Background:The molecular mechanisms regulating brain development are unclear. Results: HDAC3 deletion disrupts the organization of certain neuronal cell types and the proportions of some glial cell types in the cortex and cerebellum. Conclusion: HDAC3 regulates brain development, and other HDACs cannot compensate for its function. Significance: Our study identifies a key player in the regulation of brain development.

Recent evidence suggests that non-selective cannabinoid receptor agonists may regulate serotonin 2A (5-HT2A) receptor neurotransmission in brain. The molecular mechanisms of this regulation are unknown but could involve cannabinoid-induced enhanced interaction between 5-HT2A and dopamine D2 (D2) receptors. Here, we present experimental evidence that Sprague-Dawley rats treated with a non-selective cannabinoid receptor agonist (CP55,940, 50μg/kg, 7days, i.p.) showed enhanced co-immunoprecipitation of 5-HT2A and D2 receptors and enhanced membrane-associated expression of D2 and 5-HT2A receptors in prefrontal cortex (PFCx). Furthermore, 5-HT2A receptor mRNA levels were increased in PFCx suggesting a cannabinoid-induced upregulation of 5-HT2A receptors. To date, two cannabinoids receptors have been found in brain, CB1 and CB2 receptors. We used selective cannabinoid agonists in a neuronal cell line to study mechanisms that could mediate this 5-HT2A receptor upregulation. We found that selective CB2 receptor agonists upregulate 5-HT2A receptors by a mechanism that seems to involve activation of Gαi G-proteins, ERK1/2, and AP-1 transcription factor. We hypothesize that the enhanced cannabinoid-induced interaction between 5-HT2A and D2 receptors and in 5-HT2A and D2 receptors protein levels in the PFCx might provide a molecular mechanism by which activation of cannabinoid receptors might be contribute to the pathophysiology of some cognitive and mood disorders.

Difficulty in expressing the adrenocorticotrophin (ACTH) receptor (melanocortin 2 receptor; MC2R) after transfection of various MC2R expression vectors has been experienced by many researchers. Reproducible evidence for expression has been obtained only in the Y6/OS3 corticoadrenal cell lines or in cells expressing endogenous melanocortin receptors. In order to determine the cause of this failure of expression we have undertaken the following studies. An MC2R expression plasmid was constructed in which the green fluorescent protein (GFP) coding region had been added to the C-terminus of the mature protein. Transfection of this plasmid into Y6 cells with a cAMP-responsive reporter plasmid demonstrated normal function of this receptor. Imaging of CHO cells expressing MC2R-GFP revealed perinuclear expression, although a cholecystokinin receptor (CCKR)-GFP construct was efficiently expressed at the cell surface. Y6 cells, in contrast, showed cell surface fluorescence after transfection with MC2R-GFP. Several other cell types showed a similar pattern of GFP distribution characteristic of retention in the endoplasmic reticulum. Counterstaining with an anti-KDEL antibody confirmed this location. Coexpression of the MC2R and the CCKR-GFP did not impair CCKR trafficking to the cell surface, implying a receptor-specific impairment to trafficking in the CHO cell which was absent in the Y6 cell.

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Recent behavioral studies suggest that non-selective agonists of cannabinoid receptors may regulate serotonin 2A (5-HT2A) receptor neurotransmission. Two cannabinoids receptors are found in brain, CB1 and CB2 receptors, but the molecular mechanism by which cannabinoid receptors would regulate 5-HT2A receptor neurotransmission remains unknown. Interestingly, we have recently found that certain cannabinoid receptor agonists can specifically upregulate 5-HT2A receptors. Here, we present experimental evidence that rats treated with a non-selective cannabinoid receptor agonist (CP 55,940, 50μg/kg, 7 days) showed increases in 5-HT2A receptor protein levels, 5-HT2A receptor mRNA levels, and 5-HT2A receptor-mediated phospholipase C Beta (PLCβ) activity in prefrontal cortex (PFCx). Similar effects were found in neuronal cultured cells treated with CP 55,940 but these effects were prevented by selective CB2, but not selective CB1, receptor antagonists. CB2 receptors couple to the extracellular kinase (ERK) signaling pathway by Gαi/o class of G-proteins. Noteworthy, GP 1a (selective CB2 receptor agonist) produced a strong upregulation of 5-HT2A receptor mRNA and protein, an effect that was prevented by selective CB2 receptor antagonists and by an ERK1/2 inhibitor, PD 198306. In summary, our results identified a strong cannabinoid-induced upregulation of 5-HT2A receptor signaling in rat PFCx. Our cultured cell studies suggest that selective CB2 receptor agonists upregulate 5-HT2A receptor signaling by activation of the ERK1/2 signaling pathway. Activity of cortical 5-HT2A receptors has been associated with several physiological functions and neuropsychiatric disorders such as stress response, anxiety & depression and schizophrenia. Therefore, these results might provide a molecular mechanism by which activation of cannabinoid receptors might be relevant to the pathophysiology of some cognitive and mood disorders in humans.

We have recently reported that selective cannabinoid 2 (CB2) receptor agonists upregulate 5-HT2A receptors by enhancing ERK1/2 signaling in prefrontal cortex (PFCx). Increased activity of cortical 5-HT2A receptors has been associated with several neuropsychiatric disorders such as anxiety and schizophrenia. Here we examine the mechanisms involved in this enhanced ERK1/2 activation in rat PFCx and in a neuronal cell model. Sprague-Dawley rats treated with a non-selective cannabinoid agonist (CP55940, 50 μg/kg, 7 days, i.p.) showed enhanced co-immunoprecipitation of β-Arrestin 2 and ERK1/2, enhanced pERK protein levels, and enhanced expression of β-Arrestin 2 mRNA and protein levels in PFCx. In a neuronal cell line, we found that selective CB2 receptor agonists upregulate β-Arrestin 2, an effect that was prevented by selective CB2 receptor antagonist JTE-907 and CB2 shRNA lentiviral particles. Additionally, inhibition of clathrin-mediated endocytosis, ERK1/2, and the AP-1 transcription factor also prevented the cannabinoid receptor-induced upregulation of β-Arrestin 2. Our results suggest that sustained activation of CB2 receptors would enhance β-Arrestin 2 expression possibly contributing to its increased interaction with ERK1/2 thereby driving the upregulation of 5-HT2A receptors. The CB2 receptor-mediated upregulation of β-Arrestin 2 would be mediated, at least in part, by an ERK1/2-dependent activation of AP-1. These data could provide the rationale for some of the adverse effects associated with repeated cannabinoid exposure and shed light on some CB2 receptor agonists that could represent an alternative therapeutic because of their minimal effect on serotonergic neurotransmission.

Recent behavioral reports suggest that repeated exposure to cannabis and synthetic cannabinoid agonists is linked with mental disorders associated with dysfunction of serotonin 2A (5-HT2A) receptor neurotransmission such as anxiety and depression. Here, we studied the effect of a nonselective cannabinoid agonist, CP55940, on the activity of 5-HT2A receptors in hypothalamic paraventricular nucleus (PVN). We detected that repeated exposure to CP55940 enhanced the prolactin and corticosterone neuroendocrine responses mediated by 5-HT2A receptors and increased the membrane-associated levels of 5-HT2A receptors in PVN. Importantly, we also detected increased anxiety-like behaviors in CP55940 treated rats compared to controls. The data presented here suggest that the mechanisms mediating the cannabinoid-induced upregulation of 5-HT2A receptors would be brain-region specific, as we were unable to detect a CP55940-induced upregulation of 5-HT2A mRNA. Our results might provide insight into the molecular mechanism by which repeated exposure to cannabinoids could be associated with the pathophysiology of neuropsychiatric disorders.

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