Skip to main content
eScholarship
Open Access Publications from the University of California

Scholarly Works (6 results)

  • Thesis
  • Peer Reviewed
UC San Diego Electronic Theses and Dissertations (2022)

In neurons, intracellular calcium has the dual role of charge carrier and intracellular messenger. The signaling function of calcium has aspects that are particularly important to neurons. Synaptic transmission, the form of secretion that leads to the release of neurotransmitters, the process of learning and the formation and consolidation of memory, long term potentiation or depression of synaptic transmission, the direct coupling between depolarization of the plasma membrane and the increase of intracellular calcium are all under the control of calcium signals. Neuronal viability depends on proper functioning of all these processes and to a greater degree depend on the precise temporal and spatial regulation of calcium signals than most other eukaryotic cells. The primary focus of this work is the development of experimental and analytical tools for studies of chronic neurodegenerative disorders. This work investigates the spontaneous calcium activity in primary, dissociated cortical neurons as compared to Huntington’s disease (HD) model cells. The relative fluorescence variation of calcium-sensitive Fluo 4 is measured and analyzed using a custom, GUI-based software tool: Fluorescence Emissions Signal Analyzer (FESA). Characterization of intracellular calcium activity with well-defined parameters within emissions signal activity across several timepoints throughout incubation highlight calcium dysregulation among HD model cells. In addition, methods for optimizing the viability of long-term neuronal cultures are investigated and discussed. Furthermore, a fluorescence microscopy system capable of measuring and generating optically-coupled shear stresses via laser-induced shockwaves is described. Shear stress induced by laser-induced shockwaves is a contactless model for traumatic brain injury (TBI) in vitro that offers high levels of reproducibility and spatiotemporal precision. Results from this work have potential applications towards high-throughput methods for the discovery of novel, therapeutic avenues for chronic neurodegenerative disorders such as HD and TBI.

    Cover page: Using the Biophotonics Toolbox to Study Neurodegeneration In Vitro
    • Article
    • Peer Reviewed
    UC Irvine Previously Published Works (2017)
    Traumatic Brain Injury (TBI) occurs when an external force injures the brain. While clinical outcomes of TBI can vary widely in severity, few mechanisms of neurodegeneration following TBI have been identified for treatment. We propose a model for studying TBI using laser-induced shockwaves (LISs). An optical system was developed that allows single cells to be studied in response to LISs. Our system utilizes an optically-coupled force measurement component that allows for the visualization of shockwave dynamics. Here, the force measurement system is characterized by imaging stages over the period of violent expansion and collapse of microbubbles responsible for shockwave generation.
      Cover page: Probing mechanobiology with laser-induced shockwavesCreative Commons 'BY' version 4.0 license
      • Article
      • Peer Reviewed
      UC Irvine Previously Published Works (2021)
      Laser induced shockwave (LIS) can be utilized to subject neuronal cells to conditions similar to those occurring during a blast induced traumatic brain injury. We utilized a 532nm Coherent Flare laser to induce a shockwave near cells which had been transfected with a FRET calcium biosensor (D3CPV) so that we could monitor the immediate cellular responses. Our shockwave system was characterized with a high-speed camera to monitor cavitation bubble dynamics and calculate the shear forces cells were subjected to. We found that we could induce forces which have been previously shown to induce injury. Using both phase and fluorescence microscopy we monitored the effects of shear on our cells. We found that at distances up to 120 microns from the laser focal point cells experienced shears greater than 10kPa. At those distances cell fragmentation was observed. Cells that survived and expressed the FRET biosensor demonstrated an immediate calcium elevation irrespective of extracellular or cytoplasmic calcium concentration. Cells recovered to pre-shockwave calcium levels within ∼30s. In conclusion, LIS can be utilized to simultaneously monitor the neuronal response to shear stress and nearby cell death or injury.
        Cover page: Laser induced shockwave paired with FRET to study neuronal responses to shear stressCreative Commons 'BY' version 4.0 license
        • Article
        • Peer Reviewed
        UC Irvine Previously Published Works (2021)
        Laser-induced shockwaves (LIS) can be utilized as a method to subject cells to conditions similar to those occurring during a blast-induced traumatic brain injury. The pairing of LIS with genetically encoded biosensors allows researchers to monitor the immediate molecular events resulting from such an injury. In this study, we utilized the genetically encoded Ca2+ FRET biosensor D3CPV to study the immediate Ca2+ response to laser-induced shockwave in cortical neurons and Schwann cells. Our results show that both cell types exhibit a transient Ca2+ increase irrespective of extracellular Ca2+ conditions. LIS allows for the simultaneous monitoring of the effects of shear stress on cells, as well as nearby cell damage and death.
          Cover page: Laser-Induced Shockwave (LIS) to Study Neuronal Ca2+ Responses
          • Article
          • Peer Reviewed
          UC Irvine Previously Published Works (2020)
          The changes in intracellular calcium concentration ([Ca2+]) following laser-induced cell injury in nearby cells were studied in primary mouse astrocytes selectively expressing the Ca2+ sensitive GFAP-Cre Salsa6f fluorescent tandem protein, in an Ast1 astrocyte cell line, and in primary mouse astrocytes loaded with Fluo4. Astrocytes in these three systems exhibit distinct changes in [Ca2+] following induced death of nearby cells. Changes in [Ca2+] appear to result from release of Ca2+ from intracellular organelles, as opposed to influx from the external medium. Salsa6f expressing astrocytes displayed dynamic Ca2+ changes throughout the phagocytic response, including lamellae protrusion, cytosolic signaling during vesicle formation, vesicle maturation, and vesicle tract formation. Our results demonstrate local changes in [Ca2+] are involved in the process of phagocytosis in astrocytes responding to cell corpses and/or debris.
            Cover page: Calcium Dynamics in Astrocytes During Cell Injury
            • Article
            • Peer Reviewed
            UC Irvine Previously Published Works (2022)
            Piezo1 belongs to mechano-activatable cation channels serving as biological force sensors. However, the molecular events downstream of Piezo1 activation remain unclear. In this study, we used biosensors based on fluorescence resonance energy transfer (FRET) to investigate the dynamic modes of Piezo1-mediated signaling and revealed a bimodal pattern of Piezo1-induced intracellular calcium signaling. Laser-induced shockwaves (LIS) and its associated shear stress can mechanically activate Piezo1 to induce transient intracellular calcium (Ca[i] ) elevation, accompanied by an increase in FAK activity. Interestingly, multiple pulses of shockwave stimulation caused a more sustained calcium increase and a decrease in FAK activity. Similarly, tuning the degree of Piezo1 activation by titrating either the dosage of Piezo1 ligand Yoda1 or the expression level of Piezo1 produced a similar bimodal pattern of FAK responses. Further investigations revealed that SHP2 serves as an intermediate regulator mediating this bimodal pattern in Piezo1 sensing and signaling. These results suggest that the degrees of Piezo1 activation induced by both mechanical LIS and chemical ligand stimulation may determine downstream signaling characteristics.
              Cover page: Mechanosensor Piezo1 mediates bimodal patterns of intracellular calcium and FAK signalingCreative Commons 'BY' version 4.0 license
              Top