Molecular Implications of Repeated Aggression: Th,Dat1, Snca and Bdnf Gene Expression in the VTA of Victorious Male Mice

Background

It is generally recognized that recurrent aggression can be the result of various psychiatric disorders. The aim of our study was to analyze the mRNA levels, in the ventral tegmental area (VTA) of the midbrain, of the genes that may possibly be associated with aggression consistently shown by male mice in special experimental settings.

Methodology/Principal Findings

The genes were Th, Dat1, Snca and Bdnf; the male mice were a group of animals that had each won 20 daily encounters in succession and a group of animals that had the same winning track record followed by a no-fight period for 14 days. Increased Th, Dat1 and Snca mRNA levels were in the fresh-from-the-fight group as compared to the controls. Increased Th and Dat1 mRNA levels were in the no-fight winners as compared to the controls. Significant positive correlations were found between the level of aggression and Th and Snca mRNA levels.

Conclusions

Repeated positive fighting experience enhances the expression of the Th, Dat1 and Snca genes, which are associated with brain dopaminergic systems. The expression of the Th and Dat1 genes stays enhanced for a long time.     

Hypophagia and induction of serotonin transporter gene expression in raphe nuclei of male and female rats after short-term fluoxetine treatment

Serotonin (5-HT) is one of the regulators of feeding in humans. Drugs acting on the serotoninergic system are used to treat bulimia nervosa and to enhance the effect of hypocaloric diets in overweight subjects. They act rapidly to normalise feeding when used to treat eating-related problems. To explore the role of the 5-HT transporter (serotonin transporter (SERT)) in the short-term action of serotonin selective reuptake inhibitor fluoxetine, rats were i.p. given the drug for five consecutive days. Acute administration of fluoxetine in male and female rats produced a strong reduction in food intake, an effect that held up when daily treatment was maintained for five consecutive days. This reduction translated into a diminution of body weight that was statistically significant in the case of the males. As a reflection of the body weight change in rats killed after the fifth daily drug injection, retroperitoneal fat pad also decreased; a diminution that was statistically significant in the case of male rats. In these conditions, plasma leptin levels of both male and female rats were lower than in untreated animals. While acute fluoxetine administration did not modify SERT gene expression, subchronic drug treatment increased the content of SERT mRNA in the midbrain raphe complex of both rat genders. These findings may contribute to explain the role of SERT in fluoxetine action on binging and as an adjunct to hypocaloric diets.     

Effect of destruction of midbrain raphe nuclei on the behavioral effects of dopaminergic substances and I-tryptophan

In experiments on male albino rats, after electrolytic lesions of dorsal and median mesencephalic raphe nuclei, l-DOPA and apomorphine did not exert their stimulating effect on aggressiveness and emotional reactivity, seen in control false-operated animals. The inhibitory effect of haloperidol on emotional reactivity increased after raphectomy. The stereotype behaviour elicited by dopaminergic agents and their effects on exploratory motor activity were not changed by raphectomy. It is assumed that the effects of dopamino-potentiating agents on emotional reactivity are partly mediated by their influence on serotonergic system.     

Evidence for projections from medullary nuclei onto serotonergic and dopaminergic neurons in the midbrain dorsal raphe nucleus of the rat

Summary The anterograde tracer Phaseolus vulgaris-leucoagglutinin was injected into the medial nucleus of the solitary tract and into the rostral dorsomedial medulla. A sequential two-color immunoperoxidase staining was accomplished in order to demonstrate the co-distribution of presumed terminal axons with chemically distinct neurons in the dorsal raphe nucleus of the midbrain central gray, i.e., B7 serotonergic and A10dc dopaminergic neurons. Black-stained efferent fibers from the medial nucleus of the solitary tract and the rostral dorsomedial medulla intermingled with brown-stained serotonergic (5-hydroxytryptamine-immunoreactive) or dopaminergic (tyrosine hydroxylase-immunoreactive) neurons. Light microscopy revealed that the black-stained efferent axons exhibited numerous en passant and terminal varicosities that were often found in close apposition to brown-stained serotonergic and dopaminergic somata, and to proximal and distal dendrites and dendritic processes. The close association of immunoreactive elements suggests the presence of axo-somatic and axodendritic synaptic contacts of medullary fibers with serotonergic and dopaminergic neurons in the dorsal raphe nucleus. These projections could be involved in the modulation of dorsal raphe neurons, depending on the autonomic status of an animal.     

Features of the aggressive behavior of victorious mice in inter-male interactions

Latency of the first attack, number of attacks and total attacking time in aggressive male C57BL/6J strain mice were studied during agonistic interactions with submissive mice which had a great defeat experience. Then, aggressive mice were placed together in order to find out which will be the winner in each pair. It has been shown that males with a shorter latency of the first attack and greater total attacking time gained the victory.     

Prenatal stress decreases Bdnf expression and increases methylation of Bdnf exon IV in rats

There is ample evidence that exposure to stress during gestation increases the risk of the offspring to develop mood disorders. Brain-derived neurotrophic factor (Bdnf) plays a critical role during neuronal development and is therefore a prime candidate to modulate neuronal signaling in adult offspring of rat dams that were stressed during gestation. In the current study, we tested the hypothesis that alterations in Bdnf expression in prenatally stressed (PNS) offspring are mediated by changes in DNA methylation in exons IV and VI of the Bdnf gene. We observed decreased Bdnf expression in the amygdala and hippocampus of prenatally stressed rats both at weaning and in adulthood. This decrease in Bdnf expression was accompanied by increased DNA methylation in Bdnf exon IV in the amygdala and hippocampus, suggesting that PNS-induced reduction in Bdnf expression may, at least in part, be mediated by increased DNA methylation of Bdnf exon IV. Expression of DNA methyltransferases (Dnmt) 1 and 3a was increased in PNS rats in the amygdala and hippocampus. Our data suggest that PNS induces decreases in Bdnf expression that may at least in part be mediated by increased DNA methylation of Bdnf exon IV.     

Prenatal stress decreases Bdnf expression and increases methylation of Bdnf exon IV in rats

There is ample evidence that exposure to stress during gestation increases the risk of the offspring to develop mood disorders. Brain-derived neurotrophic factor (Bdnf) plays a critical role during neuronal development and is therefore a prime candidate to modulate neuronal signaling in adult offspring of rat dams that were stressed during gestation. In the current study, we tested the hypothesis that alterations in Bdnf expression in prenatally stressed (PNS) offspring are mediated by changes in DNA methylation in exons IV and VI of the Bdnf gene. We observed decreased Bdnf expression in the amygdala and hippocampus of prenatally stressed rats both at weaning and in adulthood. This decrease in Bdnf expression was accompanied by increased DNA methylation in Bdnf exon IV in the amygdala and hippocampus, suggesting that PNS-induced reduction in Bdnf expression may, at least in part, be mediated by increased DNA methylation of Bdnf exon IV. Expression of DNA methyltransferases (Dnmt) 1 and 3a was increased in PNS rats in the amygdala and hippocampus. Our data suggest that PNS induces decreases in Bdnf expression that may at least in part be mediated by increased DNA methylation of Bdnf exon IV.     

Hyperactivity and alteration of the midbrain dopaminergic system in maternally stressed male mice offspring

We recently demonstrated that prolonged maternal stress produces profound and long-lasting deficits in brain functions by programming a subset of target genes. We have now examined the possible effects of prenatal stress on the motility of adult offspring and dopamine (DA)-related gene expression in their midbrains, one of the target brain regions of stress hormones. Maternally stressed adult male mice showed impaired response habituation to novelty, and increased wheel-running activity associated with altered responses to DA receptor and DA transporter (DAT) blockers. Along with the behavioral changes, the expression profiles of several genes of the midbrain DAergic system appeared to be altered. Expression of DAT was reduced and expression of DA receptors and striatal DA-regulated neuropeptide genes was also affected. Taken together, the present findings indicate that maternal stress can cause hyperactivity in adult offspring associated with alterations in the midbrain DAergic system suggestive of a functional hyperdopaminergic state.     

Impaired expression of the mPer2 circadian clock gene in the suprachiasmatic nuclei of aging mice

The expression of circadian clock genes was investigated in the suprachiasmatic nuclei (SCN) of young adult and old laboratory mice. Samples were taken at two time points, which corresponded to the expected maximum (circadian time 7 [CT7]) or minimum (CT21) of mPer mRNA expression. Whereas the young mice had a stable and well-synchronized circadian activity/rest cycle, the rhythms of old animals were less stable and were phase advanced. The expression of mPer1 mRNA and mPer2 mRNA was rhythmic in both groups, with peak values at CT7. The levels of mClock and mCry1 mRNA were not different depending on the time of day and did not vary with age. In contrast, an age-dependent difference was found in the case of mPer2 (but not mPer1) mRNA expression, with the maximum at CT7 significantly lower in old mice. The decreased expression of mPer2 may be relevant for the observed differences in the overt activity rhythm of aged mice. (Chronobiology International, 18(3), 559-565, 2001)     

Testicular gene expression in male mice divergent for fertility after heat stress

Fertility losses in male mice occur approximately 18-28 d after heat stress. The objective of this study was to identify gene expression differences in males highly versus lowly fertile after heat stress. Mature male mice were exposed to heat stress (35 ± 1 °C; n = 50) or thermoneutral (21 ± 1 °C; n = 10) conditions for 24 h (Day 0) and hemicastrated (Day 1) to collect tissue for gene expression analyses. Males were subjected to a mating test from Days 18 to 26 when variation in fertility was anticipated. A fertility index was used to rank heat-stressed males and identify those males resistant and susceptible to heat stress, respectively. Microarray analyses were conducted on testis tissues from control (n = 5), heat stress resistant (n = 5), and heat stress susceptible (n = 5) males, and 225 genes were observed to be differentially expressed (P < 0.05), including genes involved in chaperone (Canx, Hspcb1, and Tcp1) and catalytic (Fkpb6, Psma7, and Idh1) activity. Expression patterns of these genes were confirmed using real-time RT-PCR. Male progeny from selected sires were similarly divergent in fertility after heat stress. Testicular expression levels of Canx, Hspcb, and Tcp1 genes were determined in these progeny. Hspcb expression was moderately heritable (0.31 ± 0.25); however, expression patterns of Canx and Tcp1 were not heritable.     

Comparative studies of the ingestive behaviors produced by microinjections of muscimol into the midbrain raphe nuclei of the ventral tegmental area of the rat

Microinjection of the GABA-A agonist muscimol into the median (MR) or dorsal (DR) raphe nuclei or the ventral tegmental area (VTA) of non-deprived rats induced intense feeding and drinking in a dose-dependent and site-specific manner. Lower doses of muscimol were required to increase food intake, spillage and water intake with injections into the MR than with injections into the other two sites. These data demonstrate that the MR is a more sensitive site for the elicitation of ingestive behavior than either the DR or the VTA.     

Food-associated estrogenic compounds induce estrogen receptor-mediated luciferase gene expression in transgenic male mice

The present paper aims at clarifying to what extent seven food-associated compounds, shown before to be estrogenic in vitro, can induce estrogenic effects in male mice with an estrogen receptor (ER)-mediated luciferase (luc) reporter gene system. The luc induction was determined in different tissues 8h after dosing the ER-luc male mice intraperitoneally (IP) or 14h after oral dosing. Estradiol-propionate (EP) was used as a positive control at 0.3 and 1mg/kg bodyweight (bw), DMSO as solvent control. The food-associated estrogenic compounds tested at non-toxic doses were bisphenol A (BPA) and nonylphenol (NP) (both at 10 and 50mg/kgbw), dichlorodiphenyldichloroethylene (p,p'-DDE; at 5 and 25mg/kgbw), quercetin (at 1.66 and 16.6mg/kgbw), di-isoheptyl phthalate (DIHP), di-(2-ethylhexyl) phthalate (DEHP) and di-(2-ethylhexyl) adipate (DEHA) all at 30 and 100mg/kgbw. In general IP dosing resulted in higher luc inductions than oral dosing. EP induced luc activity in the liver in a statistically significant dose-related way with the highest induction of all compounds tested which was 20,000 times higher than the induction by the DMSO-control. NP, DDE, DEHA and DIHP did not induce luc activity in any of the tissues tested. BPA induced luc in the liver up to 420 times via both exposure routes. BPA, DEHP and quercetin induced luc activity in the liver after oral exposure. BPA (50mg/kgbw IP) also induced luc activity in the testis, kidneys and tibia. The current study reveals that biomarker-responses in ER-luc male mice occur after a single oral exposure to food-associated estrogenic model compounds at exposure levels 10 to 10(4) times higher than the established TDI's for some of these compounds. Given the facts that (i) the present study did not include chronic exposure and that (ii) simultaneous exposure to multiple estrogenic compounds may be a realistic exposure scenario, it remains to be seen whether this margin is sufficiently high.     

Impact of exercise and a complex environment on hippocampal dendritic morphology,Bdnf gene expression,and DNA methylation in male rat pups neonatally exposed to alcohol

Alcohol exposure in utero can result in Fetal Alcohol Spectrums Disorders (FASD). Measures of hippocampal neuroplasticity, including long‐term potentiation, synaptic and dendritic organization, and adult neurogenesis, are consistently disrupted in rodent models of FASD. The current study investigated whether third trimester‐equivalent binge‐like alcohol exposure (AE) [postnatal days (PD) 4–9] affects dendritic morphology of immature dentate gyrus granule cells, and brain‐derived neurotrophic factor (Bdnf ) gene expression and DNA methylation in hippocampal tissue in adult male rats. To understand immediate impact of alcohol, DNA methylation was measured in the PD10 hippocampus. In addition, two behavioral interventions, wheel running (WR) and environmental complexity (EC), were utilized as rehabilitative therapies for alcohol‐induced deficits. AE significantly decreased dendritic complexity of the immature neurons, demonstrating the long‐lasting impact of neonatal alcohol exposure on dendritic morphology of immature neurons in the hippocampus. Both housing conditions robustly enhanced dendritic complexity in the AE animals. While Bdnf exon I DNA methylation was lower in the AE and sham‐intubated animals compared with suckle controls on PD10, alterations to Bdnf DNA methylation and gene expression levels were not present at PD72. In control animals, exercise, but not exercise followed by housing in EC, resulted in higher levels of hippocampal Bdnf gene expression and lower DNA methylation. These studies demonstrate the long‐lasting negative impact of developmental alcohol exposure on hippocampal dendritic morphology and support the implementation of exercise and complex environments as therapeutic interventions for individuals with FASD. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 77: 708–725, 2017     

Prenatal exposure of male mice to diethylstilbestrol alter the expression of the lactotransferrin gene in seminal vesicles

We have previously isolated an estrogen-inducible secretory protein, lactotransferrin (LTF), and a cDNA to its messenger RNA from the uterus of mice. In this report we determined that the level of LTF mRNA is minimal in the seminal vesicles of normal mice. In contrast, expression of LTF mRNA in the seminal vesicles of developmentally estrogenized males was both constitutive and estrogen inducible. The results suggested that this alteration may be an example of atypical gene expression after hormonal manipulation early in development.     

Neuronal activity of raphe nuclei of the brain stem in the cat

Evoked potentials were recorded in the system of raphe nuclei in experiments on unanesthetized, immobilized cats. Somatic stimulation proved to be the most effective of the different stimulations used (light flash, sound click, electrical stimulation of the skin of the limbs). Sound and light stimulation did not evoke pronounced responses, or the latter (to sound) were of a very low amplitude and irregular. In the second series of experiments on cats narcotized with nembutal (30–35 mg/kg) the spontaneous activity and activity evoked by somatic stimulation of single neurons of the caudal part of the raphe nuclei were studied. The overwhelming majority of neurons were characterized by spontaneous activity which changed (inhibited or facilitated) under the effects of somatic (especially repeated) stimulation; most of them reacted to stimulation of the skin of any limb. In the case of paired stimulation of the skin of limbs on different sides at large intervals (40–60 msec), inhibition of the test discharge occurred, whereas at small intervals summation (simple addition) of the impulses occurred. In their general characteristics the neurons of the raphe nuclei apparently differ little from the neurons of the reticular formation of the brain stem.Institute of Electrophysiology, Academy of Sciences of the Georgian SSR, Tbilisi. Translated from Neirofiziologiya, Vol. 3, No. 1, pp. 32–42, January–February, 1971.