Advancing 3Rs: The Mouse Estrus Detector (MED) as a Low-Stress, Painless, and Efficient Tool for Estrus Determination in Mice.

Determining the estrous cycle stages in mice is essential for optimizing breeding strategies, synchronizing experimental timelines, and facilitating studies in behavior, drug testing, and genetics. It is critical for reducing the production of genetically unmodified offspring in the generation and investigation of genetically modified animal models. An accurate detection of the estrus cycle is particularly relevant in the context of the 3Rs-Replacement, Reduction, and Refinement. The estrous cycle, encompassing the reproductive phases of mice, is key to refining experimental designs and addressing ethical issues related to the use of animals in research. This study presents results from two independent laboratories on the efficacy of the Mouse Estrus Detector (MED) from ELMI Ltd. (Latvia) for the accurate determination of the estrus phase. The female mice of five strains/stocks (CD1, FVB/N, C57Bl6/J, B6D2F1, and Swiss) were used. The results showed that the MEDProTM is a low-traumatic, simple, rapid, and painless method of estrus detection that supports the principles of the 3Rs. The use of the MEDProTM for estrus detection in mice caused minimal stress, enhanced mating efficiency, facilitated an increase in the number of embryos for in vitro fertilization, and allowed the production of the desired number of foster animals.

The Action of TAAR1 Agonist RO5263397 on Executive Functions in Rats.

Trace amine-associated receptor 1 (TAAR1) is a widely recognized new perspective target for the neuropsychiatric pharmacological treatment. Despite a growing number of studies investigating TAAR1 role in the animal models of different pathologies, information of TAAR1 agonists impact on executive cognitive functions is limited. The goal of the present study was to evaluate the activity of highly selective partial TAAR1 agonist RO5263397 on various executive cognitive functions. The results of the present study demonstrated that the pretreatment with RO5263397 was able to increase attention and decrease cognitive flexibility in rats. The analysis of the RO5263397 action on impulsivity demonstrated that the TAAR1 activation failed to affect premature responding but was able to slightly modify impulsive choice. Problem solving was resistant to the pharmacological intervention.

Hyperdopaminergia in rats is associated with reverse effort-cost dependent performance.

BACKGROUND: Dopamine is implicated in the effort-based control of motivational processes; however, whether tonic dopamine regulates the effort-cost impact on motivation, is still debated. AIMS: The rats lacking the dopamine transporter (DAT), which have dramatically increased levels of the synaptic dopamine, were used in the present study to elucidate the role of the synaptic dopamine in motivational processes. METHODS: To study the reward-related processes, the progressive ratio 3 (PR3) operant schedule of food reinforcement (the ratio increases by 3 after each earned reinforcer) was performed in adult male rats (DAT knockouts (DAT-KO), heterozygotes (DAT-HT) and wild-types (DAT-WT)). RESULTS: During the PR3 session, the response rate of DAT-KO rats was gradually increased following the augmented required number of responses. In contrast, the local response rate of DAT-WT and DAT-HT decreased. d-Amphetamine sulfate salt (3 mg/kg, i.p.) altered the local response rate dynamics in DAT-WT, which became similar to that of DAT-KO. Interestingly, the reduction in response rate at low effort demands was associated with decreased rate of entries into the magazine tray in DAT-WT rats treated with amphetamine (3 mg/kg) but not in DAT-KO rats. CONCLUSIONS: Our results suggest that the elevated tonic synaptic dopamine can strongly affect motivation/effort-cost relation in rodents.

C(21)-fluorinated thevinol scaffold for opioid ligands. 21,21,21-Trifluoro-6-O-nororvinols: Design, synthesis and analgesic activity.

Thevinols and their 3-O-demethylated relatives, orvinols, are derivatives of the Diels-Alder adduct of natural alkaloid thebaine with methyl vinyl ketone. Taken together, thevinols and orvinols constitute an important family of opioid receptor (OR) ligands playing an important role in both the OR mediated antinociception and OR antagonism. Herein, we disclose for the first time the OR activity of orvinols fluorinated within the pharmocophore associated with C(20) and its surrounding along with a dependence of the activity profile on the substituent at N(17). Starting from thevinone and 18,19-dihydrothevinone, a family of C(21)-fluorinated orvinols bearing methyl, cyclopropylmethyl (CPM), and allyl substituent at N(17) was synthesized. The fluorinated compounds were evaluated for OR activity. The orvinols bearing three fluorine atoms at C(21) were found to retain the properties of OR ligands and their activity profile depends on the substituent at N(17). Pilot in vivo experiments in a model of acute pain (tail-flick test in mice) revealed that 6-O-desmethyl-21,21,21-trifluoro-20-methylorvinol at doses 1.0-10.0 mg/kg (s.c.) exhibits analgesic activity at the level of morphine for a duration of 30-180 min. Its N(17)-CPM counterpart demonstrated the partial opioid agonist properties. The N(17)-allyl substituted derivative showed no analgesic activity. In vivo evaluation of an analgesic activity indicates that 21,21,21-trifluoro-20-methylorvinols represent a novel family of OR ligands related to buprenorphine, diprenorphine, etc. These compounds are promising for the structure-activity relationship studies among the thevinol/orvinol series as well as for a search for new OR ligands with potentially valuable pharmacological profiles.

Inhibition of PDE10A in a New Rat Model of Severe Dopamine Depletion Suggests New Approach to Non-Dopamine Parkinson's Disease Therapy.

Parkinson's disease is the second most common neurodegenerative pathology. Due to the limitations of existing therapeutic approaches, novel anti-parkinsonian medicines with non-dopamine mechanisms of action are clearly needed. One of the promising pharmacological targets for anti-Parkinson drug development is phosphodiesterase (PDE) 10A. The stimulating motor effects of PDE10A inhibition were detected only under the conditions of partial dopamine depletion. The results raise the question of whether PDE10A inhibitors are able to restore locomotor activity when dopamine levels are very low. To address this issue, we (1) developed and validated the rat model of acute severe dopamine deficiency and (2) tested the action of PDE10A inhibitor MP-10 in this model. All experiments were performed in dopamine transporter knockout (DAT-KO) rats. A tyrosine hydroxylase inhibitor, α-Methyl-DL-tyrosine (αMPT), was used as an agent to cause extreme dopamine deficiency. In vivo tests included estimation of locomotor activity and catalepsy levels in the bar test. Additionally, we evaluated the tissue content of dopamine in brain samples by HPLC analysis. The acute administration of αMPT to DAT-KO rats caused severe depletion of dopamine, immobility, and catalepsy (Dopamine-Deficient DAT-KO (DDD) rats). As expected, treatment with the L-DOPA and carbidopa combination restored the motor functions of DDD rats. Strikingly, administration of MP-10 also fully reversed immobility and catalepsy in DDD rats. According to neurochemical studies, the action of MP-10, in contrast to L-DOPA + carbidopa, seems to be dopamine-independent. These observations indicate that targeting PDE10A may represent a new promising approach in the development of non-dopamine therapies for Parkinson's disease.

Chronic Lithium Treatment Affects Anxious Behaviors and theExpression of Serotonergic Genes in Midbrain Raphe Nuclei of Defeated Male Mice.

There is experimental evidence that chronic social defeat stress is accompanied by the development of an anxiety, development of a depression-like state, and downregulation of serotonergic genes in midbrain raphe nuclei of male mice. Our study was aimed at investigating the effects of chronic lithium chloride (LiCl) administration on anxiety behavior and the expression of serotonergic genes in midbrain raphe nuclei of the affected mice. A pronounced anxiety-like state in male mice was induced by chronic social defeat stress in daily agonistic interactions. After 6 days of this stress, defeated mice were chronically treated with saline or LiCl (100 mg/kg, i.p., 2 weeks) during the continuing agonistic interactions. Anxiety was assessed by behavioral tests. RT-PCR was used to determine Tph2, Htr1a, Htr5b, and Slc6a4 mRNA expression. The results revealed anxiolytic-like effects of LiCl on social communication in the partition test and anxiogenic-like effects in both elevated plus-maze and social interaction tests. Chronic LiCl treatment upregulated serotonergic genes in midbrain raphe nuclei. Thus, LiCl effects depend on the treatment mode, psycho-emotional state of the animal, and experimental context (tests). It is assumed that increased expression of serotonergic genes is accompanied by serotonergic system activation and, as a side effect, by higher anxiety.

Effect of inhibiting carnitine biosynthesis on male rat sexual performance.

l-carnitine has a documented role as a cofactor in cellular energy metabolism and fatty acid beta-oxidation pathways and it has also been considered to function in reproductive biology. We investigated whether decreasing concentrations of L-carnitine using an inhibitor of its biosynthesis, mildronate (3-(2,2,2-trimethylhydrazinium)-propionate), would influence the sexual behavior or sperm quality in male rats. Mildronate treatment induced a significant decrease in carnitine concentration and an increase in gamma-butyrobetaine (GBB) concentration in both plasma and testes extracts. However, the expression of carnitine palmitoyltransferase I in testes and testosterone concentration in plasma was not changed in mildronate treated rat. Behavioral experiments demonstrated that mildronate treatment did not decrease the sexual motivation in both sexually naive and sexually experienced rats. The densities of spermatozoa in the cauda epididymis, as well as motility, were unchanged after mildronate treatment at a dose of 100 mg/kg. In conclusion, our study provides experimental evidence that mildronate induces decrease in the free carnitine concentration in rat testes, but does not decrease the sexual activity or sperm quality of male rats.

Efficacy and side effects of baclofen and the novel GABAB receptor positive allosteric modulator CMPPE in animal models for alcohol and cocaine addiction.

RATIONALE: Preclinical studies suggest that the GABAB receptor is a potential target for treatment of substance use disorders. However, recent clinical trials report adverse effects in patients treated with the GABAB receptor agonist baclofen and even question efficacy. How can the discrepancy between preclinical and clinical findings be explained? OBJECTIVE: To test efficacy and adverse effects of baclofen and the novel GABAB positive allosteric modulator (PAM) CMPPE in rat addiction models, which were developed in accordance with DSM. METHODS: We used a well-characterized rat model of long-term alcohol consumption with repeated deprivation phases that result in compulsive alcohol drinking in a relapse situation, and a rat model of long-term intravenous cocaine self-administration resulting in key symptoms of addictive behavior. We tested repeated baclofen (0, 1, and 3 mg/kg; i.p.) and CMPPE doses (0, 10, and 30 mg/kg; i.p.) in relapse-like situations, in either alcohol or cocaine addicted-like rats. RESULTS: Baclofen produced a weak anti-relapse effect at the highest dose in alcohol addicted-like rats, and this effect was mainly due to the treatment-induced sedation. CMPPE had a better profile, with a dose-dependent reduction of relapse-like alcohol drinking and without any signs of sedation. The cue-induced cocaine-seeking response was completely abolished by both compounds. CONCLUSION: Positive allosteric modulation of the GABAB receptor provides efficacy, and no observable side effects in relapse behavior whereas baclofen may cause, not only sedation, but also considerable impairment of food intake or metabolism. However, targeting GABAB receptors may be effective in reducing certain aspects of addictive-like behavior, such as cue-reactivity.

Effects of NAAG peptidase inhibitor 2-PMPA in model chronic pain - relation to brain concentration.

N-acetylated-alpha-linked-acidic peptidase (NAAG peptidase) converts N-acetyl-aspartyl-glutamate (NAAG, mGluR3 agonist) into N-acetyl-aspartate and glutamate. The NAAG peptidase inhibitor 2-PMPA (2-(phosphonomethyl)pentanedioic acid) had neuroprotective activity in an animal model of stroke and anti-allodynic activity in CCI model despite its uncertain ability to penetrate the blood-brain barrier. The NAAG concentration in brain ECF under basal conditions and its alteration in relation to the brain ECF concentration of 2-PMPA is unclear. We therefore assessed those brain concentrations after i.p. administration of 2-PMPA, using in vivo microdialysis combined with LC/MS/MS analysis. Administration of 2-PMPA (50mg/kg) produced a mean peak concentration of 2-PMPA of 29.66+/-8.1microM. This concentration is about 100,000 fold more than is needed for inhibition of NAAG peptidase, and indicates very good penetration to the brain. Application of 2-PMPA was followed by a linear increase of NAAG-concentration reaching a maximum of 2.89+/-0.42microM at the end of microdialysis. However, during the time the anti-allodynic effects of 2-PMPA were observed, the NAAG concentration in the ECF did not reach levels which are likely to have an impact on any known target. It appears therefore that the observed behavioural effects of 2-PMPA may not be mediated by NAAG nor, in turn, by mGluR3 receptors.

Optical isomers of phenibut inhibit [H(3)]-Gabapentin binding in vitro and show activity in animal models of chronic pain.

BACKGROUND: We report that R- and S-phenibut (ß-phenyl-γ-aminobutyric acid) - derivatives of GABA - bind with an affinity of c.a. 90µM to the gabapentin binding site in a competitive assay, a value comparable to that for previously claimed targets for this enantioermic molecule. This finding implied potential activity in neuropathic pain, this being one of the clinically validated indications for gabapentin. METHODS: The effect of phenibut on tactile allodynia was tested in a chronic constriction nerve injury (CCI) neuropathic pain model and against hypersensitivity following inflammation induced by inoculation using complete Freund's adjuvant (CFA) model. RESULTS: Indeed, a significant inhibitory effect on tactile allodynia was detected in rats in both employed chronic pain models with stronger and clearly dose dependent effect with R isomer. CONCLUSIONS: The results confirm activity in chronic pain models predicted from affinity for the gabapentin site and suggests, at least partially, that α2δ-subunits of presynaptic voltage-gated calcium channels are involved in mediating this effect.

Morphine-induced Straub tail reaction in mice treated with serotonergic compounds.

Constitutively active 5-HT2 receptors have been suggested to contribute to motoneuronal excitability, muscle spasms and spasticity. Accordingly, 5-HT2C receptor inverse agonists have been demonstrated in pilot experiments to reduce spasticity in animal model of spasticity and patients with spinal cord injuries. Thus, 5-HT2C receptor inverse agonists may represent a novel class of anti-spasticity agents justifying a search for compounds with robust 5-HT2C receptor inverse agonist activity either among the existing medications or via a dedicated drug discovery program. Morphine-induced Straub tail response in mice is regarded as a model of transient spasticity that may be suitable for supporting such drug discovery efforts. Subcutaneous injection of morphine (10-60mg/kg) induced a dose-dependent Straub tail reaction in male Swiss mice with maximum response obtained 15-30min after the morphine administration. When given prior to morphine, 5-HT2B/2C receptor inverse agonists cyproheptadine (1-10mg/kg, i.p.) and SB206553 (0.3-3mg/kg, i.p.) diminished Straub tail reaction dose-dependently without affecting spontaneous locomotor activity. In contrast, 5-HT2B/2C receptor antagonist methysergide (1-5.6mg/kg, i.p.) and 5-HT2C receptor antagonist SB242084 (1-5.6mg/kg, i.p.) as well as 5-HT2A receptor inverse agonist pimavanserin (1-10mg/kg, i.p.) had no appreciable effects on Straub tail response. Taken together, the findings indicate that constitutive activity of 5-HT2B/2C receptor may be involved in the mechanisms of morphine-induced spasticity. Thus, morphine-induced Straub tail response may be evaluated further as a candidate higher throughput test to identify 5-HT2C receptor inverse agonists with anti-spasticity effects in vivo.

Effects of low-affinity NMDA receptor channel blockers in two rat models of chronic pain.

In contrast to conventional opioid analgesics, antagonists acting at the N-methyl-d-aspartate (NMDA) subtype of glutamate receptors are capable of suppressing pain-related phenomena in chronic pain models while having little or no effect on acute nociception. One of the few clinically used NMDA receptor antagonists, memantine, differs from prototypic antagonists with psychotomimetic activity such as phencyclidine and (+)MK-801, in showing lower receptor affinity, faster unblocking kinetics and stronger voltage-dependency. Recently, a series of novel amino-alkyl-cyclohexanes was reported to interact with NMDA receptors in a manner similar to that of memantine. The present study aimed to evaluate the effects of these compounds as well as (+)MK-801 and memantine in two rat models of chronic pain and the rotarod test. Unlike (+)MK-801 and memantine, most of the tested compounds were inactive against tactile allodynia induced by sciatic nerve ligation. On the other hand, all tested drugs were found to inhibit formalin-induced grooming behavior-a model of chronic pain induction. In agreement with previous reports on the effects of NMDA receptor antagonists in similar assays, the late phase seemed to be inhibited to a greater extent than the early phase. For all tested compounds, inhibition of formalin-induced behaviors occurred at dose levels that were also producing significant motor deficits (rotarod test). These results confirm low efficacy of acute administration of NMDA receptor antagonists in the models of established pain states. Thus, studies on the prevention and management of chronic pain should focus on preemptive or long-term administration of NMDA receptor antagonists.

Facilitation of aggressive and sexual behaviors by saccharin deprivation in rats.

The 'deprivation effect' (DE) phenomenon is expressed as an increase in the level of free choice consumption of drugs, alcohol, or saccharin following a period of forced abstinence in humans and in several species of laboratory animals. The DE may reflect relapse-like drinking and be relevant for modeling addictive behaviors. In humans, drug or alcohol abstinence is commonly associated with the increased physical and sexual abuse. The present study aimed to study whether aggressive and sexual behaviors are affected by the conditions known to result in the DE. Adult male Wistar rats had unlimited free choice access to water and saccharin-containing solution (0.1%, w/v). After the preference for saccharin was established, animals underwent repeated 7-day-long episodes of saccharin deprivation. It was found that (i) aggressive and sexual behaviors were facilitated after 7 days of saccharin deprivation, and (ii) DE was significantly reduced in animals that were allowed to interact with the nonaggressive male or receptive female conspecifics during the last day of deprivation exposure. These results suggest that the saccharin deprivation exposures may facilitate aggressive and sexual behaviors. Alternative behavioral strategy may serve as a mechanism of coping with the deprivation state.

Facilitation of electrical brain self-stimulation behavior by abused solvents.

Animal models are needed to study the abuse-related behavioral and pharmacological effects of inhaled solvents. Previous studies have suggested that intracranial self-stimulation techniques may be successfully adapted for testing the effects of solvent exposure. The present study aimed to assess the effects of toluene, cyclohexane, acetone, and petroleum benzine (a widely used mixture of hexanes and heptanes) in rats trained to lever press or nose-poke for electrical stimulation delivered through electrodes implanted into the medial forebrain bundle. It was found that toluene, cyclohexane, and benzine but not acetone, increased rates of responding, particularly at the lower stimulation intensities. In another set of experiments utilizing an auto-titration procedure, all tested solvents significantly reduced self-stimulation thresholds. However, only for toluene and benzine were these effects observed at the exposure levels that did not impair rates of operant performance. There may not be such a clear separation of effects for acetone and cyclohexane. Thus, toluene and benzine appear to selectively affect brain reward systems in a manner similar to that for most other abused drugs. Data from intracranial self-stimulation studies of solvents may be useful in abuse potential assessment of individual compounds and for examining neural and behavioral processes involved in inhalant abuse.

Nicotine exposure throughout early development promotes nicotine self-administration in adolescent mice and induces long-lasting behavioural changes.

Maternal cigarette smoking during pregnancy can result in behavioural problems of the offspring. Although the causative agent in tobacco smoke that leads to these aberrations is not known, some studies using animal models have supported the hypothesis that nicotine may cause impairments in fatal and neonatal development. However, in many of the animal studies nicotine has been administered by subcutaneous injections, which could lead to significant fetal hypoxia; some routes of drug administration included stressful procedures to pregnant dams that could create unfavorable fetal environment. In this study, mice were exposed to nicotine via drinking solution. The effects of nicotine exposure throughout early development on behavioural measures during adolescence and adulthood were examined. Adult female dams were allowed to orally self-administer a saccharin, or nicotine plus saccharin solution during gestation and lactation. Following weaning, plasma nicotine concentrations were measured in nicotine-exposed dams, and their offspring were tested using various behavioural measures. [3H]Epibatidine binding was also measured in the cortex and hippocampus at two different time points in the nicotine-exposed adolescents. The results of the study indicate that exposure to nicotine throughout early development influenced intravenous nicotine self-administration, social interactions and performance under a forced swim test. Exposure throughout early development to nicotine however did not affect [3H]epibatidine binding in the hippocampus and cortex.

The anxiolytic and analgesic properties of fenobam, a potent mGlu5 receptor antagonist, in relation to the impairment of learning.

Fenobam [N-(3-chlorophenyl)-N'-(4,5-dihydro-1-methyl-4-oxo-1H-imidazole-2-yl)urea] was suggested to possess anxiolytic actions 30 years ago. Hoffmann-La Roche researchers recently reported that it is a selective and potent mGlu5 receptor antagonist, acting as a negative allosteric modulator. In the present study, we show that fenobam readily penetrates to the brain, reaching concentrations over 600 nM, clearly above the affinity for mGluR5 receptors. Fenobam (at 10, 30, and 100 mg/kg) did not affect horizontal locomotor activity in the open field test. Anxiolytic-like activity in the context freezing test was seen at 30 mg/kg, while fenobam was not active in the elevated plus maze test at the tested concentrations. Fenobam had antinociceptive actions in the formalin test at 10 and 30 mg/kg, but failed to attenuate mechanical allodynia in the chronic constriction injury model. Impairment of learning was revealed in the passive avoidance test at 30 mg/kg. Fenobam also impaired performance in both the Morris water maze and in the contextual fear conditioning test at the doses of 30 and 10 mg/kg, respectively. Prepulse inhibition, used as a model of psychomimetic activity, was not affected by fenobam at doses of up to 60 mg/kg. Our results indicate that the beneficial effects of fenobam occur in a similar dose range as the potential side-effects.

Comparison of the mGluR1 antagonist A-841720 in rat models of pain and cognition.

In the current study we compared the potency of the selective metabotropic glutamate receptor (mGluR1) antagonist A-841720 (7-Azepan-1-yl-4-dimethylamino-7H-9-thia-1,5,7-triaza-fluoren-8-one) in rodent models of pain with its effects in models of learning and memory, to obtain information regarding the therapeutic window of this compound. A-841720 significantly reduced formalin-induced behaviours and complete Freund's adjuvant (CFA)-induced tactile allodynia, starting at doses of 1 and 10 mg/kg, respectively. At the dose of 3 mg/kg, however, A-841720 significantly reduced the percentage of spontaneous alternations in a radial-maze task. In contextual-fear conditioning, A-841720, given at the dose of 10 mg/kg before acquisition, significantly reduced freezing behaviour tested 24 h later. In the same task, repeated treatment for 5 days did not reduce the impairing effect of the challenge dose, indicating a lack of tolerance development. In a passive-avoidance task, A-841720 at 10 mg/kg administered before acquisition, significantly reduced the latency to enter the dark box on the retention test. Given that complete Freund's adjuvant is a better measure of analgesia, these results indicate that the selective mGluR1 antagonist A-841720 has analgesic potential in a dose range at which it also produces memory impairments. This diminishes its therapeutic potential for the treatment of chronic pain.

Lowered brain stimulation reward thresholds in rats treated with a combination of caffeine and N-methyl-D-aspartate but not alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate or metabotropic glutamate receptor-5 receptor antagonists.

Previous studies suggested that adenosine A1 and A2A receptor agonists counteract behavioral effects of N-methyl-D-aspartate (NMDA) receptor antagonists while adenosine receptor antagonists may produce opposite effects enhancing the actions of NMDA receptor antagonists. To further evaluate the effects of combined administration of adenosine receptor antagonist caffeine and various NMDA and non-NMDA glutamate receptor antagonists on brain stimulation reward (discrete-trial threshold current intensity titration procedure), rats with electrodes implanted into the ventral tegmental area were tested after pretreatment with NMDA receptor channel blocker MK-801 (0.01-0.3 mg/kg), competitive antagonist D-CPPene (0.3-5.6 mg/kg), glycine site antagonist L-701,324 (1.25-5 mg/kg), alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptor antagonist GYKI-53655 (1-10 mg/kg), metabotropic glutamate receptor 5 (mGluR5) antagonist MPEP (1-10 mg/kg) alone and in combination with caffeine (1-30 mg/kg). MK-801 (0.056 and 0.1 mg/kg) was the only tested glutamate antagonist that lowered self-stimulation thresholds, while D-CPPene (5.6 mg/kg) and MPEP (5.6 and 10 mg/kg) had the opposite effects. Threshold-increasing effects of D-CPPene, but not of MPEP, however, were associated with marked impairment of operant performance, reflected by longer latencies to respond and higher rates of responding during the inter-trial intervals. Operant performance was also disrupted by the highest dose of MK-801 (0.3 mg/kg). For subsequent experiments, caffeine (1-30 mg/kg) was combined with the highest doses of NMDA receptor antagonists that did not lower the brain stimulation reward thresholds and did not impair operant performance. Caffeine had no appreciable effects on self-stimulation behavior when given alone. A low dose of caffeine (3 mg/kg) significantly lowered self-stimulation thresholds only when given together with MK-801 (0.03 mg/kg) or D-CPPene (3 mg/kg). Combined with the same antagonist drugs, higher doses of caffeine (10 and 30 mg/kg) facilitated time-out responding. These results indicate that, within a limited dose range, caffeine in combination with an NMDA receptor channel blocker and a competitive antagonist significantly lowers brain stimulation reward thresholds in rats.