5-HT1B Receptor Agonist Research Articles

5HT1BReceptor Agonists Inhibit Light-Induced Phase Shifts of Behavioral Circadian Rhythms and Expression of the Immediate–Early Genec-fosin the Suprachiasmatic Nucleus

The suprachiasmatic nucleus (SCN) is a circadian oscillator and a critical component of the mammalian circadian system. It receives afferents from the retina and the mesencephalic raphe. Retinal afferents mediate photic entrainment of the SCN, whereas the serotonergic afferents originating from the midbrain modulate photic responses in the SCN; however, the serotonin (5HT) receptor subtypes in the SCN responsible for these modulatory effects are not well characterized. In this study, we tested the hypothesis that 5HT1B receptors are located presynaptically on retinal axon terminals in the SCN and that activation of these receptors inhibits retinal input. The 5HT1B receptor agonists TFMPP and CGS 12066A, administered systemically, inhibited light-induced phase shifts of the circadian activity rhythm in a dose-dependent manner at phase delay and phase advance time points. This inhibition was not affected by previous systemic application of either the selective 5HT1A receptor antagonist (+)WAY 100135 or by the 5HT2 receptor antagonist mesulergine, whereas pretreatment with the nonselective 5HT1 antagonist methiothepin significantly attenuated the effect of TFMPP. TFMPP also produced a dose-dependent reduction in light-stimulated Fos expression in the SCN, although a small subset of cells in the dorsolateral aspect of the caudal SCN were TFMPP-insensitive. TFMPP (1 mM) infused into the SCN produced complete inhibition of light-induced phase advances. Finally, bilateral orbital enucleation reduced the density of SCN 5HT1B receptors as determined using [125I]-iodocyanopindolol to define 5HT1B binding sites. These results are consistent with the interpretation that 5HT1B receptors are localized presynaptically on retinal terminals in the SCN and that activation of these receptors by 5HT1B agonists inhibits retinohypothalamic input.

Locally formed 5-hydroxytryptamine stimulates phosphate transport in cultured opossum kidney cells and in rat kidney.

Renal proximal tubular cells have been shown to express aromatic L-amino acid decarboxylase (L-AAAD), which converts L-dopa into dopamine and 5-hydroxytryptophan [(OH)Trp] into 5-hydroxytryptamine (5-HT; serotonin). Because 5-HT receptors have been demonstrated in proximal cells, we hypothesized that 5-HT may act as an autocrine/paracrine modulator of proximal transport. We evaluated this possibility in opossum kidney (OK) cells, a renal epithelial cell line with a proximal phenotype expressing 5-HT1B receptors, and in intact anaesthetized rats. 5-HT synthesis by OK cells increased with incubation time and (OH)Trp concentration, and was abolished by benserazide, an L-AAAD inhibitor. 5-HT reversed parathyroid hormone (PTH)-induced cAMP accumulation in a pertussis toxin-sensitive manner and reduced the PTH inhibition of P(i) uptake without affecting the NaP(i)-4 mRNA level. The effects of 5-HT on cAMP generation and Na-P(i) co-transport were reproduced by (OH)Trp, except in the presence of benserazide, and by L-propranolol and dihydroergotamine, two 5-HT1B receptor agonists. In rats, (OH)Trp and dihydroergotamine decreased fractional P(i) excretion. Benserazide abolished the effect of (OH)Trp but not that of dihydroergotamine. (i) locally generated 5-HT blunts the inhibitory effect of PTH on Na-P(i) co-transport in OK cells; (ii) endogenous 5-HT decreases P(i) excretion in rats; and (iii) 5-HT is a paracrine modulator involved in the physiological regulation of renal P(i) transport.

Role of 5HT1 receptors in the control of hypoglossal motoneurons in vivo.

Recordings from serotonin (5HT)-containing neurons of the brainstem suggest that their effects on upper airway, including hypoglossal (XII), motoneurons are maximal during wakefulness and minimal during rapid eye movement (REM) sleep. Several distinct 5HT receptors are present in the XII nucleus. In this study, we assessed the effects mediated by type 1 receptors. We microinjected 5HT1 receptor agonists or antagonists into the XII nucleus of anesthetized, paralyzed, vagotomized, and artificially ventilated rats and observed the resulting changes in XII nerve activity. Both CGS-12066 and 8-OH-DPAT (5HT1B and 5HT1A receptor agonists, respectively) suppressed XII nerve activity in a dose-dependent manner by about 20%. MAN-190, a putative 5HT1A receptor antagonist, had no effect, Based on the relative potencies of the two agonists, we conclude that they may both act through 5HT1B receptors and that 5HT1A receptors are absent from the XII nucleus of adult rats. If the effects mediated by 5HT1B receptors are endogenously active, their withdrawal during REM sleep may to some degree counterbalance the disfacilitation of XII motoneurons resulting from the withdrawal of their 5HT-mediated postsynaptic excitation.

The 5-HT1B Receptor Agonist CP-94,253 Reduces Food Intake and Preserves the Behavioural Satiety Sequence

The 5-HT1B Receptor Agonist CP-94,253 Reduces Food Intake and Preserves the Behavioural Satiety Sequence

Presynaptic inhibition of glutamatergic synaptic transmission to rat motoneurons by serotonin.

1. In a brain stem slice preparation, we recorded glutamatergic excitatory postsynaptic currents (EPSCs) in hypoglossal motoneurons (HMs) evoked by extracellular stimulation in the reticular formation just ipsilateral to the hypoglossal motor nucleus (n. XII). Serotonin (5-HT) inhibited glutamatergic synaptic transmission in a dose-dependent fashion as indicated by a reduction in the evoked EPSC (eEPSC) peak amplitude to 46 +/- 2% (mean +/- SE, n = 26) of control (5-HT 10 microM). This effect was not voltage dependent, as the eEPSC reversal potential was not altered (n = 5). Additionally, 5-HT decreased the rate of rise of the eEPSC to 41 +/- 2% of control (n = 14). Blockade of N-methyl-D-aspartate-receptor-channels by D(-)-2-amino-5-phosphonopentanoic acid (50 microM) or of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid/kainate receptor-channels by 6,7-dinitro-quinoxaline (20 microM) did not alter the relative reduction of the eEPSC amplitude by 5-HT (n = 7 and 3, respectively). 2. In the presence of tetrodotoxin (1 microM), bath application of 5-HT did not reduce postsynaptic glutamate currents elicited by pressure ejection of L-glutamate (1 mM) onto HMs (n = 5), and it increased the median interevent interval of spontaneous miniature EPSCs (mEPSCs) to 178 +/- 12% of control (n = 4), suggesting that 5-HT acts presynaptically to reduce the probability of vesicle release. mEPSC amplitude was decreased slightly in three of four cells (median amplitude = 92 +/- 3% of control). 3. The specific 5-HT1B receptor agonist [3-(1,2,5,6-tetrahydropyrid-4-yl)pyrrolo[3,2-b]pyrid-5-one] (1 microM) mimicked 5-HT in its effect on eEPSCs (eEPSC amplitude reduced to 31 +/- 5% of control; rate of rise reduced to 40 +/- 4% of control, n = 10 and 5, respectively) and mEPSCs (median interevent interval increased to 231 +/- 36% of control; median mEPSC amplitude = 102 +/- 3% of control, n = 5). Additionally, 5-HT-mediated inhibition was not blocked by coapplication of 1-(2-methoxyphenyl)-4-[4-(2-phthalimido) butyl] piperazine hydrobromide (1 microM), a 5-HT1A antagonist, and 3-[2-[4-(4-flurobenzoyl)-1-piperdinyl]ethyl]-2,4(1H,3H)-quin azolinedione tartrate (1 microM), a 5-HT2A/2C antagonist (n = 4). These data indicate that the 5-HT effect is primarily 5-HT1B receptor mediated. 4. We conclude that 5-HT, acting through presynaptic 5-HT1B receptors, inhibits glutamatergic synaptic transmission by reducing the probability of vesicle release.

Promotion of cell growth by stimulation of cloned human 5-HT1D receptor sites in transfected C6-glial cells is highly sensitive to intrinsic activity at 5-HT1D receptors.

5-Hydroxytryptamine (serotonin, 5-HT), essentially known as a neurotransmitter and vasoactive agent, also functions as a mitogen in various cell types through several different second messenger systems. Stimulation of cloned human 5-HT1D receptor sites by sumatriptan in stably transfected rat C6-glial/5-HT1D cells promotes cell growth (Pauwels et al. (1996) Naunyn-Schmiedeberg's Arch Pharmacol 353:144-156). In the present study, the pharmacology of this growth response was investigated using a broad series of 5-HT receptor ligands. The data were compared with the responses obtained by measuring inhibition of forskolin-stimulated cAMP formation. 5-HT (EC50: 25 nM) promoted cell growth of C6-glial/5HT1D cells, and this in contrast to the absence of any measurable effect in pcDNA3-plasmid transfected and non-transfected C6-glial cells. The 5-HT effect could be mimicked by the following compounds (EC50 in nM): zolmitriptan (0.41), 2'-methyl-4'-(-methyl[1,2,4] oxadiazol-3-yl)biphenyl-4-carboxylic acid [4-methoxy-3-(4-methylpiperazin-1-yl)phenyl]amid (GR 127,935;0.86), naratriptan (0.92), metergoline (1.9), sumatriptan (2.9), (N,N-dimethyl-2-[5-(1,2,4-triazol-1-ylmethyl)-1H-indol-3-yl] ethylamine (MK-462; 3.0), and R(+)-8-(hydroxy-2-(di-n-propylamino)tetralin (R(+)-8-OH-DPAT; 30.7). These EC50 -values correspond to the compounds binding affinities at the human 5-HT1D receptor site and, with the exception of GR 127,935 and metergoline also to the EC50-values found by measuring over 5 min inhibition of forskolin (100 microM)-stimulated cAMP formation. Prolonged exposure of GR 127,935(3 h) and metergoline (30 min) to cells yielded EC50 values in the cAMP assay more close to those measured in the mitogenic response. The growth response to sumatriptan, 5-HT, GR 127,935 and metergoline was blocked by the apparently silent antagonist methiothepin, ritanserin and ketanserin with potencies similar to blockade of inhibition of stimulated cAMP formation. The 8-OH-DPAT effect also is likely mediated by 5-HT1D receptors; stereoselectivity was found with its enantiomers at this receptor site and the effect was blocked by ketanserin (1 microM) but not by spiperone (1 microM). Micromolar concentrations of the 5-HT1B receptor agonist 3-(1,2,5,6-tetrahydro)-4-pyridil-5-pyrrolo[3,2-b]pyril-5-one (CP 93,129) and of the 5-HT2 receptor agonist 1-(2,5-dimethoxy-4-iodophenyl_-2-aminopropane (DOI) induced cell growth with a potency that accorded with the affinity of these compounds for the human 5-HT1D receptor site. These effects were sensitive to ketanserin (1 microM) antagonism, but not to blockade by beta-adrenergic blockers and the 5-HT2 receptor antagonist 2-anilino-N-[2-(3-chlorophenoxy)-propyl] acetamidine hydroiodide (BW 501-C-67). The findings suggest that 5-HT1A, 5-HT1B and 5-HT2 receptors are not implicated in 5-HT-stimulated C6-glial/5-HT1D cell growth. In conclusion, human 5-HT1D receptors are involved in the growth of C6-glial/5-HT1D cells. This cellular response is highly sensitive to the intrinsic activity of compounds at 5-HT1D receptors.

Functional characterization of 5-HT1D autoreceptors on the modulation of 5-HT release in guinea-pig mesencephalic raphe, hippocampus and frontal cortex.

1. The aims of the present study were (i) to characterize further the pharmacology of 5-HT1D autoreceptors modulating 5-HT release in guinea-pig mesencephalic raphe, hippocampus and frontal cortex; (ii) to determine whether 5-HT1D receptors in the mesencephalic raphe are located on 5-HT neurones; (iii) to determine whether 5-HT1D autoreceptors are coupled to G proteins; and (iv) to assess their sensitivity following long-term 5-HT reuptake blockade and inhibition of type-A monoamine oxidase. 2. In mesencephalic raphe, hippocampus and frontal cortex slices, the 5-HT1D/1B receptor agonist, sumatriptan and the 5-HT1 receptor agonist, 5-methoxytryptamine (5-MeOT) but not the 5-HT1B receptor agonist, CP93129, inhibited electrically the evoked release of [3H]-5-HT in a concentration-dependent manner. This effect was antagonized by the 5-HT1D/1B receptor antagonist GR127935 in the three structures, but not by the 5-HT1A receptor antagonist, (+)-WAY100635 in mesencephalic raphe slices. These results confirm the presence of functional 5-HT1D autoreceptors controlling 5-HT release within the mesencephalic raphe as well as in terminal regions. 3. The inhibitory effect of sumatriptan on K(+)-evoked release of [3H]-5-HT was not reduced by the addition of the Na+ channel blocker, tetrodotoxin to the superfusion medium, suggesting that these 5-HT1D receptors in the mesencephalic raphe are located on 5-HT neurones and may be considered autoreceptors. 4. The in vitro treatment with the alkylating agent N-ethylmaleimide (NEM) was used to determine whether these 5-HT1D autoreceptors are coupled to G proteins. The inhibitory effect of sumatriptan on electrically evoked release of [3H]-5-HT was attenuated in NEM-pretreated slices from mesencephalic raphe, hippocampus and frontal cortex, indicating that the 5-HT1D autoreceptors activated by sumatriptan are coupled to G proteins in these three structures. Taken together with our previous results, this suggests that, in addition to the 5-HT1D autoreceptor activated by sumatriptan, another subtype of 5-HT autoreceptor is activated by 5-MeOT in the hippocampus. 5. Following a 3-week treatment with the selective 5-HT reuptake inhibitor, paroxetine (10 mg kg-1 day-1) and a 48 h washout period, the electrically evoked release of [3H]-5-HT was enhanced in mesencephalic raphe, hippocampus and frontal cortex slices. There was an attenuation of the capacity of sumatriptan to inhibit the evoked release of [3H]-5-HT from mesencephalic raphe slices but not from frontal cortex and hippocampus slices. Only in the latter structure was the suppressant effect of 5-MeOT attenuated. After a 3-week treatment with the reversible type-A monoamine oxidase inhibitor, befloxatone (0.75 mg kg-1 day-1) and 48 h washout period, the effectiveness of sumatriptan and 5-MeOT on the evoked release of [3H]-5-HT was unaltered in the same brain structures. 6. The enhancement of [3H]-5-HT release by long-term paroxetine treatment is possibly due to a desensitization of 5-HT1D autoreceptors activated by sumatriptan in mesencephalic raphe and by terminal 5-HT autoreceptors activated by 5-MeOT in hippocampus. In the case of the frontal cortex, it appears that 5-MeOT and sumatriptan may act on the same 5-HT1D autoreceptor which is not desensitized either after paroxetine or befloxatone treatment, as previously reported.

Effects of the 5‐HT2B receptor agonist, BW 723C86, on three rat models of anxiety

1. BW 723C86 (3 and 10 mg kg-1, s.c. 30 min pretest), a 5-HT2B receptor agonist, increased total interaction, but not locomotion in a rat social interaction test, a profile consistent with anxiolysis. 2. The effect of BW 723C86 in the social interaction test is likely to be 5-HT2B receptor-mediated as it was prevented by pretreatment with the 5-HT2C/2B receptor antagonist, SB 200646A, (1 and 2 mg kg-1, p.o., 1 h pretest) which did not affect basal levels of social interaction at the doses used. 3. An anxiolytic-like action was also observed in the rat Geller-Seifter conflict test, where BW 723C86 (0.5-50 mg kg-1, s.c. 30 min pretest) modestly, but significantly increased punished, but not unpublished responding. 4. In a rat 5 min elevated x-maze test, BW 723C86 (1-10 mg kg-1, s.c.) had no significant effect. 5. The maximal anxiolytic-like effect of BW 723C86 approached that of the benzodiazepine anxiolytic, chloradiazepoxide (5 mg kg-1, s.c. 30 min pretest) in the social interaction test, but was markedly less in the Geller-Siefter test. The effect of BW 723C86 was also clearly less than chlordiazepoxide in the elevated x-maze procedure where it had no significant effect. 6. In conclusion, BW 723C86 exerted an appreciable anxiolytic-like profile in a rat social interaction test, but had a weaker effect in the Geller-Siefter and was ineffective in the elevated x-maze test used. These effects are likely to be 5-HT2B receptor-mediated.

Modulation of dopamine release in the nucleus accumbens by 5-HT1B agonists: involvement of the hippocampo-accumbens pathway

Changes in extracellular levels of dopamine (DA), DA metabolites DOPAC and HVA, and the serotonin metabolite 5-HIAA, were measured by microdialysis in the rat nucleus accumbens (n. acc) after treatments with serotonin (5-HT)1A (8-OH-DPAT) or 5-HT1B (RU 24 969 and S-CM-GTNH2) receptor agonists. Subcutaneous injections of RU 24 969 (0.02–2mg/kg) dose-dependently decreased 5-HIAA levels (0 to −38%), and also induced long-lasting increases in DA levels (0 to +37%) and DOPAC (+11% at the dose 0.5mg/kg) in the shell of the n. acc, whereas 8-OH-DPAT (0.25 and 0.5mg/kg) reduced 5-HIAA levels (−25%) and very slightly increased DOPAC at the lower dose (+4%), but had no effect on DA levels. Three weeks after interruption of the subicular efferent projections, the increase in DA levels previously observed after systemic injections of RU 24 969 was abolished. Microinjections of RU 24 969 (10μg/μl) or S-CM-GTNH2 (3μg/μl) into the ventral subicular area reproduced the effects of systemic injections of RU 24 969 on DA levels and increased DOPAC (+13%; +19%, respectively) and HVA levels (+23%; +24%), with no significant change in 5-HIAA.It is concluded that: (1) serotonin interacts with the mesolimbic dopaminergic system through 5-HT1B, but not 5-HT1A, receptors; and (2) serotonin interaction with the mesolimbic dopaminergic system involves postjunctional 5-HT1B heteroreceptors located in the ventral subicular area, which modulate the activity of glutamatergic hippocampo-accumbens pathways and only secondarily alter DA levels in the n. acc. The possible relevance of these results for schizophrenia is discussed.

Effects of 5-HT agonists, selective for different receptor subtypes, on oxytocin, CCK, gastrin and somatostatin plasma levels in the rat

Adult male Sprague–Dawley rats were administered the 5-HT subtype selective receptor agonists 8-OH-DPAT (0.5–2.0mg/kg) (5-HT1B), buspirone (2–8mg/kg) (5-HT1A), TFMPP (0.125–2.0mg/kg), DOI (0.125–2.0mg/kg) (5-HT2A) and m-CPBG (1.25–20.0mg/kg) (5-HT3), subcutaneously. Oxytocin, cholecystokinin (CCK), somatostatin and gastrin plasma levels were determined by standard RIA techniques 30 and 120min after injection of the respective 5-HT receptor agonist. It was found that the 5-HT1A and the 5-HT2A/C, but not the 5-HT2B or the 5-HT3 receptor agonists produced an increase in plasma oxytocin levels and these effects were, at least partially, antagonized by the corresponding subtype selective antagonists (−)pindolol (2mg/kg) and ritanserin (2mg/kg), respectively, administered 10min before 8-OH-DPAT (0.5mg/kg) or DOI (0.5mg/kg). The maximal response to the 5-HT1A receptor agonists (approx. 120nmol/l) was from 8 to 5 times the maximal response to the 5-HT2A/C receptor agonist. In addition, 8-OH-DPAT and DOI caused a decrease in plasma CCK levels, whereas the 5-HT1B receptor agonist TFMPP gave rise to an increase in plasma CCK levels. There were no statistically significant effects by any of the 5-HT receptor agonists on plasma somatostatin or gastrin levels under the present conditions. It is suggested that the clinical effects of new anxiolytic 5-HT1A receptor agonists, such as buspirone, to an extent may be mediated via an increased release of oxytocin.

The role of various calcium and potassium channels in the regulation of somatodendritic serotonin release.

We prepared slices from midbrain containing the raphe nuclei and from hippocampus of rats. The brain slices were loaded with [3H]serotonin and superfused in order to measure the release of radioactivity at rest and in response to electrical stimulation. No difference was observed in the resting and stimulated fractional release of tritium in the somatodendritic and axon terminal parts of serotonergic neurons. The selective 5-HT1A receptor agonist 8-OH-DPAT decreased the electrically induced tritium efflux from raphe nuclei slices preloaded with [3H]serotonin, and this inhibition was reversed by the 5-HT1A receptor antagonist (+)WAY-100135. The 5-HT1B receptor agonist CGS-12066B but not 8-OH-DPAT, inhibited the stimulation-evoked tritium efflux from hippocampal slices after labeling with [3H]serotonin. The electrical stimulation-evoked tritium efflux in raphe nuclei slices incubated with [3H]serotonin was completely external Ca(2+)-dependent, and omega-conotoxin GVIA and Cd2+, but not diltiazem, inhibited the tritium overflow. In raphe nuclei slices 4-aminopyridine enhanced the electrical stimulation-induced tritium release in a concentration-dependent manner. The inhibition of tritium efflux by 8-OH-DPAT was abolished with 4-aminopyridine. Glibenclamide or tolbutamide proved to be ineffective. These data indicate that (1) different 5-HT receptor subtypes (5-HT1A and 5-HT1B) regulate dendritic and axon terminal 5-HT release; (2) serotonin release from the dendrites may be regulated by the voltage-sensitive N-type Ca2+ channels; (3) the 5-HT1A receptor-mediated inhibition of serotonin release may be due to opening of voltage-sensitive K+ channels.

Effect of a 5-HT1 receptor agonist, CP-122,288, on oedema formation induced by stimulation of the rat saphenous nerve.

Neurogenic oedema formation in the rat hind paw skin induced by electrical stimulation of the saphenous nerve and measured by extravasation of [125I]-albumin, was inhibited by the 5-HT1B receptor agonist, CP-93,129, and the novel tryptamine analogue, CP-122,288. Significant inhibition of up to 66% of control was observed with CP-122,288 (2 x 10(-14) - 2 x 10(-7) mol kg-1) and CP-93,129 (5 x 10(-7) - 5 x 10(-6) mol kg-1), with the minimum effective dose for CP-122,288 being about 10(7) fold less than that for CP-93,129. Oedema formation induced by the intradermal administration of exogenous mediators (substance P and histamine) in rat dorsal skin was not inhibited by CP-122,288 (2 x 10(-10) mol kg-1). These results suggest that CP-122,288 is a potent inhibitor of neurogenic inflammation in rat skin and that the effect may be due to a prejunctional inhibition of neuropeptide release.

Evidence that 5-hydroxytryptamine release in rat dorsal raphé nucleus is controlled by 5-HT1A, 5-HT1B and 5-HT1D autoreceptors.

Electrically stimulated 5-hydroxytryptamine (5-HT) release was monitored in slices of rat dorsal raphé nucleus (DRN) by fast cyclic voltammetry. Pseudo-single pulse stimulations (5 pulses at 100 Hz) were used to enable the effect of various receptor agonists to be seen without competition from endogenously released transmitter. The selective 5-HT1A receptor agonist, (+)-8-OH-DPAT (1.0 microM) decreased stimulated 5-HT release to 31 +/- 3% of controls. This decrease was inhibited by the 5-HT1A receptor antagonists, (+)-WAY-100135 (1.0 microM) and WAY-100635 (0.1 microM) but not by the 5-HT1D/B antagonist, GR127935 (0.05 microM). The selective 5-HT1B receptor agonist, CP-93129 (0.3 microM) decreased stimulated 5-HT release to 61 +/- 4% of control. This effect was antagonized by the 5-HT1B receptor antagonist, isamoltane (0.5 microM) but not by (+)-WAY-100135. The 5-HT1D agonist, sumatriptan (0.5 microM) decreased stimulated 5-HT release to 52 +/- 2% of controls. This decrease was blocked by GR-127935 but not by WAY-100635. These results suggest that 5-HT release in the rat DRN is under the control of 5-HT1A, 5-HT1B and 5-HT1D autoreceptors.

Presynaptic inhibition by serotonin of glycinergic inhibitory synaptic currents in the rat brain stem.

1. With the use of a thin brain stem slice preparation, we recorded in visualized neonatal rat hypoglossal motoneurons unitary glycinergic inhibitory postsynaptic currents (IPSCs) that were evoked by extracellular stimulation of nearby interneurons. We found that 10 microM serotonin (5-HT) presynaptically inhibited this glycinergic synaptic transmission by 85.5%. 2. In the somata of presynaptic interneurons, 5-HT1A receptor activation potentiated inwardly rectifying K+ channels and inhibited voltage-activated calcium channels. 3. In contrast, the 5-HT1B receptor was primarily responsible for inhibition of evoked glycinergic IPSCs; a selective 5-HT1B receptor agonist, N-(3-trifluoromethylphenyl)piperazine (TFMPP, 10 microM), inhibited synaptic transmission by 97.3%. On the other hand, 5-HT1A receptor activation by (+)-8-OH-dipropylaminotetralin (8-OHDPAT, 1 microM) inhibited IPSCs by only 24.1%. A 5-HT1A antagonist, 1-(2-methyoxyphenyl)-4-[4-(2-phthalimido)-butyl]piperazine hydrobromide (NAN-190, 1 microM), had no effect on synaptic inhibition by 5-HT. 4. In the presence of tetrodotoxin (TTX) as well as TTX with cadmium (50 microM), we found that 5-HT1B receptor activation by TFMPP reduced the frequency of spontaneous miniature IPSCs (mIPSCs) without changing their mean amplitude. The results suggested that the 5-HT1B receptors activated at the presynaptic terminal inhibited synaptic transmission independent of inhibiting calcium influx through voltage-activated calcium channels. 5. These results indicate that activation of inwardly rectifying K+ channels and inhibition of voltage-activated calcium channels by 5-HT1A receptor activation do not constitute a main pathway for presynaptic inhibition by 5-HT of glycinergic synaptic transmission.(ABSTRACT TRUNCATED AT 250 WORDS)

Chronic treatment with fluvoxamine by osmotic minipumps fails to induce persistent functional changes in central 5-HT1A and 5-HT1B receptors, as measured by in vivo microdialysis in dorsal hippocampus of conscious rats

This study investigated the alterations of the 5-HT1A and 5-HT1B autoreceptor function following chronic treatment with fluvoxamine using osmotic minipumps. The 5-HT1A and 5-HT1B autoreceptor function were studied using microdialysis in the dorsal hippocampus. The effect of the 5-HT1A receptor agonist 8-OH-DPAT (0.3 mg/kg, SC) and the 5-HT1B receptor agonist RU-24969 (100 nM through the dialysis probe for 30 min) on 5-HT release was compared with rats chronically treated with saline. 8-OH-DPAT decreased 5-HT release to 55% and 60% of baseline, while RU-24969 decreased 5-HT release to 66% and 70% of baseline value in the saline and fluvoxamine group, respectively. In both cases, differences between the saline and fluvoxamine groups were not statistically significant. Plasma levels of fluvoxamine after 21 days of treatment ranged from 3 to 5 ng/ml. Fluvoxamine concentration in rat brain during treatment was estimated between 100 and 200 nM, which approximates to the IC50 value of fluvoxamine on the 5-HT transporter in synaptosomes and is 50 times higher than the Kd value for the 5-HT reuptake site. In conclusion, no evidence was found for changes in 5-HT1A,B receptor function using 8-OH-DPAT and RU-24969 as probes after continuous treatment with fluvoxamine by means of osmotic minipumps.

5-HT moduline, a novel endogenous tetrapeptide, specifically interacts with 5-HT 1B/1D receptors

MDL 74,721 (R)-2-(N1,N1-dipropylamino)-8-methylaminosulfonylmethyl-1,2,3,4-tetrahydronaphthalene, a sulfonamidotetralin, has been found to exhibit a 10,000-fold greater potency in neurogenic versus vascular models of migraine. Sumatriptan, a relatively pure 5-HT1D/5-HT1B receptor agonist, also showed higher potency versus neurogenic inflammation. However, for sumatriptan the potency difference (100-fold) in the two pathophysiological models was less pronounced than seen for MDL 74,721. The affinity profile of MDL 74,721 at 5-HT1 receptor subtypes may in part explain its ability to differentiate these two physiological responses. MDL 74,721 demonstrated nanomolar affinity for 5-HT1A (12.7±0.3 nM) and 5-HT1D (41.3±10.9 nM) but considerably lower affinity for 5-HT1B receptors (>1,000 nM). Serotonin-like activity was seen in in vitro functional assays including inhibition of forskolin-stimulated cAMP accumulation in human 5-HT1D receptor-transfected fibroblasts or eliciting vasoconstriction in isolated human pial arteries. The intrinsic activity (relative to 5−HTEAmax) and affinity (pD2) for the human cerebrovascular 5-HT receptors was: 5-HT (100%, 7.51±0.09), sumatriptan (94%, 6.85±0.1) and MDL 74,721 (66%, 5.70±0.23). In anaesthetised cats, treatment with MDL 74,721 resulted in a dose-related reduction in the percentage of carotid flow going through the arteriovenous anastomoses to the lungs, with an ED50 of 0.3 mg/kg i.v., the same as sumatriptan. However, in the guinea pig neurogenic model, MDL 74,721 inhibited plasma protein extravasation with an ED50 of 0.023 μg/kg compared to 2.5 μg/kg for sumatriptan. MDL 74,721 was also effective in this model (in rats) after oral administration. In conclusion, MDL 74,721 demonstrates a preclinical profile consistent with anti-migraine efficacy. Its marked preference for inhibiting neurogenic inflammation makes this compound a useful tool for assessing the relative contribution of this pathophysiological mechanism to the human disease state.

Effects of 5-HT on thalamocortical synaptic transmission in the developing rat.

1. Recent immunocytochemical and receptor binding data have demonstrated a transient somatotopic patterning of serotonin (5-HT)-immunoreactive fibers in the primary somatosensory cortex of developing rats and a transient expression of 5-HT1B receptors on thalamocortical axons from the ventral posteromedial thalamic nucleus (VPM). 2. These results suggest that 5-HT should strongly modulate thalamocortical synaptic transmission for a limited time during postnatal development. This hypothesis was tested in intracellular recording experiments carried out in thalamocortical slice preparations that included VPM, the thalamic radiations, and the primary somatosensory cortex. Effects of 5-HT and analogues were monitored on membrane potentials and input resistances of cortical neurons and on the amplitude of the synaptic potentials evoked in them by stimulation of VPM. 3. Results obtained from cortical neurons in slices taken from rats during the first 2 wk of life indicated that 5-HT strongly inhibited the VPM-evoked excitatory postsynaptic potential (EPSP) recorded from cortical neurons in a dose-dependent manner. In contrast, 5-HT had no significant effects on membrane potential, input resistance, or depolarizations induced by direct application of glutamic acid to cortical cells. 4. The effects of 5-HT were mimicked by the 5-HT1B receptor agonists 1-[3-(trifluoromethyl)phenyl]-piperazine (TFMPP) and 7-trifluoromethyl-4(4-methyl-1-piperazinyl)-pyrrolo[1,2-a]-quinoxaline maleate and antagonized by the 5-HT1B receptor antagonist (-)-pindolol. The 5-HT1A agonist [(+/-)8-hydroxydipropylaminotetralin HBr] (8-OH-DPAT) had less effect on the VPM-elicited EPSP, and the effects of 5-HT upon this response were generally not antagonized by either 1-(2-methoxyphenyl)-4-[4-(2- phthalimmido)butyl]piperazine HBr (a 5-HT1A antagonist) or ketanserine (a 5-HT2 antagonist) or spiperone (a 5-HT1A and 2 antagonist). 5. The ability of 5-HT to inhibit the VPM-evoked EPSP in cortical neurons was significantly reduced in slices from animals > 2 wk of age. The effectiveness of TFMPP in such animals was even more attenuated than that of 5-HT, and the effectiveness of 8-OH-DPAT was unchanged with age. These results are consistent with the disappearance of 5-HT1B receptors from thalamocortical axons after the second postnatal week and the maintenance of 5-HT1A receptors on some neurons. 6. All of the results obtained in this study are consistent with the conclusion that 5-HT has a profound, but developmentally transient, presynaptic inhibitory effect upon thalamocortical transmission in the rat's somatosensory cortex.

An examination of the behavioural specificity of hypophagia induced by 5-HT1B, 5-HT1C and 5-HT2 receptor agonists using the post-prandial satiety sequence in rats

Previous studies have shown that administration of 5-HT1B, 5-HT1C or 5-HT2 agonists decreases food intake in rats. However, it has not been established whether these drugs induce satiety or decrease feeding by a non-specific mechanism. In the present study the post-prandial satiety sequence was used to characterise the actions of the 5-HT2 receptor agonist, 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI), the 5-HT1B/5-HT1C receptor agonists, 1-(3-chorophenyl) piperazine (mCPP) and 1-[3-(trifluoromethyl)phenyl] piperazine (TFMPP), and the 5-HT1B agonist, 5-methoxy-3-(1,2,3,6-tetrahydro-4-pyridinyl)H-indole (RU 24969), on feeding in rats. All four compounds reduced food intake in rats that had been food deprived overnight. The 5-HT1B/5-HT1C agonists, TFMPP (at a dose of 1.0 mg/kg) and mCPP (at a dose of 3.0 mg/kg), appeared to produce satiety as their effects on the satiety sequence were similar to those induced by a food pre-load. In contrast, the 5-HT1B agonist RU 24969 and the 5-HT2 agonist DOI did not produce behavioural profiles that resembled satiety. Thus, RU 24969 elevated active behaviours and did not accelerate resting whereas DOI appeared to induce hypophagia by a non-specific fragmentation of behaviour. The results suggest that simultaneous activation of 5-HT1B and 5-HT1C receptors may be sufficient to elicit behaviourally specific satiety in the rat. In contrast, selective activation of 5-HT2 receptors does not induce satiety but elicits active behaviours and decreases feeding by response competition.

Evidence that RU 24969‐induced locomotor activity in C57/B1/6 mice is specifically mediated by the 5‐HT1B receptor

1. The behavioural effects of the 5-HT1B receptor agonists, RU 24969 and CGS 12066B, have been investigated in C57/B1/6 mice. 2. RU 24969 (1-30 mg kg-1) produced intense and prolonged hyperlocomotion and other behavioural changes. 3. CGS 12066B caused similar effects, but they were much less pronounced, inconsistent and transient irrespective of whether this drug was given i.p. (1-15 mg kg-1) or i.c.v. (0.2-40 micrograms). However, CGS 12066B (7.5 and 15 mg kg-1) caused a dose-related inhibition of RU 24969 (7.5 mg kg-1)-induced hyperlocomotion indicating that the former is a 5-HT1B partial agonist. 4. RU 24969 (7.5 mg kg-1 i.p.)-induced hyperlocomotion was inhibited by the (-)-, but not (+)-isomers of pindolol (4 mg kg-1) and propranolol (20 mg kg-1) but not by metoprolol (10 mg kg-1) or ICI 118,551 (5 mg kg-1), consistent with an involvement of 5-HT1A or 5-HT1B receptors. 5. The response was not altered by the selective 5-HT1A receptor antagonist, WAY 100135 (5 mg kg-1, s.c.), the 5-HT2A/5-HT2C receptor antagonist, ritanserin (0.1 mg kg-1), the selective 5-HT3 receptor antagonist, ondansetron (1 mg kg-1) or the non-selective 5-HT receptor antagonists methysergide (3 mg kg-1) and metergoline (3 mg kg-1). 6. Although spiroxatrine (0.1 mg kg-1) and ketanserin (1 mg kg-1) inhibited RU 24969-induced hyperlocomotion, these effects were probably due to antagonism of dopamine D2 receptors and alpha 1-adrenoceptors respectively. 7. Taken together, these results indicate that RU 24969-induced hyperlocomotion results specifically from activation of central 5-HTIB receptors.8. Lesioning of 5-HT neurones with 5,7-dihydroxytryptamine (75 microg, i.c.v.) or depletion with pchlorophenylalanine(200 mg kg-1, i.p. for 14 days) had no effect on RU 24969-induced hyperlocomotiondemonstrating that the 5-HTIB receptors involved are postsynaptic and that they do not show super sensitivity.9. The involvement of other monoamine neurotransmitter systems in RU 24969-induced hyperlocomotionwas also examined. The response was inhibited by the al-adrenoceptor antagonist, prazosin(1 mg kg-1), the dopamine DI receptor antagonist, SCH 23390 (0.05 mg kg-1) and the dopamine D2 receptor antagonist, BRL 34778 (0.03 mg kg-1), but not by the M2-adrenoceptor antagonist, idazoxan(1 mg kg-1). Lesioning noradrenergic neurones with N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine(100 mg kg-1) markedly attenuated this behaviour. These results show that the hyperlocomotion is expressed via noradrenergic and dopaminergic neurones acting on alpha 1-adrenoceptors, DI and D2 receptors.10. RU 24969 decreased brain concentrations of 5-hydroxyindoleacetic acid whilst simultaneously increasing 5-HT, consistent with the reduction of 5-HT neuronal activity by activation of 5-HTlA and 5-HTIB autoreceptors. RU 24969 increased brain 3-methoxy-4-hydroxyphenylglycol, but not noradrenaline, concentrations which supports the involvement of noradrenergic neurones in the expression of hyperlocomotion. RU 24969 did not alter dopamine, dihydroxyphenylacetic acid or homovanillic acid concentrations in the nucleus accumbens suggesting that the dopaminergic neurones terminating there are not directly involved.

Time-dependent blockade of neurogenic plasma extravasation in dura mater by 5-HT1B/D agonists and endopeptidase 24.11.

1. The delayed effects of stimulating the trigeminal ganglion unilaterally in rats and guinea-pigs were assessed by measuring the leakage of radiolabelled albumin from blood into the dura mater at intervals for up to 120 min after a 5 min stimulation period (5 Hz, 0.6 mA, 5 ms). 2. [125I]-albumin (50 microCi kg-1) was injected i.v. as a tracer 0, 20, 50, 80 or 110 min after stimulation and the animals were killed 10 min later. Extravasation of plasma protein developed for up to 90 min poststimulation. 3. To examine the mechanism underlying delayed plasma protein extravasation, CP-93,129 (5-HT1B receptor agonist, 460 nmol.kg-1), sumatriptan (5-HT1B/D receptor agonist, 24 nmol kg-1), or neutral endopeptidase 24.11 (1 nmol kg-1) were administered 45 or 75 min after trigeminal stimulation and 5 min before radiolabelled albumin. The extravasation response was reduced at 45 min. CP-23,129 also blocked extravasation when injected 25 min after capsaicin administration (1 mumol kg-1). 4. If the tracer was injected 5 min prior to electrical trigeminal stimulation, endopeptidase 24.11 (1 nmol kg-1) given 10 min before stimulation blocked the leakage (as reported previously for CP-93,129 or sumatriptan). All three compounds blocked the leakage when administered 30 min (but not 60 min) poststimulation in this paradigm. 5. The data support the previously made contention that neuropeptide release from sensory fibres mediates the plasma extravasation response following trigeminal ganglion stimulation, and that release and plasma leakage continue many minutes beyond the stimulation period. Hence, drugs that inhibit neuropeptide release (CP-93,129, sumatriptan), or enhance breakdown of neuropeptide mediators(endopeptidase 24.11) block the delayed extravasation response. Extravasation developing later than 45 min poststimulation was not neurogenically mediated.