5-HT1B Autoreceptors Research Articles

Pharmacological characterisation of the electrically evoked release of monoamines from chicken brain in vitro

1. A study was conducted to develop an in vitro model for examining the basal and electrical-stimulation-induced release of [3H]monoamines from chicken hyperstriatal neurones in order to demonstrate the presence of presynaptic autoreceptors for the three main monoamine transmitters: noradrenaline, dopamine and 5-HT. 2. Two sets of experiments were carried out: the first was to evaluate the effect of calcium and tetrodotoxin (TTX, sodium channel conductance inhibitor) in order to demonstrate that evoked release of monoamines was a consequence of exocytotic processes; the second to investigate the effect of selective agonists and antagonists on neurotransmitter release. 3. Ross and Cobb broiler chickens of either sex (∼7 to 8 weeks old) were used. Slices of hyperstriatal tissue were preincubated with [3H]noradrenaline, [3H]dopamine or [3H]5-hydroxytryptamine (5-HT), washed, perfused and electrically stimulated at three time points (S1, S2 and S3) which released [3H]noradrenaline, [3H]dopamine and [3H]5-HT, as determined by scintillation spectrometry. 4. When calcium was removed from, or TTX added to, the superfusion medium prior to and including the second period of electrical stimulation (S2) the evoked releases of [3H]noradrenaline, [3H]dopamine and [3H]5-HT at S2 were abolished. 5. In the presence of the selective α 2-adrenoceptor agonist UK 14304 during the S2 period, the S2/S1 ratio was lower than the control ratio due to a reduction in the stimulated release of [3H]noradrenaline. The selective α 2-adrenoceptor antagonist RX 821002 blocked the UK 14304-induced reduction of evoked release and the S2/S1 ratio was similar to the control ratio. 6. The D2-like receptor agonist quinpirole reduced the S2/S1 ratio for [3H]dopamine release, an effect blocked by the antagonist AJ 76. The 5-HT1B receptor agonist CP 94253 during S2 reduced the S2/S1 ratio due to a reduction in evoked [3H]5-HT. This effect was blocked by the 5-HT1B receptor antagonist GR 55562. 7. The results demonstrate, for the first time, the functional presence of presynaptic α 2-adrenoceptors, presynaptic 5-HT1B autoreceptors and presynaptic D2-like autoreceptors in broiler chicken hyperstriatal neurones in vitro.

Elevated central monoamine receptor mRNA in rats bred for high endurance capacity: Implications for central fatigue

Although alteration to peripheral systems at the skeletal muscle level can contribute to one's ability to sustain endurance capacity, neural circuits regulating fatigue may also play a critical role. Previous studies demonstrated that increasing brain serotonin (5-HT) release is sufficient to hasten the onset of exercise-induced fatigue, while manipulations that increase brain dopamine (DA) release can delay the onset of fatigue. These results suggest that individual differences in endurance capacity could be due to factors capable of influencing the activity of 5-HT and DA systems. We evaluated possible differences in central fatigue pathways between two contrasting rat groups selectively bred for high (HCR) or low (LCR) capacity running. Using quantitative in situ hybridization, we measured messenger RNA (mRNA) levels of tryptophan hydroxylase (TPH), 5-HT transporter (5-HTT), 5-HT1A and 5-HT1B autoreceptors, dopamine receptor-D2 (DR-D2) autoreceptors and postsynaptic receptors, and dopamine receptor-D1 (DR-D1) postsynaptic receptors, in discrete brain regions of HCR and LCR. HCR expressed higher levels of 5-HT1B autoreceptor mRNA in the raphe nuclei relative to LCR, but similar levels of TPH, 5-HTT, and 5-HT1A mRNA in these areas. Surprisingly, HCR expressed higher levels of DR-D2 autoreceptor mRNA in the midbrain, while simultaneously expressing greater DR-D2 postsynaptic mRNA in the striatum compared to LCR. There were no differences in DR-D1 mRNA levels in the striatum or cortex between groups. These data suggest that central serotonergic and dopaminergic systems may be involved in the mechanisms by which HCR have delayed onset of exercise-induced fatigue compared to LCR.

Augmentation Strategies to Improve Treatment of Major Depression

The monoamine hypothesis has dominated almost forty years the psychopathology of depression. Yet this has not lead to antidepressants that are significantly more efficacious than the early tri- and tetracyclics from which they have evolved. Alternative hypotheses such as those involving adult neurogenesis or components of the hypothalamic-pituitaryadrenal- axis are either too premature or have not lead to drugs with improved antidepressant activity. Antidepressants may not be perfect, both in terms of efficacy and side effects, however their performance may be improved by making use of so-called augmentation strategies. Augmentation strategies that have potential to hasten and/or improve the therapeutic effect of antidepressants, in particular serotonin reuptake inhibitors, can have different forms. They can be aimed at reducing comorbid symptoms, such as anxiety, or they may be directed to processes that counteract the effect of serotonin reuptake inhibitors. Examples of the latter are those involving 5-HT1A and 5-HT1B autoreceptors, 5-HT 2C receptors and the availability of tryptophan. Another option could be exploring neuropeptide/serotonin interactions. The various augmentation strategies are reviewed in the context of literature data regarding neurobiology and pharmacotherapy of major depression.

Potentiation of Antidepressant-Like Activity with Lithium: Mechanism Involved

In the last decade, many augmentation strategies have been developed to increase the activity of antidepressant drugs or to reduce their long onset of action by acting on different targets. One of the first augmentation strategy used in psychiatric disorders is coadministration of lithium and antidepressant drugs. However, the underlaying mechanism of action involved in the potentiatory effect of lithium is still unclear and many hypotheses have been suggested such as activity on BDNF, ACTH, thyroid hormones and serotonin neurotransmission. All these systems being embedded in each other, we focused on the 5-HT neurotransmission-increase induced by lithium treatment. Based on neurobiochemical and behavioral results we tried to better understand its mechanism of action and we concluded that effect of lithium on 5-HT neurotransmission could be linked to a partial agonist activity on 5-HT1B autoreceptors, or to a modulatory activity on these receptors, located in the cortical area in the case of a short term treatment, or in the hippocampus in the case of a long term treatment. We also suggested that the anti-manic effect of lithium was linked to this activity on 5-HT1B receptors, occurring this time on 5-HT1B postsynaptic (heteroreceptors on dopaminergic pathways) receptors levels.

Basic Mechanisms of Augmentation of Antidepressant Effects with Thyroid Hormone

The thyroid hormone triiodothyronine (T3) has been used both to augment and accelerate the clinical effects of antidepressants, particularly the tricyclics. More recent work indicates that it may have similar actions with regard to the SSRIs. Two main mechanisms have been put forward to explain its antidepressant actions, (a) an action at the nuclear level involving stimulation of gene transcription, (b) an action at the cell membrane level involving potentiation of neurotransmission. In particular, there is considerable evidence for potentiation by T3 of the actions of the neurotransmitter 5-HT or serotonin. This evidence, which is mainly based on in vivo microdialysis studies, is reviewed, and evidence based on human and animal neuroendocrine studies considered. The effects of T3, alone and together with the SSRI fluoxetine, on mRNA levels for the 5-HT1A and 5-HT1B autoreceptors, which mediate serotonergic neurotransmission by feedback actions at the levels of cell firing(somatodendritic 5-HT1A autoreceptors) and neurotransmitter release (nerve terminal 5-HT1B autoreceptors) were also determined. Administration of a combination of fluoxetine and T3 induced reductions in the transcription of these autoreceptors, which may explain the clinical potentiating effects of this combination, and thus link the nuclear and neurotransmitter hypotheses of T3 action.

Compréhension du mécanisme d’action des antidépresseurs anciens ou nouveaux : apport des modèles de souris génétiquement modifiées en pharmacologie in vivo

The main hypothesis regarding the mechanism of action of antidepressant drugs is monoaminergic and mainly involves two neurotransmitters, serotonin and noradrenaline. Despite the well-recognized therapeutic efficacy of selective serotonin reuptake inhibitors (SSRIs), some disadvantages still occur. For example, they often require 4–6 weeks to achieve clinical benefits in depressed patients. In the past, some molecules that could shorten this long delay of action have been identified. The role of presynaptic autoreceptors – the activation of which leads to an inhibitory feedback control on neurotransmitter synthesis and release – has been extensively studied for antidepressant effects. In our laboratory, we studied the combined effects of an SSRI and a serotonin autoreceptor antagonist of the 5-HT1B subtype using intracerebral in vivo microdialysis in awake, freely moving mice. Important information on SSRIs has been obtained by applying this technique to genetically modified animals, such as constitutive knockout (KO) mice lacking 5-HT1B receptors (5-HT1B KO) generated by homologous recombination: we compared the effects of a combined treatment on extracellular/intrasynaptic levels of serotonin in various nerve terminals area in wild-type control and KO mice. Thus, we found that indirect activation of 5-HT1B autoreceptors limits the effects of SSRIs on dialysate 5-HT levels at serotonergic nerve terminals such as the ventral hippocampus. The study of substance P (neurokinin 1 receptor [R-NK1]) offers another example of the use of KO mice in the development of a new class of antidepressant drugs. NK1 receptor antagonists may display anxiolytic/antidepressant-like properties. The lack of selective compounds for each tachykinin receptor subtype (R-NK1, R-NK2 or R-NK3) and differences in their affinity between animal species have made R-NK1KO mice a very useful experimental tool. In collaborative work we found that genetic (R-NK1KO mice) or pharmacological (GR205171) blockade of R-NK1 is associated with several changes: the increase in cortical 5-HT outflow caused by systemic injection of paroxetine was 4- to 6-fold higher in freely moving R-NK1KO mice than in wild-type controls. The constitutive lack of NK1 receptors is associated with a functional desensitization of somatodendritic 5-HT1A autoreceptors, resembling that induced by chronic treatment with SSRI antidepressants. These results highlight the link between a neurotransmitter (serotonin) and a neuropeptide (substance P). This genetic strategy allowed us to point out that multiple targets participate to the effects of classical antidepressant drugs within the brain. We hope that, soon, some mice lines (constitutive or tissue specific, conditional rescue mice having alterations of sleep/wakefulness and/or food intake, altered central serotonin and/or noradrenaline neurotransmission, deficit in neurotrophic factors, but increases in intrasynaptic concentrations of substance P) could be a relevant model of the physiopathology of depressive disorders, and could help us understand the appearance of some symptoms. These recent findings suggest that instead of being rejected, the monoaminergic hypothesis of depression should be improved, corrected and completed by studying the role of other neurotransmitter, neuromodulatory compounds (substance P, BDNF [brain-derived neurotrophic factor]). By doing so, it thus could be possible to improve antidepressant drug treatment, i.e. shorten their long delay of action and/or to decrease treatment resistance or improve its tolerance.

Genetically Selected High Capacity Runners Have Altered Serotonergic and Dopaminergic Receptor mRNA in the Brain

Although adaptations in peripheral systems such as liver function and skeletal muscle composition can contribute to ones capacity to sustain aerobic activity, central neural circuits regulating fatigue may also play a critical role. We evaluated possible differences in central fatigue pathways, including the serotonergic (5-HT) and dopaminergic (DA) systems, between two contrasting rat-lines selectively bred for endurance treadmill running capacity. The lines were designated as Low Capacity Runners (LCR) and High Capacity Runners (HCR). Using in situ hybridization, we examined baseline differences in central serotonergic (5-HT transporter, 5-HT1A, 5-HT1B) and dopaminergic (DR-D1, DR-D2) receptor mRNA in LCR versus HCR. Rats were tested on a treadmill 3 weeks prior to sacrifice to verify trait differences. HCR ran 2 times faster,10 times farther, and 5 times longer than the LCR. Interestingly, HCR had higher levels of 5-HT1B mRNA in the dorsal and median raphe nuclei relative to LCR, but similar levels of 5-HT transporter and 5-HT1A mRNA in these areas. Furthermore, HCR expressed higher levels of DR-D2 mRNA than LCR in only the rostral-mid nucleus accumbens, but not other levels of the nucleus accumbens or caudate putamen. There was no difference in DR-D1 mRNA levels in the nucleus accumbens or caudate putamen between HCR and LCR. These data suggest that constraint of the central serotonergic and dopaminergic responses, via 5-HT1B and DR-D2 inhibitory autoreceptors, during exercise may be involved in the mechanisms by which HCR have delayed onset of fatigue.

P.1.17 Separate and combined effects of fluoxetine and a 5-HT 1A receptor agonist or antagonist on 5-HT 1A and 5-HT 1B autoreceptor activity in the rat brain

The effect of repeated treatment with the selective serotonin reuptake inhibitor fluoxetine on synthesis and turnover of 5-hydroxytryptamine (5-HT) was studied in the mouse brain in vivo. The concentration of 5-hydroxytryptophan (5-HTP), 5-hydroxyindoleacetic acid (5-HIAA) and 5-HT was measured in hypothalamus, hippocampus and frontal cortex after inhibition of the aromatic amino acid decarboxylase activity with m-hydroxybenzylhydrazine (NSD 1015). Fluoxetine 6.9 mg/kg s.c. was injected once daily for three weeks. Three days after the final daily injection of fluoxetine 5-HT synthesis (5-HTP accumulation) and turnover (5-HIAA/5-HT ratio) were significantly enhanced compared with saline-treated mice. The 5-HIAA/5-HT ratio was already significantly elevated after 3 days of fluoxetine treatment and continued to increase during treatment for 2–3 weeks. The increase in 5-HIAA/5-HT ratio was considerably larger (150–200% of controls) than the increase in 5-HTP accumulation (110–120%), which reached significance only after 3 weeks of treatment. The increase in 5-HT synthesis may be secondary to that of the turnover. The 5-HIAA/5-HT ratio returned to control values after a 14 days washout period. Simultaneous treatment with the long-acting 5-HT1B-receptor antagonist, SB 224289 for 14 days counteracted the fluoxetine-induced increase in 5-HIAA/5-HT ratio that indicates involvement of 5-HT1B autoreceptors in the development of this increase. It is proposed that the fluoxetine-induced enhancement of 5-HT turnover was evoked by the long-lasting stimulation of 5-HT1B autoreceptors that resulted in an intraneuronal compensatory adaptation of the basal 5-HT release.

Serotonin-induced increases in adult cell proliferation and neurogenesis are mediated through different and common 5-HT receptor subtypes in the dentate gyrus and the subventricular zone.

Increase in serotonin (5-HT) transmission has profound antidepressant effects and has been associated with an increase in adult neurogenesis. The present study was aimed at screening the 5-HT receptor subtypes involved in the regulation of cell proliferation in the subgranular layer (SGL) of the dentate gyrus (DG) and the subventricular zone (SVZ) and to determine the long-term changes in adult neurogenesis. The 5-HT1A, 5-HT1B, and 5-HT2 receptor subtypes were chosen for their implication in depression and their location in/or next to these regions. Using systemic administration of various agonists and antagonists, we show that the activation of 5-HT1A heteroreceptors produces similar increases in the number of bromodeoxyuridine-labeled cells in the SGL and the SVZ (about 50% over control), whereas 5-HT2A and 5-HT2C receptor subtypes are selectively involved in the regulation of cell proliferation in each of these regions. The activation of 5-HT2C receptors, largely expressed by the choroid plexus, produces a 56% increase in the SVZ, while blockade of 5-HT2A receptors produces a 63% decrease in the number of proliferating cells in the SGL. In addition to the influence of 5-HT1B autoreceptors on 5-HT terminals in the hippocampus and ventricles, 5-HT1B heteroreceptors also regulate cell proliferation in the SGL. These data indicate that multiple receptor subtypes mediate the potent, partly selective of each neurogenic zone, stimulatory action of 5-HT on adult brain cell proliferation. Furthermore, both acute and chronic administration of selective 5-HT1A and 5-HT2C receptor agonists produce consistent increases in the number of newly formed neurons in the DG and/or olfactory bulb, underscoring the beneficial effects of 5-HT on adult neurogenesis.

Effects of chronic paroxetine treatment on dialysate serotonin in 5-HT1B receptor knockout mice.

The role of serotonin (5-HT)1B receptors in the mechanism of action of selective serotonin re-uptake inhibitors (SSRI) was studied by using intracerebral in vivo microdialysis in conscious, freely moving wild-type and 5-HT1B receptor knockout (KO 5-HT1B) mice in order to compare the effects of chronic administration of paroxetine via osmotic minipumps (1 mg per kg per day for 14 days) on extracellular 5-HT levels ([5-HT]ext) in the medial prefrontal cortex and ventral hippocampus. Basal [5-HT]ext values in the medial prefrontal cortex and ventral hippocampus, approximately 20 h after removing the minipump, were not altered by chronic paroxetine treatment in both genotypes. On day 15, in the ventral hippocampus, an acute paroxetine challenge (1 mg/kg i.p.) induced a larger increase in [5-HT]ext in saline-pretreated mutant than in wild-type mice. This difference between the two genotypes in the effect of the paroxetine challenge persisted following chronic paroxetine treatment. Conversely, in the medial prefrontal cortex, the paroxetine challenge increased [5-HT]ext similarly in saline-pretreated mice of both genotypes. Such a challenge produced a further increase in cortical [5-HT]ext compared with that in saline-pretreated groups of both genotypes, but no differences were found between genotypes following chronic treatment. To avoid the interaction with raphe 5-HT1A autoreceptors, 1 micro m paroxetine was perfused locally through the dialysis probe implanted in the ventral hippocampus; similar increases in hippocampal [5-HT]ext were found in acutely or chronically treated wild-type mice. Systemic administration of the mixed 5-HT1B/1D receptor antagonist GR 127935 (4 mg/kg) in chronically treated wild-type mice potentiated the effect of a paroxetine challenge dose on [5-HT]ext in the ventral hippocampus, whereas systemic administration of the selective 5-HT1A receptor antagonist WAY 100635 did not. By using the zero net flux method of quantitative microdialysis in the medial prefrontal cortex and ventral hippocampus of wild-type and KO 5-HT1B mice, we found that basal [5-HT]ext and the extraction fraction of 5-HT were similar in the medial prefrontal cortex and ventral hippocampus of both genotypes, suggesting that no compensatory response to the constitutive deletion of the 5-HT1B receptor involving changes in 5-HT uptake capacity occurred in vivo. As steady-state brain concentrations of paroxetine at day 14 were similar in both genotypes, it is unlikely that differences in the effects of a paroxetine challenge on hippocampal [5-HT]ext are due to alterations of the drug's pharmacokinetic properties in mutants. These data suggest that there are differences between the ventral hippocampus and medial prefrontal cortex in activation of terminal 5-HT1B autoreceptors and their role in regulating dialysate 5-HT levels. These presynaptic receptors retain their capacity to limit 5-HT release mainly in the ventral hippocampus following chronic paroxetine treatment in mice.

SB-236057-A: a selective 5-HT1B receptor inverse agonist.

5-HT1B autoreceptors are involved in the control of extracellular 5-HT levels from both the terminal and cell body regions of serotonergic neurons. In this manuscript we review the pharmacological and pharmacokinetic data available for the selective and potent 5-HT1B receptor inverse agonist, SB-236057-A (1'-ethyl-5-(2'-methyl-4'-(5-methyl-1,3,4-oxadiazolyl-2-yl)biphenyl-4-carbonyl)-2,3,6,7-tetrahydrospiro (furo[2,3-f]indole-3,4'-piperidine) hydrochloride). SB 236057-A has been shown to have high affinity for human 5-HT1B receptors (pK(i) = 8.2) and displays 80 or more fold selectivity for the human 5-HT1B receptor over other 5-HT receptors and a range of additional receptors, ion channels and enzymes. In functional studies at human 5-HT1B receptors SB-236057-A displayed inverse agonism (pA(2) = 8.9) using [(35)S]GTPgammaS binding, and silent antagonism (pA(2) = 9.2) using cAMP accumulation. SB-236057-A also acted as an antagonist at the 5-HT terminal autoreceptor as measured by [3H]5-HT release from electrically stimulated guinea pig and human cortical slices. In the guinea pig, pharmacokinetic analysis demonstrated that SB-236057-A was bioavailable and according to in vivo pharmacodynamic assays it enters brain and has a long duration of action. Importantly no side effect liability was evident at relevant doses from anxiogenic, cardiovascular, sedative or migraine viewpoints. In vivo microdialysis studies demonstrated that SB-236057-A is an antagonist in the guinea pig cortex but has no effect on extracellular 5-HT levels per se. In contrast, SB-236057-A increased extracellular 5-HT levels in the guinea pig dentate gyrus. This increase in 5-HT release was comparable to that observed after 14 days of paroxetine administration. SB-236057-A has been a useful tool in confirming that, in either guinea pigs or humans, the terminal 5-HT autoreceptor is of the 5-HT1B subtype. It appears that acute 5-HT1B receptor blockade, by virtue of increased 5-HT release in the dentate gyrus, may provide a rapidly acting antidepressant.

Effects of the Selective Norepinephrine Reuptake Inhibitor Reboxetine on Norepinephrine and Serotonin Transmission in the Rat Hippocampus

Given that norepinephrine (NE) and serotonin (5-HT) neurons are implicated in the mechanisms of action of antidepressant drugs and both project to the hippocampus, the impact of acute and long-term administration of the selective NE inhibitor reboxetine was assessed on CA3 pyramidal neuron firing in this postsynaptic structure. Cumulative injections of reboxetine (1–4 mg/kg, i.v.) dose-dependently increased the recovery time of the firing of these neurons following iontophoretic applications of NE, but not 5-HT. In rats treated with reboxetine for 2.5 mg/kg/day for 21 days, a robust increase in the recovery time following NE applications was observed, and a small but significant prolongation occurred following 5-HT applications. In controls and reboxetine-treated rats, 1 and 5 Hz stimulations of the afferent 5-HT bundle to the hippocampus, which allows determination of terminal 5-HT1B autoreceptor sensitivity, produced similar frequency-dependent decreases in pyramidal neuron firing in both groups. However, after low and high doses of clonidine (10 and 400 μg/kg, i.v.), which assesses α2-adrenergic auto- and heteroreceptor sensitivity, respectively, only the effect of the high dose of clonidine was attenuated. Interestingly, administration of the selective 5-HT1A receptor antagonist WAY 100,635 induced a 140% increase in basal pyramidal neuron firing in reboxetine as compared to saline-treated rats. This increase in tonic activation of postsynaptic 5-HT1A receptors might be attributable in part to a desensitization of α2-adrenergic heteroreceptors, presumably resulting from sustained NE reuptake inhibition. These results indicate that even a selective NE reuptake inhibitor can modulate 5-HT transmission.

The role of 5-HT1B receptors in the regulation of serotonin cell firing and release in the rat brain.

The release of 5-HT in terminal areas of the rodent brain is regulated by 5-HT1B receptors. Here we examined the role of 5-HT1B receptors in the control of 5-HT output and firing in the dorsal raphe nucleus (DR), median raphe nucleus (MnR) and forebrain of the rat in vivo. The local perfusion (30-300 microM) of the selective 5-HT1B receptor agonist CP-93,129 to freely moving rats decreased 5-HT release in the DR and more markedly in the MnR. Likewise, 300 microM CP-93,129 reduced 5-HT output in substantia nigra pars reticulata, ventral pallidum, lateral habenula and the suprachiasmatic nucleus. The effect of CP-93,129 was prevented by SB-224289, but not by WAY-100635, selective 5-HT1B and 5-HT1A receptor antagonists, respectively. SB-224289 did not alter dialysate 5-HT in any raphe nuclei. The intravenous administration of the brain-penetrant selective 5-HT1B receptor agonist CP-94,253 (0.5-2.0 mg/kg) to anesthetized rats decreased dialysate 5-HT in dorsal hippocampus and globus pallidus, increased it in MnR and left it unaltered in the DR and medial prefrontal cortex. SB-224289, at a dose known to block 5-HT1B autoreceptor-mediated effects (5 mg/kg), did not prevent the effect of CP-94,253 on MnR 5-HT. The intravenous administration of CP-94,253 (0.05-1.6 mg/kg) to anesthetized rats increased the firing rate of MnR, but not DR-5-HT neurons. The local perfusion of CP-94,253 in the MnR showed a biphasic effect, with 5-HT reductions at 0.3-3 microM and increase at 300 microM. These results suggest that 5-HT cell firing and release in midbrain raphe nuclei (particularly in the MnR) are under control of 5-HT1B receptors. The activation of 5-HT1B autoreceptors (possibly located on 5-HT nerve endings and/or varicosities within DR and MnR) reduces 5-HT release. The effects of higher concentrations of 5-HT1B receptor agonists seem more compatible with the activation of 5-HT1B heteroreceptors on inhibitory neurons.

5-HT1B Autoreceptors limit the effects of selective serotonin re-uptake inhibitors in mouse hippocampus and frontal cortex.

We used knockout mice and receptor antagonist strategies to investigate the contribution of the serotonin (5-hydroxytryptamine, 5-HT) 1B receptor subtype in mediating the effects of selective serotonin re-uptake inhibitors (SSRIs). Using in vivo intracerebral microdialysis in awake mice, we show that a single systemic administration of paroxetine (1 or 5 mg/kg, i.p.) increased extracellular serotonin levels [5-HT]ext in the ventral hippocampus and frontal cortex of wild-type and mutant mice. However, in the ventral hippocampus, paroxetine at the two doses studied induced a larger increase in [5-HT]ext in knockout than in wild-type mice. In the frontal cortex, the effect of paroxetine was larger in mutants than in wild-type mice at the 1 mg/kg, but not at 5 mg/kg. In addition, either the absence of the 5-HT1B receptor or its blockade with the mixed 5-HT1B/1D receptor antagonist, GR 127935, potentiated the effect of a single administration of paroxetine on extracellular 5-HT levels more in the ventral hippocampus than in the frontal cortex. These data suggest that 5-HT1B autoreceptors limit the effects of SSRIs on dialysate 5-HT levels at serotonergic nerve terminals.

Chronic repetitive transcranial magnetic stimulation induces subsensitivity of presynaptic serotonergic autoreceptor activity in rat brain

Repetitive transcranial magnetic stimulation (rTMS) is a novel procedure which has proven effective in the treatment of major depression. We administered rTMS chronically to rats in order to determine whether this procedure affected serotonergic neurotransmission in the prefrontal cortex. Basal 5-HT levels, and the effects of challenges with the 5-HT1A receptor agonist 8-OH-DPAT and the 5-HT1B antagonist GR 127935 on 5-HT levels were determined using in vivo microdialysis. Rats which had undergone chronic rTMS showed reduced responses to both challenges, indicating subsensitivity of both the presynaptic 5-HT1A autoreceptors situated somatodendritically in the raphe nuclei and the 5-HT1B autoreceptors situated on nerve terminals. Since such subsensitivity has been demonstrated after other antidepressant treatments, our results indicate that these treatments and rTMS may have a common mechanism of action.

Identification and role of serotonin 5-HT1A and 5-HT1B receptors in primary cultures of rat embryonic rostral raphe nucleus neurons.

Autoregulatory mechanisms affecting serotonin [5-hydroxytryptamine (5-HT)] release and synthesis during the early period of development were investigated in dissociated cell cultures raised from embryonic rostral rat rhombencephalon. The presence of 5-HT1A and 5-HT1B receptors in serotoninergic neurons was assessed using binding assays. The involvement of 5-HT1A and 5-HT1B receptors in the control of the synthesis and release of [3H]5-HT was studied using biochemical approaches with several serotoninergic receptor ligands. A mean decrease of 30% in [3H]5-HT synthesis and release was observed in the presence of 5-HT (10(-8) M), the 5-HT1A agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), the 5HT1B/1A agonist 5-methoxy-3-(1,2,5,6-tetrahydro-4-pyridinyl)-1H-indole (RU 24969), the 5-HT1B agonist 3-(1,2,5,6-tetrahydropyrid-4-yl)pyrrolo[3,2-b]pyrid-5-one (CP-93,129), and the 5-HT(1D/1B) agonist sumatriptan. Inhibition of 5-HT synthesis and release induced by 8-OH-DPAT was blocked by chiral N-tert-butyl-3-[1-[1-(2-methoxy)phenyl]piperazinyl]-1-phenylpropionam ide dihydrochloride quaternary-hydrate (WAY 100135) (10(7) M) or methyl 4-[4-[4-(1,1,3-trioxo-2H-1,2-benzoisothiazol-2-yl)butyl]-1-p iperazinyl]-1Hindole-2-carboxylate (SDZ 216-525) (10(-7)M), and that of CP-93,129 was blocked by methiothepin (10(-7) M). Paradoxically, extracellular levels of [3H]5-HT increased in the presence of 8-OH-DPAT and RU 24969 at 10(-6) M. 5-HT uptake experiments showed that these two agonists interacted with the 5-HT transporter. 5-HT1 binding sites (620 fmol/mg of protein) and 5-HT1A (482 fmol/mg of protein) and 5-HT1B (127 fmol/mg of protein) receptors were detected in 12-day in vitro cell cultures. Experiments carried out with tetrodotoxin suggested that 5-HT1A receptors are located on nerve cell bodies, whereas 5-HT1B receptors are located on the nerve terminals. We concluded that autoregulatory mechanisms involving 5-HT1A and 5-HT1B autoreceptors are functionally mature in cells from rostral raphe nuclei during the early period of development.

The effect of SB-236057-A, a selective 5-HT1B receptor inverse agonist, on in vivo extracellular 5-HT levels in the freely-moving guinea-pig.

5-HT1B autoreceptors are involved in the control of extracellular 5-HT levels from both the terminal and cell body regions of serotonergic neurones. In this study we report on the effect of a selective and potent 5-HT1B receptor inverse agonist, SB-236057-A (1'-ethyl-5-(2'-methyl-4'-(5-methyl- 1,3,4-oxadiazolyl-2-yl)biphenyl-4-carbonyl)-2,3,6,7-tetrahydros piro [furo[2,3-f]indole-3,4' -piperidine] hydrochloride), on extracellular 5-HT levels in the cortex and dentate gyrus of the freely-moving guinea-pig, using the technique of in vivo microdialysis. SB-236057-A had ca. 23% bioavailability following oral drug administration. In vivo hypothermia pharmacodynamic assays demonstrated it was brain penetrant with a duration of action in excess of 18 h. SB-236057-A (0.75 mg/kg p.o.) increased extracellular 5-HT levels in the dentate gyrus to a maximum of 167+/-7% of basal but had no effect in the frontal cortex. However, a small increase in cortical 5-HT levels (117+11% of basal) was evident at 2.5 mg/kg p.o. In addition, SB-236057-A (0.75 mg/kg and 2.5 mg/kg p.o.) antagonised the sumatriptan-induced inhibition of extracellular 5-HT levels in the guinea-pig frontal cortex. These differences were attributed to MRN-innervated regions (e.g. dentate gyrus) being more responsive to 5-HT1B receptor-mediated negative feedback than DRN-innervated regions (e.g. frontal cortex). In the dentate gyrus, the increase in 5-HT release induced by SB-236057-A (0.75 mg/kg p.o.) was comparable to that after 14 days of paroxetine (10 mg/kg p.o.) administration, reaching a maximum of 183+/-13% of basal. These data suggest that acute 5-HT1B receptor blockade, by virtue of increased 5-HT release in the dentate gyrus, may provide a rapidly acting antidepressant.

Altered expression and functions of serotonin 5-HT1A and 5-HT1B receptors in knock-out mice lacking the 5-HT transporter.

By taking up serotonin (5-hydroxytryptamine, 5-HT) released in the extracellular space, the 5-HT transporter (5-HTT) regulates central 5-HT neurotransmission. Possible adaptive changes in 5-HT neurotransmission in knock-out mice that do not express the 5-HT transporter were investigated with special focus on 5-HT1A and 5-HT1B receptors. Specific labelling with radioligands and antibodies, and competitive RT-PCR, showed that 5-HT1A receptor protein and mRNA levels were significantly decreased in the dorsal raphe nucleus (DRN), increased in the hippocampus and unchanged in other forebrain areas of 5-HTT-/- vs. 5-HTT+/+ mice. Such regional differences also concerned 5-HT1B receptors because a decrease in their density was found in the substantia nigra (-30%) but not the globus pallidus of mutant mice. Intermediate changes were noted in 5-HTT+/- mice compared with 5-HTT+/+ and 5-HTT-/- animals. Quantification of [35S]GTP-gamma-S binding evoked by potent 5-HT1 receptor agonists confirmed such changes as a decrease in this parameter was noted in the DRN (-66%) and the substantia nigra (-30%) but not other brain areas in 5-HTT-/- vs. 5-HTT+/+ mice. As expected from actions mediated by functional 5-HT1A and 5-HT1B autoreceptors, a decrease in brain 5-HT turnover rate after i.p. administration of ipsapirone (a 5-HT1A agonist), and an increased 5-HT outflow in the substantia nigra upon local application of GR 127935 (a 5-HT1B/1D antagonist) were observed in 5-HTT+/+ mice. Such effects were not detected in 5-HTT-/- mice, further confirming the occurrence of marked alterations of 5-HT1A and 5-HT1B autoreceptors in these animals.

Serotonin autoreceptor function and antidepressant drug action.

This article briefly summarizes, within the context of a brief review of the relevant literature, the outcome of our recent rat microdialysis studies on (1) the relative importance of serotonin (5-HT)1A versus 5-HT1B autoreceptors in the mechanism of action of 5-HT reuptake blocking agents, including putative regional differences in this regard, and (2) autoreceptor responsiveness following chronic SSRI administration. First, our data are consistent with the primacy of 5-HT1A autoreceptors in restraining the elevation of 5-HT levels induced by SSRIs, whereas nerve terminal 5-HT1B autoreceptors appear to have an accessory role in this regard. Second, there is an important interplay between cell body and nerve terminal 5-HT autoreceptors, and recent findings suggest that this interplay may potentially be exploited to obtain regionally preferential effects on 5-HT neurotransmission in the central nervous system, even upon systemic drug administration. In particular, emerging data suggest that somatodendritic 5-HT1A autoreceptor- and nerve terminal 5-HT1B autoreceptor-mediated feedback may be relatively more important in the control of 5-HT output in dorsal raphe-frontal cortex and median raphe-dorsal hippocampus systems, respectively. Third, 5-HT autoreceptors evidently retain the capability to limit the 5-HT transmission-promoting effect of SSRIs after chronic treatment. Thus, although the responsiveness of these sites is probably somewhat reduced, residual autoreceptor capacity still remains an effective restraint on large increases in extracellular 5-HT, even after prolonged treatment. If a further increase in extracellular 5-HT is crucial to the remission of depression in patients responding only partially to prolonged administration of antidepressants, then sustained adjunctive treatment with autoreceptor-blocking drugs may consequently prove useful in the long term.

Differential sensitivity of 5-HT1B auto and heteroreceptors.

The effect of the native and rodent-selective 5-HT1B receptor agonists (5-hydroxytryptamine (5-HT) and CP93,129) on the K+-evoked overflows of [3H]5-HT, [3H]dopamine (DA) and [3H]acetylcholine (ACh) was studied in synaptosome preparations obtained from rat brain striatum or hippocampus loaded with radiolabeled neurotransmitter. The aim of the study was to compare the different potencies of the specific 5-HT1B receptor agonists to stimulate the auto and heteroreceptors and to modulate the different neurotransmitter release. Results show that under the same experimental conditions, 5-HT and CP93,129 exhibited significantly higher potencies in inhibiting the K+-evoked overflow of [3H]5-HT from synaptosomes of rat striatum (IC50=2.0+/-1.8 nM and 20.5+/-3.1 nM, respectively) than in inhibiting the K+-evoked overflow of [3H]DA from synaptosomes of the same cerebral region (IC50= 0.8+/-0.2 microM and 1.8+/-0.4 microM, respectively), or [3H]ACh from synaptosomes of hippocampus (IC50=1.7+/-0.8 microM for CP93,129). The inhibitory effects of the 5-HT1B receptor agonists on [3H] K+-overflows were antagonized by the selective 5-HT1B receptor antagonist (SB224289), further indicating that the observed effects were 5-HT1B receptor specific. Sumatriptan, a selective r5-HT1D receptor agonist, did not show any significant effect on the K+-overflow of [3H]5-HT in the range of concentrations (10(-10) to 10(-6) M), and did not affect the K+ overflow of [3H]DA or [3H]ACh at concentrations (10(-9) to 10(-4) M), which exclude the involvement of 5-HT1D receptors. These inhibitory effects of the 5-HT1B receptor agonists were highly attenuated by pertussis toxin in the three systems studied, suggesting the involvement of Gi/Go-proteins in the transduction mechanism pathway of the receptor generated signal. In conclusion, these results suggest that 5-HT1B heteroreceptors located on dopaminergic and cholinergic terminals exhibit a lower sensitivity to 5-HT1B receptor agonist and antagonist than do 5-HT1B autoreceptors. The observed difference in functional sensitivities of 5-HT1B auto- and heteroreceptors may represent important consequences in the physiological control of the release of serotonin versus that of other neurotransmitters.