The present in vitro pharmacological

The present in vitro pharmacological experiments revealed that the cholinergic up-regulation of VEGF expression in neurons and astrocytes was mainly mediated by nicotinic AChR and muscarinic AChR, respectively. In addition, neuronal VEGF expression was elevated by endogenous ACh in a manner that was completely inhibited by MCM, but incompletely by SCP, suggesting that the expression of neuronal VEGF is mainly regulated by a nicotinic AChR pathway. Several lines of evidence indicate that nicotinic AChR as well as muscarinic AChR play a significant role in cognitive performance in rodents (Picciotto and Zoli, 2002; Sadigh-Eteghad et al., 2015; Takata et al., 2017). Collectively, these findings indicate that VEGF is involved in the amelioration of cognitive deficits by nicotinic AChR agonists (Cao et al., 2004). In contrast, the astrocytic expression levels of VEGF were elevated by CCh in a manner that was reversible by SCP, but not MCM, indicating the involvement of muscarinic AChR in the regulation of VEGF expression and secretion by astrocytes. In previous studies using organotypic hippocampal slice cultures (OHSCs), we reported that a muscarinic AChR stimulation by endogenous ACh protected hippocampal neuronal Nevirapine from excitotoxicity-induced cell damage in part via signaling between astrocytic VEGF and neuronal VEGFR2 (Inada et al., 2013, 2014). Moreover, an immunohistochemical analysis demonstrated that VEGF was dominantly expressed in astrocytes and VEGFR2 in pyramidal cell layers (Inada et al., 2013, 2014). Thus, the present results support our hypothesis that a functional link between cholinergic and VEGF signaling systems is implicated in neuroprotection exerted by cholinergic mechanisms in the brain. The increases induced in VEGF mRNA expression levels by THA were also attenuated by Ro, a conventional inhibitor of PKC. Therefore, the effects of THA on neuronal VEGF expression appeared to be attenuated by the SCP blockage of muscarinic AChR coupled with the PKC cascade. These results implicate the PKC cascade triggered by the endogenous ACh stimulation of nicotinic and muscarinic AChR in the regulation of neuronal VEGF expression. This concept appears to be supported by the findings of Tang et al. (2012). They demonstrated using mesenchymal stem cells expressing nicotinic and muscarinic AChR that ACh was involved in the migration of cells via the nicotinic AChR and Ca2+/PKC/extracellular signal-related kinase (ERK) 1/2 signaling pathway. Moreover, it is important to note that CCh-induced elevations in astrocytic mRNA were inhibited by SCP, a muscarinic receptor antagonist, but were not affected by Ro. The reasons for this conflicting result on the effects of the PKC inhibitor currently remain unclear; however, VEGF expression in astrocytes may be regulated by a PKC-independent pathway or by a pathway involving other PKC subtypes, at least in astrocytes. Nevertheless, further studies are needed in order to elucidate the mechanisms underlying the regulation of VEGF expression via muscarinic AChR in astrocytes. One of the important results of the present in vitro study is that no significant changes were observed in HIF-1α mRNA expression in neuronal or astrocytic cells after the treatment with THA or CCh. Biochemical evidence suggests that HIF-1α is a downstream molecule involved in the induction of VEGF mRNA and protein under anoxia and/or inflammatory biological responses (de Lemos et al., 2013; Wang et al., 1995). Moreover, the expression of VEGF in astrocytes reportedly involves a pathway mediated by PKC/HIF-1α. Therefore, the insusceptibility of neuronal and astrocytic HIF-1α expression to cholinergic drugs suggests that the mechanisms underlying endogenous ACh- and CCh-induced VEGF expression differ from those for HIF-1α-mediated VEGF expression, which occurs as an inflammatory response. We conducted immunohistochemical studies to elucidate the relationship between cholinergic and VEGF signaling systems in the brain. Our results revealed that ChAT-immunopositive cells in primary neuronal cell cultures were also immunopositive for VEGF and that the administration of THA enhanced the expression of VEGF, particularly in medial septal cholinergic neurons, in a manner that was reversible by MCM. Previous studies by this and other laboratories demonstrated that elevations induced in endogenous ACh by the administration of THA as well as by increases in the expression levels of ChAT protected medial septal cholinergic neurons from OBX- and diabetes-induced neuronal cell damage (Inada et al., 2014; Le et al., 2013; Yamada et al., 2011; Zhao et al., 2012). Furthermore, several lines of evidence indicated that nicotinic AChR plays an important role in not only the amelioration of cognitive deficits, but also neurogenesis in animal models of dementia (Picciotto and Zoli, 2002; Sadigh-Eteghad et al., 2015; Shioda et al., 2010; Takata et al., 2017). Based on the abundance of cholinergic neurons in the medial septum, elevations in VEGF expression induced via a nicotinic AChR cascade appear to contribute to the protection of medial septal cholinergic neurons. However, it is necessary to consider the influences of ACh/VEGF on cerebrovascular function in neuroprotection. Neurons, astrocytes and endothelial cells form the neurovascular unit, which could be involved in paracrine cross talk. Bosche et al. reported that lithium affected the neurovascular unit by enhancing Ach-dependent vasorelaxation and endothelial barrier function (Bosche et al., 2016). Moreover, it is also reported that resveratrol induces sustained neurological recovery by promoting angiogenesis via VEGF expression (Hermann et al., 2015). Thus, this VEGF expression seems to ameliorate cognitive deficits in animal models of dementia.