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BACKGROUND: The opioid epidemic remains one of the most pressing public health crises facing the United States. Fentanyl and related synthetic opioid agonists have largely driven the rising rates of associated overdose deaths, in part, because of their surreptitious use as substitutes for other opioids and as adulterants in psychostimulants. Deaths involving opioids typically result from lethal respiratory depression, and it is currently unknown how co-use of psychostimulants with opioids affects respiratory toxicity. Considering psychostimulant overdoses have increased over 3-fold since 2013, and half of those co-involved opioids, this is a cardinal question. METHODS: Naloxone, d-amphetamine (AMPH), and (±)-methamphetamine (METH) were evaluated for their effects on basal and fentanyl-depressed respiration. Minute volume (MVb) was measured in awake, freely moving mice via whole-body plethysmography to quantify fentanyl-induced respiratory depression and its modulation by dose ranges of each test drug. RESULTS: Naloxone immediately reversed respiratory depression induced by fentanyl only at the highest dose tested (10 mg/kg). Both AMPH and METH exhibited bidirectional effects on MVb under basal conditions, producing significant (p ≤ 0.05) depressions then elevations of respiration as dose increased. Under depressed conditions the bidirectional effects of AMPH and METH on respiration were exaggerated, exacerbating and then reversing fentanyl-induced depression as dose increased. CONCLUSIONS: These results indicate that co-use of amphetamines with fentanyl may worsen respiratory depression, but conversely, monoaminergic components of the amphetamines may possibly be exploited to mitigate fentanyl overdose.

Background and Purpose: The opioid epidemic remains one of the most pressing public health crises facing the United States. Rising rates of opioid-involved overdose deaths are presently driven primarily by fentanyl and related synthetic opioid agonists that are resistant to reversal by naloxone and increasingly used as adulterants or clandestine substitutes in illicitly produced opioids, sedatives, and psychostimulants. Deaths involving opioids typically result from lethal respiratory depression and it is currently unknown whether co-use of psychostimulants with opioids affects respiratory toxicity. Considering psychostimulant overdoses have increased over 3-fold since 2013, and half of those co-involved opioids, this is a cardinal question. Experimental Approach: Naloxone, d-amphetamine (AMPH), and (+/-)-methamphetamine (METH), were evaluated for their effects on basal and fentanyl-depressed respiration. Minute volume (MVb) was measured in awake, freely moving mice via whole-body plethysmography to quantify fentanyl-induced respiratory depression and its modulation by dose ranges of each test drug. Key Results: Naloxone reversed respiratory depression induced by fentanyl only at the highest dose tested (10 mg•kg-1). Both AMPH and METH exhibited bidirectional effects on MVb under basal conditions, producing depressions then elevations of respiration as dose increased. Under depressed conditions the bidirectional effects of AMPH and METH on respiration were exaggerated, exacerbating and then reversing fentanyl-induced depression as dose increased. Conclusions and Implications: These results indicate that co-use of amphetamines with fentanyl may worsen respiratory depression, but conversely, monoaminergic components of the amphetamines may possibly be exploited to mitigate fentanyl overdose.

Prenatal environmental insults increase the risk of neurodevelopmental psychiatric conditions in the offspring. Structural modifications of dendritic spines are central to brain development and plasticity. Using maternal immune activation (MIA) as a rodent model of prenatal environmental insult, previous results have reported dendritic structural deficits in the frontal cortex. However, very little is known about the molecular mechanism underlying MIA-induced synaptic structural alterations in the offspring. Using prenatal (E12.5) injection with polyinosinic-polycytidylic acid potassium salt as a mouse MIA model, we show here that upregulation of the serotonin 5-HT

Prenatal environmental insults increase the risk of neurodevelopmental psychiatric conditions in the offspring. Structural modifications of dendritic spines are central to brain development and plasticity. Using maternal immune activation (MIA) as a rodent model of prenatal environmental insult, previous results have reported dendritic structural deficits in the frontal cortex. However, very little is known about the molecular mechanism underlying MIA-induced synaptic structural alterations in the offspring. Using prenatal (E12.5) injection with poly-(I:C) as a mouse MIA model, we show that upregulation of the serotonin 5-HT2A receptor (5-HT2AR) is at least in part responsible for some of the effects of prenatal insults on frontal cortex dendritic spine structure and sensorimotor gating processes. Mechanistically, we report that this upregulation of frontal cortex 5-HT2AR expression is associated with MIA-induced reduction of nuclear translocation of the glucocorticoid receptor (GR) and, consequently, a decrease in the enrichment of GR at the 5-HT2AR promoter. The translational significance of these preclinical findings is supported by data in postmortem human brain samples suggesting dysregulated nuclear GR translocation in frontal cortex of schizophrenia subjects. Repeated (twice a day for 4 days) corticosterone administration augmented frontal cortex 5-HT2AR expression and reduced GR binding to the 5-HT2AR promoter. However, virally (AAV)-mediated augmentation of GR function reduced frontal cortex 5-HT2AR expression and improved sensorimotor gating processes via 5-HT2AR. Together, these data support a negative regulatory relationship between GR signaling and 5-HT2AR expression in mouse frontal cortex that may carry implications for the pathophysiology underlying 5-HT2AR dysregulation in neurodevelopmental psychiatric disorders.

Prenatal environmental insults increase the risk of neurodevelopmental psychiatric conditions in the offspring. Structural modifications of dendritic spines are central to brain development and plasticity. Using maternal immune activation (MIA) as a rodent model of prenatal environmental insult, previous results have reported dendritic structural deficits in the frontal cortex. However, very little is known about the molecular mechanism underlying MIA-induced synaptic structural alterations in the offspring. Using prenatal (E12.5) injection with polyinosinic-polycytidylic acid potassium salt as a mouse MIA model, we show here that upregulation of the serotonin 5-HT2A receptor (5-HT2AR) is at least in part responsible for some of the effects of prenatal insults on frontal cortex dendritic spine structure and sensorimotor gating processes. Mechanistically, we report that this upregulation of frontal cortex 5-HT2AR expression is associated with MIA-induced reduction of nuclear translocation of the glucocorticoid receptor (GR) and, consequently, a decrease in the enrichment of GR at the 5-HT2AR promoter. The translational significance of these preclinical findings is supported by data in postmortem human brain samples suggesting dysregulation of GR translocation in frontal cortex of schizophrenia subjects. We also found that repeated corticosterone administration augmented frontal cortex 5-HT2AR expression and reduced GR binding to the 5-HT2AR promoter. However, virally (adeno-associated virus) mediated augmentation of GR function reduced frontal cortex 5-HT2AR expression and improved sensorimotor gating processes via 5-HT2AR. Together, these data support a negative regulatory relationship between GR signaling and 5-HT2AR expression in the mouse frontal cortex that may carry implications for the pathophysiology underlying 5-HT2AR dysregulation in neurodevelopmental psychiatric disorders. National Institutes of Health R01MH084894 (to J. G.-M.), R01MH111940 (to J. G.-M.), NIH-N01DA-17-8932 (to P. M. B.), NIH-N01DA-19-8949 (to P. M. B.), and F30MH116550 (to J. M. S.), and Basque Government IT1211-19 (to J. J. M.) participated in the funding of this study. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Synthetic narcotics have been implicated as the single greatest contributor to increases in opioid-related fatalities in recent years. This study evaluated the effects of nine fentanyl-related substances that have emerged in the recreational drug marketplace, and for which there are no existing or only limited in vivo data. Adult male Swiss Webster mice were administered fentanyl-related substances and their effects on locomotion as compared to MOR agonist standards were recorded. In locomotor activity tests, morphine (100, 180 mg/kg), buprenorphine (1, 10 mg/kg), fentanyl (1, 10 mg/kg), cyclopropylfentanyl (1, 10 mg/kg), cyclopentylfentanyl (10 mg/kg), (±)-cis-3-methylbutyrylfentanyl (0.1, 1, 10 mg/kg), ortho-methylacetylfentanyl (10 mg/kg), para-chloroisobutyrylfentanyl (100 mg/kg), ocfentanil (1, 10 mg/kg), and ortho-fluoroacrylfentanyl (0.1, 1, 10 mg/kg) elicited significant (p ≤ 0.05) dose-dependent increases in locomotion. However, 2,2,3,3-tetramethylcyclopropylfentanyl did not have any effects on locomotion, even when tested up to 100 mg/kg, and 4′-methylacetylfentanyl (10, 100 mg/kg) significantly decreased locomotion. The rank order of efficacy for stimulating locomotion (maximum effect as a % of fentanyl’s maximum effect) for fentanyl-related substances relative to MOR agonist standards was cyclopropylfentanyl (108.84 ± 20.21) > fentanyl (100 ± 15.3) > ocfentanil (79.27 ± 16.92) > morphine (75.9 ± 14.5) > (±)-cis-3-methylbutyrylfentanyl (68.04 ± 10.08) > ortho-fluoroacrylfentanyl (63.56 ± 19.88) > cyclopentylfentanyl (56.46 ± 8.54) > para-chloroisobutyrylfentanyl (22.44 ± 8.51) > buprenorphine (11.26 ± 2.30) > ortho-methylacetylfentanyl (9.45 ± 2.92) > 2,2,3,3-tetramethylcyclopropylfentanyl (6.75 ± 1.43) > 4′-methylacetylfentanyl (3.47 ± 0.43). These findings extend in vivo results from previous reports documenting additional fentanyl related-related substances that stimulate locomotion similar to known abused opioids while also identifying some anomalies.

Ulotaront (SEP-363856) is a trace amine-associated receptor 1 (TAAR1) agonist with 5-hydroxytryptamine type 1A (5-HT1A) agonist activity that is currently in Phase 3 clinical development for the treatment of schizophrenia. Unlike available antipsychotics, the efficacy of ulotaront is not mediated by blockade of dopamine D2 or serotonin 5-HT2A receptors. In a short-term randomized clinical trial, ulotaront has demonstrated significant efficacy in the treatment of adults with an acute exacerbation of schizophrenia. Given ulotaront's novel mechanism of action a series of preclinical studies were performed to evaluate its potential abuse liability.A battery of studies were conducted in male and female rats to evaluate whether ulotaront produces behavioral changes suggestive of human abuse potential. In addition, studies were undertaken to probe the potential for ulotaront to block reinstatement of cocaine-seeking behavior in male rats.Ulotaront was not self-administered by rats trained to self-administer amphetamine, cocaine, or heroin. The subjective qualities of ulotaront were distinct from those produced by amphetamine in a drug discrimination procedure. Ulotaront, and buspirone, a non-scheduled anxiolytic with 5-HT1A agonism, partially generalized to the interoceptive cue elicited by 3, 4-methylenedioxymethamphetamine (MDMA). In addition, ulotaront demonstrated a trend to reduce cocaine-primed induced reinstatement, and dose-dependently reduced cue-reinstated responding.The current results suggest that the TAAR1/5-HT1A agonist ulotaront is not likely to pose a risk for recreational abuse in humans and may have potential therapeutic utility as a treatment of substance use disorders.

Opioid-related fatalities involving synthetic opioids have reached unprecedented levels. This study evaluated the respiratory depressant effects of seven fentanyl analogs that have either emerged in the illicit drug supply or been identified in toxicological analyses following fatal or non-fatal intoxications. Adult male Swiss Webster mice were administered fentanyl analogs (isobutyrylfentanyl, crotonylfentanyl, para-methoxyfentanyl, para-methoxybutyrylfentanyl, 3-furanylfentanyl, thiophenefentanyl, and benzodioxolefentanyl) and their effects on minute volume as compared to mu-opioid receptor (MOR) agonist standards (fentanyl, morphine, and buprenorphine) were measured using whole body plethysmography (WBP). All drugs elicited significant (p ≤ 0.05) hypoventilation relative to vehicle for at least one dose tested: morphine (1, 3.2, 10, 32 mg/kg), buprenorphine, (0.032, 0.1, 0.32, 1, 3.2 mg/kg), fentanyl (0.0032, 0.01, 0.032, 0.1, 1, 32 mg/kg), isobutyrylfentanyl (0.1, 0.32, 1, 3.2, 10 mg/kg), crotonylfentanyl (0.1, 0.32, 1, 3.2, 10 mg/kg), para-methoxyfentanyl (0.1, 0.32, 1, 3.2, 10 mg/kg), para-methoxybutyrylfentanyl (0.32, 1, 3.2, 10 mg/kg), 3-furanylfentanyl (0.1, 0.32, 1, 3.2, 10 mg/kg), thiophenefentanyl (1, 3.2, 10, 32, 100 mg/kg), and benzodioxolefentanyl (3.2, 10, 32, 100 mg/kg). The ED(50) values for hypoventilation showed a rank order of potency as follows: fentanyl (ED(50) = 0.96 mg/kg) > 3-furanylfentanyl (ED(50) = 2.60 mg/kg) > crotonylfentanyl (ED(50) = 2.72 mg/kg) > para-methoxyfentanyl (ED(50) = 3.31 mg/kg) > buprenorphine (ED(50) = 10.8 mg/kg) > isobutyrylfentanyl (ED(50) = 13.5 mg/kg) > para-methoxybutyrylfentanyl (ED(50) = 16.1 mg/kg) > thiophenefentanyl (ED(50) = 18.0 mg/kg) > morphine (ED(50) = 55.3 mg/kg) > benzodioxolefentanyl (ED(50) = 10,168 mg/kg). A naloxone pretreatment (10 mg/kg) attenuated the hypoventilatory effects of all drugs. These results establish that the respiratory depressant effects of these fentanyl analogs are at least in part mediated by the MOR.

BACKGROUND: Prepulse inhibition (PPI) of startle is a sensorimotor gating phenomenon perturbed in a variety of neuropsychiatric conditions. Psychedelics disrupt PPI in rats and humans, but their effects and involvement of the serotonin 5-HT(2A) receptor (5-HT(2A)R) in mice remain unexplored. METHODS: We tested the effect of the psychedelic 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) (0.5 mg/kg, i.p.) on startle amplitude and %PPI in response to acoustic stimuli under up to four different experimental conditions that included changes in background and stimulus intensity, prepulse and pulse duration, and interstimulus interval in male and female 129S6/SvEv mice. We also evaluated the effect of the 5-HT(2A)R antagonist M100,907 (1 mg/kg, i.p.) on DOI-induced startle amplitude and %PPI, as well as the effect of the psychedelic LSD (0.24 mg/kg, i.p.) and the dopamine agonists apomorphine (5 mg/kg, s.c.) and SKF-82,958 (0.5 mg/kg, i.p.) in male 129S6/SvEv mice. RESULTS: DOI altered startle amplitude with either pulse alone or prepulse + pulse presentations in all PPI conditions, and increased %PPI in three out of four PPI conditions in male mice — an effect that was prevented by M100,907. In female mice, DOI increased %PPI without affecting startle amplitude. %PPI was positively correlated with startle amplitude in males while being negatively correlated in female mice. In male mice, LSD also increased %PPI, although it did not affect startle amplitude, whereas apomorphine and SKF-82,958 induced decreases in %PPI. CONCLUSION: Our findings highlight a distinct effect of the psychedelic DOI on PPI in 129S6/SvEv mice, suggesting 5-HT(2A)R-dependent PPI improvement in a paradigm-dependent and sex-dependent manner.

The ANKS1B gene was a top finding in genome-wide association studies (GWAS) of antipsychotic drug response. Subsequent GWAS findings for ANKS1B include cognitive ability, educational attainment, body mass index, response to corticosteroids and drug dependence. We review current human association evidence for ANKS1B, in addition to functional studies that include two published mouse knockouts. The several GWAS findings in humans indicate that phenotypically relevant variation is segregating at the ANKS1B locus. ANKS1B shows strong plausibility for involvement in CNS drug response because it encodes a postsynaptic effector protein that mediates long-term changes to neuronal biology. Forthcoming data from large biobanks should further delineate the role of ANKS1B in CNS drug response.

Background Fentanyl and its structurally related compounds have emerged as the most significant contributors to opioid overdose fatalities in recent years. While there is abundant information about the pharmacological effects of fentanyl, far less is known of its more recently abused analogs. The objective of this study was to determine whether fentanyl and several fentanyl-related substances would engender oxycodone-like responding in a mouse model of oxycodone discrimination. Oxycodone was selected as the training drug due to its high selectivity for mu opioid receptors. Compounds that elicited oxycodone-like responding in this procedure would likely evoke overlapping subjective experiences. Methods Adult male C57BL/6 mice were trained to discriminate 1.3 mg/kg oxycodone from vehicle in a food-reinforced, two-lever choice procedure. Generalization tests were conducted with fentanyl and the following fentanyl-related compounds: ocfentanil, 3-furanyl fentanyl, crotonylfentanyl, and valerylfentanyl. Results Fentanyl and each of its analogs completely generalized to the 1.3 mg/kg oxycodone discriminative stimulus and naltrexone pretreatment significantly decreased oxycodone-like responding for each compound. Rank order potency for engendering oxycodone-appropriate responding was ocfentanil > fentanyl > 3-furanyl fentanyl ≈ crotonylfentanyl > oxycodone > valerylfentanyl. Drug doses that evoked full substitution also significantly suppressed response rates compared to vehicle. Conclusions These results indicate that the discriminative stimulus, and by extension, the interoceptive and subjective effects of the tested fentanyl analogs, overlap with those of oxycodone. These observations consequentially support the prediction that they would also engender the likelihood for abuse similar to oxycodone. This article is part of the Special Issue entitled ‘New Vistas in Opioid Pharmacology’.

Background Antagonism of peripheral opioid receptors by methylnaltrexone (MNTX) was recently proposed as a potential mechanism to attenuate the development of opioid analgesic tolerance based on experiments conducted in mice. However, reports indicate that MNTX is demethylated to naltrexone (NTX) in mice, and NTX may subsequently cross the blood-brain barrier to antagonize centrally-mediated opioid effects. The goal of this study was to determine whether MNTX alters centrally-mediated behaviors elicited by the opioid analgesics, morphine and oxycodone, and to quantify concentrations of MNTX and NTX in blood and brain following their administration in mice. Methods Combinations of MNTX and morphine were tested under acute and chronic conditions in thermal nociceptive assays. Effects of MNTX and NTX pretreatment were assessed in an oxycodone discrimination operant procedure. Blood and brain concentrations of these antagonists were quantified after their administration using liquid chromatography-mass spectrometry. Results MNTX dose-dependently attenuated acute and chronic morphine antinociception. MNTX and NTX dose-dependently antagonized the discriminative stimulus effects of oxycodone. MNTX and NTX were detected in both blood and brain after administration of MNTX, confirming its demethylation and demonstrating that MNTX itself can cross the blood-brain barrier. Conclusions These results provide converging behavioral and analytical evidence that MNTX administration in mice attenuates centrally-mediated effects produced by opioid analgesics and results in functional concentrations of MNTX and NTX in blood and brain. Collectively, these findings indicate that MNTX cannot be administered systemically in mice for making inferences that its effects are peripherally restricted.

Background Nociceptin/orphanin FQ (NOP) receptor ligands have shown efficacy as putative analgesics and can modulate the abuse-related effects of opioids, suggesting therapeutic applications. The discriminative stimulus effects of a drug are related to their subjective effects, a predictor of abuse potential. To determine whether activation of NOP receptors could alter the subjective effects of an abused opioid analgesic, a novel oxycodone discrimination was established in mice, characterized with positive and negative controls, and its expression evaluated with a NOP receptor agonist. Methods Adult male C57BL/6 mice were trained to discriminate 1.3 mg/kg oxycodone from vehicle in a two-lever operant procedure. The discrimination was characterized with naloxone challenge, and generalization tests with the μ-opioid receptor agonists, heroin and morphine, and the κ-opioid receptor selective agonist, U50488. Subsequently, effects of the NOP agonist Ro64-6198 were evaluated with and without oxycodone. Results Oxycodone generalization occurred in a dose-dependent manner and was reversed by naloxone pretreatment. Heroin and morphine, but not U50488, substituted for oxycodone. Co-treatment of 1 mg/kg Ro64-6198 with the oxycodone training dose reduced % oxycodone lever responding (%OLR) and restored response rates to vehicle control levels. J-113397, a NOP antagonist, reversed these effects. Co-administration of 1 mg/kg Ro64-6198 with a range of oxycodone doses resulted in rightward dose-effect curve shifts in %OLR and response rates compared to oxycodone alone. Conclusions These results provide additional evidence that NOP receptor activation can modulate the subjective effects of opioid analgesics and represent the first characterization of oxycodone’s discriminative stimulus effects in mice.

Synthetic opioids have been implicated as the single greatest contributor to rising drug-related fatalities in recent years. This study evaluated mu-opioid receptor (MOR) mediated effects of seven fentanyl-related substances that have emerged in the recreational drug marketplace, and for which there are no existing or only limited in vivo data. Adult male Swiss Webster mice were administered fentanyl-related substances and their effects on nociception and locomotion as compared to MOR agonist standards were observed. In locomotor activity tests, morphine (100, 180 mg/kg), fentanyl (1, 10 mg/kg), beta-methylfentanyl (10 mg/kg), para-methoxyfentanyl (10 mg/kg), fentanyl carbamate (100 mg/kg), and 3-furanylfentanyl (10 mg/kg), elicited significant (p ≤ 0.05) dose-dependent increases in locomotion. However, para-methylfentanyl and beta′-phenylfentanyl did not produce significant effects on locomotion at doses up to 100 mg/kg and phenylfentanyl (100 mg/kg) significantly decreased locomotion. In warm-water tail-withdrawal tests, all substances produced significant dose-dependent increases in antinociception with increasing ED50 values (95% CI) of fentanyl [0.08 mg/kg (0.04–0.16)] > para-methoxyfentanyl [0.43 mg/kg (0.23–0.77)] > 3-furanylfentanyl [0.51 mg/kg (0.36–0.74)] > beta-methylfentanyl [0.74 mg/kg (0.64–0.85)] > para-methylfentanyl [1.92 mg/kg (1.48–2.45)] > fentanyl carbamate [5.59 mg/kg (4.11–7.54)] > morphine [7.82 mg/kg (5.42–11.0)] > beta′-phenylfentanyl [19.4 mg/kg (11.0–34.4)] > phenylfentanyl [55.2 mg/kg (33.5–93.0)]. Naltrexone (1 mg/kg) increased ED50 values several fold with decreasing magnitudes of para-methylfentanyl (63.1×) > para-methoxyfentanyl (22.5×) > beta′-phenylfentanyl (21.0×) > 3-furanylfentanyl (20.6×) > beta-methylfentanyl (19.2×) > phenylfentanyl (5.23×) > fentanyl (3.95×) > fentanyl carbamate (2.21×) > morphine (1.48×). These findings expand upon in vivo results from previous studies and establish that the effects of these fentanyl related-related substances are at least in part mediated by the MOR.

Substantial challenges exist for investigating the cannabinoid receptor type 1 (CB1)-mediated discriminative stimulus effects of the endocannabinoids, 2-arachidonoylglycerol (2-AG) and N-arachidonoylethanolamine (anandamide; AEA), compared with exogenous CB1 receptor agonists, such as Δ9-tetrahydrocannabinol (THC) and the synthetic cannabinoid CP55,940. Specifically, each endocannabinoid is rapidly degraded by the respective hydrolytic enzymes, monoacylglycerol lipase (MAGL) and fatty acid amide hydrolase (FAAH). Whereas MAGL inhibitors partially substitute for THC and fully substitute for CP55,940, FAAH inhibitors do not substitute for either drug. Interestingly, combined FAAH-MAGL inhibition results in full THC substitution, and the dual FAAH-MAGL inhibitor SA-57 serves as its own discriminative training stimulus. Because MAGL inhibitors fully substitute for SA-57, we tested whether the selective MAGL inhibitor MJN110 would serve as a training stimulus. Twelve of 13 C57BL/6J mice learned to discriminate MJN110 from vehicle, and the CB1 receptor antagonist rimonabant dose-dependently blocked its discriminative stimulus. CP55,940, SA-57, and another MAGL inhibitor JZL184, fully substituted for MJN110. In contrast, the FAAH inhibitor PF-3845 failed to substitute for the MJN110 discriminative stimulus, but produced a 1.6 (1.1-2.2; 95% confidence interval) leftward shift in the MJN110 dose-response curve. Inhibitors of other relevant enzymes (i.e., ABHD6, COX-2) and nicotine did not engender substitution. Diazepam partially substituted for MJN110, but rimonabant failed to block this partial effect. These findings suggest that MAGL normally throttles 2-AG stimulation of CB1 receptors to a magnitude insufficient to produce cannabimimetic subjective effects. Accordingly, inhibitors of this enzyme may release this endogenous brake producing effects akin to those produced by exogenously administered cannabinoids.

Opioid use disorder is a growing concern in the United States. Mice were used to investigate the mechanisms involving opioid physical dependence and for evaluating medications for treating opioid use disorders. While there are many preclinical reports describing protocols for inducing physical dependence upon morphine, there are fewer preclinical reports describing more contemporary abused prescription opiates. The goal of this study was to characterize and validate a mouse model of oxycodone dependence. Male C57BL/6J mice were injected with saline or increasing doses of oxycodone (9–33 mg/kg) twice daily for 8 days. On the 9th day, mice were challenged with 1 mg/kg naloxone and observed for somatic signs. Mice were pretreated with oxycodone (17, 33, or 75 mg/kg) prior to withdrawal to determine if it could attenuate somatic withdrawal signs. Additional mouse groups were pretreated with 1 mg/kg clonidine. Lastly, we measured somatic signs for 6, 24, and 48 h post-withdrawal during spontaneous and precipitated withdrawal. Pretreating with oxycodone or clonidine dose-dependently prevented the emergence of withdrawal signs. Mice chronically treated with oxycodone exhibited more withdrawal signs than vehicle at 24 h after the final injection during spontaneous withdrawal. In contrast, mice that received repeated naloxone challenges showed peak withdrawal signs at 6 h, and withdrawal signs were significantly greater at all time points compared to vehicle. Reversal of withdrawal effects by positive controls, and establishing spontaneous and precipitated withdrawal paradigms, serve as validation of this model and provide a means to examine novel therapeutics to treat opioid withdrawal.

There are no FDA-approved pharmacotherapies for cocaine use disorder, indicating a need to identify novel reagents with therapeutic potential. Ibudilast is an anti-inflammatory glial attenuator and non-selective phosphodiesterase inhibitor currently undergoing clinical evaluations for methamphetamine, opiate, and alcohol abuse disorders. We previously showed that twice daily (b.i.d.) ibudilast reduces the development of methamphetamine sensitization in male mice. However, nothing is known about the ability of ibudilast to modulate the expression of sensitization that occurs after drug re-exposure during abstinence, effects on cocaine-mediated behaviors, or potentially sexually dimorphic effects. Male and female rats were administered cocaine for 7 days and expression of sensitization was assessed by cocaine challenge after 21 days abstinence. On test days, 15 mg/kg i. p. cocaine was evaluated, whereas 30 mg/kg was administered on intervening days. Lower test doses avoid competition of non-motor behaviors with locomotion. In all measures where sensitization was expressed, ibudilast (7.5 and 10 mg/kg, i. p., b. i.d. for 3 days and once on test day) reversed this behavior to levels seen after acute exposure, but not below. There were some intriguing sexually dimorphic effects that were not a function of estrous cycle. Specifically, distance travelled in the center of the test arena and rearing only sensitized in male rats, and ibudilast reversed these behaviors to levels seen after acute cocaine exposure. In females, center distance travelled was reduced below acute cocaine levels by 7.5 mg/kg ibudilast. Increased distance travelled in the center versus periphery is thought to model anxiolytic-like behavior due to increased predation risk. Taken together, these data suggest that the clinical evaluation of ibudilast could be extended to cocaine use disorder.

Whereas the inhibition of fatty acid amide hydrolase (FAAH) or monoacylglycerol lipase (MAGL), the respective major hydrolytic enzymes of N-arachidonoyl ethanolamine (AEA) and 2-arachidonoylglycerol (2-AG), elicits no or partial substitution for Δ(9)-tetrahydrocannabinol (THC) in drug-discrimination procedures, combined inhibition of both enzymes fully substitutes for THC, as well as produces a constellation of cannabimimetic effects. The present study tested whether C57BL/6J mice would learn to discriminate the dual FAAH-MAGL inhibitor SA-57 (4-[2-(4-chlorophenyl)ethyl]-1-piperidinecarboxylic acid 2-(methylamino)-2-oxoethyl ester) from vehicle in the drug-discrimination paradigm. In initial experiments, 10 mg/kg SA-57 fully substituted for CP55,940 ((-)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl)cyclohexanol), a high-efficacy CB1 receptor agonist in C57BL/6J mice and for AEA in FAAH (-/-) mice. Most (i.e., 23 of 24) subjects achieved criteria for discriminating SA-57 (10 mg/kg) from vehicle within 40 sessions, with full generalization occurring 1 to 2 hours postinjection. CP55,940, the dual FAAH-MAGL inhibitor JZL195 (4-​nitrophenyl 4-​(3-​phenoxybenzyl)piperazine-​1-​carboxylate), and the MAGL inhibitors MJN110 (2,5-dioxopyrrolidin-1-yl 4-(bis(4-chlorophenyl)methyl)piperazine-1-carboxylate) and JZL184 (4-[Bis(1,3-benzodioxol-5-yl)hydroxymethyl]-1-piperidinecarboxylic acid 4-nitrophenyl ester) fully substituted for SA-57. Although the FAAH inhibitors PF-3845 ((N-3-pyridinyl-4-[[3-[[5-(trifluoromethyl)-2-pyridinyl]oxy]phenyl]methyl]-1-piperidinecarboxamide) and URB597 (cyclohexylcarbamic acid 3'-(aminocarbonyl)-[1,1'-biphenyl]-3-yl ester) did not substitute for SA-57, PF-3845 produced a 2-fold leftward shift in the MJN110 substitution dose-response curve. In addition, the CB1 receptor antagonist rimonabant blocked the generalization of SA-57, as well as substitution of CP55,940, JZL195, MJN110, and JZL184. These findings suggest that MAGL inhibition plays a major role in the CB1 receptor-mediated SA-57 training dose, which is further augmented by FAAH inhibition.

SUMMARY Clinical evidence suggests that rapid and sustained antidepressant action can be attained with a single exposure to psychedelics. However, the biological substrates and key mediators of psychedelics’ enduring action remain unknown. Here, we show that a single administration of the psychedelic DOI produces fast-acting effects on frontal cortex dendritic spine structure and acceleration of fear extinction via the 5-HT2A receptor. Additionally, a single dose of DOI leads to changes in chromatin organization, particularly at enhancer regions of genes involved in synaptic assembly that stretch for days after the psychedelic exposure. These DOI-induced alterations in the neuronal epigenome overlap with genetic loci associated with schizophrenia, depression, and attention deficit hyperactivity disorder. Together, these data support that epigenomic-driven changes in synaptic plasticity sustain psychedelics’ long-lasting antidepressant action but also warn about potential substrate overlap with genetic risks for certain psychiatric conditions., In brief de la Fuente Revenga et al. characterize in-depth molecular changes and behavioral adaptations following exposure to the psychedelic drug DOI. Their findings provide a molecular framework to understand the lingering effects of psychedelics in synaptic plasticity and rodent models of depression., Graphical Abstract

The emergence of several fentanyl-related substances in the recreational drug marketplace has resulted in a surge of opioid overdose deaths in the United States. Many of these substances have never been examined in living organisms under controlled conditions. In the present study, seven fentanyl-related substances were tested in adult male Swiss Webster mice for their effects on locomotion and antinociception and compared to those of fentanyl and morphine. In locomotor activity tests, fentanyl (1, 10 mg/kg), morphine (100, 180 mg/kg), isobutyrylfentanyl (10 mg/kg), crotonylfentanyl (10 mg/kg), para-fluorobutyrylfentanyl (10, 100 mg/kg), para-methoxybutyrylfentanyl (10 mg/kg), thiophenefentanyl (100 mg/kg), and benzodioxolefentanyl (0.1 mg/kg) produced significant (p ≤ 0.05) dose-dependent increases in locomotion. Valerylfentanyl, however, was without effects on locomotion up to 100 mg/kg. In warm-water tail-withdrawal tests, all substances produced significant (p ≤ 0.05) dose-dependent increases in antinociception with increasing ED50 values (CI) of isobutyrylfentanyl [0.0768 mg/kg (0.044–0.128)] > fentanyl [0.0800 mg/kg (0.0403–0.164)] > para-methoxybutyrylfentanyl [0.106 mg/kg (0.0516–0.195)] > crotonylfentanyl [0.226 mg/kg (0.176–0.292)] > para-fluorobutyrylfentanyl [0.908 mg/kg (0.459–1.58)] > thiophenefentanyl [4.66 mg/kg (3.65–5.95)] > valerylfentanyl [6.43 mg/kg (3.91–10.5)] > morphine [7.82 mg/kg (5.42–11.0)] > benzodioxolefentanyl [46.3 mg/kg (25.8–83.4)]. Naltrexone (1 mg/kg) increased antinociceptive ED50 values several fold in decreasing magnitudes of isobutyrylfentanyl (233x) > para-methoxybutyrylfentanyl (37.7x) > thiophenefentanyl (34.6x) > valerylfentanyl (11.9x) > para-fluorobutyrylfentanyl (10.9x) > benzodioxolefentanyl (8.42x) > crotonylfentanyl (6.27x) > fentanyl (3.95x) > morphine (1.48x). These findings establish that locomotor and antinociceptive effects of several fentanyl-related substances are similar to those of morphine and fentanyl and are mediated by opioid receptors. This article is part of the Special Issue entitled ‘New Vistas in Opioid Pharmacology’.

Opioid abuse has been a global menace for centuries, but the proliferation of synthetic opioids and their use within this current decade have created epidemic-level harms in some countries. According to the United Nations Office on Drugs and Crime, almost 12 million years were estimated loss of "healthy" life resulting in premature death and disability attributable to global opioid abuse just in 2015. Law enforcement and regulatory authorities have been particularly challenged abating the spread of synthetic opioids because soon after controlling the currently recognized abused opioids, their structures are tweaked, and new entities replace them. Drug racketeers have most often exploited the fentanyl phenylpiperidine structure in this regard, but non-fentanyl-like and classical morphinan-like structures have been pirated as well. A growing number of anecdotal reports identify respiratory depression induced by these newer synthetic opioids to be especially refractive to reversal by antagonists, with consequently high levels of lethality. This review examines three of these synthetic opioids representing three chemical classes (U-47700, MT-45, and acetylfentanyl) that have emerged to be of such menace that they have been brought under international control in recent years and addresses factors that could make synthetic opioids especially untreatable by opioid antagonists.

Incidence of prescription opioid abuse and overdose, often led by oxycodone, continues to increase, producing twice as many overdose deaths as heroin. Surprisingly, preclinical reports relevant to oxycodone’s abuse-related effects are relatively sparse considering its history and patient usage. The goal of this study was to characterize dose- and time-dependent effects of acute and repeated oxycodone administration in a frequency-rate intracranial self-stimulation (ICSS) procedure, an assay often predictive of drug-related reinforcing effects, in male Sprague-Dawley rats. We hypothesized that oxycodone would produce a biphasic profile of rate-increasing and rate-decreasing effects maintained by ICSS similar to μ-opioid receptor agonists. Oxycodone (0.03, 0.3, 1, and 3 mg/kg, s.c.) produced dose- and time-dependent alterations on ICSS, with the predicted biphasic profile of rate-increasing effects at lower stimulation frequencies followed by rate-decreasing effects at higher frequencies. Peak effects were observed between 30 and 60 minutes, which were reversed by naloxone pretreatment (30 minutes). Tolerance to rate-decreasing effects was observed over a 5-day period when rats were treated with 1 mg/kg oxycodone twice a day. Subsequently, the dosing regimen was increased to 3 mg/kg twice a day over 10 days, although further marked tolerance did not develop. When then challenged with 10 mg/kg naloxone, a significant suppression below baseline levels of ICSS-maintained responding occurred indicative of dependence that recovered to baseline within 5 hours. The results of this study provide the first report of acute and chronic effects of oxycodone on responding maintained by ICSS presentation and the use of ICSS-maintained responding to characterize its tolerance and dependence effects.

Preclinical studies revealed contribution of N-methyl-D-aspartate receptors (NMDARs) to a variety of neuropsychiatric diseases including alcoholism, but development of NMDAR antagonists for therapeutic use has been a challenge, in part due to severe side effects. One of the key intracellular events resulting from stimulation of NMDAR is activation of calpains—calcium-dependent cysteine proteases. Here we studied whether inhibition of calpains would produce therapeutic-like effects of NMDAR antagonists but without their NMDAR-mediated side-effect profile. The calpain inhibitor A-705253 (3–10 mg/kg) was tested in a model of cue-induced reinstatement of alcohol-seeking behavior in post-dependent Wistar rats and in an alcohol deprivation effect (ADE) model in long-term alcohol drinking Wistar rats, two behavioral models for alcohol-seeking and relapse, respectively. We also tested the effect of A-705253 on the saccharine deprivation effect (SDE) as a selectivity measure. Acute treatment with A-705253 dose-dependently reduced cue-induced reinstatement of alcohol-seeking behavior. Repeated administration of A-705253 caused significant reductions of relapse-like excessive alcohol intake during the post-abstinence drinking days, an effect that persisted during two more successive drug-free drinking weeks, which was selective for the ADE as the SDE was unaffected. However, A-705253 did not produce psychostimulant, cognition impairing (delayed-matching-to-position), or psychotomimetic effects (specifically, phencyclidine discriminative stimulus effects). Taken together, these results demonstrate the involvement of calpains in alcohol-seeking and relapse and present a rationale for a novel pharmacological intervention that may reduce craving and relapse with minimal side effects in alcohol-dependent patients.

Haloperidol is an effective antipsychotic drug for treatment of schizophrenia, but prolonged use can lead to debilitating side effects. To better understand the effects of long-term administration, we measured global metabolic changes in mouse brain following 3 mg/kg/day haloperidol for 28 days. These conditions lead to movement-related side effects in mice akin to those observed in patients after prolonged use. Brain tissue was collected following microwave tissue fixation to arrest metabolism and extracted metabolites were assessed using both liquid and gas chromatography mass spectrometry (MS). Over 300 unique compounds were identified across MS platforms. Haloperidol was found to be present in all test samples and not in controls, indicating experimental validity. Twenty-one compounds differed significantly between test and control groups at the p < 0.05 level. Top compounds were robust to analytical method, also being identified via partial least squares discriminant analysis. Four compounds (sphinganine, N-acetylornithine, leucine and adenosine diphosphate) survived correction for multiple testing in a non-parametric analysis using false discovery rate threshold < 0.1. Pathway analysis of nominally significant compounds (p < 0.05) revealed significant findings for sphingolipid metabolism (p = 0.015) and protein biosynthesis (p = 0.024). Altered sphingolipid metabolism is suggestive of disruptions to myelin. This interpretation is supported by our observation of elevated N-acetyl-aspartyl-glutamate in the haloperidol-treated mice (p = 0.004), a marker previously associated with demyelination. This study further demonstrates the utility of murine neurochemical metabolomics as a method to advance understanding of CNS drug effects.

The plenary session at the 2016 Behavior, Biology and Chemistry: Translational Research in Addiction Conference focused on glia as potential players in the development, persistence and treatment of substance use disorders. Glia partake in various functions that are important for healthy brain activity. Drugs of abuse alter glial cell activity producing several perturbations in brain function that are thought to contribute to behavioral changes associated with substance use disorders. Consequently, drug-induced changes in glia-driven processes in the brain represent potential targets for pharmacotherapeutics treating substance use disorders.Four speakers presented preclinical and clinical research illustrating the effects that glial modulators have on abuse-related behavioral effects of psychostimulants and opioids. This review highlights some of these findings and expands its focus to include other research focused on drug-induced glia abnormalities and glia-focused treatment approaches in substance use disorders.Preclinical findings show that drugs of abuse induce neuroinflammatory signals and disrupt glutamate homeostasis through their interaction with microglia and astrocytes. Preclinical and clinical studies testing the effects of glial modulators show general effectiveness in reducing behaviors associated with substance use disorders.The contribution of drug-induced glial activity continues to emerge as an intriguing target for substance use disorder treatments. Clinical investigations of glial modulators have yielded promising results on substance use measures and indicate that they are generally safe and well-tolerated. However, results have not been entirely positive and more questions remain for continued exploration in the development and testing of glial-directed treatments for substance use disorders.

Substantial challenges exist for investigating the cannabinoid receptor type 1 (CB1)-mediated discriminative stimulus effects of the endocannabinoids, 2-arachidonoylglycerol (2-AG) and N-arach-idonoylethanolamine (anandamide; AEA), compared with exogenous CB1 receptor agonists, such as Δ9-tetrahydrocannabinol (THC) and the synthetic cannabinoid CP55,940. Specifically, each endocannabinoid is rapidly degraded by the respective hydrolytic enzymes, monoacylglycerol lipase (MAGL) and fatty acid amide hydrolase (FAAH). Whereas MAGL inhibitors partially substitute for THC and fully substitute for CP55,940, FAAH inhibitors do not substitute for either drug. Interestingly, combined FAAH-MAGL inhibition results in full THC substitution, and the dual FAAH-MAGL inhibitor SA-57 serves as its own discriminative training stimulus. Because MAGL inhibitors fully substitute for SA-57, we tested whether the selective MAGL inhibitor MJN110 would serve as a training stimulus. Twelve of 13 C57BL/6J mice learned to discriminate MJN110 from vehicle, and the CB1 receptor antagonist rimonabant dose-dependently blocked its discriminative stimulus. CP55,940, SA-57, and another MAGL inhibitor JZL184, fully substituted for MJN110. In contrast, the FAAH inhibitor PF-3845 failed to substitute for the MJN110 discriminative stimulus, but produced a 1.6 (1.1–2.2; 95% confidence interval) leftward shift in the MJN110 dose-response curve. Inhibitors of other relevant enzymes (i.e., ABHD6, COX-2) and nicotine did not engender substitution. Diazepam partially substituted for MJN110, but rimonabant failed to block this partial effect. These findings suggest that MAGL normally throttles 2-AG stimulation of CB1 receptors to a magnitude insufficient to produce cannabimimetic subjective effects. Accordingly, inhibitors of this enzyme may release this endogenous brake producing effects akin to those produced by exogenously administered cannabinoids.

Objective We explored the antiviral therapeutic potential of ibudilast (AV411, MN-166) and its amino analog, AV1013. Methods We analyzed whether Ibudilast, a nonselective cyclic AMP phosphodiesterase inhibitor that has been used clinically in Asia for bronchial asthma, poststroke dizziness, and ocular allergies, and AV1013, attenuate HIV-1 replication and the synergistic interactions seen with opiate abuse-HIV-1 comorbidity in neuronal death and inflammation. Results AV411 and AV1013 inhibited replication by HIV-1 in microglia and significantly suppressed Tat ± morphine-induced tumor necrosis factor-α and MIF production, the activation of the nuclear factor-kappa B subunit p65, and neuronal death. AV411 and AV1013 prevented HIV-1 replication, and attenuated tumor necrosis factor-α and MIF release at concentrations of 100 nmol/l and 1 μmol/l, which are likely achievable at clinical doses. More importantly, co-exposure with morphine did not negate the inhibitory actions of AV411. Conclusion Collectively, our data suggest that AV411 and its amino analog, AV1013, may be useful neuroprotective agents counteracting neurotoxicity caused by infected and activated glia, and implicate them as potential therapies for the management of HIV-associated neurocognitive disorders in an opioid-abusing population.

For thousands of years opioids have been the first-line treatment option for pain management. However, the tolerance and addiction potential of opioids limit their applications in clinic. NFP, a MOR/KOR dual-selective opioid antagonist, was identified as a ligand that significantly antagonized the antinociceptive effects of morphine with lesser withdrawal effects than naloxone at similar doses. To validate the potential application of NFP in opioid addiction treatment, a series of in vitro and in vivo assays were conducted to further characterize its pharmacological profile. In calcium mobilization assays and MOR internalization studies, NFP showed the apparent capacity to antagonize DAMGO-induced calcium flux and etorphine-induced MOR internalization. In contrast to the opioid agonists DAMGO and morphine, cells pretreated with NFP did not show apparent desensitization and down regulation of the MOR. Though in vitro bidirectional transport studies showed that NFP might be a P-gp substrate, in warm-water tail-withdrawal assays it was able to antagonize the antinociceptive effects of morphine indicating its potential central nervous system activity. Overall these results suggest that NFP is a promising dual selective opioid antagonist that may have the potential to be used therapeutically in opioid use disorder treatment.

Behavioral sensitization has been widely studied in animal models and is theorized to reflect neural modifications associated with human psychostimulant addiction. While the mesolimbic dopaminergic pathway is known to play a role, the neurochemical mechanisms underlying behavioral sensitization remain incompletely understood. In this study, we conducted the first metabolomics analysis to globally characterize neurochemical differences associated with behavioral sensitization. Methamphetamine (MA)-induced sensitization measures were generated by statistically modeling longitudinal activity data for eight inbred strains of mice. Subsequent to behavioral testing, nontargeted liquid and gas chromatography-mass spectrometry profiling was performed on 48 brain samples, yielding 301 metabolite levels per sample after quality control. Association testing between metabolite levels and three primary dimensions of behavioral sensitization (total distance, stereotypy and margin time) showed four robust, significant associations at a stringent metabolome-wide significance threshold (false discovery rate, FDR

The human ANKS1B gene encodes an activity-dependent effector of post-synaptic signaling. It was recently associated with neuropsychiatric phenotypes in genome-wide studies. While the biological function of ANKS1B has been partly elucidated, its role in behavior is poorly understood. Here, we breed and characterize a full knockout (KO) for murine Anks1b. We found that the homozygous KO genotype was partially lethal, showing significant deviation from expected segregation ratios at weaning. Behaviorally, KOs exhibited no difference in baseline acoustic startle response, but showed deficits in prepulse inhibition (PPI). KOs also exhibited locomotor hyperactivity and increased stereotypy at baseline. Administration of ketamine, a non-competitive NMDA-receptor antagonist, greatly exacerbated locomotor activity in the KOs at lower doses, but genotype groups were almost indistinguishable as dose increased. Stereotypy showed a complex response to ketamine in the KOs, with elevated stereotypy at lower doses and markedly less at high doses, compared to wild type. Our study is the first to probe the behavioral phenotypes associated with ablation of Anks1b. Deficits in PPI, locomotor hyperactivity, elevated stereotypy and altered response to NMDA receptor antagonism are murine behavioral outcomes with translational relevance for psychiatric disorders. These findings are also consistent with the role of Anks1b as an effector of glutamatergic signaling. As an intermediary between post-synaptic receptor stimulation and long-term changes to neuronal protein expression, further investigation of Anks1b is warranted.

Abuse of prescription opioids, such as oxycodone, has markedly increased in recent decades. While oxycodone’s antinociceptive effects have been detailed in several preclinical reports, surprisingly few preclinical reports have elaborated its abuse-related effects. This is particularly surprising given that oxycodone has been in clinical use since 1917. In a novel oral operant self-administration procedure, C57BL/6J mice were trained to self-administer water before introducing increasing concentrations of oxycodone (0.056–1.0 mg/ml) under post-prandial conditions during daily, 3-h test sessions. As the concentration of oxycodone increased, the numbers of deliveries first increased, then decreased in an inverted U-shape fashion characteristic of the patterns of other drugs self-administered during limited access conditions. After post-prandial conditions were removed, self-administration at the highest concentration was maintained suggesting oral oxycodone served as a positive reinforcer. In other mice, using a novel regimen of physical dependence, mice were administered increasing doses of oxycodone (9.0–33.0 mg/kg, s.c.) over 9 days, challenged with naloxone (0.1–10.0 mg/kg, s.c.), and then observed for 30 min. Naloxone dose-dependently increased the observed number of somatic signs of withdrawal, suggesting physical dependence of oxycodone was induced under this regimen. This is the first report demonstrating induction of oral operant self-administration of oxycodone and dose-dependent precipitations of oxycodone withdrawal in C57BL/6J mice. The use of oral operant self-administration as well as the novel physical dependence regimen provides useful approaches to further examine the abuse- and dependence-related effects of this highly abused prescription opioid.

Over 800,000 Americans abuse the psychomotor stimulant, methamphetamine, yet its abuse is without an approved medication. Methamphetamine induces hypermotor activity, and sensitization to this effect is suggested to represent aspects of the addiction process. Methamphetamine’s regulation of 3'-5'-cyclic adenosine monophosphate (cAMP) levels may be partially responsible for its behavioral effects, and compounds that inhibit phosphodiesterase (PDE), the enzyme that degrades cAMP, can alter methamphetamine-induced behaviors. Methamphetamine also activates glial cells and causes a subsequent increase in pro-inflammatory cytokine levels. Modulation of glial cell activation is associated with changes in behavioral responses, and substances that oppose inflammatory activity can attenuate drug-induced behaviors. Ibudilast (aka AV411; 3-isobutyryl-2-isopropylpyrazolo-[1,5-a]pyridine), inhibits both PDE and glial pro-inflammatory activity. Ibudilast’s amino analogue, AV1013, modulates similar glial targets but negligibly inhibits PDE. The present study determined whether ibudilast and AV1013 would attenuate methamphetamine-induced locomotor activity and its sensitization in C57BL/6J mice. Mice were treated b.i.d. with ibudilast (1.8-13 mg/kg), AV1013 (10-56mg/kg) or their vehicles intraperitoneally for 7 days, beginning 48 h before 5 days of daily 1-h locomotor activity tests. Each test was initiated by either a methamphetamine (3 mg/kg) or a saline injection. Ibudilast significantly (P

Taranabant (aka, MK-0364) was tested in rhesus monkeys that were trained to self-administer cocaine and in rats that were trained to discriminate Δ9-Tetrahydrocannabinol (THC) from vehicle in order to facilitate inferences about taranabant's potential to produce subjective effects similar to THC and to be used for recreational purposes (i.e., for its potential abuse liability). Four rhesus monkeys with chronically implanted intravenous catheters were trained to lever press according to fixed-ratio 50 (FR50) schedules reinforced with infusions of 0.03 or 0.01 mg/kg cocaine during daily, 1-h experimental sessions. During substitution tests, cocaine was replaced with a test dose (0.001–0.1 mg/kg) of taranabant for four, consecutive daily sessions. Additionally, 10 adult male Long-Evans hooded rats were trained to discriminate 3 mg/kg i.p. THC from vehicle using a food-reinforced, lever press procedure. Following training, doses of THC (0.1–10 mg/kg i.p.) and taranabant (0.3–17 mg/kg i.p.) were tested. None of the four monkeys self-administered taranabant above vehicle-control levels, and for three of the monkeys levels of responding were, at times, lower than those of vehicle itself. During discrimination tests, increases in the dose of THC or taranabant resulted in ED50 values (±95% CI) of 9.65 mg/kg (7.03–13.27) and of 20.51 mg/kg (8.13–51.64), respectively, for reducing response rates. THC, but not taranabant, dose-dependently generalized to the 3-mg/kg THC discriminative stimulus at levels of 80% and above. These results indicate that taranabant does not exhibit THC-like agonistic activity in vivo, and consequentially suggest it would be unlikely that it would have a THC-like abuse potential. Drug Dev Res 70:577–584, 2009. © 2009 Wiley-Liss, Inc.

Beta2 subunit containing nicotinic acetylcholine receptors (beta2(*)nAChRs; asterisk ((*)) denotes assembly with other subunits) are critical for nicotine self-administration and nicotine-associated dopamine (DA) release that supports nicotine reinforcement. The alpha6 subunit assembles with beta2 on DA neurons where alpha6beta2(*)nAChRs regulate nicotine-stimulated DA release at neuron terminals. Using local infusion of alpha-conotoxin MII (alpha-CTX MII), an antagonist with selectivity for alpha6beta2(*)nAChRs, the purpose of these experiments was to determine if alpha6beta2(*)nAChRs in the nucleus accumbens (NAc) shell are required for motivation to self-administer nicotine. Long-Evans rats lever-pressed for 0.03 mg/kg, i.v., nicotine accompanied by light+tone cues (NIC) or for light+tone cues unaccompanied by nicotine (CUEonly). Following extensive training, animals were tested under a progressive ratio (PR) schedule that required an increasing number of lever presses for each nicotine infusion and/or cue delivery. Immediately before each PR session, rats received microinfusions of alpha-CTX MII (0, 1, 5, or 10 pmol per side) into the NAc shell or the overlying anterior cingulate cortex. alpha-CTX MII dose dependently decreased break points and number of infusions earned by NIC rats following infusion into the NAc shell but not the anterior cingulate cortex. Concentrations of alpha-CTX MII that were capable of attenuating nicotine self-administration did not disrupt locomotor activity. There was no effect of infusion on lever pressing in CUEonly animals and NAc infusion alpha-CTX MII did not affect locomotor activity in an open field. These data suggest that alpha6beta2(*)nAChRs in the NAc shell regulate motivational aspects of nicotine reinforcement but not nicotine-associated locomotor activation.

Despite considerable evidence that chronic opiate use selectively affects the pathophysiologic consequences of human immunodeficiency virus type 1 (HIV-1) infection in the nervous system, few studies have examined whether neuro-acquired immune deficiency syndrome (neuroAIDS) might intrinsically alter the pharmacologic responses to chronic opiate exposure. This is an important matter because HIV-1 and opiate abuse are interrelated epidemics, and HIV-1 patients are often prescribed opiates as a treatment of HIV-1-related neuropathic pain. Tolerance and physical dependence are inevitable consequences of frequent and repeated administration of morphine. In the present study, mice expressing HIV-1 Tat in a doxycycline (DOX)-inducible manner [Tat(+)], their Tat(-) controls, and control C57BL/6 mice were chronically exposed to placebo or 75-mg morphine pellets to explore the effects of Tat induction on morphine tolerance and dependence. Antinociceptive tolerance and locomotor activity tolerance were assessed using tail-flick and locomotor activity assays, respectively, and physical dependence was measured with the platform-jumping assay and recording of other withdrawal signs. We found that Tat(+) mice treated with DOX [Tat(+)/DOX] developed an increased tolerance in the tail-flick assay compared with control Tat(-)/DOX and/or C57/DOX mice. Equivalent tolerance was developed in all mice when assessed by locomotor activity. Further, Tat(+)/DOX mice expressed reduced levels of physical dependence to chronic morphine exposure after a 1-mg/kg naloxone challenge compared with control Tat(-)/DOX and/or C57/DOX mice. Assuming the results seen in Tat transgenic mice can be generalized to neuroAIDS, our findings suggest that HIV-1-infected individuals may display heightened analgesic tolerance to similar doses of opiates compared with uninfected individuals and show fewer symptoms of physical dependence.

The N-methyl-D-aspartate (NMDA) receptor has been implicated in mediating the reinforcing effects of abused drugs. Some reports indicate the uncompetitive NMDA antagonist, memantine, modulates the conditioned and unconditioned effects of stimulants in rats.The objective of this study was to evaluate the effects of memantine on the primary and conditioned reinforcing effects of cocaine in the rhesus monkey.Rhesus monkeys were trained to press levers reinforced with either cocaine-associated stimuli (brief stimuli, BS) or 30-microg/kg cocaine infusion during daily, 75-min experimental sessions in which the reinforcers were independently available in separate components according to identical progressive ratio (PR) schedules. Memantine (0.3-10 mg/kg), and as comparators, haloperidol (0.001-0.1 mg/kg) and cocaine (0.01-1 mg/kg), were administered 5 min before experimental sessions.Memantine (0.3-3 mg/kg) produced decreases in responding maintained by BS presentations at some doses which failed to affect cocaine self-administration when measured during equivalent periods early in the experimental session. Memantine (3 mg/kg) increased cocaine self-administration, however, later in the session. A low dose of haloperidol (0.001 mg/kg) increased the number of BS presentations, whereas higher doses decreased their number. Cocaine self-administration was not significantly affected by haloperidol until a behaviorally suppressant dose (0.1 mg/kg) was administered. Pretreatment with high doses of cocaine (0.3 and 1 mg/kg) decreased responding maintained by both reinforcers.These results suggest that while memantine may attenuate the conditioned reinforcing effects of cocaine-associated stimuli, it may also occasion increase levels of cocaine self-administration. These findings support the hypothesis that the NMDA receptor can play a role in modulating the conditioned and primary reinforcing effects of cocaine.

Stress and depression have been linked to relapse of cocaine abuse. Antagonism of the kappa opioid receptor (KOR) has been reported to attenuate some effects of stressors, and antagonism of the KOR has been reported to have antidepressant-like properties. Our objective was to determine whether the potent and selective KOR antagonist, (3R)-7-hydroxy-N-{(1S)-1-{[(3R,4R)-4-(3-hydroxyphenyl)-3,4-dimethyl-1-piperidinyl]methyl}-2-methylpropyl}-1,2,3,4-tetrahydro-3-isoquinoline-carboxamide (JDTic), can reduce the ability of a stressor (intermittent footshock) to reinstate cocaine-seeking behavior and to have antidepressant-like effects in the forced swim test (FST). Male Long–Evans hooded rats were trained to lever-press, reinforced with 0.5 mg/kg i.v. infusion of cocaine, according to fixed ratio 1 reinforcement schedules during daily 2-h experimental sessions. After performance had stabilized, lever pressing was extinguished for 12 consecutive sessions, and doses of 0 (vehicle), 3, 10, and 30 mg/kg JDTic were then administered i.g. to separate groups of 12 rats. Twenty four hours later, the rats were given 15 min of intermittent footshock (0.87 mA, 0.5 s activation time, average interactivation interval of 40 s) or a 17-mg/kg i.p. administration of cocaine prime followed by a 2-h reinstatement test session. JDTic was also evaluated for its ability to block diuresis induced by the KOR agonist, U50,488H (10 mg/kg, s.c.), during 5-h test sessions beginning 1 h after footshock reinstatement tests to verify its KOR antagonist activity. In the FST, male Sprague–Dawley rats were treated with either nor-binaltorphimine (nor-BNI) or JDTic (both at 0.3, 1, 3, or 10 mg/kg, injected s.c. 23 h before), or desipramine (5.6, 10, or 17 mg/kg, injected i.p. 23, 5, and 1 h before) and placed in a cylinder of water, during which the predominance of immobility, swimming, and climbing were scored during 5-s intervals for 5 min. The 10- and 30-mg/kg doses of JDTic significantly reduced footshock-induced reinstatement of responding previously reinforced by cocaine and significantly attenuated U50,488H-induced diuresis. In contrast, JDTic did not affect cocaine-prime-induced reinstatement. Both nor-BNI and JDTic decreased immobility and increased swimming time in the FST, similar to the antidepressant desipramine. Depression and stress are two states during cocaine abstinence which users identify as precipitating relapse, and JDTic may have properties which attenuate both.

Relatively little is known about the role of dose, duration, and pattern of nicotine exposure in the development of dependence. Disruption of learned behavior during antagonist-precipitated withdrawal can be a sensitive, quantitative measure of behavioral dependence.The present study sought to determine whether behavioral dependence upon nicotine could be induced in rats and, if so, what exposure conditions were essential for inducing it. Our primary focus was on whether continuous exposure over several days was necessary to produce dependence.Male Sprague-Dawley rats were trained to lever press under fixed-ratio 10 schedules of food reinforcement during daily, 15-min experimental sessions. Nicotine was then administered s.c. via osmotic minipumps that delivered various nicotine dosage regimens, some including 24-h nicotine-free periods, to manipulate pattern of exposure. The presence of dependence was tested with challenges with the nicotinic acetylcholine receptor antagonist, mecamylamine, or during spontaneous withdrawal.After 7 days of 3, 6, and 12 mg kg(-1) day(-1) nicotine administration, response rates were significantly reduced in nicotinized, but not in saline-treated rats following mecamylamine challenges. Subsequent studies demonstrated that 4 days, but not 3 days, of cumulative 3 mg kg(-1) day(-1) nicotine administration was sufficient to induce dependence. The induction of dependence could be prevented by imposing a nicotine-free period between the first and second days during these 4-day regimens but not at other times.Behavioral dependence upon nicotine can be induced in the rat, and its induction is dependent upon its cumulative duration and pattern of exposure suggesting that tobacco dependencies could be controlled by similar determinants.

Reports have indicated that administration of nicotine inhibits, while withdrawal of chronically administered nicotine augments effects of serotonergic 5HT2A/2C agonists.It was our objective to determine whether 5HT2A/2C agonists can modulate the discriminative stimulus effects of nicotine in rats or its locomotor activity effects in mice.Adult male Sprague-Dawley rats were trained to discriminate 0.3 mg/kg nicotine base from saline in a two-lever, fixed-ratio (FR10), food-reinforced, operant-conditioning task during daily (Monday-Friday) 15-min experimental sessions. After characterizing a dose-response curve for nicotine, we tested the ability of the 5HT(2A/2C) agonists (+/-)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane HCL (DOI; 0.18-1.0 mg/kg) and 1-(4-bromo-2, 5-dimethoxyphenyl)-2-aminopropane (DOB; 0.1-1.0 mg/kg), the 5HT2C agonist 6-chloro-2-(1-piperazinyl)pyrazine hydrochloride (MK 212; 0.1 mg/kg-1.0 mg/kg), and the 5HT1A agonist (+/-)-8-hydroxy-2-(di-n-propylamino)tetralin hydrobromide (8-OH-DPAT; 0.01 mg/kg-1.0 mg/kg) to modulate nicotine's discriminative stimulus effects. After finding that DOI was able to attenuate the percentage nicotine lever responding (%NLR), we tested for it to also reverse nicotine's effects on locomotor activity in mice.The 5HT2A/2C agonists-in particular DOI-dose dependently attenuated %NLR. The effects of DOI were reversed by the 5HT2A/2C antagonist ketanserin. MK 212 and 8-OH-DPAT had irregular effects among rats and only reduced %NLR to below 50% levels at doses markedly suppressing responding. DOI also dose dependently blocked nicotine's acute rate-lowering locomotor activity effects.These results indicate that activation of serotonin 5HT2A/2C receptors can blunt the discriminative stimulus and locomotor activity effects of nicotine and presents the possibility that activation of these receptors might also be able to attenuate other effects of nicotine.

The noncompetitive NMDA receptor antagonist MK801 (dizocilpine) produces behavioral stimulation mediated, in part, through indirect activation of the dopamine (DA) system. Previous reports indicate that D2/3 agonists inhibit MK801-induced stereotypies; however, it is unclear if these agonists also attenuate MK801-induced locomotion. As such, the ability of the D2/3 agonists, quinelorane and quinpirole, and the partial D3 agonist, BP897, to attenuate the locomotor activating effects of MK801 was examined in mice. MK801 (0.1-1.0 mg/kg) produced a biphasic effect on total distance traveled with the intermediate dose of 0.3 mg/kg producing the greatest stimulation. The increase in MK801-induced total distance traveled was attenuated by the coadministration of quinelorane and quinpirole at doses that alone had no effect on activity. Similarly, the partial D3 agonist, BP897, blocked the effects of MK801. The D3-preferring antagonist, nafadotride, reversed the attenuation of quinelorane and partially reversed the attenuation of quinpirole. The D2-preferring antagonist, eticlopride, reversed the attenuating effects of quinelorane, but was not effective against quinpirole. Nafadotride and eticlopride were ineffective against the attenuating effects of BP897 on MK801-induced locomotion. Because BP897 is a partial agonist it was tested against quinelorane/MK801 and quinpirole/MK801 combinations. BP897 reversed the attenuating effects of quinelorane, but not those of quinpirole on MK801's effects. These results demonstrate that the DA system, through D2/3 receptor activation, modulates the locomotor activating effects produced by noncompetitive NMDA receptor blockade.

In our effort to develop high-affinity ligands for the dopamine transporter which might find potential use as cocaine medication, a polar hydroxy substituent was introduced into the piperidine ring of one of our disubstituted lead analogues derived from 1-[2-(diphenylmethoxy)-ethyl]-4-(3-phenylpropyl)piperazine (GBR 12935). Both cis- and trans-3-hydroxy derivatives were synthesized and the racemic trans isomer, (+/-)-5, was further resolved into two enantiomers. Newly synthesized compounds were characterized for their binding affinity at the dopamine, serotonin, and norepinephrine transporter systems in rat brain. The two enantiomers (+)-5 and (-)-5 exhibited marked differential affinities at the dopamine transporter with (+)-5 being 122-fold more potent than (-)-5 in inhibiting radiolabeled cocaine analogue binding (IC(50); 0.46 vs 56.7 nM) and 9-fold more active for inhibiting dopamine uptake (IC(50); 4.05 vs 38.0 nM). Furthermore, the most active (+)-5 was 22-fold more potent at the dopamine transporter compared to the standard GBR 12909. Absolute configuration of one of the enantiomers was determined unambiguously by X-ray structural analysis. In in vivo locomotor activity studies, the enantiomer (+)-5 and the racemic (+/-)-5, but not (-)-5, exhibited stimulant activity with a long duration of effect. All three compounds, (+)-5, (-)-5, and (+/-)-5, within the dose range tested, partially (50%) but incompletely (80%) produced cocaine-like responses in mice trained to discriminate 10 mg/kg ip cocaine from vehicle. Compound (-)-5 was distinctive in this regard in that, unlike (+)-5 and (+/-)-5, it did not affect locomotor activity yet, but similar to them, was able to engender (albeit incompletely) cocaine-like responses.

All drugs entering the central nervous system (CNS) may initially be viewed as having a potential risk for abuse. As there is no single test or assessment procedure likely to offer a complete characterization of abuse liability, preclinical assessment uses several experimental approaches. This chapter discusses intracranial self-stimulation (ICSS) as a tool that could complement existing assessment strategies. Many drugs of abuse facilitate ICSS in laboratory animals prepared with electrodes implanted into brain “reward” areas. In addition to fairly robust predictive validity, ICSS may have a number of important advantages relative to other procedures in studying: (1) drug–drug interactions; (2) quantitative comparisons among drugs; (3) complex drug formulations, especially those intended to be abuse resistant; and (4) repeated drug exposures. This chapter argues for the standardization of ICSS experimental protocols to enable a definitive assessment of the usefulness of ICSS in predicting the abuse potential of drugs.

Glutamate receptor-dependent neural plasticity is thought to be implicated in memory processes. Ionotropic N -methyl- d -aspartate- (NMDA) sensitive and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate- (AMPA) sensitive glutamate receptors have been particularly studied for their role in synaptic plasticity. Drugs can alter AMPA and NMDA receptor neurotransmission by competing for the glutamate site or other sites on these receptor proteins. Variants of the protein subunits forming AMPA and NMDA heteromers contribute to the complexity of pharmacological activity at these receptors. The NMDA receptor has numerous modulatory centers, including the glycine binding site, NR2B protein specific binding site, and an intrachannel (PCP) binding site. In this study, the accuracy and rate of rats performing under a Fixed Consecutive Number (FCN) operant task were measured after administrations of site-selective AMPA and NMDA receptor modulators. Test compounds included two glycine site NMDA agonists [(+)HA 966 and d -cycloserine], two NR2-B site NMDA antagonists (eliprodil and ifenprodil), an NMDA channel blocking antagonist (MK 801), and a competitively acting AMPA receptor antagonist (NBQX). The accuracy of FCN performance was not affected by response-rate-altering doses of (+) HA 966, d -cycloserine, eliprodil, ifenprodil, or NBQX. MK 801, on the other hand, reduced performance accuracy at several doses. These results are consistent with earlier studies suggesting that AMPA antagonists minimally affect working memory and that glycine and NR2B protein-specific modulatory sites may have advantages as targets for the development of medications intended to alter NMDA receptor-mediated transmission.