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

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

mouse was placed, individually, in a clear plastic cage and 2.1. Animals illuminated for 30 min at 60 fc, unless noted otherwise. The retinas were then isolated and frozen in liquid Handling of animals conformed with principles regard- nitrogen. ing the care and use of animals adopted by the American Physiological Society and Society for Neuroscience. 2.4. Catecholamine analysis Normal BALB c mice, mutant homozygous rds rds 020 A mice, and heterozygous rds 1 mice were studied. DA, DOPAC and DOPA levels were analyzed by high A breeding colony of BALB c and rds rds mice is pressure liquid chromatography HPLC with electrochem- maintained at the Animal Facility of the University of ical EC detection. Two retinas of each mouse were Texas Health Science Center. Heterozygous rds 1 mice pooled for one sample. HPLC analysis was carried out as F1 were produced by breeding BALB c and rds rds previously described [39]. Briefly, frozen retinas were mice. The mice are housed in light-proof rooms on a 12 h homogenized in ice cold 0.1 M perchloric acid containing I . Nir et al. Brain Research 884 2000 13 –22 15 10 mM ascorbic acid and 20 ng ml 3,4-dihydroxybenzyl- 3.1. DA metabolism in young rds 1 and rds rds mice amine DHBA, an internal standard. Homogenates were centrifuged and aliquots of each supernatant fraction were One to two month old BALB c, rds 1 and rds rds injected into a Beckman Ultrasphere-ODS reverse phase mice were studied. By this age, photoreceptor differentia- column. DA and metabolites were eluted with a mobile tion is completed, photoreceptor cell loss is not yet phase consisting of 100 mM phosphoric acid, 0.1 mM measurable in the rds 1 retinas [18], and only the initial EDTA, 0.45 mM sodium octylsulphate and 6 acetonitrile wave of photoreceptor cell loss occurred in the rds rds at pH 2.6–2.7 and were detected amperometrically. Chro- retina [38,44]. The mutation phenotypes, as expressed in matographic peaks were identified by relative retention the organization of the photoreceptor outer segments, are times compared to those of external standards. Concen- depicted in Fig. 1. While normal BALB c photoreceptors trations were determined by comparing peak areas of outer segments consist of stacks of photopigment-con- unknowns with those of standards. Values are corrected for taining disc membranes Fig. 1A, in the homozygous the recovery of DHBA. Retinal protein was determined by rds rds mutant photoreceptors disc membranes are not the method of Lowry [31]. formed Fig. 1B. The heterozygous rds 1 outer segments are characterized by malformed disorganized disc mem- branes Fig. 1C.

3. Results