Leukemia inhibitory factor regulates galanin/galanin message-associated peptide expression in cultured mouse dorsal root ganglia; with a note on in situ hybridization methodology
Section snippets
Animals
LIF-deficient (−/−), heterozygous (+/−) and wild-type (+/+) mice were bred (see [11]), and genetic typing was performed on four-week-old animals. Approximately 5–10 mm of the mouse tail was cut off and placed in an Eppendorf tube containing 700 μl lysis buffer (50 mM Tris, pH 8; 100 mM EDTA; 100 mM NaCl; 1% sodium dodecyl sulphate, 350 μg proteinase K) and incubated overnight with shaking at 55°C. DNA was extracted with 500 μl isopropanol. The precipitate was transferred to Eppendorf tubes containing
Determination of genotype
As shown in Fig. 1, the genotype of four-week-old mice was determined by the presence only of the wild-type LIF gene in wild-type mice (+/+), indicated by an amplified band at 651 bp after electrophoresis on the agarose gel. The LIF knockout mice (−/−) had only the reporter gene, indicated by a band at 822 bp. Heterozygous (+/−) animals carry both the wild-gene and one copy of the reporter gene, and had both bands amplified (651 bp and 822 bp). Control reaction without DNA showed an absence of both
Regulation of galanin/galanin message-associated peptide expression is leukemia inhibitory factor dependent
Galanin, VIP and some other peptides are strongly up-regulated in DRG neurons after peripheral axotomy (see [20] and [75] and Introduction). In most studies rats have been analysed, but an equally strong up-regulation has been found in mice in vivo,[6] suggesting that similar mechanisms operate in both species. VIP,[38] and GMAP and galanin[30] levels also increase dramatically, when rat DRG neurons are dispersed in culture. These findings suggest that DRG neurons in culture are in an
Conclusions
In conclusion, considering all aspects of the four approaches used here, it is clear that the fastest and perhaps most reliable approach to detect galanin/GMAP-synthesizing neurons is immunohistochemistry. In this particular case it also appears to be as sensitive as in situ hybridization expression, which is often not the case, for example, when studying various peptides in brain sections. The non-radioactive approaches are fast and simple, whereby the most reproducible results were obtained
Acknowledgements
This work was supported by Marianne and Marcus Wallenbergs Stiftelse, the Swedish MRC (04X-2887) and the European Commission (BMH4-CT95-0172). We thank Professor Tamás Bártfai and Dr Katarina Bedecs, Stockholm University, Stockholm, Sweden, for generous supply of GMAP peptide and GMAP antiserum, and Dr Elvar Theodorsson, University of Linköping, Linköping, Sweden, for galanin antiserum. We thank Professor Philippe Brulet, Pasteur Institute, Paris, France, for the generous gift of the
References (75)
- et al.
Retrograde axonal transport of LIF is increased by peripheral nerve injury: correlation with increased LIF expression in distal nerve
Neuron
(1994) - et al.
Nerve growth factor counteracts the neurophysiological and neurochemical effects of chronic sciatic nerve section
Brain Res.
(1985) - et al.
Herpes simplex virus infection induces a selective increase in the proportion of galanin-positive neurons in mouse sensory ganglia
Expl Neurol.
(1992) - et al.
Increase of galanin-like immunoreactivity in rat dorsal root ganglion cells after peripheral axotomy
Neurosci. Lett.
(1987) - et al.
Messenger plasticity in primary sensory neurons following axotomy and its functional implications
Trends Neurosci.
(1994) - et al.
Galanin message-associated peptide (GMAP)- and galanin-like immunoreactivities: overlapping and differential distributions in the rat
Neurosci. Lett.
(1992) - et al.
Axonal blockade induces the expression of vasoactive intestinal polypeptide and galanin in rat dorsal root ganglion neurons
Brain Res.
(1992) - et al.
Co-localized but target-unrelated expression of vasoactive intestinal polypeptide and galanin in rat dorsal root ganglion neurons after peripheral nerve crush injury
Brain Res.
(1992) - et al.
The effect of NGF, BDNF and bFGF on expression of galanin in cultured rat dorsal root ganglia
Brain Res.
(1997) - et al.
Neuropeptide expression in cultures of adult sensory neurons: modulation of substance P and calcitonin gene-related peptide levels by nerve growth factor
Neuroscience
(1989)
Differential expression of galanin immunoreactivities in the primary sensory neurons following partial and complete sciatic nerve injuries
Neuroscience
Neuropeptide expression by newborn and adult rat sensory neurons in culture: effects of nerve growth factor and other neurotrophic factors
Neuroscience
Primary sensory neurons exhibit altered gene expression in a rat model of neuropathic pain
Pain
Prepro-VIP and preprotachykinin mRNAs in the rat dorsal root ganglion cells following peripheral axotomy
Molec. Brain Res.
α-CGRP and β-CGRP mRNAs are differentially regulated in the rat spinal cord and dorsal root ganglia
Molec. Brain Res.
Leukemia inhibitory factor mediates an injury response but not a target-directed developmental transmitter switch in sympathetic neurons
Neuron
Effect of ciliary neurotrophic factor (CNTF) and depolarization on neuropeptide expression in cultured sympathetic neurons
Devl. Biol.
Nonradioactive labelling of oligonucleotides in vitro with the hapten digoxigenin by tailing with terminal transferase
Analyt. Biochem.
Vasoactive intestinal polypeptide (VIP) increases in the spinal cord after peripheral axotomy of the sciatic nerve originate from primary afferent neurons
Brain Res.
Signal triggering the induction of leukemia inhibitory factor in sympathetic superior cervical ganglia and their nerve trunks after axonal injury
Molec. cell. Neurosci.
GP130 cytokines, leukemia inhibitory factor and interleukin-6, induce neuropeptide expression in intact adult rat sensory neurons in vivo: time-course, specificity and comparison with sciatic nerve axotomy
Neuroscience
Neuropeptide expression in rat dorsal root ganglion cells and spinal cord after peripheral nerve injury with special reference to galanin
Neuroscience
Increased neuropeptide (NPY)-like immunoreactivity in rat sensory neurons following peripheral axotomy
Neurosci. Lett.
Effects of peripheral nerve injuries and tissue inflammation on the levels of neuropeptide Y-like immunoreactivity in rat primary afferent neurons
Brain Res.
Peripheral axotomy increases the expression of galanin message-associated peptide (GMAP) in dorsal root ganglion cells and alters the effects of intrathecal GMAP on the flexor reflex in the rat
Neuropeptides
Pituitary adenylate cyclase activating peptide (PACAP) expression in the rat dorsal root ganglia: up-regulation after peripheral nerve injury
Neuroscience
Expression in brain of messenger RNA encoding a novel neuropeptide homologous to calcitonin gene-related peptide
Science
Regulation of HILDA/LIF gene expression in activated human monocytic cells
J. Immunol.
mRNA encoding IL-1β, IL-1 receptor and IL-1 receptor antagonist are induced following sympathetic axotomy
Soc. Neurosci. Abstr.
Major changes in the expression of the mRNAs for cholinergic differentiation factor/leukemia inhibitory factor and its receptor after injury to adult peripheral nerves and ganglia
Proc. natn. Acad. Sci. U.S.A.
LIF is an autocrine factor for sympathetic neurons
Molec. cell. Neurosci.
Influence of leukemia inhibitory factor on galanin/GMAP and neuropeptide Y expression in mouse primary sensory neurons after axotomy
Expl Brain Res.
NGF and LIF both regulate galanin gene expression in primary DRG cultures
NeuroReport
Sensitive mRNA detection using unfixed tissue: combined radioactive and non-radioactive in situ hybridization histochemistry
Histochemistry
Differential regulation of calcitonin gene-related peptide (CGRP) in regenerating rat facial nucleus and dorsal root ganglion
Eur. J. Neurosci.
Leukaemia inhibitory factor is necessary for maintenance of haemotapoietic stem cells and thymocyte stimulation
Nature
Increased levels of GMAP, VIP and nitric oxide synthase, and their mRNAs, in lumbar dorsal root ganglia of the rat following systemic resiniferatoxin treatment
NeuroReport
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Present address: Laboratoire de Biologie Cellulaire, Université Bordeaux II, Bâtiment 3B, 3ème étage; 146, rue Léo Saignat, 33076 Bordeaux Cedex, France.