Yoichi Kushima

Interleukin-6 improves the survival of mesencephalic catecholaminergic and septal cholinergic neurons from postnatal, two-week-old rats in cultures

Interleukin-6 (human recombinant) supported the survival of cultured mesencephalic, catecholaminergic and septal cholinergic neurons from postnatal, two-week-old (P13-P15) rats. Significantly, more catecholaminergic neurons, stained by monoclonal anti-tyrosine hydroxylase antibody, were found in cultures supplemented with interleukin-6 at a concentration of 5 ng/ml than in cultures not treated with interleukin-6. The optimal dose used was 50 ng/ml. The survival effect of interleukin-6 on postnatal rat, tyrosine hydroxylase-positive neurons was observed both in cultures using serum-containing and serum-free medium. Contents of dopamine and noradrenaline in cultures with interleukin-6 were also larger than in control cultures. Interleukin-6 also increased the survival of cultured embryonic (E17) rat midbrain tyrosine hydroxylase-positive neurons. The effect on these neurons was, however, smaller, and the optimal dose of interleukin-6 was nearly 5 ng/ml. Interleukin-6 also supported the survival of cultured postnatal (P13) rat septal cholinergic neurons, visualized by acetylcholinesterase staining. The concomitant addition of mouse nerve growth factor (100 ng/ml) and interleukin-6 (50 ng/ml) had a synergetic effect on the survival of acetylcholinesterase-positive neurons in culture. Our data suggest that the survival of cultured tyrosine hydroxylase-positive, mesencephalic, and acetylcholinesterase-positive, septal neurons from postnatal two-week-old rats was supported by interleukin-6, just as there was a different dose dependency of interleukin-6 on the cultured postnatal neurons compared with embryonic neurons.

Developmentally regulated expression of a brain specific species of chondroitin sulfate proteoglycan, neurocan, identified with a monoclonal antibody 1G2 in the rat cerebrum

The mammalian brain contains many species of proteoglycan. To identify each proteoglycan species, we have raised monoclonal antibodies against soluble chondroitin sulfate proteoglycans purified from 10-day-old rat brains. One monoclonal antibody, named monoclonal antibody 1G2, recognized two proteoglycan species with 220,000 and 150,000 mol. wt core glycoproteins (chondroitin sulfate proteoglycan-220 and chondroitin sulfate proteoglycan-150). Partial amino acid sequences of N-termini of their core proteins coincided with those of neurocan, a brain-unique chondroitin sulfate proteoglycan species, whose complete coding sequence was recently reported [Rauch et al.(1992) J. biol. Chem.269, 19,536–19,547]. Western blots revealed that chondroitin sulfate proteoglycan-220 became detectable in the rat cerebrum on embryonic day 14, and that it disappeared from the brain around postnatal day 30. In contrast, a fairly large amount of chondroitin sulfate proteoglycan-150 remained in the mature brain. Immunohisto-chemical studies revealed that 1G2 antigen was first localized in the preplate zone, then both in the marginal zone and in the subplate of the rat cerebrum on embryonic day 16, prior to arrival of the first thalamic afferents at the cortex. On embryonic day 20, immunolabeling with monoclonal antibody 1G2 began to spread from the subplate into the developing cortical plate. On postnatal day 10, the neuropil of the cerebrum, except for the barrel field, was diffusely stained with the antibody, intensely in the hippocampus and superficial layers (I–III) of the cerebral cortex and weakly elsewhere. The barrel hollows were stained very weakly compared with the barrel walls at this stage. The immunoreactivity in the hippocampus and superficial cortical layers was weakened in the mature brain, so that no particular staining pattern, but weak and diffuse staining was observed in the adult rat cerebrum. The 1G2 antigen was immunohistochemically associated largely with glial fibrillary acidic protein-positive cells in primary cultures of the neonatal rat cerebrum. Both chondroitin sulfate proteoglycan-220 and chondroitin sulfate proteoglycan-150 were detected in the conditioned media not only of highly enriched cultures of fetal rat cortical neurons but also of pure cultures of mature astrocytes; more (12- to 20-fold) in the astrocyte conditioned media. Astrocytes, in addition to neurons, may be a cellular source of neurocan in brain at least under certain physiological conditions. The spaciotemporal expression pattern of 1G2 epitope-bearing proteoglycan, or neurocan, suggests that this proteoglycan species plays some roles at least in forming the elongation pathway for early cortical afferent fibers as well as the functional barrel structure in the somatosensory cortex.

Interleukin-6 as a neurotrophic factor for promoting the survival of cultured catecholaminergic neurons in a chemically defined medium from fetal and postnatal rat midbrains

Interleukin-6 (IL-6, human recombinant) promoted the survival of catecholaminergic neurons from fetal and postnatal rat midbrains as assessed by an immunohistochemical staining for tyrosine hydroxylase (TH) in culture using a chemically defined medium. The maximal dose of IL-6 for the cell survival of postnatal P15 rat mesencephalic TH-positive neurons in culture for 7 days was 50 ng/ml. The survival-promoting effects on P15 cultures were observed both in high- and low-density cultures. The survival effect of IL-6 on the cultured P15 TH-positive neurons was significant for only 4-15 days in vitro. However, the viable number of TH-positive neurons with IL-6 was less than that of the control at early points in the culture process (1-2 days in vitro). Continuous presentation of IL-6 was required for promoting survival. The optimal dose of IL-6 for the survival of fetal E16 midbrain TH-positive neurons was 5 ng/ml, and the survival promoting effect was less than that for the P15 cultures. The maximal dose of IL-6 for the survival of P2 TH-positive neurons was 5 ng/ml and that of P8 was 50 ng/ml, indicating that the response of rat mesencephalic TH-positive neurons to IL-6 changes during the first postnatal week.

Interleukin-6 and leukemia inhibitory factor promote the survival of acetylcholinesterase-positive neurons in culture from embryonic rat spinal cord

Interleukin-6 (IL-6) and leukemia inhibitory factor (LIF) promoted the survival of acetylcholinesterase (AChE)-positive neurons in culture from embryonic E15 rat spinal cord. Half of the AChE-positive neurons died during 3-7 days in culture in the absence of IL-6 and LIF. However, IL-6 at a concentration of 5 ng/ml completely prevented the death of AChE-positive neurons. LIF at a concentration of 5 U/ml also stimulated the survival of neurons, although to a lesser extent than IL-6. IL-6 and LIF also increased the numbers of process-bearing neuron-like cells in culture. The dose-dependencies of IL-6 and LIF with regard to the survival of total neuron-like cells were different from those for AChE-positive neurons.

Effects of nerve growth factor and basic fibroblast growth factor on survival of cultured septal cholinergic neurons from adult rats.

We have established a primary culture technique for neuronal cells from rat basal forebrain from postnatal day 58 (P58) to study the effects of neurotrophic factors on the neurons. The survival of acetylcholinesterase (AChE)-positive neurons of 2-week-old rat septum has already been reported to be strongly supported by nerve growth factor (NGF) in culture. In this culture study of neurons from adult rat brains, the survival of AChE-positive neurons from P58 rat septum was slightly improved by NGF, although low affinity NGF receptor expression was also observed on cultured P58 rat septum neurons as well as on those from 2-week-old rats. The addition of basic fibroblast growth factor (bFGF) improved the survival of AChE-positive neurons cultured from P58 rat septum, but did not promote the survival of neurons from P12 rat septum. These results suggest that NGF changes to a maintenance factor in adult rat brain from a survival factor in postnatal 2-week-old rats. The survival of cholinergic neurons in culture of adult rat septum might be supported by factor(s) other than NGF, such as bFGF.

Nerve-growth-factor-dependent and cell-density-independent survival of septal cholinergic neurons in culture from postnatal rats

We have established a primary neuronal cell culture technique from the postnatal (P11 to P15) rat CNS to study the nerve growth factor (NGF) response to basal forebrain cholinergic neurons. The survival of septal cholinergic neurons in culture was monitored both by the determination of choline acetyltransferase activity and by counting acetylcholinesterase-positive cells. Cells obtained from postnatal septal regions were found to require a plentiful oxygen supply during the dissociation of the cells. NGF-mediated survival of the septal cholinergic neurons was similarly observed in the cultures by using different plating cell densities up to 12.5 X 10(5) cells/cm2. These results suggest that the promotion by NGF of cell survival in culture is independent of plating cell density.

High oxygen atmosphere for neuronal cell culture with nerve growth factor. I. Primary culture of basal forebrain cholinergic neurons from fetal and postnatal rats

Cholinergic neurons cultured from postnatal days 11-13 (P11-P13) rat basal forebrain showed better survival in the culture condition using a 50% O2 atmosphere with and without nerve growth factor (NGF) than in a low (10 or 20%) O2 atmosphere. Except for the culture at a low cell density, the beneficial effect of the highly oxidized culture condition was found in the culture from P3 neurons, but not from embryonic day 18 neurons. The survival of microtubule-associated protein 2 (MAP2)-positive neurons in culture from P3 basal forebrain regions was more enhanced in a 50% O2 atmosphere than in 20% and also 10% O2 atmosphere. The viable number of the MAP2-positive neurons in a 10% O2 condition was about half of that in a 20% condition. These results suggest that the response of the cultured neurons to an incubator O2 concentration changes during the neuronal development in CNS from fetal to postnatal stages.

Detection of Alzheimer's β-amyloid precursor related proteins bearing chondroitin sulfate both in the juvenile rat brain and in the conditioned medium of primary cultured astrocytes

Alzheimers beta-amyloid precursor related proteins bearing chondroitin sulfate chains were detected in the conditioned media of primary cultured astrocytes obtained from fetal rat brains by Western blotting using the monoclonal antibody 22C11 against Alzheimers beta-amyloid precursor protein (APP), but not in the media of cortical neurons. The chondroitin sulfate proteoglycan form of APP was also detectable in a soluble proteoglycan fraction prepared from 10-day-old rat brains. However, the amount of proteoglycan form of APP in the brain was very small compared to non-proteoglycan forms at all the developmental stages from embryonic day 14 to 2 years. These observations suggest that astrocytes are one cellular source of the proteoglycan form of APP in the brain.

Culture of neuronal cells from postnatal rat brain : application to the study of neurotrophic factors

1. The authors developed a primary culture technique for neuronal cells from postnatal rat brains and studied the effects of neurotrophic factors on the naturally developed neurons. 2. We demonstrated changes in the neurotrophic role of nerve growth factor (NGF) during the developmental stages of the rat: NGF was shown to act as a differentiation factor in the early stages and as a survival factor later. 3. It appeared that interleukin-6 (IL-6) supported the survival of septal cholinergic neurons obtained from 10-day-old rats. IL-6, however, did not induce the differentiation of embryonic rat septal cholinergic neurons. IL-6 improved the survival of mesencephalic catecholaminergic neurons from postnatal and embryonic rat brains, which have known not to be response to NGF.

Expression and localization of smg p25A (= rab3A) in cultured rat hippocampal cells

We have studied the expression of smg p25A and synaptophysin in cultured hippocampal neurons isolated from 5-day-old rat brain by an immunocytochemical technique. In a dispersed cell culture seeded on astrocyte monolayers, well-branching neurite proliferation was observed along with age in culture. The synaptophysin immunoreactivity was present in the neuronal cell bodies and neurites at 1 and 5 days in vitro (DIV) and was eventually localized to discrete areas along neurites at 15 DIV while the immunoreactivity in cell bodies became less prominent. On the other hand, the smg p25A immunoreactivity was observed in the neuronal cell bodies and neurites at 1 through 15 DIV. The immunoreactivity for smg p25A or synaptophysin was not observed in astrocytes and this finding was confirmed by an immunoblot analysis. These results indicate that smg p25A as well as synaptophysin is present exclusively in neurons and suggest that these two synapse-associated proteins have different sites of function and different kinetics of synthesis, transport, and/or turnover in cultured hippocampal neurons.