Development of Quantitative Methods for the Evaluation of the Entity of Coexistence of Neuroactive Substances in Nerve Terminal Populations in Discrete Areas of the Central Nervous System: Evidence for Hormonal Regulation of Cotransmission
We have recently developed several methods for the quantitative evaluation of coexistence of neuroactive substances in nerve cell bodies, namely the overlap method and the occlusion method (Agnati et al. 1982, Agnati et al. 1983, Fuxe et al. 1982, 1983, Agnati et al. 1984). In the overlap method the image analyzer can record the position and the perimeter of each nerve cell body visualized by means of the antiserum anti A in a Cartesian plan. Using an adjacent thin section or the same section following mild elution of anti A immunoreactivity the staining against antibody B is performed. The image analyzer then again records the position and the perimeter of the nerve cell bodies showing B immunoreactivity in the same Cartesion plan as used for the demonstration of anti A immunoreactivity. The positions in the Cartesian plan of the neurons are then compared by the computer and coexistence is considered to take place when an anti-A immuno- reactive area and an anti-B immuno reactive area overlap by at least 30 %. Thus, a 30 % overlap is considered to be a threshold value below which coexistence is not considered to exist. When using thin adjacent sections a correction factor must be calculated since some A and B immuno re active nerve cell bodies disappear as they move from one section to the other. Provided a mild elution technique can be used such as electrophoretic elution safe values of coexistence can be obtained with the overlap method. This method can also be performed using a binary coding of the images to reach the quantitative evaluation of three sets: the anti-A positive population, the anti-B positive population and the (anti-A) plus (anti-B) positive population, i.e. the unitary set. (see Jonsson this Symposium). The advantages of the overlap method is its ability to allow the assessment of coexistence of neuro- active substances in each individual nerve cell body of a nerve cell body population, (see Agnati et al. 1983a, 1984a). However, this method has the disadvantage of not being able to be used for studies on coexistence in nerve terminal populations.
KeywordsSucrose Dopamine Adrenaline Catecholamine Sine
Unable to display preview. Download preview PDF.
- Agnati, L.F., Fuxe, K., Locatelli, V., Benfenati, F., Zini, I., Panerai, A.E., El Etreby, M.F., Hökfelt, T. (1982). Neuroanatomical methods for the quantitative evaluation of coexistence of transmitters in nerve cells. Analysis of the ACTH- and beta- endorphin immunoreactive nerve cell bodies of the mediobasal hypothalamus of the rat. J Neurosci Methods 5, 203–214.PubMedGoogle Scholar
- Agnati, L.F., Fuxe, K., Benfenati, F., Calza, L., Battistini, N., Ögren, S.O. (1983a). Receptor-receptor interactions: possible new mechanisms for the action of some antidepressant drugs. In: Frontiers in Neuropsychiatric Research. (eds. E. Usdin, M. Goldstein, A.J. Friedhoff & A. Georgotas). MacMillan Press.Google Scholar
- Agnati, L.F., Fuxe, K., Benfenati, F., Battistini, N., Härfstrand, A., Tatemoto, K., Hökfelt, T., Mutt, V. (1983b). Neuropeptide Y in vitro selectively increases the number of a2-adrenergic binding sites in membranes of the medulla oblongata of the rat. Acta Physiol Scand., 118, 293–295.CrossRefPubMedGoogle Scholar
- Agnati, L.F., Fuxe, K., Benfenati, F., Zini, I., Zoli, M., Fabbri, L., Härfstrand, A. (1984a). Computer assisted morphometry and microdensitometry of transmitter-identified neurons with special reference to the mesostriatal dopamine pathway. I. Methodological aspects. Acta Physiol Scand., Suppl. 532, 5–32.Google Scholar
- Agnati, L.F., Fuxe, K., Giardino, L., Calza, L., Zoli, M., Battistini, N., Benfenati, F., Vanderhaeghen, J.J., Guidolin, D., Ruggeri, M., Goldstein, M. (1984b). Evidence for cholecystokinin- dopamine receptor interactions in the central nervous system of the dult and old rat. Studies on their functional meaning. New York Academy paper, in press.Google Scholar
- Fuxe, K., Agnati, L.F., Ganten, D., Lang, R., Calza, E., Poulsen, K., Infantellina, F. (1982). Morphometrical evaluation of the coexistence of renin-like and oxytocin-like immunoreactivity in nerve cells of the paraventricular hypothalamic nucleus of the rat. Neurosci Lett 33, 19–24.CrossRefPubMedGoogle Scholar
- Fuxe, K., Agnati, L.F., Andersson, K., Calza, L., Benfenati, F., Zini, I., Battistini, N., Köhler, C., Ögren, S.O., Hökfelt, T. (1983). Analysis of transmitter-identified neurons by morphometry and quantitative microfluorimetry. Evaluation of the actions of psychoactive drugs, especially sulpiride. In: Special aspects of psychopharmacology (eds. M. Ackenheil & N. Matussek), pp. 13–32, Espansion scientifique francaise, Paris.Google Scholar
- Fuxe, K., Gustafsson, J-Å., Agnati, L.F., Härfstrand, A., Yu, Z.Y., Wikström, A.C., Wrange, Ö, Zoli, M. (1984). Mapping out of glucocorticoid receptor immunoreactive neurons in the rat tel-and diencephalon using a monoclonal antibody against rat liver glucocorticoid receptor. Endocrinology, submitted.Google Scholar
- Goldstein, M., Lew, J.Y., Matsumoto, Y., Hokfelt, T., Fuxe, K. (1978). Localization and function of PNMT in the central nervous system. In: Psychopharmacology: A Generation of Progress (eds. M.A. Lipton, A. DiMascio & K.F. Killam. Raven Press, New York.Google Scholar
- Hökfelt, T., Skirboll, L., Rehfeld, J.F., Goldstein, M., Markey, K., Dann, O. (1980). A subpopulation of mesencephalin dopamine neurons projecting to limbic areas contains a cholecystokinin-like peptide: Evidence from immunohistochemistry combined with retrograde tracing. Neurosci 5, 2093–2124.CrossRefGoogle Scholar
- De Robertis E., Pellegrino de Iraldi, A., Rodrigues de Lores Arnaiz, G., Salganicoff L. (1969). Electron microscope observations on nerve endings isolated from rat brain. Anat Rec 139, 220.Google Scholar
- Sternberger, L.A. (1979). Immunocytochemistry. 2nd ed., Wiley, New York.Google Scholar