Impact of estrogen receptor alpha and beta agonists on delayed alternation in middle-aged rats

Estrogens act in the adult brain to modulate cognition, enhancing performance on some learning tests and impairing performance on others. Our previous research has revealed an impairing effect of chronic 17β-estradiol treatment in young and aged rats on a prefrontally-mediated working memory task, delayed spatial alternation (DSA). Little is known about the mechanisms of these impairing effects. The current study examined the effects of selective estrogen receptor (ER) α or ERβ activation on DSA performance in middle-aged female rats. Ovariectomized 12 month old Long–Evans (LE) rats were treated by subcutaneous injection with the ERα agonist propyl pyrazole triol (PPT) or the ERβ agonist diarylpropionitrile (DPN) at 0.02, 0.08, or 0.20 mg/kg/day, or with oil vehicle and tested on an operant variable delay DSA task. A 17β-estradiol group (10% in cholesterol) was included as a positive control group. We replicated our previous finding of a 17β-estradiol induced deficit on DSA performance and this effect was paralleled by low dose (0.02 mg/kg/day) DPN treatment. Higher doses of DPN failed to produce a significant change in performance. The highest dose of PPT (0.20 mg/kg/day) also impaired performance, but this effect was subtle and limited to the longest delay during the final block of testing. These data confirm our earlier findings that chronic 17β-estradiol treatment has an impairing effect on the DSA task, and suggest that ERβ activation may underlie the deficit.     

Impact of estrogen receptor alpha and beta agonists on delayed alternation in middle-aged rats

Estrogens act in the adult brain to modulate cognition, enhancing performance on some learning tests and impairing performance on others. Our previous research has revealed an impairing effect of chronic 17β-estradiol treatment in young and aged rats on a prefrontally-mediated working memory task, delayed spatial alternation (DSA). Little is known about the mechanisms of these impairing effects. The current study examined the effects of selective estrogen receptor (ER) α or ERβ activation on DSA performance in middle-aged female rats. Ovariectomized 12 month old Long–Evans (LE) rats were treated by subcutaneous injection with the ERα agonist propyl pyrazole triol (PPT) or the ERβ agonist diarylpropionitrile (DPN) at 0.02, 0.08, or 0.20 mg/kg/day, or with oil vehicle and tested on an operant variable delay DSA task. A 17β-estradiol group (10% in cholesterol) was included as a positive control group. We replicated our previous finding of a 17β-estradiol induced deficit on DSA performance and this effect was paralleled by low dose (0.02 mg/kg/day) DPN treatment. Higher doses of DPN failed to produce a significant change in performance. The highest dose of PPT (0.20 mg/kg/day) also impaired performance, but this effect was subtle and limited to the longest delay during the final block of testing. These data confirm our earlier findings that chronic 17β-estradiol treatment has an impairing effect on the DSA task, and suggest that ERβ activation may underlie the deficit.     

Voluntary exercise impairs initial delayed spatial alternation performance in estradiol treated ovariectomized middle-aged rats

Estrogens differentially modulate behavior in the adult female rodent. Voluntary exercise can also impact behavior, often reversing age associated decrements in memory processes. Our research group has published a series of papers reporting a deficit in the acquisition of an operant working memory task, delayed spatial alternation (DSA), following 17β-estradiol treatment to middle-aged ovariectomized (OVX) rats. The current study examined if voluntary exercise could attenuate the 17β-estradiol induced deficits on DSA performance. OVX 12-month old Long–Evans rats were implanted with a Silastic capsule containing 17β-estradiol (10% in cholesterol: low physiological range) or with a blank capsule. A subset of the 17β-estradiol and OVX untreated rats were given free access to a running wheel in their home cage. All rats were tested for 40 sessions on the DSA task. Surprisingly, we found running wheel access to impair initial acquisition of the DSA task in 17β-estradiol treated rats, an effect not seen in OVX untreated rats given running wheel access. This deficit was driven by an increase in perseverative responding on a lever no longer associated with reinforcement. We also report for the first time a 17β-estradiol induced impairment on the DSA task following a long intertrial delay (18-sec), an effect revealed following more extended testing than in our previous studies (15 additional sessions). Overall, running wheel access increased initial error rate on the DSA task in 17β-estradiol treated middle-aged OVX rats, and failed to prevent the 17β-estradiol induced deficits in performance of the operant DSA task in later testing sessions.     

Donepezil treatment restores the ability of estradiol to enhance cognitive performance in aged rats: Evidence for the cholinergic basis of the critical period hypothesis

Recent studies suggest that the ability of estradiol to enhance cognitive performance diminishes with age and/or time following loss of ovarian function. We hypothesize that this is due, in part, to a decrease in basal forebrain cholinergic function. This study tested whether donepezil, a cholinesterase inhibitor, could restore estradiol effects on cognitive performance in aged rats that had been ovariectomized as young adults. Rats were ovariectomized at 3 months of age, and then trained on a delayed matching to position (DMP) T-maze task, followed by a configural association (CA) operant condition task, beginning at 12-17 or 22-27 months of age. Three weeks prior to testing, rats started to receive either donepezil or vehicle. After one week, half of each group also began receiving estradiol. Acclimation and testing began seven days later and treatment continued throughout testing. Estradiol alone significantly enhanced DMP acquisition in middle-aged rats, but not in aged rats. Donepezil alone had no effect on DMP acquisition in either age group; however, donepezil treatment restored the ability of estradiol to enhance DMP acquisition in aged rats. This effect was due largely to a reduction in the predisposition to adopt a persistent turn strategy during acquisition. These same treatments did not affect acquisition of the CA task in middle-aged rats, but did have small but significant effects on response time in aged rats. The data are consistent with the idea that estrogen effects on cognitive performance are task specific, and that deficits in basal forebrain cholinergic function are responsible for the loss of estradiol effect on DMP acquisition in aged ovariectomized rats. In addition, the data suggest that enhancing cholinergic function pharmacologically can restore the ability of estradiol to enhance acquisition of the DMP task in very old rats following long periods of hormone deprivation. Whether donepezil has similar restorative effects on other estrogen-sensitive tasks needs to be explored.     

Basal forebrain cholinergic neurons are necessary for estrogen to enhance acquisition of a delayed matching-to-position T-maze task

Intraseptal injections of the selective cholinergic immunotoxin 192 IgG-saporin (SAP) were performed to determine whether basal forebrain cholinergic neurons are necessary for hormone-mediated enhancement of acquisition in a delayed matching-to-position (DMP) T-maze task. The DMP task is a simple spatial learning task. Studies have shown that continuous estradiol replacement enhances acquisition of the DMP task in young ovariectomized rats and that long-term treatment with either estradiol or estradiol + progesterone can prevent a deficit in DMP acquisition in old rats. In the present study, continuous estradiol replacement significantly enhanced acquisition of the DMP task by non-SAP-treated, ovariectomized rats. In contrast, neither continuous estradiol nor weekly administration of estradiol + progesterone significantly enhanced acquisition of the DMP task in rats that received intraseptal injections of either a high dose (1.0 microg) or a low dose (0.22 microg) of SAP. Animals that reached criterion were significantly impaired by rotating the maze 180 degrees regardless of treatment, suggesting that animals in all groups used extramaze cues to at least some degree to solve the task. SAP-treated animals were slightly more sensitive to increasing the intertrial delay than non-SAP-treated controls, suggesting that the SAP lesions produced a modest deficit in spatial working memory. Immunohistochemistry confirmed the loss of cholinergic neurons in specific regions of the basal forebrain of SAP-treated animals. In addition, DMP acquisition correlated significantly with ChAT activity in the hippocampus and frontal cortex. The data suggest that basal forebrain cholinergic projections are necessary for hormone-mediated enhancement of DMP acquisition.     

Alterations in the proestrous pattern of median eminence LHRH, serum LH, FSH, estradiol and progesterone concentrations in middle-aged rats

The purpose of the following study was to assess the changes in the proestrous hormone profile in middle-aged cycling rats to better understand the inter-relationship and possible interaction of these hormones during the transition to estrous acyclicity. Median eminence LHRH concentrations and serum LH, FSH, estradiol and progesterone concentrations were measured in young (3-4 months old) and middle-aged (8-10 months old) proestrous rats at 0900, 1200, 1500 and 1800h. The data demonstrate that (1) baseline hormone concentrations prior to the surge at 0900h are the same in middle-aged and young rats; (2) the proestrous gonadotropin surge is temporally delayed in middle-aged rats; (3) this delay is preceded by lower median eminence LHRH concentrations and serum estradiol concentrations at 1200h; (4) serum progesterone concentrations are lower in middle-aged rats during the preovulatory gonadotropin surge (at 1500 and 1800h) probably as a consequence of the delayed LH surge.     

Galanthamine plus estradiol treatment enhances cognitive performance in aged ovariectomized rats

We hypothesize that beneficial effects of estradiol on cognitive performance diminish with age and time following menopause due to a progressive decline in basal forebrain cholinergic function. This study tested whether galanthamine, a cholinesterase inhibitor used to treat memory impairment associated with Alzheimer's disease, could enhance or restore estradiol effects on cognitive performance in aged rats that had been ovariectomized in middle-age. Rats were ovariectomized at 16–17 months of age. At 21–22 months of age rats began receiving daily injections of galanthamine (5 mg/day) or vehicle. After one week, half of each group also received 17ß-estradiol administered subcutaneously. Rats were then trained on a delayed matching to position (DMP) T-maze task, followed by an operant stimulus discrimination/reversal learning task. Treatment with galanthamine + estradiol significantly enhanced the rate of DMP acquisition and improved short-term delay-dependent spatial memory performance. Treatment with galanthamine or estradiol alone was without significant effect. Effects were task-specific in that galanthamine + estradiol treatment did not significantly improve performance on the stimulus discrimination/reversal learning task. In fact, estradiol was associated with a significant increase in incorrect responses on this task after reversal of the stimulus contingency. In addition, treatments did not significantly affect hippocampal choline acetyltransferase activity or acetylcholine release. This may be an effect of age, or possibly is related to compensatory changes associated with long-term cholinesterase inhibitor treatment. The data suggest that treating with a cholinesterase inhibitor can enhance the effects of estradiol on acquisition of a DMP task by old rats following a long period of hormone deprivation. This could be of particular benefit to older women who have not used hormone therapy for many years and are beginning to show signs of mild cognitive impairment. Potential mechanisms for these effects are discussed.     

Developmental pattern of tachykinins during aging in several organs: effect of exogenous melatonin

Mammalian neurokinin A (NKA) and substance P (SP) are neuropeptides widely distributed in the body; they are potential regulators of the basal blood flow and therefore of the function of many organs and tissues. In the present investigation, we studied the age-dependent changes in NKA and SP in ovary, liver, pancreas and spleen as well as the role of exogenous melatonin on these changes. Female rats of 5, 15 or 25 months of age were studied. In the ovary, NKA concentrations did not change during aging. SP concentrations in the control group were significantly higher (P<0.01) in old rats than in the other two age groups studied. Melatonin treatment resulted in reduced concentrations as compared with those of the control old rats. In the pancreas, NKA and SP concentrations increased during aging, the young rats showing significantly lower values (P<0.01) than middle-aged and old rats for NKA and significantly lower (P<0.01) than the old rats for SP. After melatonin treatment the differences in NKA concentrations disappeared and SP decreased in middle-aged as compared with those in old rats. In the liver, NKA and SP concentrations in the control and melatonin-treated groups did not differ significantly for the three age groups studied. Splenic NKA in control and melatonin-treated groups increased from young to middle-age up to old ages. SP concentrations showed similar values at all ages except in melatonin-treated old rats; in these animals there were significantly higher concentrations than in young melatonin-treated rats. The effect of melatonin was mainly observed on the ovary and pancreas in old rats, with a reduction in the concentrations as compared with those observed in the young groups.     

Chronic estradiol treatment increases CA1 cell survival but does not improve visual or spatial recognition memory after global ischemia in middle-aged female rats

Transient global ischemia induces selective, delayed neuronal death in the hippocampal CA1 and cognitive deficits. Physiological levels of 17β-estradiol ameliorate ischemia-induced neuronal death and cognitive impairments in young animals. In view of concerns regarding hormone therapy in postmenopausal women, we investigated whether chronic estradiol treatment initiated 14 days prior to ischemia attenuates ischemia-induced CA1 cell loss and impairments in visual and spatial memory, in ovariohysterectomized (OVX), middle-aged (9-11 months) female rats. To determine whether the duration of hormone withdrawal affects the efficacy of estradiol treatment, hormone treatment was initiated immediately (0 week), 1 week, or 8 weeks after OVX. Age-matched, OVX and gonadally intact females were studied at each OVX interval. Ischemia was induced 1 week after animals were pretested on a variety of behavioral tasks. Global ischemia produced significant neuronal loss in the CA1 and impaired performance on visual and spatial recognition. Chronic estradiol modestly but significantly increased the number of surviving CA1 neurons in animals at all OVX durations. However, in contrast with previous results in young females, estradiol did not preserve visual or spatial memory performance in middle-aged females. All animals displayed normal locomotion, spontaneous alternation and social preference, indicating the absence of global behavioral impairments. Therefore, the neuroprotective effects of estradiol are different in middle-aged than in young rats. These findings highlight the importance of using older animals in studies assessing potential treatments for focal and global ischemia.     

Hypothalamic luteinizing hormone-releasing hormone (LHRH) and LH secretion in aging female rats

Age-related changes in hypothalamic luteinizing hormone-releasing hormone (LHRH) and luteinizing hormone (LH) secretion were studied in young (6 months), middle-aged (12 months) and old (18 months) female rats. The LHRH levels in the mid-hypothalamic area were higher in intact middle-aged and old females than in young ones. Additionally, there was no age difference in the hypothalamic LHRH levels in male rats. In order to clarify the significance of this age-related increase in female rats, we examined the effects of progesterone treatment in estrogen-primed ovariectomized young and old rats on the LHRH levels in the median eminence (ME) and on plasma LH levels. We found phasic changes in ME-LHRH and plasma LH levels in estrogen-primed rats following progesterone treatment in rats of both ages, but the progesterone-induced change in ME-LHRH levels tended to be delayed in old rats compared with young females. This delay may correspond to the delayed onset, slow and low magnitude of plasma LH increase in old females. The ME-LHRH levels were generally higher in old rats than in young rats. Nevertheless, we found that the increase in plasma LH in response to progesterone treatment in estrogen-primed ovariectomized females was smaller in old rats than young rats. These results suggest that the LHRH secretory mechanism changes with age in female rats. Such alterations may result in the accumulation of LHRH in the mid-hypothalamic area and an increase in ME-LHRH.     

Delayed response task performance as a function of age in cynomolgus monkeys (Macaca fascicularis)

We compared delayed response task performance in young, middle-aged, and old cynomolgus monkeys using three memory tests that have been used with non-human primates. Eighteen cynomolgus monkeys—6 young (4–9 years), 6 middle-aged (10–19 years), and 6 old (above 20 years)—were tested. In general, the old monkeys scored significantly worse than did the animals in the two other age groups. Longer delays between stimulus presentation and response increased the performance differences between the old and younger monkeys. The old monkeys in particular showed signs of impaired visuo-spatial memory and deteriorated memory consolidation and executive functioning. These results add to the body of evidence supporting the utility of Macaca fascicularis in studies of cognition and as a potential translational model for age-associated memory impairment/dementia-related disorders.     

Prior parity positively regulates learning and memory in young and middle-aged rats

Reproductive experience in female rats modifies acquired behaviors, induces long-lasting functional neuroadaptations and can also modify spatial learning and memory. The present study supports and expands this knowledge base by employing the Morris water maze, which measures spatial memory. Age-matched young adult (YNG) nulliparous (NULL; nonmated) and primiparous (PRIM; one pregnancy and lactation) female rats were tested 15 d after the litter's weaning. In addition, corresponding middle-aged (AGD) PRIM (mated in young adulthood so that pregnancy, parturition, and lactation occurred at the same age as in YNG PRIM) and NULL female rats were tested at 18 mo of age. Behavioral evaluation included: 1) acquisition of reference memory (platform location was fixed for 14 to 19 d of testing); 2) retrieval of this information associated with extinction of the acquired response (probe test involving removal of the platform 24 h after the last training session); and 3) performance in a working memory version of the task (platform presented in a novel location every day for 13 d, and maintained in a fixed location within each day). YNG PRIM outperformed NULL rats and showed different behavioral strategies. These results may be related to changes in locomotor, mnemonic, and cognitive processes. In addition, YNG PRIM exhibited less anxiety-like behavior. Compared with YNG rats, AGD rats showed less behavioral flexibility but stronger memory consolidation. These data, which were obtained by using a well-documented spatial task, demonstrate long lasting modifications of behavioral strategies in both YNG and AGD rats associated with a single reproductive experience.     

Donepezil plus estradiol treatment enhances learning and delay-dependent memory performance by young ovariectomized rats with partial loss of septal cholinergic neurons

Effects of estrogen therapy on cognitive performance appear to diminish with age and time following the loss of ovarian function. We hypothesize that this is due to a reduction in basal forebrain cholinergic function and that treatment with a cholinergic enhancer can reverse the effect. This study tested whether combining the cholinesterase inhibitor donepezil with estradiol treatment can enhance/restore estradiol effects on cognitive performance in young ovariectomized rats with selective lesions of septal cholinergic neurons. 192IgG-saporin was injected directly into the medial septum to produce selective cholinergic lesions. Rats were then treated with donepezil (Don, daily injections of 3 mg/kg/day, i.p.) or vehicle, and then with 17β-estradiol (E2, administered by silastic capsule implanted s.c.) or an empty capsule. Rats were trained on a delayed matching-to-position (DMP) T-maze task which previous studies have shown is sensitive to ovariectomy and estrogen replacement. Results show that neither estradiol nor donepezil alone significantly enhanced acquisition of the DMP task in rats with cholinergic lesions. Combination therapy was effective, however, depending on the severity of the lesion. Don + E2 significantly enhanced acquisition of the task in rats with partial lesions (< 50% loss of cholinergic neurons), but not in rats with severe lesions. This effect was due largely to a reduction in perseverative behavior. Don + E2 also improved working memory in rats with partial lesions, as evidenced by significantly better performance than controls during increased intertrial delays. These findings suggest that even partial loss of septal cholinergic neurons can reduce effects of estrogen therapy on cognitive performance, and demonstrate that combining a cholinesterase inhibitor with estrogen therapy can help to restore beneficial effects on performance. We propose that combination therapy may have similar beneficial effects in women, particularly in older women who have not used estrogen therapy for many years and are beginning to show signs of cognitive impairment or early Alzheimer's disease.     

Galanthamine plus estradiol treatment enhances cognitive performance in aged ovariectomized rats

We hypothesize that beneficial effects of estradiol on cognitive performance diminish with age and time following menopause due to a progressive decline in basal forebrain cholinergic function. This study tested whether galanthamine, a cholinesterase inhibitor used to treat memory impairment associated with Alzheimer's disease, could enhance or restore estradiol effects on cognitive performance in aged rats that had been ovariectomized in middle-age. Rats were ovariectomized at 16–17 months of age. At 21–22 months of age rats began receiving daily injections of galanthamine (5 mg/day) or vehicle. After one week, half of each group also received 17ß-estradiol administered subcutaneously. Rats were then trained on a delayed matching to position (DMP) T-maze task, followed by an operant stimulus discrimination/reversal learning task. Treatment with galanthamine + estradiol significantly enhanced the rate of DMP acquisition and improved short-term delay-dependent spatial memory performance. Treatment with galanthamine or estradiol alone was without significant effect. Effects were task-specific in that galanthamine + estradiol treatment did not significantly improve performance on the stimulus discrimination/reversal learning task. In fact, estradiol was associated with a significant increase in incorrect responses on this task after reversal of the stimulus contingency. In addition, treatments did not significantly affect hippocampal choline acetyltransferase activity or acetylcholine release. This may be an effect of age, or possibly is related to compensatory changes associated with long-term cholinesterase inhibitor treatment. The data suggest that treating with a cholinesterase inhibitor can enhance the effects of estradiol on acquisition of a DMP task by old rats following a long period of hormone deprivation. This could be of particular benefit to older women who have not used hormone therapy for many years and are beginning to show signs of mild cognitive impairment. Potential mechanisms for these effects are discussed.     

Age- and gender-related difference of ACE2 expression in rat lung

Epidemiologic data suggested that there was an obvious predominance of young adult patients with a slight female proneness in severe acute respiratory syndrome (SARS). The angiotensin-converting enzyme 2 (ACE2) was very recently identified as a functional receptor for SARS virus and is therefore a prime target for pathogenesis and pharmacological intervention. Rats of both genders at three distinct ages (young-adult, 3 months; middle-aged, 12 months; old, 24 months) were evaluated to determine the characteristic of ACE2 expression in lung and the effect of aging and gender on its expression. ACE2 was predominantly expressed in alveolar epithelium, bronchiolar epithelium, endothelium and smooth muscle cells of pulmonary vessels with similar content, whereas no obvious signal was detected in the bronchiolar smooth muscle cells. ACE2 expression is dramatically reduced with aging in both genders: young-adult vs. old P < 0.001 (by 78% in male and 67% in female, respectively) and middle-aged vs. old P < 0.001 (by 71% in male rats and 59% in female rats, respectively). The decrease of ACE2 content was relatively slight between young-adult and middle-aged groups (by 25% in male and 18% in female, respectively). Although there was no gender-related difference of ACE2 in young-adult and middle-aged groups, a significantly higher ACE2 content was detected in old female rats than male. In conclusion, the more elevated ACE2 in young adults as compared to aged groups may contribute to the predominance in SARS attacks in this age group.     

Male Age Affects Female Mate Preference and Reproductive Performance in the Cabbage Beetle, <Emphasis Type="Italic">Colaphellus bowringi</Emphasis>

The influence of male age on female mate preference and reproductive performance in the cabbage beetle, Colaphellus bowringi was examined, using male and female adults of varying ages (young, middle-aged and old) after a single mating. In a simultaneous choice test, females of all age class preferentially mated with middle-aged males. Mating duration was positively related to male age. Longevity of females was not significantly affected by male age. Young females paired to middle-aged males had significantly higher egg production than those paired to old males, and the eggs of females paired to middle-aged males exhibited significantly higher hatching success than the eggs of females mated to young or old males. These results suggest that middle-aged males are more advantageous for female fitness than young and old males.     

Acute, nongenomic vasodilatory action of estradiol is attenuated by chronic estradiol treatment

Deficiency of estradiol or chronic estrogen treatment may alter the responses to this hormone in many tissues. A possible interaction between the acute nongenomic and the chronic effects of estradiol on microvessels have not been investigated yet. In the present study we have investigated whether acute in vitro vasodilatory action of estradiol on a small artery is altered by chronic estradiol pretreatment. Female rats were surgically ovariectomized and subjected to either estradiol replacement therapy (estradiol propionate, 450 micrograms/kg/week) or vehicle administration for 5 weeks. Cylindrical segments of the saphenous artery were studied using videocomputerized microarteriography in vitro. Estradiol, in concentrations of 10(-6) to 10(-4) M relaxed norepinephrine precontracted vessel segments in a dose-dependent manner. Magnitude of relaxation observed in arteries of estradiol replaced animals was significantly smaller at all concentrations than that of nonreplaced ovariectomized rats; maximal relaxation in the control ovariectomized group was 64.5% +/- 3.6%, while it was 34.3% +/- 4.2% only in the ovariectomized and estradiol replaced group (P < 0.001). Comparison of acute relaxations in response to papaverine and nifedipine failed to prove a reduced activity of the general relaxation machinery in estradiol replaced animals. We conclude that chronic estradiol replacement can downregulate the acute nongenomic vasorelaxation effect of this hormone in small arteries of ovariectomized rats.     

Ovarian hormone replacement to aged ovariectomized female rats benefits acquisition of the morris water maze

Ovarian steroids have been suggested to aid in preserving cognitive functioning during aging in both humans and other animals. Spatial memory relies heavily on the hippocampus, a structure that is sensitive to the influence of both ovarian hormones and aging. The present study investigated the outcome of ovarian hormone replacement during aging on performance in a spatial version of the Morris water maze. Female rats were ovariectomized at 14 months of age and received one of three types of replacement prior to testing at 16 months: acute estrogen replacement (2 days), chronic estrogen replacement (28 days), or chronic replacement of both estrogen and progesterone (28 days). Control animals, which did not receive replacement hormones, displayed significant overnight forgetting during acquisition of the task. Ovarian hormone replacement, both acute and chronic, prevented forgetting. Previous studies have demonstrated that high levels of ovarian hormones are detrimental to performance of young adult female rats on this task (Warren and Juraska, 1997; Chesler and Juraska, 2000). The current study found an opposite effect during aging: ovarian hormone replacement was beneficial. This suggests that animal models of menopause, aimed at exploring the protective effects of hormone replacement therapy on cognition during human female aging, require the use of aged female animals.     

Expression of Vesicular Glutamate Transporter 2 (vGluT2) on Large Dense-Core Vesicles within GnRH Neuroterminals of Aging Female Rats

The pulsatile release of GnRH is crucial for normal reproductive physiology across the life cycle, a process that is regulated by hypothalamic neurotransmitters. GnRH terminals co-express the vesicular glutamate transporter 2 (vGluT2) as a marker of a glutamatergic phenotype. The current study sought to elucidate the relationship between glutamate and GnRH nerve terminals in the median eminence—the site of GnRH release into the portal capillary vasculature. We also determined whether this co-expression may change during reproductive senescence, and if steroid hormones, which affect responsiveness of GnRH neurons to glutamate, may alter the co-expression pattern. Female Sprague-Dawley rats were ovariectomized at young adult, middle-aged and old ages (~4, 11, and 22 months, respectively) and treated four weeks later with sequential vehicle + vehicle (VEH + VEH), estradiol + vehicle (E₂ + VEH), or estradiol + progesterone (E₂+P₄). Rats were perfused 24 hours after the second hormone treatment. Confocal microscopy was used to determine colocalization of GnRH and vGluT2 immunofluorescence in the median eminence. Post-embedding immunogold labeling of GnRH and vGluT2, and a serial electron microscopy (EM) technique were used to determine the cellular interaction between GnRH terminals and glutamate signaling. Confocal analysis showed that GnRH and vGluT2 immunofluorescent puncta were extensively colocalized in the median eminence and that their density declined with age but was unaffected by short-term hormone treatment. EM results showed that vGluT2 immunoreactivity was extensively associated with large dense-core vesicles, suggesting a unique glutamatergic signaling pathway in GnRH terminals. Our results provide novel subcellular information about the intimate relationship between GnRH terminals and glutamate in the median eminence.     

Changes in androgen receptor, estrogen receptor alpha, and sexual behavior with aging and testosterone in male rats

Reproductive aging in males is characterized by a diminution in sexual behavior beginning in middle age. We investigated the relationships among testosterone, androgen receptor (AR) and estrogen receptor alpha (ERα) cell numbers in the hypothalamus, and their relationship to sexual performance in male rats. Young (3 months) and middle-aged (12 months) rats were given sexual behavior tests, then castrated and implanted with vehicle or testosterone capsules. Rats were tested again for sexual behavior. Numbers of AR and ERα immunoreactive cells were counted in the anteroventral periventricular nucleus and the medial preoptic nucleus, and serum hormones were measured. Middle-aged intact rats had significant impairments of all sexual behavior measures compared to young males. After castration and testosterone implantation, sexual behaviors in middle-aged males were largely comparable to those in the young males. In the hypothalamus, AR cell density was significantly (5-fold) higher, and ERα cell density significantly (6-fold) lower, in testosterone- than vehicle-treated males, with no age differences. Thus, restoration of serum testosterone to comparable levels in young and middle-aged rats resulted in similar preoptic AR and ERα cell density concomitant with a reinstatement of most behaviors. These data suggest that age-related differences in sexual behavior cannot be due to absolute levels of testosterone, and further, the middle-aged brain retains the capacity to respond to exogenous testosterone with changes in hypothalamic AR and ERα expression. Our finding that testosterone replacement in aging males has profound effects on hypothalamic receptors and behavior has potential medical implications for the treatment of age-related hypogonadism in men.