Effects of ciliary neurotrophic factor and leukemia inhibiting factor on oxytocin and vasopressin magnocellular neuron survival in rat and mouse hypothalamic organotypic cultures

Abstract

Organotypic cultures of mouse and rat magnocellular neurons (MCNs) in the hypothalamo-neurohypophysial system (HNS) have served as important experimental models for the molecular and physiological study of this neuronal phenotype. However, it has been difficult to maintain significant numbers of the MCNs, particularly vasopressin MCNs, in these cultures for long periods. In this paper, we describe the use of the neurotrophic factors, leukemia inhibiting factor (LIF) and ciliary neurotrophic factor (CNTF) to rescue rat vasopressin (Avp)- and oxytocin (Oxt) – MCNs from axotomy-induced, programmed cell death in vitro. Quantitative data are presented for the efficacy of the LIF family of neurotrophic factors on the survival of MCNs in three nuclei, the paraventricular (PVN), supraoptic (SON), and accessory (ACC) nuclei in the mouse and rat hypothalamus.

Neuronal activity and axonal sprouting differentially regulate CNTF and CNTF receptor complex in the rat supraoptic nucleus

“…These data are consistent with the observations of Rusnak, et al (2003) who indicated that addition of CNTF to dissociated cultures of magnocellular neurons did not result in increased neuronal survival. However, when CNTF is applied to stationary organotypic cultures of the SON and PVN, survival of axotomized magnocellular neurons is increased significantly (House, et al, 2009, Rusnak, et al, 2002, Rusnak, et al, 2003, Vutskits, et al, 1998, Vutskits, et al, 2003) indicating that CNTF is acting through astrocytes or other glial elements. The precise mechanism(s) by which CNTF influences neuronal survival and axonal sprouting remains unclear.…”

“…CNTF signals through the tripartite receptor complex consisting of the specific receptor for CNTF, CNTF receptor alpha (CNTFRα), and the gp130 and LIFRβ receptor subunits, to promote the survival of multiple neuronal phenotypes affected by injury across numerous species (Albrecht, et al, 2002, Arakawa, et al, 1990, Ip, et al, 1991, Larkfors, et al, 1994, Lehwalder, et al, 1989, Magal, et al, 1991, Sendtner, et al, 1990). Moreover, CNTF is a potent promoter of hypothalamic magnocellular neuron survival in vitro (House, et al, 2009, Rusnak, et al, 2002, Rusnak, et al, 2003, Vutskits, et al, 1998, Vutskits, et al, 2003). In our studies we have demonstrated an increase in CNTF-immunoreactivity (Watt, et al, 2006) and CNTFRα mRNA expression (Watt, et al, 2009) within the non-injured contralateral SON, which contains the sprouting and metabolically active magnocellular neurons.…”

Inhibition of the Jak-STAT pathway prevents CNTF-mediated survival of axotomized oxytocinergic magnocellular neurons in organotypic cultures of the rat supraoptic nucleus

“…Organotypic cultures were prepared using a modification of techniques described previously (House, et al, 2009, House, et al, 2006, House, et al, 1998, Rusnak, et al, 2002, Rusnak, et al, 2003, Shahar, et al, 2004). Briefly, 6-day-old Sprague Dawley rat pups were decapitated and their brains were removed and placed in chilled Geys Balanced Salt Solution (Gibco, Grand Island, NY) enriched with glucose (5 mg/ml; Sigma).…”

“…The explants were then incubated for 36 hours at 4°C in highly specific monoclonal mouse antibodies against OT-neurophysin (PS 38, 1:500; a gift from Dr. Harold Gainer) or VP-neurophysin (PS 41, 1:500; a gift from Dr. Harold Gainer). These antibodies were characterized by Ben-Barak et al (1985) and have been consistently utilized to label OT and VP neurons and their processes within organotypic cultures (House, et al, 2009, House, et al, 2006, House, et al, 1998, Rusnak, et al, 2002, Rusnak, et al, 2003, Shahar, et al, 2004, Vutskits, et al, 1998, Vutskits, et al, 2003). Next the cultures were incubated in horse anti-mouse biotinylated secondary antibody (1:500; Vector), followed by avidin-biotin complex (ABC; 10 μl/ml in PBS; Vector ABC Elite kit) for 1 hour at room temperature.…”

“…CNTF has been previously implicated in hypothalamic magnocellular neuron sprouting in vitro (Vutskits, et al, 1998) and has been demonstrated to promote motor neuron sprouting in vivo (Gurney, et al, 1992, Guthrie, et al, 1997, Kwon and Gurney, 1994, Oyesiku and Wigston, 1996, Siegel, et al, 2000, Simon, et al, 2010, Ulenkate, et al, 1994, Wright, et al, 2007, Xu, et al, 2009). Moreover, multiple reports have demonstrated that CNTF promotes survival of magnocellular neurons in the PVN and SON following axotomy in organotypic cultures (House, et al, 2009, Rusnak, et al, 2002, Rusnak, et al, 2003, Vutskits, et al, 1998, Vutskits, et al, 2003). However, the specific intracellular signal transduction pathway(s) which mediate these distinct responses have not yet been elucidated.…”