Immunogold Detection of Co-localized Neuropeptides : Methodological Aspects
INSERM E 0358, Université Victor Segalen Bordeaux, Institut François Magendie, Bordeaux, France (ML), and Laboratoire de Neurobiologie des Signaux Intercellulaires, UMR CNRS 7101, Université Pierre et Marie Curie Paris 6, Paris, France (EVP,AC)
Correspondence to: Marc Landry, INSERM E 0358, Institut François Magendie, 1, rue Camille Saint-Saëns, 33 077 Bordeaux Cedex, France. E-mail: marc.landry{at}u-bordeaux2.fr
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Summary |
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(J Histochem Cytochem 52:617627, 2004)
Key Words: hypothalamus vasopressin galanin electron microscopy immunogold post-embedding quantification rat
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Introduction |
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Most of the many authors who have used post-embedding immunogold methods on endocrine or neuroendocrine materials, such as the hypothalamoposthypophyseal tract, attempted to refine the immunodetection procedures. Current protocols now allow immunolabeling of individual secretory granules and study of their intragranular content. Improving the sensitivity is essential for the reliability of quantitative analysis, which is a prerequisite to the study of dynamic cell biology processes. In particular, neuropeptide maturation mechanisms and pathways analysis require an accurate assessment of secretory granule content in various subcellular compartments. In addition, qualitative aspects, particularly the lack of labeling of some neurosecretory granules, would also profit from an increase in method sensitivity. Are these granules negative because of limited method sensitivity or because of an actual lack of antigen? Although their presence has rarely been discussed, unlabeled granules have been occasionally described, in particular by Merighi (1992) and Bendayan (1995)
, and technical problems were then postulated. Alternatively, El Majdoubi et al. (1996)
also proposed several hypothesis (see Discussion).
Because gold-conjugated antibodies act as surface markers on resin-embedded tissue, one must first consider the access of the granules to the surface of the section (Bendayan 1995). Although granules have diameters larger than the thickness of ultrathin sections, some of them do not reach the surface of the section, because of their random distribution within the tissue (see Figure 1)
. Consequently, these granules remained unstained after a single immunogold application (Figure 1I) but, according to Bendayan (1982)
, they could be marked by a second incubation on the other side of the section (Figure 1II) if they do contain the target antigen. Such a double-face labeling might increase labeling intensity and would minimize errors in quantitative study of the proportion of labeled granules and the labeling intensity. Finally, this method is likely to precisely reveal weak variations of neuropeptide amounts related to the physiological state or to the subcellular localization.
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Materials and Methods |
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Vibratome sections were embedded at 30C in hydrophilic Lowicryl K4M resin (Sigma; St Louis, MO) by the progressive temperature-lowering technique using a Reichert AFS system (Leica; Vienna, Austria) according to the instruction manual.
Immunoelectron Microscopy
Ultrathin sections (60100-nm) from Lowicryl blocks were mounted on gold grids (300-mesh) and submitted to the postembedding immunogold procedure for the detection of vasopressin (AVP) or galanin (GAL) as described elsewhere (Ozawa et al. 1994; VilaPorcile and Barret 1996
). Briefly, the grids were successively floated on drops of 50 mM ammonium chloride in TBS (Tris-HCl buffer 50 mM, NaCl 150 mM, pH 7.45) for 15 min and of TBS supplemented with 1% bovine serum albumin (TBS/BSA) for 1 hr. Grids were then incubated overnight at 4C on drops of the primary antibody (anti-AVP; Chemicon, Temecula, CA, diluted 1:4000 in TBS/BSA, or anti-GAL (a gift from Dr. G. Tramu, diluted 1:200 in TBS/BSA). After several washings on large drops of TBS/BSA, the grids were incubated for 1 hr at RT on goat anti-rabbit colloidal gold conjugate (GAR 10 or 20 nm, Biocell, Cardiff, UK; diluted 1:501:100 in TBS/BSA). They were then rinsed once with TBS/BSA, twice with TBS, and twice with distilled water (5 min each) and air-dried.
After complete drying, the procedure was repeated on the other side of the grid (Bendayan 1982) using the same specific primary antibody and a colloidal gold conjugate of either the same or a different size. Finally, a rapid (1012 sec) staining was performed on the grid side supporting the sections with 2% uranyl acetate in 50% ethanol.
As controls for immunogold specificity, primary antibodies were omitted or replaced by normal rabbit serum, gold conjugates being applied as usual.
Two types of double-labeling experiments were performed with the same anti-AVP (or anti-GAL) antibody, using either the same 10-nm gold particles on both sides of the grids or two differently sized particles (10 and 20 nm), to detect "newly" labeled granules, i.e., granules labeled only after the second labeling round.
Grids were observed under a JEOL 100 CX II electron microscope at a x20,000 magnification.
On standard micrographs (x40,000), the diameters of a sample (n=280) of labeled and unlabeled neurosecretory granules were measured in the three neuron compartments.
Quantification
Quantification of labeled neurosecretory granules in the different compartments of magnocellular hypothalamic neurons (i.e., perikarya, neurites, and posterior pituitary nerve endings) was performed on printed micrographs (x40,000) overlaid with a squared transparent sheet. Labeled granules were counted manually by point counting. Results were expressed as percentages of labeled granules related to the total number of granules (± SEM) in a given compartment. SEM was calculated with the one-way ANOVA program, including Fischer's, Scheffé's and t-tests, p<0.01 being considered significant.
In addition, on the same micrographs, the number of gold particles present on secretory granules was also counted and reported to the total number of labeled granules, thus providing a mean density of labeling (mean number of particles per granule ± SEM) in each neuron compartment. Frequencies of granule labeling within the three compartments were evaluated and expressed as percentages of granules distributed according to the number of gold particles they hold.
A comparison with single-labeled sections was systematically established for each size of gold particles used in the different experiments.
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Results |
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Results from quantitative analysis indicated that not only the number of labeled granules but also the number of gold particles per granule was higher after double-face labeling for AVP, up to 2022 gold particles per granule in perikarya. The mean number of gold particles per granule reached 9.2 in perikarya compared to 5.7 after single-face labeling (Figure 6a) . The efficiency of double-face labeling appeared slighter in dendrites and PN (Figure 6a). For intensity of gold labeling for GAL, a slight augmentation was observed after double staining in dendrites, and more markedly in the terminals, with respect to single labeling. Moreover, in contrast to the results obtained after single staining, the double labeling revealed that the density of gold particles remained constant, regardless of the subcellular compartment considered, and did not decrease from the perikarya to neural lobe (Figure 6b).
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Discussion |
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The double-face method used in this study allowed us to improve the immunodetection at two levels, i.e., the number of responsive granules and the number of gold particles over each positive granule. Given the better sensitivity of this approach, the immunodetection threshold was lowered and this method is likely to provide more reliable quantitative results. The resin and the embedding procedure should be carefully evaluated because, in our hands, embedding in acrylic resin (Lowicryl) with a progressive lowering of temperature protocol provided the best compromise between antigenicity and preservation of morphology.
However, the increase was rather moderate both in percentage and intensity and depended on the target peptide and/or the antibody used. It is known that a non-negligible part of immunogold signal is lost during double-labeling procedures (VilaPorcile and Corvol 1998), even when a postfixation with glutaraldehyde has followed the colloidal gold application. This loss could represent a limiting factor for maximal enhancement of the staining. In our present work, other methodological attempts were performed, i.e., a tyramide signal amplification (Mayer and Bendayan 1999
), without improving our final results.
This technique has nevertheless allowed us to detect AVP in all the granules present in some perikarya. Moreover, unlabeled granules were never found within the trans-Golgi saccules, either in these well-labeled cells or in lesser responsive ones. Hence, we can infer that all nascent granules do contain AVP. Only intragranular maturation processes could lead to an under-detection of the peptide. This is in agreement with the decrease in the labeling intensity for AVP.
The better sensitivity of the double-face approach demonstrates that a higher percentage of GAL-labeled granules was detected in dendrites, in agreement with our previous immunodetections.
Because all experiments were performed on the same material with the same set of techniques, all differences in the results of the double-face protocol do not rely on methodological hindrance. They might instead reflect specific biological features of the molecules or compartments under investigation.
The lack of changes in GAL labeling intensity suggests that this particular neuropeptide remains uniformly detectable in all compartments. In that case, the double-face protocol appears well suited to assess the preferential localization of GAL-positive granules in the dendritic compartment, as previously reported for this peptide (Landry et al. 2003). It further supports the hypothesis of a preferential routing of GAL towards dendritic processes and suggests possible dendritic release of GAL within the SON (Landry et al. 2003
). Such a somatodendritic release has been described in the same hypothalamic neurons for the major neurohormones (Pow and Morris 1989
; Ludwig and Landgraf 1992
; Morris and Pow 1993
; Morris et al. 1998
). Neuropeptide dendritic release in the hypothalamus is calcium-dependent (Di ScalaGuénot et al. 1987
) and is stimulated by intracellular calcium stores (Ludwig et al. 2002
). Moreover, this process is regulated by the hypothalamic neuropeptides themselves (Lambert et al. 1994
) and was recently shown to depend on physiological conditions, e.g., lactation (de Kock et al. 2003
).
Despite the use of a double-face approach, unlabeled granules were nevertheless observed in most ultrathin sections. This lack of labeling confirms that AVP remains undetectable in a subpopulation of secretory granules of the vasopressinergic magnocellular neurons. In the same hypothalamohypophyseal model, El Majdoubi et al. (1996) have found neurosecretory granules not responsive to chromogranin A (CGA) detection. They have raised several hypotheses to elucidate the apparent absence of CGA, i.e., the absence of peptide or its very low amount, or even its "extensive processing" into derivatives no longer recognizable by their antibody. These hypotheses could also be applied to AVP and GAL peptides.
A complete absence of AVP cannot be ruled out in some neurosecretory granules. The unreactive granules may contain only some unrelated molecules, such as GAL. Indeed, in addition to AVP, its precursor and its breakdown products (Pow and Morris 1991), many non-vasopressin-related components were demonstrated to be localized in neurosecretory granules of vasopressinergic magnocellular neurons. In addition to GAL, previously demonstrated to be partly co-localized with AVP in these neurons (Melander et al. 1986
; Gayman and Martin 1989
; Landry et al. 2003
), some of these granular constituents are listed in Table 1. They were shown to be co-localized with AVP in granules of well-identified vasopressinergic neurons either by immunogold or by co-fractionation. In addition, the previously shown existence of granules exclusively labeled for GAL is a potent argument for the possibility that such non-vasopressin-related components may constitute the core of these still vasopressin-negative granules. However, actual technical difficulties or immunoreaction hindrance must also be considered. All molecules concentrated in structures as small as secretory granules could build a mesh so tight that antibodies may have no access to the target antigen, even though the neuropeptide might still be present.
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Various processes of aggregation/condensation can be considered, depending on the studied peptide. This aggregation of non-related components might occur in addition to the possible aggregation of AVP mentioned above. Compared to AVP, the intensity of GAL labeling seems to remain rather stable from NSO to dendrites or PN. This suggests the absence of GAL proteolysis and aggregation through specific mechanisms of maturation for GAL.
In conclusion, the double-face approach used in this study offers an improvement of neuropeptideimmunogold detection while confirming the lack of immunoreactivity in some secretory granules. Such an approach could be useful for fine quantitative studies at the electron microscopic level. Although moderate, the signal increase has enabled us to demonstrate specific variations of peptidergic content along the secretory pathway of magnocellular neurons, providing new clues to possible sites of release and action of neuropeptides.
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Acknowledgments |
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Footnotes |
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Literature Cited |
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Bendayan M (1982) Double immunocytochemical labeling applying the protein Agold technique. J Histochem Cytochem 28:12511254
Bendayan M (1984) Protein A-gold electron microscopic immunocytochemistry: methods, applications and limitations. J Electron Microsc Techn 1:243270
Bendayan M (1989) Protein A-gold and protein G-gold postembedding immunoelectron microscopy. In Hayat MA, ed. Colloidal Gold: Principles, Methods and Applications. Vol 1. New York, Academic Press, 3394
Bendayan M (1995) Colloidal gold post-embedding immunocytochemistry. Prog Histochem Cytochem 29:1163[Medline]
Bendayan M (2000) A review of the potential and versatility of colloidal gold cytochemical labeling for molecular morphology. Biotech Histochem 75:203242[Medline]
Brownstein MJ, Russel JT, Gainer H (1980) Synthesis, transport and release of posterior pituitary hormones. Science 207:373378[Medline]
Di ScalaGuénot D, Strosser MT, Richard P (1987) Electrical stimulations of perifused magnocellular nuclei in vitro elicit Ca2+-dependent, tetrodotoxin-insensitive release of oxytocin and vasopressin. Neurosci Lett 76:209214[Medline]
de Kock CPJ, Wierda KDB, Bosman LWJ, Min R, Koksma JJ, Mansvelder HD, Vrhage M, et al. (2003) Somatodendritic secretion in oxytocin neurons is upregulated during the female reproductive cycle. J Neurosci 23:27262734
Duong LT, Fleming PJ, Russel JT (1984) An identical cytochrome b561 is present in bovine adrenal chromaffin vesicles and posterior pituitary neurosecretory vesicles. J Biol Chem 259:48854889
Egger C, Kirchmair R, Kapelari S, FischerColbrie R, HogueAngeletti R, Winkler H (1994) Bovine posterior pituitary: presence of p65 (synaptotagmin), PC1, PC2 and secretoneurin in large dense core vesicles. Neuroendocrinology 59:169175[Medline]
El Majdoubi M, MetzBoutigue MH, GarciaSablone P, Théodosis DT, Aunis D (1996) Immunocytochemical localization of chromogranin A in the normal and stimulated hypothalamo-neurohypophysial system of the rat. J Neurocytol 25:405416[Medline]
Faulk WP, Taylor GM (1971) An immunocolloid method for the electron microscope. Immunocytochemistry 8:10811083
Fricker LD (1988) Carboxypeptidase E. Annu Rev Physiol 50:309321[Medline]
Gayman W, Martin R (1989) Immunoreactive galanin-like material in magnocellular hypothalamo-neurohypophysial neurones of the rat. Cell Tissue Res 255:139147[Medline]
Lambert RC, Dayanithi G, Moos F, Richard P (1994) A rise in the intracellular Ca2+ concentration of isolated rat supraoptic cells in response to oxytocin. J Physiol 478:275288[Abstract]
Landry M, Hökfelt T (1998) Subcellular localization of preprogalanin messenger RNA in perikarya and axons of hypothalamo-posthypophyseal magnocellular neurons: an in situ hybridization study. Neuroscience 84:897912[Medline]
Landry M, VilaPorcile E, Hökfelt T, Calas A (2003) Differential routing of coexisting neuropeptides in vasopressin neurons. Eur J Neurosci 17:579589
Ludwig M, Landgraf R (1992) Does the release of vasopressin within the supraoptic nucleus of the rat brain depend upon changes in osmolality and Ca2+/K+? Brain Res 576:231234[Medline]
Ludwig M, Sabatier N, Bull PM, Landgraf R, Dayanithi G, Lang G (2002) Intracellular calcium stores regulate activity-dependent neuropeptide release from dendrites. Nature 418:8589[Medline]
Mahata SK, Mahata M, Steiner HJ, FischerColbrie R, Winkler H (1992) In situ hybridization: mRNA levels of secretogranin II, neuropeptides and carboxypeptidase H in brains of salt-loaded and Brattleboro rats. Neuroscience 48:669680[Medline]
Mayer G, Bendayan M (1999) Immunogold signal amplification: application of the CARD approach to electron microscopy. J Histochem Cytochem 47:421429
Mejia S, Morales MA, Zetina ME, Martinez de la Escalera G, Clapp C (1997) Immunoreactive prolactin forms colocalize with vasopressin in neurons of the hypothalamic paraventricular and supraoptic nuclei. Neuroendocrinology 66:151159[Medline]
Melander T, Hökfelt T, Rokaeus A, Cuello AC, Oertel WH, Verhofstad A, Goldstein M (1986) Coexistence of galanin-like immunoreactivity with catecholamines, 5-hydroxytryptamine, GABA and neuropeptides in the rat CNS. J Neurosci 6:36403654[Abstract]
Merighi A (1992) Post-embedding electron microscopic immunocytochemistry. In Polak JM, Priestley JV, eds. Electron Microscopic Immunocytochemistry: Principles and Practice. New York, Oxford University Press, 5187
Morris JF, Pow DV (1993) New anatomical insights into the inputs and outputs from hypothalamic magnocellular neurons. Ann NY Acad Sci 689:1633[Medline]
Morris JF, Budd TC, Epton MJ, Ma D, Pow DV, Wang H (1998) Functions of the perikaryon and dendrites in magnocellular vasopressin-secreting neurons: new insights from ultrastructural studies. Prog Brain Res 119:2130[Medline]
Nakamura S, Naruse M, Naruse K, Shioda S, Nakai Y, Uemura H (1993) Colocalization of immunoreactive endothelin-1 and neurohypophysial hormones in the axons of the neural lobe of the rat pituitary. Endocrinology 132:530533[Abstract]
Nordmann JJ, Morris JF (1984) Method for quantitating the molecular content of a subcellular organelle: hormone and neurophysin content of newly formed and aged neurosecretory granules. Proc Natl Acad Sci USA 81:180184[Abstract]
Ozawa H, Picart R, Barret A, Tougard C (1994) Heterogeneity in the pattern of distribution of the specific hormonal product and secretogranins within the secretory granules of the rat prolactin cells. J Histochem Cytochem 42:10971107
Pierre K, Rougon G, Allard M, Bonhomme R, Gennarini G, Poulain DA, Théodosis DT (1998) Regulated expression of the cell adhesion glycoprotein F3 in adult hypothalamic magnocellular neurons. J Neurosci 18:53335343
Pow DV, Morris JF (1989) Dendrites of hypothalamic magnocellular neurons release neurohypophysial peptides by exocytosis. Neuroscience 32:435439[Medline]
Pow DV, Morris JF (1991) Membrane routing during exocytosis and endocytosis in neuroendocrine neurones and endocrine cells: use of colloidal gold particles and immunocytochemical discrimination of membrane compartments. Cell Tissue Res 264:299316[Medline]
Roth J (1986) Postembedding immunolabeling. J Microsc 143:125138[Medline]
Roth J, Bendayan M, Orci L (1978) Ultrastructural localization of intracellular antigens by the use of protein Agold complex. J Histochem Cytochem 26:10741081[Abstract]
Sonnemans MAF, Evans DAP, Burbach JPH, van Leeuwen FW (1996) Immunocytochemical evidence for the presence of vasopressin in intermediate sized neurosecretory granules of solitary neurohypophyseal terminals in the homozygous Brattleboro rat. Neuroscience 72:225231[Medline]
Théodosis DT, Pierre K, Poulain DA (2000) Differential expression of two adhesion molecules of the immunoglobulin superfamily, F3 and polysialylated NCAM, in hypothalamic magnocellular neurones capable of plasticity. Exp Physiol 85:187S196S[Abstract]
Van den Pol AN, Decavel C, Levi A, Paterson B (1989) Hypothalamic expression of a novel gene product, VGF: immunocytochemical analysis. J Neurosci 9:41224137[Abstract]
VilaPorcile E, Barret A (1996) Structural and functional differences between prolactin cells from the inner and outer zones of the male rat anterior pituitary. Cell Tissue Res 284:247259[Medline]
VilaPorcile E, Corvol P (1998) Angiotensinogen, prorenin, and renin are co-localized in the secretory granules of all glandular cells of the rat anterior pituitary: an immunoultrastructural study. J Histochem Cytochem 46:301311
WalshSolimena C, Takei K, Marek KL, Midyett K, Südhof TC, De Camilli P, Jahn R (1993) Synaptotagmin: a membrane constituent of neuropeptide-containing large dense core vesicles. J Neurosci 13:38953903[Abstract]
Whitnall MH, Castel M, Key S, Gainer H (1985a) Immunocytochemical identification of dynorphin-containing vesicles in Brattleboro rats. Peptides 6:241247[Medline]
Whitnall MH, Gainer H, Cox BM, Molineaux CJ (1985b) Dynorphin-A-(18) is contained within vasopressin neurosecretory vesicles in rat pituitary. Science 222:11371139
Zhang X, Äman K, Hökfelt T (1995) Secretory pathways of neuropeptides in rat lumbar dorsal root ganglion neurons and effects of peripheral axotomy. J Comp Neurol 352:481500[Medline]