The Activation of M₁Muscarinic Receptor Signaling Induces Neuronal Differentiation in Pyramidal Hippocampal Neurons

Abstract

Muscarinic receptors have been proposed to play an important role during brain development by regulating cell survival, proliferation, and differentiation. This study investigated the effect of muscarinic receptor activation on prenatal rat hippocampal pyramidal neuron differentiation and the signal transduction pathways involved in this effect. The cholinergic agonist carbachol, after 24 h in vitro, increased the length of the axon, without affecting the length of minor neurites. Carbachol-induced axonal growth was also observed in pyramidal neurons from the neocortex but not in granule neurons from the cerebellum. The effect of carbachol was mediated by the M 1 subtype of muscarinic receptors. The Ca 2ϩ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,NЈ,NЈ-tetraacetic acid-acetoxymethyl ester, the two protein kinase -32-0432), and the extracellular signal-regulated kinase (ERK)1/2 inhibitors 2Ј-amino-3Ј-methoxyflavone (PD98059) and 1,4-diamino-2,3-dicyano-1,4-bis(methylthio)butadiene (U0126) all blocked carbachol-induced axonal outgrowth. In addition, downregulation of ERK1/2 with small interfering RNA abolished the neuritogenic effect of carbachol. These data suggest an involvement of Ca 2ϩ , PKC, and ERK1/2 in carbachol-induced axonal growth. Carbachol indeed increased the release of Ca 2ϩ from intracellular stores and induced PKC and ERK1/2 activation. Additional experiments showed that PKC, but not Ca 2ϩ , is involved in carbachol-induced ERK1/2 activation. Together, these results show that cholinergic stimulation of prenatal hippocampal pyramidal neurons accelerates axonal growth through the induction of Ca 2ϩ mobilization and the activation of PKC and especially of ERK1/2.Neuronal differentiation is an essential event in brain development and begins with the sprouting of neurites followed by the elongation of axons and dendrites. Neuronal axons can project over very long distances to reach their final targets. Growth cones, located at the edges of growing axons and dendrites, are directed by extracellular cues that can repel or attract neurite growth in a given direction. These cues can be contact-mediated or soluble, secreted molecules. Contact-mediated molecules include extracellular matrix proteins and cell adhesion molecules; soluble molecules include neurotrophins and growth factors that activate several signal transduction pathways leading to the rearrangement of cytoskeletal proteins. Several of the signals directing neurite outgrowth derive from glial cells surrounding neurons, whereas others can derive from neurons themselves (TessierLavigne and Goodman, 1996).

Reduction of β-Amyloid Levels by Novel Protein Kinase Cϵ Activators

“…Muscarinic agonists rescue 3ϫ transgenic Alzheimer disease mice from cognitive deficits and reduce A␤ and tau pathologies (36), in part by activating the TACE/ADAM17 nonamyloidogenic pathway. Muscarinic receptor signaling is closely tied to PKC: muscarinic receptor mRNA is regulated by PKC (37), and neuronal differentiation produced by M1 muscarinic receptor activation is mediated by PKC (38).…”

Ethanol-induced changes in poly (ADP ribose) polymerase and neuronal developmental gene expression

“…Mice on embryonic day 18 (E18) were used for the primary cortical neuronal cultures. The techniques for the neuronal cultures were performed as previously described in detail (Guizzetti et al, 2008; VanDeMark et al, 2009). Briefly, the fetuses were decapitated and the brains immediately transferred into Hank’s balanced salt solution (HBSS) (Life Technologies, Carlsbad, CA).…”

Ethanol impairs muscarinic receptor-induced neuritogenesis in rat hippocampal slices: Role of astrocytes and extracellular matrix proteins

“…We had previously found that carbachol could exert a dual effect on hippocampal neuron neuritogenesis: one indirect, mediated by activation of M3 muscarinic receptors on astrocytes, and the other direct, mediated by M1 muscarinic receptors on neurons [17,29]. As in the hippocampal slice carbachol would be expected to activate both receptors on both cell types, we sought to determine which of the two muscarinic receptors, and hence which cell type, may be primarily involved in carbachol-induced neuritogenesis.…”

“…In addition to this astrocyte-mediated effect, carbachol has also been shown to increase neurite outgrowth through a direct action on hippocampal neurons; such direct effect was mediated by M1 muscarinic receptors, and was limited to an increase in axonal length, with no changes in the length of minor neurites [29]. …”