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Journal of Cerebral Blood Flow & Metabolism logoLink to Journal of Cerebral Blood Flow & Metabolism
. 2015 Jun 10;35(11):1812–1818. doi: 10.1038/jcbfm.2015.129

Lack of age-dependent decrease in dopamine D3 receptor availability: a [11C]-(+)-PHNO and [11C]-raclopride positron emission tomography study

Shinichiro Nakajima 1,2,3,4, Fernando Caravaggio 1,5, Isabelle Boileau 3,6, Jun K Chung 1,5, Eric Plitman 1,5, Philip Gerretsen 1,2,3,5, Alan A Wilson 3,5,6, Sylvain Houle 3,6, David C Mamo 1,2,3,7, Ariel Graff-Guerrero 1,2,3,6,*
PMCID: PMC4635236  PMID: 26058690

Abstract

Positron emission tomography with antagonist radiotracers has showed that striatal dopamine D2/3 receptor (D2/3R) availability decreases with age. However, no study has specifically assessed whether D2/3R availability decreases with age in healthy persons as measured with agonist radiotracers. Moreover, it is unknown whether D3R availability changes with age in healthy humans. Thus, we explored the relationship between age and D2/3R availability in healthy humans using the D3 receptor (D3R)-preferential agonist radiotracer [11C]-(+)-PHNO (n=72, mean±s.d. age=40±15, range=18 to 73) and the antagonist [11C]-Raclopride (n=70, mean±s.d. age =40±14, range=18 to 73) (both, n=33). The contribution of D3R to the [11C]-(+)-PHNO signal varies across regions of interest; the substantia nigra and hypothalamus represent D3R-specific regions, the ventral pallidum, globus pallidus, and ventral striatum represent D2/3R-mixed regions, and the caudate and putamen represent D2 receptor (D2R)-specific regions. With [11C]-(+)-PHNO, a negative correlation was observed between age and nondisplaceable binding potential (BPND) in the caudate (r(70)=−0.32, P=0.005). No correlations were observed in the other regions. With [11C]-Raclopride, negative correlations were observed between age and BPND in the caudate (r(68)=−0.50, P<0.001), putamen (r(68)=−0.41, P<0.001), and ventral striatum (r(68)=−0.43, P<0.001). In conclusion, in contrast with the age-dependent decrease in D2R availability, these findings suggest that D3R availability does not change with age.

Keywords: aging, dopamine, D2 receptor, D3 receptor, positron emission tomography, [11C]-(+)-PHNO, [11C]-Raclopride

Introduction

The dopamine (DA) system in healthy individuals is generally thought to decline with age. Postmortem autoradiography and in vivo neuroimaging studies, using positron emission tomography (PET) and single-photon emission computed tomography (SPECT), suggest an age-dependent decline in the DA system. Postmortem1, 2 and [11C]-SCH23390 PET3 studies indicate age-related decreases in DA D1 receptors in the frontal 1 and occipital 3 cortices, striatum (caudate2 and putamen2), globus pallidus,2 and substantia nigra.2

Postmortem4 and in vivo studies employing [11C]-Raclopride,5, 6 [11C]NMSP,5, 7 and [11C]-FLB4578, 9 PET, and [123I]-iodobenzofuran SPECT10 indicate an age-related decrease in D2/3 receptors in the frontal, temporal, parietal, occipital, and anterior cingulate cortices,8, 9 striatum5 (caudate7, 10 and putamen10), substantia nigra,4 hippocampus,8, 9 amygdala,8 and thalamus.8, 9 Both D1 and D2/3 receptors decrease from early to late adulthood with an estimated decline of 3.2% to 8.6% and 2.2% to 13.8% per decade, respectively.11 Dopamine transporters also decline with age; postmortem12 and in vivo studies, employing [11C]-β-CFT13 and D-threo-[11C]-methylphenidate6 PET and [123I]beta-CIT SPECT,14 indicate an age-related loss between 4.4% and 11.2% per decade of the DA transporter in the striatum6, 14 (caudate13 and putamen13) and substantia nigra.12 Moreover, postmortem15 and [18F]-FDOPA PET16 studies have shown that the activity of DOPA-decarboxylase, which is the enzyme responsible for nigrostriatal DA synthesis, decreases with age. The mechanisms underlying the decrease of these DA system elements have been attributed to age-related alterations in neuronal number, synapses, and protein concentrations.11

A few studies in rodents have investigated whether there are age-related changes in DA D3 receptor expression. The results, however, are inconclusive. Wallace et al17 showed that D3 receptor density measured by [3H]-(+)-7-OH-DPAT is increased by 102% and 29% in the striatum and nucleus accumbens, respectively, in 37-month-old rats, in comparison with 4-month-old rats. In contrast, Ricci et al18 showed that D3 receptor density measured by [3H]-spiroperidol decreased with age without any change in the affinity for this radiotracer in the rat cerebellar cortex. Thus, the age-dependent change in D3 receptors warrants further investigation.

Our group has developed the PET radiotracer [11C]-(+)4-Propyl-3,4,4a,5,6,10b-hexahydro-2H-naphtho[1,2-b][1,4]oxazin-9-ol (henceforth, [11C]-(+)-PHNO). [11C]-(+)-PHNO is a DA agonist radiotracer exhibiting preferential selectivity in vitro and in vivo for D3 over D2 receptors.19 Previous occupancy studies with [11C]-(+)-PHNO employing D3 receptor antagonists in the human brain indicate that the fraction of the [11C]-(+)-PHNO signal attributable to D3 receptors varies among different brain regions: a high fraction in the substantia nigra (100%), hypothalamus (100%), ventral pallidum (75%), and globus pallidus (65% to 90%), a lower fraction in the ventral striatum (30% to 62%), and a negligible D3 contribution in the dorsal caudate and putamen.19 Therefore, [11C]-(+)-PHNO is a suitable PET radiotracer for the quantification of D3 receptors in the substantia nigra, hypothalamus, ventral pallidum, and globus pallidus, and is also a preferential D2 receptor PET radiotracer in the dorsal caudate and putamen.19

To date, there is no in vivo study specifically investigating the effects of age on D3 receptor availability in healthy humans. Thus far, the quantification of age-related changes in the D2/3 receptor availability in humans has been performed using radiotracers unable to differentiate between D2 and D3 receptors, such as [11C]-Raclopride, [11C]NMSP, and [11C]-FLB457.5, 6, 8, 9 In the present study, we explored the effects of age on [11C]-(+)-PHNO and [11C]-Raclopride nondisplaceable binding potential (BPND) in healthy individuals using PET. The primary objective was to investigate the effects of normal aging on the D3 receptor availability. We also sought to replicate previous findings that D2/3 receptor availability decreases with age in the striatum using the antagonist radiotracer [11C]-Raclopride.

Materials and methods

Subjects

All procedures were approved by the Centre for Addiction and Mental Health Research Ethics Board and complied with the 1975 Helsinki Declaration (5th revision, 2000). The sample was composed of 112 healthy volunteers ([11C]-(+)-PHNO, n=72; [11C]-Raclopride, n=70; and both, n=33). The participants were right-handed and free of any major medical or psychiatric disorder as determined by clinical interview, the Mini-International Neuropsychiatric Interview, and electrocardiography. Participants were required to have a negative urine screen for drugs of abuse and/or pregnancy at inclusion and before the PET scan. The sample analyzed for the current study was collected by our laboratory from various PET studies that were approved by the Research Ethics Board of the Centre for Addiction and Mental Health, Toronto. All participants provided written informed consent.

Positron Emission Tomography Imaging with [11C]-(+)-PHNO and [11C]-Raclopride

The radiosynthesis of [11C]-(+)-PHNO and [11C]-Raclopride, along with the acquisition of PET images have been described in detail elsewhere.19 Briefly, images were acquired with the use of a high-resolution, head-dedicated PET camera system (CPS-HRRT; Siemens Molecular Imaging, Munich, Germany), which measures radioactivity in 207 brain slices with a thickness of 1.2 mm each. The in-plane resolution was ∼2.8 mm full-width at half-maximum. Transmission scans were acquired using a 137Cs (T1/2= 30.2 years, energy=662 KeV) single-photon point source to provide attenuation correction, and the emission data were acquired in list mode. The raw data were reconstructed by filtered back-projection. [11C]-(+)-PHNO scanning data were acquired for 90 minutes after injection. Once scanning was complete, the data were redefined into 30 frames (1 to 15 of 1-minute duration and 16 to 30 of 5-minute duration). The mean±standard deviation (s.d.) radioactivity dose of [11C]-(+)-PHNO was 9.1±1.5 mCi, with a specific activity of 1,088.1±389.1 mCi/μmoL and an injected mass of 2.1±0.4 μg. No correlation was found between the injected mass of [11C]-(+)-PHNO and age (r(70)=0.02, P=0.88) (Supplementary Figure). [11C]-Raclopride data were acquired for 60 minutes and redefined into 28 frames (1 to 5 of 1-minute duration, 6 to 25 of 2-minute duration, and 26 to 28 of 5-minute duration). The mean±s.d. radioactivity dose of [11C]-raclopride was 9.7±1.1 mCi, with a specific activity of 1,271.1±621.2 mCi/μmoL and an injected mass of 3.7±2.1 μg. Participants also provided a structural magnetic resonance image (MRI), acquired on a 1.5 -T Signa-GE scanner. These images were used for the analysis of the PET scans. The PET scans were collected over several years (2004 to 2012). Controlling for the year and the month when scans were acquired did not significantly affect any of the reported results (data not shown).

Image Analysis

The region of interest (ROI)-based analysis for [11C]-(+)-PHNO and [11C]-Raclopride has been described in detail elsewhere.20 Briefly, time-activity curves from ROIs were obtained from the dynamic PET images in native space with reference to each subject's coregistered MRI. The coregistration of each subject's MRI to PET space was performed with the use of the normalized mutual information algorithm, as implemented in SPM2 (SPM2, The Wellcome Department of Cognitive Neurology, London; http://www.fil.ion.ucl.ac.uk/spm).21 The time-activity curves were analyzed by means of the SRTM (Simplified Reference Tissue Method), with the cerebellum, excluding vermix, and midline structures, used as the reference region, to derive a quantitative estimate of binding: BPND.22 The basis function implementation of the SRTM was applied to the dynamic PET images to generate parametric voxelwise BPND maps by means of PMOD (v2.7; PMOD Technologies, Zurich, Switzerland).23 These images were spatially normalized into MNI (Montreal Neurological Institute) brain space by nearest neighbor interpolation with a voxel size fixed in 2 × 2 × 2 mm3 by means of SPM2. Regional BPND estimates were then derived from ROIs defined in MNI space. The ventral striatum and dorsal striatum (dorsal caudate, hereafter caudate; dorsal putamen, hereafter putamen) were defined according with Mawlawi et al.24 The globus pallidus, ventral pallidum, and hypothalamus ROIs were defined according to the criteria of Tziortzi et al.25 The definition was made over the participant's MRI slices oriented in the coronal plane. For [11C]-Raclopride scans, BPND in the substantia nigra ROI was not obtainable because binding in this region falls within noise levels.20

Statistical Analysis

The analysis was performed using the Statistical Program for the Social Sciences (version 12.0; SPSS, Chicago, IL, USA). Nondisplaceable binding potential is presented as mean±s.d. Correlation analyses were performed to determine the association between age and BPND with each radiotracer in each ROI. Partial correlation analyses were performed to determine the association between age and BPND with each radiotracer in each ROI, controlling for gender or gray matter volume (GMV).

Results

Data from 112 healthy volunteers was analyzed. Data from these participants have been previously reported.26, 27, 28 Seventy-two subjects (26 females; 39.6±15.3 (18 to 73) years old) were scanned with [11C]-(+)-PHNO, while 70 subjects (25 females; 39.7±14.1 (18 to 73) years old) were scanned with [11C]-Raclopride. The number of subjects within each age range in the [11C]-(+)-PHNO group was 5 for 11 to 20 years, 21 for 21 to 30 years, 19 for 31 to 40 years, 10 for 41 to 50 years, 6 for 51 to 60 years, 8 for 61 to 70 years, and 3 for 71 to 80 years. The number of subjects within each age range in the [11C]-Raclopride group was 6 for 11 to 20 years, 13 for 21 to 30 years, 23 for 31 to 40 years, 12 for 41 to 50 years, 8 for 51 to 60 years, 6 for 61 to 70 years, and 2 for 71 to 80 years. Thirty-three subjects were scanned with both radiotracers. Seventeen subjects were first scanned with [11C]-(+)-PHN and sixteen with [11C]-Raclopride. Therefore, the scanning order between the radiotracers was counter-balanced (11 female, age: 43±17 years old). The time between scan acquisition ranged from 1 to 6 days (24 subjects) to 16 to 121 days (9 subjects). No subject was scanned with both tracer in the same day to avoid any potential carry-over effect. Controlling for scan order did not significantly affect any of our reported results (data not shown). Among those who successfully underwent [11C]-(+)-PHNO scans, nobody experienced serious adverse events such as vomiting which required them to interrupt the scan.

The BPNDs of [11C]-(+)-PHNO and [11C]-Raclopride within each region are described in Table 1. Correlations between age and the BPNDs of [11C]-(+)-PHNO are summarized in Table 2 and Figure 1A negative correlation was found between age and [11C]-(+)-PHNO binding in the caudate (r(70)=−0.32, P=0.005). No correlations were found between age and [11C]-(+)-PHNO binding in the putamen, ventral striatum, substantia nigra, hypothalamus, ventral pallidum, or globus pallidus (r(70)=−0.19, P=0.12; r(70)=−0.23, P=0.05; r(70)=−0.00, P=0.99; r(58)=−0.10, P=0.45; r(58)=0.19, P=0.15; r(70)=−0.07, P=0.58, respectively). Controlling for gender, a negative correlation was found between age and [11C]-(+)-PHNO binding in the caudate (r(69)=−0.33, P=0.006). No correlations were found between age and [11C]-(+)-PHNO binding in the putamen, ventral striatum, substantia nigra, hypothalamus, ventral pallidum, or globus pallidus (r(69)=−0.19, P=0.12; r(69)=−0.23, P=0.06; r(69)=0.00, P=0.99; r(57)=−0.11, P=0.43; r(57)=0.23, P=0.08; r(69)=−0.07, P=0.58, respectively). There was no correlation between the injected mass of [11C]-(+)-PHNO and age (r(70)=−0.02, P=0.88), and controlling for mass injected did not significantly affect any of our reported results (data not shown).

Table 1. Age distribution and BPND of [11C]-(+)-PHNO and [11C]-Raclopride.

Range of age (years) 11–20 21–30 31–40 41–50 51–60 61–70 71–80 Total
Age distribution and BPND of [11C]-(+)-PHNO
 Substantia nigra 1.57±0.90 1.87±0.55 1.96±0.57 2.26±0.89 1.86±0.49 1.47±0.58 2.26±0.83 1.90±0.66
 Hypothalamus 2.15±0.70 2.98±1.43 2.13±1.41 2.91±2.12 1.97±1.10 2.42±0.76 2.13±0.54 2.50±1.36
 Ventral pallidum 3.13±0.77 4.22±1.79 3.73±1.96 4.07±1.31 5.46±5.08 5.98±5.12 4.76±2.75 4.22±2.45
 Globus pallidus 4.09±0.61 4.27±0.72 3.88±0.76 4.52±1.01 4.21±0.78 3.93±0.80 3.93±0.89 4.13±0.79
 Ventral striatum 3.56±0.34 3.68±0.41 3.39±0.63 3.46±0.84 3.29±0.48 3.23±0.21 3.32±0.72 3.47±0.55
 Caudate 2.27±0.37 2.35±0.50 1.98±0.33 2.14±0.46 2.06±0.33 1.90±0.44 1.78±0.44 2.12±0.44
 Putamen 3.08±0.30 3.11±0.49 2.79±0.36 2.76±0.52 3.02±0.57 2.81±0.29 2.84±0.10 2.92±0.44
                 
Age distribution and BPND of [11C]-Raclopride
 Globus pallidus 2.40±0.88 2.01±0.41 2.17±0.51 2.11±0.51 2.31±0.57 1.96±0.46 1.61±0.08 2.13±0.53
 Ventral striatum 3.76±0.58 3.55±0.45 3.53±0.48 3.11±0.37 3.24±0.53 2.96±0.52 2.72±0.24 3.37±0.52
 Caudate 3.75±0.40 3.75±0.73 3.44±0.68 3.00±0.45 3.20±0.42 2.74±0.44 2.48±0.34 3.33±0.67
 Putamen 4.83±0.51 4.68±0.71 4.47±0.65 4.02±0.59 4.18±0.46 3.87±0.43 4.00±0.50 4.36±0.65
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BPND, binding potential nondisplaceable. BPND is presented as mean±s.d.

Table 2. Correlations between age and BPND of [11C]-(+)-PHNO.

  Substantia nigra Hypothalamus Ventral pallidum Globus pallidus Ventral striatum Caudate Putamen
Correlations between age and BPND of [11C]-(+)-PHNO
 r 0.00 −0.10 0.19 −0.07 −0.23 −0.32 −0.19
 P-value 0.99 0.45 0.15 0.58 0.05 0.005 0.12
               
Partial correlations between age and BPND of [11C]-(+)-PHNO controlling for gender
 r 0.00 −0.11 0.23 −0.07 −0.23 −0.33 −0.19
 P-value 0.99 0.43 0.08 0.58 0.06 0.006 0.12
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BPND, binding potential nondisplaceable. Correlation analyses were performed to determine the association between age and BPND with [11C]-(+)-PHNO in each region, including the substantia nigra and hypothalamus, which represent specific D3 receptor regions, the globus pallidus and ventral pallidum, which represent preferential D3 receptor regions, and the caudate and putamen, which represent specific D2 receptor regions. Partial correlation analyses were performed to determine the association between age and BPND with [11C]-(+)-PHNO in each region, controlling for gender. The numbers of subjects were 72 for the substantia nigra, globus pallidus, ventral striatum, caudate, and putamen, and 60 for the hypothalamus and ventral pallidum. Bold characters=statistically significant.

Figure 1.

Figure 1

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Correlations between age and the BPNDs of [11C]-(+)-PHNO. Correlation analyses were performed to determine the association between age and BPND with each radiotracer in each region of interest (ROI). A negative correlation was found between age and [11C]-(+)-PHNO binding in the caudate (r(70)=−0.32, P=0.005). No correlations were found between age and [11C]-(+)-PHNO binding in the substantia nigra (SN), hypothalamus, ventral pallidum, or globus pallidus (r(70)=−0.00, P=0.99; r(58)=−0.10, P=0.45; r(58) =0.19, P=0.15; r(70)=−0.07, P=0.58, respectively). BPND, nondisplaceable binding potential; D2/3R, dopamine D2/3 receptors.

Correlations between age and the BPNDs of [11C]-Raclopride are summarized in Table 3 and Figure 2. Negative correlations were found between age and [11C]-Raclopride binding in the caudate, putamen, and ventral striatum (r(68)=−0.50, P<0.001; r(68)=−0.41, P<0.001; r(68)=−0.43, P<0.001, respectively). Controlling for gender, negative correlations were found between age and [11C]-Raclopride binding in the caudate, putamen, and ventral striatum (r(67)=−0.49, P<0.001; r(67)=−0.39, P<0.001; r(67)=−0.41, P<0.001, respectively).

Table 3. Correlations between age and BPND of [11C]-Raclopride.

  Globus pallidus Ventral striatum Caudate Putamen
Correlations between age and BPND of [11C]-Raclopride
 r −0.08 −0.43 −0.50 −0.41
 P-value 0.52 <0.001 <0.001 <0.001
         
Partial correlations between age and BPND of [11C]-Raclopride controlling for gender
 r −0.04 −0.41 −0.49 −0.39
 P-value 0.77 <0.001 <0.001 0.001
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BPND, binding potential nondisplaceable. Correlation analyses were performed to determine the association between age and BPND with [11C]-Raclopride in each region, including the substantia nigra and hypothalamus, which represent specific D3 receptor regions, the globus pallidus and ventral pallidum, which represent preferential D3 receptor regions, and the caudate and putamen, which represent specific D2 receptor regions. Partial correlation analyses were performed to determine the association between age and BPND with [11C]-Raclopride in each region, controlling for gender. The numbers of subjects were 70 for each region. Bold characters=statistically significant.

Figure 2.

Figure 2

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Correlations between age and the BPNDs of [11C]-Raclopride. Correlation analyses were performed to determine the association between age and BPND with each radiotracer in each region of interest (ROI). Negative correlations were found between age and [11C]-Raclopride binding in the caudate, putamen, and ventral striatum (r(68)=−0.50, P<0.001; r(68)=−0.41, P<0.001; r(68)=−0.43, P<0.001, respectively). BPND, nondisplaceable binding potential.

Since we did not control for menstrual cycle in women, we separately examined the relationship between age and BPND in only the male subjects. [11C]-(+)-PHNO BPND was negatively correlated with age in the caudate (r(44)=−0.47, P=0.001) and ventral striatum (r(44)=−0.37, P=0.01), there was a trend relationship in the putamen (r(44)=−0.25, P=0.09), and no significant relationship in the substantia nigra (r(44)=−0.11, P=0.46), globus pallidus (r(44)=−0.16, P=0.30), hypothalamus (r(35)=0.06, P=0.72), and ventral pallidum (r(35) =0.22, P=0.18). [11C]-Raclopride BPND was negatively correlated with age in the caudate (r(43)=−0.55, P<0.001), putamen (r(43)=−0.46, P=0.002), and ventral striatum (r(43)=−0.41, P =0.006), but not in the globus pallidus (r(43)=−0.17, P=0.26).

Within the subjects scanned with [11C]-(+)-PHNO, age was negatively correlated with GMV, which was extracted from each ROI using the DARTEL pipeline provided with SPM8, in the caudate (r(71)=−0.26, P=0.03), putamen (r(71)=−0.34, P=0.004), and substantia nigra (r(71)=−0.27, P=0.02), but not in the ventral striatum (r(71)=−0.20, P=0.09) or globus pallidus (r(71)=0.01, P=0.94). Within the subjects scanned with [11C]-Raclopride, age was negatively correlated with GMV in the caudate (r(55)=−0.57, P<0001) and ventral striatum (r(55)=−0.37, P=0.005), but not in putamen (r(55)=−0.21, P=0.12) or globus pallidus (r(55)=0.17, P=0.22). Controlling for GMV, age was negatively correlated with [11C]-(+)-PHNO BPND in the caudate (r(68)=−0.33, P=0.006) and ventral striatum (r(68)=−0.28, P=0.02), but not in the putamen (r(68)=−0.17, P=0.16), globus pallidus (r(68)=−0.07, P=0.58), or substantia nigra (r(68)=0.02, P=0.89). Relationships between age, GMV, and [11C]-(+)-PHNO BPND were depicted in Supplementary Figure. Controlling for GMV, age was negatively correlated with [11C]-Raclopride BPND in the caudate (r(52)=−0.35, P=0.009), putamen (r(52)=−0.36, P=0.008), and ventral striatum (r(52)=−0.34, P =0.01), but not in the globus pallidus (r(52)=−0.15, P=0.28). Overall, the relationships between age and [11C]-(+)-PHNO BPND, and age and [11C]-Raclopride BPND including GMV as covariate did not affect our results.

Discussion

To our knowledge, this is the first PET study examining the change in D3 receptor availability over the lifespan in healthy human brains in vivo using [11C]-(+)-PHNO. We showed that [11C]-(+)-PHNO binding did not change with age in D3 receptor-specific regions (the substantia nigra and hypothalamus). Although we did not observe correlations with age in the D3 receptor-rich mixed regions (the globus pallidus and ventral pallidum), we observed a trend for a decrease in BPND with age in the ventral striatum. Notably, for the D2R-specific regions, [11C]-(+)-PHNO BPND decreased with age in the caudate but not in the putamen. Replicating previous findings, we observed an age-related decline in [11C]-Raclopride BPND in the caudate and putamen. Thus, our results suggest that D3 receptor availability in healthy humans may not decrease with aging. Due to the lack of an age-dependent decrease in D3 receptor availability, our findings with [11C]-Raclopride, which cannot differentiate between D2 and D3 receptors, suggest that only D2 receptor availability (relative to D3 receptor availability) decreases with age in healthy humans.

Our results with [11C]-(+)-PHNO are contrasted by previous findings of age-related decrease in D2/3 receptor availability, shown with [11C]-Raclopride in our sample and in other samples using antagonist D2/3 radiotracers.5, 6, 8, 9 In humans in vivo, [11C]-(+)-PHNO has been shown to be more sensitive to fluctuations in endogenous DA levels than [11C]-Raclopride.28 This increased sensitivity may, at least in part, explain some of the null findings with [11C]-(+)-PHNO, particularly in the putamen. Specifically, larger decreases in endogenous DA than decreases in D2 receptor number may result in a null change in tracer binding by age. [11C]-(+)-PHNO also has preferential affinity for D3 over D2 receptors.20 The lack of age-related changes in [11C]-(+)-PHNO binding in D3 receptor-specific regions (the substantia nigra and hypothalamus) may indicate no change in D3 receptor expression with aging. One previous postmortem study reported an age-related decrease in [3H]-spiperone BPND in the caudate and substantia nigra.4 It was reported that there are no detectable DA axon terminals in the rodent substantia nigra and that there is no synaptic DA release into this region.29 Given that [3H]-spiperone cannot differentiate between D2 and D3 receptors, the age-related decrease in [3H]-spiperone BPND in the substantia nigra may also be attributable to a decrease in D3 receptors, not D2 receptors or synaptic DA. In combination with no age-related change in [11C]-(+)-PHNO BPND within this region in our study, these findings support the notion of no age-related change in D3 receptor availability in the substantia nigra in healthy humans. Furthermore, endogenous DA has been reported to decrease with age.30 Taking into consideration a potential decrease in the competition with endogenous DA, this finding may suggest an increase in the affinity or density of D3 receptors in hypothalamus associated with aging.

D3 receptors are associated with cognitive functioning in healthy individuals. For example, Cole et al31 has directly investigated whether differences in midbrain D3 receptor availability are associated with functional interactions between large-scale networks and brain regions involved in cognition in healthy individuals.31 Combining [11C]-(+)-PHNO PET and resting-state functional magnetic resonance imaging, they showed that high midbrain D3 receptor availability is associated with reduced functional connectivity between the orbitofrontal cortex and frontoparietal networks, which are implicated in executive control and salience processing.32 This result suggests that D3 receptor availability can modulate the pathway underlying cognitive control in healthy individuals. In addition, genetic studies have reported relationships between the D3 receptor gene (DRD3) polymorphisms and cognitive function in healthy individuals, though these findings are inconsistent.33 For example, the DRD3 Ser/Ser genotype was linked to fewer perseverative errors during the Wisconsin Card Sorting Test.34 In contrast, elderly individuals carrying the DRD3 Ser/Gly genotype had more benefit from multimodal cognitive training than the carriers of the Ser/Ser genotype.35 Importantly, preclinical studies suggest that D3 receptor blockade appears to enhance cognitive function, including memory, attention, learning, processing speed, social recognition, and executive function (see review by Nakajima et al), while D3 receptor agonism seems to impair it.33 D3 receptor antagonists may exert their pro-cognitive effect by enhancing the release of acetylcholine in the prefrontal cortex, disinhibiting the activity of DA neurons projecting to the nucleus accumbens or prefrontal cortex, or activating CREB signaling in the hippocampus.33 Thus, further research is needed to explore relationships between the absence of a decrease in the D3 receptor availability and age-related cognitive decline in healthy humans.

The present study has to be considered in the light of several limitations. First, its cross-sectional between-subject study design limits our interpretation of the data, which clearly warrants longitudinal within-subject studies to confirm our findings. Second, although our sample size is large compared with other previous PET studies, we have not sampled persons above the age of 73. Further research is needed that includes a larger age range of subjects, and more subjects within older age groups, to avoid any potential bias associated with uneven age distribution. Third, the radiotracers employed in this study are not entirely selective for D2 or D3 receptors. [11C]-(+)-PHNO is the best and only available PET radiotracer that is able to differentiate between D2 and D3 receptors given particular ROIs. The current findings should be replicated in future studies when other selective PET radiotracers are available. Fourth, limited spatial resolution may make PET studies prone to partial volume effects, although it is expected that the HRRT high-resolution scanner is less sensitive to partial volume effects.36 Fifth, we did not sample enough females to adequately examine gender differences. Moreover, we did not examine menstrual cycle and gonadal steroid hormone levels in female subjects, which have been reported to influence DA receptor density.37, 38 Indeed, it is reported that the binding rate constant to D2 receptors fluctuates during the normal menstrual cycle, with a lower constant in the follicular phase and a higher constant in the periovulatory and luteal phases. However, no study has explored relationships between menstrual cycle and gonadal steroid hormone levels, and D3 receptor availability in female subjects, which necessitates further research. Sixth, it has been noted that the injected mass of [11C]-(+)-PHNO is not within ideal radiotracer conditions (i.e., <1.5 ng/kg). The specific activity required to obtain tracer conditions is not possible with the available radiosynthesis method to avoid any influence of injected ligand mass. Thus, we may be underestimating BPND, especially in the D3 receptor-rich regions.39 For our current study, controlling for the mass injected did not affect any of our reported relationships between age and [11C]-(+)-PHNO. With [11C]-(+)-PHNO injection, side effects such as nausea and vomiting have been more frequently observed with an injected mass of >3 μg. Although all our subjects analyzed had an injected mass of <3 μg, we did not explicitly record from all participants if they experienced nausea upon tracer injection and none of the subjects presented vomiting that would require interrupting the scan. While the injected mass of [11C]-(+)-PHNO is an important limitation that needs to be considered by future studies, we do not believe it substantially changes our results. We do not know whether the nausea side effect is more or less common in elderly versus younger subjects. Seventh, previous research has suggested that [11C]-(+)-PHNO BPND in D3 receptor-rich regions is underestimated if SRTM quantification is used in conjunction with 90 minutes of data acquisition.40 Therefore, it is possible that D3 receptor availability might be underestimated in the current study. However, this potential underestimation should be similar for younger and older subjects, since the mass of [11C]-(+)-PHNO was not correlated with age. Therefore, we have no reason to assume that any potential underestimation of D3 receptor availability should be differentially weighted with aging. Future studies investigating how D3 receptor expression changes with aging should employ arterial plasma-based kinetic models after 120 minutes of emission data for quantifying [11C]-(+)-PHNO BPND in D3 receptor-rich regions,40 controlling the injected mass of [11C]-(+)-PHNO as much as possible. Finally, no cognitive functions were assessed in this study. Preclinical studies and human genetic studies have suggested that there are relationships between D3 receptors and cognitive function. Thus, future PET studies should include cognitive measures to shed light on these relationships in healthy individuals.

In conclusion, this study showed no age-related change in [11C]-(+)-PHNO binding in D3 receptor-specific regions, which support a lack of a change in D3 receptor availability in human brains with age. However, this study revealed age-related decline in [11C]-(+)-PHNO binding and [11C]-Raclopride binding in D2 receptor-specific regions, which replicate previous findings indicating an age-related decline of D2 receptor availability.5, 6, 8, 9 The D3 receptor has been implicated in cognitive function in healthy individuals and D3 receptor blockade may enhance cognitive function.33 Further research is required to elucidate the mechanisms underlying this lack of age-related decline in D3 receptor availability and to explore relationships between D3 receptor availability and age-related cognitive decline in healthy subjects.

Footnotes

Supplementary Information accompanies the paper on the Journal of Cerebral Blood Flow & Metabolism website (http://www.nature.com/jcbfm)

This study was supported by the Canadian Institutes of Health Research (CIHR): MOP-114989, MOP-157739, MOP-97946 MOP 102731; the National Institutes of Health (NIH): RO1MH084886-01A2; the Ontario Mental Health Foundation (OMHF); and Parkinson Society Canada (PSC): 2010-04.

Dr Nakajima has received fellowship grants from the Canadian Institute of Health Research (CIHR), Japan Society for the Promotion of Science, and Nakatomi Foundation, and manuscript fees from Dainippon Sumitomo Pharma and Kyowa Hakko Kirin. Dr Boileau has received research support from the following external funding agencies: the CIHR, Ontario Mental Health Foundation (OMHF), and Parkinson Society Canada. Dr Gerretsen has received fellowship support from the CAMH Foundation, OMHF, and CIHR. Dr Graff-Guerrero has received research support from the following external funding agencies: the CIHR, US NIH, OMHF, Brain and Behavior Research Foundation (BBRF), Mexico ICyTDF, CONACyT, and W Garfield Weston Foundation.

Supplementary Material

Supplementary Figures
Click here for additional data file. (186.5KB, doc)

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