Antibodies to catecholamines and serotonin during alcoholic intoxication in animals with different predispositions to the development of experimental alcoholism

Experiments on C57Bl/6, CBA and DBA/2 mice characterized by different preferences for ethanol have shown that during chronic administration of alcohol to animals with natural ethanol motivation (strain C57Bl/6) the level of antibodies to catecholamines and serotonin was increased on the 3rd month of ethanol intoxication, with the voluntary alcohol consumption in mice decreased by this time. On the contrary in mice rejecting alcohol (strains DBA/2, CBA) no antibodies to catecholamines and serotonin have been found.     

Genetic variations of noradrenaline synthesis and reception in the mouse brain and the animal behavior in the new environment]

C57BL mice were found to have the highest locomotion and the lowest emotionality under novel environment out of three strains of mice. Their brain stem TH activity was increased whereas the density of alpha2-ARs and beta-ARs were decreased in their cortex and hypothalamus. The BALB mice were twice as virulent as the CBA mice whereas the emotionality was the same in both strains. In general, low emotionality and high locomotion in novel environment were found in mice with increased activity of norepinephrine synthesis and decreased amount of adrenergic receptors in the brain.     

Genetic-neurochemical analysis of the role of brain catecholamines in the manifestation of dominance in male mice in micropopulations

The ability of male mice to dominate in micropopulations, in connection with brain catecholamines was studied. It was shown that the F1 obtained from breeding PT and CBA/Lac which have respectively large and small numbers of high rank males were of high level of dominance in micropopulations. Moreover, they had high levels of noradrenaline and dopamine in hippocampus, striatum and brain stem, and low--in hypothalamus.     

Tyrosine hydroxylase activity and expression of its gene in mice with contrasting capacity to dominate in social stress]

Changes of tyrosine hydroxylase (TH) activity and level of mRNA of TH gene in PT and CBA/Lac mouse strains, which are contrast by ability to dominate in heterogenous populations, were investigated. It was established, that the activity of TH both in dominate PT and subordinate CBA/Lac mice in hypothalamus, hippocampus and brain stem elevated in one hour after forming of micropopulations. But the appearance of this increase was different: activation of TH in hypothalamus and brain stem of PT mice was stronger then one in CBA/Lac mice. Moreover, the beginning of the reaction in brain stem of PT mice was earlier then that of CBA/Lac mice. MRNA level of TH gene in hypothalamus and brain stem in one hour was elevated only in PT mice for 50% and 200%, respectively. No changing in expression TH gene was found in hippocampus. In conclusion, it was suggested that the activation of catecholamine biosynthesis under social stress in hypothalamus and brain stem of male mice was due to the TH activation and increase of its gene expression.     

Correlation between the rate of ethanol elimination and the psychophysiological characteristics of rats

Ethanol elimination from the blood of rats with different psychophysiological features was studied using gas chromatographic head-space analysis in the general complex of tests aimed at determination of ethanol consumption. The selection of animals with different levels of the initial alcohol motivation was performed according to modified Porsolt's method. It was shown that the initial level of predisposition to depression-like states is in a dose-dependent correlation with the high rate of ethanol elimination. This is suggested to be one of the genetic indications which promotes the formation of the initial alcohol motivation and the development of experimental alcoholism.     

Serotonin content in different sections of the brain, liver, intestines and blood of rats predisposed and not predisposed to alcohol consumption

Experiments were made with white random-bred rats (males) exposed to ethanol. The content of serotonin measured by spectrofluorometry was higher in the hypothalamus, brain stem and intestine, and was lower in the thalamus, striatum liver and blood in the animals predisposed to voluntary alcohol consumption and with lateral position duration 62 +/- 18 min as compared with the animals not predisposed to alcohol consumption and with lateral position duration 196 +/- 23 min, the dose of ethanol being 4.5 g/kg i. p. Thirty minutes after ethanol administration in a dose of 2.5 g/kg i. p. to the alcohol-predisposed rats there was a lowering of the serotonin content in the hypothalamus and an increase in the thalamus, brain stem, liver and blood. Meanwhile in the rats not predisposed to alcohol consumption, the serotonin content rose in the hypothalamus, brain stem, liver, intestine and blood and fell in the thalamus and striatum. It is assumed that the serotoninergic system of the brain may play a role in the formation of "positive" or "negative" attitudes to ethanol in the population of white random-bred rats.     

Interstrain differences in the serotonin and 5-hydroxyindoleacetic acid levels in the postnatal ontogeny of C57BL/6J and BALB/cLac strain mice

The content of serotonin and 5-hydroxy-indole-acetic acid (5-HIAA) was determined in the brain stem and hemispheres in 1 and 3 months old C57BL/6J and BALB/cLac mice. The characteristic dynamics of serotonin and its metabolite content related to the age was found in different brain regions and proved to be similar in both the strains, but the rate of serotonin system development in C57BL/6J mice was higher than in BALB/c Lac mice. An intensive catabolism of serotonin, possibly, related to the reaction to new environment was noted in the newborn animals. Sex differences in the rate of serotonin system maturation and serotonin and 5-HIAA content were shown for 12--16 days old mice: 12 days old males were characterized by more intensive metabolism than females while 16 days old males had less serotonin than females.     

Response of the serotoninergic brain system to social stress of various duration in male mice C57BL/6J and CBA/Lac]

The effects of social stress caused by experience of defeats in mice during 3 or 10 consecutive days of intermale confrontations on serotonergic brain activity (5-HT, 5-HIAA levels and 5-HIAA/5-HT ratio) in some brain regions of CBA/Lac (CBA) and C57BL/6J (C57) inbred mice have been studied. It was revealed the significant changes in 5-HT methabolism in the brain regions of defeated mice (losers) of CBA strain after 3 intermale confrontations. However, after 10 days of social stress these changes (excluded amygdala) turned to the control measures testifying to the adaptive mechanisms of serotonergic system in CBA losers. In C57 strain, the three-day social stress produced the mild changes in the brain serotonergic activity both quantitatively as well as qualitatively. Nevertheless, losers subjected to ten-day intermale confrontations had more expressed changes in 5-HT, 5-HIAA levels of 5-HIAA/5-HT ratios in the brain regions studied. It seems that long lasting social stress induced the development of disbalance of the brain serotonergic activity in C57 losers: it was shown the hyperactivity in the hypothalamus and hypoactivity in the amygdala and nucl. accumbens. Apparently, this cause leads to the development of the pronounced anxiety shown earlier in this mouse strain.     

Involvement of the serotonergic cerebral system in microwave-induced analgesia in mice of different genotypes

Under conditions of the formalin test, we studied changes in the level of analgesia induced by the action of low-intensity microwaves on the antinociceptive acupuncture point (AP) E36 in mice of strains CBA/CaLac (CBA) and C57BL/6j (C57) and in albino mongrel mice. Measurements were performed under control conditions and with experimentally induced decrease in the serotonin level in the brain (by injections of DL-parachlorophenylalanine, p-CPA). In the latter cases, the duration of the pain behavioral reaction increased despite irradiation of the AP E36. In mongrel, CBA, and C57 mice, the intensity of pain manifestations was 114.4, 29.0, and 21.1% greater, respectively, than in mice of these groups with no injections of p-CPA. These facts show that the serotonergic brain system is profoundly involved in the formation of analgesia after irradiation of the AP by low-intensity microwaves, and this involvement significantly depends on the genotype of the animals. Neirofiziologiya/Neurophysiology, Vol. 38, Nos. 5/6, pp. 495–497, September–December, 2006.     

Influence of the maternal effect on allogenic inhibition of hematopoietic stem cells]

Bone marrow cells (0,5-10(6)) of female mice of CBA or C57BL strains were injected intravenously to lethally irradiated CBA, C57BL/6, (femaleCBA X maleC57BL/6)F1 and (femaleC57BL/6 X maleCBA)F1 mice. Spleen of recipients as assayed for colony count on the 9th day after bone marrow transplantation by the method of Till and McCullouch. Stem cells of CBA mice demonstrated failure of allogenic inhibition in (CBA X C57BL/6)F1 hybrid mice and formed the same number of colonies as in the spleen of syngenic recipients. The level of allogenic inhibition of CBA stem cells transplanted to (C57BL/6 X X CBA)F1 hybrid mice was 50%. Bone marrow cells of C57BL/6 mice formed colonies in spleen of (CBA X C57BL/6)F1 mice at least in 20 times less than in syngenic combination. In the transplantation of bone marrow from C57BL/6 mice to (C57BL/6 X CBA)F1 hybrid mice the allogenic inhibition was less pronounced (77-85%) as compared with the transfer of cells to (CBA X C57BL/6)F1 hybrid mice (95%). The sex of a recipient did not influence the number of formed colonies. The different level of allogenic inhibition of parental stem cells can not be explained by the effect of linkage with sex as the female of reciprocal hybrid mice have identical structure of sex chromosomes (X(CBA)XC57BL/6). The data obtained indicate that the maternal effect affects allogenic inhibition of stem cells in parent--F1 system. It is possible that the maternal influence may be determined by cytoplasmic factors of inheritance which affect the expressivity of recessive genes Hh, controlling the inheritance of specific haematopoietic cell antigens.     

Effect of ethanol on the concentration of serotonin in the brain of the rat and the excretion of 5-hydroxyindoleacetic acid

It is found that serotonin content in the brain areas and heart of rats with low alcohol motivation decreases after 5 months of chronic consumption of 48% ethanol solution in a dose of 4 g/kg; in animals with high alcohol motivation serotonin content decreases only in the hypothalamus. Under chronic alcoholization for 1 and 12 months no considerable changes were found in serotonin level of the studied tissues. 60 min after intraperitoneal administration of 20% ethanol solution in a dose of 3 g/kg in intact animals there occurs an increase of serotonin content in the brain hemispheres and heart and its decrease in the hypothalamus; in rat with low alcohol motivation after taking ethanol for 5 months this administration evokes a decrease of serotonin content in the hypothalamus and truncus cerebri; in rats with high alcohol motivation--its decrease in the hypothalamus. Excretion of 5-oxyindoleacetic acid with urine decreases 10 months after alcohol intoxication. When rats were not given ethanol after its chronic taking for 3 months serotonin oxidation was intensified for the first day, which was not observed after 7-month alcoholization of animals.     

The fetal alcohol syndrome in mice: an animal model

CBA and C3H female mice were maintained on liquid diets--Metrecal plus ethanol--containing 15-35% ethanol-derived calories. These diets, which resulted in alcohol blood levels of 73-398 mg/100 ml blood in nonpregnant females, were the sole sustenance for the females for at least 30 days before and throughout gestation. Females were killed on day 18 of gestation and offspring examined for skeletal and soft tissue anomalies. Prenatal death and maldevelopment increased with the level of alcohol intake. Deficient occiput ossification, neural anomalies, and low fetal weight occurred with low ethanol diets, and cardiac and eye-lid dysmorphology with higher ethanol diets. This pattern of malformations, which exhibited both a dose-response effect and strain differences in susceptibility, indicated that chronic maternal alcoholism is embryolethal and teratogenic in mice.     

Effects of ethanol on brain monoamine content of spontaneously hypertensive rats (SHR)

Spontaneously hypertensive rats (SHR) were administered either 2.4 g/kg ethanol or an isocaloric glucose daily for 4 weeks and the levels of norepinephrine (NE), epinephrine (EP), dopamine (DA), serotonin (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) in different brain regions were determined. Results indicated a 3-fold increase in NE level in brain stem and hypothalamus and more than 2-fold increase in DA in corpus striatum in alcohol-treated rats as compared to controls. There was a significant increase in the level of DA in the corpus striatum but the levels in cerebral cortex, brain stem and hippocampus were decreased instead. Decreases in 5-HT levels were found in hypothalamus, brain stem, cortex and cerebellum of alcohol-treated brain as compared to untreated controls. These results indicate alterations of the biogenic amine contents in different regions of the SHR brain after chronic ethanol ingestion. Since stimulated release of biogenic amines in the SHR brain has been implicated in the regulation of blood pressure, changes due to ethanol ingestion may be a risk factor in hypertensive patients.     

Enkephalin and cyclic nucleotide content in the brain structures of rats at different stages of the formation and development of alcoholic dependence

Rats with increased alcohol motivation have been found to have a rise in enkephalin levels in limbic cortex and a decrease in met-enkephalin levels in the brain basal ganglia. Reduction of met-enkephalin to leu-enkephalin ratio in basal ganglia, limbic cortex and hypothalamus may serve as an index of increased inclination to ethanol in these animals. Alcohol dependence is characterized by reduced cAMP content in the majority of brain structures studied, sharply decreased met-enkephalin levels in limbic cortex and hypothalamus, and diminished cAMP and cGMP content in hypothalamus. In the third stage of experimental alcoholism the partial normalization of met-enkephalin and cAMP levels is observed in brain structures, with cGMP content increased in hypothalamus and considerably reduced in basal ganglia.     

Development of nicotine preference in C57Bl/6 and CBA mice

C57BL/6 mice were shown to consume greater amounts of nicotine solution than CBA and random-bred mice. Extensive literature data on C57BL/6 mice preference for ethanol and opiates and the experimental results obtained suggest common genetic mechanisms responsible for the inclination of C57BL/6 mice to toxicomania.     

Liver tryptophan pyrrolase. A major determinant of the lower brain 5-hydroxytryptamine concentration in alcohol-preferring C57BL mice.

The lower brain 5-hydroxytryptamine concentration in alcohol-preferring C57BL, compared with -non-preferring CBA, mice is caused by a decrease in circulating tryptophan availability to the brain secondarily to a higher liver tryptophan pyrrolase activity associated with a higher circulating corticosterone concentration. Activity or expression of liver tryptophan pyrrolase and/or their induction by glucocorticoids may be important biological determinants of predisposition to alcohol consumption.     

Migration and differentiation of gonadotropin-releasing hormone-producing neurons in the brain of mouse fetus exposed to excess of serotonin

The work has been carried out on mice of the Tg8 line with knockout of gene of monoamineoxidase A with an increase of serotonin and noradrenaline content in the brain, and on mice of the C3H line with unchanged genome and normal concentration of monoamines. An immunocytochemical study has been performed of development of neurons producing gonadotropin-releasing hormone (GnRH) under conditions of excess of serotonin and noradrenaline in the mice in embryogenesis. The GnRH-neurons were revealed at the 18th day of embryonic development in telencephalon along trajectory of their migration from olfactory bulbs to the retrochiasmatic area. In telencephalon of mouse embryos of the Tg8 line, a redistribution of the GnRH-neurons along their migration trajectory was observed as compared with embryos of the C3H line mice. The percent of the GnRH-neurons in the Tg8 mouse embryos in caudal parts of the migration trajectory was lower than in rostral parts, the opposite distribution of the neurons being observed in the C3H line mouse embryos; at the excess of serotonin and noradrenaline in the Tg8 line mouse embryos, the total amount of GnRH-neurons in the brain was lower than in the C3H mice. In males of the Tg8 line mice under conditions of excess of serotonin and noradrenaline the optical density of neurons, which correlated with the GnRH concentration in the cell, was higher than in control mice. Thus, in the Tg8 mice under conditions of the serotonin and noradrenaline excess, migration of the GnRH-neurons to their final anlage in hypothalamus is accelerated as well as the total number of the GnRH-neurons decreases, which indicates a decrease of proliferation of cells-precursors and the earlier differentiation of neurons.     

Involvement of striatum serotonergic system in the expression of genetically defined catalepsy

The activity of the rate-limiting enzyme of serotonin biosynthesis, tryptophan hydroxylase, and specific binding of [3H]ketanserin to 5-HT2A receptors and [3H]8-OH-DPAT to 5-HT1A receptors in the striatum of genetically predisposed to catalepsy rats and mice have been studied. The activity of tryptophan hydroxylase in the striatum of rats bred for many generations for predisposition to catalepsy was higher than in nonselected rats. Mice of highly susceptible to pinch-induced catalepsy CBA strain also differed from noncataleptic AKR and C57BL mouse strains by higher activity of tryptophan hydroxylase in striatum. Inhibition of tryptophan hydroxylase with p-chlorophenylalanine or p-chloromethamphetamine significantly decreased immobility time in genetically predisposed to catalepsy rats and mice. A decrease in the [3H]ketanserin specific binding in the striatum of cataleptic rats and CBA mice was found indicating a decrease in 5-HT2A receptor density. A decrease in [3H]8-OH-DPAT binding in striatum of cataleptic rats but not in CBA mice was shown. These results indicate that serotonergic system of striatum is involved in the expression of hereditary catalepsy and suggest that hereditary catalepsy may result from genetic changes in the regulation of serotonin metabolism and reception in striatum.     

Migration and differentiation of neurons producing gonadotropin-releasing hormone under conditions of serotonin excess in the brain of mouse embryos

The work has been carried out on mice of the Tg8 line with knockout of gene of monoamineoxidase A with an increase of serotonin and noradrenaline content in the brain, and on mice of the C3H line with unchanged genome and normal concentration of monoamines. An immunocytochemical study has been performed of development of neurons producing gonadotropin-releasing hormone (GnRH) under conditions of excess of serotonin and noradrenaline in the mice in embryogenesis. The GnRH-neurons were revealed at the 18th day of embryonic development in telencephalon along trajectory of their migration from olfactory bulbs to the retrochiasmatic area. In telencephalon of mouse embryos of the Tg8 line, a redistribution of the GnRH-neurons along their migration trajectory was observed as compared with embryos of the C3H line mice. The percent of the GnRH-neurons in the Tg8 mouse embryos in caudal parts of the migration trajectory was lower than in rostral parts, the opposite distribution of the neurons being observed in the C3H line mouse embryos; at the excess of serotonin and noradrenaline in the Tg8 line mouse embryos, the total amount of GnRH-neurons in the brain was lower than in the C3H mice. In males of the Tg8 line mice under conditions of excess of serotonin and noradrenaline the optical density of neurons, which correlated with the GnRH concentration in the cell, was higher than in control mice. Thus, in the Tg8 mice under conditions of the serotonin and noradrenaline excess, migration of the GnRH-neurons to their final anlage in hypothalamus is accelerated as well as the total number of the GnRH-neurons decreases, which indicates a decrease of proliferation of cells-precursors and the earlier differentiation of neurons.