Social stress in adolescents induces depression and brain-region-specific modulation of the transcription factor MAX.

MAX is a conserved constitutive small phosphoprotein from a network of transcription factors that are extensively studied in tumorigenesis and whose functions affect cell proliferation, differentiation and death. Inspired by its higher expression during development and in regions involved in emotional behaviors, we hypothesized its involvement in cerebral changes caused by early-life stress. We studied the effects of repeated social stress during adolescence on behaviors and on MAX and its putative partner MYC. Thirty-day-old C57BL/6 male mice underwent brief daily social defeat stress from an adult aggressor for 21 days. Following social stress episodes and housing in social groups after each defeat, adolescent mice exhibit depressive-like, but not anxiety-like behaviors and show higher MAX nuclear immunoreactivity in hippocampal (HC) but not prefrontal cortical (PFC) neurons. Conversely, MAX immunoreactivity is lower in the striatum (ST) of defeated adolescents. The positive correlation between MAX and MYC levels in the PFC revealed disruptions in both the HC and ST. The changes in MAX protein levels are not due to differential gene expression or protein degradation in those regions, suggesting that posttranscriptional modifications occurred. These findings indicate that repeated, brief social defeat in adolescent male mice, combined with group housing, is a useful protocol to study a subtype of depression that is dissociated from generalized (non-social) anxiety. To our knowledge, this is the first report of an association between dysregulation of the MAX-MYC network in the brain and a behavior, suggesting a novel approach for exploiting the neuroplasticity associated with depression.

Respiratory deficits in a rat model of Parkinson's disease.

Parkinson's disease (PD) is a neurodegenerative disease characterized by loss of the dopaminergic nigrostriatal pathway. In addition to deficits in voluntary movement, PD involves a disturbance of breathing regulation. However, the cause and nature of this disturbance are not well understood. Here, we investigated breathing at rest and in response to hypercapnia (7% CO2) or hypoxia (8% O2), as well as neuroanatomical changes in brainstem regions essential for breathing, in a 6-hydroxydopamine (6-OHDA) rat model of PD. Bilateral injections of 6-OHDA (24µg/µl) into the striatum decreased tyrosine hydroxylase (TH(+))-neurons in the substantia nigra pars compacta (SNpc), transcription factor phox2b-expressing neurons in the retrotrapezoid nucleus and neurokinin-1 receptors in the ventral respiratory column. In 6-OHDA-lesioned rats, respiratory rate was reduced at rest, leading to a reduction in minute ventilation. These animals also showed a reduction in the tachypneic response to hypercapnia, but not to hypoxia challenge. These results suggest that the degeneration of TH(+) neurons in the SNpc leads to impairment of breathing at rest and in hypercapnic conditions. Our data indicate that respiratory deficits in a 6-OHDA rat model of PD are related to downregulation of neural systems involved in respiratory rhythm generation. The present study suggests a new avenue to better understand the respiratory deficits observed in chronic stages of PD.

The renin-angiotensin system is modulated by swimming training depending on the age of spontaneously hypertensive rats.

AIM: To investigate the effects of swimming training on the renin-angiotensin system (RAS) during the development of hypertensive disease. MAIN METHODS: Male spontaneously hypertensive rats (SHR) were randomized into: sedentary young (SY), trained young (TY), sedentary adult (SA), and trained adult (TA) groups. Swimming was performed 5 times/wk/8wks. KEY FINDINGS: Trained young and adult rats showed both decreased systolic and mean blood pressure, and bradycardia after the training protocol. The left ventricular hypertrophy (LVH) was observed only in the TA group (12.7%), but there was no increase on the collagen volume fraction. Regarding the components of the RAS, TY showed lower activity and gene expression of angiotensinogen (AGT) compared to SY. The TA group showed lower activity of circulatory RAS components, such as decreased serum ACE activity and plasma renin activity compared to SA. However, depending on the age, although there were marked differences in the modulation of the RAS by training, both trained groups showed a reduction in circulating angiotensin II levels which may explain the lower blood pressure in both groups after swimming training. SIGNIFICANCE: Swimming training regulates the RAS differently in adult and young SHR rats. Decreased local cardiac RAS may have prevented the LVH exercise-induced in the TY group. Both groups decreased serum angiotensin II content, which may, at least in part, contribute to the lowering blood pressure effect of exercise training.

Individual vulnerability to escalated aggressive behavior by a low dose of alcohol: decreased serotonin receptor mRNA in the prefrontal cortex of male mice.

Low to moderate doses of alcohol consumption induce heightened aggressive behavior in some, but not all individuals. Individual vulnerability for this nonadaptive behavior may be determined by an interaction of genetic and environmental factors with the sensitivity of alcohol's effects on brain and behavior. We used a previously established protocol for alcohol oral self-administration and characterized alcohol-heightened aggressive (AHA) mice as compared with alcohol non-heightened (ANA) counterparts. A week later, we quantified mRNA steady state levels of several candidate genes in the serotonin [5-hydroxytryptamine (5-HT)] system in different brain areas. We report a regionally selective and significant reduction of all 5-HT receptor subtype transcripts, except for 5-HT(3), in the prefrontal cortex of AHA mice. Comparable gene expression profile was previously observed in aggressive mice induced by social isolation or by an anabolic androgenic steroid. Additional change in the 5-HT(1B) receptor transcripts was seen in the amygdala and hypothalamus of AHA mice. In both these areas, 5-HT(1B) mRNA was elevated when compared with ANA mice. In the hypothalamus, AHA mice also showed increased transcripts for 5-HT(2A) receptor. In the midbrain, 5-HT synthetic enzyme, 5-HT transporter and 5-HT receptors mRNA levels were similar between groups. Our results emphasize a role for postsynaptic over presynaptic 5-HT receptors in mice which showed escalated aggression after the consumption of a moderate dose of alcohol. This gene expression profile of 5-HT neurotransmission components in the brain of mice may suggest a vulnerability trait for alcohol-heightened aggression.

Expression of alpha-synuclein is increased in the hippocampus of rats with high levels of innate anxiety.

A genomic region neighboring the alpha-synuclein gene, on rat chromosome 4, has been associated with anxiety- and alcohol-related behaviors in different rat strains. In this study, we have investigated potential molecular and physiological links between alpha-synuclein and the behavioral differences observed between Lewis (LEW) and Spontaneously Hypertensive (SHR) inbred rats, a genetic model of anxiety. As expected, LEW rats appeared more fearful than SHR rats in three anxiety models: open field, elevated plus maze and light/dark box. Moreover, LEW rats displayed a higher preference for alcohol and consumed higher quantities of alcohol than SHR rats. alpha-Synuclein mRNA and protein concentrations were higher in the hippocampus, but not the hypothalamus of LEW rats. This result inversely correlated with differences in dopamine turnover in the hippocampus of LEW and SHR rats, supporting the hypothesis that alpha-synuclein is important in the downregulation of dopamine neurotransmission. A novel single nucleotide polymorphism was identified in the 3'-untranslated region (3'-UTR) of the alpha-synuclein cDNA between these two rat strains. Plasmid constructs based on the LEW 3'-UTR sequence displayed increased expression of a reporter gene in transiently transfected PC12 cells, in accordance with in-vivo findings, suggesting that this nucleotide exchange might participate in the differential expression of alpha-synuclein between LEW and SHR rats. These results are consistent with a novel role for alpha-synuclein in modulating rat anxiety-like behaviors, possibly through dopaminergic mechanisms. Since the behavioral and genetic differences between these two strains are the product of independent evolutionary histories, the possibility that polymorphisms in the alpha-synuclein gene may be associated with vulnerability to anxiety-related disorders in humans requires further investigation.

Anabolic-androgenic steroid treatment induces behavioral disinhibition and downregulation of serotonin receptor messenger RNA in the prefrontal cortex and amygdala of male mice.

Nandrolone is an anabolic-androgenic steroid (AAS) that is highly abused by individuals seeking enhanced physical strength or body appearance. Supraphysiological doses of this synthetic testosterone derivative have been associated with many physical and psychiatric adverse effects, particularly episodes of impulsiveness and overt aggressive behavior. As the neural mechanisms underlying AAS-induced behavioral disinhibition are unknown, we investigated the status of serotonergic system-related transcripts in several brain areas of mice receiving prolonged nandrolone administration. Male C57BL/6J mice received 15 mg/kg of nandrolone decanoate subcutaneously once daily for 28 days, and different sets of animals were used to investigate motor-related and emotion-related behaviors or 5-HT-related messenger RNA (mRNA) levels by real-time quantitative polymerase chain reaction. AAS-injected mice had increased body weight, were more active and displayed anxious-like behaviors in novel environments. They exhibited reduced immobility in the forced swim test, a higher probability of being aggressive and more readily attacked opponents. AAS treatment substantially reduced mRNA levels of most investigated postsynaptic 5-HT receptors in the amygdala and prefrontal cortex. Interestingly,the 5-HT(1B) mRNA level was further reduced in the hippocampus and hypothalamus. There was no alteration of 5-HT system transcript levels in the midbrain. In conclusion,high doses of AAS nandrolone in male mice recapitulate the behavioral disinhibition observed in abusers. Furthermore, these high doses downregulate 5-HT receptor mRNA levels in the amygdala and prefrontal cortex. Our combined findings suggest these areas as critical sites for AAS-induced effects and a possible role for the 5-HT(1B) receptor in the observed behavioral disinhibition.

Social isolation and expression of serotonergic neurotransmission-related genes in several brain areas of male mice.

Early-life events influence brain development and evoke long-lasting behavioral consequences. Postweaning social isolation in rodents induces emotional and neurochemical alterations similar to those observed among some human psychopathologies. Central serotonergic neurotransmission is intimately involved in the observed adjustments, but the impact of social deprivation on serotonergic gene expression is unknown. We investigated the effects of prolonged early social isolation on emotion-related behaviors and 5-hydroxytryptamine (5-HT)-related gene transcription in mice. After weaning, male C57BL/6J mice were reared singly or in groups of four for 6 weeks. Gene expression of 5-HT(1A), 5-HT(1B), 5-HT(2A), 5-HT(2C), 5-HT(3A), 5-HT(6) and 5-HT(7) receptors and of 5-HT transporter and tryptophan hydroxylase-2 was determined by quantitative real-time polymerase chain reaction in distinct brain areas. Single-housed mice were hyperactive in a novel environment and showed signs of aggressive behavior. Housing condition did not alter weight gain or body temperature. Isolation markedly reduced transcription of all postsynaptic 5-HT receptors in the prefrontal cortex and reduced 5-HT(1B), 5-HT(2A) and 5-HT(2C) in both hypothalamus and midbrain. In contrast, the only alteration in the hippocampus was 5-HT(6) overexpression. Neither 5-HT transporter nor synthetic enzyme gene transcription differed between housing conditions. In conclusion, early social isolation in mice induces robust changes in postsynaptic 5-HT receptors gene transcription, motor hyperactivity and behavioral disinhibition. The overall pattern of decreased gene expression in the prefrontal cortex highlights its high vulnerability to environment. Furthermore, this is the first study to present a general representation of 5-HT-related gene expression in specific brain areas after social isolation and identifies novel candidates that may be critical for underlying molecular mechanisms.

Molecular basis of aggression.

Recent pharmacological and genetic studies have dramatically expanded the list of neurotransmitters, hormones, cytokines, enzymes, growth factors, and signaling molecules that influence aggression. In spite of this expansion, serotonin (5-HT) remains the primary molecular determinant of inter-male aggression, whereas other molecules appear to act indirectly through 5-HT signaling. We review evidence of interactions among these molecules and aggressive behavior. Slight modulations in 5-HT levels, turnover, and metabolism, or in receptor subtype activation, density, and binding affinity affect aggression. Activation of specific 5-HT receptors evokes distinct, but highly interacting, second messenger systems and multiple effectors. Understanding the interactions between 5-HT receptor subtypes should lead to novel insights into the molecular mechanisms of aggression.

Brain serotonin dysfunction accounts for aggression in male mice lacking neuronal nitric oxide synthase.

Genetically engineered mice with targeted disruption of the neuronal nitric oxide synthase (nNOS) gene established the inhibitory role of nitric oxide (NO) in male impulsive aggressive behavior. This was later confirmed by using selective nNOS inhibitors in male wild-type mice. The molecular mechanisms accounting for the aggressive behavior caused by the lack of neuronally derived NO is not known. Recent studies suggest that central serotonergic neuronal circuits and particularly 5-HT(1A) and 5-HT(1B) receptors play a prominent role in the regulation of aggression. Accordingly, we investigated whether the aggressiveness caused by the lack of nNOS might be because of alterations in serotonergic function. We now demonstrate that the excessive aggressiveness and impulsiveness of nNOS knockout mice is caused by selective decrements in serotonin (5-HT) turnover and deficient 5-HT(1A) and 5-HT(1B) receptor function in brain regions regulating emotion. These results indicate an important role for NO in normal brain 5-HT function and may have significant implications for the treatment of psychiatric disorders characterized by aggressiveness and impulsivity.

A functional role for complex gangliosides: motor deficits in GM2/GD2 synthase knockout mice.

Although gangliosides are abundant molecular determinants on all vertebrate nerve cells (comprising approximately 1.5% of brain dry weight) their functions have remained obscure. We report that mice engineered to lack a key enzyme in complex ganglioside biosynthesis (GM2/GD2 synthase), and which express only the simple ganglioside molecular species GM3 and GD3, develop significant and progressive behavioral neuropathies, including deficits in reflexes, strength, coordination, and balance. Quantitative indices of motor abilities, applied at 8 and 12 months of age, also revealed progressive gait disorders in complex ganglioside knockout mice compared to controls, including reduced stride length, stride width, and increased hindpaw print length as well as a marked reduction in rearing. Compared to controls, null mutant mice tended to walk in small labored movements. Twelve-month-old complex ganglioside knockout mice also displayed significant incidence of tremor and catalepsy. These comprehensive neurobehavioral studies establish an essential role for complex gangliosides in the maintenance of normal neural physiology in mice, consistent with a role in maintaining axons and myelin (Sheikh, K. A. , J. Sun, Y. Liu, H. Kawai, T. O. Crawford, R. L. Proia, J. W. Griffin, and R. L. Schnaar. 1999. Mice lacking complex gangliosides develop Wallerian degeneration and myelination defects. Proc. Natl. Acad. Sci. USA 96: 7532-7537), and may provide insights into the mechanisms underlying certain neural degenerative diseases.

Histamine and spontaneous motor activity: biphasic changes, receptors involved and participation of the striatal dopamine system.

The time- and dose-related effects of exogenous histamine on spontaneous motor activity and receptors involved were evaluated in male rats. Intracerebroventricular administration of histamine (5.4 and 54.3 nmol) produced a biphasic effect with initial transitory hypoactivity and later hyperactivity expressed by locomotion frequency in an open-field. The rearing frequencies were only reduced by all doses of histamine used. The histamine-induced hypoactivity was inhibited by the H3-antagonist thioperamide and was also induced by the H3-agonist N-alpha-methylhistamine. The histamine-induced hyperactivity phase was blocked by the H1-antagonist mepyramine. The H2-antagonist ranitidine increased locomotion and rearing frequencies. The participation of other neurotransmitters in the persistent hypokinetic effect induced by 135.8 nmol of histamine was determined by HPLC in the striatum and hypothalamus as counter-proof. A decreased DOPAC/DA ratio was observed only in the striatum. In the hypothalamus, low levels of 5HT were detected, probably not correlated with motor activity. In conclusion, the present results suggest that the exogenous histamine-induced hypoactivity response is probably due to activation of H3-receptors as heteroreceptors reducing the activity of the striatal dopaminergic system. This effect can partially overlap with the expression of the hyperactivity induced by H1-receptor activation. The participation of H2-receptors requires further investigation.

Nitric oxide synthase activity in the dorsal periaqueductal gray of rats expressing innate fear responses.

Immunohistochemical studies have shown nitric oxide synthase (NOS)-positive neurons in the dorsolateral sector of periaqueductal gray (PAG), a neural site known to be critical for the expression of defensive responses. In the present study, we first characterized the dorsal PAG (dPAG) NOS, and then examined NOS activity and cyclic GMP (cGMP) accumulation in the dPAG of rats exposed to a predator (cat) for 15 min. NOS activity evaluated by enzymatic conversion of [3H]arginine to [3H]citrulline in dPAG of exposed rats increased 14.6% and the cGMP radioimmunoassay showed an increase of 30.6% in relation to non-exposed rats. These results suggest an involvement of the NO/cGMP pathway in the dPAG during defensive responses.

Prenatal exposure of rats to diphenhydramine: effects on physical development, open field, and gonadal hormone levels in adults.

Diphenhydramine (DPH), a classical H1 receptor antagonist, has been used in pregnancy for the treatment of allergies, nausea, and vomiting. It has been reported that 10-20% of pregnant women take antihistamine-containing preparations at some point during pregnancy. The present study analyzed the influence of prenatal exposure to DPH of rats on: 1) maternal behavior and milk production of dams; 2) physical and reflexologic development of offspring; and 3) long-term effects on open field behaviors and gonadal hormone levels in offspring. Female pregnant rats were injected s.c., daily, with 20 mg/kg DPH or saline from embryonic day (E) 0 to 21. After delivery, maternal behavior was assessed and offspring physical and reflexologic development was examined. Open field activity of male and female rats was measured at 21 and 75 days of age and plasma hormone levels were evaluated in both sexes at 120 days of age. Neither maternal behavior nor milk production was affected by DPH treatment. Treated offspring showed an accelerated pinna unfolding, eye opening, and a delay of testes descent and vaginal opening. Both righting reflex and negative geotaxis development were accelerated, but prenatal exposure to DPH did not modify offspring locomotor activity. When tested as adults, a lack of sexual dimorphism in the open field activity of males and females was observed. No differences were observed between gonadal hormone levels of control and experimental groups of either sex. The findings suggest that prenatal DPH exposure influences physical and reflex development of rat pups.

Severe reduction of rat defensive behavior to a predator by discrete hypothalamic chemical lesions.

Nonspecific lesion and stimulation methods have suggested that the hypothalamus is critical for the expression of defensive behavior, although the organization of neural circuits mediating such behavior is unclear. In the rat hypothalamus, we found that increased Fos levels were restricted to specific cell groups following presentation of a stimulus (predator) known to elicit partly innate defensive responses. The dorsal premammillary nucleus showed the most striking increase in Fos levels, and cell body-specific chemical lesions therein virtually eliminated two major components of defensive behavior but increased exploratory behavior, suggesting that this caudal hypothalamic nucleus plays a critical role in the expression of behavioral responses sometimes critical for survival of the individual. We have previously shown that the Fos-responsive cell groups in the medial hypothalamus are interconnected in a neural system distinct from those mediating reproductive and ingestive behaviors.

Effects of prenatal diphenhydramine exposure on dopaminergic function in adult rats.

Female pregnant rats were treated with 20.0 mg/kg diphenhydramine (DPH) or the same volume of saline solution (NaCl 0.9%), SC, daily during pregnancy. As adults, male pups were tested for stereotyped behavior in response to apomorphine (1 mg/kg, SC) administration. No differences between DPH-exposed and control rats were evident. In another group of rats, dopamine (DA) and homovanillic acid (HVA) levels were quantified in striatal samples. DA levels were equivalent, but increased levels of HVA were observed. Based upon these data we suggest that prenatal exposure to DPH, a histamine (H1) receptor antagonist, reduces presynaptic dopaminergic mechanisms without altering postsynaptic dopaminergic function in adulthood.

Prenatal versus postnatal effects on offspring weight gain of rats exposed to diphenhydramine: a critical evaluation of fostering procedures in rats.

1. In order to evaluate the relative contribution of fostering procedures in the analysis of the development of rats prenatally exposed to diphenhydramine (20 mg/kg/day, sc) the weight gain of litters fostered or not by their biological mothers were examined from 2-21 days of age. 2. Maternal behavior and milk production were also assessed. 3. The results showed a decreased weight gain only in offspring fostered by mothers from different prenatal treatments and a lack of effects on maternal behavior and milk production. 4. It was concluded that the reduced weight of cross-fostered litters was mediated through an interference with postnatal mother-offspring interaction, which was not herein identified.

Effects of prenatal diphenhydramine administration on sexual behavior in rats

In order to study the involvement of the brain histamine system on the sexual vehavior of rats prenatally exposed to the histamine H1 receptor blockader, diphenhydramine (DPD), the famale lordotic response and male sexual behavior were aalyzed. The results show that the lorditic response in the prenatal DPD-treated rats was increased in relation to control animals. Impairment of male sexual behavior was indicated by an invrease in ejaculation latency, in the number of mounts and a decrease in the number of ejaculations up to 30 min after the first intromission. Prenatal expossure to DPD thus appears to alter female and male sexual behavior on reaching adulthood

Effects of prenatal diphenhydramine administration on sexual behavior in rats.

In order to study the involvement of the brain histamine system on the sexual behavior of rats prenatally exposed to the histamine H1 receptor blockader, diphenhydramine (DPD), the female lordotic response and male sexual behavior were analyzed. The results show that the lordotic response in the prenatal DPD-treated rats was increased in relation to control animals. Impairment of male sexual behavior was indicated by an increase in ejaculation latency, in the number of mounts and a decrease in the number of ejaculations up to 30 min after the first intromission. Prenatal exposure to DPD thus appears to alter female and male sexual behavior on reaching adulthood.