Voltage-clamp Studies Research Articles

Cadmium ions modulate GABA induced currents in molluscan neurons.

The effect of Cd2+, as one of the most widespread toxic environmental pollutants, was studied on gamma-aminobutyric acid (GABA) evoked responses of identified neurons in the central nervous system of the pond snail, LYmnaea stagnalis L. (Gastropoda). In the experiments, the modulation of the action of GABA both on neuronal activity (current clamp recording) and on the a GABA activated membrane Cl- current (voltage clamp studies) has been shown. It was found that: 1. GABA could evoked three different various types of response in GABA sensitive neurons: i) hyperpolarization with strong inhibition of ongoing spike activity, ii) short depolarization with an increase of spike the activity, iii) biphasic respone with a short excitation followed by a more prolonged long inhibition. 2. In low-Cl- solution the inhibitory action of GABA was reduced or eliminated, but the excitatory one was not or only moderately affected. 3. CdCl2 inhibited the GABA evoked hyperpolarization, but left intact or only slightly reduced the excitation evoked by GABA. 4. The inward Cl- current evoked by GABA at a -75 mV holding potential was slightly augmented in the presence of I micromol/l Cd2+, but was reduced or blocked at higher cadmium concentrations. The effect of Cd2+ was concentration and time dependent. 5. Parallel with reducing the GABA evoked current, cadmium increased both the time to peak and the half inactivation time of the current. 6. CdCl2 alone, in 50 micromol/l concentration, induced a 1-2 nA inward current. The blocking effect of cadmium on GABA activated inhibitory processes can be an important component of the neuro-toxic effects of this heavy metal ion.

Simulation of different firing patterns in paired spider mechanoreceptor neurons: the role of Na(+) channel inactivation.

The spider VS-3 slit-sense organ contains two types of primary mechanoreceptor neurons that are morphologically similar but have different electrical behavior. Type A neurons fire only one or two action potentials in response to a mechanical or electrical step of any amplitude above the threshold, whereas type B neurons fire prolonged bursts of action potentials in response to similar stimuli. Voltage-clamp studies have shown that two voltage-activated ion currents, a noninactivating potassium current and an inactivating sodium current, dominate the firing behavior. We simulated the electrical behavior of the two neuron types, using a simplified form of Hodgkin-Huxley model based on published voltage-clamp and current-clamp recordings. Changing only two parameters of sodium inactivation, the slope of the h(infinity) curve and the time constant of recovery from inactivation, allowed a complete switch between the two firing patterns. Our simulations support previous evidence that sodium inactivation controls the firing properties of these neurons and indicate that two parameter changes are needed to achieve complete transformation between the two neuron types.

Electrophysiological analysis of cloned cyclic nucleotide-gated ion channels.

Electrophysiological studies were conducted on the cloned plant cyclic nucleotide-gated ion channels AtCNGC2 and AtCNGC1 from Arabidopsis, and NtCBP4 from tobacco (Nicotiana tobacum). The nucleotide coding sequences for these proteins were expressed in Xenopus laevis oocytes or HEK 293 cells. Channel characteristics were evaluated using voltage clamp analysis of currents in the presence of cAMP. AtCNGC2 was demonstrated to conduct K(+) and other monovalent cations, but exclude Na(+); this conductivity profile is unique for any ion channel not possessing the amino acid sequence found in the selectivity filter of K(+)-selective ion channels. Application of cAMP evoked currents in membrane patches of oocytes injected with AtCNGC2 cRNA. Direct activation of the channel by cyclic nucleotide, demonstrated by application of cyclic nucleotide to patches of membranes expressing such channels, is a hallmark characteristic of this ion channel family. Voltage clamp studies (two-electrode configuration) demonstrated that AtCNGC1 and NtCBP4 are also cyclic nucleotide-gated channels. Addition of a lipophilic analog of cAMP to the perfusion bath of oocytes injected with NtCBP4 and AtCNGC1 cRNAs induced inward rectified, noninactivating K(+) currents.

Electrophysiological Analysis of Cloned Cyclic Nucleotide-Gated Ion Channels

Electrophysiological studies were conducted on the cloned plant cyclic nucleotide-gated ion channels AtCNGC2 and AtCNGC1 from Arabidopsis, and NtCBP4 from tobacco (Nicotiana tobacum). The nucleotide coding sequences for these proteins were expressed in Xenopus laevis oocytes or HEK 293 cells. Channel characteristics were evaluated using voltage clamp analysis of currents in the presence of cAMP. AtCNGC2 was demonstrated to conduct K+ and other monovalent cations, but exclude Na+; this conductivity profile is unique for any ion channel not possessing the amino acid sequence found in the selectivity filter of K+-selective ion channels. Application of cAMP evoked currents in membrane patches of oocytes injected with AtCNGC2 cRNA. Direct activation of the channel by cyclic nucleotide, demonstrated by application of cyclic nucleotide to patches of membranes expressing such channels, is a hallmark characteristic of this ion channel family. Voltage clamp studies (two-electrode configuration) demonstrated that AtCNGC1 and NtCBP4 are also cyclic nucleotide-gated channels. Addition of a lipophilic analog of cAMP to the perfusion bath of oocytes injected with NtCBP4 and AtCNGC1 cRNAs induced inward rectified, noninactivating K+currents.

Syntheses, calcium channel agonist-antagonist modulation activities, nitric oxide release, and voltage-clamp studies of 2-nitrooxyethyl 1,4-dihydro- 2,6-dimethyl-3-nitro-4-(2-trifluoromethylphenyl)pyridine-5-carboxylate enantiomers.

The novel (-)-(S)-2 and (+)-(R)-3 enantiomers of 2-nitrooxyethyl 1,4-dihydro-2,6-dimethyl-3-nitro-4-(2-trifluoromethylphenyl)pyridine-5-carboxylate were synthesized for evaluation as calcium channel modulators. Determination of their in vitro calcium-channel-modulating activities using guinea pig left atria (GPLA) and ileum longitudinal smooth muscle (GPILSM) showed that the (-)-(S)-2 enantiomer acted as a dual cardioselective calcium channel agonist (GPLA)/smooth muscle selective calcium channel antagonist (GPILSM). In contrast, the (+)-(R)-3 enantiomer exhibited calcium channel antagonist activity on both GPLA and GPILSM. The 2-nitrooxyethyl racemate is a nitric oxide (.NO) donor that released 2.7% .NO, relative to the reference drug glyceryl trinitrate (5.3% .NO release/ONO(2) moiety), in the presence of N-acetylcysteamine. Whole-cell voltage-clamp studies using isolated guinea pig ventricular myocytes indicated that both enantiomers inhibit calcium current but that the (-)-(S)-2 enantiomer is a weaker antagonist than the (+)-(R)-3 enantiomer. These results indicate that replacement of the methyl ester substituent of (-)-(S)-methyl 1,4-dihydro-2,6-dimethyl-3-nitro-4-(2-trifluoromethylphenyl)pyridine-5-carboxylate [(-)-(S)-1] by the 2-nitrooxyethyl ester .NO donor substituent present in (-)-(S)-2 provides a useful drug design concept to abolish the contraindicated calcium channel agonist effect of (-)-(S)-1 on vascular smooth muscle. The novel (-)-(S)-2 enantiomer is a useful lead compound for drug discovery targeted toward the treatment of congestive heart failure, and it provides a useful probe to study the structure-function relationships of calcium channels.

Expression and regulation of connexins in cultured ventricular myocytes isolated from adult rat hearts.

Gap junctions were assayed during re-differentiation of adult rat cardiomyocytes in long-term culture to gain insight into the processes of remodeling. Double immunostaining allowed the localization of connexins Cx40, Cx43, and Cx45 between myocytes and demonstrated co-expression and co-localization in individual cells and gap junction plaques, respectively. Immunoblots showed differential time-dependent changes in connexin expression and phosphorylation. The total amount of connexins and the ratio of phosphorylated/non-phosphorylated isoforms gradually increased during the re-establishment of intercellular communication. Dual voltage-clamp studies showed the involvement of several types of gap junction channels. Multichannel currents yielded diverse spectra of g(j,inst)=f( V(j)) and g(j,ss)=f( V(j)) relationships ( g(j,inst): instantaneous gap junction conductance; g(j,ss): conductance at steady state; V(j): transjunctional voltage), indicative of homotypic and heterotypic channels. Single-channel currents revealed two prominent conductances reflecting gamma(j,main) and gamma(j,residual). The histograms of gamma(j,main) showed four discrete peaks (41-44, 59-61, 70-76, and 100-107 pS) attributable to a combination of Cx45-Cx45, Cx40-Cx45 and Cx43-Cx45 channels (1st peak), Cx43-Cx43 and Cx40-Cx43 channels (2nd peak), Cx43-Cx43 channels (3rd peak) and Cx40-Cx40 and Cx40-Cx43 channels (4th peak). However, the presence of heteromeric channels cannot be excluded. The data are consistent with an up-regulation of Cx45 and Cx43 during re-differentiation.

A metabolic mechanism for cardiac K+ channel remodelling.

1. Electrical remodelling of the ventricle is a common pathogenic feature of cardiovascular disease states that lead to heart failure. Experimental data suggest this change in electrophysiological phenotype is largely due to downregulation of K(+) channels involved in repolarization of the action potential. 2. Voltage-clamp studies of the transient outward current (I(to)) in diabetic cardiomyopathy support a metabolic mechanism for K(+) channel downregulation. In particular, I(to) density is significantly increased in diabetic rat isolated ventricular myocytes treated in vitro with insulin or agents that activate pyruvate dehydrogenase. Recent data suggest this mechanism is not limited to diabetic conditions, because metabolic stimuli that upregulate I(to) in diabetic rat myocytes act similarly in non- diabetic models of heart failure. 3. Depressed I(to) channel activity is also reversed by experimental conditions that increase myocyte levels of reduced glutathione, indicating that oxidative stress is involved in electrical remodelling. Moreover, upregulation of I(to) density by activators of glucose utilization is blocked by inhibitors of glutathione metabolism, supporting the premise that there is a functional link between glucose utilization and the glutathione system. 4. Electrophysiological studies of diabetic and non-diabetic disease conditions affecting the heart suggest I(to) channels are regulated by a redox-sensitive mechanism, where glucose utilization plays an essential role in maintaining a normally reduced state of the myocyte. This hypothesis has implications for clinical approaches aimed at reversing pathogenic electrical remodelling in a variety of cardiovascular disease states.

Up-regulation of K(+) channels in diabetic rat ventricular myocytes by insulin and glutathione.

The cardiac pathogenesis of diabetes mellitus involves oxidative stress that elicits profound changes in myocardial glutathione, an endogenous regulator of cell function. This study examined the role of glutathione in regulating K(+) channel activity in isolated ventricular myocytes from diabetic rats and its relationship to insulin signaling. Colorimetric analysis of extracts of ventricular tissue from Sprague-Dawley rats showed that the basal level of reduced glutathione (GSH) was significantly less in rats with experimental diabetes compared with sham controls, consistent with oxidative stress conditions. This change in GSH status paralleled a significant decrease in the activity of gamma-glutamylcysteine synthetase, a major pathway involved in GSH homeostasis. Voltage-clamp studies confirmed that, compared with control myocytes, K(+) channels carrying the transient outward current (I(to)) are down-regulated in the diabetic state and that this electrophysiological change is reversed by in vitro treatment with insulin for 2-3 h. Incubation of diabetic rat myocytes with GSH also normalized I(to) density compared with untreated myocytes, but with a longer time course than insulin. To determine if up-regulation of I(to) by insulin was mediated by alterations in myocyte GSH, insulin-responsiveness of diabetic rat myocytes was tested in the presence of 1,3-bis-chloroethyl-nitrosourea, an inhibitor of glutathione reductase, or buthionine sulfoximine, a blocker of gamma-glutamylcysteine synthetase. Neither blocker alone altered I(to) density in diabetic rat myocytes when compared with untreated cells, but each blocked the effect of insulin to up-regulate I(to). These data suggest that oxidative stress-induced alteration in GSH redox state plays an important role in regulating I(to) channel function and that GSH homeostasis in ventricular myocytes is functionally coupled to insulin signaling.

The Voltage-Dependent Proton Pumping in Bacteriorhodopsin Is Characterized by Optoelectric Behavior

The light-driven proton pump bacteriorhodopsin (bR) was functionally expressed in Xenopus laevis oocytes and in HEK-293 cells. The latter expression system allowed high time resolution of light-induced current signals. A detailed voltage clamp and patch clamp study was performed to investigate the ΔpH versus Δ ψ dependence of the pump current. The following results were obtained. The current voltage behavior of bR is linear in the measurable range between −160 mV and +60 mV. The pH dependence is less than expected from thermodynamic principles, i.e., one ΔpH unit produces a shift of the apparent reversal potential of 34 mV (and not 58 mV). The M 2–BR decay shows a significant voltage dependence with time constants changing from 20 ms at +60 mV to 80 ms at −160 mV. The linear I-V curve can be reconstructed by this behavior. However, the slope of the decay rate shows a weaker voltage dependence than the stationary photocurrent, indicating that an additional process must be involved in the voltage dependence of the pump. A slowly decaying M intermediate (decay time > 100 ms) could already be detected at zero voltage by electrical and spectroscopic means. In effect, bR shows optoelectric behavior. The long-lived M can be transferred into the active photocycle by depolarizing voltage pulses. This is experimentally demonstrated by a distinct charge displacement. From the results we conclude that the transport cycle of bR branches via a long-lived M 1* in a voltage-dependent manner into a nontransporting cycle, where the proton release and uptake occur on the extracellular side.

Modeling Temporal Behavior of Postnatal Cat Retinal Ganglion Cells

During development, mammalian retinal ganglion cells (RGCs) go through marked ontogenetic changes with respect to their excitable membrane properties. Voltage-clamp studies conducted in our laboratory have shown that the amplitude, voltage-dependence and kinetics of activation and inactivation (where present) of Na+, K+and Ca2+conductances all exhibit developmental changes during a time when the firing patterns of mammalian ganglion cells shift from being transient to being predominantly sustained in nature. In order to better understand the contribution of each conductance to the generation of spikes and spiking patterns, we have developed a model based on our experimental data. For simplicity, we have initially used experimental data obtained from postnatal ganglion cells. At this age the ontogenetic changes observed in the characteristics of the various ionic currents are complete. Utilizing the methods adopted by Hodgkin and Huxley for the giant squid axon, we have determined rate equations for the activation and inactivation properties of theIA , IK dr, INa, ICa L,ICa N , and Ileakcurrents in postnatal cat RGCs. Combining these with a simplified model of the calcium-activated potassium current (IKCa), we have solved and analysed the resulting differential equations. While spikes and spiking patterns resembling experimental data could be obtained from a model in which [Ca2+i] was averaged across the whole cell, more accurate simulations were obtained when the diffusion of intracellular Ca2+was modeled spatially. The resulting spatial calcium gradients were more effective in gating IKCa, and our simulations more accurately matched the recorded amplitude and shape of individual spikes as well as the frequency of maintained discharges observed in mammalian postnatal RGCs.

D1/D5Dopamine Receptor Activation Differentially Modulates Rapidly Inactivating and Persistent Sodium Currents in Prefrontal Cortex Pyramidal Neurons

Dopamine (DA) is a well established modulator of prefrontal cortex (PFC) function, yet the cellular mechanisms by which DA exerts its effects in this region are controversial. A major point of contention is the consequence of D(1) DA receptor activation. Several studies have argued that D(1) receptors enhance the excitability of PFC pyramidal neurons by augmenting voltage-dependent Na(+) currents, particularly persistent Na(+) currents. However, this conjecture is based on indirect evidence. To provide a direct test of this hypothesis, we combined voltage-clamp studies of acutely isolated layer V-VI prefrontal pyramidal neurons with single-cell RT-PCR profiling. Contrary to prediction, the activation of D(1) or D(5) DA receptors consistently suppressed rapidly inactivating Na(+) currents in identified corticostriatal pyramidal neurons. This modulation was attenuated by a D(1)/D(5) receptor antagonist, mimicked by a cAMP analog, and blocked by a protein kinase A (PKA) inhibitor. In the same cells the persistent component of the Na(+) current was unaffected by D(1)/D(5) receptor activation-suggesting that rapidly inactivating and persistent Na(+) currents arise in part from different channels. Single-cell RT-PCR profiling showed that pyramidal neurons coexpressed three alpha-subunit mRNAs (Nav1.1, 1.2, and 1.6) that code for the Na(+) channel pore. In neurons from Nav1.6 null mice the persistent Na(+) currents were significantly smaller than in wild-type neurons. Moreover, the residual persistent currents in these mutant neurons-which are attributable to Nav1.1/1.2 channels-were reduced significantly by PKA activation. These results argue that D(1)/D(5) DA receptor activation reduces the rapidly inactivating component of Na(+) current in PFC pyramidal neurons arising from Nav1.1/1.2 Na(+) channels but does not modulate effectively the persistent component of the Na(+) current that is attributable to Nav1.6 Na(+) channels.

NH(2)-terminal modification of a channel-forming peptide increases capacity for epithelial anion secretion.

A synthetic, channel-forming peptide, derived from the alpha-subunit of the glycine receptor (M2GlyR), has been synthesized and modified by adding four lysine residues to the NH(2) terminus (N-K(4)-M2GlyR). In Ussing chamber experiments, apical N-K(4)-M2GlyR (250 microM) increased transepithelial short-circuit current (I(sc)) by 7.7 +/- 1.7 and 10.6 +/- 0.9 microA/cm(2) in Madin-Darby canine kidney and T84 cell monolayers, respectively; these values are significantly greater than those previously reported for the same peptide modified by adding the lysines at the COOH terminus (Wallace DP, Tomich JM, Iwamoto T, Henderson K, Grantham JJ, and Sullivan LP. Am J Physiol Cell Physiol 272: C1672-C1679, 1997). N-K(4)-M2GlyR caused a concentration-dependent increase in I(sc) (k([1/2]) = 190 microM) that was potentiated two- to threefold by 1-ethyl-2-benzimidazolinone. N-K(4)-M2GlyR-mediated increases in I(sc) were insensitive to changes in apical cation species. Pharmacological inhibitors of endogenous Cl(-) conductances [glibenclamide, diphenylamine-2-dicarboxylic acid, 5-nitro-2-(3-phenylpropylamino)benzoic acid, 4,4'-dinitrostilben-2,2'-disulfonic acid, indanyloxyacetic acid, and niflumic acid] had little effect on N-K(4)-M2GlyR-mediated I(sc). Whole cell membrane patch voltage-clamp studies revealed an N-K(4)-M2GlyR-induced anion conductance that exhibited modest outward rectification and modest time- and voltage-dependent activation. Planar lipid bilayer studies yielded results indicating that N-K(4)-M2GlyR forms a 50-pS anion conductance with a k([1/2]) for Cl(-) of 290 meq. These results indicate that N-K(4)-M2GlyR forms an anion-selective channel in epithelial monolayers and shows therapeutic potential for the treatment of hyposecretory disorders such as cystic fibrosis.

Mu opiates inhibit long-term potentiation induction in the spinal cord slice.

Long-term potentiation (LTP) involves a prolonged increase in neuronal excitability following repeated afferent input. This phenomenon has been extensively studied in the hippocampus as a model of learning and memory. Similar long-term increases in neuronal responses have been reported in the dorsal horn of the spinal cord following intense primary afferent stimulation. In these studies, we utilized the spinal cord slice preparation to examine effects of the potently antinociceptive mu opioids in modulating primary afferent/dorsal horn neurotransmission as well as LTP of such transmission. Transverse slices were made from the lumbar spinal cord of 10- to 17-day-old rats, placed in a recording chamber, and perfused with artificial cerebrospinal fluid also containing bicuculline (10 microM) and strychnine (1 microM). Primary afferent activation was achieved in the spinal slice by electrical stimulation of the dorsal root (DR) or the tract of Lissauer (LT) which is known to contain a high percentage of small diameter fibers likely to transmit nociception. Consistent with this anatomy, response latencies of LT-evoked field potentials in the dorsal horn were considerably slower than the response latencies of DR-evoked potentials. Only LT-evoked field potentials were found to be reliably inhibited by the mu opioid receptor agonist [D-Ala(2), N-Me-Phe(4), Gly(5)] enkephalin-ol (DAMGO, 1 microM), although evoked potentials from both DR and LT were blocked by the AMPA/kainate glutamate receptor antagonist 6-cyano-7-nitroquinoxalene-2,3-dione. Moreover repeated stimulation of LT produced LTP of LT- but not DR-evoked potentials. In contrast, repeated stimulation of DR showed no reliable LTP. LTP of LT-evoked potentials depended on N-methyl-D-aspartate (NMDA) receptor activity, in that it was attenuated by the NMDA antagonist APV. Moreover, such LTP was inhibited by DAMGO interfering with LTP induction mechanisms. Finally, in whole cell voltage-clamp studies of Lamina I neurons, DAMGO inhibited excitatory postsynaptic current (EPSC) response amplitudes from LT stimulation-evoked excitatory amino acid release but not from glutamate puffed onto the cell and increased paired-pulse facilitation of EPSCs evoked by LT stimulation. These studies suggest that mu opioids exert their inhibitory effects presynaptically, likely through the inhibition of glutamate release from primary afferent terminals, and thereby inhibit the induction of LTP in the spinal dorsal horn.

Electrophysiological characterization of specific interactions between bacterial sensory rhodopsins and their transducers

The halobacterial phototaxis receptors sensory rhodopsin I and II (SRI, SRII) enable the bacteria to seek optimal light conditions for ion pumping by bacteriorhodopsin and/or halorhodopsin. The incoming signal is transferred across the plasma membrane by means of receptor-specific transducer proteins that bind tightly to their corresponding photoreceptors. To investigate the receptor/transducer interaction, advantage is taken of the observation that both SRI and SRII can function as proton pumps. SRI from Halobacterium salinarum , which triggers the positive phototaxis, the photophobic receptor SRII from Natronobacterium pharaonis (pSRII), as well as the mutant pSRII-F86D were expressed in Xenopus oocytes. Voltage-clamp studies confirm that SRI and pSRII function as light-driven, outwardly directed proton pumps with a much stronger voltage dependence than the ion pumps bacteriorhodopsin and halorhodopsin. Coexpression of SRI and pSRII-F86D with their corresponding transducers suppresses the proton transport, revealing a tight binding and specific interaction of the two proteins. These latter results may be exploited to further analyze the binding interaction of the photoreceptors with their downstream effectors.

Electrophysiological characterization of specific interactions between bacterial sensory rhodopsins and their transducers

The halobacterial phototaxis receptors sensory rhodopsin I and II (SRI, SRII) enable the bacteria to seek optimal light conditions for ion pumping by bacteriorhodopsin and/or halorhodopsin. The incoming signal is transferred across the plasma membrane by means of receptor-specific transducer proteins that bind tightly to their corresponding photoreceptors. To investigate the receptor/transducer interaction, advantage is taken of the observation that both SRI and SRII can function as proton pumps. SRI from Halobacterium salinarum, which triggers the positive phototaxis, the photophobic receptor SRII from Natronobacterium pharaonis (pSRII), as well as the mutant pSRII-F86D were expressed in Xenopus oocytes. Voltage-clamp studies confirm that SRI and pSRII function as light-driven, outwardly directed proton pumps with a much stronger voltage dependence than the ion pumps bacteriorhodopsin and halorhodopsin. Coexpression of SRI and pSRII-F86D with their corresponding transducers suppresses the proton transport, revealing a tight binding and specific interaction of the two proteins. These latter results may be exploited to further analyze the binding interaction of the photoreceptors with their downstream effectors.

Nociceptin/orphanin FQ and [Phe 1ψ(CH 2-NH)Gly 2]nociceptin(1–13)NH 2 modulates the activity of hypothalamic paraventricular nucleus neurons in vitro

Nociceptin, also known as orphanin FQ (N/OFQ), an endogenous ligand for the orphan opioid receptor-like 1 (ORL 1) receptor, is moderately expressed in the hypothalamic paraventricular nucleus (PVN) involved in the integrative control of the function of the endocrine and autonomic nervous systems. Our previous study demonstrated that intracerebroventricular administration of N/OFQ elicits an inhibitory action on the function of the cardiovascular and sympathetic nervous systems in conscious rats. However, the effects of N/OFQ on PVN neurons have not been examined. We investigated the effects of N/OFQ on PVN neurons using a whole-cell patch-clamp recording technique in rat brain slices. N/OFQ (30–1000 nM) hyperpolarized membrane potentials in type 1 and type 2 neurons of the PVN classified by the electrophysiological property. [Phe 1ψ(CH 2-NH)Gly 2]nociceptin(1-13)NH 2 (Pheψ) (1–9 μM), a presumed competitive antagonist of the ORL 1 receptor, also hyperpolarized membrane potential in both types of neurons. In voltage clamp studies, N/OFQ (3–3000 nM) activated a K + current concentration-dependently in 69.7% of PVN neurons with an EC 50 of 72.4±12 nM. Pheψ (100–9000 nM) also activated a K + current with an EC 50 of 818±162 nM in PVN neurons, and significantly reduced the amplitude of the N/OFQ-stimulated current. The N/OFQ-induced current was not antagonized by the classical opioid receptor antagonist naloxone and putative antagonist nocistatin. These findings suggest that N/OFQ may have a functional role in the PVN.

A model of a segmental oscillator in the leech heartbeat neuronal network.

We modeled a segmental oscillator of the timing network that paces the heartbeat of the leech. This model represents a network of six heart interneurons that comprise the basic rhythm-generating network within a single ganglion. This model builds on a previous two cell model (Nadim et al., 1995) by incorporating modifications of intrinsic and synaptic currents based on the results of a realistic waveform voltage-clamp study (Olsen and Calabrese, 1996). Due to these modifications, the new model behaves more similarly to the biological system than the previous model. For example, the slow-wave oscillation of membrane potential that underlies bursting is similar in form and amplitude to that of the biological system. Furthermore, the new model with its expanded architecture demonstrates how coordinating interneurons contribute to the oscillations within a single ganglion, in addition to their role of intersegmental coordination.

Sustained l-type calcium currents in dissociated deep dorsal horn neurons of the rat: characteristics and modulation

Deep dorsal horn neurons present plateau properties involved in non-linear integration of nociceptive inputs, in the windup of the discharge, and in the expression of long-lasting afterdischarges. In vitro experiments using intracellular recordings in a slice preparation of the rat spinal cord have established that they are supported in part by voltage-dependent calcium currents, and positively modulated by metabotropic glutamate receptor activation. In the present study, whole-cell patch-clamp recordings in acutely isolated soma of dorsal horn neurons ( n=48) were used to analyse the voltage-dependent calcium currents involved. Deep dorsal horn neurons expressed both inactivating and non-inactivating calcium currents with Ca 2+ or Ba 2+ used as a charge carrier. The non-inactivating component activated at intermediate threshold (−55 mV), and was blocked mostly by nifedipine (61±6%). Although voltage-dependent facilitation of whole-cell calcium currents could be obtained by prepulses to +100 mV, repetitive depolarization at potentials compatible with the plateau (−45 mV and −10 mV) failed to induce facilitation of calcium currents. No direct modulation of somatic calcium currents by application of (S)-3,5-dihydroxyphenylglycine, a selective group I metabotropic glutamate receptor agonist and 1S,3R-1-amino-1,3-cyclopentanedicarboxylic acid, a group I and II metabotropic glutamate receptor agonist, was found, while application of the metabotropic GABA B receptor agonist baclofen induced a significant decrease in calcium currents. Thus, the present voltage-clamp study shows that rat deep dorsal horn neurons express a non-inactivating, nifedipine sensitive, intermediate threshold (−55 mV) calcium current which could provide the depolarizing drive to generate plateau potentials near threshold. Our results also indicate that calcium currents are not sensitized following repetitive stimulation, and not modulated by metabotropic glutamate receptor activation. They provide, however, the first evidence for a direct modulation of voltage-gated calcium channels in dorsal horn neurons by GABA B receptor activation, which may contribute to the mechanism of baclofen’s antinociceptive activity.

Molecular diversity of the repolarizing voltage-gated K+ currents in mouse atrial cells.

Voltage-clamp studies on atrial myocytes isolated from adult and postnatal day 15 (P15) C57BL6 mice demonstrate the presence of three kinetically distinct Ca2+-independent, depolarization-activated outward K+ currents: a fast, transient outward current (Ito,f), a rapidly activating, slowly inactivating current (IK,s) and a non-inactivating, steady-state current (Iss). The time- and voltage-dependent properties of to,f, IK,s and Iss in adult and P15 atrial cells are indistinguishable. Pharmacological experiments reveal the presence of two components of IK,s: one that is blocked selectively by 50 microM 4-aminopyridine (4-AP), and a 4-AP-insensitive component that is blocked by 25 mM TEA; Iss is also partially attenuated by 25 mM TEA. There are also two components of IK,s recovery from steady-state inactivation. To explore the molecular correlates of mouse atrial IK,s and Iss, whole-cell voltage-clamp recordings were obtained from P15 and adult atrial cells isolated from transgenic mice expressing a mutant Kv2.1 alpha subunit (Kv2.1N216Flag) that functions as a dominant negative, and from P15 atrial myocytes exposed to (1 microM) antisense oligodeoxynucleotides (AsODNs) targeted against Kv1.5 or Kv2.1. Peak outward K+ current densities are attenuated significantly in atrial myocytes isolated from P15 and adult Kv2.1N216Flag-expressing animals and in P15 cells exposed to AsODNs targeted against either Kv1.5 or Kv2.1. Analysis of the decay phases of the outward currents evoked during long (5 s) depolarizing voltage steps revealed that IK, s is selectively attenuated in cells exposed to the Kv1.5 AsODN, whereas both IK,s and Iss are attenuated in the presence of the Kv2. 1 AsODN. In P15 and adult Kv2.1N216Flag-expressing atrial cells, mean +/- s.e.m. IK,s and Iss densities are also significantly lower than in non-transgenic atrial cells. In addition, pharmacological experiments reveal that the TEA-sensitive component IK,s is selectively eliminated in P15 and adult Kv2.1N216Flag-expressing atrial cells. Taken together, the results presented here reveal that both Kv1.5 and Kv2.1 contribute to mouse atrial IK,s, consistent with the presence of two molecularly distinct components of IK,s. In addition, Kv2.1 contributes to mouse atrial Iss.

Characterization of Ca(2+) channels in rat subthalamic nucleus neurons.

The subthalamic nucleus (STN) plays a key role in motor control. Although previous studies have suggested that Ca(2+) conductances may be involved in regulating the activity of STN neurons, Ca(2+) channels in this region have not yet been characterized. We have therefore investigated the subtypes and functional characteristics of Ca(2+) conductances in STN neurons, in both acutely isolated and slice preparations. Acutely isolated STN cells were identified by retrograde filling with the fluorescent dye, Fluoro-Gold. In acutely isolated STN neurons, Cd(2+)-sensitive, depolarization-activated Ba(2+) currents were observed in all cells studied. The current-voltage relationship and current kinetics were characteristic of high-voltage-activated Ca(2+) channels. The steady-state voltage-dependent activation curves and inactivation curves could both be fitted with a single Boltzmann function. Currents evoked with a prolonged pulse, however, inactivated with multiple time constants, suggesting either the presence of more than one Ca(2+) channel subtype or multiple inactivation processes with a single channel type in STN neurons. Experiments using organic Ca(2+) channel blockers revealed that on average, 21% of the current was nifedipine sensitive, 52% was sensitive to omega-conotoxin GVIA, 16% was blocked by a high concentration of omega-agatoxin IVA (200 nM), and the remainder of the current (9%) was resistant to the co-application of all blockers. These currents had similar voltage dependencies, but the nifedipine-sensitive current and the resistant current activated at slightly lower voltages. omega-Agatoxin IVA at 20 nM was ineffective in blocking the current. Together, the above results suggest that acutely isolated STN neurons have all subtypes of high-voltage-activated Ca(2+) channels except for P-type, but have no low-voltage-activated channels. Although acutely isolated neurons provide a good preparation for whole cell voltage-clamp study, dendritic processes are lost during dissociation. To gain information on Ca(2+) channels in dendrites, we thus studied Ca(2+) channels of STN neurons in a slice preparation, focusing on low-voltage-activated channels. In current-clamp recordings, a slow spike was always observed following termination of an injected hyperpolarizing current. The slow spike occurred at resting membrane potentials and was sensitive to micromolar concentrations of Ni(2+), suggesting that it is a low-threshold Ca(2+) spike. Together, our results suggest that STN neurons express low-voltage-activated Ca(2+) channels and several high-voltage-activated subtypes. Our results also suggest the possibility that the low-voltage-activated channels have a preferential distribution to the dendritic processes.