Citlali Trueta

Synaptic and extrasynaptic secretion of serotonin

AbstractSerotonin is a major modulator of behavior in vertebrates and invertebrates and deficiencies in the serotonergic system account for several behavioral disorders in humans.The small numbers of serotonergic central neurons of vertebrates and invertebrates produce their effects by use of two modes of secretion: from synaptic terminals, acting locally in “hard wired” circuits, and from extrasynaptic axonal and somatodendritic release sites in the absence of postsynaptic targets, producing paracrine effects.In this paper, we review the evidence of synaptic and extrasynaptic release of serotonin and the mechanisms underlying each secretion mode by combining evidence from vertebrates and invertebrates. Particular emphasis is given to somatic secretion of serotonin by central neurons.Most of the mechanisms of serotonin release have been elucidated in cultured synapses made by Retzius neurons from the central nervous system of the leech. Serotonin release from synaptic terminals occurs from clear and dense core vesicles at active zones upon depolarization. In general, synaptic serotonin release is similar to release of acetylcholine in the neuromuscular junction.The soma of Retzius neurons releases serotonin from clusters of dense core vesicles in the absence of active zones. This type of secretion is dependent of the stimulation frequency, on L-type calcium channel activation and on calcium-induced calcium release.The characteristics of somatic secretion of serotonin in Retzius neurons are similar to those of somatic secretion of dopamine and peptides by other neuron types. In general, somatic secretion by neurons is different from transmitter release from clear vesicles at synapses and similar to secretion by excitable endocrine cells.

Extrasynaptic exocytosis and its mechanisms: a source of molecules mediating volume transmission in the nervous system

We review the evidence of exocytosis from extrasynaptic sites in the soma, dendrites, and axonal varicosities of central and peripheral neurons of vertebrates and invertebrates, with emphasis on somatic exocytosis, and how it contributes to signaling in the nervous system. The finding of secretory vesicles in extrasynaptic sites of neurons, the presence of signaling molecules (namely transmitters or peptides) in the extracellular space outside synaptic clefts, and the mismatch between exocytosis sites and the location of receptors for these molecules in neurons and glial cells, have long suggested that in addition to synaptic communication, transmitters are released, and act extrasynaptically. The catalog of these molecules includes low molecular weight transmitters such as monoamines, acetylcholine, glutamate, gama-aminobutiric acid (GABA), adenosine-5-triphosphate (ATP), and a list of peptides including substance P, brain-derived neurotrophic factor (BDNF), and oxytocin. By comparing the mechanisms of extrasynaptic exocytosis of different signaling molecules by various neuron types we show that it is a widespread mechanism for communication in the nervous system that uses certain common mechanisms, which are different from those of synaptic exocytosis but similar to those of exocytosis from excitable endocrine cells. Somatic exocytosis has been measured directly in different neuron types. It starts after high-frequency electrical activity or long experimental depolarizations and may continue for several minutes after the end of stimulation. Activation of L-type calcium channels, calcium release from intracellular stores and vesicle transport towards the plasma membrane couple excitation and exocytosis from small clear or large dense core vesicles in release sites lacking postsynaptic counterparts. The presence of synaptic and extrasynaptic exocytosis endows individual neurons with a wide variety of time- and space-dependent communication possibilities. Extrasynaptic exocytosis may be the major source of signaling molecules producing volume transmission and by doing so may be part of a long duration signaling mode in the nervous system.

Somatic exocytosis of serotonin mediated by L-type calcium channels in cultured leech neurones

We studied somatic exocytosis of serotonin and its mediation by L‐type calcium (Ca2+) channels in cultured Retzius neurones of the leech. Exocytosis was induced by trains of impulses at different frequencies or by depolarisation with 40 mm potassium (K+), and was quantified by use of the fluorescent dye FM 1–43. Stimulation increased the membrane fluorescence and produced a pattern of FM 1–43 fluorescent spots of 1.28 ± 0.01 μm in diameter, provided that Ca2+ was present in the bathing fluid. Individual spots lost their stain during depolarisation with 40 mm K+. Electron micrographs showed clusters of dense core vesicles, some of which were in contact with the cell membrane. Presynaptic structures with clear vesicles were absent from the soma. The number of fluorescent spots per soma, but not their diameter or their fluorescence intensity, depended on the frequency of stimulation. Trains at 1 Hz produced 19.5 ± 5 spots per soma, 77.9 ± 13.9 spots per soma were produced at 10 Hz and 91.5 ± 16.9 spots per soma at 20 Hz. Staining patterns were similar for neurones in culture and in situ. In the presence of the L‐type Ca2+ channel blocker nimodipine (10 μm), a 20 Hz train produced only 22.9 ± 6.4 spots per soma, representing a 75 % reduction compared to control cells (P < 0.05). Subsequent incubation with 10 mm caffeine to induce Ca2+ release from intracellular stores increased the number of spots to 73.22 ± 12.5. Blockers of N‐, P‐, Q‐ or invertebrate Ca2+ channels did not affect somatic exocytosis. Our results suggest that somatic exocytosis by neurones shares common mechanisms with excitable endocrine cells.

Exocytosis of serotonin from the neuronal soma is sustained by a serotonin and calcium-dependent feedback loop

The soma of many neurons releases large amounts of transmitter molecules through an exocytosis process that continues for hundreds of seconds after the end of the triggering stimulus. Transmitters released in this way modulate the activity of neurons, glia and blood vessels over vast volumes of the nervous system. Here we studied how somatic exocytosis is maintained for such long periods in the absence of electrical stimulation and transmembrane Ca2+ entry. Somatic exocytosis of serotonin from dense core vesicles could be triggered by a train of 10 action potentials at 20 Hz in Retzius neurons of the leech. However, the same number of action potentials produced at 1 Hz failed to evoke any exocytosis. The 20-Hz train evoked exocytosis through a sequence of intracellular Ca2+ transients, with each transient having a different origin, timing and intracellular distribution. Upon electrical stimulation, transmembrane Ca2+ entry through L-type channels activated Ca2+-induced Ca2+ release. A resulting fast Ca2+ transient evoked an early exocytosis of serotonin from sparse vesicles resting close to the plasma membrane. This Ca2+ transient also triggered the transport of distant clusters of vesicles toward the plasma membrane. Upon exocytosis, the released serotonin activated autoreceptors coupled to phospholipase C, which in turn produced an intracellular Ca2+ increase in the submembrane shell. This localized Ca2+ increase evoked new exocytosis as the vesicles in the clusters arrived gradually at the plasma membrane. In this way, the extracellular serotonin elevated the intracellular Ca2+ and this Ca2+ evoked more exocytosis. The resulting positive feedback loop maintained exocytosis for the following hundreds of seconds until the last vesicles in the clusters fused. Since somatic exocytosis displays similar kinetics in neurons releasing different types of transmitters, the data presented here contributes to understand the cellular basis of paracrine neurotransmission.

Cycling of Dense Core Vesicles Involved in Somatic Exocytosis of Serotonin by Leech Neurons

We studied the cycling of dense core vesicles producing somatic exocytosis of serotonin. Our experiments were made using electron microscopy and vesicle staining with fluorescent dye FM1-43 in Retzius neurons of the leech, which secrete serotonin from clusters of dense core vesicles in a frequency-dependent manner. Electron micrographs of neurons at rest or after 1 Hz stimulation showed two pools of dense core vesicles. A perinuclear pool near Golgi apparatuses, from which vesicles apparently form, and a peripheral pool with vesicle clusters at a distance from the plasma membrane. By contrast, after 20 Hz electrical stimulation 47% of the vesicle clusters were apposed to the plasma membrane, with some omega exocytosis structures. Dense core and small clear vesicles apparently originating from endocytosis were incorporated in multivesicular bodies. In another series of experiments, neurons were stimulated at 20 Hz while bathed in a solution containing peroxidase. Electron micrographs of these neurons contained gold particles coupled to anti-peroxidase antibodies in dense core vesicles and multivesicular bodies located near the plasma membrane. Cultured neurons depolarized with high potassium in the presence of FM1-43 displayed superficial fluorescent spots, each reflecting a vesicle cluster. A partial bleaching of the spots followed by another depolarization in the presence of FM1-43 produced restaining of some spots, other spots disappeared, some remained without restaining and new spots were formed. Several hours after electrical stimulation the FM1-43 spots accumulated at the center of the somata. This correlated with electron micrographs of multivesicular bodies releasing their contents near Golgi apparatuses. Our results suggest that dense core vesicle cycling related to somatic serotonin release involves two steps: the production of clear vesicles and multivesicular bodies after exocytosis, and the formation of new dense core vesicles in the perinuclear region.

Real-Time Measurements of Synaptic Autoinhibition Produced by Serotonin Release in Cultured Leech Neurons

We studied autoinhibition produced immediately after synaptic serotonin (5-HT) release in identified leech Retzius neurons, cultured singly or forming synapses onto pressure-sensitive neurons. Cultured Retzius neurons are isopotential, thus allowing accurate recordings of synaptic events using intracellular microelectrodes. The effects of autoinhibition on distant neuropilar presynaptic endings were predicted from model simulations. Following action potentials (APs), cultured neurons produced a slow hyperpolarization with a rise time of 85.4 +/- 5.2 ms and a half-decay time of 252 +/- 17.4 ms. These inhibitory postpotentials were reproduced by the iontophoretic application of 5-HT and became depolarizing after inverting the transmembranal chloride gradient by using microelectrodes filled with potassium chloride. The inhibitory postpotentials were reversibly abolished in the absence of extracellular calcium and absent in reserpine-treated neurons, suggesting an autoinhibition due to 5-HT acting on autoreceptors coupled to chloride channels. The autoinhibitory responses increased the membrane conductance and decreased subsequent excitability. Increasing 5-HT release by stimulating with trains of ten pulses at 10 or 30 Hz produced 23 +/- 6 and 47 +/- 2% of AP failures, respectively. These failures were reversibly abolished by the serotonergic antagonist methysergide (140 muM). Moreover, reserpine-treated neurons had only 5 +/- 4% of failures during trains at 10 Hz. This percentage was increased to 35 +/- 4% by iontophoretic application of 5-HT. Increases in AP failures correlated with smaller postsynaptic currents. Model simulations predicted that the autoinhibitory chloride conductance reduces the amplitude of APs arriving at neuropilar presynaptic endings. Altogether, our results suggest that 5-HT autoinhibits its subsequent release by decreasing the excitability of presynaptic endings within the same neuron.

The role of melatonin in the neurodevelopmental etiology of schizophrenia: A study in human olfactory neuronal precursors

Dim light exposure of the mother during pregnancy has been proposed as one of the environmental factors that affect the fetal brain development in schizophrenia. Melatonin circulating levels are regulated by the environmental light/dark cycle. This hormone stimulates neuronal differentiation in the adult brain. However, little is known about its role in the fetal human brain development. Olfactory neuronal precursors (ONPs) are useful for studying the physiopathology of neuropsychiatric diseases because they mimic all the stages of neurodevelopment in culture. Here, we first characterized whether melatonin stimulates neuronal differentiation in cloned ONPs obtained from a healthy control subject (HCS). Then, melatonin effects were evaluated in primary cultures of ONPs derived from a patient diagnosed with schizophrenia (SZ) and an age‐ and gender‐matched HCS. Axonal formation was evidenced morphologically by tau immunostaining and by GSK3β phosphorylated state. Potassium‐evoked secretion was assessed as a functional feature of differentiated neurons. As well, we report the expression of MT1/2 receptors in human ONPs for the first time. Melatonin stimulated axonal formation and ramification in cloned ONPs through a receptor‐mediated mechanism and enhanced the amount and velocity of axonal and somatic secretion. SZ ONPs displayed reduced axogenesis associated with lower levels of pGSK3β and less expression of melatonergic receptors regarding the HCS ONPs. Melatonin counteracted this reduction in SZ cells. Altogether, our results show that melatonin signaling is crucial for functional differentiation of human ONPs, strongly suggesting that a deficit of this indoleamine may lead to an impaired neurodevelopment which has been associated with the etiology of schizophrenia.

The microtubular cytoskeleton of olfactory neurons derived from patients with schizophrenia or with bipolar disorder: Implications for biomarker characterization, neuronal physiology and pharmacological screening

Schizophrenia (SZ) and Bipolar Disorder (BD) are highly inheritable chronic mental disorders with a worldwide prevalence of around 1%. Despite that many efforts had been made to characterize biomarkers in order to allow for biological testing for their diagnoses, these disorders are currently detected and classified only by clinical appraisal based on the Diagnostic and Statistical Manual of Mental Disorders. Olfactory neuroepithelium-derived neuronal precursors have been recently proposed as a model for biomarker characterization. Because of their peripheral localization, they are amenable to collection and suitable for being cultured and propagated in vitro. Olfactory neuroepithelial cells can be obtained by a non-invasive brush-exfoliation technique from neuropsychiatric patients and healthy subjects. Neuronal precursors isolated from these samples undergo in vitro the cytoskeletal reorganization inherent to the neurodevelopment process which has been described as one important feature in the etiology of both diseases. In this paper, we will review the current knowledge on microtubular organization in olfactory neurons of patients with SZ and with BD that may constitute specific cytoskeletal endophenotypes and their relation with alterations in L-type voltage-activated Ca(2+) currents. Finally, the potential usefulness of neuronal precursors for pharmacological screening will be discussed.

Functional uncoupling between intracellular calcium dynamics and secretion in the ?T3-1 gonadotropic cell line

Gonadotropin releasing hormone (GnRH) stimulates both transcription and secretion of the α subunit of the gonadotropins in a Ca2+‐dependent fashion. In this study, we examined the role of Ca2+ as the signal coupling agonist occupancy of GnRH receptors to hormone secretion using the gonadotropic cell line αT3‐1. Treatment of αT3‐1 cells for 60 min with GnRH (0.1–100 nM), veratridine (50 μM) or high K+ (56 mM) was completely ineffective in stimulating secretion. The lack of effect occurred in spite of a robust, specific, and dose‐dependent biphasic [Ca2+]i response consisting of a rapid peak sensitive to thapsigargin (200 nM) followed by a smaller plateau sensitive to the extracellular application of EGTA (5 mM). On the other hand, treatment of αT3‐1 cells with the Ca2+ ionophore ionomycin resulted in a significant dose‐dependent stimulation of secretion and [Ca2+]i responses comparable to those elicited by GnRH. Binding assays revealed the presence of Ins(1,4,5)P3 receptors (Kd = 3.2 nM, Bmax = 50.5 fmol/mg protein) but not ryanodine receptors in αT3‐1 cell membranes. Together, these results show a functional uncoupling between the [Ca2+]i response and secretion in this cell line, suggesting that the increase in [Ca2+]i triggered by GnRH and depolarization may be necessary but not sufficient to stimulate exocytosis. J. Cell. Physiol. 179:347–357, 1999.

Circadian modulation of neuroplasticity by melatonin: a target in the treatment of depression

Mood disorders are a spectrum of neuropsychiatric disorders characterized by changes in the emotional state. In particular, major depressive disorder is expected to have a worldwide prevalence of 20% in 2020, representing a huge socio‐economic burden. Currently used antidepressant drugs have poor efficacy with only 30% of the patients in remission after the first line of treatment. Importantly, mood disorder patients present uncoupling of circadian rhythms. In this regard, melatonin (5‐methoxy‐N‐acetyltryptamine), an indolamine synthesized by the pineal gland during the night, contributes to synchronization of body rhythms with the environmental light/dark cycle. In this review, we describe evidence supporting antidepressant‐like actions of melatonin related to the circadian modulation of neuroplastic changes in the hippocampus. We also present evidence for the role of melatonin receptors and their signalling pathways underlying modulatory effects in neuroplasticity. Finally, we briefly discuss the detrimental consequences of circadian disruption on neuroplasticity and mood disorders, due to the modern human lifestyle. Together, data suggest that melatonins stimulation of neurogenesis and neuronal differentiation is beneficial to patients with mood disorders.