Spiperone

Recently, an increasing number of studies relied on the assumption that visually induced changes in choroidal thickness can serve as a proxy to predict future axial eye growth. The retinal signals controlling choroidal thickness are, however, not well defined. We have studied the potential roles of dopamine, released from the retina, in the choroidal response in the chicken.

Changes in retinal dopamine release and choroidal thickness changes were induced by intravitreal injections of either atropine (250 µg or 360 nMol), atropine combined with a dopamine antagonist, spiperone (500 µMol), or spiperone alone and were tracked by optical coherence tomography (OCT). To visually stimulate dopamine release, other chicks were exposed to flicker light of 1, 10, or 400 Hz (duty cycle 0.2) and choroidal thickness was tracked. In all experiments, dopamine and 3,4-Dihydroxyphenylacetic acid (DOPAC) were measured in vitreous, retina, and choroid by high-performance liquid chromatography with electrochemical detection (HLPC-ED). The distribution of the rate-limiting enzyme of dopamine synthesis, tyrosine hydroxylase (TH), neuronal nitric oxide synthase (nNOS), vascular endothelial growth factor (VEGF), and alpha2A adrenoreceptors (alpha2A-ADR) was studied in the choroid by immunofluorescence.

The choroid thickened strongly in atropine-injected eyes, less so in atropine + spiperone-injected eyes and became thinner over the day in spiperone alone-, vehicle-, or non-injected eyes. Flickering light at 20 lx, both 1 and 10 Hz, prevented diurnal choroidal thinning, compared to 400 Hz, and stimulated retinal dopamine release. Correlation analysis showed that the higher retinal dopamine levels or release, the thicker became the choroid. TH-, nNOS-, VEGF-, and alpha2A adrenoreceptor-positive nerve fibers were localized in the choroid around lacunae and in the walls of blood vessels with colocalization of TH and nNOS, and TH and VEGF.

Retinal DOPAC and dopamine levels were positively correlated with choroidal thickness. TH-positive nerve fibers in the choroid were closely associated with peptides known to play a role in myopia development. Findings are in line with the hypothesis that dopamine is related to retinal signals controlling choroidal thickness.

(+)-4-Propyl-9-hydroxynaphthoxazine ((+)PHNO) is a high affinity, preferential dopamine D₃ versus D₂ agonist employed in view of its high specificity and excellent signal-to-noise ratio as a radiotracer for positron emission tomography (PET) imaging. Surprisingly, its profile at other classes of monoamine receptor remains undocumented. In addition to hD₃ and hD_2L receptors, (+)PHNO revealed high affinity at hD_4.4 but not hD₁ or hD₅ receptors. It also revealed significant affinity for several other G protein-coupled monoaminergic receptors, in particular h5-HT_1A and h5-HT₇. (+)PHNO behaved as a full agonist at hD_4.4 and h5-HT_1A receptors with potencies comparable to its actions at hD₃ and hD_2L receptors, and with less potency at 5-HT₇ receptors. In binding assays with membranes derived from cells co-expressing hD₃ and hD_2L receptors and labeled with [³H]Nemonapride or [³H]Spiperone, the proportion of high affinity binding sites recognized by (+)PHNO was higher than an equivalent mixture of membranes from cells expressing hD₃or hD_2L receptors, suggesting that (+)PHNO promotes formation of hD₃-hD_2L heterodimers. Further, in cells co-expressing hD₃ and hD_2L receptors, (+)PHNO showed higher efficacy for inhibiting forskolin stimulated adenylyl cyclase and inducing adenylyl cyclase super-sensitization than in cells transfected with only hD_2L receptors. In conclusion, (+)PHNO is a potent agonist at hD₄.₄, h5-HT_1A and h5-HT₇ as well as hD₃ and hD_2L receptors, and it potently activates dopamine hD₃-hD_2L heterodimers. These interactions should be considered when interpreting PET studies with [¹¹C](+)PHNO and may be relevant to its functional and potential clinical properties in Parkinson's disease and other disorders.