- Open Access
Age and cognitive status dependent differences in blood steroid and thyroid hormone concentrations in intact male rats
Behavioral and Brain Functions volume 15, Article number: 10 (2019)
Age-dependent alterations of hormonal states have been considered to be involved in age related decline of cognitive abilities. Most of the studies in animal models are based on hormonal substitution in adrenal- and/or gonadectomized rodents or infusion of steroid hormones in intact rats. Moreover, the manipulations have been done timely, closely related to test procedures, thus reflecting short-term hormonal mechanisms in the regulation of learning and memory. Here we studied whether more general states of steroid and thyroid hormone profiles, independent from acute experiences, may possibly reflect long-term learning capacity. A large cohort of aged (17–18 months) intact male rats were tested in a spatial hole-board learning task and a subset of inferior and superior learners was included into the analysis. Young male adult rats (16 weeks of age) were also tested. Four to 8 weeks after testing blood plasma samples were taken and hormone concentrations of a variety of steroid hormones were measured by gas chromatography-tandem mass spectrometry or radioimmunoassay (17β-estradiol, thyroid hormones).
Aged good learners were similar to young rats in the behavioral task. Aged poor learners but not good learners showed higher levels of triiodothyronine (T3) as compared to young rats. Aged good learners had higher levels of thyroid stimulating hormone (TSH) than aged poor learning and young rats. Both aged good and poor learners showed significantly reduced levels of testosterone (T), 4-androstenedione (4A), androstanediol-3α,17β (AD), dihydrotestosterone (DHT), 17-hydroxyprogesterone (17OHP), higher levels of progesterone (Prog) and similar levels of 17β-estradiol (E2) as compared to young rats. The learning, but not the memory indices of all rats were significantly and positively correlated with levels of dihydrotestosterone, androstanediol-3α,17β and thyroxine (T4), when the impacts of age and cognitive division were eliminated by partial correlation analyses.
The correlation of hormone concentrations of individuals with individual behavior revealed a possible specific role of these androgen and thyroid hormones in a state of general preparedness to learn.
Age-dependent decline of cognitive abilities in elderly men has been mostly but not exclusively related to decreased testosterone release  and testosterone substitution is used as therapeutic intervention, however controversially discussed  and with opposing results [3,4,5]. However, testosterone is catabolized into several neuroactive and learning and memory affecting steroids such as dihydrotestosterone, which is the most potent androgen receptor agonist, and also in 17β-estradiol by the enzyme aromatase. Estrogen receptors and aromatase are present in brains of male subjects and can exert learning and memory relevant functions [6, 7]. Corticosterone (in rodents) andcortisol (in humans) are stress hormones involved in learning and memory as well as synaptic and neuronal plasticity, when modulations are timely related to the formation of long-term memories. Corticosterone application after acquisition support memory consolidation [8, 9], whereas it impairs memory retrieval when given shortly (30 min) but not hours before a memory retention test . Besides these steroid hormones, thyroid hormones could also be related to cognitive decline in men [11,12,13]. Due to this variety of neuroactive hormones that can be independently or interactively regulated, it is feasible that the individual profiles of different steroid hormones, rather than the levels of certain hormones determine the cognitive status, especially in aged subjects.
Most of the studies in animal models on steroid hormone related cognitive processes are based on hormonal substitution in adrenal- and/or gonadectomized rodents or infusion of steroid hormones in intact rats. Moreover, the manipulations have been done timely, closely related to test procedures, thus reflecting short-term hormonal mechanisms in the regulation of learning and memory. Here we were interested in a more general state of steroid hormone profiles independently from acute experiences, possibly reflecting long-term learning capacity. For this reason we tested a large cohort of aged (17–18 months) intact male rats in a spatial holeboard learning task and included a subset of inferior and superior learners into the analysis. Young male adult rats (16 weeks of age) were also tested. Four to 8 weeks after testing the blood hormone concentrations of a variety of steroid hormones were measured by gas chromatography-tandem mass spectrometry or radioimmunoassay (17β-estradiol, thyroid hormones).
The study aimed at elucidating age-related differences in hormone levels that may explain differences in cognitive states of aged and young rats and age-independent possible hormonal markers of cognitive abilities.
Aged (17–18 months) and young (4 months) male Sprague–Dawley rats, bred and maintained in the Core Unit of Biomedical Research, Division of Laboratory Animal Science and Genetics, Medical University of Vienna were used. Rats were housed in groups of three in standard Makrolon cages filled with autoclaved woodchips (temperature: 22 ± 2 °C; humidity: 55 ± 5%; 12 h artificial light/12 h dark cycle: light on at 7:00 a.m.). Tap water and food (ssniff, R/M-H Ered II, Soest, Germany) was provided ad libitum. The study was carried out according to the guidelines of the Ethics committee, Medical University of Vienna, and were approved by the Federal Ministry of Education, Science and Culture, Austria.
In order to avoid differences in steroid hormone levels due to age related different circadian rhythms blood samples were taken during the light phase at different times, but timely matched between groups. Samples were collected 4–8 weeks after the behavioral test. Animals were anesthetized with Nembutal (40 mg/kg bodyweight) and Heparin (Gilvasan Pharma GmbH, Vienna, Austria) was injected through the tail vein (1000 I.E/kg body weight). After 10 min the animals were decapitated and trunk blood was sampled and centrifuged (9000 rpm for 15 min). Plasma was aliquoted and stored at − 80 °C until measurements.
The animals were pre-screened for cognitive abilities with the hole-board test. All groups underwent this test procedure before samples were taken. The hole-board board (1 m × 1 m) was manufactured of black plastic surrounded by translucent plexiglass walls. Walls were equipped with proximal spatial cues, and surrounding room structures served as distal cues. Four out of sixteen regularly arranged holes (diameter and depth 7 cm) were baited (dustless precision pellets, 45 mg, Bioserv®, Flemington, NJ; USA) with the pattern of baited holes remained the same during the entire test. A second board below the first was provided with scattered food pellets to avoid olfactory orientation. Ten min handling sessions per day for 4 days prior to the experiment made the rats familiar to the experimenter. The following 2 days animals were habituated to the hole-board by free exploration of the maze for 15 min each day with access to food pellets. Controlled food restriction reduced the weight of the rats to reach 85% of their initial body weight. Tap water was given ad libitum. Training consisted of 3 days (five trials on day one, four trials on day 2 and a retention trial at day 3) with an intertrial interval of 20 min for individual rats. Trial duration was 120 s or until all four pellets were eaten. The apparatus was cleaned with 0.1% Incidin between trials in order to remove odor cues of individual rats. Performance of the rats was recorded by a video camera and stored on a computer. The hole visits and removals of pellets were noted for each trial. In order to compare rats with similar levels of motivation, rats with less than 40 hole visits in total over the ten trials were excluded from the analysis.
Reference memory errors were noted as the number of visits to the unbaited holes. Reference memory index (RMI) was calculated using the formula (first + revisits of baited holes)/total visits of all holes. All behavioral training/testing was performed during the light phase of the light–dark cycle. Learning index was calculated as the mean value of reference indices of trials 6–9 at day 2. Memory index is represented by the reference memory index of the retention trial 10 (day 3).
Poor learners were defined when having either learning or memory indices lower than one standard deviation from the mean and good learners when having indices one standard deviation higher than the mean. The rats analyzed in the present study were randomly chosen from good (19 animals) and poor (15 animals) performing animals from a larger cohort of rats (n = 127) with more than 40 hole visits.
Gas chromatography-tandem mass spectrometry (GC–MS/MS) was performed to measure steroid hormones. Briefly, samples were equilibrated with deuterated internal standards, extracted using Extrelut® NT columns and purified using Sephadex LH-20 mini columns. Thereafter, heptafluorobutyrate derivatives were prepared . Gas chromatography was performed on an Optima® 1-MS capillary column (25 m × 0.2 mm I.D., df 0.1 µm, Macherey–Nagel, Düren, Germany) housed in a Thermo Scientific Trace 1310 Gas Chromatograph with a TriPlus RSH Autosampler coupled to a TSQ 8000 triple quadrupole MS (Thermo Scientific, Dreieich, Germany). Helium was used as carrier gas at 1.0 mL/min. The injector temperature was 270 °C and the initial column temperature was set at 80 °C. The steroids of interest eluted at a rate of 3 °C/min until the column temperature reached 242 °C. The following MRM or m/z ratios were measured for the analytes and their corresponding internal standards: m/z 665.1 (668.1) for testosterone (T) (d3-T), m/z 482.2/482.2 (484.3/484.3) for 4-androstenedione (4A) (d2-4A), m/z 455.3/241.3 (458.3/244.4) for androstanediol-3α,17β (AD) (d3-AD), m/z 270.2/121.1 (272.2/123.1) for DHEA (d2-DHEA), m/z 414.1/185.2 (417.2/188.2), for Dihydrotestosterone (DHT) (d3-DHT), m/z 465.2/109.1 (469.1/113.1) for 17-Hydroxyprogesterone (17OHP) (d4-17OHP), m/z 467.2/253.0 (471.3/256.3) for 17-Hydroxypregnenolone (17OH5P) (d7-17OH5P), m/z 465.2/109.1 (467.2/109.1) for 11-deoxycortisol (S) (d2-S),705.1/355.1 (712.1/359.2) for corticosterone (B) (d8-B), and m/z 510.2/495.2 (/518.3/503.4) for progesterone (Prog) (d9-Prog).
Concentrations of 17β-estradiol (E2) were measured by a sequential radioimmunoassay applying tritiated tracer and an antiserum generated against E2-6-carboxymethyl oxime—BSA after extraction of the samples with toluene .
Total T3 and total T4 were measured by a radioimmunoassays (Beckman Coulter, Krefeld, Germany). TSH was measured using an immunoradiometric assay (Beckman Coulter, Krefeld, Germany).
The hormones that were quantitatively identified are listed in Table 1. For three hormones (DHEA, 17OH5P and S) all values of all groups were below the limit of detection, therefore these hormones were excluded from the analysis. Values below the detection limit were set to half of the limit value and included in the analysis. Number of measurements below the detection limit was: 4A: 8 aged good, 7 aged poor, 4 young; AD: 1 aged good, 5 aged poor, 2 young; DHT: 4 aged good, 2 aged poor, 17OHP: 5 aged good, 8 aged poor, 2 young; Prog: 8 young; TSH: 1 aged good, 8 aged poor, 9 young.
Group differences between hormone levels and behavior were analyzed by two-way multivariate general linear model (ANOVA) with hormone levels and age/performance as factors and subsequent Bonferroni post hoc tests. Correlations between individual levels of hormones and learning and memory indices were done by partial correlation analyses with age and predetermined cognitive status as controlling variables. Sample sizes: aged good (n = 10), aged poor (n = 10), young (n = 10). Analyses were done by using SPSS statistics program (V. 20).
Group differences in learning and memory
The results are summarized in Fig. 1. We could determine an overall difference in behavioral performance between groups both in learning (F2,27 = 149.4, p < 0.0001) and memory (F2,27 = 40.6, p < 0.0001) indices. Aged good-learning rats show significantly better performance in learning and memory as compared to aged poor-learning rats (p < 0.001, each) but not as compared to young rats (p = 1.0, each). Young rats performed better as compared to aged poor-learning rats (p < 0.001, each).
Age-dependent differences in hormone levels
The results are given in Figs. 2, 3. Overall significant effects between groups could be detected. Testosterone (T): F2,27 = 9.92, p = 0.001; 4-Androstenedione (4A): F2,27 = 5.34, p = 0.011; Androstanediol-3α,17β (AD): F2,27 = 7.11, p = 0.003; Dihydrotestosterone (DHT): F2,27 = 19.95, p < 0.001; 17-Hydroxyprogesterone (17OHP): F2,27 = 6.97, p = 0.004; Progesterone (Prog): F2,27 = 7.56, p = 0.002; Corticosterone (B): F2,27 = 3.46, p = 0.046; Triiodothyronine (T3): F2,27 = 4.89, p = 0.015; Thyroxine (T4): F2,27 = 5.54, p = 0.010; and Thyroid-stimulating- hormone (TSH): F2,27 = 5.65, p = 0.009, but not 17β-estradiol (E2): F2,27 = 1.37, p = 0.271.
Post-hoc tests revealed higher levels of young vs. aged good learners or aged poor learners for testosterone (p = 0.004 and p = 0.001; respectively), 4-Androstenedione (p = 0.022 and p = 0.023; respectively); Dihydrotestosterone (p < 0.001 and p < 0.001; respectively); 17-Hydroxyprogesterone (p = 0.019 and p = 0.005; respectively). Young rats showed lower levels of Progesterone as compared to aged good and aged poor rats (p = 0.003 and p = 0.021; respectively). No significant differences for these hormones could be determined between aged good learners and aged poor learning rats (p > 0.05 each). Androstanediol-3α,17β levels where higher in young vs. aged poor learners (p = 0.003) and aged good learning rats (p = 0.048), with no differences between aged good and aged poor learning rats (0.521). Triiodothyronine titers were higher in aged poor learners as compared to young (p = 0.013), but there was no difference between aged poor and aged good learners (p = 0.328) or aged good learners and young rats (p = 0.459). Thyroxine levels were lower in aged good learners as compared to young (p = 0.009), but no difference could be found when compared to aged poor learners (p = 0.944) and no difference between the two latter was observed (p = 0.102). Thyroid-stimulating-hormone levels were elevated in aged good as compared to aged poor learners (p = 0.018) and young (p = 0.025) rats, but no difference could be determined between aged poor learners and young rats (p = 1). We could not detect significant differences between single groups for Corticosterone (young vs. aged good p = 0.147; young vs. aged poor p = 0.064; aged good vs. aged poor p = 1)) and 17β-estradiol (young vs. aged good p = 1; young vs. aged poor p = 0.434; aged good vs. aged poor p = 0.559).
The results of partial correlations with learning and memory indices are summarized in Table 2. Two control variables were used, one with predetermined cognitive status dividing the sample into good (aged good and young) and poor (aged poor) learners and the other with age, dividing the sample into aged (aged good and aged poor) and young rats. Partial correlations allows to avoid misleading results if confounding variables numerically related to both variables of interest. Confounding variables here are the predetermined separation of good and poor learners and the other is the difference in age. Thus, correlations between cognitive states and hormones independently of the predetermination can be calculated. Similarly with the second control variable correlations independently of age can be determined. Significant correlations of Androstanediol-3α,17β, dihydrotestosterone and thyroxine levels could be found with learning but not with memory indices.
In Table 3 the partial correlations between individual hormone levels are presented. Whereas most of the androgenic hormone levels are positively intercorrelated and correlated with 17-Hydroxyprogesterone, the levels of T3 and T4 are positively intercorrelated but not correlated with TSH, which levels are inversely correlated with progesterone. In addition we found a positive correlation between progesterone and corticosterone levels.
In order to reveal hormone related long-term learning capacities independent from actual learning experiences blood plasma was sampled 4–8 weeks after a hole-board test, which was conducted to test for individual cognitive capacities. Although we cannot completely rule out that during this time period age related changes in hormone status may take place, this is unlikely. Tang  and Waner and Nyska  found only slight differences of thyroid hormones in male rats at ages comparable with the present study and even at larger differences in age (12–18 months). Similarly, testosterone is slightly affected during the age period considered in the present study . We found age dependent differences between steroid and thyroid hormones independently from cognitive status and also cognitive status dependent different results between aged and young animals. Namely the levels of Androstanediol-3α,17β are significantly reduced in aged poor and significantly (but close to the border of significance) in aged good learning rats as compared to young rats. The levels of TSH are significantly enhanced in aged good as compared to aged poor learners and young rats. Further, age independent positive correlations with learning but not memory indices could be detected for AD, DHT and T4.
AD, a metabolite of dihydrotestosterone (DHT), is a neuro-steroid binding to the gamma-aminobutyric acid (GABAA) receptor as a positive allosteric modulator increasing GABA responses up to 50% in hippocampal CA1 pyramidal cells effectively regulating neuronal excitability . AD has been shown to interact with cytoplasmic estrogen receptors in the brain, although to a much lesser extent than its 3β isomer . AD has been described to have rewarding and anxiolytic effects [21, 22]. AD has also effects on learning, conditioned place preference was enhanced by sub-chronic application of AD to a higher extent than by administration of DHT or testosterone . Gestational stress in male rats produced behavioral inhibition in adult life, correlated with increased levels of corticosterone and reduced levels of DHT and AD . AD but not testosterone application reinstates age-related impaired cognitive performance in aged male rats and enhanced performance in spatial learning (water maze) irrespective of age . However, intrahippocampal infusion of AD impaired water maze performance in adult male rats and decreased transcription levels of protein kinase A (PKA) . PKA is a critical mediator of spatial learning and memory and synaptic plasticity [27,28,29,30].
DHT is catabolized from testosterone by the enzyme 5α reductase and is a considerably more potent agonist of the androgen receptor than testosterone in peripheral  and brain tissue . Although, testosterone levels decline with age, there is little evidence that testosterone substitution rescues spatial cognitive abilities in aged rats, however it affects memory in young rats , whereas in humans most, but not all, of the studies report enhancement of cognition after testosterone replacement in healthy aged men . DHT is metabolized into AD by the enzyme 3α-HSD (3α-hydroxysteroid dehydrogenase). The partial correlation analysis in the present study support the view that not testosterone itself, but the metabolites DHT and AD are involved in the determination of learning capacities in an age-independent manner. The synthesis of AD may be in part independent of the availability of DHT. Although there are significantly decreased levels of DHT and AD in both aged groups compared to young rats, the significance is weak in aged good learners and the levels of AD are slightly higher as in the aged poor learners. This may be regulated by different levels or activity of 3α-HSD in aged good vs. aged poor rats. 3α-HSD hippocampal mRNA levels decrease with age in rats , which can be attenuated by environmental enrichment experience. Intrahippocampal application of indomethacin, a 3α-HSD inhibitor, impairs leaning but not memory consolidation in a spatial water maze task . However, the present group specific AD data only allow limited interpretations but should be proved in further studies.
The correlation data suggest these neuroactive steroids may represent a age independent marker for a consistently elevated learning capacity, whereas the formation of a long-term memory is probably regulated by short-term hormonal mechanisms closely related to the memory acquisition and consolidation phases and becomes independent from hormonal states long after consolidation. Scheinert et al.  found correlations over young, middle aged and aged rats of some cytokines, chemokines, corticosterone and adrenocorticotropic hormone (ACTH) from samples taken 2 weeks after water maze training with learning and memory indices and differences in concentrations in serum, hippocampus and cortex in dependence of the cognitive status of the rats. Thus, the cognitive status is reflected in some physiological parameters over a long time. Similarly, Issa et al.  found that HPA axis dysfunction in aged rats is associated with spatial memory impairments and not merely a function of age. Age dependent and independent hypothalamus–pituitary–adrenal (HPA)-axis adjustments to determine learning abilities were also found by Meijer et al. . Aged inferior but not superior male rat learners show a positive correlation of arginine vasopressin mRNA in the parvocellular nucleus of the hypothalamus with basal blood corticosterone levels, suggesting impaired glucocorticoid sensitivity. Here we did not find a correlation of corticosterone with learning and memory indices. However, corticosterone can be synthetized independently of the HPA axis activity from progesterone in rat testis [39, 40]. Thus, the levels of corticosterone, especially in aged rats, may be in part based on the highly available progesterone, which is also suggested by the positive correlation of progesterone and corticosterone at individual levels. This correlation can be also found, when young rats are excluded. Enhanced progesterone levels in aged male rats as shown here have been reported previously [41, 42]. The increased progesterone levels contribute to the suppression of gonadotropins and impaired reproductive functions in aged males .
Literature results related to effects of aging on thyroid hormones are controversial, age dependent decline of T3 and T4 in blood of male rats [16, 17, 44] or no differences  have been reported. Diminished levels of T4 but not T3 have also been found . While some found decreased levels of TSH over age  others found no difference [44, 46, 47] or increased levels of TSH in aged males .
Thyroid hormones especially improve hippocampus-dependent learning and memory and hippocampal synaptic plasticity [49,50,51] as well as hippocampal neurogenesis . Thyroxine treatment improves spatial learning in a water maze probably by induced increased cholinergic activity  and rescues spatial cognitive deficits and dentate gyrus electrical activity in a rat model of Alzheimer’s disease . T3 and T4 reduce GABA-evoked and spontaneous inhibitory synaptic currents up to 50%, whereas T4 in contrast to T3 was ineffective in decreasing extra-synaptic GABA currents . Thus, possibly T3 and T4 in conjunction with AD may effectively regulate hippocampal and probably extrahippocampal network activities to facilitate cognitive functionality during spatial learning.
TSH levels have been found to be positively related to episodic memory in aged humans (75–96 years) independently from the actual age . However, van Boxtel et al.  found a weak inverse relation of TSH and cognition in aged individuals, which was dependent on mood status. TSH shows potent neuroprotective properties. TSH injections protected against electroconvulsive disruption of memory retrieval. This effect was independent from the TSH induced levels of plasma T3 and T4 . Early thyroxine treatment improves spatial learning and memory and enlarges intra- and infrapyramidal mossy fiber projections in the hippocampus. Individual sizes of these projections were positively correlated with radial maze performance .
Thus, TSH in the present study may have cognitive enhancing functions in aged but not young rats independently of T3 and T4. Metanalytic studies in humans revealed an association of TSH with poor cognitive performance in younger but better performance in older subjects on a variety of tests, whereas thyroxine levels show such a relation only for a single test . Low TSH levels could be related to a progression of cognitive impairment to dementia .
The present study, by analyzing a large number of hormones in the same individuals, can point to some possible underlying mechanisms of hormonal learning and memory modulations in an age dependent and independent manner. Especially the role of TSH as a potential biomarker for cognitive decline in elderly but not young subjects, and the applicability of dihydrotestosterone, androstanediol-3α,17β and thyroxine as age independent biomarkers for hormone related alterations of cognitive abilities should be proved in further studies. This studies should also include a measure of these critical hormones before and after behavioral testing, which would be possible by the decreased amount of plasma that is needed for the analysis. Further measurements in brain tissue are of interest.
The major outcome of the study is that aged good learners were similar to young rats. Aged poor learners, but not good learners showed higher levels of triiodothyronine as compared to young rats. Aged good learners had higher levels of thyroid stimulating hormone than aged poor learning and young rats. Both, aged good and poor learners showed significantly reduced levels of testosterone, 4-androstenedione, androstanediol-3α,17β, dihydrotestosterone, 17-hydroxyprogesterone, higher levels of progesterone and similar levels of 17β-estradiol as compared to young rats. The learning, but not the memory indices of all rats were significantly and positively correlated with levels of dihydrotestosterone, androstanediol-3α,17β and thyroxine, when the impacts of age and cognitive division were eliminated by partial correlation analyses. Analysis of individual hormonal profiles rather than group comparisons revealed a possible specific role of these androgen and thyroid hormones in a state of general preparedness to learn.
reference memory index
protein kinase A
Wolf OT. Cognitive functions and sex steroids. Ann Endocrinol. 2003;64:158–61.
Busnelli A, Somigliana E, Vercellini P. ‘Forever Young’-Testosterone replacement therapy: a blockbuster drug despite flabby evidence and broken promises. Hum Reprod. 2017;32:719–24.
Krause W, Mueller U, Mazur A. Testosterone supplementation in the aging male: which questions have been answered? Aging Male. 2005;8:31–8.
Huang G, Wharton W, Bhasin S, Harman SM, Pencina KM, Tsitouras P, Li Z, Hally KA, Asthana S, Storer TW, Basaria S. Effects of long-term testosterone administration on cognition in older men with low or low-to-normal testosterone concentrations: a prespecified secondary analysis of data from the randomised, double-blind, placebo-controlled TEAAM trial. Lancet Diabetes Endocrinol. 2016;4:657–65.
Resnick SM, Matsumoto AM, Stephens-Shields AJ, Ellenberg SS, Gill TM, Shumaker SA, Pleasants DD, Barrett-Connor E, Bhasin S, Cauley JA, Cella D, Crandall JP, Cunningham GR, Ensrud KE, Farrar JT, Lewis CE, Molitch ME, Pahor M, Swerdloff RS, Cifelli D, Anton S, Basaria S, Diem SJ, Wang C, Hou X, Snyder PJ. Testosterone treatment and cognitive function in older men with low testosterone and age-associated memory impairment. JAMA. 2017;317:717–27.
Frye CA, Duffy CK, Walf AA. Estrogens and progestins enhance spatial learning of intact and ovariectomized rats in the object placement task. Neurobiol Learn Mem. 2007;88:208–16.
Lagunas N, Calmarza-Font I, Grassi D, Garcia-Segura LM. Estrogen receptor ligands counteract cognitive deficits caused by androgen deprivation in male rats. Horm Behav. 2011;59:581–4.
Sandi C, Loscertales M, Guaza C. Experience-dependent facilitating effect of corticosterone on spatial memory formation in the water maze. Eur J Neurosci. 2007;9:637–42.
Hui GK, Figueroa IR, Poytress BS, Roozendaal B, McGaugh JL, Weinberger NM. Memory enhancement of classical fear conditioning by post-training injections of corticosterone in rats. Neurobiol Learn Mem. 2004;81:67–74.
deQuervain DJ, Roozendaal B, McGaugh JL. Stress and glucocorticoids impair retrieval of long-term spatial memory. Nature. 1998;394:787–90.
Wahlin A, Wahlin TB, Small BJ, Bäckman L. Influences of thyroid stimulating hormone on cognitive functioning in very old age. J Gerontol B Psychol Sci Soc Sci. 1998;53:P234–9.
Beydoun MA, Beydoun HA, Shroff MR, Kitner-Triolo MH, Zonderman AB. Serum leptin, thyroxine and thyroid-stimulating hormone levels interact to affect cognitive function among US adults: evidence from a large representative survey. Neurobiol Aging. 2012;33:1730–43.
Moon JH, Park YJ, Kim TH, Han JW, Choi SH, Lim S, Park DJ, Kim KW, Jang HC. Lower-but-normal serum TSH level is associated with the development or progression of cognitive impairment in elderly: Korean Longitudinal Study on Health and Aging (KLoSHA). J Clin Endocrinol Metab. 2014;99:424–32.
Wudy SA, Schuler G, Sánchez-Guijo A, Hartmann MF. The art of measuring steroids. Principles and practice of current hormonal steroid analysis. J Steroid Biochem Mol Biol. 2018;179:88–103.
Klein R, Schams D, Failing K, Hoffmann B. Investigations on the re-establishment of the positive feedback of estradiol during anoestrus in the bitch. Reprod Domest Anim. 2003;38:13–20.
Tang F. Effect of sex and age on serum aldosterone and thyroid hormones in the laboratory rat. Horm Metab Res. 1985;17:507.
Waner T, Nyska A. Thyroxine (T4) and triiodothyronine (T3) levels in the Fischer 344 inbred rat. Lab Anim. 1998;22:276–80.
Silva MA, Villaseñor RM, Márquez SR, González MH, Jaime HB, García XG, Montiel JL. Testosterone levels and development of the penile spines and testicular tissue during the postnatal growth in wistar rats. Adv Sex Med. 2013;3:1–9.
Reddy DS, Jian K. The testosterone-derived neurosteroid androstanediol is a positive allosteric modulator of GABAA receptors. J Pharmacol Exp Ther. 2010;334:1031–41.
Ginsburg M, Maclusky NJ, Morris ID, Thomas PJ. The specificity of estrogen receptor in brain, pituitary and uterus. Br J Pharmacol. 1977;59:397–402.
Edinger KL, Frye CA. Testosterone’s anti-anxiety and analgesic effects may be due in part to actions of its 5alpha-reduced metabolites in the hippocampus. Psychoneuroendocrinology. 2005;30:418–30.
Frye CA, Babson A, Walf AA. Self-administration of 3alpha-androstanediol increases locomotion and analgesia and decreases aggressive behavior of male hamsters. Pharmacol Biochem Behav. 2007;86:415–21.
Frye CA, Park D, Tanaka M, Rosellini R, Svare B. The testosterone metabolite and neurosteroid 3alpha-androstanediol may mediate the effects of testosterone on conditioned place preference. Psychoneuroendocrinology. 2001;26:731–50.
Walf AA, Frye CA. Gestational or acute restraint in adulthood reduces levels of 5α-reduced testosterone metabolites in the hippocampus and produces behavioral inhibition of adult male rats. Front Cell Neurosci. 2012;6:40. https://doi.org/10.3389/fncel.2012.00040.
Frye CA, Edinger KL, Lephart ED, Walf AA. 3alpha-androstanediol, but not testosterone, attenuates age-related decrements in cognitive, anxiety, and depressive behavior of male rats. Front Aging Neurosci. 2010;2:15. https://doi.org/10.3389/fnagi.2010.00015.
Narenji SA, Naghdi N, Azadmanesh K, Edalat R. 3α-diol administration decreases hippocampal PKA (II) mRNA expression and impairs Morris water maze performance in adult male rats. Behav Brain Res. 2015;280:149–59.
Nguyen PV, Woo NH. Regulation of hippocampal synaptic plasticity by cyclic AMP-dependent protein kinases. Prog Neurobiol. 2003;71:401–37.
Huang T, McDonough CB, Abel T. Compartmentalized PKA signaling events are required for synaptic tagging and capture during hippocampal late-phase long-term potentiation. Eur J Cell Biol. 2006;85:635–42.
Malleret G, Alarcon JM, Martel G, Takizawa S, Vronskaya S, Yin D, Chen IZ, Kandel ER, Shumyatsky GP. Bidirectional regulation of hippocampal long-term synaptic plasticity and its influence on opposing forms of memory. J Neurosci. 2010;30:3813–25.
Park AJ, Havekes R, Choi JH, Luczak V, Nie T, Huang T, Abel T. A presynaptic role for PKA in synaptic tagging and memory. Neurobiol Learn Mem. 2014;114:101–12.
Tóth M, Zakár T. Relative binding affinities of testosterone, 19-nortestosterone and their 5 alpha-reduced derivatives to the androgen receptor and to other androgen-binding proteins: a suggested role of 5 alpha-reductive steroid metabolism in the dissociation of “myotropic” and “androgenic” activities of 19-nortestosterone. J. Steroid Biochem. 1982;17:653–60.
Naess O. Characterization of the androgen receptors in the hypothalamus, preoptic area and brain cortex of the rat. Steroids. 1976;27:167–85.
Goudsmit E, Van de Poll NE, Swaab DF. Testosterone fails to reverse spatial memory decline in aged rats and impairs retention in young and middle-aged animals. Behav Neural Biol. 1990;53:6–20.
Rossetti MF, Varayoud J, Moreno-Piovano GS, Luque EH, Ramos JG. Environmental enrichment attenuates the age-related decline in the mRNA expression of steroidogenic enzymes and reduces the methylation state of the steroid 5α-reductase type 1 gene in the rat hippocampus. Mol Cell Endocrinol. 2015;412:330–8.
AssadianNarenji S, Naghdi N, Oryan S, Azadmanesh K. Effect of 3α-androstanediol and indomethacin on acquisition, consolidation and retrieval stage of spatial memory in adult male rats. Iran Biomed J. 2012;16:145–55.
Scheinert RB, Asokan A, Rani A, Kumar A, Foster TC, Ormerod BK. Some hormone, cytokine and chemokine levels that change across lifespan vary by cognitive status in male Fischer 344 rats. Brain Behav Immun. 2015;49:216–32.
Issa AM, Rowe W, Gauthier S, Meaney MJ. Hypothalamic-pituitary-adrenal activity in aged, cognitively impaired and cognitively unimpaired rats. J Neurosci. 1990;10:3247–54.
Meijer OC, Topic B, Steenbergen PJ, Jocham G, Huston JP, Oitzl MS. Correlations between hypothalamus-pituitary-adrenal axis parameters depend on age and learning capacity. Endocrinology. 2005;146:1372–81.
Juneja HS, Murthy SK, Ganguly J. The effect of vitamin A deficiency on the biosynthesis of steroid hormones in rats. Biochem. J. 1966;99:138–45.
Maeda N, Tahata S, Yagi T, Tanaka E, Masu K, Sato M, Haeno S, Onaga T, Yokota H. Assessment of testicular corticosterone biosynthesis in adult male rats. PLoS ONE. 2015;10:e0117795. https://doi.org/10.1371/journal.pone.0117795.
Simpkins JW, Kalra PS, Kalra SP. Alterations in daily rhythms of testosterone and progesterone in old male rats. Exp Aging Res. 1981;7:25–32.
Gruenewald DA, Hess DL, Wilkinson CW, Matsumoto AM. Excessive testicular progesterone secretion in aged male Fischer 344 rats: a potential cause of age-related gonadotropin suppression and confounding variable in aging studies. J. Gerontol. 1992;47:B164–70.
Gruenewald DA, Naai MA, Marck BT, Matsumoto AM. Age-related decrease in hypothalamic gonadotropin-releasing hormone (GnRH) gene expression, but not pituitary responsiveness to GnRH, in the male Brown Norway rat. J Androl. 2000;21:72–84.
Greeley GH Jr, Lipton MA, Kizer JS. Serum thyroxine, triiodothyronine, and TSH levels and TSH release after TRH in aging male and female rats. Endocr Res Commun. 1982;9:169–77.
Bolzán AD, Brown OA, Goya RG, Bianchi MS. Hormonal modulation of antioxidant enzyme activities in young and old rats. Exp Gerontol. 1995;30:169–75.
Goya RG, Brooks K, Meites J. A comparison between hormone levels and T lymphocyte function in young and old rats. Mech Ageing Dev. 1991;61:275–85.
Moreira DG, Marassi MP, da Corrêa Costa VM, Carvalho DP, Rosenthal D. Effects of ageing and pharmacological hypothyroidism on pituitary-thyroid axis of Dutch-Miranda and Wistar rats. Exp Gerontol. 2005;40:330–4.
Goya RG, Quigley KL, Takahashi S, Sosa YE, Meites J. Changes in somatotropin and thyrotropin secretory patterns in aging rats. Neurobiol Aging. 1990;11:625–30.
Alzoubi KH, Gerges NZ, Alkadhi KA. Levothyroxin restores hypothyroidism-induced impairment of LTP of hippocampal CA1: electrophysiological and molecular studies. Exp Neurol. 2005;195:330–41.
Rivas M, Naranjo JR. Thyroid hormones, learning and memory. Genes Brain Behav. 2007;6(Suppl 1):40–4.
Artis AS, Bitiktas S, Taşkın E, Dolu N, Liman N, Suer C. Experimental hypothyroidism delays field excitatory post-synaptic potentials and disrupts hippocampal long-term potentiation in the dentate gyrus of hippocampal formation and Y-maze performance in adult rats. J Neuroendocrinol. 2012;24:422–33.
Desouza LA, Ladiwala U, Daniel SM, Agashe S, Vaidya RA, Vaidya VA. Thyroid hormone regulates hippocampal neurogenesis in the adult rat brain. Mol Cell Neurosci. 2005;29:414–26.
Smith JW, Evans AT, Costall B, Smythe JW. Thyroid hormones, brain function and cognition: a brief review. Neurosci Biobehav Rev. 2002;26:45–60.
Shabani S, Sarkaki A, Ali-Mard S, Ahangarpour A, Khorsandi L, Farbood Y. Central and peripheral administrations of levothyroxine improved memory performance and amplified brain electrical activity in the rat model of Alzheimer’s disease. Neuropeptides. 2016;59:111–6.
Puia G, Losi G. Thyroid hormones modulate GABA(A) receptor-mediated currents in hippocampal neurons. Neuropharmacology. 2011;60:1254–61.
vanBoxtel MP, Menheere PP, Bekers O, Hogervorst E, Jolles J. Thyroid function, depressed mood, and cognitive performance in older individuals: the Maastricht Aging Study. Psychoneuroendocrinology. 2004;29:891–8.
Stwertka SA, Vincent GP, Gamzu ER, MacNeil DA, Verderese AG. TRH protection against memory retrieval deficits is independent of endocrine effects. Pharmacol Biochem Behav. 1992;41:145–52.
Schwegler H, Crusio WE, Lipp HP, Brust I, Mueller GG. Early post-natal hyperthyroidism alters hippocampal circuitry and improves radial-maze learning in adult mice. J Neurosci. 1991;11:2102–6.
The authors thank Maike Schäfer and Carmen Gregor for their technical assistance.
Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
Ethics approval and consent to participate
The study was carried out according to the guidelines of the Ethics committee, Medical University of Vienna, and were approved by the Federal Ministry of Education, Science and Culture, Austria.
Consent for publication
We grant BMC Behavioral and Brain Functions consent for the publication of the present article, in accordance to the license agreement in https://www.biomedcentral.com/about/policies/license-agreement.
The authors declare that they have no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.