5HT7 receptors in the rodent suprachiasmatic nucleus

Serotonergic modulation of circadian rhythms in rodent model preparations has received considerable attention over the past decade. Investigators have also been trying to determine which of the many serotonin receptor subtypes may be mediating the effects of serotonin in the suprachiasmatic nucleus, the location of the biological clock that generates the circadian rhythms. A single study in 1993 using the in vitro rat hypothalamic slice preparation suggested that serotonergic modulation of circadian rhythms at the level of the suprachiasmatic nucleus was acting via the newly discovered 5HT7 receptor subtype. Since that initial claim, serotonin modulation of circadian rhythms at the level of the suprachiasmatic nucleus has generally been attributed to 5HT7 receptor activation. However, when trying to cite relevant literature in support of 5HT7 involvement, it becomes evident that attributing rhythm-related serotonin activity in the suprachiasmatic nucleus to 5HT7 receptors may be somewhat premature. There are issues related to pharmacological specificity, species-specific results, and significant knowledge gaps that necessitate a careful review of the literature to make a judgment as to whether 5HT7 receptors are responsible for serotonergic activity in the rodent suprachiasmatic nucleus. In addition, there is sufficient data available at present to make an initial determination as to the degree of 5HT7 receptor involvement at any level in the generation or modulation of circadian rhythms in rodent species.     

Mitogenic action of hypoxia upon cutaneous neuroepithelial cells in developing zebrafish

In zebrafish, cutaneous neuroepithelial cells (NECs) contain serotonin (5‐HT) and are believed to initiate physiological and behavioral responses to hypoxia during embryonic and early larval development, when mature gills and O₂ chemoreceptors are not yet present. The number of skin NECs rapidly declines as embryos develop into larvae, but acclimation to hypoxia leads to retention of a greater number of these cells. We hypothesized that reduction of the partial pressure of oxygen (P _O2) in water would stimulate mitosis in cutaneous NECs in zebrafish. Zebrafish were exposed to 5‐bromo‐2′‐deoxyuridine (BrdU) and immunolabeled with antibodies against serotonin and BrdU to identify mitotic skin cells, including NECs. Cells were imaged and quantified using confocal microscopy. From embryonic to larval stages, we observed an overall increase in the number of BrdU‐positive cells in the skin, but a decrease in BrdU‐positive serotonergic NECs. Exposure of larvae to hypoxia (P _O2 = 30 mmHg) in vivo for 24 h produced a 1.7‐fold increase in the number of NECs labeled with BrdU. We conclude that under normal environmental P _O2 the population of cutaneous NECs declines due to a decrease in mitotic activity. During environmental hypoxia, the number of NECs undergoing cell division in the skin is increased, and this promotes retention of NECs under these conditions. These data demonstrate the direct action of hypoxia upon the cell cycle of cutaneous NECs in developing zebrafish, and support the notion that cutaneous NECs are embryonic O₂ chemoreceptors. © 2017 Wiley Periodicals, Inc. Develop Neurobiol 77: 789–801, 2017     

Adenoviral vectors for highly selective gene expression in central serotonergic neurons reveal quantal characteristics of serotonin release in the rat brain

Background  

5-hydroxytryptamine (5 HT, serotonin) is one of the key neuromodulators in mammalian brain, but many fundamental properties of serotonergic neurones and 5 HT release remain unknown. The objective of this study was to generate an adenoviral vector system for selective targeting of serotonergic neurones and apply it to study quantal characteristics of 5 HT release in the rat brain.     

Flux coupling in the human serotonin transporter

The serotonin (5-hydroxytryptamine; 5HT) transporter (SERT) catalyzes the movement of 5HT across cellular membranes. In the brain, SERT clears 5HT from extracellular spaces, modulating the strength and duration of serotonergic signaling. SERT is also an important pharmacological target for antidepressants and drugs of abuse. We have studied the flux of radio-labeled 5HT through the transporter stably expressed in HEK-293 cells. Analysis of the time course of net transport, the equilibrium 5HT gradient sustained, and the ratio of the unidirectional influx to efflux of 5HT indicate that mechanistically, human SERT functions as a 5HT channel rather than a classical carrier. This is especially apparent at relatively high [5HT](out) (> or =10 microM), but is not restricted to this regime of external 5HT.     

Monitoring mouse serotonin transporter internalization in stem cell-derived serotonergic neurons by confocal laser scanning microscopy

In the central nervous system serotonergic neurotransmission is terminated by the rapid removal of serotonin (5-hydroxytryptamine, 5HT) out of the extra-cellular space back into the presynaptic neuron. This task is fulfilled by a specific serotonin transporter (SERT) protein which controls the concentration of extra-cellular 5HT. Consequently, one mechanism to regulate the efficacy of serotonergic neurotransmission is via modulation of the density of SERT molecules on the cell membrane.In this regard it has been shown, that chronic activation of the p38 mitogen-activated protein kinase (p38 MAPK) leads to enhanced SERT surface expression whereas activation of protein kinase C (PKC) reduces SERT surface expression. In addition, it has been reported that exposure to selective serotonin re-uptake inhibitors (SSRIs) leads to a down-regulation of SERT expression in vivo and in vitro in different cellular systems. Here, we have studied interactions between kinase- and SSRI-induced SERT internalization in mouse stem cell-derived serotonergic neurons expressing the native SERT allele in its natural surroundings. Therefore we established a method to quantify the amount of cell surface-expressed SERT molecules on individual cells by antibody detection combined with confocal laser scanning microscopy.Using this methodology we could show that activation of PKC, inhibition of the p38 MAPK as well as exposure to the SSRI citalopram each induced a significant reduction of cell surface-expressed SERT over time. Combinations of PKC activation, p38 MAPK inhibition and SSRI exposure led to a more pronounced down-regulation of SERT surface expression depending on the time of drug exposure.     

A simplified method to generate serotonergic neurons from mouse embryonic stem and induced pluripotent stem cells

We have developed a new simple method to induce serotonergic neurons from embryonic stem (ES) and induced pluripotent stem cells. When ES or induced pluripotent stem cells were cultured on a thick gel layer of Matrigel, most colonies extended TuJ1-positive neurites. We found that noggin, a known antagonist of bone morphogenic protein, induces ES cells to express genes involved in serotonergic differentiation, such as Nkx2.2, Pet-1, Sonic hedgehog, tryptophan hydroxylase 2, and serotonin transporter, as well as increases high potassium-induced release of serotonin. To concentrate serotonergic neurons, ES cells carrying Pet-1-enhancer-driven enhanced green fluorescent protein were differentiated and sorted into about 80% pure cultures of serotonergic neurons. Whole cell voltage-clamp recordings showed a voltage-dependent current in dissociated neurons. This simplified method provides an alternative option for serotonergic differentiation of pluripotent stem cells and will likely contribute a deeper understanding regarding the nature of serotonergic neurons and open new therapeutic perspectives for the treatment of psychiatric disorders.     

Serotonin in body fluids: Characterization of human plasmatic and cerebrospinal fluid pools by means of a new HPLC method

A new HPLC technique for the analysis of picomolar amounts of serotonin (5HT) in plasma and cerebrospinal fluid (CSF) is described. Bufotenin is used as internal standard. Detection is achieved electrochemically or fluorimetrically. The detection limit can be estimated as 50 pg 5HT/mL of either fluid (0.3 picomolar). The method is used to characterize a non-particulate pool of 5HT which is clearly distinct of the platelet pool. Administration of parachlorophenylalanine (PCPA) 300 mg/kg to rats leads to a 90% reduction in the plasmatic pool whereas platelet 5HT is only slightly decreased (3rd day after PCPA) or even increased (7th day after PCPA). Human concentration (n=15) of 5HT in plasma is 2.6 ± 0.9 ng/mL (x ± S.D.). The application of the method to CSF of neurological patients reveals 5HT concentrations ranging from 93 to 962 pg/mL.     

Serotonin (5HT), fluoxetine, imipramine and dopamine target distinct 5HT receptor signaling to modulate Caenorhabditis elegans egg-laying behavior

Drugs that target the serotonergic system are the most commonly prescribed therapeutic agents and are used for treatment of a wide range of behavioral and neurological disorders. However, the mechanism of the drug action remain a conjecture. Here, we dissect the genetic targets of serotonin (5HT), the selective 5HT reuptake inhibitor (SSRI) fluoxetine (Prozac), the tricyclic antidepressant imipramine, and dopamine. Using the well-established serotonergic response in C. elegans egg-laying behavior as a paradigm, we show that action of fluoxetine and imipramine at the 5HT reuptake transporter (SERT) and at 5HT receptors are separable mechanisms. Even mutants completely lacking 5HT or SERT can partially respond to fluoxetine and imipramine. Furthermore, distinct mechanisms for each drug can be recognized to mediate these responses. Deletion of SER-1, a 5HT1 receptor, abolishes the response to 5HT but has only a minor effect on the response to imipramine and no effect on the response to fluoxetine. In contrast, deletion of SER-4, a 5HT2 receptor, confers significant resistance to imipramine while leaving the responses to 5HT or fluoxetine intact. Further, fluoxetine can stimulate egg laying via the Gq protein EGL-30, independent of SER-1, SER-4, or 5HT. We also show that dopamine antagonizes the 5HT action via the 5HT-gated ion channel MOD-1 signaling, suggesting that this channel activity couples 5HT and dopamine signaling. These results suggest that the actions of these drugs at specific receptor subtypes could determine their therapeutic efficacy. SSRIs and tricyclic antidepressants may regulate 5HT outputs independently of synaptic levels of 5HT.     

Serotonergic control of prolactin release in male rats.

To further clarify the relationship between the central serotonergic system and the control of prolactin secretion, we studied the effect of dorsal raphe' lesions, electrical stimulation of the midbrain raphe' nucleus and treatment with parachlorophenylalanine (PCPA) on prolactin secretion. Radio frequency destruction of serotonergic cell bodies in the midbrain dorsal raphe' nucleus or PCPA decreased forebrain serotonin (5HT) and 5-hydroxyindoleacetic acid (5HIAA) concentration and prolactin secretion. Electrical stimulation of the raphe' increased forebrain serotonin turnover and prolactin secretion. These observations indicate that serotonergic neurons located in the raphe' nuclei may be involved in regulating prolactin secretion in male rats.     

Deletion of the serotonin 2c receptor from transgenic mice overexpressing leptin does not affect their lipodystrophy but exacerbates their diet-induced obesity

The binding of leptin to hypothalamic neurons elicits inhibition of orexigenic NPY/AgRP neurons and stimulation of anorexigenic POMC/CART neurons. Projections of serotonergic neurons onto POMC neurons suggest that leptin and serotonin converge onto POMC neurons to regulate body weight. We probed the interaction of these pathways by generating transgenic mice overexpressing leptin (LepTg) without 5HT2c receptors. On a chow diet, the lean phenotype of LepTg mice was unaffected by the absence of 5HT2c receptors, whereas on a high fat diet, LepTg/5HT2c receptors knockout mice showed an exacerbation of diet-induced obesity. POMC mRNA levels were low in LepTg, 5HT2c receptors knockout and LepTg/5HT2c receptors knockout mice, demonstrating that perturbations of the 5HT2c receptor and leptin pathways, either alone or in combination, negatively impact on POMC expression. Thus, on a chow diet, leptin action is independent of 5HT2c receptors whereas on a high fat diet 5HT2c receptors are required for the attenuation of obesity.     

Lateral hypothalamic serotonergic responsiveness to food intake in rat obesity as measured by microdialysis.

The hypothalamic serotonergic system is involved in the regulation of food ingestion and energy metabolism. Since disturbances of both energy intake and expenditure can contribute to obesity, the objective of the present study was to evaluate the serotonergic response stimulated by food ingestion in two different models of obesity: the hyperphagic Zucker and the hypophagic and hypometabolic, monosodium glutamate (MSG) obese Wistar rat. For this we used microdialysis to examine the release of 5-hydroxytryptamine (serotonin, 5HT) and 5-hydroxyindoleacetic acid (5HIAA) in the lateral hypothalamus. Daily intake of MSG-obese rats was 40% lower while that of Zucker obese rats was 60% higher than that of the respective lean controls. In overnight-fasted animals, 20-min microdialysate samples were collected before (basal release) and during a 2-h period of access to a balanced palatable food mash. The animals began to eat during the first 20 min of food access, and food consumption was similar among the four groups in all six individual 20-min periods recorded. Ingestion of food increased 5HT release in all groups. In MSG-obese and lean Wistar rats, 5HT levels were similarly elevated during the whole experimental period. In the Zucker strain, 5HT increments of basal release tended to be higher in obese than in lean rats at 20 and 40 min, and a significantly higher increment was observed at 60 min after food access (40 and 135% for lean and obese, respectively). The area under the curve relating serotonin levels to the 120 min of food availability was significantly higher in Zucker obese (246.7 +/- 23.3) than MSG-obese (152.7 +/- 13.4), lean Wistar (151.9 +/- 11.1), and lean Zucker (173.5 +/- 24.0) rats. The present observation, of a food-induced serotonin release in the lateral hypothalamus of lean Wistar and Zucker rats, evidences that 5HT in the lateral hypothalamus is important in the normal response to feeding. In obese animals, the serotonin response was similar to (in the hypophagic-hypometabolic MSG model) or even higher than (in the hyperphagic Zucker model) that seen in the respective lean controls. This result indicates that the energy homeostasis disturbances of both these obesity models may not be ascribed to an impairment of the acute lateral hypothalamic serotonin response to a dietary stimulus.     

Development and sensitivity to serotonin of Drosophila serotonergic varicosities in the central nervous system

Serotonin is a classical small-molecule neurotransmitter with known effects on developmental processes. Previous studies have shown a developmental role for serotonin in the fly peripheral nervous system. In this study, we show that serotonin can modulate the development of serotonergic varicosities within the fly central nervous system. We have developed a system to examine the development of serotonergic varicosities in the larval CNS. We use this method to describe the normal serotonergic development in the A7 abdominal ganglion. From first to third instar larvae, the volume of the neuropil and number of serotonergic varicosities increase substantially while the varicosity density remains relatively constant. We hypothesize that serotonin is an autoregulator for serotonergic varicosity density. We tested the sensitivity of serotonergic varicosities to serotonin by adding neurotransmitter at various stages to isolated larval ventral nerve cords. Addition of excess exogenous serotonin decreases native varicosity density in older larvae, and these acute effects are reversible. The effects of serotonin appear to be selective for serotonergic varicosities, as dopaminergic and corazonergic varicosities remain qualitatively intact following serotonin application.     

Relationship between serotonergic measures in periphery and the brain of mouse.

Circadian rhythm and the relationship between the concentration of serotonin (5HT) and related substances (5-hydroxyindoleacetic acid; 5HIAA and tryptophan; Trp) in mouse brain, stomach and blood have been studied. All factors underwent circadian changes in the brain and blood. 5HT and 5HIAA levels in the stomach showed no circadian fluctuation. The concentrations of 5HT in the brain and blood did not correlate. Significant correlations were found between other serotonergic parameters analyzed in brain, stomach and blood. A significant negative correlation was observed between brain 5HIAA and blood 5HIAA. The concentration of tryptophan in the brain was correlated with the plasma total tryptophan level. There was fairly significant correlation (p less than 0.06) between brain serotonin and plasma tryptophan levels. The brain serotonin and tryptophan levels were strongly correlated (R = 0.410, p less than 0.03). Significant negative correlation was found between serotonin in the blood and serotonin in the stomach as well as between its level in the brain and in the stomach. The significance of these findings and their relationship to the use of peripheral serotonergic system as a model of neurons are discussed.     

Conditions for the use of specific antibodies for immunohistochemical visualization of serotonin and melatonin in the pineal gland of sheep

Having prepared antisera to serotonin and to melatonin, the authors were able to show that, in the pineal gland, the behaviour of these two antisera in immunohistochemical studies differs. The antiserum raised against 5HT, actually bound to the molecule formed by condensation of formaldehyde on 5HT and therefore could be used to reveal 5HT in tissue fixed with formaldehyde. On the other hand, the anti-melatonin antiserum bound to melatonin, and could therefore be used to reveal its presence in fresh tissue.     

Reversible site-selective labeling of membrane proteins in live cells

Chemical and biological labeling is fundamental for the elucidation of the function of proteins within biochemical cellular networks. In particular, fluorescent probes allow detection of molecular interactions, mobility and conformational changes of proteins in live cells with high temporal and spatial resolution. We present a generic method to label proteins in vivo selectively, rapidly (seconds) and reversibly, with small molecular probes that can have a wide variety of properties. These probes comprise a chromophore and a metal-ion-chelating nitrilotriacetate (NTA) moiety, which binds reversibly and specifically to engineered oligohistidine sequences in proteins of interest. We demonstrate the feasibility of the approach by binding NTA-chromophore conjugates to a representative ligand-gated ion channel and G protein-coupled receptor, each containing a polyhistidine sequence. We investigated the ionotropic 5HT(3) serotonin receptor by fluorescence measurements to characterize in vivo the probe-receptor interactions, yielding information on structure and plasma membrane distribution of the receptor.     

Occlusion of the human serotonin transporter is mediated by serotonin-induced conformational changes in the bundle domain

The human serotonin transporter (hSERT) terminates neurotransmission by removing serotonin (5HT) from the synaptic cleft, an essential process for proper functioning of serotonergic neurons. Structures of the hSERT have revealed its molecular architecture in four conformations, including the outward-open and occluded states, and show the transporter’s engagement with co-transported ions and the binding mode of inhibitors. In this study, we investigated the molecular mechanism by which the hSERT occludes and sequesters the substrate 5HT. This first step of substrate uptake into cells is a structural change consisting of the transition from the outward-open to the occluded state. Inhibitors such as the antidepressants citalopram, fluoxetine, and sertraline inhibit this step of the transport cycle. Using molecular dynamics simulations, we reached a fully occluded state, in which the transporter-bound 5HT becomes fully shielded from both sides of the membrane by two closed hydrophobic gates. Analysis of 5HT-triggered occlusion showed that bound 5HT serves as an essential trigger for transporter occlusion. Moreover, simulations revealed a complex sequence of steps and showed that movements of bundle domain helices are only partially correlated. 5HT-triggered occlusion is initially dominated by movements of transmembrane helix 1b, while in the final step, only transmembrane helix 6a moves and relaxes an intermediate change in its secondary structure.     

Synthesis and characterization of photolabile derivatives of serotonin for chemical kinetic investigations of the serotonin 5-HT(3) receptor

A series of photolabile o-nitrobenzyl derivatives of serotonin (caged serotonin) were synthesized: the amine-linked serotonin derivatives N-(2-nitrobenzyl) serotonin (Bz-5HT) and N-(alpha-carboxy-2-nitrobenzyl) serotonin (N-CNB-5HT), and O-alpha-carboxy-2-nitrobenzyl) serotonin (O-CNB-5HT), which has the caging group attached to the phenolic OH group. All the derivatives released free serotonin when excited by 308-nm or 337-nm laser pulses. The time constant of serotonin release from N-CNB-5HT was 1. 2 ms, with a quantum yield of 0.08. This is too slow for rapid chemical kinetic measurements. O-CNB-5HT is suitable for transient kinetic investigations of the serotonin 5-HT(3) receptor. It released serotonin with a time constant of 16 micros and a quantum yield of 0.03. The biological properties of O-CNB-5HT were evaluated, and the applicability of the compound for kinetic studies of the 5-HT(3) receptor was demonstrated. O-CNB-5HT does not activate the 5-HT(3) receptor by itself, nor does it modulate the response of a cell when co-applied with serotonin. When irradiated with a 337-nm laser pulse, O-CNB-5HT released free serotonin that evoked 5-HT(3) receptor-mediated whole-cell currents in NIE-115 mouse neuroblastoma cells.     

The mouse 5HT5 receptor reveals a remarkable heterogeneity within the 5HT1D receptor family.

Serotonin (5-HT) is a neuromodulator that mediates a wide range of physiological functions by activating multiple receptors. Using a strategy based on amino acid sequence homology between 5-HT receptors that interact with G proteins, we have isolated a cDNA encoding a new serotonin receptor from a mouse brain library. Amino acid sequence comparisons revealed that this receptor was a distant relative of all previously identified 5-HT receptors; we therefore named it 5HT5. When expressed in Cos-7 cells and NIH-3T3 cells, the 5HT5 receptor displayed a high affinity for the serotonergic radioligand [125I]LSD. Surprisingly, its pharmacological profile resembled that of the 5HT1D receptor, which is a 5-HT receptor subtype which has been shown to inhibit adenylate cyclase and which is predominantly expressed in basal ganglia. However, unlike 5HT1D receptors, the 5HT5 receptor did not inhibit adenylate cyclase and its mRNA was not found in basal ganglia. On the contrary, in situ hybridization experiments revealed that the 5HT5 mRNA was expressed predominantly in cerebral cortex, hippocampus, habenula, olfactory bulb and granular layer of the cerebellum. Our results therefore demonstrate that the 5HT1D receptors constitute a heterogeneous family of receptors with distinct intracellular signalling properties and expression patterns.