Western Blotting using Chemiluminescent Substrates

Alegria-Schaffer, Alice

doi:10.1016/b978-0-12-420119-4.00019-7

Cited by 24 publications

(10 citation statements)

References 2 publications

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“…Blocked membranes were incubated either with anti‐GSK3β, anti‐phospho‐GSK3β, or anti‐MEL‐1A/B‐R antibodies (Santa Cruz Biotechnology). Specific secondary antibodies coupled to peroxidase and luminol/peroxide as substrate reagent was used for chemiluminescent reaction (ECL) as described . GAPDH was detected as a load control with a monoclonal antibody (Millipore).…”

Section: Methodsmentioning

confidence: 99%

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

Galván-Arrieta

Trueta²,

Cercós³

et al. 2017

Journal of Pineal Research

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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.

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Section: Methodsmentioning

confidence: 99%

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

Galván-Arrieta

Trueta²,

Cercós³

et al. 2017

Journal of Pineal Research

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“…Phosphorylated GSK‐3β (Ser9) was identified with an anti‐phospho‐GSK‐3β antibody (1:1300, Cell Signaling Technology Inc., #9336, Danvers, MA, USA) and a peroxidase‐conjugated donkey anti‐rabbit IgG (Jackson Immunoresearch, #711‐005‐152) diluted 1:10,000. Detection was performed by chemiluminiscence. After mild stripping, total GSK‐3β and GAPDH were identified using antibodies for GSK‐3β (1:1400, Santa Cruz Biotech, #SC‐9166, Dallas, Tx, USA) and GAPDH (1:20,000, EMD Millipore Corp., MAB374, Billerica, MA, USA).…”

Section: Methodsmentioning

confidence: 99%

Olanzapine‐induced early cardiovascular effects are mediated by the biological clock and prevented by melatonin

Romo‐Nava

Buijs

Valdés-Tovar

et al. 2017

Journal of Pineal Research

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Second generation antipsychotics (SGA) are associated with adverse cardiometabolic side effects contributing to premature mortality in patients. While mechanisms mediating these cardiometabolic side effects remain poorly understood, three independent studies recently demonstrated that melatonin was protective against cardiometabolic risk in SGA-treated patients. As one of the main target areas of circulating melatonin in the brain is the suprachiasmatic nucleus (SCN), we hypothesized that the SCN is involved in SGA-induced early cardiovascular effects in Wistar rats. We evaluated the acute effects of olanzapine and melatonin in the biological clock, paraventricular nucleus and autonomic nervous system using immunohistochemistry, invasive cardiovascular measurements, and Western blot. Olanzapine induced c-Fos immunoreactivity in the SCN followed by the paraventricular nucleus and dorsal motor nucleus of the vagus indicating a potent induction of parasympathetic tone.The involvement of a SCN-parasympathetic neuronal pathway after olanzapine administration was further documented using cholera toxin-B retrograde tracing and vasoactive intestinal peptide immunohistochemistry. Olanzapine-induced decrease in blood pressure and heart rate confirmed this. Melatonin abolished olanzapineinduced SCN c-Fos immunoreactivity, including the parasympathetic pathway and cardiovascular effects while brain areas associated with olanzapine beneficial effects including the striatum, ventral tegmental area, and nucleus accumbens remained activated. In the SCN, olanzapine phosphorylated the GSK-3β, a regulator of clock activity, which melatonin prevented. Bilateral lesions of the SCN prevented the effects of olanzapine on parasympathetic activity. Collectively, results demonstrate the SCN as a key region mediating the early effects of olanzapine on cardiovascular function and show melatonin has opposing and potentially protective effects warranting additional investigation. K E Y W O R D Sblood pressure, cardiovascular, melatonin, olanzapine, suprachiasmatic nucleus

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“…After four washes, chemiluminiscent reaction with luminol peroxidase substrate reagent (Santa Cruz Biotechnology) was allowed for 1 min. Exposed films were revealed and fixed as previously described [ 71 ]. Fluorogram densitometric analysis was done using a GS800 densitometer and Quantity One Software (Bio-Rad, Hercules, CA, USA).…”

Section: Methodsmentioning

confidence: 99%

Melatonin Stimulates Dendrite Formation and Complexity in the Hilar Zone of the Rat Hippocampus: Participation of the Ca++/Calmodulin Complex

Domínguez-Alonso

Valdés-Tovar

Solís‐Chagoyán

et al. 2015

IJMS

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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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Western Blotting using Chemiluminescent Substrates

Cited by 24 publications

References 2 publications

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

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

Olanzapine‐induced early cardiovascular effects are mediated by the biological clock and prevented by melatonin

Melatonin Stimulates Dendrite Formation and Complexity in the Hilar Zone of the Rat Hippocampus: Participation of the Ca++/Calmodulin Complex

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