D2 Receptor Function Research Articles

Anabolic-androgenic steroids (AAS) increase sensitivity to uncertainty by inhibition of dopamine D1 and D2 receptors.

Anabolic-androgenic steroid abuse is implicated in maladaptive behaviors such as impaired cognition in humans. In a rat model, our lab has shown that testosterone decreases preference for a large/uncertain reward in probability discounting. Other studies have shown that androgens decrease dopamine D1 and D2 receptors in the nucleus accumbens shell, a region important for decision-making behavior in probability discounting. Thus, we attempted to restore selection of the large/uncertain reward in testosterone-treated rats by administering the D2 receptor agonist quinpirole or the D1 receptor agonist SKF81297 and testing probability discounting. Adolescent male Long-Evans rats were treated chronically with high-dose testosterone (7.5mg/kg) or vehicle (13% cyclodextrin in water), and tested for probability discounting after injections of saline, 0.1 and 0.5mg/kg of quinpirole or SKF81297. Rats chose between a small/certain reward (1 sugar pellet, 100% probability) and a large/uncertain reward (4 pellets, decreasing probability: 100, 75, 50, 25, 0%). Testosterone-treated rats selected the large/uncertain reward significantly less than vehicle-treated controls after saline injection. However, acute injection with 0.1mg/kg quinpirole increased large/uncertain reward preference in testosterone-treated rats only, indicated by a testosterone × quinpirole interaction. At 0.5mg/kg, quinpirole increased large/uncertain reward preference in all rats. Acute injection with SKF81297 at 0.1 or 0.5mg/kg rescued large/uncertain reward preference in testosterone-treated rats by eliminating the difference between groups. It appears that altered probability discounting behavior in testosterone-treated rats is due to both decreased D1 and D2 receptor function.

The direct basal ganglia pathway is hyperfunctional in focal dystonia.

See Fujita and Eidelberg (doi:10.1093/brain/awx305) for a scientific commentary on this article. Focal dystonias are the most common type of isolated dystonia. Although their causative pathophysiology remains unclear, it is thought to involve abnormal functioning of the basal ganglia-thalamo-cortical circuitry. We used high-resolution research tomography with the radioligand 11C-NNC-112 to examine striatal dopamine D1 receptor function in two independent groups of patients, writer’s cramp and laryngeal dystonia, compared to healthy controls. We found that availability of dopamine D1 receptors was significantly increased in bilateral putamen by 19.6–22.5% in writer’s cramp and in right putamen and caudate nucleus by 24.6–26.8% in laryngeal dystonia (all P ≤ 0.009). This suggests hyperactivity of the direct basal ganglia pathway in focal dystonia. Our findings paralleled abnormally decreased dopaminergic function via the indirect basal ganglia pathway and decreased symptom-induced phasic striatal dopamine release in writer’s cramp and laryngeal dystonia. When examining topological distribution of dopamine D1 and D2 receptor abnormalities in these forms of dystonia, we found abnormal separation of direct and indirect pathways within the striatum, with negligible, if any, overlap between the two pathways and with the regions of phasic dopamine release. However, despite topological disorganization of dopaminergic function, alterations of dopamine D1 and D2 receptors were somatotopically localized within the striatal hand and larynx representations in writer’s cramp and laryngeal dystonia, respectively. This finding points to their direct relevance to disorder-characteristic clinical features. Increased D1 receptor availability showed significant negative correlations with dystonia duration but not its severity, likely representing a developmental endophenotype of this disorder. In conclusion, a comprehensive pathophysiological mechanism of abnormal basal ganglia function in focal dystonia is built upon upregulated dopamine D1 receptors that abnormally increase excitation of the direct pathway, downregulated dopamine D2 receptors that abnormally decrease inhibition within the indirect pathway, and weakened nigro-striatal phasic dopamine release during symptomatic task performance. Collectively, these aberrations of striatal dopaminergic function underlie imbalance between direct and indirect basal ganglia pathways and lead to abnormal thalamo-motor-cortical hyperexcitability in dystonia.

The role of dopamine D2 receptors in the nucleus accumbens during taste-aversive learning and memory extinction after long-term sugar consumption

The nucleus accumbens (NAcc) is a forebrain region that may significantly contribute to the integration of taste and visceral signals during food consumption. Changes in dopamine release in the NAcc have been observed during consumption of a sweet taste and during compulsive consumption of dietary sugars, suggesting that NAcc dopaminergic transmission is strongly correlated with taste familiarity and the hedonic value content. NAcc core and shell nuclei are differentially involved during and after sugar exposure and, particularly, previous evidence suggests that dopamine D2 receptors could be related with the strength of the latent inhibition (LI) of conditioned taste aversion (CTA), which depends on the length of the taste stimulus pre-exposure. Thus, the objective of this work was to evaluate, after long-term exposure to sugar, the function of dopaminergic D2 receptors in the NAcc core during taste memory retrieval preference test, and during CTA. Adult rats were exposed during 14days to 10% sugar solution as a single liquid ad libitum. NAcc core bilateral injections of D2 dopamine receptor antagonist, haloperidol (1μg/μL), were made before third preference test and CTA acquisition. We found that sugar was similarly preferred after 3 acute presentations or 14days of continued sugar consumption and that haloperidol did not disrupt this appetitive memory retrieval. Nevertheless, D2 receptors antagonism differentially affects aversive memory formation after acute or long-term sugar consumption. These results demonstrate that NAcc dopamine D2 receptors have a differential function during CTA depending on the degree of sugar familiarity.

Mechanisms of Kappa Opioid Receptor Potentiation of Dopamine D2 Receptor Function in Quinpirole-Induced Locomotor Sensitization in Rats.

BackgroundIncreased locomotor activity in response to the same stimulus is an index of behavioral sensitization observed in preclinical models of drug addiction and compulsive behaviors. Repeated administration of quinpirole, a D2/D3 dopamine agonist, induces locomotor sensitization. This effect is potentiated and accelerated by co-administration of U69593, a kappa opioid receptor agonist. The mechanism underlying kappa opioid receptor potentiation of quinpirole-induced locomotor sensitization remains to be elucidated.MethodsImmunofluorescence anatomical studies were undertaken in mice brain slices and rat presynaptic synaptosomes to reveal kappa opioid receptor and D2R pre- and postsynaptic colocalization in the nucleus accumbens. Tonic and phasic dopamine release in the nucleus accumbens of rats repeatedly treated with U69593 and quinpirole was assessed by microdialysis and fast scan cyclic voltammetry.ResultsAnatomical data show that kappa opioid receptor and D2R colocalize postsynaptically in medium spiny neurons of the nucleus accumbens and the highest presynaptic colocalization occurs on the same dopamine terminals. Significantly reduced dopamine levels were observed in quinpirole, and U69593-quinpirole treated rats, explaining sensitization of D2R. Presynaptic inhibition induced by kappa opioid receptor and D2R of electrically evoked dopamine release was faster in U69593-quinpirole compared with quinpirole-repeatedly treated rats.ConclusionsPre- and postsynaptic colocalization of kappa opioid receptor and D2R supports a role for kappa opioid receptor potentiating both the D2R inhibitory autoreceptor function and the inhibitory action of D2R on efferent medium spiny neurons. Kappa opioid receptor co-activation accelerates D2R sensitization by contributing to decrease dopamine release in the nucleus accumbens.

GSK-3β Interacts with Dopamine D1 Receptor to Regulate Receptor Function: Implication for Prefrontal Cortical D1 Receptor Dysfunction in Schizophrenia.

Impaired dopamine D1 receptor (D1R) function in prefrontal cortex (PFC) is believed to contribute to the PFC hypofunction that has been hypothesized to be associated with negative symptoms and cognitive deficits in schizophrenia. It is therefore critical to understand the mechanisms for modulation of D1R function. To investigate the physical interaction and functional modulation between D1R and GSK-3β. D1R and GSK-3β physically interact in cultured cells and native brain tissues. This direct interaction was found to occur at the S(417)PALS(421) motif in the C-terminus of D1R. Inhibition of GSK-3β impaired D1R activation along with a decrease in D1R-GSK-3β interaction. GSK-3β inhibition reduced agonist-stimulated D1R desensitization and endocytosis, the latter associated with the reduction of membrane translocation of β-arrestin-2. Similarly, inhibition of GSK-3β in rat PFC also resulted in impaired D1R activation and association with GSK-3β. Moreover, in a NMDA antagonist animal model of schizophrenia, we detected a decrease in prefrontal GSK-3β activity and D1R-GSK-3β association and decreased D1R activation in the PFC. The present work identified GSK-3β as a new interacting protein for D1R functional regulation and revealed a novel mechanism for GSK-3β-regulated D1R function which may underlie D1R dysfunction in schizophrenia.

The Roles of Dopamine D1 Receptor on the Social Hierarchy of Rodents and Nonhuman Primates.

Background:Although dopamine has been suggested to play a role in mediating social behaviors of individual animals, it is not clear whether such dopamine signaling contributes to attributes of social groups such as social hierarchy.Methods:In this study, the effects of the pharmacological manipulation of dopamine D1 receptor function on the social hierarchy and behavior of group-housed mice and macaques were investigated using a battery of behavioral tests.Results:D1 receptor blockade facilitated social dominance in mice at the middle, but not high or low, social rank in the groups without altering social preference among mates. In contrast, the administration of a D1 receptor antagonist in a macaque did not affect social dominance of the drug-treated animal; however, relative social dominance relationships between the drug-treated and nontreated subjects were altered indirectly through alterations of social affiliative relationships within the social group.Conclusions:These results suggest that dopamine D1 receptor signaling may be involved in social hierarchy and social relationships within a group, which may differ between rodents and primates.

Loss of dopamine D1 receptors and diminished D1/5 receptor-mediated ERK phosphorylation in the periaqueductal gray after spinal cord lesion

Neuropathic pain resulting from spinal cord injury is often accompanied by maladaptive plasticity of the central nervous system, including the opioid receptor-rich periaqueductal gray (PAG). Evidence suggests that sensory signaling via the PAG is robustly modulated by dopamine D1- and D2-like receptors, but the effect of damage to the spinal cord on D1 and D2 receptor protein expression and function in the PAG has not been examined. Here we show that 21days after a T10 or C6 spinothalamic tract lesion, both mice and rats display a remarkable decline in the expression of D1 receptors in the PAG, revealed by western blot analysis. These changes were associated with a significant reduction in hindpaw withdrawal thresholds in lesioned animals compared to sham-operated controls. We investigated the consequences of diminished D1 receptor levels by quantifying D1-like receptor-mediated phosphorylation of ERK1,2 and CREB, events that have been observed in numerous brain structures. In naïve animals, western blot analysis revealed that ERK1,2, but not CREB phosphorylation was significantly increased in the PAG by the D1-like agonist SKF 81297. Using immunohistochemistry, we found that SKF 81297 increased ERK1,2 phosphorylation in the PAG of sham animals. However, in lesioned animals, basal pERK1,2 levels were elevated and did not significantly increase after exposure to SKF 81297. Our findings provide support for the hypothesis that molecular adaptations resulting in a decrease in D1 receptor expression and signaling in the PAG are a consequence of SCL.

Timing of amphetamine exposure in relation to puberty onset determines its effects on anhedonia, exploratory behavior, and dopamine D1 receptor expression in young adulthood

Non-medical use of amphetamine (AMPH) among adolescents is prevalent, which is problematic given the potential consequences of developmental drug exposure on brain function and behavior. Previously we found in adult male rats that AMPH exposure starting before puberty induces a persistent decrease in dopamine D1 receptor (D1R) function in the medial prefrontal cortex (mPFC). Here we investigated if this dysfunction was associated with changes in D1R expression in the mPFC and nucleus accumbens (NAc). We also determined if starting drug exposure well before or near the onset of puberty would influence AMPH-induced changes in D1R expression and behavior. Male and female Sprague–Dawley rats were treated once every other day (10 injections total) with saline or 3mg/kg AMPH (i.p.) from either postnatal day (P) 27 to 45 (pre-puberty groups; Pre-P) or P37 to 55 (peri-puberty groups; Peri-P). After 1, 7 and 21days of withdrawal, sucrose preference tests were performed to assess anhedonia. Exploratory behavior was studied in an open-field arena and on an elevated plus maze (EPM). Rats were then sacrificed for Western blot analysis of D1R expression. We found that AMPH withdrawal induced decreases in sucrose preference that persisted in rats with Peri-P onset treatment. Pre-P onset AMPH exposure led to increased open-arm exploration in the EPM test, as well as a decreased D1R level in the mPFC but not NAc. Our results demonstrated that AMPH exposure starting at different developmental stages resulted in distinct neurobehavioral abnormalities, suggesting an important role of exposure timing in drug-induced plasticity.

Binge-Like Alcohol Exposure During Adolescence Disrupts Dopaminergic Neurotransmission in the Adult Prelimbic Cortex

Repeated binge-like exposure to alcohol during adolescence has been reported to perturb prefrontal cortical development, yet the mechanisms underlying these effects are unknown. Here we report that adolescent intermittent ethanol exposure induces cellular and dopaminergic abnormalities in the adult prelimbic cortex (PrL-C). Exposing rats to alcohol during early-mid adolescence (PD28–42) increased the density of long/thin dendritic spines of layer 5 pyramidal neurons in the adult PrL-C. Interestingly, although AIE exposure did not alter the expression of glutamatergic proteins in the adult PrL-C, there was a pronounced reduction in dopamine (DA) D1 receptor modulation of both intrinsic firing and evoked NMDA currents in pyramidal cells, whereas D2 receptor function was unaltered. Recordings from fast-spiking interneurons also revealed that AIE reduced intrinsic excitability, glutamatergic signaling, and D1 receptor modulation of these cells. Analysis of PrL-C tissue of AIE-exposed rats further revealed persistent changes in the expression of DA-related proteins, including reductions in the expression of tyrosine hydroxylase and catechol-O-methyltransferase (COMT). AIE exposure was associated with hypermethylation of the COMT promoter at a conserved CpG site in exon II. Taken together, these findings demonstrate that AIE exposure disrupts DA and GABAergic transmission in the adult medial prefrontal cortex (mPFC). As DA and GABA work in concert to shape and synchronize neuronal ensembles in the PFC, these alterations could contribute to deficits in behavioral control and decision-making in adults who abused alcohol during adolescence.

Epilepsy, amyloid-β, and D1 dopamine receptors: a possible pathogenetic link?

Experimental and clinical observations indicate that amyloid-β1–42 (Aβ1–42) peptide not only represents a major actor in neurodegenerative mechanisms but also induce hyperexcitation in individual neurons and neural circuits. In this abnormal excitability, possibly leading to seizures, the D1 dopamine (DA) receptors may play a role. Cerebrospinal fluid levels of Aβ1–42 were measured in patients with late-onset epilepsy of unknown etiology. Moreover, the effect of amyloid peptide on the hippocampal epileptic threshold and synaptic plasticity and its link to D1 receptor function were tested in experimental mouse model of cerebral amyloidosis and in acute model of Aβ1–42–induced neurotoxicity. Among 272 evaluated epileptic patients, aged >55 years, 35 suffered from late-onset epilepsy of unknown etiology. In these subjects, cerebrospinal fluid Aβ1–42 levels were measured. The effects of Aβ1–42, amyloid oligomers, and D1 receptor modulation on epileptic threshold were analyzed by electrophysiological recordings in the dentate gyrus of mice hippocampal slices. We found that Aβ1–42 levels were significantly decreased in cerebrospinal fluid of patients with late-onset epilepsy of unknown etiology with respect to controls suggesting the cerebral deposition of this peptide in these patients. Aβ1–42 enhanced epileptic activity in mice through a mechanism involving increased surface expression of D1 receptor, and this effect was mimicked by D1 receptor stimulation and blocked by SCH 23390, a D1 receptor antagonist. Aβ1–42 may contribute to the pathophysiology of late-onset epilepsy of unknown origin. Our preclinical findings indicate that the D1 receptor is involved in mediating the epileptic effects of Aβ1–42. This novel link between Aβ1–42 and D1 receptor signaling might represent a potential therapeutic target.

RGS2 modulates the activity and internalization of dopamine D2 receptors in neuroblastoma N2A cells

Dysregulated expression and function of dopamine D2 receptors (D2Rs) are implicated in drug addiction, Parkinson's disease and schizophrenia. In the current study, we examined whether D2Rs are modulated by regulator of G protein signaling 2 (RGS2), a member of the RGS family that regulates G protein signaling via acceleration of GTPase activity. Using neuroblastoma 2a (N2A) cells, we found that RGS2 was immunoprecipitated by aluminum fluoride-activated Gαi2 proteins. RGS2 siRNA knockdown enhanced membrane [35S] GTPγS binding to activated Gαi/o proteins, augmented inhibition of cAMP accumulation and increased ERK phosphorylation in the presence of a D2/D3R agonist quinpirole when compared to scrambled siRNA treatment. These data suggest that RGS2 is a negative modulator of D2R-mediated Gαi/o signaling. Moreover, RGS2 knockdown slightly increased constitutive D2R internalization and markedly abolished quinpirole-induced D2R internalization assessed by immunocytochemistry. RGS2 knockdown did not compromise agonist-induced β-arrestin membrane recruitment; however, it prevents β-arrestin dissociation from the membrane after prolonged quinpirole treatment during which time β-arrestin moved away from the membrane in control cells. Additionally, confocal microscopy analysis of β-arrestin post-endocytic fate revealed that quinpirole treatment caused β-arrestin to translocate to the early and the recycling endosome in a time-dependent manner in control cells whereas translocation of β-arrestin to these endosomes did not occur in RGS2 knockdown cells. The impaired β-arrestin translocation likely contributed to the abolishment of quinpirole-stimulated D2R internalization in RGS2 knockdown cells. Thus, RGS2 is integral for β-arrestin-mediated D2R internalization. The current study revealed a novel regulation of D2R signaling and internalization by RGS2 proteins.

Renal rescue of dopamine D2 receptor function reverses renal injury and high blood pressure.

Dopamine D2 receptor (DRD2) deficiency increases renal inflammation and blood pressure in mice. We show here that long-term renal-selective silencing of Drd2 using siRNA increases renal expression of proinflammatory and profibrotic factors and blood pressure in mice. To determine the effects of renal-selective rescue of Drd2 expression in mice, the renal expression of DRD2 was first silenced using siRNA and 14 days later rescued by retrograde renal infusion of adeno-associated virus (AAV) vector with DRD2. Renal Drd2 siRNA treatment decreased the renal expression of DRD2 protein by 55%, and DRD2 AAV treatment increased the renal expression of DRD2 protein by 7.5- to 10-fold. Renal-selective DRD2 rescue reduced the expression of proinflammatory factors and kidney injury, preserved renal function, and normalized systolic and diastolic blood pressure. These results demonstrate that the deleterious effects of renal-selective Drd2 silencing on renal function and blood pressure were rescued by renal-selective overexpression of DRD2. Moreover, the deleterious effects of 45-minute bilateral ischemia/reperfusion on renal function and blood pressure in mice were ameliorated by a renal-selective increase in DRD2 expression by the retrograde ureteral infusion of DRD2 AAV immediately after the induction of ischemia/reperfusion injury. Thus, 14 days after ischemia/reperfusion injury, the renal expression of profibrotic factors, serum creatinine, and blood pressure were lower in mice infused with DRD2 AAV than in those infused with control AAV. These results indicate an important role of renal DRD2 in limiting renal injury and preserving normal renal function and blood pressure.

Eye blink rate predicts reward decisions in adolescents.

The ventral striatum displays hyper-responsiveness to reward in adolescents relative to other age groups, and animal research on the developmental trajectory of the dopaminergic system suggests that dopamine may underlie adolescent sensitivity to reward. However, practical limitations prevent the direct measurement of dopamine in healthy adolescents. Eye blink rate (EBR) shows promise as a proxy measure of striatal dopamine D2 receptor function. We investigated developmental differences in the relationship between EBR and reward-seeking behavior on a risky decision-making task. Increasing EBR was associated with greater reward maximization on the task for adolescent but not adult participants. Furthermore, adolescents demonstrated greater sensitivity to reward value than adults, as evinced by shifts in decision patterns based on increasing potential reward. These findings suggest that previously observed adolescent behavioral and neural hypersensitivity to reward may in fact be due to greater dopamine receptor activity, as represented by the relationship of blink rate and reward-seeking behavior. They also demonstrate the feasibility and utility of using EBR as a proxy for dopamine in healthy youth in whom direct measurements of dopamine are prohibitively invasive.

Intramolecular allosteric communication in dopamine D2 receptor revealed by evolutionary amino acid covariation

The structural basis of allosteric signaling in G protein-coupled receptors (GPCRs) is important in guiding design of therapeutics and understanding phenotypic consequences of genetic variation. The Evolutionary Trace (ET) algorithm previously proved effective in redesigning receptors to mimic the ligand specificities of functionally distinct homologs. We now expand ET to consider mutual information, with validation in GPCR structure and dopamine D2 receptor (D2R) function. The new algorithm, called ET-MIp, identifies evolutionarily relevant patterns of amino acid covariations. The improved predictions of structural proximity and D2R mutagenesis demonstrate that ET-MIp predicts functional interactions between residue pairs, particularly potency and efficacy of activation by dopamine. Remarkably, although most of the residue pairs chosen for mutagenesis are neither in the binding pocket nor in contact with each other, many exhibited functional interactions, implying at-a-distance coupling. The functional interaction between the coupled pairs correlated best with the evolutionary coupling potential derived from dopamine receptor sequences rather than with broader sets of GPCR sequences. These data suggest that the allosteric communication responsible for dopamine responses is resolved by ET-MIp and best discerned within a short evolutionary distance. Most double mutants restored dopamine response to wild-type levels, also suggesting that tight regulation of the response to dopamine drove the coevolution and intramolecular communications between coupled residues. Our approach provides a general tool to identify evolutionary covariation patterns in small sets of close sequence homologs and to translate them into functional linkages between residues.

Adolescent Maturation of Dopamine D1 and D2 Receptor Function and Interactions in Rodents.

Adolescence is a developmental period characterized by heightened vulnerability to illicit drug use and the onset of neuropsychiatric disorders. These clinical phenomena likely share common neurobiological substrates, as mesocorticolimbic dopamine systems actively mature during this period. Whereas prior studies have examined age-dependent changes in dopamine receptor binding, there have been fewer functional analyses. The aim of the present study was therefore to determine whether the functional consequences of D1 and D2-like activation are age-dependent. Adolescent and adult rats were given direct D1 and D2 agonists, alone and in combination. Locomotor and stereotypic behaviors were measured, and brains were collected for analysis of mRNA expression for the immediate early genes (IEGs), cfos and arc. Adolescents showed enhanced D2-like receptor control of locomotor and repetitive behaviors, which transitioned to dominant D1-like mechanisms in adulthood. When low doses of agonists were co-administered, adults showed supra-additive behavioral responses to D1/D2 combinations, whereas adolescents did not, which may suggest age differences in D1/D2 synergy. D1/D2-stimulated IEG expression was particularly prominent in the bed nucleus of the stria terminalis (BNST). Given the BNST’s function as an integrator of corticostriatal, hippocampal, and stress-related circuitry, and the importance of neural network dynamics in producing behavior, an exploratory functional network analysis of regional IEG expression was performed. This data-driven analysis demonstrated similar developmental trajectories as those described in humans and suggested that dopaminergic drugs alter forebrain coordinated gene expression age dependently. D1/D2 recruitment of stress nuclei into functional networks was associated with low behavioral output in adolescents. Network analysis presents a novel tool to assess pharmacological action, and highlights critical developmental changes in functional neural circuitry. Immature D1/D2 interactions in adolescents may underlie their unique responses to drugs of abuse and vulnerability to psychopathology. These data highlight the need for age-specific pharmacotherapy design and clinical application in adolescence.

Dopamine D3 Receptor Mediates Preadolescent Stress-Induced Adult Psychiatric Disorders.

Several studies have shown that repeated stressful experiences during childhood increases the likelihood of developing depression- and anxiety-related disorders in adulthood; however, the underlying mechanisms are not well understood. We subjected drd3-EGFP and drd3-null mice to daily, two hour restraint stress episodes over a five day period during preadolescence (postnatal day 35 to 39), followed by social isolation. When these mice reached adulthood (post-natal day > 90), we assessed locomotor behavior in a novel environment, and assessed depression-related behavior in the Porsolt Forced Swim test. We also measured the expression and function of dopamine D3 receptor in limbic brain areas such as hippocampus, nucleus accumbens and amygdala in control and stressed drd3-EGFP mice in adulthood. Adult male mice subjected to restraint stress during preadolescence exhibited both anxiety- and depression-related behaviors; however, adult female mice subjected to preadolescent restraint stress exhibited only depression-related behaviors. The development of preadolescent stress-derived psychiatric disorders was blocked by D3 receptor selective antagonist, SB 277011-A, and absent in D3 receptor null mice. Adult male mice that experienced stress during preadolescence exhibited a loss of D3 receptor expression and function in the amygdala but not in hippocampus or nucleus accumbens. In contrast, adult female mice that experienced preadolescent stress exhibited increased D3 receptor expression in the nucleus accumbens but not in amygdala or hippocampus. Our results suggest that the dopamine D3 receptor is centrally involved in the etiology of adult anxiety- and depression-related behaviors that arise from repeated stressful experiences during childhood.

Abstract 19279: Rescue Of D2R Function in Mouse Kidney Using AAV9 Vector Abrogates the Renal Injury and High Blood Pressure Induced by D2R Silencing

In humans decreased dopamine D2 receptor (D2R) expression and function is associated hypertension and inflammation and fibrosis in renal proximal tubule cells. In mice selective D2R silencing in the kidney results in renal inflammation, injury and hypertension. We hypothesized that these alterations are ameliorated by rescue of renal D2R function. To test this hypothesis we silenced renal D2R expression by sub-capsular infusion of D2R siRNA (D2RsiRNA) using osmotic mini pumps (3 μg/day, n=6 per group). Mice treated with non-silencing siRNA (NS siRNA) served as controls. Two weeks after starting siRNA treatment, mice were treated with control AAV (CAAV) or AAV carrying wild-type D2R (D2RAAV) (n=3 per group, 1e+11 vgp/mouse). Two weeks following AAV treatment, blood pressure was measured and organs were collected. D2R expression was decreased in D2RsiRNA-treated kidneys compared with NSsiRNA-treated kidneys (54 ± 0.8 vs 100± 22 %; P<0.05). D2RAAV treatment increased D2R expression (7.5-11-fold; P<0.01) in both D2RsiRNA and NS siRNA-treated mice. Mice with silenced D2R expression (D2RsiRNA+CAAV) had increased systolic blood pressure (121±3 mmHg; P<0.05) in comparison with the D2R rescued group ( D2RsiRNA+D2RAAV) (100±6 mm Hg), NSsiRNA+CAAV (101±4 mm Hg), or NS siRNA +D2RAAV (101±1 mm Hg). D2R silencing caused an increase in the expression of TNF-α, TGF-β1 and its downstream target fibronectin-1, as well as kidney injury molecule-1 and ki-67, a marker of cell proliferation. By contrast, the expression of these proteins was decreased or reversed to normal in the D2R-rescued group. Masson trichrome staining showed tubular atrophy, interstitial fibrosis and extensive areas of scaring in D2R silenced mice while these abnormalities were significantly reduced in the D2R rescued group. T cells infiltration was significantly higher in D2R silenced group compared to the D2R-rescued group (309 ± 38 vs 217± 17 %; P<0.05). These results show that D2R re-expression prevented the progression of the lesions and demonstrate that increased blood pressure and renal injury due to D2R silencing could be rescued by over expression of D2R in the kidney using AAV9 vector. Furthermore, these data provide the basis for designing novel therapies for kidney disease.

A novel neuropeptide regulator of behavioral sensitization to cocaine

Aims: We have previously demonstrated in male rats that repeated exposure to amphetamine (AMPH) from earlyto midadolescence [postnatal day (P) 27 to 45] persistently alters the function of dopamine D1 receptors in the medial prefrontal cortex (mPFC). Here, we determined if this functional change was associated with altered D1 expression in the mPFC and nucleus accumbens (NAc), whether the timing of drug exposure (earlyvs. late-adolescence) influenced drug-induced plasticity, and whether adolescent AMPH exposure altered anxietyand depression-like behavior. Methods: Male and female Sprague-Dawley rats (n=78) were treatedwith salineor3mg/kgAMPH(i.p.) everyotherday (10 injections total) during early (P27 to 45) or late adolescence (P37 to 55). Sucrose preference (SP) tests were then performed after 1, 7, and 21 days of withdrawal. For each test, the consumption of water and 1% sucrose was measured for 48h. Before the last SP test, anxietylike behavior was assessed by measuring activity in an open-field arena and an elevated plus maze (EPM). Rats were sacrificed after finishing the last SP test, the mPFC and NAc were collected, and D1 protein levels were measured using Western blot analysis. Results: AMPH exposure induced a decrease of SP during the first test. In the EPM, early, but not late, AMPH treatment increased time spent in open arms. Lastly, we found decreased D1 levels in the mPFC following early AMPH exposure. Conclusions: Our preliminary findings suggest that adolescent AMPH exposure induces lasting changes in mPFC D1 expression, which may play a role in the anhedonia and “risk taking” behavior we observed. Importantly, these drug-induced changes are more robust following earlycompared to late-adolescent exposure, suggesting a heightened vulnerability to AMPH in early adolescence. Financial support: This study is supported byNIH (DA 029815).

Amphetamine exposure during adolescence alters anxiety- and depression-like behaviors and prefrontal cortex dopamine receptor expression in adulthood

Aims: We have previously demonstrated in male rats that repeated exposure to amphetamine (AMPH) from earlyto midadolescence [postnatal day (P) 27 to 45] persistently alters the function of dopamine D1 receptors in the medial prefrontal cortex (mPFC). Here, we determined if this functional change was associated with altered D1 expression in the mPFC and nucleus accumbens (NAc), whether the timing of drug exposure (earlyvs. late-adolescence) influenced drug-induced plasticity, and whether adolescent AMPH exposure altered anxietyand depression-like behavior. Methods: Male and female Sprague-Dawley rats (n=78) were treatedwith salineor3mg/kgAMPH(i.p.) everyotherday (10 injections total) during early (P27 to 45) or late adolescence (P37 to 55). Sucrose preference (SP) tests were then performed after 1, 7, and 21 days of withdrawal. For each test, the consumption of water and 1% sucrose was measured for 48h. Before the last SP test, anxietylike behavior was assessed by measuring activity in an open-field arena and an elevated plus maze (EPM). Rats were sacrificed after finishing the last SP test, the mPFC and NAc were collected, and D1 protein levels were measured using Western blot analysis. Results: AMPH exposure induced a decrease of SP during the first test. In the EPM, early, but not late, AMPH treatment increased time spent in open arms. Lastly, we found decreased D1 levels in the mPFC following early AMPH exposure. Conclusions: Our preliminary findings suggest that adolescent AMPH exposure induces lasting changes in mPFC D1 expression, which may play a role in the anhedonia and “risk taking” behavior we observed. Importantly, these drug-induced changes are more robust following earlycompared to late-adolescent exposure, suggesting a heightened vulnerability to AMPH in early adolescence. Financial support: This study is supported byNIH (DA 029815).

Reduced dopamine and glutamate neurotransmission in the nucleus accumbens of quinpirole-sensitized rats hints at inhibitory D2 autoreceptor function.

Dopamine from the ventral tegmental area and glutamate from several brain nuclei converge in the nucleus accumbens (NAc) to drive motivated behaviors. Repeated activation of D2 receptors with quinpirole (QNP) induces locomotor sensitization and compulsive behaviors, but the mechanisms are unknown. In this study, in vivo microdialysis and fast scan cyclic voltammetry in adult anesthetized rats were used to investigate the effect of repeated QNP on dopamine and glutamate neurotransmission within the NAc. Following eight injections of QNP, a significant decrease in phasic and tonic dopamine release was observed in rats that displayed locomotor sensitization. Either a systemic injection or the infusion of QNP into the NAc decreased dopamine release, and the extent of this effect was similar in QNP-sensitized and control rats, indicating that inhibitory D2 autoreceptor function is maintained despite repeated activation of D2 receptors and decreased dopamine extracellular levels. Basal extracellular levels of glutamate in the NAc were also significantly lower in QNP-treated rats than in controls. Moreover, the increase in NAc glutamate release induced by direct stimulation of medial prefrontal cortex was significantly lower in QNP-sensitized rats. Together, these results indicate that repeated activation of D2 receptors disconnects NAc from medial prefrontal cortex and ventral tegmental area. Repeated administration of the dopamine D2 receptor agonist quinpirole (QNP) induces locomotor sensitization. We found that the NAc of QNP-sensitized rats has reduced glutamate levels coming from prefrontal cortex together with a decreased phasic and tonic dopamine neurotransmission but a conserved presynaptic D2 receptor function. We suggest that locomotor sensitization is because of increased affinity state of D2 post-synaptic receptors.