Development of synaptic transmission at autonomic synapses in vitro revealed by cytochrome oxidase histochemistry

Abstract

We have studied the development of synaptic transmission by innervating sympathetic neurons in vitro and monitoring synaptic activity with both physiological recording and cytochrome oxidase histochemistry. The onset of synaptic transmission was reflected in increased cytochrome oxidase reaction product within individual neurons. Within 24 hours of co-culture, relatively low frequency suprathreshold potentials were recorded in approximately 20% of the innervated neurons. At this stage the cytochrome oxidase activity of innervated neurons, as assessed by optical density of the histochemical reaction product, was increased twofold compared with uninnervated neurons. Over the next 2-4 days of innervation, changes in the pattern and extent of synaptic activity and superthreshold events were accompanied by a net fourfold increase in cytochrome oxidase activity levels compared with noninnervated neurons. The increase in density of cytochrome oxidase reaction product observed after innervation was reversed completely by blockade of synaptic transmission. Differences in the efficacy of synaptic input provided to the sympathetic neurons by appropriate versus inappropriate presynaptic sources was determined by co-culturing sympathetic neurons with explants that contained either preganglionic neurons or somatic motor neurons. Although sympathetic neurons innervated by motor neuron explants had increased levels of cytochrome oxidase activity compared with noninnervated controls, the density of cytochrome oxidase reaction product was even greater in sympathetic neurons innervated by preganglionic explants. We conclude that both the onset of innervation of sympathetic neurons as well as the subsequent maturation of synaptic function is directly reflected in graded increases in cytochrome oxidase reaction product.

Target‐derived factors regulate the expression of Ca(2+)‐activated K+ currents in developing chick sympathetic neurones.

“…Innervation appears to induce the expression of several proteins important for the physiological function of chick sympathetic neurones. For example, Role and co-workers have found that innervation by spinal preganglionic fibres regulates the expression of functional nicotinic acetylcholine receptors both in vitro (Role, 1988;Mawe, Gardette, D'Agostaro & Role, 1990;Gardette, Listerud, Brussard & Role, 1991) and in situ (Moss & Role, 1993).…”

“…By contrast, IK(ca) density was invariably less than 0-01 mA cm-2 in each of sixteen neurones cultured in the absence of NGF (Fig. 6B) In previous studies, we have shown that the density of IA in sympathetic neurones is maximal by E13 (Raucher & Dryer, 1994 (Dourado & Dryer, 1992 Ebendal, 1992;Yamamori, 1992 (Role, 1988;Mawe, Gardette, D'Agostaro & Role, 1990;Gardette, Listerud, Brussard & Role, 1991) and in situ (Moss & Role, 1993). We have previously shown that interactions with spinal cord explants can also induce the expression of IA in chick sympathetic neurones developing in vitro (Raucher & Dryer, 1994) and that innervation is essential for IK(ca) expression in parasympathetic ciliary ganglion neurones developing in situ (Raucher & Dryer, 1994).…”

Enhanced ACh sensitivity is accompanied by changes in ACh receptor channel properties and segregation of ACh receptor subtypes on sympathetic neurons during innervation in vivo

“…bright-field images of sections were captured and displayed on the Gideosystem, and the perimeters of individual neurons randomly selected from all lumbar ganglia (Ll-L5) were traced with a cursor. The optical density (OD) of the circumscribed area directly correlates with the density of reaction product and hence the level of CO activity in the individual cell (Mawe et al, 1990).…”

“…To evaluate whether the arrival of presynaptic fibers was associated with functional innervation of the neurons, we assessed their electrical activity by cytochrome oxidase (CO) histochemistry (Mawe et al, 1990). Both techniques indicate a significant increase in innervation of lumbar ganglia between embryonic day (ED) 11 and ED 17.…”

Effects of development and altered gravity conditions on cytochrome oxidase activity in a vestibular nucleus of the larval teleost brain: A quantitative electronmicroscopical study

“…Other studies have shown that the onset of synaptic transmission in sympathetic neurons in vitro was reflected by an increase of cytochrome oxidase reaction product in individual neurons. A further innervation of these neurons increased the cytochrome oxidase reactivity and even more if the neurons were activated by preganglionic neurons (Mawe et al, 1990). Mawe et al concluded that the onset of innervation, as well as the subsequent maturation of the synaptic function, stimulate cytochrome oxidase activity.…”