Behavioral and transcriptional effects of repeated electroconvulsive seizures in the neonatal MK-801-treated rat model of schizophrenia.

RATIONALE: Electroconvulsive therapy (ECT) is an effective treatment modality for schizophrenia. However, its antipsychotic-like mechanism remains unclear. OBJECTIVES: To gain insight into the antipsychotic-like actions of ECT, this study investigated how repeated treatments of electroconvulsive seizure (ECS), an animal model for ECT, affect the behavioral and transcriptomic profile of a neurodevelopmental animal model of schizophrenia. METHODS: Two injections of MK-801 or saline were administered to rats on postnatal day 7 (PN7), and either repeated ECS treatments (E10X) or sham shock was conducted daily from PN50 to PN59. Ultimately, the rats were divided into vehicle/sham (V/S), MK-801/sham (M/S), vehicle/ECS (V/E), and MK-801/ECS (M/E) groups. On PN59, prepulse inhibition and locomotor activity were tested. Prefrontal cortex transcriptomes were analyzed with mRNA sequencing and network and pathway analyses, and quantitative real-time polymerase chain reaction (qPCR) analyses were subsequently conducted. RESULTS: Prepulse inhibition deficit was induced by MK-801 and normalized by E10X. In M/S vs. M/E model, Egr1, Mmp9, and S100a6 were identified as center genes, and interleukin-17 (IL-17), nuclear factor kappa B (NF-κB), and tumor necrosis factor (TNF) signaling pathways were identified as the three most relevant pathways. In the V/E vs. V/S model, mitophagy, NF-κB, and receptor for advanced glycation end products (RAGE) pathways were identified. qPCR analyses demonstrated that Igfbp6, Btf3, Cox6a2, and H2az1 were downregulated in M/S and upregulated in M/E. CONCLUSIONS: E10X reverses the behavioral changes induced by MK-801 and produces transcriptional changes in inflammatory, insulin, and mitophagy pathways, which provide mechanistic insight into the antipsychotic-like mechanism of ECT.

Resistance Temperature Detectors Fabricated via Dual Fused Deposition Modeling of Polylactic Acid and Polylactic Acid/Carbon Black Composites.

Planar-type resistance temperature detectors (P-RTDs) were fabricated via fused deposition modeling by dual nozzle extrusion. The temperature-sensing element of the fabricated sensor was printed with electrically conductive polylactic acid/carbon black (PLA/CB) composite, while the structural support was printed with a PLA insulator. The temperature-dependent resistivity change of PLA/CB was evaluated for different stacking sequences of PLA/CB layers printed with [0°/0°], [-45°/45°], and [0°/90°] plies. Compared to a PLA/CB filament used as 3D printing source material, the laminated structures exhibited a response over 3 times higher, showing a resistivity change from -10 to 40 Ω∙cm between -15 and 50 °C. Then, using the [0°/90°] plies stacking sequence, a P-RTD thermometer was fabricated in conjunction with a Wheatstone bridge circuit for temperature readouts. The P-RTD yielded a temperature coefficient of resistance of 6.62 %/°C with high stability over repeated cycles. Fabrication scalability was demonstrated by realizing a 3 × 3 array of P-RTDs, allowing the temperature profile detection of the surface in contact with heat sources.

Electroconvulsive Seizures Induce Autophagy by Activating the AMPK Signaling Pathway in the Rat Frontal Cortex.

BACKGROUND: It is uncertain how electroconvulsive therapy-induced generalized seizures exert their potent therapeutic effects on various neuropsychiatric disorders. Adenosine monophosphate-activated protein kinase (AMPK) plays a major role in maintaining metabolic homeostasis and activates autophagic processes via unc-51-like kinase (ULK1). Evidence supports the involvement of autophagy system in the action mechanisms of antidepressants and antipsychotics. The effect of electroconvulsive therapy on autophagy-related signaling requires further clarification. METHODS: The effect of electroconvulsive seizure on autophagy and its association with the AMPK signaling pathway were investigated in the rat frontal cortex. Electroconvulsive seizure was provided once per day for 10 days (E10X), and compound C or 3-methyadenine was administered through an intracerebroventricular cannula. Molecular changes were analyzed with immunoblot, immunohistochemistry, and transmission electron microscopy analyses. RESULTS: E10X increased p-Thr172-AMPKα immunoreactivity in rat frontal cortex neurons. E10X increased phosphorylation of upstream effectors of AMPK, such as LKB1, CaMKK, and TAK1, and of its substrates, ACC, HMGR, and GABABR2. E10X also increased p-Ser317-ULK1 immunoreactivity. At the same time, LC3-II and ATG5-ATG12 conjugate immunoreactivity increased, indicating activation of autophagy. An intracerebroventricular injection of the AMPK inhibitor compound C attenuated the electroconvulsive seizure-induced increase in ULK1 phosphorylation as well as the protein levels of LC3-II and Atg5-Atg12 conjugate. Transmission electron microscopy clearly showed an increased number of autophagosomes in the rat frontal cortex after E10X, which was reduced by intracerebroventricular treatment with the autophagy inhibitor 3-methyadenine and compound C. CONCLUSIONS: Repeated electroconvulsive seizure treatments activated in vivo autophagy in the rat frontal cortex through the AMPK signaling pathway.

Electroconvulsive Seizure Alters the Expression and Daily Oscillation of Circadian Genes in the Rat Frontal Cortex.

OBJECTIVE: Electroconvulsive therapy (ECT) is the most effective treatment for mood disorders. Accumulating evidence has suggested the important role of circadian genes in mood disorders. However, the effects of ECT on circadian genes have not been systemically investigated. METHODS: We examined the expression and daily oscillation of major circadian genes in the rat frontal cortex after electroconvulsive seizure (ECS). RESULTS: Firstly, mRNA and protein level were investigated at 24 hr after single ECS (E1X) and repeated ECS treatements for 10 days (E10X), which showed more remarkable changes after E10X than E1X. mRNA expression of Rorα, Bmal1, Clock, Per1, and Cry1 was decreased, while Rev-erbα expression was increased at 24 hr after E10X compared to sham. The proteins showed similar pattern of changes. Next, the effects on oscillation and rhythm properties (mesor, amplitude, and acrophase) were examined, which also showed more prominent changes after E10X than E1X. After E10X, mesor of Rorα, Bmal1, and Cry1 was reduced, and that of Rev-erbα was increased. Five genes, Rev-erbα, Bmal1, Per1, Per2, and Cry2, showed earlier acrophase after E10X. CONCLUSION: The findings suggest that repeated ECS induces reduced expression and phase advance of major circadian genes in the in vivo rat frontal cortex.

The antipsychotic agent clozapine induces autophagy via the AMPK-ULK1-Beclin1 signaling pathway in the rat frontal cortex.

Clozapine, a representative atypical antipsychotic, has superior efficacy compared to other antipsychotic agents and is used for the treatment of severe psychotic disorders. Therefore, studies on its mechanisms of action are important for understanding the mechanisms of therapeutic approaches to psychosis. Adenosine monophosphate-activated protein kinase (AMPK) is a serine-threonine kinase that plays a major role in maintaining metabolic homeostasis. Unc-51-like kinase 1 (ULK1) and Beclin1 are downstream substrates of AMPK and activate the autophagic process. In this study, we examined the effects of clozapine on the AMPK-ULK1-Beclin1 signaling pathway and autophagy in the frontal cortex of the rat. Clozapine (10mg/kg) administration increased the immunoreactivity of p-Thr172-AMPKα in the rat frontal cortex at 1, 2, and 4h after injection, as we previously reported. The immunoreactivity of p-Ser317-ULK1 and p-Ser93-Beclin1 was also increased at 2 and 4h after clozapine injection. At the same time, the immunoreactivity of LC3-II and the Atg5-Atg12 conjugate, which indicate activation of autophagy, was increased. Transmission electron microscopy clearly showed an increase in autophagosome number in the rat frontal cortex at 2h after clozapine injection. To investigate the role of AMPK in clozapine-induced autophagy, the effects of intracerebroventricular injection of compound C, an AMPK inhibitor, were examined. Administration of compound C attenuated the clozapine-induced increase in ULK1 and Beclin1 phosphorylation, as well the protein levels of LC3-II and the Atg5-Atg12 conjugate in the frontal cortex. In summary, the results showed that clozapine activates autophagy through the AMPK-ULK1-Beclin1 signaling pathway in the frontal cortex of the rat.

Selective Manipulation of Neural Circuits.

Unraveling the complex network of neural circuits that form the nervous system demands tools that can manipulate specific circuits. The recent evolution of genetic tools to target neural circuits allows an unprecedented precision in elucidating their function. Here we describe two general approaches for achieving circuit specificity. The first uses the genetic identity of a cell, such as a transcription factor unique to a circuit, to drive expression of a molecule that can manipulate cell function. The second uses the spatial connectivity of a circuit to achieve specificity: one genetic element is introduced at the origin of a circuit and the other at its termination. When the two genetic elements combine within a neuron, they can alter its function. These two general approaches can be combined to allow manipulation of neurons with a specific genetic identity by introducing a regulatory gene into the origin or termination of the circuit. We consider the advantages and disadvantages of both these general approaches with regard to specificity and efficacy of the manipulations. We also review the genetic techniques that allow gain- and loss-of-function within specific neural circuits. These approaches introduce light-sensitive channels (optogenetic) or drug sensitive channels (chemogenetic) into neurons that form specific circuits. We compare these tools with others developed for circuit-specific manipulation and describe the advantages of each. Finally, we discuss how these tools might be applied for identification of the neural circuits that mediate behavior and for repair of neural connections.

Repeated treatment with electroconvulsive seizures induces HDAC2 expression and down-regulation of NMDA receptor-related genes through histone deacetylation in the rat frontal cortex.

The enzymatic activity of histone deacetylases (HDACs) leads to a histone deacetylation-mediated condensed chromatic structure, resulting in transcriptional repression, which has been implicated in the modifications of neural circuits and behaviors. Repeated treatment with electroconvulsive seizure (ECS) induces changes in histone acetylation, expression of various genes, and intrabrain cellular changes, including neurogenesis. In this study, we examined the effects of repeated ECS on the expression of class I HDACs and related changes in histone modifications and gene expression in the rat frontal cortex. Ten days of repeated ECS treatments (E10X) up-regulated HDAC2 expression at the mRNA and protein levels in the rat frontal cortex compared with sham-treated controls; this was evident in the nuclei of neuronal cells in the prefrontal, cingulate, orbital, and insular cortices. Among the known HDAC2 target genes, mRNA expression of N-methyl-d-aspartate (NMDA) receptor signaling-related genes, including early growth response-1 (Egr1), c-Fos, glutamate receptor, ionotropic, N-methyl d-aspartate 2A (Nr2a), Nr2b, neuritin1 (Nrn1), and calcium/calmodulin-dependent protein kinase II alpha (Camk2α), were decreased, and the histone acetylation of H3 and/or H4 proteins was also reduced by E10X. Chromatin immunoprecipitation analysis revealed that HDAC2 occupancy in the promoters of down-regulated genes was increased significantly. Moreover, administration of sodium butyrate, a HDAC inhibitor, during the course of E10X ameliorated the ECS-induced down-regulation of genes in the rat frontal cortex. These findings suggest that induction of HDAC2 by repeated ECS treatment could play an important role in the down-regulation of NMDA receptor signaling-related genes in the rat frontal cortex through histone modification.

Role of MKP-1 (DUSP1) in clozapine-induced effects on the ERK1/2 signaling pathway in the rat frontal cortex.

RATIONALE: Clozapine affects the extracellular signal-regulated kinase 1/2 (ERK1/2) pathway in the brain, which plays an important role in its antipsychotic action. However, previous findings are inconsistent, and related molecular mechanisms require further clarification. OBJECTIVES: Time- and dose-dependent effects of clozapine on the ERK1/2 pathway and its regulatory mechanism were investigated in rat frontal cortex. METHODS AND RESULTS: At 15, 30, 60, and 120 min after intraperitoneal injection of clozapine (5, 10, and 20 mg/kg), changes in ERK1/2, its upstream canonical kinases (Raf1 and mitogen-activated protein kinase kinase 1/2 [MEK1/2]), and its downstream molecule (p90 ribosomal S6 kinase [p90RSK]) were investigated in rat frontal cortex. At 15 min, p-Raf1, p-MEK1/2, p-ERK1/2, and p-p90RSK all increased dose-dependently. At 30 min, p-ERK1/2 and p-p90RSK showed no significant changes, while dose-dependent increases in p-Raf1 and p-MEK1/2 were found. At 60 and 120 min, although p-ERK1/2 and p-p90RSK decreased, increases in p-Raf1 and p-MEK1/2 were maintained. A clozapine-induced reduction in ERK1/2 phosphorylation was evident at both tyrosine and threonine residues, suggesting the involvement of dual specificity phosphatases (DUSPs; mitogen-activated protein kinase phosphatases [MKPs]). mRNA expression of seven Dusps that can dephosphorylate ERK1/2 were examined; Mkp-1 (Dusp1) mRNA increased following clozapine treatment. Moreover, MKP-1 protein and phosphatase activity increased, and binding of MKP-1 to ERK1/2 was also upregulated by clozapine administration. CONCLUSIONS: In rat frontal cortex, clozapine regulates ERK1/2 phosphorylation via MKP-1, which induces uncoupling between Raf1-MEK1/2 and ERK1/2-p90RSK activity. These findings suggest an important role of MKP-1 in the mechanism of action of clozapine.

Intracerebroventricular administration of ouabain, a Na/K-ATPase inhibitor, activates mTOR signal pathways and protein translation in the rat frontal cortex.

Intracerebroventricular (ICV) injection of ouabain, a specific Na/K-ATPase inhibitor, induces behavioral changes in rats in a putative animal model of mania. The binding of ouabain to Na/K-ATPase affects signaling molecules in vitro, including ERK1/2 and Akt, which promote protein translation. We have also reported that ERK1/2 and Akt in the brain are involved in the ouabain-induced hyperactivity of rats. In this study, rats were given an ICV injection of ouabain, and then their frontal cortices were examined to determine the effects of ouabain on the mTOR/p70S6K/S6 signaling pathway and protein translation, which are important in modifications of neural circuits and behavior. Rats showed ouabain-induced hyperactivity up to 8h following injection, and increased phosphorylation levels of mTOR, p70S6K, S6, eIF4B, and 4E-BP at 1, 2, 4, and 8h following ouabain injection. Immunohistochemical analyses revealed that increased p-S6 immunoreactivity in the cytoplasm of neurons by ouabain was evident in the prefrontal, cingulate, and orbital cortex. These findings suggested increased translation initiation in response to ouabain. The rate of protein synthesis was measured as the amount of [(3)H]-leucine incorporation in the cell-free extracts of frontal cortical tissues, and showed a significant increase at 8h after ouabain injection. These results suggest that ICV injection of ouabain induced activation of the protein translation initiation pathway regulated by ERK1/2 and Akt, and prolonged hyperactivity in rats. In conclusion, protein translation pathway could play an important role in ouabain-induced hyperactivity in a rodent model of mania.

Macrophage migration inhibitory factor mediates the antidepressant actions of voluntary exercise.

Voluntary exercise is known to have an antidepressant effect. However, the underlying mechanism for this antidepressant action of exercise remains unclear, and little progress has been made in identifying genes that are directly involved. We have identified macrophage migration inhibitory factor (MIF) by analyzing existing mRNA microarray data and confirmed the augmented expression of selected genes under two experimental conditions: voluntary exercise and electroconvulsive seizure. A proinflammatory cytokine, MIF is expressed in the central nervous system and involved in innate and adaptive immune responses. A recent study reported that MIF is involved in antidepressant-induced hippocampal neurogenesis, but the mechanism remains elusive. In our data, tryptophan hydroxylase 2 (Tph2) and brain-derived neurotrophic factor (Bdnf) expression were induced after MIF treatment in vitro, as well as during both exercise and electroconvulsive seizure in vivo. This increment of Tph2 was accompanied by increases in the levels of total serotonin in vitro. Moreover, the MIF receptor CD74 and the ERK1/2 pathway mediate the MIF-induced Tph2 and Bdnf gene expression as well as serotonin content. Experiments in Mif(-/-) mice revealed depression-like behaviors and a blunted antidepressant effect of exercise, as reflected by changes in Tph2 and Bdnf expression in the forced swim test. In addition, administration of recombinant MIF protein produced antidepressant-like behavior in rats in the forced swim test. Taken together, these results suggest a role of MIF in mediating the antidepressant action of exercise, probably by enhancing serotonin neurotransmission and neurotrophic factor-induced neurogenesis in the brain.

The effect of clozapine on the AMPK-ACC-CPT1 pathway in the rat frontal cortex.

Clozapine is an antipsychotic drug that has a greater efficacy than other medications in some contexts, especially for the treatment of treatment-resistant schizophrenia. However, clozapine induces more metabolic side-effects involving abnormality in lipid metabolism compared to other antipsychotics. AMP-activated protein kinase (AMPK) plays a central role in controlling lipid metabolism through modulating the downstream acetyl CoA carboxylase (ACC) and carnitine palmitoyl transferase 1 (CPT1) pathway. In this study, we investigated the effect of a single intraperitoneal injection of clozapine on the AMPK-ACC-CPT1 pathway in the rat frontal cortex, which has been implicated as a target site for this antipsychotic drug. At 2 h after injection, the clinically relevant dose of clozapine had activated AMPK, with increased phosphorylation of AMPKα at Thr(172), and had inactivated ACC, with increased phosphorylation of ACC at Ser(79). In addition, clozapine activated the brain-specific isoform of CPT1, CPT1c, whose activity is inhibited by unphosphorylated ACC, in the rat frontal cortex. Immunohistochemistry and immunofluorescence analysis showed that clozapine induced an increase in number of p-AMPKα (Thr(172))- and p-ACC (Ser(79))-positive cells among the neurons of the rat frontal cortex. Taken together, these results show that clozapine activated the AMPK-ACC-CPT1 pathway in the neurons of the rat frontal cortex. These findings indicate that the antipsychotic agent clozapine affects the lipid regulatory system of neurons in the brain.

The effect of cyclosporine A on the phosphorylation of the AMPK pathway in the rat hippocampus.

Cyclosporine A (CsA), an immunosuppressant and calcineurin inhibitor, induces hyperlipidemia in humans and animals. AMP-activated protein kinase (AMPK) is involved in metabolic homeostasis and lipid metabolism through modulating downstream molecules acetyl CoA carboxylase (ACC) and 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoAR). AMPK activity is regulated by the phosphorylation at the Thr-172 residue by its upstream liver kinase B 1 (LKB1), Ca(2+)/calmodulin-dependent protein kinase kinase ß (CaMKKß) or transforming growth-factor-ß-activated kinase 1 (TAK1). AMPK can be deactivated through dephosphorylation by protein phosphatase 2Cα (PP2Cα). In this study, we demonstrated that phosphorylation at Thr-172-AMPK increased with a concurrent increase in the phosphorylation of Ser-431-LKB1 and Thr-184/187-TAK1 in the rat hippocampus at 5 h after an intraperitoneal CsA (50 mg/kg) injection. CsA did not affect the phosphorylation of Thr-196-Ca(2+)/calmodulin-dependent protein kinase 4 (CaMK4) and the amount of PP2Cα. An increased phosphorylation of Ser-79-ACC and Ser-872-HMG-CoAR was also observed. In conclusion, our data indicate that CsA activates the AMPK pathway in the rat hippocampus, which suggests that CsA affects the regulatory signaling pathway of lipid metabolism in the rat brain.

Intracerebroventricular administration of ouabain, a Na/K-ATPase inhibitor, activates tyrosine hydroxylase through extracellular signal-regulated kinase in rat striatum.

Alteration in dopamine neurotransmission has been reported to be involved in the mania of bipolar disorder. Tyrosine hydroxylase (TH) is the rate-limiting enzyme that is crucial for dopamine biosynthesis, and its activity is tightly regulated by phosphorylation at multiple N-terminal serine residues. Previously, we have reported that intracerebroventricular (ICV) injection of ouabain, a selective Na/K-ATPase inhibitor, induces hyperactivity in rats that mimics manic symptoms related to the activation of extracellular signal-regulated protein kinase1/2 (ERK1/2), which plays crucial roles in the modulation of TH phosphorylation. In this study, we investigated the effects of ICV injection of ouabain on TH phosphorylation in rat striatum and the involvement of ERK1/2 in ouabain-induced TH activation. ICV ouabain induced an acute dose-dependent increase in locomotor activity and in TH phosphorylation in rat striatum. TH phosphorylation at Ser19 was significantly increased with 100, 500, and 1000µM ouabain, and phosphorylation at Ser31 and Ser40 was significantly increased with 500 and 1000µM. We also found that ICV pretreatment with U0126, a specific MEK1/2 inhibitor, attenuated the 1000µM ouabain-induced increase in TH phosphorylation at Ser19, Ser31, and Ser40, as well as the hyperactivity of rats. Moreover, the increased phosphorylation of TH (Ser19, Ser31, and Ser40) was maintained until 8h after single administration ouabain was accompanied by increased phosphorylation of ERK1/2 (Thr202/Tyr204) and p90RSK (Thr359/Ser363). These findings imply that TH activation of the ERK1/2 signal pathway could play an important role in ouabain-induced hyperactivity of rats, a mania model.

The effect of systemic injection of cyclosporin A on the phosphorylation of the PKC substrates MARCKS and GAP43 in the rat hippocampus.

Cyclosporin A (CsA) is an inhibitor of calcineurin, a calcium/calmodulin dependent serine/threonine phosphatase. Protein kinase C (PKC) is a family of serine/threonine kinases. Both calcineurin and PKC are implicated in psychiatric diseases and the therapeutic mechanisms of treatment agents. It has been reported that calcineurin interacts with components of PKC signaling pathways. We administrated 50mg/kg CsA into rats by intraperitoneal injection and examined the acute effect of single systemic CsA on the locomotor activity of rats and the phosphorylation of PKC and its substrates GAP43 and MARCKS. Systemic CsA increased locomotor activity beginning 1h after injection. The immunoreactivity of p-MARCKS(S152/156) was higher in the CsA group 1h after injection, whereas p-GAP43(S41) immunoreactivity was increased by CsA after 5h. The immunoreactivity of p-PKC pan was increased by CsA at both 1 and 5h after administration. Our data suggest that activation of the PKC pathway might be related to CsA-induced hyperlocomotion.

Repeated electroconvulsive seizure treatment in rats reduces inducibility of early growth response genes and hyperactivity in response to cocaine administration.

Regulated expression of immediate early genes (IEGs) in the brain reflects neuronal activity in response to various stimuli and recruits specific gene programs involved in long-term neuronal modification and behavioral alterations. Repeated electroconvulsive seizure (ECS) treatment reduces the expression level of several IEGs, such as c-fos, which play important roles in psychostimulant-induced behavioral changes. In this study, we investigated the effects of repeated ECS treatment on the basal expression level of IEGs and its effects on cocaine-induced activation of IEGs and locomotor activity in rats. Repeated ECS treatment for 10days (E10×) reduced Egr1, Egr2, Egr3, and c-fos mRNA and protein levels in the rat frontal cortex at 24h after the last ECS treatment, and these changes were evident in the neuronal cells of the prefrontal cortex. In particular, downregulation of Egr1 and c-fos was evident until 5days after the last ECS treatment. Moreover, E10× pretreatment attenuated the cocaine-induced increase in Egr1, Egr2, and c-fos expression in the rat frontal cortex, whereas phosphorylation of ERK1/2, one of the representative upstream activators of these genes, increased significantly following cocaine treatment. Additionally, E10× pretreatment attenuated the increase in locomotor activity in response to a cocaine injection. In conclusion, repeated ECS treatment reduced the expression and inducibility of Egrs and c-fos, which could attenuate the response of the brain to psychostimulants.

Activation of Akt signaling in rat brain by intracerebroventricular injection of ouabain: a rat model for mania.

Intracerebroventricular (ICV) injection of ouabain, a specific Na-K ATPase inhibitor, induces behavioral changes in rats resembling the manic phenotypes of bipolar disorder. The binding of ouabain to the Na-K ATPase affects signal events in vitro including Akt, a possible molecular target of mood disorders. However, the effects of ouabain on Akt in the brain need further clarification. In this study, we investigated changes in the phosphorylation state of Akt in the rat brain after ICV injection of ouabain. Consistent with our previous report, the locomotor activity of rats within 30 min after ouabain ICV injection changed according to the dose with higher doses of ouabain, 0.5 and 1 mM, inducing significant hyperactivity. In addition, ouabain administration induced a dose-dependent increase in the immunoreactivity of p-Akt (Ser473) in the frontal cortex, striatum, and hippocampus after 30 min, and reached statistical significance with 1mM of ouabain. Phosphorylation of GSK-3beta (Ser9), FOXO1 (Ser256), and eNOS (Ser1177), which are downstream molecules of Akt, was also increased in a dose-dependent manner within the same brain regions. Moreover, hyperactivity was seen for 8h after a single 1mM injection of ouabain and increased phosphorylation of Akt (Ser473), GSK-3beta (Ser9), FOXO1 (Ser256), and eNOS (Ser1177) was also observed in the cortex, striatum, and hippocampus. Thus, intrabrain injection of ouabain induces activation of Akt signaling accompanied by hyperactivity, suggesting the possible role of Akt in ouabain rat model of mania.

Effects of neonatal MK-801 treatment on p70S6K-S6/eIF4B signal pathways and protein translation in the frontal cortex of the developing rat brain.

Systemic injections of MK-801, a selective NMDAR antagonist, into neonatal rats induces long-term neurochemical and behavioural changes. It has been suggested that these changes form the neurodevelopmental basis for schizophrenia-like behaviour in rats. In this study, 7-d-old rats were treated with MK-801, and their frontal cortices were examined to investigate the effects on p70S6K-S6 signal pathway and on protein translation, which play crucial roles in the neurodevelopmental process. MK-801, in doses of 0.5 and 1.0 mg/kg, induced a decrease in phosphorylation of p70S6K and its substrates, S6 and eIF4B, in the first 8 h, and no change at 24 and 48 h. These effects were more prominent after two injections of MK-801 than one. Decreased S6 phosphorylation by MK-801 was evident in the prefrontal, cingulate, and insular cortex. In two representative upstream p70S6K-S6 pathways related to ERK1/2 and Akt, changes in ERK1/2-p90RSK phosphorylation were accompanied by changes of p70S6K-S6. Although two MK-801 injections induced a dose-dependent decrease in phosphorylation of Akt and mTOR at 4 and 8 h, a single injection did not produce a significant effect. Protein synthesis rate, measured by [3H]leucine incorporation in frontal cortical tissue, was reduced until 24 h after two MK-801 (1.0 mg/kg) injections. In summary, this study found that neonatal MK-801 treatment induced dysregulation in the p70S6K-S6/eIF4B pathway and protein translation in the frontal cortex of the developing rat brain, which may suggest an important role of protein translation machinery in the MK-801 neurodevelopmental animal model of schizophrenia.

Electroconvulsive seizure increases phosphorylation of PKC substrates, including GAP-43, MARCKS, and neurogranin, in rat brain.

Protein kinase C (PKC) has been suggested as a molecular target related to the pathogenetic and therapeutic mechanisms of mood disorders in which electroconvulsive seizure (ECS) is effective. However, the reports concerning the effects of ECS on PKC are anecdotal and need further clarification. In this study, we examined the effects of ECS treatment on the phosphorylation of PKC substrates, including GAP-43, MARCKS, and neurogranin. Immunoblot using anti-p-PKC substrate antibodies revealed that a single ECS treatment induced temporal changes in the phosphorylation level of PKC substrates in rat brain, reflecting the effects on PKC activity. Phosphorylation of GAP-43 and MARCKS, representative PKC substrates related to synaptic remodeling, increased from 5 to 30 min, after a transient decrease at 0 min immediately after ECS, and returned to basal levels at 60 min in rat frontal cortex, hippocampus, and cerebellum. Phosphorylation of neurogranin, another PKC substrate, showed a similar pattern of temporal changes in the frontal cortex and hippocampus. Immunohistochemical analysis revealed that p-GAP-43 and p-MARCKS were densely stained throughout the neuronal cells of the prefrontal cortex and hippocampus, and the Purkinje cells of cerebellum, after ECS treatment. Brief and transient activation of PKC may be translated into long-term biochemical changes, resulting in synaptic plasticity. Taken together, the acute effects of ECS on PKC activity, which could be an underpinning of long-term biochemical changes induced by ECS, may contribute to understand the molecular mechanism of ECS.

Dose-dependent effect of intracerebroventricular injection of ouabain on the phosphorylation of the MEK1/2-ERK1/2-p90RSK pathway in the rat brain related to locomotor activity.

Intracerebroventricular (ICV) injection of ouabain, a specific Na-K ATPase inhibitor, induced behavioral changes in rats, a putative animal model for bipolar disorder. The binding of ouabain to Na-K ATPase is known to affect signaling molecules in vitro such as extracellular signal-regulated kinase1/2 (ERK1/2). Although ERK has been suggested to be related to the behavioral alterations induced by various psychotomimetics, the effect of ouabain on ERK in the brain related to behavioral changes has not been examined. After ICV injection of ouabain in rats, we investigated changes in the phosphorylation of mitogen-activated protein kinase kinase1/2 (MEK1/2), ERK1/2, and p90 ribosomal s6 kinase (p90RSK) in rat striatum, frontal cortex, and hippocampus along with changes in locomotor activity. Ouabain induced the following biphasic dose-dependent changes in locomotor activity: no change with 10(-6) M, a statistically significant decrease with 10(-5) M, no change with 10(-4) M, and a statistically significant increase with 0.5x10(-3) and 10(-3) M. The phosphorylation level of MEK1/2, ERK1/2, and p90RSK in rat striatum showed dose-dependent changes similar to those observed in locomotor activity with relatively high correlation. The phosphorylation of these molecules in rat frontal cortex and hippocampus also changed in a similar dose-dependent pattern. Taken together, ouabain induced biphasic dose-dependent changes in locomotor activity and the phosphorylation of the ERK1/2 pathway. These findings suggest a possible relationship between ouabain-induced behavioral changes and ERK activity in the brain and suggest an important role of ERK in regulating locomotor activity and mood state.

Haloperidol regulates the phosphorylation level of the MEK-ERK-p90RSK signal pathway via protein phosphatase 2A in the rat frontal cortex.

Haloperidol, a classical antipsychotic drug, affects the extracellular signal-regulated kinase (ERK) pathway in the brain. However, findings are inconsistent and the mechanism by which haloperidol regulates ERK is poorly understood. Therefore, we examined the ERK pathway and the related protein phosphatase 2A (PP2A) in detail after haloperidol administration. Haloperidol (0.5 and 1 mg/kg) induced biphasic changes in the phosphorylation level of mitogen-activated protein kinase kinase (MEK), ERK, and p90 ribosomal S6 kinase (p90RSK) without changing Raf-1 phosphorylation. Fifteen minutes after haloperidol administration, MEK-ERK-p90RSK phosphorylation increased, whilst PP2A activity decreased. At 60 min, the reverse was observed and the binding of PP2A to MEK and ERK increased. Higher dosages of haloperidol (2 and 4 mg/kg), affected neither MEK-ERK-p90RSK phosphorylation nor PP2A activity. Accordingly, PP2A regulates acute dose- and time-dependent changes in MEK-ERK-p90RSK phosphorylation after haloperidol treatment. These findings suggest the involvement of a dephosphorylating mechanism in the acute action of haloperidol.