CAMP promotes the differentiation of neural progenitor cells in vitro via modulation of voltage-gated calcium channels

Guilherme Lepski,Guido Nikkhah,Cinthia E Jannes,Josef Bischofberger

doi:10.3389/fncel.2013.00155

Abstract

The molecular mechanisms underlying the differentiation of neural progenitor cells (NPCs) remain poorly understood. In this study we investigated the role of Ca2+ and cAMP (cyclic adenosine monophosphate) in the differentiation of NPCs extracted from the subventricular zone of E14.5 rat embryos. Patch clamp recordings revealed that increasing cAMP-signaling with Forskolin or IBMX (3-isobutyl-1-methylxantine) significantly facilitated neuronal functional maturation. A continuous application of IBMX to the differentiation medium substantially increased the functional expression of voltage-gated Na+ and K+ channels, as well as neuronal firing frequency. Furthermore, we observed an increase in the frequency of spontaneous synaptic currents and in the amplitude of evoked glutamatergic and GABAergic synaptic currents. The most prominent acute effect of applying IBMX was an increase in L-type Ca2+currents. Conversely, blocking L-type channels strongly inhibited dendritic outgrowth and synapse formation even in the presence of IBMX, indicating that voltage-gated Ca2+ influx plays a major role in neuronal differentiation. Finally, we found that nifedipine completely blocks IBMX-induced CREB phosphorylation (cAMP-response-element-binding protein), indicating that the activity of this important transcription factor equally depends on both enhanced cAMP and voltage-gated Ca2+-signaling. Taken together, these data indicate that the up-regulation of voltage-gated L-type Ca2+-channels and early electrical excitability are critical steps in the cAMP-dependent differentiation of SVZ-derived NPCs into functional neurons. To our knowledge, this is the first demonstration of the acute effects of cAMP on voltage-gated Ca+2channels in NPC-derived developing neurons.

Highlights

Previous studies have reported the generation of functional neurons from neural stem cells/progenitor cells derived from fetal (Auerbach et al, 2000), neonatal (Scheffler et al, 2005), and adult (Song et al, 2002) brain tissue
We present several novel findings pertaining to the differentiation and maturation of SVZ-derived neural progenitor cells (NPCs)
We found that increasing cAMP levels by inhibiting phosphodiesterase activity is sufficient to support the rapid functional maturation of neuronal progenitors into fully www.frontiersin.org cAMP promotes neurogenesis via Ca2+-signaling functional neurons; this maturation involves the high-density expression of voltage-gated ion channels, the high-frequency firing of APs and the formation of functional glutamatergic and GABAergic synaptic contacts

Summary

Introduction

Previous studies have reported the generation (in vitro) of functional neurons from neural stem cells/progenitor cells derived from fetal (Auerbach et al, 2000), neonatal (Scheffler et al, 2005), and adult (Song et al, 2002) brain tissue. The activation of many G-protein-coupled receptors stimulates cAMP production as well as CREB [cAMP response element (CRE) binding protein] phosphorylation, two processes believed to be important for NPC proliferation and differentiation (Dworkin and Mantamadiotis, 2010). For adult neural stem cells, (Fujioka et al, 2004) showed that prolonged cAMP elevation induced by Forskolin increases the length and number of dendritic branches as well as the number of MAP2ab (microtubule-associated-protein 2ab) positive cells during the first 2 weeks of differentiation. Whereas phosphorylated CREB (pCREB) is basically absent in stem cells, it is transiently up-regulated in NPCs and young neurons during the first few weeks of differentiation (Merz et al, 2011). The amount of available pCREB during this time period correlates with the total dendritic length and the morphological maturity of the young neurons (Giachino et al, 2005), while the expression of dominant negative pCREB has been shown to strongly interfere with their survival and differentiation (Herold et al, 2011)

Methods

Results

Conclusion