Dopamine D2R Research Articles

Changes in dopamine release and dopamine D2/3 receptor levels with the development of mild obesity

Dopamine (DA) neurotransmission is an important modulator of both homeostatic food consumption mediated by the hypothalamus, and reward-related/ hedonic food consumption mediated by the brain reward circuit (Lutter and Nestler, 2009; Schwartz et al., 2000; Wise, 2008). Although there are monogenetic mutations that affect the regulation of body weight, the majority of cases of human obesity appear to result from the interaction of polygenetic predispositions to obesity with an environment in which highly palatable foods are readily available leading to excessive consumption of such foods (Walley et al. 2009). There have been two major hypotheses regarding the role of DA neurotransmission in the development of obesity. The first postulates a rewarddeficient state leading to compensatory overeating to remedy an anhedonic state; decreased brain reward circuit DA neurotransmission is hypothesized to be a significant factor in this reward-deficient state (Blum et al., 2000; Geiger et al., 2008). The second hypothesis postulates that increased behavioral salience of food produces excessive intake of highly palatable foods leading to obesity; this increased behavioral salience is believed to be mediated by increased ventral striatal DA release (Berridge et al., 2009). Although studies of obese animals and obesity prone animals have consistently shown decreased striatal DA release and DA D2/3 receptor levels (DA D2r), to date there have been no published data in humans on changes in DA release in the evolution of obesity and varying results on changes in DA D2r with the development of obesity (Eisenstein et al., 2013; Geiger et al., 2008; Haltia et al., 2007; Johnson and Kenny, 2010; Wang et al., 2001). The purpose of this study was to evaluate the changes in DA release and DA D2r with the development of mild obesity. Baseline [F]fallypride PET studies were performed in 33 healthy subjects (M5 18, F515, mean age5 25.8, range 18–35; mean BMI5 24.8, range 19–35) as previously described to determine regional DA D2r (Kessler, 2009). To evaluate regional DA release, 16 of the 33 subjects having the baseline [F]fallypride positron emission tomography (PET) study (8M, 8F; mean age5 24.3, age range5 21–32; mean BMI5 25.2, body mass index (BMI) range of 19–35) had a second [F]fallypride PET study 3 h following oral administration of 0.43 mg/kg d-amphetamine.(Riccardi et al., 2006). Regions believed to be involved in the development of obesity including ventral striatum, ventral midbrain/substantia nigra, amygdala, caudate, and putamen were specifically examined. High-resolution T1-weighted MRI scans and PET scans of the brain were performed and coregistered to each other. Regions of interest were delineated on the coregistered MRI, and regional estimates of DA D2r (BPND’s) and amphetamine-induced DA release were calculated as previously described (Kessler, 2009; Riccardi et al., 2006). Correlations of regional DA D2r and DA release with BMI were performed using a Pearson product moment correlation covaried for age. Correlations of DA D2r with BMI covaried for age demonstrated small negative correlations in the regions examined (Table 1). Correlations in the right

Dopamine D2R Agonist-Induced Cardiovascular Effects in Healthy Male Subjects: Potential Implications in Clinical Settings.

Dopamine D2 receptor agonists represent a first line treatment option in young patients with signs and symptoms of idiopathic Parkinson's disease. An association between the use of D2 receptor agonists in Parkinson's disease patients and heart failure has been reported. The identification of the underlying mechanism is needed to minimize the resultant cardiovascular morbidity. In a phase I clinical trial, a D2 receptor agonist (pramipexole) was administered to 52 healthy male subjects following a dose escalation scheme. Serial measurements of resting blood pressure, heart rate, and derived parameters including pulse pressure, pulsatile stress, and rate pressure product were analysed. Statistically significant and clinically relevant increases in most of the assessed parameters were found. Ten subjects were removed prematurely from the trial because of clinically significant increases in blood pressure and/or heart rate requiring immediate intervention with IV rescue medications including a selective β-1 blocker. The observed drug-related changes in vital signs were of clinical relevance and might explain some of the cardiovascular morbidity reported in patients receiving D2 receptor agonist in clinical settings. We suggest that the additional use of a β-1 blocking agent might mitigate the risk of cardiovascular morbidity among patients receiving long-term D2 receptor agonists.

Dopamine receptor dysregulation in hippocampus of aged rats underlies chronic pulsatile l-Dopa treatment induced cognitive and emotional alterations

l-Dopa is the major symptomatic therapy for Parkinson's disease, which commonly occurs in elderly patients. However, the effects of chronic use on mood and cognition in old subjects remain elusive. In order to compare the effects of a chronic pulsatile l-Dopa treatment on emotional and cognitive functions in young (3 months) and old (18 months) intact rats, an l-Dopa/carbidopa treatment was administered every 12 h over 4 weeks. Rats were assessed for behavioural despair (repeated forced swimming test, RFST), anhedonia (sucrose preference test, SPT) and spatial learning (Morris water maze, MWM) in the late phase of treatment (T). Neuronal expression of Fos in the hippocampus at the early and late phases of T, as well as after MWM was studied. The density and ratio of dopamine D5r, D3r and D2r receptors were also evaluated in the hippocampus using immunohistochemistry and confocal microscopy. Young rats showed similar patterns during behavioural tests, whereas aged treated rats showed increased immobility counts in RFST, diminished sucrose liquid intake in SPT, and spatial learning impairment during MWM. Fos expression was significantly blunted in the aged treated group after MWM. The density of D5r, D3r and D2r was increased in both aged groups. The treatment reduced the ratio of D5r/D3r and D5r/D2r in both groups. Moreover, aged treated subjects had significant lower values of D5r/D3r and higher values of D5r/D2r when compared with young treated subjects. These results indicate that chronic l-Dopa treatment in itself could trigger emotional and cognitive dysfunctions in elderly subjects through dopamine receptor dysregulation.

Roles of Adrenergic α1 and Dopamine D1 and D2 Receptors in the Mediation of the Desynchronization Effects of Modafinil in a Mouse EEG Synchronization Model

BackgroundSynchronized electroencephalogram (EEG) activity is observed in pathological stages of cognitive impairment and epilepsy. Modafinil, known to increase the release of catecholamines, is a potent wake-promoting agent, and has shown some abilities to desynchronize EEG,but its receptor mechanisms by which modafinil induces desynchoronization remain to be elucidated. Here we used a pharmacological EEG synchronization model to investigate the involvement of adrenergic α1 receptors (R, α1R) and dopamine (DA) D1 and D2 receptors (D1Rs and D2Rs) on modafinil-induced desynchronization in mice.Methodology/Principal FindingsMice were treated with cholinergic receptor antagonist scopolamine and monoamine depletor reserpine to produce experimental EEG synchronization characterized by continuous large-amplitude synchronized activity, with prominent increased delta and decreased theta, alpha, and beta power density. The results showed that modafinil produced an EEG desynchronization in the model. This was characterized by a general decrease in amplitude of all the frequency bands between 0 and 20 Hz, a prominent reduction in delta power density, and an increase in theta power density. Adrenergic α1R antagonist terazosin (1 mg/kg, i.p.) completely antagonized the EEG desynchronization effects of modafinil at 90 mg/kg. However, DA D1R and D2R blockers partially attenuated the effects of modafinil. The modafinil-induced decrease in the amplitudes of the delta, theta, alpha, and beta waves and in delta power density were completely abolished by pretreatment with a combination of the D1R antagonist SCH 23390 (30 µg/kg) and the D2R antagonist raclopride (2 mg/kg, i.p.).Conclusions/SignificanceThese results suggest that modafinil-mediated desynchronization may be attributed to the activation of adrenergic α1R, and dopaminergic D1R and D2R in a model of EEG synchronization.

Estradiol and striatal dopamine receptor antagonism influence memory system bias in the female rat

Estradiol (E2) has been shown to influence learning and memory systems used by female rats to find a reward. Rats with high levels of E2 tend to use allocentric, or place, memory while rats with low levels of E2 use egocentric, or response, memory. It has been shown that systemic dopamine receptor antagonism interacts with E2 to affect which memory system is used. Here, dopamine antagonists were administered directly into either the dorsal striatum or nucleus accumbens to determine where in the brain this interaction takes place. Seventy-four young adult, female, Sprague-Dawley rats were trained and tested in a modified plus-maze. All rats were ovariectomized, received a subcutaneous low E2 implant, and were implanted with bilateral cannulae into either the dorsal striatum or the nucleus accumbens. Additionally, high E2 rats received daily injections of E2 in a sesame oil solution while low E2 rats received daily injections of vehicle. After reaching criterion levels of performance in a plus-maze task, rats were administered microinjections of either a dopamine D1 receptor (SCH 23390; 0.1μg/ml and 0.01μg/ml) or D2 receptor (raclopride; 2μg/ml and 0.5μg/ml) antagonist or a vehicle control (saline) in a counterbalanced manner. High E2 rats exhibited a trend towards a place memory bias while low E2 rats showed a response memory bias. Dorsal striatal administration of a D1, but not D2, dopamine receptor antagonist caused a switch in the memory system used by both high and low E rats. There was no significant effect of dopamine receptor antagonism in the nucleus accumbens group. Thus, E2 determined which memory system controlled behavior in a plus-maze task. Moreover, this effect was modulated by dopamine D1R antagonism in the dorsal but not ventral striatum suggesting that memory systems are, in part, mediated by E2 and dopamine in this region.

Dopamine Receptors Modulate Cytotoxicity of Natural Killer Cells via cAMP-PKA-CREB Signaling Pathway

Dopamine (DA), a neurotransmitter in the nervous system, has been shown to modulate immune function. We have previously reported that five subtypes of DA receptors, including D1R, D2R, D3R, D4R and D5R, are expressed in T lymphocytes and they are involved in regulation of T cells. However, roles of these DA receptor subtypes and their coupled signal-transduction pathway in modulation of natural killer (NK) cells still remain to be clarified. The spleen of mice was harvested and NK cells were isolated and purified by negative selection using magnetic activated cell sorting. After NK cells were incubated with various drugs for 4 h, flow cytometry measured cytotoxicity of NK cells against YAC-1 lymphoma cells. NK cells expressed the five subtypes of DA receptors at mRNA and protein levels. Activation of D1-like receptors (including D1R and D5R) with agonist SKF38393 enhanced NK cell cytotoxicity, but activation of D2-like receptors (including D2R, D3R and D4R) with agonist quinpirole attenuated NK cells. Simultaneously, SKF38393 elevated D1R and D5R expression, cAMP content, and phosphorylated cAMP-response element-binding (CREB) level in NK cells, while quinpirole reduced D3R and D4R expression, cAMP content, and phosphorylated CREB level in NK cells. These effects of SKF38393 were blocked by SCH23390, an antagonist of D1-like receptors, and quinpirole effects were abolished by haloperidol, an antagonist of D2-like receptors. In support these results, H89, an inhibitor of phosphokinase A (PKA), prevented the SKF38393-dependent enhancement of NK cells and forskolin, an activator of adenylyl cyclase (AC), counteracted the quinpirole-dependent suppression of NK cells. These findings show that DA receptor subtypes are involved in modulation of NK cells and suggest that D1-like receptors facilitate NK cells by stimulating D1R/D5R-cAMP-PKA-CREB signaling pathway and D2-like receptors suppress NK cells by inhibiting D3R/D4R-cAMP-PKA-CREB signaling pathway. The results may provide more targets of therapeutic strategy for neuroimmune diseases.

Development of sensitization to methamphetamine in offspring prenatally exposed to morphine, methadone and buprenorphine

Heroin use among young women of reproductive age has drawn much attention around the world. However, there is lack of information on the long-term effects of prenatal exposure to opioids on their offspring. Our previous study demonstrated that prenatally buprenorphine-exposed offspring showed a marked change in the cross-tolerance to morphine compared with other groups. In the current study, this animal model was used to study effects of methamphetamine (METH)-induced behavioral sensitization in the offspring at their adulthood. The results showed no differences in either basal or acute METH-induced locomotor activity in any of the groups of animals tested. When male offspring received METH injections of 2 mg/kg, i.p., once a day for 5 days, behavioral sensitization was induced, as determined by motor activity. Furthermore, the distance and rate of development (slope) of locomotor activity and conditioned place preference induced by METH were significantly increased in the prenatally buprenorphine-exposed animals compared with those in other groups. The dopamine D1 R in the nucleus accumbens of the prenatally buprenorphine-exposed offspring had lower mRNA expression; but no significant changes in the μ-, κ-opioid, nociceptin, D2 R and D3 R receptors were noted. Furthermore, significant alterations were observed in the basal level of cAMP and the D1 R agonist enhanced adenylyl cyclase activity in the prenatally buprenorphine-exposed group. Overall, the study demonstrates that D1 R and its downregulated cAMP signals are involved in enhancing METH-induced behavioral sensitization in prenatally buprenorphine-exposed offspring. The study reveals that prenatal exposure to buprenorphine caused long-term effects on offspring and affected the dopaminergic system-related reward mechanism.

Dopamine Is Involved in Food-Anticipatory Activity in Mice

When food is available during a restricted and predictable time of the day, mammals exhibit food-anticipatory activity (FAA), an increase in locomotor activity preceding the presentation of food. Although many studies have attempted to locate the food-entrainable circadian oscillator in the central nervous system, the pathways that mediate food entrainment are a matter of controversy. The present study was designed to determine the role of dopaminergic and histaminergic systems on FAA. Mice were given access to food for 2 h (ZT12-ZT14), and FAA was defined as the locomotor activity that occurred 2 h before the availability of food. Dopamine D(1) receptor (R), D(2)R, and histamine H(1)R-specific antagonists were used to clarify the role of dopamine and histamine receptors in FAA induced by food restriction (FR). FAA was monitored by infrared locomotor activity sensors. Mice were sacrificed at ZT12 on the 14th day of FR, and monoamine concentrations were determined by high-performance liquid chromatography coupled to electrochemical detection (HPLC-ECD). The results showed that pretreatment with the D(1)R antagonist SCH23390 at 1, 3, or 10 µg/kg significantly reduced FAA by 19% (p < 0.05), 26% (p < 0.05), or 19% (p < 0.01), respectively, and the D(2)R antagonist raclopride at 22, 67, or 200 µg/kg significantly reduced FAA by 16% (p < 0.05), 36% (p < 0.01), or 41% (p < 0.01), respectively, as compared with vehicle control. Moreover, coadministration of SCH23390 (10 µg/kg) and raclopride (200 µg/kg) synergistically inhibited FAA by 57% (p < 0.01) as compared with vehicle control. Consistently, the levels of dopamine and its metabolites in the striatum and midbrain were significantly increased during FAA, even with the pretreatment of D(1)R and D(2)R antagonists. However, pretreatment with pyrilamine at 2.5, 5, or 10 mg/kg did not significantly reduce FAA, although it reduced the locomotor activity during the dark period in ad libitum mice. These results strongly indicate that the dopaminergic system plays an essential role in the FAA in mice.

Selectivity and activation of dopamine D3R from molecular dynamics

D(3) receptor, a member of dopamine (DA) D(2)-like receptor family, which belongs to class A of G-protein coupled receptors (GPCRs), has been reported to play a critical role in neuropsychiatric disorders. Recently, the crystal structure of human dopamine D3 receptor was reported, which facilitates structure-based drug discovery of D3R significantly. We dock D3R-selective compounds into the crystal structure of D3R and homology structure of D2R. Then we perform 20 ns molecular dynamics (MD) of the receptor with selective compounds bound in explicit lipid and water. Our docking and MD results indicate the important residues related to the selectivity of D3R. Specifically, residue Thr(7.39) in D3R may contribute to the high selectivity of R-22 with D3R. Meanwhile, the 4-carbon linker and phenylpiperazine of R-22 improve the binding affinity and the selectivity with D3R. We also dock the agonists, including dopamine, into D3R and perform MD. Our molecular dynamics results of D3R with agonist bound show strong conformational changes from TM5, TM6, and TM7, outward movement of intracellular part of TM6, fluctuation of "ionic lock" motif and conformational change of Tyr(7.53), which is consistent with recent crystal structures of active GPCRs and illustrates the dynamical process during activation. Our results reveal the mechanism of selectivity and activation for D3R, which is important for developing high selective antagonists and agonists for D3R.

P.6.f.002 Attenuated behavioural responses to acute and chronic cocaine in adenosine 2A overexpressed transgenic rats

Striatal dopamine D2R homodimerization is increased in the dorsal striatum after acute amphetamine challenge and in the amphetamine-induced sensitized state, a well-known animal model of schizophrenia. Therefore, it was tested if the increase in D2R homoreceptor complexes found after acute amphetamine challenge in the saline or the amphetamine sensitized state leads to changes in the antagonistic adenosine A2AR-D2R interactions in the striatum. [3H]-raclopride binding was performed in membrane preparations from the ventral and dorsal striatum involving competition with the D2R like agonist quinpirole. In the ventral striatum CGS 21680 produced a significant increase of the KiH values (p < 0.05) in the amphetamine sensitized group when expressed in percent versus the corresponding values in saline sensitized rats after amphetamine challenge. However, in the dorsal striatum a significant change did not develop in the KiH values when expressed in percent of the corresponding values in saline sensitized rats after amphetamine challenge. In fact, the non-significant change was in the opposite direction towards a reduction of the KiH values. Taken together, a reduced affinity of the high affinity D2 agonist binding site (KiH value) developed in the ventral but not in the dorsal striatum as a result of increased antagonistic allosteric A2AR-D2R interactions in the amphetamine-induced sensitized state versus the saline sensitized state after an acute amphetamine challenge. The selective reappearance of antagonistic A2AR-D2R receptor-receptor interactions in the ventral striatum after amphetamine challenge in the amphetamine sensitized rat may give one possible mechanism for the atypical antipsychotic-like actions of A2AR receptor agonists.

Requirement for the endocannabinoid system in social interaction impairment induced by coactivation of dopamine D1 and D2 receptors in the piriform cortex

The dopamine receptor family consists of D1-D5 receptors (D1R-D5R), and we explored the contributions of each dopamine receptor subtype in the piriform cortex (PirC) to social interaction impairment (SII). Rats received behavioral tests or electrophysiological recording of PirC neuronal activity after injection of the D1R/D5R agonist SKF38393, the D2R/D3R/D4R agonist quinpirole, or both, with or without pretreatment with dopamine receptor antagonists, D1R or D5R antisense oligonucleotides, the cannabinoid CB1 receptor antagonist AM281, or the endocannabinoid transporter inhibitor VDM11. Systemic injection of SKF38393 and quinpirole together, but not each one alone, induced SII and increased PirC firing rate, which were blocked by D1R or D2R antagonist. Intra-PirC microinfusion of SKF38393 and quinpirole together, but not each one alone, also induced SII, which was blocked by D1R antisense oligonucleotides or D2R antagonist but not by D3R or D4R antagonist or D5R antisense oligonucleotides. SII induced by intra-PirC SKF38393/quinpirole was blocked by AM281 and enhanced by VDM11, whereas neither AM281 nor VDM11 alone affected social interaction behavior. Coadministration of SKF38393 and quinpirole produced anxiolytic effects without significant effects on locomotor activity, olfaction, and acquisition of olfactory short-term memory. These findings suggest that SII induced by coactivation of PirC D1R and D2R requires the endocannabinoid system.

The protective effect of the insulin against paraquat-induced PC12 cells

Objective To observe dopamine (DA) D2R protein expression in the cultured PC 12 cells and the effect of insulin on the survival rate,morphology,and DA of paraquat (PQ)-induced PC12 cells. Methods Immunoprecipitate Western blotting method was performed to observe DA D2R protein expression in PC 12 cells,MTT assay was used to analyze the changes in viability and morphology of PC 12 ceils which were exposed to different concentrations of PQ and insulin. Results (1) DA D2R protein was expressed in PC 12 ceils. (2) Normal PCI2 cells bodies showed fusiform shape and the synapses were in-tegrity. The cells which were exposed to the 600 μmol/L PQ became ball-like, vacuolar degeneration oc-urred,and the synapse became shorter or disappeared. But the morphology of PC12 cells had a little difference between the normal PC12 and the insulin groups,except that the cells were fusiform shape or a-nomalism but not round shape,and the synapses grew. (3) With the increase of the concentration of PQ, the viability of the cells was decreased. Insulin increased the viability of the ceils which were exposed to 600 μmol/L PQ. Insulin elevated DA concentration both in the normal PC12 cells and those exposed to 600 μmol/L PQ,but there was no significant difference ( P > 0.05 ). Conclusion Insulin could protect the PC12 dopaminergic neurons from injury induced by PQ. Key words: Parkinson's disease; Paraquat; Insulin; PC12 cells

Role of receptor heterodimers in the development of l-dopa-induced dyskinesias in the 6-hydroxydopamine rat model of Parkinson's disease

Chronic l-dopa administration is associated with development of dyskinesias. The molecular mechanisms of these side-effects, however, remain elusive. Dopamine (DA) receptors interact with other receptors to form highly organized complexes where their activity is finely tuned by several proteins. The DA D1R forms a heteromeric complex with the NMDA receptor (NMDAR) and this interaction influences the trafficking of both receptors. Using the 6-hydroxydopamine rat model of Parkinson's disease, we report a correlation between the development of l-dopa-induced dyskinesias and changes in synaptic D1R/NMDAR complexes.

Ultrastructural analysis of prefrontal cortical inputs to the rat amygdala: spatial relationships to presumed dopamine axons and D1 and D2 receptors

Projections from the prefrontal cortex (PFC) to the amygdala (AMG) regulate affective behaviors in a manner that is modulated by dopamine (DA). Although PFC and DA inputs overlap within the basolateral nucleus (BLA) and intercalated cell masses (ICMs), the spatial relationship between these afferents has not been investigated, nor is it known how DA D1 (D1R) and D2 (D2R) receptors are localized in relationship to PFC terminals. We therefore combined tract-tracing from the rat PFC to the AMG with immunocytochemical labeling of tyrosine hydroxylase (TH) to identify presumed DA axons or D1R and D2R. In both the ICMs and BLA, PFC terminals formed asymmetric synapses onto spines that typically did not receive secondary synaptic inputs. TH-immunoreactive (-ir) fibers in the adjacent neuropil typically contacted different structures. Although PFC and TH-ir axons were sometimes apposed to the same dendrites or to each other, PFC terminals only rarely synapsed onto dendrites that also received synapses from TH-ir axons. D1R-ir spines and dendrites were observed commonly within the ICMs but less frequently within the BLA, and PFC axons in the ICMs occasionally synapsed onto D1R-ir spines. Within both regions, D2R-ir spines, dendrites, and axons were observed. PFC terminals occasionally contained presynaptic labeling for D2R but were not observed to synapse onto D2R-ir targets. The infrequent observation of synaptic convergence between PFC and presumed DA terminals within the AMG suggests that DA modulates PFC inputs primarily via extrasynaptic mechanisms, a conclusion supported by the localization of D2R within and D1R postsynaptic to PFC terminals.

Contributions of Dopamine D1, D2, and D3 Receptor Subtypes to the Disruptive Effects of Cocaine on Prepulse Inhibition in Mice

Deficits in prepulse inhibition (PPI) of startle, an operational measure of sensorimotor gating, are characteristics of schizophrenia and related neuropsychiatric disorders. Previous studies in mice demonstrate a contribution of dopamine (DA) D(1)-family receptors in modulating PPI and DA D(2) receptors (D2R) in mediating the PPI-disruptive effects of amphetamine. To examine further the contributions of DA receptor subtypes in PPI, we used a combined pharmacological and genetic approach. In congenic C57BL/6 J wild-type mice, we tested whether the D1R antagonist SCH23390 or the D2/3R antagonist raclopride would attenuate the effects of the indirect DA agonist cocaine (40 mg/kg). Both the D1R and D2/3R antagonists attenuated the cocaine-induced PPI deficit. We also tested the effect of cocaine on PPI in wild-type and DA D1R, D2R, or D3R knockout mice. The cocaine-induced PPI deficit was influenced differently by the three DA receptor subtypes, being absent in D1R knockout mice, partially attenuated in D2R knockout mice, and exaggerated in D3R knockout mice. Thus, the D1R is necessary for the PPI-disruptive effects of cocaine, while the D2R partially contributes to these effects. Conversely, the D3R appears to inhibit the PPI-disruptive effects of cocaine. Uncovering neural mechanisms involved in PPI will further our understanding of substrates of sensorimotor gating and could lead to better therapeutics to treat complex cognitive disorders such as schizophrenia.

Dopamine receptors in equine ovarian tissues

Dopamine (DA) agonist and antagonist treatments can affect ovarian reproductive events in the mare. To support our theory that DA produces these effects by acting directly on the ovary, we analyzed equine ovarian tissues for the presence of dopamine receptor-1 (D1r) and dopamine receptor-2 (D2r) mRNA by reverse transcription polymerase chain reaction (RT-PCR) and D1r and D2r proteins by Western blot and immunohistochemistry (IHC). RT-PCR was performed on RNA isolated from ovarian cortex, medulla, granulosa/theca or corpus luteum (CL) tissues and from pituitary (D2r control) and renal artery (D1r control). D1r and D2r specific primers were designed from partial DNA sequences known for the horse (D2r) or conserved sequences from other species (D1r). Western blot analyses were conducted on CL, cortex and granulosa/theca samples and IHC was performed on CL tissues using D1r or D2r specific antibodies. The incidence of positive D2r mRNA was high in CL and ovarian cortex, low in granulosa/theca, and not detectable in ovarian medulla. Dopamine D1r mRNA incidence was high (50%) only in CL tissues. D1r and D2r antibody staining was positive for each tissue type analyzed by Western blot procedures. All CL tissues prepared by IHC showed positive staining for D1r and D2r proteins. Both DA receptor proteins appeared uniformly distributed throughout the CL tissue. These results indicate that equine ovarian tissues do possess D1r and D2r, and suggests that DA can act directly on ovarian tissues through its interaction with DA receptors.

Effect of perinatal asphyxia on tyrosine hydroxylase and D2 and D1 dopamine receptor mRNA levels expressed during early postnatal development in rat brain

This study was designed to investigate the postnatal developmental plasticity of the mesostriatal and mesolimbic dopamine systems that occurs following perinatal asphyxia. The time course and patterning of the changes in levels of tyrosine hydroxylase (TH), and D1 and D2 dopamine receptor (R) mRNA in the cell body region, substantia nigra and ventral tegmental area (SN/VTA), and projection fields, striatum and limbic regions at the age of 6 and 24 h, and 1 week after asphyxia were studied with a quantitative reverse transcription polymerase chain reaction method with appropriate internal cRNA standard. In Caesarean-delivered control rats (Sprague–Dawley), TH, D2R and D1R mRNA levels showed regional and temporal specificity in both absolute levels and developmental kinetics during the first week of life. TH mRNA levels were >10-fold higher in SN/VTA than in striatum and limbic regions. Compared to Caesarean delivered controls, severe asphyxia (15–20 min) induced an increase of TH and D2R mRNA in SN/VTA 6 h and 1 week after birth. In addition, asphyxia induced an increase of TH mRNA in the projection fields, striatum and limbic regions, at 1 week. Perinatal asphyxia did not appear to exert any effect on D1R mRNA levels. No differences in any of the parameters were observed between spontaneous- and Caesarean-delivered animals. The present results indicate that perinatal asphyxia triggers coordinated changes in the expression of TH, and dopamine receptor mRNA in SN/VTA, striatum and limbic regions. These changes may affect differently dopamine D2R and D1R expression along development, contributing to long-term neurocircuitry imbalances.

Dopamine modulates inwardly rectifying potassium currents in medial prefrontal cortex pyramidal neurons.

Dopamine (DA) modulation of excitability in medial prefrontal cortex (mPFC) pyramidal neurons has attracted considerable attention because of the involvement of mPFC DA in several neuronal disorders. Here, we focused on DA modulation of inwardly rectifying K(+) current (IRKC) in pyramidal neurons acutely dissociated from rat mPFC. A Cs(+)-sensitive whole-cell IRKC was elicited by hyperpolarizing voltage steps from a holding potential of -50 mV. DA (20 microm) reduced IRKC amplitude, as did selective stimulation of DA D(1) or D(2) class receptors (D(1)Rs and D(2)Rs). D(1)Rs activate, whereas D(2)Rs inhibit, the adenylyl cyclase-cAMP-protein kinase A (PKA) signaling pathway. Suppression of IRKC by D(2)R stimulation was attributable to decreased PKA activity because similar inhibition was observed with PKA inhibitors, whereas enhancing PKA activity increased IRKC. This suggests that the DA D(1)R suppression of IRKC occurred through a PKA phosphorylation-independent process. Using outside-out patches of mPFC pyramidal neurons, which preclude involvement of cytosolic signaling molecules, we observed a Cs(+)-sensitive macroscopic IRKC that was suppressed by the membrane-permeable cyclic nucleotide Sp-cAMP but was unaffected by non-nucleotide modulators of PKA, suggesting direct interactions of the cyclic nucleotides with IRK channels. Our results indicate that DA suppresses IRKC through two mechanisms: D(1)R activation of cAMP and direct interactions of the nucleotide with IRK channels and D(2)R-mediated dephosphorylation of IRK channels. The DA modulation of IRKC indicates that ambient DA would tend to increase responsiveness to excitatory inputs when PFC neurons are near the resting membrane potential and may provide a mechanism by which DA impacts higher cognitive function.

Endogenous dopamine maintains synchronous oscillation of intracellular calcium in primary cultured-mouse midbrain neurons.

We demonstrated synchronous oscillation of intracellular Ca2+ in cultured-mouse mid-brain neurons. This synchronous oscillation was thought to result from spontaneous and synchronous neural bursts in a synaptic neural network. We also examined the role of endogenous dopamine in neural networks showing synchronous oscillation. Immunocytochemical study revealed a few tyrosine hydroxylase (TH)-positive dopaminergic neurons, and that cultured neurons expressed synaptophysin and synapsin I. Western blot analyses comfirmed synaptophysin, TH, and 2 types of dopamine receptor (DR), D1R and D2R expression. The synchronous oscillation in midbrain neurons was abolished by the application of R(-)-2-amino-5-phosphonopentanoic acid (AP-5) as an N-methyl-D-aspartate receptor (NMDAR) antagonist. This result suggests that the synchronous oscillation in midbrain neurons requires glutamatergic transmissions, as was the case in previously reported cortical neurons. SCH-12679, a D1R antagonist, inhibited synchronous oscillation in midbrain neurons, while raclopride, a D2R antagonist, induced a transient increase of intracellular Ca2+ and inhibited synchronous oscillation. We consider that endogenous dopamine maintains synchronous oscillation of intracellular Ca2+ through D1R and D2R, and that these DRs regulate intracellular Ca2+in distinctly different ways. Synchronous oscillation of midbrain neurons would be a useful tool for in vitro researches into various neural disorders directly or indirectly caused by dopaminergic neurons.

Different effects of five dopamine receptor subtypes on nuclear factor-kappaB activity in NG108-15 cells and mouse brain.

We previously showed that dopamine receptors D1R and D2R expressed in NG108-15 cells activated protein kinase A and extracellular signal-regulated kinase (ERK) respectively, resulting in differential activation of nuclear factor (NF)-kappaB activity. To investigate whether other dopamine receptor subtypes regulate NF-kappaB, we established NG108-15 cells stably expressing D3R, D4R and D5R (NGD3R, NGD4R and NGD5R). D5R stimulation with SKF 38393 decreased NF-kappaB luciferase reporter activity in NGD5R cells, similar to D1R stimulation in NGD1R cells. However, D3R or D4R stimulation with quinpirole showed no change in NF-kappaB-Luci activity, although forskolin-induced cyclic AMP responsive element-Luci activation was attenuated by quinpirole treatment in NGD2LR, NGD3R and NGD4R cells. As expected, activation of ERK or serum responsive element-luciferase reporter not observed following stimulation with quinpirole in D3R- or D4R-expressing cells. We further examined the effects of haloperidol and risperidone, which are typical and atypical antipsychotic drugs respectively, on NF-kappaB activity by gel shift assay in mouse frontal cortex. Haloperidol treatment slightly attenuated basal NF-kappaB activity. By contrast, risperidone treatment enhanced NF-kappaB activity. Taken together, D2R and D1R/D5R had opposite effects on NF-kappaB activity in NG108-15 cells. Risperidone up-regulated and haloperidol down-regulated NF-kappaB activity in mouse brain. This effect may be related to the atypical antipsychotic properties of risperidone.