Primary Cultures of Dissociated Sympathetic Neurons

Mains, Richard E.; Patterson, Paul H.

doi:10.1083/jcb.59.2.329

Cited by 400 publications

(93 citation statements)

References 43 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Like primary sympathetic neurons (12,25,26), NGF-treated PC12 cells adhere poorly to plastic tissue culture dishes and well to collagen-coated substrates. By 7 days of exposure to NGF (50 ng/ml) cell multiplication ceases ( Fig.…”

Section: Resultsmentioning

confidence: 99%

Establishment of a noradrenergic clonal line of rat adrenal pheochromocytoma cells which respond to nerve growth factor.

Greene¹,

Tischler²

1976

Proc. Natl. Acad. Sci. U.S.A.

5,017

3,231

View full text Add to dashboard Cite

A single cell clonal line which responds reversibly to nerve growth factor (NGF) subjected to three cycles of washing (with phosphate-buffered saline) and pelleting (500 X g for 5 min) in order to free them from cell debris, and were resuspended in growth medium and plated on plastic tissue culture dishes (Falcon Plastics). The following day, the lightly-adhering pheochromocytoma cells were mechanically dislodged from the plates by forceful aspiration and expulsion of the medium with a pasteur pipette, and replated on culture dishes which were coated with rat tail collagen (10). The cells were subsequently passaged two more times on collagen-coated culture dishes and three more times on plastic culture dishes. This strategy was employed for two reasons. First, newly dissociated pheochromocytoma cells adhered very poorly to plastic culture dishes. After several passages on collagen-coated dishes (to which the cells more firmly attached), the cells had adapted to culture and could be passaged onto plastic dishes. Second, the dissociated tumors contained, in addition to the cells of interest, other cell types which grew much more rapidly than the pheochromocytoma cells and which overgrew the cultures. Such cells adhered very firmly to plastic and collagen-coated substrates and tended to be left behind on the culture dishes after mechanical dislodgement. By the means described, the slowly growing pheochromocytoma cells could be adapted to culture without being overgrown.

show abstract

Section: Resultsmentioning

confidence: 99%

Establishment of a noradrenergic clonal line of rat adrenal pheochromocytoma cells which respond to nerve growth factor.

Greene¹,

Tischler²

1976

Proc. Natl. Acad. Sci. U.S.A.

5,017

3,231

View full text Add to dashboard Cite

show abstract

“…Proliferation of the cardiac cells was suppressed after 1-2 days by y-irradiation (60Co; 5000 rads in 25-30 sec, where one rad equals 1 X 10-2 J/kg). One to 5 days later, principal neurons were dissociated from superior cervical ganglia of newborn rats (Charles River CD) as previously described (1,18), and plated at a density such that many islands received only one or a few neurons. The cultures were grown in L-15 CO2 medium (1) containing 5% adult rat serum or 10% fetal calf serum (Microbiological Associates, 14-414), but lacking bovine serum albumin and Methocel.…”

mentioning

confidence: 99%

“…Neurons cultured in the near absence of nonneuronal cells develop adrenergic functions including synthesis, storage, release, and uptake of norepinephrine (NE); these neurons form synapses of adrenergic appearance (i.e., containing small granular vesicles) on each other (1)(2)(3)(4). Adrenergic transmission has not yet been detected at such synapses, apparently because of the insensitivity of the neurons to NE (unpublished; see ref.…”

mentioning

confidence: 99%

“…5). Moreover, acetylcholine (AcCh) synthesis is usually negligible, and cholinergic transmission is rarely seen (1,6,7,8). In contrast, when the neurons are cultured in the presence of nonneuronal cells (e.g., from ganglia or heart), or in medium conditioned by such cells, the neuronal population synthesizes both AcCh and NE (2,9), and many neurons form nicotinic cholinergic synapses on each other (6-8, 10, 11).…”

mentioning

confidence: 99%

See 1 more Smart Citation

Chemical transmission between rat sympathetic neurons and cardiac myocytes developing in microcultures: evidence for cholinergic, adrenergic, and dual-function neurons.

Furshpan¹,

MacLeish²,

O'Lague³

et al. 1976

Proc. Natl. Acad. Sci. U.S.A.

341

173

View full text Add to dashboard Cite

Electrophysiological studies were made on microcultures (300-500 Ism in diameter) in which solitary sympathetic principal neurons from newborn rats grew on previously dissociated rat heart cells. (1,6,7,8). In contrast, when the neurons are cultured in the presence of nonneuronal cells (e.g., from ganglia or heart), or in medium conditioned by such cells, the neuronal population synthesizes both AcCh and NE (2, 9), and many neurons form nicotinic cholinergic synapses on each other (6-8, 10, 11). These effects are graded. A higher proportion of conditioned medium or a greater number of nonneuronal cells gives a higher ratio of AcCh synthesis to catecholamine synthesis (12), a higher incidence of cholinergic transmission, and a higher proportion of synapses which lack small granular vesicles (13 (MacLeish, unpublished). He found that the incidence of detectable neuronmyocyte interaction was too low to be useful, perhaps in part because the endings of each neuron were sparsely distributed in the large field of myocytes; the few cases found all appeared to be cholinergic.It was plausible that the incidence of detectable interaction would increase if the innervation field of a given neuron was concentrated on a few cardiac myocytes. Thus, we made microcultures containing a single neuron and a small number of myocytes in an area only a fraction of a millimeter in diameter. In this paper, we report physiological observations on such microcultures; in the accompanying paper, Landis (15) reports electron microscopic observations on the same microcultures.In two previous studies (16, 17), explants of sympathetic ganglia were found to make functional contacts with explants of heart. METHODSSeveral methods for making microcultures suitable for electrophysiology and microscopy were successful, but none routinely so. The simplest method was, in brief, to apply 25 to 50 equally spaced droplets of dissolved collagen to a nonwetting polystyrene surface. When dried, these produced a grid (ca 50 mm2) of collagen islands, each island 300-500 Atm in diameter.Cardiac cells (myocytes and fibroblasts) were dissociated from hearts of newborn rats by use of collagenase (EC 3.4.24.3) (Worthington Type I; 1 mg/ml), and allowed to settle on the grid for about 2 hr. Almost all cells not adhering to the collagen islands could then be washed away with medium. Proliferation of the cardiac cells was suppressed after 1-2 days by y-irradiation (60Co; 5000 rads in 25-30 sec, where one rad equals 1 X 10-2 J/kg). One to 5 days later, principal neurons were dissociated from superior cervical ganglia of newborn rats (Charles River CD) as previously described (1,18), and plated at a density such that many islands received only one or a few neurons. The cultures were grown in L-15 CO2 medium (1) containing 5% adult rat serum or 10% fetal calf serum (Microbiological Associates, 14-414), but lacking bovine serum albumin and Methocel. Six platings (about 30 dishes per plating) were used in experiments reported here.For electrophysiological recordi...

show abstract

“…A number of studies have indicated that environmental factors can influence sympathetic neurons to acquire either adrenergic or cholinergic characteristics (Mains and Patterson 1973;O'Lague et al 1974 ;Patterson and Chun 1974;Rees and Bunge 1974;Walicke et al 1977;Kobayashi et al 1982Kobayashi et al , 1984.…”

mentioning

confidence: 99%

The effects of elevated potassium on sympathetic ganglion cells in culture.

Watanabe

Handa

Shimizu

1989

Tohoku J. Exp. Med.

View full text Add to dashboard Cite

Effects of a high potassium (40 mM) medium on the survival and differentiation of sympathetic ganglion cells from chick embryos were studied in dissociated cell culture. In the high potassium medium, survival of the sympathetic ganglion neurons was improved and catecholamine fluorescence of the nerve fibers increased with several days in culture, while acetylcholinesterase activity was slightly positive. In contrast, in the control medium, catecholamine fluorescence was only faintly observed, while acetylcholinesterase became strongly positive. Catecholamine fluorescence was intensified by increasing the potassium concentration in a medium, while it was diminished by reversing the potassium level back to the normal one. The effect of the high potassium medium on catecholamine fluorescence was reduced by Ca++ influx inhibitors, diltiazem or Mg.++ It is suggested that the high potassium medium increased the survival rate and prevented the sympathetic neurons from becoming cholinergic and allowed them to develop their adrenergic properties presumably through an increased level of the intracellular Ca++ due to Ca++ entry. sympathetic ganglion ; potassium ; cell culture ; differentiation Membrane potential is known to be an important factor involved in the mechanism and regulation of neuronal transmission. A question remains, however, if it has any trophic effects on neurons themselves. It has been shown that chronic depolarization of the cell membrane by an elevated extracellular potassium (K+) level increases the number of surviving neurons in culture of several cells, such as cerebellar neurons, ciliary ganglion neurons and dorsal root ganglion neurons

show abstract

Primary Cultures of Dissociated Sympathetic Neurons

Cited by 400 publications

References 43 publications

Establishment of a noradrenergic clonal line of rat adrenal pheochromocytoma cells which respond to nerve growth factor.

Establishment of a noradrenergic clonal line of rat adrenal pheochromocytoma cells which respond to nerve growth factor.

Chemical transmission between rat sympathetic neurons and cardiac myocytes developing in microcultures: evidence for cholinergic, adrenergic, and dual-function neurons.

The effects of elevated potassium on sympathetic ganglion cells in culture.

Contact Info

Product

Resources

About