Effect of intrauterine position on sex differences in the gabaergic system and behavior of rats.

In multiparous species such as the rat (in this case the albino Wistar strain), steroid influence during fetal growth is affected by the relative intrauterine position of male and female fetuses and is stronger when the potential effects of contiguity and caudal position are combined. The effect of intrauterine position on gonadal steroid levels in neonatal and adult animals was examined using radioimmunoassay techniques. Since the organizing effect of prenatal steroids may influence the postnatal GABA content, HPLC was used to determine the gabaergic content in several hypothalamic and limbic areas in the adult rat. The effects of intrauterine position on adaptive behavior were examined by recording exploratory behavior (using the corridor and hole board tests) and intraspecific aggression (induced by isolation). Female pups influenced by males during development produced more testosterone. In adult males, those that developed closer to the cervix (and with no influence from other fetuses) produced more testosterone and less estradiol. The same animals also produced more hypothalamic GABA and showed greater exploratory capacity. No significant differences were seen between any experimental groups with respect to aggression. These results show increased variability between males with respect to adult exploratory behavior, and in the neurochemical and endocrine systems involved, due to intrauterine position during development. The effect of this physiological phenomenon on the structure of rodent populations is discussed.

Role of maternal adrenal glands on the developing serotoninergic and aminoacidergic systems of the postnatal rat brain.

Serotonin, gamma-aminobutyric acid and glutamate, which are regulated by glucocorticoids in the central nervous system, are involved in neuroendocrine functions and the development of the brain. The present study investigates the effect of maternal adrenalectomy on the developing serotoninergic, GABAergic and glutamatergic systems. Neurotransmitter levels were measured in four brain areas of both male and female offspring on postnatal days 1, 8, 12 and 22. At postnatal day 1 and 8, the pups of adrenalectomized dams showed higher concentrations of serotonin than controls in all the brain areas studied. Serotonin levels decreased significantly in males at postnatal day 22 in the hippocampus and cortex. During the first 2 weeks of postnatal life, the lack of maternal corticosterone produced an increase in glutamate and a reduction in gamma-aminobutyric acid concentrations, mainly in males. Further, on postnatal day 1, increased serotonin and glutamate levels and lower levels of gamma-aminobutyric were observed in the hypothalamus of male pups born to adrenalectomized dams. The absence of maternal corticosterone affects the pattern of development of the serotoninergic system, especially in the hippocampus and cortex, and particularly in males. A delay in the maturation of the aminoacidergic systems, mainly of the GABAergic system and in males, was also seen. A sexually dimorphic response to the removal of maternal glucocorticoids was seen in terms of neurotransmitter levels, mainly in the hippocampus and hypothalamus.

Effect of maternal delta 9-tetrahydrocannabinol on developing serotonergic system.

In this study we investigated the effects of maternal delta 9-tetrahydrocannabinol on the developing serotonergic system. A daily dose of delta 9-tetrahydrocannabinol (5 mg/kg body weight) was administered p.o. to pregnant rats from gestational day 5 to postnatal day 1. Levels of indolamines were measured in four brain areas of the offspring on the day before or after birth. Levels of indolamines depended on the cerebral area, sex and pre- or postnatal age. Maternal exposure to delta 9-tetrahydrocannabinol decreased diencephalic levels of 5-hydroxytryptamine (5-HT), males being more susceptible than females. These perinatal changes could be responsible for the long-term neurophysiological alterations produced by cannabinoids.

Effects of maternal lead administration on monoaminergic, GABAergic and glutamatergic systems.

The effects of perinatal exposure to lead (300 mg/l) on the development of monoaminergic and aminoacidergic systems were evaluated in the striatum, cerebral cortex (Cx), dorsal hippocampus (d-Hipp) and basal-medial hypothalamus. Maternal exposure to lead produced regional alterations in monoamine content, with increases in dopamine and serotonin or their metabolites. Further, decreased glutamate levels were seen in all brain regions studied, while GABA content decreased only in the Cx. Together, these results show that lead causes alterations to neurotransmitter systems during development. These may be related to lead-induced neurobehavioral impairment.

Maternal exposure to delta 9-tetrahydrocannabinol (delta 9-THC) alters indolamine levels and turnover in adult male and female rat brain regions.

Perinatal exposure to delta 9-THC has been shown to produce effects on brain development. In this study we evaluated the changes induced by maternal exposure to delta 9-THC (5 mg/kg per day) from gestational day 5 to postnatal day 24 in eight discrete brain areas on the central serotoninergic system in both adult male and female rats. These result show that maternal exposure to delta 9-THC from gestational day 5 to postnatal day 24 affects development of the various central indoleaminergic system of the offsprings brain. Perinatal exposure to delta 9-THC decreased the levels of 5-HT in hypothalamus and rostral neostriatum in exposed males, and also decreased the levels of 5-HT in ventral hippocampus, septum, and midbrain raphe nuclei in both exposed males and females. Perinatal exposure to delta 9-THC increased the levels of 5-HIAA in dorsal hippocampus, hypothalamus, septum, midbrain raphe nuclei, and rostral neostriatum in exposed males and females. We have also found differences between nonexposed males and females in several brain regions. Our results confirm a regional and sexual specificity in endogenous levels of indoleamine after perinatal delta 9-THC treatment, being the midbrain raphe nuclei the most affected area.

Deprenyl protects from MPTP-induced Parkinson-like syndrome and glutathione oxidation in rat striatum.

An intrastriatal injection with 18.8 nmoles of the neurotoxic agent 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) induced in rats a progressive parkinsonism characterized by a major loss of striatum dopamine (DA) levels and an increased turnover of this neurotransmitter 96 h after the administration. In addition, the intrastriatal administration of MPTP produced an alteration in various behavioral markers of motor activity. Loss of DA was accompanied by a significant decrease of reduced glutathione (GSH) and an increase in GSH oxidation in the striatum. When deprenyl (10 mg/kg) was i.p. administered 2 h before the intrastriatal injection of MPTP, DA, GSH, glutathione redox status and the indexes of motor activity were not altered. These results show that MPTP increases striatum oxidative stress leading to cellular and in vivo degenerative changes which are prevented by pretreatment with deprenyl.

Study of the neurochemical alterations produced in discrete brain areas by perinatal low-level lead exposure.

Although the neurotoxic effects of Pb are well documented, the subcellular mechanisms of this action in the central nervous system are not fully understood. The present work examines some neurochemical parameters in discrete brain areas of pups whose mothers were intoxicated via drinking water with lead (300 mg/L), from day 1 of pregnancy until postnatal day 12. Lead intoxication produced a significant reduction in the activity of the enzymes alkaline phosphatase and ATP-ase in the brain. Furthermore, the levels of adenine nucleotides were significantly altered by treatment, the striatum being the area more affected, whereas lead did not vary the levels of ATP, ADP and AMP in the hypothalamus. On the other hand, there was a general decrease in neurotransmitter levels in all areas, specially in the hippocampus. These data suggest that gestational and lactational exposure to low dose of lead could produce neurochemical changes in discrete brain areas which can be responsible for the neurophysiological and behavioral changes described in lead-intoxicated animals.

Effect of maternal adrenal deprivation on the content of catecholamines in fetal brain.

Previous studies performed in our laboratory showed the importance of the effects that the absence of maternal adrenal hormones have on fetal brain. In the present study we investigated the effect of adrenal deprivation during gestation on the fetal catecholamines development in several cerebral areas. Fetuses from both control and adrenalectomized mothers from the first day of gestation were removed on the 20th embryonary day. Plasma corticosterone levels were significantly lower in the maternal serum of adrenalectomized rats, while the contents were non significantly higher in the adrenalectomized-mothers group of fetuses. Catecholamine contents in diencephalon, metencephalon, mesencephalon and telencephalon were measured by HPLC-ED. The results obtained showed that when the development of the catecholaminergic systems was previous enough to the fetal adrenal function, and under maternal adrenal deprivation conditions, the lack of corticosterone promotes an increase in the level of the catecholamines, as observed in the diencephalic NA, the earlier in maturational process. In those areas where the maturation starts at the same time than the fetal adrenal hypersecretion, no changes were observed. In the cortex, where both DA and NA develop later, the corticosterone produces an inhibition in the proliferation of the catecholaminergic neurons, showing decreased telencephalic levels of both catecholamines.

Influence of amygdala catecholamines on ovarian and adrenal medullary secretion.

The amygdaloid complex participates in the modulation of endocrine functions, and contains measurable amounts of noradrenaline (NA) and dopamine (DA). This study examined the contribution of the amygdaloid catecholaminergic systems to the regulation of the adrenal medulla and the ovary. To accomplish this the neurotoxin 6-hydroxydopamine (6-OHDA) was bilaterally injected into the basolateral nucleus of the amygdala (ABL) in cycling rats. The contents of NA and DA in right and left amygdala decreased significantly in lesioned animals with respect to sham lesioned animals, but hypothalamic levels were not different between groups. Administration of 6-OHDA to rats increased the NA, DA and adrenaline (A) contents of the adrenals compared to vehicle treated rats. In addition, lesioned animals showed a significant increase of NA and DA contents in the ovary, although A levels did not differ between groups. Serum oestradiol (O) concentrations were significantly lower in lesioned animals than in controls. These data suggest that the amygdaloid catecholaminergic systems exert an inhibitory effect on catecholamine content of the adrenals and the ovary, and influence the ovarian oestradiol secretion mechanism.

Effect of metyrapone administration in pregnant rats on monoamine concentration in fetal brain.

Studies performed in our laboratory indicate that the adrenal deprivation during gestation can greatly influence the fetal catecholamines development in several cerebral areas. The present study was undertaken to determine whether the administration of metyrapone to pregnant rats affects the content of monoamines in fetal brain at term. To test wether the content of monoamines in fetal brain is regulated, at least in part, by endogenous glucocorticoids, pregnant rats were injected for 5 days prior to delivery with metyrapone, an adrenal 11-beta-steroid hidroxylase inhibitor which crosses the placenta and blocks endogenous glucocorticoid synthesis, or saline. On day 21 of gestation, delivery of all animals was accomplished by cesarean section. The encephalons were extracted and immediately dissected in metencephalon, mesencephalon, diencephalon and telencephalon. Monoamine determination was carried out using HPLC-ED. The results obtained indicate that the metyrapone treatment increases both DA and 5-HT and their metabolites in the brain studied areas.

Maternal adrenalectomy affects development of adrenal medulla.

This work investigates the effects of maternal adrenalectomy (ADX) on the development of the adrenal medulla. Adrenal catecholamines (AC) were measured at postnatal day (PN) 1, 8, 12 and 22 in rat offspring of ADX dams and in pups of control dams. The pups of ADX rats showed a reduction in AC concentrations in the adrenal medulla at PN 1, 12 and 22, although these were higher than in the pups of sham dams at PN 8. Further, in the pups of control mothers, there was an increase in ACs during the first two weeks of life whereas pups of ADX mothers only showed increases in noradrenaline, dopamine and adrenaline levels at day 8. These results suggest that maternal absence of corticosterone affects the medulla catecholamine content during development. These data support the idea that a maternal glucocorticoids are involved in the differentiation or/and maturation of the adrenal medulla.

Neonatal catecholaminergic influence on behaviour and sexual hormones.

There is evidence of a sexually dimorphic effect of serotonin administration during the critical period of sexual differentiation on gonadal hormone secretion in adulthood. To investigate the possible involvement of catecholamines on these mechanisms, we have injected dopamine or noradrenaline intraventricularly into neonatal male and female rats to examine the influence, during the critical period, of this single treatment on the adulthood. Gonadal sex hormone contents, sexually dimorphic behaviours, and catecholaminergic distribution in hypothalamic and extrahypothalamic areas were studied. Both catecholaminergic treatments in females resulted in a reduced striatal dopaminergic activity and an increase in the hypothalamic noradrenergic ratio, while a reduction in the open field activity occurred in the same groups. These results suggest the possible involvement of striatal dopamine and hypothalamic noradrenaline in the differentiation of exploratory activity in females. A reduction in copulatory behaviour was shown in adults of both sexes after neonatal dopaminergic administration, but gonadal hormone levels were not affected in the same way. This indicates the existence of different facets of sexual differentiation, with striatal dopamine and hypothalamic noradrenaline playing important roles in neurobehavioural differentiation.

Sexual differences in the neonatal serotonin effect on hormone secretion in rats.

A number of authors in the literature have reported facilitatory or inhibitory effects of serotonin (5-HT) on gonadotropin secretion, sexual hormones content or sexual behavior, but little information has been reported about the possible role of serotonin administered during the critical period of sexual differentiation. To test this possibility, we have injected a 5-HT intraventricularly to neonate male and female rats in order to examine the influence during the critical period of this single treatment on the adult sexual hormone content and sexual behavior. Neonatal administration of 5-HT in the brain decreases significantly estradiol content of adult females, without affecting testosterone level in males. Neither male nor female sexual behavior was affected by 5-HT injection on day 1 of life. These data evidence a sexual difference of serotonin administration during the critical period on gonadal hormones secretion in adulthood.

Effect of delta 9-tetrahydrocannabinol on short-term memory in the rat.

We have reported that marihuana and its principal psycoactive compound, delta 9-tetrahydrocannabinol (delta 9-THC) produce alterations in several cerebral areas after acute treatment. Based on the involvement of 5-hydroxytryptamine (5-HT) on memory and learning and the reported effects of delta 9-THC on short-term memory, we designed an experiment to evaluate the memory performance and its possible relationship with serotonergic alterations after delta 9-THC administration. Male Wistar rats received an acute oral dose of THC (5 mg/kg). Short-Term memory was tested on a radial 8-arm maze with a 5 s delay, after 35 days of training. The animals were food deprived and adjusted for growth. 5-HT and its metabolite, 5-HIAA, levels were measured in cerebral cortex, dorsal hippocampus, ventral hippocampus, rostral neoestriatum and amygdala basal nucleus, by HPLC-ED. The experiment indicates an impairment of short-term memory in the radial maze test after delta 9-THC administration. The control group performed the test without errors, while the treated group made a significant number of errors (Z = 0.019, Mann-Whitney test). This behavioral effect did not seem to be related to serotonergic alterations, as the 5-HT turnover rate was not different between treated and control animals.

The effect of perinatal exposure to estrogens on the sexually dimorphic response to novelty.

In this study we investigated the sexually dimorphic anxiety response to a novel environment in the absence of estrogens neonatally or in adulthood. There was a sexual dimorphism in the plus-maze test after the open-field test, females being more active and less anxious. In the absence of estrogens neonatally but not in the adulthood, the activity levels were similar to those shown by females, while the anxiety level was similar to males. These results suggest the need of a normal estrogen environment during the critical period of development for the normal differentiation of female anxiety responses to a novel environment.

Neurochemical changes in newborn rat's brain after gestational cadmium and lead exposure.

Gestational administration of cadmium (10 mg/l) and lead (300 mg/l) produced a strong decrease in proteins at birth (-17%) and on day 5 (-31%) as well as in brain lipid amount on both days (-11 and -23%, respectively). At day 5 postnatal the exposure also produced a marked decrease in DNA and RNA concentrations with respect to the control group. On the other hand, we found a significant increase of indoleamine content in all brain areas studied in the cadmium-lead group and so the dopamine and its metabolite in mesencephalon, whereas dopamine levels in metencephalon decreased significantly. This data suggests that gestational and early lactational exposure to low dose of cadmium and lead could produce alterations in monoaminergic metabolism that can place the exposed animal to a significant risk in adulthood.

Injection of an aromatase inhibitor after the critical period of sexual differentiation.

We have investigated the possible role of the second week of life in the differentiation of sexually dimorphic behaviours, dependent on the androgenic aromatization, and its possible relationship with the serotonergic systems. For this purpose, 5 mg/kg of a suspension of an aromatase inhibitor, LY43578, has been intraventricularly injected to males on day 12 of life. Studies have been made in adulthood on exploratory and motor activities, anxiety, sexual motivation, and sexual performance. Indoleamine levels in the hypothalamus and corpus striatum have been measured. Sexual behaviour, exploration, and serotonergic metabolism were not affected by the treatment. Sex partner preference and anxiety in the plus-maze showed a feminized tendency in the treated group that, however, did not reach statistical significance. From these results we have confirmed the restriction of the critical period of androgenic aromatization for the organization of reproductive and exploratory behaviour.

Role of monoamines in the male differentiation of the brain induced by androgen aromatization.

Cerebral androgen aromatization has been described as a mechanism responsible for masculinization of the brain, and monoamines seem to be involved in sexual differentiation of the brain. The aim of this study was to investigate the possible implication of monoamines in the masculinization of the brain induced by cerebral androgen aromatization not only in the classic hypothalamic areas but also in some extrahypothalamic ones. For this purpose, 1-day-old male Wistar rats were injected intraventricularly with 5 mg/kg of a suspension of an aromatase inhibitor, LY43578. Saline was administered to male and female control groups. At adulthood, open-field, heterotypical, and homotypical sexual behavior tests were performed and cerebral amines were determined by HPLC-ED. Behavioral tests revealed feminine-like exploratory activity and defecation rate in the treated group, as well as an 89% lordotic response and decreased number of mounts plus intromissions. Testosterone levels were not affected by the treatment. Striatal and limbic serotonergic metabolism showed a sexual dimorphism, higher in males than females, that disappeared in the treated group. From these results, we suggest a possible role of extrahypothalamic serotonin in the mediation of the estrogen-induced mechanisms of behavioral sexual differentiation.

Extrahypothalamic serotonergic modification after masculinization induced by neonatal gonadal hormones.

Exposure to gonadal steroids during the critical period exerts an organizational effect on the CNS. This hormonal effect could be mediated, at least in part, by neurotransmitters. Traditionally, the main place involved in the aminergic sexual differentiation has been the hypothalamus. The aim of this work was to examine the possible long-term effect of cerebral administration of testosterone or estradiol on sexual behavior and hypothalamic/extrahypothalamic monoaminergic systems in the adult rat. For this purpose, female Wistar rats were intraventricularly injected during the first 24 h of life with testosterone (T) or estradiol benzoate (EB) (200 micrograms/kg) (male and female control groups were vehicle treated) and sexual behavior and monoaminergic mediobasal hypothalamic, striatal, and limbic metabolism in adult rats were studied. Receptive behavior was not affected, whereas a masculinizing effect (% mounts) was observed in the animals treated with both gonadal hormones. Only testosterone-treated females showed a male-like serotonergic ratio in corpus striatum and limbic system. A possible extrahypothalamic serotonergic role could be suggested in the mechanisms of sexual differentiation.

The effects of acute treatment with delta9-THC on exploratory behaviour and memory in the rat.

The aim of the present study was to evaluate the effects of delta9-tetrahydrocannabinol (delta9-THC) on exploratory behaviour and memory, independent of its locomotor suppressive effects. Dopamine (DA) and noradrenaline (NA) contents were determined in the areas of the brain directly related to such behaviours (hippocampus, striatum and amygdala). An acute dose of delta9-THC led to a decrease in exploratory parameters and motor activity during the holeboard test. The radial arm maze was used to evaluate the effects of this cannabinoid substance on memory. Animals treated with delta9-THC committed more errors in the maze test compared to control, particularly when the retention process was put to test. Furthermore, treatment with delta9-THC led to reduced NA contents in the hippocampus and increased DA contents in the amygdala, without changes in the striatum.