Matching Items (3)
Description
Gene manipulation techniques, such as RNA interference (RNAi), offer a powerful method for elucidating gene function and discovery of novel therapeutic targets in a high-throughput fashion. In addition, RNAi is rapidly being adopted for treatment of neurological disorders, such as Alzheimer's disease (AD), Parkinson's disease, etc. However, a major challenge in both of the aforementioned applications is the efficient delivery of siRNA molecules, plasmids or transcription factors to primary cells such as neurons. A majority of the current non-viral techniques, including chemical transfection, bulk electroporation and sonoporation fail to deliver with adequate efficiencies and the required spatial and temporal control. In this study, a novel optically transparent biochip is presented that can (a) transfect populations of primary and secondary cells in 2D culture (b) readily scale to realize high-throughput transfections using microscale electroporation and (c) transfect targeted cells in culture with spatial and temporal control. In this study, delivery of genetic payloads of different sizes and molecular characteristics, such as GFP plasmids and siRNA molecules, to precisely targeted locations in primary hippocampal and HeLa cell cultures is demonstrated. In addition to spatio-temporally controlled transfection, the biochip also allowed simultaneous assessment of a) electrical activity of neurons, b) specific proteins using fluorescent immunohistochemistry, and c) sub-cellular structures. Functional silencing of GAPDH in HeLa cells using siRNA demonstrated a 52% reduction in the GAPDH levels. In situ assessment of actin filaments post electroporation indicated a sustained disruption in actin filaments in electroporated cells for up to two hours. Assessment of neural spike activity pre- and post-electroporation indicated a varying response to electroporation. The microarray based nature of the biochip enables multiple independent experiments on the same culture, thereby decreasing culture-to-culture variability, increasing experimental throughput and allowing cell-cell interaction studies. Further development of this technology will provide a cost-effective platform for performing high-throughput genetic screens.
ContributorsPatel, Chetan (Author) / Muthuswamy, Jitendran (Thesis advisor) / Helms Tillery, Stephen (Committee member) / Jain, Tilak (Committee member) / Caplan, Michael (Committee member) / Vernon, Brent (Committee member) / Arizona State University (Publisher)
Created2012
Description
A direct Magnetic Resonance (MR)-based neural activity mapping technique with high spatial and temporal resolution may accelerate studies of brain functional organization.
The most widely used technique for brain functional imaging is functional Magnetic Resonance Image (fMRI). The spatial resolution of fMRI is high. However, fMRI signals are highly influenced by the vasculature in each voxel and can be affected by capillary orientation and vessel size. Functional MRI analysis may, therefore, produce misleading results when voxels are nearby large vessels. Another problem in fMRI is that hemodynamic responses are slower than the neuronal activity. Therefore, temporal resolution is limited in fMRI. Furthermore, the correlation between neural activity and the hemodynamic response is not fully understood. fMRI can only be considered an indirect method of functional brain imaging.
Another MR-based method of functional brain mapping is neuronal current magnetic resonance imaging (ncMRI), which has been studied over several years. However, the amplitude of these neuronal current signals is an order of magnitude smaller than the physiological noise. Works on ncMRI include simulation, phantom experiments, and studies in tissue including isolated ganglia, optic nerves, and human brains. However, ncMRI development has been hampered due to the extremely small signal amplitude, as well as the presence of confounding signals from hemodynamic changes and other physiological noise.
Magnetic Resonance Electrical Impedance Tomography (MREIT) methods could have the potential for the detection of neuronal activity. In this technique, small external currents are applied to a body during MR scans. This current flow produces a magnetic field as well as an electric field. The altered magnetic flux density along the main magnetic field direction caused by this current flow can be obtained from phase images. When there is neural activity, the conductivity of the neural cell membrane changes and the current paths around the neurons change consequently. Neural spiking activity during external current injection, therefore, causes differential phase accumulation in MR data. Statistical analysis methods can be used to identify neuronal-current-induced magnetic field changes.
The most widely used technique for brain functional imaging is functional Magnetic Resonance Image (fMRI). The spatial resolution of fMRI is high. However, fMRI signals are highly influenced by the vasculature in each voxel and can be affected by capillary orientation and vessel size. Functional MRI analysis may, therefore, produce misleading results when voxels are nearby large vessels. Another problem in fMRI is that hemodynamic responses are slower than the neuronal activity. Therefore, temporal resolution is limited in fMRI. Furthermore, the correlation between neural activity and the hemodynamic response is not fully understood. fMRI can only be considered an indirect method of functional brain imaging.
Another MR-based method of functional brain mapping is neuronal current magnetic resonance imaging (ncMRI), which has been studied over several years. However, the amplitude of these neuronal current signals is an order of magnitude smaller than the physiological noise. Works on ncMRI include simulation, phantom experiments, and studies in tissue including isolated ganglia, optic nerves, and human brains. However, ncMRI development has been hampered due to the extremely small signal amplitude, as well as the presence of confounding signals from hemodynamic changes and other physiological noise.
Magnetic Resonance Electrical Impedance Tomography (MREIT) methods could have the potential for the detection of neuronal activity. In this technique, small external currents are applied to a body during MR scans. This current flow produces a magnetic field as well as an electric field. The altered magnetic flux density along the main magnetic field direction caused by this current flow can be obtained from phase images. When there is neural activity, the conductivity of the neural cell membrane changes and the current paths around the neurons change consequently. Neural spiking activity during external current injection, therefore, causes differential phase accumulation in MR data. Statistical analysis methods can be used to identify neuronal-current-induced magnetic field changes.
ContributorsFu, Fanrui (Author) / Sadleir, Rosalind (Thesis advisor) / Kodibagkar, Vikram (Committee member) / Kleim, Jeffrey (Committee member) / Muthuswamy, Jitendran (Committee member) / Helms Tillery, Stephen (Committee member) / Arizona State University (Publisher)
Created2019
Description
Intracellular voltage recordings from single neurons in vitro and in vivo have been fundamental to our understanding of neuronal function. Conventional electrodes and associated positioning systems for intracellular recording in vivo are large and bulky, which has largely restricted their use to single-channel recording from anesthetized animals. Further, intracellular recordings are very cumbersome, requiring a high degree of skill not readily achieved in a typical laboratory. This dissertation presents a robotic, head-mountable, MEMS (Micro-Electro-Mechanical Systems) based intracellular recording system to overcome the above limitations associated with form-factor, scalability and highly skilled and tedious manual operations required for intracellular recordings. This system combines three distinct technologies: 1) novel microscale, polycrystalline silicon-based electrode for intracellular recording, 2) electrothermal microactuators for precise microscale navigation of the electrode and 3) closed-loop control algorithm for autonomous movement and positioning of electrode inside single neurons. First, two distinct designs of polysilicon-based microscale electrodes were fabricated and tested for intracellular recordings. In the first approach, tips of polysilicon microelectrodes were milled to nanoscale dimensions (<300 nm) using focused ion beam (FIB) to develop polysilicon nanoelectrodes. Polysilicon nanoelectrodes recorded >1.5 mV amplitude, positive-going action potentials and synaptic potentials from neurons in the abdominal ganglion of Aplysia Californica. In the second approach, polysilicon microelectrodes were integrated with miniaturized glass micropipettes filled with electrolyte to fabricate glass-polysilicon microelectrodes. These electrodes consistently recorded high fidelity intracellular potentials from neurons in the abdominal ganglion of Aplysia Californica (Resting Potentials < -35 mV, Action Potentials > 60 mV) as well as the rat motor cortex (Resting Potentials < -50 mV). Next, glass-polysilicon microelectrodes were coupled with microscale electrothermal actuators and controller for autonomous intracellular recordings from single neurons in the abdominal ganglion. Consistent resting potentials (< -35 mV) and action potentials (> 60 mV) were recorded after each successful penetration attempt with the controller and microactuated glass-polysilicon microelectrodes. The success rate of penetration and quality of recordings achieved using electrothermal microactuators were comparable to that of conventional positioning systems. Finally, the feasibility of this miniaturized system to obtain intracellular recordings from single neurons in the motor cortex of rats in vivo is also demonstrated. The MEMS-based system offers significant advantages: 1) reduction in overall size for potential use in behaving animals, 2) scalable approach to potentially realize multi-channel recordings and 3) a viable method to fully automate measurement of intracellular recordings.
ContributorsSampath Kumar, Swathy (Author) / Muthuswamy, Jit (Thesis advisor) / Abbas, James (Committee member) / Hamm, Thomas (Committee member) / Christen, Jennifer Blain (Committee member) / Buneo, Christopher (Committee member) / Arizona State University (Publisher)
Created2018