Physiological Electrical Signals Promote Chain Migration of Neuroblasts by Up-Regulating P2Y1 Purinergic Receptors and Enhancing Cell Adhesion
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Neuroblasts migrate as directed chains of cells during development and following brain damage. A fuller understanding of the mechanisms driving this will help define its developmental significance and in the refinement of strategies for brain repair using transplanted stem cells. Recently, we reported that in adult mouse there are ionic gradients within the extracellular spaces that create an electrical field (EF) within the rostral migratory stream (RMS), and that this acts as a guidance cue for neuroblast migration. Here, we demonstrate an endogenous EF in brain slices and show that mimicking this by applying an EF of physiological strength, switches on chain migration in mouse neurospheres and in the SH-SY5Y neuroblastoma cell line. Firstly, we detected a substantial endogenous EF of 31.8 ± 4.5 mV/mm using microelectrode recordings from explants of the subventricular zone (SVZ). Pharmacological inhibition of this EF, effectively blocked chain migration in 3D cultures of SVZ explants. To mimic this EF, we applied a physiological EF and found that this increased the expression of N-cadherin and β-catenin, both of which promote cell-cell adhesion. Intriguingly, we found that the EF up-regulated P2Y purinoceptor 1 (P2Y1) to contribute to chain migration of neuroblasts through regulating the expression of N-cadherin, β-catenin and the activation of PKC. Our results indicate that the naturally occurring EF in brain serves as a novel stimulant and directional guidance cue for neuronal chain migration, via up-regulation of P2Y1.
KeywordsNeuroblasts Chain migration P2Y1 β-catenin Extracellular electrical gradients
P2Y purinoceptor 1
Rostral migratory stream
Central nervous system
Protein kinase C
The migration of neuronal precursor cells is essential in CNS development and repair. In postnatal mammalian brain, neuroblasts from the subventricular zone (SVZ) migrate within the rostral migratory stream (RMS) in intimate cell-cell contact with one another, forming a chain-like organization without a glial scaffold [1, 2, 3].
The persistence of chain migration depends on the interplay of directional cell movement and biased cell to cell contact . For example, cell–cell interactions mediated via selective adhesion molecules establish and maintain chain migration. N-cadherin is expressed abundantly in chain migrating cells in the SVZ and RMS, but is down-regulated after cells exit these regions . Another cell adhesion molecule, NCAM, is expressed at high levels on neuronal precursor cells migrating towards the olfactory bulb (OB) [6, 7] and girdin, α6β1 integrin and miR-9 (brain-specific microRNA-9) are additional intrinsic factors regulating chain migration along the RMS [8, 9, 10]. However, it remains poorly understood how neuroblasts use locally acting extracellular cues and intrinsic molecular machinery to coordinate their behaviour during chain migration within the RMS .
Extracellular electrical signals also regulate neuronal migration, nerve growth cone guidance, nerve sprouting and nerve regeneration and are widespread in developing and regenerating tissues, including brain and spinal cord [11, 12]. They are present during neurulation, are expressed continuously across the wall of the differentiating neural tube and disrupting them, by chemical, physical, or electrical means causes major developmental abnormalities in the formation of the nervous system [13, 14, 15, 16, 17, 18]. In addition, epileptic seizure, stroke, ischaemia, migraine and acute damage to the hippocampus all induce extracellular electrical signals in brain that persist for hours to days [19, 20, 21, 22, 23]. Recently, we measured directly an electrical gradient of 5.7 ± 1.2 mV/mm within the extracellular spaces along the RMS ex vivo . Using an applied EF of similar strength (~5 mV/mm), directional migration of epithelial cells, neonatal neurones and neuroblasts is induced in vitro and in vivo [24, 25, 26, 27, 28].
Neuronal chain migration involves coordinated cell migrations. Collective migration keeps tissues intact during remodelling, allows mobile cells to carry immobile cells along with them and allows migrating cells to influence each other to ensure appropriate cell distribution and shaping of a tissue . Neuroblasts from the SVZ and newly divided neurons are recruited to the striatum following a stroke  and neurogenesis and neuronal stem cell migrations occur in the cortex and the hippocampus after ischaemic insults  which cause local EFs. Here, we found that the extracellular EF contributes to chain migration of neuroblasts. This new perspective indicates that the development of new therapeutic strategies involving electrical guidance may be useful to treat brain injury and disease.
Methods and Materials
Adult mice (C57BL/6j, 6–8 weeks, male or female), purchased from Charles River Laboratories, were euthanized in a CO2 chamber. The method for culturing neurospheres has been described previously [32, 33]. In brief, blocks of SVZ tissue were dissected from mouse brain and digested to release cells. Then a density gradient (OptiPrep density 1.32, Sigma) was used to purify the cells and cultures were suspended in Neurobasal medium (Life technology), supplemented with 2 mM l-glutamine, 2 % B-27 (without Retinyl acetate, Life technology), 20 ng/ml EGF (Life technology), 20 ng/ml FGF-2 (Life technology) and penicillin-streptomycin mixture (Life technologies) at 37 °C in humidified air containing 5 % CO2.
Electrotaxis Detection With/Without Extracellular ATP Treatment
Methods of exposing cells to an applied electric field in an electrotaxis detection chamber have been described . The SVZ neuroblasts were seeded in an electrotaxis chamber created in Falcon tissue culture dishes (BD Biosciences) which had been coated with 2 % Matrigel (BD Biosciences) [35, 36]. The cells were allowed to settle and adhere on the base of the chamber for 1 h. A roof of coverglass was applied and sealed with high vacuum silicone grease (Dow Corning Corporation) to the side walls of the chamber . The final dimensions of the shallow chamber, through which the electric current was passed, were 20 mm × 10 mm × 0.3 mm. We applied EFs 10 mV/mm to neurospheres and 5–100 mV/mm to SH-SY5Y cells with/without 100 μM ATP (Sigma) treatment through agar-salt bridges that connected silver/silver chloride electrodes in beakers containing Steinberg’s solution to pools of culture medium at either side of the chamber. The dish was placed on a Zeiss Axiovert 100 microscope with a stage incubator controlling temperature at 37 °C. Images of cells were recorded every 10 min and analyzed with Digital Pixel and MetaMorph imaging systems (Zeiss Axiovert 100 microscope) [18, 37]. Assessment of migration directedness (cosine θ) was used to quantify how directionally a cell migrated in the field, where θ is the angle between the EF vector and a straight line connecting the start and end positions of a cell . Migration rate was analyzed using the following two parameters. Trajectory rate (Tt/T) is the total length of the migration trajectory of a cell (Tt) divided by the given period of time (T). Displacement rate (Td/T) is the straight-line distance between the start and end positions of a cell (Td) divided by the time (T).
Cell lysates were collected for Western blot experiments. Western blotting was performed as described previously . Primary antibodies used included anti-β-Catenin (Cell Signalling), anti-N-cadherin (Abcam), anti-P2Y1 (Cell Signalling), anti-p-PKC (Cell Signalling) and anti-GAPDH (Santa Cruz). Immunoblots were detected by Luminata Forte Western HRP substrate (Millipore).
To knock-down P2Y1 in mouse derived neurospheres and SH-SY5Y cells, we used 100 nM of siRNA SMARTpools (a mixture of four siRNA duplexes each) using Dharmafect 1 (Thermo Scientific) according to the manufacturer’s specifications. siRNAp2y1 was comprised of: 5′-CUAUUGGUUUUAAUCUGUU-3′, 5′-GUUGAAACUUGUAAAUCUC-3′, 5′-GUAUUUAUUGAAGAGGUUU-3′, 5′- GUACUAGUGUAAAUUCUAU-3′. Under these conditions, 70–90 % of transfection efficiency was achieved, as judged by siGloGreen control (Thermo Scientific). After 72 h transfection, cells were exposed to the EF (no EF application as a control) and cell lysates were collected to confirm the final knock-down effect by Western blotting.
Cultured neurospheres were fixed with 4 % paraformaldehyde in PBS for 20 min. After incubation with blocking solution for 90 min at 37 °C, cells or tissue sections were incubated overnight with primary antibodies at 4 °C. Primary antibody dilutions used were as follows: Anti-N-Cadherin, 1:200 (Millipore), anti-β-Catenin 1:200 (BD Biosciences) and anti-P2Y1 1:200 (cell signalling). Samples then were incubated for 2 h with a secondary antibody. After washing, counterstaining with DAPI and mounting in anti-fading medium, they were visualized using a Zeiss LSM 510 Meta confocal microscope (Zeiss).
Brain Slice Culture
SVZ explants were prepared from P7 neonatal mice as described previously . Briefly, brains taken from mice were sliced using a tissue chopper (Mickle Laboratory Engineering)or with a blade under dissecting microscope at 300-500 μm thickness. Then, SVZ tissue was dissected and cut into small pieces under the stereomicroscope to be collected in cold Hanks’ balanced salt solution (Life technology). These pieces were embedded on glass-bottom dishes (Matsunami Glass) in a 3:1 mixture of Matrigel (BD Bioscience) and Neurobasal medium (Life technology) supplemented with 2 mM l-glutamine, 2 % B-27 (Life technology), and penicillin-streptomycin mixture (Life technology) at 37 °C in humidified air containing 5 % CO2 . For inhibition experiments, drugs were added to the medium at the beginning of culture and used at the following concentrations: 10 μM digoxin (Calbiochem) and 10 μM ouabain (Calbiochem).
Electrophysiological experiments to measure the electrical field in brain explants were conducted using sharp borosilicate glass microelectrodes. Electrodes were filled with artificial cerebrospinal fluid (ACSF) and had resistances of 2–20 MΩ. All recordings were made at room temperature (20–25 ºC) with an Axoclamp 2A switching amplifier. After balancing the amplifier with respect to ground (0 mV), a pipette was inserted into a 2 × 1 × 0.4 mm brain slice using a Narishige 3D micro-manipulator (Fig. 4b). The brain explants were bathed in ACSF at pH 7.4. The potential difference distribution in the brain slice was determined consistently at 15 s after the electrode was positioned in the tissue and recorded continually for at least 2 min by Scope v3.6-10. At least 4 slices in control and in each treatment group were measured and all experiments were repeated four times. For inhibitor experiments, cells were pre-incubated with 10 μM ouabain or 10 μM digoxin (both are Na+/K+-ATPase inhibitors, Sigma), for the time indicated and then exposed to the EF.
A minimum of three replicates was analysed for each experiment presented. Data are presented as the mean ± s.e.m. Student’s t test was used to assess the significant difference. Differences were considered as statistically significant with a p value <0.05.
Extracellular EFs regulate chain migration in explants from the SVZ of mice
Mimicking the endogenous electrical cue induced chain migration in cultured mouse neuroblasts
Next, we detected two molecules known as markers of typical chain migration in neuroblasts, N-cadherin  and β-catenin. Up-regulation of N-cadherin and β-catenin was seen only within the migrating chains exposed to an EF (Fig. 2b). In contrast, there was significantly lower expression of N-cadherin and β-catenin in no EF treated control cells (Fig. 2b). Protein analysis with Western blotting confirmed this (Fig. 2c), indicating that an applied physiological EF induced the formation of chain migration in cultured mouse neuroblasts by increasing cell-cell interaction. This was achieved by an EF-induced increased expression of N-cadherin and β-catenin which promoted and maintained cell-cell contacts and the extension of processes over the other cells in the migrating chains.
A physiological EF increased cell-cell contacts and directed migration in SH-SY5Y cells
P2Y1 Mediated the Chain Migration Induced by a Physiological EF
Extracellular ATP Increased EF-Directed Neuronal Migration
In adult brain, neuroblasts are generated and complete their initial differentiation in the SVZ. They then migrate directionally as neuronal chains, sliding along each other in the RMS from SVZ to OB [53, 54, 55]. Upon arriving at the OB, the new neurons differentiate into olfactory interneurons, and integrate into the olfactory processing system. Chain migration is regulated by multiple cellular and molecular cues, the coordination of which is still unclear [4, 10, 56]. We report a novel mechanism in which extracellular electrical signals contribute to directed chain migration of neuroblasts through P2Y1 receptor signalling.
Early recordings showed that the cortical surface was 0.5 to 5.5 mV positive to the ventricle . In addition, synchronous neuronal discharges within highly laminar structures such as the hippocampus generate substantial extracellular field potentials . Turner et al. found extracellular EFs ranging from 6 to 31 mV/mm in adult rat hippocampus, whilst EFs ranged from 13 to 43 mV/mm in adult turtle cerebellum . In the developing embryonic neural tube also, extracellular EFs of 10–100 mV mm−1 have been recorded [13, 15, 16, 59]. Here, we detected an extracellular EF of 31.8 ± 4.5 mV/mm in neonatal mouse SVZ slices (Fig. 1). Previously, we have measured directly an electric gradient of 5.7 ± 1.2 mV/mm in the extracellular spaces along the rostral migratory stream . Clearly extracellular electrical gradients are widespread in different developing, neonatal and adult brain locations although their physiological roles remain little recognised and poorly understood.
EFs are widespread also in other developing and regenerating tissues where they regulate cell division and directed cell migration [11, 12, 25, 38]. Intriguingly, most cell migrations in developing and damaged brain occur through tissues in which steady electrical signals exist . For example, epileptic seizure, stroke, ischaemia, migraine and acute damage to the hippocampus all induce extracellular electrical signals in brain that persist for hours [19, 20, 21, 22, 23]. Thus, the endogenous EFs represent a novel and powerful signalling mechanism with the potential to guide cell migration. In addition, EF-induced directional re-orientation of the leading edge cells of large epithelial sheets is E-cadherin dependent . To migrate at high speed over long distance within neuronal chains, the collective of neuroblasts needs to slide along each other efficiently  and for this, a consistent direction and maintained cell-cell contacts are essential. Here, we found that a physiological EF induced cathodal-directed migration and increased the expression of N-cadherin and β-catenin to promote and maintain the cell-cell connections between neuroblasts. Consistent with this, inhibition of the extracellular electrical signal in SVZ slices effectively disrupted the formation and directed migration of neuroblast chains. This is the first evidence that the natural extracellular electrical gradients of the brain contribute to the formation, maintenance and directed migration of chains of neuroblasts.
Several proteins that regulate chain formation in RMS have been identified. For example, mice lacking the cell-surface receptors ErbB4 and ApoER2 lost chain migration in the RMS and new neurons arriving in the OB were reduced [62, 63]. Extracellular matrix-related molecules such as α6β1-integrin and ADAM2 protease also are involved [64, 65, 66], indicating that both cellular receptor and extracellular matrix elements regulate neuroblast chain formation in brain. Additionally, extracellular signals such as ATP dynamically reorganize the cytoskeleton of each migrating neuroblast and regulate chemotaxis of microglia via Gi/o-coupled P2Y receptors [67, 68]. Early studies showed that electrical stimulation of axons liberated ATP [69, 70] and that extracellular ATP induces excitation and increases in calcium in neurons [49, 50, 71, 72], Erk activation , and calcium wave propagation . This calcium signalling is essential for directed cell migration induced by an applied EF . Furthermore, an applied EF transiently elevated extracellular ATP and caused Akt phosphorylation that was additive to insulin and inhibited by suramin (inhibitor of P2Y receptors) . Tran et al. demonstrated a role for extracellular ATP, purinergic receptors and protein kinase signalling in enhancing N-cadherin expression indicating a role in cell-cell interactions . We found that an applied electrical signal which mimicked that found in brain up-regulated the expression of P2Y1 receptors to increase expression of N-cadherin and β-catenin. Therefore, the mechanism of EF-induced chain migration of neuroblasts most likely involves activation of the ATP/P2Y1 signalling pathway through up-regulation of P2Y1 expression and increased release of ATP.
Increased neurogenesis and migration of progenitor cells have been observed in animal models of epilepsy, stroke, trauma, Alzheimer’s disease, Parkinson’s disease and Huntington’s disease . In addition, high expression of P2Y1 is required for neuroblast migration in brain . We found that the exogenous EF promoted chain migration of neuroblasts through increasing the expression of P2Y1. This indicates that it may be possible to deliver neuroblasts to sites of brain injury and disease by directing their migration using an applied DC electric field. EFs have been used clinically in the treatment of human spinal cord lesions  and extending their use to brain lesions therefore is feasible.
We showed previously that there is an extracellular EF between SVZ and the OB which contributes to guidance of neuroblast migration along the RMS . Here, we show further that this endogenous EF regulates chain migration of neuroblasts by promoting and maintaining cell-cell connections by up-regulating the expression of N-cadherin and β-catenin. In addition, we found that up-regulation of the P2Y1 receptor contributes to EF-induced chain migration. Our data indicate that this naturally occurring electrical gradient within the extracellular spaces of the RMS acts as a guidance cue directing chain migration of neuroblasts between the SVZ and the OB. These findings present a highly novel perspective of one of the mechanisms controlling and guiding neuroblast migration in a specific part of the mammalian brain.
This work was supported by a grant from NHS Grampian.
Conflict of Interest
The authors declare that they have no conflicts of interest.
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