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Single neuron activity in the pupillary system
BRAIN RESEARCH
219
S I N G L E N E U R O N ACTIVITY IN T H E P U P I L L A R Y SYSTEM
JAMES D. SMITH, GERALD A. MASEK, LESTER Y. ICI-IINOSE, TAKESHI WATANABE AND LAWRENCE STARK Department of Electrical Engineering, University of Southern California, Los Angeles, Calif. 90007, Department of Biomedical Engineering, Presbyterian-St. Luke's Hospital and College of Engineering, University of Illinois, Chicago, Ill. 60680, Department of Physiological Optics, University of California, Berkeley, Calif. (U.S.A.) and Department of Physiology, Tokyo Medical and Dental University, Tokyo (Japan)
(Accepted June 10th, 1970)
INTRODUCTION Investigations of the pupillary system have been reported by many authors from various disciplines for more than a century. Studies have included anatomy 6,8,~9,3~, pharmacology3,18,19, electrical stimulation 1,2,5,11, light stimulation 9,21,24,26-28,31,34, isolated muscle experiments 8, single neuron experiments 14,2°-z2,31 and control theory applications12,zs,27,3L In this investigation responses of pupil area and oculomotor neuron activity are reported for the first time during light stimulation; preliminary aspects of this study have been reported already 2~,22. In these experiments the pupil light reflex system is intact and functioning. Pupil dilatation and constriction SCHEMATIC OF AND ELECTRONICS
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responses to light are simultaneously recorded with correlated single neuron activity. The pupillary pathway is shown in Fig. 1. The anterior portion of the oculomotor nucleus, the Edinger-Westphal nucleus, can be considered as a multiple input-multiple output neural site. Light induced activity from the pretectal area and blur induced activity from the cerebral cortex are presumably influencing the Edinger-Westphal nucleus cells to send motor signals which activate the lens and pupil musculature. The purpose of this investigation was not to exhaustively classify oculomotor neurons of the pupillary system but to determine the frequency response of neurons correlated with the pupil area during response of the system to changes in light stimulation. In one communication prior to this 2° both single neuron and pupil activity have been published but only steady state data were reported. This communication reports on time varying pupil and single neuron activity in response to repetitive, transient light stimulation. Another objective of this study was to focally stimulate at the site of single neuron recordings to compare light and electrical stimulation and to further investigate a possible causal relationship between the stimulated neurons and pupil area response. In this study measures are made of single neuron activity concurrently recorded with the pupil area light response. The frequency response of the averaged neural and pupil responses are compared. This comparison helps resolve the question of whether the neural control signals for the iris musculature are limited by the pupil muscle itself or if the frequency content of the neural signal does not extend beyond the limits of pupil muscle. A class of neurons near the anterior oculomotor nucleus has been found whose change in firing rate is highly correlated with pupil area response during retinal light stimulation. Focal electrical stimulation of the brain at the site of these single neurons provided comparative information concerning the relation between the anterior oculomotor nucleus and the pupillary musculature. The single unit data reported here confirm earlier suggestions that the pupil musculature limits the frequency response of the system24,27; the neural delay times are shorter than the pupil area response delay times and the time constants of neural responses are smaller than the pupil muscle response time constants. In another p a p e d 2, we will compare the neural and pupil area responses in the frequency domain but this approach is beyond the scope of the present paper. METHODS
Fifteen cats were used for these studies. Each animal was injected with an intraperitoneal dose of sodium pentobarbital (15-30 mg/kg); surgical procedures were accompanied by infiltration of wounds with Xylocaine. A stereotaxic frame was used to position the animal's skull; the atlas of Snider and Niemer 23 was used to aim the recording electrodes. One eye of the animal was maximally dilated with cyclopentolate hydrochloride and the consensual pupil response was driven from this input eye. A glow modulator tube was the light source, as shown in Fig. 1 and a voltage driven pulse-width modulaBrain Research, 24 (1970) 219-234
SINGLE NEURONS IN THE PUPILLARY SYSTEM
221
Fig. 2. Histological verification. This photomicrograph is an example of histological verification performed after neural recording and electrical stimulation experiments. Shown here is a frozen section of midbrain with electrode track and a coagulation point, below recording area, labeled. Figs. 3 and 12 are maps composed from similar sections. tion circuit was used to control the glow modulator tube. This resulted in a constant spectral density of the light stimulus at different intensities. Pupil area response was measured by a reflection technique. The contralateral or 'output' eye was illuminated with infrared light and a photodiode in front of this eye monitored changes in the amount o f infrared light reflected from the iris. The photodiode signal was calibrated during observation of the pupil with a television monitor 32 which magnified the image by a factor of 15. In order to eliminate eye movement, an artifact in this measuring system, the animal was continuously infused with succinylcholine chloride which paralyzed the skeletal musculature and therefore respired artificially with a respirator. The microelectrodes, insulated tungsten wird, were used for extracellular recording, focal electrical stimulation and coagulation of the recordlng and stimulation sites. The waveforms of single neurons recorded in the pretectal area were triphasic and the recordings from the oculomotor dilatation units were biphasic. Recording sites were verified histologically and Fig. 2 is an example of histological control from frozen unstained sections 5 / , m thick; a modified KliiverBarrera stain 10 was also used in preparing sections. Data processing was done with digital computer (IBM 1800). Each stimulus period was divided into 500 sample periods. During each of these sample periods or bins the voltage corresponding to the pupil area was sampled, stored on the computer and averaged. The neural spike trains were also averaged but in a less straightforward manner due to the discontinuous nature of the data. The neural firing rate was computed for each bin. During the experiment a simple analog computer circuit (an RC circuit) was used to low-pass filter a series of standard pulses by the spike train. This signal was proportional to firing rate within limits determined by direct calculation. Brain Research, 24 (1970) 219-234
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(1) Dilatation correlated neurons
Dilatation neurons were recorded in and near the anterior o c u l o m o t o r nucleus. A dilatation neuron is defined as an o c u l o m o t o r neuron with a firing rate that increases during pupil dilatation and decreases during constriction. These modulations o f the neuron's steady firing rate lead the pupil area responses to light by 0.2 sec, the neuromuscular delay time reportedl,Zl, 31 for pupil area responses to electrical stimulation of the Edinger-Westphal nucleus and the short ciliary nerves. A map shows the location o f these units in Fig. 3. A total o f 53 neurons were recorded in the o c u l o m o t o r area o f 15 cats. N o n e o f these neurons sustained steady changes in firing rate in response to different levels o f light stimulation. The average firing rate during steady illumination varied f r o m unit to unit f r o m approximately 15 to 50 pitts*. All but one o f the units recorded exhibited its correlation with pupil area during periodic light stimulation. The average firing rate o f a dilatation n e u r o n is shown with concurrently averaged pupil area response to light in Fig. 4. The average firing rate varies f r o m 1 to 55 pitts. The average neural response changes f r o m one rate to another more rapidly than the pupil area changes. * Pitts equals spikes/sec, named after the late Walter Pitts, a colleague of the late Warren McCulloch. Brain Research, 24 (1970) 219-234
223
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T I M E (seconds) Fig. 4. Dilatation correlated neuron (No. 1), averaged spike train and pupil responses. In lower trace is stimulus waveform; light flux at input eye ranged from 0.01 to 10 mlumens. Middle waveform is firing rate of a dilatation unit averaged over 44 periods of light stimulation. At top is averaged pupil area response. Light stimulus was repeated at a frequency of 0.25 c/sec. For all figures; time in seconds and dilatation is upward.
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TIME (seconds) Fig. 5. Dilatation neuron (No. 1) with pupil and model response. Upper trace is same light stimulus as in Fig. 4. Immediately below is extracellular recording from dilatation unit. Third trace is a low-pass filtered version ( a second order, model with a time constant = 300 msec) of spike train above. Fourth trace shows pupil area; constriction is downward.
Brain Research, 24 (1970) 219-234
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Fig. 6. Dilatation neuron (No. 2), instantaneous neural and pupil responses. Pupil and neural response to square wave light stimulation at 0.25 c/sec. Stimulus not shown. Pupil area decreases (downward deflection) in response to light stimulation. Neural spike train has been low-pass filtered by model described in text for Fig. 5. Notice cyclic variations in neural response at frequencies from 0.5 to 3.0 c/sec. Time in sec.
The single neuron responds to a step increase of light excitation with an average delay of 30 msec while the pupil area response follows the light stimulus with an average delay of 250 msec. The neural response to light off is 60 msec; the pupil area response delay is 360 msec. The spike train response of this neuron is shown in Fig. 5. Also shown is the response of a simple neuromuscular model (RC) circuit to this spike train. The use of such lag models of neuromuscular response is supported by frequency analysis studies of skeletal16,17 as well as pupil muscle dynamics1,21, al. This second order model is accurate in predicting pupil area response to light given the dilatation neuron response. The actual pupil area response follows this model response with a delay of 250-300 msec. It is hypothesized that a neural noise generator exists in the pupillary servomechanism z4 and is responsible for the stochastic fluctuations in pupil area shown to be identical in both eyes of cats 12 and humans 27. This relationship between noise in oculomotor firing rates and in pupil area is not understood. Signals from different neurons have different amounts of noise, not necessarily correlated with pupil noise. The model response of another (more noisy) dilatation neuron is shown compared with pupil area response to a square wave of light (not shown) in Fig. 6. Both this filtered response (v ~ 0.05) and the pupil area response show a consistent component of 0.25 c/sec, the frequency of the light stimulation waveform. Note the high frequency noise in the filtered neural response which oscillates from 1.5 to 3.0 c/sec; this neural noise does not appear to be correlated at all with the concurrent pupil area recording. Another dilatation neuron response to light stimulation (neuron 3) is shown in Fig. 7. Ten cycles of the spike train response are averaged and compared with the average in Fig. 4 (neuron 1). It appears that the average neuronal rate of response during constriction is slower in Fig. 7 than in Fig. 4. Some dilatation neurons responded only to light stimulation while others (Fig. 8) exhibited firing rate changes during small fluctuations of pupil area under constant illumination. Periodic light stimulation (0.1-10 mlumens square wave at 0.1 c/sec) Brain Research, 24 (1970) 219-234
225
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TIME 0.0 10.0 20.0 300 40.0 Fig. 8. Dilatation neuron (No. 5), neural and pupil responses. Activity of a dilatation neuron that exhibited no apparent response to light. However, during steady light stimulation variations in pupil area closely followed the activity of this cell. Top trace is pupil area (dilatation upward). Below is a low-pass filtered version of spike train; with time constant of filter sufficiently small so that each separate spike can be seen.
w h i c h c a u s e d a p u p i l response d i d n o t result in a n y a p p a r e n t changes in firing rate. In Fig. 8 bursts o f n e u r a l activity regularly p r o c e e d increases in p u p i l area. This d i l a t a t i o n neuron, u n r e s p o n s i v e to light, m a y be a m e m b e r o f a class o f cells t h a t generate the p u p i l noise signal. P e a k s o f p u p i l d i l a t a t i o n f o l l o w the p e a k s o f neural activity with an average d e l a y o f 1.16 sec for 7 d i l a t a t i o n s in Fig. 8. This delay is m u c h l o n g e r t h a n those o b s e r v e d in p u p i l response to p e r i o d i c light s t i m u l a t i o n (0.2-0.25 sec). P e r i o d i c Brain Research, 24 (1970) 219-234
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Fig. 9. Dilatation neuron (No. 5), auditory response. Record taken immediately after record of Fig. 8. Pupil dilatation was elicited 3 successive times during which the dilatation neuron ceased firing entirely. Approximate times of auditory stimulus onset are marked (± 0.3 sec).
pupil dilatation responses to light and electrical stimulation can take place after section of the sympathetic innervation of iris muscle; however the sympathetic iris innervation of this animal was intact and this neuron may have been connected to the dilator muscles of the sympathetic system. We have observed pupil dilatation delay times of about 1 sec after electrical stimulation of spinal cord and cerebral cortex. These dilatations may have involved neurons similar to the one in Fig. 8. This same unit exhibited an opposite type of coupling with pupil dilatation (Fig. 9) during a psychosensory response. The animal was stimulated with 3 brief and intense auditory stimuli, hissing noises. The pupil responded with a dilatation each time. The neuron abruptly ceased firing during most of the dilatation response and for as long as 2 sec after the psychosensory evoked dilatation was completed. The delay time of the auditory response was not precisely measured but was about 0.5 sec.
(2) Other types of units Several other types of units were observed in addition to the dilatation correlated units. A majority of units showed no correlation with the pupil area response; some units showed positive correlation (dilatation) with the pupil area in an intermittent fashion; these units would track the pupil area response and then would randomly switch to some spontaneous firing rate ostensibly unrelated to the pupil activity. In one case a noisy constriction unit response was recorded, the neuron was lost before averaging techniques could be used to process the signal. The fact that no confirmed constriction-correlated units were found in the region of the anterior oculomotor nucleus is difficult to explain. Sillito z0 has reported on the activity of oculomotor neurons with higher average firing rates during periods of smaller pupil diameter in cats.
Brain Research, 24 (1970) 219-234
SINGLE NEURONS IN THE PUPILLARY SYSTEM
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Fig. 10. Map of pupil area responses to electrical stimulation. Sites in midbrain where stimulation evoked pupil responses are marked. Some recording sites of dilatation correlated single units also are listed. Note two examples of loci where both single unit recording and then focal electrical stimulation were performed.
(3) Electrical stimulation." deterministic and stochastic responses Electrical stimulation in the region of the anterior oculomotor nucleus (Fig. 10) produced both dilatation and constriction responses in pupil a r e a l Stimulus pulses were f r o m 5-10 FA (5-10 • 10 -6 A), lasted for 1 msec and were given at the rate of 50 pulses/sec. The stimulus currents were twice the threshold stimulus necessary to produce a pupil response. Pupil dilatation responses were always observed in response to electrical stimulation at the site of dilatation correlated neurons (5 animals). In all experiments constriction responses resulted only from stimulation of a restricted area. The recording site of the constriction unit shown in Fig. 3 was in this area. There is good neuroanatomical evidence 15 to suggest that the preganglionic cells of the pupil constriction pathway are in the anterior part of the third cranial nucleus and the synapse in the ciliary ganglion. The neurons at this locus are the F C P (final c o m m o n pathway) cells of the Edinger-Westphal nucleus which send preganglionic fibers from the anterior portion of the third nucleus to the ciliary ganglion and possibly the episcleral ganglia. Electrical stimulation of the F C P cells results in pupil constriction (Fig. 11). The delay time for constriction is from 300 to 350 msec and the redilatation delay time is the same. N o asymmetry in delay time occurs as was observed during pupil response to light stimulation. Pupil dilatation responses (Figs. 12 and 13) to electrical stimulation were obtained above the F C P cells. In some cases moving the
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Fig. 12. Pupil dilatation to midbrain, electrical stimulation. Lower part of figure is stimulus waveform. Stimulus intensity and rate is same as in preceding figures. Middle shows 9 consecutive traces of pupil area response superimposed; records shifted vertically in order to superimpose all traces 3.75 sec after stimulus onset. Average pupil area response shown above. Electrode was 1.5 mm dorsolateral from Edinger-Westphal nucleus (anterior 5.0, lateral 1.0, vertical 2.0) at an angle of 18°.
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Fig. 13. Pupil dilatation response,electrical stimulation. Ten consecutive responses of pupil area shown driven by square waves of electrical pulse train excitation. Pulse rate is 50 pulses/sec, pulse heights are 10 pA. Note that secondary fluctuations of pupil area appear to be unaffected by stimulus. This record is from the same electrode penetration as in Fig. 12 at a site 0.25 mm ventrolateral. electrode only 0.5 mm reversed the pupil response from dilatation to constriction.When electrically stimulating at the site of a dilatation unit (Results, part 1) the pupil never responded with constriction although in several cases the electrode was within 0.5 mm of the FCP cells. The stimulating current was thus highly localized in these cases and presumably only depolarized neurons effecting pupil area increase. The pupil can be actively dilated by stimulation of the sympathetic fibers that innervate the dilator muscle of the iris 31. In a control experiment it was found that the dilatation responses to focal midbrain stimulation were of equal amplitude before and after surgical section o f the sympathetic fibers. Therefore, this pupil dilatation response to focal electrical stimulation is believed to be due to inhibition of firing of the FCP cells mediated by the stimulated dilatation neurons. Often during light and electrical stimulation, variations in pupil area occurred that were not correlated with the stimulus, i.e., a non-deterministic response. Fig. 11 shows an example of several superimposed constriction responses to an electrical pulse train. During the influence of the stimulus, the pupil area shows a constriction response of similar shape each period. The remainders of each period are less predictable. This randomness can be represented by using the idea o f a neural noise generator 24 which
Brain Research, 24 (1970) 219-234
J.D. SMITHet al.
230
sends identical stochastic signals to each pupil. These random variations appear to be attenuated during the constriction response. We propose that the FCP cells undergo a temporal and spatial synchronization such that the neural noise signal is suppressed during the influence of the stimulus. Electrical pulse train stimulation 1.0 mm above the FCP cells produces a repeatable pupil dilatation (Fig. 12). The noise in the pupil area record appears to be attenuated during the influence of the stimulus. Random fluctuations in pupil area resume after the stimulus stops. These stimulated cells may normally generate a signal that inhibits the FCP cells randomly and thus produces pupil noise. The noise signal of these neurons would be suppressed during this stimulation which synchronizes their firing times. In Fig. 13 several periods of pupil area response are superimposed during stimulation in the same experimental animal 0.25 mm below the dilatation point in Fig. 12. At this site electrical stimulation results in repeatable pupil dilatation. There is no suppression of pupil noise during stimulation of this (different) class of cells. The stimulation of cells firing at this site results in inhibition of the FCP cells and in pupil dilatation. The cycle to cycle variation in pupil area remained constant throughout the response. Presumably the midbrain cells (candidate noise generator cells) which had been stimulated before (Fig. 12) were not synchronized by stimulation during Fig. 13. Therefore their asynchronous activity during the recording in Fig. 13 may be contributing to the pupil noise throughout the cycle duration. It must be emphasized also that the noise generator evidence above rests on visual observations of a qualitative nature (Figs. 12 and 13).
(4) Delay times and frequency response of neural and pupil response Average delay times of the neuron response to onset and termination of pulses of light have been measured. The neural 'on' delay time was 30 msec and the 'off' delay time was 60 msec; the pupil delay times were 250 and 360 msec. The neural and pupil area delay times to 'off steps' of light are longer (150 ~ ) than the delay time to 'on steps' (0.01-10 mlumens) as shown in Fig. 4. A straight line approximation to the change in average rate of firing to an on-step of light (during the first 200 msec) has a negative slope of 420 pitts/sec; the rate of change developed in response to the off-step of light is 140 pitts/sec. The average firing rate during constriction and during dilatation can be considered separately. A first order approximation of neural activity during constriction indicates that the average firing rate is frequency limited at about 6 c/sec. Tc~ = 0.025 sec (first order time constant, constriction) We1 = 1/Tc = 1/0.025 = 40 radians/sec BF¢I ---- Wc/2z~ = 40/6.28 = 6 c/sec (break frequency). The break frequency approximation to firing rate changes during dilatation can be similarly derived. TD1 = 0.1 sec (first order time constant, dilatation) WD1 1/TD : 1/0.1 : 10 radians/sec BFD1 : WD/2:r : 10/6.38 : 1.5 c/sec (break frequency). :
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TABLE I Time constants (sec)
Neural
Pupil area
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3 7 2.0 0.3
Neuron Fig. Tc = TD =
1 4 0.3 0.9
3 7 1.5 2.5
Dn-on = 0.03 Dn-oft = 0.06
0.05 0.02
DC = DD =
0.25 0.36
0.35 0.55
Time delays (sec)
Light-on Light-off
The table above summarizes the time delays and time constants of the neural and pupil response to light in Figs. 4 and 7. I f the average neural response of neuron number 1 (Fig. 4) is a fair approximation of the total deterministic neural signal transmitted to the iris, the pupil musculature would low-pass filter (frequency limit) this signal as the pupil model predicts in Fig. 5. All delay times and time constants for the pupil response are from 6-10 times larger than the neural response except for the time constant of neuron 3 during pupil constriction. DISCUSSION The pupil dilatation neurons described in this paper correlate with the contralateral pupil response to light stimulation and lead it by 250 msec; a dilatation response was always produced (5 different animals) when the recording site of these correlating neurons was electrically stimulated. These neurons were found 0.5-2.0 m m above and below the Edinger-Westphal preganglionic cells. This distance was confirmed by advance of the microelectrode to a constriction producing locus operationally defined as the Edinger-Westphal nucleus. Histological preparations show that the anterior oculomotor nucleus coincides with this operationally defined neural station (see the stereotaxic maps of Figs. 3 and 6). Both the delay times and time constants cited in Table I for 2 different dilatation neurons reflect that the neural response to light is 6-10 times faster than the pupil response. The dilatation neuron in Fig. 7 has a time constant during constriction (light on) that is larger than the time constant of the pupil response. Although this time constant is too large to be considered causal with respect to the pupil constriction response, the dilatation time constant of the cell (0.3 sec) is about 8 times faster than the pupil dilatation time constant (2.5 sec). This apparent lack of correlation with pupil dynamics may be attributed to a distribution o f dynamics among these dilatation neurons that we postulate relay light information from the pretectal area to the constrictor cells (final c o m m o n pathways) of the EdingerWestphal nucleus. We propose that a time averaged version of the total dilatation neuron signal (all dilatation neurons in the loop) would have a time constant 6-10 times smaller than the concurrent pupil response time constant. Brain Research, 24 (1970) 219-234
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The dilatation neurons reported herein are considered causal with respect to pupil area due to their prediction of pupil area response (Figs. 5 and 9), the pupil dilatation response to focal electrical stimulation (small pulses, 10 -~ A) and because of the neural time constants and delays that are appropriately smaller but match the asymmetry of the pupil response characteristics (Table I). The pupil responses to focal electrical stimulation in the midbrain also suggest that there are two classes of cells whose stimulation results in dilatation. One class of these cells is implicated in generation of pupil noise. During synchronous stimulation of these ceils (noise cells) the cycle to cycle variation of pupil response (pupil noise) is attenuated. During stimulation of the other class of cells (deterministic cells), as a different locus, the pupil noise is the same during the pupil dilatation and afterwards. We propose that the 'noise cell' signal is undisturbed during stimulation of the 'deterministic cells' and contribute a constant 'noise' signal to the pupil muscles via the constriction cells of the Edinger-Westphal nucleus. Because the dilatation responses to midbrain stimulation also occur after section of the sympathetic nerve innervating the pupil (the preganglionic fibers adjacent to the vagus nerve) we believe these responses were caused by inhibition of the constriction producing FCP motoneurons of the Edinger-Westphal nucleus. Dilatation neurons as well as dilatation stimulation sites were recorded dorsal to the Edinger-Westphal nucleus. An oculomotor accessory nucleus 6 dorsal to the Edinger-Westphal nucleus and inhibitory to somatic as well as visceral third nucleus cells 3° has been described. This group of cells may be the locus of the dilatation neuron cell bodies. One of the dilatation neurons recorded showed correlation with pupil area, did not respond to light stimulation but did respond to auditory stimulation (Fig. 8). More important is the finding that there are two classes of recorded dilatation cells. One group responds to light with delay times longer than those of pretectal neurons and leads the pupil response to light by about 0.25 sec, the delay time recorded for pupil response to stimulation of the midbrain. These cells display the same asymmetry in delay times and time constants (both smaller for light-on responses) that the pupil muscle response displays; the pupil response delay times and time constants are, however, 6-8 times larger in magnitude. A second group ofceUs may be represented by the recordings in Figs. 8 and 9. This neuron does not respond to light but leads pupil dilatation by about 1.0 sec; and, its low-pass filtered response (stimulation of pupil muscle dynamics) predicts the noise in pupil area with a fair degree of accuracy. Stimulation of the sites of both classes of dilatation cells always resulted in pupil dilatation, even in cases where later stimulation 0.5 mm away resulted in pupil constriction (the constriction cells of the Edinger-Westphal nucleus). A model of the human pupil system has been proposed 24 which suggests that a neural process common to both pupils provides a stochastic signal that results in identical pupil area variations for both pupils during steady illumination of the retinae. Pupil noise has been shown to be identical in both pupils of the cat lz as it was found to be in humans 27. Clearly the neuromuscular process is eliminated as a source of the noise; and, anatomical evidence29,~3 suggests that separate bilateral groups of FCP cells activity drive the constriction of each pupil. These data lead to the proposal that a common neural Brain Research, 24 (1970) 219-234
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233
process provides the noise signal before the division of the pupil pathway to the right and left eyes. The results of both single neuron recording and focal electrical stimulation experiments support the idea that two classes of dilatation neurons cause pupil dilatation. We postulate that both classes of cells, 'deterministic' and 'noise' cells, inhibit the final c o m m o n pathway neurons, constriction cells (as defined by electrical stimulation) of the Edinger-Westphal nucleus. Furthermore the evidence suggests that the 'noise' cells do not respond to light stimulation. SUMMARY (1) In cats with a light-driven pupillary response, dilatation-correlated single neurons have been recorded near, but not in, the Edinger-Westphal nucleus. The firing rates of these units range from 1 to 80 pitts (spikes/sec). (2) Focal electrical stimulation was performed confirming the dilatation action of these cells as well as the constriction activity of the Edinger-Westphal region itself. (3) During light stimulation the rate of change of the average neural response is faster than the concurrently averaged pupil area response. ACKNOWLEDGEMENTS The authors are grateful for the assistance of Dr. George Moore in reading the manuscript and making a number o f helpful suggestions. This work was partially supported by the W. Clement and Jesse V. Stone Foundation, N I H Grants NB-07803, NB-06197, NB-06487; and GM-01436, PHS MH-8396 and O N R N-0014.
REFERENCES 1 BAKER,F. H., AND STARK,L., Black Box Description and PhysicalElement Identification in the Papil System, M.I.T., Cambridge, Mass., 1963, pp. 247-250. 2 BOCHEFONTAINE,M., Contribution b. l'6tude des effets produit par rexcitation 61ectrique du cerveau, C.R. Soc. Biol. (Paris), 17 (1875) 324-329. 3 DENNISON,B. L., A mathematical model for the isolated cat iris sphincter muscle, Proc. 19th Ann. Conf. Eng. reed. Biol., 8 (1966) 194. 4 DENNISON,B. L., A mathematical model for isolated iris dilator muscle, Proc. 20th Ann. Conf. Eng. reed. Biol., 9 (1967) 207. 5 HOPES,R., ANOMAGOtrN,H. W., Autonomic responses to electrical stimulation of the forebrain and midbrain with special reference to the pupil, J. comp. NeuroL, 76 (1942) 169-191. 6 HOGG,I. D., Observations of the development of the nucleus of Edinger-Westphal in man and the albino rat, J. comp. NeuroL, 126 (1966) 567-584. 7 HUBEL, D. H., Tungsten microelectrode for recording from single units, Science, 125 (1957) 549-550. 8 LOWENSTEIN,O., MURPHY,S. B., ANDLOWENFELD,I. E., Functional evaluation of the pupillary light reflex pathways. Experimental pupillographic studies in cats, Arch. Ophthal., 49 (1953) 656-670. 9 LOWENSTEIN,O., ANDLOWENFELD,I. E., Influence of retinal adaptation upon the pupillary reflex Brain Research, 24 (1970) 219-234
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to light in normal man. I. Effect of adaption to bright light on the pupillary threshold, Amer. J. Ophthal., 48 (1959) 536-549. 10 MARGOLIS,G., AND PICKETT, J. P., New applications of the Luxol Fast Blue myelin stain, Lab. Invest., 5 (1956) 464-478. 11 MAGOLrY,H. W., Maintenance of the light reflex after destruction of the superior colliculus in the cat, Amer. J. Physiol., 111 (1935) 91-98. 12 MASEK,G. A., SMITH,J. D., WATANABE,T., ANDSTARK,L., System analysis of the neural control of the pupil light reflex, in preparation. 13 NATHAN,P. W., AND TURNER, J. W. A., The efferent pathway for pupillary contraction, Brain, 65 (1942) 343-351. 14 NISIDA, I., OKADA,H., AND NAKANO, O., Electrical activity of the pretectal region of the cat to visual stimulus, Yonago Acta reed., 4 (1959) 7-18. 15 OKADA,H., NAKANO,O., OKAMOTO,K., NAKAYAMA,K., AND NISHIDA, I., The central path of the light reflex via the sympathetic nerve in the cat, Jap. J. Physiol., 10 (1960) 646-658. 16 PARTRIDGE,L. D., Modifications of neural output signals by muscles: a frequency response study, J. appl. Physiol., 20 (1965) 150-156. 17 REINHART,R. L., AND ZUBER,B. L., Horizontal eye movements from abducens nerve stimulation in cat, IEEE Trans biomed. Eng., 17 (1970) 11-14. 18 SCHAEPPI,U., Postganglionic nature of parasympathetic innervation of pig iris sphincter, Amer. J. Physiol., 210 (1966) 91-98. 19 SCHAEPPI,U., RUBIN, R., ANDKOELLA,W. P., Electrical stimulation of the isolated cat iris, Amer. J. Physiol., 210 (1966) 1165-1169. 20 SILLITO,A. M., The location and activity of pupilloconstrictor neurones in the midbrain of the cat, J. Physiol. (Lond.), 194 (1968) 39-40. 21 SMITH,J. D., MASEK,G. A., ]CHINOSE, L. Y., WATANABE,T., AND STARK, L., Midbrain single units correlating with pupil response to light, Science, 162 (1968) 1302-1303. 22 SMITH,J. D., MASEK,G. A., ICHINOSE,L. Y., WATANABE,T., AND STARK,L., Single unit dynamics in the cat pupillary control system, 8th Int. Congr. Med. Biol. Eng., (1969). 23 SNIDER,R. S., AND NIEMER, W. T., A Stereotaxic Atlas of the Cat Brain, Univ. Chicago Press, Chicago, Ill., 1961. 24 STANTEN,S. F., AND STARK, L., A statistical analysis of pupil noise, IEEE Trans. biomed. Eng., 13 (1966) 140-152. 25 STARK, L., AND SHERMAN, P. M., A servoanalytic study of consensual pupil reflex to light, J. Neurophysiol., 20 (1957) 17-26. 26 STARK,L., CAMPBELL,F. W., AND ATWOOD,J., Pupil unrest: An example of noise in a biological servomechanism, Nature (Lond.), 182 (1958) 857-858. 27 STARK, L., Stability, oscillation and noise in the human pupil servomechanism, Proc. IRE, 47 (1959) 1925-1939. 28 STARK, L., Neurological Control Systems: Studies in Bioengineering, Plenum Press, New York, 1968, pp. 73-183. 29 SZEt~TAGOTI-tAI,J., Die zentrale Leitungsbahn des Lichtreflexes der Pupillen, Arch. Psychiat. Nervenkr., 115 (1942) 136-156. 30 SZ~NTAGOTHAI,J., Anatomical Aspects of Inhibitory Pathways and Synapses. Nervous Inhibition (Florey), Pergamon, New York, 1961, pp. 32-39. 31 TERDIMAN,J., SMITH, J. D., AND STARK, L., Pupil response to light and electrical stimulation: Static and dynamic characteristics, Brain Research, 16 (1969) 288-292. 32 Television Monitoring System made available by Biosystems and S.S.I. Division of Whittaker Corporation. 33 WARWICK, R., Oculomotor organization, Ann. roy. Coll. Surg. Engl., 19 (1956) 36-52. 34 YOUNG, F. A., AND BIERSDORF,W. R., Pupillary contraction and dilatation in light and darkness, J. eomp. physiol. Psyehol., 47 (1954) 264-268.
Brain Research, 24 (1970) 219-234
219
S I N G L E N E U R O N ACTIVITY IN T H E P U P I L L A R Y SYSTEM
JAMES D. SMITH, GERALD A. MASEK, LESTER Y. ICI-IINOSE, TAKESHI WATANABE AND LAWRENCE STARK Department of Electrical Engineering, University of Southern California, Los Angeles, Calif. 90007, Department of Biomedical Engineering, Presbyterian-St. Luke's Hospital and College of Engineering, University of Illinois, Chicago, Ill. 60680, Department of Physiological Optics, University of California, Berkeley, Calif. (U.S.A.) and Department of Physiology, Tokyo Medical and Dental University, Tokyo (Japan)
(Accepted June 10th, 1970)
INTRODUCTION Investigations of the pupillary system have been reported by many authors from various disciplines for more than a century. Studies have included anatomy 6,8,~9,3~, pharmacology3,18,19, electrical stimulation 1,2,5,11, light stimulation 9,21,24,26-28,31,34, isolated muscle experiments 8, single neuron experiments 14,2°-z2,31 and control theory applications12,zs,27,3L In this investigation responses of pupil area and oculomotor neuron activity are reported for the first time during light stimulation; preliminary aspects of this study have been reported already 2~,22. In these experiments the pupil light reflex system is intact and functioning. Pupil dilatation and constriction SCHEMATIC OF AND ELECTRONICS
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220
J.D. SMITHet al.
responses to light are simultaneously recorded with correlated single neuron activity. The pupillary pathway is shown in Fig. 1. The anterior portion of the oculomotor nucleus, the Edinger-Westphal nucleus, can be considered as a multiple input-multiple output neural site. Light induced activity from the pretectal area and blur induced activity from the cerebral cortex are presumably influencing the Edinger-Westphal nucleus cells to send motor signals which activate the lens and pupil musculature. The purpose of this investigation was not to exhaustively classify oculomotor neurons of the pupillary system but to determine the frequency response of neurons correlated with the pupil area during response of the system to changes in light stimulation. In one communication prior to this 2° both single neuron and pupil activity have been published but only steady state data were reported. This communication reports on time varying pupil and single neuron activity in response to repetitive, transient light stimulation. Another objective of this study was to focally stimulate at the site of single neuron recordings to compare light and electrical stimulation and to further investigate a possible causal relationship between the stimulated neurons and pupil area response. In this study measures are made of single neuron activity concurrently recorded with the pupil area light response. The frequency response of the averaged neural and pupil responses are compared. This comparison helps resolve the question of whether the neural control signals for the iris musculature are limited by the pupil muscle itself or if the frequency content of the neural signal does not extend beyond the limits of pupil muscle. A class of neurons near the anterior oculomotor nucleus has been found whose change in firing rate is highly correlated with pupil area response during retinal light stimulation. Focal electrical stimulation of the brain at the site of these single neurons provided comparative information concerning the relation between the anterior oculomotor nucleus and the pupillary musculature. The single unit data reported here confirm earlier suggestions that the pupil musculature limits the frequency response of the system24,27; the neural delay times are shorter than the pupil area response delay times and the time constants of neural responses are smaller than the pupil muscle response time constants. In another p a p e d 2, we will compare the neural and pupil area responses in the frequency domain but this approach is beyond the scope of the present paper. METHODS
Fifteen cats were used for these studies. Each animal was injected with an intraperitoneal dose of sodium pentobarbital (15-30 mg/kg); surgical procedures were accompanied by infiltration of wounds with Xylocaine. A stereotaxic frame was used to position the animal's skull; the atlas of Snider and Niemer 23 was used to aim the recording electrodes. One eye of the animal was maximally dilated with cyclopentolate hydrochloride and the consensual pupil response was driven from this input eye. A glow modulator tube was the light source, as shown in Fig. 1 and a voltage driven pulse-width modulaBrain Research, 24 (1970) 219-234
SINGLE NEURONS IN THE PUPILLARY SYSTEM
221
Fig. 2. Histological verification. This photomicrograph is an example of histological verification performed after neural recording and electrical stimulation experiments. Shown here is a frozen section of midbrain with electrode track and a coagulation point, below recording area, labeled. Figs. 3 and 12 are maps composed from similar sections. tion circuit was used to control the glow modulator tube. This resulted in a constant spectral density of the light stimulus at different intensities. Pupil area response was measured by a reflection technique. The contralateral or 'output' eye was illuminated with infrared light and a photodiode in front of this eye monitored changes in the amount o f infrared light reflected from the iris. The photodiode signal was calibrated during observation of the pupil with a television monitor 32 which magnified the image by a factor of 15. In order to eliminate eye movement, an artifact in this measuring system, the animal was continuously infused with succinylcholine chloride which paralyzed the skeletal musculature and therefore respired artificially with a respirator. The microelectrodes, insulated tungsten wird, were used for extracellular recording, focal electrical stimulation and coagulation of the recordlng and stimulation sites. The waveforms of single neurons recorded in the pretectal area were triphasic and the recordings from the oculomotor dilatation units were biphasic. Recording sites were verified histologically and Fig. 2 is an example of histological control from frozen unstained sections 5 / , m thick; a modified KliiverBarrera stain 10 was also used in preparing sections. Data processing was done with digital computer (IBM 1800). Each stimulus period was divided into 500 sample periods. During each of these sample periods or bins the voltage corresponding to the pupil area was sampled, stored on the computer and averaged. The neural spike trains were also averaged but in a less straightforward manner due to the discontinuous nature of the data. The neural firing rate was computed for each bin. During the experiment a simple analog computer circuit (an RC circuit) was used to low-pass filter a series of standard pulses by the spike train. This signal was proportional to firing rate within limits determined by direct calculation. Brain Research, 24 (1970) 219-234
222
J.D. SMITH et al.
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(1) Dilatation correlated neurons
Dilatation neurons were recorded in and near the anterior o c u l o m o t o r nucleus. A dilatation neuron is defined as an o c u l o m o t o r neuron with a firing rate that increases during pupil dilatation and decreases during constriction. These modulations o f the neuron's steady firing rate lead the pupil area responses to light by 0.2 sec, the neuromuscular delay time reportedl,Zl, 31 for pupil area responses to electrical stimulation of the Edinger-Westphal nucleus and the short ciliary nerves. A map shows the location o f these units in Fig. 3. A total o f 53 neurons were recorded in the o c u l o m o t o r area o f 15 cats. N o n e o f these neurons sustained steady changes in firing rate in response to different levels o f light stimulation. The average firing rate during steady illumination varied f r o m unit to unit f r o m approximately 15 to 50 pitts*. All but one o f the units recorded exhibited its correlation with pupil area during periodic light stimulation. The average firing rate o f a dilatation n e u r o n is shown with concurrently averaged pupil area response to light in Fig. 4. The average firing rate varies f r o m 1 to 55 pitts. The average neural response changes f r o m one rate to another more rapidly than the pupil area changes. * Pitts equals spikes/sec, named after the late Walter Pitts, a colleague of the late Warren McCulloch. Brain Research, 24 (1970) 219-234
223
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T I M E (seconds) Fig. 4. Dilatation correlated neuron (No. 1), averaged spike train and pupil responses. In lower trace is stimulus waveform; light flux at input eye ranged from 0.01 to 10 mlumens. Middle waveform is firing rate of a dilatation unit averaged over 44 periods of light stimulation. At top is averaged pupil area response. Light stimulus was repeated at a frequency of 0.25 c/sec. For all figures; time in seconds and dilatation is upward.
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TIME (seconds) Fig. 5. Dilatation neuron (No. 1) with pupil and model response. Upper trace is same light stimulus as in Fig. 4. Immediately below is extracellular recording from dilatation unit. Third trace is a low-pass filtered version ( a second order, model with a time constant = 300 msec) of spike train above. Fourth trace shows pupil area; constriction is downward.
Brain Research, 24 (1970) 219-234
224
J. D. SMITH et
al.
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Fig. 6. Dilatation neuron (No. 2), instantaneous neural and pupil responses. Pupil and neural response to square wave light stimulation at 0.25 c/sec. Stimulus not shown. Pupil area decreases (downward deflection) in response to light stimulation. Neural spike train has been low-pass filtered by model described in text for Fig. 5. Notice cyclic variations in neural response at frequencies from 0.5 to 3.0 c/sec. Time in sec.
The single neuron responds to a step increase of light excitation with an average delay of 30 msec while the pupil area response follows the light stimulus with an average delay of 250 msec. The neural response to light off is 60 msec; the pupil area response delay is 360 msec. The spike train response of this neuron is shown in Fig. 5. Also shown is the response of a simple neuromuscular model (RC) circuit to this spike train. The use of such lag models of neuromuscular response is supported by frequency analysis studies of skeletal16,17 as well as pupil muscle dynamics1,21, al. This second order model is accurate in predicting pupil area response to light given the dilatation neuron response. The actual pupil area response follows this model response with a delay of 250-300 msec. It is hypothesized that a neural noise generator exists in the pupillary servomechanism z4 and is responsible for the stochastic fluctuations in pupil area shown to be identical in both eyes of cats 12 and humans 27. This relationship between noise in oculomotor firing rates and in pupil area is not understood. Signals from different neurons have different amounts of noise, not necessarily correlated with pupil noise. The model response of another (more noisy) dilatation neuron is shown compared with pupil area response to a square wave of light (not shown) in Fig. 6. Both this filtered response (v ~ 0.05) and the pupil area response show a consistent component of 0.25 c/sec, the frequency of the light stimulation waveform. Note the high frequency noise in the filtered neural response which oscillates from 1.5 to 3.0 c/sec; this neural noise does not appear to be correlated at all with the concurrent pupil area recording. Another dilatation neuron response to light stimulation (neuron 3) is shown in Fig. 7. Ten cycles of the spike train response are averaged and compared with the average in Fig. 4 (neuron 1). It appears that the average neuronal rate of response during constriction is slower in Fig. 7 than in Fig. 4. Some dilatation neurons responded only to light stimulation while others (Fig. 8) exhibited firing rate changes during small fluctuations of pupil area under constant illumination. Periodic light stimulation (0.1-10 mlumens square wave at 0.1 c/sec) Brain Research, 24 (1970) 219-234
225
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TIME 0.0 10.0 20.0 300 40.0 Fig. 8. Dilatation neuron (No. 5), neural and pupil responses. Activity of a dilatation neuron that exhibited no apparent response to light. However, during steady light stimulation variations in pupil area closely followed the activity of this cell. Top trace is pupil area (dilatation upward). Below is a low-pass filtered version of spike train; with time constant of filter sufficiently small so that each separate spike can be seen.
w h i c h c a u s e d a p u p i l response d i d n o t result in a n y a p p a r e n t changes in firing rate. In Fig. 8 bursts o f n e u r a l activity regularly p r o c e e d increases in p u p i l area. This d i l a t a t i o n neuron, u n r e s p o n s i v e to light, m a y be a m e m b e r o f a class o f cells t h a t generate the p u p i l noise signal. P e a k s o f p u p i l d i l a t a t i o n f o l l o w the p e a k s o f neural activity with an average d e l a y o f 1.16 sec for 7 d i l a t a t i o n s in Fig. 8. This delay is m u c h l o n g e r t h a n those o b s e r v e d in p u p i l response to p e r i o d i c light s t i m u l a t i o n (0.2-0.25 sec). P e r i o d i c Brain Research, 24 (1970) 219-234
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Fig. 9. Dilatation neuron (No. 5), auditory response. Record taken immediately after record of Fig. 8. Pupil dilatation was elicited 3 successive times during which the dilatation neuron ceased firing entirely. Approximate times of auditory stimulus onset are marked (± 0.3 sec).
pupil dilatation responses to light and electrical stimulation can take place after section of the sympathetic innervation of iris muscle; however the sympathetic iris innervation of this animal was intact and this neuron may have been connected to the dilator muscles of the sympathetic system. We have observed pupil dilatation delay times of about 1 sec after electrical stimulation of spinal cord and cerebral cortex. These dilatations may have involved neurons similar to the one in Fig. 8. This same unit exhibited an opposite type of coupling with pupil dilatation (Fig. 9) during a psychosensory response. The animal was stimulated with 3 brief and intense auditory stimuli, hissing noises. The pupil responded with a dilatation each time. The neuron abruptly ceased firing during most of the dilatation response and for as long as 2 sec after the psychosensory evoked dilatation was completed. The delay time of the auditory response was not precisely measured but was about 0.5 sec.
(2) Other types of units Several other types of units were observed in addition to the dilatation correlated units. A majority of units showed no correlation with the pupil area response; some units showed positive correlation (dilatation) with the pupil area in an intermittent fashion; these units would track the pupil area response and then would randomly switch to some spontaneous firing rate ostensibly unrelated to the pupil activity. In one case a noisy constriction unit response was recorded, the neuron was lost before averaging techniques could be used to process the signal. The fact that no confirmed constriction-correlated units were found in the region of the anterior oculomotor nucleus is difficult to explain. Sillito z0 has reported on the activity of oculomotor neurons with higher average firing rates during periods of smaller pupil diameter in cats.
Brain Research, 24 (1970) 219-234
SINGLE NEURONS IN THE PUPILLARY SYSTEM
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(3) Electrical stimulation." deterministic and stochastic responses Electrical stimulation in the region of the anterior oculomotor nucleus (Fig. 10) produced both dilatation and constriction responses in pupil a r e a l Stimulus pulses were f r o m 5-10 FA (5-10 • 10 -6 A), lasted for 1 msec and were given at the rate of 50 pulses/sec. The stimulus currents were twice the threshold stimulus necessary to produce a pupil response. Pupil dilatation responses were always observed in response to electrical stimulation at the site of dilatation correlated neurons (5 animals). In all experiments constriction responses resulted only from stimulation of a restricted area. The recording site of the constriction unit shown in Fig. 3 was in this area. There is good neuroanatomical evidence 15 to suggest that the preganglionic cells of the pupil constriction pathway are in the anterior part of the third cranial nucleus and the synapse in the ciliary ganglion. The neurons at this locus are the F C P (final c o m m o n pathway) cells of the Edinger-Westphal nucleus which send preganglionic fibers from the anterior portion of the third nucleus to the ciliary ganglion and possibly the episcleral ganglia. Electrical stimulation of the F C P cells results in pupil constriction (Fig. 11). The delay time for constriction is from 300 to 350 msec and the redilatation delay time is the same. N o asymmetry in delay time occurs as was observed during pupil response to light stimulation. Pupil dilatation responses (Figs. 12 and 13) to electrical stimulation were obtained above the F C P cells. In some cases moving the
Brain Research, 24 (1970) 219-234
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Fig. 13. Pupil dilatation response,electrical stimulation. Ten consecutive responses of pupil area shown driven by square waves of electrical pulse train excitation. Pulse rate is 50 pulses/sec, pulse heights are 10 pA. Note that secondary fluctuations of pupil area appear to be unaffected by stimulus. This record is from the same electrode penetration as in Fig. 12 at a site 0.25 mm ventrolateral. electrode only 0.5 mm reversed the pupil response from dilatation to constriction.When electrically stimulating at the site of a dilatation unit (Results, part 1) the pupil never responded with constriction although in several cases the electrode was within 0.5 mm of the FCP cells. The stimulating current was thus highly localized in these cases and presumably only depolarized neurons effecting pupil area increase. The pupil can be actively dilated by stimulation of the sympathetic fibers that innervate the dilator muscle of the iris 31. In a control experiment it was found that the dilatation responses to focal midbrain stimulation were of equal amplitude before and after surgical section o f the sympathetic fibers. Therefore, this pupil dilatation response to focal electrical stimulation is believed to be due to inhibition of firing of the FCP cells mediated by the stimulated dilatation neurons. Often during light and electrical stimulation, variations in pupil area occurred that were not correlated with the stimulus, i.e., a non-deterministic response. Fig. 11 shows an example of several superimposed constriction responses to an electrical pulse train. During the influence of the stimulus, the pupil area shows a constriction response of similar shape each period. The remainders of each period are less predictable. This randomness can be represented by using the idea o f a neural noise generator 24 which
Brain Research, 24 (1970) 219-234
J.D. SMITHet al.
230
sends identical stochastic signals to each pupil. These random variations appear to be attenuated during the constriction response. We propose that the FCP cells undergo a temporal and spatial synchronization such that the neural noise signal is suppressed during the influence of the stimulus. Electrical pulse train stimulation 1.0 mm above the FCP cells produces a repeatable pupil dilatation (Fig. 12). The noise in the pupil area record appears to be attenuated during the influence of the stimulus. Random fluctuations in pupil area resume after the stimulus stops. These stimulated cells may normally generate a signal that inhibits the FCP cells randomly and thus produces pupil noise. The noise signal of these neurons would be suppressed during this stimulation which synchronizes their firing times. In Fig. 13 several periods of pupil area response are superimposed during stimulation in the same experimental animal 0.25 mm below the dilatation point in Fig. 12. At this site electrical stimulation results in repeatable pupil dilatation. There is no suppression of pupil noise during stimulation of this (different) class of cells. The stimulation of cells firing at this site results in inhibition of the FCP cells and in pupil dilatation. The cycle to cycle variation in pupil area remained constant throughout the response. Presumably the midbrain cells (candidate noise generator cells) which had been stimulated before (Fig. 12) were not synchronized by stimulation during Fig. 13. Therefore their asynchronous activity during the recording in Fig. 13 may be contributing to the pupil noise throughout the cycle duration. It must be emphasized also that the noise generator evidence above rests on visual observations of a qualitative nature (Figs. 12 and 13).
(4) Delay times and frequency response of neural and pupil response Average delay times of the neuron response to onset and termination of pulses of light have been measured. The neural 'on' delay time was 30 msec and the 'off' delay time was 60 msec; the pupil delay times were 250 and 360 msec. The neural and pupil area delay times to 'off steps' of light are longer (150 ~ ) than the delay time to 'on steps' (0.01-10 mlumens) as shown in Fig. 4. A straight line approximation to the change in average rate of firing to an on-step of light (during the first 200 msec) has a negative slope of 420 pitts/sec; the rate of change developed in response to the off-step of light is 140 pitts/sec. The average firing rate during constriction and during dilatation can be considered separately. A first order approximation of neural activity during constriction indicates that the average firing rate is frequency limited at about 6 c/sec. Tc~ = 0.025 sec (first order time constant, constriction) We1 = 1/Tc = 1/0.025 = 40 radians/sec BF¢I ---- Wc/2z~ = 40/6.28 = 6 c/sec (break frequency). The break frequency approximation to firing rate changes during dilatation can be similarly derived. TD1 = 0.1 sec (first order time constant, dilatation) WD1 1/TD : 1/0.1 : 10 radians/sec BFD1 : WD/2:r : 10/6.38 : 1.5 c/sec (break frequency). :
Brain Research, 24 (1970) 219-234
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TABLE I Time constants (sec)
Neural
Pupil area
Light-on Light-off
Neuron 1 Fig. 4 T n - o n = 0.025 T n - o t r = 0.1
3 7 2.0 0.3
Neuron Fig. Tc = TD =
1 4 0.3 0.9
3 7 1.5 2.5
Dn-on = 0.03 Dn-oft = 0.06
0.05 0.02
DC = DD =
0.25 0.36
0.35 0.55
Time delays (sec)
Light-on Light-off
The table above summarizes the time delays and time constants of the neural and pupil response to light in Figs. 4 and 7. I f the average neural response of neuron number 1 (Fig. 4) is a fair approximation of the total deterministic neural signal transmitted to the iris, the pupil musculature would low-pass filter (frequency limit) this signal as the pupil model predicts in Fig. 5. All delay times and time constants for the pupil response are from 6-10 times larger than the neural response except for the time constant of neuron 3 during pupil constriction. DISCUSSION The pupil dilatation neurons described in this paper correlate with the contralateral pupil response to light stimulation and lead it by 250 msec; a dilatation response was always produced (5 different animals) when the recording site of these correlating neurons was electrically stimulated. These neurons were found 0.5-2.0 m m above and below the Edinger-Westphal preganglionic cells. This distance was confirmed by advance of the microelectrode to a constriction producing locus operationally defined as the Edinger-Westphal nucleus. Histological preparations show that the anterior oculomotor nucleus coincides with this operationally defined neural station (see the stereotaxic maps of Figs. 3 and 6). Both the delay times and time constants cited in Table I for 2 different dilatation neurons reflect that the neural response to light is 6-10 times faster than the pupil response. The dilatation neuron in Fig. 7 has a time constant during constriction (light on) that is larger than the time constant of the pupil response. Although this time constant is too large to be considered causal with respect to the pupil constriction response, the dilatation time constant of the cell (0.3 sec) is about 8 times faster than the pupil dilatation time constant (2.5 sec). This apparent lack of correlation with pupil dynamics may be attributed to a distribution o f dynamics among these dilatation neurons that we postulate relay light information from the pretectal area to the constrictor cells (final c o m m o n pathways) of the EdingerWestphal nucleus. We propose that a time averaged version of the total dilatation neuron signal (all dilatation neurons in the loop) would have a time constant 6-10 times smaller than the concurrent pupil response time constant. Brain Research, 24 (1970) 219-234
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The dilatation neurons reported herein are considered causal with respect to pupil area due to their prediction of pupil area response (Figs. 5 and 9), the pupil dilatation response to focal electrical stimulation (small pulses, 10 -~ A) and because of the neural time constants and delays that are appropriately smaller but match the asymmetry of the pupil response characteristics (Table I). The pupil responses to focal electrical stimulation in the midbrain also suggest that there are two classes of cells whose stimulation results in dilatation. One class of these cells is implicated in generation of pupil noise. During synchronous stimulation of these ceils (noise cells) the cycle to cycle variation of pupil response (pupil noise) is attenuated. During stimulation of the other class of cells (deterministic cells), as a different locus, the pupil noise is the same during the pupil dilatation and afterwards. We propose that the 'noise cell' signal is undisturbed during stimulation of the 'deterministic cells' and contribute a constant 'noise' signal to the pupil muscles via the constriction cells of the Edinger-Westphal nucleus. Because the dilatation responses to midbrain stimulation also occur after section of the sympathetic nerve innervating the pupil (the preganglionic fibers adjacent to the vagus nerve) we believe these responses were caused by inhibition of the constriction producing FCP motoneurons of the Edinger-Westphal nucleus. Dilatation neurons as well as dilatation stimulation sites were recorded dorsal to the Edinger-Westphal nucleus. An oculomotor accessory nucleus 6 dorsal to the Edinger-Westphal nucleus and inhibitory to somatic as well as visceral third nucleus cells 3° has been described. This group of cells may be the locus of the dilatation neuron cell bodies. One of the dilatation neurons recorded showed correlation with pupil area, did not respond to light stimulation but did respond to auditory stimulation (Fig. 8). More important is the finding that there are two classes of recorded dilatation cells. One group responds to light with delay times longer than those of pretectal neurons and leads the pupil response to light by about 0.25 sec, the delay time recorded for pupil response to stimulation of the midbrain. These cells display the same asymmetry in delay times and time constants (both smaller for light-on responses) that the pupil muscle response displays; the pupil response delay times and time constants are, however, 6-8 times larger in magnitude. A second group ofceUs may be represented by the recordings in Figs. 8 and 9. This neuron does not respond to light but leads pupil dilatation by about 1.0 sec; and, its low-pass filtered response (stimulation of pupil muscle dynamics) predicts the noise in pupil area with a fair degree of accuracy. Stimulation of the sites of both classes of dilatation cells always resulted in pupil dilatation, even in cases where later stimulation 0.5 mm away resulted in pupil constriction (the constriction cells of the Edinger-Westphal nucleus). A model of the human pupil system has been proposed 24 which suggests that a neural process common to both pupils provides a stochastic signal that results in identical pupil area variations for both pupils during steady illumination of the retinae. Pupil noise has been shown to be identical in both pupils of the cat lz as it was found to be in humans 27. Clearly the neuromuscular process is eliminated as a source of the noise; and, anatomical evidence29,~3 suggests that separate bilateral groups of FCP cells activity drive the constriction of each pupil. These data lead to the proposal that a common neural Brain Research, 24 (1970) 219-234
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process provides the noise signal before the division of the pupil pathway to the right and left eyes. The results of both single neuron recording and focal electrical stimulation experiments support the idea that two classes of dilatation neurons cause pupil dilatation. We postulate that both classes of cells, 'deterministic' and 'noise' cells, inhibit the final c o m m o n pathway neurons, constriction cells (as defined by electrical stimulation) of the Edinger-Westphal nucleus. Furthermore the evidence suggests that the 'noise' cells do not respond to light stimulation. SUMMARY (1) In cats with a light-driven pupillary response, dilatation-correlated single neurons have been recorded near, but not in, the Edinger-Westphal nucleus. The firing rates of these units range from 1 to 80 pitts (spikes/sec). (2) Focal electrical stimulation was performed confirming the dilatation action of these cells as well as the constriction activity of the Edinger-Westphal region itself. (3) During light stimulation the rate of change of the average neural response is faster than the concurrently averaged pupil area response. ACKNOWLEDGEMENTS The authors are grateful for the assistance of Dr. George Moore in reading the manuscript and making a number o f helpful suggestions. This work was partially supported by the W. Clement and Jesse V. Stone Foundation, N I H Grants NB-07803, NB-06197, NB-06487; and GM-01436, PHS MH-8396 and O N R N-0014.
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