In vivo continuous and simultaneous monitoring of brain energy substrates with a multiplex amperometric enzyme-based biosensor device.

Enzyme-based amperometric biosensors are widely used for monitoring key biomarkers. In experimental neuroscience there is a growing interest in in vivo continuous and simultaneous monitoring of metabolism-related biomarkers, like glucose, lactate and pyruvate. The use of multiplex biosensors will provide better understanding of brain energy metabolism and its role in neuropathologies such as diabetes, ischemia, and epilepsy. We have developed and characterized an implantable multiplex microbiosensor device (MBD) for simultaneous and continuous in vivo monitoring of glucose, lactate, and pyruvate. First, we developed and characterized amperometric microbiosensors for monitoring lactate and pyruvate. In vitro evaluation allowed us to choose the most suitable biosensors for incorporation into the MBD, along with glucose and background biosensors. Fully assembled MBDs were characterized in vitro. The calculated performance parameters (LOD, LR, LRS, IMAX and appKM) showed that the multiplex MBD was highly selective and sensitive (LRS≥100 nA/mM) for each analyte and within an adequate range for in vivo application. Finally, MBDs were implanted in the mPFC of anesthetized adult male Wistar rats for in vivo evaluation. Following an equilibration period, baseline brain levels of glucose (1.3±0.2 mM), lactate (1.5±0.4 mM) and pyruvate (0.3±0.1 mM) were established. Subsequently, the MBDs recorded the responses of the animals when submitted to hyperglycemic (40% glucose i.v.) and hypoglycemic (5 U/kg insulin i.v.) challenges. Afterwards, MBDs were recalibrated to convert electrochemical readings into accurate substrate concentrations and to assess biofouling. The presented MBD can monitor simultaneously multiple biomarkers in vivo.

Effects of amphetamine on dopamine release in the rat nucleus accumbens shell region depend on cannabinoid CB1 receptor activation.

The psychostimulant drug amphetamine is often prescribed to treat Attention-Deficit/Hyperactivity Disorder. The behavioral effects of the psychostimulant drug amphetamine depend on its ability to increase monoamine neurotransmission in brain regions such as the nucleus accumbens (NAC) and medial prefrontal cortex (mPFC). Recent behavioral data suggest that the endocannabinoid system also plays a role in this respect. Here we investigated the role of cannabinoid CB1 receptor activity in amphetamine-induced monoamine release in the NAC and/or mPFC of rats using in vivo microdialysis. Results show that systemic administration of a low, clinically relevant dose of amphetamine (0.5mg/kg) robustly increased dopamine and norepinephrine release (to ∼175-350% of baseline values) in the NAC shell and core subregions as well as the ventral and dorsal parts of the mPFC, while moderately enhancing extracellular serotonin levels (to ∼135% of baseline value) in the NAC core only. Although systemic administration of the CB1 receptor antagonist SR141716A (0-3mg/kg) alone did not affect monoamine release, it dose-dependently abolished amphetamine-induced dopamine release specifically in the NAC shell. SR141716A did not affect amphetamine-induced norepinephrine or serotonin release in any of the brain regions investigated. Thus, the effects of acute CB1 receptor blockade on amphetamine-induced monoamine transmission were restricted to dopamine, and more specifically to mesolimbic dopamine projections into the NAC shell. This brain region- and monoamine-selective role of CB1 receptors is suggested to subserve the behavioral effects of amphetamine.

Evaluation of permselective membranes for optimization of intracerebral amperometric glutamate biosensors.

Monitoring of extracellular brain glutamate concentrations by intracerebral biosensors is a promising approach to further investigate the role of this important neurotransmitter. However, amperometric biosensors are typically hampered by Faradaic interference caused by the presence of other electroactive species in the brain, such as ascorbic acid, dopamine, and uric acid. Various permselective membranes are often used on biosensors to prevent this. In this study we evaluated the most commonly used membranes, i.e. nafion, polyphenylenediamine, polypyrrole, polyaniline, and polynaphthol using a novel silica-based platinum electrode. First we selected the membranes with the highest sensitivity for hydrogen peroxide in vitro and an optimal selectivity against electrochemical interferents. Then we evaluated the performances of these membranes in a short lasting (3-4h) in vivo experiment. We found that best in vitro performance was accomplished with biosensors that were protected by a poly(m-phenylenediamine) membrane deposited onto the platinum electrode by cyclic voltammetry. However, post-implantation evaluation of these membranes showed poor selectivity against dopamine. Combination with a previously applied nafion layer did not protect the sensors against acute biofouling; indeed it was even counter effective. Finally, we investigated the ability of our biosensors to monitor the effect of glutamate transport blocker DL-TBOA on modulating glutamate concentrations in the prefrontal cortex of anaesthetized rats. The optimized biosensors recorded a rapid 35-fold increase in extracellular glutamate, and are considered suitable for further exploration in vivo.

Direct effect of nicotine on mesolimbic dopamine release in rat nucleus accumbens shell.

Nicotine stimulates dopamine (DA) cell firing via a local action at somatodendritic sites in the ventral tegmental area (VTA), increasing DA release in the nucleus accumbens (NAcc). Additionally, nicotine may also modulate DA release via a direct effect in the NAcc. This study examined the contribution of the latter mechanism on NAcc DA release by applying nicotine systemically, as well as locally in the VTA and NAcc shell region in rats. Furthermore, the effect of i.v. nicotine on cell firing rate of dopaminergic neurons in the VTA was measured. Systemic administration of nicotine (0.32mg/kg s.c.) increased extracellular DA levels in the NAcc to ∼1.5 fold of baseline, while DA levels in the VTA remained unaffected. A similar DA increase was observed after local NAcc infusion of nicotine (1µM and 10µM). However, 10-1000-fold higher nicotine concentrations were required in the VTA to produce a comparable 150% increase in extracellular DA levels in the ipsilateral NAcc. Additionally, electrophysiological experiments showed that the dopaminergic firing rate in the VTA showed a trend towards an increase after a nicotine dose of 0.1mg/kg i.v. Taken together these data indicate that the effects of nicotine on DA release at the level of the NAcc might be more important for the rewarding effects than originally proposed.

Tramadol increases extracellular levels of serotonin and noradrenaline as measured by in vivo microdialysis in the ventral hippocampus of freely-moving rats.

Tramadol is an atypical opioid with monoamine re-uptake inhibition properties. The aim of the current study was to compare, using in vivo microdialysis, the effect of tramadol on extracellular serotonin (5-HT) and noradrenaline (NA) levels in the rat ventral hippocampus with the effects of the dual 5-HT/NA inhibitors (SNRIs) duloxetine and venlafaxine, the tricyclic antidepressant clomipramine, the selective 5-HT re-uptake inhibitor (SSRI) citalopram, and the selective NA re-uptake inhibitor (NRI) reboxetine. It was found that tramadol, duloxetine and venlafaxine increased extracellular levels of both, 5-HT and NA, in a dose-dependent manner. Clomipramine also increased extracellular 5-HT and NA levels, however not dose-dependently in the tested dose range. Citalopram selectively increased extracellular 5-HT levels. Reboxetine increased extracellular NA levels and also to a minimal degree 5-HT levels. It can be concluded that, albeit less efficacious, the effects of tramadol on serotonergic and noradrenergic neurotransmission resemble those of the dual 5-HT and NA re-uptake inhibitors duloxetine, venlafaxine, and clomipramine, and are different from those of the SSRI citalopram and the NRI reboxetine.

Pre-clinical properties of the alpha4beta2 nicotinic acetylcholine receptor partial agonists varenicline, cytisine and dianicline translate to clinical efficacy for nicotine dependence.

BACKGROUND AND PURPOSE: Smoking cessation trials with three high-affinity partial agonists of alpha4beta2 neuronal nicotinic acetylcholine receptors (nAChRs) have demonstrated differences in their clinical efficacy. This work examines the origin of the differences by taking into account brain exposure and pharmacological effects at human alpha4beta2 nAChRs. EXPERIMENTAL APPROACH: Rat plasma and brain pharmacokinetics were characterized and used to predict human steady-state plasma and brain concentrations following recommended doses of each of the three compounds. The pharmacological characterization included in vitro affinities at different nAChR subtypes, functional efficacies and potencies at the human alpha4beta2 nAChR, as well as in vivo effects on rat mesolimbic dopamine turn-over. KEY RESULTS: A comparison of predicted human brain concentrations following therapeutic doses demonstrated that varenicline and nicotine, but not dianicline and cytisine, can extensively desensitize and, to a lesser extent, activate alpha4beta2 nAChRs. The limited clinical efficacy of dianicline may be accounted for by a combination of weak functional potency at alpha4beta2 nAChRs and moderate brain penetration, while recommended doses of cytisine, despite its high in vitro potency, are predicted to result in brain concentrations that are insufficient to affect alpha4beta2 nAChRs. CONCLUSIONS AND IMPLICATIONS: The data provide a plausible explanation for the higher abstinence rate in smoking cessation trials following treatment with varenicline than with the two other alpha4beta2 nAChR partial agonists. In addition, this retrospective analysis demonstrates the usefulness of combining in vitro and in vivo parameters with estimated therapeutic human brain concentrations for translation to clinical efficacy.

A study in male and female 5-HT transporter knockout rats: an animal model for anxiety and depression disorders.

Human studies have shown that a reduction of 5-HT transporter (SERT) increases the vulnerability for anxiety and depression. Moreover, women are more vulnerable to develop depression and anxiety disorders than men. For that reason we hypothesized that homozygous 5-HT transporter knockout rat (SERT(-/-)) models, especially female, are valuable and reliable animal models for humans with an increased vulnerability for anxiety- and depression-related disorders. As rats are extensively used in neuroscience research, we used the unique 5-HT transporter knockout rat, that was recently generated using N-ethyl-N-nitrosurea (ENU) -driven mutagenesis, to test this hypothesis. Behavioral testing revealed that male and female SERT(-/-) rats spent less time in the center of the open field and spent less time on the open arm of the elevated plus maze compared with wild-type 5-HT transporter knockout rats (SERT(+/+)). In the novelty suppressed feeding test, only male SERT(-/-) rats showed a higher latency before starting to eat in a bright novel arena compared with SERT(+/+) controls. Both male and female SERT(-/-) rats showed a higher escape latency from their home cage than SERT(+/+) littermates. Moreover, SERT(-/-) rats were less mobile in the forced swim test, and sucrose consumption was reduced in SERT(-/-) rats relative to SERT(+/+) rats. Both effects were sex-independent. Neurochemically, basal extracellular 5-HT levels were elevated to a similar extent in male and female SERT(-/-) rats, which was not influenced by the selective 5-HT reuptake inhibitor citalopram. 5-HT immunostaining revealed no difference between SERT(+/+) and SERT(-/-) rats in the dorsal raphe nuclei, in both males and females. These findings demonstrate that SERT(-/-) rats show anxiety and depression-related behavior, independent of sex. Genetic inactivation of the SERT has apparently such a great impact on behavior, that hardly any differences are found between male and female rats. This knockout rat model may provide a valuable model to study anxiety- and depression-related disorders in male and female rats.

Characterization of the serotonin transporter knockout rat: a selective change in the functioning of the serotonergic system.

Serotonergic signaling is involved in many neurobiological processes and disturbed 5-HT homeostasis is implicated in a variety of psychiatric and addictive disorders. Here, we describe the functional characterization of the serotonin transporter (SERT) knockout rat model, that is generated by N-ethyl-N-nitrosurea (ENU)-driven target-selected mutagenesis. Biochemical characterization revealed that SERT mRNA and functional protein are completely absent in homozygous knockout (SERT-/-) rats, and that there is a gene dose-dependent reduction in the expression and function of the SERT in heterozygous knockout rats. As a result, 5-HT homeostasis was found to be severely affected in SERT-/- rats: 5-HT tissue levels and depolarization-induced 5-HT release were significantly reduced, and basal extracellular 5-HT levels in the hippocampus were ninefold increased. Interestingly, we found no compensatory changes in in vitro activity of tryptophan hydroxylase and monoamine oxidase, the primary enzymes involved in 5-HT synthesis and degradation, respectively. Similarly, no major adaptations in non-serotonergic systems were found, as determined by dopamine and noradrenaline transporter binding, monoamine tissue levels, and depolarization-induced release of dopamine, noradrenaline, glutamate and GABA. In conclusion, neurochemical changes in the SERT knockout rat are primarily limited to the serotonergic system, making this novel rat model potentially very useful for studying the behavioral and neurobiological consequences of disturbed 5-HT homeostasis.

Monitoring extracellular glutamate in hippocampal slices with a microsensor.

The direct local assessment of glutamate in brain slices may improve our understanding of glutamatergic neurotransmission significantly. However, an analytical technique that monitors glutamate directly in brain slices is currently not available. Most recording techniques either monitor derivatives of glutamate or detect glutamate that diffuses out of the slice. Microsensors provide a promising solution to fulfill this analytical requirement. In the present study we have implanted a 10 microm diameter hydrogel-coated microsensor in the CA1 area of hippocampal slices to monitor extracellular glutamate levels. The influence of several pharmacological agents, which facilitate glutamate release from neurons or astrocytes, was investigated to explore the applicability of the microsensor. It was observed that KCl, veratradine, alpha-latrotoxine (LTX), DL-threo-beta-benzyloxyaspartate (dl-TBOA) and L-cystine rapidly increased the extracellular glutamate levels. As far as we know this is the first study in which a microsensor is applied to monitor dynamic changes of glutamate in brain slices and in our opinion this type of research may contribute greatly to improve our understanding of the physiology of glutamatergic neurotransmission.

In vivo monitoring of extracellular glutamate in the brain with a microsensor.

Recent discoveries have revealed that glutamatergic neurotransmission in the central nervous system is mediated by a dynamic interplay between neurons and astrocytes. To enhance our understanding of this process, the study of extracellular glutamate is crucial. At present, microdialysis is the most frequently used analytical technique to monitor extracellular glutamate levels directly in the brain. However, the neuronal and physiological origin of the detected glutamate levels is questioned as they do not fulfil the classical release criteria for exocytotic release, such as calcium dependency or response to the sodium channel blocker tetrodotoxine (TTX). It is hypothesized that an analytical technique with a higher spatial and temporal resolution is required. Glutamate microsensors provide a promising analytical solution to meet this requirement. In the present study, we applied a 10 micro m diameter hydrogel-coated glutamate microsensor to monitor extracellular glutamate levels in the striatum of anesthetized rats. To explore the potential of the microsensor, different pharmacological agents were injected in the vicinity of the sensor at an approximate distance of 100 micro m. It was observed that KCl, exogenous glutamate, kainate and the reuptake inhibitor DL-threo-beta-benzyloxyaspartate (DL-TBOA) increased the extracellular glutamate levels significantly. TTX decreased the basal extracellular glutamate levels approximately 90%, which indicates that the microsensor is capable of detecting neuronally derived glutamate. This is one of the first studies in which a microsensor is applied in vivo on a routine base, and it is concluded that microsensor research can contribute significantly to improve our understanding of the physiology of glutamatergic neurotransmission in the brain.

Changes in dopamine levels and locomotor activity in response to selection on virgin lifespan in Drosophila melanogaster.

Among various other mechanisms, genetic differences in the production of reactive oxygen species are thought to underlie genetic variation for longevity. Here we report on possible changes in ROS production related processes in response to selection for divergent virgin lifespan in Drosophila. The selection lines were observed to differ significantly in dopamine levels and melanin pigmentation, which is associated with dopamine levels at eclosion. These findings confirm that variation in dopamine levels is associated with genetic variation for longevity. Dopamine has previously been implied in ROS production and in the occurrence of age-related neurodegenerative diseases. In addition, we propose a possible proximate mechanism by which dopamine levels affect longevity in Drosophila: We tested if increased dopamine levels were associated with a "rate-of-living" syndrome of increased activity and respiration levels, thus aggravating the level of oxidative stress. Findings on locomotor activity and oxygen consumption of short-lived flies were in line with expectations. However, the relation is not straightforward, as flies of the long-lived lines did not show any consistent differences in pigmentation or dopamine levels with respect to the control lines. Moreover, long-lived flies also had increased locomotor activity, but showed no consistent differences in respiration rate. This strongly suggests that the response for increased and decreased lifespan may be obtained by different mechanisms.

Influence of a selective histamine H3 receptor antagonist on hypothalamic neural activity, food intake and body weight.

OBJECTIVE: This study was conducted to elucidate whether antagonistic targeting of the histamine H3 receptor increases hypothalamic histamine levels, in parallel with decreases in food intake and body weight. METHODS: The competitive antagonist potency of a recently synthesized histamine H3 receptor antagonist, NNC 38-1049, was studied in intact HEK293 cells expressing human or rat histamine H3 receptor, in which NNC 38-1049 was allowed to antagonize the effect of the H3 receptor agonist R-alpha-methylhistamine on isoprenaline-induced accumulation of cAMP. The affinity of NNC 38-1049 for a number of variants of the histamine receptor was also determined. Following single dosing of normal rats with NNC 38-1049, hypothalamic histamine levels were assessed by means of microdialysis. Plasma and brain levels of NNC 38-1049 and acute effects on food intake and energy expenditure were followed after oral doses of 3-60 mg/kg. Potential side effects were examined with rat models of behaviour satiety sequence (BSS), pica behaviour and conditioned taste aversion (CTA). Intakes of food and water together with body weight were recorded for 15 days during daily dosing of dietary obese rats. RESULTS: NNC 38-1049 was found to be a highly specific and competitive antagonist towards both human and rat histamine H3 receptors, and measurable amounts of NNC 38-1049 were found in the plasma of rats following single oral doses of 3-60 mg/kg and in the brain after 15-60 mg/kg. Following single intraperitoneal injections of NNC 38-1049 (20 mg/kg), significant increases in extracellular histamine concentrations were observed. The same dose did not change BSS or pica behaviour acutely, nor did it induce CTA following repeated administration for 7 days. Reductions in food intake were seen very soon after administration, and occurred in a dose-dependent fashion. Energy expenditure was unchanged, but the respiratory quotient (RQ) tended to decrease at higher doses, indicating an increase in lipid oxidation. Twice daily administration of 20 mg/kg of NNC 38-1049 in old and dietary obese rats resulted in sustained reduction of food intake throughout a 2-week study, and was associated with a highly significant (P<0.01) decrease in body weight compared with controls (-18.4+/-3.4 vs +0.4+/-2.7 g). The same dose of NNC 38-1049 produced an acute decrease of water intake, but 24 h intakes were not significantly changed. CONCLUSIONS: The results of this study strongly support the idea that an increase in the hypothalamic concentration of histamine produces a specific reduction of food intake and that this effect can be translated into a decrease in body weight.

HPLC conditions are critical for the detection of GABA by microdialysis.

In microdialysis studies, neither exocytotic release of gamma-aminobutyric acid (GABA), nor the presence of GABA type B (GABA(B)) autoreceptors, have been clearly established. It was investigated whether the chromatographic separation of GABA may have contributed to discrepancies in the literature. After extending the profile of the HPLC chromatogram to a retention time of 60 min, it was observed that various unknown compounds of biological origin co-eluted near the GABA peak. The retention time of GABA appeared to be extremely sensitive to pH; even at a retention time of around 60 min there was only a small pH window (5.26 +/- 0.01) where GABA was consistently well separated from co-eluting compounds. GABA determined by the improved assay was sensitive to tetrodotoxin (TTX), calcium depletion and the GABA(B) autoreceptor agonist baclofen. The present results illustrate that if the proper analytical conditions are applied, extracellular GABA can be sampled and quantified by microdialysis in free-moving animals. However, when the time-curves are considered, there is a striking delay of about 15-30 min before the effects of TTX, calcium depletion or baclofen are observed, as compared to the reported response of neurotransmitters such as dopamine (less than 5 min). It is assumed that the glial cells serve as a buffer between the GABA synapse and the microdialysis probes. It is proposed that microdialysis samples measure synaptic GABA indirectly, through glial cells surrounding the synapses.

Regional distribution of biogenic amines, amino acids and cholinergic markers in the CNS of the C57BL/6 strain.

A reliable extrapolation of neurochemical alterations from a mouse model to human metabolic brain disease requires knowledge of neurotransmitter levels and related compounds in control mouse brain. C57BL/6 is a widely used background strain for knockout and transgenic mouse models. A prerequisite for reliable extrapolation from mouse brain to the human condition is the existence of analogous distribution patterns of neurotransmitters and related compounds in control mouse and human brain. We analysed regional distribution patterns of biogenic amines, neurotransmitter and non-neurotransmitter amino acids, and cholinergic markers. Distribution patterns were compared with known neurotransmitter pathways in human brain. The present study provides a reference work for future analyses of neurotransmitters and related compounds in mouse models bred in a C57BL/6 background strain.

Is the beneficial antidepressant effect of coadministration of pindolol really due to somatodendritic autoreceptor antagonism?

BACKGROUND: We investigated the combination of selective serotonin reuptake inhibitors (SSRIs) with the beta-adrenoceptor/serotonin 1A (5-HT(1A)) antagonist pindolol, based on the concept that 5-HT(1A) receptor blockade would eliminate the need for desensitization of presynaptic 5-HT(1A) receptors and therefore hasten the onset of action and improve the efficacy of SSRIs. However, since pindolol plasma levels after 2.5 mg three times a day are about 60 nmol/L, and the K(i) for the 5-HT(1A) receptor is 30 nmol/L, it is questionable whether pindolol levels in the brain would be sufficient to antagonize 5-HT(1A) receptors. Using microdialysis in the guinea pig, we correlated brain and plasma levels of pindolol with its capability of augmenting paroxetine-induced increases in brain 5-HT levels. In addition, central beta-receptor antagonism of pindolol was studied by investigating blockade of beta-agonist-induced increases in brain cyclic adenosine monophosphate (cAMP) formation. METHODS: Using microdialysis and jugular vein catheterization, we studied the ability of systemically administered pindolol to antagonize central 5-HT(1A) and beta-adrenoceptors, while simultaneously monitoring pindolol plasma and brain concentrations. RESULTS: Augmentation of paroxetine-induced increases in extracellular 5-HT levels in the ventral hippocampus was only observed at steady state plasma levels exceeding 7000 nmol/L (concurrent brain levels 600 nmol/L). In contrast, antagonism of beta-agonist-induced increases of brain cAMP levels was already observed at pindolol plasma levels of 70 nmol/L (concurrent brain levels < 3 nmol/L). CONCLUSIONS: At plasma levels that are observed in patients after 2.5 mg three times a day ( approximately 60 nmol/L), pindolol produces only a partial blockade of presynaptic 5-HT(1A) autoreceptors and does not augment the SSRI-induced 5-HT increase in the guinea pig brain. It is therefore very unlikely that the favorable effects of combining pindolol with SSRIs, as reported in a number of clinical studies, are due to 5-HT(1A) antagonism. Since pindolol completely blocks central beta-adrenoreceptors at clinically relevant plasma levels, it is possible that beta-adrenoceptor antagonism is involved in mediating pindolol's beneficial effects.

Acute and chronic effects of citalopram on postsynaptic 5-hydroxytryptamine(1A) receptor-mediated feedback: a microdialysis study in the amygdala.

Microdialysis was used to assess the involvement of postsynaptic 5-hydroxytryptamine(1A) (5-HT(1A)) receptors in the regulation of extracellular 5-HT in the amygdala. Local infusion of the 5-HT(1A) receptor agonist flesinoxan (0.3, 1, 3 microM) for 30 min into the amygdala maximally decreased 5-HT to 50% of basal level. Systemic administration of citalopram (10 micromol/kg) increased 5-HT to 175% of basal level. Local infusion of 1 microM of the 5-HT(1A) receptor antagonist WAY 100.635 into the amygdala augmented the effect of citalopram to more than 500% of basal 5-HT level. 5-HT(1A) receptor responsiveness after chronic citalopram treatment was determined in two ways. First, by local infusion of 1 microM flesinoxan for 30 min into the amygdala, which showed a significant 63% reduction in response (area under the concentration-time curve; AUC) for the citalopram group compared to the saline group. Second, by systemic administration of citalopram (10 micromol/kg), which increased 5-HT to 350% of basal level. The effect was larger than in untreated animals, but more important, local infusion of 1 microM WAY 100.635 into the amygdala now failed to augment the effect of citalopram. Both the flesinoxan and WAY 100.635 data suggest an involvement of postsynaptic 5-HT(1A) receptor-mediated feedback in the amygdala, which diminishes following chronic citalopram treatment.

Augmentation with a 5-HT(1A), but not a 5-HT(1B) receptor antagonist critically depends on the dose of citalopram.

Pharmacokinetic and pharmacodynamic parameters of the selective serotonin reuptake inhibitor 1-(3-dimethylaminopropyl)-1-(4-fluorophenyl)-5-phtalancarbonitril (citalopram) were determined in order to find optimal conditions for augmentation of its effect on extracellular serotonin [5-hydroxytryptamine (5-HT)] through blockade of 5-HT(1A) and 5-HT(1B) autoreceptors. Citalopram dose-dependently (0.3-10 micromol/kg s.c.) increased serotonin levels in ventral hippocampus of conscious rats. At plasma levels above approximately 0.15 microM, the effect of citalopram on extracellular 5-HT was augmented by both a 5-HT(1A) [N-[2-[4-(2-mehoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridil) cyclohexa necarboxamide trihydrochloride (Way 100635), 1 micromol/kg s.c.] and a 5-HT(1B) receptor antagonist (2'-methyl-4'-(5-methyl-[1,2, 4]oxadiazol-3-yl)biphenyl-4-carboxylic acid [4-methoxy]-3-(4-methylpiperazin-1-yl)phenyl]amide (GR 127935), 1 micromol/kg s.c.). However, at plasma levels of the selective serotonin reuptake inhibitor below 0.15 microM, the effects of the antagonists diverged viz. the 5-HT(1B) receptor antagonist was still able to potentiate citalopram's effect on extracellular 5-HT, while the 5-HT(1A) receptor antagonist was no longer effective. These results suggest that in contrast to 5-HT(1B) autoreceptors, indirect activation of 5-HT(1A) autoreceptors by citalopram is critically related to the dose of selective serotonin reuptake inhibitor administered. The latter may have consequences for selective serotonin reuptake inhibitor augmentation strategies with 5-HT(1A) receptor antagonists in the therapy of depression and anxiety disorders.

Desensitisation of 5-HT autoreceptors upon pharmacokinetically monitored chronic treatment with citalopram.

Rats were chronically treated with the selective serotonin re-uptake inhibitor citalopram [1-(3-dimethylaminopropyl)-1-(4-fluorophenyl)-5-phtalancarbonitril ], by means of osmotic minipumps. Using an infusion concentration of 50 mg/ml citalopram, steady-state plasma concentrations of approximately 0.3 mcM citalopram were maintained for 15 days. Citalopram plasma levels dropped below pharmacologically active concentrations 48 h after removal of the minipumps. Although chronic treatment with citalopram did induce an attenuated response by extracellular levels of 5-hydroxytryptamine (5-HT) after systemic administration of the 5-HT(1A) receptor agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), no effect of chronic citalopram treatment was observed when 5-HT(1B) receptor function was evaluated with a local infusion of 5-HT(1B/D) receptor agonist, sumatriptan (3-[2-dimethylamino]ethyl-N-methyl-1H-indole-5methane sulphonamide). Controversially, no augmentation of the increase of 5-HT levels was observed upon systemic administration of citalopram. It is concluded that, although chronic treatment with citalopram does induce desensitisation of 5-HT(1A) receptors, the absence of augmented effects of citalopram on 5-HT levels indicates that other mechanisms compensate for the loss of autoreceptor control.

New selective and potent 5-HT(1B/1D) antagonists: chemistry and pharmacological evaluation of N-piperazinylphenyl biphenylcarboxamides and biphenylsulfonamides.

A series of new analogues of N-[4-methoxy-3-(4-methylpiperazin-1-yl)phenyl] 2'-methyl-4'-(5-methyl-1,2,4-oxadiazol-3-yl)biphenyl-4-carboxamide (1; GR127935) as potent and selective 5-HT(1B/1D) antagonists were synthesized and evaluated pharmacologically. Their receptor binding profiles were comparable to that of 1. The 1,3,4-oxadiazole isomer 2 and the 4'-aminocarbonyl and 4'-amidinyl analogues (9 and 10) of 1 had higher affinities at the rat 5-HT(1B) receptor (IC(50) = 0.93, 1. 3, and 0.5 nM, respectively) and calf 5-HT(1D) receptor (IC(50) = 37, 10, and 3 nM, respectively) than did 1 (1.6 and 52 nM for rat 5-HT(1B) and calf 5-HT(1D) receptors, respectively). In the functional in vitro testing of 5-HT(1B/1D) antagonistic properties, 2, 9, 10, 11b (O-demethylated derivative of 2), 13a (O-methylsulfonyl analogue of 2), and 16 (which differs from 2 with a sulfonamide linker) showed more pronounced effects in the K(+)-induced 5-HT release in the cortex of guinea pig than did 1 and 3 (SB224289). Compounds 2, 9, and 10 were equally potent as 1 in rabbit saphenous vein model (pA(2) > 9). A biochemical study of 2 with in vivo microdialysis in the rat brain showed that it is capable of augmenting citalopram (a selective serotonin reuptake inhibitor, SSRI) induced 5-HT release in rat ventral hippocampus, while preventing the decrease in acetylcholine release elicited by citalopram administration. The molecular structure of 2 was determined by single-crystal X-ray analysis. The log P and log D values of these compounds were calculated. This study contributes to the SAR study of N-piperazinylphenyl biphenylcarboxamides as selective and potent 5-HT(1B/1D) antagonists.

Use of microdialysis for monitoring sympathetic and parasympathetic innervation of heart in conscious rats.

A microdialysis method was developed to sample norepinephrine and acetylcholine from the heart of freely moving rats. A flexible dialysis fiber (length 14 mm), with a copper wire inserted inside, was implanted into the heart. Extracellular norepinephrine was detectable for at least 72 h after implantation. Basal output levels 24 h after surgery were 140 pg/ml when corrected for in vitro recovery. Evidence was provided that the major part of norepinephrine in dialysates is derived from local neurotransmission. Acetylcholine was only detectable in cardiac dialysates when an esterase inhibitor was infused. Corrected basal output levels 24 h after surgery were 223 pg/ml when neostigmine was coinfused in a concentration of 100 mumol/l. In addition, the presence of local muscarinic autoreceptors on cholinergic neurons in the heart was shown. It is concluded that microdialysis is a reliable method that can be used to study the innervation of the heart in subchronic preparations in freely moving rats.