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