Background
The neurotransmitter dopamine (DA) exerts powerful control over brain circuits that regulate reward, attention, and locomotor activity [
1‐
3]. Accordingly, DA dysfunction is believed to contribute to the etiology of several neuropsychiatric disorders, including attention-deficit/hyperactivity disorder (ADHD) [
4,
5], bipolar disorder (BPD) [
6], schizophrenia [
7,
8], and Parkinson’s disease [
9‐
11]. Interestingly, increasing evidence supports findings that patients with these diseases exhibit impaired retinal and visual functions, suggesting that altered DA signaling in the retina may be under the control of the same molecular perturbations that support the etiology of these disorders [
12] and that assessment of retinal DA signaling might offer a novel window into the diagnosis and treatment of neuropsychiatric disorders.
In the retina, DA mediates light adaptation and exhibits circadian rhythms of synthesis and release, such that DA signaling is higher during the daytime and during light exposure [
13,
14]. DA is secreted by amacrine neurons in the inner nuclear layer of the retina, and it mediates feedback of photic information to the outer retina from the inner retina [
15,
16]. DA-secreting amacrine cells influence other retinal neurons through volume conduction [
17]. Among the retinal targets of DA, the influence of DA on electrical synapses is well described. Specifically, DA uncouples the gap junctions between horizontal cells [
18], AII amacrine cells [
19,
20], and rods and cones [
21], leading to a reduction of receptive field size and blockade of rod signaling to ganglion cells. As a result, retinal circuits are reconfigured to a light-adapted state with increased light-induced response amplitudes in the presence of background light and enhanced acuity [
12]. Retinal DA signaling is reflected in the amplitude of the photopic electroretinogram (ERG) with retinal-specific DA depletion producing decreased ERG amplitudes and rescue of retinal DA levels with L-DOPA restoring ERG amplitudes [
12]. In addition, contrast sensitivity, spatial acuity, and circadian rhythms of light-adapted responses are all compromised in absence of retinal DA, further confirming that DA is important for light-adaptive mechanisms [
12]. DA exerts its action on target neurons and circuits through D
1-like (D
1 and D
5) and D
2-like (D
2, D
3, and D
4) receptors. In the retina, D
4 receptor-medicated signaling pathways modulate light-adapted ERG rhythms and contrast sensitivity, whereas D
1 receptor signaling contributes to high light-adapted ERG b-wave amplitudes and high spatial resolution [
12].
The DA transporter (DAT, SLC6A3) is a key determinant of DA signaling capacity in the brain, limiting the action of the neurotransmitter through high-affinity clearance of extracellular DA, with recycling of DA into the presynaptic cytosol [
22]. In the absence of DAT, extracellular DA levels are elevated in the striatum [
23] whereas intra-neuronal levels of DA are decreased [
22,
24]. The psychostimulant amphetamine (AMPH), structurally similar to DA, competes with extracellular DA at DAT and also induces DAT-mediated, non-vesicular release of cytosolic DA, providing two routes for elevation of extracellular DA levels. AMPH formulations and other agents that elevate extracellular DA (e.g., methylphenidate, MPH, Ritalin™) are also commonly prescribed for the treatment of ADHD (e.g., Adderall [
25,
26]). In addition to its significant expression in the brain [
27], DAT is also expressed in the somata and processes of dopaminergic amacrine cells in rat and mouse retina [
28,
29]. In DAT knockout mice, a significant decrease in retinal sensitivity is observed under dark-adapted (scotopic) conditions [
30]. DAT has also been suggested to play a role in form-deprivation myopia, as DAT binding in myopic retinas is lower than that in the normal control eyes [
31].
Genetic variation in DAT has functional consequences for brain DA signaling and behavior. Recently, we identified a rare human DAT coding substitution (DAT Ala559Val) in two male siblings diagnosed with ADHD [
5]. The Val559 variant had been previously identified in a female subject with bipolar disorder (BPD) [
32] and following our ADHD report, was identified in two unrelated male subjects with autism spectrum disorder (ASD) [
33]. In both heterologous expression studies [
34,
35] and in the DAT Val559 knock-in mouse model [
34,
36], there was anomalous dopamine efflux (ADE) consistent with changes in DAT function. In the mouse model, we observed an altered pattern of locomotion with decreased vertical activity and increased horizontal locomotion speed (darting) in response to imminent handling, significantly elevated extracellular levels of striatal DA under basal conditions without a change in DA tissue content, and a blunted response to AMPH or MPH paralleled by reduced locomotor activation by these psychostimulants [
36]. We have previously proposed [
34] that ADHD drugs containing AMPH formulation block the ADE of DAT Val559, which is distinct from blocking reuptake. In the former case, normal excitation coupling to vesicular release is restored, whereas in the latter case, the coupling to release is not modulated, only the amplitude of the response. We propose that it is the “noise” from leak that is more of a problem, at least for a subset of subjects, and thus, release of DA cannot be sensed appropriately. Ex vivo brain slice studies revealed tonic presynaptic DA receptor activation that supported a blunting of depolarization-evoked DA release. Altogether, our findings in the DAT Val559 model reveal a state of tonic hyperdopaminergia that leads to changes in locomotor patterns and anomalous responses to psychostimulants.
Although rare, the DAT Val559 variant may represent a genetic form of a functional state common to the broader etiology of idiopathic ADHD. If true, non-invasive tests of DA action that can be employed in ADHD subjects demonstrating ADE may allow for improved ADHD diagnosis and/or treatment. In the current study, we sought to evaluate DA-sensitive measures in the retina that can be detected using ERG. Specifically, we determined whether the DAT Val559 allele alters light-adapted retinal responses under basal conditions and as a consequence of AMPH administration. We observed DAT Val559 animals exhibit retinal responses consistent with the reported role of the variant in elevating tonic dopaminergic signaling and blunted responses to AMPH. Moreover, we observed differential retinal responses dependent on sex, of interest given the sex bias in ADHD diagnoses [
37].
Methods
Animal usage and care
All animal protocols were approved and in accordance with the guidelines established by the Vanderbilt University Animal Care Division and the National Institutes of Health Guide for the Care and Use of Laboratory Animals. WT, homozygous (HOM), and heterozygous (HET) Val559 DAT littermates with a hybrid background (∼ 75% 129S6/SvEvTac and ∼ 25% C57BL/6 J) [
36] were reared in a 12-h-light and 12-h-dark lighting condition. Only animals aged postnatal day 40 (P40) to P120 were subject to further tests. Unless otherwise noted, mice were tested or humanely killed during the middle of light phase of their light cycles (10:00 A.M.–2:00 P.M, Central Standard Time). The light intensity of the housing room was 100 ± 15 lx, provided by fluorescent bulbs. Mice were provided with water and food ad libitum.
ERG
The ERG was used to measure global retinal responses to light stimuli using the LKC Technologies UTAS visual electrodiagnostic test system (Gaithersburg, MD). Scotopic and photopic ERG recordings were performed as previously described [
12,
38]. All animals were dark-adapted overnight (~ 16–20 h) and tested during 4–8 h after subjective light onset (6:00 A.M., Central Standard Time). Mice were anesthetized with an intraperitoneal injection (IP injection) of ketamine (70 mg/kg) and xylazine (7 mg/kg), and their pupils dilated with 1% tropicamide (AKORN, NDC17478–102-12, Lake Forest, IL) under dim red light (Kodak GBX-2 Safelight, Rochester, NY). Their eyes were kept moist with 1% carboxymethyl-cellulose sodium eye drops (CVS, Extra Strength Lubricant Gel Drops Dry Eye Relief, Woonsocket, RI), and core body temperature was maintained at ∼ 37.0 °C using a thermostatically controlled heating pad regulated by a rectal temperature feedback probe (CWE, Model TC-1000 Temperature Controller, Ardmore, PA). Needle electrodes placed in the middle of forehead and the base of the tail served as reference and ground leads, respectively. A gold contact lens electrode was used for recording ERG responses (LKC Technologies; #N30).
Scotopic ERG responses were differentially amplified and filtered (bandwidth 0.3–500 Hz), with responses digitized at 1024 Hz. The recording epoch was 250 ms, with a 20-ms prestimulation baseline. Stimulus flashes were presented in an LKC BigShot ganzfeld. A total of 15 stimulus intensities, ranging from − 6.50 to 2.00 log cd*s/m2, were used under dark-adapted conditions. Each flash duration was 20 μs, and stimuli were presented in order of increasing intensity. As flash intensity increased, retinal dark adaptation was maintained by increasing the interstimulus interval from 30 to 180 s.
For photopic ERGs, mice were first given two flashes (−0.1 log cd*s/m2) under dark-adapted conditions to assess for a normal retinal response. A steady background-adapting field (40 cd/m2) inside the UTAS BigShot ganzfeld followed to saturate rod photoreceptors, and simultaneously, 0.90 log cd*s/m2 bright light flashes were presented at 0.75 Hz for a light adaptation session of 16 min. Data were collected and averaged in 2-min bins, totaling 8 bins. All other test parameters were similar to the scotopic ERG.
For the photopic ERG rescue experiment, IP injection of 1 mg/kg D1 receptor agonist (SKF38393, Sigma-Aldrich, Cat# D047, St. Louis, MO) and 1 mg/kg D4 receptor agonist (PD168077, Tocris Bioscience, Cat# 1065, Bristol, United Kingdom) were administered to WT mice 1 h before testing. Mice were injected under dim red light and returned to dark box until testing.
For the photopic ERG suppression experiment, IP injection of 1 mg/kg selective D4 DA receptor antagonist (L-745,870, Tocris Bioscience, Cat# 1002) was administered to HOM mice for 5 days 30 min prior to light onset in the morning. On the fifth day, animals were subject to photopic ERG tests.
The effects of D-AMPH on light-adapted ERG were explored by IP injections of 4 mg/kg AMPH to HOM and WT mice 15 min before the testing.
The scotopic a-wave was measured from the onset of flashes to the trough of the first negative deflection and b-wave was from the trough of the a-wave to the peak of the b-wave amplitude. Regarding photopic recordings, only b-wave amplitude could be reliably measured, which was defined as the difference from onset of stimuli to the peak of the b-wave.
HPLC determination of DA and its metabolites
Animals from all groups were dark-adapted overnight (~ 16-20 h) and then sacrificed under either dark or light conditions. Retinas were collected, immediately frozen in liquid nitrogen in 1.5-mL tubes, and stored at − 80 °C until processed for HPLC analysis. Under dark conditions, mouse retinas were dissected from the whole eye and separated from the retinal pigment epithelium in the presence of dim red light (Kodak GBX-2 Safelight). Under light conditions, after approximately 15 min lighting exposure, retinas were obtained in the presence of room lighting similar to the background light during the photopic ERG test. HPLC analyses were conducted in the Vanderbilt Brain Institute Neurochemistry Core.
Retinas were homogenized, using a tissue dismembrator, in 100–750 μL of 0.1 M TCA, which contained 10
−2 M sodium acetate, 10
−4 M EDTA, 5 ng/mL isoproterenol (as internal standard), and 10.5% methanol (pH 3.8). Samples were spun in a microcentrifuge at 10,000
g for 20 min. The supernatant was removed and stored at − 80 °C [
39]. The pellet was saved for protein analysis. Supernatant was thawed and spun for 20 min, and samples of the supernatant were then analyzed for biogenic monoamines. Retinal biogenic amines were determined by HPLC using an Antec Decade II (oxidation 0.65) electrochemical detector operated at 33 °C. Twenty microliter samples of the supernatant were injected using a Water 2707 autosampler onto a Phenomenex Kinetex (2.6μm, 100Å) C18 HPLC column (100 × 4.60 mm). Biogenic amines were eluted with a mobile phase consisting of 89.5% 0.1 M TCA, 10
−2 M sodium acetate, 10
−4 M EDTA, and 10.5% methanol (pH 3.8). Solvent was delivered at 0.6 mL/min using a Waters 515 HPLC pump. Using this HPLC solvent, the following biogenic amines elute in the following order: dihydroxyphenylacetic acid (DOPAC), DA and homovanillic acid (HVA) [
40,
41]. HPLC control and data acquisition are managed by Empower software. In this report, retinal biogenic amine analyses are represented as ng/mg protein. Total retinal protein concentration was determined using BCA Protein Assay Kit (Thermo Scientific, Cat# 23225, Waltham, MA). Ten-microliter tissue homogenate was distributed into a 96-well plate and 200 μL of mixed BCA reagent (25 mL of protein reagent A is mixed with 500 μL of protein reagent B) was added. Incubate the plate at room temperature for 2 h for the color development. A bovine serum albumin standard curve was generated at the same time, spanning the concentration range of 20–2000 μg/mL. Absorbance of standards and samples were measured at 562 nm. The inter-day variation of biogenic amine analysis using HPLC with electrochemical detection has been determined for the following analytes as: DOPAC, 2.3%; DA, 1.2%; 5-HIAA, 4.3%; HVA, 2.6%; 5-HT, 8.6%; and 3-MT, 10.2%. The intra-day variation for these analytes are DOPAC, 2.7%; DA, 0.8%; 5-HIAA, 1.2%; HVA, 2.6%; 5-HT, 8.8%; and 3-MT, 7.1%.
Statistical analysis
Two-tailed t test and one- and two-way ANOVAs were used where applicable and reported. Post hoc analyses followed ANOVAs to confirm the difference among groups. Significance levels were set at P < 0.05 and represented as means ± SEM as indicated in each graph (Graphpad, La Jolla, CA and Sigmaplot, San Jose, CA).