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

Abstract

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of cholinergic transmission, and a higher proportion of synapses which lack small granular vesicles (13 (MacLeish, unpublished).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...

Growth cones of cultured sympathetic neurons contain adrenergic vesicles.

“…Sympathetic neurons dissociated from the superior cervical ganglia of newborn rats and grown for several weeks in the culture' conditions used in the present experiments synthesize, store, and release norepinephrine (18,19,20). Single sympathetic neurons grown in microcultures have been shown with physiological techniques to secrete norepinephrine onto cardiac myocytes (12), and the same neurons are found to contain vesicles with granular deposits in synaptic varicosities after permanganate fixation (17). The present study has demonstrated that some of the vesicles in the growth cones of these dissociated adrenergic neurons contain dense granules after permanganate fixation.…”

Sympathetic Sprouting Drives Hippocampal Cholinergic Reinnervation That Prevents Loss of a Muscarinic Receptor-Dependent Long-Term Depression at CA3–CA1 Synapses

“…How sympathetic fibers with an adrenergic phenotype restore cholinergic function in hippocampus is unknown. A small percentage (Ͻ1%) of SCG neurons are cholinergic (Schafer et al, 1998), and sympathetic neurons, including those in the SCG, are capable of gaining a cholinergic phenotype or switching their phenotype altogether from adrenergic to cholinergic (Furshpan et al, 1976;Potter et al, 1981;Landis and Keefe, 1983;Wolinsky and Patterson, 1983;Schafer et al, 1997Schafer et al, , 1998Francis and Landis, 1999;Yang et al, 2002;Weihe et al, 2005). Therefore, we entertained the possibility that sprouting from the SCG might restore cholinergic function, and thus mLTD expression, by replacing lost central cholinergic innervation to hippocampus, potentially by any one, or all, of these mechanisms.…”

“…However, the rapid loss of these fibers, which occurs within 5-7 d after unilateral ganglionectomy performed at a time when reinnervation is well established, strongly suggests that the SCG is directly responsible for, and likely the source of, the cholinergic reinnervation. Sympathetic neurons, including those in the SCG, have the unique ability to gain a cholinergic phenotype and/or functionally alter their phenotype from adrenergic to cholinergic (Furshpan et al, 1976;Potter et al, 1981;Landis and Keefe, 1983;Wolinsky and Patterson, 1983;Schafer et al, 1997Schafer et al, , 1998Francis and Landis, 1999;Yang et al, 2002;Weihe et al, 2005). Thus, the VAChT-positive fibers in hippocampus may arise from a gain of cholinergic function or an adrenergic to cholinergic phenotype switch.…”

Cellular synthesis and axonal transport of gamma‐aminobutyric acid in a photoreceptor cell of the barnacle.

“…Therefore, if both of these vesicles are synaptic vesicles, the transmitter substance of the cell might not only be GABA. Among tissue-cultured rat sympathetic neurones, there were some which released both ACh and norepinephrine (Furshpan, MacLeish, O'Lague & Potter, 1976). Multiple transmitter substances in a neurone were also suggested in molluscs (Brownstein, Saavedra, Axelrod, Zeman & Carpenter, 1974;Cottrell, 1976).…”