Activation of Excitatory Amino Acid Receptors in the Rat Hippocampal Slice Increases Intracellular Cl− and Cell Volume

Abstract: The effects of glutamatergic excitotoxins on intracellular Cl^? were investigated in the CA1 pyramidal cell layer of the hippocampal slice. Hippocampal slices from rats (14–19 days old) were loaded with 6-methoxy-N-ethylquinolinium chloride (MEQ), a Cl^?-sensitive fluorescent probe with a fluorescence intensity that correlates inversely with intracellular [Cl^?]. Slices were exposed for at least 10 min at 26–28°C to N-methyl-d -aspartate (NMDA; 100 µM) or α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA; 50 µM). A UV laser scanning confocal microscope was used to measure changes in MEQ fluorescence within area CA1 pyramidal cell soma. Both glutamate receptor agonists produced a rapid decrease in MEQ fluorescence that persisted after washout following a 10-min exposure. The effects of NMDA and AMPA were prevented by the competitive antagonists 2-amino-5-phosphonopentanoic acid and 6,7-dinitroquinoxaline-2,3-dione, respectively. Neither tetrodotoxin nor picrotoxin prevented the effect of NMDA or AMPA, indicating the lack of involvement of presynaptic mechanisms. The effects of NMDA and AMPA on MEQ fluorescence were dependent on the levels of extracellular Cl^?, but only NMDA responses were dependent on the levels of extracellular Na⁺. Removal of Ca²⁺ from the superfusion medium did not alter the effects of NMDA or AMPA on MEQ fluorescence. In addition, neither the Ca²⁺ ionophore ionomycin nor the L-type voltage-gated Ca²⁺ channel agonist (Bay K 8644) decreased MEQ fluorescence. The effects of NMDA and AMPA on cell (somal) volume were also assessed with the fluorescent probe calcein acetoxymethyl ester. Both NMDA and AMPA decreased calcein fluorescence (indicating an increased cell volume), but this was preceded by the decrease in MEQ fluorescence (equivalent to an intracellular accumulation of ～20 mM Cl^?). Thus, excitotoxins may cause Cl^? influx via an anion channel other than the GABA_A receptor and/or reduce Cl^? efflux mechanisms to produce cell swelling. Such anionic shifts may promote neuronal excitability and cell death following an excitotoxic insult to the hippocampal slice.     

Tyrosine Phosphorylation in a Model of Ischemia Using the Rat Hippocampal Slice: Specific, Long-Term Decrease in the Tyrosine Phosphorylation of the Postsynaptic Glycoprotein PSD-GP180

Abstract: The effects of the exposure of hippocampal slices to brief periods of ischemic-like conditions on the tyrosine phosphorylation of proteins and glycoproteins were investigated. Freshly prepared hippocampal slices contained a range of tyrosine-phosphorylated proteins and two prominent tyrosine-phosphorylated glycoproteins of apparent M_r 110,000 (GP110) and 180,000, which we have previously shown to correspond to the postsynaptic density (PSD)-associated glycoprotein PSD-GP180. When hippocampal slices were incubated in oxygenated Krebs-Ringer buffer containing 10 mM glucose (KRB), there was a transient increase in the tyrosine phosphorylation of a protein of M_r 42,000 (p42) and a pronounced increase in the tyrosine phosphorylation of GP110. After these initial changes, the tyrosine phosphorylation of all proteins remained constant for at least 60 min. In vitro “ischemia” was achieved by transferring slices that had been preincubated for 60 min in KRB to KRB that had been equilibrated with N₂ instead of O₂ and that did not contain glucose. Tyrosine-phosphorylated GP110 and PSD-GP180 could no longer be detected after 10 min of exposure of the slices to ischemic-like conditions. GP110 was rapidly rephosphorylated on tyrosine after transfer of slices back to oxygenated, glucose-containing buffer. In contrast, short periods of ischemia (5 or 10 min) resulted in the long-term loss of phosphotyrosine [Tyr(P)]-PSD-GP180 so that it was not detected even after 60 min of reincubation in oxygenated KRB. The sustained decrease in tyrosine phosphorylation of PSD-GP180 after ischemia was Ca²⁺ dependent, the levels of Tyr(P)-PSD-GP180 slowly increasing to preischemic values if Ca²⁺ was omitted from the incubation media. Reoxygenation of ischemic slices also resulted in the Ca²⁺-dependent, transient tyrosine phosphorylation of p42. The major PSD-associated, tyrosine-phosphorylated glycoprotein of molecular mass 180 kDa has recently been identified as the NR2B subunit of the NMDA receptor. The results suggest that changes in tyrosine phosphorylation after an ischemic insult may modulate the NMDA receptor or signal transduction pathways in the postsynaptic cell and are consistent with a role for tyrosine phosphorylation in the sequence of events leading to neuronal cell damage and death.     

An Ibotenate-Selective Metabotropic Glutamate Receptor: Mediates Protein Phosphorylation in Cultured Hippocampal Pyramidal Neurons

Abstract: Previous results showed that within 30 s after glutamate stimulation of cultured rat hippocampal pyramidal neurons there occurred an elevation of Ca²⁺ and diacylglycerol, and the phosphorylation of three acidic protein kinase C substrates, i.e., an 87-kDa protein known as myristoylated alanine-rich C kinase substrate and a 120-and a 48-kDa protein. In addition, it was suggested that a metabotropic-type glutamate receptor might be responsible for the phosphorylation observed. This work examines the ability of metabotropic and ionotropic glutamate receptor agonists to quickly activate phospholipases in 1.26 mM versus 50 nM extracellular Ca²⁺ by measuring the generation of inositol phosphates. NMDA, quisqualate, and trans-(±)-1-amino-1,3-cyclopentanedicarboxylic acid did not stimulate the generation of inositol phosphates in the presence of normal or low extracellular Ca²⁺ in pyramidal neurons. Kainate stimulated the production of inositol phosphates in the presence of 1.26 mM extracellular Ca²⁺ but not in 50 nM extracellular Ca²⁺. Other than glutamate, only ibotenate was able to stimulate the generation of inositol phosphates in both normal and low extracellular Ca²⁺. The maximal response to ibotenate was approximately equal to that of glutamate, when pyramidal neurons were stimulated in 50 nM extracellular Ca²⁺. The generation of inositol phosphates by glutamate and ibotenate could be partially blocked (50–60% reduction) by pretreatment of neurons with pertussis toxin (250 ng/ml),-suggesting that a GTP-binding protein might be involved. In addition, ibotenate stimulated the immediate phosphorylation of the same three protein kinase C substrates as glutamate. The NMDA receptor blocker MK-801 had no effect on this phosphorylation. These results suggest that the stimulation of phosphorylation in pyramidal neurons by glutamate occurs predominantly through the activation of an ibotenate-selective metabotropic glutamate receptor.     

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.     

Agonist-Evoked Ca2+ Mobilization from Stores Expressing Inositol 1,4,5-Trisphosphate Receptors and Ryanodine Receptors in Cerebellar Granule Neurones

Abstract: The mechanisms involved in Ca²⁺ mobilization evoked by the muscarinic cholinoceptor (mAChR) agonist carbachol (CCh) and N-methyl-d -aspartate (NMDA) in cerebellar granule cells have been investigated. An initial challenge with caffeine greatly reduced the subsequent intracellular Ca²⁺ concentration ([Ca²⁺]_i) response to CCh (to 45 ± 19% of the control), and, similarly, a much reduced caffeine response was detectable after prior stimulation with CCh (to 27 ± 6% of the control). CCh-evoked [Ca²⁺]_i responses were inhibited by preincubation with thapsigargin (10 µM), 2,5-di(tert-butyl)-1,4-benzohydroquinone (BHQ; 25 µM), ryanodine (10 µM), or dantrolene (25 µM). BHQ pretreatment was found to have no effect on the sustained phase of the NMDA-evoked [Ca²⁺]_i response. Both CCh (1 mM) and 1-aminocyclopentane-1S,3R-dicarboxylic acid (ACPD; 200 µM) evoked a much diminished increase in [Ca²⁺]_i in granule cells pretreated with CCh for 24 h compared with vehicle-treated control cells (CCh, 23 ± 14%; ACPD, 27 ± 1% of respective control values). In contrast, a 24-h CCh pretreatment decreased the subsequent inositol 1,4,5-trisphosphate (InsP₃) response to CCh to a much greater extent compared with responses evoked by metabotropic glutamate receptor (mGluR) agonists; this suggests that the former effect on Ca²⁺ mobilization represents a heterologous desensitization of the mGluR-mediated response distal to the pathway second messenger. Furthermore, [Ca²⁺]_i responses to caffeine and NMDA were unaffected by a 24-h pretreatment with CCh. This study indicates that ryanodine receptors, as well as InsP₃ receptors, appear to be crucial to the mAChR-mediated [Ca²⁺]_i response in granule cells. As BHQ apparently differentiates between the CCh- and NMDA-evoked responses, it is possible that the directly InsP₃-sensitive pool is physically different from the ryanodine receptor pool. Also, activation of InsP₃ receptors may not contribute significantly to NMDA-evoked elevation of [Ca²⁺]_i in cerebellar granule cells. A model for the topographic organization of cerebellar granule cell Ca²⁺ stores is proposed.     

Mechanism of the persistent sodium current activator veratridine-evoked Ca2+ elevation: implication for epilepsy

Although the role of Na⁺ in several aspects of Ca²⁺ regulation has already been shown, the exact mechanism of intracellular Ca²⁺ concentration ([Ca²⁺]_i) increase resulting from an enhancement in the persistent, non‐inactivating Na⁺ current (I_Na,P), a decisive factor in certain forms of epilepsy, has yet to be resolved. Persistent Na⁺ current, evoked by veratridine, induced bursts of action potentials and sustained membrane depolarization with monophasic intracellular Na⁺ concentration ([Na⁺]_i) and biphasic [Ca²⁺]_i increase in CA1 pyramidal cells in acute hippocampal slices. The Ca²⁺ response was tetrodotoxin‐ and extracellular Ca²⁺‐dependent and ionotropic glutamate receptor‐independent. The first phase of [Ca²⁺]_i rise was the net result of Ca²⁺ influx through voltage‐gated Ca²⁺ channels and mitochondrial Ca²⁺ sequestration. The robust second phase in addition involved reverse operation of the Na⁺–Ca²⁺ exchanger and mitochondrial Ca²⁺ release. We excluded contribution of the endoplasmic reticulum. These results demonstrate a complex interaction between persistent, non‐inactivating Na⁺ current and [Ca²⁺]_i regulation in CA1 pyramidal cells. The described cellular mechanisms are most likely part of the pathomechanism of certain forms of epilepsy that are associated with I_Na,P. Describing the magnitude, temporal pattern and sources of Ca²⁺ increase induced by I_Na,P may provide novel targets for antiepileptic drug therapy.     

Sulphur-Containing Amino Acids Modulate Noradrenaline Release from Hippocampal Slices

Abstract: The l - and d -enantiomers of the sulphur-containing amino acids (SAAs)—homocysteate, homocysteine sulphinate, cysteate, cysteine sulphinate, and S-sulphocysteine—stimulated [³H]noradrenaline release from rat hippocampal slices in a concentration-dependent manner. The relative potencies of the l -isomers (EC₅₀ values of 1.05–1.96 mM) were of similar order to that of glutamate (1.56 mM), which was 10-fold lower than that of NMDA (0.15 mM), whereas the d -isomers exhibited a wider range of potencies (0.75 to >5 mM). All stimulatory effects of the SAAs were significantly inhibited by the voltage-sensitive Na⁺ channel blocker tetrodotoxin (55–71%) and completely blocked by addition of Mg²⁺ or Co²⁺ to the incubation medium. All SAA-evoked responses were concentration-dependently antagonized by the selective NMDA receptor antagonist d -(?)-2-amino-5-phosphonopentanoic acid (IC₅₀ values of 3.2–49.5 µM). 6-Cyano-7-nitroquinoxaline-2,3-dione (CNQX), a non-NMDA receptor antagonist, at 100 µM inhibited the [³H]noradrenaline release induced by glutamate and NMDA (65 and 76%, respectively) and by all SAAs studied (65–85%), whereas 10 µM CNQX only inhibited the effects of S-sulpho-l -cysteine and l - and d -homocysteate (33, 32, and 44%, respectively). However, the more selective AMPA/kainic acid receptor antagonist 6-nitro-7-sulphamoylbenzo(f)quinoxaline-2,3-dione (100 µM), which did not antagonize the [³H]noradrenaline release induced by glutamate and NMDA, reduced only the S-sulpho-l -cysteine-evoked response (25%). Thus, the stimulation of Ca²⁺-dependent[³H]noradrenaline release from hippocampal slices elicited by the majority of the SAAs appears to be mediated by the NMDA receptor.     

High Calcium Permeability of Serotonin 5-HT3 Receptors on Presynaptic Nerve Terminals from Rat Striatum

Abstract: The serotonin 5-HT₃ receptor, a ligand-gated ion channel, has previously been shown to be present on a subpopulation of brain nerve terminals, where, on activation, the 5-HT₃ receptors induce Ca²⁺ influx. Whereas postsynaptic 5-HT₃ receptors induce depolarization, being permeant to Na⁺ and K⁺, the basis of presynaptic 5-HT₃ receptor-induced calcium influx is unknown. Because the small size of isolated brain nerve terminals (synaptosomes) precludes electrophysiological measurements, confocal microscopic imaging has been used to detect calcium influx into them. Application of 100 nM 1-(m-chlorophenyl)biguanide (mCPBG), a highly specific 5-HT₃ receptor agonist, induced increases in internal free Ca²⁺ concentration ([Ca²⁺]_i) and exocytosis in a subset of corpus striatal synaptosomes. mCPBG-induced increases in [Ca²⁺]_i ranged from 1.3 to 1.6 times over basal values and were inhibited by 10 nM tropisetron, a potent and highly specific 5-HT₃ receptor antagonist, but were insensitive to the removal of external free Na⁺ (substituted with N-methyl-d -glucamine), to prior depolarization induced on addition of 20 mM K⁺, or to voltage-gated Ca²⁺ channel blockade by 10 µM Co²⁺/Cd²⁺ or by 1 µMω-conotoxin MVIIC/1 µMω-conotoxin GVIA/200 nM agatoxin TK. In contrast, the Ca²⁺ influx induced by 5-HT₃ receptor activation in NG108-15 cells by 1 µM mCPBG was substantially reduced by 10 µM Co²⁺/Cd²⁺ and was completely blocked by 1 µM nitrendipine, an L-type Ca²⁺ channel blocker. We conclude that in contrast to the perikaryal 5-HT₃ receptors, presynaptic 5-HT₃ receptors appear to be uniquely calcium-permeant.     

Time Course and Involvement of Protein Kinase C-Mediated Phosphorylation of F1/GAP-43 in Area CA3 After Mossy Fiber Stimulation

1. Protein kinase C (PKC) activity and phosphorylation of F1/growth associated protein (GAP)-43, a PKC substrate, have been proposed to play key roles in the maintenance of long-term potentiation (LTP) at the synapses of Schaffer collateral/commissural on pyramidal neurons in CA1 (Akers et al., 1986). We have studied in the involvement of PKC and PKC-dependent protein phosphorylation of F1/GAP-3 in in vitro LTP observed at the synapses of mossy fiber (MF) on CA3 pyramidal neurons of rat hippocampus by post hoc in vitro phosphorylation.2. After LTP was induced in CA3 in either the presence or absence of D-2-amino-5-phosphonovaleric acid (AP5), an NMDA receptor antagonist, the CA3 region was dissected for in vitro phosphorylation assay. In vivo phosphorylation of F1/GAP-43 was increased in membranes at 1 and 5 min after tetanic stimulation (TS) but not at 60 min after TS.3. The degree of phosphorylation of F1/GAP-43 in the cytosol was inversely related to that in membranes at each time point after LTP.4. The similar biochemical changes obtained from either control slices or AP5-treated slices indicate that LTP and the underlying biochemical changes are independent of the NMDA receptor. Immunoreactivity of the phophorylated F1/GAP-43 in LTP slices was not significantly different from control, indicating that results from western blotting and post hoc in vitro phosphorylation are consistent.5. Post hoc in vitro phosphorylation of F1/GAP-43 was PKC-mediated since phosphorylation of F1/GAP-43 was altered by the PKC activation cofactors, Ca²⁺, phosphatidylserine and phorbol ester.6. Calmodulin (CaM) at >5 M inhibited phosphorylation, consistent with the presence of CaM-binding activity at the site on F1/GAP-43 acted upon by PKC.7. We conclude that phosphorylation of F1/GAP-43 is associated with the induction but not the maintenance phase of MF-CA3 LTP.     

Competition between ligands of glycosyltransferases and horseradish peroxidase for binding sites on intracellular and plasma membranes of HeLa cells

Summary A micro-method for the semi-quantitation of surface-bound horseradish peroxidase (HRP) was developed and was applied to study the competition between ligands of glycosyltransferases and HRP for binding sites on the surface of HeLa cells. Dried coverslip cultures of HeLa cells, fixed in methanol, were placed on 0.3 ml of the incubation medium on parafilm and were incubated for 45 min at 37° C. The incubation medium contained HRP, lysozyme and Ca²⁺ in HEPES buffer, pH 7.2. After washing, the cells were incubated for 60 min at 37° C in HEPES buffer containing 20 mM Ca²⁺. After this treatment, the plasma membranes showed a strong cytochemical reaction for HRP. Most of the HRP was released into buffer solution during a 5 h incubation at 37° C in the absence of Ca²⁺, and was measured by spectrophotometry. The addition of 20 mM Ca²⁺ to the buffer solution prevented the release of most of the HRP from the plasma membranes thus showing that the binding of HRP required Ca²⁺. Ligands of glycosyltransferases were added to the incubation medium with HRP. The amount of HRP released from the cells decreased in relation to the competing potency and concentration of these ligands. The method was applied to estimate the concentration of some ligands of galactosyltransferase and sialyltransferase that caused a 50% decrease in the release of previously-bound HRP. CMP-neuraminic acid and gangliosides showed a higher competing potency to the surface binding of HRP than UDP-galactose and chitotriose. The spectrophotometric analysis was correlated (on duplicate samples) with cytochemical observations. When dried HeLa cells, fixed in methanol, were incubated with HRP, lysozyme and Ca²⁺, without being subsequently incubated with Ca²⁺-containing buffer solution, HRP was also bound to membranes of intracellular granules. Cytochemical observations showed that UDP-galactose and chitotriose competed with the binding of HRP to most of these intracellular membranes whereas CMP-neuraminic acid and gangliosides did not. The possible binding of HRP to galactosyltransferase or sialyltransferase on cellular membranes is discussed.     

Calcium Buffering and Free Ca2+ in Rat Brain Synaptosomes

Abstract: The effects of K⁺ depolarization and of stimulation by veratridine on apparent cytosolic free Ca²⁺ ([Ca²⁺]_cyt) and net Ca²⁺ accumulation were measured in isolated rat brain presynaptic nerve terminals (synaptosomes). [Ca²⁺]_cyt was determined with fura-2, and Ca²⁺ accumulation was measured with tracer ⁴⁵Ca. [Ca²⁺]_cyt was ～ 325 nM in synaptosomes incubated in the normal physiological salt solution under resting conditions. When [K⁺]₀, was increased from the normal 5 mM to 30 or 50 mM, ⁴⁵Ca uptake and [Ca²⁺]_cyt both increased within 1 s. Both increases were directly related to [Ca²⁺]₀ for [Ca²⁺]₀= 0.02–1.2 mM; however, the increase in ⁴⁵Ca uptake greatly exceeded the increase in [Ca²⁺]_cyt. With small Ca²⁺ loads ≤100 μmol/L of cell water, equivalent to the Ca²⁺ entry during a train of ≤60 impulses), the ⁴⁵Ca uptake exceeded the increase in [Ca²⁺]_cyt by a factor of nearly 1,000. This indicates that ～99.9% of the entering Ca²⁺ was buffered and/or sequestered within ～ 1 s. With larger Ca²⁺ loads, a larger fraction of the entering Ca²⁺ was buffered; ～99.97% of the load was buffered with loads of 250–425 μmol/L of cell water. The ratio between the total Ca²⁺ entry and the increase in [Ca²⁺]_cyt, the “calcium buffer ratio”β, was therefore ～ 3,500:1. This ratio was somewhat lower than the ratio of total intraterminal calcium: [Ca²⁺]_cyt, which ranged between ～7,300:1 and 12,800:1. When the synaptosomes were activated with 10 μM veratridine ([Ca²⁺]₀= 0.2–0.6 mM), ⁴⁵Ca influx and [Ca²⁺]_cyt increased progressively for ～10 s (β= 2,700:13,050:1) and then leveled off. Application of 10 μM tetrodotoxin before the introduction of veratridine prevented the increases in ⁴⁵Ca influx and [Ca²⁺]_cyt. Application of 10 μM tetrodotoxin after 5–10 s of exposure to veratridine caused both the [Ca²⁺]_cyt and the veratridine-stimulated ⁴⁵Ca within the terminals to decline, thereby demonstrating that the Ca²⁺ loading is reversible in the presence of extracellular Ca²⁺. These data show that synaptosomes are capable of buffering and metabolizing Ca²⁺ in a manner expected for intact neurons.     

Development of depolarization‐induced calcium transients in insect glial cells is dependent on the presence of afferent axons

Changes in the intracellular Ca²⁺ concentration ([Ca²⁺]_i) induced by depolarization have been measured in glial cells acutely isolated from antennal lobes of the moth Manduca sexta at different postembryonic developmental stages. Depolarization of the glial cell membrane was elicited by increasing the external K⁺ concentration from 4 to 25 mM. At midstage 5 and earlier stages, less than 20% of the cells responded to 25 mM K⁺ (1 min) with a transient increase in [Ca²⁺]_i of approximately 40 nM. One day later, at late stage 5, 68% of the cells responded to 25 mM K⁺, the amplitude of the [Ca²⁺]_i transients averaging 592 nM. At later stages, all cells responded to 25 mM K⁺ with [Ca²⁺]_i transients with amplitudes not significantly different from those at late stage 5. In stage 6 glial cells isolated from deafferented antennal lobes, i.e., from antennal lobes chronically deprived of olfactory receptor axons, only 30% of the cells responded with [Ca²⁺]_i transients. The amplitudes of these [Ca²⁺]_i transients averaged 93 nM and were significantly smaller than those in normal stage 6 glial cells. [Ca²⁺]_i transients were greatly reduced in Ca²⁺‐free, EGTA‐buffered saline, and in the presence of the Ca²⁺ channel blockers cadmium and verapamil. The results suggest that depolarization of the cell membrane induces Ca²⁺ influx through voltage‐activated Ca²⁺ channels into antennal lobe glial cells. The development of the depolarization‐induced Ca²⁺ transients is rapid between midstage 5 and stage 6, and depends on the presence of afferent axons from the olfactory receptor cells in the antenna. © 2002 Wiley Periodicals, Inc. J Neurobiol 52: 85–98, 2002     

NMDA Receptors with Different Sensitivities to Magnesium and Ifenprodil Control the Release of [14C]Acetylcholine and [3H]Spermidine from Rat Striatal Slices in Vitro

Abstract: KCI (20–100 mM) and W-methyl-D-aspartate (NMDA, 100–1,000 μM) produce concomitant concentration-dependent increases in the release of previously captured [¹⁴C]acetylcholine and [³H]spermidine from rat striatal slices in vitro. The effects of NMDA (300μM) on striatal [¹⁴C]acetylcholine and [³H]spermidine release were blocked with equal potencies by the competitive NMDA antagonist CGP 37849, the glycine site antagonist L-689,560, and the NMDA channel blocker dizocilpine. In contrast, although NMDA-evoked [¹⁴C]acetylcholine release was antagonized by ifenprodil (IC₅₀= 5.3 μM) and MgCl₂, (IC₅₀= 200 μM), neither compound antagonized the NMDA-evoked release of [³H]spermidine at concentrations up to 100 μM (ifenprodil) or 1 mM (MgCl₂). Distinct NMDA receptor subtypes with different sensitivities to magnesium and ifenprodil therefore exist in the rat striaturn.     

Antagonistic Effect of Na+ and Mg2+ on P2z Purinoceptor-Associated Pores in Dibutyryl Cyclic AMP-Differentiated NG108-15 Cells

Abstract: The effect of replacement of extracellular Na⁺ with N-methyl-d -glucamine (NMG) on P₂ receptor signaling pathways was investigated in dibutyryl cyclic AMP-differentiated NG108-15 cells. Benzoylbenzoic ATP (BzATP) dose-dependently increased the cytosolic Ca²⁺ concentration ([Ca²⁺]_i) with an EC₅₀ value of 230 µM. Replacement of Na⁺ with NMG as well as removal of Mg²⁺ from the bathing buffer potentiated ethidium bromide uptake, [Ca²⁺]_i increase, and ⁴⁵Ca²⁺ uptake in response to ATP or BzATP. In contrast, in the presence of 5 mM Mg²⁺ to limit the amount of ATP^4?, replacement of Na⁺ with NMG had no effect on the ATP-induced [Ca²⁺]_i increase but caused a markedly larger [Ca²⁺]_i increase when the calculated concentration of ATP^4? was >10 µM. The calculated EC₅₀ value for ATP^4? stimulation of the [Ca²⁺]_i increase was 23 µM in NG108-15 cells. In vascular smooth muscle cells, intracellular Ca²⁺ release was the major pathway for the ATP-induced [Ca²⁺]_i increase; both removal of Mg²⁺ and replacement of Na⁺ with NMG did not affect the action of ATP. These data suggest that ATP^4?-promoted pores are antagonized by Na⁺ and Mg²⁺ in dibutyryl cyclic AMP-differentiated NG108-15 cells.     

Catalytically Dead αCaMKII K42M Mutant Acts as a Dominant Negative in the Control of Synaptic Strength

During long-term potentiation (LTP) of excitatory synapses, Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) is activated by Ca²⁺ influx through NMDA receptors that potentiate AMPA receptor currents by insertion of additional GluR1-containing receptors at the synapse and by increasing AMPA channel conductance, as well as by stimulating structural changes. CaMKII is also involved in the maintenance of LTP and contributes to maintenance of behavioral sensitization by cocaine or amphetamine. Recent studies show that transient expression of catalytically dead αCaMKII K42M mutant after exposure to amphetamine persistently reverses the behavioral effects of the addiction. A suggested interpretation is that this mutant acts as a dominant negative in the control of synaptic strength, but this interpretation has not been physiologically tested. Here we investigate the effect of αCaMKII K42M mutant expressed in single CA1 pyramidal neurons on basal excitatory neurotransmission in cultured rat hippocampal organotypic slices. The mutant caused nearly 50% reduction in the basal CA3–CA1 transmission, while overexpression of the wild-type αCaMKII had no effect. This result is consistent with the dominant negative hypothesis, but there are complexities. We found that the decrease in basal transmission did not occur when activity in the slices was suppressed after transfection by TTX or when NMDA receptors were blocked by APV. Thus, the dominant negative effect requires neural activity for its expression.     

Apamin Boosting of Synaptic Potentials in CaV2.3 R-Type Ca2+ Channel Null Mice

SK2- and K_V4.2-containing K⁺ channels modulate evoked synaptic potentials in CA1 pyramidal neurons. Each is coupled to a distinct Ca²⁺ source that provides Ca²⁺-dependent feedback regulation to limit AMPA receptor (AMPAR)- and NMDA receptor (NMDAR)-mediated postsynaptic depolarization. SK2-containing channels are activated by Ca²⁺ entry through NMDARs, whereas K_V4.2-containing channel availability is increased by Ca²⁺ entry through SNX-482 (SNX) sensitive Ca_V2.3 R-type Ca²⁺ channels. Recent studies have challenged the functional coupling between NMDARs and SK2-containing channels, suggesting that synaptic SK2-containing channels are instead activated by Ca²⁺ entry through R-type Ca²⁺ channels. Furthermore, SNX has been implicated to have off target affects, which would challenge the proposed coupling between R-type Ca²⁺ channels and K_V4.2-containing K⁺ channels. To reconcile these conflicting results, we evaluated the effect of SK channel blocker apamin and R-type Ca²⁺ channel blocker SNX on evoked excitatory postsynaptic potentials (EPSPs) in CA1 pyramidal neurons from Ca_V2.3 null mice. The results show that in the absence of Ca_V2.3 channels, apamin application still boosted EPSPs. The boosting effect of Ca_V2.3 channel blockers on EPSPs observed in neurons from wild type mice was not observed in neurons from Ca_V2.3 null mice. These data are consistent with a model in which SK2-containing channels are functionally coupled to NMDARs and K_V4.2-containing channels to Ca_V2.3 channels to provide negative feedback regulation of EPSPs in the spines of CA1 pyramidal neurons.     

Dual role of ryanodine-sensitive calcium stores in central neurons

The ryanodine-sensitive intracellular Ca²⁺ stores are known to play a major role in excitation-contraction coupling in muscles. Although these stores are also abundantly present in central neurons, their functional role in these cells remains unclear. Using fluorometric digital imaging of the intracellular Ca²⁺ concentration ([Ca²⁺] _i ) in rat hippocampal slices, we investigated the dynamic properties of the ryanodine-sensitive Ca²⁺ stores inCA1 hippocampal pyramidal cells. We found that at rest the ryanodine-sensitive Ca²⁺ stores are functioning predominantly as a “sink” for Ca ions responding to an increase in [Ca²⁺] _i with an increase in the amount of Ca ions accumulated inside the stores. If, however, [Ca²⁺] _i increases significantly, as happens during strong neuronal discharges, the ryanodine-sensitive Ca²⁺ stores respond with Ca²⁺ release, thus acting as an amplifier of the intracellular Ca²⁺ signal.     

Characterization of Agonist-Induced Down-Regulation of NMDA Receptors in Cerebellar Granule Cell Cultures

Abstract: Exposure of cerebellar granule cells to NMDA in culture at 5 days in vitro, when cells are not yet vulnerable to NMDA, evoked a pronounced reduction in NMDA receptor activity, measured by NMDA-induced ⁴⁵Ca²⁺ influx, and counteracted the normal developmental increase in NMDA receptors. The effect was concentration and time dependent, the half-maximal effect being reached at about 45 µM and by 4–5 h. The decrease in NMDA receptor function was accompanied by a significant reduction in the protein level of the obligatory NMDA receptor subunit (NR) NR1. Both parameters remained at a low level as long as the agonist was present. However, receptor down-regulation was reversible, as receptor protein levels and NMDA responses were restored to control values upon NMDA removal, this process requiring protein synthesis. NMDA treatment also elicited a decrease in NR1, NR2A, and NR2B subunit messenger RNA (mRNA) levels. However, in comparison with NMDA receptor proteins, the decrease was faster, and NMDA receptor mRNA content recovered to control levels within 24 h in spite of the presence of NMDA. Concerning the mechanisms of agonist-induced regulation of NMDA receptor expression, it seems that protein kinase C-mediated protein phosphorylation is not involved, whereas inhibition of Ca²⁺/calmodulin-dependent kinase II/IV by KN-62 does depress NMDA receptor expression even in the absence of NMDA.     

Ca2+ effects on ethylene, carbon dioxide and 1-aminocyclopropane-1-carboxylic acid synthase activity

The response of pericarp disks from ripening tomato (Lycopersicon esculentum Mill. cv. Traveler‘76) to CaCl₂, additions was studied to determine the effect of Ca²⁺ on ethylene and CO₂ production. Application of 5 mM CaCl₂ resulted in a 2, 20, 33, 39, and 50% increase in ethylene production in disks obtained from preclimacteric minimum, climacteric rise, climacteric peak, one, and two days postclimacteric fruit, respectively. CaCl₂ concentrations of 10 and 50 mM gave no additional stimulation of ethylene production; CO₂ production at 5 mM CaCl₂ was not different from controls, but is increased at 10 and 50mM CaCl₂. CaCl₂ also increased ethylene production in disks treated with 1-aminocyclopropane-1-carboxylic acid (ACC) or aminoethoxy-vinylglycine. Chloride salts of K⁺, Na⁺, Mg²⁺, Sr²⁺ and La³⁺ did not stimulate ethylene production. SrCl₂ stimulated ethylene production to a lesser degree than CaCl₂. Disks from potato (Solanum tuberosum L. cv. Katahdin) tubers produced greater quantities of ethylene and ACC when 5 mM CaCl₂ was included in the incubation medium (K. B. Evensen, 1983. Physiol. Plant. 60:125–128). Ca²⁺-treated disks had more than three times as much ACC synthase activity as control disks after 18 to 24 h incubation, when ethylene and ACC were maximal. The apparent K_m for S-adenosylmethionine was 13 μM at 29°C, pH 8.0 in extracts from both Ca²⁺-treated and control disks. Inclusion of 1 to 50 mM CaCl₂ in the assay medium did not significantly affect enzyme activity. ACC synthase extracted from control and Ca²⁺-treated disks had a pH optimum of 8.5 and an apparent molecular weight of 72 kdalton, estimated by gel filtration. It is likely that the presence of Ca²⁺ in the buffer allows greater synthesis of ACC synthase as part of the wound-healing response in potato, while in tomato the predominant effect is on membrane stabilization.     

Real-Time Effects of N-Methyl-d-Aspartic Acid on Dopamine Release in Slices of Rat Caudate Putamen: A Study Using Fast Cyclic Voltammetry

Abstract: The functional role of N-methyl-d -aspartic acid (NMDA) glutamate receptors in the real-time regulation of single electrical pulse (1 p)-stimulated endogenous dopamine release was investigated in slices of rat caudate putamen using fast cyclic voltammetry at a carbon fibre electrode. In the presence of Mg²⁺, 20 µM NMDA had a weak effect on background signals but did not affect 1 p-stimulated dopamine release. Removal of Mg²⁺ increased the background and doubled 1 p-stimulated dopamine release. In the absence of Mg²⁺, 20 µM NMDA caused a transient “release” of dopamine and decreased the background signal. The 1 p-stimulated dopamine release was subsequently reduced. In the presence of 1 µM (±)-3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP), superfusion with 20 µM NMDA did not cause a transient “release” of dopamine, and 1 p-stimulated dopamine release was not subsequently attenuated. In the presence of 1 µM tetrodotoxin, 1 p-stimulated dopamine release was abolished, but 20 µM NMDA still caused a transient “release” of dopamine. Removal of Ca²⁺ from the artificial CSF abolished 1 p-stimulated dopamine release and resulted in a decline in the baseline but did not affect dopamine “release” when 20 µM NMDA was added. The dopamine release-inducing effect of 20 µM NMDA was less pronounced in sites in the caudate putamen where dopamine release increased with frequency of electrical stimulation (hot spots) than in sites where there was little frequency-dependent dopamine release (cold spots). Subsequent 1 p-stimulated dopamine release was less attenuated in cold spots than in hot spots. We conclude that in the absence of Mg²⁺, NMDA induces release of dopamine by acting at CPP-sensitive NMDA receptors in a Ca²⁺-independent manner. This transient release depletes dopamine from a storage site from which dopamine is released by 1 p electrical stimulation. These real-time observations of the effects of NMDA on electrical stimulus-independent and -dependent dopamine release may explain the apparently conflicting observations of the effects of NMDA on dopamine release made in previous studies. They also indicate that dopamine release and storage are heterogeneous at different sites in the rat caudate putamen.