Alterations in light-evoked dopamine metabolism in dystrophic retinas of mutant rds mice

BRAIN RESEARCH ELSEVIER

Brain Research 649 (1994) 85-94

Research Report

Alterations in light-evoked dopamine metabolism in dystrophic retinas of mutant rds mice Izhak Nir a, p. Michael Iuvone b,, " Department of Pathology, University of Texas Health Science Center, San Antonio, TX 78284, USA h Department Pharmacology, Emory UniL,ersitySchool, School of Medicine, Atlanta, GA 30322, USA

(Accepted 10 March 1994)

Abstract In dystrophic retinas of rds mice, which are devoid of photoreceptor outer segments, high steady state levels of dopamine were found in dark and light periods. These levels were similar to those observed in normal, BALB/c mouse retinas. Major differences were determined, however, between dopamine turnover in normal and dystrophic retinas. While substantial light-evoked elevation of dopamine synthesis and utilization was observed in normal retinas, dopamine synthesis and metabolism in rds retinas was very low and response to light was depressed. Retinal dopamine metabolism was already depressed in 2 week old rds mice, prior to the onset of photoreceptor cell death, relative to that in age-matched BALB/c mice. At 1 month of age, robust light/dark differences in retinal dopamine metabolism were observed in BALB/c mice, while no significant effect of light was seen in rds mice. The limited ability of the dopaminergic system in rds retinas to respond to light may be due to the absence of normal outer segments. Interestingly, in old rds retinas, although most photoreceptor cells had degenerated, a small but significant light-evoked increase in dopamine metabolism was measured. The presence of relatively high steady state levels of dopamine in rds retinas, despite the reduced dopamine synthetic activity, is maintained by a compensatory reduction in dopamine utilization. Thus, although a considerable amount of dopamine is present in the rds retina, it might not be available to exert its biological functions. Key words: Dopamine; Retina; Mouse; Retinal dystrophy; Light/dark

I. Introduction Photoreceptors in the retinal degeneration slow (rds) mouse retina differentiate normally for the first few postnatal days. Inner segments project an extended cilium but outer segments fail to develop and only rudimentary discs are formed at the distal ciliary tip. Photoreceptor cell death is gradual and completed by 1 year [27,47,48]. The c D N A which codes for the abnormal protein in the rds mouse was cloned by Travis et al. [52]. The normal gene encodes a protein of 39 kDa, which is highly homologous to the bovine peripherin gene product [13]. The peripherin protein was localized to the rim of discs in cone and rods, and it appears to be

* Corresponding author. Dept. of Pharmacology, Emory University School of Medicine, 1510 Cliffton Road, Room 5102, Rollins Research Building, Atlanta, GA 30322-3090. Fax: (1) (404) 727-0365. 0006-8993/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0006-8993(94)00314-3

important in outer segment disc morphogenesis [3,35]. The role of the mutated p e r i p h e r i n / r d s gene in causing the photoreceptor abnormalities was shown recently in transgenic mice in which insertion of a normal p e r i p h e r i n / r d s gene resulted in development of normal photoreceptors [53]. Recent molecular genetic analysis of families with an autosomal dominant retinitis pigmentosa (ADRP) identified a mutation in the photoreceptor p e r i p h e r i n / r d s gene of some patients [22,29]. The mutation in the p e r i p h e r i n / r d s gene accounts for the defect in outer segment disc morphogenesis, but it is not clear why the absence of outer segments results in photoreceptor cell death. In studies of events which might lead from the primary lesion to the secondary photoreceptor cell death we previously investigated the expression of photoreceptor specific genes in rds retinas [2,40,41]. In the course of these studies we discovered a profound secondary effect of the mutation

86

L Nir. P.M. h, om' / Brain Research O4g (1994) 85 ~)4

on a r r e s t i n g e n e e x p r e s s i o n in t h e rds r e t i n a [1]. A r restin m R N A levels in n o r m a l m o u s e r e t i n a s a r e u n d e r d i u r n a l c o n t r o l w i t h levels h i g h e r in t h e light p e r i o d a n d l o w e r d u r i n g t h e d a r k p e r i o d [7,14,33]. In t h e rds r e t i n a a r r e s t i n m - R N A levels w e r e h i g h b o t h in light and dark. T h i s o b s e r v a t i o n r a i s e d t h e q u e s t i o n w h e t h e r loss o f d i u r n a l c o n t r o l of a r r e s t i n g e n e e x p r e s s i o n is d u e to c h a n g e s in c l o c k m e c h a n i s m s in t h e r e t i n a w h i c h r e g u l a t e d i u r n a l r h y t h m s . T o e x p l o r e this issue w e t u r n e d o u r a t t e n t i o n to d o p a m i n e ( D A ) , a r e t i n a l synaptic neurotransmitter and paracrine neuromodulator w h i c h a f f e c t s c e l l u l a r r h y t h m s in t h e r e t i n a [5,6,20,58]. R e t i n a l D A is r e l e a s e d by a s u b s e t of a m a c r i n e o r i n t e r p l e x i f o r m cells [19,31,37,56]. A d i u r n a l r h y t h m of D A m e t a b o l i s m was r e p o r t e d in v a r i o u s a n i m a l species. In the m a m m a l i a n r e t i n a , D A a n d / o r D A m e t a b o l i t e levels a r e h i g h e r in i l l u m i n a t e d r e t i n a s o f rats [12,24,34,57], rabbits [42,43] a n d h u m a n s [18]. S t i m u l a tion o f D A b i o s y n t h e s i s in light [15,45] is d u e to activation o f t y r o s i n e h y d r o x y l a s e ( T H ) [26] to p r o d u c e 3,4dihydroxyphenylalanine (DOPA) and induction of arom a t i c L - a m i n o a c i d d e c a r b o x y l a s e ( A A A D ) [25], w h i c h catalyzes the formation of DA. Previous studies of DA in d y s t r o p h i c r e t i n a s o f t h e rd m o u s e r e v e a l e d t h a t f l u o r e s c e n c e o f d o p a m i n e r g i c cells r e m a i n e d unc h a n g e d a f t e r p h o t o r e c e p t o r d e g e n e r a t i o n [30]. H o w ever, in t h e d y s t r o p h i c R C S rat r e t i n a a l t e r a t i o n s in t h e s t e a d y state o f D A w e r e r e p o r t e d [23]. In t h e p r e s e n t study we c a r r i e d o u t a d e t a i l e d analysis o f d i u r n a l D A m e t a b o l i s m in n o r m a l B A L B / c a n d d y s t r o p h i c rds m i c e in light a n d d a r k . T h e s t u d y i n c l u d e d y o u n g and old m i c e at v a r i o u s s t a g e s o f r e t i n a l d y s t r o p h y . T h e f o l l o w i n g p a r a m e t e r s w e r e i n v e s t i g a t e d : (1) S t e a d y s t a t e levels o f D A a n d a n d its m a j o r m e t a b o l i t e , 3,4-dihyd r o x y p h e n y l a c e t i c a c i d ( D O P A C ) ; (2) D A synthesis as i n d i c a t e d by c h a n g e s in D O P A levels a f t e r i n h i b i t i o n o f A A A D ; (3) D A t u r n o v e r as e s t i m a t e d f r o m t h e d e c l i n e o f D A levels a f t e r i n h i b i t i o n o f A A A D o r T H .

2. Material and Methods 2.1. Animals Normal BALB/c mice and mutant homozygous rds (020/A) mice were studied. A breeding colony of rds mice is maintained at the Animal Facility of the University of Texas Health Science Center. The mice are housed in light proof rooms on a 12 h dark/light cycle, with light on at 8 a.m. Lighting is provided by fluorescent tubes. Light intensity at cage levels is 3-5 foot candles. BALB/c mice were bred locally or purchased from Harlen (Indianapolis, IN). The later were entrained for 1-2 weeks under the local light cycle before use. Mice were euthanized by cervical dislocation. Following enucleation the anterior structures of the eye and the lens were removed and retina dissected from the posterior eye cup. In the dark period all procedures were carried out under dim red light (Kodak safety light filter 1A).

2.2. Enzyme inhibition.s For AAAD inhibition mice were treated with m-hydroxybenzylhydrazine (NSD-1015; Sigma Chemical Co.) in light or dark periods. The drug was dissolved in phosphate-buffered saline at pH 7.0 and injected i.p. (15(I mg/kg). Retinas were isolated 3(} rain after injection. For inhibition of TH mice were treated with c~-methyl-i~l-ptyrosine methyl esther (AMPT-Sigma Chemical Co.). The drug was dissolved in saline and injected i.p. (300 mg/kgl. Relinas werc isolated 0, 30, and 60 min after injection. 2.3. Catecholamine analysis DA. DOPAC and DOPA levels were analyzed by high pressure liquid chromatography (HPLC) with electrochemical (EC) detection. Isolated retinas were wrapped in thin aluminum foil, quickly frozen in liquid nitrogen and kept at -70°C. Groups of 6-10 mice were analyzed at each treatment. Two retinas of each mouse were pooled to one sample. Frozen retinas were homogenized in ice cold 0.1 M perchloric acid, 10/~M ascorbic acid, 0.1 mM EDTA, and 20 ng/ml of 3A-dihydroxybenzylaniine (DHBA), an internal standard. A 20 pA aliquot of each supernatant fraction was injected into a Beckman Ultrasphere-ODS reverse phase column (5 ~m particle size, 25 × 0.46 cm; Beckman Instruments, San Ramon, CA). Catecholamines and catecholamine metabolites were eluted at a flow rate of 1.5 mt/min with a mobile phase consisting of 100 mM phosphoric acid, 0.1 mM EDTA, 0.45 mM sodium octylsulphate, 6% acetonitrile, adjusted to pH 2.6-2.7 with NaOH. Eluted catecholamines were quantitated by amperometric detection at a glassy carbon, thin layer electrode with an applied potential of 0.56 V vs. an Ag/AgCI reference electrode. Catecholamine peaks were identified by relative retention times compared to those of extracted standards. Concentrations were determined by comparing peak areas of unknown samples with those of standards using a programmed integrator interfaced with the detector unit. Values are corrected for the recovery of DHBA. Since photoreceptor cell loss resulted in lower protein content in rds retinas, expressing catecholamine content on a per mg protein basis results in artificially higher values in dystrophic retinas. As cell loss in the rds retina is limited to photoreceptors [27,47] and does not encompass inner nuclear and ganglion cells, expressing the data on a per retina basis is a more accurate presentation of data which compares normal and rds retinas. 2.4. Electron microscopy Following enucleation the anterior eye structures and lens were removed. The posterior eye cup was fixed in 4% formaldehyde and 1% glutaraldehyde in 0.1 M phosphate buffer at pH 7.0 for 90 min at room temperature. The posterior eye cup was bisected through the optic nerve and post fixed with 1% OsO 4 in 0.15 M phosphate buffer for 60 min at room temperature. The tissue was dehydrated and embedded in epoxy resin. Thin sections were cut from different regions of the retina along the central-peripheral axis and stained with uranyl and lead salts before viewing.

3. Results 3.1. Steady state levels o f D A a n d D O P A C light

in d a r k a n d

T h e d i u r n a l p a t t e r n o f D A m e t a b o l i s m was s t u d i e d in 1 - m o n t h - o l d m i c e . A t this a g e p h o t o r e c e p t o r differe n t i a t i o n is c o m p l e t e d in n o r m a l m i c e . In l - m o n t h - o l d

I. Nir, P.M. lut~one / Brain Research 649 (1994) 85-94

DA dark [ ] light

• o5 •~

0.4.

c w

.t, c

< a

3.2. DA synthesis and utilization in normal and rds retinas

0.2 0.0

BALB/c

rds

DOPAC

0"081 A (0 e= m

•

0.06

dark

I/--] l i g h t

t=

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0.02

B

In dark adapted normal retinas, levels of D O P A C were low, but increased by 2-fold in illuminated retinas (Fig. 1B). In rds retinas DOPAC levels were also low in the dark, however, unlike the normal retina there was no significant increase in light. Furthermore, the light levels of D O P A C in rds retinas were lower than the dark level of D O P A C in normal retinas (Fig. 1B).

0.3

0.1 A

87

ooo BALB/c

rds

Fig. 1. Steady state levels of D A and D O P A C in B A L B / c and rds retinas in dark and light periods. Mice, 1-month-old, were euthanized 4 h into the dark period and 4 h into the light period. Catecholamines were determined by HPLC-EC. Each value is the m e a n _+S.E,M. from m e a s u r e m e n t s of 8-10 mice (n = 8 - 1 0 ) . (A) DA: in B A L B / c and rds retinas there was no difference in steady state levels of D A in dark and light. There was a small non-significant decrease in D A levels in rds retinas, both in light and dark, compared normal retinas. (B) D O P A C : in B A L B / c retinas D O P A C levels increased 2-fold upon light exposure ( P < 0.05) while in rds retinas there was only a negligible increase in D O P A C levels in light. D O P A C levels in illuminated rds retinas were 3.5-fold lower in comparison with illuminated normal retinas ( P < 0.05).

rds retina about 60% of the abnormal photoreceptors are still viable [41]. Steady state levels of DA and D O P A C in retinas of B A L B / c and rds mice were determined 4 h into the light period and 4 h into the dark period of the imposed 12 h light/12 h dark cycle. The results are presented in Fig. 1. In normal B A L B / c retinas, steady state levels of DA in dark-adapted retinas were similar to those of light-adapted retinas. Comparable levels of DA in dark and light were measured also in rds retinas. DA levels in the rds retina in light and dark were slightly (14-16%) lower from levels in normal retinas (Fig. 1A).

B A L B / c and rds mice, 1 month old, were studied 3 h into the light period. Mice were injected with NSD1015 and euthanized after 30 min in light. Non-injected B A L B / c and rds mice were used as controls. The results are presented in Fig. 2. In non-treated mice, both B A L B / c and rds, D O P A was low and near the detection limit of the assay. In mice treated with NSD1015 there was an increase in D O P A levels both in the B A L B / c and rds retinas. However, levels of D O P A in normal B A L B / c retinas were 4.5 fold higher than in rds retinas (Fig. 2A). DA utilization was estimated from the decline in DA levels during a 30-rain period in light after inhibition of D O P A decarboxylase. In treated normal retinas there was a 6-fold decrease in DA levels as compared with non-treated retinas. In rds retinas there was only a slight, non-significant reduction in the DA level in treated retinas (Fig. 2B). In order to determine the diurnal variations of DA metabolism in B A L B / c and rds retinas, 1-month-old normal and rds mice were studied at 3-4 h into the light or dark periods after treatment with NSD-1015 (Fig. 3). In normal mice D O P A accumulated in the dark period, indicating the presence of substantial DA synthesis in the dark. There was, however, a 2-fold increase in D O P A accumulation in the light period. In rds mice, a very limited accumulation of D O P A was seen in mice treated in the dark, indicating a low level of DA synthesis. Furthermore, illumination of rds mice during the light period did not increase the D O P A accumulation (Fig. 3A). It is noteworthy that accumulation of D O P A in B A L B / c retinas treated in the dark was 3-fold higher than D O P A accumulation in rds retinas treated in the light. Treatment of B A L B / c mice with NSD-1015 resulted in a decline in DA levels in the dark period (Fig. 3B). A light-stimulated utilization of DA was indicated by the further decline in DA levels in the light period. In the rds retina there were no differences in dark and light levels of D O P A in NSD-1015 treated mice. Furthermore, DA levels in treated rds retina were only slightly reduced from DA levels in untreated rds mice in light and dark. DA turnover rates in 1-month-old normal and rds retinas were calculated from the decline in DA levels

88

I. Nit, P.M. lucom' / Brain Research 649 (l<)94) ,~¢5-94

DOPA 1.0 ~;~J c o n t r o l

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Ultrastructure of photoreceptors was studied in 12month-old rds retinas and 8-month-old normal retinas. In the old mutant only few remnants of photoreceptor structures were observed (Fig. 5). Steady state levels of DA were studied 3 - 4 h into the light period and compared with mice treated with NSD-1015 for 30 min in light. Steady state levels of DA were higher in old normal retinas with a 33% increase

0.4

< 0.2

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treated

rd$ control

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0.0 BALB/c

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Fig. 2. DOPA and DA levels in illuminated B A L B / c and rds retinas after treatment with NSD-1015 (150 mg/kg). Control mice were not treated. Mice, 1-month-old, were studied 3-4 h into the light period. Animals were euthanized 30 min after injection of the drug in light. Each value is the mean _+S.E.M from measurements of 10 mice (n = 10). (A) DOPA: in untreated mice DOPA levels were near the sensitivity of the assay. In NSD-1015 treated B A L B / c retinas DOPA levels were about 4-fold higher than levels in treated rds retinas (P < 0.05). (B) DA: there was a 5.8-fold decline in DA in treated normal retina (P < 0.05). In treated rds retina the decline in DA levels was not significant.

C m

v

<

B

after inhibition of T H with A M P T (Fig. 4). The half-life of D A was 7-fold lower in rds retina in comparison to normal. D A synthesis rate, calculated from these data, was 8-fold lower in the mutant retina.

3.3. DA synthesis, utilization and photoreceptor structure in old mice As rds mice age and cell loss progresses, residual light input invariably decreases further. To study D A metabolism under conditions of prolonged light deprivation, 12-14-month-old rds mice were investigated. In addition, 8- and 12-month-old normal mice were studied and compared to 1.5-month-old normal retinas.

u.v

BALB/c treated

rds

treated

rds

control

Fig. 3. DOPA and DA levels in B A L B / c and rds retinas in dark and light periods after treatment with NSD-1015. Control rds mice were not treated. Mice were studied 3-4 h into the light or dark periods, Each value is the mean+S.E.M, from measurements of 6 mice (n = 6). 9A) DOPA: In normal retinas there was a significant accumulation of DOPA in the dark, about half of the light level (P < 0.05). In rds retinas there was no difference between the dark and light levels of DOPA. In untreated rds retinas, DOPA was not detected either in light or dark. 9B) DA: there was a considerable decline in DA levels in treated normal retinas in the dark. However, a still larger decrease was measured in illuminated retinas. The levels of DA in normal retinas treated in the light was 2.5-fold lower than in normal retinas treated in the dark ( P < 0.05). In rds retinas there was a small, not statistically significant reduction in DA levels in retinas treated in dark and light. However, there was no difference in DA levels between rds mice treated in light and dark.

L Nir, P.M. luvone/Brain Research 649 (1994) 85-94

1.000"

_= u

<

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0 0,01 0

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Synthesis rate pg/mln/retlna

BALB/c

12.4

40.9

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82.8

5.15

Fig. 4. Dopamine turnover and synthetic rates in 1-month-old BALB/c and rds retinas Mice were studied 3 h into the light period. Animals were treated with AMPT (300 mg/kg) in the light and euthanized 0, 30 or 60 min later. Each group was of 6-7 mice. Synthesis rates and half-life were calculated from the decline in DA levels after treatment with AMPT [8].

89

in DA level in 8-month-old retinas over levels in 1.5 month old retinas. A further 16% increase was measured in 12-month-old normal retinas (Fig. 6A). In 12-month-old rds retinas steady state levels of DA were comparable to those of old normal retinas. Although DA steady state levels were high in old rds retinas, DA utilization in light was low in comparison with normal retinas. Inhibition of D O P A decarboxylase with NSD-1015 caused only a 36% decline in DA level in 12-month-old rds retinas whereas a 78% decline was measured in 8-month-old normal retinas (Fig. 6A). D O P A accumulation in light after treatment with NDS-1015 was 3-fold lower in old mutants in comparison with old B A L B / c retinas (Fig. 6B). In order to determine whether remaining DA synthetic capacity in old rds retinas is sensitive to light exposure, light/dark differences in D O P A C and D O P A levels were examined in old rds and B A L B / c retinas. Steady state levels of D O P A C were significantly higher in retinas of old B A L B / c mice in light compared with those in darkness (Table 1). The magnitude of the light/dark differences in D O P A C levels of old B A L B / c was comparable to that seen in young normal mice. Similarly, retinal D O P A accumulation in old B A L B / c mice was higher in light than in darkness, and was similar to that at 1 month of age. In retinas of

Fig. 5. Ultrastructure of 8-month-old BALB/c retina (A) and 12-month-old rds retina (B). (A) In old BALB/c retina intact photoreceptor cells are seen which consist of outer segments (OS) inner segments (IS) and nuclei (ONL). The outer segments are juxtaposed against the pigment epithelium (PE). The subretinal space between the pigment epithelium and outer segments is occupied by pigment epithelium microvilli (*). (B) In old rds retina most of the photoreceptor cells degenerated. In the region depicted in this figure there are no photoreceptors. Nuclei of inner retinal layers (INL) are juxtaposed against the pigment epithelium (PE) microvilli (*).

1. Nir, P.M. ha'one/Brain Research 649 (1904) 85-q4

9(}

Table 1 l)ay-night variations in DOPAC levels and I)OPA accumulation in retinas of young and old B A L B / c and rds mice

DA 0.8"

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1 2m

BALB/c 1 2m

DOPA 1.2-

1.0. r(g

0.8,

[ ] control

e-

0.6.

[ ] treated

v

0.4. 0.2.

B o.o P'22 BALB/e 1.5m

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12m

Fig. 6. DA and D O P A levels in old retinas during the light period after treatment with NSD-1015. Twelve-month-old rds mice were studied together with 8-month-old rds mice and 1.5-month-old B A L B / c mice. Control mice were not treated. A group of 12-monthold B A L B / c retinas were analyzed only for steady state level of DA. Mice were studied 3-4 h into the light period. Each value is the mean ± S.E.M. from measurements of 6 mice (n = 6). (A) DA: an increase with age in steady state levels of DA in normal retinas was observed. DA levels in 8-month-old B A L B / c retina was higher than in 1.5-month-old ( P < 0.05), while levels in 12-month-old normal retinas were still higher than in 8-month-old ( P < 0.05). High steady state levels of DA were measured also in 12-month-old rds retinas. These were not significantly different from level in normal retinas of-the same age. A 7.5-8-fold decline in DA levels were measured in both 1.5- and 8-month-old normal retinas treated with NSD-1015 ( P < 0.05). In 12 m old rds retinas a much smaller (1.5-fold) decline in DA level was measured in treated retinas (P < 0.05). (B) DOPA: large accumulation of DOPA was measured in normal retinas with no significant difference between 1.5- and 8-month-old mice. In 12-month-old rds retina accumulation of DOPA was limited, about 3-fold smaller than in 8-month-old B A L B / c retinas (P < 0.05). In this experiment improved sensitivity of the analytical procedure resulted in detection of DOPA in untreated retinas. The DOPA levels are very low and a small difference between old normal and rds retinas is not statistically significant.

Condition

Age (months)

rds Light Dark Light Dark

2 2 12-14" 12 14

BALB / c Light Dark Light Dark

I I 8-12 a 8-12

I)OPAC :' 59± 3t~+ 92± 54+

DOPA ~'

(7 4" 7 7"

[75~14 150+ 10" 270±24 190±34"

203 + 16 83_+ 4 " 180 + 43 90± 7 "

1150 ~ 97 590±46" 1092 + 3 I 478±72"

Data at different ages were compiled from 4 independent experiments with different litters of mice. All measurements were made 3 h into the light phase (day) or the dark phase (night) of the imposed 12 h light/12 h dark cycle. ~' Steady-state levels of DOPAC were measured in retinas of untreated mice. ~' Retinal DOPA levels were measured 30 rain after injection of NSD-1015 (150 mg/kg, ip). DOPA accumulation was determined in 12 mo old rds mice; DOPAC levels in 14 mo old mice. d DOPA accumulation was determined in 8 mo old B A L B / c mice: DOPAC levels in 12 mo old mice. Data are expressed as m e a n + S.E.M. in pg/retina. N = 6 - 1 0 . * P < 0 . 0 5 dark vs light, in rds retinas DOPAC levels both in light and dark were signifcantly higher in old retinas P < 0.05.

l-month-old rds mice, steady state DOPAC levels and DOPA accumulation were low compared to those in B A L B / c retinas, and no significant light/dark differences were observed (see Fig. IB and Fig. 3A). Remarkably, steady state levels of D O P A C both in light and dark increased significantly with age in rds retinas, although not attaining levels comparable to those in B A L B / c retinas by 1 year of age (Table 1). In retinas of 2 month and 1 year old rds mice small light/dark differences in D O P A C levels were measured. Steady state levels of D O P A C were significantly higher in 14-month-old rds retinas than in 2-month-old rds retinas, both in light and dark. Furthermore, a significant light/dark difference in D O P A levels was observed in retinas of old rds mice but not in young rds mice (Table 1; Fig. 3); this observation was confirmed in a seperate experiment with other 1-year-old mice (data not shown). 3.4. DA synthesis and utilization in immature retinas The onset of significant photoreceptor cell loss in the rds retina occurs during the third postnatal week. In order to study DA metabolism in a mutant retina in which most of photoreceptors are still viable, 14-day-old mice were analyzed. Normal B A L B / c retinas of similar age were also studied. In normal mouse retina photoreceptors are already differentiated by 14 days,

I. Nir, P.M. luvone / Brain Research 649 (1994) 85-94

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< D

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l

c

14d

08] 0.6'

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DOPA

[ ] control [ ] treated

k.

C

0.4

v

< 0.2

B

0

normal. In treated rds retinas even such limited decline in DA level was not observed (Fig. 7A). DA synthesis as determined by accumulation of DOPA in NSD-1015 treated mice was very limited in 14-day-old normal retinas. DOPA accumulation was much lower than that in 1-month-old normal retinas. DOPA levels in treated 14-day-old rds retinas were even lower than the levels in normal 14-day-old retinas. Thus, already at a very early stage of the retinal dystrophy alterations in DA metabolism might be evident in the rds retina (Fig. 7B).

¢Lf./ ' / F ,j R~Z n I

0.0 14d

A IB e" m

91

. rds 14d

0

~ BALB/c 14d

BALB/c lm

Fig. 7. D A and D O P A levels in immature B A L B / c and rds retinas after treatment with NSD-1015 in light. Control mice were not treated. Mice were studied 3 - 4 h into the light period. Each value is the m e a n +_S.E.M. from m e a s u r e m e n t s of 6 mice (n = 6). (A) DA: steady state levels of D A were about 4.5-fold lower in 14-day-old B A L B / c than in 1-month-old B A L B / c retina ( P < 0.05). Similarly low D A levels were measured in untreated 14-day-old rds retina. A small, not statistically significant decline in D A levels was measured in treated 14-day B A L B / c retinas. In rds retinas there was no decline in D A level after treatment with NSD-1015. Note that the low D A levels in treated 1-month-old normal retinas are similar to the steady state levels of D A in 14-day-old retinas. (B) D O P A : in treated 14-day-old B A L B / e retina D O P A levels were 3-fold lower than levels in 1-month treated B A L B / c retinas ( P < 0.05). Levels of D O P A in treated 14-day rds retinas were still lower ( P < 0,05). D O P A was not detectable in control retinas.

although outer segments would not reach their full mature length until 1 month of age. DA levels in 14-day-old normal retinas were approximately 20% of those in 1-month-old normal mice (Fig. 7A). The low steady state level of DA in 14-day-old normal retinas was similar to that of 14-day-old rds retinas. DA utilization, as determined by decline in DA levels 30 min after treatment with NSD-1015 in light, showed very little decline in DA level in 14-dayold normal retinas in comparison with 1-month-old

4. Discussion

A number of metabolic processes in the retina demonstrate diurnal rhythms: cellular activities such as disc shedding and retinomotor movements [5]; hormone levels [49]; changes in the position of interphotoreceptor matrix components [55]; and expression of several genes in the photoreceptor cells [7,14,33]. In studies of disc shedding and retinomotor movements in amphibian and fish retinas DA was found to promote the light adaptive phase of the diurnal cycle [4-6,16,44]. In the present study of normal mouse retinas, diurnal variations in the steady state levels of DA were found to be small, with only a slight increase in DA levels in the light period. In the study of another mammalian species an increase of 13-20% in DA level in the light was reported in the rabbit retina [42,43]. Similar increases in light levels of DA were reported for rat retina [57]. A larger increase in light levels of DA in rat retina was observed in one study [34], but in others no dark and light differences in the levels of DA were observed [12,15]. Experimental conditions such as light intensity and duration of illumination might account for the reported differences in the diurnal levels of DA. In the dystrophic rds retina, steady state levels of DA were only slightly reduced from DA levels observed in normal mouse retinas both in light and dark. The major impact of photoreceptor degeneration was manifested by the reduced rates of DA turnover as both synthesis and utilization were greatly reduced. DA synthesis was 8-fold lower in the rds retina. Apparently reduced light perception by aberrant photoreceptors in rds retina results in lower trans-synaptic activation of TH in dopaminergic cells in the retina. As DA release from dopaminergic stores is also light dependent [31], lower light input in the rds retina similarly affected the rate of DA turnover. In young rds retinas very limited light-evoked changes in dopamine synthesis and turnover were measured. Interestingly, in old rds mice a small but significant light-evoked elevation in dopamine metabolism was measured, although very few photoreceptors nuclei

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remained in old rds retinas [47,48]. Ultrastructural analysis of old rds retinas which were utilized in the present study revealed only few remnants of photoreceptor structures. The increase in light-evoked D A metabolism in old mice might reflect an adaptation of dopaminergic system in the retina to function under conditions of very low illumination. Previously, low levels of the photopigment opsin were detected in photoreceptor remnants in old rds retinas [41]. Apparently photoreception mediated by remaining opsin molecules may account for light-evoked T H activity in very late stages of photoreceptor degeneration. In 14-day-old normal mouse retinas, 2 days after eye opening, steady state levels of DA were considerably lower than adult levels. DA synthesis and utilization, as measured by D O P A and D A levels in NSD-1015 treated mice, were also much lower than those in adult retinas. In the normal 14-day-old retinas photoreceptor outer segments are of substantial length, although not at their full adult level. Hence, whereas the mechanism for light perception is available, D A metabolic responsiveness to light is not yet developed. By 6 weeks steady state levels of D A were still significantly lower in the young retinas in comparison with old retinas. However, at this age both DA utilization and D O P A accumulation were comparable or even slightly higher in the younger retinas. This indicates that by 6 weeks the metabolic machinery was fully developed and the lower steady state levels of D A are probably due to limited DA storage capacity. These observations are compatible with the development of DA synthetic and storage machinery in the rat retina [15,36]. In 14-day-old rds retinas D A levels and turnover were also greatly reduced. However, already at this age differences were observed between mutants and normal retinas. Hence, reduced DA synthetic capability and utilization could be detected at an early age in the rds retina, prior to onset of significant photoreceptor cell loss (at the third postnatal week). Clearly, limited light reception in the absence of outer segments is the underlying cause of malfunctions in D A metabolism in rds retinas. Reduced turnover of D A in mutant retinas indicates that although high steady state levels of DA are maintained, it might not be available to exert its biological functions. The alterations in D A metabolism may affect a variety of retinal functions. D A modifies the activity of horizontal cells [21,58] and ganglion cells [28]. There is a growing body of evidence on the role of DA in photoreceptor metabolism. D A receptors were found on retinal photoreceptors. In mammalian retinas D2-like D A receptors were detected on photoreceptor cells in rat retina [51,54] and human retina [17]. The direct role of DA in regulation of photoreceptor metabolism was indicated by the specific effects of D A receptor agonists and antagonists on photoreceptor

functions. D2-1ike DA receptors were associated with retinomotor movements in fish and amphibian retinas [16,32,44]. In the amphibian retina, DA and D2 receptor agonists were shown to inhibit disc shedding through a D2 receptor mediated mechanism [5]. Inhibition of DA synthesis during early light phase caused a significant dampening of light response in disc shedding in the rat [46]. DA and DA agonists also inhibit melatonin synthesis and release from amphibian photoreceptors [9]. In the mouse retina, DA and D2 receptor agonists were shown to reduce a light sensitive pool of cAMP in photoreceptors [10]. DA D4 receptors, a D2-related subtype, might be involved in modulating cAMP levels within mouse photoreceptors [11]. It was recently suggested that DA may mediate the effect of light on accumulation of S-antigen (arrestin) m R N A and possibly other messenger RNA species in the rat retina [51]. Changes in opsin levels in rat photoreceptors following unilateral optic nerve section may be mediated by DA through a second messenger effect on gene expression [50], In view of reported and implied role of DA in photoreceptor metabolism it will be of interest to determined the consequences of DA abnormalities in the rds retina on photoreceptor functions and possibly cell death.

Acknowledgments Thanks are extended to Nancy Ransom and Bonnie Johnson for excellent technical help and Kim Hicks and Marian Osborne for help in preparing the manuscript. The research was supported by N I H (EY6892 and EY-4864) and a grant from The University of Texas Health Science Center at San Antonio. Parts of the data were previously published in abstract form [38,39].

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