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.

Figure 1

Representative low power views of rat hypothalamic organotypic culures after 14 days in vitro. The Oxt and Avp expressing neurons are identified by immunohistochemistry using antibodies against Oxt- (top panel) or Avp- (bottom panel) associated neurophysins (see Methods). All three hypothalamic nuclei containing the magnocellular neurons (MCNs) are present in these slice-explants, and are identified here as ACC (accessory nucleus), paraventricular nucleus (PVN) and the supraoptic nucleus (SON). Another nucleus in the explant that is shown is the suprachiasmatic nucleus (SCN) which expresses Avp but not Oxt, and in neurons that do not belong to the MCN phenotype. The scale line shown represents 1 mm in both panels.

Figure 2

Effect of culturing rat hypothalamic slices in the absence (control) and presence of either 10ng/ml CNTF or 10ng/ml LIF in the culture medium for 14 days, on the total number of immunohistochemically identified Oxt and Avp neurons in the organotypic cultures (see Table 1 for the numbers of neurons in the individual nuclei under these conditions). Data are expressed as means ± SEM, with n=20 for controls and 18 each for CNTF- and LIF-treated cultures. Statistical differences, shown over columns by asterisks represent p values < 0.01 (compared to control), were calculated by ANOVA, followed by Fischer’s protected LSD test.

Figure 3

Effect of culturing mouse hypothalamic slices in the absence (control) and presence of either 10ng/ml CNTF or 10ng/ml LIF in the culture medium for 14 days, on the total number of immunohistochemically identified Oxt and Avp neurons in the organotypic cultures (see Table 2 for the numbers of neurons in the individual nuclei under these conditions). Data are expressed as means ± SEM, with n=20 for controls, 15 for CNTF- and 16 for LIF-treated cultures. Statistical differences, shown over columns by asterisks represent p values < 0.01 (compared to control), were calculated by ANOVA, followed by Fischer’s protected LSD test.