Loss of leukemia inhibitory factor receptor beta or cardiotrophin-1 causes similar deficits in preganglionic sympathetic neurons and adrenal medulla

Abstract

Leukemia inhibitory factor (LIF) receptor beta (LIFRbeta) is a receptor for a variety of neurotrophic cytokines, including LIF, ciliary neurotrophic factor (CNTF), and cardiotrophin-1 (CT-1). These cytokines play an essential role for the survival and maintenance of developing and postnatal somatic motoneurons. CNTF may also serve the maintenance of autonomic, preganglionic sympathetic neurons (PSNs) in the spinal cord, as suggested by its capacity to prevent their death after destruction of one of their major targets, the adrenal medulla. Although somatic motoneurons and PSNs share a common embryonic origin, they are distinct in several respects, including responses to lesions. We have studied PSNs in mice with targeted deletions of the LIFRbeta or CT-1 genes, respectively. We show that LIF, CNTF, and CT-1 are synthesized in embryonic adrenal gland and spinal cord and that PSNs express LIFRbeta. In embryonic day 18.5 LIFRbeta (-/-) and CT-1 (-/-) mice, PSNs were reduced by approximately 20%. PSNs projecting to the adrenal medulla were more severely affected (-55%). Although LIFRbeta (-/-) mice revealed normal numbers of adrenal chromaffin cells and axons terminating on chromaffin cells, levels of adrenaline and numbers of adrenaline-synthesizing cells were significantly reduced. We conclude that activation of LIFRbeta is required for normal development of PSNs and one of their prominent targets, the adrenal medulla. Thus, both somatic motoneurons and PSNs in the spinal cord depend on LIFRbeta signaling for their development and maintenance, although PSNs seem to be overall less affected than somatic motoneurons by LIFRbeta deprivation.

Figure 1.

LIFRβ-IR in the adult rat and mouse spinal cord. A, Overview showing the distribution of LIFRβ in the ventral horn (circled), dorsal horn, and region of the mouse intermediolateral column (circled). Scale bar, 500 μm. B, C, Higher magnifications of the circled areas in A show LIFRβ-positive neurons in both locations. Scale bar, 50 μm. D, Overview showing the distribution of LIFRβ-IR in the ventral horn, dorsal horn, and region of the rat intermediolateral column (circled). Scale bar, 1000 μm. E, LIFRβ immunostaining (red) and DAPI staining of neurons within the IML. F, Retrograde tracing with FluoroGold (FG; green) from the adrenal medulla shows positive neurons combined with DAPI labeling. Comparison of E and F suggests that cells marked by white * and # are LIFRβ-positive PSNs projecting to adrenal chromaffin cells. Scale bars: E, F, 20 μm. VH, Ventral horn; DH, dorsal horn; cc, central canal; IML, intermediolateral column.

Figure 2.

Expression of LIFRβ ligands in the mouse adrenal gland and spinal cord. A, Expression of CNTF, LIF, CT-1, CNTF receptor α (CNTFRα), and LIFRβ mRNAs, revealed by RT-PCR, in the adrenal gland (ag) and spinal cord (sc) at E14, E15, E16.5, and adult. B–D, CNTF expression in Schwann cells of the adult mouse adrenal medulla. The arrows identify two examples of clearly double-labeled cells. B, CNTF-IR. C, S100-IR. D, Overlay. Scale bars, 100 μm. E, CNTF-IR in adrenal medullary cells isolated from P6 rat adrenal glands. F, TH-IR. Most cells that are immunoreactive for CNTF express TH-IR, indicating that they are chromaffin cells. Two cells with CNTF-IR (arrows) are TH negative and possibly represent Schwann cells. Scale bars, 20 μm.

Figure 3.

Quantification of AChE-positive, Nissl-stained PSNs within an area of 120 × 240 μm of the intermediate gray in the spinal cord at E18.5. A, Overview showing AChE-positive neurons (brown) in the intermediolateral column (IML), ventral horn (VH), and dorsal root ganglia (DRG). DH, Dorsal horn; CC, central canal. Scale bar, 200 μm. B, Higher magnification of the IML indicated by the asterisk in A. Scale bar, 25 μm. C, D, Quantification of PSNs in LIFRβ-deficient (C) and CT-1-deficient mice (D) reveals reductions of −22.5 and −18.5%, respectively. Data are given as mean ± SEM.

Figure 4.

Counts of ChAT mRNA-positive PSNs in E13.5 spinal cord. A, ChAT in situ shows positive motoneurons in the ventral horn (VH) and, more dorsally, PSNs predominantly located in the intermediolateral column (IML). CC, Central canal. Scale bar, 100 μm. B, Higher magnification of the IML indicated by the asterisk in A. Scale bar, 25 μm. Quantification of PSNs in LIFRβ-deficient mice reveals a 9% loss compared with wild-type littermates.

Figure 5.

Retrograde tracing with the lipophilic dye DiI from the adrenal gland to the spinal cord in E18.5 LIFRβ (+/+) and LIFRβ (−/−) mice. A, The arrows point to the site where the DiI crystal was inserted. B, Quantitative determination of DiI-traced PSN numbers of LIFRβ (+/+) and LIFRβ (−/−) mice. The numbers of positive PSNs are reduced by 55% in LIFRβ mutants. C, E, DiI-traced PSNs. Positive neurons are located within the intermediolateral column. They extend processes into the area of the intercalated nucleus and central nucleus (arrows). D, F, DAPI staining of the area shown in C and E. Scale bars: C–F, 100 μm. Data are given as mean ± SEM. IML, Intermediolateral column; IC, intercalated nucleus; NC, central nucleus; cc, central canal.

Figure 6.

TH- and PNMT-immunoreactive cells in the adrenal gland of E18.5 LIFRβ (+/+) and LIFRβ (−/−) mice. Although the numbers of TH-positive cells (A, B) do not seem to be overtly altered, the numbers of PNMT-positive cells (C, D) are clearly reduced. am, Adrenal medulla; ac, adrenal cortex. Scale bars, 100 μm. E, Quantification of TH- and PNMT-immunoreactive cells in LIFRβ (+/+)- and LIFRβ-deficient mice. Although the numbers of TH-positive cells are not altered, the numbers of PNMT-positive cells are reduced by >50%. Data are given as mean ± SEM.

Figure 7.

Quantitative determinations of catecholamines by HPLC and electrochemical detection in adrenal glands of E18.5 LIFRβ, CT-1, and CNTF knock-out mice and respective wild-type littermates. A, Quantification of adrenal catecholamines LIFRβ (+/+) and LIFRβ (−/−) mice. Although the levels of noradrenaline are not altered, the levels of adrenaline are decreased by ∼65%. B, Quantifications of noradrenaline and adrenaline in CT-1- and CNTF-deficient mice fail to reveal significant changes compared with wild-type littermates. Data are given as mean ± SEM.

Figure 8.

Electron microscopy of nerve terminals on adrenal chromaffin cells in E18.5 LIFRβ (+/+) and LIFRβ (−/−) mice. A, B, Axon terminals abutting chromaffin cells show clear synaptic vesicles and no overt morphological differences when comparing LIFRβ (+/+) (A) and LIFRβ (−/−) (B) adrenal medullae. The asterisks mark evaluated axonal terminals. Scale bars, 1 μm. Quantification of nerve terminals on chromaffin cells failed to reveal any significant difference between LIFRβ (−/−) mice and wild-type littermates. Six wild-type and eight LIFRβ knock-out mice were analyzed.