D2 Receptor Knockout Mice Research Articles

Increased Hippocampal CREB Phosphorylation in Dopamine D3 Receptor Knockout Mice Following Passive Avoidance Conditioning

Dopamine D3 receptors (D3Rs) are implicated in synaptic plasticity and memory processes. Previously we have shown that D3Rs mediate inhibitory effects on learning, since D3R knockout (D 3 (-/-) ) mice display enhanced performance in the passive avoidance task (PA). Formation of new memories is known to require de novo synthesis of proteins related to synaptic function through the activation of signaling pathways including the mitogen-activated protein kinases (MAPKs) and activation of the nuclear transcription factor cAMP response element binding protein (CREB). However, there are no clear indications regarding the specific involvement of D3Rs in the activation of these signaling cascades after acquisition of PA. Therefore, in this study we assessed whether phosphorylation levels of several MAPKs, Akt and CREB were differentially affected by PA in both wild-type (WT) and D 3 (-/-) mice hippocampi. Animals were divided in Naïve, unconditioned stimulus trained, conditioned stimulus trained and conditioned animals. Phosphorylation of extracellular signal-regulated kinase 1/2 (ERK 1/2), c-Jun-N-terminal kinase (JNK) and p38, as well as of Akt and CREB were determined. Acquisition of PA significantly increased pCREB levels both in WT and D 3 (-/-) mice. The extent of PA-driven increase in pCREB levels was significantly higher in mice lacking D3Rs. Similarly, pERK 1/2 was further augmented in trained D 3 (-/-) mice as compared to trained WTs, whereas JNK and p38 phosphorylation was not affected neither by PA nor by genetic background. Finally, Akt activation was observed in D 3 (-/-) mice, but not in response to PA. In conclusion, these data supports the notion that D3Rs might modulate CREB phosphorylation after acquisition of PA, probably via activation of ERK signaling.

The Dopamine D3 Receptor Knockout Mouse Mimics Aging-Related Changes in Autonomic Function and Cardiac Fibrosis

Blood pressure increases with age, and dysfunction of the dopamine D3 receptor has been implicated in the pathogenesis of hypertension. To evaluate the role of the D3 receptor in aging-related hypertension, we assessed cardiac structure and function in differently aged (2 mo, 1 yr, 2 yr) wild type (WT) and young (2 mo) D3 receptor knockout mice (D3KO). In WT, systolic and diastolic blood pressures and rate-pressure product (RPP) significantly increased with age, while heart rate significantly decreased. Blood pressure values, heart rate and RPP of young D3KO were significantly elevated over age-matched WT, but similar to those of the 2 yr old WT. Echocardiography revealed that the functional measurements of ejection fraction and fractional shortening decreased significantly with age in WT and that they were significantly smaller in D3KO compared to young WT. Despite this functional change however, cardiac morphology remained similar between the age-matched WT and D3KO. Additional morphometric analyses confirmed an aging-related increase in left ventricle (LV) and myocyte cross-sectional areas in WT, but found no difference between age-matched young WT and D3KO. In contrast, interstitial fibrosis, which increased with age in WT, was significantly elevated in the D3KO over age-matched WT, and similar to 2 yr old WT. Western analyses of myocardial homogenates revealed significantly increased levels of pro- and mature collagen type I in young D3KO. Column zymography revealed that activities of myocardial MMP-2 and MMP-9 increased with age in WTs, but in D3KO, only MMP-9 activity was significantly increased over age-matched WTs. Our data provide evidence that the dopamine D3 receptor has a critical role in the emergence of aging-related cardiac fibrosis, remodeling, and dysfunction.

Regulation of intraocular pressure in mice: Structural analysis of dopaminergic and serotonergic systems in response to cabergoline

Elevated intraocular pressure (IOP) is the main recognized risk factor of glaucoma. To investigate the contribution of dopaminergic and serotonergic systems in IOP regulation, we used cabergoline, a mixed dopamine and serotonin agonist, in C57BL/6J WT and dopamine D3 receptor knock-out (D3R−/−) mice with normal eye pressure or steroid-induced ocular hypertension. Furthermore, we studied the structural basis of the cabergoline-mediated activation of the dopaminergic and serotonergic systems by molecular modeling. Topical application of cabergoline, significantly decreased, in a dose-dependent manner, the intraocular pressure in WT mice, both in an ocular normotensive group (−9, −5 and −2mmHg with 5%, 1%, and 0.1%, respectively) and an ocular hypertensive group, with a prolonged effect in this latter group. No change of intraocular pressure was observed after topical application of cabergoline in D3R−/− mice. We modeled and optimized, with molecular dynamics, structures of hD3, h5HT1A and h5HT2A–C receptors; thereafter we carried out molecular docking of cabergoline. Docking revealed that binding of cabergoline into D3 and 5HT1A receptors is associated with a better desolvation energy in comparison to 5HT2A–C binding. In conclusion, the present study support the hypothesis that dopaminergic system is pivotal to regulate IOP and that D3R represents an intriguing target in the treatment of glaucoma. Furthermore, the structure-based computational approach adopted in this study is able to build and refine structure models of homologous dopaminergic and serotonergic receptors that may be of interest for structure-based drug discovery of ligands, with dopaminergic selectivity or with multi-pharmacological profile, potentially useful to treat optic neuropathies.

Intranasal and Subcutaneous Administration of Dopamine D3 Receptor Agonists Functionally Restores Nigrostriatal Dopamine in MPTP-Treated Mice

Parkinson's disease (PD) is a neurodegenerative disease with a hallmark motor defect caused by the death of dopaminergic neurons in the substantia nigra. Intranasal drug administration may be useful for Parkinson's treatment because this route avoids first-pass metabolism and increases bioavailability in the brain. In this study, we investigated the neuroprotection/neurorestoration effect of dopamine D3 receptor (D3R) agonists administered via both intranasal and subcutaneous routes in the MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine)-induced PD mouse model. Furthermore, we employed D3R knock-out mice to validate the dependence on D3R signaling. We found that in wild-type mice, but not D3 receptor knockout mice, both intranasal and subcutaneous administration of D3R agonists rescue dopamine (DA) depletion in the striatum as well as DA neuronal death in the substantia nigra after MPTP treatment. Moreover, subcutaneous 7-OH-DPAT administration significantly improved gait performance (stride length and overall running speed) of MPTP-lesioned mice after 7 and 14days of recovery. In addition, the distribution of D3 agonist 7-OH-DPAT was measured in designated brain areas by mass spectrometry analysis after subcutaneous and intranasal administration. Our data suggest that intranasal administration of D3R agonist would be a practical approach to treat PD.

Pharmacological and genetic evidence for pre- and postsynaptic D2 receptor involvement in motor responses to nociceptin/orphanin FQ receptor ligands

A combined pharmacological and genetic approach was undertaken to investigate the contribution of endogenous dopamine to the motor actions of nociceptin/orphanin FQ (N/OFQ) receptor (NOP receptor) ligands. Motor activity was evaluated by a battery of behavioural tests in mice. The involvement of the various DA receptor subtypes in the motor effects of N/OFQ and NOP receptor antagonists was evaluated pharmacologically, using D1/D5 (SCH23390), D2/D3 (raclopride, amisulpride) and D3 (S33084) receptor antagonists, and by using D2 receptor knockout mice. Low doses of N/OFQ and NOP receptor antagonists promoted movement whereas higher doses inhibited it. Motor facilitation was selectively prevented by raclopride while motor inhibition was prevented by amisulpride. Amisulpride also attenuated the hypolocomotion induced by the D2/D3 receptor agonist pramipexole and dopamine precursor l-3,4-dihydroxyphenylalanine, whereas raclopride (and S33084) worsened it. To dissect out the contribution of pre- and postsynaptic D2 receptors, mice lacking the D2 receptor (D2R−/−) or its long isoform (D2L−/−) were used. Motor facilitation induced by N/OFQ and NOP receptor antagonists was lost in D2R−/− and D2L−/− mice whereas motor inhibition induced by NOP receptor antagonists (and pramipexole) was lost in D2R−/− but preserved in D2L−/− mice. N/OFQ-induced hypolocomotion was observed in both genotypes. We demonstrate that motor actions of NOP receptor ligands rely on the modulation of endogenous dopamine. Motor facilitation induced by NOP receptor antagonists as well as low dose N/OFQ is mediated through D2L postsynaptic receptors whereas motor inhibition observed with higher doses of N/OFQ occurs by direct inhibition of mesencephalic DA neurons. Motor inhibition seen with high doses of NOP receptor antagonists appears to be mediated through the D2 presynaptic autoreceptors. These data confirm that endogenous N/OFQ is a powerful modulator of dopamine transmission in vivo and that the effects of NOP receptor antagonists on motor function reflect the blockade of this endogenous N/OFQ tone.

Dopamine D3 receptor deletion or blockade attenuates cocaine-induced conditioned place preference in mice

The dopamine (DA) D3 receptor (D3R) has received much attention in medication development for treatment of addiction. However, the functional role of the D3R in drug reward and addiction has been a matter of debate. We recently reported that D3 receptor-knockout (D3−/−) mice display increased vulnerability to cocaine self-administration, which we interpret as a compensatory response to attenuated cocaine reward after D3R deletion. Here we report that D3−/− mice displayed attenuated cocaine-induced conditioned place response (CPP) compared to wild-type mice. Similarly, blockade of brain D3Rs by YQA-14, a novel DA D3 receptor antagonist, significantly and dose-dependently inhibits acquisition and expression of cocaine-induced CPP in WT mice, but not in D3−/− mice. These findings suggest that: 1) D3Rs play an important role in mediating cocaine's rewarding effects; and 2) YQA-14 is a highly potent and selective D3R antagonist in vivo, which deserves further study as a candidate for treatment of cocaine addiction.

Dopamine D1 receptors are not critical for opiate reward but can mediate opiate memory retrieval in a state-dependent manner

Although D1 receptor knockout mice demonstrate normal morphine place preferences, antagonism of basolateral amygdala (BLA) D1 receptors only during drug-naive rat conditioning has been reported to inhibit the expression of a morphine place preference. One possible explanation for this result is state-dependent learning. That is, the omission of the intra-BLA infusion cue during testing — which acts as a potent discriminative stimulus — may have prevented the recall of a morphine–environment association and therefore, the consequent expression of a morphine place preference. To examine this possibility, we tested whether intra-BLA infusion of the D1-receptor antagonist SCH23390 during both training and testing might reveal a morphine place preference. Our results suggest that in previously drug-naive animals, D1 receptor antagonism during testing restores the opiate conditioned place preference that is normally absent when D1 receptors are blocked only during training, suggesting that BLA D1 receptors can mediate state-dependent memory retrieval.

Role of dopamine D2 receptors in plasticity of stress-induced addictive behaviours

Dopaminergic systems are implicated in stress-related behaviour. Here we investigate behavioural responses to chronic stress in dopamine D2 receptor knockout mice and find that anxiety-like behaviours are increased compared with wild-type mice. Repeated stress exposure suppresses cocaine-induced behavioural sensitization, cocaine-seeking and relapse behaviours in dopamine D2 receptor knockout mice. Cocaine challenge after drug withdrawal in cocaine-experienced wild-type or dopamine D2 receptor knockout mice is associated with inhibition of long-term depression in the nucleus accumbens, and chronic stress during withdrawal prevents inhibition after cocaine challenge in cocaine-experienced dopamine D2 receptor knockout mice, but not in wild-type mice. Lentiviral-induced knockdown of dopamine D2 receptors in the nucleus accumbens of wild-type mice does not affect basal locomotor activity, but confers stress-induced inhibition of the expression of cocaine-induced behavioural sensitization. Stressed mice depleted of dopamine D2 receptors do not manifest long-term depression inhibition. Our results suggest that dopamine D2 receptors have roles in regulating synaptic modification triggered by stress and drug addiction.

Effects of immobilization stress on emotional behaviors in dopamine D3 receptor knockout mice

A central problem in understanding the dopamine system in anxiety and depression is to specify functions of different members of the dopamine receptor family. Recent studies have reported that the dopamine D2/D3 receptor agonist pramipexole exerts an antidepressant-like effect in the chronic mild stress model and in the behavioral despair model, suggesting dopamine D3 receptor may be an important target for antidepressant actions. The aim of the present study was to examine the role of dopamine D3 receptor on the anxiety-like and depression-like behaviors induced by immobilization stress. We subjected D3 receptor knockout (D3KO) mice to a series of behavioral paradigms after acute (1h) or chronic (1h a day for 14 days) immobilization stress. The results showed that immobilization stress significantly altered the anxiety-like behaviors (open field test and elevated plus maze) and depression-like behaviors (tail suspension test) in both D3KO mice and their wild-type littermates. Moreover, further analysis of the data indicated that the D3KO mice, but not their littermates, failed to show a change in immobility time in the tail suspension test after the acute and chronic stress as compared to intact controls, suggesting an increased resistance to the immobilization stress given before behavioral tests. Although our study did not suggest a significant role of D3 receptor in regulating basal anxiety-like and depression-like behaviors, it demonstrated the mice lacking D3 receptor might be more resistant to stressful procedure than their WT littermates.

Hippocampal Neurofibromin and Amyloid Precursor Protein Expression in Dopamine D3 Receptor Knock-out Mice Following Passive Avoidance Conditioning

Passive avoidance (PA) conditioning is a fear motivated task able to initiate a cascade of altered gene expression within the hippocampus, a structure critical to learning and memory. We have previously shown that neurofibromin (NF1) and amyloid precursor protein (APP), two genes implicated in cognitive function, are differentially expressed in brain of dopamine D3 receptor knock-out mice (D(3)R(-/-)), suggesting that the receptor might have a role in their trascriptional regulation. Here in this study, we hypothesized that during acquisition of PA conditioning the expression of NF1 and APP genes could be influenced by D(3)Rs. To address this issue, we analyzed the expression of NF1 and APP in the hippocampus of both wild-type (WT) and D(3)R(-/-) mice subjected to the single trial step-through PA paradigm. Our finding demonstrated that (1) D(3)R(-/-) mice exhibit increased cognitive performance as compared to WT mice in the step-through PA trial; (2) acquisition of PA increased D(3)R and NF1, but not APP expression in WT mice hippocampus; (3) PA-driven NF1 induction in WT was abrogated in D(3)R(-/-) mice and finally that (4) the heightened basal APP expression observed in naive D(3)R(-/-) mice was totally reversed by acquisition of PA. In conclusion, the present finding show for the first time that both D(3)R and NF1 genes are upregulated following PA conditioning and suggest that hippocampal D(3)Rs might be relevant to NF1 transcriptional regulation in the hippocampus.

Pharmacological or genetic blockade of the dopamine D3 receptor increases cell proliferation in the hippocampus of adult mice

Dopamine plays an important role in cellular processes controlling the functional and structural plasticity of neurons, as well as their generation and proliferation, both in the developing and the adult brain. The precise roles of individual dopamine receptors subtypes in adult neurogenesis remain poorly defined, although D3 receptors are known to be involved in neurogenesis in the subventricular zone. By contrast, very few studies have addressed the influence of dopamine and D3 receptors upon neurogenesis in the subgranular zone of the hippocampus, an issue addressed herein employing constitutive D3 receptor knockout mice, or chronic exposure to the preferential D3 receptor antagonist, S33138. D3 receptor knockout mice revealed increased baseline levels of cell proliferation and ongoing neurogenesis, as measured both using Ki-67 and doublecortin, whereas there was no difference in cell survival as measured by BrdU (5-bromo-2'-deoxyuridine). Chronic administration of S33138 was shown to be functionally active in enhancing levels of the plasticity-related molecule, delta-FosB, in the D3 receptor-rich nucleus accumbens. In accordance with the stimulated neurogenesis seen in D3 receptor knockout mice, S33138 increased proliferation in wild-type mice. These observations suggest that D3 receptors exert a tonic, constitutive inhibitory influence upon adult hippocampal neurogenesis.

Cocaine self-administration in dopamine D₃ receptor knockout mice.

The dopamine D₃ receptor has received attention over the last two decades as a target for medications development for substance abuse disorders. Results have remained mixed. Despite emergence of more D₃-selective ligands, possible attribution of observed effects to D₂ receptors remains a concern. Knockout mice may help shed light on mechanisms. Here we evaluated the effect of constitutive D₃ receptor inactivation ("knockout") on the reinforcing effects of cocaine. We tested D₃ wild-type (WT), heterozygous (D₃⁺/⁻), and knockout (D₃⁻/⁻), mice in acquisition and maintenance of intravenous self-administration across a broad range of cocaine doses, using a fixed ratio (FR) 1 and a progressive ratio (PR) schedule of reinforcement, along with parallel food-reinforced studies. Generally, D₃⁻/⁻ mice showed cocaine self-administration comparable to WT controls across assays. Moderate and nonsignificant trends toward lesser reinforcing effects of a low cocaine dose (0.32 mg/kg) were apparent in acquisition and PR studies, consistent with the idea that the D₃ receptor may play a subtle role in the reinforcing effects of low cocaine doses under low FR conditions. However, those effects with cocaine self-administration were more subtle than the lower responding of D₃ knockout mice observed with food-maintained behavior. In addition, the D₃ antagonist PG01037 failed to affect cocaine self-administration under an FR 1 schedule in WT mice. The present data do not support a necessary role for the D₃ receptor in the direct reinforcing effects of cocaine.

L-DOPA-induced increase in TH-immunoreactive striatal neurons in parkinsonian mice: Insights into regulation and function

Tyrosine hydroxylase (TH)-immunoreactive (ir) neurons have been found in the striatum after dopamine depletion; however, little is known about the mechanism underlying their appearance or their functional significance. We previously showed an increase in striatal TH-ir neurons after l-DOPA treatment in mice with unilateral 6-OHDA lesions in the striatum. In the present study, we further examined the time-course and persistence of the effects of chronic l-DOPA treatment on the appearance and regulation of TH-ir neurons as well as their possible function. We found that the l-DOPA-induced increase in striatal TH-ir neurons is dose-dependent and persists for days after l-DOPA withdrawal, decreasing significantly 10days after l-DOPA treatment ends. Using hemiparkinsonian D1 receptor knock-out (D1R−/−) and D2 receptor knock-out (D2R−/−) mice, we found that the D1R, but not the D2R, is required for the l-DOPA-induced appearance of TH-ir neurons in the dopamine-depleted striatum. Interestingly, our experiments in aphakia mice, which lack Pitx3 expression in the brain, indicate that the l-DOPA-dependent increase in the number of TH-ir neurons is independent of Pitx3, a transcription factor necessary for the development of mesencephalic dopaminergic neurons. To explore the possible function of l-DOPA-induced TH-ir neurons in the striatum, we examined dopamine overflow and forelimb use in l-DOPA-treated parkinsonian mice. These studies revealed a tight spatio-temporal correlation between the presence of striatal TH-ir neurons, the recovery of electrically stimulated dopamine overflow in the lesioned striatum, and the recovery of contralateral forelimb use with chronic l-DOPA treatment. Our results suggest that the presence of TH-ir neurons in the striatum may underlie the long-duration response to l-DOPA following withdrawal. Promotion of these neurons in the early stages of Parkinson's disease, when dopamine denervation is incomplete, may be beneficial for maintaining motor function.

The dopamine D1 but not D3 receptor plays a fundamental role in spatial working memory and BDNF expression in prefrontal cortex of mice

Although dopamine within the prefrontal cortex has been implicated in working memory, how different dopamine receptor subtypes contribute to this process need to be further characterized. Previous studies have suggest the importance of dopamine receptors signaling in regulating the brain-derived neurotrophic factor (BDNF) function that is associated with synaptic plasticity underlying normal memory formation. Changes in BDNF expression through the dopamine receptors within the prefrontal cortex may accompany and mediate the spatial working memory. To test the possibility, dopamine D1 and D3 receptor mutant mice were tested in Morris water maze for spatial working memory. We found that trial-dependent, matching-to-sample, learning of the platform location, an index of short-term spatial working memory in mice, was significantly impaired in D1 receptor knockout mice compared to wild-type mice, and regular performance of D3 receptor mutants was observed in the similar working memory task. BDNF protein was significantly decreased in prefrontal cortex, though not in hippocampus, of the D1 receptor knockout mice, whereas no changes were found in both prefrontal cortex and hippocampus of D3 receptor knockout mice. These data suggest that dopamine D1 but not D3 receptors are critical for prefrontal cortex BDNF expression which may be related to spatial working memory processes.

The dopamine D3 receptor knockout mouse models aging‐related changes in hypertension and cardiac fibrosis

The increased prevalence of hypertension with aging is well established, as is a role for the dopamine D3 receptor in the general pathophysiology of hypertension. To determine the specific effects of D3 dysfunction on autonomic output and cardiac involvement in aging associated hypertension, non‐invasive blood pressure measurements, echocardiography, and histochemistry were used to compare cardiac structure and function in differently‐aged wild‐type (WT) animals (10 weeks, 1 year, 2 year) and D3 receptor knockout mice (D3KO). In WT animals, aging‐related increases in blood pressure and cardiac function, accompanied by bradycardia in the oldest animals were observed. Intriguingly, 2 month‐old D3KO mice displayed blood pressure and heart rate values that were significantly increased over their age‐matched WT controls, and that were comparable to those of the 2 year‐old WT group. Echocardiography did not reveal significant differences between age matched young WT and D3KO mice, but immunohistology demonstrated fibrosis in the young D3KO mice that was comparable to the age‐related fibrosis seen in 2 year old WT mice. Taken together, these data suggest that the D3KO animal may serve as a model to better understand the role of the dopamine D3 receptor in the aging heart, and in particular its role in the pathophysiology of cardiovascular fibrosis. Funded by Brody School of Medicine (SC), and NIH NHLBI HL081720 (DAT).

Role of dopamine D3 receptors in basal nociception regulation and in morphine-induced tolerance and withdrawal

Repeated administration of opioids such as morphine leads to the development of tolerance to their pain-relieving effects as well as to physical dependence. Although the association between the dopamine system and the molecular mechanisms of morphine-induced antinociceptive tolerance has been studied, the possible interaction between morphine-induced tolerance and D3 receptors has not been investigated. In the present study, male mice lacking the dopamine D3 receptor gene were used to investigate the function of D3 receptors in the development of morphine-induced tolerance and withdrawal. Compared with wild-type (WT) mice, the dopamine D3 receptor knockout (D3R KO) mice showed pronounced hypoalgesia. The D3R KO mice clearly developed lower morphine-induced tolerance and showed attenuated withdrawal signs compared with the WT mice. These results suggest that D3 receptors regulate basal nociception and are involved in the development of morphine-induced tolerance and withdrawal.

Analyses of motor activity and striatal GABAergic synaptic transmission in dopamine D1 and D2 receptor knock-out mice

Analyses of motor activity and striatal GABAergic synaptic transmission in dopamine D1 and D2 receptor knock-out mice

YQA14: a novel dopamine D3 receptor antagonist that inhibits cocaine self‐administration in rats and mice, but not in D3 receptor‐knockout mice

The dopamine (DA) D3 receptor is posited to be importantly involved in drug reward and addiction, and D3 receptor antagonists have shown extraordinary promise as potential anti-addiction pharmacotherapeutic agents in animal models of drug addiction. SB-277011A is the best characterized D3 receptor antagonist in such models. However, the potential use of SB-277011A in humans is precluded by pharmacokinetic and toxicity problems. We here report a novel D3 receptor antagonist YQA14 that shows similar pharmacological properties as SB-277011A. In vitro receptor binding assays suggest that YQA14 has two binding sites on human cloned D3 receptors with K(i-High) (0.68 × 10(-4) nM) and K(i-Low) (2.11 nM), and displays > 150-fold selectivity for D3 over D2 receptors and > 1000-fold selectivity for D3 over other DA receptors. Systemic administration of YQA14 (6.25-25 mg/kg) or SB-277011A (12.5-25 mg/kg) significantly and dose-dependently reduced intravenous cocaine self-administration under both low fixed-ratio and progressive-ratio reinforcement conditions in rats, while failing to alter oral sucrose self-administration and locomotor activity, suggesting a selective inhibition of drug reward. However, when the drug dose was increased to 50 mg/kg, YQA14 and SB-277011A significantly inhibited basal and cocaine-enhanced locomotion in rats. Finally, both D3 antagonists dose-dependently inhibited intravenous cocaine self-administration in wild-type mice, but not in D3 receptor-knockout mice, suggesting that their action is mediated by D3 receptor blockade. These findings suggest that YQA14 has a similar anti-addiction profile as SB-277011A, and deserves further study and development.

Dopamine D2-receptor knockout mice are protected against dopaminergic neurotoxicity induced by methamphetamine or MDMA

Methamphetamine (METH) and 3,4-methylenedioxymethamphetamine (MDMA), amphetamine derivatives widely used as recreational drugs, induce similar neurotoxic effects in mice, including a marked loss of tyrosine hydroxylase (TH) and dopamine transporter (DAT) in the striatum. Although the role of dopamine in these neurotoxic effects is well established and pharmacological studies suggest involvement of a dopamine D2-like receptor, the specific dopamine receptor subtype involved has not been determined. In this study, we used dopamine D2 receptor knock-out mice (D2R−/−) to determine whether D2R is involved in METH- and MDMA-induced hyperthermia and neurotoxicity. In wild type animals, both drugs induced marked hyperthermia, decreased striatal dopamine content and TH- and DAT-immunoreactivity and increased striatal GFAP and Mac-1 expression as well as iNOS and interleukin 15 at 1 and 7days after drug exposure. They also caused dopaminergic cell loss in the SNpc. Inactivation of D2R blocked all these effects. Remarkably, D2R inactivation prevented METH-induced loss of dopaminergic neurons in the SNpc. In addition, striatal dopamine overflow, measured by fast scan cyclic voltammetry in the presence of METH, was significantly reduced in D2R−/− mice. Pre-treatment with reserpine indicated that the neuroprotective effect of D2R inactivation cannot be explained solely by its ability to prevent METH-induced hyperthermia: reserpine lowered body temperature in both genotypes, and potentiated METH toxicity in WT, but not D2R−/− mice. Our results demonstrate that the D2R is necessary for METH and MDMA neurotoxicity and that the neuroprotective effect of D2R inactivation is independent of its effect on body temperature.

Operant Sensation Seeking in the Mouse

Operant methods are powerful behavioral tools for the study of motivated behavior. These 'self-administration' methods have been used extensively in drug addiction research due to their high construct validity. Operant studies provide researchers a tool for preclinical investigation of several aspects of the addiction process. For example, mechanisms of acute reinforcement (both drug and non-drug) can be tested using pharmacological or genetic tools to determine the ability of a molecular target to influence self-administration behavior. Additionally, drug or food seeking behaviors can be studied in the absence of the primary reinforcer, and the ability of pharmacological compounds to disrupt this process is a preclinical model for discovery of molecular targets and compounds that may be useful for the treatment of addiction. One problem with performing intravenous drug self-administration studies in the mouse is the technical difficulty of maintaining catheter patency. Attrition rates in these experiments are high and can reach 40% or higher. Another general problem with drug self-administration is discerning which pharmacologically-induced effects of the reinforcer produce specific behaviors. For example, measurement of the reinforcing and neurological effects of psychostimulants can be confounded by their psychomotor effects. Operant methods using food reinforcement can avoid these pitfalls, although their utility in studying drug addiction is limited by the fact that some manipulations that alter drug self-administration have a minimal impact on food self-administration. For example, mesolimbic dopamine lesion or knockout of the D1 dopamine receptor reduce cocaine self-administration without having a significant impact on food self-administration. Sensory stimuli have been described for their ability to support operant responding as primary reinforcers (i.e. not conditioned reinforcers). Auditory and visual stimuli are self-administered by several species, although surprisingly little is known about the neural mechanisms underlying this reinforcement. The operant sensation seeking (OSS) model is a robust model for obtaining sensory self-administration in the mouse, allowing the study of neural mechanisms important in sensory reinforcement. An additional advantage of OSS is the ability to screen mutant mice for differences in operant behavior that may be relevant to addiction. We have reported that dopamine D1 receptor knockout mice, previously shown to be deficient in psychostimulant self-administration, also fail to acquire OSS. This is a unique finding in that these mice are capable of learning an operant task when food is used as a reinforcer. While operant studies using food reinforcement can be useful in the study of general motivated behavior and the mechanisms underlying food reinforcement, as mentioned above, these studies are limited in their application to studying molecular mechanisms of drug addiction. Thus, there may be similar neural substrates mediating sensory and psychostimulant reinforcement that are distinct from food reinforcement, which would make OSS a particularly attractive model for the study of drug addiction processes. The degree of overlap between other molecular targets of OSS and drug reinforcers is unclear, but is a topic that we are currently pursuing. While some aspects of addiction such as resistance to extinction may be observed with OSS, we have found that escalation is not observed in this model. Interestingly, escalation of intake and some other aspects of addiction are observed with self-administration of sucrose. Thus, when non-drug operant procedures are desired to study addiction-related processes, food or sensory reinforcers can be chosen to best fit the particular question being asked. In conclusion, both food self-administration and OSS in the mouse have the advantage of not requiring an intravenous catheter, which allows a higher throughput means to study the effects of pharmacological or genetic manipulation of neural targets involved in motivation. While operant testing using food as a reinforcer is particularly useful in the study of the regulation of food intake, OSS is particularly apt for studying reinforcement mechanisms of sensory stimuli and may have broad applicability to novelty seeking and addiction.