Antidepressant Low Doses of Ketamine and Melatonin in Combination Produce Additive Neurogenesis in Human Olfactory Neuronal Precursors.

Melatonin (MEL), an indolamine with diverse functions in the brain, has been shown to produce antidepressant-like effects, presumably through stimulating neurogenesis. We recently showed that the combination of MEL with ketamine (KET), an NMDA receptor antagonist, has robust antidepressant-like effects in mice, at doses that, by themselves, are non-effective and have no adverse effects. Here, we show that the KET/MEL combination increases neurogenesis in a clone derived from human olfactory neuronal precursors, a translational pre-clinical model for effects in the human CNS. Neurogenesis was assessed by the formation of cell clusters > 50 µm in diameter, positively stained for nestin, doublecortin, BrdU and Ki67, markers of progenitor cells, neurogenesis, and proliferation. FGF, EGF and BDNF growth factors increased the number of cell clusters in cultured, cloned ONPs. Similarly, KET or MEL increased the number of clusters in a dose-dependent manner. The KET/MEL combination further increased the formation of clusters, with a maximal effect obtained after a triple administration schedule. Our results show that the combination of KET/MEL, at subeffective doses that do not produce adverse effects, stimulate neurogenesis in human neuronal precursors. Moreover, the mechanism by which the combination elicits neurogenesis is meditated by melatonin receptors, CaM Kinase II and CaM antagonism. This could have clinical advantages for the fast treatment of depression.

Specific Localization of an Auto-inhibition Mechanism at Presynaptic Terminals of Identified Serotonergic Neurons.

Auto-regulation mechanisms in serotonergic neurons regulate their electrical activity and secretion. Since these neurons release serotonin from different structural compartments - including presynaptic terminals, soma, axons and dendrites - through different mechanisms, autoregulation mechanisms are also likely to be different at each compartment. Here we show that a chloride-mediated auto-inhibitory mechanism is exclusively localized at presynaptic terminals, but not at extrasynaptic release sites, in serotonergic Retzius neurons of the leech. An auto-inhibition response was observed immediately after intracellular stimulation with an electrode placed in the soma, in neurons that were isolated and cultured retaining an axonal stump, where presynaptic terminals are formed near the soma, but not in somata isolated without axon, where no synaptic terminals are formed, nor in neurons in the nerve ganglion, where terminals are electrotonically distant from the soma. Furthermore, no auto-inhibition response was detected in either condition during the longer time course of somatic secretion. This shows that the auto-inhibition effects are unique to nerve terminals. We further determined that serotonin released from peri-synaptic dense-core vesicles contributes to auto-inhibition in the terminals, since blockade of L-type calcium channels, which are required to stimulate extrasynaptic but not synaptic release, decreased the amplitude of the auto-inhibition response. Our results show that the auto-regulation mechanism at presynaptic terminals is unique and different from that described in the soma of these neurons, further highlighting the differences in the mechanisms regulating serotonin release from different neuronal compartments, which expand the possibilities of a single neuron to perform multiple functions in the nervous system.

Purinergic Signaling Pathway in Human Olfactory Neuronal Precursor Cells.

Extracellular ATP and trophic factors released by exocytosis modulate in vivo proliferation, migration, and differentiation in multipotent stem cells (MpSC); however, the purinoceptors mediating this signaling remain uncharacterized in stem cells derived from the human olfactory epithelium (hOE). Our aim was to determine the purinergic pathway in isolated human olfactory neuronal precursor cells (hONPC) that exhibit MpSC features. Cloning by limiting dilution from a hOE heterogeneous primary culture was performed to obtain a culture predominantly constituted by hONPC. Effectiveness of cloning to isolate MpSC-like precursors was corroborated through immunodetection of specific protein markers and by functional criteria such as self-renewal, proliferation capability, and excitability of differentiated progeny. P2 receptor expression in hONPC was determined by Western blot, and the role of these purinoceptors in the ATP-induced exocytosis and changes in cytosolic Ca2+ ([Ca2+]i) were evaluated using the fluorescent indicators FM1-43 and Fura-2 AM, respectively. The clonal culture was enriched with SOX2 and OCT3/4 transcription factors; additionally, the proportion of nestin-immunopositive cells, the proliferation capability, and functionality of differentiated progeny remained unaltered through the long-term clonal culture. hONPC expressed P2X receptor subtypes 1, 3-5, and 7, as well as P2Y2, 4, 6, and 11; ATP induced both exocytosis and a transient [Ca2+]i increase predominantly by activation of metabotropic P2Y receptors. Results demonstrated for the first time that ex vivo-expressed functional P2 receptors in MpSC-like hONPC regulate exocytosis and Ca2+ signaling. This purinergic-triggered release of biochemical messengers to the extracellular milieu might be involved in the paracrine signaling among hOE cells.

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 melatonin's stimulation of neurogenesis and neuronal differentiation is beneficial to patients with mood disorders. LINKED ARTICLES: This article is part of a themed section on Recent Developments in Research of Melatonin and its Potential Therapeutic Applications. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.16/issuetoc.

Abnormally Increased Secretion in Olfactory Neuronal Precursors from a Case of Schizophrenia Is Modulated by Melatonin: A Pilot Study.

The alterations that underlie the pathophysiology of schizophrenia (SCZ) include the dysregulation of structural and functional properties of neurons. Among these, the secretion of neurotransmitters and hormones, which plays a key role for neuronal communication and development, is altered. Neuronal precursors from the human olfactory epithelium have been recently characterized as a reliable model for studying the etiopathogenesis of neuropsychiatric diseases. Our previous work has shown that melatonin enhances the development of morphological and functional features of cloned olfactory neuronal precursors (ONPs) from a healthy subject. In this work we found that primary cultures of ONPs obtained from a schizophrenic patient display an increased potassium-evoked secretion, when compared with ONPs from an age- and gender-matched healthy control subject (HCS). Secretion was evaluated by FM1-43 fluorescence cumulative changes in response to depolarization. Interestingly, a 12 h-melatonin treatment modulated the abnormally increased secretion in SCZ ONPs and brought it to levels similar to those found in the HCS ONPs. Our results suggest that the actin cytoskeleton might be a target for melatonin effects, since it induces the thickening of actin microfilament bundles. Further research will address the mechanisms by which melatonin modulates neurochemical secretion from ONPs.

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 GSK3ß 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 pGSK3ß 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.

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 Ca(2+) 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 Ca(2+) transients, with each transient having a different origin, timing and intracellular distribution. Upon electrical stimulation, transmembrane Ca(2+) entry through L-type channels activated Ca(2+)-induced Ca(2+) release. A resulting fast Ca(2+) transient evoked an early exocytosis of serotonin from sparse vesicles resting close to the plasma membrane. This Ca(2+) 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 Ca(2+) increase in the submembrane shell. This localized Ca(2+) 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 Ca(2+) and this Ca(2+) 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.

Regulación de la liberación de serotonina en distintos compartimientos neuronales / Regulation of serotonin release from different neuronal compartments

Serotonin is fundamental for the modulation of social behavior, emotions and a wide variety of physiological functions. The functions of serotonergic systems have been highly conserved along the evolutionary scale and in general small numbers of neurons innervate virtually all the nervous system, and exert multiple effects depending on the site of release. Synaptic pools produce fast and local effects, while extrasynaptic pools in the soma, dendrites, axons and the periphery of synapses produce diffuse effects, characteristic of mood modulation. Serotonin release from synaptic terminals is produced by exocytosis of small clear vesicles and is activated by single or low-frequency impulses, while increases in the stimulation frequency produce synaptic facilitation and depression. In contrast, release from the soma is produced by exocytosis of dense-cored vesicles and requires stimulation at high frequencies, the activation of L-type calcium channels and calcium-induced calcium release from intracellular stores. Serotonin released from the presynaptic terminals immediately activates auto-receptors in the same terminals, locally decreasing the subsequent excitability, firing frequency and release. Differential regulation of serotonin release in different cell compartments allows the same neuron to produce different types of effects depending on the firing rate.

La serotonina es fundamental para la modulación de la conducta social, las emociones y una gran cantidad de funciones fisiológicas. La función de los sistemas serotonérgicos se ha conservado a lo largo de la escala evolutiva y, en general, números pequeños de neuronas inervan prácticamente todo el Sistema Nervioso. Estas neuronas son capaces de ejercer múltiples efectos, dependiendo de si liberan serotonina de pozas sinápticas, que ejercen efectos rápidos y locales o de pozas extrasinápticas en la periferia de las sinapsis, el axon, el cuerpo celular o las dendritas, con lo que se producen efectos lentos y difusos, característicos de los estados de ánimo. La liberación de serotonina en las terminales sinápticas es producida por la exocitosis de vesículas claras pequeñas y se activa con impulsos sencillos o a baja frecuencia. La estimulación con trenes de impulsos a frecuencias crecientes produce facilitación y depresión sináptica. En contraste, la liberación a partir del soma es producida por la exocitosis de vesículas de núcleo denso y requiere de la estimulación a frecuencias altas, la activación de canales de calcio tipo L y de la liberación de calcio de los depositos intracelulares. La serotonina liberada por las terminales sinápticas activa de manera inmediata autorreceptores en las propias terminales que la liberaron, disminuyendo la excitabilidad subsiguiente y, por lo tanto, la frecuencia de disparo y la liberación de manera localizada. La regulación diferencial de la liberación en cada compartimiento celular permite que la misma neurona produzca diferentes tipos de efectos dependiendo de la frecuencia de disparo.

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.