Current-clamp Experiments Research Articles

Computational Sophistication at a Single GABAergic Connection

Diverse computational roles of GABAergic inhibition are often assumed to reflect heterogeneity in the sources of GABA and in the receptors sensing the neurotransmitter. New data suggest that distinct effects on integration of excitatory inputs by cerebellar granule cells might result from different modes of signaling by individual interneurons.

Complex Intrinsic Membrane Properties and Dopamine Shape Spiking Activity in a Motor Axon

We studied the peripheral motor axons of the two pyloric dilator (PD) neurons of the stomatogastric ganglion in the lobster, Homarus americanus. Intracellular recordings from the motor nerve showed both fast and slow voltage- and activity-dependent dynamics. During rhythmic bursts, the PD axons displayed changes in spike amplitude and duration. Pharmacological experiments and the voltage dependence of these phenomena suggest that inactivation of sodium and A-type potassium channels are responsible. In addition, the "resting" membrane potential was dependent on ongoing spike or burst activity, with more hyperpolarized values when activity was strong. Nerve stimulations, pharmacological block and current clamp experiments suggest that this is due to a functional antagonism between a slow after-hyperpolarization (sAHP) and inward rectification through hyperpolarization-activated current (IH). Dopamine application resulted in modest depolarization and "ectopic" peripheral spike initiation in the absence of centrally generated activity. This effect was blocked by CsCl and ZD7288, consistent with a role of IH. High frequency nerve stimulation inhibited peripheral spike initiation for several seconds, presumably due to the sAHP. Both during normal bursting activity and antidromic nerve stimulation, the conduction delay over the length of the peripheral nerve changed in a complex manner. This suggests that axonal membrane dynamics can have a substantial effect on the temporal fidelity of spike patterns propagated from a spike initiation site to a synaptic target, and that neuromodulators can influence the extent to which spike patterns are modified.

Activation of neurokinin 3 receptor increases Nav1.9 current in enteric neurons

The intrinsic primary afferent neurons (IPANs) of the guinea pig enteric nervous system express Na(v)1.9 sodium channels that produce a persistent TTX-resistant current having a low activation threshold and slow gating kinetics. These neurons receive slow EPSPs induced mainly by the activation of neurokinin 3 receptors (NK3r). Here, we demonstrate that senktide, a specific NK3r agonist, potentiates the Na(v)1.9 current (I(Nav1.9)) in IPANs. Using whole-cell patch-clamp recordings from IPANs in duodenum longitudinal muscle/myenteric plexus preparations, we show that short (1-5 s) and long (up to 1 min) applications of senktide, increase the I(Nav1.9) peak current up to 13-fold. The effect, blocked by a NK3r antagonist SB235375 is transient, lasting approximately 2 min and is due to a negative shift of the activation voltage by approximately 20 mV and of fast inactivation by approximately 10 mV. As a consequence, the window current resulting from the product of the activation and fast inactivation curves is shifted and enlarged. The transient effect of senktide is likely to be due to the fast desensitization of NK3r. Protein kinase C (PKC) activation with phorbol or oleoyl acetylglycerol also increases I(Nav1.9), although persistently, by inducing similar voltage-dependent changes. Current-clamp experiments showed that I(Nav1.9) modulation by senktide lowers action potential threshold and increases excitability. The increase in I(Nav1.9) by NK3r activation is also likely to amplify slow EPSPs generated in the IPANs. These changes in excitability potentially have a profound effect on the entire enteric synaptic circuit and ultimately on gut motility and secretion.

Plasma Current Start-up in a Spherical Tokamak

The various plasma current start-up techniques and related topics in a spherical tokamak (ST) device are described. The Ohmic heating coil current clamp experiments in NSTX are described and discussed, and the plasma current start-up experiments in the STOR-M tokamak with iron core and the outer vertical field coil is presented as one of technique for a plasma current start-up in a ST.

The muscarinic inhibition of the potassium M-current modulates the action-potential discharge in the vestibular primary-afferent neurons of the rat

There is consensus that muscarinic and nicotinic receptors expressed in vestibular hair cells and afferent neurons are involved in the efferent modulation of the electrical activity of the afferent neurons. However the underlying mechanisms of postsynaptic control in neurons are not well understood. In our work we show that the activation of muscarinic receptors in the vestibular neurons modulates the potassium M-current modifying the activity of afferent neurons.Whole-cell patch-clamp recordings were made on vestibular-afferent neurons isolated from Wistar rats (postnatal days 7–10) and held in primary culture (18–24 h). The M-current was studied during its deactivation after depolarizing voltage-clamp pulses. In 68% of the cells studied, those of larger capacitance, the M-current antagonists linopirdine and XE-991 reduced the amplitude of the M-current by 54%±7% and 50%±3%. The muscarinic-receptor agonist oxotremorine-M also significantly reduced the M-current by 58%±12% in the cells. The action of oxotremorine-M was blocked by atropine, thus indicating its cholinergic nature. The erg-channel blocker E-4031 did not significantly modify the M-current amplitude. In current-clamp experiments, linopirdine, XE-991, and oxotremorine-M modified the discharge response to current pulses from single spike to multiple spiking, reducing the adaptation of the electrical discharge. Our results indicate that large soma-size cultured vestibular-afferent neurons (most probably calyx-bearing neurons) express the M-current and that the modulation of this current by activation of muscarinic-receptor reduces its spike-frequency adaptation.

ASIC3, a sensor of acidic and primary inflammatory pain

Acid-sensing ion channels (ASICs) are cationic channels activated by extracellular acidosis that are expressed in both central and peripheral nervous systems. Although peripheral ASICs seem to be natural sensors of acidic pain (e.g., in inflammation, ischaemia, lesions or tumours), a direct demonstration is still lacking. We show that approximately 60% of rat cutaneous sensory neurons express ASIC3-like currents. Native as well as recombinant ASIC3 respond synergistically to three different inflammatory signals that are slight acidifications (approximately pH 7.0), hypertonicity and arachidonic acid (AA). Moderate pH, alone or in combination with hypertonicity and AA, increases nociceptors excitability and produces pain suppressed by the toxin APETx2, a specific blocker of ASIC3. Both APETx2 and the in vivo knockdown of ASIC3 with a specific siRNA also have potent analgesic effects against primary inflammation-induced hyperalgesia in rat. Peripheral ASIC3 channels are thus essential sensors of acidic pain and integrators of molecular signals produced during inflammation where they contribute to primary hyperalgesia.

Kv4 Accessory Protein DPPX (DPP6) is a Critical Regulator of Membrane Excitability in Hippocampal CA1 Pyramidal Neurons

A-type K+ currents have unique kinetic and voltage-dependent properties that allow them to finely tune synaptic integration, action potential (AP) shape and firing patterns. In hippocampal CA1 pyramidal neurons, Kv4 channels make up the majority of the somatodendritic A-type current. Studies in heterologous expression systems have shown that Kv4 channels interact with transmembrane dipeptidyl-peptidase-like proteins (DPPLs) to regulate the surface trafficking and biophysical properties of Kv4 channels. To investigate the influence of DPPLs in a native system, we conducted voltage-clamp experiments in patches from CA1 pyramidal neurons expressing short-interfering RNA (siRNA) targeting the DPPL variant known to be expressed in hippocampal pyramidal neurons, DPPX (siDPPX). In accordance with heterologous studies, we found that DPPX downregulation in neurons resulted in depolarizing shifts of the steady-state inactivation and activation curves, a shallower conductance-voltage slope, slowed inactivation, and a delayed recovery from inactivation for A-type currents. We carried out current-clamp experiments to determine the physiological effect of the A-type current modifications by DPPX. Neurons expressing siDPPX exhibited a surprisingly large reduction in subthreshold excitability as measured by a decrease in input resistance, delayed time to AP onset, and an increased AP threshold. Suprathreshold DPPX downregulation resulted in slower AP rise and weaker repolarization. Computer simulations supported our experimental results and demonstrated how DPPX remodeling of A-channel properties can result in opposing sub- and suprathreshold effects on excitability. The Kv4 auxiliary subunit DPPX thus acts to increase neuronal responsiveness and enhance signal precision by advancing AP initiation and accelerating both the rise and repolarization of APs.

On the mechanisms of arrhythmias in the myocardium of mXinα-deficient murine left atrial-pulmonary veins

We have previously shown that left atrial-pulmonary vein tissue (LA-PV) can generate reentrant arrhythmias (atrial fibrillation, AF) in wild-type (mXinα+/+) but not in mXinα-null (mXinα−/−) mice. With the present experiments, we investigated the arrhythmogenic activity and the underlying mechanisms in mXinα+/+ vs. mXinα−/− LA-PV. Electrical activity and conduction velocity (CV) were recorded in LA-PV by means of a MED64 system. CV was significantly faster in mXinα+/+ than in mXinα−/− LA-PV and it was increased by 1 μM isoproterenol (ISO). AF could be induced by fast pacing in the mXinα+/+ but not in mXinα−/− LA-PV where automatic rhythms could occur. ISO increased the incidence of AF in Xinα+/+ whereas it increased that of automatic rhythms in mXinα−/− LA-PV. In LA-PV with the right atrium attached (RA-LA-PV), automatic rhythms occurred in all preparations. In mXinα+/+ RA-LA-PV simultaneously treated with ISO, strophanthidin and atropine, the incidence of the automatic rhythm was about the same, but AF increased significantly. In contrast, in mXinα−/− RA-LA-PV under the same condition, the automatic rhythm was markedly enhanced, but still no AF occurred. Conventional microelectrode techniques showed a longer APD 90 and a less negative maximum diastolic potential (MDP) in mXinα−/− than mXinα+/+ LA-PV tissues. Whole-cell current clamp experiments also showed a less negative MDP in mXinα−/− vs. mXinα+/+ LA-PV cardiomyocytes. The fact that AF could be induced by fast pacing under several conditions in mXinα+/+ but not in mXinα−/− LA-PV preparations appears to be due to a slower CV, a prolonged APD 90, a less negative MDP and possibly larger areas of conduction block in mXinα−/− myocardial cells. In contrast, the non-impairment of automatic and triggered rhythms in mXinα−/− preparations may be due to the fact that the mechanisms underlying these rhythms do not involve cell-to-cell conduction.

TRPV1 Gene Required for Thermosensory Transduction and Anticipatory Secretion from Vasopressin Neurons during Hyperthermia

Increases in core body temperature promote thermoregulatory cooling by stimulating sweat production and preemptive renal water reabsorption through the release of vasopressin (VP, antidiuretic hormone). The mechanism by which the hypothalamus orchestrates this anticipatory VP release during hyperthermia is unknown but has been linked to a central thermosensory mechanism. Here, we report that thermal stimuli spanning core body temperatures activate a calcium-permeable, ruthenium red- and SB366791-sensitive nonselective cation conductance in hypothalamic VP neurons. This response is associated with a depolarizing receptor potential and an increase in action potential firing rate, indicating that these neurons are intrinsically thermosensitive. The thermosensitivity of VP neurons isolated from trpv1 knockout (Trpv1(-/-)) mice was significantly lower than that of wild-type counterparts. Moreover, Trpv1(-/-) mice showed an impaired VP response to hyperthermia in vivo. Channels encoded by the trpv1 gene thus confer thermosensitivity in central VP neurons and contribute to the thermal control of VP release in vivo.

Small-Conductance Calcium-Activated Potassium Channels Control Excitability and Firing Dynamics in Gonadotropin-Releasing Hormone (GnRH) Neurons

The cellular mechanisms determining the firing patterns of GnRH neurons are presently under intense investigation. In this study, we used GnRH-green fluorescent protein transgenic mice and perforated-patch electrophysiology to examine the role of small conductance calcium-activated potassium (SK) channels in determining the electrical excitability and burst-firing characteristics of adult GnRH neurons. After establishing an appropriate protocol for examining the afterhyperpolarization potential (AHP) currents in GnRH neurons, the highly selective SK channel blocker apamin was used to demonstrate that all GnRH neurons express functional SK channels (35.7 +/- 2.7 pA, mean decay time constant = 2167 msec, apamin IC(50) = 9.6 nm) and that this channel underlies approximately 90% of the AHP in these cells. Current-clamp experiments showed that apamin-sensitive SK channels were tonically active in the majority (74%) of GnRH neurons, with apamin (100 nm) administration resulting in a mean 6.9 +/- 0.5 mV membrane depolarization. Apamin also elevated the firing rate of GnRH neurons, including increased burst frequency and duration in spontaneously bursting cells as well as the ability of GnRH neurons to fire action potentials in response to current injection. In GnRH neurons activated by current injection, apamin significantly enhanced the amplitude of the afterdepolarization potential after a single action potential and eliminated spike frequency adaptation. Together, these studies show that apamin-sensitive SK channels play a key role in restraining GnRH neuron excitability. Through direct modulation of the AHP and indirect actions on the afterdepolarization potential, the SK channel exerts a powerful tonic influence upon the firing dynamics of GnRH neurons.

Functional role of cyclic nucleotide‐gated channels in rat medial vestibular nucleus neurons

Although cyclic nucleotide-gated (CNG) channels are expressed in numerous brain areas, little information is available on their functions in CNS neurons. The aim of the present study was to define the distribution of CNG channels in the rat medial vestibular nucleus (MVN) and their possible involvement in regulating MVN neuron (MVNn) excitability. The majority of MVNn expressed both CNG1 and CNG2 A subunits. In whole-cell current-clamp experiments carried out on brainstem slices containing the MVNn, the membrane-permeant analogues of cyclic nucleotides, 8-Br-cGMP and 8-Br-cAMP (1 mM), induced membrane depolarizations (8.9 +/- 0.8 and 9.2 +/- 1.0 mV, respectively) that were protein kinase independent. The cGMP-induced depolarization was associated with a significant decrease in the membrane input resistance. The effects of cGMP on membrane potential were almost completely abolished by the CNG channel blockers, Cd(2+) and L-cis-diltiazem, but they were unaffected by blockade of hyperpolarization-activated cyclic nucleotide-gated channels. In voltage-clamp experiments, 8-Br-cGMP induced non-inactivating inward currents (-22.2 +/- 3.9 pA) with an estimated reversal potential near 0 mV, which were markedly inhibited by reduction of extracellular Na(+) and Ca(2+) concentrations. Membrane depolarization induced by CNG channel activation increased the firing rate of MVNn without changing the action potential shape. Collectively, these findings provide novel evidence that CNG channels affect membrane potential and excitability of MVNn. Such action should have a significant impact on the function of these neurons in sensory-motor integration processes. More generally, it might represent a broad mechanism for regulating the excitability of different CNS neurons.

The Chemokine CXCL1/Growth Related Oncogene Increases Sodium Currents and Neuronal Excitability in Small Diameter Sensory Neurons

BackgroundAltered Na+ channel expression, enhanced excitability, and spontaneous activity occur in nerve-injury and inflammatory models of pathological pain, through poorly understood mechanisms. The cytokine GRO/KC (growth related oncogene; CXCL1) shows strong, rapid upregulation in dorsal root ganglion in both nerve injury and inflammatory models. Neurons and glia express its receptor (CXCR2). CXCL1 has well-known effects on immune cells, but little is known about its direct effects on neurons.ResultsWe report that GRO/KC incubation (1.5 nM, overnight) caused marked upregulation of Na+ currents in acutely isolated small diameter rat (adult) sensory neurons in vitro. In both IB4-positive and IB4-negative sensory neurons, TTX-resistant and TTX-sensitive currents increased 2- to 4 fold, without altered voltage dependence or kinetic changes. These effects required long exposures, and were completely blocked by co-incubation with protein synthesis inhibitor cycloheximide. Amplification of cDNA from the neuronal cultures showed that 3 Na channel isoforms were predominant both before and after GRO/KC treatment (Nav 1.1, 1.7, and 1.8). TTX-sensitive isoforms 1.1 and 1.7 significantly increased 2 – 3 fold after GRO/KC incubation, while 1.8 showed a trend towards increased expression. Current clamp experiments showed that GRO/KC caused a marked increase in excitability, including resting potential depolarization, decreased rheobase, and lower action potential threshold. Neurons acquired a striking ability to fire repetitively; IB4-positive cells also showed marked broadening of action potentials. Immunohistochemical labelling confirmed that the CXCR2 receptor was present in most neurons both in dissociated cells and in DRG sections, as previously shown for neurons in the CNS.ConclusionMany studies on the role of chemokines in pain conditions have focused on their rapid and indirect effects on neurons, via release of inflammatory mediators from immune and glial cells. Our study suggests that GRO/KC may also have important pro-nociceptive effects via its direct actions on sensory neurons, and may induce long-term changes that involve protein synthesis.

Effects of the muscarinic agonist, 5-methylfurmethiodide, on contraction and electrophysiology of Ascaris suum muscle

Contraction and electrophysiological effects of 5-methylfurmethiodide (MFI), a selective muscarinic agonist in mammals, were tested on Ascaris suum muscle strips. In a contraction assay, MFI produced weak contraction and was less potent than levamisole and acetylcholine. Atropine (3μM) a non-selective muscarinic antagonist in mammalian preparations, did not affect contractions produced by MFI. Mecamylamine (3μM) a nicotinic antagonist in A. suum preparations, blocked the MFI contractions indicating that MFI had weak nicotinic agonist actions. In two-micropipette current-clamp experiments MFI, at concentrations greater than 10μM, produced concentration-dependent depolarizations and small increases in membrane conductance. The depolarizing effects were not abolished by perfusing the preparation in a calcium-free Ascaris Ringer solution to block synaptic transmission, suggesting that MFI effects were mediated by receptors on the muscle and were calcium-independent. A high concentration of mecamylamine, 30μM, only reduced the depolarizing responses by 42%, indicating that MFI also had effects on non-nicotinic receptors. Three non-nicotinic effects in the presence of 30μM mecamylamine were identified using voltage-clamp techniques: (i) MFI produced opening of mecamylamine-resistant non-selective-cation channel currents; (ii) MFI inhibited opening of voltage-activated potassium currents; and (iii) MFI increased the threshold of voltage-activated calcium currents. We suggest that a drug that is more selective for voltage-activated potassium currents, without effects on other channels like MFI, may be exploited pharmacologically as a novel anthelmintic or as an agent to potentiate the action of levamisole. In a larval migration assay we demonstrated that 4-aminopyridine (4-AP: a potassium channel blocker) potentiated the effects of levamisole but MFI did not.

Inhibition of T‐type Calcium Current in the Reticular Thalamic Nucleus by a Novel Neuroactive Steroid

Neurons of the nucleus reticularis of the thalamus (nRT) serve as an important inhibitory gate that controls trafficking of thalamocortical sensory signals and states of sleep, arousal, and epilepsy. T-type calcium channels in nRT play a crucial role in the subthreshold excitability of these neurons, but their modulation by neuroactive steroids has not been previously studied. Here we explored the effect of (3beta,5beta,17beta)-3-hydroxyandrostane-17-carbonitrile (3beta-OH), a novel neuroactive steroid on T-type currents recorded from nRT neurons in intact brain slices of young rats. We found in voltage-clamp experiments that 3beta-OH potently and reversibly decreased T-type Ca(2+) current amplitude and stabilized inactive states of the channels. In current-clamp experiments, 3beta-OH significantly decreased the frequency of action potential firing from negative membrane potentials and minimally changed passive membrane properties. Our results indicate that 5beta-reduced neuroactive steroids, through the mechanisms of inhibition of T-type Ca(2+) currents and diminished spike firing in nRT neurons, may be important agents in control of sensory information processing in physiological conditions and possibly pathological brain states associated with increased cellular excitability such as epilepsy and/or tissue ischemia/hypoxia.

Kv1 and Kir2 potassium channels are expressed in rat brain endothelial cells

The endothelial cells of the brain microvasculature, which constitute the blood-brain barrier, secrete K+ into brain interstitial fluid. K+ channels are predicted to have a central role to play in this process. The aim of the following study was to characterise K+ channels in primary cultures of endothelial cells isolated from rat brain microvessels by whole-cell patch clamp and real-time polymerase chain reaction. In the 4 h after plating, the rat brain endothelial cells expressed predominantly a depolarisation-activated delayed-rectifying outward K+ conductance and a time-independent inwardly rectifying K+ conductance prominent at hyperpolarising potentials. The outward current was inhibited by 1 mM 4-aminopyridine (4AP), 10 nM margatoxin and 100 nM dendrotoxin-K, indicating the involvement of Kv1 channels. The half maximal activation voltage and time constants of activation and inactivation of the 4AP-sensitive current were similar to Kv1.3. The inwardly rectifying conductance was inhibited by Ba2+ in a dose- and voltage-dependent fashion; the kinetics of which resembled Kir2 channels. Quantification of messenger ribonucleic acid transcripts revealed Kv1.3>1.2=1.4=1.5=1.6 and Kir2.1=2>2.3. In current-clamp experiments, both 4AP and Ba2+ depolarised the membrane potential. In conclusion, rat brain endothelial cells express Kv1 and Kir2 K+ channels, both of which participate in setting membrane potential and could mediate K+ secretion into the brain interstitial fluid.

Voltage-gated potassium currents within the dorsal vagal nucleus: Inhibition by BDS toxin

Voltage-gated potassium (Kv) channels are essential components of neuronal excitability. The Kv3.4 channel protein is widely distributed throughout the central nervous system (CNS), where it can form heteromeric or homomeric Kv3 channels. Electrophysiological studies reported here highlight a functional role for this channel protein within neurons of the dorsal vagal nucleus (DVN). Current clamp experiments revealed that blood depressing substance (BDS) and intracellular dialysis of an anti-Kv3.4 antibody prolonged the action potential duration. In addition, a BDS sensitive, voltage-dependent, slowly inactivating outward current was observed in voltage clamp recordings from DVN neurons. Electrical stimulation of the solitary tract evoked EPSPs and IPSPs in DVN neurons and BDS increased the average amplitude and decreased the paired pulse ratio, consistent with a presynaptic site of action. This presynaptic modulation was action potential dependent as revealed by ongoing synaptic activity. Given the role of the Kv3 proteins in shaping neuronal excitability, these data highlight a role for homomeric Kv3.4 channels in spike timing and neurotransmitter release in low frequency firing neurons of the DVN.

Vertebrate rod photoreceptors express both BK and IK calcium‐activated potassium channels, but only BK channels are involved in receptor potential regulation

In salamander rods, Ca(2+)-activated K(+) current (I(KCa)) provides an effective "clamp" of the dark membrane potential to its normal resting level. By a combination of electrophysiological, pharmacological, and immunohistochemical approaches, we show that salamander rods functionally express large-conductance Ca(2+)- and voltage-dependent potassium (BK) channel and intermediate-conductance Ca(2+)-dependent potassium (IK) channel, but not small-conductance Ca(2+)-dependent potassium channel (SK) subtypes. Application of 100 nM iberiotoxin and 100 nM clotrimazole reduced net I(KCa) to 36% and 63%, respectively, whereas the current was unaffected by application of 1 microM apamin. Consistently, anti- SK1, -SK2, and -SK3 antibodies were unable to stain rod photoreceptors, whereas both anti-BK and -SK4/ IK1 antibodies heavily stained the ellipsoid region of the inner segments of the rods. Moreover, by using current-clamp experiments, it was clearly seen that the strong clamping effect of the total I(KCa) was lost when IbTx, but not CLTZ, was applied to the bath. This behavior strongly suggests that of BK and IK channels, only the former are responsible for the clamping effect on the photoreceptor membrane potential.

Local inflammation in rat dorsal root ganglion alters excitability and ion currents in small-diameter sensory neurons.

Chronic pain conditions may result from peripheral nerve injury, chronic peripheral inflammation, or sensory ganglia inflammation. However, inflammatory processes may also contribute to peripheral nerve injury responses. To isolate the contribution of local inflammation of sensory ganglia to chronic pain states, the authors previously developed a rat model in which long-lasting pain is induced by inflaming sensory ganglia without injuring the neurons. This results in prolonged mechanical pain, local increases in proinflammatory cytokines, increased neuronal hyperexcitability, and abnormal spontaneous activity. The authors used whole cell patch clamp in acutely isolated small-diameter neurons to determine how localized inflammation (3-5 days) of L4 and L5 ganglia altered voltage-gated K and Na currents. Tetrodotoxin-sensitive Na currents increased twofold to threefold in neurons from inflamed ganglia. Tetrodotoxin-resistant Na currents increased more than twofold, but only in cells that bound isolectin B4. These increases occurred without shifts in voltage dependence of activation and inactivation. Similar results are seen in models of peripheral inflammation, except for the large magnitudes. Unlike most pain models, localized inflammation increased rather than decreased voltage-gated K currents, due to increased amplitudes of the sustained (delayed rectifier) and fast-inactivating transient components. The overall effect in current clamp experiments was an increase in excitability as indicated by decreased rheobase and lower action potential threshold. Neuronal inflammation per se, in the absence of nerve injury, causes large increases in Na channel density and enhanced excitability. The unusual finding of increased K current may reflect regulation of excitability in the face of such large increases in Na current.

Characterization of the Heteromeric Potassium Channel Formed by Kv2.1 and the Retinal Subunit Kv8.2 in Xenopus Oocytes

Kv8.2 (KCNV2) subunits do not form homotetrameric potassium channels, although they coassemble with Kv2.1 to constitute functional heteromers. High expression of Kv8.2 was reported in the human retina and its mutations were linked to the visual disorder "cone dystrophy with supernormal rod electroretinogram." We detected abundant Kv8.2 expression in the photoreceptor layer of mouse retina, where Kv2.1 is also known to be present. When the two subunits were coexpressed in Xenopus oocytes in equal amounts, Kv8.2 abolished the current of Kv2.1. If the proportion of Kv8.2 was reduced then the current of heteromeric channels emerged. Kv8.2 shifted the steady-state activation of Kv2.1 to more negative potentials, without affecting the voltage dependence of inactivation. This gave rise to a window current within the -40 to -10 mV membrane potential range. Ba2+ inhibited the heteromeric channel and shifted its activation to more positive potentials. These electrophysiological and pharmacological properties resemble those of the voltage-gated K+ current (named I Kx) described in amphibian retinal rods. Furthermore, oocytes expressing Kv2.1/Kv8.2 developed transient hyperpolarizing overshoots in current-clamp experiments, whereas those expressing only Kv2.1 failed to do so. Similar overshoots are characteristic responses of photoreceptors to light flashes. We demonstrated that Kv8.2 G476D, analogous to a disease-causing human mutation, eliminated Kv2.1 current, if the subunits were coexpressed equally. However, Kv8.2 G476D did not form functional heteromers under any conditions. Therefore we suggest that the custom-tailored current of Kv2.1/Kv8.2 functionally contributes to photoreception, and this is the reason that mutations of Kv8.2 lead to a genetic visual disorder.

Resurgent Na+ current in pyramidal neurones of rat perirhinal cortex: axonal location of channels and contribution to depolarizing drive during repetitive firing

The perirhinal cortex (PRC) is a supra-modal cortical area that collects and integrates information originating from uni- and multi-modal neocortical regions and directed to the hippocampus. The mechanisms that underlie the specific excitable properties of the different PRC neuronal types are still largely unknown, and their elucidation may be important in understanding the integrative functions of PRC. In this study we investigated the expression and properties of resurgent Na(+) current (I(NaR)) in pyramidal neurones of rat PRC area 35 (layer II). Patch-clamp experiments in acute PRC slices were first carried out. A measurable I(NaR) was expressed by a large majority of neurones (31 out of 35 cells). I(NaR) appeared as an inward, slowly decaying current elicited upon step repolarization after depolarizations sufficient to induce nearly complete inactivation of the transient Na(+) current (I(NaT)). I(NaR) had a peak amplitude of approximately 2.5% that of I(NaT), and showed the typical biophysical properties also observed in other neuronal types (i.e. cerebellar Purkinje and granule cells), including a bell-shaped current-voltage relationship with a peak at approximately -40 mV, and a characteristic acceleration of activation and decay speed at potentials negative to -45 mV. Current-clamp experiments were then carried out in which repetitive action-potential discharge at various frequencies was induced with depolarizing current injection. The voltage signals thus obtained were then used as command waveforms for voltage-clamp recordings. These experiments showed that a Na(+) current identifiable as I(NaR) activates in the early interspike phase even at relatively high firing frequencies (20 Hz), thereby contributing to the depolarizing drive and possibly enhancing repetitive discharge. In acutely dissociated area 35 layer II neurones, as well as in nucleated patches from the same neurones, I(NaR) was never observed, despite the presence of typical I(NaT)s. Since in both preparations neuronal processes are lost, we carried out experiments of focal tetrodotoxin (TTX) application in slices to verify whether the channels responsible for I(NaR) are located in compartment(s) different from the soma. We found that TTX preferentially inhibited I(NaR) when applied close to the site of axon emergence from soma, whereas application to the apical pole of the soma had a significantly smaller effect on I(NaR). Our results indicate that in area 35 pyramidal cells I(NaR) is largely generated in the axon initial segment, where it may participate in setting the coding properties of these neurones.