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1 From the Department of Psychology, University of Sheffield; and the 2 Institute of Ophthalmology, University College London, United Kingdom.
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Abstract |
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METHODS. Rats aged 3 and 7 months were tested with a general neurologic examination that assessed visual, auditory, tactile, and whisker displacement responses. Photophobic responses and visual discrimination were also measured.
RESULTS. Dystrophic RCS rats failed the visual presentation tests, even at 3 months of age, and showed diminished performance on tactile tests. Auditory and whisker displacement performances were normal. Albino rats also showed diminished performance on the visual test, particularly to stimuli presented in the upper visual field. Photophobic responses were diminished in the dystrophic RCS rats compared with the pigmented control animals. Albino animals showed heightened photophobia. The dystrophic rats failed to reach criterion levels of performance on the visual discrimination test even with gratings of 0.045 cyc/deg.
CONCLUSIONS. The tests used discriminate deteriorated complex visual functions in RCS rats at ages when some simple reflexes can still be demonstrated. As such, they provide easily executed tests for screening for the effects of reparative treatments such as transplantation, administration of growth factors, and gene transfer technology. The integrity of whisker and auditory function are important when using tests requiring polysensory inputs. The somatosensory defect is surprising but may be useful in searching for the gene locus of the retinal disorder. The aberrations seen in the albino rats may be attributable to the effects of light damage and unfiltered light.
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Introduction |
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The animal is of considerable interest in that it shows some parallels with certain degenerative diseases of the human retina. One of these, age related macular degeneration, is a leading cause of blindness in western countries and, although not directly comparable to the RCS rat, shows similarities, in that it is apparently due to malfunction of the RPE cell layer.10 11 Transplantation has been attempted as a way of stabilizing the disorder in patients with advanced age-related macular degeneration,12 13 and although no clear success has yet been reported, the studies point to the need to develop the animal model further so that it more closely parallels the human situation. This requires a more comprehensive assessment of noninvasive tests of visual function. Furthermore, because some tests require interaction of visual and nonvisual sensory inputs, it is imperative, because the exact locus of the RCS defect is unknown, to be sure that the potential for deficits beyond those in the visual system are investigated. The present study addressed these issues.
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Methods |
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Neurologic Examination
Animals were subjected to a neurologic examination of the kind
developed by Marshall et al.14
All animals were placed on
a table top for this series of tests. A number of tests were performed
to determine visual, auditory, tactile, and whisker responses. The
visual stimulus was a card approximately 5 x 6 cm with a square
wave grating on it. The visual stimulus was presented either in front
(Vis F), above (Vis Ab), to the side but stationary (Vis Stat), or to
the side but moving (Vis Mot). The auditory stimulus was a hand-held
clicker of 70-dB intensity delivered for approximately 200 msec. There
were two tactile tests: a soft tactile (Tact S) test that was performed
by brushing the flank of the rat with a paint brush and a hard tactile
(Tact H) test that was performed by touching the animal with the paint
brush handle. Finally, the whisker test was performed by brushing
across the whiskers with the paint brush. Animals were scored on a 0 to
4 scale. The scores indicated the following response from an animal: 0,
no response from the animal to stimulus presentation; 1, ongoing
behavior interrupted but no orientation to the stimulus; 2, a slight
(less than 20°) orientation toward the stimulus; 3, orientation
toward the stimulus; and, 4, the maximal responses recorded for any
animal when the subject oriented toward the stimulus and moved toward
the stimulus to investigate it. Scores 1 through 3 indicate
intermediate levels of responsiveness. With the exception of the Vis F
and Vis Ab tests, each test was repeated if the animal did not score a
maximum response (i.e., 4) to take into account attentional
distractions.
Photophobia
The animals studied in the clinical tests were also tested for
photophobia using a method previously used for the assessment of
intracerebral graft function.15
All animals were tested at
3 and 7 months of age. They were placed in a circular arena with a
cover above, which was divided into three segments: dark plexiglas,
clear plexiglas, and an open segment that allowed access for placing
the animal into the arena.
The animals were placed in the open field for 5 minutes on four separate occasions: under red light conditions and with three levels of illumination with white light. The luminance levels recorded under the clear plexiglas and open segments were as follows: with red light, 5 fc; with level one white-light illumination, 560 fc; level two, 2,400 fc; and level three, 3,500 fc. The order of these tests was counterbalanced across subjects. The experiment was designed to establish whether animals would exhibit photophobic behavior by seeking out the dark quadrant. The total time spent under the dark segment during each 4-minute exposure was recorded.
Visual Discrimination
Visual discrimination was measured using an operant paradigm
developed by Dean and Redgrave.16
The animals were first
trained to lick a stainless steel tube for a sucrose solution reward.
Once the animals spent at least 60% of the half-hour testing session
licking the tube, the tube was withdrawn by 1-cm steps until it was 2.5
cm outside the box. This allowed the consistent placement of the eyes
necessary for calculating acuity. Animals were rewarded with a drop of
sucrose solution for on average every 3.2 seconds they spent in contact
with the tube. The schedule was in fact a variable contact time (VCT)
schedule, with reward appearing after 0.4 to 6.4 seconds of contact
with the tube. When the rats licked reliably at the tube in this
position, training to detect gratings began.
A daily session consisted of 100 trials, during each of which a slide was presented for 20 seconds. For 92 of the trials, neutral density filters were projected; for the remaining 8, a square wave grating was shown. The locations of the grating slides were varied randomly from day to day. Vertical square wave gratings were projected onto the screen, which was 12 cm from the rats’ eyes when their heads were in position licking the tube. This gave a grating of fundamental frequency 0.045 cyc/deg, occupying 87° of visual field and had the same mean luminance (500–800 candelas [cd]/m2) as the uniform field. If the rat licked the tube for more than 8 seconds during a grating trial, it received a 0.5-second foot shock, delivered with the grating still on the screen. Reward was withheld during grating trials to facilitate learning. To check whether the animal was using absence of a reward as a cue, eight neutral-density trials, chosen at random, were also made nonreward trials. The criterion for learning was that all shocks were avoided in a session, and contact time during nonrewarded safe trials averaged 12 seconds or more. To ensure the latter, the VCT was increased in stages. The levels of shock used were the minimum for effective performance.
Analysis
For the neuropsychological tests, separate Kruskal–Wallis one way
analyses of variance were performed on the various tests. Post hoc
analysis was performed on significant differences using Student’s
t-test. The photophobic data (i.e., time spent under the
dark segment) were subjected to a two-way analysis of variance with
repeated measures: group x illumination (repeated). For visual
discrimination, the number of shocks avoided was subjected to a two-way
analysis of variance: group x days (repeated). Post hoc analysis
of variance was performed on significant effects for both photophobia
and acuity results.
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Results |
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Photophobia
The nondystrophic RCS, LH, and SD rats showed similar results at 3
and 7 months, and so data for the two time points were collapsed
together for each group. Both nondystrophic RCS and LH rats spent
significantly more time under the dark segment than under the other
segments. Perhaps not surprisingly, the SD rats spent even longer in
the dark under all lighting conditions. By contrast, the dystrophic RCS
rats spent significantly less time under the dark segment under all
lighting conditions (group: F2,18 = 45.53;
P < 0.001) than did all other groups of animals and
furthermore spent less time at 7 months than at 3 months (see Fig. 2
). The performance of 7-month-old dystrophic RCS rats was random, with
approximately 33% of total time spent under the dark segment. As
lighting levels increased, all animals, including dystrophic RCS rats,
spent progressively more time under the dark segment
(F3,54 = 9.341; P < 0.001).
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Visual Discrimination
All animals quickly learned to lick the tube for the sucrose
reward, reaching criterion response within 4 days. Animals were then
trained to detect square wave gratings. Both nondystrophic RCS and LH
animals quickly learned to stop licking the tube on presentation of the
grating slide, avoiding all eight possible foot shock deliveries in the
battery of 100 tests within 7 days of training. In contrast, dystrophic
RCS rats at both 3 and 7 months of age never reached criterion levels
of performance. Three-month-old dystrophic RCS rats avoided
approximately 50% of shocks over the training period, whereas
7-month-old dystrophic RCS rats avoided approximately 25% of shocks.
Although neither reached criterion levels, the better performance at 3
months suggested that there is some residual level of visual
discrimination, which may be demonstrable under different testing
conditions, such as increased contrast levels. To establish whether the
inability of the dystrophic animals to learn the task with a visual cue
was an inability to see the grating or a deficit in learning per se, an
auditory signal was substituted for the grating slide. All animals
including the dystrophic RCS rats rapidly learned to stop licking the
tube during the onset of the auditory signal. Within 4 days, all groups
of animals avoided all eight shocks (Fig. 3)
. This result indicates that dystrophic animals could learn the task
when an auditory stimulus was used to indicate foot shock but were
unable to use a visual cue when it was a square wave grating of 0.045
cyc/deg (well within the visual capacity of nondystrophic animals,
which can discriminate stripes up to 0.5 cy/deg or better; see Dean and
Redgrave16
).
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Discussion |
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Despite these failures to detect visual activity in dystrophic RCS rats, these animals are not totally nonresponsive to light. Although there is a progressive loss in responses recorded in the superior colliculus to focal stimulation by spots of light presented throughout the visual field continuing from 6 weeks of age at the optic nerve head to include the whole retina by 6 months, responses to whole-eye stimulation can still be recorded over most of the colliculus at 6 months.18 Furthermore, a number of behavioral responses to light can still be elicited, albeit with reduced sensitivity and elevated threshold, well beyond 6 months of age. Both pupillary light reflex19 20 21 and conditioned suppression responses22 23 have been recorded in rats more than 1 year old. The present study shows that photophobia can also be recorded in these animals up to at least 7 months of age. These results point to the fact that deteriorated visual function in the dystrophic RCS rat is task dependent. Tasks such as the photophobic response or pupilloconstriction that depend on whole-eye stimulation continue through a time when it is extremely difficult to identify remaining photoreceptors. Tasks that require high resolution or positional information such as the orienting or visual discrimination tests are failed at an early stage.
Of the nonvisual tasks, the absence of a defect in auditory responsiveness is important from two points of view. It emphasizes that the RCS rat is unlikely to bear any homology with Usher’s disease, in which blindness and deafness are combined (see Algvere et al.24 ). It also means that tests of visual function in the RCS rat using an auditory component either as part of the test or as a control stimulus are not likely to be compromised by hearing loss. The evidence of sensory neglect to tactile stimuli is quite unexpected. In the light of the literature on the impact of visual loss both in animals and humans, it might be expected that dystrophic RCS rats would be more rather than less sensitive to tactile signals. For example, in the mole rat, an animal with a naturally diminished optic input, the somatosensory representation extends into the visual cortex in the absence of a visual signal to the visual cortex.25 Expansion of the somatosensory representation has been seen after neonatal eye enucleation (see Rauschecker26 ). In patients who have been blind for long periods, positron emission tomography (PET) scans recorded during Braille reading causes activation of the visual cortex, a phenomenon not seen in sighted Braille readers.27 Our result suggests either a central defect of visual attention or a defect of peripheral receptors.
Previous work has shown that sensory neglect, both visual and somatosensory, can be achieved by lesions of the superior colliculus or hypothalamus.14 28 29 It is possible in the dystrophic RCS rat that as the visual input to the superior colliculus becomes less competent as the photoreceptor degeneration progresses, there is some sort of gating effect on the somatosensory pathway so that the final common pathway in the orienting response cannot be accessed. Alternatively, it is conceivable that the RCS defect besides affecting cells that are essential for photoreceptor function may also involve other cells, such as those encapsulating somatosensory nerve endings producing an analogous result in the cutaneous receptor system to that seen in the retina—namely, the function and integrity of a primary sensory neuron being compromised by a malfunctioning supporting cell. It should be noted that although neglect is elicited by cutaneous stimulation, it is not elicited by whisker displacement. Transplantation studies may provide some insight into whether the cutaneous sensory neglect is due to central or peripheral sensory dysfunction. If part of the retina is rescued by transplantation of RPE cells, then it may be expected that the sensory neglect on the appropriate side would be eliminated if it were due to a gating phenomenon, but not if it were due to a peripheral sensory defect. This is presently under examination.
The results presented in the current study offer a further contribution to assessing the efficacy of transplantation and other potential therapeutic approaches. It is clear that different visual functions have different threshold sensitivities. Responses such as the pupillary light reflex still work (albeit at a reduced level) at more than a year of age. This means that although effects of transplantation can be detected,21 very careful quantitative studies must be undertaken to separate them from residual baseline activity. By contrast, both visual orienting and visual discrimination responses are lost completely by 3 months of age. This means that transplantation effects occur over a background of zero function, and therefore these tests should be the ones of choice in screening transplantation efficacy. It should also be pointed out that the goal of transplantation in humans is to maintain conscious vision. Although various of the reflexes play a supporting role, it is only by measuring cortical visual functions such as acuity that direct comparisons can be made between animal studies and human investigation in examining the conditions necessary for optimal recovery of vision.
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Footnotes |
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Submitted for publication November 17, 1999; revised April 26, 2000; accepted June 8, 2000.
Commercial relationships policy: N.
Corresponding author: Peter J. Coffey, Department of Psychology, University of Sheffield, Western Bank, Sheffield S10 2TP, UK. p.coffey{at}sheffield.ac.uk
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