• The Korean Journal of Physiology and Pharmacology
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• v.5 no.3
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• pp.199-204
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• 2001

In order to understand the phenomenon in a living cell correctly, it has been required to obtain intact responses from the cell membrane without disrupting the cytoplasmic circumstances. Gramicidin perforated patch configuration allows the electrical access to the whole cell with a minimal dialysis of cytoplasm and preventing the loss of native intracellular constituents, such as $Cl^-.$ Here, we would like to show the background of this method and the actual application of the gramicidin perforated patch recording mode on the dissociated neurons.

• Animal cells and systems
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• v.13 no.4
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• pp.357-362
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• 2009

Hesperidin, the most abundant polyphenolic compound found in citrus fruits, has been known to possess neuroprotective, sedative, and anticonvulsive effects on the nervous system. In a recent electrophysiological study, it was reported that hesperidin induced biphasic change in population spike amplitude in hippocampal CA1 neurons in response to both single spike stimuli and theta-burst stimulation depending on its concentration. However, the precise mechanism by which hesperidin acts on neuronal functions has not been fully elucidated. Here, using whole-cell patch-clamp recording, we revealed that hesperidin did not affect excitatory synaptic activities such as basal synaptic transmission and theta-burst LTP. Moreover, in a current injection experiment, spike number, resting membrane potential and action potential threshold also remained unchanged. Taken together, these results indicate that the effects of hesperidin on the neuronal functions such as spiking activity might not be attributable to either modification of excitatory synaptic transmissions or changes in membrane excitability in hippocampal CA1 neuron.

• Proceedings of the Korean Biophysical Society Conference
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• 2002.06b
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• pp.38-38
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• 2002

Whole-cell recordings from adult rat sympathetic neurons demonstrate that calcium current is comprised of at least three types, N, L '||'&'||' R. We are using cell-attached patch recording to identify the single calcium channels that underlie these macroscopic currents. Single channels were resolved the presence of 100 mM Ba$\^$2＋/ and l${\mu}$M BayK 8644 over the voltage range -40 to $\^$＋/50 mV.(omitted)

• International Journal of Oral Biology
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• v.30 no.2
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• pp.71-76
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• 2005

The mesencephalic trigeminal nucleus (Mes V) contains cell bodies of primary afferent sensory neurons that relay proprioceptive information from the periodontium and masticatory muscles and function as typical sensory neurons or potentially as integrative interneurons. In the present study, we studied these two potential functions using combined experimental approaches of retrograde labeling and whole cell patch clamp recording. Mes V neurons that presumably originate from periodontal nerve fibers in subsets of Mes V nucleus were identified by retrograde labeling with a fluorescent dye, DiI, which was applied onto inferior alveolar nerve. These cells were elliptical perikarya shaped cells about $40{\mu}m$ in diameter. In these neurons, we measured high voltage-activated calcium channel (HVACC) currents. $GABA_B$ agonist, baclofen, inhibited calcium currents, and the HVACC currents inhibition by baclofen was voltage-dependent, exhibited prepulse facilitation, indicating that it was mediated by $G_i/_G_o$ protein. Taken together, our results demonstrate that Mes V neurons not only have cell bodies originating from periodontium, but also receive synaptic inputs including GABAergic neurons suggesting that Mes V neurons function as both primary sensory neurons and integrative interneurons.

• Proceedings of the Korean Biophysical Society Conference
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• 1998.06a
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• pp.38-38
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• 1998

Resting membrane potential of atrial myocytes is less negative than K+ equilibrium potential, suggesting the presence of ion channels carrying inward currents. We investigated the background Na$\^$＋/ current in rat atrial myocytes using both conventional whole cell voltage clamp technique and single channel recording.(omitted)

• The Korean Journal of Physiology and Pharmacology
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• v.1 no.4
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• pp.355-366
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• 1997

Although cerebellar Purkinje cells display spontaneous electrical activity in vivo and in slice experiments, the mechanism of the spontaneous activity generation has not been clearly understood. The aim of this study was to investigate whether cerebellar Purkinje cells of postnatal rats generate spontaneous electrical activity without synaptic inputs. Dissociated cerebellar Purkinje cells were used for reducing synaptic inputs in the present study. Cerebellar Purkinje cells with dendrites were dissociated from postnatal rats using enzymatic treatment followed by mechanical trituration. Spontaneous electrical activities were recorded from dissociated cells without any stimulus using whole-cell patch clamp configuration. Two types, spontaneously firing or quiescent, of dissociated Purkinje cells were observed in postnatal rats. Both types of cells were identified as Purkinje cells using immunocytochemical staining technique with anti-calbindin after recording. Spontaneously active cells displayed two patterns of firing, repetitive and burst firings. Two thirds of dissociated Purkinje cells displayed repetitive firing and the rest of them did burst firing under same recording condition. Repetitive firing activities were maintained even after further isolation using either physical or pharmacological techniques. Neither high magnessium solution nor excitatory synaptic blockers, AP-5 and DNQX, block the spontaneous activity. These results demonstrate that spontaneous electrical activity of isolated cerebellar Purkinje cells in postnatal rats is generated by intrinsic membrane properties rather than synaptic inputs.

• The Korean Journal of Physiology and Pharmacology
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• v.7 no.3
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• pp.131-142
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• 2003

We have studied mutant forms of Kir2.1 in which an aspartate residue (D172), important for gating by intracellular polyamines, is replaced by one of three basic residues (Arg, Lys or His). Such channels are highly selective for $K^+$, but show inward rectification that is a shallow function of voltage compared with that found in wild type. This inward rectification occurs with a reduced affinity for spermine and persists in the absence of polyamines. Though the unitary current-voltage relation shows some inward rectification, it is insufficient to account for that seen under whole cell recording. Channels open and shut under single channel recording, and changes of $P_{open}$ appear to generate inward rectification. In D172H, the reduction in affinity for spermine is greater when His is protonated at low $pH_i$. The effective valency for spermine is reduced from $3.09{\pm}0.07$ in wild type to $1.95{\pm}0.09$ in D172H at $pH_i$ 6.3. In the presence of dual mutants of Kir2.1, where E224 is also replaced, spermine affinity becomes undetectable. However, channels still show inward rectification and open and shut under hyper- and depolarisation, respectively. We suggest that Kir2.1 channel are able to undergo conformation changes; these changes may be important physiologically in generating inward rectification, the normal parameters of which are set by the binding of polyamines such as spermine.

• Molecules and Cells
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• v.22 no.2
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• pp.198-202
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• 2006

The $P2X_7$ receptor, an ATP-gated cation channel, induces cell death in immune cells and is involved in neurodegenerative diseases. Although the receptor plays various roles in these diseases, the cellular mechanisms involved are poorly understood and antagonists are limited. Here, the development of a cell-based assay for human $P2X_7$ receptor is reported. We established permanent lines of HEK 293 cells expressing a high level of $hP2X_7$ receptor. Functional activity of the $hP2X_7$ receptor was confirmed by whole-cell patch recording of ATP-induced ion currents. Prolonged exposure to ATP resulted in death of the $hP2X_7$-expressing HEK 293 cells and this cell death could be quantified. Two known $P2X_7$ antagonists, PPADS and KN-62, blocked ATP-induced death in a concentration-dependent manner. Thus, this assay can be used to screen for new antagonists of $hP2X_7$ receptors.

• The Korean Journal of Physiology and Pharmacology
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• v.4 no.3
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• pp.169-175
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• 2000

To learn the developmental changes in intrinsic electrophysiological properties of the second order taste neurons, whole cell recordings from the developing nucleus of the solitary tract neurons were done in brainstem slices of postnatal rats. Rats aged from postnatal 0 to 21 days (P0-P21) were used, being divided into 3 age groups: postnatal first week (P0-P7 days), second week (P8-P14 days), and third week (P15-P21 days). Slices containing gustatory NTS were cut horizontally in the thickness of $300\;{\mu}m.$ Whole cell recordings were obtained from neurons in response to a series of hyperpolarizing and depolarizing current pulses. The intrinsic electrophysiological properties of the rostral NTS (rNTS) neurons were compared among the age groups. Depolarizing current pulses evoked a train of action potentials in all neurons of all age groups. The resting membrane potential and input resistance of the neurons did not show any significant differences during the postnatal 3 weeks. The time constant, however, decreased during the development. Duration of action potential measured at half maximum amplitude was longer in younger age groups. Both the maximum rate of rise and the maximum rate of fall in the action potential increased during the first 3 weeks postnatal. Electrophysiologically more than half neurons were type III. In summary, it is suggested that developmental changes in electrophysiological properties in rNTS occur during the first three weeks in rats.

• Journal of Ginseng Research
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• v.39 no.2
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• pp.169-177
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• 2015

Background: Ginsenoside Rd (GSRd), one of the most abundant ingredients of Panax ginseng, protects the heart via multiple mechanisms including the inhibition of $Ca^{2+}$ influx.We intended to explore the effects of GSRd on L-type $Ca^{2+}$ current ($I_{Ca,L}$) and define the mechanism of the suppression of $I_{Ca,L}$ by GSRd. Methods: Perforated-patch recording and whole-cell voltage clamp techniques were applied in isolated rat ventricular myocytes. Results: (1) GSRd reduced $I_{Ca,L}$ peak amplitude in a concentration-dependent manner [half-maximal inhibitory concentration $(IC_{50})=32.4{\pm}7.1{\mu}mol/L$] and up-shifted the current-voltage (I-V) curve. (2) GSRd ($30{\mu}mol/L$) significantly changed the steady-state activation curve of $I_{Ca,L}$ ($V_{0.5}:-19.12{\pm}0.68$ vs. $-6.26{\pm}0.38mV$; n = 5, p < 0.05) and slowed down the recovery of $I_{Ca,L}$ from inactivation [the time content (${\zeta}$) from 91 ms to 136 ms, n = 5, p < 0.01]. (3) A more significant inhibitive effect of GSRd ($100{\mu}mol/L$) was identified in perforated-patch recording when compared with whole-cell recording [$65.7{\pm}3.2%$ (n = 10) vs. $31.4{\pm}5.2%$ (n = 5), p < 0.01]. (4) Pertussis toxin ($G_i$ protein inhibitor) completely abolished the $I_{Ca,L}$ inhibition induced by GSRd. There was a significant difference in inhibition potency between the two cyclic adenosine monophosphate elevating agents (isoprenaline and forskolin) prestimulation [$55{\pm}7.8%$ (n = 5) vs. $17.2{\pm}3.5%$ (n = 5), p < 0.01]. (5) 1H-[1,2,4]Oxadiazolo[4,3-a]-quinoxalin-1-one (a guanylate cyclase inhibitor) and N-acetyl-$\small{L}$-cysteine (a nitric oxide scavenger) partly recovered the $I_{Ca,L}$ inhibition induced by GSRd. (6) Phorbol-12-myristate-13-acetate (a protein kinase C activator) and GF109203X (a protein kinase C inhibitor) did not contribute to the inhibition of GSRd. Conclusion: These findings suggest that GSRd could inhibit $I_{Ca,L}$ through pertussis toxin-sensitive G protein ($G_i$) and a nitric oxide-cyclic guanosine monophosphate-dependent mechanism.