Marta Imbesi

Impaired hippocampal long-term potentiation in melatonin MT2 receptor-deficient mice

The pineal product melatonin that acts on specific melatonin receptors has been implicated in pathobiological mechanisms of neuropsychiatric disorders including Alzheimers disease. We used mice lacking melatonin MT(2) receptors (MT(2) knockouts) to investigate the role of these receptors in synaptic plasticity and learning-dependent behavior. In field CA1 of hippocampal slices from wild-type mice, theta burst stimulation induced robust and stable long-term potentiation that was smaller and decremental in slices from MT(2) knockouts. Tested in an elevated plus-maze on two consecutive days, wild-type mice showed shorter transfer latencies to enter a closed arm on the second day; this experience-dependent behavior did not occur in MT(2) knockouts. These results suggest that MT(2) receptors participate in hippocampal synaptic plasticity and in memory processes.

The melatonin receptor MT1 is required for the differential regulatory actions of melatonin on neuronal 'clock' gene expression in striatal neurons in vitro.

Abstract:  Through inhibitory G protein‐coupled melatonin receptors, melatonin regulates intracellular signaling systems and also the transcriptional activity of certain genes. Clock genes are proposed as regulatory factors in forming dopamine‐related behaviors and mood and melatonin has the ability to regulate these processes. Melatonin‐mediated changes in clock gene expression have been reported in brain regions, including the striatum, that are crucial for the development of dopaminergic behaviors and mood. However, it is not known whether melatonin receptors present in striatum mediate these effects. Therefore, we investigated the role of the melatonin/melatonin receptor system on clock gene expression using a model of primary neuronal cultures prepared from striatum. We found that melatonin at the receptor affinity range (i.e., nm) affects the expression of the clock genes mPer1, mClock, mBmal1 and mNPAS2 (neuronal PAS domain protein 2) differentially in a pertussis toxin‐sensitive manner: a decrease in Per1 and Clock, an increase in NPAS2 and no change in Bmal1 expression. Furthermore, mutating MT1 melatonin receptor (i.e., MT1 knockouts, MT1−/−) reversed melatonin‐induced changes, indicating the involvement of MT1 receptor in the regulatory action of melatonin on neuronal clock gene expression. Therefore, by controlling clock gene expression we propose melatonin receptors (i.e., MT1) as novel therapeutic targets for the pathobiologies of dopamine‐related behaviors and mood.

Minocycline increases phosphorylation and membrane insertion of neuronal GluR1 receptors.

The tetracycline antibiotic minocycline beneficially affects neuronal functioning and also inhibits the enzyme 5-lipoxygenase (5-LOX). We hypothesized that similar to 5-LOX inhibitors, minocycline may increase phosphorylation and membrane insertion of the glutamate receptor GluR1. The experiments were performed in primary cultures of mouse striatal neurons and in the prefrontal cortex and striatum of minocycline-treated mice. In vitro, low micromolar minocycline concentrations increased GluR1 phosphorylation at Ser845 and Ser831 and increased the surface content of GluR1. Minocycline also increased GluR1 phosphorylation in vivo. Increased GluR1 phosphorylation and minocycline treatment have been associated with antidepressant and memory-enhancing activities. Direct consequences of minocycline-increased GluR1 phosphorylation are yet to be established.

Role of melatonin receptors in the effects of melatonin on BDNF and neuroprotection in mouse cerebellar neurons.

Although melatonin affects developing neurons and is neuroprotective, a role of melatonin receptors termed MT1 and MT2 in these actions is unclear. We investigated the effects of melatonin on the levels of the brain derived neurotrophic factor (BDNF) in the developing cerebellum and cerebellar granule cells (CGC) of wild-type (WT), MT1- and MT2-knockout mice. A model of low-potassium CGC toxicity was used to evaluate neuroprotection. A 14-day-old pups and CGC cultures were treated with melatonin; 0.01 mg/kg intraperitoneally and 1 nM in vitro, respectively. Treatment of WT pups and CGC with melatonin did not alter BDNF levels. The absence of MT2 but not MT1 receptors enabled melatonin to increase cerebellar and CGC BDNF content. Nanomolar melatonin was neuroprotective in MT2-knockout but not WT CGC. We propose that CGC from MT2-knockout mice could serve as a model for studying the influence of melatonin on human CGC, which express MT1 but not MT2 receptors.

Stimulatory effects of a melatonin receptor agonist, ramelteon, on BDNF in mouse cerebellar granule cells.

Melatonin receptor activation has been linked to the regulation of neurotrophic factors, including the brain-derived neurotrophic factor (BDNF). To further characterize the effects of melatonin receptor stimulation on neuronal BDNF, we used a clinically available novel agonist for MT1 and MT2 melatonin receptors, ramelteon. Primary cultures of cerebellar granule cells (CGC) have been established as an in vitro model for studying neuronal BDNF. We took advantage of the availability of MT1- and MT2-deficient (knockout; KO) mice to employ primary CGC prepared from wild type (WT), MT1 KO, and MT2 KO mice. We investigated the effects of ramelteon on BDNF protein and mRNA content. Administered in a low nanomolar range, ramelteon increased BDNF protein content in all three types of mouse CGC. This ramelteon-triggered BDNF protein elevation was not preceded by a BDNF mRNA increase. However, it was prevented by treatment of cultures with a protein synthesis inhibitor cycloheximide. These results demonstrated that the MT1/MT2 melatonin receptor agonist ramelteon is capable of increasing BDNF protein in neurons expressing either of the two melatonin receptor types and that this action of ramelteon involves translational mechanisms. Further research is needed to explore the putative influence of ramelteon on BDNF-associated neuroplasticity.

Melatonin signaling in mouse cerebellar granule cells with variable native MT1 and MT2 melatonin receptors

Although G protein-coupled MT1 and MT2 melatonin receptors are expressed in neurons of the mammalian brain including in humans, relatively little is known about the influence of native MT1 and MT2 melatonin receptors on neuronal melatonin signaling. Whereas human cerebellar granule cells (CGC) express only MT1 receptors, mouse CGC express both MT1 and MT2. To study the effects of altered neuronal MT1/MT2 receptors, we used CGC cultures prepared from immature cerebella of wild-type mice (MT1/MT2 CGC) and MT1- and MT2-knockout mice (MT2 and MT1 CGC, respectively). Here we report that in MT1/MT2 cultures, physiological (low nanomolar) concentrations of melatonin decrease the activity (phosphorylation) of extracellular-signal-regulated kinase (ERK) whereas a micromolar concentration was ineffective. Both MT1 and MT2 deficiencies transformed the melatonin inhibition of ERK into melatonin-induced ERK activation. In MT1/MT2 CGC, 1 nM melatonin inhibited serine/threonine kinase Akt, whereas in MT1 and MT2 CGC, this concentration was ineffective. Under these conditions, both MT1 and MT2 deficiencies prevented melatonin from inhibiting forskolin-stimulated cAMP levels and cFos immunoreactivity. We demonstrated that selective removal of native neuronal MT1 and MT2 receptors has a profound effect on the intracellular actions of low/physiological concentrations of melatonin. Since the expression of MT1 and MT2 receptors is cell-type-specific and species-dependent, we postulate that the pattern of expression of neuronal melatonin receptor types in different brain areas and cells could determine the capabilities of endogenous melatonin in regulating neuronal functioning.

Effects of MK-886, a 5-lipoxygenase activating protein (FLAP) inhibitor, and 5-lipoxygenase deficiency on the forced swimming behavior of mice

A common biological pathway may contribute to the comorbidity of atherosclerosis and depression. Increased activity of the enzymatic 5-lipoxygenase (5-LOX, 5LO) pathway is a contributing factor in atherosclerosis and a 5-LOX inhibitor, MK-886, is beneficial in animal models of atherosclerosis. In the brain, MK-886 increases phosphorylation of the glutamate receptor subunit GluR1, and the increased phosphorylation of this receptor has been associated with antidepressant treatment. In this work, we evaluated the behavioral effects of MK-886 in an automated assay of mouse forced swimming, which identifies antidepressant activity as increased climbing behavior and/or decreased rest time. Whereas a single injection of MK-886 (3 and 10 mg/kg) did not affect forced swimming behaviors assayed 30 min later, six daily injections of 3 mg/kg MK-886 slightly increased climbing and significantly reduced rest time in wild-type mice but not in 5-LOX-deficient mice. A diet delivery of MK-886, 4 micro/(100 mg(body-weight)day), required 3 weeks to affect forced swimming; it increased climbing behavior. Climbing behavior was also increased in naive 5-LOX-deficient mice compared to naive wild-type controls. These results suggest that 5-LOX inhibition and deficiency may be associated with antidepressant activity. Increased climbing in a forced swimming assay is a typical outcome of antidepressants that increase noradrenergic and dopaminergic activity. Interestingly, 5-LOX deficiency and MK-886 treatment have been shown to be capable of increasing the behavioral effects of a noradrenaline/dopamine-potentiating drug, cocaine. Future research is needed to evaluate the clinical relevance of our findings.

Caffeic acid attenuates the decrease of cortical BDNF transcript IV mRNA induced by swim stress in wild-type but not in 5-lipoxygenase-deficient mice

Caffeic acid is a natural compound that inhibits 5-lipoxygenase (5-LOX). In mice, caffeic acid produces antidepressant-like effects and attenuates the decrease in cortical brain-derived neurotrophic factor (BDNF) mRNA induced by forced swimming. We used wild-type and 5-LOX-deficient mice and found that swimming reduced the cortical content of BDNF exon IV but not exon I mRNA. The BDNF transcript IV decrease was attenuated by caffeic acid in wild-type but not in 5-LOX-deficient mice, suggesting a role for 5-LOX in BDNF regulation.

5-Lipoxygenase inhibitor MK-886 increases GluR1 phosphorylation in neuronal cultures in vitro and in the mouse cortex in vivo

Modifications of AMPA glutamate receptor GluR1 phosphorylation are critical for neuroplastic mechanisms. Previous in vitro studies in brain slices employed MK-886, a functional inhibitor of the enzyme 5-lipoxygenase (5-LOX), and found increased GluR1 phosphorylation. Since slice preparations have accompanying postmortem phosphorylation changes, e.g., decreased GluR1 phosphorylation, it remains to be clarified whether MK-886 can affect GluR1 phosphorylation in intact neurons and in the brain in vivo. We used primary neuronal cultures prepared from embryonic mouse brain and in vivo drug administration to investigate the effects of MK-886 on GluR1 phosphorylation using quantitative Western immunoblotting assays. In vitro, MK-886 increased GluR1 phosphorylation at both serine 831 and serine 845. In vivo, repeated but not a single MK-886 injection increased GluR1 phosphorylation in the prefrontal cortex. These findings indicate that MK-886 has an intrinsic effect on neuronal phosphorylation both in vitro and in vivo and support the use of MK-886 as a pharmacological tool in studies of not only the 5-LOX pathway but also neuronal GluR1 functioning.

The pineal gland and anxiogenic-like action of fluoxetine in mice.

Fluoxetine produces initial paradoxical anxiogenic effect in some patients. In an elevated plus-maze (EPM), fluoxetine triggers an anxiogenic-like effect in rodents. Behavioral responses to psychoactive drugs can be modified by the pineal gland. We assessed the actions of fluoxetine in the EPM in melatonin-proficient C3H mice, melatonin-deficient C57BL6 mice, and in sham-operated and pinealectomized mice. Mice were assayed 30 min after the first injection and on day 14. Protracted fluoxetine treatment reduced the time on the anxiogenic open arms and increased the entries into the safe closed arms in sham-operated C3H mice. Fluoxetine was ineffective in pinealectomized C3H or C57BL6 mice. It is possible that the pineal system contributes to the previously observed anxiogenic action of fluoxetine in humans.