mRNAs encoding five genetically distinct muscarinic ACh receptors are present in the CNS. Because of their pharmacological similarities, it has not been possible to detect the individual encoded proteins; thus, their physiological functions are not well defined. To characterize the family of proteins, a panel of subtype-selective antibodies was generated against recombinant muscarinic receptor proteins and shown to bind specifically to each of the cloned receptors. Using immunoprecipitation, three receptor proteins (m1, m2, and m4) accounted for the vast majority of the total solubilized muscarinic binding sites in rat brain. These receptor subtypes had marked differences in regional and cellular localization as shown by immunocytochemistry. The m1-protein was present in cortex and striatum and was localized to cell bodies and neurites, consistent with its role as a major postsynaptic muscarinic receptor. The m2-receptor protein was abundant in basal forebrain, scattered striatal neurons, mesopontine tegmentum, and cranial motor nuclei; this distribution is similar to that of cholinergic neurons and suggests that m2 is an autoreceptor. However, m2 was also present in noncholinergic cortical and subcortical structures, providing evidence that this subtype may presynaptically modulate release of other neurotransmitters and/or function postsynaptically. The m4-receptor was enriched in neostriatum, olfactory tubercle, and islands of Calleja, indicating an important role in extrapyramidal function. These results clarify the roles of these genetically defined receptor proteins in cholinergic transmission in brain.(ABSTRACT TRUNCATED AT 250 WORDS)

60th passage. The cultured organisms retained their characteristic helical morpholo-

Five or more dopine receptor genes are expressd in brain. However, the phacological similarities of the encoded DI-Ds receptors have hindered studies of the localizion and functions of the subtypes. To better understand the roles of the individual receptors, antibodies were raised apinst recombinant D1 and D2 proteins and were shown to bind to the receptor subtpes spefically in Western blot and immunoprecipitation studies. Each antibody reacted selectively with the r ive receptor protein expressed both in cells transfected with the cDNAs and in brain. By immunocytochemistry, D1 and D2 had similr reonal distibutions in rat, monkey, and human brain, with the most intense saning in sriatum, olfactory bulb, and substantia nigra. Within each region, however, the precise distributions ofeach subtype were dstinct and often complementary. Di and D2 were differentidy enriched in striatal patch and matrix compwrtments, in selective layers of the olfactory bulb, and in either substantia nigra pars compacta or reticulata. Electron microscopy demonstrated that D1 and D2 also had highly selective subcellular distributions. In the rat nestiatum, the majority of D1 and D2 immunoreactivity was localized in postsynaptic sites in subsets of spiny dendrites and spine heads in rat nosriatum. Presynaptic D1 and D2 receptors were also observed, indicating both ubtypes may regulate neurotransmitter rebase. DI was also present in axon terminals in the substantia nigra. These results provide a morphologkal substrate for understanding the preand postsynaptic functions of the genetically defined DI and D2 receptors in discrete neuronal circuits in mammalin brain.A family of at least five dopamine receptor genes, D1-D5, has been identified in both rodents and humans (1,2 1047-1339 (13) encoding the C-terminal 97 aa] and D2i3 [nt 661-1020 (14) encoding 120 aa of the i3 loop from the D2s splice variant] were subcloned into pGEX-2T, expressed as soluble fusion proteins in bacteria, and affinity-purified as described (12,15). The recombinant plasmids were confirmed by sequence analysis and encode a 27.5-kDa polypeptide fragment of glutathione S-transferase (GST) fused to the receptor polypeptide (Dic-GST and D%-GST). High yields (10-25 mg/ liter) ofpurified DIC-GST and D2v-GST fusion proteins were obtained.Antisera. Two female New Zealand White rabbits were immunized with each fusion protein. Animals received 100 jg of affinity-purified fusion protein in Freund's adjuvant followed by secondary injections at 3 weeks with the same dose, and then monthly booster injections with 25 ,ug of protein.Antibodies were affinity-purified on the respective purified fusion proteins conjugated to Affi-Gel (Bio-Rad) as described (12). Sera were preadsorbed with GST and bacterial lysates prior to affinity purification to remove antibodies reactive with the nondopamine receptor portion ofthe fusion proteins. The two antisera for each receptor yielded similar results.Immunoblot Analysis. SDS/PAGE was used to fractionate total protein from bacteria...

Within the basal ganglia, acetylcholine and dopamine play a central role in the extrapyramidal control of motor function. The physiologic effects of these neurotransmitters are mediated by a diversity of receptor subtypes, several of Which have now been cloned. Muscarinic acetylcholine receptors are encoded by five genes (ml-mS), and of the two known dopamine receptor subtypes (Dl and D2) the D2 receptor gene has been characterized. To gain insight into the physiological roles of each of these receptor subtypes, we prepared oligodeoxynucleotide probes to localize receptor subtype mRNAs within tlie rat striatum and substantia nigra by in situ hybridization histochemistry. Within the striatum, three muscarinic (ml, m2, m4) receptor mRNAs and the D2 receptor mRNA were detected.The ml mRNA was expressed in most neurons (>80%); the m2 mRNA, in neurons which were both very large and rare; and the m4 and D2 mRNAs, in 40-50% of the neurons, one-third of which express both mRNAs. Within the substantia nigra, pars compacta, only the m5 and D2 mRNAs were detected, and most neurons expressed both mRNAs. These data provide anatomical evidence for the identity of the receptor subtypes which mediate the diverse effects of muscarinic and dopaminergic drugs on basal ganglia function.The maintenance of a balance between cholinergic and dopaminergic tone within the basal ganglia has long been appreciated as being central to the clinical management of many extrapyramidal motor disorders (1-3). For example, muscarinic antagonists and dopamine agonists have both been used in the treatment of Parkinson disease (2, 3).Unfortunately, both types of drugs exert many untoward side effects (2), particularly in later phases of the disease. The recent discovery of a heterogeneity of muscarinic and dopaminergic receptor subtypes has led to the suggestion that these subtypes may mediate distinct aspects of cholinergic and dopaminergic function. On the basis of pharmacologic data, muscarinic receptors have been divided into three subtypes (Ml, M2, and M3) (4) and dopaminergic receptors into two (Dl and D2) (5, 6). Molecular cloning efforts have identified five genetically distinct muscarinic receptor subtypes (ml-m5) (7-10). Functional expression of these genes has indicated a correlation between the genetically and pharmacologically defined subtypes, where the Ml = ml, m4, and m5; the M2 = m2; and the M3 = m3 (11). A dopamine D2 receptor has also recently been cloned (12-15). Because the available pharmacologic tools do not discriminate among all the receptor subtypes, and due to the limited anatomic resolution that receptor autoradiographic procedures allow, we have prepared oligodeoxynucleotide probes to determine which cells within the basal ganglia express each receptor subtype mRNA. ¶ These data should provide a rational basis for the development of subtype-selective drugs for the management of movement disorders. MATERIALS AND METHODSOligodeoxynucleotide Probes. Three 48-base oligodeoxynucleotide probes for each of the five muscar...

We used in situ hybridization histochemistry with synthetic oligodeoxyribonucleotide probes to identify cells that synthesize mRNAs encoding tyrosine hydroxylase in the mesencephalon and substance P, enkephalin, and dynorphin in the rat forebrain. Dopaminergic cells in the mesencephalon project to the forebrain and influence neuropeptide levels. We examined the effect of unilateral 6-hydroxydopamine lesions (which eliminated tyrosine hydroxylase mRNA-containing cells in the mesencephalon) on substance P, enkephalin, and dynorphin mRNA levels. Substance P mRNA levels were depressed, whereas enkephalin mRNA levels were elevated in consecutive sections from striatal areas in all animals. The effects of the lesions on dynorphin mRNA levels were less robust, and considerable variation between animals was observed. Changes were evident in the levels of message in individual cells but not in the numbers of labeled cells. These effects were not uniform throughout the dopamine-innervated areas, suggesting degrees of control not apparent with RNA blot-hybridization or dotblot analyses.Dopamine is a principal transmitter of the mesostriatal and mesocortical (or mesolimbic) components of the mesencephalic pathways to the forebrain (1), which exert major influences on an animal's behavior. In rats, lesions of these pathways may lead to behavioral deficits, such as akinesia, adipsia, and aphagia, while stimulation leads to increased motor activity and stereotyped behaviors such as sniffing, licking, and gnawing (2, 3). Side effects of neuroleptics such as acute dystonia and tardive dyskinesia and neurological diseases such as Parkinson's disease, in which a deficiency in mesostriatal dopamine is accompanied by movement disorders such as bradykinesia and tremor, demonstrate the importance of dopamine in human motor control (4).Undoubtedly, dopamine, in part, exerts its influence and is influenced in turn by neuropeptide transmitter systems. Extensive evidence exists for complex topographical relationships between these systems in the basal ganglia (5-9). Broadly, the caudate-putamen (CP) system receives input from the substantia nigra (SN) and ventral tegmental area (VTA), while the nucleus accumbens (NA) and olfactory tubercle (OT) are primarily innervated by the VTA. However, there is some overlap in the innervations of these striatal areas by the SN and VTA. Also, the dopamine cell group A8 projects to the NA and ventral CP. These dopamine-innervated areas contain substance P (SP) (10), enkephalin (Enk) (ref. 11 and references within), and dynorphin (Dyn) (12, 13) cell bodies, which project in characteristic ways out of the striatum. In the rat, SP (14-16) and Dyn (17) cells project principally to the SN, especially the pars reticulata, whereas Enk cells project to the globus pallidus (18-21).Changes in striatal neuropeptide biosynthesis result from lesioning mesencephalic dopamine neurons with 6-hydroxydopamine or chronic administration of neuroleptics. SP and SP mRNA levels in the striatum are depressed by neurole...