1.Development of a Laboratory-safe and Low-cost Detection Protocol for SARS-CoV-2 of the Coronavirus Disease 2019(COVID-19)
Joungha WON ; Solji LEE ; Myungsun PARK ; Tai Young KIM ; Mingu Gordon PARK ; Byung Yoon CHOI ; Dongwan KIM ; Hyeshik CHANG ; Won Do HEO ; V. Narry KIM ; C. Justin LEE
Experimental Neurobiology 2020;29(5):402-402
2.Flow Shear Stress Enhances the Proliferative Potential of Cultured Radial Glial Cells Possibly Via an Activation of Mechanosensitive Calcium Channel.
Min Gu PARK ; Heeyeong JANG ; Sang Hoon LEE ; C Justin LEE
Experimental Neurobiology 2017;26(2):71-81
Radial glial cells (RGCs) which function as neural stem cells are known to be non-excitable and their proliferation depends on the intracellular calcium (Ca²⁺) level. It has been well established that Inositol 1,4,5-trisphosphate (IP3)-mediated Ca²⁺ release and Ca²⁺ entry through various Ca²⁺ channels are involved in the proliferation of RGCs. Furthermore, RGCs line the ventricular wall and are exposed to a shear stress due to a physical contact with the cerebrospinal fluid (CSF). However, little is known about how the Ca²⁺ entry through mechanosensitive ion channels affects the proliferation of RGCs. Hence, we hypothesized that shear stress due to a flow of CSF boosts the proliferative potential of RGCs possibly via an activation of mechanosensitive Ca²⁺ channel during the embryonic brain development. Here, we developed a new microfluidic two-dimensional culture system to establish a link between the flow shear stress and the proliferative activity of cultured RGCs. Using this microfluidic device, we successfully visualized the artificial CSF and RGCs in direct contact and found a significant enhancement of proliferative capacity of RGCs in response to increased shear stress. To determine if there are any mechanosensitive ion channels involved, a mechanical stimulation by poking was given to individual RGCs. We found that a poking on radial glial cell induced an increase in intracellular Ca²⁺ level, which disappeared under the extracellular Ca²⁺-free condition. Our results suggest that the shear stress by CSF flow possibly activates mechanosensitive Ca²⁺ channels, which gives rise to a Ca²⁺ entry which enhances the proliferative capacity of RGCs.
Brain
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Calcium Channels*
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Calcium*
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Cerebrospinal Fluid
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Ependymoglial Cells*
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Inositol 1,4,5-Trisphosphate
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Ion Channels
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Lab-On-A-Chip Devices
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Microfluidics
;
Neural Stem Cells
3.Development of a Low-cost, Comprehensive Recording System for Circadian Rhythm Behavior
Jea KWON ; Min Gu PARK ; Seung Eun LEE ; C Justin LEE
Experimental Neurobiology 2018;27(1):65-75
Circadian rhythm is defined as a 24-hour biological oscillation, which persists even without any external cues but also can be re-entrained by various environmental cues. One of the widely accepted circadian rhythm behavioral experiment is measuring the wheel-running activity (WRA) of rodents. However, the price for commercially available WRA recording system is not easily affordable for researchers due to high-cost implementation of sensors for wheel rotation. Here, we developed a cost-effective and comprehensive system for circadian rhythm recording by measuring the house-keeping activities (HKA). We have monitored animal's HKA as electrical signal by simply connecting animal housing cage with a standard analog/digital converter: input to the metal lid and ground to the metal grid floor. We show that acquired electrical signals are combined activities of eating, drinking and natural locomotor behaviors which are well-known indicators of circadian rhythm. Post-processing of measured electrical signals enabled us to draw actogram, which verifies HKA to be reliable circadian rhythm indicator. To provide easy access of HKA recording system for researchers, we have developed user-friendly MATLAB-based software, Circa Analysis. This software provides functions for easy extraction of scalable “touch activity” from raw data files by automating seven steps of post-processing and drawing actograms with highly intuitive user-interface and various options. With our cost-effective HKA circadian rhythm recording system, we have estimated the cost of our system to be less than $150 per channel. We anticipate our system will benefit many researchers who would like to study circadian rhythm.
Animals
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Circadian Rhythm
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Cues
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Drinking
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Eating
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Electrical Equipment and Supplies
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Housing, Animal
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Information Storage and Retrieval
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Mice
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Rodentia
4.Visuosocial Preference Memory, but Not Avoidance Memory, Requires PLCγ1 in the CA2 Hippocampus
Sunpil KIM ; Jeongyeon KIM ; Yongmin Mason PARK ; Pann-Ghill SUH ; C. Justin LEE
Experimental Neurobiology 2022;31(5):332-342
Visuosocial memory is defined as stored visual information containing social context. Primates have a powerful ability to associate visuosocial memory with episodic memory. However, the existence of visuosocial memory in mice remains unclear. Here, we design a novel vision-specific social memory test using a portrait picture or mirrored self-image and demonstrate that mice can distinguish conspecific from other species by forming a visuosocial memory. Because CA2 hippocampus has been reported as a critical brain region for social memory, we develop CA2-specific blockade of memory formation through deletion of phospholipase C gamma 1 (PLCγ1), which is a key molecule in the brain-derived neurotrophic factor (BDNF) signaling pathway. Interestingly, these mice have intact sociability but impaired social memory in three chamber test and five-trial social memory test, which is highly dependent on visual information. Finally, PLCγ1 deletion in CA2 impairs visuosocial preference memory, but not avoidance memory, whereas non-social object recognition is intact. Our study proposes that mice have visuosocial memory, just as primates and humans.
5.NKCC1 in Neonatal Cochlear Support Cells Reloads Ions Necessary for Cochlear Spontaneous Activity
Kwon-Woo KANG ; Kushal SHARMA ; Shi-Hyun PARK ; Jae Kwang LEE ; Justin C. LEE ; Eunyoung YI
Experimental Neurobiology 2024;33(2):68-76
In the auditory system, the spontaneous activity of cochlear inner hair cells (IHCs) is initiated by the release of ATP from inner supporting cells (ISCs). This ATP release sets off a cascade, activating purinergic autoreceptors, opening of Ca 2+ -activated Cl- channel TMEM16A, Cl- efflux and osmotic cell shrinkage. Then, the shrunken ISCs efficiently regain their original volume, suggesting the existence of mechanisms for refilling Cl- and K + , priming them for subsequent activity. This study explores the potential involvement of NKCCs (Na+ -K+ -Cl- cotransporters) and KCCs (K+ -Cl- cotransporters) in ISC spontaneous activity, considering their capability to transport both Cl- and K+ ions across the cell membrane. Employing a combination of immunohistochemistry, pharmacological interventions, and shRNA experiment, we unveiled the pivotal role of NKCC1 in cochlear spontaneous activity. Immunohistochemistry revealed robust NKCC1 expression in ISCs, persisting until the 2nd postnatal week. Intriguingly, we observed a developmental shift in NKCC1 expression from ISCs to synaptophysin-positive efferent terminals at postnatal day 18, hinting at its potential involvement in modulating synaptic transmission during the post-hearing period. Experiments using bumetanide, a well-known NKCC inhibitor, supported the functional significance of NKCC1 in ISC spontaneous activity. Bumetanide significantly reduced the frequency of spontaneous extracellular potentials (sEP) and spontaneous optical changes (sOCs) in ISCs. NKCC1-shRNA experiments conducted in cultured cochlear tissues further supported these findings, demonstrating a substantial decrease in event frequency and area. Taken together, we revealed the role of NKCC1 in shaping the ISC spontaneous activity that govern auditory pathway development.
6.Digital Tomosynthesis for Patient Alignment System Using Half-fan Mode CBCT Projection Images.
Justin C PARK ; Sung Ho PARK ; Jin Sung KIM ; Yongyih HAN ; Sang Gyu JU ; Eunhyuk SHIN ; Jung Suk SHIN ; Hee Chul PARK ; Yong Chan AHN ; Willian Y SONG
Korean Journal of Medical Physics 2010;21(4):360-366
To generate on-board digital tomosynthesis (DTS) for three-dimensionalimage-guided radiation therapy (IGRT) as an alternative to conventional portal imaging or on-board cone-beam computed tomography (CBCT), two clinical cases (liver and bladder) were selected to illustrate the capabilities of on-board DTS for IGRT. DTS images were generated from subsets of CBCT projection data (45, 162 projections) using half-fan mode scanning with a Feldkamp-type reconstruction algorithm. Digital tomosynthesis slices appeared similar to coincident CBCT planes and yielded substantially more anatomic information. Improved bony and soft-tissue visibility in DTS images is likely to improve target localization compared with radiographic verification techniques and might allow for daily localization of a soft-tissue target. Digital tomosynthesis might allow targeting of the treatment volume on the basis of daily localization.
Cone-Beam Computed Tomography
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Humans
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Patient Positioning
7.Development of a Laboratory-safe and Low-cost Detection Protocol for SARS-CoV-2 of the Coronavirus Disease 2019 (COVID-19)
Joungha WON ; Solji LEE ; Myungsun PARK ; Tai Young KIM ; Mingu Gordon PARK ; Byung Yoon CHOI ; Dongwan KIM ; Hyeshik CHANG ; V. Narry KIM ; C. Justin LEE
Experimental Neurobiology 2020;29(2):107-119
The severe acute respiratory coronavirus 2 (SARS-CoV-2), which emerged in December 2019 in Wuhan, China, has spread rapidly to over a dozen countries. Especially, the spike of case numbers in South Korea sparks pandemic worries. This virus is reported to spread mainly through personto- person contact via respiratory droplets generated by coughing and sneezing, or possibly through surface contaminated by people coughing or sneezing on them. More critically, there have been reports about the possibility of this virus to transmit even before a virus-carrying person to show symptoms. Therefore, a low-cost, easy-access protocol for early detection of this virus is desperately needed. Here, we have established a real-time reverse-transcription PCR (rtPCR)-based assay protocol composed of easy specimen self-collection from a subject via pharyngeal swab, Trizolbased RNA purification, and SYBR Green-based rtPCR. This protocol shows an accuracy and sensitivity limit of 1-10 virus particles as we tested with a known lentivirus. The cost for each sample is estimated to be less than 15 US dollars. Overall time it takes for an entire protocol is estimated to be less than 4 hours. We propose a cost-effective, quick-and-easy method for early detection of SARS-CoV-2 at any conventional Biosafety Level II laboratories that are equipped with a rtPCR machine. Our newly developed protocol should be helpful for a first-hand screening of the asymptomatic virus-carriers for further prevention of transmission and early intervention and treatment for the rapidly propagating virus.
8.AAV-Mediated Astrocyte-Specific Gene Expression under Human ALDH1L1 Promoter in Mouse Thalamus.
Wuhyun KOH ; Yongmin Mason PARK ; Seung Eun LEE ; C Justin LEE
Experimental Neurobiology 2017;26(6):350-361
Adeno-associated virus (AAV)-mediated gene delivery has been proposed to be an essential tool of gene therapy for various brain diseases. Among several cell types in the brain, astrocyte has become a promising therapeutic target for brain diseases, as more and more contribution of astrocytes in pathophysiology has been revealed. Until now, genetically targeting astrocytes has been possible by utilizing the glial fibrillary acidic protein (GFAP) promoter. In some brain areas including thalamus, however, the GFAP expression in astrocytes is reported to be low, making it difficult to genetically target astrocytes using GFAP promoter. To study the function of astrocytes in thalamus, which serves as a relay station, there is a great need for identifying an alternative astrocyte-specific promoter in thalamus. Recently, a new astrocyte-specific promoter of ALDH1L1 has been identified. However, it has not been examined in thalamus. Here we developed and characterized an AAV vector expressing Cre recombinase under the human ALDH1L1 promoter, AAV-hALDH1L1-Cre. To test the cell-type specific expression of AAV-hALDH1L1-Cre, AAV virus was injected into several brain regions of Ai14 (RCL-tdTomato) mouse, which reports Cre activity by tdTomato expression. In thalamus, we observed that tdTomato was found mostly in astrocytes (91.71%), with minimal occurrence in neurons (2.67%). In contrast, tdTomato signal was observed in both neurons and astrocytes of the amygdala (neuron: 68.13%, astrocyte: 28.35%) and hippocampus (neuron: 76.25%, astrocyte: 18.00%), which is consistent with the previous report showing neuronal gene expression under rat ALDH1L1 promoter. Unexpectedly, tdTomato was found mostly in neurons (91.98%) with minimal occurrence in astrocytes (6.66%) of the medial prefrontal cortex. In conclusion, hALDH1L1 promoter shows astrocyte-specificity in thalamus and may prove to be useful for targeting thalamic astrocytes in mouse.
Amygdala
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Animals
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Astrocytes
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Brain
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Brain Diseases
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Dependovirus
;
Gene Expression*
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Genetic Therapy
;
Glial Fibrillary Acidic Protein
;
Hippocampus
;
Humans*
;
Mice*
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Neurons
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Prefrontal Cortex
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Rats
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Recombinases
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Thalamus*
;
Ventral Thalamic Nuclei
9.Pharmacological Dissection of Intrinsic Optical Signal Reveals a Functional Coupling between Synaptic Activity and Astrocytic Volume Transient
Junsung WOO ; Young Eun HAN ; Wuhyun KOH ; Joungha WON ; Min Gu PARK ; Heeyoung AN ; C Justin LEE
Experimental Neurobiology 2019;28(1):30-42
The neuronal activity-dependent change in the manner in which light is absorbed or scattered in brain tissue is called the intrinsic optical signal (IOS), and provides label-free, minimally invasive, and high spatial (~100 µm) resolution imaging for visualizing neuronal activity patterns. IOS imaging in isolated brain slices measured at an infrared wavelength (>700 nm) has recently been attributed to the changes in light scattering and transmittance due to aquaporin-4 (AQP4)-dependent astrocytic swelling. The complexity of functional interactions between neurons and astrocytes, however, has prevented the elucidation of the series of molecular mechanisms leading to the generation of IOS. Here, we pharmacologically dissected the IOS in the acutely prepared brain slices of the stratum radiatum of the hippocampus, induced by 1 s/20 Hz electrical stimulation of Schaffer-collateral pathway with simultaneous measurement of the activity of the neuronal population by field potential recordings. We found that 55% of IOSs peak upon stimulation and originate from postsynaptic AMPA and NMDA receptors. The remaining originated from presynaptic action potentials and vesicle fusion. Mechanistically, the elevated extracellular glutamate and K⁺ during synaptic transmission were taken up by astrocytes via a glutamate transporter and quinine-sensitive K2P channel, followed by an influx of water via AQP-4. We also found that the decay of IOS is mediated by the DCPIB- and NPPB-sensitive anion channels in astrocytes. Altogether, our results demonstrate that the functional coupling between synaptic activity and astrocytic transient volume change during excitatory synaptic transmission is the major source of IOS.
Action Potentials
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alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid
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Amino Acid Transport System X-AG
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Astrocytes
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Brain
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Electric Stimulation
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Glutamic Acid
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Hippocampus
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Jupiter
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Neurons
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Receptors, N-Methyl-D-Aspartate
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Synaptic Transmission
;
Water
10.Astrocyte Specificity and Coverage of hGFAP-CreERT2 Tg(GFAP-Cre/ERT2)13Kdmc Mouse Line in Various Brain Regions.
Yongmin Mason PARK ; Heejung CHUN ; Jeong Im SHIN ; C Justin LEE
Experimental Neurobiology 2018;27(6):508-525
Astrocyte is the most abundant cell type in the central nervous system and its importance has been increasingly recognized in the brain pathophysiology. To study in vivo function of astrocyte, astrocyte-specific gene-targeting is regarded as a powerful approach. Especially, hGFAP-CreERT2, which expresses tamoxifen-inducible Cre recombinase under the human GFAP promoter, has been developed and characterized from several research groups. However, one of these mouse lines, [Tg(GFAP-Cre/ERT2)13Kdmc] from Ken McCarthy group has not been quantitatively analyzed, despite its frequent use. Here, we performed comprehensive characterization of this mouse line with quantitative analysis. By crossing this mouse line with Ai14 (RCL-tdTomato), a very sensitive Cre reporter mouse line, we visualized the Cre-expressing cells in various brain regions. For quantitative analysis, we immunostained S100β as an astrocytic marker and NeuN, tyrosine hydroxylase or calbindin as a neuronal marker in different brain regions. We calculated ‘astrocyte specificity’ as the proportion of co-labelled S100β and tdTomato positive cells in the total number of tdTomato positive cells and the ‘astrocyte coverage’ as the proportion of co-labelled S100β and tdTomato positive cells in the total number of S100β positive cells. Interestingly, we found varying degree of astrocyte specificity and coverage in each brain region. In cortex, hypothalamus, substantia nigra pars compacta and cerebellar Purkinje layer, we observed high astrocyte specificity (over 89%) and relatively high astrocyte coverage (over 70%). In striatum, hippocampal CA1 layer, dentate gyrus and cerebellar granule layer, we observed high astrocyte specificity (over 80%), but relative low astrocyte coverage (50–60%). However, thalamus and amygdala showed low astrocyte specificity (about 65%) and significant neuron specificity (over 30%). This hGFAP-CreERT2 mouse line can be useful for genetic modulations of target gene either in gain-of-function or loss-of-function studies in the brain regions with high astrocyte specificity and coverage. However, the use of this mouse line should be restricted to gain-of-function studies in the brain regions with high astrocyte specificity but low coverage. In conclusion, hGFAP-CreERT2 mouse line could be a powerful tool for gene-targeting of astrocytes in cortex, striatum, hippocampus, hypothalamus, substantia nigra pars compacta and cerebellum, but not in thalamus and amygdala.
Amygdala
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Animals
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Astrocytes*
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Brain*
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Calbindins
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Central Nervous System
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Cerebellum
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Dentate Gyrus
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Hippocampus
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Humans
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Hypothalamus
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Mice*
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Neurons
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Pars Compacta
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Recombinases
;
Sensitivity and Specificity*
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Thalamus
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Tyrosine 3-Monooxygenase