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Research ArticleApril 13, 2016

Brief Isoflurane Anesthesia Produces Prominent Phosphoproteomic Changes in the Adult Mouse Hippocampus
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ACS Chemical Neuroscience

Cite this: ACS Chem. Neurosci. 2016, 7, 6, 749–756

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https://doi.org/10.1021/acschemneuro.6b00002

Published April 13, 2016

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Abstract

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Anesthetics are widely used in medical practice and experimental research, yet the neurobiological basis governing their effects remains obscure. We have here used quantitative phosphoproteomics to investigate the protein phosphorylation changes produced by a 30 min isoflurane anesthesia in the adult mouse hippocampus. Altogether 318 phosphorylation alterations in total of 237 proteins between sham and isoflurane anesthesia were identified. Many of the hit proteins represent primary pharmacological targets of anesthetics. However, findings also enlighten the role of several other proteins—implicated in various biological processes including neuronal excitability, brain energy homeostasis, synaptic plasticity and transmission, and microtubule function—as putative (secondary) targets of anesthetics. In particular, isoflurane increases glycogen synthase kinase-3β (GSK3β) phosphorylation at the inhibitory Ser⁹ residue and regulates the phosphorylation of multiple proteins downstream and upstream of this promiscuous kinase that regulate diverse biological functions. Along with confirmatory Western blot data for GSK3β and p44/42-MAPK (mitogen-activated protein kinase; reduced phosphorylation of the activation loop), we observed increased phosphorylation of microtubule-associated protein 2 (MAP2) on residues (Thr^1620,1623) that have been shown to render its dissociation from microtubules and alterations in microtubule stability. We further demonstrate that diverse anesthetics (sevoflurane, urethane, ketamine) produce essentially similar phosphorylation changes on GSK3β, p44/p42-MAPK, and MAP2 as observed with isoflurane. Altogether our study demonstrates the potential of quantitative phosphoproteomics to study the mechanisms of anesthetics (and other drugs) in the mammalian brain and reveals how already a relatively brief anesthesia produces pronounced phosphorylation changes in multiple proteins in the central nervous system.

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The discovery and development of anesthetics revolutionized and humanized surgical procedures. Each year millions of people undergo medical treatment requiring anesthesia(s). Anesthetics are also commonly used in veterinary medicine and in animal research. Volatile anesthetics, such as halogenated hydrocarbons isoflurane and sevoflurane, are among the most commonly used general anesthetics today. These drugs produce concentration-dependent rapid general anesthesia (unconsciousness, insensateness, analgesia, and amnesia) that is quickly recovered after the discontinuation of drug administration. The depth of anesthesia can be monitored using cortical electroencephalogram (EEG) whereby deep surgical anesthesia is characterized by intermittent shifts between almost flatline EEG and “packages” of spiking activity (i.e., EEG burst suppression). (1)

The mechanisms underlying the neurobiological actions and effects of anesthetics remain poorly understood. Early studies demonstrated that the anesthetic potency increases with mere lipid solubility and thus the effects of anesthetics were thought to rise through unspecific effects on cellular lipids. (2) According to a more recent theory, anesthetics act predominantly by facilitating the opening of ligand-gated GABA_A (γ-aminobutyric acid) ion channel receptors and/or by dampening the opening of ligand-gated NMDA (N-methyl-d-aspartate) receptors. (2, 3) GABA_A receptors are responsible for the fast neuronal inhibition mediated by hyperpolarizing chloride (Cl^–) currents in the adult mammalian CNS. (4) NMDA receptors are activated by glutamate, the most abundant excitatory neurotransmitter in the nervous system. Although these pharmacological mechanisms are clearly demonstrated for anesthetic agents, anesthetics target, indirectly or directly, several other proteins as well. (2) Surprisingly little is known about the downstream intracellular changes and global intracellular signaling level alterations set fourth by the primary effects of anesthetics.

The functional consequences of short and long-term exposures to anesthesia have received considerable interest in recent decades. Anesthetics and sedatives have been shown to produce neuroapoptosis and atrophic neuronal changes, particularly during early development. (5) However, the effects of anesthetics strongly depend on brain maturation stage, and the adult brain seems to be much less vulnerable for the deleterious effects of anesthetics. Yet, exposure to anesthetics in adult life may produce reversible amnesia and have been suggested to produce cognitive problems and even accelerate Alzheimer’s disease pathology. (6, 7) On the other hand, brief deep anesthesia has also been shown to produce therapeutic effects against depression, stroke, and brain trauma. (8-11) The neurobiological mechanisms and neurophysiological correlates underlying all of these effects of anesthetics remain poorly understood.

Protein phosphorylation is an important—transient—posttranslational modification that dynamically regulates the activity, folding, protein–protein interaction and cellular trafficking and targeting of many, if not most, proteins. (12, 13) Indeed, quantitative phosphoproteomics is becoming very efficient tool to investigate global signaling-level changes in biological systems. (12) However, to our knowledge, no study has utilized this method to investigate phosphoproteomic alterations in samples obtained from the mammalian brain following anesthesia. We have here employed this method to study the effects of a single and brief isoflurane anesthesia on protein phosphorylation in the adult mouse hippocampus.

Results and Discussion

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Isoflurane anesthesia has been shown to produce a myriad of central effects, ranging from therapeutic to pathological alterations, yet the neurobiological correlates for these effects (as for those underlying anesthesia in general) are poorly understood. To elucidate this issue using quantitative phosphoproteomics, we first utilized pharmaco-EEG to validate an isoflurane treatment procedure that brings persistent and reproducible state of anesthesia in adult male mice. As shown in Figure 1, the treatment protocol consisting of 4% induction phase and maintenance at 2% of isoflurane strongly and reproducibly reduced cortical EEG power spectra during the 30 min treatment period and rapidly and sustainably led to burst suppression pattern characterized by intermittent periods of low and high (bursts) amplitude electrical activity. Induction and maintenance with 2% isoflurane produced similar, albeit less pronounced, EEG alterations in some but not all animals (data not shown). Notably, rapid anesthesia induction phase comprising initial high isoflurane concentration is commonly employed in animal experiments requiring surgical anesthesia (e.g., implantation of microdialysis probes or optogenetic wires).

Figure 1. Effects of isoflurane anesthesia (4% induction, 2% maintenance) on cortical EEG spectrogram and burst suppression. (A) EEG spectrogram during awake, non-REM (NREM) sleep, and under the influence of isoflurane (20–30 min recording). Representative traces of burst suppressing EEG shown in inset (A2) and (A3). (B) Quantitation of delta (1–4 Hz) (B1), theta (4–8 Hz) (B2), alpha (8–12 Hz) (B3), beta (12–30 Hz) (B4), and gamma (30–60 Hz) (B5) frequencies under the influence of isoflurane, during awake and NREM sleep. Deep burst-suppressing isoflurane anesthesia significantly reduces spectral power in frequency bands below 30 Hz when compared to baseline NREM sleep. EEG spectra were normalized to total EEG power for all frequencies within the baseline period recording. N = 5; *p < 0.05, **p < 0.01, ***p < 0.001; one-way ANOVA followed by Dunnett’s multiple comparison test.

Next the treatment protocol producing reproducible deep anesthesia was used for a new cohort of naïve mice and their hippocampi were collected for the phosphoproteomic study (Figure 2). We focused our analysis to the hippocampus to ensure enough tissue for protein extraction and to ensure reproducible dissection. This is to our knowledge the first attempt to investigate the effects of anesthesia on global level protein phosphorylation in the adult mammalian brain using quantitative phosphoproteomics. The adult brain is rich in lipids, which need to be removed before analysis. Therefore, our protocol included lipid elimination followed by protein digestion and TiO₂-based phosphopeptide enrichment (selectivity: phosphoserine/threonine > phosphotyrosine). The heat map schematically depicting phosphopeptide alterations in individual animals subjected to either sham and isoflurane anesthesia demonstrates prominent differences between the treatment groups (Figure 2). Altogether 318 significant phosphoproteomic alterations in total of 237 different proteins between sham and anesthesia were identified (Supporting Information). These proteins are associated and functionally clustered into various biological processes, and the great majority of them are associated with protein phosphorylation, which also partially validates our analysis (Supporting Information).

Figure 2. Phosphoproteomic workflow (A) and heat map (B) depicting differentially regulated phosphoproteins after sham (C1–C3) or 30 min isoflurane anesthesia (I1–I3) (4% induction, 2% maintenance). N = 3/group.

According to prevailing theory, volatile anesthetics primary act by facilitating the inhibitory tone mediated by GABA_A receptors and/or dampening excitatory neurotransmission. (2) Although the exact binding site of isoflurane to GABA_A receptors remains unknown, α and β subunits are presumable targets. (3) The phosphorylations of α₁ (Gabra1; Ser³⁷³, Thr³⁶⁶), which governs the sedative effects of benzodiazepines, (14) and β₃ (Gabrb3; Ser³⁹⁴) subunits are regulated by isoflurane in our study (Table 1). The phosphorylation of Grin2B, the NMDA receptor subunit serving as the binding site for glutamate, is also regulated by isoflurane into two residues (Ser¹⁰³⁶, Tyr¹⁰³⁹) (Table 1). In addition, three metabotropic glutamate receptors (Grm1, Grm5, Grm7) show phosphorylation changes in serine residues (Table 1). Several members of solute carriers, some of which regulate neurotransmitter reuptake (Slc1a2, Slc12a6, Slc4a8, Slc6a17, Slc9a1) are also differentially phosphorylated in response to isoflurane anesthesia (Supporting Information).

Table 1. Isoflurane Anesthesia (4% Induction, 2% Maintenance; 30 min) Induced Phosphorylation Changes on Selected Plasma Membrane Channels and Receptors^a

^{Table a}

Green up triangle = increased phosphorylation; red down triangle = reduced phosphorylation.

Several other ion channels have been also implicated as targets of anesthetics. These include glycine receptors, nicotinic acetylcholine receptors, 5-HT₃ receptors and several potassium (K⁺), sodium (Na⁺) and calcium (Ca²⁺) channels. Isoflurane anesthesia produced phosphorylation changes in four K⁺ channels (Hcn2, Kcnj6, Kcnb1–2) (Table 1). Of note, most anesthetics have been shown to block Hcn channel mediated I_h currents (15-18) and Hcn1 channels (although not found as a significant hit in the current study) have been shown to contribute to the hypnotic effects of anesthetics. (15, 19) Our data further suggests that anesthetics affect multiple mechanisms regulating [Ca²⁺]_i (Table 2), including markedly altered phosphorylation of Ca²⁺-dependent secretion activator (Cadps), voltage dependent Ca²⁺ channels (Canb4, Cacna1a), glutamate receptors, junctophilin 3 (Jph3), ryanodine receptor 2, stromal interaction molecule 1 (Stim1), and a number of inositol trisphosphate (IP₃) related kinases and phosphatases. Phosphorylation-dependent alteration of the functionality of these regulators and effectors of Ca²⁺ signaling might result in the triggering of a variety of intracellular cascades leading into diverse outcomes, which is also supported by our data showing numerous Ca²⁺ regulated targets with modified phosphorylation profiles. The intracellular release of Ca²⁺ is primarily dependent on two mechanisms, the inositol 1,4,5-trisphosphate receptors (InsP₃Rs) and the ryanodine receptors (RyRs), (20) but other possible mediators are likely involved as well. (21) The InsP₃Rs are regulated by the binding of IP₃ that is generated by the exposure of cells to diverse stimuli, such as neurotrophic factors and neurotransmitters, which activate signaling through G-protein coupled receptors. (22) The RyRs, however, are regulated directly by increases in [Ca²⁺]_i. Aside from neuronal Ca²⁺ signaling, glial cells utilize Ca²⁺ in even more complex manner. (23, 24) Approximately half of the adult mammalian brain is comprised of astrocytes that participate in a wide array of functions, including synaptic transmission, neurotrophic signaling and structural support. (25)

Table 2. Isoflurane Anesthesia (4% Induction, 2% Maintenance; 30 min) Induced Phosphoproteomic Changes on Selected Proteins Implicated in Intracellular Calcium Regulation and Homeostasis^a

^{Table a}

Green up triangle = increased phosphorylation; red down triangle = reduced phosphorylation.

Some evidence indicate that PDZ (PSD-95/SAP90, Dlg, ZO-1) domain mediated proteome disruption in the mechanisms of actions of anesthetics. (26) Interestingly, isoflurane anesthesia produced differential phosphorylation in three PSD (postsynaptic density) protein family members (Dlgap1–3) (Table 3), which act as scaffolding proteins in synaptic sites and orchestrate the functions and localization of proteins important for synaptic plasticity and neuronal transmission (e.g., NMDA receptors). According to our data search, several identified “hit” proteins are also associated with PDZ domain functionality (Shank2–3, Rims1, MIIt4, Arhgap21–23, Pclo, Lrrc7, Sipa1, Mast1) (Table 3, Supporting Information). Our data further shows that isoflurane anesthesia produces phosphorylation changes on several other proteins intimately implicated in synaptic plasticity, synapse formation, and function (Table 3).

Table 3. Isoflurane Anesthesia (4% Induction, 2% Maintenance; 30 min) Induced Phosphoproteomic Changes on Selected Proteins Implicated in Synaptic Plasticity and Synapse Function^a

^{Table a}

Green up triangle = increased phosphorylation; red down triangle = reduced phosphorylation.

Our study further strengthens previous studies pointing glycogen synthase 3β (GSK3β) as a target of anesthetics (27) (Table 4). GSK3β is a promiscuous (28) central serine-threonine kinase in several biological pathways critical for brain energy metabolism, microtubule stability, neuronal development, synaptic plasticity, and inflammation. (29, 30) Specifically, GSK3β phosphorylation at the inhibitory residue Ser⁹ is significantly increased after isoflurane anesthesia. Alteration of GSK3β-mediated signaling is further supported by phosphoproteomic data demonstrating differential phosphorylation of multiple proteins acting downstream or upstream of GSK3β including protein phosphatase 1 (PP1), (29) microtubule-associated proteins 1–2 (MAP1–2), mitogen-activated protein kinase 3 (Mapk3, better known as p44-mitogen-activated protein kinase (p44-MAPK)), phosphoinositide kinase, amyloid β precursor protein, catenin Δ1, dynamin 1, dystrophin, muscular dystrophy, phosphorylase kinase α₂, and microtubule-associated protein tau (Table 4). (30) Importantly, in addition to GSK3β, we confirmed two of these isoflurane-induced phosphoprotein alterations, reduced phosphorylation of p44/42-MAPK^{Thr202/Tyr204} and increased phosphorylation of p-MAP2^{Thr1620/Thr1623}, with commercially available antibodies in complementary animal experiments and Western blot analyses (Figure 3A).

Table 4. Isoflurane Anesthesia (4% Induction, 2% Maintenance; 30 min) Induced Phosphoproteomic Changes on Selected Proteins Implicated in Glycogen Synthase Kinase 3β (GSK3β) Signaling and Microtubule and Actin Dynamics^a

^{Table a}

Green up traingle = increased phosphorylation; red down triangle = reduced phosphorylation.

Figure 3. Diverse anesthetics produce similar acute phosphorylation changes on p44/42-MAPK^{Thr202/Tyr204}, GSK3β^Ser9, and MAP2^{Thr1620/Thr1623} in the adult mouse hippocampus. (A) Effects of isoflurane anesthesia (4% induction, 2% maintenance; 30 min) (N = 10/group). (B) Effects of sevoflurane anesthesia (6% induction, 4.5% maintenance; 30 min) (N = 6/group). (C) Effects of urethane anesthesia (2.0 g/kg, i.p.; 30 min) (N = 4/control group, N = 6/urethane group). (D) Effects of subanesthetic ketamine (100 mg/kg, i.p.; 30 min) (N = 6/group). *p < 0.05, **p < 0.01, ****p < 0.0001; two-tailed unpaired t test with Welch’s correction. Abbreviations: MAPK, mitogen activated protein kinase; GSK3β, glycogen synthase kinase 3β; MAP2, microtubule-associated protein 2.

Many of the hit proteins regulate actin cytoskeleton and microtubule dynamics (Table 4). Microtubules coordinate with other cytoskeletal components including microtubule associated proteins (MAPs) and actin filaments and play a critical role in neuronal cell morphology by establishing axons, dendrites and synapses, and maintaining them. (32) This bundling activity of microtubules forms the basis for the scaffolding of the neuron and the synaptic architecture. MAPs, like MAP2 (Figure 3), are heavily regulated by phosphorylation, which affects their ability to bind and stabilize microtubules. (31) Thus, the phosphorylation of MAPs can give rise to a variety of effects from the regulation of organelle transport to the anchorage of important signaling molecules, such as protein kinases and phosphatases. MAP2 has also been proposed to participate in the outgrowth of neuronal processes, synaptic plasticity and cellular death. (31) Both GSK3β and MAPKs are known to phosphorylate MAP2^Thr1620/1623 in vitro (33, 34) and thereby cause MAP2 to lose its ability to effectively associate with the microtubules. (35) However, our data, that shows significant phosphorylation at the inhibitory Ser9 residue of GSK3β and dephosphorylation of p44/42-MAPK at the activating loop residues Thr202/Tyr204, suggests alternative pathways for the phosphorylation of MAP2 following isoflurane anesthesia. For example MAP/microtubule affinity regulating kinases (MARKs), cyclin-dependent kinases (CDKs) and protein kinase A and C have been shown to regulate MAP2 phosphorylation. (33, 35, 36) Regulation of the phosphorylation of MAP2 could also happen through the inhibition of specific phosphatases. Notably, our proteomic data shows phosphorylation changes in several members of MARKs and CDK family of kinases (Mark1, Mark2, Cdk11b, Cdk14 and Cdkl5) as well as proteins that regulate phosphatase (inhibitor) activity (e.g., Elfn2, Ppp1r1a and Ensa) (Table 4, Supporting Information).

In order to deepen the mechanistic insights of isoflurane-induced rapid phosphoproteomic alterations on GSK3β^Ser9, p44/42-MAPK^{Thr202/Tyr204} and MAP2^{Thr1620/Thr1623}, we decided to study the effects of pharmacologically diverse anesthetics on these same residues. First, we investigated sevoflurane, a structurally and pharmacologically close relative of isoflurane, which produced similar phosphorylation changes on all three proteins (Figure 3B). Next, we assessed the effects of ethyl carbamate (better known as urethane). Urethane is commonly used in experimental animals because it produces strong analgesia and anesthesia but has minimal effects on cardiovascular/respiratory systems and baseline electrophysiological properties. However, also urethane anesthesia increased GSK3β and MAP2 phosphorylation and reduced p44/42-MAPK phosphorylation (Figure 3C). The mechanism of action of urethane is poorly understood, but it has been shown to regulate several ion channels, including GABA_A and NMDA receptors. (37) Ketamine on the other hand specifically binds to and blocks NMDA receptors and thereby produces so-called dissociative anesthesia. Ketamine also produces very minor effects on cardiovascular/respiratory systems. However, it is more difficult to achieve deep anesthesia with ketamine monotherapy in experimental animals. Intriguingly, ketamine produced essentially similar changes on GSK3β, p44/42-MAPK and MAP2 phosphorylation already at a sedative dose (100 mg/kg, i.p.) (Figure 3D).

Few proteomic studies have been performed to investigate the central effects of anesthetics but only a handful of proteins appear sustainably and reproducibly regulated by anesthetics. (41, 42) Protein phosphorylation is instead a very dynamic posttranslational modification that often critically alters the function of a given protein. To our knowledge this is the first study investigating phosphoproteomic changes induced by anesthesia in the adult mammalian brain. Although we took specific experimental focus (e.g., deep surgical anesthesia) and methodology (e.g., TiO₂ more readily enriches phospho-serine/threonine peptides compared to phospho-tyrosine), significant number of phosphorylation events appeared in known targets of anesthetics. However, the biological functions of many of the identified phosphorylation sites have not been described and further experiments and tools (e.g., antibodies) need to be performed and developed, respectively, to confirm and extend our observations.

Nevertheless, our study rationalizes the use of quantitative phosphoproteomics to investigate the neurobiological mechanisms and targets of anesthetics (and drugs in general) in the mammalian brain and demonstrates unexpectedly pronounced phosphorylation changes in multiple proteins in the central nervous system following already a brief anesthesia. Most interestingly, our pharmacological follow-up and confirmatory experiments clearly demonstrate that anesthesia (or sedation)—irrespective of the pharmacological means inducing it—produce rapid (inhibitory) phosphorylation changes on GSK3β and p44/42-MAPK, and phosphorylation alterations on MAP2 that render its dissociation from microtubules. Although the precise neurobiological basis and overall significance of these findings remain unclear it is important to note that regulation of GSK3β signaling and MAP2 has been implicated in the mechanisms of actions of neuroplastic treatments, antidepressants and mood stabilizers. (38-40)

Methods

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Animals

The animal experiments were carried out according to the guidelines of the Society for Neuroscience and were approved by the County Administrative Board of Southern Finland (License: ESAVI/10527/04.10.07/2014). Adult male C57BL/6JRccHsd mice (Harlan Laboratories, Venray, Netherland) were used in the study. Animals were maintained in the animal facility of University of Helsinki, Finland, under standard laboratory conditions (21 °C, 12 h light–dark cycle, lights on at 6 A.M.) with free access to food and water.

Drug Treatments

Unless otherwise stated the isoflurane (Vetflurane, Virbac) anesthesia was induced with 4% isoflurane for 2 min, after which the anesthetic concentration was gradually reduced to 2% for 30 min duration (oxygen flow: 0.3–0.5 l/min). Sevoflurane (Sevorane, Baxter) anesthesia was induced with 6%, after which the anesthetic concentration was gradually reduced to 4.5% for 30 min duration (oxygen flow: 0.3–0.5 l/min). Sham mice were kept in the induction chamber without any oxygen or isoflurane/sevoflurane flow for 2 min. Urethane (2 g/kg; kindly provided by Dr. Kai Kaila, University of Helsinki) and ketamine (100 mg/kg; Ketaminol, Intervet) were injected intraperitoneally. Saline was injected in similar volume for the control group. Body temperature was maintained with a heat pad throughout the treatments.

EEG and EMG Recordings

For the implantation of electroencephalographic (EEG) and electromyographic (EMG) electrodes, mice were anesthetized with isoflurane anesthesia (4% induction, 1.5–2% maintenance). Lidocaine (10 mg/mL) was used for local analgesia, and buprenorphine (0.1 mg/kg, s.c.) for postoperative care. Two epidural screw EEG electrodes were placed above the fronto-parietal cortex. A further screw served as mounting support. Two silver wire electrodes were implanted in the nuchal muscles to monitor the EMG. After surgery, rats were single-housed in Plexiglas boxes. After a recovery period of 1 week, animals were connected to flexible counterbalanced cables for EEG/EMG recording and habituated to recording cables for 2 days. The EEG and EMG signals were amplified (EMG gain setting, 5 K; EEG gain setting, 10 K) and filtered (high pass, 1 Hz; low pass, 100 Hz) with a 16-channel AC amplifier (A-M System, model 3500), sampled at 254 Hz (EEG) or 70 Hz (EMG) with 1401 unit (CED), and recorded using Spike2 (version 6, Cambridge Electronic Devices). The processing of the EEG data was obtained using Spike2 (version 6, Cambridge Electronic Devices). EEG files were manually scored in 4 s epochs with Spike2 sleep scoring script Sleepscore 1.01 (CED) according to standard criteria: Non-REM sleep was recognized as high-amplitude EEG associated with low-voltage EMG and presence of slow delta (1.0–4 Hz) oscillations in the EEG, REM sleep as low-amplitude, high-frequency EEG with absence of EMG and presence of prominent EEG theta (4–8 Hz), and waking as low-amplitude, high-frequency EEG with high-voltage EMG. Artifacts or epochs with mixed states were marked and excluded from power spectral analysis. EEG power spectra were calculated within the 1–60 Hz frequency range by fast Fourier transform (FFT = 256, Hanning window, 1.0 Hz resolution).

Phosphoprotein Enrichment and Quantitative Mass Spectrometry

Mice were killed by rapid cervical dislocation immediately after the treatment. Both hippocampi were dissected on a cooled dish as described and processed further for phosphoproteomic or confirmatory Western blot analyses. Phosphoproteomic analyses have been conducted essentially as described. (43) Briefly, proteins were extracted from the brain samples by cutting the tissue with a razor blade and vortexing in the presence of glass beads (0.5–5 mm) in a detergent free lysis buffer containing broad-range protease and phosphatase inhibitors. After centrifugation protein concentrations were determined and sample tubes with equal amounts of total protein were prepared. Samples were then treated with ice-cold acetone to precipitate the proteins and to get rid of lipids that might hinder the subsequent analysis. The precipitates were dried, resuspended and prepared for digestion into peptides by trypsin. Phosphopeptides were enriched by titanium dioxide (TiO₂) using self-prepared microcolumns and analyzed subsequently by liquid chromatography tandem mass spectrometry (LC-MS/MS) method to define relative phosphoproteome changes. Notably, the phosphopeptide enrichment step multiplies the sensitivity to detect phosphoproteins, particularly serine and threonine residues.

Bioinformatics

Progenesis software (Nonlinear Dynamics, UK) was used for peptide quantification and the corresponding peptide identifications were performed using Mascot search through Proteome Discoverer (Thermo Fisher Scientific). The Progenesis-normalized peptide abundances of phosphorylated peptides were selected for differential expression analysis. The phosphosite locations in the peptide sequences were matched to locations in full protein sequence and peptides of the same protein having same phosphosites were summed. Abundances therefore represent phosphorylated proteins that have a specific combination of phosphosites. Significant differences in the abundances between the treatment groups were detected using reproducibility-optimized test statistic (ROTS), which selects a statistic from a family of t-type statistics based on the maximal overlap of top-ranked results in resamplings of the original data set. (44) Phosphoproteomic alterations with false discovery rate (FDR) below 0.05 were considered as significant. Functional analyses were carried out using the DAVID tool, which is a biological knowledgebase integrated with different analytical tools. (45) The functional annotation terms that were enriched in the list of differentially expressed phosphoproteins were determined using a modified Fisher’s exact test (EASE score). Benjamini-Hochberg correction for multiple testing was used to control false discovery rate.

Western Blot

Western blotting was performed essentially as previously described. (46, 47) Briefly, the brain samples were homogenized in NP buffer (137 mM NaCl, 20 mM Tris, 1% NP-40, 10% glycerol, 48 mM NaF, H₂O, Complete inhibitor mix (Roche), PhosphoStop (Roche)), incubated on ice, and centrifuged (16 000g, 15 min, +4 °C), and the resulting supernatant collected for further analysis. Sample protein concentrations were measured using Bio-Rad DC protein assay (Bio-Rad Laboratories, Hercules, CA). Samples (40 μg protein) were separated with SDS-PAGE under reducing conditions and blotted to a PVDF (polyvinylidene difluoride) membrane. Membranes were incubated with the following primary antibodies: anti-p-GSK3β^Ser9 (#9336, 1:1000, Cell Signaling Technology, (CST)), anti-p-p44/42-MAPK^Thr202/Y204 (#9106, 1:1000, CST), anti-p-MAP2^Thr1620/1623 (#4544, 1:1000, CST), anti-GSK3β (#9315, 1:1000, CST), anti-p44/42-MAPK (#9102, 1:1000, CST), and anti-GAPDH (sc-25778, 1:10 000, Santa Cruz Biotechnology). Further, the membranes were washed with TBS/0.1% Tween (TBST) and incubated with horseradish peroxidase conjugated secondary antibodies (1:10 000 in nonfat dry milk, 1 h at room temperature; Bio-Rad). After subsequent washes, secondary antibodies were visualized using enhanced chemiluminescence (ECL Plus, ThermoScientific, Vantaa, Finland) for detection by Bio-Rad ChemiDoc MP camera (Bio-Rad Laboratories, Helsinki, Finland).

Statistics

The statistical analyses for the phosphoproteomic data were done using the R project (http://www.r-project.org) and Bioconductor (http://www.bioconductor.org). For the EEG data, one-way ANOVA followed by Dunnett‘s multiple comparison test was used. For the Western blot data, Student’s t test was used. Statistical significance was set to <0.05.

Supporting Information

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The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acschemneuro.6b00002.

Table 1: Differentially regulated phosphoproteins after sham and isoflurane anesthesia (4% induction, 2% maintenance; 30 min) (N = 3/group). Phosphopeptide changes arranged based on statistical significance (FDR < 0.05). Table 2: Functional annotation clusters arranged based on the statistical significance (FDR < 0.05). (XLSX)

cn6b00002_si_001.xlsx (64.83 kb)

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Author Information

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Corresponding Author
- Tomi Rantamäki - †Faculty of Biological and Environmental Sciences, Department of Biosciences, Division of Physiology and Neuroscience, and ‡Neuroscience Center, University of Helsinki, FI-00014 Helsinki, Finland; Turku Centre for Biotechnology, University of Turku, FI-20014 Turku, Finland; Institute of Biomedicine, University of Helsinki, FI-00014 Helsinki, Finland; Email: [email protected]
Authors
- Samuel Kohtala - †Faculty of Biological and Environmental Sciences, Department of Biosciences, Division of Physiology and Neuroscience, and ‡Neuroscience Center, University of Helsinki, FI-00014 Helsinki, Finland; Turku Centre for Biotechnology, University of Turku, FI-20014 Turku, Finland; Institute of Biomedicine, University of Helsinki, FI-00014 Helsinki, Finland
- Wiebke Theilmann - †Faculty of Biological and Environmental Sciences, Department of Biosciences, Division of Physiology and Neuroscience, and ‡Neuroscience Center, University of Helsinki, FI-00014 Helsinki, Finland; Turku Centre for Biotechnology, University of Turku, FI-20014 Turku, Finland; Institute of Biomedicine, University of Helsinki, FI-00014 Helsinki, Finland
- Tomi Suomi - Turku Centre for Biotechnology, University of Turku, FI-20014 Turku, Finland
- Henna-Kaisa Wigren - Institute of Biomedicine, University of Helsinki, FI-00014 Helsinki, Finland
- Tarja Porkka-Heiskanen - Institute of Biomedicine, University of Helsinki, FI-00014 Helsinki, Finland
- Laura L. Elo - Turku Centre for Biotechnology, University of Turku, FI-20014 Turku, Finland
- Anne Rokka - Turku Centre for Biotechnology, University of Turku, FI-20014 Turku, Finland
Author Contributions
W.T. and T.S.: Equal contribution. S.K., W.T., T.S., H-K.W. performed the studies; S.K., W.T. and T.S., analyzed the data; S.K., W.T. and T.R. prepared the figures and tables. T.R. generated the original idea of the study. T.P-H., L.E., A.R. and T.R. planned and supervised the studies; S.K. and T.R. wrote the manuscript.
Funding
This study has been supported by the Academy of Finland (T.R.; grant no: 284569 and 276333) and Orion Research Foundation (S.K.)
Notes
The authors declare no competing financial interest.

Acknowledgment

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Mass spectrometry analysis was performed at the Turku Proteomics Facility, University of Turku and Åbo Akademi University. The facility is supported by Biocenter Finland. The authors would like to thank Maria Partanen for excellent technical assistance.

References

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General anesthetic actions on GABAA receptors
Garcia, Paul S.; Kolesky, Scott E.; Jenkins, Andrew
Current Neuropharmacology (2010), 8 (1), 2-9CODEN: CNUEAN; ISSN:1875-6190. (Bentham Science Publishers Ltd.)
A review. General anesthetic drugs interact with many receptors in the nervous system, but only a handful of these interactions are crit. for producing anesthesia. Over the last 20 years, neuropharmacologists have revealed that one of the most important target sites for general anesthetics is the GABAA receptor. In this review we will discuss what is known about anesthetic - GABAA receptor interactions.
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Blaesse, P., Airaksinen, M. S., Rivera, C., and Kaila, K. (2009) Cation-chloride cotransporters and neuronal function Neuron 61, 820– 838 DOI: 10.1016/j.neuron.2009.03.003
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Cation-chloride cotransporters and neuronal function
Blaesse, Peter; Airaksinen, Matti S.; Rivera, Claudio; Kaila, Kai
Neuron (2009), 61 (6), 820-838CODEN: NERNET; ISSN:0896-6273. (Cell Press)
A review. Recent years have witnessed a steep increase in studies on the diverse roles of neuronal cation-chloride cotransporters (CCCs). The versatility of CCC gene transcription, posttranslational modification, and trafficking are on par with what is known about ion channels. The cell-specific and subcellular expression patterns of different CCC isoforms have a key role in modifying a neuron's electrophysiol. phenotype during development, synaptic plasticity, and disease. While having a major role in controlling responses mediated by GABAA and glycine receptors, CCCs also show close interactions with glutamatergic signaling. A cross-talk among CCCs and trophic factors is important in short-term and long-term modification of neuronal properties. CCCs appear to be multifunctional proteins that are also involved in shaping neuronal structure at various stages of development, from stem cells to synaptogenesis. The rapidly expanding work on CCCs promotes our understanding of fundamental mechanisms that control brain development and functions under normal and pathophysiol. conditions.
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Vutskits, L. (2012) General anesthesia: a gateway to modulate synapse formation and neural plasticity? Anesth. Analg. 115, 1174– 1182 DOI: 10.1213/ANE.0b013e31826a1178
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Review Article: General Anesthesia: A Gateway to Modulate Synapse Formation and Neural Plasticity?
Vutskits, Laszlo
Anesthesia & Analgesia (Hagerstown, MD, United States) (2012), 115 (5), 1174-1182CODEN: AACRAT; ISSN:0003-2999. (Lippincott Williams & Wilkins)
A review. Appropriate balance between excitatory and inhibitory neural activity patterns is of utmost importance in the maintenance of neuronal homeostasis. General anesthetic-induced pharmacol. interference with this equil. results not only in a temporary loss of consciousness but can also initiate long-term changes in brain function. Although these alterations were initially considered deleterious, recent observations suggest that at least under some specific conditions, they may eventually improve neural function. The goal of this review is to provide insights into the mechanisms underlying these dual effects. Basic science issues on the important role of crit. periods during neural circuitry assembly will be discussed to better understand how even brief exposures to general anesthetics could initiate context-dependent lasting changes in neuronal structure and function. Recent series of observations suggesting a developmental stage-dependent impact of these drugs on synaptogenesis will then be summarized together with currently known mol. mechanisms underlying these effects. Particular emphasis will be placed on how anesthetic drugs modulate neural plasticity in the adult brain and how this may improve neural function under some pathol. states. The ensemble of these new observations strongly suggests that general anesthetics should not merely be considered toxic drugs but rather acknowledged as robust, context-dependent modulators of neural plasticity.
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Kapila, A. K., Watts, H. R., Wang, T., and Ma, D. (2014) The impact of surgery and anesthesia on post-operative cognitive decline and Alzheimer’s disease development: biomarkers and preventive strategies J. Alzheimer's Dis. 41, 1– 13 DOI: 10.3233/JAD-132258
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The Impact of Surgery and Anesthesia on Post-Operative Cognitive Decline and Alzheimer's Disease Development: Biomarkers and Preventive Strategies
Kapila, Ayush K.; Watts, Helena R.; Wang, Tianlong; Ma, Daqing
Journal of Alzheimer's Disease (2014), 41 (1), 1-13CODEN: JADIF9; ISSN:1387-2877. (IOS Press)
A review. Alzheimer's disease (AD) is a major social and clin. burden in the elderly, affecting 5% of people aged over 65 and 20% aged over 80. Despite improved management, a cure has not been found and hence anal. of predisposing factors to identify preventive strategies has become increasingly important. Surgery and anesthesia have been proposed to increase the incidence of post-operative cognitive decline (POCD) and AD. This is hypothesized to be the result of a malignant neuroinflammatory response and subsequent synaptic impairment in the elderly and susceptible individuals. As a result, strategies are being explored to prevent surgery and anesthesia induced cognitive impairment. Whereas previously the diagnosis of AD was primarily dependent on clin. examn., biomarkers such as inflammatory cytokines, amyloid-β, and tau deposition in the cerebrospinal fluid have received increased attention. Nonetheless, AD is currently still treated symptomatically with acetylcholinesterase inhibitors and NMDA antagonists to improve cholinergic transmission and prevent glutamatergic excitotoxicity. Therapeutic success is, however, often not achieved, since these treatment methods do not address the ongoing neuroinflammatory processes and hence novel therapeutic and protective strategies are urgently needed. This review provides an insight into the current understanding of age-related cognitive impairment post-surgery and reflects on novel markers of AD pathogeneses exploring their use as targets for treatment. It gives a summary of recent efforts in preventing and treating POCD or AD with regards to the choice and depth of anesthesia, surgical strategy, and peri-operative medication, and discusses the mechanism of action and therapeutic prospects of novel agents.
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Whittington, R. A., Bretteville, A., Dickler, M. F., and Planel, E. (2013) Anesthesia and tau pathology Prog. Neuro-Psychopharmacol. Biol. Psychiatry 47, 147– 155 DOI: 10.1016/j.pnpbp.2013.03.004
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Anesthesia and tau pathology
Whittington, Robert A.; Bretteville, Alexis; Dickler, Maya F.; Planel, Emmanuel
Progress in Neuro-Psychopharmacology & Biological Psychiatry (2013), 47 (), 147-155CODEN: PNPPD7; ISSN:0278-5846. (Elsevier Inc.)
A review. Alzheimer's disease (AD) is the most common form of dementia and remains a growing worldwide health problem. As life expectancy continues to increase, the no. of AD patients presenting for surgery and anesthesia will steadily rise. The etiol. of sporadic AD is thought to be multifactorial, with environmental, biol. and genetic factors interacting together to influence AD pathogenesis. Recent reports suggest that general anesthetics may be such a factor and may contribute to the development and exacerbation of this neurodegenerative disorder. Intra-neuronal neurofibrillary tangles (NFT), composed of hyperphosphorylated and aggregated tau protein are one of the main neuropathol. hallmarks of AD. Tau pathol. is important in AD as it correlates very well with cognitive dysfunction. Lately, several studies have begun to elucidate the mechanisms by which anesthetic exposure might affect the phosphorylation, aggregation and function of this microtubule-assocd. protein. Here, we specifically review the literature detailing the impact of anesthetic administration on aberrant tau hyperphosphorylation as well as the subsequent development of neurofibrillary pathol. and degeneration.
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Langer, G., Neumark, J., Koinig, G., Graf, M., and Schönbeck, G. (1985) Rapid psychotherapeutic effects of anesthesia with isoflurane (ES narcotherapy) in treatment-refractory depressed patients Neuropsychobiology 14, 118– 120 DOI: 10.1159/000118216
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Engelhardt, W., Carl, G., and Hartung, E. (1993) Intra-individual open comparison of burst-suppression-isoflurane-anaesthesia versus electroconvulsive therapy in the treatment of severe depression Eur. J. Anaesthesiol. 10, 113– 118
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Intra-individual open comparison of burst-suppression-isoflurane-anaesthesia versus electroconvulsive therapy in the treatment of severe depression
Engelhardt W; Carl G; Hartung E
European journal of anaesthesiology (1993), 10 (2), 113-8 ISSN:0265-0215.
Isoflurane anaesthesia was proposed instead of electro-convulsive therapy (ECT) in patients with treatment-refractory depression. This open study compared burst-suppression-isoflurane-anaesthesia (BSIA) and ECT in 12 severely depressed patients. A series of 6 BSIA was administered in every patient. If improvement was insufficient or only temporary, a series of up to 12 ECT was given. A marked improvement of the depression was shown after both BSIA and ECT. Three patients were discharged from hospital after BSIA, nine patients were treated with BSIA and then ECT. The therapeutic effect of both regimens was equal as evidenced by the Hamilton-depression-rating-scale, a visual-analog-scale and the clinical global impression. BSIA requires more time and monitoring than ECT. Our exclusions of coronary, cerebral and peripheral vascular disease, untreated hypertension and focal neurological disease are strongly recommended. Due to the ease of application, ECT remains the standard treatment in depressed patients, but we consider BSIA a valuable alternative at least in patients who object to ECT.
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Langer, G., Karazman, R., Neumark, J., Saletu, B., Schönbeck, G., Grünberger, J., Dittrich, R., Petricek, W., Hoffmann, P., and Linzmayer, L. (1995) Isoflurane narcotherapy in depressive patients refractory to conventional antidepressant drug treatment. A double-blind comparison with electroconvulsive treatment Neuropsychobiology 31, 182– 194 DOI: 10.1159/000119190
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Doyle, P. W. and Matta, B. F. (1999) Burst suppression or isoelectric encephalogram for cerebral protection: evidence from metabolic suppression studies Br. J. Anaesth. 83, 580– 584 DOI: 10.1093/bja/83.4.580
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Burst suppression or isoelectric encephalogram for cerebral protection: evidence from metabolic suppression studies
Doyle P W; Matta B F
British journal of anaesthesia (1999), 83 (4), 580-4 ISSN:0007-0912.
Metabolic suppression may have a role in cerebral protection. It is often assumed that the cerebral metabolic and protective effects of qualitative burst suppression are similar to those of the isoelectric encephalogram (EEG). We have examined the effect of different degrees of EEG suppression on blood flow and oxygen difference during general anaesthesia. We studied 11 patients undergoing general anaesthesia for resection of acoustic neuromas. The study was performed after surgery with propofol and remifentanil anaesthesia. Transcranial Doppler ultrasonography and jugular bulb venous saturations were measured at values of EEG suppression: 0%, 50% and 100% (isoelectric EEG). Data from nine patients were suitable for analysis. There were no significant differences in mean arterial pressure, heart rate or PaCO2 during EEG activity, 50% burst suppression ratio or isoelectric EEG. There was a significant decrease in middle cerebral artery flow velocity (vmca) with increasing EEG suppression (0% suppression, mean 38 (SEM 4) cm s-1; 50% suppression, 29 (3) cm s-1; and 100% suppression, 24 (2) cm s-1; P < 0.05). Jugular bulb venous saturations did not change consistently with the change in EEG activity, indicating intact flow-metabolism coupling. We conclude that the degree of EEG suppression had a significant effect on blood flow. If flow-metabolism coupling is maintained, the assumption that cerebral metabolism during 50% EEG burst suppression is equivalent to isoelectric EEG may not be justified. If cerebral protection is related to brain metabolism, then an isoelectric EEG may give more cerebral protection than 50% burst suppression.
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Liu, Y. and Chance, M. R. (2014) Integrating phosphoproteomics in systems biology Comput. Struct. Biotechnol. J. 10, 90– 97 DOI: 10.1016/j.csbj.2014.07.003
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Integrating phosphoproteomics in systems biology
Liu Yu; Chance Mark R
Computational and structural biotechnology journal (2014), 10 (17), 90-7 ISSN:2001-0370.
Phosphorylation of serine, threonine and tyrosine plays significant roles in cellular signal transduction and in modifying multiple protein functions. Phosphoproteins are coordinated and regulated by a network of kinases, phosphatases and phospho-binding proteins, which modify the phosphorylation states, recognize unique phosphopeptides, or target proteins for degradation. Detailed and complete information on the structure and dynamics of these networks is required to better understand fundamental mechanisms of cellular processes and diseases. High-throughput technologies have been developed to investigate phosphoproteomes in model organisms and human diseases. Among them, mass spectrometry (MS)-based technologies are the major platforms and have been widely applied, which has led to explosive growth of phosphoproteomic data in recent years. New bioinformatics tools are needed to analyze and make sense of these data. Moreover, most research has focused on individual phosphoproteins and kinases. To gain a more complete knowledge of cellular processes, systems biology approaches, including pathways and networks modeling, have to be applied to integrate all components of the phosphorylation machinery, including kinases, phosphatases, their substrates, and phospho-binding proteins. This review presents the latest developments of bioinformatics methods and attempts to apply systems biology to analyze phosphoproteomics data generated by MS-based technologies. Challenges and future directions in this field will be also discussed.
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Walaas, S. I. and Greengard, P. (1991) Protein phosphorylation and neuronal function Pharmacol. Rev. 43, 299– 349
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Protein phosphorylation and neuronal function
Walaas, Sven Ivar; Greengard, Paul
Pharmacological Reviews (1991), 43 (3), 299-349CODEN: PAREAQ; ISSN:0031-6997.
A review, with ∼750 refs. Topics discussed include: protein kinases in brain, phosphoprotein phosphatases in brain, phosphoproteins in neurotransmission, and phosphoproteins in clin. disorders.
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McKernan, R. M., Rosahl, T. W., Reynolds, D. S., Sur, C., Wafford, K. A., Atack, J. R., Farrar, S., Myers, J., Cook, G., Ferris, P., Garrett, L., Bristow, L., Marshall, G., Macaulay, A., Brown, N., Howell, O., Moore, K. W., Carling, R. W., Street, L. J., Castro, J. L., Ragan, C. I., Dawson, G. R., and Whiting, P. J. (2000) Sedative but not anxiolytic properties of benzodiazepines are mediated by the GABA(A) receptor alpha1 subtype Nat. Neurosci. 3, 587– 592 DOI: 10.1038/75761
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Sedative but not anxiolytic properties of benzodiazepines are mediated by the GABAA receptor α1 subtype
McKernan, R. M.; Rosahl, T. W.; Reynolds, D. S.; Sur, C.; Wafford, K. A.; Atack, J. R.; Farrar, S.; Myers, J.; Cook, G.; Ferris, P.; Garrett, L.; Bristow, L.; Marshall, G.; Macaulay, A.; Brown, N.; Howell, O.; Moore, K. W.; Carling, R. W.; Street, L. J.; Castro, J. L.; Ragan, C. I.; Dawson, G. R.; Whiting, P. J.
Nature Neuroscience (2000), 3 (6), 587-592CODEN: NANEFN; ISSN:1097-6256. (Nature America Inc.)
Inhibitory neurotransmission in the brain is largely mediated by GABAA receptors. Potentiation of GABA receptor activation through an allosteric benzodiazepine (BZ) site produces the sedative, anxiolytic, muscle relaxant, anticonvulsant and cognition-impairing effects of clin. used BZs such as diazepam. We created genetically modified mice (α1 H101R) with a diazepam-insensitive α1 subtype and a selective BZ site ligand, L-838,417, to explore GABAA receptor subtypes mediating specific physiol. effects. These two complimentary approaches revealed that the α1 subtype mediated the sedative, but not the anxiolytic effects of benzodiazepines. This finding suggests ways to improve anxiolytics and to develop drugs for other neurol. disorders based on their specificity for GABAA receptor subtypes in distinct neuronal circuits.
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Zhou, C., Liang, P., Liu, J., Ke, B., Wang, X., Li, F., Li, T., Bayliss, D. A., and Chen, X. (2015) HCN1 Channels Contribute to the Effects of Amnesia and Hypnosis but not Immobility of Volatile Anesthetics Anesth. Analg. 121, 661– 666 DOI: 10.1213/ANE.0000000000000830
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HCN1 Channels Contribute to the Effects of Amnesia and Hypnosis but not Immobility of Volatile Anesthetics
Zhou, Cheng; Liang, Peng; Liu, Jin; Ke, Bowen; Wang, Xiaojia; Li, Fengshan; Li, Tao; Bayliss, Douglas A.; Chen, Xiangdong
Anesthesia & Analgesia (Hagerstown, MD, United States) (2015), 121 (3), 661-666CODEN: AACRAT; ISSN:0003-2999. (Lippincott Williams & Wilkins)
Background: Hyperpolarization-activated, cyclic nucleotide-gated (HCN) subtype 1 (HCN1) channels have been identified as targets of ketamine to produce hypnosis. Volatile anesthetics also inhibit HCN1 channels. However, the effects of HCN1 channels on volatile anesthetics in vivo are still elusive. This study uses global and conditional HCN1 knockout mice to evaluate how HCN1 channels affect the actions of volatile anesthetics. Methods: Min. alveolar concns. (MACs) of isoflurane and sevoflurane that induced immobility (MAC of immobility) and/or hypnosis (MAC of hypnosis) were detd. in wild-type mice, global HCN1 knockout (HCN1) mice, HCN1 channel gene with 2 lox-P sites flanking a region of the fourth exon of HCN1 (HCN1) mice, and forebrain-selective HCN1 knockout (HCN1: cre) mice. Immobility of mice was defined as no purposeful reactions to tail-clamping stimulus, and hypnosis was defined as loss of righting reflex. The amnestic effects of isoflurane and sevoflurane were evaluated by fear-potentiated startle in these 4 strains of mice. Results: All MAC values were expressed as mean ± SEM. For MAC of immobility of isoflurane, no significant difference was found among wild-type, HCN1, HCN1, and HCN1: cre mice (all ∼1.24%-1.29% isoflurane). For both HCN1 and HCN1: cre mice, the MAC of hypnosis for isoflurane (each ∼1.05% isoflurane) was significantly increased over their nonknockout controls: HCN1 vs. wild-type (0.86% ± 0.03%, P < 0.001) and HCN1: cre vs. HCN1 mice (0.84% ± 0.03%, P < 0.001); no significant difference was found between HCN1 and HCN1: cre mice. For MAC of immobility of sevoflurane, no significant difference was found among wild-type, HCN1, HCN1, and HCN1: cre mice (all ∼2.6%-2.7% sevoflurane). For both HCN1 and HCN1: cre mice, the MAC of hypnosis for sevoflurane (each ∼1.90% sevoflurane) was significantly increased over their nonknockout controls: HCN1 vs. wild-type (1.58% ± 0.05%, P < 0.001) and HCN1: cre vs. HCN1 mice (1.56% ± 0.05%, P < 0.001). No significant difference was found between HCN1 and HCN1: cre mice. By fear-potentiated startle expts., amnestic effects of isoflurane and sevoflurane were significantly attenuated in HCN1 and HCN1: cre mice (both P < 0.002 vs. wild-type or HCN1 mice). No significant difference was found between HCN1 and HCN1: cre mice. Conclusions: Forebrain HCN1 channels contribute to hypnotic and amnestic effects of volatile anesthetics, but HCN1 channels are not involved in the immobilizing actions of volatile anesthetics.
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Bojak, I., Day, H. C., and Liley, D. T. J. (2013) Ketamine, propofol, and the EEG: a neural field analysis of HCN1-mediated interactions Front. Comput. Neurosci. 7, 22 DOI: 10.3389/fncom.2013.00022
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Ketamine, Propofol, and the EEG: A Neural Field Analysis of HCN1-Mediated Interactions
Bojak Ingo; Day Harry C; Liley David T J
Frontiers in computational neuroscience (2013), 7 (), 22 ISSN:.
Ketamine and propofol are two well-known, powerful anesthetic agents, yet at first sight this appears to be their only commonality. Ketamine is a dissociative anesthetic agent, whose main mechanism of action is considered to be N-methyl-d-aspartate (NMDA) antagonism; whereas propofol is a general anesthetic agent, which is assumed to primarily potentiate currents gated by γ-aminobutyric acid type A (GABAA) receptors. However, several experimental observations suggest a closer relationship. First, the effect of ketamine on the electroencephalogram (EEG) is markedly changed in the presence of propofol: on its own ketamine increases θ (4-8 Hz) and decreases α (8-13 Hz) oscillations, whereas ketamine induces a significant shift to beta band frequencies (13-30 Hz) in the presence of propofol. Second, both ketamine and propofol cause inhibition of the inward pacemaker current I h, by binding to the corresponding hyperpolarization-activated cyclic nucleotide-gated potassium channel 1 (HCN1) subunit. The resulting effect is a hyperpolarization of the neuron's resting membrane potential. Third, the ability of both ketamine and propofol to induce hypnosis is reduced in HCN1-knockout mice. Here we show that one can theoretically understand the observed spectral changes of the EEG based on HCN1-mediated hyperpolarizations alone, without involving the supposed main mechanisms of action of these drugs through NMDA and GABAA, respectively. On the basis of our successful EEG model we conclude that ketamine and propofol should be antagonistic to each other in their interaction at HCN1 subunits. Such a prediction is in accord with the results of clinical experiment in which it is found that ketamine and propofol interact in an infra-additive manner with respect to the endpoints of hypnosis and immobility.
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Carr, D. B., Andrews, G. D., Glen, W. B., and Lavin, A. (2007) alpha2-Noradrenergic receptors activation enhances excitability and synaptic integration in rat prefrontal cortex pyramidal neurons via inhibition of HCN currents J. Physiol. 584, 437– 450 DOI: 10.1113/jphysiol.2007.141671
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α2-Noradrenergic receptors activation enhances excitability and synaptic integration in rat prefrontal cortex pyramidal neurons via inhibition of HCN currents
Carr, David B.; Andrews, Glenn D.; Glen, William B.; Lavin, A.
Journal of Physiology (Oxford, United Kingdom) (2007), 584 (2), 437-450CODEN: JPHYA7; ISSN:0022-3751. (Blackwell Publishing Ltd.)
Stimulation of α2-noradrenergic (NA) receptors within the PFC improves working memory performance. This improvement is accompanied by a selective increase in the activity of PFC neurons during delay periods, although the cellular mechanisms responsible for this enhanced response are largely unknown. Here we used current and voltage clamp recordings to characterize the response of layer V-VI PFC pyramidal neurons to α2-NA receptor stimulation. α2-NA receptor activation produced a small hyperpolarization of the resting membrane potential, which was accompanied by an increase in input resistance and evoked firing. Voltage clamp anal. demonstrated that α2-NA receptor stimulation inhibited a cesium and ZD7288-sensitive hyperpolarization-activated (HCN) inward current. Suppression of HCN current by α2-NA stimulation was not dependent on adenylate cyclase but instead required activation of a PLC-PKC linked signaling pathway. Similar to direct blockade of HCN channels, α2-NA receptor stimulation produced a significant enhancement in temporal summation during trains of distally evoked EPSPs. These dual effects of α2-NA receptor stimulation - membrane hyperpolarization and enhanced temporal integration - together produce an increase in the overall gain of the response of PFC pyramidal neurons to excitatory synaptic input. The net effect is the suppression of isolated excitatory inputs while enhancing the response to a coherent burst of synpatic activity.
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Chen, X., Shu, S., Kennedy, D. P., Willcox, S. C., and Bayliss, D. A. (2009) Subunit-specific effects of isoflurane on neuronal Ih in HCN1 knockout mice J. Neurophysiol. 101, 129– 140 DOI: 10.1152/jn.01352.2007
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Subunit-specific effects of isoflurane on neuronal Ih in HCN1 knockout mice
Chen, Xiangdong; Shu, Shaofang; Kennedy, Dylan P.; Willcox, Sarah C.; Bayliss, Douglas A.
Journal of Neurophysiology (2009), 101 (1), 129-140CODEN: JONEA4; ISSN:0022-3077. (American Physiological Society)
The ionic mechanisms that contribute to general anesthetic actions have not been elucidated, although increasing evidence has pointed to roles for subthreshold ion channels, such as the HCN channels underlying the neuronal hyperpolarization-activated cationic current (Ih). Here, the authors used conventional HCN1 knockout mice to test directly the contributions of specific HCN subunits to effects of isoflurane, an inhalational anesthetic, on membrane and integrative properties of motor and cortical pyramidal neurons in vitro. Compared with wild-type mice, residual Ih from knockout animals was smaller in amplitude and presented with HCN2-like properties. Inhibition of Ih by isoflurane previously attributed to HCN1 subunit-contg. channels (i.e., a hyperpolarizing shift in half-activation voltage [V1/2]) was absent in neurons from HCN1 knockout animals; the remaining inhibition of current amplitude could be attributed to effects on residual HCN2 channels. The authors also found that isoflurane increased temporal summation of excitatory postsynaptic potentials (EPSPs) in cortical neurons from wild-type mice; this effect was predicted by simulation of anesthetic-induced dendritic Ih inhibition, which also revealed more prominent summation accompanying shifts in V1/2 (an HCN1-like effect) than decreased current amplitude (an HCN2-like effect). Accordingly, anesthetic-induced EPSP summation was not obsd. in cortical cells from HCN1 knockout mice. In wild-type mice, the enhanced synaptic summation obsd. with low concns. of isoflurane contributed to a net increase in cortical neuron excitability. In summary, HCN channel subunits account for distinct anesthetic effects on neuronal membrane properties and synaptic integration; inhibition of HCN1 in cortical neurons may contribute to the synaptically mediated slow-wave cortical synchronization that accompanies anesthetic-induced hypnosis.
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Zhou, C., Douglas, J. E., Kumar, N. N., Shu, S., Bayliss, D. A., and Chen, X. (2013) Forebrain HCN1 channels contribute to hypnotic actions of ketamine Anesthesiology 118, 785– 795 DOI: 10.1097/ALN.0b013e318287b7c8
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There is no corresponding record for this reference.
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Henzi, V. and MacDermott, A. B. (1992) Characteristics and function of Ca(2+)- and inositol 1,4,5-trisphosphate-releasable stores of Ca2+ in neurons Neuroscience 46, 251– 273 DOI: 10.1016/0306-4522(92)90049-8
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There is no corresponding record for this reference.
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Takeshima, H., Venturi, E., and Sitsapesan, R. (2015) New and notable ion-channels in the sarcoplasmic/endoplasmic reticulum: do they support the process of intracellular Ca(2+) release? J. Physiol. 593, 3241– 3251 DOI: 10.1113/jphysiol.2014.281881
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New and notable ion-channels in the sarcoplasmic/endoplasmic reticulum: do they support the process of intracellular Ca2+ release?
Takeshima, Hiroshi; Venturi, Elisa; Sitsapesan, Rebecca
Journal of Physiology (Oxford, United Kingdom) (2015), 593 (15), 3241-3251CODEN: JPHYA7; ISSN:1469-7793. (Wiley-Blackwell)
Intracellular Ca2+ release through ryanodine receptor (RyR) and inositol trisphosphate receptor (IP3R) channels is supported by a complex network of addnl. proteins that are located in or near the Ca2+ release sites. In this review, we focus, not on RyR/IP3R, but on other ion-channels that are known to be present in the sarcoplasmic/endoplasmic reticulum (ER/SR) membranes. We review their putative physiol. roles and the evidence suggesting that they may support the process of intracellular Ca2+ release, either indirectly by manipulating ionic fluxes across the ER/SR membrane or by directly interacting with a Ca2+-release channel. These channels rarely receive scientific attention because of the general lack of information regarding their biochem. and/or electrophysiol. characteristics makes it difficult to predict their physiol. roles and their impact on SR Ca2+ fluxes. We discuss the possible role of SR K+ channels and, in parallel, detail the known biochem. and biophys. properties of the trimeric intracellular cation (TRIC) proteins and their possible biol. and pathophysiol. roles in ER/SR Ca2+ release. We summarize what is known regarding Cl- channels in the ER/SR and the non-selective cation channels or putative 'Ca2+ leak channels', including mitsugumin23 (MG23), pannexins, presenilins and the transient receptor potential (TRP) channels that are distributed across ER/SR membranes but which have not yet been fully characterized functionally.
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Ghosh, A. and Greenberg, M. E. (1995) Calcium signaling in neurons: molecular mechanisms and cellular consequences Science 268, 239– 247 DOI: 10.1126/science.7716515
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Calcium signaling in neurons: molecular mechanisms and cellular consequences
Ghosh, Anirvan; Greenberg, Michael E.
Science (Washington, D. C.) (1995), 268 (5208), 239-47CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
A review with >66 refs. Neuronal activity can lead to marked increases in the concn. of cytosolic calcium, which then functions as a second messenger that mediates a wide range of cellular responses. Calcium binds to calmodulin and stimulates the activity of a variety of enzymes, including calcium-calmodulin kinases and calcium-sensitive adenylate cyclases. These enzymes transduce the calcium signal and effect short-term biol. responses, such as the modification of synaptic proteins and long-lasting neuronal responses that require changes in gene expression. Recent studies of calcium signal-transduction mechanisms have revealed that, depending on the route of entry into a neuron, calcium differentially affects processes that are central to the development and plasticity of the nervous system, including activity-dependent cell survival, modulation of synaptic strength, and calcium-mediated cell death.
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Verkhratsky, A. J. and Petersen, O. H. (1998) Neuronal calcium stores Cell Calcium 24, 333– 343 DOI: 10.1016/S0143-4160(98)90057-4
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Verkhratsky, A., Orkand, R. K., and Kettenmann, H. (1998) Glial calcium: homeostasis and signaling function Physiol. Rev. 78, 99– 141
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There is no corresponding record for this reference.
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Agulhon, C., Petravicz, J., McMullen, A. B., Sweger, E. J., Minton, S. K., Taves, S. R., Casper, K. B., Fiacco, T. A., and McCarthy, K. D. (2008) What is the role of astrocyte calcium in neurophysiology? Neuron 59, 932– 946 DOI: 10.1016/j.neuron.2008.09.004
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What is the role of astrocyte calcium in neurophysiology?
Agulhon, Cendra; Petravicz, Jeremy; McMullen, Allison B.; Sweger, Elizabeth J.; Minton, Suzanne K.; Taves, Sarah R.; Casper, Kristen B.; Fiacco, Todd A.; McCarthy, Ken D.
Neuron (2008), 59 (6), 932-946CODEN: NERNET; ISSN:0896-6273. (Cell Press)
A review. Astrocytes comprise approx. half of the vol. of the adult mammalian brain and are the primary neuronal structural and trophic supportive elements. Astrocytes are organized into distinct nonoverlapping domains and extend elaborate and dense fine processes that interact intimately with synapses and cerebrovasculature. The recognition in the mid-1990s that astrocytes undergo elevations in intracellular Ca2+ concn. following activation of G protein-coupled receptors by synaptically released neurotransmitters demonstrated not only that astrocytes display a form of excitability but also that astrocytes may be active participants in brain information processing. The roles that astrocytic Ca2+ elevations play in neurophysiol. and esp. in modulation of neuronal activity have been intensely researched in recent years. Here, the authors summarize the current understanding of the function of astrocytic Ca2+ signaling in neurophysiol. processes and discuss areas where the role of astrocytes remains controversial and will therefore benefit from further study.
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Fang, M., Tao, Y.-X., He, F., Zhang, M., Levine, C. F., Mao, P., Tao, F., Chou, C.-L., Sadegh-Nasseri, S., and Johns, R. A. (2003) Synaptic PDZ Domain-mediated Protein Interactions Are Disrupted by Inhalational Anesthetics J. Biol. Chem. 278, 36669– 36675 DOI: 10.1074/jbc.M303520200
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Li, X., Friedman, A. B., Roh, M.-S., and Jope, R. S. (2005) Anesthesia and post-mortem interval profoundly influence the regulatory serine phosphorylation of glycogen synthase kinase-3 in mouse brain J. Neurochem. 92, 701– 704 DOI: 10.1111/j.1471-4159.2004.02898.x
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Anesthesia and post-mortem interval profoundly influence the regulatory serine phosphorylation of glycogen synthase kinase-3 in mouse brain
Li, Xiaohua; Friedman, Ari B.; Roh, Myoung-Sun; Jope, Richard S.
Journal of Neurochemistry (2005), 92 (3), 701-704CODEN: JONRA9; ISSN:0022-3042. (Blackwell Publishing Ltd.)
Glycogen synthase kinase-3 (GSK3) is a crucial enzyme contributing to the regulation of neuronal structure, plasticity and survival, is implicated as a contributory factor in prevalent diseases such as Alzheimer's disease and mood disorders and is regulated by a wide range of signaling systems and pharmacol. agents. Therefore, factors regulating GSK3 in vivo are currently of much interest. GSK3 is inhibited by phosphorylation of serine-9 or serine-21 in GSK3β and GSK3α, resp. This study found that accurate measurements of phospho-Ser-GSK3 in brain are confounded by a rapid post-mortem dephosphorylation, with ∼90% dephosphorylation of both GSK3 isoforms occurring within 2 min post-mortem. Furthermore, three anesthetics, pentobarbital, halothane and chloral hydrate, each caused large in vivo increases in the serine phosphorylation of both GSK3β and GSK3α in several regions of mouse brain. Thus, studies of the phosphorylation state of GSK3 in brain, and perhaps in other tissues, need to take into account post-mortem changes and the effects of anesthetics and there is a direct correlation between anesthesia and high levels of serine-phosphorylated GSK3.
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Linding, R., Jensen, L. J., Ostheimer, G. J., van Vugt, M. A. T. M., Jørgensen, C., Miron, I. M., Diella, F., Colwill, K., Taylor, L., Elder, K., Metalnikov, P., Nguyen, V., Pasculescu, A., Jin, J., Park, J. G., Samson, L. D., Woodgett, J. R., Russell, R. B., Bork, P., Yaffe, M. B., and Pawson, T. (2007) Systematic discovery of in vivo phosphorylation networks Cell 129, 1415– 1426 DOI: 10.1016/j.cell.2007.05.052
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Systematic discovery of in vivo phosphorylation networks
Linding, Rune; Jensen, Lars Juhl; Ostheimer, Gerard J.; van Vugt, Marcel A. T. M.; Jorgensen, Claus; Miron, Ioana M.; Diella, Francesca; Colwill, Karen; Taylor, Lorne; Elder, Kelly; Metalnikov, Pavel; Nguyen, Vivian; Pasculescu, Adrian; Jin, Jing; Park, Jin Gyoon; Samson, Leona D.; Woodgett, James R.; Russell, Robert B.; Bork, Peer; Yaffe, Michael B.; Pawson, Tony
Cell (Cambridge, MA, United States) (2007), 129 (7), 1415-1426CODEN: CELLB5; ISSN:0092-8674. (Cell Press)
Protein kinases control cellular decision processes by phosphorylating specific substrates. Thousands of in vivo phosphorylation sites have been identified, mostly by proteome-wide mapping. However, systematically matching these sites to specific kinases is presently infeasible, due to limited specificity of consensus motifs, and the influence of contextual factors, such as protein scaffolds, localization, and expression, on cellular substrate specificity. We have developed an approach (NetworKIN) that augments motif-based predictions with the network context of kinases and phosphoproteins. The latter provides 60%-80% of the computational capability to assign in vivo substrate specificity. NetworKIN pinpoints kinases responsible for specific phosphorylations and yields a 2.5-fold improvement in the accuracy with which phosphorylation networks can be constructed. Applying this approach to DNA damage signaling, we show that 53BP1 and Rad50 are phosphorylated by CDK1 and ATM, resp. We describe a scalable strategy to evaluate predictions, which suggests that BCLAF1 is a GSK-3 substrate.
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Peineau, S., Bradley, C., Taghibiglou, C., Doherty, A., Bortolotto, Z. A., Wang, Y. T., and Collingridge, G. L. (2008) The role of GSK-3 in synaptic plasticity Br. J. Pharmacol. 153, S428– S437 DOI: 10.1038/bjp.2008.2
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The role of GSK-3 in synaptic plasticity
Peineau, S.; Bradley, C.; Taghibiglou, C.; Doherty, A.; Bortolotto, Z. A.; Wang, Y. T.; Collingridge, G. L.
British Journal of Pharmacology (2008), 153 (Suppl. 1), S428-S437CODEN: BJPCBM; ISSN:0007-1188. (Nature Publishing Group)
A review. Glycogen synthase kinase-3 (GSK-3), an important component of the glycogen metab. pathway, is highly expressed in the CNS. It has been implicated in major neurol. disorders including Alzheimer's disease, schizophrenia and bipolar disorders. Despite its central role in these conditions it was not known until recently whether GSK-3 has neuronal-specific functions under normal conditions. However recent work has shown that GSK-3 is involved in the regulation of, and cross-talk between, two major forms of synaptic plasticity, N-methyl-D-aspartate receptor (NMDAR)-dependent long-term potentiation (LTP) and NMDAR-dependent long-term depression (LTD). The present article summarizes this recent work and discusses its potential relevance to the treatment of neurol. disorders.
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Sutherland, C. and Sutherland, C. (2011) What Are the bona fide GSK3 Substrates? Int. J. Alzheimer's Dis. 2011, 505607 DOI: 10.4061/2011/505607
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What Are the bona fide GSK3 Substrates?
Sutherland Calum
International journal of Alzheimer's disease (2011), 2011 (), 505607 ISSN:.
Nearly 100 proteins are proposed to be substrates for GSK3, suggesting that this enzyme is a fundamental regulator of almost every process in the cell, in every tissue in the body. However, it is not certain how many of these proposed substrates are regulated by GSK3 in vivo. Clearly, the identification of the physiological functions of GSK3 will be greatly aided by the identification of its bona fide substrates, and the development of GSK3 as a therapeutic target will be highly influenced by this range of actions, hence the need to accurately establish true GSK3 substrates in cells. In this paper the evidence that proposed GSK3 substrates are likely to be physiological targets is assessed, highlighting the key cellular processes that could be modulated by GSK3 activity and inhibition.
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Sánchez, C., Díaz-Nido, J., and Avila, J. (2000) Phosphorylation of microtubule-associated protein 2 (MAP2) and its relevance for the regulation of the neuronal cytoskeleton function Prog. Neurobiol. 61, 133– 168 DOI: 10.1016/S0301-0082(99)00046-5
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Phosphorylation of microtubule-associated protein 2 (MAP2) and its relevance for the regulation of the neuronal cytoskeleton function
Sanchez, C.; Diaz-Nido, J.; Avila, J.
Progress in Neurobiology (Oxford) (2000), 61 (2), 133-168CODEN: PGNBA5; ISSN:0301-0082. (Elsevier Science Ltd.)
A review with many refs. Neurons, the basic information processing units of the nervous system, are characterized by a complex polar morphol. which is essential for their function. To attain their precise morphol., neurons extend cytoplasmic processes (axons and dendrites) and establish synaptic connections in a highly regulated way. Addnl., neurons are also subjected to small plastic changes at the adult stage which serve to regulate synaptic transmission. Every step of neuronal development is genetically controlled by endogenous determinants, as well as by environmental signals including intercellular contacts, extracellular matrix and diffusible signals. Cytoskeletal components are among the main protein targets modified in response to most of those extracellular signals which ultimately det. neuronal morphol. One of the major mechanisms controlling the neuronal cytoskeleton is the modification of the phosphorylation state of cytoskeletal proteins via changes in the relative activities of protein kinases and phosphatases within neurons. In particular, the microtubule-assocd. protein 2 (MAP2) family of proteins is an abundant group of cytoskeletal components which are predominantly expressed in neurons and serve as substrates for most of protein kinases and phosphatases present in neurons. MAP2 phosphorylation seems to control its assocn. with the cytoskeleton and it is developmentally regulated. Moreover, MAP2 may perform many functions including the nucleation and stabilization of microtubules (and maybe microfilaments), the regulation of organelle transport within axons and dendrites, as well as the anchorage of regulatory proteins such as protein kinases which may be important for signal transduction. These putative functions of MAP2 have also been proposed to play important roles in the outgrowth of neuronal processes, synaptic plasticity and neuronal cell death. Thus, MAP2 constitutes an interesting case to understand the regulation of neuronal function by the alteration of the phosphorylation state of cytoskeletal proteins in response to different extracellular signals. Here we will review the current knowledge about the regulation of MAP2 function through phosphorylation/dephosphorylation and its relevance in the broader context of neuronal function.
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Baas, P. W., Rao, A. N., Matamoros, A. J., and Leo, L. (2016) Stability properties of neuronal microtubules Cytoskeleton DOI: 10.1002/cm.21286
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Sánchez, C., Tompa, P., Szücs, K., Friedrich, P., and Avila, J. (1996) Phosphorylation and dephosphorylation in the proline-rich C-terminal domain of microtubule-associated protein 2 Eur. J. Biochem. 241, 765– 771 DOI: 10.1111/j.1432-1033.1996.00765.x
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Phosphorylation and dephosphorylation in the proline-rich C-terminal domain of microtubule-associated protein 2
Sanchez, Carlos; Tompa, Peter; Szucs, Kornelia; Friedrich, Peter; Avila, Jesus
European Journal of Biochemistry (1996), 241 (3), 765-771CODEN: EJBCAI; ISSN:0014-2956. (Springer)
The C-terminal domain of microtubule-assocd. protein 2 (MAP2) contains a Pro-rich region and the tubulin-binding domain. Antibodies were generated to follow the phosphorylation state of the Pro-rich domain. One of these antibodies (no. 305) was raised against a synthetic peptide P (sequence RTPGTPGTPSY) phosphorylated at the Thr residues. This sequence is present in the Pro-rich region of MAP2 and is phosphorylated in vitro by at least 3 different Pro-directed protein kinases: p42mpk, p34cdc2-, and GSK3 (glycogen-synthase kinase 3) α/β. The MAP2 sites phosphorylated by these kinases are different, although all of them phosphorylate the C-terminal domain of MAP2 as detd. by Staphylococcus aureus VS protease mapping. Nonphosphorylated peptide P can be phosphorylated in vitro by all 3 kinases studied with similar efficiency. In high-mol.-mass MAP2, this sequence is highly phosphorylated in vivo at the late stages of rat development. This motif can be rapidly dephosphorylated in vitro by protein-phosphatase 1 (PP1) and 2A (PP2A) catalytic subunits but not by PP2B.
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Sánchez, C., Díaz-Nido, J., and Avila, J. (1998) Regulation of a site-specific phosphorylation of the microtubule-associated protein 2 during the development of cultured neurons Neuroscience 87, 861– 870 DOI: 10.1016/S0306-4522(98)00195-X
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Sánchez, C., Pérez, M., and Avila, J. (2000) GSK3beta-mediated phosphorylation of the microtubule-associated protein 2C (MAP2C) prevents microtubule bundling Eur. J. Cell Biol. 79, 252– 260 DOI: 10.1078/S0171-9335(04)70028-X
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GSK3β-mediated phosphorylation of the microtubule-associated protein 2C (MAP2C) prevents microtubule bundling
Sanchez, Carlos; Perez, Mar; Avila, Jesus
European Journal of Cell Biology (2000), 79 (4), 252-260CODEN: EJCBDN; ISSN:0171-9335. (Urban & Fischer Verlag)
A major determinant of neuronal morphol. is the cytoskeleton. And, one of the main regulatory mechanisms of cytoskeletal proteins is the modification of their phosphorylation state via changes in the relative activities of protein kinases and phosphatases in neurons. In particular, the microtubule-assocd. protein 2 (MAP2) family of proteins are abundant cytoskeletal components predominantly expressed in neurons and have been found to be substrates for most of the protein kinases and phosphatases present in neurons, including glycogen synthase kinase 3 (GSK3). It has been suggested that changes in GSK3-mediated MAP phosphorylation may modify MT stability and could control neuronal development. We have previously shown that MAP2 is phosphorylated in vitro and in situ by GSK3 at Thr1620 and Thr1623, located in the proline-rich region of MAP2 and recognized by antibody 305. However, the function of the phosphorylation of this site of MAP2 is still unknown. In this study, non-neuronal COS-1 cells have been co-transfected with cDNAs encoding MAP2C and either wild type or mutated GSK3β to analyze possible effects on microtubule stability and on the assocn. of MAP2 with microtubules. We have found that GSK3β phosphorylates MAP2C in co-transfected cells. Moreover, this phosphorylation is inhibited by the specific GSK3 inhibitor lithium chloride. Addnl., the formation of microtubule bundles, which is obsd. after transfection with MAP2C, was decreased when MAP2C was co-transfected with GSK3β wild type. Microtubule bundles were not obsd. in cells expressing MAP2C phosphorylated at the site recognized by antibody 305. The absence of microtubule bundles was reverted after treatment of MAP2C/GSK3β wild type transfected cells with lithium chloride. Highly phosphorylated MAP2C species, which were phosphorylated at the site recognized by antibody 305, appeared in cells co-transfected with MAP2C and GSK3β wild type. Interestingly, these MAP2C species were enriched in cytoskeleton-unbound protein prepns. These data suggests that GSK3-mediated phosphorylation of MAP2 may modify its binding to microtubules and regulate microtubule stability.
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Ebneth, A., Drewes, G., Mandelkow, E. M., and Mandelkow, E. (1999) Phosphorylation of MAP2c and MAP4 by MARK kinases leads to the destabilization of microtubules in cells Cell Motil. Cytoskeleton 44, 209– 224 DOI: 10.1002/(SICI)1097-0169(199911)44:3<209::AID-CM6>3.0.CO;2-4
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Phosphorylation of MAP2c and MAP4 by MARK kinases leads to the destabilization of microtubules in cells
Ebneth, A.; Drewes, G.; Mandelkow, E.-M.; Mandelkow, E.
Cell Motility and the Cytoskeleton (1999), 44 (3), 209-224CODEN: CMCYEO; ISSN:0886-1544. (Wiley-Liss, Inc.)
Microtubules serve as transport tracks in mol. mechanisms governing cellular shape and polarity. Rapid transitions between stable and dynamic microtubules are regulated by several factors, including microtubule-assocd. proteins (MAPs). We have shown that MAP/microtubule affinity regulating kinases (MARK) can phosphorylate the microtubule-assocd.-proteins MAP4, MAP2c, and tau on their microtubule-binding domain in vitro. This leads to their detachment from microtubules (MT) and an increased dynamic instability of MT. Here, we show that MARK protein kinases phosphorylate MAP2 and MAP4 on their microtubule-binding domain in transfected CHO cells. In CHO cells expressing MARK1 or MARK2 under control of an inducible promoter, MARK2 phosphorylates an endogenous MAP4-related protein. Prolonged expression of MARK2 results in microtubule-disruption, detachment of cells from the substratum, and cell death. Concomitant with microtubule disruption, we also obsd. a breakdown of the vimentin network, whereas actin fibers remained unaffected. Thus, MARK seems to play an important role in controlling cytoskeletal dynamics.
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Hara, K. and Harris, R. A. (2002) The anesthetic mechanism of urethane: the effects on neurotransmitter-gated ion channels Anesth. Analg. 94, 313– 318 DOI: 10.1213/00000539-200202000-00015
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The anesthetic mechanism of urethane: the effects on neurotransmitter-gated ion channels
Hara, Koji; Harris, R. Adron
Anesthesia & Analgesia (Baltimore, MD, United States) (2002), 94 (2), 313-318CODEN: AACRAT; ISSN:0003-2999. (Lippincott Williams & Wilkins)
Urethane is widely used as an anesthetic for animal studies because of its minimal effects on cardiovascular and respiratory systems and maintenance of spinal reflexes. Despite its usefulness in animal research, there are no reports concerning its mol. actions. We designed this study to det. whether urethane affects neurotransmitter-gated ion channels. We examd. the effects of urethane on recombinant GABAA, glycine, N-methyl-D-aspartate, α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid, and neuronal nicotinic acetylcholine receptors expressed in Xenopus oocytes. Urethane potentiated the functions of neuronal nicotinic acetylcholine, γ-aminobutyric acidA, and glycine receptors, and it inhibited N-methyl-D-aspartate and α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors in a concn.-dependent manner. At concns. close to anesthetic 50% effective concn., urethane had modest effects on all channels tested, suggesting the lack of a single predominant target for its action. This may account for its usefulness as a veterinary anesthetic. However, a large concn. of urethane exerts marked effects on all channels. These findings not only give insight into the mol. mechanism of anesthetics but also caution that neurophysiol. measurements from animals anesthetized with urethane may be complicated by the effects of urethane on multiple neurotransmitter systems. Our results also suggest that small changes in multiple receptor systems can produce anesthesia.
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Seira, O. and Del Río, J. A. (2014) Glycogen synthase kinase 3 beta (GSK3β) at the tip of neuronal development and regeneration Mol. Neurobiol. 49, 931– 944 DOI: 10.1007/s12035-013-8571-y
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Glycogen Synthase Kinase 3 Beta (GSK3β) at the Tip of Neuronal Development and Regeneration
Seira, Oscar; del Rio, Jose Antonio
Molecular Neurobiology (2014), 49 (2), 931-944CODEN: MONBEW; ISSN:0893-7648. (Humana Press Inc.)
A review. Gaining a basic understanding of the inhibitory mols. and the intracellular signaling involved in axon development and repulsion after neural lesions is of clear biomedical interest. In recent years, numerous studies have described new mols. and intracellular mechanisms that impair axonal outgrowth after injury. In this scenario, the role of glycogen synthase kinase 3 beta (GSK3β) in the axonal responses that occur after central nervous system (CNS) lesions began to be elucidated. GSK3β function in the nervous tissue is assocd. with neural development, neuron polarization, and, more recently, neurodegeneration. In fact, GSK3β has been considered as a putative therapeutic target for promoting functional recovery in injured or degenerative CNS. In this review, we summarize current understanding of the role of GSK3β during neuronal development and regeneration. In particular, we discuss GSK3β activity levels and their possible impact on cytoskeleton dynamics during both processes.
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Li, X. and Jope, R. S. (2010) Is glycogen synthase kinase-3 a central modulator in mood regulation? Neuropsychopharmacology 35, 2143– 2154 DOI: 10.1038/npp.2010.105
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Is glycogen synthase kinase-3 a central modulator in mood regulation?
Li, Xiaohua; Jope, Richard S.
Neuropsychopharmacology (2010), 35 (11), 2143-2154CODEN: NEROEW; ISSN:0893-133X. (Nature Publishing Group)
A review. Little is known regarding the mechanisms underlying the complex etiol. of mood disorders, represented mainly by major depressive disorder and bipolar disorder. The 1996 discovery that lithium inhibits glycogen synthase kinase-3 (GSK3) raised the possibility that impaired inhibition of GSK3 is assocd. with mood disorders. This is now supported by evidence from animal biochem., pharmacol., mol., and behavioral studies and from human post-mortem brain, peripheral tissue, and genetic studies that are reviewed here. Mood disorders may result in part from impairments in mechanisms controlling the activity of GSK3 or GSK3-regulated functions, and disruptions of these regulating systems at different signaling sites may contribute to the heterogeneity of mood disorders. This substantial evidence supports the conclusion that bolstering the inhibitory control of GSK3 is an important component of the therapeutic actions of drugs used to treat mood disorders and that GSK3 is a valid target for developing new therapeutic interventions.
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Bianchi, M. and Baulieu, E.-E. (2012) 3β-Methoxy-pregnenolone (MAP4343) as an innovative therapeutic approach for depressive disorders Proc. Natl. Acad. Sci. U. S. A. 109, 1713– 1718 DOI: 10.1073/pnas.1121485109
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There is no corresponding record for this reference.
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Kalenka, A., Hinkelbein, J., Feldmann, R. E., Kuschinsky, W., Waschke, K. F., and Maurer, M. H. (2007) The Effects of Sevoflurane Anesthesia on Rat Brain Proteins: A Proteomic Time-Course Analysis Anesth. Analg. 104, 1129– 1135 DOI: 10.1213/01.ane.0000260799.37107.e6
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The Effects of Sevoflurane Anesthesia on Rat Brain Proteins: A Proteomic Time-Course Analysis
Kalenka, Armin; Hinkelbein, Jochen; Feldmann, Robert E., Jr.; Kuschinsky, Wolfgang; Waschke, Klaus F.; Maurer, Martin H.
Anesthesia & Analgesia (Hagerstown, MD, United States) (2007), 104 (5), 1129-1135CODEN: AACRAT; ISSN:0003-2999. (Lippincott Williams & Wilkins)
Background: Recent studies showed changes in cerebral protein expression up to 3 days after desflurane anesthesia in rats. In the present study, we investigated the existence of persisting changes on the proteome level after sevoflurane anesthesia that persisted for as long as 28 days after anesthesia. Methods: Rats were anesthetized by spontaneous inhalation of 2.4% sevoflurane in air for 3 h. Animals (n = 6 for each group) were killed either directly, 72 h, or 28 days after anesthesia. Brains were removed and subjected to global protein expression profiling based on two-dimensional gel electrophoresis and mass spectrometry. Expression factors were compared to results from untreated conscious animals at each time point. Data were statistically analyzed by ANOVA (P < 0.01) and a cut of more than two-fold change in the expression factor. Results: We found 11 protein spots differentially regulated directly after anesthesia. Seventeen proteins were differentially expressed 72 h after the anesthesia. Only one spot was differentially regulated 28 days after anesthesia. The plausible targets of these differentially regulated proteins can be attributed to synaptic vesicle handling and cell-cell communication. Conclusions: Sevoflurane induced relevant changes in protein expression profiles directly and 72 h after an anesthesia with 1 MAC. Twenty-eight days after the anesthesia, all proteins except one had returned to baseline levels of abundance.
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Kalenka, A., Gross, B., Maurer, M. H., Thierse, H.-J., and Feldmann, R. E. (2010) Isoflurane Anesthesia Elicits Protein Pattern Changes in Rat Hippocampus J. Neurosurg. Anesthesiol. 22, 144– 154 DOI: 10.1097/ANA.0b013e3181cb7cb8
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Isoflurane anesthesia elicits protein pattern changes in rat hippocampus
Kalenka Armin; Gross Benjamin; Maurer Martin H; Thierse Hermann-Josef; Feldmann Robert E Jr
Journal of neurosurgical anesthesiology (2010), 22 (2), 144-54 ISSN:.
Postoperative cognitive dysfunction (POCD) is a known phenomenon occurring after anesthesia with volatile anesthetics (VA), such as isoflurane. Recent reports suggest that VA interact with neurodegenerative disease-associated proteins including compounds with pathogenic relevance in Alzheimer disease (AD) and induce processes that may be linked to AD neuropathology. Unfortunately, our present understanding of the exact anesthetics' molecular mechanisms of action, their side effects on the brain, and their catenation with AD pathology is still limited. The present study analyzes the differential proteome of the hippocampus immediately after and 3 days after a 3-hour 1 minimal alveolar concentration isoflurane anesthesia in rats. Differential 2-dimensional electrophoresis, mass spectrometry, and functional network mapping were used to identify and functionally classify 12 different hippocampal proteins, which were significantly regulated after isoflurane anesthesia (6 up-regulated, 11 down-regulated with P<0.01). Induction of differential expression ranged from 0.05 (25-fold down-regulation) to 4.4 (4.4-fold up-regulation). Ten proteins were regulated immediately after and 7 proteins 3 days after isoflurane exposure. The proteome displays isoflurane-responsive protein candidates, which have also been shown to play a role in AD. They were grouped according to their key biologic activities, which showed that isoflurane affects selected biologic processes including synaptic plasticity, stress response, detoxification, and cytoskeleton in early and late recovery phases after anesthesia. These processes are also affected in AD. Results are discussed in view of AD, the toxicity mechanisms of isoflurane as well as the implications for our present understanding and conduction of clinical anesthesia.
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Tan, H., Wu, Z., Wang, H., Bai, B., Li, Y., Wang, X., Zhai, B., Beach, T. G., and Peng, J. (2015) Refined phosphopeptide enrichment by phosphate additive and the analysis of human brain phosphoproteome Proteomics 15, 500– 507 DOI: 10.1002/pmic.201400171
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Elo, L. L., Filén, S., Lahesmaa, R., and Aittokallio, T. (2008) Reproducibility-optimized test statistic for ranking genes in microarray studies IEEE/ACM Trans. Comput. Biol. Bioinf. 5, 423– 431 DOI: 10.1109/tcbb.2007.1078
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Reproducibility-optimized test statistic for ranking genes in microarray studies
Elo, Laura L.; Filen, Sanna; Lahesmaa, Riitta; Aittokallio, Tero
IEEE/ACM Transactions on Computational Biology and Bioinformatics (2008), 5 (3), 423-431CODEN: ITCBCY; ISSN:1545-5963. (IEEE Computer Society)
A principal goal of microarray studies is to identify the genes showing differential expression under distinct conditions. In such studies, the selection of an optimal test statistic is a crucial challenge, which depends on the type and amt. of data under anal. Although previous studies on simulated or spike-in data sets do not provide practical guidance on how to choose the best method for a given real data set, we introduce an enhanced reproducibility-optimization procedure, which enables the selection of a suitable gene-ranking statistic directly from the data. In comparison with existing ranking methods, the reproducibility-optimized statistic shows good performance consistently under various simulated conditions and on Affymetrix spike-in data set. Further, the feasibility of the novel statistic is confirmed in a practical research setting using data from an inhouse cDNA microarray study of asthma-related gene expression changes. These results suggest that the procedure facilitates the selection of an appropriate test statistic for a given data set without relying on a priori assumptions, which may bias the findings and their interpretation. Moreover, the general reproducibility-optimization procedure is not limited to detecting differential expression only but could be extended to a wide range of other applications as well.
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Huang, D. W., Sherman, B. T., and Lempicki, R. A. (2009) Bioinformatics enrichment tools: paths toward the comprehensive functional analysis of large gene lists Nucleic Acids Res. 37, 1– 13 DOI: 10.1093/nar/gkn923
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Bioinformatics enrichment tools: paths toward the comprehensive functional analysis of large gene lists
Huang, Da Wei; Sherman, Brad T.; Lempicki, Richard A.
Nucleic Acids Research (2009), 37 (1), 1-13CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)
A review. Functional anal. of large gene lists, derived in most cases from emerging high-throughput genomic, proteomic and bioinformatics scanning approaches, is still a challenging and daunting task. The gene-annotation enrichment anal. is a promising high-throughput strategy that increases the likelihood for investigators to identify biol. processes most pertinent to their study. Approx. 68 bioinformatics enrichment tools that are currently available in the community are collected in this survey. Tools are uniquely categorized into three major classes, according to their underlying enrichment algorithms. The comprehensive collections, unique tool classifications and assocd. questions/issues will provide a more comprehensive and up-to-date view regarding the advantages, pitfalls and recent trends in a simpler tool-class level rather than by a tool-by-tool approach. Thus, the survey will help tool designers/developers and experienced end users understand the underlying algorithms and pertinent details of particular tool categories/tools, enabling them to make the best choices for their particular research interests.
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Rantamäki, T., Hendolin, P., Kankaanpää, A., Mijatovic, J., Piepponen, P., Domenici, E., Chao, M. V., Männistö, P. T., and Castrén, E. (2007) Pharmacologically diverse antidepressants rapidly activate brain-derived neurotrophic factor receptor TrkB and induce phospholipase-Cgamma signaling pathways in mouse brain Neuropsychopharmacology 32, 2152– 2162 DOI: 10.1038/sj.npp.1301345
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Pharmacologically Diverse Antidepressants Rapidly Activate Brain-Derived Neurotrophic Factor Receptor TrkB and Induce Phospholipase-Cγ Signaling Pathways in Mouse Brain
Rantamaki, Tomi; Hendolin, Panu; Kankaanpaa, Aino; Mijatovic, Jelena; Piepponen, Petteri; Domenici, Enrico; Chao, Moses V.; Mannisto, Pekka T.; Castren, Eero
Neuropsychopharmacology (2007), 32 (10), 2152-2162CODEN: NEROEW; ISSN:0893-133X. (Nature Publishing Group)
Previous studies suggest that brain-derived neurotrophic factor and its receptor TrkB are critically involved in the therapeutic actions of antidepressant drugs. We have previously shown that the antidepressants imipramine and fluoxetine produce a rapid autophosphorylation of TrkB in the rodent brain. In the present study, we have further examd. the biochem. and functional characteristics of antidepressant-induced TrkB activation in vivo. We show that all the antidepressants examd., including inhibitors of monoamine transporters and metab., activate TrkB rapidly in the rodent anterior cingulate cortex and hippocampus. Furthermore, the results indicate that acute and long-term antidepressant treatments induce TrkB-mediated activation of phospholipase-Cγ1 (PLCγ1) and increase the phosphorylation of cAMP-related element binding protein, a major transcription factor mediating neuronal plasticity. In contrast, we have not obsd. any modulation of the phosphorylation of TrkB Shc binding site, phosphorylation of mitogen-activated protein kinase or AKT by antidepressants. We also show that in the forced swim test, the behavioral effects of specific serotonergic antidepressant citalopram, but not those of the specific noradrenergic antidepressant reboxetine, are crucially dependent on TrkB signaling. Finally, brain monoamines seem to be crit. mediators of antidepressant-induced TrkB activation, as antidepressants reboxetine and citalopram do not produce TrkB activation in the brains of serotonin- or norepinephrine-depleted mice. In conclusion, our data suggest that rapid activation of the TrkB neurotrophin receptor and PLCγ1 signaling is a common mechanism for all antidepressant drugs.
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Rantamäki, T., Vesa, L., Antila, H., Di Lieto, A., Tammela, P., Schmitt, A., Lesch, K.-P., Rios, M., and Castrén, E. (2011) Antidepressant drugs transactivate TrkB neurotrophin receptors in the adult rodent brain independently of BDNF and monoamine transporter blockade PLoS One 6, e20567 DOI: 10.1371/journal.pone.0020567
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Antidepressant drugs transactivate TrkB neurotrophin receptors in the adult rodent brain independently of BDNF and monoamine transporter blockade
Rantamaki, Tomi; Vesa, Liisa; Antila, Hanna; Di Lieto, Antonio; Tammela, Paivi; Schmitt, Angelika; Lesch, Klaus-Peter; Rios, Maribel; Castren, Eero
PLoS One (2011), 6 (6), e20567CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)
Background: Antidepressant drugs (ADs) have been shown to activate BDNF (brain-derived neurotrophic factor) receptor TrkB in the rodent brain but the mechanism underlying this phenomenon remains unclear. ADs act as monoamine reuptake inhibitors and after prolonged treatments regulate brain BDNF mRNA levels indicating that monoamine-BDNF signaling regulate AD-induced TrkB activation in vivo. However, recent findings demonstrate that Trk receptors can be transactivated independently of their neurotrophin ligands. Methodol.: In this study we examd. the role of BDNF, TrkB kinase activity and monoamine reuptake in the AD-induced TrkB activation in vivo and in vitro by employing several transgenic mouse models, cultured neurons and TrkB-expressing cell lines. Principal Findings: Using a chem.-genetic TrkBF616A mutant and TrkB overexpressing mice, we demonstrate that ADs specifically activate both the maturely and immaturely glycosylated forms of TrkB receptors in the brain in a TrkB kinase dependent manner. However, the tricyclic AD imipramine readily induced the phosphorylation of TrkB receptors in conditional BDNF-/- knock-out mice (132.4 ± 8.5% of control; P = 0.01), indicating that BDNF is not required for the TrkB activation. Moreover, using serotonin transporter (SERT) deficient mice and chem. lesions of monoaminergic neurons we show that neither a functional SERT nor monoamines are required for the TrkB phosphorylation response induced by the serotonin selective reuptake inhibitors fluoxetine or citalopram, or norepinephrine selective reuptake inhibitor reboxetine. However, neither ADs nor monoamine transmitters activated TrkB in cultured neurons or cell lines expressing TrkB receptors, arguing that ADs do not directly bind to TrkB. Conclusions: The present findings suggest that ADs transactivate brain TrkB receptors independently of BDNF and monoamine reuptake blockade and emphasize the need of an intact tissue context for the ability of ADs to induce TrkB activity in brain.

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Abstract
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Figure 1
Figure 1. Effects of isoflurane anesthesia (4% induction, 2% maintenance) on cortical EEG spectrogram and burst suppression. (A) EEG spectrogram during awake, non-REM (NREM) sleep, and under the influence of isoflurane (20–30 min recording). Representative traces of burst suppressing EEG shown in inset (A2) and (A3). (B) Quantitation of delta (1–4 Hz) (B1), theta (4–8 Hz) (B2), alpha (8–12 Hz) (B3), beta (12–30 Hz) (B4), and gamma (30–60 Hz) (B5) frequencies under the influence of isoflurane, during awake and NREM sleep. Deep burst-suppressing isoflurane anesthesia significantly reduces spectral power in frequency bands below 30 Hz when compared to baseline NREM sleep. EEG spectra were normalized to total EEG power for all frequencies within the baseline period recording. N = 5; *p < 0.05, **p < 0.01, ***p < 0.001; one-way ANOVA followed by Dunnett’s multiple comparison test.
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Figure 2
Figure 2. Phosphoproteomic workflow (A) and heat map (B) depicting differentially regulated phosphoproteins after sham (C1–C3) or 30 min isoflurane anesthesia (I1–I3) (4% induction, 2% maintenance). N = 3/group.
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Figure 3
Figure 3. Diverse anesthetics produce similar acute phosphorylation changes on p44/42-MAPK^{Thr202/Tyr204}, GSK3β^Ser9, and MAP2^{Thr1620/Thr1623} in the adult mouse hippocampus. (A) Effects of isoflurane anesthesia (4% induction, 2% maintenance; 30 min) (N = 10/group). (B) Effects of sevoflurane anesthesia (6% induction, 4.5% maintenance; 30 min) (N = 6/group). (C) Effects of urethane anesthesia (2.0 g/kg, i.p.; 30 min) (N = 4/control group, N = 6/urethane group). (D) Effects of subanesthetic ketamine (100 mg/kg, i.p.; 30 min) (N = 6/group). *p < 0.05, **p < 0.01, ****p < 0.0001; two-tailed unpaired t test with Welch’s correction. Abbreviations: MAPK, mitogen activated protein kinase; GSK3β, glycogen synthase kinase 3β; MAP2, microtubule-associated protein 2.
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  Doyle P W; Matta B F
  British journal of anaesthesia (1999), 83 (4), 580-4 ISSN:0007-0912.
  Metabolic suppression may have a role in cerebral protection. It is often assumed that the cerebral metabolic and protective effects of qualitative burst suppression are similar to those of the isoelectric encephalogram (EEG). We have examined the effect of different degrees of EEG suppression on blood flow and oxygen difference during general anaesthesia. We studied 11 patients undergoing general anaesthesia for resection of acoustic neuromas. The study was performed after surgery with propofol and remifentanil anaesthesia. Transcranial Doppler ultrasonography and jugular bulb venous saturations were measured at values of EEG suppression: 0%, 50% and 100% (isoelectric EEG). Data from nine patients were suitable for analysis. There were no significant differences in mean arterial pressure, heart rate or PaCO2 during EEG activity, 50% burst suppression ratio or isoelectric EEG. There was a significant decrease in middle cerebral artery flow velocity (vmca) with increasing EEG suppression (0% suppression, mean 38 (SEM 4) cm s-1; 50% suppression, 29 (3) cm s-1; and 100% suppression, 24 (2) cm s-1; P < 0.05). Jugular bulb venous saturations did not change consistently with the change in EEG activity, indicating intact flow-metabolism coupling. We conclude that the degree of EEG suppression had a significant effect on blood flow. If flow-metabolism coupling is maintained, the assumption that cerebral metabolism during 50% EEG burst suppression is equivalent to isoelectric EEG may not be justified. If cerebral protection is related to brain metabolism, then an isoelectric EEG may give more cerebral protection than 50% burst suppression.
12. 12
  Liu, Y. and Chance, M. R. (2014) Integrating phosphoproteomics in systems biology Comput. Struct. Biotechnol. J. 10, 90– 97 DOI: 10.1016/j.csbj.2014.07.003
  12
  Integrating phosphoproteomics in systems biology
  Liu Yu; Chance Mark R
  Computational and structural biotechnology journal (2014), 10 (17), 90-7 ISSN:2001-0370.
  Phosphorylation of serine, threonine and tyrosine plays significant roles in cellular signal transduction and in modifying multiple protein functions. Phosphoproteins are coordinated and regulated by a network of kinases, phosphatases and phospho-binding proteins, which modify the phosphorylation states, recognize unique phosphopeptides, or target proteins for degradation. Detailed and complete information on the structure and dynamics of these networks is required to better understand fundamental mechanisms of cellular processes and diseases. High-throughput technologies have been developed to investigate phosphoproteomes in model organisms and human diseases. Among them, mass spectrometry (MS)-based technologies are the major platforms and have been widely applied, which has led to explosive growth of phosphoproteomic data in recent years. New bioinformatics tools are needed to analyze and make sense of these data. Moreover, most research has focused on individual phosphoproteins and kinases. To gain a more complete knowledge of cellular processes, systems biology approaches, including pathways and networks modeling, have to be applied to integrate all components of the phosphorylation machinery, including kinases, phosphatases, their substrates, and phospho-binding proteins. This review presents the latest developments of bioinformatics methods and attempts to apply systems biology to analyze phosphoproteomics data generated by MS-based technologies. Challenges and future directions in this field will be also discussed.
13. 13
  Walaas, S. I. and Greengard, P. (1991) Protein phosphorylation and neuronal function Pharmacol. Rev. 43, 299– 349
  13
  Protein phosphorylation and neuronal function
  Walaas, Sven Ivar; Greengard, Paul
  Pharmacological Reviews (1991), 43 (3), 299-349CODEN: PAREAQ; ISSN:0031-6997.
  A review, with ∼750 refs. Topics discussed include: protein kinases in brain, phosphoprotein phosphatases in brain, phosphoproteins in neurotransmission, and phosphoproteins in clin. disorders.
14. 14
  McKernan, R. M., Rosahl, T. W., Reynolds, D. S., Sur, C., Wafford, K. A., Atack, J. R., Farrar, S., Myers, J., Cook, G., Ferris, P., Garrett, L., Bristow, L., Marshall, G., Macaulay, A., Brown, N., Howell, O., Moore, K. W., Carling, R. W., Street, L. J., Castro, J. L., Ragan, C. I., Dawson, G. R., and Whiting, P. J. (2000) Sedative but not anxiolytic properties of benzodiazepines are mediated by the GABA(A) receptor alpha1 subtype Nat. Neurosci. 3, 587– 592 DOI: 10.1038/75761
  14
  Sedative but not anxiolytic properties of benzodiazepines are mediated by the GABAA receptor α1 subtype
  McKernan, R. M.; Rosahl, T. W.; Reynolds, D. S.; Sur, C.; Wafford, K. A.; Atack, J. R.; Farrar, S.; Myers, J.; Cook, G.; Ferris, P.; Garrett, L.; Bristow, L.; Marshall, G.; Macaulay, A.; Brown, N.; Howell, O.; Moore, K. W.; Carling, R. W.; Street, L. J.; Castro, J. L.; Ragan, C. I.; Dawson, G. R.; Whiting, P. J.
  Nature Neuroscience (2000), 3 (6), 587-592CODEN: NANEFN; ISSN:1097-6256. (Nature America Inc.)
  Inhibitory neurotransmission in the brain is largely mediated by GABAA receptors. Potentiation of GABA receptor activation through an allosteric benzodiazepine (BZ) site produces the sedative, anxiolytic, muscle relaxant, anticonvulsant and cognition-impairing effects of clin. used BZs such as diazepam. We created genetically modified mice (α1 H101R) with a diazepam-insensitive α1 subtype and a selective BZ site ligand, L-838,417, to explore GABAA receptor subtypes mediating specific physiol. effects. These two complimentary approaches revealed that the α1 subtype mediated the sedative, but not the anxiolytic effects of benzodiazepines. This finding suggests ways to improve anxiolytics and to develop drugs for other neurol. disorders based on their specificity for GABAA receptor subtypes in distinct neuronal circuits.
15. 15
  Zhou, C., Liang, P., Liu, J., Ke, B., Wang, X., Li, F., Li, T., Bayliss, D. A., and Chen, X. (2015) HCN1 Channels Contribute to the Effects of Amnesia and Hypnosis but not Immobility of Volatile Anesthetics Anesth. Analg. 121, 661– 666 DOI: 10.1213/ANE.0000000000000830
  15
  HCN1 Channels Contribute to the Effects of Amnesia and Hypnosis but not Immobility of Volatile Anesthetics
  Zhou, Cheng; Liang, Peng; Liu, Jin; Ke, Bowen; Wang, Xiaojia; Li, Fengshan; Li, Tao; Bayliss, Douglas A.; Chen, Xiangdong
  Anesthesia & Analgesia (Hagerstown, MD, United States) (2015), 121 (3), 661-666CODEN: AACRAT; ISSN:0003-2999. (Lippincott Williams & Wilkins)
  Background: Hyperpolarization-activated, cyclic nucleotide-gated (HCN) subtype 1 (HCN1) channels have been identified as targets of ketamine to produce hypnosis. Volatile anesthetics also inhibit HCN1 channels. However, the effects of HCN1 channels on volatile anesthetics in vivo are still elusive. This study uses global and conditional HCN1 knockout mice to evaluate how HCN1 channels affect the actions of volatile anesthetics. Methods: Min. alveolar concns. (MACs) of isoflurane and sevoflurane that induced immobility (MAC of immobility) and/or hypnosis (MAC of hypnosis) were detd. in wild-type mice, global HCN1 knockout (HCN1) mice, HCN1 channel gene with 2 lox-P sites flanking a region of the fourth exon of HCN1 (HCN1) mice, and forebrain-selective HCN1 knockout (HCN1: cre) mice. Immobility of mice was defined as no purposeful reactions to tail-clamping stimulus, and hypnosis was defined as loss of righting reflex. The amnestic effects of isoflurane and sevoflurane were evaluated by fear-potentiated startle in these 4 strains of mice. Results: All MAC values were expressed as mean ± SEM. For MAC of immobility of isoflurane, no significant difference was found among wild-type, HCN1, HCN1, and HCN1: cre mice (all ∼1.24%-1.29% isoflurane). For both HCN1 and HCN1: cre mice, the MAC of hypnosis for isoflurane (each ∼1.05% isoflurane) was significantly increased over their nonknockout controls: HCN1 vs. wild-type (0.86% ± 0.03%, P < 0.001) and HCN1: cre vs. HCN1 mice (0.84% ± 0.03%, P < 0.001); no significant difference was found between HCN1 and HCN1: cre mice. For MAC of immobility of sevoflurane, no significant difference was found among wild-type, HCN1, HCN1, and HCN1: cre mice (all ∼2.6%-2.7% sevoflurane). For both HCN1 and HCN1: cre mice, the MAC of hypnosis for sevoflurane (each ∼1.90% sevoflurane) was significantly increased over their nonknockout controls: HCN1 vs. wild-type (1.58% ± 0.05%, P < 0.001) and HCN1: cre vs. HCN1 mice (1.56% ± 0.05%, P < 0.001). No significant difference was found between HCN1 and HCN1: cre mice. By fear-potentiated startle expts., amnestic effects of isoflurane and sevoflurane were significantly attenuated in HCN1 and HCN1: cre mice (both P < 0.002 vs. wild-type or HCN1 mice). No significant difference was found between HCN1 and HCN1: cre mice. Conclusions: Forebrain HCN1 channels contribute to hypnotic and amnestic effects of volatile anesthetics, but HCN1 channels are not involved in the immobilizing actions of volatile anesthetics.
16. 16
  Bojak, I., Day, H. C., and Liley, D. T. J. (2013) Ketamine, propofol, and the EEG: a neural field analysis of HCN1-mediated interactions Front. Comput. Neurosci. 7, 22 DOI: 10.3389/fncom.2013.00022
  16
  Ketamine, Propofol, and the EEG: A Neural Field Analysis of HCN1-Mediated Interactions
  Bojak Ingo; Day Harry C; Liley David T J
  Frontiers in computational neuroscience (2013), 7 (), 22 ISSN:.
  Ketamine and propofol are two well-known, powerful anesthetic agents, yet at first sight this appears to be their only commonality. Ketamine is a dissociative anesthetic agent, whose main mechanism of action is considered to be N-methyl-d-aspartate (NMDA) antagonism; whereas propofol is a general anesthetic agent, which is assumed to primarily potentiate currents gated by γ-aminobutyric acid type A (GABAA) receptors. However, several experimental observations suggest a closer relationship. First, the effect of ketamine on the electroencephalogram (EEG) is markedly changed in the presence of propofol: on its own ketamine increases θ (4-8 Hz) and decreases α (8-13 Hz) oscillations, whereas ketamine induces a significant shift to beta band frequencies (13-30 Hz) in the presence of propofol. Second, both ketamine and propofol cause inhibition of the inward pacemaker current I h, by binding to the corresponding hyperpolarization-activated cyclic nucleotide-gated potassium channel 1 (HCN1) subunit. The resulting effect is a hyperpolarization of the neuron's resting membrane potential. Third, the ability of both ketamine and propofol to induce hypnosis is reduced in HCN1-knockout mice. Here we show that one can theoretically understand the observed spectral changes of the EEG based on HCN1-mediated hyperpolarizations alone, without involving the supposed main mechanisms of action of these drugs through NMDA and GABAA, respectively. On the basis of our successful EEG model we conclude that ketamine and propofol should be antagonistic to each other in their interaction at HCN1 subunits. Such a prediction is in accord with the results of clinical experiment in which it is found that ketamine and propofol interact in an infra-additive manner with respect to the endpoints of hypnosis and immobility.
17. 17
  Carr, D. B., Andrews, G. D., Glen, W. B., and Lavin, A. (2007) alpha2-Noradrenergic receptors activation enhances excitability and synaptic integration in rat prefrontal cortex pyramidal neurons via inhibition of HCN currents J. Physiol. 584, 437– 450 DOI: 10.1113/jphysiol.2007.141671
  17
  α2-Noradrenergic receptors activation enhances excitability and synaptic integration in rat prefrontal cortex pyramidal neurons via inhibition of HCN currents
  Carr, David B.; Andrews, Glenn D.; Glen, William B.; Lavin, A.
  Journal of Physiology (Oxford, United Kingdom) (2007), 584 (2), 437-450CODEN: JPHYA7; ISSN:0022-3751. (Blackwell Publishing Ltd.)
  Stimulation of α2-noradrenergic (NA) receptors within the PFC improves working memory performance. This improvement is accompanied by a selective increase in the activity of PFC neurons during delay periods, although the cellular mechanisms responsible for this enhanced response are largely unknown. Here we used current and voltage clamp recordings to characterize the response of layer V-VI PFC pyramidal neurons to α2-NA receptor stimulation. α2-NA receptor activation produced a small hyperpolarization of the resting membrane potential, which was accompanied by an increase in input resistance and evoked firing. Voltage clamp anal. demonstrated that α2-NA receptor stimulation inhibited a cesium and ZD7288-sensitive hyperpolarization-activated (HCN) inward current. Suppression of HCN current by α2-NA stimulation was not dependent on adenylate cyclase but instead required activation of a PLC-PKC linked signaling pathway. Similar to direct blockade of HCN channels, α2-NA receptor stimulation produced a significant enhancement in temporal summation during trains of distally evoked EPSPs. These dual effects of α2-NA receptor stimulation - membrane hyperpolarization and enhanced temporal integration - together produce an increase in the overall gain of the response of PFC pyramidal neurons to excitatory synaptic input. The net effect is the suppression of isolated excitatory inputs while enhancing the response to a coherent burst of synpatic activity.
18. 18
  Chen, X., Shu, S., Kennedy, D. P., Willcox, S. C., and Bayliss, D. A. (2009) Subunit-specific effects of isoflurane on neuronal Ih in HCN1 knockout mice J. Neurophysiol. 101, 129– 140 DOI: 10.1152/jn.01352.2007
  18
  Subunit-specific effects of isoflurane on neuronal Ih in HCN1 knockout mice
  Chen, Xiangdong; Shu, Shaofang; Kennedy, Dylan P.; Willcox, Sarah C.; Bayliss, Douglas A.
  Journal of Neurophysiology (2009), 101 (1), 129-140CODEN: JONEA4; ISSN:0022-3077. (American Physiological Society)
  The ionic mechanisms that contribute to general anesthetic actions have not been elucidated, although increasing evidence has pointed to roles for subthreshold ion channels, such as the HCN channels underlying the neuronal hyperpolarization-activated cationic current (Ih). Here, the authors used conventional HCN1 knockout mice to test directly the contributions of specific HCN subunits to effects of isoflurane, an inhalational anesthetic, on membrane and integrative properties of motor and cortical pyramidal neurons in vitro. Compared with wild-type mice, residual Ih from knockout animals was smaller in amplitude and presented with HCN2-like properties. Inhibition of Ih by isoflurane previously attributed to HCN1 subunit-contg. channels (i.e., a hyperpolarizing shift in half-activation voltage [V1/2]) was absent in neurons from HCN1 knockout animals; the remaining inhibition of current amplitude could be attributed to effects on residual HCN2 channels. The authors also found that isoflurane increased temporal summation of excitatory postsynaptic potentials (EPSPs) in cortical neurons from wild-type mice; this effect was predicted by simulation of anesthetic-induced dendritic Ih inhibition, which also revealed more prominent summation accompanying shifts in V1/2 (an HCN1-like effect) than decreased current amplitude (an HCN2-like effect). Accordingly, anesthetic-induced EPSP summation was not obsd. in cortical cells from HCN1 knockout mice. In wild-type mice, the enhanced synaptic summation obsd. with low concns. of isoflurane contributed to a net increase in cortical neuron excitability. In summary, HCN channel subunits account for distinct anesthetic effects on neuronal membrane properties and synaptic integration; inhibition of HCN1 in cortical neurons may contribute to the synaptically mediated slow-wave cortical synchronization that accompanies anesthetic-induced hypnosis.
19. 19
  Zhou, C., Douglas, J. E., Kumar, N. N., Shu, S., Bayliss, D. A., and Chen, X. (2013) Forebrain HCN1 channels contribute to hypnotic actions of ketamine Anesthesiology 118, 785– 795 DOI: 10.1097/ALN.0b013e318287b7c8
  There is no corresponding record for this reference.
20. 20
  Henzi, V. and MacDermott, A. B. (1992) Characteristics and function of Ca(2+)- and inositol 1,4,5-trisphosphate-releasable stores of Ca2+ in neurons Neuroscience 46, 251– 273 DOI: 10.1016/0306-4522(92)90049-8
  There is no corresponding record for this reference.
21. 21
  Takeshima, H., Venturi, E., and Sitsapesan, R. (2015) New and notable ion-channels in the sarcoplasmic/endoplasmic reticulum: do they support the process of intracellular Ca(2+) release? J. Physiol. 593, 3241– 3251 DOI: 10.1113/jphysiol.2014.281881
  21
  New and notable ion-channels in the sarcoplasmic/endoplasmic reticulum: do they support the process of intracellular Ca2+ release?
  Takeshima, Hiroshi; Venturi, Elisa; Sitsapesan, Rebecca
  Journal of Physiology (Oxford, United Kingdom) (2015), 593 (15), 3241-3251CODEN: JPHYA7; ISSN:1469-7793. (Wiley-Blackwell)
  Intracellular Ca2+ release through ryanodine receptor (RyR) and inositol trisphosphate receptor (IP3R) channels is supported by a complex network of addnl. proteins that are located in or near the Ca2+ release sites. In this review, we focus, not on RyR/IP3R, but on other ion-channels that are known to be present in the sarcoplasmic/endoplasmic reticulum (ER/SR) membranes. We review their putative physiol. roles and the evidence suggesting that they may support the process of intracellular Ca2+ release, either indirectly by manipulating ionic fluxes across the ER/SR membrane or by directly interacting with a Ca2+-release channel. These channels rarely receive scientific attention because of the general lack of information regarding their biochem. and/or electrophysiol. characteristics makes it difficult to predict their physiol. roles and their impact on SR Ca2+ fluxes. We discuss the possible role of SR K+ channels and, in parallel, detail the known biochem. and biophys. properties of the trimeric intracellular cation (TRIC) proteins and their possible biol. and pathophysiol. roles in ER/SR Ca2+ release. We summarize what is known regarding Cl- channels in the ER/SR and the non-selective cation channels or putative 'Ca2+ leak channels', including mitsugumin23 (MG23), pannexins, presenilins and the transient receptor potential (TRP) channels that are distributed across ER/SR membranes but which have not yet been fully characterized functionally.
22. 22
  Ghosh, A. and Greenberg, M. E. (1995) Calcium signaling in neurons: molecular mechanisms and cellular consequences Science 268, 239– 247 DOI: 10.1126/science.7716515
  22
  Calcium signaling in neurons: molecular mechanisms and cellular consequences
  Ghosh, Anirvan; Greenberg, Michael E.
  Science (Washington, D. C.) (1995), 268 (5208), 239-47CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
  A review with >66 refs. Neuronal activity can lead to marked increases in the concn. of cytosolic calcium, which then functions as a second messenger that mediates a wide range of cellular responses. Calcium binds to calmodulin and stimulates the activity of a variety of enzymes, including calcium-calmodulin kinases and calcium-sensitive adenylate cyclases. These enzymes transduce the calcium signal and effect short-term biol. responses, such as the modification of synaptic proteins and long-lasting neuronal responses that require changes in gene expression. Recent studies of calcium signal-transduction mechanisms have revealed that, depending on the route of entry into a neuron, calcium differentially affects processes that are central to the development and plasticity of the nervous system, including activity-dependent cell survival, modulation of synaptic strength, and calcium-mediated cell death.
23. 23
  Verkhratsky, A. J. and Petersen, O. H. (1998) Neuronal calcium stores Cell Calcium 24, 333– 343 DOI: 10.1016/S0143-4160(98)90057-4
  There is no corresponding record for this reference.
24. 24
  Verkhratsky, A., Orkand, R. K., and Kettenmann, H. (1998) Glial calcium: homeostasis and signaling function Physiol. Rev. 78, 99– 141
  There is no corresponding record for this reference.
25. 25
  Agulhon, C., Petravicz, J., McMullen, A. B., Sweger, E. J., Minton, S. K., Taves, S. R., Casper, K. B., Fiacco, T. A., and McCarthy, K. D. (2008) What is the role of astrocyte calcium in neurophysiology? Neuron 59, 932– 946 DOI: 10.1016/j.neuron.2008.09.004
  25
  What is the role of astrocyte calcium in neurophysiology?
  Agulhon, Cendra; Petravicz, Jeremy; McMullen, Allison B.; Sweger, Elizabeth J.; Minton, Suzanne K.; Taves, Sarah R.; Casper, Kristen B.; Fiacco, Todd A.; McCarthy, Ken D.
  Neuron (2008), 59 (6), 932-946CODEN: NERNET; ISSN:0896-6273. (Cell Press)
  A review. Astrocytes comprise approx. half of the vol. of the adult mammalian brain and are the primary neuronal structural and trophic supportive elements. Astrocytes are organized into distinct nonoverlapping domains and extend elaborate and dense fine processes that interact intimately with synapses and cerebrovasculature. The recognition in the mid-1990s that astrocytes undergo elevations in intracellular Ca2+ concn. following activation of G protein-coupled receptors by synaptically released neurotransmitters demonstrated not only that astrocytes display a form of excitability but also that astrocytes may be active participants in brain information processing. The roles that astrocytic Ca2+ elevations play in neurophysiol. and esp. in modulation of neuronal activity have been intensely researched in recent years. Here, the authors summarize the current understanding of the function of astrocytic Ca2+ signaling in neurophysiol. processes and discuss areas where the role of astrocytes remains controversial and will therefore benefit from further study.
26. 26
  Fang, M., Tao, Y.-X., He, F., Zhang, M., Levine, C. F., Mao, P., Tao, F., Chou, C.-L., Sadegh-Nasseri, S., and Johns, R. A. (2003) Synaptic PDZ Domain-mediated Protein Interactions Are Disrupted by Inhalational Anesthetics J. Biol. Chem. 278, 36669– 36675 DOI: 10.1074/jbc.M303520200
  There is no corresponding record for this reference.
27. 27
  Li, X., Friedman, A. B., Roh, M.-S., and Jope, R. S. (2005) Anesthesia and post-mortem interval profoundly influence the regulatory serine phosphorylation of glycogen synthase kinase-3 in mouse brain J. Neurochem. 92, 701– 704 DOI: 10.1111/j.1471-4159.2004.02898.x
  27
  Anesthesia and post-mortem interval profoundly influence the regulatory serine phosphorylation of glycogen synthase kinase-3 in mouse brain
  Li, Xiaohua; Friedman, Ari B.; Roh, Myoung-Sun; Jope, Richard S.
  Journal of Neurochemistry (2005), 92 (3), 701-704CODEN: JONRA9; ISSN:0022-3042. (Blackwell Publishing Ltd.)
  Glycogen synthase kinase-3 (GSK3) is a crucial enzyme contributing to the regulation of neuronal structure, plasticity and survival, is implicated as a contributory factor in prevalent diseases such as Alzheimer's disease and mood disorders and is regulated by a wide range of signaling systems and pharmacol. agents. Therefore, factors regulating GSK3 in vivo are currently of much interest. GSK3 is inhibited by phosphorylation of serine-9 or serine-21 in GSK3β and GSK3α, resp. This study found that accurate measurements of phospho-Ser-GSK3 in brain are confounded by a rapid post-mortem dephosphorylation, with ∼90% dephosphorylation of both GSK3 isoforms occurring within 2 min post-mortem. Furthermore, three anesthetics, pentobarbital, halothane and chloral hydrate, each caused large in vivo increases in the serine phosphorylation of both GSK3β and GSK3α in several regions of mouse brain. Thus, studies of the phosphorylation state of GSK3 in brain, and perhaps in other tissues, need to take into account post-mortem changes and the effects of anesthetics and there is a direct correlation between anesthesia and high levels of serine-phosphorylated GSK3.
28. 28
  Linding, R., Jensen, L. J., Ostheimer, G. J., van Vugt, M. A. T. M., Jørgensen, C., Miron, I. M., Diella, F., Colwill, K., Taylor, L., Elder, K., Metalnikov, P., Nguyen, V., Pasculescu, A., Jin, J., Park, J. G., Samson, L. D., Woodgett, J. R., Russell, R. B., Bork, P., Yaffe, M. B., and Pawson, T. (2007) Systematic discovery of in vivo phosphorylation networks Cell 129, 1415– 1426 DOI: 10.1016/j.cell.2007.05.052
  28
  Systematic discovery of in vivo phosphorylation networks
  Linding, Rune; Jensen, Lars Juhl; Ostheimer, Gerard J.; van Vugt, Marcel A. T. M.; Jorgensen, Claus; Miron, Ioana M.; Diella, Francesca; Colwill, Karen; Taylor, Lorne; Elder, Kelly; Metalnikov, Pavel; Nguyen, Vivian; Pasculescu, Adrian; Jin, Jing; Park, Jin Gyoon; Samson, Leona D.; Woodgett, James R.; Russell, Robert B.; Bork, Peer; Yaffe, Michael B.; Pawson, Tony
  Cell (Cambridge, MA, United States) (2007), 129 (7), 1415-1426CODEN: CELLB5; ISSN:0092-8674. (Cell Press)
  Protein kinases control cellular decision processes by phosphorylating specific substrates. Thousands of in vivo phosphorylation sites have been identified, mostly by proteome-wide mapping. However, systematically matching these sites to specific kinases is presently infeasible, due to limited specificity of consensus motifs, and the influence of contextual factors, such as protein scaffolds, localization, and expression, on cellular substrate specificity. We have developed an approach (NetworKIN) that augments motif-based predictions with the network context of kinases and phosphoproteins. The latter provides 60%-80% of the computational capability to assign in vivo substrate specificity. NetworKIN pinpoints kinases responsible for specific phosphorylations and yields a 2.5-fold improvement in the accuracy with which phosphorylation networks can be constructed. Applying this approach to DNA damage signaling, we show that 53BP1 and Rad50 are phosphorylated by CDK1 and ATM, resp. We describe a scalable strategy to evaluate predictions, which suggests that BCLAF1 is a GSK-3 substrate.
29. 29
  Peineau, S., Bradley, C., Taghibiglou, C., Doherty, A., Bortolotto, Z. A., Wang, Y. T., and Collingridge, G. L. (2008) The role of GSK-3 in synaptic plasticity Br. J. Pharmacol. 153, S428– S437 DOI: 10.1038/bjp.2008.2
  29
  The role of GSK-3 in synaptic plasticity
  Peineau, S.; Bradley, C.; Taghibiglou, C.; Doherty, A.; Bortolotto, Z. A.; Wang, Y. T.; Collingridge, G. L.
  British Journal of Pharmacology (2008), 153 (Suppl. 1), S428-S437CODEN: BJPCBM; ISSN:0007-1188. (Nature Publishing Group)
  A review. Glycogen synthase kinase-3 (GSK-3), an important component of the glycogen metab. pathway, is highly expressed in the CNS. It has been implicated in major neurol. disorders including Alzheimer's disease, schizophrenia and bipolar disorders. Despite its central role in these conditions it was not known until recently whether GSK-3 has neuronal-specific functions under normal conditions. However recent work has shown that GSK-3 is involved in the regulation of, and cross-talk between, two major forms of synaptic plasticity, N-methyl-D-aspartate receptor (NMDAR)-dependent long-term potentiation (LTP) and NMDAR-dependent long-term depression (LTD). The present article summarizes this recent work and discusses its potential relevance to the treatment of neurol. disorders.
30. 30
  Sutherland, C. and Sutherland, C. (2011) What Are the bona fide GSK3 Substrates? Int. J. Alzheimer's Dis. 2011, 505607 DOI: 10.4061/2011/505607
  30
  What Are the bona fide GSK3 Substrates?
  Sutherland Calum
  International journal of Alzheimer's disease (2011), 2011 (), 505607 ISSN:.
  Nearly 100 proteins are proposed to be substrates for GSK3, suggesting that this enzyme is a fundamental regulator of almost every process in the cell, in every tissue in the body. However, it is not certain how many of these proposed substrates are regulated by GSK3 in vivo. Clearly, the identification of the physiological functions of GSK3 will be greatly aided by the identification of its bona fide substrates, and the development of GSK3 as a therapeutic target will be highly influenced by this range of actions, hence the need to accurately establish true GSK3 substrates in cells. In this paper the evidence that proposed GSK3 substrates are likely to be physiological targets is assessed, highlighting the key cellular processes that could be modulated by GSK3 activity and inhibition.
31. 31
  Sánchez, C., Díaz-Nido, J., and Avila, J. (2000) Phosphorylation of microtubule-associated protein 2 (MAP2) and its relevance for the regulation of the neuronal cytoskeleton function Prog. Neurobiol. 61, 133– 168 DOI: 10.1016/S0301-0082(99)00046-5
  31
  Phosphorylation of microtubule-associated protein 2 (MAP2) and its relevance for the regulation of the neuronal cytoskeleton function
  Sanchez, C.; Diaz-Nido, J.; Avila, J.
  Progress in Neurobiology (Oxford) (2000), 61 (2), 133-168CODEN: PGNBA5; ISSN:0301-0082. (Elsevier Science Ltd.)
  A review with many refs. Neurons, the basic information processing units of the nervous system, are characterized by a complex polar morphol. which is essential for their function. To attain their precise morphol., neurons extend cytoplasmic processes (axons and dendrites) and establish synaptic connections in a highly regulated way. Addnl., neurons are also subjected to small plastic changes at the adult stage which serve to regulate synaptic transmission. Every step of neuronal development is genetically controlled by endogenous determinants, as well as by environmental signals including intercellular contacts, extracellular matrix and diffusible signals. Cytoskeletal components are among the main protein targets modified in response to most of those extracellular signals which ultimately det. neuronal morphol. One of the major mechanisms controlling the neuronal cytoskeleton is the modification of the phosphorylation state of cytoskeletal proteins via changes in the relative activities of protein kinases and phosphatases within neurons. In particular, the microtubule-assocd. protein 2 (MAP2) family of proteins is an abundant group of cytoskeletal components which are predominantly expressed in neurons and serve as substrates for most of protein kinases and phosphatases present in neurons. MAP2 phosphorylation seems to control its assocn. with the cytoskeleton and it is developmentally regulated. Moreover, MAP2 may perform many functions including the nucleation and stabilization of microtubules (and maybe microfilaments), the regulation of organelle transport within axons and dendrites, as well as the anchorage of regulatory proteins such as protein kinases which may be important for signal transduction. These putative functions of MAP2 have also been proposed to play important roles in the outgrowth of neuronal processes, synaptic plasticity and neuronal cell death. Thus, MAP2 constitutes an interesting case to understand the regulation of neuronal function by the alteration of the phosphorylation state of cytoskeletal proteins in response to different extracellular signals. Here we will review the current knowledge about the regulation of MAP2 function through phosphorylation/dephosphorylation and its relevance in the broader context of neuronal function.
32. 32
  Baas, P. W., Rao, A. N., Matamoros, A. J., and Leo, L. (2016) Stability properties of neuronal microtubules Cytoskeleton DOI: 10.1002/cm.21286
  There is no corresponding record for this reference.
33. 33
  Sánchez, C., Tompa, P., Szücs, K., Friedrich, P., and Avila, J. (1996) Phosphorylation and dephosphorylation in the proline-rich C-terminal domain of microtubule-associated protein 2 Eur. J. Biochem. 241, 765– 771 DOI: 10.1111/j.1432-1033.1996.00765.x
  33
  Phosphorylation and dephosphorylation in the proline-rich C-terminal domain of microtubule-associated protein 2
  Sanchez, Carlos; Tompa, Peter; Szucs, Kornelia; Friedrich, Peter; Avila, Jesus
  European Journal of Biochemistry (1996), 241 (3), 765-771CODEN: EJBCAI; ISSN:0014-2956. (Springer)
  The C-terminal domain of microtubule-assocd. protein 2 (MAP2) contains a Pro-rich region and the tubulin-binding domain. Antibodies were generated to follow the phosphorylation state of the Pro-rich domain. One of these antibodies (no. 305) was raised against a synthetic peptide P (sequence RTPGTPGTPSY) phosphorylated at the Thr residues. This sequence is present in the Pro-rich region of MAP2 and is phosphorylated in vitro by at least 3 different Pro-directed protein kinases: p42mpk, p34cdc2-, and GSK3 (glycogen-synthase kinase 3) α/β. The MAP2 sites phosphorylated by these kinases are different, although all of them phosphorylate the C-terminal domain of MAP2 as detd. by Staphylococcus aureus VS protease mapping. Nonphosphorylated peptide P can be phosphorylated in vitro by all 3 kinases studied with similar efficiency. In high-mol.-mass MAP2, this sequence is highly phosphorylated in vivo at the late stages of rat development. This motif can be rapidly dephosphorylated in vitro by protein-phosphatase 1 (PP1) and 2A (PP2A) catalytic subunits but not by PP2B.
34. 34
  Sánchez, C., Díaz-Nido, J., and Avila, J. (1998) Regulation of a site-specific phosphorylation of the microtubule-associated protein 2 during the development of cultured neurons Neuroscience 87, 861– 870 DOI: 10.1016/S0306-4522(98)00195-X
  There is no corresponding record for this reference.
35. 35
  Sánchez, C., Pérez, M., and Avila, J. (2000) GSK3beta-mediated phosphorylation of the microtubule-associated protein 2C (MAP2C) prevents microtubule bundling Eur. J. Cell Biol. 79, 252– 260 DOI: 10.1078/S0171-9335(04)70028-X
  35
  GSK3β-mediated phosphorylation of the microtubule-associated protein 2C (MAP2C) prevents microtubule bundling
  Sanchez, Carlos; Perez, Mar; Avila, Jesus
  European Journal of Cell Biology (2000), 79 (4), 252-260CODEN: EJCBDN; ISSN:0171-9335. (Urban & Fischer Verlag)
  A major determinant of neuronal morphol. is the cytoskeleton. And, one of the main regulatory mechanisms of cytoskeletal proteins is the modification of their phosphorylation state via changes in the relative activities of protein kinases and phosphatases in neurons. In particular, the microtubule-assocd. protein 2 (MAP2) family of proteins are abundant cytoskeletal components predominantly expressed in neurons and have been found to be substrates for most of the protein kinases and phosphatases present in neurons, including glycogen synthase kinase 3 (GSK3). It has been suggested that changes in GSK3-mediated MAP phosphorylation may modify MT stability and could control neuronal development. We have previously shown that MAP2 is phosphorylated in vitro and in situ by GSK3 at Thr1620 and Thr1623, located in the proline-rich region of MAP2 and recognized by antibody 305. However, the function of the phosphorylation of this site of MAP2 is still unknown. In this study, non-neuronal COS-1 cells have been co-transfected with cDNAs encoding MAP2C and either wild type or mutated GSK3β to analyze possible effects on microtubule stability and on the assocn. of MAP2 with microtubules. We have found that GSK3β phosphorylates MAP2C in co-transfected cells. Moreover, this phosphorylation is inhibited by the specific GSK3 inhibitor lithium chloride. Addnl., the formation of microtubule bundles, which is obsd. after transfection with MAP2C, was decreased when MAP2C was co-transfected with GSK3β wild type. Microtubule bundles were not obsd. in cells expressing MAP2C phosphorylated at the site recognized by antibody 305. The absence of microtubule bundles was reverted after treatment of MAP2C/GSK3β wild type transfected cells with lithium chloride. Highly phosphorylated MAP2C species, which were phosphorylated at the site recognized by antibody 305, appeared in cells co-transfected with MAP2C and GSK3β wild type. Interestingly, these MAP2C species were enriched in cytoskeleton-unbound protein prepns. These data suggests that GSK3-mediated phosphorylation of MAP2 may modify its binding to microtubules and regulate microtubule stability.
36. 36
  Ebneth, A., Drewes, G., Mandelkow, E. M., and Mandelkow, E. (1999) Phosphorylation of MAP2c and MAP4 by MARK kinases leads to the destabilization of microtubules in cells Cell Motil. Cytoskeleton 44, 209– 224 DOI: 10.1002/(SICI)1097-0169(199911)44:3<209::AID-CM6>3.0.CO;2-4
  36
  Phosphorylation of MAP2c and MAP4 by MARK kinases leads to the destabilization of microtubules in cells
  Ebneth, A.; Drewes, G.; Mandelkow, E.-M.; Mandelkow, E.
  Cell Motility and the Cytoskeleton (1999), 44 (3), 209-224CODEN: CMCYEO; ISSN:0886-1544. (Wiley-Liss, Inc.)
  Microtubules serve as transport tracks in mol. mechanisms governing cellular shape and polarity. Rapid transitions between stable and dynamic microtubules are regulated by several factors, including microtubule-assocd. proteins (MAPs). We have shown that MAP/microtubule affinity regulating kinases (MARK) can phosphorylate the microtubule-assocd.-proteins MAP4, MAP2c, and tau on their microtubule-binding domain in vitro. This leads to their detachment from microtubules (MT) and an increased dynamic instability of MT. Here, we show that MARK protein kinases phosphorylate MAP2 and MAP4 on their microtubule-binding domain in transfected CHO cells. In CHO cells expressing MARK1 or MARK2 under control of an inducible promoter, MARK2 phosphorylates an endogenous MAP4-related protein. Prolonged expression of MARK2 results in microtubule-disruption, detachment of cells from the substratum, and cell death. Concomitant with microtubule disruption, we also obsd. a breakdown of the vimentin network, whereas actin fibers remained unaffected. Thus, MARK seems to play an important role in controlling cytoskeletal dynamics.
37. 37
  Hara, K. and Harris, R. A. (2002) The anesthetic mechanism of urethane: the effects on neurotransmitter-gated ion channels Anesth. Analg. 94, 313– 318 DOI: 10.1213/00000539-200202000-00015
  37
  The anesthetic mechanism of urethane: the effects on neurotransmitter-gated ion channels
  Hara, Koji; Harris, R. Adron
  Anesthesia & Analgesia (Baltimore, MD, United States) (2002), 94 (2), 313-318CODEN: AACRAT; ISSN:0003-2999. (Lippincott Williams & Wilkins)
  Urethane is widely used as an anesthetic for animal studies because of its minimal effects on cardiovascular and respiratory systems and maintenance of spinal reflexes. Despite its usefulness in animal research, there are no reports concerning its mol. actions. We designed this study to det. whether urethane affects neurotransmitter-gated ion channels. We examd. the effects of urethane on recombinant GABAA, glycine, N-methyl-D-aspartate, α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid, and neuronal nicotinic acetylcholine receptors expressed in Xenopus oocytes. Urethane potentiated the functions of neuronal nicotinic acetylcholine, γ-aminobutyric acidA, and glycine receptors, and it inhibited N-methyl-D-aspartate and α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors in a concn.-dependent manner. At concns. close to anesthetic 50% effective concn., urethane had modest effects on all channels tested, suggesting the lack of a single predominant target for its action. This may account for its usefulness as a veterinary anesthetic. However, a large concn. of urethane exerts marked effects on all channels. These findings not only give insight into the mol. mechanism of anesthetics but also caution that neurophysiol. measurements from animals anesthetized with urethane may be complicated by the effects of urethane on multiple neurotransmitter systems. Our results also suggest that small changes in multiple receptor systems can produce anesthesia.
38. 38
  Seira, O. and Del Río, J. A. (2014) Glycogen synthase kinase 3 beta (GSK3β) at the tip of neuronal development and regeneration Mol. Neurobiol. 49, 931– 944 DOI: 10.1007/s12035-013-8571-y
  38
  Glycogen Synthase Kinase 3 Beta (GSK3β) at the Tip of Neuronal Development and Regeneration
  Seira, Oscar; del Rio, Jose Antonio
  Molecular Neurobiology (2014), 49 (2), 931-944CODEN: MONBEW; ISSN:0893-7648. (Humana Press Inc.)
  A review. Gaining a basic understanding of the inhibitory mols. and the intracellular signaling involved in axon development and repulsion after neural lesions is of clear biomedical interest. In recent years, numerous studies have described new mols. and intracellular mechanisms that impair axonal outgrowth after injury. In this scenario, the role of glycogen synthase kinase 3 beta (GSK3β) in the axonal responses that occur after central nervous system (CNS) lesions began to be elucidated. GSK3β function in the nervous tissue is assocd. with neural development, neuron polarization, and, more recently, neurodegeneration. In fact, GSK3β has been considered as a putative therapeutic target for promoting functional recovery in injured or degenerative CNS. In this review, we summarize current understanding of the role of GSK3β during neuronal development and regeneration. In particular, we discuss GSK3β activity levels and their possible impact on cytoskeleton dynamics during both processes.
39. 39
  Li, X. and Jope, R. S. (2010) Is glycogen synthase kinase-3 a central modulator in mood regulation? Neuropsychopharmacology 35, 2143– 2154 DOI: 10.1038/npp.2010.105
  39
  Is glycogen synthase kinase-3 a central modulator in mood regulation?
  Li, Xiaohua; Jope, Richard S.
  Neuropsychopharmacology (2010), 35 (11), 2143-2154CODEN: NEROEW; ISSN:0893-133X. (Nature Publishing Group)
  A review. Little is known regarding the mechanisms underlying the complex etiol. of mood disorders, represented mainly by major depressive disorder and bipolar disorder. The 1996 discovery that lithium inhibits glycogen synthase kinase-3 (GSK3) raised the possibility that impaired inhibition of GSK3 is assocd. with mood disorders. This is now supported by evidence from animal biochem., pharmacol., mol., and behavioral studies and from human post-mortem brain, peripheral tissue, and genetic studies that are reviewed here. Mood disorders may result in part from impairments in mechanisms controlling the activity of GSK3 or GSK3-regulated functions, and disruptions of these regulating systems at different signaling sites may contribute to the heterogeneity of mood disorders. This substantial evidence supports the conclusion that bolstering the inhibitory control of GSK3 is an important component of the therapeutic actions of drugs used to treat mood disorders and that GSK3 is a valid target for developing new therapeutic interventions.
40. 40
  Bianchi, M. and Baulieu, E.-E. (2012) 3β-Methoxy-pregnenolone (MAP4343) as an innovative therapeutic approach for depressive disorders Proc. Natl. Acad. Sci. U. S. A. 109, 1713– 1718 DOI: 10.1073/pnas.1121485109
  There is no corresponding record for this reference.
41. 41
  Kalenka, A., Hinkelbein, J., Feldmann, R. E., Kuschinsky, W., Waschke, K. F., and Maurer, M. H. (2007) The Effects of Sevoflurane Anesthesia on Rat Brain Proteins: A Proteomic Time-Course Analysis Anesth. Analg. 104, 1129– 1135 DOI: 10.1213/01.ane.0000260799.37107.e6
  41
  The Effects of Sevoflurane Anesthesia on Rat Brain Proteins: A Proteomic Time-Course Analysis
  Kalenka, Armin; Hinkelbein, Jochen; Feldmann, Robert E., Jr.; Kuschinsky, Wolfgang; Waschke, Klaus F.; Maurer, Martin H.
  Anesthesia & Analgesia (Hagerstown, MD, United States) (2007), 104 (5), 1129-1135CODEN: AACRAT; ISSN:0003-2999. (Lippincott Williams & Wilkins)
  Background: Recent studies showed changes in cerebral protein expression up to 3 days after desflurane anesthesia in rats. In the present study, we investigated the existence of persisting changes on the proteome level after sevoflurane anesthesia that persisted for as long as 28 days after anesthesia. Methods: Rats were anesthetized by spontaneous inhalation of 2.4% sevoflurane in air for 3 h. Animals (n = 6 for each group) were killed either directly, 72 h, or 28 days after anesthesia. Brains were removed and subjected to global protein expression profiling based on two-dimensional gel electrophoresis and mass spectrometry. Expression factors were compared to results from untreated conscious animals at each time point. Data were statistically analyzed by ANOVA (P < 0.01) and a cut of more than two-fold change in the expression factor. Results: We found 11 protein spots differentially regulated directly after anesthesia. Seventeen proteins were differentially expressed 72 h after the anesthesia. Only one spot was differentially regulated 28 days after anesthesia. The plausible targets of these differentially regulated proteins can be attributed to synaptic vesicle handling and cell-cell communication. Conclusions: Sevoflurane induced relevant changes in protein expression profiles directly and 72 h after an anesthesia with 1 MAC. Twenty-eight days after the anesthesia, all proteins except one had returned to baseline levels of abundance.
42. 42
  Kalenka, A., Gross, B., Maurer, M. H., Thierse, H.-J., and Feldmann, R. E. (2010) Isoflurane Anesthesia Elicits Protein Pattern Changes in Rat Hippocampus J. Neurosurg. Anesthesiol. 22, 144– 154 DOI: 10.1097/ANA.0b013e3181cb7cb8
  42
  Isoflurane anesthesia elicits protein pattern changes in rat hippocampus
  Kalenka Armin; Gross Benjamin; Maurer Martin H; Thierse Hermann-Josef; Feldmann Robert E Jr
  Journal of neurosurgical anesthesiology (2010), 22 (2), 144-54 ISSN:.
  Postoperative cognitive dysfunction (POCD) is a known phenomenon occurring after anesthesia with volatile anesthetics (VA), such as isoflurane. Recent reports suggest that VA interact with neurodegenerative disease-associated proteins including compounds with pathogenic relevance in Alzheimer disease (AD) and induce processes that may be linked to AD neuropathology. Unfortunately, our present understanding of the exact anesthetics' molecular mechanisms of action, their side effects on the brain, and their catenation with AD pathology is still limited. The present study analyzes the differential proteome of the hippocampus immediately after and 3 days after a 3-hour 1 minimal alveolar concentration isoflurane anesthesia in rats. Differential 2-dimensional electrophoresis, mass spectrometry, and functional network mapping were used to identify and functionally classify 12 different hippocampal proteins, which were significantly regulated after isoflurane anesthesia (6 up-regulated, 11 down-regulated with P<0.01). Induction of differential expression ranged from 0.05 (25-fold down-regulation) to 4.4 (4.4-fold up-regulation). Ten proteins were regulated immediately after and 7 proteins 3 days after isoflurane exposure. The proteome displays isoflurane-responsive protein candidates, which have also been shown to play a role in AD. They were grouped according to their key biologic activities, which showed that isoflurane affects selected biologic processes including synaptic plasticity, stress response, detoxification, and cytoskeleton in early and late recovery phases after anesthesia. These processes are also affected in AD. Results are discussed in view of AD, the toxicity mechanisms of isoflurane as well as the implications for our present understanding and conduction of clinical anesthesia.
43. 43
  Tan, H., Wu, Z., Wang, H., Bai, B., Li, Y., Wang, X., Zhai, B., Beach, T. G., and Peng, J. (2015) Refined phosphopeptide enrichment by phosphate additive and the analysis of human brain phosphoproteome Proteomics 15, 500– 507 DOI: 10.1002/pmic.201400171
  There is no corresponding record for this reference.
44. 44
  Elo, L. L., Filén, S., Lahesmaa, R., and Aittokallio, T. (2008) Reproducibility-optimized test statistic for ranking genes in microarray studies IEEE/ACM Trans. Comput. Biol. Bioinf. 5, 423– 431 DOI: 10.1109/tcbb.2007.1078
  44
  Reproducibility-optimized test statistic for ranking genes in microarray studies
  Elo, Laura L.; Filen, Sanna; Lahesmaa, Riitta; Aittokallio, Tero
  IEEE/ACM Transactions on Computational Biology and Bioinformatics (2008), 5 (3), 423-431CODEN: ITCBCY; ISSN:1545-5963. (IEEE Computer Society)
  A principal goal of microarray studies is to identify the genes showing differential expression under distinct conditions. In such studies, the selection of an optimal test statistic is a crucial challenge, which depends on the type and amt. of data under anal. Although previous studies on simulated or spike-in data sets do not provide practical guidance on how to choose the best method for a given real data set, we introduce an enhanced reproducibility-optimization procedure, which enables the selection of a suitable gene-ranking statistic directly from the data. In comparison with existing ranking methods, the reproducibility-optimized statistic shows good performance consistently under various simulated conditions and on Affymetrix spike-in data set. Further, the feasibility of the novel statistic is confirmed in a practical research setting using data from an inhouse cDNA microarray study of asthma-related gene expression changes. These results suggest that the procedure facilitates the selection of an appropriate test statistic for a given data set without relying on a priori assumptions, which may bias the findings and their interpretation. Moreover, the general reproducibility-optimization procedure is not limited to detecting differential expression only but could be extended to a wide range of other applications as well.
45. 45
  Huang, D. W., Sherman, B. T., and Lempicki, R. A. (2009) Bioinformatics enrichment tools: paths toward the comprehensive functional analysis of large gene lists Nucleic Acids Res. 37, 1– 13 DOI: 10.1093/nar/gkn923
  45
  Bioinformatics enrichment tools: paths toward the comprehensive functional analysis of large gene lists
  Huang, Da Wei; Sherman, Brad T.; Lempicki, Richard A.
  Nucleic Acids Research (2009), 37 (1), 1-13CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)
  A review. Functional anal. of large gene lists, derived in most cases from emerging high-throughput genomic, proteomic and bioinformatics scanning approaches, is still a challenging and daunting task. The gene-annotation enrichment anal. is a promising high-throughput strategy that increases the likelihood for investigators to identify biol. processes most pertinent to their study. Approx. 68 bioinformatics enrichment tools that are currently available in the community are collected in this survey. Tools are uniquely categorized into three major classes, according to their underlying enrichment algorithms. The comprehensive collections, unique tool classifications and assocd. questions/issues will provide a more comprehensive and up-to-date view regarding the advantages, pitfalls and recent trends in a simpler tool-class level rather than by a tool-by-tool approach. Thus, the survey will help tool designers/developers and experienced end users understand the underlying algorithms and pertinent details of particular tool categories/tools, enabling them to make the best choices for their particular research interests.
46. 46
  Rantamäki, T., Hendolin, P., Kankaanpää, A., Mijatovic, J., Piepponen, P., Domenici, E., Chao, M. V., Männistö, P. T., and Castrén, E. (2007) Pharmacologically diverse antidepressants rapidly activate brain-derived neurotrophic factor receptor TrkB and induce phospholipase-Cgamma signaling pathways in mouse brain Neuropsychopharmacology 32, 2152– 2162 DOI: 10.1038/sj.npp.1301345
  46
  Pharmacologically Diverse Antidepressants Rapidly Activate Brain-Derived Neurotrophic Factor Receptor TrkB and Induce Phospholipase-Cγ Signaling Pathways in Mouse Brain
  Rantamaki, Tomi; Hendolin, Panu; Kankaanpaa, Aino; Mijatovic, Jelena; Piepponen, Petteri; Domenici, Enrico; Chao, Moses V.; Mannisto, Pekka T.; Castren, Eero
  Neuropsychopharmacology (2007), 32 (10), 2152-2162CODEN: NEROEW; ISSN:0893-133X. (Nature Publishing Group)
  Previous studies suggest that brain-derived neurotrophic factor and its receptor TrkB are critically involved in the therapeutic actions of antidepressant drugs. We have previously shown that the antidepressants imipramine and fluoxetine produce a rapid autophosphorylation of TrkB in the rodent brain. In the present study, we have further examd. the biochem. and functional characteristics of antidepressant-induced TrkB activation in vivo. We show that all the antidepressants examd., including inhibitors of monoamine transporters and metab., activate TrkB rapidly in the rodent anterior cingulate cortex and hippocampus. Furthermore, the results indicate that acute and long-term antidepressant treatments induce TrkB-mediated activation of phospholipase-Cγ1 (PLCγ1) and increase the phosphorylation of cAMP-related element binding protein, a major transcription factor mediating neuronal plasticity. In contrast, we have not obsd. any modulation of the phosphorylation of TrkB Shc binding site, phosphorylation of mitogen-activated protein kinase or AKT by antidepressants. We also show that in the forced swim test, the behavioral effects of specific serotonergic antidepressant citalopram, but not those of the specific noradrenergic antidepressant reboxetine, are crucially dependent on TrkB signaling. Finally, brain monoamines seem to be crit. mediators of antidepressant-induced TrkB activation, as antidepressants reboxetine and citalopram do not produce TrkB activation in the brains of serotonin- or norepinephrine-depleted mice. In conclusion, our data suggest that rapid activation of the TrkB neurotrophin receptor and PLCγ1 signaling is a common mechanism for all antidepressant drugs.
47. 47
  Rantamäki, T., Vesa, L., Antila, H., Di Lieto, A., Tammela, P., Schmitt, A., Lesch, K.-P., Rios, M., and Castrén, E. (2011) Antidepressant drugs transactivate TrkB neurotrophin receptors in the adult rodent brain independently of BDNF and monoamine transporter blockade PLoS One 6, e20567 DOI: 10.1371/journal.pone.0020567
  47
  Antidepressant drugs transactivate TrkB neurotrophin receptors in the adult rodent brain independently of BDNF and monoamine transporter blockade
  Rantamaki, Tomi; Vesa, Liisa; Antila, Hanna; Di Lieto, Antonio; Tammela, Paivi; Schmitt, Angelika; Lesch, Klaus-Peter; Rios, Maribel; Castren, Eero
  PLoS One (2011), 6 (6), e20567CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)
  Background: Antidepressant drugs (ADs) have been shown to activate BDNF (brain-derived neurotrophic factor) receptor TrkB in the rodent brain but the mechanism underlying this phenomenon remains unclear. ADs act as monoamine reuptake inhibitors and after prolonged treatments regulate brain BDNF mRNA levels indicating that monoamine-BDNF signaling regulate AD-induced TrkB activation in vivo. However, recent findings demonstrate that Trk receptors can be transactivated independently of their neurotrophin ligands. Methodol.: In this study we examd. the role of BDNF, TrkB kinase activity and monoamine reuptake in the AD-induced TrkB activation in vivo and in vitro by employing several transgenic mouse models, cultured neurons and TrkB-expressing cell lines. Principal Findings: Using a chem.-genetic TrkBF616A mutant and TrkB overexpressing mice, we demonstrate that ADs specifically activate both the maturely and immaturely glycosylated forms of TrkB receptors in the brain in a TrkB kinase dependent manner. However, the tricyclic AD imipramine readily induced the phosphorylation of TrkB receptors in conditional BDNF-/- knock-out mice (132.4 ± 8.5% of control; P = 0.01), indicating that BDNF is not required for the TrkB activation. Moreover, using serotonin transporter (SERT) deficient mice and chem. lesions of monoaminergic neurons we show that neither a functional SERT nor monoamines are required for the TrkB phosphorylation response induced by the serotonin selective reuptake inhibitors fluoxetine or citalopram, or norepinephrine selective reuptake inhibitor reboxetine. However, neither ADs nor monoamine transmitters activated TrkB in cultured neurons or cell lines expressing TrkB receptors, arguing that ADs do not directly bind to TrkB. Conclusions: The present findings suggest that ADs transactivate brain TrkB receptors independently of BDNF and monoamine reuptake blockade and emphasize the need of an intact tissue context for the ability of ADs to induce TrkB activity in brain.
Supporting Information
Supporting Information
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acschemneuro.6b00002.
- Table 1: Differentially regulated phosphoproteins after sham and isoflurane anesthesia (4% induction, 2% maintenance; 30 min) (N = 3/group). Phosphopeptide changes arranged based on statistical significance (FDR < 0.05). Table 2: Functional annotation clusters arranged based on the statistical significance (FDR < 0.05). (XLSX)
- cn6b00002_si_001.xlsx (64.83 kb)
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Brief Isoflurane Anesthesia Produces Prominent Phosphoproteomic Changes in the Adult Mouse Hippocampus
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Brief Isoflurane Anesthesia Produces Prominent Phosphoproteomic Changes in the Adult Mouse HippocampusClick to copy article linkArticle link copied!

ACS Chemical Neuroscience

Abstract

Results and Discussion

Figure 1

Figure 2

Figure 3

Methods

Animals

Drug Treatments

EEG and EMG Recordings

Phosphoprotein Enrichment and Quantitative Mass Spectrometry

Bioinformatics

Western Blot

Statistics

Supporting Information

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Acknowledgment

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ACS Chemical Neuroscience

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Brief Isoflurane Anesthesia Produces Prominent Phosphoproteomic Changes in the Adult Mouse Hippocampus
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