Nadia Benturquia

Allosteric modulation of the effect of escitalopram, paroxetine and fluoxetine : in-vitro and in-vivo studies

Clinical and preclinical studies have shown that the effect of citalopram on serotonin (5-HT) reuptake inhibition and its antidepressant activity resides in the S-enantiomer. In addition, using a variety of in-vivo and in-vitro paradigms, it was shown that R-citalopram counteracts the effect of escitalopram. This effect was suggested to occur via an allosteric modulation at the level of the 5-HT transporter. Using in-vitro binding assays at membranes from COS-1 cells expressing the human 5-HT transporter (hSERT) and in-vivo electrophysiological and microdialysis techniques in rats, the present study was directed at determining whether R-citalopram modifies the action of selective serotonin reuptake inhibitors (SSRIs) known to act on allosteric sites namely escitalopram, and to a lesser extent paroxetine, compared to fluoxetine, which has no affinity for these sites. In-vitro binding studies showed that R-citalopram attenuated the association rates of escitalopram and paroxetine to the 5-HT transporter, but had no effect on the association rates of fluoxetine, venlafaxine or sertraline. In the rat dorsal raphe nucleus, R-citalopram (250 microg/kg i.v.) blocked the suppressant effect on neuronal firing activity of both escitalopram (100 microg/kg i.v.) and paroxetine (500 microg/kg i.v.), but not fluoxetine (10 mg/kg i.v.). Interestingly, administration of R-citalopram (8 mg/kg i.p.) attenuated the increase of extracellular levels of 5-HT ([5-HT]ext) in the ventral hippocampus induced by both escitalopram (0.28 microM) and paroxetine (0.75 microM), but not fluoxetine (10 microM). In conclusion, the present in-vitro and in-vivo studies show that R-citalopram counteracts the activity of escitalopram and paroxetine, but not fluoxetine, by acting at the allosteric binding site of the 5-HT transporter, either located in the dorsal raphe nucleus or post-synaptically in the ventral hippocampus. This conclusion is strengthened by the observation that the inhibitory effect of fluoxetine, which has no stabilizing effect on the radioligand/hSERT complex, was not blocked by co-administration of R-citalopram.

Involvement of D1 dopamine receptor in MDMA-induced locomotor activity and striatal gene expression in mice

3,4-Methylenedioxymethamphetamine (MDMA), a widely used recreational drug with psychoactive properties, induces both serotonin and dopamine release in the brain. In rats and mice MDMA induces behavioural changes and has rewarding effects but little is known about its cellular effects. We have previously shown that the ERK pathway is important for the changes in gene expression observed in mice striatum after treatment with this psychostimulant. In this study we investigated the role of D1 receptors in MDMA-induced locomotor hyperactivity and regulation of immediate-early genes (Fos, Fosb, Egr1 and Egr2) mRNA levels requiring ERK activity in mice striatum. We used the selective D1 receptor antagonist, SCH23390 at a dose (0.05 mg/kg) that did not influence locomotor activity. This dose totally blocked MDMA-induced locomotor activity but only partially the increase in transcription levels of Fos, Fosb, Egr1 and Egr2 (24%, 23%, 22% and 29% respectively). In conclusion, our results showed that D1 receptors play a key role in the acute MDMA-induced hyperlocomotion and that ERK pathway is partially under D1 receptors control to induce Fos, FosB, Egr1 and Egr2 transcription.

Use of preproenkephalin knockout mice and selective inhibitors of enkephalinases to investigate the role of enkephalins in various behaviours

RationaleThe most simple and efficient method to study the physiological role of enkephalins is to increase the lifetime of these endogenous opioid peptides by inhibiting their inactivating enzymes. Enkephalins are degraded by the concomitant action of two metallopeptidases: neutral endopeptidase (NEP, EC3.4.21.11) and aminopeptidase N (APN, EC3.4.11.2), both enzymes releasing inactive metabolites.ObjectivesPotent dual inhibitors have been developed, such as RB101. However, NEP and APN have a broad specificity and can cleave various peptides in vitro. Therefore, it was essential to investigate the specific involvement of enkephalins in the various pharmacological responses induced by dual inhibitors.Materials and methodsWe compared the pharmacological responses induced by RB101 in wild-type and preproenkephalin-deficient mice (Penk1−/−) using several behavioural assays.ResultsIn all the tests used (hot plate test, force swim test, castor-oil-induced diarrhoea), RB101 induced strong effects in wild-type animals, whereas slight effects were observed in Penk1−/− animals. These residual effects are blocked by pre-administration of the opioid antagonist naloxone, supporting the involvement of the opioid receptors in the responses observed.ConclusionsThe pharmacological effects induced by dual inhibitors acting on both NEP and APN are mainly due to the protection of the endogenous enkephalins at supraspinal and peripheral levels. It could be speculated that the residual effects observed in Penk1−/− mice after RB101 administration could be due to the direct action of other opioid peptides or through an indirect effect involving the protection of other peptide substrates of NEP or APN, as substance P or angiotensin.

In Vivo Visualization of Delta Opioid Receptors upon Physiological Activation Uncovers a Distinct Internalization Profile

G-protein-coupled receptors (GPCRs) mediate numerous physiological functions and represent prime therapeutic targets. Receptor trafficking upon agonist stimulation is critical for GPCR function, but examining this process in vivo remains a true challenge. Using knock-in mice expressing functional fluorescent delta opioid receptors under the control of the endogenous promoter, we visualized in vivo internalization of this native GPCR upon physiological stimulation. We developed a paradigm in which animals were made dependent on morphine in a drug-paired context. When re-exposed to this context in a drug-free state, mice showed context-dependent withdrawal signs and activation of the hippocampus. Receptor internalization was transiently detected in a subset of CA1 neurons, uncovering regionally restricted opioid peptide release. Importantly, a pool of surface receptors always remained, which contrasts with the in vivo profile previously established for exogenous drug-induced internalization. Therefore, a distinct response is observed at the receptor level upon a physiological or pharmacological stimulation. Altogether, direct in vivo GPCR visualization enables mapping receptor stimulation promoted by a behavioral challenge and represents a powerful approach to study endogenous GPCR physiology.

Short- and long-lasting behavioral and neurochemical adaptations: relationship with patterns of cocaine administration and expectation of drug effects in rats.

Cocaine dependence is a significant public health problem, characterized by periods of abstinence. Chronic exposure to drugs of abuse induces important modifications on neuronal systems, including the dopaminergic system. The pattern of administration is an important factor that should be taken into consideration to study the neuroadaptations. We compared the effects of intermittent (once daily) and binge (three times a day) cocaine treatments for 1 (WD1) and 14 (WD14) days after the last cocaine injection on spontaneous locomotor activity and dopamine (DA) levels in the nucleus accumbens (Nac). The intermittent treatment led to a spontaneous increase in DA (WD1/WD14), and in locomotor activity (WD1) at the exact hour which rats were habituated to receive a cocaine injection. These results underline that taking into consideration the hours of the day at which the experiments are performed is crucial. We also investigated these behavioral and neurochemical adaptations in response to an acute cocaine challenge on WD1 and WD14. We observed that only the binge treatment led to sensitization of locomotor effects of cocaine, associated to a DA release sensitization in the Nac, whereas the intermittent treatment did not. We demonstrate that two different patterns of administration induced distinct behavioral and neurochemical consequences. We unambiguously demonstrated that the intermittent treatment induced drug expectation associated with higher basal DA level in the Nac when measured at the time of chronic cocaine injection and that the binge treatment led to behavioral and sensitization effects of cocaine.

Characteristics of dual specificity phosphatases mRNA regulation by 3,4-methylenedioxymethamphetamine acute treatment in mice striatum.

3,4-methylenedioxymethamphetamine (MDMA) is a popular recreational drug that has rewarding properties in rodents but little is known about its effects at the cellular and molecular levels. We have previously shown that the ERK pathway is important for the regulation in gene expression observed in mice striatum after acute treatment with MDMA. Interestingly, three dual specificity phosphatases were found among the genes modulated by MDMA acute treatment. In this study we investigated the signalling pathways leading to the up-regulation of these three mRNAs and the kinetics of their regulation. We found that the increase in Dusp14 mRNA depends on the activation of ERK and lasts longer than those of Dusp1 and Dusp5. The modulation of the three studied Dusps depends partially on the activation of D1 receptors but is independent of the activation of D2 receptors. These results suggest that at least two separate signalling cascades lead to the up-regulation of MAPK phosphatase mRNAs. The increase of Dusp1 and Dusp5 mRNAs is not controlled by ERK activation while that of Dusp14 is a direct negative-feedback mechanism of MDMA-induced ERK signalling. Both mechanisms converge to increase the expression levels of phosphatases able to inactivate ERK.

Temporal Regulation of Peripheral BDNF Levels during Cocaine and Morphine Withdrawal: Comparison with a Natural Reward

Background: Brain-derived neurotrophic factor (BDNF) is a neurotrophin that has long been studied in the field of addiction and its importance in regulating drug addiction–related behavior has been widely demonstrated. The aim of our study was to analyze the consequences of a repeated exposure to drugs of abuse or natural reward on plasma BDNF levels during withdrawal. Methods: Rats were chronically injected with morphine (subcutaneously, 5mg/kg) or cocaine (intraperitoneally, 20mg/kg) or fed with a butter biscuit (per os, 4g) once per day for 14 days. Blood collection was performed on the 1st (withdrawal day 1 or WD1) or on (WD14), either at the same time point rats had been exposed to drugs or natural reward or at a different time point (used to quantify basal brain-derived neurotrophic factor levels). Results: Cocaine treatment led to a rapid (WD1) and persistent (WD14) decrease of basal BDNF levels compared with saline-treated animals, whereas morphine induced an increase on WD14 without any alteration on WD1. On the contrary, the natural reward induced a significant increase of basal brain-derived neurotrophic factor levels only on WD1. The analysis of BDNF levels at the usual time point at which animals had been exposed showed that both drugs, but not the natural reward, increased BDNF levels compared with basal levels. Conclusion: Our data highlight that only drugs of abuse are able to persistently alter BDNF levels and to induce specific variations of this neurotrophic factor at the usual hour of injection.

Relationship between vulnerability to reinforcing effects of morphine and activity of the endogenous cholecystokinin system in Lewis and Fischer rats.

A great number of studies have shown the presence of physiological interactions between brain neurotransmitter systems in behavioural responses. This is the case for opioid, cholecystokinin (CCK) and dopamine systems. However, so far the role that the CCK system may play in vulnerability to consumption of drugs of abuse is not clear. This was investigated in this study using Lewis rats that are more sensitive to the reinforcing properties of drugs of abuse than Fischer rats. The extraneuronal CCK8 levels and brain CCK2 receptors were found higher in Fischer than in Lewis rats in the nucleus accumbens, one of the most important structures involved in drug consumption. Moreover, pharmacological modulation of the CCK system by administration of a selective CCK2 agonist blocked, in the conditioned place preference, the reinforcing effects of morphine in Lewis rats, whereas a selective CCK2 antagonist revealed reinforcing effects of the alkaloid in Fischer rats. These results obtained following systemic administrations of the CCK ligands were confirmed following microinjection into the nucleus accumbens. Thus, a low level of CCK efflux in the nucleus accumbens could be one of the many factors involved in drug reinforcing effects, whereas a high level of CCK efflux could attenuate it.

Neurobehavioral effects of lithium in the rat: Investigation of the effect/concentration relationships and the contribution of the poisoning pattern

&NA; Severity of lithium poisoning depends on the ingested dose, previous treatment duration and renal function. No animal study has investigated neurobehavioral differences in relation to the lithium poisoning pattern observed in humans, while differences in lithium pharmacokinetics have been reported in lithium‐pretreated rats mimicking chronic poisonings with enhanced brain accumulation in rats with renal failure. Our objectives were: 1)‐to investigate lithium‐related effects in overdose on locomotor activity, anxiety‐like behavior, spatial recognition memory and anhedonia in the rat; 2)‐to model the relationships between lithium‐induced effects on locomotion and plasma, erythrocyte, cerebrospinal fluid and brain concentrations previously obtained according to the poisoning pattern. Open‐field, elevated plus‐maze, Y‐maze and sucrose consumption tests were used. In acutely lithium‐poisoned rats, we observed horizontal (p < 0.001) and vertical hypolocomotion (p < 0.0001), increased anxiety‐like behavior (p < 0.05) and impaired memory (p < 0.01) but no altered hedonic status. Horizontal (p < 0.01) and vertical (p < 0.001) hypolocomotion peaked more markedly 24 h after lithium injection and was more prolonged in acute‐on‐chronically vs. acutely lithium‐poisoned rats. Hypolocomotion in chronically lithium‐poisoned rats with impaired renal function did not differ from acutely poisoned rats 24 h after the last injection. Interestingly, hypolocomotion/concentration relationships best fitted a sigmoidal Emax model in acute poisoning and a linear regression model linked to brain lithium in acute‐on‐chronic poisoning. In conclusion, lithium overdose alters rat behavior and consistently induces hypolocomotion which is more marked and prolonged in repeatedly lithium‐treated rats. Our data suggest that differences between poisoning patterns regarding lithium‐induced hypolocomotion are better explained by the duration of lithium exposure than by its brain accumulation. HighlightsLithium overdose consistently induces hypolocomotion in the ratLithium overdose increases anxiety‐like behaviour and impairs memory but not hedonic statusLocomotion/concentration relationships fit a sigmoidal Emax model in acute poisoningLocomotion/concentration relationships fit a linear regression model in acute‐on‐chronic poisoningDifferences in locomotion between patterns are better explained by duration of lithium exposure than brain accumulation