The effects of early life stress on the excitatory/inhibitory balance of the medial prefrontal cortex.

Aversive environmental conditions during early life are known to cause long-lasting social deficits, similar to those observed in patients with neurodevelopmental disorders. However, the mechanism of how early life stress can cause social deficits is not well understood. To clarify how being in an aversive environment during development affects sociability, we conducted various analyses focusing on the excitatory and inhibitory (E/I) balance in the medial prefrontal cortex (mPFC) and how it is related to social deficits, with young adult male rats that had been exposed to maternal separation (MS). In our MS procedure, part of the pups were separated from each dam for 3 h, twice a day, during postnatal days 2-20, and then were used for each analysis at 9 weeks old. We identified that MS mainly reduced pre- and post-synaptic protein expression of inhibitory neurons in the mPFC, and that decreased the number of GAD67-positive interneurons and inhibitory synapses in the mPFC. Furthermore, MS impaired social behavior related to social recognition, which is closely linked to the mPFC, in the three-chamber sociability and social novelty test (3-CST). With relation to this social deficit, immunohistological analysis revealed that c-fos-positive cells in the mPFC of rats exposed to MS decreased during the 3-CST. Considering that inhibitory neurons in the mPFC play a role in synchronizing neural activation for information processing, our findings demonstrate that MS-induced E/I imbalance associated with cell activity in the mPFC leads to deficits in social recognition.

Early postnatal repeated maternal deprivation causes a transient increase in OMpg and BDNF in rat cerebellum suggesting precocious myelination.

Repetitive maternal deprivation (MD) of neonatal rats during early life is known as one of the strongest stressors to pre-weaned animals. There is increasing evidence that the cerebellum is involved in cognition and emotion. In the present study, we examined how neurotrophic factors and myelin-associated molecules and their receptors (NGF, BDNF, OMgp, TrkA, TrkB, p75 NTR, and NgR) in the cerebellum are affected by early postnatal maternal separation. Rat pups were separated from their mothers for 3h/day during postnatal days (PND) 10-15. At PND 16 and 30, the levels of mRNA and protein in the cerebellum were determined using real-time PCR and Western blot analysis. Cerebellar mRNA and protein levels of BDNF, TrkB, and OMgp were significantly increased in MD rats at PND 16. However, by PND 30 these variables normalized to control levels. In contrast, the levels of mRNA and protein for NGF, TrkA, p75 NTR, and NgR were unchanged at both ages examined. Transient enhancement of neurotrophic system and myelin-associated molecule expression may cause interference of normal development of the cerebellum such as precocious myelination, which may lead to functional and cognitive deficits later in life.

Short-term ethanol exposure causes imbalanced neurotrophic factor allocation in the basal forebrain cholinergic system: a novel insight into understanding the initial processes of alcohol addiction.

Alcohol ingestion affects both motor and cognitive functions. One brain system that is influenced by ethanol is the basal forebrain (BF) cholinergic projection system, which projects to diverse neocortical and limbic areas. The BF is associated with memory and cognitive function. Our primary interest is the examination of how regions that receive BF cholinergic projections are influenced by short-term ethanol exposure through alterations in the mRNA levels of neurotrophic factors [nerve growth factor/TrkA, brain-derived neurotrophic factor/TrkB, and glial-derived neurotrophic factor (GDNF)/GDNF family receptor α1]. Male BALB/C mice were fed a liquid diet containing 5 % (v/v) ethanol. Pair-fed control mice were maintained on an identical liquid diet, except that the ethanol was isocalorically substituted with sucrose. Mice exhibiting signs of ethanol intoxication (stages 1-2) were used for real-time reverse transcription-polymerase chain reaction analyses. Among the BF cholinergic projection regions, decreased levels of GDNF mRNA and increased levels of TrkB mRNA were observed in the basal nucleus, and increased levels of TrkB mRNA were observed in the cerebral cortex. There were no significant alterations in the levels of expression of relevant neurotrophic factors in the septal nucleus and hippocampus. Given that neurotrophic factors function in retrograde/anterograde or autocrine/paracrine mechanisms and that BF cholinergic projection regions are neuroanatomically connected, these findings suggested that an imbalanced allocation of neurotrophic factor ligands and receptors is an initial phenomenon in alcohol addiction. The exact mechanisms underlying this phenomenon in the BF cholinergic system are unknown. However, our results provide a novel notion for the understanding of the initial processes in alcohol addiction.

Ameliorative effects of yokukansan on behavioral deficits in a gerbil model of global cerebral ischemia.

The aim of this study was to investigate the neuroprotective effects of yokukansan, a traditional Kampo medicine, on the behavioral dysfunction induced by cerebral ischemia/reperfusion injury in gerbils. Gerbils were treated with yokukasan by oral gavage for 30 days, once per day, until the day before induction of ischemia, which was induced by occluding the bilateral common carotid artery for 5 min. The effects of yokukansan (50, 100 and 300 mg/kg) were examined by measuring neuronal damage and behavioral deficits (locomotor activity, 8-arm radial maze task). The anti-inflammatory and anti-oxidant properties of yokukansan were also examined. Administration of yokukansan at 300 mg/kg significantly reduced hippocampal neuronal death after brain ischemia, inhibited the ischemia-induced inflammatory response and DNA oxidative damage. Yokukansan also reduced ischemia-induced locomotor hyperactivity and improved memory impairment. These findings suggest that yokukansan can inhibit the inflammatory response, oxidative damage and subsequent neuronal death induced by cerebral ischemia/reperfusion injury, and also can contribute to improvement in neurological deficits following such injury.

Early postnatal maternal separation causes alterations in the expression of ß3-adrenergic receptor in rat adipose tissue suggesting long-term influence on obesity.

The effects of early postnatal maternal deprivation on the biological characteristics of the adipose tissue later in life were investigated in the present study. Sprague-Dawley rats were classified as either maternal deprivation (MD) or mother-reared control (MRC) groups. MD was achieved by separating the rat pups from their mothers for 3h each day during the 10-15 postnatal days. mRNA levels of mitochondrial uncoupling protein 1 (UCP-1), ß3-adrenergic receptor (ß3-AR), and prohibitin (PHB) in the brown and white adipose tissue were determined using real-time RT-PCR analysis. UCP-1, which is mediated through ß3-AR, is closely involved in the energy metabolism and expenditure. PHB is highly expressed in the proliferating tissues/cells. At 10 weeks of age, the body weight of the MRC and MD rats was similar. However, the levels of the key molecules in the adipose tissue were substantially altered. There was a significant increase in the expression of PHB mRNA in the white adipose tissue, while the ß3-AR mRNA expression decreased significantly, and the UCP-1 mRNA expression remained unchanged in the brown adipose tissue. Given that these molecules influence the mitochondrial metabolism, our study indicates that early postnatal maternal deprivation can influence the fate of adipose tissue proliferation, presumably leading to obesity later in life.

Stage- and region-specific cyclooxygenase expression and effects of a selective COX-1 inhibitor in the mouse amygdala kindling model.

In an attempt to elucidate the involvement of cyclooxygenase (COX) enzymes, particularly COX-1, in epileptogenesis, the localization of COX-1 and COX-2 expression in the mouse kindling model was analyzed by immunohistochemistry. COX-2 was predominantly observed in brain neurons and its concentration in the hippocampus increased with progressing seizures, as reported previously. COX-1 was predominant in microglia and its concentration was also enhanced in the hippocampus and areas around the third ventricle during the progression of seizures. These regions are thought to play an important role in the propagation of limbic seizures. Moreover, the administration of SC-560 (a selective COX-1 inhibitor) or indomethacin (a non-selective COX inhibitor) retarded the progress of seizures. Although the precise function of COX-positive cells in microglia and elsewhere is not clear, our results suggest that COX-1 as well as COX-2 may be involved in epileptogenesis, and that certain COX inhibitors can potentially prevent the occurrence of seizures.

Early postnatal ethanol exposure induces fluctuation in the expression of BDNF mRNA in the developing rat hippocampus.

Effects of early postnatal ethanol exposure on brain-derived neurotrophic factor (BDNF) mRNA expression in the rat hippocampus were investigated. Wistar rats were assigned to either ethanol treatment (ET) separation control (SC) or mother-reared control (MRC) groups. Ethanol exposure was achieved by a vapor inhalation method for 3 hours a day between postnatal days (PND) 1015. On PND 16, 20, 30, and 60, the expression of BDNF mRNA in the hippocampus was determined using real-time RT-PCR analysis. There was a significant age-related increase in the BDNF mRNA expression between PND 3060 in MRC animals. The BDNF mRNA expression in ET rats was increased at both PND 16 and 20 and thereafter decreased at PND 60 compared to SC animals. Such age-related fluctuation in the expression of BDNF mRNA differed from that of MRC animals. The exact functional implications, if any, of these ethanol-induced changes in BDNF mRNA expression remain unknown although it can be speculated that they may have an effect on the behaviors known to be influenced by the hippocampal formation.

Ethanol neurotoxicity and dentate gyrus development.

Maternal alcohol ingestion during pregnancy adversely affects the developing fetus, often leading to fetal alcohol syndrome (FAS). One of the most severe consequences of FAS is brain damage that is manifested as cognitive, learning, and behavioral deficits. The hippocampus plays a crucial role in such abilities; it is also known as one of the brain regions most vulnerable to ethanol-induced neurotoxicity. Our recent studies using morphometric techniques have further shown that ethanol neurotoxicity appears to affect the development of the dentate gyrus in a region-specific manner; it was found that early postnatal ethanol exposure causes a transitory deficit in the hilus volume of the dentate gyrus. It is strongly speculated that such structural modifications, even transitory ones, appear to result in developmental abnormalities in the brain circuitry and lead to the learning disabilities observed in FAS children. Based on reports on possible factors deciding ethanol neurotoxicity to the brain, we review developmental neurotoxicity to the dentate gyrus of the hippocampal formation.

The effect of prenatal X-irradiation on the developing cerebral cortex of rats. II: A quantitative assessment of glial cells in the somatosensory cortex.

The developing central nervous system is known to be highly vulnerable to X-irradiation. Although glial cells are involved in various brain functions, knowledge on the effects of X-irradiation on glial cells is limited. Therefore, the purpose of the present study was to evaluate the effects of prenatal X-irradiation on glial cells. Pregnant Wistar rats were exposed to X-irradiation at a dose of 1.0 Gy on day 15 of gestation. Their offspring were examined at 7 weeks of age. The forebrain weight of X-irradiated rats was significantly lower than that of the age-matched controls. Histological quantification with stereology of the somatosensory cortex (SC) revealed no significant difference in the numerical density of glial cells between the X-irradiated and control rats. However, the glial cells in the X-irradiated animals had significantly larger nuclear size. We had previously reported that a similar X-irradiation paradigm resulted in no significant change in the numerical density of neurons in the SC. According to the results of the present study, there were no significant differences in the glial cell-to-neuron ratios between the X-irradiated and control animals. Taken together, it is speculated that prenatal X-irradiation has an equal effects on the numerical densities of glial cells and neurons.

Unilateral ibotenic acid lesions of the prefrontal cortex reduce rotational behavior in 6-hydroxydopamine-lesioned rats.

Rats with 6-hydroxydopamine (6-OHDA)-induced lesions of the substantia nigra are used as a model of Parkinson.s disease (PD), and these "lesioned" rats exhibit a rotational behavior when further injected with apomorphine (APO). We examined whether lesions in the prefrontal cortex (PFC) could modify the rotational behavior in PD model rats. Rats initially received unilateral lesions of the substantia nigra by 6-OHDA injection, and then their rotational behavior was measured. Two PFC lesions were achieved by intracerebral infusions of ibotenic acid, followed by measurement of APO-induced rotation. Rotation was reduced by approximately 30% after PFC injury. The PFC may have functional influence on the basal ganglia and may be involved in the pathophysiology of the rotational behavior of PD model rats.

Vascular changes in the rat brain during chronic hypoxia in the presence and absence of hypercapnia.

Changes in brain vascularity in adult rats during adaptation to chronic normobaric hypoxia with or without elevated CO(2) were morphometrically investigated. Immunohistochemistry with anti-rat endothelial cell antigen (RECA-1) antibody was carried out for the vascular analysis. After the rats were subjected to hypoxia for 2 to 8 weeks (wks)(10 percent O(2) in N(2)), the total area of blood vessels was measured in 6 brain regions. After 2 wks of hypoxia, the blood vessel area was found to be significantly increased in the frontal cortex, striatum, hippocampus, thalamus, cerebellum, and medulla oblongata, by 44% , 96% , 65% , 50% , 102% and 97% , respectively. The ratio of large vessels with an area > 500 micro m(2) was also increased in all brain regions. Hypoxic adaptation in brain vascularity did not change during 8 wks of hypoxia, and the hypoxia-induced levels measured in the vasculature returned to control levels 2 wks after the termination of hypoxia in areas of the brain other than the cortex and thalamus. In addition, hypoxia-induced changes in terms of the total vascular area and vessel size distribution were significantly inhibited by the elevation in CO(2), whereas chronic hypercapnia without hypoxia had no effect on brain vascularity. These findings suggested that adaptations in brain vascularity in response to hypoxia are rapidly induced, and there are regional differences in the reversibility of such vascular changes. Carbon dioxide is a potent suppressor of hypoxia-induced vascular changes, and may play an important role in vascular remodeling during the process of adaptation to chronic hypoxia.

Mechanisms of the anti-ischemic effect of vagus nerve stimulation in the gerbil hippocampus.

The neuroprotective mechanisms of cervical vagus nerve stimulation (VNS) in transient ischemia were investigated. Left VNS (0.4 mA, 40 Hz) was performed during 5 min ischemia in gerbils. About 50% of the hippocampal neurons were rescued from ischemic insult by VNS, and this effect was prevented by transection of the vagus nerve centrally to the site of cervical stimulation. VNS significantly attenuated both ischemia-induced glutamate release and transient increase of hippocampal blood flow during reperfusion. Hyperemia as well as excessive glutamate release after ischemia is regarded as an important factor in ischemic brain damage as it leads to generate considerable reactive oxygen species. Thus, VNS might protect neurons from ischemia-induced glutamate excitotoxicity and reperfusion injury via the afferent path-way of the vagus.

[The influence of severe long-term exercise on the mouse hippocampus].

To determine whether severe long-term exercise affects on the brain, we investigated the mice brain after 12-week treadmill exercise. The mice (ddN, male, 25-35 g in body weight) were divided into severe, mild, and non-exercise group. Mice in severe groups ran on a treadmill at a speed of 25 m/min for 12 weeks and mice in mild group ran on a treadmill at a speed of 10 m/min for 12 weeks. The mice were killed by transcardial perfusion with 0.1 M phosphate-buffered saline (PBS) followed by ice-cold 4% paraformaldehyde in 0.1 M PB. The another sets of mice were fixed by 2.5% glutaraldehyde-2% osmium tetroxide for electromicroscope (EM). The brains were serially sectioned in the coronal plane at a thickness of 20-microns with a vibratome and then processed for histology, by means of hematoxylin-eosin (HE) staining and immunohistochemistry. Fifty % of mice in severe exercise showed hyperchromatic and shrunken nerve cells with nuclear pyknosis (dark neuron) in the hippocampus, but not in mild exercise and non-exercise groups. The immunoreactivity of microtuble associated protein-2 (MAP-2) decreased, while the heat-shock protein/cognate 70 (HSP/C 70) increased in the hippocampus of severe exercise group. Many destroyed mitochondria were observed in dark neurons by Electron micrograph. These findings suggested that severe long-term exercise might damage hippocampal neurons.