Serotonin is a common thread linking different classes of antidepressants.

Depression pathology remains elusive. The monoamine hypothesis has placed much focus on serotonin, but due to the variable clinical efficacy of monoamine reuptake inhibitors, the community is looking for alternative therapies such as ketamine (neurogenesis theory of antidepressant action). There is evidence that different classes of antidepressants may affect serotonin levels; a notion we test here. We measure hippocampal serotonin in mice with voltammetry and study the effects of acute challenges of escitalopram, fluoxetine, reboxetine, and ketamine. We find that pseudo-equivalent doses of these drugs similarly raise ambient serotonin levels, despite their differing pharmacodynamics because of differences in Uptake 1 and 2, rapid SERT trafficking, and modulation of serotonin by histamine. These antidepressants have different pharmacodynamics but have strikingly similar effects on extracellular serotonin. Our findings suggest that serotonin is a common thread that links clinically effective antidepressants, synergizing different theories of depression (synaptic plasticity, neurogenesis, and the monoamine hypothesis).

Serotonin is a Common Thread Linking Different Classes of Antidepressants.

Depression pathology remains elusive. The monoamine hypothesis has placed much focus on serotonin, but due to the variable clinical efficacy of monoamine reuptake inhibitors, the community is looking for alternative therapies such as ketamine (synaptic plasticity and neurogenesis theory of antidepressant action). There is evidence that different classes of antidepressants may affect serotonin levels; a notion we test here. We measure hippocampal serotonin in mice with voltammetry and study the effects of acute challenges of antidepressants. We find that pseudo-equivalent doses of these drugs similarly raise ambient serotonin levels, despite their differing pharmacodynamics because of differences in Uptake 1 and 2, rapid SERT trafficking and modulation of serotonin by histamine. These antidepressants have different pharmacodynamics but have strikingly similar effects on extracellular serotonin. Our findings suggest that serotonin is a common thread that links clinically effective antidepressants, synergizing different theories of depression (synaptic plasticity, neurogenesis and the monoamine hypothesis).

Voltammetric Approach for Characterizing the Biophysical and Chemical Functionality of Human Induced Pluripotent Stem Cell-Derived Serotonin Neurons.

Depression is quickly becoming one of the world's most pressing public health crises, and there is an urgent need for better diagnostics and therapeutics. Behavioral models in animals and humans have not adequately addressed the diagnosis and treatment of depression, and biomarkers of mental illnesses remain ill-defined. It has been very difficult to identify biomarkers of depression because of in vivo measurement challenges. While our group has made important strides in developing in vivo tools to measure such biomarkers (e.g., serotonin) in mice using voltammetry, these tools cannot be easily applied for depression diagnosis and drug screening in humans due to the inaccessibility of the human brain. In this work, we take a chemical approach, ex vivo, to introduce a human-derived system to investigate brain serotonin. We utilize human induced pluripotent stem cells differentiated into serotonin neurons and establish a new ex vivo model of real-time serotonin neurotransmission measurements. We show that evoked serotonin release responds to stimulation intensity and tryptophan preloading, and that serotonin release and reuptake kinetics resemble those found in vivo in rodents. Finally, after selective serotonin reuptake inhibitor (SSRI) exposure, we find dose-dependent internalization of the serotonin reuptake transporters (a signature of the in vivo response to SSRI). Our new human-derived chemical model has great potential to provide an ex vivo chemical platform as a translational tool for in vivo neuropsychopharmacology.

Activation of the glucocorticoid receptor rapidly triggers calcium-dependent serotonin release in vitro.

AIMS: Glucocorticoids rapidly provoke serotonin (5-HT) release in vivo. We aimed to investigate molecular mechanisms of glucocorticoid receptor (GR)-triggered 5-HT release. METHODS: Employing 1C11 cells to model 5-HT neurotransmission, immunofluorescence and Pearson's Correlation Coefficient were used to analyze colocalization of GR, 5-HT, vesicle membrane protein synaptotagmin 1 and vesicle dye FM4-64FX. FFN511 and FM4-64FX dyes as well as calcium imaging were used to visualize vesicular 5-HT release upon application of GR agonist dexamethasone, GR antagonist mifepristone and voltage-gated calcium channel (VGCC) inhibitors. RESULTS: GR, 5-HT, synaptotagmin 1 and FM4-64FX showed overlapping staining patterns, with Pearson's Correlation Coefficient indicating colocalization. Similarly to potassium chloride, dexamethasone caused a release of FFN511 and uptake of FM4-64FX, indicating vesicular 5-HT release. Mifepristone, calcium depletion and inhibition of L-type VGCC significantly diminished dexamethasone-induced vesicular 5-HT release. CONCLUSIONS: In close proximity to 5-HT releasing sites, activated GR rapidly triggers L-type VGCC-dependent vesicular 5-HT release. These findings provide a better understanding of the interrelationship between glucocorticoids and 5-HT release.

Routine Optical Clearing of 3D-Cell Cultures: Simplicity Forward.

Three-dimensional cell cultures, such as spheroids and organoids, serve as increasingly important models in fundamental and applied research and start to be used for drug screening purposes. Optical tissue clearing procedures are employed to enhance visualization of fluorescence-stained organs, tissues, and three-dimensional cell cultures. To get a more systematic overview about the effects and applicability of optical tissue clearing on three-dimensional cell cultures, we compared six different clearing/embedding protocols on seven types of spheroid- and chip-based three-dimensional cell cultures of approximately 300 µm in size that were stained with nuclear dyes, immunofluorescence, cell trackers, and cyan fluorescent protein. Subsequent whole mount confocal microscopy and semi-automated image analysis were performed to quantify the effects. Quantitative analysis included fluorescence signal intensity and signal-to-noise ratio as a function of z-depth as well as segmentation and counting of nuclei and immunopositive cells. In general, these analyses revealed five key points, which largely confirmed current knowledge and were quantified in this study. First, there was a massive variability of effects of different clearing protocols on sample transparency and shrinkage as well as on dye quenching. Second, all tested clearing protocols worked more efficiently on samples prepared with one cell type than on co-cultures. Third, z-compensation was imperative to minimize variations in signal-to-noise ratio. Fourth, a combination of sample-inherent cell density, sample shrinkage, uniformity of signal-to-noise ratio, and image resolution had a strong impact on data segmentation, cell counts, and relative numbers of immunofluorescence-positive cells. Finally, considering all mentioned aspects and including a wish for simplicity and speed of protocols - in particular, for screening purposes - clearing with 88% Glycerol appeared to be the most promising option amongst the ones tested.

Human dopamine transporter: the first implementation of a combined in silico/in vitro approach revealing the substrate and inhibitor specificities.

Parkinson's disease (PD) is characterized by the loss of dopamine-generating neurons in the substantia nigra and corpus striatum. Current treatments alleviate PD symptoms rather than exerting neuroprotective effect on dopaminergic neurons. New drugs targeting the dopaminergic neurons by specific uptake through the human dopamine transporter (hDAT) could represent a viable strategy for establishing selective neuroprotection. Molecules able to increase the bioactive amount of extracellular dopamine, thereby enhancing and compensating a loss of dopaminergic neurotransmission, and to exert neuroprotective response because of their accumulation in the cytoplasm, are required. By means of homology modeling, molecular docking, and molecular dynamics simulations, we have generated 3D structure models of hDAT in complex with substrate and inhibitors. Our results clearly reveal differences in binding affinity of these compounds to the hDAT in the open and closed conformations, critical for future drug design. The established in silico approach allowed the identification of promising substrate compounds that were subsequently analyzed for their efficiency in inhibiting hDAT-dependent fluorescent substrate uptake, through in vitro live cell imaging experiments. Taken together, our work presents the first implementation of a combined in silico/in vitro approach enabling the selection of promising dopaminergic neuron-specific substrates.

Methyl-4-phenylpyridinium (MPP+) differentially affects monoamine release and re-uptake in murine embryonic stem cell-derived dopaminergic and serotonergic neurons.

1-Methyl-4-phenyl-1,2,5,6-tetrahydropyridine (MPTP) is known to selectively damage dopaminergic (DA) cells in the substantia nigra and to produce symptoms which are alike to those observed in Parkinson's disease (PD). Based on the similarity between MPTP-induced neurotoxicity and PD-related neuropathology, application of MPTP or its metabolite methyl-4-phenylpyridinium (MPP+) was successfully established in experimental rodent models to study PD-related neurodegenerative events. MPP+ is taken up by the dopamine transporter (DAT) into DA neurons where it exerts its neurotoxic action on mitochondria by affecting complex I of the respiratory chain. MPP+ is also a high affinity substrate for the serotonin transporter (SERT), however little is known about possible toxic effects of MPP+ on serotonergic (5-HT) neurons. In order to compare cell type-specific effects of MPP+ treatment, we have differentiated mouse embryonic stem (ES) cells into DA and 5-HT neurons and studied the impact of MPP+ treatment on both types of monoaminergic neurons in vitro. MPP+ treatment impacts on mitochondrial membrane potential in DA as well as 5-HT ES cell-derived neurons. Although mitochondria metabolisms are similarly affected, synaptic vesicle cycling is only impaired in DA ES cell-derived neurons. Most importantly we show that MPP+ induces DAT externalization in DA neurons, but internalization of SERT in 5-HT neurons. This diverse MPP+-induced transporter trafficking is reflected by elevated substrate uptake in DA neurons, and diminished substrate uptake in 5-HT neurons. In summary, our experimental data point toward differential effects of MPP+ intoxication on neurotransmitter release and re-uptake in different types of monoaminergic neurons.

The allosteric citalopram binding site differentially interferes with neuronal firing rate and SERT trafficking in serotonergic neurons.

Citalopram is a clinically applied selective serotonin re-uptake inhibitor for antidepressant pharmacotherapy. It consists of two enantiomers, S-citalopram (escitalopram) and R-citalopram, of which escitalopram exerts the antidepressant therapeutic effect and has been shown to be one of the most efficient antidepressants, while R-citalopram antagonizes escitalopram via an unknown molecular mechanism that may depend on binding to a low-affinity allosteric binding site of the serotonin transporter. However, the precise mechanism of antidepressant regulation of the serotonin transporter by citalopram enantiomers still remains elusive. Here we investigate escitalopram׳s acute effect on (1) serotonergic neuronal firing in transgenic mice that express the human serotonin transporter without and with a mutation that disables the allosteric binding site, and (2) regulation of the serotonin transporter׳s cell surface localization in stem cell-derived serotonergic neurons. Our results demonstrate that escitalopram inhibited neuronal firing less potently in the mouse line featuring a mutation that abolishes the function of the allosteric binding site and induced serotonin transporter internalization independently of the allosteric binding site mechanism. Furthermore, citalopram enantiomers dose-dependently induced serotonin transporter internalization. In conclusion, this study provides new insight into antidepressant effects exerted by citalopram enantiomers in presence and absence of a functional allosteric binding site.

Protein Kinases Alter the Allosteric Modulation of the Serotonin Transporter In Vivo and In Vitro.

AIM: Studies using S- and R-enantiomers of the SSRI citalopram have shown that R-citalopram exerts an antagonistic effect on the efficacy of the antidepressant S-citalopram (escitalopram) through an interaction at an allosteric modulator site on the serotonin transporter (SERT). Here, we show that protein kinase signaling systems are involved in the allosteric modulation of the SERT in vivo and in vitro. METHODS: We assessed the effects of nonspecific protein kinase inhibitor staurosporine in the action of escitalopram and/or R-citalopram using electrophysiological and behavioral assays in rats and cell surface SERT expression measures in serotoninergic cells. RESULTS: Acute administration of R-citalopram counteracted the escitalopram-induced suppression of the serotonin (5-HT) neuronal firing activity and increase of the head twitches number following L-5-hydroxytryptophan injection. Importantly, these counteracting effects of R-citalopram were abolished by prior systemic administration of staurosporine. Interestingly, the preventing effect of staurosporine on 5-HT neuronal firing activity was abolished by direct activation of protein kinase C with phorbol 12-myristate 13-acetate. Finally, in vitro, quantification of the amount of cell surface-expressed SERT molecules revealed that R-citalopram prevented escitalopram-induced SERT internalization that was completely altered by staurosporine. CONCLUSION: Taken together, these results highlight for the first time an involvement of protein kinases in the allosteric modulation of SERT function.

Outside the brain: an inside view on transgenic animal and stem cell-based models to examine neuronal serotonin-dependent regulation of HPA axis-controlled events during development and adult stages.

Recently, Trista North and colleagues showed that neuronal synthesis of serotonin is an essential key process for embryonic hematopoietic stem (HPS) cell production in zebrafish. Using their experimental design, they were able to show that neuronal serotonin activates the stress-responsive hypothalamic-pituitary-adrenal (HPA) axis and glucocorticoid receptor activity which in turn induces HPS cell formation. In our perspective, we give a short overview on established experimental approaches for serotonergic neurotransmission in vivo and in vitro and their potential to address putative contributions of serotonergic neurotransmission to physiological processes beyond the central nervous systems (CNS). We briefly introduce common features of brain serotonin-depleted, tryptophan hydroxylase-2 knockout mice, which can be applied to investigate the contribution of brain-derived serotonin to developmental and adult physiological processes outside the CNS. These models allow to analyzing gender-specific, HPA axis-dependent processes in female and male knockout mice during developmental and adult stages. We also highlight the application of human and mouse stem cell-derived serotonergic neurons as an independent research model as well as complementary experimental approach to transgenic animal models. In case of human serotonergic neurotransmission, human in vitro-generated neurons present a very promising and highly valuable experimental approach to address characteristics of human neuronal serotonin signaling on a molecular and cellular level. The combination of transgenic animal models and newly established stem cell technologies will provide powerful research platforms, which will help to answer yet unsolved mysteries of serotonergic neurotransmission.

Differential Uptake Mechanisms of Fluorescent Substrates into Stem-Cell-Derived Serotonergic Neurons.

The actions of the neurotransmitters serotonin, dopamine, and norepinephrine are partly terminated by diffusion and in part by their uptake into neurons via the selective, high-affinity transporters for serotonin (SERT), dopamine (DAT), and norepinephrine (NET), respectively. There is also growing evidence that all three monoamines are taken up into neurons by low-affinity, high-capacity organic cation transporters (OCT) and the plasma membrane monoamine transporter (PMAT). Pharmacological characterization of these low-affinity recombinant transporter proteins in heterologous expression systems has revealed that they are not antagonized by classical inhibitors of SERT, DAT, or NET but that decynium-22 (D22) antagonizes OCT3 and PMAT, whereas corticosterone and progesterone selectively inhibit OCT3. Here, we show that SERT, PMAT, and OCT3, but not OCT1 and OCT2, are coexpressed in murine stem cell-derived serotonergic neurons. Using selective antagonists, we provide evidence that uptake of the fluorescent substrates FFN511, ASP+, and 5-HT into stem cell-derived serotonergic neurons is mediated differentially by these transporters and also involves an as yet unknown transport mechanism.

Serotonin stimulates secretion of exosomes from microglia cells.

Microglia are resident immune cells in the brain and exert important functions in the regulation of inflammatory processes during infection or cellular damage. Upon activation, microglia undergo complex morphological and functional transitions, including increased motility, phagocytosis and cytokine secretion. Recent findings indicate that exosomes, small vesicles that derive from fusion of multivesicular bodies with the plasma membrane, are involved in secretion of certain cytokines. The presence of specific receptors on the surface of microglia suggests communication with neurons by neurotransmitters. Here, we demonstrate expression of serotonin receptors, including 5-HT2a,b and 5-HT4 in microglial cells and their functional involvement in the modulation of exosome release by serotonin. Our data demonstrate the involvement of cAMP and Ca(2+) dependent signaling pathways in the regulation of exosome secretion. Co-culture of microglia with embryonic stem cell-derived serotonergic neurons further demonstrated functional signaling between neurons and microglia. Together, these data provide evidence for neurotransmitter-dependent signaling pathways in microglial cells that regulate exosome release.

Adult AMPA GLUA1 receptor subunit loss in 5-HT neurons results in a specific anxiety-phenotype with evidence for dysregulation of 5-HT neuronal activity.

Both the glutamatergic and serotonergic (5-HT) systems are implicated in the modulation of mood and anxiety. Descending cortical glutamatergic neurons regulate 5-HT neuronal activity in the midbrain raphe nuclei through α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and N-methyl-D-aspartate (NMDA) receptors. To analyze the functional role of GLUA1-containing AMPA receptors in serotonergic neurons, we used the Cre-ERT2/loxP-system for the conditional inactivation of the GLUA1-encoding Gria1 gene selectively in 5-HT neurons of adult mice. These Gria1(5-HT-/-) mice exhibited a distinct anxiety phenotype but showed no alterations in locomotion, depression-like behavior, or learning and memory. Increased anxiety-related behavior was associated with significant decreases in tryptophan hydroxylase 2 (TPH2) expression and activity, and subsequent reductions in tissue levels of 5-HT, its metabolite 5-hydroxyindoleacetic acid (5-HIAA), and norepinephrine in the raphe nuclei. However, TPH2 expression and activity as well as monoamine levels were unchanged in the projection areas of 5-HT neurons. Extracellular electrophysiological recordings of 5-HT neurons revealed that, while α1-adrenoceptor-mediated excitation was unchanged, excitatory responses to AMPA were enhanced and the 5-HT1A autoreceptor-mediated inhibitory response to 5-HT was attenuated in Gria1(5-HT-/-) mice. Our data show that a loss of GLUA1 protein in 5-HT neurons enhances AMPA receptor function and leads to multiple local molecular and neurochemical changes in the raphe nuclei that dysregulate 5-HT neuronal activity and induce anxiety-like behavior.

Visualization of neurotransmitter uptake and release in serotonergic neurons.

BACKGROUND: To study serotonergic volume neurotransmission at cellular level it needs to investigate neurotransmitter release and re-uptake sites in serotonergic neurons. However, due to the low number of cell bodies in the raphe nuclei and their widely branching neurites, serotonergic neuronal cultures are not accessible ex vivo. NEW METHOD: We have combined differentiation protocols for the generation of stem cell-derived serotonergic neurons together with confocal microscopy to study the uptake and release of fluorescent substrates known to be selectively taken up by monoaminergic neurons. These substances include: (i) 4-(4-(dimethylamino)styryl)-N-methylpyridiunium (ASP+), an analog of the neurotoxin MPP+; (ii) the fluorescent false neurotransmitter (FFN511); and (iii) serotonin (5-hydroxytryptamine; 5-HT) itself, which is known to emit fluorescence upon excitation at 320-460nm. RESULT: ASP+ is taken up into living serotonergic neurons through the serotonin transporter, but not accumulated into synaptic vesicles; FFN511 diffuses in a SERT-independent way into serotonergic neurons and accumulated into synaptic vesicles. KCl-induced release of FFN511 and 5-HT can be visualized and quantified in living serotonergic neurons. COMPARISON WITH EXISTING METHODS: Application of ASP+ so far has been used to investigate substrate/transporter interactions; studies on FFN511 uptake and release have only been performed in dopaminergic neurons; quantitative studies on uptake and release of 5-HT in living serotonergic neurons have not been reported yet. CONCLUSION: The differentiation protocols for the generation of stem cell-derived serotonergic neurons combined with the application of different fluorescent dyes allow to quantify neurotransmitter uptake and release in living serotonergic neurons in vitro.

Nongenomic, glucocorticoid receptor-mediated regulation of serotonin transporter cell surface expression in embryonic stem cell derived serotonergic neurons.

Depressive disorders have been linked to the combined dysregulation of the hypothalamus-pituitary-adrenal (HPA)-axis and the serotonergic system. The HPA-axis and serotonergic (5-HT) neurons exert reciprocal regulatory actions. It has been reported that glucocorticoid-glucocorticoid receptor (GR) signaling influences serotonin transporter (5-HTT) transcription but data also points to the fact that 5-HTT expression is regulated nongenomically via redistribution of 5-HTT from the cell surface into intracellular compartments. In order to analyze the acute effects of glucocorticoids on 5-HTT cell surface localization we differentiated serotonergic neurons from mouse embryonic stem (ES) cells derived from the C57BL/6N blastocysts. These postmitotic 5-HT neurons express all relevant serotonergic markers following the application of a growth factor-based differentiation protocol. Increasing concentrations of the GR agonist dexamethasone (Dex) resulted in enhanced, dose-dependent 5-HTT cell surface localization in the presence of the protein synthesis inhibitor cycloheximide already 1h after incubation. Inhibition of GR function by the specific GR-antagonist mifepristone abolished the increase in 5-HTT cell surface localization. Hence, our data account for a nongenomic upregulation of 5-HTT cell surface expression by glucocorticoid-GR interaction which likely constitutes a rapid physiological response to increased levels of glucocorticoids as seen during stress. Taken together, we provide a cellular model to analyze and dissect glucocorticoid-5HTT interactions on a molecular level that corresponds to in vivo animal models using C57BL/6N mice.

Electroconvulsive therapy induces neurogenesis in frontal rat brain areas.

Electroconvulsive therapy (ECT) is an effective therapy for several psychiatric disorders, including severe major depression, mania and certain forms of schizophrenia. It had been proposed that ECT acts by modulating local plasticity via the stimulation of neurogenesis. In fact, among antidepressant therapies, ECT is the most robust enhancer of neurogenesis in the hippocampus of rodents and non-human primates. The existence of ECT-triggered neurogenesis in other brain areas, particularly in those adjacent to the other main locus of neurogenesis, the subventricular zone (SVZ), had so far remained unknown. Here we show that ECT also strongly enhances neurogenesis in frontal brain areas, especially in the rostro-medial striatum, generating specific, small-size calretinin-positive interneurons. We provide here the first evidence that ECT stimulates neurogenesis in areas outside the hippocampus. Our data may open research possibilities that focus on the plastic changes induced by ECT in frontal limbic circuitry.

Differential expression of neuronal dopamine and serotonin transporters DAT and SERT in megakaryocytes and platelets generated from human MEG-01 megakaryoblasts.

In the central nervous system, serotonergic and dopaminergic signaling is terminated by the activity of specialized transporter proteins for serotonin (SERT) and dopamine (DAT). These transporter proteins are found both on the cell surface and in intracellular transport vesicles. Trafficking between these subcellular domains regulates the efficiency of removal of extracellular neurotransmitters and hence the efficacy of neuronal signaling. Therefore, it is of high interest to gain more insight into the regulatory mechanisms of the human DAT and SERT cell surface expression in their natural surroundings, i.e., in human cells. Because it is not possible to cultivate human neuronal cells expressing these transporter proteins, there is a need to find other human cells expressing these neuronal proteins. Here, we have investigated the expression of human SERT and DAT on developing megakaryocytes and platelet-like particles derived from the megakaryocyte progenitor cell line MEG-01 upon differentiation by valproic acid (VPA) and all-trans retinoic acid (ATRA). Our results show that MEG-01 cells express SERT and DAT and that VPA and ATRA induce a significant increase of transporter expression on developing megakaryocytes and platelets. As compared to ATRA, VPA more efficiently induced SERT expression but not DAT expression. Comparable to naïve platelets and neurons, SERT was localized to both the cell surface and intracellular compartments. Hence, VPA and ATRA-treated MEG-01 cells provide a model well-suited to studying neuronal monoamine transporter expression, not only during transcription and translation but also with respect to protein trafficking to and from the cell surface.