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Effects of Ageing on Spatial Aspects of the Pattern Electroretinogram in Male and Female Quail
e
Pergamon
VisionRes,, Vol.37,No.5, pp.505–514,1997 01997 ElsevierScienceLtd.All rightsreserved Printedin GreatBritain 0042-6989/97 $17.CKI + 0.00
PII:S0042-6989(96)00159-9
Effects of Ageing on Spatial Aspects of the Pattern Electroretinogram in Male and Female Quail I
JEE-YAU LEE,*7 LESLIE A. HOLDEN,* MUSTAFA B. A. DJAMGOZ* Received 7November 1995; in revisedform 6 March 1996; infinalform 8 May 1996
Thi8 study deals with age-relatedfunctionalchange8in the eye of quail. Measurementsof lens refractivestate and transmission,as well as pupil diameter,showedthat the quail’seye became more myopicwith increasingage, but no changecouldbe detectedin lens transmissionand pupil diameter.Threeaspectsof the patternelectroretinogram(PERG) were studiedaftercorrectingfor ocular refraction:(i) spatialcharacteristics(visualacuity and contrastsensitivity);(ii) maximal amplitude;and (iii) peak latency. The PERG results suggestedthat the visuaJacuity was ageindependentfor both sexes.However,the contrastsensitivityof the old quail (16-20 months)was muchlower (1.5-3 times)thanthat of the young(3-6 months)at low to intermediate(<2 c/d), but not at highspatialfrequencies.The peaklatencyof the PERGresponsewas3-4 mseclongerin the old birds comparedwith the young,whilethe maximalamplitudeof the PERG responsewas ageindependent.These results clearly suggestedthat at least some of the age-inducedchangesare locatedin the neuralretina. @ 1997ElsevierScienceLtd. All rightsreserved Ageing
Quail retina
Pattern electroretinogram
INTRODUCTION There is considerable evidence to suggest that the visual system of the pigeon shows patterns of change with age that are strikingly similar to those observed in humans (Hodos et al., 1988, 1991a; Porciatti et al., 1991). In a series of psychophysical,morphologicaland electrophysiological experiments on pigeons, Hodos et al. (1991a) and Porciatti et al. (1991) found a progressivedecline in visual acuity with increasing age of birds. Some retinal pathological(Fite & Bengston, 1989)and psychophysical data (Hodos et al., 1991b) have shown comparable agedependentchanges in quail, but occurring at a faster rate. In a behavioral experiment on female Japanese quail, Hodos et al. (1991b) obtained similar results as with pigeons. On the other hand, a morphologicalstudy on the quail retina found no profound age-dependentchange in the quantity or morphologyof photoreceptors(PCs) (Fite & Bengston, 1989). Quail are sexually dimorphic; females are reported to live for considerably shorter periods than males if they are maintained on long day (12 hr or more) illumination, when the females ovulate continuously (Woodard & Abplanalp, 1971). Ichi!chik and Austin (1978) reported
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that some 50% cumulativemortalityoccurred for females (VS38% for males) by 1 yr of age, and this increased to 95% mortality by 100 weeks for females (VS60% for males). Thus, the rate of ageing of quail appears not only to be faster than that of pigeon, but also highly sexdependent. The presentstudy adoptedthe quail as an animalmodel for characterizingthe effects of ageing and sex on retinal mechanisms of vision. Thus, pattern electroretinogram (PERG) measurements have been made to explore several different aspects of visual functional changes. Riggs et al. (1964) first introduced the use of PERG to study the electricalresponsesof the human eye. Evidence has been accumulated that the PERG has multiple cellular sources (Berninger & Schuurmans, 1985; Baker et al., 1988). In the pigeon, Holden and Vaegan (1983) recorded the PERG with intra-retinal microelectrodes and reported a maximum amplitude in the inner nuclear layer (INL). Furthermore, the PERG of the pigeon was shown to remain intact after optic nerve section (Bagnoli et al., 1984).These results suggest that the PERG of the pigeon is likely to arise from retinal neurones. METHODS
*Department of Biology, Neurobiology Group, Imperial College of Science, Technologyand Medicine, LondonSW72BB, U.K. ~To whom all correspondenceshould be addressed c/o Prof. Ban-Dar Hsu, Departmentof Life Sciences, National Tsing-HuaUniversity, Hsin-Chu, 30043,Taiwan [Tel 886-35-715131ext 3445; Fax88635-721591;[email protected]].
Subjects
Quail (Coturnzk coturnix japonica) were kept on a light/darkcycle of 12/12hr; light was given from 8 am to 8 pm. For anesthesia, 10% ketamine (0.06 ml/kg) was first injected intramuscularly and was followed 10-
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20 min later by 20’%urethane (0.1 ml/100 g) injected into the peritoneum. Before each experiment, the quail was weighed and an ocular fundus examination was carried out after anesthesiawith an ophthalmoscopeto determine the clarity of the ocular media. The bird was then wrapped with cotton to keep it warm for 1–1.5hr until it was completely anesthetized. All the procedures used on living animals described in this study were monitored carefully and adequate measures were taken to minimize pain or discomfort. The experiments were carried out under a personal license to Jee-Yau Lee (Home Office No. PIL: 70/10132, U.K.). PERG measurements
The anesthetized quail was mounted in a stereotaxic apparatus.The head positionwas adjustedso that the eye corresponded to the stimulus center, similar to that used in pigeon (Hayes et al., 1987). The PERG was recorded from the left eye by a small stainlesssteel needle inserted in the conjunctivalof the lower fornix. Another similar electrode, placed at the same position on the right eye, served as reference (Porciatti et al., 1989). Patterned stimuli were black and white stripes produced on a 19-inch studio quality TV monitor (Melford Electronics, DUI-20) controlled by a grating pattern generator (T221). A screen distanceof 50 cm was used. The stimuli comprised vertical sinusoidal wave gratings that varied in both contrast and spatial frequency. The gratings were modulated in counterphase at 7.7 Hz. Recordings were obtained after at least 40 min of light adaption to the background luminance of 50 cd/m2.An amplifierwith a FET head stage, gain of lOOOx and a bandpass response frequency of 1–50 Hz was used. The output of the amplifierwas connectedto an oscilloscope (Hewlett-Packard, 1200B) to monitor the recorded signals. The oscilloscope also provided further amplificationof the signals(1OOX).A functiongenerator (Hewlett-Packard, 331OA)was used to synchronize the grating generator and the oscilloscope.The signals were then averaged with a Hewlett-Packard signal analyzer (5’480B).10-bit A-D conversion was provided by the averager, and 1024responseswere averagedroutinelyfor each reading. The amplitude of the averaged response was read from a digital voltmeter (Racal-Dana Trms, 4002A) connected to the signal analyzer. The peak latency (the latency of the signal peak) was estimated directly from the display of the signal analyzer. An example of PERG waveform is presented in Fig. 1. The noise level of the recording was determined with the stimulus grating turned off and set to the mean luminance. Measurement of the light intensity of the TV screen using a photometer showed that the real 70 contrast was the same as the apparent Yocontrastgiven by the machine reading in the range G85% and then saturated up to 100VOapparent contrast. In order to study and compare the various visual parameters in the quail’s PERG as a function of age, the
FIGURE 1. An example of PERG waveform shown op the screen of signal averager after 1024 readings. The amplitude and the peak latency of the signal are defined as shown in the figure.
birds were generally divided into three age groups, as follows: (i) 3–6 months (“young”), (ii) 7–15 months (“middle-aged”)and (iii) 16 months and older (“old”). Refractive state measurements
A bird under anesthesia would lose the ability to accommodate,and a spectaclelens of +2 dioptres(D), for thescreen distanceof 50 cm, shouldtheoreticallybe used to insure that the screen was conjugate with the retina, assumingemmetropiain the lateralvisual field.However, it was found that most quail eyes were, in fact, ametropic. Ocular refractive states were determined routinely, therefore, for individual birds by PERG measurement to find the spectacle lens which projected the sharpest image onto the retina. Spectaclelenses varying from +10 to –10 D were tested on each bird and the respective amplitudes of PERG signals were recorded. The grating pattern used in the examination was set at a spatial frequency of 2 cld with 85% contrast. The correct spectacle lens was then determined by locating the peak position of the graph of PERG amplitude vs lens power. The refractive state of the eye was defined as the difference between the power of the correct lens minus +2 D. Visual acuity measurements
The spatial frequency of the stimulus was varied from 0.25 to 5 cld in 12 steps with the contrastfixedat 85%. The amplitude and the peak latency (defined as the time from the onset of stimulus to the peak of response)were then recorded for the various spatialfrequencies.A graph of amplitude vs spatial frequency was plotted. Linear —
VISUALAGEING I: PERG
regression analysis was then done. Data points 21.2x noise level were used. However, since it was not a linear function, some data points for low spatial frequencies (0.25--0.75c/d) usually digressed from the regression line. They were excluded. Visual acuity was determined as the spatial frequency correspondingto the intersection point of the linear regression line and the horizontalline of noise level. Contrast sensitivip measurements
Contrast (70) was defined conventionally as (b,. – where L stands for luminance. The contrast of the stimulus at a given frequency was varied from 5 to 85% in 11 steps. The amplitude and the peak latency were recorded for various contrasts. A graph of amplitude vs contrast was plotted and second-order regression was used to fit the data points best. The contrast threshold was determined as the value of the contrast (70) corresponding to the intersection of the regression line and the horizontalline of noise level. The contrast sensitivitywas then deduced as the inverseof the contrast threshold. The same procedure was repeated for various spatial frequencies (0.25–5 c/d). Data sets were plotted as contrast sensitivity vs spatial frequency. The contrast sensitivity measurements (which usually lasted for 1–1.5 hr) were concluded routinely with a check of the maximal PERG amplitude (1 c/d at 85% contrast). If this remained above 85% of the initial value, the birds were assumed to still be in good physiologicalcondition. Lmin)/(&mx + &iJ,
Pupil diameter measurements
Post-mortemdissectionwas carried out after the PERG measurements. The quail eyeball was removed and its pupil diameter was measured by a micrometer. (a)
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Lenses were removed from the eyeballs and temporarily stored in a freezer for later measurement.They were thawed immediately before measurement. The spectral absorbance characteristics of the lenses were measured accordingto the method of Douglas (1989). Briefly,each lens was firstput in a hole, drilled to the same size of the lens, in an aluminum square screen column. The column was then mounted onto the window of an integration sphere fitted in the sample chamber of a spectrophotometer (Shimadzu UV-240). With the presence of the integration sphere, the true transmission of the lens could be measured without interference from light scattering. The spectral transmission was measured in the range 200-700 nm. Data analysis
Quantitative data were determined as means & standard errors. Statistical significance was tested using a two-way analysis of variance (ANOVA), with age and gender as independentvariables.
RESULTS Refractive state
The refractive state of the quail’s eye was agedependent in both sexes. On the whole, more myopic spectacle lenses were required for the older birds. The averaged values of the refractive states are given in Fig. 2. The refractive states of young quail were on the negative side of the theoretical value ( + 2 D), with average values of –0.77 f 0.54 D (n = 16) and –0.59 f 0.83 D (n = 15) for males and females, respectively. For males, the refraction dropped to –3.5 f 1.05 D (b)
Female
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FIGURE2. The refractive state as a functionof age of (a) male and (b) female quail.The error bars represent standarderrors. n is sample size and D is Dioptre. In each case (a and b), the lower panel denotes average data groupedfor “young”(3-6 months), “middle-aged”(7-15 months) and “old” (>16 months) quail.
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(n= 9) and –3.25 ~ 0.84 D (n = 6) in middle- and oldage, respectively.The females were somewhat different. There was a continuous decline from young (–0.59 f 0.83 D; n = 15), to middle (–1.5 f 0.61 D; n = 7) and then to old (–3.16 f 0.64 D; n = 19) age. A two-way analysis of variance (ANOVA) was carried out on these data, with age and gender as independent variables. For age, the results were F(2, 65) = 5.13, P = 0.009. The post-hoc Scheffe test indicatedthat when gender is disregarded, the refractive state of young birds was different from that of older birds (P= 0.003). However, the distinction between male and female was not significantbetween young and old quail. Lens transmission
Typical absorption spectra covering the spectral range from 200 to 700 nm are shown in Fig. 3. It can be seen that the quail’slenseswere almostcompletelytransparent (100% transmission) in the visible range (400-700 rim). There were significant absorbance wavelengths below 400 nm and a very sharp increase occurred at around 310 nm. However, age and sex appearedto have no major effect on the transmission of the lenses.
Pupil diameter
Since pupil size could decrease during light adaption, the pupil diameters of the anesthetizedquails’ eyes were also checked both before and after the PERG recordings to determine if there was any change induced by the grating stimuli. The result revealed that there was no significantchange for both sexes (data not shown). The pupil diameters of the different age groups after postmortem dissectionare shown in Fig. 4. For both sexes of the middle-age group, the averaged pupil diameter [2.62 t 0.1 mm for males (n = 6) and 2.58 f 0.12 mm for females (n = 5)] was slightly smaller than the values [2.6 Y 0.08 mm for males (n= 9) and 2.67 t 0.1 mm for the females (n = 6)] for the young. The distinction between the middle-age and the old was also minimal [2.62 ~ 0.1 mm (n = 6) vs 2.56 & 0.11 mm (n = 14) for males, and 2.58 f 0.12 mm (n = 5) vs 2.56 ~ 0.08 mm (n= 16) for females]. A two-way ANOVA was performed on these data with gender as one factor and age as the other. The results of ANOVA revealed no significant effect for age [F(2, 50) = 0.229, P = 0.796] and gender [I’(1, 50) = 0.005, P = 0.946]. The results would suggest that pupil diameter, and hence retinal
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FIGURE 4. The uuuil diameter as a function of age of (a) male; and (b) female quail.”T~eerror bars represent starrdarderrors. n is sample size. Age is counted in months and pupil diameter is in mm. The “young” quail (3-6 months), “middle-aged” quail (7–15 months) and “old” quail (>16 months).
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FIGURE 6. The contrast sensitivity as a function of the spatial frequencyof the grating stimuli of (a) male; and (b) female quail. The data of both young and old quail are shown. The error bars represent standarderrors. The contrast sensitivitiesof the youngand the old quail are significantly different at spatial frequencies below 2 c/d for the males and femrdes.n is sample size.
Female
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are given in Fig. 5. For males, the averaged values were 4.82 & 0.22,4.83 t 0.18 and 4.9 ~ 0.21 c/d (for young, middle and old; n = 16, 9, 6), respectively. For females, the average values of the visual acuity for the three age groups were 4.73 t 0.33, 4.26 ~ 0.3 and 4.54 A 0.17 c/d (n= 15, 7, 19), respectively. A two-way ANOVA was performed on these visual acuity data with age and gender as independentvariables. No significant effect was found for age [F(2, 67) = 0.393, P = 0.677] and gender IF(l, 67) = 2.295, P = 0.135]. Contrast sensitivity
Since it was difficultto acquire quail of known ages in large quantities, contrast sensitivity measurement could only be done on groups of “young” and “old” birds. The contrast sensitivitydata obtained for young and old quail YOUNG MIDDLE OLD YOUNG MIDDLE OLO are compared in Fig. 6(a) for male; an~ Fig. 6@~for FIGURE 5. Visual acuity as a function of age of (a) male; and females.-Thedata for the youngbirds were fitt~dbestby a (b) female quail. The error bars represent standard errors. n is sample parabolic curve, and showed that contrast sensitivity size. Age is in monthsand the unit of visual acuity is c/deg. In each case (aandb), thelowerpaneldenotes averagedatagroupedfor’’young’’(3A declined continuouslywith increasing spatial frequency. months), “middle-aged”(7–15 months) and “old” (>16 months) quail. The data obtainedfrom the old birds were also fitted by a parabolic curve. For the old birds, there was a slight rise in contrastsensitivityas the spatialfrequency approached illumination, play no major role in any age-dependent 0.5 c/d from the lower side. However, a two-way ANOVA performed on these slight rise data, with gender change in the visual functions of the quail. and spatial frequencies as independentvariables showed no significant effect. A further increase in spatial Visual acuity frequency then resulted in a continuous decline in the Visual acuity was measured after correcting for the contrast sensitivityof the old birds. A two-way ANOVA refractive state of the eye in each case. The results of the (with age and gender as independentvariables) indicated visual acuity measurements in the different age groups that there was a significantdifference [1.5–3 times; F(l,
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FIGURE 7. The amplitude of PERG signals as a function of age of (a) male; and (b) female quail. These were the averages of the data recorded from the measurement of visual acuity with spatial frequencies below (and including) 1 c/d, representingthe values when quail’s visuaf response was the greatest. The error bars represent standard errors. n is sample size. The noise has been subtracted from the amplitude. Age is in months, amplitude is in #V. In each case (a and b), the lower panel denotes average data groupedfor “young”(3+ months), “middle-aged”(7–15 months) and “old” (>16 months) quail.
8)= 24.901, P = 0.001] between the young and the old when contrast sensitivity was measured at low (<1 c/d) spatial frequencies. The difference was significantup to intermediate frequencies [<2 c/d; ~(1, 8) = 8.362, p = 0.02]. At higher frequencies [24 c/d, F(l, 8)= 1.275, P = 0.292), the “young” and “old” curves gradually converged. No significant main effect was found for gender. It was clear from these data, that the contrastsensitivity of quail, as opposed to the visual acuity, is age-related. The decline was most significant at lower spatial frequencies. The maximal amplitude and the peak latency of PERG signals
The maximal amplitudes and the peak latencies of the PERG signals were recorded and averaged for the spatial frequencies 0.25, 0.5, 0.75 and 1 c/d. These data are shown in Figs 7 and 8, respectively. For young male quail, the averaged maximal amplitudeof the PERG was 7.2 ~ 0.4 pV (n= 16). This changed to 7.3 i 0.6 pV (n= 9) in middle-aged and 7.5 f 0.6 pV (n = 6) in the old birds. For the females, the values were 6.0 + 0.4 pV (n= 15) for the young, 7.1 t 1.0 pV (n= 7) for the middle-aged and 6.3 t 0.4 pV (n = 19) for the old. However, a two-way ANOVA showed no effect for age [F(2, 63)= 0.567, P = 0.57] and gender [F’(1, 63) = 3.426, P = 0.07]. On the other hand, the peak latency of the male and the female quail increased with time up to middle age. They
MIDDLE
OLD
FIGURE8. The peak latency of PERG signals as a function of age of (a) male; and (b) female quail. These were the averages of the data recorded from the measurement of visual acuity with spatiaf frequencies below (and including) 1 c/d. The error bars represent standarderrors. n is sample size. In each case (a and b), the lower panel denotes average data grouped for “young” (3-6 months), “middleaged” (7–15 months) and “old” (>16 months) quail.
increased from 44.3 ~ 0.4 msec (n = 16) for young males and 43.7 t 0.4 msec (n = 15) for young females to 47.1 t 0.2 msec (n = 9) for middle-aged males and 47.6 t 0.6 msec (n = 7) for middle-aged females. A two-way ANOVA was carried out on the peak latency data, with age and gender as independent variables. No significanteffect was found for gender.For age, however, the resultswere F(2, 67) = 34.032, P <0.001. Individual post-hoc Scheffe tests indicated that when gender is disregarded,the increases from the young to the middleaged birds were significant(P < 0.001), but there was no further increase in response latency in old quail (P> 0.71).
DISCUSSION Considerable quantitativevariability was experienced in the measurementsof the visual parametersof quail of a given age and sex, possibly reflecting differences in the ageing process and/or the general physiological conditions of the animals. It was necessary, therefore, to maximize the sample size when performing a given measurementand to make comparisonsamongst specific age groups. Thus, it was found that the refractive state of the quail’s eyes became more myopic with increasing age, but no change could be detected in lens transmission and pupil diameter. From the PERG measurements (precededby ocular refractive correction),several effects of ageing on visual functions were found, as follows: (i) The contrastsensitivitydeclinedwith age. The &Cl& was most significantat low to intermediate (<2 c/d), but
VISUALAGEING I: PERG
not at high spatial frequencies.This would imply that the age-relatedimpairmentin visionwas more pronouncedin the visual pathways responsiblefor the perceptionof low spatial frequencies. (ii) There was no substantial change in visual acuitywith increasingage. (iii) The peak latency of PERG responseincreased up to middle-age.(iv) There was no change in the maximal amplitude. None of the visual functions studied and the ageing effects described showed sex differences. Refractive state
In the present study, the old quail were approximately –2.5 D more myopic than the young. Myopia thus was quite prominent in the old quail, possibly due to an increase in the axial length of the eye (Stone et al., 1989) and/or an increase in the extent of the anterior chamber with age (McBrien & Norton, 1992).This result is in line with the observation of Hodos et al. (1991b) suggesting, from viewing distance measurements, the presence of progressively increasing myopia with age in quail. The result, however, is different from the finding in aged pigeons (aged 10-16 yr) assessed by refractometry (Hodos et al., 1991a) and viewing distance measurement (Fitzke et al., 1985);these birds were found generally to be more hypermetropic than the young (aged 2 yr). The difference may be rooted in their different lifestyle. While pigeons are good at flight, quail spend almost all the time on the ground pecking food. When comparison was made between the two sexes, we found that there was a difference between the refractive states of the males (–3.5 ~ 1.05 D) and the females (–1.5 ~ 0.61 D) in middle-age. The reason behind this is not clear yet. Lens transmission
It has been found that the human lens becomes yellow when aged due to excessive absorption of UV light (Weale, 1963, 1988). An increase in the proportion and confirmational changes of crystallineproteinsin the lens has also been detected (Takemoto et al., 1989;Srivastava et al., 1992). These changes are likely to be involved in the opacification of the lens found with ageing and can consequently decrease the luminance on the retinal surface, as well as causing blurring of the image. However, there is no reported evidence of lens yellowing with age in any species of bird that has been studied so far. In transmission measurements on quail lenses, also, no difference between the young and the old, as well as the male and the female quail could be found (Fig. 3). These results are consistentwith the report of Hodos et al. (1991a), who found that the optical density of lens and cornea did not change with increasing age in pigeons. The spectral transmissionmeasurementsin quail showed that the lens remains completelytransparentin the visible range (400-700 rim). There was some absorbance at wavelengths below 400 nm and a sharp increase in absorbance occurred at around 310 nm, probably due to the presence of proteins and nucleic acids. If cumulative UV exposure is the main causative agent of lens
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darkening, then birds may have evolved a protective mechanism against this so as to maintain their ability to detect and discriminate UV light (Bowmaker, 1979; Remy & Emmerton, 1989). Pupil diameter
It was found that the pupil size of the quail remained the same following the PERG measurements, indicating that the light from the grating stimuliitselfwas not strong enough to cause any pupillary response. It also implied that the retinal illuminance remained the same throughout the course of the PERG measurements. Changes in pupil diameter have been demonstrated as the adult grows older (Weale, 1963; Sekuler, 1982; Sekuler & Owsley, 1983), and senile iris degeneration is most often held as the reason (Fisher, 1973).However, in quail, the averaged pupil diameter did not depend significantly on age, so the age-related changes in the visual functions observed in this study could not be attributed to the decrease of retinal illuminance or the increase of diffraction due to pupillary changes. Visual acuity and contrast sensitivi~
In humans, a modest decline in visual acuity prior to age 60 years, followed by a rapid decline from 60 to 80 years of age has been documented (Weale, 1982; Ordy, 1986).From behavioral and ele.ctrophysiologicalstudies (Hodos et al., 1988, 1991a;Porciatti et al., 1991), it was found that the visual acuity of pigeons declined with age. Also in a behavioral test of female quail, Hodos et al. (1991b) showed a decline in visual acuity with age. Nevertheless, no age-related change in the visual acuity of the quail could be found in the present study. The PERG is a retinal response, while the behavioral tests involve the whole visual pathway to the brain, postretinal factors may contribute to the difference. Furthermore, Hodos et al. (1991b) did not carry out refractive correction before the visual acuity measurements.Image blurring caused by refractive error might affect the perception in high spatial frequency vision (BodisWollner & Camisa, 1980).We had also measured visual acuity of quail by PERG without refractive correction and found that the visual acuity of quail declinedwith age (Hodos et al., 1996). However, we noticed later that the averagedrefractivestate of the old quailwas significantly more myopic than that of the young (Fig. 2), so image blurring would be more serious in the old quail without refractive correction, which in turn could result in a relative underestimation of visual acuity. With proper refractive correction performed in the present study, this age-dependentdecline of visual acuity disappeared. It is worth noting that visual acuity is a measure of visual discrimination of fine details with high contrast. Since fine details can only be discriminated by the macula, visual acuity relates mainly to macular function (Frisen, 1980). Therefore; “visual Xwity’’-asqgemrally---- —xmeasured cannot altogetherbe relied upon as a complete measure of spatial sensitivity. A more complete assessment of ageing effects comes from contrast sensitivity
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measurements, because the contrast sensitivity function describes sensitivity to a broad range of target sizes. Thus, the contrast sensitivityfunction provides information about visual status above and beyond that provided by visual acuity measurements (Sekuler, 1982). The existing literature on the effects of age on contrast sensitivity in humans suggests some age-dependent deficitsat high but not at low spatialfrequencies(Owsley et aZ., 1983; Scialfa et al., 1988). In these reports, static gratings were used in the tests. If, however, the display was temporally modulated, age differences then became apparent also at low spatial frequencies (Sekuler & Hutman, 1980; Owsley et al., 1983; Kline et al., 1990; Burton et al., 1993). Sekuler & Hutman (1980) found with grating stimuli modulated at 6 Hz, that the older observers had contrast sensitivity decreased by about three times at low and intermediate spatial frequencies compared with the young, but had similar sensitivity at high frequencies. The similarity observed at the high spatial frequencies for the two age groups may not be surprisingsince the two groups were very similar in their acuity levels. The human data, reviewed briefly above, are in good agreement with the result of the contrast sensitivity measurements obtained in the present study. The grating stimuli used was modulated at a temporal frequency of 7.7 Hz and did reveal significant age-related contrast sensitivity decline (1.5–3 times) at low to intermediate spatial frequencies (<2 c/d) in old quail compared with the young. Since optical factors would tend to affect responses to high spatial frequencies more than low (Bodis-Wollner& Camisa, 1980),the reduced sensitivity of the older observers to low spatial frequencies cannot be explained in terms of optical factors and/or as the result of ocular pathology. It would seem more probable that this effect reflects a selective loss in retinal mechanisms controlling low spatial frequency signal detection and temporally transient targets (Selculeret al., 1980).
study showed that there is no significant difference in pupil diameter and lens transmissionbetween young and old quail, and hence optical factors could not play any role in the visual dysfunction occurring during senescence in quail, as in pigeons and humans. We would propose, therefore, that age-related deterioration is caused primarily by changeswithin the retina and/orcentralvisual areas. However, since the PERG is a retinal response, the ageing effects that we have described in the current study must be due to retinal factors. Several studies on a variety of species, including birds and mammals, have shown that ageing is accompanied by loss of photoreceptor cells (e.g. Fite & Bengston,1989;Hodos et aZ., 1991a,b;Gao & Hollyfield, 1992; Fite et al., 1993). It is interesting, therefore, that there was no decrease in the peak amplitudeof the PERG in the present study. One possibility would be that any reductionin photoreceptoractivitycould be compensated by neuromodulatoryeffects in the post-receptoralretina. Among various neurotransmitters,it is well established that dopamine (DA) has an important role in the retina (for review, see Djamgoz & Wagner, 1992) and affects various retinal visual functions including spatialtemporal signaling (Nguyen-kgros, 1984; Piccolino et al., 1987; Piccolino, 1988). Depletion of retinal DA was found to significantly increase the VEP latency in rat (Dyer et al., 1981), and increased synaptic delay (McGeer & McGeer, 1976; Cotman, 1981; Fibiger, 1981). A connection between DA and form-deprivation myopia has also been shown in experimental chicks (Stone et al., 1989;Li et al., 1992).Moreover,suppressed contrast sensitivity has been reported in parkinsonian patients with normal visual acuity (Bulens et al., 1986; Bodis-Wollner et al., 1987) and the retinal DA levels were found to be significantly lower in those patients comparedto the normal (Harnois& Di Paolo, 1990).The possible role of DA in the visual dysfunctionsobserved during ageing in quail is examined in an accompanying paper (Lee & Djamgoz, 1996).
The maximal amplitude and the peak latency of PERG signals
From the PERG measurement, an age-related lengthening of the peak latency (by 34 msec), but no significant change in the averaged maximal amplitude was found between the young and the old quail. These results were parallel with the measurements of the P1OO of the visual evoked potential (VEP) in humans (Morrison & Reilly, 1989; Tobimatsu et al., 1993). These authors reported that the latency of the P1OO componentwas longer in older individuals,but there was no significanteffect on the amplitude. Possible causes of age-related changes in visual finction
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Bodis-Wollner, I. & Camisa, J. M. (1980). Contrast sensitivity measurement in clinical diagnosis. In Lessen, S. & Van Dalen, J. T. W. (Eds), Neuro-ophthdmology (Vol. 1, pp. 373--401).
Amsterdam: Excerpta Medica. Many investigators (Morrison & McGrath, 1985; Bodis-Wollner, I., Marx, M. S., Mitra, S., Boback, P., Mylin, L. & Sloane et al., 1988; Hodos et al., 1991a) supported the Yshr, M. (1987). Visual dysfunctionin Parkinson’sdisease: loss in idea that the morphologicalchanges in optical media and spatiotemporalcontrast sensitivity. Brain, 110, 1657–1698. retina do not appear to be sufficientto account for all of Bowrnsker,J. K (1979).Visualpigmentsand oil dropletsin the pigeon retina, as measured by microspectrophotometryand their relationthe visual deficits associated with age. Also, the present
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Press. Bulens, C., Meerwaldt, J. D., van der Wildt, G. J. & Keemink, C. K. (1986). Contrast sensitivity in Parkinson’s disease. Neurolo~, 36, 1121–1125. Burton, K. B., Owsley, C. & Sloane, M. E. (1993).Ageing and neural spatial contrast sensitivity. Vision Research, 33, 939–946. Cotman, C. W. (1981). Synaptic stmcture and plasticity. In Schimke, R. (Ed), Biological mechanisms in ageing (pp. 575–582).Bethesda, MD: NIH. Djamgoz, M. B.A. & Wagner, H.-J. (1992).Locrdizationand function of dopamine in the adult vertebrate retina. Neurochemistry International, 20, 139–191.
Douglas,R. H. (1989).The spectral transmissionof the lens and cornea of the brown trout (Salmo trutta) and goldfish (Carassius auratu.s): effect of age and implications for ultra violet vision. Vision Research, 29, 861-869.
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Fibiger, H. C. (1981).Neurotransmittersduringageing. In Schimke,R. (Ed), Biological mechanisms in ageing (pp.567-577). Bethesda, MD: NIH. Fisher, R. F. (1973). Presbyopia and changes with age in human crystalline lens. Journal of Physiology, 228, 765-779. Fite, K. V. & Bengston, L. (1989).Ageing and sex-related changes in the outer retina of Japanese quail. Current Eye Research 8, 10391048.
Fite, K. V., Bengston, L. & Donaghey,B. (1993). Experimental light damage increases lipofuscin in the retinal pigment epitheliumsof Japauese quail (Coturnix cotarnix japonica). Experimental Eye Research, 57, 449460.
Fitzke, F. W., Hayes, B. P., Hodos, W., Holden, A. L. & Low, J. C. (1985). Refractive sectors in the visual field of the pigeon eye. Journal of Physiology, 369, 17–31.
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McGeer, ~. G. & McGeer, P. L. (1976). Neurotransmittermetabolism in ageing brain. In Terry, R. D. & Gershon, S. (Eds), Neurobiology of ageing (pp. 389403). New York: Raven Press. Morrison, J. D. & McGrath, C. (1985). Assessment of the optical contributions to the age-related deterioration in vision. Quarterly Journal of Experimental Sciences, 70, 249-269.
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Morrison, J. D. & Reilly, J. (1989). The pattern of visual evoked cortical response in human ageing. Quarterly Journal of Experimental Physiology and Cognitive Medical Sciences, 74, 311–328.
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Ordy, J. M. (1986). Recent progress in development of valid animal models for human type memory loss in ageing senile dementia and Alzheimer’s disease. American Chemical SocieV (Abstract), 191, 17. Owsley, C., Sekrder, R. & Siemsen, D. (1983). Contrast sensitivity throughoutadulthood. Vision Research, 23, 689-699. Picccdino,M. (1988).La vision et la dopamine. Recherche, 19, 14561464.
Piccolino, M., Witkovsky, P. & Trimarchi, C. (1987). Dopaminergic mechanisms underlyingthe reduction of electric coupling between horizontal cells of the turtle retina induced by rr-amphetamine, bicuculline and veratridine.Journal ofNeuroscience, 7,2273-2284. Porciatti, V., Fontanesi, G. & Bagnoli, P. (1989). The electroretinogram of the Iitie owl. Vision Research, 29, 1693-1698. Porciatti, V., Hodos, W., Signonni, G. & Bramrmti, F. (1991). Electroretinographicchanges in aged pigeons. Vision Research, 31, 661-668.
Frisen, L. (1980).The neurologyof visual acuity.Brain, 103,639-670. Remy, M. & Emmerton,J. (1989).Behavioral spectral sensitivities of different retinal areas in pigeons. Behavioral Neuroscience, 103, Gao, H. & Hollyfield, J. G. (1992). Ageing of the human retina: 170-177. differential loss of neurons and retinal pigment epithelial cell.% Investigative Ophthalmolo~ and Visual Science, 33, 1-17. Riggs, L. A., Johnson, E. P. & Schick, A. M. C. (1964). Electrical responses of the human eye to moving stimulus patterns. Science, Harnois, C. & Di Paolo, T. (1990).Decreased dopaminein the retinas 144,567. of patients with Parkinson’s disease. Investigative Ophthalmology Scialfa, C. T., Tyrrell, R. A., Garvey,P. M., Deenng, L. M., Leibowitz, and Visual Science, 31, 2473-2475. H. W. & Goebel, C. C. (1988). Age differences in Vistech near Hayes, B. P., Hodos, W., Holden, A. L. & Low, J. C. (1987). The contrast sensitivity. American Journal of Optometry and Physioloprojection of the visual field upon the retina of the pigeon. Vision Research 27, 31-40.
Hodos, W., Holden, A. L., Lee, J.-Y., Djamgoz, M. B. A. & Porciatti, V. (1996). Gender differences and life-span changes in the visual acuity of Japanese quail. Neurobiology ofAging, in press. Hodos, W., Miller, R. F. & Fite, K. V. (1988). Visual acuity losses in aged pigeons. Social Neuroscience (Abstract), 14, 392. Hodos, W., Miller, R. F. & Fite, K. V. (1991a). Age-dependent changes in visual acuity and retinal morphologyin pigeons. Vision Research 31, 669-677.
Hodos,W., Miller, R. F., Fite, K. V., Porciatti, V., Holden,A. L., Lee, J.-Y. & Djamgoz,M. B. A. (1991b).Life-span changes in the visual acuity and retina in Birds. In Bagnoli, P. & Hodos, W. (Eds), The changing visual system (pp. 137–148).New York: Plenum Press. Holden, A. L. & Vaegan. (1983). Vitreal and intraretinal responses to contrast reversing patterns in the pigeon eye. Vision Research, 23, 561–572.
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Sekuler,R. (1982).Vision as a source of simple and reliable marker for ageing. In Reff, M. E. & Schneider,E. L. @ds), Biological markers of ageing (pp. 220-227). Washington, DC: U.S. Department of Health and Human Service. Sekuler, R. & Hutman, L. P. (1980). Spatird vision and ageing. 1: Contrast sensitivity.Journul of Gerontology, 35,692-699. Sekuler, R., Hutman, L. P. & Owsley, C. J. (1980).Human ageing and spatial vision. Science, 209, 1255-1256. Sekuler, R. & Owsley, C. (1983). Visual manifestations of biological aging. Experimental Aging Research, 9, 253-255. Sloane, M. E., Owsley, C. & Alvarez, S. L. (1988). Aging, senile miosis and spatial contrast sensitivity at low luminanw. Vision Research 28, 1235–1246.
Snvastava, O. P., McEntire, J. E. & Srivastava, K. (1992). Identification of a 9 kDa r- crystalline fragment in human lenses. Experimental Eye Research, 54,893-901.
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Takemoto, L., Straatsma, B. & Horwitz, J. (1989). Immunochemical characterization of the major low molecular weight polypeptide (10 K) from human cataractous lens. Experimental Eye Research, 48, 261–270.
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Weale, R. A. (1963). In The ageing eye. London:H. K. Lewis & Co. Weale, R. A. (1982). InA biography of the eye: development, growth and age. London: H. K. Lewis & Co. Weale, R. A. (1988). Age and the transmittance of the human crystalline lens. Journal of Physiology, 395, 577-587.
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Acknowledgements—We are grateful to ProfessorsWilliam Hodos and
Vittorio Porciatti for many useful discussions. This study was supportedby a grant from Fight For Sight (U.K.).
Pergamon
VisionRes,, Vol.37,No.5, pp.505–514,1997 01997 ElsevierScienceLtd.All rightsreserved Printedin GreatBritain 0042-6989/97 $17.CKI + 0.00
PII:S0042-6989(96)00159-9
Effects of Ageing on Spatial Aspects of the Pattern Electroretinogram in Male and Female Quail I
JEE-YAU LEE,*7 LESLIE A. HOLDEN,* MUSTAFA B. A. DJAMGOZ* Received 7November 1995; in revisedform 6 March 1996; infinalform 8 May 1996
Thi8 study deals with age-relatedfunctionalchange8in the eye of quail. Measurementsof lens refractivestate and transmission,as well as pupil diameter,showedthat the quail’seye became more myopicwith increasingage, but no changecouldbe detectedin lens transmissionand pupil diameter.Threeaspectsof the patternelectroretinogram(PERG) were studiedaftercorrectingfor ocular refraction:(i) spatialcharacteristics(visualacuity and contrastsensitivity);(ii) maximal amplitude;and (iii) peak latency. The PERG results suggestedthat the visuaJacuity was ageindependentfor both sexes.However,the contrastsensitivityof the old quail (16-20 months)was muchlower (1.5-3 times)thanthat of the young(3-6 months)at low to intermediate(<2 c/d), but not at highspatialfrequencies.The peaklatencyof the PERGresponsewas3-4 mseclongerin the old birds comparedwith the young,whilethe maximalamplitudeof the PERG responsewas ageindependent.These results clearly suggestedthat at least some of the age-inducedchangesare locatedin the neuralretina. @ 1997ElsevierScienceLtd. All rightsreserved Ageing
Quail retina
Pattern electroretinogram
INTRODUCTION There is considerable evidence to suggest that the visual system of the pigeon shows patterns of change with age that are strikingly similar to those observed in humans (Hodos et al., 1988, 1991a; Porciatti et al., 1991). In a series of psychophysical,morphologicaland electrophysiological experiments on pigeons, Hodos et al. (1991a) and Porciatti et al. (1991) found a progressivedecline in visual acuity with increasing age of birds. Some retinal pathological(Fite & Bengston, 1989)and psychophysical data (Hodos et al., 1991b) have shown comparable agedependentchanges in quail, but occurring at a faster rate. In a behavioral experiment on female Japanese quail, Hodos et al. (1991b) obtained similar results as with pigeons. On the other hand, a morphologicalstudy on the quail retina found no profound age-dependentchange in the quantity or morphologyof photoreceptors(PCs) (Fite & Bengston, 1989). Quail are sexually dimorphic; females are reported to live for considerably shorter periods than males if they are maintained on long day (12 hr or more) illumination, when the females ovulate continuously (Woodard & Abplanalp, 1971). Ichi!chik and Austin (1978) reported
Dopamine
that some 50% cumulativemortalityoccurred for females (VS38% for males) by 1 yr of age, and this increased to 95% mortality by 100 weeks for females (VS60% for males). Thus, the rate of ageing of quail appears not only to be faster than that of pigeon, but also highly sexdependent. The presentstudy adoptedthe quail as an animalmodel for characterizingthe effects of ageing and sex on retinal mechanisms of vision. Thus, pattern electroretinogram (PERG) measurements have been made to explore several different aspects of visual functional changes. Riggs et al. (1964) first introduced the use of PERG to study the electricalresponsesof the human eye. Evidence has been accumulated that the PERG has multiple cellular sources (Berninger & Schuurmans, 1985; Baker et al., 1988). In the pigeon, Holden and Vaegan (1983) recorded the PERG with intra-retinal microelectrodes and reported a maximum amplitude in the inner nuclear layer (INL). Furthermore, the PERG of the pigeon was shown to remain intact after optic nerve section (Bagnoli et al., 1984).These results suggest that the PERG of the pigeon is likely to arise from retinal neurones. METHODS
*Department of Biology, Neurobiology Group, Imperial College of Science, Technologyand Medicine, LondonSW72BB, U.K. ~To whom all correspondenceshould be addressed c/o Prof. Ban-Dar Hsu, Departmentof Life Sciences, National Tsing-HuaUniversity, Hsin-Chu, 30043,Taiwan [Tel 886-35-715131ext 3445; Fax88635-721591;[email protected]].
Subjects
Quail (Coturnzk coturnix japonica) were kept on a light/darkcycle of 12/12hr; light was given from 8 am to 8 pm. For anesthesia, 10% ketamine (0.06 ml/kg) was first injected intramuscularly and was followed 10-
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20 min later by 20’%urethane (0.1 ml/100 g) injected into the peritoneum. Before each experiment, the quail was weighed and an ocular fundus examination was carried out after anesthesiawith an ophthalmoscopeto determine the clarity of the ocular media. The bird was then wrapped with cotton to keep it warm for 1–1.5hr until it was completely anesthetized. All the procedures used on living animals described in this study were monitored carefully and adequate measures were taken to minimize pain or discomfort. The experiments were carried out under a personal license to Jee-Yau Lee (Home Office No. PIL: 70/10132, U.K.). PERG measurements
The anesthetized quail was mounted in a stereotaxic apparatus.The head positionwas adjustedso that the eye corresponded to the stimulus center, similar to that used in pigeon (Hayes et al., 1987). The PERG was recorded from the left eye by a small stainlesssteel needle inserted in the conjunctivalof the lower fornix. Another similar electrode, placed at the same position on the right eye, served as reference (Porciatti et al., 1989). Patterned stimuli were black and white stripes produced on a 19-inch studio quality TV monitor (Melford Electronics, DUI-20) controlled by a grating pattern generator (T221). A screen distanceof 50 cm was used. The stimuli comprised vertical sinusoidal wave gratings that varied in both contrast and spatial frequency. The gratings were modulated in counterphase at 7.7 Hz. Recordings were obtained after at least 40 min of light adaption to the background luminance of 50 cd/m2.An amplifierwith a FET head stage, gain of lOOOx and a bandpass response frequency of 1–50 Hz was used. The output of the amplifierwas connectedto an oscilloscope (Hewlett-Packard, 1200B) to monitor the recorded signals. The oscilloscope also provided further amplificationof the signals(1OOX).A functiongenerator (Hewlett-Packard, 331OA)was used to synchronize the grating generator and the oscilloscope.The signals were then averaged with a Hewlett-Packard signal analyzer (5’480B).10-bit A-D conversion was provided by the averager, and 1024responseswere averagedroutinelyfor each reading. The amplitude of the averaged response was read from a digital voltmeter (Racal-Dana Trms, 4002A) connected to the signal analyzer. The peak latency (the latency of the signal peak) was estimated directly from the display of the signal analyzer. An example of PERG waveform is presented in Fig. 1. The noise level of the recording was determined with the stimulus grating turned off and set to the mean luminance. Measurement of the light intensity of the TV screen using a photometer showed that the real 70 contrast was the same as the apparent Yocontrastgiven by the machine reading in the range G85% and then saturated up to 100VOapparent contrast. In order to study and compare the various visual parameters in the quail’s PERG as a function of age, the
FIGURE 1. An example of PERG waveform shown op the screen of signal averager after 1024 readings. The amplitude and the peak latency of the signal are defined as shown in the figure.
birds were generally divided into three age groups, as follows: (i) 3–6 months (“young”), (ii) 7–15 months (“middle-aged”)and (iii) 16 months and older (“old”). Refractive state measurements
A bird under anesthesia would lose the ability to accommodate,and a spectaclelens of +2 dioptres(D), for thescreen distanceof 50 cm, shouldtheoreticallybe used to insure that the screen was conjugate with the retina, assumingemmetropiain the lateralvisual field.However, it was found that most quail eyes were, in fact, ametropic. Ocular refractive states were determined routinely, therefore, for individual birds by PERG measurement to find the spectacle lens which projected the sharpest image onto the retina. Spectaclelenses varying from +10 to –10 D were tested on each bird and the respective amplitudes of PERG signals were recorded. The grating pattern used in the examination was set at a spatial frequency of 2 cld with 85% contrast. The correct spectacle lens was then determined by locating the peak position of the graph of PERG amplitude vs lens power. The refractive state of the eye was defined as the difference between the power of the correct lens minus +2 D. Visual acuity measurements
The spatial frequency of the stimulus was varied from 0.25 to 5 cld in 12 steps with the contrastfixedat 85%. The amplitude and the peak latency (defined as the time from the onset of stimulus to the peak of response)were then recorded for the various spatialfrequencies.A graph of amplitude vs spatial frequency was plotted. Linear —
VISUALAGEING I: PERG
regression analysis was then done. Data points 21.2x noise level were used. However, since it was not a linear function, some data points for low spatial frequencies (0.25--0.75c/d) usually digressed from the regression line. They were excluded. Visual acuity was determined as the spatial frequency correspondingto the intersection point of the linear regression line and the horizontalline of noise level. Contrast sensitivip measurements
Contrast (70) was defined conventionally as (b,. – where L stands for luminance. The contrast of the stimulus at a given frequency was varied from 5 to 85% in 11 steps. The amplitude and the peak latency were recorded for various contrasts. A graph of amplitude vs contrast was plotted and second-order regression was used to fit the data points best. The contrast threshold was determined as the value of the contrast (70) corresponding to the intersection of the regression line and the horizontalline of noise level. The contrast sensitivitywas then deduced as the inverseof the contrast threshold. The same procedure was repeated for various spatial frequencies (0.25–5 c/d). Data sets were plotted as contrast sensitivity vs spatial frequency. The contrast sensitivity measurements (which usually lasted for 1–1.5 hr) were concluded routinely with a check of the maximal PERG amplitude (1 c/d at 85% contrast). If this remained above 85% of the initial value, the birds were assumed to still be in good physiologicalcondition. Lmin)/(&mx + &iJ,
Pupil diameter measurements
Post-mortemdissectionwas carried out after the PERG measurements. The quail eyeball was removed and its pupil diameter was measured by a micrometer. (a)
5
Normal
Lenses were removed from the eyeballs and temporarily stored in a freezer for later measurement.They were thawed immediately before measurement. The spectral absorbance characteristics of the lenses were measured accordingto the method of Douglas (1989). Briefly,each lens was firstput in a hole, drilled to the same size of the lens, in an aluminum square screen column. The column was then mounted onto the window of an integration sphere fitted in the sample chamber of a spectrophotometer (Shimadzu UV-240). With the presence of the integration sphere, the true transmission of the lens could be measured without interference from light scattering. The spectral transmission was measured in the range 200-700 nm. Data analysis
Quantitative data were determined as means & standard errors. Statistical significance was tested using a two-way analysis of variance (ANOVA), with age and gender as independentvariables.
RESULTS Refractive state
The refractive state of the quail’s eye was agedependent in both sexes. On the whole, more myopic spectacle lenses were required for the older birds. The averaged values of the refractive states are given in Fig. 2. The refractive states of young quail were on the negative side of the theoretical value ( + 2 D), with average values of –0.77 f 0.54 D (n = 16) and –0.59 f 0.83 D (n = 15) for males and females, respectively. For males, the refraction dropped to –3.5 f 1.05 D (b)
Female
0
o !i
4?
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(3
0. % e –lo
0
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o 0
507
5
10 Age
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25
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25
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“=9
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-6
–6 YOUNG
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OLD
YOUNG
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FIGURE2. The refractive state as a functionof age of (a) male and (b) female quail.The error bars represent standarderrors. n is sample size and D is Dioptre. In each case (a and b), the lower panel denotes average data groupedfor “young”(3-6 months), “middle-aged”(7-15 months) and “old” (>16 months) quail.
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2.0 1.5 1,0 0.5 0.0
n’ I
(a) Male
(4.5
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n =4 I
I
I
(b)
1.5 Male
1.0 0.5
m)
n =2
I
t
I I
I
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a
1.0 0.5
(5.2
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n =4
1-
0.0
1 I
I
I
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1.0
m)
n=3
0.5 i0.0 200
(1 8,9
1 I
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400
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Wavelength
600
700
(rim)
FIGURE 3. The average absorptionspectra of the lens of (a) young male; (b) old male; (c) young female; and (d) old female quail. n is sample size and m is months.
(n= 9) and –3.25 ~ 0.84 D (n = 6) in middle- and oldage, respectively.The females were somewhat different. There was a continuous decline from young (–0.59 f 0.83 D; n = 15), to middle (–1.5 f 0.61 D; n = 7) and then to old (–3.16 f 0.64 D; n = 19) age. A two-way analysis of variance (ANOVA) was carried out on these data, with age and gender as independent variables. For age, the results were F(2, 65) = 5.13, P = 0.009. The post-hoc Scheffe test indicatedthat when gender is disregarded, the refractive state of young birds was different from that of older birds (P= 0.003). However, the distinction between male and female was not significantbetween young and old quail. Lens transmission
Typical absorption spectra covering the spectral range from 200 to 700 nm are shown in Fig. 3. It can be seen that the quail’slenseswere almostcompletelytransparent (100% transmission) in the visible range (400-700 rim). There were significant absorbance wavelengths below 400 nm and a very sharp increase occurred at around 310 nm. However, age and sex appearedto have no major effect on the transmission of the lenses.
Pupil diameter
Since pupil size could decrease during light adaption, the pupil diameters of the anesthetizedquails’ eyes were also checked both before and after the PERG recordings to determine if there was any change induced by the grating stimuli. The result revealed that there was no significantchange for both sexes (data not shown). The pupil diameters of the different age groups after postmortem dissectionare shown in Fig. 4. For both sexes of the middle-age group, the averaged pupil diameter [2.62 t 0.1 mm for males (n = 6) and 2.58 f 0.12 mm for females (n = 5)] was slightly smaller than the values [2.6 Y 0.08 mm for males (n= 9) and 2.67 t 0.1 mm for the females (n = 6)] for the young. The distinction between the middle-age and the old was also minimal [2.62 ~ 0.1 mm (n = 6) vs 2.56 & 0.11 mm (n = 14) for males, and 2.58 f 0.12 mm (n = 5) vs 2.56 ~ 0.08 mm (n= 16) for females]. A two-way ANOVA was performed on these data with gender as one factor and age as the other. The results of ANOVA revealed no significant effect for age [F(2, 50) = 0.229, P = 0.796] and gender [I’(1, 50) = 0.005, P = 0.946]. The results would suggest that pupil diameter, and hence retinal
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FIGURE 4. The uuuil diameter as a function of age of (a) male; and (b) female quail.”T~eerror bars represent starrdarderrors. n is sample size. Age is counted in months and pupil diameter is in mm. The “young” quail (3-6 months), “middle-aged” quail (7–15 months) and “old” quail (>16 months).
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FIGURE 6. The contrast sensitivity as a function of the spatial frequencyof the grating stimuli of (a) male; and (b) female quail. The data of both young and old quail are shown. The error bars represent standarderrors. The contrast sensitivitiesof the youngand the old quail are significantly different at spatial frequencies below 2 c/d for the males and femrdes.n is sample size.
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are given in Fig. 5. For males, the averaged values were 4.82 & 0.22,4.83 t 0.18 and 4.9 ~ 0.21 c/d (for young, middle and old; n = 16, 9, 6), respectively. For females, the average values of the visual acuity for the three age groups were 4.73 t 0.33, 4.26 ~ 0.3 and 4.54 A 0.17 c/d (n= 15, 7, 19), respectively. A two-way ANOVA was performed on these visual acuity data with age and gender as independentvariables. No significant effect was found for age [F(2, 67) = 0.393, P = 0.677] and gender IF(l, 67) = 2.295, P = 0.135]. Contrast sensitivity
Since it was difficultto acquire quail of known ages in large quantities, contrast sensitivity measurement could only be done on groups of “young” and “old” birds. The contrast sensitivitydata obtained for young and old quail YOUNG MIDDLE OLD YOUNG MIDDLE OLO are compared in Fig. 6(a) for male; an~ Fig. 6@~for FIGURE 5. Visual acuity as a function of age of (a) male; and females.-Thedata for the youngbirds were fitt~dbestby a (b) female quail. The error bars represent standard errors. n is sample parabolic curve, and showed that contrast sensitivity size. Age is in monthsand the unit of visual acuity is c/deg. In each case (aandb), thelowerpaneldenotes averagedatagroupedfor’’young’’(3A declined continuouslywith increasing spatial frequency. months), “middle-aged”(7–15 months) and “old” (>16 months) quail. The data obtainedfrom the old birds were also fitted by a parabolic curve. For the old birds, there was a slight rise in contrastsensitivityas the spatialfrequency approached illumination, play no major role in any age-dependent 0.5 c/d from the lower side. However, a two-way ANOVA performed on these slight rise data, with gender change in the visual functions of the quail. and spatial frequencies as independentvariables showed no significant effect. A further increase in spatial Visual acuity frequency then resulted in a continuous decline in the Visual acuity was measured after correcting for the contrast sensitivityof the old birds. A two-way ANOVA refractive state of the eye in each case. The results of the (with age and gender as independentvariables) indicated visual acuity measurements in the different age groups that there was a significantdifference [1.5–3 times; F(l,
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FIGURE 7. The amplitude of PERG signals as a function of age of (a) male; and (b) female quail. These were the averages of the data recorded from the measurement of visual acuity with spatial frequencies below (and including) 1 c/d, representingthe values when quail’s visuaf response was the greatest. The error bars represent standard errors. n is sample size. The noise has been subtracted from the amplitude. Age is in months, amplitude is in #V. In each case (a and b), the lower panel denotes average data groupedfor “young”(3+ months), “middle-aged”(7–15 months) and “old” (>16 months) quail.
8)= 24.901, P = 0.001] between the young and the old when contrast sensitivity was measured at low (<1 c/d) spatial frequencies. The difference was significantup to intermediate frequencies [<2 c/d; ~(1, 8) = 8.362, p = 0.02]. At higher frequencies [24 c/d, F(l, 8)= 1.275, P = 0.292), the “young” and “old” curves gradually converged. No significant main effect was found for gender. It was clear from these data, that the contrastsensitivity of quail, as opposed to the visual acuity, is age-related. The decline was most significant at lower spatial frequencies. The maximal amplitude and the peak latency of PERG signals
The maximal amplitudes and the peak latencies of the PERG signals were recorded and averaged for the spatial frequencies 0.25, 0.5, 0.75 and 1 c/d. These data are shown in Figs 7 and 8, respectively. For young male quail, the averaged maximal amplitudeof the PERG was 7.2 ~ 0.4 pV (n= 16). This changed to 7.3 i 0.6 pV (n= 9) in middle-aged and 7.5 f 0.6 pV (n = 6) in the old birds. For the females, the values were 6.0 + 0.4 pV (n= 15) for the young, 7.1 t 1.0 pV (n= 7) for the middle-aged and 6.3 t 0.4 pV (n = 19) for the old. However, a two-way ANOVA showed no effect for age [F(2, 63)= 0.567, P = 0.57] and gender [F’(1, 63) = 3.426, P = 0.07]. On the other hand, the peak latency of the male and the female quail increased with time up to middle age. They
MIDDLE
OLD
FIGURE8. The peak latency of PERG signals as a function of age of (a) male; and (b) female quail. These were the averages of the data recorded from the measurement of visual acuity with spatiaf frequencies below (and including) 1 c/d. The error bars represent standarderrors. n is sample size. In each case (a and b), the lower panel denotes average data grouped for “young” (3-6 months), “middleaged” (7–15 months) and “old” (>16 months) quail.
increased from 44.3 ~ 0.4 msec (n = 16) for young males and 43.7 t 0.4 msec (n = 15) for young females to 47.1 t 0.2 msec (n = 9) for middle-aged males and 47.6 t 0.6 msec (n = 7) for middle-aged females. A two-way ANOVA was carried out on the peak latency data, with age and gender as independent variables. No significanteffect was found for gender.For age, however, the resultswere F(2, 67) = 34.032, P <0.001. Individual post-hoc Scheffe tests indicated that when gender is disregarded,the increases from the young to the middleaged birds were significant(P < 0.001), but there was no further increase in response latency in old quail (P> 0.71).
DISCUSSION Considerable quantitativevariability was experienced in the measurementsof the visual parametersof quail of a given age and sex, possibly reflecting differences in the ageing process and/or the general physiological conditions of the animals. It was necessary, therefore, to maximize the sample size when performing a given measurementand to make comparisonsamongst specific age groups. Thus, it was found that the refractive state of the quail’s eyes became more myopic with increasing age, but no change could be detected in lens transmission and pupil diameter. From the PERG measurements (precededby ocular refractive correction),several effects of ageing on visual functions were found, as follows: (i) The contrastsensitivitydeclinedwith age. The &Cl& was most significantat low to intermediate (<2 c/d), but
VISUALAGEING I: PERG
not at high spatial frequencies.This would imply that the age-relatedimpairmentin visionwas more pronouncedin the visual pathways responsiblefor the perceptionof low spatial frequencies. (ii) There was no substantial change in visual acuitywith increasingage. (iii) The peak latency of PERG responseincreased up to middle-age.(iv) There was no change in the maximal amplitude. None of the visual functions studied and the ageing effects described showed sex differences. Refractive state
In the present study, the old quail were approximately –2.5 D more myopic than the young. Myopia thus was quite prominent in the old quail, possibly due to an increase in the axial length of the eye (Stone et al., 1989) and/or an increase in the extent of the anterior chamber with age (McBrien & Norton, 1992).This result is in line with the observation of Hodos et al. (1991b) suggesting, from viewing distance measurements, the presence of progressively increasing myopia with age in quail. The result, however, is different from the finding in aged pigeons (aged 10-16 yr) assessed by refractometry (Hodos et al., 1991a) and viewing distance measurement (Fitzke et al., 1985);these birds were found generally to be more hypermetropic than the young (aged 2 yr). The difference may be rooted in their different lifestyle. While pigeons are good at flight, quail spend almost all the time on the ground pecking food. When comparison was made between the two sexes, we found that there was a difference between the refractive states of the males (–3.5 ~ 1.05 D) and the females (–1.5 ~ 0.61 D) in middle-age. The reason behind this is not clear yet. Lens transmission
It has been found that the human lens becomes yellow when aged due to excessive absorption of UV light (Weale, 1963, 1988). An increase in the proportion and confirmational changes of crystallineproteinsin the lens has also been detected (Takemoto et al., 1989;Srivastava et al., 1992). These changes are likely to be involved in the opacification of the lens found with ageing and can consequently decrease the luminance on the retinal surface, as well as causing blurring of the image. However, there is no reported evidence of lens yellowing with age in any species of bird that has been studied so far. In transmission measurements on quail lenses, also, no difference between the young and the old, as well as the male and the female quail could be found (Fig. 3). These results are consistentwith the report of Hodos et al. (1991a), who found that the optical density of lens and cornea did not change with increasing age in pigeons. The spectral transmissionmeasurementsin quail showed that the lens remains completelytransparentin the visible range (400-700 rim). There was some absorbance at wavelengths below 400 nm and a sharp increase in absorbance occurred at around 310 nm, probably due to the presence of proteins and nucleic acids. If cumulative UV exposure is the main causative agent of lens
511
darkening, then birds may have evolved a protective mechanism against this so as to maintain their ability to detect and discriminate UV light (Bowmaker, 1979; Remy & Emmerton, 1989). Pupil diameter
It was found that the pupil size of the quail remained the same following the PERG measurements, indicating that the light from the grating stimuliitselfwas not strong enough to cause any pupillary response. It also implied that the retinal illuminance remained the same throughout the course of the PERG measurements. Changes in pupil diameter have been demonstrated as the adult grows older (Weale, 1963; Sekuler, 1982; Sekuler & Owsley, 1983), and senile iris degeneration is most often held as the reason (Fisher, 1973).However, in quail, the averaged pupil diameter did not depend significantly on age, so the age-related changes in the visual functions observed in this study could not be attributed to the decrease of retinal illuminance or the increase of diffraction due to pupillary changes. Visual acuity and contrast sensitivi~
In humans, a modest decline in visual acuity prior to age 60 years, followed by a rapid decline from 60 to 80 years of age has been documented (Weale, 1982; Ordy, 1986).From behavioral and ele.ctrophysiologicalstudies (Hodos et al., 1988, 1991a;Porciatti et al., 1991), it was found that the visual acuity of pigeons declined with age. Also in a behavioral test of female quail, Hodos et al. (1991b) showed a decline in visual acuity with age. Nevertheless, no age-related change in the visual acuity of the quail could be found in the present study. The PERG is a retinal response, while the behavioral tests involve the whole visual pathway to the brain, postretinal factors may contribute to the difference. Furthermore, Hodos et al. (1991b) did not carry out refractive correction before the visual acuity measurements.Image blurring caused by refractive error might affect the perception in high spatial frequency vision (BodisWollner & Camisa, 1980).We had also measured visual acuity of quail by PERG without refractive correction and found that the visual acuity of quail declinedwith age (Hodos et al., 1996). However, we noticed later that the averagedrefractivestate of the old quailwas significantly more myopic than that of the young (Fig. 2), so image blurring would be more serious in the old quail without refractive correction, which in turn could result in a relative underestimation of visual acuity. With proper refractive correction performed in the present study, this age-dependentdecline of visual acuity disappeared. It is worth noting that visual acuity is a measure of visual discrimination of fine details with high contrast. Since fine details can only be discriminated by the macula, visual acuity relates mainly to macular function (Frisen, 1980). Therefore; “visual Xwity’’-asqgemrally---- —xmeasured cannot altogetherbe relied upon as a complete measure of spatial sensitivity. A more complete assessment of ageing effects comes from contrast sensitivity
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measurements, because the contrast sensitivity function describes sensitivity to a broad range of target sizes. Thus, the contrast sensitivityfunction provides information about visual status above and beyond that provided by visual acuity measurements (Sekuler, 1982). The existing literature on the effects of age on contrast sensitivity in humans suggests some age-dependent deficitsat high but not at low spatialfrequencies(Owsley et aZ., 1983; Scialfa et al., 1988). In these reports, static gratings were used in the tests. If, however, the display was temporally modulated, age differences then became apparent also at low spatial frequencies (Sekuler & Hutman, 1980; Owsley et al., 1983; Kline et al., 1990; Burton et al., 1993). Sekuler & Hutman (1980) found with grating stimuli modulated at 6 Hz, that the older observers had contrast sensitivity decreased by about three times at low and intermediate spatial frequencies compared with the young, but had similar sensitivity at high frequencies. The similarity observed at the high spatial frequencies for the two age groups may not be surprisingsince the two groups were very similar in their acuity levels. The human data, reviewed briefly above, are in good agreement with the result of the contrast sensitivity measurements obtained in the present study. The grating stimuli used was modulated at a temporal frequency of 7.7 Hz and did reveal significant age-related contrast sensitivity decline (1.5–3 times) at low to intermediate spatial frequencies (<2 c/d) in old quail compared with the young. Since optical factors would tend to affect responses to high spatial frequencies more than low (Bodis-Wollner& Camisa, 1980),the reduced sensitivity of the older observers to low spatial frequencies cannot be explained in terms of optical factors and/or as the result of ocular pathology. It would seem more probable that this effect reflects a selective loss in retinal mechanisms controlling low spatial frequency signal detection and temporally transient targets (Selculeret al., 1980).
study showed that there is no significant difference in pupil diameter and lens transmissionbetween young and old quail, and hence optical factors could not play any role in the visual dysfunction occurring during senescence in quail, as in pigeons and humans. We would propose, therefore, that age-related deterioration is caused primarily by changeswithin the retina and/orcentralvisual areas. However, since the PERG is a retinal response, the ageing effects that we have described in the current study must be due to retinal factors. Several studies on a variety of species, including birds and mammals, have shown that ageing is accompanied by loss of photoreceptor cells (e.g. Fite & Bengston,1989;Hodos et aZ., 1991a,b;Gao & Hollyfield, 1992; Fite et al., 1993). It is interesting, therefore, that there was no decrease in the peak amplitudeof the PERG in the present study. One possibility would be that any reductionin photoreceptoractivitycould be compensated by neuromodulatoryeffects in the post-receptoralretina. Among various neurotransmitters,it is well established that dopamine (DA) has an important role in the retina (for review, see Djamgoz & Wagner, 1992) and affects various retinal visual functions including spatialtemporal signaling (Nguyen-kgros, 1984; Piccolino et al., 1987; Piccolino, 1988). Depletion of retinal DA was found to significantly increase the VEP latency in rat (Dyer et al., 1981), and increased synaptic delay (McGeer & McGeer, 1976; Cotman, 1981; Fibiger, 1981). A connection between DA and form-deprivation myopia has also been shown in experimental chicks (Stone et al., 1989;Li et al., 1992).Moreover,suppressed contrast sensitivity has been reported in parkinsonian patients with normal visual acuity (Bulens et al., 1986; Bodis-Wollner et al., 1987) and the retinal DA levels were found to be significantly lower in those patients comparedto the normal (Harnois& Di Paolo, 1990).The possible role of DA in the visual dysfunctionsobserved during ageing in quail is examined in an accompanying paper (Lee & Djamgoz, 1996).
The maximal amplitude and the peak latency of PERG signals
From the PERG measurement, an age-related lengthening of the peak latency (by 34 msec), but no significant change in the averaged maximal amplitude was found between the young and the old quail. These results were parallel with the measurements of the P1OO of the visual evoked potential (VEP) in humans (Morrison & Reilly, 1989; Tobimatsu et al., 1993). These authors reported that the latency of the P1OO componentwas longer in older individuals,but there was no significanteffect on the amplitude. Possible causes of age-related changes in visual finction
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Acknowledgements—We are grateful to ProfessorsWilliam Hodos and
Vittorio Porciatti for many useful discussions. This study was supportedby a grant from Fight For Sight (U.K.).