Showing papers by "Harold Gainer published in 1998"

Stationary organotypic cultures of oxytocin and vasopressin magnocellular neurones from rat and mouse hypothalamus.

Abstract: Rat and mouse hypothalami from postnatal animals containing highly differentiated neurones survive very well in long-term (>15 days in vitro, DIV) stationary organotypic cultures. Magnocellular oxytocin (OT) and vasopressin (VP) neurones are present in identifiable paraventricular (PVN), supraoptic (SON) and accessory (ACC) nuclei in these cultures. After 15 DIV in standard medium immunocytochemistry revealed 427 +/- 63 OT cells and 217 +/- 27 VP cells per cultured rat hypothalamus, and 380 +/- 72 OT cells and 622 +/- 91 VP cells per cultured mouse hypothalamus. Following a 7-day adaptation period in standard culture medium containing serum, the rat slice-explants survived very well after subsequent transfer to defined, serum- free media (SFM) for an additional 8 days. The number of OT cells surviving in SFM was 612 +/- 147 OT cells per cultured rat hypothalamus. Only 0.5% of the magnocellular OT and VP neurones in the cultures appeared to express both peptides. Experiments on c-fos gene expression in these cultures showed that while only 12% of the magnocellular OT and VP neurones contained barely detectable Fos protein in their nuclei under control conditions, potassium depolarization of these cultures for 3 h produced intense c-fos expression in 87-91% of these cells. Thus, magnocellular neurones in these cultures are sufficiently stable and responsive to permit long-term physiological and gene expression studies to be done under defined media conditions.

Cell-specific gene expression in oxytocin and vasopressin magnocellular neurons.

Abstract: The oxytocin (OT) and vasopressin (VP) expressing magnocellular neurons in the hypothalamic-neurohypophysial system (HNS) have been the most studied of all the neuroendocrine cell-types. Despite this, our understanding of the mechanisms that underly the cell-specific expression of the peptide genes in these neurons has remained obscure. Part of the reason for this may be related to the close apposition of the OT and VP genes in the chromosomal locus, the genes being separated by as little as 3.5 kb in the mouse, and their interactions which are critical for cell-specific expression of the genes. Recent studies using intact rat OT and VP constructs in transgenic mice, and rat and mouse VP genes with CAT inserts in exon III as reporters in transgenic rats and mice, respectively, have suggested the presence of cell-specific enhancer elements in the 3’ downstream (intergenic region, IGR) region of the VP gene. Evidence in favor of this view is presented from transgenic mouse studies on the expression of mouse OT- and VP-CAT gene constructs. Oxytocin and vasopressin phenotypes in the magnocellular neuronal population have traditionally been assessed by either immunocytochemical or in situ hybridization histochemical methods leading to the view that these genes are never coexpressed. However, more sensitive methods show that most OT cells also express some VP mRNA, and most VP cells contain some OT mRNA. A third phenotype containing equivalent levels of both OT and VP mRNA can also be found under some conditions, thereby complicating our analysis of cell-specificity. A continuing problem hindering studies of the regulation of OT and VP gene expression in neurons, is the absence of an appropriate cell line to examine these issues. We have found that stationary slice-explant cultures allow for excellent preservation of highly differentiated magnocellular neurons in long-term culture, and that these culture can be used for physiological and pharmacological studies and analysis of gene expression.

Molecular diversity in neurosecretion: reflections on the hypothalamo-neurohypophysial system.

Abstract: 1. The diversity of molecules involved in various aspects of neurosecretion, such as proprotein processing, axonal transport of large dense core vesicles (LDCVs), and regulated secretion, is discussed in the context of the hypothalamo-neurohypophysial system (HNS). 2. Recent studies have uncovered a family of at least seven processing enzymes known as proprotein convertases (PCs) which are involved in proteolytically cleaving protein precursors at paired basic amino acid motifs to yield biologically active peptides. Three of these, PC1(3), 2, and 5, are found in neurons and are involved in producing regulated secretory peptide products. 3. The axonal transport of LDCVs occurs on microtubule tracks by still unknown mechanisms. There are over 11 distinct kinesin-related molecules that have now been identified as possible microtubule motor candidates. 4. Calcium channels in the nervous system are known to be derived from at least five alpha-subunit and four beta-subunit genes with multiple alternatively spliced isoforms in each case. These could account, in part, for the varied calcium currents found in the HNS. 5. The large number of proteins and isoforms now demonstrated to be involved in regulated secretion are discussed, with a focus on LDCV compositions and the synaptotagmin gene family.

Special Issue: Modulating Mechanisms of Neuroendocrine Cell Activity

Abstract: This Special Issue focuses on recent developments in our understanding of cellular and molecular mechanisms involved in the regulation of neurosecretory activity. In recent years, a wealth of information has been gathered about the various mechanisms involved in neurosecretion. These include data derived from explorations of issues such as the molecular regulation of neuropeptide gene expression and the roles of neurotransmitters and hormones, second messenger, and immediate-early genes involved in transcriptional translational and secretory activity. Recent advances in our understanding of the molecular machinery involved in packaging of neurosecretory products in secretory vesicles (or granules) and the subsequent axonal transport of these vesicles to their release sites for exocytosis of their contents have allowed for new hypotheses about neurosecretion. The role of ion channels in secretion, particularly those selective for calcium, is now becoming better understood. The kinetics of calcium ion permeability and its temporal correlation with capacitance changes in the secretory membrane are providing interesting insights into the mechanisms of secretion and its regulation. Finally, the role of long-term mechanisms such as light-darkness cycles and circadian and seasonal rhythms and the plastic changes related to previous activity and/or during development are adding new components to the rich repertoire of regulatory mechanisms continuously adjusting the activity of secretory neurons. The articles in this issue consider a wide range of mechanisms of regulation in a variety of secretory systems. It contains papers that present various physiological and network aspects of the regulation of neurosecretory cell activity. Bourque et al, describe some ionic properties of the phasic patterning of electrical activity in the neurosecretory cell body in hypothalamic vasopressin secreting neurons, focusing on the role of depolarizing afterpotentials. Hatton and Li provide an account of the role of intracellular calcium in the patterning of activity of magnocellular hypothalamic cells. Misgeld et al., offer new views on mechanisms generating bursting electrical activity in networks of cultured