Androgen receptor is a negative regulator of contextual fear memory in male mice

Hormones and Behavior 106 (2018) 10–18

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Androgen receptor is a negative regulator of contextual fear memory in male mice Firyal Ramzana, Amber B. Azamb, D. Ashley Monksa,b, Iva B. Zovkica, a b

T

⁎

Department of Psychology, University of Toronto Mississauga, Mississauga, ON, Canada Department of Cell and Systems Biology, University of Toronto Mississauga, Mississauga, ON, Canada

A R T I C LE I N FO

A B S T R A C T

Keywords: Fear conditioning Androgen Testosterone Flutamide Gonadectomy Gene expression Hippocampus

Although sex-hormones have a well-documented role in memory formation, most literature has focused on estrogens, whereas the role of androgens and their receptor (the androgen receptor; AR) in fear memory is relatively unexplored. To address this gap, we used a transgenic mouse model of AR overexpression (CMV-AR) to determine if AR regulates fear memory, and if this effect can be reversed either by the removal of circulating androgens via gonadectomy, or by antagonising AR activity with flutamide. We found that AR overexpression results in reduced freezing in response to foot shock, and that this difference is reversed with both gonadectomy and flutamide treatment. Differences between genotypes were reinstated by testosterone replacement in gonadectomized mice, suggesting that reduced fear memory in mutants results from AR activation by testosterone and is not secondary to group differences in circulating testosterone. Potential transcriptional mechanisms by which CMV-AR exerts its effects on fear memory were assessed by quantitating the expression of memory-related genes in area CA1 of the hippocampus. Several genes that are altered with AR inhibition and activation, including genes that encode for the histone variant H2A.Z, cholinergic receptors, glutamate receptors, and brainderived neurotrophic factor. Overall, our findings suggest that AR is a negative regulator of fear memory and identify potential gene targets through which AR may mediate this effect.

1. Introduction Hormones powerfully regulate emotional and cognitive systems that are involved in establishing both normal and pathological forms of associative fear memory, which manifest in conditions such as posttraumatic stress disorder (PTSD) at different rates in men and women (Aikey et al., 2002; Dalla and Shors, 2009; Edinger and Frye, 2004, 2005; Jovanovic et al., 2015; Kranz et al., 2015; McHenry et al., 2014; Morgan and Pfaff, 2001; Suzuki et al., 2013). For example, PTSD is characterized by excessive fear and intrusive memories that disproportionately affect women compared to men who are exposed to trauma (Breslau et al., 1998; Holbrook et al., 2002), suggesting that testosterone is protective against PTSD, as it is against other types of affective disorders (Kaminetsky, 2005; Veras and Nardi, 2010). Despite evidence that androgens impact cognition and emotional memory, the nature of their influence is unclear (Burkitt et al., 2007; Galea et al., 2008; Gouchie and Kimura, 1991; Janowsky, 2006; Moffat and Hampson, 1996). Indeed, the majority of research on the role of sex hormones has focused primarily on estrogenic mechanisms (Frick et al.,

2017; Galea et al., 2008, 2017; Sherwin, 1988, 2005), whereas the role of the androgen receptor (AR) in hippocampus-dependent fear memory remains unclear. AR is expressed in brain regions that regulate contextual fear memory, most notably in areas CA1 and CA3 of the hippocampus (Kerr et al., 1995; Raskin et al., 2009). Studies have reported mixed effects of AR and its ligands, testosterone and dihydrotestosterone (DHT), on fear memory. For example, some studies report no effects of gonadectomy on fear memory in male rodents (Anagnostaras et al., 1998), whereas others report distinct effects of androgenic manipulations on contextual and cued fear memory (Chen et al., 2014; Edinger et al., 2004; Frye et al., 2008; MacLusky et al., 2005; Rubin, 2011; Zhang et al., 2014). Moreover, some evidence shows that gonadectomy reduces fear memory (Edinger et al., 2004; McDermott et al., 2012) and that this deficit is rescued by treatment with either testosterone or 17β estradiol, but not with DHT (Edinger et al., 2004). Given that DHT is a direct AR agonist, whereas testosterone can be further aromatized into 17β estradiol to activate estrogen receptors (Hojo et al., 2004; Mukai et al., 2010; Ooishi et al., 2012), these data suggest that testosterone may

⁎ Corresponding author at: Department of Psychology, University of Toronto Mississauga, CCT Building, Rm 4071, 3359 Mississauga Road, Mississauga, Ontario L5L 1C6, Canada. E-mail address: [email protected] (I.B. Zovkic).

https://doi.org/10.1016/j.yhbeh.2018.08.012 Received 13 May 2018; Received in revised form 21 August 2018; Accepted 29 August 2018 0018-506X/ © 2018 Elsevier Inc. All rights reserved.

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of-function mutations are invaluable in determining the necessity of AR, AR overexpressing mice take advantage of increased androgen response as a result of increased receptor expression to elucidate phenotypes that emerge at the high range of androgenic signaling. This approach has revealed functions of high AR levels in disease and sexual differentiation that do not always correlate with predictions made from loss-of-function mutant studies (Coome et al., 2017; Ramzan et al., 2015; Swift-Gallant et al., 2016b), demonstrating a unique utility of this mouse line for studying AR function in fear memory. In the present study, our goal was to investigate the role of AR in fear memory by addressing four questions: 1) Does increased AR density affect fear memory; 2) Is this effect mediated by testosterone; 3) Can effects of AR density on fear memory be reversed by blocking AR receptors, either indirectly (through gonadectomy) or directly (using the AR antagonist, flutamide); and 4) Given that AR is a ligand-activated transcription factor, does increased AR density alter the expression of memory-related genes in area CA1 of the hippocampus?

influence fear memory through AR-independent actions. In contrast, both DHT and testosterone improved performance on the inhibitory avoidance test, in which mice learn to avoid an environment associated with shock (Edinger et al., 2004), supporting a direct role of AR in regulating fear-based tasks. Androgenic regulation of morphological and functional plasticity in the hippocampus is also complex, with dissociable effects of androgen manipulations on excitability, spine density and neurotrophin production, and evidence for both androgenic and estrogenic mechanisms of gonadal testosterone (see Atwi et al., 2016 for review). For example, neural-specific AR deletion in mice reduces LTP induced by high-frequency stimulation, without impacting LTP induced by theta-burst stimulation (Picot et al., 2016), indicating that AR has selective effects on different types of hippocampal plasticity. Spine density is reduced in the medial prefrontal cortex (mPFC) of Tfm rats (Hajszan et al., 2007), and either testosterone or DHT maintain CA1 hippocampal spine synapse density in castrated male rats (Leranth et al., 2003). Androgens also increase hippocampal neurogenesis through modulation of survival of new neurons (see Galea et al., 2013 for review). Long-term testosterone/DHT replacement consistently ameliorates the impaired survival of new neurons in castrated males (Hamson et al., 2013; Spritzer et al., 2011b; Wainwright et al., 2011), while short-term androgen replacement shows mixed results (Carrier and Kabbaj, 2012; Spritzer et al., 2011a, 2011b; Wainwright et al., 2016). Further, survival is also impaired upon AR blocking with flutamide (Hamson et al., 2013). Given the complexity of androgenic compound actions, it can be difficult to distinguish between androgenic compounds that act through the AR compared to alternative modes of action. That is, testosterone can activate AR directly, or through its metabolite, DHT. In addition, DHT can be further metabolized to 3α-androstanediol to act on the GABA-A receptors, and testosterone can also be aromatized to estradiol to activate the estrogen receptor, which typically potentiates fear memory (Maeng and Milad, 2015). Thus, to specifically address the role of AR in fear memory, we utilized an AR-overexpressing mouse line (Swift-Gallant et al., 2016b), which we previously used to reveal novel insights into AR function in several behavioural processes, including the resident-intruder paradigm, sexual and aggressive behaviors, and partner preference (Swift-Gallant et al., 2016a, 2016b). Whereas lossWT

(a)

CMV-AR

Arbitrary Units

AR 100 kD

Actin

50 kD

37 kD

% Freezing

Male C57Bl/6 mice bred in our colony were pair housed after weaning and were assigned to testing groups at 60–90 days of age. Mice were housed in 7.5 × 11.5 × 5 in cages and maintained on a 12 h light cycle, with ad libitum access to standard mouse chow (Harlan Teklad, Madison, WI) and water. Transgenic mice containing a CMV-stop-AR transgene were crossed with a CMV-Cre line to obtain tissue-wide AR overexpression, as previously described (Swift-Gallant et al., 2016b). Briefly, CMV-stop-AR mice contain a cytomegalovirus (CMV) promoter coupled to the human androgen receptor (hAR) gene, separated by a floxed stop sequence (Fig. 1a), which is excised when mice are crossed with the CMV-Cre line to allow for hAR transcription, beginning ~ embryonic day 8.5, when CMV expression occurs (Baskar et al., 1996). Details on mouse generation can be found in (Swift-Gallant et al., 2016b). All of the procedures were approved by the University of Toronto Mississauga animal care committee and complied with institutional guidelines and the Canadian Council on Animal Care. Fig. 1. AR activation impairs fear memory in CMV-AR mice. (a) Representative immunoblot demonstrating that CMV-AR have higher AR protein levels in the hippocampus compared to WT controls. Quantification of the western blot (N = 2/ group) is shown on the right. (b) Freezing behaviour during the training session (left) and during the memory test (right), conducted 24 h after training. N = 13/group for WT and CMV-AR in the control condition and 5/group for WT and CMV-AR in the flutamide condition. *p ≤ 0.05.

*

0 .8 0 .6 0 .4 0 .2 0 .0

WT

80

80

60

60

*

40

CMV-AR

Memory test

Training day

*

WT CMV-AR

*

40 20

20 0

2.1. Animals

1 .0

150 kD

(b)

2. Methods

Control

Flutamide Control

Before shock

Flutamide

0

Control

Flutamide

After shock

11

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Table 1 DNA primers for genotyping. Gene

Forward

Reverse

neostop AR Cre

5′-AGGATCTCCTGTCATCTCACCTTGCTCCTG 5′-ACCGAGGAGCTTTCCAGAAT 5′-AGGTGTAGAGAAGGCACTTA

5′-AAGAACTCGTCAAGAAGGCGATAGAAGGCG 5′-CTCATCCAGGACCAGGTAGC 5′-CTAATCGCCATCTTCCAGCA

(Coome et al., 2017). Using the same implant specifications in C57Bl6 mice, Bowen et al. (2012) found that this replacement protocol results in 15.4 ± 1.2 ng/mL plasma T levels. Control animals received an empty Silastic implant. The animals were administered Anafen (5 mg/ kg) on the day of the surgery and for 2 days after surgery. Wound clips were removed if still present after 7 days. Animals underwent 3 days of handling (30 s/mouse) beginning 11 days after surgery and were trained 14 days after surgery.

2.2. Genotyping Genotyping was carried out at weaning for each mouse used in the experiments, as previously described (Swift-Gallant et al., 2016b). Briefly, the presence of the CMV-STOP-AR transgene was identified by amplifying within the Neo stop sequence. However, because this Neo stop sequence is deleted in mice expressing both CMV-Cre and CMVSTOP-AR, we also amplified a unique portion of the CMV-STOP-AR transgene within the hAR coding region (see Table 1 for a list of genotyping primers). To ensure that AR overexpression extends to area CA1 of the hippocampus, we used qPCR to confirm the presence of hAR mRNA in CMV-AR compared to WT mice in this region for each mouse included in the experiments.

2.6. Tissue collection For RNA tissue analysis, mice were cervically dislocated and decapitated. Animal brains were immediately collected and snap frozen in ice-cold isopentane and stored at −80 °C until processing.

2.3. Fear conditioning 2.7. Western blotting Associative fear memory was assessed with contextual fear conditioning, a hippocampus-dependent task in which mice learn to associate a novel context with exposure to shock. To facilitate habituation to the experimenter and the non-associative aspects of the training procedure, mice were transported to the testing room and handled for 30–60 s daily for 3 days before testing. On the training and test days, mice were transported to the testing room and placed into test chambers (9.8 in boxes; designed for mice) equipped with an electrified grid floor (Coulbourn Instruments, Holliston, MA, USA). During training, mice were given 2 min to explore the apparatus before receiving a foot shock (0.5 mA, 2 s), followed by an additional minute of exploration. Memory was assessed 24 h later by re-placing the mouse into the training apparatus without shock and measuring freezing behavior for a total of 3 min. Freezing was recorded with a camera placed directly in the chamber ceiling and facing down towards the mouse and scored by automated software (FreezeFrame, Coulbourn Instruments).

Tissues were homogenized using a dounce homogenizer in RIPA buffer (50 mM Tris HCl pH 7.4, 150 mM NaCl, 10% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) supplemented with Protease Inhibitor Cocktail (Cell Signaling, Danvers, MA, USA). Homogenates were incubated for 20 min on ice and flicked every 5 min, centrifuged at maximum speed at 4 °C for 15 min, and the supernatant was collected. Proteins were separated on 10% SDS-PAGE and transferred to a PVDF membrane. Membranes were blocked in TBS-T (TBS with 0.1% Tween20) containing 5% skim milk and incubated with rabbit monoclonal anti-AR (1:500, Abcam, ab133273 RRID, Cambridge, UK) and rabbit anti-β-Actin (1:10.000, Cell Signaling, 4967S, Danvers, MA, USA) overnight at 4 °C. After three 5 min TBS-T washes, membranes were incubated with anti-rabbit-HRP secondary antibody (1:10.000 Life Technologies, ThermoFisher, Waltham, MA, USA) for 1 h at RT. Detection was performed by enhanced chemiluminescence and quantification was done using ImageJ (NIH) to quantify Area Under the Curve, with AR normalized to actin.

2.4. Flutamide administration 5 days before handling (at 60–90 days of age), a subset of intact male mice were injected subcutaneously with the AR antagonist flutamide (8 mg/day) and injections continued daily for a total of 13 days to ensure continued AR inhibition until tissue collection. Flutamide (Sigma # F9397, Oakville, Canada) was prepared daily immediately prior to use, by first dissolving it in 100% ethanol, then in sesame oil (Sigma # S3547, Oakville, Canada). The ethanol was removed using an Eppendorf Vacufuge Plus (spun for 20 min at 30 °C on the V-AL setting), producing the final concentration of 53 mg/mL. Mice were administered 0.15 mL through subcutaneous injection in the nape of the neck. Vehicle-treated control mice received sesame oil on the same schedule as the flutamide-treated mice.

2.8. mRNA expression and RT-PCR Mice were killed by cervical dislocation at least 2 weeks after the final training session and area CA1 was dissected. RNA was extracted using BioBasic RNA Extraction Kit (BioBasic #BS82322-250, Amherst, NY). Complementary DNA was synthesized using High Capacity cDNA Reverse Transcription Kit (Applied BioSystems #4368814, Folster, CA, USA). Primers were designed in the lab and used to detect levels of the indicated transcripts, and data were normalized to GAPDH. Comparison of group differences was conducted using relative enrichment analysis, whereby data for experimental animals were normalized to controls using the following formula: 2^−(ΔΔCT). The list of primer sequences is provided in Table 2.

2.5. Gonadectomy and testosterone (T) replacement 2.9. Statistics At 60–90 days of age, a subset of mice underwent castration under inhalant anaesthesia (1–2% isoflurane). A midline incision was made in the scrotum to allow for the removal of both testes, after which the incision was closed with wound clips. Immediately after castration, animals were subcutaneously implanted at the nape of the neck with a Silastic capsule containing T (10 mm of crystalline T; Silastic tube 1.02 mm id/2.16 mm od) and sealed with Silastic adhesive, as in

Analyses were conducted with SPSS Version 24 and consisted of independent-samples t-test for comparison of Genotype (WT, CMV-AR). In cases in which there were 2 independent variables, we used a two-way ANOVA with Genotype (WT, CMV-AR) and Treatment (Control, Testosterone; or Vehicle, Flutamide) as independent variables. To compare freezing before and after shock during the training session, we used a 12

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Table 2 cDNA primers used in gene expression studies. Gene

Forward

Reverse

Melting temperature (°C)

Actb Bdnf4 Chrna7 Chrm3 hAR Gapdh Gria1 Gria4 Grin1 H2afz

5′-AGATCAAGATCATTGCTCCTCCT 5′-CCAGAGCAGCTGCCTTGCTGTTTA 5′-GCAACATCTGATTCCGTGCC 5′-GACAGTCGCTGTCTCCGAAC 5′-CTTCGCCCCTGATCTGGTTT 5′-GTGGAGTCATACTGGAACATGTAG 5′-ATGTGGAAGCAAGGACTCCG 5′-GGACAAGACGAGTGCCTTGA 5-AAACCTCGACCAACTGTCCT 5-CACCGCAGAGGTACTTGAGTT

5′-ACGCAGCTCAGTAACAGTCC 5′-TGCCTTCTCCGTGGACGTTTACTT 5′-TGATCCTGGTCCACTTAGGC 5-GGTCATATCTGGCAGCCGTG 5′-GAGAGAGGTGCCTCATTCGG 5-AATGGTGAAGGTCGGTGTG 5′-ACAGAAACCCTTCATCCGCT 5-GCTTCGGAAAAAGTCAGCTTCA 5′-GTCGTCCTCGCTTGCAGAAA 5-TCCTTTCTTCCCGATCAGCG

58 60 58 58 56 60 58 56 55 58

ƞp2 = 0.40), whereby freezing increased in the 1 min period after shock exposure compared to the 2-min period before shock exposure in control (t25 = 7.48, p < 0.0001, d = 1.47) and flutamide-treated (t9 = 10.46, p < 0.0001, d = 3.31) mice. In addition, flutamidetreated mice froze more than controls after shock exposure (t34 = 3.94, p < 0.0001, d = 1.46), irrespective of genotype (Fig. 1b). Fear memory was assessed by comparing freezing behaviour in control and flutamide-treated CMV-AR and WT mice 24 h after training. There was a significant interaction (F1,32 = 4.54, p = 0.04, ƞp2 = 0.12) between Genotype (WT, CMV-AR) and Treatment (Control, Flutamide), whereby Control CMV-AR mice exhibited reduced freezing compared to WT controls (t24 = 3.91, p = 0.001, d = 1.53). In contrast, no difference in freezing behaviour between genotypes was found for flutamidetreated mice, such that flutamide increased freezing behaviour compared to control treatment in CMV-AR (t16 = 3.71, p = 0.002, d = 1.95), but not in WT mice (Fig. 1b). These data demonstrate that AR overexpression reduces fear memory and that this effect is reversed by blocking the AR with flutamide.

mixed-measures ANOVA, with Genotype and Treatment as the between group factors and Shock (before shock; after shock) as the within-group factor. Follow-up analyses were conducted using independent-samples ttest or paired-samples t-test only when the omnibus test was significant, thus precluding the need to correct for multiple comparisons. Effect sizes were calculated using the publicly available effect size calculator, found at http://www.campbellcollaboration.org/escalc/html/EffectSizeCalculatorSMD1.php. Significance was set at p ≤ 0.05. 3. Results 3.1. Validation of CMV-AR in the hippocampus Generation and validation of AR overexpression in CMV-AR mice has been previously reported (Swift-Gallant et al., 2016b). Here, we confirmed that AR overexpression extends to the hippocampus using qPCR for each mouse (see Methods section), as well as immunoblotting (t2 = 12.4, p = 0.006, d = 12.74) to confirm differences in protein levels (Fig. 1a).

3.3. Gonadectomy removes genotype effects and testosterone restores them 3.2. CMV-AR reduces fear memory To determine if the effects of CMV-AR on fear memory are due to differences in sensitivity to circulating androgens, mice were gonadectomised and implanted with a Silastic tube containing either T or an empty control. Although T can be aromatized into estradiol, we chose to replace T instead of a non-aromatizable androgen as an initial test of whether an androgen would affect the system, and also because our interest was in testing how AR overexpression influences behavior in response to the dominant source of androgen. During training, all mice exhibited significantly increased freezing after shock than before shock (Main effect of Shock: F1,48 = 70.28, p < 0.001; ƞp2 = 0.59), but there were no interactions or main effects of Treatment and Genotype, indicating that these factors did not influence within-session learning (Fig. 2). Gonadectomy (GDX) reversed the effects of AR-overexpression on fear memory 24 h after training, as evidenced by a significant

To determine if AR influences fear memory, we compared freezing behavior in WT and CMV-AR mice. We reasoned that any differences between the genotypes that are directly attributable to AR overexpression would be eliminated by blocking the activity of AR, prompting us to compare freezing behaviour in control WT and CMVAR mice to the behaviour of mice treated with the AR antagonist, flutamide. We found no differences between mice that did not receive any injection and mice that received a vehicle injection during training (F1,22 = 1.88, p = 0.18) or on the test day (F1,22 = 1.69, p = 0.21), so these were combined into a single control group. We first assessed within-session learning by comparing freezing behaviour before and after exposure to shock during the training day. We found a significant Shock (before shock, after shock) × Treatment (Control, Flutamide) interaction (F1,32 = 20.86, p < 0.001,

Training day

% Freezing

WT

80

80

*

60

60

*

40

CMV-AR

*

40 20

20 0

Memory test

Control

Testosterone

Before shock

Control

Testosterone

0 Control

Testosterone

After shock

13

Fig. 2. Gonadectomy removes differences between genotypes on fear memory and T replacement restores them. Freezing behaviour during the training session (left) and during the memory test (right), conducted 24 h after training. N = 9–19/group (Vehicle treated: N = 19 WT and 10 CMVAR; Testosterone treated: N = 14 WT and 9 CMV-AR). *p ≤ 0.05.

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Genotype × Treatment (Control, Testosterone) interaction (F1,48 = 5.41, p = 0.02, ƞp2 = 0.10), whereby differences between WT and CMV-AR mice were not evident in GDX mice without hormone replacement, suggesting that the removal of circulating AR ligands (i.e., testosterone) eliminates the effect of CMV-AR on fear memory. Consistent with this observation, T replacement reduced fear memory only in CMV-AR mice (t17 = 5.05, p < 0.0001, d = 2.31), thus reinstating the difference between genotypes, whereby T-replaced CMVAR mice froze less than T-replaced WT mice (t21 = 2.07, p = 0.05, d = 0.94). Overall, these data suggest that testosterone-mediated AR activation reduces fear memory (Fig. 2).

H2afz

2.0

*

WT CMV-AR

*

1.5

*

1.0 0.5 0.0

2.0

* *

Chrm3

1.5

3.4. Effects of AR on gene expression in area CA1 of the hippocampus Given that AR is a ligand-activated transcription factor, we explored potential transcriptional outcomes from all the groups that were utilized in behavioural studies, including intact (i.e., control or flutamidetreated mice that were not gonadectomized) and gonadectomized (with or without T replacement) mice. We focused on genes that were previously implicated in memory formation, including H2afz, a gene encoding the histone variant H2A.Z, which we recently identified as a memory suppressor (Stefanelli et al., 2018; Zovkic et al., 2014), and which is positively regulated by AR in prostate cancer (Dryhurst and Ausió, 2014). For intact mice, H2afz levels depended on both Genotype and Treatment (Genotype × Treatment interaction: F3,24 = 4.87, p = 0.04, ƞ2p = 0.17), whereby H2afz expression was elevated in control-treated CMV-AR compared to WT mice (t16 = 2.44, p = 0.03, d = 1.16). In contrast, the effect of genotype was not evident in mice treated with flutamide, as H2afz expression was reduced to WT levels in CMV-AR mice treated with flutamide compared to CMV-AR controls (t13 = 3.03, p = 0.01, Cohen's d = 1.66). Similarly, the effect of genotype in gonadectomized mice depended on hormonal status (Genotype × Treatment interaction: F1,36 = 8.10, p = 0.007, ƞ2p = 0.18), whereby differences between the genotypes were not evident in gonadectomized mice without hormone replacement, and were restored with T replacement. Specifically, T-replaced CMV-AR mice had higher H2afz expression compared to T-treated WT mice (t17 = 2.83, p = 0.01, d = 1.30), indicating that high levels of AR promote H2afz expression in the hippocampus (Fig. 3). Some evidence suggests that testosterone mediates cholinergic receptors (Bleisch et al., 1982), leading us to investigate effects on a muscarinic (Chrm3) and nicotinic (Chrna7) acetylcholine receptor expression. In intact mice, the interaction of Genotype and Treatment approached significance (F1,23 = 3.77, p = 0.06, ƞ2p = 0.14) for Chrm3, leading us to conduct follow-up analyses to test our hypothesis that differences in expression between genotypes would be eliminated by AR blockade. In control mice, Chrm3 levels were higher in CMV-AR compared to WT mice (t15 = 2.76, p = 0.02, d = 1.34), whereas this difference was not found in flutamide-treated mice. Similarly, Chrm3 expression in gonadectomized mice depended on hormonal status (Genotype × Treatment interaction: F1,37 = 5.06, p = 0.03, ƞ2p = 0.12), whereby gonadectomized mice without T replacement had similar Chrm3 expression. T treatment only increased Chrm3 expression in CMV-AR mice (t17 = 3.25, p = 0.005, d = 1.49), such that T-treated CMV-AR mice had higher Chrm3 expression than T-treated WT (t17 = 2.46, p = 0.025, d = 1.13). For Chrna7, no differences were found in intact mice, but there was a significant increase in Chrna7 expression in response to T treatment in gonadectomized mice (Main effect of Treatment: F1,37 = 7.86, p = 0.008). There is also evidence for a potential influence of T on glutamatergic transmission in the hippocampus (Picot et al., 2016), leading us to investigate a potential role of CMV-AR on the expression of Gria1 (encodes glutamate receptor AMPA1 subunit), Gria4 (encodes glutamate receptor AMPA4 subunit), and Grin1 (encodes the NMDA1 receptor subunit). There were no differences in Gria1 or Gria4 expression in intact mice, but for both genes, T replacement in gonadectomized

1.0 0.5 0.0 2 .0

*

Chrna7

1 .5 1 .0 0 .5 0 .0

*

2 .0

Gria1

1 .5 1 .0 0 .5 0 .0 2 .0

*

Gria4

1 .5 1 .0 0 .5 0 .0

*

2 .0

*

*

Grin1

1 .5 1 .0 0 .5 0 .0 2 .0

* 1 .5

Bdnf4

*

1 .0 0 .5 0 .0

Control

Flutamide

Control Testosterone (caption on next page)

14

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Fig. 3. Effects of genotypes, flutamide, and gonadectomy on gene expression in area CA1 of the hippocampus. Gene expression is shown as relative enrichment for intact (left) and gonadectomised (right) mice for H2afz, Chrm3, Chrna7, Gria1, Gria4, Grin1, and Bdnf4. The Y axis indicates the gene name for which relative enrichment was calculated. *p ≤ 0.05.

et al., 2016b), as evidenced by reduced bouts of chasing and tumbling behaviors in response to a male intruder. In light of the current finding of reduced fear memory, these findings suggest a potential relationship between reduced fear, sensitivity to predator odor, and aggression. Our results indicate that increased androgen signaling via AR overexpression impairs memory potentially through effects on various memory-related genes, including synaptic genes and the histone variant H2A.Z. This is in stark contrast with data from studies of estradiol, which typically produces enhanced memory in females through actions on the estrogen receptor β (ERβ) (Frick et al., 2017; Kim and Frick, 2017). Although estradiol similarly regulates memory in males, little is known about the mechanisms by which this occurs and whether these effects are distinct from females (Frick et al., 2017). Interestingly, the histone variant H2A.Z is a vital modulator of estrogen and androgen receptor signaling (Gevry et al., 2009), suggesting that both steroid receptors may interact with similar epigenetic factors, though the nature of their interaction is likely to differ. Although fear memory provides an index of hippocampal function, it also serves as an index of PTSD (Zovkic and Sweatt, 2013), such that AR modulation of fear memory may be distinct from AR modulation of other forms of memory. Indeed, testosterone has been reported to have anxiolytic effects (Edinger and Frye, 2004, 2005; Fenchel et al., 2015; Frye et al., 2008) and as such, it may have an inhibitory effect on fear memory while promoting other forms of memory. Other studies have reported that testosterone improves non-fear based hippocampus-dependent memory, such as the Morris water maze and memory for temporal order in which objects are presented (Naghdi et al., 2001, 2005; Picot et al., 2016), suggesting that AR may be protective against fear memory.

mice increased gene expression, irrespective of genotype (Gria1: F1,37 = 10.56, p = 0.002, ƞ2p = 0.22; Gria4: F1,37 = 6.30, p = 0.02 ƞ2p = 0.15). For Grin1 expression in intact mice, there was a Genotype × Treatment interaction (F1,24 = 5.25, p = 0.03, ƞ2p = 0.18), whereby Grin1 expression was higher in control-treated CMV-AR compared to control-treated WT mice (t16 = 4.12, p = 0.001, d = 1.98). There were no differences between genotypes when AR was blocked with flutamide. Differences between genotypes were also not evident when mice were gonadectomized without hormone treatment, but were restored by T replacement (Genotype × Treatment interaction: F1,37 = 5.44, p = 0.03, ƞ2p = 0.13), such that T-replaced CMV-AR mice had higher Grin1 expression than T-replaced WT mice (t17 = 2.72, p = 0.015, d = 1.25) (Fig. 3). Finally, androgens have also been implicated in regulating BDNF expression (Skucas et al., 2013), leading us to examine the expression of BDNF exon 4 (Bdnf4). In intact mice, a main effect of Treatment (F1,24 = 8.13, p = 0.009, ƞ2p = 0.25) demonstrated reduced levels of Bdnf4 in response to AR blockade with flutamide. In gonadectomized mice, Bdnf4 expression depended on genotype and hormone treatment (Genotype × Treatment interaction: F1,37 = 7.60, p = 0.009, ƞ2p = 0.17), such that T only increased Bdnf4 expression in CMV-AR mice (t17 = 5.80, p < 0.0001, d = 2.67), although the difference between WT and CMV-AR mice treated with T missed significance (t17 = 2.03, p = 0.059, d = 0.93) (Fig. 3).

4.1. Effects of AR on gene expression

4. Discussion

Contextual fear memory is heavily reliant on the hippocampus. Given that AR is a transcriptional regulator, we examined the effects of CMV-AR on mRNA levels of several memory-related genes in area CA1. CMV-AR had a robust effect on H2afz, a gene encoding the histone variant H2A.Z. We previously showed that H2A.Z is a memory suppressor, whereby virally-mediated depletion of H2afz enhanced fear memory (Stefanelli et al., 2018; Zovkic et al., 2014). Here, we show that H2afz expression is elevated in CMV-AR mice and that this difference is not evident when AR activity is blocked by gonadectomy or by flutamide, and is restored by testosterone replacement, thus providing converging evidence for AR-mediated upregulation of H2A.Z levels. These findings are consistent with reports in prostate cancer, where high levels of AR activity (via the AR agonist R1881) enhance H2A.Z expression (Dryhurst et al., 2012). Given that H2A.Z depletion (Stefanelli et al., 2018; Zovkic et al., 2014) promotes fear memory, these data generate the hypothesis that at least some of the effects of AR overexpression on memory may be mediated by heightened H2A.Z expression. Cholinergic receptors are also important regulators of hippocampal fear memory (e.g. Sahdeo et al., 2014) and testosterone mediates muscarinic receptor expression in the rat epididymis, hypothalamus, and amygdala, as well as in vocalization-related regions of the quail brain (Al-Daham and Thomas, 1987; Ball and Balthazart, 1990; Maróstica et al., 2005). Here, we show that Chrm3, a gene encoding the M3 muscarinic receptor, is upregulated in CMV-AR mice and that this difference is eliminated by manipulations that block AR activity and is restored with testosterone replacement, indicating that high AR density promotes Chrm3 expression. In contrast, Chrna7, a gene that encodes the nicotinic receptor 7 alpha, was not affected by CMV-AR, but was elevated by testosterone independently of genotype. These findings are consistent with studies in muscle that show reduced density of nicotinic receptors in response to testosterone deprivation (Souccar et al., 1991) and suggest that muscarinic and nicotinic receptors may be differentially sensitive to regulation by AR, whereby the nicotinic receptor may

Different lines of evidence presented here converge on the conclusion that AR is a negative regulator of fear memory. Specifically, as differences between genotypes in gonadectomized mice were restored with equivalent replacement dose of testosterone, our data suggest that reduced freezing in CMV-AR mice is due to differential responses to circulating androgens rather than to different levels of circulating androgens in the two groups. This conclusion is reinforced by our prior reports of distinct behavioural outcomes in WT and CMV-AR mice despite similar levels of circulating testosterone (Swift-Gallant et al., 2016a, 2016b). Indeed, the lack of sensitivity to gonadectomy in WT mice is consistent with others who found that gonadectomy did not affect fear memory (Anagnostaras et al., 1998). Nonetheless, androgen receptor overexpression reduced fear memory only in the presence of ligand, suggesting that sufficiently high androgenic signaling can inhibit fear memory. A key implication of these data is that the ability of AR to modulate fear memory may at least in part depend on individual differences in AR density. In support of this hypothesis, a study of predator scent-induced fear memory showed that rats with an extreme response to this type of stress had lower levels of AR in area CA1 compared to rats that had a minimal response to predator stress (Fenchel et al., 2015), suggesting that individual differences in AR density may alter sensitivity to fear-inducing stimuli. Moreover, the same study showed that testosterone treatment reduced contextual fear memory, but only when administered 7 days after predator scent exposure. If testosterone was administered shortly after stress exposure (within 1 h), it increased fear memory, suggesting that the timing of testosterone treatment is important. In our study, mice received chronic testosterone replacement, indicating that AR can also inhibit memory under conditions of stable activation. Altered sensitivity to predator scent is particularly interesting in light of our previous data, which showed that CMV-AR males have reduced aggression compared to WT mice (Swift-Gallant 15

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and Raber, 2009). Additionally, androgens promote neurogenesis and spine growth in area CA1 of the hippocampus (Leranth et al., 2003; MacLusky et al., 2006; Swift-Gallant et al., 2018), suggesting that androgens have a complex role on memory-related systems, which can translate to unique behavioural outcomes for different forms of memory. Ultimately, our goal is to draw conclusions about the function of AR during the normal formation of fear memory, particularly in relation to individual and sex differences. The extent to which our data reflect such endogenous variability in AR-related outcomes is not clear, but our findings provide an important first step to demonstrate the capacity of AR to inhibit the formation of fear memory. In upcoming studies, we hope to strengthen our findings of AR-mediated regulation of fear memory by focusing on individual differences in fear memory in relation to variable AR expression in strains with varying magnitudes of fear memory formation (Brinks et al., 2007; Siegmund and Wotjak, 2007a, 2007b).

be affected by testosterone in an AR-independent manner. 17β estradiol is a potent regulator of nicotinic receptors in tumor tissues (Lee et al., 2011), suggesting that aromatized testosterone may be acting through estrogen receptors to promote Chrna7 expression. Interestingly, Gria1 and Gria4, genes that encode AMPA receptor subunits, were similar to the nicotinic receptor, in that we did not find any differences associated with genotype, but the expression of both increased with testosterone replacement. In contrast, Grin1 (glutamate ionotropic receptor NMDA subunit 1) expression was higher in CMV-AR mice, this difference was eliminated by blocking AR activity, and restored by testosterone replacement, directly implicating AR in promoting NMDA receptor expression. Previous studies suggest that AR deletion impacts hippocampal plasticity through effects on NMDA receptor function, although no differences were found in receptor density in that study (Picot et al., 2016). However, AR stimulation with DHT increased NMDA receptor number in area CA1 (Romeo et al., 2005), which is consistent with increased Grin1 expression in CMV-AR mice. However, the lack of effect in WT mice suggests that these transcriptional effects occur selectively in cases of high AR sensitivity, or by alternate mechanisms associated with potential organizational effects of germline AR overexpression. Finally, the neurotrophic factor BDNF exon 4 expression is reduced by flutamide treatment irrespective of genotype, but is increased by testosterone only in CMV-AR mice, suggesting that AR activation promotes Bdnf4 expression. This contrasts with evidence that gonadectomy decreases BDNF protein levels in the mossy fiber pathway from the dentate gyrus to the CA3 (Skucas et al., 2013), although studies in birds have documented a positive effect of testosterone on BDNF expression in several brain regions (rev in Brenowitz, 2013). Overall, the use of CMV-AR mice allowed us to identify 2 major categories of genes that exhibit differential sensitivity to AR regulation: 1) genes that are upregulated selectively by CMV-AR, suggesting that their expression is increased by the presence of high levels of AR; and 2) genes that are not affected by AR density, but are nevertheless sensitive to testosterone treatment, implicating alternate pathways by which testosterone may influence their expression, such as the activation of estrogen receptors. These AR-sensitive genes, particularly genes encoding H2A.Z and NMDA receptor subunits, provide strong candidates for investigating the mechanism by which AR influences fear memory. In particular, we are actively pursuing the link between AR and H2A.Z, given the strong evidence for the regulation of this memory suppressor by AR.

5. Conclusions The differences in fear memory observed in groups with differing levels of AR expression have implications for certain types of resilience, particularly as a protective factor against PTSD. Although fear conditioning is a widely-used index of hippocampus-dependent associative memory, it is also utilized as a model of PTSD because of the strong aversive memory that is produced by exposure to a single traumatic event (Zovkic and Sweatt, 2013). In Dutch military veterans, low testosterone levels pre-deployment predicted increased likelihood of developing PTSD 1–2 years post-deployment (Reijnen et al., 2015), consistent with the observation that PTSD is more common among women than men exposed to trauma (Maddox et al., 2018). In addition, testosterone has been associated with reduced anxiety in humans (McHenry et al., 2014) and in rodent models (Edinger and Frye, 2004; Frye et al., 2008; McDermott et al., 2012), as well as reduced fear responses to a predator scent (King et al., 2005). Moreover, individual differences in sensitivity to predator scent exposure are associated with differences in AR in CA1 (Fenchel et al., 2015), suggesting that high levels of AR expression may protect against the development of fearrelated disorders. Disclosure The authors report no conflicts of interest in this work.

4.2. Limitations Funding We focused our investigation of gene expression on the hippocampus, but we cannot exclude the possible contribution of other brain regions or non-neural tissues to mediating behavioural difference in fear memory, given that AR in this model is overexpressed across brain regions and tissue types. Indeed, interpretations of our data would be greatly improved by direct manipulations of AR in specific brain regions of interest, particularly the hippocampus, which is a target of future studies in our lab. Moreover, we cannot exclude potential developmental factors as mediators of differences between genotypes. Indeed, some studies have found opposite LTP effects in brain-specific AR knockout mice compared to pharmacological inhibition of AR (Harley et al., 2000; Picot et al., 2016), indicating that AR overexpression may have unique effects on the adult brain as a result of altered development. Furthermore, our studies focused specifically on fear conditioning in male mice, so it is not clear if our findings reflect a generalized learning deficit, or a specific reduction in memory for aversive stimuli. Indeed, castration impairs working memory in the Morris Water Maze, without affecting spatial reference memory, or memory in tests of novel object recognition and passive avoidance. In addition, DHT replacement selectively recovered spatial memory in castrated male mice without impacting other forms of memory (Benice

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