Guanosine Prevents Spatial Memory Impairment and Hippocampal Damage Following Amyloid-ß1-42 Administration in Mice.

Alzheimer's disease (AD) is a progressive neurodegenerative illness responsible for cognitive impairment and dementia. Accumulation of amyloid-beta (Aß) peptides in neurons and synapses causes cell metabolism to unbalance, and the production of reactive oxygen species (ROS), leading to neuronal death and cognitive damage. Guanosine is an endogenous nucleoside recognized as a neuroprotective agent since it prevents glutamate-induced neurotoxicity by a mechanism not yet completely elucidated. In this study, we evaluated behavioral and biochemical effects in the hippocampus caused by the intracerebroventricular (i.c.v.) infusion of Aß1-42 peptide (400 pmol/site) in mice, and the neuroprotective effect of guanosine (8 mg/kg, i.p.). An initial evaluation on the eighth day after Aß1-42 infusion showed no changes in the tail suspension test, although ex vivo analyses in hippocampal slices showed increased ROS production. In the second protocol, on the tenth day following Aß1-42 infusion, no effect was observed in the sucrose splash test, but a reduction in the recognition index in the object location test showed impaired spatial memory. Analysis of hippocampal slices showed no ROS production and mitochondrial membrane potential alteration, but a tendency to increase glutamate release and a significant lactate release, pointing to a metabolic alteration. Those effects were accompanied by decreased cell viability and increased membrane damage. Guanosine treatment prevented behavioral and biochemical alterations evoked by Aß1-42, suggesting a potential role against behavioral and biochemical damage evoked by Aß in the hippocampus.

Neonatal Isoflurane Exposure in Rats Impairs Short-Term Memory, Cell Viability, and Glutamate Uptake in Slices of the Frontal Cerebral Cortex, But Not the Hippocampus, in Adulthood.

Neonatal exposure to general anesthetics has been associated with neurotoxicity and morphologic changes in the developing brain. Isoflurane is a volatile anesthetic widely used in pediatric patients to induce general anesthesia, analgesia, and perioperative sedation. In the present study, we investigated the effects of a single neonatal isoflurane (3% in oxygen, 2 h) exposure in rats at postnatal day (PND) 7, in short-term (24 h - PND8) and long-term (adulthood) protocols. In PND8, ex vivo analysis of hippocampal and frontal cortex slices evaluated cell viability and susceptibility to in vitro glutamate challenge. In adult rats, behavioral parameters related to anxiety-like behavior, short-term memory, and locomotor activity (PND60-62) and ex vivo analysis of cell viability, membrane permeability, glutamate uptake, and susceptibility to in vitro glutamate challenge in hippocampal and cortical slices from PND65. A single isoflurane (3%, 2 h) exposure at PND7 did not acutely alter cell viability in cortical and hippocampal slices of infant rats (PND8) per se and did not alter slice susceptibility to in vitro glutamate challenge. In rat's adulthood, behavioral analysis revealed that the neonatal isoflurane exposure did not alter anxiety-like behavior and locomotor activity (open field and rotarod tests). However, isoflurane exposure impaired short-term memory evaluated in the novel object recognition task. Ex vivo analysis of brain slices showed isoflurane neonatal exposure selectively decreased cell viability and glutamate uptake in cortical slices, but it did not alter hippocampal slice viability or glutamate uptake (PND65). Isoflurane exposure did not alter in vitro glutamate-induced neurotoxicity to slices, and isoflurane exposure caused no significant long-term damage to cell membranes in hippocampal or cortical slices. These findings indicate that a single neonatal isoflurane exposure did not promote acute damage; however, it reduced cortical, but not hippocampal, slice viability and glutamate uptake in the adulthood. Additionally, behavioral analysis showed neonatal isoflurane exposure induces short-term recognition memory impairment, consolidating that neonatal exposure to volatile anesthetics may lead to behavioral impairment in the adulthood, although it may damage brain regions differentially.

Guanosine prevents depressive-like behaviors in rats following bilateral dorsolateral striatum lesion induced by 6-hydroxydopamine.

The dorsolateral striatum (DLS) processes motor and non-motor functions and undergoes extensive dopaminergic degeneration in Parkinson's disease (PD). Beyond the nigrostriatal pathway, dopaminergic degeneration also affects other brain areas including the pre-frontal cortex (PFC) and hippocampus, which have been associated with the appearance of anhedonia and depression at pre-motor phases of PD. Herein, using behavioral and biochemical approaches, we investigated the protective effects of guanosine (GUO) (7.5 mg/kg, i.p.) against emotional impairments and cellular events in cortical, striatal and hippocampal slices of rats submitted to a bilateral infusion of 6-OHDA (10 µg/hemisphere) into the DLS. 6-OHDA-lesioned rats displayed anhedonic- and depressive-like behaviors addressed in the splash and forced swimming tests (at 8 and 21 days after lesion, respectively). In addition, no alterations in motor performance in the open field test and social interaction were observed. Biochemical analyses were performed 22 days after 6-OHDA lesions. 6-OHDA lesion induced hippocampal mitochondrial membrane potential disruption. However, intra-striatal 6-OHDA administration did not alter the ROS levels measured in cortical, striatal and hippocampal slices. GUO treatment attenuated anhedonic- and depressive-like behaviors in 6-OHDA-lesioned rats and protected hippocampal slices against the mitochondrial membrane potential disruption. These results indicate antidepressant-like effects of GUO in a rat model of PD, indicating the potential of GUO for the treatment of depression associated with PD.

Long-Term Neurobehavioral Consequences of a Single Ketamine Neonatal Exposure in Rats: Effects on Cellular Viability and Glutamate Transport in Frontal Cortex and Hippocampus.

The neonatal exposure to general anesthetics has been associated with neuronal apoptosis and dendritic spines morphologic changes in the developing brain. Ketamine, a noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist, is widely used in pediatric patients to induce general anesthesia, analgesia, and perioperative sedation. In the present study, we investigated short- and long-term effects of a single ketamine (20 mg/kg, s.c.) neonatal exposure at postnatal day 7 in rats on the hippocampal and frontal cortical cellular viability. Additionally, putative neurochemical alterations and neurobehavioral impairments were evaluated in the adulthood. Ketamine neonatal administration selectively decreased cellular viability in the hippocampus, but not in the frontal cortex, 24 h after the treatment. Interestingly, a single ketamine neonatal exposure prevented the vulnerability to glutamate-induced neurotoxicity in the frontal cortex of adult rats. No short- or long-term damage to cellular membranes, as an indicative of cell death, was observed in hippocampal or cortical slices. However, ketamine induced a long-term increase in hippocampal glutamate uptake. Regarding behavioral analysis, neonatal ketamine exposure did not alter locomotor activity and anxiety-related parameters evaluated in the open-field test. However, ketamine administration disrupted the hippocampal-dependent object recognition ability of adult rats, while improved the motor coordination addressed on the rotarod. These findings indicate that a single neonatal ketamine exposure induces a short-term reduction in the hippocampal, but not in cortical, cellular viability, and long-term alterations in hippocampal glutamate transport, improvement on motor performance, and short-term recognition memory impairment.

Behavioral and Neurochemical Consequences of Pentylenetetrazol-Induced Kindling in Young and Middle-Aged Rats.

(1) Objectives: Epilepsy disorder is likely to increase with aging, leading to an increased incidence of comorbidities and mortality. In spite of that, there is a lack of information regarding this issue and little knowledge of cognitive and emotional responses in aging subjects following epileptogenesis. We investigated whether and how aging distress epilepsy-related behavioral and biochemical outcomes are associated with cognition and emotion. (2) Methods: Young and middle-aged Wistar rats (3 or 12 months old) were treated with pentylenetetrazol (PTZ, 35 mg/kg) and injected on alternated days for 20 (young rats) and 32 days (middle-aged rats). Kindling was reached after two consecutive stages 4 plus one stage 5 or 6 in Racine scale. Control and kindled rats were evaluated in the elevated plus-maze (EPM) and object-recognition tests and their hippocampus was collected 24 h later for mitogen-activated protein kinases (MAPK) dosage. (3) Results: Middle-aged rats presented a higher resistance to develop kindling, with a decrease in the seizure severity index observed following the 4th and 9th PTZ injections. Middle-aged rats displayed an increased duration of the first myoclonic seizure and an increased latency to the first generalized seizure when compared to younger rats. The induction of kindling did not impair the animals' performance (regardless of age) in the object-recognition task and the EPM test as well as it did not alter the hippocampal levels of MAPKs. (4) Significance: Our findings reveal that, despite age-related differences during epileptogenesis, middle-aged rats evaluated after kindling performed similarly during discriminative learning and emotional tasks in comparison to young animals, with no alteration of hippocampal MAPKs. Additional investigation must be carried out to explore the electrophysiological mechanisms underlying these responses, as well as the long-term effects displayed after kindling.

Atorvastatin and Fluoxetine Prevent Oxidative Stress and Mitochondrial Dysfunction Evoked by Glutamate Toxicity in Hippocampal Slices.

Atorvastatin has been shown to exert a neuroprotective action by counteracting glutamatergic toxicity. Recently, we have shown atorvastatin also exerts an antidepressant-like effect that depends on both glutamatergic and serotonergic systems modulation. Excitotoxicity is involved in several brain disorders including depression; thus, it is suggested that antidepressants may target glutamatergic system as a final common pathway. In this study, a comparison of the mechanisms involved in the putative neuroprotective effect of a repetitive atorvastatin or fluoxetine treatment against glutamate toxicity in hippocampal slices was performed. Adult Swiss mice were treated with atorvastatin (10 mg/kg, p.o.) or fluoxetine (10 mg/kg, p.o.), once a day during seven consecutive days. On the eighth day, animals were killed and hippocampal slices were obtained and subjected to an in vitro protocol of glutamate toxicity. An acute treatment of atorvastatin or fluoxetine was not neuroprotective; however, the repeated atorvastatin or fluoxetine treatment prevented the decrease in cellular viability induced by glutamate in hippocampal slices. The loss of cellular viability induced by glutamate was accompanied by increased D-aspartate release, increased reactive oxygen species (ROS) and nitric oxide (NO) production, and impaired mitochondrial membrane potential. Atorvastatin or fluoxetine repeated treatment also presented an antidepressant-like effect in the tail suspension test. Atorvastatin or fluoxetine treatment was effective in protecting mice hippocampal slices from glutamate toxicity by preventing the oxidative stress and mitochondrial dysfunction.

Involvement of PI3K/Akt/GSK-3ß and mTOR in the antidepressant-like effect of atorvastatin in mice.

Atorvastatin is a cholesterol-lowering statin that has been shown to exert several pleiotropic effects in the nervous system as a neuroprotective and antidepressant-like agent. Antidepressant-like effect of atorvastatin in mice is mediated by glutamatergic and serotoninergic receptors, although the precise intracellular signaling pathways involved are unknown. PI3K/Akt/GSK-3ß/mTOR signaling pathway has been associated to neurobiology of depression and seems to be modulated by some pharmacological antidepressant strategies. The present study investigated the participation of the PI3K/Akt/GSK-3ß/mTOR signaling pathway in the antidepressant-like effect of an acute atorvastatin treatment in mice. Atorvastatin sub-effective (0.01 mg/kg) or effective (0.1 mg/kg) doses in the tail suspension test (TST) was administered orally alone or in combination with PI3K, GSK-3ß or mTOR inhibitors. The administration of PI3K inhibitor, LY294002 (10 nmol/site, i.c.v) completely prevented the antidepressant-like effect of atorvastatin (0.1 mg/kg, p.o.). The participation of GSK-3ß in the antidepressant-like effect of atorvastatin was demonstrated by co-administration of a sub-effective dose of atorvastatin (0.01 mg/kg, p.o.) with AR-A014418 (0.01 µg/site, i.c.v., a selective GSK-3ß inhibitor) or with lithium chloride (10 mg/kg, p.o., a non-selective GSK-3ß inhibitor). The mTOR inhibitor, rapamycin (0.2 nmol/site, i.c.v.) was also able to prevent atorvastatin (0.1 mg/kg, p.o.) antidepressant-like effect. These behavioral findings were supported by neurochemical observations, as atorvastatin treatment increased the immunocontent of the phosphorylated isoforms of Akt, GSK-3ß and mTOR in the hippocampus of mice. Taken together, our results suggest an involvement of the PI3K/Akt/GSK-3ß/mTOR signaling pathway in the antidepressant-like effect of atorvastatin in mice.

Atorvastatin evokes a serotonergic system-dependent antidepressant-like effect in mice.

Atorvastatin is a statin largely used in the treatment of hypercholesterolemia and recently revealed as a neuroprotective agent. The antidepressant-like effect of acute atorvastatin treatment in mice has been previously demonstrated by our laboratory. The purpose of this study was to explore the contribution of the serotonergic system in the antidepressant-like effect of atorvastatin in mice. Data demonstrate that the serotonin (5-HT) depleting agent p-chlorophenylalanine methyl ester (PCPA, 100 mg/kg, i.p.) completely abolished atorvastatin (0.1 mg/kg, p.o.) antidepressant-like effect. Besides atorvastatin, fluoxetine (10 mg/kg, p.o.), a serotonin selective reuptake inhibitor (SSRI) was able to exert an antidepressant-like effect, but any of them changed 5-HT content in the hippocampus or frontal cortex. The 5H-T1A (WAY100635, 0.1 mg/kg, s.c) or the 5-HT2A/2C (ketanserin, 5 mg/kg, s.c.) receptor antagonists prevented atorvastatin antidepressant-like effect. In addition, a combinatory antidepressant-like effect was observed when mice received the co-administration of sub-effective doses of atorvastatin (0.01 mg/kg, p.o.) and the SSRI fluoxetine (5 mg/kg, p.o.), paroxetine (0.1 mg/kg, p.o.) or sertraline (1 mg/kg, p.o.). Taken together, these results indicate that the antidepressant-like effect of atorvastatin depends on the serotonergic system modulation.

Inibição da atividade da citrato sintase cerebral em um modelo animal de sepse / Inhibition of brain citrate synthase activity in an animal model of sepsis

OBJETIVO: Um amplo corpo de evidência oriundo de estudos experimentais indica que a sepse se associa com um aumento da produção de espécies de oxigênio reativo, depleção de antioxidantes, e acúmulo de marcadores de estresse oxidativo. Além disto, a disfunção mitocondrial foi implicada na patogênese da síndrome de disfunção de múltiplos órgãos. A citrato sintase é uma enzima que se localiza no interior das células, na matriz mitocondrial, sendo uma etapa importante do ciclo de Krebs; esta enzima foi utilizada como um marcador enzimático quantitativo da presença de mitocôndrias intactas. Assim, investigamos a atividade da citrato sintase no cérebro de ratos submetidos ao modelo sepse com de ligadura e punção do ceco. MÉTODOS: Em diferentes horários (3, 6, 12, 24 e 48 horas) após cirurgia de ligadura e punção do ceco, seis ratos foram sacrificados por decapitação, sendo seus cérebros removidos e dissecados o hipocampo, estriato, cerebelo, córtex cerebral e córtex pré-frontal, e utilizados para determinação da atividade de citrato sintase. RESULTADOS: Verificamos que a atividade de citrato sintase no córtex pré-frontal estava inibida após 12, 24 e 48 horas da ligadura e punção do ceco. No córtex cerebral, esta atividade estava inibida após 3, 12, 24 e 48 horas da ligadura e punção do ceco. Por outro lado a citrato sintase não foi afetada no hipocampo, estriato e cerebelo até 48 horas após a ligadura e punção do ceco. CONCLUSÃO: Considerando-se que é bem descrito o comprometimento da energia decorrente da disfunção mitocondrial na sepse, e que o estresse oxidativo desempenha um papel essencial no desenvolvimento da sepse, acreditamos que o comprometimento da energia pode também estar evolvido nestes processos. Se a inibição da citrato sintase também ocorre em um modelo de sepse, é tentador especular que a redução do metabolismo cerebral pode provavelmente estar relacionada com a fisiopatologia desta doença.

OBJECTIVE: An extensive body of evidence from experimental studies indicates that sepsis is associated with increased reactive oxygen species production, depletion of antioxidants, and accumulation of markers of oxidative stress. Moreover, mitochondrial dysfunction has been implicated in the pathogenesis of multiple organ dysfunction syndrome (MODS). Citrate synthase is an enzyme localized in the mitochondrial matrix and an important component of the Krebs cycle; consequently, citrate synthase has been used as a quantitative enzyme marker for the presence of intact mitochondria. Thus, we investigated citrate synthase activity in the brains of rats submitted to a cecal ligation puncture model of sepsis. METHODS: At several times points (3, 6, 12, 24 and 48 hours) after the cecal ligation puncture operation, six rats were killed by decapitation. Their brains were removed, and the hippocampus, striatum, cerebellum, cerebral cortex and prefrontal cortex were dissected and used to determine citrate synthase activity. RESULTS: We found that citrate synthase activity in the prefrontal cortex was inhibited 12, 24 and 48 hours after cecal ligation puncture. In the cerebral cortex, citrate synthase activity was inhibited 3, 12, 24 and 48 hours after cecal ligation puncture. Citrate synthase was not affected in the hippocampus, striatum or cerebellum up to 48 hours after cecal ligation puncture. CONCLUSION: Considering that energy impairment due to mitochondrial dysfunction in sepsis has been well described and that oxidative stress plays a crucial role in sepsis development, we believe that energy impairment may also be involved in these processes. If citrate synthase inhibition also occurs in a sepsis model, it is tempting to speculate that a reduction in brain metabolism may be related to the pathophysiology of this disease.

Inhibition of brain citrate synthase activity in an animal model of sepsis. / Inibição da atividade da citrato sintase cerebral em um modelo animal de sepse.

OBJECTIVE: An extensive body of evidence from experimental studies indicates that sepsis is associated with increased reactive oxygen species production, depletion of antioxidants, and accumulation of markers of oxidative stress. Moreover, mitochondrial dysfunction has been implicated in the pathogenesis of multiple organ dysfunction syndrome (MODS). Citrate synthase is an enzyme localized in the mitochondrial matrix and an important component of the Krebs cycle; consequently, citrate synthase has been used as a quantitative enzyme marker for the presence of intact mitochondria. Thus, we investigated citrate synthase activity in the brains of rats submitted to a cecal ligation puncture model of sepsis. METHODS: At several times points (3, 6, 12, 24 and 48 hours) after the cecal ligation puncture operation, six rats were killed by decapitation. Their brains were removed, and the hippocampus, striatum, cerebellum, cerebral cortex and prefrontal cortex were dissected and used to determine citrate synthase activity. RESULTS: We found that citrate synthase activity in the prefrontal cortex was inhibited 12, 24 and 48 hours after cecal ligation puncture. In the cerebral cortex, citrate synthase activity was inhibited 3, 12, 24 and 48 hours after cecal ligation puncture. Citrate synthase was not affected in the hippocampus, striatum or cerebellum up to 48 hours after cecal ligation puncture. CONCLUSION: Considering that energy impairment due to mitochondrial dysfunction in sepsis has been well described and that oxidative stress plays a crucial role in sepsis development, we believe that energy impairment may also be involved in these processes. If citrate synthase inhibition also occurs in a sepsis model, it is tempting to speculate that a reduction in brain metabolism may be related to the pathophysiology of this disease.