- Email: [email protected]
The slow ERG potentials in hereditary retinal dystrophy of albino and pigmented rats
Exp. Eye Res. (19i6) 22, 493-503
The Slow ERG Potentials in Hereditary Retinal Dystrophy of Albino and Pigmented Rats* EUGEXE L.
PATTLER?
AXD
\~ERSER
K.
~OELL
Nemosensory Laboratory, School oj Medic&. Statr University of Xew York cct Ry@lo. N.Y.. 7’S.A. The slow ERG potentials of different sites of generation-PI, PII-related and Slow PIII~erc st.udied at different ages in normal and mutant rats reared in a dark environment. The manifestations of these potentials in the normal rat were similar to the adult rabbit although their amplitudes in comparison to the b-wave were lower. They were peculiarly dependent on age and were highest during the fifth week of age. In the adult, normal pigmented rats, early PI components slightly exceeded Slow PI11 but Slow PI11 dominated in the records from adult albinos, In the mutant albino or pigmented rats, all slow ERG components were evident at the age of 21 days except the late posit’ive transient of PI. The negative transient of PI disappeared after 26 days but a slow c-wave survived beyond 5 weeks. The iodate-isolated Slow PI11 was better preserved than any PI component but it also was sluggish in response to the cessation of illumination. Responses to azide and thiocyanate were well preserved in 4-month-old mutants. The preservation of PI was generally better at any age for the pigmented than the albino mutants. An interpretation of the findings, based upon previous analyses in the rabbit-Slow PI11 originating in the distal Miiller cell process, PI generated by the pigment epithelium-leads to the conclusions (a) that the electrical properties of the pigment, epithelium are not primarily affected in the dystrophic rat and (b) that the accumulation of debris impedes the chemical communication between still functioning photoreceptors and pigment epit,helium as well as d&al Miiller cell processes.
1. Introduction Hereditary visual cell degeneration (also called retinal dystrophy) in the rat is one of several useful models providing information which may enrich our understanding of degenerative retinal diseasein man. The rat disease.first described by Bourne et al. (1938) is characterized by the selective deterioration and death of photoreceptor cells commencing during the third week of life (Dowling and Sidman, 1962; Noell. 1963, 1965). Accompanying visual cell degeneration is the accumulation and preservation of rhodopsin-rich, lipid-depleted membranous material. mainly of outer segment origin. called debris, between pigment epithelium (PE) and the surviving retinal layers when the animals are kept continuously in a dark environment (LaVail, Sidman and O’Neil, 1972; Organisciak and Noell. 1976: Delmelle, Noel1 and Organisciak. 1975). The progressof any diffuse visual cell disorder is easily monitored by the measurement of the trans-ocular potentials. Recordings of this kind have been restricted to the measurementsof the a- and b-waves of the ERG in the rat disease,while in human patients, the electro-oculogram (EOG) is an additional and easily applied diagnostic tool. The latter provides information on slow potential changes or d.c. potential shifts with a step change in illumination. The interest in these slow shifts in d.c. potentia.1 relates to the experimental * Supported by NIH Grant. t Permanent address: Department (‘oIlins, Colorado 80581, U.S.A.
of Physiology
and Biophysics.
Colorado
State
University,
Fort
A!14
I‘::. I,. l’.41.7’1,Kl: ASD \v. K. pu‘OlcI,I,
obticrration on niainnia1s t,hat the (l.c. transretinal current8 and the slow potential changes in response to light 011 or of are mainly generated by the PE and involve’ t,he action of light on the photoreceptors (Noell, 1953; Faber, 1969). *Measurement of the slow potential changes in conjunction with the analvsis of the II- and C-wavc~s thus may provide information on cert,ain aspects of the’ function of PE as well as on the “conllllullicatior1” between phot,oreccptors and PE. The means by which this eonnnunication operates is still unknown but it must involve a chemical linkage through the intercellular space between the two cell layers. The present study reports on characteristic features of the slow potentials in normal and dystrophic rats. The nomenclature to be used follows the original designations of ERG component,s (PI. II, III) by Granit (1947) and distinguishes between three different generating systems for the slow changes (Faber, 1969).
2. Methods A11 pigmented and albino rats (normal aud abnormal) of this study were from long established colonies of the laboratorv. They had been reared in a (lark environmerit (Delmelle et al., 1975). Controls were mainly phenon~enologically normal heterozygous litter mates from back-crosses selected by electroretinographic criteria. Connnerciall~ purchased adult albino (Charles River Laboratory) aud pigmented rats (Lcng Evans strain) were additionally used as adult. controls. The abnormal rats will be rzferretl to as ‘mutants”; they are genetically of the same origin as t,he Ba (albino) and B (pigmented) at.raius described by Delmelle et al. (1975). For ERG recordings, rats, 3-7 weeks old, were anesthetizedwith 20 mg/kg of Nembutal”. This was usually sufficient to minimize overt movemems. For adult rats 35 mg/kg of Nembutal was used supplement,ed by small additions as necessary for surgical procedures. Local anesthesia (2% Xylocaine”) was applied around the eye. The pupils of pigmented rats were dilated with 1:/h &opine su1fat.e. Recordings were made by cot,torl wicks threaded through a small polyethyleue tube filled with saline, connectiug t,o a Ag AgCl wire. One of these electrodes u-as iusertetl behind the palpebral conjunrtiva for a distance of 223 mm, the other placed on the cornea. A needle electrode under the scalp conuect.ed to ground. Responses were recorded from a Tetronix 5103N oscilloscope aft,er pre-amplificat’ion by a Grass P16 d.c. amplifier. The light sources employed were a Grass Photostimulator (Pa) and a Sylvania R1131C glow modulated t,ube electronically controlled. The maximal illumination (I = 0) of the glow stimulat.or was about 4.5 log units above the 20 p,V h-wave threshold of normal rats at the age of 5 weeks. It was reduced by neutral density filters in 1 log-unit steps. The light was channeled directly to the eye bv fiber optics. The brief and int’ense flash of the Grass stimulator was used for the analysis of n- arid b-waves while the glow stimulator served in the measurements of the slow components. The stimuli were presented in 3.u ordered sequence at npproximatelv 2-niiu intervals. Testing was performed under a lo\\-intensity red background light. Glowlight, stimulation was for 5 and 25 see. Azide and thiocyanate respouees were elicited by a rapid intravenous (externnl juguklr vein) iujection of 0.3 ml of 1.6 mM-sodium nzide and 60 m>f-sodium thiocyaunte. respeatively. Both salts were dissolved in a bicarbonate buffered solution, replacing NaCl as needed for normal osmo1arit.y. At. least oue eye was taken from each animal group for histological examination. 3. Young
norrd
albino
The slow ERG amplitude during
Results
rats
waves elicited with stimuli of 5-set duration reached their highest the fifth week after birth in normal, albino or pigmented ra.ts reared
SLOW
ERG
IN
RETIIL’AL
DYSTROPHIC
495
RATS
and continuously maintained in darkness. Depending upon the intensity and duration of illumination, at least three components of different time course and, presumably, of different origin were apparent at this age. gt a low intensity of illumination (l-5 log units above b-wave threshold) the dominant component of the slow potentials was a steadily maintained positivit,y appearing almost immediately upon the rise of the &wave [Fig. l(a)]. Upon the cessation of illumination, this potential rapidly disappeared, as indicated by a steplike change from positivity to the control (l.c. level. This step change at the cessation I=-3
I:-2
,)I -III0 r I\ ’ I if”’ 0.2
mV
#‘~a. 1. Tracings (a)-(d) are from a 31-day-old normal albino rat: responses are elicited by flashes of increasing intensity. Stimulus intensity is expressed in log units (I); I = 0 is the maximal stimulus, approximately 4.5 log units above b-wave threshold. Stimulus duration is indicated by the heavy bars beneath the tracings. Calibration is the same for all tracings and indicated by the vertical bar (0.2 mV) at the right-hand corner. Tracings (e) and (f) are recorded from 34-day-old normal albino rats at slow sweep speed (5 cm/set) and illustrate the late negative PI transient (e). and Slow PI11 (f) after the elimination of PI components by previous iodate administration.
of illumination was the main or sole indication of the presence of this component at high flash intensity because other slow potentials then dominated in amplitude. It was absent when b-wave generation was blocked by deep anesthesia or anoxia, while other slow potentials and the a-wave were preserved. The potential will be referred to as the PII-related component (c.f. Brown, 1968). A slowly rising positivity was superimposed upon the PII-related component when flash intensity was more than 3 log units above b-wave threshold [Fig. l(b)-(d)]. This slowly rising positivity of the rat ERG had the same time course as the c-wave of the rabbit (Noell, 1953), although its amplitude never reached the same height in relation to the b-wave. Following its peak at about 3 see, this c-wave declined almost linearly to below the baseline to be succeeded by a slow negativity [Fig. l(e)].
4198
E. L. PAUTLEK.
AXD
11’. Ii.
SOE:LL
In the dark-adapted rabbit, this negativity reaches it:: maximum about, 40 XX after the beginning of continuous moderate illumination: the potential then slowly rise? again, passes through the control t1.c. level at about 2.5 niin of illumination, ant1 reaches after additional 5-10 min of continued illumination a posit’ive level. 0.5 t’o 2 mV above the d.c. level during the control period (Noell. 1953). In our experiments on the rat,, illumination was terminated no later than at 25 M:C [Fig. l(e)]. At this time, the slow negativity was still developing. Clessation of illumination then was followed. within lo-20 sec. by a slow rise to above the baseline [Fig. 4(a), (c)l. This late positivity reached its peak about 1 min after the termination of t’he 25 set flash and was about as high as the earlier c-wave. Previous studies on the rabbit provide evidence that (1) the c-wave, (2) t.he slow negative transient and (3) the late positivity are all dependent upon the integrity of the pigment epithelium (Noell, 1953). W e will refer to these potentials as the PI component(s) of the response (Faber, 1969). 1=-l
I=-2
11
11
11
11
(J/Y.++
1 I
+- ;‘ei0.2mv \
I\ O
/
\
Off
L
‘zq\/--+w -W I
’
1
’
11 +I
11 I-+
1 lsec
I
11
I”“1
II +5sec
FIG. 2. Tracings (a)-(d) are from one mutant albino dark-reared rat, 21 days old, recorded as in Fig. 1. Flash intensity (I) varies. (d) is the response of the dark-reared rat, for the first time, to a sbrong flash of light of 5 set duration; this “first” response was typically lower than subsequent responses in the mutants; to a lesser degree this was also observed in normals. Sweep speed is slower in (e); the second tracing (marked “2”) is an additional sweep following immediately upon the first sweep. The onset of the flash is indicated by the a- and b-wave deflections in the first tracing; cessation is at “off” in the same tracing.
The third major slow component of the rat’s ERG became evident when the PI components were eliminated by the administration (i.p.) of sodium iodate, 15-24 hr prior to the measurements. As illustrated in Fig. l(f), a negativity of considerably larger amplitude than the c-wave was the dominant feature of the response.It reached its maximum usually within 5-15 set depending upon stimulus intensity. When the preparation was very responsive and the stimulus strong, as in Fig. l(f), it had an early maximum around 3 set and a less negative, almost steady level thereafter. Upon cessation of strong illumination 25 set after “on”, the negativity decayed initially almost as fast as it developed but tended to persist partially for one minute or more.
SLOW
ERG
Ip; KETIXAL
DFSTROPHIC’
RATS
497
In accordance with previous studies on the rabbit, the negativity after iodate poisoning will be referred to as Slow PI11 (Faber, 1969). It was identified by extensive depth profile measurements. It is a normal component of the ERG but masked to varying degree by the PI potentials. It is most probably of Miiller cell origin, manifesting a hyper-polarization of the distal Miiller cell processes. The simultaneous PI component providing cornea-positivity (c-wave) most probably results from the hyper-polarization of the apical PE membrane (Steinberg. Schmidt and Brown. 19X).
The earliest stage at which animals of t,he mutant strain mere examined was at 21 day3 after birth. At this age, the mutants differed little in a- and b-wave amplitudes from controls. The histological signs of the degenerative process also were apparent Normal
albino
Mutant
0 (*)I
0 !f-
albino
I 0.2
mV
(h). d-
-4 Lf-
FIG. 3. (a)-(d): c-wave occurrence at the age of 26 days Recording is as in Fig. 1. Tracings (e), (f) are from Z-day-old ones. Calibration is the same for all tracings.
in normal and mutant albino litter-mates. litter-mates. and (g), (h) from 76dday-old
only to a slight degree over most of the retina. This includes the signs of visual cell death and debris accumulation in front of the PE (Delmelle et al., 1975). As illustrated in Fig. 2, the slow potentials were, at this age, not strikingly different from those of the normal rat. Nevertheless, the following deviations from the best normal records were consistently observed : (1) a slowing of the c-wave, (2) a slower than normal decay of the negative PI transient, and (3) a weak or missing late PI positivity.
1!H
12. L. l’AI~‘l‘LR~t
AK I) tv. K. NOELL
Figure 3 compares the records of normal and mutant albino rats in resporise to light stimuli of 5-set duration at the ages of 26, 32 and 76 days. ;1t the age of 26 days the cl-wave of the nn&nt was significantlv reduced (I>elmelle et al.. 1975). The steacly (l.c. dark potential was within the same amplitude range, :! 4 IIIV. as in the cont~rols but the c-wave clearly differed from normal. It rose at. a.much slower rate and deeavetl less rapidly after t,he end of illuniinat,ion. 111fact, it tended to grow after cesqation of illumination. Its amplitude relative to the h-wave was slightly rctluced 5 ties aft,er “on”, cspeciallv in response to a weak flash [Fig. 3(b)]. Normal I-O,
Mutantolblna
albino 26 days
(b)
I
32 days
(0.2 If
mV
1 ,-
I”
FIG. 4. Recordings from the same animals sweeps in each tracing follolv immediately
”
”
’ ‘J b-5 set
as in Fig. 3 with the lower sweep speed (5 cmjsec). upon the preceding sweep as in WK. 3(e).
Atlditionaf
The development of the c-wave in the normal dark-reared animal reached its peak around 32-37 days [Fig. 3(e)]. In th e mutants, the c-wave appearance also was somewhat greater at 32 days [Fig. 3(e)] than at 26 days [(d)], despite enhanced b-wave deterioration. At 76 days [Fig. 3(h)]? mainly a small b-wave had survived together with a PII-related potential which at all ages of the mutant was a,s well evident as the b-wave [Fig. 3(b), (d), (f)]. Evidence for the behaviour of late negativity and late positivity is provided in Fig. 4 and Fig. 5(a), (b). Compared to the controls of the sameage, the late positivity (late increase above baseline) had disappeared already at the age of 26 days. It was absent also in the records from the 21-day-old mutant of Fig. 2. Although it was observed at 21 days, the late negativity was missing at 32 days. Thus, the PI components seemedto he represented exclusively by a slowly rising and falling c-wave after 26-28 days. The preservation of Slow PI11 at the age of 32 days was suggested by a sharp negativity interposed between the b-wave and the c-wave [Fig. 4(d)].
SLOW
ERG
In- RETINAL
DTSTKOPHIC
499
RATS
More precise information on the preservation of slow PI11 was obtained by studying the mutant rats after iodate injection [Fig. 5(c)-(e)]. Slow PI11 was surprisingly well preserved at the age of 35 days when the a-wave was at best one-third of the normal amplitude. Highest PIIIs after iodate poisoning at this age were half the amplitude measured in controls. Slow PIII, however, was sluggish, especially its decay to the baseline after the end of the maximal (I = 0) illumination. It, was faster at a reduced int,ensity (I = 1). Mutant
albino
I=0 (a)
I
0.2 mV
(d)
PK. 5. (a); the slow rise and decay of the c-wave at the age of 31 days illustrated by successive sweeps; the tlash was turned off during the first sweep as indicated. (b) is recorded with slow sweep speed in a 2%day-old animal in which the b-wave was much reduced due to rxceasirc Nembutal; note that the sudden d.r. shift with the cessation of illumination is also much reduced; the initial negative transient at the start of illumination is a manifestation of Fast and Slow PIII. Recordings (c) and (d), (e) are from two animals aged 35 and 28 days respectively, which were injected with sodium iodate, 24 hr before t&kg; they illustrate the preservation of Slow PIII: the h-wavr was absent in these animals as a result of the iodste poisoning.
Figure 6 illustrates the slow potentials in the pigmented mutant rats. Essentially the same results were obtained as in the mutant albinos illustrated in Figs 2-5. The strong impression was gained, however, that the deterioration of the slow potentials in the pigmented animals proceeded slower than in albinos, although both strains were maintained in the dark environment. Especially, the sluggishness of the reactions to the cessation of illumination was less pronounced than in the albinos. This was true for the decay of the c-wave after “off” as well as for the decay of the iodate-isolated PHI. Comparison
of adult normal
albino and hoodedrats
Dodt and Echte (1961) reported that the adult pigmented rat has a c-wave of up to O-6mV, whereas no such wave is evident in the al’)ino rat. In our experiments,
500
Il. L. I’ACTLEK
AND
\\‘.
K. XOELL
alhino as well as pigmented animals generated c-waves at the age of 3:! days which approached 1.2 mV in amplitude during illumination approximat.cly 4.5 log 1lnit.s ahove b-wave threshold. Figure 7(a). (1~).showsthe records from 4months-oh1 albino and pigmented rats. The commercially purchased animals had been in t,he dark environment for lessthan one week. In responseto the standard illumination (I = 0). Slow PI11 negativity dominated in the albino. rvhereas in the pigmented animal, a c-wave followed the b-wave, and reached approximately 50% of the h-wave atnplit~ude. These were t,ypical findings. Mutant
pigmented
1~0, 26 days
29 days (e). -4 I
tf A 34days 4 ?J
10.2
mV
FIG. 6. All tracings are from pigmented mutants of different ages. Left column (a), (c) are recorded at 1 see/cm sweep speed; right column (b), (d) are from same animals using 5 secjcm sweeps. Tracings (c) and (f) are obtained after t,he administration of iodatr 24 hr preriously (different animals).
In order to ascertain whether or not the electrophysiological properties of PE were innately different, the two strains were compared for their ability to generat.e an electrical responseto the rapid injections of sodium azide and sodium thiocyanate (Noell. 1963 and unpublished). The responsesto both ions [seeFig. 7B, (a), (h)] were in 4 out of 5 albino animals 20% lower than for the average of 5 hooded rats. Figure 7(h), (d) illustrates two exceptional findings. Tracing (h) (A) showsthe ERG of an albino rat in which the initial negativity manifesting Slow PI11 is followed hy a delayed c-wave rise to positivity which wasnot attained in the other four albino rats. Interestingly, the responsesto azide and thiocyanate [(c) (B)] were also greater in this animal than in any of the others. Record (d) (A) is from a hooded animal. The ERG was characterized by an exceptional PI11 negativity. The responsesto the rapid injections of the anions illustrated by records (d) (B) also were higher than in the other pigmented rats, as if the resistive network for the transocular current distribution was more favorable than usual for recording slow potentials of high amplitude.
SLOW Normal
ERG
IN
RETINAL
Normal
albino
I (a’ II “‘w
L
Azidr--
I0 ‘tThlocyanate
RATS
561
pigmented
h Yv--
I=0 A
DYBTROPHIC
A(cl
(b
b-
P-, ( e 1 Mutant
Cd)
i-
IT
V
7r-/+-
5mV
Ssec
FIG. 7. Comparison of adult normal albino and hooded rats. Upper (A) (a) and (c) typify the difference between the two strains; (b) and (d) are exceptional records (see text). The loner part of the figure (B) illustrates the responses to 1.6 rn>r-aside and 60 mivr-thiocyanate injected rapidly into the jugular vein, for the same animals as in A. B(e) is from a mutant albino rat, 3 months old; its ERG is extinct (compare with B(a)). The responses to aside are illustrated in the upper row of B; those to t~hiocyanate in the lower row. As indicated by the calibrat,ion line (5 m\‘), the responses to these anion injections range from 6 to 15 mv.
4. Discussion The transretinally measured electrical potentials of slow time course generated in response to a change in retinal illumination are rarely studied for probably several reasons: (I) as with all d.c. measurements within the range of l--d mV, their recording requires attention to numerous details; (2) the rate of change of some of these components is very slow ; a steady-state d.c. level may not be reached within 30 min after a single step change in illumination ; (3) they are variable from one preparation to another even when conditions appear well controlled; (4) they are very dependent upon the state of the animal (or the eye) e.g. the dose of the anesthesia agent, the state of circulation and ventilation ; (5) differences among animal species are much greater than for CL-or b-wave. even between mammals and different strains, and (6) they are complex in origin. The observations of OUT study illustrate several of these points. Nevertheless. the slow potentials provide information on important aspects of ret,ina,l physiology and pathology. As analyzed in the rabbit, they reveal, first of all. that a change in retinal illumination elicits photoreceptor reactions which affect the chemical environment of pigment epithelium and Miiller cells. The pigment epithelium reacts to these changes with the generation of a complex series of electrical transcellular potential changes, of which the cornea-positive c-wave of the classical ERG literat’ure is just one manifestation. It is well established that the c-wave of rabbk
302
E. L. PAY1‘1,EII
ANT1
\V. K. KOELT.
anal cat is caused by a hyperpolarization of the apical plasma membrane of PH. but little is known of the mechanism involved. From studies on the rabbit, it is known that the slow potentials which follow the c-wave when illumination is continued, a transient negativity, and a late poxitivitp to levels often higher than the c-wave, arc prol)abl\ also generated by the PE as is much of the steady (“resting”) t1.c. potential under steady-state conditions of adaptations. Acute destruction of the pigment epithelium in vivo by sodium iodate isolates a component, Slow PIII, upon which normally the pigment epithelial responses are superimposed. Depth profile measurements by Faber (1969) established that the current of Slow PI11 is distributed to the inner surface of the rabbit’s retina in the same manner as the b-wave current. Therefore, the origin of Slow PI11 is most probably the Miiller cell&.e. a hyperpolarization of the distal Miiller cell processes. coinciding, to somedegree, with the hyperpolarization of the apical membrane of PE. The present study on rats demonstrates that the slow ERG potentials generated in this speciesare similar to those of the rabbit. They differ from the rabbit, however. by a peculiar age dependence of the PI potentials, being highest in dark-reared animals around 5 weeks of age. At that age PI dominates normally over Slow PHI. but at 10 weeks already Slow PIII overrides PI. more so in the albino than in tht~ pigmented rat. A difference between such strains was also noted by Dodt and Echte (1961) who concluded that albino rats lack a c-wave. They also found that the maximal b-wave of albinos is only half as high as that of pigmented rats and that the manifestations of vitamin A deficiency are much less evident in pigmented than in albino rats. The findings of Dodt and Echte may partly result from the vulnerability of the rat’s photoreceptors to strong light against which pigmented rats are more protected than albinos (Noell, Delmelle and Albrecht, 1971). All slow potentials are extinct in the dystrophic rat at an age when the b-wave is also very small or absent. Virtually the whole space between the outer limiting membrane and PE is then occupied by debris in place of outer and inner segments. The pigment epithelium is preserved light-microscopically, and probably also t,he distal and apical processesof the Xiiller cells and PE, respectively (cf. LaVail et al., 1972). Furthermore, typical transocular responses,assumedto depend upon the PE, can be elicited almost as high as in the normal rat by a rapid i.v. injection of azide or thiocyanat’e (Noell, 1965; and Fig. 7R, (e): this paper). In the rabbit, these azide and thiocyanate responsesappear to manifest depolarization and hyperpolarization. respectively. of the basal PE membrane, as the result of transient diffusion potentials of these anions across the basal plasma membrane (Noell, unpublished). Their presence in the dystrophic rat, after photoreceptor clegeneration strongly suggeststhat relative ionic permeabilities acrossthis membrane are preserved. The junctional coupling between the epithelial cells and a high transepithelial resistance across the intercellular space also cannot have disappeared in the disease.In addition, the intracellular potential of the PE of the dystrophic rat cannot be much different from normal. Nevertheless, the sequential failure of the PI components, and their sluggishness before failure, also shown by Slow PIII. suggeststhat these components are more affected than can be accounted for simply by photoreceptor loss. This is particularly evident in comparison to the b-wave which maintains its time course to a much better degree during its failure in amplitude. Three possibilities clearly exist : (1) a failure on.the part of the degenerating photoreceptors to provide the appropriate stimuli
SLOW
ERG
IZT RETISI\L
DYSTROPHIC
RAT'S
503
for the initiation of the slow components; (2) a failure in the “communication” system between receptors and generator membranes due to the accumulation of debris ; and (3) a failure in the responsiveness of these membranes. All three possibilities may be involved but the sluggishness of both the Slow PI1 and the PI components. despite t.heir different rate of amplitude failure. suggests that possibility (2) may be the major factor for the observations. In a similar way, the debris seemed to impedcx the transfer of retinol from bleached rhodopsin into PE, although retinol product,ion from retinal wa,q also slowed (Delmelle et, a,l.. 19i6). ACKNOWLEDGMBNT This
work was supported by
an N.
I. H. Grant.
REFERESCES Brown, K. T. (1968). The electroretinogram: its components and their origins. IYis. Res. 8,633-77. Delmelle, &I., Noel& W. K. and Organisciak, D. T. (1975). Hereditary retinal dystrophy in the rat : rhodopsin, retinal, vitamin A deficiency. Exp. Eye Res. 22, 369-80. Dodt,, E. and Echte, K. (1961). Darkand light-adaptation in pigmented and white rats as measured by electroretinogram threshold. J. Neurophysiol. 24, 42745. Dowling, J. E. and Sidman. R. L. (1962). Inherited retinal dystrophy in the rat. J. Cell Biol. 14, T::--ll)g. Faber. D. S. (1969). Analysis of the slow transretinal potentials in response to light. Ph.D. thesis. SFSY/Buffalo. Granit, R. (1947). Sensory Mechanisms of the Retina. Oxford University Press, London, Xew York and Toronto. LaVail, M. AI., Sidman, R. L. and O’Seil, D. (1972). Photoreceptor-pigment epithelial cell relationships in rats with inherited retinal degeneration. J. (Tell Biol. 53, 185-209. Noell. \V. K. (1953). Studies on the elertrophysiology and metabolism of the retina. rSAF 9d.M Project 21-12~~1-0004. h:oell, w. K. (1963). Cellular physiology of the retina. J. Opt. Sot. Amer. 53, 3648. Noell, W. K. (1965). Aspects of experimental and hereditary degeneration. First Symposium o/l tlw Biochemistry of the Retina. (Ed. Graymore, G. S.). Pp. 51-74. Academic Press, London. Noell, \V. K., Delmelle, 11. C. and Albrecht,, R. (19il). Vitamin A deficiency effect on the retina: dependence on light. Science 172, 72-5. Soell, 1%‘. K., Walker, V. S., Kang, B. S. and Berman, S. (1966). Retinal damage by light in rath. In/*eat. Ophthnlmol. 5, 460-73. Organisciak, D. T. and Noell, W. K. (1976). Hereditary retinal dystrophy in the rat: lipid composition of debris. Exp. Eye Res. 22, 101-13. Steinberg, R. H.. Schmidt, R. and Brown, K. T. (1970). Intracellular responses to light from rat pigment epithelium; origin of the electroretinogram c-wave. Sature 227, 728-30.
The Slow ERG Potentials in Hereditary Retinal Dystrophy of Albino and Pigmented Rats* EUGEXE L.
PATTLER?
AXD
\~ERSER
K.
~OELL
Nemosensory Laboratory, School oj Medic&. Statr University of Xew York cct Ry@lo. N.Y.. 7’S.A. The slow ERG potentials of different sites of generation-PI, PII-related and Slow PIII~erc st.udied at different ages in normal and mutant rats reared in a dark environment. The manifestations of these potentials in the normal rat were similar to the adult rabbit although their amplitudes in comparison to the b-wave were lower. They were peculiarly dependent on age and were highest during the fifth week of age. In the adult, normal pigmented rats, early PI components slightly exceeded Slow PI11 but Slow PI11 dominated in the records from adult albinos, In the mutant albino or pigmented rats, all slow ERG components were evident at the age of 21 days except the late posit’ive transient of PI. The negative transient of PI disappeared after 26 days but a slow c-wave survived beyond 5 weeks. The iodate-isolated Slow PI11 was better preserved than any PI component but it also was sluggish in response to the cessation of illumination. Responses to azide and thiocyanate were well preserved in 4-month-old mutants. The preservation of PI was generally better at any age for the pigmented than the albino mutants. An interpretation of the findings, based upon previous analyses in the rabbit-Slow PI11 originating in the distal Miiller cell process, PI generated by the pigment epithelium-leads to the conclusions (a) that the electrical properties of the pigment, epithelium are not primarily affected in the dystrophic rat and (b) that the accumulation of debris impedes the chemical communication between still functioning photoreceptors and pigment epit,helium as well as d&al Miiller cell processes.
1. Introduction Hereditary visual cell degeneration (also called retinal dystrophy) in the rat is one of several useful models providing information which may enrich our understanding of degenerative retinal diseasein man. The rat disease.first described by Bourne et al. (1938) is characterized by the selective deterioration and death of photoreceptor cells commencing during the third week of life (Dowling and Sidman, 1962; Noell. 1963, 1965). Accompanying visual cell degeneration is the accumulation and preservation of rhodopsin-rich, lipid-depleted membranous material. mainly of outer segment origin. called debris, between pigment epithelium (PE) and the surviving retinal layers when the animals are kept continuously in a dark environment (LaVail, Sidman and O’Neil, 1972; Organisciak and Noell. 1976: Delmelle, Noel1 and Organisciak. 1975). The progressof any diffuse visual cell disorder is easily monitored by the measurement of the trans-ocular potentials. Recordings of this kind have been restricted to the measurementsof the a- and b-waves of the ERG in the rat disease,while in human patients, the electro-oculogram (EOG) is an additional and easily applied diagnostic tool. The latter provides information on slow potential changes or d.c. potential shifts with a step change in illumination. The interest in these slow shifts in d.c. potentia.1 relates to the experimental * Supported by NIH Grant. t Permanent address: Department (‘oIlins, Colorado 80581, U.S.A.
of Physiology
and Biophysics.
Colorado
State
University,
Fort
A!14
I‘::. I,. l’.41.7’1,Kl: ASD \v. K. pu‘OlcI,I,
obticrration on niainnia1s t,hat the (l.c. transretinal current8 and the slow potential changes in response to light 011 or of are mainly generated by the PE and involve’ t,he action of light on the photoreceptors (Noell, 1953; Faber, 1969). *Measurement of the slow potential changes in conjunction with the analvsis of the II- and C-wavc~s thus may provide information on cert,ain aspects of the’ function of PE as well as on the “conllllullicatior1” between phot,oreccptors and PE. The means by which this eonnnunication operates is still unknown but it must involve a chemical linkage through the intercellular space between the two cell layers. The present study reports on characteristic features of the slow potentials in normal and dystrophic rats. The nomenclature to be used follows the original designations of ERG component,s (PI. II, III) by Granit (1947) and distinguishes between three different generating systems for the slow changes (Faber, 1969).
2. Methods A11 pigmented and albino rats (normal aud abnormal) of this study were from long established colonies of the laboratorv. They had been reared in a (lark environmerit (Delmelle et al., 1975). Controls were mainly phenon~enologically normal heterozygous litter mates from back-crosses selected by electroretinographic criteria. Connnerciall~ purchased adult albino (Charles River Laboratory) aud pigmented rats (Lcng Evans strain) were additionally used as adult. controls. The abnormal rats will be rzferretl to as ‘mutants”; they are genetically of the same origin as t,he Ba (albino) and B (pigmented) at.raius described by Delmelle et al. (1975). For ERG recordings, rats, 3-7 weeks old, were anesthetizedwith 20 mg/kg of Nembutal”. This was usually sufficient to minimize overt movemems. For adult rats 35 mg/kg of Nembutal was used supplement,ed by small additions as necessary for surgical procedures. Local anesthesia (2% Xylocaine”) was applied around the eye. The pupils of pigmented rats were dilated with 1:/h &opine su1fat.e. Recordings were made by cot,torl wicks threaded through a small polyethyleue tube filled with saline, connectiug t,o a Ag AgCl wire. One of these electrodes u-as iusertetl behind the palpebral conjunrtiva for a distance of 223 mm, the other placed on the cornea. A needle electrode under the scalp conuect.ed to ground. Responses were recorded from a Tetronix 5103N oscilloscope aft,er pre-amplificat’ion by a Grass P16 d.c. amplifier. The light sources employed were a Grass Photostimulator (Pa) and a Sylvania R1131C glow modulated t,ube electronically controlled. The maximal illumination (I = 0) of the glow stimulat.or was about 4.5 log units above the 20 p,V h-wave threshold of normal rats at the age of 5 weeks. It was reduced by neutral density filters in 1 log-unit steps. The light was channeled directly to the eye bv fiber optics. The brief and int’ense flash of the Grass stimulator was used for the analysis of n- arid b-waves while the glow stimulator served in the measurements of the slow components. The stimuli were presented in 3.u ordered sequence at npproximatelv 2-niiu intervals. Testing was performed under a lo\\-intensity red background light. Glowlight, stimulation was for 5 and 25 see. Azide and thiocyanate respouees were elicited by a rapid intravenous (externnl juguklr vein) iujection of 0.3 ml of 1.6 mM-sodium nzide and 60 m>f-sodium thiocyaunte. respeatively. Both salts were dissolved in a bicarbonate buffered solution, replacing NaCl as needed for normal osmo1arit.y. At. least oue eye was taken from each animal group for histological examination. 3. Young
norrd
albino
The slow ERG amplitude during
Results
rats
waves elicited with stimuli of 5-set duration reached their highest the fifth week after birth in normal, albino or pigmented ra.ts reared
SLOW
ERG
IN
RETIIL’AL
DYSTROPHIC
495
RATS
and continuously maintained in darkness. Depending upon the intensity and duration of illumination, at least three components of different time course and, presumably, of different origin were apparent at this age. gt a low intensity of illumination (l-5 log units above b-wave threshold) the dominant component of the slow potentials was a steadily maintained positivit,y appearing almost immediately upon the rise of the &wave [Fig. l(a)]. Upon the cessation of illumination, this potential rapidly disappeared, as indicated by a steplike change from positivity to the control (l.c. level. This step change at the cessation I=-3
I:-2
,)I -III0 r I\ ’ I if”’ 0.2
mV
#‘~a. 1. Tracings (a)-(d) are from a 31-day-old normal albino rat: responses are elicited by flashes of increasing intensity. Stimulus intensity is expressed in log units (I); I = 0 is the maximal stimulus, approximately 4.5 log units above b-wave threshold. Stimulus duration is indicated by the heavy bars beneath the tracings. Calibration is the same for all tracings and indicated by the vertical bar (0.2 mV) at the right-hand corner. Tracings (e) and (f) are recorded from 34-day-old normal albino rats at slow sweep speed (5 cm/set) and illustrate the late negative PI transient (e). and Slow PI11 (f) after the elimination of PI components by previous iodate administration.
of illumination was the main or sole indication of the presence of this component at high flash intensity because other slow potentials then dominated in amplitude. It was absent when b-wave generation was blocked by deep anesthesia or anoxia, while other slow potentials and the a-wave were preserved. The potential will be referred to as the PII-related component (c.f. Brown, 1968). A slowly rising positivity was superimposed upon the PII-related component when flash intensity was more than 3 log units above b-wave threshold [Fig. l(b)-(d)]. This slowly rising positivity of the rat ERG had the same time course as the c-wave of the rabbit (Noell, 1953), although its amplitude never reached the same height in relation to the b-wave. Following its peak at about 3 see, this c-wave declined almost linearly to below the baseline to be succeeded by a slow negativity [Fig. l(e)].
4198
E. L. PAUTLEK.
AXD
11’. Ii.
SOE:LL
In the dark-adapted rabbit, this negativity reaches it:: maximum about, 40 XX after the beginning of continuous moderate illumination: the potential then slowly rise? again, passes through the control t1.c. level at about 2.5 niin of illumination, ant1 reaches after additional 5-10 min of continued illumination a posit’ive level. 0.5 t’o 2 mV above the d.c. level during the control period (Noell. 1953). In our experiments on the rat,, illumination was terminated no later than at 25 M:C [Fig. l(e)]. At this time, the slow negativity was still developing. Clessation of illumination then was followed. within lo-20 sec. by a slow rise to above the baseline [Fig. 4(a), (c)l. This late positivity reached its peak about 1 min after the termination of t’he 25 set flash and was about as high as the earlier c-wave. Previous studies on the rabbit provide evidence that (1) the c-wave, (2) t.he slow negative transient and (3) the late positivity are all dependent upon the integrity of the pigment epithelium (Noell, 1953). W e will refer to these potentials as the PI component(s) of the response (Faber, 1969). 1=-l
I=-2
11
11
11
11
(J/Y.++
1 I
+- ;‘ei0.2mv \
I\ O
/
\
Off
L
‘zq\/--+w -W I
’
1
’
11 +I
11 I-+
1 lsec
I
11
I”“1
II +5sec
FIG. 2. Tracings (a)-(d) are from one mutant albino dark-reared rat, 21 days old, recorded as in Fig. 1. Flash intensity (I) varies. (d) is the response of the dark-reared rat, for the first time, to a sbrong flash of light of 5 set duration; this “first” response was typically lower than subsequent responses in the mutants; to a lesser degree this was also observed in normals. Sweep speed is slower in (e); the second tracing (marked “2”) is an additional sweep following immediately upon the first sweep. The onset of the flash is indicated by the a- and b-wave deflections in the first tracing; cessation is at “off” in the same tracing.
The third major slow component of the rat’s ERG became evident when the PI components were eliminated by the administration (i.p.) of sodium iodate, 15-24 hr prior to the measurements. As illustrated in Fig. l(f), a negativity of considerably larger amplitude than the c-wave was the dominant feature of the response.It reached its maximum usually within 5-15 set depending upon stimulus intensity. When the preparation was very responsive and the stimulus strong, as in Fig. l(f), it had an early maximum around 3 set and a less negative, almost steady level thereafter. Upon cessation of strong illumination 25 set after “on”, the negativity decayed initially almost as fast as it developed but tended to persist partially for one minute or more.
SLOW
ERG
Ip; KETIXAL
DFSTROPHIC’
RATS
497
In accordance with previous studies on the rabbit, the negativity after iodate poisoning will be referred to as Slow PI11 (Faber, 1969). It was identified by extensive depth profile measurements. It is a normal component of the ERG but masked to varying degree by the PI potentials. It is most probably of Miiller cell origin, manifesting a hyper-polarization of the distal Miiller cell processes. The simultaneous PI component providing cornea-positivity (c-wave) most probably results from the hyper-polarization of the apical PE membrane (Steinberg. Schmidt and Brown. 19X).
The earliest stage at which animals of t,he mutant strain mere examined was at 21 day3 after birth. At this age, the mutants differed little in a- and b-wave amplitudes from controls. The histological signs of the degenerative process also were apparent Normal
albino
Mutant
0 (*)I
0 !f-
albino
I 0.2
mV
(h). d-
-4 Lf-
FIG. 3. (a)-(d): c-wave occurrence at the age of 26 days Recording is as in Fig. 1. Tracings (e), (f) are from Z-day-old ones. Calibration is the same for all tracings.
in normal and mutant albino litter-mates. litter-mates. and (g), (h) from 76dday-old
only to a slight degree over most of the retina. This includes the signs of visual cell death and debris accumulation in front of the PE (Delmelle et al., 1975). As illustrated in Fig. 2, the slow potentials were, at this age, not strikingly different from those of the normal rat. Nevertheless, the following deviations from the best normal records were consistently observed : (1) a slowing of the c-wave, (2) a slower than normal decay of the negative PI transient, and (3) a weak or missing late PI positivity.
1!H
12. L. l’AI~‘l‘LR~t
AK I) tv. K. NOELL
Figure 3 compares the records of normal and mutant albino rats in resporise to light stimuli of 5-set duration at the ages of 26, 32 and 76 days. ;1t the age of 26 days the cl-wave of the nn&nt was significantlv reduced (I>elmelle et al.. 1975). The steacly (l.c. dark potential was within the same amplitude range, :! 4 IIIV. as in the cont~rols but the c-wave clearly differed from normal. It rose at. a.much slower rate and deeavetl less rapidly after t,he end of illuniinat,ion. 111fact, it tended to grow after cesqation of illumination. Its amplitude relative to the h-wave was slightly rctluced 5 ties aft,er “on”, cspeciallv in response to a weak flash [Fig. 3(b)]. Normal I-O,
Mutantolblna
albino 26 days
(b)
I
32 days
(0.2 If
mV
1 ,-
I”
FIG. 4. Recordings from the same animals sweeps in each tracing follolv immediately
”
”
’ ‘J b-5 set
as in Fig. 3 with the lower sweep speed (5 cmjsec). upon the preceding sweep as in WK. 3(e).
Atlditionaf
The development of the c-wave in the normal dark-reared animal reached its peak around 32-37 days [Fig. 3(e)]. In th e mutants, the c-wave appearance also was somewhat greater at 32 days [Fig. 3(e)] than at 26 days [(d)], despite enhanced b-wave deterioration. At 76 days [Fig. 3(h)]? mainly a small b-wave had survived together with a PII-related potential which at all ages of the mutant was a,s well evident as the b-wave [Fig. 3(b), (d), (f)]. Evidence for the behaviour of late negativity and late positivity is provided in Fig. 4 and Fig. 5(a), (b). Compared to the controls of the sameage, the late positivity (late increase above baseline) had disappeared already at the age of 26 days. It was absent also in the records from the 21-day-old mutant of Fig. 2. Although it was observed at 21 days, the late negativity was missing at 32 days. Thus, the PI components seemedto he represented exclusively by a slowly rising and falling c-wave after 26-28 days. The preservation of Slow PI11 at the age of 32 days was suggested by a sharp negativity interposed between the b-wave and the c-wave [Fig. 4(d)].
SLOW
ERG
In- RETINAL
DTSTKOPHIC
499
RATS
More precise information on the preservation of slow PI11 was obtained by studying the mutant rats after iodate injection [Fig. 5(c)-(e)]. Slow PI11 was surprisingly well preserved at the age of 35 days when the a-wave was at best one-third of the normal amplitude. Highest PIIIs after iodate poisoning at this age were half the amplitude measured in controls. Slow PIII, however, was sluggish, especially its decay to the baseline after the end of the maximal (I = 0) illumination. It, was faster at a reduced int,ensity (I = 1). Mutant
albino
I=0 (a)
I
0.2 mV
(d)
PK. 5. (a); the slow rise and decay of the c-wave at the age of 31 days illustrated by successive sweeps; the tlash was turned off during the first sweep as indicated. (b) is recorded with slow sweep speed in a 2%day-old animal in which the b-wave was much reduced due to rxceasirc Nembutal; note that the sudden d.r. shift with the cessation of illumination is also much reduced; the initial negative transient at the start of illumination is a manifestation of Fast and Slow PIII. Recordings (c) and (d), (e) are from two animals aged 35 and 28 days respectively, which were injected with sodium iodate, 24 hr before t&kg; they illustrate the preservation of Slow PIII: the h-wavr was absent in these animals as a result of the iodste poisoning.
Figure 6 illustrates the slow potentials in the pigmented mutant rats. Essentially the same results were obtained as in the mutant albinos illustrated in Figs 2-5. The strong impression was gained, however, that the deterioration of the slow potentials in the pigmented animals proceeded slower than in albinos, although both strains were maintained in the dark environment. Especially, the sluggishness of the reactions to the cessation of illumination was less pronounced than in the albinos. This was true for the decay of the c-wave after “off” as well as for the decay of the iodate-isolated PHI. Comparison
of adult normal
albino and hoodedrats
Dodt and Echte (1961) reported that the adult pigmented rat has a c-wave of up to O-6mV, whereas no such wave is evident in the al’)ino rat. In our experiments,
500
Il. L. I’ACTLEK
AND
\\‘.
K. XOELL
alhino as well as pigmented animals generated c-waves at the age of 3:! days which approached 1.2 mV in amplitude during illumination approximat.cly 4.5 log 1lnit.s ahove b-wave threshold. Figure 7(a). (1~).showsthe records from 4months-oh1 albino and pigmented rats. The commercially purchased animals had been in t,he dark environment for lessthan one week. In responseto the standard illumination (I = 0). Slow PI11 negativity dominated in the albino. rvhereas in the pigmented animal, a c-wave followed the b-wave, and reached approximately 50% of the h-wave atnplit~ude. These were t,ypical findings. Mutant
pigmented
1~0, 26 days
29 days (e). -4 I
tf A 34days 4 ?J
10.2
mV
FIG. 6. All tracings are from pigmented mutants of different ages. Left column (a), (c) are recorded at 1 see/cm sweep speed; right column (b), (d) are from same animals using 5 secjcm sweeps. Tracings (c) and (f) are obtained after t,he administration of iodatr 24 hr preriously (different animals).
In order to ascertain whether or not the electrophysiological properties of PE were innately different, the two strains were compared for their ability to generat.e an electrical responseto the rapid injections of sodium azide and sodium thiocyanate (Noell. 1963 and unpublished). The responsesto both ions [seeFig. 7B, (a), (h)] were in 4 out of 5 albino animals 20% lower than for the average of 5 hooded rats. Figure 7(h), (d) illustrates two exceptional findings. Tracing (h) (A) showsthe ERG of an albino rat in which the initial negativity manifesting Slow PI11 is followed hy a delayed c-wave rise to positivity which wasnot attained in the other four albino rats. Interestingly, the responsesto azide and thiocyanate [(c) (B)] were also greater in this animal than in any of the others. Record (d) (A) is from a hooded animal. The ERG was characterized by an exceptional PI11 negativity. The responsesto the rapid injections of the anions illustrated by records (d) (B) also were higher than in the other pigmented rats, as if the resistive network for the transocular current distribution was more favorable than usual for recording slow potentials of high amplitude.
SLOW Normal
ERG
IN
RETINAL
Normal
albino
I (a’ II “‘w
L
Azidr--
I0 ‘tThlocyanate
RATS
561
pigmented
h Yv--
I=0 A
DYBTROPHIC
A(cl
(b
b-
P-, ( e 1 Mutant
Cd)
i-
IT
V
7r-/+-
5mV
Ssec
FIG. 7. Comparison of adult normal albino and hooded rats. Upper (A) (a) and (c) typify the difference between the two strains; (b) and (d) are exceptional records (see text). The loner part of the figure (B) illustrates the responses to 1.6 rn>r-aside and 60 mivr-thiocyanate injected rapidly into the jugular vein, for the same animals as in A. B(e) is from a mutant albino rat, 3 months old; its ERG is extinct (compare with B(a)). The responses to aside are illustrated in the upper row of B; those to t~hiocyanate in the lower row. As indicated by the calibrat,ion line (5 m\‘), the responses to these anion injections range from 6 to 15 mv.
4. Discussion The transretinally measured electrical potentials of slow time course generated in response to a change in retinal illumination are rarely studied for probably several reasons: (I) as with all d.c. measurements within the range of l--d mV, their recording requires attention to numerous details; (2) the rate of change of some of these components is very slow ; a steady-state d.c. level may not be reached within 30 min after a single step change in illumination ; (3) they are variable from one preparation to another even when conditions appear well controlled; (4) they are very dependent upon the state of the animal (or the eye) e.g. the dose of the anesthesia agent, the state of circulation and ventilation ; (5) differences among animal species are much greater than for CL-or b-wave. even between mammals and different strains, and (6) they are complex in origin. The observations of OUT study illustrate several of these points. Nevertheless. the slow potentials provide information on important aspects of ret,ina,l physiology and pathology. As analyzed in the rabbit, they reveal, first of all. that a change in retinal illumination elicits photoreceptor reactions which affect the chemical environment of pigment epithelium and Miiller cells. The pigment epithelium reacts to these changes with the generation of a complex series of electrical transcellular potential changes, of which the cornea-positive c-wave of the classical ERG literat’ure is just one manifestation. It is well established that the c-wave of rabbk
302
E. L. PAY1‘1,EII
ANT1
\V. K. KOELT.
anal cat is caused by a hyperpolarization of the apical plasma membrane of PH. but little is known of the mechanism involved. From studies on the rabbit, it is known that the slow potentials which follow the c-wave when illumination is continued, a transient negativity, and a late poxitivitp to levels often higher than the c-wave, arc prol)abl\ also generated by the PE as is much of the steady (“resting”) t1.c. potential under steady-state conditions of adaptations. Acute destruction of the pigment epithelium in vivo by sodium iodate isolates a component, Slow PIII, upon which normally the pigment epithelial responses are superimposed. Depth profile measurements by Faber (1969) established that the current of Slow PI11 is distributed to the inner surface of the rabbit’s retina in the same manner as the b-wave current. Therefore, the origin of Slow PI11 is most probably the Miiller cell&.e. a hyperpolarization of the distal Miiller cell processes. coinciding, to somedegree, with the hyperpolarization of the apical membrane of PE. The present study on rats demonstrates that the slow ERG potentials generated in this speciesare similar to those of the rabbit. They differ from the rabbit, however. by a peculiar age dependence of the PI potentials, being highest in dark-reared animals around 5 weeks of age. At that age PI dominates normally over Slow PHI. but at 10 weeks already Slow PIII overrides PI. more so in the albino than in tht~ pigmented rat. A difference between such strains was also noted by Dodt and Echte (1961) who concluded that albino rats lack a c-wave. They also found that the maximal b-wave of albinos is only half as high as that of pigmented rats and that the manifestations of vitamin A deficiency are much less evident in pigmented than in albino rats. The findings of Dodt and Echte may partly result from the vulnerability of the rat’s photoreceptors to strong light against which pigmented rats are more protected than albinos (Noell, Delmelle and Albrecht, 1971). All slow potentials are extinct in the dystrophic rat at an age when the b-wave is also very small or absent. Virtually the whole space between the outer limiting membrane and PE is then occupied by debris in place of outer and inner segments. The pigment epithelium is preserved light-microscopically, and probably also t,he distal and apical processesof the Xiiller cells and PE, respectively (cf. LaVail et al., 1972). Furthermore, typical transocular responses,assumedto depend upon the PE, can be elicited almost as high as in the normal rat by a rapid i.v. injection of azide or thiocyanat’e (Noell, 1965; and Fig. 7R, (e): this paper). In the rabbit, these azide and thiocyanate responsesappear to manifest depolarization and hyperpolarization. respectively. of the basal PE membrane, as the result of transient diffusion potentials of these anions across the basal plasma membrane (Noell, unpublished). Their presence in the dystrophic rat, after photoreceptor clegeneration strongly suggeststhat relative ionic permeabilities acrossthis membrane are preserved. The junctional coupling between the epithelial cells and a high transepithelial resistance across the intercellular space also cannot have disappeared in the disease.In addition, the intracellular potential of the PE of the dystrophic rat cannot be much different from normal. Nevertheless, the sequential failure of the PI components, and their sluggishness before failure, also shown by Slow PIII. suggeststhat these components are more affected than can be accounted for simply by photoreceptor loss. This is particularly evident in comparison to the b-wave which maintains its time course to a much better degree during its failure in amplitude. Three possibilities clearly exist : (1) a failure on.the part of the degenerating photoreceptors to provide the appropriate stimuli
SLOW
ERG
IZT RETISI\L
DYSTROPHIC
RAT'S
503
for the initiation of the slow components; (2) a failure in the “communication” system between receptors and generator membranes due to the accumulation of debris ; and (3) a failure in the responsiveness of these membranes. All three possibilities may be involved but the sluggishness of both the Slow PI1 and the PI components. despite t.heir different rate of amplitude failure. suggests that possibility (2) may be the major factor for the observations. In a similar way, the debris seemed to impedcx the transfer of retinol from bleached rhodopsin into PE, although retinol product,ion from retinal wa,q also slowed (Delmelle et, a,l.. 19i6). ACKNOWLEDGMBNT This
work was supported by
an N.
I. H. Grant.
REFERESCES Brown, K. T. (1968). The electroretinogram: its components and their origins. IYis. Res. 8,633-77. Delmelle, &I., Noel& W. K. and Organisciak, D. T. (1975). Hereditary retinal dystrophy in the rat : rhodopsin, retinal, vitamin A deficiency. Exp. Eye Res. 22, 369-80. Dodt,, E. and Echte, K. (1961). Darkand light-adaptation in pigmented and white rats as measured by electroretinogram threshold. J. Neurophysiol. 24, 42745. Dowling, J. E. and Sidman. R. L. (1962). Inherited retinal dystrophy in the rat. J. Cell Biol. 14, T::--ll)g. Faber. D. S. (1969). Analysis of the slow transretinal potentials in response to light. Ph.D. thesis. SFSY/Buffalo. Granit, R. (1947). Sensory Mechanisms of the Retina. Oxford University Press, London, Xew York and Toronto. LaVail, M. AI., Sidman, R. L. and O’Seil, D. (1972). Photoreceptor-pigment epithelial cell relationships in rats with inherited retinal degeneration. J. (Tell Biol. 53, 185-209. Noell. \V. K. (1953). Studies on the elertrophysiology and metabolism of the retina. rSAF 9d.M Project 21-12~~1-0004. h:oell, w. K. (1963). Cellular physiology of the retina. J. Opt. Sot. Amer. 53, 3648. Noell, W. K. (1965). Aspects of experimental and hereditary degeneration. First Symposium o/l tlw Biochemistry of the Retina. (Ed. Graymore, G. S.). Pp. 51-74. Academic Press, London. Noell, \V. K., Delmelle, 11. C. and Albrecht,, R. (19il). Vitamin A deficiency effect on the retina: dependence on light. Science 172, 72-5. Soell, 1%‘. K., Walker, V. S., Kang, B. S. and Berman, S. (1966). Retinal damage by light in rath. In/*eat. Ophthnlmol. 5, 460-73. Organisciak, D. T. and Noell, W. K. (1976). Hereditary retinal dystrophy in the rat: lipid composition of debris. Exp. Eye Res. 22, 101-13. Steinberg, R. H.. Schmidt, R. and Brown, K. T. (1970). Intracellular responses to light from rat pigment epithelium; origin of the electroretinogram c-wave. Sature 227, 728-30.