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Journal of Neuroscience, Vol 6, 3229-3241, Copyright © 1986 by Society for Neuroscience
Noradrenergic neurons from the locus ceruleus in dissociated cell culture: culture methods, morphology, and electrophysiology
S Masuko, Y Nakajima, S Nakajima and K Yamaguchi
We have developed a dissociated primary cell culture of noradrenergic
neurons from the locus ceruleus of postnatal (1- to 5-d-old) mice or rats.
Slices of the brain stem were made on a Vibratome. Then the region of locus
ceruleus, which was identified by observing the slices under a dissecting
microscope, was dissected out from the slices. The removed fragments of
brain slices were dissociated and cultured up to 3 weeks on a non-neuronal
feeder layer, which consisted predominantly of astroglial cells, or on a
fibronectin-treated collagen substratum. After 2 weeks of culture, about
70% of total neuronlike cells revealed positive catecholamine
histofluorescence, indicating that they were probably noradrenergic
neurons. About 98% of large- and medium-sized cultured neurons (soma
diameter greater than or equal to 20 microns) was histofluorescence
positive. The fluorescence-positive cells had long processes rich in
varicosities, and the shape of their soma was either multipolar or
fusiform. Electron microscopy using permanganate fixation revealed that the
varicosities along their processes had small granular vesicles, which may
contain norepinephrine. Physiological properties of these noradrenergic
neurons were investigated with intracellular microelectrodes or with the
whole-cell version of the patch clamp. We observed that many cells were
producing spontaneous firing. Many of these spontaneously firing cells had
no obvious contact with neighboring cells. The neurons were depolarized
when glutamate was applied by pressure ejection. They also responded to
GABA and glycine with either hyperpolarization or depolarization, and these
responses were antagonized by picrotoxin and strychnine. Application of
substance P generally produced depolarization with an increase in input
resistance. The neurons responded with hyperpolarization to somatostatin,
beta-endorphin, and enkephalin. This culture system will become a useful
tool for elucidating the cellular and molecular properties of the central
noradrenergic neurons.
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