Characteristics of the Pupillary Light Reflex in the Macaque Monkey: Metrics

Milton Pong and Albert F. Fuchs

Regional Primate Research Center and Department of Physiology and Biophysics, University of Washington, Seattle, Washington 98195-7330


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Pong, Milton and Albert F. Fuchs. Characteristics of the Pupillary Light Reflex in the Macaque Monkey: Metrics. J. Neurophysiol. 84: 953-963, 2000. To investigate whether the simian light reflex is a reasonable model for the human light reflex, we elicited pupillary responses in three behaving rhesus macaques. We measured the change in pupillary area in response to brief (100 ms), intermediate (1 s), and long (3-5 s) light flashes delivered by light-emitting diodes while the monkey fixated a stationary target. Individual responses in the same monkey to either 100-ms or 1-s stimuli of the same light intensity were quite variable. Nevertheless, in response to the 100-ms stimulus, average pupillary constriction and peak constriction velocity increased and latency decreased linearly with the log of stimulus luminance. The minimum average constriction latency across monkeys for the brightest flash was 136 ms. A linear decrease of constriction latency with stimulus luminance also occurs in humans, but their latencies are ~70 ms longer. In addition, peak constriction velocity was highly correlated with the decrease in pupillary area. Dilation metrics were not as well related to stimulus luminance as were constriction metrics. The latency from flash offset to the onset of dilation was relatively constant, averaging ~480 ms. Peak dilation velocity was also correlated, but less well, with the increase in pupillary area. Constriction generally was greater and of longer duration for 1-s light pulses than for 100-ms pulses of equal luminance. The initial time courses of the responses to the two stimuli of different durations were identical until ~150 ms after response onset. Human pupillary responses for long and short flashes also have identical initial time courses. For very long (3-5 s) and very bright constant-luminance stimuli, the simian pupil underwent oscillations at frequencies of 0.9-1.6 Hz. Similar oscillations, called hippus, occur in the human pupillary light reflex. Like humans, the monkeys also exhibited consensual and binocular pupillary responses. Except for response latency, the pupillary responses in the two primate species are otherwise quite similar. Therefore any knowledge we gain about the neuronal substrate of the simian light reflex can be expected to have considerable relevance when extrapolated to humans.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The pupillary light reflex causes the pupil to reduce its area in response to a bright light, thereby providing the earliest control of light intensity reaching the retina. Because it is easy to elicit and observe, the light reflex has been a handy clinical tool with which to evaluate damage to the CNS and to assess the level of consciousness in humans. The most direct pathway underlying the light reflex is well documented. Its input arises from ganglion cells in the retina, its interneurons lie in the pretectum, its premotor neurons are located in the Edinger-Westphal (EW) nucleus, and its motoneurons have their cell bodies in the ciliary ganglion. Although this anatomy has been confirmed in a number of species (humans: Loewenfeld 1993; monkey: Carpenter and Pierson 1973; cat: Ranson and Magoun 1953; pigeon: Gamlin et al. 1984; rat: Clarke and Ikeda 1985), the discharge patterns of its individual elements in the alert animal have received scant attention. We have begun to remedy this situation by studying the activity of pretectal neurons believed to be the interneurons of the direct light reflex in the monkey. The results of those studies are presented in a companion paper (Pong and Fuchs 2000). In the current paper, we will examine the metrics of the pupillary light reflex in the monkey as a basis for understanding the behaviors of its constituent neurons.

With the exception of one recent study (Gamlin et al. 1998), data on the monkey pupillary light reflex have been limited to qualitative observations. In contrast, the pupillary light reflex of humans has come under considerable quantitative scrutiny. The human light reflex has been characterized by measuring pupillary diameter under different light stimulus conditions. As stimulus intensity increases, the reflex constriction increases in size and speed (Reeves 1920) and the latency decreases (Lowenstein and Loewenfeld 1969). The change in pupillary diameter increases linearly with logarithmic changes in luminance (Loewenfeld 1993). Stimuli 9 log units above the scotopic threshold make the pupil constrict maximally (Loewenfeld 1993). When a bright light stimulus persists, the pupil remains constricted. However, a dim stimulus light will not keep the pupil constricted. The failure to maintain constriction with a dim light stimulus (step change from 1 × 10-4 to 0.035 ft-L) (Sun and Stark 1983) is called pupillary escape (Lowenstein and Loewenfeld 1959).

Under steady photopic illumination, the pupil will oscillate in size (Lowenstein and Loewenfeld 1969). These oscillations, which have been called pupillary unrest or hippus, are absent in dim light or darkness and can be abolished when the pathway of the light reflex is interrupted or the iris is damaged (Lowenstein and Loewenfeld 1969). Hippus is more noticeable in the young and the pattern of oscillations (speed, size, and frequency) is the same in identical twins but not necessarily in fraternal twins (Loewenfeld 1993). Pupillary oscillations also occur in fatigued subjects but are slower and larger in amplitude (Loewenfeld 1993). The source of the oscillations is unknown.

When only one eye alone is stimulated with light, the pupil of the opposite eye constricts as well. In humans, the two pupils constrict equally (Lowenstein 1954; Lowenstein and Friedman 1942). The reaction of the opposite pupil is called the consensual light reflex, and the response of the pupil in the stimulated eye is called the direct light reflex. When both eyes are stimulated with light simultaneously, the pupil constricts more than when either eye alone is stimulated (Doesschate and Alpern 1967).

To determine whether the monkey's light reflex is a reasonable model for the human light reflex, we measured it under several of the same conditions described in the preceding text. These conditions include stimuli of different durations and light intensities and stimuli delivered to one or both eyes.

This paper constitutes part of M. Pong's PhD thesis in the Department of Physiology and Biophysics at the University of Washington.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Behavioral control of eye position

Three juvenile male rhesus macaques (Macaca mulatta; 2.5-3 kg) served as subjects. Target spots were presented on a tangent screen in front of the monkeys. The tangent screen subtended ±36° horizontally and ±26° vertically. To ensure that the target eye was stationary, we controlled eye position by rewarding the monkeys for fixating a small 0.25° red laser spot on the screen. Eye movements were monitored via the scleral search coil technique described by Fuchs and Robinson (1966). In brief, an eye coil was implanted around the monkey's left eye in a sterile surgical procedure. The eye coil leads were led off the eye and beneath the skin to the recording equipment through a dental acrylic cap attached to the monkey's head. The cap also provided insertion points for stabilization bars that allowed us to hold the monkey's head still. When the animals were placed within alternating magnetic fields, a voltage proportional to the horizontal and vertical components of eye position was induced in the coil. Eye movements could be resolved to within 0.25°. Details of the surgical procedures and the eye movement transducer can be found elsewhere (Fuchs et al. 1993).

During experimental sessions, the monkeys sat in a primate chair with their heads held by stabilization bars. The monkey and chair were in a sound-deadened and light-proof enclosure.

The surgical and behavioral training procedures used in these experiments are well documented (e.g., Fuchs et al. 1993) and have been approved by the Animal Care and Use Committee at the University of Washington. The animals were cared for by the veterinary staff of the Regional Primate Research Center. Between experimental sessions, the animals were housed under conditions that complied with NIH standards as stated in the Guide for the Care and Use of Laboratory Animals (DHEW Publication NIH85-23, 1985) and with recommendations from the Institute of Laboratory Animal Resources and the American Association for Accreditation of Laboratory Animal Care for animals of this species.

Measurement of pupillary size

Pupillary area was measured with the device schematized in Fig. 1. We measured pupillary area rather than the more traditional pupillary diameter for two reasons. The role of pupillary constriction is to control the total intensity of light falling on the retina and that depends on pupillary area, not diameter. Second, the change in pupillary area is actually a more sensitive measure than changes in pupillary diameter because the area changes as the square of the diameter.



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Fig. 1. Schematic of the pupillometer. Infrared (IR) light is reflected off the eye and imaged onto an array of charge-coupled devices (CCD) via a mirror. The image frame is collapsed into a single voltage value that reflects the light level of the image. The pupil dominates the image so any decrease in the pupillary area increases the voltage output from the pupillometer. Therefore in this and all subsequent figures, an upward deflection of a pupillary area trace indicates pupillary constriction. Full description in text.

From a position 30° lateral to straight-ahead gaze, the monkey's eye was bathed with infrared (IR) light from a three-by-four array of light-emitting diodes (LEDs; Active Electronics LD271, 12 mW output at 940 nm, 50° beam spread). The IR light was pulsed at a rate of 1 kHz or 60 Hz to match the capture rates of an array of IR-sensitive charge-coupled devices (CCD; Texas Instruments, Model No. TC211), which was the primary light-sensing component. The IR image reflected from the iris was redirected by an IR mirror (Edmund Scientific) angled at 45° to the monkey's straight-ahead gaze and positioned in front of the monkey's eye. The redirected image entered a tele-macro camera lens with an IR-passing filter to keep out stray visible light. The tele-macro lens (focal length: 210 mm) focused the image onto the CCD array. With appropriate positioning and focus, an image consisting mainly of the pupil filled the entire array surface. The positioning was made possible by requiring the monkey to fixate the laser spot while the lens was adjusted.

The image frames were sampled at 1 kHz and processed into a single voltage level per sample by summing the light signals reported by the individual elements in the CCD array. Because the optics were arranged so that the pupil was the dominant object in the image, any change in the signal from the pupillometer was the result of changes in the pupil per se. Increases in the signal represented pupillary constriction because more IR light was reflected from the iris. Decreases in the signal represented enlargement of the pupil because it filled more of the image frame and reflected less of the IR light. The net pupillometer signal was passed through a 25-Hz low-pass filter to remove high-frequency noise.

Photographs or video images of two pupillary states, one with maximum dilation and one with maximum constriction, were taken for each monkey. Before each recording session, the dilated state was measured after 15 min of dark-adaptation and the constricted state during a sustained (>5 s) light flash of 28,800 cd/m2 from a single LED directed at each eye. The diameter of the iris, measured with a ruler, was used as a reference to calibrate the pupillometer signal according to the actual pupillary sizes revealed on either the pictures or video. We assumed that the pupil's response range would be similar from session to session.

This calibration is valid if there is a linear relation between pupillometer voltage and pupillary area over the entire range of pupillary areas. The linearity of the pupillometer was tested by using the device to measure the area of holes drilled out of the halves of table tennis balls, which were placed where the monkeys' eyes would be in the apparatus. The voltage output of the pupillometer was linearly related to the area of the holes, which ranged from 2 to 80 mm2 (correlation coefficient, r = 0.996, P < 0.01). The juvenile monkeys tested here displayed pupillary sizes ranging from 17.8 to 59.17 mm2.

The sensitivity of the pupillometer was limited by the noise introduced by the magnetic field coils, by the range of the pupillometer signal, which was determined by the level of reflected IR light, and by the extent to which the dilated pupil filled the image frame. Consequently the noise varied from animal to animal and day to day. The voltage range from the smallest to largest pupil was usually ~5 V, and noise measured <200 mV peak-to-peak for a theoretical best signal-to-noise ratio of 25:1. With the noise level at ~200 mV, the smallest change in pupillary area that we could detect for monkey R was 1.7 mm2. With a maximum change in pupillary area of 33.7 mm2 at the highest light intensity, the operational signal-to-noise ratio for monkey R was 33.7/1.7 = 19.8.

Blinks and other lid movements also caused changes in the pupillometer signal. The blinks and saccadic eye movements could be detected easily as changes in eye position and were not analyzed. To ascertain that the measurement of pupillary area was not corrupted by subtle lid movements, we did two things. First, we presented the light flashes at regular intervals so the monkey would not produce a startle response with its attendant eye blink or palpebral narrowing. Second, we viewed the actual pupillary constriction through the video monitor by sampling the frames at 60 Hz. Unfortunately, we could not view the pupil simultaneously with the video monitor and with the pupillometer. However, during trials in which we viewed the pupil with the monitor, the stimulus seldom elicited any noticeable changes in lid position. In particular, the lid never occluded part of the pupil when the animal was alert and not blinking.

Testing pupillary behavior

During testing of the pupillary light reflex, the monkeys fixated the laser spot, which was positioned 10° above straight ahead. After the animals had been dark adapted for 15 min, light flashes of 100-ms duration and occasionally 1-s duration were presented from a single LED, which was positioned 50° temporal of straight-ahead and 7 cm away from the targeted eye. This LED (Stanley HPG5066X) emitted green light (560 nm peak wavelength) that ranged in luminance from 5 to 28,800 cd/m2 (averages of 3 measurements with a Minolta LS100 luminance meter). For the 5- to 28,800-cd/m2 luminances used here, we estimate that the light intensity entering the eye ranged from 10-4 to 0.6 candela. The light spread 10° from the LED and covered the entire eye but traveled no further than the nasal side of the orbit. The monkey's head was fixed relative to the LED so there was no change in illumination during testing. For binocular stimulation, an LED was positioned 50° temporal-ward of each eye. The light intensities to each eye were equal. The duration and intensity of the light flash were specified with a Nuclear Chicago stimulator that controlled the LED circuitry. Flashes were presented every 2.5-3.3 s.

The stimulus LED was connected in series with a second green LED, which was directed away from the monkey toward a light detector. Because both LEDs were activated together, the light detector monitored light onset and offset without obstructing any part of the stimulus directed at the pupil.

Data analysis

The horizontal and vertical components of eye and target position, pupillary area and a signal proportional to light intensity were recorded on magnetic video tape with the use of a VCR system that employs pulse-code modulation (Vetter 2000) or on FM tape (Honeywell 5600). These signals then were digitized at 1 kHz by a National Instruments A/D conversion board connected to an Apple Macintosh IIfx computer. The pupillary area traces were smoothed twice with a program that employed a seventh-order binomial smoothing filter (Marchand and Marmet 1983), which used the seven points before and after a datum to determine its value in the smoothed series. The half transmission frequency, at which amplitude was reduced by 50%, was 100 Hz. The data were analyzed off-line by means of an interactive computer program, which displayed pupillary area, the light stimulus, and horizontal eye position on a computer monitor. In addition, the program calculated and displayed the rate of change of pupillary area, which it smoothed by replacing each point with an average of itself and the points before and after it. The first author used a cursor to mark the beginning and end of the various movements from which the program calculated salient features of the movements such as their amplitude, duration, peak velocity, and latency relative to the visual stimulus. The data were displayed at 5 ms/pixel, allowing the data to be marked with a timing accuracy of ±5 ms. Pupillary responses accompanied by eye movements or blinks or preceded by pupillary dilation were not analyzed. Nor were responses made when the animal was clearly sleepy, as indicated by either slow oscillations in the pupillary area or slowly drifting eye movements.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Direct light reflex to a brief light pulse

GENERAL PROPERTIES. In response to a brief 100-ms pulse of light to one eye, the pupil of the stimulated eye constricted relatively rapidly and then, after reaching its peak constriction, immediately began a slower dilation (Fig. 2). Because the pupil did not start to constrict until after the pulse was over, the entire stimulus reached the retina before the pupil began to close.



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Fig. 2. Example of direct pupillary light reflex. Response of pupil in monkey R to 100-ms LED flash to the measured pupil. The constriction velocity departed from zero 187 ms after the onset of the light stimulus. Pupillary area changed by 12.4 mm2, peak constriction velocity was 102 mm2/s, and constriction duration was 300 ms. Dilation was considered to begin when constriction velocity fell to 0. The pupil dilated 10.4 mm2, peak dilation velocity was 35.8 mm2/s, and dilation duration was 1,040 ms. The dilation break indicates the point of separation between the 2 phases of the dilation.

We separated the pupillary response into a constriction and dilation phase and analyzed them separately. The start of pupillary constriction was marked as the time of the first positive change in the rate of change of pupillary area, henceforth called pupillary velocity, and the end as the time when pupillary velocity returned to zero (Fig. 2). The start of dilation was marked as the time of the first negative change in pupillary velocity and the end of dilation as the time when pupillary velocity returned to zero. During dilation, there often was a noticeable genuflection in the pupillary area trace (the dilation break), at which time there was a clear drop in dilation velocity. A similar two-phase dilation is seen in the human pupillary light reflex (Loewenfeld 1993).

PUPILLARY CONSTRICTION. Effect of light intensity on pupillary area. Pupillary area decreased as the eye was exposed to more intense light pulses but the basic shape of the pupillary response remained the same as shown by the average responses in Fig. 3, A and B. The change in pupillary area increased with the log of stimulus luminance for each animal. Although there was considerable trial-by-trial variation in the responses to the same light luminance, the average relation between the change in pupillary area and the log of stimulus luminance could be reasonably fit by an increasing straight line for each monkey (Fig. 4). The correlation coefficients (r) were 0.741, 0.742, and 0.618 for monkeys R, E, and T, respectively (P < 0.01). The sensitivity of the pupil to differences in luminance, measured as the slope of the change-in-area versus log luminance relation, was 4.89, 3.85, and 3.49 mm2/log of luminance for monkeys R, E, and T, respectively.



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Fig. 3. Average pupillary responses to 100-ms light flashes with different luminances for monkey R. A: pupillary constriction increases with light intensity. Each trace is the average of 24-36 responses to 4 different luminances (right of figure) in the same session. Traces are aligned on the onset of the constriction. B: peak constriction velocity increases with light luminance. Average velocity traces from the data in A, aligned on pupillary constriction onset. ···, peaks of responses associated with the 4 luminances. C: latency of pupillary constriction decreases with increases in light intensity. Responses aligned on the onset of the light pulse.



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Fig. 4. Relation of the change in pupillary area (PA) with stimulus luminance (I) for all 3 monkeys. Horizontal line at the top of each panel represents the average constriction in response to binocular calibration stimuli (28,800 cd/m2). Across all 3 monkeys the average pupillary size from dark adaptation to the calibration stimulus ranged from 49.0 mm2 dilated to 19.5 mm2 for an average area change of 29.5 mm2. Data are fit with log/linear regression lines. Regression fits are PA = 4.89 log I - 4.02 (r = 0.74), PA = 3.85 log I - 0.87 (r = 0.74), and PA = 3.49 log I - 3.79 (r = 0.62) for monkeys R, E, and T, respectively. The number of data points for monkeys R, E, and T are 574, 212, and 726, respectively.

The light intensities used in this experiment did not cause saturation in the change of pupillary area. Indeed the pupil was capable of constricting further as evidenced by the response to the binocular calibration stimulus (28,800 cd/m2 to both eyes; Fig. 4: calibration level, horizontal bars). Unfortunately, the amount of light generated by our single LED was insufficient to produce the maximum monocular constriction response.

At low light intensities, a pupil constriction was considered to be a response and not just a spontaneous fluctuation associated with hippus if the constriction occurred at a relatively constant latency relative to the presentation of repeated light stimuli (see following text).

Effect of light intensity on peak constriction velocity. Like the change in pupillary area, peak constriction velocity increased with stimulus intensity (Fig. 3B). The peak velocities increased with the log of stimulus luminance in each animal and were fit by straight lines (Fig. 5) with r values of 0.744, 0.574, and 0.572 for monkeys R, E, and T, respectively. The velocity sensitivity of the pupil to different luminances, i.e., the slope of the peak velocity-log luminance relation, was 29.6, 18.2, and 21.0 mm2/s/log luminance for monkeys R, E, and T, respectively. Again, there was no indication that peak velocity saturated at the maximum stimulus luminances (23,600 cd/m2) that we were able to produce with our monocular LED stimulus.



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Fig. 5. Relation between peak constriction velocity (PCV) and stimulus luminance (I). Data are fit with log/linear regression lines. Regression fits are PCV = 29.57 log I - 15.61 (r = 0.74), PCV = 18.15 log I + 6.48 (r = 0.57), and PCV = 21.03 log I - 14.83 (r = 0.53) for monkeys R, E, and T, respectively.

Effect of light intensity on constriction latency. The latency to pupillary constriction decreased with light intensity (Fig. 3C). In each animal, the latency decreased with the log of stimulus luminance and could be fit by a straight line (Fig. 6). The r values for the fits were -0.694, -0.515, and -0.548 for monkeys R, E, and T, respectively (P < 0.01). The sensitivity of pupillary latency to changes in luminance, i.e., the slope of the latency-log luminance relation, was -24.4, -20.7, and -28.2 ms/log luminance for monkeys R, E, and T, respectively. The shortest average latency to the brightest stimulus (23,600 cd/m2) that we could present to one eye was 128.6 ± 24.4 (SD) ms in monkey T. As we mentioned earlier, logistical constraints prevented us from presenting the calibration stimulus intensity to a single eye.



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Fig. 6. Relation between constriction latency (L) and stimulus luminance (I). Latency is the time from flash onset to start of pupillary movement as marked by the first author at a resolution of 5 ms/pixel. Data are fit with log/linear regression lines. Regression fits are L = -24.39 log I + 265.03 (r = -0.69), L = -20.74 log I + 221.24 (r = -0.52), and L = -28.16 log I + 264.95 (r = -0.55) for monkeys R, E, and T, respectively.

The brighter calibration stimuli (28,800 cd/m2 to both eyes) could not be used to measure either latency or constriction velocity because, under those conditions, the animals always blinked or made eye movements away from the fixation point. The change in pupillary area could be measured eventually, however, because the animal would resume fixation while the stimulus was still on for periods of >= 2 s.

Effect of light intensity on other constriction metrics. Constriction duration and time-to-peak constriction velocity were not well related to stimulus luminance. Constriction durations showed no significant relation with stimulus luminance for monkey T (P > 0.20) and although the relations were significant for monkeys E and R (P < 0.05), the correlation coefficients were only 0.37 and 0.10, respectively.

Similarly the time to reach peak constriction velocity showed no significant relation to stimulus luminance for monkey T (P > 0.50), and although the relations were significant for monkeys E and R (P < 0.01), the correlation coefficients were only -0.27 and -0.20 (Fig. 3), respectively. The ratio of the time to reach peak velocity to the constriction duration, which indicates how early peak velocity was reached in the constriction phase, averaged 29.6 ± 8.1, 27.2 ± 10.0, and 32.1 ± 10.1% (means ± SD) for all the responses from monkeys R, E, and T, respectively. Therefore peak velocity consistently occurred in the first third of the constriction phase.

Correlations between movement attributes. Some of the movement parameters were correlated not only with stimulus luminance but with each other as well.

The correlation between peak velocity and the change in pupillary area was particularly robust (Fig. 7). The data are nicely fit with linear relations, with r values of 0.962, 0.812, and 0.894 (P < 0.01) and slopes of 5.84, 5.79, and 4.96 mm2/s/mm2 for monkeys R, E, and T, respectively. The partial correlation coefficient (Snedecor and Cochran 1967) between the change in pupillary area and peak velocity with luminance fixed was calculated to determine whether the relation exists only because both parameters change with luminance. The partial correlation coefficients were 0.915, 0.703, and 0.839 for monkeys R, E, and T, respectively (P < 0.01). Comparison of the partial coefficients with the overall coefficients shows that the relation between constriction peak velocity and the change in pupillary area still was strong when luminance was fixed and therefore was not due solely to a covariance with luminance.



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Fig. 7. Relation between peak constriction velocity (PCV) and the change in pupillary area (PA). Data are fit with a linear regression line. Regression fits are PCV = 29.57 log PA - 15.61 (r = 0.74), PCV = 18.15 log PA + 6.48 (r = 0.57), and PCV = 21.03 log PA - 14.83 (r = 0.53) for monkeys R, E, and T, respectively.

Constriction latency decreased with the change in pupillary area. Linear fits to the data indicated that the relation was less robust (r = -0.729, -0.567, and -0.602 for monkeys R, E, and T, respectively (P < 0.01), than the relation between peak velocity and the change in pupillary area. The slopes of this relation were -5.480, -3.877, and -4.405 ms/mm2. Furthermore, the partial correlation coefficients with luminance fixed were only -0.444, -0.322, and -0.398 for monkeys R, E, and T, respectively (P < 0.01), indicating that the relation between latency and amplitude can be attributed largely to the relation of both amplitude and latency to luminance.

Constriction duration and the change in pupillary area also showed significant, but even weaker, correlations. The slopes of the relation between change in area and duration were 0.035, 0.026, and 0.014 mm2/ms, with correlation coefficients of 0.271, 0.434, and 0.259. The partial correlation coefficients with luminance fixed were 0.253, 0.365, and 0.295 for monkeys R, E, and T, respectively (P < 0.01).

DILATION. As seen in Fig. 2, dilation takes longer and reaches much lower peak velocities than does constriction. Dilation metrics were not related as robustly to luminance as were constriction metrics. The increase in pupillary area for dilation was related to the log of luminance with slopes of 3.353, 2.887, and 3.526 mm2/log of luminance for monkeys R, E, and T, respectively (r values of 0.606, 0.601, and 0.524, P < 0.01). Peak pupillary velocity was related to the log of luminance [slopes of 4.683, 4.736, and 4.294 mm2/s/log of luminance; r values of 0.440, 0.432, and 0.279 (all P < 0.01) for monkeys R, E, and T, respectively]. The worst relation with the log of luminance was for the time from stimulus offset to the start of dilation [slopes of -18.65, 2.038, and -32.66 ms/log of luminance; r values of 0.182, 0.019, and 0.276 (P < 0.01, P > 0.50, and P < 0.01) for monkeys R, E, and T, respectively]. Indeed, the latency of dilation from the stimulus offset was very similar across luminances and across monkeys: average latencies were 489 ± 99, 482 ± 60, and 467 ± 83 (SD) ms for monkeys R, E, and T, respectively.

Several dilation metrics were related. As with constriction, peak dilation velocity was linearly related to the change in dilation area. The partial correlation coefficients for the relation with luminance fixed were 0.486, 0.608, and 0.727 for monkeys R, E, and T, respectively (all P < 0.01). This correlation was not as strong as that between constriction peak velocity and the change in pupillary area (Fig. 7). Dilation duration also increased with the increase in pupillary area. The partial correlation coefficients with luminance fixed for that relation were 0.538, 0.547, and 0.481, for monkeys R, E, and T, respectively (P < 0.01).

VARIABILITY OF PUPILLARY RESPONSES. As can be seen from the data of individual responses in Figs. 4-6, the variability of pupillary constrictions to the same stimulus intensity was striking. The variability occurred throughout the entire time course of the constriction and throughout the dilation as well. Data at four stimulus intensities are shown in Fig. 8. For the brighter light intensities, the shapes of the responses at each light intensity remained similar but the amplitude varied substantially from trial to trial. The source of the variability is unclear. However, it is not the result of fatigue, previous stimulus luminance, or the nature of the previous response. For example, detailed tests in monkey T revealed no significant correlations between the amplitude of the pupillary constriction and any of these factors.



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Fig. 8. Variability in the pupillary light reflex. Families of pupillary responses to 100-ms pulse stimuli of different luminances from monkey R. Average of traces indicated by thicker line.

Monocular light reflex to prolonged light stimuli

To determine the steady-state pupillary response, we also presented 1-s LED light flashes of seven different intensities to two of the animals. Figure 9A compares the average responses of one animal aligned on constriction onset to both 100-ms (pulse) and 1-s (long) flashes of the same luminance. The initial part of pupillary constriction was identical for both the brief and prolonged flashes. For the 1-s stimulus, however, the pupil exhibited a sustained constriction for the duration of the stimulus.



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Fig. 9. Pupillary responses to stimuli of different durations. A: comparison between responses to 100-ms and 1-s monocular stimuli. B: response of the same monkey to a >5-s calibration stimulus of 28,800 cd/m2. Constriction and dilation in left and right panels, respectively.

The response to a prolonged stimulus differed from that to a brief stimulus in several ways. Responses were measured at stimulus intensities of 20, 47, 70, 130, 410, 1,360, and 1,420 cd/m2. Constrictions after long stimuli had significantly (P < 0.05) larger average constriction amplitudes at luminances of 70, 410, 1,360, and 1,420 cd/m2 than did constrictions to short stimuli. On the other hand, at the lowest luminance (20 cd/m2), the responses to brief stimuli were significantly (P < 0.05) larger than the responses to long stimuli. At two other intensities (47 and 130 cd/m2), the averages were not significantly different for brief and long stimuli (P > 0.05). These latter intensities were among the lower intensities tested, and the responses were more variable. At the higher intensities, the constrictions for the longer stimuli consistently were larger than those to brief stimuli.

At three of the four intensities that produced larger constriction amplitudes for long-duration stimuli, the average constriction duration also was significantly longer (P < 0.05). For the longer-duration stimuli, the constriction duration increased with luminance (r = 0.652 and 0.549, P < 0.01, for monkeys R and E, respectively). The increase in duration with luminance was 156 and 104 ms/log luminance, which was much larger than the increase in duration with luminance shown for the same monkeys with 100-ms flashes (5 and 33 ms/log luminance, respectively).

For long-duration stimuli, the correlation between peak constriction velocity and the change in pupillary area was significant for both monkey R (r = 0.733, P < 0.01) and monkey E (r = 0.821, P < 0.05). The slopes of this relation, 3.21 and 3.26 mm2/s/mm2 for monkeys R and E, respectively, were lower than the slopes for 100-ms flash responses. This relative difference can be seen in the data in Fig. 9A, which shows that two different constriction amplitudes can have essentially identical peak velocities.

When the light stimulus was maintained for several seconds, as when the calibration stimulus of 28,800 cd/m2 was presented to both eyes, the pupil developed oscillations, which continued after the light was turned off (Fig. 9B, right). At least one oscillation also was visible in response to the 1-s stimulus (Fig. 9A).

The magnitude and frequency of the oscillations were variable. We measured the change in peak-to-peak pupillary area over 20 different calibration sessions covering all three animals. During these sessions, we analyzed all oscillations that occurred after the large initial pupillary constriction across all three monkeys. The data set consisted of 309 single cycles of oscillation at light-off and 328 single cycles of oscillation at light-on. The oscillations, which averaged 2.3 ± 1.1 (SD) mm2 (n = 637), were not significantly different in average amplitude whether measured in the light or the dark. However, the oscillations in the light had shorter average periods in 17 of 20 calibration sessions, with 10 of the 17 session averages being significantly shorter (P < 0.05). In the light, average periods ranged from 630 to 901 ms, corresponding to frequencies of 1.6-1.1 Hz (96-66/min); in the dark, they ranged from 891 to 1,082 ms (1.1-0.9 Hz, 66-54/min).

Consensual light reflex

Thus far we have described pupillary constrictions that were elicited by light stimuli presented to the same eye as the pupil being measured, i.e., the direct light reflex. In all three monkeys, the pupil also constricted when 100-ms stimuli were presented to the opposite eye to elicit the consensual light reflex. The shape of the time course of the consensual response was similar to that of the direct light reflex response (Fig. 10A). However, it was both slower and smaller than the direct response. The change in pupillary area during the consensual response, like that of the direct light reflex, increased linearly with the log of stimulus luminance. The averages of the consensual response at each luminance were consistently less than those of the direct responses (Fig. 10B) but the data were so variable that the differences were not always significant. Only averages of >= 10 responses were compared. The consensual and direct responses were significantly different for four of eight luminances (P < 0.05) in monkey R, two of five luminances in monkey T, and one of five in monkey E. The average consensual response at any luminance ranged from 17 to 88% of the direct response (average = 52 ± 31%) in monkey R (Fig. 10B) and from 57 to 108% of the direct response (median of 92%, average 87 ± 17%) in monkey T. In monkey E, for which only one luminance was tested, the consensual response was 79% of the direct.



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Fig. 10. Comparison of direct and consensual pupillary light reflexes. A: average change in constriction area of 17 pupillary responses in the left eye (consensual) and 8 in the right eye (direct) during 100-ms pulse (890 cd/m2) stimulation of the right eye of monkey R. Right pupil measured first, then the left, on the same day. B: change in pupillary area for consensual and direct responses as a function of luminance. Data are fit with log/linear regression lines. Regression fits are PA = 6.04 log I - 9.22 (r = 0.82) for consensual responses (---) and PA = 7.27 log I - 7.82 (r = 0.77) for direct responses (- - -).

When both eyes were stimulated at the same time in the one monkey tested (T), the shape of the pupillary response was similar in time course to the direct monocular stimulus response (Fig. 11A). However, both the amplitude and peak velocity were greater. Like the monocular responses, the change in pupillary area and latency of the binocular stimulus responses varied with stimulus luminance (Fig. 11, B and C). At the three luminances tested in monkey T, stimulating both eyes led to an average change in pupillary area that was 87, 90, and 39% larger (P < 0.05) than the change elicited by the direct light reflex (Fig. 11B). Because monkey T's consensual reflex averaged ~92% of the direct reflex, an essentially linear addition of the direct and consensual responses did occur at two of the light intensities, 362 and 2836 cd/m2, but not at 7377 cd/m2.



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Fig. 11. Pupillary responses to binocular and monocular stimulation. A: average change in constriction area of 16 direct pupillary responses and 38 responses to binocular stimuli in monkey T; stimulus luminance was 2,840 cd/m2. Binocular stimuli produced a greater constriction than monocular stimuli. B: comparison of the change in pupillary area as a function of stimulus luminance for direct and binocular responses. Data are fit with log/linear regression lines. Regression fits are PA = 5.13 log I - 5.92 (r = 0.49) and PA = 4.77 log I - 9.26 (r = 0.66) for direct and binocular stimuli, respectively. C: comparison of constriction latency as a function of luminance for direct and binocular responses. Data are fit with log/linear regression lines. Regression fits are L = -52.40 log I + 377.51 (r = -0.59) and L = -23.34 log I + 262.53 (r = -0.45) for direct and binocular stimuli, respectively. In both B and C, - - - and --- are fits through data obtained from direct and binocular stimulations, respectively.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

We have tested the pupillary light reflex of nonhuman primates under conditions similar to those that have been used to measure the human light reflex. In our macaques, pupillary constriction and peak constriction velocity increased and latency decreased with the log of the luminance of a 100-ms light pulse. However, individual responses in the same monkey to the same light intensity were quite variable. In addition, peak constriction velocity was highly correlated with the decrease in pupillary area. Peak dilation velocity was also correlated, but less well, with the increase in pupillary area. For light pulses of 1 s, constriction generally was greater and of longer duration than for 100-ms pulses of equal luminance. For long (>1 s) and constant-luminance stimuli, the simian pupil underwent oscillations at frequencies of 0.9-1.6 Hz. The monkeys also exhibited consensual and binocular pupillary responses. As we will now discuss, the human and simian pupillary responses are qualitatively quite similar except in their response latencies.

Relation of response metrics to light intensity

In monkeys, the change in pupillary area (Fig. 4) and peak constriction velocity (Fig. 5) increased with stimulus luminance while the latency of the reflex decreased (Fig. 6). Similar relations have been reported in humans (Ellis 1981; Loewenfeld 1993; Reeves 1920). In addition to the relations with luminance, the change in pupillary area exhibited a tight linear correlation with peak constriction velocity in the monkey (Fig. 7). In humans, where the change in pupillary diameter was measured, the fit of this relation was described by a second-order polynomial because peak constriction velocity increased more slowly with larger response amplitudes (Ellis 1981). The monkey data do not show this saturation in peak constriction velocity, probably because our stimulus range was limited. In our study, the stimulus LED was positioned 50° temporal to the animal's straight-ahead line of sight, whereas the stimulus used in the human experiments (Ellis 1981) was positioned closer, i.e., 7° 31' temporal, allowing a more intense exposure of the retina.

The strength of the correlation between the change in pupillary area and peak constriction velocity contrasts strongly with the extreme variability of individual responses to stimuli of the same luminance (Figs. 4 and 5). We were unable to attribute the variability to either the condition of the pupil prior to the response, the light level of the previous stimulus, or animal fatigue. Hence we conclude that the strong correlation between the change of constriction amplitude and peak constriction velocity was likely due to a process occurring at the motor limb of the reflex arc. The signals being sent to the motor limb of the reflex may be variable, and this variability would account for the range of responses to stimuli of the same luminance. However, each signal that reaches the motor limb then appears to elicit a stereotyped change in peak constriction velocity for a given change in constriction area from the sphincter muscle. In contrast to that of the monkey, the human light reflex apparently is less variable (Alpern et al. 1963; Ellis 1981), although a direct comparison of the data are confounded somewhat because the human studies involved measurements of diameter, whereas we measured change in area.

Perhaps the variability is not so surprising because pupillary constriction in response to a bright light is a simple protective response that need not be accurate or reproducible. Although the reflex serves to regulate the amount of light that falls on the retina, most of the adjustment of the 1010 visual operating range is done by chemical and neuronal processes (Dowling 1987).

The pupillary responses observed in these monkeys had shorter latencies than those observed in humans. The shortest human latencies average ~200 ms in most studies (Feinberg and Podolak 1965), although there have been reports of latencies as low as 120 ms (Dolének 1960). Across our three monkeys, the shortest average latency at the highest stimulus luminances averaged 136 ms. Although they gave no absolute latency measures, Gamlin et al. (1998) also mentioned that pupillary response latencies were 80-100 ms shorter in their monkey subjects than in the human subject they tested.

Dilation parameters were not as well related to luminance as constriction parameters. Curiously, the latency of dilation from the time that the light was turned off seemed to be constant over luminances and across animals. In our monkeys, the average dilation latency of ~480 ms was less than has been shown for humans (~600 ms) (Gamlin et al. 1998, measured from their Fig. 3B) but greater than had been shown for monkeys responding to a 250-ms light pulse of 24 cd/m2 (~300 ms) (Gamlin et al. 1998).

Responses to sustained stimuli

As in humans, the simian pupil exhibits hippus (Fig. 9B). The oscillations are of similar frequency in both species (Loewenfeld 1993). Unlike the human pupil (Lowenstein and Loewenfeld 1969), the monkey pupil oscillated in darkness. However, those oscillations were significantly lower in frequency than oscillations with the light on. The oscillations in the dark were unlikely to be caused by fatigue because they were always observed during calibration at the beginning of the experimental session. The cause for hippus in humans is unknown.

Disparity between the direct and consensual responses

In early literature it was assumed that normal consensual and direct responses in humans would be equal (Lowenstein 1954; Lowenstein and Friedman 1942), and unequal responses were taken as a sign of CNS pathology (Lowenstein 1954). However, subsequent studies found that the consensual response may be smaller than the direct response even in healthy subjects (Smith et al. 1979; Wyatt and Musselman 1981). In our study, the consensual responses were less than the direct responses in the two monkeys where a distinction could be drawn (Fig. 10), ranging between ~ 63 and 82% of the direct response. Any conclusion that the consensual response is less than the direct response in nonhuman primates must be viewed with caution, however, because we were unable to measure the same pupil for both responses; thus a calibration error was possible. When we attempted to measure the same eye for the direct and consensual responses, the animals invariably became distracted and failed to fixate when we tried to redirect the light source at the other eye.

The consistently smaller consensual responses can be explained by an unequal distribution of nerve efferents from the pretectal olivary nucleus (PON), the presumed first relay in the light reflex after the retina, to the EW nucleus, the site of preganglionic neurons. Others also have suggested an unequal signal distribution in the light reflex pathway (Smith et al. 1979; Wyatt and Musselman 1981). In our study, the pupil was stimulated from 50° temporal to the monkey's straight-ahead line of sight, activating receptors in the nasal retina. From the nasal retina, most of the visual input crosses to the contralateral PON, assuming that the projections to the PON are arranged like the projections to the lateral geniculate nucleus (Malpeli and Baker 1975). Because each EW nucleus projects only to the ipsilateral ciliary ganglion, the direct response can be larger only if the distribution of fibers from the PON to the EW nucleus or their activation favors the EW nucleus contralateral to the PON. This would mean that the directly stimulated pupil would have a larger pupillary response. Indeed, anatomical studies show a consistently stronger contralateral than ipsilateral projection from the PON to the EW nucleus (Büttner-Ennever et al. 1996; Gamlin and Clarke 1995; Steiger and Büttner-Ennever 1979).

An asymmetry in the reflex pathway has been disputed because the pupil can be made to respond equally to stimulation on any part of the retina (Lowenstein and Loewenfeld 1969). This issue cannot be resolved with the current data because we did not determine or control for the specific areas of retinal stimulation.

One way to test the unequal projection from the PON would be to stimulate electrically at one PON and compare the effects on each pupil. If there is an unequal distribution of fibers, the pupil contralateral to the stimulation site should constrict more than the ipsilateral pupil.

Conclusion

This work has shown many similarities between the monkey and human pupillary light reflexes. The only difference is that simian constriction latencies are shorter, a finding confirmed by others (Gamlin et al. 1998). The similarities make the simian light reflex a reasonable model for the human light reflex and support the transfer of findings in monkeys to humans. In particular, findings about the neural substrate of the pupillary light reflex revealed in the companion paper can be expected to have relevance when extrapolated to humans.


    ACKNOWLEDGMENTS

The pupillometer electronics were designed primarily by E. Fonzo, formerly of the Regional Primate Center engineering staff. We are grateful for the deft editorial assistance of K. Elias.

This work was supported by National Institutes of Health Grants EY-00745 and RR-00166. M. Pong was supported by Vision Training Grant EY-07031.

Present address of M. Pong: Barrow Neurological Institute, Phoenix, AZ 85013.


    FOOTNOTES

Address for reprint requests: A. F. Fuchs, Regional Primate Research Center, Box 357330, University of Washington, Seattle, WA 98195-7330 (E-mail: fuchs{at}u.washington.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 13 September 1999; accepted in final form 11 May 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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