Maternal exposure to fluoxetine during gestation and lactation does not alter plasma concentrations of testosterone, oestrogen or corticosterone in peripubertal offspring.

Antidepressants are widely used around the world, primarily for the treatment of mood disorders, anxiety and pain syndromes. Women who use antidepressants often continue to use them during pregnancy. Selective serotonin reuptake inhibitors, including fluoxetine, are the main class of antidepressants prescribed to pregnant women. It is known that fluoxetine crosses the placental-blood barrier and is excreted in breast milk. Consequently, indirect exposure of the infant occurs. Knowing that fluoxetine alters the balance of neurotransmitters in the central nervous system, several studies have shown that maternal exposure to this drug leads to various adverse effects on the nervous, reproductive and cardiovascular systems of the offspring. The aim of the present study was to evaluate the effects of exposure to fluoxetine during gestation and lactation on parameters related to steroid hormones in prepubertal and pubertal male and female rats. The endpoints evaluated were date of puberty onset, plasma testosterone and oestrogen concentrations before and after puberty onset and corticosterone concentration before and after adrenocorticotrophin stimulus. None of the parameters was affected by fluoxetine exposure.

Anxiolytic-like effect of noradrenaline microinjection into the dorsal periaqueductal gray of rats.

The brain noradrenergic system has been implicated in the expression of defensive behaviors elicited by acute stress. The dorsal periaqueductal gray area (dPAG) is a key structure involved in the behavioral and cardiovascular responses elicited by fear and anxiety situations. Although there are noradrenergic terminals in the dPAG, few studies have investigated the role of noradrenaline (NA) in the dPAG on anxiety modulation. The aim of this study was to evaluate the effect of NA microinjection into the dPAG of rats subjected to two animal models of anxiety, the elevated plus-maze and the Vogel conflict test. Male Wistar rats implanted with a guide cannula aimed at the dPAG received microinjections of NA (3, 15, or 45 nmol/0.05 microl) or artificial cerebral spinal fluid into the dPAG immediately before being exposed to the elevated plus-maze or the Vogel conflict test. NA increased the exploration of the open arms and the number of enclosed arm entries in the elevated plus-maze. The increase in open arm exploration remained significant after being subjected to an analysis of covariance using the latter variable as covariate. Moreover, the NA microinjection into the dPAG did not increase general exploratory activity of animals subjected to the open-field test, indicating that the increase in open arm exploration cannot be attributed to a nonspecific increase in exploratory activity. In the Vogel test, the NA microinjection into the dPAG increased the number of punished licks without changing the number of nonpunished licks or interfering with the tail-flick test. The results, therefore, indicate that the NA microinjection into the dPAG produces anxiolytic-like effects, suggesting its possible involvement in the anxiety modulation.

Intrauterine and lactational exposure to fluoxetine enhances endothelial modulation of aortic contractile response in adult female rats.

The study aimed to evaluate if maternal exposure to fluoxetine (FLX) during pregnancy and lactation would result in altered aortic reactivity in adult offspring. We also sought to understand the role of endothelium derived relaxing factors in aortic response. Wistar rats (75­80 days old), whose progenitors had received FLX (5 mg/kg, FLX offspring) or tap water (control offspring) during pregnancy and lactation were anesthetized, after which the aorta was removed and cut into two rings, one with (Endo+) and the other without (Endo-) endothelium. Concentration-effect curves for acetylcholine (ACh), sodium nitroprusside (SNP), and phenylephrine (Phe) were performed. The vasodilation to ACh and SNP was similar between control and FLX groups in both male and female offspring. In male rats, the response to Phe was similar between the FLX and control groups on Endo+ and Endo- rings. The response to Phe was reduced on Endo+ rings from female FLX when compared with the control group. The endothelium removal, as well as L-NAME, indomethacin, and tranylcypromine incubation corrected the reduced Phe-induced contraction in the aorta from the female FLX group. On the other hand, catalase, NS-398, and L-NIL did not interfere with the vasoconstriction. The aortic level of nitric oxide (NO) was higher in the female FLX than the control group. Although endothelial NO synthase isoform and cyclooxygenase (COX)-1 expressions were similar between the groups, there was a notable increment in neuronal NO synthase expression in the aorta of FLX-exposed female rats, suggesting an important role of this enzyme in the higher levels of NO. Our results show that developmental exposure to FLX causes sex-specific alteration in aortic function through a mechanism involving endothelial factors, probably NO and COX-1 products.

Intrauterine and lactation exposure to fluoxetine blunted in the offspring the aortic adaptive response induced by acute restraint stress.

Selective serotonin reuptake inhibitors are the most widely prescribed antidepressants to women during pregnancy. Maternal treatment with fluoxetine can expose fetuses and neonates to higher levels of serotonin that plays a role in stress response. Thus, the aim of the study was to evaluate whether maternal treatment with fluoxetine interferes with aorta reactivity of adult male offspring after acute restraint stress. Wistar rats were gavaged with fluoxetine (5mg/kg/day) or water (control) during pregnancy and lactation. The experiments were performed in adult male offspring, treated or not with reserpine (4mg/Kg, ip, 28h before the experimental protocol). Fluoxetine and control rats were submitted to a single restraint stress session (ST) for 1h. Curves to phenylephrine were performed in thoracic aorta with endothelium. Aortic nitric oxide (NOx) were evaluated by the Griess method. The aortic contraction induced by phenylephrine was similar between control and fluoxetine rats. The acute stress reduced contraction in aorta of control ST compared to control, and L-NAME equaled this response. In fluoxetine rats, ST did not change the aortic constriction. Reserpine treatment restored the vasoconstriction in control ST, but did not interfere with aortic contraction in control, fluoxetine or fluoxetine ST. The NOx concentration was higher in aortas from control ST than control rats, and reserpine reduced NOx levels of control ST. The NOx concentration was similar between fluoxetine and fluoxetine ST rats, treated or not with reserpine. In conclusion, maternal treatment with fluoxetine blunted acute restraint stress-induced NO system activation and aortic adaptation in adult offspring.

Acute stress affects the global DNA methylation profile in rat brain: modulation by physical exercise.

The vulnerability of epigenetic marks of brain cells to environmental stimuli and its implication for health have been recently debated. Thus, we used the rat model of acute restraint stress (ARS) to evaluate the impact of stress on the global DNA methylation and on the expression of the Dnmt1 and Bdnf genes of hippocampus, cortex, hypothalamus and periaqueductal gray (PAG). Furthermore, we verified the potential of physical exercise to modulate epigenetic responses evoked by ARS. Sedentary male Wistar rats were submitted to ARS at the 75th postnatal day (PND), whereas animals from a physically active group were previously submitted to swimming sessions (35-74th PND) and to ARS at the 75th PND. Global DNA methylation profile was quantified using an ELISA-based method and the quantitative expression of the Dnmt1 and Bdnf genes was evaluated by real-time PCR. ARS induced a decrease in global DNA methylation in hippocampus, cortex and PAG of sedentary animals and an increased expression of Bdnf in PAG. No change in DNA methylation was associated with ARS in the exercised animals, although it was associated with abnormal expression of Dnmt1 and Bdnf in cortex, hypothalamus and PAG. Our data reveal that ARS evokes adaptive changes in global DNA methylation of rat brain that are independent of the expression of the Dnmt1 gene but might be linked to abnormal expression of the Bdnf gene in the PAG. Furthermore, our evidence indicates that physical exercise has the potential to modulate changes in DNA methylation and gene expression consequent to ARS.

Age-related changes in the global DNA methylation profile of leukocytes are linked to nutrition but are not associated with the MTHFR C677T genotype or to functional capacities.

Global DNA methylation of peripheral blood leukocytes has been recently proposed as a potential biomarker for disease risk. However, the amplitude of the changes in DNA methylation associated with normal aging and the impacts of environmental changes on this variation are still unclear. In this context, we evaluated the association of global DNA methylation with nutritional habits, tobacco smoking, body mass index (BMI), clinical laboratory parameters, polymorphism C677T MTHFR, functional cognition and the daily practice of physical activity in a cancer-free older population. Leukocyte global DNA methylation from 126 older individuals was quantified using a high-throughput ELISA-based method. Global DNA hypomethylation was observed in older individuals when compared to a younger population (p = 0.0469), confirming changes in DNA methylation in the aging process. Furthermore, the methylation profile of elders was correlated with the daily ingestion of carbohydrates (p = 0.0494), lipids (p = 0.0494), vitamin B6 (p = 0.0421), magnesium (p = 0.0302), and also to the serum levels of total protein (p = 0.0004), alpha 2 globulin (p = 0.0013) and albumin (p = 0.0015). No statistically significant difference was observed when global DNA methylation were stratified according to C677T MTHFR genotypes (p = 0.7200), BMI (p = 0.1170), smoking habit (p = 0.4382), physical activity in daily life (p = 0.8492), scored cognitive function (p = 0.7229) or depression state (p = 0.8301). Our data indicate that age-related variations in the global DNA methylation profile of leukocytes might be modulated by the daily intake of carbohydrates, lipids, vitamin B6, and magnesium and be associated with serum protein levels, however it is independent of C677T MTHFR genotype and not correlated with BMI, smoking habit, cognitive function or the routine physical activities.

Paraventricular nucleus mediates pressor response to noradrenaline injection into the dorsal periaqueductal gray area.

The dorsal periaqueductal gray area (dPAG) is involved in cardiovascular modulation. In a previous study, we reported that noradrenaline (NA) microinjection into the dPAG of rats caused pressor response that was mediated by vasopressin release. Vasopressin is synthesized by magnocellular neurons in the hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei. In the present study, we verified which nuclei mediated the cardiovascular response to NA as well as the existence of direct neural projection from the dPAG to hypothalamic nuclei. Then, we studied the effect of treating either PVN or SON with the nonselective synaptic blocker cobalt chloride (1mM) on the cardiovascular response to NA (15 nmol) microinjection into dPAG. Attempting to identify neural projections from dPAG to hypothalamic nuclei, we microinjected the neuronal tracer biotinylated-dextran-amine (BDA) into the dPAG and searched varicosity-containing nerve terminals in the PVN and SON. Unilateral cobalt-induced inhibition of synapses in the SON did not affect the cardiovascular response to NA. However, unilateral inhibition of PVN significantly reduced the pressor response to NA. Moreover, cobalt-induced inhibition of synapses in both PVN blocked the pressor response caused by NA microinjected into the dPAG. Microinjection of BDA into the dPAG evidenced presence of varicosity-containing neuronal fibers in PVN but not in SON. The results from cobalt treatment indicated that synapses in PVN mediate the vasopressin-induced pressor response caused by NA microinjection into the dPAG. In addition, the neuroanatomical results from BDA microinjection into the dPAG pointed out the existence of direct neural projections from the dPAG site to the PVN.

Rostrocaudal somatotopy in the neural connections between the lateral hypothalamus and the dorsal periaqueductal gray of the rat brain.

1. The lateral hypothalamus (LH) and the dorsal periaqueductal gray area (dPAG) are two important brain structures involved in central cardiovascular control. 2. In the present study, we searched for possible rostrocaudal somatotopy in the neural connections from the three subdivisions of the LH (anterior-LHa; tuberal-LHt and posterior-LHp) to the different rostrocaudal portions of the dPAG. 3. The bidirectional neuronal tracer biotinylated-dextran-amine (BDA) was microinjected into different rostrocaudal coordinates of the dPAG (AP 3.4-2.7 mm) of male Wistar rats. One week later, animals were sacrificed and brain slices were processed and analyzed to detect neuronal efferent projections from the LH to the dPAG. 4. Neuronal cell body staining was observed along all the rostrocaudal axis of the LH when BDA was microinjected in more rostral dPAG coordinates. When the BDA was microinjected into more caudal dPAG regions, labeled neurons were observed only in the caudal portion of the LH. 5. Efferent projections from the LHa were directed only to the rostral portion of the dPAG. Projections from the rostral and medial portions of the LHt were also directed to the rostral dPAG, whereas both rostral and caudal dPAG received projections from the caudal portion of the LHt. Efferent projections from the anterior portion of the LHp were directed to both rostral and caudal dPAG, whereas projections from the caudal LHp were only directed to the rostral portion of the dPAG.6. The results suggest a somatotopic correlation in LH projections to the dPAG with main connections to the rostral dPAG, which are efferent from the three divisions of the LH. More caudal regions of the dPAG received afferents only from posterior sites in the LH. 7. Moreover, the results point out to extensive and complex neural somatotopic projections from all LH subdivisions to different rostrocaudal portions of the dPAG, reinforcing the idea of significant functional interactions between the brain structures.