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

Highlights

• Testosterone replacement restored spatial working memory in castrated male rats.

• Testosterone replacement had no effect on reference memory in castrated male rats.

• Testosterone replacement improved long-term memory in castrated male rats.

• High and low physiological doses of testosterone had positive effects on memory.

• A supra-physiological dose of testosterone had some positive effects on memory.

Abstract

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.

Introduction

Spatial memory, the ability to recall the locations and relative spatial relationships of stimuli in one’s environment, shows a robust sex difference in humans (Voyer et al., 1995), with men performing better than women on a variety of spatial tasks (Kaufman, 2007; Woolley et al., 2010). Similarly, male rats have better spatial memory than females based on performance in the Morris water maze (MWM) (Markowska, 1999), radial-arm maze (RAM) (Gibbs and Johnson, 2008), and object location memory task (OLMT) (Cost et al., 2012). Considerable evidence indicates that male sex steroids (androgens) play an important role in producing sex differences in spatial memory. Experiments with humans and rats have shown that testosterone has organizational effects upon the brain early in development that enhance spatial learning and memory (Bull et al., 2010; Isgor and Sengelaub, 1998). Whether androgens have activational effects that enhance spatial memory in adult men remains controversial (Holland et al., 2011; Ulubaev et al., 2009), but some studies have shown a positive correlation between circulating testosterone levels and performance on a variety of spatial memory tasks both in younger men, under 30 years old (Christiansen and Knussmann, 1987; Silverman et al., 1999; Thilers et al., 2006), and in older men, over 50 years old (Barrett-Connor et al., 1999; Moffat et al., 2002; Yaffe et al., 2002). Among patients with prostate cancer, androgen deprivation therapy caused significant impairments in spatial ability (Cherrier et al., 2009). However, testosterone replacement given to healthy elderly men has produced mixed effects on spatial cognition (Emmelot-Vonk et al., 2008), so the therapeutic benefits of testosterone replacement remain unclear.

Studies with rats have shown that castration in adulthood impairs spatial memory (Gibbs and Johnson, 2008; Kritzer et al., 2001; Spritzer et al., 2008), and exogenous testosterone replacement restores the spatial memory of castrated adult male rats to normal (Bimonte-Nelson et al., 2003; Spritzer et al., 2011). Additionally, testosterone seems to differentially influence the working and reference components of spatial memory. Within this context, working memory is defined as a form of short-term memory that involves storage of information from a particular task for as long as it is useful to complete the task, and reference memory is defined as the long-term storage of memories that are used from one task to the next (Cowan, 2008; Olton and Papas, 1979). Although most studies show no effect of castration on reference memory on the RAM (Gibbs and Johnson, 2008; Spritzer et al., 2008), some experiments involving the MWM indicate that testosterone replacement can improve spatial reference memory in adult male rats (Khalil et al., 2005; Spritzer et al., 2011). In contrast, testosterone replacement has consistently been shown to improve spatial working memory on various versions of the RAM (Bimonte-Nelson et al., 2003; Gibbs and Johnson, 2008; Spritzer et al., 2008). However, the reference vs. working memory dichotomy does not fully capture the variety of spatial memory tasks that have been employed with rodents. For example, two experiments have shown that testosterone injections improve performance on the OLMT with a 2 h delay interval (Jacome et al., 2016; McConnell et al., 2012), suggesting that testosterone improves reference (i.e., long-term) memory when the retention period is relatively short. Thus, although the most consistent activational benefits of testosterone have been demonstrated using working-memory tasks, testosterone may also improve some forms of long-term memory.

It has been speculated that the relationship between testosterone and spatial memory is curvilinear, with both unusually low and high levels of testosterone causing memory impairments. In support of this hypothesis, long-term use of anabolic-androgenic steroids has been shown to impair memory in humans (Kaufman et al., 2015). Similarly, intra-hippocampal injections of supra-physiological doses of testosterone impair spatial memory in male rats (Emamian et al., 2010). Some studies with men have supported the optimum-testosterone hypothesis (Muller et al., 2005; Nowak et al., 2014), but few studies have tested a broad range of testosterone doses on spatial memory using rodents. In our previous work, we found that a broad range of testosterone doses improved performance on the MWM relative to castrated control rats, and certain doses (0.250 and 1.00 mg/rat) impaired reversal learning (Spritzer et al., 2011). Another study using the MWM demonstrated 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).

In the current study, we tested the effects of both testosterone elimination and replacement, using four doses of testosterone, ranging from physiological to supra-physiological, on the performance of adult male rats on two different tasks: a working-reference memory version of the eight-arm RAM and an OLMT. For our RAM experiment, we predicted that testosterone replacement would restore working memory in castrated male rats while having minimal impact on reference memory (Spritzer et al., 2011). The OLMT has been used in relatively few studies testing the relationship between testosterone and memory (Jacome et al., 2016; McConnell et al., 2012). Two advantages of this task are that it avoids the stressors associated with other tasks and that the inter-trial interval can be easily varied to test different memory durations. We used a 2 h inter-trial interval on the OLMT for comparison with the longer retention period (24 h) that we used for RAM testing. Thus, these experiments replicate and expand upon past research designed to assess the cognitive benefits of androgen replacement.