Perinatal Exposure of Bisphenol A Differently Affects Dendritic Spines of Male and Female Grown-Up Adult Hippocampal Neurons.

Perinatal exposure to Bisphenol A (BPA) at a very low dose may modulate the development of synapses of the hippocampus during growth to adulthood. Here, we demonstrate that perinatal exposure to 30 µg BPA/kg per mother's body weight/day significantly altered the dendritic spines of the grownup rat hippocampus. The density of the spine was analyzed by imaging of Lucifer Yellow-injected CA1 glutamatergic neurons in adult hippocampal slices. In offspring 3-month male hippocampus, the total spine density was significantly decreased by BPA exposure from 2.26 spines/µm (control, no BPA exposure) to 1.96 spines/µm (BPA exposure). BPA exposure considerably changed the normal 4-day estrous cycle of offspring 3-month females, resulting in a 4∼5 day estrous cycle with 2-day estrus stages in most of the subjects. In the offspring 3-month female hippocampus, the total spine density was significantly increased by BPA exposure at estrus stage from 2.04 spines/µm (control) to 2.25 spines/µm (BPA exposure). On the other hand, the total spine density at the proestrus stage was moderately decreased from 2.33 spines/µm (control) to 2.19 spines/µm (BPA exposure). Thus, after the perinatal exposure to BPA, the total spine density in males became lower than that in females. Concerning the BPA effect on the morphology of spines, the large-head spine was significantly changed with its significant decrease in males and moderate change in females.

Prenatal bisphenol A exposure is associated with medial amygdala neuron hyperresponsiveness to predator odor in rats.

Perinatal exposure to bisphenol A (BPA) causes several alterations in brain function and behavior. In previous studies, we showed that prenatal treatment with low-level BPA impaired gender-specific behavior, enhanced depression-like behavior, and augmented behavioral responses to predator odor in rats. On this premise, we hypothesized that BPA-treated rats were more susceptible to predator odor stress. To test the potential neural mechanism underlying this effect, we conducted an electrophysiological study of neurons in the medial amygdala-a regional component of the olfactory pathway with high estrogen and androgen receptor expression, and thus a potential target of BPA-in rats exposed to BPA. Extracellular recordings were obtained during the presentation of 3 plant odors and 3 predator odorants. Odor-responsive neurons in BPA-exposed rats showed greater activity in response to fox odor than did those in control rats. This finding complements the results of our previous behavioral study in which BPA-exposed rats exhibited enhanced avoidance behavior in response to fox odor. Given the close relationship between olfactory signaling and the stress response system, we suspect that BPA modifies the olfactory pathway at the level of the medial amygdala and thus modulates the corresponding stress response.

The functional link between tail-pinch-induced food intake and emotionality and its possible role in stress coping in rats.

Tail pinch facilitates eating in rats. We investigated an unidentified link between tail-pinch-induced eating behavior and individual emotionality in male Sprague-Dawley rats. Anxiety-like behavior was assessed on the elevated plus maze (EPM) and in the open field test (OFT). Tail-pinch-induced eating was observed as follows: After a 30-min habituation period, the tail pinch was applied for 5 min, followed by a 30-min recovery period. During the habituation and recovery periods, rats were allowed to access food ad libitum. During the recovery period, 14 of 24 rats ate more food than during the habituation period. Thus, we named them "high responders" and the others as "low responders". The food intake was significantly greater, while the times spent in the open arms in the EPM and in the center area in the OFT were significantly shorter in high responders than in low responders. This result suggests that the rats consuming more food after mild stress have higher anxiety.

Water spray-induced grooming is negatively correlated with depressive behavior in the forced swimming test in rats.

Rodents show grooming, a typical self-care behavior, under stress and non-stress conditions. Previous studies revealed that grooming under stress conditions such as the open-field test (OFT) or the elevated plus-maze test (EPM) is associated with anxiety, but the roles of grooming under non-stress conditions are not well understood. Here, we examined spray-induced grooming as a model of grooming under a non-stress condition to investigate the relationship between this grooming and depression-like behavior in the forced swim test (FST) and tail suspension test, and we compared spray-induced grooming with OFT- and EPM-induced grooming. The main finding was that the duration of spray-induced grooming, but not that of OFT/EPM-induced grooming, was negatively correlated with the duration of immobility in the FST, an index of depression-like behavior. The results suggest that spray-induced grooming is functionally different from the grooming in the OFT and EPM and is related to reduction of depressive behavior.

Development of a direct exposure system for studying the mechanisms of central neurotoxicity caused by volatile organic compounds.

Many volatile organic compounds (VOCs) used in work places are neurotoxic. However, it has been difficult to study the cellular mechanisms induced by a direct exposure to neurons because of their high volatility. The objective of this study was to establish a stable system for exposing brain slices to VOCs. With a conventional recording system for brain slices, it is not possible to keep a constant bath concentration of relatively highly volatile solvents, e.g. 1-bromopropane (1-BP). Here we report a new exposure system for VOCs that we developed in which a high concentration of oxygen is dissolved to a perfused medium applying a gas-liquid equilibrium, and in which the tubing is made of Teflon, non adsorptive material. Using our system, the bath concentration of the perfused 1-BP remained stable for at least 2 h in the slice chamber. Both 6.4 and 2.2 mM of 1-BP did not change the paired-pulse response, but fully suppressed long-term potentiation in the dentate gyrus (DG) of hippocampal slices obtained from rats, suggesting that 1-BP decreases synaptic plasticity in the DG at the concentrations tested. Our new system can be applicable for investigating the underlying mechanisms of the neurotoxicity of VOCs at the cellular level.

Prenatal low-dose bisphenol A enhances behavioral responses induced by a predator odor.

Bisphenol A (BPA) is an environmental endocrine disrupter (EED). Previous studies by our group showed that pre- and postnatal administration of low-level BPA induced depression-like behavior in rats. In this study, we evaluated the effects of prenatal BPA on behavioral responses to a predator odor by using a novel cross-form apparatus consisting of 4 plastic chambers. On the first day, nothing was placed into the chambers (Session 1). On the second day, a predator odor (fox odor) was located in separate chambers at 2 opposite corners of the apparatus (Session 2). Pregnant Wistar rats were exposed to low-dose BPA (less than the reference dose) during the 7 days just before birth, and the offspring of the treated rats were evaluated as adults. The locomotor activity and avoidance response of each rat on both test days were compared. The control and BPA groups showed reduced locomotor activity in the presence of the predator odor, but the odor-avoidance response was significant only in the BPA rats. The BPA-exposed rats were obviously sensitive to the predator odor. These results suggest that prenatal BPA exposure has an amplifying effect on avoidance responses to predator odor stress.

Brief neonatal handling alters sexually dimorphic behaviors in adult rats.

Several effects of neonatal handling on brain and behavior have been reported. We investigated the effects of neonatal handling on behaviors that have been shown to be sexually dimorphic in rats using an open-field test. "Gender differences" were observed in locomotor activity, exploratory behavior and grooming in the handled group. However, clear gender differences in these behaviors were not observed in the non-handled group. Our findings show that brief daily handling sessions (~ 1 min) in the first 2 weeks of postnatal life increased locomotor activity and exploratory behavior, and that these effects were more pronounced in females. Moreover, many rats in the non-handling group exhibited an increase in defecation relative to the handling group during the 10-min observation period. This suggests that the non-handling group experienced more stress in response to the novel open-field arena, and that this resulted in the absence of gender differences. Notably, this anxiety-related response was attenuated by neonatal handling. Our study underscores the impact of brief neonatal handling on sexually dimorphic behaviors, and indicates that caution should be exercised in controlling for the effects of handling between experimental groups, particularly in neurotoxicological studies that evaluate gender differences.

Postnatal exposure to low-dose bisphenol A influences various emotional conditions.

Bisphenol A (BPA) is one of the 'environmental endocrine disrupters' (EEDs) released by plastics and resin known to interfere with hormonal responses. In this study, female Wistar rats were exposed to low-dose BPA (24 µg/kg/day) during 7 days after giving birth. The male and female offspring, exposed to the BPA through lactation, were evaluated using an open field test (OFT) at the age of six weeks, an elevated plus maze test (EPM) at seven weeks, and a forced swimming test (FST) at nine weeks. The OFT indicated that females were more active than males, and that BPA selectively increased rearing duration in males, thereby eliminating the gender effect. The results of EPM showed that BPA did not enhance the anxiety-like action; rather, it was associated with an anxiolytic-like action in females. In the FST, not only there was an increase in the immobility time, but also there was reduction of latency observed in BPA rats. It indicated that the depression-like responses were clearly enhanced by the postnatal exposure. Altogether, these data suggest that low-dose BPA ingested by neonates through breastfeeding may cause persistent aberrant behaviors that are relevant to emotions.

Multisensory interaction mediates the social transmission of avoidance in rats: dissociation from social transmission of fear.

Social interaction enables animals to transmit various types of sensory information that can modulate learned avoidance behavior and fear responses, which are important to survival. We previously reported that, under a passive avoidance paradigm, avoidance behavior is facilitated when a rat observes another rat (demonstrator) receiving a shock when performing a specific behavior. However, the sensory mechanisms underlying this 'social facilitation of avoidance' are not well understood. The present study examined the role of sensory pathways for social transmission of avoidance, focusing on the olfactory and visual systems. The olfactory ability of observer rats was blocked by an intranasal application of ZnSO4, and their visual ability was blocked by an opaque partition placed between observer and demonstrator rats. We found that blocking either olfactory or visual input drastically diminished the social transmission of avoidance. Interestingly the social transmission of fear responses remained intact even when olfactory or visual information was blocked. These results indicate that the social transmission of avoidance is mediated not by any single sensory modality but by multisensory interaction in rats, suggesting a distinct sensory mechanism from that underlying the social transmission of fear.

Penicillin-induced epileptiform activity elevates focal brain temperature in anesthetized rats.

To elucidate a relationship between changes in focal brain temperature and severity of abnormal brain activity, epileptiform discharges and behavioral seizures were induced by Penicillin G in anesthetized rats, and focal brain-temperature was measured. Penicillin G was injected into the right primary sensorimotor cortex (400IU/µl). After the injection, epileptiform discharges induced a temperature increase gradually by 0.65±0.24°C. Moreover, when behavioral seizures were induced by reducing the anesthesia level, the temperature was raised by 0.26±0.22°C. These results suggest that elevation of the focal brain temperature is associated with the severity of epileptic activity.

Neurotoxin A2NTX Blocks Fast Inhibitory and Excitatory Transmitter Release From Presynaptic Terminals.

Our recent study showed a possibility that newly developed A2 type botulinum toxin (A2NTX) inhibits both spontaneous and evoked transmitter release from inhibitory (glycinergic or GABAergic) and excitatory (glutamatergic) nerve terminals using rat spinal sacral dorsal commissural nucleus neurons. In the present study, to determine the modulatory effect of A2NTX on glycinergic and glutamatergic release probabilities, we tested the effects of A2NTX on a single inhibitory or excitatory nerve ending adherent to a dissociated neuron that was activated by paired-pulse stimuli by using the focal electrical stimulation technique. The results of the present paired-pulse experiments showed clearly that A2NTX enhanced paired-pulse facilitation of evoked glycinergic inhibitory postsynaptic currents and glutamatergic excitatory postsynaptic currents and increased the failure rate (Rf) of the first postsynaptic currents (P1) and both the responses. These effects of A2NTX on the amplitude and Rf of the P1 and the second postsynaptic currents (P2) and paired-pulse ratio were rescued by application of 4-aminophthalimide. In summary, the present results showed that A2NTX acts purely presynaptically and inhibits the release machinery of transmitters such as glycine and glutamate, and the transmitter release machinery became less sensitive to intracellular free-Ca2+ in A2NTX poisoned nerve terminals.

Neurotoxin A2NTX blocks fast inhibitory and excitatory transmitter release from presynaptic terminals.

Our recent study showed a possibility that newly developed A2 type botulinum toxin (A2NTX) inhibits both spontaneous and evoked transmitter release from inhibitory (glycinergic or GABAergic) and excitatory (glutamatergic) nerve terminals using rat spinal sacral dorsal commissural nucleus neurons. In the present study, to determine the modulatory effect of A2NTX on glycinergic and glutamatergic release probabilities, we tested the effects of A2NTX on a single inhibitory or excitatory nerve ending adherent to a dissociated neuron that was activated by paired-pulse stimuli by using the focal electrical stimulation technique. The results of the present paired-pulse experiments showed clearly that A2NTX enhanced paired-pulse facilitation of evoked glycinergic inhibitory postsynaptic currents and glutamatergic excitatory postsynaptic currents and increased the failure rate (Rf) of the first postsynaptic currents (P(1)) and both the responses. These effects of A2NTX on the amplitude and Rf of the P(1) and the second postsynaptic currents (P(2)) and paired-pulse ratio were rescued by application of 4-aminophthalimide. In summary, the present results showed that A2NTX acts purely presynaptically and inhibits the release machinery of transmitters such as glycine and glutamate, and the transmitter release machinery became less sensitive to intracellular free-Ca(2+) in A2NTX poisoned nerve terminals.

Ca2+/calmodulin-dependent protein kinase II and protein kinase C activities mediate extracellular glucose-regulated hippocampal synaptic efficacy.

To define how extracellular glucose levels affect synaptic efficacy and long-term potentiation (LTP), we evaluated electrophysiological and neurochemical properties in hippocampal CA1 regions following alterations in glucose levels in the ACSF. In rat hippocampal slices prepared in ACSF with 3.5mM glucose, fEPSPs generated by Schaffer collateral/commissural stimulation markedly increased when ACSF glucose levels were increased from 3.5 to 7.0mM. The paired-pulse facilitation reflecting presynaptic transmitter release efficacy was significantly suppressed by elevation to 7.0mM glucose because of potentiation of the input-output relationship (I/O relationship) of fEPSPs by single pulse stimulation. Prolonged potentiation of fEPSPs by elevation to 7.0mM glucose coincided with increased autophosphorylation both of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) and protein kinase Cα (PKCα). The increased I/O relationship of fEPSPs was also associated with markedly increased synapsin I phosphorylation by CaMKII. Transmitter-evoked postsynaptic currents were also measured in CA1 neurons by electrophoretical application of NMDA and AMPA to the apical dendrites of pyramidal neurons. NMDA- and AMPA-evoked currents were significantly augmented by elevation to 7.0mM. Notably, high frequency stimulation of the Schaffer collateral/commissural pathway failed to induce LTP in the CA1 region at 3.5mM glucose but LTP was restored dose-dependently by increasing glucose levels to 7.0 and 10.0mM. LTP induction in the presence of 7.0mM glucose was closely associated with further increases in CaMKII autophosphorylation without changes in PKCα autophosphorylation. Taken together, CaMKII and PKC activation likely mediate potentiation of fEPSPs by elevated glucose levels, and CaMKII activity is also associated with LTP induction in the hippocampal CA1 region.

Lesions of the medial prefrontal cortex enhance social modulation of avoidance.

Our previous study showed that social interactions induce inhibitory and facilitatory modulations of avoidance behavior under both safe and dangerous situations in rats. To understand the neural mechanisms for these phenomena, we investigated the effects of bilateral lesions of the medial prefrontal cortex (mPFC) on the social modulation of avoidance behavior. We found that the lesions did not impair but actually augmented both social inhibition and facilitation of avoidance. These results suggest that the mPFC in rodents plays a suppressive role in the social modulation of avoidance.

Prandial increase of leptin in the brain activates spatial learning and memory.

Leptin is well known to be involved in the control of feeding, thermogenesis, reproduction and neuroendocrine functions through its actions on the rodents hypothalamic receptors. Leptin facilitated the presynaptic transmitter release and postsynaptic sensitivity to the transmitters in the hippocampal CA1 neurons. Thus long-term potentiation (LTP) and the phosphorylation of Ca(2+)/calmodulin protein kinase II (CaMKII) were facilitated in the CA1 neurons. Therefore behavioral performance related to spatial learning and memory was improved by leptin in vivo applications. We also investigated LTP and spatial learning and memory function in two leptin receptor-deficient rodents, Zucker fatty rats and db/db mice. The CA1 region of both strains showed impairments of LTP and leptin application did not improve these impairments. These strains showed lower basal levels of CaMKII activity in the CA1 region than the respective controls, and the levels did not respond to a brief tetanic stimulation. These strains also showed impaired spatial learning and memory. The present studies suggest that leptin signaling in the brain may have important implications for cognitive function.

Social transmission of avoidance behavior under situational change in learned and unlearned rats.

BACKGROUND: Rats receive information from other conspecifics by observation or other types of social interaction. Such social interaction may contribute to the effective adaptation to changes of environment such as situational switching. Learning to avoid dangerous places or objects rapidly occurs with even a single conditioning session, and the conditioned memory tends to be sustained over long periods. The avoidance is important for adaptation, but the details of the conditions under which the social transmission of avoidance is formed are unknown. We demonstrate that the previous experience of avoidance learning is important for the formation of behaviors for social transmission of avoidance and that the experienced rats adapt to a change of situation determined by the presence or absence of aversive stimuli. We systematically investigated social influence on avoidance behavior using a passive avoidance test in a light/dark two-compartment apparatus. METHODOLOGY/PRINCIPAL FINDINGS: Rats were divided into two groups, one receiving foot shocks and another with no aversive experience in a dark compartment. Experienced and inexperienced rats were further divided into subjects and partners. In Experiment 1, each subject experienced (1) interaction with an experienced partner, (2) interaction with an inexperienced partner, or (3) no interaction. In Experiment 2, each subject experienced interaction with a partner that received a shock. The entering latency to a light compartment was measured. The avoidance behavior of experienced rats was inhibited by interaction with inexperienced or experienced partners in a safely-changed situation. The avoidance of experienced rats was reinstated in a dangerously-changed situation by interaction with shocked rats. In contrast, the inexperienced rats were not affected by any social circumstances. CONCLUSIONS/SIGNIFICANCE: These results suggest that transmitted information among rats can be updated under a situational change and that the previous experience is crucial for social enhancement and inhibition of avoidance behavior in rats.

Prandial increases of leptin and orexin in the brain modulate spatial learning and memory.

Leptin is well known to be involved in the inhibition of feeding, hermogenesis, reproduction and neuroendocrine functions through its actions on the rodents hypothalamic receptors. Leptin facilitated the presynaptic transmitter release and postsynaptic sensitivity to the transmitters in the hippocampal CA1 neurons. Thus long-term potentiation (LTP) and the phosphorylation of Ca2+/calmodulin protein kinase II (CaMK II) were facilitated in the CA1 neurons. Therefore behavioral performance related to spatial learning and memory was improved by leptin in vivo applications. Orexin-A produced by glucose-sensitive neurons in the lateral hypothalamic area (LHA) and released during food intake facilitates feeding. Orexin-A suppressed LTP and CaMK II phosphorylation without affecting the presynaptic transmitter release. Therefore behavioral performance on learning and memory was impaired. The present studies suggest that leptin and orexin signalings in the brain may have important implications for cognitive function.

Dynamical properties of the two-process model for sleep-wake cycles in infantile autism.

The two-process model is a scheme for the timing of sleep that consists of homeostatic (Process S) and circadian (Process C) variables. The two-process model exhibits abnormal sleep patterns such as internal desynchronization or sleep fragmentation. Early infants with autism often experience sleep difficulties. Large day-by-day changes are found in the sleep onset and waking times in autistic children. Frequent night waking is a prominent property of their sleep. Further, the sleep duration of autistic children is often fragmented. These sleep patterns in infants with autism are not fully understood yet. In the present study, the sleep patterns in autistic children were reproduced by a modified two-process model using nonlinear analysis. A nap term was introduced into the original two-process model to reproduce the sleep patterns in early infants. The nap term and the time course of Process S are mentioned in the present study. Those parameters led to bifurcation of the sleep-wake cycle in the modified two-process model. In a certain range of these parameter sets, a small external noise was amplified, and an irregular sleep-wake cycle appeared. The short duration of sleep led to another irregular sleep onset or waking. Consequently, an irregular sleep-wake cycle appeared in early infantile autism.

Categorization of biologically significant objects, food and gender, in rhesus monkeys. I. Behavioral study.

Macaque monkeys have a highly evolved visual system comparable to that of humans. One of the important visual functions is performing discriminations among biologically significant objects such as food or heterosexual partners. In the present study, we examined whether rhesus monkeys could categorize two-dimensional images related to food or gender using a visual discrimination task. Three rhesus monkeys were trained to make distinctions of food from non-food items, and between male and female monkeys, using 60 or more different pictures of each category. After more than 9 months of training, the monkeys discriminated a variety of foods from non-food and different males from females with more than 80% accuracy, even when the stimuli were used for the first time or presented only once in a session. The proportion of correct responses and response latencies showed better performance in discrimination of food/non-food than that of gender. The results suggest that rhesus monkeys are able to perform visual discrimination of highly abstract biologically significant categories with better performance in a food-related category than a gender-related one, using two-dimensional visual information.

Role of substantia innominata in cerebral blood flow autoregulation.

Ascending projections from the substantia innominata (SI) may have an important role in the regulation of cerebral blood flow (CBF). However, several reports have suggested that unilateral lesion of the SI does not affect CBF autoregulation. On the other hand, it is also reported that several cortical and subcortical functions may be regulated not only by ipsilateral SI, but also by contralateral SI. Thus, the objective of this study is to test the hypothesis that bilateral lesions of the SI affect CBF autoregulation. Experiments were performed on anesthetized male Sprague-Dawley rats. Ibotenic acid or physiological saline was microinjected into bilateral SI. Rats were classified into four groups as follows: bilateral SI lesion rats (ibotenic acid was injected bilaterally), left or right SI lesion rats (ibotenic acid was injected into the unilateral SI and saline into the contralateral SI), and control rats (saline was injected bilaterally). Ten days after injection, CBF in the left frontal cortex was measured by laser-Doppler flowmetry during stepwise controlled hemorrhagic hypotension. In bilateral SI lesion rats, CBF was started to decrease significantly at 80 mm Hg (p<0.01). In the other three groups, CBF was well maintained until 50 mm Hg. Changes in CBF through stepwise hypotension in bilateral SI lesion rats were significantly different from the other groups (p<0.01). These results suggest that bilateral SI regulates cortical vasodilator mechanisms during hemorrhagic hypotension. Under unilateral SI lesion, some compensatory effects from the contralateral SI may maintain CBF autoregulation.