Complex role of 5-HT in the regulation of TSH secretion in the male rat

The role of 5-hydroxytryptamine (5-HT) in the regulations of TSH secretion was studied in male rats using both peripheral and central administration of the drugs. Basal TSH levels were not modified by moderate doses of 5-HT (subcutaneously) or its precursors or antagonists (intraperitoneally) given 1 h before decapitation. The cold-stimulated TSH secretion was decreased by L-tryptophan (L-TRP, 400 mg/kg i.p.), quipazine (10 mg/kg i.p.) and 5-HT (1 or 5 mg/kg s.c. or i.v.) as well as by p-chlorophenylalanine (pCPA, 20 or more mg/kg i.p.) when the drugs were given 1 h before sampling. pCPA (100-400 mg/kg i.p.) was active 24-48 h after the injection but repetitive administration did not affect TSH levels. 5-HT (5 mg/kg s.c.) was effective also in pinealectomized animals. L-TRP and 5-hydroxytryptophan potentiated the TRH-stimulated TSH secretion when given 1 h before killing. 5-HT (10 microgram/rat) infused into the third ventricle enhanced the cold-stimulated TSH secretion when given 30-45 min before sampling. When injected into the medial basal hypothalamus, 50-HT (1-10 microgram/rat) had no effect on basal or stimulated TSH levels. The results suggest: (1) 5-HT does not play any role in the regulation of basal TSH secretion; (2) in the cold-stimulated TSH secretion 5-HT has a stimulatory action evidently inside the blood-brain barrier and also an inhibitory effect obviously outside this barrier.     

Cloning, functional expression and role in cell growth regulation of a hamster 5-HT2 receptor subtype

We have isolated a hamster fibroblast cDNA clone that encodes a serotoninergic receptor whose deduced amino acid sequence displays 94% identity with the rat brain serotonin (5-HT) type 2 receptor. When expressed in Xenopus oocytes, the hamster receptor efficiently couples to the phosphoinositide second messenger system and leads to intracellular Ca2+ mobilization in response to 5-HT. To determine the pharmacological properties of this receptor, and to evaluate the role of phospholipase C (PLC) activation in growth modulation by 5-HT, we have expressed it in hamster fibroblasts. Transfected cells that express 5-HT receptors were selected using a novel method based on coexpression of the Na+/H+ antiporter gene as a selectable marker. After co-transfection of the 5-HT receptor and Na+/H+ antiporter cDNAs in fibroblasts lacking antiporter activity (variants of the CCL39 line), 50% of the clones resistant to an acute acid load express functional receptors. The pharmacological profile of the transfected receptor is consistent with it being of the 5-HT2 subtype, and the extent of 5-HT-stimulated PLC activation in independent clones correlates with their relative level of cRNA expression. In cells in where addition of 5-HT leads to strong activation of PLC, and inhibition of adenylate cyclase via endogenous 5-HT1b receptors, 5-HT alone has little effect on DNA synthesis stimulation. Thus we conclude that activation of the PLC signalling pathway in these cells is not sufficient to trigger G0/G1 to S phase transition. Strong activation of PLC via 5-HT2 receptors does however contribute to the synergy observed between 5-HT (Gi-coupled pathway) and fibroblast growth factor (tyrosine kinase-activated pathway) on DNA synthesis reinitiation in transfected cells.     

Effects of 5-HT1A and 5-HT2A receptor stimulation on temporal differentiation performance in the fixed-interval peak procedure

We examined the effects of the 5-HT1A receptor agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) and 5-HT(2A/2C) receptor agonist 2,5-dimethoxy-4-iodoamphetamine (DOI) on performance on the fixed-interval peak procedure, and the sensitivity of these effects to 5-HT1A and 5-HT2A receptor antagonists (N-[2-(4-[2-methoxyphenyl]-1-piperazinyl]ethyl]-N-2-pyridinylcyclohexanecarboxamide [WAY-100635] and ketanserin). Rats were trained to press a lever for food reinforcement in 50 min sessions consisting of 32 trials in which the lever was continuously available, separated by 10 s inter-trial intervals. In 16 trials, reinforcement was delivered following the first response after 30 s had elapsed since trial onset (fixed-interval 30 s). In 16 randomly interposed (peak/probe) trials, reinforcement was omitted, and the lever remained in the operant chamber for 120 s. Response rate in probe trials was plotted against time from trial onset. Time to peak response rate (t(peak)) and the Weber fraction were derived from modified Gaussian curves fitted to each rat's data. 8-OH-DPAT (0.05 mg kg(-1)) reduced t(peak) and increased the Weber fraction; the effect on t(peak) was antagonized by WAY-100635 (0.1 mg kg(-1)). DOI (0.25 mg kg(-1)) also reduced t(peak) and increased the Weber fraction; the reduction of t(peak) was antagonized by ketanserin (2 mg kg(-1)). Stimulation of 5-HT1A and 5-HT2A receptors alters temporal differentiation in qualitatively similar ways.     

The role of putative 5-HT1A and 5-HT1B receptors in the control of feeding in rats

8-hydroxy-2(di-n-propylamino)tetraline (8-OH-DPAT) and 5-methoxy-3(1,2,3,6-tetrahydro-4-pyridinyl)1H indole succinate (RU 24969), two agonists on the putative serotonin 1A and serotonin 1B receptors, were used for exploring the role of these sites in the inhibitory effect of serotonin (5-HT) on feeding. In free-feeding rats, 2.5-5 mg/kg RU 24969 significantly reduced food intake while doses of 8-OH-DPAT ranging from 0.125 to 0.5 mg/kg increased eating. The effects of the highest doses were associated with hyperlocomotion and hyperreactivity for RU 24969 and a typical motor syndrome (flat body posture and forepaw treading) for 8-OH-DPAT. The motor syndrome caused by 0.5 mg/kg 8-OH-DPAT was much more obvious in food-deprived rats in which food intake was also markedly reduced. RU 24969 1.25 and 5 mg/kg reduced food intake by food-deprived rats and caused hyperlocomotion not different from that in free-feeding animals. Pretreatment with metergoline (2 mg/kg i.p.) prevented the effect of 5 mg/kg RU 24969 on food intake by food-deprived rats but had no effect on the reduction of eating caused by 0.5 mg/kg 8-OH-DPAT. The motor syndrome caused by 8-OH-DPAT was not changed by metergoline but the hyperlocomotion caused by RU 24969 was potentiated. Haloperidol (0.1 mg/kg i.p.) completely blocked the hyperlocomotion but did not change the reduction of food intake caused by RU 24969 in food-deprived rats. It is suggested that the putative serotonin 1B receptors specifically mediate the inhibitory effect of 5-HT on feeding whereas serotonin 1A sites act by enhancing eating only in free-feeding animals.     

DHA prevents altered 5-HT1A, 5-HT2A,CB1 and GABAA receptor binding densities in the brain of male rats fed a high-saturated-fat diet

Low levels of docosahexaenoic acid (DHA) have been linked to a number of mental illnesses such as memory loss, depression and schizophrenia. While supplementation of DHA is beneficial in improving memory and cognition, the influence of dietary fats on the neurotransmitters and receptors involved in cognitive function is still not known. The aim of this study was to investigate serotonin receptor (5-HT_1A and 5-HT_2A), cannabinoid receptor (CB1) and gamma-aminobutyric acid type A (GABA_A) receptor binding densities in the brain of male rats fed a high-saturated-fat (HF) diet, as well as the effect of DHA supplementation on HF diet. Alterations of these receptors in the post-mortem rat brain were detected by [³H]-WAY-100635, [³H]-ketanserin, [³H]-CP-55,940 and [³H]-muscimol binding autoradiography, respectively. In the hippocampus, the 5-HT_1A, CB1 and GABA_A receptor binding densities significantly increased in response to an HF diet, while in the hypothalamus, 5-HT_1A and CB1 binding densities significantly increased in HF-fed rats. Importantly, DHA supplementation prevented the HF-induced increase of receptors binding density in the hippocampus and hypothalamus. Furthermore, DHA supplementation attenuated 5-HT_2A receptor binding density in the caudate putamen, anterior cingulate cortex and medial mammillary nucleus, which was also increased in HF group. This study showed that an HF diet increased 5-HT_1A, 5-HT_2A, CB1 and GABA_A receptor binding densities in the brain regions involved in cognitive function and that dietary DHA can attenuate such alterations. These findings provide insight into the mechanism by which DHA supplementation ameliorates reduced cognitive function associated with an HF diet.     

5-HT2A and 5-HT2C receptors and their atypical regulation properties

The 5-HT(2A) and 5-HT(2C) receptors belong to the G-protein-coupled receptor (GPCR) superfamily. GPCRs transduce extracellular signals to the interior of cells through their interaction with G-proteins. The 5-HT(2A) and 5-HT(2C) receptors mediate effects of a large variety of compounds affecting depression, schizophrenia, anxiety, hallucinations, dysthymia, sleep patterns, feeding behaviour and neuro-endocrine functions. Binding of such compounds to either 5-HT(2) receptor subtype induces processes that regulate receptor sensitivity. In contrast to most other receptors, chronic blockade of 5-HT(2A) and 5-HT(2C) receptors leads not to an up- but to a (paradoxical) down-regulation. This review deals with published data involving such non-classical regulation of 5-HT(2A) and 5-HT(2C) receptors obtained from in vivo and in vitro studies. The underlying regulatory processes of the agonist-induced regulation of 5-HT(2A) and 5-HT(2C) receptors, commonly thought to be desensitisation and resensitisation, are discussed. The atypical down-regulation of both 5-HT(2) receptor subtypes by antidepressants, antipsychotics and 5-HT(2) antagonists is reviewed. The possible mechanisms of this paradoxical down-regulation are discussed, and a new hypothesis on possible heterologous regulation of 5-HT(2A) receptors is proposed.     

The role and predicted propensity of conserved proline residues in the 5-HT3 receptor

5-HT3 receptors possess a number of highly conserved proline residues. We changed each of these to alanine, expressed the mutants as homomeric 5-HT3A receptors in HEK293 cells, and analyzed them with radioligand binding, electrophysiology, and immunocytochemistry. Mutation of Pro56, Pro104, Pro123, and Pro170 resulted in ablation of radioligand binding, whereas mutation of Pro257 and Pro301 did not. Only the latter were expressed at the plasma membrane but were non-functional. Thus the former, which are in the N-terminal domain, may be involved in forming correct receptor structure, while those in the transmembrane region (Pro257 and Pro301) are necessary for the function of the protein. To explore the conformational preference (propensity) of these residues we examined the proportion of cis-prolines and the influence of adjacent residues in known protein structures. 4.7% of prolines in the protein data base were in the cis conformation, and the distribution of amino acids adjacent to cis-prolines was not randomly distributed. Comparison of the proportion of each amino acid residue adjacent to a cis-proline revealed that aromatic and bend-facilitating residues were favored while those with beta-branched chains were not. Thus five residues (Gly, Pro, Tyr, Trp, Phe) and three residues (Pro, Tyr, Phe) were found more frequently than expected before and after cis-prolines respectively, whereas five residues (Val, Ile, Leu, Asp, Thr) and two residues (Asp, Glu) were found less frequently. Of the 20 proline residues in the 5-HT3A receptor subunit only Pro170 has adjacent residues that are favorable. Mutating these to non-favorable residues resulted in ablation of ligand binding, whereas replacement with alternative favorable residues did not. We therefore propose that Pro170, which is part of the characteristic cys-loop found in this family of proteins, may be in the cis conformation.     

Hypothalamic 5-HT functional regulation through 5-HT1A and 5-HT2C receptors during pancreatic regeneration

5-HT receptors are predominantly located in the brain and are involved in pancreatic function and cell proliferation through sympathetic nervous system. The objective of this study was to investigate the role of hypothalamic 5-HT, 5-HT1A and 5-HT2C receptor binding and gene expression in rat model of pancreatic regeneration using 60% pancreatectomy. The pancreatic regeneration was evaluated by 5-HT content, 5-HT1A and 5-HT2C receptor gene expression in the hypothalamus of sham operated, 72 h and 7 days pancreatectomised rats. 5-HT content was quantified by HPLC. 5-HT1A receptor assay was done by using specific agonist [3H]8-OH DPAT. 5-HT2C receptor assay was done by using specific antagonist [3H]mesulergine. The expression of 5-HT1A and 5-HT2C receptor gene was analyzed by RT-PCR. 5-HT content was higher in the hypothalamus of 72 h pancreatectomised rats. 5-HT1A and 5-HT2C receptors were down-regulated in the hypothalamus. RT-PCR analysis revealed decreased 5-HT1A and 5-HT2C receptor mRNA expression. The 5-HT1A and 5-HT2C receptors gene expression in the 7 days pancreatectomised rats reversed to near sham level. This study is the first to identify 5-HT1A and 5-HT2C receptor gene expression in the hypothalamus during pancreatic regeneration in rats. Our results suggest the hypothalamic serotonergic receptor functional regulation during pancreatic regeneration.     

The role of lipophilicity in determining binding affinity and functional activity for 5-HT2A receptor ligands

The lipophilicity of a set of 5-HT(2A) ligands was determined using immobilized-artificial-membrane chromatography, a method that generates values well correlated with octanol-water partition coefficients. For agonists, a highly significant linear correlation was observed between binding affinity and lipophilicity. For ligands exhibiting partial agonist or antagonist properties, the lipophilicity was consistently higher than would be expected for an agonist of comparable affinity. The results suggest a possible method for distinguishing agonists from antagonists in high-throughput screening when a direct assay for functional activity is either unavailable or impractical.     

Cloning of the human serotonin 5-HT2 and 5-HT1C receptor subtypes.

We report the cloning and the deduced amino acid sequence of cDNAs encoding both the human serotonin 5-HT2 and 5-HT1C receptors. The human 5-HT2 and 5-HT1C receptors shared 87% and 90% amino acid homology, respectively, with their rat counterparts. The most divergent regions of the 5-HT2 receptor between human and rat were the N-terminal extracellular domain (75% homology) and the C-terminal intracellular domain (67% homology between amino acids 426-474). The greatest variability between the human and rat 5-HT1C receptors were at the N-terminal extracellular domain (78% homology) and the third cytoplasmic loop (71% homology). The availability of the cloned human 5-HT2 and 5-HT1C receptors will help facilitate the further understanding of the molecular pharmacology and physiology of these receptors.     

The regulation of male sexual behavior by the sex hormones

The article provides a review of the clinical and experimental data, both published and the author's own observations. They demonstrate that the hormone regulation of sex behaviour takes place at the central and the peripheral levels. The major role is played by androgenes, of which testosterone is the main regulator of sex appeal (libido, courtship behaviour in animals), while the most important regulator of ejaculation, in particular, of the time of ejaculation, is the non-aromatized androgene dihydrotestosterone (DHT). There is no rigid correlation between the level of sex activity and the level of androgenes, if the latter are within the normal individual and physiological range. However, both in case of hypo- and hyperendrogeny, the observations indicate depression of sexuality, though the mechanism of the depression are different. The author established the optimal levels of androgenes, which maintain the individual elements of the sexual act. The role of estrogene in regulation of the male sex appeal is not yet clear. As a rule, use of estrogenes results in depression of the male sexuality. At the same time, combination of DHT and estradiole (in laboratory and productive animals) leads to normalisation of erection and ejaculation disorders. These result is, probably, due to the central effect of estrogene, which prolongs the DHT effect and censures its accelerated penetration into cellular neuronal structures, as well as to the peripheral effect of DHT.     

Homology modelling of the serotoninergic 5-HT2c receptor

Since its discovery, 5-hydroxytryptamine, more usually called serotonin, has been an elusive candidate as a major mood regulator. This capacity gives it a great importance in the treatment of depression. It is within this framework that our work takes place, as it is related more particularly to a new therapeutic class whose leader is agomelatine. This compound binds to the melatoninergic receptors and to the serotoninergic 5-HT2c receptor, giving rise to the MASSA concept (Melatonin Agonist and Selective Serotonin Antagonist). Like the majority of the serotoninergic receptors, the sub-type 5-HT2c is a G-protein coupled receptor (GPCR). The three-dimensional structure of 5-HT2c is not experimentally known, and we thus resorted to comparative homology modelling to build a model allowing us to study its interactions with agomelatine.     

Administration of an oxytocin receptor antagonist attenuates sexual motivation in male rats

In male rats, oxytocin impacts both sexual arousal and certain types of consummatory sexual behaviors. However, the role of oxytocin in the motivational aspects of sexual behavior has received limited attention. Given the role that oxytocin signaling plays in consummatory sexual behaviors, it was hypothesized that pharmacological attenuation of oxytocin signaling would reduce sexual motivation in male rats. Sexually experienced Long-Evans male rats were administered either an oxytocin receptor antagonist (L368,899 hydrochloride; 1 mg/kg) or vehicle control into the intraperitoneal cavity 40 min prior to placement into the center chamber of a three-chambered arena designed to assess sexual motivation. During the 20-minute test, a sexually experienced stimulus male rat and a sexually receptive stimulus female rat were separately confined to smaller chambers that were attached to the larger end chambers of the arena. However, physical contact between test and stimulus rats was prevented by perforated dividers. Immediately following the sexual motivation test, test male rats were placed with a sexually receptive female to examine consummatory sexual behaviors. Although both drug and vehicle treated rats exhibited a preference for the female, treatment with an oxytocin receptor antagonist decreased the amount of time spent with the female. There were no differences between drug and vehicle treated rats in either general activity, exploratory behaviors, the amount of time spent near the stimulus male rat, or consummatory sexual behaviors. Extending previous findings, these results indicate that oxytocin receptors are involved in sexual motivation in male rats.     

Activation of 5-HT2A/C receptors counteracts 5-HT1A regulation of n-methyl-D-aspartate receptor channels in pyramidal neurons of prefrontal cortex

Abnormal serotonin-glutamate interaction in prefrontal cortex (PFC) is implicated in the pathophysiology of many mental disorders, including schizophrenia and depression. However, the mechanisms by which this interaction occurs remain unclear. Our previous study has shown that activation of 5-HT(1A) receptors inhibits N-methyl-D-aspartate (NMDA) receptor (NMDAR) currents in PFC pyramidal neurons by disrupting microtubule-based transport of NMDARs. Here we found that activation of 5-HT(2A/C) receptors significantly attenuated the effect of 5-HT(1A) on NMDAR currents and microtubule depolymerization. The counteractive effect of 5-HT(2A/C) on 5-HT(1A) regulation of synaptic NMDAR response was also observed in PFC pyramidal neurons from intact animals treated with various 5-HT-related drugs. Moreover, 5-HT(2A/C) stimulation triggered the activation of extracellular signal-regulated kinase (ERK) in dendritic processes. Inhibition of the beta-arrestin/Src/dynamin signaling blocked 5-HT(2A/C) activation of ERK and the counteractive effect of 5-HT(2A/C) on 5-HT(1A) regulation of NMDAR currents. Immunocytochemical studies showed that 5-HT(2A/C) treatment blocked the inhibitory effect of 5-HT(1A) on surface NR2B clusters on dendrites, which was prevented by cellular knockdown of beta-arrestins. Taken together, our study suggests that serotonin, via 5-HT(1A) and 5-HT(2A/C) receptor activation, regulates NMDAR functions in PFC neurons in a counteractive manner. 5-HT(2A/C), by activating ERK via the beta-arrestin-dependent pathway, opposes the 5-HT(1A) disruption of microtubule stability and NMDAR transport. These findings provide a framework for understanding the complex interactions between serotonin and NMDARs in PFC, which could be important for cognitive and emotional control in which both systems are highly involved.     

The role of alpha-2u-globulin in corticosteroidogenesis in male rats

Administration of estradiol-17 beta for 7 days to the adult male rat results in adrenal hyperplasia, decreased serum corticosterone along with elevation in serum ACTH and inhibition of adrenal 5-ene-3 beta-hydroxysteroid dehydrogenase activity (5-ene-3 beta-HSD). Treatment with alpha 2u-globulin for following 14 days of estrogen-treated rats reversed the effects of estrogen while in normal rats alpha 2u-globulin treatment increased adrenal 5-ene-3 beta-HSD activity and serum corticosterone level while causing a fall in serum ACTH. It is concluded that alpha 2u-globulin may play a role in ACTH secretion by inducing corticosterone synthesis.     

Lordotic behavior in male rats: genetic and hormonal regulation of sexual differentiation

The male offspring of Long-Evans rats treated with the aromatization inhibitor ATD (1,4,6-androstatriene-3,17-dione) during pregnancy show high levels of lordotic behavior when treated with estrogen and progesterone in adulthood. The male offspring of Sprague-Dawley dams treated in the same way show only a slight facilitation of lordotic potential. These strain differences could reflect strain differences in gestation length and therefore differences in the timing of the sensitive period of sexual differentiation; they could reflect differences in the sensitivity to the defeminizing actions of gonadal hormones; or they could reflect differences in the sensitivity to ATD treatment. We therefore directly compared the effects of prenatal and early postnatal treatment with ATD on the potential of male Long-Evans and Sprague-Dawley rats to show lordosis when given estrogen and progesterone in adulthood. In both strains ATD treatment facilitated adult lordotic behavior. Treatment appeared to have a greater effect in the Long-Evans strain. However, control Long-Evans males were substantially more responsive to hormone treatment in adulthood than were Sprague-Dawley males. In the Long-Evans strain short-term ATD treatment (Days 20-23 of pregnancy) was as effective as long-term treatment (Days 10-23). In the Sprague-Dawley strain, ATD treatment was most effective when given prenatally and postnatally. Strain differences in hormonal sensitivity best account for the present findings.     

Estrogen-activated sexual behavior in male rats

Daily injections of 100 μg estradiol benzoate activated the whole pattern of sexual behavior in castrated sexually experienced male rats. If compared to rats treated daily with 100 μg testosterone propionate, the estrogen-treated males tended to have longer latencies and more mounts and intromissions prior to ejaculation. Fifty micrograms of estradiol benzoate stimulated the display of mounts and intromissions in prepuberally castrated male rats. No peripheral effects of the estrogen treatment were noted. These results suggest that estrogen has central “androgen-like” effects, but no such effects in the periphery. Estrogen treatment (5, 50, and 200 μg/kg for 3 weeks) of intact sexually experienced male rats resulted in testicular atrophy and loss of body weight, but had no significant effects on the sexual behavior.