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.     

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.     

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.     

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.     

Lead-induced alterations of apoptosis and neurotrophic factor mRNA in the developing rat cortex, hippocampus, and cerebellum

Previous reports have recently shown the prototypic neurotoxicant, lead, to induce apoptosis in the brains of developing organisms. In the current study, timed-pregnant rats were exposed to lead acetate (0.2% in the drinking water) 24 h following birth at postnatal day 1 (PND 1). Dams and pups were continuously exposed to lead through the drinking water of the dam until PND 20. Postnatal exposure in the pups resulted in altered mRNA levels of the following apoptotic and neurotrophic factors: caspase 2 and 3, bax, bcl-x, brain-derived neurotrophic factor (BDNF). Ribonuclease protection assays were conducted to measure the factors simultaneously at the following postnatal time points: 9, 12, 15, 20, 25, days. Our results suggest a brain region- and time-specific response following lead acetate exposure. The region most vulnerable to alterations occurs in the hippocampus with alterations beginning at PND 12, in which caspase 3, bcl-x, BDNF increase with lead exposure. Significant treatment effects were not observed for both the cortex and cerebellum.     

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.     

CCL28 in the mouse hippocampal CA1 area and the dentate gyrus during and after pilocarpine-induced status epilepticus

The present study showed a wide presence of CCL28 in mouse CNS, including cerebral, cerebellum, brain stem and spinal cord. In hippocampus, the expression of CCL28 at both mRNA and protein level was clarified. The CCL28 expression was mainly localized in pyramidal cells of CA area, granular cells of dentate gyrus and some interneurons in CA area and hilus. Double-labelling immunocytochemistry revealed that most of calbindin, calretinin and parvalbumin immunopositive neurons expressed CCL28. During and after pilocarpine induced status epilepticus (SE), a down-regulated expression of CCL28 in hippocampal interneurons in the CA1 area and in the hilus of the dentate gyrus was demonstrated. The present study may, therefore provide evidence that CCL28 may have a novel role in CNS and may be involved in the loss of hippocampal interneurons, and subsequent disinhibition of pyramidal neurons.     

In vivo hippocampal microdialysis reveals impairment of NMDA receptor–cGMP signaling in APPSW and APPSW/PS1L166P Alzheimer’s transgenic mice

Transgenic (Tg) mice overexpressing human amyloid precursor protein (APP) mutants reproduce features of early Alzheimer’s disease (AD) including memory deficit, presence of β-amyloid (Aβ) oligomers, and age-associated formation of amyloid deposits. In this study we used hippocampal microdialysis to characterize the signaling of N-methyl-d-aspartic acid receptors (NMDA-Rs) in awake and behaving AD Tg mice. The NMDA-R signaling is central to hippocampal synaptic plasticity underlying memory formation and several lines of evidence implicate the role of Aβ oligomers in effecting NMDA-R dysfunction. CA1 NMDA-Rs were stimulated by NMDA infused through reverse microdialysis while changes in the cyclic guanosine monophosphate (cGMP) concentration in the brain interstitial fluid (ISF) were used to determine NMDA-Rs responsiveness. While 4 months old wild type C57BL/6 mice mounted robust cGMP response to the NMDA challenge, the same stimulus failed to significantly change the cGMP level in 4 and 15 months old APP_SW and 4 months old APP_SW/PS1_L166P Tg mice, which were all on C57BL/6 background. Lack of response to NMDA in AD Tg mice occurred in the absence of changes in expression levels of several synaptic proteins including synaptophysin, NR1 NMDA-R subunit and postsynaptic density protein 95, which indicates lack of profound synaptic degeneration. Aβ oligomers were detected in all three AD Tg mice groups and their concentration in the hippocampus ranged from 40.5 ± 3.6 ng/g in 4 months old APP_SW mice to 60.8 ± 15.9 ng/g in 4 months old APP_SW/PS1_L166P mice. Four months old APP_SW mice had no Aβ amyloid plaques, while the other two AD Tg mice groups showed evidence of incipient Aβ amyloid plaque formation. Our studies describes a novel approach useful to study the function of NMDA-Rs in awake and behaving AD Tg mice and demonstrate impairment of NMDA-R response in the presence of endogenously formed Aβ oligomers but predating onset of Aβ amyloidosis.