Adult rat hepatocytes have been previously isolated and maintained in monolayer culture, but attempts to stimulate DNA synthesis have been unsuccessful. Hormonal conditions are now described which induce DNA synthesis in cultured hepatocytes from partially hepatectomized rats. DNA synthesis was determined autoradiographically by the incorporation of [3H~thymidine into nuclei of morphologically distinct hepatocytes. Insulin (4-4000 nM) or epidermal growth factor (10 ng/ml) alone caused significant increases in the labeling index. The two hormones together acted synergistically to produce labeling indices of 35-50% on the third day of culture, compared with 2-7% in control cultures. The addition of glucagon (400 nM) further increased the labeling index. Dexamethasone (80 ng/ml) inhibited DNA synthesis but, under certain conditions, enhanced cell attachment. Growth hormone and triiodothyronine had no significant effect on DNA synthesis. The mixture of epidermal growth factor, insulin, and glucagon also stimulated incorporation of [3H~thymidine into phenol-extracted DNA. Although DNA synthesis was stimulated, cell division occurred infrequently. These data suggest a prominent role for epidermal growth factor in promoting hepatic DNA synthesis by acting in concert with insulin and glucagon.Liver regeneration after partial hepatectomy has been employed widely as an experimental model of mammalian cell division (1, 2). However, the regulatory mechanisms that stimulate quiescent hepatocytes to proliferate are poorly understood. Cross circulation studies between partially hepatectomized rats and intact animals suggest the involvement of humoral stimuli (3). Although several hormones have been implicated (4-9), definitive studies have been difficult to carry out because of complex hormonal interactions in vivo. The development of cell cultures of normal adult hepatocytes would provide a system to characterize those factors necessary for DNA synthesis and cell division. However, while fetal liver (10) and hepatoma cells (11) can be grown in culture, there have been, as yet, no successful attempts to induce DNA synthesis or cell division in adult liver cells in culture. Studies with hepatocytes from partially hepatectomized rats have shown that DNA synthesis declines progressively with the time in culture (12), and that only 50-60% of the isolated hepatocytes adhere to the substratum.We report conditions that increase to 90% the number of hepatocytes that adhere to flasks as well as conditions which stimulate DNA synthesis. Epidermal growth factor (EGF), in combination with insulin and glucagon, has been found to greatly enhance DNA synthesis. MATERIALS AND METHODSIsolation and Plating of Liver Cells. Male rats (150-300 g, Sprague-Dawley, Madison, Wisc.) were subjected to partial hepatectomy (13) and then starved for 18-24 hr. The remaining liver fragments were perfused with collagenase and hyaluronidase as previously described (14), except that after catheterization of the portal and superior vena cava veins the fr...

Recurrent inhibition in olfactory bulb mitral cells is mediated via reciprocal dendrodendritic synapses with granule cells. Although GABAergic granule cells express both NMDA and non-NMDA glutamate receptors, dendrodendritic inhibition (DDI) relies on the activation of NMDA receptors. Using whole-cell recordings from rat olfactory bulb slices, we now show that olfactory NMDA receptors have a dual role; they depolarize granule cell spines, and they provide a source of calcium that can evoke GABA exocytosis. We demonstrate that exogenous NMDA can trigger GABA release after blockade of voltagedependent calcium channels (VDCCs) with Cd. We also find that postsynaptic depolarization alone can evoke GABA release via a separate mechanism that relies on calcium influx through Cd-sensitive VDCCs. By selectively manipulating postsynaptic responses in granule cells with high-K or low-Na extracellular solutions, we show that endogenous glutamate can elicit GABA release via both NMDA receptor-and VDCC-dependent pathways. Finally, we find that blockade of Na channels in granule cells with tetrodotoxin enhances DDI, presumably by reducing the depolarization of granule cells during DDI and thereby increasing the driving force for Ca entry through NMDA receptors. These results provide evidence of a novel mechanism for evoked transmitter release that depends on Ca influx through ionotropic receptors and provides a new potential site for synaptic plasticity in the olfactory bulb.

A simple model is depicted below that suggests some unifying principals in the action of cyclic nucleotides in the GO-to-G+ interconversion, differentiation, and transformation (see article). The letters with G+ subscripts (AG+ through EG+) represent cell states at different increasing levels of "determination" (see sect. vE). Cells in each of these states are continuously reproducing themselves through cell division (i.e., they are in G+). As an alternative to cell reporduction, cells at each level may move toward or enter GO, which is conceived of not only as a quiescent state but also as a state in which differentiated properties are more fully expressed. This state is designated by letters with GO subscripts (AGO through EGO). Entrance into this more expressed state will usually be reversible (nerve cells and red blood cells are two exceptions). Sometimes movement toward GO and full expression may require a number of cell divisions. Ultimately, however, there usually will be a slowing or cessation of cell division. The transformed state, according to this model, is one in which cells have lost the ability to enter the "expressed" CO state. However, they do remain differentiated in the sense that they have maintained their level of determination and can be induced to enter into the expressed state, as for example in the case of DBcAMP treatment of transformed fibroblasts. In most cases, cAMP appears to stimulate cells to proceed toward XGO (where X = A,B,C,D, or E) and toward fuller expression of their differentiated functions. It is not the sole mediator of this transition. In cell types where cAMP plays this role, transformation may arise through a defect in the ability to raise cAMP levels in response to growth-regulatory signals or in a defect in the cell's ability to respond to cAMP. In other cell types, cAMP may not be involved in the CO-to-C+ transition or may act in the opposite direction (see sect. II). It remains to be seen whether these situations are ture exceptions or whether different loci of regulation are involved. For example it is possible that in certain cases where cAMP has been shown to stimulate growth that it is stimulating growth toward a more expressed state. Other actions of cAMP relating to cell-cycle traverse have been discussed (sect. III). Investigations of the action of cGMP are still at a preliminary stage of development. There is evidence consistent with the idea that cGMP mediates conversion toward the G+ state in some cell types (see sect. II) under certain conditions. However, further studies are required to establish this as a fact. There has been little or no reported evidence relating to a role for cGMP in expression of differentiated properties, nor has there been any significant evidence as yet for other cell-cycle roles of cGMP. It should be apparent that the areas of biology covered in this review are only beginning to evolve biochemically...

Odor perception depends on a constellation of molecular, cellular, and network interactions in olfactory brain areas. Recently, there has been better understanding of the cellular and molecular mechanisms underlying the odor responses of neurons in the olfactory epithelium, the first-order olfactory area. In higher order sensory areas, synchronized activity in networks of neurons is known to be a prominent feature of odor processing. The perception and discrimination of odorants is associated with fast (20–70 Hz) electroencephalographic oscillations. The cellular mechanisms underlying these fast network oscillations have not been defined. In this study, we show that synchronous fast oscillations can be evoked by brief electrical stimulation in the rat olfactory bulb in vitro, partially mimicking the natural response of this brain region to sensory input. Stimulation induces periodic inhibitory synaptic potentials in mitral cells and prolonged spiking in GABAergic granule cells. Repeated stimulation leads to the persistent enhancement in both granule cell activity and mitral cell inhibition. Prominent oscillations in field recordings indicate that stimulation induces high-frequency activity throughout networks of olfactory bulb neurons. Network synchronization results from chemical and electrical synaptic interactions since both glutamate-receptor antagonists and gap junction inhibitors block oscillatory intracellular and field responses. Our results demonstrate that the olfactory bulb can generate fast oscillations autonomously through the persistent activation of networks of inhibitory interneurons. These local circuit interactions may be critically involved in odor processing in vivo.