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Effects of dehydroepiandrosterone and its sulfate on brain tissue in culture and on memory in mice
Brain Research, 406 (1987) 357-362 Elsevier
357
BRE 22110
Effects of dehydroepiandrosterone and its sulfate on brain tissue in culture and on memory in mice Eugene Roberts 1, Liane Bologa 1'*, James
F. F l o o d 2 and
Gary E. Smith 2
1Department of Neurobiochemistry, Beckman Research Institute of the City of Hope, Duarte, CA 91010 (U.S.A.) and 2psychobiology Research Laboratory and Geriatric Research, Education and Clinical Center, Veterans Administration Hospital, Sepulveda, CA 91343 (U.S.A.)
(Accepted 18 November 1986) Key words: Dehydroepiandrosterone; Dehydroepiandrosterone sulfate; Tissue culture; Memory
Low concentrations of dehydroepiandrosterone (DHEA) and dehydroepiandrosterone sulfate (DHEAS) enhanced neuronal and glial survival and/or differentiation in dissociated cultures of 14-day mouse embryo brain. Posttrial intracisternal injection into the brains of mice undergoing active avoidance training alleviated amnesia and enhanced long-term memory. By minimizingdegenerative changes in injured nerve tissue and facilitating plastic changes, DHEA and DHEAS may be of use in treatment of neurodegenerative and memory disorders in man. Incoordinations in a number of bodily systems with age generally occur coincidentally with decrements in levels of circulating sex hormones 22. Among the major blood serum steroid classes, monotonic decreases after puberty in both males and females occur only in mean levels of dehydroepiandrosterone (DHEA) and dehydroepiandrosterone sulfate (DHEAS) 18, largely adrenally derived substances which serve as precursors for both androgens and estrogens 19. The stimulatability of release of D H E A and D H E A S by A C T H is reduced markedly in the aging human organism 2°. Administration of D H E A and D H E A S may exert ameliorative effects in such different conditions as diabetes, obesity, autoimmune disease, cancer, and connective tissue and nervous system disorders, possibly by releasing diverse programs of metabolic machinery necessary for effective intracellular and intercellular communication to take place (see ref. 22 for review). D H E A , D H E A S , and the sex-related steroids that derive from them may pass into the brain through microcapillaries, the endothelial cells of which may serve as conduits for, as
well as consumers of, steroids 12'16A7. Inadequate availability of sex steroids and/or their precursors could have grave effects on structure and function of nerve tissue 14 since, in general, steroid hormones are believed to form complexes with specific receptor proteins in the cytoplasm which, after undergoing appropriate transformations, pass into the nucleus where they regulate gene expression 3'21. Without currently being concerned about the partitioning of D H E A , DHEAS, and their metabolites among various cell types and the detailed aspects of the effects of these substances on m R N A transcription, we reasoned that they generally might strengthen metabolic and communication apparatuses in nerve tissue, so as to increase regenerative capacity when injury occurs and facilitate plastic changes that take place during learning. In the present experiments we observed (1) the effects of low concentrations of D H E A and D H E A S on survival and development of dissociated mouse embryo brain in culture, which we assume in some respects to be akin to injured nerve tissue, and (2) the effects of intracisternal injection of these substances in a well-established learning paradigm in
* Present address: L. Bologa, University of Zfirich School of Medicine, Department of Pathology/Neuropathology, 8091 Ziirich, Switzerland. Correspondence.. E. Roberts, Department of Neurobiochemistry, Beckman Research Institute of the City of Hope, Duarte, CA 91010, U.S.A. 0006-8993/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
358
mice l°, d u r i n g t h e a c h i e v e m e n t o f w h i c h significant,
l i e v e d to o c c u r ~. T h e results r e p o r t e d b e l o w e n c o u r
hut as yet u n c h a r a c t e r i z e d , plastic c h a n g e s are be-
age f u r t h e r w o r k a l o n g t h e s e lines.
Fig. 1. Effects of DHEA and DHEAS on embryonic mouse brain cultures, 9 days in vitro (see text), lmmunostained for NF: A: control; B: 10-7 M DHEA; C: 10-8 M DHEAS. lmmunostained for GFAP: D: control; E: 11)-7 M DHEA; F: l(I -~ M DHEAS. Bar = 34.7 /~m. Cultures were prepared from finely chopped, mechanically dissociated brains from 14-day-old Swiss mouse embryos2. Cells were plated at a density of 3 x 105 cells/cm2 on poly-D-lysine-coated glass cover slips and maintained at 37 °C in atmosphere of 5% CO2/80% humidity in Dulbecco-modified Minimum Essential Medium supplemented with 10% fetal calf serum. The medium was first changed at 5 days of culture, at which time solutions of test substances adjusted to pH 7.3 were added. Control cultures received only normal medium. On the 9th day the cultures were fixed and immunostained. Staining was achieved by an indirect immunofluorescence technique, as adapted for brain cell cultures2. Cultures were fixed by successive incubations at room temperature with 3.7% buffered formaldehyde alone and containing 0.2% Triton X-100, each for 10 min. The cultures were rinsed briefly at 4 °C successively with 50%, 100%, and 50% acetone. Incubation with appropriately diluted antisera (usually 1/20-1/30) was carried out at room temperature for 30 min. Incubation with antisera was followed by washings and incubation with fluorescein isothiocyanate-labeled goat anti-rabbit immunoglobulin. Rabbit antiserum to neuron-specific neurofilament triplet of proteins (NF) was used to identify neurons 7, and rabbit antiserum to glial-specific fibrillary acidic protein (GFAP) I was employed to visualize astrocytes. Stained coverslips were mounted in buffered glycerol and examined with an Olympus Vanox microscope equipped with an automatic camera.
359 Dissociated brains of 14-day-old mouse embryos were maintained in culture for 5 days 2, at which time they were found to contain closely similar numbers of cells per dish. Fresh medium alone or medium containing D H E A or DHEAS (10-5-10 -8 M) then was added, and after another 4 days the cultures were fixed and fluorescently immunostained for neurofilament proteins (NF) and glial fibrillary acidic protein (GFAP) 1,7. The preparations were examined thoroughly by fluorescence and phase-contrast microscopy. Positive staining for NF or GFAP is considered to be evidence that cells are either differentiated neurons or astrocytes, respectively. In control cultures, rather sparsely distributed small NF-positive cells with short processes were observed, either isolated from each other or in clusters consisting of a few cells (Fig. 1A). The staining was, at most, of moderate intensity. In media containing D H E A or DHEAS, remarkable increases were found in numbers of NFpositive neurons in comparison with control cultures (Fig. 1B, C). Under higher power (not shown), processes of the NF-positive cells appeared to be greatly extended, thickened, and intertwined. Numerous connections were noted between isolated neurons and greatly enlarged neuronal clusters. The brilliance of the fluorescence shown by the neurons and their processes was greatly enhanced in D H E A or DHEAS-containing media by comparison with the rather dull fluorescent signal observed in the controls, indicating the presence of increased content of NF protein per unit area of cell surface. Many of the NF-positive neurons in the experimental cultures were larger than those seen in the controls. Maximal enhancements were seen at 10-7 M D H E A and 10-8 M DHEAS, respectively, among the concentrations tested, the neuronal clusters being somewhat larger with DHEAS. Highly significant increases over control values were found in numbers of NF-positive cells per 380 mm 2 coverslip (P < 0.01; 24 determinations in 3 separate experiments): control, 107 + 10 x 103; D H E A (10 -7 M), 983 + 53 x 103; DHEAS (10 -s M), 997 + 49 x 103. The latter increases could be attributable to enhanced neuronal survival and/or to an acceleration of the differentiation into neurons of precursor cells, but not to mitosis of NF-positive cells, themselves, since separate experiments (to be reported elsewhere) showed no incorporation of [3H]thymidine, whatsoever, to have taken place into
such cells under standard conditions of study. Remarkable increases over the controls (Fig. 1D) were also noted in the presence of D H E A (Fig. 1E) and DHEAS (Fig. 1F) in numbers of GFAP-positive astrocytes, in the extensions of their processes, and in the brilliance of their fluorescent staining, indicative of increased amounts of total GFAP as well as amount per unit cell surface. Since a number of experiments showed clearly that incorporation of [3H]thymidine was less in the cultures with D H E A and DHEAS than in the controls, the results suggest that the steroids enhance the survival of initially plated astrocytes and/or the differentiation of GFAPpositive astrocytes from stem cells, while decreasing mitosis. Taken together, the above results show that in both neurons and glia, D H E A and DHEAS enhance the expression of properties related to postmitotic differentiated states. In this regard, it is of great interest that estradiol-17fl and testosterone, both metabolites of D H E A , elicited growth and aborization of neurites in cultures of new-born mouse brain hypothalamic/preoptic area and that the neuritic response to estradiol occurred in [3H]estradiol-accumulating cells28-3°. Quantitative measurements are being made in experiments similar to those above of contents of NF, GFAP, and other neuronal and glial specific proteins as well as of relevant mRNAs. Experiments were performed employing an active avoidance behavioral paradigm (buzzer footshock) in a T-maze 9 with groups of 15 mice per dose. Injections of test solutions were made within 3 min after training. One week after training, retention for Tmaze training was tested. It was established that 2 ~1 intracerebroventricular (i.c.v.) injections of solutions containing D H E A or DHEAS had enhancing effects on long-term memory retention (Fig. 2). DHEA, not readily soluble in saline, was dissolved in dimethyl sulfoxide (DMSO), which was shown in preliminary experiments to cause amnesia when 2/A was injected. Taking advantage of the latter observation, we used well-trained animals to determine whether or not D H E A dissolved in DMSO could prevent the DMSO-induced amnesia. Training was performed under conditions that maximize learning (see legend to Fig. 2A), and the mice were retested one week later. Mice making a successful avoidance in 3 trials were classed as remembering the original training, a criterion providing optimal separation between
360 A
80
SALINE
6C 4C
• p > 005 p < 0.05 ® p < 001
20 - - DMSO ALONE
:~
0 8O
DHEAS IN SALINE./~ 6O 40
20
SALINE A L O N E -1~2
-I1
' -I0
-9
LOG DOSE (moles/mouse, ICV)
Fig. 2. Effects of intracerebroventricularly injected DHEA and DHEAS on memory retention in mice (see refs. 9 and I0 for further experimental details). A: DHEA in DMSO in welltrained animals: 5 training trials in the T-maze; the buzzer, loud; intertrial interval, 45 s; footshock level, 0.35 mA. B: DHEAS in saline in poorly trained animals: 4 training trials; buzzer, muffled; intertrial interval, 30 s; footshock level, 0.30 mA. Injections of test solutions were made within 3 min after training. Retention was tested one week after the training trials. naive and well-trained animals9; and the percentages of animals meeting the criterion (recall scores) were recorded. Because standard deviations vary among identical mean values calculated in this fashion, the same mean recall scores may show different P-values when tested for significance of changes from the controls. The saline control group showed good retention (80% recall) (Fig. 2A), while the group receiving D M S O alone was reduced to 20%, Dunnett's ttest showing a significant difference between them (P < 0.01). An A N O V A of results for all groups showed a significant enhancing effect of D H E A on m e m o r y retention (F14,210 = 5.13, P < 0.001). Individually, 10 of the 13 doses improved retention relative to the D M S O group at P < 0.01, one at P < 0.05, and 3 were not statistically significant. Doses between 8.4 × 10 -11 and 5.4 × 10 -l° mol/mouse gave results similar to those with saline controls, completely overcoming the amnesic effect of the D M S O in which they were contained. The two plateaus in the d o s e - response curve (Fig. 2A) suggest that more than one rate-limiting process may be affected under the experimental conditions employed.
Experiments with D H E A S (Fig. 2B) were performed similarly to those above, with the exception that training conditions were adjusted so that initial recall in the saline controls was only 20%, making it easier to detect whether or not there was an enhancing effect on memory. A n A N O V A showed a significant effect (F8,12 6 = 2.59, P < 0.025) over all 8 doses tested, which corresponded to the higher levels of D H E A tested. Preliminary experiments showed lower doses of D H E A S to be ineffective. Injection of D H E A S significantly increased the recall scores at 4 concentrations between 4.4 x 10-1° and 6.9 x 10-1° mol/mouse. The memory-enhancing effects of D H E A and D H E A S were maximal at 5.4 x 10 -1° and 6.2 x 10 -1° mol/mouse, respectively, and decreased at higher levels in a manner typical for most substances that improve memory ~5. The effective concentration range for D H E A was much broader than that for D H E A S . The results for D H E A and D H E A S taken together indicate that these closely related substances can enhance m e m o r y and alleviate amnesia by their actions in the central nervous system, either directly or via their metabolites. 'In an overwhelming majority of cases, treatments that attenuate experimental amnesia also enhance learning and memory H3. Experiments are in progress to determine whether or not parenterally or orally administered D H E A and D H E A S can also alleviate amnesia and/or enhance learning. Living organisms are programmed to attain certain goals, survival and reproduction. Their functions are aimed at achieving and maintaining maximal behavioral flexibility in reacting to and acting upon the environment. A key organizing principle of adaptive function is the coupling of variability generation to functional demand 23. While cycling freely through all of their operational modes, healthy living systems use their functional capabilities to an extent which is sufficient to ensure a high probability of achieving solutions to the problems with which they are faced. Disease may be said to occur when there is continued uncoupling, for whatever reason, between environmental pressures on living systems and their abilities to adapt to them. When such uncoupling occurs locally or globally in the nervous system as a result of disease, injury, or aging, the whole functional terrain may be disturbed. In such instances, major therapeutic goals are amelioration of resultant functional
361 inadequacies by minimizing progression of degenerative processes and maximizing self-repair by appropriately manipulating rate-limiting factors. Mutually shaping interactions among intracellular, intercellular, and extracellular elements must occur freely in order to enable a system to self-organize into health. We are concerned with the roles of fundamental factors such as pH, pO2 and steroid availability in neuronal/glial survival and differentiation, and in regeneration of injured nerve tissue. Previously we have shown the importance of maintenance of a freeflowing protonic e c o n o m y in experiments on neural membrane transport 24'25 as well as in studies of neuronal survival and neuritogenesis in chick embryo ganglia 26, and injured rat spinal cord n. D H E A and D H E A S were found in the present work markedly to enhance neuronal and glial survival and/or differentiation in dissociated mouse embryo brain culture and memory retention in mice. Although there are many reports of studies with these substances in whole animals and in non-neural tissues and some in neural tissue 4-6'27, definitive molecular functions cannot yet be assigned to them in any instance. Further experiments are now in progress to determine wheth-
er the effects observed by us are specific to D H E A and D H E A S themselves, or whether their precursors or one or more of the several steroids that can be derived from them metabolically are also effective. Nonetheless, it is now not unreasonable to suppose that supplementation with D H E A or D H E A S , normal constituents of the blood, might exert salutary effects by helping reestablish effective communication within and among neural subsystems that are inadequately functional in aging individuals and in those with major nervous system disorders. With the above in mind, clinical studies with orally administered D H E A have been begun in patients with Alzheimer's disease and multiple sclerosis.
1 Bignami, A. and Dahl, D., Specificity of the glial fibrillary acidic protein for astroglia, J. Histochem. Cytochem., 25 (1977) 466-469. 2 Bologa, L., Joubert, R., Bisconte, J.-C., Margules, S., Deugnier, M.-A., Derbin, C. and Herschkowitz, N., Development of immunologically identified brain cells in culture: quantitative aspects, Exp. Brain Res., 53 (1983)
3080-3085. 9 Flood, J.F., Bennett, E.L., Orme, A.E. and Rosenzweig, M.R., Effects of protein synthesis inhibition on memory for active avoidance training, Physiol. Behav., 14 (1975) 177-184. 10 Flood, J.F., Smith, G.E. and Cherkin, A., Hydergine enhances memory in mice, J. Pharmacol., 16 (Suppl. 3) (1985) 39-49. 11 Guth, L., Barrett, C.P., Donati, E.J., Smith, M.V., Lifson, M. and Roberts, E., Enhancement of axonal growth into a spinal lesion by topical application of triethanolamine and cytosine arabinoside, Exp. Neurol., 88 (1985) 44-55. 12 Harrison, R.L. and McKee, P.A., Estrogen stimulates von Willebrand factor production by cultured endothelial cells, Blood, 63 (1984) 657-665. 13 Martinez Jr., J.L., Jensen, R.A. and McGaugh, J.L., Attenuation of experimentally-induced amnesia, Prog. NeurobioL, 16 (1981) 155-186. 14 McEwen, B.S., Biegon, A., Fischette, C.T., Luine, V.N., Parsons, B. and Rainbow, T.C., Toward a neurochemical basis of steroid hormone action. In L. Martini and W.F. Ganong (Eds.), Front. Neuroendocrinology, VoL 8, Raven Press, New York, 1984, pp. 153-176. 15 McGaugh, J.L., Drug facilitation of learning and memory, Ann. Rev. Pharmacol., 13 (1973) 229-241. 16 McGill Jr., H.C. and Sheridan, P.J., Nuclear uptake of sex steroid hormones in the cardiovascular system of the baboon, Orc. Res., 48 (1981) 238-244. 17 Milewich, L., Bagheri, A., Shaw, C.B. and Johnson, A.R., Metabolism of androsterone and 5a-androstane-3a,17fl-
163-167.
3 Bourgeois, S., Pfahl, M. and Baulieu, E.-E., DNA binding properties of glucocorticosteroid receptors bound to the steroid antagonist RU-486, EMBO J., 3 (1984) 751-755. 4 Carette, B. and Poulain, P., Excitatory effect of dehydroepiandrosterone, its sulphate ester and pregnenolone sulphate, applied by iontophoresis and pressure, on single neurones in the septo-preoptic area of the guinea pig, Neurosci. Lett., 45 (1984) 205-210. 5 Corpechot, C., Robel, P., Axelson, M., Sjovall, J. and Baulieu, E.-E., Characterization and measurement of dehydroepiandrosterone sulfate in rat brain, Proc. Natl. Acad. Sci. U.S.A., 78 (1981) 4704-4707. 6 Corpechot, C., Synguelakis, M., Talha, S., Axelson, M., Sjovall, J., Vihko, R., Baulieu, E.-E. and Robel, P., Pregnenolone and its sulfate ester in the rat brain, Brain Research, 270 (1983) 119-125. 7 Dahl, D. and Bignami, A., Preparation of antisera to neurofilament protein from chicken brain and human sciatic nerve, J. Comp. Neurol., 176 (1977) 645-658. 8 Eisenstein, E.M., Altman, H.J., Barraco, D.A., Barraco, R.A. and Lovell, K.L., Brain protein synthesis and memory: the use of antibiotic probes, Fed. Proc., 42 (1983)
Work of E.R. and L.B. was supported in part by the G. Harold and Lila Y. Mathers Charitable Foundation, The Robert W o o d Johnson 1962 Charitable Trust, and that of J.F.F. and G.E.S. by the Medical Research Service of the Veterans Administration, and by the Sepulveda Geriatric Research, Education and Clinical Center ( G R E C C ) . We are indebted to Dr. Doris Dahl for generous gifts of antisera to NF proteins.
362 diol in human lung tissue and in pulmonary endothelial cells in culture, J. Clin. Endocrinol. Metabol.. 60 (1985) 244-250. 18 Orentreich, N., Brind, J.L., Rizer, R.L. and Vogelman, J.H., Age changes and sex differences in serum dehydroepiandrosterone sulfate concentrations throughout adulthood, J. Clin. Endocrinol. Metabol., 59 (1984) 551-555. 19 Parker, L.N. and Odell, W.D., Control of adrenal androgen secretion, Endocrinol. Rev., I (1980) 392-410. 20 Parker, L.N., Levin, E.R. and Lifrak, E.T., Evidence for adrenocortical adaptation to severe illness, J. Clin. Endocrinol. Metabol., 60 (1985) 947-952. 21 Pfahl, M., McGinnis, D., Hendricks, M., Groner, B. and Hynes, N.E., Correlation of glucocorticoid receptor binding sites on MMTV proviral DNA with hormone inducible transcription, Science, 222 (1983) 1341-1343. 22 Roberts, E., Guides through the labyrinth of AD: dehydroepiandrosterone, potassium channels and the C4 component of complement. In T. Crook, R.T. Bartus, S. Ferris and S. Gershon (Eds.), Treatment Development Strategies for Alzheimer's Disease, Mark Powley and Associates, Madison, CT, 1986, pp. 173-219. 23 Roberts, E., What do GABA neurons really do? They make possible variability generation in relation to demand, Exp. Neurol., 93 (1986) 279-290. 24 Roberts, E., Liron, Z. and Wong, E., Potentiation of Na +-
25
26
27 28
29
30
dependent uptake of v-aminobutyric acid in mouse brain particles by buffer-mediated proton removal, Neurochem. Res., 10 (1985) 1025-1046. Roberts, E., Liron, Z., Wong, E. and Schroeder, F., Roles of proton removal and membrane fluidity in Na ÷- and CI-dependent uptake of ~-aminobutyric acid by mouse brain particles, Exp. Neurol., 88 (1985) 13-26. Sisken, B.F., Roberts, E. and Goetz, I., Triethanolamine, Tris, Hepes, and cytosine arabinoside show neuritogenic activity in cultured chick embryo ganglia, Exp. Neurol., 88 (1985) 27-43. Sonka, J., Dehydroepiandrosterone. Metabolic effects, Acta Univ. Carol. Med. Monogr., 71 (1976) 1-171. Toran-Allerand, C.D., Sex steroids and the development of the newborn mouse hypothalamus and preoptic area in vitro: implications for sexual differentiation, Brain Research, 106 (1976) 407-412. Toran-Allerand, C.D., Sex steroids and development of the newborn mouse hypothalamus and preoptic area in vitro: II. Morphological correlates and hormonal specificity, Brain Research, 189 (1980) 413-427. Toran-Allerand, C.D., Gerlach, J.L. and McEwen, B.S., Autoradiographic localization of [3H]estradiol related to steroid responsiveness in cultures of the newborn mouse hypothalamus and preoptic area, Brain Research, 184 (1980) 517-522.
357
BRE 22110
Effects of dehydroepiandrosterone and its sulfate on brain tissue in culture and on memory in mice Eugene Roberts 1, Liane Bologa 1'*, James
F. F l o o d 2 and
Gary E. Smith 2
1Department of Neurobiochemistry, Beckman Research Institute of the City of Hope, Duarte, CA 91010 (U.S.A.) and 2psychobiology Research Laboratory and Geriatric Research, Education and Clinical Center, Veterans Administration Hospital, Sepulveda, CA 91343 (U.S.A.)
(Accepted 18 November 1986) Key words: Dehydroepiandrosterone; Dehydroepiandrosterone sulfate; Tissue culture; Memory
Low concentrations of dehydroepiandrosterone (DHEA) and dehydroepiandrosterone sulfate (DHEAS) enhanced neuronal and glial survival and/or differentiation in dissociated cultures of 14-day mouse embryo brain. Posttrial intracisternal injection into the brains of mice undergoing active avoidance training alleviated amnesia and enhanced long-term memory. By minimizingdegenerative changes in injured nerve tissue and facilitating plastic changes, DHEA and DHEAS may be of use in treatment of neurodegenerative and memory disorders in man. Incoordinations in a number of bodily systems with age generally occur coincidentally with decrements in levels of circulating sex hormones 22. Among the major blood serum steroid classes, monotonic decreases after puberty in both males and females occur only in mean levels of dehydroepiandrosterone (DHEA) and dehydroepiandrosterone sulfate (DHEAS) 18, largely adrenally derived substances which serve as precursors for both androgens and estrogens 19. The stimulatability of release of D H E A and D H E A S by A C T H is reduced markedly in the aging human organism 2°. Administration of D H E A and D H E A S may exert ameliorative effects in such different conditions as diabetes, obesity, autoimmune disease, cancer, and connective tissue and nervous system disorders, possibly by releasing diverse programs of metabolic machinery necessary for effective intracellular and intercellular communication to take place (see ref. 22 for review). D H E A , D H E A S , and the sex-related steroids that derive from them may pass into the brain through microcapillaries, the endothelial cells of which may serve as conduits for, as
well as consumers of, steroids 12'16A7. Inadequate availability of sex steroids and/or their precursors could have grave effects on structure and function of nerve tissue 14 since, in general, steroid hormones are believed to form complexes with specific receptor proteins in the cytoplasm which, after undergoing appropriate transformations, pass into the nucleus where they regulate gene expression 3'21. Without currently being concerned about the partitioning of D H E A , DHEAS, and their metabolites among various cell types and the detailed aspects of the effects of these substances on m R N A transcription, we reasoned that they generally might strengthen metabolic and communication apparatuses in nerve tissue, so as to increase regenerative capacity when injury occurs and facilitate plastic changes that take place during learning. In the present experiments we observed (1) the effects of low concentrations of D H E A and D H E A S on survival and development of dissociated mouse embryo brain in culture, which we assume in some respects to be akin to injured nerve tissue, and (2) the effects of intracisternal injection of these substances in a well-established learning paradigm in
* Present address: L. Bologa, University of Zfirich School of Medicine, Department of Pathology/Neuropathology, 8091 Ziirich, Switzerland. Correspondence.. E. Roberts, Department of Neurobiochemistry, Beckman Research Institute of the City of Hope, Duarte, CA 91010, U.S.A. 0006-8993/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
358
mice l°, d u r i n g t h e a c h i e v e m e n t o f w h i c h significant,
l i e v e d to o c c u r ~. T h e results r e p o r t e d b e l o w e n c o u r
hut as yet u n c h a r a c t e r i z e d , plastic c h a n g e s are be-
age f u r t h e r w o r k a l o n g t h e s e lines.
Fig. 1. Effects of DHEA and DHEAS on embryonic mouse brain cultures, 9 days in vitro (see text), lmmunostained for NF: A: control; B: 10-7 M DHEA; C: 10-8 M DHEAS. lmmunostained for GFAP: D: control; E: 11)-7 M DHEA; F: l(I -~ M DHEAS. Bar = 34.7 /~m. Cultures were prepared from finely chopped, mechanically dissociated brains from 14-day-old Swiss mouse embryos2. Cells were plated at a density of 3 x 105 cells/cm2 on poly-D-lysine-coated glass cover slips and maintained at 37 °C in atmosphere of 5% CO2/80% humidity in Dulbecco-modified Minimum Essential Medium supplemented with 10% fetal calf serum. The medium was first changed at 5 days of culture, at which time solutions of test substances adjusted to pH 7.3 were added. Control cultures received only normal medium. On the 9th day the cultures were fixed and immunostained. Staining was achieved by an indirect immunofluorescence technique, as adapted for brain cell cultures2. Cultures were fixed by successive incubations at room temperature with 3.7% buffered formaldehyde alone and containing 0.2% Triton X-100, each for 10 min. The cultures were rinsed briefly at 4 °C successively with 50%, 100%, and 50% acetone. Incubation with appropriately diluted antisera (usually 1/20-1/30) was carried out at room temperature for 30 min. Incubation with antisera was followed by washings and incubation with fluorescein isothiocyanate-labeled goat anti-rabbit immunoglobulin. Rabbit antiserum to neuron-specific neurofilament triplet of proteins (NF) was used to identify neurons 7, and rabbit antiserum to glial-specific fibrillary acidic protein (GFAP) I was employed to visualize astrocytes. Stained coverslips were mounted in buffered glycerol and examined with an Olympus Vanox microscope equipped with an automatic camera.
359 Dissociated brains of 14-day-old mouse embryos were maintained in culture for 5 days 2, at which time they were found to contain closely similar numbers of cells per dish. Fresh medium alone or medium containing D H E A or DHEAS (10-5-10 -8 M) then was added, and after another 4 days the cultures were fixed and fluorescently immunostained for neurofilament proteins (NF) and glial fibrillary acidic protein (GFAP) 1,7. The preparations were examined thoroughly by fluorescence and phase-contrast microscopy. Positive staining for NF or GFAP is considered to be evidence that cells are either differentiated neurons or astrocytes, respectively. In control cultures, rather sparsely distributed small NF-positive cells with short processes were observed, either isolated from each other or in clusters consisting of a few cells (Fig. 1A). The staining was, at most, of moderate intensity. In media containing D H E A or DHEAS, remarkable increases were found in numbers of NFpositive neurons in comparison with control cultures (Fig. 1B, C). Under higher power (not shown), processes of the NF-positive cells appeared to be greatly extended, thickened, and intertwined. Numerous connections were noted between isolated neurons and greatly enlarged neuronal clusters. The brilliance of the fluorescence shown by the neurons and their processes was greatly enhanced in D H E A or DHEAS-containing media by comparison with the rather dull fluorescent signal observed in the controls, indicating the presence of increased content of NF protein per unit area of cell surface. Many of the NF-positive neurons in the experimental cultures were larger than those seen in the controls. Maximal enhancements were seen at 10-7 M D H E A and 10-8 M DHEAS, respectively, among the concentrations tested, the neuronal clusters being somewhat larger with DHEAS. Highly significant increases over control values were found in numbers of NF-positive cells per 380 mm 2 coverslip (P < 0.01; 24 determinations in 3 separate experiments): control, 107 + 10 x 103; D H E A (10 -7 M), 983 + 53 x 103; DHEAS (10 -s M), 997 + 49 x 103. The latter increases could be attributable to enhanced neuronal survival and/or to an acceleration of the differentiation into neurons of precursor cells, but not to mitosis of NF-positive cells, themselves, since separate experiments (to be reported elsewhere) showed no incorporation of [3H]thymidine, whatsoever, to have taken place into
such cells under standard conditions of study. Remarkable increases over the controls (Fig. 1D) were also noted in the presence of D H E A (Fig. 1E) and DHEAS (Fig. 1F) in numbers of GFAP-positive astrocytes, in the extensions of their processes, and in the brilliance of their fluorescent staining, indicative of increased amounts of total GFAP as well as amount per unit cell surface. Since a number of experiments showed clearly that incorporation of [3H]thymidine was less in the cultures with D H E A and DHEAS than in the controls, the results suggest that the steroids enhance the survival of initially plated astrocytes and/or the differentiation of GFAPpositive astrocytes from stem cells, while decreasing mitosis. Taken together, the above results show that in both neurons and glia, D H E A and DHEAS enhance the expression of properties related to postmitotic differentiated states. In this regard, it is of great interest that estradiol-17fl and testosterone, both metabolites of D H E A , elicited growth and aborization of neurites in cultures of new-born mouse brain hypothalamic/preoptic area and that the neuritic response to estradiol occurred in [3H]estradiol-accumulating cells28-3°. Quantitative measurements are being made in experiments similar to those above of contents of NF, GFAP, and other neuronal and glial specific proteins as well as of relevant mRNAs. Experiments were performed employing an active avoidance behavioral paradigm (buzzer footshock) in a T-maze 9 with groups of 15 mice per dose. Injections of test solutions were made within 3 min after training. One week after training, retention for Tmaze training was tested. It was established that 2 ~1 intracerebroventricular (i.c.v.) injections of solutions containing D H E A or DHEAS had enhancing effects on long-term memory retention (Fig. 2). DHEA, not readily soluble in saline, was dissolved in dimethyl sulfoxide (DMSO), which was shown in preliminary experiments to cause amnesia when 2/A was injected. Taking advantage of the latter observation, we used well-trained animals to determine whether or not D H E A dissolved in DMSO could prevent the DMSO-induced amnesia. Training was performed under conditions that maximize learning (see legend to Fig. 2A), and the mice were retested one week later. Mice making a successful avoidance in 3 trials were classed as remembering the original training, a criterion providing optimal separation between
360 A
80
SALINE
6C 4C
• p > 005 p < 0.05 ® p < 001
20 - - DMSO ALONE
:~
0 8O
DHEAS IN SALINE./~ 6O 40
20
SALINE A L O N E -1~2
-I1
' -I0
-9
LOG DOSE (moles/mouse, ICV)
Fig. 2. Effects of intracerebroventricularly injected DHEA and DHEAS on memory retention in mice (see refs. 9 and I0 for further experimental details). A: DHEA in DMSO in welltrained animals: 5 training trials in the T-maze; the buzzer, loud; intertrial interval, 45 s; footshock level, 0.35 mA. B: DHEAS in saline in poorly trained animals: 4 training trials; buzzer, muffled; intertrial interval, 30 s; footshock level, 0.30 mA. Injections of test solutions were made within 3 min after training. Retention was tested one week after the training trials. naive and well-trained animals9; and the percentages of animals meeting the criterion (recall scores) were recorded. Because standard deviations vary among identical mean values calculated in this fashion, the same mean recall scores may show different P-values when tested for significance of changes from the controls. The saline control group showed good retention (80% recall) (Fig. 2A), while the group receiving D M S O alone was reduced to 20%, Dunnett's ttest showing a significant difference between them (P < 0.01). An A N O V A of results for all groups showed a significant enhancing effect of D H E A on m e m o r y retention (F14,210 = 5.13, P < 0.001). Individually, 10 of the 13 doses improved retention relative to the D M S O group at P < 0.01, one at P < 0.05, and 3 were not statistically significant. Doses between 8.4 × 10 -11 and 5.4 × 10 -l° mol/mouse gave results similar to those with saline controls, completely overcoming the amnesic effect of the D M S O in which they were contained. The two plateaus in the d o s e - response curve (Fig. 2A) suggest that more than one rate-limiting process may be affected under the experimental conditions employed.
Experiments with D H E A S (Fig. 2B) were performed similarly to those above, with the exception that training conditions were adjusted so that initial recall in the saline controls was only 20%, making it easier to detect whether or not there was an enhancing effect on memory. A n A N O V A showed a significant effect (F8,12 6 = 2.59, P < 0.025) over all 8 doses tested, which corresponded to the higher levels of D H E A tested. Preliminary experiments showed lower doses of D H E A S to be ineffective. Injection of D H E A S significantly increased the recall scores at 4 concentrations between 4.4 x 10-1° and 6.9 x 10-1° mol/mouse. The memory-enhancing effects of D H E A and D H E A S were maximal at 5.4 x 10 -1° and 6.2 x 10 -1° mol/mouse, respectively, and decreased at higher levels in a manner typical for most substances that improve memory ~5. The effective concentration range for D H E A was much broader than that for D H E A S . The results for D H E A and D H E A S taken together indicate that these closely related substances can enhance m e m o r y and alleviate amnesia by their actions in the central nervous system, either directly or via their metabolites. 'In an overwhelming majority of cases, treatments that attenuate experimental amnesia also enhance learning and memory H3. Experiments are in progress to determine whether or not parenterally or orally administered D H E A and D H E A S can also alleviate amnesia and/or enhance learning. Living organisms are programmed to attain certain goals, survival and reproduction. Their functions are aimed at achieving and maintaining maximal behavioral flexibility in reacting to and acting upon the environment. A key organizing principle of adaptive function is the coupling of variability generation to functional demand 23. While cycling freely through all of their operational modes, healthy living systems use their functional capabilities to an extent which is sufficient to ensure a high probability of achieving solutions to the problems with which they are faced. Disease may be said to occur when there is continued uncoupling, for whatever reason, between environmental pressures on living systems and their abilities to adapt to them. When such uncoupling occurs locally or globally in the nervous system as a result of disease, injury, or aging, the whole functional terrain may be disturbed. In such instances, major therapeutic goals are amelioration of resultant functional
361 inadequacies by minimizing progression of degenerative processes and maximizing self-repair by appropriately manipulating rate-limiting factors. Mutually shaping interactions among intracellular, intercellular, and extracellular elements must occur freely in order to enable a system to self-organize into health. We are concerned with the roles of fundamental factors such as pH, pO2 and steroid availability in neuronal/glial survival and differentiation, and in regeneration of injured nerve tissue. Previously we have shown the importance of maintenance of a freeflowing protonic e c o n o m y in experiments on neural membrane transport 24'25 as well as in studies of neuronal survival and neuritogenesis in chick embryo ganglia 26, and injured rat spinal cord n. D H E A and D H E A S were found in the present work markedly to enhance neuronal and glial survival and/or differentiation in dissociated mouse embryo brain culture and memory retention in mice. Although there are many reports of studies with these substances in whole animals and in non-neural tissues and some in neural tissue 4-6'27, definitive molecular functions cannot yet be assigned to them in any instance. Further experiments are now in progress to determine wheth-
er the effects observed by us are specific to D H E A and D H E A S themselves, or whether their precursors or one or more of the several steroids that can be derived from them metabolically are also effective. Nonetheless, it is now not unreasonable to suppose that supplementation with D H E A or D H E A S , normal constituents of the blood, might exert salutary effects by helping reestablish effective communication within and among neural subsystems that are inadequately functional in aging individuals and in those with major nervous system disorders. With the above in mind, clinical studies with orally administered D H E A have been begun in patients with Alzheimer's disease and multiple sclerosis.
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3 Bourgeois, S., Pfahl, M. and Baulieu, E.-E., DNA binding properties of glucocorticosteroid receptors bound to the steroid antagonist RU-486, EMBO J., 3 (1984) 751-755. 4 Carette, B. and Poulain, P., Excitatory effect of dehydroepiandrosterone, its sulphate ester and pregnenolone sulphate, applied by iontophoresis and pressure, on single neurones in the septo-preoptic area of the guinea pig, Neurosci. Lett., 45 (1984) 205-210. 5 Corpechot, C., Robel, P., Axelson, M., Sjovall, J. and Baulieu, E.-E., Characterization and measurement of dehydroepiandrosterone sulfate in rat brain, Proc. Natl. Acad. Sci. U.S.A., 78 (1981) 4704-4707. 6 Corpechot, C., Synguelakis, M., Talha, S., Axelson, M., Sjovall, J., Vihko, R., Baulieu, E.-E. and Robel, P., Pregnenolone and its sulfate ester in the rat brain, Brain Research, 270 (1983) 119-125. 7 Dahl, D. and Bignami, A., Preparation of antisera to neurofilament protein from chicken brain and human sciatic nerve, J. Comp. Neurol., 176 (1977) 645-658. 8 Eisenstein, E.M., Altman, H.J., Barraco, D.A., Barraco, R.A. and Lovell, K.L., Brain protein synthesis and memory: the use of antibiotic probes, Fed. Proc., 42 (1983)
Work of E.R. and L.B. was supported in part by the G. Harold and Lila Y. Mathers Charitable Foundation, The Robert W o o d Johnson 1962 Charitable Trust, and that of J.F.F. and G.E.S. by the Medical Research Service of the Veterans Administration, and by the Sepulveda Geriatric Research, Education and Clinical Center ( G R E C C ) . We are indebted to Dr. Doris Dahl for generous gifts of antisera to NF proteins.
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