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Effects of aging on testosterone and androgen receptors in the mesocorticolimbic system of male rats
Hormones and Behavior 120 (2020) 104689
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Effects of aging on testosterone and androgen receptors in the mesocorticolimbic system of male rats
T
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Katelyn L. Lowa,b, , Ryan J. Tomma, Chunqi Maa, Daniel J. Tobianskya, Stan B. Florescoa, Kiran K. Somaa,b a b
Department of Psychology and Djavad Mowafaghian Centre for Brain Health, University of British Columbia, Vancouver, BC, Canada Department of Zoology, University of British Columbia, Vancouver, BC, Canada
A R T I C LE I N FO
A B S T R A C T
Keywords: Brain Executive function Medial prefrontal cortex Neurosteroid Nucleus accumbens Orbitofrontal cortex Ventral tegmental area
As males age, systemic testosterone (T) levels decline. T regulates executive function, a collection of cognitive processes that are mediated by the mesocorticolimbic system. Here, we examined young adult (5 months) and aged (22 months) male Fischer 344 × Brown Norway rats, and measured systemic T levels in serum and local T levels in microdissected nodes of the mesocorticolimbic system (ventral tegmental area (VTA), nucleus accumbens (NAc), medial prefrontal cortex (mPFC), and orbitofrontal cortex (OFC)). We also measured androgen receptor (AR) immunoreactivity (-ir) in the mesocorticolimbic system. As expected, systemic T levels decreased with age. Local T levels in mesocorticolimbic regions – except the VTA – also decreased with age. Mesocorticolimbic T levels were higher than serum T levels at both ages. AR-ir was present in the VTA, NAc, mPFC, and OFC and decreased with age in the mPFC. Taken together with previous results, the data suggest that changes in androgen signaling may contribute to changes in executive function during aging.
1. Introduction In male humans, non-human primates, and rodents, aging is associated with declines in several cognitive functions, which coincide with a decline in systemic levels of androgens (Ghanadian et al., 1975; Kaler and Neaves, 1981; Vermeulen, 1991). Androgens such as testosterone (T) play an important role in cognition (Janowsky, 2006; Tobiansky et al., 2018b) and their decline may be an important factor in cognitive changes during aging (Lund et al., 1999). Aging is characterized in part by declines in executive functions (Alexander et al., 2012), which are cognitive processes involved in coordinating complex behaviors and goal-directed actions. Executive functioning is modulated by prefrontal cortical structures, such as the medial prefrontal cortex (mPFC) and orbitofrontal cortex (OFC) (Janowsky, 2006; Gallagher et al., 2011; Beas et al., 2013). For example, both regions facilitate different forms of cognitive flexibility; the mPFC mediates extradimensional attentional set-shifts, while the OFC mediates reversal shifts between different stimulus-reward associations (Dalley et al., 2004; Gallagher et al., 2011). In rats, the mPFC also governs working memory, a form of shortterm memory involving the maintenance and manipulation of information for obtaining goals (Goldman-Rakic, 1995; Funahashi, 2006). Androgens regulate executive functions, including working memory
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and set-shifting (Janowksy et al., 2000; Bimonte-Nelson et al., 2003; Aubele and Kritzer, 2011; Tobiansky et al., 2018b). T administration and T deprivation affect performance on PFC-dependent tasks (Janowksy et al., 2000; Kritzer et al., 2001; Daniel et al., 2003). One major pathway through which androgens exert effects on executive function is the mesocorticolimbic system (Tobiansky et al., 2018b). The mesocorticolimbic system is critical for reward seeking, motivation, and higher-order cognitive functions (Fibiger and Phillips, 1988; Kritzer, 1997) and consists of dopaminergic neurons in the ventral tegmental area (VTA), with projections to the nucleus accumbens (NAc), mPFC, and OFC (Le Moal and Simon, 1991; Ikemoto, 2007). Androgens are secreted from the testes into the general circulation to reach the brain, and androgens are also produced by the brain itself, including nodes within the mesocorticolimbic system. Steroidogenic enzymes are expressed in human, rodent and songbird brain, including the VTA, NAc and mPFC (Soma et al., 1999; Schumacher et al., 2003; Hojo et al., 2004; Hojo et al., 2009; Tobiansky et al., 2018a). Furthermore, even 6 wk. after gonadectomy, T is detectable in the mesocorticolimbic system (but not blood) of male rats with liquid chromatography tandem mass spectrometry (Tobiansky et al., 2018a). Notably, T levels in the VTA of the gonadectomized rats were similar to T levels in the blood of intact rats (Tobiansky et al., 2018a).
Corresponding author at: Department of Psychology and Djavad Mowafaghian Centre for Brain Health, University of British Columbia, Vancouver, BC, Canada. E-mail address: [email protected] (K.K. Soma).
https://doi.org/10.1016/j.yhbeh.2020.104689 Received 21 July 2019; Received in revised form 23 November 2019; Accepted 10 January 2020 0018-506X/ © 2020 Elsevier Inc. All rights reserved.
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received sugar pellets during behavioral testing, and were fed their daily ration of chow in their home cage immediately after testing. Subjects were euthanized 3–5 h after completion of behavioral testing (i.e., the set-shifting task). All subjects were euthanized between 4:00–6:00 pm. All procedures complied with the Canadian Council on Animal Care and were approved by the University of British Columbia Animal Care Committee.
Androgen effects are mediated in part through the androgen receptor (AR) (Janowksy et al., 2000). AR acts as ligand-dependent transcription factors (Evans, 1988) and also act via non-genomic mechanisms (Heinlein and Chang, 2002; Rahman and Christian, 2007). AR is present in multiple brain regions, and its expression is regulated by circulating androgen levels (Menard and Harlan, 1993), suggesting that age-related declines in circulating T could impact brain AR. Although several studies have examined age-related changes in brain AR, these studies have focused on the hypothalamus (Chambers et al., 1991; Wu et al., 2009), amygdala (Chambers et al., 1991) and hippocampus (Tohgi et al., 1995; Kerr et al., 1995). No studies have examined the effects of age on AR immunoreactivity (-ir) in mesocorticolimbic regions, although AR is present in the VTA, NAc, and PFC (Kritzer, 1997; Kritzer, 2004; Low et al., 2017). Furthermore, NAc- and PFC-projecting dopaminergic cells in the VTA contain AR (Simerly et al., 1990; Kritzer, 1997; Creutz and Kritzer, 2004; Kritzer and Creutz, 2008). Here, we compared young adult (5 months) and aged (22 months) male Fischer 344 × Brown Norway rats. We collected serum and the brain and measured systemic T levels in the serum and local T levels in microdissected regions of the mesocorticolimbic system. In addition, we used a sensitive immunohistochemical protocol to measure AR-ir in the mesocorticolimbic system.
2.2. Tissue collection and fixation To reduce the effects of stress on T levels, rats were rapidly and deeply anesthetized with isoflurane and then euthanized by rapid decapitation (within 3 min of onset of anesthesia). Trunk blood was collected into a microcentrifuge tube and kept on wet ice until centrifugation at 10,000 rpm for 10 min, and then serum was collected and stored at −80 °C. Brains were removed and then bisected using a rat brain matrix and a razor blade. Brains were cut along the mid-sagittal plane, and one half of the brain (left or right hemisphere; counterbalanced) was immediately fixed for immunohistochemical processing, and the other half was immediately snap frozen on powdered dry ice and stored at −80 °C. For immunohistochemistry, half of the brain was immersion fixed in 4% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS, pH 7.4) for 4 h at room temperature, washed in PBS for 1.5 h (3 × 30 min), and then cryoprotected in 30% sucrose solution at 4 °C for 48 h or until it sank (Tomm et al., 2018). The immersion-fixed brains were then snap frozen on powdered dry ice and stored at −80 °C. Brains were serially sectioned on a cryostat into five series of 40 μm coronal sections. Sections were stored in a cryoprotectant solution (5 g polyvinylpyrrolidone, 150 g sucrose, 150 mL ethylene glycol, and 250 mL 0.1 M PBS, adjusted to 500 mL with dH2O) at −20 °C until immunohistochemical processing. Of the five series obtained, three series were used in immunohistochemical staining in a prior study (Tomm et al., 2018), and one series was used in the present study for immunohistochemical staining of AR.
2. Materials and methods 2.1. Subjects Subjects were male Fischer 344 × Brown Norway F1 hybrid (F344/ BN) rats (Rattus norvegicus) consisting of young adult rats (3 months old at arrival; 5 months at euthanasia) and aged rats (20 months old at arrival, 22 months at euthanasia) (n = 12 per age; n = 24 total). Rats were obtained from the National Institute on Aging (Taconic Farms, NY) and were sexually naïve. Rats were group-housed with rats of similar age for the first week after arrival and given ad libitum access to food (Rat Diet 5012; LabDiet, St. Louis, MO) and water. In the following weeks, rats were single-housed and mildly food-restricted to 85–90% of their free-feeding weight with ad libitum access to water throughout behavioral testing. All cages contained aspen chip bedding (Nepco, Warrensburg, NY) and a PVC pipe for environmental enrichment. Rats were kept on a 12 h light:dark cycle (lights on at 07:00 am) at an average temperature of 21–22 °C and relative humidity of 40–50%. Two weeks after arrival, rats were subjected to behavioral training. Behavioral procedures and results have been reported in detail in Tomm et al., 2018, which tested the same subjects as examined here. Briefly, subjects were trained on a radial arm maze task and then a strategy set-shifting task in operant chambers (Tomm et al., 2018). Rats
VTA
2.3. T measurement in serum and brain To measure T levels in the mesocorticolimbic system, tissue was microdissected from 4 brain regions: VTA, NAc, mPFC, and OFC. Frozen brain tissue was sectioned into 300 μm coronal sections in a cryostat. Brain regions were identified using major neuroanatomical landmarks and a rat brain atlas (Paxinos and Watson, 2009) and collected via Palkovits punch (Charlier et al., 2010; Taves et al., 2011; Tobiansky et al., 2018a; Fig. 1). A stainless steel cannula (1 mm inner diameter; 0.245 mg/punch; Fine Science Tools, #18035–01) was used to collect
NAc
mPFC
OFC
Fig. 1. Representative location of brain punches (1 mm diameter) for neural T measurement. From left: ventral tegmental area (VTA), nucleus accumbens (NAc), medial prefrontal cortex (mPFC), orbitofrontal cortex (OFC). Tissue was microdissected from 6 to 7 adjacent sections, depending on the region. Each region was not subdivided (e.g. the mPFC was not subdivided into prelimbic and infralimbic subregions) due to the size of the punch. 2
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VTA
PrL
NAcS
NAcC
LOFC
IL
MOFC
Fig. 2. Representative placement of region of interest for quantification of AR-immunoreactivity in mesolimbic regions and prefrontal cortical regions. Abbreviations: ventral tegmental area (VTA), nucleus accumbens core (NAcC), nucleus accumbens shell (NAcS), prelimbic mPFC (PrL), infralimbic mPFC (IL), lateral orbitofrontal cortex (LOFC), medial orbitofrontal cortex (MOFC).
incubated for 4 h at room temperature. Then 200 μL of tracer was added and incubated overnight at room temperature. The following day, 50 μL of precipitant (secondary antibody) was added to all tubes and incubated for 1 h at 37 °C in a water bath. Tubes were then centrifuged for 30 min at 3200 rpm at 4 °C, decanted, and counted for 2 min on a gamma counter. Final values were corrected for recovery. Recovery percentages were calculated for each sample type (serum or brain tissue) by comparing unspiked samples to samples spiked with a known amount of steroid. Recoveries were 88% for serum, and 113% for brain tissue.
tissue from 6 to 7 sections (1.470–1.715 mg of tissue total), depending on the region. One punch was collected per section. The correct placement of punches was verified by Nissl staining. Subjects with incorrectly placed punches were excluded from analysis (VTA: 4 subjects, NAc: 2 subjects, mPFC: 1 subject). The microdissected tissue was collected into microcentrifuge tubes. Tissue was homogenized with 1 mL 84% ice-cold methanol, using a bead homogenizer (Omni International Inc., Kennesaw, GA). Homogenized tissue samples were left overnight at −20 °C. The following day, samples were centrifuged for 5 min and the supernatant was collected and diluted in 10 mL deionized water. For T measurement in serum, 40 μL of serum was diluted in 10 mL deionized water. Steroids were extracted from the samples using solid phase extraction (SPE) with C18 columns (Agilent Bond Elut LRC-C18 OH, #12113045) mounted on a glass vacuum manifold (UCT #VMF024GL). After column conditioning and equilibration, samples were loaded onto the SPE columns. Then, samples were washed with 10 mL of 40% HPLC-grade methanol (Taves et al., 2010), steroids were eluted from columns with 5 mL of 90% HPLC-grade methanol, and eluates were dried in a vacuum centrifuge at 40 °C. T was measured using a sensitive and specific commercial RIA kit (#07189102; MP Biomedicals, Solon, OH), with modifications described by Overk et al. (2013) and Ferris et al. (2015). Dried eluates were re-suspended in 100% ethanol, and then diluted with the buffer provided in RIA kit. The amount of ethanol in samples did not exceed 1%. From the original samples, a portion (90% for the serum, and 75% for the brain) was used for T measurement. Serum samples were run in duplicate, and brain samples were run as singletons. 200 μL of primary antibody was added to the re-suspended sample and standards, and
2.4. Immunohistochemistry for AR Immunohistochemistry for AR was performed as described in detail by Low et al. (2017), using free-floating tissue sections in 24-well plates, with each plate containing tissue from one young and one aged rat. Sections were washed in 0.1 M Tris-buffered saline (TBS; pH 7.4), and then incubated in 2% hydrogen peroxide (H2O2) for 30 min. Sections were then washed in 0.1 M TBS and then in 0.1 M PBS with 0.03% Triton X-100 and 0.1% gelatin (PBS-GT). Next, sections were incubated in 8% tryptone (J859; Amresco, Inc.) in PBS for 2 h to block nonspecific reactivity. Directly following the block, sections were incubated in primary antibody solution (1:200 in PBS-GT) for 24 h at room temperature. The primary antibody was a monoclonal anti-AR antibody [EPR1535(2)] raised in rabbit (ab133273; Abcam, Inc.) and used at a concentration of [1:200]. This primary antibody has been extensively validated (Hamson et al., 2013; Low et al., 2017). Then sections were washed in PBS-GT and incubated in biotin-SP-conjugated affinity-purified donkey anti-rabbit secondary antibody (1:2000 in 0.8% tryptone 3
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circularity and size were uniformly applied.
in PBS; 711-065-152; Jackson ImmunoResearch Laboratories, RRID: AB_2340593) for 1 h at room temperature and then overnight at 4 °C. Sections were washed in PBS-GT, incubated in avidin-biotin peroxidase (AB) (in 0.8% tryptone in PBS; VectaStain ABC Kit; PK-6100 Standard) for 30 min at room temperature, and washed in PBS-GT. Signal was amplified by incubation in biotinylated tyramide for 10 min (Adams, 1992). Sections were washed in PBS-GT, incubated in AB solution for 30 min, and washed in PBS-GT. Sections were washed quickly in TBS, and then were incubated with 3,3′-Diaminobenzidine and nickel (Vector Labs Peroxidase Substrate Kit SK-4100) for 20 min at room temperature. Lastly, sections were mounted onto charged slides, dehydrated, and cover-slipped.
2.6. Statistical analysis The effect of Age on T levels was analyzed using one-way ANOVAs, and data were log-transformed prior to analyses to reduce heteroscedasticity. The ratio of Brain T: Serum T (T-ratio) was calculated by dividing the T level in the brain region by the serum T level, for qualitative analysis only. The effect of Age on AR-ir was analyzed using one-way ANOVAs. Effect size (Cohen's d) was also calculated for T and AR-ir comparisons to assess the standardized difference between the young and aged group means. Pearson's correlations were used to assess the relationship between AR-ir and brain T. Correlations were assessed separately for young rats and aged rats. For T analyses, we could not subdivide the mPFC, OFC and NAc, and thus for this correlation, we did not subdivide these regions with respect to AR-ir. Analyses were conducted using SPSS Statistics software (IBM, Armonk, NY) ver. 23.0 (MacOSX) and ver. 24.0 (Windows) and R ver. 3.5.1 (2018-07-02, “Feather Spray”).
2.5. Quantification of AR immunoreactivity AR-ir was assessed in regions of the mesocorticolimbic system (Fig. 2). The mesolimbic regions were: (1) the VTA, for which we focused on the parabrachial pigmented (PBP) and paranigral (PN) nuclei, (2) the NAc core (NAcC), and (3) the NAc shell (NAcS). The prefrontal cortical regions were: (4) the prelimbic portion (PrL) of the mPFC, (5) the infralimbic (IL) portion of the mPFC; (6) the lateral orbitofrontal cortex (LOFC), and (7) the medial orbitofrontal cortex (MOFC). The regions were identified by major neuroanatomical landmarks (Paxinos and Watson, 2009). For each brain region, photomicrographs were captured using a Nikon Digital Sight DS-U1 camera and Nikon Eclipse 90i microscope (at the 20× objective) interfaced with NIS-Elements Basic Research software. Images were taken from two to five serial sections depending on the region (Table 1). In some cases, individual sections were damaged, and these were excluded. All images were captured at identical brightness and contrast settings with the exception of the NAc, for which brightness and contrast were increased consistently across all subjects. All images were captured at maximum resolution (2560 pixels × 1920 pixels). Importantly, slides were coded, and all photomicroscopy and image analysis was conducted by an experimenter (KLL) who was blind to the age of each subject. Images were quantified in a manner similar to Heimovics et al. (2012). For each image, a background mean intensity value was calculated by averaging six intensity measurements taken from locations within each brain area where specific staining was not present. Next, the background intensity value for each image was used to generate a quantification threshold value, either 1.25× or 1.5× the background, depending on the region. The selected threshold value (1.25× or 1.5×) was used uniformly for all subjects for a given region. Once thresholds were calculated for all images, they were applied to a rectangular region of interest (ROI) with area 0.135 mm2 for each brain region. In NIS-Elements, the ROI was superimposed onto each photomicrograph within the limits of each brain region, to measure the percentage of the ROI that was above the threshold (% AR-ir). Restrictions on object
3. Results 3.1. Serum and brain T levels In serum and all brain regions – except the VTA – there was a significant main effect of Age on T levels (Fig. 3). In particular, T levels were significantly lower in aged rats than in young rats in the serum (F1,22 = 12.24, p = 0.002; d = −1.15), NAc (F1,20 = 7.84, p = 0.011; d = −1.11), mPFC (F1,21 = 5.57, p = 0.028; d = −0.92), and OFC (F1,22 = 7.38, p = 0.013; d = −1.03). In contrast, the effect of Age was not significant in the VTA (F1,17 = 1.87, p = 0.190; d = −0.60). In young and aged animals, the T-ratio was > 1 in all brain regions, indicating that T levels were higher in the brain than in the serum. 3.2. AR immunoreactivity The overall distribution of AR-ir was similar to previous studies (Simerly et al., 1990; Sar et al., 1990). In the VTA (Fig. 4), there were few (but strongly) labeled cells, primarily in the PBP and PN subregions, as described by Kritzer (1997). In the PFC (Fig. 5), AR-ir was observed in a bilaminar pattern, as before (Low et al., 2017). In mesolimbic brain regions (VTA and NAc), there were no significant main effects of Age on AR-ir (Figs. 4, 6A). Regions examined included the VTA (F1,16 = 0.70, p = 0.417; d = 0.40), NAcC (F1,20 = 1.97, p = 0.176; d = 0.60) and NAcS (F1,18 = 0.18, p = 0.675; d = 0.19). In contrast, in prefrontal cortical brain regions, there were significant effects of Age on AR-ir (Figs. 5, 6B). Aged rats had
Table 1 Number of photomicrographs analyzed per region per subject for quantification of androgen receptor immunoreactivity. Region
Number of brain sections examined
Number of photos taken per section
Total number of photos analyzed
VTA NAcC NAcS PrL IL LOFC MOFC
3 5 5 4 4 5 2
2 1 1 1 1 1 1
6 5 5 4 4 5 2
Fig. 3. Testosterone (T) levels in young (5 months) and aged (22 months) male rats. T levels were significantly lower in aged rats than young rats, except in the VTA. n = 9–12 (young), 10–12 (aged). *p ≤ 0.05, **p ≤ 0.01. Values are reported as mean ± SEM. Abbreviations: VTA, ventral tegmental area; NAc, nucleus accumbens; mPFC, medial prefrontal cortex; OFC, orbitofrontal cortex.
VTA, ventral tegmental area; NAcC, nucleus accumbens core; NAcS, nucleus accumbens shell; PrL, prelimbic region of the medial prefrontal cortex; IL, infralimbic region of the prefrontal cortex; LOFC, lateral orbitofrontal cortex; MOFC, medial orbitofrontal cortex. 4
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Fig. 4. Representative photomicrographs of AR-immunoreactivity (AR-ir) from young (5 months; A, C, and E) and aged (22 months; B, D, and F) male rats in mesolimbic regions. (A–B) VTA, ventral tegmental area; (C–D) NAcC, nucleus accumbens core; (E–F) NAcS, nucleus accumbens shell. Scale bars represent 100 μm. All images were taken at the same magnification.
age, except in the VTA. Brain T levels were higher than serum T levels in both ages. The AR-ir in the VTA, NAc and OFC did not change with age. In contrast, AR-ir in the PFC (mPFC-PrL and mPFC-IL) decreased with age.
significantly lower AR-ir than young rats in the mPFC, including the PrL (F1,20 = 6.25; p = 0.021; d = −1.07) and IL (F1,19 = 9.11, p = 0.007; d = −1.31). However, the effect of Age was not significant in the LOFC (F1,20 = 2.41; p = 0.136; d = −0.66) or MOFC (F1,19 = 1.34; p = 0.261; d = −0.51).
4.1. Age-related changes in testosterone levels
3.3. Correlations between neural T and AR immunoreactivity
Age-related declines in serum T occur in males of several species, including humans (Feldman et al., 2002), rhesus monkeys (Downs and Urbanski, 2006), mice (Flood et al., 1995), and multiple strains of rats (Roselli et al., 1986; Wu et al., 2009; Ghanadian et al., 1975; Bernardi et al., 1998; Gruenewald et al., 1994; Rosario et al., 2009; Chambers et al., 1991; McGinnis and Yu, 1995; Luine et al., 2007). Serum T levels in male rats vary with strain, sexual experience, and time of day, with levels of 1–10 ng/mL in young rats (Wu and Gore, 2010) and 0.2–1.5 ng/mL in aged rats (Ghanadian et al., 1975; Chambers et al., 1991). The serum T levels measured here are consistent with these ranges. In addition, we measured T in microdissected regions of the mesocorticolimbic system. Brain T levels declined with age in the NAc, mPFC and OFC but not in the VTA. The ratio of brain T to serum T was > 1 in all regions in both young and aged rats. These data are consistent with our previous study reporting steroidogenic enzyme expression in the mesocorticolimbic system, higher T levels in the mesocorticolimbic
In young rats, neural T and AR-ir were significantly positively correlated in the VTA (r = 0.70, p = 0.05). However, the correlations were not statistically significant in the NAc (r = −0.52, p = 0.12), mPFC (r = 0.46, p = 0.15), and OFC (r = 0.21, p = 0.54). In aged rats, correlations between neural T and AR-ir were not statistically significant in any region: VTA (r = −0.002, p = 0.97), NAc (r = 0.06, p = 0.90), mPFC (r = 0.41, p = 0.28), and OFC (r = 0.48, p = 0.16). 4. Discussion This study measured serum T levels, neural T levels, and neural ARir in young adult (5 months) and aged (22 months) F344/BN male rats. Androgens influence executive functions via effects on the mesocorticolimbic system (Kritzer et al., 2007; Tobiansky et al., 2018b), so we were interested in T and AR in the VTA, NAc, mPFC and OFC. Serum T levels declined with age, as expected. Brain T levels also declined with 5
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Fig. 5. Representative photomicrographs of AR-immunoreactivity (AR-ir) from young (5 months; A, C, E, and G) and aged (22 months; B, D, F, and H) male rats in prefrontal cortical regions. (A–B) PrL, prelimbic subregion of the medial prefrontal cortex; (C–D) IL, infralimbic subregion of the medial prefrontal cortex; (E–F) LOFC, lateral orbitofrontal cortex; (G–H) MOFC, medial orbitofrontal cortex. Scale bars represent 100 μm. All images were taken at the same magnification.
4.2. Age-related changes in AR immunoreactivity
et al., 2003). Age-related changes in neural AR have been reported in rodents (Haji et al., 1981; Chambers et al., 1991; McGinnis and Yu, 1995; Kerr et al., 1995; Xiao and Jordan, 2002; Wu et al., 2009; Munetomo et al., 2015), and the effects of aging are region-specific (Wu et al., 2009). Although previous work focused on the hypothalamus, amygdala, and hippocampus (Wood, 2008; Wu et al., 2009), the present study is distinguished in that it examined the effects of age on AR in the mesocorticolimbic system.
AR staining was consistent with previous reports of predominantly nuclear immunoreactivity in rodent brain (Lu et al., 1998; Zhou et al., 1994; Fernandez-Guasti et al., 2003), although AR-ir is also present at extranuclear sites (Tabori et al., 2005; Sarkey et al., 2008; DonCarlos
4.2.1. Mesolimbic AR immunoreactivity The VTA contains dopaminergic neurons that project to the PFC and NAc (Ikemoto, 2007), and AR mRNA and protein are present in this
system than in the blood of young adult rats, and the presence of T in the mesocorticolimbic system of long-term gonadectomized rats (Tobiansky et al., 2018a). Future studies should examine age-related changes in steroidogenic enzyme mRNA and activity in the mesocorticolimbic system.
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report an age-associated decrease (Munetomo et al., 2015) or no change (Kerr et al., 1995; Kumar and Thakur, 2004). To our knowledge, the present results are the first to characterize age-related changes in AR specifically in the PFC. AR-ir in the PrL and IL subregions of the mPFC decreased with age. In contrast, AR-ir in the LOFC and MOFC did not significantly change with age. The decreased mPFC AR-ir in aged rats is unlikely to be fully accounted for by age-related cell loss. In one study, during normal aging, the mPFC does not show loss of neurons (Finch, 2003; Zamzow et al., 2014). In other studies, there is some neuron loss in the PFC of the rat (Yates et al., 2008) and monkey (Smith et al., 2004), but the losses are subregion-specific and are not substantial enough to fully explain the present results. Moreover, previous work has shown an agerelated decrease in brain AR binding without accompanying loss of neurons (McGinnis and Yu, 1995). Collectively, these data show that age-associated reductions in AR-ir within the frontal lobes show some anatomical specificity, with medial (cingulate) regions being more affected than orbital regions of the PFC. 4.3. Androgen regulation of AR AR is generally thought to be an auto-regulated protein (Lu et al., 1998) and neural AR-ir is regulated by circulating T levels (FernandezGuasti et al., 2003). Castration strongly reduces neural AR-ir levels, and AR-ir is restored with androgen replacement (Menard and Harlan, 1993; Lu et al., 1998; Xiao and Jordan, 2002; Fernandez-Guasti et al., 2003). Supraphysiological doses of androgens increase AR-ir in the brain (Wood and Newman, 1993; Lu et al., 1998; Fernandez-Guasti et al., 2003). Androgen manipulations can also affect intracellular partitioning of AR (Wood and Newman, 1993). Consequently, decreases in systemic T levels may be expected to down-regulate AR, although this may be region-specific. The decreased AR-ir in the mPFC in aged animals along with decreased circulating T is consistent with this autoregulation model. Moreover, the positive correlation between local T levels and AR-ir in the mPFC suggests that, in addition to systemic T, local T levels also regulate AR. Our results show that ligand and receptor concentrations do not always change in parallel in aging. While the mPFC had both decreased T and AR-ir, in contrast the NAc and OFC had decreased T and no change in AR-ir. These data suggest that AR is regulated differently across brain regions. Our analysis is limited to the nuclear AR. Androgens could also regulate neural activity through membrane AR. A membrane AR has been characterized (Thomas et al., 2014), but its distribution in the brain is not known. Our results are also limited to changes in the amount of AR-ir and not necessarily AR binding or function.
Fig. 6. Mesolimbic and prefrontal cortical AR immunoreactivity (AR-ir) in young (5 months old) and aged (22 months old) male rats. (A) There were no significant effects of Age on AR-ir in the ventral tegmental area (VTA), nucleus accumbens core (NAcC), or nucleus accumbens shell (NAcS). n = 8–11 (young), 5–11 (aged). (B) AR-ir levels were significantly lower in aged rats compared to young rats in the prelimbic (PrL) and infralimbic (IL) subregions of the medial prefrontal cortex (mPFC). In the lateral (LOFC) and medial (MOFC) subregions of the orbitofrontal cortex (OFC), the effect of age was not significant. n = 11 (young), 10–11 (aged). *p ≤ 0.05, **p ≤ 0.01.
nucleus (Simerly et al., 1990; Kritzer, 1997; Tobiansky et al., 2018b). Moreover, some VTA dopaminergic neurons express AR and thus are direct targets of androgens (Kritzer, 1997). We observed sparsely distributed but strongly labeled AR-ir cells in the VTA, with the majority of AR+ cells in the PBP and PN. Yet AR-ir did not significantly change with age in the VTA. This lack of an effect of age is in keeping with a report by Menard and Harlan (1993) that examined the effects of castration and androgen replacement on the number of AR-ir cells in male rats. There were no effects of androgen manipulations on AR-ir in the VTA, while AR-ir in other regions was affected. The NAc is a key subcortical projection target of VTA dopaminergic neurons. In the NAc, previous studies have reported that AR-ir is sparse (Wood, 2008; Sato et al., 2010, but see Fernandez-Guasti et al., 2003). Here, using a very sensitive protocol that minimizes background staining (Low et al., 2017), we clearly detected AR-ir in the NAc, especially in the NAcS. Yet, as with the VTA, AR-ir in the NAc was not affected by age.
4.4. Implications for aging and executive functions Androgen modulation of mesocorticolimbic dopamine signaling and PFC-dependent executive function is supported by both behavioral and neurochemical studies (Aubele and Kritzer, 2011; Tobiansky et al., 2018b). First, behavioral studies have shown that gonadectomy impairs and, in some cases, T administration restores performance on working memory tasks (Kritzer et al., 2001; Bimonte-Nelson et al., 2003; Aubele et al., 2008; Sandstrom et al., 2006; Spritzer et al., 2008). In addition, androgen manipulation regulates behavioral flexibility through changes in perseverative behaviors (Andrew and Rogers, 1972; Archer, 1977; Thompson and Wright, 1979; van Hest et al., 1989; Neese and Schantz, 2012; Wallin and Wood, 2015). Androgens also influence cost/benefit decision-making by altering the discounting of a large reward (versus a small reward) when the effort or delay required to obtain the large reward is varied (Wallin et al., 2015). Most of these behavioral studies focus on young adult animals, and it remains unclear whether similar effects are present in aged animals. Second, neurochemical studies have shown that gonadectomy alters
4.2.2. Prefrontal cortical AR immunoreactivity There is increasing appreciation for the large number of AR+ cells (primarily neurons) in the cerebral cortex (DonCarlos et al., 2006; Low et al., 2017; Tobiansky et al., 2018b). AR+ cells are present in the PFC of primates (Finley and Kritzer, 1999) and rats (Kritzer, 2004). Some studies have examined AR mRNA in the entire cerebral cortex and 7
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immunoreactivity of tyrosine hydroxylase (TH), a catecholamine-synthesizing enzyme, in the PFC (Adler et al., 1999; Kritzer, 2000) and increases extracellular dopamine levels in the PFC (Aubele and Kritzer, 2010). Normal dopamine neurotransmission is critical to maintain executive functions (Floresco and Magyar, 2006). The present results provide additional support for the notion that androgens modulate executive functioning by acting on specific nodes within the mesocorticolimbic system. We have previously observed agerelated changes in executive function (Tomm et al., 2018), as well as markers of androgen signaling in the mesocorticolimbic system. The age-related decreases in neural T levels and mPFC AR-ir might impact TH, dopamine synthesis and executive function (Tomm et al., 2018). It remains unclear whether androgens also influence executive functions through indirect effects on regions that project to the PFC.
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5. Conclusions We demonstrate age-related changes in T and AR in male rats. Compared to young rats (5 months old), aged rats (22 months old) have lower T levels in the serum, NAc, mPFC, and OFC, but not in the VTA. At both ages, T levels are higher in the mesocorticolimbic system than in the serum. The AR-ir in the mPFC decreases with age. Taken together, these results demonstrate age-associated changes in androgen signaling and suggest that changes in T and AR in the mesocorticolimbic system may contribute to age-related changes in executive function. Declaration of competing interest The authors have no conflicts of interest to declare. Acknowledgements The authors gratefully acknowledge funding from the Canadian Institutes of Health Research (Operating Grant 133606 to K.K.S); Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery Grant (to S.B.F.); the Michael J. Quinn Award and UBC Science Undergraduate Research Experience Award (to K.L.L); the NSERC USRA, UBC Aboriginal Graduate Fellowship, UBC 4 Year Fellowship and NSERC PGS-D Fellowship (to R.J.T.); and the Bluma Tischler Post-Doctoral Fellowship (to D.J.T.). The authors thank Maric Tse, Anastasia Korol, Sarah Marjanovic, and Madison Grist for assistance with data collection. References Adams, J.C., 1992. Biotin amplification of biotin and horseradish peroxidase signals in histochemical stains. J. Histochem. Cytochem. 40, 1457–1463. Adler, A., Vescovo, P., Robinson, J.K., Kritzer, M.F., 1999. Gonadectomy in adult life increases tyrosine hydroxylase immunoreactivity in the prefrontal cortex and decreases open field activity in male rats. Neuroscience 89, 939–954. Alexander, G.E., Ryan, L., Bowers, D., Foster, T.C., Bizon, J.L., Geldmacher, D.S., Glisky, E.L., 2012. Characterizing cognitive aging in humans with links to animal models. Front. Aging Neurosci. 4. Andrew, R.J., Rogers, L.J., 1972. Testosterone, search behaviour and persistence. Nature 237, 343. Archer, J., 1977. Testosterone and persistence in mice. Anim. Behav. 25, 479–488. Aubele, T., Kritzer, M.F., 2010. Gonadectomy and hormone replacement affects in vivo basal extracellular dopamine levels in the prefrontal cortex but not motor cortex of adult male rats. Cereb. Cortex 21, 222–232. Aubele, T., Kritzer, M.F., 2011. Androgen influence on prefrontal dopamine systems in adult male rats: localization of cognate intracellular receptors in medial prefrontal projections to the ventral tegmental area and effects of gonadectomy and hormone replacement on glutamate-stimulated extracellular dopamine level. Cereb. Cortex 22, 1799–1812. Aubele, T., Kaufman, R., Montalmant, F., Kritzer, M.F., 2008. Effects of gonadectomy and hormone replacement on a spontaneous novel object recognition task in adult male rats. Horm. Behav. 54, 244–252. Beas, B.S., Setlow, B., Bizon, J.L., 2013. Distinct manifestations of executive dysfunction in aged rats. Neurobiol. Aging 34, 2164–2174. Bernardi, F., Salvestroni, C., Casarosa, E., Nappi, R.E., Lanzone, A., Luisi, S., Purdy, R.H.,
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Effects of aging on testosterone and androgen receptors in the mesocorticolimbic system of male rats
T
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Katelyn L. Lowa,b, , Ryan J. Tomma, Chunqi Maa, Daniel J. Tobianskya, Stan B. Florescoa, Kiran K. Somaa,b a b
Department of Psychology and Djavad Mowafaghian Centre for Brain Health, University of British Columbia, Vancouver, BC, Canada Department of Zoology, University of British Columbia, Vancouver, BC, Canada
A R T I C LE I N FO
A B S T R A C T
Keywords: Brain Executive function Medial prefrontal cortex Neurosteroid Nucleus accumbens Orbitofrontal cortex Ventral tegmental area
As males age, systemic testosterone (T) levels decline. T regulates executive function, a collection of cognitive processes that are mediated by the mesocorticolimbic system. Here, we examined young adult (5 months) and aged (22 months) male Fischer 344 × Brown Norway rats, and measured systemic T levels in serum and local T levels in microdissected nodes of the mesocorticolimbic system (ventral tegmental area (VTA), nucleus accumbens (NAc), medial prefrontal cortex (mPFC), and orbitofrontal cortex (OFC)). We also measured androgen receptor (AR) immunoreactivity (-ir) in the mesocorticolimbic system. As expected, systemic T levels decreased with age. Local T levels in mesocorticolimbic regions – except the VTA – also decreased with age. Mesocorticolimbic T levels were higher than serum T levels at both ages. AR-ir was present in the VTA, NAc, mPFC, and OFC and decreased with age in the mPFC. Taken together with previous results, the data suggest that changes in androgen signaling may contribute to changes in executive function during aging.
1. Introduction In male humans, non-human primates, and rodents, aging is associated with declines in several cognitive functions, which coincide with a decline in systemic levels of androgens (Ghanadian et al., 1975; Kaler and Neaves, 1981; Vermeulen, 1991). Androgens such as testosterone (T) play an important role in cognition (Janowsky, 2006; Tobiansky et al., 2018b) and their decline may be an important factor in cognitive changes during aging (Lund et al., 1999). Aging is characterized in part by declines in executive functions (Alexander et al., 2012), which are cognitive processes involved in coordinating complex behaviors and goal-directed actions. Executive functioning is modulated by prefrontal cortical structures, such as the medial prefrontal cortex (mPFC) and orbitofrontal cortex (OFC) (Janowsky, 2006; Gallagher et al., 2011; Beas et al., 2013). For example, both regions facilitate different forms of cognitive flexibility; the mPFC mediates extradimensional attentional set-shifts, while the OFC mediates reversal shifts between different stimulus-reward associations (Dalley et al., 2004; Gallagher et al., 2011). In rats, the mPFC also governs working memory, a form of shortterm memory involving the maintenance and manipulation of information for obtaining goals (Goldman-Rakic, 1995; Funahashi, 2006). Androgens regulate executive functions, including working memory
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and set-shifting (Janowksy et al., 2000; Bimonte-Nelson et al., 2003; Aubele and Kritzer, 2011; Tobiansky et al., 2018b). T administration and T deprivation affect performance on PFC-dependent tasks (Janowksy et al., 2000; Kritzer et al., 2001; Daniel et al., 2003). One major pathway through which androgens exert effects on executive function is the mesocorticolimbic system (Tobiansky et al., 2018b). The mesocorticolimbic system is critical for reward seeking, motivation, and higher-order cognitive functions (Fibiger and Phillips, 1988; Kritzer, 1997) and consists of dopaminergic neurons in the ventral tegmental area (VTA), with projections to the nucleus accumbens (NAc), mPFC, and OFC (Le Moal and Simon, 1991; Ikemoto, 2007). Androgens are secreted from the testes into the general circulation to reach the brain, and androgens are also produced by the brain itself, including nodes within the mesocorticolimbic system. Steroidogenic enzymes are expressed in human, rodent and songbird brain, including the VTA, NAc and mPFC (Soma et al., 1999; Schumacher et al., 2003; Hojo et al., 2004; Hojo et al., 2009; Tobiansky et al., 2018a). Furthermore, even 6 wk. after gonadectomy, T is detectable in the mesocorticolimbic system (but not blood) of male rats with liquid chromatography tandem mass spectrometry (Tobiansky et al., 2018a). Notably, T levels in the VTA of the gonadectomized rats were similar to T levels in the blood of intact rats (Tobiansky et al., 2018a).
Corresponding author at: Department of Psychology and Djavad Mowafaghian Centre for Brain Health, University of British Columbia, Vancouver, BC, Canada. E-mail address: [email protected] (K.K. Soma).
https://doi.org/10.1016/j.yhbeh.2020.104689 Received 21 July 2019; Received in revised form 23 November 2019; Accepted 10 January 2020 0018-506X/ © 2020 Elsevier Inc. All rights reserved.
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received sugar pellets during behavioral testing, and were fed their daily ration of chow in their home cage immediately after testing. Subjects were euthanized 3–5 h after completion of behavioral testing (i.e., the set-shifting task). All subjects were euthanized between 4:00–6:00 pm. All procedures complied with the Canadian Council on Animal Care and were approved by the University of British Columbia Animal Care Committee.
Androgen effects are mediated in part through the androgen receptor (AR) (Janowksy et al., 2000). AR acts as ligand-dependent transcription factors (Evans, 1988) and also act via non-genomic mechanisms (Heinlein and Chang, 2002; Rahman and Christian, 2007). AR is present in multiple brain regions, and its expression is regulated by circulating androgen levels (Menard and Harlan, 1993), suggesting that age-related declines in circulating T could impact brain AR. Although several studies have examined age-related changes in brain AR, these studies have focused on the hypothalamus (Chambers et al., 1991; Wu et al., 2009), amygdala (Chambers et al., 1991) and hippocampus (Tohgi et al., 1995; Kerr et al., 1995). No studies have examined the effects of age on AR immunoreactivity (-ir) in mesocorticolimbic regions, although AR is present in the VTA, NAc, and PFC (Kritzer, 1997; Kritzer, 2004; Low et al., 2017). Furthermore, NAc- and PFC-projecting dopaminergic cells in the VTA contain AR (Simerly et al., 1990; Kritzer, 1997; Creutz and Kritzer, 2004; Kritzer and Creutz, 2008). Here, we compared young adult (5 months) and aged (22 months) male Fischer 344 × Brown Norway rats. We collected serum and the brain and measured systemic T levels in the serum and local T levels in microdissected regions of the mesocorticolimbic system. In addition, we used a sensitive immunohistochemical protocol to measure AR-ir in the mesocorticolimbic system.
2.2. Tissue collection and fixation To reduce the effects of stress on T levels, rats were rapidly and deeply anesthetized with isoflurane and then euthanized by rapid decapitation (within 3 min of onset of anesthesia). Trunk blood was collected into a microcentrifuge tube and kept on wet ice until centrifugation at 10,000 rpm for 10 min, and then serum was collected and stored at −80 °C. Brains were removed and then bisected using a rat brain matrix and a razor blade. Brains were cut along the mid-sagittal plane, and one half of the brain (left or right hemisphere; counterbalanced) was immediately fixed for immunohistochemical processing, and the other half was immediately snap frozen on powdered dry ice and stored at −80 °C. For immunohistochemistry, half of the brain was immersion fixed in 4% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS, pH 7.4) for 4 h at room temperature, washed in PBS for 1.5 h (3 × 30 min), and then cryoprotected in 30% sucrose solution at 4 °C for 48 h or until it sank (Tomm et al., 2018). The immersion-fixed brains were then snap frozen on powdered dry ice and stored at −80 °C. Brains were serially sectioned on a cryostat into five series of 40 μm coronal sections. Sections were stored in a cryoprotectant solution (5 g polyvinylpyrrolidone, 150 g sucrose, 150 mL ethylene glycol, and 250 mL 0.1 M PBS, adjusted to 500 mL with dH2O) at −20 °C until immunohistochemical processing. Of the five series obtained, three series were used in immunohistochemical staining in a prior study (Tomm et al., 2018), and one series was used in the present study for immunohistochemical staining of AR.
2. Materials and methods 2.1. Subjects Subjects were male Fischer 344 × Brown Norway F1 hybrid (F344/ BN) rats (Rattus norvegicus) consisting of young adult rats (3 months old at arrival; 5 months at euthanasia) and aged rats (20 months old at arrival, 22 months at euthanasia) (n = 12 per age; n = 24 total). Rats were obtained from the National Institute on Aging (Taconic Farms, NY) and were sexually naïve. Rats were group-housed with rats of similar age for the first week after arrival and given ad libitum access to food (Rat Diet 5012; LabDiet, St. Louis, MO) and water. In the following weeks, rats were single-housed and mildly food-restricted to 85–90% of their free-feeding weight with ad libitum access to water throughout behavioral testing. All cages contained aspen chip bedding (Nepco, Warrensburg, NY) and a PVC pipe for environmental enrichment. Rats were kept on a 12 h light:dark cycle (lights on at 07:00 am) at an average temperature of 21–22 °C and relative humidity of 40–50%. Two weeks after arrival, rats were subjected to behavioral training. Behavioral procedures and results have been reported in detail in Tomm et al., 2018, which tested the same subjects as examined here. Briefly, subjects were trained on a radial arm maze task and then a strategy set-shifting task in operant chambers (Tomm et al., 2018). Rats
VTA
2.3. T measurement in serum and brain To measure T levels in the mesocorticolimbic system, tissue was microdissected from 4 brain regions: VTA, NAc, mPFC, and OFC. Frozen brain tissue was sectioned into 300 μm coronal sections in a cryostat. Brain regions were identified using major neuroanatomical landmarks and a rat brain atlas (Paxinos and Watson, 2009) and collected via Palkovits punch (Charlier et al., 2010; Taves et al., 2011; Tobiansky et al., 2018a; Fig. 1). A stainless steel cannula (1 mm inner diameter; 0.245 mg/punch; Fine Science Tools, #18035–01) was used to collect
NAc
mPFC
OFC
Fig. 1. Representative location of brain punches (1 mm diameter) for neural T measurement. From left: ventral tegmental area (VTA), nucleus accumbens (NAc), medial prefrontal cortex (mPFC), orbitofrontal cortex (OFC). Tissue was microdissected from 6 to 7 adjacent sections, depending on the region. Each region was not subdivided (e.g. the mPFC was not subdivided into prelimbic and infralimbic subregions) due to the size of the punch. 2
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VTA
PrL
NAcS
NAcC
LOFC
IL
MOFC
Fig. 2. Representative placement of region of interest for quantification of AR-immunoreactivity in mesolimbic regions and prefrontal cortical regions. Abbreviations: ventral tegmental area (VTA), nucleus accumbens core (NAcC), nucleus accumbens shell (NAcS), prelimbic mPFC (PrL), infralimbic mPFC (IL), lateral orbitofrontal cortex (LOFC), medial orbitofrontal cortex (MOFC).
incubated for 4 h at room temperature. Then 200 μL of tracer was added and incubated overnight at room temperature. The following day, 50 μL of precipitant (secondary antibody) was added to all tubes and incubated for 1 h at 37 °C in a water bath. Tubes were then centrifuged for 30 min at 3200 rpm at 4 °C, decanted, and counted for 2 min on a gamma counter. Final values were corrected for recovery. Recovery percentages were calculated for each sample type (serum or brain tissue) by comparing unspiked samples to samples spiked with a known amount of steroid. Recoveries were 88% for serum, and 113% for brain tissue.
tissue from 6 to 7 sections (1.470–1.715 mg of tissue total), depending on the region. One punch was collected per section. The correct placement of punches was verified by Nissl staining. Subjects with incorrectly placed punches were excluded from analysis (VTA: 4 subjects, NAc: 2 subjects, mPFC: 1 subject). The microdissected tissue was collected into microcentrifuge tubes. Tissue was homogenized with 1 mL 84% ice-cold methanol, using a bead homogenizer (Omni International Inc., Kennesaw, GA). Homogenized tissue samples were left overnight at −20 °C. The following day, samples were centrifuged for 5 min and the supernatant was collected and diluted in 10 mL deionized water. For T measurement in serum, 40 μL of serum was diluted in 10 mL deionized water. Steroids were extracted from the samples using solid phase extraction (SPE) with C18 columns (Agilent Bond Elut LRC-C18 OH, #12113045) mounted on a glass vacuum manifold (UCT #VMF024GL). After column conditioning and equilibration, samples were loaded onto the SPE columns. Then, samples were washed with 10 mL of 40% HPLC-grade methanol (Taves et al., 2010), steroids were eluted from columns with 5 mL of 90% HPLC-grade methanol, and eluates were dried in a vacuum centrifuge at 40 °C. T was measured using a sensitive and specific commercial RIA kit (#07189102; MP Biomedicals, Solon, OH), with modifications described by Overk et al. (2013) and Ferris et al. (2015). Dried eluates were re-suspended in 100% ethanol, and then diluted with the buffer provided in RIA kit. The amount of ethanol in samples did not exceed 1%. From the original samples, a portion (90% for the serum, and 75% for the brain) was used for T measurement. Serum samples were run in duplicate, and brain samples were run as singletons. 200 μL of primary antibody was added to the re-suspended sample and standards, and
2.4. Immunohistochemistry for AR Immunohistochemistry for AR was performed as described in detail by Low et al. (2017), using free-floating tissue sections in 24-well plates, with each plate containing tissue from one young and one aged rat. Sections were washed in 0.1 M Tris-buffered saline (TBS; pH 7.4), and then incubated in 2% hydrogen peroxide (H2O2) for 30 min. Sections were then washed in 0.1 M TBS and then in 0.1 M PBS with 0.03% Triton X-100 and 0.1% gelatin (PBS-GT). Next, sections were incubated in 8% tryptone (J859; Amresco, Inc.) in PBS for 2 h to block nonspecific reactivity. Directly following the block, sections were incubated in primary antibody solution (1:200 in PBS-GT) for 24 h at room temperature. The primary antibody was a monoclonal anti-AR antibody [EPR1535(2)] raised in rabbit (ab133273; Abcam, Inc.) and used at a concentration of [1:200]. This primary antibody has been extensively validated (Hamson et al., 2013; Low et al., 2017). Then sections were washed in PBS-GT and incubated in biotin-SP-conjugated affinity-purified donkey anti-rabbit secondary antibody (1:2000 in 0.8% tryptone 3
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circularity and size were uniformly applied.
in PBS; 711-065-152; Jackson ImmunoResearch Laboratories, RRID: AB_2340593) for 1 h at room temperature and then overnight at 4 °C. Sections were washed in PBS-GT, incubated in avidin-biotin peroxidase (AB) (in 0.8% tryptone in PBS; VectaStain ABC Kit; PK-6100 Standard) for 30 min at room temperature, and washed in PBS-GT. Signal was amplified by incubation in biotinylated tyramide for 10 min (Adams, 1992). Sections were washed in PBS-GT, incubated in AB solution for 30 min, and washed in PBS-GT. Sections were washed quickly in TBS, and then were incubated with 3,3′-Diaminobenzidine and nickel (Vector Labs Peroxidase Substrate Kit SK-4100) for 20 min at room temperature. Lastly, sections were mounted onto charged slides, dehydrated, and cover-slipped.
2.6. Statistical analysis The effect of Age on T levels was analyzed using one-way ANOVAs, and data were log-transformed prior to analyses to reduce heteroscedasticity. The ratio of Brain T: Serum T (T-ratio) was calculated by dividing the T level in the brain region by the serum T level, for qualitative analysis only. The effect of Age on AR-ir was analyzed using one-way ANOVAs. Effect size (Cohen's d) was also calculated for T and AR-ir comparisons to assess the standardized difference between the young and aged group means. Pearson's correlations were used to assess the relationship between AR-ir and brain T. Correlations were assessed separately for young rats and aged rats. For T analyses, we could not subdivide the mPFC, OFC and NAc, and thus for this correlation, we did not subdivide these regions with respect to AR-ir. Analyses were conducted using SPSS Statistics software (IBM, Armonk, NY) ver. 23.0 (MacOSX) and ver. 24.0 (Windows) and R ver. 3.5.1 (2018-07-02, “Feather Spray”).
2.5. Quantification of AR immunoreactivity AR-ir was assessed in regions of the mesocorticolimbic system (Fig. 2). The mesolimbic regions were: (1) the VTA, for which we focused on the parabrachial pigmented (PBP) and paranigral (PN) nuclei, (2) the NAc core (NAcC), and (3) the NAc shell (NAcS). The prefrontal cortical regions were: (4) the prelimbic portion (PrL) of the mPFC, (5) the infralimbic (IL) portion of the mPFC; (6) the lateral orbitofrontal cortex (LOFC), and (7) the medial orbitofrontal cortex (MOFC). The regions were identified by major neuroanatomical landmarks (Paxinos and Watson, 2009). For each brain region, photomicrographs were captured using a Nikon Digital Sight DS-U1 camera and Nikon Eclipse 90i microscope (at the 20× objective) interfaced with NIS-Elements Basic Research software. Images were taken from two to five serial sections depending on the region (Table 1). In some cases, individual sections were damaged, and these were excluded. All images were captured at identical brightness and contrast settings with the exception of the NAc, for which brightness and contrast were increased consistently across all subjects. All images were captured at maximum resolution (2560 pixels × 1920 pixels). Importantly, slides were coded, and all photomicroscopy and image analysis was conducted by an experimenter (KLL) who was blind to the age of each subject. Images were quantified in a manner similar to Heimovics et al. (2012). For each image, a background mean intensity value was calculated by averaging six intensity measurements taken from locations within each brain area where specific staining was not present. Next, the background intensity value for each image was used to generate a quantification threshold value, either 1.25× or 1.5× the background, depending on the region. The selected threshold value (1.25× or 1.5×) was used uniformly for all subjects for a given region. Once thresholds were calculated for all images, they were applied to a rectangular region of interest (ROI) with area 0.135 mm2 for each brain region. In NIS-Elements, the ROI was superimposed onto each photomicrograph within the limits of each brain region, to measure the percentage of the ROI that was above the threshold (% AR-ir). Restrictions on object
3. Results 3.1. Serum and brain T levels In serum and all brain regions – except the VTA – there was a significant main effect of Age on T levels (Fig. 3). In particular, T levels were significantly lower in aged rats than in young rats in the serum (F1,22 = 12.24, p = 0.002; d = −1.15), NAc (F1,20 = 7.84, p = 0.011; d = −1.11), mPFC (F1,21 = 5.57, p = 0.028; d = −0.92), and OFC (F1,22 = 7.38, p = 0.013; d = −1.03). In contrast, the effect of Age was not significant in the VTA (F1,17 = 1.87, p = 0.190; d = −0.60). In young and aged animals, the T-ratio was > 1 in all brain regions, indicating that T levels were higher in the brain than in the serum. 3.2. AR immunoreactivity The overall distribution of AR-ir was similar to previous studies (Simerly et al., 1990; Sar et al., 1990). In the VTA (Fig. 4), there were few (but strongly) labeled cells, primarily in the PBP and PN subregions, as described by Kritzer (1997). In the PFC (Fig. 5), AR-ir was observed in a bilaminar pattern, as before (Low et al., 2017). In mesolimbic brain regions (VTA and NAc), there were no significant main effects of Age on AR-ir (Figs. 4, 6A). Regions examined included the VTA (F1,16 = 0.70, p = 0.417; d = 0.40), NAcC (F1,20 = 1.97, p = 0.176; d = 0.60) and NAcS (F1,18 = 0.18, p = 0.675; d = 0.19). In contrast, in prefrontal cortical brain regions, there were significant effects of Age on AR-ir (Figs. 5, 6B). Aged rats had
Table 1 Number of photomicrographs analyzed per region per subject for quantification of androgen receptor immunoreactivity. Region
Number of brain sections examined
Number of photos taken per section
Total number of photos analyzed
VTA NAcC NAcS PrL IL LOFC MOFC
3 5 5 4 4 5 2
2 1 1 1 1 1 1
6 5 5 4 4 5 2
Fig. 3. Testosterone (T) levels in young (5 months) and aged (22 months) male rats. T levels were significantly lower in aged rats than young rats, except in the VTA. n = 9–12 (young), 10–12 (aged). *p ≤ 0.05, **p ≤ 0.01. Values are reported as mean ± SEM. Abbreviations: VTA, ventral tegmental area; NAc, nucleus accumbens; mPFC, medial prefrontal cortex; OFC, orbitofrontal cortex.
VTA, ventral tegmental area; NAcC, nucleus accumbens core; NAcS, nucleus accumbens shell; PrL, prelimbic region of the medial prefrontal cortex; IL, infralimbic region of the prefrontal cortex; LOFC, lateral orbitofrontal cortex; MOFC, medial orbitofrontal cortex. 4
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Fig. 4. Representative photomicrographs of AR-immunoreactivity (AR-ir) from young (5 months; A, C, and E) and aged (22 months; B, D, and F) male rats in mesolimbic regions. (A–B) VTA, ventral tegmental area; (C–D) NAcC, nucleus accumbens core; (E–F) NAcS, nucleus accumbens shell. Scale bars represent 100 μm. All images were taken at the same magnification.
age, except in the VTA. Brain T levels were higher than serum T levels in both ages. The AR-ir in the VTA, NAc and OFC did not change with age. In contrast, AR-ir in the PFC (mPFC-PrL and mPFC-IL) decreased with age.
significantly lower AR-ir than young rats in the mPFC, including the PrL (F1,20 = 6.25; p = 0.021; d = −1.07) and IL (F1,19 = 9.11, p = 0.007; d = −1.31). However, the effect of Age was not significant in the LOFC (F1,20 = 2.41; p = 0.136; d = −0.66) or MOFC (F1,19 = 1.34; p = 0.261; d = −0.51).
4.1. Age-related changes in testosterone levels
3.3. Correlations between neural T and AR immunoreactivity
Age-related declines in serum T occur in males of several species, including humans (Feldman et al., 2002), rhesus monkeys (Downs and Urbanski, 2006), mice (Flood et al., 1995), and multiple strains of rats (Roselli et al., 1986; Wu et al., 2009; Ghanadian et al., 1975; Bernardi et al., 1998; Gruenewald et al., 1994; Rosario et al., 2009; Chambers et al., 1991; McGinnis and Yu, 1995; Luine et al., 2007). Serum T levels in male rats vary with strain, sexual experience, and time of day, with levels of 1–10 ng/mL in young rats (Wu and Gore, 2010) and 0.2–1.5 ng/mL in aged rats (Ghanadian et al., 1975; Chambers et al., 1991). The serum T levels measured here are consistent with these ranges. In addition, we measured T in microdissected regions of the mesocorticolimbic system. Brain T levels declined with age in the NAc, mPFC and OFC but not in the VTA. The ratio of brain T to serum T was > 1 in all regions in both young and aged rats. These data are consistent with our previous study reporting steroidogenic enzyme expression in the mesocorticolimbic system, higher T levels in the mesocorticolimbic
In young rats, neural T and AR-ir were significantly positively correlated in the VTA (r = 0.70, p = 0.05). However, the correlations were not statistically significant in the NAc (r = −0.52, p = 0.12), mPFC (r = 0.46, p = 0.15), and OFC (r = 0.21, p = 0.54). In aged rats, correlations between neural T and AR-ir were not statistically significant in any region: VTA (r = −0.002, p = 0.97), NAc (r = 0.06, p = 0.90), mPFC (r = 0.41, p = 0.28), and OFC (r = 0.48, p = 0.16). 4. Discussion This study measured serum T levels, neural T levels, and neural ARir in young adult (5 months) and aged (22 months) F344/BN male rats. Androgens influence executive functions via effects on the mesocorticolimbic system (Kritzer et al., 2007; Tobiansky et al., 2018b), so we were interested in T and AR in the VTA, NAc, mPFC and OFC. Serum T levels declined with age, as expected. Brain T levels also declined with 5
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Fig. 5. Representative photomicrographs of AR-immunoreactivity (AR-ir) from young (5 months; A, C, E, and G) and aged (22 months; B, D, F, and H) male rats in prefrontal cortical regions. (A–B) PrL, prelimbic subregion of the medial prefrontal cortex; (C–D) IL, infralimbic subregion of the medial prefrontal cortex; (E–F) LOFC, lateral orbitofrontal cortex; (G–H) MOFC, medial orbitofrontal cortex. Scale bars represent 100 μm. All images were taken at the same magnification.
4.2. Age-related changes in AR immunoreactivity
et al., 2003). Age-related changes in neural AR have been reported in rodents (Haji et al., 1981; Chambers et al., 1991; McGinnis and Yu, 1995; Kerr et al., 1995; Xiao and Jordan, 2002; Wu et al., 2009; Munetomo et al., 2015), and the effects of aging are region-specific (Wu et al., 2009). Although previous work focused on the hypothalamus, amygdala, and hippocampus (Wood, 2008; Wu et al., 2009), the present study is distinguished in that it examined the effects of age on AR in the mesocorticolimbic system.
AR staining was consistent with previous reports of predominantly nuclear immunoreactivity in rodent brain (Lu et al., 1998; Zhou et al., 1994; Fernandez-Guasti et al., 2003), although AR-ir is also present at extranuclear sites (Tabori et al., 2005; Sarkey et al., 2008; DonCarlos
4.2.1. Mesolimbic AR immunoreactivity The VTA contains dopaminergic neurons that project to the PFC and NAc (Ikemoto, 2007), and AR mRNA and protein are present in this
system than in the blood of young adult rats, and the presence of T in the mesocorticolimbic system of long-term gonadectomized rats (Tobiansky et al., 2018a). Future studies should examine age-related changes in steroidogenic enzyme mRNA and activity in the mesocorticolimbic system.
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report an age-associated decrease (Munetomo et al., 2015) or no change (Kerr et al., 1995; Kumar and Thakur, 2004). To our knowledge, the present results are the first to characterize age-related changes in AR specifically in the PFC. AR-ir in the PrL and IL subregions of the mPFC decreased with age. In contrast, AR-ir in the LOFC and MOFC did not significantly change with age. The decreased mPFC AR-ir in aged rats is unlikely to be fully accounted for by age-related cell loss. In one study, during normal aging, the mPFC does not show loss of neurons (Finch, 2003; Zamzow et al., 2014). In other studies, there is some neuron loss in the PFC of the rat (Yates et al., 2008) and monkey (Smith et al., 2004), but the losses are subregion-specific and are not substantial enough to fully explain the present results. Moreover, previous work has shown an agerelated decrease in brain AR binding without accompanying loss of neurons (McGinnis and Yu, 1995). Collectively, these data show that age-associated reductions in AR-ir within the frontal lobes show some anatomical specificity, with medial (cingulate) regions being more affected than orbital regions of the PFC. 4.3. Androgen regulation of AR AR is generally thought to be an auto-regulated protein (Lu et al., 1998) and neural AR-ir is regulated by circulating T levels (FernandezGuasti et al., 2003). Castration strongly reduces neural AR-ir levels, and AR-ir is restored with androgen replacement (Menard and Harlan, 1993; Lu et al., 1998; Xiao and Jordan, 2002; Fernandez-Guasti et al., 2003). Supraphysiological doses of androgens increase AR-ir in the brain (Wood and Newman, 1993; Lu et al., 1998; Fernandez-Guasti et al., 2003). Androgen manipulations can also affect intracellular partitioning of AR (Wood and Newman, 1993). Consequently, decreases in systemic T levels may be expected to down-regulate AR, although this may be region-specific. The decreased AR-ir in the mPFC in aged animals along with decreased circulating T is consistent with this autoregulation model. Moreover, the positive correlation between local T levels and AR-ir in the mPFC suggests that, in addition to systemic T, local T levels also regulate AR. Our results show that ligand and receptor concentrations do not always change in parallel in aging. While the mPFC had both decreased T and AR-ir, in contrast the NAc and OFC had decreased T and no change in AR-ir. These data suggest that AR is regulated differently across brain regions. Our analysis is limited to the nuclear AR. Androgens could also regulate neural activity through membrane AR. A membrane AR has been characterized (Thomas et al., 2014), but its distribution in the brain is not known. Our results are also limited to changes in the amount of AR-ir and not necessarily AR binding or function.
Fig. 6. Mesolimbic and prefrontal cortical AR immunoreactivity (AR-ir) in young (5 months old) and aged (22 months old) male rats. (A) There were no significant effects of Age on AR-ir in the ventral tegmental area (VTA), nucleus accumbens core (NAcC), or nucleus accumbens shell (NAcS). n = 8–11 (young), 5–11 (aged). (B) AR-ir levels were significantly lower in aged rats compared to young rats in the prelimbic (PrL) and infralimbic (IL) subregions of the medial prefrontal cortex (mPFC). In the lateral (LOFC) and medial (MOFC) subregions of the orbitofrontal cortex (OFC), the effect of age was not significant. n = 11 (young), 10–11 (aged). *p ≤ 0.05, **p ≤ 0.01.
nucleus (Simerly et al., 1990; Kritzer, 1997; Tobiansky et al., 2018b). Moreover, some VTA dopaminergic neurons express AR and thus are direct targets of androgens (Kritzer, 1997). We observed sparsely distributed but strongly labeled AR-ir cells in the VTA, with the majority of AR+ cells in the PBP and PN. Yet AR-ir did not significantly change with age in the VTA. This lack of an effect of age is in keeping with a report by Menard and Harlan (1993) that examined the effects of castration and androgen replacement on the number of AR-ir cells in male rats. There were no effects of androgen manipulations on AR-ir in the VTA, while AR-ir in other regions was affected. The NAc is a key subcortical projection target of VTA dopaminergic neurons. In the NAc, previous studies have reported that AR-ir is sparse (Wood, 2008; Sato et al., 2010, but see Fernandez-Guasti et al., 2003). Here, using a very sensitive protocol that minimizes background staining (Low et al., 2017), we clearly detected AR-ir in the NAc, especially in the NAcS. Yet, as with the VTA, AR-ir in the NAc was not affected by age.
4.4. Implications for aging and executive functions Androgen modulation of mesocorticolimbic dopamine signaling and PFC-dependent executive function is supported by both behavioral and neurochemical studies (Aubele and Kritzer, 2011; Tobiansky et al., 2018b). First, behavioral studies have shown that gonadectomy impairs and, in some cases, T administration restores performance on working memory tasks (Kritzer et al., 2001; Bimonte-Nelson et al., 2003; Aubele et al., 2008; Sandstrom et al., 2006; Spritzer et al., 2008). In addition, androgen manipulation regulates behavioral flexibility through changes in perseverative behaviors (Andrew and Rogers, 1972; Archer, 1977; Thompson and Wright, 1979; van Hest et al., 1989; Neese and Schantz, 2012; Wallin and Wood, 2015). Androgens also influence cost/benefit decision-making by altering the discounting of a large reward (versus a small reward) when the effort or delay required to obtain the large reward is varied (Wallin et al., 2015). Most of these behavioral studies focus on young adult animals, and it remains unclear whether similar effects are present in aged animals. Second, neurochemical studies have shown that gonadectomy alters
4.2.2. Prefrontal cortical AR immunoreactivity There is increasing appreciation for the large number of AR+ cells (primarily neurons) in the cerebral cortex (DonCarlos et al., 2006; Low et al., 2017; Tobiansky et al., 2018b). AR+ cells are present in the PFC of primates (Finley and Kritzer, 1999) and rats (Kritzer, 2004). Some studies have examined AR mRNA in the entire cerebral cortex and 7
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immunoreactivity of tyrosine hydroxylase (TH), a catecholamine-synthesizing enzyme, in the PFC (Adler et al., 1999; Kritzer, 2000) and increases extracellular dopamine levels in the PFC (Aubele and Kritzer, 2010). Normal dopamine neurotransmission is critical to maintain executive functions (Floresco and Magyar, 2006). The present results provide additional support for the notion that androgens modulate executive functioning by acting on specific nodes within the mesocorticolimbic system. We have previously observed agerelated changes in executive function (Tomm et al., 2018), as well as markers of androgen signaling in the mesocorticolimbic system. The age-related decreases in neural T levels and mPFC AR-ir might impact TH, dopamine synthesis and executive function (Tomm et al., 2018). It remains unclear whether androgens also influence executive functions through indirect effects on regions that project to the PFC.
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5. Conclusions We demonstrate age-related changes in T and AR in male rats. Compared to young rats (5 months old), aged rats (22 months old) have lower T levels in the serum, NAc, mPFC, and OFC, but not in the VTA. At both ages, T levels are higher in the mesocorticolimbic system than in the serum. The AR-ir in the mPFC decreases with age. Taken together, these results demonstrate age-associated changes in androgen signaling and suggest that changes in T and AR in the mesocorticolimbic system may contribute to age-related changes in executive function. Declaration of competing interest The authors have no conflicts of interest to declare. Acknowledgements The authors gratefully acknowledge funding from the Canadian Institutes of Health Research (Operating Grant 133606 to K.K.S); Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery Grant (to S.B.F.); the Michael J. Quinn Award and UBC Science Undergraduate Research Experience Award (to K.L.L); the NSERC USRA, UBC Aboriginal Graduate Fellowship, UBC 4 Year Fellowship and NSERC PGS-D Fellowship (to R.J.T.); and the Bluma Tischler Post-Doctoral Fellowship (to D.J.T.). The authors thank Maric Tse, Anastasia Korol, Sarah Marjanovic, and Madison Grist for assistance with data collection. References Adams, J.C., 1992. Biotin amplification of biotin and horseradish peroxidase signals in histochemical stains. J. Histochem. Cytochem. 40, 1457–1463. Adler, A., Vescovo, P., Robinson, J.K., Kritzer, M.F., 1999. Gonadectomy in adult life increases tyrosine hydroxylase immunoreactivity in the prefrontal cortex and decreases open field activity in male rats. Neuroscience 89, 939–954. Alexander, G.E., Ryan, L., Bowers, D., Foster, T.C., Bizon, J.L., Geldmacher, D.S., Glisky, E.L., 2012. Characterizing cognitive aging in humans with links to animal models. Front. Aging Neurosci. 4. Andrew, R.J., Rogers, L.J., 1972. Testosterone, search behaviour and persistence. Nature 237, 343. Archer, J., 1977. Testosterone and persistence in mice. Anim. Behav. 25, 479–488. Aubele, T., Kritzer, M.F., 2010. Gonadectomy and hormone replacement affects in vivo basal extracellular dopamine levels in the prefrontal cortex but not motor cortex of adult male rats. Cereb. Cortex 21, 222–232. Aubele, T., Kritzer, M.F., 2011. Androgen influence on prefrontal dopamine systems in adult male rats: localization of cognate intracellular receptors in medial prefrontal projections to the ventral tegmental area and effects of gonadectomy and hormone replacement on glutamate-stimulated extracellular dopamine level. Cereb. Cortex 22, 1799–1812. Aubele, T., Kaufman, R., Montalmant, F., Kritzer, M.F., 2008. Effects of gonadectomy and hormone replacement on a spontaneous novel object recognition task in adult male rats. Horm. Behav. 54, 244–252. Beas, B.S., Setlow, B., Bizon, J.L., 2013. Distinct manifestations of executive dysfunction in aged rats. Neurobiol. Aging 34, 2164–2174. Bernardi, F., Salvestroni, C., Casarosa, E., Nappi, R.E., Lanzone, A., Luisi, S., Purdy, R.H.,
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