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. Author manuscript; available in PMC: 2019 Oct 1.
Published in final edited form as: Exp Eye Res. 2018 Jun 5;175:32–41. doi: 10.1016/j.exer.2018.06.006

Expression of dopamine D2 and D3 receptors in the human retina revealed by positron emission tomography and targeted mass spectrometry

Fernando Caravaggio a,b,*, Enzo Scifo c,d, Etienne L Sibille c,e, Sergio E Hernandez-Da Mota f, Philip Gerretsen a,b, Gary Remington a,b, Ariel Graff-Guerrero a,b
PMCID: PMC6167174  NIHMSID: NIHMS974121  PMID: 29883636

Abstract

Dopamine D2 receptors (D2R) are expressed in the human retina and play an important role in the modulation of neural responses to light-adaptation. However, it is unknown whether dopamine D3 receptors (D3R) are expressed in the human retina. Using positron emission tomography (PET), we have observed significant uptake of the D3R-preferring agonist radiotracer [11C]-(+)-PHNO into the retina of humans in vivo. This led us to examine whether [11C]-(+)-PHNO binding in the retina was quantifiable using reference tissue methods and if D3R are expressed in human post-mortem retinal tissue. [11C]-(+)-PHNO data from 49 healthy controls (mean age: 39.96 ± 14.36; 16 female) and 12 antipsychotic-naïve patients with schizophrenia (mean age: 25.75 ± 6.25; 4 female) were analyzed. We observed no differences in [11C]-(+)-PHNO binding in the retina between first-episode, drug-naïve patients with schizophrenia and healthy controls. Post-mortem retinal tissues from four healthy persons (mean age: 59.75 ± 9.11; 2 female) and four patients with schizophrenia (mean age: 54 ± 17.11; 2 female) were analyzed using a targeted mass spectrometry technique: parallel reaction monitoring (PRM) analysis. Using targeted mass spectrometry, we confirmed that D3R are expressed in human retinal tissue ex vivo. Notably, there was far greater expression of D2R relative to D3R in the healthy human retina (~12:1). Moreover, PRM analysis revealed reduced D2R, but not D3R, expression in the retinas of non-first episode patients with schizophrenia compared to healthy controls. We confirm that D3R are expressed in the human retina. Future studies are needed to determine what proportion of the [11C]-(+)-PHNO signal in the human retina in vivo is due to binding to D3R versus D2R. Knowledge that both D2R and D3R are expressed in the human retina, and potentially quantifiable in vivo using [11C]-(+)-PHNO, poses new research avenues for better understanding the role of retinal dopamine in human vision. This work may have important implications for elucidating pathophysiological and antipsychotic induced visual deficits in schizophrenia.

1. Introduction

Dopamine (DA) is a major neurotransmitter in the modulation of neural adaptations to light in the retina of mammals (Frederick et al., 1982; Hirasawa et al., 2015; Jackson et al., 2012; Popova, 2014; Tian et al., 2015; Witkovsky, 2004; Zhang et al., 2007). In this regard, DA D2 receptors (D2R) are thought to mediate several important functional roles. For example, the retinal circadian clock controls the extent and strength of rod-cone coupling (ensuring dim-light signals from rods are received by cones at night, but not in the day) through activation of D2R during the daytime (Ribelayga et al., 2008). Moreover, D2R are expressed on Müller glial cells (Biedermann et al., 1995) and may affect photoreceptor survival by regulating the release of neuroprotective factors (De Melo Reis et al., 2008), especially during stress (Chen et al., 2013).

It has been established that DA D2 receptors (D2R) are expressed in the retinas of all vertebrate species examined to date (Djamgoz and Wagner, 1992; Nguyen-Legros et al., 1999; Witkovsky and Dearry, 1991), including humans (Dearry et al., 1991; Stormann et al., 1990). Whether this is also true for the DA D3 receptor (D3R) is unclear. Direct quantification of D3R has been difficult due to a lack of compounds and antibodies selective for D3R as opposed to D2R (Le Foll et al., 2014). While evidence of D3R mRNA has been observed in the retina of zebrafish (Boehmler et al., 2004), this has not been observed in the retina of rodents (Cohen et al., 1992; Derouiche and Asan, 1999); with several rodent studies employing non-specific antibodies or radioligands for D2R and D3R (Tran and Dickman, 1992; Veruki, 1997; Zarbin et al., 1986). Moreover, whether D3R are expressed in the retina of humans remains unclear. An un-published abstract suggests that D3R mRNA could not be detected in human post-mortem retinal tissue (Prünte et al., 1992). Moreover, autoradiographic studies have employed nonspecific radioligands for D2R and D3R (Denis et al., 1990; Zarbin et al., 1986).

The positron emission tomography (PET) radiotracer [11C]-(+)-PHNO (Wilson et al., 2005) is an agonist for DA D2R and D3R. Notably, [11C]-(+)-PHNO has been shown to have greater in vivo selectivity for D3R relative to D2R than other D2/3R PET tracers such as [18F]fallypride and [11C]raclopride (Doot et al., 2018; Narendran et al., 2006; Searle et al., 2010, 2013; Tziortzi et al., 2011). This in vivo preference of [11C]-(+)-PHNO to bind to D3R relative to D2R is unique, and occurs despite the fact that other radiotracers like [18F]-fallypride demonstrate greater in vitro affinity for D3R than [11C]-(+)-PHNO (Mukherjee et al., 2015). Using a high-resolution head-dedicated PET camera, we observed what appeared to be significant uptake of [11C]-(+)-PHNO in the retinas of human participants (see Fig. 1). Notably, we did not observe similar uptake into the retina with the antagonist radiotracer [11C]-raclopride, which does not have significant binding to D3R in vivo (Narendran et al., 2006).

Fig. 1.

Fig. 1

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Averaged [11C]-(+)-PHNO Simplified Reference Tissue Model (SRTM) parametric binding potential non-displaceable (BPND) maps in healthy controls (n=49). These four averaged parametric BPND maps correspond to contiguous slices.

In this investigation, we set out to determine whether the [11C]-(+)-PHNO signal in the retina could be meaningfully quantified using typical pharmacokinetic modelling approaches. Given growing evidence of multiple retinal abnormalities in patients with schizophrenia (Adams and Nasrallah, 2017), we further explored whether [11C]-(+)-PHNO binding in the retina was different between first-episode, drug-naïve patients with schizophrenia, and age- and sex-matched healthy controls. Using targeted mass spectrometry, we then set out to determine whether D3R are expressed in post-mortem human retinal tissue. This included retinal samples from healthy persons and age- and sex-matched persons with schizophrenia, to complement our in vivo data with [11C]-(+)-PHNO.

2. Material and methods

2.1. [11C]-(+)-PHNO participants

PET data from healthy persons and drug-naive persons with schizophrenia, previously reported in the literature, were re-analyzed for the current investigation (Graff-Guerrero et al., 2008, 2009; Mizrahi et al., 2011). These studies were approved by the Research Ethics Board of the Centre for Addiction and Mental Health (CAMH), Toronto. Healthy participants were right-handed adults free of any major medical or psychiatric disorders as determined by clinical interview, the Mini International Neuropsychiatric Interview-Plus (MINI-Plus), basic clinical laboratory tests, and electrocardiography. Participants with schizophrenia were antipsychotic-naïve and met criteria for the diagnosis of schizophrenia or schizoaffective disorder based on the DSM-IV and the MINI-Plus. Participants were excluded if they had a current diagnosis of substance abuse or dependence, a history of clinically significant physical illness, or metal implants precluding the ability to be scanned with MRI. At inclusion and before the PET scan, participants were required to have a negative urine screen for drugs of abuse and/or pregnancy. All participants provided written informed consent. The average age of illness onset for the patient sample was 24.42 ± 6.75 (range: 17–42), with an average duration of illness of 16.33 ± 14.74 (range: 2–48) months.

2.2. [11C]-(+)-PHNO synthesis & acquisition

A full description of the radiosynthesis of [11C]-(+)-PHNO, as well as how PET images were acquired, can be found in previous publications (Graff-Guerrero et al., 2008; Wilson et al., 2005). Briefly, [11C]-propionyl chloride was reacted with 9-hydroxynaph-thoxazine to generate a [11C]-amide. This was subsequently reduced by lithium aluminium hydride. Purification by HPLC and formulation gave radio-chemically pure [11C]-(+)-PHNO as a sterile, pyrogen-free solution. PET images were acquired using a high-resolution head-dedicated PET camera system (CPS-HRRT; Siemens Molecular Imaging, USA) (Graff-Guerrero et al., 2008). The CPS-HRRT measures radioactivity in 207 brain slices, each with a thickness of 1.2 mm (Graff-Guerrero et al., 2008). The in-plane resolution was approximately 2.8 mm full-width at half-maximum (FWHM) (Graff-Guerrero et al., 2008). Notably, significant [11C]-(+)-PHNO uptake into the retina could not be observed on scans collected using a high-resolution PET CT, Siemens-Biograph HiRez XVI (Siemens Molecular Imaging, Knoxville, TN, USA) operating in 3D mode with an in-plane resolution of approximately 4.6 mm FWHM (almost half the resolution of the CPS-HRRT). Thus, PET camera systems of ~2.8 mm FWHM or better are likely required to have the appropriate resolution to image [11C]-(+)-PHNO uptake into the retina. Transmission scans were acquired using a 137Cs (T1/2=30.2 yr, E=662 KeV) single photon point source to provide attenuation correction, and the emission data were acquired in list mode (Graff-Guerrero et al., 2008). The raw data were reconstructed by filtered-back projection and the emission data were re-binned into a series of 3D sonograms (Graff-Guerrero et al., 2008). Scanning time was 90-min in length, wherein 30 frames were defined: 1 to 15 of 1-min duration and 16 to 30 of 5-min duration. A custom-fitted thermoplastic mask (Tru-Scan Imaging, Annapolis) was created for each subject and used with a head fixation system during PET scans to reduce movement during the acquisition (Graff-Guerrero et al., 2008). [11C]-(+)-PHNO was injected as a bolus followed by a flush of 2 mL saline into an intravenous line placed in an antecubital vein (Graff-Guerrero et al., 2008). The mean radioactivity dose was 9.38(±1.34)mCi, with a specific activity of 1160.82(±401.58)mCi/µmol, an injected mass of 2.07(±0.46)µg, and an injected mass per kilogram of 0.03(±0.008)µg/Kg. None of the participants included in this sample reported nausea associated with [11C]-(+)-PHNO injection (Mizrahi et al., 2009, 2010; Rabiner and Laruelle, 2010).

2.3. MRI imaging

Subjects provided a T1-weighted MRI image (TE=17, TR=6000, FOV=22 cm 2D, 256×256, slice thickness of 2 mm, NEX=2) acquired on a 1.5 T Signa scanner (General Electric Medical Systems, Milwaukee, WI). These images were used to enhance the analysis of PET scans, via co-registration (described in detail below, under [11C]-(+)-PHNO Image Analyses).

2.4. [11C]-(+)-PHNO Image Analyses

Two complementary approaches were employed for quantifying [11C]-(+)-PHNO binding in the retina. For the first approach (termed “manual”), retina ROIs were drawn manually on each subjects averaged dynamic [11C]-(+)-PHNO image in native-space (on every slice where the retina could be observed), employing previous PET guidelines (Bauer et al., 2017) (See Fig. 2). These manually drawn ROIs were then used to extract time activity curves from each subjects' dynamic PET image in native-space (See Fig. 3). This was done with reference to each subjects co-registered T1-weighted MRI image, to confirm the placement of the retina ROIs. The co-registration of each subjects MRI to PET space was done using the normalized mutual information algorithm (Studholme et al., 1997) as implemented in SPM2 (SPM2, Wellcome Department of Cognitive Neurology, London; http://www.fil.ion.ucl.ac.uk/spm). The TACs from the manually drawn retina ROIs were analyzed using both the Simplified Reference Tissue Method (SRTM) (Lammertsma and Hume, 1996) and Ichise's Multilinear Reference Tissue Model (MRTM)(Ichise et al., 2003), both of which have been validated for use with [11C]-(+)-PHNO (Ginovart et al., 2007). For both models, the cerebellum was used as the reference region to derive a quantitative estimate of binding – binding potential relative to the non-displaceable compartment (BPND) – as defined by the consensus nomenclature for in vivo imaging of reversibly binding radioligands (Innis et al., 2007). It has been noted for in vivo human studies with [11C]-(+)-PHNO that there is a small displaceable signal in cerebellum (BPND=~0.4) representing binding to D3R (Searle et al., 2013) in cerebellar lobes IX and X (Barik and de Beaurepaire, 1996). Thus, while we have done our best to exclude lobes IX and X in our cerebellar ROI, using the cerebellum as the reference region with [11C]-(+)-PHNO may result in some underestimation of BPND at D3R (Searle et al., 2013). Moreover, we recognize that use of the SRTM and MRTM is predicated on the assumption that blood flow into and out of the target and reference regions are similar (Salinas et al., 2015). This assumption may not be true for both the blood-brain barrier (cerebellar reference region) and the retina-brain barrier (retina target region), thus biasing our estimate of BPND. This poses a limitation in quantification which can only be avoided using arterial sampling of [11C]-(+)-PHNO, which is both invasive and costly. For the second approach (termed “automated”), the basis function implementation of the SRTM (Gunn et al., 1997) was applied to the dynamic PET images to generate parametric voxel-wise BPND maps using PMOD (v2.7, PMOD Technologies, Zurich, Switzerland). These images were spatially normalized into MNI brain space by Nearest Neighbour Interpolation with a voxel size fixed in 2 × 2 × 2 mm3 using SPM2. Regional BPND estimates were then derived from a manually drawn retina ROI defined in MNI space. The drawing of this ROI was aided by superimposing the average of the parametric voxel-wise BPND maps of all the subjects onto an MNI template.

Fig. 2.

Fig. 2

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Example of a retina ROI delineation on a single healthy subjects' averaged dynamic [11C]-(+)-PHNO image (90-min) in native space. The image contrast is presented both normally and inverted to aide in visual delineation of the ROI.

Fig. 3.

Fig. 3

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Averaged standardized [11C]-(+)-PHNO uptake values from manually drawn ROIs.

2.5. Post-mortem retinal tissue samples

Post-mortem retinal tissues from four patients with schizophrenia, and four age- and sex-matched healthy controls, were acquired from the St. Michael's Hospital Human Eye Biobank (http://www.stmichaelshospital.com/eye-biobank/; Toronto, Ontario, Canada) (See Table 3). Samples were formalin-fixed paraffin-embedded (FFPE).

Table 3.

Participant characteristics of post-mortem retinal tissue.

Cases Schizophrenia Patients Controls
Age Sex Age Sex
1 66 F 59 F
2 69 F 50 F
3 49 M 58 M
4 32 M 72 M
Mean ± S.D. 54 ± 17.11 59.75 ± 9.11
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2.6. Sample preparation and high-resolution parallel reaction monitoring (PRM) analyses

Total sample homogenates (approx. 2 µg) were subjected to a modified filter-aided sample preparation (FASP) protocol as previously described (Scifo et al., 2015), with additional precipitation using an equal volume of 2M KCL for depletion of residual detergents. Lys-C and tryptic peptides were combined and processed on Pierce C18 Tips reversed phase resin (Thermo Scientific) for desalting and concentration. High-resolution parallel reaction monitoring (HR-PRM) analyses were performed on a Q Exactive HF quadrupole–orbitrap mass spectrometer (Thermo Fisher Scientific) coupled to an EASY nanoflow liquid chromatography (EASY-nLC™) system (Thermo Fisher Scientific). Peptides were separated on a 50 cm column (75 µm inner diameter) packed with PepMap®RSLC C18 resin at 60 °C, using a flow rate of 250 nl/min on a 0%–42% acetonitrile (ACN) in 0.1% formic acid (FA) gradient and Buffer B (80% ACN with 0.1% FA) over 60 min. Column washes were performed on a 30%–90% acetonitrile in 0.1% formic acid gradient for 2 min. The IonMax electrospray ion source settings were: spray voltage, +1900 V and capillary temperature, +250 °C. In the PRM method, we only observed charge states as described in the inclusion list and skyline file. The targeted MS/MS was run at an Orbitrap resolution of 30,000 at m/z 200, an AGC target value of 2 × 105, and maximum fill times of 100 ms. The targeted peptide was isolated using a 0.8 m/z unit window. Fragmentation was performed with normalized collision energy (NCE) of 32 eV. In the PRM method, we only observed charge states as described in the inclusion list and skyline file. Seven peptides and their corresponding heavy peptides (Table 4) were comprised in the inclusion list. Target peptides were selected based on in silico cleavage of the proteins using the Peptide Mass feature of ExPASy bioinformatics portal (http://web.expasy.org/peptide_mass/). The following settings were used: enzyme (trypsin); 0 missed cleavage and display the peptides with a mass bigger than 500 Da.

Table 4.

Targeted peptides included in the PRM method. Four and three unique peptides for D2 and D3 receptors, respectively were included in the PRM method. Target peptides were selected in silico using the ExPASy bioinformatics portal. Unique peptides (light and heavy), their corresponding parent ions, top 3 most intense fragment ions and retention times, are indicated in the table.

Protein/Unique Peptide (a,b) Parent ion Top three most intense ions (c) Retention times (min)
DRD2
MDPLNLSWYDDDLER (light) 941.4198++ (y9, y11, y8) 44.8
MDPLNLSWYDDDLER (heavy) 946.4240++ (y9, y11, y8) 44.8
YTAVAMPMLYNTR (light)d 765.8758++ (y7, y9, y8) 36.8
YTAVAMPMLYNTR (heavy) 770.8799++ (y7, y9, y8) 36.8
GNCTHPEDMK (light) 594.7422++ (y5, b8, b5) 16.1
GNCTHPEDMK (heavy) 598.7493++ (y5, b8, b5) 16.1
AQELEMEMLSSTSPPER (light) 967.9453++ (y7, y9, y8) 34.9
AQELEMEMLSSTSPPER (heavy) 972.9495++ (y7, y9, y8) 34.9
DRD3
QNSQCNSVRPGFPQQTLSPDPAHLELK (light) 1016.8367+++ (y7, y9, y10) 30.9
QNSQCNSVRPGFPQQTLSPDPAHLELK (heavy) 1019.5081+++ (y7, y9, y10) 30.9
YYSICQDTALGGPGFQER (light) 1031.4704++ (y14, y11, y9) 34.5
YYSICQDTALGGPGFQER (heavy) 1036.4745++ (y14, y11, y9) 34.5
NSLSPTIAPK (light)d 514.2928++ (y6, y7, b7) 24.3
NSLSPTIAPK (heavy) 518.2999++ (y6, y7, b7) 24.3
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a

Heavy peptides are labelled at C-terminal arginine (R) or Lysine (K).

b

All cysteines are carbamidomethylated.

c

Fragments used for quantification are indicated and they are singly charged.

d

Peptides used for quantitation are indicated in bold.

2.7. PRM data analysis

Mass spectrometry analysis was performed at the SPARC Biocentre (The Hospital for Sick Children; SickKids, Toronto). We attempted to quantify 4 and 3 unique peptides for D2R and D3R, respectively (see Table 4). However, because of the relatively low protein yield in our FFPE slide samples, only a single unique peptide of either protein was detected and subsequently used for identification. Data analysis and quantification of PRM runs was performed using Skyline software (MacLean et al., 2010). Relative quantification of D2R and D3R peptides from schizophrenia patient and control samples was based on the sum of the area under the curves (AUC) of the top three most intense peptide fragments. The use of a subset of three to six fragment ions (with acceptable peak purity) for peptide quantitation has been previously suggested to yield more accurate results than the entire set (Gallien and Domon, 2015; Sundberg et al., 2016). Endogenous peptides were matched to heavy isotope peptides based on their similar retention times and transition patterns.

2.8. Statistical analyses

Statistical analyses were performed using IBM SPSS (v.20) and GraphPad (v.5.0; GraphPad Software, La Jolla California). Normality of variables was determined using the D'Agostino-Pearson test. The significance level for all tests was set at p < .05 (two-tailed).

3. Results

[11C]-(+)-PHNO scans from 49 healthy participants (16 female; mean ± S.D. age: 36.96 ± 14.36; range: 18–73) were analyzed. Using both manual delineation techniques and automated methods, it was possible to quantify [11C]-(+)-PHNO binding (BPND) in the retina using commonly employed reference tissue models. The estimated parameters and model fits for [11C]-(+)-PHNO BPND in the retina are presented in Tables 1 and 2. Notably, the estimated [11C]-(+)-PHNO BPND in the retina, and associated model fits, fell within previously accepted ranges for other regions of interest (ROIs), such as the thalamus (average BPND, 95% CI): 0.36, 0.26–0.49 (Tziortzi et al., 2011); 0.44, 0.35–0.54 (Searle et al., 2013).

Table 1.

Estimated parameters and model fits for manually drawn [11C]-(+)-PHNO retina ROIs using the Simplified Reference Tissue Model (SRTM) (n = 49).

Estimated Parameters Parameter Fits
SRTM %Covariance
R1 K2 BPND K2′ K2a R1 K2 BPND K2′ K2a χ2
Mean .25 .02 .73 .07 .01 2.88 5.95 31.29 8.10 13.76 1.62
S.D. (.05) (.004) (.58) (.02) (.003) (.83) (1.83) (27.68) (2.19) (6.06) (.87)
Minimum .17 .01 .04 .04 .00 1.82 2.74 6.76 4.34 5.40 .52
Maximum .42 .03 2.32 .12 .02 6.54 10.33 141.24 13.38 42.74 4.28
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Table 2.

Estimated parameters and model fits for manually drawn [11C]-(+)-PHNO retina ROIs using Ichise's Multilinear Reference Tissue Model (MRTM) (n = 49).

Estimated Parameters Parameter Fits
MRTM %Covariance
BPND K2′ -Vt/(Vt'b) 1/b -Vt/(Vt'k2′b) BPND K2′ -Vt/(Vt'b) 1/b -Vt/(Vt'k2′b) χ2
Mean .81 .07 .0003 −.0002 .1806 33.37 45.24 11.36 22.52 38.93 .93
S.D. (.57) (.05) (.0001) (.0001) (.36) (27.29) (83.08) (10.96) (17.50) (81.65) (.34)
Min. .05 −.09 – – – 7.06 5.42 3.10 6.22 2.09 .35
Max. 2.69 .26 – – – 140.82 400.62 44.60 84.10 372.26 1.92
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A Wilcoxon matched-pairs signed rank test demonstrated that the % covariance in manually derived retina BPND did not differ between choice of reference tissue model employed: the SRTM or MRTM (W=163, p=.42). However, the chi-square (χ2) total model fit was significantly better for the MRTM than the SRTM (W=−949, p < .0001), suggesting that the MRTM was better than the SRTM at modelling [11C]-(+)-PHNO BPND in the retina overall. This is similar to what has been suggested with other ROIs for [11C]-(+)-PHNO (Mizrahi et al., 2011). The MRTM produced larger retina BPND estimates than the SRTM (W=515, p=.001).

The average measure interclass correlation coefficient (ICC) between manually drawn SRTM and MRTM retina BPND estimates was 0.91, with a 95% CI from 0.84 to 0.95 (F(48,48)=11.70, p < .0001). The average measure ICC between the manually and automatically derived SRTM retina BPND estimates was 0.53, with a 95% CI from 0.19 to 0.73 (F(48,48)=2.23, p < .003), suggesting that the automatic, parametric SRTM method was less reliable.

The size of the manually drawn retina ROIs (mean ± S.D.) were 4217.22(±624.97) voxels, with an area of 6276.92(±930.21)mm2, and a volume of 7657.64(±1134.86)mm3.

We examined whether retina BPND was correlated with the size of the manually drawn ROIs from which they were derived. Retina SRTM BPND was not correlated with the total number of voxels (r(47)=0.18, p=.22), area (mm2) (r (47)=0.18, p=.22), nor the volume (mm3) (r(47)=0.18, p=.22) of the manually drawn retina ROIs. Similarly, retina MRTM BPND was not correlated with the total number of voxels (r(47)=0.16, p=.27), area (mm2) (r(47)=0.16, p=.27), nor the volume (mm3) (r(47)=0.16, p=.27) of the manually drawn retina ROIs. We examined whether retina BPND, from the manually drawn ROIs, was correlated with the injection parameters of [11C]-(+)-PHNO. Retina SRTM BPND was not correlated with the radioactivity dose (mCi) (r(47)=0.01, p=.94), specific activity (mCi/µmol) (r(47)=-0.11, p=.46), injected mass (µg) (r(47)=0.08, p=.57), nor the injected mass per kilogram (µg/Kg) (r(47)=0.18, p=.24) of [11C]-(+)-PHNO. Similarly, retina MRTM BPND was not correlated with the radioactivity dose (mCi) (r(47)=-0.05, p=.76), specific activity of (mCi/µmol) (r (47)=-0.20, p=.16), injected mass (µg) (r(47)=0.20, p=.16), nor the injected mass per kilogram (µg/Kg) (r(47)=0.21, p=.17) of [11C]-(+)-PHNO.

The χ2 total model fit did not differ between males and females (SRTM: U=213, p=.28; MRTM: U=258, p=.90). While males tended to have higher retina BPND's than females (SRTM: 0.81 ± 0 .66 vs 0.56 ± 0 .31; MRTM: 0.91 ± 0 .64 vs 0.60 ± 0 .35), this was not significant (SRTM: U=224.5, p=.41; MRTM: U=197, p=.16). Age was not correlated with the %covariance BPND for the SRTM (r (47)=0.20, p=.18), nor the MRTM (r(47)=0.12, p=.42). Age was not correlated with the χ2 total model fit for the SRTM (r(47)=0.15, p=.30), nor the MRTM (r(47)=0.11, p=.44). Age was not correlated with manually derived retina BPND, using the SRTM (r(47)=-0.07, p=.65) nor the MRTM (r(47)=-0.10, p=.49).

Since it was possible to quantify [11C]-(+)-PHNO BPND in the retina, we calculated retinal BPND values from 12 antipsychotic-naïve, first-episode patients with schizophrenia (4 female; mean ± S.D. age: 25.75 ± 6.25; range: 19–42). Retina BPND did not differ between patients and age- and sex-matched healthy controls measured with the SRTM (W=2, p=.97) and MRTM (W=16, p=.57) (See Fig. 4). In the first-episode patients, age of illness onset and illness duration were not correlated with [11C]-(+)-PHNO BPND in the retina (SRTM: age, r (9)=0.16, p=.63, duration r(9)=-0.27, p=.42; MRTM: age, r (9)=0.14, p=.68, duration r(9)=-0.22, p=.53).

Fig. 4.

Fig. 4

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Comparing manually delineated [11C]-(+)-PHNO BPND's in the retina between drug-naïve patients with schizophrenia & age- and sex-matched healthy controls (n=12, per group).

We employed high-resolution PRM to determine the availability and relative amounts of DA D2R and D3R receptors in our human retina samples. The choice for PRM analysis was motivated by its sensitivity and selectivity in detecting/quantifying target proteins in complex samples, that are otherwise undetectable by Western blot analysis. Total protein was extracted from the FFPE human retina tissue slides, consisting of 4 biological replicates from schizophrenia patients and control subjects, and subsequently used to generate peptides for PRM analysis. Given the low protein yield of FFPE samples, we did not perform technical replicates for this analysis. Peptide intensities were normalized based on the total ion chromatogram (TIC) intensity of the samples measured in shotgun LC-MS/MS mode, in order to correct for variations in the total peptide amount injected for the different samples. We included 4 and 3 target peptides in our PRM method (Table 4) to probe D2R and D3R, respectively. We detected one unique peptide for D2R (YTAVAMPMLYNTR) based on matching co-elution of heavy/light peptides at 36.8 min, similar transition patterns and mass errors within the −5.6 - +2.8 ppm ppm range (see Supplementary Material). A single unique peptide for D3R (NSLSPTIAPK) was also detected at 24.3 min and with mass errors within −0.1- +2.4 ppm range (see Supplementary Material).

These unique peptide detections indicated that D2R and D3R are expressed in the human retina. Notably, D2R was significantly decreased in patients with schizophrenia compared to healthy controls (t (6)=2.57, p=.04). However, there was no statistical difference with regards to levels of D3R (t(6)=1.18, p=.28) (see Fig. 5). Fold-change ratios for D2R and D3R in patients relative to controls are presented in the Supplementary Material.

Fig. 5.

Fig. 5

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Expression of dopamine D2 and D3 receptors in schizophrenia and control subjects. Quantitation is based on analysis of single unique peptides for D2 (YTAVAMPMLYNTR) and D3 (NSLSPTIAPK) receptors derived from four controls and four schizophrenia patients, in Skyline software (Tables 3 and 4). Relative peptide intensities are indicated in arbitrary units and error bars represent standard deviation.

4. Discussion

We observed significant uptake of the agonist D2/3R radiotracer [11C]-(+)-PHNO into the retina of humans. We examined whether this uptake could be meaningfully quantified using simple pharmacokinetic modelling. We observed that this uptake could be quantified, suggesting that [11C]-(+)-PHNO may be binding to D2R and/or D3R expressed in the human retina. Moreover, we found no difference in [11C]-(+)-PHNO binding in the retinas of first-episode, drug-naive patients with schizophrenia and healthy controls.

To ascertain whether D3R are expressed in the human retina, we employed targeted mass spectrometry in post-mortem retinal tissue. High-resolution PRM analysis revealed that indeed both D2R and D3R are expressed in the human retina at significant levels. Moreover, D2R expression in the retina was reduced in older, chronically treated patients with schizophrenia compared to healthy controls. There was also a non-significant reduction in D3R expression observed in patients relative to controls.

Collectively, our post-mortem data demonstrates that both D2R and D3R are expressed in the human retina and that, on average, the expression of D3R is 11–12-times less than that of D2R. Given that D2R expression in the retina is several orders of magnitude higher than D3R expression, it is possible that zero percent of the uptake in the retina is due to [11C]-(+)-PHNO binding to D3R. However, it is important to note that since [11C]-(+)-PHNO has ~25- to 45-fold greater selectivity for D3R versus D2R in vivo (Gallezot et al., 2012) it is possible that the majority of the [11C]-(+)-PHNO signal in a brain region can be attributable to D3R, despite having greater levels of D2R expressed therein. For example, in the substantia nigra it can be estimated ex vivo that there is roughly 3- to 4-times greater D2R expression compared to D3R (Gurevich and Joyce, 1999). However, in vivo practically 100% of the [11C]-(+)-PHNO signal in the substantia nigra is due to binding to D3R (Searle et al., 2010, 2013; Tziortzi et al., 2011). Unfortunately, without a blocking study employing a highly D3R-prefering antagonist, we are currently unable to estimate what proportion of the [11C]-(+)-PHNO signal in the retina, if any, is due to binding to D3R. Moreover, we could only observe significant uptake of [11C]-(+)-PHNO on our highest resolution PET camera – the HRRT, not the Siemens Biograph HiRez PETCT. Unfortunately, previously published D3R blocking studies with [11C]-(+)-PHNO aquired scans using a Siemens Biograph HiRez PETCT (with Truepoint Gantry, 3D-mode) (Searle et al., 2010, 2013; Tziortzi et al., 2011). Thus, significant uptake into the retina could also not be observed on these scans (personal communication, Dr. Graham Searle, April 17, 2018). Thus, future [11C]-(+)-PHNO blocking studies – acquired on the HRRT and employing highly selective D3R-preferring antagonists – are required to determine whether [11C]-(+)-PHNO is binding to retinal D3R and/or D2R in vivo.

Collectively, our data also suggests that both D2R and D3R may be reduced in older, treated patients with schizophrenia. However, this may not be the case in first-episode, drug-naïve patients. This appears prima facie consistent with evidence that DA receptor blockade by antipsychotics may cause degenerative retinopathies in patients with schizophrenia (Fornaro et al., 2002). Reductions in the retinal nerve fiber layer, macula, ganglion cell layer, and the inner plexiform layer is present in chronically treated patients with schizophrenia (Adams and Nasrallah, 2017; Ascaso et al., 2015; Celik et al., 2016; Lee et al., 2013b; Yilmaz et al., 2016) – although this has not been observed in all studies (Chu et al., 2012; Silverstein et al., 2017). In this regard, it is worth noting that thinning of the retinal nerve fiber layer and outer retina layers has been associated with neurodegenerative disorders such as Alzheimer's disease (Lu et al., 2010) and frontotemporal degeneration (Kim et al., 2017a). Thus, it will be important to elucidate the relationship between D2/3R expression and structural integrity of the retina across various neuropsychiatric diseases. However, it is currently unclear how chronic antipsychotic exposure affects D2R and D3R expression in retina, and in turn visual functioning. For example, while several studies have demonstrated contrast sensitivity deficits in patients with schizophrenia (Cadenhead et al., 2013; Cimmer et al., 2006; Skottun and Skoyles, 2007; Slaghuis, 2004), reports on the effects of antipsychotics on contrast sensitivity have been mixed. Some studies report abnormally low contrast sensitivity in unmedicated patients relative to medicated patients (Chen et al., 2003), while others report abnormally high baseline sensitivity which is reduced by short-term antipsychotic treatment (Kelemen et al., 2013). Further in vivo and ex vivo studies are required to examine the relationships between antipsychotic exposure on D2/3R expression and contrast sensitivity in patients with schizophrenia. While these aspects could not be examined in our current investigation, we believe this work marks as a first step towards elucidating this kind of information in the future.

Given that dopaminergic functioning in the retina may play diverse roles besides light adaptation (Zhang et al., 2007), such as contributing to the development of myopia (Nebbioso et al., 2014; Sun et al., 2018), it will be important to determine whether deficits in visual/retinal functioning in schizophrenia are inherent to the pathophysiology of the disease, or related to chronic D2/3R blockade by antipsychotics (Adams and Nasrallah, 2017). For example, it has been well documented that the adverse ocular effects of chlorpromazine include miosis, blurred vision (caused by paralysis of accommodation), pigmentary changes in the retina, attenuation of retinal vessels, and optic nerve pallor (Oshika, 1995; Siddall, 1965). Moreover, given that loss of retinal DA has been observed in patients with Parkinson's disease (Harnois and Di Paolo, 1990), it will be important to determine whether D3R expression is altered in vivo and ex vivo, and how this may relate to visual functioning in these patients.

In contrast, evidence suggest that agonism of both DA D1 receptors (D1R) and D2R may be neuroprotective (Wakakura, 2001) and a potentially effective intervention for ischemic or degenerative disorders in the retina and optic nerve (Li et al., 2012; Yamauchi et al., 2003). Notably, D3R agonism has been suggested to mediate neuroprotective effects on striatal DA neurons (Fiorentini et al., 2015; Kim et al., 2017b); although pro-cognitive effects of D3R antagonists in general has also been suggested (Nakajima et al., 2013). Since D1R and D3R form heterodimers in the striatum (Fiorentini et al., 2015), it will be important to determine, i) whether D1R and D3R form heterodimers in the retina, and, ii) whether selective D3R agonism can mediate neuroprotective effects against neurodegeneration in the retina. To add to this complexity, it has been well established that D2R expression decreases with age in the striatum (Karrer et al., 2017) – even in patients with schizophrenia (Graff-Guerrero et al., 2015) – while D3R expression may increase with age (Matuskey et al., 2016; Nakajima et al., 2015). Larger in vivo and ex vivo studies are required to determine whether or not D3R increases, while D2R decreases, with age in the retina, similar to what has been proposed for the striatum.

There are several limitations with the current investigation. First, our PET analysis was retrospective. Thus, we did not acquire information about visual/retinal functioning in our participants. This important information can be acquired by future prospective [11C]-(+)-PHNO studies. Notably, both our first-episode sample and our post-mortem retina samples were relatively small. While both of these samples are difficult to recruit/obtain, it will be important for future studies to employ larger samples in order to be sufficiently powered to detect important associations between retinal D2/3R availability and clinical/cognitive variables. Moreover, future [11C]-(+)-PHNO studies examining the retina should recruit a patient group with an established diagnosis of schizophrenia and/or schizoaffective disorder, to compare with first-episode and healthy controls. Second, in quantifying [11C]-(+)-PHNO BPND in the retina we employed a reference tissue model, which requires several assumptions to be met between the reference and target tissue (i.e., the retina and cerebellum) (Salinas et al., 2015). Thus, it will be important to further validate our quantification of BPND in the retina using arterial sampling (Lammertsma, 2017). This in turn would help overcome any potential issues regarding underestimation of binding to D3R in the retina due to specific binding to D3R in the cerebellum. Third, the characterization of our participants in the post-mortem retina sample was incomplete. Namely, information about history of psychotic symptoms, history of antipsychotic drug usage, and cause of death was unavailable. Fourth, our retina tissue samples were formalin-fixed paraffin-embedded. It will be important to attempt to replicate our findings using unfixed, fresh tissue. Fifth, it will be important to determine where, and on which cell types, D3R are expressed in the human retina. However, this knowledge may be contingent upon future advances in the development of D3R selective antibodies and radioligands. Finally, due to limitations in radiosynthesis methods, all [11C]-(+)-PHNO scans are acquired under non-tracer conditions (Gallezot et al., 2012; Searle et al., 2013). Specifically, tracer conditions (less than 10% D3R occupancy by (+)-PHNO) would require an injected mass of 4.5 ng/kg, or 0.3 µg of (+)-PHNO in a 70 kg person (Rabiner and Laruelle, 2010; Searle et al., 2013). Thus, given non-tracer conditions, the signal from D3R blockade may be underestimated (Girgis et al., 2011) and physiological side-effects from tracer injection may occur (Mizrahi et al., 2009, 2010; Rabiner and Laruelle, 2010). Notably, it has been reported that a 40 ng/kg mass dose of [11C]-(+)-PHNO is associated with 50% occupancy of D3R (Searle et al., 2013). In our current investigation, the average injected mass was 30 ng/kg. Thus, our [11C]-(+)-PHNO scans were also conducted under non-tracer conditions, likely resulting in significant occupancy of D3R (≤50%). While this is a currently unavoidable limitation of [11C]-(+)-PHNO studies, none of the participants in this report experienced physiological side-effects (i.e. nausea or vomiting) in response to the injection of [11C]-(+)-PHNO. It is currently unclear what makes certain participants more susceptible to experiencing physiological side-effects in response to [11C]-(+)-PHNO injection, even at relatively lower reported averaged injected masses with constant infusion (Lee et al., 2013a).

The demonstration of D3R expression in the human retina, and the potential to quantify retinal D3R availability in vivo with [11C]-(+)-PHNO, opens several new avenues of inquiry for elucidating the role of retinal DA in visual processing, in health and disease.

Supplementary Material

1
2

Acknowledgments

The authors would like to thank the SPARC Biocentre at The Hospital for Sick Children (SickKids) for all their help and support. The authors would also like to acknowledge the insightful discussions and input from Dr. Timour Al-Khindi and Dr. Raúl Velez Montoya in the writing of this manuscript.

Funding/support

Funding for this study was provided by the Canadian Institutes of Health Research (MOP-114989) and U.S. National Institute of Health (RO1MH084886-01A2). These funding sources had no further role in study design, in the collection, analysis and interpretation of data, in the writing of the report, and in the decision to submit the paper for publication.

Footnotes

Financial disclosures

The authors declare no conflicts of interest with regards to this work.

Appendix A. Supplementary data

Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.exer.2018.06.006.

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