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Melatonin stimulates dendrite formation and complexity in the hilar zone of the rat hippocampus: participation of the Ca++/Calmodulin complex.

Domínguez-Alonso A, Valdés-Tovar M, Solís-Chagoyán H, Benítez-King G - Int J Mol Sci (2015)

Bottom Line: We found that the CaMKII inhibitor, KN-62, abolished the MEL stimulatory effects on dendritogenesis and that MEL increased the relative amount of CaM in the soluble fraction of hippocampal slices.Our results indicate that MEL stimulates dendrite formation through CaMKII and the translocation of CaM to the soluble fraction.Data strongly suggest that MEL could repair the loss of hippocampal dendrites that occur in neuropsychiatric disorders by increasing CaM levels and activation of CaMKII.

View Article: PubMed Central - PubMed

Affiliation: Laboratorio de Neurofarmacología, Subdirección de Investigaciones Clínicas, Instituto Nacional de Psiquiatría Ramón de la Fuente Muñiz, Calzada México-Xochimilco No. 101, Col. San Lorenzo-Huipulco, CP 14370 Tlalpan, DF, Mexico. aline.dmgzalonso@gmail.com.

ABSTRACT
Melatonin (MEL), the main product synthesized by the pineal gland, stimulates early and late stages of neurodevelopment in the adult brain. MEL increases dendrite length, thickness and complexity in the hilar and mossy neurons of hippocampus. Dendrite formation involves activation of Ca2+/Calmodulin (CaM)-dependent kinase II (CaMKII) by CaM. Previous work showed that MEL increased the synthesis and translocation of CaM, suggesting that MEL activates CaM-dependent enzymes by this pathway. In this work we investigated whether MEL stimulates dendrite formation by CaMKII activation in organotypic cultures from adult rat hippocampus. We found that the CaMKII inhibitor, KN-62, abolished the MEL stimulatory effects on dendritogenesis and that MEL increased the relative amount of CaM in the soluble fraction of hippocampal slices. Also, PKC inhibition abolished dendritogenesis, while luzindole, an antagonist of MEL receptors (MT1/2), partially blocked the effects of MEL. Moreover, autophosphorylation of CaMKII and PKC was increased in presence of MEL, as well as phosphorylation of ERK1/2. Our results indicate that MEL stimulates dendrite formation through CaMKII and the translocation of CaM to the soluble fraction. Dendritogenesis elicited by MEL also required PKC activation, and signaling through MT1/2 receptors was partially involved. Data strongly suggest that MEL could repair the loss of hippocampal dendrites that occur in neuropsychiatric disorders by increasing CaM levels and activation of CaMKII.

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Participation of Melatonin receptors on dendrite formation elicited by Melatonin. To evaluate the involvement of MEL receptors, hippocampal organotypic cultures were incubated with the MT1/2 receptor antagonist, luzindole. Thus, rat brain hippocampus was cut in 400 µm slices and cultured in Neurobasal® media for 7 days. Then they were incubated for 6 h with either the vehicle (A); 100 nM melatonin (B); or pre-incubated with 100 µM luzindole followed by 6 h incubation with the vehicle (C) or 100 nM melatonin (D). After the incubation time, slices were cut into 50 µM sections and immunostained for the specific marker of dendrites MAP2. Images were acquired with a camera coupled to a light microscope with the NIS-Elements software. Scale bar = 100 µm.
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ijms-16-01907-f006: Participation of Melatonin receptors on dendrite formation elicited by Melatonin. To evaluate the involvement of MEL receptors, hippocampal organotypic cultures were incubated with the MT1/2 receptor antagonist, luzindole. Thus, rat brain hippocampus was cut in 400 µm slices and cultured in Neurobasal® media for 7 days. Then they were incubated for 6 h with either the vehicle (A); 100 nM melatonin (B); or pre-incubated with 100 µM luzindole followed by 6 h incubation with the vehicle (C) or 100 nM melatonin (D). After the incubation time, slices were cut into 50 µM sections and immunostained for the specific marker of dendrites MAP2. Images were acquired with a camera coupled to a light microscope with the NIS-Elements software. Scale bar = 100 µm.

Mentions: Stimulation of MEL receptors (MT1/2) causes PKC activation that has been shown to be involved in physiological responses such as phase shifts in circadian rhythms [39]. Also it has been suggested that MEL directly stimulates PKC activity to modulate cytoskeletal assembly [41]. Thus, we studied the participation of MEL receptors upstream PKC in the MEL signaling pathway involved in dendrite formation. Therefore, the organotypic hippocampal slices were incubated with 100 µM luzindole, a MT1/2 competitive antagonist. Figure 6 shows images of hippocampal slices cultured with the MT1/2 receptor antagonist in presence of either the vehicle or MEL and stained with the MAP2 antibody. Luzindole had no effect on dendrite formation and similar dendrite staining pattern was obtained in the vehicle-incubated cultures with or without the MEL receptor antagonist (Figure 6A,C). By contrast, the organotypic cultures pre-incubated with luzindole followed by MEL showed fewer but enlarged dendrites in comparison with slices incubated only with MEL (Figure 6B,D). Histogram analysis of the effects of luzindole on the three stages of dendritogenesis is shown in Figure 7. Parameters of dendrite formation and growth were similar in the vehicle-incubated slices cultured with or without luzindole. (Figure 7A,B). However, the index of maturation determined as the number of secondary dendrites was stimulated by luzindole itself by 40% (Figure 7C). By contrast, in presence of MEL, luzindole blocked dendrite formation and enlargement by 50% and 65%, respectively (Figure 7A,B); while dendrite complexity evaluated through both the number and length of secondary dendrites was only decreased by nearly 30% (Figure 7C,D).


Melatonin stimulates dendrite formation and complexity in the hilar zone of the rat hippocampus: participation of the Ca++/Calmodulin complex.

Domínguez-Alonso A, Valdés-Tovar M, Solís-Chagoyán H, Benítez-King G - Int J Mol Sci (2015)

Participation of Melatonin receptors on dendrite formation elicited by Melatonin. To evaluate the involvement of MEL receptors, hippocampal organotypic cultures were incubated with the MT1/2 receptor antagonist, luzindole. Thus, rat brain hippocampus was cut in 400 µm slices and cultured in Neurobasal® media for 7 days. Then they were incubated for 6 h with either the vehicle (A); 100 nM melatonin (B); or pre-incubated with 100 µM luzindole followed by 6 h incubation with the vehicle (C) or 100 nM melatonin (D). After the incubation time, slices were cut into 50 µM sections and immunostained for the specific marker of dendrites MAP2. Images were acquired with a camera coupled to a light microscope with the NIS-Elements software. Scale bar = 100 µm.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4307341&req=5

ijms-16-01907-f006: Participation of Melatonin receptors on dendrite formation elicited by Melatonin. To evaluate the involvement of MEL receptors, hippocampal organotypic cultures were incubated with the MT1/2 receptor antagonist, luzindole. Thus, rat brain hippocampus was cut in 400 µm slices and cultured in Neurobasal® media for 7 days. Then they were incubated for 6 h with either the vehicle (A); 100 nM melatonin (B); or pre-incubated with 100 µM luzindole followed by 6 h incubation with the vehicle (C) or 100 nM melatonin (D). After the incubation time, slices were cut into 50 µM sections and immunostained for the specific marker of dendrites MAP2. Images were acquired with a camera coupled to a light microscope with the NIS-Elements software. Scale bar = 100 µm.
Mentions: Stimulation of MEL receptors (MT1/2) causes PKC activation that has been shown to be involved in physiological responses such as phase shifts in circadian rhythms [39]. Also it has been suggested that MEL directly stimulates PKC activity to modulate cytoskeletal assembly [41]. Thus, we studied the participation of MEL receptors upstream PKC in the MEL signaling pathway involved in dendrite formation. Therefore, the organotypic hippocampal slices were incubated with 100 µM luzindole, a MT1/2 competitive antagonist. Figure 6 shows images of hippocampal slices cultured with the MT1/2 receptor antagonist in presence of either the vehicle or MEL and stained with the MAP2 antibody. Luzindole had no effect on dendrite formation and similar dendrite staining pattern was obtained in the vehicle-incubated cultures with or without the MEL receptor antagonist (Figure 6A,C). By contrast, the organotypic cultures pre-incubated with luzindole followed by MEL showed fewer but enlarged dendrites in comparison with slices incubated only with MEL (Figure 6B,D). Histogram analysis of the effects of luzindole on the three stages of dendritogenesis is shown in Figure 7. Parameters of dendrite formation and growth were similar in the vehicle-incubated slices cultured with or without luzindole. (Figure 7A,B). However, the index of maturation determined as the number of secondary dendrites was stimulated by luzindole itself by 40% (Figure 7C). By contrast, in presence of MEL, luzindole blocked dendrite formation and enlargement by 50% and 65%, respectively (Figure 7A,B); while dendrite complexity evaluated through both the number and length of secondary dendrites was only decreased by nearly 30% (Figure 7C,D).

Bottom Line: We found that the CaMKII inhibitor, KN-62, abolished the MEL stimulatory effects on dendritogenesis and that MEL increased the relative amount of CaM in the soluble fraction of hippocampal slices.Our results indicate that MEL stimulates dendrite formation through CaMKII and the translocation of CaM to the soluble fraction.Data strongly suggest that MEL could repair the loss of hippocampal dendrites that occur in neuropsychiatric disorders by increasing CaM levels and activation of CaMKII.

View Article: PubMed Central - PubMed

Affiliation: Laboratorio de Neurofarmacología, Subdirección de Investigaciones Clínicas, Instituto Nacional de Psiquiatría Ramón de la Fuente Muñiz, Calzada México-Xochimilco No. 101, Col. San Lorenzo-Huipulco, CP 14370 Tlalpan, DF, Mexico. aline.dmgzalonso@gmail.com.

ABSTRACT
Melatonin (MEL), the main product synthesized by the pineal gland, stimulates early and late stages of neurodevelopment in the adult brain. MEL increases dendrite length, thickness and complexity in the hilar and mossy neurons of hippocampus. Dendrite formation involves activation of Ca2+/Calmodulin (CaM)-dependent kinase II (CaMKII) by CaM. Previous work showed that MEL increased the synthesis and translocation of CaM, suggesting that MEL activates CaM-dependent enzymes by this pathway. In this work we investigated whether MEL stimulates dendrite formation by CaMKII activation in organotypic cultures from adult rat hippocampus. We found that the CaMKII inhibitor, KN-62, abolished the MEL stimulatory effects on dendritogenesis and that MEL increased the relative amount of CaM in the soluble fraction of hippocampal slices. Also, PKC inhibition abolished dendritogenesis, while luzindole, an antagonist of MEL receptors (MT1/2), partially blocked the effects of MEL. Moreover, autophosphorylation of CaMKII and PKC was increased in presence of MEL, as well as phosphorylation of ERK1/2. Our results indicate that MEL stimulates dendrite formation through CaMKII and the translocation of CaM to the soluble fraction. Dendritogenesis elicited by MEL also required PKC activation, and signaling through MT1/2 receptors was partially involved. Data strongly suggest that MEL could repair the loss of hippocampal dendrites that occur in neuropsychiatric disorders by increasing CaM levels and activation of CaMKII.

Show MeSH
Related in: MedlinePlus