Elsevier

Differentiation

Volume 77, Issue 2, February 2009, Pages 188-198
Differentiation

Neurotransmitter responsiveness during early maturation of neural progenitor cells

https://doi.org/10.1016/j.diff.2008.10.005Get rights and content

Abstract

Neurotransmitters are potential regulators of proliferation and differentiation of neural progenitor cells (NPC). To gain insight into the dynamics of neurotransmitter responsiveness, neurospheres were prepared from the lateral ventricles of postnatal day 6/7 mice. Individual NPCs migrating out from spheres were simultaneously monitored using Ca2+ imaging, during the initial 8 days of differentiation, at an area between the inner edge of the sphere and outer periphery of the area of migration. At the first day of differentiation most cells showed metabotropic responses (Ca2+ discharge from stores) to glutamate (pharmacologically identified as metabotropic glutamate receptor 5, mGluR 5), norepinephrine (NE), acetylcholine (Ach) and ATP, and a smaller proportion of cells also responded to substance P (SP). When outside the neurosphere, many of mGluR5 responding cells gained immunostaining for markers of neuronal lineage (Tuj-1 and NeuN). The number of cells responding through mGluR5 (and responses to Ach, NE and SP) showed during subsequent days of differentiation (day 2–3 onwards) a decline with time and progressively disappeared at the outer periphery of the area of migration. Conversely the number ionotropic glutamate responses as well as responses to depolarization increased in this area. After 5–8 days of differentiation mGluR5 responses could only be observed at the very inner edge of the neurosphere. At 8 days the migrated cells showed very robust ionotropic responses to glutamate, NMDA and depolarization comparable to mature neurons. Taken together, the data presented here suggest that differentiation of NPCs is a dynamic process triggered by cell migration, which leads to a loss of regulatory influences imposed by the inner milieu of the neurosphere. The subsequent switch or loss of metabotropic responses to glutamate, SP, NE, Ach and ATP with the gain of excitable characteristics such as ionotropic responses appears to be a key event in the final differentiation process.

Introduction

The mechanisms involved in determining the differentiation of neural stem/progenitor cells (NPCs) into mature functional neurons have not been fully resolved. Intrinsic characteristics of NPCs as well as their microenvironment shape the properties of NPCs and define their potential. The stem cell niches provide a microenvironment that allows stem cells to self-renew and keep the differentiation programs on hold (Fuchs et al., 2004). Changes in the environmental cues and in the intrinsic behavior of NPCs cause alterations in the fate determination of NPCs and may lead to differentiation of NPCs. Progenies generated by NPCs change over the course of development in vivo (Merkle and Alvarez-Buylla, 2006). There is evidence that, in addition to growth factors, neurotransmitters play a role in neuronal differentiation (Emerit et al., 1992; Nguyen et al., 2001; Hagg, 2005). Elevated intracellular Ca2+ is also a typical trigger of plastic changes in neurons (Spitzer et al., 2004; D’Ascenzo et al., 2006; Deisseroth et al., 2004). Neurotransmitters typically activate ionotropic or G-protein-coupled receptors (GPCRs) to cause local or global changes in intracellular Ca2+. One of the primary G-protein-linked mechanisms involves the Gq protein which, when activated, couples to phospholipase C (PLC), leading to hydrolysis of phospatidyl-inositol-bis-phosphate (PIP2). The diacylglycerol (DAG) product activates protein kinase C (PKC), while the phosphorylated sugar moiety inositol-1,4,5-phospate (IP3) mobilizes Ca2+ from intracellular stores (Berridge et al., 2000). Both Ca2+ and PKC have profound tropic effects (Berridge et al., 2000). Local and global changes in intracellular free Ca2+ play an important role in differentiation of NPCs (Ciccolini et al., 2003) and determine their neurotransmitter phenotype (Spitzer et al., 2004). Changes in Ca2+ concentration may thus represent a common target for factors and signals that regulate NPC differentiation and acquisition of specific neuronal phenotypes.

Several neurotransmitters acting via GPCRs such as acetylcholine (Ach), norepinephrine (NE) and neuropeptides have been implicated in the control of neurogenesis (reviewed in Hagg, 2005). Metabotropic glutamate receptors (mGluRs) mediate the effects of glutamate via G-protein-linked mechanisms. Group I mGluR subtypes mGluR1 and mGluR5 are coupled to polyphosphoinositide (PI) hydrolysis. Both Group I mGluRs and ionotropic glutamate receptors have been shown to promote neuronal survival and neurogenesis (Di Giorgi Gerevini et al., 2004; Simonyi et al., 2005; Brazel et al., 2005; Baskys et al., 2005; Poulsen et al., 2005; Gandhi et al., 2008). The mGluR5 is expressed in the regions of active neurogenesis in the embryonic and postnatal brain and its expression has also been shown in embryonic stem (ES) cells and in NPCs. Very little information is available concerning the neurotransmitter responsiveness of individual NPCs in relation to differentiation. We have previously distinguished two major functional cell types during differentiation of NPCs derived from early prenatal brain by analyzing intracellular Ca2+ responses to glutamate (Castrén et al., 2005). Type I cells show robust metabotropic Ca2+ elevations mediated exclusively via mGluR5 and little Ca2+ elevation in response to depolarization, while type II cells only show ionotropic responses to glutamate but robust responses to depolarization. The two cell types may represent two stages of the maturation process.

Elucidation of the neurotransmitter responsiveness at certain stages of differentiation should aid studies aimed at delineation of the role of neurotransmitters in determining the fate of differentiating progenitors. Neurotransmitters may be involved in modulating the regenerative capacity of NPC:s. We therefore analyzed the functional properties of cells derived from neurospheres, cultured from the lateral ventricles of postnatal (P6/P7) mice. We used Ca2+ imaging to monitor the spatial and dynamic pattern of the neurotransmitter responsiveness of the individual NPC:s migrating out from neurospheres. During early stages of differentiation distinct cell populations could be distinguished based on their reactivity to these neurotransmitters. Cells that migrate out from the neurosphere progressively loose their metabotropic responses to glutamate and gain ionotropic responses concomitant with the development of a neuronal phenotype.

Section snippets

Cell cultures

NPCs were propagated from the brain cortex of postnatal day 6 or 7 (P6/P7) mice and grown as free-floating aggregates termed “neurospheres” as previously described (Castrén et al., 2005). Postnatal mice of this age were chosen as their cells have regained a significant potential for neuronal differentiation. All animal experiments were performed according to the guidelines of The Society for Neuroscience and were approved by the Experimental Animal Ethics Committee of the National Laboratory

Neurotransmitter responses of NPCs propagated from postnatal mice during differentiation for 1–4 days

NPCs were propagated from brains of P6 or P7 mice and multiplied as neurospheres in the presence of EGF and FGF-2. After initiation of differentiation by removing the mitogens, cells migrated out from the neurosphere. Already after 1 day a significant number of cells could be found outside the sphere. Responses to neurotransmitter agonists known to mobilize intracellular Ca2+ were monitored at the edge and periphery of the neurosphere during 1–4 days of differentiation (an approximate area for

Discussion

Neurotransmitters regulate the differentiation of neural stem/progenitor cells (NPC:s) (reviewed in Hagg, 2005) and may thus have a central role in the regenerative capacity of these cells in the central nervous system. Very little is known about the responsiveness of individual NPC:s to neurotransmitters in relation to differentiation. The present study shows the dynamic patterns of neurotransmitter responses during early differentiation of postnatal NPCs. Several lines of evidence indicate

Acknowledgements

The authors are grateful for the laboratory assistance provided by Veera Pevgonen, A.I. Virtanen Institute. This study was supported by the Academy of Finland, the Sigrid Jusélius Foundation and Arvo and Lea Ylppö Foundation.

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