The effect of direct current electric fields on hippocampal neurons in vitro
Initiation and effectiveness of neuron migration to the right places are critical for the development and repair of central nervous system. It is now confirmed that mammalian, including human central nervous system (CNS) contains stem cells and nerve progenitor cells. Upon brain injury either from ischemia or trauma, those cells must be able to proliferate and migrate to the damaged parts in order to repair the damage. To understand the underlying mechanisms and find new techniques of directing neuron growth are of both scientific and medical significance. Endogenous electric fields (EFs) are widespread in developing and regenerating tissues. During embryonic development, endogenous EFs exist rostrocaudally and mediolaterally at the neural plate and neural fold stages. The size, location and developmental timing of EFs correlate with active neuron migration. Therefore, electric fields may provide a signal to direct cellular behavior of nerve cells during nervous system development and reparation. Application of EFs directs cell migration of many types of cells. However, whether neurons migrate directionally in applied electric fields has not been demonstrated. I have investigated the effects of applied physiological EFs on cultured hippocampal neurons and the underlying mechanisms. Hippocampal neurons from rats and mice were cultured on poly-L-lysine and laminin coated dishes and were identified with MAP-2 (a specific marker for neuron) staining. The neurons were exposed to small applied electric fields and the migration and division were recorded with an imaging system. 1. In a DC electric field, dissociated rat hippocampal neurons migrate to the cathode. The migration direction was reversed when the EF polarity was reversed. Neuronal migration in EFs involes the same steps as that in non-EF cultures, namely leading process extension, nuclear translocation and retraction of trailing process. 2. The cathodal migration of hippocampal neurons is voltage and time dependent. A DC EF does not have significant influence on neuronal migration speed. This result shows that the effect of EFs is different on neuronal migration speed and direction of migration. 3. The guidance effect of EFs is also seen in neuronal migration from hippocampal micro-explants and neuronal migration along glial process. Both types of migration are towards the cathode. 4. Electric fields direct growth cone path finding and neurite orientation. EF induced neurite orientation showed time and voltage dependence. Leading process branching, turning and swapping over of leading and trailing processes are the main types of neurite re-orientation. Guided neuronal migration and neuron leading process orientation are not accompanied with neuronal size change. 5. In an applied EF, MAP-2 (microtubule associated protein 2), p-Akt, Golgi apparatus and centrosome redistribute asymmetrically to the cathode facing side of hippocampal neurons. 6. Inhibition of ROCK (Rho-associated protein kinase) and Pi3k (phosphoinositide-3 kinase) with inhibitors decreases leading neurite orientation and Golgi polarization in the neurons in response to the EFs. They also decrease the directedness and speed of guided neuronal migration in EFs. 7. Null mutation of p110gamma, which encodes the catalytic domain of Pi3k gamma significantly decreased the electric field directed neuron migration. These results suggest that ROCK and Pi3k regulate EFs directed neuronal migration. 8. Application of electric fields also affects cell division of postnatal hippocampal neurons. The neuronal phenotype of divided cells was confirmed with MAP-2 and GFAP staining. Most cells divided with a cleavage plane perpendicular to the EF vector. 9. Using vibrating probe techniques, we show that there is a significant change in electric currents upon wounding hippocampus in vitro. In conclusion, this study demonstrates that hippocampal neurons from both rat and mice respond to applied EFs by directional migration and directional division. The electric field directed neuronal migration involves ROCK and Pi3k signaling. This raises the possibilities that 1) there may be a neglected role for endogenous electric fields in directing neuron migration and division; 2) electric fields may be used as a potential cue to direct neuronal migration in repair of the central nervous system.