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.     

A-type K+ current dominates somatic excitability of delayed firing neurons in rat substantia gelatinosa

Substantia gelatinosa neurons display three main types of intrinsic firing behavior: tonic, adapting, and delayed onset. Here, voltage‐gated currents expressed by delayed firing neurons were studied in nucleated patches obtained in spinal cord slices of 3–5 weeks‐old rats. Inward Na⁺ current was negligible under these conditions and was usually occluded by superposition of much larger outward currents. Two kinds of outward currents were found, an A‐type (K_A) and delayed rectifier (K_DR) potassium currents. K_A activated rapidly (<1.5 ms at >?20 mV) and operated at subthreshold membrane potentials; voltages of steady‐state half‐maximal activation and inactivation were ?38.7 and ?87.2 mV, respectively. Inactivation was biexponential with a dominant fast component (～90%, time constant ～8 ms). K_DR activated more slowly (<8 ms at >?20 mV), half‐maximal activation was ?23.6 mV, and decayed mono‐exponentially with a time constant 70–110 ms. Maximal amplitudes of K_A were almost 10‐times larger than those of K_DR, their respective densities were 8.5 and 0.97 μS μm^?2. Tetraethylammonium, 5 mM, blocked K_DR but not K_A, whereas both currents were depressed by 5 mM 4‐aminopyridine. In current‐clamp recordings, 4‐action potential but not tetraethylammonium abolished firing delay suggesting the causative role of K_A. Thus, the predominance of fast K_A over other somatic currents is a distinctive feature of delayed firing neurons among all other types of substantia gelatinosa neurons and likely explains the appearance of their typical firing delay. Synapse, 2011. © 2010 Wiley‐Liss, Inc.     

A TASK3 channel (KCNK9) mutation in a genetic model of absence epilepsy

Childhood absence epilepsy is an idiopathic, generalized, nonconvulsive epilepsy with a multifactorial genetic etiology. The KCNK9 gene coding for the TASK3 (Twik-like acid-sensitive K相似文献   

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.     

Sensitivity of thalamic GABAergic currents to clonazepam does not differ between control and genetic absence epilepsy rats

Mutations in GABA-A receptor subunits have been reported in a number of idiopathic generalized epilepsies including childhood absence epilepsy. One of these mutations is located within a high-affinity benzodiazepine-binding domain, and clonazepam is clinically used as an anti-absence drug. The intrathalamic loop consisting of the GABAergic neurons of the nucleus reticularis thalami (NRT) and the thalamocortical (TC) neurons of sensory thalamic nuclei plays an essential role in spike and wave discharges. In a well-established genetic model of absence epilepsy (Genetic Absence Epilepsy rat from Strasbourg, GAERS), systemic injections of benzodiazepines have been shown to suppress spike-and-waves discharges. The aim of this study, therefore, was to determine whether the sensitivity of GABAergic synaptic currents to clonazepam in NRT and TC neurons was different in GAERS and non-epileptic control (NEC) rats. In both pre-seizure GAERS and NEC clonazepam (100 nM) had no effect on the mIPSCs recorded from TC neurons while it increased the decay time constant of the mIPSCs recorded in NRT neurons by a similar amount in GAERS (54.5+/-5%) and NEC (50.7+/-5%). Similar results have been obtained in the presence of 100 microM Cd2+, showing that the effect of clonazepam did not occur via modulation of voltage-activated Ca2+ currents. These results are relevant to understand that in GAERS, the clonazepam anti-absence actions cannot be fully explained by the enhancement of the intra-NRT inhibition and the modulation of the GABAergic synaptic currents in other brain areas, in particular the cortex, must be taken into consideration.     

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.     

Effect of charybdotoxin and leiurotoxon I on potassium currents in bullfrog sympathetic ganglion and hippocampal neurons

The effects of charybdotoxin and leiurotoxin I were examined on several classes of K⁺ currents in bullfrog sympathetic ganglion and hippocampal CA1 pyramidal neurons. Highly purified preparations of charybdotoxin selectively blocked a large voltage- and Ca²⁺-dependent K⁺ current (I_c) responsible for action potential repolarization (IC₅₀ = 6 nM) while leiurotoxin I selectively blocked a small Ca²⁺-dependent K⁺ conductance (I_AHP) responsible for the slow afterhyperpolarization following an action potential (IC₅₀ = 7.5 nM) in bullfrog sympathetic ganglion neurons. Neither of the toxins had a significant effects on other K⁺ currents (M-current [I_M], A-current [I_A] and the delayed rectifier [I_KD] present in these cells. Leiurotoxin I at a concentration of 20 nM had no detectable effect on currents in hippocampal CA1 pyramidal neurons. This lack of effect on I_AHP in central neurons suggests that the channels underlying slow AHPs in those neurons are pharmacologically distinct from analogous channels in peripheral neurons.     

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.     

Nucleus-specific modulation of phasic and tonic inhibition by endogenous neurosteroidogenesis in the murine thalamus

Neurosteroids are potent allosteric modulators of GABA_A receptors (GABA_ARs). Although the effects of exogenous neurosteroids on GABA_AR function are well documented, less is known about effects of neurosteroids produced by local endogenous biosynthesis. The neurosteroidogenic enzymes 5α-reductase and 3α-hydroxysteroid dehydrogenase are expressed in two nuclei of somatosensory thalamus, the thalamic reticular nucleus (nRT) and ventrobasal nucleus (VB). Here, the effects of acute blockade of neurosteroidogenesis by the 5α-reductase inhibitor finasteride on phasic and tonic GABA_AR-mediated currents were examined in nRT and VB of mice. In nRT, finasteride altered the decay and amplitude, but not the frequency, of phasic currents, with no effect on tonic inhibition. In VB neurons, by contrast, finasteride reduced both the size and frequency of phasic currents, and also reduced the degree of tonic inhibition. These studies thus provide novel evidence for endogenous modulation of GABA_AR function by 5α-reduced neurosteroids in the mature thalamus.     

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.     

Differential expression of voltage-gated K+ and Ca2+ currents in bipolar cells in the zebrafish retinal slice

Whole-cell voltage-gated currents were recorded from bipolar cells in the zebrafish retinal slice. Two physiological populations of bipolar cells were identified. In the first, depolarizing voltage steps elicited a rapidly activating A-current that reached peak amplitude ≤ 5 ms of step onset. I_A was antagonized by external tetraethylammonium or 4-aminopyridine, and by intracellular caesium. The second population expressed a delayed rectifying potassium current (I_K) that reached peak amplitude ≥ 10 ms after step onset and did not inactivate. I_K was antagonized by internal caesium and external tetraethylammonium. Bipolar cells expressing I_K also expressed a time-dependent h-current at membrane potentials < – 50 mV. I_h was sensitive to external caesium and barium, and was also reduced by Na⁺-free Ringer. In both groups, a calcium current (I_Ca) and a calcium-dependent potassium current (I_K(Ca)) were identified. Depolarizing voltage steps > – 50 mV activated I_Ca, which reached peak amplitude between – 20 and – 10 mV. I_Ca was eliminated in Ca⁺²-free Ringer and blocked by cadmium and cobalt, but not tetrodotoxin. In most cells, I_Ca was transient, activating rapidly at – 50 mV. This current was antagonized by nickel. The remaining bipolar cells expressed a nifedipine-sensitive sustained current that activated between – 40 and – 30 mV, with both slower kinetics and smaller amplitude than transient I_Ca. I_K(Ca) was elicited by membrane depolarizations > – 20 mV. Bipolar cells in the zebrafish retinal slice preparation express an array of voltage-gated currents which contribute to non-linear I–V characteristics. The zebrafish retinal slice preparation is well-suited to patch clamp analyses of membrane mechanisms and provides a suitable model for studying genetic defects in visual system development.     

Calcium spikes and calcium currents in neurons from the medial preoptic nucleus of rat

Ca²⁺ spikes, their contribution to firing patterns, and the underlying Ca²⁺ currents in neurons of the medial preoptic nucleus of rat were investigated by tight-seal whole-cell recordings in a slice preparation. Two different types of spikes were recorded: Low-threshold spikes were generated from membrane potentials <−75 mV. High-threshold spikes were recorded when K⁺ currents were reduced, and were readily evoked from membrane potentials near −40 mV. Both types of spikes were blocked by substitution of Co²⁺ for Ca²⁺ in the external medium, but were insensitive to 2.0 μM TTX. Under voltage-clamp conditions, two main types of Ca²⁺ currents were characterized: low-threshold currents that activated at membrane potentials >−60 mV, and high-threshold currents that activated at potentials >−30 mV. The low-threshold current and the low-threshold spike were more sensitive to block by external Ni²⁺ than to block by Cd²⁺, whereas the high-threshold current and the high-threshold spike were more sensitive to block by external Cd²⁺ than to block by Ni²⁺. Significant fractions of the high-threshold currents were blocked by 10 μM nifedipine, 1.0 μM ω-conotoxin GVIA, 50 nM ω-agatoxin IVA and 1.0 μM ω-conotoxin MVIIC, suggesting the presence of L-, N-, P- and Q-type Ca²⁺ channels. There were also a high-threshold current component insensitive to the above mentioned toxins. It is proposed that the low-threshold current serves as a trigger for short bursts of fast spikes from hyperpolarized levels, whereas the high-threshold current is involved in the Cd²⁺-sensitive burst firing seen in relatively depolarized neurons.     

Comparing GABAergic cell populations in the thalamic reticular nucleus of normal and genetic absence epilepsy rats from Strasbourg (GAERS)

The GABAergic neurons of the thalamic reticular nucleus (TRN) play a critical role in the generation and control of spike-and-wave discharges (SWDs) in absence epilepsy. We have used the disector method to count the GABA+ve and GABA?ve neurons in the intermediate TRN sector of genetic absence epilepsy rats from Strasbourg (GAERS) and of Wistar rats during postnatal (P) development at P10, P20, P30, and P60 days. The same part of TRN was removed from each animal, the GABAergic neurons were labelled using light-microscopical GABA immunohistochemistry and the data were statistically analysed. Both the GAERS and Wistar animals showed an increase in the density of GABA+ve and GABA?ve cells from P10 to P20. From P20 to P60, Wistar animals showed no significant differences for either cell type, but in the GAERS a progressive decrease from P20 to P60 was observed in both GABA+ve and GABA?ve cells. The decrease of the GABA?ve cells was more pronounced than that of the GABA+ve cells. There were no significant differences between cell sizes for GAERS and Wistar rats at any developmental age. The lower density GABA+ve and GABA?ve neurons at P30 and P60 of GAERS compared to Wistar animals may contribute to the generation of SWDs in absence epilepsy.     

HERG- and IRK-like Inward Rectifier Currents are Sequentially Expressed During Neuronal Development of Neural Crest Cells and their Derivatives

Quail neural crest cells were cultured in a differentiative medium to study the inward K⁺ channel profile in neuronal precursors at various stages of maturation. Between 12 and 24 h of culture, neural crest-derived neurons displayed, in addition to the previously described outward depolarization-activated K⁺ currents, an inward current enhanced in high K⁺ medium. A biophysical and pharmacological analysis led us to conclude that this inward K⁺ current is identical to that previously demonstrated in mouse and human neuroblastoma cell lines (I_IR). This current (quail I_IR or ql_lR), which is active at membrane potentials positive to -35 mV, was blocked by Cs⁺ and by class Ill antiarrhythmic drugs, thus resembling the K⁺ current encoded by the human ether-a-go-go-related gene (HERG). At later stages of incubation (>48 h), neural crest-derived neurons underwent morphological and biochemical differentiation and expressed fast Na⁺ currents. At this stage the cells lost qll_R, displaying instead a classical inward rectifier K⁺ (IRK) current (quail I_IRK= qI_IRK). This substitution was reflected in the resting potential (V_REST), which became hyperpolarized by >20 mV compared with the 24 h cells. Neurons were also harvested from peripheral ganglia and other derivatives originating from the migration of neural crest cells, viz. ciliary ganglia, dorsal root ganglia, adrenal medulla and sympathetic chain ganglia. After brief culture following harvesting from young embryos, ganglionic neurons always expressed qi_lR. On the other hand, when ganglia were explanted from older embryos (7–12 days), briefly cultured neurons displayed the IRK-like current. Again, in all the above derivatives the ql_lR substitution by ql_lRK was accompanied by a 20 mV hyperpolarization of V_REST. Together, these data indicate that the V_REST of normal neuronal precursors is sequentially regulated by HERG- and IRK-like currents, suggesting that HERG-like channels mark an immature and transient stage of neuronal differentiation, probably the same stage frozen in neuroblastomas by neoplastic transformation.     

Comparison of numbers of interneurons in three thalamic nuclei of normal and epileptic rats

The inhibitory sources in the thalamic nuclei are local interneurons and neurons of the thalamic reticular nucleus. Studies of models of absence epilepsy have shown that the seizures are associated with an excess of inhibitory neurotransmission in the thalamus. In the present study, we used light-microscopic gamma-aminobutyric acid (GABA) immunocytochemistry to quantify the interneurons in the lateral geniculate (LGN), ventral posteromedial (VPM), and ventral posterolateral (VPL) thalamic nuclei, and compared the values from normal Wistar rats and genetic absence epilepsy rats from Strasbourg (GAERS). We found that in both Wistar rats and GAERS, the proportion of interneurons was significantly higher in the LGN than in the VPM and VPL. In the LGN of Wistar rats, 16.4% of the neurons were interneurons and in the GAERS, the value was 15.1%. In the VPM, the proportion of interneurons was 4.2% in Wistar and 14.9% in GAERS; in the VPL the values were 3.7% for Wistar and 11.1% for the GAERS. There was no significant difference between Wistar rats and the GAERS regarding the counts of interneurons in the LGN, whereas the VPM and VPL showed significantly higher counts in GAERS. Comparison of the mean areas of both relay cells and interneuronal profiles showed no significant differences between Wistar rats and GAERS. These findings show that in the VPL and the VPM there are relatively more GABAergic interneurons in GAERS than in Wistar rats. This may represent a compensatory response of the thalamocortical circuitry to the absence seizures or may be related to the production of absence seizures.     

Low-voltage-activated calcium channel subunit expression in a genetic model of absence epilepsy in the rat

The Genetic Absence Epilepsy Rats from Strasbourg (GAERS) are an inbred strain of rats that display many of the characteristics of human absence epilepsy. In these rats, reciprocal thalamocortical projections play a critical role in the generation of spike-and-wave discharges that characterize absence seizures. When compared to those of the non-epileptic control strain, juvenile animals of the GAERS strain reportedly possess higher-amplitude T-type calcium currents in neurons of the thalamic reticular nucleus (nRt). We hypothesized that differences in calcium currents seen between GAERS and controls result from differences in expression of genes for low-voltage-activated calcium channels. Quantitative in situ hybridization was used to compare expression of alpha1G, alpha1H, alpha1I, and alpha1E calcium channel subunit mRNAs from adult and juvenile animals of the two strains. We found higher levels of alpha1H mRNA expression in nRt neurons of juvenile animals (34.9+/-2. 3 vs. 28.4+/-1.8 grains/10(3) pixels, p<0.05), perhaps accounting in part for earlier reports of elevated T-type current amplitude in those cells. In adult GAERS animals, we found elevated levels of alpha1G mRNA in neurons of the ventral posterior thalamic relay nuclei (64.8+/-3.5 vs. 53.5+/-1.7 grains/10(3) pixels, p<0.05), as well as higher levels of alpha1H mRNA in nRt neurons (32.6+/-0.8 vs. 28.2+/-1.6 grains/10(3) pixels, p<0.05). These results suggest that the epileptic phenotype apparent in adult GAERS may result in part from these significant, albeit small ( approximately 15-25%), elevations in T-type calcium channel mRNA levels.     

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).     

Experimental status epilepticus alters γ-aminobutyric acid type A receptor function in CA1 pyramidal neurons

There is a reduction of β-aminobutyric acid (GABA)-mediated inhibition in the CA1 pyramidal region of the hippocampus during status epilepticus (SE). The cellular basis of this loss of GABA-mediated inhibition is not known. This study tested the possibility that GABA type A (GABA_A) receptor function in CA1 pyramidal neurons was reduced or blocked during SE, at least in part by postsynaptic cellular mechanisms. GABA_A receptor currents (I_GABA) were studied by whole-cell patch-clamp techniques in CA1 pyramidal neurons acutely dissociated from rats undergoing lithium/pilocarpine-induced limbic status epilepticus (SE neurons) and from naive rats (naive neurons). SE neurons had more depolarized resting membrane potential (?17.3 mV) compared with naive neurons (?56 mV). I_GABA was absent in 47% of SE neurons and reduced in 55% of the remainder, compared with naive neurons. The reduction in I_GABA in SE neurons resulted from a combination of factors, including reduced potency and reduced efficacy of GABA in activating chloride channels, and diminished driving force for the GABA-induced chloride currents once activated. These postsynaptic cellular mechanisms resulted in a net reduction or loss in GABA-mediated inhibition and may explain previous in vivo findings reporting a loss of inhibition in hippocampus during limbic SE.     

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.