Effects of gonadectomy on performance in operant tasks measuring prefrontal cortical function in adult male rats

Previous studies have shown that gonadectomy in adult male rats influences the acquisition and performance of spatial and other working memory tasks that depend in part on the medial prefrontal cortex and its dopamine innervation. Stimulated by previous findings that gonadectomy alters dopamine axon density in not only medial but several other prefrontal fields, the present studies asked whether gonadectomy might also broadly impact dopamine-dependent prefrontal functions, and whether these effects bore any relation to hormone modulation of mesoprefrontal dopamine afferents. Specifically, control, gonadectomized, and gonadectomized rats given estradiol or testosterone propionate were tested on a series of operant tasks that together measured medial prefrontal functions of spatial working memory, impulsivity and extradimensional set shifting and orbital prefrontal functions of reversal learning/perseveration and motivation. Afterwards, animals were sacrificed, their bulbospongiosus muscles were removed and weighed, their brains were processed for immunocytochemistry for the dopamine-synthesizing enzyme tyrosine hydroxylase, and axon densities were measured in orbital and medial prefrontal fields. Statistical evaluations of group effects on behavior and regression analyses comparing individual performance with muscle weights and axon density measures revealed androgen-reversible effects of gonadectomy on acquisition of spatial working memory and extradimensional set shifting that were correlated with bulbospongiosus weight and medial prefrontal dopamine axon density, estrogen-sensitive influences of gonadectomy on motivation and response withholding that were correlated with bulbospongiosus weight but not with dopamine innervation, and still other prefrontal functions, i.e., impulsivity, reversal learning, that were insensitive to gonadectomy and unrelated to gonadectomy-induced changes in muscle weight or prefrontal dopamine innervation.     

Effects of gonadectomy and hormone replacement on a spontaneous novel object recognition task in adult male rats

Recent studies in adult male rats have shown that gonadal hormones influence performance on certain working memory and other types of cognitive tasks that are sensitive to lesions of the medial and/or orbital prefrontal cortices. This study asked whether gonadal hormone modulation of prefrontal cortical function in males also extends to the perirhinal division of the rat prefrontal cortex. Specifically, sham-operated control, gonadectomized, and gonadectomized rats supplemented with testosterone propionate or estradiol were tested on a spontaneous novel object recognition task, a paradigm where performance has been shown to be impaired by perirhinal cortical lesions. Using analyses of variance, regression analyses and post-hoc testing to evaluate group differences, it was found that during both the sample and test trials of the task all four groups spent similar absolute and proportional amounts of time ambulating, rearing, stationary, and exploring the two objects present. All groups also explored each of the two identical objects present during sample trials equally. However, during the test trials, only the control and gonadectomized rats given testosterone showed the expected increase in exploration of the novel objects presented, whereas the gonadectomized and gonadectomized, estradiol-supplemental groups continued to explore the novel and familiar objects equally. That regression analyses also identified significant correlations between low bulbospongiosus muscle weight and impaired novel vs. familiar object discrimination further indicates that gonadectomy in adult male rats adversely affects spontaneous novel object recognition in an androgen-sensitive, estrogen-insensitive manner.     

Testosterone impairs the acquisition of an operant delayed alternation task in male rats

The current study examined the effects of gonadectomy (GDX) and subsequent testosterone treatment of male Long-Evans rats on an operant variable delay spatial alternation task (DSA). Gonadally-intact rats (intact-B), GDX rats receiving implants that delivered a physiological level of testosterone (GDX-T), and GDX rats receiving blank implants (GDX-B) were tested for 25 sessions on a DSA task with variable inter-trial delays ranging from 0 to 18 s. Acquisition of the DSA task was found to be enhanced following GDX in a time and delay dependent manner. Both the GDX-T and the intact-B rats had lower performance accuracies across delays initially, relative to GDX-B rats, and this deficit persisted into subsequent testing sessions at longer delays. The GDX-T and intact-B rats also had a tendency to commit more perseverative errors during the early testing sessions, with both groups persisting in pressing a lever which had not been associated with reinforcement for at least two consecutive trials. However, both the GDX-T and intact-B groups were able to achieve performance accuracy similar to that of the GDX-B rats by the final sessions of testing. Overall, these results suggest that castration of adult male rats enhances their acquisition of an operant DSA task.     

Testosterone and estradiol produce different effects on cognitive performance in male rats

The effects of castration and hormone treatment on cognitive performance were evaluated in male rats. Castrated animals received either testosterone or estradiol and were compared with gonadally intact animals and with castrated controls. Results revealed a dissociation between the effects of testosterone and estradiol on cognitive performance in male rats. Specifically, estradiol enhanced acquisition of a delayed matching-to-position spatial task, similar to previously published observations in females. In contrast, neither castration nor testosterone treatment had any significant effect on acquisition of the delayed matching-to-position task, but did appear to affect delay-dependent working memory. None of the treatments had any significant effect on acquisition of a configural association negative patterning task, suggesting that effects on the delayed matching-to-position task were not due to effects on motivational factors. These data demonstrate that, as in females, gonadal hormones influence cognitive performance in males and suggest that estradiol and testosterone affect distinct cognitive domains.     

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.     

Effects of bilateral electrolytic lesions of the medial nucleus accumbens on exploration and spatial learning.

Rats with electrolytic lesions of the medial part of the nucleus accumbens, comprising the shell region, were compared to sham-operated rats in tests of exploration in a T-maze, in a hole-board, and in an elevated (+)-maze and in a test of water maze spatial learning. Rats with medial nucleus accumbens lesions had higher choice latencies than sham-operated controls during the beginning of the spontaneous alternation test. A higher number of hole pokes was found in the lesioned group, but only during the beginning of the second day of testing. In the elevated (+)-maze, lesioned rats had a higher number of closed and total arm entries and spent more time in the center region. The lesioned group did not differ from the control group for the number of alternations in the T-maze, for horizontal and vertical motor activity in the hole-board, and for acquisition or reversal of spatial learning in the Morris water maze. These results indicate that lesions of the medial nucleus accumbens slowed down decision time during spontaneous alternation testing and increased exploration in a time and test-specific manner without altering acquisition of a reference memory task.     

Does neonatal gonadectomy affect the sexual differentiation of quail?

This experiment was designed to determine the contribution, if any, of posthatching gonadal hormones to sexual differentiation of behavior in Japanese quail (Coturnix coturnix japonica). Males and females were gonadectomized or sham-operated (controls) prior to age 7 days posthatching. At age 4-9 weeks controls were gonadectomized. All birds were then given 2 weeks of testosterone propionate injections and tested for sexual behavior with female partners. Neonatally gonadectomized females exhibited more male-typical copulatory behavior than control females, but this effect was not statistically significant. Neonatal gonadectomy had no effect on males, and neonatally gonadectomized males exhibited significantly more male-typical copulatory behavior than neonatally gonadectomized females. Although the process of sexual differentiation may extend to a minor degree into the posthatching period in females, nonetheless it is largely complete at hatching in this species.     

Evidence for an alternation strategy in time-place learning

Many different conclusions concerning what type of mechanism rats use to solve a daily time-place task have emerged in the literature. The purpose of this study was to test three competing explanations of time-place discrimination. Rats (n = 10) were tested twice daily in a T-maze, separated by approximately 7 h. Food was available at one location in the morning and another location in the afternoon. After the rats learned to visit each location at the appropriate time, tests were omitted to evaluate whether the rats were utilizing time-of-day (i.e., a circadian oscillator) or an alternation strategy (i.e., visiting a correct location is a cue to visit the next location). Performance on this test was significantly lower than chance, ruling out the use of time-of-day. A phase advance of the light cycle was conducted to test the alternation strategy and timing with respect to the light cycle (i.e., an interval timer). There was no difference between probe and baseline performance. These results suggest that the rats used an alternation strategy to meet the temporal and spatial contingencies in the time-place task.     

Follicle-stimulating hormone secretion in monosodium glutamate-lesioned rats: response to unilateral gonadectomy or porcine follicular fluid (inhibin)

The purpose of this study is to determine the acute response of pituitary FSH and LH release to unilateral gonadectomy in the MSG-treated rat, and to determine whether pFF (inhibin) can act effectively on pituitary FSH secretion in the MSG-lesioned rat. MSG (4 mg/kg B.W.) or saline was injected subcutaneously on postnatal days 2, 4, 6, 8, and 10 to male and female littermates which were used in the experiments after postnatal day 60. In the first experiment male and female littermates were bilaterally gonadectomized and bled serially for the next 72 h. At 0 h plasma FSH concentrations in MSG-treated rats were lower (p less than 0.05) than those in saline-treated controls, and for the 72 h immediately following bilateral gonadectomy FSH levels increased parallel to those of the controls, but after a significant delay. In the second experiment, MSG-treated male and female littermates were injected with 0.5 ml of pFF at several intervals following bilateral gonadectomy and decapitated 6 hours later. Injection of pFF significantly suppressed circulating FSH titers in all groups without affecting LH levels. In a third experiment, rats were unilaterally gonadectomized and blood samples were obtained at various intervals for 48 h. Following unilateral gonadectomy there was a significant transient increase in FSH levels in male or female MSG-treated rats as compared to their 0 h values; however, the absolute levels attained were barely equal to the basal concentrations observed in the saline-treated control rats. The conclusions from these data are: insufficient FSH secretion in response to unilateral gonadectomy may be responsible for the lack of compensatory gonadal hypertrophy in MSG-lesioned rats, pituitary response to inhibin is apparently unaltered by MSG toxicity, and the MSG-lesioned rat is a useful model to study the differential control mechanisms of FSH and LH secretion.     

The influence of oestradiol on androgen- and oestrogen-dependent enzyme activities of hepatic steroid metabolism in rats.

Five sexually differentiated enzyme activities of hepatic steroid metabolism (cytoplasmic 17 beta-hydroxysteroid dehydrogenase, 5 beta-reductase; microsomal 3 alpha- and 3 beta-hydroxysteroid dehydrogenase and 5 alpha-reductase) were investigated in intact, gonadectomized and hypophysectomized rats after administration of a single dose of oestradiol valerate. Oestradiol administration caused a partial or complete feminization of these activities in intact male rats. The influence of oestradiol on these activities in gonadectomized rats was determined by the mode of sex hormone-dependent regulation of the individual activity: the most prominent effects were seen in the oestrogen-dependent activities (17 beta-hydroxysteroid dehydrogenase, 5 beta-reductase); no effect was seen in the completely androgen-dependent 3 alpha-hydroxysteroid dehydrogenase because gonadectomy alone was sufficient to cause complete feminization of the activity. Oestradiol administration had no effect on the activities of hypophysectomized rats. The fact that oestrogen administration to intact male rats caused greater changes than prepuberal gonadectomy demonstrates that oestrogen action is more than simple suppression of testicular function.     

Chronic treatment with estrogen receptor agonists restores acquisition of a spatial learning task in young ovariectomized rats

Previous work has shown that continuous estradiol replacement in young ovariectomized rats enhances acquisition of a delayed matching-to-position (DMP) T-maze task over that of ovariectomized controls. The mechanism by which estradiol confers this benefit has not been fully elucidated. This study examined the role of selective estrogen receptor agonists of ERα, ERβ, and GPR30 in the enhancement of spatial learning on a DMP task by comparing continuous estradiol replacement with continuous administration of PPT (an agonist of ERα), DPN (an agonist of ERβ), or G-1 (an agonist of GPR30) relative to gonadally intact and ovariectomized vehicle-treated controls. It was found that ovariectomy impaired acquisition on this task, whereas all ER selective agonists restored the rate of acquisition to that of gonadally intact controls. These data suggest that estradiol can work through any of several estrogen receptors to enhance the rate of acquisition on this task.     

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.     

Gradual translocation of spatial correlates of neuronal firing in the hippocampus toward prospective reward locations

In a continuous T-maze alternation task, CA1 complex-spike neurons in the hippocampus differentially fire as the rat traverses overlapping segments of the maze (i.e., the stem) repeatedly via alternate routes. The temporal dynamics of this phenomenon were further investigated in the current study. Rats learned the alternation task from the first day of acquisition and the differential firing pattern in the stem was observed accordingly. More importantly, we report a phenomenon in which spatial correlates of CA1 neuronal ensembles gradually changed from their original firing locations, shifting toward prospective goal locations in the continuous T-maze alternation task. The relative locations of simultaneously recorded firing fields, however, were preserved within the ensemble spatial representation during this shifting. The within-session shifts in preferred firing locations in the absence of any changes in the environment suggest that certain cognitive factors can significantly alter the location-bound coding scheme of hippocampal neurons.     

Effects of gonadectomy on polymorphism in stored and circulating gonadotropins in the bullfrog, Rana catesbeiana. I. Clearance profiles

Marked differences were observed between the clearance profiles of immunoreactive plasma gonadotropins in gonadectomized and intact male bullfrogs (Rana catesbeiana). The disappearance patterns of endogenously secreted follicle-stimulating hormone (FSH) and luteinizing hormone (LH) from plasma of intact animals following chronic (1-4 days) infusion with gonadotropin releasing hormone (GnRH) showed multiple components, but the initial few half-lives were relatively short (less than 1 h) and about 90% of both gonadotropins were cleared from the plasma within 6 h. Hypophysectomy had no effect on gonadotropin clearance rates following the termination of GnRH infusion. Clearance profiles of exogenous gonadotropins after chronic (6 h) infusion of bullfrog pituitary extract were similar to those observed after GnRH infusion. Gonadectomized frogs also cleared these infused pituitary gonadotropins at the same rate as intact animals, confirming that gonadectomy did not impair peripheral clearance mechanisms. Relatively rapid clearance rates were also observed for endogenous FSH and LH in normal untreated frogs. By comparison, the disappearance rates of FSH and LH from plasma of six long-term gonadectomized males following hypophysectomy were extremely slow: first half-lives for FSH and LH were 25.6 h and 17.2 h, respectively, and subsequent half-lives were even longer. Several weeks were required to clear fully the FSH and LH from the circulation in these males. Thus, a significant change in the physicochemical form of the circulating gonadotropins after gonadectomy in the male bullfrog is postulated; the corresponding changes in clearance rates were considerably greater than have been observed in any other species.     

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.     

Both PKMζ and KIBRA are closely related to reference memory but not working memory in a T-maze task in rats

Protein kinase M zeta (PKMζ) and the kidney and brain protein (KIBRA) play important roles in various forms of memories. However, whether they are involved in performing the T-maze task is still unknown. In this study, the delayed nonmatch-to-sample (DNMS) task in a T-maze was given to rats. The percentage of correct choices denoting the performance accuracy was calculated and the protein levels of PKMζ and KIBRA in rat’s prefrontal cortex were measured. The results showed significantly increased performance accuracy after the training phase, which was maintained on the next day in groups with a delay of 10 s but not 30 s, indicating that 30 s is too long for rats to maintain working memory. As for the expressions of PKMζ and KIBRA, significant increases were observed 1 day after the training phase, indicating that the formation of reference memory accompanies an increase in PKMζ and KIBRA. No significant difference was found among groups with various delay intervals, indicating that the expressions of PKMζ and KIBRA exert no effects on the performance of working memory. These results provide the first evidence that KIBRA as well as PKMζ is closely related to reference memory but not working memory in rats.     

Estrogen replacement enhances acquisition of a spatial memory task and reduces deficits associated with hippocampal muscarinic receptor inhibition

A delayed matching-to-position (DMP) T-maze task was used to examine the effects of estrogen replacement on spatial learning and memory, as well as the ability of estrogen replacement to reduce performance deficits produced by acute systemic and intrahippocampal muscarinic cholinergic inhibition. Two experiments were performed. In Experiment 1, ovariectomized animals were trained to criterion on the DMP task and then tested with increased intertrial delays and following systemic scopolamine administration. The animals then received either continuous estrogen replacement or sham surgery and were retested beginning 10 days later. In Experiment 2, ovariectomized animals received guide cannulae implanted bilaterally into the hippocampus. Half of these animals also began receiving continuous estrogen replacement. Two months later, the animals were trained on the DMP task and then tested with increased intertrial delays and following systemic as well as intrahippocampal scopolamine administration. Animals received the same test battery 8 months later and were then immediately trained on a reversal task. The results indicate that estrogen-treated animals acquired the DMP task at a significantly faster rate than the ovariectomized, non-estrogen-treated controls. In addition, estrogen replacement significantly reduced deficits in DMP performance produced by intrahippocampal, but not systemic, scopolamine administration. This occurred when animals were tested after 3.5 months, as well as after 12 months, of continuous estrogen replacement. No evidence for an effect of estrogen replacement on spatial working memory or reversal learning was detected. These findings demonstrate that estrogen replacement can enhance acquisition of a spatial memory task and reduce performance deficits associated with hippocampal cholinergic impairment.     

Regulation of neural oxytocin gene expression by gonadal steroids in pubertal rats

We have previously demonstrated that neuronal oxytocin mRNA increases during the pubertal development of female rats. In this paper we have examined the factors that regulate this developmental increase in both male and female rats. Northern blot analysis demonstrated that neural oxytocin mRNA increased 5- to 10-fold from postnatal day 20 (P20) to P60 in animals of both sexes, coincident with puberty. Mature male rats and females at all stages of the estrous cycle expressed similar levels of neural oxytocin mRNA. Pubertal up-regulation of oxytocin mRNA was largely, but not completely, inhibited by prepubescent gonadectomy, indicating a requirement for intact gonads as well as some other as yet undefined factor(s). Pubertal treatment of gonadectomized animals with estradiol or testosterone abolished the effects of gonadectomy; treated animals expressed levels of neural oxytocin mRNA similar to those in controls. However, treatment of prepubertal animals with estradiol or testosterone from P10 to P20 had no effect on oxytocin mRNA levels, suggesting that neural maturation or other factors are necessary requisites for steroid sensitivity. To determine whether neural activin played any role in regulating oxytocin mRNA during puberty, we examined levels of inhibin/activin beta A-chain mRNA. This mRNA was expressed at similar levels in all brain regions and did not vary as a function of gonadectomy or steroid treatment, making it unlikely that activin mediates the observed changes. Together, these data indicate that neural oxytocin mRNA is induced by gonadal steroids during puberty, and suggest a mechanism for coordinating development of reproductive functions with other pubertal changes.     

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.