The pathways governing signal transduction in the mesocortical and nigrostriatal dopamine systems of the brain are of central importance in a variety of drug actions and neurological diseases. We have analyzed the regional, cellular, and subcellular distribution of the closely related D1 and D5 subtypes of dopamine receptors in the cerebral cortex and selected subcortical structures of rhesus monkey using subtype specific antibodies. The distribution of D1 and D5 receptors was highly differentiated in subcortical structures. In the neostriatum, both D1 and to a lesser extent D5 antibodies labeled medium spiny neurons, while only D5 antibodies labeled the large aspiny neurons typical of cholinergic interneurons. In the caudate nucleus, D1 labeling was concentrated in the spines and shafts of projection neurons, whereas D5 antibodies predominantly labeled the shafts, and less commonly, the spines of these cells. The D1 receptor was abundantly expressed in the neuropil of the substantia nigra pars reticulata while the D5 antibodies labeled only a few scattered cell bodies in this structure. Conversely, D5 antibodies labeled cholinergic neurons in the basal forebrain more intensely than D1 antibodies. Within the cerebral cortex and hippocampus, D1 and D5 antibody labeling was prominent in pyramidal cells. Double-label experiments revealed that the two receptors were frequently coexpressed in neurons of both structures. Ultrastructurally, D1 receptors were especially prominent in dendritic spines whereas dendritic shafts were more prominently labeled by the D5 receptor. The anatomical segregation of the D1 and D5 receptors at the subcellular level in cerebral cortex and at the cellular level in subcortical areas suggest that these closely related receptors may be preferentially associated with different circuit elements and may play distinct regulatory roles in synaptic transmission.

Dopamine receptors are the principal targets of drugs used in the treatment of schizophrenia. Among the five mammalian dopamine-receptor subtypes, the D4 subtype is of particular interest because of its high affinity for the atypical neuroleptic clozapine. Interest in clozapine stems from its effectiveness in reducing positive and negative symptoms in acutely psychotic and treatment-resistant schizophrenic patients without eliciting extrapyramidal side effects. We have produced a subtype-specific antibody against the D4 receptor and localized it within specific cellular elements and synaptic circuits of the central nervous system. The D4-receptor antibody labelled GABAergic neurons in the cerebral cortex, hippocampus, thalamic reticular nucleus, globus pallidus and the substantia nigra (pars reticulata). Labelling was also observed in a subset of cortical pyramidal cells. Our findings suggest that clozapine's beneficial effects in schizophrenia may be achieved, in part, through D4-mediated GABA modulation, possibly implicating disinhibition of excitatory transmission in intrinsic cortical, thalamocortical and extrapyramidal pathways.

Muscarinic ml receptors traditionally are considered to be postsynaptic to cholinergic fibers, while m2 receptors are largely presynaptic receptors associated with axons. We have examined the distribution of these receptor proteins in the monkey cerebral cortex and obtained results that are at odds with this expectation. Using immunohistochemistry with specific antibodies to recombinant ml and m2 muscarinic receptor proteins, we have demonstrated that both ml and m2 receptors are prominently associated with noncholinergic asymmetric synapses as well as with the symmetric synapses that characterize the cholinergic pathways in the neocortex. At asymmetric synapses, both ml and m2 receptor immunoreactivity is observed postsynaptically within spines and dendrites; the m2 receptor is also found in presynaptic axon terminals which, in the visual cortex, resemble the parvicellular geniculocortical pathway. In addition, m2 labeling was also found in a subset of nonpyramidal neurons. These rmdings establish that the m2 receptor is located postsynapticafly as well as presynapticafly. The association of ml and m2 receptors with asymmetric synapses in central pathways, which use excitatory amino acids as neurotransmitters, provides a morphological basis for cholinergic modulation of excitatory neurotransmission.Muscarinic receptors in the central nervous system have been implicated in normal learning and memory (1-3), arousal (4-6), and motor and sensory modulation (4, 5, 7) as well as in conditions such as normal aging, Alzheimer and Parkinson diseases, schizophrenia, and depression (8)(9)(10)(11). Recently, the cloning of five different genes encoding structurally similar muscarinic receptor proteins (ml-m5) (12, 13) has led to production of subtype-specific antibodies against recombinant muscarinic receptor proteins (14, 15). These antibodies have enabled us to identify the neuronal elements containing particular receptors in the cerebral cortex at a level of resolution and specificity not possible in previous autoradiographic studies of these receptors (16, 17) or in previous immunohistochemical analyses using nonselective muscarinic antibodies (18). Our findings have revealed an unexpected synaptic association ofml and m2 receptors with fiber systems utilizing excitatory transmitters in the primate cerebral cortex. MATERIALS AND METHODSTissue and Fixation. The results of this study are based on light and electron microscopic analysis of frontal and occipital lobe tissue from three adult macaque monkeys (Macaca mulatta). The monkeys were anesthetized with an intraveThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. nous overdose of sodium pentobarbital (Nembutal) (100 mg/kg) and after initial saline perfusion, one monkey was perfused with 4% paraformaldehyde in 0.1 M phosphate buffer (PB; pH 7.4) and two monkeys were perfused with 4% paraformaldehyde/0.08...

As a result of alternative splicing, the D2 gene of the dopamine receptor family exists in two isoforms. The D2 long is characterized by the insertion of 29 amino acids in the third cytoplasmic loop, which is absent in the short isoform. We have produced subtype-specific antibodies against both the D2 short and D2 long isoforms and found a unique compartmentalization between these two isoforms in the primate brain. The D2 short predominates in the cell bodies and projection axons of the dopaminergic cell groups of the mesencephalon and hypothalamus, whereas the D2 long is more strongly expressed by neurons in the striatum and nucleus accumbens, structures targeted by dopaminergic fibers. These results show that the splice variants of the dopamine D2 receptor are differentially distributed and possess distinct functions. The strategic localization of the D2 short isoform in dopaminergic cell bodies and axons strongly suggests that this isoform is the likely dopamine autoreceptor, whereas the D2 long isoform is primarily a postsynaptic receptor.Among all the neurotransmitter receptors cloned to date, the D2 subtype of dopamine receptors has been the one most closely linked to the positive symptoms of schizophrenia (1, 2) and often implicated in extrapyramidal side effects (3, 4). Neuroanatomical (5, 6) and physiological (7,8) studies indicate that this receptor has both autoreceptor and postsynaptic functions. Further, recent evidence of loss of autoreceptor function in D2-deficient mice (8) but sparing of this function in D3 mutant mice (9) strongly suggests that the D2 subtype is the only autoreceptor within the dopamine system. However, the D2 receptor exists in two isoforms (10, 11), and it is not known if both isoforms contribute to autoreceptor function. With the use of recently developed subtype-specific antibodies to dopamine D2 short (D2S) and D2 long (D2L) isoforms, we have found prevalent labeling of the D2S isoform in the cell bodies and axons of brain stem dopamine neurons, suggesting that this isoform is the autoreceptor of the dopamine system. METHODSPreparation of Antibodies. The D2S peptide TPLKDAAR and D2L peptide SNGSFPVNRRRM corresponding to residues 238-245 and 259-270 (10, 11), respectively, were derived from the third cytoplasmic loop of the receptor. The D2S peptide was arranged by adopting four amino acids from each side of the insertion site to differentiate between D2S and D2L isoforms. A similar procedure has previously been used for the preparation of antibodies to ␥-aminobutyric acid (GABA) receptors (12). The peptides were coupled to keyhole limpet hemocyanin (KLH) protein. Peptide-KLH conjugate (100 g) emulsified in complete Freund's adjuvant was injected into rabbits for antibody development. Affinity purification of the antisera was as described elsewhere (12). In brief, peptide (5 mg) was coupled to 1 g of activated thiopropyl-Sepharose 6B (Pharmacia LKB). Phosphate-buffered saline (PBS) diluted antiserum (1:5) was circulated through the column. After washing with PBS, th...