Agedependent expression of glucocorticoid and ... - Wiley Online Library

Aging Cell (2004) 3, pp363–371

Doi: 10.1111/j.1474-9728.2004.00130.x

Age-dependent expression of glucocorticoid- and mineralocorticoid receptors on neural precursor cell populations in the adult murine hippocampus

Blackwell Publishing, Ltd.

Ana Garcia,1,3 Barbara Steiner,2 Golo Kronenberg,1,3 Anika Bick-Sander2 and Gerd Kempermann1,2 1

Max Delbrück Center for Molecular Medicine (MDC) Berlin-Buch, Robert-Rössle-Str. 10, 13125 Berlin, Germany 2 Department of Neurology, Charité University Medicine Berlin, Schumannstr. 20/21, 10117 Berlin, Germany 3 Department of Psychiatry, Charité University Medicine Berlin, Eschenallee 3, 14050 Berlin, Germany

Summary Steroid hormones are regulators of adult hippocampal neurogenesis and are central to hypotheses regarding adult neurogenesis in age-related and psychiatric disturbances associated with altered hippocampal plasticity – most notably dementias and major depression. Using immunohistochemistry, we examined the expression of glucocorticoid (GR) and mineralocorticoid (MR) receptors during adult hippocampal neurogenesis. In young mice only 27% of dividing cells in the subgranular zone expressed GR, whereas 4 weeks after division 87% had become positive for GR and MR. GR was expressed by 50% of the radial glia-like type-1 and type-2a progenitor cells, whereas MR was expressed only by mature calbindin-positive granule cells. Doublecortin-positive neuronal progenitor cells (type-2b) and early postmitotic calretinin-positive neurons were devoid of GR and MR expression. Fifty per cent of the intermediate type-3 cells showed GR expression, possibly reflecting cells terminating maturation. Thus, all subpopulations of dividing precursor cells showed an identical receptor profile (50% GR, no MR), except for type-2b cells, which expressed neither receptor. There was also no overlap between calretinin and GR early postnatally (P8) or after physical activity or exposure to an enriched environment, both of which are potent neurogenic stimuli. In contrast, in old age calretinin-positive young neurons became GR and MR positive, suggesting increased steroid sensitivity. Age also increased the expression of GR in type-1 and type-2a precursor cells. Other intermediates were so rare in old age that they could not be studied. This course and variability of receptor

Correspondence Dr Gerd Kempermann, Max Delbrück Center for Molecular Medicine (MDC), Berlin-Buch, Robert-Rössle-Str. 10, 13125 Berlin, Germany. Tel.: +49 30 94062362; fax: +49 30 94063814; e-mail: [email protected] Accepted for publication 30 July 2004 © Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland 2004

expression in aging might help to explain differential vulnerability of adult neural precursor cells to corticoidmediated influences. Key words: corticosterone; cortisol; neurogenesis; progenitor cell; stem cell; stress.

Introduction Age is the strongest known negative regulator of adult hippocampal neurogenesis, but most of the age-dependent decline in the number of newly generated neurons occurs very early in the life of a rodent (Altman & Das, 1965; Kuhn et al., 1996; Kempermann et al., 1998; Cameron & McKay, 1999; Bizon & Gallagher, 2003). Because adult hippocampal neurogenesis adds new granule cells to the dentate gyrus (the first relay station within the essentially trisynaptic circuit of the hippocampus), and does so in an activity-dependent way, relevance to hippocampal function is now generally assumed for the newly generated neurons (Kempermann et al., 2004b). The new cells become electrophysiologically indistinguishable from older granule cells (van Praag et al., 2002), but in their earliest stage they are particularly sensitive to the induction of long-term potentiation, the assumed electrophysiological equivalent of learning (Wang et al., 2000; Schmidt-Hieber et al., 2004). Our hypothesis is that adult hippocampal neurogenesis allows a cumulative optimization of the mossy fibre tract connecting the dentate gyrus to area CA3 (Kempermann, 2002; Kempermann & Wiskott, 2004). In rodents, the dentate gyrus grows with increasing age (Altman & Das, 1965; Bayer et al., 1982; Boss et al., 1985), but most of the growth occurs relatively early in life, when levels of neurogenesis are highest. In old age, levels of adult neurogenesis are very low, but can be induced strongly by environmental stimuli, such as experiences of complexity and novelty (‘enriched environment’) (Kempermann et al., 1998). It seems that in old age, the dentate gyrus, faced with a novel situation, mobilizes its entire remaining potential for neurogenesis in order to meet some as yet unknown functional needs. It therefore seems that even the low levels of hippocampal neurogenesis in old individuals are functionally relevant. In humans, adult hippocampal neurogenesis has been detected in subjects as old as 72 years (Eriksson et al., 1998). Corticosteroids have been identified as having strong negative regulatory effects on adult hippocampal neurogenesis (Cameron & Gould, 1994; Gould et al., 1997; Cameron et al., 1998; Cameron & McKay, 1999; Montaron et al., 1999, 2003). Adrenalectomy, which removes endogenous corticosterone, restores or maintains neurogenesis in aging rats to levels corresponding to an earlier age (Cameron & McKay, 1999). 363

364 GR and MR in adult hippocampal neurogenesis, A. Garcia et al.

However, an enriched environment in old age has a similar positive effect on adult neurogenesis in mice, despite elevated corticosterone levels (Kempermann et al., 2002). Similarly, data on more acute regulation are difficult to interpret. Acute and strong stress, which dramatically increases corticosterone levels, reduces neurogenesis (Gould et al., 1997), but physical activity, which also is associated with higher serum corticosterone (Makatsori et al., 2003; Adlard & Cotman, 2004), leads to more new neurons in the dentate gyrus (van Praag et al., 1999). Consequently, there does not seem to be a simple relationship between corticosterone levels and adult neurogenesis (Heine et al., 2004). This is of particular consequence in those cases where a dysregulation or failure of adult hippocampal neurogenesis may be associated with a pathogenic mechanism. This applies not only to dementias and major depression, but also to the more general cognitive decline during aging, which is not attributable to a defined clinical entity. Cameron & McKay (1999) suggested that the chronically elevated corticosterone levels during aging might be detrimental to precursor cell function in the adult dentate gyrus and may reduce adult neurogenesis. To reconcile this hypothesis with the other partially conflicting evidence we set out to study the expression of corticosterone receptors during adult hippocampal neurogenesis. There has been one study with this goal before, from the same group (Cameron et al., 1993). At that time, however, little was known about the heterogeneity of proliferating cells in the adult dentate gyrus and the time-course of neuronal development in the adult. Our hypothesis was that heterogeneity in receptor expression in the course of neuronal development in the adult and aging hippocampus might help to explain the seemingly contradictory findings regarding how glucocorticoids are involved in the control of adult hippocampal neurogenesis. We have previously identified stages of neuronal development in the adult hippocampus (Kempermann et al., 2004a), presumably originating from a radial glia-like cell, identified by Seri et al. (2001) as the stem cell of this brain region. This type-1 cell has numerous astrocytic features (Filippov et al., 2003) and probably gives rise to a population of transiently amplifying progenitor cells. Dependent on the expression pattern of nestin [detected with green fluorescent protein, GFP, under the nestin promoter in a transgenic mouse model (Yamaguchi et al., 2000)] and doublecortin [DCX (Brown et al., 2003)], the following cell types can be distinguished: type-2a (nestin-GFP-positive, but DCX-negative), type-2b (nestin-GFP-positive and DCX-positive) and type-3 (nestin-GFP-negative, but DCX-positive). Numerical expansion occurs at the level of progenitor cells (type-2 and -3), not in the radial glia-like type-1 cells (Kronenberg et al., 2003). Early postimitotic cells transiently express calretinin that is later replaced by calbindin (Brandt et al., 2003). During the calretininpositive stage the newly generated cells are recruited into the local neuronal network or otherwise are eliminated by apoptotic mechanisms (Biebl et al., 2000). The action of corticosterone is mediated through two types of receptors, the glucocorticoid receptor (GR) and the mineralocorticoid receptor (MR). The MR has a higher affinity to corticosterone

than the GR, and in the brain is primarily found in the limbic structures, including the hippocampus (Van Eekelen et al., 1988). The GR is expressed ubiquitously. The two receptors, which can dimerize, act as hormone-activated transcription factors. Whereas GR activation is involved in acute, for example stress-induced, suppressive effects, MR activation is thought to contribute to more tonic, long-term and permissive effects (Sapolsky et al., 2000). Generally, adrenalectomy, i.e. the removal of all endogenous corticosteroids, stimulates adult neurogenesis, suggesting that corticosterone inhibits neurogenesis and precursor cell activity (Cameron & Gould, 1994; Cameron & McKay, 1999). This effect can be counteracted by exogenous corticosteroids. Montaron et al. (2003) have shown that in adrenalectomized rats, MR stimulation is sufficient to restore overall cell proliferation, but stimulation of the GR is necessary to reconstitute the expression of the polysialated form of the neural cell adhesion molecule (PSA-NCAM), a molecule whose expression overlaps with DCX. In addition to many other open questions in this context, it is not known how far such observations reflect direct or indirect effects on precursor cells, and how potentially altered receptor expression due to the adrenalectomy might influence the results. We reasoned that during physiological regulation of adult neurogenesis a differential expression of GR and MR on precursor cells and during the postmitotic selection period might explain the complexity of corticosterone effects. The present study was designed to prepare the grounds for molecular investigations by immunohistochemically mapping GR and MR expression in the adult and aged mouse dentate gyrus. We focused on cells within the lineage of neuronal development and thus concentrated on putative direct effects.

Results GR and MR expression by proliferating cells in the dentate gyrus We first examined the expression of GR and MR on proliferative cells in the adult dentate gyrus at various points after cell division, independent of the exact nature of the dividing cell. Dividing cells were labelled with immunohistochemically detectable thymidine analogue bromodeoxyuridine (BrdU) and brain sections were studied at 4 h, 1 day, 3 days, 7 days and 4 weeks after a single injection of BrdU. Because BrdU is only incorporated into the DNA of a dividing cell during the S-phase of the mitosis, the 4-h value can be considered as roughly reflecting cells currently in division. The other time-points reflect different survival timepoints of the progeny of these initially dividing cells. Triple-channel immunohistochemistry for BrdU, GR and MR allowed the identification of nuclei expressing the receptors (Fig. 1). The fluorescent signal was not graded; the cells rather fell into categories of being labelled or not. Nevertheless, immunohistochemical detection of protein expression remains a qualitative statement that is based on thresholds set by the investigator. The time-course of MR and GR expression in BrdU-labelled cells is shown in Fig. 2. MR expression was first seen at 3 days after © Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland 2004

GR and MR in adult hippocampal neurogenesis, A. Garcia et al. 365

BrdU in about 4% of the BrdU-positive cells, and this increased to over 90% at 4 weeks. There were no MR-expressing cells at 4 h, suggesting that cells in the process of division do not express the MR. The GR in contrast was found on 13% of the dividing cells at 4 h after BrdU injection. No BrdU-positive cells expressing only the MR were found at any time-point. These data indicate that with increasing time after the initial cell division GR and MR expression increase. Mature cells (beginning as early as 3 days after division) are positive for both receptors.

Different stages of neuronal development are associated with different receptor profiles We next aimed to determine which cell types in the course of adult hippocampal neurogenesis corresponded to this profile based on the time after division. To provide a better description of neuronal development in the adult hippocampus, we have devised a schematic of six milestones (Kempermann et al., 2004a). Use of a transgenic mouse expressing GFP under the nestin promoter allowed us to identify morphologically putative precursor cells. Cells remain proliferative at different stages during development and thus might incorporate BrdU at each stage. BrdU-incorporating cells are not homogeneous. However, they progress through different developmental stages with increasing time after BrdU incorporation. These stages can be studied independently of BrdU incorporation. Combining this analysis with the markers DCX and PSA-NCAM (expressed in neuronal progenitor cells and immature neurons), calretinin (CR) (expressed in early postmitotic granule cells) and calbindin (expressed in mature granule cells) allows cells to be categorized according to the schematic outlined in the top half of Fig. 3 and explained in detail elsewhere (Brandt et al., 2003; Kronenberg et al., 2003; Kempermann et al., 2004a; Steiner et al., 2004). Note that this model and nomenclature is meant as a useful tool, not a definitive concept of neuronal development in the adult hippocampus. Combining the above markers with the detection of GR or MR yielded the data depicted in Fig. 3(A). In young mice, radial glia-like type-1 cells expressed GR in roughly 50% of the cases without any obvious pattern or spatial preference (Fig. 1B; all numbers are means ± standard error). This also applied to the nestin-GFP-positive type-2a cells. Type-2 cells constitute the largest fraction of dividing cells in the adult dentate gyrus. Type-2b cells, however, which differ from type-2a cells in their expression of DCX and PSA-NCAM, were never found to be GR- or MR-positive (Fig. 1C). Analysis of the receptor expression in type-2b and type-3 cells was based on the expression of PSA-NCAM and the absence or presence of nestin-GFP. DCX could not be used because both the DCX and the MR antibody are derived from rabbit. Roughly half of the type-3 cells (negative for nestin-GFP, but PSA-NCAMpositive) expressed GR but not MR. By the CR stage no cells expressed GR or MR. All CB-positive granule cells expressed GR. Only CB-positive granule cells expressed the MR. In accordance with the data obtained in the time-course study, we did not detect any MR expression during the proliferative stages. © Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland 2004

At early postnatal age, GR and MR expression on developing granule cells reflect the situation in the adult Around postnatal day 8 granule cell production peaks in the murine dentate gyrus and the transition from embryonic and early postnatal granule cell development to adult neurogenesis is underway. Apart from Joseph Altman and Shirley Bayer’s pioneering descriptions, no detailed analysis of this transition is as yet available (Altman & Bayer, 1990). A future study with nestin-GFP reporter gene mice will further address this issue; for the present context, we intended to examine whether the CR-positive stage of granule cell development at this age would be devoid of GR as well as of MR expression. At P8, we found no overlap between CR and GR or MR immunoreactivity in the dentate gyrus (Fig. 1). An interesting additional finding was the general abundance of GR expression at this stage of development. Also, note the gradient of GR and MR expression from no expression in the subgranular zone to full expression in mature cells, similar to the pattern found in the adult.

In old age, a shift of receptor expression towards less mature stages is found Overall levels of adult neurogenesis drastically decrease in old age (Kuhn et al., 1996; Kempermann et al., 1998; Cameron & McKay, 1999). Time course experiments with single injections of BrdU cannot be performed in old age, because the numbers of BrdUlabelled cells are too low. Nestin-GFP-expressing cells are more abundant than BrdU-labelled cells after single injections in old age. When we analysed GR and MR expression during adult hippocampal neurogenesis at the age of 16 or 20 months, the overall pattern seen in young animals was replicated. However, there were two important exceptions. The percentage of type-1 and type-2 cells expressing GR had increased to about 80%, and almost half of the CR-positive cells showed expression of both GR and MR (Figs 1 and 3). Owing to their very low abundance in the old mice, type-2b and type-3 cells could not be identified in sufficient numbers to permit an analysis of receptor expression. These results indicate a shift of both GR and MR expression towards more immature stages of neuronal development following neurogenesis in the adult hippocampus.

No shift of receptor expression is found with physical activity and environmental enrichment Both environmental enrichment and voluntary physical activity induce neurogenesis (van Praag et al., 1999; Kempermann et al., 2002), but can also be associated with elevated corticosteroid levels. We thus hypothesized that the lack of receptor expression on CR-positive neurons found in young–adult mice would not be affected by these experimental protocols. This was indeed the case: in mice with unlimited access to a running wheel for 7 days and in mice living in an enriched environment for 35 days (Steiner et al., 2004), no MR or GR expression was found in CR-positive cells.


Fig. 1 Glucocorticoid- and mineralocorticoid receptor expression during adult neurogenesis. (A) Proliferating cells in the dentate gyrus of mice express the glucocorticoid receptor (GR) but not the mineralocorticoid receptor (MR). At 4 h after BrdU injection BrdU-labelled, acutely dividing cells (red) are detected in the subgranular zone of the dentate gyrus. Some of these cells express the GR (arrow in insert, green), but none the MR (blue). Scale bar, 200 µm. (B) In nestin-GFP transgenic mice nestin-expressing cells are constitutively visualized. According to the scheme depicted in Fig. 3(A) (for details see Kempermann et al., 2004a), nestin-GFP- and PSA-NCAM-expressing cells can be separated into four categories. Roughly 50% of type-1, type-2a and type-3 cells express the GR, but type-2b cells are generally devoid of GR expression. Nestin-GFP: green; PSA-NCAM: red; GR: blue. Scale bar (in A), 20 µm for the large panel. (C) Analogous examination of MR expression in nestin-GFP-expressing cells showed that all progenitor cells are negative for MR. Nestin-GFP: green; PSA-NCAM: red; MR: blue. Scale bar (in A), 10 µm for the large panel. (D) Early postmitotic neurons express the calcium-binding protein calretinin (CR, red). In young mice, CR-positive cells in the subgranular zone (SGZ) (arrow) were all devoid of GR (green) and MR (blue) immunoreactivity. Scale bar (in A), 100 µm. (E) At postnatal day 8, CR-expressing cells (red) in hilus and SGZ are free of GR and MR immunoreactivity, whereas CR-positive cells in the molecular layer (upper right corner) are GR-positive (green). Granule cell maturation occurs in an inside–outside fashion with the most mature cells being located at the outer rim of the granule cell layer. This is reflected in the pattern of MR expression (blue), which is expressed late during neuronal development. The same pattern has been found for hippocampal neurogenesis beyond P21 (data of the present study; compare Figs 2 and 3). GR, in contrast, can be found at earlier stages of granule cell development and is thus detected in cells closer to the hilus. Scale bar (in A), 50 µm. (F) In contrast to (D) and (E), in old mice CR-positive cells (red) can express GR and MR (arrows). Scale bar (in A), 80 µm. © Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland 2004


Fig. 2 GR and MR expression at different time-points after BrdUincorporation. BrdU-positive cells in the subgranular zone of the dentate gyrus were counted whether they expressed the GR, MR or both. A, absolute numbers; B, relative numbers. No BrdU-positive cells expressed only MR. Most dividing cells were negative for both receptors, but with increasing time after BrdU incorporation the percentage of GR-expressing cells grew. GR- and MRpositive cells were first seen 3 days after BrdU incorporation. Their absolute number decreased between 3 days and 4 weeks.

Discussion We found that the expression of corticosteroid receptors on the precursor cells involved in adult hippocampal neurogenesis is not homogeneous, and with older age it increases overall and shifts to less mature cells in the maturational process that newly formed cells undergo. Thus, the calretinin-expressing early postmitotic neurons, which were devoid of GR or MR expression in younger mice, acquired receptor expression in aged animals. This might help to explain a putative increased sensitivity to corticosterone action at older ages. The mechanisms of this shift and the up-regulation in less mature cells remain unknown at present, but deserve to be studied. Generally, corticosteroid receptor expression underlies a feedback regulation, linking it tightly to the levels of the hormone itself (Kalman & Spencer, 2002). Increased hormone levels should thus lead to desensitization through a down-regulation of receptor expression. At the same time, GR expression is highly correlated with the biological effects of the hormone (Vanderbilt et al., 1987). If age causes chronically elevated corticosterone levels (Sapolsky, 1992) and receptor expression, the overall corticosterone effect on adult neurogenesis will increase (Cameron & McKay, 1999). The number of precursor cells in adult neurogenesis is expanded in an activity-dependent manner (Kronenberg et al., © Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland 2004

Fig. 3 GR and MR expression in putative precursor cells of the adult dentate gyrus. (A) As shown in Fig. 1, GR and MR expression varies during the proposed stages of development during adult hippocampal neurogenesis (all numbers are mean ± standard error). Type-2b and CR-stage cells are devoid of GR and MR expression in young mice. In old mice (16 months), GR and MR are also found at the CR stage. There were too few type-2b and type-3 cells in old mice to evaluate receptor expression in these cells. In general, a shift towards higher receptor expression at earlier stages of neuronal development was found. (B) We hypothesize that the DCX- and PSA-NCAMpositive stage during adult neurogenesis is essentially free of GR and MR expression. We propose that those cells at the type-3 stage that express the GR are those that will exit from development and are likely to be eliminated by apoptosis. In aged mice, CR-positive cells acquire GR and MR expression, which makes them more sensitive to corticosteroid action. This hypothesis remains to be tested.

2003). Cells from this expanded pool become CR-positive and begin to extend their dendrites and axons (Brandt et al., 2003). Of these only a subset is recruited into function and persists long-term (Kempermann et al., 2004a). Thus, the CR-positive stage of adult neurogenesis is an important phase of neuronal development during which most of the quantitative regulation occurs. Regulation of survival is activity-dependent but is underlaid by strong genetic control (Kempermann et al., 1997). It is conceivable that corticosteroid receptor expression is part of this


inherited predisposition. Because the acute effects of corticosteroids seem to be anti-neurogenic, lacking receptor expression of GR and MR during this phase would make regulation of neurogenesis at this stage independent of the direct effects of corticosteroids. Whereas in young mice the MR was only expressed on mature calbindin-positive granule cells but not during the preceding neuronal development, in old mice both MR and GR were found at the calretinin stage. In postnatal mice and under situations of neurogenic stimulation, as in young mice, no overlap of receptor expression was found. Consequently, owing to the increased receptor expression in old age during a vulnerable selection period of neuronal development, relatively more cells would become sensitive to anti-neurogenic influences. Removing steroids, for example through adrenalectomy, as in the experiment by Cameron & McKay (1999), would counteract this increased sensitivity by reducing the receptor ligand. Regulation of survival of the newborn cells, which is normally independent of direct corticosteroid action, would come under the influence of the hypothalamo–pituitary–adrenal (HPA) system, which uses corticosterone as its mediator. The HPA system is thought to be the major source of endocrine control in acute and chronic stress responses. The second suggestive finding in our study was the lack of corticosteroid receptor expression on type-2b cells. Type-2 cells are among the transiently amplifying progenitor cells during adult hippocampal neurogenesis, and respond strongly to, for example, physical activity. Their initial description was based on the expression of nestin (or to be precise the presence of GFP expressed under the nestin promoter in a transgenic reporter mouse model) and the absence of radial glia morphology (type1) (Filippov et al., 2003). However, it soon became obvious that type-2 cells are more heterogeneous. For example, depending on the expression of DCX, type-2a and type-2b cells can be distinguished (Kronenberg et al., 2003). This nomenclature is preliminary, because it is not clear whether type-2b cells are more closely related to other nestin-positive or to the other DCX-positive cells. Regardless, they represent an intermediate, which is characterized by the first appearance of neuronal characteristics in the developing cells (Brown et al., 2003; Kronenberg et al., 2003). DCX expression is associated with early neuronal features such as sodium currents, although many of the DCX-expressing cells are still proliferative. During the DCXpositive stage the cells become postmitotic, migrate and acquire a maturing morphology (Brandt et al., 2003; Kempermann et al., 2004a). DCX expression is maintained essentially throughout the remaining maturation stage and thus shows a large overlap with calretinin (Brandt et al., 2003). DCX plays an important role in neuronal migration, but possibly has other functions during neuronal development as well. Mutations in the DCX genes cause deficits in cortical migration and layer formation. Whereas proliferative type-3 cells are rounded, the postmitotic cells, which also express calretinin, send out branching dendrites towards the molecular layer and have a more mature phenotype. Because the selection process apparently sets in only after the cells have become postmitotic, the DCX-positive proliferative cell, and

type-2b and type-3 cells are all in a prominent position at the point at which crucial decisions for neurogenesis are made. We thus found the absence of GR and MR expression exactly on the transition stage of development at which DCX is switched on. Type-3 cells lie between type-2b cells and the postmitotic, CRpositive stage. Surprisingly, half of these cells did express the GR. This result allows two interpretations, between which we cannot at present distinguish: either a transient GR-positive phase occurs between two GR-negative stages or GR expression identifies that subset of type-3 cells that does not continue differentiation. We favour the second idea (Fig. 3B), but do not yet have further evidence to support it. A current study aims at characterizing type-3 cells in greater detail in vivo and in vitro. By morphological analysis we did not find obvious differences between GR-positive and GR-negative type-3 cells. Because in old age type-2b cells are too rare it was not possible to state with confidence whether they expressed corticosteroid receptors. If our hypothesis is correct that GR-positive type-3 cells are those that have exited from the developmental path, all DCXexpressing cells within the process of differentiation would be strictly negative for corticosteroid receptors. Extending the argument used above for the CR-positive cells, we therefore suggest that both the pivotal steps of neuronal development following the expansion of neuronal progenitor cells, i.e. the decision to exit from the cell cycle and initiate maturation, as well as selection for functional integration at the postmitotic stage should be independent of direct effects of corticosteroid. Unfortunately, this hypothesis is difficult to test because no longitudinal observation of neuronal development in vivo is possible. In contrast to some far-reaching conclusions drawn by others from the pioneering studies by Cameron and Gould, who first showed that corticosteroids can act negatively on adult hippocampal neurogenesis (Gould et al., 1992; Cameron et al., 1993; Cameron & Gould, 1994), there is no general and strict correlation between corticosterone levels and adult neurogenesis (Kempermann & Gage, 2002; Heine et al., 2004; Holmes et al., 2004). Explanations for the variance of net effects would include changes in GR and MR expression patterns, plus the predominance of indirect effects mediated by other components of the stem cell niche. Present-day microscopical tools, limited to three parallel channels of confocal analysis, do not allow the examination of precursor cells plus their neighbours in the same section. Thus, interaction among the different cells in the niche (besides the precursor cells these are astrocytes, microglia or macrophages, and endothelia) relative to their receptor profiles is unknown. In contrast to the predictions from our findings, Montaron et al. (2003) found that the MR agonist aldosterone normalized cell proliferation in adrenalectomized rats. Consistent with our data showing MR expression on mature cells, however, the same group reported that low doses of aldosterone were sufficient to prevent the adrenalectomy-induced death of mature cells. In addition, expression of PSA-NCAM, which almost completely overlaps with DCX expression, returned only after treatment with the GR agonist RU 28362 (Montaron et al., 2003). Again, © Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland 2004


indirect effects related to other constituents of the stem cell niche might be responsible for this regulation. Surprisingly, there are few in vitro data on corticosteroid actions in precursor cells. In fetal hippocampal progenitor cells, the GR agonist dexamethasone was sufficient to suppress cell proliferation, whereas activation of GR and MR caused defects in cell proliferation and differentiation (Yu et al., 2004). The relevant targets of GR and MR activation have not yet been identified in this context. Consequently, no concrete theories exist that would explain precisely how corticosterone interacts with the different stages of neuronal development during adult hippocampal neurogenesis. The changing receptor profile in the course of adult hippocampal neurogenesis and its age-dependent modification, however, provides an initial insight into the more macroscopic principles governing this regulation. Our data suggest that many acute regulatory effects are likely to be indirect, because the relevant cells do not express GR or MR. On the other hand, increasing expression of corticosteroid receptors with increasing age might explain an increased vulnerability to the direct effects of corticosteroids. Regardless, our results support the view that the putative role of corticosteroids in the regulation of adult neurogenesis is complex and tightly controlled via receptor expression.

Experimental procedures Animals and housing conditions Mice used in the present experiment were part of a previously published study (Steiner et al., 2004). Seven-week-old female C57BL6 mice were purchased from Charles River Laboratory, Germany. Thirty animals were randomly assigned to five experimental groups, six per group, to allow the investigation of the newborn cells at different time points. Animals were killed at five different time points (4 h, 1 day, 3 days, 7 days, 4 weeks) after BrdU injection. In a second experiment we used nestin-GFPexpressing transgenic reporter gene mice, originally developed by Masahiro Yamaguchi, University of Tokyo (Yamaguchi et al., 2000). The general principle and detail of immunohistochemical analysis in these mice has been described previously (Filippov et al., 2003; Kronenberg et al., 2003). For the nestin-GFP transgenic mice, female animals were used: n = 5 at the age of 7 weeks, and n = 3 at the age of 20 months. In addition, GR and MR expression in CR-positive cells was assessed in 16-month-old C57Bl / 6 mice ( n = 3). All mice were held three per cage under standard laboratory housing conditions with a light/ dark cycle of 12 h each and free access to food and water. GR and MR expression was also investigated in female C57BL / 6 mice at postnatal day P8, n = 3.

Tissue preparation Mice were anaesthetized with ketamine and perfused transcardially with 4% paraformaldehyde (PFA) in cold 0.1 M phosphate buffer. The brains were removed from the skulls, postfixed in 4% PFA at 4 °C overnight and transferred into 30% sucrose. Coronal brain sections of 40 µm were cut on a dry-ice-cooled copper block with a sliding microtome (Leica, Bensheim) and stored at −20 °C in cryoprotectant consisting of 25% ethylene glycol and 25% glycerin in 0.05 M phosphate buffer. P8 brains were only immersion fixed.

Immunofluorescence Primary and secondary antibodies were diluted in Tris-buffered saline (TBS) containing 0.1% TritonX-100, 0.1% Tween 20 and 3% donkey serum (TBS-plus). Sections were incubated with the primary antibody against GR and MR for 48 h at 4 °C, washed with TBS and postfixed with 4% paraformaldehyde in phosphatebuffered saline (PBS) for 15 min. If BrdU was to be detected in the sections, this step was followed by denaturing in 2 N HCl for 30 min at 37 °C. Afterwards sections were rinsed in 0.1 M borate buffer followed by TBS-plus and the anti-BrdU antibody. After an additional washing step, the sections were incubated with the secondary antibodies in TBS-plus for 4 h at room temperature, again washed with TBS and coverslipped in polyvinyl alcohol with diazabicyclo-octane (DABCO) as anti-fading agent. The primary antibodies were applied in the following concentrations: anti-BrdU (rat, 1 : 500, Biozol), anti-calretinin (rabbit, 1 : 250, or mouse, 1 : 250, Swant), anti-calbindin (rabbit, 1 : 250, Swant), anti-Doublecortin (goat, 1 : 200, Santa Cruz Biotechnologies), anti-PSA-NCAM (mouse IgM, Chemicon), anti-MR (goat, 1 : 50, Santa Cruz Biotechnologies), anti-GR (rabbit, 1 : 50, Abcam). Secondary antibodies used were: anti-rat RhodamineX, anti-guinea-pig Cy5, anti-rabbit FITC, anti-mouse FITC, anti-goat Cy5 (all 1 : 250, Jackson Immuno, distributed by Dianova).

Quantification of double- and triple-labelled cells One-in-12 series of hippocampal sections from each animal (at least five per group) were triple-labelled as described. Fifty BrdUpositive cells within the granule cell layer were analysed for co-expression of BrdU, MR and GR using a Leica TCS SP2 confocal microscope. The images were taken in sequential scanning mode and processed in Adobe Photoshop 7.0 for Macintosh. Only general contrast adaptations were made and figures were not otherwise manipulated.

BrdU immunohistochemistry BrdU injections BrdU (Sigma, Germany) was dissolved in 0.9% NaCl and filtered. The animals in the time-course study received a single intraperi−1 toneal (i.p.) injection of 50 mg kg body weight. The nestinGFP-expressing mice did not receive BrdU. © Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland 2004

Endogenous tissue peroxidases were blocked with 0.6% H2O2. Sections were then washed and incubated with 2 N HCl for 30 min at 37 °C and rinsed in borate buffer as described above. After incubation with the primary anti-BrdU antibody overnight at 4 °C, the sections were rinsed in TBS and TBS-plus and


incubated with the secondary antibody for 2 h at room temperature. ABC reagent (Vectastain Elite, Vector Laboratories) was −1 applied for 1 h at a concentration of 9 µL mL . Diaminobenzidine (DAB, Sigma, Germany) was used as chromogen at the con−1 centration of 0.25 mg mL in TBS with 0.01% H2O2 and 0.04% nickel chloride.

Quantification of BrdU-labelled cells Total numbers of BrdU-positive cells in the same tissue had been determined in our previously published study (Steiner et al., 2004). Briefly, one-in-six series of sections (240 µm apart) from all animals had been DAB-stained, and BrdU-positive cells had been counted throughout the rostro-caudal extent of the granule cell layer and multiplied by six to obtain the estimated total number of BrdU-positive cells per granule cell layer (Kempermann et al., 2003).

Acknowledgements We would like to thank Ruth Segner and Irene Thun for technical support. This study was funded by Volkswagenstiftung and Deutsche Forschungsgemeinschaft (DFG). A.G. was supported by DFG Research Training Group 429 ‘Neuropsychiatry and Psychology: Psychic Potentials and Limits in Old Age’.

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