The novel adenosine A2A antagonist Lu AA47070 reverses the motor and motivational effects produced by dopamine D2 receptor blockade.

Dopamine D2 and adenosine A(2A) receptors interact to regulate aspects of motor and motivational function, and it has been suggested that adenosine A(2A) antagonists could be useful for the treatment of parkinsonism and depression. The present experiments were performed to characterize the effects of Lu AA47070, which is a phosphonooxymethylene prodrug of a potent and selective adenosine A(2A) receptor antagonist, for its ability to reverse the motor and motivational effects of D2 antagonism. In the first group of studies, Lu AA47070 (3.75-30 mg/kg IP) was assessed for its ability to reverse the effects of the D2 receptor antagonist pimozide (1.0 mg/kg IP) using several measures of motor impairment, including catalepsy, locomotion, and tremulous jaw movements, which is a rodent model of parkinsonian tremor. Lu AA47070 produced a significant reversal of the effects of pimozide on all three measures of parkinsonian motor impairment. In addition, Lu AA47070 was able to reverse the effects of a low dose of the D2 antagonist haloperidol on a concurrent lever pressing/chow feeding task that is used as a measure of effort-related choice behavior. The ability of Lu AA47070 to reverse the effects of D2 receptor blockade suggests that this compound could have potential utility as a treatment for parkinsonism, and for some of the motivational symptoms of depression.

Involvement of a subpopulation of neuronal M4 muscarinic acetylcholine receptors in the antipsychotic-like effects of the M1/M4 preferring muscarinic receptor agonist xanomeline.

Disturbances in central dopaminergic neurotransmission are believed to be centrally involved in the pathogenesis of schizophrenia. Central dopaminergic and cholinergic systems interact and the cholinergic muscarinic agonist xanomeline has shown antipsychotic effects in clinical studies. Preclinical studies indicate that the M(4) muscarinic cholinergic receptor subtype (mAChR) modulates the activity of the dopaminergic system and that this specific mAChR subtype is involved in mediating the antipsychotic-like effects of xanomeline. A specific neuronal subpopulation that expresses M(4) mAChRs together with D(1) dopamine receptors seems to be especially important in modulating dopamine-dependent behaviors. Using mutant mice that lack the M(4) mAChR only in D(1) dopamine receptor-expressing cells (D1-M4-KO), we investigated the role of this neuronal population in the antipsychotic-like effects of xanomeline in amphetamine-induced hyperactivity and apomorphine-induced climbing. Interestingly, the antipsychotic-like effects of xanomeline in the two models were almost completely abolished in D1-M4-KO mice, suggesting that M(4) mAChRs colocalized with D(1) dopamine receptors are centrally involved in mediating the antipsychotic-like effects of xanomeline. This is consistent with the hypothesis that activation of the M(4) mAChR represents a potential target for the future medical treatment of psychosis.

Stimulant effects of adenosine antagonists on operant behavior: differential actions of selective A2A and A1 antagonists.

RATIONALE: Adenosine A(2A) antagonists can reverse many of the behavioral effects of dopamine antagonists, including actions on instrumental behavior. However, little is known about the effects of selective adenosine antagonists on operant behavior when these drugs are administered alone. OBJECTIVE: The present studies were undertaken to investigate the potential for rate-dependent stimulant effects of both selective and nonselective adenosine antagonists. METHODS: Six drugs were tested: two nonselective adenosine antagonists (caffeine and theophylline), two adenosine A(1) antagonists (DPCPX and CPT), and two adenosine A(2A) antagonists (istradefylline (KW6002) and MSX-3). Two schedules of reinforcement were employed; a fixed interval 240-s (FI-240 sec) schedule was used to generate low baseline rates of responding and a fixed ratio 20 (FR20) schedule generated high rates. RESULTS: Caffeine and theophylline produced rate-dependent effects on lever pressing, increasing responding on the FI-240 sec schedule but decreasing responding on the FR20 schedule. The A(2A) antagonists MSX-3 and istradefylline increased FI-240 sec lever pressing but did not suppress FR20 lever pressing in the dose range tested. In fact, there was a tendency for istradefylline to increase FR20 responding at a moderate dose. A(1) antagonists failed to increase lever pressing rate, but DPCPX decreased FR20 responding at higher doses. CONCLUSIONS: These results suggest that adenosine A(2A) antagonists enhance operant response rates, but A(1) antagonists do not. The involvement of adenosine A(2A) receptors in regulating aspects of instrumental response output and behavioral activation may have implications for the treatment of effort-related psychiatric dysfunctions, such as psychomotor slowing and anergia in depression.

Tumor necrosis factor α primes cerebral endothelial cells for erythropoietin-induced angiogenesis.

Erythropoietin (EPO) enhances angiogenesis in the ischemic brain. Stroke induces secretion of tumor necrosis factor α (TNF-α). We investigated the effect of TNF-α on EPO-induced in vitro angiogenesis in cerebral endothelial cells. Using a capillary-like tubular formation assay, we found that transient incubation of primary rat cerebral microvascular endothelial cells (RECs) with TNF-α substantially upregulated EPO receptor (EPOR) expression and addition of EPO into TNF-α-treated RECs significantly augmented the capillary-like tube formation. Blockage of TNF receptor 1 (TNFR1) suppressed TNF-α-upregulated EPOR expression and abolished EPO-induced tube formation. Attenuation of endogenous EPOR with small interfering RNA (siRNA) also inhibited EPO-enhanced tube formation. Treatment of RECs with EPO activated nuclear factor-kappa B (NF-κB) and Akt. Incubation of the TNF-α-treated endothelial cells with EPO activated vascular endothelial growth factor (VEGF), VEGF receptor 2 (VEGFR2), angiopoietin 1 (Ang1), and Tie2. Blockage of VEGFR2 and Tie2 resulted in reduction of EPO-augmented tube formation. These data indicate that interaction of TNF-α with TNFR1 sensitizes cerebral endothelial cells for EPO-induced angiogenesis by upregulation of EPOR, which amplifies the effect of EPO on activation of the VEGF/VEGFR2 and Ang1/Tie2 pathways. Our results provide the evidence for crosslink between TNF and EPOR to coordinate the onset of angiogenesis in cerebral endothelial cells.

Effects of posttraumatic carbamylated erythropoietin therapy on reducing lesion volume and hippocampal cell loss, enhancing angiogenesis and neurogenesis, and improving functional outcome in rats following traumatic brain injury.

OBJECT: Carbamylated erythropoietin (CEPO) is a modified erythropoietin molecule that does not affect hematocrit. In this study, the authors compared the efficacy of a single dose with a triple dose of CEPO treatment for traumatic brain injury (TBI) in rats. METHODS: Traumatic brain injury was induced by controlled cortical impact over the left parietal cortex. Carbamylated erythropoietin (50 µg/kg) was administered intraperitoneally in rats with TBI at 6 hours (CEPO × 1) or at 6, 24, and 48 hours (CEPO × 3) postinjury. Neurological function was assessed using a modified neurological severity score and foot fault and Morris water maze tests. Animals were killed 35 days after injury, and brain sections were stained for immunohistochemical analysis to assess lesion volume, cell loss, cell proliferation, angiogenesis, and neurogenesis after CEPO treatment. RESULTS: Compared with the vehicle treatment, single treatment of CEPO (6 hours) significantly reduced lesion volume and hippocampal cell loss, enhanced angiogenesis and neurogenesis in the injured cortex and hippocampus, and significantly improved sensorimotor functional recovery and spatial learning in rats after TBI. Importantly, triple dosing of CEPO (6, 24, and 48 hours) further reduced lesion volume and improved functional recovery and neurogenesis compared with the CEPO × 1 group. CONCLUSIONS: The authors' results indicate that CEPO has considerable therapeutic potential in TBI and related pathologies and furthermore that repeated dosing in the subacute phase might have important pharmacological relevance.

A subpopulation of neuronal M4 muscarinic acetylcholine receptors plays a critical role in modulating dopamine-dependent behaviors.

Acetylcholine (ACh) regulates many key functions of the CNS by activating cell surface receptors referred to as muscarinic ACh receptors (M(1)-M(5) mAChRs). Like other mAChR subtypes, the M(4) mAChR is widely expressed in different regions of the forebrain. Interestingly, M(4) mAChRs are coexpressed with D(1) dopamine receptors in a specific subset of striatal projection neurons. To investigate the physiological relevance of this M(4) mAChR subpopulation in modulating dopamine-dependent behaviors, we used Cre/loxP technology to generate mutant mice that lack M(4) mAChRs only in D(1) dopamine receptor-expressing cells. The newly generated mutant mice displayed several striking behavioral phenotypes, including enhanced hyperlocomotor activity and increased behavioral sensitization following treatment with psychostimulants. These behavioral changes were accompanied by a lack of muscarinic inhibition of D(1) dopamine receptor-mediated cAMP stimulation in the striatum and an increase in dopamine efflux in the nucleus accumbens. These novel findings demonstrate that a distinct subpopulation of neuronal M(4) mAChRs plays a critical role in modulating several important dopamine-dependent behaviors. Since enhanced central dopaminergic neurotransmission is a hallmark of several severe disorders of the CNS, including schizophrenia and drug addiction, our findings have substantial clinical relevance.

Oral tremor induced by the muscarinic agonist pilocarpine is suppressed by the adenosine A2A antagonists MSX-3 and SCH58261, but not the adenosine A1 antagonist DPCPX.

Tremulous jaw movements in rats, which can be induced by dopamine (DA) antagonists, DA depletion, and cholinomimetics, have served as a useful model for studies of tremor. Although adenosine A(2A) antagonists can reduce the tremulous jaw movements induced by DA antagonists and DA depletion, there are conflicting reports about the interaction between adenosine antagonists and cholinomimetic drugs. The present studies investigated the ability of adenosine antagonists to reverse the tremorogenic effect of the muscarinic agonist pilocarpine. While the adenosine A(2A) antagonist MSX-3 was incapable of reversing the tremulous jaw movements induced by the 4.0mg/kg dose of pilocarpine, both MSX-3 and the adenosine A(2A) antagonist SCH58261 reversed the tremulous jaw movements elicited by 0.5mg/kg pilocarpine. Systemic administration of the adenosine A(1) antagonist DPCPX failed to reverse the tremulous jaw movements induced by either an acute 0.5mg/kg dose of the cholinomimetic pilocarpine or the DA D2 antagonist pimozide, indicating that the tremorolytic effects of adenosine antagonists may be receptor subtype specific. Behaviorally active doses of MSX-3 and SCH 58261 showed substantial in vivo occupancy of A(2A) receptors, but DPCPX did not. The results of these studies support the use of adenosine A(2A) antagonists for the treatment of tremor.

Differential actions of adenosine A1 and A2A antagonists on the effort-related effects of dopamine D2 antagonism.

Adenosine and dopamine receptors in striatal areas interact to regulate a number of different functions, including aspects of motor control and motivation. Recent studies indicate that adenosine A(2A) receptor antagonists can reverse the effects of dopamine (DA) D(2) antagonists on instrumental tasks that provide measures of effort-related choice behavior. The present experiments compared the ability of the adenosine A(2A) antagonist KW6002, the nonselective adenosine antagonist caffeine, and the adenosine A(1) receptor selective antagonist DPCPX, to reverse the behavioral effects of the DA D(2) antagonist haloperidol. For these studies, a concurrent choice procedure was used in which rats could select between lever pressing on a fixed ratio 5 schedule for a preferred food or approaching and consuming a less preferred lab chow that was concurrently available in the chamber. Under baseline or control conditions, rats show a strong preference for lever pressing, and eat little of the chow; IP injections of 0.1 mg/kg haloperidol significantly reduced lever pressing and substantially increased chow intake. The adenosine A(2A) antagonist KW6002 (0.125-0.5 mg/kg IP) and the nonselective adenosine antagonist caffeine (5.0-20.0 mg/kg) significantly reversed the effects of haloperidol. However, the adenosine A(1) antagonist DPCPX (0.1875-0.75 mg/kg IP) failed to reverse the effects of the D(2) antagonist. The rank order of effect sizes in the reversal experiments was KW6002>caffeine>DPCPX. None of these drugs had any effect on behavior when they were injected in the absence of haloperidol. These results indicate that the ability of an adenosine antagonist to reverse the effort-related effects of a D(2) antagonist depends upon the subtype of adenosine receptor being blocked. Together with other recent results, these experiments indicate that there is a specific interaction between DA D(2) and adenosine A(2A) receptors, which could be related to the co-localization of these receptors on the same population of striatal neurons.

Striatal extracellular dopamine levels and behavioural reversal in MPTP-lesioned mice.

Intoxication induced by MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) in mice results in a significant loss of nigrostriatal dopamine (DA) neurons. This is accompanied by a change in behavioural phenotype that can be reversed by L-DOPA (3,4-dihydroxy-L-phenylalanine) treatment. Here, we examined the extracellular levels of DA, behavioural deficits and the response to L-DOPA treatment in severely intoxicated mice. The MPTP intoxication produced more than a 90% reduction in tissue DA and a 65% decline in extracellular DA levels. In-vivo binding did not show any increased raclopride binding to the D2 receptor. Administration of L-DOPA, 5 or 20 mg/kg (subcutaneously), significantly increased dialysate DA levels and both doses of L-DOPA reversed the behavioural deficit. Interestingly, only 5 mg/kg L-DOPA normalized DA levels to 56% of controls showing that only a minor increase in DA levels is sufficient to yield motor recovery.

Therapeutic window for nonerythropoietic carbamylated-erythropoietin to improve motor function following multiple infarct ischemic strokes in New Zealand white rabbits.

Carbamylated erythropoietin (CEPO) is a novel neuroprotective agent that does not bind to the classical erythropoietin receptor or affect hematocrit. Since CEPO has not been systematically studied in a fully blinded and randomized manner in an embolic stroke model, we determined if CEPO would be useful to attenuate clinical deficits associated with multiple infarct ischemia using the rabbit small clot embolic stroke model (RSCEM). Rabbits were embolized and treated with vehicle or CEPO within 6 h of embolization and behavioral analysis was conducted 48 h after embolization. Using quantal analysis, we determined the quantity of blood clot (mg) in brain that produce neurologic dysfunction in 50% of the rabbits (P(50)), with intervention considered beneficial if it increased the P(50) compared to controls. CEPO administered between 5 min and 3 h after embolization significantly (p<0.05) improved behavioral function and increased the P(50) value by 55-216%. However, CEPO administration did not improve behavior when administered 6 h following embolization. In conclusion, in the RSCEM, CEPO had a therapeutic window of at least 3 h, where it effectively improved clinical rating scores and motor function. Our results suggest that CEPO may be useful to treat acute ischemic stroke and supports the study of CEPO in stroke patients.

Effects of acute and chronic aripiprazole treatment on choice between cocaine self-administration and food under a concurrent schedule of reinforcement in rats.

RATIONALE: Dopamine D2-like partial agonists such as aripiprazole have received some attention as potential pharmacotherapies for the treatment of psychostimulant addiction. However, the preclinical evaluations so far have focused on acute effects of aripiprazole. OBJECTIVES: We tested the hypothesis that aripiprazole, both as acute and as chronic treatment, would preferentially decrease cocaine self-administration while sparing behavior maintained by a natural reinforcer, resulting in a shift in the allocation of behavior from cocaine-taking towards the alternative reinforcer. MATERIALS AND METHODS: Rats were trained to self-administer intravenous cocaine in a concurrent choice procedure, with a palatable food as the competing reinforcer, under a fixed ratio (FR) 1 FR 5 chain schedule. Aripiprazole was then administered as continuous infusion by osmotic minipumps for 5 days, during which performance in the choice procedure was assessed daily. RESULTS: An intermediate dose of aripiprazole decreased cocaine self-administration and shifted the cocaine choice curve to the right as an acute treatment. However, as a chronic treatment, aripiprazole failed to decrease cocaine self-administration or cocaine choice, despite a dose-dependent decrease in overall response rates and food-maintained behavior. CONCLUSIONS: Our results confirm and extend earlier findings and indicate that acute administration of aripiprazole can decrease cocaine self-administration. However, based on the present data, chronic treatment with aripiprazole does not show much promise as a potential pharmacotherapy for cocaine addiction. Both acute and chronic treatment data are in agreement with published clinical findings, suggesting that the concurrent choice procedure in rats has predictive validity of efficacy in humans.

Tremorolytic effects of adenosine A2A antagonists: implications for parkinsonism.

Drug-induced tremulous jaw movements in rats have been used as a model of parkinsonian tremor. Because adenosine A2A antagonists have antiparkinsonian effects, the present experiments were conducted to study the ability of adenosine A2A antagonism to reverse the tremulous jaw movements produced by the antipsychotic drugs pimozide, haloperidol and reserpine. In one group of studies, rats received daily injections of the dopamine antagonist pimozide, and on day 8 they received injections of pimozide plus various doses of the A2A antagonists KW 6002 or MSX-3. KW 6002 and MSX-3 suppressed pimozide-induced tremulous jaw movements, reduced catalepsy, and increased locomotion. MSX-3 also suppressed the jaw movements induced by haloperidol and reserpine. In addition, local injections of MSX-3 into the ventrolateral neostriatum suppressed pimozide-induced tremulous jaw movements. Thus, adenosine A2A antagonism can reverse the tremulous movements induced by antipsychotic drugs, which is consistent with the hypothesis that antagonism of adenosine A2A receptors can result in antiparkinsonian effects. Adenosine A2A antagonists may be useful for their tremorolytic effects, and may help in treating both idiopathic and antipsychotic-induced parkinsonian symptoms.

Aripiprazole blocks acute self-administration of cocaine and is not self-administered in mice.

RATIONALE: The novel antipsychotic aripiprazole in use for treatment of schizophrenia is a partial agonist at dopamine D2 receptors with actions at a variety of other receptors as well. Cocaine is believed to exert an important part of its rewarding effect by increasing extracellular levels of dopamine that subsequently act at dopamine D2 receptors. OBJECTIVES: As a partial agonist, aripiprazole may antagonize effects at D2 receptors and we accordingly tested whether aripiprazole could antagonize self-administration of cocaine. Because D2-like receptor agonists are self-administered, a D2 receptor partial agonist like aripiprazole might itself be reinforcing. Thus, we also assessed whether mice would acquire self-administration of aripiprazole. MATERIALS AND METHODS: A single session, mouse self-administration procedure was used. RESULTS: Oral pretreatment with aripiprazole dose-dependently decreased cocaine self-administration under a fixed ratio 1 schedule at the peak cocaine dose (0.03 mg/kg/infusion), reaching significance at 0.2 and 0.4 mg/kg of aripiprazole. Using 0.4 mg/kg, aripiprazole decreased rates of cocaine self-administration without shifting the peak of the dose-response function. There was no effect of aripiprazole per se, suggesting that its inhibitory action was due to effects on cocaine self-administration rather than non-specific motor effects. Aripiprazole was not found to be self-administered in the tested dose range (0.0003-0.3 mg/kg/infusion). The three highest doses (0.03, 0.1, and 0.3 mg/kg/infusion) even caused significant decreases in nose-poking activity, possibly due to extrapyramidal side effects. CONCLUSIONS: These data are consistent with a potential role for aripiprazole in treatment of cocaine addiction without abuse potential per se.

Effects of postnatal anoxia on striatal dopamine metabolism and prepulse inhibition in rats.

Various evidence indicate that schizophrenia is a neurodevelopmental disorder. Epidemiological observations point to oxygen deficiencies during delivery as one of the early risk factors for developing schizophrenia. The aim of the present study was to examine the effect of postnatal anoxia in rats. Anoxia was experimentally induced by placing 9-day-old rat pups for 6 min in a chamber saturated with 100% nitrogen (N(2)). Exposure to anoxia on postnatal day (PND) 9 resulted in significantly reduced subcortical dopamine metabolism and turnover, as measured by striatal 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) concentrations. Furthermore, in the anoxic group only, striatal HVA concentrations were negatively correlated to prefrontal cortical N-acetylaspartate (NAA) levels. Similar findings of distorted prefrontal-subcortical interactions have recently been reported in schizophrenic patients. There was no effect of postnatal anoxia on either baseline or d-amphetamine-induced deficit in the prepulse inhibition (PPI) paradigm in adulthood. Accordingly, although oxygen deficiency early in life has been discussed as vulnerability factor in developing schizophrenia, exposure to postnatal anoxia in the rat does not show clear-cut phenomenological similarities with the disorder.