Brain-derived neurotrophic factor increases the motility of a particular N-methyl-d-aspartate /GABA-responsive subset of neural progenitor cells
Graphical abstract
Highlights
► BDNF increases the motility of a E14 SVZ neurosphere-derived neuronal cell population. ► BDNF increases the distance TrkB-positive neuronal cells travel from their origin. ► Most of BDNF-responsive cells respond to NMDA and GABA with calcium elevations. ► The movement of the cells is phasic with bursts of motility and periods of stalling. ► BDNF reduces phases with low motility and enhances bursts of motility.
Introduction
Migration of newly born neurons from the ventricular walls to form the cerebral cortex requires multiple molecular signals for selection of the correct pathway and final destination in the cortical plate (Rakic, 1988). External cues, interpreted by receptors and activating intracellular signal pathways, thus provide signals for the control of neuroblast movement along the migratory pathway (reviewed in Ayala et al., 2007). The intracellular signals are thought to be relayed to the migratory motor consisting of the cytoskeleton and associated structures and changes in intracellular Ca2+ concentrations are generally considered to be the essential signal that promotes neuronal motility (reviewed in Bolsover, 2005, Komuro and Kumada, 2005, Zheng and Poo, 2007). Time-lapse microscopy has revealed a heterogeneous pattern of migration in brain slices consisting of radial migration guided by radial glial fibers and independent tangential migration (Nadarajah et al., 2003). Neurotrophins like brain-derived neurotrophic factor (BDNF) acting on tropomyosin-related kinase B (TrkB) receptors promotes the migration of neural progenitor cells (NPCs) (reviewed in Dicou, 2009). BDNF-promoted chemotaxis is blocked by tyrosin kinase inhibitors such as K252a, inhibition of phosphatidyl inositol-3-kinase, and buffering of intracellular Ca2+ (Behar et al., 1997, Polleux et al., 2002, Chiaramello et al., 2007). The target for the action of BDNF seems to be a specific subpopulation (Ohmiya et al., 2002) of tangentially migrating progenitors (Polleux et al., 2002, Alcántara et al., 2005). Neurotransmitters, in particular glutamate and GABA, also play a role in neural progenitor proliferation, migration, and differentiation (reviewed in Nguyen et al., 2001, Hagg, 2005, Sclett, 2006, Nakamichi et al., 2009). In vitro studies on cultured NPCs have demonstrated receptor-mediated responses in differentiating progenitors. Ionotropic N-methyl-d-aspartate (NMDA) receptors are Ca2+-permeable ion channels and are present on differentiating hippocampal progenitors (Ciccolini et al., 2003, Deisseroth et al., 2004). AMPA receptors are expressed during very early phases of NPC differentiation (Scherer and Gallo, 1998, Maric et al., 2000, Suzuki et al., 2006, Whitney et al., 2008, Jansson et al., 2011) and cause rises in intracellular Ca2+ mainly through a Ca2+-permeable philantotoxin-sensitive receptor channel (Jansson et al., 2011). The permeability of AMPA receptor channels to Ca2+ is regulated by RNA editing (Sommer et al., 1991, Burnashev et al., 1992) and Ca2+-permeable unedited AMPA receptors dominate in immature neural progenitors (Whitney et al., 2008). The main functionally identified metabotropic glutamate receptor in embryonic and adult neural precursors is metabotropic glutamate receptor 5 (mGluR5) (Castrén et al., 2005, Kärkkäinen et al., 2009). The mGluR5 receptor is expressed in regions of active neurogenesis in vivo in embryonic and postnatal brain and interference with its function impairs progenitor cell proliferation (Di Giorgi-Gerevini et al., 2004, Gandhi et al., 2008). GABA and GABA(A) receptors are expressed early during neurogenesis (Behar et al., 1998, Maric et al., 2001, Ge et al., 2006). In neural progenitor cells activation of these receptors leads to depolarization and Ca2+ elevations. Several studies suggest a role for GABA in migration and differentiation (reviewed in Nguyen et al., 2001, Hagg, 2005, Sclett, 2006, Ge et al., 2007, Nakamichi et al., 2009). A connection between neurotrophin and glutamate/GABA signaling pathways is suggested as glutamate and GABA induce the expression of BDNF (Mattson, 2008, Porcher et al., 2011).
A population of migrating neurosphere-derived NPCs expresses TrkB receptors (about 20%) and responds to BDNF by Ca2+ elevations (Louhivuori et al., 2011). The aim of the present study was to delineate the functional properties of BDNF-responsive neural progenitor cells. We show that BDNF increases the motility of a Calbindin- and neuron-specific nuclear protein (NeuN)-positive cell population, which migrates freely independently of radial glial cells, and reduces phases of slow progress and prolongs phases of high motility. The BDNF-stimulated cell population responds to both NMDA and GABA with robust Ca2+ elevations.
Section snippets
Cell culture and neuronal differentiation
Neural progenitor cells were isolated from the walls of lateral ventricles of embryonic day 14 (E14) FVB mice as described previously (Castrén et al., 2005, Kärkkäinen et al., 2009). Mice were handled in accordance with the institutional animal care policy of the University of Helsinki, performed in accordance with the guidelines of the National Institute of Health Guide for the Care and Use of Laboratory Animals and carried out under protocols approved by the Experimental Animal Ethical
Quantification of cell motility – effect of BDNF
Time-lapse imaging during the initial period of neurosphere-derived cell migration was performed using the Cell-IQ® system. Images of neurosphere cultures were captured every 15 min after plating and removal of mitogens. Analysis of the data was performed over a period of 36 h. Examples of monitored cells are shown in Fig. 1A and the mother neurosphere at time = 0 in Fig. 1B. During the first hours there was an extension of thick processes from the sphere, which at later time points could be
Discussion
The present study utilizes the neurosphere model to delineate the functional properties of cells, whose migration is enhanced by BDNF. The advantage of this model for in vitro studies is that the point of reference of each cell with respect to the mother neurosphere or point of origin is obtainable. Using time-lapse imaging of cell migration the main types of cell movements could be disclosed. Initially processes of GLAST-positive radial glial cells emerge from the neurosphere, forming a
Conclusion
It is concluded that the neurosphere model offers a possibility to in vitro explore the mechanisms by which neural progenitor cell migration is regulated by external factors. Taken together, these results show that BDNF increases the motility of and the distance travelled by a specific subpopulation of neuron-like cells. BDNF also increases the number of TrkB-positive cells, neuronal marker (Calbindin, MAP-2, and NeuN) expression, and cellular responsiveness to NMDA/GABA at the outer layer of
Acknowledgements
The authors like to thank Mr. Jarno Hörhä for laboratory assistance. We thank the Biomedicum Imaging Unit (BIU) (Biomedicum Helsinki, Finland) for providing microscopy services. This study was supported by the Academy of Finland, the Sigrid Jusélius Foundation, the Magnus Ehrnrooth Foundation, the Finnish Medical Society, the Swedish Cultural Foundation in Finland, and the University of Helsinki Funds.
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