Nystatin-perforated Patch Clamp Research Articles

Antidepressants modulate glycine action in rat hippocampus.

Antidepressants are drugs that relieve symptoms of depressive disorders. Fluoxetine, tianeptine, and milnacipran are different types of antidepressants, and they have widely been used for relieving of depression symptoms. In the present study, the effects of fluoxetine, tianeptine, and milnacipran on the glycine-induced ion current by nystatin-perforated patch clamp and on the amplitude of field potential in the hippocampal CA1 region by multichannel extracellular recording, MED64, system, were studied. In the present results, fluoxetine, tianeptine, and milnacipran reduced glycine-induced ion current in the hippocampal CA1 neurons in nystatin-perforated patch clamp method. These drugs enhanced the amplitude of the field potential in the hippocampal CA1 region in MED64 system. These results suggest that antidepressants may increase neuronal activity by enhancing field potential through inhibition on glycine-induced ion current.

Abstract 18503: Geranylgeranylacetone (gga) And Gga-derivatives Inhibit Acetylcholine-activated K+ Current In Mouse Atrial Cardiomyocyte ; Possible Mechanism For Af Suppression

Atrial fibrillation (AF) is the most common sustained arrhythmia. AF is caused by inflammation, oxidative stress, and various structural heart diseases. An important key factor in AF promotion is that atrial cardiomyocytes have constitutively active acetylcholine-activated K+ (IKACh) current that is enhanced by the tachycardia. Furthermore, it is known that heat shock proteins (HSPs) are involved in the protection against different forms of cellular stress and recent investigators reported that the HSP inducing compound geranylgeranylacetone (GGA, also known as teprenone) prevents atrial remodeling and attenuates the promotion of AF. Whether GGA and GGA-derivatives prevent the acetylcholine-activated K+ current is unknown. Methods: We examined the effect of HSP inducer GGA and 5 derivatives on IKACh current in mouse atrial cardiomyocytes. In the present study, the IKACh current was recorded using a nystatin-perforated whole cell patch-clamp technique following activation by acetylcholine (10 μM for 2 min). After the measurement of the baseline IKACh current, atrial cardiomyocytes were treated with GGA 10μM or a derivative of GGA (NYK9274, NYK9308, NYK9354, NYK9356 or NYK9376) for 10min and the IKACh current was re-measured. Results: IKACh current in mouse atrial cardiomyocyte treated with acetylcholine (10 μM) was significantly increased, which was markedly attenuated by GGA 10μM treatment (n=5, peak; 54±11.2%, quasi-steady-state; 58±11.4%, p<0.001). In addition, the effects of the GGA derivatives on IKACh were evaluated with same experimental protocol. Our results showed that next to GGA, also NYK9308 and NYK9356 significantly inhibit both peak and quasi-steady-state IKACh. In contrast, NYK9274, NYK9354 and NYK9376 had no effect on IKACh. Conclusion: This study provides important evidences that the HSP inducer GGA facilitates its potent antiarrhythmic effect by inhibiting IKAch, which underlies electrical remodeling in mammalian atrial cardiomyocytes. Furthermore, two GGA derivatives, NYK9308 and NYK9356, with improved pharma-chemical properties compared to GGA, reveal similar protective effects compared to GGA, and provide important background information for the use of GGA and GGA-like compounds in patients with AF.

Attenuation of acetylcholine activated potassium current (I KACh) by simvastatin, not pravastatin in mouse atrial cardiomyocyte: possible atrial fibrillation preventing effects of statin.

Statins, 3-hydroxy-3-methyl-glutaryl-CoA reductase inhibitors, are associated with the prevention of atrial fibrillation (AF) by pleiotropic effects. Recent clinical trial studies have demonstrated conflicting results on anti-arrhythmia between lipophilic and hydrophilic statins. However, the underlying mechanisms responsible for anti-arrhythmogenic effects of statins are largely unexplored. In this study, we evaluated the different roles of lipophilic and hydrophilic statins (simvastatin and pravastatin, respectively) in acetylcholine (100 µM)-activated K+ current (IKACh, recorded by nystatin-perforated whole cell patch clamp technique) which are important for AF initiation and maintenance in mouse atrial cardiomyocytes. Our results showed that simvastatin (1–10 µM) inhibited both peak and quasi-steady-state IKACh in a dose-dependent manner. In contrast, pravastatin (10 µM) had no effect on IKACh. Supplementation of substrates for the synthesis of cholesterol (mevalonate, geranylgeranyl pyrophosphate or farnesyl pyrophosphate) did not reverse the effect of simvastatin on IKACh, suggesting a cholesterol-independent effect on IKACh. Furthermore, supplementation of phosphatidylinositol 4,5-bisphosphate, extracellular perfusion of phospholipase C inhibitor or a protein kinase C (PKC) inhibitor had no effect on the inhibitory activity of simvastatin on I KACh. Simvastatin also inhibits adenosine activated IKACh, however, simvastatin does not inhibit IKACh after activated by intracellular loading of GTP gamma S. Importantly, shortening of the action potential duration by acetylcholine was restored by simvastatin but not by pravastatin. Together, these findings demonstrate that lipophilic statins but not hydrophilic statins attenuate IKACh in atrial cardiomyocytes via a mechanism that is independent of cholesterol synthesis or PKC pathway, but may be via the blockade of acetylcholine binding site. Our results may provide important background information for the use of statins in patients with AF.

Modulation of Amygdalin on Glycine- and Glutamate-induced Ion Currents in Rat Periaqueductal Gray Neurons

Amygdalin is known as vitamain B17, and it was called laetrile. Amygdalin is com posed of two molecules of glucose, one molecule of benzaldehyde which induces an analgesic action, and one molecule of hydrocyanic acid which is an anti-neoplastic compound. Amygdalin had been used to treat cancers and relieve pain. In order to evaluate whether the analgesic action of amygdalin is related with descending pain control system, we performed patch clamp study. In the present study, the modulatory effects of amygdalin on glycine- and glutamate-induced ion currents in periaqueductal gray (PAG) neurons were investigated using the nystatin-perforated patch clamp method. Continuous application of lipopolysaccharides (LPS) on PAG neurons resulted in increased glycine-induced ion current, and in decreased glutamate-induced ion current. In contrast, continuous application of amygdalin with LPS resulted in decreased glycine-induced ion current increased by LPS, and increased glutamateinduced ion current decreased by LPS in concentration- and time-dependent fashion. These results demonstrate that amygdalin modulates neuronal activity of PAG by modulation of glycine and glutamate. Based on the present results, it can be suggested that amygdalin participates in the regulation of the descending pain control system in the level of PAG neurons. The present study demonstrated that activation of the descending pain control system is one of the possible analgesic mechanisms of amygdalin. words: cyclooxygenase-2, periaqueductal gray neurons (PAG), glycine, glutamate, patch clamp, amygdalin

Effects of fluid flow on voltage-dependent calcium channels in rat vascular myocytes: Fluid flow as a shear stress and a source of artifacts during patch-clamp studies

We examined the effects of fluid flow on L-type voltage-dependent Ca 2+ channel (VDCC L) currents in rat vascular myocytes using the nystatin perforated patch-clamp technique. The effect of fluid flow on the liquid (bathing solution)–metal (Ag/AgCl ground electrode) junction potential was also studied. With a fluid flow of 0–10 ml/min, changes in the junction potential of up to 5 mV were observed in proportion to the flow rate. Accordingly, fluid flow shifted the current–voltage ( I– V) relationship of the recorded VDCC L currents in a positive direction. In addition to these shifts, fluid flow also increased the peak VDCC L current, suggesting some modulatory role for fluid flow in VDCC L currents. The use of a 3-M KCl agar-bridge between the ground electrode and bathing solution abnegated the potential shifts, and fluid flow increased the VDCC L currents in a voltage-independent manner. These results suggest that the bathing fluid flow can be both a source of erroneous voltage shift between liquid and metal junctions in the patch-clamp configuration and an important shear stress for the cells. The facilitation of VDCC L currents by fluid flow in vascular myocytes may contribute to the myogenic contraction of blood vessels. The mechanism by which fluid flow causes the voltage shift is vigorously discussed.

Somatostatin decreases voltage-gated Ca2+currents in GH3 cells through activation of somatostatin receptor 2

The secretion of growth hormone (GH) is inhibited by hypothalamic somatostatin (SRIF) in somatotropes through five subtypes of the somatostatin receptor (SSTR1-SSTR5). We aimed to characterize the subtype(s) of SSTRs involved in the Ca2+ current reduction in GH3 somatotrope cells using specific SSTR subtype agonists. We used nystatin-perforated patch clamp to record voltage-gated Ca2+ currents, using a holding potential of -80 mV in the presence of K+ and Na+ channel blockers. We first established the presence of T-, L-, N-, and P/Q-type Ca2+ currents in GH3 cells using a variety of channel blockers (Ni+, nifedipine, omega-conotoxin GVIA, and omega-agatoxin IVA). SRIF (200 nM) reduced L- and N-type but not T- or P/Q-type currents in GH3 cells. A range of concentrations of each specific SSTR agonist was tested on Ca2+ currents to find the maximal effective concentration. Activation of SSTR2 with 10(-7) and 10(-8) M L-797,976 decreased the voltage-gated Ca2+ current and abolished any further decrease by SRIF. SSTR1, SSTR3, SSTR4, and SSTR5 agonists at 10(-7) M did not modify the voltage-gated Ca2+ current and did not affect the Ca2+ current response to SRIF. These results indicate that SSTR2 is involved mainly in regulating voltage-gated Ca2+ currents by SRIF, which contributes to the decrease in intracellular Ca2+ concentration and GH secretion by SRIF.

Modulation of ATP-Induced Inward Currents by Docosahexaenoic Acid and Other Fatty Acids in Rat Nodose Ganglion Neurons

The effects of docosahexaenoic acid (DHA) and other fatty acids on P2X-receptor-mediated inward currents in rat nodose ganglion neurons were studied using the nystatin perforated patch-clamp technique. DHA accelerated the desensitization rate of the ATP-induced current. DHA showed use-dependent inhibition of the peak ATP-induced current. Other polyunsaturated fatty acids, such as arachidonic acid and eicosapentaenoic acid, displayed a similar use-dependent inhibition. The inhibitory effects of saturated fatty acids including palmitic acid and arachidic acid were weaker than those of polyunsaturated fatty acids. The results suggest that fatty acids may modulate the P2X receptor-mediated response when the channel is in the open-state.

Carbachol induces burst firing of dopamine cells in the ventral tegmental area by promoting calcium entry through L‐type channels in the rat

Enhanced activity of the central dopamine system has been implicated in many psychiatric disorders including schizophrenia and addiction. Besides terminal mechanisms that boost dopamine levels at the synapse, the cell body of dopamine cells enhances terminal dopamine concentration through encoding action potentials in bursts. This paper presents evidence that burst firing of dopamine cells in the ventral tegmental area was under cholinergic control using nystatin-perforated patch clamp recording from slice preparations. The non-selective cholinergic agonist carbachol excited the majority of recorded neurones, an action that was not affected by blocking glutamate and GABA ionotropic receptors. Twenty per cent of dopamine cells responded to carbachol with robust bursting, an effect mediated by both muscarinic and nicotinic cholinoceptors postsynaptically. Burst firing induced as such was completely dependent on calcium entry as it could be blocked by cadmium and more specifically the L-type blocker nifedipine. In the presence of the sodium channel blocker tetrodotoxin, carbachol induced membrane potential oscillation that had similar kinetics and frequency as burst firing cycles and could also be blocked by cadmium and nifedipine. Direct activation of the L-type channel with Bay K8644 induced strong bursting which could be blocked by nifedipine but not by depleting internal calcium stores. These results indicate that carbachol increases calcium entry into the postsynaptic cell through L-type channels to generate calcium-dependent membrane potential oscillation and burst firing. This could establish the L-type channel as a target for modulating the function of the central dopamine system in disease conditions.

Somatostatin Increases Voltage-Gated K+ Currents in GH3 Cells through Activation of Multiple Somatostatin Receptors

The secretion of GH by somatotropes is inhibited by somatostatin (SRIF) through five specific membrane receptors (SSTRs). SRIF increases both transient outward (IA) and delayed rectifying (IK) K+ currents. We aim to clarify the subtype(s) of SSTRs involved in K+ current enhancement in GH3 somatotrope cells using specific SSTR subtype agonists. Expression of all five SSTRs was confirmed in GH3 cells by RT-PCR. Nystatin-perforated patch clamp was used to record voltage-gated K+ currents. We first established the presence of IA and IK type K+ currents in GH3 cells using different holding potentials (-40 or -70 mV) and specific blockers (4-aminopirimidine and tetraethylammonium chloride). SRIF (200 nM) increased the amplitude of both IA and IK in a fully reversible manner. Various concentrations of each specific SRTR agonist were tested on K+ currents to find the maximal effective concentration. Activation of SSTR2 and SSTR4 by their respective agonists, L-779,976 and L-803,087 (10 nM), increased K+ current amplitude without preference to IA or IK, and abolished any further increase by SRIF. Activation of SSTR1 and SSTR5 by their respective agonists, L-797,591 or L-817,818 (10 nM), increased K+ current amplitude, but SRIF evoked a further increase. The SSTR3 agonist L-797,778 (10 nM) did not affect the K+ currents or the response to SRIF. These results indicate that SSTR1, -2, -4, and -5 may all be involved in the enhancement of K+ currents by SRIF but that only the activation of SSTR2 or -4 results in the full activation of K+ current caused by SRIF.

KATPchannel conductance of descending vasa recta pericytes

Using nystatin-perforated patch-clamp and whole cell recording, we tested the hypothesis that K(ATP) channels contribute to resting conductance of rat descending vasa recta (DVR) pericytes and are modulated by vasoconstrictors. The K(ATP) blocker glybenclamide (Glb; 10 microM) depolarized pericytes and inhibited outward currents of cells held at -40 mV. K(ATP) openers pinacidil (Pnc; 10 microM) and P-1075 (1 microM) hyperpolarized pericytes and transiently augmented outward currents. All effects of Pnc and P-1075 were fully reversed by Glb. Inward currents of pericytes held at -60 mV in symmetrical 140 mM K(+) were markedly augmented by Pnc and fully reversed by Glb. Ramp depolarizations in symmetrical K(+), performed in Pnc and Pnc + Glb, yielded a Pnc-induced, Glb-sensitive K(ATP) difference current that lacked rectification and reversed at 0 mV. Immunostaining identified both K(IR)6.1, K(IR)6.2 inward rectifier subunits and sulfonurea receptor subtype 2B. ANG II (1 and 10 nM) and endothelin-1 (10 nM) but not vasopressin (100 nM) significantly lowered holding current at -40 mV and abolished Pnc-stimulated outward currents. We conclude that DVR pericytes express K(ATP) channels that make a significant contribution to basal K(+) conductance and are inhibited by ANG II and endothelin-1.

Ghrelin Reduces Voltage-Gated Potassium Currents in GH<SUB>3</SUB> Cells Via Cyclic GMP Pathways

Ghrelin is an endogenous growth hormone secretagogue (GHS) causing release of GH from pituitary somatotropes through the GHS receptor. Secretion of GH is linked directly to intracellular free Ca(2+) concentration ([Ca(2+)]i), which is determined by Ca(2+) influx and release from intracellular Ca(2+) storage sites. Ca(2+) influx is via voltage-gated Ca(2+) channels, which are activated by cell depolarization. Membrane potential is mainly determined by transmembrane K(+) channels. The present study investigates the in vitroeffect of ghrelin on membrane voltage-gated K(+) channels in the GH3 rat somatotrope cell line. Nystatin-perforated patch clamp recording was used to record K(+) currents under voltage-clamp conditions. In the presence of Co(2+) (1 mM, Ca(2+) channel blocker) and tetrodotoxin (1 microM, Na(+) channel blocker) in the bath solution, two types of voltage-gated K(+) currents were characterized on the basis of their biophysical kinetics and pharmacological properties. We observed that transient K(+) current (IA) represented a significant proportion of total K(+) currents in some cells, whereas delayed rectifier K(+) current (IK) existed in all cells. The application of ghrelin (10 nM) reversibly and significantly decreased the amplitude of both IA and IK currents to 48% and 64% of control, respectively. Application of apamin (1 microM, SK channel blocker) or charybdotoxin (1 microM, BK channel blocker) did not alter the K(+) current or the response to ghrelin. The ghrelin-induced reduction in K(+) currents was not affected by PKC and PKA inhibitors. KT5823, a specific PKG inhibitor, totally abolished the K+ current response to ghrelin. These results suggest that ghrelin-induced reduction of voltage-gated K(+) currents in GH3 cells is mediated through a PKG-dependent pathway. A decrease in voltage-gated K(+) currents may increase the frequency, duration, and amplitude of action potentials and contribute to GH secretion from somatotropes.

Excitatory effects of ATP on rat dorsomedial hypothalamic neurons

We investigated P2X purinoceptors in rat dorsomedial hypothalamic (DMH) neurons using nystatin-perforated patch-clamp recordings and fura-2 microfluorometry. Adenosine triphosphate (ATP) concentration-dependently evoked an inward current and increased cytosolic Ca 2+ ([Ca] i). The rise in [Ca] i was dependent on external Ca 2+ and Na +, was blocked by Ca 2+ channel antagonists and had pharmacological properties consistent with P2X2 receptors. These results suggest that P2X receptor-mediated depolarization activates voltage-gated Ca 2+ channels, resulting in an increase in [Ca] i.

Interaction of angiotensin II with the angiotensin type 2 receptor inhibits the cardiac transient outward potassium current.

The Ca2+ -independent transient outward K+ current (Ito) plays a crucial role in shaping the cardiac action potential. In the present study, we examined whether angiotensin II (AngII) regulated the Ito as well as the putative intracellular cascade responsible for the effects. Ito was recorded in rat ventricular myocytes using the nystatin-perforated patch-clamp configuration. AngII (0.1 microM) inhibited Ito (21.9+/-4.8% at +40 mV), but not the IK1, in a voltage- and time-independent manner. The inhibition decreased at concentrations higher than 1 microM resulting in a bell-shaped dose-response curve (IC50 = 3.1+/-1.5 microM). The blocking effects were abolished in the presence of the type 2 AngII receptor (AT2R) antagonist PD123319, but not in the presence of the selective type 1 AngII receptor (AT1R) antagonist candesartan. Moreover, the selective AT2R agonist CGP42112A completely reproduced the effects of AngII (20.5+/-2.4% of block at +40 mV), indicating that AngII-induced Ito block was mediated via stimulation of AT2R. Furthermore, selective stimulation of AT2R by CGP42112A significantly prolonged the rat atrial action potentials recorded using conventional microelectrode techniques. The AngII-induced inhibition of I(to) was not modified by either Npi-nitro-L-arginine-methyl ester (L-NAME) or eicosatetrayonic acid (ETYA), indicating that neither the nitric oxide (NO)-guanosine 3',5'-cyclic monophosphate (cGMP) system nor the arachidonic acid cascade was implicated in the effects of AngII on Ito. However, the AngII-induced Ito inhibition was completely abolished by the serine/threonine phosphatase type 2A (PP2A) inhibitors, okadaic acid and cantharidin, but not by the inactive analog of okadaic acid, 1-norokadaone. Intracellular application of PP2A decreased Kv4.2 currents recorded in transiently transfected Chinese hamster ovary cells (CHO). These results indicate that AngII activates PP2A through the stimulation of the AT2R, resulting in a decrease of the Ito amplitude.

A Rapid Increase in the Total Number of Cell Surface Functional GABAA Receptors Induced by Brain-derived Neurotrophic Factor in Rat Visual Cortex

The number of postsynaptic gamma-aminobutyric acid type A (GABAA) receptors is a fundamental determinant of the variability of inhibitory synaptic responses in the central nervous system. In rat visual cortex, [3H]SR-95531 binding assays revealed that brain-derived neurotrophic factor (BDNF), one of the neurotrophins, induced a rapid increase in the total number of cell surface GABAA receptors, through the activation of Trk B receptor tyrosine kinases. We also demonstrated that BDNF rapidly induced a sustained potentiation of GABAA receptor-mediated currents, using nystatin-perforated patch clamp recordings, in visual cortical layer 5 pyramidal neurons freshly isolated from P14 rats. The potentiation was caused by the activation of Trk B receptor tyrosine kinase and phospholipase C-gamma. In addition, intracellular Ca2+ was important for the potentiation of GABAA responses induced by BDNF. The selective increase in mean miniature inhibitory postsynaptic (mIPSC) current amplitude without effects on mIPSC time courses supports the idea that BDNF rapidly induces an increase in the total number of cell surface functional GABAA receptors in visual cortical pyramidal neurons. These results suggest that BDNF could alter the number of cell surface GABAA receptors in a region-specific manner.

Modulation of rabbit corneal epithelial cell proliferation by growth factor-regulated K(+) channel activity.

We characterized the dependence of the mitogenic response by rabbit corneal epithelial (RCE) cells to serum containing growth factors on K(+) channel activation. Using both cell-attached and nystatin-perforated patch-clamp configurations, a K(+) channel was identified whose current-voltage relationship is linear with a single-channel conductance of 31 pS. Its activity was barely detectable following 24 h serum starvation. Exposure of starved cells to either 10% FBS, 5 ng/ml epidermal growth factor (EGF) or 2 n M endothelin-1 (ET-1) continuously increased its activity within 30 min by 40%, 54% and 29%, respectively. EGF and ET-1 in combination had additive effects on such activity. Application of 100 micro M 4-aminopyridine (4-AP), a K(+) channel blocker, inhibited serum-stimulated K(+) channel activity by 85%. DNA synthesis was markedly stimulated by serum, whereas incubation with either 4-AP (200 micro M) or Ba(2+) (1 m M) suppressed this increase by 51% and 23%, respectively, whereas 5 m M tetra ethyl ammonium (TEA) had no effect. Taken together, growth factor-induced increases in proliferation are dependent on K(+) channel stimulation. As the increases in K(+) channel activity induced by ET-1 and EGF were additive, these mitogens may stimulate K(+) channel activity through different signaling pathways linked to their cognate receptors.

Chlorotoxin-sensitive Ca2+-activated Cl- channel in type R2 reactive astrocytes from adult rat brain.

Astrocytes express four types of Cl(-) or anion channels, but Ca(2+)-activated Cl(-) (Cl(Ca)) channels have not been described. We studied Cl(-) channels in a morphologically distinct subpopulation ( approximately 5% of cells) of small (10-12 micro m, 11.8 +/- 0.6 pF), phase-dark, GFAP-positive native reactive astrocytes (NRAs) freshly isolated from injured adult rat brains. Their resting potential, -57.1 +/- 4.0 mV, polarized to -72.7 +/- 4.5 mV with BAPTA-AM, an intracellular Ca(2+) chelator, and depolarized to -30.7 +/- 6.1 mV with thapsigargin, which mobilizes Ca(2+) from intracellular stores. With nystatin-perforated patch clamp, thapsigargin activated a current that reversed near the Cl(-) reversal potential, which was blocked by Cl(-) channel blockers, 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) and Zn(2+), by I(-) (10 mM), and by chlorotoxin (EC(50) = 47 nM). With conventional whole-cell clamp, NPPB- and Zn(2+)-sensitive currents became larger with increasing [Ca(2+)](i) (10, 150, 300 nM). Single-channel recordings of inside-out patches confirmed Ca(2+) sensitivity of the channel and showed open-state conductances of 40, 80, 130, and 180 pS, and outside-out patches confirmed sensitivity to chlorotoxin. In primary culture, small phase-dark NRAs developed into small GFAP-positive bipolar cells with chlorotoxin-sensitive Cl(Ca) channels. Imaging with biotinylated chlorotoxin confirmed the presence of label in GFAP-positive cells from regions of brain injury, but not from uninjured brain. Chlorotoxin-tagged cells isolated by flow cytometry and cultured up to two passages exhibit positive labeling for GFAP and vimentin, but not for prolyl 4-hydroxylase (fibroblast), A2B5 (O2A progenitor), or OX-42 (microglia). Expression of a novel chlorotoxin-sensitive Cl(Ca) channel in a morphologically distinct subpopulation of NRAs distinguishes these cells as a new subtype of reactive astrocyte.

The action of BDNF on GABA(A) currents changes from potentiating to suppressing during maturation of rat hippocampal CA1 pyramidal neurons.

During the development of the hippocampus, the action of GABA shifts from depolarizing to hyperpolarizing, and brain-derived neurotrophic factor (BDNF) has important roles in GABAergic transmission. We demonstrate that BDNF (20 ng ml-1) rapidly and reversibly potentiates postsynaptic GABAA receptor-mediated currents (by 80.5 +/- 14.3 %, n = 10) in hippocampal CA1 pyramidal neurons isolated from postnatal day (P)6 rats, using nystatin-perforated patch-clamp recordings. This potentiation is caused by an elevation of intracellular Ca2+ that occurs in response to the activation of Trk B receptor tyrosine kinase and phospholipase C-gamma. The modulation of the GABAA responses by BDNF in hippocampal CA1 pyramidal neurons isolated from P10 rats was more diverse (from potentiating to inhibitory), and at P14, BDNF induced a long-lasting inhibition. In addition, Ca2+/calmodulin-dependent protein kinase 2 plays important roles in the potentiating, but not in the inhibitory effect, of BDNF on the GABAA responses. These results suggest that changes in the intracellular signalling pathway could contribute to the developmental shift of the actions of BDNF on inhibitory systems.

Roles of protein kinase A and C in the opioid potentiation of the GABA A 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, γ-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 μ-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 μ-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.

Brain-derived neurotrophic factor induces long-lasting Ca2+-activated K+ currents in rat visual cortex neurons.

Brain-derived neurotrophic factor (BDNF) increases postsynaptic intracellular Ca2+ and modulates synaptic transmission in various types of neurons. Ca2+-activated K+ currents, opened mainly by intracellular Ca2+ elevation, contribute to hyperpolarization following action potentials and modulate synaptic transmission. We asked whether BDNF induces Ca2+-activated K+ currents by postsynaptic elevation of intracellular Ca2+ in acutely dissociated visual cortex neurons of rats. Currents were analysed using the nystatin-perforated patch clamp technique and imaging of intracellular Ca2+ mobilization with fura-2. At a holding potential of -50 mV, BDNF application (20 ng/mL) for 1-2 min induced an outward current (IBDNF-OUT; 80.0 +/- 29.0 pA) lasting for more than 90 min without attenuation in every neuron tested. K252a (200 nm), an inhibitor of Trk receptor tyrosine kinase, and U73122 (3 microm), a specific phospholipase C (PLC)-gamma inhibitor, suppressed IBDNF-OUT completely. IBDNF-OUT was both charybdotoxin- (600 nm) and apamin- (300 nm) sensitive, suggesting that this current was carried by Ca2+-activated K+ channels. BAPTA-AM (150 microm) gradually suppressed IBDNF-OUT. Fura-2 imaging revealed that a brief application of BDNF elicited a long-lasting elevation of intracellular Ca2+. These results show that BDNF induces long-lasting Ca2+-activated K+ currents by sustained intracellular Ca2+ elevation in rat visual cortex neurons. While BDNF, likely acting through the Trk B receptor, was necessary for the induction of long-lasting Ca2+-activated K+ currents via intracellular Ca2+ elevation, BDNF was not necessary for the maintenance of this current.

Modulation of cyclooxygenase-2 on glycine- and glutamate-induced ion currents in rat periaqueductal gray neurons

Cyclooxygenase (COX) is a key enzyme in the conversion of arachidonic acid to prostaglandins. Two isoforms of COX are known: COX-1 and COX-2. In the present study, the modulatory effect of COX-2 on glycine- and glutamate-induced ion currents in periaqueductal gray (PAG) neurons was investigated using the nystatin-perforated patch clamp method. Continuous application of lipopolysaccharides on PAG neurons resulted in increased glycine-induced ion current and decreased glutamate-induced ion current. In contrast, continuous application of celecoxib, selective COX-2 inhibitor, resulted in decreased glycine-induced ion current and increased glutamate-induced ion current. These results demonstrate that COX-2 modulates neuronal activity of PAG, and it can be suggested that COX-2 participates in the regulation of the descending pain control system in the level of PAG neurons.