Mesostriatal projections, which arise from dopaminergic and non- dopaminergic neurons in the ventral tegmental area, substantia nigra, and retrorubral area, are compartmentally organized in the striatum. Anterograde axonal tract tracing with Phaseolus vulgaris- leucoagglutinin (PHA-L), combined with immunohistochemical localization of tyrosine hydroxylase (TH) and autoradiographic localization of mu- opiate receptor binding sites, shows that midbrain projections to the striatum are distributed to either the mu-opiate receptor-rich “patch” or the receptor-poor “matrix” striatal compartments. Three morphologically distinct mesostriatal afferent fiber types are labeled. The first type, type A, forms a plexus of relatively thin (0.1–0.4 micron), smooth fibers with small varicosities (0.3–0.6 micron). A second type, type B, is similar to the first in forming a plexus of fibers, but is slightly thicker (0.2–0.6 micron), with more frequent varicosities (0.4–1.0 micron) that give this fiber type a crinkled appearance. The third type, type C, constitutes a minority of striatal afferents and is characterized by its large caliber (0.4–0.7 micron) with large bulbous varicosities (1.2–2.0 micron). Projections of the ventral tegmental area (A10 cell group) are primarily dopaminergic type A fibers directed to the matrix of the ventromedial striatum, including the nucleus accumbens. The retrorubral area (A8 cell group) also provides predominantly dopaminergic type A fibers to the striatal matrix, but these are distributed dorsally. The substantia nigra contains a mixed population of neurons that project to the striatum. Some, located in the dorsal tier of the pars compacta (dorsal A9 cell group), provide dopaminergic type A fibers to the striatal matrix. Others, in the ventral tier of the pars compacta (ventral A9 cell group) and in the ventral tier of the pars reticulata (displaced A9 cells), provide dopaminergic type B fibers to the striatal patches. An additional set of substantia nigra neurons that are non-dopaminergic is the source of type C fibers to the striatal matrix. Thus, distinct dorsal and ventral sets of midbrain dopaminergic neurons project, respectively, to striatal matrix and patches, and there is a non- dopaminergic mesostriatal projection to the matrix.

Of the five known dopamine receptors, DlA and D2 represent the major subtypes expressed in the striatum of the adult brain. Within the striatum, these two subtypes are differentially distributed in the two main neuronal populations that provide direct and indirect pathways between the striatum and the output nuclei ofthe basal ganglia. Movement The pivotal role played by dopamine receptors in the pathophysiology and treatment ofParkinson disease (1) and schizophrenia (2) and in the mode of action of addictive drugs such as amphetamine and cocaine (3, 4) is well established. Of the five known dopamine receptor subtypes (5), the DlA and D2 receptors account for the vast majority of dopamine receptors (6) expressed in the striatum. The DlA (also known as D, in the primate system) and D2 receptor subtypes are expressed mainly by spiny projection neurons, which account for 90-95% of the striatal neuron population (7). These striatal neurons may be subdivided into two major types on the basis of their axonal projections. One type provides a direct projection to the output nuclei of the basal ganglia: the substantia nigra and entopeduncular nucleus (the internal segment of the globus pallidus in primates). The other type provides projections to the globus pallidus (the external segment of the primate globus pallidus). As this latter type is connected indirectly to the output nuclei of the basal ganglia through connections with the subthalamic nucleus, the two output pathways are referred to as the direct and indirect output systems. Striatal neurons giving rise to the direct pathway express high levels of the DlA dopamine receptor subtype and the neuropeptides substance P and dynorphin, whereas neurons giving rise to the indirect pathway express high levels of the D2 dopamine receptor and the peptide enkephalin (7). The levels of peptide expression in these neurons provide an assay oftheir activity (7), as neuropeptide levels correlated with fuing rates in target neurons (1).Current models suggest that imbalanced activity in the direct and indirect pathways is responsible for clinical movement disorders (8). A number of studies have demonstrated that dopamine oppositely effects these two output pathways through their differential expression ofthe DlA and D2 receptors (7). For example, depletion of striatal dopamine with lesions of the nigrostriatal dopamine pathway in animal models of Parkinson disease results in reduced expression of substance P in direct output neurons and increased enkephalin expression in indirect striatal output neurons. Moreover, these changes may be selectively reversed with selective dopamine receptor agonist treatments, so that D1 agonist treatment normalizes substance P levels whereas D2 agonist treatment normalizes enkephalin levels (9). While these studies have demonstrated the differential role of DlA and D2 receptors in striatal function, important questions concerning the interaction between these neuronal pathways remain. To provide an experimental animal model to address the...

ABSTRACTderived from the 129/sv strain (Clontech) was screened using a mouse D3 cDNA probe (17). A positive clone encompassing exon 2 of the murine D3 gene was isolated and further characterized. A 7-kb Xho I-Asp718 fragment was engineered for targeted mutagenesis by introducing the GKNeo cassette (16) in antisense orientation at the Sal I site in exon 2 (17). Integration of sequences derived from the pGKNeo cassette generates a novel open reading frame, resulting in the following peptide sequence appended after Arg-148: PASDGIRT-WQNNTENEVYVEQRLLISFFRL Opal (Stop). The sequence of the mutant allele was confirmed by direct sequencing of reverse transcription-PCR (rPCR) products derived from brain mRNAs of -/-and +/-mice (data not shown).Transfection ofES Cells and Embryo Manipulations. J-1 ES cells (a kind gift of R. Jaenisch, Massachussetts Institute of Technology) at passage 13 were grown on mitomycin C-treated embryonic fibroblasts derived from a homozygous neomycin (Neo)-resistant transgenic mouse (16). Cells (2 x 107) were electroporated in a 1-ml cuvette (path length-0.2 cm) at 0.4 kV and 25 ,uF. Cells were plated onto 40 gelatin-coated Petri dishes (6 cm) on embryonic feeder cells. Selection with G418 (0.3 mg/ml; active concentration of 0.66 ,vg/mg of dry powder; GIBCO) was applied 24 hr after plating and was continued for 7-9 days. Individual Neo-resistant colonies were picked using a dissection microscope and expanded as described (16). Genomic DNA was prepared from an aliquot of cells for each clone using previously described techniques and analyzed by Southern blotting (18). Recovery, microinjection, and transfer of 3.5 day postcoitus embryos was performed as described (16).

The effects of the indirect dopamine receptor agonist cocaine in the striatum on levels of mRNAs of the immediate-early gene c-fos and the neuropeptides dynorphin, substance P, and enkephalin were analyzed with quantitative in situ hybridization histochemistry. Both single (acute) and repeated (twice a day for 4 d) systemic injections of cocaine (3.75-30 mg/kg) to rats resulted in dose-dependent, regionally specific elevations of mRNA expression in striatal neurons. A single drug treatment elevated c-fos mRNA expression, whereas repeated treatments resulted in little c-fos expression but elevated dynorphin mRNA levels. Both the regional and temporal patterns of gene expression revealed an inverse relationship between dynorphin and c-fos expression. This relationship was examined in a time course experiment in which cocaine (30 mg/kg) was administered for 1, 2, 3 or 4 d. Basal levels of dynorphin expression were relatively high in the ventral striatum, including the nucleus accumbens, a ventrolateral region, and an area along the medial bank of the striatum. A single injection of cocaine induced c-fos mRNA in striatal areas with low basal expression of dynorphin. Thus, c-fos mRNA induction was highest in the dorsal central striatum, where basal dynorphin mRNA levels were lowest. In this region, dynorphin mRNA expression increased on subsequent treatment days parallel to diminished c-fos mRNA induction. Changes in substance P. mRNA levels appeared to match directly both the temporal and regional patterns of c-fos induction. Enkephalin mRNA expression was altered, but only slightly, by these cocaine treatments. Statistical analysis of the regional patterns of basal and altered mRNA levels shows a unique inverse relationship between basal dynorphin expression and c-fos induction by cocaine. Further evidence of this relationship is provided by the dose-dependent blockade of cocaine-induced c-fos expression by spiradoline, a dynorphin agonist. Together, these results suggest that the restricted regional pattern of cocaine-induced c-fos expression is related, in part, to the basal level of dynorphin expression, and that cocaine treatment elevates dynorphin expression in striatal regions with a strong c-fos response, thereby limiting subsequent c-fos induction by cocaine. These findings lead to the hypothesis that dynorphin acts to regulate the responsiveness of striatal neurons to dopamine stimulation.

Dopamine regulation of the levels of dynorphin, enkephalin, and substance P messenger RNAs in rat striatal neurons was analyzed with in situ hybridization histochemistry (ISHH). Relative levels of peptide mRNA expression in the patch and matrix compartments of the dorsolateral striatum were compared among control rats, rats treated for 10 d with apomorphine, rats with unilateral 6-hydroxydopamine (6-OHDA) lesions of the nigrostriatal dopaminergic system, and rats with nigrostriatal dopaminergic lesions followed 2 weeks later by 10 d of apomorphine treatment. Image analysis of ISHH labeling demonstrated that the number of neurons expressing each peptide mRNA remained constant, whereas the relative level of peptide mRNA per neuron changed significantly, depending on the experimental treatment. Dynorphin mRNA expression increased following chronic apomorphine treatment: striatal patch neurons increased to an average of 100% above control values, whereas striatal matrix neurons showed only a 25% increase. Dynorphin mRNA expression decreased following 6-OHDA lesions: patch neurons showed an average 75% reduction in expression, whereas matrix neurons showed no significant change. In animals with 6-OHDA lesions followed by apomorphine treatment, both patch and matrix neurons showed an average increase in dynorphin expression of 300% above control levels. Changes in dynorphin mRNA levels with these treatments were matched by qualitative changes in dynorphin immunoreactivity both in the striatum and in striatonigral terminals in the substantia nigra. Neither substance P nor enkephalin mRNA levels showed a significant difference between the striatal patch and matrix compartments in any experimental condition (in the dorsolateral striatum). Substance P mRNA expression was increased an average of 50% after 10 d of apomorphine treatment and showed an average decrease of 75% following 6-OHDA lesions of the mesostriatal system. There was no significant change in the expression of substance P mRNA in striatal neurons compared to control values in rats with combined 6-OHDA lesion and apomorphine treatment. Enkephalin mRNA expression was not significantly altered by chronic apomorphine treatment but showed an average increase per cell of some 130% above control levels following 6-OHDA-induced lesions of the mesostriatal system. In animals with a 6-OHDA lesion and apomorphine treatment, enkephalin mRNA was also elevated but not significantly above the levels produced by the lesions alone. These data show that the expression of dynorphin, enkephalin, and substance P is differentially regulated by the mesostriatal dopaminergic system and, further, suggests that the mechanisms by which this regulation occurs may be different for the 3 peptide families.