The ERK phosphorylation levels in the amygdala predict anxiety symptoms in humans and MEK/ERK inhibition dissociates innate and learned defensive behaviors in rats.

We demonstrate that the rate of extracellular signal-related kinase phosphorylation (P-ERK1,2/Total-ERK1,2) in the amygdala is negatively and independently associated with anxiety symptoms in 23 consecutive patients with drug-resistant mesial temporal lobe epilepsy that was surgically treated. In naive Wistar rats, the P-ERK1,2/Total-ERK1,2 ratio in the amygdala correlates negatively with innate anxiety-related behavior on the elevated plus maze (n = 20) but positively with expression of defensive-learned behavior (i.e., freezing) on Pavlovian aversive (fear) conditioning (n = 29). The microinfusion of ERK1/2 inhibitor (FR180204, n = 8-13/group) or MEK inhibitor (U0126, n = 8-9/group) into the basolateral amygdala did not affect anxiety-related behavior but impaired the evocation (anticipation) of conditioned-defensive behavior (n = 9-11/group). In conclusion, the P-ERK1,2/Total-ERK1,2 ratio in the amygdala predicts anxiety in humans and the innate anxiety- and conditioned freezing behaviors in rats. However, the ERK1/2 in the basolateral AMY is only required for the expression of defensive-learned behavior. These results support a dissociate ERK-dependent mechanism in the amygdala between innate anxiety-like responses and the anticipation of learned-defensive behavior. These findings have implications for understanding highly prevalent psychiatric disorders related to the defensive circuit manifested by anxiety and fear. HIGHLIGHTS: The P-ERK1,2/Total-ERK1,2 ratio in the amygdala (AMY) correlates negatively with anxiety symptoms in patients with mesial temporal lobe epilepsy. The P-ERK1,2/Total-ERK1,2 in the amygdala correlates negatively with the anxiety-like behavior and positively with freezing-learned behavior in naive rats. ERK1,2 in the basolateral amygdala is required for learned-defensive but not for the anxiety-like behavior expression in rats.

Amygdala levels of the GluA1 subunit of glutamate receptors and its phosphorylation state at serine 845 in the anterior hippocampus are biomarkers of ictal fear but not anxiety.

Fear is a conscious state caused by exposure to real or imagined threats that trigger stress responses that affect the body and brain, particularly limbic structures. A sub-group of patients with mesial temporal lobe epilepsy related to hippocampus sclerosis (MTLE-HS) have seizures with fear, which is called ictal fear (IF), due to epileptic activity within the brain defensive survival circuit structures. Synaptic transmission efficacy can be bi-directionally modified through potentiation (long-term potentiation (LTP)) or depression (long-term depression (LTD)) as well as the phosphorylation state of Ser831 and Ser845 sites at the GluA1 subunit of the glutamate AMPA receptors, which has been characterized as a critical event for this synaptic plasticity. In this study, GluA1 levels and the phosphorylation at Ser845 and Ser831 in the amygdala (AMY), anterior hippocampus (aHIP) and middle gyrus of temporal neocortex (CX) were determined with western blots and compared between MTLE-HS patients who were showing (n = 06) or not showing (n = 25) IF. Patients with IF had an 11% decrease of AMY levels of the GluA1 subunit (p = 0.05) and a 21.5% decrease of aHIP levels of P-GluA1-Ser845 (p = 0.009) compared to patients not showing IF. The observed associations were not related to imbalances in the distribution of other concomitant types of aura, demographic, clinical or neurosurgical variables. The lower levels of P-GluA1-Ser845 in the aHIP of patients with IF were not related to changes in the levels of the serine/threonine-protein phosphatase PP1-alpha catalytic subunit or protein kinase A activation. Taken together, the GluA1 subunit levels in AMY and P-GluA1-Ser845 levels in the aHIP show an overall accuracy of 89.3% (specificity 95.5% and sensitivity 66.7%) to predict the presence of IF. AMY levels of the GluA1 subunit and aHIP levels of P-GluA1-Ser845 were not associated with the psychiatric diagnosis and symptoms of patients. Taken together with previous findings in MTLE-HS patients with IF who were evaluated by stereotactic implanted depth electrodes, we speculate our findings are consistent with the hypothesis that AMY is not a centre of fear but together with other sub-cortical and cortical structures integrates the defensive circuit that detect and respond to threats. This is the first report to address neuroplasticity features in human limbic structures connected to the defensive survival circuits, which has implications for the comprehension of highly prevalent psychiatric disorders and symptoms.

Sodium selenite protects from 3-nitropropionic acid-induced oxidative stress in cultured primary cortical neurons.

Selenium (Se) is an essential trace element for humans; its intake is needed to allow the proper synthesis of 25 different selenoproteins that are necessary to the normal functioning of several organs, including the brain. Accordingly, decreased Se levels have been associated with neurological disorders. In the present study, we investigated the potential beneficial effects of Se, as sodium selenite, against 3-nitropropionic acid (3-NP)-induced oxidative stress in primary cultures of mouse cortical neurons. 3-NP treatment caused a significant decrease in cellular viability, which was accompanied by decreases in mitochondrial complex II activity and reduced glutathione (GSH) content, as well as increases in reactive oxygen species (ROS) generation and oxidized glutathione (GSSG) levels. Sodium selenite pretreatment (6 days) attenuated 3-NP-induced decrease in cell viability. In addition, sodium selenite pretreatment significantly protected against 3-NP-induced increase in ROS generation and decrease in GSH/GSSG ratio. Of note, sodium selenite pretreatment did not change 3-NP-induced decrease of mitochondrial complex II activity, suggesting that Se modulates secondary events resultant from 3-NP-induced mitochondrial dyshomeostasis. In addition, sodium selenite pretreatment significantly increased glutathione peroxidase (GPx) activity. Our data provide insights into the mechanism of protection by sodium selenite, which is related, at least in part, to GPx induction.

Lipopolysaccharide-Induced Striatal Nitrosative Stress and Impaired Social Recognition Memory Are Not Magnified by Paraquat Coexposure.

Systemic inflammation triggered by lipopolysaccharide (LPS) administration disrupts blood-brain barrier (BBB) homeostasis in animal models. This event leads to increased susceptibility of several encephalic structures to potential neurotoxicants present in the bloodstream. In this study, we investigated the effects of alternate intraperitoneal injections of LPS on BBB permeability, social recognition memory and biochemical parameters in the striatum 24 h and 60 days after treatments. In addition, we investigated whether the exposure to a moderate neurotoxic dose of the herbicide paraquat could potentiate LPS-induced neurotoxicity. LPS administration caused a transient disruption of BBB integrity, evidenced by increased levels of exogenously administered sodium fluorescein in the striatum. Also, LPS exposure caused delayed impairment in social recognition memory (evaluated at day 38 after treatments) and increase in the striatal levels of 3-nitrotyrosine. These events were observed in the absence of significant changes in motor coordination and in the levels of tyrosine hydroxylase (TH) in the striatum and substantia nigra. PQ exposure, which caused a long-lasting decrease of striatal mitochondrial complex I activity, did not modify LPS-induced behavioral and striatal biochemical changes. The results indicate that systemic administration of LPS causes delayed social recognition memory deficit and striatal nitrosative stress in adult mice and that the coexposure to a moderately toxic dose of PQ did not magnify these events. In addition, PQ-induced inhibition of striatal mitochondrial complex I was also not magnified by LPS exposure, indicating the absence of synergic neurotoxic effects of LPS and PQ in this experimental model.

Glutamatergic system and mTOR-signaling pathway participate in the antidepressant-like effect of inosine in the tail suspension test.

Glutamatergic system and mTOR signaling pathway have been proposed to be important targets for pharmacological treatment of major depressive disorder. Previous studies have shown that inosine, an endogenous purine, is able to exert a remarkable antidepressant-like effect in mice. Nevertheless, the role of glutamatergic system and mTOR in this effect was not previously determined. This study was designed to investigate the possible modulation of NMDA receptors (NMDAR), AMPA receptors (AMPAR) and mTOR complex 1 (mTORC1) signaling pathway in the inosine anti-immobility effect in the tail suspension test (TST) in mice. Pre-treatment of mice with NMDA (0.1 pmol/mouse, NMDAR agonist, i.c.v.) and D-serine (30 µg/mouse, NMDAR co-agonist, i.c.v.) prevented inosine (10 mg/kg, i.p.) anti-immobility effect in the TST. In addition, a synergistic antidepressant-like effect was observed when a sub-effective dose of inosine (0.1 mg/kg, i.p.) was combined with sub-effective doses of NMDAR antagonists MK-801 (0.001 mg/kg, p.o.) or ketamine (0.1 mg/kg, i.p.). Conversely, the antidepressant-like effect elicited by inosine was not altered by pre-treatment with AMPAR antagonist, DNQX (2.5 µg/mouse, i.c.v.). The mTORC1 inhibitor rapamycin (0.2 nmol/mouse, i.c.v.) prevented the inosine anti-immobility effect in the TST. Noteworthy, inosine treatment did not change the immunocontent of the synaptic proteins PSD95, GluA1 and synapsin I. Mice locomotor activity assessed by open-field test, was not altered by treatments. Taken together, this study shows a pivotal role of NMDAR inhibition and mTORC1 activation for inosine antidepressant-like effect and extends the knowledge concerning the molecular mechanism and potential of inosine for antidepressant strategies.

Signaling pathways underlying the antidepressant-like effect of inosine in mice.

Inosine is a purine nucleoside formed by the breakdown of adenosine that elicits an antidepressant-like effect in mice through activation of adenosine A1 and A2A receptors. However, the signaling pathways underlying this effect are largely unknown. To address this issue, the present study investigated the influence of extracellular-regulated protein kinase (ERK)1/2, Ca2+/calmoduline-dependent protein kinase (CaMKII), protein kinase A (PKA), phosphoinositide 3-kinase (PI3K)/Akt, and glycogen synthase kinase 3beta (GSK-3ß) modulation in the antiimmobility effect of inosine in the tail suspension test (TST) in mice. In addition, we attempted to verify if inosine treatment was capable of altering the immunocontent and phosphorylation of the transcription factor cyclic adenosine monophosphatate (cAMP) response-binding element protein (CREB) in mouse prefrontal cortex and hippocampus. Intracerebroventricular administration of U0126 (5 µg/mouse, MEK1/2 inhibitor), KN-62 (1 µg/mouse, CaMKII inhibitor), H-89 (1 µg/mouse, PKA inhibitor), and wortmannin (0.1 µg/mouse, PI3K inhibitor) prevented the antiimmobility effect of inosine (10 mg/kg, intraperitoneal (i.p.)) in the TST. Also, administration of a sub-effective dose of inosine (0.1 mg/kg, i.p.) in combination with a sub-effective dose of AR-A014418 (0.001 µg/mouse, GSK-3ß inhibitor) induced a synergic antidepressant-like effect. None of the treatments altered locomotor activity of mice. Moreover, 24 h after a single administration of inosine (10 mg/kg, i.p.), CREB phosphorylation was increased in the hippocampus. Our findings provided new evidence that the antidepressant-like effect of inosine in the TST involves the activation of PKA, PI3K/Akt, ERK1/2, and CaMKII and the inhibition of GSK-3ß. These results contribute to the comprehension of the mechanisms underlying the purinergic system modulation and indicate the intracellular signaling pathways involved in the antidepressant-like effect of inosine in a preclinical test of depression.

Differential Activation of Mitogen-Activated Protein Kinases, ERK 1/2, p38(MAPK) and JNK p54/p46 During Postnatal Development of Rat Hippocampus.

Mitogen-activated protein kinases (MAPKs) are a group of serine-threonine kinases, including p38(MAPK), ERK 1/2 and JNK p54/p46, activated by phosphorylation in response to extracellular stimuli. The early postnatal period is characterized by significant changes in brain structure as well as intracellular signaling. In the hippocampus MAPKs have been involved in the modulation of development and neural plasticity. However, the temporal profile of MAPK activation throughout the early postnatal development is incomplete. An understanding of this profile is important since slight changes in the activity of these enzymes, in response to environmental stress in specific developmental windows, might alter the course of development. The present study was undertaken to investigate the hippocampal differential activation of MAPK during postnatal period. MAPK activation and total content were evaluated by Western blotting of hippocampal tissue obtained from male Wistar rats at postnatal days (P) 1, 4, 7, 10, 14, 21, 30 and 60. The total content and phosphorylation of each MAPK was expressed as mean ± SEM and then calculates as a percentile compared to P1 (set at 100 %). The results showed: (1) phosphorylation peaks of p38(MAPK) at PN4 (p = 0.036) and PN10 to PN60; (2) phosphorylation of ERK1 and ERK2 were increased with age (ERK1 p = 0.0000005 and ERK2 p = 0.003); (3) phosphorylation profile of JNK p54/p46 was not changed during the period analyzed (JNKp56 p = 0.716 and JNKp46 p = 0.192). Therefore, the activity profile of ERK 1/2 and p38(MAPK) during postnatal development of rat hippocampus are differentially regulated. Our results demonstrate that ERK 1/2 and p38(MAPK) are dynamically regulated during postnatal neurodevelopment, suggesting temporal correlation of MAPK activity with critical periods when programmed cell death and synaptogenesis are occurring. This suggests an important role for these MAPKs in postnatal development of rat hippocampus.

Mitochondrial Respiration Chain Enzymatic Activities in the Human Brain: Methodological Implications for Tissue Sampling and Storage.

Mitochondrial respiratory chain complexes enzymatic (MRCCE) activities were successfully evaluated in frozen brain samples. Epilepsy surgery offers an ethical opportunity to study human brain tissue surgically removed to treat drug resistant epilepsies. Epilepsy surgeries are done with hemodynamic and laboratory parameters to maintain physiology, but there are no studies analyzing the association among these parameters and MRCCE activities in the human brain tissue. We determined the intra-operative parameters independently associated with MRCCE activities in middle temporal neocortex (Cx), amygdala (AMY) and head of hippocampus (HIP) samples of patients (n = 23) who underwent temporal lobectomy using multiple linear regressions. MRCCE activities in Cx, AMY and HIP are differentially associated to trans-operative mean arterial blood pressure, O2 saturation, hemoglobin, and anesthesia duration to time of tissue sampling. The time-course between the last seizure occurrence and tissue sampling as well as the sample storage to biochemical assessments were also associated with enzyme activities. Linear regression models including these variables explain 13-17 % of MRCCE activities and show a moderate to strong effect (r = 0.37-0.82). Intraoperative hemodynamic and laboratory parameters as well as the time from last seizure to tissue sampling and storage time are associated with MRCCE activities in human samples from the Cx, AMYG and HIP. Careful control of these parameters is required to minimize confounding biases in studies using human brain samples collected from elective neurosurgery.

Time course evaluation of behavioral impairments in the pilocarpine model of epilepsy.

Epilepsy is a brain function disorder characterized by unpredictable and recurrent seizures. The majority of patients with temporal lobe epilepsy (TLE), which is the most common type of epilepsy, have to live not only with seizures but also with behavioral alterations, including anxiety, psychosis, depression, and impaired cognitive functioning. The pilocarpine model has been recognized as an animal model of TLE. However, there are few studies addressing behavioral alterations in the maturation phase when evaluating the time course of the epileptogenic process after pilocarpine administration. Therefore, the present work was designed to analyze the neurobehavioral impairments of male adult Wistar rats during maturation and chronic phases in the pilocarpine model of epilepsy. Behavioral tests included: open-field tasks, olfactory discrimination, social recognition, elevated plus maze, and the forced swimming test. The main behavioral alterations observed in both maturation and chronic phases of the pilocarpine model were olfactory and short-term social memory deficits and decrease in the immobility time in the forced swimming test. Moreover, increased anxiety-like responses were only observed in the maturation phase. These findings indicate that early behavioral impairments can be observed in the pilocarpine model during the maturation phase, and these behavioral deficits also occur during the acquired epilepsy (chronic phase). Several of the neurobehavioral impairments that are associated with epilepsy in humans were observed in the pilocarpine-treated rats, thus, rendering this animal model a useful tool to study neuroprotective strategies as well as neurobiological and psychopathological mechanisms associated with epileptogenesis.

ConBr, a lectin from Canavalia brasiliensis seeds, modulates signaling pathways and increases BDNF expression probably via a glycosylated target.

In the central nervous system, many receptors, ion channels and neurotransmitter transporters are glycoproteins, where the glycan chains are modulator elements. Lectins are proteins, which recognize and bind carbohydrate complexes. We have previously shown that ConBr, a lectin purified from Canavalia brasiliensis seeds, produced antidepressant-like effect and blocked hippocampal neurotoxicity induced by quinolinic acid and glutamate. Noteworthy, all these effects occurred in a dependence of its carbohydrate recognition domain. Therefore, the present study was undertaken in order to elucidate intracellular signaling pathways regulated by ConBr that may be potentially associated with the antidepressant and neuroprotective effects previously reported to be dependent on carbohydrate interaction. ConBr (10 µg/site) was injected into the ventricle (i.c.v.) of mice, and the hippocampi were removed 0.5, 1, 3, 6, 8, 12, 18, and 24 h after treatment. Our results showed that in the period of 0.5-3 h, ConBr induced activation of the protein kinases Akt, ERK1, and PKA. Furthermore, the phosphorylation of CREB-Ser133 was stimulated by ConBr (1-6 h), while brain-derived neurotrophic factor (BDNF) mRNA was increased at 12 h and BDNF protein at 18-24 h. Our data suggest that an early activation of protein kinases may trigger CREB-dependent BDNF transcription, resulting in a subsequent increase of BDNF protein in response to ConBr. Later, increment of Akt phosphorylation was observed 24 h after ConBr administration, possibly due to BDNF/TrkB-dependent activation of Akt. Our findings indicate that ConBr is a multifunctional molecule capable to activate signaling pathways involved in neuroplasticity and neuroprotection.

Time-dependent modulation of AMPA receptor phosphorylation and mRNA expression of NMDA receptors and glial glutamate transporters in the rat hippocampus and cerebral cortex in a pilocarpine model of epilepsy.

The pilocarpine model in rodents reproduces the main features of mesial temporal lobe epilepsy related to hippocampus sclerosis (MTLE-HS) in humans. It has been demonstrated in this model that the phosphorylation of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor GluR1 subunit is increased 1 h after pilocarpine treatment. Moreover, alterations in the levels of glutamate transporters have been associated with chronic epilepsy in humans. Despite these studies, the profile of these changes has not yet been addressed. We analyzed the protein content and phosphorylation profile of the AMPA receptor GluR1 subunit by western blotting. We also used quantitative real-time polymerase chain reaction to analyze the expression of glial glutamate transporters and the N-methyl-D-aspartate receptor NR1 subunit in the hippocampus (Hip) and cerebral cortex (Ctx) at different time points after pilocarpine-induced status epilepticus (Pilo-SE) in male adult Wistar rats. Biochemical analysis was performed in the Hip and Ctx at 1, 3, 12 h (acute period), 5 days (latent period), and 50 days (chronic period) after Pilo-SE. Key findings include an increase in the phosphorylation of GluR1-Ser(845) in the Ctx and GluR1-Ser(831) in the Hip at different times during the acute period, and a decrease in the total content of the GluR1 subunit in the Ctx in the latent period. There was a down-regulation of the mRNA expression and protein levels of EAAT1 and EAAT2, and a decrease of the NR1 mRNA expression, in the Ctx during the latent period. Notably, during the chronic period, the EAAT2 mRNA expression and protein levels decreased while the NR1 mRNA levels increased in the Hip. Taken together, our findings suggest a time- and structure-dependent imbalance of glutamatergic transmission in response to Pilo-SE, which might be associated with either epileptogenesis or the seizure threshold in MTLE-HS.

Time-dependent modulation of mitogen activated protein kinases and AKT in rat hippocampus and cortex in the pilocarpine model of epilepsy.

The epileptogenesis may involve a variety of signaling events that culminate with synaptic reorganization. Mitogen-activated protein kinases (MAPKs) and AKT may be activated by diverse stimulus including neurotransmitter, oxidative stress, growth factors and cytokines and are involved in synaptic plasticity in the hippocampus and cerebral cortex. The pilocarpine model in rodents reproduces the main features of mesial temporal lobe epilepsy related to hippocampus sclerosis (MTLE-HS) in humans. We analyze the phosphorylation profile of MAPKs (ERK1/2, p38(MAPK), JNK1/2/3) and AKT by western blotting in the hippocampus (Hip) and cortex (Ctx) of male adult wistar rats in different periods, after pilocarpine induced status epilepticus (Pilo-SE) and compared with control animals. Biochemical analysis were done in the Hip and Ctx at 1, 3, 12 h (acute period), 5 days (latent period) and 50 days (chronic period) after Pilo-SE onset. Hence, the main findings include increased phosphorylation of ERK1 and p38(MAPK) in the Hip and Ctx 1 and 12 h after the Pilo-SE onset. The JNK2/3 isoform (54 kDa) phosphorylation was decreased at 3 h after the Pilo-SE onset and in the chronic period in the Hip and Ctx. The AKT phosphorylation increased only in the Hip during the latent period. Our study demonstrates, in a systematic manner, the profile of MAPKs and AKT modulation in the hippocampus and cerebral cortex in response to pilocarpine. Based in the role of each signaling enzyme is possible that these changes may be related, at least partially, to modifications in the intrinsic neuronal physiology and epileptogenic synaptic network that appears in the MTLE-HS.

AMPAr GluA1 Phosphorylation at Serine 845 in Limbic System Is Associated with Cardiac Autonomic Tone.

The central autonomic network, which is connected to the limbic system structures including the amygdala (AMY) and anterior hippocampus (aHIP), regulates the sympathetic and parasympathetic modulation of visceromotor, neuroendocrine, pain, and behavior manifestations during stress responses. Heart rate variability (HRV) is useful to estimate the cardiac autonomic tone. The levels of phosphorylation on the Ser831 and Ser845 sites of the GluA1 subunit of the AMPAr (P-GluA1-Ser845 and P-GluA1-Ser831) are useful markers of synaptic plasticity. The relation between synaptic plasticity in the human limbic system structures and autonomic regulation in humans is unknown. This study investigated the association between HRV and neurochemistry biomarkers of synaptic plasticity in AMY and aHIP. HRV indices were obtained from the resting state electrocardiogram of patients with drug-resistant mesial temporal lobe epilepsy (MTLE, n = 18) and the levels of P-GluA1-Ser845 and P-GluA1-Ser831 in the AMY and aHIP resected during the epilepsy surgery. A backward stepwise multiple linear regression models were used to analyze the association between HRV and synaptic plasticity biomarkers controlling for imbalances in the distribution of sociodemographic, clinical, neuroimaging, and neurosurgical variables. P-GluA1-Ser845 levels in AMY show a negative association (p < 0.05) with the 3 investigated parasympathetic autonomic HRV indices (SDNN, rMSSD, and HF) predicting 24 to 40% of their variation. The final multiple linear regression models include disease duration and levels of P-GluA1-Ser845 and predict 24 to 56% of cardiac autonomic tone variation (p < 0.01). P-GluA1-Ser845 levels in AMY and aHIP are negatively associated with the resting HRV in MTLE-HS indicating that increased synaptic efficiency in amygdala is associated with a parasympathetic cardiac autonomic tone impairment. The results suggest that specific changes in synaptic plasticity may be involved in the brain-heart axis regulation by the limbic system.

Glutathione in Chlorpyrifos-and Chlorpyrifos-Oxon-Induced Toxicity: a Comparative Study Focused on Non-cholinergic Toxicity in HT22 Cells.

Chlorpyrifos (CPF) is a neurotoxic organophosphorus (OP) insecticide widely used for agricultural purposes. CPF-mediated neurotoxicity is mainly associated with its anticholinesterase activity, which may lead to a cholinergic syndrome. CPF metabolism generates chlorpyrifos-oxon (CPF-O), which possesses higher anticholinesterase activity and, consequently, plays a major role in the cholinergic syndrome observed after CPF poisoning. Recent lines of evidence have also reported non-cholinergic endpoints of CPF- and CPF-O-induced neurotoxicities, but comparisons on the non-cholinergic toxic properties of CPF and CPF-O are lacking. In this study, we compared the non-cholinergic toxicities displayed by CPF and CPF-O in cultured neuronal cells, with a particular emphasis on their pro-oxidant properties. Using immortalized cells derived from mouse hippocampus (HT22 line, which does present detectable acetylcholinesterase activity), we observed that CPF-O was 5-fold more potent in decreasing cell viability compared with CPF. Atropine, a muscarinic acetylcholine receptor antagonist, protected against acetylcholine (ACh)-induced toxicity but failed to prevent the CPF- and CPF-O-induced cytotoxicities in HT22 cells. CPF or CPF-O exposures significantly decreased the levels of the antioxidant glutathione (GSH); this event preceded the significant decrease in cell viability. Pretreatment with N-acetylcysteine (NAC, a GSH precursor) protected against the cytotoxicity induced by both CPF and CPF-O. The present study indicates that GSH depletion is a non-cholinergic event involved in CPF and CPF-O toxicities. The study also shows that in addition of being a more potent AChE inhibitor, CPF-O is also a more potent pro-oxidant molecule when compared with CPF, highlighting the role of CPF metabolism (bioactivation to CPF-O) in the ensuing non-cholinergic toxicity.

New Probucol Analogues Inhibit Ferroptosis, Improve Mitochondrial Parameters, and Induce Glutathione Peroxidase in HT22 Cells.

Probucol, a hypocholesterolemic compound, is neuroprotective in several models of neurodegenerative diseases but has serious adverse effects in vivo. We now describe the design and synthesis of two new probucol analogues that protect against glutamate-induced oxidative cell death, also known as ferroptosis, in cultured mouse hippocampal (HT22) cells and in primary cortical neurons, while probucol did not show any protective effect. Treatment with both compounds did not affect glutathione depletion but still significantly decreased glutamate-induced production of oxidants, mitochondrial superoxide generation, and mitochondrial hyperpolarization in HT22 cells. Both compounds increase glutathione peroxidase (GPx) 1 levels and GPx activity, also exhibiting protection against RSL3, a GPx4 inactivator. These two compounds are therefore potent activators of GPx activity making further studies of their neuroprotective activity in vivo worthwhile.

Effects of perinatal exposure to n-3 polyunsaturated fatty acids and methylmercury on cerebellar and behavioral parameters in mice.

Fish and shellfish, which represent important sources of nutrients (i.e., n-3 fatty acids), can contain significant amounts of methylmercury (MeHg), a neurotoxic compound. We investigated the potential neuroprotective effects of perinatal treatment with dietary n-3 fatty acids against MeHg-induced neurotoxicity. Pregnant mice were divided in 4 groups: (i) Control; (ii) MeHg; (iii) n-3 enriched diet and (iv) n-3 enriched diet + MeHg. The treatments were performed from gestational day 1 to postnatal day 21. Twenty-four hours after treatments, motor-related behavioral tests, as well as the analyses of cerebellar biochemical, histological and immunohistochemical parameters related to neuronal and glial homeostasis, were performed. Maternal exposure to MeHg induced motor coordination impairment and cerebellar MeHg accumulation in the offspring and n-3 fatty acids treatment did not prevent these effects. The immunocontent of proteins related to synaptic homeostasis, glial fibrillary acidic protein immunostaining and morphology were not significantly altered in the pups perinatally exposed to MeHg and/or n-3 diet. The results indicate that perinatal exposure to MeHg causes motor coordination impairment even with no evident changes on the evaluated cerebellar biochemical and histological parameters. The performed exposure protocol was unable to show beneficial effects of n-3 fatty acids supplementation against MeHg-induced motor coordination.

Neuropsychological functioning and brain energetics of drug resistant mesial temporal lobe epilepsy patients.

Interictal hypometabolism is commonly measured by 18-fluoro-deoxyglucose Positron Emission Tomography (FDG-PET) in the temporal lobe of patients with mesial temporal lobe epilepsy (MTLE-HS). Left temporal lobe interictal FDG-PET hypometabolism has been associated with verbal memory impairment, while right temporal lobe FDG-PET hypometabolism is associated with nonverbal memory impairment. The biochemical mechanisms involved in these findings remain unknown. In comparison to healthy controls (n=21), surgically treated patients with MTLE-HS (n=32, left side=17) had significant lower scores in the Rey Auditory Verbal Learning Test (RAVLT retention and delayed), Logical Memory II (LMII), Boston Naming test (BNT), Letter Fluency and Category Fluency. We investigated whether enzymatic activities of the mitochondrial enzymes Complex I (C I), Complex II (C II), Complex IV (C IV) and Succinate Dehydrogenase (SDH) from the resected samples of the middle temporal neocortex (mTCx), amygdala (AMY) and hippocampus (HIP) were associated with performance in the RAVLT, LMII, BNT and fluency tests of our patients. After controlling for the side of hippocampus sclerosis, years of education, disease duration, antiepileptic treatment and seizure outcome after surgery, no independent associations were observed between the cognitive test scores and the analyzed mitochondrial enzymatic activities (p>0.37). Results indicate that memory and language impairment observed in MTLE-HS patients are not strongly associated with the levels of mitochondrial CI, CII, SDH and C IV enzymatic activities in the temporal lobe structures ipsilateral to the HS lesion.

Knockdown of Carboxypeptidase A6 in Zebrafish Larvae Reduces Response to Seizure-Inducing Drugs and Causes Changes in the Level of mRNAs Encoding Signaling Molecules.

Carboxypeptidase A6 (CPA6) is an extracellular matrix metallocarboxypeptidase that modulates peptide and protein function by removal of hydrophobic C-terminal amino acids. Mutations in the human CPA6 gene that reduce enzymatic activity in the extracellular matrix are associated with febrile seizures, temporal lobe epilepsy, and juvenile myoclonic epilepsy. The characterization of these human mutations suggests a dominant mode of inheritance by haploinsufficiency through loss of function mutations, however the total number of humans with pathologic mutations in CPA6 identified to date remains small. To better understand the relationship between CPA6 and seizures we investigated the effects of morpholino knockdown of cpa6 mRNA in zebrafish (Danio rerio) larvae. Knockdown of cpa6 mRNA resulted in resistance to the effect of seizure-inducing drugs pentylenetetrazole and pilocarpine on swimming behaviors. Knockdown of cpa6 mRNA also reduced the levels of mRNAs encoding neuropeptide precursors (bdnf, npy, chga, pcsk1nl, tac1, nts, edn1), a neuropeptide processing enzyme (cpe), transcription factor (c-fos), and molecules implicated in glutamatergic signaling (grin1a and slc1a2b). Treatment of zebrafish embryos with 60 mM pilocarpine for 1 hour led to reductions in levels of many of the same mRNAs when measured 1 day after pilocarpine exposure, except for c-fos which was elevated 1 day after pilocarpine treatment. Pilocarpine treatment, like cpa6 knockdown, led to a reduced sensitivity to pentylenetetrazole when tested 1 day after pilocarpine treatment. Taken together, these results add to mounting evidence that peptidergic systems participate in the biological effects of seizure-inducing drugs, and are the first in vivo demonstration of the molecular and behavioral consequences of cpa6 insufficiency.

Agmatine produces antidepressant-like effects by activating AMPA receptors and mTOR signaling.

The activation of AMPA receptors and mTOR signaling has been reported as mechanisms underlying the antidepressant effects of fast-acting agents, specially the NMDA receptor antagonist ketamine. In the present study, oral administration of agmatine (0.1mg/kg), a neuromodulator that has been reported to modulate NMDA receptors, caused a significant reduction in the immobility time of mice submitted to the tail suspension test (TST), an effect prevented by the administration of DNQX (AMPA receptor antagonist, 2.5µg/site, i.c.v.), BDNF antibody (1µg/site, i.c.v.), K-252a (TrkB receptor antagonist, 1µg/site, i.c.v.), LY294002 (PI3K inhibitor, 10nmol/site, i.c.v.) or rapamycin (selective mTOR inhibitor, 0.2nmol/site, i.c.v.). Moreover, the administration of lithium chloride (non-selective GSK-3ß inhibitor, 10mg/kg, p.o.) or AR-A014418 (selective GSK-3ß inhibitor, 0.01µg/site, i.c.v.) in combination with a sub-effective dose of agmatine (0.0001mg/kg, p.o.) reduced the immobility time in the TST when compared with either drug alone. Furthermore, increased immunocontents of BDNF, PSD-95 and GluA1 were found in the prefrontal cortex of mice just 1h after agmatine administration. These results indicate that the antidepressant-like effect of agmatine in the TST may be dependent on the activation of AMPA and TrkB receptors, PI3K and mTOR signaling as well as inhibition of GSK-3ß, and increase in synaptic proteins. The results contribute to elucidate the complex signaling pathways involved in the antidepressant effect of agmatine and reinforce the pivotal role of these molecular targets for antidepressant responses.

Enhancement of memory consolidation by the histone deacetylase inhibitor sodium butyrate in aged rats.

Here we show that a systemic injection of the histone deacetylase inhibitor (HDACi) sodium butyrate (NaB) immediately after training in a step-down inhibitory avoidance task produced an enhancement of memory consolidation that persisted across consecutive retention tests during 14 days in aged rats, while it did not significantly affect memory in young adults. Control aged and young adult rats showed comparable basal levels of memory retention. Our results suggest that HDACis can display memory-enhancing effects specific for aged animals, even in the absence of age-related memory impairment.