Accepted Manuscript Lithium ions in nanomolar concentration modulate glycine-activated chloride current in rat hippocampal neurons E.I. Solntseva, J.V. Bukanova, R.V. Kondratenko, V.G. Skrebitsky PII:
S0197-0186(16)30016-X
DOI:
10.1016/j.neuint.2016.02.007
Reference:
NCI 3821
To appear in:
Neurochemistry International
Received Date: 30 December 2015 Revised Date:
5 February 2016
Accepted Date: 9 February 2016
Please cite this article as: Solntseva, E.I., Bukanova, J.V., Kondratenko, R.V., Skrebitsky, V.G., Lithium ions in nanomolar concentration modulate glycine-activated chloride current in rat hippocampal neurons, Neurochemistry International (2016), doi: 10.1016/j.neuint.2016.02.007. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Lithium ions in nanomolar concentration modulate glycine-activated chloride current in rat hippocampal neurons E.I.Solntseva, J.V.Bukanova, R.V.Kondratenko, V.G.Skrebitsky Research Center of Neurology, Moscow, Russia Corresponding author: Elena I. Solntseva, [email protected]; [email protected];
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tel: +79096427758, fax: +74959172382
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Mail address: 105064 per.Obukha,5, Research Center of Neurology Moscow Russia
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Abstract
Lithium salts are successfully used to treat bipolar disorder. At the same time, according to recent data lithium may be considered as a candidate medication for the treatment of neurodegenerative disorders. The mechanisms of therapeutic action of lithium have not been fully elucidated. In particular, in the literature there are no data on the effect of lithium on the glycine receptors. In the present study we investigated the effect of Li+ on glycine-activated
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chloride current (IGly) in rat isolated pyramidal hippocampal neurons using patch-clamp technique. The effects of Li+ were studied with two glycine concentrations: 100 µM (EC50) and 500 µM (nearly saturating). Li+ was applied to the cell in two ways: first, by 600 ms coapplication with glycine through micropipette (short application), and, second, by addition to an
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extracellular perfusate for 10 min (longer application). Li+ was used in the range of
concentrations of 1 nM – 1 mM. Short application of Li+ caused two effects: (1) an acceleration
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of desensitization (a decrease in the time of half-decay, or “τ”) of IGly induced by both 100 µM and 500 µM glycine, and (2) a reduction of the peak amplitude of the IGly, induced by 100 µM, but not by 500 µM glycine. Both effects were not voltage-dependent. Dose-response curves for both effects were N-shaped with two maximums at 100 nM and 1mM of Li+ and a minimum at 1 µM of Li+. This complex form of dose-response may indicate that the process activated by high concentrations of lithium inhibits the process that is sensitive to low concentrations of lithium.
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Longer application of Li+ caused similar effects, but in this case 1µM lithium was effective and the dose-effect curves were not N-shaped. The inhibitory effect of lithium ions on glycineactivated current suggests that lithium in low concentrations is able to modulate tonic inhibition in the hippocampus. This important property of lithium should be considered when using this
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drug as a therapeutic agent.
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Key words: Glycine receptor; Hippocampus; Li+; patch clamp
Introduction
For many decades lithium salts have been successfully used to treat bipolar disorder (BPD) (for a review: Curran and Ravindran, 2014). At the same time, over recent years evidence of the positive effect of lithium ions in treatment of other diseases, including neuro-, cardio- and nephroprotection has been accumulated (for a review: Malhi et al., 2013; Plotnikov et al., 2014; Vo et al., 2015). The possibility of neuroprotective effects of lithium is particularly relevant. According to recent data lithium may be considered as a candidate medication for the treatment of the most significant neurodegenerative disorders such as Alzheimer’s and Parkinson’s diseases
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(for a review: Vo et al., 2015). In in vitro experiments lithium has been shown to facilitate neural plasticity (Gray and McEwen, 2013) and regulate intracellular calcium levels as well as calcium turnover (Sourial-Bassillious et al., 2009). However, to date no definitive mechanism for lithium effects has been established. It has been proposed that lithium exerts its therapeutic effects by interfering with signal transduction through G-protein-coupled receptor (GPCR) pathways or by direct inhibition of specific targets in signaling systems, including inositol monophosphatase and
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glycogen synthase kinase-3 (GSK-3) (for a review: Beaulieu et al., 2009; Plotnikov et al., 2014). Ionic channels of neuronal membrane are also considered as potential targets for lithium ions. The experimental results show that lithium can modulate both ligand-activated and voltagegated channels. However, in some cases the effective lithium concentrations exceeded
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therapeutic dose of 0.6-1.2 mM in neurons, with the concentrations >2 mM considered toxic (Plotnikov et al., 2014). Lithium at 10 mM was found to facilitate AMPAR currents in hippocampal CA1 cells by selectively increasing the probability of channel opening (Gebhardt
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and Cull-Candy, 2010). Na+-dependent K+ current responsible for after-hyperpolarization was shown to be reduced in the presence of 1-10 mM lithium, which resulted in increased neuronal excitability (Safronov and Vogel, 1996; Butler-Munro et al., 2010). In adrenal chromaffin cells, lithium inhibited voltage-dependent sodium channels in a concentration-dependent manner (IC50=23.4 mM) (Yanagita et al., 2007). Finally, dual regulation of G-protein-activated K+-
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channels (GIRK) was described in mouse hippocampal neurons where 1-2 mM Li+ increased GIRK basal current but attenuated neurotransmitter-evoked GIRK-current (Tselnicker et al., 2014). In our work, we demonstrate for the first time, that Li+ at nanomolar concentrations can modulate glycine-activated chloride current in rat hippocampus neurons.
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Glycine is a crucial inhibitory neurotransmitter acting on specific glycine receptors (GlyRs), whose localization can be both synaptic and extra-synaptic (Lynch 2009; Song et al.,
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2006). Extra-synaptic GlyRs in hippocampus provide a tonic inhibition (Xu and Gong, 2010), which is very important for information processing within a neuronal network and disturbance of which can contribute to many pathophysiological processes. Numerous structurally diverse compounds have been shown to modulate GlyRs, including neurosteroids, alcohols, anesthetics, cannabinoids, antagonists of 5-HT3 receptor, ginkgolide B, cyclothiazide and quercetin (for a review: Xu and Gong, 2010; Yevenes and Zeilhofer, 2011). Recently we demonstrated the ability of cyclic nucleotides (cAMP and cGMP) and beta-amyloid peptide to exert extracellular modulation of GlyRs (Bukanova et al., 2014a, 2014b). A lot of attention was given to the study of GlyRs modulation by cations (for a review: Lynch, 2004; Xu and Gong, 2010). Divalent cation Zn2+ was shown to modulate GlyRs extracellularly in a bidirectional manner depending on its concentration. There is no convincing evidence of the ability of other metal ions to mimic the
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biphasic action of zinc. However, the potentiating site is recognized by several metal ions with the following potency sequence: zinc > lanthanide > lead > cobalt, whereas the inhibitory site exhibits a different potency sequence: zinc > copper > nickel (for a review: Lynch, 2004). 100 µM of Ba2+, Sr2+, Mn2+, Co2+, Cd2+ and Al3+ were shown to have no effect on GlyR currents in rat septal neurons (Kumamoto and Murata, 1996). We have shown in our previous study conducted in rat hippocampus neurons that Fe2+ and Fe3+ can modulate glycine-evoked current
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(IGly) in a similar manner. These ions in micromolar concentrations caused an acceleration of desensitization and a decrease of peak amplitude of IGly (Solntseva et al., 2015). We could not find any data on Li+ influence on IGly in the available literature. Thus, our data on the effect of lithium on the IGly can help to elucidate the mechanisms of therapeutic action of lithium, and to
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understand the functional properties of glycine receptors. 2. Material and methods
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2.1. Cell preparation
All procedures were performed in accordance with the institutional guidelines on the care and use of experimental animals set by the Russian Academy of Sciences. The cells were isolated from transverse hippocampal slices as described in detail elsewhere (Vorobjev 1991). Briefly, the slices (200-500 µm) of Wistar rats (11-14 days of age) hippocampus were incubated at room
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temperature for at least 2 h in a solution containing the following components (in mM): 124 NaCl, 3 KC1, 2 CaCl2, 2 MgSO4, 25 NaHCO3, 1.3 NaH2PO4, 10 D-glucose, pH 7.4. The saline was continuously stirred and bubbled with carbogen (95% 02 + 5% CO2). Single pyramidal neurons from CA3 were isolated from the stratum pyramidale by a vibrating fused glass pipette
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with a spherical tip (Vorobjev 1991).
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2.2. Current recordings
Glycine-activated currents in isolated neurons were induced by a step application of agonist for 600 ms with 30-40 s intervals. Transmembrane currents were recorded using a conventional patch-clamp technique in the whole-cell configuration. Patch-clamp electrodes had a tip resistance of ~2 MΩ. The solution in the recording pipette contained the following (in mM): 40 CsF, 100 CsCl, 0.5 CaCl2, 5 EGTA, 3 MgCl2, 4 NaATP, 5 HEPES, 4 ATP, pH 7.3. The composition of extracellular solution was as follows (in mM): 140 NaCl, 3 KCl, 3 CaCl2, 3 MgCl2, 10 D-glucose, 10 HEPES hemisodium, pH 7.4. The speed of perfusion was 0.6 ml/min. Recording of the currents was performed using EPC7 patch-clamp amplifier (HEKA Electronik, Germany). Unless noted otherwise, the holding potential was maintained at -70 mV.
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Transmembrane currents were filtered at 3 kHz, stored and analyzed with IBM-PC computer, using homemade software. 2.3. Drug application Glycine was applied through glass capillary, 0.1 mm in diameter, which could be rapidly displaced laterally under control of home-made software (Vorobjev et al., 1996). The system
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allows a complete exchange of external solution surrounding the neuron within 20 ms. Li+ was applied to the cell in two ways. In the first set of experiments Li+ was co-applied with glycine through micropipette during 600 ms (short application), and in the second series of experiments, Li+ was added in an extracellular perfusate for 10 min (longer application) using two reservoirs
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system. The speed of perfusion was 0.6 ml/min. To avoid the reduction in the concentration of lithium during the application of glycine, we added lithium in corresponding concentration also
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to the glycine-containing pipette. 2.3. Reagents
All the drugs were purchased from “Sigma”. LiCl was used as the source of Li+. The tested substances were dissolved in distilled water to make 0.1-1mM stock solution, which was
2.4. Data analysis
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dissolved in external saline to their final concentration immediately before the experiments.
All statistical analysis was performed with the help of Prism Graphpad software. All comparisons were made with one-way repeated measures ANOVA at a significance level of p =
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0.05. In results descriptions, mean and standard error of mean (SEM) are specified. The meanings of asterisks (probability levels) in figures is the following: *P < 0.05, **P < 0.01, ***P
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< 0.001. Results
3.1. Glycine activates chloride currents in rat hippocampal neurons Short application of glycine for 600 ms on pyramidal neurons evoked chloride currents (IGly) which characteristics were described in details in our previous works (Bukanova et al., 2014a, 2014b). Shortly, the amplitude of IGly depended on glycine concentration with EC50 value of 90 ± 7 µM. The average value of the reversal potential of IGly -9.8 ± 0.9 mV matched well the chloride reversal potential calculated for the chloride concentrations used (-9.5 mV). In this paper, the effects of Li+ were studied with two glycine concentrations: 100 µM and 500 µM. Short (600 ms) and longer (10 min) applications of Li+ were used.
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3.2. The effect of short application of Li+ on IGly induced by 100 µM glycine Application of Li+ alone in different concentrations (1 nM – 1 mM) did not evoke any direct membrane response. When co-applied with 100 µM glycine, Li+ rapidly, reversibly and in a dose-dependent manner changed the IGly in all cells tested (n=12). With this glycine concentration, Li+ reduced the IGly peak amplitude and accelerated the desensitization of the current. To quantitatively assess the alteration of current kinetics, we measured the time of half-
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decay of the IGly (τ) in the absence and presence of Li+. Figure 1 shows representative traces of IGly, induced by 100 µM glycine, obtained in control and in the presence of various
concentrations of lithium from 1 nM to 1 mM (Fig.1A), as well as concentration dependence of Li+ effect on the normalized peak amplitude (Fig.1B) and normalized τ of IGly (Fig.1C). It can be
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seen that both dose-response curves are N-shaped, which is due to the fact that the lithium concentration of 1 µM is ineffective. Such a deflection on the dose-response suggests that there
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are at least two binding sites for lithium with different affinity, and low-affinity site inhibits the high affinity site. Accordingly, each curve has two maximums at 100 nM and 1 mM lithium. At these Li+ concentrations, the corresponding values of the peak amplitude of IGly are reduced to 0.83 ± 0.03 (P < 0.001) and 0.85 ±0.05 (P < 0.05) of the control value. The corresponding values of τ are reduced to 0.59 ± 0.04 (P < 0.001) and 0.42 ± 0.04 (P < 0.001). Fig.2 shows the time
applied with glycine.
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course of one typical experiment where different concentrations of Li+ (full circles) were co-
3.3. The effect of short application of Li+ on IGly induced by 500 µM glycine
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In contrast to the experiments with 100 µM glycine, where lithium caused a reduction both in the peak amplitude and τ of the IGly, in the experiments with 500 µM glycine, lithium induced only acceleration of glycine current desensitization and did not change its peak amplitude (Fig.3,
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n=8). The effect of Li+ on IGly desensitization was similar in experiments with 500 µM glycine and 100 µM glycine. A concentration dependence of Li+ effect on the normalized τ of IGly induced by 500 µM glycine was also N-shaped with deflection at 1 µM Li+ (Fig.3A and C). Two maximums were at 100 nM and 1mM Li+, and the values of τ were reduced to 0.46 ± 0.07 (P < 0.001) and 0.40 ± 0.05 (P < 0.001), correspondingly. 3.4 Voltage-independence of Li+ effects on the IGly . It was found in the experiments with 100 µM glycine, that the effect of Li+ on both peak amplitude and τ of the IGly did not depend on membrane holding potential (Fig.4). Figure 4A shows representative traces of IGly, induced by 100 µM glycine, recorded from one cell. The
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currents were obtained in control (left) and in the presence of 100 nM Li+ (right) at different membrane potentials varying from -90 mV to +30 mV. It can be seen that the changes, induced by Li+, are equally pronounced at negative and positive membrane potentials. The average values (n=6) of the peak amplitude and τ of glycine current recorded at various membrane potentials are shown in Fig.4B and 4C. It is important to note that reversal potential of the IGly peak (-9.8 ± 0.9 mV) remained unchanged in the presence of Li+ (Fig.4B).
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3.5. The effects of longer application of Li+ on the IGly.
In the second series of experiments, Li+ was added to an extracellular perfusate for 10 min (n=8). It has been found that, in general, Li+ has the same effect on the IGly during the short (600 ms)
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and long-term (10 min) exposure. Prolonged application of Li+ also caused a decrease in the peak amplitude and in the time of half-decay of the IGly induced by 100 µM glycine, and a decrease in the time of half-decay without changing the peak amplitude of the IGly induced by 500 µM
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glycine. These effects were reversible after 10-15 minute washing with control solution. At the same time, prolonged application of lithium revealed one distinctive feature: the dose-effect curve was smooth with this method of application. Figure 5 shows concentration dependence of Li+ effects on the normalized IGly peak and τ measured 10 min after Li+ addition to the perfusate. It can be seen that in contrast to short applications, when 1 µM of lithium was not effective,
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continuous application of lithium in this concentration caused a significant effect on the IGly, namely, a reduction of peak amplitude to 0.88 ± 0.04 (P < 0.05) (Fig.5A) and of τ to 0.65 ± 0.05 (P < 0.001) (Fig.5C) of the control value in experiments with 100 µM glycine, and a reduction of
(Fig.5D).
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Discussion
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τ to 0.61 ± 0.05 (P < 0.001) of the control value in the experiments with 500 µM glycine
In the present paper, we studied chloride current activated by 100 and 500 µM glycine (IGly). These concentrations of glycine look too high compared to its physiological concentration in the hippocampus, which do not exceed a few micromoles. However, the GlyRs can be also activated by other agonists, taurine and beta-alanine, which concentration in hipppocampus are relatively high (Mori et al., 2002; Shibanoki et al., 1993). The total concentration of these three agonists of GlyRs may approach high micromolar level in hippocampus. In our experiments, we demonstrate for the first time the capability of Li+ to modulate IGly. The threshold effective concentration of Li+ was 10 nM, which is significantly lower than therapeutic doses of the drug (0.6-1.2 mM, Plotnikov et al., 2014). We tested Li+ in the range of concentrations of 1 nM – 1mM and studied it using two concentrations of glycine, 100 µM and
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500 µM. Two effects of Li+ on IGly were observed: a reduction of the peak amplitude and an acceleration of desensitization, which is quantified by a decrease in the time of half-decay (τ) of IGly. The second effect was quantitatively stronger than the first one. Both effects were voltageindependent, so the possibility that Li+ ions modulate IGly by affecting electrostatic forces at the channel pore seems unlikely. The effect of acceleration of desensitization was similar for currents induced by 100 µM and 500 µM glycine. Therefore, this effect of Li+ did not depend on
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glycine concentration and may be considered noncompetitive, suggesting that lithium ions affect glycine receptor-channel complex at a site distinct from glycine binding site. Another
characteristic of lithium action was observed with regard to the peak current amplitude. Reduction of the peak amplitude by lithium was observed only for 100 µM glycine, and with 500
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µM glycine the effect disappeared. Therefore, this effect can be regarded as a competitive, and the site responsible for it may be located near the glycine binding site. Thus, the two described
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effects of lithium appear to be realized through the sites located in different loci. Dependence of the effect of certain other substances on the GlyR-induced currents on the concentration of glycine was described by other authors. For example, it was shown that the cannabinoids augment the IGly at low doses of the glycine (EC50; Lozovaya et al., 2005; Hejazi et al., 2006; Yang et al., 2008). The same dependence was observed in experiments with ginkgolide B (BN52021)
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(Kondratskaya et al., 2002).
In experiments with short application of Li+ the dose-effect curves were N-shaped for both the peak amplitude and τ. A deflection on the dose-response was at 1 µM Li+ for both effects. This complex form of dose-response may indicate that the process activated by high
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concentrations of lithium inhibits the process that is sensitive to low concentrations of lithium. This assumption applies to both observed effects, reduction of the peak amplitude and
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acceleration of desensitization. For each of the two described effects one can assume the existence of a pair of sites binding lithium, and in each pair the low affinity site inhibits the high affinity site. However, in experiments with prolonged application of lithium (10 min), doseresponse curves lost their N-shaped form, because the concentration of lithium of 1 µM became efficient. So, in this case other more complicated mechanisms may be at work. Literature data point to a complex dose-effect relationship of other drugs which modulate the IGly. Thus, the concentration dependence for both potentiating and inhibitory effects of ethanol on the IGly has been shown to have a bell-shaped appearance (Ye et al., 2001a and 2001b). In our previous works we have shown that short application of some other substances, namely, cyclic nucleotides (cAMP and cGMP) (Bukanova et al., 2014a), beta-amyloid
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peptide(25-35) (Bukanova et al., 2014b) and Fe ions (Solntseva et al., 2015) also cause the effect of acceleration of desensitization of IGly in hippocampal neurons. Interestingly, the effects of the above substances were in some ways similar to the effect of lithium found in this work. Firstly, these effects did not decrease with increasing concentration of glycine and therefore can be considered non-competitive. Secondly, the dose-response relationships of all of these drugs were not smooth and had a deflection at a certain point: at 1nM for beta-amyloid peptide (25-35), at 1
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µM for cyclic nucleotides, and 1 µM for Fe ions. As described above, this point exists for lithium at the concentration of 1 µM. Similar profile of the effects of different substances on the IGly allows to suggest the existence of non-specific sites on GlyRs which can influence the mechanisms of desensitization and can be activated by different ligands, possessing different
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affinities for these sites. Literature data indicate that different ligands can share binding sites on GlyR. For example, it was shown that binding sites for Zn2+, Cu2+ and H+ overlap (Chen et al.,
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2006; Chen and Huang, 2007).
We have previously demonstrated that Fe ions reduced the peak amplitude of the IGly (Solntseva et al., 2015). However, in contrast to the effects of lithium, Fe effect did not diminish with increasing concentration of glycine and can be considered non-competitive. For many decades lithium salts have been successfully used to treat bipolar disorder (BPD) (for a review: Curran and Ravindran, 2014). Recent literature data supports the suggestion
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that lithium can also be considered as a candidate medication for the treatment of the most significant neurodegenerative disorders such as Alzheimer’s and Parkinson’s diseases (for a review: Vo et al., 2015). The inhibitory effect of lithium ions on glycine-activated current described in the present study suggests that lithium in low concentrations is able to modulate
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tonic inhibition in the hippocampus. This important property of lithium should be considered
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when using this drug as a therapeutic agent.
Acknowledgments
This work was supported by Grants 16-04-00205 and 14-04-00391 from Russian Foundation for Basic Research, and Grant 9045.2016.4 from the Foundation for Support of Russian Scientific Schools.
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Figure Legends
Fig.1. The effects of short application of lithium on the IGly induced by 100 µM glycine. (A)
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Representative traces of IGly obtained in control and in the presence of 1, 10 and 100 nM Li+ (left), and 1, 10, 100 and 1000 µM Li+ (right). (B and C) Concentration dependence of Li+ effect
between data points were drawn.
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on the averaged normalized peak amplitude (B) and τ (C) of the IGly (n=12). The direct lines
Fig.2. The time course of one experiment where different concentrations of Li+ (full circles) were co-applied with 100 µM glycine. Responses to glycine are shown by open circles. Numbers under full circles show concentrations of Li+. The changes in the peak amplitude of IGly are
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shown in nA (A), and the changes in the time of the half-decay (τ) of IGly are shown in ms (B). Fig.3. The effects of short application of lithium on the IGly induced by 500 µM glycine. (A) Representative traces of IGly obtained in control and in the presence of 1, 10 and 100 nM Li+
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(left), and 1, 10, 100 and 1000 µM Li+ (right). (B and C) Concentration dependence of Li+ effect on the averaged normalized peak amplitude (B) and τ (C) of the IGly.(n=8). The direct lines
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between data points were drawn. Fig.4. Voltage-independence of the IGly modulation by Li+. (A) The family of representative traces of IGly induced by 100 µM glycine at holding potentials of -90, -70, -50, -30, -10, +10 and +30 mV, obtained in control solution (left) and in presence of 100 nM Li+ (right).(B) Averaged normalized current-voltage relationships for IGly peak obtained in control and in the presence of 100 nM Li+ (n=6). Data are plotted as a fraction of IGly peak at -70 mV in control solution. (C) Averaged values of the time of half-decay (τ) of IGly at different holding potentials in control solution and in the presence of 100 nM Li+. Fig.5. The effect of longer application of lithium on the IGly .(A and B) Concentration dependence of Li+ effect on the averaged normalized peak amplitude of IGly induced by 100 µM
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glycine (A) and 500 µM glycine (B) (n=8). (C and D) Concentration dependence of Li+ effect on the averaged normalized τ of IGly induced by 100 µM glycine (C) and 500 µM glycine (D) (n=8). Unlike short application, continuous application of 1 µM lithium causes significant effect,
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• The effects of Li+ on glycine-activated current (IGly) were studied using patch-clamp • Li+ caused two effects on the IGly with threshold effective concentration of 10 nM • An acceleration of desensitization of the IGly was noncompetitive
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• A reduction of the peak amplitude of the IGly was competitive
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• Dose-response relationships were different at short and long applications of Li+
S0197-0186(16)30016-X
DOI:
10.1016/j.neuint.2016.02.007
Reference:
NCI 3821
To appear in:
Neurochemistry International
Received Date: 30 December 2015 Revised Date:
5 February 2016
Accepted Date: 9 February 2016
Please cite this article as: Solntseva, E.I., Bukanova, J.V., Kondratenko, R.V., Skrebitsky, V.G., Lithium ions in nanomolar concentration modulate glycine-activated chloride current in rat hippocampal neurons, Neurochemistry International (2016), doi: 10.1016/j.neuint.2016.02.007. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Lithium ions in nanomolar concentration modulate glycine-activated chloride current in rat hippocampal neurons E.I.Solntseva, J.V.Bukanova, R.V.Kondratenko, V.G.Skrebitsky Research Center of Neurology, Moscow, Russia Corresponding author: Elena I. Solntseva, [email protected]; [email protected];
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tel: +79096427758, fax: +74959172382
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Mail address: 105064 per.Obukha,5, Research Center of Neurology Moscow Russia
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Abstract
Lithium salts are successfully used to treat bipolar disorder. At the same time, according to recent data lithium may be considered as a candidate medication for the treatment of neurodegenerative disorders. The mechanisms of therapeutic action of lithium have not been fully elucidated. In particular, in the literature there are no data on the effect of lithium on the glycine receptors. In the present study we investigated the effect of Li+ on glycine-activated
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chloride current (IGly) in rat isolated pyramidal hippocampal neurons using patch-clamp technique. The effects of Li+ were studied with two glycine concentrations: 100 µM (EC50) and 500 µM (nearly saturating). Li+ was applied to the cell in two ways: first, by 600 ms coapplication with glycine through micropipette (short application), and, second, by addition to an
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extracellular perfusate for 10 min (longer application). Li+ was used in the range of
concentrations of 1 nM – 1 mM. Short application of Li+ caused two effects: (1) an acceleration
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of desensitization (a decrease in the time of half-decay, or “τ”) of IGly induced by both 100 µM and 500 µM glycine, and (2) a reduction of the peak amplitude of the IGly, induced by 100 µM, but not by 500 µM glycine. Both effects were not voltage-dependent. Dose-response curves for both effects were N-shaped with two maximums at 100 nM and 1mM of Li+ and a minimum at 1 µM of Li+. This complex form of dose-response may indicate that the process activated by high concentrations of lithium inhibits the process that is sensitive to low concentrations of lithium.
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Longer application of Li+ caused similar effects, but in this case 1µM lithium was effective and the dose-effect curves were not N-shaped. The inhibitory effect of lithium ions on glycineactivated current suggests that lithium in low concentrations is able to modulate tonic inhibition in the hippocampus. This important property of lithium should be considered when using this
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drug as a therapeutic agent.
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Key words: Glycine receptor; Hippocampus; Li+; patch clamp
Introduction
For many decades lithium salts have been successfully used to treat bipolar disorder (BPD) (for a review: Curran and Ravindran, 2014). At the same time, over recent years evidence of the positive effect of lithium ions in treatment of other diseases, including neuro-, cardio- and nephroprotection has been accumulated (for a review: Malhi et al., 2013; Plotnikov et al., 2014; Vo et al., 2015). The possibility of neuroprotective effects of lithium is particularly relevant. According to recent data lithium may be considered as a candidate medication for the treatment of the most significant neurodegenerative disorders such as Alzheimer’s and Parkinson’s diseases
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(for a review: Vo et al., 2015). In in vitro experiments lithium has been shown to facilitate neural plasticity (Gray and McEwen, 2013) and regulate intracellular calcium levels as well as calcium turnover (Sourial-Bassillious et al., 2009). However, to date no definitive mechanism for lithium effects has been established. It has been proposed that lithium exerts its therapeutic effects by interfering with signal transduction through G-protein-coupled receptor (GPCR) pathways or by direct inhibition of specific targets in signaling systems, including inositol monophosphatase and
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glycogen synthase kinase-3 (GSK-3) (for a review: Beaulieu et al., 2009; Plotnikov et al., 2014). Ionic channels of neuronal membrane are also considered as potential targets for lithium ions. The experimental results show that lithium can modulate both ligand-activated and voltagegated channels. However, in some cases the effective lithium concentrations exceeded
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therapeutic dose of 0.6-1.2 mM in neurons, with the concentrations >2 mM considered toxic (Plotnikov et al., 2014). Lithium at 10 mM was found to facilitate AMPAR currents in hippocampal CA1 cells by selectively increasing the probability of channel opening (Gebhardt
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and Cull-Candy, 2010). Na+-dependent K+ current responsible for after-hyperpolarization was shown to be reduced in the presence of 1-10 mM lithium, which resulted in increased neuronal excitability (Safronov and Vogel, 1996; Butler-Munro et al., 2010). In adrenal chromaffin cells, lithium inhibited voltage-dependent sodium channels in a concentration-dependent manner (IC50=23.4 mM) (Yanagita et al., 2007). Finally, dual regulation of G-protein-activated K+-
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channels (GIRK) was described in mouse hippocampal neurons where 1-2 mM Li+ increased GIRK basal current but attenuated neurotransmitter-evoked GIRK-current (Tselnicker et al., 2014). In our work, we demonstrate for the first time, that Li+ at nanomolar concentrations can modulate glycine-activated chloride current in rat hippocampus neurons.
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Glycine is a crucial inhibitory neurotransmitter acting on specific glycine receptors (GlyRs), whose localization can be both synaptic and extra-synaptic (Lynch 2009; Song et al.,
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2006). Extra-synaptic GlyRs in hippocampus provide a tonic inhibition (Xu and Gong, 2010), which is very important for information processing within a neuronal network and disturbance of which can contribute to many pathophysiological processes. Numerous structurally diverse compounds have been shown to modulate GlyRs, including neurosteroids, alcohols, anesthetics, cannabinoids, antagonists of 5-HT3 receptor, ginkgolide B, cyclothiazide and quercetin (for a review: Xu and Gong, 2010; Yevenes and Zeilhofer, 2011). Recently we demonstrated the ability of cyclic nucleotides (cAMP and cGMP) and beta-amyloid peptide to exert extracellular modulation of GlyRs (Bukanova et al., 2014a, 2014b). A lot of attention was given to the study of GlyRs modulation by cations (for a review: Lynch, 2004; Xu and Gong, 2010). Divalent cation Zn2+ was shown to modulate GlyRs extracellularly in a bidirectional manner depending on its concentration. There is no convincing evidence of the ability of other metal ions to mimic the
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biphasic action of zinc. However, the potentiating site is recognized by several metal ions with the following potency sequence: zinc > lanthanide > lead > cobalt, whereas the inhibitory site exhibits a different potency sequence: zinc > copper > nickel (for a review: Lynch, 2004). 100 µM of Ba2+, Sr2+, Mn2+, Co2+, Cd2+ and Al3+ were shown to have no effect on GlyR currents in rat septal neurons (Kumamoto and Murata, 1996). We have shown in our previous study conducted in rat hippocampus neurons that Fe2+ and Fe3+ can modulate glycine-evoked current
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(IGly) in a similar manner. These ions in micromolar concentrations caused an acceleration of desensitization and a decrease of peak amplitude of IGly (Solntseva et al., 2015). We could not find any data on Li+ influence on IGly in the available literature. Thus, our data on the effect of lithium on the IGly can help to elucidate the mechanisms of therapeutic action of lithium, and to
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understand the functional properties of glycine receptors. 2. Material and methods
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2.1. Cell preparation
All procedures were performed in accordance with the institutional guidelines on the care and use of experimental animals set by the Russian Academy of Sciences. The cells were isolated from transverse hippocampal slices as described in detail elsewhere (Vorobjev 1991). Briefly, the slices (200-500 µm) of Wistar rats (11-14 days of age) hippocampus were incubated at room
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temperature for at least 2 h in a solution containing the following components (in mM): 124 NaCl, 3 KC1, 2 CaCl2, 2 MgSO4, 25 NaHCO3, 1.3 NaH2PO4, 10 D-glucose, pH 7.4. The saline was continuously stirred and bubbled with carbogen (95% 02 + 5% CO2). Single pyramidal neurons from CA3 were isolated from the stratum pyramidale by a vibrating fused glass pipette
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with a spherical tip (Vorobjev 1991).
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2.2. Current recordings
Glycine-activated currents in isolated neurons were induced by a step application of agonist for 600 ms with 30-40 s intervals. Transmembrane currents were recorded using a conventional patch-clamp technique in the whole-cell configuration. Patch-clamp electrodes had a tip resistance of ~2 MΩ. The solution in the recording pipette contained the following (in mM): 40 CsF, 100 CsCl, 0.5 CaCl2, 5 EGTA, 3 MgCl2, 4 NaATP, 5 HEPES, 4 ATP, pH 7.3. The composition of extracellular solution was as follows (in mM): 140 NaCl, 3 KCl, 3 CaCl2, 3 MgCl2, 10 D-glucose, 10 HEPES hemisodium, pH 7.4. The speed of perfusion was 0.6 ml/min. Recording of the currents was performed using EPC7 patch-clamp amplifier (HEKA Electronik, Germany). Unless noted otherwise, the holding potential was maintained at -70 mV.
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Transmembrane currents were filtered at 3 kHz, stored and analyzed with IBM-PC computer, using homemade software. 2.3. Drug application Glycine was applied through glass capillary, 0.1 mm in diameter, which could be rapidly displaced laterally under control of home-made software (Vorobjev et al., 1996). The system
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allows a complete exchange of external solution surrounding the neuron within 20 ms. Li+ was applied to the cell in two ways. In the first set of experiments Li+ was co-applied with glycine through micropipette during 600 ms (short application), and in the second series of experiments, Li+ was added in an extracellular perfusate for 10 min (longer application) using two reservoirs
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system. The speed of perfusion was 0.6 ml/min. To avoid the reduction in the concentration of lithium during the application of glycine, we added lithium in corresponding concentration also
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to the glycine-containing pipette. 2.3. Reagents
All the drugs were purchased from “Sigma”. LiCl was used as the source of Li+. The tested substances were dissolved in distilled water to make 0.1-1mM stock solution, which was
2.4. Data analysis
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dissolved in external saline to their final concentration immediately before the experiments.
All statistical analysis was performed with the help of Prism Graphpad software. All comparisons were made with one-way repeated measures ANOVA at a significance level of p =
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0.05. In results descriptions, mean and standard error of mean (SEM) are specified. The meanings of asterisks (probability levels) in figures is the following: *P < 0.05, **P < 0.01, ***P
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< 0.001. Results
3.1. Glycine activates chloride currents in rat hippocampal neurons Short application of glycine for 600 ms on pyramidal neurons evoked chloride currents (IGly) which characteristics were described in details in our previous works (Bukanova et al., 2014a, 2014b). Shortly, the amplitude of IGly depended on glycine concentration with EC50 value of 90 ± 7 µM. The average value of the reversal potential of IGly -9.8 ± 0.9 mV matched well the chloride reversal potential calculated for the chloride concentrations used (-9.5 mV). In this paper, the effects of Li+ were studied with two glycine concentrations: 100 µM and 500 µM. Short (600 ms) and longer (10 min) applications of Li+ were used.
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3.2. The effect of short application of Li+ on IGly induced by 100 µM glycine Application of Li+ alone in different concentrations (1 nM – 1 mM) did not evoke any direct membrane response. When co-applied with 100 µM glycine, Li+ rapidly, reversibly and in a dose-dependent manner changed the IGly in all cells tested (n=12). With this glycine concentration, Li+ reduced the IGly peak amplitude and accelerated the desensitization of the current. To quantitatively assess the alteration of current kinetics, we measured the time of half-
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decay of the IGly (τ) in the absence and presence of Li+. Figure 1 shows representative traces of IGly, induced by 100 µM glycine, obtained in control and in the presence of various
concentrations of lithium from 1 nM to 1 mM (Fig.1A), as well as concentration dependence of Li+ effect on the normalized peak amplitude (Fig.1B) and normalized τ of IGly (Fig.1C). It can be
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seen that both dose-response curves are N-shaped, which is due to the fact that the lithium concentration of 1 µM is ineffective. Such a deflection on the dose-response suggests that there
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are at least two binding sites for lithium with different affinity, and low-affinity site inhibits the high affinity site. Accordingly, each curve has two maximums at 100 nM and 1 mM lithium. At these Li+ concentrations, the corresponding values of the peak amplitude of IGly are reduced to 0.83 ± 0.03 (P < 0.001) and 0.85 ±0.05 (P < 0.05) of the control value. The corresponding values of τ are reduced to 0.59 ± 0.04 (P < 0.001) and 0.42 ± 0.04 (P < 0.001). Fig.2 shows the time
applied with glycine.
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course of one typical experiment where different concentrations of Li+ (full circles) were co-
3.3. The effect of short application of Li+ on IGly induced by 500 µM glycine
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In contrast to the experiments with 100 µM glycine, where lithium caused a reduction both in the peak amplitude and τ of the IGly, in the experiments with 500 µM glycine, lithium induced only acceleration of glycine current desensitization and did not change its peak amplitude (Fig.3,
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n=8). The effect of Li+ on IGly desensitization was similar in experiments with 500 µM glycine and 100 µM glycine. A concentration dependence of Li+ effect on the normalized τ of IGly induced by 500 µM glycine was also N-shaped with deflection at 1 µM Li+ (Fig.3A and C). Two maximums were at 100 nM and 1mM Li+, and the values of τ were reduced to 0.46 ± 0.07 (P < 0.001) and 0.40 ± 0.05 (P < 0.001), correspondingly. 3.4 Voltage-independence of Li+ effects on the IGly . It was found in the experiments with 100 µM glycine, that the effect of Li+ on both peak amplitude and τ of the IGly did not depend on membrane holding potential (Fig.4). Figure 4A shows representative traces of IGly, induced by 100 µM glycine, recorded from one cell. The
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currents were obtained in control (left) and in the presence of 100 nM Li+ (right) at different membrane potentials varying from -90 mV to +30 mV. It can be seen that the changes, induced by Li+, are equally pronounced at negative and positive membrane potentials. The average values (n=6) of the peak amplitude and τ of glycine current recorded at various membrane potentials are shown in Fig.4B and 4C. It is important to note that reversal potential of the IGly peak (-9.8 ± 0.9 mV) remained unchanged in the presence of Li+ (Fig.4B).
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3.5. The effects of longer application of Li+ on the IGly.
In the second series of experiments, Li+ was added to an extracellular perfusate for 10 min (n=8). It has been found that, in general, Li+ has the same effect on the IGly during the short (600 ms)
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and long-term (10 min) exposure. Prolonged application of Li+ also caused a decrease in the peak amplitude and in the time of half-decay of the IGly induced by 100 µM glycine, and a decrease in the time of half-decay without changing the peak amplitude of the IGly induced by 500 µM
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glycine. These effects were reversible after 10-15 minute washing with control solution. At the same time, prolonged application of lithium revealed one distinctive feature: the dose-effect curve was smooth with this method of application. Figure 5 shows concentration dependence of Li+ effects on the normalized IGly peak and τ measured 10 min after Li+ addition to the perfusate. It can be seen that in contrast to short applications, when 1 µM of lithium was not effective,
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continuous application of lithium in this concentration caused a significant effect on the IGly, namely, a reduction of peak amplitude to 0.88 ± 0.04 (P < 0.05) (Fig.5A) and of τ to 0.65 ± 0.05 (P < 0.001) (Fig.5C) of the control value in experiments with 100 µM glycine, and a reduction of
(Fig.5D).
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Discussion
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τ to 0.61 ± 0.05 (P < 0.001) of the control value in the experiments with 500 µM glycine
In the present paper, we studied chloride current activated by 100 and 500 µM glycine (IGly). These concentrations of glycine look too high compared to its physiological concentration in the hippocampus, which do not exceed a few micromoles. However, the GlyRs can be also activated by other agonists, taurine and beta-alanine, which concentration in hipppocampus are relatively high (Mori et al., 2002; Shibanoki et al., 1993). The total concentration of these three agonists of GlyRs may approach high micromolar level in hippocampus. In our experiments, we demonstrate for the first time the capability of Li+ to modulate IGly. The threshold effective concentration of Li+ was 10 nM, which is significantly lower than therapeutic doses of the drug (0.6-1.2 mM, Plotnikov et al., 2014). We tested Li+ in the range of concentrations of 1 nM – 1mM and studied it using two concentrations of glycine, 100 µM and
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500 µM. Two effects of Li+ on IGly were observed: a reduction of the peak amplitude and an acceleration of desensitization, which is quantified by a decrease in the time of half-decay (τ) of IGly. The second effect was quantitatively stronger than the first one. Both effects were voltageindependent, so the possibility that Li+ ions modulate IGly by affecting electrostatic forces at the channel pore seems unlikely. The effect of acceleration of desensitization was similar for currents induced by 100 µM and 500 µM glycine. Therefore, this effect of Li+ did not depend on
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glycine concentration and may be considered noncompetitive, suggesting that lithium ions affect glycine receptor-channel complex at a site distinct from glycine binding site. Another
characteristic of lithium action was observed with regard to the peak current amplitude. Reduction of the peak amplitude by lithium was observed only for 100 µM glycine, and with 500
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µM glycine the effect disappeared. Therefore, this effect can be regarded as a competitive, and the site responsible for it may be located near the glycine binding site. Thus, the two described
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effects of lithium appear to be realized through the sites located in different loci. Dependence of the effect of certain other substances on the GlyR-induced currents on the concentration of glycine was described by other authors. For example, it was shown that the cannabinoids augment the IGly at low doses of the glycine (EC50; Lozovaya et al., 2005; Hejazi et al., 2006; Yang et al., 2008). The same dependence was observed in experiments with ginkgolide B (BN52021)
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(Kondratskaya et al., 2002).
In experiments with short application of Li+ the dose-effect curves were N-shaped for both the peak amplitude and τ. A deflection on the dose-response was at 1 µM Li+ for both effects. This complex form of dose-response may indicate that the process activated by high
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concentrations of lithium inhibits the process that is sensitive to low concentrations of lithium. This assumption applies to both observed effects, reduction of the peak amplitude and
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acceleration of desensitization. For each of the two described effects one can assume the existence of a pair of sites binding lithium, and in each pair the low affinity site inhibits the high affinity site. However, in experiments with prolonged application of lithium (10 min), doseresponse curves lost their N-shaped form, because the concentration of lithium of 1 µM became efficient. So, in this case other more complicated mechanisms may be at work. Literature data point to a complex dose-effect relationship of other drugs which modulate the IGly. Thus, the concentration dependence for both potentiating and inhibitory effects of ethanol on the IGly has been shown to have a bell-shaped appearance (Ye et al., 2001a and 2001b). In our previous works we have shown that short application of some other substances, namely, cyclic nucleotides (cAMP and cGMP) (Bukanova et al., 2014a), beta-amyloid
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peptide(25-35) (Bukanova et al., 2014b) and Fe ions (Solntseva et al., 2015) also cause the effect of acceleration of desensitization of IGly in hippocampal neurons. Interestingly, the effects of the above substances were in some ways similar to the effect of lithium found in this work. Firstly, these effects did not decrease with increasing concentration of glycine and therefore can be considered non-competitive. Secondly, the dose-response relationships of all of these drugs were not smooth and had a deflection at a certain point: at 1nM for beta-amyloid peptide (25-35), at 1
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µM for cyclic nucleotides, and 1 µM for Fe ions. As described above, this point exists for lithium at the concentration of 1 µM. Similar profile of the effects of different substances on the IGly allows to suggest the existence of non-specific sites on GlyRs which can influence the mechanisms of desensitization and can be activated by different ligands, possessing different
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affinities for these sites. Literature data indicate that different ligands can share binding sites on GlyR. For example, it was shown that binding sites for Zn2+, Cu2+ and H+ overlap (Chen et al.,
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2006; Chen and Huang, 2007).
We have previously demonstrated that Fe ions reduced the peak amplitude of the IGly (Solntseva et al., 2015). However, in contrast to the effects of lithium, Fe effect did not diminish with increasing concentration of glycine and can be considered non-competitive. For many decades lithium salts have been successfully used to treat bipolar disorder (BPD) (for a review: Curran and Ravindran, 2014). Recent literature data supports the suggestion
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that lithium can also be considered as a candidate medication for the treatment of the most significant neurodegenerative disorders such as Alzheimer’s and Parkinson’s diseases (for a review: Vo et al., 2015). The inhibitory effect of lithium ions on glycine-activated current described in the present study suggests that lithium in low concentrations is able to modulate
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tonic inhibition in the hippocampus. This important property of lithium should be considered
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when using this drug as a therapeutic agent.
Acknowledgments
This work was supported by Grants 16-04-00205 and 14-04-00391 from Russian Foundation for Basic Research, and Grant 9045.2016.4 from the Foundation for Support of Russian Scientific Schools.
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Figure Legends
Fig.1. The effects of short application of lithium on the IGly induced by 100 µM glycine. (A)
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Representative traces of IGly obtained in control and in the presence of 1, 10 and 100 nM Li+ (left), and 1, 10, 100 and 1000 µM Li+ (right). (B and C) Concentration dependence of Li+ effect
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Fig.2. The time course of one experiment where different concentrations of Li+ (full circles) were co-applied with 100 µM glycine. Responses to glycine are shown by open circles. Numbers under full circles show concentrations of Li+. The changes in the peak amplitude of IGly are
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shown in nA (A), and the changes in the time of the half-decay (τ) of IGly are shown in ms (B). Fig.3. The effects of short application of lithium on the IGly induced by 500 µM glycine. (A) Representative traces of IGly obtained in control and in the presence of 1, 10 and 100 nM Li+
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(left), and 1, 10, 100 and 1000 µM Li+ (right). (B and C) Concentration dependence of Li+ effect on the averaged normalized peak amplitude (B) and τ (C) of the IGly.(n=8). The direct lines
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between data points were drawn. Fig.4. Voltage-independence of the IGly modulation by Li+. (A) The family of representative traces of IGly induced by 100 µM glycine at holding potentials of -90, -70, -50, -30, -10, +10 and +30 mV, obtained in control solution (left) and in presence of 100 nM Li+ (right).(B) Averaged normalized current-voltage relationships for IGly peak obtained in control and in the presence of 100 nM Li+ (n=6). Data are plotted as a fraction of IGly peak at -70 mV in control solution. (C) Averaged values of the time of half-decay (τ) of IGly at different holding potentials in control solution and in the presence of 100 nM Li+. Fig.5. The effect of longer application of lithium on the IGly .(A and B) Concentration dependence of Li+ effect on the averaged normalized peak amplitude of IGly induced by 100 µM
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glycine (A) and 500 µM glycine (B) (n=8). (C and D) Concentration dependence of Li+ effect on the averaged normalized τ of IGly induced by 100 µM glycine (C) and 500 µM glycine (D) (n=8). Unlike short application, continuous application of 1 µM lithium causes significant effect,
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• The effects of Li+ on glycine-activated current (IGly) were studied using patch-clamp • Li+ caused two effects on the IGly with threshold effective concentration of 10 nM • An acceleration of desensitization of the IGly was noncompetitive
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• A reduction of the peak amplitude of the IGly was competitive
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• Dose-response relationships were different at short and long applications of Li+
