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Proliferation and programmed cell death are important in the formation of morphologic structures and functional activity during CNS development. We used immunohistochemical and TUNEL methods to examine the proliferation and differentiation of neural cells in, distribution of apoptotic cells in, and microglial cell involvement in the removal of apoptotic cells from the fetal cerebral cortex of cynomolgus monkeys. At embryonic day (E) 50 and E80, the neuroepithelium contained many mitotic cells. Cells staining for PCNA (a nuclear marker of proliferating cells) were prominent in the proliferative zone, whereas cells positive for NeuN (a neuron-specific marker) were absent. GFAP staining for glial cells was positive in the neuroepithelium and radial glial fibers. Iba1-positive cells (that is, macrophages and microglia) were distributed throughout all regions at all time points but accumulated especially in the ventricular zone at E80. Apoptotic morphology (at E80) and TUNEL-positive cells (that is, containing DNA fragmentation; at E50 and E80) were observed also. At E120 and E150, most PCNA-positive cells were in the ventricular zone, and NeuN-positive cells were prominent in all layers except layer I-II at E120. GFAP immunoreactivity was detected mainly in cells with fine processes in the white matter. Neither apoptosis nor TUNEL-positive cells were detected at either E120 or E150. These results suggest that proliferation, migration, and neural cell death occur during midgestation (that is, E50 to E80) in fetal brain of cynomolgus macaques, whereas differentiation and maturation of neural cells occur after midgestation (E80).

We demonstrated synchronous oscillation of intracellular Ca2+ in cultured-mouse mid-brain 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.

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.

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.

We have developed a cytoplasmic replicating virus vector of Sendai virus (SeV) that infects and replicates in most mammalian cells, including neurons, and directs high-level gene expression. To investigate the protective effect of SeV vector-mediated gene transfer of glial cell line-derived neurotrophic factor (GDNF) on the delayed neuronal death caused by transient global ischemia in gerbils, SeV vectors carrying either GDNF (SeV/GDNF) or enhanced green fluorescent protein gene (SeV/GFP) were stereotaxically microinjected into the lateral ventricle. Four days after injection, occlusion of the bilateral common carotid arteries for 5 min produced transient global forebrain ischemia. Treatment with SeV/GDNF significantly decreased the delayed neuronal death of the hippocampal CA1 pyramidal neurons observed 6 days after the operation. TUNEL staining demonstrated that SeV/GDNF treatment markedly reduced the number of apoptotic cells in the hippocampal CA1 neurons, indicating that SeV/GDNF treatment prevented apoptosis. Furthermore, delayed neuronal death on the contralateral side of the hippocampal CA1 was also prevented to a similar extent as that on the ipsilateral side. These results suggest that SeV/GDNF prevents the delayed neuronal death induced by ischemia and is potentially useful for gene therapy for stroke.

N. Kimura, K. Yanagisawa, K. Terao, F. Ono, I. Sakakibara, Y. Ishii, S. Kyuwa and Y. Yoshikawa (2004)Neuropathology and Applied Neurobiology30,000–000, doi: 10.1111/j.1365-2990.2004.00624.xAge-related changes of intracellular Aβ in cynomolgus monkey brainsTo confirm the intracellular accumulation of amyloidβ-protein (Aβ), we carefully performed immunohistochemistry using brains of cynomolgus monkeys of various ages. Cortical neurones and their large neurites were immunostained with antibodies against Aβ in young monkey brains. In aged monkey brains, intracellular Aβ localized within cortical neurones; no clear association was found between the presence of intracellular Aβ and senile plaques (SPs). Interestingly, we did not observe Aβ-immunoreactive cortical neurones in brains fixed with neutral buffered formalin. Western blot analyses of microsomal and nerve ending fractions derived from the brains of young to aged monkeys revealed that intracellular Aβ generation changed with age. In the microsomal fraction, the amount of Aβ42 significantly increased in brains from older monkeys (> 30 years of age), and the amount of Aβ43 significantly decreased with age in the microsomal fraction. The amount of Aβ40 remained the same regardless of age. Biochemical analyses also showed that intracellular levels of each of these Aβ molecules significantly increased with age in nerve ending fractions. As we previously observed that a similar accumulation of presenilin1,β-amyloid precursor protein (APP) and APP C-terminal fragment cleaved byβ-secretase in the nerve ending fractions obtained from brains with SPs, the accumulation of intracellular Aβ in this fraction may be closely related to formation of spontaneous SPs with age. Taken together, these results suggest that intensive investigation of age-related changes in the nerve ending will contribute to a better understanding of the pathogenesis of age-related neurodegenerative disorders such as sporadic Alzheimer's disease. [ABSTRACT FROM AUTHOR]