Abstract
The basal forebrain is located close to the medial and ventral surfaces of the cerebral hemispheres that develop from the sub-pallium. It regulates multiple processes including attention, learning, memory and sleep. Dysfunction and degeneration of basal forebrain cholinergic neurons (BFCNs) are believed to be involved in many disorders of the brain such as Alzheimer’s disease (AD), schizophrenia, sleep disorders and drug abuse (Mobley et al., 1986). Primary cultures of BFCNs will provide an important tool for studying the mechanism of these diseases. This protocol provides a detailed description of experimental procedures in establishing in vitro primary culture of rat embryonic BFCNs.
Keywords: Basal forebrain cholinergic neuron, Nerve growth factor, Alzheimer’s disease, in vitro culture, Microfluidic chamber, Axonal transport, Neurodegeneration
Background
The basal forebrain cholinergic system innervates the cerebral cortex and hippocampus. The normal function of the BFCNs is essential for normal sleeping, learning and memory. And the atrophy of BFCNs is considered as the early event of Alzheimer’s disease. Thus, the primary BFCNs culture will be the ideal cell model for AD research. In previous studies, primary BFCNs cultures were rarely used. Here, we present a reliable method to isolate and culture BFCNs from the embryonic rat septum which is simple, less time consuming than the previous method (Schnitzler et al., 2008). Our method will greatly facilitate studies of many critical aspects of BFCN function and cell biology.
Materials and Reagents
Note: Materials #3-9 can be from various suppliers.
Equipment
Procedure
Data analysis
Neurons can be cultured in microfluidic chamber (Figure 1A, left panel: a DIC image; right panel: an image of immunostaining with anti-TrkA antibody) to separate neuronal soma from long axons to study axonal transport. Neurons can also be grown in mass culture on coated coverglasses for other applications. For instance, immunostaining was used to confirm the presence of cholinergic (at DIV7), NGF-responsive neurons in the culture with specific antibodies against choline acetyltransferase (ChAT), a marker for cholinergic neurons, and TrkA, the receptor for NGF (Figure 1B). The majority of neurons (> 90%) were positive for both markers (Xu et al., 2016). Figure 1. Cultures of rat E18 basal forebrain cholinergic neurons (BFCNs). A. Representative images of primary BFCNs (DIV7) (left: DIC; right: anti-TrkA immunostaining) cultured in microfluidic chamber. Scale bars = 50 μm. B. Representative images of primary BFCNs (DIV7) were costained for the cholinergic neuronal marker ChAT (red) and the NGF receptor TrkA (green). Scale bars = 10 μm. Note: For representative photos and results please refer to (Xu et al., 2016).
Recipes
Acknowledgments
This protocol was adapted from methods described by Schnitzler et al. (2008). The study is supported by the following grants: NIH (PN2EY016525), NIH UCSD ADRC P50 Pilot grant, Down Syndrome Research and Treatment Foundation, Larry L. Hillblom Foundation, Tau Consortium, the Ministry of Science and Technology of the People’s Republic of China (2014CB965002, 2012BAI10B03), National Natural Science Foundation of China (81171200), Science and Technology Commission of Shanghai Municipality (13JC1401502, 13140904000), Shanghai Municipal Education Commission (12ZZ115).
References
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