons. 4. weaver cerebellar granule cells could be rescued from cell death by the GIRK2wv cationic channel blocker, QX-314. 5. QX-314 lowered resting intracellular Ca2+ levels in weaver cerebellar granule cells. 6. These results suggest that changes in resting [Ca2+]i levels and alterations in K+ channel function are most likely to contribute to the developmental abnormalities and increased cerebellar cell death observed in weaver mice.

To investigate the effect of cell-to-cell variation in store-operated calcium entry (SOCE) on the evaluation of data from stable cell clones selected following gene transfection, we measured SOCE in 2700 individual HEK-293 cells from the parent population and in 1900 individual cells from a clonal subpopulation of HEK-293 cells. We applied statistical resampling techniques to model conditions where one would compare the average SOCE in n control clones to the average SOCE in n experimental clones (n = 1-200). For an overexpression experiment with n = 1, there is a 27% chance of observing a 100% or higher difference in SOCE between clones, with n = 10 there is a 34% probability of observing a 20% or greater difference in SOCE, and with n = 100, there is less than a 10% chance of seeing a 10% or greater difference in SOCE, based solely on random selection of clones from the parent HEK-293 cell population. To assure that the degree of cell-to-cell variation was predictive of the degree of clone-to-clone variation, we measured SOCE in 270 clones, each arising from a single cell, and found the variation to be very similar to that observed for individual cells.

The murine weaver ( wv ) mutation is characterized by a genetically determined loss of several neuronal populations, which include the nigrostriatal dopaminergic neurons. Animals homozygous for the wv gene exhibit marked deficits in dopaminergic morphological and neurochemical parameters. The wv gene shows incomplete dominance in that heterozygous ( wv /+) mice exhibit moderate reductions in midbrain dopaminergic neuron number. It is unclear whether the dopaminergic neuronal loss in homozygous and heterozygous animals results from an effect of the wv gene solely on the dopaminergic neurons or is due to a failure of interaction of dopaminergic neurons with target cells of the striatum. This issue has been addressed utilizing three-dimensional reaggregate tissue cultures to determine whether the wv gene acts directly on the mesencephalic dopaminergic neurons. Embryonic mesencephalon and striatum from wv /+ and wild-type (+/+) brains were dissociated and the cells recombined into four mesencephalic-striatal aggregate combinations: (1) mesencephalic (+/+) -striatal (+/+) aggregates; (2) mesencephalic (wv/+) -striatal (wv/+) aggregates; (3) mesencephalic (wv/+) -striatal (+/+) aggregates; and (4) mesencephalic (+/+) -striatal (wv/+) aggregates. At 29 days and 57 days of culture, the number of dopaminergic neurons and dopamine content from mesencephalic-striatal aggregates consisting of mixed genotype or from only wv /+ tissue were quantitated and compared with that from mesencephalic-striatal cultures prepared from +/+ tissue alone. At both culture time points, aggregates containing wv /+ mesencephalon coaggregated with either wv /+ or +/+ striatum contained fewer dopaminergic neurons than mesencephalic-striatal cultures composed of only +/+ cells. Coaggregation of +/+ mesencephalon with wv /+ striatum did not have a detrimental effect on dopaminergic cell number. The findings demonstrate that the difference in the number of mesencephalic dopaminergic neurons between wv /+ and +/+ animals seen in vivo can be reproduced in three-dimensional reaggregate culture. Since the coculture of +/+ striatum with wv /+ mesencephalon did not appear to rescue wv /+ dopaminergic neurons in the aggregates as compared to wv /+ striatum and, wv /+ striatum proved to be a perfectly adequate target for +/+ mesencephalic dopaminergic neurons, it appears that the effect of the wv gene is on the dopaminergic neurons themselves.

Three-dimensional (3D) reaggregate tissue culture provides a means of quantitatively assessing neuronal cell survival under a variety of experimental conditions. A method is presented for estimation of, and comparison between, the total numbers of cells of a given neurochemical type within individual experimental flasks. The method involves cell counting from random reaggregate sections, determination of sectional volumes, and of 3D reaggregate volumes by a novel image-analysis computer system.

1. The rotation-mediated three-dimensional reaggregate culture system is uniquely suited for studies on developmental neurotoxicity. In this system, it is possible to reconstruct central neuronal pathways and follow their development. 2. Exposure to drugs of abuse including methamphetamine and methylenedioxyamphetamine or the appetite suppressant, fenfluramine, reduces monoamines in the cultures in a dose-dependent manner and interrupts normal monoaminergic development. 3. While the monoaminergic neurones may attain normal rates of development following drug removal, the affected neurones are not capable of overcoming the drug-induced insults and a deficiency in monoamines persists throughout development. 4. In addition, the production of immortalized monoclonal hybrid cells obtained by fusion of fetal mesencephalic neurones with a neuroblastoma has yielded cell lines expressing a dopaminergic phenotype. 5. Such cells have been useful in establishing the relationship of neurotoxicity to cell lineage and can serve as models for the study of the cellular and molecular mechanisms of neurotoxicity.

Three-dimensional, rotation-mediated reaggregate tissue cultures composed of rostral mesencephalic cells and corpus striatal cells were used to examine the short-term and persistent effects of methamphetamine on developing monoamine-containing neurons. Reaggregates were exposed to drug for one week. Reductions in reaggregate endogenous dopamine and serotonin levels occurred following treatment with methamphetamine during days 15-22 of culture over the concentration range 10(-7) to 10(-4) M. The highest methamphetamine concentration reduced dopamine and serotonin levels to 29 and 33%, respectively, of control values. Monoamine levels were reduced from control values after 3 days of exposure to 10(-4) M methamphetamine. No further reduction resulted from 4 additional days of drug treatment. In order to determine whether monoaminergic neurons would recover from the drug-induced deficit, reaggregates were exposed to 10(-4) M methamphetamine for 7 days and then grown in drug-free media for an additional 20 days. During the 20 day recovery period, monoamine levels in the control group increased with time in culture. After an initial rapid increase (recovery days 0-9), the level of monoamines in the recovery group remained at a constant proportion to the level in the control group suggesting that the monoaminergic neurons return to a rate of development similar to that seen in untreated cultures. However, this rate was not sufficient to overcome the reduction in monoamine levels produced by 7 days of methamphetamine treatment. The results indicate that the effects of methamphetamine on developing monoaminergic neurons are marked and persistent.

The study of cellular interactions during the development of the nervous system is facilitated by the use of the reaggregate tissue culture system. In this system, embryonic neurons are dissociated from one another and allowed to reaggregate in rotatory culture, where they resume their normal differentiation. Reaggregates provide a model for studying neuronal development and cell interactions under conditions which allow for the control of a large number of variables, such as the numbers of cells which are allowed to interact, the presence or absence of appropriate target cells, and the chemical environment in which the neurons develop. For quantitative purposes, it is necessary to estimate the numbers of cells of a given neurochemical or morphologic type. Here we describe a computer-assisted counting system which allows one to determine the numbers of neurochemically identified neurons within reaggregate cultures from the counting of such neurons in a randomly selected sample of thirty 10-μm sections from the reaggregates in a given flask. These sections represent less than 0.5% of the total number of sections derived from the reaggregates in a single flask. In addition, the system allows one to quantitatively compare differences in the numbers of such neurons between experimental flasks utilizing appropriate statistical methods. A comparison of the number of central dopaminergic neurons estimated by this method with the direct counting of such neurons in serial sections of single reaggregates resulted in excellent agreement as to the number of dopaminergic neurons within a given experimental flask. The method of assessing and comparing the number of neurons of a given neurochemical type outlined here can be applied to problems of natural cell survival as well as to survival following exposure to a variety of potentially toxic agents.