Physiology & Behavior 106 (2012) 229237

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Physiology & Behavior

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Short-term testosterone manipulations modulate visual recognition memory and some aspects of emotional reactivity in male rhesus monkeys
Agns Lacreuse , Heather E. Gore, Jeemin Chang, Emily R. Kaplan
Department of Psychology, University of Massachusetts, Amherst, MA 01003, United States

a r t i c l e

i n f o

a b s t r a c t
The role of testosterone (T) in modulating cognitive function and emotion in men remains unclear. The paucity of animal studies has likely contributed to the slow progress in this area. In particular, studies in nonhuman primates have been lacking. Our laboratory has begun to address this issue by pharmacologically manipulating T levels in intact male rhesus monkeys, using blind, placebo-controlled, crossover designs. We previously found that T-suppressed monkeys receiving supraphysiological T for 4 weeks had lower visual recognition memory for long delays and enhanced attention to videos of negative social stimuli (Lacreuse et al., 2009, 2010) compared to when treated with oil. To further delineate the conditions under which T affects cognition and emotion, the present study focused on the short-term effects of physiological T. Six intact males were treated with the gonadotropin-releasing hormone antagonist degarelix (3 mg/kg) for 7 days and received one injection of T enanthate (5 mg/kg) followed by one injection of oil vehicle 7 days later (n = 3), or the reverse treatment (n = 3). Performance on two computerized tasks, the Delayed-nonmatching-to-sample (DNMS) with random delays and the object-Delayed Recognition Span test (objectDRST) and one task of emotional reactivity, an approach/avoidance task of negative, familiar and novel objects, was examined at baseline and 35 days after treatment. DNMS performance was signicantly better when monkeys were treated with T compared to oil, independently of the delay duration or the nature (emotional or neutral) of the stimuli. Performance on the object-DRST was unaffected. Interestingly, subtle changes in emotional reactivity were also observed: T administration was associated with fewer object contacts, especially on negative objects, without overt changes in anxious behaviors. These results may reect increased vigilance and alertness with high T. Altogether, the data suggest that changes in general arousal may underlie the benecial effects of T on DNMS performance. This hypothesis will require further study with objective measures of physiological arousal. 2012 Elsevier Inc. All rights reserved.

Article history: Received 13 January 2012 Accepted 7 February 2012 Keywords: Androgens Arousal Cognition Emotion Macaque Memory

1. Introduction Sex hormones have far-reaching effects on brain function and modulate several aspects of cognition [1,2] and emotion [3,4]. While considerable progress has been made towards understanding the neurocognitive and emotional effects of estrogens in females [510], much less research has been carried out to determine whether androgens have similar effects in males. Neurobiological and cellular data suggest that androgens have the same potential as estrogens to affect cognitive and affective processes: androgen receptors are present in brain areas important for cognition and emotion such as the hippocampus, prefrontal cortex and amygdala [1113]. Secondly, androgens modulate synaptic plasticity in the hippocampus and frontal

Corresponding author at: Department of Psychology, 135 Hicks Way, University of Massachusetts, Amherst, MA 01003, United States. Tel.: + 1 413 545 2183; fax: + 1 413 545 0996. E-mail address: alacreuse@psych.umass.edu (A. Lacreuse). 0031-9384/$ see front matter 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.physbeh.2012.02.008

cortex [1417]. Third, not unlike estradiol, androgens have a myriad of neuroprotective and neurotrophic effects [18]. Evidence for an effect of androgens, in particular testosterone (T) on cognitive function in men is mixed. While positive associations have consistently been observed between endogenous T and various tests of visuospatial ability in young men [1928], inconsistent results have been provided regarding the cognitive effects of exogenous T. In one study in which T was administered for 8 weeks to both eugonadal and hypogonadal men [29], supraphysiological T was found to decrease spatial ability and increase verbal uency. Two studies using pharmacological manipulations of T found no effect of T administration on executive function, memory, spatial cognition [30] or visuospatial ability [31]. In contrast, Cherrier et al. [32] reported that T depletion induced by a synthetic progestin was associated with a decline in verbal memory that was restored by T supplementation. Many more studies have been conducted in older men and the majority reported improvements in visuospatial ability, verbal memory, verbal uency or working memory following T administration [29,3339]. Yet, there are many inconsistencies, with other studies

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nding no effect [30,4044] or even a negative effect of T on cognition [45]. Multiple factors may account for the differences across studies, including the variety of cognitive domains examined, differences in health status and demographic factors of the population studied and differences in the specic T regimen used (i.e., dose, duration, formulation). In addition, some of the cognitive effects of T may be mediated by changes in emotional states, but emotional variables have rarely been incorporated in the studies mentioned above. The development of a new paradigm for exogenous T administration in humans, which raises T levels for only a few hours, has allowed the investigation of the inuence of T on human emotional processing [4]. These studies have repeatedly shown that T modulates anxiety, social vigilance and aggression [46]. Importantly, because of health risk posed by T administration in eugonadal men, these studies have been conducted in women. Thus, it remains unclear whether androgens have similar effects in men. Studies in animal models may be best suited to examine the effects of T on cognition and emotion in the male. The male rhesus monkey is an excellent model for such investigations because the endocrine control of testicular function is, in many respects, similar in men and male macaques [47,48] and because rhesus monkeys share many cognitive, emotional and physiological characteristics with humans [49]. In previous studies in this model, we found that supraphysiological T administered for 4 weeks had a negative impact on visual recognition memory [50] and an enhancing effect on selective attention to threatening social stimuli [51]. The present study was conducted to further delineate the conditions under which T affects cognition and emotion in males, by using an acute T administration paradigm more directly comparable to that used in recent human studies [4]. Based on these data, we tested the hypothesis that short-term treatment (35 days) with a physiological dose of T would enhance cognitive function [52] and reduce anxiety [53]. Endogenous T was suppressed with a GnRh antagonist prior to T administration to avoid supraphysiological increases in T levels and prevent reciprocal actions of the environment on endogenous T levels. Monkeys were tested in two memory tasks and an approach/avoidance task 35 days after administration of a physiological dose of TE (5 mg/kg) or oil vehicle. We predicted that T would improve performance on the memory tasks and reduce anxiety in the approach/ avoidance task. 2. Methods 2.1. Subjects Six young (8 years old) male rhesus monkeys (Macaca mulatta) participated in the study. All monkeys were housed in stainless steel primate cages in the same room at constant 12:12 h lighting conditions. Four monkeys were singly housed and two monkeys were housed together. All but one male were surrogate peer-reared [54]. The monkeys were humanely treated in accordance with the standards of the PHS policy on Humane Care and Use of Laboratory Animals. The study was approved by the Institutional Animal Care and Use Committee of the University of Massachusetts. The monkeys were procient with the touchscreen and had participated in several cognitive and emotional studies prior to the current experiment [50,51,55]. 2.2. General procedure and treatments The design of the study can be seen in Fig. 1. The experiment involved four week-long phases. The baseline phase (Week 1) was a period during which monkeys were tested on the battery of tasks in the absence of any drug treatment. The degarelix phase (degarelix only, Week 2) started 7 days after the onset of the baseline phase and

involved the administration of degarelix (Ferring Pharmaceuticals, Inc.), a newly FDA approved GnRh antagonist for use in humans with advanced prostate cancer. Degarelix rapidly suppresses T as early as 3 days after administration in humans, without the T are associated with GnRh agonists [5659]. Degarelix was injected subcutaneously at a dose of 3 mg/kg which suppresses gonadal function for up to 16 weeks in male rhesus monkeys (Dr. Wolfgang Koechling, pers. comm.). The goal of the degarelix phase was to ensure suppression of gonadal hormone activity before the onset of treatment with TE and oil. Monkeys were not tested on the tasks during this phase. Seven days after the degarelix injection, 3 monkeys received one I.M. injection of TE (5 mg/kg) and 3 monkeys received one I.M. injection of oil vehicle. Seven days after receiving their rst treatment, monkeys switched to the alternate treatment. TE administered at 5 mg/kg to adult male rhesus monkeys yields T levels that increase sharply after 24 h, peak on day 3 and decrease progressively afterwards [60]. The half-time of the TE terminal elimination phase has been shown to be 5 days in male rhesus monkeys [61]. The experimenters were blind to the treatment assignments. Monkeys performed the cognitive tasks 3 days per week (MondayWednesday, i.e., from days 3 to 5 post-treatment) on a computerized touchscreen system with a 17-inch color monitor that was rolled in front of their home cages. The emotional reactivity assessments were also performed 3 days per week in the home cage, several hours following the cognitive tests. 2.2.1. Delayed non matching to sample (DNMS) The DNMS is a classic task of visual recognition memory, in which monkeys are required to discriminate a new from a previously seen stimulus after various delays. The task was created with customdesigned Java-based software. Monkeys had previously performed a version of the task with a 1 s delay [55]. The stimuli were 6 6 cm black and white images. The images were either social images, consisting of neutral and emotional (lipsmacks or threats) portraits of rhesus monkey faces, or nonsocial images, consisting of neutral (shoes, cage locks and lab wall xtures) and emotional pictures of objects such as capture gloves and syringes (negative), or apples, bananas and grapes (positive). Animals were presented with 30 trials of the DNMS per testing day, consisting of 6 trials per delay. Images were drawn from a set of 288 images, 48 images for each emotion (negative, neutral, positive) in each category (social, nonsocial). Images were never repeated within a testing session (60 images), but were repeated twice on average during the experiment. The accuracy and response times (RT) were recorded. Monkeys performed the DNMS 3 days per week. The task began with the presentation of a start button that the animals had to touch to begin the session. Then, one sample image was presented in the center of a black screen, which the animal was required to touch in order to proceed through the trial. Following a specic delay (0, 5, 10, 15 or 30 s, randomly selected) during which the screen was blanked, two images were presented side-by-side, centered on the y-axis of the screen. One image was the same as the sample image and one image was a novel image from the same category (Fig. 2). Animals were rewarded with a fruit-avored 190 mg pellet (Test Diet, Inc) for touching the novel image. A touch on the image matching the sample or failure to respond within 60 s triggered a time-out of 15 s signaled by a green screen. 2.2.2. Object-Delayed Recognition Span Test The object-DRST required subjects to select the new stimulus among an increasing array of stimuli appearing one at a time at different locations on the screen (Fig. 2). For this task, created with Javabased software, the screen was programmed to display 15 nonoverlapping positions, arranged in a 3 5 matrix. The stimuli were clip-art images (randomly drawn at each testing session from a database of 1500 clip-art images), presented on a black background. On

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Onset of Experiment Day 7 Day 14 Day 21

Cognitive and emotional testing Day 28

Baseline

Degarelix

Treatment 1

Treatment 2

Degarelix 3 mg/kg (n=6)

TE 5 mg/kg (n=3) or Oil (n=3)

Oil (n=3) or TE 5 mg/kg (n=3)

Fig. 1. Design of the experiment.

the rst trial of a sequence, one image appeared in one of the 15 possible positions. The monkey had to touch the image to obtain a pellet. The screen was cleared and 1 s later, two images were presented: a novel image and the same image reappearing in a new location. The monkey was required to touch the novel image to obtain the reward. Each successive correct response was followed by the addition of new image until the monkey made an error, or up to a maximum of 8 images. An error a touch on an image previously seen was followed by a green display signaling the onset of a 5 s timeout. Ten trials were presented in one session. The exact position of each image at each trial was determined by a random sequence that was unique within and across sessions. The dependent variables were the mean number of images correctly identied before making an error (object memory span). Chance performance on this task is 1.72. 2.2.3. Approach/avoidance For this task, monkeys were tested 3 times a week during each phase of the experiment, hours apart from the cognitive tests (e.g., in late afternoon). Monkeys were tested in the top quadrant of their home cage (80 84 79 cm). A session consisted of a 2 min exposure to either a familiar, a novel or a threatening object. Different objects were used for each phase of the experiment (Table 1) and the order of object type was counterbalanced across weeks. At the start of a session, an object baited with a grape was placed on a familiar foraging board (39 15 1 cm) directly in front and in the middle axis of the monkey's cage opening (15 7.7 cm or 15 5 cm depending on the cage). A digital video camera (Panasonic PV-GS300) recorded behaviors elicited during each trial. Recording began as soon as the object and grape were placed on the foraging board. An ethogram was developed to measure the proportion and duration of the behaviors elicited in response to the objects (Table 2). The ethogram was constructed based on behaviors observed in a previous study [51] and were grouped into 3 general categories of behaviors: afliative, aggressive, and anxious behaviors [62,63]. The monkeys' behavior was decoded by two observers (HG and EK) blind to the treatment assignments, using JWatcher Video version 1 [64]. The mean percent agreement between the two observers was above 90%. Given the limited number of aggressive and afliative behaviors that were exhibited during the task, only the proportion of anxious behaviors (number of anxious behaviors out of the total number of behaviors exhibited during the session) was submitted to the analysis. A ratio was used as opposed to the actual number of anxious behaviors in order to account for potential changes in overall activity across days. In addition, two other variables, the latencies to touch the grape and the number of touches on the object were analyzed. 2.3. Blood samples and assays Blood samples (~4 ml/sample) were collected once a week (i.e., on Fridays) during the experiment. Blood samples were drawn from a saphenous vein between 8 h and 9 h from monkeys anesthetized

with ketamine (610 mg/kg I.M.) or Telazol (3 mg/kg). Serum was removed by centrifugation and frozen. Serum samples were analyzed for total T using commercially available RIA kits (TKTT kit, Siemens Healthcare Diagnostics Inc, NJ) and protocols in use in the laboratory of Dr. Jerrold Meyer at the University of Massachusetts. 3. Statistical analyses For all measures, the baseline phase was analyzed separately from the treatment phase. The treatment phase was analyzed using mixed model repeated measures of variance (ANOVA). For the DNMS, prior to any analyses, trials with RT exceeding two standard deviations above or below the mean were eliminated from the data set. An initial analysis was performed to determine if there were signicant differences between the left and right side of stimuli presentation, using paired t-tests. As no signicant difference was observed, the two sides were combined in further analyses. Accuracy and RT were analyzed using Treatment (TE, oil), Delays (0, 5, 10, 15, 30), Stimulus Type (social, nonsocial) and Emotional Valence (neutral, negative, positive) as within-subject factors and Sequence of Treatment (TE rst, oil rst) as a between-subject factor. For the object-DRST, the memory span was analyzed with a repeated measure ANOVA with Treatment as a within-subject factor and Sequence of Treatment as a between-subject factor. The dependent measures for the approach/avoidance task (proportion of anxious behaviors, latencies to touch the grape and number of touches on the object) were not normally distributed. Square root transformations (for the proportion of anxious behaviors and number of touches) and log transformations (for the latencies) were applied before submission to ANOVAs with Object Type (familiar, novel, negative) and Treatment as withinsubject factors and Sequence of Treatment as a between-subject factor. Planned contrasts were used to examine differences between factor levels. Nontransformed values were used for illustration purposes. Potential associations between T levels and behavior were examined using mixed linear regression models. For the DNMS and DRST analyses, the models included Treatment and Sequence of Treatment as xed factors and T levels as a covariate. Object Type as a xed factor was added to the models for the measures of the Approach/avoidance task. 4. Results 4.1. T levels T levels for each phase of the experiment are represented in Fig. 3. T levels could not be obtained from 2 serum samples and these missing data were replaced by the mean of the group. A repeated measure ANOVA with Phase and Sequence of Treatment was used to compare T levels between the baseline, degarelix, oil and TE phases. This analysis revealed a signicant effect of Phase (F(3,12) = 50.44, p = 0.001). Paired t-tests indicated that T levels were signicantly higher during

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Sample

DNMS: Negative Social

Delay (0, 5, 10, 15 or 30s)

Non-Matching

Time

Object-DRST

+ +

Time

Fig. 2. Representation of a DNMS trial with negative social stimuli (top) and the three rst steps of a trial in the object-DRST. The cross represents the rewarded stimulus.

the TE phase than during the baseline phase (t(5) = 3.23, p = 0.023), the oil phase (t(5) = 3.99, p = .001) and the degarelix phase (t(5) = 3.86, p = 0.012). T levels tended to be lower during the degarelix phase than the baseline phase (t(5) = 2.13, p = 0.086). However, T levels during oil treatment were not signicantly different from those

of baseline (t(5) = 1.52, p = 0.19). As can be seen in Fig. 3, baseline T levels were particularly low, likely due to seasonal effects, as the monkeys were tested from mid-April to May, a season typically characterized by low T levels in male rhesus monkeys [65,66]. Sequence of Treatment (F(1,4) = 11.39, p = 0.028) and the interaction between

A. Lacreuse et al. / Physiology & Behavior 106 (2012) 229237 Table 1 Objects used in each phase of the experiment. Baseline Familiar Novel Negative Red kong toy Blue boppero toy Green rubber snake Treatment 1 Blue cylinder toy Red pin toy Green costume mask with large eyes Treatment 2 Black and yellow ball toy Blue sphere toy Brown costume mask with large eyes

233

16 14 12
Group receiving TE first Group receiving Oil first

T levels (ng/ml)

10 8 6 4 2 0 Baseline Degarelix Oil TE

Phase and Sequence of Treatment (F(3,12) = 15.10, p = 0.001) were also signicant. This indicated that, unexpectedly, monkeys receiving TE as their second treatment had lower T levels than monkeys receiving TE as their rst treatment and this difference was signicant for both the TE (t(4) = 3.85, p = 0.02) and the oil phases (t(4) = 3.34, p = 0.029). As a result, Sequence of Treatment was consistently included as a factor in the analyses of the cognitive and emotional measures, and associations between T levels and behavior were systematically examined. 4.2. DNMS data Monkeys completed a total of 90 trials per phase at the DNMS. The elimination of trials with RT above 2 SDs from the mean (4% of trials) resulted in a dataset of 1555 trials. Before treatment, monkeys obtained 77% (4.9 SEM) of correct responses and responded in an average of 1.97 s (0.12 SEM). None of the main effects was signicant for either accuracy or RT, but a signicant interaction between Delay and Emotional Valence was revealed for both accuracy (F(8,16) = 2.55, p = 0.028) and RT (F(8,16) = .26, p = 0.049). This indicated an effect of delay for negative stimuli only, with performance being both worse and slower for shorter delays (0 and 5 s) than longer delays. No relationship was found between T levels at baseline and accuracy (F(1, 4.03) = 0.23, p = 0.66) or RT (F(1, 4.01) = 0.01, p = 0.93). During the treatment period, monkeys obtained an average of 76.5% (5.0 SEM) of correct responses with a mean RT of 1.89 s (0.78 SEM). The ANOVA revealed a main effect of Treatment for accuracy (F(1,4)=9.77, p=0.035), indicating that performance at the DNMS was signicantly higher when monkeys were treated with TE (M=78.6%5.1) compared to when treated with oil (M=74.4%5.0; Fig. 4). Treatment did not interact with any other factors. In particular, the interaction between Treatment and Delay was not signicant (F(4,16)=1.39, p=0.28). Accuracy tended to decrease with increasing
Table 2 Ethogram used for the approach/avoidance task (from Lacreuse et al., 2010). Category Anxious Behavior Self-directed behavior Locomotor stereotypies Teeth gnashing Coo vocalization Yawn Aggressive Cage shake Mouth-threat/lunge Aggressing the object Lip smack Presentation Grunt vocalization Denition Scratches, grooms, holds any part of the body Activities (rock/sway/jump) repeated 3 or more Chewing motion without food in mouth High-pitched, low intensity vocalization Opens mouth wide baring upper teeth Vigorously shakes cage for >1 s Opens mouth slightly or head/body lunge Biting, hitting or displacing the object with intensity Purses and alternatively closes and opens lips Presents hindquarters with tail up Low pitched, low intensity vocalization

Phase of the experiment

Fig. 3. Mean T levels (+SEM) in each phase of the experiment for monkeys receiving TE rst and monkeys receiving oil rst. Monkeys in the latter group had lower T levels than monkeys receiving TE as their rst treatment. T levels in the TE phase were signicantly higher than in the baseline, degarelix and oil phases.

delays (F(4,16)=2.32, p=0.10). Specic contrasts indicated that accuracy was lower at 30 s delay compared to both 1 s delay (F(1,4)=14.30, pb 0.02) and 5 s delay (F(1,4)=53.16, pb 0.01) but was not signicantly different from accuracy obtained at the intermediate delays of 10 s (F(1,4)=0.46, p=0.54) and 15 s (F(1,4)=0.66, p=0.46). The other main effects and interactions were not signicant for accuracy. There was no effect of Treatment on RT (F(1,4) = 0.21, p = 0.67) but a signicant effect of Delay (F(4,16) = 5.43, p = 0.006), reecting slower responses as the delays increased from 1 s to 30 s (F(1,4) = 25.31, p b 0.01). The effect of Stimulus Type approached signicance for both accuracy (F(1,4) = 5.72, p = 0.075) and RT (F(1,4) = 5.33, p = 0.082), indicating that monkeys tended to perform better and to respond faster to object compared to face stimuli, as previously found in a 1 s delay version of the test [55]. Sequence of Treatment was not signicant for either accuracy (F(1,4) = .75, p = 0.43) or RT (F(1,4) = 1.87, p = 0.24) and did not interact with any other factors. T levels were not signicant predictors of accuracy (F(1, 4.05) = 0.004, p = 0.95) or RT (F(1, 4.68) = 0.99, p = 0.36).

100

Degarelix + Oil Degarelix + TE

Percentage of correct responses

90

80

70

60

50

10

15

20

Afliative

Delays (s)
Fig. 4. Percentage of correct responses on the DNMS as a function of delays and treatment phase. Monkeys were signicantly better at the task when treated with TE compared to when treated with oil, independently of delays.

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4.3. Object-DRST Monkeys correctly identied an average of 2.64 0.18 stimuli at baseline. There was no signicant relationship between memory span and T levels at baseline (Pearson's correlation, r = 0.36, p = 0.48). The memory span obtained during oil treatment (M= 2.74 0.19) was not signicantly different from the memory span obtained during TE treatment (M= 2.84 0.31; F(1,4) = 0.33, p = 0.59). The effect of Sequence of Treatment was not signicant (F(1,4)= 3.53, p = 0.13) and did not interact with Treatment (F(1,4) = 1.90, p = 0.24). T levels were not a signicant predictor of the memory span during the treatment phase (F(1, 4.39) = 5.41, p = 0.08). 4.4. Approach/avoidance 4.4.1. Proportion of anxious behaviors At baseline, the proportion of anxious behaviors differed signicantly according to Object Type (F(2,10) = 4.13, p = 0.049), with a trend for monkeys to display more anxious behaviors during exposure to a negative compared to the two other types of objects (F(1,5) = 5.74, p = 0.06). The proportion of anxious behaviors was similar between familiar and novel objects (F(1,5) = 0.91, p = 0.38). T levels were not a signicant predictor of the proportion of anxious behaviors during baseline (F(1,4) = 1.30, p = 0.32). During treatment, the effect of Object Type approached signicance (F(2,8) = 4.33, p = 0.053). Monkeys exhibited more anxious behaviors during exposure to a negative compared to the other types of objects (F(1,4) = 8.27, p = 0.045). The proportion of anxious behaviors did not differ between familiar and novel objects (F(1,4) = 0.44, p = 0.54). There was no effect of Treatment (F(1,4) = .48, p = 0.52; Fig. 5a), or Sequence of Treatment (F(1,4) = 4.58, p = 0.10). None of the interaction was signicant. T levels were not a signicant predictor of the proportion of anxious behaviors during the treatment phase (F(1,29.52) = 0.12, p = 0.73). 4.4.2. Latencies to touch the reward The latencies to touch the grape were not signicantly different between the 3 types of objects at baseline (F(2,10) = 1.15, p = 0.35). T levels were not a signicant predictor of the latencies to touch the reward at baseline (F(1,14) = 0.26, p = 0.62). During the treatment phase, the latencies to touch the grape differed according to Object Type (F(2,8) = 4.87, p = 0.041). The latencies to touch the grape tended to be longer for negative vs. other types of objects (F(1,4) = 4.53, p = 0.10), and were signicantly longer for novel compared to familiar objects (F(1,4) = 11.99, p = 0.026). The effect of Treatment (F(1,4) = 0.028, p = 0.87) and Sequence of Treatment (F(1,4) = .22, p = 0.66) were not signicant. None of the interaction was signicant (Fig. 5b). T levels were not a signicant predictor of the latencies to touch the reward during the treatment phase (F(1,30) = 2.13, p = 0.15). 4.4.3. Number of touches on the object At baseline, the effect of Object Type on the number of touches approached signicance (F(2,10) = 3.97, p = 0.054). Negative objects tended to be touched less than other types of objects (F(1,5) = 7.50, p = 0.067), with no difference found between familiar and novel object (F(1,5) = 1.22, p = 0.32). T levels were not a signicant predictor of performance at the number of touches at baseline (F(1,4) = 0.08, p = 0.79). During the treatment phase, the effect of Object Type was not signicant (F(2,8)=2.33, p=.016). However, the number of touches on objects was signicantly lower when monkeys were treated with T compared to oil (F(1,4)=11.2, p=0.029). In addition, an interaction of marginal signicance between Object Type and Treatment (F(1,4)=3.27, p=0.09) suggested that the effect of treatment depended on the type of object. Planned contrasts for the interaction terms indicated that the effect of

a
Proportion of anxious behavior

1.0

0.8

Oil TE

0.6

0.4

0.2

0.0 Familiar Novel Negative

Object Type

b
Latencies (s) to touch the grape

120

100

Oil TE

80

60

40

20

0 Familiar Novel Negative

Object Type

c
Number of touches on object

12

Oil TE
10

0 Familiar Novel Negative

Object Type
Fig. 5. a. Proportion of anxious behavior + SEM as a function of object type and treatment; b. Latencies to touch the grape + SEM as a function of object type and treatment; c. Number of touches on object + SEM as a function of object type and treatment.

treatment was signicant when comparing negative objects relative to other types of objects (F(1,4)=38.77, p=0.01), while no effect of treatment was seen when comparing familiar and novel objects (F(1,4)=

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0.44, p=0.54; Fig. 5c). T levels were not a signicant predictor of the number of touches on the objects (F(1,29.77)=0.49, p=0.55).

5.3. Mnemonic vs. non-mnemonic effects Importantly, although DNMS performance was sensitive to delays, the benecial effect of T administration on DNMS accuracy was independent of delay duration. This suggests specic effects of T on nonmnemonic processes, such as attention, motivation or alertness. It is unlikely that T modulated attention because T did not interact with the social relevance or emotional valence of the stimuli, consistent with the results of a recent study in which monkeys were tested on an attentional task (dot-probe task) with emotional stimuli [55]. Rather, T may have modulated motivation and/or alertness. In support of this interpretation, T administration was associated with faster response times in our previous study [55]. The rodent and human literature also suggests that T inuences motivational behavior. Men with androgen deciency often suffer from lack of motivation and low energy [72,73] and androgens have been shown to modulate the sensitivity of brain reward systems in both rodents [74] and humans [75]. In addition, a recent study in women found that T administration facilitates the motivation to act [76]. Additional evidence for an effect of T on motivational drive or alertness may be provided by examining differences in tasks characteristics between the DNMS, which was sensitive to T manipulations, and the object-DRST, which was insensitive. The objectDRST is a task that typically generates high rates of responding from the monkeys, likely due to the rapid successive appearance of new stimuli as the monkey advances in the task. In contrast, the DNMS requires the monkeys to wait for an unpredictable delay between the sample presentation and the appearance of comparison stimuli, which increases chances for distraction, loss of motivation and refusals to perform the trial. In this context, it is possible that T helps monkeys to remain engaged in the task and motivated to perform as accurately as possible, despite the occurrence of delays. If our interpretation is correct, performance on tasks with long delays or a high degree of difculty should benet from T administration. This interpretation of the data would be consistent with effects of T at the arousal level, dened as by a greater alertness to sensory stimuli, greater motor activity, and greater emotional reactivity [77]. 5.4. Emotional reactivity The Approach/Avoidance task was conducted to specically examine T effects on emotional reactivity. In contrast to our previous study, in which monkeys were tested in an experimental cage outside of their living quarters, monkeys were tested in their home cage for a rapid assessment of emotional reactivity in the absence of overwhelmingly anxious responses. We found that the type of object exposure signicantly affected the experimental variables and observed a higher proportion of anxious behaviors and longer latencies to touch the grape for negative compared to other types of objects. Also in line with our previous ndings [51], no evidence was found for an effect of T on anxious behaviors or latencies to touch the reward. However, T signicantly reduced the number of touches on the objects, and the difference was signicant when contrasting the negative to the other types of objects. This nding could be interpreted either as an increase in vigilant behavior, which would be reected by enhanced visual scanning and watchfulness of objects, or an increase in fearfulness, which would be manifested by avoidance, freezing, fear grimacing and/or fear barks. The lack of treatment effect on latencies to touch the reward and anxious behaviors, as well as the absence of fear grimaces, freezing or fear barks throughout the study, suggest that T increased vigilance rather than fearfulness. Unfortunately, because monkeys were tested in their home cage, we were unable to accurately measure their gaze towards the objects. Therefore, our hypothesis that T increased vigilance towards objects perceived as potentially threatening will require validation at a ner level of analysis. The nding that T affected the responsiveness to objects differs from that obtained in two earlier studies in male rhesus monkeys.

5. Discussion Six male rhesus monkeys were tested for visual recognition memory, object memory and emotional reactivity at baseline, and following a short period of chemical castration using the GnRh blocker degarelix with add-back of TE (1 week) or oil vehicle (1 week). Monkeys were tested on the tasks 3 to 5 days after treatment. Accuracy scores on the DNMS were greater with T compared to oil treatment, independent of the delays, the social relevance (faces or objects) or the emotional valence (neutral, positive or negative) of the stimuli. In contrast, performance on the object-DRST was not affected by treatment. Emotional reactivity, as assessed by an approach/avoidance task of 3 types of objects in the home cage showed only subtle changes. While the proportion of anxious behaviors did not vary as a function of treatment, monkeys touched the objects relatively less when treated with T compared to oil, particularly in the case of negative objects. Altogether, the results suggest that acute changes in T levels do not affect memory per se but may rather modulate arousal levels. Comparisons of these ndings with those of our prior studies using more long-term T administration highlight some of the conditions under which T modulates cognitive and socioemotional function. 5.1. Low vs. high T levels Monkeys treated with physiological T levels in the present study beneted from T treatment with regard to visual recognition memory. In contrast, we previously found an impairment at the longest delay of the DNMS when monkeys were treated with supraphysiological T [50]. These results support the idea that the cognitive effects of T vary as a function of T concentrations. Specically, cognitive benets of T may be observed at intermediate T levels between low and high thresholds that remain to be specied. Direct evidence supporting this hypothesis was provided by a study in older men comparing the effects of low, moderate and supraphysiological T increases on cognition [67]: benecial effects of T on spatial and verbal memory were only observed for the group with moderate T increases, while the low and high groups exhibited no cognitive change. Other human studies have found detrimental effects of supraphysiological T on spatial ability [29] and verbal memory [45]. Thus, supraphysiological levels of T may be detrimental to certain cognitive abilities, whereas physiological levels may provide benets. It is likely that these effects characterize specic cognitive domains. Since the studies mentioned above used tasks mostly dependent on the medial temporal lobe, we hypothesize that moderate T levels improve performance on tasks involving the medial temporal lobe. 5.2. Duration of T exposure The present study focused on acute effects (35 days) of T treatment, as opposed to our prior studies in which 4 weeks of treatment were used. The inuence of chronic vs. acute T treatment on cognition is not completely understood. It has been recently suggested that prolonged T exposure is necessary to observe benecial effects of T on selective aspects of spatial abilities in male rats [68]. Yet, studies in humans indicate that acute T administration (few hours post administration) is sufcient to enhance cognitive abilities such as mental rotation [69], object location memory [52], decision-making [70] and selective attention to emotional stimuli [71]. It is important to note that these studies have used women participants. Our data further suggest that acute T administration enhances visual recognition memory in males.

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A. Lacreuse et al. / Physiology & Behavior 106 (2012) 229237 [13] Abdelgadir SE, Roselli CE, Choate JV, Resko JA. Androgen receptor messenger ribonucleic acid in brains and pituitaries of male rhesus monkeys: studies on distribution, hormonal control, and relationship to luteinizing hormone secretion. Biol Reprod 1999;60: 12516. [14] Leranth C, Petnehazy O, MacLusky NJ. Gonadal hormones affect spine synaptic density in the CA1 hippocampal subeld of male rats. J Neurosci 2003;23:158892. [15] Leranth C, Prange-Kiel J, Frick KM, Horvath TL. Low CA1 Spine synapse density is further reduced by castration in male non-human primates. Cereb Cortex 2004;14:50310. [16] Hajszan T, Maclusky NJ, Johansen JA, Jordan CL, Leranth C. Effects of androgens and estradiol on spine synapse formation in the prefrontal cortex of normal and testicular feminization mutant male rats. Endocrinology 2007;148:19637. [17] MacLusky NJ, Hajszan T, Prange-Kiel J, Leranth C. 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We previously found that T affected the monkeys' reactivity to social stimuli (videoclips of conspecics) but not to objects [51]; similarly, Richards et al. [78] reported that castrated and intact adolescent male rhesus monkeys differed in their responsiveness to facial stimuli but not in their approach/avoidance behavior towards objects. It is possible that the lack of T effects on reactivity to objects in these two studies was directly related to high anxiety levels generated by the testing context. We hypothesize that testing the subjects in their home cages reduced anxiety and unmasked subtle differences in reactivity to objects according to T treatment. In conclusion, we found that acute T administration (35 days post treatment) modulated visual recognition memory and subtle aspects of emotional reactivity in T-suppressed male rhesus monkeys. In contrast, performance on an object memory task was unchanged. Importantly, the benecial effects of T on visual recognition memory were independent of the delay duration of the stimuli, suggesting that T may modulate non-mnemonic processes, such as motivation or alertness. T also reduced the number of object contacts in the approach/avoidance task, especially for negative compared to other types of objects, consistent with the idea that T increases vigilance towards negative stimuli. Our interpretation of these results is that acute T manipulations may modulate generalized arousal in male rhesus monkeys, with T treatment leading to greater alertness in both the cognitive and the approach/avoidance tasks. This hypothesis will require further validation with objective measures of physiological arousal suitable for macaques, such as heart rate [79], skin conductance [80] or pupil dilatation [81]. Acknowledgments We thank Dr. Wolfgang Koechling for his input regarding degarelix treatment in male macaques. We also thank Christian Rose, Stephen Ferrigno, Kari Phan, Jenna Cavanaugh and Lorena Alves for their help with data collection and entry. We are grateful to Dr. Jerrold Meyer and the UMass animal care staff, veterinary staff and shop staff for their assistance. References
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