Identification of Molecular Markers of Clozapine Action in Ketamine-Induced Cognitive Impairment: A GPCR Signaling PathwayFinder Study.

BACKGROUND: Cognitive disorders associated with schizophrenia are closely linked to prefrontal cortex (PFC) dysfunction. Administration of the non-competitive NMDA receptor antagonist ketamine (KET) induces cognitive impairment in animals, producing effects similar to those observed in schizophrenic patients. In a previous study, we showed that KET (20 mg/kg) induces cognitive deficits in mice and that administration of clozapine (CLZ) reverses this effect. To identify biochemical mechanisms related to CLZ actions in the context of KET-induced impairment, we performed a biochemical analysis using the same experimental paradigm-acute and sub-chronic administration of these drugs (0.3 and 1 mg/kg). METHODS: Since the effect of CLZ mainly depends on G-protein-related receptors, we used the Signaling PathwayFinder Kit to identify 84 genes involved in GPCR-related signal transduction and then verified the genes that were statistically significantly different on a larger group of mice using RT-PCR and Western blot analyses after the administration of acute and sub-chronic drugs. RESULTS: Of the 84 genes involved in GPCR-related signal transduction, the expression of six, ßarrestin1, ßarrestin2, galanin receptor 2 (GalR2), dopamine receptor 2 (DRD2), metabotropic glutamate receptor 1 (mGluR1), and metabotropic glutamate receptor 5 (mGluR5), was significantly altered. Since these genes affect the levels of other signaling proteins, e.g., extracellular signal-regulated kinase 1/2 (ERK1/2), G protein-coupled receptor kinase 2 (Grk2), and G protein-gated inwardly rectifying potassium 3 (Girk3), we determined their levels in PFC using Western blot. Most of the observed changes occurred after acute treatment with 0.3 mg/kg CLZ. We showed that acute treatment with CLZ at a lower dose significantly increased ßarrestin1 and ERK1/2. KET treatment induced the upregulation of ßarrestin1. Joint administration of these drugs had no effect on the ßarrestin1 level. CONCLUSION: The screening kit we used to study the expression of GPCR-related signal transduction allowed us to select several important genes affected by CLZ. However, the obtained data do not explain the mechanism of action of CLZ that is responsible for reversing KET-induced cognitive impairment.

Restraint Stress in Mice Alters Set of 25 miRNAs Which Regulate Stress- and Depression-Related mRNAs.

In the present study, we aim to identify the effect of restrain stress (RS) on the expression of miRNAs in mouse serum. We used three genotypes of animals (mice with knock-out of the gene-encoding norepinephrine transporter, NET-KO; C57BL/6J, and SWR/J) which had previously been shown to display different sensitivity to RS, and focused on miRNAs which were altered by RS in the serum of all three genotypes. An analysis of miRNAs expression allowed for the identification of a set of 25 differentially expressed miRNAs; 10 were down-regulated compared to an appropriate control group of animals, while 15 were up-regulated. The application of DIANA-miRPath v. 3.0 allowed for the identification of selected pathways (KEGG) and Gene Ontology (GO) categories that were significantly controlled by these miRNAs, while miRWalk v. 3.0-the platform that used the machine learning based algorithm, TaRPmiR-was used to find their targets. The results indicate that 25 miRNAs, identified as altered upon RS in three genotypes of mice, are responsible for regulation of mRNA-encoding proteins that are key for the main hypotheses of depression; therefore, they may help to understand the link between stress and depression at the molecular level.

Effects on brain-derived neurotrophic factor signalling of chronic mild stress, chronic risperidone and acute intracranial dopamine receptor challenges.

We have previously reported the effects of intracranial injections of dopamine D1, D2 and D3 ligands in animals subjected to the Novel Object Recognition (NOR) test following exposure to chronic mild stress (CMS) and chronic treatment with risperidone (RSP). Here, we present some molecular biological data from the same animals. It was predicted that brain-derived neurotrophic factor (BDNF) signalling in the prefrontal cortex (PFC) would reflect behavioural performance, implying an increase following acute administration of a D2 agonist or a D3 antagonist, blockade of this effect by CMS and its restoration by chronic RSP. In separate cohorts, animals were injected within the PFC or the hippocampus (HPC) with either the D1 agonist SKF-81297, the D2 agonist quinpirole or the D3 antagonist SB-277,011, following exposure to control conditions or CMS and chronic treatment with saline or RSP. Intracranial injections followed an exposure trial in the NOR test, with a retention trial 24 h later. Immediately afterwards, the animals were killed and expression of BDNF and TRKß protein, and their respective mRNAs, was measured in PFC and HPC samples. CMS decreased the expression of TRKß in both PFC and HPC. Several effects associated with intracranial injection were noted, but they were inconsistent and unrelated to CMS exposure. The effects of CMS on TRKß are consistent with a decrease in BDNF signalling, albeit that expression of BDNF itself did not change significantly. There was no evidence for an involvement of the BDNF-TRKß system in responses to RSP or dopamine ligands in animals exposed to CMS. However, there was a 24 h delay between the intracranial injection and tissue harvesting, meaning that brief early drug effects could have been missed.

Discovering the mechanisms underlying serotonin (5-HT)2A and 5-HT2C receptor regulation following nicotine withdrawal in rats.

We have previously demonstrated that nicotine withdrawal produces depression-like behavior and that serotonin (5-HT)2A/2C receptor ligands modulate that mood-like state. In the present study we aimed to identify the mechanisms (changes in radioligand binding, transcription or RNA-editing) related to such a behavioral outcome. Rats received vehicle or nicotine (0.4 mg/kg, s.c.) for 5 days in home cages. Brain 5-HT2A/2C receptors were analyzed on day 3 of nicotine withdrawal. Nicotine withdrawal increased [(3)H]ketanserin binding to 5-HT2A receptors in the ventral tegmental area and ventral dentate gyrus, yet decreased binding in the nucleus accumbens shell. Reduction in [(3)H]mesulergine binding to 5-HT2C receptors was seen in the ventral dentate gyrus. Profound decrease in the 5-HT2A receptor transcript level was noted in the hippocampus and ventral tegmental area. Out of five 5-HT2C receptor mRNA editing sites, deep sequencing data showed a reduction in editing at the E site and a trend toward reduction at the C site in the hippocampus. In the ventral tegmental area, a reduction for the frequency of CD 5-HT2C receptor transcript was seen. These results show that the reduction in the 5-HT2A receptor transcript level may be an auto-regulatory response to the increased receptor density in the hippocampus and ventral tegmental area during nicotine withdrawal, while decreased 5-HT2C receptor mRNA editing may explain the reduction in receptor labeling in the hippocampus. Serotonin (5-HT)2A/2C receptor ligands alleviate depression-like state in nicotine-withdrawn rats. Here, we show that the reduction in 5-HT2A receptor transcript level may be an auto-regulatory response to the increased receptor number in the hippocampus and ventral tegmental area during nicotine withdrawal, while attenuated 5-HT2C receptor mRNA editing in the hippocampus might explain reduced inverse agonist binding to 5-HT2C receptor and suggest a shift toward a population of more active receptors. 5-HT, serotonin; 5-HT2A R, 5-HT2A receptor; 5-HT2C R, 5-HT2C receptor.

Pro-cognitive effect of acute imipramine administration correlates with direct interaction of BDNF with its receptor, Trkß.

Given the important role of brain-derived neurotrophic factor (BDNF)-mediated Trkß signalling in the mechanism of action of antidepressants (ADs), we examined ligand-receptor interactions in the rat cingulate cortex using a proximity ligation assay (PLA) in response to acute and repeated administration of imipramine (IMI), followed by various drug-free periods. Both the acute and chronic administration of IMI increased the BDNF-Trkß interaction observed 3 h after drug administration. Withdrawal of IMI for 72 h or 7 days did not alter BDNF-Trkß interaction. A significant reduction in this interaction after chronic IMI administration followed by 21 drug-free days was observed, but it returned to the control value when a new dose of IMI was given after this time. The level of mRNA encoding BDNF or Trkß did not change in the experimental groups of animals, so one can conclude that alterations in the BDNF-Trkß interaction depend not on acute vs. repeated treatment with IMI but on the presence of the drug in the body. This effect correlates well with the strong pro-cognitive effect of acute IMI, assessed by the novel object recognition (NOR) test.

Effect of chronic mild stress and imipramine on the proteome of the rat dentate gyrus.

The present study uses a proteomic approach to examine possible alterations of protein expression in the hippocampus of rats that are subjected to chronic mild stress (CMS). These rats serve as an animal model that was developed to mimic anhedonia, which is one of the core symptoms of depression. As antidepressant treatment is effective after a few weeks of administration, we also aimed to identify changes that were linked to chronic (once daily for 4 weeks) and 'pulse' (once a week) administration of imipramine. Fifteen differential proteins were identified with 2D electrophoresis followed by mass spectrometry. Although both methods of imipramine administration restored normal sucrose consumption in rats that were subjected to CMS, the molecular mechanisms of these two therapies were different. CMS-induced changes in the levels of dynactin 2, Ash 2, non-neuronal SNAP25 and alpha-enolase were reversed by chronic imipramine, but 'pulse' treatment was not that effective.

Genetic variants in dopamine receptors influence on heterodimerization in the context of antipsychotic drug action.

Human dopamine D2 receptor (D2R) gene has polymorphic variants, three of them alter its amino acid sequence: Val96Ala, Pro310Ser and Ser311Cys. Their functional role never became the object of extensive studies, even though there are some evidence that they correlate with schizophrenia. The present work reviews data indicating that these mutations play a role in dimer formation with dopamine D1 receptor (D1R), with the strongest effect observed for Ser311Cys variant. Similarly, the affinity for antipsychotic drugs of this genetic variant depends on whether it is expressed together with D1R or not. Better understanding of altered ability of genetic variants of D2R to form dimers with D1R, as well as of altered affinity for antipsychotic drugs, depending on the absence or presence of the second dopamine receptor is of great importance-since these two receptors are not always co-expressed in the same cell. It may well be that targeting new compounds toward the D1R-D2R dimers, which the most probably form under conditions of excessive dopamine release, will result in antipsychotic drugs devoid of serious side effects.

Serum Level of miR-1 and miR-155 as Potential Biomarkers of Stress-Resilience of NET-KO and SWR/J Mice.

In the present study, we used three strains of mice with various susceptibility to stress: mice with knock-out of the gene encoding norepinephrine transporter (NET-KO), which are well characterized as displaying a stress-resistant phenotype, as well as two strains of mice displaying two different stress-coping strategies, i.e., C57BL/6J (WT in the present study) and SWR/J. The procedure of restraint stress (RS, 4 h) was applied, and the following behavioral experiments (the forced swim test and sucrose preference test) indicated that NET-KO and SWR/J mice were less sensitive to RS than WT mice. Then, we aimed to find the miRNAs which changed in similar ways in the serum of NET-KO and SWR/J mice subjected to RS, being at the same time different from the miRNAs found in the serum of WT mice. Using Custom TaqMan Array MicroRNA Cards, with primers for majority of miRNAs expressed in the serum (based on a preliminary experiment using the TaqMan Array Rodent MicroRNA A + B Cards Set v3.0, Thermo Fisher Scientific, Waltham, MA, USA) allowed the identification of 21 such miRNAs. Our further analysis focused on miR-1 and miR-155 and their targets-these two miRNAs are involved in the regulation of BDNF expression and can be regarded as biomarkers of stress-resilience.

Genomic Screening of Wistar and Wistar-Kyoto Rats Exposed to Chronic Mild Stress and Deep Brain Stimulation of Prefrontal Cortex.

Stress, a major precipitant of depression, and antidepressants have major impact on synaptic integrity and plasticity in brain areas, such as hippocampus (HPC) and prefrontal cortex (PFC). We have recently shown that, unlike Wistar rats, rats of the Wistar-Kyoto (WKY) strain fail to respond to chronic antidepressant treatment after exposure to chronic mild stress (CMS) procedure. However, deep brain stimulation (DBS) of PFC was effective in both strains. We aimed to identify genes that were affected by CMS, to determine whether their expression was normalized by DBS, and to establish whether common changes could be identified in antidepressant responsive (Wistar) and antidepressant-resistant (WKY) strains. Male Wistar and WKY rats were exposed chronically to CMS then treated acutely with DBS. A battery of behavioural tests was used to monitor recovery, followed by TaqMan screening of a panel of genes known to be involved in stress and antidepressant action. WKY showed over-expression of five genes in dorsal HPC and under-expression of seven genes in ventral HPC. Expression of three genes, Egr1, Htr7 and Mmp9 was decreased by CMS and normalized by DBS in the ventral HPC of Wistar rats. Some other changes in gene expression were identified in dorsal HPC and PFC, particularly in Wistars, that were not normalized by DBS. No effects were identified that were common to both Wistars and WKY. The difference between Wistars and WKY in the balance of overall gene expression in HPC may be relevant to the resistance of WKY rats to antidepressant drug treatment.

Clozapine administered repeatedly following pretreatment with ketamine enhances dopamine D2 receptors in the dopamine mesolimbic pathway in mice brain.

The mechanisms underlying the beneficial effects of clozapine (CLZ) in the treatment of schizophrenia still remains far from clear. In the present work we studied the effect of CLZ on the dopamine D2 receptors (D2R) in the mouse brain. CLZ was administered after ketamine (KET) in a paradigm strictly matching the one used in KET-induced attentional set-shifting task (ASST). It has been shown previously that CLZ reversed KET-induced cognitive impairments. In the present study we used in situ hybridization to estimate the level of mRNA, together with specific D2R radioligand, [3H]domperidone binding in the ventral tegmental area (VTA) as well as in the striatum, and observed an increase in the [3H]domperidone binding in the striatum and an increase in D2R mRNA level in the VTA following repeated (but not acute) CLZ administration in mice pre-treated repeatedly with KET. The obtained results allow for conclusion that CLZ in this experimental paradigm enhances biosynthesis of presynaptic D2R.

Understanding GPCR dimerization.

Initially G protein-coupled receptors, GPCRs, were thought to act as monomers, but recently strong evidence has been gathered indicating that they are capable of forming homo- and heterodimers or higher order oligomeric complexes, and that the dimerization phenomenon can modulate the pharmacological response and function of these receptors. In this chapter we point to the great potential of alternative therapeutic approach targeted at GPCR dimers, which is especially important in the field of neuropsychopharmacology. We also included a brief description of methods used for studying the phenomenon of GPCR oligomerization, with particular attention paid to the proximity ligation assay, PLA, the procedure which allows the study of interactions between receptors not only in vitro but also in vivo, with good anatomical resolution, what is especially important in the studies of various GPCRs involved in central neurotransmission.

Neuroadaptive changes in the rat brain GABA(B) receptors after withdrawal from cocaine self-administration.

We examined neuroadaptive changes in gamma-aminobutyric acid (GABA)(B) receptor binding following cocaine self-administration and its withdrawal in several rat brain structures using a "yoked" procedure and a quantitative autoradiographic analysis. In order to estimate the distribution of GABA(B) receptors in several brain areas, we used ([S-(R,R)]-[3-[[1-(3,4-diclorophenyl)ethyl]amino]-2-hydroxypropyl]([3,4-(3)H]-cyclohexylmethyl) phosphinic acid) ([(3)H]CGP 54626), a GABA(B) receptor antagonist. The binding of [(3)H]CGP 54626 in the nucleus accumbens subareas and the amygdala was decreased by ca. 20% in rats that actively self-administered cocaine. Similar decreases in the nucleus accumbens were seen in the animals passively receiving cocaine. These animals also showed a reduction in GABA(B) receptor binding in the prefrontal and frontal cortices, septum and dorsal striatum. The [(3)H]CGP 54626 binding in several rat brain areas was decreased after 10-day withdrawal from self-administered cocaine. In summary, the decreases in the GABA(B) receptor binding seem to reflect the effects of chronic administration of cocaine per se and not the motivated process of reinforced responding. Furthermore, withdrawal from cocaine self-administration evoked changes in the GABA(B) receptor binding in several rat brain areas that may indicate neurobiological adaptations.

The role of D1-D2 receptor hetero-dimerization in the mechanism of action of clozapine.

Clozapine is effective although still not perfect drug used to treat schizophrenia. The precise mechanism of its action is not known. Here we show that there are two binding sites for clozapine at the dopamine D1 and D2 receptors, and the affinity of D1R strongly depended on whether the receptor was present alone or together with D2R (or its genetic variant D2Ser311Cys) in the cell membrane, pointing to the role of receptor hetero-dimerization in the observed phenomenon. The use of fluorescence resonance energy transfer (FRET) technology, observed via fluorescence lifetime microscopy of the single cell, indicated that low concentration of clozapine (10(-9) M), sufficient to saturate the high affinity site, uncoupled the D1R-D2R hetero-dimers. Therefore it has been concluded that clozapine might antagonize the effect of concomitant stimulation of both dopamine receptors, which has been shown previously to enhance the formation of hetero-dimers and to stimulate the calcium signaling pathway.

Alterations in gamma-aminobutyric acid(B) receptor binding in the rat brain after reinstatement of cocaine-seeking behavior.

We examined neuroadaptive changes in GABA(B) receptor binding following reinstatement of cocaine-seeking behavior in rat brain structures using a "yoked" procedure and quantitative autoradiographic analysis. To estimate the distribution of GABA(B) receptors in several brain areas, we used [(3)H]CGP 54626, a GABA(B) receptor antagonist. The binding of [(3)H]CGP 54626 in the nucleus accumbens and the amygdaloid complex was decreased by about 20% in rats that actively administered cocaine. Similar decreases were seen in the animals that were passively administered cocaine; these rats also demonstrated decreased GABA(B) receptor binding in the prefrontal and frontal cortices, septum and dorsal striatum. The binding of [(3)H]CGP 54626 in several rat brain areas was decreased during 10-day withdrawal from self-administered cocaine. The cocaine-priming dose (10 mg/kg, ip) induced a significant increase of [(3)H]CGP 54626 binding in the core of the nucleus accumbens, substantia nigra (reticular part), prefrontal and frontal cortices and septum in rats withdrawn from cocaine self-administration. The presentation of the conditioned stimulus (tone + light) associated with previous cocaine self-administration induced a significant decrease of [(3)H]CGP 54626 binding in the mediodorsal thalamic nucleus and amygdaloid complex in the rats withdrawn from cocaine self-administration. Increases in GABA(B) receptor binding in limbic regions during cocaine-induced reinstatement likely reflect motivational states that were present during active drug self-administration.

Repeated Clozapine Increases the Level of Serotonin 5-HT1AR Heterodimerization with 5-HT2A or Dopamine D2 Receptors in the Mouse Cortex.

G-protein-coupled receptor (GPCR) heterodimers are new targets for the treatment of schizophrenia. Dopamine D2 receptors and serotonin 5-HT1A and 5-HT2A receptors play an important role in neurotransmission and have been implicated in many human psychiatric disorders, including schizophrenia. Therefore, in this study, we investigated whether antipsychotic drugs (clozapine (CLZ) and haloperidol (HAL)) affected the formation of heterodimers of D2-5-HT1A receptors as well as 5-HT1A-5-HT2A receptors. Proximity ligation assay (PLA) was used to accurately visualize, for the first time, GPCR heterodimers both at in vitro and ex vivo levels. In line with our previous behavioral studies, we used ketamine to induce cognitive deficits in mice. Our study confirmed the co-localization of D2/5-HT1A and 5-HT1A/5-HT2A receptors in the mouse cortex. Low-dose CLZ (0.3 mg/kg) administered repeatedly, but not CLZ at 1 mg/kg, increased the level of D2-5-HT1A and 5-HT1A-5-HT2A heterodimers in the mouse prefrontal and frontal cortex. On the other hand, HAL decreased the level of GPCR heterodimers. Ketamine affected the formation of 5-HT1A-5-HT2A, but not D2-5-HT1A, heterodimers.

Paroxetine and Low-dose Risperidone Induce Serotonin 5-HT1A and Dopamine D2 Receptor Heteromerization in the Mouse Prefrontal Cortex.

Recently, it has been shown that serotonin 5-HT1A receptor interacts with dopamine D2 receptor in vitro. However, the existence of 5-HT1A-D2 heteromers in native tissue remains unexplored. In the present study, we investigated 5-HT1A-D2 receptor heteromerization in mice treated acutely or chronically with paroxetine (10 mg/kg) or risperidone (0.05 mg/kg). Receptor heteromerization was visualized and quantified in the mouse brain by in situ proximity ligation assay (PLA). Additionally, we aimed to determine the cellular localization of 5-HT1A-D2 receptor heteromers in mouse adult primary neuronal cells by immunofluorescent staining with markers for astrocytes (GFAP) and neurons (NeuN and MAP2). The results from the current study demonstrated that 5-HT1A and D2 receptor co-localization and heteromerization occurred in the mouse prefrontal cortex. Counterstaining after PLA confirmed neuronal (pyramidal and GABAergic) as well as astrocytal localization of 5-HT1A-D2 receptor heteromers. Chronic administration of paroxetine or risperidone increased the level of 5-HT1A-D2 receptor heteromers in the prefrontal cortex. These changes were not accompanied by any changes in the expression of mRNAs (measured by in situ hybridization) or densities of 5-HT1A and D2 receptors (quantified by receptor autoradiography with [3H]8-OH-DPAT and [3H]domperidone, respectively), what all indicated that paroxetine and risperidone facilitated 5-HT1A-D2 heteromer formation independently of the receptor expression. In vitro homogenous time-resolved FRET (HTRF) study confirmed the ability of tested drugs to influence the human 5-HT1A-D2 heteromer formation. The obtained data indicate that the increase in 5-HT1A-D2 receptor heteromerization is a common molecular characteristic of paroxetine and low-dose risperidone treatment.

Regulation of somatostatin receptor 2 in the context of antidepressant treatment response in chronic mild stress in rat.

RATIONALE: The role of somatostatin and its receptors for the stress-related neuropsychiatric disorders has been widely raised. Recently, we have also demonstrated the involvement of somatostatin receptor type 2-sst2R and dopamine receptor type 2-D2R in stress. OBJECTIVE: In this context, we decided to find if these receptors are involved in response to antidepressant treatment in animal model of depression-chronic mild stress (CMS). METHODS: Here, we report data obtained following 7-week CMS procedure. The specific binding of [125I]Tyr3-Octreotide to sst2R and [3H]Domperidone to D2R was measured in the rat brain, using autoradiography. Additionally, the level of dopamine and metabolites was measured in the rat brain. RESULTS: In the final baseline test after 7 weeks of stress, the reduced consumption of sucrose solution was observed (controls vs the stressed animals (6.25 0.16 vs. 10.39 0.41; p < 0.05). Imipramine was administered for the next 5 weeks, and it reversed anhedonia in majority of animals (imipramine-reactive); however, in some animals, it did not (imipramine-non-reactive). Two-way repeated measures ANOVA revealed significant effects of stress and treatment and time interaction [F(16, 168) = 3.72; p < 0.0001], n = 10 per groups. We observed decreased binding of [125I]Tyr3-Octreotide in most of rat brain regions in imipramine non-reactive groups of animals. The decrease of D2R after stress in striatum and nucleus accumbens and no effect of imipramine were observed. In the striatum and prefrontal cortex, the significant role of stress and imipramine in dopamine levels was observed. CONCLUSIONS: The results obtained in binding assays, together with dopamine level, indicate the involvement of sst2R receptors for reaction to antidepressant treatment. Besides, the stress context itself changes the effect of antidepressant drug.

Active versus passive cocaine administration: differences in the neuroadaptive changes in the brain dopaminergic system.

There is considerable evidence that chronic exposure to cocaine is associated with low striatal dopamine D2 receptor availability. In the present study we wished to determine whether neuroadaptive changes in densities of D2 receptors were due to direct pharmacological actions of cocaine or they reflected motivational states that were present when cocaine injection depended on active drug-seeking behavior and whether these changes were related to the actual expression of D2 mRNA. To achieve this goal we utilized a "yoked" procedure in which rats were tested simultaneously in groups of three, with only one rat actively self-administering cocaine while the other two received yoked injections of either cocaine or saline. Only passively administered cocaine produced a decrease in dopamine D2 receptor levels in the anterior and central regions of caudate/putamen, and both the shell and core of the nucleus accumbens, as measured by in vitro quantitative autoradiography. In contrast, examination of D2 receptor gene expression using in situ hybridization analysis revealed that there was an increase in D2 receptor mRNA levels in the ventral tegmental area of rats actively self-administered cocaine. We conclude that the reductions in striatal D2 receptor densities may be related to the chronic administration of cocaine per se and not to the motivated process of reinforced responding. Our results also suggest that increases in D2 receptor mRNA levels in limbic regions do not necessarily result in increased receptor densities and these changes likely reflect motivational states that were present when cocaine injection dependent on active drug self-administration.

Expression of proopiomelanocortin, proenkephalin and prodynorphin genes in porcine theca and granulosa cells.

Previous studies have demonstrated the presence of endogenous opioid peptides (EOP) in the ovary and suggested their implication in local interactions within ovarian structures. Nevertheless, data pertaining to the expression of genes, coding for the opioid precursors, in ovarian cells are still rudimentary and not available for the pig. The study was undertaken to test whether genes of the opioid precursors - proopiomelanocortin (POMC), proenkephalin (PENK) and prodynorphin (PDYN) - are expressed in non-treated and gonadotropin-treated theca and granulosa cells isolated from ovarian follicles of the pig. The cells were isolated from small (days 15-16 of the estrous cycle) and large (days 19-20) porcine follicles. Dispersed cells were cultured in Eagle's medium under the water saturated atmosphere of 95% air and 5% CO(2), in the presence or absence of respective gonadotropin; theca cells with LH (100 ng/ml) and granulosa cells with FSH (100 ng/ml). Following 24h-incubation, the cells were harvested and the total RNA was isolated. The expression of genes coding for opioid precursors was estimated by the semi-quantitative RT-PCR technique involving co-amplification of the target cDNA (POMC, PENK or PDYN) and control cDNA (beta-actin or 18S rRNA). Specificities of PCR products were confirmed by Southern analysis and sequencing. In theca cells the expression of opioid precursors appeared to be gonadotropin-dependent except for PENK in the cells isolated from large follicles. In turn, granulosa cells exhibited the expression of POMC and PENK genes independently on treatment with FSH. This gonadotropin induced the expression of PDYN gene in granulosa cells isolated from small and large follicles and significantly increased POMC mRNA content in the cells from the large ones. The present studies indicate that porcine follicular cells (especially granulosa cells) may produce opioid peptides and that gonadotropins may modulate gene expression of their precursors in these cells. Moreover, our results support a participation of opioid peptides in the local regulations within ovarian follicle.

Effect of citalopram in the modified forced swim test in rats.

The present study examined the effect of citalopram (7.5 and 15 mg/kg) in the modified forced swim test (FST) in Wistar rats, in comparison to the effect of desipramine at the same doses. The citalopram at both doses increased swimming behavior, at the cost of climbing and immobility. The administration of desipramine increased climbing behavior while immobility counts were decreased. The modified FST is indeed more sensitive than the conventional FST in describing precisely the behavioral effects of antidepressant drugs, allowing to roughly estimate the contribution of individual neurotransmitter system to the mechanism of action of the studied drug.