Early activation of Ca2+‐permeable AMPA receptors reduces neurite outgrowth in embryonic chick retinal neurons

Calcium entry through Ca²⁺‐permeable AMPA/kainate receptors may activate signaling cascades controlling neuronal development. Using the fluorescent Ca²⁺‐indicator Calcium Green 1‐AM we showed that the application of kainate or AMPA produced an increase of intracellular [Ca²⁺] in embryonic chick retina from day 6 (E6) onwards. This Ca²⁺ increase is due to entry through AMPA‐preferring receptors, because it was blocked by the AMPA receptor antagonist GYKI 52466 but not by the N‐methyl‐D ‐aspartic acid (NMDA) receptor antagonist AP5, the voltage‐gated Ca²⁺ channel blockers diltiazem or nifedipine, or by the substitution of Na⁺ for choline in the extracellular solution to prevent the depolarizing action of kainate and AMPA. In dissociated E8 retinal cultures, application of glutamate, kainate, or AMPA reduced the number of neurites arising from these cells. The effect of kainate was prevented by the AMPA/kainate receptor antagonist CNQX and by GYKI 52466 but not by AP5, indicating that the reduction in neurite outgrowth resulted from the activation of AMPA receptors. Blocking Ca²⁺ influx through L‐type voltage‐gated Ca²⁺ channels with diltiazem and nifedipine prevented the effect of 10–100 μM kainate but not that of 500 μM kainate. In addition, joro spider toxin‐3, a blocker of Ca²⁺‐conducting AMPA receptors, prevented the effect of all doses of kainate. Neither GABA, which is depolarizing at this age in the retina, nor the activation of metabotropic glutamate receptors with tACPD mimicked the effects of AMPA receptor activation. Calcium entry via AMPA receptor channels themselves may therefore be important in the regulation of neurite outgrowth in developing chick retinal cells. © 2001 John Wiley & Sons, Inc. J Neurobiol 49: 200–211, 2001     

Kainate responses of leech Retzius neurons in situ and in vitro

Responses to the ionotropic glutamate receptor agonist kainate were measured in Retzius cells (RCs) of intact segmental ganglia (in situ), acutely isolated RCs, and cultured RCs (in vitro) of the leech Hirudo medicinalis. RCs in intact ganglia responded to kainate (5–20 μM) with depolarizations up to 30 mV or with an inward current under voltage-clamp that reversed near -10 mV. The membrane conductance increased by a factor of 2.5 at a holding potential of -70 mV in the presence of 20 μM kainate. In RCs in situ the membrane responses to 5 μM kainate increased when applied repeatedly 3-5 times. After this potentiation, the amplitude and time course of the membrane responses to 5 μM kainate were similar to the membrane response to 20 μM kainate. In current-clamp experiments kainate evoked an increase in intracellular calcium concentrations ([Ca²⁺]¡) only when the membrane depolarized beyond -40 mV. In voltage-clamped RCs at a holding potential of -70 mV, kainate caused no significant rise in [Ca²⁺]¡, indicating that the Ca²⁺ permeability of these kainate-gated ion channels appears to be negligible. The potentiation of the kainate-induced responses in RCs in situ was also present in voltage-clamped cells, where no or only small changes in [Ca²⁺]¡ occurred, suggesting that the underlying mechanism seemed to be independent of intracellular Ca²⁺ changes. In addition, the potentiation of the kainate-induced membrane responses was unaffected by cyclothiazide (100 μM), concanavalin A (0.5 mg/mL), and in the presence of extracellular low-Ca²⁺ and high-Mg²⁺ concentrations to suppress synaptic transmission in the ganglion. During whole-cell patch-clamp recordings (up to 50 min) potentiation remained the same indicating that small intracellular messenger molecules, which would be expected to dissipate, were not likely to be involved in mediating this potentiation. In acutely isolated RCs kainate induced no or only very small voltage responses. A potentiation of the kainate response was never observed in acutely isolated RCs. In cultured RCs (2–7 days in vitro) kainate evoked membrane responses with no apparent potentiation. Cultured RCs also responded with Ca²⁺ transients only when depolarized beyond -40 mV. The results show that RCs respond differently to kainate when kept isolated in culture compared to RCs in intact ganglia. The mechanism underlying the potentiation of the kainate response of RCs in situ, however, could not yet be identified. © 1996 John Wiley & Sons, Inc.     

Glutamate receptor agonists evoked Ca2+-dependent and Ca2+-independent release of [3H]d-Aspartate from cultured chick retina cells

We studied the release of [³H]d-aspartate evoked by glutamate receptor agonists from monolayer cultures of chick retina cells, and found that activation of the glutamate receptors can evoke both Ca²⁺-dependent and Ca²⁺-independent release of [³H]d-aspartate. In Ca²⁺-free (no added Ca²⁺) Na⁺ medium, the agonists of the glutamate receptors induced the release of [³H]d-aspartate with the following rank order of potency: kainate>α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)∼N-methyl-d-aspartate (NMDA). In media containing 1 mM CaCl₂ the release of [³H]d-aspartate evoked by NMDA, kainate and AMPA was increased by about 112%, 20% and 39%, respectively, as compared to the release evoked by the same agonists in Ca²⁺-free medium. NMDA was the most potent agonist in stimulating the Ca²⁺-dependent release of [³H]d-aspartate, possibly by exocytosis, and AMPA was as potent as kainate. The Ca²⁺-dependent release of [³H]d-aspartate evoked by kainate was dependent on the influx of Ca²⁺ through the receptor associated channel, as well as through the N- (ω-Conotoxin GVIA-sensitive) and L- (nitrendipine-sensitive)type voltage-sensitive Ca²⁺ channels (VSCC). The exocytotic release of [³H]d-aspartate evoked by AMPA relied exclusively on Ca²⁺ entry through the L-type VSCC, whereas the effect of NMDA was partially mediated by the influx of Ca²⁺ through the receptor-associated channel, but not through L- or N-type VSCC. Thus, activation of these different glutamate receptors under physiological conditions is expected to cause the release of cytosolic and vesicular glutamate, and the routes of Ca²⁺ entry modulating vesicular release may be selectively recruited.     

Voltage-dependent and Src-mediated regulation of NMDA receptor single channel outward currents in cortical neurons

A voltage-dependent but Ca²⁺-independent regulation of N-methyl-D-aspartate (NMDA) receptor outward activity was studied at the single channel level using outside-out patches of cultured mouse cortical neurons. Unlike the inward activity associated with Ca²⁺ and Na⁺ influx, the NMDA receptor outward K⁺ conductance was unaffected by changes in Ca²⁺ concentration. Following a depolarizing pre-pulse, the single channel open probability (NP _o), amplitude, and open duration of the NMDA inward current decreased, whereas the same pre-depolarization increased those parameters of the NMDA outward current (pre-pulse facilitation). The outward NP _o was increased by the pre-pulse facilitation, disregarding Ca²⁺ changes. The voltage–current relationships of the inward and outward currents were shifted by the pre-depolarization toward opposite directions. The Src family kinase inhibitor, PP1, and the Src kinase antibody, but not the anti-Fyn antibody, blocked the pre-pulse facilitation of the NMDA outward activity. On the other hand, a hyperpolarizing pre-pulse showed no effect on NMDA inward currents but inhibited outward currents (pre-pulse depression). Application of Src kinase, but not Fyn kinase, prevented the pre-pulse depression. We additionally showed that a depolarization pre-pulse potentiated miniature excitatory synaptic currents (mEPSCs). The effect was blocked by application of the NMDA receptor antagonist AP-5 during depolarization. These data suggest a voltage-sensitive regulation of NMDA receptor channels mediated by Src kinase. The selective changes in the NMDA receptor-mediated K⁺ efflux may represent a physiological and pathophysiological plasticity at the receptor level in response to dynamic changes in the membrane potential of central neurons.     

Properties of AMPA Receptors Expressed in Rat Cerebellar Granule Cell Cultures: Ca2+ Influx Studies

Abstract: Cultured cerebellar granule cells become vulnerable to excitatory amino acids, especially to NMDA and kainate, by 9 days in vitro. In the same time, the sensitivity of cells to (RS)-α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA), in terms of AMPA-induced toxicity or ⁴⁵Ca²⁺ uptake, was very low. The low AMPA responsiveness was due to receptor desensitization, because agents known to block desensitization, cyclothiazide and the lectins concanavalin A and wheat germ agglutinin, rendered granule cells vulnerable to AMPA and produced a pronounced stimulation of ⁴⁵Ca²⁺ accumulation. ⁴⁵Ca²⁺ influx was induced specifically by AMPA-receptor stimulation, because it was blocked virtually completely by 2,3-dihydroxy-6-nitro-7-sulfamoylbenzoquinoxaline (NBQX) and the benzodiazepine GYKI 52466 (selective non-NMDA receptor antagonists). Nevertheless, indirect routes activated by cellular responses to AMPA-receptor stimulation contributed significantly to the overall ⁴⁵Ca²⁺ influx. These included Ca²⁺ uptake through NMDA-receptor channels, voltage-sensitive Ca²⁺ channels, and via Na⁺/Ca²⁺ exchange. However, nearly one-fifth of the total ⁴⁵Ca²⁺ influx remained unaccounted for and this estimate was similar to ⁴⁵Ca²⁺ influx observed under Na⁺-free conditions. This observation suggested that a significant proportion of the Ca²⁺ flux passes through the AMPA-receptor channel proper, a view supported by Co²⁺ uptake into nearly all granule cells on exposure to AMPA in the presence of cyclothiazide. Results are discussed in light of the reported AMPA receptor-subunit composition of cerebellar granule cells in vitro.     

Potentiation of voltage-dependent calcium channel currents by NMDA receptor agonists

The effects of NMDA receptor agonists on voltage-dependent Ca²⁺ channels were studied in pyramidal neurons freshly dissociated from theCA3 region of the rat hippocampus. In a fraction of investigated cells (18 of 26), application of NMDA receptor agonists resulted in a rapid increase in the amplitude of whole-cell Ca²⁺ channel currents (Ca²⁺CC). This effect immediately disappeared on return to the control solution. The current-voltage relationship for the whole-cell Ca²⁺ channel currents was not shifted under this action of NMDA receptor agonists. It was shown that neither T-, nor L-type Ca²⁺CC were facilitated by NMDA receptor agonists. The experiments with specific blockers of various types (ω-CgTxGVIA, ω-Aga-IVA, and ω-CgTxMVIIC) showed that N-, P-, and Q-types of Ca²⁺ channels were not potentiated by NMDA receptor agonists. The involvement of other types of Ca²⁺ CC (R type, in particular) in the modulatory action of NMDA receptor agonists is considered.     

Ionotropic glutamate receptor subunit distribution on hypoglossal motoneuronal pools in the rat

The expression of ionotropic glutamate receptor subunits in the motoneuronal pools of the hypoglossal nucleus was studied using specific antibodies against subunits of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA), kainate and N-methyl-D-aspartate (NMDA) subtypes. The highest numbers of intensely immunolabelled motoneurons were found in the dorsal tier and caudoventromedial part of the hypoglossal nucleus with all antibodies except that against the GluR1 AMPA subunit. Labelling for the GluR1 subunit was weak except for caudally located groups of motoneurons which innervate tongue muscles related to respiratory activity. By contrast, most motoneurons were intensely immunostained with antibodies against GluR2/3 and GluR4 subunits of the AMPA subtype. The low staining observed using an antibody specific for the GluR2 subunit (which prevents Ca²⁺-entry through AMPA channels) strongly suggests that AMPA receptors in hypoglossal motoneurons are Ca²⁺-permeable. Immunolabelling for the GluR5/6/7 kainate receptor subunits was found in many motoneuronal somata as well as in thin axon-like profiles and puncta that resembled synaptic boutons. Most motoneurons were intensely immunostained for the NMDA receptor subunit NR1. These results show that the hypoglossal nucleus contains five heterogeneous pools of motoneurons which innervate functionally defined groups of tongue muscles. The uneven expression of the different receptor subunits analysed here could reflect diverse phenotypic properties of hypoglossal motoneurons which might be expected to generate different patterns of motor responses under different physiological or pathological conditions.     

Differential Contribution of L-, N-, and P/Q-type Calcium Channels to [Ca<Superscript>2+</Superscript>]<Subscript>i</Subscript> Changes Evoked by Kainate in Hippocampal Neurons

We investigated the contribution of L-, N- and P/Q-type Ca²⁺ channels to the [Ca²⁺]_i changes, evoked by kainate, in the cell bodies of hippocampal neurons, using a pharmacological approach and Ca²⁺ imaging. Selective Ca²⁺ channel blockers, namely nitrendipine, ω-Conotoxin GVIA (ω-GVIA) and ω-Agatoxin IVA (ω-AgaIVA) were used. The [Ca²⁺]_i changes evoked by kainate presented a high variability, and were abolished by NBQX, a AMPA/kainate receptor antagonist, but the N-methyl-d-aspartate (NMDA) receptor antagonist, D-AP5, was without effect. Each Ca²⁺ channel blocker caused differential inhibitory effects on [Ca²⁺]_i responses evoked by kainate. We grouped the neurons for each blocker in three subpopulations: (1) neurons with responses below 60% of the control; (2) neurons with responses between 60% and 90% of the control, and (3) neurons with responses above 90% of the control. The inhibition caused by nitrendipine was higher than the inhibition caused by ω-GVIA or ω-AgaIVA. Thus, in the presence of nitrendipine, the percentage of cells with responses below 60% of the control was 41%, whereas in the case of ω-GVIA or ω-AgaIVA the values were 9 or 17%, respectively. The results indicate that hippocampal neurons differ in what concerns their L-, N- and P/Q- type Ca²⁺ channels activated by stimulation of the AMPA/kainate receptors. Special issue article in honor of Dr. Ricardo Tapia.     

NMDA and Non-NMDA Receptor-Mediated Release of [3H]GABA from Granule Cell Dendrites of Rat Olfactory Bulb

Abstract: We have studied the effect of glutamate and the glutamatergic agonists N-methyl-d -aspartate (NMDA), kainate, and α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) on [³H]GABA release from the external plexiform layer of the olfactory bulb. The GABA uptake blocker nipecotic acid significantly increased the basal [³H]GABA release and the release evoked by a high K⁺ concentration, glutamate, and kainate. The glutamate uptake blocker pyrrolidine-2,4-dicarboxylate (2,4-PDC) inhibited by 50% the glutamate-induced [³H]GABA release with no change in the basal GABA release. The glutamatergic agonists NMDA, kainate, and AMPA also induced a significant [³H]GABA release. The presence of glycine and the absence of Mg²⁺ have no potentiating effect on NMDA-stimulated release; however, when the tissue was previously depolarized with a high K⁺ concentration, a significant increase in the NMDA response was observed that was potentiated by glycine and inhibited by the NMDA receptor antagonist 2-amino-5-phosphonoheptanoic acid (AP-7). The kainate and AMPA effects were antagonized by the non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) but not by AP-7. The glutamate effect was also inhibited by CNQX but not by the NMDA antagonist 2-amino-5-phosphonopentanoic acid (AP-5); nevertheless, in the presence of glycine, [³H]GABA release evoked by glutamate was potentiated, and this response was significantly antagonized by AP-5. Tetrodotoxin inhibited glutamate- and kainate-stimulated [³H]GABA release but not the NMDA-stimulated release. The present results show that in the external plexiform layer of the olfactory bulb, glutamate is stimulating GABA release through a presynaptic, receptor-mediated mechanism as a mixed agonist on NMDA and non-NMDA receptors; glutamate is apparently also able to induce GABA release through heteroexchange.     

Evolution of mechanisms of Ca2+-signalization. Role of Ca2+ in regulation of specialized cell functions

The review describes peculiarities of Ca²⁺-signalization in electro-excitable cells of higher eukaryotes. The light has been shed on problems of Ca²⁺-dependent mechanisms of regulation of muscle contractility and of neuronal synaptic plasticity in the higher vertebrate animals. A particular attention has been paid to analysis of contribution of such poorly studied components of Ca²⁺-signalization as non-selective TRPC-channels, Orai channels, sensory STIM1 proteins, Ca²⁺-controlled K⁺-channels of large and small conductance, and neuronal Ca²⁺-sensors (NCS).     

Metabotropic Glutamate Receptor in C6BU-1 Glioma Cell Has NMDA Receptor-Ion Channel Complex-Like Properties and Interacts with Serotonin2 Receptor-Stimulated Signal Transduction

Abstract: We found in cultured glioma (C6BU-1) cells that excitatory amino acids (EAAs) such as glutamate, N-methyl-d -aspartate (NMDA), aspartate, and metabotropic glutamate receptor agonist trans-(±)-1-amino-1,3-cyclopentanedicarboxylate caused an increase in the inositol 1,4,5-trisphosphate formation and the intracellular Ca²⁺ concentration ([Ca²⁺]_i) in the absence of extracellular Mg²⁺ and Ca²⁺. Pertussis toxin treatment abolished this glutamate-induced [Ca²⁺]_i increase. Various antagonists against NMDA receptor-ion channel complex, such as Mg²⁺, d -2-amino-5-phosphonovalerate (d -APV), HA-966, and MK-801, also inhibited the increase in [Ca²⁺]_i induced by glutamate. These results indicate that these metabotropic EAA receptors coupled to pertussis toxin-susceptible GTP-binding protein and phospholipase C system in C6BU-1 glioma cells have the pharmacological properties of NMDA receptor-ion channel complexes. We also found that in the presence of Mg²⁺ these metabotropic receptors resemble the NMDA receptor-ion channel complex interacted with 5-hydroxytryptamine₂ (5-HT₂) receptor signaling. EAAs inhibited 5-HT₂ receptor-mediated intracellular Ca²⁺ mobilization and inositol 1,4,5-trisphosphate formation in a concentration-dependent manner. The inhibitory effect of glutamate was reversed by various NMDA receptor antagonists (d -APV, MK-801, phencyclidine, and HA-966), but l -APV failed to block the inhibitory effect of glutamate. The same result was observed in the absence of extracellular Ca²⁺. In addition, this inhibitory effect on 5-HT₂ receptor-mediated signal transduction was abolished by treatment of C6BU-1 cells with pertussis toxin, whereas 5-HT₂ receptor-mediated [Ca²⁺]_i increase was not abolished by pertussis toxin treatment. We can, therefore, conclude that the inhibitory effect of glutamate is not a result of the influx of Ca²⁺ through the ion channel and that it operates via metabotropic glutamate receptors, having NMDA receptor-ion channel complex-like properties and being coupled with pertussis toxin-sensitive GTP-binding protein and phospholipase C.     

Pharmacological and functional characterization of excitatory amino acid mediated cytotoxicity in cerebral cortical neurons

The cytotoxic action of the excitatory amino acids (EAAs) glutamate, N-methyl- D-aspartate (NMDA), quisqualate (QA), kainate (KA) and (RS)-2-amino-3(3-hydoxy-5-methylisoxazol-4-yl) propionate (AMPA) was studied in cerebral cortical neurons in culture. The pharmacological profile of these actions was characterized using the NMDA selective antagonist D-(-)-2-amino-5- phosphonopentanoate (APV) and the non-NMDA selective antagonists 6.7- dinitroquinoxaline-2,3-dione (DNQX), 2-amino-3[3-(carboxymethoxy)-5- methylisoxazol-4-yl]-propionate (AMOA) and 2-amino-3-[2-(3-hydroxy-5- methylisoxazol-4-yl)methyl-3-methyl-3-oxoisoxazolin-4-yl] propionate (AMNH). The role of intracellular Ca⁺⁺ homeostasis and cGMP production for development of EAA mediated cytotoxicity was assessed by measurements of changes in [Ca⁺⁺]i using the flourescent Ca⁺⁺ chelator Fluo-3 and in cGMP concentrations using a conventional radioimmune assay. It was found that glutamate toxicity involves both NMDA and non-NMDA receptor activation and that aberrations in Ca⁺⁺ homeostasis brought about by Ca⁺⁺ influx and/or liberation of Ca⁺⁺ from internal stores aare important for development of toxicity. The drug dantrolene which prevents release of Ca⁺⁺ from such stores can prevent toxicity induced by glutamate, NMDA and QA completely but has no effect on KA and AMPA toxicity. Changes in cGMP levels appear to play a role for development of glutamate, NMDA and KA toxicity but does not seem to be involved in that triggered by QA and AMPA.Abbreviations AMNH: (2-amino-3-[2-(3-hydroxy-5-methylisoxazol-4-yl)methyl-5-methyl-3-oxoisoxazolin-4-yl]propionate) - AMOA: (2-amino-3[3-(carboxymethoxy)-5-methylisoxazol-4-yl]propinate) - AMPA: ( (RS) —2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propinate) - APV: (D-(-)-2-amino-5-phosphonopentanoate) - DNQX: (6,7-dinitroquinoxaline-2,3-dione) - KA (kinate) - QA (quisqualate)     

Non-NMDA excitatory amino acid receptors in a subcellular fraction enriched in cerebellar glomeruli

In the internal granular layer of the cerebellar cortex the polysynaptic complexes called glomeruli consist mainly of homogeneous populations of glutamatergic and GABAergic synapses, both located on granule cell dendrites. A subcellular fraction enriched in glomeruli was prepared from rat cerebellum, and the distribution of the different types of NMDA and non-NMDA glutamate binding sites was studied in the membranes derived from this fraction (fraction G) as compared to that in the membranes prepared from a total cerebellar homogenate (fraction T). Cl^–/Ca²⁺ independent [³H]glutamate binding sites were not abundant and could be reliably measured only in fraction G. Cl^– dependent/Ca²⁺ activated [³H]glutamate binding sites were more abundant and exhibited a single K _d in both fractions G and T. Quisqualate, NMDA, kainate, L-AP4 andtrans-ACPD inhibited [³H]glutamate binding to different extents in the two membrane fractions. Quisqualate sensitive sites were predominant in all cases but more abundant in fraction T than in fraction G. An opposite distribution was observed for the NMDA sensitive binding sites while kainate sensitive binding sites were scarce everywhere.Trans-ACPD, a ligand presumed selective for metabotropic glutamate binding sites, displaced [³H]glutamate from fraction T but nor from fraction G, suggesting the absence of these sites from glomeruli. Similarly, no L-AP4 sensitive sites were present in fraction G while they were abundant in fraction T. Binding sites associated with ionotropic receptors of the quisqualate type were determined by measuring [³H]AMPA binding. The density of the high affinity [³H]AMPA binding sites in fraction T was twice as high as in fraction G, indicating that these sites are abundant in structures other than glomeruli. High-affinity [³H]kainate binding sites are more abundant in fraction G than in fraction T; the same, but with smaller differences, occurs for the distribution of the low affinity [³H]kainate binding sites. The density of the latter sites is close to that of the high affinity [³H]AMPA binding sites confirming the presence of quisqualate/kainate receptors on granule cells, as previously hypothesized (for review, see Gallo et al., 1990). Taken together, these results indicate a segregation of the glutamate binding sites types at specialized synapses or neuronal cell types in the cerebellar network.Abbreviations AMPA (RS)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid - DL-AP4 dl-2-amino-4-phosphonobutyric acid - D-AP5 d-2-amino-5-phosphonovaleric acid - EAA excitatory amino acid - EGTA ethylene glycol-bis(-aminoethyle ether) N,N,N,N-tetracetic acid - NMDA N-methyl-D-aspartate - Quisqualate -[3,5-dioxo-1,2,4-oxadiazolidin-2-yl]-L-alanine - trans-ACPD trans-1-amino-cyclopentyl-1,3-dicarboxylic acid     

Glutamate receptor channels in rat DRG neurons: activation by kainate and quisqualate and blockade of desensitization by Con A

Primary afferent C fibers in rat dorsal roots are depolarized by the excitatory amino acids kainate and domoate. Under whole-cell voltage clamp, kainate and domoate increase membrane conductance in a subpopulation of freshly dissociated DRG neurons. In contrast to kainate currents observed in CNS neurons, responses to kainate and domoate in DRG cells desensitize with prolonged agonist exposure. Half-maximal activation is achieved with much lower concentrations of kainate and domoate in sensory neurons than in CNS neurons from cerebral cortex. Rapid applications of glutamate, quisqualate, and AMPA evoke a transient current in DRG neurons and desensitize cells to subsequent applications of kainate or domoate. Brief incubation with the lectin concanavalin A eliminates desensitization to excitatory amino acids; after treatment with concanavalin A, all five agonists gate sustained currents of similar amplitude via the same receptor.     

Control of spontaneous synchronous Ca<Superscript>2+</Superscript> oscillations in hippocampal neurons by GABAergic neurons containing kainate receptors without desensitization

Previously we have shown that in culture of rat hippocampal neurons, the calcium responses of individual cells (changes of cytoplasmic free Ca²⁺ concentration in response to agonists of glutamate kainate receptors) differed in shape and amplitude (Kononov A.V., Bal’ N.V., Zinchenko V.P. 2011. Biochemistry (Moscow) Suppl. Series A: Membrane and Cell Biology. 5 (2), 162–170). In the majority of neurons, the amplitudes of calcium response were regularly distributed, although there were a small number of cells that generated the desensitization-free signals of far greater amplitudes. In these cells, the desensitization inhibitors did not increase the amplitude of calcium response. We identified these neurons and revealed their function. The agonists of kainate receptors inhibited the synchronized spontaneous Ca²⁺ oscillations, decreased the baseline calcium level in the majority of neurons, and considerably elevated it in some of them. After washout of the agonists, the oscillations were restored in all neurons only after a certain time lag determined by the period needed for calcium concentration to decrease to subbasal level in specific neurons with high calcium signal amplitude. This observation indicates the command role of these neurons in synchronizing the activity of the entire population. To identify the subtype of KA receptors in these neurons, we used especially selective agonists and showed that KA receptors of the neurons characterized with desensitization-free calcium signals of unusually great amplitude contained GluR5/GLUK1 subunits. These receptors are known to be located mostly in the presynaptic membrane, where they promote exocytosis of neurotransmitters due to elevation of the Ca²⁺ conductivity. Having marked the positions of these neurons, we fixed the preparation and stained the cells with fluorescently labeled antibodies raised against glutamate decarboxylase, an enzyme which is selectively expressed in GABAergic neurons. The experiments demonstrated that antibodies were localized only in the neurons, where the kainate receptor agonist evoked desensitization-free calcium responses of especially large amplitude. Thus, GABAergic neurons control the synchronous activity of a large number of neurons via glutamate-evoked activation of specific presynaptic kainate receptors with GluR5/GLUK1 subunits leading to desensitization-free calcium signals of especially large amplitude.     

Glutamatergic and GABAnergic control in the tentacle effector systems of Hydra vulgaris

In addition to their role in orchestrating body and tentacle contractions, hydra’s nerves control the behavior of nematocysts; precisely how is still a work in progress. There are strong indications that the classical neurotransmitters, glutamate and GABA (γ-amino-butyric acid), play an essential role in effecting stenotele and desmoneme discharge. In experiments on isolated tentacles of Hydra vulgaris, in which cnidocils were mechanically deflected with a piezo-electrically-driven glass micropipette, stenoteles and desmonemes respond to differences in applied force in a dose-dependent manner. GABA, working through its metabotropic receptor, appears to be involved with the recruitment of desmonemes. Desmonemes in distant battery cells or in another part of a given battery cell were discharged by stimulating a desmoneme cnidocil in the presence of bath-applied GABA or its metabotropic agonist, baclofen. The effect was blocked by phaclofen, its metabotropic antagonist. Neither GABA nor baclofen affected stenotele discharge. GABA_A agonists had no effect on nematocyst discharge. Glutamate caused a significant increase in number of stenoteles responding to direct mechanical stimuli, but did not effect desmoneme discharge. The effect was mimicked by NMDA (n-methyl-d-aspartate) together with kainate, or by NMDA plus AMPA (amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid), but not with any ionotropic agonist alone. The effect was blocked by D-AP 5 (d- (−)–2-amino–5-phosphopentanoic acid), a specific NMDA antagonist, or CNQX (6-cyano-7-nitroquinoxaline-2,3-dione), a specific kainate/AMPA antagonist. A glutamatergic mechanism working through ionotropic glutamate receptors appears to lower the firing threshold of stenoteles, perhaps␣by permitting the entry of Ca²⁺ into the cell through the early evolved NMDA/kainite/AMPA mechanism.     

Identification and characterization of inward K+ -channels in plasma membranes ofArabidopsis root cortex cells

Patch clamping whole-cell recording techniques were applied to study the inward K⁺ -channels inArabidopsis root cortex cells. The inward K⁺ -channels in the plasma membranes of the root cortex cell protoplasts were activated by hyperpolarized membrane potentials. The channels were highly selective for K⁺ ions over Na⁺ ions. The channel activity was significantly inhibited by the external TEA⁺ or Ba²⁺. The changes in cytoplasmic Ca²⁺ concentrations did not affect the whole-cell inward K⁺ -currents. The possible association between the channel selectivity to K⁺ and Na⁺ ions and plant salt-tolerance was also discussed.     

Ca++- and Na+, K+-ATPase activities in the fungiform papilla of the tongue of Rana Esculenta (Anura Ranidae)

In the fungiform papilla of Rana esculenta (Anura Ranidae), the Ca⁺⁺-ATPase is mainly distributed on the basolateral membrane of the sensory area cells (i.e., neuroepithelial, supporting, and mucous cells). Apical membranes of all cells facing the surface present a slight enzymatic activity. Lateral wall cells have a strong Ca⁺⁺-ATPase activity on basolateral and apical membranes. Strong Na⁺, K⁺-ATPase activity occurs on the apical surface of neuroepithelial cells. Ca⁺⁺-ATPase activity is absent on the surface of endothelial cells of the capillaries located under the sensory area. These observations lead us to conclude that the sensory area of fungiform papilla is the selective way for calcium influx. Furthermore the absence of ATPase activity on the surface of the endothelial cells indicates that there is no functional barrier to calcium influx into capillary, and that calcium can be removed by vessels from the sensory area.     

Streptozotocin-Induced Diabetes Mellitus and Further Nimodipine Treatment Do Not Change Calcium Currents in Rat Sensory Neurons within Early Stages of the Disease

Earlier, considerable prolongation of the depolarization-induced Ca²⁺ transients was demonstrated in primary sensory neurons of rats with streptozotocin (STZ)-induced diabetes mellitus. To analyze the nature of this effect, we examine possible changes in the characteristics of voltage-operated calcium channels. Neither the amplitude of Ca²⁺ currents provided by both high- and low-voltage activated calcium channels nor the respective current densities significantly changed within the early stages of diabetes mellitus. In rats treated with nimodipine, also no significant changes in the calcium channel activity were observed. Only in the case of a decrease in the external calcium concentration was some drop in the Ca²⁺ current amplitude observed. We conclude that within the early stages of diabetes mellitus there are no significant modifications in the structure of the membrane of primary sensory neurons manifested in the expression of Ca²⁺ channels, which might be responsible for the observed rapidly occurring changes in calcium signalling, cytosolic Ca²⁺ accumulation, and synaptic plasticity.