Takayuki Negishi

Attention-deficit and hyperactive neurobehavioural characteristics induced by perinatal hypothyroidism in rats.

Thyroid hormone is essential for the proper development of the mammalian central nervous system (CNS). In the present study, we examined behavioural alterations caused by transient perinatal hypothyroidism induced by an anti-thyroid drug, propylthiouracil (PTU). This drug produces perinatal disruption of the thyroid system and subsequent behavioural changes, which we investigated using a series of behavioural tests and focusing particularly on attention-deficit/hyperactivity disorder (ADHD)-like behaviours. In the open field test, both male and female rats that had experienced perinatal hypothyroidism (HT rats) showed an increased percent of locomotion behaviour and reduced grooming behaviour, suggesting that HT rats may be hyperactive and show fewer anxiety characteristics. Neither male nor female HT rats showed retention in the passive avoidance test. Male HT rats showed a significantly lower rate of correct avoidance responses than control rats in earlier sessions in the active avoidance test. In addition, we observed significant increases in the number of times that rats crossed the partition during inter-trial intervals and the percent of failure of avoidance during 5 s electrical stimuli in HT rats, suggesting that HT rats are restless, have a shortened attention span and panic easily. In measuring spontaneous motor activity during a period of darkness, male HT rats appeared to plunge into active phase with short, quick steps, while male control rats showed only long active phases during a stress-free period of darkness. These abnormal behavioural characteristics in HT rats might coincide with those found in some cases of ADHD.

Effects of perinatal exposure to bisphenol A on the behavior of offspring in F344 rats

The objective of this investigation is to evaluate whether perinatal maternal exposure to bisphenol A (BPA) at 4, 40, and 400 mg/kg per day affects the behavior of offspring in F344 rats. Perinatal BPA exposure inhibited the body weight increases of male and female offspring in a dose-dependent manner, which continued after weaning. Spontaneous activity analyses revealed that BPA elongated immobile time during the dark phase in female offspring. At 4 weeks of age, male offspring exposed to BPA at 40 and 400 mg/kg per day performed avoidance responses significantly higher in the shuttlebox avoidance test. At 8 weeks of age, however, male offspring only at 4 mg/kg per day showed significantly lower responses. In the open-field behavior test at 8 weeks of age, male offspring exposed to BPA only at 4 mg/kg per day showed a higher percent of grooming than the control male offspring. In conclusion, perinatal exposure to BPA caused the behavioral alterations in the offspring.

Inhibition of staurosporine-induced neuronal cell death by bisphenol A and nonylphenol in primary cultured rat hippocampal and cortical neurons

We examined whether bisphenol A (BPA) and 4-nonylphenol (NP) influenced staurosporine-induced neuronal cell death in primary cultured rat hippocampal and cortical neurons. In hippocampal neurons, 17beta-estradiol (E2) (1 nM and 10 microM) and BPA (10 microM) significantly inhibited the staurosporine-induced release of lactate dehydrogenase (LDH). In cortical neurons, BPA significantly inhibited the LDH release, while E2 did not. In hippocampal neurons, E2 and BPA significantly inhibited the staurosporine-induced increase in caspase-3 activity. In cortical neurons, BPA and NP significantly inhibited the increase in caspase-3 activity, while E2 did not. Furthermore, low-dose BPA (10 nM) also significantly inhibited the increase in caspase-3 activity in both hippocampal and cortical neurons. BPA and NP might impede normal brain development by inhibiting even desirable neuronal cell death, interfering with caspase-3 activation.

Primary culture of cortical neurons, type-1 astrocytes, and microglial cells from cynomolgus monkey (Macaca fascicularis) fetuses

We established selective primary cultures of neurons, astrocytes, and microglial cells from cryopreserved fetal cerebral cortex of cynomolgus monkeys (Macaca fascicularis). At 14 days in serum-containing medium, the cell cultures of the fetal cerebral cortex consisted primarily of neurons, astrocytes, and floating microglial cells. At 21 days, we observed a small number of myelin basic protein (MBP)-positive oligodendrocytes. The addition of cytosine arabinoside (a selective DNA synthesis inhibitor) at 2 days in culture eliminated proliferative glial cells, allowing adequate numbers of neurons to survive selectively. A chemically defined serum-free medium successfully supported neuronal survival at a level equivalent to that supported by the serum-containing medium. Brain-derived neurotrophic factor (BDNF) significantly affected the survival of primate neurons. Glutamate induced a significant degree of neuronal cell death against primate neurons and MK-801, a selective N-methyl-D-aspartate receptor (NMDAR) antagonist, blocked cell death, which suggests that primate cortical neurons have NMDAR and the glutamate-induced cell toxicity is mediated by NMDAR. In the serum-free medium, type-1 astrocytes responded to dibutyryl cyclic AMP and showed a process-bearing morphology. The growth of type-1 astrocytes in the serum-free medium was stimulated by epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and hydrocortisone, which are known growth factors in rat type-1 astrocytes. Cultured microglial cells expressed CD68, a monocyte marker. Macrophage-colony stimulating factor (M-CSF) stimulated microglial cell growth in the serum-free medium. These selective primary culture systems of primate cerebral cortical cells will be useful in issues involving species specificity in neuroscience.

Endogenous dopamine maintains synchronous oscillation of intracellular calcium in primary cultured-mouse midbrain neurons.

We demonstrated synchronous oscillation of intracellular Ca2+ in cultured-mouse midbrain neurons. This synchronous oscillation was thought to result from spontaneous and synchronous neural bursts in a synaptic neural network. We also examined the role of endogenous dopamine in neural networks showing synchronous oscillation. Immunocytochemical study revealed a few tyrosine hydroxylase (TH)-positive dopaminergic neurons, and that cultured neurons expressed synaptophysin and synapsin I. Western blot analyses comfirmed synaptophysin, TH, and 2 types of dopamine receptor (DR), D1R and D2R expression. The synchronous oscillation in midbrain neurons was abolished by the application of R(−)-2-amino-5-phosphonopentanoic acid (AP-5) as an N-methyl-D-aspartate receptor (NMDAR) antagonist. This result suggests that the synchronous oscillation in midbrain neurons requires glutamatergic transmissions, as was the case in previously reported cortical neurons. SCH-12679, a D1R antagonist, inhibited synchronous oscillation in midbrain neurons, while raclopride, a D2R antagonist, induced a transient increase of intracellular Ca2+ and inhibited synchronous oscillation. We consider that endogenous dopamine maintains synchronous oscillation of intracellular Ca2+ through D1R and D2R, and that these DRs regulate intracellular Ca2+in distinctly different ways. Synchronous oscillation of midbrain neurons would be a useful tool for in vitro researches into various neural disorders directly or indirectly caused by dopaminergic neurons.

Astroglial responses against Aβ initially occur in cerebral primary cortical cultures: species differences between rat and cynomolgus monkey

In the present study, we investigated how amyloid beta (Abeta) peptides initially affect neuronal cells in primary cerebral cortical cultures from rat and cynomolgus monkey. In these cultures, complicated interactions between glial and neuronal cells occur; moreover, synaptic interactions similar to those observed in vivo also occur between neuronal cells in these cultures. In this study, we applied low concentrations of Abeta to these well-characterized primary cultures to investigate how Abeta initially affects neurons or astroglial cells. In both rat and monkey cortical cultures, treatment with low concentrations of Abeta failed to drastically change or damage of neurons. Abeta treatment, however, significantly activated astrocytes, resulting in increased apolipoprotein E (ApoE) production. Rat astrocytes were more sensitive to Abeta than monkey astrocytes, and responded to Abeta via a different mechanism. In monkey astrocyte cultures, only direct treatment with Abeta increased ApoE production. In rat astrocyte cultures, however, treatment with conditioned media from cortical cultures grown with Abeta increased ApoE production, indicating that some sort of neuron-derived soluble factor(s) was also involved in activating rat astrocytes. These species differences suggest that monkey cortical cultures would be more useful as an in vitro model system to understand the details of how Abeta accumulates in the human brain, since monkeys are phylogenetically more similar to humans.

Dopamine Receptor 2 Regulates L-Type Voltage-Gated Calcium Channel in Primary Cultured Mouse Midbrain Neural Network

Abstract1. Synchronous oscillation of intracellular Ca2+ in the central nervous system is essential for neural development. We previously reported that endogenous dopamine was involved with synchronous Ca2+ oscillation of primary cultured midbrain neurons, and that regulation of dopamine in synchronous oscillation was distinctly different through dopamine receptor 1 (D1R) and 2 (D2R): the action of dopamine through D1R or D2R was facilitative or suppressive, respectively, to the Ca2+ influx of synchronous oscillation.2. In the present study, we confirmed that the suppressive effects of D2R were mediated by the regulation of the L-type voltage-gated Ca2+ channel, not by the regulation of NMDA receptor on the Ca2+ influx in the midbrain neural network showing synchronous oscillation.3. This evidence promotes better understanding of the regulation of neural activity by endogenous dopamine in networked neurons.

Cryopreservation and primary culture of cerebral neurons from cynomolgus monkeys (Macaca fascicularis).

We established the procedures for cryopreservation and primary culture of fetal cerebral neurons of cynomolgus monkeys (Macaca fascicularis). Three developmental stages of fetuses (80, 93, and 102 days of gestation) were compared to determine the optimal stage of cerebrum development for primary culture. Among the three fetuses, the 80-day-old fetus produced the most process-rich neurons with the highest survival. The number of total recovery cells from the cryopreserved 80-day-old fetus corresponded to 83.4% of that from fresh tissue. Besides, synchronous oscillations of intracellular calcium were first seen in primate cerebral neurons, which suggested the formation of synapse-networks. Cultured neurons expressed synaptophysin protein. Successful cryopreservation and subsequent cell culture of primate neurons would be useful tools for neuroscience research with species specificity.

Gene expression profiling of mouse embryonic stem cell progeny differentiated by Lumelsky's protocol.

Successful conversion of embryonic stem (ES) cells into insulin-producing cells has been reported by Lumelsky et al. (Science 2001;292:1389–1394); however, it remains controversial. In this study, we investigated the properties of ES cell progeny-induced differentiation according to Lumelsky’s protocol by immunocytochemistry, oligonucleotide microarray and real-time RT-PCR. Insulin-positive cells were observed at stages 3, 4 and 5. Microarray analysis demonstrated upregulation and appearance of some genes involved in pancreatic development but not β-cell-specific functional genes in cells at stage 5. Similarly, real-time RT-PCR revealed that expression of β-cell-specific functional genes such as islet amyloid polypeptide, insulin I and II was not increased in cells at stage 5. These results suggest that terminal differentiation of ES cell progeny toward functional pancreatic β-cell is insufficient. This study also demonstrates the usefulness of multiple time-course expression profiles for validating differentiation fates of ES cell progeny.

Glutamate Regulates the Frequency of Spontaneous Synchronized Ca2+ Spikes Through Group II Metabotropic Glutamate Receptor in Cultured Mouse Cortical Networks

Abstract1. Synchronized spontaneous intracellular Ca2+ spikes in networked neurons are believed to play a major role in the development and plasticity of neural circuits. Glutamate-induced signals through the ionotropic glutamate receptors (iGluRs) are profoundly involved in the generation of synchronized Ca2+ spikes.1 2. In this study, we examined the involvement of metabotropic glutamate receptors (mGluRs) in cultured mouse cortical neurons. We pharmacologically revealed that glutamate-induced signals through inclusive mGluRs decreased the frequency of Ca2+ spikes. Further experiments indicated that this suppressive effect on the spike frequency was mainly due to the signal through group II mGluR, inactivation of adenylate cyclase-cAMP-PKA signaling pathway. Group I mGluR had little involvement in the spike frequency.3. Taken together, glutamate generates the synchronized Ca2+ spikes through iGluRs and modulates simultaneously their frequency through group II mGluR–adenylate cyclase–cAMP–PKA signaling pathway in the present in vitro neural network. These results provide the evidence of the profound role of group II mGluR in the spontaneous and synchronous neural activities.