The dopamine D3 receptor and drug addiction.

Hedonic and reinforcing properties of drugs of abuse are closely related to brain dopamine neuron activity. All these drugs increase dopamine release in the shell of the nucleus accumbens, a brain region in which neurons co-express the D1 (D1R) and D3 (D3R) dopamine receptor subtypes, that converging pharmacological, human post-mortem and genetic studies suggest to be implicated in drug addiction. The D3R through a cross-talk with the D1R, is involved in induction and expression of behavioral sensitization to levodopa in rats bearing unilateral lesions of dopamine neurons. Behavioral sensitization, a cardinal feature of addiction arises from repeated administration of drugs of abuse thought to play a role in intensification of reinforcing efficacy of these drugs observed under certain conditions. Stimulation of the D3R also appears to enhance the reinforcing effect of cocaine in rats. By interacting with these processes, D3R agents have potential therapeutic applications for treating drug addiction. BP 897 (N-[4-(4-(2-methoxyphenyl)piperazin-1-yl) butyl] naphtalen 2-carboxamide dichlorhydrate), a partial and highly selective D3R agonist in vitro, behaves as an agonist or an antagonist in vivo depending on the response considered. BP 897 has the unprecedented property to reduce cocaine-seeking behavior induced by presentation of a cocaine-associated cue, without having any intrinsic reinforcing effect. As drug-associated cues maintain drug-seeking in animals and elicit craving and relapse in humans, D3R agents like BP 897 may represent new medications for drug addiction, with minimal liability to maintaining dependence.

Involvement of the direct striatonigral pathway in levodopa-induced sensitization in 6-hydroxydopamine-lesioned rats.

Induction of dopamine D3 receptor gene expression in 6-hydroxydopamine-lesioned rats by repeated administration of levodopa had been suggested to be responsible for behavioural sensitization developing in these animals. Using double in situ hybridization techniques, we show that D3 receptor mRNA induction after repeated administration of levodopa took place mainly in dynorphin/substance P-expressing neurons of the direct striatonigral pathway. In agreement, induction of D3 receptor binding sites was evidenced, using 7-[3H]hydroxy-N,N-di-propyl-2-aminotetralin ([3H]7-OH-DPAT), in substantia nigra pars reticulata, the projection area of the direct nigrostriatonigral pathway. Changes in D3 receptor binding and behavioural sensitization during intermittent administration of levodopa paralleled changes in prodynorphin/preprotachykinin rather than preproenkephalin/prodynorphin and preproenkephalin/preprotachykinin mRNA ratios. Behavioural sensitization, induction of D3 receptor binding and changes in prodynorphin/preprotachykinin ratio were all prevented together when levodopa was continuously delivered or intermittently delivered in combination with R-(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4, 5-tetrahydro-1H-3-benzazepine (SCH 23390), a selective D1 receptor antagonist. Our results indicate that functional changes of the direct striatal output pathway, possibly through an interaction between D1 and D3 receptors at the level of terminals in the substantia nigra pars reticulata, are important for the development of behavioural sensitization.

Coexpression of dopamine D1 and D3 receptors in islands of Calleja and shell of nucleus accumbens of the rat: opposite and synergistic functional interactions.

Using double in situ hybridization, we found extensive coexpression of dopamine D1 and D3 receptor (D1R and D3R) mRNAs in neurons of the island of Calleja major (ICjM) and ventromedial shell of nucleus accumbens (ShV), respectively. Thus, at least 79 and 63% of D3R mRNA-expressing neurons in ICjM and ShV also expressed the D1R mRNA. Coexpression of D1R and D3R mRNAs was found to occur in substance P (SP) mRNA-expressing neurons in both areas, suggesting SP mRNA as a marker of the activity of coexpressing neurons. Administration of SKF 38393, a D1R receptor agonist, increased c-fos mRNA in ICjM, whereas administration of quinpirole, a D2R/D3R agonist, decreased it; SCH 23390, a D1 R antagonist and nafadotride, a preferential D3R antagonist, given alone, had effects opposite to those of the corresponding agonists. These data indicate that basal c-fos expression in ICjM is maintained by endogenous dopamine acting tonically upon two receptor subtypes subserving opposite effects on the same cell. However, in ShV, whereas SKF 38393 also increased c-fos mRNA, quinpirole had no effect, a difference presumably reflecting the lower fraction of neurons coexpressing D1R and D3R in this area. In contrast, in ShV from reserpine-treated rats, SKF 38393 increased SP mRNA and quinpirole potentiated this effect. These contrasting interactions of D1R- and D3R-mediated signalling events, i.e. in either opposite or synergistic directions, most likely occurring at the single cell level, may serve to increase the dopamine response threshold of the target cells in ICjM and to maintain a strong tonic activity of ShV neurons.

Functional implications of multiple dopamine receptor subtypes: the D1/D3 receptor coexistence.

The D3 dopamine receptor, a D2-like receptor, is selectively expressed in the ventral striatum, particularly in the shell of nucleus accumbens and islands of Calleja, where it is found in medium sized substance P neurons. The latter co-express the D1 receptor whose interaction with the D3 receptor was studied by treating rats with selective agonists and antagonists. In agreement with the opposite cAMP response, they mediate in cultured neuroblastoma cells, the D1 and D3 receptors exerted opposite influences on c-fos expression in islands of Calleja. However, in agreement with the synergistic influence of cAMP on D3 receptor-mediated mitogenesis on the same cultured cells, D1 and D3 receptor stimulation in vivo synergistically enhanced preprotachykinin mRNA in the shell of accumbens. This indicates that the two receptor subtypes may affect neurons in either synergy or opposition according to the cell or signal generated. Levodopa-induced behavioral sensitization in hemiparkinsonian rats is another example of D1/D3 receptor interaction. Hence repeated levodopa administration induces the ectopic appearance of the D3 receptor in substance P/dynorphin, striatonigral neurons of the dorsal striatum. This induction is secondary to D1 receptor stimulation in neurons of the denervated side and fully accounts for the sensitization, i.e. the increased behavioral responsiveness to levodopa. During brain development, a similar process could operate to control the late appearance of the D3 receptor in D1-receptor bearing neurons of the ventral striatum at a time at which they start to be innervated by dopamine neurons. Finally, taking into account a variety of genetic, developmental, neuroimaging and pharmacological data, we postulate that imbalances between the levels of D1 and D3 receptors in the same neurons could be responsible for schizophrenic disorders.

[Function and therapeutic potential of the dopamine D3 receptor]. / Fonction et potentialités thérapeutiques du récepteur D3 de la dopamine.

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.

Selective expression of dopamine D3 receptor mRNA in proliferative zones during embryonic development of the rat brain.

We studied by in situ hybridization histochemistry the expression of D3 receptor (D3R) mRNA at various stages of rat brain development. The first expression of D3R mRNA was detected at embryonic day 14 (E14) in the striatal and rhinencephalic neuroepithelia and throughout the tectal neuroepithelium. From E16 to E19 D3R mRNA expression extended along a rostrocaudal axis to additional proliferative ventricular zones of the basal forebrain, including the neuroepithelia of the olfactory bulb, nucleus accumbens, septum, and amygdala, whereas D1 and D2 receptor (D1R and D2R) mRNAs were expressed predominantly by migrating neuroblasts and/or differentiating striatal neurons. Only a few neuroblasts, migrating in the lateral cortical stream or developing as cerebellar Purkinje cells, expressed D3R mRNA from E18. At birth D3R expression mRNA appeared in differentiating neuronal fields of the nucleus accumbens and medial mamillary body primordia and on P5 reached a distribution similar to that found in adult. In addition, a transient upregulation was detected on P5 in the medial mamillary bodies, parietofrontal cortex, and olfactory tubercle. In the adult brain D3R gene expression continued in the striatal proliferative subventricular zone. The late expression D3R mRNA in neurons, after achievement of dopamine innervation, supports the existence of a regulating factor released from dopamine neurons, as suggested by denervation studies in the adult. The sustained and abundant D3R gene expression, predominantly in germinative neuroepithelial zones actively involved in neurogenesis of most basal forebrain structures, supports the hypothesis of a neurogenetic but minor morphogenetic modulatory role for the D3R during CNS development.

Induction of dopamine D3 receptor expression as a mechanism of behavioral sensitization to levodopa.

In rats with unilateral lesions of the nigrostriatal dopamine pathway with 6-hydroxydopamine, the motor stimulating effects of levodopa, an indirect dopamine receptor agonist, evidenced by contraversive rotations, become enhanced upon repeated intermittent administration. However, the mechanisms of this behavioral sensitization are essentially unknown. We show that development of sensitization is accompanied by a progressive appearance of D3 receptor mRNA and binding sites, visualized by in situ hybridization and 7-[3H] hydroxy-N,N-di-n-propyl-2-aminotetralin autoradiography, respectively, occurring in the denervated caudate putamen, a brain area from which this receptor subtype is normally absent. Development and decay of these two processes occur with closely parallel time courses, whereas there were no marked changes in D1 or D2 receptor mRNAs. D3 receptor induction by levodopa is mediated by repeated D1 receptor stimulation, since it is prevented by the antagonist SCH 33390 and mimicked by the agonist SKF 38393, but not by two D2 receptor agonists. The enhanced behavioral response to levodopa is mediated by the newly synthesized D3 receptor, since it is antagonized by nafadotride, a preferential D3 receptor antagonist, in low dosage, which has no such effect before D3 receptor induction. D3 receptor induction and behavioral sensitization are also accompanied by a sustained enhancement of prodynorphin mRNA level and a progressively decreasing expression of the preprotachykinin gene. We propose that imbalance between dynorphin and substance P release from the same striatonigral motor efferent pathway, related to D3 receptor induction, is responsible for behavioral sensitization.

Hyperinsulinemia and smooth muscle cell proliferation.

Diabetic and obese subjects run a greater risk of developing atherosclerosis than the rest of the population. Several epidemiological studies suggest that hyperinsulinemia, which characterizes both obese and insulin resistant diabetic subjects, may be involved in atherosclerosis. Because insulin stimulates proliferation of arterial smooth muscle cells (SMC) in culture, it was supposed that insulin may exert an atherogenic role by promoting proliferation of SMC in the intima which is considered as one of the most important initial steps in atherogenesis. However, direct evidence of the growth-promoting effect of insulin on SMC in vivo is still lacking. Using an animal model of arterial injury with a balloon catheter (deendothelialization), we have investigated the short and long-term effect of high circulating insulin, associated or not with plasma glucose variations on aortic SMC proliferative response to endothelial denudation. SMC proliferation was appreciated by measuring the incorporation of 3H-thymidine in the DNA of intima-media layers. In a first series of experiments, we induced concomitant variations of plasma glucose and insulin concentrations by submitting rats with experimental aortic injury to fasting and refeeding. In control rats, the proliferative response to endothelial denudation was dramatically increased on day 2 after injury, remained high on day 4, and progressively declined thereafter. In fasted-refed (FR) rats, the increase on day 2 was less pronounced than in controls; concomitantly a sharp decrease in plasma glucose and insulin concentrations was observed. Refeeding was associated with a significantly less marked decline in SMC mitotic activity since day 4 after injury; at the same time, plasma glucose and insulin concentrations were sharply increased. These results suggested an involvement of variations of both glucose and insulin in SMC proliferation. However, when the surge of glucose and insulin after refeeding was prevented by adding acarbose to the diet, the SMC mitotic activity remained higher in FR rats than in controls, casting doubt as to whether hyperglycemia and hyperinsulinemia could be of importance in this process. Moreover, when deendothelialized rats were rendered either euglycemic-hyperinsulinemic or hyperglycemic-hyperinsulinemic by simultaneous infusion of glucose and insulin, no difference with controls, in the SMC proliferation, was observed. Taken together these data indicate that the acute mitotic response of SMC to endothelial denudation is not influenced by insulin associated or not with high levels of glucose. The long-term of long-lasting hyperinsulinemia was investigated using the obese Zucker rat model which is characterized especially by early spontaneous development of hyperinsulinemia and insulin resistance. Compared to controls, the SMC mitotic activity was not significantly increased from day 2 to day 7 after injury, but, from day 14 to day 30 after endothelial denudation, SMC proliferation was significantly less decreased in obese than in lean rats. Moreover, on day 30, aortic thickness, as measured by histomorphometric technique, was much higher in obese than in lean rats. This difference was entirely due to an increase in SMC number within the intima.(ABSTRACT TRUNCATED AT 400 WORDS)

Increased SMC proliferation after endothelial injury in hyperinsulinemic obese Zucker rats.

The long-term effect of long-lasting hyperinsulinemia on aortic smooth muscle cell (SMC) proliferation after endothelial injury was investigated using the obese Zucker rat model, which is characterized especially by early spontaneous development of hyperinsulinemia and insulin resistance. SMC proliferation was provoked by the passage of an embolectomy catheter with a tightly inflated balloon and was assessed by measuring the incorporation of [3H]thymidine in the DNA of intima-media layers. Compared with controls, the SMC mitotic activity was not significantly increased from day 2 to day 7 after injury, but from day 14 to day 30 after endothelial denudation, SMC proliferation was significantly less decreased in obese than in lean rats [on day 14, DNA synthesis = 107 +/- 18 counts.min-1.micrograms DNA-1 in lean and 345 +/- 44 counts.min-1.micrograms DNA-1 in obese rats (P = 0.003); and on day 30, DNA synthesis = 74 +/- 18 counts.min-1.micrograms DNA-1 in lean and 133 +/- 19 counts.min-1.micrograms DNA-1 in obese rats (P = 0.0055)]. As a result, the intima-media DNA content was higher in obese than in lean rats on day 14 and even more so on day 30, suggesting a higher amount of SMCs in the intima-media. Moreover, on day 30, the aortic thickening, as measured by a histomorphometric technique, was much higher in obese than in lean rats. This difference was entirely due to an increase in SMC content of the intima, mainly resulting from a dramatic increment in the number of nuclei and nuclear number density.(ABSTRACT TRUNCATED AT 250 WORDS)

In vivo effect of insulin on the acute proliferative response of the rat aorta to injury.

The objective of our study was to investigate the effect of hyperinsulinemia associated with either euglycemia, hypoglycemia, or hyperglycemia on the short-term mitotic activity of arterial smooth muscle cells (SMCs) after aortic injury. The proliferative reaction of arterial SMCs was provoked by the passage of an embolectomy catheter with a tightly inflated balloon. DNA synthesis was measured as DNA specific activity after incubation of the aorta in a medium containing 3[H]thymidine. Unrestrained rats were rendered hyperinsulinemic (4-7 nM versus 0.3 nM in controls) immediately after aortic injury by insulin infusion (10 units/day) and either euglycemic (about 5.6 mM), hyperglycemic (14-17 mM), or hypoglycemic (about 2.8 mM) by adjusting the flow rate of hypertonic glucose (30% wt/vol) that was infused simultaneously. The infusion was performed via a catheter inserted into the jugular vein and lasted 2 or 4 days after the aortic injury. After the injury, SMC mitotic activity was dramatically increased on day 2 in control rats with deendothelialized aortas and declined between days 2 and 4. In euglycemic-hyperinsulinemic and hyperglycemic-hyperinsulinemic rats, SMC proliferation showed the same pattern as in controls. At no time was a significant difference observed among the three groups of rats. In hypoglycemic-hyperinsulinemic rats, SMC mitotic activity increased to a lesser extent than in controls on day 2 after deendothelialization. These data indicate that hyperinsulinemia with or without hyperglycemia does not stimulate the early stages of arterial SMC proliferation in the rat although a long-lasting effect of hyperinsulinemia cannot be excluded.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of fasting and refeeding on the proliferative response of rat aorta to injury.

To explore the interplay between the mitotic activity of arterial smooth muscle cells and the variations of plasma glucose and insulin concentrations, we have studied over 14 days the response of thoracic aorta to injury with a balloon catheter in rats submitted to fasting and refeeding. Animals were fasted from the day before until the third day after injury. The proliferative reaction of intima-media was assessed 2, 3.5, 4, 6, 8, and 14 days after injury, comparing freely fed with fasted-refed rats. Fasting decreased plasma glucose and insulin concentrations and DNA synthesis by intima-media, whereas refeeding increased these three variables transiently. The DNA content of intima-media at any time during the response to injury and the intimal thickening on day 14 were not influenced by the sequence of fasting and refeeding, which suggests that the early decrease in DNA synthesis induced by fasting had been compensated for by the later increase in DNA synthesis induced by refeeding. In conclusion, besides hormonal influences (such as insulin), metabolic influences (such as the availability of energetic fuels) are likely to act on the proliferative response of arteries to injury.