Prenatal and perinatal phthalate exposure is associated with sex-dependent changes in hippocampal miR-15b-5p and miR-34a-5p expression and changes in testicular morphology in rat offspring.

MicroRNAs are a large group of non-coding nucleic acids, usually 20-22 nt long, which bind to regulatory sections of messenger RNA (mRNA) and inhibit gene expression. However, genome activity is also regulated by hormones. Endocrine disruptors such as those from the phthalate group imitate or block these hormonal effects, and our previous study showed a long-lasting decrease in plasma testosterone levels in rat offspring exposed to a mixture of three phthalates in utero and postnatally. These effects were also observed at the behavioural level. To shed more light on these findings, in this new study we compared testicular tissue morphology between control and phthalatetreated males and investigated possible persistent changes and sex differences in the expression of two hippocampal microRNAs - miR- 15b-5p and miR-34a-5p - participating in the transcription of steroidogenic genes. Histologically observed changes in testicular tissue morphology of phthalate-exposed males compared to control support testosterone drop observed in the previous study. At the microRNA level, we observed more significant changes in phthalate-treated females than in males. However, we are unable to relate these effects to the previously observed behavioural changes.

Effect of 17ß-estradiol on the daily pattern of ACE2, ADAM17, TMPRSS2 and estradiol receptor transcription in the lungs and colon of male rats.

Covid-19 progression shows sex-dependent features. It is hypothesized that a better Covid-19 survival rate in females can be attributed to the presence of higher 17ß-estradiol (E2) levels in women than in men. Virus SARS-CoV-2 is enabled to enter the cell with the use of angiotensin converting enzyme 2 (ACE2). The expression of several renin-angiotensin system components has been shown to exert a rhythmic pattern, and a role of the circadian system in their regulation has been implicated. Therefore, the aim of the study is to elucidate possible interference between E2 signalling and the circadian system in the regulation of the expression of ACE2 mRNA and functionally related molecules. E2 was administered at a dosage of 40 µg/kg/day for 7 days to male Wistar rats, and sampling of the lungs and colon was performed during a 24-h cycle. The daily pattern of expression of molecules facilitating SARS-CoV-2 entry into the cell, clock genes and E2 receptors was analysed. As a consequence of E2 administration, a rhythm in ACE2 and TMPRSS2 mRNA expression was observed in the lungs but not in the colon. ADAM17 mRNA expression showed a pronounced rhythmic pattern in both tissues that was not influenced by E2 treatment. ESR1 mRNA expression exerted a rhythmic pattern, which was diminished by E2 treatment. The influence of E2 administration on ESR2 and GPER1 mRNA expression was greater in the lungs than in the colon as a significant rhythm in ESR2 and GPER1 mRNA expression appeared only in the lungs after E2 treatment. E2 administration also increased the amplitude of bmal1 expression in the lungs, which implicates altered functioning of peripheral oscillators in response to E2 treatment. The daily pattern of components of the SARS-CoV-2 entrance pathway and their responsiveness to E2 should be considered in the timing of pharmacological therapy for Covid-19.

Food reward induction of rhythmic clock gene expression in the prefrontal cortex of rats is accompanied by changes in miR-34a-5p expression.

The current study is focused on mechanisms by which the peripheral circadian oscillator in the prefrontal cortex (PFC) participates in food reward-induced activity. The experimental group of male Wistar rats was trained to receive a food reward with a low hedonic and caloric value. Afterwards, animals were exposed to a 5 h phase advance. Experimental animals could access a small food reward as they had been accustomed to, while control rats were exposed to the same phase shift without access to a food reward. When synchronisation to a new light:dark cycle was accompanied by intake of food reward, animals exerted more exact phase shift compared to the controls. In rats with access to a food reward, a rhythm in dopamine receptors types 1 and 2 in the PFC was detected. Rhythmic clock gene expression was induced in the PFC of rats when a food reward was provided together with a phase shift. The per2 and clock genes are predicted targets of miR-34a-5p. The precursor form of miR-34a-5p (pre-miR-34a-5p) showed a daily rhythm in expression in the PFC of the control and experimental groups. On the other hand, the mature form of miR-34a-5p exerted an inverted rhythm compared to pre-miR-34a-5p and negative correlation with per and clock genes expression only in the PFC of rewarded rats. A difference in the pattern of mature and pre-miR-34a-5p values was not related to expression of enzymes drosha, dicer and dgcr8. A role of the clock genes and miR-34a-5p in reward-facilitated synchronisation has been hypothesised.

Dim light at night attenuates circadian rhythms in the cardiovascular system and suppresses melatonin in rats.

AIMS: Cardiovascular parameters exhibit significant 24-h variability, which is coordinated by the suprachiasmatic nucleus (SCN), and light/dark cycles control SCN activity. We aimed to study the effects of light at night (ALAN; 1-2 lx) on cardiovascular system control in normotensive rats. MAIN METHODS: Heart rate (HR) and blood pressure (BP) were measured by telemetry during five weeks of ALAN exposure. From beat-to-beat telemetry data, we evaluated spontaneous baroreflex sensitivity (sBRS). After 2 (A2) and 5 (A5) weeks of ALAN, plasma melatonin concentrations and the response of BP and HR to norepinephrine administration were measured. The expression of endothelial nitric oxide synthase (eNOS) and endothelin-1 was determined in the aorta. Spontaneous exploratory behaviour was evaluated in an open-field test. KEY FINDINGS: ALAN significantly suppressed the 24-h variability in the HR, BP, and sBRS after A2, although the parameters were partially restored after A5. The daily variability in the BP response to norepinephrine was reduced after A2 and restored after A5. ALAN increased the BP response to norepinephrine compared to the control after A5. Increased eNOS expression was found in arteries after A2 but not A5. Endothelin-1 expression was not affected by ALAN. Plasma melatonin levels were suppressed after A2 and A5. Spontaneous exploratory behaviour was reduced. SIGNIFICANCE: ALAN decreased plasma melatonin and the 24-h variability in the haemodynamic parameters and increased the BP response to norepinephrine. A low intensity ALAN can suppress circadian control of the cardiovascular system with negative consequences on the anticipation of a load.

Testosterone enhancement during pregnancy influences social coping and gene expression of oxytocin and vasopressin in the brain of adult rats.

Steroid hormones are important mediators of prenatal maternal effects and play an important role in fetal programming. The aim of our study was to investigate how testosterone enhancement during pregnancy influences neurobehavioral aspects of social coping of rat offspring in adulthood. Pregnant rat dams were exposed to depot form of testosterone during the last third of pregnancy (i.e., beginning on the 14th day of pregnancy). Their adult offspring were later tested in a social interaction test and expression of oxytocin and arginine-vasopressin mRNA in the hypothalamic nuclei was evaluated. Our research showed that prenatal exposure to higher levels of testosterone activated socio­cohesive and socio­aversive interactions, but only in males. The testosterone­exposed group also showed decreased oxytocin mRNA expression in the supraoptic and paraventricular nuclei of the hypothalamus, and increased arginine-vasopressin mRNA expression in the supraoptic and suprachiasmatic nuclei as compared to controls. However, we did not observe any sex differences in the expression of oxytocin and arginine­vasopressin mRNA in these regions. Our findings show that testosterone enhancement in pregnancy could have long­lasting effects on oxytocin and arginine-vasopressin levels in the brain of adult animals, but lead to changes in behavioral aspects of coping strategies only in males.

Effect of oral contraceptives intake on postural stability in young healthy women throughout the menstrual cycle.

The purpose of the study was to investigate the effect of oral contraceptives on static postural stability in young healthy women during their menstrual cycle. Twenty-three women with the regular menstrual cycle, using or not using oral contraceptives, participated in this study. Salivary progesterone and estradiol levels were measured during one menstrual cycle. Measurements of balance were performed during a quiet stance on a firm and foam surface by the force platform, with eyes either opened or closed, on day 2, 7, 14, 21 and 28 of the cycle. Results of stability on a firm surface with eyes opened showed a significant effect in the amplitude of body sway in the anterior-posterior direction since women using oral contraceptives had a lower amplitude compared to control women on day 28. During stance on a firm surface with eyes closed we showed only impact of the menstrual cycle on postural stability of women. In condition of stance on a foam surface with the eyes opened or closed no significant effects were found. Our results showed that oral contraceptives intake can improve the static postural stability before the onset of menstruation and decrease a risk of injury of young healthy women.

Prenatal exposure to angiotensin II increases blood pressure and decreases salt sensitivity in rats.

Renin angiotensin aldosterone system (RAAS) plays an essential role in the homeostatic control of arterial blood pressure, perfusion of tissues, and control of extracellular fluid. Its components are highly expressed in the developing kidney, general vasculature, brain, and heart. A modified intrauterine environment alters mechanisms controlling blood pressure (BP) and can lead to hypertension in the adult offspring and developmentally programmed RAAS can be involved in this process. There are very little data about the effects of increased angiotensin II (Ang II) concentrations during pregnancy on in utero development of the fetus. In our study, we administered Ang II to pregnant female rats via osmotic mini-pumps and evaluated the postnatal development and BP control in the offspring. To estimate possible developmental changes in sensitivity to salt, we exposed the offspring to a diet with increased salt content and measured plasma aldosterone levels and plasma renin activity. Increased Ang II during pregnancy raised BP in the offspring; however, salt sensitivity was decreased in comparison to controls. Relative weight of the left ventricle was decreased in the offspring prenatally exposed to Ang II, while relative kidney weight was reduced only in female offspring. Prenatal treatment led to increased aldosterone levels and decreased plasma renin activity, suggesting a complex physiological response. Our results suggest that conditions leading to upregulation of RAAS during pregnancy can influence the cardiovascular system of the fetus and have a long-term impact on the offspring's health.

Effect of maternal renin-angiotensin-aldosterone system activation on social coping strategies and gene expression of oxytocin and vasopressin in the brain of rat offspring in adulthood.

The intrauterine condition in which the mammalian foetus develops has an important role in prenatal programming. The aim of this study was to determine the extent to which activation of the maternal renin-angiotensin-aldosterone system (RAAS) could influence social behaviour strategies in offspring via changes in social neurotransmitters in the brain. Pregnant female Wistar rats were implanted with osmotic minipumps which continually released angiotensin II for 14 days at concentration of 2 µg/kg/h. The adult offspring (angiotensin and control groups) underwent a social interaction test. The mRNA expression of vasopressin, oxytocin and the oxytocin receptor in selected brain areas was measured by in situ hybridisation. Prenatal exposure to higher levels of angiotensin II resulted in a strong trend toward decreased total social interaction time and significantly decreased time spent in close proximity and frequency of mutual sniffing. The angiotensin group showed no changes in oxytocin mRNA expression in the hypothalamic paraventricular or supraoptic nuclei, but this group had reduced vasopressin mRNA expression in the same areas. We concluded that maternal activation of RAAS (via higher levels of angiotensin II) caused inhibition of some socio-cohesive indicators and decreased vasopressinergic activity of offspring. Taken together, these results suggest a reactive rather than proactive social coping strategy.

Angiotensin II enhancement during pregnancy influences the emotionality of rat offspring (Rattus norvegicus) in adulthood. Potential use of the Rat Grimace Scale.

OBJECTIVES: One of the systems, which can be prenatally reprogrammed, is the renin-angiotensin-aldosterone system (RAAS). The aim of our experiment was to determine how prenatal activation of RAAS via exposure to elevated levels of angiotensin II (Ang II) influences the rat offspring's emotionality. METHODS: Pregnant female rats were implanted with osmotic minipumps that continually released Ang II and oval object of the same shape and size was implanted into control dams. The adult offspring (AngII and control groups) were tested in rat grimace scale (RGS), open field test (OF) and elevated plus maze (EPM). RESULTS: Psychological stress increased the RGS score in both groups of animals. AngII animals had significantly lower RGS score (i.e. less negative emotions) in the home cage but higher index of emotional reactivity in RGS. AngII animals had also significantly lower frequency of defecation in OF and had no effect on changes in anxiety-like behaviour. CONCLUSION: We concluded that maternal activation of RAAS modified some aspect of emotionality of experimental animals and led to an enhanced emotional response to stress situation.

Increased anxiety-like behaviour and altered GABAergic system in the amygdala and cerebellum of VPA rats - An animal model of autism.

Anxiety is one of the associated symptoms of autism spectrum disorder. According to the literature, increases in anxiety are accompanied by GABAergic system deregulation. The aim of our study, performed using an animal model of autism in the form of rats prenatally treated with valproic acid (VPA rats), was to investigate changes in anxiety-like behaviour and the gene expression of molecules that control levels of the inhibitory neurotransmitter γ-aminobutyric acid (GABA) in the brain. Anxiety-like behaviours were investigated using zone preferences in the open field test. The levels of the 65 and 67kDa enzymes of l-glutamic acid decarboxylase (GAD) mRNAs and type 1 GABA transporter (GAT1) were evaluated in the amygdala, as well as GABA producing enzymes in the cortex layer of the cerebellum. Our research showed that adult VPA rats spent less time in the inner zone of the testing chamber and more time in the outer zone of the testing chamber in the open field test. We also found that adult VPA rats had increased expression of GAT1 in the amygdala, as well as decreased levels of GAD65 and GAD67 mRNA in the cerebellum compared to control animals. These findings support the existence of a relationship between increased anxiety-like behaviour and changes in the regulation of the GABAergic system in VPA rats.

Increased sociability and gene expression of oxytocin and its receptor in the brains of rats affected prenatally by valproic acid.

Autism is a neurodevelopmental disorder characterised by the disruption of social interactions. Autistic animal models play a crucial role in neurophysiologic research on this disorder. One of these models is based on rats that have been prenatally treated with valproic acid - VPA rats. The aim of our study performed with this model was to investigate changes in sociability and gene expression of neuropeptides and receptors involved in regulating social behaviour. We focused on gene expression in the hypothalamus, where the neuropeptides oxytocin (OT) and arginine-vasopressin (AVP) are produced, as well as oxytocin receptors (OTR) in certain neuronal structures involved in the creation of social abilities. Our research showed that VPA rats spent more time in the part with an unknown animal and less time in the central part of a three chamber sociability test apparatus than control animals. The latency period of VPA rats before initiating social contact was decreased. In addition, during weaning, VPA female rats spent more time in direct interaction with an unknown rat. We also found that adult VPA rats had an increased expression of OT in the hypothalamic supraoptic and paraventricular nuclei and of OTR in the medial prefrontal cortex, piriform cortex, cortex-amygdala transition zone and the region of the basolateral and basomedial amygdaloid nuclei compared with controls. To sum up, we observed that a single prenatal injection of VPA increased social behaviour and gene expression of OT and OTR in neurological structures connected with the social behaviour of rats. One unanticipated finding was the absence of one of the core symptoms of autism in VPA rats, suggesting a decreased ability to understand intraspecific communication signals.

Diurnal changes of the postural control in young women without and with hormonal contraceptive treatment.

OBJECTIVES: The aim of this study was to investigate diurnal changes of the postural control in young women without and with hormonal contraceptive treatment. METHODS: The postural activity was assessed during stance from two accelerometers positioned at the level of the lumbar (L5) and thoracic (Th4) vertebra in twenty healthy young women non-using (13) and using (7) hormonal contraception. RESULTS: We observed a significant increase of trunk tilts in the morning in the group of women with hormonal contraception compared to control. Women with hormonal contraception showed the significant decrease of trunk tilts and their velocity in the evening in relation to increased morning data at the L5 in anterior-posterior direction during stance on foam. Measurements at Th4 showed higher variability of lateral trunk tilts in conditions with altered somatosensory inputs. Distinct reduction of velocity of lateral trunk tilts in the evening related to morning measurements were present in the control group at the L5 in conditions with altered somatosensory inputs and at the Th4 in all experimental conditions in both groups. CONCLUSION: We demonstrated diurnal changes of the postural control in young women. Women using hormonal contraceptives showed a weakened postural stability compared with the control group in the morning and the normalization of postural stability in the evening to the values of the control group. These findings suggest that the time of day and the use of hormonal contraception affect postural stability of women.

Habituation of exploratory behaviour in VPA rats: animal model of autism.

Autism is a neurodevelopmental disorder with multifactorial aetiology, represented as impairment in social behaviour, communication and the occurrence of repetitive activities, which can be observed in the early life. The core features are frequently accompanied by other manifestations, including limited environmental exploration. The aim of the presented study, realised on an animal model of autism - VPA rats, i.e. animals prenatally affected with valproic acid on gestation day 12.5, was to investigate the habituation process of exploratory activity (manifested by a gradual decrease in the intensity of locomotor activity), which reflects the stage of the central nervous system. VPA rats were tested in open-field in three developmental periods - weaning (postnatal day 21 - PND 21), puberty (PND 42) and adulthood (PND 72). In each period of ontogenesis, the rapidity of habituation was evaluated by using the method of linear regression. Compared to controls, VPA rats showed a significant decrease in the intensity and an increase in the rapidity of exploratory activity habituation during puberty and adulthood. Our results indicate that the animal model of autism, i.e. VPA rats, showed disabilities in the development of the nervous system. These findings can help confirm not only the validity of this animal model of autism but can also help better understand neuronal changes in humans with autism.

Animal models of autism with a particular focus on the neural basis of changes in social behaviour: an update article.

Research on autism has been gaining more and more attention. However, its aetiology is not entirely known and several factors are thought to contribute to the development of this neurodevelopmental disorder. These potential contributing factors range from genetic heritability to environmental effects. A significant number of reviews have already been published on different aspects of autism research as well as focusing on using animal models to help expand current knowledge around its aetiology. However, the diverse range of symptoms and possible causes of autism have resulted in as equally wide variety of animal models of autism. In this update article we focus only on the animal models with neurobehavioural characteristics of social deficit related to autism and present an overview of the animal models with alterations in brain regions, neurotransmitters, or hormones that are involved in a decrease in sociability.

Influence of up-regulated renin-angiotensin system on the exploration, anxiety-related behavior and object recognition.

The renin-angiotensin system (RAS) plays an important role in the development of hypertension and has serious consequences on behaviour. The aim of our study was to investigate the effect of hypertension, induced by up-regulated RAS, on the exploration, anxiety-related behaviour and object recognition in laboratory rats. In the experiment, 12 weeks old normotensive Sprague-Dawley (SD) and hypertensive TGR(mREN2)27 (TGR) male rats with up-regulated RAS were used. In the open-field test, the TGR rats were less active in ambulating, rearing and sniffing and more active in self-grooming and urinating than SD ones. In the elevated plus-maze test, the TGR rats showed lower frequency of total arm entries, closed arm entries and higher frequency of defecation than in controls. In the emergence test, TGR rats did not show significant differences. In the novel object recognition task, the TGR rats spent less time with exploration of both familiar and unfamiliar objects but preferred the novel object over the familiar one and exhibited higher percentage of the total exploring time spent with novel object exploration than SD rats. Our results indicate that the TGR rats are less actively exploring, show some modifications of emotional/anxiety-related behavior and exhibited better recognition abilities.

Testosterone enhancement during pregnancy influences the 2D:4D ratio and open field motor activity of rat siblings in adulthood.

In humans, the relationship between the prenatal testosterone exposure and the ratio of the second and the fourth digits (2D:4D) has been extensively studied. Surprisingly, data on this relationship have thus far been lacking in experimental animals such as rats. We studied the effect of maternal testosterone enhancement during pregnancy on the digit ratio and open field activity of adult progeny in Wistar rats. Elevated levels of maternal testosterone resulted in lower 2D:4D ratios and an elongated 4D on the left and right forepaws in both males and females. We found no sex difference in 2D:4D in control animals. In the open field test, control females were more active than control males and testosterone females, while the activity of testosterone females did not differ from that of control males. We found a positive correlation between motor activity and the right forepaw 2D:4D ratio of control males and females. Prenatal exposure to testosterone resulted in the disappearance of this correlation in both males and females. Our results show that elevated levels of testosterone during the prenatal period can influence forepaw 4D length, 2D:4D ratio, and open field motor activity of rats, and that these variables are positively correlated. Thus, this approach represents a noninvasive and robust method for evaluating the effects of prenatal testosterone enhancement on anatomical and physiological parameters.

Effect of melatonin on exploration and anxiety in normotensive and hypertensive rats with high activity of renin-angiotensin system.

OBJECTIVES: The purpose of the study was to investigate effects of melatonin (MEL) on exploration and anxiety in normotensive Sprague-Dawley (SD) and hypertensive TGR(mREN2)27 (TGR) rats with high activity of renin-angiotensin system. METHODS: Mature control (n=20) and hypertensive (n=20) rats were used. Half of each group was treated with MEL in drinking water (40 microg/ml) for 3 weeks. The influence of MEL on exploration was measured in the open field test (OF) and on anxiety in the elevated plus maze test (EPM). RESULTS: Hypertensive TGR rats showed a lower level of ambulation (p<0.05) and higher level of urination (p<0.001) in OF. In EPM they spent more time in closed arms (p<0.05) and showed low frequency of total arm entries (p<0.01) than SD rats. MEL treated SD rats exhibited increased ambulation (p<0.01), sniffing (p<0.05) and decreased creeping (p<0.05) in OF than SD controls and did not exhibit differences in behaviour observed in EPM. MEL treated TGR rats exhibited a decrease in creeping, defecation and urination (p<0.05) in OF and spent less time in closed arms (p<0.05) and increased frequency of total arm entries (p<0.05) in EPM than untreated TGR animals. CONCLUSION: Our results suggest that MEL decreased anxiety related behaviours in hypertensive rats with an up regulated renin-angiotensin system and stimulated active exploration of control animals.

Some assessments of the amygdala role in suprahypothalamic neuroendocrine regulation: a minireview.

The amygdala is a complex structure playing primary role in the processing and memorizing of emotional reactions. The amygdalae send impulses to the hypothalamus for activation of the sympathetic nervous system, to the reticular nucleus for increasing reflexes, to the nuclei of the trigeminal nerve and facial nerve for facial expressions of fear, and to the ventral tegmental area, locus coeruleus, and laterodorsal tegmental nucleus for activation of dopamine, norepinephrine and epinephrine release. The amygdala plays a key role in what has been called the "general-purpose defense response control network" and reacts in response to unpleasant sights, sensations, or smells. Anger, avoidance, and defensiveness are emotions activated largely by the amygdale. The amygdala is responsible for activating ancestral signs of distress such as "tense-mouth" and defensive postures such as crouching. Poor functioning of amygdala has also been associated with anxiety, autism, depression, narcolepsy, post-traumatic stress disorder, phobias, frontotemporal dementia, and schizophrenia. Impairment of emotional event memory in patients with Alzheimer's disease also correlates with the intensity of amygdalar damage. All these events speak out for the importance to preserve the normal function of the amygdala which can only be achieved by constant deepening of our knowledge about this unique structure.

Influence of maternal testosterone on the strategies in the open field behaviour of rats.

OBJECTIVES: The purpose of the present study was to characterize the influence of testosterone administered to pregnant females on offsprings postnatal behavioral strategies in the open field. METHODS: The influence of maternal testosterone on behaviour of 23 day old male and female offsprings was studied in a 20-minute open field test. A total of 9 behavioural events were compared between a control (male n=12, female n=8) and a testosterone group (male n=9, female n=9). Dynamics and patterns of association of these behavioural events were analyzed. The testosterone group was prenatally exposed to testosterone (a single intramuscular injection of 2.5 mg testosteroni isobutyras on gestation day 14). RESULTS: Male offsprings exposed prenatally to testosterone displayed significantly high levels of ambulation (P<0.05), sniffing (P<0.01), sniffing the air (P<0.05), urination (P<0.05) and significantly lower level of vocalization (P<0.05) than control. Female offsprings exposed prenatally to testosterone displayed significantly higher level of sniffing the air (P<0.05) than control. Significant differences in the dynamics of habituation process were registered only in the group of male. They were from 5 to 10 min in ambulation (P<0.05), from 10 to 15 min in sniffing (P<0.05) and vocalization (P<0.05), from 10 to 15 min and from 15 to 20 min in sniffing the air (P<0.05) and from 0 to 5 min in defecation (P<0.05) and urination (P<0.01). CONCLUSION: Our results suggest that maternal testosterone may influence especially male postnatal open field strategies.