Journal of Neuroscience Research 28:583-600 (1991)

Quantitative Image Analysis With Densitometry for Immunohistochemistry and Autoradiography of Receptor Binding Sites-Methodological Considerations R. Peretti-Renucci, C. Feuerstein, M. Manier, P. Lorimier, M. Savasta, J. Thibault, N. Mons, and M. Geffard Laboratoire de Physiologie Section Neurophysiologie (LAPSEN). INSERM U 3 18, Departement des Neurosciences Cliniques et Biologiques, Pavillon de Neurologie, CHU de Grenoble, BP 217 X, F-38043 Grenoble Cedex, ( R . P . - R . , C.F., M . M . , M . S . . P.L.). Laboratoire de Biochimie Cellulaire, College de France, F-75231 Paris Cedex 05, (J.T.). and Laboratoire de Neuroimmunologie, Institut de Biochimie Cellulaire et Neurochimie (IBCN), CNRS F-33077 Bordeaux Cedex ( N . M . , M.G.), France

Major technical progress in the development of computer-based image analysis has made possible the entry of autoradiography and immunohistochemistry into a new era where quantification by densitometry has become easily accessible. Autoradiography could become quantitative and displayed adequate reproducibility with the help of emulsion-coated films and the use of scales of standards of known radioactivity exposed and analyzed in parallel to the tissue sections. Immunohistochemistry after revelation by a colorbased enzymatic technique can also become quantitative, providing that standardization of the crucial steps of the procedure and calibration through a parallel treatment of a scale of antigen standards can be ensured. Such an approach is described here in the rat with reference to tyrosine hydroxylase (TH), the main synthesizing enzyme for catecholamines, and with dopamine (DA) itself, a catecholaminergic neurotransmitter. The different parts of the procedure, which can influence the results, such as the fixation of the animals by perfusion and the evaluation of the fluctuations via the calibration curve, are discussed in detail. Biological validation of the proposed procedure is described by reference to experiments already well documented biochemically, such as the induction effect of reserpine on TH in the rat locus coeruleus and the depleting effect of a-methyltyrosine (AMPT), a well-known blocker of TH activity, on rat striatal DA content. Finally the importance of restricting the measurements to the (pseudo)linear portion of the calibration curve is illustrated by the autoradiographic identification of the differential intrastriatal repartition of the dopaminergic D, and D, receptor 0 1991 Wiley-Liss, Inc.

sites, particularly the dual patch-matrix compartments. Key words: image analysis, tyrosine hydroxylase, dopamine, dopaminergic D, and D, receptors INTRODUCTION Recent progress in the development of computer technology has brought image analysis into a new era of high interest for neuroscience. Not only has such a technology facilitated all kinds of morphometric studies, but it has also made possible measurements of the contents of labeled markers at histological and cytological levels. Due to the large number of commercially available radioactive compounds with high specific activity, autoradiography (Kuhar, 1981) has rapidly taken advantage of this technology (Gallistel et al., 1982; Goochee et al., 1983). The development of calibrated standards of predetermined radioactive content has thus provided the quantitative step. These standards are processed in parallel to the exposed labeled tissue sections and allow one to obtain a calibration curve for each experiment or each radiographic film (Altar et al., 1984; Palacios et al., 1981a; Unnerstall et al., 1982). Scales of radioactive standards are now also available from commercial sources, which renders quantitative analysis easier. Immunohistochemistry has become a powerful and

Received January 31, 1990: revised July 16, 1990; accepted July 17, 1990. Address reprint requests to C. Feuerstein. Laboratoire de Physiologie section Neurophysiologie (LAPSEN). INSERM U 3 18, Departement des Neurosciences Cliniques et Biologiques. Pavillon de Neurologie, CHU de Grenoble, BP 217 X , F-38043 Grenoble Cedex, France.

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widely used technique for the visualization of most of the containing 30 mg BSA/ml. The saturated membrane was biochemical markers at the histological and cytological then transferred into another PBS BSA bath (supplelevels. The sensitivity and the resolution provided by mented with 0.1% sodium azide), in which it could be such color-based immunohistochemical detections (im- left overnight at room temperature and stored frozen at munoenzymatic colorations or immunofluorescence) -20°C. Thus, a full scale of eight different known (Coons, 1958; Sternberger, 1979) are now of such a high amounts of adsorbed TH standards was obtained after quality that the quantitative approach appears as a nec- cutting a strip of membrane including one of the 12 rows. essary step. Recently radioimmunohistochemistry has Such a strip could be further processed throughout the been introduced and it represents such a tentative step. In various steps of the immunohistochemical technique, in this case, calibration is ensured via the use of either parallel with the tissue sections. Dopamine (DA). DA hydrochloride (Sigma) was radioactive scales with commercially available standards or a set of known amounts of antigen adsorbed onto conjugated via glutaraldehyde bridge to BSA (Sigma), paper filter strips or nitrocellulose membranes (Cuello et according to a previously described method (Chagnaud a].. 1982; Correa et al., 1988; Hunt and Mantyh, 1984; et al., 1987; Geffard et al., 1984). The molar coupling Lewis and Watson, 1989; McLean et al., 1985; Weiss- ratio of the DA-G-BSA preparation used here was 23 (as mann et al., 1988, 1989). When considering the high calculated with the help of a known minute amount 'Hresolution of the images provided by the enzymatic- DA (Amersham) diluted in the DA hydrochloride solubased color detection of the immunohistochemical reac- tion before conjugation to BSA). The preparation was tion, the crude autoradiographic images generated by ra- diluted in 100 p1 PBS containing 1% sodium metdioimmunohistochemistry appear then to have limited abisulfite (SMB, Sigma, PB-SMB) to obtain different microscopical resolution and high background. In this concentrations of DA, from 25 to 3,000 ng (as DA base) context, the aim of our own study was to develop a in 100 p1. As for TH standards, each DA-G-BSA solusimple procedure for the realization of scales of stan- tion was complemented with BSA to provide each samdards of antigens adaptable to imniunoenzymatic color- ple with a constant total protein content. The DA-G-BSA ation procedures. This should offer new opportunities for dilutions were spotted under mild vacuum onto a prequantitative calibration of computer-based image analy- washed (in PB-SMB) nitrocellulose BA 85 membrane. Rinsing was carried out with 100 (11 PB-SMB, followed sis systems. by immersion of the nitrocellulose membrane for 1 hr at room temperature in a PB-SMB solution containing 30 mg BSA/ml. Strips of nitrocellulose membrane could MATERIALS AND METHODS then be stored frozen in a fresh PB-SMB solution conAntigen Scales of Adsorbed Standards taining 30 mg BSA/ml. To evaluate the effective amount Tyrosine hydroxylase (TH). Purified TH from rat of DA bound to nitrocellulose, recovery of the binding to phaeochromocytoma tumors has been obtained accord- the membrane was estimated by cutting some of the ing to a previously published procedure (Thibault et al., spots, digesting them for 3 hr at 50°C in NaOH 0.5 N 1981). TH was estimated 99% pure by SDS gel electro- before neutralization by nitric acid 2 N and further scinphoresis, and its amount was estimated by the Bradford tillation counting of 'H-DA content. assay (Bradford, 1976) by using bovine serum albumin (BSA, Sigma Chemical Co, St. Louis, Missouri, USA) as a standard. Different dilutions of purified TH were Animals Studies were performed with male Wistar rats (Iffa then prepared in 0 . l M phosphate buffer pH 7.4 containing 0.9% NaCl (PBS). Their range extended from 25 to Credo, Les Oncins. L' Arbresle, France) weighing 1803,000 ng per 100 p1 of PBS. BSA was added in each TH 250 g and housed in a temperature controlled environsolution to ensure a constant total protein content of ment (23 ? 1°C) with food and water ad libitum and a 12 3.000 ng per sample. These TH+BSA preparations hr-on/ 12 hr-off dark-light cycle. For reserpine treatments, the animals received a were then spotted under mild vacuum onto a prewashed (in PBS) nitrocellulose membrane (BA 85, Schleicher subcutaneous injection of 10 mg/kg of the drug (Serpasil, and Schuell, Inc., Keene, New Hampshire, USA), with Ciba-Geigy, Basel, Switzerland) 3 days prior to sacrithe use of a 96 well filtration system. Accordingly. for fice. AMPT treatments consisted of intrapcritoneal ineach nitrocellulose sheet, eight different samples of jections of 250 mg/kg of a-methyl- 1-tyrosine methylTH+BSA were distributed along 12 rows (100 p1 per ester (AMPT, Regis Chemical Co., Morton Groves, spot). Each well was then rinsed with 100 (1.1 PBS. Illinois, USA) 2 hr 30 min before perfusion of the aniThereafter, nitrocellulose was immersed for 1 hr at room mals for immunohistochemistry (see below). Control rats temperature through gentle agitation into a PBS solution received saline injections.

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brane were mounted onto microscope glass slides and coverslipped in XAM-Neutral (Improved white, Gurr, Tyrosine hydroxylase. The animals were anes- Hopkin and Williams, Essex, Great Britain), or in imthetized by intraperitoneal injection of a mixture contain- mersion oil, immediately before examination under the ing chloral hydrate and nembutal. They were perfused microscope and further image analysis. transcardially with 15 ml saline (NaCI 0.9%) followed Dopamine. Animals were perfused transcardially by 600 ml of 4% paraformaldehyde (PFA) in 0.1 M with 15 ml saline containing 1% SMB followed by 600 sodium phosphate buffer (pH 7.4) delivered by a peri- ml of 5% glutaraldehyde (EM grade, 25% aqueous sostaltic pump (Mastoflex) at 150 ml/min for 2 rnin fol- lution, TAAB Laboratories Equipment, Reading, Berks, lowed by a lower rate at 50 ml/min for 6 min. The brain Great Britain) in 0.1M sodium phosphate buffer (pH 7.4) was removed from the skull and postfixed in the same containing 1% SMB. The peristaltic pump was adjusted fixative for 3 hr at 4"C, in parallel with a strip of ad- to the same rates as those described above for TH. After sorbed TH standards (TH scale). Thereafter, tissue was removal of the brain, blocks of tissue were postfixed in dipped overnight at 4°C in a sucrose solution (15%) and the same fixative for 30 min at 4°C and transferred into the brain was frozen in isopentane (chilled down to a 15% sucrose solution containing 1% SMB for over-35°C with dry ice) and further cut with a microtome night incubation at 4°C. whereas the DA scale was postcryostat (Microm, RFA) at -20°C into 20 pm-thick fixed for 1 hr at 4°C in the fixative and rinsed for 2 hr (or coronal sections, according to the atlas of Paxinos and overnight) at 4°C in sodium phosphate buffer containing Watson ( 1982). Tissue sections and TH scale (rinsed 2 hr I % SMB. Coronal tissue sections (20 k m thick, as dein PBS) were processed altogether, as free-floating sec- scribed above) and DA scale were processed all together tions, for PAP immunostaining, as described elsewhere for immunohistochemistry as free-floating sections, as (Mouchet et al., 1986; Manier et al., 1987). Briefly, described elsewhere (Chagnaud et al., 1987). Briefly, tissue sections and nitrocellulose membrane were incu- tissue sections and DA scale were incubated for 1 hr in bated for 1 hr in 0.02M PBS containing 1% normal 0.01M Tris-HC1 buffer containing 1% SMB (TB-SMB, swine serum (NSW, Dakopatts, Denmark). After wash- pH 7.2) and 3% normal rabbit serum (NRS, Miles Labing (2 X 10 min) in PBS containing 0.3% Triton X-100 oratories, Naperville, Illinois, USA). After washing ( 2 (PBST, Sigma), sections and nitrocellulose membrane X 10 min) in TB-SMB containing 0.3% Triton X-100 were incubated in anti-TH antiserum (Thibault et al., (TB-TSMB), sections and nitrocellulose membrane were 1981) diluted at 1:2,000 in PBST with 1% NSW and incubated in anti-DA monoclonal antibody (Chagnaud et 0.1% sodium azide for 36 hr at 4°C. After washing in al., 1987) diluted at 1:10,000 in TB-TSMB containing PBST (2 X 10 min), the following steps consisted of 1% NRS for 36 hr at 4°C. After washing in saline Trisincubation ( 1 hr 30 min) in swine antirabbit serum (Da- HCI buffer (TBS, 0.05M, pH 7.5) containing 0.3%Trikopatts, Denmark) diluted at 1: 100 in PBST containing ton X-100 (TBST, 2 X 10 min), the following steps 1% NSW, followed by washing (2 X 10 min) in PBST consisted of incubation ( 1 hr 30 min) in rabbit antimouse and incubation ( 1 hr) in rabbit PAP complex (Dakopatts) serum (Dakopatts) diluted at 1 :100 in TBST containing diluted at 1:400 in PBS followed by one rinse in PBS (10 I% NRS, followed by washing (2 x 10 min) in TBST min) and terminated by final rinsing in 0.05 M Tris-HCI and incubation (1 hr) in mouse PAP complex (Dakopatts) buffer pH 7.5. The brown peroxidase color was revealed diluted at 1:lOO in TBS. All the subsequent steps were with 0.025% 3,3'-diaminobenzidine hydrochloride similar to those described for TH immunolabeling. (DAB, Sigma) and 0.01% H,O, in Tris-HC1 buffer pH 7.5. The concentration of DAB was decreased here to Autoradiography of the Dopaminergic and Mu 0.025% (instead of the usual 0.05% concentration) be- Opiate Binding Sites cause of the very intense and saturating coloration obThe animals were decapitated; their brains were tained for striatal TH immunostaining. Severe control of quickly removed and frozen in isopentane at -35°C. the duration of the incubation in the DAB-H,O, solution Coronal tissue sections (15 pm thick) were prepared at was requested (5 rnin here) to ensure comparable color- -20°C in a microtome cryostat, according to the atlas of ation procedure between tissue sections and TH scale. Paxinos and Watson (1982). Sections were thawAfter rinsing in Tris-HCI buffer (10 min) and a further mounted onto gelatine-coated microscope slides. After washing in 0.02M PBS, sections were mounted onto drying under mild vacuum at room temperature for 20 gelatine-coated glass slides and dehydrated simulta- min, slides were stored frozen at -80°C until use. neously with the nitrocellulose TH scale as following: 10 After a 20 min preincubation of the sections at rnin in 35% ethanol, 10 rnin in 70% ethanol, 10 rnin in room temperature in 0.05M Tris-HCI buffer pH 7.4 con95% ethanol, 10 min in 100% ethanol, and 10 min in taining various salts (NaCl 120 niM, KCI 5 mM, CaCI, xylene. Both tissue sections and nitrocellulose mem- 2 mM, MgCI, 1 mM), the binding to D, and D2 dopa-

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minergic receptor sites was performed by incubation for 1 hr at room temperature in the same buffer containing either 0.4 nM of 'H-spiperone (77 Ci/mmol, Amersham) for D, sites or 1 nM of 'H-SCH 23390 (85 Ci/mmol, Amersham) for D, sites according to our usual procedure (Savasta et al., 1986, 1987). Nonspecific binding was defined with 1 pM of ( + ) butaclamol (Research Biochemical Inc., Wayland, Massachusetts, USA) for D, sites and in presence of 10 p M ketanserine (Janssen Pharmaceutica, Beerse, Belgium) to prevent the fixation of 'H-spiperone to 5 HT, sites. For D, sites, the nonspecific binding was performed with 1 pM of a-flupentixol (Lundbeck, Copenhagen, Denmark) and in presence of 10 pM sulpiride (Sigma) to block the D, sites. Thereafter, tissue sections were briefly dipped in cold distilled water and rinsed by two successive 5 min washes in 0.05M ice-cold Tris-HC1 buffer. In the case of mu opiate binding sites, preincubation was conducted for 30 min at room temperature in 0.05M phosphate buffer pH 7.5 containing 120 mM NaCl, followed by incubation for 30 rnin at room temperature in the same buffer containing 0.8 nM of 'Hetorphine (30 Ciimmol, Amersham), nonspecific binding being defined with 1 pM of naloxone (Sigma). Tissue sections were further briefly dipped in cold distilled water and rinsed by six successive 20 sec washes in 0.05M ice-cold phosphate buffer. In all cases, after a short immersion in cold distilled water, the slides were dried at room temperature under a cold air stream. Scintillation counts of digested striatal sections scraped with a razor blade revealed that the specific binding accounted for 85% of the total binding in the case of D, sites, for 95% in the case of D, ones, and for 90% in the case of mu opiate sites. Tissue sections were then apposed to 'HUltrofilm (LKB. Broma, Sweden) or 'H-Hyperfilm (Amersham), in parallel with 'H-microscales (ref RPA 506 and 507, Amersham). After 2-3 weeks of exposition at 4°C in light-proof Kodak X-OMATIC cassettes, films were developed in Kodak D- 19 for 1 min at 19"C, rinsed, and fixed as usual. Protein evaluation. In some experiments, coronal sections of striatum cut at 15 pm for autoradiography or at 20 pm for immunohistochemistry were thaw-mounted onto glass slides. The respective striatal sections were then scraped with a razor blade into a glass tube and digested overnight in 100 p1 of 0.5N NaOH. The tissue suspension was then further sonicated for 1 min at 20 kHz at 40 W (Sonimasse 75 Ti, Ultrasons Annemasse, France). Reference standards were obtained by diluting in 0.5N NaOH a solution of calibrated concentration of bovine serum albumin. Tissue preparations and standards were analyzed through the colorimetric procedure of Bradford (1976) with Bio Rad Reagents (Bio Rad Protein Assay kit, Bio Rad Laboratories, Richmond,

California, USA). The coloration was then quantified by spectrophotometry at 595 nm and the tissue protein content was expressed by reference to the bovine serum albumin calibration curve. Computer-based image analysis. The analyzer used was the system SAMBA@,versions 200 and 2005 (Thomson T.I.T.N., CGE-Alcatel, Grenoble, France). The SAMBA 200@version consists of a microscope with a motorized scanning stage and autofocus, equipped with an electro-optical image scanner including a photomultiplier tube, connected to a command and control processor for image acquisition, an image analysis and pattern recognition processor, a hardwired operator for color processing, a data analysis processor, a color image display. and a text-and-graphics terminal with printer. When using a 2.5 objective, the whole surface occupied by the rat corpus striatum could be scanned in one step by the software-controlled scanning stage of the microscope and visualized on the full-scale color image display (256 X 256 dot matrix). With this magnification the size of each image point (pixel) was 16 pm. The recent SAMBA 2005@version derived from the 200 one, except that a CCD monochromatic camera (CP-9003, 625 lines, 500 (H) X 582 (V) picture elements, Tokina Optical Co., Tokyo, Japan) was mounted on the microscope in place of the photomultiplier. This system is by far quicker than the SAMBA 200@ version and color image display is a 5 12 x 5 12 dot matrix. At 1.25 magnification on a Zeiss Axiophot microscope, the whole striatum could be visualized on the color image display without the need for scanning. Through either photomultiplier or CCD camera, optical densities (O.D.) were calculated by common logarithmic transform of the ratio of incident to transmitted light. The digitalized signal allowed a precision better than 1% O.D. The whole O.D. range (0-+2) was divided into 256 intervals (0-255). Through interaction with the software, it was possible to affect a given color of the image display to a selected set of O.D. values. Therefore the gray-toned images on the autoradiographic film could be reconstructed into a pseudocolor-coded image, according to O.D. intervals. Three filters (red, blue, green) could be used to select optimal wavelengths. In the case of the brown peroxidase coloration, a blue filter at 450 t 4 nm (Schott Glaswerke, Wiesbaden, Germany) was inserted, whereas none was introduced for analysis of the black and white gray-toned autoradiographic films. For each set of experiments. the respective scales of standards processed in parallel were first analyzed to measure the mean O.D. values (i.e., O.D. per pixel in a 0-255 range) for each standard spot minus background value of the surrounding nitrocellulose membrane. These values were then plotted against the corresponding

Quantitative Immunohistochemistry

amounts of standard antigen or radioactivity and the best statistical fit (polynomial adjustment up to an order of 5 ) was calculated. Subsequent analyses of the tissue sections were restricted to values strictly belonging to the pseudolinear portion of the calibration curve. With the computerized system, it was then possible to automatically delineate the portions of the image belonging to a selected window of O.D. The surface of those selected regions (i,e., number of pixels within a defined O.D. interval) and their mean O.D. could be evaluated by the computer and further converted into molar quantities by means of the respective calibration curves. Thus number of binding sites or antigen content could be estimated. For calibration of the 3H radioactive standards, the correspondence in nCi/mg protein of gray matter given by the manufacturer (Amersham) was used and further converted into fmol/mg protein, according to the specific activity of the radioactive material used (after eventual correction for time-related tritium decay factor). Variation of the quenching of tritium according to the abundance of white matter in the different parts of the brain (Alexander et al., 1981; Geary and Wooten, 1985; Herkenham and Sokoloff. 1984; Kuhar and Unnerstall, 1982; Rainbow et a]., 1984) was taken into account when comparing results obtained from different cerebral regions or even from distinct subregions within the corpus striatum itself, although they were negligible in the latter case (Dubois et al., 1986). Densitometric subtraction of two images, pixel by pixel, after their adequate superimposition, allowed, if necessary, the extraction of a resulting image that corresponded to specific binding (subtraction of the densitometric image of the nonspecific binding from that of the total binding). Different interactive devices, such as joystick or computer mouse, allowed the drawing of masks of various shapes and sizes on the display, thus delineating zones of the image where the desired measurements (mean O.D., surface) could be properly obtained. For the calibration of the antigen scales, as revealed by the immunoperoxidase-DAB brown coloration, each spot on the nitrocellulose sheet was thoroughly dehydrated and freshly mounted in XAM-Neutral medium or in immersion oil to obtain a translucid background of the white nitrocellulose membrane. This became necessary for adequate examination of the membrane by transillumination through the microscope. The image analysis system allowed then measurement of the surface (in pixels) and determination of the mean O.D. value of each spot, after subtraction of the mean O.D. of the surrounding background. Since antigen contents had to be estimated relatively to striatal protein content, the respective volume of each spot (surface of the spot X thickness of the membrane) with its related c

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protein equivalence to the studied brain structure had to be calculated. The mean protein content of the studied structure in each anatomical plane was then estimated biochemically from a 20 pm scraped section, as described here above. The analysis of an immunohistochemically stained section at the same plane allowed then the measurement of the surface of the structure at the same anatomical level. When dividing the value of the protein content measured biochemically by the number of pixels corresponding to the related surface of the structure. one could get an estimation of the mean amount of protein contained in the precise volume of tissue of the studied structure delimited by a surface of 1 pixel and a thickness of 20 pm. By taking into account the respective surface (in pixels) of each spot of antigen standard, the latter value (protein content per pixel) allowed evaluation of the equivalent protein content of the corresponding spot of antigen. In fact, since the nitrocellulose sheet is about six times thicker than the 20 pm-thick tissue sections, the latter result was then corrected by this factor to allow expression of the units of the calibration curve directly in ng of antigen per mg of equivalent protein of the studied structure. This could be achieved by dividing the known absolute amount of antigen spotted onto the nitrocellulose sheet (corrected by the estimated recovery of the binding of the antigen to the membrane) by the equivalent protein value calculated for each spot. Measurement of the mean O.D. values for each spot and plotting these results against the respective contents of spotted antigens (in ng/mg protein) resulted then in obtaining a calibration curve to be used as external standard for each immunohistochemical experiment.

RESULTS Calibration Curves of Standards and Analysis of the Main Factors of Their Variations By plotting mean O.D. from each rectangular graytoned image on the film (subtracted by the surrounding mean O.D. value as background) against the respective amount of radioactivity of the corresponding scale, a typical calibration curve could be obtained for autoradiography (Fig. 1A). When adequate exposition time was defined, the calibration curve displayed a pseudolinear section limited at both extremes by curvilinear portions showing asymptotic progression. While many mathematical tools are able to describe adequately the whole calibration curve (specially via polynomial fitting of high order), it appeared best to restrict ourselves to the pseudolinear portion of each curve. Effectively, any attempt to convert measured O.D. values into absolute amounts of radioactive material through incurvated portions of the calibration curve or through sections with asymptotic trend appeared to entail undesirably large er-

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rors. Adapting the duration of the exposition time against the 3H-sensitive film according to the working range of radioactivity for strict measurements within the linear part of the calibration curve appeared then to be the most valuable means for appropriate quantitative autoradiography. On the same grounds, calibration of the i n m u n o histochemical reaction with the use of known amounts of antigen processed in parallel was expected, in this study, to allow indirect evaluation of the amounts of tissue antigen in different brain regions and to provide a reliable control for the procedure. The striking resemblance of the calibration curve of Figure IS obtained for the immunohistochemically stained brown-colored standards of antigen with that of Figure 1A favors such an assumption. The reproducibility of the quantitative immunohistochemical procedure, within each set of experiments, could directly be estimated by the evaluation of the variability of the O.D. measures made on the various spots of each scale of standards (or even on spots from multiple sets of scales processed in parallel). Sensitivity, as for usual spectrophotometric biochemical assays, could be determined by checking the theoretical amount of antigen corresponding to an O.D. value (after background subtraction) equal to the background.

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Besides the attention to be given to carefully adjusting the concentration of the DAB-H,O, mixture and to strictly controlling the timing of the enzymatic reaction for each section, as for any biochemical enzymatic assay, it appeared possible to get information on the range of the quantitative procedure with the help of the calibration curve. Effectively, the extent of (pseudo)linearity of the plotting curve allowed one to control whether the concentration of the primary antibody (and, subsequently, that of the secondary antiserum and the PAP complex) was high enough as to saturate all the antigen sites to be assayed in the experiment. It is obvious that a too high dilution of antibody could lead to a sharp reduction of the amplitude of the linear relationship of the antigen-antibody reaction. This clearly manifests itself as a lowering effect on the level of the upper saturating plateau of the calibration curve (Fig. 1C). Accordingly, the use of extremely high concentrations of antiserum might appear to be theoretically convenient for most biological situations. However, such a systematic procedure still remains inappropriate not only because of cost consequences but also because of a parallel increase of background levels and of nonspecific detections. Therefore, optimization of the dilution to be used for each antiserum had to be performed. The dilutions of

Fig. I . A: Example of calibration curve obtained with radio- cording to the 0-255 intervals). The O.D. values measured active scales as used in quantitative autoradiography for the from the immunolabeled brown coloration of the tissue secmeasurement of the density of receptor binding sites. The tions (run in parallel to the antigen scales throughout the whole curve results from a polynomial adjustment of the plotting procedure) can be deducted from the calibration curve, thereby points of the mean O.D. values measured on the gray-toned allowing the evaluation of the immunolabeling into ng of anfilm against the respective known amounts of radioactivity on tigen per mg of tissue protein. C: Theoretical curves reconthe scales. The autoradiographic gray-toned image of the stan- structed from experimental data and evidencing the influence dard scales apposed to the film is represented near the curve. of the dilution of the primary antibody (A.B.), as illustrated by The autoradiographic scales and the radioactive tissue sections the incidence on the calibration curves and the extent of their are always exposed in parallel against the same film sheet. In linear portion. This allows one to control whether a given our hands, nonsaturating conditions arc obtained when the concentration (X) of the primary antibody used is high enough measured O.D. are restricted to 10-65 O.D. units (according to saturate all the reacting antigenic sites to be measured. A to the 0-255 intervals of the full O.D. range). Accordingly too-high dilution of the primary antibody (X/2 and X/4 here) correspondence between the O.D. values measured on the film clearly produces an important reduction of the range of the for the images of the tissue sections and the respective amounts pseudolinear portion of the calibration curve, through a lowof radioactivity or density of binding sites (expressed in fmol ering effect on the level of the saturating plateau, the too-low per mg of tissue or of protein) can be deducted from such a amount of antibody being thus unable to react with all the calibration curve. B: Example of calibration curve obtained antigens available in the preparation. D: Theoretical curves with known amounts of tyrosine hydroxylase spotted onto ni- reconstructed from experimental data and evidencing the introcellulose sheets and processed through immunohistochem- fluence of the concentration of the DP.B-H20, mixture on the istry with enzymatic coloration (see Materials and Methods for peroxidase coloration step. The enzymatic reaction speed being details of the procedure). As in A, the calibration curve is lower. the amount of brown-colored precipitate formed during obtained after polynomial adjustment of the plotting of the a given time becomes lower, thereby allowing the saturating mean O.D., measured for each brown-colored spot, against the plateau level to be reached at a similar O.D. level but for respective amounts of antigen adsorbed to the nitrocellulose higher amounts of antigen. providing that the amounts of ansheet. The original image of the set of immunolabeled spots is tibody used are kept high enough to saturate all the antigen represented near the curve. In our hands, nonsaturating mea- sites available in the preparation. A lowering effect on the surements (pseudolinear portion of the calibration curve) are slope of the pseudolinear portion of the calibration curve is obtained when O.D. is restricted to 5-30 O.D. units (ac- then obtained.

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and Watson, 1982), whereas Figure 2B and F represent the same section but after conversion into a pseudocolorcoded reconstructed image, according to the TH calibration curve obtained from TH standards processed in parallel (Fig. 1B). Such a heterogeneous labeling is clearly exemplified here by quantitative immunohistochemistry with enzymatic coloration. To facilitate comparison with published biochemical data from the whole locus coeruleus, TH contents were calculated in three different rats from images displaying the extent of the whole locus coeruleus surface per coronal section taken from a set of at least six different coronal sections distributed rostrocaudally along the locus coeruleus. The values obtained were then of 137 ? 8 ng/mg protein (mean k SEM). Three days after a single subcutaneous injection of 10 mg/kg of reserpine, the brown-colored immunostaining of the locus coeruleus appeared stronger (Fig. 2C and G ) , which was confirmed through the TH-related calibration curve (Fig. 2D and H). Although sections from control animals, from animals treated with reserpine, and a set of TH standard scales were systematically processed in parallel. one might object that some variations in the intensity of the labeling could be related to fluctuations in the fixation procedure of the rats, particularly during the perfusion step. Therefore, to evaluate the perfusion variability, O.D. measures of TH in catecholaminergic regions other than the locus coeruleus itself and known for being not affected or poorly affected by the related reserpine treatment were systematically ensured. Such measures could serve as internal standard for the control of eventual fixation variations between animals. When TH contents were calculated on the whole locus coeruleus through six different coronal sections along the rostrocaudal axis of three reserpinized rats, values obtained were 275 ? 12 ng/mg protein (mean -+ SEM), which corresponded to two times the mean value measured in control animals. Interestingly reserpine had no effect on TH content within the cells of the ventral tegmental area or on those Biological Validation of the Quantitative of the substantia nigra pars compacta (Fig. 3 ) . In the Immunohistochemical Procedure latter case, the immunohistochemical staining of TH To know whether the proposed quantitative immu- found in the various cell bodies (Fig. 3A, B, E, F for nohistochemical procedure might have reliable support, control animals) remained unaffected by the reserpine biological experiments leading to biochemically well- treatment (Fig. 3C. D, G , H for reserpinized animals). documented variations of the contents of TH or DA have TH contents were 145 -+ 9 ng/mg protein (mean SEM) been performed through our own procedure. in the substantia nigra of control rats and 151 ? 10 ng/mg protein in the case of reserpinized ones. Induction of TH in the Rat Locus Coeruleus by Reserpine Blockade of TH Activity by Methyl Tyrosine (AMPT) and its Consequences for Striatal The original brown-colored immunolabeling of TH DA Content in the locus coeruleus of a control rat is presented in Figure 2A and E from a coronal section taken at the level The immunolabeling of DA in the rat striatum at of plane 33 of the atlas of Paxinos and Watson (Paxinos coronal plane 14 (according to Paxinos and Watson,

anti-TH antiserum (1:2,000) and of the anti-DA one (1: 10,000)used here allowed the detection of a large range of amounts of antigen within the (pseudo)linear part of the calibration curve (Fig. IB), even if the antigen contents increased by a factor of two or three over the basal values. In some cases, although concentrations of the various antibodies appear well adapted to the effective amounts of antigen to be detected in the tissue, the upper saturating plateau of the calibration curve could be reached for even lower amounts of antigen than what might have been expected. This could occur in the case of very intense brown coloration (as it is the case for TH and DA in the striatum for example) and it corresponded to a saturation effect of the highly sensitive analyzer, as for conventional spectrophotometry. This differs from most visual enhancements which require intense coloration for optimal contrast. One of the solutions to such a problem would have been to reduce the thickness of the tissue sections, but this is not always available. To circumvent such a saturation effect due to over-intense colorations, the DAB-H,O, concentration could be optimized. Effectively, the final brown coloration being the end product of an enzymatic reaction, speed of this reaction is related to the concentration of substrate. By lowering the concentration of DAB-H,O,, lower amounts of the brown end product were then formed in a given time, thereby leading to a lower slope of the (pseudo)linear portion of the curve, the maximal O.D. values of the upper saturating plateau remaining unchanged (Fig. ID). It allowed then the saturating plateau to be reached for higher amounts of antigen, providing that the dilutions of the antisera still remained compatible with such high amounts of antigens (Fig. 1C and D). Interestingly, after such an optimization, manipulation and reproducibility of the coloration step became even easier, a 5 min time-controlled reaction (as chosen here for TH and DA (Fig. IB) being more easily reproducible from one section to the other than if this step had only lasted a few seconds.

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Quantitative Immunohistochemistry

1982) and its quantitative repartition within this structure after display of the image in pseudocolor, according to the corresponding DA calibration curve, are illustrated in Figure 4A and 4B respectively. The uneven distribution of DA (mean 2 SEM = 99 ? 18 ng/mg protein, when considering evaluation from various sections along the whole striatal rostrocaudal extent) revealed a possible emergence of the two richest zones, a lateral aspect (mean -+ SEM = 120 2 28 ng/mg protein) and a small dorsomedial strip (mean ? SEM = 85 17 n g h g protein) along the border of the ventricle, separated by a zone of lower density (mean SEM = 69 2 18 n g h g protein) where most of the myelinated fibers originating in the cortex cross the striaturn. After a single intraperitoneal injection of 250 mg/ kg AMPT, striatal DA contents sharply dropped down, the decrease of DA in the whole striatal extent being 54 13% as compared to the DA content found in control striata. The decrease of DA content appeared more pronounced (70 8%) in the lateral aspect than in the dorsomedial one (27 ? 9%), being only 10 ? 9% in the remaining central part, evidencing then some differential DA turnover rates between these striatal subregions (Fig. 4C and D). In addition, islands of higher DA content were more discernible after AMPT treatment than in control striatal sections, revealing that the patch-matrix compartmentation of DA in the rat striatum. with its differential DA turnover rate, is still observable in the adult rat.

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Differential Striatal D, and D, Dopaminergic Receptor Site Repartition Although D, and D, sites were already known to be present in high amounts in the striatum, proper use of a calibration curve of standards for autoradiography has been rarely ensured and the subtle differential patterns of the striatal repartition of these two receptor sites have been mostly obscured. Figure 4F and G display the reconstructed image in pseudocolor of the autoradiographic films of the binding of D , and D2 dopaminergic binding sites respectively. taken from serial coronal sections of the same rat at plane 14 (according to Paxinos and Watson, 1982). The color-coded reconstructed image refers only to the (pseudo)linear portion of the autoradiographic calibration curve (Fig. 1A), nonspecific binding being represented in black (background), blue, and cyan colors, whereas true specific binding (total minus nonspecific binding) appears in green, yellow, red, magenta, and white (in line with increasing contents). The differential niediolateral pattern of repartition for these two DA receptor sites is clearly evidenced. D, sites are in register with the repartition pattern found for DA content, i.e., a subset of two zones of higher labeling, one, the largest, in the lateral aspect and corresponding

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to the putamen region, the other rather restricted to the dorsomedial part of the striatum being related merely to the caudate nucleus, both zones of high D, sites density being separated by a central portion of rather lower density (Fig. 4G). In contrast, D, binding sites have a more widespread striatal repartition, without the low-density central zone identified for D, sites, with a heavily labeled portion in the dorsomedial part and in the lateral aspect, as for D, sites, but with a more ventrolateral location than for D, ones for the latter subregion. A particular pattern emerged, clearly evidenced here only for D, sites, showing that the richest striatal zones with D, sites appear as islands or patches (in magenta) superimposed on a less intensely labeled background or matrix (in green) (Fig. 4F). Some of these islands are in register with those classically found for mu opiate binding sites labeled at the same striatal levels with 'H-etorphine (Fig. 4E). Visualization of such a patchy organization of the striatal D , binding sites could be facilitated here because quantitation was strictly restricted to nonsaturating 0.D. levels, without the masking effects related to overexposition of the films.

DISCUSSION By referring to the commonly used procedure for quantitation of autoradiographic films by computer-assisted image analysis, where scales of standards of known radioactivity are processed in parallel (Altar et al., 1984; Davenport and Hall, 1988; Palacios et a]., 1981a; Unnerstall et al., 1982), a similar approach was proposed here for immunohistochemistry with enzymatic (peroxidase-antiperoxidase) coloration. It is based on calibration of the procedure with the help of scales of standards obtained from known amounts of the respective antigens spotted onto nitrocellulose sheets and processed in parallel with the tissue sections. Due to the high recovery of nitrocellulose membranes for the attachment of proteins, this easy and reproducible medium was chosen in place of another matrix such as gelatin or activated agar (Mize et al., 1988; Nabors et a]., 1988). their use being more tedious than dot or slot blotting. These standards allow then the building of a calibration curve after densitometric scanning of the scale spots. It is worth noting that the standards are represented by the antigens themselves (and not only by a set of colored standards or of radioactive material) and that they are also reacted with the same fixative as that used for the tissue itself, thereby ensuring a biochemical antigen-antibody reaction close to that occurring in the fixed tissue itself. Thus, such a scale can be considered as an effective internal standard for each immunohistochemical experiment, while, within a given experiment, it represents only an external standard for each tissue section. There-

Fig. 2.

Quantitative Immunohistochemistry

fore two factors appear crucial: first, the reproducibility of the procedure between samples within each experiment where they are all run in parallel, and second, the reproducibility of the fixation via perfusion of the different animals, the standards being obviously not submitted to this initial critical step. The first factor can be effectively evaluated through the use of various sets of standards processed in parallel to the tissue sections, since the reproducibility

Fig. 2. A: Photomicrograph of the brown tyrosine hydroxylase immunolabeling (TH) as observable through PAP technique in the rat locus coeruleus of a control rat (coronal plane 33 according to the atlas of Paxinos and Watson, 1982). Horizontal bar = SO pm. B: Visualization of the TH immunolabeling observed in A after densitometry and pseudocolor image reconstruction along the pseudolinear portion of the TH calibration curve (color scale). Such a colored representation provides an easier identification of the various densitometric variations. A scale of known amounts of antigen is processed in parallel and allows one to then convert O.D. values measured from the colored immunoenzymatic reaction into ng of spotted tyrosine hydroxylase per mg of tissue protein. The mean 5 SEM of TH in the extent of the locus coeruleus, as calculated from a set of at least six different coronal sections from three different rats, is about 137 5 8 ngimg protein. The fourth ventricle. in black on the picture, is located at the upper right corner. Horizontal bar = SO pm. C: Photomicrograph of the brown TH immunolabeling as observable through PAP technique, in a rat treated with a single S.C. injection of 10 ingikg of reserpine, 3 days prior to sacrifice. All tissue sections (from three control and three treated rats) together with the calibration scale were processed in parallel. Horizontal bar = SO km. D: Visualization of the TH immunolabeling observed in C after densitometry and pseudocolor image reconstruction, the color scale being the same as in B . Note the enhancement of the labeling which is about twice as high as the control value. This is made visible by the appearance of a large number of cells in red (corresponding to the high densities of TH immunolabeling) according to the nearby color scale. The mean ? SEM concentration calculated in the extent of the locus coeruleus from three different rats is about 275 2 12 ngimg protein. The fourth ventricle. in black, is located at the upper right corner. Horizontal bar = SO km. E: Same situation as in A but at a higher magnification. Note the high resolution obtainable through such a colored immunoenzymatic procedure. Horizontal bar = SO pm. F: Densitonietric reconstruction of the immunolabeling displayed in E. At such a magnification, measurement can be easily performed at a cellular level. Horizontal bar = 50 e m . G : Photomicrograph corresponding to the same situation as in C but at a higher magnification. Horizontal bar = SO pm. H: Densitometric reconstruction of the immunolabeling observed in G. The enhanced labeling is evidenced by the extent of the regions in white and red (corresponding to the highest densities of TH immunolabeling) according to the nearby color scale. Horizontal bar = 50 pm.

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between samples within each experiment similarly affects the standards and the sections. E.L.I.S.A. procedures are routinely used for immunochemical assays with enzymatic coloration and are endowed with good reproducibility; the same may hold true for inimunohistochemistry applied to tissue sections, providing that adaptation and optimization of the procedure based on standards run in parallel are ensured. Such adaptation and optimization are effectively dependent on the quality and the specificity of the primary antibody used, and this concerns any common immunohistochemical procedure by itself, even when quantitation is not required. In addition, testing the adequate concentration of the primary antibody to be used in accordance with the conditions employed for incubation (duration, temperature) and in reference to the expected amount of antigen to be measured in the tissue represents a key factor to be checked in each new situation. Concentrations of the second antibody and of the PAP complex need then to be adjusted accordingly. Finally, the enzymatic reaction step of the peroxidase reaction has to be performed with the same scrupulous control on both the substrate concentration and the time-controlled duration of the reaction, as is usually the case in any biochemical enzymatic assay. All these steps allow then convenient quantitation of the immunohistochemical reaction revealed by indirect imniunoperoxidase methods, as with conventional spectrophotometry (Nibbering and Van Furth, 1987). Benno and coworkers (Benno et a]., 1982a) attempted to quantify T H via peroxidase-related immunohistochemistry but difficulties were encountered because of a lack of reproducibility of the coloration, in the absence of standards run in parallel, between different sets of experiments. The second factor concerns the perfusion of the animals for fixation. It represents one of the most critical step, since standards are not submitted to this initial part of the procedure. Obviously, rigorous conditions for this step should be drastically respected: rapidity and reproducibility of the surgical procedure, control of the capnia, control of arterial dilation (anticoagulant treatment), absence of any air bubble within the catheter, control of the volume of the prefixative rinsing solution and of the fixative, reproducibility of the speed and the strength of the perfusion via a peristaltic pump set at a fixed rate with control of the duration and the volume of the injected fixative, performance of all the perfusions of the animals of a given experiment (both control and treated ones) by the same hands with the same material, etc. Even though drastic experimental conditions are respected, some variations may still persist. Accordingly, densitometric measurement of the immunolabeling should be performed, as a control, within areas of the tissue sections where the antigen is known to be present at levels in the same range of magnitude as in the studied

Fig. 3.

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structure, but known to be unaffected by the considered experimental treatment. Such measures can thus serve either as a control of the eventual fluctuations of the fixation between animals or, eventually, as a means for correcting the measures in the studied structure relative to this particular region. Accordingly, the remaining uncertainties affecting the fixation step are circumvented and quantitation of immunohistochemical reaction on fixed tissue with enzymatic colorations become feasible, allowing then quantitative studies at cellular levels. Autoradiographic revelation of the immunohistochemical reaction has previously been proposed for quantitation with radioactive scales. Such procedures named radioimmunohistochemistry have been described for use with monoclonal antibodies incorporating a radioactive aminoacid (Cuello et al., 1982; Lewis and Watson, 1989), with secondary radiolabeled antibodies (McLean et a]., 1985; Lewis and Watson, 1989). or with secondary antibodies to which a radioactive material can be attached, such as biotin (Hunt and Mantyh, 1984; Lewis and Watson, 1989) or protein A (Correa et al.,

Fig. 3. A: Photomicrograph of the brown tyrosine hydroxylase (TH) immunolabeling as observable through PAP technique in the rat substantia nigra pars compacta of a control rat (coronal plane 25 according to the atlas of Paxinos and Watson, 1982). Horizontal bar = 50 p m . B: Visualization of the TH immunolabeling observed in A after densitometry and pseudocolor image reconstruction along the pseudolinear portion of the TH calibration curve (color scale). Such a colored representation provides an easier identification of the various densitometric variations. A scale of known amounts of antigen is processed in parallel and allows one then to convert O.D. values measured from the colored immunoenzyniatic reaction into ng of spotted tyrosine hydroxylase per mg of tissue protein. Horizontal bar = 50 p m . C: Photomicrograph of the brown TH immunolabeling as observable through PAP technique. in a rat treated with a single S.C. injection of 10 mg/kg of reserpine, 3 days prior to sacrifice. All tissue sections (from three control and three treated rats) together with the calibration scale were processed in parallel. Horizontal bar = 50 p m . D: Visualization of the TH immunolabeling observed in C after densitometry and pseudocolor image reconstruction, the color scale being the same as in B. No enhancement of the labeling does appear when compared to the corresponding control in B. Horizontal bar = 50 pm. E: Same situation as in A but at a higher magnification. Horizontal bar = 50 p m . F: Densitometric reconstruction of the immunolabeling displayed in E. At such a magnification, measurement can be easily performed at a cellular level. Horizontal bar = 50 p m . G : Photomicrograph corresponding to the same situation as in C but at a higher magnification. Horizontal bar = 50 pm. H: Densitometric reconstruction of the immunolabeling observed in G. Horizontal bar = 50 pin.

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1988; Lewis and Watson, 1989; Weissmann et al., 1988, 1989). Some of these quantitative radioimmunohistochemical procedures have been calibrated by comparison with radioimmunoassays on serial sections of the fixed tissue (Correa et a]., 1988), by reference to antigen standards spotted onto filter paper strips (McLean et al., 1985), or by running biochemically precalibrated antigen with unfixed tissue on parallel nitrocellulose sheets, according to conventional assays via Western blotting (Weissmann et al., 1988, 1989). Obviously, the use of unfixed tissue directly transferred onto nitrocellulose sheets renders such a quantitative radioimmunohistochemical procedure much more comparable to biochemical reactions and the whole protocol is then more closely related to a biochemical Western assay of proteins adapted to tissue sections than to an immunohistochemical technique per se. Although such an approach would appear biochemically more rigorous than the others, the absence of fixation incontestably limits its resolution to rough anatomical regions, preventing any quantitative and morphometrical study at the cellular level. Such a limitation of the cytological resolution is not only relevant to the absence of fixation but also to the use of radioactive material inherent to any radioimmunohistochemical procedure. This contrasts with color-based revelation made quantitative here through our procedure. Whatever the quantitative immunohistochemistry procedure used, there still remain intrinsic problems which make absolute quantification difficult: these are due to limitations on the accuracy of antigen detection by antibodies in tissue sections. Effectively, accurate quantitation of tissue antigens depends upon the assumption that no loss or modification of antigens occurs during fixation or subsequent steps and that the accessibility of the antigen to antibody is unrestricted, i.e., free penetration and lack of sterial hindrance. Nevertheless, accurate relative quantitation probably suffices for most comparative experiments where changes in protein, peptide, or hapten content are requested. To validate the quantitative feasibility of our o w n approach, biological examples of biochemically wellknown experimental models have been tested. One concerns the reserpine-inducing effect on TH locus coeruleus content. Induction could readily be detected here, 3 days after a unique subcutaneous injection of reserpine (10 mg/kg). The amplitude of the observed effect is in accordance with most biochemical studies performed with a comparable dose of reserpine and with similar delay after the injection (Faucon-Biguet et a]., 1986; Labatut et al., 1988; Reis et al., 1974; Weissmann et a]. , 1988, 1989). Its range is, however, only weaker than that already reported with another immunohistochemical peroxidase procedure performed in absence of reference to standards (Benno et al., 1982a,b).

Fig. 4.

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T h e other model concerns the decrease o f D A striatal content 2 hr 30 tnin after a single intraperitoneal injection o f 250 mg/kg A M P T , a known inhibitor o f TH activity (Anden et a l . , 1966: Corrodi and Hansson. 1966). T h e D A content measured in the control striatum according t o our procedure is consistent with most reported striatal D A values assayed with biochemical methods (Agnati e t a l . , 1979, 1982; Brownstein et al., 1974; Coyle and Henry, 1973; DiPaolo e t al., 1982; Koslow et al.. 1974; Redgrave and Mitchell. 1982; Tassin e t a l . , 1976). T h e striatal subregions of rich D A content, evidenced here in the lateral portion and the dorsomedial part of the striaturn along the lateral ventricle, might be reminiscent respectively of the putamen and the caudate nucleus, t w o striatal nuclei structurally well individualized in animals of higher phylogenetic order. T h e effects of A M P T treatment on striatal D A contents are also in accordance with those found through biochemical assays (Costa and Neff, 1966; Javoy and Glowinski, 1971). T h e y confirm that the effects of

AMPT are unevenly distributed, as already suggested from visual inspection of data obtained via histochemistry and related techniques (Doucet et al., 1986: Graybiel, 1984; Olson e t a l . , 1972). Accordingly, our procedure provides quantitative validation of the notion of differential turnover rates for D A in the rat striatum, opposing then t h e limbic portion t o the nonlimbic one, t h e patches or islands of DA with low turnover rates t o the matrix (Olson e t al., 1972; Agnati et al., 1979, 1982; Donoghue and Herkenham, 1986; Graybiel, 1984; Gerfen et al., 1987a,b; Kelley et al., 1982; Nastuk and Graybiel, 1985). It is worth noting that the quantitative immunohistochemical technique presented here is applicable to serial sections of the same structure and from the s a m e animal, allowing serial evaluation of different antigens, such as the (synthesizing) enzyme(s) of a given neurotransmitter and the transmitter itself, such as D A here. Finally, whatever the method used (quantitative receptor autoradiography or quantitative immunohistochemistry) the use of calibration curves with identifica-

Fig. 4. A: Photomicrograph of the brown dopamine (DA) inimunolabeling in the striatum of a control rat, as observable through PAP technique in the rat striatum (coronal plane 14 of the atlas of Paxinos and Watson. 1982). Horizontal bar = 400 pm. B: Visualization of the DA immunolabeling observed in A after densitometry and pseudocolor image reconstruction along the pseudolinear portion of the DA calibration curve (color scale). The immunolabeling does not extend homogeneously through the striatum. some regions, appearing in red and white, being of richer DA content than others. Such regions are located mostly in the dorsolateral area. A calibration scale is processed in parallel and allows one then to convert O.D. values measured from the colored irnmunoenzyniatic reaction into ng of DA per mg of striatal protein. The mean 2 SEM DA concentration in the extent of the striatum, as calculated from a set of at least six different coronal sections from three different rats, is about 99 ? 18 ngimg protein. Horizontal bar = 400 p m . C: Same procedure as in A from a rat treated with a single 1.P. injection of a-methyl tyrosine (150 mglkg) 2 hr 30 min prior to sacrifice. All tissue sections (from three control and three treated rats) together with the calibration scale were processed in parallel. Horizontal bar = 400 p m . D: Visualization of the DA immunolabeling in the striatum of an amethyl-tyrosine-treated rat (as described in C ) . Such a treatment is responsible for an important decrease of the labeling. This is clearly revealed by the massive disparition of the red and white colors, most striatal sub-regions and particularly the lateral aspect appearing then in blue (corresponding to low labeled areas). This contrasts with the more medial portions of the striatum which are poorly affected by this treatment. Some islandic organization of DA fibers emerges from the extrastriosomal surrounding after such a treatment. The mean 2 SEM DA concentration in the striatum of the three treated rats, as calculated from a set of at least six different coronal sections,

is about 53 2 13 ngimg protein. Horizontal bar = 400 pni. E: Densitonietric reconstruction of the autoradiographic image of mu opiate receptor binding sites in the rat striatum (coronal plane 14 of the atlas of Paxinos and Watson. 1982) as labeled with 0.8 nM of 311-etorphine. The colored image in green, red, and white (from the Samba 200@analyzer) represents the specific binding. whereas the blue and cyan colors correspond to nonspecific binding. The corresponding color scale is located near the image. The color scale is restricted to the pseudolinear portion of the calibration curve. F: Densitometric reconstruction of the autoradiographic image of dopaminergic D, receptor binding sites in the rat striatum (coronal plane 14 of the atlas of Paxinos and Watson. 1982) as labeled with I nM of 'H-SCH 23390. The colored image (from the Samba 200@ analyzer) represents the specific binding, nonspecific binding having been subtracted from the values of the total binding. The corresponding color scale is located near the image. The color scale is restricted to the pseudolinear portion of the calibration curve. Two zones are discernible on the image: one of high density of D, doparninergic binding sites, appearing in magenta, the other. of lower density of D , sites, corresponding to the green region. White arrows point to some islands of D , binding sites in register with those rich of mu opiate sites and pointed at in Figure 4E. Horizontal bar = 500 Fni. G : Densitonietric reconstruction of the autoradiographic image (from the Samba 200@analyzer) of dopaminergic Dz receptor binding sites in the rat striatum (coronal plane 14 of the atlas of Paxinos and Watson, 1982), as labeled with 0.4 nM of 3H-spiperone. in presence of 10 nM ketanserine. The regions in blue and cyan correspond to nonspecific binding, whereas those in green. yellow, red, and magenta are related to specific binding. The richest zones, in magenta, are clearly visible in the lateral part of the structure and also within the dorsomedial part of the striatum. Horizontal bar = 500 p m .

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tion of their upper and lower limits should be taken into account. The saturation effect found for intense coloration closely resembles that usually found in biochemical spectrophotometry where it is routinely taken into account. The same holds true for histochemistry and the examples shown here of the autoradiography of striatal D, and D, dopaminergic binding sites illustrate this aspect. Both these receptor sites are enriched in the striatum but their respective heterogeneous distribution in the rat striatum partly differs, being thus consistent with other reports (Aiso et al., 1987; Altar et al., 1984; Altar and Marien, 1987; Beckstead et al., 1988; Besson et al., 1988; Camps et al., 1989; Dawson et al., 1986, 1988a. 1988b; Dubois et al., 1986; Feuerstein et al., 1989; Joyce et al., 1986; Joyce and Marshall, 1987; Martres et al., 1985; Palacios et al., 1981b; Savasta et al., 1986; Wamsley et al., 1989). Also the reconstruction of the images with respect to the upper and lower limits of the (pseudo)linear portion of the calibration curve clearly made possible obtaining evidence that the widespread striatal repartition of the rat D, receptor sites can be subdivided not only into a rich lateral aspect and a dense dorsomedial portion but also into patch-like zones which emerge from a surrounding extrastriosomal matrix-like zone, where D, sites are of lower density. Such a dual heterogeneous striatal pattern, which has been evidenced in the striatum of animals of higher phylogenetic order (Besson et al., 1988), has not been easily identified previously in the adult rat, probably because of masking effects related to saturated conditions (overexposed films): in such circumstances, OD of the slightly less radioactive portions of the extrastriosomal matrix-like zone were unable to appear significantly different from OD measured in the rich patch-like ones, because of the saturating plateau effect encountered at too elevated OD values. In conclusion, the adequate use of scales of standards for the calibration of computer-based image analysis techniques, both for autoradiography and immunohistochemistry , allows the neuroscientist to be provided with sensitive quantitative biochemical tools to be used at anatomical, histological, or even cytological resolution levels. Accordingly, modulatory effects can be evaluated with respect to the heterogeneity of brain organization. Amplitude of the studied effects becomes even more pronounced since such effects are evaluated at the precise site where they happen, without the flattening influence of the unresponsive surroundings.

ACKNOWLEDGMENTS This work was supported by INSERM U 318, Universite Joseph Fourier de Grenoble, Commission Recherche de la Region RhGne-Alpes and Centre Hospi-

talier Regional de Grenoble. We are much devoted to Lundbeck company for generous supply of a-flupentixol and piflutixol. Our thanks go to Marie-Jeanne Gallet for kindly typing the manuscript and to the C.G.E. AlcatelT.I.T.N. staff for their kind help in operating the computer-based image analysis system SAMBA@ and for giving us free access to information concerning both the hardware and the software of the system.

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