Specificity and actions of an arylaspartate inhibitor of glutamate transport at the Schaffer collateral-CA1 pyramidal cell synapse

In this study we characterized the pharmacological selectivity and physiological actions of a new arylaspartate glutamate transporter blocker, L-threo-ß-benzylaspartate (L-TBA). At concentrations up to 100 µM, L-TBA did not act as an AMPA receptor (AMPAR) or NMDA receptor (NMDAR) agonist or antagonist when applied to outside-out patches from mouse hippocampal CA1 pyramidal neurons. L-TBA had no effect on the amplitude of field excitatory postsynaptic potentials (fEPSPs) recorded at the Schaffer collateral-CA1 pyramidal cell synapse. Excitatory postsynaptic currents (EPSCs) in CA1 pyramidal neurons were unaffected by L-TBA in the presence of physiological extracellular Mg²⁺ concentrations, but in Mg²⁺-free solution, EPSCs were significantly prolonged as a consequence of increased NMDAR activity. Although L-TBA exhibited approximately four-fold selectivity for neuronal EAAT3 over glial EAAT1/EAAT2 transporter subtypes expressed in Xenopus oocytes, the L-TBA concentration-dependence of the EPSC charge transfer increase in the absence of Mg²⁺ was the same in hippocampal slices from EAAT3 +/+ and EAAT3 −/− mice, suggesting that TBA effects were primarily due to block of glial transporters. Consistent with this, L-TBA blocked synaptically evoked transporter currents in CA1 astrocytes with a potency in accord with its block of heterologously expressed glial transporters. Extracellular recording in the presence of physiological Mg²⁺ revealed that L-TBA prolonged fEPSPs in a frequency-dependent manner by selectively increasing the NMDAR-mediated component of the fEPSP during short bursts of activity. The data indicate that glial glutamate transporters play a dominant role in limiting extrasynaptic transmitter diffusion and binding to NMDARs. Furthermore, NMDAR signaling is primarily limited by voltage-dependent Mg²⁺ block during low-frequency activity, while the relative contribution of transport increases during short bursts of higher frequency signaling.     

Cyclic ADP ribose-dependent Ca2+ release by group I metabotropic glutamate receptors in acutely dissociated rat hippocampal neurons

Group I metabotropic glutamate receptors (group I mGluRs; mGluR1 and mGluR5) exert diverse effects on neuronal and synaptic functions, many of which are regulated by intracellular Ca²⁺. In this study, we characterized the cellular mechanisms underlying Ca²⁺ mobilization induced by (RS)-3,5-dihydroxyphenylglycine (DHPG; a specific group I mGluR agonist) in the somata of acutely dissociated rat hippocampal neurons using microfluorometry. We found that DHPG activates mGluR5 to mobilize intracellular Ca²⁺ from ryanodine-sensitive stores via cyclic adenosine diphosphate ribose (cADPR), while the PLC/IP₃ signaling pathway was not involved in Ca²⁺ mobilization. The application of glutamate, which depolarized the membrane potential by 28.5±4.9 mV (n = 4), led to transient Ca²⁺ mobilization by mGluR5 and Ca²⁺ influx through L-type Ca²⁺ channels. We found no evidence that mGluR5-mediated Ca²⁺ release and Ca²⁺ influx through L-type Ca²⁺ channels interact to generate supralinear Ca²⁺ transients. Our study provides novel insights into the mechanisms of intracellular Ca²⁺ mobilization by mGluR5 in the somata of hippocampal neurons.     

An inhibitory effect of extracellular Ca2+ on Ca2+-dependent exocytosis

Aim

Neurotransmitter release is elicited by an elevation of intracellular Ca²⁺ concentration ([Ca²⁺]_i). The action potential triggers Ca²⁺ influx through Ca²⁺ channels which causes local changes of [Ca²⁺]_i for vesicle release. However, any direct role of extracellular Ca²⁺ (besides Ca²⁺ influx) on Ca²⁺-dependent exocytosis remains elusive. Here we set out to investigate this possibility on rat dorsal root ganglion (DRG) neurons and chromaffin cells, widely used models for studying vesicle exocytosis.

Results

Using photolysis of caged Ca²⁺ and caffeine-induced release of stored Ca²⁺, we found that extracellular Ca²⁺ inhibited exocytosis following moderate [Ca²⁺]_i rises (2–3 µM). The IC₅₀ for extracellular Ca²⁺ inhibition of exocytosis (ECIE) was 1.38 mM and a physiological reduction (∼30%) of extracellular Ca²⁺ concentration ([Ca²⁺]_o) significantly increased the evoked exocytosis. At the single vesicle level, quantal size and release frequency were also altered by physiological [Ca²⁺]_o. The calcimimetics Mg²⁺, Cd²⁺, G418, and neomycin all inhibited exocytosis. The extracellular Ca²⁺-sensing receptor (CaSR) was not involved because specific drugs and knockdown of CaSR in DRG neurons did not affect ECIE.

Conclusion/Significance

As an extension of the classic Ca²⁺ hypothesis of synaptic release, physiological levels of extracellular Ca²⁺ play dual roles in evoked exocytosis by providing a source of Ca²⁺ influx, and by directly regulating quantal size and release probability in neuronal cells.     

KCa2 channels activation prevents [Ca2+]i deregulation and reduces neuronal death following glutamate toxicity and cerebral ischemia

Exacerbated activation of glutamate receptor-coupled calcium channels and subsequent increase in intracellular calcium ([Ca²⁺]_i) are established hallmarks of neuronal cell death in acute and chronic neurological diseases. Here we show that pathological [Ca²⁺]_i deregulation occurring after glutamate receptor stimulation is effectively modulated by small conductance calcium-activated potassium (K_Ca2) channels. We found that neuronal excitotoxicity was associated with a rapid downregulation of K_Ca2.2 channels within 3 h after the onset of glutamate exposure. Activation of K_Ca2 channels preserved K_Ca2 expression and significantly reduced pathological increases in [Ca²⁺]_i providing robust neuroprotection in vitro and in vivo. These data suggest a critical role for K_Ca2 channels in excitotoxic neuronal cell death and propose their activation as potential therapeutic strategy for the treatment of acute and chronic neurodegenerative disorders.     

The effect of OPA1 on mitochondrial Ca²⁺ signaling

The dynamin-related GTPase protein OPA1, localized in the intermembrane space and tethered to the inner membrane of mitochondria, participates in the fusion of these organelles. Its mutation is the most prevalent cause of Autosomal Dominant Optic Atrophy. OPA1 controls the diameter of the junctions between the boundary part of the inner membrane and the membrane of cristae and reduces the diffusibility of cytochrome c through these junctions. We postulated that if significant Ca²⁺ uptake into the matrix occurs from the lumen of the cristae, reduced expression of OPA1 would increase the access of Ca²⁺ to the transporters in the crista membrane and thus would enhance Ca²⁺ uptake. In intact H295R adrenocortical and HeLa cells cytosolic Ca²⁺ signals evoked with K⁺ and histamine, respectively, were transferred into the mitochondria. The rate and amplitude of mitochondrial [Ca²⁺] rise (followed with confocal laser scanning microscopy and FRET measurements with fluorescent wide-field microscopy) were increased after knockdown of OPA1, as compared with cells transfected with control RNA or mitofusin1 siRNA. Ca²⁺ uptake was enhanced despite reduced mitochondrial membrane potential. In permeabilized cells the rate of Ca²⁺ uptake by depolarized mitochondria was also increased in OPA1-silenced cells. The participation of Na⁺/Ca²⁺ and Ca²⁺/H⁺ antiporters in this transport process is indicated by pharmacological data. Altogether, our observations reveal the significance of OPA1 in the control of mitochondrial Ca²⁺ metabolism.     

Mg2+ as an indicator of nutritional status in marine bacteria

Cells maintain an osmotic pressure essential for growth and division, using organic compatible solutes and inorganic ions. Mg²⁺, which is the most abundant divalent cation in living cells, has not been considered an osmotically important solute. Here we show that under carbon limitation or dormancy native marine bacterial communities have a high cellular concentration of Mg²⁺ (370–940 m) and a low cellular concentration of Na⁺ (50–170 m). With input of organic carbon, the average cellular concentration of Mg²⁺ decreased 6–12-fold, whereas that of Na⁺ increased ca 3–4-fold. The concentration of chlorine, which was in the range of 330–1200 m and was the only inorganic counterion of quantitative significance, balanced and followed changes in the concentration of Mg²⁺+Na⁺. In an osmotically stable environment, like seawater, any major shift in bacterial osmolyte composition should be related to shifts in growth conditions, and replacing organic compatible solutes with inorganic solutes is presumably a favorable strategy when growing in carbon-limited condition. A high concentration of Mg²⁺ in cells may also serve to protect and stabilize macromolecules during periods of non-growth and dormancy. Our results suggest that Mg²⁺ has a major role as osmolyte in marine bacteria, and that the [Mg²⁺]/[Na⁺] ratio is related to its physiological condition and nutritional status. Bacterial degradation is a main sink for dissolved organic carbon in the ocean, and understanding the mechanisms limiting bacterial activity is therefore essential for understanding the oceanic C-cycle. The [Mg²⁺]/[Na⁺]-ratio in cells may provide a physiological proxy for the transitions between C-limited and mineral nutrient-limited bacterial growth in the ocean''s surface layer.     

Effects of Weak Environmental Magnetic Fields on the Spontaneous Bioelectrical Activity of Snail Neurons

We examined the effects of 50-Hz magnetic fields in the range of flux densities relevant to our current environmental exposures on action potential (AP), after-hyperpolarization potential (AHP) and neuronal excitability in neurons of land snails, Helix aspersa. It was shown that when the neurons were exposed to magnetic field at the various flux densities, marked changes in neuronal excitability, AP firing frequency and AHP amplitude were seen. These effects seemed to be related to the intensity, type (single and continuous or repeated and cumulative) and length of exposure (18 or 20 min). The extremely low-frequency (ELF) magnetic field exposures affect the excitability of F1 neuronal cells in a nonmonotonic manner, disrupting their normal characteristic and synchronized firing patterns by interfering with the cell membrane electrophysiological properties. Our results could explain one of the mechanisms and sites of action of ELF magnetic fields. A possible explanation of the inhibitory effects of magnetic fields could be a decrease in Ca²⁺ influx through inhibition of voltage-gated Ca²⁺ channels. The detailed mechanism of effect, however, needs to be further studied under voltage-clamp conditions.     

An endogenous elevation of cGMP increases the excitability of identified insect neurosecretory cells

In the moth, Manduca sexta, the neuropeptide, eclosion hormone, triggers a dramatic rise in the levels of intracellular cGMP within a group of 50 neurons. The cells within this group include the segmentally repeated neurosecretory cell, Cell 27. In this study the effect of cGMP on the excitability of Cell 27 was investigated using intracellular recordings. Prior to its normal elevation in cGMP, Cell 27 exhibited a high spike threshold, but this was lowered dramatically when intracellular cGMP levels increased. The latter was also associated with spontaneous action potentials. This change in excitability did not correspond with changes in either resting potential, input resistance, or action potential amplitude. A similar lowering of threshold was induced by perfusion of 8-bromo-cGMP, whereas 8-bromo-cAMP caused the threshold to increase. Intracellular recordings using various ion substitution paradigms and channel blockers provided evidence which suggests indirectly that Ca²⁺ is mostly responsible for the depolarizing phase of the action potential while a Ca²⁺-activated K⁺ current contributes to the hyperpolarization. The results of these manipulations are consistent with the hypothesis that cGMP may partially increase excitability in Cell 27 by enhancing an inward Ca²⁺ current. Accepted: 31 October 1996     

Lithium reduces apoptosis and autophagy after neonatal hypoxia�Cischemia

Lithium is used in the treatment of bipolar mood disorder. Reportedly, lithium can be neuroprotective in models of adult brain ischemia. The purpose of this study was to evaluate the effects of lithium in a model of neonatal hypoxic–ischemic brain injury. Nine-day-old male rats were subjected to unilateral hypoxia–ischemia (HI) and 2 mmol/kg lithium chloride was injected i.p. immediately after the insult. Additional lithium injections, 1 mmol/kg, were administered at 24-h intervals. Pups were killed 6, 24 or 72 h after HI. Lithium reduced the infarct volume from 24.7±2.9 to 13.8±3.3 mm³ (44.1%) and total tissue loss (degeneration + lack of growth) from 67.4±4.4 to 38.4±5.9 mm³ (43.1%) compared with vehicle at 72 h after HI. Injury was reduced in the cortex, hippocampus, thalamus and striatum. Lithium reduced the ischemia-induced dephosphorylation of glycogen synthase kinase-3β and extracellular signal-regulated kinase, the activation of calpain and caspase-3, the mitochondrial release of cytochrome c and apoptosis-inducing factor, as well as autophagy. We conclude that lithium could mitigate the brain injury after HI by inhibiting neuronal apoptosis. The lithium doses used were in the same range as those used in bipolar patients, suggesting that lithium might be safely used for the avoidance of neonatal brain injury.     

Salinomycin induces calpain and cytochrome c-mediated neuronal cell death

Salinomycin is a polyether antibiotic with properties of an ionophore, which is commonly used as cocciodiostatic drug and has been shown to be highly effective in the elimination of cancer stem cells (CSCs) both in vitro and in vivo. One important caveat for the potential clinical application of salinomycin is its marked neural and muscular toxicity. In the present study we show that salinomycin in concentrations effective against CSCs exerts profound toxicity towards both dorsal root ganglia as well as Schwann cells. This toxic effect is mediated by elevated cytosolic Na⁺ concentrations, which in turn cause an increase of cytosolic Ca²⁺ by means of Na⁺/Ca²⁺ exchangers (NCXs) in the plasma membrane as well as the mitochondria. Elevated Ca²⁺ then leads to calpain activation, which triggers caspase-dependent apoptosis involving caspases 12, 9 and 3. In addition, cytochrome c released from depolarized mitochondria directly activates caspase 9. Combined inhibition of calpain and the mitochondrial NCXs resulted in significantly decreased cytotoxicity and was comparable to caspase 3 inhibition. These findings improve our understanding of mechanisms involved in the pathogenesis of peripheral neuropathy and are important to devise strategies for the prevention of neurotoxic side effects induced by salinomycin.     

Carbenoxolone blocks the light-evoked rise in intracellular calcium in isolated melanopsin ganglion cell photoreceptors

Background

Retinal ganglion cells expressing the photopigment melanopsin are intrinsically photosensitive (ipRGCs). These ganglion cell photoreceptors send axons to several central targets involved in a variety of functions. Within the retina ipRGCs provide excitatory drive to dopaminergic amacrine cells via glutamatergic signals and ipRGCs are coupled to wide-field GABAergic amacrine cells via gap junctions. However, the extent to which ipRGCs are coupled to other retinal neurons in the ganglion cell layer via gap junctions is unclear. Carbenoxolone, a widely employed gap junction inhibitor, greatly reduces the number of retinal neurons exhibiting non-rod, non-cone mediated light-evoked Ca²⁺ signals suggesting extensive intercellular coupling between ipRGCs and non-ipRGCs in the ganglion cell layer. However, carbenoxolone may directly inhibit light-evoked Ca²⁺ signals in ipRGCs independent of gap junction blockade.

Methodology/Principal Findings

To test the possibility that carbenoxolone directly inhibits light-evoked Ca²⁺ responses in ipRGCs, the light-evoked rise in intracellular Ca²⁺ ([Ca²⁺]_i) was examined using fura-2 imaging in isolated rat ipRGCs maintained in short-term culture in the absence and presence of carbenoxolone. Carbenoxolone at 50 and 100 µM concentrations completely abolished the light-evoked rise in [Ca²⁺]_i in isolated ipRGCs. Recovery from carbenoxolone inhibition was variable.

Conclusions/Significance

We demonstrate that the light-evoked rise in [Ca²⁺]_i in isolated mammalian ganglion cell photoreceptors is inhibited by carbenoxolone. Since the light-evoked increase in [Ca²⁺]_i in isolated ipRGCs is almost entirely due to Ca²⁺ entry via L-type voltage-gated calcium channels and carbenoxolone does not inhibit light-evoked action potential firing in ipRGCs in situ, carbenoxolone may block the light-evoked increase in [Ca²⁺]_i in ipRGCs by blocking L-type voltage-gated Ca²⁺ channels. The ability of carbenoxolone to block evoked Ca²⁺ responses must be taken into account when interpreting the effects of this pharmacological agent on retinal or other neuronal circuits, particularly if a change in [Ca²⁺]_i is the output being measured.     

Effects of different ions on resting polarization and on the mass receptor potential of carotid body chemosensors

The carotid body and its own nerve were removed from cats anesthetized with sodium pentobarbital and placed in an air gap system; the carotid body was bathed in modified Locke's solution equilibrated with 50% O₂ in N₂, pH 7.43 at 35°C. The sensory discharges, changes in “resting” receptor polarization and the mass receptor potential evoked by ACh or NaCN were recorded with nonpolarizable electrodes placed across the gap. Receptor potentials and sensory discharges evoked by ACh showed an appreciable increase in amplitude and frequency when the preparation was bathed in eserinized Locke. Eserine did not change appreciably the responses evoked by NaCN. Excessive depolarization elicited by either ACh or NaCN was accompanied by sensory discharge block. Removal of K⁺ ions from the bathing solution induced receptor hyperpolarization and an increase in the amplitude of the evoked receptor potentials. An increase of K⁺ concentration had the opposite effect. Reduction of Na⁺ or NaCl to one half, or total removal of this salt, induced an initial reduction and later disappearance of the sensory discharges, some receptor hyperpolarization and a reduction in the amplitude of the evoked receptor potentials. Reduction or removal of Ca⁺⁺ produced receptor depolarization, a marked depression of the evoked receptor potentials, an increase in the frequency of the sensory discharges and a reduction in the amplitude of the nerve action potentials. High Ca⁺⁺ or Mg⁺⁺ had little or no effect on action potential amplitude or resting polarization, but decreased sensory discharge frequency and the evoked receptor potentials. Total or partial replacement of Ca⁺⁺ with Mg⁺⁺ induced complex effects: (1) receptor depolarization which occurred in low Ca⁺⁺, was prevented by addition of Mg⁺⁺ ions; (2) the amplitude of the evoked receptor potentials was depressed; (3) the nerve discharge frequency was reduced as it was in high Mg⁺⁺ solutions; and (4) the amplitude of the nerve action potentials was reduced as it was in low Ca⁺⁺ solutions. Temperature had a marked effect on the chemoreceptors since a t high temperatures the receptors were depolarized and the discharge frequency increased. The baseline discharge and responses evoked by ACh or NaCN were depressed at low temperatures. The results are discussed in terms of possible receptor mechanisms influenced by the different ions.     

Minocycline inhibition of monocyte activation correlates with neuronal protection in SIV neuroAIDS

Background

Minocycline is a tetracycline antibiotic that has been proposed as a potential conjunctive therapy for HIV-1 associated cognitive disorders. Precise mechanism(s) of minocycline''s functions are not well defined.

Methods

Fourteen rhesus macaques were SIV infected and neuronal metabolites measured by proton magnetic resonance spectroscopy (¹H MRS). Seven received minocycline (4 mg/kg) daily starting at day 28 post-infection (pi). Monocyte expansion and activation were assessed by flow cytometry, cell traffic to lymph nodes, CD16 regulation, viral replication, and cytokine production were studied.

Results

Minocycline treatment decreased plasma virus and pro-inflammatory CD14+CD16+ and CD14^loCD16+ monocytes, and reduced their expression of CD11b, CD163, CD64, CCR2 and HLA-DR. There was reduced recruitment of monocyte/macrophages and productively infected cells in axillary lymph nodes. There was an inverse correlation between brain NAA/Cr (neuronal injury) and circulating CD14+CD16+ and CD14^loCD16+ monocytes. Minocycline treatment in vitro reduced SIV replication CD16 expression on activated CD14+CD16+ monocytes, and IL-6 production by monocytes following LPS stimulation.

Conclusion

Neuroprotective effects of minocycline are due in part to reduction of activated monocytes, monocyte traffic. Mechanisms for these effects include CD16 regulation, reduced viral replication, and inhibited immune activation.     

Inhibitory control over Ca(2+) sparks via mechanosensitive channels is disrupted in dystrophin deficient muscle but restored by mini-dystrophin expression

Background

In dystrophic skeletal muscle, osmotic stimuli somehow relieve inhibitory control of dihydropyridine receptors (DHPR) on spontaneous sarcoplasmic reticulum elementary Ca²⁺ release events (ECRE) in high Ca²⁺ external environments. Such ‘uncontrolled’ Ca²⁺ sparks were suggested to act as dystrophic signals. They may be related to mechanosensitive pathways but the mechanisms are elusive. Also, it is not known whether truncated dystrophins can correct the dystrophic disinhibition.

Methodology/Principal Findings

We recorded ECRE activity in single intact fibers from adult wt, mdx and mini-dystrophin expressing mice (MinD) under resting isotonic conditions and following hyper-/hypo-osmolar external shock using confocal microscopy and imaging techniques. Isotonic ECRE frequencies were small in wt and MinD fibers, but were markedly increased in mdx fibers. Osmotic challenge dramatically increased ECRE activity in mdx fibers. Sustained osmotic challenge induced marked exponential ECRE activity adaptation that was three times faster in mdx compared to wt and MinD fibers. Rising external Ca²⁺ concentrations amplified osmotic ECRE responses. The eliminated ECRE suppression in intact osmotically stressed mdx fibers was completely and reversibly resuscitated by streptomycine (200 µM), spider peptide GsMTx-4 (5 µM) and Gd³⁺ (20 µM) that block unspecific, specific cationic and Ca²⁺ selective mechanosensitive channels (MsC), respectively. ECRE morphology was not substantially altered by membrane stress. During hyperosmotic challenge, membrane potentials were polarised and a putative depolarisation through aberrant MsC negligible excluding direct activation of ECRE through tubular depolarisation.

Conclusions/Significance

Dystrophin suppresses spontaneous ECRE activity by control of mechanosensitive pathways which are suggested to interact with the inhibitory DHPR loop to the ryanodine receptor. MsC-related disinhibition prevails in dystrophic muscle and can be resuscitated by transgenic mini-dystrophin expression. Our results have important implications for the pathophysiology of DMD where abnormal MsC in dystrophic muscle confer disruption of microdomain Ca²⁺ homeostasis. MsC blockers should have considerable therapeutic potential if more muscle specific compounds can be found.     

Heat and exercise acclimation increases intracellular levels of Hsp72 and inhibits exercise-induced increase in intracellular and plasma Hsp72 in humans

In order to verify the effects of heat and exercise acclimation (HA) on resting and exercise-induced expression of plasma and leukocyte heat shock protein 72 (Hsp72) in humans, nine healthy young male volunteers (25.0 ± 0.7 years; 80.5 ± 2.0 kg; 180 ± 2 cm, mean ± SE) exercised for 60 min in a hot, dry environment (40 ± 0°C and 45 ± 0% relative humidity) for 11 days. The protocol consisted of running on a treadmill using a controlled hyperthermia technique in which the work rate was adjusted to elevate the rectal temperature by 1°C in 30 min and maintain it elevated for another 30 min. Before and after the HA, the volunteers performed a heat stress test (HST) at 50% of their individual maximal power output for 90 min in the same environment. Blood was drawn before (REST), immediately after (POST) and 1 h after (1 h POST) HST, and plasma and leukocytes were separated and stored. Subjects showed expected adaptations to HA: reduced exercise rectal and mean skin temperatures and heart rate, and augmented sweat rate and exercise tolerance. In HST1, plasma Hsp72 increased from REST to POST and then returned to resting values 1 h POST (REST: 1.11 ± 0.07, POST: 1.48 ± 0.10, 1 h POST: 1.22 ± 0.11 ng mL⁻¹; p < 0.05). In HST2, there was no change in plasma Hsp72 (REST: 0.94 ± 0.08, POST: 1.20 ± 0.15, 1 h POST: 1.17 ± 0.16 ng mL⁻¹; p > 0.05). HA increased resting levels of intracellular Hsp72 (HST1: 1 ± 0.02 and HST2: 4.2 ± 1.2 density units, p < 0.05). Exercise-induced increased intracellular Hsp72 expression was observed on HST1 (HST1: REST, 1 ± 0.02 vs. POST, 2.9 ± 0.9 density units, mean ± SE, p < 0.05) but was inhibited on HST2 (HST2: REST, 4.2 ± 1.2 vs. POST, 4.4 ± 1.1 density units, p > 0.05). Regression analysis showed that the lower the pre-exercise expression of intracellular Hsp72, the higher the exercise-induced increase (R = −0.85, p < 0.05). In conclusion, HA increased resting leukocyte Hsp72 levels and inhibited exercise-induced expression. This intracellular adaptation probably induces thermotolerance. In addition, the non-increase in plasma Hsp72 after HA may be related to lower stress at the cellular level in the acclimated individuals.     

Requirement of a Relatively High Threshold Level of Mg2+ for Cell Growth of a Rhizoplane Bacterium, Sphingomonas yanoikuyae EC-S001

Mg²⁺ is one of the essential elements for bacterial cell growth. The presence of the magnesium cation (Mg²⁺) in various concentrations often affects cell growth restoration in plant-associating bacteria. This study attempted to determine whether Mg²⁺ levels in Sphingomonas yanoikuyae EC-S001 affected cell growth restoration in the host plant and what the threshold level is. S. yanoikuyae EC-S001, isolated from the rhizoplane of spinach seedlings grown from surface-sterilized seeds under aseptic conditions, displayed uniform dispersion and attachment throughout the rhizoplane and phylloplane of the host seedlings. S. yanoikuyae EC-S001 did not grow in potato-dextrose broth medium but grew well in an aqueous extract of spinach leaves. Chemical investigation of the growth factor in the spinach leaf extract led to identification of the active principle as the magnesium cation. A concentration of ca. 0.10 mM Mg²⁺ or more allowed S. yanoikuyae EC-S001 to grow in potato-dextrose broth medium. Some saprophytic and/or diazotrophic bacteria used in our experiment were found to have diverse threshold levels for their Mg²⁺ requirements. For example, Burkholderia cepacia EC-K014, originally isolated from the rhizoplane of a Melastoma sp., could grow even in Mg²⁺-free Hoagland's no. 2 medium with saccharose and glutamine (HSG medium) and requires a trace level of Mg²⁺ for its growth. In contrast, S. yanoikuyae EC-S001, together with Bacillus subtilis IFO12113, showed the most drastic restoring responses to subsequent addition of 0.98 mM Mg²⁺ to Mg²⁺-free HSG medium. Our studies concluded that Mg²⁺ is more than just the essential trace element needed for cell growth restoration in S. yanoikuyae EC-S001 and that certain nonculturable bacteria may require a higher concentration of Mg²⁺ or another specific essential element for their growth.     

On conduction in a bacterial sodium channel

Voltage-gated Na⁺-channels are transmembrane proteins that are responsible for the fast depolarizing phase of the action potential in nerve and muscular cells. Selective permeability of Na⁺ over Ca²⁺ or K⁺ ions is essential for the biological function of Na⁺-channels. After the emergence of the first high-resolution structure of a Na⁺-channel, an anionic coordination site was proposed to confer Na⁺ selectivity through partial dehydration of Na⁺ via its direct interaction with conserved glutamate side chains. By combining molecular dynamics simulations and free-energy calculations, a low-energy permeation pathway for Na⁺ ion translocation through the selectivity filter of the recently determined crystal structure of a prokaryotic sodium channel from Arcobacter butzleri is characterised. The picture that emerges is that of a pore preferentially occupied by two ions, which can switch between different configurations by crossing low free-energy barriers. In contrast to K⁺-channels, the movements of the ions appear to be weakly coupled in Na⁺-channels. When the free-energy maps for Na⁺ and K⁺ ions are compared, a selective site is characterised in the narrowest region of the filter, where a hydrated Na⁺ ion, and not a hydrated K⁺ ion, is energetically stable.     

Rem2-targeted shRNAs reduce frequency of miniature excitatory postsynaptic currents without altering voltage-gated Ca²⁺ currents

Ca²⁺ influx through voltage-gated Ca²⁺ channels (VGCCs) plays important roles in neuronal cell development and function. Rem2 is a member of the RGK (Rad, Rem, Rem2, Gem/Kir) subfamily of small GTPases that confers potent inhibition upon VGCCs. The physiologic roles of RGK proteins, particularly in the brain, are poorly understood. Rem2 was implicated in synaptogenesis through an RNAi screen and proposed to regulate Ca²⁺ homeostasis in neurons. To test this hypothesis and uncover physiological roles for Rem2 in the brain, we investigated the molecular mechanisms by which Rem2 knockdown affected synaptogenesis and Ca²⁺ homeostasis in cultured rat hippocampal neurons. Expression of a cocktail of shRNAs targeting rat Rem2 (rRem2) reduced the frequency of miniature excitatory postsynaptic currents (mEPSCs) measured 10 d after transfection (14 d in vitro), but did not affect mEPSC amplitude. VGCC current amplitude after rRem2-targeted knockdown was not different from that in control cells, however, at either 4 or 10 d post transfection. Co-expression of a human Rem2 that was insensitive to the shRNAs targeting rRem2 was unable to prevent the reduction in mEPSC frequency after rRem2-targeted knockdown. Over-expression of rRem2 resulted in 50% reduction in VGCC current, but neither the mEPSC frequency nor amplitude was affected. Taken together, the observed effects upon synaptogenesis after shRNA treatment are more likely due to mechanisms other than modulation of VGCCs and Ca²⁺ homeostasis, and may be independent of Rem2. In addition, our results reveal a surprising lack of contribution of VGCCs to synaptogenesis during early development in cultured hippocampal neurons.     

Regulation of Hippocampal and Behavioral Excitability by Cyclin-Dependent Kinase 5

Cyclin-dependent kinase 5 (Cdk5) is a proline-directed serine/threonine kinase that has been implicated in learning, synaptic plasticity, neurotransmission, and numerous neurological disorders. We previously showed that conditional loss of Cdk5 in adult mice enhanced hippocampal learning and plasticity via modulation of calpain-mediated N-methyl-D-aspartic acid receptor (NMDAR) degradation. In the present study, we characterize the enhanced synaptic plasticity and examine the effects of long-term Cdk5 loss on hippocampal excitability in adult mice. Field excitatory post-synaptic potentials (fEPSPs) from the Schaffer collateral CA1 subregion of the hippocampus (SC/CA1) reveal that loss of Cdk5 altered theta burst topography and enhanced post-tetanic potentiation. Since Cdk5 governs NMDAR NR2B subunit levels, we investigated the effects of long-term Cdk5 knockout on hippocampal neuronal excitability by measuring NMDAR-mediated fEPSP magnitudes and population-spike thresholds. Long-term loss of Cdk5 led to increased Mg²⁺-sensitive potentials and a lower threshold for epileptiform activity and seizures. Biochemical analyses were performed to better understand the role of Cdk5 in seizures. Induced-seizures in wild-type animals led to elevated amounts of p25, the Cdk5-activating cofactor. Long-term, but not acute, loss of Cdk5 led to decreased p25 levels, suggesting that Cdk5/p25 may be activated as a homeostatic mechanism to attenuate epileptiform activity. These findings indicate that Cdk5 regulates synaptic plasticity, controls neuronal and behavioral stimulus-induced excitability and may be a novel pharmacological target for cognitive and anticonvulsant therapies.