ARTICLE |
Correspondence to: Lutgarde Arckens, Laboratory of Neuroendocrinology and Immunological Biotechnology, Katholieke Uni-versiteit Leuven, Naamsestr. 59, B-3000 Leuven, Belgium. E-mail: Lut.Arckens@bio.kuleuven.ac.be
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Summary |
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We developed a novel antibody against cat Fos by immunizing rabbits with a 26-amino-acid peptide. Immunocytochemistry on visual cortex of cats undergoing different visual manipulations was applied to test the reliability and the efficacy of this antiserum. One hour of light stimulation after an overnight dark adaptation resulted in strongly induced Fos expression in supra- and infragranular layers of cat primary visual cortex. Short-term monocular deprivation changed the Fos expression profile into a columnar immu-nostaining related to ocular dominance columns. Fos expression has also been analyzed in cats in which visual input was confined to the right hemisphere by sectioning the left optic tract and the corpus callosum. In the right hemisphere, visual stimulation elicited Fos induction, whereas in the contralateral hemisphere a very low Fos signal was observed. The specificity of this newly synthesized antibody was confirmed by Western blotting. To further establish the applicability of this Fos antiserum, we performed immunostaining on monkey and rat visual cortex. This new cat Fos antibody appears to be excellent for study of Fos expression as a marker for mapping neuronal activity in mammalian brain. (J Histochem Cytochem 48:671684, 2000)
Key Words: immediate early gene (IEG), antibody production, Western blotting, immunocytochemistry, Fos expression, visual cortex, cat, monkey
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Introduction |
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It is now well established that the study of the expression of immediate early genes (IEGs) in the brain has become a powerful tool for the identification of neuronal activity in brain regions of different species after sensory stimulation (
Previous immunocytochemical studies of cat visual cortex in our laboratory have been hampered by the lack of reliable antisera.
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Materials and Methods |
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Animals
All animal experiments were carried out in accordance with the European Communities Council Directive of 24 November, 1986 (86/609/EEC). All efforts were made to minimize animals' discomfort and to reduce the number of animals used. Ten adult cats (Felis catus), four adult rats (PFD:Wist), and one adult rhesus monkey (Macaca mulatta) were used in this study. All animals were housed in the animal house (Animal Facilities; Katholieke Universiteit Leuven, Leuven, Belgium). Five cats, two rats, and one monkey were placed overnight in a dark room. The next day they received a binocular light stimulation for 1 hr and were sacrificed immediately thereafter. A light exposure for 1 hr after a period of darkness results in an increase in Fos expression in the mammalian visual system (
As a control, one cat and two rats were sacrificed after a long-term exposure to light for more than 8 hr to reveal the basal expression level of Fos (
Tissue Preparation
All animals were sacrificed at the same moment of the day (1100 hr) with an overdose of Nembutal (20 mg/kg IV). All animals but two were perfused transcardially with 0.9% NaCl, followed by 4% paraformaldehyde (Sigma; St Louis, MO) in PBS (0.15 M; pH 7.4). The brains were postfixed in the same fixative for 24 hr, then rinsed in water for another 24 hr. Frontal sections (50-µm) were cut on a vibratome, collected in 12-well plates, and processed for immunocytochemistry.
The brains of two cats (a 1-hr and a long-term light-stimulated cat) were rapidly removed and immediately frozen by immersion in liquid nitrogen-cooled isopentane (Merck; Leuven, Belgium) and stored at -70C. Brain tissue of these cats was used to prepare the nuclear extraction for the Western blotting.
Preparation of the Conjugate and Immunization
A 26-amino-acid peptide of cat Fos (NH2 -Gly-Lys-Val-Glu-Gln-Leu-Ser-Pro-Glu-Glu-Glu-Glu-Lys-Arg-Arg-Ile-Arg-Gly- Glu-Arg-Asn-Lys-Met-Ala-Ala-Ala-COOH) was produced in our laboratory by continuous-flow peptide synthesis (
For immunization, 1 ml conjugate containing 1 mg conjugated peptide was thoroughly mixed with 1 ml complete Freund's adjuvant (Difco; Detroit, MI) and used to immunize two rabbits. The emulsion was injected SC at four separate sites. This procedure was repeated twice at 3-week intervals. For the following injections, similar emulsions were prepared using incomplete Freund's adjuvant (Difco). From the fourth injection on, blood samples were collected 6 days after injection. The samples were centrifuged the day after and the immune response was monitored immunocytochemically on brain sections of a lens-fitted cat and a visually stimulated cat. This revealed that the blood samples from the tenth injection of one rabbit provided the best staining. All results and photographs presented here were therefore obtained with this antiserum. A fraction of this antiserum was then affinity-purified on a CNBr-activated Sepharose 4B gel column (Amersham Pharmacia Biotech; Roosendaal, The Netherlands) coupled with the synthetic Fos peptide. The coupling ratio was 5 mg peptide/ml gel. The unabsorbed fraction of the serum was collected, and the absorbed antibodies were eluted separately from the column with 0.1 M glycine buffer (pH 2.8). Both fractions were tested in Western blotting experiments.
Western Blotting
A Western blotting experiment was carried out on visual cortex from two cats to determine whether the new cat Fos antibody recognized a protein band of the predicted size for cat Fos. One cat was stimulated with light for 1 hr after a period of darkness and the second cat was sacrificed under non-stimulation conditions (see above). Nuclear fractions from cortical gray matter of both experimental conditions were prepared according to
Immunocytochemistry
Single Staining.
Free-floating vibratome sections of four rats, one monkey, four cats, two lens-fitted, and two isolated-hemisphere cats were incubated overnight with the primary antibody (Fos 1:20,000 for cat brain tissue; Fos 1:5000 for rat and monkey brain tissue). In addition, free-floating vibratome sections of two light-stimulated cats and one monkey were incubated overnight with both commercial Fos antisera: Oncogene Science (1:1000) and Santa Cruz Biotechnology (1:1000). For the detection of Fos antibodies biotinylated goat anti-rabbit IgGs (DAKO; 1:500, 30 min) and peroxidase-conjugated streptavidin (DAKO; 1:600, 30 min) were used. All the dilutions were made in Tris-saline (0.01 M, pH 7.4) and all incubations were performed at RT under gentle agitation. The reaction product was visualized as a black precipitate using the glucose oxidasediaminobenzidinenickel method (
Double Staining.
Vibratome sections of two visually stimulated cats were treated the same way as the single-stained sections but detected with the Envision+ System Peroxidase (DAB) (DAKO), resulting in an intensified brown color (
Cresyl Violet Counterstaining
Some immunostained sections for Fos from each animal were counterstained with Cresyl violet (1%) (Fluka Chemical; Buchs, Switzerland) to determine which layers of the visual cortex contained Fos-immunoreactive nuclei and whether only neurons or also glial cells are involved in the immunoreaction (
Quantitative Analysis
Fos-immunoreactive nuclei were counted semiautomatically using a Leitz DM RBE microscope equipped with a color video camera (Optronics Engineering; Goleta, CA) attached to a computer-aided image analyzing system (Bioquant; R&M Biometrics, Nashville, TN). Each section was viewed at a magnification of x20. The quantitative analyses were performed within the medial bank of the lateral gyrus (Area 17). Quantitative data were obtained from DABnickel-intensified sections at three different HorsleyClarke levels from two visually stimulated animals. Adjacent sections counterstained with Cresyl violet (1%) (Fluka) were used to distinguish the supragranular layers in Area 17 to which the measurements have been restricted. We were compelled to quantify only in Layers II/III because the immunostaining pattern with the Fos antisera is most pronounced in these layers. A sampling frame, subdivided into 10 equally sized boxes, was positioned over cortical Layers II/III along Area 17. Three frames in each section stained with either our cat Fos antibody or the Fos antibody from Santa Cruz were screened. No quantitative analysis could be done for Fos stained with the Oncogene Science antibody. Because of a very low signal:noise ratio, the computer-aided image analyzing system could detect only background staining and no Fos-positive nuclei. We quantified the number of neuronal profiles, corresponding to complete or partially cut cells in the sections. Immunoreactive neuronal profiles were selected using a semiautomatic threshold procedure, based on the optical density of the neuronal profiles. The optical density corresponded to the intensity of the transmitted light and was measured on a scale from 0 (100% transmitted light) to 255 (0% transmitted light) for each pixel. Because there is a difference in intensity with which the nuclei were immunostained, we used the lowest optical density found over clearly stained nuclei as threshold. Criteria of object inclusion were the shape and the size of the neuronal nuclei and a strong immunoreactive status (
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Results |
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Immunocytochemical Screening
Two rabbits were immunized with the cat-specific Fos synthetic peptide. In one rabbit, Fos antibody was clearly detectable in blood samples after the tenth injection, using immunocytochemical screening of vibratome sections of cat visual cortex. The nuclear Fos staining observed in the cell was the result of the antigenantibody binding capacity. Identification of the cell type containing the Fos-positive nuclei was established in two ways. First, immunoreactive cells appeared to be neurons on the basis of observations of sections labeled for Fos and counterstained with Cresyl violet (Fig 1A). Fos immunoreactivity was found exclusively in nuclei of neurons and was not present in cytoplasm, nucleolus, axons, or dendrites. Second, double staining for Fos and glial fibrillary acidic protein (GFAP), a constitutive intermediate filament found in astrocytes, showed a negative correlation (Fig 1B). No double-labeled cells were observed, indicating that Fos immunoreactivity is restricted to neurons.
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Western Blotting
The specificity of the immunoreactivity of this new anti-cat Fos antiserum was confirmed by Western blotting analysis of different nuclear fractions of cat visual cortex (Fig 2). Immunodetection with the affinity-purified fraction revealed an intense and specific band at 62 kD, corresponding to the expected molecular weight of Fos protein (Fig 2, Lane 2), but this band was not detectable when the collected unabsorbed fraction of the serum was applied to the immunoblot (Fig 2, Lane 1). The 62-kD band was much more intense in the nuclear fraction of a visually stimulated cat than the faint band in the nuclear extract from a nonstimulated cat (Fig 2, Lane 3). As a verification of our results, we applied two commercially available antisera on a 5 µl nuclear fraction of cat visual cortex. The antisera from Santa Cruz Biotechnology (Fig 2, Lane 4) and from Oncogene Science (Fig 2, Lane 5) recognize the same intense band at 62 kD. Both our cat Fos antibody and the commercially applied antisera also identified additional bands at 46 kD, 35 kD, and 30 kD (Fig 2, Lanes 25). These bands correspond to the well-known Fos-related antigens (Fras) that represent a family of DNA binding proteins related to Fos. It is not unexpected that both Fos and Fras are recognized by this novel antibody, because the amino acid sequence of our synthetic peptide deduced from cat Fos shows a high homology to the three other Fos-related proteins (Fig 3).
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The anti-cat Fos antiserum therefore recognizes proteins with apparent molecular masses of 62, 46, 35, and 30 kD. Although we mention "immunoreactive Fos" in this article, the anti-Fos antibody against the 26-amino-acid sequence of cat Fos in fact recognizes both Fos (62 kD) and Fos-related antigens (46, 35, and 30 kD).
Immunocytochemical Detection of Fos in Cat Visual Cortex
Qualitative Analysis.
The IEG c-fos is expressed at very low basal levels under nonstimulatory conditions, whereas visual stimulation upregulates c-fos in cortical neurons. Therefore, we first examined the expression profile of Fos induction after 1-hr visual experience following an overnight dark adaptation in adult cat visual cortex. Fig 4A shows that the labeled nuclei were located almost solely above and below Layer IV, respectively, in Layers II/III and V/VI. In Layer I, Fos-immunoreactivity was rarely observed, reflecting the sparse cell density in this layer. Immunocytochemistry for Fos in the visual cortex of a lens-fitted cat resulted in a completely different pattern for the Fos expression profile. Whereas binocular visual stimulation led to a homogeneous distribution of Fos-positive nuclei in infra- and supragranular layers along the cortical surface (Fig 4A), monocular deprivation resulted in a columnar pattern for Fos in the primary visual cortex (Fig 4B and Fig 4C). Highly stained columns perpendicular to the pial surface alternate with columns that show lower Fos immunoreactivity throughout Layers II/III, V, and VI.
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As a control, we analyzed Fos immunoreactivity in a cat in which visual input was confined to one hemisphere by sectioning of the left optic tract and the corpus callosum (Fig 5). An overnight dark adaptation in combination with a binocular light exposure (1 hr) resulted in detection of Fos-immunostained neurons in the infra- and supragranular layers of the right hemisphere, which is driven by normal visual input (Fig 5A). In contrast, sectioning of the left optic tract and the corpus callosum resulted in very low to undetectable Fos levels in the same layers of the left visually deprived hemisphere (Fig 5B). These results show that visual stimulation induces alterations in Fos protein expression levels.
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Fig 6A to 6F compare the immunocytochemical staining pattern for Fos as detected with our novel cat Fos antibody and the two commercial Fos antisera on cat primary visual cortex. Marked differences in the labeling pattern were observed among sections stained for each Fos antiserum. The cat-specific Fos antibody used at a very low concentration (1:20,000) resulted in strong labeling of neurons in Layers II/III and V/VI and very low background staining (Fig 6A and Fig 6B). However, Fos staining with the Santa Cruz antibody was markedly lower in supra- and infragranular layers, resulting in a lower signal-to-noise ratio (Fig 6C and Fig 6D), despite the higher antibody concentration (1:1000) and much longer chromogen color reaction times (30 min). In addition, the Santa Cruz antibody induced more intense background staining at the tissue edges. The third Fos antibody from Oncogene Science (1:1000) showed little or no detection of Fos, with moderate to high nonspecific binding in Layers II/III and V/VI along the visual cortex in Area 17 ( Fig 6E and Fig 6F ).
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Quantitative Analysis. Because differences in the signal-to-noise ratio for the three Fos antibodies were consistently observed, we conducted semiquantitative analyses to determine the relative extent of the Fos labeling with the different antisera. This quantitative analysis revealed statistically significant (p<0.001) differences between the numbers of Fos-positive nuclei stained with the cat Fos antibody and the Santa Cruz Biotechnology antibody (Fig 7). In both cats, the mean number of Fos-immunoreactive neuronal profiles per mm2 detected with the cat Fos antibody was almost three times that of the Santa Cruz Fos antibody. No quantitative analysis was performed for Fos stained with the Oncogene Science antibody, because the computer-aided image analyzing system detected only background staining and no Fos-positive nuclei.
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Immunocytochemical Detection of Fos in Monkey and Rat Visual Cortex
To test the applicability of this cat Fos antibody to other mammalian species, we performed immunocytochemistry on monkey (Macaca mulatta ) and rat primary visual cortex. Immunodetection for Fos showed significant staining in Layers II/III, IV, and VI of monkey V1 after a binocular light input subsequent to a period of darkness (Fig 8A). Layers II/III (Fig 8B ) and VI contained the most Fos-immunostained nuclei. Layer IV of macaque monkey V1 is subdivided into four laminae. Fos immunoreactivity was prominent in Layers IVA and IVB. In addition, a band of intensely labeled cells in the lowest part of Layer IV, especially Layer IVCß, was also discernible. Layer I was devoid of Fos staining, whereas Layer V contained a small number of scattered Fos-immunopositive neurons (Fig 8C). Sections of monkey V1 were also processed for IEG immunocytochemistry with two commercially available antisera from Oncogene Science and Santa Cruz Biotechnology. Using the Santa Cruz antibody, no Fos staining was detected in monkey Layer V1 (Fig 8D); only intense nonspecific binding (cytoplasmic staining) and widespread background staining could be observed. Fair Fos staining in upper Layer II and moderate to high nonspecific binding in the other layers was detected with the Oncogene Science antibody (Fig 8E), as was the case with cat brain tissue.
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Fig 9 shows photomicrographs of the primary visual cortex of rats exposed to light either for 1 hr after darkness or for a prolonged period (8 hr). Fos was expressed at higher levels in the nuclei in cortical Layers II/III, IV, and VI, but minimally in Layer V after a 1-hr light stimulation (Fig 9A). Visual cortical Area 17 of control rats sacrificed under nonstimulatory conditions (Fig 9B) revealed a low signal related to the basal expression of Fos in the same layers. Laminar distribution of Fos expression in rat and monkey visual cortex was determined from adjacent Cresyl violet-stained sections. The neuronal nature of these Fos nuclei in both rat and monkey was assessed by combining Fos immunostaining with Cresyl violet counterstaining (not shown).
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Discussion |
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In this article we report the synthesis and characterization of a novel polyclonal antibody recognizing the protein product of the IEG c-fos in mammalian brain tissue. This Fos antibody was directed against amino acids 127152 of cat Fos in analogy to the polyclonal antibody against the M-peptide region of rat c-fos (
We next wanted to assess the applicability of this new antibody on brain tissue from other species, i.e., monkey and rat. Before the recent paper from
In addition, the detection of intense vs low Fos expression in primary visual cortex of a visually stimulated and a nonstimulated rat respectively (
In conclusion, the supporting evidence provided in the present study from Western blotting and immunodetection of Fos with this antibody give certainty of the successful development of a novel Fos antibody that is specific to cat Fos. This antibody clearly allows the investigator to identify the cellular localization of Fos in neurons activated under different physiological conditions in cat and monkey visual cortex.
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Acknowledgments |
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Supported by grants from the Belgian Queen Elisabeth Medical Foundation, the Belgian program of Inter-University Poles of Attraction P4/22, and the Fund for Scientific Research (Flanders, Belgium). Lutgarde Arckens was supported as a postdoctoral fellow of the Fund for Scientific Research, Flanders (Belgium, F.W.O.).
We thank Luc Grauwels for synthesis of the peptide, Kathy Cromphout for excellent technical assistance, and Dr Steve Raiguel for comments on the manuscript.
Received for publication July 23, 1999; accepted November 24, 1999.
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