RGS mRNA expression in rat striatum: modulation by dopamine receptors and effects of repeated amphetamine administration

Single injections of cocaine, amphetamine, or methamphetamine increased RGS2 mRNA levels in rat striatum by two- to fourfold. The D1 dopamine receptor-selective antagonist SCH-23390 had no effect by itself but strongly attenuated RGS2 mRNA induction by amphetamine. In contrast, the D2 receptor-selective antagonist raclopride induced RGS2 mRNA when administered alone and greatly enhanced stimulation by amphetamine. To examine the effects of repeated amphetamine on RGS2 expression, rats were treated with escalating doses of amphetamine (1.0-7.5 mg/kg) for 4 days, followed by 8 days of multiple daily injections (7.5 mg/kg/2 h x four injections). Twenty hours after the last injection the animals were challenged with amphetamine (7.5 mg/kg) or vehicle and killed 1 h later. In drug-naive animals, acute amphetamine induced the expression of RGS2, 3, and 5 and the immediate early genes c-fos and zif/268. RGS4 mRNA levels were not affected. Prior repeated treatment with amphetamine strongly suppressed induction of immediate early genes and RGS5 to a challenge dose of amphetamine. In sharp contrast, prior exposure to amphetamine did not reduce the induction of RGS2 and RGS3 mRNAs to a challenge dose of amphetamine, indicating that control of these genes is resistant to amphetamine-induced tolerance. These data establish a role for dopamine receptors in the regulation of RGS2 expression and suggest that RGS2 and 3 might mediate some aspects of amphetamine-induced tolerance.     

Modulation of dopamine D1 receptor binding by neurotensin in the rat striatum

The effect of neurotensin on binding characteristics of dopamine D1 receptors was examined in the rat striatal membranes through radioreceptor assay. Neurotensin or its analogs were added to incubation medium of[³H]SCH 23390 saturation or dopamine/[³H]SCH 23390 inhibition experimental systems. Neurotensin did not modulate D1 antagonist binding but converted a part of D1 agonist high affinity binding sites to a low affinity state. Neurotensin_8–13 had the same potency as neurotensin itself, whereas neurotensin_1–8 had only weak activity in modulating D1 agonist binding. GTP and neurotensin had the same effect on D1 agonist binding. However, when both neurotensin and GTP were added, the result was the same as with either alone.

These data suggest that neurotensin modulates the functional state of D1 receptors probably via a GTP binding protein in the rat striatum.     

Significance of glutamate and dopamine neurons in the ventral pallidum in the expression of behavioral sensitization to amphetamine

To explore the significance of ventral pallidum (VP) during the amphetamine sensitization, we first investigated if there are neurochemical alterations in the VP during amphetamine withdrawal period. Chronic amphetamine-treated (5 mg/kg x 14 days) rats displayed an apparent locomotion sensitization as compared with saline controls when challenged with 2 mg/kg amphetamine at withdrawal days 10-14. A microdialysis analysis revealed that output of the dopamine metabolites, 3,4-dihydroxyphenylacetic acid and homovanillic acid, in the VP of amphetamine-sensitized rats increased approximately two-fold as compared to controls at both pre- and post-amphetamine challenge period. On the other hand, the in vivo glutamate output in the VP increased upon amphetamine challenge in the behaviorally sensitized rats, but not in the controls. To evaluate if drug manipulation in the VP would affect the behavioral sensitization, we treated both groups of rats with NMDA receptor antagonist, MK-801 (5 microg/microl for 5 days; bilateral) in the VP during withdrawal days 6-10. Animals were challenged with 2 mg/kg amphetamine at withdrawal day 11. The behavioral profile exhibited that MK-801 pre-treatment significantly blocked the locomotion hyperactivity in amphetamine-sensitized rats. Taken together, the current results suggest that the excitatory amino acid in the VP plays a significant role during the expression of behavioral sensitization to amphetamine.     

High doses of amphetamine augment, rather than disrupt, exocytotic dopamine release in the dorsal and ventral striatum of the anesthetized rat

High doses of amphetamine (AMPH) are thought to disrupt normal patterns of action potential-dependent dopaminergic neurotransmission by depleting vesicular stores of dopamine (DA) and inducing robust non-exocytotic DA release or efflux via dopamine transporter (DAT) reversal. However, these cardinal AMPH actions have been difficult to establish definitively in vivo. Here, we use fast-scan cyclic voltammetry (FSCV) in the urethane-anesthetized rat to evaluate the effects of 10 and 20 mg/kg AMPH on vesicular DA release and DAT function in dorsal and ventral striata. An equivalent high dose of cocaine (40 mg/kg) was also examined for comparison to psychostimulants acting preferentially by DAT inhibition. Parameters describing exocytotic DA release and neuronal DA uptake were determined from dynamic DA signals evoked by mild electrical stimulation previously established to be reinforcing. High-sensitivity FSCV with nanomolar detection was used to monitor changes in the background voltammetric signal as an index of DA efflux. Both doses of AMPH and cocaine markedly elevated evoked DA levels over the entire 2-h time course in the dorsal and ventral striatum. These increases were mediated by augmented vesicular DA release and diminished DA uptake typically acting concurrently. AMPH, but not cocaine, induced a slow, DA-like rise in some baseline recordings. However, this effect was highly variable in amplitude and duration, modest, and generally not present at all. These data thus describe a mechanistically similar activation of action potential-dependent dopaminergic neurotransmission by AMPH and cocaine in vivo. Moreover, DA efflux appears to be a unique, but secondary, AMPH action.     

Retinal dopamine D1 and D2 receptors: Characterization by binding or pharmacological studies and physiological functions

1. In the retinal inner nuclear layer of the majority of species, a dopaminergic neuronal network has been visualized in either amacrine cells or the so-called interplexiform cells. 2. Binding studies of retinal dopamine receptors have revealed the existence of both D1- as well D2-subtypes. The D1-subtype was characterized by labeled SCH 23390 (Kd ranging from 0.175 to 1.6 nM and Bmax from 16 to 482 fmol/mg protein) and the D2-subtype by labelled spiroperidol (Kd ranging from 0.087 to 1.35 nM and Bmax from 12 to 1500 fmol/mg protein) and more selectively by iodosulpiride (Kd 0.6 nM and Bmax 82 fmol/mg protein) or methylspiperone (Kd 0.14 nM and Bmax 223 fmol/mg protein). 3. Retinal dopamine receptors have been also shown to be positively coupled with adenylate cyclase activity in most species, arguing for the existence of D1-subtype, whereas in some others (lower vertebrates and rats), a negative coupling (D2-subtype) has been also detected in peculiar pharmacological conditions implying various combinations of dopamine or a D2-agonist with a D1-antagonist or a D2-antagonist in the absence or presence of forskolin. 4. A subpopulation of autoreceptors of D2-subtype (probably not coupled to adenylate cyclase) also seems to be involved in the modulation of retinal dopamine synthesis and/or release. 5. Light/darkness conditions can affect the sensitivity of retinal dopamine D1 and/or D2-receptors, as studied in binding or pharmacological experiments (cAMP levels, dopamine synthesis, metabolism and release). 6. Visual function(s) of retinal dopamine receptors were connected with the regulation of electrical activity and communication (through gap junctions) between horizontal cells mediated by D1 and D2 receptor stimulation. Movements of photoreceptor cells and migration of melanin granules in retinal pigment epithelial cells as well as synthesis of melatonin in photoreceptors were on the other hand mediated by the stimulation of D2-receptors. 7. Other physiological functions of dopamine D1-receptors respectively in rabbit and in embryonic avian retina would imply the modulation of acetylcholine release and the inhibition of neuronal growth cones.     

Dopamine D1 and D2 receptors in spontaneously hypertensive rat brain striatum

Since it has been reported that dopamine D2 receptors are elevated in the brain striatum of spontaneously hypertensive (SH) rats, and since both D1 and D2 receptors may interact with one another, we measured the densities of both these receptors in SH rat striatum, as well as those in the normotensive Wistar-Kyoto rat striatum. The D1 receptor density in both strains was virtually the same, 72.9 +/- 2.2 and 71.3 +/- 3.2 pmol/g, respectively (mean +/- SD). The D2 receptor densities were also almost identical, 16.3 +/- 0.6 and 16.8 +/- 1.0 pmol/g, respectively (mean +/- SD). Thus, these data do not support the concept of a dopamine receptor related role in spontaneous hypertension.     

Distinct target size of dopamine D-1 and D-2 receptors in rat striatum

Frozen rat striatal tissue was exposed to 10 MeV electrons from a linear accelerator. Based on the theory of target size analysis, the molecular weights of dopamine D-1 receptors (labelled by 3H-piflutixol) and dopamine D-2 receptors (labelled by 3H-spiroperidol) were 79,500 daltons and 136,700 daltons, respectively. The size of the dopamine-stimulated adenylate cyclase was 202,000 daltons. The estimated molecular sizes were deduced by reference to proteins with known molecular weights which were irradiated in parallel. The results showed that the molecular entities for 3H-piflutixol binding and 3H-spiroperidol binding were not identical. The present results do not allow conclusions as to whether D-1 and D-2 receptors are two distinct proteins in the membrane, or whether the receptors are located on the same protein. In the latter case the binding of 3H-spiroperidol needs the presence of a second molecule.     

Evidence for the involvement of dopamine D1 and D2 receptors in mediating the decrease of food intake during repeated treatment with amphetamine

Repeated treatment with amphetamine (AMPH), a well-known anorectic agent, into animals could induce anorexia on day 1 and produce a gradual reversion of food intake (tolerant anorexia) on the following days. It is unknown whether these feeding changes are related to dopamine (DA) and/or noradrenergic neurotransmission. Thus, the present study investigated the subtype of receptor mediating AMPH-induced anorexia. Daily food intake was measured after various drugs were given. Pretreatment with haloperidol, an antagonist of DA receptors, may lead to inhibition of AMPH-induced anorexia. However, pretreatment with the alpha-adrenoceptor antagonist phentolamine, and the beta-adrenoceptor antagonist propranolol, failed to modify the action of AMPH, suggesting the involvement of DA receptors but not adrenoceptors in the action of AMPH-induced anorexia. Furthermore, pretreatment with SCH 23390 at a dose sufficient to block D(1) receptors or pimozide at a dose sufficient to inhibit D(2) receptors blocked AMPH-induced anorexia, indicating the involvement of D(1) and D(2) receptors. In a study of tolerant anorexia, repeated treatment with the D(1)/D(2) agonist apomorphine, but not the D(1) agonist SKF 38393 or D(2) agonist quinpirole, induced an AMPH-like tolerant feeding response, providing evidence for conjoint action of D(1) and D(2) receptors in the effect. The present results suggest that both D(1) and D(2) receptors are involved in anorexia and tolerant anorexia induced by chronic intermittent administration of AMPH.     

Acute amphetamine down-regulates RGS4 mRNA and protein expression in rat forebrain: distinct roles of D1 and D2 dopamine receptors

Administration of psychostimulants modulates mRNA of several regulators of guanine nucleotide-binding protein signaling (RGSs) proteins in the brain. In the present study, the regulation of amphetamine-induced decrease of RGS4 expression in the rat forebrain was evaluated. RGS4 mRNA was reduced by amphetamine in an inverse, dose-dependent manner. The lowest dose (2.5 mg/kg) decreased RGS4 mRNA in caudate putamen for up to 6 h after injection whereas the decrease in several frontal cortical areas was detected at 3 h only. Analysis of RGS4 immunoreactivity by western blotting revealed a decrease 3 h after amphetamine solely in the caudate putamen. Systemic administration of D(1) (SCH23390) or D(2) (eticlopride) receptor antagonists blocked amphetamine-induced locomotion but amphetamine augmented both the SCH23390-induced increase and the eticlopride-induced decrease in RGS4 mRNA in the caudate putamen. Further, the down-regulation of RGS4 immunoreactivity by eticlopride was robust whereas the effect of SCH23390 was blunted as compared with its effect on mRNA. These data suggest that, by decreasing RGS4 expression in the caudate putamen via D(1) receptors, acute amphetamine could disinhibit RGS4-sensitive guanine nucleotide-binding protein alpha-subunit i- and/or q-coupled signaling pathways and favor mechanisms that counterbalance D(1) receptor stimulation.     

Regional distribution of monoamines and dopamine D1-and D2-receptors in the striatum of the rat

Dopamine (DA) D₁-and D₂-receptor densities were determined in 18 discrete areas of the caudate-putamen-globus pallidus of male Wistar rats and compared to local DA concentrations. All three parameters were found to decrease caudally. The globus pallidus was distinguished by the low concentration of DA and its receptors and high noradrenaline, (NA) content. While there were no mediolateral differences in DA or DA D₁-receptors, a clear mediolateral gradient was observed for DA D₂-receptors which extended over several sections of the brain. The ratio of DA D₁-to D₂-receptors was significantly higher in the dorsal than in the ventral areas of the mediolateral and caudal striatum. This is the first report of clear dorsoventral differences in parameters relating to DA activity in the striatum. These findings may be of particular significance in understanding the functional dichotomy between the dorsal and ventral striatum.     

Adenosine and dopamine receptor antagonist binding in the rat ventral and dorsal striatum: lack of changes after a neonatal bilateral lesion of the ventral hippocampus.

There is experimental evidence from radioligand binding experiments for the existence of strong antagonistic interactions between different subtypes of adenosine and dopamine receptors in the striatum, mainly between adenosine A1 and dopamine D1 and between adenosine A2A and dopamine D2 receptors. These interactions seem to be more powerful in the ventral compared to the dorsal striatum, which might have some implications for the treatment of schizophrenia. The binding characteristics of different dopamine and adenosine receptor subtypes were analysed in the different striatal compartments (dorsolateral striatum and shell and core of the nucleus accumbens), by performing saturation experiments with the dopamine D1 receptor antagonist [125I]SCH-23982, the dopamine D2-3 receptor antagonist [3H]raclopride, the adenosine A1 receptor antagonist [3H]DPCPX and the adenosine A2A receptor antagonist [3H]SCH 58261. The experiments were also performed in rats with a neonatal bilateral lesion of the ventral hippocampus (VH), a possible animal model of schizophrenia. Both dopamine D2-3 and adenosine A2A receptors follow a similar pattern, with a lower density of receptors (40%) in the shell of the nucleus accumbens compared with the dorsolateral caudate-putamen. A lower density of adenosine A1 receptors (20%) was also found in the shell of the nucleus accumbens compared with the caudate-putamen. On the other hand, dopamine D1 receptors showed a similar density in the different striatal compartments. Therefore, differences in receptor densities cannot explain the stronger interactions between adenosine and dopamine receptors found in the ventral, compared to the dorsal striatum. No statistical differences in the binding characteristics of any of the different adenosine and dopamine receptor antagonists used were found between sham-operated and VH-lesioned rats.     

Selective analyzers of dopamine D2 receptors modulate serotonin metabolism in the striatum and nucleus accumbens of the rat brain during blockade of dopaminergic impulse flow]

Effects of D2 dopamine receptor selective agonists: quinpirole (0.1, 0.3 and 1 mg/kg, i. p.), pergolide (0.3 mg/kg, i. p.), lisuride (0.1 mg/kg, i. p.) and antagonist raclopride (1.2 mg/kg, i. p.) on the metabolism and synthesis of DA and serotonin in the rat brain striatum and nucleus accumbens after GBL treatment were studied. GBL as well as dopamine D2 receptor selective drugs were shown not only to change neurochemical parameters of dopaminergic brain systems, but also to modulate serotonin metabolism without affecting its biosynthesis.     

Agonist binding fraction of dopamine D2/3 receptors in rat brain: A quantitative autoradiographic study

There has arisen considerable interest in the study of dopamine D_2/3 agonist binding sites by positron emission tomography (PET), based on the claim that agonist sites represent a functional subset of the total number of sites labeled by more conventional antagonist ligands. To test the basis of this claim, we used quantitative autoradiography to measure the abundance of binding sites of a dopamine D_2/3 agonist ([³H]NPA) and an antagonist ([³H]raclopride) in cryosections of rat brain. Saturation binding studies revealed that the B_max for [³H]NPA was nearly identical to that of [³H]raclopride in dorsal brain regions, but was 25% less in the ventral striatum and 56% less in the olfactory tubercle. We also tested the displacement of the two ligands by the hallucinogen LSD, which is known to have dopamine agonist properties. Whereas displacement of [³H]raclopride by increasing LSD concentrations was monophasic, displacement of [³H]NPA was biphasic, suggesting an action of LSD via a subset of dopamine D_2/3 agonist binding sites. Addition of the stable GTP analogue Gpp(NH)p to the medium abolished 90% of the [³H]NPA binding, and increased [³H]raclopride binding by 10%, with a shift to the right in the LSD competition curve, suggesting retention of endogenous dopamine in washed cryostat sections. Thus [³H]NPA and [³H]raclopride binding sites have nearly identical abundances in rat dorsal striatum, but are distinct in the ventral striatum, and with respect to their displacement by LSD.     

Extended treatment with typical and atypical antipsychotic drugs differential effects on the densities of dopamine D2-like and GABAA receptors in rat striatum

In situ radioligand binding and quantitative autoradiography have been used to measure the density of striatal D1-like, D2-like, and GABAA receptors in rats treated with haloperidol at 0.01 or 0.1 mg/kg/ day or chlorpromazine, olanzapine or clozapine at 0.1 or 1.0 mg/kg/day for 1, 3 or 7 months. [3H]SCH23390 binding to D1-like receptors was not changed by any drug treatments. There were significant increases in [3H]nemonapride binding to D2-like receptors at different time points due to treatment with haloperidol, chlorpromazine and olanzapine. By contrast, treatment with clozapine and olanzapine caused a time-dependent decrease in [3H]muscimol binding to the GABAA receptor. These data suggest that treatment with atypical antipsychotic drugs, but not typical antipsychotic drugs, affect striatal GABAergic neurons. In addition, it would appear that clozapine might be unique in that it does not increase dopamine-D2 like receptor density at doses which would be predicted to have antipsychotic effects in humans. The extent to which such changes are involved in the therapeutic effects of drugs such as olanzapine and clozapine remains to be determined.     

Transient and selective overexpression of dopamine D2 receptors in the striatum causes persistent abnormalities in prefrontal cortex functioning

Increased activity of D2 receptors (D2Rs) in the striatum has been linked to the pathophysiology of schizophrenia. To determine directly the behavioral and physiological consequences of increased D2R function in the striatum, we generated mice with reversibly increased levels of D2Rs restricted to the striatum. D2 transgenic mice exhibit selective cognitive impairments in working memory tasks and behavioral flexibility without more general cognitive deficits. The deficit in the working memory task persists even after the transgene has been switched off, indicating that it results not from continued overexpression of D2Rs but from excess expression during development. To determine the effects that may mediate the observed cognitive deficits, we analyzed the prefrontal cortex, the brain structure mainly associated with working memory. We found that D2R overexpression in the striatum impacts dopamine levels, rates of dopamine turnover, and activation of D1 receptors in the prefrontal cortex, measures that are critical for working memory.     

Characterization of D2 receptors and dopamine levels in the thalamus of the rat.

We kinetically characterized D2 receptors in thalami pooled from a group of Sprague-Dawley rats and then determined thalamic levels of dopamine (DA), homovanillic acid (HVA), dihydroxyphenylacetic acid (DOPAC), and norepinephrine (NE) in relation to a measure of thalamic DA D2 receptor densities in another group of rats. The equilibrium dissociation constant (kd) was estimated as 0.1 nM by three independent methods, while the Bmax for thalamic D2 receptors was found to be 6.4 fmol/mg p using 3H-spiperone as ligand and ketanserin to occlude 5HT2 binding. Kinetic constants were in agreement with previously reported kinetic data from rodent caudate-putamen. This suggests that thalamic D2 receptors are similar to D2 receptors from other brain areas. Mean thalamic levels of DA (22.6 ng/mg p), DOPAC (1.19 ng/mg p) and HVA (0.31 ng/mg p) concur with previous reports of a sparse distribution of thalamic DA neurons. D2 receptor densities were positively correlated with DA metabolites DOPAC (P less than .05; r = 0.423) and HVA (P less than .05; r = 0.368), but not DA or NE. These results establish fundamental characteristics of thalamic DA neurotransmission to assist in the investigation of behavioral pharmacology of this area.