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Calbindin distribution in male, female and lactating rat pituitary
Experimental Neurology 193 (2005) 141 – 148 www.elsevier.com/locate/yexnr
Calbindin distribution in male, female and lactating rat pituitary Vincenzo Cimini* Department of Biomorphological and Functional Sciences, Chair of Histology, Medical School of Federico II University of Naples, Edificio 20, Via Pansini 5-80131 NAPOLI, Italy Received 12 July 2004; revised 19 October 2004; accepted 23 November 2004
Abstract Calcium binding proteins such as calbindin and calretinin have been studied in the pituitary gland, but information on them is still incomplete. To investigate the localization, distribution and role of calbindin in the pituitary, several antibodies to calbindin and to other pituitary markers, such as calretinin and tyrosine–hydroxylase, have been used in male, female and lactating rats. Calbindin has not been localised to a specific endocrine cell population unlike calretinin in the thyrotrophs. There was occasional localization in somatotrophs, thyrotrophs and luteotrophs, but not in corticotrophs or lactotrophs. However, there are sex differences in the expression of this protein as the number of calbindin-immunoreactive cells is higher in the male than in the female pituitary. Furthermore, the number of calbindin containing cells, not lactotrophs, increases in lactating rats and decreases after removal of the pups. It is concluded that calbindin expression may be altered by physiological and endocrine events such lactation, even though it is still unclear why the protein is not related to a specific cell type. The simultaneous use of monoclonal and polyclonal antisera to calbindin revealed that the rabbit antibody recognizes nuclear and cytoplasmic calbindin, while the monoclonal one binds only to the cytoplasmic calbindin. The suggestion is that calbindin may have a secondary role that is not simply to bind calcium. D 2004 Elsevier Inc. All rights reserved. Keywords: Calbindin; Pituitary; Calretinin
Introduction The Ca2+ binding protein, spot 35-calbindin (27 kDa), has been localized to rat anterior pituitary in normal male and female (Abe et al., 1990), but its localization in pituitary cells is still unclear, as well as its role. Our previous studies have shown that another calcium-binding protein, calretinin, is present in thyrotrophs of rat pituitary, its expression being related to the metabolism of thyroid gland and of course to the hypothalamic thyrotrophin releasing hormone (TRH) function (Cimini et al., 1997). Buffa et al. (1989) were unable to confirm the localization of 28 kDa calbindin (CB) in thyrotrophs of several mammals, while, together with occasional gonadotroph and thyrotroph immunoreactivity, rather strong positivity was found in the nuclei of human pituitary CLIPimmunoreactive cells (Buffa et al., 1990). * Fax: +39 81 746 3427. E-mail address: [email protected]. 0014-4886/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.expneurol.2004.11.029
Spot 35-CB is also expressed transiently in immature pituicytes of rat fetuses between days 11 and 18. CBimmunoreactive nerve fibers appear in the posterior lobe on the 16th day (Abe et al., 1991). Literature reports are incomplete and raise the question of the true localization of CB immunoreactivity in the pituitary of mammals. We here reexamined this in view of our recent results on calretinin localization in rat pituitary thyrotrophs and have further characterized the localization of CB within the intermediate lobe (IL) and posterior lobe (PL) of the rat pituitary by employing double immunofluorescence methods. We have also tried to clarify whether the expression of CB can be influenced by physiological events, such as lactation.
Material and methods Adult female and male Sprague–Dawley rats (250–300 g), five for each group, were obtained from Zivic Labs
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(Pittsburgh, PA, USA). Females rats (aged 60 days) were housed under controlled light/dark cycle and temperature (218C). Only proestrus animals with regular 4-day cycles were used in this study. Daily vaginal smears monitored the oestrous cyclicity. One group of lactating females was sacrificed within the first 48 h of lactation, 1 h after pup removal. The second group of lactating rats was left 1 week far from pups. The number of pups ranged between 10 and 14. Three male pituitaries were also studied after stalk transection as previously described (Cimini et al., 1997). All animals were treated according to NIH guidelines. For immunocytochemistry, rats were anaesthetised with chloropent (0.3 ml/100 g body weight), perfused with 0.5% sodium nitrite and then with 10% buffered formalin. Pituitary glands were carefully removed and kept for a further 2–3 h in the same fixative. Specimens were then left under refrigeration at 48C in 30% sucrose in PBS for 48 h and embedded in OCT medium before freezing and cryostat sectioning. Immunocytochemistry Ten-micrometer-thick cryostat sections were processed for the indirect immunofluorescence method (Coons, 1971). The following primary antisera were used to localise CB: mouse anti-CB (gift of D. Jacobowitz; Celio et al., 1990), diluted 1:5000; rabbit anti-CB, diluted 1:5000 (gift of D. Jacobowitz); and rabbit anti-CB (Swant), diluted 1:7500–
Fig. 1. CB immunoreactivity of a male rat anterior pituitary cell. Cell nucleus and cytoplasm exhibit green fluorescence. The primary antibody was produced in rabbit (diluted 1:7500). Magnification: 1000. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 2. Distribution of 28 kDa CB-ir cells in the anterior pituitary. CB containing cells are scattered in the pituitary parenchyma and are often clustered around blood vessels (Fig. 3) or at the gland periphery (arrows) with different aspects between male (Fig. 4) and female (Fig. 5). The specific antibody was produced in rabbit (diluted 1:5000). Magnifications: 100; Fig. 3, 240; Fig. 4, 150; Fig. 5, 100.
10000. In order to prove a true localisation of CB, the monoclonal and polyclonal antibodies together were used for double immunostaining. For negative controls, the mixture of the primary antisera with 0.1 AM recombinant rat CB (Swant) was used instead of the first layer. As a further control, the monoclonal antiserum was cross adsorbed with 0.1 AM recombinant calretinin (Swant). Double immunofluorescence staining was also carried out by using goat anti-calretinin (1:1000, Swant) and mouse or rabbit anti-CB antisera. Fluorescein isothiocyanate (FITC, Jackson ImmunoResearch) and Texas red (Jackson ImmunoResearch)-conjugated donkey anti-mouse and/or antirabbit immunoglobulins were used to reveal the antigens as needed. Occasionally, rabbit anti-tyrosine hydroxylase (TH) antiserum (1:2000, Incstar) was used together with both anti-CB antisera to show the coexistence of CB and calretinin within dopaminergic nerves. Double immunofluorescence staining used mouse anti-CB serum together with rabbit antisera (1:2000) against pituitary hormones, namely, anti-PRL, anti-ACTH, anti-LH, anti-FSH, anti-TSH and anti-GH (gift from NIDDK). The sections were then mounted with phenylenediamine/glycerine/PBS medium and viewed with a Leitz Diaplan fluorescence microscope provided with a fluores-
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CB-immunoreactive (ir) cells were present in male AP (Fig. 1). They were sparse throughout the parenchyma of the endocrine gland (Fig. 2) and around blood vessels (Fig. 3). Clusters of cells were frequently found at the periphery of the pituitary ventral portion (Fig. 4). The number of CB-ir cells was definitely lower in the female AP gland (Figs. 5 and 6), most of them showing less intense fluorescence. Small clusters of immunoreactive cells were also present in the female pituitary, particularly at the periphery of the gland, but they were not comparable in size with those found in males (Figs. 7 and 8). In lactating rats, the general feature of CB-IR was similar to that of male pituitary, although peripheral clusters of CB-ir cells were not observed (Fig. 9). Thus, with a lactating female whose pups were removed on the day of birth, the number of positive cells appeared to be increased with respect to normal female, as well as the average intensity of fluorescence, which however was not evaluated in this study. On the contrary, in lactating female 1 week after removal of the pups, CB-IR was more like that of a normal female (Fig. 6). Intermediate lobe (IL)
Fig. 3. Distribution of 28 kDa CB-ir cells in the anterior pituitary. CB containing cells are scattered in the pituitary parenchyma (Fig. 2) and are often clustered around blood vessels or at the gland periphery (arrows) with different aspects between male (Fig. 4) and female (Fig. 5). The specific antibody was produced in rabbit (diluted 1:5000). Magnifications: Fig. 2, 100; 240; Fig. 4, 150; Fig. 5, 100.
The IL showed both immunoreactive cells and nerve fibers. No differences were detected between male,
cence filter system allowing simultaneous emission of green and red wavelengths. Methods for controls of double staining cross-reactivity have been described previously (Cimini et al., 1989). Quantification Photographs of 10-Am-thick immunostained sections were taken by a Leitz Diaplan fluorescence microscope provided with a Leica DC-200 digital camera. The number of CB-immunoreactive cells were counted in five fields/ section/animal/experimental group. Mean values and standard deviation for each group were calculated with reference to the histological surface area of 1 mm2, which is considered the unit area in this study. The significance of differences between groups was established by calculation of Bonferroni’s P values.
Results Anterior pituitary (AP) Immunoreactivity (IR) to CB was present not only in the cytoplasm, but also in the nucleus. Intensely positive
Fig. 4. Distribution of 28 kDa CB-ir cells in the anterior pituitary. CB containing cells are scattered in the pituitary parenchyma (Fig. 2) and are often clustered around blood vessels (Fig. 3) or at the gland periphery (arrows) with different aspects between male and female (Fig. 5). The specific antibody was produced in rabbit (diluted 1:5000). Magnifications: Fig. 2, 100; Fig. 3, 240; 150; Fig. 5, 100.
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Fig. 7. This panel shows a comparison between the patterns of peripheral clustered CB cells in normal male, normal female (Fig. 8) and lactating female (Fig. 9) at level of the pituitary ventral surface. The primary antibody was produced in rabbit (diluted 1:10000). Magnification: 150.
Immunostaining of the intermediate lobe after pituitary stalk section showed a complete absence of nerve fibers IR (Figs. 11 and 12). Posterior lobe (PL)
Fig. 5. Distribution of 28 kDa CB-ir cells in the anterior pituitary. CB containing cells are scattered in the pituitary parenchyma (Fig. 2) and are often clustered around blood vessels (Fig. 3) or at the gland periphery (arrows) with different aspects between male (Fig. 4) and female. The specific antibody was produced in rabbit (diluted 1:5000). Magnifications: Fig. 2, 100; Fig. 3, 240; Fig. 4, 150; 100.
female and lactating CB-IR in the IL. All the CB-ir cells had nuclear positivity while very few had immunoreactivity in the cytoplasm. In addition, a fine network of immunoreactive varicose nerve fibers was observed in perilobular and intralobular tissue of IL (Fig. 10). The nerve fibers corresponded to TH-containing nerves, as previously shown in the hypothalamus (Jacobowitz et al., 1998).
Fig. 6. Statistical analysis of CB-immunoreactive cells in the anterior pituitary of male, female, lactating same day (SD) and lactating 1 week later (OWL) rats. *P b 0.05 male vs. female, male vs. OWL and SD vs. OWL.
PL-IR to CB antisera showed in male pituitary a very intense network of nerve fibers with variable thickness and length (Fig. 10). A more intense fluorescence was observed in the lactating female as compared with the normal female, but indeed the increase was not estimated. As for the IL, in the PL double immunofluorescence revealed the coexistence of CB and TH. Double immunostaining and controls Immunofluorescence with the monoclonal antibody to CB was quenched by mouse ascites fluid in all animals used, when used instead of the primary antibody. Staining was also absent when normal rabbit serum was used instead of the CB polyclonal antibody. In order to verify the rabbit polyclonal antibody tissue specificity, double immunostaining with both monoclonal and polyclonal antisera was carried out. In the male pituitary, while cell cytoplasm was positive for both antibodies, cell nuclei immunoreacted to polyclonal serum only (Fig. 13). Some cells were entirely stained by the polyclonal antibody
Fig. 8. This panel shows a comparison between the patterns of peripheral clustered CB cells in normal male (Fig. 7), normal female and lactating female (Fig. 9) at level of the pituitary ventral surface. The primary antibody was produced in rabbit (diluted 1:10000). Magnification: 150.
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Fig. 9. This panel shows a comparison between the patterns of peripheral clustered CB cells in normal male (Fig. 7), normal female (Fig. 8) and lactating female at level of the pituitary ventral surface. The primary antibody was produced in rabbit (diluted 1:10000). Magnification: 150.
only, and none was found to be immunoreactive to the monoclonal antibody alone. In females, cells immunoreactive only to the monoclonal antibody were seen as well as cells immunoreactive only to the polyclonal one, these latter showing both cytoplasmic and nuclear positivity, while the former showed only cytoplasmic positivity. Furthermore, some cells with polyclonal-immunoreactive nuclei had monoclonal positive cytoplasm. In very few cases the nuclei of cells with cytoplasmic and nuclear Fig. 11. The immunoreactivity of intermediate lobe (IL) nerves to CB (arrows) completely disappears after pituitary stalk section (Fig. 12). A rabbit polyclonal antibody to calbindin was used in this experiment (diluted 1:10000). Magnification: 150.
Fig. 10. 28 kDa CB in rat male pituitary localized by the antibody produced in rabbit (diluted 1:10000). The three portions of the pituitary are shown: adenohypophysis with sparse CB-ir cells (AP), intermediate lobe with its predominant nuclear IR and a fine network of immunoreactive nerve fibres (IL) and the posterior lobe with its rich CB-ir network of nerve fibres (PL). Magnification: 150.
polyclonal CB-positivity were also immunoreactive to monoclonal anti-CB. Preabsorption of CB antisera with recombinant calretinin did not show any difference in staining pattern compared with CB antiserum. The absorption of both CB antisera with recombinant CB completely quenched the immunostaining. Double immunostaining with monoclonal antiserum to CB and polyclonal goat antiserum to calretinin revealed three cell types: one only immunoreactive to CB, one only immunoreactive to calretinin and one immunoreactive to both CB and calretinin, the latter exhibiting more frequently nuclear CB-IR and cytoplasmic CR- and/or CB-IR. In double immunostaining with antibodies to pituitary hormones, faint CB-ir cells with negative nuclei corresponded mostly to LH cells, while CB-ir cells with positive nuclei did not correspond to LH cells in all cases studied. Occasional colocalisation with faintly CB-ir PRL cells occurred in male and female anterior pituitary, but not with CB-ir cells containing positive nuclei. Similarly, occasional coexistence with GH was found in both sexes. Coexistence with TSH was found in the male only. No coexistence was found with ACTH. In the IL, there were abundant cells with CB-ir nuclei with no calretinin colocalization (Fig. 14) while in the
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Fig. 12. The immunoreactivity of intermediate lobe (IL) nerves to CB (Fig. 11, arrows) completely disappears after pituitary stalk section. A rabbit polyclonal antibody to calbindin was used in this experiment (diluted 1:10000). Magnification: 150.
nerves of the intermediate lobe the two proteins were found together. In the PL the nerves showed partial colocalization of calretinin and CB (Fig. 15). No difference was noticed between the different primary antibodies to CB in the IL and PL.
Discussion In this study the presence of CB 28 kDa was detected in the cytoplasm and nuclei of endocrine cells of rat pituitary with one monoclonal antibody and two polyclonal ones. Nuclear staining for spot 35 protein has already been reported in chicken (Jande et al., 1981; Thorens et al., 1982) as well as nuclear staining for calbindin in cat thyroid C cells and dog islets (Buffa et al., 1989). These authors mentioned the possible presence of calbindin in the pituitary TSH cells but did not specify the species or the sex of the animals. However, their hypothesis can be explained now on the basis of our results on calretinin localization in TSH cells of rat anterior pituitary (Cimini et al., 1997) because their antiserum raised against chick intestinal calbindin cross-reacted with calretinin. The nuclear localization of calbindin, with particular reference to CLIP-producing cells of human pituitary (Buffa et al., 1990), has been confirmed by our results on the IL of
the rat pituitary. Its significance is still unknown at least in the pituitary, although the presence of CB has been related to the ability of taking up 3H-1,25(OH)2 vitamin D, and most interestingly, nuclear CB has been recently related to the regulation of nuclear calcium signals in nerve cells (German et al., 1997). To rule out possible cross-reactivity with calretinin, we used a mixture of CB antibody with recombinant calretinin, as primary antibody, and anti-CB antisera on a serial section. The two sections showed the same staining pattern. Our results on sexual differences of CB-IR in the anterior pituitary gland confirm the previous report on distribution of spot 35-calbindin (Abe et al., 1990). This is due to its 79% homology of amino acid sequence with 28 kDa calbindin. We have also found occasional coexistence of CB with TSH, PRL and GH in both sexes by double immunofluorescence on the same section, while the previous results showing supposed spot 35-CB coexistence with all classical pituitary hormones were based on mirror-image sections and were therefore not perfectly reliable. In addition, only occasional GH cells seem to contain CB in lactating rats. The results of double immunofluorescence confirm that some of the CB cells immunoreactive to the polyclonal antiserum (Swant) are calretinin cells because of its (admittedly low) cross-reactivity or, according to Schiffmann et al. (1999), the anti-calretinin antiserum might recognize CB protein when highly saturated with Ca2+. Therefore, the differences between sexes are related to the number of CB expressing cells not to the cell types, and in addition a variation of the endocrine status, such as lactation, might alter calcium-linked activity of some anterior pituitary cells. In man, nuclear staining of melanotrophs and occasional staining of gonadotrophs and thytrophs was reported (Buffa et al., 1990), but again age and sex were not mentioned. The presence of 28 kDa CB in IL has been studied here for the first time in the rat, thus confirming the results previously obtained on human tissue (Buffa et al., 1990), while the coexistence of CB with calretinin within the pars nervosa was previously established by light and electron microscopy (Miyata et al., 2000). In our results, the nuclear and less prominent cytoplasmic immunoreactivity in the IL are not related to sex or endocrine status. This is in contrast with the transient expression of spot 35-CB in the immature pituicytes of embryonic rats (Abe et al., 1991). The different results could be explained by the different antisera used, but also by calcium-dependent conformational changes. Thus, the hypothesis can be made that at least two different CB molecular conformations, one nuclear and one cytoplasmic, could be present in the pituitary gland. However, this hypothesis has been widely discussed in the literature and is based on the suggestion that antibody recognition of calcium binding proteins may depend on their calcium-
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Fig. 15. Double immunolabelling of calretinin and CB within the posterior lobe of normal male rat pituitary. There is a partial coexistence of the two calcium-binding protein as some nerve fibres exhibit only green fluorescence (CB), some orange fluorescence (CB and calretinin) and few red (calretinin). Antibodies used were the same as for Fig. 14. Magnification: 250.
Fig. 13. Double labelling of anterior pituitary cells with monoclonal and polyclonal antibodies to CB. Nuclei exhibiting green fluorescence are labelled with the polyclonal antibody (diluted 1:10000), while cytoplasm exhibiting orange fluorescence is labelled with both CB antibodies. The monoclonal antibody to CB was revealed by Texas red-conjugated immunoglobulins. Magnification: 650.
binding status (Dutar et al., 1991; Olive and Ferrer, 1994; Winsky and Kuznicki, 1996). The lack of difference between the sexes in the IL and PL CB-immunoreactive nerves implies that CB is a constitutive protein of pituitary and, of course, hypothalamic nerves, and its expression is not apparently altered by the complex functional changes of the hypothalamus. The coexistence of CB with TH in all conditions studied may suggest that both CB and CR, two calcium binding proteins, might participate in the neurosecretory activity of PL nerve terminals because it has previously been shown that CR and TH do colocalize in the PL nerves (Cimini et al., 1997). Interestingly, THimmunoreactive nerve terminals are reported to contain serotonin too in the intermediate lobe of rat pituitary (Vanhatalo et al., 1995). In conclusion, we have reviewed CB-IR in the pituitary of rat. There are sex-related differences and endocrine status-related differences mainly in the anterior pituitary, where the calcium-binding protein colocalizes with somatotrophs, thyrotrophs and luteotrophs. CB might have different configurations in the nucleus and in the cytoplasm of anterior and intermediate pituitary cells. It is present in nerves of IL and PL and colocalizes with TH. Fig. 14. Colocalisation of CB and calretinin within the intermediate lobe of male rat pituitary. Green fluorescent nuclei represent CB, and orangelabelled nerve fibres show coexistence of CB and calretinin. CB immunoreactivity was revealed by the rabbit antiserum (diluted 1:10000) and calretinin IR by the goat antiserum. Magnification: 250.
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Acknowledgments The author wish to thank David M. Jacobowitz who made possible the realization of this project, most of it being performed in his lab. The author is indebted to Susan Van Noorden for useful comments and language revision and to Dr. Giovanna G. Altobelli and Alfredo Sales for lab assistance. Many thanks are also due to Dr. Parlow (NIDDK) for the gift of antisera to pituitary hormones. Departmental funds from the University of Naples Federico II were also used.
References Abe, H., Amano, O., Yamakuni, T., Takahashi, Y., Kondo, H., 1990. Localization of spot 35-calbindin (rat cerebellar calbindin) in the anterior pituitary of the rat: developmental and sexual differences. Arch. Histol. Cytol. 53, 585 – 591. Abe, H., Amano, O., Yamakuni, T., Kuwano, R., Takahashi, Y., Kondo, H., 1991. Transient expression of a calcium-binding protein (spot 35calbindin) and its mRNA in the immature pituicytes of embryonic rats. Cell Tissue Res. 266, 325 – 330. Buffa, R., Mare’, P., Salvadore, M., Solcia, E., Furness, J.B., Lawson, D.E., 1989. Calbindin 28 kDa in endocrine cells of known or putative calcium-regulating function. Thyro-parathyroid C cells, gastric ECL cells, intestinal secretin and enteroglucagon cells, pancreatic glucagon, insulin and PP cells, adrenal medullary NA cells and some pituitary (TSH?) cells. Histochemistry 91, 107 – 113. Buffa, R., Mare’, P., Salvadore, M., Gini, A., 1990. Immunohistochemical detection of 28 kDa calbindin in human tissues. Adv. Exp. Med. Biol. 269, 205 – 210 (review). Celio, M.R., Baier, W., Scharer, L., Gregersen, H.J., de Viragh, P.A., Norman, A.W., 1990. Monoclonal antibodies directed against the calcium binding protein calbindin D-28k. Cell Calcium 11, 599 – 602. Cimini, V., Van Noorden, S., Polak, J.M., 1989. Co-localisation of substance P-, bombesin- and peptide histidine isoleucine (PHI)-like
peptides in gut endocrine cells of the dogfish Scyliorhinus stellaris. Anat. Embryol. 179, 605 – 614. Cimini, V., Isaacs, K.R., Jacobowitz, D.M., 1997. Calretinin in the rat pituitary: colocalization with thyroid-stimulating hormone. Neuroendocrinology 65, 179 – 188. Coons, A.H., 1971. The development of immunohistochemistry. Ann. N. Y. Acad. Sci. 177, 5 – 9. Dutar, P., Potier, B., Lamour, Y., Emson, P.C., Senut, M.C., 1991. Loss of calbindin-28K immunoreactivity in hippocampal slices from aged rats: a role for calcium? Eur. J. Neurosci. 3, 839 – 849. German, D.C., Ng, M.C., Liang, C.L., McMahon, A., Iacopino, A.M., 1997. Calbindin-D28k in nerve cell nuclei. Neuroscience 81, 735 – 743. Jacobowitz, D.M., Isaacs, K.R., Cimini, V., 1998. Triple colocalization of tyrosine hydroxylase, calretinin, and calbindin D-28k in the periventricular–hypophyseal dopaminergic neuronal system. Adv. Pharmacol. 42, 37 – 40. Jande, S.S., Tolnai, S., Lawson, D.E., 1981. Immunohistochemical localization of vitamin D-dependent calcium-binding protein in duodenum, kidney, uterus and cerebellum of chickens. Histochemistry 71, 99 – 116. Miyata, S., Nakai, S., Kiyohara, T., Hatton, G.I., 2000. Calbindin-D28k and calretinin in the rat posterior pituitary; light and electron microscopic localization and upregulation with dehydration. J. Neurocytol. 29, 5 – 17. Olive, M., Ferrer, I., 1994. Parvalbumin immunohistochemistry in denervated skeletal muscle. Neuropathol. Appl. Neurobiol. 20, 495 – 500. Schiffmann, S.N., Cheron, G., Lohof, A., d’Alcantara, P., Meyer, M., Parmentier, M., Schurmans, S., 1999. Impaired motor coordination and Purkinje cell excitability in mice lacking calretinin. Proc. Natl. Acad. Sci. U. S. A. 96, 5257 – 5262. Thorens, B., Roth, J., Norman, A.W., Perrelet, A., Orci, L., 1982. Immunocytochemical localization of the vitamin D-dependent calcium binding protein in chick duodenum. J. Cell Biol. 94, 115 – 122. Vanhatalo, S., Soinila, S., Kaartinen, K., Back, N., 1995. Colocalization of dopamine and serotonin in the rat pituitary gland and in the nuclei innervating it. Brain Res. 669, 275 – 284. Winsky, L., Kuznicki, J., 1996. Antibody recognition of calcium-binding proteins depends on their calcium-binding status. J. Neurochem. 66, 764 – 771.
Calbindin distribution in male, female and lactating rat pituitary Vincenzo Cimini* Department of Biomorphological and Functional Sciences, Chair of Histology, Medical School of Federico II University of Naples, Edificio 20, Via Pansini 5-80131 NAPOLI, Italy Received 12 July 2004; revised 19 October 2004; accepted 23 November 2004
Abstract Calcium binding proteins such as calbindin and calretinin have been studied in the pituitary gland, but information on them is still incomplete. To investigate the localization, distribution and role of calbindin in the pituitary, several antibodies to calbindin and to other pituitary markers, such as calretinin and tyrosine–hydroxylase, have been used in male, female and lactating rats. Calbindin has not been localised to a specific endocrine cell population unlike calretinin in the thyrotrophs. There was occasional localization in somatotrophs, thyrotrophs and luteotrophs, but not in corticotrophs or lactotrophs. However, there are sex differences in the expression of this protein as the number of calbindin-immunoreactive cells is higher in the male than in the female pituitary. Furthermore, the number of calbindin containing cells, not lactotrophs, increases in lactating rats and decreases after removal of the pups. It is concluded that calbindin expression may be altered by physiological and endocrine events such lactation, even though it is still unclear why the protein is not related to a specific cell type. The simultaneous use of monoclonal and polyclonal antisera to calbindin revealed that the rabbit antibody recognizes nuclear and cytoplasmic calbindin, while the monoclonal one binds only to the cytoplasmic calbindin. The suggestion is that calbindin may have a secondary role that is not simply to bind calcium. D 2004 Elsevier Inc. All rights reserved. Keywords: Calbindin; Pituitary; Calretinin
Introduction The Ca2+ binding protein, spot 35-calbindin (27 kDa), has been localized to rat anterior pituitary in normal male and female (Abe et al., 1990), but its localization in pituitary cells is still unclear, as well as its role. Our previous studies have shown that another calcium-binding protein, calretinin, is present in thyrotrophs of rat pituitary, its expression being related to the metabolism of thyroid gland and of course to the hypothalamic thyrotrophin releasing hormone (TRH) function (Cimini et al., 1997). Buffa et al. (1989) were unable to confirm the localization of 28 kDa calbindin (CB) in thyrotrophs of several mammals, while, together with occasional gonadotroph and thyrotroph immunoreactivity, rather strong positivity was found in the nuclei of human pituitary CLIPimmunoreactive cells (Buffa et al., 1990). * Fax: +39 81 746 3427. E-mail address: [email protected]. 0014-4886/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.expneurol.2004.11.029
Spot 35-CB is also expressed transiently in immature pituicytes of rat fetuses between days 11 and 18. CBimmunoreactive nerve fibers appear in the posterior lobe on the 16th day (Abe et al., 1991). Literature reports are incomplete and raise the question of the true localization of CB immunoreactivity in the pituitary of mammals. We here reexamined this in view of our recent results on calretinin localization in rat pituitary thyrotrophs and have further characterized the localization of CB within the intermediate lobe (IL) and posterior lobe (PL) of the rat pituitary by employing double immunofluorescence methods. We have also tried to clarify whether the expression of CB can be influenced by physiological events, such as lactation.
Material and methods Adult female and male Sprague–Dawley rats (250–300 g), five for each group, were obtained from Zivic Labs
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(Pittsburgh, PA, USA). Females rats (aged 60 days) were housed under controlled light/dark cycle and temperature (218C). Only proestrus animals with regular 4-day cycles were used in this study. Daily vaginal smears monitored the oestrous cyclicity. One group of lactating females was sacrificed within the first 48 h of lactation, 1 h after pup removal. The second group of lactating rats was left 1 week far from pups. The number of pups ranged between 10 and 14. Three male pituitaries were also studied after stalk transection as previously described (Cimini et al., 1997). All animals were treated according to NIH guidelines. For immunocytochemistry, rats were anaesthetised with chloropent (0.3 ml/100 g body weight), perfused with 0.5% sodium nitrite and then with 10% buffered formalin. Pituitary glands were carefully removed and kept for a further 2–3 h in the same fixative. Specimens were then left under refrigeration at 48C in 30% sucrose in PBS for 48 h and embedded in OCT medium before freezing and cryostat sectioning. Immunocytochemistry Ten-micrometer-thick cryostat sections were processed for the indirect immunofluorescence method (Coons, 1971). The following primary antisera were used to localise CB: mouse anti-CB (gift of D. Jacobowitz; Celio et al., 1990), diluted 1:5000; rabbit anti-CB, diluted 1:5000 (gift of D. Jacobowitz); and rabbit anti-CB (Swant), diluted 1:7500–
Fig. 1. CB immunoreactivity of a male rat anterior pituitary cell. Cell nucleus and cytoplasm exhibit green fluorescence. The primary antibody was produced in rabbit (diluted 1:7500). Magnification: 1000. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 2. Distribution of 28 kDa CB-ir cells in the anterior pituitary. CB containing cells are scattered in the pituitary parenchyma and are often clustered around blood vessels (Fig. 3) or at the gland periphery (arrows) with different aspects between male (Fig. 4) and female (Fig. 5). The specific antibody was produced in rabbit (diluted 1:5000). Magnifications: 100; Fig. 3, 240; Fig. 4, 150; Fig. 5, 100.
10000. In order to prove a true localisation of CB, the monoclonal and polyclonal antibodies together were used for double immunostaining. For negative controls, the mixture of the primary antisera with 0.1 AM recombinant rat CB (Swant) was used instead of the first layer. As a further control, the monoclonal antiserum was cross adsorbed with 0.1 AM recombinant calretinin (Swant). Double immunofluorescence staining was also carried out by using goat anti-calretinin (1:1000, Swant) and mouse or rabbit anti-CB antisera. Fluorescein isothiocyanate (FITC, Jackson ImmunoResearch) and Texas red (Jackson ImmunoResearch)-conjugated donkey anti-mouse and/or antirabbit immunoglobulins were used to reveal the antigens as needed. Occasionally, rabbit anti-tyrosine hydroxylase (TH) antiserum (1:2000, Incstar) was used together with both anti-CB antisera to show the coexistence of CB and calretinin within dopaminergic nerves. Double immunofluorescence staining used mouse anti-CB serum together with rabbit antisera (1:2000) against pituitary hormones, namely, anti-PRL, anti-ACTH, anti-LH, anti-FSH, anti-TSH and anti-GH (gift from NIDDK). The sections were then mounted with phenylenediamine/glycerine/PBS medium and viewed with a Leitz Diaplan fluorescence microscope provided with a fluores-
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CB-immunoreactive (ir) cells were present in male AP (Fig. 1). They were sparse throughout the parenchyma of the endocrine gland (Fig. 2) and around blood vessels (Fig. 3). Clusters of cells were frequently found at the periphery of the pituitary ventral portion (Fig. 4). The number of CB-ir cells was definitely lower in the female AP gland (Figs. 5 and 6), most of them showing less intense fluorescence. Small clusters of immunoreactive cells were also present in the female pituitary, particularly at the periphery of the gland, but they were not comparable in size with those found in males (Figs. 7 and 8). In lactating rats, the general feature of CB-IR was similar to that of male pituitary, although peripheral clusters of CB-ir cells were not observed (Fig. 9). Thus, with a lactating female whose pups were removed on the day of birth, the number of positive cells appeared to be increased with respect to normal female, as well as the average intensity of fluorescence, which however was not evaluated in this study. On the contrary, in lactating female 1 week after removal of the pups, CB-IR was more like that of a normal female (Fig. 6). Intermediate lobe (IL)
Fig. 3. Distribution of 28 kDa CB-ir cells in the anterior pituitary. CB containing cells are scattered in the pituitary parenchyma (Fig. 2) and are often clustered around blood vessels or at the gland periphery (arrows) with different aspects between male (Fig. 4) and female (Fig. 5). The specific antibody was produced in rabbit (diluted 1:5000). Magnifications: Fig. 2, 100; 240; Fig. 4, 150; Fig. 5, 100.
The IL showed both immunoreactive cells and nerve fibers. No differences were detected between male,
cence filter system allowing simultaneous emission of green and red wavelengths. Methods for controls of double staining cross-reactivity have been described previously (Cimini et al., 1989). Quantification Photographs of 10-Am-thick immunostained sections were taken by a Leitz Diaplan fluorescence microscope provided with a Leica DC-200 digital camera. The number of CB-immunoreactive cells were counted in five fields/ section/animal/experimental group. Mean values and standard deviation for each group were calculated with reference to the histological surface area of 1 mm2, which is considered the unit area in this study. The significance of differences between groups was established by calculation of Bonferroni’s P values.
Results Anterior pituitary (AP) Immunoreactivity (IR) to CB was present not only in the cytoplasm, but also in the nucleus. Intensely positive
Fig. 4. Distribution of 28 kDa CB-ir cells in the anterior pituitary. CB containing cells are scattered in the pituitary parenchyma (Fig. 2) and are often clustered around blood vessels (Fig. 3) or at the gland periphery (arrows) with different aspects between male and female (Fig. 5). The specific antibody was produced in rabbit (diluted 1:5000). Magnifications: Fig. 2, 100; Fig. 3, 240; 150; Fig. 5, 100.
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Fig. 7. This panel shows a comparison between the patterns of peripheral clustered CB cells in normal male, normal female (Fig. 8) and lactating female (Fig. 9) at level of the pituitary ventral surface. The primary antibody was produced in rabbit (diluted 1:10000). Magnification: 150.
Immunostaining of the intermediate lobe after pituitary stalk section showed a complete absence of nerve fibers IR (Figs. 11 and 12). Posterior lobe (PL)
Fig. 5. Distribution of 28 kDa CB-ir cells in the anterior pituitary. CB containing cells are scattered in the pituitary parenchyma (Fig. 2) and are often clustered around blood vessels (Fig. 3) or at the gland periphery (arrows) with different aspects between male (Fig. 4) and female. The specific antibody was produced in rabbit (diluted 1:5000). Magnifications: Fig. 2, 100; Fig. 3, 240; Fig. 4, 150; 100.
female and lactating CB-IR in the IL. All the CB-ir cells had nuclear positivity while very few had immunoreactivity in the cytoplasm. In addition, a fine network of immunoreactive varicose nerve fibers was observed in perilobular and intralobular tissue of IL (Fig. 10). The nerve fibers corresponded to TH-containing nerves, as previously shown in the hypothalamus (Jacobowitz et al., 1998).
Fig. 6. Statistical analysis of CB-immunoreactive cells in the anterior pituitary of male, female, lactating same day (SD) and lactating 1 week later (OWL) rats. *P b 0.05 male vs. female, male vs. OWL and SD vs. OWL.
PL-IR to CB antisera showed in male pituitary a very intense network of nerve fibers with variable thickness and length (Fig. 10). A more intense fluorescence was observed in the lactating female as compared with the normal female, but indeed the increase was not estimated. As for the IL, in the PL double immunofluorescence revealed the coexistence of CB and TH. Double immunostaining and controls Immunofluorescence with the monoclonal antibody to CB was quenched by mouse ascites fluid in all animals used, when used instead of the primary antibody. Staining was also absent when normal rabbit serum was used instead of the CB polyclonal antibody. In order to verify the rabbit polyclonal antibody tissue specificity, double immunostaining with both monoclonal and polyclonal antisera was carried out. In the male pituitary, while cell cytoplasm was positive for both antibodies, cell nuclei immunoreacted to polyclonal serum only (Fig. 13). Some cells were entirely stained by the polyclonal antibody
Fig. 8. This panel shows a comparison between the patterns of peripheral clustered CB cells in normal male (Fig. 7), normal female and lactating female (Fig. 9) at level of the pituitary ventral surface. The primary antibody was produced in rabbit (diluted 1:10000). Magnification: 150.
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Fig. 9. This panel shows a comparison between the patterns of peripheral clustered CB cells in normal male (Fig. 7), normal female (Fig. 8) and lactating female at level of the pituitary ventral surface. The primary antibody was produced in rabbit (diluted 1:10000). Magnification: 150.
only, and none was found to be immunoreactive to the monoclonal antibody alone. In females, cells immunoreactive only to the monoclonal antibody were seen as well as cells immunoreactive only to the polyclonal one, these latter showing both cytoplasmic and nuclear positivity, while the former showed only cytoplasmic positivity. Furthermore, some cells with polyclonal-immunoreactive nuclei had monoclonal positive cytoplasm. In very few cases the nuclei of cells with cytoplasmic and nuclear Fig. 11. The immunoreactivity of intermediate lobe (IL) nerves to CB (arrows) completely disappears after pituitary stalk section (Fig. 12). A rabbit polyclonal antibody to calbindin was used in this experiment (diluted 1:10000). Magnification: 150.
Fig. 10. 28 kDa CB in rat male pituitary localized by the antibody produced in rabbit (diluted 1:10000). The three portions of the pituitary are shown: adenohypophysis with sparse CB-ir cells (AP), intermediate lobe with its predominant nuclear IR and a fine network of immunoreactive nerve fibres (IL) and the posterior lobe with its rich CB-ir network of nerve fibres (PL). Magnification: 150.
polyclonal CB-positivity were also immunoreactive to monoclonal anti-CB. Preabsorption of CB antisera with recombinant calretinin did not show any difference in staining pattern compared with CB antiserum. The absorption of both CB antisera with recombinant CB completely quenched the immunostaining. Double immunostaining with monoclonal antiserum to CB and polyclonal goat antiserum to calretinin revealed three cell types: one only immunoreactive to CB, one only immunoreactive to calretinin and one immunoreactive to both CB and calretinin, the latter exhibiting more frequently nuclear CB-IR and cytoplasmic CR- and/or CB-IR. In double immunostaining with antibodies to pituitary hormones, faint CB-ir cells with negative nuclei corresponded mostly to LH cells, while CB-ir cells with positive nuclei did not correspond to LH cells in all cases studied. Occasional colocalisation with faintly CB-ir PRL cells occurred in male and female anterior pituitary, but not with CB-ir cells containing positive nuclei. Similarly, occasional coexistence with GH was found in both sexes. Coexistence with TSH was found in the male only. No coexistence was found with ACTH. In the IL, there were abundant cells with CB-ir nuclei with no calretinin colocalization (Fig. 14) while in the
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Fig. 12. The immunoreactivity of intermediate lobe (IL) nerves to CB (Fig. 11, arrows) completely disappears after pituitary stalk section. A rabbit polyclonal antibody to calbindin was used in this experiment (diluted 1:10000). Magnification: 150.
nerves of the intermediate lobe the two proteins were found together. In the PL the nerves showed partial colocalization of calretinin and CB (Fig. 15). No difference was noticed between the different primary antibodies to CB in the IL and PL.
Discussion In this study the presence of CB 28 kDa was detected in the cytoplasm and nuclei of endocrine cells of rat pituitary with one monoclonal antibody and two polyclonal ones. Nuclear staining for spot 35 protein has already been reported in chicken (Jande et al., 1981; Thorens et al., 1982) as well as nuclear staining for calbindin in cat thyroid C cells and dog islets (Buffa et al., 1989). These authors mentioned the possible presence of calbindin in the pituitary TSH cells but did not specify the species or the sex of the animals. However, their hypothesis can be explained now on the basis of our results on calretinin localization in TSH cells of rat anterior pituitary (Cimini et al., 1997) because their antiserum raised against chick intestinal calbindin cross-reacted with calretinin. The nuclear localization of calbindin, with particular reference to CLIP-producing cells of human pituitary (Buffa et al., 1990), has been confirmed by our results on the IL of
the rat pituitary. Its significance is still unknown at least in the pituitary, although the presence of CB has been related to the ability of taking up 3H-1,25(OH)2 vitamin D, and most interestingly, nuclear CB has been recently related to the regulation of nuclear calcium signals in nerve cells (German et al., 1997). To rule out possible cross-reactivity with calretinin, we used a mixture of CB antibody with recombinant calretinin, as primary antibody, and anti-CB antisera on a serial section. The two sections showed the same staining pattern. Our results on sexual differences of CB-IR in the anterior pituitary gland confirm the previous report on distribution of spot 35-calbindin (Abe et al., 1990). This is due to its 79% homology of amino acid sequence with 28 kDa calbindin. We have also found occasional coexistence of CB with TSH, PRL and GH in both sexes by double immunofluorescence on the same section, while the previous results showing supposed spot 35-CB coexistence with all classical pituitary hormones were based on mirror-image sections and were therefore not perfectly reliable. In addition, only occasional GH cells seem to contain CB in lactating rats. The results of double immunofluorescence confirm that some of the CB cells immunoreactive to the polyclonal antiserum (Swant) are calretinin cells because of its (admittedly low) cross-reactivity or, according to Schiffmann et al. (1999), the anti-calretinin antiserum might recognize CB protein when highly saturated with Ca2+. Therefore, the differences between sexes are related to the number of CB expressing cells not to the cell types, and in addition a variation of the endocrine status, such as lactation, might alter calcium-linked activity of some anterior pituitary cells. In man, nuclear staining of melanotrophs and occasional staining of gonadotrophs and thytrophs was reported (Buffa et al., 1990), but again age and sex were not mentioned. The presence of 28 kDa CB in IL has been studied here for the first time in the rat, thus confirming the results previously obtained on human tissue (Buffa et al., 1990), while the coexistence of CB with calretinin within the pars nervosa was previously established by light and electron microscopy (Miyata et al., 2000). In our results, the nuclear and less prominent cytoplasmic immunoreactivity in the IL are not related to sex or endocrine status. This is in contrast with the transient expression of spot 35-CB in the immature pituicytes of embryonic rats (Abe et al., 1991). The different results could be explained by the different antisera used, but also by calcium-dependent conformational changes. Thus, the hypothesis can be made that at least two different CB molecular conformations, one nuclear and one cytoplasmic, could be present in the pituitary gland. However, this hypothesis has been widely discussed in the literature and is based on the suggestion that antibody recognition of calcium binding proteins may depend on their calcium-
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Fig. 15. Double immunolabelling of calretinin and CB within the posterior lobe of normal male rat pituitary. There is a partial coexistence of the two calcium-binding protein as some nerve fibres exhibit only green fluorescence (CB), some orange fluorescence (CB and calretinin) and few red (calretinin). Antibodies used were the same as for Fig. 14. Magnification: 250.
Fig. 13. Double labelling of anterior pituitary cells with monoclonal and polyclonal antibodies to CB. Nuclei exhibiting green fluorescence are labelled with the polyclonal antibody (diluted 1:10000), while cytoplasm exhibiting orange fluorescence is labelled with both CB antibodies. The monoclonal antibody to CB was revealed by Texas red-conjugated immunoglobulins. Magnification: 650.
binding status (Dutar et al., 1991; Olive and Ferrer, 1994; Winsky and Kuznicki, 1996). The lack of difference between the sexes in the IL and PL CB-immunoreactive nerves implies that CB is a constitutive protein of pituitary and, of course, hypothalamic nerves, and its expression is not apparently altered by the complex functional changes of the hypothalamus. The coexistence of CB with TH in all conditions studied may suggest that both CB and CR, two calcium binding proteins, might participate in the neurosecretory activity of PL nerve terminals because it has previously been shown that CR and TH do colocalize in the PL nerves (Cimini et al., 1997). Interestingly, THimmunoreactive nerve terminals are reported to contain serotonin too in the intermediate lobe of rat pituitary (Vanhatalo et al., 1995). In conclusion, we have reviewed CB-IR in the pituitary of rat. There are sex-related differences and endocrine status-related differences mainly in the anterior pituitary, where the calcium-binding protein colocalizes with somatotrophs, thyrotrophs and luteotrophs. CB might have different configurations in the nucleus and in the cytoplasm of anterior and intermediate pituitary cells. It is present in nerves of IL and PL and colocalizes with TH. Fig. 14. Colocalisation of CB and calretinin within the intermediate lobe of male rat pituitary. Green fluorescent nuclei represent CB, and orangelabelled nerve fibres show coexistence of CB and calretinin. CB immunoreactivity was revealed by the rabbit antiserum (diluted 1:10000) and calretinin IR by the goat antiserum. Magnification: 250.
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Acknowledgments The author wish to thank David M. Jacobowitz who made possible the realization of this project, most of it being performed in his lab. The author is indebted to Susan Van Noorden for useful comments and language revision and to Dr. Giovanna G. Altobelli and Alfredo Sales for lab assistance. Many thanks are also due to Dr. Parlow (NIDDK) for the gift of antisera to pituitary hormones. Departmental funds from the University of Naples Federico II were also used.
References Abe, H., Amano, O., Yamakuni, T., Takahashi, Y., Kondo, H., 1990. Localization of spot 35-calbindin (rat cerebellar calbindin) in the anterior pituitary of the rat: developmental and sexual differences. Arch. Histol. Cytol. 53, 585 – 591. Abe, H., Amano, O., Yamakuni, T., Kuwano, R., Takahashi, Y., Kondo, H., 1991. Transient expression of a calcium-binding protein (spot 35calbindin) and its mRNA in the immature pituicytes of embryonic rats. Cell Tissue Res. 266, 325 – 330. Buffa, R., Mare’, P., Salvadore, M., Solcia, E., Furness, J.B., Lawson, D.E., 1989. Calbindin 28 kDa in endocrine cells of known or putative calcium-regulating function. Thyro-parathyroid C cells, gastric ECL cells, intestinal secretin and enteroglucagon cells, pancreatic glucagon, insulin and PP cells, adrenal medullary NA cells and some pituitary (TSH?) cells. Histochemistry 91, 107 – 113. Buffa, R., Mare’, P., Salvadore, M., Gini, A., 1990. Immunohistochemical detection of 28 kDa calbindin in human tissues. Adv. Exp. Med. Biol. 269, 205 – 210 (review). Celio, M.R., Baier, W., Scharer, L., Gregersen, H.J., de Viragh, P.A., Norman, A.W., 1990. Monoclonal antibodies directed against the calcium binding protein calbindin D-28k. Cell Calcium 11, 599 – 602. Cimini, V., Van Noorden, S., Polak, J.M., 1989. Co-localisation of substance P-, bombesin- and peptide histidine isoleucine (PHI)-like
peptides in gut endocrine cells of the dogfish Scyliorhinus stellaris. Anat. Embryol. 179, 605 – 614. Cimini, V., Isaacs, K.R., Jacobowitz, D.M., 1997. Calretinin in the rat pituitary: colocalization with thyroid-stimulating hormone. Neuroendocrinology 65, 179 – 188. Coons, A.H., 1971. The development of immunohistochemistry. Ann. N. Y. Acad. Sci. 177, 5 – 9. Dutar, P., Potier, B., Lamour, Y., Emson, P.C., Senut, M.C., 1991. Loss of calbindin-28K immunoreactivity in hippocampal slices from aged rats: a role for calcium? Eur. J. Neurosci. 3, 839 – 849. German, D.C., Ng, M.C., Liang, C.L., McMahon, A., Iacopino, A.M., 1997. Calbindin-D28k in nerve cell nuclei. Neuroscience 81, 735 – 743. Jacobowitz, D.M., Isaacs, K.R., Cimini, V., 1998. Triple colocalization of tyrosine hydroxylase, calretinin, and calbindin D-28k in the periventricular–hypophyseal dopaminergic neuronal system. Adv. Pharmacol. 42, 37 – 40. Jande, S.S., Tolnai, S., Lawson, D.E., 1981. Immunohistochemical localization of vitamin D-dependent calcium-binding protein in duodenum, kidney, uterus and cerebellum of chickens. Histochemistry 71, 99 – 116. Miyata, S., Nakai, S., Kiyohara, T., Hatton, G.I., 2000. Calbindin-D28k and calretinin in the rat posterior pituitary; light and electron microscopic localization and upregulation with dehydration. J. Neurocytol. 29, 5 – 17. Olive, M., Ferrer, I., 1994. Parvalbumin immunohistochemistry in denervated skeletal muscle. Neuropathol. Appl. Neurobiol. 20, 495 – 500. Schiffmann, S.N., Cheron, G., Lohof, A., d’Alcantara, P., Meyer, M., Parmentier, M., Schurmans, S., 1999. Impaired motor coordination and Purkinje cell excitability in mice lacking calretinin. Proc. Natl. Acad. Sci. U. S. A. 96, 5257 – 5262. Thorens, B., Roth, J., Norman, A.W., Perrelet, A., Orci, L., 1982. Immunocytochemical localization of the vitamin D-dependent calcium binding protein in chick duodenum. J. Cell Biol. 94, 115 – 122. Vanhatalo, S., Soinila, S., Kaartinen, K., Back, N., 1995. Colocalization of dopamine and serotonin in the rat pituitary gland and in the nuclei innervating it. Brain Res. 669, 275 – 284. Winsky, L., Kuznicki, J., 1996. Antibody recognition of calcium-binding proteins depends on their calcium-binding status. J. Neurochem. 66, 764 – 771.