Interaction between Low Voltage-activated Currents in Reticular Thalamic Neurons in a Rat Model of Absence Epilepsy

A transient potassium (K⁺) outward current (I_A) contributes to the distinctive patterns of low-threshold spike firing observed in various classes of thalamic neurons through a functional interaction with a calcium (Ca²⁺)-mediated inward current (I_T). The present study was undertaken to investigate the properties of transient K⁺ currents and their interaction with I_T in neurons of the reticular thalamic nucleus, and to compare these properties in reticular thalamic nucleus neurons from a rat model of absence epilepsy, designated the Genetic Absence Epilepsy Rat from Strasbourg (GAERS), with those from a Non-epileptic Control strain (NEC). This comparative approach appeared to be particularly important in view of the recent finding of a selective increase in I_T in reticular thalamic nucleus neurons from GAERS. Neurons were acutely isolated from the reticular thalamic nucleus through enzymatic procedures, and identified by morphological and immunocytochemical criteria. Ionic currents were analysed using whole-cell patch-clamp techniques. Transient K⁺ currents in reticular thalamic nucleus neurons with properties indicative of I_A activated at ～?55 mV (with half-activation at ?27 and ?33 mV in NEC and GAERS respectively), declined rapidly with a voltage-dependent time constant (τ= 4 ms at +45 mV), were 50% steady-state-inactivated at ?81 and ?86 mV in the two strains of rats respectively, and rapidly recovered from inactivation with a monoexponential time course (τ= 31 and 37 ms respectively). No significant differences in I_A properties or densities were found between reticular thalamic nucleus neurons from GAERS and NEC rats. Analysis of the interaction between I_A and I_T indicated a shift in the balance between the two opposing membrane conductances towards the generation of a low-voltage-activated inward current in reticular thalamic nucleus neurons from GAERS compared with NEC, and a lack of I_A to functionally compensate for this shift, which in turn may contribute to pathological forms of low-threshold spike firing characterizing spike-and-wave discharges.     

Ionic mechanisms of action of prion protein fragment PrP(106–126) in rat basal forebrain neurons

Prion diseases are neurodegenerative disorders that are characterized by the presence of the misfolded prion protein (PrP). Neurotoxicity in these diseases may result from prion‐induced modulation of ion channel function, changes in neuronal excitability, and consequent disruption of cellular homeostasis. We therefore examined PrP effects on a suite of potassium (K⁺) conductances that govern excitability of basal forebrain neurons. Our study examined the effects of a PrP fragment [PrP(106–126), 50 nM] on rat neurons using the patch clamp technique. In this paradigm, PrP(106–126) peptide, but not the “scrambled” sequence of PrP(106–126), evoked a reduction of whole‐cell outward currents in a voltage range between –30 and +30 mV. Reduction of whole‐cell outward currents was significantly attenuated in Ca²⁺‐free external media and also in the presence of iberiotoxin, a blocker of calcium‐activated potassium conductance. PrP(106–126) application also evoked a depression of the delayed rectifier (I_K) and transient outward (I_A) potassium currents. By using single cell RT‐PCR, we identified the presence of two neuronal chemical phenotypes, GABAergic and cholinergic, in cells from which we recorded. Furthermore, cholinergic and GABAergic neurons were shown to express K_v4.2 channels. Our data establish that the central region of PrP, defined by the PrP(106–126) peptide used at nanomolar concentrations, induces a reduction of specific K⁺ channel conductances in basal forebrain neurons. These findings suggest novel links between PrP signalling partners inferred from genetic experiments, K⁺ channels, and PrP‐mediated neurotoxicity. © 2010 Wiley‐Liss, Inc.     

Adult rat optic nerve oligodendrocyte progenitor cells express a distinct repertoire of voltage- and ligand-gated ion channels

Cultured oligodendrocyte progenitor cells derived from the developing central nervous system (CNS) express a pattern of ion channels that is distinct from mature oligodendrocytes and other cell types of the CNS. In the present study, we used the whole-cell patch-clamp technique and the fura-2-based Ca⁺⁺ imaging system to study the ion channel expression of oligodendrocyte progenitor cells derived from the optic nerves of adult rats. We found that the adult oligodendrocyte progenitor cell membrane is dominated by K⁺ currents, both delayed outward and inward rectifying. The inwardly rectifying K⁺ currents were often as large as the outward delayed rectifying K⁺ currents. The delayed rectifying outward currents were partially blocked by 50 mM tetraethylammonium or 1 mM 4-aminopyridine, but not by 2 or 5 mM BaCl₂. This suggests that the delayed rectifier channels expressed by adult progenitor cells are different from those expressed by perinatal cells. Most adult oligodendrocyte progenitor cells showed no or only small A-type K⁺ currents. Both Ca⁺⁺ and Na⁺ channels were also detected in these cells. Furthermore, adult progenitor cells responded to the neurotransmitters GABA and kainate and the pharmacology of these responses indicated that these cells express GABA_A receptors and kainate receptors that are Ca⁺⁺ -permeable. Our study suggests that adult oligodendrocyte progenitor cells are electrophysiologically distinct and that these cells share electrophysiological characteristics with both perinatal progenitor cells and immature oligodendrocytes. © 1995 Wiley-Liss, Inc.     

Blockade of bepridil on IA and IK in acutely isolated hippocampal CA1 neurons

The effects of bepridil, an antianginal agent with antiarrhythmic action, on voltage-dependent K⁺ currents in the CA1 pyramidal neurons acutely isolated from rat hippocampus were studied by means of whole-cell patch clamp techniques. Current recordings were made in the presence of TTX to block Na⁺ current. Depolarizing test pulses activated two components of outward K⁺ currents: a rapidly activating and inactivating component, I_A; and a delayed component, I_K. Results showed that bepridil reduced the amplitude of I_A and I_K, and exerted its inhibitory action in time- and dose-dependent manner. Half-blocking concentrations (IC₅₀) of bepridil on I_A and I_K were 17.8 μM and 1.7 μM, respectively. 10 μM bepridil suppressed I_A and I_K by 46.7% and 77.1% at +30 mV of depolarization, respectively. When I_K was activated nearly uncontaminated with I_A by holding at −50 mV, 10 μM bepridil inhibited I_K by 71.6% at +30 mV of depolarization; 10 μM bepridil positively shifted the voltage-dependent of activation curves of I_A and I_K 12.1 mV and 28.7 mV, respectively. These results suggested that blockade on K⁺ currents by bepridil is preferential for I_K, and contributes to the protection brain against ischemic damage.     

Ionic currents on type-I cells of the rabbit carotid body measured by voltage-clamp experiments and the effect of hypoxia

Type-I cells (from rabbit embryos) in primary culture were studied in voltage-clamp experiments using the whole cell arrangement of the patch-clamp technique. With a pipette solution containing 130 mM K⁺ and 3 mM Mg-ATP, large outward currents were obtained positive to a threshold of about −30 mV by clamping cells from −50 mV to different test pulses (−80 to 50 mV). Negative to −30 mV, the slope conductance was low (outward rectification). The outward currents were blocked by external Cs⁺ (5 mM) and partially blocked by TEA (5 mM) and Co²⁺ (1 mM). The initial part of the outward currents during depolarizing voltage pulses exhibited a transient Ca²⁺ inward component partially superimposed to a Ca²⁺-dependent outward current. Inward currents were further characterized by replacing K⁺ with Cs⁺ in the intra- and extracellular solution in order to minimize the outward component and by using 1.8 mM Ca²⁺ or 10.8 mM Ba²⁺ as charge carrier. Slow-inactivating inward currents were recorded at test potentials ranging from −50 to 40 mV (holding potential −80 mV). The maximal amplitude, measured at 10 mV in the U-shaped I–V curve, amounted to 247 ± 103pA(n = 3). This inward current was insensitive to 3 μM TTX, but blocked by 1 mM Co²⁺ and partially reduced by 10 μM D600 and 3 μM PN 200-110. In contrast to outward currents, the inward currents exhibited a ‘run-down’ within about 10 min. Lowering the pO₂ from the control of 150 Torr (air-gassed medium) to 28 Torr had no apparent effect on inward currents, but depressed reversibly outward currents by 28%. In conclusion, it is suggested that type-I cells possess voltage-activated K⁺ and Ca²⁺ channels which might be essential for chemoreception in the carotid body.     

The effects of pentylenetetrazol on molluscan neurons II. Voltage clamp studies

The effects of pentylenetetrazol (PTZ) upon the steady and transient outward ionic currents during PTZ-induced prolonged depolarizations were investigated using voltage clamp techniques. PTZ causes a 5–35% reduction in g_L and a 40–60% reduction in steady-state g_K. There is also a marked reduction in the activation of g_A of Connor and Stevens⁶ at all clamp potentials; a shortening of the time constant for the inactivation of g_A; and a 10–15 mV shift in the depolarizing direction of the curve relating the steady-state inactivation of g_A to membrane potential. The equillibrium potentials for both g_A and g_K are depolarized by 20 mV in PTZ solution. Equation and voltage clamp data for normal repetitive firing were integrated with the normal and PTZ-altered data. Solution to these equations demonstrated: (1) normal repetitive firing in response to a constant current stimulus; and (2) PTZ-altered repetitive firing that was in the direction of, and for the most part, similar to the observed behavior.     

Endogenous voltage-gated potassium channels in human embryonic kidney (HEK293) cells

Endogenous voltage-gated potassium currents were investigated in human embryonic kidney (HEK293) and Chinese hamster ovary (CHO) cells using whole-cell voltage clamp recording. Depolarizing voltage steps from −70 mV triggered an outwardly rectified current in nontransfected HEK293 cells. This current had an amplitude of 296 pA at +40 mV and a current density of 19.2 pA/pF. The outward current was eliminated by replacing internal K⁺ with Cs⁺ and suppressed by the K⁺ channel blockers tetraethylammonium and 4-aminopyridine. Raising external K⁺ attenuated the outward current and shifted the reversal potential towards positive potentials as predicted by the Nernst equation. The current had a fast activation phase but inactivated slowly. These features implicate delayed rectifier (I_K)-like channels as mediators of the observed current, which was comparable in size to I_K currents in many other cells. A small native inward rectifier current but no transient outward current I_A, the M current I_M, or Ca²⁺-dependent K⁺ currents were detected in HEK293 cells. In contrast to these findings in HEK293 cells, little or no I_K-like current was detected in CHO cells. The difference in endogenous voltage-activated currents in HEK293 and CHO cells suggest that CHO cell lines are a preferred system for exogenous K⁺ channel expression. J. Neurosci. Res. 52:612–617, 1998. © 1998 Wiley-Liss, Inc.     

Distinct Populations of Identified Glial Cells in the Developing Rat Spinal Cord Slice: Ion Channel Properties and Cell Morphology

Four types of glial cells could be distinguished in the grey matter of rat spinal cord slices at postnatal days 1-19 (P1-P19), based on their pattern of membrane currents as revealed by the whole cell patch clamp technique, and by their morphological and immunocytochemical features. The recorded cells were labelled with Lucifer Yellow, which allowed the subsequent identification of cells using cell-type-specific markers. Astrocytes were identified by positive staining for glial fibrillary acidic protein (GFAP). These were morphologically characterized by multiple, very fine and short processes and electrophysiologically by symmetrical, non-decaying K⁺ selective currents. Oligodendrocytes were identified by a typical oligodendrocyte-like morphology, lack of GFAP staining and positive labelling with a combination of O1 and O4 antibodies (markers of the oligodendrocyte lineage), and their membrane was dominated by symmetrical, passive, decaying K⁺ currents. The third population of glial cells was also characterized by positive staining for O1/O4 or only for O4 antigens, lack of GFAP staining and, in some cells, oligodendrocyte-like morphology. However, these cells could be distinguished by the presence of inwardly rectifying (K_DR), delayed outwardly rectifying (K_DR) and A-type K⁺ currents (K_A), representing the most likely glial precursor cells of the oligodendrocyte lineage. The fourth population of glial cells had small somata and a widespread network of long processes with no apparent orientation preference. In one case, processes were positively labelled with GFAP, while 30% were characterized by faint, diffuse staining. These cells expressed a complex pattern of voltage-gated channels, namely Na⁺, K_DR, K_A and K_IR channels. In contrast to neurons, the amplitude of Na⁺ currents was at least one order of magnitude smaller than the K⁺ currents, and none of these cells showed the ability to generate action potentials in the current clamp mode. Since none of these cells could be labelled by oligodendrocyte markers we assume that they were either astrocytes or glial precursor cells of the astrocyte lineage. The four cell types were found in all regions of the grey matter. When randomly accessing the glial cells, the probability of recording from the oligodendrocyte precursor cells and the glial cells with Na⁺ currents decreased during development. At P1-P3, 50% of the cells revealed the Na⁺ current, while at P13-P15 only 18% did. Concomitantly, the number of glial cells with astrocyte- and oligodendrocyte-like membrane currents increased from 19 and 12% to 41 and 35.5% respectively. We conclude that the glial cells in the spinal cord slices possess distinct morphological, immunohistochemical and physiological properties, and that the glial populations undergo changes during postnatal development.     

Bicarbonate efflux via GABA(A) receptors depolarizes membrane potential and inhibits two-pore domain potassium channels of astrocytes in rat hippocampal slices

Increasing evidence indicates the functional expression of ionotropic γ‐aminobutyric acid receptor (GABA_A‐R) in astrocytes. However, it remains controversial in regard to the intracellular Cl^? concentration ([Cl^?]_i) and the functional role of anion‐selective GABA_A‐R in astrocytes. In gramicidin perforated‐patch recordings from rat hippocampal CA1 astrocytes, GABA and GABA_A‐R‐specific agonist THIP depolarized astrocyte membrane potential (V_m), and the THIP‐induced currents reversed at the voltages between ?75.3 and ?78.3 mV, corresponding to a [Cl^?]_i of 3.1–3.9 mM that favored a passive distribution of Cl^? anions across astrocyte membrane. Further analysis showed that GABA_A‐R‐induced V_m depolarization was ascribed to HCO₃^? efflux, while a passively distributed Cl^? mediated no net flux or influx of Cl^? that leads to an unchanged or hyperpolarized V_m. In addition to a rapidly activated GABA_A‐R current component, GABA and THIP also induced a delayed inward current (DIC) in 63% of astrocytes. The DIC became manifest after agonist withdrawal and enhanced in amplitude with increasing agonist application duration or concentrations. Astrocytic two‐pore domain K⁺ channels (K2Ps), especially TWIK‐1, appeared to underlie the DIC, because (1) acidic intracellular pH, as a result of HCO₃^? efflux, inhibited TWIK‐1, (2) the DIC remained in the Cs⁺ recording solutions that inhibited conventional K⁺ channels, and (3) the DIC was completely inhibited by 1 mM quinine but not by blockers for other cation/anion channels. Altogether, HCO₃^? efflux through activated GABA_A‐R depolarizes astrocyte V_m and induces a delayed inhibition of K2Ps K⁺ channels via intracellular acidification. © 2012 Wiley Periodicals, Inc.     

The voltage‐dependent conductances of rat neocortical layer I neurons

Whole cell patch-clamp techniques were used to study voltage-dependent sodium (Na⁺), calcium (Ca²⁺), and potassium (K⁺) conductances in acutely isolated neurons from cortical layer I of adult rats. Layer I cells were identified by means of γ-aminobutyric acid (GABA) immunocytochemistry. Positive stainings for the Ca²⁺-binding protein calretinin in a subset of cells, indicated the presence of Cajal–Retzius (C-R) cells. All investigated cells displayed a rather homogeneous profile of voltage-dependent membrane currents. A fast Na⁺ current activated at about −45 mV, was half-maximal steady-state inactivated at −66.6 mV, and recovery from inactivation followed a two-exponential process (τ₁ = 8.4 ms and τ₂ = 858.8 ms). Na⁺ currents declined rapidly with two voltage-dependent time constants, reaching baseline current after some tens of milliseconds. In a subset of cells (< 50%) a constant current level of < 65 pA remained at the end of a 90 ms step. A transient outward current (I_fast) activated ≈–40 mV, declined rapidly with a voltage-insensitive time constant (τ≈ 350 ms) and was relatively insensitive to tetraethylammonium (TEA, 20 mm ). I_fast was separated into two components based on their sensitivity to 4-aminopyridine (4-AP): one was blocked by low concentrations (40 μm ) and a second by high concentrations (6 mm ). After elimination of I_fast by a conditioning prepulse (50 ms to −50 mV), a slow K⁺ current (I_KV) could be studied in isolation. I_KV was only moderately affected by 4-AP (6 mm ), while TEA (20 mm ) blocked most (> 80%) of the current. I_KV activated at about −40 mV, declined monoexponentially in a voltage-dependent manner (τ≈ 850 ms at −30 mV), and revealed an incomplete steady-state inactivation. In addition to I_fast and I_KV, indications of a Ca²⁺-dependent outward current component were found. When Na⁺ currents, I_fast, and I_KV were blocked by tetrodotoxin (TTX, 1 μm ), 4-AP (6 mm ) and TEA (20 mm ) an inward current carried by Ca²⁺ was found. Ca²⁺ currents activated at depolarized potentials at about −30 mV, were completely blocked by 50 μm cadmium (Cd²⁺), were sensitive to verapamil (≈ 40% block by 10 μm ), and were not affected by nickel (50 μm ). During current clamp recordings, isolated layer I neurons displayed fast spiking behaviour with short action potentials (≈ 2 ms, measured at half maximal amplitude) of relative small amplitude (≈ 83 mV, measured from the action potential threshold).     

Arachidonic acid potently inhibits both postsynaptic-type Kv4.2 and presynaptic-type Kv1.4 IA potassium channels

Arachidonic acid (AA) is a free fatty acid membrane‐permeable second messenger that is liberated from cell membranes via receptor‐ and Ca²⁺‐dependent events. We have shown previously that extremely low [AA]_i (1 pm ) inhibits the postsynaptic voltage‐gated K⁺ current (I_A) in hippocampal neurons. This inhibition is blocked by some antioxidants. The somatodendritic I_A is mediated by Kv4.2 gene products, whereas presynaptic I_A is mediated by Kv1.4 channel subunits. To address the interaction of AA with these α‐subunits we studied the modulation of A‐currents in human embryonic kidney 293 cells transfected with either Kv1.4 or Kv4.2 rat cDNA, using whole‐cell voltage‐clamp recording. For both currents 1 pm [AA]_i inhibited the conductance by > 50%. In addition, AA shifted the voltage dependence of inactivation by ?9 (Kv1.4) and +6 mV (Kv4.2), respectively. Intracellular co‐application of Trolox C (10 μm ), an antioxidant vitamin E derivative, only slowed the effects of AA on amplitude. Notably, Trolox C shifted the voltage dependence of activation of Kv1.4‐mediated I_A by ?32 mV. Extracellular Trolox for > 15 min inhibited the AA effects on I_A amplitudes as well as the effect of intracellular Trolox on the voltage dependence of activation of Kv1.4‐mediated I_A. Extracellular Trolox further shifted the voltage dependence of activation for Kv4.2 by +33 mV. In conclusion, the inhibition of maximal amplitude of Kv4.2 channels by AA can explain the inhibition of somatodendritic I_A in hippocampal neurons, whereas the negative shift in the voltage dependence of inactivation apparently depends on other neuronal channel subunits. Both AA and Trolox potently modulate Kv1.4 and Kv4.2 channel α‐subunits, thereby presumably tuning presynaptic transmitter release and postsynaptic somatodendritic excitability in synaptic transmission and plasticity.     

Differential Kv1.3, KCa3.1, and Kir2.1 expression in “classically” and “alternatively” activated microglia

Microglia are highly plastic cells that can assume different phenotypes in response to microenvironmental signals. Lipopolysaccharide (LPS) and interferon‐γ (IFN‐γ) promote differentiation into classically activated M1‐like microglia, which produce high levels of pro‐inflammatory cytokines and nitric oxide and are thought to contribute to neurological damage in ischemic stroke and Alzheimer's disease. IL‐4 in contrast induces a phenotype associated with anti‐inflammatory effects and tissue repair. We here investigated whether these microglia subsets vary in their K⁺ channel expression by differentiating neonatal mouse microglia into M(LPS) and M(IL‐4) microglia and studying their K⁺ channel expression by whole‐cell patch‐clamp, quantitative PCR and immunohistochemistry. We identified three major types of K⁺ channels based on their biophysical and pharmacological fingerprints: a use‐dependent, outwardly rectifying current sensitive to the K_V1.3 blockers PAP‐1 and ShK‐186, an inwardly rectifying Ba²⁺‐sensitive K_ir2.1 current, and a Ca²⁺‐activated, TRAM‐34‐sensitive K_Ca3.1 current. Both K_V1.3 and K_Ca3.1 blockers inhibited pro‐inflammatory cytokine production and iNOS and COX2 expression demonstrating that K_V1.3 and K_Ca3.1 play important roles in microglia activation. Following differentiation with LPS or a combination of LPS and IFN‐γ microglia exhibited high K_V1.3 current densities (～50 pA/pF at 40 mV) and virtually no K_Ca3.1 and K_ir currents, while microglia differentiated with IL‐4 exhibited large K_ir2.1 currents (～ 10 pA/pF at ?120 mV). K_Ca3.1 currents were generally low but moderately increased following stimulation with IFN‐γ or ATP (～10 pS/pF). This differential K⁺ channel expression pattern suggests that K_V1.3 and K_Ca3.1 inhibitors could be used to inhibit detrimental neuroinflammatory microglia functions. GLIA 2016;65:106–121     

Effects of Acetazolamide on Transient K+ Currents and Action Potentials in Nodose Ganglion Neurons of Adult Rats

The aim of the present study was to determine whether acetazolamide (AZ) contributes to the inhibition of the fast inactivating transient K⁺ current (I_A) in adult rat nodose ganglion (NG) neurons. We have previously shown that pretreatment with either AZ or 4‐AP attenuated or blocked the CO₂‐induced inhibition of slowly adapting pulmonary stretch receptors in in vivo experiments. The patch‐clamp experiments were performed by using the isolated NG neurons. In addition to this, the RT‐PCR of mRNA and the expression of voltage‐gated K⁺ (Kv) 1.4, Kv 4.1, Kv 4.2, and Kv 4.3 channel proteins from nodose ganglia were examined. We used NG neurons sensitive to the 1 mM AZ application. The application of 1 mM AZ inhibited the I_A by approximately 27% and the additional application of 4‐AP (1 mM) further inhibited I_A by 48%. The application of 0.1 μM α‐dendrotoxin (α‐DTX), a slow inactivating transient K⁺ current (I_D) blocker, inhibited the baseline I_A by approximately 27%, and the additional application of 1 mM AZ further decreased the I_A by 51%. In current clamp experiments, AZ application (1 mM) increased the number of action potentials due to the decreased duration of the depolarizing phase of action potentials and/or due to a reduction in the resting membrane potential. Four voltage‐gated K⁺ channel proteins were present, and most (80–90%) of the four Kv channels immunoreactive neurons showed the co‐expression of carbonic anhydrase‐II (CA‐II) immunoreactivity. These results indicate that the application of AZ causes the reduction in I_A via the inhibition of four voltage‐gated K⁺ channel (Kv) proteins without affecting I_D.     

Dopamine neuron membrane physiology: Characterization of the transient outward current (IA) and demonstration of a common signal transduction pathway for IA and IK

Dopamine neurons derived from the mesencephalon of embryonic rats were maintained in primary culture, identified and studied with whole-cell patch recording techniques. These neurons demonstrated a rapidly activating and inactivating voltage-dependent outward current which required the presence of K⁺ ions. This current was termed I_A because of its transient nature. It was elicited by step depolarizations from holding potentials more negative than -50 mV and exhibited steady-state inactivation at a membrane potential more positive than -40 mV and half-maximal inactivation observed at -65 mV. This current rapidly achieved peak activation in less than 8 msec and decayed with a time constant (τ) of 58±5 msec. This current was observed in the presence of tetraethylammonium but was readily blocked by 4-aminopyridine (2-4 mM). This current was also observed to be modulated by stimulation of D₂ dopamine receptors (DA autoreceptors) located on the dopamine neurons. Thus, both DA and the D₂ receptor agonist quinpirole enhanced the peak I_A observed, while the partial D₁ receptor agonist SKF 38393 was without effect. The enhancement of I_A was confirmed to be due to the activation of D2 receptors as the effects of either DA or quinpirole were blocked by the D₂ receptor antagonists eticlopride and sulpiride, but not by the D₁ receptor antagonist SCH 23390. Since we have previously demonstrated that the I_K present: in these cells is also enhanced by D₂ receptor stimulation, we investigated the signal transduction pathways involved in coupling DA autoreceptors to both I_A and I_K. The response of both these potassium currents to DA autoreceptor stimulation was completely abolished by the preincubation of cultures with pertussis toxin, indicating the possible involvement of the G proteins G_i and G_O. In an attempt to further characterize which G protein may be involved, additional experiments were performed. The ability of DA autoreceptor stimulation to augment both currents was also blocked completely when G protein activation was prevented by the intracellular application of GDPßS (100 μM). In contrast, irreversible activation of G proteins by intracellular application of the nonhydrolyzable GTP analog GTPγS (100 μM) mimicked the effects of DA autoreceptor stimulation on both I_A and I_K. In addition, the intracellular application of a polyclonal antibody that was selective for the β-subunit of G_O completely abolished the DA autoreceptor modulation of both currents while preimmune serum was without effect. Taken together, these data demonstrate that the enhancement of I_A and I_K in response to stimulation of DA autoreceptors is dependent upon the activation of G_O and appears to involve a G_Oα subunit. © 1994 Wiley-Liss, Inc.     

Properties of the Hyperpolarization-activated Cation Current lh in Rat Midbrain Dopaminergic Neurons

Intracellular electrophysiological recordings in current- and voltage-clamp mode were obtained from dopaminergic neurons of the rat mesencephalon in an in vitro slice preparation. In current-clamp mode, a time-dependent anomalous rectification (TDR) of the membrane was observed in response to hyperpolarizing current pulses. In single-electrode voltage-clamp mode, a slowly developing inward current (l_h) underlying the TDR was studied by hyperpolarizing voltage commands from a holding potential of -50 to -60 mV. l_h started to be activated at -69 mV, was fully activated at -129 to -141 mV, with half-maximal activation at -87 mV, and showed no inactivation with time. The time course of development of Ih followed a single exponential, and its time constant was voltage-dependent. At -81 mV, l_h activated with a time constant of 379 2 47.6 ms, whereas at -129 mV l_h activated with a time constant of 65 ? 2.2 ms. Its estimated reversal potential was -35 ± 4 mV. Raising the extracellular concentration of K⁺ from 2.5 to 6.5 and to 12.5 mM increased the amplitude of l_h while reducing the extracellular concentration of Na⁺ from 153.2 to 27.2 mM caused a reduction in amplitude of l_h. Bath application of caesium (1–5 mM) reversibly reduced or blocked the TDR/l_h. Perfusion of tetrodotoxin (0.5–1 μM), tetraethylammonium (10–20 mM) or barium (0.3–2 mM) did not significantly affect l_h. l_h was also present in cells impaled with CsCI-filled electrodes. When l_h was substantially reduced by extracellular caesium (1 mM) the firing rate of the dopaminergic cells, which consisted of a spontaneous pacemaker discharge of action potentials, was not clearly changed. In addition, the holding current in voltage-clamp experiments at -50 to -60 mV was not affected by 1 mM caesium. We conclude that although the l_h current is a typical feature of the dopaminergic neurons, it is neither a significant factor underlying the spontaneous pacemaker activity nor does it contribute substantially to the setting of the normal resting potential level of the membrane. On the other hand, since l_h starts at voltages lower than or equal to -69 mV (below firing threshold), it may play a modulatory role in the cell's excitability by limiting the amplitude and duration of any prolonged hyperpolarizing events in the dopaminergic cells.     

Electrophysiological and morphological properties of light and dark cells isolated from mudpuppy taste buds

Isolated Necturus taste receptor cells were studied by giga-seal whole-cell recording and electron microscopy to correlate electrophysiological properties with taste cell structural features. Dark (type I) cells were identified by the presence of dense granular packets in the supranuclear and apical regions of the cytoplasm. In response to a series of depolarizing voltage commands from a holding potential of ?80 mV, these cells exhibited a transient, TTX-sensitive inward Na⁺ current, a sustained outward K⁺ current, and a slowly inactivating inward Ca⁺⁺ current. Light (type II) cells were identified by a lack of granular packets and by an abundance of smooth endoplasmic reticulum distributed throughout the cell. In addition, isolated light cells had clear vesicular inclusions in the cytoplasm and blebs on the plasma membrane. Light cells were divided into two functional populations based upon electrophysiological criteria: cells with inward and outward currents, and cells with outward currents only. Light cells with inward and outward currents had voltage-activated Na⁺, K⁺, and Ca⁺⁺ currents with properties similar to those of dark cells. In contrast, the second group of light cells had only voltageactivated outward K⁺ currents in response to depolarizing voltage commands. These data suggest that dark cells and light cells with inward and outward currents are capable of generating action potentials and releasing neurotransmitters onto gustatory afferent neurons in response to taste stimulation. In contrast, light cells with outward currents only likely serve a different function in the taste bud. © 1994 Wiley-Liss, Inc.     

Mechanism of the medium‐duration afterhyperpolarization in rat serotonergic neurons

Most serotonergic neurons display a prominent medium‐duration afterhyperpolarization (mAHP), which is mediated by small‐conductance Ca²⁺‐activated K⁺ (SK) channels. Recent ex vivo and in vivo experiments have suggested that SK channel blockade increases the firing rate and/or bursting in these neurons. The purpose of this study was therefore to characterize the source of Ca²⁺ which activates the mAHP channels in serotonergic neurons. In voltage‐clamp experiments, an outward current was recorded at ?60 mV after a depolarizing pulse to +100 mV. A supramaximal concentration of the SK channel blockers apamin or (‐)‐bicuculline methiodide blocked this outward current. This current was also sensitive to the broad Ca²⁺ channel blocker Co²⁺ and was partially blocked by both ω‐conotoxin and mibefradil, which are blockers of N‐type and T‐type Ca²⁺ channels, respectively. Neither blockers of other voltage‐gated Ca²⁺ channels nor DBHQ, an inhibitor of Ca²⁺‐induced Ca²⁺ release, had any effect on the SK current. In current‐clamp experiments, mAHPs following action potentials were only blocked by ω‐conotoxin and were unaffected by mibefradil. This was observed in slices from both juvenile and adult rats. Finally, when these neurons were induced to fire in an in vivo‐like pacemaker rate, only ω‐conotoxin was able to increase their firing rate (by ~30%), an effect identical to the one previously reported for apamin. Our results demonstrate that N‐type Ca²⁺ channels are the only source of Ca²⁺ which activates the SK channels underlying the mAHP. T‐type Ca²⁺ channels may also activate SK channels under different circumstances.     

Neurokinins inhibit low threshold inactivating K currents in capsaicin responsive DRG neurons

Neurokinins (NK) released from terminals of dorsal root ganglion (DRG) neurons may control firing of these neurons by an autofeedback mechanism. In this study we used patch clamp recording techniques to determine if NKs alter excitability of rat L4-S3 DRG neurons by modulating K⁺ currents. In capsaicin (CAPS)-responsive phasic neurons substance P (SP) lowered action potential (AP) threshold and increased the number of APs elicited by depolarizing current pulses. SP and a selective NK₂ agonist, [βAla⁸]-neurokinin A (4–10) also inhibited low threshold inactivating K⁺ currents isolated by blocking non-inactivating currents with a combination of high TEA, (−) verapamil and nifedipine. Currents recorded under these conditions were heteropodatoxin-sensitive (Kv4 blocker) and α-dendrotoxin-insensitive (Kv1.1 and Kv1.2 blocker). SP and NKA elicited a > 10 mV positive shift of the voltage dependence of activation of the low threshold currents. This effect was absent in CAPS-unresponsive neurons. The effect of SP or NKA on K⁺ currents in CAPS-responsive phasic neurons was fully reversed by an NK₂ receptor antagonist (MEN10376) but only partially reversed by a PKC inhibitor (bisindolylmaleimide). An NK₁ selective agonist ([Sar⁹, Met¹¹]-substance P) or direct activation of PKC with phorbol 12,13-dibutyrate, did not change firing in CAPS-responsive neurons, but did inhibit various types of K⁺ currents that activated over a wide range of voltages. These data suggest that the excitability of CAPS-responsive phasic afferent neurons is increased by activation of NK₂ receptors and that this is due in part to inhibition and a positive voltage shift in the activation of heteropodatoxin-sensitive Kv4 channels.     

Activation of outward current by pituitary adenylate cyclase activating polypeptide in mouse microglial cells

In order to investigate the interaction between the nervous and immune systems, we have analyzed the effect of one of the neuropeptides, pituitary adenylate cyclase activating polypeptide (PACAP), on microglia cells by the patch-clamp method. Puff application of PACAP38 onto mouse microglial cells induced an outward current in a dose-dependent manner. Reversal potentials of the outward current were dependent on external K⁺ concentrations ([K⁺]_o) and independent of [Cl⁻]_o. Ion channel blockers of potassium currents, quinine (1 mM), tetraethylammonium (TEA, 20 mM) and 4-aminopyridine (4-AP, 5 mM), suppressed the outward current with a potency order of quinine>TEA>4-AP. PACAP27 also induced outward current less effectively than PACAP38. A fragment of PACAP38 [PACAP(6-38)], known as an inhibitor for PACAP38, suppressed the outward current. These data suggest that PACAP38 activates a quinine-sensitive K⁺ outward current and modulates activities in microglia. They indicate that the immune system in the brain can be modulated by neurotransmitters, the mediators of neurons. J. Neurosci. Res. 51:382–390, 1998. © 1998 Wiley-Liss, Inc.     

Ionic channel currents in cultured neurons from human cortex

Ionic channels in human cortical neurons have not been studied extensively. HCN-1 and HCN-1A cells, which recently were established as continuous cultures from human cortical tissue, have been shown by histochemical and immunochemical methods to exhibit a neuronal phenotype, but expression of functional ionic channels was not demonstrated. For the present study, HCN-1 and HCN-1A cells were cultured in Dulbecco's modified Eagle's medium with 15% fetal calf serum, in some cases supplemented with 10 ng/ml nerve growth factor, 10 μM forskolin, and 1 mM dibutyryl cyclic adenosine monophosphate to promote differentiation. Cells or membrane patches were voltage clamped using conventional patch clamp techniques. In HCN-1A cells, we identified a tetrodotoxin-sensitive Na⁺ current, two types of Ca²⁺ channel current, including L-type current and a second type that in some respects resembled N-type current, and four types of K⁺ current, including a delayed outward rectifier that showed voltage-dependent inactivation, two types of noninactivating Ca²⁺-activated K⁺ channels with slope conductances of 146 and 23 pS (K⁺ _iK⁺ _o 145 mM/5 mM), and less frequently, a noninactivating, intermediate conductance channel that was not sensitive to internal Ca²⁺. When HCN-1A cells were examined after 3 days of exposure to differentiating agents, pronounced morphological changes were evident but no differences in ionic currents were apparent. HCN-1 cells also exhibited K⁺ and Ca²⁺ channel currents, but Na⁺ currents were not detected in these cells. Our data provide additional evidence indicating a functional neuronal phenotype for HCN-1A cells, and represent the most comprehensive survey to date of the variety of ionic channels expressed by human cortical neurons. © 1993 Wiley-Liss, Inc.