Noradrenergic neurons from the locus ceruleus in dissociated cell culture: culture methods, morphology, and electrophysiology

Abstract

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.

Substitution of a mutant α 2a -adrenergic receptor via “hit and run” gene targeting reveals the role of this subtype in sedative, analgesic, and anesthetic-sparing responses in vivo

“…Coronal brain slices of 200 m thickness containing the LC (20) were prepared from B6,129 mice Ϸ2-3 weeks old using techniques described (21). Spontaneous action potentials, observed in LC neurons using the perforated patch technique, were amplified using an Axopatch 1D amplifier (Axon Instruments, Foster City, CA) and were recorded using the PCLAMP program (Axon Instruments) on a computer and on a Grass Instruments (Quincy, MA) chart recorder and PCM͞VCR.…”

Activation of G protein‐coupled inward rectifier K+ channels in brain neurons requires association of G protein βγ subunits with cell membrane

“…Experiments were conducted on large neurons (soma diameter 28 + 3 gM; mean + S.D. ), which were very likely noradrenergic [4]. The patch pipette solution (external solution)contained: 156 mM KC1; 2.4 mM CaC12; 1.3 mM MgCI2; 5 mM HEPES-NaOH; and 0.5 gM tetrodotoxin (pH 7.4) with or without 0.5 gM somatostatin.…”

“…We developed dissociated cultures of noradrenergic neurons flom the locus coeruleus; these neurons responded well to s,~matostatin and met-enkephalin with an increase in Kit(G) c,mductance [4][5][6]. The single channel properties of this endogenous brain Kir(G) were recently analyzed, and it was fi,und that this brain Kit(G) differed from the cardiac I~ir(G) in several respects (for instance, the degree of inward rectification of the brain Kir(G) is milder than that of the cardiac Kir(G)) [7].…”

“…The prenylation of 7 s~tbunit is a prerequisite for the 137 subunit to be anchored to the cell membrane [8,9], and to regulate the activity of adeny-1~1 cyclases [10]. In this report, we will show that prenylation o r 7 subunit is also necessary for the 137 subunit to be able to [4] was used with some modifications. Briefly, rats were anesthetized with ether, and brain slices were sectioned from the excised brainstems.…”

Possível envolvimento do monóxido de carbono na modulação do comportamento emocional em ratos: o papel do locus coeruleus

“…Um estudo anterior mostrou que 70% das células do LC possivelmente apresentam imunorreatividade para catecolaminas, além de apresentarem outras características de neurônios noradrenérgicos (Masuko et al, 1986). Além disso, neste mesmo estudo constatou-se que aproximadamente 77% dessas células imunorreativas as catecolaminas são multipolares.…”