Dopamine D2 receptors in discrimination learning and spine enlargement.

Dopamine D2 receptors (D2Rs) are densely expressed in the striatum and have been linked to neuropsychiatric disorders such as schizophrenia1,2. High-affinity binding of dopamine suggests that D2Rs detect transient reductions in dopamine concentration (the dopamine dip) during punishment learning3-5. However, the nature and cellular basis of D2R-dependent behaviour are unclear. Here we show that tone reward conditioning induces marked stimulus generalization in a manner that depends on dopamine D1 receptors (D1Rs) in the nucleus accumbens (NAc) of mice, and that discrimination learning refines the conditioning using a dopamine dip. In NAc slices, a narrow dopamine dip (as short as 0.4 s) was detected by D2Rs to disinhibit adenosine A2A receptor (A2AR)-mediated enlargement of dendritic spines in D2R-expressing spiny projection neurons (D2-SPNs). Plasticity-related signalling by Ca2+/calmodulin-dependent protein kinase II and A2ARs in the NAc was required for discrimination learning. By contrast, extinction learning did not involve dopamine dips or D2-SPNs. Treatment with methamphetamine, which dysregulates dopamine signalling, impaired discrimination learning and spine enlargement, and these impairments were reversed by a D2R antagonist. Our data show that D2Rs refine the generalized reward learning mediated by D1Rs.

AMPA and NMDA receptors in dentate gyrus mediate memory for sucrose in two port discrimination task.

Although the phenomenon of memory formation and recall associated with the use of psychotropic drugs has been extensively studied, mechanisms underlying memories for natural reward have not been clarified. Herein, we test the hypothesis that glutamatergic receptors in the dentate gyrus play a role in memories associated with sucrose. We used pellet self-administration protocol to generate memories in two-port nose-poke discrimination task using male Wistar rats. During non-rewarded probe trial, the conditioned animals readily discriminated the active port versus inactive port and showed massive increase in mRNA expression of AMPA receptor subunit genes (gria2, gria3) as well as c-Fos protein in the DG. Access to sweet pellet further enhanced c-Fos expression in the DG. However, animals pre-treated with AMPA receptor antagonist CNQX (intra-DG), on exposure to operant chamber (no pellet), showed decreased discrimination as well as c-Fos expression. We suggest that AMPA receptors in DG mediate recall and consolidation of memories associated with sucrose consumption. CNQX pre-treated animals, if presented with sweet pellet on nose poke, exhibited high discrimination index coupled with increased c-Fos expression. In these CNQX treated rats, the DI was again decreased following administration of NMDA receptor antagonist AP5. We suggest that, although AMPA receptors are blocked, the access to sweet pellet may induce surge of glutamate in the DG, which in turn may reinstate memories via activation of erstwhile silent synapses in NMDA dependant manner.

Generalization of a positive-feature interoceptive morphine occasion setter across the rat estrous cycle.

INTRODUCTION: Interoceptive stimuli elicited by drug administration acquire conditioned modulatory properties of the induction of conditioned appetitive behaviours by exteroceptive cues. This effect may be modeled using a drug discrimination task in which the drug stimulus is trained as a positive-feature (FP) occasion setter (OS) that disambiguates the relation between an exteroceptive light conditioned stimulus (CS) and a sucrose unconditioned stimulus (US). We previously reported that females are less sensitive to generalization of a FP morphine OS than males, so we investigated the role of endogenous ovarian hormones in this difference. METHODS: Male and female rats received intermixed injections of 3.2 mg/kg morphine or saline before each daily training session. Training consisted of 8 presentations of the CS, each followed by access to sucrose on morphine, but not saline sessions. Following acquisiton, rats were tested for generalization of the morphine stimulus to 0, 1.0, 3.2, and 5.4 mg/kg morphine. Female rats were monitored for estrous cyclicity using vaginal cytology throughout the study. RESULTS: Both sexes acquired stable drug discrimination. A gradient of generalization was measured across morphine doses and this behaviour did not differ by sex, nor did it differ across the estrous cycle in females. CONCLUSIONS: Morphine generalization is independent of fluctuations in levels of sex and endogenous gonadal hormones in females under these experimental conditions.

Discriminative stimulus properties of Cannabis sativa terpenes in rats.

Cannabis is a pharmacologically complex plant consisting of hundreds of potentially active compounds. One class of compounds present in cannabis that has received little attention are terpenes. Traditionally thought to impart aroma and flavor to cannabis, it has become increasingly recognized that terpenes might exert therapeutic effects themselves. Several recent reports have also indicated terpenes might behave as cannabinoid type 1 (CB1) receptor agonists. This study aimed to investigate whether several terpenes present in cannabis produce discriminative stimulus effects similar to or enhance the effects of Δ 9 -tetrahydrocannabinol (THC). Subsequent experiments explored other potential cannabimimetic effects of these terpenes. Rats were trained to discriminate THC from vehicle while responding under a fixed-ratio 10 schedule of food presentation. Substitution testing was performed with the CB receptor agonist JWH-018 and the terpenes linalool, limonene, γ-terpinene and α-humulene alone. Terpenes were also studied in combination with THC. Finally, THC and terpenes were tested in the tetrad assay to screen for CB1-receptor agonist-like effects. THC and JWH-018 dose-dependently produced responding on the THC-paired lever. When administered alone, none of the terpenes produced responding predominantly on the THC-paired lever. When administered in combination with THC, none of the terpenes enhanced the potency of THC, and in the case of α-humulene, decreased the potency of THC to produce responding on the THC-paired lever. While THC produced effects in all four tetrad components, none of the terpenes produced effects in all four components. Therefore, the terpenes examined in this report do not have effects consistent with CB1 receptor agonist properties in the brain.

Serotonergic Innervations of the Orbitofrontal and Medial-prefrontal Cortices are Differentially Involved in Visual Discrimination and Reversal Learning in Rats.

Cross-species studies have identified an evolutionarily conserved role for serotonin in flexible behavior including reversal learning. The aim of the current study was to investigate the contribution of serotonin within the orbitofrontal cortex (OFC) and medial prefrontal cortex (mPFC) to visual discrimination and reversal learning. Male Lister Hooded rats were trained to discriminate between a rewarded (A+) and a nonrewarded (B-) visual stimulus to receive sucrose rewards in touchscreen operant chambers. Serotonin was depleted using surgical infusions of 5,7-dihydroxytryptamine (5,7-DHT), either globally by intracebroventricular (i.c.v.) infusions or locally by microinfusions into the OFC or mPFC. Rats that received i.c.v. infusions of 5,7-DHT before initial training were significantly impaired during both visual discrimination and subsequent reversal learning during which the stimulus-reward contingencies were changed (A- vs. B+). Local serotonin depletion from the OFC impaired reversal learning without affecting initial discrimination. After mPFC depletion, rats were unimpaired during reversal learning but slower to respond at the stimuli during all the stages; the mPFC group was also slower to learn during discrimination than the OFC group. These findings extend our understanding of serotonin in cognitive flexibility by revealing differential effects within two subregions of the prefrontal cortex in visual discrimination and reversal learning.

Chemogenetics Reveal an Anterior Cingulate-Thalamic Pathway for Attending to Task-Relevant Information.

In a changing environment, organisms need to decide when to select items that resemble previously rewarded stimuli and when it is best to switch to other stimulus types. Here, we used chemogenetic techniques to provide causal evidence that activity in the rodent anterior cingulate cortex and its efferents to the anterior thalamic nuclei modulate the ability to attend to reliable predictors of important outcomes. Rats completed an attentional set-shifting paradigm that first measures the ability to master serial discriminations involving a constant stimulus dimension that reliably predicts reinforcement (intradimensional-shift), followed by the ability to shift attention to a previously irrelevant class of stimuli when reinforcement contingencies change (extradimensional-shift). Chemogenetic disruption of the anterior cingulate cortex (Experiment 1) as well as selective disruption of anterior cingulate efferents to the anterior thalamic nuclei (Experiment 2) impaired intradimensional learning but facilitated 2 sets of extradimensional-shifts. This pattern of results signals the loss of a corticothalamic system for cognitive control that preferentially processes stimuli resembling those previously associated with reward. Previous studies highlight a separate medial prefrontal system that promotes the converse pattern, that is, switching to hitherto inconsistent predictors of reward when contingencies change. Competition between these 2 systems regulates cognitive flexibility and choice.

ß4-Nicotinic Receptors Are Critically Involved in Reward-Related Behaviors and Self-Regulation of Nicotine Reinforcement.

Nicotine addiction, through smoking, is the principal cause of preventable mortality worldwide. Human genome-wide association studies have linked polymorphisms in the CHRNA5-CHRNA3-CHRNB4 gene cluster, coding for the α5, α3, and ß4 nicotinic acetylcholine receptor (nAChR) subunits, to nicotine addiction. ß4*nAChRs have been implicated in nicotine withdrawal, aversion, and reinforcement. Here we show that ß4*nAChRs also are involved in non-nicotine-mediated responses that may predispose to addiction-related behaviors. ß4 knock-out (KO) male mice show increased novelty-induced locomotor activity, lower baseline anxiety, and motivational deficits in operant conditioning for palatable food rewards and in reward-based Go/No-go tasks. To further explore reward deficits we used intracranial self-administration (ICSA) by directly injecting nicotine into the ventral tegmental area (VTA) in mice. We found that, at low nicotine doses, ß4KO self-administer less than wild-type (WT) mice. Conversely, at high nicotine doses, this was reversed and ß4KO self-administered more than WT mice, whereas ß4-overexpressing mice avoided nicotine injections. Viral expression of ß4 subunits in medial habenula (MHb), interpeduncular nucleus (IPN), and VTA of ß4KO mice revealed dose- and region-dependent differences: ß4*nAChRs in the VTA potentiated nicotine-mediated rewarding effects at all doses, whereas ß4*nAChRs in the MHb-IPN pathway, limited VTA-ICSA at high nicotine doses. Together, our findings indicate that the lack of functional ß4*nAChRs result in deficits in reward sensitivity including increased ICSA at high doses of nicotine that is restored by re-expression of ß4*nAChRs in the MHb-IPN. These data indicate that ß4 is a critical modulator of reward-related behaviors.SIGNIFICANCE STATEMENT Human genetic studies have provided strong evidence for a relationship between variants in the CHRNA5-CHRNA3-CHRNB4 gene cluster and nicotine addiction. Yet, little is known about the role of ß4 nicotinic acetylcholine receptor (nAChR) subunit encoded by this cluster. We investigated the implication of ß4*nAChRs in anxiety-, food reward- and nicotine reward-related behaviors. Deletion of the ß4 subunit gene resulted in an addiction-related phenotype characterized by low anxiety, high novelty-induced response, lack of sensitivity to palatable food rewards and increased intracranial nicotine self-administration at high doses. Lentiviral vector-induced re-expression of the ß4 subunit into either the MHb or IPN restored a "stop" signal on nicotine self-administration. These results suggest that ß4*nAChRs provide a promising novel drug target for smoking cessation.

Pharmacological Comparison of Mitragynine and 7-Hydroxymitragynine: In Vitro Affinity and Efficacy for µ-Opioid Receptor and Opioid-Like Behavioral Effects in Rats.

Relationships between µ-opioid receptor (MOR) efficacy and effects of mitragynine and 7-hydroxymitragynine are not fully established. We assessed in vitro binding affinity and efficacy and discriminative stimulus effects together with antinociception in rats. The binding affinities of mitragynine and 7-hydroxymitragynine at MOR (Ki values 77.9 and 709 nM, respectively) were higher than their binding affinities at κ-opioid receptor (KOR) or δ-opioid receptor (DOR). [35S]guanosine 5'-O-[γ-thio]triphosphate stimulation at MOR demonstrated that mitragynine was an antagonist, whereas 7-hydroxymitragynine was a partial agonist (Emax = 41.3%). In separate groups of rats discriminating either morphine (3.2 mg/kg) or mitragynine (32 mg/kg), mitragynine produced a maximum of 72.3% morphine-lever responding, and morphine produced a maximum of 65.4% mitragynine-lever responding. Other MOR agonists produced high percentages of drug-lever responding in the morphine and mitragynine discrimination assays: 7-hydroxymitragynine (99.7% and 98.1%, respectively), fentanyl (99.7% and 80.1%, respectively), buprenorphine (99.8% and 79.4%, respectively), and nalbuphine (99.4% and 98.3%, respectively). In the morphine and mitragynine discrimination assays, the KOR agonist U69,593 produced maximums of 72.3% and 22.3%, respectively, and the DOR agonist SNC 80 produced maximums of 34.3% and 23.0%, respectively. 7-Hydroxymitragynine produced antinociception; mitragynine did not. Naltrexone antagonized all of the effects of morphine and 7-hydroxymitragynine; naltrexone antagonized the discriminative stimulus effects of mitragynine but not its rate-decreasing effects. Mitragynine increased the potency of the morphine discrimination yet decreased morphine antinociception. Here we illustrate striking differences in MOR efficacy, with mitragynine having less than 7-hydroxymitragynine. SIGNIFICANCE STATEMENT: At human µ-opioid receptor (MOR) in vitro, mitragynine has low affinity and is an antagonist, whereas 7-hydroxymitragynine has 9-fold higher affinity than mitragynine and is an MOR partial agonist. In rats, intraperitoneal mitragynine exhibits a complex pharmacology including MOR agonism; 7-hydroxymitragynine has higher MOR potency and efficacy than mitragynine. These results are consistent with 7-hydroxymitragynine being a highly selective MOR agonist and with mitragynine having a complex pharmacology that combines low efficacy MOR agonism with activity at nonopioid receptors.

Antinociceptive and Discriminative Stimulus Effects of Six Novel Psychoactive Opioid Substances in Male Rats.

Compounds with novel or fentanyl-like structures continue to appear on the illicit drug market and have been responsible for fatalities, yet there are limited preclinical pharmacological data available to evaluate the risk of these compounds to public health. The purpose of the present study was to examine acetyl fentanyl, butyryl fentanyl, 3,4-dichloro-N-[[1-(dimethylamino)cyclohexyl]methyl]benzamide (AH-7921), 1-cyclohexyl-4-(1,2-diphenylethyl)piperazine (MT-45), 4-chloro-N-[1-(2-phenylethyl)-2-piperidinylidene]-benzenesulfonamide (W-15), and 4-chloro-N-[1-[2-(4-nitrophenyl)ethyl]-2-piperidinylidene]-benzenesulfonamide (W-18) for their relative potency to reference opioids and their susceptibility to naltrexone antagonism using the 55oC warm-water, tail-withdrawal assay of antinociception and a morphine drug discrimination assay in male, Sprague-Dawley rats. In the antinociception assay, groups of 8 rats per drug were placed into restraining tubes, their tails were immersed into 40o or 55oC water, and the latency for tail withdrawal was measured with a cutoff time of 15 seconds. In the drug discrimination assay, rats (n = 11) were trained to discriminate between 3.2 mg/kg morphine and saline, subcutaneously, in a two-choice, drug discrimination procedure under a fixed ratio-5 schedule of sucrose pellet delivery. Morphine, fentanyl, and four of the synthetic opioids dose dependently produced antinociception and fully substituted for morphine in the drug discrimination assay with the following rank order of potency: fentanyl > butyryl fentanyl > acetyl fentanyl > AH-7921 > MT45 > morphine. All drugs that produced antinociception or morphine-like discriminative stimulus effects were blocked by naltrexone. W-15 and W-18 did not show antinociceptive or morphine-like discriminative stimulus effects at the doses tested supporting a lack of opioid activity for these two compounds. These findings suggest that butyryl fentanyl, acetyl fentanyl, AH-7941, and MT-45 have abuse liability like other opioid agonists. SIGNIFICANCE STATEMENT: As novel psychoactive substances appear on the illicit drug market, preclinical pharmacological testing is required to assist law enforcement, medical professionals, and legal regulators with decisions about potential public health risks. In this study, four synthetic opioids, acetyl fentanyl, butyryl fentanyl, AH-7921, and MT-45 produced effects similar to fentanyl and morphine and were blocked by naltrexone. These data suggest the four synthetic opioids possess similar abuse liability risks as typical opioid agonists.

Acute vagus nerve stimulation enhances reversal learning in rats.

Cognitive flexibility is a prefrontal cortex-dependent neurocognitive process that enables behavioral adaptation in response to changes in environmental contingencies. Electrical vagus nerve stimulation (VNS) enhances several forms of learning and neuroplasticity, but its effects on cognitive flexibility have not been evaluated. In the current study, a within-subjects design was used to assess the effects of VNS on performance in a novel visual discrimination reversal learning task conducted in touchscreen operant chambers. The task design enabled simultaneous assessment of acute VNS both on reversal learning and on recall of a well-learned discrimination problem. Acute VNS delivered in conjunction with stimuli presentation during reversal learning reliably enhanced learning of new reward contingencies. Enhancement was not observed, however, if VNS was delivered during the session but was not coincident with presentation of to-be-learned stimuli. In addition, whereas VNS delivered at 30 HZ enhanced performance, the same enhancement was not observed using 10 or 50 Hz. Together, these data show that acute VNS facilitates reversal learning and indicate that the timing and frequency of the VNS are critical for these enhancing effects. In separate rats, administration of the norepinephrine reuptake inhibitor atomoxetine also enhanced reversal learning in the same task, consistent with a noradrenergic mechanism through which VNS enhances cognitive flexibility.

Discriminative-Stimulus Effects of Synthetic Cathinones in Squirrel Monkeys.

BACKGROUND: Synthetic cathinones display overlapping behavioral effects with psychostimulants (e.g., methamphetamine [MA]) and/or entactogens (e.g., 3,4-methylenedioxymethaphetamine [MDMA])-presumably reflecting their dopaminergic and/or serotonergic activity. The discriminative stimulus effects of MDMA thought to be mediated by such activity have been well characterized in rodents but have not been fully examined in nonhuman primates. METHODS: The present studies were conducted to systematically evaluate the discriminative stimulus effects of 5 abused synthetic cathinones (methylenedioxypyrovalerone [MDPV], α-pyrrolidinovalerophenone [α-PVP], methcathinone [MCAT], mephedrone, and methylone) in adult male squirrel monkeys trained to distinguish intramuscular injections of MA (0.1 mg/kg; n = 4) or MDMA (0.6 mg/kg; n = 4) from vehicle. RESULTS: Each training drug produced dose-dependent effects and, at the highest dose, full substitution. MDMA produced predominantly vehicle-like responding in the MA-trained group, whereas the highest dose of MA (0.56 mg/kg) produced partial substitution (approximately 90% appropriate lever responding in one-half of the subjects) in the MDMA-trained group. MDPV, α-PVP, and MCAT produced full substitution in MA-trained subjects, but, at the same or higher doses, only substituted for MDMA in one-half of the subjects, consistent with primarily dopaminergically mediated interoceptive effects. In contrast, mephedrone and methylone fully substituted in MDMA-trained subjects but failed to fully substitute for the training drug in MA-trained subjects, suggesting a primary role for serotonergic actions in their interoceptive effects. CONCLUSIONS: These findings suggest that differences in the interoceptive effects of synthetic cathinones in nonhuman primates reflect differing compositions of monoaminergic actions that also may mediate their subjective effects in humans.

Nicotinic aspects of the discriminative stimulus effects of arecoline.

Despite the evidence that the muscarinic agonist arecoline is a drug of abuse throughout Southeast Asia, its stimulus characteristics have not been well studied. The goal of this work was to understand more about the mediation of discriminative stimulus effects of arecoline. Arecoline (1.0 mg/kg s.c.) was trained as a discriminative stimulus in a group of eight rats. The ability of various cholinergic agonists and antagonists to mimic or antagonize the discriminative stimulus effects of arecoline and to modify its rate-suppressing effects was evaluated. A muscarinic antagonist, but neither of two nicotinic antagonists, was able to modify the discriminative stimulus effects of arecoline, suggesting a predominant muscarinic basis of arecoline's discriminative stimulus effects in this assay. However, both nicotine itself and two nicotine agonists with selective affinity for the α4ß2* receptor (ispronicline and metanicotine) produced full arecoline-like discriminative stimulus effects in these rats. The discriminative stimulus effects of the selective nicotine agonists were blocked by both the general nicotine antagonist mecamylamine and by the selective α4ß2* antagonist, dihydro-beta-erythroidine (DHßE). Surprisingly, only DHßE antagonized the rate-suppressing effects of the selective nicotine agonists. These data indicate a selective α4ß2* nicotine receptor component to the behavioral effects of arecoline. Although the nicotinic aspects of arecoline's behavior effects could suggest that abuse of arecoline-containing material (e.g. betel nut chewing) is mediated through nicotinic rather than muscarinic actions, further research, specifically on the reinforcing effects of arecoline, is necessary before this conclusion can be supported.

Behavioral effects of four novel synthetic cathinone analogs in rodents.

A new generation of novel cathinone compounds has been developed as stimulant substitutes to avoid drug control laws and detection of use by blood tests. Dipentylone, N-ethylhexedrone, 4-chloroethcathinone (4-CEC), and 4'-methyl-α-pyrrolidinohexiophenone (MPHP) were tested for in vivo psychostimulant-like effects to assess their abuse liability. Locomotor activity was assessed in an open-field assay using Swiss-Webster mice to screen for locomotor stimulant effects and to identify behaviorally-active dose ranges, times of peak effect, and durations of action. Discriminative stimulus effects were assessed in separate groups of Sprague-Dawley rats trained to discriminate cocaine or methamphetamine from vehicle. Dipentylone, N-ethylhexedrone, 4-CEC, and MPHP dose-dependently increased locomotor activity. Dipentylone, N-ethylhexedrone, and MPHP produced maximal stimulant effects similar to cocaine and methamphetamine. 4-CEC was less efficacious, producing peak stimulant effects of about 74% of that of methamphetamine. The compounds were less potent than methamphetamine and approximately equipotent with cocaine. The doses of cocaine, methamphetamine, dipentylone, and 4-CEC that produced peak effects lasted 2 to 3 h, the peak dose of N-ethylhexedrone lasted 4 h, and the peak dose of MPHP lasted 6 h. All four compounds fully substituted for the discriminative stimulus effects of methamphetamine and cocaine, although full substitution by 4-CEC occurred at doses that substantially decreased response rate. Only 4-CEC fully substituted for MDMA. These data provide evidence that the novel cathinone compounds dipentylone, N-ethylhexedrone, 4-CEC, and MPHP demonstrate potential for abuse as psychostimulants, given their ability to stimulate locomotor activity and their substitution for the discriminative stimulus effects of methamphetamine and cocaine.

Low-dose alcohol: Interoceptive and molecular effects and the role of dentate gyrus in rats.

Alcohol abuse and dependence are world-wide health problems. Most research on alcohol use focuses on the consequences of moderate to high levels of alcohol. However, even at low concentrations, alcohol is capable of producing effects in the brain that can ultimately affect behavior. The current studies seek to understand the effects of low-dose alcohol (blood alcohol levels of ≤10mM). To do so, these experiments utilize a combination of behavioral and molecular techniques to (1) assess the ability of the interoceptive effects of a low dose of alcohol to gain control over goal-tracking behavior in a Pavlovian discrimination task, (2) determine brain regional differences in cellular activity via expression of immediate early genes (IEGs), and (3) assess the role of the dentate gyrus in modulating sensitivity to the interoceptive effects of a low dose of alcohol. Here, we show that intragastric administration of a dose of 0.8 g/kg alcohol produces blood alcohol levels ≤10mM in both male and female Long-Evans rats and can readily be trained as a Pavlovian interoceptive drug cue. In rats trained on this procedure, this dose of alcohol also modulates expression of the IEGs c-Fos and Arc in brain regions known to modulate expression of alcohol interoceptive effects. Finally, pharmacological inactivation of the dentate gyrus with GABA agonists baclofen and muscimol disrupted the ability of a low dose of alcohol to serve as an interoceptive cue. Together, these findings demonstrate behavioral and molecular consequences of low-dose alcohol.

Hesperidin protects against behavioral alterations and loss of dopaminergic neurons in 6-OHDA-lesioned mice: the role of mitochondrial dysfunction and apoptosis.

Hesperidin is a flavonoid glycoside that is frequently found in citrus fruits. Our group have demonstrated that hesperidin has neuroprotective effect in 6-hydroxydopamine (6-OHDA) model of Parkinson's disease (PD), mainly by antioxidant mechanisms. Although the pathophysiology of PD remains uncertain, a large body of evidence has demonstrated that mitochondrial dysfunction and apoptosis play a critical role in dopaminergic nigrostriatal degeneration. However, the ability of hesperidin in modulating these mechanisms has not yet been investigated. In the present study, we examined the potential of a 28-day hesperidin treatment (50 mg/kg/day, p.o.) in preventing behavioral alterations induced by 6-OHDA injection via regulating mitochondrial dysfunction, apoptosis and dopaminergic neurons in the substantia nigra pars compacta (SNpc) in C57BL/6 mice. Our results demonstrated that hesperidin treatment improved motor, olfactory and spatial memory impairments elicited by 6-OHDA injection. Moreover, hesperidin treatment attenuated the loss of dopaminergic neurons (TH+ cells) in the SNpc and the depletion of dopamine (DA) and its metabolities 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) in the striatum of 6-OHDA-lesioned mice. Hesperidin also protected against the inhibition of mitochondrial respiratory chain complex-I, -IV and V, the decrease of Na + -K + -ATPase activity and the increase of caspase-3 and -9 activity in the striatum. Taken together, our findings indicate that hesperidin mitigates the degeneration of dopaminergic neurons in the SNpc by preventing mitochondrial dysfunction and modulating apoptotic pathways in the striatum of 6-OHDA-treated mice, thus improving behavioral alterations. These results provide new insights on neuroprotective mechanisms of hesperidin in a relevant preclinical model of PD.

Moderate Prenatal Alcohol Exposure Impairs Visual-Spatial Discrimination in a Sex-Specific Manner: Effects of Testing Order and Difficulty on Learning Performance.

BACKGROUND: Exposure to high levels of alcohol during development leads to alterations in neurogenesis and deficits in hippocampal-dependent learning. Evidence suggests that even more moderate alcohol consumption during pregnancy can have negative impacts on the cognitive function of offspring. Methods for assessing impairments differ greatly across species, complicating translation of preclinical findings into potential therapeutics. We have demonstrated the utility of a touchscreen operant measure for assessing hippocampal function in mice. METHODS: Here, we integrated a well-established "drinking-in-the-dark" exposure model that produces reliable, but more moderate, levels of maternal intoxication with a trial-unique, delayed nonmatching-to-location (TUNL) task to examine the effects of prenatal alcohol exposure (PAE) on hippocampal-sensitive behavior directly analogous to those used in clinical assessment. PAE and SAC offspring mice were trained to touch a single visual stimulus ("sample phase") in one of 10 possible spatial locations (2 × 5 grid) in a touchscreen operant system. After a delay, animals were simultaneously presented with the original stimulus and a rewarded stimulus in a novel location ("choice phase"). PAE and saccharin (SAC) control mice were trained on a series of problems that systematically increased the difficulty by decreasing the separation between the sample and choice stimuli. Next, a separate cohort of PAE and SAC animals were given a brief training and then tested on a challenging variant where both the separation and delay varied with each trial. RESULTS: We found that PAE mice were generally able to perform at levels similar to SAC control mice at progressively more difficult separations. When tested on the most difficult unpredictable variant immediately, PAE showed a sex-specific deficit with PAE females performing worse during long delays. CONCLUSIONS: Taken together, these data demonstrate the utility of the TUNL task for examining PAE related alterations in hippocampal function and underline the need to examine sex-by-treatment interactions in these models.

The discriminative stimulus effects of epibatidine in C57BL/6J mice.

The α4ß2* nicotinic acetylcholine receptor (nAChR) subtypes are targeted for the development of smoking cessation aids, and the use of drug discrimination in mice provides a robust screening tool for the identification of drugs acting through nAChRs. Here, we established that the α4ß2* nAChR agonist epibatidine can function as a discriminative stimulus in mice. Male C57BL/6J mice discriminated epibatidine (0.0032 mg/kg, subcutaneously) and were tested with agonists varying in selectivity and efficacy for α4ß2* nAChRs. The discriminative stimulus effects of epibatidine were characterized with the nonselective, noncompetitive nicotinic antagonist mecamylamine, with the selective ß2-substype-containing nAChR antagonist dihydro-ß-erythroidine hydrobromide (DHßE), and the α7 antagonist methyllycaconitine (MLA). Nicotine (0.32-1.0 mg/kg, subcutaneously), the partial nAChR agonist cytisine (1.0-5.6 mg/kg, subcutaneously), and the α7 nAChR agonist N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]-4-chlorobenzamide (10-56 mg/kg, intraperitoneally) produced no more than 33% epibatidine-appropriate responding. The partial α4ß2* nAChR agonists varenicline and 2'-fluoro-3'-(4-nitro-phenyl)deschloroepibatidine produced 61 and 69% epibatidine-appropriate responding, respectively. DHßE and mecamylamine, but not MLA, significantly antagonized the discriminative stimulus effects of epibatidine. These results show that epibatidine may be trained as a discriminative stimulus in mice and has utility in elucidating the in-vivo pharmacology of α4ß2* nAChR ligands.

Methylenedioxymethamphetamine-like discriminative stimulus effects of seven cathinones in rats.

Synthetic cathinone derivatives are commonly considered quasi-legal alternatives for stimulant drugs, such as cocaine and methamphetamine, but some derivatives are increasingly being detected in club drug formulations of Ecstasy or 'Molly' as substitutes for methylenedioxymethamphetamine (±-MDMA). Although several studies have evaluated the psychostimulant-like effects of synthetic cathinones, few cathinone compounds have been assessed for MDMA-like activity. In order to determine their likelihood of interchangeability with entactogenic club drugs, the discriminative stimulus effects of methcathinone, 4-fluoromethcathinone, 4-methylmethcathinone, 4-methylethcathinone, 3-fluoromethcathinone, pentedrone, and ethylone were assessed in Sprague-Dawley rats trained to discriminate 1.5 mg/kg racemic methylenedioxymethamphetamine (±-MDMA) from vehicle. Methamphetamine and the cathinones 4-fluoromethcathinone, 4-methylmethcathinone, 4-methylethcathinone, 3-fluoromethcathinone, pentedrone, and ethylone fully substituted for the discriminative stimulus effects of ±-MDMA. In contrast, methcathinone produced a maximum of only 43% ±-MDMA-appropriate responding and higher doses suppressed responding. Most, but not all of the cathinone compounds tested have discriminative stimulus effects similar to those of MDMA as well as psychostimulant-like effects; however, the potency of MDMA versus psychostimulant substitution varies substantially among the compounds, suggesting that a subset of synthetic cathinones are more MDMA-like than psychostimulant-like. These findings further highlight the highly-variable pharmacology of this class of compounds and suggest that those cathinones with MDMA-like effects may also have increased use as club drugs.

Comparison of the muscarinic antagonist effects of scopolamine and L-687,306.

This study aimed to use central and peripheral assays to compare the effects of the muscarinic antagonist scopolamine with those of a novel muscarinic antagonist, L-687,306 [(3R,4R)-3-(3-cyclopropyl-1,2,4,oxadiazol[5-yl]-1-azabicyclo[2.2.1]heptane. Groups of rats were trained to discriminate the stimulus effects of the muscarinic agonist, arecoline (1.0 mg/kg); concomitant measures of response rate were recorded. Separate groups were prepared with telemetery devices for recording bradycardia induced by arecoline (10 mg/kg). Methyl arecoline and arecoline were nearly equally potent in producing a brief but profound bradycardia, indicative of an equivalent effect in the heart. L-687,306 and scopolamine were both able to block this peripheral effect of arecoline. L-687,306 produced a surmountable antagonism of both the discriminative and rate-suppressing effects of arecoline. Scopolamine, however, was unable to antagonize the rate-reducing effects of arecoline in the discrimination assay. This limited the number of rats that could respond to the discriminative stimulus effects of arecoline, as well as the amount of arecoline stimulus effects they were able to report. The data suggest that L-687,306 may be a more generally effective muscarinic antagonist than scopolamine and support earlier reports that this antagonist has less direct effect on behavior.

Re-evaluation of the discriminative stimulus effects of lysergic acid diethylamide with male and female Sprague-Dawley rats.

Recent discoveries from clinical trials with psychedelic-assisted therapy have led to a resurgence of interest in the psychopharmacology of lysergic acid diethylamide (LSD). Preclinical drug discrimination is an invaluable tool to investigate the neurochemical mechanisms underlying subjective drug effects. The current study extends previous drug discrimination research by including both sexes. Adult female (n = 8) and male (n = 8) Sprague-Dawley rats were trained to discriminate 0.08 mg/kg LSD from saline under a fixed ratio 20 schedule of food reinforcement. Substitution tests were conducted with several substances, including other serotonergic hallucinogens, psychostimulants, mixed psychedelic-stimulants and synthetic cathinones. Stimulus antagonist tests were conducted with selected serotonin and dopamine antagonists. LSD-substitution with serotonergic hallucinogens was comparable between sexes. Modest but intriguing differences were observed between male and female rats in the extent of partial substitution by 3,4-methylenedioxymethamphetamine and 3,4-methylenedioxyamphetamine enantiomers and the synthetic cathinones, 3,4-methylenedioxypyrovalerone and 4-methylmethcathinone. Dopamine antagonists failed to block the LSD cue in both sexes and exerted stronger rate suppressant effects in male rats. The 5-hydroxytryptamine antagonist, (R)-(+)-a-(2,3-dimethoxyphenyl)-1-[2-(4-fluorophenyl) ethyl]-4-piperidinemethanol (MDL 100 907) blocked LSD discrimination in both sexes, although complete blockade was evident at lower doses in male rats. These results support previous findings regarding the prominent role of serotonergic activities underlying LSDs discriminative stimulus effects in male rats and generalize these findings to female rats. In consideration of the rising popularity in psychedelic-assisted psychotherapy, further research may be warranted to evaluate possible sex differences in the behavioral and subjective effects of LSD.