312
Hmn Reseurc~h. 55-l (I9Yl) 312-315 0 lY91 Elsevier Science Publishers B.V. (K)O6-8993/91/$03,iO ADONIS
00068993912473XS
B RES 24738
Effects of aldosterone or RU28362 treatment on adrenalectomy-induced cell death in the dentate gyrus of the adult rat Catherine
S. Woolley, Elizabeth Gould, Randall R. Sakai, Robert L. Spencer and Bruce S. McEwen
Laboratory of Neuroendocrinology,
Rockefeller
University, 1230 York Ave., New York, NY 10021 (U.S.A.)
(Accepted 9 April 1991) Key words: Hippocampus;
Granule cell; Pyknotic cell; Glucocorticoid receptor; Mineralocorticoid
receptor; Aldosterone;
RU28362
Previous studies have shown that granule cells of the adult dentate gyrus require adrenal steroids for their survival. In order to investigate whether activation of type I or type II adrenal steroid receptors can mediate granule cell survival, we have analyzed the density of pyknotic cells in the granule cell, CA1 and CA3 pyramidal cell layers in Nissl stained hippocampal sections from adult male rats which were either sham operated, adrenalectomized, or adrenalectomized and treated with aldosterone as a specific type I receptor agonist or RU28362 as a specific type II receptor agonist. Aldosterone treatment completely protected the dentate gyrus from adrenalectomy-induced cell death, while treatment with RU28362 resulted in only a partial protection against cell death in this region. These results indicate that type I adrenal steroid receptor activation is sufficient to protect against adrenalectomy-induced cell death.
Granule cells of the adult rat dentate gyrus require adrenal steroids for their survival as both short- and long-term adrenalectomy have been shown to result in degeneration within the granule cell layer. Animals adrenalectomized for a period of 3-7 days show a substantial increase in the density of degenerating, or pyknotic, cells within the granule cell layer4, while near obliteration of the granule cell layer has been observed 3-4 months following adrenalectomy”. In either case, low dose replacement of corticosterone prevents granule cell degeneration. Because dentate gyrus granule cells contain both type I and type II adrenal steroid receptors’, s,14 and because corticosterone is capable of binding to either receptor type3, it is unclear whether it is activation of type I or type II receptors that is required for survival of these cells. In order to distinguish between these possibilities, we have compared the density of pyknotic cells within the granule cell layer of the dentate gyrus in sham operated rats, adrenalectomized rats and adrenalectomized rats which have been treated with either aldosterone at a dose which binds selectively to the type I receptor or the synthetic steroid, RU28362, a selective type II receptor agonist. For this study, young adult male Sprague-Dawley rats (220-250 g) were assigned to one of the following treatment groups: (i) sham operated, (ii) adrenalectoCorrespondence:
U.S.A.
C.S. Woolley, Laboratory of Neuroendocrinology,
mized, (iii) adrenalectomized with aldosterone delivered by osmotic minipump (Alzet) at a rate of 1 &h, or (iv) adrenalectomized with RU28362 delivered by osmotic minipump (Alzet) at a rate of 10 &h. Surgery was performed under Metofane anesthesia. Minipumps were implanted subcutaneously in the midscapular region of the back immediately following surgery. All adrenalectomized rats received 0.9% NaCl in their drinking water. Seven days following surgery, all rats were deeply anesthetized with ketamine. In order to validate adrenalectomy, a small amount of blood was reserved for radioimmunoassay of serum corticosterone levels using B21-42 antiserum (Endocrine Sciences, Tarzana, CA). Assay sensitivity was 10 pg of corticosterone. Rats were transcardially perfused with 100-150 ml 4% paraformaldehyde in 0.1 M sodium phosphate buffer with 1.5% (v/v) picric acid (pH 7.4). Brains were removed from the cranial cavities and postfixed for 24 h. Following postfixation, 50 pm thick coronal sections through the rostra1 hippocampal formation were cut using a Vibratome. These sections were mounted onto gelatinized slides and stained for Nissl using a Cresyl violet stain. All slides were coded prior to quantitative analysis and the code was not broken until the analysis was complete. For all groups in which rats had been adrenalectomized, analysis was only performed on brains from animals in
Box 290, 1230 York Ave., Rockefeller University, New York, NY 10021,
313
which
serum
corticosterone
levels were below 0.40 pg/lOO
ml. The density of pyknotic cells in the granule cell layer of the dentate gyrus from these brains was determined as follows: (i) the number of pyknotic cells (characterized
6-
by condensed chromatin, lack of nuclear membrane and pale or absent cytoplasm”) within the granule cell layer
5-
(8 half-sections
4-
subgranular
per
animal),
including
those
in the
zone, i.e. at the border of the cell layer and
the hilus, was counted at 1000x ; (ii) the granule cell layer was traced at 50x using a camera lucida drawing tube and
3-
its cross-sectional area was determined from these drawings using the Southern Micro Instruments image analysis
2-
program;
l-
pressed
(iii) the mean as number
density
of pyknotic
determined for each animal. adrenalectomy group resulted
of pyknotic
cells (ex-
cells per lo6 pm*) was Large variability in heterogeneity
of the of vari-
ance between treatment groups. In order to achieve homogeneity of variance between treatment groups, we performed a log transformation of the raw data. Transformed data were then subjected to one-way analysis of variance followed by Tukey HSD post hoc comparisons. The selectivity of aldosterone and RU28362 for hippocampal type I and type II receptors, respectively, at the doses used in this study was examined in a separate pilot study. Type I and type II receptor binding on whole hippocampal cytosol was measured according to the method of Spencer et al.‘*. Receptor binding in adrenalectomized rats that were given either aldosterone (1 ,q/h) or RU28362 (10 pg/h) treatment for 7 days was compared to receptor binding in rats that were adrenalectomized for a period of 18 h (a sufficient time to allow clearance of endogenous adrenal steroids; n = 4). Animals treated with aldosterone showed a significant decrease in type I receptor binding (mean + S.E.M. = 80.5 f 10.7 fmoVmg protein) relative to that in animals adrenalectomized for a period of 18 h (217.8 + 14.1 fmol/mg protein), indicating 60% occupation and/or downregulation of the type I receptor. In contrast, there was no decrease in type II receptor binding with aldosterone treatment. The selective type II receptor agonist, RU28362, produced a significant decrease in type II receptor binding (163.9 + 26.1 fmol/mg protein) relative to adrenalectomy for a period of 18 h (385.7 f 7.5 fmol/mg protein) indicating approximately 60% occupation of the type II receptor. This treatment produced no decrease in type I receptor binding. Quantitative analysis of Nissl stained brain sections revealed significant overall differences in the density of pyknotic cells in the granule cell layer of the dentate gyrus with treatment (F3,15 = 86.461, P < 0.001). As previously reported4, we observed a low density of pyknotic cells in the granule cell layer of sham operated animals which was significantly increased in animals
O-l-
-r
SHAM
ADX
ALDO
NJ26362
Fig. 1. Values represent mean + S.E.M. log (# pyknotic cells/lo6 pm’) in the granule cell layer of the dentate gyrus of animals that were either sham adrenalectomized (SHAM; n = 5); adrenalectomized (ADX; n = 4); adrenalectomized and treated with aldosterone (ALDO; n = 4) or adrenalectomized and treated with RU28362 (RU28362; n = 6). (*) indicates a significant difference from SHAM and ALDO; P < 0.001.
adrenalectomized for a period of 7 days (P < 0.001; Fig. 1). Aldosterone treatment completely prevented the effect of adrenalectomy in that pyknotic cell density in these animals was significantly lower than that observed in adrenalectomized animals which received no hormone replacement (P < 0.001; Fig. 1) and virtually indistinguishable from that in sham operated animals (P > 0.1; Fig. 1). On the other hand, treatment of adrenalectomized rats with RU28362 resulted in only a partial prevention of the adrenalectomy-induced increase in pyknotic cell density in the granule ceil layer, i.e. pyknotic cell density in RU28362 treated animals was intermediate between that observed in sham operated and adrenalectomized animals. While treatment of adrenalectomized rats with RU28362 resulted in a trend toward a decrease in pyknotic cell density compared to adrenalectomized animals which received no drug (P < 0.1,Fig. l), this RU28362 treatment was unable to effectively prevent adrenalectomy-induced pyknosis (Figs. 1, 2). That is, the density of pyknotic cells in the RU28362 treated animals remained significantly greater than in the sham operated animals (P < 0.001, Fig. 1). As previously reported4, the density of pyknotic cells in the CA1 and CA3 pyramidal cell layers was extremely low and adrenalectomy had no significant effect on pyknotic cell density in either the CA1 (F3,15 = 1.954, P > 0.1) or CA3 (F3,15 = 2.006, P > 0.1) pyramidal cell population (data not shown). Previous studies have shown that removal of circulat-
314
Fig. 2. Photomicrographs of a pyknotic cell in the granule cell layer of either an adrenalectomized animal which received no steroid treatment (A) or an adrenaiectomized animal which was treated with RU28362 (B) following surgery. Arrows indicate granules of condensed chromatin which are characteristic of pyknotic cells. Scale bar equals 20 pm and applies to both frames.
ing adrenal steroids by adrenalectomy results in granule cell degeneration and death4.“. The results presented here demonstrate that selective type 1 receptor activation is sufficient to protect dentate gyrus granule cells from this degeneration. The data also suggest that type II receptor activation could have some effect on granule cell survival. However, previous reports regarding occupation of the type II receptor in the normal animal indicate that it is probably not stimulation of type II receptors
normal
hippocampal
function’.7,x.
Our observation
that
specific type I receptor occupation by aldosterone is sufficient to completely protect against the effect of adrenalectomy, i.e. to restore the normal condition, is fully consistent with this hypothesis. However, this apparent effect of type I receptor activation on granule cell survival does not appear to be generalizable to other
which is responsible for the maintenance of granule cells in this case. While our hippocampal cytosolic binding assays indicate that the RU28362 treatment used in this
cell types. Hippocampal pyramidal cells which also express high levels of type I receptor7%’ do not degenerate in response to adrenalectomy” and thus do not appear to require type I receptor activation for survival. Further studies investigating different responses of hippocampal
study occupies nearly 60% of type II receptor sites and does not affect the type I receptor, we observe that this
neuron types to alterations in adrenal steroid levels may help to clarify their apparently different survival require-
treatment results in only a partial protection against the effect of adrenalectomy. Cytosolic binding assays indicate that the type II receptor is only approximately 10% occupied at the lowest point in the diurnal cycle of glucocorticoid secretion7 and only 50-75% occupied at the peak of this diurnal cycle7 or at the peak of the adrenocortical stress response7,‘*. Thus, if the substantial occupation of the type II receptor achieved by our RU28362 regimen was unable to protect against the effects of adrenalectomy and, in the normal hippocampus, type II receptors are only intermittently occupied to such an extent, it seems unlikely that it is activation of this receptor type which is required for survival of granule cells. Our data suggest, on the other hand, that it is activation of the type I receptor which is more likely to be necessary for granule cell survival. Because, even at the lowest point in the diurnal cycle of glucocorticoid secretion, type 1 receptors in the hippocampus are as much as 90% occupied, it has been suggested that this receptor type has a tonic influence over hippocampal activity and could thus be involved in maintenance of
ments. The fact that replacement doses of either aldosterone or corticosterone are capable of completely protecting granule cells from the damaging effects of short-term adrenalectomy may have important implications for the development of the dentate gyrus. Recently, we have demonstrated that the dentate gyrus undergoes a period of naturally occurring cell death during early postnatal development5. We have found that the greatest degree of cell death occurs during the ‘stress hyporesponsive period” when levels of both aldosterone and corticosterone are naturally very low. Our findings with neonatal aldosterone and corticosterone manipulations indicate that these adrenal steroids may play an important role in regulating cell death as well as cell birth during this developmental period (unpublished observations). The presence of particularly high levels of type I receptors in the hippocampus7~“~‘* as well as the observation that hippocampal neurons appear to be able to bind aldosterone even in the presence of much higher concentrations of circulating corticosteroneh have been
315 difficult to reconcile
with the lack of any specific function
known for the type I receptor in the hippocampus. Our data indicate that type I receptor activation is probably very important for dentate gyrus granule cell survival and thus shed some light on possible functions for this receptor
type
in maintenance
of normal
granule
cell
1 Collier, T.J., Miller, J.S., Travis, J. and Routtenberg, A., Dentate gyrus granule cells and memory: electrical stimulation disrupts memory for places rewarded, Behav. Neural Biol., 34 (1984) 227-239. 2 Corini, H., Magarinos, A.M., De Nicola, A.F., Rainbow, T.C. and McEwen, B.S., Further studies of brain aldosterone binding sites employing new mineralocorticoid and glucocorticoid receptor markers in vitro, Bruin Research, 361 (1985) 212-216. 3 De Kloet, E.R. and Reul, J.M.H., Feedback action and tonic influence of corticosteroids on brain function: a concept arising from the heterogeneity of brain receptor systems, Psychoneuroendocrinology, 12 (1987) 83-105. 4 Gould, E., Woolley, C.S. and McEwen, B.S., Short-term glucocorticoid manipulations affect neuronal morphology and survival in the adult rat dentate gyrus, Neuroscience, 37 (1990) 367-375. 5 Gould, E., Woolley, C.S. and McEwen, B.S., Naturally occur-
ring cell death in the developing dentate gyrus of the rat, J. Comp. Neurof., 304 (1991) 408-418. 6 McEwen, B.S., Lamdin, L.T., Rainbow, T.C. and De Nicola, A.F., Aldosterone effects on salt appetite in adrenalectomized rats, Neuroendocrinofogy, 43 (1986) 38-43. 7 Reul, J.M.H. and De Kloet, E.R., Two receptor systems for glucocorticoids in the rat brain: microdistribution and differential occupation, Endocrinology, 117 (1985) 2505-2511. 8 Reul, J.M.H., Van der Bosch, F.R. and De Kloet, E.R., Relative occupation of type-1 and type-II corticosteroid receptors in rat brain following stress and dexamethasone treatment:
functions
which
include
processes
of
learning
and
memory’T’5.
This work was supported Lounsbery Foundation.
by MH 41256 and the Richard
functional implications, J. Endocrinol., 115 (1987) 459-467. 9 Sapolsky, R.M. and Meaney, M.J., Maturation of the adrenocortical stress response: neuroendocrine control mechanisms and the stress hyporesponsive period, Bruin Res. Rev., 11 (1986) 65-76. 10 Sengelaub, D.R. and Finley, B.L., Cell death in the mammalian visual system during normal development: I. Retinal ganglion cells, J. Comp. Neural., 204 (1982) 311-317. 11 Sloviter, R.S., Valiquette, G., Abrams, G.M., Ronk, E.C., Sollas, A.I., Paul, L.A. and Neubort, S.L., Selective loss of hippocampal granule cells in the mature rat brain after adrenalectomy, Science, 243 (1989) 535-538. 12 Spencer, R.L., Young, E.A., Choo, P.H. and McEwen, B.S., Adrenal steroid type I and type II receptor binding: estimates of in vivo receptor number, occupancy, and activation with varying level of steroid, Brain Research, 514 (1990) 37-48. 13 Sutanto, W. and De Kloet, E.R., Species-specificity of corticosteroid receptors in hamster and rat brains, Endocrinology, 121 (1987) 1405-1411.
14 Van Eekelen, J.A.M., Jiang, W., De Kloet, E.R. and Bohn, M.C., Distribution of the mineralocorticoid and the glucocorticoid receptor mRNAs in the rat hippocampus, J. Neurosci. Merh., 21 (1988) 88-94.
15 Walsh, T.J., Schulz, D.W., Tilson, H.A. and Schmechel, D.E., Colchicine-induced granule cell loss in rat hippocampus: selective behavioral and histological alterations, Brain Research, 398 (1986) 23-36.
Hmn Reseurc~h. 55-l (I9Yl) 312-315 0 lY91 Elsevier Science Publishers B.V. (K)O6-8993/91/$03,iO ADONIS
00068993912473XS
B RES 24738
Effects of aldosterone or RU28362 treatment on adrenalectomy-induced cell death in the dentate gyrus of the adult rat Catherine
S. Woolley, Elizabeth Gould, Randall R. Sakai, Robert L. Spencer and Bruce S. McEwen
Laboratory of Neuroendocrinology,
Rockefeller
University, 1230 York Ave., New York, NY 10021 (U.S.A.)
(Accepted 9 April 1991) Key words: Hippocampus;
Granule cell; Pyknotic cell; Glucocorticoid receptor; Mineralocorticoid
receptor; Aldosterone;
RU28362
Previous studies have shown that granule cells of the adult dentate gyrus require adrenal steroids for their survival. In order to investigate whether activation of type I or type II adrenal steroid receptors can mediate granule cell survival, we have analyzed the density of pyknotic cells in the granule cell, CA1 and CA3 pyramidal cell layers in Nissl stained hippocampal sections from adult male rats which were either sham operated, adrenalectomized, or adrenalectomized and treated with aldosterone as a specific type I receptor agonist or RU28362 as a specific type II receptor agonist. Aldosterone treatment completely protected the dentate gyrus from adrenalectomy-induced cell death, while treatment with RU28362 resulted in only a partial protection against cell death in this region. These results indicate that type I adrenal steroid receptor activation is sufficient to protect against adrenalectomy-induced cell death.
Granule cells of the adult rat dentate gyrus require adrenal steroids for their survival as both short- and long-term adrenalectomy have been shown to result in degeneration within the granule cell layer. Animals adrenalectomized for a period of 3-7 days show a substantial increase in the density of degenerating, or pyknotic, cells within the granule cell layer4, while near obliteration of the granule cell layer has been observed 3-4 months following adrenalectomy”. In either case, low dose replacement of corticosterone prevents granule cell degeneration. Because dentate gyrus granule cells contain both type I and type II adrenal steroid receptors’, s,14 and because corticosterone is capable of binding to either receptor type3, it is unclear whether it is activation of type I or type II receptors that is required for survival of these cells. In order to distinguish between these possibilities, we have compared the density of pyknotic cells within the granule cell layer of the dentate gyrus in sham operated rats, adrenalectomized rats and adrenalectomized rats which have been treated with either aldosterone at a dose which binds selectively to the type I receptor or the synthetic steroid, RU28362, a selective type II receptor agonist. For this study, young adult male Sprague-Dawley rats (220-250 g) were assigned to one of the following treatment groups: (i) sham operated, (ii) adrenalectoCorrespondence:
U.S.A.
C.S. Woolley, Laboratory of Neuroendocrinology,
mized, (iii) adrenalectomized with aldosterone delivered by osmotic minipump (Alzet) at a rate of 1 &h, or (iv) adrenalectomized with RU28362 delivered by osmotic minipump (Alzet) at a rate of 10 &h. Surgery was performed under Metofane anesthesia. Minipumps were implanted subcutaneously in the midscapular region of the back immediately following surgery. All adrenalectomized rats received 0.9% NaCl in their drinking water. Seven days following surgery, all rats were deeply anesthetized with ketamine. In order to validate adrenalectomy, a small amount of blood was reserved for radioimmunoassay of serum corticosterone levels using B21-42 antiserum (Endocrine Sciences, Tarzana, CA). Assay sensitivity was 10 pg of corticosterone. Rats were transcardially perfused with 100-150 ml 4% paraformaldehyde in 0.1 M sodium phosphate buffer with 1.5% (v/v) picric acid (pH 7.4). Brains were removed from the cranial cavities and postfixed for 24 h. Following postfixation, 50 pm thick coronal sections through the rostra1 hippocampal formation were cut using a Vibratome. These sections were mounted onto gelatinized slides and stained for Nissl using a Cresyl violet stain. All slides were coded prior to quantitative analysis and the code was not broken until the analysis was complete. For all groups in which rats had been adrenalectomized, analysis was only performed on brains from animals in
Box 290, 1230 York Ave., Rockefeller University, New York, NY 10021,
313
which
serum
corticosterone
levels were below 0.40 pg/lOO
ml. The density of pyknotic cells in the granule cell layer of the dentate gyrus from these brains was determined as follows: (i) the number of pyknotic cells (characterized
6-
by condensed chromatin, lack of nuclear membrane and pale or absent cytoplasm”) within the granule cell layer
5-
(8 half-sections
4-
subgranular
per
animal),
including
those
in the
zone, i.e. at the border of the cell layer and
the hilus, was counted at 1000x ; (ii) the granule cell layer was traced at 50x using a camera lucida drawing tube and
3-
its cross-sectional area was determined from these drawings using the Southern Micro Instruments image analysis
2-
program;
l-
pressed
(iii) the mean as number
density
of pyknotic
determined for each animal. adrenalectomy group resulted
of pyknotic
cells (ex-
cells per lo6 pm*) was Large variability in heterogeneity
of the of vari-
ance between treatment groups. In order to achieve homogeneity of variance between treatment groups, we performed a log transformation of the raw data. Transformed data were then subjected to one-way analysis of variance followed by Tukey HSD post hoc comparisons. The selectivity of aldosterone and RU28362 for hippocampal type I and type II receptors, respectively, at the doses used in this study was examined in a separate pilot study. Type I and type II receptor binding on whole hippocampal cytosol was measured according to the method of Spencer et al.‘*. Receptor binding in adrenalectomized rats that were given either aldosterone (1 ,q/h) or RU28362 (10 pg/h) treatment for 7 days was compared to receptor binding in rats that were adrenalectomized for a period of 18 h (a sufficient time to allow clearance of endogenous adrenal steroids; n = 4). Animals treated with aldosterone showed a significant decrease in type I receptor binding (mean + S.E.M. = 80.5 f 10.7 fmoVmg protein) relative to that in animals adrenalectomized for a period of 18 h (217.8 + 14.1 fmol/mg protein), indicating 60% occupation and/or downregulation of the type I receptor. In contrast, there was no decrease in type II receptor binding with aldosterone treatment. The selective type II receptor agonist, RU28362, produced a significant decrease in type II receptor binding (163.9 + 26.1 fmol/mg protein) relative to adrenalectomy for a period of 18 h (385.7 f 7.5 fmol/mg protein) indicating approximately 60% occupation of the type II receptor. This treatment produced no decrease in type I receptor binding. Quantitative analysis of Nissl stained brain sections revealed significant overall differences in the density of pyknotic cells in the granule cell layer of the dentate gyrus with treatment (F3,15 = 86.461, P < 0.001). As previously reported4, we observed a low density of pyknotic cells in the granule cell layer of sham operated animals which was significantly increased in animals
O-l-
-r
SHAM
ADX
ALDO
NJ26362
Fig. 1. Values represent mean + S.E.M. log (# pyknotic cells/lo6 pm’) in the granule cell layer of the dentate gyrus of animals that were either sham adrenalectomized (SHAM; n = 5); adrenalectomized (ADX; n = 4); adrenalectomized and treated with aldosterone (ALDO; n = 4) or adrenalectomized and treated with RU28362 (RU28362; n = 6). (*) indicates a significant difference from SHAM and ALDO; P < 0.001.
adrenalectomized for a period of 7 days (P < 0.001; Fig. 1). Aldosterone treatment completely prevented the effect of adrenalectomy in that pyknotic cell density in these animals was significantly lower than that observed in adrenalectomized animals which received no hormone replacement (P < 0.001; Fig. 1) and virtually indistinguishable from that in sham operated animals (P > 0.1; Fig. 1). On the other hand, treatment of adrenalectomized rats with RU28362 resulted in only a partial prevention of the adrenalectomy-induced increase in pyknotic cell density in the granule ceil layer, i.e. pyknotic cell density in RU28362 treated animals was intermediate between that observed in sham operated and adrenalectomized animals. While treatment of adrenalectomized rats with RU28362 resulted in a trend toward a decrease in pyknotic cell density compared to adrenalectomized animals which received no drug (P < 0.1,Fig. l), this RU28362 treatment was unable to effectively prevent adrenalectomy-induced pyknosis (Figs. 1, 2). That is, the density of pyknotic cells in the RU28362 treated animals remained significantly greater than in the sham operated animals (P < 0.001, Fig. 1). As previously reported4, the density of pyknotic cells in the CA1 and CA3 pyramidal cell layers was extremely low and adrenalectomy had no significant effect on pyknotic cell density in either the CA1 (F3,15 = 1.954, P > 0.1) or CA3 (F3,15 = 2.006, P > 0.1) pyramidal cell population (data not shown). Previous studies have shown that removal of circulat-
314
Fig. 2. Photomicrographs of a pyknotic cell in the granule cell layer of either an adrenalectomized animal which received no steroid treatment (A) or an adrenaiectomized animal which was treated with RU28362 (B) following surgery. Arrows indicate granules of condensed chromatin which are characteristic of pyknotic cells. Scale bar equals 20 pm and applies to both frames.
ing adrenal steroids by adrenalectomy results in granule cell degeneration and death4.“. The results presented here demonstrate that selective type 1 receptor activation is sufficient to protect dentate gyrus granule cells from this degeneration. The data also suggest that type II receptor activation could have some effect on granule cell survival. However, previous reports regarding occupation of the type II receptor in the normal animal indicate that it is probably not stimulation of type II receptors
normal
hippocampal
function’.7,x.
Our observation
that
specific type I receptor occupation by aldosterone is sufficient to completely protect against the effect of adrenalectomy, i.e. to restore the normal condition, is fully consistent with this hypothesis. However, this apparent effect of type I receptor activation on granule cell survival does not appear to be generalizable to other
which is responsible for the maintenance of granule cells in this case. While our hippocampal cytosolic binding assays indicate that the RU28362 treatment used in this
cell types. Hippocampal pyramidal cells which also express high levels of type I receptor7%’ do not degenerate in response to adrenalectomy” and thus do not appear to require type I receptor activation for survival. Further studies investigating different responses of hippocampal
study occupies nearly 60% of type II receptor sites and does not affect the type I receptor, we observe that this
neuron types to alterations in adrenal steroid levels may help to clarify their apparently different survival require-
treatment results in only a partial protection against the effect of adrenalectomy. Cytosolic binding assays indicate that the type II receptor is only approximately 10% occupied at the lowest point in the diurnal cycle of glucocorticoid secretion7 and only 50-75% occupied at the peak of this diurnal cycle7 or at the peak of the adrenocortical stress response7,‘*. Thus, if the substantial occupation of the type II receptor achieved by our RU28362 regimen was unable to protect against the effects of adrenalectomy and, in the normal hippocampus, type II receptors are only intermittently occupied to such an extent, it seems unlikely that it is activation of this receptor type which is required for survival of granule cells. Our data suggest, on the other hand, that it is activation of the type I receptor which is more likely to be necessary for granule cell survival. Because, even at the lowest point in the diurnal cycle of glucocorticoid secretion, type 1 receptors in the hippocampus are as much as 90% occupied, it has been suggested that this receptor type has a tonic influence over hippocampal activity and could thus be involved in maintenance of
ments. The fact that replacement doses of either aldosterone or corticosterone are capable of completely protecting granule cells from the damaging effects of short-term adrenalectomy may have important implications for the development of the dentate gyrus. Recently, we have demonstrated that the dentate gyrus undergoes a period of naturally occurring cell death during early postnatal development5. We have found that the greatest degree of cell death occurs during the ‘stress hyporesponsive period” when levels of both aldosterone and corticosterone are naturally very low. Our findings with neonatal aldosterone and corticosterone manipulations indicate that these adrenal steroids may play an important role in regulating cell death as well as cell birth during this developmental period (unpublished observations). The presence of particularly high levels of type I receptors in the hippocampus7~“~‘* as well as the observation that hippocampal neurons appear to be able to bind aldosterone even in the presence of much higher concentrations of circulating corticosteroneh have been
315 difficult to reconcile
with the lack of any specific function
known for the type I receptor in the hippocampus. Our data indicate that type I receptor activation is probably very important for dentate gyrus granule cell survival and thus shed some light on possible functions for this receptor
type
in maintenance
of normal
granule
cell
1 Collier, T.J., Miller, J.S., Travis, J. and Routtenberg, A., Dentate gyrus granule cells and memory: electrical stimulation disrupts memory for places rewarded, Behav. Neural Biol., 34 (1984) 227-239. 2 Corini, H., Magarinos, A.M., De Nicola, A.F., Rainbow, T.C. and McEwen, B.S., Further studies of brain aldosterone binding sites employing new mineralocorticoid and glucocorticoid receptor markers in vitro, Bruin Research, 361 (1985) 212-216. 3 De Kloet, E.R. and Reul, J.M.H., Feedback action and tonic influence of corticosteroids on brain function: a concept arising from the heterogeneity of brain receptor systems, Psychoneuroendocrinology, 12 (1987) 83-105. 4 Gould, E., Woolley, C.S. and McEwen, B.S., Short-term glucocorticoid manipulations affect neuronal morphology and survival in the adult rat dentate gyrus, Neuroscience, 37 (1990) 367-375. 5 Gould, E., Woolley, C.S. and McEwen, B.S., Naturally occur-
ring cell death in the developing dentate gyrus of the rat, J. Comp. Neurof., 304 (1991) 408-418. 6 McEwen, B.S., Lamdin, L.T., Rainbow, T.C. and De Nicola, A.F., Aldosterone effects on salt appetite in adrenalectomized rats, Neuroendocrinofogy, 43 (1986) 38-43. 7 Reul, J.M.H. and De Kloet, E.R., Two receptor systems for glucocorticoids in the rat brain: microdistribution and differential occupation, Endocrinology, 117 (1985) 2505-2511. 8 Reul, J.M.H., Van der Bosch, F.R. and De Kloet, E.R., Relative occupation of type-1 and type-II corticosteroid receptors in rat brain following stress and dexamethasone treatment:
functions
which
include
processes
of
learning
and
memory’T’5.
This work was supported Lounsbery Foundation.
by MH 41256 and the Richard
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