Kir and Kv channels regulate electrical properties and proliferation of adult neural precursor cells
Introduction
Adult brain neurogenesis occurs constitutively throughout life in a wide variety of animals. Adult multipotent neural precursor cells (NPCs), including self-renewing neural stem cells, in the rodent forebrain subventricular zone (SVZ) are capable of forming spherical proliferative cell clusters, known as “neurospheres”, when cultured in the presence of growth factors. These neurospheres can be passaged for an extended period of time and produce progeny that can differentiate into neurons and glial cells, such as astrocytes and oligodendrocytes (Morshead et al., 1994, Reynolds and Weiss, 1992). Using neurosphere assays, numerous laboratories have investigated how epigenetic factors, such as growth factors or cytokines, affect the proliferation and differentiation of NPCs. Although there are some reports with regard to cell-intrinsic (i.e. electrophysiological) properties of postnatal and adult NPCs, the functional role of the electrical properties of these cells in the regulation of proliferation and differentiation has been largely overlooked.
The membrane electrical properties of postnatal astrocytic NPCs have been investigated using the patch-clamp recording technique in acute brain cortical slice preparations of glial fibrillary acidic protein (GFAP)-green fluorescent protein (GFP) transgenic mice (Liu et al., 2005, Liu et al., 2006). GFAP-expressing astrocytic NPCs in the SVZ of a cortical slice exhibit a resting membrane potential (RMP) of ~ − 85 mV with an input resistance (Rin) of ~ 300 MΩ (Liu et al., 2006). In contrast, embryonic human and rat NPCs exhibit more depolarized RMPs of ~ − 40 mV and a higher Rin of ~ 1 GΩ (Cai et al., 2004, Piper et al., 2000), which are somewhat similar to those of neuroblasts in the postnatal and juvenile rodent SVZ (Bolteus and Bordey, 2004, Stewart et al., 1999, Walker et al., 2007, Wang et al., 2003). Postnatal and adult NPCs are inexcitable but express functional voltage-gated K+ (Kv) channels, as well as GABAA and glycine receptors (Liu et al., 2005, Liu et al., 2006, Nguyen et al., 2002). The GFAP-expressing astrocytic NPCs also exhibit voltage-independent inward K+ currents, which are pharmacologically distinct from inwardly rectifying K+ (Kir) channel currents in astrocytes (Liu et al., 2006). At the protein level, however, Kir2.1 and Kir4.1, which are abundant in astrocytes, have been identified immunohistochemically in the GFAP-expressing astrocytic NPCs (Liu et al., 2006).
In the present study, we identify the Kir and Kv channel currents expressed in astrocytic NPCs dissociated from neurospheres derived from the adult mouse SVZ. We also investigate the role of these K+ channels in NPC proliferation. Our findings suggest that Kir and Kv channels may play a significant role in adult neurogenesis in the SVZ. Preliminary reports of these findings have been published, in part, in abstract form (Yasuda et al., 2006).
Section snippets
Immunocytochemical identification of astrocytic NPCs dissociated from neurospheres
NPCs dissociated from non-passaged primary (P0) or passaged (P1 or P2) neurospheres derived from the adult mouse SVZ were used for electrophysiological studies and proliferation assays. Dissociated cells were plated and used within 2 days in vitro (DIV0–2). During short-term culture in the presence of epidermal growth factor (EGF) and fibroblast growth factor (FGF-2), a large number of cells died quickly or were not firmly attached to coverslips. No neuronal or glial differentiation was
Discussion
This is the first comprehensive study of K+ channels identified in adult NPCs and their functional significance. The passive membrane properties of the neurosphere-derived adult NPCs, including a hyperpolarized RMP of − 82.4 mV and a low Rin of 167 MΩ, are consistent with those of GFAP-expressing astrocytic NPCs in adult mouse SVZ slices (Liu et al., 2006). This indicates that the adult SVZ-derived NPCs in our neurosphere cultures essentially preserve the electrophysiological properties of
Preparation of neurosphere-derived adult NPCs
Adult mouse NPCs were obtained from primary neurospheres derived from the forebrain SVZ. Animals were treated in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes, and all experiments were approved by the University of Queensland Animal Ethics Committee. CBA mice (~ 8 weeks old) were killed by cervical dislocation and their brains were immediately removed. The forebrain SVZ was isolated, diced and then enzymatically dissociated with 0.05%
Acknowledgments
This study was funded by a National Health and Medical Research Council program (P.F.B.) and project (D.J.A.) grants. We thank Mark Stafford and John Normyle for assistance with PCR experiments, Brent Reynolds for constructive criticism on drafts of the manuscript, and Rowan Tweedale for editorial assistance.
References (45)
- et al.
Ion channel expression by white matter glia: the type-1 astrocyte
Neuron
(1990) - et al.
Subventricular zone astrocytes are neural stem cells in the adult mammalian brain
Cell
(1999) - et al.
Role of potassium channels in mitogenesis
Prog. Biophys. Mol. Biol.
(1993) - et al.
GABA and glutamate depolarize cortical progenitor cells and inhibit DNA synthesis
Neuron
(1995) - et al.
Neural stem cells in the adult mammalian forebrain: a relatively quiescent subpopulation of subependymal cells
Neuron
(1994) The A-current: how ubiquitous a feature of excitable cells is it?
Trends Neurosci.
(1985)- et al.
PSD-95 mediates formation of a functional homomeric Kir5.1 channel in the brain
Neuron
(2002) - et al.
Ion channel expression by white matter glia: I. Type 2 astrocytes and oligodendrocytes
Glia
(1988) - et al.
GABA release and uptake regulate neuronal precursor migration in the postnatal subventricular zone
J. Neurosci.
(2004) - et al.
Differential inhibition of glial K+ currents by 4-AP
J. Neurophysiol.
(1999)