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.     

Exocytosis and Selective Neurite Calcium Responses in Rat Cerebellar Granule Cells During Field Stimulation

The free calcium concentration, [Ca²⁺]_c, in fura-2-loaded rat cerebellar granule cells was investigated by digital imaging during trains of uniform field stimuli in order to compare the ability of calcium channels in somata and neurites to respond to brief, physiologically relevant depolarizations. Very few somata responded to 20 Hz trains of 1 ms pulses, while virtually all neurites showed an extensive increase which was rapidly reversed when stimulation was terminated. In contrast, both somata and neurites responded when cells were depolarized with 50 mM KCl. The field stimuli evoked a tetrodotoxin-sensitive increase in Na⁺ concentration in both somata and neurites. When 4-aminopyridine, which inhibits delayed K⁺ currents in these cells, was present during the field stimulus both somata and neurites increased their [Ca²⁺]_c, suggesting that prolongation of the duration of depolarization is required for somatic Ca²⁺ channel activation. The neurite response did not depend on the orientation of the neurite relative to the applied field. The neurite response was insensitive to nifedipine (1 μM) and ω-agatoxin-IVA (30 nM) but was uniformly inhibited by ω-conotoxin-GVIA (30% inhibition at 1 μM) and ω-conotoxin-MVIIC (44% inhibition at 5 μM). The two inhibitors were not additive. The neurite [Ca²⁺]_c response was insensitive to the combination of ionotropic glutamate receptor antagonists. Field stimulation caused the exocytosis of the fluorescent probe FM1-43 previously loaded during KCl depolarization, suggesting that presynaptic Ca²⁺ channels contribute to the field-evoked neurite response.     

Glutamate Depresses Release by Activating Non-conventional Glutamate Receptors at Crayfish Nerve Terminals

The present study shows that release of glutamate from crayfish nerve terminals is inhibited at low depolarizing current pulses by glutamate, N -methyl-D-aspartate (NMDA) and quisqualate. These agonists elicit inhibitory effects at concentrations as low as 10^-8 M (quisqualate) and 10^-7 M (glutamate and NMDA). The NMDA-mediated inhibition is blocked by (±)-2-amino-5-phosphonovaleric acid (APV). The quisqualate-mediated inhibition is blocked by 6-cyano-7-nitroquinoxaline-2, 3-dione (CNQX). Both CNQX and APV are needed to block glutamate-mediated inhibition. The inhibition of release is not accompanied by a detectable change in presynaptic membrane conductance at the secondary branch. Using fura-2, Ca²⁺ accumulation during repetitive stimulation (100 Hz) was monitored in single release boutons. Inhibition of release, elicited by 10^-4 M glutamate, was not associated with a reduction in the accumulation of Ca²⁺. We show that the glutamate released from a single or a few release boutons during normal activity acts similarly to glutamate added externally, i.e. it inhibits its own release.     

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.     

Glutamate Receptors on type I Vestibular Hair Cells of Guinea-pig

Afferent nerve calyces which surround type I vestibular hair cells (VHCI) have recently been shown to contain synaptic-like vesicles and to be immunoreactive to glutamate antibodies. In order to understand the physiological significance of these observations, the presence of glutamate receptors on type I vestibular sensory cells has been investigated. The effect of excitatory amino acids applied by iontophoresis was examined by spectrofluorimetry using fura-2 sensitive dye. Glutamate application caused a rapid and transient increase in intracellular calcium concentration ([Ca²⁺]ⁱ), in a dose-dependent manner. The ionotropic glutamate receptors agonists N -methyl- d -aspartic acid (NMDA), α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) and quisqualic acid (QA) induced an increase of [Ca²⁺]_i. The NMDA receptor antagonist 2-amino-5-phosphonovaleric acid and the AMPA receptor antagonist 6,7-dinitro-quinoxaline-2,3-dione partially blocked the glutamate response, by 39 ± 10 and 53 ± 11% respectively. Metabotropic receptors were also revealed by the specific agonist trans -1-amino-cyclopentyl-1,3-dicarboxylate. The presence of different glutamate receptors on the VHCI membrane suggests two kinds of feedback, (i) At the base of the sensory cell, autoreceptors may locally control the synaptic transmission, (ii) At the apex, postsynaptic receptors may modulate sensory transduction from glutamate release at the upper part of the afferent nerve calyx. These feedbacks suggest presynaptic modulation of the vestibular hair cell response which could affect its sensitivity.     

Activation of Nucleotide Receptors Inhibits High-threshold Calcium Currents in NG108-15 Neuronal Hybrid Cells

A P_2U (UTP-sensitive) nucleotide receptor has previously been cloned from NG108-15 neuroblastoma × glioma hybrid cells and it has been shown that activation of this receptor inhibits the M-type K⁺-current. We now report that UTP also inhibits Ca²⁺-currents in differentiated NG 108-15 cells, but probably through a different nucleotide receptor. UTP (100 μM) inhibited the peak of the high-threshold current by 28.4 ± 3.1% ( n = 38) with no effect on the low-threshold current. Two components of high-threshold current were identified: one inhibited by 100 nM ω-conotoxin (CgTx) and one inhibited by 2 μM nifedipine and enhanced by 1 μM BAY K8644. UTP inhibited the former by 31.0 ± 3.1%, with an IC₅₀ of 2.8 ± 1.1 μM, and the latter by 34.2 ± 6.1% with an IC₅₀ of 1.7 ± 1.3 μM. Pertussis toxin pretreatment prevented inhibition of the CgTx-sensitive, nifedipine-resistant but not CgTx-resistant current. Inhibition was not prevented by intracellular BAPTA (20 mM) or CAMP (1 mM). Effects of UTP on both currents were imitated by UDP, ATP, ADP, AP₄A and ATPγS but weakly or not at all by 2-MeSATP, GTP, AMP-CPP or ITP. Since the receptors which inhibit Ca²⁺-currents are activated by ATP, it is suggested that they might mediate auto-inhibition of transmitter release by ATP if present on purinergic nerve terminals.     

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.     

Group I mGluRs modulate calcium currents in rat GP: Functional implications

The modulation of voltage-dependent calcium currents strongly affects the firing pattern of central neurons. Changes in the intrinsic firing properties of mammalian globus pallidus cells (external pallidus in humans) are indicated as underlying the development of movement disorders. Pallidal neurons receive an excitatory input from the subthalamus, supposed to activate both ionotropic and metabotropic glutamate receptors. Since the activation of glutamate metabotropic receptors in rodent basal ganglia affects dopamine-mediated motor behaviors, we examined whether agonists at metabotropic sites modulate high-threshold calcium currents in pallidus. The broad agonist 1S,3R-ACPD produced a 22% reduction of calcium currents, which was mimicked by the group I agonist DHPG. These two responses were not additive; furthermore, the ACPD- and DHPG-mediated inhibition of high-threshold calcium currents were prevented by the cycloglycine MCPG, suggesting the involvement of a group I mGluR. The modulation was fast, saturating in less than 3 sec, partially voltage-dependent, in that about one-third was relieved by facilitation, and G-protein-mediated, since it was largely suppressed by NEM. Finally, the response was antagonized by ω-conotoxin-GVIA and ω-agatoxin-IVA, supporting the involvement of N- and P-type channels. The observed reduction of calcium signals might shape pallidal excitability, influencing the physiological balancing between globus pallidus and subthalamus. In pathological conditions such as parkinsonism, characterized by the putative increase of the endogenous release of glutamate from subthalamic neurons, the inhibition of high-threshold calcium currents in pallidus might modify the firing pattern of pallidal neurons and partially counteract the excitatory drive from STN. Nevertheless, the putative mGluR-induced reduction of intrinsic excitability might turn out to decrease the transmitter release from pallidal axon terminals, leading to further disinhibition of the output stations of the basal ganglia. Synapse 30:424–432, 1998. © 1998 Wiley-Liss, Inc.     

Receptors and Second Messengers Involved in Long-term Depression in Rat Cerebellar Slices In Vitro: a Reappraisal

In patch-clamped Purkinje cells (PCs), bath application of the ionotropic glutamate receptor antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) prevents induction of long-term depression (LTD) of parallel fibre (PF)-mediated EPSPs by a pairing protocol between Ca²⁺ spike firing and PF stimulation whereas bath application of ( RS )-α-methyl-4-carboxyphenylglycine (MCPG), a metabotropic glutamate (mGLU) receptor antagonist, does not. On the other hand, LTD can be also induced by pairing direct depolarization of PCs with activation of mGLU receptors by 1 S,3 R -aminocyclopentyl-dicarboxylate (1S, 3 R -ACPD), even in the presence of CNQX. In this case, LTD induction is not consistently blocked by bath application of the nitric oxide synthase inhibitor, N _G-methyl- l -arginine ( l -NMMA), whereas it is strongly blocked when the protein kinase C inhibitor peptide 19-36 is dialysed into PCs. These results are at variance with LTD induced by a pairing protocol between Ca²⁺ spikes and PF-mediated EPSPs which depends to the same extent on both cascades. Finally, thapsigargin, which depletes most intracellular Ca²⁺ pools, does not block induction of LTD by a pairing protocol between Ca²⁺ spikes and PF-mediated EPSPs whereas it prevents the induction of LTD depending on strong mGLU receptor activation.     

Locatization of Voltaae-sensitive Ca2+ Fluxes and Neuropeptide Y Immunoreactivity to Varicosities in SH-SY5Y Human Neuroblastoma Cells Differentiated by Treatment with the Protein Kinase Inhibitor Staurosporine

The distribution of voltage-sensitive elevations of the level of Ca²⁺ in untreated SH-SY5Y cells and cells that had been induced to differentiate with staurosporine was investigated by monitoring fura-2 fluorescence in cell suspensions, and by using microfluorometry and quantitative fluorescence imaging on cell bodies and on cellular processes. Cell bodies of both types of cells displayed small Ca²⁺ elevations, which were composed of transient and sustained components. Elevations were partially sensitive to the L- and N-channel blockers nifedipine (1 μM) and ω-conotoxin GVIA (100 nM) respectively. Up to ten times higher Ca²⁺ elevations were observed in varicosities of treated cells than in cell bodies of treated and untreated cells. These elevations were insensitive to compounds known to release Ca²⁺ from intracellular stores. Elevations of Ca²⁺ were sustained, and they were insensitive to 5 pM nifedipine, 100 nM ω-agatoxin IVA and 100 nM ω-conotoxin GVIA, and partially sensitive to 2 pM ω-conotoxin GVIA, indicating predominance of non-L-type, non-N-type, non-P-type channel activity. The intracellular localization of neuropeptide Y, a marker of differentiation in these cells, was also investigated by fluorescence immunocytochemistry. Varicosities of treated cells displayed marked fluorescence when viewed in a confocal microscope. These findings show that the varicosities of staurosporine-treated cells exhibit some of the functional properties of nerve terminals. The varicosities resemble boutons en passant nerve endings and they seem to express Ca²⁺ channels different from those in the cell body.     

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.     

Cholinergic Modulation of the Action Potential in Rat Hippocampal Neurons

The cholinergic input to the hippocampus from the medial septum is important for modulating hippocampal activity and functions, including theta rhythm and spatial learning. Neuromodulation by transmitters in central nervous system neurons usually affects cell excitability by modifying the membrane potential, discharge pattern and spike frequency. Here we describe another type of neuromodulation: changing the action potential waveform. During intracellular recordings from CA1 pyramidal cells in hippocampal slices from rats, the cholinergic agonist carbachol caused several reversible changes in the action potential: low doses (2 μM) caused an increase in spike duration; high doses (10–40 μM) or long-lasting applications also reduced the spike amplitude and rate of rise, and raised the spike threshold. These effects are similar to those of metabotropic glutamate receptor agonists or phorbol esters, both of which activate protein kinase C. The effects were blocked by the muscarinic antagonist atropine, and were prevented by Ca²⁺-free medium and by Ca²⁺-channel blockers. However, the cholinergic spike modulation was not occluded or mimicked by blocking the Ca²⁺-dependent K⁺ currents I_c or I_AHP, suggesting that these K⁺ currents are not involved in the modulation.     

HIV-1 Envelope Protein gp120 Potentiates NMDA-evoked Noradrenaline Release by a Direct Action at Rat Hippocampal and Cortical Noradrenergic Nerve Endings

Exposure of rat or human neocortical or hippocampal tissue to glutamate receptor agonists elicits a Ca²⁺-dependent, exocytotic-like release of previously accumulated [³H]noradrenaline through activation of both N -methyl- d -aspartate (NMDA) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors colocalized on the noradrenergic axon terminals. Here we show that the NMDA (100 μM)-evoked release of [³H]noradrenaline from superfused thin layers of isolated rat hippocampal or cortical nerve endings was potentiated when the human immunodeficiency virus type 1 coat protein gp120 was added to the superfusion medium concomitantly with NMDA. The effect of gp120 (10 pM to 3 nM) on the 100 μM NMDA-evoked release of [³H]noradrenaline was concentration-dependent; the maximal effect (-140% potentiation) was reached at 100 pM of gp120. The protein was inactive on its own. The [³H]noradrenaline release evoked by NMDA (100 μM) + gp120 (100 μM) was prevented by classical NMDA receptor antagonists, as well as by 10 μM memantine. Neither the release evoked by NMDA nor that elicited by NMDA + gp120 was sensitive to the nitric oxide synthase inhibitor N ^G -nitro- l -arginine, suggesting no involvement of nitric oxide. The [³H]noradrenaline release elicited by 100 nM AMPA was unaffected by gp120. The protein potentiated the release evoked by 100 nM glutamate; the effect of 100 pM gp120 was quantitatively identical to that of 1 μM glycine, with no apparent additivity between gp120 and glycine. The antagonism by 1 μM 7-chloro-kynurenic acid of the NMDA-induced [³H]noradrenaline release was reversed by glycine or gp120. The data are compatible with gp120 acting directly as a powerful positive allosteric modulator at a neuronal NMDA receptor.     

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.     

Protein SUMOylation modulates calcium influx and glutamate release from presynaptic terminals

Posttranslational modification by small ubiquitin-like modifier (SUMO) proteins is emerging as an important regulatory mechanism for neuronal function and dysfunction. Although multiple potential presynaptic SUMOylation substrate proteins have been proposed from sequence analysis the functional consequences of presynaptic SUMOylation have not been determined. Here we show that SUMOylation of presynaptic proteins modulates neurotransmitter release. Increasing protein SUMOylation by entrapping recombinant SUMO-1 in synaptosomes decreased glutamate release evoked by KCl whereas decreasing SUMOylation with the SUMO-specific protease SENP-1 enhanced KCl-evoked release. In contrast, SUMO increased and SENP-1 decreased synaptosomal glutamate release evoked by kainate stimulation. Consistent with these results, SENP-1 increased Ca²⁺ influx into synaptosomes evoked by KCl whereas it decreased kainate-induced Ca²⁺ influx. These results demonstrate that, in addition to postsynaptic effects, protein SUMOylation acts to modulate neurotransmitter release and thereby regulate synaptic function.     

Metabotropic Glutamate Receptors in the Rat Nucleus Accumbens

The effects of glutamate metabotropic receptors (mGluRs) on excitatory transmission in the nucleus accumbens were investigated using electrophysiological techniques in rat nucleus accumbens slices. The broad-spectrum mGluR agonist (1S,3 R )-1-aminocyclopentyl-1,3-dicarboxylate, the mGluR group 2 selective agonists (S)-4-carboxy-3-hydroxyphenylglycine, (1S,3S)-ACPD) and (2S,1'S,2'S)-2-(2'-carboxycyclopropyl)glycine (L-CCG1), and the mGluR group 3 specific agonist L-2-amino-4-phosphonobutyrate (L-AP4) all reversibly inhibited evoked excitatory synaptic responses. The specific group 1 mGluR agonist (R,S)-3,5-dihydroxyphenylglycine [(R,S)-DHPG] did not depress transmission. Dose-response curves showed that the rank order of agonist potencies was: L-CCGI > L-AP4 > (1 S,3S)-ACPD. Group 2 and 3 mGluRs inhibited transmission via a presynaptic mechanism, as they increased paired-pulse facilitation, decreased the frequency of miniature excitatory postsynaptic currents and had no effect on their amplitude. The mGluRs did not inhibit transmitter release by reducing voltage-dependent Ca²⁺ currents through N- or P-type Ca²⁺ channels, as inhibition persisted in the presence of a-conotoxin-GVIA or Aga-IVA. The depression induced by mGluRs was not affected by specific antagonists of dopamine D1, GABA-B or adenosine A1 receptors, indicating direct effects. Finally, (13,s)-DHPG specifically blocked the postsynaptic afterhyperpolarization current (I_ahp). Our results represent the first direct demonstration of functional mGluRs in the nucleus accumbens of the rat.     

Developmental Expression and Self-regulation of Ca2+ Entry via AMPA/KA Receptors in the Embryonic Chick Retina

Excessive activation of glutamate receptors in the late embryonic and adult retina leads to excitotoxic cell death through an increase in intracellular calcium concentration. Here we use the cobalt-staining technique of Pruss et al. to investigate the developmental expression of Ca²⁺-permeable α-amino-3–hydroxy-5–methyl-isoxazole-4-propionic acid/kainate (AMPNKA) receptors in the embryonic chick retina, and the effects of AMPNKA receptor activation on cell survival and AMPNKA receptor expression. Ca²⁺-permeable AMPNKA receptors are present in the retina as early as embryonic day 6 (E6). While sustained activation of these receptors with KA led to massive cell death in explant and dissociated cultures of the chick retina late in development, continuous application of high doses of KA from early times was not excitotoxic. Cell survival in KA is correlated with both a reduction in cobalt staining and the KA-evoked membrane current, and thus with a reduction in the Ca²⁺entry into cells via AMPNKA receptors. The effects of KA could be blocked by the non- N -methyl- d -aspartic acid (NMDA) receptor antagonist 6-cyano-7-nitro-quinoxaline-2,3–dione (CNQX), but not by the NMDA receptor antagonist D-2-amino-5-phosphonovalerate (AP5) nor the L-type Ca²⁺ channel blockers diltiazem and nifedipine. The action of AP5 was mimicked by exposure to glutamate but not by the metabotropic receptor agonist 1S,3R-1–aminocyclopentane-1,3–dicarboxylic acid. Thus exposure of retinal neurons to glutamate early in development may protect them from its excitotoxic actions later on.     

Relationship Between Intracellular Free Calcium Concentration and NMDA-induced Cerebellar Granule Cell Survival In Vitro

The survival of cerebellar granule cells in culture is stimulated by activation of the N -methyl- d -aspartate (NMDA) class of glutamate receptors. Activation of these receptors at the key period for cell survival in vitro (3 days; 3DIV) resulted in a sustained elevation of intracellular free calcium concentration [Ca²⁺]_i over the same concentration range of NMDA that led to granule cell survival. Agents that release Ca²⁺ from intracellular stores led to only small, transient elevations of [Ca²⁺]_i and were unable to stimulate granule cell survival. Addition of the Ca²⁺ ionophore ionomycin to granule cell cultures at 3DIV resulted in increased granule cell number at 7DIV. The ability of ionomycin to stimulate granule cell survival was related to the [Ca²⁺]_i elicited, indicating that a rise in [Ca²⁺]_i is sufficient to activate the processes leading to granule cell survival and that the extent of the elevation in [Ca²⁺]_i is crucially important in determining granule cell fate.     

Protein Kinase C Facilitation of Acetylcholine Release at the Rat Neuromuscular Junction

Protein kinase C (PKC) is a Ca²⁺-dependent enzyme involved in synaptic transmission, which can be experimentally activated by the phorbol ester, phorbol 12-myristate-13-acetate (TPA). We studied the effects of TPA application on acetylcholine (ACh) release at the rat neuromuscular junction by means of the focal recording technique; possible effects of TPA at the postsynaptic site had been ruled out in preliminary studies. In extracellular solutions containing 2 mM Ca²⁺ and at the stimulation frequency of 0.1 Hz, TPA increased endplate current (EPC) amplitude. In non-stimulated preparations spontaneous current frequency was increased at a similar rate. The similar time course of TPA action on evoked and spontaneous currents suggests that an increased presynaptic Ca²⁺ efficacy can be considered to be the probable mechanism of action. The interactions of PKC with ACh release were further investigated. In 0.1 mM Ca²⁺ extracellular solutions, TPA enhanced evoked currents only at stimulation frequencies (e.g. 40 Hz) that were themselves capable of inducing facilitation. This facilitation is classically associated with presynaptic Ca²⁺ accumulation, indicating that PKC interacts synergistically with Ca²⁺ to facilitate ACh release. In particular, since mean quantum size and release probability remained almost unchanged during TPA facilitation, it was concluded that PKC acted by enlarging the immediately available store. Interestingly, TPA also increased the presynaptic currents that were observed to be largely brought about by Ca²⁺-dependent K⁺ currents: evidence was obtained to suggest that increases in these currents provide negative feedback against excess release activation rather than being an expression of enhanced Ca²⁺ influx.     

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.