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.     

p21ras Oncogene Protein Selectively Increases Low-voltage-activated Ca2+ Current Density in Embryonic Chick Dorsal Root Ganglion Neurons

p21^ras protein resembles the α subunit of trimeric G-proteins, which regulate ion channel function. We now report a modulation of Ca²⁺ channels in vertebrate sensory neurons by p21^ras in addition to its role in cell growth and differentiation. Quantitative microinjection of oncogenic p21-H-ras into embryonic chick dorsal root ganglion neurons was performed. After 4 h the current density of the low-voltage-activated (LVA; T-type) Ca²⁺ channels was increased. However, in contrast to trimeric G-proteins, which inhibit high-voltage-activated (HVA) Ca²⁺ channels in chick dorsal root ganglion neurons, p21^ras did not significantly affect HVA Ca²⁺ currents. To study the time course of p21^ras action, guanosine triphosphate-preloaded p21^ras was added to the patch pipette. Full-length ras was effective only after a delay of 20 -30 min. C-terminal modification by cellular enzymes is required to activate full-length ras, and can account for the observed delay. Unexpectedly, C-terminal-truncated p2l^ras, which was found to be inactive in biological assays, enhanced LVA Ca²⁺ currents within minutes. This suggests a G-protein-like modulation of the LVA Ca²⁺ channel by p21^ras. In an early phase of neuronal differentiation, dorsal root ganglion neurons express only LVA Ca²⁺ currents. The regulatory role of p21^ras on LVA channels may therefore be particularly important during differentiation.     

Mechanisms for Synchronous Calcium Oscillations in Cultured Rat Cerebellar Neurons

Removal of Mg²⁺ caused oscillations of the cytosolic Ca²⁺ concentration ([Ca²⁺]_i) and the membrane potential in cultured cerebellar granule neurons. Oscillations of [Ca²⁺]_i were synchronous in all the cells, and were restricted to the neurons (immunocytochemically identified) that responded to exogenous N -methyl-D-aspartate (NMDA). Oscillations were blocked by Ca²⁺ removal, nickel, NMDA receptor antagonists, ω-agatoxin IVA, tetrodotoxin, sodium removal and γ-aminobutyric acid, but not by dihydropyridines, ω-conotoxin M VIIA or by emptying the intracellular Ca²⁺ stores with thapsigargin or ionomycin. The upstroke of the [Ca²⁺]_i oscillations coincided in time with an increase in manganese permeability of the plasma membrane. Propagation of the [Ca²⁺]_i wave followed more than one pathway and the spatiotemporal pattern changed with time. Membrane potential oscillations consisted of transient slow depolarizations of ˜20 mV with faster phasic activity superimposed. We propose that the synchronous [Ca²⁺]_i oscillations are the expression of irradiation of random excitation through a neuronal network requiring generation of action potentials and functional glutamatergic synapses. Oscillations of [Ca²⁺]_i are due to cyclic Ca²⁺ entry through NMDA receptor channels activated by synaptic release of glutamate, which requires Ca²⁺ entry through P-type Ca²⁺ channels activated by action potentials at the presynaptic terminal.     

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.     

Muscarinic Regulation of Ca2+ Currents in Rat Sensory Neurons: Channel and Receptor Types, Dose - response Relationships and Cross-talk Pathways

We studied, in rat sensory neurons, the modulation of high voltage-activated Ca²⁺ currents (I_Ca mediated by the pertussis toxin-sensitive activation of muscarinic receptors, which were found to be of subtypes M₂, or M₄. Muscarine reversibly blocked somatic Ca²⁺ spikes but strong predepolarizations only partially relieved the inhibited Ca²⁺ current. On the other hand, the putative coupling messenger could not rapidly diffuse towards channels whose activity was recorded from a macro-patch. The perforated patch technique virtually prevented the response rundown present during whole-cell experiments. Both ω-conotoxin GVIA (ω-CgTx)-sensitive channels and ω-CgTx- and dihydropyridine-resistant channels are coupled to the muscarinic receptor, but not the L-channel. When measured in the same neuron, dose - response relationships for the first and subsequent agonist applications differed; maximal inhibition, the reciprocal of half-maximal concentration and the Hill coefficient were always highest in the first trial. Muscarine and oxotremorine exhibited monotone dose - response curves, but oxotremorine-M showed non-linear relationships which became monotonic when cells were intracellularly perfused with inhibitors of protein kinase A (PKA) and C (PKC), suggesting that either PKA or receptor-induced PKC could phosphorylate and thus inactivate G-proteins or other unknown proteins involved in inhibitory muscarinic actions on I_Ca. In summary, these data provide a preliminary pharmacological characterization of the muscarinic inhibition of the Ca²⁺ channels in sensory neurons, with implications about agonist specificity and the interplay between signalling pathways.     

Effect of Thiol Reagents on Phosphoinositide Hydrolysis in Rat Brain Synaptoneurosomes

Some divalent ions, such as Cd²⁺ and Zn²⁺, are able to stimulate phosphoinositide (PI) breakdown and to inhibit receptor-mediated PI metabolism. These ions are also known to react with the free – SH groups of proteins. This prompted us to investigate the effects of more potent sulphhydryl reagents, Hg²⁺ and p -chloromercuric benzosulphonic acid (PCMBS), on the inositol phosphate (IP) accumulation triggered by the neuroactive substances: glutamate, carbachol and K⁺, using synaptoneurosomes from 8-day-old rat forebrains. Hg²⁺ and PCMBS, depending on their concentration, had two distinct effects on IP accumulation: at low doses, Hg²⁺ (from 1 to 10 μM) and PCMBS (0.1 mM) by themselves stimulated PI breakdown, inhibited glutamate-elicited IP accumulation and had additive effects with respect to carbachol-induced IP stimulation. At higher doses, Hg²⁺ (from 0.01 to 1 mM) inhibited both basal and neuroactive substance-stimulated IP accumulation. PCMBS (1 mM), provoked only an inhibition of the agonist-stimulated IP formation. Monitoring membrane potential and intracellular Ca²⁺ with the fluorescent dyes diSC2(5) and fura2, respectively, indicated that these mercurials could strongly depolarize the synaptoneurosomal membrane and produce a Ca²⁺ influx dependent on extracellular Ca²⁺. The stimulatory effects of low concentrations of mercurials on PI turnover could be linked to the depolarization they provoke and the subsequent Ca²⁺ rise, which in turn is known to stimulate some phospholipase C enzymes. The inhibitory effects observed at high concentrations might be due to a loss of activity of proteins involved in PI breakdown, as all receptor-mediated IP accumulations were inhibited.     

Effects of Phenytoin, Carbamazepine, and Gabapentin on Calcium Channels in Hippocampal Granule Cells from Patients with Temporal Lobe Epilepsy

Summary: Purpose: The anticonvulsants phenytoin (PHT), carbamazepine (CBZ), and gabapentin (GBP) are commonly used in the treatment of temporal lobe epilepsy. Ca²⁺ current modulation has been proposed to contribute to the antiepileptic activity of these drugs. The purpose of this study was to determine the effects of these anticonvulsants on voltage-dependent calcium channels in pathologically altered neurons from patients with chronic temporal lobe epilepsy.
Methods : Acutely isolated human hippocampal granule cells were examined by using the whole-cell configuration of the patch-clamp technique.
Results : PHT and CBZ produced a reversible, concentrationdependent inhibition of high-voltagectivated (HVA) Ca²⁺ currents without affecting voltage-dependent activation. The concentration-response curves of PHT and CBZ indicated maximal inhibition of 35 and 65%, respectively, with halfmaximal inhibition being obtained at 89 and 244 μ M , respectively. At therapeutic cerebrospinal fluid (CSF) concentrations, HVA currents were not significantly altered by PHT and CBZ. However, PHT but not CBZ showed a reduction of HVA currents of 16% at a therapeutic whole-brain concentration of 80 μ M . In ontrast to CBZ, PHT produced a small hyperpolarizing shift in the voltage dependence of steady-state inactivation. PHT, 80 μ4, shifted the potential of half-maximal inactivation by -3.1 ± 0.5 mV (p < 0.05). GBP, which was recently found to bind to the ಠsubunit of a neuronal Ca²⁺ channel, showed no modulation of Ca²⁺ conductances.
Conclusions : These results suggest that, in contrast to GBP and CBZ, modulation of postsynaptic Ca2+ channels can contribute to the anticonvulsant action of PHT in human hippocampal granule cells.     

The patterns of spontaneous Ca2+ signals generated by ventral spinal neurons in vitro show time-dependent refinement

Embryonic spinal neurons maintained in organotypic slice culture are known to mimic certain maturation-dependent signalling changes. With such a model we investigated, in embryonic mouse spinal segments, the age-dependent spatio-temporal control of intracellular Ca²⁺ signalling generated by neuronal populations in ventral circuits and its relation with electrical activity. We used Ca²⁺ imaging to monitor areas located within the ventral spinal horn at 1 and 2 weeks of in vitro growth. Primitive patterns of spontaneous neuronal Ca²⁺ transients (detected at 1 week) were typically synchronous. Remarkably, such transients originated from widespread propagating waves that became organized into large-scale rhythmic bursts. These activities were associated with the generation of synaptically mediated inward currents under whole-cell patch-clamp. Such patterns disappeared during longer culture of spinal segments: at 2 weeks in culture, only a subset of ventral neurons displayed spontaneous, asynchronous and repetitive Ca²⁺ oscillations dissociated from background synaptic activity. We observed that the emergence of oscillations was a restricted phenomenon arising together with the transformation of ventral network electrophysiological bursting into asynchronous synaptic discharges. This change was accompanied by the appearance of discrete calbindin immunoreactivity against an unchanged background of calretinin-positive cells. It is attractive to assume that periodic oscillations of Ca²⁺ confer a summative ability to these cells to shape the plasticity of local circuits through different changes (phasic or tonic) in intracellular Ca²⁺.     

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.     

Ca2+ Channel Expression in the Oligodendrocyte Lineage

The development of oligodendrocytes from their precursor cells through different developmental stages can be studied in vitro. These stages can be distinguished by specific monoclonal antibodies and by a characteristic K⁺ channel profile. In this study we demonstrate that the occurrence of Ca²⁺ currents also undergoes marked changes during the development of mouse oligodendrocytes. Immature precursor cells which can develop into astrocytes or oligodendrocytes expressed two different types of voltage-activated Ca²⁺ channels. The expression of Ca²⁺ channels in precursor cells was strongly correlated with the expression of Na⁺ channels. When cells started to express the O1 antigen and were committed to the oligodendrocyte lineage, Ca²⁺ and Na⁺ currents could no longer be detected. Large Ca²⁺ currents were, however, recorded later in the development of the oligodendrocytes, correlated with the expression of the O10 antigen. The Ca²⁺ channels were classified as high and low voltage-activated Ca²⁺ channels according to their range of activation, and are further described by their kinetic and pharmacological properties.     

Shaker Mutants Lack Post-tetanic Potentiation at Motor End-plates

The two-electrode voltage clamp technique was employed to measure end-plate currents in larval neuromuscular junctions of wild-type (Canton-S) and of three different Drosophila Shaker mutants: Shaker^KS133, Shaker¹⁰² and f⁵Shaker⁵. In the Shaker mutants, nerve-evoked end-plate currents (neepc) were 4–5-fold larger than those measured in Canton-S. Shaker motor end-plates were found to lack post-tetanic potentiation (PTP), but could undergo facilitation. Moreover, PTP but not facilitation was lost in wild-type larvae if the neuromuscular junction was exposed to 4-aminopyridine (4-AP), a blocker of Shaker A-type K⁺ currents. End-plate currents were depressed by Ca²⁺ channel blockers like Mg²⁺, at millimolar concentrations, and Co²⁺ and Cd²⁺, at micromolar concentrations, but not by nifedipine (100 nM) and verapamil (100 nM). After exposure to Ca²⁺ channel blockers, Shaker end-plates exhibited PTP. In particular, Cd²⁺ was most effective in depressing neepes and in restoring PTP in all Shaker mutants. The results obtained indicate the abnormal function of Shaker K⁺ channels at motor nerves specifically abolishes PTP in Drosophila larval neuromuscular junctions.     

HIV-1 Envelope Proteins gp120 and gp160 Potentiate NMDA-induced [Ca2+]i Increase, Alter [Ca2+]i Homeostasis and Induce Neurotoxicity in Human Embryonic Neurons

The envelope glycoprotein gp120 of the human immunodeficiency virus HIV-1 has been proposed to cause neuron death in developing murine hippocampal cultures and rat retinal ganglion cells. In the present study, cultured human embryonic cerebral and spinal neurons from 8- to 10-week-old embryos were used to study the neurotoxic effect of gp120 and gp160. Electrophysiological properties as well as N -methyl- d -aspartate (NMDA)-induced currents were recorded from neurons maintained in culture for 10–30 days. Neither voltage-activated sodium or calcium currents nor NMDA-induced currents were affected by exposure of neurons to 250 pM gp120 or gp160. In contrast, when neurons were subjected to photometric measurements using the calcium dye indo-1 to monitor the intracellular free Ca²⁺ concentration ([Ca²⁺]_i), gp120 and gp160 (20–250 pM) potentiated the large rises in [Ca²⁺]_i induced by 50 μM NMDA. The potentiation of NMDA-induced Ca²⁺ responses required the presence of Ca²⁺ in the medium, and was abolished by the NMDA antagonist d -2-amino-5-phosphonovalerate (AP5) and the voltage-gated Ca²⁺ channel inhibitor nifedipine. Moreover, exposure of a subpopulation of spinal neurons (25% of the cells tested) to 20–250 pM gp120 or gp160 resulted in an increase in [Ca²⁺]_i that followed three patterns: fluctuations not affected by AP5, a single peak, and the progressive and irreversible rise of [Ca²⁺]_i. The neurotoxicity of picomolar doses of gp120 and gp160 cultures was estimated by immuno-fluorescence and colorimetric assay. Treatment of cultures with AP5 or nifedipine reduced gp120-induced toxicity by 70 and 100% respectively.     

Is Protein Kinase C Activity Required for the N-Methyl-d-Aspartate-evoked Rise in Cytosolic Ca2+ in Mouse Striatal Neurons?

The present study investigates the roles of protein kinase C (PKC) and A (PKA) activities in NMDA-mediated Ca²⁺ entry in primary cultures of mouse striatal neurons. Inhibitors of protein kinases, such as sphingosine, RO 31 – 8220 and staurosporine inhibited the NMDA- but also the KCI-induced rise in cytosolic Ca²⁺. However, the PKA antagonist Rp-adenosine-3',5'monophosphothioate (Rp-cAMPS) did not alter the NMDA + d -serine response, whereas it completely suppressed the KCI response. The NMDA + d -serine-evoked rise in cytosolic Ca²⁺, observed in the absence of external Mg²⁺, was potentiated by the PKC activator phorbol 12-myristate 13-acetate (PMA) only when submaximal effective concentrations of this agonist and co-agonist were used. In addition, the PKC activator did not alter the NMDA + d -serine-evoked response in the presence of varying concentrations of Mg²⁺. Confirming the dependence on PKC activity, desensitization of PKC resulting from long-term PMA treatment led to an impairment of the NMDA response, leaving the KCI-induced response intact. We therefore propose that PKC not only potentiates but is also required for the NMDA-evoked elevation in cytosolic Ca²⁺ in mouse striatal neurons.     

Complete brain-type creatine kinase deficiency in mice blocks seizure activity and affects intracellular calcium kinetics

Purpose:   Brain-type creatine kinase (CK-B) and ubiquitous mitochondrial creatine kinase (UbCKmit) act as components of local phosphocreatine ATP shuttles that help in the compartmentalization and maintenance of pools of high-energy phosphate molecules in both neurons and glial cells. We investigated the role of these brain-type creatine kinases during extreme energy-demanding conditions in vivo (generalized tonic–clonic seizures) and in vitro.
Methods:   The physiologic response of wild-types and mice lacking both CK-B and UbCKmit (CK--/--mice) to pentylenetetrazole (PTZ)–induced seizures was measured using electroencephalography (EEG) recordings and behavioral monitoring. In vitro intracellular Ca²⁺ kinetics in hippocampal granule neurons were monitored upon single and repetitive depolarizations.
Results:   PTZ induced in only a few CK--/-- mice PTZ seizure-like behavior, but in all wild-types a full-blown seizure. EEG analysis showed that preseizure jerking was associated with high-amplitude discharges. Wild-type EEG recordings showed continuous runs of rhythmic 4–6 Hz activity, whereas no rhythmic EEG activities were observed in the few CK--/-- mice that developed a behavioral seizure. All other CK--/-- mice displayed a sudden postictal depression without any development of a generalized seizure. Hippocampal granule neurons of CK--/-- mice displayed a higher Ca²⁺ removal speed following repetitive KCl-induced depolarizations.
Discussion:   Deficiency for creatine kinase is affecting brain energy metabolism and will likely contribute to the disturbance of seizure development. Because CK--/-- hippocampal neurons exhibited an increase in Ca²⁺ removal rate of elevated intracellular levels, we conclude that altered Ca²⁺ clearance in CK--/-- neurons could play a role in the abnormal EEG and seizure activity.     

Pronase Acutely Modifies High Voltage-activated Calcium Currents and Cell Properties of Lymnaea Neurons

Pronase E ('pronase') is one of the proteolytic enzymes that are used in preparative procedures such as cell isolation and to soften the sheath of invertebrate ganglia. Although several effects of proteolytic enzymes on the physiology of non-neuronal tissues have been described, the effects of these enzymes on central neurons have received little attention. We examined the effects of bath-applied pronase on neurons in the Lymnaea central nervous system and in vitro . Pronase caused action potential broadening in neurons that exhibit a shoulder on the repolarization phase of their action potentials. This effect of pronase was accompanied by, although unrelated to, a depolarization and decrease in action potential interval. Some, but not all, effects of pronase in the central nervous system were reversible. For example, the decreases in membrane potential and action potential interval were both reversed after ∼1 h of washing with saline. However, the effect of pronase on the action potential duration was not reversed after a period of 90 min. The modulation of action potential width prompted us to examine Ca²⁺ currents. Exposure to pronase resulted in an increase in both peak and late high voltage-activated Ca²⁺ currents in isolated neurons. Pronase neither changed the inactivation rate nor caused a shift in the current-voltage relationship of the current. The changes in action potential duration could be prevented by application of 0.1 mM Cd²⁺, indicating that the action potential broadening caused by pronase depends on Ca²⁺ influx. This is the first systematic study of the acute and direct actions of pronase on Ca²⁺ currents and cell properties both in the CNS and in vitro .     

Calcium-Dependent Regulation of Genetically Determined Spike and Waves by the Reticular Thalamic Nucleus of Rats

Summary: The pathophysiologic role of the reticular thalamic nucleus (RT) in rat generalized nonconvulsive epilepsy was investigated in the selected strain GAERS (genetic absence epilepsy rats from Strasbourg). After the RT was lesioned by the excitotoxic agent ibotenic acid stereotaxically injected in previously callosotomized rats, a disruption of ipsilateral spike and wave discharges (SWD) was observed in freely moving animals. In a second group of animals Cd²⁺ (0.5–1.5 μl, 1 m M ), which is known to block Ca²⁺ and Ca²⁺-dependent K⁺ conductances (gK⁺ (Ca²⁺), was injected into the thalamus. Cd²⁺reversibly suppressed ipsilateral SWD when injected in RT, whereas it slightly reduced SWD expression when injected in the ventrobasal (VB) complex. The difference was highly significant. We conclude that Ca²⁺-dependent oscillatory properties of the RT are critical for expression of genetically determined SWD in GAERS.     

Depolarization amplitude and Ca2+-inflow control the time course of quantal releases at murine motor nerve terminals

In order to test whether the time courses of quantal releases after a depolarization pulse are affected by the depolarization amplitude, time courses were measured for small depolarization pulses that elicited close to threshold releases and for large depolarizations that elicited releases approaching saturation level. Diaphragms of young mice were excised and superfused with Bretag's solution at 18°C. Synaptic currents were elicited and recorded through a perfused macropatch pipette. Releases elicited by threshold depolarizations rose earlier than releases elicited by saturation depolarizations. The short delays in the rising phases of release after large depolarizations may be due to the shift of Ca²⁺ currents flowing during the pulse to tail currents. After its peak, release decayed with a time constant τ. For saturation depolarizations τ was about 0.3 ms, and for threshold depolarizations τ increased up to 0.8 ms. In order to differentiate between the effects of variations in Ca²⁺ inflow and in depolarization, the amplitudes of large depolarization pulses were held constant while the amount of release was depressed by halving the Ca²⁺ concentration at the terminal. The time course of the lowered releases was slightly delayed while τ remained at 0.3 ms as typical for saturation depolarizations. Double pulse facilitation unexpectedly revealed a short phase of depression of release after the pulse. This depression may contribute to the rapid decay (τ) of release after large depolarizations. The dependence of τ on depolarization amplitude indicates that the final phase of the time course of release is largely controlled by the amplitude of the preceding depolarization.     

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.     

Calcium Channel Currents in Undifferentiated Human Neuroblastoma (SH-SY5Y) Cells: Actions and Possible Interactions of Dihydropyridines and ω-Conotoxin

Ca²⁺ channel currents were recorded in undifferentiated human neuroblastoma (SH-SY5Y) cells with the whole-cell patch-clamp technique, using 10 mM Ba²⁺ as charge carrier. Currents were only evoked by depolarizations to -30 mV or more positive (holding potential -80 mV), inactivated partially during 200 ms depolarizing steps, and were abolished by 150 μMCd²⁺. Currents could be enhanced by Bay K-8644 and partially inhibited by nifedipine, suggesting that they arose in part due to activation of L-type Ca²⁺ channels. Currents were also inhibited by the marine snail peptide ω-conotoxin GVIA (ω-CgTx). At a concentration of 10 nM inhibition by ω-CgTx was reversible, but at higher concentrations blockade was always irreversible. Although current inhibition by nifedipine was maximal at 1μM, supramaximal concentrations reduced the inhibitory actions of ω-CgTx in a concentration-dependent manner. Ca²⁺ channel currents evoked from a holding potential of -50 mV showed no inactivation during 200 ms depolarizations but declined in amplitude with successive depolarizing steps (0.2 Hz). Current amplitudes could be restored by returning the holding potential to -80 mV. Currents evoked from -50 mV were inhibited by nifedipine and ω-CgTx to a similar degree as those evoked from -80 mV. Our results indicate that undifferentiated SH-SY5Y cells possess L- and N-type Ca²⁺ channels which can be distinguished pharmacologically but cannot be separated by using depolarized holding potentials. Furthermore, these data suggest that nifedipine has a novel action to inhibit blockade of N-type channels by ω-CgTx.     

Serotonin enhances a low-voltage-activated calcium current in rat spinal motoneurons

Calcium currents and the effects of 5-HT on these currents were investigated in visually identified motoneurons using whole-cell recording in the neonatal rat spinal cord slice preparation. In current-clamp recording, step depolarizations from a holding potential of about -90 mV produced a low-threshold transient depolarizing response and a high-threshold long-lasting spike. In voltage-clamp recording, low (LVA) and high (HVA) voltage-activated Ca2+ currents were recorded in response to depolarizing voltage steps. Low concentration of Cd2+ (50 microM) did not reduce the amplitude of the LVA current but markedly diminished the HVA current. Bath application of 5-HT (10-50 microM) markedly increased the amplitude of the LVA current without causing a shift in the current (I)-voltage (V) relation. In contrast, 5-HT did not appreciably affect the amplitude of the HVA current. We conclude that 5-HT specifically enhances the LVA Ca2+ current and that this effect together with the previously reported 5-HT-induced inward current (Takahashi and Berger, 1990), would facilitate the excitation of motoneurons.