Extinction of auditory fear conditioning requires MAPK/ERK activation in the basolateral amygdala

Whereas the neuronal substrates underlying the acquisition of auditory fear conditioning have been widely studied, the substrates and mechanisms mediating the acquisition of fear extinction remain largely elusive. Previous reports indicate that consolidation of fear extinction depends on the mitogen-activated protein kinase/extracellular-signal regulated kinase (MAPK/ERK) signalling pathway and on protein synthesis in the medial prefrontal cortex (mPFC). Based on experiments using the fear-potentiated startle paradigm suggesting a role for neuronal plasticity in the basolateral amygdala (BLA) during fear extinction, we directly addressed whether MAPK/ERK signalling in the basolateral amygdala is necessary for the acquisition of fear extinction using conditioned freezing as a read-out. First, we investigated the regional and temporal pattern of MAPK/ERK activation in the BLA following extinction learning in C57Bl/6J mice. Our results indicate that acquisition of extinction is associated with an increase of phosphorylated MAPK/ERK in the BLA. Moreover, we found that inhibition of the MAPK/ERK signalling pathway by intrabasolateral amygdala infusion of the MEK inhibitor, U0126, completely blocks acquisition of extinction. Thus, our results indicate that the MAPK/ERK signalling pathway is required for extinction of auditory fear conditioning in the BLA, and support a role for neuronal plasticity in the BLA during the acquisition of fear extinction.     

Active Transition of Fear Memory Phase from Reconsolidation to Extinction through ERK-Mediated Prevention of Reconsolidation

The retrieval of fear memory induces two opposite memory process, i.e., reconsolidation and extinction. Brief retrieval induces reconsolidation to maintain or enhance fear memory, while prolonged retrieval extinguishes this memory. Although the mechanisms of reconsolidation and extinction have been investigated, it remains unknown how fear memory phases are switched from reconsolidation to extinction during memory retrieval. Here, we show that an extracellular signal-regulated kinase (ERK)-dependent memory transition process after retrieval regulates the switch of memory phases from reconsolidation to extinction by preventing induction of reconsolidation in an inhibitory avoidance (IA) task in male mice. First, the transition memory phase, which cancels the induction of reconsolidation, but is insufficient for the acquisition of extinction, was identified after reconsolidation, but before extinction phases. Second, the reconsolidation, transition, and extinction phases after memory retrieval showed distinct molecular and cellular signatures through cAMP responsive element binding protein (CREB) and ERK phosphorylation in the amygdala, hippocampus, and medial prefrontal cortex (mPFC). The reconsolidation phase showed increased CREB phosphorylation, while the extinction phase displayed several neural populations with various combinations of CREB and/or ERK phosphorylation, in these brain regions. Interestingly, the three memory phases, including the transition phase, showed transient ERK activation immediately after retrieval. Most importantly, the blockade of ERK in the amygdala, hippocampus, or mPFC at the transition memory phase disinhibited reconsolidation-induced enhancement of IA memory. These observations suggest that the ERK-signaling pathway actively regulates the transition of memory phase from reconsolidation to extinction and this process functions as a switch that cancels reconsolidation of fear memory.SIGNIFICANCE STATEMENT Retrieval of fear memory induces two opposite memory process; reconsolidation and extinction. Reconsolidation maintains/enhances fear memory, while extinction weakens fear memory. It remains unknown how memory phases are switched from reconsolidation to extinction during retrieval. Here, we identified an active memory transition process functioning as a switch that inhibits reconsolidation. This memory transition phase showed a transient increase of extracellular signal-regulated kinase (ERK) phosphorylation in the amygdala, hippocampus and medial prefrontal cortex (mPFC). Interestingly, inhibition of ERK in these regions at the transition phase disinhibited the reconsolidation-mediated enhancement of inhibitory avoidance (IA) memory. These findings suggest that the transition memory process actively regulates the switch of fear memory phases of fear memory by preventing induction of reconsolidation through the activation of the ERK-signaling pathway.     

Prefrontal infusion of PD098059 immediately after fear extinction training blocks extinction-associated prefrontal synaptic plasticity and decreases prefrontal ERK2 phosphorylation

A previous study has demonstrated that disruption of fear extinction-induced long-term potentiation (LTP) in the medial prefrontal cortex (mPFC) is associated with the return of fear responding. Given that immediate posttraining infusion of PD098059, an inhibitor of extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) cascade, into the mPFC also promotes recovery of fear, we investigated whether impairment of mPFC ERK/MAPK cascade also interferes with development of extinction-related LTP in the mPFC in rats. In Experiment 1, extinction training consisting of repetitive presentations of a tone previously associated with eyelid-shock application induced LTP-like changes at hippocampal inputs to the mPFC that were evident for approximately 2 h following fear extinction. Infusion of PD098059 into the mPFC immediately after extinction training abolished training-related prefrontal LTP and impaired retention of extinction memory tested on the following day. In Experiment 2, immunoblotting assays revealed that posttraining infusion of PD098059 into the mPFC produced a significant reduction of mPFC ERK2. These data, along with previous findings, suggest that low levels of ERK2 phosphorylation in the mPFC may interfere with mechanisms of retention of extinction training. The involvement of mPFC LTP in fear extinction is discussed.     

Early postnatal stress alters extracellular signal-regulated kinase signaling in the corticolimbic system modulating emotional circuitry in adult rats

The present study elucidated whether early life stress alters the extracellular signal-regulated kinase (ERK) pathway that underlies fear retrieval and fear extinction based on a contextual fear conditioning paradigm, using a juvenile stress model. Levels of phospho-ERK (pERK), the active form of ERK, increased after fear retrieval in the hippocampal CA1 region but not in the medial prefrontal cortex (mPFC). ERK activation in the CA1 following fear retrieval was not observed in adult rats who received aversive footshock (FS) stimuli during the second postnatal period (2wFS), which exhibited low levels of freezing. In fear extinction, pERK levels in the CA1 were increased by repeated extinction trials, but they were not altered after extinction retrieval. In contrast, pERK levels in the mPFC did not change during extinction training, but were enhanced after extinction retrieval. These findings were compatible in part with electrophysiological data showing that synaptic transmission in the CA1 field and mPFC was enhanced during extinction training and extinction retrieval, respectively. ERK activation in the CA1 and mPFC associated with extinction processes did not occur in rats that received FS stimuli during the third postnatal period (3wFS), which exhibited sustained freezing behavior. The repressed ERK signaling and extinction deficit observed in the 3wFS group were ameliorated by treatment with the partial N-methyl-D-aspartate receptor agonist D-cycloserine. These findings suggest that early postnatal stress induced the downregulation of ERK signaling in distinct brain regions through region-specific regulation, which may lead to increased behavioral abnormalities or emotional vulnerabilities in adulthood.     

ERK1/2 activation in reactive astrocytes of mice with pilocarpine-induced status epilepticus

Abstract

Objective: To investigate the activation pattern of extracellular signal-regulated kinase 1/2 (ERK1/2) in the hippocampus of mice during pilocarpine-induced status epilepticus (SE) and its relationship with reactive astrogliosis.

Methods: Status epilepticus (SE) models were established by intraperitoneal injection of pilocarpine. The intervention group received the ERK1/2 signaling pathway inhibitor SL327 before the pilocarpine injection. We evaluated the SE model group, the intervention group and the control saline-treated group, at 6 hours and 3 days after initiation of the seizure. Phosphorylated activated ERK1/2 and glial fibrillary acidic protein (GFAP) were labeled with both single-labeling and sequential single-labeling immunohistochemical techniques.

Results: Among the pilocarpine-treated (SE model) mice, strong immunohistochemical staining of phospho-ERK1/2 was observed in the neurons and astrocytes of the hippocampus at 6 hours after initiation of SE, whereas staining on the third day of SE was not different from the control saline-treated mice. In the SL327-treated mice (intervention group), SL327 effectively blocked the ERK1/2 activation and little gliosis could be detected at 6 hours and 3 days after initiation of SE; the levels of phospho-ERK1/2 remained low, but the level of gliosis was similar to that of SE mice.

Conclusion: The ERK1/2 signaling pathway plays an important role in the early stage of reactive astrogliosis in mice with pilocarpine-induced SE.     

Cotinine enhances the extinction of contextual fear memory and reduces anxiety after fear conditioning

Posttraumatic stress disorder (PTSD) is an anxiety disorder triggered by traumatic events. Symptoms include anxiety, depression and deficits in fear memory extinction (FE). PTSD patients show a higher prevalence of cigarette smoking than the general population. The present study investigated the effects of cotinine, a tobacco-derived compound, over anxiety and contextual fear memory after fear conditioning (FC) in mice, a model for inducing PTSD-like symptoms. Two-month-old C57BL/6J mice were separated into three experimental groups. These groups were used to investigate the effect of pretreatment with cotinine on contextual fear memory and posttreatment on extinction and stability or retrievability of the fear memory. Also, changes induced by cotinine on the expression of extracellular signal-regulated kinase (ERK)1/2 were assessed after extinction in the hippocampus. An increase in anxiety and corticosterone levels were found after fear conditioning. Cotinine did not affect corticosterone levels but enhanced the extinction of contextual fear, decreased anxiety and the stability and/or retrievability of contextual fear memory. Cotinine-treated mice showed higher levels of the active forms of ERK1/2 than vehicle-treated mice after FC. This evidence suggests that cotinine is a potential new pharmacological treatment to reduce symptoms in individuals with PTSD.     

Spontaneous recovery of fear differs among early – late adolescent and adult male mice

Adolescence is a vulnerable period for developing anxiety-related mental disorders such as post-traumatic stress disorder (PTSD), which requires a long-term course of therapy when a traumatic event has been experienced during childhood. However, the biological mechanism underlying these age-dependent characteristics remains unclear. In the present study, we used early adolescent, late adolescent and adult (4-, 8-, and 15-week old) male mice to examine age differences in fear memory, fear extinction, and spontaneous recovery of fear. We also measured the activation of extracellular signal-regulated kinase (ERK) 2 in the dorsal hippocampus (dHip) and the basolateral amygdala (BLA) following a spontaneous recovery test. Our major findings were as follows: (1) early adolescent and adult mice did not recover the fear response; only late adolescent mice recovered the fear response. (2) The ERK2 in the dHip was more activated after the spontaneous recovery test in late adolescent mice than in adult mice, and the ERK2 in the BLA was more activated after the spontaneous recovery test in adult mice than in late adolescent mice. These results suggest that there exists a unique period in which spontaneous recovery occurs and that these late adolescent behavioral signatures may be related to alteration in the ERK2 phosphorylation in the dHip and BLA.     

Ketamine plus imipramine treatment induces antidepressant-like behavior and increases CREB and BDNF protein levels and PKA and PKC phosphorylation in rat brain

A growing body of evidence has pointed to the N-methyl-d-aspartate (NMDA) receptor antagonists as a potential therapeutic target for the treatment of major depression. The present study investigated the possibility of synergistic interactions between antidepressant imipramine with the uncompetitive NMDA receptor antagonist ketamine. Wistar rats were acutely treated with ketamine (5 and 10 mg/kg) and imipramine (10 and 20 mg/kg) and then subjected to forced swimming tests. The cAMP response element bindig (CREB) and brain-derived neurotrophic factor (BDNF) protein levels and protein kinase C (PKC) and protein kinase A (PKA) phosphorylation were assessed in the prefrontal cortex, hippocampus and amygdala by imunoblot. Imipramine at the dose of 10 mg/kg and ketamine at the dose of 5 mg/kg did not have effect on the immobility time; however, the effect of imipramine (10 and 20 mg/kg) was enhanced by both doses of ketamine. Ketamine and imipramine alone or in combination at all doses tested did not modify locomotor activity. Combined treatment with ketamine and imipramine produced stronger increases of CREB and BDNF protein levels in the prefrontal cortex, hippocampus and amygdala, and PKA phosphorylation in the hippocampus and amygdala and PKC phosphorylation in prefrontal cortex. The results described indicate that co-administration of antidepressant imipramine with ketamine may induce a more pronounced antidepressant activity than treatment with each antidepressant alone. This finding may be of particular importance in the case of drug-resistant patients and could suggest a method of obtaining significant antidepressant actions whilst limiting side effects.     

Time-dependent involvement of the dorsal hippocampus in trace fear conditioning in mice

Hippocampal and amygdaloid neuroplasticity are important substrates for Pavlovian fear conditioning. The hippocampus has been implicated in trace fear conditioning. However, a systematic investigation of the significance of the trace interval has not yet been performed. Therefore, this study analyzed the time-dependent involvement of N-methyl-D-aspartate (NMDA) receptors in the dorsal hippocampus in one-trial auditory trace fear conditioning in C57BL/6J mice. The NMDA receptor antagonist APV was injected bilaterally into the dorsal hippocampus 15 min before training. Mice were exposed to tone (conditioned stimulus [CS]) and footshock (unconditioned stimulus [US]) in the conditioning context without delay (0 s) or with CS-US (trace) intervals of 1-45 s. Conditioned auditory fear was determined 24 h after training by the assessment of freezing and computerized evaluation of inactivity in a new context; 2 h later, context-dependent memory was tested in the conditioning context. NMDA receptor blockade by APV markedly impaired conditioned auditory fear at trace intervals of 15 s and 30 s, but not at shorter trace intervals. A 45-s trace interval prevented the formation of conditioned tone-dependent fear. Context-dependent memory was always impaired by APV treatment independent of the trace interval. The results indicate that the dorsal hippocampus and its NMDA receptors play an important role in auditory trace fear conditioning at trace intervals of 15-30-s length. In contrast, NMDA receptors in the dorsal hippocampus are unequivocally involved in contextual fear conditioning independent of the trace interval. The results point at a time-dependent role of the dorsal hippocampus in encoding of noncontingent explicit stimuli. Preprocessing of long CS-US contingencies in the hippocampus appears to be important for the final information processing and execution of fear memories through amygdala circuits.     

Changes in apical dendritic structure correlate with sustained ERK1/2 phosphorylation in medial prefrontal cortex of a rat model of dopamine D1 receptor agonist sensitization

Rats lesioned with 6-hydroxydopamine (6-OHDA) as neonates exhibit behavioral and neurochemical abnormalities in adulthood that mimic Lesch-Nyhan disease, schizophrenia, and other developmental disorders of frontostriatal circuit dysfunction. In these animals a latent sensitivity to D1 agonists is maximally exposed by repeated administration of dopamine agonists in the postpubertal period (D1 priming). In neonate-lesioned, adult rats primed with SKF-38393, we found selective, persistent alterations in the morphology of pyramidal neuron apical dendrites in the prelimbic area of the medial prefrontal cortex (mPFC). In these animals, dendrite bundling patterns and the typically straight trajectories of primary dendritic shafts were disrupted, whereas the diameter of higher-order oblique branches was increased. Although not present in neonate-lesioned rats treated with saline, these morphological changes persisted at least 21 days after repeated dosing with SKF-38393, and were not accompanied by markers of neurodegenerative change. A sustained increase in phospho-ERK immunoreactivity in wavy dendritic shafts over the same period suggested a relationship between prolonged ERK phosphorylation and dendritic remodeling in D1-primed rats. In support of this hypothesis, pretreatment with the MEK1/2-ERK1/2 pathway inhibitors PD98059 or SL327, prior to each priming dose of SKF-38393, prevented the morphological changes associated with D1 priming. Together, these findings demonstrate that repeated stimulation of D1 receptors in adulthood interacts with the developmental loss of dopamine to profoundly and persistently modify neuronal signaling and dendrite morphology in the mature prefrontal cortex. Furthermore, sustained elevation of ERK activity in mPFC pyramidal neurons may play a role in guiding these morphological changes in vivo.     

Role of NMDA receptor in homocysteine-induced activation of Mitogen-Activated Protein Kinase and Phosphatidyl Inositol 3-Kinase pathways in cultured human vascular smooth muscle cells

Introduction

Exposure of vascular smooth muscle cells (VSMC) to homocysteine, at concentrations associated with an increased risk of cardiovascular events, enhances synthesis and secretion of Matrix Metalloproteinase-2 (MMP-2), which is involved in atherosclerotic plaque instabilization. This effect was prevented by inhibitors of Mitogen Activated Protein Kinase (MAPK) and Phosphatidylinositol 3-Kinase (PI3-K) pathways, allowing to hypothesize that homocysteine activates both these pathways, likely via a receptor-mediated mechanism. One possible receptor is N-methyl-D-aspartate receptor (NMDAr), which is expressed in VSMC and is involved in homocysteine effects in other cell types.

Materials and Methods

VSMC exposed to DL-homocysteine or NMDA (100 μmol/L for both; 5 min-8 hours), were investigated by measuring: i) phosphorylation of ERK1/2, p38MAPK (signaling molecules of MAPK pathway) and Akt and p70S6K (signaling molecules of PI3-K pathway) by western blot; ii) synthesis and secretion of MMP-2 (western blot); iii) activation of MMP-2 (gelatin zimography). To evaluate NMDAr involvement in the homocysteine effects, the experiments were repeated in the presence of a non-competitive NMDAr-antagonist MK-801 (50 μmol/L) or L-glycine (10 μmol/L), which inhibits NMDAr function by promoting its internalization.

Results

DL-homocysteine and NMDA time-dependently increased: i) the phosphorylation of ERK1/2, p38 MAPK, Akt and p70S6K (ANOVA, p < 0.0001); ii) the synthesis, secretion and activation of MMP-2. DL-homocysteine and NMDA effects were prevented by VSMC pre-incubation with MK-801 or high L-glycine concentrations.

Conclusions

In human VSMC homocysteine-at concentrations associated with increased cardiovascular risk- activates MAPK and PI3-K pathways and MMP-2 synthesis and secretion through NMDA receptor, a potential mechanism involved in intracellular signaling in response to homocysteine in VSMC.     

NMDA receptor antagonism in the basolateral but not central amygdala blocks the extinction of Pavlovian fear conditioning in rats

Glutamate receptors in the basolateral complex of the amygdala (BLA) are essential for the acquisition, expression and extinction of Pavlovian fear conditioning in rats. Recent work has revealed that glutamate receptors in the central nucleus of the amygdala (CEA) are also involved in the acquisition of conditional fear, but it is not known whether they play a role in fear extinction. Here we examine this issue by infusing glutamate receptor antagonists into the BLA or CEA prior to the extinction of fear to an auditory conditioned stimulus (CS) in rats. Infusion of the α‐amino‐3‐hydroxyl‐5‐methyl‐4‐isoxazole‐propionate (AMPA) receptor antagonist, 2,3‐dihydroxy‐6‐nitro‐7‐sulfamoyl‐benzo[f]quinoxaline‐2,3‐dione (NBQX), into either the CEA or BLA impaired the expression of conditioned freezing to the auditory CS, but did not impair the formation of a long‐term extinction memory to that CS. In contrast, infusion of the N‐methyl‐d ‐aspartate (NMDA) receptor antagonist, d,l ‐2‐amino‐5‐phosphonopentanoic acid (APV), into the amygdala, spared the expression of fear to the CS during extinction training, but impaired the acquisition of a long‐term extinction memory. Importantly, only APV infusions into the BLA impaired extinction memory. These results reveal that AMPA and NMDA receptors within the amygdala make dissociable contributions to the expression and extinction of conditioned fear, respectively. Moreover, they indicate that NMDA receptor‐dependent processes involved in extinction learning are localized to the BLA. Together with previous work, these results reveal that NMDA receptors in the CEA have a selective role acquisition of fear memory.     

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 10 days (E10×) reduced Egr1, Egr2, Egr3, and c-fos mRNA and protein levels in the rat frontal cortex at 24 h 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 5 days 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.     

Lack of hippocalcin causes impairment in Ras/extracellular signal-regulated kinase cascade via a Raf-mediated activation process

Hippocalcin (Hpca) is a member of the neuronal calcium sensor protein family and is highly expressed in hippocampal neurons. Hpca-deficient (Hpca(-/-)) mice display a defect in cAMP response element-binding protein (CREB) activation associated with impaired spatial and associative memory. Here we examine the involvement of Hpca in the extracellular signal-regulated kinase (ERK) cascade leading to CREB activation, because application of PD98059, a broad ERK cascade inhibitor, has resulted in similar levels of CREB activation in Hpca(-/-) hippocampus. N-methyl-D-aspartate (NMDA)- and KCl-induced phosphorylation of ERK was significantly attenuated in Hpca(-/-) hippocampal slices, as was ionomycin-induced phosphorylation of ERK, whereas forskolin and 12-O-tetradecanoyl-phorbol-13-acetate (TPA) stimulation yielded indistinguishable levels of ERK phosphorylation in both wild-type and Hpca(-/-) slices. In an in vitro reconstitution assay system, recombinant Hpca affected neither Raf-1 protein kinase activity with recombinant MEK-1 as a substrate nor MEK-1 kinase activity with ERK2 as a substrate. Activation of Ras by NMDA and KCl stimulation of hippocampal slices showed no obvious changes between the two genotypes; however, phosphorylation of Raf-1 was significantly lower in Hpca(-/-) slices. These results suggest that Hpca plays an important role in the activation of Raf conducted by Ras.     

Interaction of basolateral amygdala,ventral hippocampus and medial prefrontal cortex regulates the consolidation and extinction of social fear

Background

Following a social defeat, the balanced establishment and extinction of aversive information is a beneficial strategy for individual survival. Abnormal establishment or extinction is implicated in the development of mental disorders. This study investigated the time course of the establishment and extinction of aversive information from acute social defeat and the temporal responsiveness of the basolateral amygdala (BLA), ventral hippocampus (vHIP) and medial prefrontal cortex (mPFC) in this process.

Methods

Mouse models of acute social defeat were established by using the resident–intruder paradigm. To evaluate the engram of social defeat, the intruder mice were placed into the novel context at designated time to test the social behavior. Furthermore, responses of BLA, vHIP and mPFC were investigated by analyzing the expression of immediate early genes, such as zif268, arc, and c-fos.

Results

The results showed after an aggressive attack, aversive memory was maintained for approximately 7 days before gradually diminishing. The establishment and maintenance of aversive stimulation were consistently accompanied by BLA activity. By contrast, vHIP and mPFC response was inhibited from this process. Additionally, injecting muscimol (Mus), a GABA receptor agonist, into the BLA alleviated the freezing behavior and social fear and avoidance. Simultaneously, Mus treatment decreased the zif268 and arc expression in BLA, but it increased their expression in vHIP.

Conclusion

Our data support and extend earlier findings that implicate BLA, vHIP and mPFC in social defeat. The time courses of the establishment and extinction of social defeat are particularly consistent with the contrasting BLA and vHIP responses involved in this process.

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Extracellular HIV-1 Tat upregulates TNF-α dependent MCP-1/CCL2 production via activation of ERK1/2 pathway in rat hippocampal slice cultures: inhibition by resveratrol, a polyphenolic phytostilbene

Human immunodeficiency virus-1 (HIV-1) associated dementia (HAD) has been attributed to an encephalitis resulting from intense infiltration of monocytes. Evidence suggests that the viral protein Tat, which is released actively from HIV-1 infected cells, can contribute significantly to this process. Therefore, the principal objective of this study was to evaluate the potential molecular basis for the role of extracellular HIV-1 Tat in the induction of monocyte chemotactic protein-1 (MCP-1/CCL2) in the hippocampus, which is primarily linked to cognitive function and most commonly damaged in HAD. We also attempted to identify the mechanism by which resveratrol (trans-3,5,4′-trihydroxystilbene) modulates MCP-1 release in hippocampal tissues exposed to Tat. An ex vivo study using rat hippocampal slices demonstrated a time- and dose-dependent increase in MCP-1 production from Tat-treated hippocampal tissues. This increase was accompanied by the activation of the MEK/ERK pathway and TNF-α production. Tat-induced MCP-1 release was abrogated by inhibitors of tyrosine kinases (TK), herbimycin A or genistein, a finding that supports the MAPK signaling mechanism. The inhibition of the ERK1/2 pathway with SL327 induced a near-complete abolition of the observed Tat-induced effects. Furthermore, anti-TNF-α antibodies suppressed Tat-induced MCP-1 release. Resveratrol, to a level similar to that of SL327, downregulated Tat-induced proinflammatory responses via the inactivation of ERK1/2. These results indicate that the activation of the ERK1/2 pathway and TK are critical factors in the production of TNF-α and MCP-1 in the Tat-exposed hippocampus. Additionally, the inhibition of Tat-induced production of MCP-1 and TNF-α via the inactivation of the ERK1/2 pathway may represent the anti-inflammatory mechanism of resveratrol in the hippocampus.     

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), Ca²⁺/calmodulin-dependent protein kinase kinase β (CaMKKβ) or transforming growth-factor-β-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-Ca²⁺/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.     

Enhancement of acoustic prepulse inhibition by contextual fear conditioning in mice is maintained even after contextual fear extinction

Prepulse inhibition (PPI) of the acoustic startle response is one of the few and major paradigms for investigating sensorimotor gating systems in humans and rodents in a similar fashion. PPI deficits are observed not only in patients with schizophrenia, but also in patients with anxiety disorders. Previous studies have shown that PPI in rats can be enhanced by auditory fear conditioning. In this study, we evaluated the effects of contextual fear conditioning (FC) for six times a day and fear extinction (FE) for seven days on PPI in mice. C57BL/6J mice (male, 8–12 weeks) were divided into three groups; no-FC (control), FC and FC + FE. We measured PPI at the following three time points, (1) baseline before FC, (2) after FC, and (3) after FE. The results showed that PPI was increased after FC. Moreover, the enhanced PPI following FC was observed even after FE with decreased freezing behaviors. These results suggested contextual fear conditioning could enhance acoustic PPI, and that contextual fear extinction could decrease freezing behaviors, but not acoustic PPI.     

Differential roles of basolateral and central amygdala on the effects of uncontrollable stress on hippocampal synaptic plasticity

The amygdala is considered central in mediating stress-related changes of hippocampal functions. However, it remains unclear whether different amygdala subnuclei have different roles in coordinating stress effects. Here, we report that stress exposure caused an immediate increase of extracellular signal-regulated kinase (ERK)1/2 phosphorylation in the hippocampal area CA1 and the basolateral amygdala (BLA) and after a delay in the central amygdala (CEA). Exposure to the novel environment following stress increased ERK1/2 phosphorylation in the CEA, but reversed the stress-induced increase of ERK1/2 phosphorylation in the hippocampal area CA1 and the BLA. Either ERK1/2 inhibitor U0126 or N-methyl-D-aspartate (NMDA) receptor antagonist DL-(-)-2-amino-5-phosphonopentanoic acid (APV) administration into the BLA, but not the CEA, blocked the stress effects on hippocampal long-term potentiation (LTP) and long-term depression. Novelty-exploration-induced reversal of stress effects was prevented when animals were injected U0126 or APV into the CEA, but not the BLA, before subjected to the novel environment. The ability of novelty exploration to reverse the stress effects was mimicked by intra-CEA infusion of NMDA. These findings suggest that BLA ERK1/2 signaling pathway is critical to mediate the stress effects on hippocampal synaptic plasticity; the activation of CEA ERK1/2, in contrast, appears to mediate the reversal of stress effects.     

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 1 mM of ouabain. Phosphorylation of GSK-3β (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 8 h after a single 1 mM injection of ouabain and increased phosphorylation of Akt (Ser473), GSK-3β (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.