Fig 2 - uploaded by Sehoon Jeong
Content may be subject to copyright.
Live/dead staining of cultured neurons without astrocytes before and after electrical stimulation for 3 days. a, c Live/dead staining b, d Hoechst staining for nuclei. After stimulation, cellular necrosis (red dots in c) was observed around stimulating electrodes in neuron-only cultures and a number of live neurons (green cells in a, c) were diminished remarkably after electrical stimulation. The white squares at the centers indicate the position of the stimulating electrode. The inside of the dotted line is the area for counting neurons. Scale bar 50 lm
Source publication
Recently, we found that electrical stimulation can induce neuronal migration in neural networks cultured for more than 3 weeks on microelectrode arrays. Immuno-cytochemistry data showed that the aggregation of neurons was related to the emergence of astrocytes in culture. In this study, when neurons were cocultured with astrocytes, electrical stimu...
Citations
... It has been widely used for the characterization of the surface properties of the membrane, as well as for the separation of uniform cell subpopulations in a cell mixture [9]. In addition, electric fields are used for axonal guidance [10,11], and migration of stem cells [12] or neurons within neural networks [13]. ...
Cellular biomechanics can be manipulated by strong electric fields, manifested by the field-induced membrane deformation and migration (galvanotaxis), which significantly impacts normal cellular physiology. Artificial giant vesicles that mimic the phospholipid bilayer of the cell membrane have been used to investigate the membrane biomechanics subjected to electric fields. Under a strong direct current (DC) electric field, the vesicle membrane demonstrates various patterns of deformation, which depends on the conductivity ratio between the medium and the cytoplasm. The vesicle exhibits prolate elongation along the direction of the electric field if the cytoplasm is more conductive than the medium. Conversely, the vesicle exhibits an oblate deformation if the medium is more conductive. Unlike a biological cell, artificial vesicles do not migrate in the strong DC electric field. To reconcile the kinematic difference between a cell and a vesicle under a strong DC electric field, we proposed a structure that represents a low-conductive, “shell-like” membrane. This membrane separates the extracellular medium from the cytoplasm. We computed the electric field, induced surface charge and mechanical pressure on the fixed membrane surface. We also computed the overall translational forces imposed on the structure for a vesicle and a cell. The DC electric field generated a steady-state radial pressure due to the interaction between the local electric field and field-induced surface charges. The radial pressure switches its direction from “pulling” to “compressing” when the medium becomes more conductive than the cytoplasm. However, this switch can happen only if the membrane becomes extremely conductive under the strong electric field. The induced surface charges do not contribute to the net translational force imposed on the structure. Instead, the net translational force generated on the shell structure depends on its intrinsic charges. It is zero for the neutrally-charged, artificial vesicle membrane. In contrast, intrinsic charges in a biological cell could generate translational force for its movement in a DC electric field. This work provides insights into factors that affect cellular/vesicle biomechanics inside a strong DC electric field. It provides a quantitative explanation for the distinct kinematics of a spherical cell verses a vesicle inside the field.
... As such, neuronal development, and the morphological and functional specifications of neuronal sub-domains are distinctly regulated by cellular excitability and the synaptic transmission it enables [121]. Neuronal differentiation and migration depend heavily on electrical excitability and stimulation acting in concert with transcriptional and translational regulation of molecular guidance cues and glial and axonal scaffolds [122]. ...
Large-scale genetic sequencing studies have identified a wealth of genes in which mutations are associated with autism spectrum disorder (ASD). Understanding the biological function of these genes sheds light onto the neurodevelopmental basis of ASD. To this end, we defined functional categories representing brain development – (1) Cell Division and Survival, (2) Cell Migration and Differentiation, (3) Neuronal Morphological Elaboration, (4) Development and Regulation of Cellular Excitability, and (5) Synapse Formation and Function – and place 100 high confidence ASD-associated genes yielding at least 50 published PubMed articles into these categories based on keyword searches. We compare the categorization of ASD genes to genes associated with developmental delay (DD) and systematically review the published literature on the function of these genes. We find evidence that ASD-associated genes have important functions that span the neurodevelopmental continuum. Further, examining the temporal expression pattern of these genes using the BrainSpan Atlas of the Developing Human Brain supports their function across development. Thus, our analyses and review of literature on ASD gene function support a model whereby differences in brain development – from very early stages of macroarchitectural patterning to late stages of activity-dependent sculpting of synaptic connectivity – may lead to ASD. It will be important to keep investigating potential points of mechanistic convergence which could explain a common pathophysiological basis of ASD behind this disparate array of genes.
... The DRG explant contains neurons and support cells, which primarily consist of fibroblasts, pre-Schwann cells, and Schwann cells. Previously, the application of EF supported the migration of neuronal cell bodies [50]. However, the cells migrating from the explant in our study did not label for neurofilament and therefore the emigrating cells were not neuronal cells. ...
Objective:
To improve peripheral nerve repair, new techniques to increase the speed of regeneration are required. Studies have shown that electrical stimulation can enhance nerve regeneration; however, stimulation parameters that regulate growth increases are unknown. The objective of this work was to examine dorsal root ganglion (DRG) neurite extension, directionality, and density after using methods to specifically control ac electrical field (EF) intensity and frequency exposure.
Methods:
Chick DRG explants were exposed to 20 Hz, 200 Hz, 1 MHz, and 20 MHz sinusoidal electric field of 17.86 V/m, and tissue parameters were measured.
Results:
Results show that neurite extension and directionality were influenced by frequency, however, the ratio of support cell emigration with respect to neurite extension from the DRG body was not. These results were further verified through finite element modeling of intracellular calcium, which show that higher frequencies have minimal effect on intracellular calcium.
Conclusion:
In conclusion, these results demonstrate that (1) directional growth of neurites within EFs can be achieved, (2) high frequency stimulation in the MHz does not enhance or impair neurite growth, and (3) low frequency stimulation affects growth and directionality.
Significance:
The significance of this work is the direct comparison of neurite extension after high stimulation frequencies (MHz) with typical low frequency fields (20 and 200 Hz) and modeling the results with finite element modeling.
... 55−57 Furthermore, by accessing information about the neurons and the network configuration distant from microelectrodes we are filling a gap in the MEA spatial resolution. Moreover, the automatic retrieval of the neurons and microelectrode position may be useful in studies related to neurons' migrations, such as those described in Ref. 58. ...
Microelectrode Arrays (MEA) are devices for long term electrophysiological recording of extracellular spontaneous or evocated activities on in vitro neuron culture. This work proposes and develops a framework for quantitative and morphological analysis of neuron cultures on MEAs, by processing their corresponding images, acquired by fluorescence microscopy. The neurons are segmented from the fluorescence channel images using a combination of segmentation by thresholding, watershed transform, and object classification. The positioning of microelectrodes is obtained from the transmitted light channel images using the circular Hough transform. The proposed method was applied to images of dissociated culture of rat dorsal root ganglion (DRG) neuronal cells. The morphological and topological quantitative analysis carried out produced information regarding the state of culture, such as population count, neuron-to-neuron and neuron-to-microelectrode distances, soma morphologies, neuron sizes, neuron and microelectrode spatial distributions. Most of the analysis of microscopy images taken from neuronal cultures on MEA only consider simple qualitative analysis. Also, the proposed framework aims to standardize the image processing and to compute quantitative useful measures for integrated image-signal studies and further computational simulations. As results show, the implemented microelectrode identification method is robust and so are the implemented neuron segmentation and classification one (with a correct segmentation rate up to 84%). The quantitative information retrieved by the method is highly relevant to assist the integrated signal-image study of recorded electrophysiological signals as well as the physical aspects of the neuron culture on MEA. Although the experiments deal with DRG cell images, cortical and hippocampal cell images could also be processed with small adjustments in the image processing parameter estimation.
... Primary hippocampal neurons were obtained by referring to Ref. [10]. Whole brains were isolated from fetuses of embryonic day 17 Sprague-Dawley rats (Samtako, Osan, Korea) and were placed in sterile buffered saline solution (BSS). ...
Since in vitro neural recording and imaging applications based on a surface plasmon resonance (SPR) technique have expanded dramatically in recent years, cytotoxicity assessment to ensure the biosafety and biocompatibility for those applications is crucial. Here, we report the cytotoxicity of the SPR substrate incorporating a flint glass whose refractive index is larger than that of a conventional crown glass. A high refractive index glass substrate is essential in neural signal detection due to the advantages such as high sensitivity and wide dynamic range. From experimental data using primary hippocampal neurons, it is found that a lead-based flint glass is not appropriate as a neural recording template although the neuron cells are not directly attached to the toxic glass. We also demonstrate that the adhesion layer between the glass substrate and the gold film plays an important role in achieving the substrate stability and the cell viability.
... Primary hippocampal neurons are obtained by referring to the works by Jeong et al. [12]. Whole brains are isolated from fetuses of embryonic day 17 Sprague-Dawley rats (Samtako, Korea) and are placed in sterile buffered saline solution (BSS). ...
We demonstrate the proof-of-concept for developing a multi-color fluorescence imaging system based on plasmonic wavelength selection and double illumination by white light source. This technique is associated with fluorescence excitation by transmitted light via a diffraction of propagating surface plasmons. Since double illumination through both sides of isosceles triangle prism in the Kretschmann configuration enables multiple transmission beams of different wavelengths to interact with the specimen, our approach can be an alternative to conventional fluorescence detection owing to alignment stability and functional expandability. After fabricating a plasmonic wavelength splitter and integrating it with microscopic imaging system, we successfully confirm the performance by visualizing in vitro neuron cells labeled with green and red fluorescence dyes. The suggested method has a potential that it could be combined with plasmonic biosensor scheme to realize a multi-functional platform which allows imaging and sensing of biological samples at the same time.
... as evidenced, electrical currents have been detected around damaged nerves in the spinal cord (Borgens et al. 1980), where they are believed to enhance axonal regeneration (Mccaig et al. 2005). an in vitro study showed that cultured progenitor cells, derived from the rat cNs, migrate toward the area of electrical stimulation (Jeong et al. 2009). ...
... With regard to the lFs, beneficial effects have been reported by cortical stimulation with 30-40 hz for pain relief as well as in rehabilitation therapies in stroke patients (tsubokawa et al. 1991a, b, 1993Nguyen et al. 1999). In addition, in vitro studies using rat cNs derived cultured progenitor cells showed that 20-50 hz electrical stimulation induced neuronal migration (Jun et al. 2007;Jeong et al. 2009;li et al. 2010). In addition, we applied 330 hz as hFs based on the beneficial effects of this frequency on motor function in an animal model of stroke (salimi et al. 2008). ...
Clinical and preclinical investigations suggest that epidural stimulation of the motor cortex (MC) can improve stroke-induced neurological deficits. The mechanisms involved in stimulation-induced recovery are not well understood and might involve neurogenesis-related processes. Here, we addressed the question whether MC stimulation influences processes of migration and differentiation of neuronal progenitor cells in vivo. Epidural stimulation electrodes were implanted at the level of the MC in rats, and electrical current was applied for a period of 1 month. Increased cell proliferation was observed in the subventricular zone (SVZ). We also found evidences for enhanced cell migration toward the source of current, a process known as electrotaxis. Some of these cells expressed the neuronal marker, NeuN. In addition, our results indicate that MC stimulation enhances neuronal activity of the dorsal raphe nucleus, leading to an increase in the expression of 5-hydroxytryptamine in the SVZ. It is known that such an increase can promote formation of new cells in the SVZ. Our findings suggest that epidural MC stimulation influences neurogenesis-related processes in animal models.
... Potential role for glia in DBS effect V Vedam-Mai et al Neurogenic astrocytes-stimulation of neural precursor proliferation by DBS? A study by Jeong et al. 77 in a co-culture of neurons and astrocytes on microelectrode arrays demonstrated that electrical stimulation led to migration of neuronal cell bodies, and was enhanced by the presence of astrocytes near stimulating electrodes. This report also provided evidence that electrical stimulation could induce an increase in spontaneous proliferative activity. ...
Deep brain stimulation (DBS) has emerged as a powerful surgical therapy for the management of treatment-resistant movement disorders, epilepsy and neuropsychiatric disorders. Although DBS may be clinically effective in many cases, its mode of action is still elusive. It is unclear which neural cell types are involved in the mechanism of DBS, and how high-frequency stimulation of these cells may lead to alleviation of the clinical symptoms. Neurons have commonly been a main focus in the many theories explaining the working mechanism of DBS. Recent data, however, demonstrates that astrocytes may be active players in the DBS mechanism of action. In this review article, we will discuss the potential role of reactive and neurogenic astrocytes (neural progenitors) in DBS.
... In addition, we used a monopolar setting to reduce the risk of tissue damage (Temel et al., 2004). The stimulation frequencies were low (30 Hz) (Jeong et al., 2009;Nguyen et al., 1999;Tsubokawa et al., 1993) or high (330 Hz) (Salimi et al., 2008), based on findings on cell mobility and regenerative processes, respectively. We applied low amplitude current, approximately 80 µA/contact, in the range, which is observed in physiological conditions in neuronal networks in vivo (Borgens and Shi, 1995). ...
Electrical brain stimulation used to treat a variety of neurological and psychiatric diseases is entering a new period. The technique is well established and the potential complications are well known and generally manageable. Recent studies demonstrated that electrical fields (EFs) can enhance neuroplasticity-related processes. EFs applied in the physiological range induce migration of different neural cell types from different species in vitro. There are some evidences that also the speed and directedness of cell migration are enhanced by EFs. However, it is still unclear how electrical signals from the extracellular space are translated into intracellular actions resulting in the so-called electrotaxis phenomenon. Here, we aim to provide a comprehensive review of the data on responses of cells to electrical stimulation and the relation to functional recovery.
