In Vitro and in Vivo Methods for Evaluating Actions of Cytokines on Nerve Growth Factor Production in Central Nervous System

[3]

In Vitro and in Vivo Methods for Evaluating Actions of Cytokines on Nerve Growth Factor Production in Central Nervous System Dan Lindholm, Bastian Hengerer, and Eero Castrén

Introduction Neurotrophic factors are important regulators of survival and differentiation of specific populations of nerve cells during development (1). Nerve growth factor (NGF) was the first neurotrophic molecule to be characterized in terms of its physiology and action in the peripheral nervous system, where NGF acts as a neurotrophic factor for developing sympathetic and some sensory neurons (2). However, NGF is also present in the central nervous system (CNS), and the cholinergic neurons in the basal forebrain are responsive to this factor (3). The molecular cloning of brain-derived neurotrophic factor (BDNF), which is structurally related to NGF, demonstrated the existence of a new family of neurotrophic factors (4). This gene family includes NGF, BDNF, neurotrophin-3, and neurotrophin-4 (5). However, compared with NGF little is known about the action and physiology of these novel neurotrophins. Cytokines, which were first described as immunoregulatory molecules, have also been implicated in many host defense reactions in brain, including glial cell activation. Of the cytokines studied, interleukin 1 (IL-1) and tumor necrosis factor (TNF-α) are expressed by astrocytes and microglial cells in culture (6-8) and contribute to their expression of major histocompatibility complex (MHC) class II antigens. However, IL-1 is also increased after brain injury in vivo and stimulates astrogliosis (9). Interferon y (IFN-γ) is another cytokine that induces MHC expression on cultured glial cells and on microglial cells in vivo (10). On the other hand, transforming growth factor ßx (TGF-/3), which is produced by many cells in culture, inhibits MHC class II expression on cultured astrocytes (11) and reduces astrocyte proliferation induced by IL-1 and other growth factors (12). Transforming growth factor ß levels are low in normal, intact brain, but its mRNA is upregulated following brain injury (13). The functional link between the various cytokines and neurotrophic factors, such as NGF, became apparent when it was demonstrated that IL-1 Methods in Neurosciences, Volume 17 Copyright © 1993 by Academic Press, Inc. All rights of reproduction in any form reserved.

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II CENTRAL NERVOUS SYSTEM ACTIONS

contributes to the enhanced synthesis of NGF in the injured sciatic nerve (14). These studies were subsequently extended to the CNS, where it was shown that both IL-1 and TGF-ß increase NGF production by cultured rat astrocytes (15, 16). Likewise, injections of either factor into the brain of neonatal rats elevated the levels of NGF mRNA in hippocampus. Transforming growth factor ß mRNA was shown to increase in brain following a stab wound, and it precedes an increase in the levels of NGF mRNA in vivo (12). The role played by various cytokines in the production of different neurotrophins after brain injury and in development remains an active area of research with many functional implications. In this article we give a brief description of methods used to study the influence of IL-1 and TGF-/3 on NGF production in cultured brain cells and in vivo. Although we focus on NGF synthesis, these methods can also be extended to other neurotrophins for which sequence data are now available, and can be used to study the more general question of the mutual interaction between cytokines and neurotrophic factors.

Methods Cell Culture Techniques Glial Cell Cultures Newborn rats were used to prepare primary brain cell cultures. Brains were removed and placed in 35-mm petri dishes containing calcium- and magnesium-free phosphate-buffered saline (PBS). To avoid contamination with fibroblasts, the méninges were carefully removed from the brains. The tissues were then dissociated by trituration, using fire-polished Pasteur pipettes, filtered through a nylon mesh, and collected by centrifugation (900 g for 5 min). The cells were resuspended in Dulbecco's modified Eagle's medium (DMEM, pH 7.4) supplemented with 10% fetal calf serum (FCS), streptomycin (100 ^tg/ml), and penicillin (100 U/ml), plated onto plastic culture dishes, and grown in 10% CO2-90% air at 37°C. The medium was changed every 4 days, and the cells were confluent after about 10 days. To remove microglial and oligodendroglial cells from the astrocyte monolayers, the culture flasks were shaken on a rotary shaker overnight at 180 rpm at 37°C. Detached cells were used to isolate microglial cells (see below). Astrocytes were further purified by removing them from the culture dishes with 20 mM ethylenediaminetetraacetic acid (EDTA) (20 min) and collecting the cells by centrifugation. The cells were resuspended in DMEM containing 10% FCS and allowed to adhere to plastic culture dishes for 30 min. The unattached cells representing purified astrocytes were gently washed off and plated onto culture dishes precoated with poly-L-lysine. Confluent astrocytes

[3] ACTIONS OF CYTOKINES ON NGF PRODUCTION

39

subcultured two to three times were usually used for the experiments. Figure 1 shows a phase-contrast micrograph and glialfibrillaryacidic protein (GFAP) staining (marker for astrocytes) of a sparse astrocyte culture. To obtain microglial cells the detached cells removed by shaking from the astrocyte cultures were plated onto 35-mm Falcon (Los Angeles, CA) dishes. The rapidly adhering cells, representing microglial cells, were maintained in DMEM supplemented with 10% FCS. Addition of a one-tenth volume of conditioned medium from astrocyte cultures, as a source of growth factors, was found to improve the viability of the microglial cells. The microglial cells were identified by their unspecific esterase reaction and by their typical morphology in culture (Fig. 2). Embryonic Neuronal Cultures Pregnant rats of 17 days gestation were anesthetized and embryos rapidly removed by abdominal surgery. Brains were removed and hippocampi or other brain areas were dissected under a stereomicroscope, using fine forceps. The tissue was incubated for 20 min at 37°C in PBS containing 10 mM glucose, albumin (1 mg/ml), DNase (6 ^cg/ml), and papain (12 U/ml) (reagents from Sigma, St. Louis, MO). The cells were then washed in medium without papain and dissocated by 10 passages through a fire-polished Pasteur pipette. Cells were then collected by low-speed centrifugation (900 g for 5 min), and resuspended in DMEM supplemented with 10% FCS. Following cell counting, the neurons were plated onto plastic culture dishes that had been precoated with poly-DL-ornithine (0.5 mg/ml). The density of neurons plated varied according to the experiments, but was usually 0.5 x 106 cells/35-mm dish. After 3 hr the medium was replaced by an enriched serum-free medium that lacked glutamate but contained other supplements, as described by Brewer and Cotman (17). The experiments were usually performed during the first week after plating and cells remained viable for up to 3 weeks in this medium. The number of contaminating astrocytes increased during the second week of incubation, but remained lower than 5% of total cells as revealed by GFAP staining. Addition of cytosine arabinose during the first 2 days after plating reduced the number of astrocytes. Figure 3 shows a phase-contrast view of hippocampal neurons (low density) grown for 4 days. The neurons exhibit long neuntes.

Isolation and Hybridization of RNA RNA from cultured cells or tissue was prepared according to the method of Chomczynski and Sacchi (18). The samples were supplemented with 10 pg of a shortened (510 bp) NGF cRNA before extraction. The details of the procedure used are given in Appendix 1.

FIG. 1 Morphological characterization of astrocyte cultures. (A) Phase-contrast micrograph of the cells. Bar = 50 /xm. (B) Staining with glial fibrillary acidic protein (GFAP) as a marker for astrocytes. Cells were fixed with cold (-20°C) 5% acetic acid-ethanol, labeled with a mouse monoclonal anti-GFAP antibody (Boehringer, Mannheim, Germany), and astrocytes were observed with a fluorescence microscope. Bar = 50 μπι. (Courtesy of M. Spranger.)

[3] ACTIONS OF CYTOKINES ON NGF PRODUCTION

41

FIG. 2 Phase-contrast micrograph of microglial cells. The cells were recovered from primary glial cell cultures by shaking, as described in Methods. Note that some cells extend a long process. Magnification x200. (Courtesy of U. Tontsch.)

To analyze RNA by Northern blot, purified RNA (usually 20-40 μg of total cellular RNA) was glyoxylated, electrophoresed through a 1% or 1.5% agarose gel, and the transferred to Hybond N filters (Amersham, Arlington Heights, IL) (see Appendix 1 for details). Following prehybridization for 2-3 hr, the filters were hybridized overnight at 65°C in hybridization solution (see Appendix 1) together with the specific complementary RNA (cRNA) probes (2-5 x 106 cpm/ml). The cRNA probes used were prepared by in vitro transcription of the corresponding cDNAs (NGF, IL-1, TGF-/3,) subcloned into pGemini or Bluescript vectors (Stratagene, La Jolla, CA) with [32P] UTP (3000 Ci/mmol; Amersham) as label. The filters were washed as described in Appendix 1, and were then exposed to X-ray film (Fuji, Tokyo, Japan) for various time periods. The autoradiograms were analyzed with an LKB (Bromma, Sweden) laser scanner and the absolute amount of specific mRNA present was estimated by comparing the intensity of the bands with those of the recovery standard, and with the cRNA calibration standards coelectrophoresed in separate lanes with the sample RNA.

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FIG. 3 Phase-contrast view of hippocampal neurons grown for 4 days in culture. Note the extension of neuntes and the well-preserved structure of the neurons. Magnification x400.

Quantitative Polymerase Chain Reaction A quantitative polymerase chain reaction (PCR) method was used to estimate the low levels of NGF transcripts in cultured neurons. Total RNA extracted from 0.2-0.5 x 106 cells was first reverse transcribed into cDNA and subjected to PCR, using specific oligonucleotide primers for NGF. To assay for recovery, 30 fg of a shortened NGF cRNA standard was added to the samples before extraction and coamplified in the same tube. The NGF cRNA standard was designed to give a shorter amplification product (153 bp) compared with that of cellular NGF mRNA (203 bp), while still retaining the same oligonucleotide binding sites. To avoid possible interference with contaminating genomic DNA in the PCR reaction the specific oligonucleotides used, that is, the 5' primer (24-mer: 5'-CAGCATGGTGGAGTTTTGGCCTGT-3'), and the 3' primer (24-mer: 5'-TGTACGCCGATCAAAAACGCAGTG-3') hybridized to different exons in the NGF gene. Following PCR the reaction products were resolved by electrophoresis on a 3% NuSieve/agarose gel. The details of the PCR method as well as the source of the materials used are described in Appendix 2.

[3]

43

ACTIONS OF CYTOKINES ON NGF PRODUCTION 70

60 H 0)

H 50 c 3

Uj 4 0 Ü Z < 00

ce

o

c/> OÙ

<

30 A 20

io H

L^

— i —

0.12

1.2

12.0

120

— i —

1200

RNA ( f g ) FIG. 4 Quantitative analysis of NGF mRNA, using PCR. The incubation was carried out as described in detail in Appendix 2, using different amounts of NGF RNA made in vitro. A shortened NGF RNA standard was coamplified with the samples to estimate the recovery as well as the efficiency of the priming reaction. Insert: The original autoradiogram, using 20 PCR cycles and 0.12 fg to 1.2 pg of NGF RNA (upper band) and 1.2 pg of NGF standard in each tube (lower band) as starting material. The curves depict the signal intensities obtained with the different amounts of NGF RNA, using 30 (upper curve, Δ) or 20 cycles (lower curve, · ) of PCR. Note that the absorbance increases linearly between about 10 fg and 1.2 pg of RNA. The values on the x axis are shown on a logarithmic scale.

As shown in Fig. 4, there is a linear relationship between the amount of NGF cRNA present (range, 10-1200 fg) and the signal observed on the autoradiogram. The inset in Fig. 4 shows that at 1.2 pg of NGF cRNA (the highest concentration tested), the signal intensity of the recovery standard was already reduced. This is probably because of competition of binding of the primers to the NGF cRNA and the shorter standard. We therefore subjected total cellular RNA to no more than 17 to 20 PCR cycles, which gave a linear signal for both NGF mRNA and the shorter standard (data not shown). Applying the PCR method to RNA isolated from hippocampal neu-

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NGF-

Standard

C +DEXA

FIG. 5 Application of quantitative PCR method to NGF mRNA isolated from hippocampal neurons treated with dexamethasone. Total RNA was extracted from cultured hippocampal neurons and subjected to PCR. Right: C, Controls; DEXA, treated with 0.5 mM dexamethasone for various time periods. Left: Various amounts of NGF standards.

rons revealed that treatment with dexamethasone, a synthetic glucocorticoid hormone, increased the NGF mRNA levels in these neurons (see Fig. 5).

In Situ Hybridization The in situ hybridization method was used to study the expression of NGF and various cytokines in brain. Because the mRNA levels of IL-1 and TGFß are normally low in brain, the choice of the probes is important and both cRNA and single-stranded cDNA probes have been tested. The method used for the in situ hybridization with cRNA probes is a modification of a method described by Angerer et al. (19) and Simmons et al. (20), and is described in detail in Appendix 3. cRNA probes often produce nonspecific, Nissl staining-type hybridization, which is most apparent in cell-rich areas in the hippocampus and in the cerebellar granule cell layer. This nonspecific hybridization is not a problem when relatively high-abundance mRNAs are analyzed, but when the exposure times have to be extended to detect low-abundance messages, this can be a serious problem. To circumvent this problem we used single-stranded cDNA probes transcribed from the corresponding cRNA (in sense orientation), using reverse transcriptase and random priming. Our method is a modification of that published by Schnüren and Risau (21) and is described in detail in Appendix 4. These probes produce less background problems than cRNA probes, and

[3] ACTIONS OF CYTOKINES ON NGF PRODUCTION

45

the probes can be labeled to high specific activity, which allows shorter exposure times than those needed for oligonucleotides and cRNA probes. Because random priming is used in the reverse transcriptase reaction (significantly higher specific activity is obtained with random priming than when using a single specific oligonucleotide primer), care must be taken to make sure that there is no cross-hybridization with other mRNAs in the same gene family. We have successfully used this method to detect mRNAs for different members of the neurotrophin gene family. Single-stranded cDNA probes for NGF, BDNF, and NT-3 produce distinct hybridization patterns without any apparent cross-hybridization (E. Castrén, unpublished observations). A control probe transcribed from corresponding cRNA in the antisense orientation consistently produces a faint background hybridization. Moreover 32P-labeled probes, produced and hybridized in a manner identical to that described in Appendix 4 for 35S probes, produce only the expected band(s) in Northern blot hybridization. However, when a new single-stranded cDNA probe is used, we recommend careful specificity testing. Figure 6 shows an example of m situ hybridization with TGF-ßj cRNA probes. Transforming growth factor mRNA increases in rat brain following a stab wound injury and it localizes mainly to cells (macrophages and possible microglial cells) in the vicinity of the wound (12). The sense probe revealed no clear signal over any particular cell type in this situation (Fig. 6).

Nerve Growth Factor Protein Determination Nerve growth factor protein in brain samples or in culture medium was measured by a sensitive two-site enzyme-linked immunosorbent assay (ELIS A) (15). The details of the NGF ELIS A as well as the sources of the reagents are given in detail in Appendix 5.

Results and Discussion Interleukin 1 plays an important role in many reactions associated with brain injury and reactive gliosis (see Table I, and other articles in this volume and in Volume 16). Interleukin 1 is also present in normal brain and its mRNA localizes to neurons, especially in hippocampus (22). Granule neurons in the dentate gyrus have also been shown to possess IL-1 receptors, opening up the possibility that IL-1 might act locally in brain (see articles by De Souza and colleagues in Volume 16). As for the peripheral nervous system (14), IL-1 increases NGF mRNA in the CNS, in neonatal hippocampus, and in cultured rat astrocytes (15). Whether IL-1 also has an effect on NGF synthesis

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FIG. 6 In situ hybridization of TGF-ßj in brain cortex 4 days after lesion, (a) Cells in the vicinity of the brain wound strongly express TGF-ß!· (b) Staining with OX42, a marker for macrophage/microglial cells, reveals a pattern of immunostaining similar to that of TGF-ß expression, (c) TGF-ßj sense probe gave no specific labeling, (d) Higher magnification of the sections shows the presence of TGF-/3r specific grains in some but not all cells in the wound area. Bar = 20 μ,πι. (Reproduced from the Journal of Cell Biology, 1992,117, p. 395 by copyright permission of the Rockefeller University Press.) in neurons is currently under investigation, using the quantitative PCR method described (Figs. 4 and 5). Transforming growth factor β is a potent cytokine with many diverse actions. It is synthesized by many cells, at least in culture, although in

[3] ACTIONS OF CYTOKINES ON NGF PRODUCTION TABLE I

47

Interleukin 1 in Brain Tissue

Induced after brain lesion Involved in reactive gliosis Induces astrocyte production of eicosanoids (prostaglandin and leukotrienes) Regulates amyloid β-protein precursor mRNA and arantichymotrypsin Increased IL-1 immunoreactivity in Alzheimer's disease and Down syndrome (glial cells) Induces neurotrophic activity, NGF mRNA Expressed in some neurons in intact brain

a latent and biologically inactive form. The mechanism(s) regulating the availability of active TGF-/3 are not fully understood, but TGF-ß is thought to be active during wound repair. In situ hybridization experiments, as used here after brain injury, localized TGF-/3 to macrophages and/or microglial cells surrounding the stab wound area (Fig. 6). Subsequent to the lesioninduced increase in TGF-/3 mRNA in vivo, the NGF mRNA levels also increased in the injured brain (12), suggesting a possible functional link between these phenomena. It remains to be determined whether TGF-/3 is also able to upregulate the synthesis of other neurotrophic factors in brain cells, and whether TGF-/3 acts in conjunction with other cytokines, such as IL-1, in stimulating NGF production in vivo. It has previously been shown that TGF-ß elevates NGF mRNA in neonatal rat hippocampus. The methods described in this article provide a useful and general framework with which to study the interaction between neurotrophic factors and cytokines.

Appendix 1 RNA Isolation and Northern Hybridization RNA Isolation Prepare according to Chomczynski and Sacchi (18). 1. Weigh the tissues (<100 mg). 2. Homogenize in buffer D (see Solutions, below) in a small, RNase-free glass homogenizer. a. For tissues, use 800 μΐ and transfer to a 2-ml Eppendorf. b. For cells, use 600 μΐ and transfer to a 1.5-ml Eppendorf. c. From this point on, always keep the tubes on ice.

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3. Add the following: Component

Volume

Tissues (μ,Ι)

Cells (μΐ)

Sodium acetate (2 M), pH 4 Phenol-H20 Chloroform-isoamyl alcohol (49:1) Recovery standard (10 pg of NGF or BDNF)

0.1 1 0.25

80 800 200

60 600 150

4. Vortex each tube for 15 sec, and keep on ice for 15 min. 5. Centrifuge at > 12,000 rpm for 30 min at 4°C. 6. Transfer the aqueous (upper) phase to another tube. Note: Avoid DNA at the interphase. 7. Add 1 vol of 2-propanol (stored in -20°C) and vortex. 8. Precipitate at -20°C for 1 hr. 9. Centrifuge for 30 min (> 12,000 rpm, 4°C). 10. Remove the supernatant. 11. Resuspend the pellet to 100-150 μΐ in buffer D. Note: If 2-ml tubes were used, transfer to 1.5-ml tubes at this point. 12. Add 100 μΐ of cold 2-propanol; vortex. 13. Precipitate again for 1 hr to overnight. 14. Centrifuge again (> 12,000 rpm, 30 min, 4°C). 15. Remove the supernatant and add 500 μλ of 80% RNase-free ethanol. Vortex. 16. Centrifuge for 5 min. 17. Carefully remove the supernatant with a pipette. 18. Dry the pellet in a Speed Vac (Savant, Hicks ville, NY) for 5-10 min. 19. Resuspend the pellet to 10 μΐ in RNase-free water. 20. Add 20 μ,Ι of fresh glyoxal solution (see Solutions, below). 21. Incubate for 60 min at 50-60°C. 22. Add 3 μΐ of RNA loading buffer and load on a Northern gel. Northern Gel 1. For a medium-sized gel box, combine the following: Gel Component

1.5%

1%

Agarose NaHP0 4 (1 M), pH 7 H20

2g 1.3 ml 130 ml

1.3 g 1.3 ml 130 ml

[3] ACTIONS OF CYTOKINES ON NGF PRODUCTION

2. 3. 4. 5. 6.

49

Running buffer (1.5 liters): 10 mM N a H P 0 4 , pH 7 Run at 70 mV, or 15 mV overnight. Circulate the buffer through a pump. Blot by vacuum (3 hr) or by capillary blotting to Hybond N. Fix the filter with UV, 3.5 min for each side.

Comments 1. When using cells for Northern blotting, steps 10-14 can be omitted. 2. Isolation procedure may be interrupted after step 8 or 13 (keep overnight at -20°C), or after step 18 (keep overnight at -70°C. 3. Repeat steps 15-17 if salt precipates. Hybridization 1. Preincubate the filters in 5-20 ml of hybridization solution at 65°C for 1-3 hr. 2. Add the probe to fresh hybridization medium, 5 x 106 cpm/ml. 3. Hybridize overnight at 65°C. 4. Wash twice (10 min each) in sodium citrate buffer (2x SSC: 0.3 M NaCl, 0.03 M sodium citrate)/0.1% sodium dodecyl sulfate (SDS) at room temperature. 5. Wash 10-20 min in 0.2x SSC/0.1% SDS at 70-75°C (check the activity with a Geiger counter after 10 min). 6. Expose to X-ray film between two intensifying screens at -70°C. Preparation of cRNA Probes 1. Pipette into RNase-free Eppendorf tubes at room temperature in the following order: Transcription buffer (5 x) Dithiothreitol (DTT) (1 M) RNase inhibitor ATP, CTP, GTP (25 mM) Linearized template plasmid T3/T7/Sp6 RNA polymerase [32P] UTP H 2 0 diethylpyrocarbonate (DEPC)

5 μί 1 μλ 1 μ,Ι 1 μΐ each 1 /xg 1 μ,Ι 5 μ\ to 25 /xl

2. Incubate at 37°C for 60 min. 3. Add 75 μΐ of TE buffer plus 0.1% SDS (TES) 4. Purify by Spun column equilibrated with 100 μΐ of TES (see Maniatis)

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II CENTRAL NERVOUS SYSTEM ACTIONS

Solutions Buffer D (denaturing buffer): Guanidium thiocyanate (4 M) Sodium citrate (25 mM), pH 7 Sarkosyl lauryl sulfate (0.5%) Mercaptoethanol (0.1 M) 1. Mix guanidium, sodium citrate, and sarkosyl, and store in 50-ml Falcon tubes. 2. Before use, add 360 μΐοί 2-mercaptoethanol/50 ml. Sodium acetate (2 M), pH 4 Chloroform-isoamyl alcohol, 49:1 Water-saturated phenol 2-Propanol Glyoxal solution Component

Stock

For 300 μ\

For 600 μ\

NaHP0 4 (100 mM), pH 7 Deionized glyoxal Dimethyl sulfoxide

100 mM

30 μ\ 70 μ,Ι 200 μ\

60 μ\ 140 μ,Ι 400 μ\

Hybridization solution Component

Stock

For 20 ml

For 40 ml

Deionized formamide (50%) N a H P 0 4 buffer (50 mM), pH 7 SSC(3x) SDS (0.5%) Na 2 EDTA (5 mM) ssDNA (250 Aig/ml) Denhardt's solution (5x)

100% IM 20 x 10% 0.5 M 10 mg/ml 50 x

10 ml 1 ml 6 ml 1 ml 200 μ\ 500 μ\ 2 ml

20 ml 2 ml 12 ml 2 ml 400 μ\ 1 ml 4 ml

Appendix 2 Quantitative Polymerase Chain Reaction RNA Extraction Extract RNA from cells or tissues as described in Appendix 1, but resuspend the pellet into 50 μλ of H 2 0.

51

[3] ACTIONS OF CYTOKINES ON NGF PRODUCTION

Reverse Transcriptase-Polymerase Chain Reaction 1. Dilute the calibration standard (500, 100, 50, 25, 10, and 5 fg/5 μΐ) 2. Label 0.5-μ1 Eppendorf tubes for PCR. 3. Prepare RT-PCR master mix: For each RT-PCR reaction, add the following (do not forget the negative control, and add one extra aliquot for pipetting errors): Water (no DEPC) PCR buffer (lOx) dNTP mix (25 mM each) 5'-01igo (50 μΜ) 3'Oligo (50 μΜ) AMV reverse transcriptase ( -18 υ/μϊ) RNasin (Promega, Madison, WI) Taq DNA polymerase

13.5 μ\ 2.5 μΐ 0.25 μΐ 1 μΐ 1 μΐ 0.25 μΐ 0.5 μ\ 1 μ\ 20 μ\

4. Pipette 5 μΐ of RNA solution (or calibration standard or water for a negative control) into the corresponding tube. 5. Add 20 μΐ of RT-PCR master mix. 6. Overlay the reaction with two drops of light mineral oil. 7. Run the RT-PCR reactions in a thermocycler: Reverse transcription, 41°C for 30 min RNA/DNA denaturation, 92°C for 10 min PCR denaturation, 92°C for 1 min Cooling, 50°C for 10 sec Primer annealing, 55°C for 1 min Primer extension, 72°C for 1 min Run 17 cycles of the last 4 steps. Note: optimize the temperature profile for RT-PCR. 8. If contaminating chromosomal DNA interferes with the RT-PCR, the RNA can be treated with DNase I before the reverse transcription: RNA PCR buffer ( 10 x) Water DNase I (RNase free; RQ-DNase, Promega)

5 μ\ 1 μ\ 3 μΐ 10 μΐ

a. Overlay the reaction with two drops of light mineral oil. b. Incubate for 15 min at 37°C.

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c. Heat for 10 min on a boiling water bath to destroy the DNase I activity. d. Add 20 μλ of a modified master mix containing 2 μ,Ι of 10 x PCR buffer and 14 μ,Ι of water per reaction. e. Run the PCR as described above. Gel Electrophoresis 1. Prepare agarose gel: 3% NuSieve-SeaKem (3 : 1) in TBE buffer. 2. Remove the mineral oil; add 5 μ,Ι of loading buffer. 3. Run the gel. 4. Denature the DNA for 30 min in 0.4 N NaOH. 5. Vacuum blot to the Hybond N plus (Amersham) with 20x SSC for 3 hr; no neutralization. 6. Fix the DNA to the filter with 0.4 N NaOH for 3 min. 7. Wash the filter in 5x SSC for 10 sec; air dry.

Appendix 3 In Situ Hybridization with cRNA Probes Proceed according to Angerer et al. (fl9). 1. Dissect tissues, using sterile technique whenever possible. Freeze in Tissue-Tek on dry ice and keep at -70°C or perfuse with 4% paraformaldehyde and postfix in 4% PFA/10% sucrose overnight. Then freeze and keep at -70°C, if necessary. 2. Coat glass slides with TESPA (#09326, Fluka, Switzerland), 2% in acetone and wash twice in acetone; dry and store dust free. Cut the tissues into 10 to 12-μ,πι thick sections in a cryostat and mount on glass slides coated with Tespa. Dry and store desiccated at -80°C. 3. On the day of hybridization, take the sections from the freezer and allow to warm to room temperature. 4. Fix in fresh 4% paraformaldehyde/PBS (pH 7.4) for 5 min and rinse twice (2 min each) in PBS/DEPC; dip quickly in H 2 0/DEPC. 5. Dehydration: Ethanol Ethanol Ethanol Ethanol Ethanol

(50%), 2 min (70%), 2 min (95%), 1 min (100%), 10 min (95%), 1 min

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[3] ACTIONS OF CYTOKINES ON NGF PRODUCTION

6. Dry. 7. Hybridization:

Conc.

Buffer Buffer A Formamide Dextran S 0 4 NaCl Denhardt's Tris, pH 8 EDTA, pH 8 H 2 0/DEPC Buffer B tRNA DTT Probe

Per 1 ml

50% 10% 0.3 M lx 2mM 1 mM

500 μ\ 200 μ\ 60 μ\ 20 μλ 2μ\ \0μ\ 8μ1

500 μ^πύ 10-100 mM 5 x 10"6 to 10"7 cpm/ml

H 2 0/DEPC

Per 5 ml 2.5 ml 1 ml 300 μ\ 100 μ\ 10 μ\ 50 μ\ 40 μ\

50 μ\ 10-100 μ\

250 50-500

μ\ μ\

To 200 μ\

To 1

ml

Stock

50% 1 M 50 x 0.5 M

10 mg/ml 1 M

a. Store buffer A frozen at -20°C and add buffer B. The ratio of A:B = 8:2. Heat to 65°C for 10 min, and spin briefly (2000-4000 rpm). b. Take an aliquot of 1-2 μ,Ι for cpm counting. c. Apply 100 μΐ/glass slide (50 μΐ/brain slice) d. Cover with a strip of Parafilm. e. Place the slides into a box humidified with 2x SSC. f. Hybridization at 58°C overnight. 8. Dip the sections into 4x SSC and remove the Parafilm coverslips. 9. Wash four times (5 min each) in 4x SSC; the first two washes will become radioactive. 10. Treat with RNase (30 min at 37°C):

Component

Per 25 ml

Per 10 ml

RNase A (Boehringer) NaCl (0.5 M) Tris (10 mM), pH 8 EDTA (1 mM), pH 8 Water

50 μ\ 2.5 ml μ\ 250 50 μ\ 22.15 ml

μ\ 20 1 ml 100 μ\ μ\ 20 8.86 ml

Stock 10 mg. 5Μ 1 M 0.5 M

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II CENTRAL NERVOUS SYSTEM ACTIONS

11. Washes: Time

Buffer

5 min (twice) 5 min 5 min 30 min 1 min

SSC (2x) SSC(lx) SSC (0.5x) SSC (0.1 x) SSC (0.1 x)

Temperature Room Room Room 60°C Room

temperaure temperature temperature temperature

12. Dehydrate with 70% ethanol (1 min) and 100% ethanol (2 min). 13. Let the tissues dry, arrange the slides in an X-ray cassette, and expose to Hyperfilm for 1-5 days. If a positive signal is detected, dip into Kodak NTB-2 emulsion. a. Dilute the emulsion 1: 1 with water and aliquot to black film vials, 20 ml each. b. Store the vials in the dark, at 4°C. c. Melt one vial on a water bath at 40°C in a darkroom. d. Dip two empty slides to remove air bubbles. e. Dip the sections in the emulsion for 5 sec. f. Dry for 2 hr in the dark on damp paper towels. g. Transfer into black slide boxes with desiccant and seal. h. Expose in the dark, at 4°C for three to five times the time needed for Hyperfilm. i. Develop: D19 developer, 3 min Water, 30 sec Sodium thiosulfate (5%), 6 min Water, 20 min Counterstain with cresyl violet or toluidine blue.

Appendix 4 In Situ Hybridization with cDNA Probes This method uses single-stranded cDNA probes that are prepared by a reverse transcriptase reaction, using the respective sense cRNA as a template and random hexanucleotides as primers.

[3] ACTIONS OF CYTOKINES ON NGF PRODUCTION

55

Preparation of cRNA Template 1. The gene must be subcloned into a Bluescript (or analogous) vector containing T3 and T7 RNA polymerase primers. 2. Linearize the plasmid so that the cRNA will be in the sense orientation. Component Transcription buffer Dithiothreitol RNase inhibitor ATP, CTP, GTP, UTP Linearized plasmid T3 or T7 RNA polymerase H 2 0 (RNase free)

Stock 5x 1M 25 mM each 1 μ%/μ\

Volume 5 1 1 1 1 1 12

μ\ μ\ μ\ μ\ μ\ μ\ μ\

3. Incubate for 60 min at 37°C. 4. Take 4-5 μΐ for the gel (see the next section). 5. To the rest add 2 μΐ of 3 M sodium acetate and 60 μΐ of 100% ethanol. 6. Precipitate >1 hr at -20°C and centrifuge (12,000 rpm 30 min, 4°C). 7. Remove the supernatant and wash the pellet with 80% ethanol. 8. Centrifuge (12,000 rpm for 5 min), remove the ethanol, and dry the pellet. 9. Resuspend to 4-10 μ\ in RNase-free H 2 0, or use the pellet directly for cDNA synthesis, depending on the cRNA yield (estimate on the gel). Formaldehyde Minigelfor Checking cRNA Component

13-lane box

6-lane box

Agarose (1.5% gel) 10x MOPS H 2 0 (boil to dissolve) Formaldehyde, 37% Ethidium bromide, 10 mg/ml

600 mg 5 ml 33.8 ml 2.16 ml 1 μ\

300 mg 2 ml 16.9 ml 1.08 ml 1 μ\

1. To the cRNA and RNA size marker (3 μΐ), add 1 μΐ of loading buffer and bring the volume to 10 μ,Ι with RNase-free H 2 0. 2. Run the gel in l x MOPS. 3. Photograph under UV light.

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II CENTRAL NERVOUS SYSTEM ACTIONS

Preparation of the Single-Stranded cDNA Probe 6/nl Reverse transcriptase buffer (5x) 1-2 μΐ Template RNA 4μ1 Random primer (10 Aig/μΐ) (heat to 90°C for 1 min, cool on ice) RNase inhibitor 1/U 3μ1 Dithiothreitol (100 mM) Ιμΐ dCTP, dGTP, dTTP (10 mM each) Ιμΐ Actinomycin D 10 μΐ [35S]dATP (Amersham) MMLV reverse transcriptase (BRL) 1/Λ RNase-free H 2 0 To 30 μΐ 1. Incubate at 37°C for 1-2 hr. 2. Add: NaOH (0.4 N) (heat to 65°C for 5 min) HC1 (0.4 Λ0 Tris (1 M), pH 7.5 3. 4. 5. 6.

30 μΐ 60 μ\ 1 μΐ

Vortex, and take 1 μ\ for scintillation counting. Purify the probe with a G50 spin column equilibrated with TE. Take 1 μΐ for scintillation counting. Percent incorporation = cpm after column purification/cpm before column purification; should be >30%.

In Situ Hybridization with cDNA Probes Sections and Pretreatments 1. Cut 12-μτη thick sections in the cryostat, thaw-mount on TESPA-coated slides, and keep the slides inside the cryostat after cutting. 2. Bring the sections to room temperature quickly by blowing cool air on them with a hair dryer. 3. Fix the sections for 30 min in 4% paraformaldehyde/PBS at 4°C. 4. Wash twice for 2 min in PBS (RNase free). 5. Wash once in 0.1 M triethanolamine, pH 8 (RNase free). 6. Incubate for 15 min in 0.25% acetic anhydride-TEA. 7. Wash once in PBS (RNase free).

[3] ACTIONS OF CYTOKINES ON NGF PRODUCTION

57

8. Dehydrate in ethanol: 70%, 1 min 95%, 1 min 100%, 2 min 9. Let the sections dry. Hybridization Hybridization mix Component

Stock

Amount

Formamide (50%) SSC (4x) Denhardt's ( l x ) Lauryl sarkosine (1%) Sodium phosphate (20 mM), pH 7.0 Dextran sulfate (10%)

100% 20 x 50 x 20% 0.2 M

5 ml 2 ml 200 μΐ 500 μ\ 1 ml lg

1. Weigh dextran sulphate in an RNase-free Falcon tube, add other components, and dissolve by warming to 37°C. 2. Store frozen at -20°C in 900-μΙ aliquots. Hybridization buffer Hybridization mix 900 μ\ tRNA (50 mg/ml) 5.5 μ\ ssDNA (10 mg/ml) (boil for 5 min to denaturate) 50 μ\ 35 S-Labeled probe 10,000 cpm/μΐ To 1000 μΐ, but at least 12 μ\ Dithiothreitol (5 M) Hybridize overnight at 42°C in an air-tight box humified with 50% formamide-4x SSC. Washing 1. Wash four times (15 min each) in l x SSC/10 mM 2-mercaptoethanol at room temperature. 2. Wash four times (15 min each) in 0.5x SSC/10 mM 2-mercaptoethanol at 60°C. 3. Wash for 15 min in 0.5x SSC/10 mM 2-mercaptoethanol at room temperature.

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4. Dehydrate: Ethanol (70%), Ethanol (95%), Ethanol (100%),

30 sec 30 sec 2 min

5. Air dry. 6. Expose to Amersham Hyperfilm /3-max or dip into Kodak NTB2 emulsion.

Appendix 5 Nerve Growth Factor ELISA Method Coating a 96-Well Plate with Antibody 1. Dilute 0.8 μg of antibody 27/21 in 1 ml of coating buffer. 2. Pipette 50 μΐ/well. 3. Incubate overnight at 4°C. Blocking 1. Wash twice with washing buffer. 2. Add 100 μΐ/well of coating buffer-1% BSA. 3. Incubate for 2 hr at room temperature. 4. Wash three times with washing buffer. Preparation of Tissues, Media, and Standards Tissues 1. Homogenize the tissue in 10 vol of homogenization buffer. The homogenate is stable at -70°C. 2. Divide the homogenized tissue into two tubes and add 100 pg of NGF/ ml as a recovery standard in one of the two tubes. 3. Centrifuge (10,000 rpm, 15 min, 4°C). 4. Collect the supernatant and dilute it 1:1 with dilution buffer. 5. Pipette 50 μΐ/well. 6. Incubate overnight at 4°C. Media 1. Dilute in homogenization buffer containing 0.2% Triton X-100 and 10 mM MgCl2. 2. Pipette 50 μΐ/well. 3. Incubate overnight at 4°C.

[3] ACTIONS OF CYTOKINES ON NGF PRODUCTION

59

Standards 1. Dissolve standards in homogenization buffer containing 0.2% Triton X-100 and 10mMMgCl 2 . 2. Dilute different standard concentrations (250 pg/ml —> 1 pg/ml) by stepwise 1: 1 dilutions in incubation buffer. 3. Pipette three wells/concentration, 50 μΐ/well. 4. Incubate overnight at 4°C. 5. Wash three times in washing buffer. ß-Galactosidase 1. Dilute in incubation buffer to a concentration of 0.1 U/ml. 2. Incubate overnight at 4°C. 3. Wash three times with washing buffer. 4. Wash twice with substrate buffer. Substrate 1. Dilute 4-methylumbellifrenyl-/3-D-galactoside in substrate buffer to 200 μΜ (sonicate to dissolve). 2. Add 50 μΐ/well. 3. Incubate at room temperature. 4. Measure the fluorescence after 1, 2, and 3 hr. 5. Stop the reaction by adding stop buffer. Materials Antibody: Anti-mouse ß-NGF-ß-Gal (Cat. No. 1008234; Boehringer) ß-Galactosidase (Cat. No. 105031; Boehringer) Substrate: 4-Methylumbellifrenyl-/3-D-galactoside (Cat. No. 1633; Sigma) Buffers Coating buffer: 0.05 M NaC0 3 , pH 9.7 (mix Na 2 C0 3 and NaHC03) Incubation buffer: Combine 50 mM Tris (pH 7.0), 150 mM NaCl, 5 mM MgCl2 ; autoclave; just before use add 0.1% Triton X-100 and 1% BSA Washing buffer: Combine 50 mM Tris (pH 7.0), 150 mM NaCl, 5 mM MgCl2; autoclave; just before use add 0.1% Triton X-100 Homogenization buffer: 100 mM Tris (pH 7.0), 300 mM NaCl, 2% BSA Dilution buffer (pH 7.0): 10 mM MgCl2, 0.2% Triton X-100 Substrate buffer: 100 mM sodium phosphate buffer (pH 7.3), lmMMgCl2 Stop buffer: 150 mM glycine, pH 10.5

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Acknowledgments We thank Professor Hans Thoenen for his continuous support, and Drs. F. Zafra and R. Kiefer for collaboration and stimulating discussions. E.C. is an Alexander von Humboldt Fellow.

References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22.

Y.-A. Barde, Neuron 2, 1525 (1989). R. Levi-Montalcini, EMBO J. 6, 1145 (1987). F. Hefti and W. J. Weiner, Ann. Neurol. 20, 275 (1986). H. Thoenen, Trends N euro s ci. 14, 165 (1991). F. Hallböök, C. F. Ibanez, and H. Persson, Neuron 6, 845 (1991). D. Giulian, T. J. Baker, L. Shih, and L. B. Lachman, J. Exp. Med. 164,594 (1986). K. Frei, U. V. Malipiero, T. P. Leist, R. M. Zinkernagel, M. E. Schwab, and A. Fontana, Eur. J. Immunol. 19, 689 (1989). M. Sawada, N. Kondo, A. Suzumura, and T. Marunouchi, Brain Res. 491, 394 (1989). D. Giulian, J. Woodward, D. G. Young, J. F. Krebs, and L. B. Lachman, J. Neurosci. 8, 2485 (1988). K. Vass and H. Lassmann, Am. J. Pathol. 137, 789 (1990). H. J. Schluesener, / . Neuroimmunol. 27, 41 (1990). D. Lindholm, E. Castrén, R. Kiefer, F. Zafra, and H. Thoenen, J. Cell Biol. 117, 39 (1992). N. R. Nichols, N. J. Laping, J. R. Day, and C. E. Finch, / . Neurosci. Res. 28, 134 (1991). D. Lindholm, R. Heumann, M. Meyer, and H. Thoenen, Nature (London) 330, 658 (1987). M. Spranger, D. Lindholm, C. Bandtlow, R. Heumann, H. Gnahn, M. NäherNoe, and H. Thoenen, Eur. J. Neurosci. 2, 69 (1990). D. Lindholm, B. Hengerer, F. Zafra, and H. Thoenen, NeuroReport 1, 9 (1990). G. J. Brewer and C. W. Cotman, Brain Res. 497, 65 (1989). P. Chomzcynski and N. Sacchi, Anal. Biochem. 162, 156 (1987). L. M. Angerer, K. H. Cox, and R. C. Angerer, in ''Methods in Enzymology" (S. Berger and A. Kimmel, eds.), Vol. 152, p. 649. Academic Press, Orlando, FL, 1987. D. M. Simmons, J. L. Arriza, and L. W. Swanson, J. Histotechnol. 12,169 (1989). H. Schnüren and W. Risau, Development (Cambridge, UK) 111, 1143 (1991). C. Bandtlow, M. Meyer, D. Lindholm, M. Spranger, R. Heumann, and H. Thoenen, J. Cell Biol. I l l , 1701 (1990).