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132 expression in the hippocampus of rats subjected to olfactory bulbectomy
Molecular and cellular Male rats were assigned to the following experimental groups: Vehicle + Vehicle (VEH+VEH); Vehicle + Haloperidol 0.8 mg/kg (VEH+HAL); Vehicle + GBR12909 30 mg/kg (VEH+GBR); Vehicle + Caffeine 40 mg/kg (VEH+CAF); Vehicle + Nicotine 1.5 mg/kg (VEH+NIC); CAF+HAL; NIC+HAL. Drugs were dissolved in saline, adjusted to physiological pH and intraperiotoneally injected. The second injection was made 20 min after the first one. After 150 min, animals were sacrificed and brains were frozen at −80ºC. Half of the brains were dissected and lysed for Western Blotting (WB) analysis. The others were cut at the cryostat in order to obtain coronal sections of 12 m for in Situ Hybridization (ISHH). Early genes such as Homer1a and Arc had a predominant expression in the striatum and nucleus accumbens after HAL administration, respectively. Arc expression was significantly reduced by CAF in the ventromedial striatum. The constitutive gene Homer1b was increased by NIC+HAL in the medial striatum and by NIC only in the nucleus accumbens. In the cortex, Homer1a expression was significantly reduced by CAF+HAL in the anterior cingulate cortex, while Arc expression was significantly induced by HAL in the motor and insular cortices. Homer1b expression was increased by NIC+HAL in almost all cortical regions and by GBR in the medial agranular and somatosensory cortices. Homer1a/Homer1b mRNA expression ratio was shifted toward Homer1b expression in all cortical regions by all treatments. In the striatum, HAL shifted the ratio toward Homer1a expression in all regions. CAF, NIC and CAF+HAL shifted the ratio toward Homer1b expression in all striatal regions. WB analysis revealed a significant overexpression of Arc and Homer1a proteins in the CAF+HAL group compared to vehicle. The data show that caffeine and nicotine are able to modulate genes involved in the dopamineglutamate postsynaptic interaction. Their combination with antipsychotics induces a different modulation of gene and protein expression compared to that produced by individual drugs in specific brain areas. Notably, when evaluating results of Western Blot analysis, relative ratio of Homer1a/Homer1b protein levels were shifted toward Homer1a by CAF+HAL and NIC, and shifted toward Homer1b expression by HAL, GBR, and CAF. These results indicate an uncoupling between the transcriptome and the proteome level, that may exert differential biological actions. Otherwise, negative feedbacks may reciprocally regulate mRNA expression and protein levels, possibly by post-transcriptional mechanisms or modulation of protein degradation. These results open the way for the definition of the molecular signature underlying the
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use of coffee and tobacco and their combined effects in psychiatric patients under pharmacological treatment. Reference(s) [1] Thoma, P., Daum, I., 2013. Comorbid substance use disorder in schizophrenia: a selective overview of neurobiological and cognitive underpinnings. Psychiatry Clin Neurosci. 67(6), 367−83. [2] Iasevoli, F., Tomasetti, C., Buonaguro, E.F., de Bartolomeis, A., 2014. The glutamatergic aspects of schizophrenia molecular pathophysiology: role of the postsynaptic density, and implications for treatment. Curr Neuropharmacol. 12(3), 219−38. P.1.011 Changes in miR-212/132 expression in the hippocampus of rats subjected to olfactory bulbectomy P. Misztak1,2 ° , P. Pa´nczyszyn-Trzewik2 , G. Nowak1,2 , M. Sowa-Ku´cma2 . 1 Jagiellonian University Medical College, Pharmacology, Krakow, Poland; 2 Institute of Pharmacology − Polish Academy of Science, Neurobiology, Krakow, Poland Background: Depression is a severe psychiatric disorder characterized by depressed mood, anxiety, disturbances in sleep and appetite and a dangerous sense of guilt with an accompanying low self-esteem of the patient. An increasing number of studies has shown that brainderived neurotrophic factor (BDNF) as well as cAMP response binding element (CREB) may play crucial roles in the pathophysiology and treatment of this disease [1]. It also appears that some small non-coding RNA (miRNA), especially cluster miR-212/132, may be involved in the regulation of BDNF and CREB expression, as well as other cellular processes that are important in the development of depression [2]. Purpose of the study: The aim of our study was to investigate the effects of olfactory bulbectomy (OB) − the most validated animal model of depression − on the expression of the cluster miR-212/132 in the hippocampus of rats. Furthermore, the effects of 14-day treatment with amitriptyline (10 mg/kg), fluoxetine (10 mg/kg), venlafaxine (10 mg/kg) or olanzapine (2 mg/kg) were examined in the OB model. Material and Methods: The OB procedure was carried out on male Sprague-Dawley rats according to our published procedure [3]. Fourteen days after surgery, the open-field test was performed to examine depressionlike behavior (hyperactivity) induced by OB. OB rats with hyperactivity were selected for drug treatment. All of the compounds mentioned above were administered
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Molecular and cellular
to both sham- and OB-operated rats, repeatedly once daily intraperitoneally (i.p.). Controls received saline (0.9% NaCl). After 14 days of treatment, the open field test was repeated to elucidate the effect of the drug on the rats’ behavior. 24 h after the test, rats were decapitated and the hippocampus was collected for realtime PCR analysis. Differences between the experimental and control groups were assessed using one-way analysis of variance (ANOVA) and Bonferroni’s post hoc test. A p-value of >0.05 was considered statistically significant. Results: The olfactory bulbectomy procedure significantly increased the levels of miR-132 (by 72%) and miR-212 (by 131%) in the hippocampus of OB-operated rats compared to sham-operated rats. These changes were correlated with hyperactivity in the OB group. Treatment with venlafaxine or olanzapine (but not fluoxetine) resulted in statistically significant decreases of miR-132 (by 118% and 94%, respectively) and miR-212 (by 127% and 143%, respectively), in OB rats (vs OB–saline rats). On the other hand, amitryptyline administration caused only a decrease in the miR-212 (by 152%) and no changes in miR-132 expression in the OB group. The changes in miRNA expression after treatment with venlafaxine or amitryptyline (but not olanzapine) were associated with reduced hyperactivity. None of the compounds tested had impact on locomotor activity and miRNA levels in the sham-operated group. Conclusions: Our findings confirm the role of the miR-212/132 cluster in the development and treatment of depression. However, the observed alterations seem to be drug-specific and characteristic only for drugs with a specific mechanism of action, and not for antidepressants. Reference(s) [1] Keller, M.C., Nesse, R.M., 2006. The evolutionary significance of depressive symptoms: different adverse situations lead to different depressive symptom patterns. J Pers Soc Psychol 91(2), 316–330. [2] Wanet, A., Tacheny, A., Arnould, T., Renard, P. 2012. miR-212/132 expression and functions: within and beyond the neuronal compartment. Nucleic Acids Research. Vol. 40, No. 11. [3] Pochwat, B., Sowa-Kucma, M., Kotarska, K., Misztak, P., Nowak, G., Szewczyk, B. 2014. Antidepressant-like activity of magnesium in the olfactory bulbectomy model is associated with the AMPA/BDNF pathway. Psychopharmacology (Berl). 232(2): 355−67. Disclosure statement: This study was partially supported by grant UMO-2013/09/D/NZ7/02520
P.1.012 Epigenetic regulation of oxytocinergic system during social fear conditioning R. Menon1 ° , D.A. Slattery1 , I.D. Neumann1 . 1 University of Regensburg Institute for Zoology, Department of Behavioral and Molecular Neurobiology, Regensburg, Germany Maladaptation of fear responses can lead to a number of anxiety disorders including social anxiety disorder (SAD), which is defined as persistent fear and avoidance of social situations. Treatment of SAD is rather unspecific and is only affective in 38% of patients with a high rate of relapse [1]. In order to reveal the molecular and neuronal underpinnings of SAD, we have established a mouse model of social fear using a Social Fear Conditioning (SFC) paradigm [2]. The neuropeptide OXT has been proposed as a potential therapeutic agent for SAD due to its pro-social, anxiolytic and stress-attenuating effects [3]. Our previous results have shown that social fear conditioned (SFC+ ) mice showed increased oxytocin receptor (OXTR) binding in the dorso-lateral septum (DLS) together with decreased local OXT release in the paraventricular nucleus (PVN) when compared with control (SFC− ) mice [3]. Importantly, we could then reveal that intra-DLS infusion of OXT abolished fear expression in SFC+ mice. Therefore, the aims of the present study were (1) to investigate, whether SFC alters the oxytocinergic system (i.e. Oxt and Oxtr) at mRNA level, (2) determining if these changes are mediated via epigenetic mechanisms and (3) to determine, if local DLS infusion of a specific histone deacetylase inhibitor (MS275) affected social fear extinction. SFC+ mice showed elevated Oxtr mRNA levels in the DLS (p < 0.01, n = 6), but neither in the dorsal hippocampus nor amygdala compared with SFC− mice, as measured using quantitative real-time PCR 120 min after fear acquisition. Similarly, the attenuated OXT release in SFC+ mice was reflected by reduced Oxt mRNA levels in the PVN 120 min after social fear extinction compared with SFC− (p < 0.001, n = 6). SFC exposure also increased Hdac1 mRNA expression in the DLS of SFC+ mice 120 min after fear acquisition (p < 0.01, n = 6), and this increase returned to basal levels following fear extinction on day 2 (p < 0.01, n = 6). Stimulation of Neuro 2A cells for 24 h with MS275 (2 mM), a potent HDAC1 inhibitor, increased Oxtr mRNA (p < 0.01, n = 4) expression by 150% and conversely, stimulating them with C646 (2.5mM) a CBP/p300 inhibitor brought about 40% decrease in Oxtr mRNA (p < 0.01, n = 5) expression. In line with this, chromatin immunoprecipitation revealed an enrichment of H3K27ac (p < 0.001, n = 3) and depletion
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use of coffee and tobacco and their combined effects in psychiatric patients under pharmacological treatment. Reference(s) [1] Thoma, P., Daum, I., 2013. Comorbid substance use disorder in schizophrenia: a selective overview of neurobiological and cognitive underpinnings. Psychiatry Clin Neurosci. 67(6), 367−83. [2] Iasevoli, F., Tomasetti, C., Buonaguro, E.F., de Bartolomeis, A., 2014. The glutamatergic aspects of schizophrenia molecular pathophysiology: role of the postsynaptic density, and implications for treatment. Curr Neuropharmacol. 12(3), 219−38. P.1.011 Changes in miR-212/132 expression in the hippocampus of rats subjected to olfactory bulbectomy P. Misztak1,2 ° , P. Pa´nczyszyn-Trzewik2 , G. Nowak1,2 , M. Sowa-Ku´cma2 . 1 Jagiellonian University Medical College, Pharmacology, Krakow, Poland; 2 Institute of Pharmacology − Polish Academy of Science, Neurobiology, Krakow, Poland Background: Depression is a severe psychiatric disorder characterized by depressed mood, anxiety, disturbances in sleep and appetite and a dangerous sense of guilt with an accompanying low self-esteem of the patient. An increasing number of studies has shown that brainderived neurotrophic factor (BDNF) as well as cAMP response binding element (CREB) may play crucial roles in the pathophysiology and treatment of this disease [1]. It also appears that some small non-coding RNA (miRNA), especially cluster miR-212/132, may be involved in the regulation of BDNF and CREB expression, as well as other cellular processes that are important in the development of depression [2]. Purpose of the study: The aim of our study was to investigate the effects of olfactory bulbectomy (OB) − the most validated animal model of depression − on the expression of the cluster miR-212/132 in the hippocampus of rats. Furthermore, the effects of 14-day treatment with amitriptyline (10 mg/kg), fluoxetine (10 mg/kg), venlafaxine (10 mg/kg) or olanzapine (2 mg/kg) were examined in the OB model. Material and Methods: The OB procedure was carried out on male Sprague-Dawley rats according to our published procedure [3]. Fourteen days after surgery, the open-field test was performed to examine depressionlike behavior (hyperactivity) induced by OB. OB rats with hyperactivity were selected for drug treatment. All of the compounds mentioned above were administered
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Molecular and cellular
to both sham- and OB-operated rats, repeatedly once daily intraperitoneally (i.p.). Controls received saline (0.9% NaCl). After 14 days of treatment, the open field test was repeated to elucidate the effect of the drug on the rats’ behavior. 24 h after the test, rats were decapitated and the hippocampus was collected for realtime PCR analysis. Differences between the experimental and control groups were assessed using one-way analysis of variance (ANOVA) and Bonferroni’s post hoc test. A p-value of >0.05 was considered statistically significant. Results: The olfactory bulbectomy procedure significantly increased the levels of miR-132 (by 72%) and miR-212 (by 131%) in the hippocampus of OB-operated rats compared to sham-operated rats. These changes were correlated with hyperactivity in the OB group. Treatment with venlafaxine or olanzapine (but not fluoxetine) resulted in statistically significant decreases of miR-132 (by 118% and 94%, respectively) and miR-212 (by 127% and 143%, respectively), in OB rats (vs OB–saline rats). On the other hand, amitryptyline administration caused only a decrease in the miR-212 (by 152%) and no changes in miR-132 expression in the OB group. The changes in miRNA expression after treatment with venlafaxine or amitryptyline (but not olanzapine) were associated with reduced hyperactivity. None of the compounds tested had impact on locomotor activity and miRNA levels in the sham-operated group. Conclusions: Our findings confirm the role of the miR-212/132 cluster in the development and treatment of depression. However, the observed alterations seem to be drug-specific and characteristic only for drugs with a specific mechanism of action, and not for antidepressants. Reference(s) [1] Keller, M.C., Nesse, R.M., 2006. The evolutionary significance of depressive symptoms: different adverse situations lead to different depressive symptom patterns. J Pers Soc Psychol 91(2), 316–330. [2] Wanet, A., Tacheny, A., Arnould, T., Renard, P. 2012. miR-212/132 expression and functions: within and beyond the neuronal compartment. Nucleic Acids Research. Vol. 40, No. 11. [3] Pochwat, B., Sowa-Kucma, M., Kotarska, K., Misztak, P., Nowak, G., Szewczyk, B. 2014. Antidepressant-like activity of magnesium in the olfactory bulbectomy model is associated with the AMPA/BDNF pathway. Psychopharmacology (Berl). 232(2): 355−67. Disclosure statement: This study was partially supported by grant UMO-2013/09/D/NZ7/02520
P.1.012 Epigenetic regulation of oxytocinergic system during social fear conditioning R. Menon1 ° , D.A. Slattery1 , I.D. Neumann1 . 1 University of Regensburg Institute for Zoology, Department of Behavioral and Molecular Neurobiology, Regensburg, Germany Maladaptation of fear responses can lead to a number of anxiety disorders including social anxiety disorder (SAD), which is defined as persistent fear and avoidance of social situations. Treatment of SAD is rather unspecific and is only affective in 38% of patients with a high rate of relapse [1]. In order to reveal the molecular and neuronal underpinnings of SAD, we have established a mouse model of social fear using a Social Fear Conditioning (SFC) paradigm [2]. The neuropeptide OXT has been proposed as a potential therapeutic agent for SAD due to its pro-social, anxiolytic and stress-attenuating effects [3]. Our previous results have shown that social fear conditioned (SFC+ ) mice showed increased oxytocin receptor (OXTR) binding in the dorso-lateral septum (DLS) together with decreased local OXT release in the paraventricular nucleus (PVN) when compared with control (SFC− ) mice [3]. Importantly, we could then reveal that intra-DLS infusion of OXT abolished fear expression in SFC+ mice. Therefore, the aims of the present study were (1) to investigate, whether SFC alters the oxytocinergic system (i.e. Oxt and Oxtr) at mRNA level, (2) determining if these changes are mediated via epigenetic mechanisms and (3) to determine, if local DLS infusion of a specific histone deacetylase inhibitor (MS275) affected social fear extinction. SFC+ mice showed elevated Oxtr mRNA levels in the DLS (p < 0.01, n = 6), but neither in the dorsal hippocampus nor amygdala compared with SFC− mice, as measured using quantitative real-time PCR 120 min after fear acquisition. Similarly, the attenuated OXT release in SFC+ mice was reflected by reduced Oxt mRNA levels in the PVN 120 min after social fear extinction compared with SFC− (p < 0.001, n = 6). SFC exposure also increased Hdac1 mRNA expression in the DLS of SFC+ mice 120 min after fear acquisition (p < 0.01, n = 6), and this increase returned to basal levels following fear extinction on day 2 (p < 0.01, n = 6). Stimulation of Neuro 2A cells for 24 h with MS275 (2 mM), a potent HDAC1 inhibitor, increased Oxtr mRNA (p < 0.01, n = 4) expression by 150% and conversely, stimulating them with C646 (2.5mM) a CBP/p300 inhibitor brought about 40% decrease in Oxtr mRNA (p < 0.01, n = 5) expression. In line with this, chromatin immunoprecipitation revealed an enrichment of H3K27ac (p < 0.001, n = 3) and depletion