Biomedical subjects

Da-Ting Lin

Publications and source records attributed to Da-Ting Lin.

5 recordsLinked to original sources

PubMed2006

Synapse-specific regulation of AMPA receptor function by PSD-95.

PSD-95 is a major protein found in virtually all mature excitatory glutamatergic synapses in the brain. Here, we have addressed the role of PSD-95 in controlling glutamatergic synapse function by generating and characterizing a PSD-95 KO mouse. We found that the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)subtype of glutamate receptor (AMPAR)-mediated synaptic transmission was reduced in these mice. Two-photon (2P) uncaging of MNI-glutamate onto individual spines suggested that the decrease in AMPAR function in the PSD-95 KO mouse stems from an increase in the proportion of "silent" synapses i.e., synapses containing N-methyl-d-aspartate (NMDA) receptors (NMDARs) but no AMPARs. Unexpectedly, the silent synapses in the KO mouse were located onto morphologically mature spines. We also observed that a significant population of synapses appeared unaffected by PSD-95 gene deletion, suggesting that the functional role of PSD-95 displays synapse-specificity. In addition, we report that the decay of NMDAR-mediated current was slower in KO mice: The contribution of NR2B subunit containing receptors to the NMDAR-mediated synaptic current was greater in KO mice. The greater occurrence of silent synapses might be related to the greater magnitude of potentiation after long-term potentiation induction observed in these mice. Together, these results suggest a synapse-specific role for PSD-95 in controlling synaptic function that is independent of spine morphology.

Animals↗

PubMed2006

Multiphoton laser scanning microscopy as a tool for Xenopus oocyte research.

Multiphoton laser scanning microscopy (MPLSM) has become an increasingly invaluable tool in fluorescent optical imaging. There are several distinct advantages to implementing MPLSM as a Xenopus oocyte research tool. MPLSM increases signal-to-noise ratio and therefore increases image quality because there is no out-of-focus fluorescence as would be created in conventional or confocal microscopy. All the light that is generated can be collected and used to generate an image because point detection of descanned fluorescence is not required. This is particularly useful when imaging deep into tissue sections, as is necessary for Xenopus oocytes, which are notoriously large (approximately 1-mm diameter). Because multiphoton lasers use pulsed energy in the infrared wavelengths, the energy can also travel further into tissues with much less light scattering. Because there is no out-of-focus excitation, phototoxicity, photodamage, and photobleaching are significantly reduced, which is particularly important for long-term experiments that require the same region to be scanned repeatedly. Finally, multiple fluorophores can be simultaneously excited because of the broader absorption spectra of multiphoton dyes. In this chapter, we describe the advantages and disadvantages of using MPLSM to image Xenopus oocytes as compared to conventional and confocal microscopy. The practical application of imaging oocytes is demonstrated with specific examples.

Animals↗

PubMed2005

Ca2+ signaling, mitochondria and sensitivity to oxidative stress in aging astrocytes.

Age-related changes in astrocytes that could potentially affect neuroprotection have been largely unexplored. To test whether astrocyte function was diminished during the aging process, we examined cell growth, Ca2+ signaling, mitochondrial membrane potential (DeltaPsi) and neuroprotection of NGF-differentiated PC12 cells. We observed that cell growth was significantly slower for astrocytes cultured from old (26-29 months) mice as compared to young (4-6 months) mice. DeltaPsis in old astrocytes were also more depolarized (lower) than in young astrocytes and old astrocytes showed greater sensitivity to the oxidant tert-butyl hydrogen peroxide (t-BuOOH). ATP-induced Ca2+ responses in old astrocytes were consistently larger in amplitude and more frequently oscillatory than in young astrocytes, which may be attributable to lower mitochondrial Ca2+ sequestration. Finally, NGF-differentiated PC12 cells that were co-cultured with old astrocytes were significantly more sensitive to t-BuOOH treatment than co-cultures of NGF-differentiated PC12 cells with young astrocytes. Together, these data demonstrate that astrocyte physiology is significantly altered during the aging process and that the astrocyte's ability to protect neurons is compromised.

Aging↗

PubMed2002

Mitochondrial targeted cyclophilin D protects cells from cell death by peptidyl prolyl isomerization.

Cyclophilin D (CyPD) is thought to sensitize opening of the mitochondrial permeability transition pore (mPTP) based on the findings that cyclosporin A (CsA), a pseudo-CyPD substrate, hyperpolarizes the mitochondrial membrane potential (DeltaPsi) and inhibits apoptosis. We provide evidence that contrasts with this model. Using live cell imaging and two photon microscopy, we report that overexpression of CyPD desensitizes HEK293 and rat glioma C6 cells to apoptotic stimuli. By site-directed mutagenesis of CyPD that compromises peptidyl-prolyl cis-trans isomerase (PPIase) activity, we demonstrate that the mechanism involved in this protective effect requires PPIase activity. Furthermore, we show that, under resting conditions, DeltaPsi is hyperpolarized in CyPD wild type-overexpressing cells but not in cells overexpressing mutant forms of CyPD that lack PPIase activity. Finally, in glutathione S-transferase (GST) pull-down assays, we demonstrate that CyPD binding to the adenine nucleotide translocator (ANT), which is considered to be the core component of the mPTP, is not affected by the loss of PPIase activity. Collectively, our data suggest that CyPD should be viewed as a cell survival-signaling molecule and indicate a protective role of CyPD against apoptosis that is mediated by one or more targets other than the ANT.

Animals↗

PubMed2002

Multi-photon laser scanning microscopy using an acoustic optical deflector.

Multi-photon laser scanning microscopes have many advantages over single-photon systems. However, the speed and flexibility of currently available multi-photon microscopes are limited by the use of mechanical mirrors to steer pulsed radiation for fluorophore excitation. Here, we describe the multi-photon adaptation of a confocal microscope that uses an acoustic optical deflector (AOD) for beam steering. AODs are capable of very rapid scanning and, in addition, offer the flexibility of zooming, panning, and being adjustable for slow image acquisition. Because of the highly dispersive nature of AODs, pulsed radiation must be temporally compressed by introducing negative dispersion into the beam path. More critically, pulsed radiation must also be spatially compressed by introducing lateral dispersion into the beam path. This was accomplished by using two prisms in the external beam path and by introducing a third prism element subsequent to the AOD. The end result is an AOD-based multi-photon microscope that is capable of rapid imaging of physiological events as well as slow detection of weakly fluorescent biological samples.

Animals↗