Caffeine - treat or trigger? Disparate behavioral and long-term dopaminergic changes in control and schizophrenia-like Wisket rats.

The influence of caffeine on behavioral functions in both healthy and schizophrenic subjects is controversial. Here we aimed to reveal the effects of repeated caffeine pre- and post-training treatments on motor and exploratory activities and cognitive functions in a reward-based test (Ambitus) along with a brain region-specific dopamine D2 receptor profile in control and schizophrenia-like WISKET model rats. In the control animals, pre-treatment caused temporary enhancement in motor activity, while permanent improvement in learning function was detected in the WISKET animals. Post-treatment produced significant impairments in both groups. Caffeine caused short-lasting hyperactivity followed by a rebound in the inactive phase determined in undisturbed circumstance. Caffeine treatment substantially enhanced the dopamine D2 receptor mediated G-protein activation in the prefrontal cortex and olfactory bulb of both groups, while it increased in the dorsal striatum and cerebral cortex only in the WISKET animals. Caffeine enhanced the maximal binding capacity in the hippocampus and cerebral cortex of WISKET animals, but it decreased in the prefrontal cortex of the control animals. Regarding the dopamine D2 receptor mRNA expression, caffeine treatment caused significant enhancement in the prefrontal cortex of WISKET animals, while it increased the hippocampal dopamine D2 receptor protein amount in both groups. This study highlights the disparate effects of caffeine pre- versus post-training treatments on behavioral parameters in both control and schizophrenia-like animals and the prolonged changes in the dopaminergic system. It is supposed that the delayed depressive effects of caffeine might be compensated by frequent coffee intake, as observed in schizophrenic patients.

Impaired GAD1 expression in schizophrenia-related WISKET rat model with sex-dependent aggressive behavior and motivational deficit.

After peri-adolescence isolation rearing (IS) and subchronic ketamine (KET) treatment, adult, selectively bred Wistar rats (named WISKET) mimic abnormal behaviors reminiscent of human schizophrenia, including reduced prepulse-inhibition of startle reflex, disturbances in cognition, locomotor activity and thermoregulation, decreased pain sensitivity and electrophysiological alterations. To further validate our WISKET rat line, regarding its translational utility in schizophrenia research, we examined their social behavior and introduced a short and simple holeboard (HB)-like test to investigate their motivational deficit that predicts the cognitive disturbance. Sex-dependent alterations in schizophrenia may yield important insights into its etiology; thus, male and female WISKET rats were also investigated and compared with their naive Wistar counterparts. Considering the contribution of the hippocampal and cortical GABAergic inhibitory circuitry in these behavioral alterations, molecular-biology studies were also performed regarding the GAD1 gene products. Impaired social activity with increased aggression, stress-related behavior, active social withdrawal, motivation deficit and decreased exploration were observed, especially in male WISKET rats, compared with Wistar ones and their corresponding females. These alterations were accompanied by sex-dependent alterations regarding GAD67 mRNA and protein expression in the prefrontal cortex and hippocampus. In conclusion, the WISKET animals are valuable tools for animal-based preclinical drug discovery studies for predictive screening of novel compounds improving negative symptoms with potential antipsychotic efficacy.

Detection of stress and the effects of central nervous system depressants by gastrointestinal smooth muscle electromyography in wakeful rats.

AIMS: The altered gut-brain interaction can be in the background of functional gastrointestinal (GI) disorders. In the GI tract, the slow-wave myoelectric signals can be detected by electromyography (EMG). The aims of our study were to follow up the stress induced alteration in the GI tract by smooth muscle EMG in wakeful rats. MAIN METHODS: The GI tract myoelectric activity of male rats was measured by an electrode pair under the abdominal skin, the responses were detected and analyzed by a software using fast Fourier transformation. Animals were immobilized and treated with either diazepam or haloperidol. The plasma corticosterone level was determined by ELISA kit, the levels of drugs were measured by HPLC, while the direct GI effects of the compounds were tested in an organ bath. KEY FINDINGS: Significant correlation (r2 = 0.52) was found between the immobilization induced increase in the EMG spectra of the GI tract segments and the increase in corticosterone plasma levels. The stress-reducing effects of diazepam and haloperidol were also detectable by smooth muscle EMG in the GI tract. No direct smooth muscle actions of the drugs were found in organ bath studies. SIGNIFICANCE: The smooth muscle EMG instrument can measure the level of acute stress and is applicable for the investigation of central nervous system affecting drugs through the GI tract in awake rats. This is the first tool to measure the stress response via the GI tract reactions. The technique may open a new perspective in the diagnosis and therapy of psychosomatic disorders.

Alteration in expressions of RhoA and Rho-kinases during pregnancy in rats: their roles in uterine contractions and onset of labour.

Activation of RhoA and Rho-associated kinases (ROCKs) is known to play a pivotal role in the regulation of smooth muscle contraction via phosphorylation of myosin-light chain and myosin phosphatase. There are few data on the RhoA and ROCKs expression levels in rat uteri. Therefore, our aim was to investigate the mRNA and protein concentration of RhoA and ROCKs in rat uterus during pregnancy, during parturition and post-partum using real time PCR and Western blot analysis. The other purpose was to evaluate the effects of the ROCK (Y-27632, fasudil and RKI 1441) and RhoA inhibitors (simvastatin) on uterine contractility in isolated organ bath experiments. The mRNA and protein levels of RhoA decreased on the 5th day of pregnancy to day 22, then a sharp increase was detected at term. The mRNA and protein concentration of ROCKs was down-regulated in the early stage of pregnancy, while it sharply increased during parturition. The RhoA-inhibitor simvastatin relaxed the uterus contractions, although its inhibitory effects were not followed by the alteration of RhoA. The strongest inhibitory effect of non-selective ROCK inhibitor fasudil was found on non-pregnant uterus, while it elicited milder relaxation on day 22, during parturition and postpartum day 1. The maximum relaxing effects of Y-27632 and RKI 1441 were altered in a proportional way with the target protein expressions. The RhoA/ROCK signalling pathway might be a potential target for the development of new tocolytic agents; however, high specificity to RhoA, ROCK I or ROCK II seems to be fundamental to the high efficacy of uterine relaxation.

Tocopherol inhibits the relaxing effect of terbutaline in the respiratory and reproductive tracts of the rat: the role of the oxidative stress index.

AIMS: Reactive oxygen species play a role in the signal transduction of beta-adrenergic receptors. We investigated whether an antioxidant (tocopherol) can reduce the effect of terbutaline in beta-2-adrenergic receptor (ß2-AR)-regulated smooth muscles. MAIN METHODS: Contractility of the tissues from nonpregnant (trachea) and 22-day-pregnant (myometrium and cervix) rats was investigated in an isolated organ bath. The tracheal and uterine ß2-AR expressions were increased by 17-beta-estradiol valerate (E2) and progesterone (P4), respectively. The accumulation of cyclic-AMP (cAMP), and the total oxidant (TOS) and total antioxidant status (TAS) were also measured. The oxidative stress index (OSI) was defined as the ratio of TOS and TAS. KEY FINDINGS: Terbutaline (10(-10)-10(-5)M) decreased the contractions in the nontreated and the P4-pretreated myometria, but tocopherol (10(-7)M) did not alter these actions. Terbutaline (10(-6)M) increased the cervical resistance both in the nontreated and in the P4-treated samples, while tocopherol reduced this action only in the P4-treated cervices. Terbutaline (10(-9)-10(-4)M) reduced the tracheal tones both in the nontreated and in the E2-treated tissues, while tocopherol reduced these effects. The changes in the intracellular cAMP levels of the tissues were in harmony with the isolated organ results. The OSI was highest in the trachea and lowest in the pregnant myometrium. SIGNIFICANCE: A higher OSI is linked to a higher tocopherol sensitivity of beta-mimetic-induced relaxation. Our results suggest that the antiasthmatic effect of beta-mimetics may worsen, while their tocolytic effect may remain unchanged during parallel tocopherol administration.

Androgenic effect of honeybee drone milk in castrated rats: roles of methyl palmitate and methyl oleate.

ETHNOPHARMACOLOGICAL RELEVANCE: Numerous honeybee (Apis mellifera) products have been used in traditional medicine to treat infertility and to increase vitality in both men and women. Drone milk (DM) is a relatively little-known honeybee product with a putative sexual hormone effect. The oestrogenic effect of a fraction of DM has recently been reported in rats. However, no information is available on the androgenic effects of DM. The purpose of the present study was to determine the androgen-like effect of DM in male rats and to identify effective compounds. MATERIALS AND METHODS: A modified Hershberger assay was used to investigate the androgenic effect of crude DM, and the plasma level of testosterone was measured. The prostatic mRNA and protein expression of Spot14-like androgen-inducible protein (SLAP) were also examined with real-time PCR and Western blot techniques. GC-MS and NMR spectroscopic investigations were performed to identify the active components gained by bioactivity-guided fractionation. RESULTS: The crude DM increased the relative weights of the androgen-dependent organs and the plasma testosterone level in castrated rats and these actions were flutamide-sensitive. DM increased the tissue mRNA and protein level of SLAP, providing further evidence of its androgen-like character. After bioactivity-guided fractionation, two fatty acid esters, methyl palmitate (MP) and methyl oleate (MO), were identified as active compounds. MP alone showed an androgenic effect, whereas MO increased the weight of androgen-sensitive tissues and the plasma testosterone level only in combination. CONCLUSION: The experimental data of DM and its active compounds (MO and MP) show androgenic activity confirming the traditional usage of DM. DM or MP or/and MO treatments may project a natural mode for the therapy of male infertility.

Ontogeny of sulfonylurea-binding regulatory subunits of K(ATP) channels in the pregnant rat myometrium.

ATP-sensitive potassium channels (K(ATP) channels) are composed of sulfonylurea receptors (SURs) and potassium inward rectifiers (Kir(6.x)) that assemble to form a large octameric channel. This study was designed to examine the expression and role of sulfonylurea-binding regulatory subunits 1 (SUR1 (ABCC8)) and 2 (SUR2 (ABCC9)) of the K(ATP) channels in the pregnant rat myometrium with particular regard to the contractility. RT-PCR and western blot analyses were performed to detect the presence of SUR1 and SUR2. The SUR1 levels were markedly increased in the early stages of pregnancy. The highest level was detected on day 6 of pregnancy, whereas in the late stages, the levels of SUR1 were significantly decreased. The SUR2 level remained unchanged throughout pregnancy. The SUR non-selective diazoxide and the SUR2-selective pinacidil inhibited oxytocin-induced contractions. Glibenclamide, a K(ATP) channel blocker, antagonized both pinacidil- and diazoxide-induced relaxations. It was established that SURs are responsible for pharmacological reactivity of K(ATP) channel openers. We conclude that both SURs are involved in the K(ATP) channel in the pregnant rat myometrium. It may further be concluded that 'pinacidil-like' K(ATP) channel openers may be of therapeutic relevance as tocolytic agents in the future.

Use of antisense oligonucleotides to verify the role of the alpha(1A)-adrenergic receptor in the contractility of the rat uterus post partum.

The adrenergic system plays a major role in the regulation of the contractility of the uterus during pregnancy. This study investigated the role of the alpha(1A)-adrenergic receptor (AR) in this regulation. The use of partial phosphorothioate antisense oligodeoxynucleotides (AONs) permitted the sequence-selective inhibition of AR gene expression. AONs were injected together with a cationic liposomal carrier agent into the post partum rat uterus. Incubation for 12 or 24 h with the most effective AON (480-AON) caused a 58.7 or 53.0% inhibition, respectively, of the expression of the alpha(1A)-AR density, whereas incubation for 36 or 48 h resulted in only a 38.8 or 26.7% inhibition, respectively. The decrease of the alpha(1A)-AR density by 480-AON was demonstrated by Western blot analysis and a radioreceptor binding assay on rat uterus preparations 24 h after delivery. The changes in the contractility of the uterus after AON treatment were measured on isolated rat uterine tissue by electric field stimulation. The significant decrease in the ability of the uterus to contract was indicated by the area under the curve method. The electric field studies revealed that the specific alpha(1A)-blockers 5-methylurapidil and WB 4101 inhibited the rhythmic contraction by about 74 and 70% in the control uteri and by 25 and 20% in 480-AON-treated uteri, respectively. The curves for the beta-mimetic (terbutaline) and alpha(1D)-antagonist (BMY7370) inhibitors were unchanged after 480-AON treatment of the uteri. These results suggest the importance of the alpha(1A)-AR in the tocolytic effect exerted by the alpha(1)-antagonist, although high concentrations of antagonists can not exclude the role of alpha(1D)-ARs, too. Additionally, these prove that the knockdown transformation by AONs offers a useful animal model for the investigation of receptors controlling the function of uterine tissue.

[Pharmacological models to investigate the role of the adrenergic system on post-partum rat uterus]. / Farmakológiai modell az adrenerg rendszer szerepének vizsgálatára post partum patkány uteruson.

The adrenergic system plays a major role in the regulation of the pregnant uterine contractility. Our aim was to develop an experimental animal model to study the role of the alpha 1A-adrenergic receptor (AR) in uterine motor activity by antisense oligonucleotides (AONs). AONs were injected with DOTAP and pluronic gel into the uterine lumen of post-partum rats 2-3 hours after delivery. The decrease of the alpha 1A-AR density by AON was demonstrated by RT-PCR method, Western blot analysis and radioligand binding assay on rat uterus preparations 24 h after delivery. The changes in the contractility of the uterus were measured on isolated rat uterine tissue by electric field stimulation (EFS). The EFS investigation demonstrated that the effect of the specific alpha 1A-blocker 5-methylurapidil and WB4101 was significantly decreased in the AON-treated rat uterus as compared to the control group but the effect of the beta-mimetic terbutalin and alpha 1D-antagonist BMY7378 was unchanged. Our result suggest that the alpha 1A-ARs play a very important role in the regulation of uterine contractility, and may serve as the basis for a subsequent new group of tocolytics (uterus selective alpha 1-antagonists), which may lead to more selective therapy than currently used beta-mimetics.