Rat sympathetic neurons and cardiac myocytes developing in microcultures: correlation of the fine structure of endings with neurotransmitter function in single neurons.

Abstract

Microcultures containing single sympathetic principal neurons and small numbers of dissociated heart myocytes were prepared from newborn rats. After the transmitter properties of the neuron were studied by electrophysiological experiments, the microculture was examined with the electron microscope. Single neurons of either putative cholinergic or putative adrenergic character made morphological synapses on themselves (autapses), although only cholinergic autapses were detected electrophysiologicaily. Numerous axonal varicosities were present adjacent to the myocytes but no synaptic specializations were evident. After permanganate fixation to localize endogenous norepinephrine, the endings of neurons which appeared to secrete catecholamines contained many small granular vesicles, while the endings of neurons which appeared to secrete acetylcholine contained none. The endings of neurons which apparently secreted both catecholamines and acetylcholine contained only occasional small granular vesicles.If sympathetic neurons, dissociated from the superior cervical ganglia of newborn rats, are grown in the absence of non-neuronal cells, then the cultures are predominantly adrenergic in character (1). However, if these same neurons are grown in the presence of non-neuronal cells or in medium conditioned by them, the cultures synthesize significant amounts of acetylcholine (AcCh) as well as catecholamines; if sufficiently high concentrations of conditioned medium are used, then the cultures become predominantly cholinergic (2-5). An interesting question is whether single neurons in these cultures display either exclusively adrenergic or cholinergic properties or whether single neurons possess mixed properties. In a preliminary series of experiments (6), 3-week-old cultures that synthesized predominantly catecholamines, predominantly AcCh, or significant amounts of both were obtained by using varying proportions of conditioned medium; the incidence of cholinergic synapse formation was estimated electrophysiologically and, varicosities were examined with the electron microscope in sister cultures fixed with permanganate to localize norepinephrine (NE) in small granular vesicles (SGV) (7). In predominantly adrenergic cultures, SGV were absent from only 1% of the endings, but as the amount of AcCh synthesized and the fraction of neurons shown to interact cholinergically increased, the proportion of terminals in the culture lacking SGV also increased. Because some terminals containing numerous SGV and resembling those in predominantly adrenergic cultures were also observed in mixed cultures, it seemed likely that the cultures contained at least two populations of neurons. On the other hand, several observations raised the possibility that some neurons were not exclusively adrenergic or cholinergic: (i) in some cultures, the proportion of terminals which contained no SGV and were presumably cholinergic was less than the proportion of neurons interacting cholinergically; (ii) some terminals with SGV contained relativel...

Myocardial Infarction Causes Transient Cholinergic Transdifferentiation of Cardiac Sympathetic Nerves via gp130

“…Sensitive HPLC assays for NE and other catecholamines have been available for many years, but it was not possible to quantify ACh directly until the recent development of a sensitive mass spectrometry assay. Therefore, cholinergic function in the past was assayed indirectly by measuring ChAT activity in tissue homogenates (Schotzinger and Landis, 1988) or by the lack of a potassium permanganate precipitate in EM analysis of vesicles (Landis, 1976;Kanazawa et al, 2010). The advent of a sensitive assay for ACh has allowed us to quantify both NE and ACh in the same tissue and thus track relative changes over time.…”

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.…”

Massive Autaptic Self-Innervation of GABAergic Neurons in Cat Visual Cortex

“…The existence of autapses has been reported primarily from cultures (Crain, 1971;Landis, 1976;Bekkers and Stevens, 1991;Segal, 1991), and their formation has been considered an artifact because of the lack of appropriate postsynaptic targets. Occasional autapses on neurons have been reported in anatomical studies from various brain regions (Held, 1897;Chan-Palay, 1971;Scheibel and Scheibel, 1971;Shkol'nik-Yarros, 1971; Van der Loos and Glaser, 1972;DiFiglia et al, 1976;Karabelas and Purpura, 1980;Peters and Proskauer, 1980;Preston et al, 1980;Kuffler et al, 1987;Shi and Rayport, 1994;Lübke et al, 1996), but in view of our difficulty predicting the existence of autapses on the basis of light microscopy, some of the previous predictions will require reexamination, including EM or physiology.…”