Effects of pre- and postnatal exposure to 1880-1900MHz DECT base radiation on development in the rat.

In the present study, to evaluate the effects of wireless 1880-1900MHz Digital Enhanced Communication Telephony (DECT) base radiation on fetal and postnatal development, Wistar rats were exposed at an average electric field intensity of 3.7V/m, 12h/day, during pregnancy. After parturition, a group of dams and offspring were similarly exposed for another 22days. Controls were sham-exposed. The data showed that DECT base radiation exposure caused heart rate increase in the embryos on the 17th day of pregnancy. Moreover, significant changes on the newborns' somatometric characteristics were noticed. Pyramidal cell loss and glia fibrilliary acidic protein (GFAP) over-expression were detected in the CA4 region of the hippocampus of the 22-day old pups that were irradiated either during prenatal life or both pre- and postnatally. Changes in the integrity of the brain in the 22-day old pups could potentially be related to developmental behavioral changes during the fetal period.

The levels of the GluN2A NMDA receptor subunit are modified in both the neonatal and adult rat brain by an early experience involving denial of maternal contact.

The composition of the N-methyl-d-aspartate receptor receptor in GluN2A/GluN2B subunits is important in determining its characteristics and its role in plasticity, a property of the brain which is known to be critically affected by early experiences. In the present work we employed an early experience model involving either receipt (RER) or denial (DER) of the expected reward of maternal contact within the context of learning by the pups of a T-maze on postnatal days (PND) 10-13. We investigated the effects of the RER and DER early experiences on GluN1, GluN2A and GluN2B levels in the prefrontal cortex (PFC), hippocampus and amygdala of the rat. We show that on PND13 the DER animals had lower GluN2A levels in the PFC. In adulthood DER males had higher GluN2A levels in the hippocampus, both under basal conditions and after exposure to a novel environment. The early experiences did not affect the response to the novelty. After exposure to a novel environment animals of all three groups (DER, RER, Control) responded with an increase in GluN2A levels in the brain areas examined. We did not detect any effects on GluN1 or GluN2B levels. The alterations in GluN2A levels observed in the DER animals could in part be responsible for their behavioral phenotype, described previously, which includes an increased susceptibility for the expression of depressive-like behavior.

Effects of an early experience of reward through maternal contact or its denial on the dopaminergic system of the rat brain.

The mesolimbic/mesocortical dopaminergic pathway plays a pivotal role in the reward system. During the neonatal period the mother is the main source of rewarding stimuli. We have developed an experimental model in which rat pups learn a T-maze during the neonatal period (postnatal day (PND) 10-13) using contact with the mother as the reward. One group of animals is allowed contact with the mother (receipt of expected reward, RER) while the other was denied (denial of expected reward, DER). We determined the effects of these two early experiences in the prefrontal cortex (PFC) and the nucleus accumbens (nAc), the levels of dopamine (DA) and its metabolites [3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA)] by high-performance liquid chromatography and those of D1 and D2 receptors by autoradiographic in vitro binding both on PND 13 and in adulthood. On PND13, 2h after the end of training, the RER experience resulted in higher DA, HVA and D1 receptor levels in the nAc, while the DER in lower DA and its metabolites (DOPAC and HVA) in the PFC. These results could be related to the reward the RER pups received through the contact with their mother. The RER and DER early experience had long-term sex-dependent effects: The RER-induced activation of the dopaminergic system in the nAc was also evident in adult female rats. In contrast, adult DER males, similar to PND13 animals, had reduced dopamine in the PFC. Our results document that early experiences, a key determinant of adult brain function, affect the dopaminergic system which is disturbed in many psychiatric diseases.

Sexually dimorphic long-term effects of an early life experience on AMPA receptor subunit expression in rat brain.

Neonatal handling, an experimental model of early life experiences, is known to affect hypothalamic-pituitary-adrenal (HPA) axis function, thus increasing adaptability, coping with stress, cognitive abilities and in general brain plasticity-related processes. AMPA receptors (AMPARs) mediate fast synaptic transmission at excitatory glutamatergic synapses in the CNS and are crucial during neuronal development, synaptic plasticity and structural remodeling. AMPARs are composed of four types of subunits, designated as AMPA glutamate receptor subunits (GluA1, GluA2, GluA3 and GluA4), which combine to form tetramers. The present study addressed the question of whether neonatal handling (15min daily maternal separation from postnatal day 1 (PND1) to PND21) might have an effect on GluA1-4 mRNA levels in adult rat male and female brain using in situ hybridization. We have identified selective sexually dimorphic effects of neonatal handling on the mRNA expression levels of AMPAR subunits in adult rat hippocampus and nuclei of the amygdaloid complex. In the dorsal hippocampus GluA1 mRNA levels were increased in handled males, while they were decreased in handled female animals. In the ventral hippocampus and the amygdaloid complex GluA2 mRNA was lower in handled females, while no effect was observed in handled males. Furthermore, we observed that neonatal handling induced in both sexes decreases of GluA2 mRNA in the dorsal hippocampus, as well as in the somatosensory and occipital cortex, of GluA3 mRNA in most hippocampal areas, amygdaloid complex and cortical regions studied, and of GluA4 mRNA in the ventral hippocampus. These results show that glutamatergic transmission is markedly affected by an early experience. The neonatal handling-induced alterations in AMPAR subunit composition are in line with the increased brain plasticity, the more effective HPA axis function, and in general the more adaptive behavioral phenotype known to characterize the handled animals.

Effects of interaction of an early experience of reward through maternal contact or its denial with social stress during adolescence on the serotonergic system and the stress responsiveness of adult female rats.

Experiences during critical periods, such as the neonatal and adolescence, play a critical role in determining adult stress-coping behavior. Based on the aforementioned we developed an experimental protocol, which included a neonatal experience and a social stress during adolescence. The serotonergic system is known as an important modulator of coping ability and, in general, emotional balance in both normal and pathological states, such as depression and anxiety, for which females are more vulnerable. Thus in the present work we used female rats and determined 5-HT, 5-hydroxyindoleacetic acid (5-HIAA), and 5-hydroxytryptamine receptor type 1A (5-HT(1A)) receptor levels in the prefrontal cortex (PFC) and the amygdala (AMY). During postnatal days 10-13 (PND 10-13) rat pups were exposed to a T-maze, one arm of which lead to the mother. One group of animals was allowed contact with the mother (rewarded-receiving expected reward (RER)), whereas the other was denied the expected reward (DER). High performance liquid chromatography (HPLC) analysis revealed that in both the PFC and in AMY, adult RER animals had higher basal 5-HT levels. Furthermore, in the AMY of this group of animals, higher levels of 5-HT(1A) receptors were detected by Western blot analysis. In adulthood rats were exposed to the Forced Swimming Test/Stress (FST/S). RER animals not exposed to the adolescent stress exhibited longer immobility time during both the first and second day of FST. Corticosterone levels following the FST fell faster in the DER animals. Adolescent stress affected the responses to the adult FSS only in the DER animals, which had decreased 5-HT in the AMY and increased immobility time on both days of the FST, compared with the DER, not stressed in adolescence. The phenotype of the DER animals is in line with the "match-mismatch" hypothesis, which states that if two events during critical periods of life "match" in being mildly stressful, their interaction can be adaptive.

Reward or its denial during the neonatal period affects adult spatial memory and hippocampal phosphorylated cAMP response element-binding protein levels of both the neonatal and adult rat.

Early life experiences, particularly mother-infant interactions, have been shown to influence adult coping and learning abilities via gene-environment interactions. We have developed a paradigm, in which mother contact is used as either a positive or a negative reinforcer in a T-maze, during postnatal days 10-13. In both neonates receiving (RER) or denied (DER) the expected reward, exposure to the memory test in the absence of the mother resulted in a remarkable increase in the number of pCREB immunopositive cells, when compared to their corresponding levels 2 h after the completion of the training process, but also to the levels of naïve animals. In the CA3 area, the pattern of pCREB immunoreactivity, when evaluated 2 h after the completion of the training on postnatal day 13 seemed to distinguish between the two different neonatal experiences in the T-maze, with the DER pups showing higher levels of pCREB immunopositive cells than the RER. Exposure to the Morris Water Maze (MWM) during adulthood revealed a memory advantage of the DER animals compared to the RER and the animals not exposed to the neonatal experience. Relevantly, in the DER animals an increased number of pCREB immunopositive cells was observed in the CA3 area even 24 h after the end of MWM training. When also measured after exposure to the probe trial, the number of pCREB immunopositive cells was again higher in the DER compared to the RER animals. In conclusion, we show that a learning experience involving discrepancy during the particularly plastic neonatal period is able to induce long-term effects, which result in enhanced adult hippocampal dependent spatial memory. Furthermore, our data document a role of plasticity molecules like pCREB in mediating hippocampal dependent learning and detection of novelty not only in adulthood, but also more importantly in the neonatal period of the rat.

Short-term memory in mice is affected by mobile phone radiation.

The effects of mobile phone electromagnetic fields (EMFs) were studied on a non-spatial memory task (Object Recognition Task - ORT) that requires entorhinal cortex function. The task was applied to three groups of mice Mus musculus C57BL/6 (exposed, sham-exposed and control) combined with 3 different radiation exposure protocols. In the first protocol designated "acute exposure", mice 45 days old (PND45 - postnatal day 45) were exposed to mobile phone (MP) radiation (SAR value 0.22W/kg) during the habituation, the training and the test sessions of the ORT, but not during the 10min inter-trial interval (ITI) where consolidation of stored object information takes place. On the second protocol designated "chronic exposure-I", the same mice were exposed for 17 days for 90min/per day starting at PND55 to the same MP radiation. ORT recognition memory was performed at PND72 with radiation present only during the ITI phase. In the third protocol designated "chronic exposure-II", mice continued to be exposed daily under the same conditions up to PND86 having received radiation for 31 days. One day later the ORT test was performed without irradiation present in any of the sessions. The ORT-derived discrimination indices in all three exposure protocols revealed a major effect on the "chronic exposure-I" suggesting a possible severe interaction of EMF with the consolidation phase of recognition memory processes. This may imply that the primary EMF target may be the information transfer pathway connecting the entorhinal-parahippocampal regions which participate in the ORT memory task.

Whole body exposure with GSM 900MHz affects spatial memory in mice.

Extended work has been performed worldwide on the effects of mobile phone radiation upon rats' cognitive functions, however there is great controversy to the existence or not of deficits. The present work has been designed in order to test the effects of mobile phone radiation on spatial learning and memory in mice Mus musculus Balb/c using the Morris water maze (a hippocampal-dependent spatial memory task), since there is just one other study on mice with very low SAR level (0.05W/kg) showing no effects. We have applied a 2h daily dose of pulsed GSM 900MHz radiation from commercially available mobile phone for 4 days at SAR values ranging from 0.41 to 0.98W/kg. Statistical analysis revealed that during learning, exposed animals showed a deficit in transferring the acquired spatial information across training days (increased escape latency and distance swam, compared to the sham-exposed animals, on the first trial of training days 2-4). Moreover, during the memory probe-trial sham-exposed animals showed the expected preference for the target quadrant, while the exposed animals showed no preference, indicating that the exposed mice had deficits in consolidation and/or retrieval of the learned spatial information. Our results provide a basis for more thorough investigations considering reports on non-thermal effects of electromagnetic fields (EMFs).

Selective effects of neonatal handling on rat brain N-methyl-D-aspartate receptors.

Neonatal handling, an experimental model of early life experiences, is known to affect the hypothalamic-pituitary-adrenal axis function thus increasing adaptability, coping with stress, cognitive abilities and in general brain plasticity-related processes. A molecule that plays a most critical role in such processes is the N-methyl-D-aspartate (NMDA) receptor, a tetramer consisting of two obligatory, channel forming NR1 subunits and two regulatory subunits, usually a combination of NR2A and NR2B. Since the subunit composition of the NMDA receptor affects brain plasticity, in the present study we investigated the effect of neonatal handling on NR1, NR2A and NR2B mRNA levels using in situ hybridization, and on NR2B binding sites, using autoradiography of in vitro binding of [(3)H]-ifenprodil, in adult rat limbic brain areas. We found that neonatal handling specifically increased NR2B mRNA and binding sites, while it had no effect on the NR1 and NR2A subunits. More specifically, neonatally handled animals, both males and females, had higher NR2B mRNA and binding sites in the dorsal CA1 hippocampal area, as well as the prelimbic, the anterior cingulate and the somatosensory cortex, compared to the non-handled. Moreover NR2B binding sites were increased in the dorsal CA3 area of handled animals of both sexes. Furthermore, neonatal handling had a sexually dimorphic effect, increasing NR2B mRNA and binding sites in the central and medial amygdaloid nuclei only of the females. The neonatal handling-induced increase in the NR2B subunit of the NMDA receptor could underlie the higher brain plasticity, which neonatally handled animals exhibit.

Sex differences in water maze performance and cortical neurotrophin levels of LHX7 null mutant mice.

Mice lacking both alleles of the LIM-homeobox gene Lhx7 display dramatically reduced number of forebrain cholinergic neurons. Given the fact that sex differences are consistently observed in forebrain cholinergic function, in the present study we investigated whether the absence of LHX7 differentially affects water maze performance in the two sexes. Herein we demonstrate that LHX7 null mutants display a sex-dependent impairment in water maze, with females appearing more affected than males. Moreover, neurotrophin assessment revealed a compensatory increase of brain-derived neurotrophic factor and neurotrophin 3 in the neocortex of both male and female mutants and an increase of nerve growth factor levels only in the females. Nevertheless, the compensatory increase of cortical neurotrophin levels did not restore cognitive abilities of Lhx7 homozygous mutants. Finally, our analysis revealed that cortical neurotrophin levels correlate negatively with water maze proficiency, indicating that there is an optimal neurotrophin level for successful cognitive performance.

Effect of neonatal handling on adult rat spatial learning and memory following acute stress.

Brief neonatal handling permanently alters hypothalamic-pituitary-adrenal axis function resulting in increased ability to cope with stress. Since stress is known to affect cognitive abilities, in the present study we investigated the effect of brief (15 min) handling on learning and memory in the Morris water maze, following exposure to an acute restraint stress either before training or recall. Exposure of non-handled rats to the acute stress prior to training resulted in quicker learning of the task, than in the absence of the stressor. When acute stress preceded acquisition, male handled rats showed an overall better learning performance, and both sexes of handled animals were less impaired in the subsequent memory trial, compared to the respective non-handled. In addition, the number of neurons immunoreactive for GR was higher in all areas of Ammon's horn of the handled rats during the recall. In contrast, the number of neurons immunoreactive for MR was higher in the CA1 and CA2 areas of the non-handled males. When the acute restraint stress was applied prior to the memory test, neonatal handling was not effective in preventing mnemonic impairment, as all animal groups showed a similar deficit in recall. In this case, no difference between handled and non-handled rats was observed in the number of GR positive neurons in the CA2 and CA3 hippocampal areas during the memory test. These results indicate that early experience interacts with sex and acute stress exposure in adulthood to affect performance in the water maze. Hippocampal corticosterone receptors may play a role in determining the final outcome.

Cellular mechanisms underlying the effect of a single exposure to neonatal handling on neurotrophin-3 in the brain of 1-day-old rats.

Neurotrophin-3 (NT-3) has an important role in brain development and is thus a good candidate molecule to be involved in the cellular mechanisms mediating the effects of early experiences on the brain. In the present work we employed the model of neonatal handling, which is known to affect the ability of the adult organism to respond to stressful stimuli, and determined its effects on NT-3 levels in the rat hippocampus and cortex 2, 4 and 8 h after handling on postnatal day 1. We also recorded maternal behavior during the 8 h following handling. At both the 4 and 8 h time-points there was an increase in NT-3 positive cells in field 1 of Ammon's horn (CA1 area of the hippocampus) and parietal cortex of the handled animals. In the parietal cortex NT-3 levels increased with time following handling: at 8 h there were more NT-3 positive cells than at 4 h. During the 4 h following the end of handling, handled pups were subject to more maternal licking, indicating that the more intense maternal care could underlie the handling-induced increase in NT-3. In the hippocampus, the handling induced increase in NT-3 was cancelled by inhibition of N-methyl-D-aspartate (NMDA), AMPA/kainate, or GABA-A receptors, as well as L-type voltage-gated Ca(2+) channels. It thus appears that neonatal handling activates these neurotransmitter receptors and channels, leading to increased intracellular Ca(2+) and increased NT-3 expression. NT-3 can then activate downstream effectors and exert its morphogenetic actions and thus imprint the effects of handling on the brain.

Effects of neonatal handling on the basal forebrain cholinergic system of adult male and female rats.

Neonatal handling is an early experience which results in improved function of the hypothalamic-pituitary-adrenal axis, increased adaptability and coping as a response to stress, as well as better cognitive abilities. In the present study, we investigated the effect of neonatal handling on the basal forebrain cholinergic system, since this system is known to play an important role in cognitive processes. We report that neonatal handling results in increased number of choline-acetyl transferase immunopositive cells in the septum/diagonal band, in both sexes, while no such effect was observed in the other cholinergic nuclei, such as the magnocellular preoptic nucleus and the nucleus basalis of Meynert. In addition, neonatal handling resulted in increased M1 and M2 muscarinic receptor binding sites in the cingulate and piriform cortex of both male and female rats. A handling-induced increase in M1 muscarinic receptor binding sites was also observed in the CA3 and CA4 (fields 3 and 4 of Ammon's horn) areas of the hippocampus. Furthermore, a handling-induced increase in acetylcholinesterase staining was found only in the hippocampus of females. Our results thus show that neonatal handling acts in a sexually dimorphic manner on one of the cholinergic parameters, and has a beneficial effect on BFCS function, which could be related to the more efficient and adaptive stress response and the superior cognitive abilities of handled animals.

Effect of neonatal handling on serotonin 1A sub-type receptors in the rat hippocampus.

Serotonin 1A sub-type receptors play an important role in the etiopathogenesis of depression, which is known to occur more often in females than males. Early experiences can be a predisposing factor for depression; however, the underlying cellular processes remain unknown. In an effort to address such issues, we employed neonatal handling, an experimental model of early experience, which has been previously shown to render females more vulnerable to display enhanced depression-like behavior in response to chronic stress, while it increases the ability of males to cope. In rat pre-pubertal (30 days of age) and adult (90 days) hippocampus, of both males and females, the effect of neonatal handling on serotonin 1A sub-type receptor mRNA and protein levels was determined by in situ hybridization and immunohistochemistry, respectively, while the number of binding sites was determined by in vitro autoradiography using [(3)H]8-hydroxy-2(di-n-propylamino)tetralin as the ligand. Our results revealed a significant sex difference in serotonin 1A sub-type receptor mRNA, protein and binding sites, with females having higher levels than males. Handling resulted in statistically significant decreased numbers of cells positive for serotonin 1A sub-type receptor mRNA or protein, as well as [(3)H]8-hydroxy-2(di-n-propylamino)tetralin binding sites in the area 4 of Ammon's horn and dentate gyrus of both pre-pubertal males and females. In adult animals the number of serotonin 1A sub-type receptor mRNA positive cells was increased as a result of handling in the area 1 of Ammon's horn, area 4 of Ammon's horn and dentate gyrus of males, while it was decreased only in the area 4 of Ammon's horn of females. Furthermore, the number of serotonin sub-type 1A receptor immunopositive cells, as well as [(3)H]8-hydroxy-2(di-n-propylamino)tetralin binding sites was increased in the area 1 of Ammon's horn, area 4 of Ammon's horn and dentate gyrus of handled males, whereas it was decreased in these same brain areas in the handled females. We can thus infer that neonatal handling results in alterations in postsynaptic serotonergic neurotransmission, which may contribute to the sex dimorphic effects of handling as to the vulnerability toward depression-like behavior in response to chronic stressful stimuli.

Sexually dimorphic effects of the Lhx7 null mutation on forebrain cholinergic function.

It has been reported recently that mice lacking both alleles of the LIM-homeobox gene Lhx7, display dramatically reduced number of forebrain cholinergic neurons. In the present study, we investigated whether the Lhx7 mutation affects male and female mice differently, given the fact that gender differences are consistently observed in forebrain cholinergic function. Our results show that in adult male as well as female Lhx7 homozygous mutants there is a dramatic loss of choline acetyltransferase immunoreactive forebrain neurons, both projection and interneurons. The reduction of forebrain choline acetyltransferase immunoreactive neurons in Lhx7 homozygous mutants is accompanied by a decrease of acetylcholinesterase histochemical staining in all forebrain cholinergic neuron target areas of both male and female homozygous mutants. Furthermore, there was an increase of M1-, but not M2-, muscarinic acetylcholine receptor binding site density in the somatosensory cortex and basal ganglia of only the female homozygous mutant mice. Such an increase can be regarded as a mechanism acting to compensate for the dramatically reduced cholinergic input, raising the possibility that the forebrain cholinergic system in female mice may be more plastic and responsive to situations of limited neurotransmitter availability. Finally, our study provides additional data for the sexual dimorphism of the forebrain cholinergic system, as female mice appear to have a lower density of M1-muscarinic acetylcholine receptors in the striatal areas of the basal ganglia and a higher density of M2-muscarinic acetylcholine receptors, in a number of cortical areas, as well as the striatal areas of the basal ganglia.

Control of neuronal nitric oxide synthase and brain-derived neurotrophic factor levels by GABA-A receptors in the developing rat cortex.

Gamma-aminobutyric acid (GABA) plays an important morphogenetic role, acting through GABA-A receptors, which are depolarizing in the developing rat brain. Other molecules with major morphogenetic roles are the nitric oxide free radical (NO(.)) and brain-derived neurotrophic factor (BDNF), both of which are involved in the control of synaptic plasticity and apoptosis. In the present work, we investigated the effect of GABA-A receptor activation on neuronal NO(.) synthase (nNOS) and BDNF immunoreactivity in the developing cortex of 5-day-old rats. We also determined the effect of GABA-A receptor activation on phosphorylated cAMP-response element binding protein (pCREB) immunoreactivity in an effort to elucidate the molecular mechanisms involved. Our results show that activation of GABA-A receptors leads to increased numbers of nNOS, BDNF and pCREB, as well as nNOS-pCREB and BDNF-pCREB doubly immunopositive cells. This effect is abolished when L-type Ca(2+) channels are blocked. These results indicate that the following mechanism could be operating: depolarization following GABA-A receptor activation leads to opening of L-type voltage-gated calcium channels, resulting in an increased Ca(2+) influx, which in turn leads to phosphorylation and, thus, activation, of the transcription factor CREB; the phosphorylated CREB can then induce BDNF, as well as nNOS.

Effects of neonatal handling on basal and stress-induced monoamine levels in the male and female rat brain.

Neonatal handling has pervasive effects on the rat brain leading to increased ability to cope with and adapt to stressful stimuli. We determined the effects of neonatal handling on the dopaminergic and serotonergic system, in the male and female rat brain, under basal conditions before and after puberty and after short- and long-term forced swimming stress. Exposure of animals to neonatal handling resulted in sex-dependent changes in the concentration and turnover of monoamines in the different brain areas. In the prepubertal brain, the effect of neonatal handling was manifested as an increase in dopamine turnover in the females, particularly in the hypothalamus, an increase in serotonin levels and a decrease in its turnover in all three brain regions examined of both males and females. Certain of the handling-induced effects observed in the prepubertal brain were reversed in the postpubertal animals. Thus, in the postpubertal brain, the handling-induced changes in serotonin levels and its turnover observed in both sexes before puberty were abolished. On the other hand, the handling-induced increase in hypothalamic dopamine turnover was maintained. After exposure to short-term stress, the effect of handling was manifested on one hand as decreased striatal dopamine levels in the females, and decreased dopamine turnover in the hypothalamus of both males and females, and on the other, as increased serotonin levels in the hypothalamus. After exposure to long-term stress, handled females had decreased dopamine turnover in the hypothalamus and the striatum, but there was no effect of handling on the serotonergic system. Our results provide some neurobiological evidence supporting the determinant role of the mother-infant relationship in the development of psychopathology. Neonatal handling, which modifies normal mother-pup interactions, results in alterations in brain dopaminergic and serotonergic systems, both of which are involved in the etiopathogenesis of major psychoses. Exposure to either short- or long-term stress in adult life results in sex-dependent changes in brain monoamines, which are affected by handling thus making coping more efficient and rendering the stressful stimulus less noxious.

Sex differences in the effects of neonatal handling on the animal's response to stress and the vulnerability for depressive behaviour.

Neonatal handling is known to affect the programming of the hypothalamic-pituitary-adrenal axis and, as a result, the ability of the organism to respond to stress. We determined the effect of neonatal handling on the animal's response in three animal models of depression, as well as to either (a) acute or (b) chronic forced swimming stress. Neonatal handling resulted in a significant increase in the immobility time in the Porsolt forced swimming test in both sexes, and in the 8-hydroxy-2-(di-n-propylamino) tetralin-induced hypothermia in the males. On the other hand, handling had sex-dependent effects when animals were exposed to a chronic stressor. After exposure to chronic restraint stress, statistically more handled than non-handled females failed to adapt, while no such difference was found in the males. In the chronic forced swimming stress, handled males had shorter immobility times, and higher plasma corticosterone levels, while the opposite held true in the females. Furthermore, neonatal handling significantly decreased basal plasma corticosterone levels in both pre- and post-pubertal animals. Thus, the early experience of handling provides males with a greater capacity to actively face chronic stressors, while in the females it increases their susceptibility to express 'depressive' behaviour since they are unable to cope and adopt a 'passive, despaired' behaviour.

Protooncogene c-fos Involvement in the Molecular Mechanism of Rat Brain Sexual Differentiation.

Brain sexual differentiation is mediated through testosterone, which acts during the perinatal period in the form of both 5alpha-dihydrotestosterone and estradiol. In order to gain insight into the molecular mechanisms involved, we studied induction of c-fos, an index of functional neuronal activation, in the 2-day-old female rat brain after injection of a masculinizing dose of testosterone. Administration of testosterone resulted in induction of c-fos gene expression in the hypothalamus, as determined by Northern analysis. Following immunocytochemistry, we demonstrated an increase in the number of Fos-positive nuclei in the median and medial preoptic nucleus, the medial preoptic area extending to the lateral preoptic area, and the peri- and paraventricular area. In an effort to see whether testosterone acted as 5alpha-dihydrotestosterone or as estradiol, we injected either steroid and looked at fos induction. Estradiol mimicked the effect of testosterone, while 5alpha-dihydrotestosterone was without effect. Furthermore, injection of an estrogen receptor blocker, clomiphene, together with testosterone, abolished the testosterone-induced increase in Fos-positive nuclei, thus confirming the finding that testosterone induces c-fos by acting through estrogen receptors. Electrophoretic mobility shift assays showed that nuclear extracts from 2-day-old female hypothalamus contain a protein, most probably the estrogen receptor, which binds specifically to oligodeoxynucleotides with the sequence of either vitERE, the consensus estrogen-responsive element (ERE) found in the vittelogenin gene, or fosERE, the ERE found in the 3'-untranslated region of the mouse c-fos gene. This suggests that the effect of testosterone-derived estradiol on c-fos expression is a direct one, mediated by binding of estrogen receptors to an ERE in the c-fos gene-regulatory regions.

In utero radiation-induced apoptosis and p53 gene expression in the developing rat brain.

PURPOSE: This study addressed the question of the role of the p53 gene in prenatal low-dose radiation-induced apoptosis in the neuroepithelium, in an effort to elucidate molecular mechanisms involved in the extreme radiosensitivity of the developing brain. MATERIALS AND METHODS: Pregnant Wistar rats were exposed to a single dose of 10, 20 or 40 cGy of X-rays on day 15 or 17 of gestation. Animals were sacrificed 4 or 24h after exposure. Apoptosis was studied by gel electrophoresis of isolated DNA and in situ by the TUNEL reaction. Expression of the p53 gene was studied by immunocytochemistry and Western analysis, as well as Northern analysis, for the detection of the protein and mRNA respectively. RESULTS: In utero low-dose irradiation led to apoptosis and an increase of p53 gene expression in the developing rat brain. Apoptotic as well as p53 immunopositive cells were detected among proliferating, migratory and post-mitotic neurones in the developing neuroepithelium following prenatal irradiation, even after only l0 cGy. In addition to the p53 protein, p53 mRNA brain levels were also increased following prenatal irradiation. CONCLUSIONS: Low-dose prenatal irradiation of the developing brain led to p53 induction and cell death by apoptosis.