A comparison of imputation procedures and statistical tests for the analysis of two-dimensional electrophoresis data.

BACKGROUND: Numerous gel-based softwares exist to detect protein changes potentially associated with disease. The data, however, are abundant with technical and structural complexities, making statistical analysis a difficult task. A particularly important topic is how the various softwares handle missing data. To date, no one has extensively studied the impact that interpolating missing data has on subsequent analysis of protein spots. RESULTS: This work highlights the existing algorithms for handling missing data in two-dimensional gel analysis and performs a thorough comparison of the various algorithms and statistical tests on simulated and real datasets. For imputation methods, the best results in terms of root mean squared error are obtained using the least squares method of imputation along with the expectation maximization (EM) algorithm approach to estimate missing values with an array covariance structure. The bootstrapped versions of the statistical tests offer the most liberal option for determining protein spot significance while the generalized family wise error rate (gFWER) should be considered for controlling the multiple testing error. CONCLUSIONS: In summary, we advocate for a three-step statistical analysis of two-dimensional gel electrophoresis (2-DE) data with a data imputation step, choice of statistical test, and lastly an error control method in light of multiple testing. When determining the choice of statistical test, it is worth considering whether the protein spots will be subjected to mass spectrometry. If this is the case a more liberal test such as the percentile-based bootstrap t can be employed. For error control in electrophoresis experiments, we advocate that gFWER be controlled for multiple testing rather than the false discovery rate.

Structural and functional effects of developmental exposure to ethanol on the zebrafish heart.

BACKGROUND: Fetal alcohol exposure during development results in a host of cardiac abnormalities including atrial and ventricular septal defects, teratology of Fallot, d-transposition of the great arteries, truncus arteriosus communis, and aortico-pulmonary window. The mechanisms behind these ethanol-induced deficits are unknown. The purpose of this study was to determine whether the zebrafish, a simple model in which heart development and the sequence of gene expression is well elucidated and comparable to that in higher vertebrates, is sensitive to developmental exposure of pharmacologically relevant concentrations of ethanol. METHODS: Zebrafish eggs of the AB strain were raised in egg water or in 0.5% (v/v) ethanol solution for either 54 hpf (hours postfertilization) or 72 hpf. Heart pathology and volumes were evaluated on the latter group at 5 dpf (days postfertilization) on tissue sections from fixed larvae embedded in glycolmethacrylate. Heart rates were determined in embryos of 54 hpf and larvae of 5 dpf. The functional maturity of the heart's conducting system was measured by determining the response of ethanol-treated and control embryos and larvae to the adrenergic agonist, isoproterenol, and the cholinergic agonist, carbachol. RESULTS: Ethanol-induced alterations occurred in heart morphology and heart volume. A developmental lag in the isoproterenol response and the absence of carbachol-mediated bradycardia were also observed following ethanol treatment. CONCLUSIONS: These results show that exposure of the zebrafish to ethanol during development results in structural and functional changes in the heart that mimic malformations that occur in patients with fetal alcohol syndrome (FAS). These findings promote the zebrafish heart as a future model for investigating the mechanisms responsible for ethanol's adverse effects on vertebrate heart development.

Statins decrease expression of the proinflammatory neuropeptides calcitonin gene-related peptide and substance P in sensory neurons.

Clinical and experimental observations suggest that statins may be useful for treating diseases presenting with predominant neurogenic inflammation, but the mechanism(s) mediating this potential therapeutic effect are poorly understood. In this study, we tested the hypothesis that statins act directly on sensory neurons to decrease expression of proinflammatory neuropeptides that trigger neurogenic inflammation, specifically calcitonin gene-related peptide (CGRP) and substance P. Reverse transcriptase-polymerase chain reaction, radioimmunoassay, and immunocytochemistry were used to quantify CGRP and substance P expression in dorsal root ganglia (DRG) harvested from adult male rats and in primary cultures of sensory neurons derived from embryonic rat DRG. Systemic administration of statins at pharmacologically relevant doses significantly reduced CGRP and substance P levels in DRG in vivo. In cultured sensory neurons, statins blocked bone morphogenetic protein (BMP)-induced CGRP and substance P expression and decreased expression of these neuropeptides in sensory neurons pretreated with BMPs. These effects were concentration-dependent and occurred independent of effects on cell survival or axon growth. Statin inhibition of neuropeptide expression was reversed by supplementation with mevalonate and cholesterol, but not isoprenoid precursors. BMPs signal via Smad activation, and cholesterol depletion by statins inhibited Smad1 phosphorylation and nuclear translocation. These findings identify a novel action of statins involving down-regulation of proinflammatory neuropeptide expression in sensory ganglia via cholesterol depletion and decreased Smad1 activation and suggest that statins may be effective in attenuating neurogenic inflammation.

d-LSD-induced c-Fos expression occurs in a population of oligodendrocytes in rat prefrontal cortex.

Induction of mRNA or protein for immediate-early genes, such as c-fos, is used to identify brain areas, specific cell types, and neuronal circuits that become activated in response to various stimuli including psychoactive drugs. The objective of the present study was to identify the cell types in the prefrontal cortex in which lysergic acid diethylamide (d-LSD) induces c-Fos expression. Systemic administration of d-LSD resulted in a dose-dependent increase in c-Fos immunoreactivity. Although c-Fos-positive cells were found in all cortical layers, they were most numerous in layers III, IV, and V. d-LSD-induced c-Fos immunoreactivity was found in cells co-labeled with anti-neuron-specific enolase or anti-oligodendrocyte Oligo1. The Oligo1-labeled cells had small, round bodies and nuclear diameters characteristic of oligodendrocytes. Studies using confocal microscopy confirmed colocalization of c-Fos-labeled nuclei in NeuN-labeled neurons. Astrocytes and microglia labeled with glial fibrillary acidic protein antibody and OX-42 antibody, respectively, did not display LSD-induced c-Fos expression. Pyramidal neurons labeled with anti-neurofilament antibody also did not show induction of c-Fos immunoreactivity after systemic d-LSD administration. The present study demonstrates that d-LSD induced expression of c-Fos in the prefrontal cortex occurs in subpopulations of neurons and in oligodendrocytes, but not in pyramidal neurons, astrocytes, and microglia.

Ocular deficits associated with alcohol exposure during zebrafish development.

Approximately 90% of fetal alcohol syndrome cases are accompanied by ocular abnormalities. The zebrafish (Danio rerio) is a well-known developmental model that provides an opportunity for better understanding the histological and cytological effects of developmental exposure to ethanol on the vertebrate eye. The purpose of the present study was to determine the gross, microscopic, and ultrastructual effects of developmental exposure to ethanol in the zebrafish model. Eggs were obtained from WT outbred zebrafish and exposed to 0%, 0.1%, 0.2%, 0.4%, 0.5%, or 1.0% (v/v) ethanol to assess viability and the effect of dose and duration of exposure on eye size. Light and electron microscopy were performed on ethanol-treated and control larvae. Results showed that ethanol treatment decreased viability by about 20% at concentrations of 0.1-0.5% ethanol and by 50% at 1.0% ethanol. Ethanol-related decreases in eye size were recorded at 6 days postfertilization (dpf) and were dose dependent. There were significant decreases in the volumes of the photoreceptor, inner nuclear, and ganglionic layers and in the lens of 9 dpf ethanol-exposed compared with control larvae. Ultrastructural examination showed signs of developmental lags in the ethanol-treated fish as well as abnormal retinal apoptosis in the 6 dpf ethanol-treated larvae compared with their controls. These results demonstrate that the developing zebrafish eye is sensitive to perturbation with ethanol and displays some of the eye deficits present in fetal alcohol syndrome.

Minimizing variability in two-dimensional electrophoresis gel image analysis.

For reliable protein identification and quantitation, it is important to minimize the variability associated with two-dimensional electrophoresis (2-DE) analysis. Since experimental factors contribute largely to the variability observed in 2-DE, most studies have focused on reducing this variability with modest concern to the variability associated with post-experimental analyses. Although often ignored, software analyses of 2-DE gel images present a considerable source of variability in the analysis of proteins. In particular, cropping of gel images prior to quantitative 2-DE analysis has been shown to contribute a significant amount of variability in image analysis. To address this problem, we propose a simple, reliable, and objective method of cropping 2-DE gel images to consequently minimize the variability in 2-DE analysis.

Mechanism of ethanol enhancement of apoptosis and caspase activation in serum-deprived PC12 cells.

Neuronal death is one of the most prominent consequences of alcohol exposure during development. Ethanol-induced neuronal death appears to involve apoptosis. The objective of the present study was to characterize the effect of ethanol on neuronal cell viability and to determine the mechanism by which ethanol enhances apoptosis in neural cells. For these studies the rat pheochromocytoma (PC12) cells were used. PC12 cells were incubated for 24 h in the presence or absence of 100 mM ethanol. Apoptosis was induced by serum withdrawal. Ethanol in the presence of serum-containing media did not alter cell viability, while incubation of PC12 cells in serum-free media resulted in a significant increase in cell death that was further significantly increased by 35% in cells exposed to ethanol. The temporal response of the PC12 cells to serum withdrawal was studied over a period of 22 h. At least 18 h of ethanol exposure was necessary to observe a significant increase in death for cells incubated in serum-free media. An increase in the caspase-3 activity in PC12 cells deprived of serum was observed that was further increased by ethanol exposure. This increase of caspase-3 activity was correlated with an enhancement of caspase-9 activity. Ethanol exposure increased the amount of cytosolic cytochrome c in PC12 cells incubated in serum-free media but did not alter the level of cytochrome c in cells incubated in serum. Finally, a 26% increase was observed in the number of cells with depolarized mitochondria due to ethanol treatment. The present study implicates an increase in the mitochondrial outer membrane permeability as a potential mechanism of enhancement of apoptosis in serum-deprived PC12 cells by ethanol.

Evaluating peptide mass fingerprinting-based protein identification.

Identification of proteins by mass spectrometry (MS) is an essential step in proteomic studies and is typically accomplished by either peptide mass fingerprinting (PMF) or amino acid sequencing of the peptide. Although sequence information from MS/MS analysis can be used to validate PMF-based protein identification, it may not be practical when analyzing a large number of proteins and when high- throughput MS/MS instrumentation is not readily available. At present, a vast majority of proteomic studies employ PMF. However, there are huge disparities in criteria used to identify proteins using PMF. Therefore, to reduce incorrect protein identification using PMF, and also to increase confidence in PMF-based protein identification without accompanying MS/MS analysis, definitive guiding principles are essential. To this end, we propose a value-based scoring system that provides guidance on evaluating when PMF-based protein identification can be deemed sufficient without accompanying amino acid sequence data from MS/MS analysis.

Effects of chronic ethanol administration on brain protein levels: a proteomic investigation using 2-D DIGE system.

The effects of chronic ethanol treatment on the brain proteome were investigated in the long-fin striped strain of zebrafish Danio rerio. Prolonged exposure to 0.5% (v/v) ethanol resulted in the development of tolerance to the ethanol-induced disruption of normal swimming behavior. This behavioral tolerance was manifested after two weeks of continuous treatment and was maintained for an additional three weeks. After four weeks of ethanol treatment, zebrafish brains were divided into 40,000 g supernatant and pellet fractions, and an Ettan 2-D fluorescence difference gel electrophoresis (DIGE) system was used to detect ethanol-induced alterations in the level of protein expression. Protein identification was carried out using matrix assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry and the Mascot and ProFound search engines. In the present study, we have identified some novel protein targets as well as substantiated some putative previous targets of chronic ethanol exposure.

Hallucinogen-like actions of 2,5-dimethoxy-4-(n)-propylthiophenethylamine (2C-T-7) in mice and rats.

RATIONALE: Few studies have examined the effects of 2,5-dimethoxy-4-(n)-propylthiophenethylamine (2C-T-7) in vivo. OBJECTIVES: 2C-T-7 was tested in a drug-elicited head twitch assay in mice and in several drug discrimination assays in rats; 2C-T-7 was compared to the phenylisopropylamine hallucinogen R(-)-1-(2,5-dimethoxy-4-methylphenyl)-2aminopropane (DOM) in both assays, with or without pretreatment with the selective 5-HT2A antagonist (+)-(2,3-dimethoxyphenyl)-1-[2-(4-fluorophenylethyl)]-4-piperidine-methanol (M100907). Finally, the affinity of 2C-T-7 for three distinct 5-HT receptors was determined in rat brain. METHODS: Drug-elicited head twitches were quantified for 10 min following administration of various doses of either 2C-T-7 or R(-)-DOM, with and without pretreatments of 0.01 mg/kg M100907. In rats trained to discriminate lysergic acid diethylamide (LSD), 2C-T-7 and R(-)-DOM were tested for generalization. In further studies, rats were trained to discriminate 2C-T-7 from saline, then challenged with 0.05 mg/kg M100907. In competition binding studies, the affinity of 2C-T-7 was assessed at 5-HT2A receptors, 5-HT1A receptors, and 5-HT2C receptors. RESULTS: 2C-T-7 and R(-)-DOM induced similar head twitch responses in the mouse that were antagonized by M100907. In the rat, 2C-T-7 produced an intermediate degree of generalization (75%) to the LSD cue and served as a discriminative stimulus; these interoceptive effects were attenuated by M100907. Finally, 2C-T-7 had nanomolar affinity for 5-HT2A and 5-HT2C receptors and lower affinity for 5-HT1A receptors. CONCLUSIONS: 2C-T-7 is effective in two rodent models of 5-HT2 agonist activity and has affinity at receptors relevant to hallucinogen effects. The effectiveness with which M100907 antagonizes the behavioral actions of 2C-T-7 strongly suggests that the 5-HT2A receptor is an important site of action for this compound.

Adult blood lead epidemiology and surveillance--United States, 1998-2001.

PROBLEM/CONDITION: Elevated blood lead levels (BLLs) in adults can damage the cardiovascular, central nervous, reproductive, hematologic, and renal systems. The majority of cases are workplace-related. U.S. Department of Health and Human Services recommends that BLLs among all adults be reduced to < 25 microg/dL. The highest BLL acceptable by standards of the U.S. Occupational Safety and Health Administration is 40 microg/dL. The mean BLL of adults in the United States is < 3 microg/dL. REPORTING PERIOD: This report covers cases of adults (aged > or = 16 years) with BLLs > or = 25 microg/dL, as reported by 25 states during 1998-2001. DESCRIPTION OF SYSTEM: Since 1987, CDC has sponsored the state-based Adult Blood Lead Epidemiology and Surveillance (ABLES) program to track cases of elevated BLLs and provide intervention consultation and other assistance. Overall ABLES program data were last published in 1999 for the years 1994-1997. This report provides an update with data from 25 states reporting for > or = 2 years during 1998-2001. During that period, the ABLES program funded surveillance in 21 states - Alabama, Arizona, Connecticut, Iowa, Maryland, Massachusetts, Michigan, Minnesota, New Jersey, New York, North Carolina, Ohio, Oklahoma, Oregon, Pennsylvania, Rhode Island, South Carolina, Texas, Washington, Wisconsin, and Wyoming. Four additional states - California, Nebraska, New Hampshire, and Utah contributed data without CDC funding. RESULTS: During 1998-2001, the overall program's annual mean state prevalence rate for adults with BLLs > or = 25 microg/dL was 13.4/100,000 employed adults. This compares with 15.2/100,000 for 1994-1997. Yearly rates were 13.8 (1998), 12.9 (1999), 14.3 (2000), and 12.5 (2001). For adults with BLLs > or = 40 microg/dL, the overall program's annual mean state prevalence rare during 1998-2001 was 2.9/ 100,000 employed adults. This compares with 3.9/100,000 for 1994-1997. Yearly rates were 3.3 (1998), 2.5 (1999), 2.9 (2000), and 2.8 (2001). INTERPRETATION: Although certain limitations exist, the overall ABLES data indicate a declining trend in elevated BLLs among employed adults. PUBLIC HEALTH ACTIONS: ABLES-funded states increased from 21 to 35 in 2002, and more detailed reporting requirements were put into effect. These, and other improvements, will enable the ABLES program to work more effectively toward its 2010 target of eliminating all cases of BLLs > or = 25 microg/dL in adults caused by workplace exposures.

Characterization of a novel effect of serotonin 5-HT1A and 5-HT2A receptors: increasing cGMP levels in rat frontal cortex.

Elucidating the mechanisms of action of hallucinogens has become an increasingly important area of research as their abuse has grown in recent years. Although serotonin receptors appear to play a role in the behavioral effects of the phenethylamine and indoleamine hallucinogens, the signaling pathways activated by these agents are unclear. Here it is shown that administration of serotonin (5-hydroxytryptamine, 5-HT) increased cyclic guanosine monophosphate (cGMP) production in frontal cortical slices of rat brain. The effect of 5-HT was greater than that of N-methyl-D-aspartate (NMDA), a stimulant of cGMP formation in the central nervous system. The 5-HT(2A/2C) receptor phenethylamine agonist, 2,5-dimethoxy-4-methylamphetamine (DOM), increased cGMP content in the slices. Additionally 8-hydroxy-2-(di-n-propylamino)tetralin (DPAT), a 5-(HT1A/7) receptor agonist also increased cGMP production. Stimulation of cGMP formation by DOM was prevented by a 5-HT(2A/2C) receptor antagonist, pirenperone, as well as by a 5-HT2A receptor selective antagonist, MDL100907. A 5-HT2C receptor antagonist, SB242084, did not block the effect of DOM. Stimulation of cGMP production by DPAT was blocked by the 5-HT1A receptor antagonist, WAY100635. Stimulation of cGMP formation by serotonin could be prevented by pirenperone or WAY100635. In summary, activation of serotonin 5-HT1A and 5-HT2A receptors increase brain cGMP levels.

Role of serotonergic 5-HT2A receptors in the psychotomimetic actions of phencyclidine.

The psychotomimetic phencyclidine (PCP) alters various behavioural responses involving the serotonergic system including potentiating the discriminative stimulus effects of the phenethylamine hallucinogen, 2,5-dimethoxy-4-methylamphetamine (DOM). The present study was undertaken to test the hypothesis that PCP directly interacts with the 5-HT2A receptor. PC12 cells, a neuronal cell line, were stably transfected with the cDNA encoding the rat 5-HT2A receptor (PC12-5-HT2A). In these cells PCP and the related compounds, ketamine and dizocilpine, did not increase [3H]inositol phosphate generation nor did they alter 5-HT-stimulated phosphoinositide hydrolysis. These compounds also did not display appreciable affinity for the 5-HT2A receptor labelled with [3H]ketanserin. The present study indicates that the behavioural responses to PCP, ketamine and dizocilpine do not involve a direct interaction of these compounds with the 5-HT2A receptor.

Partial generalization of (-)DOM to fluvoxamine in the rat: implications for SSRI-induced mania and psychosis.

Recent reports have implicated selective serotonin-reuptake inhibitors in the induction of psychosis and mania when SSRIs are given in combination with neuroleptics. We hypothesize that the partial substitution of fluvoxamine for the hallucinogen, (-)DOM, in the rat provides evidence for a 5-HT(2)-mediated effect of fluvoxamine which may in turn account for the adverse effects observed in humans. Male Fischer-344 rats were trained with (-)DOM (0.56 mg/kg) as a discriminative stimulus using standard operant procedures. Tests of generalization were then conducted with fluvoxamine either alone or in combination with the 5-HT(1A) antagonist, WAY-100635, the 5-HT(2) antagonist, pirenperone, and the neuroleptics, fluphenazine, chlorpromazine, thioridazine, loxapine, risperidone, and clozapine. In rats trained with (-)DOM, fluvoxamine at a dose of 20 mg/kg yielded a maximum 58% (-)DOM-appropriate response. This partial generalization was potentiated by treatment with WAY-100635 and antagonized by pirenperone, loxapine, risperidone, and clozapine. The present data are compatible with a 5-HT(2)-mediated effect of fluvoxamine which may play a role in SSRI-induced mania and psychosis. It is predicted by the results of this study that the probability of these adverse effects will be increased by the concurrent use of antagonists at 5-HT(1A) receptors and decreased by neuroleptics with antagonistic activity at 5-HT(2) receptors.

Lysergic acid diethylamide and [-]-2,5-dimethoxy-4-methylamphetamine increase extracellular glutamate in rat prefrontal cortex.

The ability of hallucinogens to increase extracellular glutamate in the prefrontal cortex (PFC) was assessed by in vivo microdialysis. The hallucinogen lysergic acid diethylamide (LSD; 0.1 mg/kg, i.p.) caused a time-dependent increase in PFC glutamate that was blocked by the 5-HT(2A) antagonist M100907 (0.05 mg/kg, i.p.). Similarly, the 5-HT(2A/C) agonist [-]-2,5-dimethoxy-4-methylamphetamine (DOM; 0.6 mg/kg, i.p.), which is a phenethylamine hallucinogen, increased glutamate to 206% above saline-treated controls. When LSD (10 microM) was directly applied to the PFC by reverse dialysis, a rapid increase in PFC glutamate levels was observed. Glutamate levels in the PFC remained elevated after the drug infusion was discontinued. These data provide direct evidence in vivo for the hypothesis that an enhanced release of glutamate is a common mechanism in the action of hallucinogens.

Cellular mechanisms of serotonin 5-HT2A receptor-mediated cGMP formation: the essential role of glutamate.

The current study explores the mechanisms by which activation of serotonin(2A) (5-HT(2A)) receptors increase production of cyclic guanosine monophosphate (cGMP) in slices of rat frontal cortex. Contrary to results in cortical slices, stimulation of 5-HT(2A) receptors in cells stably expressing this serotonin receptor did not alter cGMP levels. In cortical slices, stimulation of cGMP formation by 2,5-dimethoxy-4-methylamphetamine (DOM), a 5-HT(2A/2C) receptor agonist, was blocked by tetanus toxin, a substance that prevents vesicular neurotransmitter release. However, this stimulation was not altered by tetrodotoxin, an agent that inhibits depolarization-induced neurotransmitter release. Addition of an N-methyl-d-aspartate (NMDA) receptor antagonist, d-AP-7, but not of an AMPA/kainate receptor antagonist CNQX, completely inhibited DOM-mediated cGMP production in the slices. Combined application of maximally effective concentrations of NMDA and DOM elicited a greater increase in cGMP content than either drug alone. The present study shows that 5-HT(2A) receptors do not directly stimulate cGMP formation, but rather that 5-HT(2A) receptor-mediated cGMP production is dependent on extracellular glutamate activating NMDA receptors. The results indicate that 5-HT(2A) receptor-mediated cGMP production could be at least partially attributed to potentiation of NMDA receptor-mediated cGMP formation.

Ethanol effects on three strains of zebrafish: model system for genetic investigations.

The effects of acute and chronic ethanol administration on the wild-type (WT), long-fin striped (LFS), and blue long-fin (BLF) strains of zebrafish were investigated. In the LFS strain, acute exposure to 0.25% (v/v) ethanol inhibited the startle reaction and increased both the area occupied by a group of subjects and the average distance between each fish and its nearest neighbor. Similar effects were found in the WT fish although higher concentrations of ethanol were required. No effects on the behavior of the BLF fish were observed with up to 1.0% (v/v) ethanol. Brain alcohol levels were comparable among the three strains precluding a pharmacokinetic explanation for the behavioral results. In LFS zebrafish, behavioral tolerance was observed after 1 week of continual exposure to ethanol. Conversely, chronic ethanol exposure of the WT fish for up to 2 weeks did not result in the development of tolerance, but rather appeared to increase the disruptive action of the drug. The present results suggest the observed strain differences in the effects of ethanol reflect genotypic differences in both the response of the central nervous system (CNS) to ethanol as well as the ability of the CNS to adapt to ethanol exposure. Although preliminary, the present study indicates that the zebrafish is an excellent model system to investigate the genetic determinants involved in regulating the responses to ethanol.

5-HT2A receptor-stimulated phosphoinositide hydrolysis in the stimulus effects of hallucinogens.

The role of 5-HT2A-mediated stimulation of phosphoinositide hydrolysis in the discriminative effects of hallucinogens was investigated in PC12 cells stably expressing the rat 5-HT2A receptor (PC12-5-HT2A cells). The hallucinogenic compounds, D-lysergic acid diethylamide (LSD), (-)2,5-dimethoxy-4-methylamphetamine (DOM), psilocybin, N,N-dimethyltryptamine (DMT), 5-methoxy-N,N-dimethyltryptamine (MDMT) and N,N-diethyltryptamine (DET), all caused a concentration-dependent increase in the generation of [3H]inositol phosphates. The nonhallucinogenic compounds, 6-fluoro-N,N-diethyltryptamine (6-F-DET), lisuride and quipazine, also displayed significant efficacy in stimulating phosphoinositide hydrolysis, while 2-bromo-lysergic acid diethylamide (BOL), which is not a hallucinogen, did not alter inositol phosphate generation. The beta-carbolines, harmaline and harmane, also did not alter phosphoinositide hydrolysis. Comparison of these results with previous drug discrimination studies indicated the apparent lack of correlation between the degree of substitution in LSD- and DOM-trained animals and efficacy in stimulating phosphoinositide hydrolysis. The present study indicates that 5-HT2A-mediated stimulation of phosphoinositide hydrolysis does not appear to be the sole critical signaling mechanism involved in the discriminative effects of hallucinogens.

Characterization of the discriminative stimulus properties of centrally administered (-)-DOM and LSD.

Despite the plausible assumption that the effects of hallucinogens predominantly arise in the central nervous system, most studies of these drugs in intact subjects have been conducted following systemic administration. The objective of the present investigation was to characterize the stimulus effects of (-)2,5-dimethoxy-4-methylamphetamine ((-)-DOM) following intracerebroventricular administration. Chronic indwelling cannulae were implanted into the lateral ventricle of male Fischer 344 rats trained to discriminate systemically administered (-)-DOM or lysergic acid diethylamide (LSD) from saline. Time-course and dose-response relationships for (-)-DOM and LSD administered intracerebroventricularly were established. For both LSD and (-)-DOM, central administration did not change the pretreatment times required for the maximal stimulus effects to occur. However, the onset of the stimulus effect was more rapid following intracerebroventricular administration. Following pretreatment periods that maximize drug-appropriate responding, central administration of (-)-DOM and LSD was approximately 2.4- and 1.5-times more potent, respectively, than systemic administration. The results of this study are consistent with the assumption that the stimulus effects of (-)-DOM and LSD are centrally mediated.