Jin-Young Koh

University of Iowa, Iowa City, Iowa, United States

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Publications (8)17.89 Total impact

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    ABSTRACT: Synaptic vesicles in functional nerve terminals undergo exocytosis and endocytosis. This synaptic vesicle recycling can be effectively analyzed using styryl FM dyes, which reveal membrane turnover. Conventional protocols for the use of FM dyes were designed for analyzing neurons following stimulated (evoked) synaptic activity. Recently, protocols have become available for analyzing the FM signals that accompany weaker synaptic activities, such as spontaneous or miniature synaptic events. Analysis of these small changes in FM signals requires that the imaging system is sufficiently sensitive to detect small changes in intensity, yet that artifactual changes of large amplitude are suppressed. Here we describe a protocol that can be applied to evoked, spontaneous, and miniature synaptic activities, and use cultured hippocampal neurons as an example. This protocol also incorporates a means of assessing the rate of photobleaching of FM dyes, as this is a significant source of artifacts when imaging small changes in intensity.
    Journal of Visualized Experiments 01/2014;
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    ABSTRACT: Images in biomedical imaging research are often affected by non-specific background noise. This poses a serious problem when the noise overlaps with specific signals to be quantified, e.g. for their number and intensity. A simple and effective means of removing background noise is to prepare a filtered image that closely reflects background noise and to subtract it from the original unfiltered image. This approach is in common use, but its effectiveness in identifying and quantifying synaptic puncta has not been characterized in detail. We report on our assessment of the effectiveness of isolating punctate signals from diffusely distributed background noise using one variant of this approach, "Difference of Gaussian(s) (DoG)" which is based on a Gaussian filter. We evaluated immunocytochemically stained, cultured mouse hippocampal neurons as an example, and provided the rationale for choosing specific parameter values for individual steps in detecting glutamatergic nerve terminals. The intensity and width of the detected puncta were proportional to those obtained by manual fitting of two-dimensional Gaussian functions to the local information in the original image. DoG was compared with the rolling-ball method, using biological data and numerical simulations. Both methods removed background noise, but differed slightly with respect to their efficiency in discriminating neighboring peaks, as well as their susceptibility to high-frequency noise and variability in object size. DoG will be useful in detecting punctate signals, once its characteristics are examined quantitatively by experimenters.
    Journal of neuroscience methods 12/2013; · 2.30 Impact Factor
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    ABSTRACT: Although recent developments in methodologies for light microscopy have enabled imaging of fine biological structures, such imaging is often accompanied by two types of problems. One is a tilting of the specimen with respect to the x-y plane (i.e. rotation around the x- or y-axis) such that the sample is not perpendicular to the optical z-axis, and the other is rotation around the z-axis that precludes optimal orientations for imaging and experimentation. These rotation problems can cause optical aberrations and hamper imaging experiments, even when the angular difference from the ideal position is small. New Method: In order to correct for these practical issues, we have developed a specimen holder with 3-axis (x-y-z) rotation for an inverted light microscope. This allows for full-range rotations of 2-4° for x-, y-axes, ∼24° for z-axis, and a small-angle control of <0.1° for either axis. Using this device, we observed the cultured hippocampal neurons stained by immunofluorescence for a dendritic marker, or the sub-resolution fluorescent beads plated on a glass coverslip. The rotations and associated problems could be manipulated, while viewing the specimens by laser-scanning confocal microscopy. Comparison with Existing Methods: This tilting/rotation device is easily manufactured and installed on a conventional microscope stage without requiring changes to the existing optical components. Similar devices with full capability have not been available. It will be useful for imaging experiments with biomedical applications.
    Journal of neuroscience methods 09/2013; · 2.30 Impact Factor
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    ABSTRACT: Presynaptic functions of the mammalian central neurons are regulated by a network of protein interactions. Synaptic vesicle recycling in and neurotransmitter release from the presynaptic nerve terminals are altered when a glutamate-deleting mutation is present in the torsinA protein (ΔE-torsinA). This mutation is linked with a hereditary form of the movement disorder dystonia known as DYT1 dystonia. Although torsinA expression is prevalent throughout the central nervous system, its subcellular localization - in particular with respect to presynaptic nerve terminals - remains unclear. This information would be useful in narrowing down possible models for how wild-type torsinA affects presynaptic function, as well as the nature of the presynaptic dysfunction that arises in the context of ΔE-torsinA mutation. Here we report on an analysis of the presynaptic localization of torsinA in cultured neurons obtained from a knock-in mouse model of DYT1 dystonia. Primary cultures of neurons were established from heterozygous and homozygous ΔE-torsinA knock-in mice, as well as form their wild-type littermates. Neurons were obtained from the striatum, cerebral cortex and hippocampus of these mice, and were subjected to immunocytochemistry. This analysis revealed expression of both proteins in the somata and dendrites. However, neither the nerve terminals nor axonal shafts were immunoreactive. These results were confirmed by fluorogram-based quantitation. Our findings indicate that neither the wild-type nor the ΔE-torsinA mutant protein is present at substantial levels in the presynaptic structures of cultured neurons. Thus, the effects of torsinA, in wild-type and mutant forms, appear to influence presynaptic function indirectly, without residing in presynaptic structures.
    Neuroscience 09/2013; · 3.12 Impact Factor
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    ABSTRACT: Increased activities of cytoplasmic calcium and the excitatory neurotransmitter glutamate have been independently implicated in dystonia pathophysiology. However, cellular-level evidence linking these two features is not available. Here we show that glutamate-dependent changes in neuronal calcium dynamics occur in a knock-in mouse model of DYT1 dystonia, the most common hereditary form of this disorder. Fluorescence-based analysis of the dynamics of cytoplasmic calcium concentration ([Ca(2+)]c) in cultured hippocampal neurons shows that electrical stimulation depolarizes the neurons and increases the dendritic [Ca(2+)]c, which then decays slowly to the pre-stimulus level. Whereas the peak amplitude of [Ca(2+)]c was not affected, the decay period was prolonged in neurons of heterozygous mice whose genotype reflects the human condition. We found that this effect was blocked by the antagonists of ionotropic glutamate receptors, and confirmed that glutamate receptors are present in these neurons. As the [Ca(2+)]c is readout and regulator of neuronal excitability, its abnormality represents an important cellular phenotype of dystonia.
    Neuroscience Letters 06/2013; · 2.03 Impact Factor
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    ABSTRACT: DYT1 dystonia is the most common hereditary form of primary torsion dystonia. This autosomal-dominant disorder is characterized by involuntary muscle contractions that cause sustained twisting and repetitive movements. It is caused by an in-frame deletion in the TOR1A gene, leading to the deletion of a glutamic acid residue in the torsinA protein. Heterozygous knock-in mice, which reproduce the genetic mutation in human patients, have abnormalities in synaptic transmission at the principal GABAergic neurons in the striatum, a brain structure that is involved in the execution and modulation of motor activity. However, whether this mutation affects the excitability of striatal GABAergic neurons has not been investigated in this animal model. Here, we examined the excitability of cultured striatal neurons obtained from heterozygous knock-in mice, using calcium imaging as indirect readout. Immunofluorescence revealed that more than 97% of these neurons are positive for a marker of GABAergic neurons, and that more than 92% are also positive for a marker of medium spiny neurons, indicating that these are mixed cultures of mostly medium spiny neurons and a few (~5%) GABAergic interneurons. When these neurons were depolarized by field stimulation, the calcium concentration in the dendrites increased rapidly and then decayed slowly. The amplitudes of calcium transients were larger in heterozygous neurons than in wild-type neurons, resulting in ~15% increase in cumulative calcium transients during a train of stimuli. However, there was no change in other parameters of calcium dynamics. Given that calcium dynamics reflect neuronal excitability, these results suggest that the mutation only slightly increases the excitability of striatal GABAergic neurons in DYT1 dystonia.
    PLoS ONE 01/2013; 8(11):e80793. · 3.53 Impact Factor
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    ABSTRACT: TorsinA is an evolutionarily conserved AAA+ ATPase, and human patients with an in-frame deletion of a single glutamate (ΔE) codon from the encoding gene suffer from autosomal-dominant, early-onset generalized DYT1 dystonia. Although only 30-40% of carriers of the mutation show overt motor symptoms, most experience enhanced excitability of the central nervous system. The cellular mechanism responsible for this change in excitability is not well understood. Here we show the effects of the ΔE-torsinA mutation on miniature neurotransmitter release from neurons. Neurotransmitter release was characterized in cultured hippocampal neurons obtained from wild-type, heterozygous, and homozygous ΔE-torsinA knock-in mice using two approaches. In the first approach, patch-clamp electrophysiology was used to record glutamate-mediated miniature excitatory postsynaptic currents (mEPSCs) in the presence of the Na⁺ channel blocker tetrodotoxin (TTX) and absence of GABA(A) receptor antagonists. The intervals between mEPSC events were significantly shorter in neurons obtained from the mutant mice than in those obtained from wild-type mice. In the second approach, the miniature exocytosis of synaptic vesicles was detected by imaging the unstimulated release of FM dye from the nerve terminals in the presence of TTX. Cumulative FM dye release was higher in neurons obtained from the mutant mice than in those obtained from wild-type mice. The number of glutamatergic nerve terminals was also assessed, and we found that this number was unchanged in heterozygous relative to wild-type neurons, but slightly increased in homozygous neurons. Notably, in both heterozygous and homozygous neurons, the unitary synaptic charge during each mEPSC event was unchanged. Overall, our results suggest more frequent miniature glutamate release in neurons with ΔE-torsinA mutations. This change may be one of the underlying mechanisms by which the excitability of the central nervous system is enhanced in the context of DYT1 dystonia. Moreover, qualitative differences between heterozygous and homozygous neurons with respect to certain synaptic properties indicate that the abnormalities observed in homozygotes may reflect more than a simple gene dosage effect.
    Synapse 05/2012; 66(9):807-22. · 2.31 Impact Factor
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    ABSTRACT: Early-onset generalized dystonia, DYT1, is caused by a mutation in the gene encoding the evolutionarily conserved AAA+ ATPase torsinA. Synaptic abnormalities have been implicated in DYT1 dystonia, but the details of the synaptic pathophysiology are only partially understood. Here, we demonstrate a novel role for torsinA in synaptic vesicle recycling, using cultured hippocampal neurons from a knock-in mouse model of DYT1 dystonia (ΔE-torsinA) and live-cell imaging with styryl FM dyes. Neurons from heterozygous ΔE-torsinA mice released a larger fraction of the total recycling pool (TRP) during a single round of electrical stimulation than did wild-type neurons. Moreover, when the neurons were subjected to prior high activity, the time course of release was shortened. In neurons from homozygous mice, these enhanced exocytosis phenotypes were similar, but in addition the size of the TRP was reduced. Notably, when release was triggered by applying a calcium ionophore rather than electrical stimuli, neither a single nor two ΔE-torsinA alleles affected the time course of release. Thus, the site of action of ΔE-torsinA is at or upstream of the rise in calcium concentration in nerve terminals. Our results suggest that torsinA regulates synaptic vesicle recycling in central neurons. They also indicate that this regulation is influenced by neuronal activity, further supporting the idea that synaptic abnormalities contribute to the pathophysiology of DYT1 dystonia.
    Synapse 12/2011; 66(5):453-64. · 2.31 Impact Factor

Publication Stats

25 Citations
17.89 Total Impact Points


  • 2013
    • University of Iowa
      • Department of Molecular Physiology and Biophysics
      Iowa City, Iowa, United States