Simultaneous serotonin and dopamine monitoring across timescales by rapid pulse voltammetry with partial least squares regression.

Many voltammetry methods have been developed to monitor brain extracellular dopamine levels. Fewer approaches have been successful in detecting serotonin in vivo. No voltammetric techniques are currently available to monitor both neurotransmitters simultaneously across timescales, even though they play integrated roles in modulating behavior. We provide proof-of-concept for rapid pulse voltammetry coupled with partial least squares regression (RPV-PLSR), an approach adapted from multi-electrode systems (i.e., electronic tongues) used to identify multiple components in complex environments. We exploited small differences in analyte redox profiles to select pulse steps for RPV waveforms. Using an intentionally designed pulse strategy combined with custom instrumentation and analysis software, we monitored basal and stimulated levels of dopamine and serotonin. In addition to faradaic currents, capacitive currents were important factors in analyte identification arguing against background subtraction. Compared to fast-scan cyclic voltammetry-principal components regression (FSCV-PCR), RPV-PLSR better differentiated and quantified basal and stimulated dopamine and serotonin associated with striatal recording electrode position, optical stimulation frequency, and serotonin reuptake inhibition. The RPV-PLSR approach can be generalized to other electrochemically active neurotransmitters and provides a feedback pipeline for future optimization of multi-analyte, fit-for-purpose waveforms and machine learning approaches to data analysis.

The role of uptake and degradation in the regulation of peripheral serotonin dynamics in Gulf toadfish, Opsanus beta.

The neurotransmitter serotonin (5-hyroxytryptamine, 5-HT) is involved in a variety of peripheral processes. Arguably most notable is its role as a circulating vasoconstrictor in the plasma of vertebrates. Plasma 5-HT is maintained at constant levels under normal conditions through the processes of cellular uptake, degradation, and excretion, known collectively as clearance. However, the degree to which each individual component of clearance contributes to this whole animal response remains poorly understood. The goal of this experiment was to determine the extent to which transporter-mediated uptake and intracellular degradation contribute to 5-HT clearance in the model teleost Gulf toadfish (Opsanus beta). Fish that were treated with the 5-HT transport inhibitors fluoxetine, buproprion, and decynium-22 had 1.47-fold higher plasma 5-HT concentrations and a 40% decrease in clearance rate compared to control fish. In contrast, fish treated with the MAO inhibitor clorgyline had a 1.54-fold increase in plasma 5-HT with no change in clearance rate. The results show that transporter-mediated 5-HT uptake plays an important role in controlling circulating 5-HT and whole body 5-HT homeostasis.

Serotonergic modulation of basolateral amygdala nucleus in the extinction of reward-driven learning: The role of 5-HT bioavailability and 5-HT1A receptor.

Serotonin (5-HT) neurotransmission has been associated with reward-related behaviour. Moreover, the serotonergic system modulates the basolateral amygdala (BLA), a structure involved in reward encoding, and reward prediction error. However, the role played by 5-HT on BLA during a reward-driven task has not been fully elucidated. In this paper, we investigated whether serotonergic modulation of the BLA is involved in reward-driven learning. To this end, we trained Long Evans rats in an operant conditioning task, and examined the effects of fluoxetine treatment (a selective serotonin reuptake inhibitor, 10 mg/kg) in combination with BLA lesions with NMDA (20 mg/mL) on extinction learning. We also investigated whether intra-BLA injection of the serotonergic 5-HT1A receptor agonist 8-OH DPAT, or antagonist WAY-100635, alters extinction performance. We found that fluoxetine treatment strongly accelerated extinction learning, while BLA lesions partially reverted this effect and slightly impaired consolidation of extinction. Stimulation and inhibition of 5-HT1A receptors in BLA induced opposite effects to those of fluoxetine, impairing or accelerating extinction performance, respectively. Our findings suggest that 5-HT modulates reward-driven learning, and 5-HT1A receptors located in the BLA are relevant for extinction.

Efficiency of Penetration of Dopamine and Serotonin Peptide Derivatives through the Artificial Membranes.

A mass spectrometric method has been developed for determining the content of dopamine and serotonin derivatives, which allows evaluating the efficiency of their penetration through artificial membranes depending on the structure of their peptide fragment. In this case, the diffusion of dopamine and serotonin derivatives through the membrane occurred as a result of competitive interactions. It was shown which compounds in this mixture more easily penetrate through artificial membranes. It was found that the most promising in terms of overcoming the BBB are Boc-Pro-Srt and Boc-Pro-DOPA.

Increasing serotonin bioavailability in preweaned dairy calves impacts hematology, growth, and behavior.

Peripheral serotonin has been shown to regulate important physiological functions such as energy homeostasis and immunity, particularly in rodent and humans, but its role is poorly understood in livestock species. Herein, we tested the safety and effectiveness of increasing serotonin bioavailability in preweaned dairy calves by oral supplementation of a serotonin precursor (5-hydroxytryptophan, 5-HTP) or a serotonin reuptake inhibitor (fluoxetine, FLX). Bull Holstein calves (21 ± 2 d old; N = 24) were fed milk replacer (8 L/d) supplemented with either saline as control (CON, 8 mL/d, n = 8), FLX (40 mg/d, approx. 0.8 mg/kg; n = 8), or 5-HTP (90 mg/d, approx. 1.8 mg/kg; n = 8) for 10 consecutive days in a complete randomized block design. Heart rate (HR), respiration rate, rectal temperature, and health scores were recorded daily. Hip height and body weight were measured at d 1, 5, and 10 relative to initiation of supplementation. Blood samples were collected once before the supplementation period (d 1), during the 10-d supplementation period (daily), and during a 14-d withdrawal period (d 2, 3, 4, 7, and 14 relative to initiation of withdrawal). Cerebrospinal fluid and muscle tissue were collected from a subset of calves (n = 12) that were euthanized after the 10-d supplementation or 14-d withdrawal period. Whole blood serotonin concentrations increased in 5-HTP calves and decreased in FLX calves compared with CON (P < 0.001), indicating that serotonin bioavailability was increased in both groups. Whole blood serotonin concentrations of 5-HTP and FLX calves returned to CON levels after 7 d of withdrawal. All calves grew and were considered healthy throughout the study. In fact, calves fed 5-HTP had higher average daily gain compared with CON (0.87 vs 0.66 ± 0.12 kg/d, P = 0.05). Calves fed FLX had lower HR (P = 0.02) and greater red blood cells and hemoglobin counts on d 10 of supplementation compared with CON (P < 0.01). After the 14-d withdrawal period, FLX was not detected in circulation of FLX calves, but was still present in the muscle tissue. Our results demonstrate that manipulation of the serotonin pathway by supplementing FLX or 5-HTP is a feasible and safe approach in preweaned dairy calves; however, it takes more than 14 d for FLX to be completely withdrawn from the body.

Isobolographic analysis of the cutaneous antinociceptive interaction between bupivacaine co-injected with serotonin in rats.

BACKGROUND: The aim of this experiment was to investigate a long-lasting local anesthetic bupivacaine combined with serotonin at inducing cutaneous antinociception. METHODS: The skin antinociception, characterized by an inhibition of the cutaneous trunci muscle reflex (CTMR) following the pinprick on the dorsal skin of rats, was evaluated. The cutaneous antinociceptive effects of bupivacaine alone, serotonin alone, or bupivacaine co-injected with serotonin in a dose-dependent fashion were constructed, while the drug-drug interactions were evaluated by isobologram. RESULTS: Subcutaneous serotonin, as well as the local anesthetic bupivacaine provoked dose-related cutaneous antinociception. On an equipotent basis (50% effective dose [ED50]), the relative potency was bupivacaine (0.43 [0.37-0.50] µmol)>serotonin (1.27 [1.15-1.40] µmol) (p<0.01). At the equi-anesthetic doses (ED75, ED50 and ED25), the duration of bupivacaine was similar to that of serotonin at producing cutaneous antinociceptive effects. Co-administration of bupivacaine and serotonin displayed a synergistic antinociception. CONCLUSIONS: The preclinical data demonstrated that serotonin is less potent in eliciting cutaneous antinociceptive effects but has the similar duration of action, compared with bupivacaine. We also found a more significant depth of the sensory block with bupivacaine+serotonin than bupivacaine alone.

Cranial dural permeability of inflammatory nociceptive mediators: Potential implications for animal models of migraine.

Background Application of inflammatory mediators to the cranial dura has been used as a method to activate and sensitize neurons in the meningeal sensory pathway in preclinical behavioral studies of headache mechanisms. However, the relatively high concentrations and volumes used in these studies raise the question of whether the applied agents might pass through the dura to act directly on central neurons, thus bypassing the dural afferent pathway. Methods We used a radiolabeling approach to quantify the meningeal permeability of two of the inflammatory mediators, 5-HT and PGE2, when applied to the cranial dura as part of an inflammatory mixture used in preclinical headache models. Results Both agents could be detected in samples taken four hours after dural application in the cerebrospinal fluid (CSF) and, in measurements made only for PGE2, in the central nervous system (CNS) as well. Based on our measurements, we made estimates of the CSF and CNS levels that would be attained with the higher concentrations and volumes of 5HT and PGE2 that were exogenously applied in previous pre-clinical headache studies. These estimated levels were comparable to or larger than normal endogenous levels, potentially large enough to have physiological effects. Conclusions The finding that the cranial meninges are permeable to the two tested inflammatory mediators PGE2 and 5-HT raises some uncertainty about whether the behavioral changes observed in prior pre-clinical headache studies with these as well as other agents can be attributed entirely to the activation of dural nociceptors, particularly when the agents are applied at concentrations several orders of magnitude above physiological levels.

The antidepressant bupropion is a negative allosteric modulator of serotonin type 3A receptors.

The FDA-approved antidepressant and smoking cessation drug bupropion is known to inhibit dopamine and norepinephrine reuptake transporters, as well as nicotinic acetylcholine receptors (nAChRs) which are cation-conducting members of the Cys-loop superfamily of ion channels, and more broadly pentameric ligand-gated ion channels (pLGICs). In the present study, we examined the ability of bupropion and its primary metabolite hydroxybupropion to block the function of cation-selective serotonin type 3A receptors (5-HT3ARs), and further characterized bupropion's pharmacological effects at these receptors. Mouse 5-HT3ARs were heterologously expressed in HEK-293 cells or Xenopus laevis oocytes for equilibrium binding studies. In addition, the latter expression system was utilized for functional studies by employing two-electrode voltage-clamp recordings. Both bupropion and hydroxybupropion inhibited serotonin-gated currents from 5-HT3ARs reversibly and dose-dependently with inhibitory potencies of 87 µM and 112 µM, respectively. Notably, the measured IC50 value for hydroxybupropion is within its therapeutically-relevant concentrations. The blockade by bupropion was largely non-competitive and non-use-dependent. Unlike its modulation at cation-selective pLGICs, bupropion displayed no significant inhibition of the function of anion-selective pLGICs. In summary, our results demonstrate allosteric blockade by bupropion of the 5-HT3AR. Importantly, given the possibility that bupropion's major active metabolite may achieve clinically relevant concentrations in the brain, our novel findings delineate a not yet identified pharmacological principle underlying its antidepressant effect.

Antidepressant-like Effects of LPM580153, A Novel Potent Triple Reuptake Inhibitor.

The purpose of this study was to characterize a novel compound, 4-[2-(dimethylamino)-1-(1-hydroxycyclohexyl) ethyl] phenyl 3-nitrophenyl ether, designated LPM580153. We used several well-validated animal models of depression to assess the antidepressant-like activity of LPM580153, followed by a neurotransmitter uptake assay and a corticosterone-induced cell injury model to explore its mechanism of action. In mice, LPM580153 reduced immobility time in the tail suspension test, and in rats subjected to chronic unpredictable mild stress it reversed reductions in body weight gain and ameliorated anhedonia. The neurotransmitter uptake assay results demonstrated that LPM580153 inhibited the uptake of serotonin, norepinephrine and dopamine. Furthermore, LPM580153 protected the SH-SY5Y cells against the cytotoxic activity of corticosterone, an action that might be related to the role of LPM580153 in increasing the protein levels of BDNF, p-ERK1/2, p-AKT, p-CREB and p-mTOR. Together, these findings indicate that LPM580153 is a novel triple reuptake inhibitor with robust antidepressant-like effects.

A novel isoquinoline compound abolishes chronic unpredictable mild stress-induced depressive-like behavior in mice.

Chronic unpredictable mild stress (CUMS) elicits aspects of cognitive and behavioral alterations that can be used to model comparable aspects of depression in humans. The aim of the present study was to investigate the antidepressant-like potential of 7-fluoro-1,3-diphenylisoquinoline-1-amine (FDPI), a novel isoquinoline compound, in CUMS, a model that meets face, construct and predictive criteria for validity. Swiss mice were subjected to different stress paradigms daily for a period of 35 days to induce the depressive-like behavior. The animals received concomitant FDPI (0.1 and 1mg/kg, intragastric) or paroxetine (8mg/kg, intraperitoneal) and CUMS. The behavioral tests (splash test, tail suspension test, modified forced swimming test and locomotor activity) were performed. The levels of cytokines, corticosterone and adrenocorticotropic (ACTH) hormones were determined in the mouse prefrontal cortex and serum. The synaptosomal [(3)H] serotonin (5-HT) uptake, nuclear factor (NF)-κB, tyrosine kinase receptor (TrkB) and pro-brain-derived neurotrophic factor (BDNF) levels were determined in the mouse prefrontal cortex. CUMS induced a depressive-like behavior in mice, which was demonstrated in the modified forced swimming, tail suspension and splash tests. FDPI at both doses prevented depressive-like behavior induced by CUMS, without altering the locomotor activity of mice. FDPI at the highest dose prevented the increase in the levels of NF-kB, pro-inflammatory cytokines, corticosterone and ACTH and modulated [(3)H]5-HT uptake and the proBDNF/TrkB signaling pathway altered by CUMS. The present findings demonstrated that FDPI elicited an antidepressant-like effect in a model of stress-induced depression.

The new psychoactive substances 5-(2-aminopropyl)indole (5-IT) and 6-(2-aminopropyl)indole (6-IT) interact with monoamine transporters in brain tissue.

In recent years, use of psychoactive synthetic stimulants has grown rapidly. 5-(2-Aminopropyl)indole (5-IT) is a synthetic drug associated with a number of fatalities, that appears to be one of the newest 3,4-methylenedioxymethamphetamine (MDMA) replacements. Here, the monoamine-releasing properties of 5-IT, its structural isomer 6-(2-aminopropyl)indole (6-IT), and MDMA were compared using in vitro release assays at transporters for dopamine (DAT), norepinephrine (NET), and serotonin (SERT) in rat brain synaptosomes. In vivo pharmacology was assessed by locomotor activity and a functional observational battery (FOB) in mice. 5-IT and 6-IT were potent substrates at DAT, NET, and SERT. In contrast with the non-selective releasing properties of MDMA, 5-IT displayed greater potency for release at DAT over SERT, while 6-IT displayed greater potency for release at SERT over DAT. 5-IT produced locomotor stimulation and typical stimulant effects in the FOB similar to those produced by MDMA. Conversely, 6-IT increased behaviors associated with 5-HT toxicity. 5-IT likely has high abuse potential, which may be somewhat diminished by its slow onset of in vivo effects, whereas 6-IT may have low abuse liability, but enhanced risk for adverse effects. Results indicate that subtle differences in the chemical structure of transporter ligands can have profound effects on biological activity. The potent monoamine-releasing actions of 5-IT, coupled with its known inhibition of MAO A, could underlie its dangerous effects when administered alone, and in combination with other monoaminergic drugs or medications. Consequently, 5-IT and related compounds may pose substantial risk for abuse and serious adverse effects in human users.

Escherichia coli Nissle 1917 enhances bioavailability of serotonin in gut tissues through modulation of synthesis and clearance.

Accumulating evidence shows indigenous gut microbes can interact with the human host through modulation of serotonin (5-HT) signaling. Here we investigate the impact of the probiotic Escherichia coli Nissle 1917 (EcN) on 5-HT signalling in gut tissues. Ex-vivo mouse ileal tissue sections were treated with either EcN or the human gut commensal MG1655, and effects on levels of 5-HT, precursors, and metabolites, were evaluated using amperometry and high performance liquid chromatography with electrochemical detection (HPLC-EC). Exposure of tissue to EcN cells, but not MG1655 cells, was found to increase levels of extra-cellular 5-HT. These effects were not observed when tissues were treated with cell-free supernatant from bacterial cultures. In contrast, when supernatant recovered from untreated ileal tissue was pre-incubated with EcN, the derivative cell-free supernatant was able to elevate 5-HT overflow when used to treat fresh ileal tissue. Measurement of 5-HT precursors and metabolites indicated EcN also increases intracellular 5-HTP and reduces 5-HIAA. The former pointed to modulation of tryptophan hydroxylase-1 to enhance 5-HT synthesis, while the latter indicates an impact on clearance into enterocytes through SERT. Taken together, these findings show EcN is able to enhance 5-HT bioavailability in ileal tissues through interaction with compounds secreted from host tissues.

Immobility responses between mouse strains correlate with distinct hippocampal serotonin transporter protein expression and function.

Mouse strain differences in immobility and in sensitivity to antidepressants have been observed in the forced swimming test (FST) and the tail suspension test (TST). However, the neurotransmitter systems and neural substrates that contribute to these differences remain unknown. To investigate the role of the hippocampal serotonin transporter (5-HTT), we measured baseline immobility and the immobility responses to fluoxetine (FLX) in the FST and the TST in male CD-1, C57BL/6, DBA and BALB/c mice. We observed strain differences in baseline immobility time, with CD-1 mice showing the longest and DBA mice showing the shortest. In contrast, DBA and BALB/c mice showed the highest sensitivity to FLX, whereas CD-1 and C57BL/6 mice showed the lowest sensitivity. Also we found strain differences in both the total 5-HTT protein level and the membrane-bound 5-HTT level (estimated by V max) as follows: DBA>BALB/c>CD-1=C57BL/6. The uptake efficiency of the membrane-bound 5-HTT (estimated by 1/K m) was highest in DBA and BALB/c mice and lowest in CD-1 and C57BL/6 mice. A correlation analysis of subregions within the hippocampus revealed that immobility time was negatively correlated with V max and positively correlated with K m in the hippocampus. Therefore a higher uptake capacity of the membrane-bound 5-HTT in the hippocampus was associated with lower baseline immobility and greater sensitivity to FLX. These results suggest that alterations in hippocampal 5-HTT activity may contribute to mouse strain differences in the FST and the TST.

Reduced computational models of serotonin synthesis, release, and reuptake.

Multiscale computational models can provide systemic evaluation and prediction of neuropharmacological drug effects. To date, little computational modeling work has been done to bridge from intracellular to neuronal circuit level. A complex model that describes the intracellular dynamics of the presynaptic terminal of a serotonergic neuron has been previously proposed. By systematically perturbing the model's components, we identify the slow and fast dynamical components of the model, and the reduced slow or fast mode of the model is computationally significantly more efficient with accuracy not deviating much from the original model. The reduced fast-mode model is particularly suitable for incorporating into neurobiologically realistic spiking neuronal models, and hence for large-scale realistic computational simulations. We also develop user-friendly software based on the reduced models to allow scientists to rapidly test and predict neuropharmacological drug effects at a systems level.

N-terminus regulation of VMAT2 mediates methamphetamine-stimulated efflux.

The 20 amino acid (AA) N-terminus of the vesicular monoamine transporter 2 (VMAT2) was examined as a regulator of VMAT2 function. Removal of the first 16 or 19 AAs of the N-terminus resulted in a molecule with reduced ability to sequester [(3)H]-5HT. A glutathione-S-transferase-construct of the N-terminus underwent phosphorylation in the presence of PKC at serines 15 and 18. These putative phosphorylation sites were examined for effects on function. Phospho-mimetic substitution of serines 15 and 18 with aspartate in the full-length VMAT2 resulted in reduced [(3)H]-5HT sequestration and reduced methamphetamine (METH)-stimulated efflux of preloaded [(3)H]-5HT. In contrast, mutation of serines 15 and 18 to alanines maintained intact net substrate sequestration but eliminated METH-stimulated efflux of pre-accumulated [(3)H]-5HT. In summary, these data suggest a model in which the VMAT2 N-terminus regulates monoamine sequestration.

Iptakalim attenuates self-administration and acquired goal-tracking behavior controlled by nicotine.

Iptakalim is an ATP-sensitive potassium channel opener, as well as an α4ß2-containing nicotinic acetylcholine receptor (nAChR) antagonist. Pretreatment with iptakalim diminishes nicotine-induced dopamine (DA) and glutamate release in the nucleus accumbens. This neuropharmacological profile suggests that iptakalim may be useful for treatment of nicotine dependence. Thus, we examined the effects of iptakalim in two preclinical models. First, the impact of iptakalim on the interoceptive stimulus effect of nicotine was evaluated by training rats in a discriminated goal-tracking task that included intermixed nicotine (0.4 mg/kg, SC) and saline sessions. Sucrose was intermittently presented in a response-independent manner only on nicotine sessions. On intervening test days, rats were pretreated with iptakalim (10, 30, 60 mg/kg, IP). Results revealed that iptakalim attenuated nicotine-evoked responding controlled by the nicotine stimulus in a dose-dependent manner. In a separate study, the impact of iptakalim on the reinforcing effects of nicotine was investigated by training rats to lever-press to self-administer nicotine (0.01 mg/kg/infusion) [Dosage error corrected]. Results revealed that pretreatment with iptakalim (1, 3, 6 mg/kg, IV) decreased nicotine intake (i.e., less active lever responding). Neither behavioral effect was due to a non-specific motor effect of iptakalim, nor to an ability of iptakalim to inhibit DA transporter (DAT) or serotonin transporter (SERT) function. Together, these finding support the notion that iptakalim may be an effective pharmacotherapy for increasing smoking cessation and a better understanding of its action could contribute to medication development.

p38 MAP kinase activation does not stimulate serotonin transport in rat brain: Implications for sickness behaviour mechanisms.

AIMS: Several studies suggested an association between dysregulation of immune mediators and behavioural, neuroendocrine and neurochemical features of depression. Available data showed that cytokines affect the serotonin transporter (SERT) activity through p38 MAP kinase (MAPK)-dependent mechanisms in some cell lines and mice neurons (Zhu et al., Neuropsychopharmacology, 2006; 31:2121-31). The aim of this study was to investigate the interaction of Interleukin-1ß (IL-1ß) or p38 MAPK with SERT activity in rat brain and cell lines. MAIN METHODS: Synaptosomes or cells were treated with IL-1ß or the p38 MAPK activator anisomycin at different concentrations and end-points and the modulation of SERT activity as Km and Vmax was evaluated. KEY FINDINGS: Treatments with IL-1ß or anisomycin did not affect serotonin uptake and p38 MAPK activation in rat synaptosomes, in contrast to reports in mice (Zhu et al., Neuropsychopharmacology, 2010; 35:2510-20). The same treatments activated p38 MAPK phosphorylation in HeLa cells used as positive controls. Similarly, no changes after anisomycin treatment could be detected in [(3)H]serotonin uptake rate in LLC-PK cells expressing human SERT, although phosphorylated p38 MAPK levels augmented significantly. Direct cytokine release in brain was induced by intracerebroventricular administration of bacterial lipopolysaccaride. Although pro-inflammatory cytokines, such as IL-1ß, IL6, and Tumor Necrosis Factor α, showed significant increases in brain cortex, modulation of SERT activity in term of Km and Vmax was not detected. SIGNIFICANCE: These results imply that the stimulation of serotonin uptake by cytokines may not be a unique and fundamental mechanism in the pathology of depression induced by altered immune response.

Serotonin uptake is largely mediated by platelets versus lymphocytes in peripheral blood cells.

The serotonin transporter (SERT), a primary target for many antidepressants, is expressed in the brain and also in peripheral blood cells. Although platelet SERT function is well accepted, lymphocyte SERT function has not been definitively characterized. Due to their small size, platelets often are found in peripheral blood mononuclear cell preparations aimed at isolating lymphocytes, monocytes, and macrophages. The presence of different cells makes it difficult to assign SERT expression and function to specific cell types. Here, we use flow cytometry and IDT307, a monoamine transporter substrate that fluoresces after uptake into cells, to investigate SERT function in lymphocyte and platelet populations independently, as well as simultaneously without prior isolation. We find that murine lymphocytes exhibit temperature-dependent IDT307 transport but uptake is independent of SERT. Lack of measurable SERT function in lymphocytes was corroborated by chronoamperometry using serotonin as a substrate. When we examined rhesus and human mixed blood cell populations, we found that platelets, and not lymphocytes, were primary contributors to SERT function. Overall, these findings indicate that lymphocyte SERT function is minimal. Moreover, flow cytometry, in conjunction with the fluorescent transporter substrate IDT307, can be widely applied to investigate SERT in platelets from populations of clinical significance.

A novel spirocyclic tropanyl-Δ²-isoxazoline derivative enhances citalopram and paroxetine binding to serotonin transporters as well as serotonin uptake.

A group of spirocyclic tropanyl-Δ(2)-isoxazolines was synthesized exploiting the 1,3-dipolar cycloaddition of nitrile oxides to olefins. Their interaction with the dopamine and serotonin transporters (DAT and SERT, respectively) was evaluated through binding experiments. The majority of the compounds had no inhibitory effects (IC(50) >> 10 µM), while some had an IC(50) value in the range 5-10 µM (8a-c, 10b and 11c on DAT, 12b on SERT). Unexpectedly, one of the tertiary amines under investigation, that is 3'-methoxy-8-methyl-spiro{8-azabicyclo[3.2.1]octane-3,5'(4'H)-isoxazole 7a, was able to enhance at a concentration of 10 µM both [(3)H]citalopram and [(3)H]paroxetine binding to SERT in rat brain homogenate (up to 25%, due to an increase of B(max)) and [(3)H]serotonin uptake (up to 30%) in cortical synaptosomes. This peculiar pharmacological profile of 7a suggests it binds to an allosteric site on SERT, and positions derivative 7a as a very useful tool to investigate SERT machinery.