Absence Of External Mg2 Research Articles

Purinergic modulation of the excitatory synaptic input onto rat striatal neurons

There is no in situ evidence hitherto for a modulation by ATP of the glutamatergic excitatory transmission onto medium spiny neurons (MSNs) in the rat striatum. In order to resolve this question, we used the patch-clamp technique in brain slice preparations to record excitatory postsynaptic currents (EPSCs) evoked by intrastriatal electrical stimulation and applied N-methyl-d-aspartate (NMDA) or α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) to activate transmembrane currents of MSNs. In the absence of external Mg2+, ATP caused a higher maximum inhibition of the EPSCs than adenosine. Only P1 (A1), but not P2 receptor antagonists interfered with the effects of both ATP and adenosine. Moreover, A1 receptor antagonists were less potent in blocking the inhibition by ATP than that by adenosine. Eventually, adenosine deaminase (ADA) almost abolished the adenosine-induced inhibition, but only moderately decreased the ATP-induced inhibition. Antagonists of A1 receptors (but not of P2 receptors) counteracted the depression by ATP of the current responses to exogenous NMDA, without altering those to AMPA. It is suggested that ATP indirectly, via its degradation product adenosine, stimulates presynaptic inhibitory A1 receptors situated at glutamatergic nerve terminals of striatal afferents; these nerve terminals are devoid of P2 receptors. However, ATP, in contrast to adenosine, also activates postsynaptic A1 receptors at the MSN neurons themselves. The resulting negative interaction with NMDA receptors requires localized extracellular catabolism of ATP by ectonucleotidases.

Glutamatergic calcium dynamics and deregulation of rat retinal ganglion cells

A rise in intracellular calcium levels ([Ca(2+)](i)) is a key trigger for the lethal effects of the excitatory neurotransmitter glutamate in various central neurons, but a consensus has not been reached on the pathways that mediate glutamate-dependent increases of [Ca(2+)](i) in retinal ganglion cells (RGCs). Using Ca(2+) imaging techniques we demonstrated that, in the absence of external Mg(2+), the Ca(2+) signal evoked by glutamate was predominantly mediated by NMDA-type glutamate receptors (NMDA-Rs) in immunopanned RGCs isolated from neonatal or adult rats. Voltage-gated Ca(2+) channels and AMPA/kainate-Rs contributed a smaller portion of the Ca(2+) response at saturating concentrations of glutamate. Consistent with NMDA-R involvement, extracellular Mg(2+) inhibited RGC glutamate responses, while glycine had a potentiating effect. With Mg(2+) present externally, the effect of AMPA/kainate-R antagonists was enhanced and both NMDA- and AMPA/kainate-R antagonists greatly reduced the glutamate-induced increases of RGC [Ca(2+)](i). This finding indicates that the primary contribution of AMPA/kainate-Rs to RGC glutamatergic Ca(2+) dynamics is through the depolarization-dependent relief of the Mg(2+) block of NMDA-R channels. The effect of glutamate receptor antagonists on glutamatergic Ca(2+) signals from RGCs in adult rat retinal wholemounts yielded results similar to those obtained using immunopanned RGCs. Additional experiments on isolated RGCs revealed that during a 1 h glutamate (10-1000 microm) exposure, 18-28% of RGCs exhibited delayed Ca(2+) deregulation (DCD) and the RGCs that underwent DCD were positive for the death marker annexin V. RGCs with larger glutamate-evoked Ca(2+) signals were more likely to undergo DCD, and NMDA-R blockade significantly reduced the occurrence of DCD. Identifying the mechanisms underlying RGC excitotoxicity aids in our understanding of the pathophysiology of retinal ischaemia, and this work establishes a major role for NMDA-R-mediated increases in [Ca(2+)](i) in glutamate-related RGC death.

Mutant Presenilin 1 Alters Synaptic Transmission in Cultured Hippocampal Neurons

Mutations in presenilins are the major cause of familial Alzheimer disease, but the precise pathogenic mechanism by which presenilin (PS) mutations cause synaptic dysfunction leading to memory loss and neurodegeneration remains unclear. Using autaptic hippocampal cultures from transgenic mice expressing human PS1 with the A246E mutation, we demonstrate that mutant PS1 significantly depressed the amplitude of evoked alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid and N-methyl-D-aspartate receptor-mediated synaptic currents. Analysis of the spontaneous miniature synaptic activity revealed a lower frequency of miniature currents but normal miniature amplitude. Both alterations could be rescued by the application of a gamma-secretase blocker. On the other hand, the application of synthetic soluble Abeta42 in wild-type neurons induced the PS1 mutant phenotype on synaptic strength. Together, these findings strongly suggest that the expression of mutant PS1 in cultured neurons depresses synaptic transmission by causing a physical reduction in the number of synapses. This hypothesis is consistent with morphometic and semiquantitative immunohistochemical analysis, revealing a decrease in synaptophysin-positive puncta in PS1 mutant hippocampal neurons.

Early expression of glycine and GABAA receptors in developing spinal cord neurons. Effects on neurite outgrowth

Using fluorometric and immunocytochemical techniques, we found that high glycine concentrations or blockade of glycine receptors increases neurite outgrowth in developing mouse spinal cord neurons. Glycine- and GABAA-activated currents were demonstrated during applications of glycine and GABA (50–100 μM) in 5 days in vitro (DIV) neurons. Long application (≥10 min) of 100 μM glycine desensitized the membrane response by more than 95%. Application of glutamate in the absence of external Mg2+, at several membrane potentials, did not produce any detectable membrane response in these cells. Immunocytochemical studies with NR1 and GluR1 antibodies showed a delayed appearance of N-methyl-D-aspartate (NMDA) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors respectively. Spontaneous synaptic activity was readily observed in 5 DIV neurons. The use of various receptor antagonists (strychnine, bicuculline, DL-2-amino-5-phosphonovalerate [APV], 6-cyano-7-nitroquinoxaline-2,3-dione [CNQX]) revealed that this activity was predominantly glycinergic, and to a smaller extent, GABAergic. In the presence of bicuculline, APV and CNQX, we detected abundant spontaneous depolarizing potentials which often reached the action potential threshold. Further evidence for functional synaptic activity was provided by the detection of co-localization of gephyrin and synaptophysin at 5 DIV using confocal microscopy. Fluorometric studies with Fluo-3, a Ca2+ indicator, in 5 DIV cultures showed the presence of spontaneous fluctuations associated with tetrodotoxin-sensitive synaptic events. The number of neurons displaying these fluctuations was significantly increased (>100%) when the cells were bathed in a strychnine-containing solution. On the other hand, these synaptically mediated Ca2+ events were blocked by the co-application of strychnine and bicuculline. This suggests that glycine and GABAA receptors provide a fundamental regulation of both neuronal excitability and intracellular Ca2+ at this early time of development.The neurotrophic effects of agonists and antagonists for glycine, GABAA and glutamate receptors were examined in neurons cultured for 2 or 5 DIV. From all the agonists used, only high concentrations of glycine increased neurite outgrowth in 5 DIV neurons. We found that strychnine also increased neurite outgrowth, whereas tetrodotoxin (1 μM), nimodipine (4 μM) and bicuculline (20 μM) completely blocked it. On the other hand, APV (50 μM) and CNQX (20 μM) were unable to affect neurite outgrowth.These data suggest that spinal glycine receptors depress neurite outgrowth by shunting neuronal excitability. Outgrowth induction possibly results from the enhanced activity found after the inhibition of glycinergic activity. We postulate that this resets the intracellular calcium at a concentration that favors neurite outgrowth.

Calcium handling and cell contraction in rat cardiomyocytes depleted of intracellular magnesium.

Depressed levels of cardiac Mg have been found in patients with ischaemic heart disease or heart failure, but it is not known whether low intracellular free [Mg2+] ([Mg2+]i) is a causal factor in such myocardial dysfunction. The aims for the present study were to develop a method of lowering [Mg2+]i in myocytes isolated from normal rat hearts, and so to determine whether a low [Mg2+]i itself would cause abnormalities of intracellular Ca2+ ([Ca2+]i) homeostasis or myocyte contractile function in absence of any cardiac disease. Rat ventricular myocytes were loaded with mag-indo-1/AM or indo-1/AM for determination of total [Mg2+]i and [Ca2+]i, respectively. Mitochondrial [Ca2+] was determined by selective loading of indo-1/AM into the mitochondria. Cell contraction was measured using an edge-tracking device. Myocytes were depleted of [Mg2+]i by incubation in absence of external Mg2+. This resulted in a decrease in [Mg2+]i from about 1.3 to 0.3 mM. In subsequent experiments, 1.2 mM MgCl2 was again present in the superfusate. Under basal conditions (low rate of stimulation, 0.2 Hz, and 1 mM external [Ca2+]), the Mg-depleted cells showed very similar changes in [Ca2+] to control cells, despite an increase in the amplitude of cell contraction. But in presence of high external [Ca2+] (4 mM) and 5 Hz stimulation rate, the Mg-depleted cells showed defects in systolic Ca2+ handling and in cell contraction; in particular, they were unable to increase systolic [Ca2+] in response to the stimulus, unlike control cells. Despite these alterations in total [Ca2+]i, mitochondrial Ca2+ uptake was unchanged in the Mg-depleted cells. A low [Mg2+]i can itself cause significant cardiomyocyte dysfunction in absence of any contributing disease state.

Phenotype of a recombinant store-operated channel: highly selective permeation of Ca2+.

1. Genes related to trp (transient receptor potential) are proposed to encode store-operated channels. We examined the ionic permeation of recombinant channels formed by stable and transient expression of the TRP homologue bCCE1 in Chinese hamster ovary (CHO) cells (CHO(CCE1)) and rat basophilic leukaemia (RBL) cells, respectively. 2. Store-operated currents were activated in CHO(CCE1) cells by internal dialysis of IP3 under strong buffering of intracellular Ca2+. The action of IP3 was mimicked by thapsigargin but not by IP4. 3. With extracellular Ca2+, Na+ and Mg2+, the store-operated currents of CHO(CCE1) rectified inwardly in the presence of internal Cs+. Outward currents were not detected below +80 mV. Identical currents were recorded with external Ba2+ and also with no external Na+ and Mg2+. In the absence of external Mg2+, the inward currents showed an anomalous mole fraction behaviour between Ca2+ and Na+. Half-maximal inhibition of Na+ currents was observed with approximately 100 nM and full block with 2-5 microM external Ca2+. 4. In the parental CHO(-) cells, IP3 dialysis evoked inward currents that also displayed anomalous mole fraction behaviour between Ca2+ and Na+. However, half-maximal block of Na+ currents required 5 times higher Ca2+ concentrations in CHO(-) cells. Additionally, the density of Ca2+ and Na+ currents at -80 mV was 5 and 2 times larger in CHO(CCE1) cells, respectively. 5. In RBL cells, dialysis of IP3 evoked store-operated currents that showed 1.4-fold larger densities at -80 mV in cells expressing bCCE1. 6. The enhanced density of store-operated currents in CHO(CCE1) cells and in bCCE1-transfected RBL cells probably reflects the phenotype of CCE1. These results suggest a highly selective permeation of Ca2+ through recombinant channels formed by CCE1 either alone or in combination with endogenous channel proteins.

Blockade of M2-like muscarinic receptors enhances long-term potentiation at corticostriatal synapses

Acetylcholine (ACh) exerts a crucial role in learning and memory. The striatum contains the highest concentration of this transmitter in the brain. This structure expresses two different forms of synaptic plasticity, long-term depression (LTD) and long-term potentiation (LTP), which might contribute to the storage of motor skills and some cognitive processes. We have investigated the role of M2-like muscarinic receptors in striatal LTP by utilizing intracellular recordings in vitro from a rat corticostriatal slice preparation. Methoctramine (250 nm), an antagonist of M2-like muscarinic receptors, enhanced striatal LTP induced in the absence of external magnesium (Mg2+) by high-frequency stimulation (HFS) of corticostriatal fibres. Methoctramine did not affect the amplitude of excitatory postsynaptic potentials (EPSPs) when bath applied either before or after the conditioning tetanus suggesting that a critical increase of ACh concentrations is produced only during HFS. Methoctramine per se failed to enhance the NMDA-mediated EPSPs recorded in the absence of external Mg2+ and in the presence of 10 μm CNQX. Methoctramine antagonized the presynaptic inhibitory action of neostigmine, an inhibitor of ACh-esterase, and oxotremorine, an agonist of M2-like muscarinic receptors. These data indicate that the activation of M2-like muscarinic receptors exerts a negative influence on striatal LTP, probably by reducing the release of glutamate from corticostriatal fibres and they suggest a complex modulatory effect of ACh in striatal synaptic plasticity.

Blockade of M2-like muscarinic receptors enhances long-term potentiation at corticostriatal synapses.

Acetylcholine (ACh) exerts a crucial role in learning and memory. The striatum contains the highest concentration of this transmitter in the brain. This structure expresses two different forms of synaptic plasticity, long-term depression (LTD) and long-term potentiation (LTP), which might contribute to the storage of motor skills and some cognitive processes. We have investigated the role of M2-like muscarinic receptors in striatal LTP by utilizing intracellular recordings in vitro from a rat corticostriatal slice preparation. Methoctramine (250 nM), an antagonist of M2-like muscarinic receptors, enhanced striatal LTP induced in the absence of external magnesium (Mg2+) by high-frequency stimulation (HFS) of corticostriatal fibres. Methoctramine did not affect the amplitude of excitatory postsynaptic potentials (EPSPs) when bath applied either before or after the conditioning tetanus suggesting that a critical increase of ACh concentrations is produced only during HFS. Methoctramine per se failed to enhance the NMDA-mediated EPSPs recorded in the absence of external Mg2+ and in the presence of 10 microM CNQX. Methoctramine antagonized the presynaptic inhibitory action of neostigmine, an inhibitor of ACh-esterase, and oxotremorine, an agonist of M2-like muscarinic receptors. These data indicate that the activation of M2-like muscarinic receptors exerts a negative influence on striatal LTP, probably by reducing the release of glutamate from corticostriatal fibres and they suggest a complex modulatory effect of ACh in striatal synaptic plasticity.

Dendritic glutamate receptor channels in rat hippocampal CA3 and CA1 pyramidal neurons.

1. Properties of dendritic glutamate receptor (GluR) channels were investigated using fast application of glutamate to outside-out membrane patches isolated from the apical dendrites of CA3 and CA1 pyramidal neurons in rat hippocampal slices. CA3 patches were formed (15-76 microns from the soma) in the region of mossy fibre (MF) synapses, and CA1 patches (25-174 microns from the soma) in the region of Schaffer collateral (SC) innervation. 2. Dual-component responses consisting of a rapidly rising and decaying component followed by a second, substantially slower, component were elicited by 1 ms pulses of 1 mM glutamate in the presence of 10 microM glycine and absence of external Mg2+. The fast component was selectively blocked by 2-5 microM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and the slow component by 30 microM D-2-amino-5-phosphonopentanoic acid (D-AP5), suggesting that the fast and slow components were mediated by the GluR channels of the L-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) and NMDA type, respectively. The peak amplitude ratio of the NMDA to AMPA receptor-mediated components varied between 0.03 and 0.62 in patches from both CA3 and CA1 dendrites. Patches lacking either component were rarely observed. 3. The peak current-voltage (I-V) relationship of the fast component was almost linear, whereas the I-V relationship of the slow component showed a region of negative slope in the presence of 1 mM external Mg2+. The reversal potential for both components was close to 0 mV. 4. Kainate-preferring GluR channels did not contribute appreciably to the response to glutamate. The responses to 100 ms pulses of 1 mM glutamate were mimicked by application of 1 mM AMPA, whereas 1 mM kainate produced much smaller, weakly desensitizing currents. This suggests that the fast component is primarily mediated by the action of glutamate on AMPA-preferring receptors. 5. The mean elementary conductance of AMPA receptor channels was about 10 pS, as estimated by non-stationary fluctuation analysis. The permeability of these channels to Ca2+ was low (approximately 5% of the permeability to Cs+). 6. The elementary conductance of NMDA receptor channels was larger, with a main conductance state of about 45 pS. These channels were 3.6 times more permeable to Ca2+ than to Cs+.(ABSTRACT TRUNCATED AT 400 WORDS)

Effect of magnesium on cytosolic free sodium in human lymphocytes.

Cytosolic free sodium concentration ([Na+]i) was measured in intact human lymphocytes using the novel sodium-sensitive fluorescent dye sodium-binding benzofuran-isophthalate. In the presence of 1 mmol/l external Mg2+ the resting [Na+]i was significantly lower compared to the value in the absence of external Mg2+ (22.7 +/- 1.1 mmol/l vs. 37.6 +/- 1.4 mmol/l; p < 0.0001). The thapsigargin induced [Na+]i increase was significantly lower in the presence of 1 mmol/l external Mg2+ compared to the value in the absence of external Mg2+ (73.4 +/- 6.0 mmol/l vs. 120.7 +/- 5.8 mmol/l; p < 0.001). Since Mg2+ is known to be a cofactor of the membrane Na+,K(+)-ATPase these measurements in intact lymphocytes indicate that deprivation of external Mg2+ causes an increase of [Na+]i.

Measurement of matrix free Mg2+ concentration in rat heart mitochondria by using entrapped fluorescent probes

1. The concentration of free Mg2+ ([Mg2+]m) within the matrix of isolated rat heart mitochondria was measured after loading of the mitochondria with the fluorescent Mg2+ indicators mag-indo-1 and mag-fura-2. No detectable change in total mitochondrial magnesium content occurred during loading with the indicators. Apparent Kd values for Mg2+ of 3.7 mM and 2.3 mM were obtained for mag-indo-1 and mag-fura-2 respectively within mitochondria permeabilized to bivalent cations with ionomycin and the uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone. These values are 2.7- and 1.8-fold greater respectively than those obtained for the free acid forms of the dyes in incubation medium. 2. Based on the above Kd values, mitochondrial matrix Mg2+ concentrations were found to lie in the range 0.8-1.5 mM in the absence, or immediately after the addition, of a respiratory substrate. 3. Incubation of mitochondria in the presence of respiratory substrate, but in the absence of external Mg2+, led to a time-dependent decline in [Mg2+]m to about half the initial values after 5 min. This was accompanied by a fall in the total mitochondrial magnesium content from 12.7 to 7.0 nmol/mg of protein. 4. ADP (0.5 mM), ATP (0.5 mM) or 10 mM-NaCl had no significant effect on the fall in [Mg2+], whereas 1 microM-nigericin blocked, and 0.3 microM-valinomycin accelerated, the fall. 5. External Mg2+ concentrations above 1 mM progressively inhibited and reversed the decline in free and total mitochondrial Mg2+.

Intracellular free magnesium in frog skeletal muscle fibres measured with ion‐selective micro‐electrodes.

Intracellular free Mg2+ concentration [( Mg2+]i) was measured in frog skeletal muscle fibres, using Mg2+-selective micro-electrodes based on the neutral ligand ETH-1117. In calibration solutions the electrodes showed significant interference from K+, and to a lesser extent from Na+, at concentrations found intracellularly. Therefore, in order to calibrate the electrodes properly, it was necessary first to obtain an accurate value for intracellular free Na+ and K+ concentrations ([Na+]i and [K+]i), using the appropriate liquid ion exchanger micro-electrodes. In fibres from muscles maintained in Ringer solution, the mean value for [Na+]i was 6.2 +/- 0.4 mM (S.E. of mean; n = 20 fibres in five muscles), while [K+]i was 104 +/- 1.7 mM (range 83-122 mM; n = 25 fibres in eight muscles). Due to the substantial variability found for [K+]i, not only between fibres from different muscles, but also between fibres belonging to the same muscle, it was necessary to measure [Mg2+]i and [K+]i simultaneously in the same fibre to determine as accurately as possible the degree of K+ interference on Mg2+-selective micro-electrode response. In nineteen fibres from six muscles maintained in Ringer solution, the mean [K+]i was 91.7 +/- 2.7 mM (range 71-110 mM), while the mean [Mg2+]i was 0.80 +/- 0.07 mM (range 0.2-1.2 mM). The mean resting potential was -79.3 +/- 0.4 mV (S.E. of mean). In fifteen fibres from four muscles equilibrated in Ringer solution containing 0.5 mM-Mg2+, the mean [K+]i was 115.5 +/- 0.1 mM (range 97-129 mM) and the mean [Mg2+]i measured simultaneously in the same fibres was 1.69 +/- 0.21 mM (range 0.2-2.7 mM). The mean resting potential was -83 +/- 0.7 mV. The mean [K+]i and [Mg2+]i found in these fibres was significantly higher (P less than 0.0001) than those measured in fibres from muscles maintained in standard Ringer solution (i.e. without external Mg2+). Possible explanations for this finding are discussed. Whether in the presence (0.5 mM) or in the absence of external Mg2+, our values for [Mg2+]i are distinctly lower than those previously reported by others, using the same type of Mg2+-selective micro-electrodes but calibrated simply from assumptions about the actual level of K+ and Na+ interference on Mg2+-selective micro-electrode response.(ABSTRACT TRUNCATED AT 400 WORDS)

The role of Mg2+ in the regulation of the structural and functional steady-states in rat liver mitochondria

A possible relationship between mitochondrial Mg2+ levels, structural configurations, and functional steady states has been studied in rat liver mitochondria. The results show that the concentration of mitochondrial Mg2+ in respiratory state 4 is definitely higher than in respiratory state 3. The metabolic transition from state 3 to state 4 and vice-versa is associated with reversible influx-efflux of about 10 nmol of Mg2+ per mg protein. The net uptake of this aliquot of Mg2+ is a necessary condition in order for the metabolic transition to state 4, both structurally and functionally, to occur. This process requires a threshold concentration of external Mg2+ greater than 5 mM. The phosphorylative mechanism does not appear to depend on the presence or absence of external Mg2+. The role of Mg2+ on the attainment and maintenance of the structural and functional steady state 4 seems to be correlated with its regulatory effect on the concentration of the mitochondrial P(i).