Different alcohol exposures induce selective alterations on the expression of dynorphin and nociceptin systems related genes in rat brain.

Molecular mechanisms of adaptive transformations caused by alcohol exposure on opioid dynorphin and nociceptin systems have been investigated in the rat brain. Alcohol was intragastrically administered to rats to resemble human drinking with several hours of exposure: water or alcohol (20% in water) at a dose of 1.5 g/kg three times daily for 1 or 5 days. The development of tolerance and dependence were recorded daily. Brains were dissected 30 minutes (1- and 5-day groups) or 1, 3 or 7 days after the last administration for the three other 5-day groups (groups under withdrawal). Specific alterations in opioid genes expression were ascertained. In the amygdala, an up-regulation of prodynorphin and pronociceptin was observed in the 1-day group; moreover, pronociceptin and the kappa opioid receptor mRNAs in the 5-day group and both peptide precursors in the 1-day withdrawal group were also up-regulated. In the prefrontal cortex, an increase in prodynorhin expression in the 1-day group was detected. These data indicate a relevant role of the dynorphinergic system in the negative hedonic states associated with multiple alcohol exposure. The pattern of alterations observed for the nociceptin system appears to be consistent with its role of functional antagonism towards the actions of ethanol associated with other opioid peptides. Our findings could help to the understanding of how alcohol differentially affects the opioid systems in the brain and also suggest the dynorphin and nociceptin systems as possible targets for the treatment and/or prevention of alcohol dependence.

Olanzapine counteracts stress-induced anxiety-like behavior in rats.

Atypical antipsychotics, such as olanzapine, have been reported to display anxiolytic properties as shown in several preclinical and clinical studies. Furthermore, several experimental evidences have shown that olanzapine reduces fear and anxiety in activated anxiety-like behavior test such as Geller-Seifter test, ultrasonic vocalization test and stress-induced EtOH consumption. Here, we hypothesized that the anxiolytic action of olanzapine might be due to via an indirect activation of the gamma-amino butyric acid (GABA)-ergic system through 3alpha-hydroxy-5alpha-pregnan-20-one [allopregnanolone (ALLO)], a potent neuroactive steroid that positively modulates the benzodiazepine-gamma-aminobutyric acid type A (GABA(A))/benzodiazepine receptors complex. To address this question, we used a preclinical animal test to screen for novel anxiolytic compounds - the elevated plus-maze (EPM) - in basal condition and after 45 min restrain stress after acute or repeated (21 days) administration of olanzapine (0.5mg/kg, i.p.). In this condition, we therefore study the effect of the 5-alpha-reductase inhibitor finasteride (FIN) (50mg/kg) after co-administration with olanzapine. FIN is an inhibitor of steroidogenic enzymes which acts by inhibiting type II 5-alpha reductase, the enzyme that converts into 5-alpha-reduced metabolites like the GABA(A) positive neuroactive steroid ALLO. Results showed an anxiolytic effect of the acute, but not of the chronic, treatment with olanzapine only in stressed rats. This anxiolytic effect was counteracted by the co-administration with FIN. These evidences suggest that the anxiolytic effects of olanzapine might be due to possible action of olanzapine on steroid function via activation of GABA system.

Water T-maze, an improved method to assess spatial working memory in rats: Pharmacological validation.

The present study was performed to validate a spatial working memory task using pharmacological manipulations. The water escape T-maze combines the advantages of the Morris water maze and the T-maze while minimizing the disadvantages. Scopolamine (1mg/kg), a drug that affects cognitive function in spatial working memory tasks, significantly decreased the rats' performance in the present delayed alternation task. Glutamate neurotransmission plays an important role in the maintenance of working memory; rats treated with dizocilpine (MK-801; 0.125-0.25mg/kg), a N-methyl-d-aspartate (NMDA) receptor antagonist, were impaired in this task. In agreement with evidence showing a functional interaction between ionotropic and metabotropic glutamatergic receptors, 2-methyl-6-(phenylethynyl)-pyridine (MPEP), a mGlu(5) receptor antagonist, at a dose (1mg/kg) which by itself had no significant effects, enhanced MK-801-induced impairments of spatial working memory. These evidences suggest that the water escape T-maze might be a valid method to assess spatial working memory, sensitive to pharmacological manipulations.

Determination of Olanzapine in rat brain using liquid chromatography with coulometric detection and a rapid solid-phase extraction procedure.

A sensitive and selective method was developed for the determination of the antipsychotic drug Olanzapine levels in rat brain tissue, based on HPLC with electrochemical detection. The analyses were carried out on a C8 reversed phase column (150 mm x 4.6 mm, 5 microm), using a mobile phase composed of methanol and a phosphate buffer (44.0 mM, pH 3.5), containing triethylamine (21:79, v/v), flowing at 1.2 mL min(-1). A high sensitivity coulometric detection analytical cell containing two flow-through low volume working electrodes was used: electrode 1 was set at +0.350 V and electrode 2 at -0.200 V. Olanzapine, administered to rats in different doses or in different times, was extracted from tissue homogenate of either the whole brain or specific areas (cortex, hyppocampus, nucleus striatum) with a rapid solid phase extraction procedure (SPE) on Oasis HLB cartridges. The method provided a high extraction yield of Olanzapine and internal standard (2-methylolanzapine) from brain tissue homogenate with absolute recovery values higher than 90.0%. The detector response was linear over a concentration range of 0.2-100.0 ng mL(-1) of Olanzapine. The limit of quantification (LOQ) was 0.2 ng mL(-1). Precision results, expressed by the intra-day and the inter-day relative standard deviation values, were satisfactory, better than 4.6%. Accuracy was satisfactory as well. This method proved to be suitable for the analysis of Olanzapine in rat brain tissues and for the study of distribution and pharmacokinetics of Olanzapine in rat brain after a single treatment with the antipsychotic drug.

Repeated administration of the novel antipsychotic olanzapine does not modulate NMDA-sensitive glutamate and 5HT2 serotonin receptors in rats.

Antipsychotic drugs reportedly show a common property in facilitating glutamatergic transmission in rat cerebral cortex. Since the binding of the radiolabelled channel blocker [3H]-MK801 is generally considered an affordable index of N-methyl-d-aspartate (NMDA)-sensitive glutamate receptor activation, we examined the effects of clinically effective treatment (3 weeks, daily administration) of the atypical antipsychotic drug olanzapine (32 micromol/kg/5 ml) on the specific binding of [3H]-MK801 specific binding and on the strychnine-insensitive glycine sites (glycine B) in synaptic plasma membranes (SPM) prepared from the medial prefrontal cortex (mPFC) of rats sacrificed after different (24, 60, 120 h) washout periods. We studied also the effects of repeated olanzapine administration on [3H]-ketanserin binding to 5HT2A receptors to verify whether, consistent with previously reported paradoxical effects of repeated administration of 5HT2A antagonists, this drug decreases 5HT2A receptor density without changing the apparent affinity. Neither single nor repeated olanzapine administration changed the kinetic characteristics of [3H]-MK801 or [3H]-glycine specific binding. When rats were sacrificed 120 h after the last olanzapine administration, both single or repeated treatment had failed to change the kinetic characteristics of [3H]-ketanserin binding, while the apparent affinity of 5HT2A receptors was increased in animals sacrificed at shorter (60 h) washout periods. Owing to the long half-life of olanzapine (24 h), and since the drug concentrations in mPFC of rats sacrificed 60 h after a single olanzapine administration (about 50 nM) are high enough to induce changes in 5HT2A receptor affinity, it is concluded that this modification, probably unrelated to the therapeutic efficacy, could be due to some drug still present in the brain at the time of the sacrifice.

An ionotropic but not a metabotropic glutamate agonist potentiates the pharmacological effects of olanzapine in the rat.

This study aimed to evaluate the possible potentiating action of ionotropic or metabotropic (metabotropic glutamate receptor type 5) glutamate agonists on pharmacological effects induced in rats by the atypical antipsychotic olanzapine. The administration of doses of olanzapine, which did not affect spontaneous motility, inhibited behaviors induced by the selective stimulation of 5HT(2A) and D(2) receptors. In particular, 0.03 or 0.06 mg/kg of olanzapine was sufficient to reduce, respectively, head shakes induced by the 5HT(2A) agonist 1-2,5-dimethoxy-4-iodophenyl-2-aminopropane (1 mg/kg) or hypermotility elicited by the D(2) stimulant quinpirole (0.15 mg/kg). Behavioral responses to a D(1)/D(2) agonist (apomorphine-induced stereotypies) were inhibited by doses of olanzapine that also influenced spontaneous behavior. The concomitant administration of D-cycloserine, an agonist at the glycine site on the N-methyl-D-aspartate receptor complex, given at a dose (3 mg/kg) that did not affect behavior, increased the inhibitory effect of olanzapine on the responses produced by 5HT2A, D(2) and D(1)/D(2) receptor stimulation. The concomitant administration of 2-chloro-5-hydroxyphenylglycine, an agonist of metabotropic glutamate receptor type 5, increased the inhibitory effect of olanzapine on the behaviors induced by the stimulation of D(2), but not 5HT2A or D(1)/D(2) receptors. As the effect on the serotonergic system seems important for the unusual pharmacological profile of atypical antipsychotics, the present results suggest that N-methyl-D-aspartate, but not metabotropic glutamate receptor type 5 agonists could be seen as promising therapeutic agents for increasing the pharmacological effects of olanzapine.