Gerardo Ramírez-Rodríguez

Melatonin Modulates Cell Survival of New Neurons in the Hippocampus of Adult Mice

Regulation of adult hippocampal neurogenesis is influenced by circadian rhythm, affected by the manipulation of sleep, and is disturbed in animal models of affective disorders. These observations and the link between dysregulation of the circadian production of melatonin and neuropsychiatric disorders prompted us to investigate the potential role of melatonin in controlling adult hippocampal neurogenesis. In vitro, melatonin increased the number of new neurons derived from adult hippocampal neural precursor cells in vitro by promoting cell survival. This effect was partially dependent on the activation of melatonin receptors as it could be blocked by the application of receptor antagonist luzindole. There was no effect of melatonin on cell proliferation. Similarly, in the dentate gyrus of adult C57BL/6 mice in vivo, exogenous melatonin (8 mg/kg) also increased the survival of neuronal progenitor cells and post-mitotic immature neurons. Melatonin did not affect precursor cell proliferation in vivo and also did not influence neuronal and glial cell maturation. Moreover, melatonin showed antidepressant-like effects in the Porsolt forced swim test. These results indicate that melatonin through its receptor can modulate the survival of newborn neurons in the adult hippocampus, making it the first known exogenously applicable substance with such specificity

Cannabinoid receptor CB1 mediates baseline and activity-induced survival of new neurons in adult hippocampal neurogenesis

BackgroundAdult neurogenesis is a particular example of brain plasticity that is partially modulated by the endocannabinoid system. Whereas the impact of synthetic cannabinoids on the neuronal progenitor cells has been described, there has been lack of information about the action of plant-derived extracts on neurogenesis. Therefore we here focused on the effects of Δ9-tetrahydrocannabinol (THC) and Cannabidiol (CBD) fed to female C57Bl/6 and Nestin-GFP-reporter mice on proliferation and maturation of neuronal progenitor cells and spatial learning performance. In addition we used cannabinoid receptor 1 (CB1) deficient mice and treatment with CB1 antagonist AM251 in Nestin-GFP-reporter mice to investigate the role of the CB1 receptor in adult neurogenesis in detail.ResultsTHC and CBD differed in their effects on spatial learning and adult neurogenesis. CBD did not impair learning but increased adult neurogenesis, whereas THC reduced learning without affecting adult neurogenesis. We found the neurogenic effect of CBD to be dependent on the CB1 receptor, which is expressed over the whole dentate gyrus. Similarly, the neurogenic effect of environmental enrichment and voluntary wheel running depends on the presence of the CB1 receptor. We found that in the absence of CB1 receptors, cell proliferation was increased and neuronal differentiation reduced, which could be related to CB1 receptor mediated signaling in Doublecortin (DCX)-expressing intermediate progenitor cells.ConclusionCB1 affected the stages of adult neurogenesis that involve intermediate highly proliferative progenitor cells and the survival and maturation of new neurons. The pro-neurogenic effects of CBD might explain some of the positive therapeutic features of CBD-based compounds.

Chronic treatment with melatonin stimulates dendrite maturation and complexity in adult hippocampal neurogenesis of mice

Abstract:  In the course of adult hippocampal neurogenesis, the postmitotic maturation and survival phase is associated with dendrite maturation. Melatonin modulates the survival of new neurons with relative specificity. During this phase, the new neurons express microtubule‐associated protein doublecortin (DCX). Here, we show that the entire population of cells expressing DCX is increased after 14 days of treatment with melatonin. As melatonin also affects microtubule polymerization which is important for neuritogenesis and dendritogenesis, we studied the consequences of chronic melatonin administration on dendrite maturation of DCX‐positive cells. Treatment with melatonin increased the number of DCX‐positive immature neurons with more complex dendrites. Sholl analysis revealed that melatonin treatment lead to greater complexity of the dendritic tree. In addition, melatonin increased the total volume of the granular cell layer. Besides its survival‐promoting effect, melatonin thus also increases dendritic maturation in adult neurogenesis. This might open the opportunity of using melatonin as an adjuvant in attempts to extrinsically stimulate adult hippocampal neurogenesis in neuropsychiatric disease, dementia or cognitive ageing.

Melatonin stimulates calmodulin phosphorylation by protein kinase C

Abstract:  Calmodulin (CaM)‐dependent processes can be modulated by the availability of Ca+2, the subcellular distribution of both CaM and its target proteins, CaM antagonism, and post‐translational modifications such as CaM phosphorylation. Melatonin, the pineal secretory product synthesized during the dark phase of the photoperiod is an endogenous CaM antagonist. This indolamine causes CaM subcellular redistribution in epithelial MDCK and MCF‐7 cells, and selectively activates protein kinase C alpha (PKC α) in neuronal N1E‐115 cells. In the present work we have characterized the phosphorylation of CaM mediated by PKC α and its stimulation by melatonin in an in vitro reconstituted enzyme system. Additionally, the participation of MAPK and ERKs, downstream kinases of the PKC signaling pathway, was explored utilizing MDCK cell extracts as source of these kinases. Phosphorylation of CaM was characterized in the whole cells by MDCK cell metabolic labeling with [32P]‐orthoposhospate, and CaM separation by sodium dodecyl sulphate‐polyacrylamide gel electrophoresis, as well as by immunocolocalization of phosphorylated threonine/serine residues and CaM in cultured cells incubated with melatonin. Our results show that melatonin increased CaM phosphorylation by PKC α with an EC50 of 10−8 m in the presence of the phorbol ester, phorbol‐12‐myristate‐13‐acetate (PMA) in the in vitro reconstituted enzyme system. An increase in phosphorylated CaM was also observed in cells cultured with melatonin, or PMA for 2 hr, while, PKC, MAPK, or ERK inhibitors abolished CaM phosphorylation elicited by melatonin in MDCK cell extracts. Our data show that melatonin can stimulate phosphorylation of CaM by PKC α in the in vitro reconstituted system and suggest that in MDCK cells this phosphorylation is accomplished by PKC. Modification of CaM by melatonin can be another route to inhibit CaM interaction with its target enzymes.

Synaptic network activity induces neuronal differentiation of adult hippocampal precursor cells through BDNF signaling

Adult hippocampal neurogenesis is regulated by activity. But how do neural precursor cells in the hippocampus respond to surrounding network activity and translate increased neural activity into a developmental program? Here we show that long-term potentiation (LTP)-like synaptic activity within a cellular network of mature hippocampal neurons promotes neuronal differentiation of newly generated cells. In co-cultures of precursor cells with primary hippocampal neurons, LTP-like synaptic plasticity induced by addition of glycine in Mg2+-free media for 5 min, produced synchronous network activity and subsequently increased synaptic strength between neurons. Furthermore, this synchronous network activity led to a significant increase in neuronal differentiation from the co-cultured neural precursor cells. When applied directly to precursor cells, glycine- and Mg2+-free solution did not induce neuronal differentiation. Synaptic plasticity-induced neuronal differentiation of precursor cells was observed in the presence of GABAergic neurotransmission blockers but was dependent on NMDA-mediated Ca2+ influx. Most importantly, neuronal differentiation required the release of brain-derived neurotrophic factor (BDNF) from the underlying substrate hippocampal neurons as well as TrkB receptor phosphorylation in precursor cells. This suggests that activity-dependent stem cell differentiation within the hippocampal network is mediated via synaptically evoked BDNF signaling.

ROCK‐regulated cytoskeletal dynamics participate in the inhibitory effect of melatonin on cancer cell migration

Abstract:  Cell movement is generated by a driving force provided by dynamic cytoskeletal organization. Two main cytoskeletal‐dependent features, essential for migration, are the highly cell polarized structure and focal adhesion complexes. Cell migration and substrate anchorage are finely regulated by external signaling exerted by growth factors and hormones. In particular, the serine threonine kinase activated by the small GTPase Rho, the Rho‐associated protein kinase (ROCK), participate in both processes through regulation of actin rearrangements in lamellipodia, filopodia, ruffles, and stress fibers. Melatonin, the main product secreted by the pineal gland has oncostatic properties. In MCF‐7 cells, 1 nm melatonin reduces migration and invasiveness through increased expression of two cell surface adhesion proteins, E‐cadherin and β1‐integrin. In this work, we studied the microfilament and microtubule rearrangements elicited by melatonin in migrating leader MCF‐7 cells by a wound‐healing assay. Additionally, cell anchorage was estimated by quantification of focal adhesions in MCF‐7 cells cultured with melatonin. ROCK participation in the indole effects on anchorage and migration was explored by inhibition of the kinase activity with the specific inhibitor of ROCK, the Y‐27632 compound. The results indicate that ROCK participates in the melatonin inhibitory effects on cell migration by changing cytoskeletal organization of leader MCF‐7 cells. Also, they indicated that indole increased the number of focal contacts through ROCK. These results support the notion that melatonin inhibits cancer cell invasion and metastasis formation via ROCK‐regulated microfilament and microtubule organization that converge in a migration/anchorage switch.

Melatonin induces neuritogenesis at early stages in N1E-115 cells through actin rearrangements via activation of protein kinase C and Rho-associated kinase

Abstract:  Melatonin increases neurite formation in N1E‐115 cells through microtubule enlargement elicited by calmodulin antagonism and vimentin intermediate filament reorganization caused by protein kinase C (PKC) activation. Microfilament rearrangement is also a necessary process in growth cone formation during neurite outgrowth. In this work, we studied the effect of melatonin on microfilament rearrangements present at early stages of neurite formation and the possible participation of PKC and the Rho‐associated kinase (ROCK), which is a downstream kinase in the PKC signaling pathway. The results showed that 1 nm melatonin increased both the number of cells with filopodia and with long neurites. Similar results were obtained with the PKC activator phorbol 12‐myristate 13‐acetate (PMA). Both melatonin and PMA increased the quantity of filamentous actin. In contrast, the PKC inhibitor bisindolylmaleimide abolished microfilament organization elicited by either melatonin or PMA, while the Rho inhibitor C3, or the ROCK inhibitor Y27632, abolished the bipolar neurite morphology of N1E‐115 cells. Instead, these inhibitors prompted neurite ramification. ROCK activity measured in whole cell extracts and in N1E‐115 cells was increased in the presence of melatonin and PMA. The results indicate that melatonin increases the number of cells with immature neurites and suggest that these neurites can be susceptible to differentiation by incoming extracellular signals. Data also indicate that PKC and ROCK are involved at initial stages of neurite formation in the mechanism by which melatonin recruits cells for later differentiation.

Melatonin supplementation delays the decline of adult hippocampal neurogenesis during normal aging of mice

Melatonin modulates adult hippocampal neurogenesis in adult mice. Also, plasma melatonin levels and new neuron formation decline during aging probably causing cognitive alterations. In this study, we analyzed the impact of exogenous supplementation with melatonin in three key events of hippocampal neurogenesis during normal aging of mice. The analysis was performed in rodents treated with melatonin during 3, 6, 9 or 12 months. We found an increase in cell proliferation in the dentate gyrus of the hippocampus after 3, 6 and 9 months of treatment (>90%). Additionally, exogenous melatonin promoted survival of new cells in the dentate gyrus (>50%). Moreover, melatonin increased the number of doublecortin-labeled cells after 6 and 9 months of treatment (>150%). In contrast, melatonin administered during 12 months did not induce changes in hippocampal neurogenesis. Our results indicate that melatonin also modulates the neurogenic process in the hippocampus during normal aging of mice. Together, the data support melatonin as one of the positive endogenous regulators of neurogenesis during aging.

Melatonin increases dendritogenesis in the hilus of hippocampal organotypic cultures

Abstract:  Neuropsychiatric disorders are characterized by hippocampus decreased volume and loss of dendrite arborizations in the subiculum and prefrontal cortex. These structural changes are associated with diminished memory performance. Hilar neurons of the hippocampus integrate spatial memory and are lost in dementia. They receive information from dentate gyrus neurons through dendrites, while they send axonal tracts to the CA3 region. Dendrites are complex structures of neurons that receive chemical information from presynaptic and postsynaptic terminals. Melatonin, the main product of the pineal gland, has neuroprotective actions through its free radical‐scavenging properties and decreases neuronal apoptosis. Recently, we found that melatonin increases dendrite maturation and complexity in new neurons formed in the dentate gyrus of mice. In addition, in N1E‐115 cultured cells, the indole stimulates early stages of neurite formation, a process that is known to antecede dendrite formation and maturation. Thus, in this study, we explored whether melatonin stimulates dendrite formation and complexity in the adult rat hippocampus in organotypic slice cultures, which is a model that preserves the hippocampal circuitry and their tridimensional organizations of connectivity. The effects of melatonin were studied in nonpathological conditions and in the absence of harmful agents. The results showed that the indole at nocturnal concentrations reached in the cerebrospinal fluid stimulates dendritogenesis at formation, growth, and maturation stages. Also, data showed that dendrites formed became competent to form presynaptic specializations. Evidence strongly suggests that melatonin may be useful in the treatment of neuropsychiatric diseases to repair the loss of dendrites and re‐establish lost synaptic connections.

Environmental enrichment induces neuroplastic changes in middle age female BalbC mice and increases the hippocampal levels of BDNF, p-Akt and p-MAPK1/2

Hippocampus is one of the brain regions in which neuroplastic changes occur. Paradigms such as environmental enrichment (ENR) have been used to prevent or delay the neuroplastic changes of the hippocampus during aging. Here, we investigated the beneficial effects of ENR on dendritic spines and hippocampal neurogenesis in middle age Balb/c mice. ENR increased the number of dendritic spines, cell survival, and intermediate stages of the hippocampal neurodevelopment process. Also, ENR alters the distribution of cells involved in the neurogenic process along the dorsal-ventral dentate gyrus. In addition, ENR increased the proportion of cells with more mature dendritic morphology and net hippocampal neurogenesis. Whole-hippocampus protein extracts revealed that ENR increases the levels of BDNF, phospho-Akt and phospho-MAPK1/2, suggesting that the positive effects of ENR on neuroplasticity in middle age Balb/c mice involve the participation of these key-signaling proteins. Our results suggest that ENR is a relevant strategy to prevent neuroplastic decline by increasing the formation of both dendritic spines and new neurons in the hippocampus during middle age.