galanin message-associated peptide expression in cultured mouse dorsal root ganglia; with a note on in situ hybridization methodology

Pergamon

PII:

Neuroscience Vol. 89, No. 4, pp. 1123–1134, 1999 Copyright  1999 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306–4522/99 $19.00+0.00 S0306-4522(98)00405-9

LEUKEMIA INHIBITORY FACTOR REGULATES GALANIN/GALANIN MESSAGE-ASSOCIATED PEPTIDE EXPRESSION IN CULTURED MOUSE DORSAL ROOT GANGLIA; WITH A NOTE ON IN SITU HYBRIDIZATION METHODOLOGY N. KEREKES,* M. LANDRY† and T. HO } KFELT Department of Neuroscience, Karolinska Institutet, Stockholm, Sweden Abstract––After transection of the sciatic nerve there is a dramatic increase in both galanin/galanin message-associated peptide-like immunoreactivities and preprogalanin messenger RNA levels in rat and mouse lumbar 4 and 5 dorsal root ganglion neurons. There is strong evidence that after nerve injury leukemia inhibitor factor is a key molecule in the control of peptide expression both in sympathetic neurons and in dorsal root ganglion neurons, although the cells of origin of endogenous leukemia inhibitory factor remain to be established. We have therefore studied the effect of leukemia inhibitory factor on galanin expression in 72 h cultured dorsal root ganglion neurons from normal mice, leukemia inhibitory factor-deficient and heterozygous mice with immunohistochemistry and in situ hybridization. In cultures of leukemia inhibitory factor-deficient (
/
) mice only 13% of the dorsal root ganglion neurons expressed galanin message-associated peptide and in cultures from heterozygous (+/
) and wild-type (+/+) mice the corresponding figures were, respectively, 24 and 40%. After addition of leukemia inhibitory factor (10 or 50 ng/ml) to the culture medium, the number of neurons expressing galanin messageassociated peptide was increased (up to 41%) in cultures from (
/
) animals after the high concentration and reached similar values in cultures from heterozygous animals incubated with the low concentration. These findings were supported by parallel analysis of prepro-galanin messenger RNA levels, where similar transcript levels and effects in the various cultures were observed in the non-radioactive in situ hybridization experiments. These results support the hypothesis that leukemia inhibitory factor is an important regulator of galanin/galanin message-associated peptide expression following axotomy, and may therefore be involved in the defence mechanisms against neuropathic pain at the level of dorsal root ganglion neurons.  1999 IBRO. Published by Elsevier Science Ltd. Key words: nerve injury, neuropeptide, pain, plasticity, sensory neuron.

Changes in neuropeptide expression have been shown to occur in several systems in response to nerve injury (see Refs 20 and 75). In dorsal root ganglia (DRGs) vasoactive intestinal polypeptide (VIP),28,44,56,70 galanin and galanin message-associated peptide (GMAP),19,28,66,69 neuropeptide tyrosine42,67,68,73 *To whom correspondence should be addressed. †Present address: Laboratoire de Biologie Cellulaire, Universite´ Bordeaux II, Baˆtiment 3B, 3e`me e´tage; 146, rue Le´o Saignat, 33076 Bordeaux Cedex, France. Abbreviations: BCIP, 5-bromo-4-chloro-3-indolylphosphate; BSA, bovine serum albumin; CGRP, calcitonin gene-related peptide; DRG, dorsal root ganglion; DTT, dithiothreitol; EDTA, ethylenediaminetetra-acetate; FITC, fluorescein isothiocyanate; GMAP, galanin message-associated peptide; hLIF, human leukemia inhibitory factor; HNPP, 2-hydroxy-3-naphtoic acid-2 phenylanilide phosphate; IL-1â, interleukin-1â; LI, like immunoreactivity; LIF, leukemia inhibitory factor; LRSC, lissamine–rhodamine B sulfonyl chloride; NBT, nitroblue tetrazolium; NGF, nerve growth factor; PBS, phosphate-buffered saline; SCG, superior cervical ganglion; SSC, standard saline citrate; VIP, vasoactive intestinal polypeptide.

and pituitary adenylate cyclase-activating peptide72,74 are up-regulated after transection of the sciatic nerve. The level of other peptides, such as substance P41 and calcitonin gene-related peptide (CGRP)10,45 in general decrease, although more recently it has been shown that these two peptides are up-regulated in small subpopulations of DRG neurons.37,42,43 These changes in neuropeptide levels do not only occur after axotomy, but an increased expression of, for example, galanin can also be seen after local application of vinblastine on the sciatic nerve,29 systemic administration of resiniferatoxin,12 herpes simplex infection18 as well as chronic constriction injury, a neuropathic pain model.36,39 The regulatory factors and mechanisms involved in galanin expression are poorly understood. One candidate factor is nerve growth factor (NGF). NGF is a target-derived factor,32 and axotomy and vinblastine therefore block NGF’s retrograde transport. NGF is important for expression of substance P and CGRP13,31,33,34,38,46 and partially inhibits the

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increase in galanin expression in the superior cervical ganglion (SCG) in vivo59,60 and in DRGs after intrathechal, continuous infusion65 as well as in dissociated DRG cultures.30 There is multiple evidence from several studies that leukemia inhibitory factor (LIF)15/the cholinergic neuronal differentiation factor,71 a neuroimmune molecule (see Ref. 47) and a lesion-induced factor,4,8,50,57 is a key factor for peptide expression both in sympathetic neurons and in DRGs: (i) LIF influences expression of substance P, VIP and somatostatin in dissociated sympathetic neuron cultures;14,40,50,51,54 (ii) in cultured SCG explants VIP induction is inhibited by antiserum against LIF;58 (iii) LIF counteracts the nerve section-induced decrease in substance P mRNA in adult DRG neurons;72 (iv) LIF inhibits NGF-stimulated expression of substance P and CGRP in adult rat sensory neurons in vitro;38 (v) LIF stimulates VIP expression in newborn rat DRG neurons in the presence of NGF;38 (vi) the dramatic increase in galanin in the SCG of wild-type mice is strongly reduced in LIFdeficient mice;50 (vii) in LIF-deficient mice there is a strong attenuation of the axotomy induced galanin/ GMAP up-regulation;6,61 and (viii) intraneural injection of LIF into the intact sciatic nerve of adult rats induced a significant increase in the percentage of galanin-positive DRG neurons.62 We have recently shown that DRG neurons in culture represent a valid model for studying mechanisms underlying nerve injury-induced regulation of galanin. Thus around 30% of all neurons express detectable galanin/GMAP and preprogalanin mRNA after three days in culture30 versus 5% in intact ganglia.66 This suggests that cultured DRG neurons are in an axotomized state.30 In the present study we have focused on galanin/ GMAP expression of sensory neurons in dispersed DRG cell cultures prepared from LIF-deficient,11 heterozygous and wild-type mice. Galanin and GMAP are both products of the same precursor, preprogalanin,52 and they have a parallel distribution in the nervous system,21 and both peptides are upregulated to the same extent in DRG neurons after peripheral nerve injury.69 The preprogalanin mRNA expression was examined with radioactive and three different non-radioactive in situ hybridization protocols, in addition to immunohistochemical detection mainly of GMAP. We also compared several different in situ hybridization protocols with regard to suitability for studies of cell cultures. EXPERIMENTAL PROCEDURES

Animals LIF-deficient (
/
), heterozygous (+/
) and wild-type (+/+) mice were bred (see Ref. 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 55C. DNA was extracted with 500 µl isopropanol. The precipitate was transferred to Eppendorf tubes containing 250 µl 75% ethanol and was centrifuged at 13,000 r.p.m. in an Eppendorf rotor. The resulting pellets were dried in a Speed Vac (Savant Instruments, Farmingdale, NY, U.S.A.) and resuspended in 250 µl Tris– EDTA, pH 8. The polymerase chain reaction was performed in a PTC-100 (Programmable Thermal Controller) machine (MI Research, Watertown, MA, U.S.A.) on 4 µl of each sample using Taq polymerase (MBI Fermentas, Vilnius, Lithuania) and the following primers: (i) LIF 1: GGG ATT GTG CCC TTA CTG CTG CTG GTT; (ii) LIF 2: CCC CAC CTG TGA CAT GGG GAC TCC ACT; (iii) â-galactosidase 1: TCG AGC TGG GTA ATA AGC GTT GGC; (iv) â-galactosidase 2: CCA GAC CAA CTG GTA ATG GTA GCG.11 The annealing temperature was 61C for the reaction. Dorsal root ganglion cell cultures DRGs from the lumbar, cervical and thoracic region were dissected from adult LIF-deficient (
/
), heterozygous (+/
) and wild-type (+/+) mice and collected into Leibowitz L15 transfer medium (GIBCO BRL, Life Technologies Ltd, Paisley, Scotland, U.K.) kept on ice. The ganglia were mechanically dissociated into small pieces, and enzymatically digested overnight at 37C by collagenase (80 U/ml; Wortington Biochemical Corporation, Freehold, NJ, U.S.A.) and dispase (1.6 mg/ml; Boehringer– Mannheim, Mannheim, Germany). After centrifugation and removal of the supernatant, cells were seeded on eight-chambered slides (Nunc, Naperville, IL, U.S.A.), which had previously been coated with laminin (Sigma Chemical Co, St Louis, MO, U.S.A.) and poly--lysine (Boehringer–Mannheim). Cultures were kept at 37C in feeding medium with 5% CO2/95% O2 atmosphere. The feeding medium was 50% minimum essential medium, 50% nutrient (HAM) F12 mixture (GIBCO BRL), to which apotransferrin, glucose, insulin, glutamine, albumin and antibiotic (0.5% penicillin–streptomycin) were added. Part of the cultures was kept in feeding medium supplemented with human LIF (hLIF) (Alomane Labs, Jerusalem, Israel). The medium (with and without hLIF) was changed every day. Mitotic inhibitors were not used in the experiments, and the cultures were not enriched for neuronal cells. For each experiment 10–14 DRGs per mouse were extirpated from 3 two mice (two for each genotype). The DRGs of the two mice with the same genotype were pooled; thus no measure for variability between the two mice could be obtained. The ganglia were then dissociated and used for six cultures (two control, untreated; two treated with 10 ng/ml; and two treated with 50 ng/ml hLIF). High (900–3000 neurons) and low (70–300 neurons)-density cultures were obtained dependent on how many ganglia could be successfully extirpated and on the tirturation procedure, thus not being related to genotype. Cultures were fixed after 72 h in 10% formalin (pH 7.0, GIBCO BRL) for 25 min at room temperature and processed for immunohistochemical experiments or were washed in phosphate-buffered saline (PBS) and dried for in situ hybridization experiments. Immunohistochemistry Fixed cultures were washed three times with PBS and incubated with antiserum to GMAP (diluted 1:400) raised in rabbits21 or with antiserum to galanin (1:400) raised in rabbit or guinea-pig (Theodorsson, unpublished observations), together with mouse monoclonal PGP 9.5 antibodies (1:1000) (UltraClone, Wellow, Isle of Wight, U.K.) for 24 h at 4C. All antisera were diluted in PBS containing 0.3% Triton X-100, 0.01% sodium azide, 0.02% bacitracin (Sigma) and 0.1% bovine serum albumin (BSA). The cultures were washed with PBS and incubated for 1 h at

LIF regulation of galanin room temperature with fluorescein isothiocyanate (FITC) or lissamine–rhodamine â-sulfonyl chloride (LRSC)conjugated donkey anti-rabbit or anti-mouse secondary antibodies (1:80, Jackson ImmunoResearch, West Grove, PA, U.S.A.). The slides were mounted in a mixture of glycerol and PBS (3:1) containing 0.1% paraphenylenediamine in order to retard fading. The cultures were studied under a Nikon Mikrophot-FX fluorescence microscope equipped with epifluorescence and proper filter combinations. FITC staining was detected with filter cube B-1E, excitation at 48010 nm with a bandpass emission filter passing 520–555 nm, LRSC and Texas Red staining with filter cube G-1B (excitation at 5465 nm with a barrier filter at 590 nm). Radioactive in situ hybridization Preparation of probe. A 48-base pair-long oligonucleotide probe complementary to nucleotides 152–199 of preprogalanin mRNA1,27 was purchased from Scandinavian Gene Synthesis AB (Ko¨ping, Sweden). The probe was labelled following published protocols.9,65,69 Briefly, the probe was labelled at the 3 end with 35S-dATP (New England Nuclear, Boston, MA, U.S.A.) using terminal deoxynucleotidyl transferase (Amersham, Amersham, U.K.) in a buffer containing 10 mM CoCl2, 1 mM dithiothreitol (DTT) (LKB, Bromma, Sweden), 300 mM Tris-base and 1.4 M potassium cacodylate, pH 7.2. Afterwards, the labelled probe was purified with QIAquick Nucleotide Removal Kit (Qiagen, Hilden, Germany) and DTT was added to a final concentration of 10 mM. The specific activity was 1–4109 c.p.m./µl. Hybridization procedure. After washing in PBS and drying, the cultures were hybridized overnight at 42C with 106 c.p.m. labelled probe per 100 µl of a hybridization mixture containing: 50% formamide (J.T. Baker Chemicals BW, Deventer, The Netherlands); 4standard saline citrate (SSC; 1SSC=0.15 M NaCl and 0.0015 M sodium citrate); 1Denhardt solution (0.02% each of polyvinyl pyrrolidone, BSA and Ficoll); 1% sarcosyl (Na-lauroylsarcosine; Sigma); 0.02 M potassium buffer, pH 7.0; 10% dextran sulphate (Pharmacia, Uppsala, Sweden); 250 µg/ml yeast tRNA (Sigma); 500 µg/ml sheared and heat denaturated salmon sperm DNA (Sigma); and 200 mM DTT (LKB). After hybridization the cultures were rinsed repeatedly (415 min) in 1SSC at 56C, then at room temperature in 1SSC and 0.5SSC followed by distilled water. They were dehydrated in 60% ethanol (1 min) followed by 95% ethanol (1 min) and dried in air. The slides were dipped in NTB2 nuclear track emulsion (Kodak, Rochester, NY, U.S.A.) diluted 1:1 with distilled water, exposed in the dark at
20C for six weeks, developed in D-19 (Kodak) for 3 min, fixed in Kodak 3000A and B for 6 min, and rinsed in running tapwater for 30 min. The slides were mounted with glycerol and coverslipped for analysis. In addition, following the radioactive in situ hybridization, Toluidine Blue staining was performed. The developed slides were analysed in a Nikon Microphot-FX microscope equipped with dark-field and bright-field condensers. Non-radioactive in situ hybridization Preparation of probes. Oligonucleotide probes were synthesized by the phosphate/phosphotriester method on an automated Applied Biosystem DNA synthesizer 381A (Foster City, CA, U.S.A.). Six oligonucleotides were used in this study for in situ detection of preprogalanin mRNA. Sequences were complementary to nucleotides 152–199; 241–273; 307–339; 361–393; 427–459; 487–519 of the cDNA encoding the rat preprogalanin.64 All oligonucleotides were chosen in regions presenting only few homologies with

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sequences of related mRNAs, and they were then checked against the Genebank database. The oligonucleotides were labelled by tailing at the 3 - end with digoxigenin-11-dUTP (Boehringer) according to published protocols.53 Briefly, 100 pmol of each probe were incubated in a final volume of 20 µl with 1 nmole of digoxigenin-11-dUTP, 9 nmoles of dATP (Sigma), 50 units of terminal deoxynucleotidyl transferase (Amersham) and 4.2 µl of cobalt-containing buffer. After 45 min at 37C the reaction was stopped before purification by ethanol precipitation and then stored at
20C. In situ hybridization procedures. After rinses in PBS and air drying, the cultures were hybridized as described above, in a hybridization solution containing 1 nM of each of the six digoxigenin-labelled oligonucleotides. For control experiments, we added to this mixture an excess of nonlabelled probes (100-fold). After hybridization, the cultures were rinsed in 1SSC (415 min) at 56C followed by 30 min at room temperature. They were immersed for 30 min in buffer A (0.1 M Tris, pH 7.5, 1 M NaCl, 2 mM MgCl2) containing 0.5% BSA (Sigma) and incubated overnight at 4C in the same solution with alkaline phosphataseconjugated anti-digoxigenin F(ab) fragment (1:5000; Boehringer Mannheim). Afterwards, they were rinsed (310 min) in buffer A, and twice in buffer C (0.1 M Tris, pH 9.5, 0.1 M NaCl, 5 mM MgCl2). Enzymatic method. Alkaline phosphatase activity was developed by incubating the cultures with 44 µl nitroblue tetrazolium (NBT) and 33 µl 5-bromo-4-chloro-3indolylphosphate (BCIP) (Gibco BRL) diluted in buffer C. The enzymatic reaction was stopped by extensive rinsing in buffer C and in distilled water. The slides were air dried, mounted in glycerol and coverslipped for analysis under bright-field conditions in a Nikon Microphot-FX microscope. Gold method. After washing, the sections were rinsed in PBS (215 min) and preincubated for 30 min in PBS containing 0.1% gelatin, 5% normal goat serum and 0.8% BSA (Sigma). The sections were then incubated for 30 min at room temperature in the same solution with 1 nm goldconjugated anti-digoxigenin antibody (1:50; Boehringer– Mannheim). After three washes in PBS (10 min each), the sections were postfixed for 30 min in 4% paraformaldehyde in PBS48 and rinsed (310 min) in double-distilled water. The silver enhancement was performed using the silver enhancement kit from Nanogold (Nanoprobes, Stony Brook, NY, U.S.A.) according to the provided instructions. The sections were extensively rinsed in double-distilled water. The slides were air dried, mounted in glycerol and coverslipped for analysis under bright-field conditions in a Nikon Microphot-FX microscope. 2-Hydroxy-3-naphtoic acid-2 -phenylanilide phosphate– fluorescein method. After incubation with alkaline phosphatase conjugated anti-digoxigenin F(ab) fragment, the cultures were washed (310 min) in buffer A and rinsed once in Tris buffer (Tris–HCl 100 mM, NaCl 100 mM, MgCl2 10 mM, pH 8.0). They were incubated for 30 min at room temperature in Tris buffer containing 10 µl/ml 2-hydroxy-3-naphtoic acid-2 -phenylanilide phosphate (HNPP) and 10 µl/ml Fast Red TR solution (HNPP Fluorescent Detection Set, Boehringer–Mannheim) filtered through a 0.2 µm nylon syringe immediately before use. This step was repeated twice and the cultures were washed once in Tris buffer between the steps (see provided instructions in Refs 25 and 26). The reaction was stopped by rinsing the cultures with water. The slides were mounted in a mixture of glycerol and PBS (3:1) containing 0.1% para-phenylenediamine (Sigma) in order to

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Fig. 1. Agarose gel showing polymerase chain reaction (PCR) products of a genetic typing experiment performed on tail samples of LIF-deficient, heterozygous and homozygous mice. Four primers were used in the PCR protocol: LIF 1 and 2, and â-galactosidase 1 and 2. The presence of the 651 bp band indicates a wild-type LIF gene (asterisk), while an 822 bp band indicates a replaced LIF gene (arrow). LIF-deficient mice have only the 822 bp band. In heterozygous animals both genes are expressed and therefore two bands are seen.

retard fading.23,49 The cultures were studied under a Nikon Mikrophot-FX fluorescence microscope. Quantification For immunohistochemistry the quantification was made in the Nikon Mikrophot-FX fluorescence microscope. First all PGP9.5-positive cells were counted in a culture providing the total number of neurons. Then the B-1E filter cube was switched to G-1B filter cube, and all the GMAP-positive neurons were counted in the same culture. Percentages presented in the Results section were calculated by dividing the number of GMAP-positive neurons by the total number of neurons in a given culture. Counting was performed with the help of the delineated area of the camera viewing field. In the quantitative analysis GMAP staining of neuronal cell bodies was considered to be specific, if a distinct Golgi complex-like staining was observed (see Ref. 30). For in situ hybridization the quantification was made as described above using the Nikon Microphot-FX microscope and the delineated area of the camera viewing field. Neurons were differentiated from Schwann cells and fibroblasts on the basis of morphological criteria. Controls The specificity of the peptide antisera was tested after preabsorption with an excess (10
6 M) of the respective peptide (galanin from Bachem, Bissendorf, Switzerland, and GMAP from Prof. T. Ba´rtfai, Stockholm University, Stockholm, Sweden). The in situ hybridization controls were carried out with an excess of cold probe (100-fold) added to the hybridization cocktail. RESULTS

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 LIF 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 bands.

Fig. 2. Immunofluorescence micrographs of a mice DRG cell culture after processing for double-staining with antiserum to GMAP (a) and PGP9.5 (b). GMAP-LI is present in several cell bodies and shows a Golgi-like staining (arrows). The GMAP-stained neurons are PGP9.5 positive (arrows). Scale bars: (a, b)=50 µm.

Galanin/galanin message-associated peptide immunohistochemistry For immunohistochemical and in situ hybridization 64 mice were used, 20 (+/+), 24 (+/
) and 20 (
/
). Both galanin- and GMAP (Fig. 2a)-positive neurons were found in the mouse DRG cultures. Double-staining experiments showed that all galanin- and GMAP-positive cell bodies reacted with

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Table 1. Number of quantified cultures for each treatment Genotypes and treatments LIF LIF LIF LIF LIF LIF LIF LIF LIF

Immunohistochemistry

In situ hybridization

5 3 1 7 5 1 3 2 4

5 2 3 5 2 3 7 5 3


/
; 0 ng/ml
/
; 10 ng/ml
/
; 50 ng/ml +/
; 0 ng/ml +/
; 10 ng/ml +/
; 50 ng/ml +/+; 0 ng/ml +/+; 10 ng/ml +/+; 50 ng/ml

mouse monoclonal PGP9.5 antibodies (compare Fig. 2b with Fig. 2a). However, many PGP9.5-positive, galanin- and GMAP-negative neurons were encountered (see below). Galanin- and GMAP-like immunoreactivities (LIs) were observed at about the same percentage. Since the GMAP antiserum was a better marker for cell bodies than the galanin antiserum,30 we selected the GMAP antiserum for most immunohistochemical experiments and for all quantitative evaluations. Quantitative evaluation of the immunohistochemical examinations was performed on at least three different occasions with the three different genotypes and three different treatments (0, 10 and 50 LIF). Forty-eight cultures were tested and counts were made on those 33 cultures, which were completely undamaged after the immunohistochemical manipulations. Both low (70–300 neurons per culture)- and high (900–3000 neurons per culture)-density cultures were quantified for all three genotypes. (The number of quantified low-density cultures was 5, 7 and 8 and high-density cultures 4, 6 and 1, prepared from, respectively, LIF
/
, +/
and +/+ mice.) No correlation was observed between total neuron numbers and percentage of GMAP-positive neurons (data not shown), i.e. the percentage of GMAPpositive neurons was approximately the same in high- and low-density cultures. The types of treatment and the results from the immunohistochemical analysis of DRG cultures are summarized in Table 1 and Fig. 3. In 72 h cultures of the untreated LIF-deficient mice 12–14% of the neurons were GMAP positive, a percentage which increased to 20% after three days of incubation with a low (10 ng/ml), and to 41% with a high (50 ng/ml) LIF concentration. The heterozygous animals showed a higher GMAP expression. Thus, in untreated cultures 21–26% of the neurons were GMAP positive, and in LIF-treated cultures (10 ng/ml) 42% of neurons were immunoreactive. There was a tendency to a further, slight increase after incubation with high dose of LIF (up to 50%). The wild-type animals had the highest expression of GMAP in control (untreated) cultures (43%), and this did not change significantly after LIF treatment. The increase in the percentage of GMAP-positive neurons seen after 10 ng/ml LIF was not considered sig-

nificant in any of the cultures, but the differences between the three genotypes were significant according to statistical analysis with an unpaired t-test. Radioactive and non-radioactive in situ hybridization The in situ hybridization analysis included 10 experiments with the three different genotypes and three different LIF concentrations (0, 10 and 50 LIF). About 180 cultures were tested, 35 of which were intact and used for quantification. They included both low- and high-density cultures. (The number of quantified low-density cultures was 2, 4 and 6 and high-density cultures 8, 6 and 9 prepared from, respectively, LIF
/
, +/
and +/+ mice. For treatments, see Table 1.) The detection of preprogalanin mRNA levels in neurons in 72 h DRG cultures from the different genotypes (Fig. 4a–f) corroborated the immunohistochemical results, with an increase in galanin mRNA transcripts after LIF treatment, as summarized in Fig. 5. The following data are means of the quantitative evaluation of the enzymatic, gold and HNPP protocols for detection of digoxigenin-labelled preprogalanin mRNA. The values obtained with the most frequently used protocol, that is the enzymatic technique, are marked on the same graph for comparison (Fig. 5). In DRG cultures of LIF-deficient mice 17% of neurons contained preprogalanin mRNA versus 26% after treatment with a low dose of LIF (10 ng/ml). In cultures from heterozygous animals 29% of all neurons expressed preprogalanin mRNA, and there was an increase to 43% following the low dose of LIF. In the wild-type control cultures, 43% of the neurons contained preprogalanin mRNA, and the corresponding percentage after LIF treatment (10 ng/ml) was 45%. LIF treatment with the high dose (50 ng/ml) resulted in a significant increase in preprogalanin mRNA levels (36–38%) in both knockout (
/
) and heterozygous mouse DRG cultures. In the wild-type cultures around 48% of the neurons were preprogalanin mRNA positive (Fig. 5). Quantitative analysis was not performed after radioactive in situ hybridization (see below). The number of neurons did not vary significantly between cultures of different animals or experiments

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Fig. 3. In the untreated DRG cultures of LIF-deficient mice (
/
) 13% of neurons were GMAP positive, and this percentage was increased to 20% (NS with unpaired t-test) with 10 ng/ml and to 41% with 50 ng/ml LIF treatment for three days in culture. The heterozygous (+/
) mice DRG cultures had a higher GMAP expression rate: in untreated cultures 23% of the neurons and in cultures treated with 10 ng/ml LIF 42% of neurons (P=n.s.) were GMAP positive which increased even more after application of 50 ng/ml LIF (51%). The wild-type animals had the highest expression of GMAP in culture, and in control (untreated) cultures 43% GMAP-positive neurons were observed, a percentage that did not change significantly neither after treatment with 10 ng/ml or with 50 ng/ml LIF (44–43%) (P=n.s.). Error bars indicate S.D., derived from the means of percentages of one to seven cultures in case of all three genotypes, treated with three different concentrations of LIF (0, 10 and 50 ng/ml).

(Kruskal–Wallis ANOVA test gave P>0.3 for cultures used in immunohistochemical, and P>0.9 for cultures used in in situ hybridization studies) (data not shown). No attempts were made to monitor for specific cell death in the cultures. Methodological experiments Radioactive as well as non-radioactive in situ hybridization using three different development protocols was performed in several experiments on these DRG cultures. Preprogalanin mRNA could be detected with all four protocols. Radioactive in situ hybridization. Using radioactive in situ hybridization grains overlying DRG neurons were observed after six weeks of exposure. There were also numerous unlabelled cells. Yet the quantification was complicated by the use of the chamber slide system, since after dipping the emulsion accumulated along the borders of the chambers, making it impossible to obtain an even distribution of the emulsion over all cells. Against this background no

attempts were made to quantify the radioactively labelled neurons. It should be emphasized that the results were obtained using only a single, labelled probe. Non-radioactive in situ hybridization. All nonradioactive in situ hybridizations were performed with a mixture of six oligonucleotide probes. As indicated in Fig. 5, similar percentages of preprogalanin mRNA-positive cells were obtained with the different protocols. The total duration of the experiments following these protocols was only two to three days. In all cases the specific reaction product could be easily identified. Using the enzymatic dioxygenin protocol there was often a contamination with substrate particles that could not be rinsed away (Fig. 4b). In contrast, both the fluorescent digoxigenin and in particular the gold digoxigenin approach showed much less background activity. Thus, gold-labelled neurons sharply contrasted with the negative neurons (Fig. 4c, d). The fluorescence staining gave a certain background signal in unlabelled cells, but could clearly be distinguished from the positive cells

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Fig. 4. (a) Bright-field micrographs of a mouse DRG cell culture after hybridization with radioactively labelled preprogalanin probe and counterstained with Toluidine Blue. Arrowheads point to the weakly Toluidine-stained neurons which do not express preprogalanin mRNA, and arrows label positive cells. (b) Hybridization according to the alkaline phosphatase protocol gave dark blue–black labelled cells (arrows) clearly distinguishable from negative (arrowheads) neurons. Contaminating dye particles (open arrows) can be seen between the cells. (c, d) Bright-field (c) and phase-contrast (d) micrographs of culture after hybridization according to the gold protocol combined with silver enhancement. Arrow marks a preprogalanin expressing neuron and the arrowhead points to a negative cell. Using the phase-contrast microscopy (d), the labelled cells can be more distinctly seen. (e, f) After hybridization according to the HNPP-fluorescence protocol, preprogalanin mRNA-labelled cells (arrows) are seen together with negative neurons (arrowheads). (f) For control, cultures were incubated with a 100-fold excess of unlabelled probe. Scale bars: (a–f)=50 µm.

(Fig. 4e, f). Some of the characteristics, advantages and disadvantages of the different hybridization procedures have been summarized in Table 2 and will be further dealt with in the Discussion.

Controls None of the fluorescent structures described above in the immunohistochemical studies was observed after incubation with antiserum preabsorbed with an

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Fig. 5. Detection of preprogalanin mRNA levels in 72 h cultured DRG neurons from LIF (
/
), LIF (+/
) and wild-type mice using non-radioactive in situ hybridization. In DRG cultures of LIF-deficient mice 17% of neurons contained preprogalanin mRNA versus 26% (P=0.333; significant) after 10 ng/ml and 35% (P=0.004; extremely significant) after 50 ng/ml LIF treatment. In cultures from heterozygous animals 29% of all neurons expressed preprogalanin mRNA, and there was an increase to 43% (P=0.0139; significant) after 10 ng/ml and to 38% after 50 ng/ml LIF treatment. In the wild-type mice control cultures 43% neurons contained preprogalanin mRNA and the corresponding percentage after LIF treatment was 45% (10 ng/ml) and 48% (50 ng/ml) (considered not significant). Results obtained using the enzymatic development (the most frequently used protocol) of the non-radioactive in situ hybridization are marked with stars. Error bars represent S.D., derived from the means of percentages of two to seven cultures per genotype per treatment.

excess of galanin and GMAP, respectively (both at 10
6 M). An excess of cold probe (100-fold) added to the hybridization cocktail completely abolished the hybridization signals described above. DISCUSSION

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 Refs 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 galanin30 levels also increase dramatically, when rat DRG neurons are dispersed in culture. These findings suggest that DRG neurons in culture are in an axotomized state and that such neurons represent a model to study mechanisms underlying phenotypic changes after axotomy.30 It has been shown that LIF represents an important factor for galanin regulation, since in LIFdeficient mice there is a marked attenuation of

galanin up-regulation both in the sympathetic SCGs and in DRGs.6,50,61 In the present study we have studied dispersed cultures from LIF-deficient (
/
), heterozygous (+/
) and control (+/+) mice. Our results show that in wild-type mice GMAP-LI increases together with preprogalanin mRNA levels in DRG cultures, similar to our previous results on rat DRG neurons in culture,30 suggesting that also in mice extirpation of DRG neurons and subsequent culturing results in up-regulation of GMAP/galanin synthesis. The increase in the wild-type mice was not affected by addition of LIF to the cultures, even at as a high concentration as 50 ng/ml. However, the number of GMAP-positive neurons in the DRG cultures was significantly reduced in heterozygous (down from 40% to 19–25%) and even more in LIF-deficient (down to 13%) mice. Thus, endogenously produced LIF is necessary for a complete up-regulation of galanin/GMAP levels, when DRG neurons are dispersed and put into culture, i.e. are axotomized. Application of 10 ng/ml LIF partly counteracted the attenuation of GMAP expression and preprogalanin mRNA levels in LIF-deficient mice and completely in heterozygous mice. The 50 ng/ml LIF treatment

LIF regulation of galanin

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Table 2. Comparison between different in situ hybridization protocols Protocols Markers

Radioactive 35

S H P (32P) 1 3

33

Number of probes Detection

Emulsion; phospho-imager; X-ray film

Advantages

Easy quantification; high sensitivity

Disadvantages

Long exposure time; uneven spread of emulsion in chamber slides; radioactivity

Enzymatic

Gold

Fluorescence

Digoxigenin Biotin Fluorescein

Digoxigenin Biotin

Digoxigenin Biotin Fluorescein

Mixture of 6

Mixture of 6

Mixture of 6

Alkaline phosphatase or peroxidase (NBT–BCIP; DAB; True Blue and others) Rapid results (three days)

Ultra small gold particles+silver enhancement

HNPP; FITC; LRSC; Cy3

Rapid results (two days); very high resolution; possibility of electron microscopy Difficulties in obtaining reproducible results

Rapid results (three days); high resolution; possibility of confocal microscopy Fading

Contamination by substrate particles

completely restored GMAP expression (but not fully preprogalanin mRNA levels) in DRG cultures of LIF-deficient mice. Earlier evidence has suggested that LIF is produced in non-neuronal cells. Thus, after transection of the sciatic nerve in vivo LIF mRNA levels increase in Schwann cells distal and proximal to the lesion.4,57 Retrograde transport of LIF within the sciatic sensory afferents has been shown with biotinylated and with iodinated LIF in in vivo experiments.17,63 LIF accumulates within neuronal somata in the DRGs,63 and in vitro experiments have shown that at least 60% of sensory neurons in cultures bind a significant amount of LIF.17 In other experiments it has been shown that DRGs are capable of a high production of LIF mRNA in explant cultures.57,61 There is recent evidence that rat DRG neurons and sympathetic neurons have detectable LIF mRNA levels under normal circumstances.5 It is possible that under normal conditions LIF is produced by neurons at low concentrations and could influence glial function and neuron–neuron interaction (see Ref. 5). After axotomy, as well as after culturing, LIF is strongly up-regulated, presumably in non-neuronal cells. Our results indicate that the endogenous levels of LIF are sufficient to support a ‘‘maximal’’ upregulation of galanin in DRG cultures from wild-type mice, since addition of exogenous LIF did not increase the percentage of galanin-expressing neurons in the cultures. In contrast, in LIF (+/
) and (
/
) mice there was a significant reduction in galaninpositive DRG neurons, suggesting that endogenous LIF levels in these mice are not able to provide a normal galanin up-regulation. To support this view the LIF levels should be monitored in the media from the DRGs of the different mouse genotypes. It is still not known whether it is LIF itself, which, after transport to the sensory neuron cell bodies,

regulates galanin/GMAP expression, or whether there are indirect control mechanisms. Interesting interactions between LIF and NGF have recently been described.7,55 Furthermore, several cytokines and growth factors induce LIF activity, for example interleukin-1â (IL-â), tumor necrosis factor-á, transforming growth factor-â, granulocyte-colony stimulating factor and basic fibroblast growth factor2,16,22 (see also Refs 24 and 35). Among these IL-1â has been detected in axotomized, but not in normal SCGs,3 and IL-1â can induce LIF production in neonatal SCG cultures.54 There is recent evidence that a protein factor(s) present in sympathetic ganglia and their nerve trunks under normal conditions is activated and/or released after axonal injury.57 This new LIF regulatory protein has a high molecular weight (>66,000), and is therefore distinctly different from the above-mentioned cytokines and growth factors, and can induce LIF mRNA in intact SCGs both in vivo and in organ cultures.57 Whether or not the same or a similar regulatory factor is present in DRGs in vivo and in vitro has so far not been demonstrated. In situ hybridization—methodological aspects The comparison of the various in situ hybridization protocols (see Table 2) revealed that all four approaches are valid and give approximately the same percentages of labelled neurons under the various experimental conditions. However, owing to problems in applying the liquid emulsion evenly on to the chamber slides, we were not able to quantify radioactively labelled cultures. It is likely that if a different type of slide is used, it should be possible to obtain quantitative results with this approach. The advantage of the radioactive approach is that a single probe is sufficient to obtain strong labelling, suggesting high sensitivity.

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With the three non-radioactive protocols, using a mixture of six probes, labelled cells could be clearly distinguished with all three approaches, whereby the alkaline phosphatase labelling almost always resulted in contamination by substrate particles. In fact, in single cases it could be difficult to distinguish these precipitates from specifically labeled cells. The best signal-to-noise ratio was obtained with the gold approach, since non-labelled cells were completely negative. However, the gold labelling was capricious, and in several experiments good and reproducible results were not obtained. The fluorescence labelling resulted in distinct background fluorescence, but it was still easy to recognize the specifically labelled cells. A disadvantage of the fluorescence approach was fading which could not be fully counteracted by the use of the antifading reagent paraphenylenediamine, even when combined with storage at
20C. According to our experience the most consistent results were still obtained with the alkaline phosphatase method. This choice is further underlined by cost aspects, which show that gold and fluorescence methodologies are more expensive than the enzymatic protocol. With regard to radioactively labelled probes the cost will depend on frequency of use and perhaps the possibility of sharing the costs of radioactivity with other groups.

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 by digoxigenin combined with alkaline phosphatase as the detection method, although problems are also associated with that, in particular the contamination by unspecific substrate particles. Finally, the omission of radioactivity is certainly an advantage in terms of safety. Acknowledgements—This work was supported by Marianne and Marcus Wallenbergs Stiftelse, the Swedish MRC (04X-2887) and the European Commission (BMH4-CT950172). We thank Professor Tama´s Ba´rtfai and Dr Katarina Bedecs, Stockholm University, Stockholm, Sweden, for generous supply of GMAP peptide and GMAP antiserum, and Dr Elvar Theodorsson, University of Linko¨ping, Linko¨ping, Sweden, for galanin antiserum. We thank Professor Philippe Brulet, Pasteur Institute, Paris, France, for the generous gift of the LIF-deficient mice.

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