The striatum, which is the major component of the basal ganglia in the brain, is regulated in part by dopaminergic input from the substantia nigra. Severe movement disorders result from the loss of striatal dopamine in patients with Parkinson's disease. Rats with lesions of the nigrostriatal dopamine pathway caused by 6-hydroxydopamine (6-OHDA) serve as a model for Parkinson's disease and show alterations in gene expression in the two major output systems of the striatum to the globus pallidus and substantia nigra. Striatopallidal neurons show a 6-OHDA-induced elevation in their specific expression of messenger RNAs (mRNAs) encoding the D2 dopamine receptor and enkephalin, which is reversed by subsequent continuous treatment with the D2 agonist quinpirole. Conversely, striatonigral neurons show a 6-OHDA-induced reduction in their specific expression of mRNAs encoding the D1 dopamine receptor and substance P, which is reversed by subsequent daily injections of the D1 agonist SKF-38393. This treatment also increases dynorphin mRNA in striatonigral neurons. Thus, the differential effects of dopamine on striatonigral and striatopallidal neurons are mediated by their specific expression of D1 and D2 dopamine receptor subtypes, respectively.

Dopamine receptor belong to a large class of neurotransmitter and hormone receptors that are linked to their signal transduction pathways through guanine nucleotide binding regulatory proteins (G proteins). Pharmacological, biochemical and physiological criteria have been used to define two subcategories of dopamine receptors referred to as D1 and D2. D1 receptors activate adenylyl cyclase and are coupled with the Gs regulatory protein. By contrast, activation of D2 receptors results in various responses including inhibition of adenylyl cyclase, inhibition of phosphatidylinositol turnover, increase in K+ channel activity and inhibition of Ca2+ mobilization. The G protein(s) linking the D2 receptors to these responses have not been identified, although D2 receptors have been shown to both copurify and functionally reconstitute with both Gi and Go related proteins. The diversity of responses elicited by D2-receptor activation could reflect the existence of multiple D2 receptor subtypes, the identification of which is facilitated by the recent cloning of a complementary DNA encoding a rat D2 receptor. This receptor exhibits considerable amino-acid homology with other members of the G protein-coupled receptor superfamily. Here we report the identification and cloning of a cDNA encoding an RNA splice variant of the rat D2 receptor cDNA. This cDNA codes for a receptor isoform which is predominantly expressed in the brain and contains an additional 29 amino acids in the third cytoplasmic loop, a region believed to be involved in G protein coupling.

The expression of mRNAs coding for alpha 1, alpha 2, alpha 3, alpha 5, and alpha 6 subunits of the GABAA neurotransmitter receptor was followed during the development of the rat CNS by in situ hybridization histochemistry. Expression of these subunit mRNAs in tissue sections of embryonic day 15 and 17 (E15, E17) whole rat and in brain at ages greater than E17 to adult were varied, transient, and region specific. Subunit mRNAs first detected at E15 were those coding for the alpha 2 and alpha 3 subunits. At E17, alpha 2, alpha 3, and alpha 5 mRNAs were present in abundance in numerous areas in the CNS, with lower but significant amounts of alpha 6 being present in the cortical neuroepithelial layers. However, alpha 6 subunit mRNA expression in the cortex declined until little or no alpha 6 mRNA was detected at E19. alpha 1 subunit mRNA first appeared at E19 in the cortex, followed by expression in the hippocampus by postnatal 5 (PN5). Particularly high expression of alpha 2 and alpha 5 subunit mRNAs was detected throughout the developing CNS, but they were most abundant in the olfactory bulb neurons. The high levels of alpha 2 and alpha 5 subunit mRNAs began to decline around PN5 to the amounts observed in adult. These results demonstrate that numerous GABAA receptor alpha-subunits are expressed before birth in a region- and age-specific manner. This complex and varied expression supports the hypothesis that GABA may play a role in cellular and synaptic differentiation.

In order to clone the D1 dopamine receptor linked to adenylyl cyclase activation, the polymerase chain reaction was used with highly degenerate primers to selectively amplify a cDNA sequence from NS20Y neuroblastoma cell mRNA. This amplification produced a cDNA fragment exhibiting considerable sequence homology to guanine nucleotidebinding (G)-protein-coupled receptors that have been cloned previously. To characterize this cDNA further, a full-length clone was isolated from a rat striatal library by using the cDNA fragment as a probe. Sequence analysis of this cDNA clone indicated that it is indeed a member of the G-protein-coupled receptor family and exhibits greatest homology with the

No abstract

We have investigated the in vivo efficacy of a systemic gene transfer method, which combines a liposomal delivery system (DLS liposomes) Systemic gene therapy by direct delivery of plasmid DNA coupled to synthetic carriers is appealing because of its simplicity, low toxicity, and potential for multiorgan targeting. Although the efficiency of in vitro transfection of DNA plasmids is limited when compared to delivery by viral vectors, recent advances, especially in liposomal delivery, have demonstrated that nonviral gene transfer offers exciting potential (1, 2). Recent attempts to deliver genetic material have provided an impetus to liposome technology (3-6), in particular formation of complexes of DNA with cationic liposomes (7-11). A few therapeutic clinical trial protocols using local administration of these complexes are ongoing but data on systemic administration are still poorly documented (12,13). In this study, we describe a liposomal formulation for gene delivery called DLS, which could be suitable for systemic administration. To obtain long-term expression from a nonintegrated transgene, episomal self-replicating plasmid vectors were constructed and tested. Using the luciferase gene (luc) as a reporter gene (14), we detected transgene expression in various mouse tissues using measurements of luciferase activity, PCR analysis, and immunohistochemistry. Our results demonstrated that a single injection of an episomal DNA vector using the DLS liposome delivery system yielded widespread and long-lasting transgene expression.MATERIALS AND METHODS Materials. Dioctadecylamidoglycylspermidine (7) (15) were deleted to remove expression of these potentially immunogenic proteins. The resultant episomal/reporter expression vector was termed pBKd2RSV-luc. In addition, a second series of pBKdl and pBKd2 vectors were made in which luc expression was placed under the control of the enhancer/promoter sequences from the immediate-early gene of the human cytomegalovirus (CMV) and the polyadenylylation signal and transcriptional termination sequences from the bovine growth hormone gene. The resultant episomal/reporter expression vectors were termed pBKdlCMV-Iuc and pBKd2CMV-Iuc, respectively. The details of these constructs will be reported elsewhere (L.C.M., unpublished data). The nonepisomal pRSV-luc containing luc under control of the RSV long terminal repeat was constructed as reported (14). pCMVintlux plasmid encodes luc under the control of human CMV immediate-early promoter with intron A (16) (generously provided by M. Mansthorpe, Vical, CA). Preparation of the DLS Liposomes. Liposomes were formed by mixing 1 mg of dioctadecylamidoglycylspermidine and 1 mg of dioleoyl phosphatidylethanolamine. After thorough stirring, the mixture was evaporated to dryness in a round-bottomed borosilicate tube using a rotary Vortex evaporator under vacuum. Then the dry lipid film was hydrated with a maximum volume (60 ,ul per mg of lipid) of a solution containing 160 ,ug of plasmid DNA and was slightly Vortex mixed. After in...

We have characterized a new synthetic gene delivery sysplasmid DNA could be detected in blood cells up to 1 h tem, termed DLS, which may be suitable for systemic gene after injection. Systemic administration of DLS-DNA therapy. DLS constitutes a lipopolyamine and a neutral yielded transgene expression in mouse tissues, such as in lipid and associated plasmid DNA in the formation of lamellung or liver. The ratio of DLS:DNA and the procedure used lar vesicles (DLS-DNA). The ratio of lipids and lipid to DNA to form DLS-DNA affected both the level and cellular as well as the method of preparation were optimized to specificity of expression of a luciferase reporter gene yield a high in vitro transfection efficiency compared with showing that in vitro transfection efficiency of DLS-DNA that previously reported for cationic lipid systems. DLSformulations cannot be easily extrapolated to an in vivo set-DNA showed a rapid cellular uptake and distribution in the ting. Optimization of the formulation of a DNA delivery syscytoplasmic and nuclear (especially in the nucleoli) comtem was critical to obtain a defined structure resulting in a partments as determined by laser-assisted confocal preparation with high reproducibility and stability, greater microscopy. There was little or no plasmid DNA degrahomogeneity of particle size and high efficacy following dation over a period of 20 min, relatively slow plasma clearsystemic gene transfer. In addition, the DLS system may ance, and effective and rapid cellular uptake of DLS-DNA be formulated for specific target tissues and may have a following intravenous administration in mice. Supercoiled wide range of applications for gene therapy.Keywords: gene transfer; liposomes; plasmid DNA; biodistribution; pharmacokinetics Synthetic gene transfer vectors are receiving increasingIntroduction study as an alternative to viral vectors since this strategy One of the main impediments to successful gene therapy appears to be safe. Potential methods of gene delivery is the generally poor efficiency of DNA delivery. Most that could be employed include the pneumatic DNA gene therapy efforts involve the use of retroviral vectors gun, 4 DNA-protein complexes 5 or lipidic particles. 6 The because of the usual efficient cell entry and stable intetypical genetic material delivered to target cells by these gration of the gene. Clinical use of retroviral vectors, methods are plasmids, although antisense oligonucleohowever, is hampered by safety issues.1-3 A first concern tides are currently being tested as well. 8 Plasmid prepis the possibility of generating an infectious wild-type arations are simple, quick, safe, inexpensive and may be virus following a recombination event. A second concern applied in combination with a synthetic carrier. Thereis the consequence of random integration of the viral fore, gene therapy by this means may be safe, durable sequence into the genome of the target cell which may and used as a drug-like therapy. The successful use of lead to a tumorigenic or a cytotoxic event. ...

Expression of central nervous system receptors for dopamine was examined by i jection of poly(A)+ RNA (mRNA) from rat striatum into oocytes from Xenopus laevis. Electrophysiological measurements in mRNA-injected oocytes indicated that addition of 100 ,IM dopamine induced an inward current (40-100 nA) that was consistent with the activation of endogenous Ca2+-dependent Cl-channels. This current was also elicited by addition of the selective DI agonist SKF 38393 but not by the selective D2 agonist quinpirole. Prior addition of the dopaminergic antagonist cis-piflutixol completely abolished dopamine-induced currents but had no effect on currents produced by serotonin. Using 4Ca2' efflux assays, addition of 100 FLM dopamine to injected oocytes stimulated efflux 2-to tTo whom reprint requests should be addressed. 2196The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" ein accordance with 18 U.S.C. §1734 solely to indicate this fact.