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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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Morphometric analysis of dendritogenesis elicited by Melatonin: participation of Ca2+/CaM Kinase II and Protein Kinase C. The three stages of dendrite formation were evaluated by measuring the number and length of primary dendrites, as well as the number and length of secondary and tertiary dendrites. Hippocampal slices were incubated for 6 h with the following treatments: vehicle (VEH), 100 nM melatonin (MEL), pre- incubation of 15 min with 5 µM of the PKC inhibitor, bisindolylmaleimide (BIS) followed by the vehicle (BIS + VEH) or melatonin (BIS + MEL) for 6 h, or pre- incubation of 15 min with 10 µM of the CaMKII inhibitor, the KN-62 compound (KN-62) followed by the vehicle (KN-62 + VEH) or melatonin (KN-62 + MEL) for 6 h. After the incubation time, slices were cut into 50 µM sections and immunostained for the specific marker of dendrites MAP2. Slices were analyzed by the modified Sholl method. Results represent the mean ± SEM of one experiment of three done by quadruplicate. Asterisks show significant differences (p < 0.05). (A) Number of primary dendrites; (B) Primary dendrite length (µm); (C) Number of secondary dendrites; (D) Secondary and tertiary dendrite length (µm).
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ijms-16-01907-f003: Morphometric analysis of dendritogenesis elicited by Melatonin: participation of Ca2+/CaM Kinase II and Protein Kinase C. The three stages of dendrite formation were evaluated by measuring the number and length of primary dendrites, as well as the number and length of secondary and tertiary dendrites. Hippocampal slices were incubated for 6 h with the following treatments: vehicle (VEH), 100 nM melatonin (MEL), pre- incubation of 15 min with 5 µM of the PKC inhibitor, bisindolylmaleimide (BIS) followed by the vehicle (BIS + VEH) or melatonin (BIS + MEL) for 6 h, or pre- incubation of 15 min with 10 µM of the CaMKII inhibitor, the KN-62 compound (KN-62) followed by the vehicle (KN-62 + VEH) or melatonin (KN-62 + MEL) for 6 h. After the incubation time, slices were cut into 50 µM sections and immunostained for the specific marker of dendrites MAP2. Slices were analyzed by the modified Sholl method. Results represent the mean ± SEM of one experiment of three done by quadruplicate. Asterisks show significant differences (p < 0.05). (A) Number of primary dendrites; (B) Primary dendrite length (µm); (C) Number of secondary dendrites; (D) Secondary and tertiary dendrite length (µm).

Mentions: Dendrite development is a complicated process that involves three steps: formation, growth and complexity [46]. All three stages can be evaluated by measuring the number of primary dendrites, the length of primary dendrites, and the number and length of secondary dendrites, as an index of formation, outgrowth and complexity, respectively [18]. Thus, we determined those parameters and as shown in Figure 3, a decrease below basal levels was observed in the presence of either the CaMKII or PKC inhibitors. Also, no increase of dendrite formation, growth or complexity was observed in organotypic cultures incubated with 100 nM MEL and either of those inhibitors. Thus, morphometric analysis shows that all three stages of dendritogenesis were abolished by either the CaMKII or PKC inhibitors, supporting that both enzymes are in the signaling pathway by which MEL elicits dendritogenesis. Since KN-62 interacts with the Ca2+/CaM binding site of CaMKII impeding its activation, the results suggest that either increased CaM affinity or availability could be involved in MEL-induced dendritogenesis. In this regard, contradictory evidence has been reported about the ability of phosphorylated CaM to interact with its binding site in the regulatory domain of CaMKII. However, serine/threonine phosphorylation of CaM by casein kinase II increases its affinity for binding to CaMKII [47,48]. Thus, increased affinity by PKC-mediated phosphorylation on CaM serine/threonine residues or increased availability of CaM by augmented synthesis via PKC/cAMP response element binding protein or by targeting to specific subcellular sites can be involved in the mechanisms by which MEL stimulates dendritogenesis [22,29,47,49,50].


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)

Morphometric analysis of dendritogenesis elicited by Melatonin: participation of Ca2+/CaM Kinase II and Protein Kinase C. The three stages of dendrite formation were evaluated by measuring the number and length of primary dendrites, as well as the number and length of secondary and tertiary dendrites. Hippocampal slices were incubated for 6 h with the following treatments: vehicle (VEH), 100 nM melatonin (MEL), pre- incubation of 15 min with 5 µM of the PKC inhibitor, bisindolylmaleimide (BIS) followed by the vehicle (BIS + VEH) or melatonin (BIS + MEL) for 6 h, or pre- incubation of 15 min with 10 µM of the CaMKII inhibitor, the KN-62 compound (KN-62) followed by the vehicle (KN-62 + VEH) or melatonin (KN-62 + MEL) for 6 h. After the incubation time, slices were cut into 50 µM sections and immunostained for the specific marker of dendrites MAP2. Slices were analyzed by the modified Sholl method. Results represent the mean ± SEM of one experiment of three done by quadruplicate. Asterisks show significant differences (p < 0.05). (A) Number of primary dendrites; (B) Primary dendrite length (µm); (C) Number of secondary dendrites; (D) Secondary and tertiary dendrite length (µm).
© Copyright Policy
Related In: Results  -  Collection

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

ijms-16-01907-f003: Morphometric analysis of dendritogenesis elicited by Melatonin: participation of Ca2+/CaM Kinase II and Protein Kinase C. The three stages of dendrite formation were evaluated by measuring the number and length of primary dendrites, as well as the number and length of secondary and tertiary dendrites. Hippocampal slices were incubated for 6 h with the following treatments: vehicle (VEH), 100 nM melatonin (MEL), pre- incubation of 15 min with 5 µM of the PKC inhibitor, bisindolylmaleimide (BIS) followed by the vehicle (BIS + VEH) or melatonin (BIS + MEL) for 6 h, or pre- incubation of 15 min with 10 µM of the CaMKII inhibitor, the KN-62 compound (KN-62) followed by the vehicle (KN-62 + VEH) or melatonin (KN-62 + MEL) for 6 h. After the incubation time, slices were cut into 50 µM sections and immunostained for the specific marker of dendrites MAP2. Slices were analyzed by the modified Sholl method. Results represent the mean ± SEM of one experiment of three done by quadruplicate. Asterisks show significant differences (p < 0.05). (A) Number of primary dendrites; (B) Primary dendrite length (µm); (C) Number of secondary dendrites; (D) Secondary and tertiary dendrite length (µm).
Mentions: Dendrite development is a complicated process that involves three steps: formation, growth and complexity [46]. All three stages can be evaluated by measuring the number of primary dendrites, the length of primary dendrites, and the number and length of secondary dendrites, as an index of formation, outgrowth and complexity, respectively [18]. Thus, we determined those parameters and as shown in Figure 3, a decrease below basal levels was observed in the presence of either the CaMKII or PKC inhibitors. Also, no increase of dendrite formation, growth or complexity was observed in organotypic cultures incubated with 100 nM MEL and either of those inhibitors. Thus, morphometric analysis shows that all three stages of dendritogenesis were abolished by either the CaMKII or PKC inhibitors, supporting that both enzymes are in the signaling pathway by which MEL elicits dendritogenesis. Since KN-62 interacts with the Ca2+/CaM binding site of CaMKII impeding its activation, the results suggest that either increased CaM affinity or availability could be involved in MEL-induced dendritogenesis. In this regard, contradictory evidence has been reported about the ability of phosphorylated CaM to interact with its binding site in the regulatory domain of CaMKII. However, serine/threonine phosphorylation of CaM by casein kinase II increases its affinity for binding to CaMKII [47,48]. Thus, increased affinity by PKC-mediated phosphorylation on CaM serine/threonine residues or increased availability of CaM by augmented synthesis via PKC/cAMP response element binding protein or by targeting to specific subcellular sites can be involved in the mechanisms by which MEL stimulates dendritogenesis [22,29,47,49,50].

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