Microfluidic capillary zone electrophoresis mass spectrometry analysis of alkaloids in Lobelia cardinalis transgenic and mutant plant cell cultures.

Application of a microfluidic CE* device for CZE-MS allows for fast, rapid, and in-depth analysis of large sample sets. This microfluidic CZE-MS device, the 908 Devices ZipChip, involves minimal sample preparation and is ideal for small cation analytes, such as alkaloids. Here, we evaluated the microfluidic device for the analysis of alkaloids from Lobelia cardinalis hairy root cultures. Extracts from wild-type, transgenic, and selected mutant plant cultures were analyzed and data batch processed using the mass spectral processing software MZmine2 and the statistical software Prism 8. In total 139 features were detected as baseline resolved peaks via the MZmine2 software optimized for the electrophoretic separations. Statistically significant differences in the relative abundance of the primary alkaloid lobinaline (C27 H34 N2 ), along with several putative "lobinaline-like" molecules were observed utilizing this approach. Additionally, a method for performing both targeted and untargeted MS/MS experiments using the microfluidic device was developed and evaluated. Coupling data-processing software with CZE-MS data acquisition has enabled comprehensive metabolomic profiles from plant cell cultures to be constructed within a single working day.

The Dietary Flavonoid Rhamnetin Inhibits Both Inflammation and Excitotoxicity During Ethanol Withdrawal in Rat Organotypic Hippocampal Slice Cultures.

BACKGROUND: Ethanol (EtOH) causes neurotoxicity via several mechanisms including neuroinflammation (during EtOH exposure), and excitotoxicity (during EtOH withdrawal [EWD]). Alpha7 nicotinic acetylcholine receptor (nAChR) selective agonists have the potential to reduce both. The aim of this study was to evaluate the anti-inflammatory and neuroprotective potential of rhamnetin, a dietary flavonoid with alpha7 nAChR selective activity, in an in vitro model of EtOH-induced neurotoxicity. METHODS: The anti-inflammatory and neuroprotective properties of rhamnetin were assessed in neonatal organotypic hippocampal slice cultures undergoing EWD (or not) and challenged with N-methyl-D-aspartate (NMDA) and/or lipopolysaccharide (LPS). Neurotoxicity was determined using propidium iodide uptake, and the inflammatory response was evaluated by measuring the release of tumor necrosis factor (TNF)-alpha (NO; quantified by ELISA) and nitric oxide (quantified by the Griess reaction) into culture media. RESULTS: As predicted, rhamnetin reduced LPS-induced release of TNF-alpha and NO both under control conditions and during EWD. Additionally, rhamnetin had no effect on NMDA-induced neurotoxicity under control conditions, but significantly reduced NMDA toxicity during EWD. In contrast, rhamnetin had no effect on neurotoxicity induced by NMDA and LPS combined despite reducing TNF-alpha and NO levels under these conditions. CONCLUSIONS: Rhamnetin is anti-inflammatory and neuroprotective during EWD and therefore has potential value in treating neurotoxicity caused by EtOH.

Altered relation between lipopolysaccharide-induced inflammatory response and excitotoxicity in rat organotypic hippocampal slice cultures during ethanol withdrawal.

BACKGROUND: Ethanol (EtOH) causes neurotoxicity by several mechanisms including excitotoxicity and neuroinflammation, but little is known about the interaction between these mechanisms. Because neuroinflammation is known to enhance excitotoxicity, we hypothesized that neuroinflammation contributes to the enhanced excitotoxicity, which is associated with EtOH withdrawal (EWD). The aim of this study was to evaluate the lipopolysaccharide (LPS)-induced inflammatory response of cultured hippocampal tissue during EWD and its effects on the enhanced N-methyl-d-aspartate (NMDA) receptor-mediated excitotoxicity, which occurs at this time. METHODS: Using a neonatal organotypic hippocampal slice culture (OHSC) model, we assessed the effects of NMDA and LPS (separately or combined) during EWD after 10 days of EtOH exposure. Neurotoxicity was assessed using propidium iodide uptake, and the inflammatory response was evaluated by measuring the release of tumor necrosis factor (TNF)-alpha (quantified by enzyme-linked immunosorbent assay) and nitric oxide (NO; quantified by the Griess reaction) into culture media. Furthermore, we explored the potential role of the microglial cell type using immortalized BV2 microglia treated with EtOH for 10 days and challenged with LPS during EWD. RESULTS: As predicted, NMDA-induced toxicity was potentiated by LPS under control conditions. However, during EWD, the reverse was observed and LPS inhibited peak NMDA-induced toxicity. Additionally, LPS-induced release of TNF-alpha and NO during EWD was reduced compared to control conditions. In BV2 microglia, following EtOH exposure, LPS-induced release of NO was reduced, whereas TNF-alpha release was potentiated. CONCLUSIONS: During EWD following chronic EtOH exposure, OHSC exhibited a desensitized inflammatory response to LPS and the effects of LPS on NMDA toxicity were reversed. This might be explained by a change in microglia to an anti-inflammatory and neuroprotective phenotype. In support, studies on BV2 microglia indicate that EtOH exposure and EWD do alter the response of these cells to LPS, but this cannot fully explain the changes observed in the OHSC. The data suggest that neuroinflammation and excitotoxicity do interact during EWD. However, the interaction is not as simple as we originally proposed. This in turn illustrates the need to assess the extent, importance, and relation of these mechanisms in models of EtOH exposure producing neurotoxicity.

Use of alpha-, beta-Estrogen Receptor as a "new tool" for detection of specific small molecule activity.

Cell-based screening methods for nuclear receptor ligands that use transgenic plant cells expressing a single human NR may have advantages over other eukaryotic systems which express multiple NRs. For example, signal-to-noise ratio might be improved because ligands would be less likely to bind to other NRs and/or less likely to cause confounding functional changes in plant cells. As a first step toward this aim we have expressed in plants truncated human estrogen receptor (ER) constructs linked to reporters, or selective markers such as luciferase, green fluorescent protein (GFP) and hygromycin. A variety of ligands for the ER (including estradiol and known phytoestrogens) have then been tested for their ability to over-express the linked marker gene(s) which could be measured (luciferase activity), visualized under fluorescent microscopy (GFP activity), or selected on antibiotic-containing media (Hygromycin B). Our results show a close association between the effects of ER ligands in the transgenic plant roots and their effects on native ERs in mammalian cells. With the stable expression of an ERalpha-GFP ligand detection system in A. thaliana, the estradiol- mediated response in transgenic roots is inhibited by an ER partial agonist (tamoxifen) and an antagonist (fulvestrant) at concentrations relevant to their use in breast cancer. We conclude that it is possible to express human NRs in plants in a form that can report on exogenous or endogenous ER ligands and that these constructs have a pharmacology which is relevant to ligands for the native NRs in human cells.

Improving the inhibitory activity of arylidenaminoguanidine compounds at the N-methyl-D-aspartate receptor complex from a recursive computational-experimental structure-activity relationship study.

Using a combination of both the partial least squares (PLS) and back-propagation artificial neural network (ANN) pattern recognition methods, several models have been developed to predict the activity of a series of arylidenaminoguanidine analogs as inhibitory modulators of the N-methyl-D-aspartate receptor complex. This was done by correlating structural and physicochemical descriptors obtained from computation software with the experimentally observed [(3)H]MK-801 displacement ability of a small library of synthesized and in vitro screened arylidenaminoguanidines. Results for the generated PLS model were r(2)=0.814, rmsd=0.208, rCV(2)=0.714, loormsd=0.261. The ANN model was created utilizing the eleven descriptors from the PLS model for comparison. The quality of the ANN model (r(2)=0.828, rmsd=0.200, rCV(2)=0.721, loormsd=0.257) is similar to the PLS model, and indicates that the feature between the inputs and the output is majorly linear. These computational models were able to predict inhibition of the NMDA receptor complex by this series of compounds in silico, affording a predictive structure-based 'pre-screening' paradigm for the arylideneaminoguanidine analogs.

Strategy for Conjugating Oligopeptides to Mesoporous Silica Nanoparticles Using Diazirine-Based Heterobifunctional Linkers.

Successful strategies for the attachment of oligopeptides to mesoporous silica with pores large enough to load biomolecules should utilize the high surface area of pores to provide an accessible, protective environment. A two-step oligopeptide functionalization strategy is examined here using diazirine-based heterobifunctional linkers. Mesoporous silica nanoparticles (MSNPs) with average pore diameter of ~8 nm and surface area of ~730 m2/g were synthesized and amine-functionalized. Tetrapeptides Gly-Gly-Gly-Gly (GGGG) and Arg-Ser-Ser-Val (RSSV), and a peptide comprised of four copies of RSSV (4RSSV), were covalently attached via their N-terminus to the amine groups on the particle surface by a heterobifunctional linker, sulfo-succinimidyl 6-(4,4'-azipentanamido)hexanoate (sulfo-NHS-LC-diazirine, or SNLD). SNLD consists of an amine-reactive NHS ester group and UV-activable diazirine group, providing precise control over the sequence of attachment steps. Attachment efficiency of RSSV was measured using fluorescein isothiocyanate (FITC)-tagged RSSV (RSSV-FITC). TGA analysis shows similar efficiency (0.29, 0.31 and 0.26 mol peptide/mol amine, respectively) for 4G, RSSV and 4RSSV, suggesting a generalizable method of peptide conjugation. The technique developed here for the conjugation of peptides to MSNPs provides for their attachment in pores and can be translated to selective peptide-based separation and concentration of therapeutics from aqueous process and waste streams.

Target-directed evolution of novel modulators of the dopamine transporter in Lobelia cardinalis hairy root cultures.

The dopamine transporter (DAT) is targeted in substance use disorders (SUDs), and "non-classical"" DAT inhibitors with low abuse potential are therapeutic candidates. Lobinaline, from Lobelia cardinalis, is an atypical DAT inhibitor lead. Chemical synthesis of lobinaline is challenging; thus, "target-directed evolution" was used for lead optimization. A target protein is expressed in plant cells, and a mutant cell population is selected under conditions where target protein functional inhibition confers a survival advantage. Surviving mutants are "mined" for the targeted activity. Applied to a mutant L. cardinalis cell population expressing the human DAT, we identified 20 mutants overproducing DAT inhibitors. Microanalysis prioritized novel lobinaline derivatives, and we first investigated the more water-soluble lobinaline N-oxide. It inhibited rat synaptosomal [3H]DA uptake with an IC50 similar to lobinaline. Against repeated DA microinjections into the rat striatum, lobinaline produced transient DA clearance reductions. In contrast, lobinaline N-oxide prolongingly increased DA peak amplitudes, particularly in the ventral striatum. Lobinaline N-oxide also produced complex changes in post-peak DA clearance inconsistent with simple DAT inhibition. This unusual DAT interaction may prove therapeutically useful for treating SUDs. This study demonstrates the value of target-directed evolution of plant cells for optimizing lead compounds difficult to synthesize chemically.

Mechanism of Mesoporous Silica Nanoparticle Interaction with Hairy Root Cultures during Nanoharvesting of Biomolecules.

Cellular uptake and expulsion mechanisms of engineered mesoporous silica nanoparticles (MSNPs) are important in their design for novel biomolecule isolation and delivery applications such as nanoharvesting, defined as using nanocarriers to transport and isolate valuable therapeutics (secondary metabolites) out of living plant organ cultures (e.g., hairy roots). Here, temperature-dependent MSNP uptake and recovery processes in hairy roots are examined as a function of surface chemistry. MSNP uptake into hairy roots and time-dependent expulsion are quantified using Ti content (present for biomolecule binding) and fluorescence spectroscopy of fluorescently tagged MSNPs, respectively. The results suggest that functionalization and surface charge (regulated by amine group attachment) play the biggest role in the effectiveness of uptake and recovery. Comparison of MSNP interactions with hairy roots at 4 and 23 °C shows that weakly charged MSNPs functionalized only with Ti are taken up and expelled by thermally activated mechanisms, while amine-modified positively charged particles are taken up and expelled mainly by direct penetration of cell walls. Amine-functionalized MSNPs move spontaneously in and out of plant cells by dynamic exchange with a residence time of 20 ± 5 min, suggesting promise as a biomolecule nanoharvesting platform for plant organ cultures.

Laboratory models available to study alcohol-induced organ damage and immune variations: choosing the appropriate model.

The morbidity and mortality resulting from alcohol-related diseases globally impose a substantive cost to society. To minimize the financial burden on society and improve the quality of life for individuals suffering from the ill effects of alcohol abuse, substantial research in the alcohol field is focused on understanding the mechanisms by which alcohol-related diseases develop and progress. Since ethical concerns and inherent difficulties limit the amount of alcohol abuse research that can be performed in humans, most studies are performed in laboratory animals. This article summarizes the various laboratory models of alcohol abuse that are currently available and are used to study the mechanisms by which alcohol abuse induces organ damage and immune defects. The strengths and weaknesses of each of the models are discussed. Integrated into the review are the presentations that were made in the symposium "Methods of Ethanol Application in Alcohol Model-How Long is Long Enough" at the joint 2008 Research Society on Alcoholism (RSA) and International Society for Biomedical Research on Alcoholism (ISBRA) meeting, Washington, DC, emphasizing the importance not only of selecting the most appropriate laboratory alcohol model to address the specific goals of a project but also of ensuring that the findings can be extrapolated to alcohol-induced diseases in humans.

Reactive oxygen species regulate alkaloid metabolism in undifferentiated N. tabacum cells.

Plants produce an immense number of natural products and undifferentiated cells from various plant tissues have long been considered an ideal source for their synthesis. However, undifferentiated plant cells often either lose their biosynthetic capacity over time or exhibit immediate repression of the required pathways once dedifferentiated. In this study, freshly prepared callus tissue was employed to further investigate the regulation of a natural product pathway in undifferentiated tobacco cells. Putrescine N-methyltransferase (PMT) is a pathway-specific enzyme required in nicotinic alkaloid production in Nicotiana species. Callus derived from transgenic Nicotiana tabacum plants harboring PMT promoter-GUS fusions were used to study factors that influence PMT expression. Under normal callus growth conditions in the presence of light and auxin, PMT promoter activity was strongly repressed. Conversely, dark conditions and the absence of auxin were found to upregulate PMT promoter activity, with light being dominant to the repressive effects of auxin. Since reactive oxygen species (ROS) are known by-products of photosynthesis and have been implicated in signaling, their involvement was investigated in transgenic callus by treatment with the ROS scavenger, dimethylthiourea, or catalase. Under highly repressive conditions for alkaloid synthesis, including normal culture conditions in the light, both ROS scavengers resulted in significant induction of PMT promoter activity. Moreover, treatment of callus with catalase resulted in the upregulation of PMT promoter activity and alkaloid accumulation in this tissue. These results suggest that ROS impact the regulation of the alkaloid pathway in undifferentiated cells and have implications for regulation of the pathway in other plant tissues.

Nanoharvesting of bioactive materials from living plant cultures using engineered silica nanoparticles.

Plant secondary metabolites are valuable therapeutics not readily synthesized by traditional chemistry techniques. Although their enrichment in plant cell cultures is possible following advances in biotechnology, conventional methods of recovery are destructive to the tissues. Nanoharvesting, in which nanoparticles are designed to bind and carry biomolecules out of living cells, offers continuous production of metabolites from plant cultures. Here, nanoharvesting of polyphenolic flavonoids, model plant-derived therapeutics, enriched in Solidago nemoralis hairy root cultures, is performed using engineered mesoporous silica nanoparticles (MSNPs, 165 nm diameter and 950 m2/g surface area) functionalized with both titanium dioxide (TiO2, 425 mg/g particles) for coordination binding sites, and amines (NH2, 145 mg/g particles) to promote cellular internalization. Intracellular uptake and localization of the nanoparticles (in Murashige and Skoog media) in hairy roots were confirmed by tagging the particles with rhodamine B isothiocyanate, incubating the particles with hairy roots, and quenching bulk fluorescence using trypan blue. Nanoharvesting of biologically active flavonoids was demonstrated by observing increased antiradical activity (using 2,2-diphenyl-1-picrylhydrazyl radical scavenging assay) by nanoparticles after exposure to hairy roots (indicating general antioxidant activity), and by the displacement of the radio-ligand [3H]-methyllycaconitine from rat hippocampal nicotinic receptors by solutes recovered from nanoharvested particles (indicating pharmacological activity specific to S. nemoralis flavonoids). Post-nanoharvesting growth suggests that the roots are viable after nanoharvesting, and capable of continued flavonoid synthesis. These observations demonstrate the potential for using engineered nanostructured particles to facilitate continuous isolation of a broad range of biomolecules from living and functioning plant cultures.

Topiramate attenuates the stress-induced increase in alcohol consumption and preference in male C57BL/6J mice.

Stress increases the risk for alcohol abuse and relapse behaviors. However, there are hardly any medications to counteract stress-induced alcoholism and relapse behaviors. The present study examined the effects of topiramate (intraperitoneal injections of 10, 20, and 30 mg/kg) in its ability to attenuate alcohol consumption on exposure to restraint stress in C57BL/6J mice on a 2-choice test procedure. Mice were either restrained for 1h/day for 5 successive days or left unrestrained. Subsequently, the effects of topiramate were studied in post-restraint days. Results showed that restrained animals increased alcohol consumption and alcohol preference significantly compared to control group on day 5. On post-restraint days, topiramate reduced alcohol consumption and alcohol preference on days 2-5 compared to saline. This experiment suggests that one mechanism of topiramate in reducing alcohol consumption and alcohol preference may involve an interaction with stress.

Difluoromethylornithine (DFMO) reduces deficits in isolation-induced ultrasonic vocalizations and balance following neonatal ethanol exposure in rats.

Neonatal ethanol (EtOH) exposure is associated with central nervous system dysfunction and neurotoxicity in rats. Increases in polyamine levels have been implicated as one underlying mechanism for some of EtOH's effects on the developing brain. In this study we addressed whether the inhibition of polyamine biosynthesis by alpha-difluoromethylornithine (DFMO) could reduce behavioral deficits induced by early EtOH exposure. Male and female rat pups received ethanol (6 g/kg/day EtOH i.g.), or isocaloric maltose (control) from postnatal days (PND) 1-8. On PND 8, animals were injected with either saline or DFMO (500 mg/kg, s.c.) immediately following the final neonatal treatment. Subjects were tested for isolation-induced ultrasonic vocalizations (USV) on PND 16; spontaneous activity in an open field apparatus on PND 20 and 21; and balance on PND 31. Animals exposed to EtOH neonatally displayed an increased latency to the first USV and reduced frequencies of USV, hyperactivity and preference for the center of the open field and poorer balance relative to controls. DFMO minimized these deficits in latency to the first USV and balance. These data provide further support that polyamines play a role in some of the functional deficits associated with EtOH exposure during early development and that reducing polyamine activity can improve outcome.

Effects of mecamylamine on alcohol consumption and preference in male C57BL/6J mice.

Alcohol and nicotine (in the form of tobacco) are 2 commonly used recreational drugs and studies show a high correlation between tobacco use and alcohol consumption. In the present study, using C57BL/6J mice, we investigated the ability of mecamylamine (a nicotinic antagonist) to reduce alcohol consumption and alcohol preference with free 24-hour access using a 2-bottle choice test drinking procedure. Male C57BL/6J mice were individually housed and acclimatized to 10% alcohol. Immediately following the last day of alcohol acclimatization, the mice (n = 5/group) received subcutaneous injections of mecamylamine (0.5, 1 and 2 mg/kg) or saline consisting of either intermittent (3 injections given every other day) or daily (injections on all 5 days) exposures. Fluid consumption (alcohol and water) was recorded daily. The results showed that mecamylamine significantly reduced alcohol consumption and alcohol preference in both phases of intermittent and daily drug exposures, while the total fluid consumption was unchanged. These results provide further support that mecamylamine is effective in reducing alcohol consumption and preference, and nicotinic-receptor-based drugs could further be explored as potential treatments for alcoholism.

Promoter analysis of the Catharanthus roseus geraniol 10-hydroxylase gene involved in terpenoid indole alkaloid biosynthesis.

Geraniol 10-hydroxylase (G10H) is an important enzyme in the biosynthetic pathway of monoterpenoid alkaloids found in diverse plant species. The Catharanthus roseus G10H controls the first committed step in biosynthesis of terpenoid indole alkaloids (TIA). The C. roseus G10H promoter sequence was isolated by a PCR-based genome walking method. Sequence analysis revealed that the G10H promoter contains several potential eukaryotic regulatory elements involved in regulation of gene expression. The major transcription start site of the promoter was mapped to an adenine 31 bp downstream of the TATA-box. For functional characterization, transcriptional fusions between the G10H promoter fragments with 5' or 3' deletions and the GUS reporter gene were generated and their expressions were analyzed in a tobacco protoplast transient expression assay. Deletion of the promoter down to -318 bp had little effect on GUS activity. However, further deletion of the promoter to position -103 resulted in approximately 5-fold reduction of GUS activity. Gain-of-function experiments revealed the presence of three potential transcriptional enhancers located in regions between -191 and -147, -266 and -188, and -318 and -266, respectively. The G10H promoter was capable of conferring stable GUS expression in transgenic tobacco plants and C. roseus hairy roots. In transgenic tobacco seedlings GUS expression was tissue-specific, restricted to leaf and actively growing cells around the root tip, and not detected in the hypocotyls, root cap and older developing areas of the root. The GUS expression in both transgenic C. roseus hairy roots and tobacco seedlings were responsive to fungal elicitor and methyljasmonate. Compared to other known promoters of TIA pathway genes, the G10H promoter contains unique binding sites for several transcription factors, suggesting that the G10H promoter may be regulated by a different transcriptional cascade.

Acamprosate: recent findings and future research directions.

This article explores the mechanisms of action and the potential responder profile of acamprosate, a compound efficacious in relapse prevention of alcoholism. New evidence at the molecular and cellular level suggests that acamprosate attenuates hyper-glutamatergic states that occur during early abstinence and involves iono (NMDA)- and metabotrotropic (mGluR5) glutamate receptors along with augmented intracellular calcium release and electrophysiological changes. Thus mutant mice with enhanced glutamate levels exhibit higher alcohol consumption than wild type mice and respond better to acamprosate, demonstrating that acamprosate acts mainly on a hyper-glutamatergic system. This mode of action further suggests that acamprosate exhibits neuroprotective properties. In rats, cue-induced reinstatement behavior is significantly reduced by acamprosate treatment whereas cue-induced craving responses in alcohol-dependent patients seem not to be affected by this treatment. An ongoing study ("Project Predict") defines specific responder profiles for an individualized use of acamprosate and naltrexone. Neurophysiological as well as psychometric data are used to define 2 groups of patients: "reward cravers" and "relief cravers". While naltrexone should work better in the first group, acamprosate is hypothesized to be efficacious in the latter where withdrawal associated and/or cue induced hyper-glutamatergic states are thought to trigger relapse. Further research should target the definition of subgroups applying endophenotypic approaches, e.g. by detecting a hyperglutamatergic syndrome using MR spectroscopy.

Age and gender differences in response to neonatal ethanol withdrawal and polyamine challenge in organotypic hippocampal cultures.

BACKGROUND: Polyamines are synthesized and released in high concentrations during CNS development. These agents can potentiate N-methyl-D-aspartate receptor (NMDAR) function and appear to play an important role in CNS development. Previous work has shown that polyamine release is increased during ethanol withdrawal (EWD). This likely promotes NMDAR overactivity and contributes to neurotoxicity during EWD, however, little is known regarding such effects in early neonatal brain. The present study compared the effects of EWD and polyamine exposure on toxicity in hippocampal slice cultures derived from postnatal day 2 (PND 2) or postnatal day 8 (PND 8) day-old rats. Due to changes in NMDAR subtypes and response to polyamines, we predicted that slices taken from PND 2 pups would be more sensitive to EWD and polyamine challenge. METHODS: Organotypic hippocampal slice cultures were obtained from neonatal rats either 2 or 8 days of age (PND 2 or PND 8). Five days after explantation, cultures were exposed to ETOH (50 mM- typically subthreshold for EWD induced cell death) for 10 days and then withdrawn from ETOH for 24-hour in the presence of 100 microM of the polyamine spermidine and/or 100 microM ifenprodil, an NMDAR antagonist that blocks the NMDAR that is the most sensitive to polyamine modulation. Cytotoxicity was measured after 24-hour by visualization of propidium iodide (PI) fluorescence. RESULTS: There were clear age and gender-dependent differences in response to EWD and to polyamines. EWD produced significant increases in PI uptake in all subregions (CA1, CA3 and DG) of cultures derived from PND 2 pups, but not PND 8 pups. Exposure of cultures to spermidine for 24-hour also produced significant increases in cytotoxicity in all 3 regions of PND 2 cultures with no gender differences. In contrast, there were both gender and region-specific differences in response to spermidine in cultures from PND 8. While the CA1 region of both sexes displayed increased cytotoxicity following spermidine exposure, only females showed increased cytotoxicity in the CA3 region while the DG appeared relatively insensitive to spermidine. Exposure to spermidine during EWD produced enhanced toxicity in all 3 hippocampal subregions in tissue from both PND 2 and PND 8 rats and this was reduced or prevented by co-exposure to ifenprodil. Of interest, the PND 2 hippocampus was significantly more sensitive than the PND 8 hippocampus to the toxic effects of EWD and to spermidine during EWD in the DG and CA3 regions. CONCLUSIONS: Hippocampal slice cultures derived from PND 2 rats were more sensitive to the toxic effects of both EWD and EWD + spermidine exposure than were those derived from PND 8 rats. These findings are similar to recent behavioral data collected from our lab showing greater sensitivity to ETOH's behavioral teratogenic effects when ETOH exposure in vivo occurred during the first postnatal week relative to the second postnatal week. Ifenprodil's ability to block the toxic effects of spermidine during EWD suggests that excess activity of NR2B subunits of the NMDAR contributed to the excitatory and cytotoxic effects of EWD plus spermidine. While no sex differences in toxicity were observed in cultures taken from pups during the first postnatal week, these data do suggest that later in neonatal life (i.e., the second postnatal week), the female hippocampus may be more sensitive to polyamine-induced neurotoxicity than males.

Alcohol withdrawal-induced hippocampal neurotoxicity in vitro and seizures in vivo are both reduced by memantine.

BACKGROUND: The ethanol withdrawal (EWD) syndrome is typically treated using benzodiazepines such as diazepam. However there is concern that benzodiazepines may not prevent neurotoxicity associated with EWD. Antagonists of glutamate/N-Methyl-D-Aspartate receptors (NMDARs) such as MK801 have been shown to be effective against both EWD-induced neurotoxicity in vitro and seizures in vivo. However, most of these agents have adverse side effects. An exception is the moderate affinity NMDAR channel blocker memantine, used in Alzheimer's dementia. The present studies examined the ability of memantine to protect against EWD-related toxicity in vitro and seizures in vivo. METHODS: Organotypic hippocampal slice cultures from neonatal rat pups were treated starting at 15 days in vitro with 100 mM ethanol for 10 days followed by a 24-hour EWD period. During the 24-hour EWD period cultures were treated with memantine (15 or 30 microM). MK801 (10 microM) was utilized as a positive control. For the in vivo studies, the ability of memantine (2, 5, 10, and 15 mg/kg) to reduce convulsions was analyzed in Swiss-Webster mice using the handling induced convulsion test paradigm. RESULTS: In vitro studies demonstrated that memantine is effective at blocking EWD-induced neurotoxicity. In vivo experiments showed that memantine also significantly reduced convulsions induced by EWD in mice. CONCLUSIONS: Memantine may be of therapeutic value during alcohol detoxification by virtue of its having neuroprotective effects in addition to anti-seizure activity. The potential role of memantine in treatment of alcoholism is deserving of further study.

Acamprosate attenuates the handling induced convulsions during alcohol withdrawal in Swiss Webster mice.

In the present study, we examined the effects of acamprosate for its ability to reduce handling induced convulsions (HICs) during alcohol withdrawal. Diazepam was used as a positive control. Swiss Webster male mice received three daily IP injections of alcohol (2.5 g/kg) or alcohol (2.5 g/kg)+methylpyrazole (4-MP) (9 mg/kg). (4-MP, being an alcohol dehydrogenase inhibitor slows down the breakdown of alcohol. 4-MP in combination with alcohol exhibits a dramatic increase in blood alcohol level compared to alcohol alone). Ten hours following the last alcohol injection, the mice were picked up by the tail and examined for their seizure susceptibility (HICs). Diazepam, a benzodiazepine known to reduce seizures during alcohol withdrawal, significantly reduced these HICs at doses of 0.25, 0.5 and 1 mg/kg (p's<0.001). Acamprosate, an anti-relapse compound used clinically in newly abstinent alcoholics, also reduced these HICs at doses of 100, 200 and 300 mg/kg (p's<0.05). This study supports the use of acamprosate during periods of alcohol withdrawal as well as during abstinence.

Methamphetamine exposure antagonizes N-methyl-D-aspartate receptor-mediated neurotoxicity in organotypic hippocampal slice cultures.

Glutamatergic systems have been increasingly recognized as mediators of methamphetamine's (METH) pharmacological effects though little is known about the means by which METH interacts with glutamate receptors. The present studies examined effects of METH (0.1-100 microM) on [3H]MK-801 binding to membranes prepared from adult rat cortex, hippocampus and cerebellum, as well as the neurotoxicity produced by 24-h exposure to N-methyl-D-aspartate (5-10 microM; NMDA) employing organotypic hippocampal slice cultures of neonatal rat. Co-incubation of [3H]MK-801 with METH (0.1-100 microM) did not reduce dextromethorphan (1 mM)-displaceable ligand binding. Exposure of slice cultures to NMDA for 24-h produced increases in uptake of the non-vital fluorescent marker propidium iodide (PI) of 150-500% above control levels, most notably, in the CA1 region pyramidal cell layer. Co-exposure to METH (>1.0 microM) with NMDA (5 microM) reduced PI uptake by approximately 50% in each subregion, though the CA1 pyramidal cell layer was markedly more sensitive to the protective effects of METH exposure. In contrast, METH exposure did not reduce PI uptake stimulated by 24-h exposure to 10 microM NMDA. Co-exposure to the NMDA receptor antagonist D-2-amino-5-phosphonovaleric acid (20 microM) prevented toxicity produced by exposure to 5 or 10 microM NMDA. These findings indicate that the pharmacological effects of short-term METH exposure involve inhibition of NMDA receptor-mediated neuronal signaling, not reflective of direct channel inhibition at an MK-801-sensitive site.