Monika Leśkiewicz

The effect of antidepressant drugs on the HPA axis activity, glucocorticoid receptor level and FKBP51 concentration in prenatally stressed rats.

Dysregulation of hypothalamic-pituitary-adrenal (HPA) axis activity is thought to be an important factor in pathogenesis of depression. In animals, stress or glucocorticoids given in prenatal period lead to long-lasting behavioral and neuroendocrine changes similar to those observed in depressed patients. However, molecular basis for HPA disturbances in animals exposed to prenatal stress - a model of depression - have been only partially recognized. Therefore, in the present study we investigated the effect of prenatal stress on behavioral changes, blood corticosterone level, concentrations of glucocorticoid receptor (GR) and its cochaperone, FKBP51, in the hippocampus and frontal cortex in adult rats. It has been found that prenatally stressed rats display high level of immobility in the Porsolt test and anxiety-like behavior. The HPA axis hyperactivity in theses animals was evidenced by corticosterone hypersecretion at the end of the light phase and 1h following acute stress. Western blot study revealed that GR level was significantly elevated in the hippocampus but not in the frontal cortex of prenatally stressed rats, whereas concentration of FKBP51 was decreased only in the former brain structure. Chronic treatment with imipramine, fluoxetine, mirtazapine and tianeptine have diminished both behavioral and biochemical alterations observed in this animal model of depression. These data indicate that the increase in hippocampal GR level and low concentration of FKBP51 in the frontal cortex may be responsible for enhanced glucocorticoid action in depression.

Antipsychotic drugs inhibit the human corticotropin-releasing-hormone gene promoter activity in neuro-2A cells-an involvement of protein kinases.

Antipsychotic drugs can regulate transcription of some genes, including those involved in regulation of hypothalamic–pituitary–adrenal (HPA) axis, whose activity is frequently disturbed in schizophrenic patients. However, molecular mechanism of antipsychotic drug action on the corticotropin-releasing hormone (CRH) gene activity has not been investigated so far. This study was undertaken to examine the influence of conventional and atypical antipsychotic drugs on the CRH gene promoter activity in differentiated Neuro-2A cell cultures stably transfected with a human CRH promoter fragment linked to the chloramphenicol acetyltransferase (CAT) reporter gene. It has been found that chlorpromazine (0.1–5.0 μM), haloperidol (0.5–5.0 μM), clozapine (1.0–5.0 μM), thioridazine (1.0–5.0 μM), promazine (5.0 and 10 μM), risperidone (5.0 and 10.0 μM), and raclopride (only at the highest used concentrations, ie 30 and 100 μM) present in culture medium for 5 days inhibited the CRH-CAT activity. Sulpiride and remoxipride had no effect. Since CRH gene activity is most potently enhanced by cAMP/protein kinase A pathway, the effect of antipsychotics on the forskolin-induced CRH-CAT activity was determined. Chlorpromazine (1.0–5.0 μM), haloperidol (1.0–5.0 μM), clozapine (1.0–5.0 μM), thioridazine (3.0 and 5.0 μM), and raclopride (30 and 100 μM), but not promazine, sulpiride, risperidone, and remoxipride, inhibited the forskolin-stimulated CRH gene promoter activity. A possible involvement of protein kinases in chlorpromazine and clozapine inhibitory action on CRH activity was also investigated. It was found that wortmannin (0.01 and 0.02 μM), an inhibitor of phosphatidylinositol 3-kinase (PI3-K), significantly attenuated the inhibitory effect of chlorpromazine and clozapine on CRH gene promoter activity. In line with these results, a Western blot study showed that these drugs increased phospho-Ser-473 Akt level, had no effect on total Akt, and decreased glycogen synthase kinase-3β level. Additionally, we found that clozapine decreased protein kinase C (PKC) level and that its action on CRH activity was attenuated by PKC activator (TPA, 0.1 μM). The obtained results indicate that inhibition of CRH gene promoter activity by some antipsychotic drugs may be a molecular mechanism responsible for their inhibitory action on HPA axis activity. Clozapine and chlorpromazine action on CRH activity operates mainly through activation of the PI3-K/Akt pathway. Moreover, PKC-mediated pathway seems to be involved in clozapine action on CRH gene activity.

Protective effects of neurosteroids against NMDA-induced seizures and lethality in mice.

The effects of some neurosteroids on N-methyl-D-aspartic acid (NMDA)-induced seizures were examined in mice. Intraperitoneal (i.p.) administration of 5 alpha-pregnan-3 alpha-ol-20-one (5, 10 and 20 mg/kg). 5 beta-pregnan-3 alpha-ol-20-one (10 and 20 mg/kg), 5 alpha-pregnan-3 alpha-ol-11,20-dione (15 mg/kg), 5 alpha-androstan-3 alpha-ol-17-one (10 mg/kg) and dehydroepiandrosterone sulfate (25 mg/kg) significantly increased the dose of NMDA necessary to induce clonic convulsions in 50% of the tested animals (CD50). Furthermore, 5 alpha-pregnan-3 alpha-ol-20-one, 5 beta-pregnan-3 alpha-ol-20-one, 5 alpha-pregnan-3 alpha-ol-11,20-dione and 5 alpha-androstan-3 alpha-ol-17-one also protected the mice against NMDA-induced mortality. Importantly, it is only at the highest doses that neurosteroids impair motor performance of the animals, as estimated by a rotorod equilibrium procedure. The other neurosteroids tested, such as 5 alpha-pregnan-3 beta-ol-20-one (5-20 mg/kg), 5 alpha-pregnan-3 alpha,21-diol-20-one (10 and 15 mg/kg), 5 alpha-pregnan-3,20-dione (15 mg/kg) and pregnenolone sulfate (12.5-100 mg/kg) had no significant effects on the measured parameters. In another set of experiments, we evaluated the effects of neurosteroids on D-[3H]-aspartate release from rat hippocampal slices. None of the neurosteroids tested exerted a significant effect on basal D-[3H]-aspartate release. On the other hand, K(+)-stimulated D-[3H]-aspartate release was significantly attenuated by 5 alpha-pregnan-3 alpha-ol-20-one, 5 beta-pregnan-3 alpha-ol-20-one, alphaxalone, pregnenolone sulfate and dehydroepiandrosterone sulfate. The effect of 5 alpha-pregnan-3 alpha-ol-20-one was the most potent and was distinctly concentration-dependent, whereas the other compounds were effective only at the highest concentrations used. The above results indicate that some neurosteroids administered in non-sedative doses can protect mice against NMDA-induced seizures and mortality; furthermore, they inhibit D-[3H]-aspartate release in rat hippocampal slices.

Protective effects of TRH and its stable analogue, RGH-2202, on kainate-induced seizures and neurotoxicity in rodents

Thyrotropin-releasing hormone (TRH) has been postulated to be involved in the regulation of seizures and neural degeneration. We examined the effects of TRH and its stable analogue, RGH-2202, on the kainate-induced seizures and excitotoxicity in mice - a model of a drug-resistant temporal lobe epilepsy. We found that TRH (2.0 and 5.0 mg/kg) and RGH-2202 (2.5 and 5 mg/kg) elevated the ED(50) for kainate-induced convulsions and tended to decrease mortality. A histological analysis showed that kainate caused a neuronal loss of CA(1) and CA(3) hippocampal fields. TRH (10, 20 and 50 mg/kg) and RGH-2202 (2.5, 7.5 and 10.0 mg/kg) markedly reduced the excitotoxic effect of kainate. Further studies showed that TRH (1-100 microM) and RGH-2202 (100 microM) significantly attenuated the kainate (150 microM)-induced lactate dehydrogenase release in a primary cortical cell culture from rat embryos. In conclusion, the present study showed that TRH and RGH-2202 attenuated the kainate-induced seizures and inhibited the kainate-evoked neurotoxicity in vivo and in vitro. These results support the hypothesis of a potential utility of TRH and its analogues in the treatment of seizures and some neurodegenerative diseases.

Effects of neurosteroids on spike-wave discharges in the genetic epileptic WAG:Rij rat

Effects of i.p. administration of the neurosteroids, allopregnanolone and pregnenolone sulfate, were studied in WAG/Rij rats, a genetic model for generalized absence epilepsy. EEG recordings showed that allopregnanolone, a positive modulator of the GABA(A) receptor, in doses ranging from 5 to 20 mg/kg, increased dose-dependently the number- and total duration of spike-wave discharges. Pregnenolone sulfate, a positive modulator of NMDA receptors, also increased those parameters, though only at the highest dose used (100 mg/kg). Significant changes in spike-wave discharges occurred during the first hour post-injection and were not accompanied with behavioral alterations. The obtained data indicate that both these neurosteroids aggravate the spike-wave activity. This finding contrasts with the anti-convulsant effects of some neurosteroids and they point to a different pharmacological profile of epilepsy with convulsive or non-convulsive seizures.

Regulation of the human corticotropin-releasing-hormone gene promoter activity by antidepressant drugs in Neuro-2A and AtT-20 cells.

Major depression is frequently associated with hyperactivity of the hypothalamic–pituitary–adrenal (HPA) axis. Clinically effective therapy with antidepressant drugs normalizes the disturbed activity of HPA axis, in part, by decreasing corticotropin-releasing hormone (CRH) synthesis, but the mechanism of this action is poorly recognized. In order to find out whether antidepressants directly affect CRH gene promoter activity, we studied their effect on undifferentiated and differentiated Neuro-2A cells, and for comparison the effect of the selected antidepressants on AtT-20 cells was also determined. The cells were stably transfected with a human CRH promoter fragment (−663 to +124 bp) linked to the chloramphenicol acetyltransferase (CAT) reporter gene. The regulation of CRH gene promoter activity is similar in Neuro-2A cells, both intact and differentiated, and in AtT-20 cell line, and cAMP/PKA-dependent pathway plays an important role in the stimulation of CRH gene. It was found that imipramine, amitryptyline, desipramine, fluoxetine, and mianserin, present in the culture medium for 5 days, in a concentration-dependent manner inhibited basal hCRH gene promoter activity in undifferentiated Neuro-2A cells, while other drugs under study (citalopram, tianeptine, moclobemide, venlafaxine, reboxetine, mirtazapine, and milnacipram) were inactive. In the differentiated cells, all examined antidepressants, except moclobemide (no effect) and tianeptine (increase), inhibited hCRH gene transcription. Moreover, in differentiated cells, the drugs acted stronger and were effective at lower concentrations. Forskolin-induced CAT activity was attenuated by imipramine and fluoxetine and to a lesser degree by amitriptyline and desipramine in differentiated cells, whereas other drugs were inactive. Moreover, imipramine and fluoxetine, but not tianeptine, showed moderate inhibitory effect on CRH gene promoter activity also in AtT-20 cell line, commonly used in CRH gene regulation studies. These results indicate that neuron-like differentiated Neuro-2A cells are a better model than pituitary and intact neuroblastoma to investigate the mechanism of psychotropic drug action. Inhibition of CRH gene promoter activity by antidepressant drugs may be a molecular mechanism by which these drugs inhibit the activity of HPA axis.

Chlorpromazine inhibits the glucocorticoid receptor-mediated gene transcription in a calcium-dependent manner.

Antipsychotic drugs can modulate transcription factors and also nuclear receptors, but their action on glucocorticoid receptors (GR)-members of the steroid/thyroid hormone receptor family has not been studied so far. In the present study we investigated effects of various antipsychotics on the glucocorticoid-mediated gene transcription in fibroblast cells, stably transfected with a mouse mammary tumor virus promoter (LMCAT cells). Chlorpromazine (3-100 microM) inhibited the corticosterone-induced gene transcription in a concentration- and time-dependent manner. Clozapine showed a similar, but less potent effect, while haloperidol acted only in high concentrations, and other antipsychotic drugs (sulpiride, raclopride, remoxipride) were without any effect. It was also found that a phorbol ester (an activator of protein kinase C (PKC)) and A-23187 (Ca(2+)-ionophore) attenuated the inhibitory effect of chlorpromazine on the GR-induced gene transcription. An antagonist of the L-type Ca(2+) channel, as well as an inhibitor of phospholipase C (PLC) inhibited the corticosterone-induced gene transcription, but had no effect on the chlorpromazine-induced changes. The involvement of a PKC/PLC pathway in the chlorpromazine action was confirmed by Western blot analysis which showed that the drug in question decreased the PLC-beta(1) protein level, and to a lesser extent that of the PKC-alpha protein in LMCAT cells. The aforementioned data suggest that inhibition of the glucocorticosteroid-induced gene transcription by chlorpromazine and clozapine may be a mechanism by which these drugs block some effects induced by glucocorticoids. The inhibitory effect of chlorpromazine on the corticosterone-induced gene transcription seems to depend on the inhibition of Ca(2+) influx and/or the inhibition of some calcium-dependent enzymes, e.g. phospholipase beta(1).

Prenatal stress decreases glycogen synthase kinase-3 phosphorylation in the rat frontal cortex

It has been postulated that hyperactive glycogen synthase kinase-3 (GSK-3) is an important factor in the pathogenesis of depression, and that this enzyme also contributes to the mechanism of antidepressant drug action. In the present study, we investigated the effect of prenatal stress (an animal model of depression) and long-term treatment with antidepressant drugs on the concentration of GSK-3beta and its main regulating protein kinase B (PKB, Akt). The concentration of GSK-3beta, its inactive form (phospho-Ser9-GSK-3beta), and the amounts of active (phospho-Akt) and total Akt were determined in the hippocampus and frontal cortex in rats. In order to verify our animal model of depression, immobility time in the forced swim test (Porsolt test) was also determined.We found that prenatally stressed rats display a high level of immobility in the Porsolt test and chronic treatment with imipramine, fluoxetine, mirtazapine and tianeptine normalize this change. Western blot analysis demonstrated that GSK-3beta levels were significantly elevated in the frontal cortex, but not in the hippocampus, of prenatally stressed rats. The concentration of its non-active form (phospho-Ser9-GSK-3beta) was decreased only in the former brain structure. No changes were found in the amounts of active (phospho-Akt) and total Akt in both studied brain structures. Chronic treatment with antidepressant drugs diminished stress-induced alterations in GSK-3beta and phospho-GSK-3beta the frontal cortex, but had no effect on the concentration of these enzymes in the hippocampus. Moreover, levels of Akt and phospho-Akt in all experimental groups remained unchanged. Since our animal model of depression is connected with hyperactivity of the HPA axis, our results suggest that GSK-3beta is an important intracellular target for maladaptive glucocorticoid action on frontal cortex neurons and in antidepressant drug effects. Furthermore, the influence of stress and antidepressant drugs on GSK-3beta does not appear to impact the kinase activity of Akt.

Inhibitory effect of imipramine on the human corticotropin-releasing-hormone gene promoter activity operates through a PI3-K/AKT mediated pathway

Antidepressant drugs inhibit the corticotropin-releasing-hormone (CRH) gene promoter activity in the differentiated Neuro-2A cells, but a molecular mechanism of their action has been poorly recognized. The aim of the present study was to elucidate the involvement of some intracellular signal transduction pathways in imipramine-induced inhibition of CRH gene activity in the differentiated Neuro-2A cells, stably transfected with a human CRH promoter fragment linked to the chloramphenicol acetyltransferase (CAT) reporter gene. It was found that wortmannin (0.1muM), an inhibitor of phosphatidylinositol 3-kinase (PI3-K) and forskolin (10, 25muM), an activator of adenylate cyclase enhanced the basal activity of CRH gene promoter, whereas inhibitors of protein kinase A, calcium/calmodulin kinase (CaMK) and mitogen-activated protein kinase (MAPK) had opposite effects. Moreover, wortmannin at a low concentration (0.01muM) significantly reversed the inhibitory effect of imipramine on CRH-CAT activity, whereas other protein kinase inhibitors were inactive or even enhanced the imipramine effects. The involvement of PI3-K/Akt pathway in the imipramine action was confirmed by Western blot study, which showed that this drug increased phospho-Ser-473 Akt level, but had no effect on total Akt and glycogen synthase kinase (GSK-3beta) levels. These results indicate that the inhibitory effect of imipramine on the CRH gene promoter activity in Neuro-2A cells is mainly connected with enhancement of PI-3K/Akt pathway.

Maternal immune activation leads to age-related behavioral and immunological changes in male rat offspring - the effect of antipsychotic drugs

BACKGROUND Prenatal immune system disturbances have been postulated to play an important role in pathogenesis of schizophrenia and related disorders. In the present study, we sought to answer the question whether behavioral changes in the neurodevelopmental model of schizophrenia in rats are accompanied by alterations in proliferative activity of splenocytes and pro- and anti-inflammatory cytokine levels. Furthermore, the effects of two antipsychotic drugs on these parameters were determined. METHODS Lipopolysaccharide (LPS) was administered subcutaneously to pregnant dams at a dose of 1 mg/kg every second day from the 7(th) day of pregnancy till delivery. Age-dependent behavioral and immunological changes were studied when control and prenatally LPS-pretreated offspring male rats were 30 and 90 days old. Chlorpromazine (10 mg/kg ip) or clozapine (10 mg/kg ip) was administered chronically (21 days) after behavioral verification to 3 months old offspring males. Changes in sensorimotor gating (prepulse inhibition, PPI), mitogen-induced proliferative activity of splenocytes ([(3)H]-thymidine incorporation) and cytokine levels (ELISA) were measured. RESULTS Prenatally LPS-pretreated rats showed PPI deficit only at 90 but not at 30 days of age, whereas an enhancement of mitogen-stimulated proliferative activity of splenocytes was observed in both time points. Additionally, the level of proinflammatory cytokines (IL-1β, IL-2, IL-6, TNF-α) in prenatally LPS-pretreated rats was enhanced when they were 30 days old and remained elevated in 90 days old offspring. No changes in IL-10 level were observed. Chronic administration of chlorpromazine or clozapine reduced the deficit in PPI deficit in prenatally LPS-treated rats. In the used model, chlorpromazine normalized both T and B lymphocyte proliferation, whereas clozapine B lymphocyte activity only. Moreover, both antipsychotics modulated the enhanced levels of IL-1β, IL-2 and TNF-α in the offspring of LPS-treated mothers. CONCLUSIONS This study indicates that in LPS-evoked model of schizophrenia, peripheral immunological changes are long-lasting and precede behavioral deficit. The disturbances in T cell-mediated immunity as well as cytokine production were attenuated by antipsychotic drug administration.