Attenuation of D-1 antagonist-induced D-1 receptor upregulation by concomitant D-2 receptor blockade

The effect of chronic selective D-1 and/or D-2 dopamine receptor blockade on regional D-1 receptor binding was studied in rat brain following chronic treatment with the specific D-1 antagonist SCH 23390 and/or the predominantly D-2 antagonist haloperidol. D-1 receptor density and affinity were evaluated by quantitative autoradiography using 125I-SCH 23982. Chronic SCH 23390 treatment increased D-1 receptor density by 30 to 40% in the striatum, accumbens and tuberculum olfactorium; receptor affinity remained unchanged. Haloperidol had no effect on D-1 receptor Bmax or Kd values, although, when administered with SCH 23390, reduced the D-1 receptor upregulation induced by the D-1 antagonist in striatum and tuberculum olfactorium, but not in nucleus accumbens. These results may be attributable to D-1/D-2 dopamine receptor interactions occurring in the striatum and tuberculum olfactorium and may have implications for the prevention and treatment of drug-induced extrapyramidal disorders.     

Neuroleptic-Induced Supersensitivity and Brain Iron: I. Iron Deficiency and Neuroleptic-Induced Dopamine D2 Receptor Supersensitivity

Previous studies have shown that nutritional iron deficiency in rats reduces brain iron content, resulting in dopamine D2 receptor subsensitivity, as indicated by a decrease in [3H]spiperone binding in caudate nucleus and in behavioral responses to apomorphine. Both phenomena can be reversed by iron supplementation. The possibility that neuroleptic-induced dopamine D2 receptor supersensitivity involves an alteration in brain iron content was investigated in nutritionally iron-deficient and control rats chronically treated with haloperidol (5 mg/kg daily for 14 or 21 days). Neuroleptic treatment was initiated either (a) concurrently with iron deficiency or (b) 2 weeks after the start of iron deficiency. The results show that dopamine D2 receptor subsensitivity, a feature of iron deficiency, is absent in haloperidol-treated, iron-deficient groups. On the contrary, these animals demonstrated biochemical and behavioral dopamine D2 receptor supersensitivity that is relatively greater than that observed with control, haloperidol-treated animals. Haloperidol (5 mg/kg daily for 21 days) as well as chlorpromazine (10 mg/kg daily for 21 days) caused a significant reduction (20-25%) in liver nonheme iron stores as compared with values in control rats. However, in iron-deficient rats, in which liver iron stores were almost totally depleted, haloperidol had no effect. The ability of chronic haloperidol treatment to prevent the reduction of dopamine D2 receptor number during iron deficiency may be associated with alteration of body iron status. Thus, less iron may result in an increase in free haloperidol available to the dopamine D2 receptor.     

Autoradiographic Localization of the Putative D4 Dopamine Receptor in Rat Brain

The putative dopamine D4 receptor protein in rat brain was labelled and quantified autoradiographically using two selective benzamides: [³H]YM-09151-2 which labels D2, D3 and D4 dopamine receptors and [³H]Raclopride which labels D2 and D3. The difference in densities of both ligands at saturable concentrations, show a regional distribution for the putative D4 receptor in the following rank order: hippocampus > caudate putamen > olfactory tubercle = substancia nigra > nucleus accumbens core > cerebral cortex > cerebellum. A calculated value of 0.34 pmol/mg protein was attributable to D4 receptor maximum capacity in caudate putamen and was obtained after subtracting the Bmax of the ligands. Our results show that the distribution of D4 receptor only partially overlaps with the D4 mRNA localization reported earlier and is not only associated to limbic structures but to motor areas as well.     

Intraventricular infusion of leupeptin decreases Bmax of the D2 receptor in the striatum of young rats.

Intraventricular infusion of a thiol protease inhibitor, leupeptin, was previously shown to induce several morphological and immunochemical manifestations of normal and pathological aging in rat brain. The present study attempted to elucidate whether this treatment also perturbs another brain function which declines in aging, dopamine D2 receptor binding in striatum. Intraventricular infusion of leupeptin (0.6 mg per day) for two weeks caused a significant (about 20%) reduction in the binding maximum (Bmax) of dopamine D2 receptors (as examined by [3H] spiperone binding) in the striatum of young male Fischer-344 rats in comparison to (saline-infused) control rats. The apparent Kd values did not differ significantly between the control and leupeptin-treated rat groups. The results suggest that decreased protein turnover may be a factor in the decline in Bmax of D2 receptors during aging.     

Differential effects of chronic treatment with haloperidol and clozapine on the level of preprosomatostatin mRNA in the striatum,nucleus accumbens,and frontal cortex of the rat

1. The goal of this work was to determine the effects of typical and atypical neuroleptics on the level of preprosomatostatin messenger RNA (mRNA) in regions of the rat brain innervated by dopaminergic neurons. 2. Quantitative in situ hybridization histochemistry was used to measure the levels of mRNA encoding preprosomatostatin in neurons of the striatum, the nucleus accumbens, and the medial and lateral agranular areas of the frontal cortex in adult rats treated with either haloperidol or clozapine. 3. In untreated animals, the density of neurons containing preprosomatostatin mRNA was higher in the nucleus accumbens than in the striatum and frontal cortex. The intensity of labeling per neuron, however, was higher in the striatum than in the two other areas examined, suggesting that the expression of preprosomatostatin mRNA is differentially regulated in these brain regions. Chronic administration of haloperidol (1 mg/kg for 28 days) induced a significant decrease in the labeling for preprosomatostatin mRNA in neurons of the nucleus accumbens, frontal cortex, and medial but not lateral striatum. Treatment with clozapine (20 mg/kg for 28 days) increased the levels of preprosomatostatin mRNA in the nucleus accumbens but not in the striatum or the frontal cortex. 4. These results support a role for dopamine in the regulation of central somatostatinergic neurons. The differences in the effects of haloperidol, a neuroleptic which induces extrapyramidal side effects, and clozapine, which does not, suggest that somatostatinergic neurons may play an important role in the regulation of motor behavior.     

Effects of long-term antipsychotic treatment on NMDA receptor binding and gene expression of subunits

Postmortem studies in schizophrenic patients revealed alterations in NMDA receptor binding and gene expression of specific subunits. Because most of the patients had been treated with antipsychotics over long periods, medication effects might have influenced those findings. We treated animals with haloperidol and clozapine in clinical doses to investigate the effects of long-term antipsychotic treatment on NMDA receptor binding and gene expression of subunits. Rats were treated with either haloperidol (1,5 mg/kg/day) or clozapine (45 mg/kg/day) given in drinking water over a period of 6 months. Quantitative receptor autoradiography with [³H]-MK-801 was used to examine NMDA receptor binding. In situ hybridization was performed for additional gene expression studies of the NR1, NR2A, NR2B, NR2C, and NR2D subunits. [³H]-MK-801 binding was found to be increased after haloperidol treatment in the striatum and nucleus accumbens. Clozapine was shown to up-regulate NMDA receptor binding only in the nucleus accumbens. There were no alterations in gene expression of NMDA subunits in any of the three regions. However, the NR2A subunit was down-regulated in the hippocampus and prefrontal cortex by both drugs, whereas only clozapine induced a down-regulation of NR1 in the dorsolateral prefrontal cortex. NR2B, 2C, and 2D subunits did not differ between treatment groups and controls. Both altered NMDA receptor binding and subunit expression strengthen a hyperglutamatergic function after haloperidol treatment and may contribute to some of our postmortem findings in antipsychotically treated schizophrenic patients. Because the effects seen in different brain areas clearly vary between haloperidol and clozapine, they may also be responsible for some of the differences in efficacy and side effects.     

Altered Dopamine Receptor and Dopamine Transporter Binding and Tyrosine Hydroxylase mRNA Expression Following Perinatal NMDA Receptor Blockade

This study examined how perinatal phencyclidine (PCP) treatment would affect dopamine D2 receptor and dopamine transporter (DAT) binding at different stages after treatment cessation. Female rat pups received injections of PCP (10 mg/kg, s.c.) or saline on postnatal day (PN)7, 9 and 11. D2 receptor and transporter binding was examined at four time-points (PN12, 18, 32 and 96) following injections. PCP treatment altered D2 receptor binding throughout development, with a final end-point of 22-33% decreased binding at adulthood in the nucleus accumbens and caudate putamen (P < 0.01), accompanied by a small but significant increase in DAT binding in the caudate putamen. Tyrosine hydroxylase mRNA expression was also significantly increased by 25% (P < 0.05) in the ventral tegmental area of adult rats, suggesting that this model may produce a long-term increase in dopamine output. This study demonstrates that early insult to the brain from NMDA receptor hypofunction alters the dopaminergic system at different stages of development.     

Regulation of D1A and D2 dopamine receptor mRNA during ontogenesis, lesion and chronic antagonist treatment.

The developmental characteristics of D1A and D2 dopamine receptor mRNA levels were determined by Northern blot analyses. Striatal D1A and D2 dopamine receptor mRNAs of male Fischer 344 rats were about 60% of adult (day 120) levels at postnatal day 1 and reached their highest levels at day 30 (126 and 139% adult levels) and then decreased by day 120 (100%). D1 and D2 dopamine receptors showed much greater quantitative changes with densities at day 30 about 6- and 14-fold higher than at day 1, respectively, while mRNA levels showed only a 2-fold increase. The highest level of D2 dopamine receptor mRNA in the midbrain was reached at day 14 (195% of adult levels) while the level at day 1 was 31% higher than that at day 120. Striatal beta-actin mRNA levels decreased gradually as the rats developed with the level at postnatal day 1 almost twice that at day 120 postpartum. Treatment of adult rats with the selective D2 dopamine receptor antagonist, haloperidol (0.5 mg/kg/day, s.c., for 2 h, 7, 14, 21 days or 21 days + 3 days withdrawal) had no effect on striatal D2 dopamine receptor mRNA levels in spite of significant increases in dopamine receptor density at the later time points. However, 21 days following a 6-hydroxydopamine lesion of the nigrostriatal pathway, striatal D2 dopamine receptor mRNA levels were increased by 53%.     

Function and therapeutic potential of the dopamine D3 receptor

The D3 receptor is recognized with high affinity by all antipsychotics and selectively expressed in limbic brain areas participating in the central of emotions, motivation and reward. In transfected cultured cells, stimulation of the D3 receptor inhibits cAMP formation and increases mitogenesis, which, in turn, is potentiated by activation of the cAMP cascade. This suggests that both opposite and synergistic interactions occur between the D3 receptor and the cydic AMP pathway, possibly underlying D1/D3 receptor interactions. In fact, D1 and D3 receptors colocalize in the islands of Calleja, in which they interact in opposition on c-fos mRNA expression, and in the shell of nucleus accumbens, in which they interact in synergy on substance P mRNA expression. The expression of the D3 receptor is highly dependent of the dopamine innervation: lesion of ascending dopamine neurons reduces D3 receptor mRNA and binding in the shell of nudeus accumbens, by deprivation of an unknown factor of dopamine neurons, distinct form dopamine and its cotransmitters. In agreement, expression of the D3 receptor in neurons during rat brain development starts after the settlement of dopamine innervation during the first postnatal week. However, in adult rats with a unilateral lesion of dopamine neurons, repeated treatment with levodopa rescues D3 receptor expression in the shell of nudeus accumbens and induces this expression in the dorsal striatum, a region controlling movements in which the D3 receptor is normally absent. This induction seems responsible for the behavioral sensitisation, i.e. increased responsiveness to levodopa. These observations suggest a role of the D3 receptor in the progressive increase in the therapeutic efficacy of levodopa in the initial treatment of Parkinson's disease, and/or its adversive motor and psychopathological effects during long-term treatment. Finally, various pharmacological and genetic data suggest a role of the D3 receptor in drug addiction and schizophrenia, the treatment of which could benefit from selective D3R agents.     

Involvement of Dopamine D1 and D2 Receptors in the Regulation of Proenkephalin mRNA Abundance in the Striatum and Accumbens of the Rat Brain

The effects of short-term treatment (6 h) with selective D1 or D2 agonists and antagonists on the mRNA for proenkephalin in the medial and anterior aspects of the caudate-putamen and the nucleus accumbens were assessed by in situ hybridization histochemistry. Proenkephalin mRNA abundance was significantly changed in the striatum and accumbens in response to D2 receptor manipulation. D2 blockade with haloperidol or raclopride increased, whereas D2 stimulation with LY-171555 (D2 agonist) decreased, striatal and accumbens proenkephalin mRNA abundance. Antagonism of D1 receptor activity with SCH-23390 significantly decreased proenkephalin mRNA abundance in all brain regions. Concurrent administration of the D1 agonist SKF-38393 prevented the SCH-23390 effect in all brain areas. The data demonstrate that acute treatment with dopaminergic D2 agonists and antagonists affects proenkephalin mRNA abundance in the striatum and accumbens via a D2 receptor mechanism, consistent with the concept that D2 receptor function inhibits the synthesis of the mRNA encoding the enkephalin peptides. Moreover, D1 receptor activity, directly or indirectly, exerts modulatory effects on proenkephalin mRNA abundance in the striatum and nucleus accumbens.     

D(3) receptor ligands modulate extracellular dopamine clearance in the nucleus accumbens

An involvement of the D(3) dopamine receptor in the regulation of extracellular dopamine has been suggested. However, the mechanisms mediating this effect are unclear. We have used the technique of no net flux microdialysis under transient conditions to examine the influence of the D(3) -preferring agonist (+)-PD128907 upon extracellular dopamine levels in the nucleus accumbens of the mouse. (+)-PD 128907 (0.1 mg/kg intraperitoneally) significantly decreased extracellular dopamine. This decrease was associated with a marked increase in the extraction fraction, which suggests an increase in dopamine clearance. The ability of D(3) -preferring compounds to modulate dopamine uptake was investigated in vitro using rotating disk electrode voltammetry. (+)-PD 128907 (10 nm) significantly increased the initial clearance rate of 3 microm dopamine in rat nucleus accumbens tissue suspensions. Kinetic analysis revealed no change in the apparent K (m) of uptake but it showed a 33% increase in V (max). In contrast, the D(3) antagonist GR 103691 (10 nm) significantly decreased dopamine uptake. Consistent with the low levels of D(3) receptors in the dorsal striatum, neither compound affected uptake in tissue suspensions from this brain region. These data indicate that D(3) receptor activation increases dopamine uptake in the nucleus accumbens and suggest that this receptor subtype can regulate extracellular dopamine by modulating the DA transporter activity.     

Effect of Chronic Typical and Atypical Neuroleptic Treatment on Proenkephalin mRNA Levels in the Striatum and Nucleus Accumbens of the Rat

We measured proenkephalin (PEK) mRNA levels in the anterior and medial aspects of the caudate-putamen (CPU) and in the nucleus accumbens (NAc) of the rat by in situ hybridization histochemistry after chronic treatment for 21 days with typical (haloperidol and prolixin) and atypical (molindone, thioridazine, and clozapine) neuroleptics. Chronic administration with these drugs resulted in PEK mRNA levels that were 60-80% higher than controls in the anterior and medial aspects of the CPU but only 25-30% over controls in the NAc. All three atypical neuroleptics studied increased PEK mRNA in the following order: anterior-CPU, thioridazine greater than clozapine and molindone; medial-CPU, thioridazine and molindone greater than clozapine; and NAc, thioridazine much greater than molindone and clozapine. Chronic treatment with the specific dopamine D2 antagonist sulpiride also caused elevation in PEK mRNA levels in all three brain regions studied whereas the specific serotonin S2 receptor blocker, cinanserin, had no significant effects on PEK mRNA levels. These results are consistent with the hypothesis that elevated levels of the enkephalins in the mesolimbic system may be necessary for antipsychotic activity. They also support the idea that the undesirable motoric signs and symptoms observed after chronic treatment with typical neuroleptics may not be the result of increased levels of enkephalins in the basal ganglia because atypical neuroleptics which are almost totally devoid of these side effects caused similar increases in PEK mRNA in the CPU.     

Interrelationships between plasma homovanillic acid and indices of dopamine turnover in multiple brain areas during haloperidol and saline administration

Haloperidol or saline was administered to rats daily for 1, 8, 15 or 22 days. During haloperidol, but not saline administration, changes in plasma homovanillic acid (HVA) concentrations were correlated with changes in nucleus accumbens HVA. Haloperidol administration also had a significant effect on the intercorrelation of dopamine (DA) concentrations and indices of DA turnover across multiple brain areas. In particular, intercorrelations of HVA concentrations among DA terminal brain areas (i.e. striatum, nucleus accumbens, and olfactory tubercle) occurred only during haloperidol treatment.     

A low protein-high carbohydrate diet decreases D2 dopamine receptor density in rat brain.

The property of D2 dopamine receptors in the rat brain was evaluated after long-term dietary manipulation. Groups of rats were pair-fed with equicaloric diet containing low protein (8%)-high carbohydrate, high protein (52%)-low carbohydrate and normal protein (20%) for 36 weeks. The low protein-high carbohydrate fed rats exhibited a significant decrease in the density (Bmax) of D2 dopamine receptor in the striatum (28%) and the mesolimbic regions (36%) with no apparent change in the receptor affinity (Kd). These findings suggest that long-term consumption of a low protein-high carbohydrate diet, by decreasing D2 dopamine receptor density, may be an important determinant of central dopaminergic function.     

Prenatal Amphetamine-Induced Dopaminergic Alteration in a Gender- and Estrogen-Dependent Manner

Prenatal exposure to amphetamine induces changes in dopamine receptors in mesolimbic areas and alters locomotor response to amphetamine during adulthood. Sex differences have been reported in amphetamine-induced brain activity and stress sensitivity. We evaluated the effects of prenatal amphetamine exposure on locomotor activity, dopamine receptors and tyrosine hydroxylase mRNA expression in nucleus accumbens and caudate-putamen in response to amphetamine challenge in adult female and male rats. The role of estrogen in the response to restraint stress was analyzed in ovariectomized, prenatally amphetamine-exposed rats. Pregnant rats were treated with d-amphetamine during days 15–21 of gestation. Nucleus accumbens and caudate-putamen were processed for mRNA determination by real-time PCR. In nucleus accumbens, higher mRNA dopamine (D3) receptor expression was found in basal and d-amphetamine-challenge conditions in female than male, and prenatal amphetamine increased the difference. No sex differences were observed in caudate-putamen. Basal saline-treated females showed higher locomotor activity than males. Amphetamine challenge in prenatally amphetamine-exposed rats increased locomotor activity in males and reduced it in females. In nucleus accumbens, estrogen diminished mRNA D1, D2 and D3 receptor expression in basal, and D1 and D3 in ovariectomized stressed rats. Estrogen prevented the increase in tyrosine hydroxylase expression induced by stress in ovariectomized prenatally exposed rats. In conclusion, estrogen modulates mRNA levels of D1, D2 and D3 receptors and tyrosine hydroxylase expression in nucleus accumbens; prenatal amphetamine-exposure effects on D3 receptors and behavioral responses were gender dependent.

     

A subacute treatment of L-methionine induces an increase in the number of [3H]spiperone binding sites in the striatum of the rat

A subacute treatment, 500 mg/kg I.P. twice daily during 5 days, by L-methionine provoked an increase in the Bmax of [3H]-spiperone binding in the striatum of the rat. This increase was associated to a decrease in membrane microviscosity. However in these conditions no changes were found in the [3H]-DHA, [3H]QNB bindings or in the brain dopamine sensitive adenylate cyclase activity. L-methionine treatment reduced the accumulation of Dopa after NSD 1015 and antagonized the decrease in striatal acetylcholine provoked by haloperidol. Thus L-methionine might be a new potential drug for Parkinson's disease treatment.     

Psychotomimetics moderately affect dopamine receptor binding in the rat brain

The hypothesis that psychotomimetics induce a rapid dopamine receptor regulation that could participate in the expression of the brain dopaminergic overactivation and in the early signs of psychotic-like behaviour, was checked by radioligand binding on rat brain cryosections. For this purpose, subchronic 7-day-d-amphetamine pretreatment was combined with acute amphetamine, phencyclidine or LSD challenge. Acute application of psychotomimetics affected only striatal and accumbens but not nigral and olfactory dopamine receptor binding after 40 min, while subchronic amphetamine expressed no effect, as revealed by two-way ANOVA. Post-hoc statistical analysis showed that only striatal and accumbens[3H]SCH 23390 binding decrease (10-12%) following phencyclidine and striatal [3H]spiperone binding increase (11%) after acute amphetamine were significant. It is assumed that such moderate dopamine receptor binding changes probably reflect the fast receptor regulation responses without important influence on a proposed drug-induced dopaminergic overactivity. The registered alterations of D1 receptor binding after phencyclidine are suggested to be capable to modify the activity of some other neural pathways in the basal ganglia and thus participate in a psychotic-like behaviour.     

Effect of chronic treatment with haloperidol on vasopressin release and behavioral changes by osmotic stimulation of the supraoptic nucleus.

Chronic treatment with dopamine D2 blockers in schizophrenic patients has been proposed as one of the causes of polydipsia and water intoxication, but this conclusion is still controversial. To investigate the relationship between dopamine D2 blockers and these syndromes, we designed a behavioral and neurochemical study using hyperosmotic stimulation in the supraoptic nucleus (SON) by microdialysis after chronic treatment with haloperidol in rats. Animals were injected with haloperidol decanoate (20 mg/kg, i.m.) or sesame oil at 2-week intervals for 8 successive weeks. During the 7th week, water-intake was increased 30-60 min after the hyperosmotic stimulation in both groups, but more so in haloperidol-treated animals compared to that in the control group. Moreover, arginine vasopressin (AVP) was released by the hyperosmotic stimulation in SON, but was not significantly different between groups. In addition, striatal dopamine levels 3-4 days after the microdialysis study showed a significant decrease in the haloperidol-treated animals. These results suggest that chronic treatment with haloperidol enhances water-intake produced by hyperosmotic stimulation in the SON but does not increase AVP levels in dialysates following hyperosmotic stimulation. Thus, these symptoms may be mediated by dopaminergic systems in brain.     

Dopamine D3 receptor stimulation promotes the proliferation of cells derived from the post-natal subventricular zone

In the adult mammalian brain, neural stem cells persist in the subventricular zone (SVZ) where dopamine D3 receptors are expressed. Here, we demonstrate that addition of 1 microm apomorphine increases cell numbers in post-natal SVZ cell cultures. This effect was prevented by a co-treatment with haloperidol, sulpiride or U-99194A, a D3-preferring antagonist, and mimicked by the dopamine D3 receptor selective agonist 7-hydroxy-dipropylaminotetralin (7-OH-DPAT). EC50 values were 4.04 +/- 1.54 nm for apomorphine and 0.63 +/- 0.13 nm for 7-OH-DPAT, which fits the pharmacological profile of the D3 receptor. D3 receptors were detected in SVZ cells by RT-PCR and immunocytochemistry. D3 receptors were expressed in numerous beta-III tubulin immunopositive cells. The fraction of apoptotic nuclei remained unchanged following apomorphine treatment, thus ruling out any possible effect on cell survival. In contrast, proliferation was increased as both the proportion of nuclei incorporating bromo-deoxyuridine and the expression of the cell division marker cyclin D1 were enhanced. These findings provide support for a regulatory role of dopamine over cellular dynamics in post-natal SVZ.