The role of muscarinic receptors has been largely documented over the past few decades. Recently we demonstrated that the activation of M2 muscarinic receptors arrested cell proliferation and induced apoptosis in glioblastoma and in other tumour types. This paper aims to evaluate the expression of the M2 muscarinic receptor subtypes in different neuroblastoma cell lines and its role in the control of cell proliferation and survival. Neuroblastoma is the most common solid extracranial tumour, appearing during childhood and displaying a differentiated clinical behaviour. Considering the high homology between muscarinic receptor subtypes, we have identified Arecaidine Propargyl Ester (APE) as a selective orthosteric agonist for M2 muscarinic receptors. Using this agonist, we demonstrate how a selective activation of the M2 receptor subtype negatively modulates cell growth without affecting cell survival in different human neuroblastoma cell lines. As similarly demonstrated in other cell types, following the M2 receptor silencing by short-interference RNA, the effects of APE are completely abolished. We conclude by confirming the ability of APE to bind selectively M2 muscarinic receptor subtypes. Moreover, for the first time we demonstrate that M2 receptor activation inhibits cell growth also in human neuroblastoma cells, indicating that M2 receptors may be an interesting therapeutic target in several solid tumours.

Chronic pain is a debilitating condition that influences the social, economic, and psychological aspects of patients’ lives. Hence, the need for better treatment is drawing extensive interest from the research community. Developmental molecules such as Wnt, ephrins, and semaphorins are acknowledged as central players in the proper growth of a biological system. Their receptors and ligands are expressed in a wide variety in both neurons and glial cells, which are implicated in pain development, maintenance, and resolution. Thereby, it is not surprising that the impairment of those pathways affects the activities and functions of the entire cell. Evidence indicates aberrant activation of their pathways in the nervous system in rodent models of chronic pain. In those conditions, Wnt, ephrin, and semaphorin signaling participate in enhancing neuronal excitability, peripheral sensitization, synaptic plasticity, and the production and release of inflammatory cytokines. This review summarizes the current knowledge on three main developmental pathways and their mechanisms linked with the pathogenesis and progression of pain, considering their impacts on neuronal and glial cells in experimental animal models. Elucidations of the downstream pathways may provide a new mechanism for the involvement of Wnt, ephrin, and semaphorin pathways in pain chronicity.

The spinal cord is the main pathway connecting brain and peripheral nervous system. Its functionality relies on the orchestrated activity of both neurons and glial cells. To date, most advancement in understanding the spinal cord inner mechanisms has been made either by in vivo exposure of its dorsal surface through laminectomy or by acute ex vivo slice preparation, likely affecting spinal cord physiology in virtue of the necessary extensive manipulation of the spinal cord tissue. This is especially true of cells immediately responding to alterations of the surrounding environment, such as microglia and astrocytes, reacting within seconds or minutes and for up to several days after the original insult. Ca2+ signaling is considered one of the most immediate, versatile, and yet elusive cellular responses of glia. Here, we induced the cell-specific expression of the genetically encoded Ca2+ indicator GCaMP3 to evaluate spontaneous intracellular Ca2+ signaling in astrocytes and microglia. Ca2+ signals were then characterized in acute ex vivo (both gray and white matter) as well as in chronic in vivo (white matter) preparations using MSparkles, a MATLAB-based software for automatic detection and analysis of fluorescence events. As a result, we were able to segregate distinct astroglial and microglial Ca2+ signaling patterns along with method-specific Ca2+ signaling alterations, which must be taken into consideration in the reliable evaluation of any result obtained in physiological as well as pathological conditions. Our study revealed a high degree of Ca2+ signaling diversity in glial cells of the murine spinal cord, thus adding to the current knowledge of the astonishing glial heterogeneity and cell-specific Ca2+ dynamics in non-neuronal networks.