Sex chromosome complement regulates Dnmt3a and Dnmt3b-gene expression in the anterior amygdala of developing mouse brain
Sosa C1, Cabrera Zapata LE1,Cisternas CD1; CambiassoMJ1, 2
1Instituto de Investigación Médica M y M Ferreyra INIMEC-CONICET-UNC. Córdoba, Argentina 2Cátedra de Biología Celular, Facultad de Odontología, Universidad Nacional de Córdoba, Córdoba, Argentina. E-mail: csosa@immf.uncor.edu
Both hormonal and genetic factors interact to induce long lasting effects on sexually dimorphic gene expression in the mouse brain. Epigenetic mechanisms, such as DNA methylation, have recently been proposed as mediators of hormonal-dependent sexual differentiation of the brain. DNA methylation involves the addition of methyl groups by DNA methyltransferase enzymes (Dnmts) as well as the recruitment of methyl-binding proteins (such as MeCP2) and usually leads to gene repression. In order to study whether DNA methylation also mediates sex chromosome-dependent factors involved in sexual differentiation of the brain we used the “four core genotypes” mouse model which allows the evaluation of gonadal sex, sex chromosome complement, and their interaction. We analyzed the mRNA expression of Dnmt3a, Dnmt3b, Dnmt1 and Mecp2 in vivo (anterior amygdala) and the interaction of hormonal and sex chromosome complement in vitro (primary neuronal cultures). Dnmt3a and Dnmt3b expression levels were higher in the anterior amygdala derived from XX embryos compared to XY, irrespectively of gonadal sex. No differences were observed in the expression of Dnmt1 and Mecp2. No significant effect of E2 or DHT on Mecp2 was seen in vitro. These results suggest that sex chromosome complement might determine a higher “de novo DNA methylation” in specific areas of the XX brain. More experiments are required to understand the role of X/Y chromosomes in the epigenetic changes involved in the sexual differentiation of brain.
β-CATENIN EXPRESSION IS UP-REGULATED IN BRACHIOCEPHALIC ARTERY AND THORACIC AORTA BEFORE TO THE CHRONIC CARDIAC CHAGAS DISEASE DEVELOPMENT
1,2 Volpini Ximena, Ph.D.; 2 Baigorrí Eliana; 2 Brugo Belén; 1 Natali Lautaro; 2 Cerbán Fabio; 2 Motrán Cristina; 1,3 Musri Melina.
1 Instituto de Investigación Médica Mercedes y Martín Ferreyra, Universidad Nacional de Córdoba. INIMEC-CONICET. Córdoba, Argentina.
2Depto. de Bioquímica Clínica. Facultad de Cs. Químicas, Universidad Nacional de Córdoba. CIBICI-CONICET. Córdoba, Argentina.
3Cátedra de Genética. Facultad de Cs. Exactas, Físicas y Naturales, Universidad Nacional de Córdoba. Córdoba, Argentina.
Wnt signaling participates in the development of the cardiovascular system by regulating processes such as cell fate, proliferation and migration. This pathway decreases in healthy adult heart and vessels but become reactivated during the development of several cardiovascular pathologies. It is well known that during Trypanosoma cruzi infection, a number of cardiovascular alterations take place, eventually triggering cardiomyopathy associated with chronic cardiac Chagas disease (CCC). However, little is known about the contribution of the systemic circulation to this condition. Previously, we demonstrated that Wnt inhibition during the acute phase of infection reduces CCC severity in BALB/c mice. In this work, we aimed to determine the expression of the canonical Wnt/β-catenin pathway in the systemic vasculature before the development of CCC. To this end, BALB/c mice were infected with 1000 tripomastigotes and β-catenin mRNA and T. cruzi satellite DNA were determined by q-RT-PCR in heart, brachiocephalic artery (BCA), thoracic (TA) and abdominal (AA) aorta at different days post-infection (dpi). A significant increase of β-catenin expression was observed in BCA (p=0.0255) and TA (p=0.0035) at 16 dpi, while no changes were observed in neither AA nor heart at any of the evaluated time points. Interestingly, β-catenin increment was consistent with parasite load in the assessed tissues and blood. In addition, parasitemia, parasite load and β-catenin in both BCA and TA decreased after 30 dpi together with the establishment of the chronic phase of the infection. We conclude that Wnt/β-catenin could be involved in early vascular alterations in response to T. cruzi infection.
PRENATAL EXPOSURE TO AMPHETAMINE: SEXUAL INFLUENCE IN BEHAVIORAL AND DOPAMINERGIC RESPONSE
Pennacchio GE1, Santonja FE1, Soaje M1,2, Bregonzio C3 .
1IMBECU, UNCuyo, CONICET, 2FCM-UNCuyo, 3 FCQ-UNC, IFEC-CONICET.
Since several evidences support that addiction is a development disorder, it becomes relevant to study how early drug abuse exposure can influence the response later in life. Moreover, there are sexual differences in the neuroadaptations to pharmacological inputs. Our aim was to determine whether prenatal amphetamine exposure (PEA) influences the response to the psychostimulant in males and females rats during adulthood. For this purpose, female Wistar rats received amphetamine (2.5 mg/kg i.p.) or saline daily during pregnancy (day 15 to 21). Their offspring were sexed and left undisturbed until day 60. The animals (males and females during estrous day) were tested for locomotor activity in response to a challenge dose of amphetamine (0.5 mg/kg i.p.) and their brains were dissected for RT-PCR determination of tyrosine hydroxylase (TH) and dopamine receptors (D1, D2 and D3) in striatum and nucleus accumbens. It was found a clear difference in locomotor activity between male and female in basal conditions and in the response to the challenge in PEA animals. In males, PEA increased the response to the challenge meanwhile in females it was decreased. The females showed significant differences in TH expression in striatum in basal and in response to the challenge in PEA conditions. Moreover, females showed increased levels of D3R in nucleus accumbens in all conditions. We conclude that PEA induces long lasting changes that affect the animal response during adulthood with a clear sexual influence.
SLUG REGULATES MICRORNAS EXPRESSION IN SMOOTH MUSCLE CELLS
Natali L1, de la Cruz-Thea B1, Ruiz Micol P1, Volpini X1, Meister G2, Musri M1,3
1Instituto de Investigación Médica M y M Ferreyra (INIMEC-CONICET-UNC). 2 RNA Biology, Facultät de VorKlinikum and Medizin, Universität Regensburg, Alemania, 3 Facultad de Ciencias Exactas Físicas y Naturales, Universidad Nacional de Córdoba.
E-mail: lautaronatali@outlook.com
Vascular Remodeling (VR) is an active process which contributes to the physiopathology of cardiovascular diseases. Smooth muscle cell (SMC) phenotypic alteration play a central role in the development of VR. We previously reported an upregulation of the transcription factor Slug in pulmonary arteries with high VR degree. We found that Slug promotes dedifferentiation and proliferation of SMC, but the underlying mechanisms are still unknown. MicroRNAs (miRNAs) are small noncoding RNAs that post-transcriptionally regulate mRNAs expression. MiRNAs are implicated in the regulation of physiological and pathological states and their expression have been found altered in arteries with VR. In this work we aimed to evaluate if Slug regulates the expression of miRNAs in SMC. To this end, we used an in vitro model of human pulmonary arteries SMC (Lonza) and analyzed miRNAs expression by deep sequencing of small RNAs (Illumina Seq) after knockdown of Slug using a specific siPool against Slug or a control siPool. Candidate miRNAs expression was validated using Northern Blot and mRNA expression by real time PCR. Our results showed a significant change in the expression of a number of miRNAs in Slug knocked down SMC vs the control cells. We validated the increase of miR-let7a, miR-23a and miR-10a, which have been previously implicated in the regulation of proliferation. In conclusion, in SMC, Slug knockdown allows the expression of miR-let7a, miR-23 and miR-10a which in turn might block SMC proliferation. These findings contribute with new knowledge to the biology of SMC.
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Is sex chromosome complement (SCC) responsible for sex differences in kidney V2 receptor (V2R) expression and desmopressin-induced antidiuresis?
Gonzalez, Lihue; Porcari, Cintia; Godino, Andrea; Vivas, Laura; Caeiro, Ximena.
INIMEC-CONICET-Universidad Nacional de Córdoba, Córdoba, Argentina.
An important number of experimental and clinical studies indicate a clear sexual difference in the antidiuretic response to desmopressin (V2R agonist). Women have a greater sensitivity to desmopressin and are more likely than men to develop hyponatremia in response to similar doses. Furthermore, in intact rats (without neutering) females express higher levels of renal mRNA and protein V2 receptor (V2R) levels, as well as a major urinary osmolarity response to desmopressin when compared to males. Moreover, in vitro studies have demonstrated that the X-linked V2R gene has high probability of escaping X-inactivation. Our present study aimed to explore the role of the SCC (XX/XY) and/or the organizational hormonal effects of gonadal steroids in the sexually dimorphic response to the antidiuretic desmopressin administration on urinary osmolarity as well as in the relative gene expression of renal V2R. For this purpose, we used male (XX and XY) and female (XX and XY) mice of the “four core genotype” model, in which the effect of gonadal sex and SCC are dissociated, allowing comparisons of sexually dimorphic traits among XX and XY females, as well as in XX and XY males. Mice aged 60-65 days old, were gonadectomized and forty-two days later were subcutaneously injected with vehicle solution or desmopressin (1 mg/kg). In a different group of animals kidneys were excised for V2R mRNA evaluation by qPCR.
As expected desmopressin administration induced a significant effect of treatment {F(1,37)=439,63} however no SCC nor organizational hormonal effects were observed in absence of the activational hormonal effects. In coincidence with this data the analysis of V2R mRNA levels showed no differences between male and female mice as well as among XX and XY mice. All this data in conjunction with previous studies highlights the importance of analyzing in further studies the contribution of the activational hormonal effects as well as the interaction with SCC and organizational hormonal effect in the potential mechanisms involved in sex-specific differences in kidney V2R expression and desmopressin-induced antidiuresis.
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Sex-related alcohol consumption induced by restraint stress in adolescent rats
Wille-Bille, A1; Pautassi, R1,2
1Insituto de Investigación Médica Mercedes y Martín Ferreyra (INIMEC-CONICET-UNC), Córdoba, Argentina.
2Faculta de Psicología, UNC, Córdoba, Argentina.
Adolescence is a developmental stage associated with neurobiological, behavioral and hormonal changes. These changes may underlie the greater vulnerability to drug abuse observed during this stage. Preclinical studies suggest that adolescents are uniquely sensitive to ethanol's pharmacological effects and that this pattern of response may put them at risk for alcohol initiation and escalation. Adolescent rats exhibit, when compared to adults, greater sensitivity to the appetitive and social facilitating effects of ethanol; yet they are less sensitive than adults to the aversive motivational effects of the drug. Stress is one of the risk factors that promotes greater engagement and escalation into alcohol consumption. Stress can enhance the appetitive reinforcing and decrease the aversive effects of ethanol; and it has been demonstrated that adolescents are more sensitive to the anxiolytic effects of alcohol than adults. Based on this background, we aimed at analyzing the permissive effects of stress on alcohol intake, in adolescents; and scrutinize possible sex-differences in this effect.
Male and female rats, 30 days-old, were subjected to five daily sessions of 120 minutes of restraint stress, using PVC tubes 20 cm long and 5-8 cm wide. Forty-eight hours later the alcohol intake test began. The two-bottle choice alcohol intake test lasted 2 weeks, with intermittent, every-other day access to the drug (18 hours per day, three times a week) taking. Alcohol solutions were ethanol 4% v/v on the first week, and 5% v/v on the second; in both cases accompanied with a bottle of vehicle (tap water). Experiment 2 replicated this procedure on female rats administrated with 10 mg/kg kappa antagonist, nor-Binaltorphimine (nor-BNI), 24 hours before the first stress session; vs. vehicle (saline solution). We registered g/kg alcohol consumed and percent preference.
The results indicated, for both g/kg and percent preference ethanol ingested, an interactive effect between sex and stress, with higher alcohol intake in stressed females than in their control counterparts. Males were unaffected by stress. Experiment 2 indicated that the facilitatory effects of stress on alcohol intake in female rats was blockaded by nor-BNI in female rats.
The relationship found in females between stress and subsequent alcohol consumption, guides to further analysis of the molecular basis underlying this permissive effect.
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Dam Early Free Access to Hypertonic NaCl Solution Induces a Long-Term Effect on Offspring Basal Chronic Brain Cell Activity
Macagno A1, Porcari C1, Vivas L1,2, Anastasia A1,3 and Godino A1,3.
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Instituto de Investigación Médica Mercedes y Martín Ferreyra (INIMEC-CONICET-Universidad Nacional de Córdoba), Córdoba, Argentina.
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Facultad de Ciencias Exactas Físicas y Naturales, Universidad Nacional de Córdoba, Córdoba, Argentina.
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Facultad de Psicología, Universidad Nacional de Córdoba, Córdoba, Argentina
Exposure to hyperosmotic environment during a pre/postnatal period can differentially program the fluid intake and excretion pattern in a way that persists until adulthood. Our results suggest that maternal voluntary ingestion of hypertonic NaCl solution during pregnancy and lactation until one week post-weaning (MP-Na) alters the offspring’s central osmoregulatory mechanisms. Our aim was to evaluate the impact of MP-Na on the basal expression of TRPV1 osmosensitive channel (by western blot) and chronic neuronal activity along the brain osmosensor areas of adult’s offspring by the immunohistochemical detection of brain Fra like protein (Fra-LI), alone or combined with vasopressin (AVP). The imprinting animals (MP-Na group) showed increased Fra-Li immunoreactivity(ir) in the organum vasculosum of the lamina terminalis (OVLT)(p=0.018) but not in the subfornical organ. Fra-AVP-ir neurons along the supraoptic nucleus(SON) and in the lateral magnocelular subdivision of the paraventricular nucleus(PaLM) show a significant decreased and increased respectively in MP-Na animals(SON p=0.03 and PaLM p=0.02).The expression of TRPV1 in OVLT and SON were not significantly different after MP.
Taking into account our previous evidence, these results indicate that the availability of a rich source of NaCl during the perinatal period induces a long-term effect on drinking and the basal neural activity along the OVLT,SON and PVN nuclei implicated in the control of hydroelectrolyte balance.
EFFECTS OF PRENATAL ETHANOL EXPOSURE ON EXPLORATORY AND ANXIETY-LIKE BEHAVIOR AND SOCIAL INTERACTION
Cabrera Vac, Porcari Ca, Férnandez Pc, Vivas Lab, Molina JCac, Miranda-Morales RSac, Godino Aac
a-Instituto de Investigación Médica Mercedes y Martín Ferreyra (INIMEC-CONICET-Universidad Nacional de Córdoba), Casilla de Correo, 389-5000 Córdoba, Argentina.
b -Facultad de Ciencias Exactas Físicas y Naturales, Universidad Nacional de Córdoba, Córdoba, Argentina.
c- Facultad de Psicología, Universidad Nacional de Córdoba, Córdoba, Argentina.
The aim of the present work was to analyze the effect of prenatal binge-like ethanol exposure to a moderate dose (2.0 g/kg; group Pre-EtOH) during the gestational days 17 to 20 on exploratory and anxiety-like behavior and social interaction of adolescent offspring (postnatal day 28-33) .
Pre-EtOH rats exhibited hypolocomotion in the open field test, showing a significant less distance travelled than control group (Mean=87,65,36). This effect was also evident when only the center area of the open field was taken into account (Mean=6,850,73). No significant differences could be observed for time spent in the center of the chamber. A significantly lower number of rearing behavior was also evidenced (Mean= 22,921,79) in Pre-EtOH animals and in males adolescents, but the interaction of the factors did not attached significance.
In the elevated plus maze, the time spent on the closed arms (Mean=255,75 11,1) and time spent in open arms (Mean=8,84,92) were significantly different as a function of prenatal treatment. PreEtOH group spent more time in closed arms than control. Head dipping behavior was also significantly lower in PreEtOH than control group (Mean=2,40,5).
The analysis of behavior in the three-chamber social interaction test did not show any significant differences in the interaction with a new congener or objet between the control and Pre-EtOH group.
In conclusion, the prenatal binge-like exposure to a moderate ethanol dose reduced exploratory motor activity and increased anxiety-like behavior during adolescence. However, the Pre-EtOH exposure did not seems to affect social interaction with novel congeners at this age.
THE ONSET OF SODIUM APPETITE: ROLE OF OXITOCINERGIC AND SEROTONERGIC CENTRAL SYSTEMS
Porcari, C.1; Mecawi, A.3; Reis L.3, Antunes-Rodrigues, J.3; Caeiro, X. 1; Vivas, L.1; Godino, A.1,2.
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INIMEC-CONICET-Universidad Nacional de Córdoba, Argentina;
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Fac de Psicología, Universidad Nacional de Córdoba, Argentina.
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Fac. Medicina de Ribeirao Preto, USP, Brazil.
A temporal dissociation exists between sodium depletion(SD) and the appearance of sodium appetite(SA), and an inhibitory modulation can be postulated. Previous studies demonstrated the inhibitory participation of serotonergic(5-HT) and oxytocinergic(OT) neurons on SA induced by SD. Our aim was to evaluate during the delay of SA appearance after SD, gene expression changes of different components of central OT and 5HT systems. Wistar rats were SD using furosemide combined with low sodium diet, and then at 2h or 24h later the animals were decapitated. Specific brain areas: dorsal raphe nucleus(DRN), subfornical organ(SFO), lateral parabrachial(LPBN) and anteroventral area of third ventricule plus supraoptic nucleus(AV3V+SON), were submitted to RT-PCR of oxytocin receptor(OTR), serotonin 2A receptor(5HT2A), tryptophan hydroxylase 2(TPH2) and serotonin transporter (SERT). OTR mRNA expression significantly increases(p=0.045) early at 2 hs after SD along AV3V+SON in comparison to control and 24 h SD groups. Also, the OTR mRNA expression along the DRN, decreasing 24 h after SD in relation to control and 2h SD groups, but this differences did not reach significant levels(p=0.06). Non-significant changes in the SERT and TPH2 mRNA expression were find along the DRN or the 5HT2A mRNA expression in the LPBN and SFO. In sum, our results suggest that OT circuit acting thought its receptors along nuclei previously involved in the regulation of sodium appetite may modulate SA appearance.
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TRPV1 OSMOSENSITIVE CHANNEL INVOLVEMENT IN THE CONTROL OF SODIUM APPETITE
Porcari (1), Susana Quirós Cognuck (2), Ximena Caeiro (1), André Mecawi (3), Luis Reis (3), José Antunes Rodrigues (2), Laura Vivas (1,4), Andrea Godino (1,5)
(1) Instituto de Investigación Médica M. y M.Ferreyra, Córdoba Argentina. (2) Fac. Med. Ribeirao Preto, USP, Brasil. (3) Universidade Federal Rural do Rio de Janeiro, Brasil. (4) Fac. de Cs. Exac., Físicas y Naturales, UNC, Argentina. (5) Fac. de Psicología, UNC, Argentina.
There is a temporal dissociation between sodium depletion (SD) and the appearance of sodium appetite (SA) behavior. After an acute SD, the natremia decreases immediately however SA takes at least 16 h to appear. Our recent results demonstrated in Wistar rats that the transient receptor potential vanilloid type 1 (TRPV1) channel, required for normal osmoregulation, is involved in SA control. The TRPV1 mRNA expression was increased in the kidney and previously involved brain nuclei, during the delay of SA (2 h after SD) and significantly decreased during the appearance of SA (24 h after SD), possibly allowing the hypertonic sodium consumption. The aim of the present work was to evaluate in TRPV1 knockout mice (KO), sodium intake and the urinary pattern of renal excretion at different times after SD. In particular, we analyzed the sodium and water intake and the renal response at 2 h and 24 h after SD induced by furosemide (50mg/kg) in combination with low sodium diet in wild type (WT) and KO mice.
After SD, the KO animals showed an increase in the sodium preference (F=8.49; p=0.006) and consumed a higher hypertonic cocktail (F=8.49; p=0.0059) in relation to WT animals, independent of the time after SD. These data suggest that KO animals, when stimulated to drink water and sodium, make a hypertonic cocktail instead of the isotonic one usually made by the control animals. The urinary volume (F=5.45; p=0.0003) and sodium excretion (F=3.99; p=0.028) induced by Furosemide at 30 minutes were both reduced in KO animals in comparison to WT. There was no change in plasma osmolality between the groups 2 h and 24 h after SD.
In sum, these data suggest that the TRPV1 channels are involved in the osmoregulatory behavioral and renal responses after acute body SD.
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