The use of direct current electric stimulation (DCS) is an effective strategy to treat disease and enhance body functionality. Thus, treatment with DCS is an attractive biomedical alternative, but the molecular underpinnings remain mostly unknown. The lack of experimental models to dissect the effects of DCS from molecular to organismal levels is an important caveat. Here, we introduce the planarian flatworm Schmidtea mediterranea as a tractable organism for in vivo studies of DCS. We developed an experimental method that facilitates the application of direct current electrical stimulation to the whole planarian body (pDCS).
Materials and Methods:
Planarian immobilization was achieved by combining treatment with anesthesia, agar embedding, and low temperature via a dedicated thermoelectric cooling unit. Electric currents for pDCS were delivered using pulled glass microelectrodes. The electric potential was supplied through a constant voltage power supply. pDCS was administered up to six hours, and behavioral and molecular effects were measured by using video recordings, immunohistochemistry, and gene expression analysis.
Results:
The behavioral immobilization effects are reversible, and pDCS resulted in a redistribution of mitotic cells along the mediolateral axis of the planarian body. The pDCS effects were dependent on the polarity of the electric field, which led to either increase in reductions in mitotic densities associated with the time of pDCS. The changes in mitotic cells were consistent with apparent redistribution in gene expression of the stem cell marker smedwi-1.
Conclusion:
The immobilization technique presented in this work facilitates studies aimed at dissecting the effects of exogenous electric stimulation in the adult body. Treatment with DCS can be administered for varying times, and the consequences evaluated at different levels, including animal behavior, cellular and transcriptional changes. Indeed, treatment with pDCS can alter cellular and transcriptional parameters depending on the polarity of the electric field and duration of the exposure.
Get full access to this article
View all access options for this article.
References
1.
AleemIS, AleemI, EvaniewN, et al.Efficacy of electrical stimulators for bone healing: A meta-analysis of randomized sham-controlled trials. Sci Rep, 2016; 6:31724.
2.
GriffinM, BayatA. Electrical stimulation in bone healing: Critical analysis by evaluating levels of evidence. Eplasty, 2011; 11:e34.
3.
HaoS, TangB, WuZ, et al.Forniceal deep brain stimulation rescues hippocampal memory in Rett syndrome mice. Nature, 2015; 526:430–434.
4.
KadoshR.Modulating and enhancing cognition using brain stimulation: Science and fiction. J Cogn Psychol, 2015; 27:141–163.
5.
KadoshR, SoskicS, IuculanoT, et al.Modulating neuronal activity produces specific and long-lasting changes in numerical competence. Curr Biol, 2010; 20:2016–2020.
6.
McCaigCD, RajnicekAM, SongB, et al.Controlling cell behavior electrically: Current views and future potential. Physiol Rev, 2005; 85:943–978.
7.
NelsonJ, McKinleyRA, PhillipsC, et al.The effects of transcranial direct current stimulation (tDCS) on multitasking throughput capacity. Front Hum Neurosci, 2016; 10:589.
8.
SankarT, ChakravartyMM, BescosA, et al.Deep brain stimulation influences brain structure in Alzheimer's disease. Brain Stimul, 2015; 8:645–654.
9.
WeaverFM, FollettK, SternM, et al.Bilateral deep brain stimulation vs best medical therapy for patients with advanced Parkinson disease: A randomized controlled trial. JAMA, 2009; 301:63–73.
10.
ObesoJA, OlanowCW, Rodriguez-OrozMC, et al.Deep-brain stimulation of the subthalamic nucleus or the pars interna of the globus pallidus in Parkinson's disease. N Engl J Med, 2001; 345:956–963.
11.
Coates McCallI, LauC, MiniellyN, et al.Owning ethical innovation: Claims about Commercial Wearable Brain Technologies. Neuron, 2019; 102:728–731.
12.
WexlerA, ReinerPB. Oversight of direct-to-consumer neurotechnologies. Science, 2019; 363:234–235.
13.
LevinM.Molecular bioelectricity: How endogenous voltage potentials control cell behavior and instruct pattern regulation in vivo. Mol Biol Cell, 2014; 25:3835–3850.
14.
MarshG, BeamsHW. Electrical control of morphogenesis in regenerating Dugesia tigrina. I. Relation of axial polarity to field strength. J Cell Comp Physiol, 1952; 39:191–213.
15.
AmirRE, Van den VeyverIB, WanM, et al.Rett syndrome is caused by mutations in X-linked MECP2, encoding methyl-CpG-binding protein 2. Nat Genet, 1999; 23:185–188.
16.
BakerSA, ChenL, WilkinsAD, et al.An AT-hook domain in MeCP2 determines the clinical course of Rett syndrome and related disorders. Cell, 2013; 152:984–996.
17.
ChahrourM, JungSY, ShawC, et al.MeCP2, a key contributor to neurological disease, activates and represses transcription. Science, 2008; 320:1224–1229.
18.
JohnsonBS, ZhaoYT, FasolinoM, et al.Biotin tagging of MeCP2 in mice reveals contextual insights into the Rett syndrome transcriptome. Nat Med, 2017; 23:1203–1214.
19.
SuginoK, HempelCM, OkatyBW, et al.Cell-type-specific repression by methyl-CpG-binding protein 2 is biased toward long genes. J Neurosci, 2014; 34:12877–12883.
20.
LuH, AshRT, HeL, et al.Loss and gain of MeCP2 cause similar hippocampal circuit dysfunction that is rescued by deep brain stimulation in a Rett syndrome mouse model. Neuron, 2016; 91:739–747.
21.
PohodichAE, YalamanchiliH, RamanAT, et al.Forniceal deep brain stimulation induces gene expression and splicing changes that promote neurogenesis and plasticity. eLife, 2018; 7:e34031.
22.
AboobakerAA.Planarian stem cells: A simple paradigm for regeneration. Trends Cell Biol, 2011; 21:304–311.
23.
KingRS, NewmarkPA. The cell biology of regeneration. J Cell Biol, 2012; 196:553–562.
24.
PellettieriJ, Sanchez AlvaradoA. Cell turnover and adult tissue homeostasis: From humans to planarians. Annu Rev Genet, 2007; 41:83–105.
25.
ReddienPW, Sánchez AlvaradoA. Fundamentals of planarian regeneration. Annu Rev Cell Dev Biol, 2004; 20:725–757.
26.
ReddienPW, OviedoNJ, JenningsJR, et al.SMEDWI-2 is a PIWI-like protein that regulates planarian stem cells. Science, 2005; 310:1327–1330.
27.
RinkJC.Stem Cells, Patterning and regeneration in planarians: Self-organization at the organismal scale. Methods Mol Biol, 2018; 1774:57–172.
28.
AdlerCE, Sanchez AlvaradoA. Types or states? Cellular dynamics and regenerative potential. Trends Cell Biol, 2015; 25:687–696.
29.
RinkJC.Stem cell systems and regeneration in planaria. Dev Genes Evol, 2013; 223:67–84.
30.
HoganBL, BarkauskasCE, ChapmanHA, et al.Repair and regeneration of the respiratory system: Complexity, plasticity, and mechanisms of lung stem cell function. Cell Stem Cell, 2014; 15:123–138.
31.
HsuYC, FuchsE. A family business: Stem cell progeny join the niche to regulate homeostasis. Nat Rev Mol Cell Biol, 2012; 13:103–114.
32.
MendelsonA, FrenettePS. Hematopoietic stem cell niche maintenance during homeostasis and regeneration. Nat Med, 2014; 20:833–846.
33.
BlackistonDJ, McLaughlinKA, LevinM. Bioelectric controls of cell proliferation: Ion channels, membrane voltage and the cell cycle. Cell Cycle, 2009; 8:3527–3536.
34.
LevinM.Large-scale biophysics: Ion flows and regeneration. Trends Cell Biol, 2007; 17:261–270.
35.
ZhaoM, SongB, PuJ, et al.Electrical signals control wound healing through phosphatidylinositol-3-OH kinase-gamma and PTEN. Nature, 2006; 442:457–460.
36.
OviedoNJ, NicolasCL, AdamsDS, et al.Establishing and maintaining a colony of planarians. CSH Protoc, 2008; 2008:pdb prot5053.
37.
GuedelhoeferOC, 4th, Sanchez AlvaradoA. Planarian immobilization, partial irradiation, and tissue transplantation. J Vis Exp 2012:4015.
38.
KingRS, NewmarkPA. In situ hybridization protocol for enhanced detection of gene expression in the planarian Schmidtea mediterranea. BMC Dev Biol, 2013; 13:8.
39.
PearsonBJ, EisenhofferGT, GurleyKA, et al.Formaldehyde-based whole-mount in situ hybridization method for planarians. Dev Dyn, 2009; 238:443–450.
40.
ForsthoefelDJ, RossKG, NewmarkPA, et al.Fixation, processing, and immunofluorescent labeling of whole mount planarians. Methods Mol Biol, 2018; 1774:353–366.
41.
AgataK, SoejimaY, KatoK, et al.Structure of the planarian central nervous system (CNS) revealed by neuronal cell markers. Zoolog Sci, 1998; 15:433–440.
42.
CollinsJJ, HouX, RomanovaEV, et al.Genome-wide analyses reveal a role for peptide hormones in planarian germline development. PLoS Biol, 2010; 8:e1000509.
43.
OviedoNJ, NicolasCL, AdamsDS, et al.Live imaging of planarian membrane potential using DiBAC4(3). CSH Protoc, 2008; 2008:pdb prot5055.
44.
StevensonCG, BeaneWS. A low percent ethanol method for immobilizing planarians. PLoS One, 2010; 5:e15310.
45.
DexterJP, TammeMB, LindCH, et al.On-chip immobilization of planarians for in vivo imaging. Sci Rep, 2014; 4:6388.
46.
GuedelhoeferOC, 4th, Sanchez AlvaradoA. Amputation induces stem cell mobilization to sites of injury during planarian regeneration. Development, 2012; 139:3510–3520.
47.
SongB, GuY, PuJ, et al.Application of direct current electric fields to cells and tissues in vitro and modulation of wound electric field in vivo. Nat Protoc, 2007; 2:1479–1489.
48.
BlattMR, SlaymanCL. KCl leakage from microelectrodes and its impact on the membrane parameters of a nonexcitable cell. J Membr Biol, 1983; 72:223–234.
49.
StonerLC, NatkeEJr., DixonMK. Direct measurement of potassium leak from single 3 M KCl microelectrodes. Am J Physiol, 1984; 246:F343–F348.
50.
BerendsonJ, SimonssonD. Electrochemical aspects of treatment of tissue with direct current. Eur J Surg Suppl 1994:111–115.
51.
BaguñàJ. Mitosis in the intact and regenerating planarian Dugesia mediterranea n.sp. I. Mitotic studies during growth, feeding and starvation. J Exp Zool, 1975; 195:53–64.
52.
PeirisTH, RamirezD, BarghouthPG, et al.Regional signals in the planarian body guide stem cell fate in the presence of genomic instability. Development, 2016; 143:1697–1709.
53.
LiWC, SoffeSR, RobertsA. A direct comparison of whole cell patch and sharp electrodes by simultaneous recording from single spinal neurons in frog tadpoles. J Neurophysiol, 2004; 92:380–386.
54.
DahanE, BizeV, LehnertT, et al.Rapid fluidic exchange microsystem for recording of fast ion channel kinetics in Xenopus oocytes. Lab Chip, 2008; 8:1809–1818.
55.
BergerS, LattmannE, Aegerter-WilmsenT, et al.Long-term C. elegans immobilization enables high resolution developmental studies in vivo. Lab Chip, 2018; 18:1359–1368.
56.
SabadoV, NagoshiE. Single-cell resolution fluorescence live imaging of drosophila circadian clocks in larval brain culture. J Vis Exp 2018:57015.
57.
GoldsteinDB, ChinJH. Interaction of ethanol with biological membranes. Fed Proc, 1981; 40:2073–2076.
58.
ChinJH, GoldsteinDB. Effects of low concentrations of ethanol on the fluidity of spin-labeled erythrocyte and brain membranes. Mol Pharmacol, 1977; 13:435–441.
59.
GoldmanY, MoradM. Measurement of transmembrane potential and current in cardiac muscle: A new voltage clamp method. J Physiol, 1977; 268:613–654.
60.
HagiwaraS, OzawaS, SandO. Voltage clamp analysis of two inward current mechanisms in the egg cell membrane of a starfish. J Gen Physiol, 1975; 65:617–644.
61.
BorgensRB, McGinnisME, VanableJWJr, et al.Stump currents in regenerating salamanders and newts. J Exp Zool, 1984; 231:249–256.
62.
ChiangMC, CragoeEJJr., VanableJWJr. Intrinsic electric fields promote epithelization of wounds in the newt, Notophthalmus viridescens. Dev Biol, 1991; 146:377–385.
63.
ZhaoM, ForresterJV, McCaigCD. A small, physiological electric field orients cell division. Proc Natl Acad Sci U S A, 1999; 96:4942–4946.
64.
MengX, ArocenaM, PenningerJ, et al.PI3K mediated electrotaxis of embryonic and adult neural progenitor cells in the presence of growth factors. Exp Neurol, 2011; 227:210–217.
65.
EricksonCA, NuccitelliR. Embryonic fibroblast motility and orientation can be influenced by physiological electric fields. J Cell Biol, 1984; 98:296–307.
66.
FengJF, LiuJ, ZhangL, et al.Electrical guidance of human stem cells in the rat brain. Stem Cell Reports, 2017; 9:177–189.
67.
ZhaoM, DickA, ForresterJV, et al.Electric field-directed cell motility involves up-regulated expression and asymmetric redistribution of the epidermal growth factor receptors and is enhanced by fibronectin and laminin. Mol Biol Cell, 1999; 10:1259–1276.
68.
BedlackRSJr., WeiM, LoewLM.Localized membrane depolarizations and localized calcium influx during electric field-guided neurite growth. Neuron, 1992; 9:393–403.
69.
YamashitaM.Electric axon guidance in embryonic retina: Galvanotropism revisited. Biochem Biophys Res Commun, 2013; 431:280–283.
70.
LiuC, BaiY, ChenY, et al.Reduction of Na/K-ATPase potentiates marinobufagenin-induced cardiac dysfunction and myocyte apoptosis. J Biol Chem, 2012; 287:16390–16398.
71.
MorganJI, CurranT. Stimulus-transcription coupling in the nervous system: Involvement of the inducible proto-oncogenes fos and jun. Annu Rev Neurosci, 1991; 14:421–451.
72.
JenningsJ, ChenD, FeldmanD. Transcriptional response of dermal fibroblasts in direct current electric fields. Bioelectromagnetics, 2008; 29:394–405.
73.
SundelacruzS, LevinM, KaplanDL. Role of membrane potential in the regulation of cell proliferation and differentiation. Stem Cell Rev Rep, 2009; 5:231–246.
74.
PaiVP, MartyniukCJ, EcheverriK, et al.Genome-wide analysis reveals conserved transcriptional responses downstream of resting potential change in Xenopus embryos, axolotl regeneration, and human mesenchymal cell differentiation. Regeneration (Oxf), 2016; 3:3–25.
75.
BarghouthPG, ThiruvalluvanM, OviedoNJ. Bioelectrical regulation of cell cycle and the planarian model system. Biochim Biophys Acta, 2015; 1848:2629–2637.
76.
SundelacruzS, LevinM, KaplanDL. Depolarization alters phenotype, maintains plasticity of predifferentiated mesenchymal stem cells. Tissue Eng Part A, 2013; 19:1889–1908.
77.
VadlamaniRA, NieY, DetwilerDA, et al.Nanosecond pulsed electric field induced proliferation and differentiation of osteoblasts and myoblasts. J R Soc Interface, 2019; 16:20190079.
78.
ConeCDJr., TongierMJr. Contact inhibition of division: Involvement of the electrical transmembrane potential. J Cell Physiol, 1973; 82:373–386.
79.
VodovnikL, MiklavcicD, SersaG. Modified cell proliferation due to electrical currents. Med Biol Eng Comput, 1992; 30:CE21–CE28.
80.
CunhaF, RajnicekAM, McCaigCD. Electrical stimulation directs migration, enhances and orients cell division and upregulates the chemokine receptors CXCR4 and CXCR2 in endothelial cells. J Vasc Res, 2019; 56:39–53.
81.
BeaneWS, MorokumaJ, AdamsDS, et al.A chemical genetics approach reveals H,K-ATPase-mediated membrane voltage is required for planarian head regeneration. Chem Biol, 2011; 18:77–89.
82.
DurantF, BischofJ, FieldsC, et al.The role of early bioelectric signals in the regeneration of planarian anterior/posterior polarity. Biophys J, 2019; 116:948–961.
83.
Emmons-BellM, DurantF, HammelmanJ, et al.Gap junctional blockade stochastically induces different species-specific head anatomies in genetically wild-type girardia dorotocephala flatworms. Int J Mol Sci, 2015; 16:27865–27896.
84.
WenemoserD, LapanSW, WilkinsonAW, et al.A molecular wound response program associated with regeneration initiation in planarians. Genes Dev, 2012; 26:988–1002.
85.
WurtzelO, CoteLE, PoirierA, et al.A Generic and cell-type-specific wound response precedes regeneration in planarians. Dev Cell, 2015; 35:632–645.
86.
WenemoserD, ReddienPW. Planarian regeneration involves distinct stem cell responses to wounds and tissue absence. Dev Biol, 2010; 344:979–991.
87.
YanT, JiangX, GuoX, et al.Electric field-induced suppression of PTEN drives epithelial-to-mesenchymal transition via mTORC1 activation. J Dermatol Sci, 2017; 85:96–105.
88.
PazmanyT, MurphySP, GollnickSO, et al.Activation of multiple transcription factors and fos and jun gene family expression in cells exposed to a single electric pulse. Exp Cell Res, 1995; 221:103–110.
89.
IvesterCT, KentRL, TagawaH, et al.Electrically stimulated contraction accelerates protein synthesis rates in adult feline cardiocytes. Am J Physiol, 1993; 265:H666–H674.
90.
HuangCW, ChenHY, YenMH, et al.Gene expression of human lung cancer cell line CL1–5 in response to a direct current electric field. PLoS One, 2011; 6:e25928.
91.
KimMS, KooH, HanSW, et al.Repeated anodal transcranial direct current stimulation induces neural plasticity-associated gene expression in the rat cortex and hippocampus. Restor Neurol Neurosci, 2017; 35:137–146.
92.
ZhangJ, NeohKG, HuX, et al.Combined effects of direct current stimulation and immobilized BMP-2 for enhancement of osteogenesis. Biotechnol Bioeng, 2013; 110:1466–1475.
93.
PaiVP, LemireJM, PareJF, et al.Endogenous gradients of resting potential instructively pattern embryonic neural tissue via Notch signaling and regulation of proliferation. J Neurosci, 2015; 35:4366–4385.
94.
D'ArcoM, DolphinAC. L-type calcium channels: On the fast track to nuclear signaling. Sci Signal, 2012; 5:pe34.
95.
D'AscenzoM, PiacentiniR, CasalboreP, et al.Role of L-type Ca2+ channels in neural stem/progenitor cell differentiation. Eur J Neurosci, 2006; 23:935–944.
96.
ZhangD, ChanJD, NogiT, et al.Opposing roles of voltage-gated Ca2+ channels in neuronal control of regenerative patterning. J Neurosci, 2011; 31:15983–15995.
Supplementary Material
Please find the following supplemental material available below.
For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.
For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.
