Ultrastructural characterization of rat neurons in primary culture.

Few studies have addressed the ultrastructure and morphology of neurons in primary pure culture. We therefore use immunohistochemistry and electron microscopy to investigate the ultrastructure of cultured neurons during extended incubation in vitro. Rat cerebral cortex neurons were cultured in Neurobasal™ medium. Adherent cells developed as networks of single neurons or clusters depending on the plating density. Almost all surviving cells were neurons as demonstrated by neurofilament immunolabeling. The number of cultured neurons increased substantially to 14-21 days in vitro (DIV) and then plateaued and subsequently declined. From DIV 1-10 neurons extended large neurites, followed by the development of fine and dense neurites, and neurones survived until DIV 30-50. Notably, numerous mitochondria were observed along fibrous elements within neurites, suggestive of active intracellular trafficking. Electron microscopy also revealed that multiple types of synapses were formed between neurons. These ultrastructural results confirm previous reports of electrophysiological activity in cultured neurons. However many neurons contained distorted mitochondria and abnormal organelles including multilamellar vesicles and multivesicular myeloid bodies. The proportion of neurons containing abnormal organelles increased significantly in culture medium supplemented with antibiotics. On long-term culture neuronal death and apoptotic nuclei were observed. Despite the presence of abnormal organelles, the ultrastructure of cultured neurons was very similar to that of in vivo neurons; in vitro culture therefore provides a useful tool for studies on neuronal development, aging, and neurotransmission.

Recent advances on endogenous Na+,K+-ATPase inhibitors: clinical investigation and purification.

Evidence exists which demonstrates the relationship between a Natriuretic Factor or Na+,K+-ATPase inhibitor and volemic expansion, both in man and animal. Patients having extracellular volume expansion have been studied for the effect of their plasma on erythrocytes 3H-ouabain binding. High levels of ouabain-like activity was found in plasma from acromegalic patients and patients with chronic renal failure. High levels were also observed in some hypertensive patients. A partial purification of such a compound was performed from urine of hypertensives. The partially purified compound inhibited to a greater extent the Na+,K+-ATPase semi-purified from dog kidney than that from sheep brain. The present data are consistent with the possible regulation of the activity or the secretion of plasma ouabain-like activity by extracellular volume.

Investigation of the endogenous Na+-pump inhibitor in essential hypertension and blood volume expansion.

The digitalis-like activities of plasma extracts from 108 patients and normal subjects were measured by their ability to compete with ouabain for binding to the digitalis sites of the Na+-pump. High levels were found in 18 of 54 untreated patients with moderate hypertension, 10 of 14 patients with end-stage renal failure and six patients with active acromegaly. These levels returned to control values after dialysis in the patients with renal insufficiency and high levels of the inhibitor, and after successful surgery and cobalt therapy in seven acromegalic patients. An increase in circulating Na+, K+-ATPase inhibitor was also found in rats after chronic sodium loading. These results indicate that levels of the circulating compound with digitalis-like properties do not result from high blood pressure but, rather, are related to blood volume and Na+ balance.