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1

Differential mRNA expression level of the Bcl2 protein family in the thalamus of rats reactive and non-reactive to chronic mild stress - the animal model of depression

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Bielawski A., Rafa-Zablocka K., Papp M., Nalepa I.
Acta Neurobiologiae Experimentalis
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2011
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tom 71
|
nr S
Apoptosis is controlled by the balance between pro- (Bax) and anti-(Bcl-2, Bcl-xl) apoptotic proteins within the cell. Bcl-2 and Bcl-xl interact with Bax in the outer mitochondrial membrane and regulate the release of cytochrome c, which activates caspases, the main executors of apoptosis. The increased ratio of pro- vs. antiapoptotic proteins is associated with an enhanced vulnerability to apoptotic activation. The chronic mild stress (CMS) procedure induces depression-like symptoms in animals. The rats subjected for a prolonged period of time to a variety of mild stressors gradually decrease their responsiveness to rewarding stimuli (e.g., consumption of sweet pellets). We aimed to investigate the expression of Bcl-2, Bcl-xl and Bax mRNAs in the thalamus of rats subjected to the 3-weeks CMS. Three groups of male Wistar rats, selected basing on behavioral test of sucrose (1% solution) consumption – sham, stress reactive and stress non-reactive, were considered. The mRNA expression was measured by quantitative RT-PCR applying TaqMan probes. We found that in the thalamus of rats developing anhedonia to sucrose consumption after the CMS, the mRNA expression of both anti-apoptotic (Bcl-2 and Bcl-xl) and pro-apoptotic (Bax) genes was significantly attenuated, though to a various extent. In the stress reactive animals, the Bcl-2 mRNA was decreased by 57% (p<0.01), Bcl-xl – by 51% (p<0.05) and Bax – by 24% (p<0.05), while no change was noticed in the stress non-reactive animals. Further analysis revealed a significant decrease in the Bcl-2/Bax and Bcl-xl/Bax ratios (respectively, by 48% and 25%; p<0.01) in the stress reactive animals, and no change in case of the stress non-reactive animals. Our results suggest that the behavioral reactiveness of rats to the CMS is associated with the enhanced susceptibility to apoptotic activation and development of apoptotic processes in the thalamus. Supported by statutory funds of the Institute of Pharmacology, PAS.
2

Evaluation of transgenic mice conditionally lacking CREB in noradrenergic neurons as a novel tool for studying its role in the antidepressant drug action

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Rafa-Zablocka K., Kreiner G., Baginska M., Nalepa I.
Acta Neurobiologiae Experimentalis
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2015
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tom 75
|
nr Supl.
BACKGROUND AND AIMS: Most of the current antidepressants modulate levels of monoamines just after administration, however, only after prolonged therapy the clinical effect may be observed. Myriad attempts tried to identify molecular factors responsible for such a delayed response, the prominent example being the cyclic AMP response element binding protein (CREB). Many research suggest that chronically given antidepressants enhance CREB levels and activity. On the other hand, CREB knock-out mice showed rather antidepressant-like behavior, however, the compensatory effects of cAMP response element modulator (CREM) in absence of CREB were not taken into account. In our study we evaluated transgenic mice with selective ablation of CREB in noradrenergic cells, maintained in CREM deficient background (CrebDBHCreCrem-/-) for elucidation of the role of CREB in antidepressant treatment. METHODS: mRNA levels of neurotrophins and α1-adrenoceptors in CrebDBHCreCrem-/- mice were investigated in hippocampus and prefrontal cortex using qPCR method. Next mice were screened in behavioral tests like: open field (OF), tail suspension test (TST) and rotarod. Preliminary TST after acute desipramine (DMI) administration (20 mg/kg, ip) was executed. RESULTS: CrebDBHCreCrem-/- mutant mice did not show any abnormalities in their basal phenotype, moreover the mRNA levels of studied genes were not changed either. However, preliminary experiments revealed that CrebDBHCreCrem-/- show a treatment-resistant phenotype after acute DMI administration in TST, (effect absent in single mutants). CONCLUSIONS: Our results provide further evidence for the important role of CREM as a compensatory factor and indicate that these mice may represent an unique tool to dissect the role of CREB in the mechanism of antidepressants. Supported by statutory funds of the Institute of Pharmacology and 2014/13/B/NZ7/02293 grant. K. Rafa-Zabłocka is a holder of scholarship from the KNOW sponsored by MSHE, Poland.
3

The effects of trehalose administration on autophagy enhancement in mice with conditional and progressive degeneration of medial spiny neurons

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Kreiner G., Rafa-Zablocka K., Baginska M., Nalepa I.
Acta Neurobiologiae Experimentalis
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2015
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tom 75
|
nr Supl.
BACKGROUND AND AIMS: In neural cells, autophagy is proposed to serve as a surveillance mechanism which helps to clear protein aggregates, and loss of autophagy leads to neurodegeneration in mice. Our previous experiments, performed on genetically engineered mice with conditional and progressive neurodegeneration of medial spiny neurons (TIF-IAD1RCre mice) mimicking the typical progression of Huntington’s disease (HD), showed that the delayed onset of neurodegeneration observed in these mutants might be associated with temporary increased autophagy. The aim of current study was to evaluate a new strategy proposed recently for the anti-HD treatment, based on enhancing autophagy by administration of trehalose, a natural alpha-linked disaccharide. METHODS: Trehalose (2%) was dissolved in water and presented to the mice as a replacement for their water bottles for 1 or 2 months prior the experiments. The effects of trehalose were compared with groups receiving maltose (2%) as well as water (vehicle). The autophagy was determined by Western blot (WB) and immunohistochemistry (IHC) with use of anti-LC3B antibody. The animals were screened for their motor coordination by accelerated rotarod, and post-mortem for selected neurodegenerative markers by WB and IHC. RESULTS: Both control and mutant mice showed enhanced autophagy after trehalose administration as revealed by WB and IHC staining. Nevertheless, further analysis of quantitative assessment of several neurodegenerative markers by WB did not reveal any significant effects in attenuating the neurodegenerative process. There have been also no differences in behavioral phenotype. CONCLUSIONS: Our results provide additional evidence for stimulation of autophagy evoked by chronic administration of trehalose. However further study is needed, the enhancement of autophagy has not yet been proved to be neuroprotective in investigated model. Supported by 2011/03/B/NZ7/05949 grant financed by National Science Center (NCN).
4

Prolonged incubation with desipramine differentially modulate A1A-and A1B-adrenoreceptor signaling in PC12 cells

100%
Chmielarz P., Kowalska M., Rafa-Zablocka K., Nalepa I.
Acta Neurobiologiae Experimentalis
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2015
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tom 75
|
nr Supl.
BACKGROUND AND AIMS: Action of tricyclic antidepressant drugs (TCA) involves inhibition of noradrenaline re-uptake, however, several of TCA also exhibit affinities for adrenergic receptors (α1-ARs). Furthermore there are reports in literature implying role of α1-AR in depression and antidepressant action. However due to scarcity of selective ligands discriminating the α1-ARs, the specific role of each of three subtypes remains elusive. Aim of current study was to investigate if and how prolonged incubation with desipramine (DMI), a TCA exhibiting affinity for α1-AR could affect the α1A- and α1B-adrenoreceptor reactiveness in vitro. METHODS: Measurements were performed in PC12 cells stably transfected with human α1A- and α1B‑AR. To assess receptor activity cells expressing either one of the receptors were incubated for 24 hours with 10 µM DMI or vehicle. After incubation receptor responsiveness to agonist was assayed by stimulation with serial dilutions of noradrenaline (form 10-9 to 10-4 M) and measurement of inositol phosphate generation with use of TR-FRET based assay. RESULTS: The 24 hours preincubation with DMI shifted the noradrenaline dose response curve rightwards in case of both α1A-AR (EC50 = 0.9 µM and EC50 = 14.5 µM for VEH and DMI preincubated cells, respectively) and α1B‑AR (EC50 = 0.5 µM and EC50 = 2.7 µM, for VEH and DMI preincubated cells, respectively). The effect of DMI was more pronounced for α1A-AR and difference between receptor subtypes was reflected in significant receptor×treatment interaction (P<0.001) in two-way ANOVA comparison. CONCLUSIONS: Our data indicate that DMI can differentially modulate activity of α1A- and α1B‑AR. The diverse susceptibility of α1A- and α1B‑AR to DMI action may be interesting in the light of reports of different role of these receptors in depressive-like behaviors in mice. This study was supported by statutory funds of Institute of Pharmacology, PAS.
5

Changed expression of apoptotic signaling-releted genes, Pmaip1 and Rock1, in the prefrontal cortex of rats treated with imipramine in chronic mild stress model of depression

88%
Bielawski A., Rafa-Zablocka K., Zelek-Molik A., Papp M., Nalepa I.
Acta Neurobiologiae Experimentalis
|
2015
|
tom 75
|
nr Supl.
BACKGROUND AND AIMS: The chronic mild stress (CMS) procedure induces depression-like symptoms in animals. In this model, rats subjected for a prolonged time to mild stressors gradually decrease their responsiveness to rewarding stimuli. This deficit can be effectively reversed by chronic antidepressant treatment. Apoptotic changes in the prefrontal cortex were shown in animal stress models, as well as degenerative changes, which are reversed by antidepressant treatment in depressed patients. We aimed to study the apoptotic signaling-related genes in the prefrontal cortex (PFC) of rats treated with imipramine (IMI) in CMS model. METHODS: First, we used the TaqMan Low Density Arrays to indentify genes in the three groups of Wistar rats: sham-saline; stressIMI-responders and stress-IMI-nonresponders (the sucrose intake score did not return to the control level). Then, these groups of rats and two additional ones (stress-saline and sham-IMI) were assessed in the PCR reactions with one TaqMan probe for detailed mRNA analysis of the identified genes. Finally, the levels of these proteins were assessed by Western Blot in all experimental groups. RESULTS: We found that CMS decreased the expression of Pmaip1 mRNA (by 18%) and the effect remained unchanged in rats nonresponding behaviorally to IMI treatment. Furthermore, in rats nonresponding to IMI treatment, the Rock1 mRNA was decreased by 40% vs. sham and IMI responding rats. However, at the Rock1 protein level its decreased expression was observed in both groups, the IMI nonresponding and IMI responding animals (by 22% and 29%, respectively). CONCLUSION: Our results suggest the involvement of apoptotic Pmaip1 and Rock1 genes in the process of response to IMI treatment in CMS model of depression in rats. Supported by statutory funds of the Institute of Pharmacology PAS and POIG.01.01.02-12-004/09-00 grant financed by European Regional Development Fund.
6

Characterization of mice with genetically evoked selective degeneration of noradrenergic system and its possible influence on dopaminergic system

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Jurga A., Kreiner G., Rafa-Zablocka K., Baginska M., Parlato R., Schuetz G., Nalepa I.
Acta Neurobiologiae Experimentalis
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2013
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tom 73
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nr 1
Parkinson’s Disease (PD) is characterized by an increased production of oxygen free radicals leading to alteration of the cellular constituents and subsequent dopaminergic cell loss within the region of substantia nigra (SN) and ventral tegmental area (VTA). However, it is well known that PD is not only associated with dopaminergic transmission. Involvement of extranigral structures in PD includes the noradrenergic system as well. Post-mortem studies of human brains revealed that neuronal loss associated with PD may proceed and is even greater in the region of locus ceruleus (LC) than SN/ VTA. In PD animal models, the loss of noradrenaline made worse the dopamine nigrostriatal damage and, in opposite, an enhanced noradrenaline level may have a neuroprotective role. The aim of this study was to determine whether genetically evoked, selective loss of noradrenergic neurons may have any long-term, negative impact on the dopaminergic system. We applied the conditional inactivation of the gene encoding transcription factor TIF-IA (essential for the regulation of rRNA synthesis) by the Cre-loxP system to induce the progressive and selective loss of noradrenergic neurons which was achieved by expressing Cre recombinase under dopamine beta-hydroxylase (DBH) promoter. Resulting TIFIADBHCre mice were born at expected rates, viable but showed clear signs of noradrenergic innervations failure e.g. ptosis, reduced locomotor activity, growth retardance and shorten life span. The animals were analyzed at 8 and 12 weeks of age. The selective loss of noradrenergic neurons was confirmed by immunofluorescent staining with the anti-tyrosine hydroxylase (TH) antibody. We observed approx. 90% reduction of TH positive cells in the LC of 8 weeks TIF-IADBHCre mice. The number of TH+ cells was not changed in the region of SN/VTA, neither in 8 nor 12 week old mutants. However, our preliminary data indicate that lack of the noradrenergic transmission may lead to enhanced expression of selected markers associated with neurodegeneration within the region of SN/VTA. Namely, we have found 1.4 fold up-regulation of mRNA encoding for glial fibrillary acidic protein (GFAP) as revealed by quantitative real-time PCR and increased level of oxidative stress shown by immunoblot detection of carbonyl groups by Western Blot in the SN/VTA of 12 weeks TIF-IADBHCre mice compared to control animals. If we provide additional evidences that selective noradrenergic degeneration affects functioning of dopaminergic neurons, TIF-IADBHCre mice may became a valuable, new model for study possible anti-PD treatment at early stages of the disease as dopaminergic neurons in these mice are not directly affected by the mutation. As for today, there are no experimental studies on a possible long-term negative impact of progressive noradrenergic degeneration on other neurotransmitter systems despite of clinically observed concomitant loss of SN/VTA and LC neurons in PD. This study was supported by the grant no 2011/03/B/NZ7/05949 financed by National Science Centre and statutory funds of the Institute of Pharmacology, Polish Academy of Sciences.
7

Impact of fear memory consolidation and retrieval on the mRNA expression of G proteins in mice brain

76%
Zelek-Molik A., Costanzi M., Rafa-Zablocka K., Kreiner G., Roman A., Cestari V., Nalepa I.
Acta Neurobiologiae Experimentalis
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2017
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tom 77
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nr Suppl.1
INTRODUCTION: Fear evoked signaling disturbances among hippocampus (HP), nuclei of amygdala (Amy) and prefrontal cortex (PFC) underlie anxiety related disorders, but their molecular mechanism remains elusive. Heterotrimeric G proteins (GP) based on intracellular activity of alpha subunit (Gα) are divided into four families: G(s) stimulating cAMP generation; G(i/o) inhibiting cAMP pathway; G(Q/11) increasing Ca++ level; G(12/13) activating monomeric GP-Rho. AIM(S): In the present study, the effects of fear memory consolidation and retrieval on the mRNA expression of Gα from all GP families were assessed in HP, Amy and PFC. METHOD(S): C57BL/6J mice were subject to 1-day fear conditioning (FC) procedure followed by contextual (CTX) or cued (Cs) retrieval test of freezing behavior. Morphine (1mg/kg/ip) injected immediately after FC was used to prevent fear consolidation process. RealTime PCR technique was adopted to measure mRNA expression of Gα subunits: 1 h after FC, 24 h later, 1 h after CTX or Cs retrieval test. RESULTS: In HP, the increased levels of Gα(s), (12) and (11) were observed 1 h after FC. The Gα(s) mRNA decreased (vs. control) when consolidation was stabilized as well after Cs retrieval. Elevated Gα(12) mRNA, as observed 1h after FC, returned to control level at fear memory stabilization and raised again with CTX retrieval. The increase in Gα(11) persisted 24 h after FC and after CTX (but not Cs) retrieval. In PFC, the CTX retrieval was accompanied by a decrease in Gα(i2) and (i3) mRNA levels. In Amy, no specific change to fear memory process was observed. CONCLUSIONS: FC evoked changes in Gα mRNA expression are observed mainly in HP and mostly connected to CTX learning. Results suggest that activated signaling pathways from Gα(s) and Gα(12) are necessary to begin fear memory consolidation process in HP while signal transduction via Gα(11) is implicated in maintenance of fear consolidation. FINANCIAL SUPPORT: Supported by statutory funds from the Institute of Pharmacology PAS.
8

Changes in alfa1-adrenergic receptor subtypes in the hippocampus of rats non-responding to imipramine treatment in the chronic mild stress model of depression

76%
Rafa-Zablocka K., Zelek-Molik A., Bielawski A., Gruca P., Lason-Tyburkiewicz M., Papp M., Nalepa I.
Acta Neurobiologiae Experimentalis
|
2011
|
tom 71
|
nr S
Despite many years of research on depression the mechanism of the disorder remains elusive. Many studies are focused on dysfunction of central monoaminergic systems and some evidence exist for the role of α1-adrenoceptor (α1-AR). There are three subtypes of this receptor - α1A, α1B and α1D, which are widely distributed in brain. The aim of this study was to assess the expression of all three α1-AR, both at the mRNA and at the protein level in the hippocampi of male Wistar rats, subjected to the chronic mild stress (CMS) procedure followed by treatment with antidepressant drug, imipramine (IMI). Five groups of animals were studied: sham-saline; stress-saline; sham-IMI; stress-IMIresponders and stress-IMI-non-responders. The latter included the stressed animals resistant to IMI treatment as indicated by anhedonia test. The mRNA level was measured using qRT-PCR and SybrGreen dye, and the protein level was assessed by Western blotting. We found that mRNA expression of all α1-AR subtypes was significantly elevated only in the IMI-non-responders group (α1A-AR by 76%; α1B-AR by 96%; α1D-AR by 50%, vs. shamsaline). Moreover, stress alone caused an increase in α1A-AR mRNA (by 41%) though the effect was statistically insignificant. Changes found in the protein level were less pronounced. The only difference between IMI-responders and non-responders was found in α1A receptor protein that was decreased by 73% vs IMIresponders. The level of α1D protein was elevated in all IMI treated groups (by about 79%, vs. sham-saline) and the change occurred independently on stress procedure. No change in the α1B protein was found. Our results indicate that although α1A-AR and α1D-AR are involved in mechanism of IMI action, only the α1A receptor seems to be engaged in the phenomenon of resistance to IMI treatment. Supported by a grant POIG.01.01.02-12-004/09-00 financed by European Regional Development Fund.
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