Molecular cloning and expression of the gene for a human D1 dopamine receptor.

The diverse physiological actions of dopamine are mediated by its interaction with two basic types of G protein-coupled receptor, D1 and D2, which stimulate and inhibit, respectively, the enzyme adenylyl cyclase. Alterations in the number or activity of these receptors may be a contributory factor in diseases such as Parkinson's disease and schizophrenia. Here we describe the isolation and characterization of the gene encoding a human D1 dopamine receptor. The coding region of this gene is intronless, unlike the gene encoding the D2 dopamine receptor. The D1 receptor gene encodes a protein of 446 amino acids having a predicted relative molecular mass of 49,300 and a transmembrane topology similar to that of other G protein-coupled receptors. Transient or stable expression of the cloned gene in host cells established specific ligand binding and functional activity characteristic of a D1 dopamine receptor coupled to stimulation of adenylyl cyclase. Northern blot analysis and in situ hybridization revealed that the messenger RNA for this receptor is most abundant in caudate, nucleus accumbens and olfactory tubercle, with little or no mRNA detectable in substantia nigra, liver, kidney, or heart. Several observations from this work in conjunction with results from other studies are consistent with the idea that other D1 dopamine receptor subtypes may exist.

Sigma receptor "antagonist" BMY 14802 increases neurotensin concentrations in the rat nucleus accumbens and caudate.

Acute and chronic treatment with antipsychotic drugs, such as haloperidol, selectively increases the concentrations of neurotensin (NT) in the nucleus accumbens and caudate of the rat. These increases in NT concentration in the nucleus accumbens and caudate have been hypothesized to underlie the therapeutic and extrapyramidal effects of antipsychotic drugs, respectively. The present study evaluates the effects of the putative antipsychotic and selective sigma receptor "antagonist" BMY 14802 on regional brain NT concentrations. NT concentrations in discrete brain regions of adult, male, Sprague-Dawley rats were measured by a sensitive and specific radioimmunoassay. Like haloperidol (1 mg/kg i.p.), acute and chronic treatment with BMY 14802 (35 mg/kg/day i.p.) produced significant increases in the concentrations of NT in the nucleus accumbens and anterior and posterior caudate. This effect was dose-dependent. Maximal increases in NT concentration were observed 18 hr after a single dose of BMY 14802. Neither acute nor chronic treatment with the sigma "agonist" (+)-SKF 10,047 (20 mg/kg i.p.), the N-methyl-D-aspartate-phencyclidine binding site antagonist MK-801 (0.25 mg/kg i.p.) or the selective D2 antagonist sulpiride (100 mg/kg i.p.), produced the pattern of NT alterations observed after the administration of BMY 14802. These findings suggest that the blockade of sigma receptors modulates NT concentrations in these brain regions.

The effect of repeated administration of antidepressant drugs on the thyrotropin-releasing hormone (TRH) content of rat brain structures.

The present study investigated the effect of repeated treatment with the antidepressant drugs imipramine, amitryptyline, citalopram and mianserin (10 mg/kg PO, twice daily for 14 days) on levels of thyrotropin-releasing hormone (TRH) in several brain structures (cerebral cortex, amygdala + pyriform cortex, hippocampus, nucleus accumbens, striatum and hypothalamus) of the rat. Amitriptyline caused a marked increase in the TRH content in the striatum and nucleus accumbens. Citalopram and mianserin produced a smaller but significant increase in the TRH content in the striatum only, while imipramine did not significantly affect the TRH concentrations in any of the brain structures. None of the antidepressant drugs administered acutely significantly affected the TRH concentrations in the nucleus accumbens or the striatum. These results indicate that changes in brain TRH induced by antidepressant drugs are not related to their therapeutic activity.

Norepinephrine stimulates behavioral activation in rats following depletion of nucleus accumbens dopamine.

Intraventricular (ICV) infusion of norepinephrine (NE) produces locomotor activation in rats that is greatly potentiated by prior depletion of whole brain catecholamines by ICV injection of 6-hydroxydopamine (6OHDA). In a series of experiments, the neural substrates of this potentiated locomotor response were examined. One group of animals received ICV infusion of 6OHDA to deplete whole brain catecholamines. Other rats were pretreated with desmethylimipramine (DMI) and then received 6OHDA infusions into the nucleus accumbens (NAC) to selectively deplete dopamine (DA) from this region. One week later, all animals were tested for their locomotor response to ICV infusion of NE. Both groups of rats exhibited a greatly potentiated locomotor response to ICV NE compared to corresponding sham-lesioned animals. Both ICV and NAC 6OHDA-injected animals also exhibited a supersensitive locomotor response to the DA receptor agonist apomorphine. These results suggest that NE-induced locomotor activation in ICV 6OHDA-treated rats results from the actions of NE on supersensitive NAC DA receptors.

Serotoninergic projections from the midbrain periaqueductal gray to the nucleus accumbens in the rat.

A combined method of retrograde tracing of horseradish peroxidase (HRP) and serotonin (5-HT) immunocytochemistry indicated that some 5-HT-like immunoreactive neurons in the midbrain periaqueductal gray (PAG) of the rat send their axons to the nucleus accumbens. These PAG neurons are mainly located in the ventromedial and ventrolateral subdivisions at the middle and caudal levels of PAG.

Apomorphine affects cholecystokinin content via preferentially D1 or D2 dopamine receptor according to the regions of the rat brain.

Cholecystokinin octapeptide-like immunoreactivity (CCK-8IR) was measured in several regions of the rat brain after the intraperitoneal administration of apomorphine, SKF-38393 (D1 agonist), LY-171555 (D2 agonist). In the medial prefrontal cortex and striatum, apomorphine and SKF 3839 decreased CCK-8IR. In the anterior and posterior nucleus accumbens, on the other hand, the inhibitory effect of apomorphine was mimicked by LY-171555. These results suggest that apomorphine affects CCK-8IR via either the D1 dopamine (DA)-receptor or D2 DA-receptor according to the brain region.

[Prolactin-secreting neurons in the dorsolateral hypothalamus in Sprague-Dawley rats]. / Neurones à prolactine dans l'hypothalamus dorso-latéral du rat Sprague-Dawley.

By means of immunocytochemical techniques ovine prolactin like immunoreactivity (oPRL-LIR) has been demonstrated in the perikarya located around fornix in the dorso-lateral part of the rat hypothalamus. No PRL-LIR was observed in the arcuate n. perikarya. Immunoreactive fibers were present in the hypothalamus, medial thalamus, accumbens and amygdaloid nuclei.

Aspartame and the rat brain monoaminergic system.

A high dose of aspartame (APM) was administered to rats to study possible effects on brain monoaminergic systems. APM and its metabolite phenylalanine (Phe) were given orally at doses of 1000 and 500 mg/kg, respectively. Significant increases were seen in brain Phe and tyrosine (Tyr) levels. Two different approaches were used to study monoaminergic systems: whole tissue measurements by HPLC-ED and in vivo voltammetry in freely moving rats. Dopamine, serotonin and their metabolites were taken as indexes of neuronal activity. In spite of the high dose used, no modification was found in monoamines or their metabolites in striatum, hippocampus and nucleus accumbens.

In vivo dopaminergic activity from nucleus accumbens, substantia nigra and ventral tegmental area in the freely moving rat: basal neurochemical output and prolactin effect.

In this paper, by means of a push-pull perfusion technique, we have examined the local effect of ovine prolactin (oPRL) on the spontaneous in vivo dopaminergic (DAergic) activity of the nucleus accumbens (NAcc), substantia nigra (SN) and ventral tegmental area (VTA) of living male rats. First, we found a 2-fold spontaneous rise of 3,4-dihydroxyphenylacetic acid (DOPAC) output during the afternoon hours of the photoperiod only in the NAcc. In addition, native oPRL (10 ng/microliters for 20 min), but not boiled oPRL, perfused into the NAcc caused a significant elevation in DOPAC output without changes in homovanillic acid or 5-hydroxyindoleacetic acid. In a similar preparation, male rats bearing push-pull cannulae in the SN or VTA were infused locally with oPRL (10 or 50 ng/microliters) for 20 min. The hormone did not alter the efflux of DOPAC from these DA cell body/dendrite areas. These results indicate that oPRL can activate the mesolimbic DA system through local actions on DA presynaptic terminals of the NAcc without affecting the SN or VTA.

A rapid and simple HPLC microassay for biogenic amines in discrete brain regions.

A rapid microassay is described for the measurement of biogenic amines using an isocratic HPLC system with electrochemical detection. Catecholamines, indoleamines and their major metabolites were extracted with 150 microliters of perchloric acid from brain tissue punches (less than 250 micrograms) using a simple one-step sample preparation method. These compounds were separated on a short (80 mm) column with 3 microns particle size packing, and electrochemically detected within a total run time of less than 6 minutes. Detection limit sensitivity was approximately 2-5 pg. This method, detailed in an easy-to-follow description, reduces assay time, minimizes the possibility for errors, maximizes efficiency, and requires only standard HPLC equipment and supplies.

Reduced cAMP-dependent phosphorylation in striatum and nucleus accumbens of aged rats: evidence of an altered functioning of D1 dopaminoceptive neurons.

Cyclic AMP-dependent phosphorylation was measured in particulate and in cytosolic fractions of two brain dopaminergic areas, i.e., striatum and nucleus accumbens, of young (3 months) and old (24 months) rats. A reduced cAMP-stimulated 32P incorporation was observed in several protein bands in both brain areas in the aged group. In the soluble fraction the reduced phosphorylation of DARPP-32, which is specific for dopaminoceptive neurons bearing D-1 receptors, may be of particular relevance in the mechanisms of age-related changes in dopaminergic transmission.

Gamma-aminobutyric acid in the medial rat nucleus accumbens: ultrastructural localization in neurons receiving monosynaptic input from catecholaminergic afferents.

Neurons containing gamma-aminobutyric acid (GABA) in the medial portion of the adult rat nucleus accumbens were characterized with respect to their ultrastructure, sites of termination, and catecholaminergic input. Antisera against GABA-conjugates and the catecholamine-synthesizing enzyme, tyrosine hydroxylase (TH), were localized within single sections by means of peroxidase-antiperoxidase (PAP) and immunoautoradiographic labeling methods. Peroxidase reaction product indicating GABA-like immunoreactivity (GABA-LI) was seen in medium-size (15-20 microns) perikarya containing either round and unindented or invaginated nuclear membranes. The cells with invaginated nuclei were few in number and usually exhibited more intense peroxidase reaction product in sections collected at the same distance from the surface of the tissue. Reaction product for GABA was also detected in proximal (1.5-3.0 microns) dendrites, axons, and terminals. Terminals with GABA-LI formed symmetric junctions on perikarya, proximal dendrites, and dendritic spines of neurons that usually lacked detectable immunoreactivity. Many of the GABAergic terminals also were apposed directly to other unlabeled terminals and to terminals exhibiting either peroxidase labeling for GABA or immunoautoradiographic labeling for TH. Many of the unlabeled terminals associated with the GABAergic axons formed asymmetric junctions on dendritic spines. From 138 TH-labeled, principally dopaminergic terminals that were examined in the medial nucleus accumbens, 4% were associated with the somata of GABAergic neurons and another 14% formed symmetric junctions with proximal dendrite showing GABA-LI. The remaining TH-immuno-reactive terminals either lacked recognizable densities or formed symmetric synapses on unlabeled dendrites and spines. A few of the unlabeled dendrites, as well as those containing GABA-LI, received symmetric synapses from both catecholaminergic and GABAergic terminals. We conclude that in the medial portion of the rat nucleus accumbens, GABA is localized to two morphologically distinct types of neurons, one or both of which receive monosynaptic input from catecholaminergic afferents, and that GABAergic terminals form symmetric synapses on other principally non-GABAergic neurons. The results also support earlier physiological evidence showing that GABA may modulate the output of other GABAergic and non-GABAergic neurons through presynaptic associations.

Neuropeptide Y in the rat nucleus accumbens: ultrastructural localization in aspiny neurons receiving synaptic input from GABAergic terminals.

The ultrastructure, afferent input, and sites of termination of neurons containing neuropeptide Y-like immunoreactivity (NPY-LI) were examined in the adult rat nucleus accumbens by using the peroxidase-antiperoxidase (PAP) method. The NPY-LI was seen in sparsely distributed, spindle-shaped perikarya having cross-sectional diameters of 15-20 microns. These perikarya exhibited highly invaginated nuclear membranes and thin rims of cytoplasm containing Golgi lamellae, dense-core vesicles, and other organelles. A few large, principally aspiny, dendrites also showed NPY-LI. The dendrites received synaptic input from unlabeled terminals forming both symmetric and asymmetric junctions. Immunolabeling for NPY was evident in other processes that were not clearly differentiated as dendrites or axons. These were seen primarily near glial processes and the basal laminae of blood vessels. A few myelinated and many unmyelinated axons and axon terminals also were labeled for NPY. These terminals contained numerous, small (40-60 nm), clear and one or more large (80-100 nm) dense core vesicles. Forty-seven percent (27 out of 57) of the terminals containing NPY-LI formed symmetric junctions with unlabeled dendrites or dendritic spines. The remainder lacked recognizable densities within single planes of section. The neurons exhibiting NPY-LI in the nucleus accumbens were characterized further with respect to their afferent input from terminals labeled for the GABA-synthesizing enzyme, glutamic acid decarboxylase (GAD). Immunogold labeling of a rabbit antiserum against NPY and PAP labeling for a sheep antiserum to GAD were sequentially applied to the same sections. The GAD-labeled terminals formed symmetric junctions primarily with the more numerous unlabeled dendrites. However, a few synaptic junctions also were detected between the GAD-labeled terminals and dendrites showing immunogold labeling for NPY. We conclude (1) that in the rat nucleus accumbens, NPY-LI is found principally in neurons of the aspiny type and (2) that the output from these presumably intrinsic neurons to other neighboring neurons or blood vessels is at least partially modulated by GABA.

Dopamine-GABA interactions in the nucleus accumbens and lateral septum of the rat.

The relationships between dopaminergic afferents and GABAergic neurones were studied at the electron microscopic level in the rat lateral septum and nucleus accumbens by coupling 6-hydroxydopamine degeneration and gamma-aminobutyric acid (GABA) immunocytochemistry. Degenerating fibres were observed in the two regions making synaptic contact with GABA-immunoreactive and non-labelled cell bodies and dendrites. It is concluded that dopaminergic afferents to the septum and the nucleus accumbens contact, among others, a population of GABAergic cells. A similar route of regulation of the basalo-cortical and septo-hippocampal cholinergic pathways by dopaminergic afferents is proposed.

The regional distribution of somatostatin and neuropeptide Y in control and Alzheimer's disease striatum.

Somatostatin-like immunoreactivity (SLI) and neuropeptide Y-like immunoreactivity (NPYLI) were measured in subdissections of both normal and Alzheimer's disease (AD) striatum at 5 coronal levels. Concentrations of both neuropeptides were relatively homogeneously distributed in the coronal and anterior-posterior planes except for a trend towards increased concentrations in the tail of the caudate and the posterior putamen. The nucleus accumbens showed 2-3-fold higher concentrations of both SLI and NPYLI than the rest of the striatum. There were no significant differences between control and AD brains. The high concentrations of SLI and NPYLI in the nucleus accumbens suggest that this region may receive somatostatin-neuropeptide Y afferents and that somatostatin and neuropeptide Y may play a role in the modulation of motor activity.