Partial compensation for N-type Ca2+ channel loss by P/Q-type Ca2+ channels underlines the differential release properties supported by these channels at cerebrocortical nerve terminals

N-type and P/Q-type Ca²⁺ channels support glutamate release at central synapses. To determine whether the glutamate release mediated by these channels exhibits distinct properties, we have isolated each release component in cerebrocortical nerve terminals from wild-type mice by specifically blocking N-type Ca²⁺ channels with ω-conotoxin-GVIA and P/Q-type Ca²⁺ channels with ω-agatoxin-IVA. In addition, we have determined the release properties at terminals from mice lacking the α_1B subunit of N-type channels (Ca_v 2.2) to test the possibility that P/Q-type channels can compensate for the loss of N-type Ca²⁺ channels. We recently demonstrated that, while evoked glutamate release depends on P/Q- and N-type channels in wild-type nerve terminals, only P/Q-type channels participate in these knockout mice. Moreover, in nerve terminals expressing solely P/Q-type channels, metabotropic glutamate receptor 7 (mGluR7) fails to inhibit the evoked Ca²⁺ influx and glutamate release. Here, we show that the failure of mGluR7 to modulate evoked glutamate release is not due to a lack of receptors, as nerve terminals from mice lacking N-type Ca²⁺ channels express mGluR7. Indeed, we show that other receptor responses, such as the inhibition of forskolin-induced release, are preserved in these knockout mice. N-type channels are more loosely coupled to release than P/Q-type channels in nerve terminals from wild-type mice, as reflected by the tighter coupling of release in knockout nerve terminals. We conclude that the glutamate release supported by N- and P/Q-type channels exhibits distinct properties, and that P/Q-type channels cannot fully compensate for the loss of N-type channels.     

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.     

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.     

Depolarization promotes GAD 65-mediated GABA synthesis by a post-translational mechanism in neural stem cell-derived neurons

Neuronal activity regulates neurogenesis and neuronal differentiation in the mammalian brain. The commencement of neurotransmitter expression establishes the neuronal phenotype and enables the formation of functional connectivity between neurons. In addition, release of neurotransmitters from differentiating neurons may modulate the behaviour of neural precursors. Here, we show that neuronal activity regulates γ-aminobutyric acid (GABA) expression in neurons generated from stem cells of the striatum and adult subventricular zone (SVZ). Differentiating neurons display spontaneous Ca²⁺ events, which are voltage-gated calcium channel (VGCC) dependent. Depolarization increases both the frequency of Ca²⁺ transients and the amount of Ca²⁺ influx in differentiating neurons. We show that depolarization-dependent GABA expression is regulated by the amplitude and not by the frequency of Ca²⁺ influx. Brief activation of VGCCs leads to Ca²⁺ influx that in turn promotes a rapid expression of GABA. Depolarization-dependent GABA expression does not require changes in gene expression. Instead, it involves cAMP-dependent protein kinase (PKA) and Ca²⁺ and phospholipid-dependent protein kinase (PKC) signalling. Activity increases the number of glutamic acid decarboxylase (GAD) 65-immunoreactive neurons in a PKA-dependent manner, without altering the expression of GAD 65, suggesting that depolarization promotes recruitment of GAD 65 by a post-translational mechanism. In line with this, depolarization does not permanently increase the expression of GABA in neurons derived from neural stem cells of the embryonic striatum, cortex and adult SVZ. Thus, neuronal activity does not merely accelerate neuronal differentiation but it may alter the mechanism of GABA synthesis in newly generated neurons.     

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.     

Neuronal Ca2+ Channels and Nicotinic ACh Receptors as Functional Targets of the Nootropic Nefiracetam

Abstract: Piracetam-like nootropics (or cognitive enhancers) have been used for the treatment of various forms of dementia, including Alzheimer's disease. The underlying mechanisms of their actions, however, are largely unknown. Our recent studies have demonstrated that nefiracetam, a nootropic agent, can markedly enhance activities of neuronal L-and N-type (α_1B) Ca²⁺ channels as well as those of presynaptic nicotinic acetylcholine (ACh) receptors, thereby increasing neurotransmitter release. Aniracetam exerted a slight facilitatory effect on Ca²⁺ channels, but no effect on nicotinic ACh receptors. Piracetam and oxiracetam have no such actions on Ca²⁺ channels and nicotinic ACh receptors. It is suggested that inhibitory G-proteins (Go/Gi) and protein kinase A (PKA) mediate the nefiracetam action on Ca²⁺ channels, whereas protein kinase C (PKC) mediates the drug action on nicotinic ACh receptors. In the hippocampus of the rodent, nefiracetam induces a long-lasting (>4 h) facilitation of synaptic transmission. The 'LTP-like' facilitation appears to result from activation of presynaptic nicotinic ACh receptors (and Ca²⁺ channels as well) by nefiracetam. In conclusion, nefiracetam is distinguished from other nootropic agents for its preferential actions on both presynaptic Ca²⁺ channels and nicotinic ACh receptors, and could therefore be of great therapeutic importance to the neurotransmission failure that contributes to the symptoms of Alzheimer's disease and associated disorders.     

Skin Sympathetic Response In Subclinical Diabetic Neuropathy

Hyperglycemia and its associated Na⁺/K⁺ pump activity has been implicated in the development of diabetic neuropathy. We recently reported that high glucose in the presence of ouabain induced a progressive increase in the delayed K⁺ current which was suppressed by a blocker of Ca²⁺-activated K⁺ channels and blockers of Ca²⁺ channels in rat single myelinated nerve fibers, suggesting an increase of cytosolic free Ca²⁺ concentration ([Ca²⁺]_i). However, the influences of high glucose with ouabain on [Ca²⁺]_i in sensory neurons remain to be elucidated. The present study was undertaken to examine the modulation of depolarization-induced Ca²⁺ transients by high glucose and ouabain in isolated adult rat dorsal root ganglion (DRG) neurons using the fluorescent Ca²⁺ indicator fura-2. Bath application of KCl (50 mM) evoked a rapid increase in [Ca²⁺]_i through voltage dependent Ca²⁺ channels ([Ca²⁺]_i: 154.2 ± 22.5 nM). This increase was enhanced under high glucose (30 mM D-glucose) in the presence of ouabain (100 M) ([Ca²⁺]_i: 764.8 ± 210.1 nM). We conclude that a combination of high glucose and decreased Na⁺/K⁺ pump activity leads to an increase in [Ca²⁺]_i in rat DRG neurons, thereby resulting in nerve dysfunction.     

Tuning the excitability of midbrain dopamine neurons by modulating the Ca2+ sensitivity of SK channels

Small conductance Ca²⁺ -activated K⁺ (SK) channels play a prominent role in modulating the spontaneous activity of dopamine (DA) neurons as well as their response to synaptically-released glutamate. SK channel gating is dependent on Ca²⁺ binding to constitutively bound calmodulin, which itself is subject to endogenous and exogenous modulation. In the present study, patch-clamp recording techniques were used to examine the relationship between the apparent Ca²⁺ affinity of cloned SK3 channels expressed in cultured human embryonic kidney 293 cells and the excitability of DA neurons in slices from rat substantia nigra using the positive and negative SK channel modulators, 6,7-dichloro-1 H -indole-2,3-dione-3-oxime and R- N -(benzimidazol-2-yl)-1,2,3,4-tetrohydro-1-naphtylamine. Increasing the apparent Ca²⁺ affinity of SK channels decreased the responsiveness of DA neurons to depolarizing current pulses, enhanced spike frequency adaptation and slowed spontaneous firing, effects attributable to an increase in the amplitude and duration of an apamin-sensitive afterhyperpolarization. In contrast, decreasing the apparent Ca²⁺ affinity of SK channels enhanced DA neuronal excitability and changed the firing pattern from a pacemaker to an irregular or bursting discharge. Both the reduction in apparent Ca²⁺ affinity and the bursting associated with negative SK channel modulation were gradually surmounted by co-application of the positive SK channel modulator. These results underscore the importance of SK channels in 'tuning' the excitability of DA neurons and demonstrate that gating modulation, in a manner analogous to physiological regulation of SK channels in vivo , represents a means of altering the response of DA neurons to membrane depolarization.     

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.     

GABAergic signaling induces divergent neuronal Ca2+ responses in the suprachiasmatic nucleus network

Intercellular communication between γ-aminobutyric acid (GABA)ergic suprachiasmatic nucleus (SCN) neurons facilitates light-induced phase changes and synchronization of individual neural oscillators within the SCN network. We used ratiometric Ca²⁺ imaging techniques to record changes in the intracellular calcium concentration ([Ca²⁺]_i) to study the role of GABA in interneuronal communication and the response of the SCN neuronal network to optic nerve stimulations that mimic entraining light signals. Stimulation of the retinohypothalamic tract (RHT) evoked divergent Ca²⁺ responses in neurons that varied regionally within the SCN with a pattern that correlated with those evoked by pharmacological GABA applications. GABA_A and GABA_B receptor agonists and antagonists were used to evaluate components of the GABA-induced changes in [Ca²⁺]_i. Application of the GABA_A receptor antagonist gabazine induced changes in baseline [Ca²⁺]_i in a direction opposite to that evoked by GABA, and similarly altered the RHT stimulation-induced Ca²⁺ response. GABA application induced Ca²⁺ responses varied in time and region within the SCN network. The NKCC1 cotransporter blocker, bumetanide, and L-type calcium channel blocker, nimodipine, attenuated the GABA-induced rise of [Ca²⁺]_i. These results suggest that physiological GABA induces opposing effects on [Ca²⁺]_i based on the chloride equilibrium potential, and may play an important role in neuronal Ca²⁺ balance, synchronization and modulation of light input signaling in the SCN network.     

Effects of the HIV-1 Envelope Glycoprotein gp120 in Cerebellar Cultures. [Ca2+]i Increases in a Glial Cell Subpopulation

The various types of cells present in cultures prepared from the postnatal rat cerebellum, identified by their gross morphology and immunocytochemistry, were loaded with the specific dye fura-2 and analysed individually for [Ca²⁺]_i changes induced by the HIV-1 envelope glycoprotein gp120 and a variety of other treatments. In granule neurons [Ca²⁺]_i increases were induced by high KCl and glutamate (mainly through the NMDA receptor) while in type-1 astrocytes this effect was observed after serotonin, carbachol and also quisqualate. In contrast, administration of gp120 was always without effect in these cells. Type-2 astrocytes (an arborized cell type responsive to agonists targeted to the glutamatergic AMPA and cholinergic receptors) were also most often unresponsive to the viral glycoprotein. However, among the cells exhibiting the arborized phenotype, a subpopulation (-13%) responded to gp120 with conspicuous [Ca²⁺]_i increases sustained by both release from intracellular stores and influx across the plasma membrane. These responses to the viral protein did not involve activation of either voltage-gated Ca²⁺ channels or glutamatergic receptors. Although not yet conclusively identified by specific cytochemical markers, the gp120-responsive cells resemble type-2 astrocytes and differ from neurons and type-1 astrocytes both in gross phenotype and in a number of receptor/channel properties: positivity to AMPA and cholinergic agonists; negativity to NMDA, serotonin and high KCl. From these results it is concluded that a subpopulation of glial cells is affected by gp120. The role of these cells in HIV brain infection and damage requires further studies to be precisely established.     

Axotomy-induced Changes in Ca2+ Homeostasis in Rat Sympathetic Ganglion Cells

Some of the marked biochemical and electrophysiological changes provoked by section of the axon in mature neurons suggest that the intracellular calcium concentration ([Ca²⁺]_i) may be increased. We have measured the [Ca²⁺]_i using the fluorescent indicator Indo-1 microinjected into rat superior cervical ganglion neurons. No differences in resting [Ca²⁺]_i levels were found between control neurons and cells which had been axotomized 7–10 days before. However, the rise in [Ca²⁺]_i evoked by orthodromic or antidromic stimulation and the recovery after the stimulating train were considerably slower in axotomized neurons than in control cells. We also found that the number of calbindin-D28k-immunopositive cells in the ganglion increases after axotomy, which could be related to the observed differences in calcium homeostasis.     

NCS-1 differentially regulates growth cone and somata calcium channels in Lymnaea neurons

Local voltage-gated calcium channels, which regulate intracellular Ca²⁺ levels by allowing Ca²⁺ influx, play an important role in guiding and shaping growth cones, and in regulating the outgrowth and branching of neurites. Therefore, elucidating the mechanisms that regulate the biophysical properties of whole-cell calcium currents in the growth cones and somata of growing neurons is important to improving our understanding of neuronal development and regeneration. In this study, taking advantage of the large size of the pedal A (PeA) neurons in Lymnaea stagnalis , we compared the biophysical properties of somata and growth cone whole-cell calcium channel currents using Ba²⁺ and Ca²⁺ as current carriers. We found that somata and growth cone currents exhibit similar high-voltage activation properties. However, Ba²⁺ and Ca²⁺ currents in growth cones and somata are differentially affected by a dominant-negative peptide containing the C-terminal amino acid sequence of neuronal calcium sensor-1 (NCS-1). The peptide selectively reduces the peak and sustained components of current densities and the slope conductance in growth cones, and shifts the reversal potential of the growth cone currents to more hyperpolarized voltages. In contrast, the peptide had no significant effect on the somata calcium channels. Thus, we conclude that NCS-1 differentially modulates Ca²⁺ currents in the somata and growth cones of regenerating neurons, and may serve as a key regulator to facilitate the growth cone calcium channel activity.     

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.     

The Effect of Barium on [3H]Noradrenalin Release from the Human Neuroblastoma SH-SY5Y

Replacement of Ca²⁺ with Ba²⁺ in HEPES-buffered saline stimulated [³H]noradrenalin release in the human neuroblastoma clone SH-SY5Y by up to 20% of the cell content in the absence of other secretory stimuli. The Ba²⁺-evoked release was inhibited by 85% by 3 μM tetrodotoxin and 95% by 5 μM nifedipine. Ba²⁺ also increased the potency of K⁺-evoked release of [³H]noradrenalin, as maximal release was observed with 60 mM K⁺ compared with the 100 mM K⁺ necessary to achieve maximal release in the presence of Ca²⁺. In contrast, replacing Ca²⁺ with Ba²⁺ had little effect on carbachol- and bradykinin-evoked release of [³H]noradrenalin. No evidence was obtained from studies on changes in [Ca²⁺]_i (in response to 100 pM carbachol) using fura-2 that Ba²⁺ could enter intracellular stores in SH-SY5Y cells. Whole-cell patch-clamp studies showed that Ba²⁺ depolarizes SH-SY5Y cells as well as enhancing inward Ca²⁺ channel currents and shifting their voltage dependence to more negative values. These results are discussed in terms of the hypothesis that Ba²⁺ blocks K⁺ channels, leading to depolarization followed by opening of voltage-sensitive Na⁺ channels. This in turn opens voltage-sensitive L-type Ca²⁺ channels, which are coupled to the release of [³H]noradrenalin in SH-SY5Y cells.     

A Decrease in [Ca2+]c but not in cAMP Mediates L-AP4 Inhibition of Glutamate Release: PKC-mediated Suppression of this Inhibitory Pathway

We investigated the mechanism of the inhibition of glutamate release by L-2-amino-4-phosphonobutyrate (L-AP4) in cerebrocortical nerve terminals from young rats (3 weeks of age). The Ca²⁺-dependent release of glutamate was reduced by L-AP4 in a concentration-dependent manner. This inhibitory effect was prevented by pertussis toxin, insensitive to staurosporine and associated with a reduction both in the depolarization-evoked increase in the cytoplasmic free Ca²⁺ concentration ([Ca²⁺]_c) and in forskolin-stimulated cAMP formation. However, the reduction in [Ca²⁺]_c but not in cAMP seemed to be responsible for the decrease in release, since inhibition by L-AP4 can also be observed in the absence of detectable changes in CAMP. The inhibitory modulation by L-AP4 was suppressed by the activation of protein kinase C with phorbol esters. The nerve terminals from young rats also exhibited a facilitatory pathway of glutamate release which was mediated by protein kinase C. Interestingly, stimulation of this pathway with the glutamate agonist (1 S,3R)-1-aminocyclopentane-1,3-dicarboxylate in the presence of arachidonic acid also abolished the inhibitory action of L-AP4. The dominance of the facilitatory pathway in its interaction with the L-AP4-mediated inhibitory control may provide some clues to understand the presynaptic changes during synaptic plasticity.     

Methoxyverapamil Reduction of Nicotine-induced Catecholamine Release Involves Inhibition of Nicotinic Acetylcholine Receptor Currents

The mechanism by which the putative Ca²⁺ channel blocker methoxyverapamil (D600) inhibits nicotine-induced catecholamine release was investigated in bovine adrenal chromaffin cells and in neurons from paravertebral sympathetic ganglia of chick embryos. We found D600 to prevent catecholamine release evoked by 30 s applications of nicotine with a significantly higher potency than the release induced either by 30 s K⁺ depolarizations or by electrical field stimulation of sympathetic neurons. Like the use-dependent action of D600 upon Ca²⁺ channels, the magnitude of inhibition of the K⁺-evoked secretion depended on the duration of stimulation (10 s to 5 min). Data on catecholamine release were supplemented by patch-clamp recordings. We found whole-cell currents in chromaffin cells evoked by (extrapolated) 0.5 s applications of nicotine to be significantly more sensitive to D600 than Ca²⁺ currents induced by a 0.5 s depolarization from -80 to 0 mV. In both instances, the potency of D600 depended on the duration of the (nicotinic and depolarizing) stimuli. Our data suggest that D600 inhibits nicotine-induced catecholamine release by reducing nicotinic acetylcholine receptor currents rather than voltage-gated Ca²⁺ currents. Hence, in chromaffin cells as well as in sympathetic neuronal preparations, D600 does not appear to be a suitable tool to investigate the part voltage-activated Ca²⁺ currents play in cellular events induced by nicotine.     

Effects of a Cognition-enhancer, Linopirdine (DuP 996), on M-type Potassium Currents (IK(M)) Some Other Voltage- and Ligand-gated Membrane Currents in Rat Sympathetic Neurons

Linopirdine is a cognition enhancer which augments depolarization-induced transmitter release in the cortex and which is under consideration for potential treatment of Alzheimer's disease. It has previously been reported to inhibit M-type K⁺ currents in rat hippocampal neurons. In the present experiments we have tested its effect on whole-cell M-currents and single M-channels, and on a range of other membrane currents, in dissociated rat superior cervical sympathetic ganglion cells. Linopirdine inhibited the whole-cell M-current with an IC50 of 3.4 μM and blocked M-channels recorded in excised outside-out membrane patches but not in inside-out patches. This suggests that linopirdine directly blocks M-channels from the outside. It was much less effective in inhibiting other voltage-gated potassium currents [delayed rectifier (I_K(v)), IC50 63 μM; transient (I_A) current, IC50 69 μM] and produced no detectable inhibition of the fast and slow Ca²⁺-activated K⁺ currents I_c and I_AHP or of a hyperpolarization-activated cation current (I_Q/I_h), at 10–30 μM. However, it reduced acetylcholine-activated nicotinic currents and GABA-activated Cl^- currents with IC50 values of 7.6 and 26 μM respectively. It is concluded that linopirdine shows some 20–fold selectivity for M-channels among different K⁺ channels but can also block some transmitter-gated channels. The relationship between M-channel block and the central actions of linopirdine are discussed.     

Ethanol enhances GABAB-mediated inhibitory postsynaptic transmission on rat midbrain dopaminergic neurons by facilitating GIRK currents

It is largely accepted that an activation of the dopaminergic system underlies the recreational and convivial effects of ethanol. However, the mechanisms of action of this drug on the dopaminergic neurons are not fully understood yet. In the present study, we have used intracellular electrophysiological techniques (current and single-electrode voltage-clamp) to investigate the actions of ethanol on the γ-aminobutyric acid (GABA)_B-mediated inhibitory postsynaptic potentials (IPSPs) in rat midbrain dopaminergic neurons. Ethanol (10–200 m m ) augmented, in a concentration-dependent and reversible manner, the amplitude of the GABA_B–IPSP. In addition, the GABA_B agonist baclofen generated G-protein-gated inward rectifying K⁺ channels (GIRK)-related membrane hyperpolarizations/outward currents that were potentiated by ethanol. The potentiating effect of ethanol persisted in tetrodotoxin (TTX)-treated neurons, suggesting a postsynaptic site of action. These effects of ethanol were not changed by manipulating adenyl cyclase, protein kinases and phospholipase C activity, or by chelating intracellular Ca²⁺ with EGTA. Interestingly, the outward current caused by the intracytoplasmatic diffusion of the irreversible G-protein activator GTPγS was transiently enhanced by ethanol. Our observations suggest that the action of ethanol occurs on activated GIRK channels downstream of the GABA_B receptors. These enhancing effects of ethanol on GABA_B-induced synaptic responses could modulate alcohol intake and the altered mental and motor performance of individuals in an acute intoxicative phase.