Mami Miyamoto

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.

Interleukin-6 as a neurotrophic factor for promoting the survival of cultured basal forebrain cholinergic neurons from postnatal rats

Human recombinant interleukin-6 (IL-6, B-cell stimulating factor-2) was capable of supporting neuronal survival in cholinergic neuron culture, prepared from 10-day-old rat brain septal region. Cell survival of the cultured cholinergic neurons was estimated by measuring the remaining choline acetyltransferase (ChAT) activities after 6 days of culture. IL-6 at a concentration of 5 ng/ml maintained a more than 3-fold higher ChAT activity in the culture as compared with that in cultures without IL-6. The maximal dose of IL-6 was near 50 ng/ml. The concomitant addition of mouse nerve growth factor (NGF) and IL-6, both at maximal doses, had a synergistic effect on cholinergic cell survival. These results indicate that IL-6 can act as a neurotrophic agent, independent of the action of NGF, supporting neuronal survival of cultured postnatal rat septal cholinergic neurons. On the other hand, IL-6 did not affect the differentiation of the cultured embryonic rat septal cholinergic neurons, differently from the differentiation action by NGF.

INTRACORTICAL REGIONALITY REPRESENTED BY SPECIFIC TRANSCRIPTION FOR A NOVEL PROTEIN, LATEXIN

The monoclonal antibody (mAb) PC3.1 recognizes a subset of neurons distributed in the infragranular layers of the lateral neocortex of the rat. Immunoaffinity chromatography with mAb PC3.1 showed that this antibody specifically binds a peptide epitope on a 29 kDa protein named latexin. To study the molecular details of the protein, we isolated four independent cDNA clones for latexin from cDNA libraries of the rat cerebral cortex and whole brain using the amino acid sequences of latexin fragments. Analysis of these cDNA clones showed that the predicted primary structure of latexin consists of 223 amino acids, and has no strict homology to any sequences so far known. Western and Northern blots demonstrated that the latexin and its mRNA were expressed predominantly in neural tissues with some expression in non‐neural tissues. The gene that encodes latexin in the rat appeared to have homologues in other mammalian species and in the chick. In situ hybridization showed that latexin mRNA is synthesized in a subset of neurons in the lateral but not the dorsal neocortex, and that the distribution profile of these neurons is quite similar to that of neurons expressing latexin. These results indicate that latexin is a novel class of neuronal protein which represents intracortical regionality, and suggest that the regional specification of the neocortex involves selective parcellation of neurons which express a particular gene.

Survival-promoting effect of NGF on in vitro septohippocampal neurons with cholinergic and GABAergic phenotypes

Recently, we demonstrated a survival-promoting effect of nerve growth factor (NGF) on cultured hippocampus-projecting neurons from developing septum/diagonal band region using fluorescent latex microspheres as a retrograde neuronal marker (Arimatsu et al., 1989). In the present study, we characterized these projection neurons by combining the retrograde cell labeling and histochemical stainings for acetylcholinesterase (AChE) activity and NGF receptor-, choline acetyltransferase- (ChAT-) and gamma-aminobutyric acid- (GABA-) immunoreactivities. The surviving microsphere-labeled neurons were, for the most part, immunoreactive for NGF receptor in the culture. A great majority (about 90%) of the microsphere-labeled neurons showed strong AChE activity and ChAT-immunoreactivity. The number of strongly AChE-positive neurons and that of ChAT-immunoreactive neurons in the culture supplemented with NGF was much greater with than without exogenous NGF. In addition, a major part (about 70%) of the microsphere-labeled neurons exhibited GABA-immunoreactivity in the presence of NGF. The number was also much greater than that without NGF. A considerable portion of cultured septal cholinergic neurons were shown to express GABA-immunoreactivity by a two-color immunofluorescence labeling experiment for ChAT and GABA. These findings are consistent with the assumption that NGF plays an important role in the development and organization of the cholinergic and GABAergic septohippocampal systems by supporting the neuronal survival, and raise a possibility that cholinergic and GABAergic fractions of the septohippocampal neurons may be developmentally correlated.

Nerve growth factor promotes survival of retrogradely labeled hippocampus-projecting neurons in the rat basal forebrain in vitro

The effect of nerve growth factor (NGF) on the survival of neurons projecting to the hippocampus from developing medial septum and vertical limb of the diagonal band was studied in vitro. The neurons had previously been labeled retrogradely in vivo with fluorescent latex microspheres. The microspheres were injected bilaterally into the hippocampus of 5-day-old rats. Twenty to 24 h after the injection, cells from the basal forebrain were dissociated with papain and cultured for 3-5 days. The number of microsphere-labeled neurons in the culture with supplementation of NGF was much greater than that without NGF. The result clearly indicates a survival-promoting effect of NGF on these projection neurons.

Colocalization of NGF receptor immunoreactivity and [3H]GABA uptake activity in developing rat septum/diagonal band neurons in vitro

Neurons in the septum (SEP) and vertical limb of the diagonal band (vDB) of 6-day-old rats were dissociated and cultured for 3 days. Cells with high affinity uptake mechanism for gamma-aminobutyric acid (GABA) were identified autoradiographically using 16 nM [3H]GABA. Cells that expressed nerve growth factor (NGF) receptor immunoreactivity were identified immunocytochemically using MC192, a monoclonal antibody against NGF receptor. Double labeling experiments combining [3H]GABA uptake with immunostaining for NGF receptor showed that 46% of the NGF receptor-immunoreactive cells accumulated [3H]GABA. The result was discussed in relation to a possible involvement of the developing septal GABAergic neurons in NGF-induced neuronal survival.

Interleukin-6 as a Neurotrophic Factor for Promoting Survival of Septal Cholinergic Neurons and Mesencephalic Catecholaminergic Neurons from Postnatal Rats

Neuronal differentiation and survival are supported by several kinds of neurotrophic factors in the central nervous system (CNS) as in the peripheral nervous system (PNS). The well-known nerve frowth factor (NGF), target derived growth factor, is synthesized1–4 and acts on cholinergic neurons5–12 in the CNS; however the action of NGF on other neurons is limited. We have been studying such factors that act on neuronal survival by using primary cultures of postnatal rat brain.

Co-Localization of Cholinergic and GABAergic Traits in in Vitro Septohippocampal Neurons from Developing Rats

It is well known that basal forebrain neurons are involved in Alzheimer’s disease. Massive neuronal loss occurs in the basal forebrain as well as in the cortical areas that are interconnected with this region. In rodents, nerve growth factor (NGF) acts on cholinergic neurons in the basal forebrain to induce choline acetyltransferase activity.1 It was suggested that survival of the basal forebrain neurons is also dependent on NGF during development2 and in adult life.3 We have offered direct in vitro evidence that NGF in fact promotes the survival of hippocampus-projecting neurons in the medial septum and vertical limb of the diagonal band of developing rats.4 The septohippocampal neurons were identified by retrograde-labeling with fluorescent latex microspheres that had been injected into the hippocampus in vivo. The number of microsphere-labeled projection neurons in the cultures supplemented with 100 ng/ml NGF was much greater than that without NGF.