Cell Proliferation and Cell Death Levels in the Dentate Gyrus Correlate with Home Range Size Among Adult Male Meadow Voles.

Neurogenesis occurs throughout adulthood within the dentate gyrus, and evidence indicates that these new neurons play a critical role in both spatial and social memory. However, a vast majority of past research on adult neurogenesis has involved experiments with captive mice and rats, making the generalizability of results to natural settings questionable. We assessed the connection between adult neurogenesis and memory by measuring the home range size of wild-caught, free-ranging meadow voles (Microtus pennsylvanicus). Adult male voles (n = 18) were captured, fitted with radio collars, and released back into their natural habitat, where each vole's home range was assessed using 40 radio-telemetry fixes over the course of 5 evenings. Voles were then recaptured, and brain tissue was collected. Cellular markers of cell proliferation (pHisH3, Ki67), neurogenesis (DCX), and pyknosis were labeled on histological sections and then quantified using either fluorescent or light microscopy. Voles with larger home ranges had significantly higher pHisH3+ cell densities within the granule cell layer and subgranular zone (GCL + SGZ) of the dentate gyrus and higher Ki67+ cell densities in the dorsal GCL + SGZ. Voles with larger ranges also had significantly higher pyknotic cell densities in the entire GCL + SGZ and in the dorsal GCL + SGZ. These results support the hypothesis that cell proliferation and cell death within the hippocampus are involved with spatial memory formation. However, a marker of neurogenesis (DCX+) was not correlated with range size, suggesting that there may be selective cellular turnover in the dentate gyrus when a vole is ranging through its environment.

Dose-dependent effects of testosterone on spatial learning strategies and brain-derived neurotrophic factor in male rats.

Studies suggest that males outperform females on some spatial tasks. This may be due to the effects of sex steroids on spatial strategy preferences. Past experiments with male rats have demonstrated that low doses of testosterone bias them toward a response strategy, whereas high doses of testosterone bias them toward a place strategy. We investigated the effect of different testosterone doses on the ability of male rats to effectively employ these two spatial learning strategies. Furthermore, we quantified concentrations of brain-derived neurotrophic factor (pro-, mature-, and total BDNF) in the prefrontal cortex, hippocampus, and striatum. All rats were bilaterally castrated and assigned to one of three daily injection doses of testosterone propionate (0.125, 0.250, or 0.500 mg/rat) or a control injection of the drug vehicle. Using a plus-maze protocol, we found that a lower testosterone dose (0.125 mg) significantly improved rats' performance on a response task, whereas a higher testosterone dose (0.500 mg) significantly improved rats' performance on a place task. In addition, we found that a low dose of testosterone (0.125 mg) increased total BDNF in the striatum, while a high dose (0.500 mg) increased total BDNF in the hippocampus. Taken altogether, these results suggest that high and low levels of testosterone enhance performance on place and response spatial tasks, respectively, and this effect is associated with changes in BDNF levels within relevant brain regions.

Testosterone and Adult Neurogenesis.

It is now well established that neurogenesis occurs throughout adulthood in select brain regions, but the functional significance of adult neurogenesis remains unclear. There is considerable evidence that steroid hormones modulate various stages of adult neurogenesis, and this review provides a focused summary of the effects of testosterone on adult neurogenesis. Initial evidence came from field studies with birds and wild rodent populations. Subsequent experiments with laboratory rodents have tested the effects of testosterone and its steroid metabolites upon adult neurogenesis, as well as the functional consequences of induced changes in neurogenesis. These experiments have provided clear evidence that testosterone increases adult neurogenesis within the dentate gyrus region of the hippocampus through an androgen-dependent pathway. Most evidence indicates that androgens selectively enhance the survival of newly generated neurons, while having little effect on cell proliferation. Whether this is a result of androgens acting directly on receptors of new neurons remains unclear, and indirect routes involving brain-derived neurotrophic factor (BDNF) and glucocorticoids may be involved. In vitro experiments suggest that testosterone has broad-ranging neuroprotective effects, which will be briefly reviewed. A better understanding of the effects of testosterone upon adult neurogenesis could shed light on neurological diseases that show sex differences.

Testosterone replacement causes dose-dependent improvements in spatial memory among aged male rats.

Testosterone has been shown to have dose-dependent effects on spatial memory in males, but the effects of aging upon this relationship remain unclear. Additionally, the mechanism by which testosterone regulates memory is unknown, but may involve changes in brain-derived neurotrophic factor (BDNF) within specific brain regions. We tested the effects of age and testosterone on spatial memory among male rats using two spatial memory tasks: an object-location memory task (OLMT) and the radial-arm maze (RAM). Castration had minimal effect on performance on the RAM, but young rats (2 months) performed significantly fewer working memory errors than aged rats (20 months), and aged rats performed significantly fewer reference memory errors. Both age and castration impaired performance on the OLMT, with only the young rats with intact gonads successfully performing the task. Subsequent experiments involved daily injections of either drug vehicle or one of four doses of testosterone propionate (0.125, 0.250, 0.500, and 1.00 mg/rat) given to castrated aged males. On the RAM, a low physiological dose (0.125 mg) and high doses (0.500-1.000 mg) of testosterone improved working memory, while an intermediate dose (0.250 mg) did not. On the OLMT, only the 0.250 mg T group showed a significant increase in exploration ratios from the exposure trials to the testing trials, indicating that this group remembered the position of the objects. Brain tissue (prefrontal cortex, hippocampus, and striatum) was collected from all subjects to assay BDNF. We found no evidence that testosterone influenced BDNF, indicating that it is unlikely that testosterone regulates spatial memory through changes in BDNF levels.

Effects of testosterone dose on spatial memory among castrated adult male rats.

Previous research on the activational effects of testosterone on spatial memory has produced mixed results, possibly because such effects are dose-dependent. We tested a wide range of testosterone doses using two spatial memory tasks: a working-reference memory version of the radial-arm maze (RAM) and an object location memory task (OLMT). Adult male Sprague-Dawley rats were castrated or sham-castrated and given daily injections of drug vehicle (Oil Sham and Oil GDX) or one of four doses of testosterone propionate (0.125, 0.250, 0.500, and 1.000 mg T) beginning seven days before the first day of behavioral tests and continuing throughout testing. For the RAM, four arms of the maze were consistently baited on each day of testing. Testosterone had a significant effect on working memory on the RAM, with the Oil Sham, 0.125 mg T, and 0.500 mg T groups performing better than the Oil GDX group. In contrast, there was no significant effect of testosterone on spatial reference memory on the RAM. For the OLMT, we tested long-term memory using a 2 h inter-trial interval between first exposure to two identical objects and re-exposure after one object had been moved. Only the 0.125 and 0.500 mg T groups showed a significant increase in exploration of the moved object during the testing trials, indicating better memory than all other groups. Testosterone replacement restored spatial memory among castrated male rats on both behavioral tasks, but there was a complex dose-response relationship; therefore, the therapeutic value of testosterone is likely sensitive to dose.

Seasonal and sex differences in cell proliferation, neurogenesis, and cell death within the dentate gyrus of adult wild-caught meadow voles.

Past research indicates that female meadow voles (Microtus pennsylvanicus) show decreased neurogenesis within the hippocampus during the breeding season relative to the non-breeding season, whereas male voles show no such seasonal changes. We expanded upon these results by quantifying a variety of endogenous cell proliferation and neurogenesis markers in wild-caught voles. Adult male and female voles were captured in the summer (breeding season) or fall (non-breeding season), and blood samples and brain tissue were collected. Four cellular markers (pHisH3, Ki67, DCX, and pyknosis) were labeled and then quantified using either fluorescent or light microscopy. The volume of the cell layers within the dentate gyrus (hilus and granule cell layer) was significantly larger in males than in females. In both sexes, all the cellular markers decreased significantly in the dentate gyrus during the breeding season relative to the non-breeding season, indicating decreased cell proliferation, neurogenesis, and pyknosis. Only the pHisH3 marker showed a sex difference, with females having a greater density of this cell proliferation marker than males. During the breeding season relative to the non-breeding season, males and females showed the predicted significant increases in testosterone and estradiol, respectively. Overall, these results suggest higher levels of neuronal turn-over during the non-breeding season relative to the breeding season, possibly due to seasonal changes in sex steroids.

Changes in the sexual behavior and testosterone levels of male rats in response to daily interactions with estrus females.

Male rat sexual behavior has been intensively studied over the past 100 years, but few studies have examined how sexual behavior changes over the course of several days of interactions. In this experiment, adult male rats in the experimental group (n=12) were given daily access to estrus females for 30 min per day for 15 consecutive days while control males (n=11) did not interact with females. Ovariectomized females were induced into estrus with hormonal injections, and males interacted with a different female each day. The amount of sexual activity (mounts, intromissions, and ejaculations) was found to cycle with a period of approximately 4 days in most male rats. Additionally, blood was collected every other day following sexual interactions to assess serum testosterone levels. Testosterone was found to peak on the first day of interaction and then fell back to near the level of control rats that did not interact with females. Following the initial peak, testosterone concentrations fluctuated less in males exposed to females than in controls. Sexual activity was not found to predict testosterone concentration. We conclude that when male rats have daily sexual interactions, sexual behavior tends to show cyclic changes and testosterone is significantly elevated only on the first day of interactions.

Testosterone influences spatial strategy preferences among adult male rats.

Males outperform females on some spatial tasks, and this may be partially due to the effects of sex steroids on spatial strategy preferences. Previous work with rodents indicates that low estradiol levels bias females toward a striatum-dependent response strategy, whereas high estradiol levels bias them toward a hippocampus-dependent place strategy. We tested whether testosterone influenced the strategy preferences in male rats. All subjects were castrated and assigned to one of three daily injection doses of testosterone (0.125, 0.250, or 0.500 mg/rat) or a control group that received daily injections of the drug vehicle. Three different maze protocols were used to determine rats' strategy preferences. A low dose of testosterone (0.125 mg) biased males toward a motor-response strategy on a T-maze task. In a water maze task in which the platform itself could be used intermittently as a visual cue, a low testosterone dose (0.125 mg) caused a significant increase in the use of a cued-response strategy relative to control males. Results from this second experiment also indicated that males receiving a high dose of testosterone (0.500 mg) were biased toward a place strategy. A third experiment indicated that testosterone dose did not have a strong influence on the ability of rats to use a nearby visual cue (floating ball) in the water maze. For this experiment, all groups seemed to use a combination of place and cued-response strategies. Overall, the results indicate that the effects of testosterone on spatial strategy preference are dose dependent and task dependent.

Effects of testosterone on spatial learning and memory in adult male rats.

A male advantage over females for spatial tasks has been well documented in both humans and rodents, but it remains unclear how the activational effects of testosterone influence spatial ability in males. In a series of experiments, we tested how injections of testosterone influenced the spatial working and reference memory of castrated male rats. In the eight-arm radial maze, testosterone injections (0.500 mg/rat) reduced the number of working memory errors during the early blocks of testing but had no effect on the number of reference memory errors relative to the castrated control group. In a reference memory version of the Morris water maze, injections of a wide range of testosterone doses (0.0625-1.000 mg/rat) reduced path lengths to the hidden platform, indicative of improved spatial learning. This improved learning was independent of testosterone dose, with all treatment groups showing better performance than the castrated control males. Furthermore, this effect was only observed when rats were given testosterone injections starting 7 days prior to water maze testing and not when injections were given only on the testing days. We also observed that certain doses of testosterone (0.250 and 1.000 mg/rat) increased perseverative behavior in a reversal-learning task. Finally, testosterone did not have a clear effect on spatial working memory in the Morris water maze, although intermediate doses seemed to optimize performance. Overall, the results indicate that testosterone can have positive activational effects on spatial learning and memory, but the duration of testosterone replacement and the nature of the spatial task modify these effects.

Prior sexual experience increases hippocampal cell proliferation and decreases risk assessment behavior in response to acute predator odor stress in the male rat.

Acute exposure to the predator odor trimethylthiazoline (TMT) induces defensive behavior in the male rat, and this response is associated with a decrease in cell proliferation within the dentate gyrus of the hippocampus. Sexual experience appears to be protective, as it exerts anxiolytic-like effects and sustains gonadal function in the face of stress. To examine the influence of sexual experience on subsequent stress-induced defensive behavior and cell proliferation in the hippocampus we exposed adult male rats to TMT odor with or without prior exposure to sexually receptive female rats. A subset of rats was injected with the DNA-synthesis marker bromodeoxyuridine (BrdU; 200 mg/kg) during TMT exposure and perfused 24 h later to provide an index of cell proliferation within the dentate gyrus. In response to TMT, sexual experience reduced the duration of stretched attend postures, but had no significant effect on defensive burying. Furthermore, TMT induced a significant increase in cell proliferation in the dentate gyrus, but only in males with sexual experience. The results demonstrate an influence of socio-sexual experience on the magnitude of the behavioral and neural responses to predator odor stress.

Castration differentially affects spatial working and reference memory in male rats.

A male advantage for spatial learning and memory tasks is well documented among humans and rodents. A possible physiological cause for this male advantage is activational effects of androgens among males. The spatial memory of eight castrated and eight sham-castrated adult male rats was compared using a working-reference memory version of the eight-arm radial arm maze followed by a reference memory version of the Morris water maze. After maze testing, blood was collected from each rat, and testosterone levels were determined using radioimmunoassay. In the radial arm maze, castrates committed significantly more working memory errors and significantly fewer reference memory errors than did shams. In the water maze, no statistically significant differences were found for acquisition or retention. There was a trend for shams with higher testosterone levels to have better retention in the water maze, but this seemed to be due to higher levels of perseverance rather than better reference memory. Castration may have affected performance in the radial arm maze and not in the water maze because the radial arm maze was a more difficult task or because the water maze was aversively motivated while the radial arm maze was appetitively motivated. Our results indicate that androgens improve working memory and may impair reference memory, but the effects of androgens on reference memory seem to be task dependent.

Testosterone and dihydrotestosterone, but not estradiol, enhance survival of new hippocampal neurons in adult male rats.

Past research suggested that androgens may play a role in the regulation of adult neurogenesis within the dentate gyrus. We tested this hypothesis by manipulating androgen levels in male rats. Castrated or sham castrated male rats were injected with 5-Bromo-2'deoxyuridine (BrdU). BrdU-labeled cells in the dentate gryus were visualized and phenotyped (neural or glial) using immunohistochemistry. Castrated males showed a significant decrease in 30-day cell survival within the dentate gyrus but there was no significant change in cell proliferation relative to control males, indicating that androgens positively affect cell survival, but not cell proliferation. To examine the role of testosterone on hippocampal cell survival, males were injected with testosterone s.c. for 30 days starting the day after BrdU injection. Higher doses (0.5 and 1.0 mg/kg) but not a lower dose (0.25 mg/kg) of testosterone resulted in a significant increase in neurogenesis relative to controls. We next tested the role of testosterone's two major metabolites, dihydrotestosterone (DHT), and estradiol, upon neurogenesis. Thirty days of injections of DHT (0.25 and 0.50 mg/kg) but not estradiol (0.010 and 0.020 mg/kg) resulted in a significant increase in hippocampal neurogenesis. These results suggest that testosterone enhances hippocampal neurogenesis via increased cell survival in the dentate gyrus through an androgen-dependent mechanism.

Gonadal hormone modulation of hippocampal neurogenesis in the adult.

Gonadal hormones modulate neurogenesis in the dentate gyrus (DG) of adult rodents in complex ways. Estradiol, the most potent estrogen, initially enhances and subsequently suppresses cell proliferation in the dentate gryus of adult female rodents. Much less is known about how estradiol modulates neurogenesis in the adult male rodent; however, recent evidence suggests that estradiol may have a moderate effect on cell proliferation but enhances cell survival in the DG of newly synthesized cells but only when estradiol is administered during a specific stage in the cell maturation cycle in the adult male rodent. Testosterone likely plays a role in adult neurogenesis, although there have been no direct studies to address this. However, pilot studies from our laboratory suggest that testosterone up-regulates cell survival but not cell proliferation in the DG of adult male rats. Progesterone appears to attenuate the estradiol-induced enhancement of cell proliferation. Neurosteroids such as allopregnalone decrease neurogenesis in adult rodents, while pregnancy and motherhood differentially regulate adult neurogenesis in the adult female rodent. Very few studies have investigated the effects of gonadal hormones on male rodents; however, studies have indicated that there is a gender difference in the response to hormone-regulated hippocampal neurogenesis in the adult. Clearly, more work needs to be done to elucidate the effects of gonadal hormones on neurogenesis in the DG of both male and female rodents.

The relationship between dominance rank and spatial ability among male meadow voles (Microtus pennsylvanicus).

Males of many mammalian species exhibit contest competition and scramble competition for mates, but the relationship between these 2 forms of competition remains poorly understood. The authors measured dominance rank and spatial ability as traits likely to be selected by contest and scramble competition, respectively, among male meadow voles (Microtus pennsylvanicus). The spatial ability of males was assessed using water maze tests, and dominance rank was determined using paired trials in a neutral arena. Dominant males had better spatial-learning ability and tended to have quicker learning speed but did not have better spatial memory than less aggressive subordinates. Therefore, the authors found no evidence that contest and scramble competition have favored alternative reproductive phenotypes among male meadow voles.