Level of neuroactive steroids in the brain and sex-related peculiarities of formation and extinction of conditioned reflex in rats

Sex-related peculiarities of dynamics of brain sex steroids in the process of learning and extinction of the conditioned reflex of passive avoidance have been studied in model experiment. Prior to learning of the conditioned reflex, female rats were found to be distinguished by manifestation of anxiety and fear as compared with male rats. At formation of the conditioned reflex, no significant sex-related differences were detected between males and females, whereas extinction of the conditioned reaction of passive avoidance in males occurred by 2–3 days faster than in females. At learning of conditioned reaction of passive avoidance, in sexually mature male rats there was revealed an increase of the testosterone content in various brain structures, especially in hippocampus and frontal cortex, while its level in blood plasma remained unchanged. Also shown was an elevation of estradiol concentration in female amygdale, whereas at extinction of the conditioned reaction of passive avoidance, a rise of estradiol values was noted in hippocampus and cingular cortex. At the same time, the testosterone level in blood plasma did not change, whereas after extinction of the conditioned reflex the estradiol concentration decreased statistically significantly. Different dynamics of changes of the sex steroid levels in brain and blood plasma can indicate a possibility of their formation in the nervous tissue. The performed correlation analysis confirms the concept of selective involvement of testosterone and estradiol of individual brain structures in realization of processes of learning and memory in sexually mature male and female rats.     

A Single Neonatal Injection of Ethinyl Estradiol Impairs Passive Avoidance Learning and Reduces Expression of Estrogen Receptor α in the Hippocampus and Cortex of Adult Female Rats

Although perinatal exposure of female rats to estrogenic compounds produces irreversible changes in brain function, it is still unclear how the amount and timing of exposure to those substances affect learning function, or if exposure alters estrogen receptor α (ERα) expression in the hippocampus and cortex. In adult female rats, we investigated the effects of neonatal exposure to a model estrogenic compound, ethinyl estradiol (EE), on passive avoidance learning and ERα expression. Female Wistar-Imamichi rats were subcutaneously injected with oil, 0.02 mg/kg EE, 2 mg/kg EE, or 20 mg/kg 17β-estradiol within 24 h after birth. All females were tested for passive avoidance learning at the age of 6 weeks. Neonatal 0.02 mg/kg EE administration significantly disrupted passive avoidance compared with oil treatment in gonadally intact females. In a second experiment, another set of experimental females, treated as described above, was ovariectomized under pentobarbital anesthesia at 10 weeks of age. At 15–17 weeks of age, half of each group received a subcutaneous injection of 5 μg estradiol benzoate a day before the passive avoidance learning test. Passive avoidance learning behavior was impaired by the 0.02 mg/kg EE dose, but notably only in the estradiol benzoate-injected group. At 17–19 weeks of age, hippocampal and cortical samples were collected from rats with or without the 5 μg estradiol benzoate injection, and western blots used to determine ERα expression. A significant decrease in ERα expression was observed in the hippocampus of the estradiol-injected, neonatal EE-treated females. The results demonstrated that exposure to EE immediately after birth decreased learning ability in adult female rats, and that this may be at least partly mediated by the decreased expression of ERα in the hippocampus.     

Changes in brain serotonin metabolism and [3H]-serotonin receptor binding during recall of conditioned passive avoidance in rats

The content of serotonin and its metabolite 5-hydroxyindoleacetic acid, monoamine oxidase activity, and [3H]-serotonin radioligand receptor binding were examined in the prefrontal cortex, striatum, amygdala, hippocampus and periaqueductal gray matter at different time after one-trial passive avoidance training of rats. Changes in the serotonergic activity were observed only in rats, which showed retrieval of conditioned passive avoidance response. No serotonergic changes were found immediately and one day after training. Also, there were no changes in trained rats without retrieval of conditioned passive avoidance response or rats with experimental amnesia. The pattern of the involvement of brain structures in the retrieval process was also revealed. [3H]-serotonin binding was decreased in the amygdala, periaqueductal gray matter and striatum, whereas it did not change in the prefrontal cortex and hippocampus. At the same time, the serotonin content in these structures did not differ from that of intact rats. Deamination of serotonin by monoamine oxidase and active transport of 5-hydroxyindoleacetic acid from nerve terminals were increased in the amygdala and periaqueductal gray matter, whereas in the striatum serotonin catabolism was decreased. The obtained differences in serotonin catabo- lism suggest that the decrease in receptor binding of serotonin in these brain structures is provided by different synaptic processes: presynaptic changes in the striatum and postsynaptic receptor changes in the amygdala and periaqueductal gray matter. It is concluded that the decrease in the serotonergic activity in the amygdala and periaqueductal gray matter represents one of the mechanisms activating the emotiogenic system mediating the memory trace retrieval in inhibitory avoidance learning.     

Different roles of dopamine and serotonin in conditioned passive avoidance response of rats

Deamination of dopamine and serotonin by monoamine oxidase was studied in the prefrontal cortex, striatum, hippocampus and amygdaloid complex of the brain of rats during retrieval of conditioned passive avoidance response. Changes in the dopamine and serotonin metabolism were observed in different brain structures. A decrease in dopamine-deaminating activity of monoamine oxidase was found in the hippocampus, striatum and prefrontal cortex. At the same time, serotonin-deaminating activity of the enzyme was decreased in the striatum and increased in the amygdaloid complex, whereas it did not change in the prefrontal cortex and hippocampus. The observed changes in dopamine metabolism in the prefrontal cortex and hippocampus and serotonin metabolism in the amygdaloid complex indicate that dopamine and serotonin are involved in the regulation of two different processes mediating the memory trace retrieval. Dopamine is involved in neuronal mechanisms of information processes providing the strategy of behavior, whereas serotonin is related to emotional mechanisms of memory.     

Altered expression of NCAM in hippocampus and cortex may underlie memory and learning deficits in rats with streptozotocin-induced diabetes mellitus

Neurological and structural changes are paralleled by cognitive deficits in diabetes mellitus. The present study was designed to evaluate the expression of neural cell adhesion molecules (NCAM) in the hippocampus, cortex and cerebellum and to examine cognitive functions in diabetic rats. Diabetes was induced in male albino rats via intraperitoneal streptozotocin injection. Learning and memory behaviors were investigated using a passive avoidance test and a spatial version of the Morris water maze test. NCAM expression was detected in the hippocampus, cortex and cerebellum by an immunoblotting method. The diabetic rats developed significant impairment in learning and memory behaviours as indicated by deficits in passive avoidance and water maze tests as compared to control rats. Expression of NCAM 180 and 120 kDa were found to be higher in hippocampus and cortex of diabetic rat brains compared to those of control, whereas expression of NCAM 140 kDa decreased in these brain regions. Our findings suggest that streptozotocin-induced diabetes impairs cognitive functions and causes an imbalance in expression of NCAM in those brain regions involved in learning and memory. Altered expression of NCAM in hippocampus may be an important cause of learning and memory deficits that occur in diabetes mellitus.     

An experimental study of anti-amnestic activity of piracetam in orchidectomized rats]

The decrease of learning ability by 50% as assessed by passive avoidance test and the increase 28% of microviscosity of synaptosomal membranes from brain cortex were observed in rats in a month after orchidectomy as compared with that of intact or sham--operated animals. The treatment of orchidectomized rats with piracetam either by single application or in the course of 10 days applications in the daily dose of 250 mg/kg intraperitoneally failed to improve learning ability of animals in passive avoidance test and to produce a normalizing effect on the structure of membrane lipid matrix. The testosterone content in blood of orchidectomized rats was 10 times lower as compared with control and was not affected by treatment with piracetam.     

Brain and plasma levels of testosterone, dihydrotestosterone and estradiol in the one-day-old rat.

Current evidence indicates that the secretion of testosterone during perinatal life is essential in organizing the male brain which subsequently directs the male pattern of gonadotrophin (GTH) secretion and adult male sexual behavior in the rat. It has been hypothesized that testosterone is converted into estradiol enzymatically in the brain prior to its action. In the absence of testosterone and with the resultant low levels of estradiol, female patterns of gonadotrophin secretion and behavior result. In order to investigate this hypothesis further, the endogenous levels of gonadal steroids in the plasmas and brains of 24–48 hr old male and female rats were determined. Pooled samples were analyzed for testosterone, dihydrotestosterone and estradiol by radioimmunoassay. Testosterone levels in male brain and plasma samples were significantly (10-fold) higher than those in the female brain and plasma samples. Brain levels of estradiol were significantly higher in the male than in the female neonate, while plasma levels were identical. Whether the higher level of estradiol in the male brain is due to enzymatic conversion from testosterone within the brain differences in permeability or some other mechanism cannot be stated at this point. The significantly higher brain levels of both testosterone and estradiol in male neonates do fit the pattern predicted by the present concept of sexual differentiation. Dihydrotestosterone levels in brain and plasma of male rats were about 25% of those of testosterone. However in females the brain levels of dihydrotestosterone were significantly higher than testosterone even though the plasma levels of these hormones were identical. This may reflect a protective mechanism through which permeability of testosterone is lowered in the neonatal female brain during the critical period or simply a functional conversion of testosterone to dihydrotestosterone in the female during this period.     

Analysis of the participation of rat brain noradrenergic terminals in elaboration of conditioned passive avoidance response]

A change in a noradrenaline concentration in the noradrenergic terminals was investigated by the fluorescent histochemical method in pars ventralis nuc. interstitialis striae terminalis, nuc. septi lateralis and in nuc. preopticus medialis of male Wistar rats in the process of reproduction of conditioned passive avoidance response. In all the investigated structures of the preliminarily trained animals the reproduction of a conditioned response (without any electrical stimulation) led to a statistically reliable decrease in the noradrenaline concentration. The data obtained testified to the participation of the noradrenergic brain system in the reproductive process of the conditioned passive avoidance response.     

Hormonal modulation of extinction responses induced by sexual steroid hormones in rats

A functional interrelation between the nervous and endocrine systems has been established. However, few studies have dealt with the effects of sexual steroids on learning and memory. The aim of this work was to determine whether sexual steroid hormones could modulate the extinction response of a passive avoidance conditioning in rats. Male Wistar rats, randomly assigned to five groups, two controls and three experimental groups, were submitted to a one-trial passive avoidance conditioning and tested for their retention 24 hr after and during 10 weeks. One control group received no treatment at all, the other received vegetable oil, and the three experimental received 20 mg of testosterone enanthate, 0.8 mg estradiol valerate, or 4 mg nandrolone decanoate, respectively. All substances were applied in a 0.3 ml volume, 24 hours before training and before testing for retention each week during 10 weeks. Results indicate that the extinction process is modulated by these hormones, since testosterone and estradiol facilitate extinction, whereas the anabolic androgen produced a resistance to the extinction process.     

Conditional Reflex Reaction of the Passive Avoidance in Female and Male Rats Exposed to Hypoxia at Different Terms of Prenatal Development

Elaboration of the conditional reflex reaction of passive avoidance was studied in the 42–46-day old offsprings of two groups of female rats: intact (control) and exposed to action of hypoxia at the 13th, 16th or 19th day of pregnancy. The parameter of learning was the time of stay of the rats in a dark camera, in which they obtained an electroshock. It is shown that the prenatal hypoxia has different effect on the ability to elaborate the conditional reflex reaction of passive avoidance in female and male rats. By this parameter, no differences were revealed in the male group between the control and the animals exposed to hypoxia at different terms of intrauterine development. In female exposed to hypoxia at the 19th day of gestation, it was not possible to elaborate the conditional reflex reaction of passive avoidance. In females exposed to hypoxia at the 13th day of gestation the parameters of learning were lower, whereas in the females exposed to hypoxia at the 16th gestation day they were higher than in control animals.     

The effect of prenatal stress on activity of the neurosteroids synthesis enzyme in the "critical period" of brain sexual differentiation in male rats

The effect of daily immobillisation stress in female rats on the 15th to 18th days of pregnancy upon synthesis enzyme for neurosteroids of alpha-reductase in their male offspring brain, was studied. A decrease in the enzyme activity in the cortex and hypothalamus of male foetuses occurred within 24 hr following the latest stress, whereas it was increased in the cortex of newborn offspring. An enhancement of the 5 alpha-reductase activity in the cortex, hippocampus and hypothalamus was also found in prenatally stressed males on the 5th day of life. A decrease in the testosterone and progesterone contents in the blood plasma of the animals under study was revealed on the 19th day of their embryonic life as well as in newborn rats, the blood level of progesterone, at that, remained decreased even at the age of 5 days. A possible part ofneurosteroids in action of prenatal stress upon sexual differentiation of the brain is discussed.     

Sex hormone receptors in the hypothalamus and their role in the sexual differentiation of the brain of male rats

The content of receptors to estradiol and testosterone was determined in cytoplasmic and nuclear fractions of hypothalamus and brain cortex of male rats in the early postnatal period. Receptors to both estradiol and testosterone were revealed in cytosol and nuclear fractions, with the decrease in their concentration observed from days 1 to 5. The data obtained demonstrate that receptors to sexual hormones take part in the brain differentiation and regulation of hypophysis gonadotropic function by male or female type.     

The effect of rapid and depot testosterone and estradiol on spatial performance in water maze

Men and women differ in some cognitive functions including spatial abilities. These differences seem to be affected by sex steroids, but the results are controversial. The aim of this work is to describe the effects of rapid or depot testosterone and estradiol on spatial memory in rats. Thirty-two adult male Wistar rats were divided into 6 groups. Five groups were gonadectomized, and one group was left as control. Castrated groups received sterile oil, testosterone isobutyras, testosterone propionate, estradiol dipropionate or estradiol benzoate. We evaluated spatial performance (escape latency, overall improvement, and time in the quadrant after platform removal) of the rats in a spatial water maze. Animals receiving exogenous sex steroids showed higher plasma concentrations of the particular hormones. Experimental groups improved during the acquisition spatial trials in the water maze. No significant differences between the groups during probe trial were found. In overall improvement, the testosterone depot and estradiol depot groups showed less improvement in comparison to the control groups (P<0.05). No differences in respect to administered hormones were found in corresponding receptor gene expression in hippocampus. In conclusion, exogenous testosterone affects spatial memory of adult castrated males.     

Behavioral and biochemical characteristics of rats preferring ethanol or water

A study was made of the RNA biosynthesis intensity in the brain cortex depending on the rate of the learning of the conditioned active avoidance and food reinforcement alteration responses in ethanol or water preferring rats placed in a complex maze. The level of the brain RNA biosynthesis in ethanol preferring rats was lower as compared with that in rats preferring water. In ethanol preferring rats, the rate of the learning of the conditioned active avoidance response was lower, whereas that of the learning of the alternation response was greater than in water preferring rats. The data are discussed in accordance with the concepts that disorders in the interaction of transmitter and genetic structures of brain cells form the basis of the behavior of ethanol preferring animals.     

Circadian rhythms of the sex steroids of female Papio hamadryas in long-term hypokinesia

Radioimmunoassay was used to study the effect of restricted motor activity on circadian rhythms of blood plasma sexual steroids and cortisol in female Papio hamadryas during different phases of the menstrual cycle. Instact animals manifested clear-cut circadian rhythms of the testosterone and cortisol content during both the phases and those of estradiol and progesterone during the follicular phase of the cycle. Two-week immobilization did not produce any changes in the general pattern of circadian rhythms of cortisol and testosterone but led to a decrease in their mean daily concentrations, with that decrease being more pronounced for testosterone of females immobilized during the luteal phase. The authors established unbalance of sexual steroids with a dramatic fall of the mean daily concentrations of estradiol and progesterone and desynchronism of their circadian rhythms regardless of the cycle phase at the moment of immobilization. The mechanisms responsible for alterations in circadian rhythms of sexual steroids in hypokinetic females are discussed.     

Concentration of acid and basic proteins in the nuclei of hippocampal neurons and neuroglia of rat brain following elaboration of a passive avoidance conditioned reflex]

By means of two-wave-length visible cytophotometry, the content of acidic and basic proteins per cell nucleus was shown to increase in CA3 hippocampal neurons only 3 hours after the learning session of conditioned reflex of passive avoidance. The protein content in the nuclei of perineuronal neuroglia cells of CA3 hippocampus increased only 6 hours after the learning session. Biochemical mechanisms dealing with the process of learning are discussed.     

Inhibition of estrogen biosynthesis and its consequences on gonadotrophin secretion in the male

Of the gonadal steroids in the male, testosterone is the most important regulator of gonadotrophin secretion. However, whether testosterone affects gonadotrophin secretion directly or whether it must first be aromatized to estrogens is controversial. We have reported extensively on the endocrine and anti-tumor effects of the non-steroidal aromatase inhibitors CGS 16949A and CGS 20267 in adult female rats. In these animals, both inhibitors potently and selectively inhibit estrogen biosynthesis. Thus these agents can be effectively used in studying estrogen-dependent processes. CGS 16949A was administered for 14 days to adult male rats, over a dose range which in females suppresses estradiol and elevates LH. In male rats a suppression of estradiol was seen, however, there was no significant effect on either serum LH or on the weights of androgen-dependent organs. CGS 16949A, when administered to healthy men at a dose of 1 mg b.i.d. for 10 days, causes a significant fall in plasma estradiol and significant elevations of plasma FSH and testosterone. Dose-dependent suppression of serum estradiol and an increase in serum testosterone and LH are seen after administration of single oral doses of CGS 20267. These results indicate that in the male rat, inhibition of aromatization of testosterone to estrogens does not influence gonadotrophin secretion whereas in men the negative feedback exerted by testosterone on gonadotrophin secretion is dependent on the aromatization of testosterone to estrogens.     

Effect of thymalin on protein synthesis in the brain and conditioned-reflex activity of the progeny of neurosensitized female rats

Thymalin administration to two-week-old offspring of neurosensitized female rats prevented the development of protein synthesis disturbances in the central nervous system and the retention of conditioned reflex of passive avoidance. Thymalin injection to the offspring of intact female rats improved conditioned reflex retention and did not affect brain protein synthesis. A possible mechanism of thymalin effect in conditions of congenital neuroimmunopathology is discussed.     

Effect of acute melipramine administration on motor activity and defensive conditioned reflexes of passive and active avoidance in rats

The administration of threecyclic antidepressant melipramine to Wistar rats (15 mg/kg, intraperitonaly, 2 h before of experiments) increases time of an "open field" centre leaving. Thus melipramine does not influence horizontal and vertical activity, and also the number of bolus. At the development of a passive avoidance conditioned reflex melipramine significantly slows down realization of a unconditional mink reflex by untrained rats, increasing the latency of call in a dark compartment of the chamber. After training significant deterioration of a reflex reproduction is observed. At research of a defensive conditioned reflex of active avoidance melipramine worsens both development and reproduction of a reflex. The comparative analysis of the literary data of imipramine action on uptake of serotonine and noradrenaline and the analysis of the literary data on a role of these systems in the processes of learning and memory allows to suggest, that the effect of melipramine is connected mainly to amplification activity of serotoninergic system of a brain. It is supposed, that acute administration of melipramine creates emotionally negative state, worsens processes of learning and memory, strengthening mainly activity of a brain serotoninergic system. It specifies that serotoninergic system of a brain is system of punishment. Its activation interferes with formation and consolidation of connections between conditional and unconditional irritation.     

铅致大鼠记忆受损及海马AChE阳性纤维的改变

本实验采用明暗箱被动回避反应和穿梭箱条件性回避反应测定, 探讨了铅对大鼠学习与记忆的损害作用。应用酶组织化学方法观察了铅致大鼠海马的结构改变。行为测定显示, 大鼠饮１０％ 醋酸铅水液之前和之后, 其进洞潜伏期 （ｓｔｏｐｔｈｒｏｕｇｈ ｌａｔｅｎｃｙ, ＳＴＬ） 与对照组无明显差异, 而饮醋酸铅水液后, 其条件性回避反应率（ｃｏｎｄｉｔｉｏｎｅｄ ａｖｏｉｄａｎｃｅ ｒｅｓｐｏｎｓｅ ｒａｔｅ, ＣＡＲＲ） 较对照组明显下降（ｄ４－ ｄ１４, Ｐ＜ ００５）, 表明大鼠饮醋酸铅水液后长时记忆不受影响, 而短时记忆受到损伤。组织化学反应染色显示实验组海马腔隙分子层内ＡＣｈＥ阳性纤维面积百分数较对照组明显减少（Ｐ＜ ００５）, 且纤维变细, 膨体减少。推测铅致大鼠短时记忆能力下降, 可能与海马内胆碱能纤维减少有关。