Voltage-clamp Mode Research Articles

Orexin peptides enhance median preoptic nucleus neuronal excitability via postsynaptic membrane depolarization and enhancement of glutamatergic afferents

Subpopulations of neurons in the median preoptic nucleus (MnPO) located within the lamina terminalis contribute to thermoregulatory, cardiovascular and hydromineral homeostasis, and sleep-promotion. MnPO is innervated by lateral hypothalamic neurons that synthesize and secrete the arousal-promoting and excitatory orexin (hypocretin) neuropeptides. To evaluate the hypothesis that orexins modulate the excitability of MnPO neurons, we used patch-clamp recording techniques applied in rat brain slice preparations to assess the effects of exogenously applied orexin A and orexin B peptides on their intrinsic and synaptic properties. Whole cell recordings under current-clamp mode revealed that 11/15 tested MnPO neurons responded similarly to either orexin A or B (500–1000 nM) with a slowly rising, prolonged (10–15 min) and reversible membrane depolarization. Under voltage-clamp mode, orexin applications induced a tetrodotoxin-resistant inward current of −7.2±1.6 pA, indicating a direct (postsynaptic) activation, with a time course similar to the observed membrane depolarization. The orexin-induced responses in 4/7 neurons were associated with a significant decrease in membrane conductance and the net orexin-induced current that reversed at −99±5 mV, suggesting closure of potassium channels. Orexins did not attenuate the properties of excitatory ( n=4) or inhibitory ( n=7) postsynaptic currents evoked by subfornical organ stimulation. By contrast, orexins applications induce a significant increase in both frequency and amplitude of spontaneous glutamatergic postsynaptic currents (5/7 cells) but had no influence on spontaneous GABAergic currents (6/6 cells). Thus, in addition to a direct postsynaptic receptor-mediated excitation, orexins can also increase the excitability of MnPO neurons via increasing their excitatory inputs, presumably through an orexin receptor–mediated excitation of local glutamatergic neurons whose axons project to MnPO neurons.

B-type natriuretic peptide (BNP) attenuates the L-type calcium current and regulates ventricular myocyte function

A fundamental question in physiology is how hormones regulate the functioning of a cell or organ. It was therefore the aim of this study to investigate the effect(s) of BNP-32 on calcium handling by ventricular myocytes obtained from the rat left ventricle. We specifically tested the hypothesis that BNP-32 decreased the L-type calcium current ( I Ca,L). Perforated patch clamp technique was used to record I Ca,L and action potential (AP) in voltage and current clamp mode, respectively. Myocyte shortening was measured using a photodiode array edge-detection system and intracellular calcium transients were measured by fluorescence photometry. Western blotting was used to determine the relative change in the expression of proteins. At the concentrations tested, BNP-32 significantly decreased cell shortening in a dose-dependent manner; increased the phase II slope of the AP by 53.0%; increased the APD 50 by 16.9%; reduced the I Ca,L amplitude with a 22.9% decrease in the peak amplitude and reduced Ca 2+-dependent inactivation; increased the V 1/2 activation of the L-type calcium channel by 51.1% and decreased V 1/2 inactivation by 31.8%; and, intracellular calcium transient amplitude was significantly decreased by 32.0%, whereas the time to peak amplitude and T 1/2 were both significantly increased by 38.7% and 89.4% respectively. Sarcoplasmic reticulum Ca 2+-ATPase (SERCA2a) protein expression was reduced by BNP-32. These data suggest that BNP-32 regulates ventricular myocyte function by attenuating I Ca,L, altering the AP and reducing SERCA2a activity and/or expression. This study suggests a novel constitutive mechanism for the autocrine action of BNP on the L-type calcium channel in ventricular myocytes.

Role of sarcolemmal ATP‐sensitive K+ channels in the regulation of sinoatrial node automaticity: an evaluation using Kir6.2‐deficient mice

The role of cardiac sarcolemmal ATP-sensitive K+ (K(ATP)) channels in the regulation of sinoatrial node (SAN) automaticity is not well defined. Using mice with homozygous knockout (KO) of the Kir6.2 (a pore-forming subunit of cardiac K(ATP) channel) gene, we investigated the pathophysiological role of K(ATP) channels in SAN cells during hypoxia. Langendorff-perfused mouse hearts were exposed to hypoxic and glucose-free conditions (hypoxia). After 5 min of hypoxia, sinus cycle length (CL) was prolonged from 207 +/- 10 to 613 +/- 84 ms (P < 0.001) in wild-type (WT) hearts. In Kir6.2 KO hearts, CL was slightly prolonged from 198 +/- 17 to 265 +/- 32 ms. The CL of spontaneous action potentials of WT SAN cells, recorded in the current-clamp mode, was markedly prolonged from 410 +/- 56 to 605 +/- 108 ms (n = 6, P < 0.05) with a decrease of the slope of the diastolic depolarization (SDD) after the application of the K+ channel opener pinacidil (100 microm). Pinacidil induced a glibenclamide (1 microm)-sensitive outward current, which was recorded in the voltage-clamp mode, only in WT SAN cells. During metabolic inhibition by 2,4-dinitrophenol, CL was prolonged from 292 +/- 38 to 585 +/- 91 ms (P < 0.05) with a decrease of SDD in WT SAN cells but not in Kir6.2 KO SAN cells. Diastolic Ca2+ concentration, measured by fluo-3 fluorescence, was decreased in WT SAN cells but increased in Kir6.2 KO SAN cells after short-term metabolic inhibition. In conclusion, the present study using Kir6.2 KO mice indicates that, during hypoxia, activation of sarcolemmal K(ATP) channels in SAN cells inhibits SAN automaticity, which is important for the protection of SAN cells.

Coincidence detection of convergent perforant path and mossy fibre inputs by CA3 interneurons

We performed whole-cell recordings from CA3 s. radiatum (R) and s. lacunosum-moleculare (L-M) interneurons in hippocampal slices to examine the temporal aspects of summation of converging perforant path (PP) and mossy fibre (MF) inputs. PP EPSPs were evoked from the s. lacunosum-moleculare in area CA1. MF EPSPs were evoked from the medial extent of the suprapyramidal blade of the dentate gyrus. Summation was strongly supralinear when examining PP EPSP with MF EPSP in a heterosynaptic pair at the 10 ms ISI, and linear to sublinear at longer ISIs. This pattern of nonlinearities suggests that R and L-M interneurons act as coincidence detectors for input from PP and MF. Summation at all ISIs was linear in voltage clamp mode demonstrating that nonlinearities were generated by postsynaptic voltage-dependent conductances. Supralinearity was not detected when the first EPSP in the pair was replaced by a simulated EPSP injected into the soma, suggesting that the conductances underlying the EPSP boosting were located in distal dendrites. Supralinearity was selectively eliminated with either Ni2+ (30 microm), mibefradil (10 microm) or nimodipine (15 microm), but was unaffected by QX-314. This pharmacological profile indicates that supralinearity is due to recruitment of dendritic T-type Ca2+channels by the first subthreshold EPSP in the pair. Results with the hyperpolarization-activated (Ih) channel blocker ZD 7288 (50 microm) revealed that Ih restricted the time course of supralinearity for coincidently summed EPSPs, and promoted linear to sublinear summation for asynchronous EPSPs. We conclude that coincidence detection results from the counterbalanced activation of T-type Ca2+ channels and inactivation of Ih.

The effects of ouabain on resting membrane potential and hyperpolarization-activated current in neonatal rat nodose ganglion neurons

To determine whether the responses of resting membrane potential (RMP) and hyperpolarization-activated current (IH) are altered by the application of ouabain, one of the Na+-K+ pump inhibitors, in neonatal rat small-diameter (<30microm) nodose ganglion (NG) neurons, we examined the effects of 1microM ouabain on those responses using perforated patch-clamp techniques. In current-clamp mode, the RMP was 40.2+/-1.6mV (n=31). Twenty of 31 cells tested were depolarized by ouabain application, and these responses were associated with an increase in the cell input resistance. In the remaining 11 cells studied, 3 showed hyperpolarization in response to ouabain and 8 showed no effect on RMP. In voltage-clamp mode, 1muM ouabain application enhanced the IH in all of 10 neurons examined. These results suggest that ouabain application at 1microM is capable of setting both the RMP level and the neuronal excitability in small-diameter NG neurons.

A lack of potentiating effect of increasing temperature on the responses to chemical activators in vagal sensory neurons isolated from TRPV1‐null mice

A recent study demonstrated that the responses of rat pulmonary sensory neurons to TRPV1 activators were enhanced by increasing temperature (Ni and Lee, FASEB J., 2007), but the role of the TPRV1 channel in the potentiating effect could not be determined. In the present study, we used whole cell perforated patch‐clamp technique to study the effect of increasing temperature on the response of isolated nodose/jugular sensory neurons to non‐selective TRPV1 channel activators in TRPV1‐null (TRPV1−/−) and wild type (WT) mice. Our results showed that, in voltage‐clamp mode, the inward current evoked by 2‐aminoethoxydiphenyl borate (2‐APB), a common activator of TRPV1‐3 channels, was greatly potentiated by increasing temperature from 36 to 40.5°C in WT neurons (n = 9; P &lt; 0.01), but was not affected in TRPV1−/− neurons (n = 9; P = 0.54). Similarly, the inward current evoked by acid (pH 5.5), an activator of both TRPV1 channel and the acid sensing ion channel, was enhanced by increasing temperature (n = 7; P &lt; 0.05), and this effect was not observed in TRPV1−/ − neurons (n = 13; P = 0.11). These results demonstrated that disruption of the TRPV1 channel eliminates the potentiating effect of increasing temperature on the response of nodose/jugular neurons to non‐selective TRPV1 channel activators, suggested a possible interaction between these chemical activators and increasing temperature at the TRPV1 channel.

Suppression of carbachol-induced oscillatory Cl−secretion by forskolin in rat parotid and submandibular acinar cells

Sympathetic stimulation induces weak salivation compared with parasympathetic stimulation. To clarify this phenomenon in salivary glands, we investigated cAMP-induced modulation of Ca(2+)-activated Cl(-) secretion from rat parotid and submandibular acinar cells because fluid secretion from salivary glands depends on the Cl(-) secretion. Carbachol (Cch), a Ca(2+)-increasing agent, induced hyperpolarization of the cells with oscillatory depolarization in the current clamp mode of the gramicidin-perforated patch recording. In the voltage clamp mode at -80 mV, Cch induced a bumetanide-sensitive oscillatory inward current, which was larger in rat submandibular acinar cells than in parotid acinar cells. Forskolin and IBMX, cAMP-increasing agents, did not induce any marked current, but they evoked a small nonoscillatory inward current in the presence of Cch and suppressed the Cch-induced oscillatory inward current in all parotid acinar cells and half (56%) of submandibular acinar cells. In the current clamp mode, forskolin + IBMX evoked a small nonoscillatory depolarization in the presence of Cch and reduced the amplitude of Cch-induced oscillatory depolarization in both acinar cells. The oscillatory inward current estimated at the depolarized membrane potential was suppressed by forskolin + IBMX. These results indicate that cAMP suppresses Ca(2+)-activated oscillatory Cl(-) secretion of parotid and submandibular acinar cells at -80 mV and possibly at the membrane potential during Cch stimulation. The suppression may result in the weak salivation induced by sympathetic stimulation.

Lidocaine suppresses subthreshold oscillations by inhibiting persistent Na+ current in injured dorsal root ganglion neurons

The aim of this study was to determine the effect and mechanism of low concentration of lidocaine on subthreshold membrane potential oscillations (SMPO) and burst discharges in chronically compressed dorsal root ganglion (DRG) neurons. DRG neurons were isolated by enzymatic dissociation method. SMPO, burst discharges and single spike were elicited by whole cell patch-clamp technique in current clamp mode. Persistent Na(+) current (I(NaP)) and transient Na(+) current (I(NaT)) were elicited in voltage clamp mode. The results showed that SMPO was suppressed and burst discharges were eliminated by tetrodotoxin (TTX, 0.2 micromol/l) in current clamp mode, I(NaP) was blocked by 0.2 micromol/l TTX in voltage clamp mode. SMPO, burst discharges and I(NaP) were also suppressed by low concentration of lidocaine (10 micromol/l) respectively. However, single spike and I(NaT) could only be blocked by high concentration of lidocaine (5 mmol/l). From these results, it is suggested that I(NaP) mediates the generation of SMPO in injured DRG neurons. Low concentration of lidocaine (10 micromol/l) suppresses SMPO by selectively inhibiting I(NaP), but not I(NaT), in chronically compressed DRG neurons.

Involvement of Thromboxane A2in the Modulation of Pacemaker Activity of Interstitial Cells of Cajal of Mouse Intestine

Although many studies show that thromboxane A(2) (TXA(2)) has the action of gastrointestinal (GI) motility using GI muscle cells and tissue, there are no reports on the effects of TXA(2) on interstitial cells of Cajal (ICC) that function as pacemaker cells in GI tract. So, we studied the modulation of pacemaker activities by TXA(2) in ICC with whole cell patch-clamp technique. Externally applied TXA(2) (5microM) produced membrane depolarization in current-clamp mode and increased tonic inward pacemaker currents in voltage-clamp mode. The tonic inward currents by TXA(2) were inhibited by intracellular application of GDP-beta-S. The pretreatment of ICC with Ca(2+) free solution and thapsigargin, a Ca(2+)-ATPase inhibitor in endoplasmic reticulum, abolished the generation of pacemaker currents and suppressed the TXA(2)-induced tonic inward currents. However, chelerythrine or calphostin C, protein kinase C inhibitors, did not block the TXA(2)-induced effects on pacemaker currents. These results suggest that TXA(2) can regulate intestinal motility through the modulation of ICC pacemaker activities. This modulation of pacemaker activities by TXA(2) may occur by the activation of G protein and PKC independent pathway via extra and intracellular Ca(2+) modulation.

Adenosine A2A Receptors Are Essential for Long-Term Potentiation of NMDA-EPSCs at Hippocampal Mossy Fiber Synapses

The physiological conditions under which adenosine A2A receptors modulate synaptic transmission are presently unclear. We show that A2A receptors are localized postsynaptically at synapses between mossy fibers and CA3 pyramidal cells and are essential for a form of long-term potentiation (LTP) of NMDA-EPSCs induced by short bursts of mossy fiber stimulation. This LTP spares AMPA-EPSCs and is likely induced and expressed postsynaptically. It depends on a postsynaptic Ca2+ rise, on G protein activation, and on Src kinase. In addition to A2A receptors, LTP of NMDA-EPSCs requires the activation of NMDA and mGluR5 receptors as potential sources of Ca2+ increase. LTP of NMDA-EPSCs displays a lower threshold for induction as compared with the conventional presynaptic mossy fiber LTP; however, the two forms of LTP can combine with stronger induction protocols. Thus, postsynaptic A2A receptors may potentially affect information processing in CA3 neuronal networks and memory performance.

Mind Bomb-2 Is an E3 Ligase That Ubiquitinates the N-Methyl-d-aspartate Receptor NR2B Subunit in a Phosphorylation-dependent Manner

The N-methyl-D-aspartate receptor (NMDAR) plays a critical role in synaptic plasticity. Post-translational modifications of NMDARs, such as phosphorylation, alter both the activity and trafficking properties of NMDARs. Ubiquitination is increasingly being recognized as another post-translational modification that can alter synaptic protein composition and function. We identified Mind bomb-2 as an E3 ubiquitin ligase that interacts with and ubiquitinates the NR2B subunit of the NMDAR in mammalian cells. The protein-protein interaction and the ubiquitination of the NR2B subunit were found to be enhanced in a Fyn phosphorylation-dependent manner. Immunocytochemical studies reveal that Mind bomb-2 is localized to postsynaptic sites and colocalizes with the NMDAR in apical dendrites of hippocampal neurons. Furthermore, we show that NMDAR activity is down-regulated by Mind bomb-2. These results identify a specific E3 ubiquitin ligase as a novel interactant with the NR2B subunit and suggest a possible mechanism for the regulation of NMDAR function involving both phosphorylation and ubiquitination.

The partial α7 nicotine acetylcholine receptor agonist S 24795 enhances long-term potentiation at CA3-CA1 synapses in the adult mouse hippocampus

The effects of S 24795, a newly developed partial agonist at α7 nAChRs, were tested on synaptic transmission and plasticity using extracellular field excitatory postsynaptic potentials (fEPSPs) evoked in the CA1 region by Schaffer collateral stimulation in hippocampal slices obtained from adult mice. S 24795 reduced the amplitude of the fEPSPs in a concentration-dependent manner with an IC 50 of 127 μM and a Hill coefficient of 1.1. The reduction in amplitude of the fEPSPs started at S 24795 concentrations higher than 3 μM and reached 71% of controls at 300 μM. This effect was mediated by α7 nAChRs since it was blocked by nAChR antagonists and was not observed in α7 −/− mice. This effect was probably due to a reduction in glutamate release from presynaptic terminals since it was associated with a significant increase in the paired pulse ratio. In addition, S 24795 (100 μM) significantly reduced the frequency, but not the amplitude of spontaneous excitatory postsynaptic currents, recorded in the whole cell configuration of the patch clamp technique (in voltage clamp mode), further supporting a presynaptic site of action of S 24795. In addition, S 24795 at 3 μM, a concentration that did not affect basic synaptic transmission, potentiated LTP. This effect was mediated by α7 nAChRs since it was prevented by MLA (10 nM) and was absent in α7 −/− mice. Galantamine an allosteric modulator of nAChRs, at the concentrations of 0.3–3 μM, failed to potentiate LTP. In view of its powerful effect on LTP and on cognitive function, S 24795 can be considered a novel useful tool for the treatment of AD patients and other senile forms of dementia.

Excitatory effects of human immunodeficiency virus 1 Tat on cultured rat cerebral cortical neurons

Human immunodeficiency virus 1 (HIV-1) Tat protein is one of the neurotoxins involved in the pathogenesis of HIV-1-associated neuronal disorders. Combined electrophysiological and optical imaging experiments were undertaken to investigate whether HIV-1 Tat30-86, herein referred to as Tat30-86, acted directly or indirectly via the release of glutamate or both and to test its effect on the properties of spontaneous quantal events in cultured cortical neurons. Whole-cell patch recordings were made from cultured rat cortical neurons in either current- or voltage-clamp mode. Tat30-86 (50–1000 nM) induced in a population of cortical neurons a long-lasting depolarization, which was accompanied by a decrease of membrane resistance and persisted in a Krebs solution containing tetrodotoxin (TTX, 0.5 μM). Depolarizations were slightly reduced by pretreatment with glutamate receptor antagonists 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) (10 μM) and d-2-amino-5-phosphonovaleric acid (AP-5) (50 μM), and were markedly reduced in a Ca 2+-free Krebs solution; the differences were statistically significant. Tat30-86-induced inward currents had a reversal potential between −30 and 0 mV. While not causing a noticeable depolarization, lower concentrations of Tat30-86 (10 nM) increased membrane excitability, as indicated by increased numbers of neuronal discharge in response to a step depolarizing pulse. Tat30-86 (10 nM) increased the frequency of spontaneous miniature excitatory postsynaptic currents (mEPSCs), while not significantly affecting their amplitude. Tat30-86 (10 nM) moderately increased the frequency as well as the amplitude of spontaneous miniature inhibitory postsynaptic currents (mIPSCs). Ratiometric Ca 2+ imaging studies showed that Tat30-86 produced three types of Ca 2+ responses: 1) a fast and transitory increase, 2) Ca 2+ oscillations, and 3) a fast increase followed by a plateau; the glutamate receptor antagonists eliminated the late component of Ca 2+ response. The result suggests that Tat30-86 is an active fragment and that it excites cortical neurons directly and indirectly via releasing glutamate from adjacent neurons.

Different suppressive effect of lidocaine on persistent Na + current and transient Na + current in dorsal root ganglion neurons

Objective To investigate the different suppressive effect of lidocaine on persistent Na + current and transient Na + current in injured or uninjured dorsal root ganglion neurons. Methods Totally 23 SD rats were randomly divided into 2 groups; control group ( n = 10) and chronically compressed DRG (dorsal root ganglion) group (CCD group, n = 13). Rats were anesthetized and DRG was isolated. Single DRG neuron was isolated by enzymatic disassociation method. Persistent Na + current (I NaP) and transient Na + current (I NaT) were elicited in voltage clamp mode. Results The presence of I NaP was testified in most DRG neurons (38/46 neurons in CCD group and 31/39 neurons in control group, P>0.05). However, the current density of I NaP in CCD group (4.6 ± 0.6 pA/pF, n = 38 neurons) was greater than that in control group (2.5 ± 0.4 pA/pF, n = 31 neurons) ( P<0.05). The characteristics of I NaP was observed and found that I NaP could be blocked by 0.2 μmol/L tetrodotoxin easily. Furthermore, the does-effect relationship of Iidocaine on I NaP and I NaT were also examined. I NaP and I NaT were suppressed by different concentrations of lidocaine, the range for I NaP was 5-20 μmol/L and for I NaT was 0.05-2 mmol/L. Conclusion I NaP and I NaT were suppressed by different concentrations of lidocaine. I NaP was suppressed by very low concentration of lidocaine (5-20 μmol/L). However, I NaT could only be blocked by high concentration of lidocaine (0.05-2 mmol/L).

Activation of a G Protein–coupled Inwardly Rectifying K+Current and Suppression of I hContribute to Dexmedetomidine-induced Inhibition of Rat Hypothalamic Paraventricular Nucleus Neurons

Alpha2-adrenoceptor agonist has been reported to produce inhibition of arginine vasopressin release, diuresis, and sympatholytic effects. However, its mechanisms of central action remain incompletely understood. Hypothalamic paraventricular nucleus (PVN) neurons, which are in direct contact with noradrenergic synapses and are controlled by the hyperpolarization-activated currents, are called Ih (H current). The effect of dexmedetomidine, a highly selective and potent agonist, at alpha2 adrenoceptors on Ih is unknown. The purpose of this study was to examine the effects of dexmedetomidine on the PVN neuron, which is involved in the arginine vasopressin release and autonomic regulation. The authors investigated the effects of dexmedetomidine on the membrane properties in PVN magnocellular neurons and an Ih in PVN parvocellular neurons with a whole cell patch clamp technique using a rat brain slice preparation. Dexmedetomidine dose-dependently hyperpolarized PVN magnocellular neurons. In the voltage clamp mode, dexmedetomidine induced an outward current, with a reversal potential of -94 mV, and this was shown to depend on the external concentration of K. Pretreatment with Ba or peptide toxin tertiapin blocked hyperpolarization induced by dexmedetomidine. The effect of dexmedetomidine was blocked by an alpha2-adrenoceptor antagonist, yohimbine. Ih was suppressed dose dependently by dexmedetomidine in PVN parvocellular neurons. Pretreatment with Cs occluded the Ih suppression by dexmedetomidine. Yohimbine blocked the Ih suppression by dexmedetomidine. The Ih sensitive to dexmedetomidine was weakly modulated by intracellular cyclic adenosine monophosphate. Dexmedetomidine inhibited PVN magnocellular neurons by activation of the G protein-coupled inwardly rectifying K current and inhibited PVN parvocellular neurons by suppression of Ih.

Streptomycin and gentamicin have no immediate effect on outer hair cell electromotility

The cochlear outer hair cell (OHC), which plays a crucial role in mammalian hearing through its unique voltage-dependent motility, has been established as a primary target of the ototoxicity of aminoglycoside antibiotics. These polycationic drugs are also known to block a wide variety of ion channels, purinergic ionotropic channels, and nicotinic ACh receptors in hair cells in vitro. The OHC motor protein, prestin, is a voltage-sensitive transmembrane protein containing several negatively charged residues on both intra- and extracellular surface. The acidic sites may be susceptible to polycationic-charged aminoglycoside binding, which may result in disruption of motility. We attempted to examine whether aminoglycosides such as streptomycin and gentamicin could affect OHC motility and its electrical signature, the nonlinear capacitance (NLC) in adult gerbil OHCs. Somatic motility and NLC were measured under the whole-cell voltage-clamp mode. Streptomycin and gentamicin were applied extracellularly or intracellularly. Results show that streptomycin and gentamicin did not change either the magnitude of motility or the NLC. Theses results suggest that, although streptomycin and gentamicin can block mechanotransduction channels as well as ACh receptors in hair cells, they have no direct affect on OHC somatic motility.

Selective Vulnerability to Ischemia in the Rat Spinal Cord

Whole-cell patch-clamp recordings were performed from ventral horn (VH) and dorsal horn (DH) neurons obtained from the rat spinal cord slices. This study investigated which is more vulnerable to ischemia, spinal VH neurons or DH neurons. Spinal cord ischemia or injury sometimes causes a greater loss of motor function than of sensory function in patients. However, it is difficult to evaluate whether spinal motor neurons are more vulnerable than sensory neurons because of the anatomic complexity and a variety of physiologic factors in the spinal cord. Whole-cell patch-clamp recordings were performed from VH and DH neurons obtained from the spinal cord slices. Ischemia was simulated by superfusing an oxygen- and glucose-deprived medium (ischemia simulating medium [ISM]). Perfusion with ISM generated an agonal depolarization in all VH and DH neurons recorded in current-clamp mode. Following ISM superfusion, an agonal inward current was produced at a holding potential of -70 mV in all VH and DH neurons tested in voltage-clamp mode. The agonal inward current consisted of a slow and subsequent rapid inward current. The average latency of the rapid inward currents after ISM exposures in VH neurons was significantly shorter than that in DH neurons. The average amplitude of the agonal inward currents in VH neurons was significantly bigger than that of DH neurons. Moreover, the recovery ratio by the reintroduction of oxygen and glucose in VH neurons was smaller than that in DH neurons. These results suggest that VH neurons are more vulnerable to ischemia than DH neurons. This finding may help in achieving a better understanding of the difference between motor and sensory disturbance in spinal cord ischemia or injury patients.

NAADP Controls Cross-talk between Distinct Ca2+ Stores in the Heart

In cardiac muscle the sarcoplasmic reticulum (SR) plays a key role in the control of contraction, releasing Ca(2+) in response to Ca(2+) influx across the sarcolemma via voltage-gated Ca(2+) channels. Here we report evidence for an additional distinct Ca(2+) store and for actions of nicotinic acid adenine dinucleotide phosphate (NAADP) to mobilize Ca(2+) from this store, leading in turn to enhanced Ca(2+) loading of the SR. Photoreleased NAADP increased Ca(2+) transients accompanying stimulated action potentials in ventricular myocytes. The effects were prevented by bafilomycin A (an H(+)-ATPase inhibitor acting on acidic Ca(2+) stores), by desensitizing concentrations of NAADP, and by ryanodine and thapsigargin to suppress SR function. Bafilomycin A also suppressed staining of acidic stores with Lysotracker Red without affecting SR integrity. Cytosolic application of NAADP by means of its membrane permeant acetoxymethyl ester increased myocyte contraction and the frequency and amplitude of Ca(2+) sparks, and these effects were inhibited by bafilomycin A. Effects of NAADP were associated with an increase in SR Ca(2+) load and appeared to be regulated by beta-adrenoreceptor stimulation. The observations are consistent with a novel role for NAADP in cardiac muscle mediated by Ca(2+) release from bafilomycin-sensitive acidic stores, which in turn enhances SR Ca(2+) release by increasing SR Ca(2+) load.

Prostaglandin E2 depresses solitary tract-mediated synaptic transmission in the nucleus tractus solitarius

Prostaglandin E2 (PGE2) is a prototypical inflammatory mediator that excites and sensitizes cell bodies [Kwong K, Lee LY (2002) PGE2 sensitizes cultured pulmonary vagal sensory neurons to chemical and electrical stimuli. J Appl Physiol 93:1419–1428; Kwong K, Lee LY (2005) Prostaglandin E2 potentiates a tetrodotoxin (TTX)-resistant sodium current in rat capsaicin-sensitive vagal pulmonary sensory neurons. J Physiol 56:437–450] and peripheral nerve terminals [Ho CY, Gu Q, Hong JL, Lee LY (2000) Prostaglandin E (2) enhances chemical and mechanical sensitivities of pulmonary C fibers in the rat. Am J Respir Crit Care Med 162:528–533] of primary vagal sensory neurons. Nearly all central nerve terminals of vagal afferents are in the nucleus tractus solitarius (NTS), where they operate with a high probability of release [Doyle MW, Andresen MC (2001) Reliability of monosynaptic sensory transmission in brain stem neurons in vitro. J Neurophysiol 85:2213–2223]. We studied the effect of PGE2 on synaptic transmission between tractus solitarius afferent nerve terminals and the second-order NTS neurons in brain stem slices of Sprague–Dawley rats. Whole-cell patch recording in voltage clamp mode was used to study evoked excitatory postsynaptic glutamatergic currents (evEPSCs) from NTS neurons elicited by electrical stimulation of the solitary tract (ST). In 34 neurons, bath-applied PGE2 (200 nM) decreased the evEPSC amplitude by 49±5%. In 22 neurons, however, PGE2 had no effect. We also tested 15 NTS neurons for capsaicin sensitivity. Seven neurons generated evEPSCs that were equally unaffected by PGE2 and capsaicin. Conversely, evEPSCs of the other eight neurons, which were PGE2-responsive, were abolished by 200 nM capsaicin. Furthermore, the PGE2-induced depression of evEPSCs was associated with an increase in the paired pulse ratio and a decrease in both the frequency and amplitude of the spontaneous excitatory postsynaptic currents (sEPSCs) and TTX-independent spontaneous miniature excitatory postsynaptic currents (mEPSCs). These results suggest that PGE2 acts both presynaptically on nerve terminals and postsynaptically on NTS neurons to reduce glutamatergic responses.

The functional difference between transient and sustained K + currents on the action potentials in tetrodotoxin-resistant adult rat trigeminal ganglion neurons

To determine whether there is a difference between a transient K + current ( I A) and a sustained K + current ( I K) regarding the neuronal function in small-diameter adult rat trigeminal ganglion (TG) neurons, which were insensitive to tetrodotoxin (TTX, 1 μM), we performed two different types of experiments. Primary cultures of dissociated TG neurons were prepared, and electrophysiological recordings were performed with the whole-cell configuration using the patch-clamp technique. In the voltage-clamp mode, two distinct K + current components, ( I A) and ( I K), were identified, and two different components (59.5% and 96.3%) of I K to the total K + current were observed at a + 50 mV step-pulse. The IC 50 value for 4-aminoprydine (4-AP, 0.05–50 mM), which inhibited the I A by 50%, was 0.7 mM. That for tetraethylammonium (TEA, 0.02–20 mM) to inhibit 50% of I K was 1.5 mM. In the current-clamp mode, we used 0.5 mM 4-AP and 2 mM TEA at each concentration nearly equal to the IC 50 value. Irrespective of the absence or presence of TEA (2 mM), 0.5 mM 4-AP application increased the number of action potentials due to the decreased duration of the depolarization phase (DDP). TEA in the presence and absence of 4-AP prolonged the duration of action potentials as well as the duration of repolarization phase (DRP). These results suggested that I A and I K had independent effects regulating the intrinsic firing properties of the action potential number and timing, respectively, in adult rat TTX-R TG neurons.