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.

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.

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 CCK(8) levels and brain CCK(2) 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 CCK(2) agonist blocked, in the conditioned place preference, the reinforcing effects of morphine in Lewis rats, whereas a selective CCK(2) 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.

Individual differences in cocaine-induced conditioned place preference in male rats: Behavioral and transcriptomic evidence.

BACKGROUND: Substance use disorder emerges in a small proportion of drug users and has the characteristics of a chronic relapsing pathology. AIMS: Our study aimed to demonstrate and characterize the variability in the expression of the rewarding effects of cocaine in the conditioned place preference (CPP) paradigm. METHODS: A cocaine-CPP paradigm in male Sprague-Dawley rats with an extinction period of 12 days and reinstatement was conducted. A statistical model was developed to distinguish rats expressing or not a cocaine-induced place preference. RESULTS: Two groups of rats were identified: rats that did express rewarding effects (CPP expression (CPPE), score >102 s) and rats that did not (no CPP expression (nCPPE), score between -85 and 59 s). These two groups did not show significant differences in a battery of behavioral tests. To identify differentially expressed genes in the CPPE and nCPPE groups, a whole-transcriptome ribonucleic acid-sequencing analysis was performed in the nucleus accumbens (NAc) 24 h after the CPP test. Four immediate early genes (Fos, Egr2, Nr4a1, and Zbtb37) were differentially expressed in the NAc of CPPE rats after expression of CPP. Variability in cocaine-induced place preference persisted in the CPPE and nCPPE groups after the extinction and reinstatement phases. Transcriptomic differences observed after reinstatement were distinct from those observed immediately after expression of CPP. CONCLUSION: These new findings provide insights into the identification of mechanisms underlying interindividual variability in the response to cocaine's rewarding effects.

Translational study of the whole transcriptome in rats and genetic polymorphisms in humans identifies LRP1B and VPS13A as key genes involved in tolerance to cocaine-induced motor disturbances.

Motor disturbances strongly increase the burden of cocaine use disorder (CUDs). The objective of our translational study was to identify the genes and biological pathways underlying the tolerance to cocaine-induced motor effects. In a 5-day protocol measuring motor tolerance to cocaine in rats (N = 40), modeling the motor response to cocaine in patients, whole-genome RNA sequencing was conducted on the ventral and dorsal striatum to prioritize a genetic association study in 225 patients with severe CUD who underwent thorough phenotypic (cocaine-induced hyperlocomotion, CIH; and cocaine-induced stereotypies, CIS) and genotypic [571,000 polymorphisms (SNPs)] characterization. We provide a comprehensive description of the rat striatal transcriptomic response to cocaine in our paradigm. Repeated vs. acute cocaine binge administration elicited 27 differentially expressed genes in the ventral striatum and two in the dorsal striatum. One gene, Lrp1b, was differentially expressed in both regions. In patients, LRP1B was significantly associated with both CIS and CIH. CIH was also associated with VPS13A, a gene involved in a severe neurological disorder characterized by hyperkinetic movements. The LRP1B minor allele rs7568970 had a significant protective effect against CIS (558 SNPs, Bonferroni-corrected p = 0.02) that resisted adjustment for confounding factors, including the amount of cocaine use (adjusted beta = -0.965 and -2.35 for heterozygotes and homozygotes, respectively, p < 0.01). Using hypothesis-free prioritization of candidate genes along with thorough methodology in both the preclinical and human analysis pipelines, we provide reliable evidence that LRP1B and VPS13A are involved in the motor tolerance to cocaine in CUD patients, in line with their known pathophysiology.

The neurobehavioral effects of the designer drug naphyrone - an experimental investigation with pharmacokinetics and concentration/effect relationship in mice.

RATIONALE: The recreational use of naphyrone, a potent synthetic cathinone with a pyrovalerone structure, has raised questions about possible deleterious neurobehavioral consequences. OBJECTIVE: To investigate naphyrone-induced neurobehavioral effects and alterations in brain monoamines using two patterns of abuse, i.e., single and repeated (binge) use. METHODS: We studied naphyrone dose/induced locomotor activity relationship at 3, 10, 30, and 100 mg/kg in mice. We investigated the effects of single (30 mg/kg; acute injection) versus repeated (30 mg/kg ×3/day for 3 days; binge injection) intraperitoneal naphyrone administration on locomotor activity, anxiety-like behavior, spatial recognition memory, anhedonia, behavioral despair, and social interaction. We measured post-mortem prefrontal cortex levels of monoamines and modeled naphyrone pharmacokinetics and concentration/locomotor effect relationship. RESULTS: Both naphyrone administration patterns induced time-dependent increases in locomotor activity (p < 0.001 and p < 0.0001, respectively) and social interaction (p < 0.05 and p < 0.001, respectively) but did not alter spatial recognition memory or anhedonia. Acute naphyrone injection induced anxiety-like behavior (p < 0.01) and reduced resignation (p < 0.01) whereas binge administration induced non-anxiety-like behavior (p < 0.05) and did not alter behavioral despair. Both patterns increased the prefrontal cortex dopamine (p < 0.0001) and norepinephrine (p < 0.05 and p < 0.01, respectively) but not serotonin content. Naphyrone pharmacokinetics followed a two-compartment model with an overall elimination half-life of 0.3 h. The naphyrone concentration/locomotor effect relationship was described by an additive Emax model with an EC50 of 672 µg/L. CONCLUSIONS: Single naphyrone administration increases locomotor activity according to a direct concentration/effect relationship. The neurobehavioral effects after binge differs from those after single administration and are not explained by drug accumulation given the relatively fast elimination.

Acute and chronic neurobehavioral effects of the designer drug and bath salt constituent 3,4-methylenedioxypyrovalerone in the rat.

BACKGROUND: The substantial increase in use of 3,4-methylenedioxypyrovalerone (MDPV), a popular recreational synthetic cathinone, has raised legitimate questions about its behavioral consequences and abuse liability. AIMS: The aim of this study was to study MDPV-induced neurobehavioral effects in the rat, using different paradigms traditionally developed to study drug-attributed addictive properties. METHODS: Different patterns of intraperitoneal 3 mg/kg MDPV administration were investigated. Consequences on rat horizontal locomotion and behavior of acute, intermittent (once daily dosing over 10 days), and binge (three-time daily dosing for 3 days) MDPV administration as well as challenge after 10 day MDPV withdrawal were studied. The dopamine receptor-D1 antagonist, SCH23390, was bilaterally infused in the nucleus accumbens to determine the role of D1-receptors in MDPV-related effects on the associative memory recall using the conditioned place preference paradigm. In addition, in a separate experience using western blot, we investigated the effects of chronic MDPV administration (four injections during 24 h) on ΔFosB expression in the nucleus accumbens, caudate putamen, and prefrontal cortex. RESULTS: Acute MDPV administration increased stereotypies and open arm entries in the elevated plus maze while SCH23390 abolished MDPV-induced enhancing effects on memory consolidation. Intermittent MDPV administration resulted in sensitization of MDPV-induced locomotor effects and tolerance during the following challenge. With binge MDPV administration, locomotor activity was not altered despite tolerance onset after challenge. SCH23390 abolished MDPV-induced conditioned place preference. Chronic MDPV administration induced ΔFosB accumulation in the nucleus accumbens, caudate putamen, and prefrontal cortex. CONCLUSIONS: Our findings clearly show that MDPV produces profound behavioral alterations mediated by the activation of the dopaminergic system similarly to other amphetamines.

Is the 3,4-methylendioxypyrovalerone/mephedrone combination responsible for enhanced stimulant effects? A rat study with investigation of the effect/concentration relationships.

RATIONALE: The use of synthetic cathinones as recreational drugs frequently sold in combination has been increasing exponentially. However, the consequences of combining cathinones on the resulting stimulant effects and the pharmacokinetics have been poorly investigated. OBJECTIVE AND METHODS: To study 3,4-methylenedioxypyrovalerone (MDPV; 3 mg/kg) and mephedrone (4-MMC; 30 mg/kg)-induced effects on rat locomotor activity and pharmacokinetics, administered alone or in combination by the intragastric route. The pharmacokinetic parameters were determined using non-compartmental analysis and the relationships between the locomotor activity and drug concentrations using sigmoidal Emax modeling. RESULTS: Locomotor activity significantly increased during the first hour post-administration with the MDPV/4-MMC combination in comparison to MDPV (p < 0.001) and 4-MMC (p < 0.01) alone. The pharmacokinetic profile of MDPV, but not 4-MMC, was significantly modified with the combination resulting in decreases in Cmax (16.4 ± 5.5 versus 62.2 ± 14.2 µg/L, p < 0.05) and AUC0 → ∞ (708 ± 91 versus 3316 ± 682 µg/L/min, p < 0.01) and increases in V/F (582.6 ± 136.8 versus 115.9 ± 42.7 L/kg, p < 0.05) and Cl/F (4.6 ± 0.7 versus 1.2 ± 0.4 L/kg/min, p < 0.01) in comparison to MDPV alone. The sigmoidal Emax model fitted the observed data well; MDPV being markedly more potent than 4-MMC (EC50, 0.043 versus 0.7 µmol/L). The enhancing factor representing the MDPV contribution to the alteration in the relationships between locomotor activity and 4-MMC concentrations was 0.3. CONCLUSION: An MDPV/4-MMC combination results in enhanced stimulant effects in the rat, despite significant reduction in MDPV bioavailability. Enhanced effects could be explained by increased MDPV distribution and/or possible complementation at the brain dopaminergic targets. However, the exact consequences of the MDPV/4-MMC combination in humans remain to be clarified.

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

RATIONALE: The 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. OBJECTIVES: Potent 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 METHODS: We compared the pharmacological responses induced by RB101 in wild-type and preproenkephalin-deficient mice (Penk1-/-) using several behavioural assays. RESULTS: In 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. CONCLUSIONS: The 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.

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.

Baclofen-Induced Neuro-Respiratory Toxicity in the Rat: Contribution of Tolerance and Characterization of Withdrawal Syndrome.

Baclofen, a γ-amino-butyric acid type-B receptor agonist with exponentially increased use at high-dose to facilitate abstinence in chronic alcoholics, is responsible for increasing poisonings. Tolerance and withdrawal syndromes have been reported during prolonged treatment but their contribution to the variability of baclofen-induced neurotoxicity in overdose is unknown. We studied baclofen-induced effects on rat sedation, temperature, and ventilation and modeled baclofen pharmacokinetics and effect/concentration relationships aiming to investigate the consequences of repeated baclofen pretreatment and to characterize withdrawal syndrome. Baclofen-induced dose-dependent sedation (p <0.01), hypothermia (p <.001) and respiratory depression (p <.01) were altered in repeatedly baclofen-pretreated rats (p <.05). Repeatedly baclofen-pretreated rats did not exhibit respiratory depression following baclofen overdose due to limitations on baclofen-induced increase in inspiratory (p <.01) and expiratory times (p <.01). Only slight hypoxemia without respiratory acidosis was observed. Baclofen discontinuation resulted in hyperlocomotion and non-anxiogenic withdrawal symptoms. Regarding pharmacokinetics, repeated baclofen pretreatment increased the peak concentration (p <.05) and absorption constant rate (p <.05) and reduced the distribution volume (p <.0001) and elimination half-life (p <.05). Analysis of the effect/concentration relationships indicated that plasma baclofen concentration decreases more rapidly than all studied neuro-respiratory effects, in tolerant and non-tolerant rats. Taken together, our findings supported the role of brain distribution in baclofen-induced neurotoxicity expression and its probable involvement in tolerance-related attenuation in addition to physiological adaptations of ventilation. In conclusion, repeated pretreatment attenuates baclofen-attributed neurotoxicity in overdose and results in post-discontinuation withdrawal syndrome. Our findings suggest both pharmacodynamic and pharmacokinetic mechanisms whose relative contributions to the variability of baclofen-induced neurotoxicity in overdose remain to be established.

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

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 24h 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 24h 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.

Simultaneous determination of vigabatrin and amino acid neurotransmitters in brain microdialysates by capillary electrophoresis with laser-induced fluorescence detection.

Capillary electrophoresis with laser-induced fluorescence detection (CE-LIFD) coupled to in vivo microdialysis sampling was used in order to monitor simultaneously a drug and several neurotransmitters in the brain extracellular fluid. Determination of the antiepileptic drug vigabatrin and the amino acid neurotransmitters glutamate (Glu), l-aspartate (l-Asp) and gamma-aminobutyric acid (GABA) was performed on low-concentration samples which were derivatized with naphthalene-2,3-dicarboxaldehyde (NDA) and separated using a pH 9.2 75 mM sodium borate running buffer containing 60 mM sodium dodecyl sulfate (SDS) and 5mM hydroxypropyl-beta-cyclodextrin (HP-beta-CD). Glu, l-Asp and vigabatrin derivatized at a concentration of 1.0 x 10(-9) M, and GABA derivatized at a concentration of 5.0 x 10(-9) M, produced peaks with signal-to-noise ratios of 8:1, 8:1, 4:1 and 5:1, respectively. The nature of the neurotransmitter peaks found in rat brain microdialysates was confirmed by both electrophoretic and pharmacological validations. This method was used for monitoring vigabatrin and amino acid neurotransmitters in microdialysates from the rat striatum during intracerebral infusion of the drug and revealed rapid vigabatrin-induced changes in GABA and Glu levels. This original application of CE-LIFD coupled to microdialysis represents a powerful tool for pharmacokinetic/pharmacodynamic investigations.

Influence of cocaine administration patterns on dopamine receptor regulation.

RATIONALE: Chronic exposure to drugs of abuse induces important modifications on neuronal systems. Increasing evidence shows that the consequences to chronic cocaine exposure can be different depending on the administration pattern. OBJECTIVES: The aim of the present study was to evaluate the consequences of two cocaine administration patterns on dopaminergic receptor regulation. METHODS: Male Sprague-Dawley rats were injected with cocaine (20 mg/kg, i.p.) for 14 days according to an intermittent (one daily injection) or a binge (three daily injections) pattern. By autoradiography, we compared the modifications of dopamine D1 and D2 receptor densities in the dopaminergic systems (mesocorticolimbic and nigrostriatal) 1 (WD1) and 14 (WD14) days after the last cocaine injection. RESULTS: On WD1, we observed modifications of D1 receptors after the binge cocaine treatment pattern while no modification was observed after the intermittent pattern, suggesting that multiple daily injections are needed to induce early D1 receptor modifications. On the contrary, densities of the D2 receptors were modified by both cocaine administration patterns, and interestingly, they were opposite depending on the administration pattern. On WD14, we observed different modifications of D1 and D2 receptors depending on the administration pattern, suggesting that the cocaine administration pattern promoted long-term regulations of the dopaminergic system. CONCLUSION: Two cocaine administration patterns induce different modifications of the dopaminergic receptor densities.

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.

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.

Analysis of serotonin in brain microdialysates using capillary electrophoresis and native laser-induced fluorescence detection.

Serotonin or 5-hydroxytryptamine (5-HT) is a major neurotransmitter in the central nervous system. In this work, a method for analyzing 5-HT in brain microdialysis samples using a commercially available capillary electrophoresis (CE) system has been developed. A pH-mediated in-capillary preconcentration of samples was performed, and after separation by capillary zone electrophoresis, native fluorescence of 5-HT was detected by a 266 nm solid-state laser. The separation conditions for the analysis of 5-HT in standard solutions and microdialysates have been optimized, and this method has been validated on both pharmacological and analytical bases. Separation of 5-HT was performed using a 80 mmol/L citrate buffer, pH 2.5, containing 20 mmol/L hydroxypropyl-beta-cyclodextrin (HP-beta-CD) and +30 kV voltage. The detection limit was 2.5 x 10(-10) mol/L. This method allows the in vivo brain monitoring of 5-HT using a simple, accurate CE measurement in underivatized microdialysis samples.

In vivo simultaneous monitoring of gamma-aminobutyric acid, glutamate, and L-aspartate using brain microdialysis and capillary electrophoresis with laser-induced fluorescence detection: Analytical developments and in vitro/in vivo validations.

gamma-Aminobutyric acid (GABA), glutamate (Glu), and L-aspartate (L-Asp) are three major amino acid neurotransmitters in the central nervous system. In this work, a method for the separation of these three neurotransmitters in brain microdialysis samples using a commercially available capillary electrophoresis (CE) system has been developed. Molecules were tagged on their primary amine function with the fluorogene agent naphthalene-2,3-dicarboxaldehyde (NDA), and, after separation by micellar electrokinetic chromatography, were detected by laser-induced fluorescence using a 442 nm helium-cadmium laser. The separation conditions for the analysis of derivatized neurotransmitters in standard solutions and microdialysates have been optimized, and this method has been validated on both pharmacological and analytical basis. The separation of GABA, Glu, and L-Asp takes less than 10 min by using a 75 mmol/L borate buffer, pH 9.2, containing 70 mmol/L SDS and 10 mmol/L hydroxypropyl-beta-cyclodextrin and + 25 kV voltage. The detection limits were 3, 15 nmol/L and, 5 nmol/L for GABA, Glu, and L-Asp, respectively. Moreover, submicroliter samples can be analyzed. This method allows a simple, rapid and accurate measurement of the three amino acid neurotransmitters for the in vivo brain monitoring using microdialysis sampling.