Jong-Ju Lee

The ameliorating effect of oroxylin A on scopolamine-induced memory impairment in mice.

Oroxylin A is a flavonoid and was originally isolated from the root of Scutellaria baicalensis Georgi., one of the most important medicinal herbs in traditional Chinese medicine. The aim of this study was to investigate the ameliorating effects of oroxylin A on memory impairment using the passive avoidance test, the Y-maze test, and the Morris water maze test in mice. Drug-induced amnesia was induced by administering scopolamine (1 mg/kg, i.p.) or diazepam (1 mg/kg, i.p.). Oroxylin A (5 mg/kg) significantly reversed cognitive impairments in mice by passive avoidance and the Y-maze testing (P<.05). Oroxylin A also improved escape latencies in training trials and increased swimming times and distances within the target zone of the Morris water maze (P<.05). Moreover, the ameliorating effects of oroxylin A were antagonized by both muscimol and diazepam (0.25 mg/kg, i.p., respectively), which are GABA(A) receptor agonists. Furthermore, oroxylin A (100 microM) was found to inhibit GABA-induced inward Cl(-) current in a single cortical neuron. These results suggest that oroxylin A may be useful for the treatment of cognitive impairments induced by cholinergic dysfunction via the GABAergic nervous system.

Serotonergic Modulation of GABAergic and Glutamatergic Synaptic Transmission in Mechanically Isolated Rat Medial Preoptic Area Neurons

The medial preoptic area (MPOA) of the hypothalamus is critically involved in the regulation of male sexual behavior and has been implicated in several homeostatic processes. Serotonin (5-hydroxytryptamine, 5-HT) inhibits sexual behavior via effects in the MPOA, where there are high densities of 5-HT1A and 5-HT1B receptor subtypes. We used whole-cell recordings under voltage-clamp conditions to investigate the serotonergic modulation of γ-aminobutyric acid (GABA)ergic and glutamatergic synaptic transmission in mechanically dissociated rat MPOA neurons with native presynaptic nerve endings. Spontaneous GABAergic miniature inhibitory postsynaptic currents (mIPSCs) in the MPOA were completely blocked by bicuculline. Serotonin reversibly reduced the GABAergic mIPSC frequency without affecting the mean current amplitude. Serotonergic inhibition of mIPSC frequency was mimicked by (±)-8-hydroxy-2-dipropylaminotetralin hydrobromide, a specific 5-HT1A receptor agonist, and blocked by 1-(2-methoxyphenyl)-4-[4-(2-phthalimido)butyl] piperazine hydrobromide, a specific 5-HT1A receptor antagonist. 6-Cyano-7-nitroquinoxaline-2,3-dione completely blocked spontaneous glutamatergic miniature excitatory postsynaptic currents (mEPSCs) in the MPOA. Serotonin reversibly decreased the glutamatergic mEPSC frequency without affecting the mean current amplitude. Serotonergic inhibition of mEPSC frequency was mimicked by CGS 12066B, a specific 5-HT1B receptor agonist, and blocked by SB 216641, a specific 5-HT1B receptor antagonist. Stimulation of adenylyl cyclase with forskolin increased the frequencies of GABAergic mIPSCs and glutamatergic mEPSCs, and blocked the inhibitory effects of 5-HT. H-89, a selective protein kinase A (PKA) inhibitor, decreased the frequencies of GABAergic mIPSCs and glutamatergic mEPSCs, and blocked their reduction by 5-HT. These findings suggest that 5-HT reduces the frequency of GABAergic mIPSCs and glutamatergic mEPSCs through 5-HT1A and 5-HT1B receptor-mediated inhibition, respectively, of the PKA-dependent pathway in the presynaptic nerve terminals of MPOA neurons.

Electroacupuncture Enhancement of Natural Killer Cell Activity Suppressed by Anterior Hypothalamic Lesions in Rats

Background/Objective: Neuroendocrine hormones are derived from the hypothalamus. The central nervous system, particularly the hypothalamus, is capable of modulating the cytolytic activity of adherent natural killer (NK) cells. In addition, electroacupuncture (EA) stimulation of the Zusanli (ST36) acupoint enhances splenic NK cell and cytokine activities in rats. However, it is still unclear whether the anterior hypothalamus affects this immunomodulation. Therefore, the aim of the present study was to examine the effect of EA stimulation at the Zusanli acupoint on the NK cell activity modulated by an anterior hypothalamic area lesion. Methods: Male Sprague-Dawley rats were used. Lesions were placed by means of a direct current through a concentric electrode. The electric acupuncture stimulation was delivered for 30 min per each experiment at the right ST36 acupoint with an electrical stimulator. The NK cell activity of the spleen was measured by a fluorescence assay. Results: The NK cell activity was significantly reduced on the 2nd day after the lesion, but was restored to that of the sham group by the 7th day. However, when EA was applied for 2 days after the operation, the NK cell activity of the lesion group was restored to that of the sham group. After 7 days of EA, the NK cell activity of the lesion group was slightly higher than that of the sham group. Conclusion: From these results, we can suggest that EA enhances or restores the NK cell activity suppressed by an anterior hypothalamic area lesion.

Activation of protein kinase C antagonizes the opioid inhibition of calcium current in rat spinal dorsal horn neurons.

Spinal dorsal horn (SDH) is one of important regions in both nociceptive transmission and antinociception. Opioid peptides produce analgesia via regulation of neurotransmitter release through modulation of voltage-dependent Ca(2+) channel (VDCC) in neuronal tissues. The modulatory effect of micro-opioid receptor (MOR) activation on VDCC was investigated in acutely isolated rat SDH neurons under the conventional whole-cell patch-clamp recording mode. The Ba(2+) current passing through VDCC was reversibly inhibited by a MOR agonist, [D-Ala(2),N-MePhe(4),Gly(5)-ol]-enkephalin (DAMGO, 1 microM). Among 108 SDH neurons tested, VDCC of 39 neurons (36%) were inhibited by MOR activation, while other 69 neurons (64%) were not affected. The L-, N-, P/Q-, and R-type VDCC components shared 58.4+/-18.9%, 29.3+/-12.1%, 8.7+/-7.2%, and 3.4+/-4.8% of the total VDCC, respectively. Among VDCC subtypes inhibited by MOR activation, L- and N-types were 61.4+/-12.8% and 30.7+/-14.4%, respectively, while both P/Q- and R-types were 7.9+/-11.8%. A depolarizing pre-pulse increased the amplitude of VDCC and suppressed most of the inhibitory effect of MOR activation. Application of 1 microM phorbol-12-myristate-13-acetate completely antagonized the inhibitory effect of MOR activation without any alteration of basal VDCC amplitude. In contrast, the response of MOR activation was not altered by application of 4-alpha-phorbol (1 microM), 2-[3-Dimethylaminopropyl]indol-3-yl]-3-(indol-3-yl) maleimide (GF109203X, 1 microM), forskolin (1 microM), N-(2-[p-Bromocinnamylamino]ethyl)-5-isoquinolinesulfonamide hydrochloride (H-89, 1 microM). These results indicate that activation of MOR coupled to G-proteins inhibits VDCC, and that this G-protein-mediated inhibition is antagonized by PKC-dependent phosphorylation.

Roles of protein kinase A and C in the opioid potentiation of the GABAA response in rat periaqueductal gray neuron

The periaqueductal gray (PAG) is the main target site of the opioid-induced analgesia. The present study was designed to examine the roles of protein kinase A (PKA) and C (PKC) in the opioid-induced modulation of the currents activated by an inhibitory neurotransmitter, gamma-aminobutyric acid (GABA). The PAG neurons were acutely isolated and voltage-clamped under the nystatin-perforated patch-clamp mode. The GABA-activated current was sensitively blocked by a GABA(A) receptor antagonist, bicuculline, and selectively carried by chloride ions. The GABA(A) receptor-activated Cl(-) current was potentiated by a mu-opioid receptor agonist, [D-Ala(2),N-MePhe(4),Gly(5)-ol]-enkephalin acetate (DAMGO). The GABA response was also potentiated by phorbol-12-myristate-13-acetate (PMA). Pretreatment with PMA occluded the DAMGO potentiation. However, both chelerythrine and 2-[1-(3-dimethylaminopropyl)indol-3-yl]-3-(indol-3-yl) maleimide (GF109203X) also potentiated the GABA response. Pretreatment with chelerythrine or GF109203X also occluded the DAMGO potentiation. Meanwhile, the GABA response was potentiated by N-(2-[p-bromocinnamylamino]-ethyl)-5-isoquinolinesulfonamide (H-89), while not altered by forskolin. Pretreatment with H-89 occluded the potentiation effect of DAMGO on the GABA response. In addition, the DAMGO effect was completely blocked by pretreatment with forskolin. From the result, it can be suggested that activation of mu-opioid receptor potentiates the GABA(A) response through the mediation of PKA inhibition, and that PKC is not directly involved in the action mechanism of DAMGO.

Opioid inhibition of GABAergic neurotransmission in mechanically isolated rat periaqueductal gray neurons

The descending pain control system is activated by opioid peptides mainly at the midbrain periaqueductal gray (PAG). Although activation of presynaptic opioid receptors has been reported to inhibit gamma-aminobutyric acid (GABA) release, the exact electrophysiological mechanisms are controversial. To elucidate the mechanisms involved in the opioid modulation of presynaptic GABA release, we isolated single PAG neurons with functionally intact synaptic terminals by a mechanical dissociation in the absence of enzyme. With the conventional whole-cell recording mode under the voltage-clamp conditions, the spontaneous miniature inhibitory postsynaptic currents (mIPSCs) were recorded. Bicuculline completely and reversibly blocked mIPSCs. A specific mu-opioid agonist, [d-Ala(2),N-Me-Phe(4),Gly(5)-ol]-enkephalin (DAMGO), reversibly reduced the frequency of mIPSCs without any alteration of amplitude. The inhibitory effect of DAMGO was blocked by N-ethylmaleimide. Blockade of presynaptic Ca(2+) influx by cadmium or depletion of extracellular Ca(2+) did not alter the DAMGO inhibition. In addition, K(+) channels blockers, Ba(2+) or 4-aminopyridine, did not affect the DAMGO effect. The present study indicates that activation of presynaptic mu-opioid receptors coupled to G-proteins inhibits GABA release through unknown intracellular mechanisms downstream of Ca(2+) influx.

Effect of nitric oxide on auditory cortical neurons of aged rats

Age-related changes in the effects of nitric oxide (NO) on neurons of the auditory cortex have not been determined. We therefore evaluated the anatomical changes and neurophysiological characteristics of these neurons in rats as a function of age. The numbers of cresyl violet stained cells, the numbers and areas of NADPH-d-positive neuronal cell bodies, and their optical density, were measured in Sprague-Dawley rats aged 24 months (aged group) and 4 months (control group). The modulatory effects of NO on K(+) currents of acutely isolated rat auditory cortical neurons were also assessed. There were no between-group differences in the distribution patterns of glial cells and neurons, or in the numbers and areas of NADPH-d-positive neuronal cell bodies. However, the optical density of NADPH-d-positive neuronal cell bodies was significantly greater in the aged group than in the control group. In addition, voltage-gated K(+) currents of rat auditory cortical neurons were activated by increased levels of NO. As activation of the K(+) current likely suppresses neuronal excitability, age-associated increases in NO production can hinder the function of the acoustic center by inhibiting neuron excitability.

Role of protein kinase C in opioid modulation of glycine-gated Cl- current in rat periaqueductal gray neuron

The Role of protein kinase C in the modulatory effect of a mu-opioid receptor agonist, [D-Ala(2),N-Me-Phe(4),Gly(5)-ol]enkephalin (DAMGO), on the glycine-gated Cl(-) current was examined in acutely dissociated rat periaqueductal gray neurons. Using the nystatin-perforated patch-clamp technique, the neurons were voltage-clamped at -60 mV. The glycine-gated Cl(-) current (I(Gly)) was sensitive to strychnine. On pretreatment with 1 microM DAMGO, the 30-microM glycine response increased with time and showed a maximum amplitude of 209+/-37% of control. After a protein kinase C activator, phorbol-12-myristate-13-acetate (PMA, 0.1 microM) as pretreatment, I(Gly) increased to 138+/-6% of control. The DAMGO potentiation of I(Gly) was not altered by coapplication with PMA. Although protein kinase C inhibitors, chelerythrine (3 microM) and 2-[1-(3-dimethylaminopropyl)indol-3-yl]-3-(indol-3-yl) maleimide (GF109203X, 1 microM), did not alter I(Gly), the DAMGO-induced potentiation of I(Gly) was reduced to 161+/-21% or 164+/-31% of the control after coapplication with chelerythrine or GF109203X, respectively. These results indicate that the potentiation of I(Gly) by a mu-opioid receptor agonist is partly mediated by activation of protein kinase C.

5-HT1A receptor-mediated activation of a G-protein-coupled inwardly rectifying K+ current in rat medial preoptic area neurons.

The medial preoptic area plays an important role in the regulation of sexual behavior, and serotonin (5-hydroxytryptamine, 5-HT) exerts an inhibitory effect on sexual behavior by acting on the medial preoptic area region. This study was designed to clarify the inhibitory effect of 5-HT on the medial preoptic area neurons and to elucidate the electrophysiological mechanisms involved in the action of 5-HT. Superfusion of 100 nM 5-HT hyperpolarized the membrane potential and inhibited the action potential firing. When the membrane potential was stepped to various potentials, the inward K+ currents were potentiated in the presence of 100 nM 5-HT. When the concentration of K+ in the external solution was increased from 5 mM to 30 mM, 5-HT markedly potentiated the inward K+ currents. In the steady-state current-voltage relationship, the 5-HT-activated inward current was carried by K+ ions and showed characteristics typical of an inwardly rectifying K+ current. The 5-HT-activated K+ current was mimicked by a 5-HT1A receptor agonist, (+/-)-8-hydroxy-2-dipropylaminotetralin hydrobromide, and was reversibly blocked by a 5-HT1A receptor antagonist, 1-(2-methoxyphenyl)-4-[4-(2-phthalimido)butyl] piperazine hydrobromide, but not by a 5-HT2 receptor antagonist, ketanserin. The 5-HT-activated K+ current was sensitively blocked by Ba2+, but not by 4-aminopyridine, and was completely suppressed by N-ethylmaleimide. These results indicate that 5-HT-induced hyperpolarization of the medial preoptic area neurons occurs as a result of activation of the G-protein-coupled inwardly rectifying K+ currents by 5-HT1A receptors.

Developmental change of GABAergic postsynaptic current in rat periaqueductal gray

The present study was designed to examine developmental changes of GABAergic spontaneous miniature inhibitory postsynaptic currents (mIPSCs) in periaqueductal gray (PAG) neurons mechanically isolated from young (12- to 18-day) and adult (8- to 12-week) rats. While the frequency of mIPSCs was similar, the current amplitude in adult rats was significantly smaller than in young rats. In the study of mIPSC kinetics, all kinetic parameters except for the fast decay time in adult rats were smaller or shorter than in the case of young rats. The present study demonstrates that a decrease in the amplitude of GABAergic mIPSC during development may result from a decrease in the GABA contents of synaptic vesicles and from changes in the kinetics of postsynaptic GABA-activated Cl- channels.