Hexose uptake by hamster cells was increased five to ten fold by either substituting D-fructose for glucose or by completely omitting D-glucose from the culture medium for 24 to 48 hours. Conversely, when cycloheximide was present for 24 hours in media containing glucose, up to 20-fold decreases in hexose uptake were observed. However, these decreases in uptake activity were only observed over a narrow range of cycloheximide concentrations. After extended exposure to low concentrations of cycloheximide (0.05 to 10 mug/ml), the uptake by the fed cells decreased parallel with inhibition of protein synthesis whereas at high concentrations (greater than 50 mug/ml) uptake was increased. Cells deprived of glucose and maintained in the presence of cycloheximide did not show decreases in uptake activity. In separate experiments the high uptake rates of glucose-starved cells could be decreased by addition of glucose-free medium. The reversal was complete in 6 to 8 hours. The analog of glucose, 2-deoxy-D-glucose, did not promote the time-dependent decrease suggesting that the 6-phosphoester of glucose is not an inhibitor of transport. In addition, when cycloheximide is added at the same time as glucose, there is no decrease in uptake for at least 12 hours. We propose that turnover of components of hexose uptake systems could account for part of the control of hexose transport. Moreover, the results indicate that the turnover mechanism becomes inactive during glucose starvation and must be resynthetized following refeeding of the starved cells with glucose.

The modulation of motor behavior by protein kinase C (PKC) signaling pathways in nigrostriatal neurons was examined by using a genetic intervention approach. Herpes simplex virus type 1 (HSV-1) vectors that encode a catalytic domain of rat PKC␤II (Pkc⌬) were developed. Pkc⌬ exhibited a constitutively active protein kinase activity with a substrate specificity similar to that of rat brain PKC. As demonstrated in cultured sympathetic neurons, Pkc⌬ caused a long-lasting, activationdependent increase in neurotransmitter release. In the rat brain, microinjection of HSV-1 vectors that contain the tyrosine hydroxylase promoter targeted expression to dopaminergic nigrostriatal neurons. Expression of pkc⌬ in a small percentage of nigrostriatal neurons (ϳ0.1-2%) was sufficient to produce a long-term (Ն1 month) change in apomorphine-induced rotational behavior. Nigrostriatal neurons were the only catecholaminergic neurons that contained Pkc⌬, and the amount of rotational behavior was correlated with the number of affected nigrostriatal neurons. The change in apomorphineinduced rotational behavior was blocked by a dopamine receptor antagonist (fluphenazine). D 2 -like dopamine receptor density was increased in those regions of the striatum innervated by the affected nigrostriatal neurons. Therefore, this strategy enabled the demonstration that a PKC pathway or PKC pathways in nigrostriatal neurons modulate apomorphineinduced rotational behavior, and altered dopaminergic transmission from nigrostriatal neurons appears to be the affected neuronal physiology responsible for the change in rotational behavior.

A defective herpes simplex virus type one (HSV-1) vector that contains a 6.8-kb fragment of the rat tyrosine hydroxylase promoter (pTHlac-7kb) was examined for its capability to target catecholaminergic cell type-specific expression in the CNS. Cell type-specific expression was assessed by comparison with a control vector (pHSVlac) that uses the HSV-1 immediate early 4/5 promoter to support expression in multiple cell types.In initial experiments comparing expression in catecholaminergic and noncatecholaminergic cell lines, pTHIac-7kb supported a seven-to 20-fold increase in reporter gene expression in catecholaminergic cell lines. Four days after stereotactic injection into the midbrain of adult rats, pTHlac-7kb supported a 10-fold targeting of~3-galactosidase expression to tyrosine hydroxylase-expressing neurons in the substantia nigra pars compacta compared with pHSVlac. Expression from pTHlac-7kb was stably maintained for 6 weeks with no significant changes in the pattern of expression. Long-term expression from pTHlac-7kb was confirmed by ANA and DNA analysis. In contrast, reporter gene expression in the midbrain from pHSVlac decreased~30-fold between 4 days and 6 weeks after gene transfer. Thus, within the context of this HSV-1 vector system, the tyrosine hydroxylase promoter enhanced cell type-specific expression and contributed to stable, long-term expression of a recombinant gene product in neurons. The capability to target recombinant gene expression to catecholaminergic neurons in specific brain areas may be useful for studies on the roles of these neurons in brain physiology and behavior. Key Words: HSV-1 vector-Tyrosine hydroxylase promoter-Cell type-specific expression -In vivo gene transfer-Longterm expression-Genetic intervention.