Neurotoxicity Induced by Glutamate in Glucose-Deprived Rat Hippocampal Slices is Prevented by GMP

Guanosine-5-monophosphate (GMP) was evaluated as a neuroprotective agent against the damage induced by glutamate in rat hippocampal slices submitted to glucose deprivation. In slices maintained under physiological conditions, glutamate (0.01 to 10 mM), Kainate, alpha-amino-3-hydroxi-5-methylisoxazole-propionic acid (AMPA), N-methyl-D-aspartate (NMDA), 1S,3R-aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD), or L-2-amino-4-phosphonobutanoic acid (L-AP4) (100 M) did not alter cell membrane permeability, as evaluated by lactate dehydrogenase (LDH) release assay. In slices submitted to glucose deprivation, GMP (from 0.5 mM) prevented LDH leakage and the loss of cell viability induced by 10 mM glutamate. LDH leakage induced by Kainate, AMPA, NMDA or 1S,3R-ACPD was fully prevented by 1 mM GMP. However, glutamate uptake was not altered in slices submitted to glucose deprivation and glutamate analogues. Glucose deprivation induced a significant decrease in ATP levels which was unchanged by addition of glutamate or GMP. Our results show that glucose deprivation decreases the energetic charge of cells, making hippocampal slices more susceptible to excitotoxicity and point to GMP as a neuroprotective agent acting as a glutamatergic antagonist.     

Alternative pathway activities in aged potato tuber slices measured by three methods

To find a simple and reliable oxygen electrode-based method to estimate the values of alternative pathway activity (V _alt) and its contribution to total respiration V _alt/V _t) in aged potato (Solanum tuberosum L.) tuber slices, we compared conventional hydroxamate-inhibiting method, improved hydroxamate-inhibiting method with 2,6-dichlorophenol indophenol (DCPIP), and the oxygen isotope discrimination (OID) method. The values of V _alt and V _alt/V _t obtained with an improved hydroxamate-inhibiting method with DCPIP in 12-h- and 24-h-aged slices were about twice higher than those with the conventional hydroxamate-inhibiting method. Only a relatively small difference in the values of V _alt and V _alt/V _t obtained by the OID method and the improved hydroxamate-inhibiting method with DCPIP in 12-h and 24-h-aged slices was observed. These results indicated that the improved hydroxamate-inhibiting method with DCPIP could be considered as a new, simple, and reliable technique for the noninvasive assay of the AP activity.From Fiziologiya Rastenii, Vol. 52, No. 2, 2005, pp. 311–315.Original English Text Copyright © 2005 by Hou, Zhou, Kong, Liang, Zhang.This article was submitted by the authors in English.This revised version was published online in April 2005 with a corrected cover date.     

Cloning and expression of ligand-gated ion-channel receptor L2 in central nervous system

An orphan receptor of ligand-gated ion-channel type (L2, also termed ZAC according to the presence of zinc ion for channel activation) was identified by computer-assisted search programs on human genome database. The L2 protein shares partial homology with serotonin receptors 5HT3A and 5HT3B. We have cloned L2 cDNA derived from human caudate nucleus and characterized the exon-intron structure as follows: (1) The L2 protein has four transmembrane regions (M1-M4) and a long cytoplasmic loop between M3 and M4. (2) The sequence is conserved in species including chimpanzee, dog, cow, and opossum. (3) Nine exons form its protein-coding region and especially exon 5 corresponds to a disulfide bond region on the amino-terminal side. Our analysis using multiple tissue cDNA panels revealed that at least two splicing variants of L2 mRNA are present. The cDNA PCR amplification study revealed that L2 mRNA is expressed in tissues including brain, pancreas, liver, lung, heart, kidney, and skeletal muscle while 5HT3A mRNA could be detected in brain, heart, placenta, lung, kidney, pancreas, and skeletal muscle, and 5HT3B mRNA in brain, kidney, and skeletal muscle, suggesting different significance in tissue expression of these receptors. Regional expression of L2 mRNA and protein was examined in brain. The RT-PCR studies confirmed L2 mRNA expression in hippocampus, striatum, amygdala, and thalamus in adult brain. The L2 protein was immunolocalized by using antipeptide antibodies. Immunostained tissue sections revealed that L2-like immunoreactivity was dominantly expressed in the hippocampal CA3 pyramidal cells and in the polymorphic layer of the dentate gyrus. We analyzed the expression of L2 protein in HEK293 cells using GFP fusion protein reporter system. Western blots revealed that L2 protein confers sugar chains on the extracellular side. In transfected HEK293 cells, cellular membranes and intracellular puncta were densely labeled with GFP, suggesting selective dispatch to the final destination.     

Proteomic analysis revealed a novel synaptic proline-rich membrane protein (PRR7) associated with PSD-95 and NMDA receptor

Proteomic analyses have revealed a novel synaptic proline-rich membrane protein: PRR7 (proline rich 7), in the postsynaptic density (PSD) fraction of rat forebrain. PRR7 is 269 amino acid residues long, and displays a unique architecture, composed of a very short N-terminal extracellular region, a single membrane spanning domain, and a cytoplasmic domain possessing a proline-rich sequence and a C-terminal type-1 PDZ binding motif. A fraction of PRR7 accumulates in spines along with synapse maturation, and colocalizes with PSD-95 in a punctate pattern in rat hippocampal neural cultures. Immunoprecipitation and GST pull-down assays demonstrated that PRR7 binds to the third PDZ domain of PSD-95. In addition, the NMDA receptor subunits, NR1 and NR2B, specifically co-immunoprecipitated with PRR7. These results suggest that PRR7 is involved in modulating neural activities via interactions with the NMDA receptor and PSD-95, and PSD core formation.     

Organ slices for the evaluation of human drug toxicity

Human organ slices, an in vitro model representing the multicellular and functional features of in vivo tissue, is a promising model for characterizing mechanisms of drug-induced organ injury and for identifying biomarkers of organ injury. Target organ injury is a significant clinical issue. In vitro models, which compare human and animal tissue to improve the extrapolation of animal in vivo studies for predicting human outcome, will contribute to improving drug candidate selection and to defining species susceptibilities in drug discovery and development programs. A critical aspect to the performance and outcome of human organ slice studies is the use of high quality tissue, and the use of culture conditions that support optimum organ slice survivability, in order to accurately reproduce mechanisms of organ injury in vitro. The attribute of organ slices possessing various cell types and interactions contributes to the overall biotransformation, inflammatory response and assessment of injury. Regional differences and changes in morphology can be readily evaluated by histology and special stains, similar to tissue obtained from in vivo studies. The liver is the major organ of which slice studies have been performed, however the utility of extra-hepatic derived slices, as well as co-cultures is increasing. Recent application of integrating gene expression, with human organ slice function and morphology demonstrate the increased potential of this model for defining the molecular and biochemical pathways leading to drug-induced tissue changes. By gaining a more detailed understanding of the mechanisms of drug-induced organ injury, and by correlating clinical measurements with drug-induced effects in the in vitro models, the vision of human in vitro models to identify more sensitive and discriminating markers of organ damage is attainable.     

Increased seizure susceptibility and cortical malformation in beta-catenin mutant mice

Beta-catenin has been implicated in epilepsy because of its altered post seizure expression and the role of Wnt2 signaling in autism. To determine beta-catenin's role in seizure susceptibility, we injected penetylenetetrazol intraperitoneally in beta-catenin cerebral cortex- and hippocampus-specific knockout mice. We then analyzed the latency, number, and duration of four phases of seizure behaviors: (I) non-seizure activity, (II) myoclonic jerks, (III) generalized clonic seizures, and (IV) tonic seizures. The latencies to both death and Phase IV were significantly reduced in mutant mice. Mutant mice also spent significantly more time in Phases III and IV and showed significantly less time in the non-convulsive state (Phase I). Nissl and gold chloride staining indicated that the knockout mice had underdeveloped cortices, lacked a corpus callosum, and were missing hippocampal structures. This suggests that dysfunction of beta-catenin-mediated signaling pathways in mice leads to cortical malformation and increased seizure susceptibility.     

MGluRs regulate the expression of neuronal calcium sensor proteins NCS-1 and VILIP-1 and the immediate early gene arg3.1/arc in the hippocampus in vivo

The metabotropic glutamate receptor (mGluR) agonist (R,S)-3,5-dihydroxyphenylglycine (DHPG) is involved in several forms of hippocampal synaptic plasticity. DHPG application can induce slow-onset potentiation, a form of long-term potentiation (LTP), in the dentate gyrus and in the CA1 region in vivo. The induction of LTP correlates with increased expression levels of neuronal calcium sensor (NCS), considered as key elements for plasticity. In this study we investigated mGluR- and time-dependent changes in the expression of two different NCS proteins. Following DHPG application in vivo NCS-1 and VILIP-1 expression increased, with significant levels reached after 8 and 24h. The effect was attenuated by treatment with the group I mGluR specific antagonist S-4-carboxyphenylglycine. The immediate early gene (IEG) arg3.1/arc showed highest expression levels 2h after DHPG-treatment. Therefore, mGluRs at concentrations which induce synaptic plasticity regulate the expression of IEGs and NCS proteins in different time frames and thus contribute to late phases of synaptic plasticity.     

Sexual dimorphism in the induction of LTP: critical role of tetanizing stimulation

Numerous studies have suggested that sexual dimorphism may exist in learning and memory, particularly in types involving the hippocampus. In the present study, we examined the effects of two different tetani on the induction of long-term potentiation in the CA1 region of hippocampal slices from adult female and male rats to determine the sexual differences in their responses to tetanizing stimulation. We found that the induction of LTP is sex-dependent, and that there were clear sexual differences in the responses to different tetanus patterns, but not impulse number or stimulation frequency. Multiple trains of tetani were more effective in the indution of LTP in male rats than in female ones. These findings suggest that male rats can react to a broader range of tetanizing stimulation compared with female rats. Based on our results and the findings of other studies, we propose that the interaction of gonadal hormones with Ca2+/NMDAR and the subsequent regulation of the ERK/MAP kinase pathway are critical mechanisms for sexual dimorphism in the induction of LTP.     

Effects of Glucose, Insulin, and Supernatant from Pancreatic β-cells on Brain–Pancreas Relative Protein in Rat Hippocampus

Brain–pancreas relative protein (BPRP) is a novel protein that mainly expresses in brain and pancreas. In our previous study, we found that various stressors significantly decreased the expression of BPRP in pancreas in vivo, accompanied by changes in insulin and glucose levels, and that expression of BPRP in pancreas also decreased significantly in diabetic rats induced by Streptozocin (STZ). All these findings suggest that BPRP may be a glucose or insulin-sensitive protein. However, how the changes in insulin or glucose levels influence the expression of BPRP in hippocampus requires further study. Here, we investigated the effects of insulin or glucose on the expression of BPRP in primary cultured hippocampal neurons. We supplied hippocampal neurons with glucose, insulin, or supernatant from pancreatic β-cells, which secrete insulin into the supernatant. Our data showed that insulin had beneficial effect on the viability while no significant effect on the expression of BPRP in hippocampal neurons. On the contrary, 40 mM glucose or free glucose culture significantly decreased the expression of BPRP, while had no significant effect on the viability and apoptosis of hippocampal neurons. Further study showed that levels of insulin in the supernatant collected from pancreatic β-cells medium changed over days, and that supernatant increased the viability of hippocampal neurons, while it had no obvious effect on the expression of BPRP in hippocampal neurons. These results suggest that BPRP may be a glucose-sensitive protein.     

An interaction between benzodiazepines and neuroactive steroids at GABA A receptors in cultured hippocampal neurons

Neurosteroids are modulators of several receptors and ion channels and are implicated in the pathophysiology of several neuropsychiatric diseases including hepatic encephalopathy (HE). The neurosteroid, allopregnanolone, a positive allosteric modulator of GABA_A receptors, accumulates in the brains of HE patients where it can potentiate GABA_A receptor-mediated responses. Attenuation of the effects of neurosteroids on GABA-ergic neurotransmission is therefore of interest for the management of HE. In the present study, we determined the effect of the benzodiazepine partial inverse agonist, Ro15-4513, and the benzodiazepine antagonist, flumazenil on modulation of the GABA_A mediated chloride currents by allopregnanolone and on spontaneous synaptic activity in cultured hippocampal neurons using the patch-clamp technique. Allopregnanolone (0.03–0.3 μM), dose-dependently potentiated GABA-induced currents, an action significantly reduced by Ro15-4513 (10 μM). In contrast, flumazenil (10 μM) had no effect on the ability of allopregnanolone to potentiate GABA_A currents but it blocked the effects of Ro15-4513. The frequency of spontaneous synaptic activity was significantly reduced in the presence of allopregnanolone (0.1 μM) from 1.5 ± 0.7 to 0.1 ± 0.04 Hz. This action was partially reversed by Ro15-4513 (10 μM) but was not significantly influenced by flumazenil (10 μM). These findings suggest that the beneficial affects of Ro15-4513 in experimental HE result from attenuation of the effects of neurosteroids at GABA_A receptors. Our results may provide a rational basis for the use of benzodiazepine inverse agonists in the management and treatment of hepatic encephalopathy in patients with liver failure.