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

Highlights

• Aged rats had impaired spatial working memory relative to young rats.

• Aging and castration impaired performance on an object location memory task.

• High and low, but not intermediate, doses of testosterone improved working memory.

• An intermediate dose of testosterone improved long-term spatial memory.

• Testosterone manipulations had no effect on BDNF levels in the brains of aged rats.

Abstract

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.

Introduction

Reduced endogenous testosterone may be an important contributor to age-related memory loss in men (Handelsman et al., 2015). Testosterone decline begins as early as age 30, and hypogonadism occurs in 20 % of men 60−69 year olds and in 50 % of men over 80 (Feldman et al., 2002). Numerous studies have shown a positive correlation between testosterone levels and cognitive ability in older men, including better spatial memory, suggesting that a reduction in testosterone levels may be partially responsible for age-related memory deficits in men (Barrett-Connor et al., 1999; Yaffe et al., 2002; Moffat et al., 2002). Additionally, testosterone reduction through age-related hypogonadism or artificial androgen deprivation has been identified as a risk factor for dementia (Lv et al., 2016; Nead et al., 2017). Testosterone supplementation improved spatial memory among patients with Alzheimer’s disease or mild cognitive impairment (Cherrier et al., 2005; Tan and Pu, 2003), but the benefits of androgen replacement therapies have been inconsistent (Kenny et al., 2004; Lu et al., 2006). Studies with healthy older men have also produced mixed results regarding the effects of androgen supplementation on cognitive ability (Cherrier et al., 2001; Vaughan et al., 2007; Wolf et al., 2000). These discrepancies may be due to differences among studies in the duration of hormone exposure, doses of testosterone used, and the specific aspects of spatial memory tested. Controlled experiments with animal models provide a useful way to disentangle these variables.

Spatial memory is one cognitive process that seems to be particularly influenced by both circulating testosterone and aging (Holland et al., 2011). Involving aspects of procedural, declarative, as well as both short-term and long-term memory, spatial memory is the process of recognizing, encoding, storing, and recalling spatial information about surroundings, positions of objects, or specific routes (Moscovitch et al., 2006). Testosterone seems to differentially influence the working and reference components of spatial memory. Working memory is a form of short-term memory that involves storage of relevant information while completing a specific task, whereas reference memory involves long-term storage of information from one task to be used for the next task (Cowan, 2008). Using a rat model, we recently demonstrated that testosterone enhances spatial working memory but has no effect on spatial reference memory (Wagner et al., 2018), which corroborates previous findings (Bimonte-Nelson et al., 2003; Gibbs and Johnson, 2008; Spritzer et al., 2011, 2008). However, experiments using the object location memory task (OLMT) suggest that testosterone influences some forms of long-term memory. Specifically, testosterone replacement was shown to improve spatial memory among castrated young adult male rats on the OLMT following both a 30 min retention period (McConnell et al., 2012) and a 2 h retention period (Jacome et al., 2016; Wagner et al., 2018). Thus, the length of the retention period may be a critical variable influencing the memory-enhancing effects of testosterone.

Considerable evidence from studies with humans and rodents suggests that the relationship between circulating testosterone levels and spatial memory is non-linear (Muller et al., 2005; Nowak et al., 2014). Testing male rats on the Morris Water Maze (MWM),we previously demonstrated that certain doses of testosterone (0.250 and 1.00 mg/rat) impaired reversal learning (Spritzer et al., 2011). Using other memory tasks (RAM and OLMT), we demonstrated that high and low physiological doses of testosterone (0.125 and 0.500 mg/rat) improved spatial memory, whereas an intermediate dose (0.250 mg/rat) did not (Wagner et al., 2018). Another study with rats suggested a curvilinear relationship between testosterone and spatial reference memory, with a high physiological dose (0.750 mg/rat) of testosterone optimal for performance (Jia et al., 2013). Administering supra-physiological doses of testosterone has either no effect on memory (Gibbs, 2005) or impairs spatial memory (Emamian et al., 2010). Additionally, human studies suggest that the optimal level of testosterone for spatial memory varies with age (Holland et al., 2011), but this hypothesis has yet to be tested experimentally.

The physiological mechanism by which testosterone influences spatial memory remains unknown. The hippocampus, striatum, and prefrontal cortex have all been implicated in spatial memory (Compton et al., 1997; Liljeholm and O’Doherty, 2012), making them potential direct targets for testosterone. The hippocampus is essential for place learning (i.e., the processing of information about the positions of environmental cues relative to a goal and one’s own position), whereas the striatum is essential for response learning (i.e., the use of stimulus-response relationships to locate a goal). Initially, both the hippocampus and striatum are engaged in parallel to solve a spatial task, and then the striatum becomes more important as the task becomes habitual (Chang and Gold, 2003; Jacobson et al., 2012; Packard and McGaugh, 1996). The pre-frontal cortex seems to be more involved in response learning, and is specifically engaged during spatial working memory tasks to plan the next choice (de Bruin et al., 1997; Floresco et al., 1997). Evidence suggests that the pre-frontal cortex is not essential for performance on the OLMT (Chao et al., 2016) but is employed in parallel with other brain regions for solving RAM tasks (Floresco et al., 1997; Kolb et al., 1994). Due to its critical role in neural plasticity and spatial cognition, brain-derived neurotrophic factor (BDNF) could play a key role in the connection between testosterone and cognitive processing in select brain regions (Egan et al., 2003; Tyler et al., 2002). Some evidence indicates that male rats have higher hippocampal levels of BDNF than do females (Franklin and Perrot-Sinal, 2006). Although BDNF levels have been found to decrease with age in male rats, this decline does not seem to correlate with age-related changes in testosterone (Bimonte-Nelson et al., 2008). Consequently, the relationship between testosterone and BDNF remains unclear and, therefore, merits further investigation.

Few past studies have tested the effects of testosterone on spatial memory in aged rodents (Bimonte-Nelson et al., 2003; Goudsmit et al., 1990), and none have used a broad dose range. Building upon our past research with young rats (Wagner et al., 2018), the present study tested the effects of both age and testosterone dose on spatial memory. Initial experiments tested the effects of castration and age by comparing the performance of 20-month-old and 2-month-old male rats on the RAM and OLMT. Subsequent experiments used the same tasks to test the cognitive effects of testosterone replacement on aged males. We also quantified BDNF levels in the hippocampus, striatum, and prefrontal cortex to assess a possible mechanistic link between testosterone and BDNF.