The prototypic mineralocorticoid receptor agonist aldosterone influences neurogenesis in the dentate gyrus of the adrenalectomized rat

Brain Research 947 (2002) 290–293 www.elsevier.com / locate / bres

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The prototypic mineralocorticoid receptor agonist aldosterone influences neurogenesis in the dentate gyrus of the adrenalectomized rat Anja K. Fischer a , Philipp von Rosenstiel b , Eberhard Fuchs a , Daniel Goula b , ´ Czeh ´ a ,* Osborne F.X. Almeida b , Boldizsar a

¨ , Germany Division of Neurobiology, German Primate Center, Kellnerweg 4, 37077 Gottingen b Max Planck Institute of Psychiatry, 80804 Munich, Germany Accepted 6 May 2002

Abstract Glucocorticoid receptor activation inhibits granule cell proliferation in the hippocampus, but little is known about the role of mineralocorticoid receptors in this process. Here we administered aldosterone to adrenalectomized (ADX) rats, and monitored neurogenesis by BrdU immunohistochemistry. ADX significantly increased the number of BrdU-positive cells and aldosterone replacement further augmented BrdU-positivity. Our results indicate that aldosterone, most probably acting through mineralocorticoid receptors, may positively influence the proliferation and survival of newly-generated granule cells.  2002 Elsevier Science B.V. All rights reserved. Theme: Development and regeneration Topic: Hormones and development Keywords: Adrenalectomy; Aldosterone; Neurogenesis; Hippocampus; Dentate gyrus

New granule neurons are produced throughout adulthood in the dentate gyrus of rodents, non-human and human primates [1,7,13] under the control of a number of internal and external factors [8]. For example, it is now well documented that elevated glucocorticoid hormone levels potently suppress adult hippocampal neurogenesis [3,5], whereas removal of adrenal steroids by adrenalectomy (ADX) stimulates this process [3]; interestingly, ADX also leads to increased degeneration of dentate granule cells [15]. ADX-induced cell death can be attenuated by stimulating the mineralocorticoid receptor (MR) [17], whose presence and occupation appears to be essential for the generation and survival of granule cells [9,16]. To further explore the role of mineralocorticoids in

*Corresponding author. Tel.: 149-551-3851-134; fax: 149-551-3851307. ´ E-mail address: [email protected] (B. Czeh).

granule cell proliferation, we investigated the influence of adrenalectomy and subsequent replacement with aldosterone on adult hippocampal cell proliferation, measured by bromodeoxyuridine incorporation. To determine the phenotype of the newly-generated cells we performed double-label immunocytochemistry using either a neuronal (TuJ1) or astrocytic (GFAP) marker. The experiments were conducted in accordance with NIH guidelines on the use of laboratory animals and in accordance with local statutory regulations. Male Wistar rats (Charles River) aged 8 weeks were group-housed under standard laboratory conditions, with food and water ad libitum. Two groups of animals were adrenalectomized under halothane anesthesia and maintained on 0.9% saline for a period of 1 month. Immediately after adrenalectomy, the animals were injected daily with either oil (ADX group, n57) or aldosterone (100 mg / kg, Sigma; ADX1 ALDO group, n56). A third group of sham-adrenalectomized (adrenal-intact) animals served as controls (Control,

0006-8993 / 02 / $ – see front matter  2002 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 02 )03042-1

A.K. Fischer et al. / Brain Research 947 (2002) 290 – 293

n57); this group received oil injections. All animals were injected with 5-bromo-29-deoxyuridine (BrdU, 50 mg / kg, Sigma) 7 days before sacrifice when animals were deeply anesthetized and perfused transcardially with 4% paraformaldehyde. Serial coronal 50-mm sections were collected throughout the septo-temporal extent of the hippocampal formation with the freezing microtome. Every sixth section was slide-mounted and coded before processing for immunocytochemistry to ensure objectivity. BrdU immunocytochemistry was performed according to our published protocol [6], consisting of pretreatment with 0.01 M citric acid (pH 6.0, 95 8C, 20 min), membrane permeabilization (0.1% trypsin, 10 min), and acidification (2 M HCl, 30 min), and incubation with mouse anti-BrdU (DAKO, 1:200), followed by the avidin–biotin / diaminobenzidine visualization method (Vector Laboratories); sections were finally counterstained with hematoxylin. All BrdU-labeled cells were counted under 3400 magnification regardless of size or shape. The number of BrdU-positive cells was assessed in the granule cell layer (gcl), the subgranular zone (sgz, defined as a two-cellbody-wide zone along the border of the granule cell layer) and the hilus. Every sixth section was analyzed and the total number of BrdU-labeled cells was estimated by multiplying the number of counted cells by 6. Additionally, the volume of the granule cell layer was measured on the basis of the Cavalieri principle using the Neurolucida system (MicroBrightField). Volumes are reported as mm 3 . For double-fluorescence labeling to evaluate the phenotype of the BrdU-positive cells, sections were treated as described above before overnight incubation with a monoclonal primary rat antibody against BrdU (1:50, Accurate Chemical), and antigen visualization with Cy5-conjugated rat secondary antibody (1:100, Jackson ImmunoResearch; 4 h) before incubation with either mouse monoclonal antibody against unique b-tubulin (TuJ1, 1:500, BAbCo) or rabbit polyclonal antibody against GFAP (1:600, Chemicon). After 24 h, sections incubated in anti-TuJ1 were incubated in Alexa 488-conjugated mouse secondary antisera (1:200, Molecular Probes) for 4 h, and those incubated in antisera against GFAP were incubated for 4 h in Cy3-conjugated rabbit secondary antisera (1:200, Jackson). To examine the phenotype of BrdU-labeled cells, fluorescence double-labeling for the neuronal marker TuJ1 was verified using a confocal laser scanning microscope (BioRad MRC 1024 ES System attached to a Nikon Eclipse TE 300 microscope). A minimum of 25 BrdU-labeled cells per brain were analyzed and the number of double-labeled cells is expressed as a percentage of all BrdU-labeled cells. Results are given as the mean6S.E.M. Treatment effects were assessed with one-way ANOVA followed by Newman-Keuls post-hoc analysis for further examination of group differences. To ascertain the efficacy of adrenalectomy we compared

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thymus weights between the various groups of animals, since thymus weight serves as a sensitive index of adrenocortical activity [14]. ADX and ADX1ALDO-treated animals showed significantly higher thymus weights as compared to intact controls (data not shown). Fig. 1 displays the total number of BrdU-positive cells of the three experimental groups. Adrenalectomy significantly increased the number of BrdU-positive cells both in the granule cell layer (170%; q53.49, P,0.05) and hilus (1168%; q56.70, P,0.001). In the subgranular zone, the number of BrdU-labeled cells was mildly increased (111%, not significant). Replacement of aldosterone to adrenalectomized rats further increased the number of BrdU-positive cells in all layers: an additional 186% increase in the granular cell layer (q56.97, P,0.001), 187% in the subgranular zone (q57.39, P,0.001), and 186% in the hilus (q58.83, P,0.001). The volume of the granule cell layer was used to assess whether the ADX-induced granule cell loss and / or the increased rate of cell proliferation resulted in gross volumetric changes of the dentate gyrus. Only a mild reduction in volume of the gcl (in mm 3 ) was observed after ADX: Control: 1.6060.06; ADX: 1.4360.08, representing a nonsignificant 11% volume decrease. ALDO replacement resulted in a tendency to normalization: ADX1ALDO: 1.4760.06, representing a non significant 3% increase in volume compared to the ADX group. To determine the phenotype of the newly-born cells, double labeling was performed with BrdU and with either the neuronal marker TuJ1 or the astrocyte-specific marker GFAP (Fig. 2). At 7 days after BrdU injection, |50% of BrdU-labeled cells in the gcl and sgz were immunoreactive for TuJ1, but no such cells were found in the hilus (Table 1); less than 10% of all BrdU-positive cells in the

Fig. 1. Effects of adrenalectomy (ADX) and subsequent replacement with aldosterone (ADX1ALDO) on the number of BrdU-incorporating cells in the adult rat hippocampal dentate gyrus. The number of newly-proliferated cells (BrdU-labeled cells) in each, the granule cell layer, the subgranular zone, and hilus, were counted separately. ADX increased the number of BrdU-labeled cells in all subregions, and aldosterone substitution enhanced this effect further. Results are given as the mean number of BrdU-positive cells6S.E.M. *P,0.05, **P,0.01, *** P,0.001, versus Control; [[P,0.01, [[[P,0.001 versus ADX.

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Fig. 2. Co-localization of BrdU immunoreactivity with either a marker for immature and mature neurons TuJ1 (A) or with the astrocyte marker GFAP (B). (A) Newly generated granule cells as indicated by the co-expression of BrdU with the neuronal marker TuJ1 (arrowheads: BrdU, red; TuJ1, green). (B) Double staining with BrdU (red) and the astrocyte marker GFAP (blue) resulted in some cells expressing both markers (arrowhead), which are newly generated astrocytes, or cells that showed labeling with BrdU only (red), that maybe putative neurons. Scale bar: 10 mm. gcl, granule cell layer; str mol, stratum moleculare.

hippocampal formation showed GFAP immunoreactivity (Table 1). A recent study by Cameron and McKay [4] in young adult rats suggested that |9000 new cells are generated in the dentate gyrus each day, i.e. more than 250,000 per month. On the other hand, many newly-generated cells reportedly die within 2 weeks after their birth [12]. It has also been documented that the continuous generation of new neurons is counterbalanced by an accompanying cell death, suggesting a substantial turnover of dentate granule cells; remarkably, however, the number of newly-generated cells is considerably greater than that of the dying cells [2]. In line with previous studies, we here report that removal of circulating glucocorticoids by ADX stimulated neurogenesis in the dentate gyrus [3,10], but produced even greater neuronal degeneration, as indicated by the reduced volume of the granule cell layer. We also show that aldosterone substitution to ADX animals results in a pronounced increase in the number of BrdU-labeled cells. Table 1 Percent cells colabeled with BrdU and neuronal (TuJ1) or astrocytic (GFAP) cell-specific markers TuJ1

Control, % ADX, % ADX1ALDO, %

GFAP

gcl

sgz

Hilus

gcl

sgz

Hilus

4863 5165 5266

5064 4764 5367

061 062 061

161 161 262

161 362 362

862 964 1066

gcl, granule cell layer; sgz, subgranular zone.

As with ADX, a modest decrease in the volume of the granule cell layer was also found. Our observations show that aldosterone, most probably acting through mineralocorticoid receptors, positively influences the proliferation rate and / or enhances the survival of newly-born granule cells. Since immature granule cells reportedly do not express corticosteroid receptors [11], it is likely that MR localized in neighboring mature cells activate biochemical pathways which may contribute to the increased generation and better survival of newly-born granule cell. Since GR activation has been previously suggested to inhibit neurogenesis in the dentate gyrus, another possibility that must be considered is that the increased cell proliferation in ADX animals may, at least partially, result from the reduced occupancy of glucocorticoid receptors (GR) in the ADX animals. In summary, these observations provide new information showing that activation of mineralocorticoid receptors can contribute to the process of cell acquisition and maintenance in the granule cell layer of the adult rat hippocampus.

Acknowledgements A.K.F. was supported by a grant of the European DFG Graduiertenkolleg ‘Neuroplasticity: from Molecules to Systems’. B.C. was supported by a grant of the Bundes¨ Bildung, Wissenschaft, Forschung and ministerium fur Technologie (0311467B9), and D.G. by a Marie Curie Fellowship from the European Union (QLGA-CT-2000-

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51268). This work was partly supported by a grant from the European Union (QLG3-2000-0084).

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