5-HT2 receptor activation in the midbrain periaqueductal grey (PAG) reduces anxiety-like behaviour in mice

It is widely acknowledged that the indoleamine neurotransmitter serotonin (5-HT) plays a dual role in the regulation of anxiety, a role that in part depends upon neuroanatomical locus of action. Thus, whereas stimulation of 5-HT1A or 5-HT2 receptors in the limbic forebrain (amygdala, hippocampus) enhances anxiety-like responding in rodents, activation of corresponding receptor populations in the midbrain periaqueductal grey (PAG) more often than not reduce anxiety-like behaviour. The present study specifically concerns the anxiety-modulating influence of 5-HT2 receptors within the mouse PAG. Experiment 1 assessed the effects of intra-PAG infusions of the 5-HT2B/2C receptor agonist mCPP (0, 0.03, 0.1 or 0.3 nmol/0.1 mu l) on the behaviour of mice exposed to the elevated plus-maze. As mCPP acts preferentially at 5-HT2B and 5-HT2C receptors, Experiment 2 investigated its effects in animals pretreated with ketanserin, a preferential 5-HT2A/2C receptor antagonist. In both cases, test sessions were videotaped and subsequently, scored for anxiety-like behaviour (e.g., percentage of open arm entries and percentage of open arm time) as well as general locomotor activity (closed arm entries). The results of Experiment I showed that mCPP microinfusions (0.03 and 0.1 nmol) into the PAG of mice decreased behavioural indices of anxiety without significantly altering general activity measures. In Experiment 2, the anxiolytic-like profile of intra-PAG mCPP (0.03 nmol) was substantially attenuated by intra-PAG pretreatment with an intrinsically inactive dose of the preferential 5-HT2A/2C receptor antagonist, ketanserin (10 nmol/0.1 mu l). Together, these data suggest that 5HT(2C) receptor populations within the midbrain PAG play an inhibitory role in plus-maze anxiety in mice. (C) 2007 Elsevier B.V. All rights reserved.

Activation of paraventricular nucleus of hypothalamus 5-HT1A receptor on sodium intake

Hypothalamic paraventricular nucleus (PVN) has an important role in the regulation of water and sodium intake. Several researches described the presence of 5-HT1 receptors in the central nervous system. 5-HTIA was one of the prime receptors identified and it is found in the somatodendritic and post-synaptic forms. Therefore, the aim of this study was to investigate the participation of serotonergic 5-HT1A receptors in the PVN on the sodium intake induced by sodium depletion followed by 24 h of deprivation (injection of the diuretic furosemide plus 24 h of sodium-deficient diet). Rats (280-320 g) were submitted to the implant of cannulas bilaterally in the PVN. 5-HT injections (10 and 20 mu g/0.2 mu l) in the PVN reduced NaCl 1.8% intake. 8-OH-DPAT injections (2.5 and 5.0 fig/0.2 mu l) in the PVN also reduced NaCl 1.8% intake. pMPPF bilateral injections (5-HT1A antagonist) previously to 8-OH-DPAT injections have completely blocked the inhibitory effect over NaCl 1.8% intake. 5-HT1A antagonists partially reduced the inhibitory effect of 5-HT on NaCl 1.8% intake induced by sodium depletion. In contrast, the intake of palatable solution (2% sucrose) under body fluid-replete conditions was not changed after bilateral PVN 8-OH-DPTA injections. The results show that 5HT(1A) serotonergic mechanisms in the PVN modulate sodium intake induced by sodium loss. The finding that sucrose intake was not affected by PVN 5-HT1A activation suggests that the effects of the 5-HT1A treatments on the intake of NaCl are not due to mechanisms producing a nonspecific decrease of all ingestive behaviors. (c) 2006 Elsevier B.V. All rights reserved.

The bradycardic and hypotensive responses to serotonin are reduced by activation of GABA A receptors in the nucleus tractus solitarius of awake rats

We investigated the effects of bilateral injections of the GABA receptor agonists muscimol (GABA A) and baclofen (GABA B) into the nucleus tractus solitarius (NTS) on the bradycardia and hypotension induced by iv serotonin injections (5-HT, 2 µg/rat) in awake male Holtzman rats. 5-HT was injected in rats with stainless steel cannulas implanted bilaterally in the NTS, before and 5, 15, and 60 min after bilateral injections of muscimol or baclofen into the NTS. The responses to 5-HT were tested before and after the injection of atropine methyl bromide. Muscimol (50 pmol/50 nl, N = 8) into the NTS increased basal mean arterial pressure (MAP) from 115 ± 4 to 144 ± 6 mmHg, did not change basal heart rate (HR) and reduced the bradycardia (-40 ± 14 and -73 ± 26 bpm at 5 and 15 min, respectively, vs -180 ± 20 bpm for the control) and hypotension (-11 ± 4 and -14 ± 4 mmHg, vs -40 ± 9 mmHg for the control) elicited by 5-HT. Baclofen (12.5 pmol/50 nl, N = 7) into the NTS also increased basal MAP, but did not change basal HR, bradycardia or hypotension in response to 5-HT injections. Atropine methyl bromide (1 mg/kg body weight) injected iv reduced the bradycardic and hypotensive responses to 5-HT injections. The stimulation of GABA A receptors in the NTS of awake rats elicits a significant increase in basal MAP and decreases the cardiac Bezold-Jarisch reflex responses to iv 5-HT injections.

On a possible dual role for the lateral septal area 5-HT1A receptor system in the regulation of water intake and urinary excretion

The 5-hydroxytryptamine (5-HT)(1A) receptor system plays a prominent role in a variety of physiological functions and behavior and regulation of this responsiveness of the receptor system has been implicated in the central regulation of water intake and urinary excretion. The lateral septal area (LSA) exhibits a high density of 5-HT1A receptors, as well as a subpopulation of oxytocin (OT) receptors. Here we report the effects of pMPPF (a selective 5-HT1A antagonist), d(CH2)(5)[Tyr(Me)(2)Thr(4), Orn(5), Tyr(NH2)(9)]-vasotocin (an OT antagonist), and that 5-HT1A receptor system is regulated as a consequence of activation of the Na+ channel by veratridine. Cannulae were implanted into the LSA of rats to enable the introduction of the drugs. Injections of 8-OH-DPAT (a 5-HT1A agonist) blocked water intake and increased urinary excretion, while pMPPF or the OT antagonist injected bilaterally before 8-OH-DPAT blocked its inhibitory effect on water intake and its diuretic effect. In contrast, increases in extracellular sodium levels induced by the sodium channel modulator, veratridine, enhanced 5-HT1A responsiveness for water intake and reduced the diuretic effects induced by 8-OH-DPAT. These trials demonstrated that the responsiveness of the 5-HT1A receptor system in the LSA can be enhanced or depressed as a consequence of an induced rise in extracellular sodium. (C) 2010 Elsevier B.V. All rights reserved.

Activation of the serotonergic 5-HT1A receptor in the paraventricular nucleus of the hypothalamus inhibits water intake and increases urinary excretion in water-deprived rats

The paraventricular nucleus (PVN) may be considered as a dynamic mosaic of chemically-specified subgroups of neurons. 5-HT1A is one of the prime receptors identified and there is expressed throughout all magnocellular regions of the PVN. Several reports have demonstrated that a subpopulation of the magnocellular neurons expressing 5-HT1A receptors are oxytocin (OT) neurons and activation of 5-HT1A receptors in the PVN increases the plasma OT. Increasing evidence shows that OT inhibits water intake and increases urinary excretion in rats. The aim of this study was to investigate the role of serotonergic 5-HT1A receptors in the lateral-medial posterior magnocellular region of the PVN in the water intake and diuresis induced by 24 h of water deprivation. Cannulae were implanted in the PVN of rats. 5-HT injections in the PVN reduced water intake and increased urinary excretion. 8-OH-DPAT (a 5-HT1A agonist) injections blocked the water intake and increased urinary output in all the periods of the observation. pMPPF (a 5-HT1A antagonist) injected bilaterally before the 8-OH-DPAT blocked its inhibitory effect on water intake and its diuretic effect. We suggest that antidipsogenic and diuretic responses seem to be mediated via 5-HT1A receptors of the lateral-medial posterior magnocellular region of the PVN in water-deprived rats. (C) 2008 Elsevier B.V. All rights reserved.

Interação entre estradiol e receptores 5 - H T 1A no Núcleo Mediano da Rafe em ratas ovariectomizadas: na aquisição da informação aversiva e associação ao contexto

Pós-graduação em Biociências - FCLAS

Serotonin-2C receptors in the basolateral nucleus of the amygdala mediate the anxiogenic effect of acute imipramine and fluoxetine administration

A growing body of evidence indicates that facilitation of serotonin-2C receptor (5-HT2CR)-mediated neurotransmission in the basolateral nucleus of the amygdala (BLA) is involved in anxiety generation. We investigated here whether BLA 5-HT(2C)Rs exert a differential role in the regulation of defensive behaviours related to generalized anxiety (inhibitory avoidance) and panic (escape) disorders. We also evaluated whether activation of BLA 5-HT(2C)Rs accounts for the anxiogenic effect caused by acute systemic administration of the antidepressants imipramine and fluoxetine. Male Wistar rats were tested in the elevated T-maze after intra-BLA injection of the endogenous agonist 5-HT, the 5-HT2CR agonist MK-212 or the 5-HT2CR antagonist SB-242084. This test allows the measurement of inhibitory avoidance acquisition and escape expression. We also investigated whether intra-BLA administration of SB-242084 interferes with the acute anxiogenic effect caused by imipramine and fluoxetine in the Vogel conflict test, and imipramine in the elevated T-maze. While intra-BLA administration of 5-HT and MK-212 facilitated inhibitory avoidance acquisition, suggesting an anxiogenic effect, SB-242084 had the opposite effect. None of these drugs affected escape performance. Intra-BLA injection of a sub-effective dose of SB-242084 fully blocked the anxiogenic effect caused either by the local microinjection of 5-HT or the systemic administration of imipramine and fluoxetine. Our findings indicate that 5-HT(2C)Rs in BLA are selectively involved in the regulation of defensive behaviours associated with generalized anxiety, but not panic. The results also provide the first direct evidence that activation of BLA 5-HT(2C)Rs accounts for the short-term aversive effect of antidepressants.

5-Hydroxytryptamine-4 receptor messenger ribonucleic acid levels and densities in gastrointestinal muscle layers from healthy dairy cows

Serotonin (5-hydroxytryptamine, 5-HT) is involved in gastrointestinal tract (GIT) motor functions through binding to specific receptors located in the GIT walls. The objectives of the current study were to compare mRNA levels and binding sites of 5-HT(4) receptors (5-HTR(4)) in smooth muscle layers from the fundus abomasi, pylorus, ileum, cecum, proximal loop of the ascending colon (PLAC), and external loop of the spiral colon (ELSC) of healthy dairy cows, and to verify whether mRNA and protein expression were correlated. Smooth muscle samples were prepared by scraping the mucosa and submucosa from full-thickness intestinal wall samples. The mRNA levels of 5-HTR(4) were measured by real-time PCR and expressed relative to those of the housekeeping gene glyceraldehyde phosphate dehydrogenase. Binding studies were performed using the 5-HTR(4) antagonist [(3)H]GR113808. The mRNA levels of 5-HTR(4) were affected (P < 0.05) by location along the GIT. The mRNA levels of 5-HTR(4) in the ELSC and the ileum were greater than in the PLAC (P = 0.05 and P = 0.07, respectively) but similar to those of all other locations. The competitive binding of [(3)H]GR113808 to suspended membranes from the fundus abomasi, pylorus, cecum, and ELSC was best fit by a 2-site receptor model, whereas it was best fit by a 1-site receptor model in the ileum and PLAC. The mRNA levels and numbers of 5-HTR(4) were not correlated (r = 0.14; P = 0.71). In conclusion, mRNA and binding sites for 5-HTR(4) are present in the smooth muscle layer of the entire GIT of dairy cows and may play a role with respect to motility. The effects of activation of this receptor subtype may be different among GIT locations due to differences in the amount of high- relative to low-affinity binding sites.

Replacement of histidine in position 105 in the alpha(5) subunit by cysteine stimulates zolpidem sensitivity of alpha(5)beta(2)gamma(2) GABA(A) receptors

Zolpidem is a positive allosteric modulator of GABA(A) receptors with sensitivity to subunit composition. While it acts with high affinity and efficacy at GABA(A) receptors containing the alpha(1) subunit, it has a lower affinity to GABA(A) receptors containing alpha(2), alpha(3), or alpha(5) subunits and has a very weak efficacy at receptors containing the alpha(5) subunit. Here, we show that replacing histidine in position 105 in the alpha(5) subunit by cysteine strongly stimulates the effect of zolpidem in receptors containing the alpha(5) subunit. The side chain volume of the amino acid residue in this position does not correlate with the modulation by zolpidem. Interestingly, serine is not able to promote the potentiation by zolpidem. The homologous residues to alpha(5)H105 in alpha(1), alpha(2), and alpha(3) are well-known determinants of the action of classical benzodiazepines. Other studies have shown that replacement of these histidines alpha(1)H101, alpha(2)H101, and alpha(3)H126 by arginine, as naturally present in alpha(4) and alpha(6), leads to benzodiazepine insensitivity of these receptors. Thus, the nature of the amino acid residue in this position is not only crucial for the action of classical benzodiazepines but in alpha(5) containing receptors also for the action of zolpidem.

Characterizing New Fluorescent Tools for Studying 5-HT3 Receptor Pharmacology

The pharmacological characterization of ligands depends upon the ability to accurately measure their binding properties. Fluorescence provides an alternative to more traditional approaches such as radioligand binding. Here we describe the binding and spectroscopic properties of eight fluorescent 5-HT3 receptor ligands. These were tested on purified receptors, expressed receptors on live cells, or in vivo. All compounds had nanomolar affinities with fluorescent properties extending from blue to near infra-red emission. A fluorescein-derivative had the highest affinity as measured by fluorescence polarization (FP; 1.14 nM), flow cytometry (FC; 3.23 nM) and radioligand binding (RB; 1.90 nM). Competition binding with unlabeled 5-HT3 receptor agonists (5-HT, mCPBG, quipazine) and antagonists (granisetron, palonosetron, tropisetron) yielded similar affinities in all three assays. When cysteine substitutions were introduced into the 5-HT3 receptor binding site the same changes in binding affinity were seen for both granisetron and the fluorescein-derivative, suggesting that they both adopt orientations that are consistent with co-crystal structures of granisetron with a homologous protein (5HTBP). As expected, in vivo live imaging in anaesthetized mice revealed staining in the abdominal cavity in intestines, but also in salivary glands. The unexpected presence of 5-HT3 receptors in mouse salivary glands was confirmed by Western blots. Overall, these results demonstrate the wide utility of our new high-affinity fluorescently-labeled 5-HT3 receptor probes, ranging from in vitro receptor pharmacology, including FC and FP ligand competition, to live imaging of 5-HT3 expressing tissues.

Synthesis of photo-crosslinking probes and their application for the site-selective chemical modification of the 5-HT3 receptor

The 5-HT3 receptor (5-HT3R) is an important ion channel responsible for the transmission of nerve impulses in the CNS and PNS that is activated by the endogenous agonist serotonin (5-hydroxytryptamine, 5-HT). 5-HT3R is the only serotonin receptor belonging to the Cys-loop superfamily of neurotransmitter receptors. Different structural biology approaches can be applied, such as crystallization and x-ray analysis. Nonetheless, characterizing the exact ligand binding site(s) of these dynamic receptors is still challenging. The use of photo-crosslinking probes is an alternative validated approach allowing identification of regions in the protein that are important for the binding of small molecules. We designed our probes based on the core structure of the 5-HT3R antagonist granisetron, a FDA approved drug used for the treatment of chemotherapy-induced nausea and vomiting. We synthesized a small library of photo-crosslinking probes by conjugating diazirines and benzophenones via various linkers to granisetron. We were able to obtain several compounds with diverse linker lengths and different photo-crosslinking moieties that show nanomolar binding affinity for the orthosteric binding site. Furthermore we established a stable h5-HT3R expressing cell line and a purification protocol to yield the receptor in a high purity. Several experiments showed unambiguously that we are able to photo-crosslink our probes with the receptor site-specifically. The functionalised protein was analysed by Western blot and MS-analysis. This yielded the exact covalent modification site, corroborating current ligand binding models derived from mutagenesis and docking studies.

Elevated anxiety and antidepressant-like responses in serotonin 5-HT1A receptor mutant mice

The brain serotonin (5-hydroxytryptamine; 5-HT) system is a powerful modulator of emotional processes and a target of medications used in the treatment of psychiatric disorders. To evaluate the contribution of serotonin 5-HT1A receptors to the regulation of these processes, we have used gene-targeting technology to generate 5-HT1A receptor-mutant mice. These animals lack functional 5-HT1A receptors as indicated by receptor autoradiography and by resistance to the hypothermic effects of the 5-HT1A receptor agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT). Homozygous mutants display a consistent pattern of responses indicative of elevated anxiety levels in open-field, elevated-zero maze, and novel-object assays. Moreover, they exhibit antidepressant-like responses in a tail-suspension assay. These results indicate that the targeted disruption of the 5-HT1A receptor gene leads to heritable perturbations in the serotonergic regulation of emotional state. 5-HT1A receptor-null mutant mice have potential as a model for investigating mechanisms through which serotonergic systems modulate affective state and mediate the actions of psychiatric drugs.

Inference of binding affinity from neuronal receptors in humans

Tese de mestrado, Bioinformática e Biologia Computacional (Bioinformática), Universidade de Lisboa, Faculdade de Ciências, 2016

Using psilocybin to investigate the relationship between attention, working memory, and the serotonin 1A and 2A receptors

Increasing evidence suggests a link between attention, working memory, serotonin (5-HT), and prefrontal cortex activity. In an attempt to tease out the relationship between these elements, this study tested the effects of the hallucinogenic mixed 5-HT1A/2A receptor agonist psilocybin alone and after pretreatment with the 5-HT2A antagonist ketanserin. Eight healthy human volunteers were rested on a multiple-object tracking task and spatial working memory task under the four conditions: placebo, psilocybin (215 mu g/kg), ketanserin (50 mg), and psilocybin and ketanserin. Psilocybin significantly reduced attentional tracking ability, but had no significant effect on spatial working memory, suggesting a functional dissociation between the two tasks. Pretreatment with ketanserin did not attenuate the effect of psilocybin on attentional performance, suggestinga primary involvement of the 5-HT1A receptor in the observed defecit. Based on physiological and pharmacological data,we speculate that this impaired attentional performance may reflect a reduced ability to suppress or ignore distracting stimuli rather than reduced attentional capacity. The clinical relevance of these results is also discussed.

Modulating the rate and rhythmicity of perceptual rivalry alternations with the mixed 5-HT2A and 5-HT1A agonist psilocybin

Binocular rivalry occurs when different images are presented simultaneously to corresponding points within the left and right eyes. Under these conditions, the observer's perception will alternate between the two perceptual alternatives. Motivated by the reported link between the rate of perceptual alternations, symptoms of psychosis and an incidental observation that the rhythmicity of perceptual alternations during binocular rivalry was greatly increased 10 h after the consumption of LSD, this study aimed to investigate the pharmacology underlying binocular rivalry and to explore the connection between the timing of perceptual switching and psychosis. Psilocybin (4-phosphoryloxy-N,N-dimethyltryptamine, PY) was chosen for the study because, like LSD, it is known to act as an agonist at serotonin (5-HT)(1A) and 5-HT2A receptors and to produce an altered state sometimes marked by psychosis-like symptoms. A total of 12 healthy human volunteers were tested under placebo, low-dose ( 115 mg/kg) and high-dose ( 250 mg/kg) PY conditions. In line with predictions, under both low- and high-dose conditions, the results show that at 90 min postadministration ( the peak of drug action), rate and rhythmicity of perceptual alternations were significantly reduced from placebo levels. Following the 90 min testing period, the perceptual switch rate successively increased, with some individuals showing increases well beyond pretest levels at the final testing, 360 min postadministration. However, as some subjects had still not returned to pretest levels by this time, the mean phase duration at 360 min was not found to differ significantly from placebo. Reflecting the drug-induced changes in rivalry phase durations, subjects showed clear changes in psychological state as indexed by the 5D-ASC ( altered states of consciousness) rating scales. This study suggests the involvement of serotonergic pathways in binocular rivalry and supports the previously proposed role of a brainstem oscillator in perceptual rivalry alternations and symptoms of psychosis.