Translate this page to:
Choose Language…
In JoVE (1)
Other Publications (13)
- Conference Proceedings : ... Annual International Conference of the IEEE Engineering in Medicine and Biology Society. IEEE Engineering in Medicine and Biology Society. Conference
- Conference Proceedings : ... Annual International Conference of the IEEE Engineering in Medicine and Biology Society. IEEE Engineering in Medicine and Biology Society. Conference
- IEEE Transactions on Bio-medical Engineering
- Medical & Biological Engineering & Computing
- Journal of Neuroscience Methods
- Journal of Neural Engineering
- Medical & Biological Engineering & Computing
- Biosensors & Bioelectronics
- Journal of Neurochemistry
- Electrophoresis
- Medical & Biological Engineering & Computing
- The Biochemical Journal
- Conference Proceedings : ... Annual International Conference of the IEEE Engineering in Medicine and Biology Society. IEEE Engineering in Medicine and Biology Society. Conference
Articles by Sang Beom Jun in JoVE
JoVE Neuroscience
Vibrodissociation of Neurons from Rodent Brain Slices to Study Synaptic Transmission and Image Presynaptic Terminals
1Section on Synaptic Pharmacology/Laboratory for Integrative Neuroscience, National Institutes of Health/National Institute on Alcohol Abuse and Alcoholism, 2Department of Electronics Engineering, Ewha Womans University, 3Section on Transmitter Signaling/Laboratory of Molecular Physiology, National Institutes of Health/National Institute on Alcohol Abuse and Alcoholism
This report demonstrates a technique for mechanical isolation of individual viable neurons retaining attached presynaptic boutons. Vibrodissociated neurons have the advantages of rapid production, excellent pharmacological control and improved space-clamp without influence from neighboring cells. This method can be used for imaging of synaptic elements and patch-clamp recording.
Other articles by Sang Beom Jun on PubMed
A Cochlear Implant System with Infra-red Telemetry for Behaving Animals
A new cochlear implant system for animal experiments is proposed. The system uses a DSP-based sound processor to implement the six-channel continuous interleaved sampling (CIS) speech processing strategy. An infra-red telemetry is designed to remotely connect the power-demanding sound processor to receiver/stimulator attached on the animal. This enables the animal to move freely. The receiver/stimulator consumes about 40mW, thus can operates for about 200hours without changing battery.
Synaptic Connectivity of a Low Density Patterned Neuronal Network Produced on the Poly-L-lysine Stamped Microelectrode Array
Rectangular networks of rat hippocampal neurons have been produced on microelectrode arrays (MEAs). The crossing points of networks were located at the recording electrode sites by aligned microcontact printing (μCP) technique. Polydimethysiloxane (PDMS) stamp was fabricated to print fine poly-L-lysine (PLL) patterns of 2 -width lines for neurites and 20 -diameter circles for cell bodies. Different densities of neurons were applied on the PLL-stamped MEAs to find how a low density of neurons still has the functional connectivity. From the neural network applied with a density of 200 cells/mm2, we could observe signal propagation among spontaneous activities. Electrical responses were also evoked by 200 current pulse stimulation with 50 pulse width. Immunocytostaining was employed to identify dendrites, synapses, and nuclei in the patterned neurons.
Design for a Simplified Cochlear Implant System
A simplified cochlear implant (CI) system would be appropriate for widespread use in developing countries. Here, we describe a CI that we have designed to realize such a concept. The system implements 8 channels of processing and stimulation using the continuous interleaved sampling (CIS) strategy. A generic digital signal processing (DSP) chip is used for the processing, and the filtering functions are performed with a fast Fourier transform (FFT) of a microphone or other input. Data derived from the processing are transmitted through an inductive link using pulse width modulation (PWM) encoding and amplitude shift keying (ASK) modulation. The same link is used in the reverse direction for backward telemetry of electrode and system information. A custom receiver-stimulator chip has been developed that demodulates incoming data using pulse counting and produces charge balanced biphasic pulses at 1000 pulses/s/electrode. This chip is encased in a titanium package that is hermetically sealed using a simple but effective method. A low cost metal-silicon hybrid mold has been developed for fabricating an intracochlear electrode array with 16 ball-shaped stimulating contacts.
Electrical Stimulation-induced Cell Clustering in Cultured Neural Networks
Planar microelectrode arrays (MEAs) are widely used to record electrical activity from neural networks. However, only a small number of functional recording sites frequently show electrical activity. One contributing factor may be that neurons in vitro receive insufficient synaptic input to develop into fully functional networks. In this study, electrical stimulation was applied to neurons mimicking synaptic input. Various stimulation paradigms were examined. Stimulation amplitude and frequency were tailored to prevent cell death. Two effects of stimulation were observed when 3-week-old cultures were stimulated: (1) clusters of neural cells were observed adjacent to stimulating electrodes and (2) an increase in spontaneous neuronal activity was recorded at stimulating electrodes. Immunocytochemical analysis indicates that stimulation may cause both new neuron process growth as well as astrocyte activation. These data indicate that electrical stimulation can be used as a tool to modify neural networks at specific electrode sites and promote electrical activity.
Low-density Neuronal Networks Cultured Using Patterned Poly-l-lysine on Microelectrode Arrays
Synaptic activity recorded from low-density networks of cultured rat hippocampal neurons was monitored using microelectrode arrays (MEAs). Neuronal networks were patterned with poly-l-lysine (PLL) using microcontact printing (microCP). Polydimethysiloxane (PDMS) stamps were fabricated with relief structures resulting in patterns of 2 microm-wide lines for directing process growth and 20 microm-diameter circles for cell soma attachment. These circles were aligned to electrode sites. Different densities of neurons were plated in order to assess the minimal neuron density required for development of an active network. Spontaneous activity was observed at 10-14 days in networks using neuron densities as low as 200 cells/mm(2). Immunocytochemistry demonstrated the distribution of dendrites along the lines and the location of foci of the presynaptic protein, synaptophysin, on neuron somas and dendrites. Scanning electron microscopy demonstrated that single fluorescent tracks contained multiple processes. Evoked responses of selected portions of the networks were produced by stimulation of specific electrode sites. In addition, the neuronal excitability of the network was increased by the bath application of high K(+) (10-12 mM). Application of DNQX, an AMPA antagonist, blocked all spontaneous activity, suggesting that the activity is excitatory and mediated through glutamate receptors.
Modulation of Cultured Neural Networks Using Neurotrophin Release from Hydrogel-coated Microelectrode Arrays
Polyacrylamide and poly(ethylene glycol) diacrylate hydrogels were synthesized and characterized for use as drug release and substrates for neuron cell culture. Protein release kinetics was determined by incorporating bovine serum albumin (BSA) into hydrogels during polymerization. To determine if hydrogel incorporation and release affect bioactivity, alkaline phosphatase was incorporated into hydrogels and a released enzyme activity determined using the fluorescence-based ELF-97 assay. Hydrogels were then used to deliver a brain-derived neurotrophic factor (BDNF) from hydrogels polymerized over planar microelectrode arrays (MEAs). Primary hippocampal neurons were cultured on both control and neurotrophin-containing hydrogel-coated MEAs. The effect of released BDNF on neurite length and process arborization was investigated using automated image analysis. An increased spontaneous activity as a response to the released BDNF was recorded from the neurons cultured on the top of hydrogel layers. These results demonstrate that proteins of biological interest can be incorporated into hydrogels to modulate development and function of cultured neural networks. These results also set the stage for development of hydrogel-coated neural prosthetic devices for local delivery of various biologically active molecules.
Activity-dependent Neuronal Cell Migration Induced by Electrical Stimulation
Recently, we found that electrical stimulation can induce neuronal migration in neural networks cultured for more than 3 weeks on microelectrode arrays. Immunocytochemistry data showed that the aggregation of neurons was related to the emergence of astrocytes in culture. In this study, when neurons were cocultured with astrocytes, electrical stimulation could induce the migration of neuronal cell bodies after only 1 week in culture, while the same stimulation paradigm caused neural necrosis in neuron-only cultures. In addition, the stimulation-induced migration was inhibited by blocking action potentials in neural networks using the voltage-gated sodium channel blocker, tetrodotoxin. Immunocytochemistry was performed to monitor precisely the neuronal migration and count the number of neurons. These results indicate that neuronal migration of cell bodies is dependent on neuronal activity evoked by electrical stimulation and can be enhanced by coculturing with astrocytes. We believe this method can be employed as a means for modifying neural networks and improving the interface between electrodes and neurons.
Cell-based Olfactory Biosensor Using Microfabricated Planar Electrode
The initial event in olfactory perception is the binding of odorant molecules to specific receptor proteins in the human nose. The interaction between odorant and receptor initiates olfactory signal transduction that leads to a cation influx and change in the membrane potential of the olfactory sensory neuron. In this study, a microfabricated planar electrode was used to measure the generated membrane potential in a heterologous olfactory system. Human embryonic kidney (HEK)-293 cells expressing the olfactory receptor I7 were transfected with the gustatory cyclic nucleotide gated (CNG) channel to amplify the membrane potential. A microfabricated planar electrode was used to measure the electrical responses of odorant-receptor binding. Stimulation of the olfactory receptor with its specific odorant caused an intracellular Ca(2+) influx, which was quantitatively measured using a planar electrode. The extracellular field potential generated by the Ca(2+) influx through the CNGgust channel of the cells was approximately 10 mV. This cell-based olfactory biosensor, which uses a microfabricated planar electrode for detection, would be useful for screening specific ligands for binding to orphan olfactory receptors.
Docosahexaenoic Acid Promotes Hippocampal Neuronal Development and Synaptic Function
Docosahexaenoic acid (DHA, 22:6n-3), the major polyunsaturated fatty acid accumulated in the brain during development, has been implicated in learning and memory, but underlying cellular mechanisms are not clearly understood. Here, we demonstrate that DHA significantly affects hippocampal neuronal development and synaptic function in developing hippocampi. In embryonic neuronal cultures, DHA supplementation uniquely promoted neurite growth, synapsin puncta formation and synaptic protein expression, particularly synapsins and glutamate receptors. In DHA-supplemented neurons, spontaneous synaptic activity was significantly increased, mostly because of enhanced glutamatergic synaptic activity. Conversely, hippocampal neurons from DHA-depleted fetuses showed inhibited neurite growth and synaptogenesis. Furthermore, n-3 fatty acid deprivation during development resulted in marked decreases of synapsins and glutamate receptor subunits in the hippocampi of 18-day-old pups with concomitant impairment of long-term potentiation, a cellular mechanism underlying learning and memory. While levels of synapsins and NMDA receptor subunit NR2A were decreased in most hippocampal regions, NR2A expression was particularly reduced in CA3, suggesting possible role of DHA in CA3-NMDA receptor-dependent learning and memory processes. The DHA-induced neurite growth, synaptogenesis, synapsin, and glutamate receptor expression, and glutamatergic synaptic function may represent important cellular aspects supporting the hippocampus-related cognitive function improved by DHA.
Enhancement of Cellular Olfactory Signal by Electrical Stimulation
The binding of olfactory receptors with odorant molecules initiates olfactory signal transduction, which leads to a cationic influx. In the present study, human embryonic kidney-293 cells were recombinantly engineered to co-express olfactory receptors I7 and gustatory cyclic nucleotide gated channels, which increased the cationic influx. The odorant-induced change in the membrane potential was measured in the extracellular region using microelectrode arrays. A biphasic electrical current stimulation was applied to the cells in order to increase the intrinsic cellular activity. Upon odorants recognition, the electrical cellular activity was enhanced following each electrical stimulation pulse. Compared with odorant-only stimulation, the electrical stimulation induced up to a fivefold increase in the amplitude of the electrical signal, which was produced in response to the odorants. These results demonstrate that electrical stimulation can enhance cellular activity and increase the response signal for detecting ligand binding. This paradigm of electrical stimulation can be used as a novel method in the field of versatile cell-based biosensors for detection of specific odorants.
Optical Monitoring of Neural Networks Evoked by Focal Electrical Stimulation on Microelectrode Arrays Using FM Dyes
Patch-clamping or microelectrode arrays (MEA) are conventional methods to monitor the electrical activity in biological neural networks in vitro. Despite the effectiveness of these techniques, there are disadvantages including the limited number of electrodes and the predetermined location of electrodes in MEAs. In particular, these drawbacks raise a difficulty in monitoring a number of neurons outnumbering the electrodes. Here, we propose an optical technique to determine the effective range of focal electrical stimulation using FM dyes in neural networks grown on planar-type MEAs. After 3 weeks in culture, electrical stimulation was delivered to neural networks via an underlying electrode in the presence of FM dyes. The stimulation induced the internalization of the dye into the neurons around the stimulating electrodes. Fluorescent images of dye distribution successfully showed the effects of focal stimulation. A range of stimulus amplitudes and frequencies were examined to collect fluorescence images. FM-dye uptake after electrical stimulation resulted in the labeling of cells up to approximately 300 microm away from the stimulating electrode. Fluorescence intensity increased proportionally to stimulation amplitude. Tetrodotoxin was shown to inhibit the labeling of neurons except those located immediately adjacent (within 40 microm) from the stimulating electrode. In the presence of AMPA and NMDA receptors antagonists, the FM-dye labeling appeared within 80 microm from the electrode, indicating directly evoked neural networks via blocking of glutamatergic synaptic transmission. These results showed that FM dyes can be a useful tool for monitoring activity-dependent synaptic events and determining the effect of focal stimulation in cultured neural networks.
N-Docosahexaenoylethanolamide Promotes Development of Hippocampal Neurons
DHA (docosahexaenoic acid, C22:6,n-3) has been shown to promote neurite growth and synaptogenesis in embryonic hippocampal neurons, supporting the importance of DHA known for hippocampus-related learning and memory function. In the present study, we demonstrate that DHA metabolism to DEA (N-docosahexaenoylethanolamide) is a significant mechanism for hippocampal neuronal development, contributing to synaptic function. We found that a fatty acid amide hydrolase inhibitor URB597 potentiates DHA-induced neurite growth, synaptogenesis and synaptic protein expression. Active metabolism of DHA to DEA was observed in embryonic day 18 hippocampal neuronal cultures, which was increased further by URB597. Synthetic DEA promoted hippocampal neurite growth and synaptogenesis at substantially lower concentrations in comparison with DHA. DEA-treated neurons increased the expression of synapsins and glutamate receptor subunits and exhibited enhanced glutamatergic synaptic activity, as was the case for DHA. The DEA level in mouse fetal hippocampi was altered according to the maternal dietary supply of n-3 fatty acids, suggesting that DEA formation is a relevant in vivo process responding to the DHA status. In conclusion, DHA metabolism to DEA is a significant biochemical mechanism for neurite growth, synaptogenesis and synaptic protein expression, leading to enhanced glutamatergic synaptic function. The novel DEA-dependent mechanism offers a new molecular insight into hippocampal neurodevelopment and function.
A 1μm Diameter Tip Fiber-based Surface Plasmon Resonance System for Single Unit Optical Neural Recording
A gold-deposited optical fiber sensor system based on surface plasmon resonance (SPR) was developed for optical measurement of neuronal activity. To enhance the sensitivity of localized SPR and to make a precise and safe contact with the cellular membrane, we designed a tapered optical probe of 1μm diameter at the tip of the fiber. By wet etching and gold evaporating processes, pencil-shaped optical probes were successfully fabricated. The SPR system with the sharp optical probe was integrated with a conventional patch clamping system to realize a simultaneous optical and electrical recording on a single neuron. Although the shape of optical signal is not clear due to tiny change of intrinsic optical properties on the neuron, optical and electrical signals were simultaneously changed by capsaicin stimulation. Furthermore, our designed fiber probe can be applicable to localized optical stimulation as well as in vivo optical neuroprosthetic devices.
