Possible modulatory role of voltage-activated Ca currents determining the membrane properties of isolated pyramidal neurones of the rat dorsal cochlear nucleus

Voltage-activated Ca²⁺ currents have been studied in pyramidal cells isolated enzymatically from the dorsal cochlear nuclei of 6–11-day-old Wistar rats, using whole-cell voltage-clamp. From hyperpolarized membrane potentials, the neurones exhibited a T-type Ca²⁺ current on depolarizations positive to −90 mV (the maximum occurred at about −40 mV). The magnitude of the T-current varied considerably from cell to cell (−56 to −852 pA) while its steady-state inactivation was consistent (E₅₀=−88.2±1.7 mV, s=−6.0±0.4 mV). The maximum of high-voltage activated (HVA) Ca²⁺ currents was observed at about −15 mV. At a membrane potential of −10 mV the L-type Ca²⁺ channel blocker nifedipine (10 μM) inhibited approximately 60% of the HVA current, the N-type channel inhibitor ω-Conotoxin GVIA (2 μM) reduced the current by 25% while the P/Q-type channel blocker ω-Agatoxin IVA (200 nM) blocked a further 10%. The presence of the N- and P/Q-type Ca²⁺ channels was confirmed by immunochemical methods. The metabotropic glutamate receptor agonist (±)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (200 μM) depressed the HVA current in every cell studied (a block of approximately 7% on an average). The GABA_B receptor agonist baclofen (100 μM) reversibly inhibited 25% of the HVA current. Simultaneous application of ω-Conotoxin GVIA and baclofen suggested that this inhibition could be attributed to the nearly complete blockade of the N-type channels. Possible physiological functions of the voltage-activated Ca²⁺ currents reported in this work are discussed.     

Calcium currents in acutely isolated stellate and pyramidal neurons of rat entorhinal cortex

Calcium currents were studied in morphologically identified pyramidal and stellate neurons acutely isolated from layer II/III of rat entorhinal cortex, using the whole-cell patch-clamp configuration. The peak amplitude of high-voltage activated current (HVA) measured at +10 mV was not different in both neuron populations with 0.94±0.08 nA for pyramidal and 1.03±0.08 nA for stellate cells. Stellate neurons had a larger capacitance (14.4±1.1 pF) than pyramidal neurons (9.6±0.8 pF), indicating a 50% larger cell surface. Most striking was the difference between the current density in stellate (79±8 pA/pF) versus pyramidal neurons (113±13 pA/pF). The potential of half maximal inactivation was not different: −37±2 mV (pyramidals) and −37±3 mV (stellates). Half of the cells contained a low-voltage activated calcium current (LVA) with a peak amplitude that was twice as large in stellate as in pyramidal neurons (0.21±0.04 nA resp. 0.11±0.03 nA; at −50 mV). In contrast to the HVA component, the current density of the LVA component was not different between cell types (13±3 pA/pF vs. 13±2 pA/pF). This implies that the relative abundance of LVA and HVA currents in stellate and pyramidal neurons is different which could result in different firing characteristics. The potential of half maximal LVA inactivation was −88±4 mV (pyramidals) and −85±3 mV (stellates). The slope of the voltage dependent steady state inactivation was steeper in stellate (7±1 mV) than in pyramidal cells (10±2 mV).     

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.     

Hyposmotic activation hyperpolarizes outer hair cells of guinea pig cochlea

The electrophysiological responses of isolated guinea pig outer hair cells (OHCs) to hyposmotic activation were studied using the whole-cell patch-clamp technique. The cell swelling by hyposmotic activation hyperpolarized OHCs by 6.6 ± 2.3 mV from the resting membrane potential of −58.5 ± 5.9 mV (n = 48). This hyperpolarization was associated with an outward current ( 97.7 ± 22.2, pA, n = 15). The hyperpolarization was inhibited by 300 μM quinine, 5 mN Ba²⁺ and increasing the extracellular K⁺ to 30 mM from 5 mM. In the absence of extracellular Ca²⁺ (1 mM EGTA), the hyperpolarization during hyposmotic activation was also abolished while the following depolarization was preserved. 50 μM GdCl₃, which is known to block strecch-activated non-specific cation channels, inhibited the hyperpolarization reversibly. 50 μM GdCl₃ also inhibited [Ca²⁺]_i increase during hyposmotic activation as shown by the calcium-sensitive dye fura-2. Simultaneously, the [Ca²⁺]_i increase and the hyperpolarization during hyposmotic activation could be observed using the combined method of whole-cell patch clamp and fura-2 technique. It is concluded that the cell swelling by hyposmotic activation may activate the stretch-activated non-specific cation channels in the OHCs which allow a Ca²⁺ influx. In turn, this [Ca²⁺]_i increase leads to an activation of the Ca²⁺-activated K⁺ channels at the basolateral membrane of OHCs which results finally in a reversible hyperpolarization of OHCs by K⁺ efflux.     

Differential effects of the pyrethroid tetramethrin on tetrodotoxin-sensitive and tetrodotoxin-resistant single sodium channels

The differential effects of the pyrethroid tetramethrin on tetrodotoxin-sensitive (TTX-S) and tetrodotoxin-resistant (TTX-R) single sodium channel currents in rat dorsal root ganglion (DRG) neurons were investigated using the outside-out configuration of patch-clamp technique. Channel conductances were 10.7 and 6.3 pS for TTX-S and TTX-R sodium channels, respectively, at a room temperature of 24–26°C. The single-channel current of TTX-S sodium channels at the test potential of −30 mV was −1.27 ± 0.25 pA, and was not changed after exposure to 10 μM tetramethrin (−1.28 ± 0.23 pA). The open time histogram of TTX-S single-channel currents could be fitted by a single exponential function with a time constant of 1.27 ms. After exposure to 10 μM tetramethrin, the open time histogram could be fitted by the sum of two exponential functions with time constants of 1.36 ms (τ_fast) and 5.73 ms (τ_low). The percentage of contribution of each component to the population was 62% for the fast component representing the normal channels and 38% for the slow component representing the tetramethrin modified channels. The amplitudc of TTX-R single-channel currents was slightly changed from −0.72 ± 0.14 to −0.83 ± 0.07 pA by 10 μM tetramethrin. The open time histogram of TTX-R single-channel currents could be fitted by a single exponential function with a time constant of 1.92 ms. In the presence of 10 μM tetramethrin, the open time histogram could be fitted by the sum of two exponential functions with time constants of 2.07 ms (τ_fast) and 9.75 ms (τ_slow). The percentage of contribution of each component was 15% for the fast, unmodified component and 85% for the slow, modified component. Differential effects of tetramethrin on the open time distribution of single sodium channel currents explains the differential sensitivity of TTX-S and TTX-R sodium channels.     

Time course and temperature dependence of allethrin modulation of sodium channels in rat dorsal root ganglion cells

Key effects of the pyrethroid insecticide allethrin, delivered to or washed out from cells at 10 or 100 μM in 0.1% DMSO, on neuronal Na⁺ channel currents were studied in rat dorsal root ganglion (DRG) cells under whole-cell patch clamp. Tetrodotoxin-resistant (TTX-R) Na⁺ channels were more responsive to allethrin than tetrodotoxin-sensitive (TTX-S) Na⁺ channels. On application of 10 or 100 μM allethrin to cells with TTX-R Na⁺ channels, the Na⁺ tail current during repolarization developed a large slowly decaying component within 10 min. This slow tail developed multiphasically, suggesting that allethrin gains access to Na⁺ channels by a multiorder process. On washout (with 0.1% DMSO present), the slow tail current disappeared monophasically (exponential τ=188±44 s). Development and washout rates did not depend systematically on temperature (12°, 18°, or 27°C), but washout was slowed severely if DMSO was absent. As the duration of a depolarizing pulse was increased (range 0.32–10 ms), the amplitude of the slow component of the succeeding tail conductance first increased then decreased. Tail current amplitude had the same dependence on preceding pulse duration (at 18°) at 10 or 100 μM, consistent with allethrin modification of Na⁺ channels at rest before opening. At 10 μM, slow tail conductance was at maximum 40% of the peak conductance during the previous depolarization, independent of temperature; evidently, the fraction of open modified channels did not change. However, at low temperature, the tail is more prolonged, bringing more Na⁺ ions into a cell. In functioning neurons, this Na⁺ influx would cause a larger depolarizing afterpotential, a condition favoring the repetitive discharges, which are signatory of pyrethroid intoxication.     

Prostaglandin E2 excites neurons of the nucleus tractus solitarius by activating cation channels

Nucleus tractus solitarius (NTS) has a high density of prostaglandin E₂ (PGE₂)-binding sites. Action of PGE₂ (10⁻⁹–10⁻⁶ M) was tested on neurons in a NTS slice with patch-clamp recording under synaptic blockade. PGE₂ raised the firing rate in approximately half of the neurons in cell-attached patch mode. In whole-cell current clamp, PGE₂ depolarized membrane potential accompanied by an increase in firing rate. In whole-cell voltage clamp (−58 mV), PGE₂ induced the inward current with an increase in conductance. The current was linearly related to voltage from −100 mV to −10 mV and suppressed between −10 mV and 20 mV. The current-voltage curve remained similar under low external Cl⁻ or high internal Cl⁻ conditions and after external Na⁺ was replaced by Cs⁺. It is concluded that PGE₂ excites NTS neurons by activating cation conductance.     

Differentiation of Ionic Currents in CNS Progenitor Cells: Dependence upon Substrate Attachment and Epidermal Growth Factor

Multipotential progenitor cells grown from central nervous system (CNS) tissues in defined media supplemented with epidermal growth factor (EGF), when attached to a suitable substratum, differentiate to express neural and glial histochemical markers and morphologies. To assess the functional characteristics of such cells, expression of voltage-gated Na⁺and K⁺currents (I_Na, I_K) was studied by whole-cell patch clamp methods in progenitors raised from postnatal rat forebrain. Undifferentiated cells were acutely dissociated from proliferative “spheres,” and differentiated cells were studied 1–25 days after plating spheres onto polylysine/laminin-treated coverslips.I_NaandI_Kwere detected together in 58%,I_Naalone in 11%, andI_Kalone in 19% of differentiated cells recorded with K⁺-containing pipettes. With internal Cs⁺(to isolateI_Na),I_Naup to 45 pA/pF was observed in some cells within 1 day after plating.I_Naranged up to 150 pA/pF subsequently. Overall, 84% of cells expressedI_Na, with an average of 38 pA/pF.I_Nahad fast kinetics, as in neurons, but steady-state inactivation curves were strongly negative, resembling those of glialI_Na. Inward tail currents sensitive to [K⁺]_outwere observed upon repolarization after the 10-ms test pulse with internal Cs⁺, indicating the expression of K⁺channels in 82% of cells. In contrast to the substantial currents observed in differentiating cells, little or noI_NaorI_K-tail currents were detected in recordings from cells acutely dissociated from spheres. Thus, in the presence of EGF, ionic currents develop early during differentiation induced by attachment to an appropriate substratum. Cells switched from EGF to basic fibroblast growth factor (bFGF) when plated onto coverslips showed greatly reduced proliferation and developed less neuron-like morphologies than cells plated in the presence of EGF.I_Nawas observed in only 53% of bFGF-treated cells, with an average of 9 pA/pF. Thus, in contrast to reports that bFGF promotes neuronal differentiation in some CNS progenitor populations, our EGF-generated postnatal rat CNS progenitors do not develop neuronal characteristics when switched to medium containing bFGF. Thus, differentiated CNS progenitors can express a mix of neuronal and glial molecular, morphological, and electrophysiological properties that can be modified by culture conditions.     

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.     

Different voltage-gated sodium currents are expressed by human neuroblastoma NB69 cells when cultured in defined serum-free and in astroglial-conditioned media

Voltage-gated Na⁺ currents (I_Na) were analysed with the whole-cell patch-clamp technique in human neuroblastoma NB69 cells plated in serum-free “defined” medium (DM) or in “astroglial-conditioned” medium (CM). Cells survived in both media and expressed the microtubule associated protein 1A, indicating neuron-like differentiation. Two I_Na types with different time-, voltage-dependent properties and tetrodotoxin (TTX) sensitivities were expressed in DM and CM. The I_Na in DM-plated cells was present from day 4 and its surface density increased from 11 pA/pF (days 5–7) to 68 pA/pF (days 15–30). The underlying conductance (G_Na) half-activated (V_0A) at −24 mV. I_Na inactivation was fitted by single exponentials with 7.5 ms time constant (t_h) at the −35 mV half-inactivation voltage (V_0I). I_Na was not affected by 10 nM, was reduced (65%) by 100 nM, and not completely abolished (92%) by 300 nM tetrodotoxin (TTX). The I_Na of CM-plated cells appeared at day 3–4 and its surface density increased from 14 pA/pF (days 3–6) to 28 pA/pF (days 11–14). The G_Na V_0A was −29 mV and inactivation was fitted by single exponentials with 2.6 ms t_h at the −58 mV V_0I. This I_Na was reduced (55%) by 10 nM and totally abolished by 100 nM tetrodotoxin (TTX). In conclusion, NB69 cells displayed a slow, “TTX-resistant,” or a fast, “TTX-sensitive” I_Na in DM and CM, respectively, suggesting that the CM contained diffusible trophic factors of astroglial origin that induced the expression of a different Na⁺ channel type. About half of the CM- and DM-plated cells also displayed a persistent Na⁺ current (I_NaP). © 1997 Wiley-Liss Inc.     

Sodium and chloride-dependent high and low-affinity uptakes of GABA by brain capillary endothelial cells

The mechanisms of carrier-mediated transport of γ-aminobutyric acid (GABA) at the blood–brain barrier (BBB) were examined by investigating [ ]GABA uptake by isolated bovine brain capillaries, monolayers of primary cultured brain capillary endothelial cells (BCECs) attached to plates or suspended BCECs. The uptake of [ ]GABA was concentration-dependent and saturable. Nonlinear regression analysis of the original data indicated the existence of two distinct high and low-affinity GABA transporters on isolated brain capillaries or suspended BCECs, with K_m1, K_m2, V_m1 and V_m2 equal to 25.3 μM, 485.2 μM, 3.6 and 8.4 nmol/5 min/mg protein, respectively, for the capillaries, and 21.3 μM, 322.0 μM, 6.1 and 15.7 nmol/5 min/mg protein, respectively, for the suspended BCECs. In contrast, a single low-affinity transporter was found for monolayers of BCECs attached to plates with K_m and V_m equal to 338.7 μM and 18.8 nmol/5 min/mg protein, respectively. Subcellular location of the two distinct transporters on BCECs is discussed, suggesting that the low-affinity GABA transporter is probably localized to the luminal membrane of BCECs, and the high-affinity GABA transporter is probably localized to the antiluminal membrane. Low temperature (4°C) and metabolic inhibitors markedly diminished both high and low-affinity uptakes of [ ]GABA by isolated brain capillaries. The substitution of Na⁺ with choline⁺, K⁺ or Li⁺ with the counter anion Cl⁻ almost completely abolished both uptakes. Substitution of Cl⁻ with Br⁻, I⁻, F⁻ or NO₃⁻ in the presence of Na⁺ significantly reduced both uptakes to different extents. Alanine, leucine, phenylalanine, arginine, glutamate and pyruvate had no obvious effect on either uptake. Probenecid, amino-oxyacetic acid, β-alanine, taurine, betaine, and nipecotic acid significantly reduced both uptakes. These data suggested that both the GABA transporters at the BBB were temperature, metabolic energy, Na⁺ and Cl⁻-dependent, and may be specific and different from the known monocarboxylic acid, GABA and other amino acid transporters, which may play a role in the disposition of GABA in the brain.     

Ammonia-induced depolarization of cultured rat cortical astrocytes

Exposure of cultured rat cortical astrocytes to increased concentrations of ammonia has been shown to induce morphological and biochemical changes similar to those found in hyperammonemic (e.g., hepatic) encephalopathy in vivo. Alterations of electrophysiological properties are not well investigated. In this study, we examined the effect of ammonia on the astrocyte membrane potential by means of perforated patch recordings. Exposure to millimolar concentrations of NH₄Cl induced a slow dose-dependent and reversible depolarization. At steady state, i.e., after several tens of minutes, the cells were significantly depolarized from a resting membrane potential of −96.2±0.6 mV (n=83, S.E.M.) to −89.1±1.6 mV (n=7, S.E.M.) at 5 mM NH₄Cl, −66.3±3.6 mV (n=9, S.E.M.) at 10 mM NH₄Cl and −50.4±2.5 mV (n=12, S.E.M.) at 20 mM NH₄Cl, respectively. In order to examine the underlying depolarizing mechanisms we determined changes in the fractional ion conductances for potassium, chloride and sodium induced by 20 mM NH₄Cl. No significant changes were found in the fractional sodium or chloride conductances, but the dominating fractional potassium conductance decreased slightly from a calculated 0.86±0.04 to 0.77±0.04 (n=9, S.E.M.). Correspondingly, we found a significant fractional ammonium ion (NH₄⁺) conductance of 0.23±0.02 (n=10, S.E.M.) which was blocked by the potassium channel blocker barium and, hence, most likely mediated by barium-sensitive potassium channels. Our data suggest that the sustained depolarization induced by NH₄Cl depended on changes in intracellular ion concentrations rather than changes in ion conductances. Driven by the high membrane potential NH₄⁺ accumulated intracellularly via a barium-sensitive potassium conductance. The concomitant decrease in the intracellular potassium concentration was primarily responsible for the observed slow depolarization.     

Multiple actions of the novel anticonvulsant drug topiramate in the rat subiculum in vitro

We used an in vitro slice preparation to study whether and how the anticonvulsant drug topiramate (TPM, 50–500 μM) modulates the excitability of rat subicular neurons that generate action potential bursts mainly caused by voltage-dependent, Na⁺-electrogenesis. Subiculum is a gating structure for outputs originating from the hippocampus proper, and thus it may play a role in limbic seizures. In 28/45 neurons, TPM induced a steady hyperpolarization of the resting membrane potential (RMP) that ranged between −2 and −16 mV and was associated with a 24–62% decrease of the apparent membrane input resistance. TPM also depressed the ability of these cells to generate action potential bursts in response to brief (5–150 ms) depolarizing pulses; such an effect was characterized by an increase in the amount of intracellular depolarizing current required for eliciting action potential bursts, and it also occurred when the TPM-induced steady hyperpolarization was compensated by injecting steady depolarizing current. In addition TPM reduced by approx. 50% the regular action potential firing elicited by prolonged (350–1000 ms) depolarizing pulses (n=15 of 27 neurons). Recovery of the TPM-induced changes was not seen during washout for periods of 20–80 min (n=7). Both the steady hyperpolarization of the RMP and the input resistance decrease elicited by TPM were markedly reduced by the GABA_A receptor antagonists bicuculline methiodide (10 μM; n=6) or picrotoxin (100 μM; n=2); such an effect was associated with a reduction, but not with blockade of the depressant action exerted by TPM on burst generation. Our findings indicate that TPM reduces subicular cell excitability, and modifies bursting ability and repetitive firing properties. These effects may be ascribed to actions on voltage-gated, Na⁺ electrogenesis and GABA_A receptors. We propose that these changes in excitability may all contribute to the anticonvulsant action of TPM in limbic seizures that occur in temporal lobe epilepsy patients.     

Impairment of electrophysiological function of astrocytes by cerebrospinal fluid from a patient with Waldenström's macroglobulinemia

Patients with the Bing Neel type of Waldenström's macroglobulinemia often present with global neurological symptoms. In this case report, we investigated the effects of cerebrospinal fluid (CSF) of a such a patient (CSF-WM), who presented with seizures and psychomotor slowing, on the electrophysiological properties of cultured rat neurons and astrocytes. Membrane potential and Na⁺ and K⁺ currents of neurons were unaffected. Astrocytes, however, were significantly depolarized from − 77.6 ± 8.2 mV to − 48.0 ± 7.6 mV (38%) by CSF-WM. The depolarization was markedly reduced after CSF-WM heat inactivation or after pre-incubation of astrocytes with dexamethasone (1 μM). Astrocytes are electrophysiologically active cells, which control local ionic micro-environment. Therefore, we conclude that global neurological symptoms in the Bing Neel type of Waldenström's macroglobulinemia like generalized seizures can result from an impairment of glial cells electrophysiological functions.     

Zinc modulation of GABAA receptor-mediated chloride flux in rat hippocampal slices

We studied the effect of ZnCl₂ application on GABA_A receptor-mediated ³⁶Cl⁻ flux in microsacs prepared from whole rat hippocampus and in region-specific hippocampal slices. Slices were obtained from the dentate gyrus (DG), which contains the zinc-enriched hilar region, and from the CA1 region which contains lower levels of endogenous zinc. Muscimol (10 μM)-evoked ³⁶Cl⁻ flux was significantly reduced by ZnCl₂ (100 μM) in hippocampal microsacs. In hippocampal slices, muscimol (50 μM)-evoked ³⁶Cl⁻ efflux was higher in CA1 (112.5 ± 27.9% above basal efflux rate) than in DG slices (29.7 ± 5.6%). In the presence of ZnCl₂, the muscimol effect on efflux rate in CA1 and DG regions was decreased to 10.6 ± 5.4% and 6.9 ± 4.9%, respectively. Preincubation with the zinc chelator, tetrakis(2-pyridylmethyl)ethylenediamine (TPEN, 20 μM), caused a significant increase in muscimol-evoked ³⁶Cl⁻ efflux only in DG slices (57.2 ± 7.0%), suggesting that GABA_A receptors in the DG of rat hippocampus under physiological conditions may function under the inhibitory influence of endogenous chelatable zinc. In intracellular recordings, ZnCl₂ (100 μM) application had no effect on the responses to GABA applied perisomatically or in the dendritic region of CA1 neurons. The lack of Zn²⁺ effect on the postsynaptic GABA_A receptor-mediated responses suggests that the decreases of the ³⁶Cl⁻ efflux observed in the biochemical assays may be due to zinc action on neurons other than the principal pyramidal CA1 cells, and possibly the non-neuronal cell populations.     

Deleterious brain cell membrane effects after NMDA receptor antagonist administration to newborn piglets

Previous studies have shown that administration of the N-methyl- -aspartate (NMDA) receptor antagonist 3-(2-carboxypiperazin-4-yl)-1-phosphonic acid (CPP) reduces NMDA-mediated neurotoxicity in animal models of hypoxia/ischemia but also may induce brain tissue vacuolization and alter glucose metabolism. The present study tests the hypothesis that CPP administration alters brain cell membrane structure and function in the cerebral cortex of normoxic newborn piglets through the generation of oxygen free radicals and induction of lipid peroxidation. Twenty six anesthetized, ventilated newborn piglets—13 treated with 2 mg/kg i.v. CPP and 13 untreated controls—were studied. ATP and phosphocreatine (PCr) levels were measured as an index of cellular energy metabolism and tissue glucose levels determined. Na⁺,K⁺-ATPase activity was measured as an index of brain cell membrane function and the lipid peroxidation products conjugated dienes (CD) and fluorescent compounds (FC) measured. Free radical generation was detected on cortical biopsies homogenized with α-phenyl-N-tert-butyl-nitrone (PBN) through electron spin resonance spectroscopy. Signal height of spectrum was divided by dry tissue weight and expressed as mm/g tissue. In the two groups brain tissue ATP and PCr levels were not different. Tissue glucose levels were higher in the CPP group (24±5 mg/dl) than in controls (14±3 mg/dl), p<0.05, whereas Na⁺,K⁺-ATPase activity was lower in the CPP group than in controls (34±4 vs. 43±6 μmol Pi/mg protein/h), p<0.05. Lipid peroxidation products were higher in the CPP group (CD: 57±19 nmol/g brain, FC: 1.5±0.3 μg/g brain) than in controls (CD: 0±0 nmol/g brain, FC: 0.9±0.2 μg/g brain), p<0.05. Free radical intensity was higher in the CPP group (493±397 mm/g tissue) than in controls (51±83 mm/g tissue), p<0.05. In vitro administration of CPP to brain cell membranes did not change Na⁺,K⁺-ATPase activity or the generation of lipid peroxidation products. The data demonstrate that administration of CPP induces lipid peroxidation, results in free radical generation, decreases brain cell membrane Na⁺,K⁺-ATPase activity and alters glucose metabolism in the cerebral cortex of newborn piglets. Since CPP is a potent antagonist of the NMDA receptor, we speculate that CPP generates free radicals through a pathway independent of the NMDA receptor by altering cellular metabolism and possibly glucose utilization during normoxia in newborn piglets.     

Temperature and amiloride alter taste nerve responses to Na, K, and NH4 salts in rats

The effects of adaptation/stimulus temperature (25°C vs. 35°C) on taste nerve responses to salt stimulation and amiloride suppression were assessed in rats. We measured the integrated responses of the chorda tympani nerve to 500 mM concentrations of NaCl, Na₂SO₄, sodium acetate (NaAc), KCl, K₂SO₄, potassium acetate (KAc), NH₄Cl, (NH₄)₂SO₄, and ammonium acetate (NH₄Ac) mixed with or without 100 μM amiloride hydrochloride at 25°C and 35°C. Taste nerve responses to all Na⁺ and NH₄⁺ salts, but not K⁺ salts, were significantly smaller at 25°C than at 35°C. Amiloride significantly suppressed taste nerve responses to all salts (Na⁺ salts>K⁺ salts>NH₄⁺ salts); amiloride suppression of Na⁺ and NH₄⁺ salts was significantly greater at 25°C than at 35°C. Benzamil-HCl, a more potent Na⁺ channel blocker compared to amiloride, strongly suppressed taste nerve responses to NaCl and KCl, but not to NH₄Cl. Amiloride and benzamil suppression of NaCl responses were similar; however, amiloride suppressed KCl responses more than did benzamil. The results suggest that: (1) amiloride-sensitive Na⁺ channels are involved to varying degrees in the transduction of sodium and potassium salt taste, and (2) amiloride may inhibit membrane proteins other than passive Na⁺ channels during stimulation with potassium and ammonium salts.     

Inhibition of major cationic inward currents prevents spreading depression-like hypoxic depolarization in rat hippocampal tissue slices

Hypoxia-induced spreading depression-like depolarization (hypoxic SD, or anoxic depolarization) is accompanied by the near-loss of membrane potential, severe reduction of membrane resistance, and influx of Na⁺, Ca²⁺, Cl⁻ and water into neurons. The biophysical nature of these membrane changes is incompletely understood. In the present study we applied a pharmacological mixture (10 μM DNQX, 10 μM CPP, 1 μM TTX, and 2 mM Ni²⁺) to rat hippocampal tissue slices to inhibit major Na⁺ and Ca²⁺ inward currents. This inhibitory cocktail slightly depolarized CA1 pyramidal neurons and completely blocked all evoked potentials. In its presence severe hypoxia of up to 20 min duration failed to induce hypoxic SD and the accompanying intrinsic optical signal. Instead, only moderate, very slow negative shifts of the extracellular DC potential were observed. Following 10 min hypoxia and 1 hour wash-out of the inhibitors antidromic and orthodromic responses were still blocked but hypoxic SD with markedly delayed onset could be induced in most slices. In current-clamped CA1 pyramidal cells hypoxia induced a rapid, near-complete depolarization and decreased the input resistance by 89%. In the presence of the cocktail, however, hypoxia caused a gradual, partial depolarization, to about −40 mV; the membrane resistance decreased by only 37%. We conclude that simultaneous blockade of the known major Na⁺ and Ca²⁺ channels consistently prevents hypoxic SD. The hypothesis that SD initiation is the consequence of general loss of selective permeability or general membrane breakdown becomes unlikely. Instead, influx of Na⁺ and Ca²⁺ might play a crucial role in the generation of the rapid SD-like depolarization.     

Interactions of dextromethorphan with theN-methyl-d-aspartate receptor-channel complex: single channel recordings

The actions of dextromethorphan (DXM) on the 50 pS conductance state of theN-methyl-d-aspartate (NMDA) receptor-operated channel were studied using outside-out patches obtained from cultured rat hippocampal pyramidal neurons. DXM (5–50 μM) had no effect on the amplitudes of unitary currents but caused concentration-dependent reductions in channel mean open times and the frequency of channel openings. Channel open probability was reduced in a concentration-dependent manner by DXM and was one-half of the control value at a DXM concentration of 6 μM, with the patch potential held at −60 mV. An IC₅₀ value of 4 μM was obtained for the reduction by DXM of NMDA-evoked rises in [Ca²⁺]_i in cultured rat hippocampal pyramidal neurons loaded with Fura-2. The results were consistent with drug block of the open NMDA channel with an onward (blocking) rate constant of 7.7 × 10⁶ M⁻¹ · s⁻¹ (at −60 mV). The estimated unblocking rate constant was about 10 s⁻¹, a value considerably higher compared to the off-rate constant found for dizocilpine block of the NMDA channel.