Acute pretreatment with estradiol protects against CA1 cell loss and spatial learning impairments resulting from transient global ischemia

Estradiol can act to protect against hippocampal damage resulting from transient global ischemia, but little is known about the functional consequences of such neuroprotection. The present study examines whether acute estradiol administered prior to the induction of transient global ischemia protects against hippocampal cell death and deficits in performance on a spatial learning task. Ovariectomized female rats were primed with estradiol benzoate or oil vehicle 48 and 24 h prior to experiencing one of three durations of 4-vessel occlusion (0, 5, or 10 min). Performance on the cued and hidden platform versions of the Morris water maze was assessed 1 week following ischemia. On the cued platform task, neither hormone treatment nor ischemia significantly influenced acquisition. When tested on the hidden platform task, however, oil-treated rats exhibited impairments in spatial learning after either 5 or 10 min of ischemia while estradiol-treated rats showed no impairments after 5 min of ischemia and only mild impairments after 10 min of ischemia. Immediately following behavioral testing, rats were perfused and survival of CA1 pyramidal cells was assessed. Ischemia was associated with the loss of CA1 pyramidal cells but rats that received estradiol prior to ischemia showed less severe damage. Furthermore, the extent of cell loss was correlated with degree of spatial bias expressed on a probe trial following hidden platform training. These findings indicate that acute exposure to estradiol prior to ischemia is both neuroprotective and functionally protective.     

Transplantation of Foetal Neural Stem Cells into the Rat Hippocampus During Trimethyltin-Induced Neurodegeneration

The present study investigates the survival and fate of neural stem cells/progenitor cells (NSC/NPCs) homografted into the hippocampus of rats treated with trimethyltin (TMT), a potent neurotoxicant considered a useful tool to obtain a well characterized model of neurodegeneration, to evaluate their possible role in the reparative mechanisms that accompany neurodegenerative events. NSC/NPCs expressing eGFP by lentivirus-mediated infection were stereotaxically grafted into the hippocampus of TMT-treated animals and controls. Two weeks after transplantation surviving NSC/NPCs were detectable in 60% of TMT-treated animals and 30% of controls, while 30 days after transplantation only 40% of TMT-treated animals showed surviving grafted cells, which were undetectable in controls. At both times investigated, while grafted NSC/NPCs differentiated into neurons or astrocytes could be observed in addition to undifferentiated NSC/NPCs, we did not find evidence of structural integration of grafted cells into the main site of hippocampal lesion leading to appreciable repair. Maria Concetta Geloso and Stefano Giannetti contributed equally to this work.     

The Role of Nitric Oxide in the Neuroprotection of Limb Ischemic Preconditioning in Rats

Brief limb ischemia was reported to protect neurons against injury induced by subsequent cerebral ischemia-reperfusion, and this phenomenon is known as limb ischemic preconditioning (LIP). To explore the role of nitric oxide (NO) in neuroprotection of LIP in rats, we observed changes in the content of nitric oxide (NO) and activity of NO synthase (NOS) in the serum and CA1 hippocampus of rats after transient limb ischemic preconditioning (LIP), and the influence of N^G-nitro-l-arginine methylester (l-NAME), a NOS inhibitor, on the neuroprotection of LIP against cerebral ischemia-reperfusion injury. Results showed that NO content and NOS activity in serum increased significantly after LIP compared with the sham group. The increase showed a double peak pattern, in which the first one appeared at time 0 (immediate time point) and the second one appeared at 48 h after the LIP (P < 0.01). The NO content and NOS activity in the CA1 hippocampus in LIP group showed similar change pattern with the changes in the serum, except for the first peak of up-regulation of NO content and NOS activity appeared at 6 h after LIP. Pretreatment with l-NAME before LIP blocked the neuroprotection of LIP against subsequent cerebral ischemic insult. The blocking effect of l-NAME was abolished with pretreatment of l-Arg. These findings indicated that NO may be associated with the tolerance of pyramidal cells in the CA1 hippocampus to ischemia induced by LIP in rats.     

Effect of Radicicol Infusion on the <Emphasis Type="Italic">Src</Emphasis> Tyrosine Kinase Activity of Rat Hippocampus before and after Training in an Inhibitory Avoidance Task

The participation of protein serine/threonine kinases in memory formation and retrieval is well established. In contrast, relatively little is known on the role of protein tyrosine kinases (PTKs). Previous work showed that intra-hippocampal infusion of the Src-PTK inhibitor radicicol inhibits memory acquisition, consolidation, and retrieval of one-trial step-down inhibitory avoidance task. In this study, we investigated the possible interaction between levels of Src-PTK activity in hippocampus and memory acquisition, formation, and retrieval of this task. Radicicol (0.5 μg/ml) was infused into the CA1 region of the hippocampus of rats trained in a one-trial step-down inhibitory avoidance task. Radicicol infused 15 min before training decreased Src-PTK activity, as measured 0, 1.5, and 24 h after training, and impaired memory acquisition of the task. When given immediately after training, there was a decrease in Src-PTK activity 1.5 h, but not 0 or 24 h after training. This treatment depressed memory consolidation. Radicicol infused into CA1 10 min prior to retrieval testing inhibited hippocampal Src-PTK activity, as measured immediately after the test session. The results suggest that Src-PTKs participate in memory acquisition, consolidation, and retrieval processes, but the timing of the role of the enzyme is different in each case.     

Basic Fibroblast Growth Factor Prevents the Memory Impairment Induced by Gastrin-Releasing Peptide Receptor Antagonism in Area CA1 of the Rat Hippocampus

Increasing evidence indicates that the gastrin-releasing peptide receptor (GRPR) is implicated in regulating synaptic plasticity and memory formation in the hippocampus and other brain areas. However, the molecular mechanisms underlying the memory-impairing effects of GRPR antagonism have remained unclear. Here we report that basic fibroblast growth factor (bFGF/FGF-2) rescues the memory impairment induced by GRPR antagonism in the rat dorsal hippocampus. The GRPR antagonist [D-Tpi⁶, Leu¹³ psi(CH₂NH)-Leu¹⁴] bombesin (6–14) (RC-3095) at 1.0 μg impaired, whereas bFGF at 0.25 μg enhanced, 24 h retention of inhibitory avoidance (IA) when infused immediately after training into the CA1 hippocampal area in male rats. Coinfusion with an otherwise ineffective dose of bFGF blocked the memory-impairing effect of RC-3095. These findings suggest that the memory-impairing effects of GRPR antagonists might be partially mediated by an inhibition in the function and/or expression of neuronal bFGF or diminished activation of intracellular protein kinase pathways associated with bFGF signaling.     

Mass Spectrometrical Characterization of NDRG2 Protein (N-myc-Downstream Regulated Gene 2) and Description of Two Novel Phosphorylation Sites

Antidepressant-related protein (NDRG2) is a member of the N-myc downstream-regulated gene family and a role for differentiation and signaling has been proposed. Performing protein profiling we observed NDRG2 and decided to characterize this important biomolecule. Estrous cycle phases were determined in Sprague-Dawley rats and the hippocampus was taken. Proteins were extracted, run on two-dimensional gel electrophoresis with subsequent multi-enzyme digestion followed by MALDI-TOF-TOF and nano-LC-ESI-MS/MS analysis of spots. Spots identified as NDRG2 were quantified by specific software. Five spots were identified as NDRG2 and two novel phosphorylation sites (T330 and T334) were detected. Gender and estrous cycle-dependent NDRG2 levels were observed. Results are of importance for further qualitative and quantitative studies at the protein level as well as for the design of antibodies for immunochemical applications and for the interpretation of previous studies on NDRG2 that did not take into account different expression forms and posttranslational modifications.     

A Dietary Fat Excess Alters Metabolic and Neuroendocrine Responses Before the Onset of Metabolic Diseases

Early changes in neuroendocrine pathways are essential in the development of metabolic pathologies. Thus, it is important to have a better understanding of the signals involved in their initiation. Long-term consumption of high-fat diets induces insulin resistance, obesity, diabetes. Here, we have investigated early neural and endocrine events in the hypothalamus and hippocampus induced by a short-term high fat, low carbohydrate diet in adult male Wistar rats. The release of serotonin, which is closely associated with the actions of insulin and leptin, was measured, by electrochemical detection following reverse-phase liquid chromatography (HPLC), in the extracellular space of the medial hypothalamus and the dorsal hippocampus in samples obtained from non-anesthetized animals, by microdialysis. The high-fat diet had a specific effect on the hypothalamus. Serotonin release induced by food intake was reduced after 1 week, and effectively ceased after 6 weeks of the diet. After 1 week, there was an increased gene expression of the insulin receptor and the insulin receptor substrates IRS1 and IRS2, as measured by real-time PCR. After 6 weeks of diet, insulin gene expression increased. Leptinemia increased in all cases. This new data support the concept that high-fat diets, in addition to have peripheral effects, cause a rapid alteration in specific central mechanisms involved in energy and glucose homeostasis. The changes in the gene expression of insulin and signaling elements represent possible adaptations aimed at counterbalancing the reduced responsiveness of the serotonergic system to nutritional signals and maintaining homeostasis. Sophie M. Banas and Claude Rouch have contributed equally to this work.     

Binge Ethanol-induced Neurodegeneration in Rat Organotypic Brain Slice Cultures: Effects of PLA2 Inhibitor Mepacrine and Docosahexaenoic Acid (DHA)

Using rat organotypic hippocampal-entorhinal cortical (HEC) slice cultures, we examined whether phospholipase A2 (PLA2) activity is involved in binge alcohol (ethanol)-induced neurodegeneration, and whether docosahexaenoic acid (DHA; 22:6n-3), a fish oil-enriched fatty acid that is anti-inflammatory in brain damage models, is neuroprotective. Assessed with propidium iodide and lactate dehydrogenase (LDH) leakage, neurodamage from ethanol (6 days 100 mM ethanol with four withdrawal periods) was prevented by the PLA2 pan-inhibitor, mepacrine. Also, ethanol-dependent neurodegeneration—particularly in the entorhinal region—was significantly ameliorated by DHA supplementation (25 μM); however, adrenic acid, a 22:4n-6 analog, was ineffective. Consistent with PLA2 activation, [³H] liberation was approximately fivefold greater in [³H]arachidonic acid-preloaded HEC slice cultures during ethanol withdrawal compared to controls, and DHA supplementation suppressed [³H] release to control levels. DHA might antagonize PLA2 activity directly or suppress upstream activators (e.g., oxidative stress); however, other DHA mechanisms could be important in subdueing ethanol-induced PLA2-dependent and independent neuroinflammatory processes.