Nerve growth factor and glial cell line-derived neurotrophic factor restore the cholinergic neuronal phenotype in organotypic brain slices of the basal nucleus of Meynert

doi:10.1016/S0306-4522(00)00452-8

Neuroscience

Volume 102, Issue 1, 2 January 2001, Pages 129-138

https://doi.org/10.1016/S0306-4522(00)00452-8 Get rights and content

Abstract

Loss of cholinergic neurons is found in the medial septum and nucleus basalis of Meynert in Alzheimer’s disease. Recent observations suggest that cholinergic neurons down-regulate their phenotype and that growth factors may rescue cholinergic neurons. The aim of this study was to investigate whether cholinergic neurons of the basal nucleus of Meynert can be cultured in rat organotypic slices, and if nerve growth factor and glial cell line-derived neurotrophic factor can rescue the cholinergic phenotype. In the organotypic slices, glial cells, GABAergic and cholinergic neurons were visualized using immunohistochemistry. The number of cholinergic neurons was found to be very low in slices cultured without exogenous nerve growth factor. Analysis of nerve growth factor tissue levels by enzyme-linked immunosorbent assay revealed very low endogenous tissue levels. When slices were incubated with 100 ng/ml nerve growth factor during the initial phase of culturing, a stable expression of choline acetyltransferase was found for up to several weeks. After eight weeks in culture with nerve growth factor or two to three weeks after nerve growth factor withdrawal, numbers of detected cholinergic neurons decreased. Neurons incubated with nerve growth factor displayed a significantly enlarged cell soma compared to neurons without growth factors. In cultures incubated for up to nine weeks, it was also found that glial cell line-derived neurotrophic factor was capable of restoring the cholinergic phenotype. The low-affinity p75 and high-affinity trkA receptors, as well as the glial cell line-derived neurotrophic factor receptor GFRα-1, could be visualized in slices using immunohistochemistry.

In conclusion, it is shown that, in the axotomized organotypic slice model, the number of cholinergic neurons is decreased, but can be partly restored by nerve growth factor and glial cell line-derived neurotrophic factor.

Section snippets

Organotypic slice cultures

Organotypic cultures were established as described recently.10., 20., 37. In brief, the nBM of postnatal day 10 rats (Sprague–Dawley, Institute of Animal Breeding, Himberg, Austria) was dissected under aseptic conditions (Fig. 1), 400-μm slices were cut with a tissue chopper and the slices were placed on a Millicell-CM 0.4-μm (Millipore, Austria) culture plate (five or six slices per membrane). Slices were cultured in Petri dishes at 37°C and 5% CO₂ with 1 ml/Petri dish of the following culture

Slices cultured without growth factors

A strong glial GFAP-positive fiber network was observed within all slices cultured for two, three, four, five and six weeks (Fig. 2A). The staining appeared strongly enhanced at the borders of the slices (Fig. 2A). Very few GFAP-positive cell bodies were found within the slices; however, strong GFAP-positive astrocytes were seen at the border of the slices (Fig. 2A). No obvious changes were visible by GFAP staining at any of the observed time-points in culture (data not shown).

Discussion

The present study demonstrates that cholinergic neurons of the nBM can be cultured for several weeks in organotypic slices. NGF but not other growth factors tested supported the cholinergic phenotype during initial culturing, while NGF and GDNF restored the cholinergic phenotype in older slices. Receptors for both growth factors were detectable immunohistochemically.

Conclusions

The organotypic slice model is an isolated axotomized system and is useful to study the effects of exogenously applied growth factors on cholinergic neurons of the basal forebrain. NGF is essential to rescue the cholinergic phenotype in these neurons, at least during the initial phase of the culture. NGF withdrawal and long-term culturing lead to a decreased expression of the cholinergic phenotype, which could be partially reversed by NGF or GDNF. Both growth factors could be of major

Acknowledgements

We thank Iris Berger for excellent technical assistance, Prof. Hersh (Lexington, KY, USA) for kindly providing the ChAT antibody and Dr Kayvon Salimi for carefully reading the manuscript. This study was supported by the Austrian Science Foundation (P11956MED).

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Nerve growth factor and glial cell line-derived neurotrophic factor restore the cholinergic neuronal phenotype in organotypic brain slices of the basal nucleus of Meynert

Abstract

Section snippets

Organotypic slice cultures

Slices cultured without growth factors

Discussion

Conclusions

Acknowledgements

Neuron

Expl Neurol.

Trends Neurosci.

Cell

Int. J. devl Neurosci.

Trends Neurosci.

Neuroscience

Curr. Opin. Neurobiol.

Int. J. devl Neurosci.

Devl Brain Res.

Neuroscience

Cell

Neuroscience

Brain Res.

Brain Res.

Devl Brain Res.

Trends Neurosci.

Expl Neurol.

Brain Res. Bull.

Cell contact-mediated regulation of tyrosine hydroxylase synthesis in cultured bovine adrenal, chromaffine cells

J. biol. Chem.

Ret receptor tyrosine kinase immunoreactivity is altered in glial cell line-derived neurotrophic factor-responsive neurons following lesions of the nigrostriatal and septohippocampal pathways

Neuroscience

Nerve growth factor: a tale of two receptors

Oncogene

Neurturin responsiveness requires a GPI-linked receptor and the ret receptor tyrosine kinase

Nature

GDNF signalling through the ret receptor tyrosine kinase

Nature