Introduction The retinal degeneration slow rds mouse carrying the

Brain Research 884 2000 13–22 www.elsevier.com locate bres Research report Diurnal metabolism of dopamine in dystrophic retinas of homozygous and heterozygous retinal degeneration slow rds mice a , b b Izhak Nir , Rashidul Haque , P. Michael Iuvone a Department of Pharmacology , University of Texas Health Science Center, San Antonio, TX, USA b Department of Pharmacology , Emory University School of Medicine, Atlanta, GA, USA Accepted 15 August 2000 Abstract Dopamine metabolism was studied in dystrophic retinal degeneration slow rds mice which carry a mutation in the rds peripherin gene. RDS mutations in humans cause several forms of retinal degeneration. Dopamine synthesis and utilization were analyzed at various time points in the diurnal cycle in homozygous rds rds retinas which lack photoreceptor outer segments and heterozygous rds 1 retinas which have short malformed outer segments. Homozygous retinas exhibited depressed dopamine synthesis and utilization while the heterozygous retina retained a considerable level of activity which was, nevertheless, significantly lower than that of normal retinas. By one year, heterozygous rds 1 retinas which had lost half of the photoreceptors still maintained significant levels of dopamine metabolism. Normal characteristics of dopamine metabolism such as a spike in dopamine utilization at light onset were observed in mutant retinas. However, light intensity-dependent changes in dopamine utilization were observed in normal but not rds 1 retinas. The findings of this study suggest that human patients with peripherin rds mutations, or other mutations that result in abnormal outer segments that can still capture light, might maintain light-evoked dopamine metabolism and dopamine-dependent retinal functions during the progression of the disease, proportional to remaining levels of light capture capabilities. However, visual deficits due to reduced light-evoked dopamine metabolism and abnormal patterns of dopamine utilization could be expected in such diseased retinas.  2000 Elsevier Science B.V. All rights reserved. Theme : Sensory systems Topic : Retina and photoreceptors Keywords : Dopamine; Retina; Rds peripherin mutation; Rds mouse; Retinal degeneration

1. Introduction The retinal degeneration slow rds mouse carrying the

mutant peripherin rds gene is extensively studied as an Retinitis Pigmentosa RP is a clinical diagnosis that animal model of RP. A mutation in the peripherin rds gene encompasses a group of heterogeneous hereditary disorders in rds mice accounts for the defect in disc morphogenesis, that manifest progressive loss of photoreceptors and blind- since replacement of the gene in transgenic rds mice ness. The prevalence of the disorder is about 1 4000, corrects the defect [51]. RDS encodes peripherin rds, a which makes it a common cause of visual impairment in structural glycoprotein in the rims of outer-segment discs all age groups [2]. Mutations in multiple genes have been [12,27,50]. shown to cause blindness due to photoreceptor cell-death Photoreceptors in the homozygous rds rds mouse retina [34,52]. Over 50 mutations in the RDS gene have been differentiate normally for the first few postnatal days. Inner implicated in dominant forms of RP and macular degenera- segments project an extended cilium but outer segments tion [28,47]. fail to develop and only rudimentary discs are formed at the distal ciliary tip. Between the second and third post natal weeks the photoreceptor starts to undergo apoptotic Corresponding author. Tel.: 11-210-567-4974; fax: 11-210-567- cell death. Photoreceptor cell loss proceeds slowly until 4226. E-mail address : niruthscsa.edu I. Nir. completed by one year [23,44]. In the heterozygous rds 1 0006-8993 00 – see front matter  2000 Elsevier Science B.V. All rights reserved. P I I : S 0 0 0 6 - 8 9 9 3 0 0 0 2 8 5 5 - 9 14 I mouse retina, photoreceptors develop a distinct outer dark light cycle, with light on at 8 AM. Throughout this segment layer of moderate length, but disc structures paper, ‘‘day’’ refers to the light phase and ‘‘night’’ refers remain disarrayed and form irregular whorls. Photorecep- to the dark phase of the imposed dark light cycle. Lighting tor cell loss in the rds 1 retina is first noted by 2 months, was provided by fluorescent tubes. Light intensity at cage then it progresses more slowly than in the homozygous levels was 3–5 foot candles fc. Experiments were carried retina and by one year the photoreceptor population is out in a laboratory under selected illumination levels. reduced by half [18]. Overhead illumination was provided by cool white fluores- Reduced light responses measured in mutant rds retinas cent bulbs at a room temperature of 218C. Illumination was [10,43] are expected to affect metabolic pathways in retinal measured with a digital illuminance meter DX-200, INS neurons that are regulated by light. In a previous study we Enterprise Inc., Taiwan. The mice were euthanized by established that light-evoked metabolism of dopamine cervical dislocation. Following enucleation, the anterior DA is diminished in homozygous rds rds mice [39]. DA structures and lens were removed and the retina was is an important retinal neurotransmitter and neuro- dissected from the posterior eye cup. Procedures in the modulator [4,14,15,56]. In the mammalian retina, DA is ‘‘dark’’ were carried out under dim red light Kodak safety synthesized and released by a subset of amacrine and light filter no. 1. interplexiform cells [35,54]. Cellular responses to DA are mediated by the D1 and D2 receptor families [46]. 2.2. Electron microscopy Quantitative analysis of DA receptors in the rat retina revealed the highest concentration of D2-like receptors in Eyes were placed in 4 formaldehyde and 2 glutaral- the photoreceptor inner segments and in the outer nuclear dehyde in 0.1 M phosphate buffer pH 7.0 for fixation. layer [49]. In the mouse retina, D4 receptors, a subtype of After 30 min, the eyes were bisected along the vertical D2, were localized to the photoreceptor layer [11]. meridian and the two hemispheres were fixed for an DA release from dopaminergic neurons affects several additional 3 h. The tissue was then treated in 1 OsO4, processes in the retina, including: photoreceptor disc dehydrated and embedded in an epoxy resin. Thin sections shedding [4]; cone contraction and movement of melanin were cut at different sites along the central–peripheral axis in the pigment epithelium [13,42]; Na1,K1-ATPase of the retina and stained with uranyl and lead salts before activity in photoreceptors [48]; rod opsin gene expression viewing under the electron microscope. [1]; gap junction permeability between horizontal cells [41] and between rods and cones [30]; glutamate-gated 2.3. Enzyme inhibitions ionic conductances in horizontal cells [29] and bipolar cells [32], and ganglion cell receptive fields [25]. Synthesis of dopamine from L -tyrosine is a two step In the present study we investigated the diurnal metabo- process: 1 hydroxylation by tyrosine hydroxylase TH lism of DA in mutant homozygous rds rds and hetero- to produce L -3,4-dihydroxyphenylalanine L -DOPA; and zygous rds 1 mouse retinas. The analysis included young 2 decarboxylation of DOPA by aromatic L -amino acid and old mice to determine the consequences of a life-long decarboxylase AAAD to produce dopamine. DA syn- light deprivation on light-evoked DA metabolism. Results thesis and utilization were analyzed after inhibition of from the present study will be relevant for the evaluation AAAD with m-hydroxybenzylhydrazine NSD-1015; of abnormalities in DA metabolism and its effect on retinal Sigma Chemical Co.. Inhibition of AAAD results in functions in RP patients with peripherin rds mutations and accumulation of DOPA, which is used as an estimation of also with other mutations which affect outer segment in situ TH activity and DA synthesis. Utilization of DA is integrity and light capture. reflected by the decline in DA levels following inhibition of AAAD. The drug was dissolved in phosphate-buffered saline pH 7.0 and injected intraperitoneally 150 mg kg

2. Material and methods body weight. Groups of 5–7 mice were injected and each