Antihypertensive effect of etamicastat in dopamine D2 receptor-deficient mice.

Abnormalities of the D2R gene (DRD2) play a role in the pathogenesis of human essential hypertension; variants of the DRD2 have been reported to be associated with hypertension. Disruption of Drd2 (D2-/-) in mice increases blood pressure. The hypertension of D2-/- mice has been related, in part, to increased sympathetic activity, renal oxidative stress, and renal endothelin B receptor (ETBR) expression. We tested in D2-/- mice the effect of etamicastat, a reversible peripheral inhibitor of dopamine-ß-hydroxylase that reduces the biosynthesis of norepinephrine from dopamine and decreases sympathetic nerve activity. Blood pressure was measured in anesthetized D2-/- mice treated with etamicastat by gavage, (10 mg/kg), conscious D2-/- mice, and D2+/+ littermates, and mice with the D2R selectively silenced in the kidney, treated with etamicastat in the drinking water (10 mg/kg per day). Tissue and urinary catecholamines and renal expression of selected G protein-coupled receptors, enzymes related to the production of reactive oxygen species, and sodium transporters were also measured. Etamicastat decreased blood pressure both in anesthetized and conscious D2-/- mice and mice with renal-selective silencing of D2R to levels similar or close to those measured in D2+/+ littermates. Etamicastat decreased cardiac and renal norepinephrine and increased cardiac and urinary dopamine levels in D2-/- mice. It also normalized the increased renal protein expressions of ETBR, NADPH oxidase isoenzymes, and urinary 8-isoprostane, as well as renal NHE3 and NCC, and increased the renal expression of D1R but not D5R in D2-/- mice. In conclusion, etamicastat is effective in normalizing the increased blood pressure and some of the abnormal renal biochemical alterations of D2-/- mice.

Efficacy of Hybrid Tetrahydrobenzo[d]thiazole Based Aryl Piperazines D-264 and D-301 at D2 and D3 Receptors.

In elucidating the role of pharmacodynamic efficacy at D3 receptors in therapeutic effectiveness of dopamine receptor agonists, the influence of study system must be understood. Here two compounds with D3 over D2 selectivity developed in our earlier work, D-264 and D-301, are compared in dopamine receptor-mediated G-protein activation in striatal regions of wild-type and D2 receptor knockout mice and in CHO cells expressing D2 or D3 receptors. In caudate-putamen of D2 knockout mice, D-301 was ~3-fold more efficacious than D-264 in activating G-proteins as assessed by [(35)S]GTPγS binding; in nucleus accumbens, D-301 stimulated G-protein activation whereas D-264 did not. In contrast, the two ligands exerted similar efficacy in both regions of wild-type mice, suggesting both ligands activate D2 receptors with similar efficacy. In D2 and D3 receptor-expressing CHO cells, D-264 and D-301 appeared to act in the [(35)S]GTPγS assay as full agonists because they produced maximal stimulation equal to dopamine. Competition for [(3)H]spiperone binding was then performed to determine Ki/EC50 ratios as an index of receptor reserve for each ligand. Action of D-301, but not D-264, showed receptor reserve in D3 but not in D2 receptor-expressing cells, whereas dopamine showed receptor reserve in both cell lines. Gαo1 is highly expressed in brain and is important in D2-like receptor-G protein coupling. Transfection of Gαo1 in D3- but not D2-expressing CHO cells led to receptor reserve for D-264 without altering receptor expression levels. D-301 and dopamine exhibited receptor reserve in D3-expressing cells both with and without transfection of Gαo1. Altogether, these results indicate that D-301 has greater intrinsic efficacy to activate D3 receptors than D-264, whereas the two compounds act on D2 receptors with similar intrinsic efficacy. These findings also suggest caution in interpreting Emax values from functional assays in receptor-transfected cell models without accounting for receptor reserve.

"TAARgeting Addiction"--The Alamo Bears Witness to Another Revolution: An Overview of the Plenary Symposium of the 2015 Behavior, Biology and Chemistry Conference.

BACKGROUND: In keeping with the free-thinking tradition San Antonians are known for, the Scientific Program Committee of the Behavior, Biology and Chemistry: Translational Research in Addiction Conference chose trace amine-associated receptor 1 (TAAR1) as the focus of the plenary symposium for its 7th annual meeting held at the University of Texas Health Science Center at San Antonio on March 14 and 15, 2015. The timing of the meeting's plenary session on TAAR1 coincided with the Ides of March, an apt concurrence given the long association of this date with the overthrow of the status quo. And whether aware of the coincidence or not, those in attendance witnessed the plunging of the metaphorical dagger into the heart of the dopamine (DA) transporter (DAT)-centric view of psychostimulant action. METHODS: The symposium's four plenary presentations focused on the molecular and cellular biology, genetics, medicinal chemistry and behavioral pharmacology of the TAAR1 system and the experimental use of newly developed selective TAAR1 ligands. RESULTS: The consensus was that TAAR1 is a DA and methamphetamine receptor, interacts with DAT and DA D2 receptors, and is essential in modulating addiction-related effects of psychostimulants. CONCLUSIONS: Collectively the findings presented during the symposium constitute a significant challenge to the current view that psychostimulants such as methamphetamine and amphetamine solely target DAT to interfere with normal DA signaling and provide a novel conceptual framework from which a more complete understanding of the molecular mechanisms underlying the actions of DA and METH is likely to emerge.

Dopamine D2-Like Receptors and Behavioral Economics of Food Reinforcement.

Previous studies suggest dopamine (DA) D2-like receptor involvement in the reinforcing effects of food. To determine contributions of the three D2-like receptor subtypes, knockout (KO) mice completely lacking DA D2, D3, or D4 receptors (D2R, D3R, or D4R KO mice) and their wild-type (WT) littermates were exposed to a series of fixed-ratio (FR) food-reinforcement schedules in two contexts: an open economy with additional food provided outside the experimental setting and a closed economy with all food earned within the experimental setting. A behavioral economic model was used to quantify reinforcer effectiveness with food pellets obtained as a function of price (FR schedule value) plotted to assess elasticity of demand. Under both economies, as price increased, food pellets obtained decreased more rapidly (ie, food demand was more elastic) in DA D2R KO mice compared with WT littermates. Extinction of responding was studied in two contexts: by eliminating food deliveries and by delivering food independently of responding. A hyperbolic model quantified rates of extinction. Extinction in DA D2R KO mice occurred less rapidly compared with WT mice in both contexts. Elasticity of food demand was higher in DA D4R KO than WT mice in the open, but not closed, economy. Extinction of responding in DA D4R KO mice was not different from that in WT littermates in either context. No differences in elasticity of food demand or extinction rate were obtained in D3R KO mice and WT littermates. These results indicate that the D2R is the primary DA D2-like receptor subtype mediating the reinforcing effectiveness of food.

Trace Amine-Associated Receptor 1 Localization at the Apical Plasma Membrane Domain of Fisher Rat Thyroid Epithelial Cells Is Confined to Cilia.

BACKGROUND: The trace amine-associated receptor 1 (Taar1) is one member of the Taar family of G-protein-coupled receptors (GPCR) accepting various biogenic amines as ligands. It has been proposed that Taar1 mediates rapid, membrane-initiated effects of thyronamines, the endogenous decarboxylated and deiodinated relatives of the classical thyroid hormones T4 and T3. OBJECTIVES: Although the physiological actions of thyronamines in general and 3-iodothyronamine (T1AM) in particular are incompletely understood, studies published to date suggest that synthetic T1AM-activated Taar1 signaling antagonizes thyromimetic effects exerted by T3. However, the location of Taar1 is currently unknown. METHODS: To fill this gap in our knowledge we employed immunofluorescence microscopy and a polyclonal antibody to detect Taar1 protein expression in thyroid tissue from Fisher rats, wild-type and taar1-deficient mice, and in the polarized FRT cells. RESULTS: With this approach we found that Taar1 is expressed in the membranes of subcellular compartments of the secretory pathway and on the apical plasma membrane of FRT cells. Three-dimensional analyses further revealed Taar1 immunoreactivity in cilial extensions of postconfluent FRT cell cultures that had formed follicle-like structures. CONCLUSIONS: The results suggest Taar1 transport along the secretory pathway and its accumulation in the primary cilium of thyrocytes. These findings are of significance considering the increasing interest in the role of cilia in harboring functional GPCR. We hypothesize that thyronamines can reach and activate Taar1 in thyroid follicular epithelia by acting from within the thyroid follicle lumen, their potential site of synthesis, as part of a nonclassical mechanism of thyroid autoregulation.

Regulation of ethanol intake under chronic mild stress: roles of dopamine receptors and transporters.

Studies have shown that exposure to chronic mild stress decreases ethanol intake and preference in dopamine D2 receptor wild-type mice (Drd2 (+/+)), while it increases intake in heterozygous (Drd2 (+/-)) and knockout (Drd2 (-/-)) mice. Dopaminergic neurotransmission in the basal forebrain plays a major role in the reinforcing actions of ethanol as well as in brain responses to stress. In order to identify neurochemical changes associated with the regulation of ethanol intake, we used in vitro receptor autoradiography to measure the levels and distribution of dopamine D1 and D2 receptors and dopamine transporters (DAT). Receptor levels were measured in the basal forebrain of Drd2 (+/+), Drd2 (+/-), and Drd2 (-/-) mice belonging to one of four groups: control (C), ethanol intake (E), chronic mild stress exposure (S), and ethanol intake under chronic mild stress (ES). D2 receptor levels were higher in the lateral and medial striatum of Drd2 (+/+) ES mice, compared with Drd2 (+/+) E mice. Ethanol intake in Drd2 (+/+) mice was negatively correlated with striatal D2 receptor levels. D2 receptor levels in Drd2(+/-) mice were the same among the four treatment groups. DAT levels were lower in Drd2(+/-) C and Drd2 (-/-) C mice, compared with Drd2 (+/+) C mice. Among Drd2(+/-) mice, S and ES groups had higher DAT levels compared with C and E groups in most regions examined. In Drd2(-/-) mice, ethanol intake was positively correlated with DAT levels in all regions studied. D1 receptor levels were lower in Drd2(+/-) and Drd2(-/-) mice, compared with Drd2(+/+), in all regions examined and remained unaffected by all treatments. The results suggest that in normal mice, ethanol intake is associated with D2 receptor-mediated neurotransmission, which exerts a protective effect against ethanol overconsumption under stress. In mice with low Drd2 expression, where DRD2 levels are not further modulated, ethanol intake is associated with DAT function which is upregulated under stress leading to ethanol overconsumption.

The effect of dopamine D4 receptor density on novelty seeking, activity, social interaction, and alcohol binge drinking in adult mice.

The dopamine D4 receptor has been postulated to play a role in the pathophysiology of alcoholism. This study examined how varying levels of D4 expression and their associated behaviors in male and female mice correlate with future alcohol intake. We hypothesized that: (1) mice with low (Drd4(+/-) ) or deficient (Drd4(-/-) ) in D4 receptors would show enhanced ethanol consumption compared with control mice (Drd4(+/+) ), and (2) a specific phenotype in these mice is associated with future vulnerability for alcohol consumption. Individually housed mice were allowed free access to ethanol (20% vv) in the dark (DID). The behaviors measured in male and female mice were: novel object recognition, open-field locomotor activity, and social interaction. Correlation analyses showed that in male Drd4(-/-) mice (relative to Drd4(+/+) controls), anxiolytic behavior was significantly correlated with increased alcohol consumption. Also, in male Drd4(-/-) mice, there was a significant positive correlation between increased exploratory behavior and increased alcohol consumption. These findings were not observed in females. In conclusion, our data suggest that the dopamine D4 receptor gene has an important role in increased exploratory and anxiolytic behavior only in males and these behaviors were positively correlated with increased alcohol consumption. This interaction between sex hormones and dopamine D4 receptor genotype/function predicting future alcohol abuse and correlation with anxiolytic and exploratory behavior in male mice could have important implications for better understanding of vulnerabilities associated with addiction.

Ractopamine, a livestock feed additive, is a full agonist at trace amine-associated receptor 1.

Ractopamine (RAC) is fed to an estimated 80% of all beef, swine, and turkey raised in the United States. It promotes muscle mass development, limits fat deposition, and reduces feed consumption. However, it has several undesirable behavioral side effects in livestock, especially pigs, including restlessness, agitation, excessive oral-facial movements, and aggressive behavior. Numerous in vitro and in vivo studies suggest RAC's physiological actions begin with its stimulation of ß1- and ß2-adrenergic receptor-mediated signaling in skeletal muscle and adipose tissue; however, the molecular pharmacology of RAC's psychoactive effects is poorly understood. Using human cystic fibrosis transmembrane conductance regulator (hCFTR) chloride channels as a sensor for intracellular cAMP, we found that RAC and p-tyramine (TYR) produced concentration-dependent increases in chloride conductance in oocytes coexpressing hCFTR and mouse trace amine-associated receptor 1 (mTAAR1), which was completely reversed by the trace amine-associated receptor 1 (TAAR1)-selective antagonist EPPTB [N-(3-ethoxyphenyl)-4-pyrrolidin-1-yl-3-trifluoromethylbenzamide]. Oocytes coexpressing hCFTR and the human ß2-adrenergic receptor showed no response to RAC or TYR. These studies demonstrate that, contrary to expectations, RAC is not an agonist of the human ß2-adrenergic receptor but rather a full agonist for mTAAR1. Since TAAR1-mediated signaling can influence cardiovascular tone and behavior in several animal models, our finding that RAC is a full mTAAR1 agonist supports the idea that this novel mechanism of action influences the physiology and behavior of pigs and other species. These findings should stimulate future studies to characterize the pharmacological, physiological, and behavioral actions of RAC in humans and other species exposed to this drug.

Choice for response alternatives differing in reinforcement frequency in dopamine D2 receptor mutant and Swiss-Webster mice.

RATIONALE: A previous study showed that dopamine (DA) D2 receptors (D2Rs) are involved in the reinforcing effectiveness of food, but the specific involvement of DA D2Rs in choice among food reinforcers remains unclear. OBJECTIVES: The current study used genetic and pharmacological approaches to assess the role of D2Rs in choice among food-reinforcement frequencies using the generalized matching law (GML), which specifies that logged response and time allocation ratios vary linearly with logged reinforcer ratios. METHODS: Congenic D2R knockout (KO) and wild-type (WT) mice were exposed to concurrent variable-interval schedules of reinforcement with scheduled relative-reinforcement rates from 4:1 to 1:4. Effects of the D2R antagonist (-)-eticlopride (0.1-1.0 mg/kg) were assessed in Swiss-Webster mice. RESULTS: Response and time allocation ratios were related to obtained reinforcement ratios as predicted by the GML. GML fits accounted for ≥ 92 % of the variance in allocation ratios and did not differ in D2R KO and WT mice. Similarly, there were no significant effects of (-)-eticlopride dose on GML fits, despite effects on overall response rates. CONCLUSIONS: The current results demonstrate that neither deletion nor acute blockade of D2Rs affects choice among response alternatives varying in food-reinforcement frequencies. Because previously published results suggest a role of D2Rs in choice between response alternatives differing in reinforcer magnitude and delay or magnitude and probability, the current findings suggest that D2Rs play a role in choice only among certain parameters of reinforcement. Furthermore, these findings suggest parameters of reinforcement may only be fungible in a complex manner.

Dopamine D4 receptors in psychostimulant addiction.

Since the cloning of the D4 receptor in the 1990s, interest has been building in the role of this receptor in drug addiction, given the importance of dopamine in addiction. Like the D3 receptor, the D4 receptor has limited distribution within the brain, suggesting it may have a unique role in drug abuse. However, compared to the D3 receptor, few studies have evaluated the importance of the D4 receptor. This may be due, in part, to the relative lack of compounds selective for the D4 receptor; the early studies were mainly conducted in mice lacking the D4 receptor. In this review, we summarize the literature on the structure and localization of the D4 receptor before reviewing the data from D4 knockout mice that used behavioral models relevant to the understanding of stimulant use. We also present evidence from more recent pharmacological studies using selective D4 agonists and antagonists and animal models of drug-seeking and drug-taking. The data summarized here suggest a role for D4 receptors in relapse to stimulant use. Therefore, treatments based on antagonism of the D4 receptor may be useful treatments for relapse to nicotine, cocaine, and amphetamine use.

Pharmacological characterization of a dopamine transporter ligand that functions as a cocaine antagonist.

An N-butyl analog of benztropine, JHW007 [N-(n-butyl)-3α-[bis(4'-fluorophenyl)methoxy]-tropane], binds to dopamine transporters (DAT) but has reduced cocaine-like behavioral effects and antagonizes various effects of cocaine. The present study further examined mechanisms underlying these effects. Cocaine dose-dependently increased locomotion, whereas JHW007 was minimally effective but increased activity 24 hours after injection. JHW007 (3-10 mg/kg) dose-dependently and fully antagonized the locomotor-stimulant effects of cocaine (5-60 mg/kg), whereas N-methyl and N-allyl analogs and the dopamine (DA) uptake inhibitor GBR12909 [1-(2-[bis(4-fluorophenyl)methoxy]ethyl)-4-(3-phenylpropyl)piperazine dihydrochloride] stimulated activity and failed to antagonize effects of cocaine. JHW007 also blocked the locomotor-stimulant effects of the DAT inhibitor GBR12909 but not stimulation produced by the δ-opioid agonist SNC 80 [4-[(R)-[(2S,5R)-4-allyl-2,5-dimethylpiperazin-1-yl](3-methoxyphenyl)methyl]-N,N-diethylbenzamide], which increases activity through nondopaminergic mechanisms. JHW007 blocked locomotor-stimulant effects of cocaine in both DA D2- and CB1-receptor knockout and wild-type mice, indicating a lack of involvement of these targets. Furthermore, JHW007 blocked effects of cocaine on stereotyped rearing but enhanced stereotyped sniffing, suggesting that interference with locomotion by enhanced stereotypies is not responsible for the cocaine-antagonist effects of JHW007. Time-course data indicate that administration of JHW007 antagonized the locomotor-stimulant effects of cocaine within 10 minutes of injection, whereas occupancy at the DAT, as determined in vivo, did not reach a maximum until 4.5 hours after injection. The σ1-receptor antagonist BD 1008 [N-[2-(3,4-dichlorophenyl)ethyl]-N-methyl-2-(1-pyrrolidinyl)ethylamine dihydrobromide] blocked the locomotor-stimulant effects of cocaine. Overall, these findings suggest that JHW007 has cocaine-antagonist effects that are deviate from its DAT occupancy and that some other mechanism, possibly σ-receptor antagonist activity, may contribute to the cocaine-antagonist effect of JHW007 and like drugs.

Exploring the determinants of trace amine-associated receptor 1's functional selectivity for the stereoisomers of amphetamine and methamphetamine.

Amphetamines are widely abused drugs that interfere with dopamine transport and storage. Recently, however, another mechanism of action was identified: stereoselective activation of the GαS protein-coupled trace amine-associated receptor 1 (TAAR1). To identify structural determinants of this stereoselectivity, we functionally evaluated six mutant receptors in vitro and then used homology modeling and dynamic simulation to predict drug affinities. Converting Asp102 to Ala rendered mouse and rat TAAR1 (mTAAR1 and rTAAR1, respectively) insensitive to ß-phenylethylamine, amphetamine (AMPH), and methamphetamine (METH). Mutating Met268 in rTAAR1 to Thr shifted the concentration-response profiles for AMPH and METH isomers rightward an order of magnitude, whereas replacing Thr268 with Met in mTAAR1 resulted in profiles leftward shifted 10-30-fold. Replacing Asn287 with Tyr in rTAAR1 produced a mouselike receptor, while the reciprocal mTAAR1 mutant was rTAAR1-like. These results confirm TAAR1 is an AMPH/METH receptor in vitro and establish residues 102 (3.32) and 268 (6.55) as major contributors to AMPH/METH binding with residue 287 (7.39) determining species stereoselectivity.

Dopamine D4 receptor deficiency in mice alters behavioral responses to anxiogenic stimuli and the psychostimulant methylphenidate.

An allele of the human dopamine D4 receptor (D4R) gene (DRD4), containing seven tandem repeats of a 48-base nucleotide sequence (DRD4.7), has been reproducibly found in novelty seekers, substance abusers, and individuals with attention-deficit hyperactivity disorder. One hypothesis predicts the resultant protein product of the DRD4.7 polymorphism is deficient in G protein-coupled signaling. If attenuated D4R signaling contributes to these complex behaviors, then wild-type (WT) mice and mice completely lacking D4Rs (D4R KO) might be expected to display significantly different behavioral responses to stimuli known to affect dopamine signaling, such as novelty or psychostimulants. Adolescent male D4R KO mice exhibited greater locomotor activity and spent less time in the anxiogenic center of a novel open field environment than WT littermates. The presence of D4Rs had no effect on emergence into a novel environment from a sheltered space or exploration of a novel object. Low doses of acute methylphenidate (MP) had no effect on the exploration of a novel object, but dose-dependently increased the latency to emerge into a novel environment from a sheltered space. WT and D4R KO mice responded differently to high doses of acute MP, displaying significantly elevated locomotor activity and reduced stereotypy relative to WT mice. Chronic MP treatment produced enhanced locomotor sensitization in D4R KO mice, however this effect could not be fully recapitulated using the putative D4R antagonist L-745-870. These studies suggest that the roles of D4R signaling in novelty-seeking behaviors and the response to psychostimulants are not as clear as previously reported, and that some of these effects may be due to developmental compensatory effects as a consequence of lost D4R expression. If the DRD4.7 variant results in deficient D4R signaling in vivo, this may contribute to an elevated risk of sensitization to drugs of abuse including psychostimulants used to treat ADHD.

Paraoxonase 2 decreases renal reactive oxygen species production, lowers blood pressure, and mediates dopamine D2 receptor-induced inhibition of NADPH oxidase.

The dopamine D(2) receptor (D(2)R) regulates renal reactive oxygen species (ROS) production, and impaired D(2)R function results in ROS-dependent hypertension. Paraoxonase 2 (PON2), which belongs to the paraoxonase gene family, is expressed in various tissues, acting to protect against cellular oxidative stress. We hypothesized that PON2 may be involved in preventing excessive renal ROS production and thus may contribute to maintenance of normal blood pressure. Moreover, D(2)R may decrease ROS production, in part, through regulation of PON2. D(2)R colocalized with PON2 in the brush border of mouse renal proximal tubules. Renal PON2 protein was decreased (-33±6%) in D(2)(-/-) relative to D(2)(+/+) mice. Renal subcapsular infusion of PON2 siRNA decreased PON2 protein expression (-55%), increased renal oxidative stress (2.2-fold), associated with increased renal NADPH oxidase expression (Nox1, 1.9-fold; Nox2, 2.9-fold; and Nox4, 1.6-fold) and activity (1.9-fold), and elevated arterial blood pressure (systolic, 134±5 vs 93±6mmHg; diastolic, 97±4 vs 65±7mmHg; mean 113±4 vs 75±7mmHg). To determine the relevance of the PON2 and D(2)R interaction in humans, we studied human renal proximal tubule cells. Both D(2)R and PON2 were found in nonlipid and lipid rafts and physically interacted with each other. Treatment of these cells with the D(2)R/D(3)R agonist quinpirole (1µM, 24h) decreased ROS production (-35±6%), associated with decreased NADPH oxidase activity (-32±3%) and expression of Nox2 (-41±7%) and Nox4 (-47±8%) protein, and increased expression of PON2 mRNA (2.1-fold) and protein (1.6-fold) at 24h. Silencing PON2 (siRNA, 10nM, 48h) not only partially prevented the quinpirole-induced decrease in ROS production by 36%, but also increased basal ROS production (1.3-fold), which was associated with an increase in NADPH oxidase activity (1.4-fold) and expression of Nox2 (2.1-fold) and Nox4 (1.8-fold) protein. Inhibition of NADPH oxidase with diphenylene iodonium (10µM/30 min) inhibited the increase in ROS production caused by PON2 silencing. Our results suggest that renal PON2 is involved in the inhibition of renal NADPH oxidase activity and ROS production and contributes to the maintenance of normal blood pressure. PON2 is positively regulated by D(2)R and may, in part, mediate the inhibitory effect of renal D(2)R on NADPH oxidase activity and ROS production.

Dopaminergic signaling in the cochlea: receptor expression patterns and deletion phenotypes.

Pharmacological studies suggest that dopamine release from lateral olivocochlear efferent neurons suppresses spontaneous and sound-evoked activity in cochlear nerve fibers and helps control noise-induced excitotoxicity; however, the literature on cochlear expression and localization of dopamine receptors is contradictory. To better characterize cochlear dopaminergic signaling, we studied receptor localization using immunohistochemistry or reverse transcriptase PCR and assessed histopathology, cochlear responses and olivocochlear function in mice with targeted deletion of each of the five receptor subtypes. In normal ears, D1, D2, and D5 receptors were detected in microdissected immature (postnatal days 10-13) spiral ganglion cells and outer hair cells but not inner hair cells. D4 was detected in spiral ganglion cells only. In whole cochlea samples from adults, transcripts for D1, D2, D4, and D5 were present, whereas D3 mRNA was never detected. D1 and D2 immunolabeling was localized to cochlear nerve fibers, near the first nodes of Ranvier (D2) and in the inner spiral bundle region (D1 and D2) where presynaptic olivocochlear terminals are found. No other receptor labeling was consistent. Cochlear function was normal in D3, D4, and D5 knock-outs. D1 and D2 knock-outs showed slight, but significant enhancement and suppression, respectively, of cochlear responses, both in the neural output [auditory brainstem response (ABR) wave 1] and in outer hair cell function [distortion product otoacoustic emissions (DPOAEs)]. Vulnerability to acoustic injury was significantly increased in D2, D4 and D5 lines: D1 could not be tested, and no differences were seen in D3 mutants, consistent with a lack of receptor expression. The increased vulnerability in D2 knock-outs was seen in DPOAEs, suggesting a role for dopamine in the outer hair cell area. In D4 and D5 knock-outs, the increased noise vulnerability was seen only in ABRs, consistent with a role for dopaminergic signaling in minimizing neural damage.

Behavioral economics of food reinforcement and the effects of prefeeding, extinction, and eticlopride in dopamine D2 receptor mutant mice.

RATIONALE: Several studies have investigated the reinforcing effects of food in genetically engineered mice lacking dopamine D(2) receptors (DA D(2)Rs); however, behavioral economic analyses quantifying reinforcement have not been conducted. OBJECTIVE: The role of DA D(2)Rs in food reinforcement was examined by comparing responding under various fixed-ratio (FR) schedules of reinforcement, and effects of extinction, satiation, and the DA D(2)R antagonist eticlopride, in mice with and without genetic deletions of the receptor. RESULTS: Response rates of DA D(2)R knockout (KO) mice were generally lower than those of littermate wild-type (WT) and heterozygous (HET) mice. The demand curve (consumption vs. FR value) for KO mice decreased more steeply than that of HET or WT mice, suggesting that reinforcing effectiveness is decreased with DA D(2)R deletion. Prefeeding decreased, whereas extinction increased overall response rates as a proportion of baseline, with no significant genotype differences. Both (+)- and (-)-eticlopride dose-dependently decreased responding in all genotypes with (-)-eticlopride more potent than (+)-eticlopride in all but KO mice. The enantiomers were equipotent in KO mice, and similar in potency to (+)-eticlopride in WT and HET mice. CONCLUSIONS: That prefeeding and extinction did not vary across genotypes indicates a lack of involvement of DA D(2)Rs in these processes. Differences between (-)-eticlopride effects and extinction indicate that DA D(2)R blockade does not mimic extinction. The maintenance of responding in KO mice indicates that the DA D(2)R is not necessary for reinforcement. However, the economic analysis indicates that the DA D(2)R contributes substantially to the effectiveness of food reinforcement.

Upregulation of cannabinoid type 1 receptors in dopamine D2 receptor knockout mice is reversed by chronic forced ethanol consumption.

BACKGROUND: The anatomical proximity of the cannabinoid type 1 (CNR1/CB1R) and the dopamine D2 receptors (DRD2), their ability to form CB1R-DRD2 heteromers, their opposing roles in locomotion, and their involvement in ethanol's reinforcing and addictive properties prompted us to study the levels and distribution of CB1R after chronic ethanol intake, in the presence and absence of DRD2. METHODS: We monitored the drinking patterns and locomotor activity of Drd2+/+ and Drd2-/- mice consuming either water or a 20% (v/v) ethanol solution (forced ethanol intake) for 6 months and used the selective CB1 receptor antagonist [³H]SR141716A to quantify CB1R levels in different brain regions with in vitro receptor autoradiography. RESULTS: We found that the lack of DRD2 leads to a marked upregulation (approximately 2-fold increase) of CB1R in the cerebral cortex, the caudate-putamen, and the nucleus accumbens, which was reversed by chronic ethanol intake. CONCLUSIONS: The results suggest that DRD2-mediated dopaminergic neurotransmission and chronic ethanol intake exert an inhibitory effect on cannabinoid receptor expression in cortical and striatal regions implicated in the reinforcing and addictive properties of ethanol.