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Copyright © 2001 by Institute of Pharmacology Polish Academy of Sciences Polish Journal of Pharmacology Pol. J. Pharmacol., 2001, 53, 681684 ISSN 1230-6002 PRELIMINARY COMMUNICATION SEARCH FOR THE PRESENCE OF GLUCOCORTICOID RECEPTORS IN DOPAMINERGIC NEURONS OF RAT VENTRAL TEGMENAL AREA AND SUBSTANTIA NIGRA Anna Czyrak, Agnieszka Chocyk Laboratory of Pharmacology and Brain Biostructure, Institute of Pharmacology, Polish Academy of Sciences, Smêtna 12, PL 31-343 Kraków, Poland Search for the presence of glucocorticoid receptors in dopaminergic neurons of rat ventral tegmenal area and substantia nigra. A. CZYRAK, A. CHOCYK. Pol. J. Pharmacol., 2001, 53, 681–684. Using non-fluorescent immunocytochemical double-labelling procedure and specific antibodies visualizing GR (glucocorticoid receptors) and TH (tyrosine hydroxylase) we have been looking for the co-localization of both antigens in neurons of the rat ventral tegmental area and adjacent substantia nigra. This experimental direction has been inspired by the available data showing that alterations in the level of circulating glucocorticosteroids have distinct effects on the intensity of dopaminergic neurotransmission. Thus, it was of interest to find the anatomical background for the above interaction. It has been found that the rat ventral tegmental area and substantia nigra possess a relatively moderate number of cells with active GR, i.e. receptors which are condensed in the nuclei. Further, we found that dopaminergic neurons (TH-positive) of the ventral tegmental area and substantia nigra were not immunopositive for GR. This observation was in the sharp contrast to the results from the locus coeruleus, where the co-localization of GR with TH was a general rule. Above anatomical data indicate that glucocorticoid receptors influence the dopaminergic neurotransmission by an indirect mechanism, which possibly involves intermittent neurotransmitter. Key words: double-labelling, glucocorticoid receptors, locus coeruleus, substantia nigra, tyrosine hydroxylase, ventral tegmental area correspondence; e-mail: [email protected] A. Czyrak, A. Chocyk Glucocorticoids operating via their receptors which are hormone-activated transcription factors exert remarkable influence on various neurotransmitters in the central nervous system [4]. Above effects involve alterations of neurotransmitters synthesis and release, changes in synthesis of specific proteins (enzymes and receptors), apart from alterations of the anatomical shape of certain populations of hippocampal neurons [1, 2, 3, 4, 7]. In this respect, several arguments indicate that glucocorticoids, or specifically alterations in their circulating concentrations, exceeding their physiological levels, have profound effects on the dopaminergic neurotransmission. For example, it has been observed that corticosterone, operating via GR alters the turnover rate and release of dopamine, evokes changes in the density of dopaminergic receptors of D1 subtype, with the subsequent alteration in their mRNA level [1, 3, 7]. It has also been found that corticosterone alters the responsiveness of animals to addictive psychostimulants, which are acting via dopaminergic systems [8]. In spite of the above neurochemical and behavioral arguments for the interaction between glucocorticosteroids and dopaminergic neurotransmission, the cellular mechanism of this interaction is not fully understood. Therefore, in the present study we investigated whether corticosterone receptors of GR subtype are present in the dopaminergic cell bodies of the rat ventral tegmental area and adjacent substantia nigra, the two major groups of dopaminergic neurons forming mesolimbic, mesocortial and mesostriatal dopaminergic systems. This experimental direction was based on the assumption that presence of GR, which operates as transcripctional factors [4, 5] in dopaminergic neurons may provide anatomical evidence that corticosterone via GR may directly modify the function of dopaminergic cells and subsequently the intensity of dopaminergic neurotransmission. All the experiments were carried out on male Wistar rats (200–250 g) housed in groups (6 animals per cage) under an artificial light/dark cycle (12/12 hours, light on at 07:00. Control (vehicle-treated) and corticosterone-injected (10 mg/kg sc) rats were deeply anesthetized (1 h after the treatment) with sodium pentobarbital (100 mg/kg) and were transcardially perfused with saline (NaCl, 0.9%) followed by an ice cold 4% paraformaldehyde in a 0.1 M PBS (phosphate buffered saline). Following a 4-hour postfixation period (4% para- 682 A c VTA SN r B C D Fig. 1. Photomicrographs of the rat ventral tegmental area and substantia nigra stained for the presence of TH (brown) and ) GR (black), ( ) low power magnification (objective 4x) with marked areas presented in high power magnification photomicrographs (objective 40x) from substantia nigra pars compacta * ( + ), substantia nigra pars reticulata ( , area ( ) and ventral tegmental ); arrows neurons positive for the presence of TH, arrowheads neuronal nuclei GR-immunopositive. Abbreviations: VTA ventral tegmental area; SN substantia nigra, c pars compacta, r pars reticulata Pol. J. Pharmacol., 2001, 53, 681684 GLUCOCORTICOID RECEPTORS AND DOPAMINERGIC NEURONS ) Fig. 2. Photomicrographs of the rat locus coeruleus stained for the presence of TH (brown) and GR (black), ( ) low power magnifica- * tion (objective 10x) with marked area presented in (objective 40x) high power magnification photomicrograph ( ), TH-positive lo- cus coeruleus neurons (cytoplasm) are also immunopositive for the presence of GR receptors (nucleus). Abbreviations: LC locus coeruleus, 4v fourth ventricle formaldehyde in 0.1 M PBS, as a fixative), 50 mm thick sections were cut at the level of substantia nigra, ventral tegmental area and locus coeruleus, using a vibratome (Technical Products International, USA). The sections were rinsed extensively in 0.05 M TBS (Tris buffered saline) and then incubated for 1 h in blocking buffer containing 3% normal goat serum (Vector Lab., USA), 0.1% bovine serum albumin in TBS. Then the sections were incubated with primary polyclonal rabbit antibody (Affinity Bioreagents INC., USA) against 346–367 amino acids from N-terminus of human GR, diluted at 1:1000 in the blocking buffer (48 h at 4°C). After incubation and several washes, the sections were incubated (1 h) with biotinylated, secondary goat anti-rabbit antibody (Vector Lab.). The final incubation of the sections with a solution of the avidin-biotin horseradish peroxidase complex (Vector Lab.) was preceded by the 30 min incubation of sections in 0.6% H2O2. The reaction was visualized by nickel-glucose oxidase-diaminobenzidine method, giving the black color of staining which was in a good contrast to the brown deposits of TH (see below). After washing in 0.01 M PBS, free-floating sections were incubated for 60 min in a blocking buffer containing 2% normal horse serum, 0.2% ISSN 1230-6002 Triton X-100, and 0.1% bovine serum albumin in 0.01 M PBS. Then the sections were incubated (48 h, 4°C, continuos agitation) with primary monoclonal antibody, recognizing the TH (Chemicon, USA) diluted at 1:5000 in blocking buffer. After rinsing, the sections were incubated for 1 h with biotinylated anti-mouse, IgG (rat adsorbed) antibody (Vector Lab.) diluted in the blocking buffer. The reaction was visualized by incubation with avidin-biotin horseradish peroxidase complex (Vectastain Elite ABC Kit, Vector Lab.) and developed by diaminobenzidine-hydrogen peroxide reaction, which produced the brown staining of immunopositive material. It has been found that GR are present in the ventral tegmental area and adjacent substantia nigra (both, pars compacata and pars reticulata) as well as in the locus coeruleus. The GR protein was present in the cell nuclei, which indicates that our antibody visualized the active form of GR, i.e. receptors which are activated by corticosterone and translocated to the nucleus [4, 5]. We observed a similar degree of nuclear condensation of immunopositive material in naive and corticosterone-treated animals which suggests that in the studied brain regions, the vast majority of GR, are in the active 683 A. Czyrak, A. Chocyk form already in physiological conditions. The size of GR-immunopositive nuclei varied from relatively large ones in the locus coeruleus to large ones and moderately small in the substantia nigra and ventral tegmental area. Size of stained nuclei may indicate that in the locus coeruleus, GR are present mainly in neurons, whereas in the ventral tegmental area and substantia nigra they are present in neurons and glia cells in comparable amounts. The monoclonal antibody visualizing TH showed a typical pattern of distribution of this enzyme in all brain regions tested [5]. The double-labelling procedures revealed that TH-positive neurons of both ventral tegmental area and substantia nigra did not show GR-immunopositive material. Above data are in sharp contrast to the data collected from the locus coeruleus, which has been used as a positive control, where we observed a clear co-localization of GR with TH. In conclusion, the obtained results indicate that dopaminergic neurons of the rat ventral tegmental area and substantia nigra, i.e. neurons which contain enzyme TH, are not immunopositive for GR, although these receptors are present in above brain regions both in neurons and also glia cells. The present anatomical data indicate that the impact of glucocorticoids on dopaminergic neurotransmission is not a direct one, but possibly involves the intermittent neurotransmitter that tunes the activity of dopaminergic neurons. It will be of importance to analyze which neurotransmitter is involved in the above effects. Among the possible candidates, glutamatergic system should be taken into consideration, which innervates the ventral tegmental area and substantia nigra, originating in the cortex (among other structures). Interestingly, we observed that almost all pyramidal neurons, i.e. glutamatergic neurons in the cortex, contain GR immunoreactivity (our unpublished observations). Moreover, alterations in the glutamate outflow in the ventral tegmental area have been linked with the phenomenon of sensitization to psychostimulants [6]. Finally, it has been also found that release of glutamate is controlled by dopamine D1 receptors which are localized on the glutamatergic terminals originating in the cortex [9, 10]. Density of D1 dopamine receptors is controlled by glucocorticoids, what was 684 shown by our previous experiments [3]. Additional important information obtained in the present study is that the appearance of GRE sequences (glucocorticoid response element) in the promoter of TH gene does not indicate that GR may directly influence TH expression in every single neuron. It is apparent from our study that direct interaction at the level of single neuron would depend on the type of neurotransmitter and brain structure. REFERENCES 1. Biron D., Dauphin C., Di Paolo T.: Effects of adrenalectomy and glucocorticoids on rat brain dopamine receptors. Neuroendocrinology, 1992, 55, 468–476. 2. Brown E.S., Rush A.J., McEwen B.S.: Hippocampal remodeling and damage by corticosteroids: implications for mood disorders. Neuropsychopharmacology, 1999, 4. 474–484. 3. Czyrak A., Wêdzony K., Michalska B., Fija³ K., Dziedzicka-Wasylewska M., Maækowiak M.: The corticosterone synthesis inhibitor metyrapone decreases dopamine D1 receptors in the rat brain. Neuroscience, 1997, 79, 489–495. 4. de Kloet E.R., Reul J.M., Sutanto W.: Corticosteroids and the brain. J. Steroid Biochem. Mol. Biol., 1990, 37, 387–394. 5. Härfstrand A., Fuxe K., Cintra A., Agnati L.F., Zini I., Wikstrom A.C., Okret S., Yu Z.Y., Goldstein M., Steinbusch H.: Glucocorticoid receptor immunoreactivity in monoaminergic neurons of rat brain. Proc. Nat. Acad. Sci. USA, 1986, 83, 9779–9783. 6. Kalivas P.W., Duffy P.: Repeated cocaine administration alters extracellular glutamate in the ventral tegmental area. J. Neurochem., 1998, 70, 1497–1502. 7. Piazza P.V., Le Moal M.: Glucocorticoids as a biological substrate of reward: physiological and pathophysiological implications. Brain Res. Rev., 1997, 25, 359–372. 8. Przegaliñski E., Filip M., Siwanowicz J., Nowak E.: Effect of adrenalectomy and corticosterone on cocaine-induced sensitization in rats. J. Physiol. Pharmacol., 2000, 51, 193–204. 9. Vezina P.: D1 dopamine receptor activation is necessary for the induction of sensitization by amphetamine in the ventral tegmental area. J. Neurosci., 1996, 16, 2411–2420. 10. Wêdzony K., Czyrak A.: Presence and function of dopamine D1 receptors in the rat ventral tegmental area. Pol. J. Pharmacol., 1997, 49, 277–281. Received: October 25, 2001; in revised form: November 20, 2001. Pol. J. Pharmacol., 2001, 53, 681684