Voltage-dependent Currents in Microvillar Receptor Cells of the Frog Vomeronasal Organ

Vomeronasal receptor cells are differentiated bipolar neurons with a long dendrite bearing numerous microvilli. Isolated cells (with a mean dendritic length of 65 μm) and cells in mucosal slices were studied using whole-cell and Nystatin-perforated patch-clamp recordings. At rest, the membrane potential was −61 ± 13 mV (mean ± SD; n = 61). Sixty-four per cent of the cells had a resting potential in the range of –60 to –86 mV, with almost no spontaneous action potential. The input resistance was in the GΩ range and overshooting repetitive action potentials were elicited by injecting depolarizing current pulses in the range of 2 – 10 pA. Voltage-dependent currents were characterized under voltage-clamp conditions. A transient fast inward current activating near –45 mV was blocked by tetrodotoxin. In isolated cells, it was half-deactivated at a membrane potential near –75 mV. An outward K⁺ current was blocked by internal Cs⁺ ions or by external tetraethylammonium or Ba²⁺ ions. A calcium-activated voltage-dependent potassium current was blocked by external Cd²⁺ ions. A voltage-dependent Ca²⁺ current was observed in an iso-osmotic BaCl₂ solution. Finally, a hyperpolarization-activated inward current was recorded. Voltage-dependent currents in these microvillar olfactory receptor neurons appear qualitatively similar to those already described in ciliated olfactory receptor cells located in the principal olfactory epithelium.     

Lamotrigine Reduces Voltage-Gated Sodium Currents in Rat Central Neurons in Culture

Summary: Purpose : To study the mechanism or mechanisms of action of lamotrigine (LTG) and, in particular, to establish its effects on the function of NA⁺ channels in mammalian central neurons.
Methods : Rat cerebellar granule cells in culture were subjected to the whole-cell mode of voltage clamping under experimental conditions designed to study voltage-gated Na⁺ currents.
Results : Extracellular application of LTG (10–500 μ M , n = 21) decreased in a dose-related manner a tetrodotoxin-sensitive inward current that was elicited by depolarizing commands (from −80 to +20mV). The peak amplitude of this Na⁺-mediated current was diminished by 38.8 ± 12.2% (mean ± SD, n = 6) during application of 100 μ M LTG, and the dose-response curve of this effect indicated an IC₅₀ 145 μM. The reduction in the inward currents produced by LTG was not associate with any signficant change in the current decay, whereas the voltage dependency of the steady-state inactivation shifted toward more negative values (midpoint of the inactivation curve: –47.5 and –59.0 mV under control conditions and during application of 100 μM LTG, respectively, n = 4).
Conclusions : Our findings indicate that LTG reduces the amplitude of voltage-gated Na⁺ inward current in rat cerebellar granule cells and induces a negative shift of the steady-state inactivation curve. Both mechanisms may be instrumental in controlling the repetitive firing of action potentials (AP) that occurs in neuronal networks during seizure activity.     

Responses to Metabotropic Glutamate Receptor Activation in Cerebellar Purkinje Cells: Induction of an Inward Current

The responses to activation of metabotropic glutamate receptors (mGluRs) of Purkinje cells in rat cerebellar slice cultures were investigated using intracellular recordings in single-electrode voltage-clamp mode combined with microfluorometric measurements of cytosolic free calcium using fura-2. Purkinje cells were perfused with saline containing 0.5 μM tetrodotoxin and 10 μM bicuculline and voltage-clamped at –60 mV. Bath-applied trans-(±)-1-amino-1,3-cyclopentanedicarboxylic acid ( t -ACPD, 50–100 μM), a selective agonist of mGluRs, induced a transient inward current that was followed by an outward current. The response induced by t -ACPD was not affected by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, up to 40 μM). In contrast, inward currents caused by (RS)-α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA, 1–2 μM) were completely abolished, while inward currents caused by quisqualate (0.25 μM) were only partially depressed by CNQX (5–40μM). The inward current induced by t -ACPD was unaffected by external Ba²⁺ (1 mM), tetraethylammonium (10 mM) and Cs⁺ (1 mM), and was associated with an increase in apparent input conductance of the cell membrane. The extrapolated reversal potential of inward currents induced by t -ACPD was +18 mV while Cl⁻ currents induced by muscimol reversed at –66 mV. Inward currents induced by t -ACPD, but not those induced by AMPA, were associated with a rise in cytosolic Ca²⁺ concentration and suppressed by intracellular injection of a calcium chelator. Replacement of external Na⁺ by choline or Li⁺ depressed the inward current and resulted in a slower decay of the Ca²⁺ signal.     

Analysis of the Ion Channel Complement of the Rat Oligodendrocyte Progenitor in a Commonly Studied In vitro Preparation

We have analysed the ion channel complement of the oligodendrocyte-type 2 astrocyte (O-2A) glial cell progenitor obtained from the commonly studied neonatal rat mixed brain preparation. Ionic currents, in O-2A progenitors identified on both morphological and immunological grounds, were recorded using the whole-cell variant of the patch-clamp technique. The cells had an average resting membrane potential close to -50 mV and fired single action potentials in response to suprathreshold current injections. Using voltage-clamp methods we were able to identify and characterize a voltage-activated TTX-sensitive Na⁺ current, two classes of voltage-activated outward K⁺ currents, an inactivating inwardly rectifying K⁺ current, a voltage-activated Cl^- current and at least three classes of Ca²⁺ current.     

Sodium Current Amplitude Increases Dramatically in Human Retinal Glial Cells during Diseases of the Eye

Müller cells, the main macroglial cells of the retina, express several types of voltage and ligand-activated ion channels, including Na^- channels. Using the whole-cell voltage-clamp technique, we studied the expression of Na^- currents in acutely isolated, non-cultivated human Muller cells from retinas of healthy organ donors and patients suffering from different eye diseases. In both types of retinas transient Na+ currents could be recorded from Muller cells. The tetrodotoxin-resistant Na⁺ currents, which were not completely blocked even at a concentration of 10 $muM tetrodotoxin, had a mean current density of 3.0± 3.0 pA/pF (mean ± SD, n = 10) in Muller cells from donor retinas and of 12.2± 2 9.6 pA/pF ( n = 74) in Muller cells from patient retinas. Only 33.3% of healthy but 88.4% of pathological Muller cells depicted such currents. The G_Na+/G_K+ ratio was very high in several Muller cells from patient retinas, such that action potential-like activity could be generated after prehyperpolarizing current injection in some of these cells. Apparently, the Na'channels, due to their negative steady-state inactivation curve (V_h= -84.5 mV), do not influence the lowered membrane potential of the pathological cells, since they are inactivated at these voltages. Currently, we do not have an explanation for the increase in amplitude and frequency of Na+ currents in human Muller cells under pathological conditions. However, the up-regulation of Na channels may mirror a basic glial response to pathological conditions, since it has also been found previously in human hippocampal astrocytes from epileptic foci and in rat cortex stab wounds lined by an astrocytic scar.     

A plateau potential mediated by the activation of extrasynaptic NMDA receptors in rat hippocampal CA1 pyramidal neurons

Hippocampal pyramidal neurons express various extrasynaptic glutamate receptors. When glutamate spillover was facilitated by blocking glutamate uptake and fast synaptic transmission was blocked by antagonists of AMPA- and NMDA-type glutamate receptors and an ionotropic GABA receptor blocker, repetitive synaptic stimulation evoked a persistent membrane depolarization that consisted of an early Ca²⁺-independent component and a late Ca²⁺-dependent component. The early component, which we refer to as a plateau potential, had a half-width of 770 ± 160 ms and a steady peak level of −9.54 ± 3.50 mV. It was accompanied by an increase in membrane conductance, the I–V relationship of which showed a peak at −19.91 ± 2.18 mV and reversal of the current at −4.32 ± 2.13 mV, and was suppressed by high concentration of an NMDA receptor (NMDAR) antagonist d -APV, or an NMDAR glycine-binding site antagonist 5,7-dCK. After blocking synaptically located NMDARs using MK801, the potential was still evoked synaptically when spillover was facilitated. A sustained depolarization was evoked by iontophoretic application of glutamate in the presence or absence of a glutamate uptake blocker. This potential was not affected by Na⁺ or Ca²⁺ channel blockers, but was suppressed by 5,7-dCK, leaving an unspecified depolarizing potential. Iontophoresis of NMDA evoked a sustained depolarization that was blocked by a high concentration of d -APV or 5,7-dCK. The I–V relationship of the current during this potential was similar to that obtained during the synaptically induced plateau potentials. These results show that CA1 pyramidal neurons generate plateau potentials mediated most likely by activation of extrasynaptic NMDARs.     

Membrane Currents Influencing Action Potential Latency in Granule Neurons of the Rat Cochlear Nucleus

Granule cells are the most numerous neurons in the cochlear nucleus, but, because of their small size, little information on their membrane properties and ionic currents is available. We used an in vitro slice preparation of the rat ventral cochlear nucleus to make whole-cell recordings from these cells. Under current clamp, some granule neurons fired spontaneous action potentials and all generated a train of action potentials on depolarization (threshold current, 10–35 pA). Hyperpolarization increased the latency to the first action potential evoked during a subsequent depolarization. We examined which voltage-gated currents might underlie this latency shift. In addition to a fast inward Na⁺ current, depolarization activated two outward potassium currents. A transient current was rapidly inactivated by membrane potentials positive to -60 mV, while a second, more slowly inactivating current was observed following the decay of the transient current. No hyperpolarization-activated conductances were observed in these cells. Modelling of the currents suggests that removal of inactivation on hyperpolarization accounts for the increased action potential latency in granule cells. Such a mechanism could account for the 'pauser'-type firing patterns of the fusiform cells which receive a prominent projection from the granule cells in the dorsal cochlear nucleus.     

Sodium and Calcium Currents in Glial Cells of the Mouse Corpus Callosum Slice

We studied Na⁺ and Ca²⁺ currents in glial cells during the development of the corpus callosum in situ. Glioblasts and oligodendrocytes from frontal brain slices of postnatal day (P) 3 to P18 mice were identified based on morphological and ultrastructural features after characterization of the currents with the patch-clamp technique. Slices from P3-P8 mice contained predominantly glioblasts with immature morphological features. These cells showed Na⁺ and Ca²⁺ currents, but the population with these currents decreased between P3 and P8. Na⁺ currents were blocked in Na⁺-free bathing solution and in the presence of tetrodotoxin, Ca²⁺ currents were only observed when a high concentration of extracellular Ba²⁺ was present. The cells from the corpus callosum of P10 – P18 mice predominantly had morphological features of oligodendrocytes. In these cells, which in some cases were shown to form myelin, neither Na⁺ nor Ca²⁺ currents were detected. To compare these in situ results with those from the electrophysiologically and immunocytochemically well-characterized cultured glial cells, we determined the expression pattern of stage-specific antigens in the corpus callosum in situ. The first O4 antigen-positive glial precursors were observed at P1, the earliest stage examined. The oligodendrocytic antigens O7 and O10 appeared at P6 and P14, respectively, and prominent labelling with the corresponding markers was seen at P12 and P18, respectively. Despite the existence of numerous mature, O10-positive oligodendrocytes at P18, which expressed Ca²⁺ channels in vitro , we failed to detect Ca²⁺ currents in situ at this stage.     

Sodium, Calcium and Late Potassium Currents are Reduced in Cerebellar Granule Cells Cultured in the Presence of a Protein Complex Conferring Resistance to Excitatory Amino Acids

Whole-cell, patch-clamp recordings were used to study voltage-gated currents generated by cerebellar granule cells that were cultured in medium containing either 10% fetal calf serum (hereafter termed S + granules) or neurite outgrowth and adhesion complex (NOAC, hereafter called NOAC granules). NOAC is a protein complex found in rabbit serum that renders granules resistant to the excitotoxic action of excitatory amino acids. During depolarizing commands both S+ and NOAC granules generated Na⁺ and Ca²⁺ inward currents and an early and a late K⁺ outward currents. However, Na⁺ and Ca²⁺ Inward currents and late outward K⁺ currents recorded in NOAC granules were smaller than those seen in S+ granules. Furthermore, although of similar amplitude, early K⁺ currents displayed different kinetics in the two types of neurons. Thus, these data demonstrate that the electrophysiological properties of cerebellar granules, and probably of other neuronal populations, depend upon serum components and raise the possibility that an analogous modulation might be operative in vivo, and play a role in development, synaptic plasticity or neuropathological processes.     

Heterogeneity in the Membrane Current Pattern of Identified Glial Cells in the Hippocampal Slice

Glial cells, acutely isolated or in tissue culture, have previously been shown to express a variety of voltage-gated channels. To resolve the question whether such channels are also expressed by glial cells in their normal cellular environment, we have applied the patch-clamp technique to study glial cells in hippocampal slices of 10–12-day-old mice. Based on the membrane current pattern, we distinguished four glial cell types. One was characterized by passive, symmetrical K⁺ currents activated in depolarizing and hyperpolarizing directions. A second population showed a similar current pattern, but with a marked decay of the current during the 50-ms voltage jumps. In a third population, the decaying passive currents were superimposed with a delayed rectifier outward current and, in some cases, with a slow inward current activated by depolarization. The fourth population expressed delayed rectifying outward currents, an inward rectifier K⁺ current and fast inward currents activated by depolarization. To unequivocally identify the glial cells we combined electrophysiological and ultrastructural characterizations. Therefore, cells were filled with the fluorescent dye lucifer yellow during characterization of their membrane currents, the fluorescence of the dye was used to convert diaminobenzidine to an electron-dense material, and subsequently slices were inspected in the electron microscope. Recordings were obtained from cells in the stratum radiatum and were identified as glial by their size, the characteristic chromatin distribution, and the lack of synaptic membrane specializations.     

Cholinergic Responses in Developing Outer Hair Cells of the Rat Cochlea

Acetylcholine-evoked currents were investigated using the conventional whole-cell patch-clamp recording technique in developing outer hair cells (OHCs). The cells were isolated from the rat cochlea at different stages of postnatal development ranging from day 4 (P4) to P30. Acetylcholine-evoked currents could be recorded at P6 and P8. At this developmental stage, the majority of OHCs displayed inward nicotinic-like currents near the resting membrane potential. These cholinergic currents zeroed near 0 mV, as expected for a non-selective cation current, and could be reversibly blocked by d-tubocurarine. At P12 and adult stage, the cholinergic response of OHCs switched to an outward current reversing near E _K and displaying a bell shape peaking between -40 and -30 mV. This change in polarity of the acetylcholine response during postnatal development might be explained by progressive functional coupling between acetylcholine ionotropic receptors permeable to Ca²⁺ and nearby Ca²⁺-activated K⁺ channels at the synaptic pole of OHCs.     

Astrocytes in the hippocampus of patients with temporal lobe epilepsy display changes in potassium conductances

Functional properties of astrocytes were investigated with the patch-clamp technique in acute hippocampal brain slices obtained from surgical specimens of patients suffering from pharmaco-resistant temporal lobe epilepsy (TLE). In patients with significant neuronal cell loss, i.e. Ammon's horn sclerosis, the glial current patterns resembled properties characteristic of immature astrocytes in the murine or rat hippocampus. Depolarizing voltage steps activated delayed rectifier and transient K+ currents as well as tetrodotoxin-sensitive Na+ currents in all astrocytes analysed in the sclerotic human tissue. Hyperpolarizing voltages elicited inward rectifier currents that inactivated at membrane potentials negative to -130 mV. Comparative recordings were performed in astrocytes from patients with lesion-associated TLE that lacked significant histopathological hippocampal alterations. These cells displayed stronger inward rectification. To obtain a quantitative measure, current densities were calculated and the ratio of inward to outward K+ conductances was determined. Both values were significantly smaller in astrocytes from the sclerotic group compared with lesion-associated TLE. During normal development of rodent brain, astroglial inward rectification gradually increases. It thus appears reasonable to suggest that astrocytes in human sclerotic tissue return to an immature current pattern. Reduced astroglial inward rectification in conjunction with seizure-induced shrinkage of the extracellular space may lead to impaired spatial K+ buffering. This will result in stronger and prolonged depolarization of glial cells and neurons in response to activity-dependent K+ release, and may thus contribute to seizure generation in this particular condition of human TLE.     

A novel role for MNTB neuron dendrites in regulating action potential amplitude and cell excitability during repetitive firing

Principal cells of the medial nucleus of the trapezoid body (MNTB) are simple round neurons that receive a large excitatory synapse (the calyx of Held) and many small inhibitory synapses on the soma. Strangely, these neurons also possess one or two short tufted dendrites, whose function is unknown. Here we assess the role of these MNTB cell dendrites using patch-clamp recordings, imaging and immunohistochemistry techniques. Using outside-out patches and immunohistochemistry, we demonstrate the presence of dendritic Na⁺ channels. Current-clamp recordings show that tetrodotoxin applied onto dendrites impairs action potential (AP) firing. Using Na⁺ imaging, we show that the dendrite may serve to maintain AP amplitudes during high-frequency firing, as Na⁺ clearance in dendritic compartments is faster than axonal compartments. Prolonged high-frequency firing can diminish Na⁺ gradients in the axon while the dendritic gradient remains closer to resting conditions; therefore, the dendrite can provide additional inward current during prolonged firing. Using electron microscopy, we demonstrate that there are small excitatory synaptic boutons on dendrites. Multi-compartment MNTB cell simulations show that, with an active dendrite, dendritic excitatory postsynaptic currents (EPSCs) elicit delayed APs compared with calyceal EPSCs. Together with high- and low-threshold voltage-gated K⁺ currents, we suggest that the function of the MNTB dendrite is to improve high-fidelity firing, and our modelling results indicate that an active dendrite could contribute to a 'dual' firing mode for MNTB cells (an instantaneous response to calyceal inputs and a delayed response to non-calyceal dendritic excitatory postsynaptic potentials).     

Activation of calcium channel currents in rat sensory neurons by large depolarizations: effect of Guanine nucleotides and (-)-baclofen

Calcium channel currents have been recorded from cultured rat sensory neurons at clamp potentials of between -30 and +120 mV. At large depolarizing potentials between +50 and +120 mV, the current was outward. This outward current was shown to be largely due to ions passing through calcium channels, because it was substantially although generally incompletely blocked by Cd2+ (1 mM) and omega-conotoxin (1 microM). Internal GTP-gamma-S (100 microM) and to a lesser extent GTP (1 mM) reduced the amplitude and slowed the activation of the outward, as well as the inward calcium channel current. Baclofen (100 microM) reversibly inhibited both the inward and outward currents. These results suggest that the effect of baclofen and G protein activation on calcium channel currents is not due to a shift in the voltage-dependence of channel availability.     

Ionic mechanism mediating Mytilus inhibitory peptides elicited membrane currents in identified Helix neurons

Effects of seven, pressure applied MIP (Mytilus inhibitory peptides) had been studied on D-neurons of the CNS of Helix pomatia in voltage-clamp experiments. In physiological saline, the peptides produced a hyperpolarization usually coupled with the cessation of any spontaneous spiking activity. Clamped at the resting potential (−60 mV), peptide applications elicited an outward current, which increased its amplitude by shifting the holding potential towards depolarisation. The response was concentration-dependent and accompanied by an increased membrane conductance. Reversal potentials obtained at different [K⁺]_o were plotted with a slope of 52 mV per ten-fold change in [K⁺]_o showing that the peptide-elicited current was mainly due to the increased K⁺-conductance(s). The peptide-induced outward current could partially be blocked by Ba²⁺ (5 mM), CdCl₂ (1 mM), TEACl (10 mM) or apamin (2.5×10⁻⁵ M) or furosemide (10 mg/ml) and decreased either in Na⁺-free or Cl⁻-free solutions. 4-Aminopyridine at 5 mM concentration completely blocked the peptide-induced current. In the presence of high [K⁺]_o, the peptide(s) was still found to induce an outward current at membrane potentials beyond K⁺-reversal potential. This component was not present in Cl⁻-free saline, suggesting that the current was due to the inward flow of Cl⁻ ions. Our results show that the MIPs have at least two (three) independent actions, each associated with different voltage-, concentration-dependence and ionic mechanisms. It is suggested, that the peptide-induced currents are carried by K⁺, and Cl⁻ ions. According to our present finding, the observed effects are mediated by the same receptor, activating different second messenger systems, inducing multiple conductance changes in the membrane of neurons of the snail ganglia.     

Inward Rectification (Ih) in Immunocytochemically-ldentified Vasopressin and Oxytocin Neurons of Guinea-Pig Supraoptic Nucleus

Intracellular recordings of magnocellular neurons from the supraoptic nucleus of guinea-pigs were made with KCI/K citrate- and biocytin-filled electrodes. Fifty of 99 cells exhibited a time-dependent inward rectification (TDR). The TDR was activated during hyperpolarizing current pulses to membrane potentials more hyperpolarized than −75 mV. In voltage-clamp recordings, an inward current appeared at voltage steps more hyperpolarized than −75 mV, with properties similar to the slow inward rectifier (I_h) described in other tissues. The I_h was blocked by 2 mM CsCI. BaCI₂ (100 to 500 μM) did not block the I_h. Immunocytochemical identification of the recorded cells revealed that both vasopressin (AVP)- and oxytocin (OT)- containing neurons exhibited an I_h.     

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.     

Gene inactivation of Na+/H+ exchanger isoform 1 attenuates apoptosis and mitochondrial damage following transient focal cerebral ischemia

We investigated mechanisms underlying the Na⁺/H⁺ exchanger isoform 1 (NHE1)-mediated neuronal damage in transient focal ischemia. Physiological parameters, body and tympanic temperatures, and regional cerebral blood flow during 30 min of middle cerebral artery occlusion were similar in wild-type NHE1 (NHE1^+/+) and NHE1 heterozygous (NHE1^+/−) mice. NHE1^+/+ mice developed infarct volume of 57.3 ± 8.8 mm³ at 24 h reperfusion (Rp), which progressed to 86.1 ± 10.0 mm³ at 72 h Rp. This delayed cell death was preceded by release of mitochondrial cytochrome c (Cyt. C), nuclear translocation of apoptosis-inducing factor (AIF), activation of caspase-3, and TUNEL-positive staining and chromatin condensation in the ipsilateral hemispheres of NHE1^+/+ brains. In contrast, NHE1^+/− mice had a significantly smaller infarct volume and improved neurological function. A similar neuroprotection was obtained with NHE1 inhibitor HOE 642. The number of apoptotic cells, release of AIF and Cyt. C or levels of active caspase-3 was significantly reduced in NHE1^+/− brains. These data imply that NHE1 activity may contribute to ischemic apoptosis. Ischemic brains did not exhibit changes of NHE1 protein expression. In contrast, up-regulation of NHE1 expression was found in NHE1^+/+ neurons after in vitro ischemia. These data suggest that NHE1 activation following cerebral ischemia contributes to mitochondrial damage and ischemic apoptosis.