ARTICLE |
Correspondence to: Michel Grino, Laboratoire de Neuroendocrinologie Expérimentale, INSERM U 297, UER de Médecine Nord, Blvd P. Dramard, 13916, Marseille Cedex 20, France.
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Summary |
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We present a specific and sensitive method for simultaneous detection of three mRNA species in individual neurons. The method relies on the use of riboprobes labeled with [35S]-UTP, digoxigeninUTP, or biotinUTP. The nonradioactive probes were sequentially revealed by incubation with anti-digoxigenin immunoglobulins or streptavidin conjugated to peroxidase, followed by the use of fluorochrome-labeled tyramides as peroxidase substrates. The radioactive probe was revealed by conventional autoradiography. There was no interaction among the different probes or the various detection systems. We demonstrate the use of this method by illustrating on laser scanning confocal microscopy the co-localization of the mRNAs coding for corticotropin-releasing factor (CRF), arginine vasopressin (AVP), or peptidylglycine -amidating monooxygenase (PAM) in rat hypothalamic paraventricular nucleus (PVN) and its modulation by endogenous glucocorticoids. Our results suggest that this method could be used not only to study the regulation of the hypothalamopituitaryadrenal axis but also in various models in which mRNAs are present at low concentrations. (J Histochem Cytochem 46:753759, 1998)
Key Words:
fuorescent in situ hybridization, radioactive in situ hybridization, multiple labelings, mRNA, laser confocal microscopy, paraventricular nucleus, arginine vasopressin, corticotropin-releasing factor, peptidylglycine -amidating, monooxygenase, digoxigenin, biotin
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Introduction |
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In situ hybridization histochemistry (ISHH) is a powerful tool to study the regulation of neurohormones mRNAs. ISHH has been developed mainly using radiolabeled probes (-amidating monooxygenase (PAM), an enzyme involved in CRF and AVP biosynthesis (
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Materials and Methods |
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Chemicals
Restriction enzymes were from Gibco BRL (Eragny, France) or MBI Fermentas (distributed by Euromedex; Strasbourg, France). T3 or T7 RNA polymerase was purchased from New England Biolabs (distributed by Ozyme; Montigny-le Bretonneux, France). RNasin and RNase-free DNase I were from Promega (Charbonnieres, France). ATP, GTP, CTP, UTP, digoxigenin-11-UTP, biotin-16-UTP, RNase A, tRNA, 5(6) carboxytetramethylfluorescein-N-hydroxysuccinimide ester, and anti-digoxigenin immunoglobulins (Fab fragments) coupled to horseradish peroxidase (HRP) were from Boehringer Mannheim (Meylan, France). 5(6) Carboxytetramethylrhodamine-N-hydroxysuccinimide ester was purchased from Sigma (St Quentin Fallavier, France). Tyramide was from Aldrich (L'Isle d'Abeau Chesnes, France). [35S]-UTP (SA 1300 Ci/mmole) and streptavidin coupled to HRP were purchased from New England Nuclear (Les Ulis, France). The nuclear emulsion (K5) was from Ilford Anitec (St-Priest, France) and the photographic chemicals were from Kodak-Pathé (Paris, France). All other reagents (molecular biology grade, or the highest possible grade) were purchased either from Sigma or Aldrich.
Animals
Male SpragueDawley rats (180200 g bw) were purchased from the Centre d'Elevage R. Janvier (le Genest St. Isle, France). They were housed in our laboratory under controlled temperature (2224C) and a constant 12-hr light/dark cycle for at least 3 weeks before the experiment. They had free access to standard rat chow and tapwater. All experimental procedures were performed in accordance with local animal use regulations; studies were approved by the University Committee on the Use and Care of Animals. Bilateral sham operation or adrenalectomy (ADX, n = 4 in each group) was performed under ether anesthesia using a dorsal approach. ADX rats had free access to 0.9% NaCl in tapwater. One week later animals were sacrificed by decapitation between 1000 and 1100 hr. Brains were carefully removed, immediately frozen on dry ice, and stored at -70C until sectioning.
Probes
The CRF probe was a 770-BP BamHI fragment of the rat CRH gene (-35S]-UTP, 20 U RNasin, 25 U T7 (anti-sense probe), or T3 RNA polymerase (sense probe) in a final volume of 20 µl. The reaction mixture was subsequently incubated for 15 min at 37C with 1 U RNase-free DNase I. The riboprobes were precipitated using LiCl and ethanol and diluted in 100 µl TE [10 mM Tris, pH 7.4, 1 mM ethylenediaminetetraacetic acid (EDTA)] containing 100 mM DTT. One µl yielded about 1.5 x 106 dpm. The AVP or PAM riboprobes were synthesized by incubating for 90 min at 37C 1 µg linearized plasmid in RNA polymerase buffer, 10 mM DTT, 0.5 mM ATP/GTP/CTP, 325 µM UTP, 175 µM biotinUTP (AVP probe) or digoxigeninUTP (PAM probe), 20 U RNasin, 25 U T7 (AVP anti-sense probe, PAM sense probe), or T3 RNA polymerase (AVP sense probe, PAM anti-sense probe) in a final volume of 20 µl. The reaction mixture was subsequently incubated for 15 min at 37C with 1 U RNase-free DNase I. The riboprobes were precipitated using LiCl and ethanol. The AVP probes were diluted in 100 µl TE. The PAM probes' length was adjusted to a mass average of about 200 BP by incubation for 45 min at 60C in 40 mM NaHCO3/60 mM Na2CO3, pH 10.2 (
In Situ Hybridization
Coronal sections (6 µm) through the hypothalamic PVN were cut in a cryostat microtome at -20C. They were thaw-mounted onto gelatin twice-coated slides, dried on a slide warmer, and kept at -70C. The sections were warmed at room temperature (RT) and fixed in 4% formaldehyde in PBS, pH 7.2, for 5 min. After two washes in PBS, they were placed in 0.25% acetic anhydride in 0.1 M triethanolamine/0.9% NaCl, pH 8, for 10 min and delipidated in ethanol and chloroform. They were hybridized for 20 hr at 56C with a buffer containing 10 mM Tris, pH 7.4, 1 mM EDTA, 600 mM NaCl, 50% (v/v) formamide, 10 % (w/v) dextran sulfate, 25 µg/ml yeast tRNA, 1 x Denhardt's solution, 0.1 M DTT, and 1.5 x 107 dpm/ml CRF probe, 20 µl/ml AVP probe, and 40 µl/ml PAM probe under a glass coverslip (these various concentrations of probes have been determined in pilot experiments to saturate the mRNA to be hybridized and to give the lowest possible background). All the subsequent steps were performed at RT unless otherwise specified. Coverslips were removed in 2 x SSC (1 x SSC is 0.15 M NaCl, 0.015 M sodium citrate, pH 7.2). The sections were washed in 2 x SSC for 30 min, treated with RNase A (10 µg/ml in 2 x SSC) for 30 min at 30C, and subsequently washed in 1 x SSC/10 mM ß-mercaptoethanol (ß-ME) twice for 10 min, 0.5 x SSC/10 mM ß-ME for 10 min, 0.1 x SSC/10 mM ß-ME for 10 min, 0.1 x SSC/10 mM ß-ME twice for 30 min at 65C, and finally in 0.1 x SSC/10 mM ß-ME for 10 min. Slides were subsequently incubated for 10 min in Tris-buffered saline (0.1 M Tris, 0.9% NaCl, pH 7.4) containing 0.1% Triton X-100 (TBST) and then for 1 hr in TBST containing 3% normal sheep serum (NSS). Slides were incubated overnight in TBST containing 1% NSS and the anti-digoxigenin immunoglobulins conjugated to HRP, diluted 1:100. Slides were subsequently washed three times for 10 min in TBST and incubated for 10 min in 0.2 M Tris, 10 mM imidazole, 0.01% H2O2, pH 8.8 (TIH) containing 50 µM tyramide coupled to fluorescein (FITC). After three 5-min washes in TBST, the HRP was inactivated by incubation for 15 min in 0.1 M HCl/0.9% NaCl, and slides were further washed three times for 5 min in TBST. Then the slides were incubated for 30 min at 37C in TBS containing 0.5% DuPont blocking reagent (TBSB) and further incubated for 30 min at 37C in TBSB containing the streptavidin conjugated to HRP diluted 1:500. After three 5-min washes in TBST slides were incubated for 10 min in TIH containing 50 µM tyramide coupled to rhodamine (TRITC) and washed three times for 5 min in TBST. Then the slides were rapidly dipped in distilled H2O, followed by 70% ethanol, and dried under a stream of warm air. They were subsequently dipped in nuclear emulsion diluted 1:2 in H2O and exposed for 8 days at 4C. Slides were developed for 4 min in D-19 diluted 1:2, fixed for 5 min in Unifix, washed, and coverslipped with mowiol.
Controls included hybridization with the sense probes, inactivation of the HRP before incubation with the tyramine coupled to fluorochromes, or omission of the anti-digoxigenin immunoglobulins or the streptavidin coupled to HRP.
Confocal Microscopy
Specimens were viewed under a Leica TCS laser scanning confocal microscope coupled to a DMR inverted microscope equipped with planachromatic 20/0.4 and planapochromatic 63/1.4 objectives (Heidelberg, Germany). The argonkrypton laser generated excitation bands at 488 nm for FITC, 568 nm for TRITC, and light for differential interference contrast to detect autoradiographic silver grains. FITC and TRITC signals were recovered through bandpass filters centered at 520 and 600 nm, respectively. Simultaneous three-channel scanning was performed, adjusting the pinhole diameter to make optical sections about 1 µm thick, giving acceptable resolution on the z-axis for both the fluorescent and the autoradiographic focal planes. The power of the laser beam and the gain of each photomultiplier were optimized to minimize the injection of the signal from one channel to another. Leica Scanware software was used to store confocal images, which were transferred to a Power Macintosh computer (Apple Computer) and prepared with Photoshop software (Adobe Systems). The silver grains were inverted to white to improve visualization. Final images were printed on a Codonics NP 1600 printer.
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Results |
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To determine the specificity of our technique, we ran several control experiments using sections obtained from sham-operated rats. First, we tested a possible interaction among the various probes or among the different revelation systems. Serial sections through the hypothalamic supraoptic nucleus [SON, which is known to contain both AVP and PAM mRNAs (
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Discussion |
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This study describes a new method for labeling three distinct mRNA species within a highly heterogeneous population of neurons. Our results show that this technique is specific and sensitive, and that the different markers are well distinguishable, allowing their co-localization to be easily detected.
The specificity of the probes and the revelation systems was assessed in several ways. Sections hybridized with sense probes did not show any signal above background, demonstrating the specificity of the hybridization step. Such specificity was most probably due to the perfect homology between the probes and the mRNAs to be detected and to the high stringency of the hybridization (50% formamide, 56C) and washing (0.1 x SSC, 65C). There was no interaction between the two steps of fluorescent detection of the nonradioactive probes. Incubation with HCl after revelation of the first (FITC) fluorescent signal was effective to inactivate the HRP coupled to the anti-digoxigenin immunoglobulins. This was demonstrated by the lack of signal when the sections were treated with HCl before incubation with tyramide coupled to fluorescein. In addition, hybridization of SON sections with the AVP or the PAM probe or with both probes revealed the same distribution pattern for AVP and PAM mRNAs. Finally, the lack of silver grain clusters in triple labeled sections through the SON, a nucleus that is, under basal conditions, devoid of CRF mRNA (
The high sensitivity of our triple labeling method is illustrated by the relatively intense PAM and AVP hybridization signals in the parvocellular cell bodies of the PVN. These two mRNAs are known to be expressed at low levels in the parvocellular PVN compared with the magnocellular PVN (
A triple labeling ISHH of HRP-labeled oligonucleotides to centromeric regions of human chromosomes has been recently described by
Finally, in the present study we have used laser confocal microscopy. The simultaneous capture for several signals implies the risk for capturing a part of the signal of one channel through the other. This drawback is well controlled under laser confocal microscopy because both gain and offset of photomultipliers can be accurately adapted to the emission intensity, and high-order bandpass filters can be wedged very near the peak emission frequency of the corresponding fluorochrome. However, conventional fluorescence microscopy with good filters sets and dichroics and a high quality CCD-video camera, together with postacquisition pseudo-colorization and overlays, could provide good results.
In conclusion, this work demonstrates for the first time that two nonradioactive and one radioactive ISHH can be easily associated to allow detection of three mRNA species in the same cell body by laser scanning confocal microscopy. This technique is specific, sensitive, and can be performed in a single round of hybridization. Our results strongly suggest that our method could be used not only to study the regulation of the hypothalamopituitaryadrenal axis but also in various models in which mRNAs are present at low concentrations.
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