Discriminative-stimulus effects of cannabidiol oil in Sprague-Dawley rats.

Cannabidiol (CBD) is one of the major centrally active phytocannabinoid components of cannabis, and has been approved by the FDA only for the treatment of seizures associated with three rare disorders. It has also been touted as a potential treatment for anxiety in place of more traditional treatments like benzodiazepines. Although there is some evidence of anxiolytic effects of CBD, its suitability as a substitute for benzodiazepines is unknown. This experiment was designed to assess the extent to which CBD shares interoceptive discriminative-stimulus properties with the anxiolytic drug chlordiazepoxide (CDP), a benzodiazepine. In the present experiment, a range of doses (0-1569 mg/kg) of over-the-counter CBD oil was administered (i.g.) in male Sprague-Dawley rats trained to discriminate 5.6 mg/kg CDP from saline. Due to the long time-course effects of CBD, generalization tests were conducted at 90 and 120 min post-CBD administration. The two highest doses of CBD tested (1064 and 1569 mg/kg) were found to partially substitute for 5.6 mg/kg CDP, with mean percent responding on the CDP-associated lever reaching above 20% at time 2 (120 min post-CBD administration), suggesting that high doses of the over-the-counter CBD oils used in this experiment share interoceptive discriminative-stimulus properties to some degree with CDP. These results are novel in comparison to existing research into stimulus effects of CBD, in which substitution for benzodiazepines has not previously been observed.

Further disentangling the motivational processes underlying benzodiazepine hyperphagia.

In addition to their well-known anxiolytic functions, benzodiazepines produce hyperphagia. Previously, we reported that the benzodiazepine, chlordiazepoxide (CDP), increased consumption of both normally-preferred and normally-avoided taste stimuli during long-term (1 h) tests, primarily through changes in licking microstructure patterns associated with hedonic taste evaluation, whereas there was little effect on licking microstructure measures associated with post-ingestive feedback. In this study, we further examined the hedonic and motivational specificity of CDP effects on ingestive behavior. We tested brief access (15 s) licking responses for tastants spanning all taste qualities after treatment with either CDP (5 or 10 mg/kg) or the non-benzodiazepine anxiolytic, buspirone (1.5 or 3 mg/kg). A between-subjects, counterbalanced design compared the CDP or buspirone effects on licking responses for water and a range of weak to strong concentrations of NaCl, Q-HCl, citric acid, MSG, saccharin, and capsaicin under water-restricted (23 h) conditions; and sucrose, saccharin, and MSG under water-replete conditions. In a dose dependent manner, CDP increased licking for taste stimuli that were normally-avoided after saline treatment, with a notable exception observed for the trigeminal stimulus, capsaicin, which was not affected at any concentration or drug dose, suggesting a taste-specific effect of CDP on orosensory processing. Under water-replete conditions, CDP dose-dependently increased licking to normally-accepted concentrations of sucrose, saccharin, and MSG. There was no effect of either drug on licks for water under either water-restricted or water-replete conditions. Buspirone slowed oromotor coordination by increasing brief interlick intervals, but it did not affect licking for any concentrations of the tastants. Overall, these results indicate that benzodiazepines selectively enhance the hedonic acceptance of gustatory orosensory stimuli, independent of general anxiolytic or oromotor coordination effects, or physiological states such as thirst.

Effects of plus-maze experience and chlordiazepoxide on anxiety-like behavior and serotonin neural activity in the dorsal raphe nucleus in rats.

The extent to which rats express anxiety-like behavior on the elevated plus-maze (EPM) depends on their previous maze experience. Open-arm avoidance develops in maze-experienced rats, and is often accompanied by a diminished anxiolytic response to benzodiazepines. Regions of the dorsal raphe nucleus (DRN) were examined in male Sprague-Dawley rats using c-Fos and serotonin immunohistochemistry following a single exposure, a second exposure or no exposure to the EPM. We then examined the effect of the benzodiazepine anxiolytic chlordiazepoxide (CDP, 5 mg/kg) on EPM behavior and DRN neural activity. Enhanced open-arm avoidance was evident on the second EPM trial in both experiments. The observed pattern of c-Fos expression suggests that the first exposure to the maze activates serotonin cells in the rostral and dorsal regions of the DRN and that only the dorsal subregion is activated by a second exposure. CDP increased open-arm exploration during the first trial, which corresponded to decreased 5-hydroxytryptamine (5-HT) activity in the rostral and ventral subregions of the DRN. However, 5-HT activity in the DRN was reduced in rats on the second maze trial compared with the first trial, when CDP had no effect on open-arm exploration. These results suggest that open-arm avoidance in maze-experienced rats can be characterized as a coping response that is mediated by specific populations of 5-HT neurons in the DRN.

The elevated gradient of aversion: a new apparatus to study the rat behavior dimensions of anxiety, fear, and impulsivity

Few behavioral tests allow measuring several characteristics and most require training, complex analyses, and/or are time-consuming. We present an apparatus based on rat exploratory behavior. Composed of three different environments, it allows the assessment of more than one behavioral characteristic in a short 3-min session. Factorial analyses have defined three behavioral dimensions, which we named Exploration, Impulsivity, and Self-protection. Behaviors composing the Exploration factor were increased by chlordiazepoxide and apomorphine and decreased by pentylenetetrazole. Behaviors composing the Impulsivity factor were increased by chlordiazepoxide, apomorphine, and both acute and chronic imipramine treatments. Behaviors composing the Self-protection factor were decreased by apomorphine. We submitted Wistar rats to the open-field test, the elevated-plus maze, and to the apparatus we are proposing. Measures related to exploratory behavior in all three tests were correlated. Measures composing the factors Impulsivity and Self-protection did not correlate with any measures from the two standard tests. Also, compared with existing impulsivity tests, the one we proposed did not require previous learning, training, or sophisticated analysis. Exploration measures from our test are as easy to obtain as the ones from other standard tests. Thus, we have proposed an apparatus that measured three different behavioral characteristics, was simple and fast, did not require subjects to be submitted to previous learning or training, was sensitive to drug treatments, and did not require sophisticated data analyses.

Assessing anxiety in C57BL/6J mice: a pharmacological characterization of the open-field and light/dark tests.

INTRODUCTION: In order to assess anxiety in mammals various tests and species are currently available. These current assays measure changes in anxiety-like behaviors. The open-field and the light/dark are anxiety tests based on the spontaneous behavior of the animals, with C57BL/6J mice being a frequently used strain in behavioral studies. However, the suitability of this strain as a choice in anxiety studies has been questioned. In this study, we performed two pharmacological characterizations of this strain in both the open-field and the light/dark tests. METHODS: We examined the changes in the anxiety-like behaviors of C57BL/6J mice exposed to chlordiazepoxide (CDP), an anxiolytic drug, at doses of 5 and 10 mg/kg, picrotoxine (PTX), an anxiogenic drug, at doses of 0.5 and 1 mg/kg, and methylphenidate (MPH), a psychomotor stimulant drug, at doses of 5 and 10 mg/kg, in a first experiment. In a second experiment, we tested CDP at 2.5 mg/kg, PTX at 2 mg/kg and MPH at 2.5 mg/kg. RESULTS: Results showed an absence of anxiolytic-like effects of CDP in open-field and light/dark tests. Light/dark test was more sensitive to the anxiogenic effects of PTX than the open-field test. Finally, a clear anxiogenic effect of MPH was observed in the two tests. DISCUSSION: Although C57BL/6J mice could not be a sensitive model to study anxiolytic effects in pharmacological or behavioral interventions, it might be a suitable model to test anxiogenic effects. Further studies are necessary to corroborate these results.

Multiple processes underlie benzodiazepine-mediated increases in the consumption of accepted and avoided stimuli.

Hyperphagia is a reported side effect of anxiolytic benzodiazepines such as chlordiazepoxide (CDP). Prior research has focused primarily on the ingestive responses to sweet or solid foods. We examined CDP effects on licking for normally accepted and avoided taste solutions across a range of concentrations. The effect of CDP (10 mg/kg) versus saline on the licking patterns of water-restricted rats for water and 3 concentrations of sucrose, saccharin, NaCl, monosodium glutamate (MSG), citric acid, and quinine (Q-HCl) solutions was evaluated during 1 h tests. CDP increased meal size for all tastants except citric acid. Analysis of licking microstructure revealed 3 dissociable effects of CDP. CDP affected oromotor coordination as indicated by a uniform increase in the modal interlick interval for all stimuli. CDP increased meal size as indicated by shorter pauses during consumption of water, MSG, and weaker saccharin concentrations, and by fewer long interlick intervals (250-2000 ms) for normally avoided tastants. CDP also increased meal size by increasing burst size, burst duration, and the initial rate of licking for most solutions, suggesting increased hedonic taste evaluation. CDP did not affect variables associated with postingestive feedback such as meal duration or number of bursts, and the results also suggest that CDP did not enhance the perceived taste intensity. We hypothesize that the reduction of pause duration is consistent with an increased motivation to sample the stimulus that synergizes with changes in taste-mediated responsiveness to some but not all stimuli to yield increases in the consumption of both normally accepted and avoided taste stimuli.

Pharmacological modulation of stress-induced behavioral changes in the light/dark exploration test in male C57BL/6J mice.

Psychological stress is a major risk factor for mood and anxiety disorders. However, the phenotypic manifestation of stress effects varies across individuals, likely due, in part, to genetic variation. Modeling the behavioral and neural consequences of stress across genetically diverse inbred mouse strains is a valuable approach to studying gene × stress interactions. Recent work has shown that C57BL/6J mice exposed to ten daily sessions of restraint stress exhibited increased exploration of the aversive light compartment in the light/dark exploration (LDE) test. Here we sought to clarify the nature of this stress-induced phenotype by testing the ability of treatment with various clinically efficacious drugs of different therapeutic classes to rescue it. Ten days of restraint increased light compartment exploration, reduced body weight and sensitized the corticosterone response to swim stress. Subchronic administration (during stress and LDE testing) of fluoxetine, and to a lesser extent, lithium chloride, rescued stress-induced LDE behavior. Chronic fluoxetine treatment prior to (plus during stress and testing) failed to block the LDE stress effect. Acute administration of antipsychotic haloperidol, anti-ADHD medication methylphenidate or anxiolytic drug chlordiazepoxide, prior to LDE testing, was also unable to normalize the LDE stress effect. Collectively, these data demonstrate a treatment-selective prophylactic rescue of a restraint stress-induced behavioral abnormality in the C57BL/6J inbred strain. Further work with this novel model could help elucidate genetic and neural mechanisms mediating stress-induced changes in mouse 'emotion-relevant' behaviors and, ultimately, further understanding of the pathophysiology of stress-related neuropsychiatric disorders. This article is part of a Special Issue entitled 'Anxiety and Depression'.

Nicotine trained as a negative feature passes the retardation-of-acquisition and summation tests of a conditioned inhibitor.

Nicotine functions as a negative feature in a Pavlovian discriminated goal-tracking task. Whether withholding of responding to the conditional stimulus (CS) reflects nicotine functioning as a conditioned inhibitor is unknown. Accordingly, the present research sought to determine whether nicotine trained as a negative feature passed the retardation-of-acquisition and summation tests, thus characterizing it as a pharmacological (interoceptive) conditioned inhibitor. In the retardation test, rats received either nicotine (0.4 mg/kg) or chlordiazepoxide (5 mg/kg) negative feature training in which the drug state signaled when a 15-sec light CS would not be paired with sucrose; light was paired with sucrose on intermixed saline sessions. Following acquisition of the discrimination, both groups received nicotine CS training in which sucrose was intermittently available on nicotine but not intermixed saline sessions. Acquisition of conditioned responding to the nicotine CS was slower in the nicotine negative feature group than in the chlordiazepoxide negative feature group. In the summation test, rats were assigned to either the nicotine negative feature group or a pseudoconditioning control. In this control, the light CS was paired with sucrose on half the nicotine and half the saline sessions. Both groups also received excitatory training in which a white noise CS was paired with sucrose. The summation test consisted of presenting the white noise in conjunction with nicotine. Conditioned responding evoked by the white noise was decreased in the negative feature but not the pseudoconditioning group. Combined, the results provide the first evidence that an interoceptive stimulus (nicotine) can become a conditioned inhibitor.

Absence of repeated-trial tolerance to the anxiolytic-like effects of chlordiazepoxide in the rat triple test.

The triple test, recently developed to assess anxiety-related behaviors in rodents, combines three widely used behavioral tests: the open field (OF), elevated plus maze (EPM) and light/dark box (LDB). The EPM and LDB, individually, are normally sensitive to the anxiolytic effects of benzodiazepines only in the first trial, due to the phenomenon of one-trial tolerance, which limits their use in longitudinal studies. The main objective of the present investigation was to verify whether the anxiolytic-like effects of chlordiazepoxide (CDZ), previously observed in naive animals submitted to the triple test, would persist after repeated testing. To this end, three experiments were carried out where male Wistar rats received CDZ (10mg/kg) 30min before the triple test for 2, 3 or 20 consecutive days. Except for the first day of drug treatment following a previous test experience in an undrugged state, CDZ had enduring anxiolytic-like effects under all schedules, promoting an increase in the exploration of the EPM open arms (and in some cases of the white compartment of the LDB), without affecting the number of closed-arm entries. The finding that rats did not develop tolerance to CDZ even with chronic treatment and repeated exposures to the triple test suggests that this new device is a promising tool to be used in longitudinal studies involving pharmacological manipulations of anxiety-related behaviors.

In vitro cytotoxicity study of oxaziridines generated after chlordiazepoxide, demoxepam, and desmethylchlordiazepoxide UV irradiation.

The cytotoxicity of oxaziridines photogenerated after irradiation of chlordiazepoxide (CDZ) and its metabolites was investigated in vitro by a MTT assay on P388 leukemia and B16 melanoma cell lines and compared with that of the anticancer drug, melphalan. For the longer time-exposure experiment, oxaziridines had the same cytotoxicity as melphalan and this toxicity was higher when oxaziridines were photogenerated in the presence of cells. In conclusion, oxaziridines generated after CDZ, demoxepam, and desmethylchlordiazepoxide ultraviolet irradiation exhibited cytotoxicity activity against cancer cell lines. A possibility of CDZ use within the context of photodynamic therapy as a treatment for small, superficial tumors should not be excluded, because oxaziridines can be generated locally by skin-tumor local irradiation after CDZ topical administration.

Psychological stress suppresses innate IFN-gamma production via glucocorticoid receptor activation: reversal by the anxiolytic chlordiazepoxide.

Studies in humans and in animals indicate that psychological stress can modulate immune responses. Here we demonstrate that exposure to psychological stress (restraint stress) suppresses innate interferon (IFN)-gamma production in mice following an in vivo lipopolysaccharide (LPS) challenge. IFN-gamma signaling was also impaired by stress, as indicated by reduced STAT1 phosphorylation and reduced expression of the IFN-gamma-inducible genes, inducible nitric oxide synthase (iNOS) and IFN-gamma-inducible protein 10 (IP-10/CXCL10). Furthermore, restraint stress suppressed production of the IFN-gamma inducing cytokine interleukin (IL)-12 and increased production of the anti-inflammatory cytokine IL-10, which can inhibit both IL-12 and IFN-gamma production. However, using IL-10 knockout mice, we demonstrate that IL-10 does not mediate the suppressive effect of restraint stress on innate IFN-gamma production. Restraint stress increased corticosterone concentrations in serum and spleen, and consistent with a role for glucocorticoids in the immunosuppressive actions of stress, pre-treatment with the glucocorticoid receptor antagonist mifepristone completely blocked the stress-related suppression of innate IFN-gamma production. Addition of exogenous IL-12 to LPS-stimulated spleen cells reversed the suppressive effect of both restraint stress and corticosterone on IFN-gamma production. These data suggest that reduced IL-12 production is a key event in stress-induced suppression of innate IFN-gamma production. Finally, we demonstrate that pre-treatment with the anxiolytic drug chlordiazepoxide prevents the suppressive effect of stress on innate IFN-gamma production, and also attenuates the stress-induced increase in circulating corticosterone concentrations.

Modelling the anxiety-depression continuum in chicks.

The clinical syndromes of anxiety and depression are now thought to exist along a temporal continuum and this construct has been modelled in a preclinical setting in chicks separated from conspecifics. This research sought to further the validity of the chick anxiety-depression continuum model. Dose-response studies using two classes of anxiolytics (chlordiazepoxide: 2.5, 5.0, 10.0, 15.0 mg/kg, and clonidine: 0.1, 0.15, 0.2, 0.25 mg/kg) and three classes of antidepressants (imipramine: 1.0, 3.0, 10.0, 15.0 mg/kg, maprotoline: 2.5, 5.0, 10.0, 20.0 mg/kg and fluoxetine: 1.0, 5.0, 10.0, 20.0 mg/kg) showed an ability to detect anxiolytic activity of chlordiazepoxide, clonidine, imipramine and maprotoline in the anxiety-like phase of the model and to detect antidepressant effects of imipramine, maprotoline and fluoxetine in the depression-like phase of the model. In addition, blood plasma interleukin-6, a biomarker of stress, was found to be elevated in response to social-separation stress. Collectively, these findings further characterize the model as a simulation of the anxiety-depression continuum and begin to establish the paradigm as a high-utility adjuvant to rodent screening assays for putative anxiolytic and antidepressant compounds.

Occasion setting by drug states: Functional equivalence following similar training history.

Three experiments examined whether a drug state serving as a positive feature for pairings between a discrete conditional stimulus (CS, 15-s light or 15-s noise) and sucrose could transfer facilitative control to a CS with which it had never been presented. To do so, a CS was paired with a sucrose reward in the nicotine (0.4 mg/kg), amphetamine (AMP, 1mg/kg), or chlordiazepoxide (CDP, 5mg/kg) drug state; in separate saline sessions the CS was presented but was not followed by any reward. All three drug states facilitated responding to a discrete CS; previous studies found that this facilitation did not depend on direct associations between the drug state and sucrose. When a second discrimination was trained (e.g., CDP: light-sucrose and nicotine: noise-sucrose) the drug states facilitated responding to the CS trained in that state (nicotine: noise) as well as the CS normally presented in the other drug state (e.g., nicotine: light). A novel drug state (e.g., amphetamine) did not affect responding to either CS, indicating that the originally trained drug states had acquired functional similarity based on learning history. Also, a novel or ambiguous CS did not evoke responding in the previously trained drug state, indicating that both the features (drug states) and target conditional stimuli had to be trained in discriminations before transfer could occur.

Differential response to gepirone but not to chlordiazepoxide in malnourished rats subjected to learned helplessness

The learned helplessness (LH) paradigm is characterized by learning deficits resulting from inescapable events. The aims of the present study were to determine if protein-calorie malnutrition (PCM) alters learning deficits induced by LH and if the neurochemical changes induced by malnutrition alter the reactivity to treatment with GABA-ergic and serotonergic drugs during LH. Well-nourished (W) and PCM Wistar rats (61 days old) were exposed or not to inescapable shocks (IS) and treated with gepirone (GEP, 0.0-7.5 mg/kg, intraperitoneally, N = 128) or chlordiazepoxide (0.0-7.5 mg/kg, intraperitoneally, N = 128) 72 h later, 30 min before the test session (30 trials of escape learning). The results showed that rats exposed to IS had higher escape latency than non-exposed rats (12.6 ± 2.2 vs 4.4 ± 0.8 s) and that malnutrition increased learning impairment produced by LH. GEP increased the escape latency of W animals exposed or non-exposed to IS, but did not affect the response of PCM animals, while chlordiazepoxide reduced the escape deficit of both W and PCM rats. The data suggest that PCM animals were more sensitive to the impairment produced by LH and that PCM led to neurochemical changes in the serotonergic system, resulting in hyporeactivity to the anxiogenic effects of GEP in the LH paradigm.

Overlapping and distinct brain regions associated with the anxiolytic effects of chlordiazepoxide and chronic fluoxetine.

Little is known about the sites of action for the behavioral effects of chronic antidepressants. The novelty-induced hypophagia (NIH) test is one of few animal behavioral tests sensitive to acute benzodiazepines and chronic antidepressants. The goals of these experiments were to examine patterns of brain activation associated with the behavioral response to novelty and identify regions that could regulate the anxiolytic effects of acute benzodiazepine and chronic antidepressant treatments, measured using the NIH test. In the first experiment, rats were treated acutely with the anxiolytic, chlordiazepoxide (2.5 or 5 mg/kg, i.p.). In separate experiments, animals were implanted with osmotic minipumps delivering vehicle or fluoxetine (5 or 20 mg/kg per day s.c.) for 3 or 28 days. NIH was assessed by giving animals access to a familiar palatable food in a novel environment. Associated brain areas were identified using c-fos immunohistochemistry. NIH was mitigated by acute chlordiazepoxide and chronic fluoxetine. Both drugs reversed novelty-induced changes in c-fos expression in the lateral division of the posterolateral part of the bed nucleus of the stria terminalis (STLP), cingulate cortex (Cg), and dorsal field CA2 of the hippocampus (dCA2). Chronic fluoxetine additionally increased c-fos expression in the anterior nucleus accumbens (aAcb) and the piriform cortex (Pir). The effects of the drugs on c-fos expression in many regions correlated with anxiolytic efficacy. These findings identified brain regions where the effects of chronic antidepressants and benzodiazepines may converge to produce anxiolytic activity, as well as distinct sites of action for the two classes of drugs.

Anxiolytic effects of lavender oil inhalation on open-field behaviour in rats.

To establish a valid animal model of the effects of olfactory stimuli on anxiety, a series of experiments was conducted using rats in an open-field test. Throughout, effects of lavender oil were compared with the effects of chlordiazepoxide (CDP), as a reference anxiolytic with well-known effects on open-field behaviour. Rats were exposed to lavender oil (0.1-1.0 ml) for 30 min (Experiment 1) or 1h (Experiment 2) prior to open-field test and in the open field or injected with CDP (10 mg/kg i.p.). CDP had predicted effects on behaviour, and the higher doses of lavender oil had some effects on behaviour similar to those of CDP. In Experiment 3, various combinations of pre-exposure times and amounts of lavender oil were used. With sufficient exposure time and quantity of lavender the same effects were obtained as in Experiment 2. Experiment 4 demonstrated that these behavioural effects of lavender could be obtained following pre-exposure, even if no oil was present in the open-field test. In Experiments 2-4, lavender oil increased immobility. Together, these experiments suggest that lavender oil does have anxiolytic effects in the open field, but that a sedative effect can also occur at the highest doses.

GABA enhancement of maternal defense in mice: possible neural correlates.

Previous studies have shown that low doses of GABA(A) receptor agonists facilitate maternal defense of offspring (maternal aggression), without significantly affecting other maternal behaviors. In addition, it has been demonstrated that endogenous changes in GABAergic neurotransmission occur in association with lactation. This study investigated the effects of GABA(A) receptor agonist, chlordiazepoxide (CDP), a benzodiazepine (BDZ), on maternal behaviors including aggression, and identified brain regions with altered activity in association with treatment. Another aim of the study was to determine whether CDP injections could prevent decreases in maternal aggression that occur with pup separation. Intraperitoneal injections of 1 mg/kg of CDP significantly increased maternal defense without affecting other maternal behaviors, although a trend towards elevated nursing was noted. CDP significantly reduced c-Fos in lateral septum (LS) and caudal periaqueductal gray (cPAG) in behaviorally-experienced mice relative to vehicle-injected controls. In behaviorally-naïve subjects, CDP also decreased c-Fos in LS, but in cPAG this decrease was just above significance (p=0.051). CDP was not sufficient to "rescue" maternal aggression when pup stimulus was removed. Overall, these studies provide further insights into the role for GABA in maternal behaviors, including aggression, and how and where BDZs may act to modulate behavior.

Facilitation by drug states does not depend on acquired excitatory strength.

Three experiments examined the effects of drug-extinction when a drug state served as a conditional stimulus (CS) for sucrose delivery or as a positive feature for pairings between a discrete CS (e.g., 15-s light-on) and sucrose. Some conditioning models predict that drug state will facilitate the conditional response (CR) based on an association with sucrose whether the drug is trained as a CS or as a facilitator. If so, repeated presentation of the drug state alone (drug-extinction) should decrease the CR in both situations. Nicotine (0.4mg/kg), amphetamine (AMP, 1mg/kg), and chlordiazepoxide (CDP, 5mg/kg) facilitated a goal tracking conditioned response to the discrete CS; however, AMP and CDP did not evoke reliable responding without an interposed stimulus, suggesting that associations between these drug states and sucrose are not expressed as anticipatory food seeking (goal tracking). Repeated presentation of each drug state alone did not disrupt facilitation by nicotine, amphetamine, or CDP; suggesting that the drug states did not facilitate goal tracking based on a direct association with sucrose. This latter finding implicates a higher-order or non-associative mechanism for facilitation of anticipatory food seeking by drug states in this Pavlovian discrimination task.

Anxiolytic properties of green tea polyphenol (-)-epigallocatechin gallate (EGCG).

Naturally occurring polyphenols are potent antioxidants. Some of these compounds are also ligands for the GABA(A) receptor benzodiazepine site. This feature endows them with sedative properties. Here, the anxiolytic activity of the green tea polyphenol (-)-epigallocatechin gallate (EGCG) was investigated after acute administration in mice, using behavioral tests (elevated plus-maze and passive avoidance tests) and by electrophysiology on cultured hippocampal neurons. Patch-clamp experiments revealed that EGCG (1-10 muM) had no effect on GABA currents. However, EGCG reversed GABA(A) receptor negative modulator methyl beta-carboline-3-carboxylate (beta-CCM) inhibition on GABA currents in a concentration dependent manner. This was also observed at the level of synaptic GABA(A) receptors by recording spontaneous inhibitory synaptic transmission. In addition, EGCG consistently inhibited spontaneous excitatory synaptic transmission. Behavioral tests indicated that EGCG exerted both anxiolytic and amnesic effects just like the benzodiazepine drug, chlordiazepoxide. Indeed, EGCG in a dose-dependent manner both increased the time spent in open arms of the plus-maze and decreased the step-down latency in the passive avoidance test. GABA(A) negative modulator beta-CCM antagonized EGCG-induced amnesia. Finally, state-dependent learning was observable after chlordiazepoxide and EGCG administration using a modified passive avoidance procedure. Optimal retention was observed only when animals were trained and tested in the same state (veh-veh or drug-drug) and significant retrieval alteration was observed in different states (veh-drug or drug-veh). Moreover, EGCG and chlordiazepoxide fully generalized in substitution studies, indicating that they induced indistinguishable chemical states for the brain. Therefore, our data support that EGCG can induce anxiolytic activity which could result from an interaction with GABA(A) receptors.

Characterization of nicotine's ability to serve as a negative feature in a Pavlovian appetitive conditioning task in rats.

RATIONALE: Pavlovian feature negative discriminations have been widely used to understand inhibitory conditioning processes using exteroceptive stimuli. Comparatively little is known about inhibitory conditioning processes using a drug state as a negative feature. A negative feature signals that presentation of a conditional stimulus (CS) will not be paired with an unconditioned stimulus. OBJECTIVES: The present research examined whether nicotine served as a negative feature and started characterizing its properties. METHODS AND RESULTS: In acquisition, rats received intermixed saline and nicotine (0.4 mg/kg, base) sessions. On saline sessions, a 15-s light CS was paired with 4-s access to sucrose; the CS was presented on nicotine sessions, but sucrose was withheld. The discrimination was acquired with more goal tracking during the CS on saline sessions. Nicotine's inhibition of this conditioned response (CR) was sensitive to nicotine dose (ED50=0.225) and injection to testing interval (CR returned at 200 min). Mecamylamine pretreatment, but not hexamethonium, produced a loss of inhibitory control by nicotine suggesting a role for central nicotinic acetylcholine receptors. Amphetamine, bupropion, arecoline, and chlordiazepoxide, but not caffeine, substituted for the nicotine feature. However, in locomotor tests, amphetamine and bupropion increased activity; arecoline and chlordiazepoxide decreased activity. For this reason, the motor effects of these ligands could not be dissociated from substitution via shared stimulus properties. CONCLUSIONS: This feature negative task provides a preclinical model for studying how drug states inhibit responding, although identifying the process(es) mediating CR inhibition will require further research.