ORIGIN OF THE OCULAR LIGHT-MODULATED
STANDING POTENTIAL IN CAT

By
HARRIS R. LIEBERMAN

A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF

THE UNIVERSITY OF FLORIDA

IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE

DEGREE OF DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA
1977

ACKNOWLEDGEMENTS

I would like to thank the Chairman of ray committee,
Dr. William W. Dawson, for his guidance. He generously
shared his time providing intellectual stimulation and
advice and assistance that were invaluable to me. I would
also like to thank the other members of my committee,
Dr. Calvin K. Adams, Dr. Jay M. Enoch, Dr. Merle E. Meyer,
and Dr. Horst 0. Schwassmann, for their thoughtful criticism
and constructive advice. I am also very grateful to
Dr. Constance Fitzgerald who photocoagulated the discs of
the subjects used in the vascular lesion experiments. I
would also like to thank Minnie Hawthorne who did the
histology and Carl Litzkow who constructed the high
impedence DC amplifier. I wish to thank the Center for
Neurobiological Sciences for financial support.

Finally, my Ellice provided invaluable help actively
assisting throughout.

TABLE OF CONTENTS

Page

ACKNOWLEDGEMENTS ii

ABSTRACT v

CHAPTER 1 : INTRODUCTION 1

Significance 8

CHAPTER 2 : LITERATURE REVIEW 10

CHAPTER 3 : GENERAL METHODOLOGY 18

Subjects 18

Electrode Fabrication 18

Generation and Presentation of Stimuli 20

Signal Amplification and Processing 22

Preparation and Maintenance of Animal 22

CHAPTER 4: EXPERIMENT 1: CHARACTERISTICS OF

THE LMSP OF THE CAT 2 6

Rationale and Methodology 2 6

Results 27

Discussion 38

CHAPTER 5 : EXPERIMENT 2 : STEADY VERSUS

FLICKERING STIMULI 41

Rationale 41

Methodology 43

Results 45

Discussion 55

iii

CHAPTER 6 : EXPERIMENT 3 : ASPARTATE 57

Rationale 57

Methodology 58

Results _ 59

Discussion _ 59

CHAPTER 7: EXPERIMENT 4: VASCULAR LESION 72

Rationale 72

Methodology _ 73

Results _ 75

Discussion _ 9]_

CHAPTER 8 : ANALYSIS AND CONCLUSIONS 94

Conclusions _ 104

APPENDIX A - Subjects Used in Each Experiment 105

APPENDIX B - R1131C Glow Tube Spectral Emission 106

APPENDIX C - Formula for Paralytic Agent 107

REFERENCES 108

BIOGRAPHICAL SKETCH 115

IV

Abstract of Dissertation Presented to the Graduate Council
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

ORIGIN OF THE OCULAR LIGHT-MODULATED
STANDING POTENTIAL IN CAT

By

Harris R. Lieberman

August, 1977

Chairman: William W. Dawson
Major Department: Psychology

In many vertebrates a direct current (DC) potential or
standing potential (SP) exists across the eye with the
cornea being positive relative to the posterior pole. After
the onset of a light preceded by a period of dark adaptation
a slow damped oscillation of the potential is observed.
The initial peak of this oscillation occurs at about 6
minutes after light onset. At light cessation a similar
damped oscillation of opposite polarity and lower amplitude
results. These modulations in the standing potential in-
duced by light onset and cessation are termed the light
modulated standing potential (LMSP) . To directly record
the LMSP in animals a highly DC stable recording system was
developed. It was employed to localize the origin of the
ocular LMSP in the cat. The precise origin of the potential
is controversial although the PE and receptors are generally

considered the source. Three experiments were performed to
determine if other retinal structures participate. In one
experiment the LMSPs elicited by the onset or cessation of
a steady or flickering light of equal time averaged irradiance
were compared. The neural elements in the retina are known
to be responsive to transient changes in irradiance. There-
fore large differences in the LMSPs should be elicited by
the paired flickering and steady stimuli if these neural
elements or an associated process participate in generation
of the LMSP . No differences were found. In two other ex-
periments the inner retina (all retinal elements except the
receptors) was lesioned either chemically with sodium aspar-
tate or by occlusion of the retinal vasculature at the optic
disc. After either type of lesion the LMSP could no longer
be elicited or was reduced significantly. The b-wave of the
electroretinogram (ERG) which originates in the inner retina
was also abolished or similarly reduced while the a-wave
arising mainly in the receptors was preserved. It was con-
cluded an inner retinal component is essential for gener-
ation of the LMSP. Since the LMSP did not respond dif-
ferentially to flickering vs. steady illumination, as
expected if it was of neural origin, it was hypothesized
that the Muller cells (retinal glia) are responsible for its
generation .

CHAPTER 1
INTRODUCTION

Since the mid-nineteenth century it has been known that
there is a potential difference between the anterior and
posterior pole of the eye (Skoog, 1975). In the vertebrate
eye the cornea is positive with respect to the fundus (Skoog,
1975) . This standing potential (SP) across the eye varies
with changes in retinal illumination. Specifically, the SP
voltage increases when illuminance is increased (Fig. la)
and decreases with dark adaptation (Fig. lb). In the human
these changes resemble damped oscillations and have a period
of approximately 25 minutes (Arden and Kelsey, 1962a,b;
Skoog, 1975; Taumer, et al., 1975).

Until recently, the human SP as influenced by changes
in retinal illumination had been measured only by the in-
direct electrooculogram technique because of the technical
difficulties associated with direct current (DC) recording.
For this indirect method electrodes are placed at the inner
and outer canthus of each eye and, because the eye is a
dipole, horizontal eye movements of a standard amplitude
indirectly monitor standing potential. As the eye rhythm-
ically moves left and right a potential resembling a tri-
angular waveform is generated. The peak to trough amplitude

10 Ml N

Figure 1. The light-on (A) and light-off (B)
light modulated standing potentials (LMSPs)
elicited by the onset or cessation of the
steady unattenuated light source. Light-on
and off are indicated by arrows. Note the long
time course of the changes. In this and all
other illustrations of electrophysiological
signals an upward deflection is a cornea-positive
response.

3
of this potential is an indirect measure of the SP across
the eye.

A number of problems, however, are associated with this
technique. The subject must be cooperative and able to make
the necessary eye movements. Therefore it is not readily
applicable to children and animals. Even fully cooperative
subjects fatigue rapidly and any variance in the extent of
eye movements may be an artifact.

To avoid confusion the following terminology will be
adapted:

SP - the D.C. voltage across the eye

LMSP - the change in SP induced by an increment

or decrement in retinal illumination
EOG - indirect measure of the light modulated SP
as described above
Recently, however, it has been demonstrated that high
quality direct recordings of the light modulated SP in the
human are possible (Nilsson and Skoog, 1975; Skoog, 1975) .
Directly recording the SP is difficult because of its DC
nature. Direct current amplifiers must be substituted for
conventional capacitively coupled amplifiers and specially
constructed non-polarizing and highly stable electrodes must
be employed.

Both direct and indirect methods of recording SP have
been attempted in non-human subjects by a few investigators.
For indirect recordings mechanical devices have been em-

4
ployed to physically move the eyes of animals (Foulcls and
Ikeda, 1966; Heck and Papst, 1957).

Both direct and indirect recordings have shown that
like the human SP, the SPs of many vertebrates respond to
changes in retinal illumination and these changes resemble
damped oscillations (Kikwada, 1968).

Due perhaps to the severe technical problems there has
been little basic research on the LMSP and SP in spite of
the wide acceptance of the EOG as a clinically useful diag-
nostic test. We have been successful, however, in develop-
ing a satisfactory system for directly recording the stand-
ing potential. It has been employed here to localize the
LMSP in the cat.

The precise origins of the SP and the LMSP are contro-
versial. Generally it is believed they originate from a
pigment epithelium (PE) and receptor interaction (Arden and
Kelsey, 1962b; Arden, et al., 1962; Noell, 1954; and Taumer,
et al., 1974). It was Noell (1954) using rabbits, who
documented the importance of the PE in generating the SP
(but not the LMSP). His research was largely based on his
accidental discovery of SP responsiveness to a chemical,
sodium azide. When small amounts of this drug are intro-
duced into the circulation, a transient increase in SP
voltage results. Noell found that when the retina was
severely lesioned with iodoacetic acid the azide response of
the SP was preserved although somewhat reduced in amplitude.
However in animals with an intact retina but a PE lesioned

5
by another chemical (iodate) , the SP responsiveness to azide
was not present. Noell concluded that the PE was an es-
sential element in the generation of the SP across the eye.

The importance of the retinal receptors in the gener-
ation of the SP is evident from the human action spectrum of
the light modulated EOG. It closely resembles the CLE.
scotopic sensitivity curve (Arden and Kelsey, 1962b) . Also
in the rabbit the LMSP is totally abolished after retinal
detachment (Arden and Kelsey, 1962b; Foulds and Ikeda,
1966) .

Gouras and Carr (1965) and Gouras (1969) while con-
ceding the importance of the PE and photoreceptor in the
generation of the SP have stated that the LMSP is influenced
by retinal structures other than the receptors. This con-
clusion was based on studies of monkeys with occluded
central retinal arteries and humans with similar lesions.
Succeeding this occlusion the inner retina degenerates but
the photoreceptors and PE remain intact (Brown, 1968; Gouras
and Carr, 1965) . In monkeys Gouras and Carr (1965)
report that one to five weeks following surgery the LMSP
response to "light on" was absent or severely attenuated.
In humans with similar lesions it has been reported that the
light onset rise of the EOG is abolished (Nagaya, 1964) but
the light-off response initially disappears but eventually
returns. Gouras (1969) concluded that the light rise and
dark trough of the SP arise at different loci and that the
light rise requires the presence of a functioning inner

6
retina. Arden and Kelsey (1962a, b) also suggest that the
light peak and dark trough of the EOG have fundamentally
different properties. They based this conclusion on ap-
parent differences between the two EOG components. Other
investigators, notably Taumer et al. (1974) believe that the
light rise and dark trough of the LMSP represent the same
process.

Since we have developed a method of recording LMSP in
animals, it was employed to begin to localize the LMSP. The
overall approach taken was to determine if certain retinal
layers or processes were necessary for the generation of the
LMSP. Since the retina transmits information serially for
the most part, it seemed advisable to begin at the earliest
stages of this process and move inward.

Four experiments make up the body of this dissertation.
The first characterizes the LMSP of the cat. Specifically a
quantitative description of the time course of the light-on
and light-off LMSP is presented. In addition amplitude by
intensity functions were generated for both the light-on and
off LMSP. This function could then be compared with ampli-
tude by intensity functions at certain stages in the retinal
visual process. A second experiment compared LMSPs elicited
by steady and flickering lights of equal time averaged ir-
radiance. This experiment was designed to determine the
contribution of retinal neural activity to the LMSP as com-
pared to photopigment related processes. The neural retina
is known to be highly sensitive to time varying stimuli

7
(as reviewed by Brown, 1965) while the photopigment in the
receptors and related metabolic processes are not sensitive
to such stimuli (Campbell and Rushton, 1955).

The third and fourth experiments determined if the
LMSP is generated in the receptors and PE alone or if neural
and/or glial inner retinal components are involved. To
functionally separate the inner and outer retina two tech-
niques, one chemical and one surgical, were applied. The
chemical approach used sodium aspartate which by activity on
the horizontal and bipolar cells prevents conduction of
impulses beyond the receptors (Cervetto and MacNichol, 1972;
Murakami et al., 1972; Murakami et al., 1975). The other
technique consists of surgically occluding the blood supply
to the inner retina by lesioning the retinal vessels as they
enter the eye at the optic disc. The receptors and PE are
supported by the choroidal circulation so they are not af-
fected. This is a standard technique (Brown, 1968; Maffei
and Poppele, 1968). The effectiveness of both types of
inner retinal lesions were confirmed by recording the
electroretinogram (ERG) . The a-wave is a measure of outer
retina viability while the b-wave has an inner retinal site
of origin (Brown, 1968).

In all experiments the primary dependent variable was
the LMSP. It was examined as a function of the various
experimental conditions described individually below. In
some experiments ERGs were also recorded.

Significance

Research on various field potentials in the eye has a
long history. The major components of the electroretinogram
have been localized and this has led to a number of basic
and applied contributions. For example, because the a-wave
has been shown to reflect the activity of the rods and cones
various researchers and clinicians have used it to study the
receptors. For example, Boynton and his associates (Baron
and Boynton, 1974; Boynton and Whitten, 1970, 1972) have made
important basic contributions by using the a-wave to study
receptor information coding in primates. Their findings
concur with intracellular recordings of Normann and Werblin
(1974) in the mudpuppy.

It is not currently feasible to record from mammalian
receptors and even in other animals it is extremely dif-
ficult. Field potentials are presently the only available
technique for directly studying the electrophysiology of
receptors .

It is generally believed that the LMSP and SP are a
measure of receptor and PE metabolic activity (Arden and
Kelsey, 1962b; Arden, et al., 1962; Noell, 1954; Taumer, et
al., 1974). This viewpoint has therefore channeled basic re-
search and clinical application regarding the SP and LMSP
in a particular direction. As discussed above this may not
be a correct formulation since Gouras and Carr (1965) have
apparently demonstrated an inner retinal contribution to the
light modulated SP . Recent basic research of Skoog (1975),

9
Nilsson and Skoog (1975) and Taumer , et al., (1974), for
example, are based on the assumption the LMSP is a measure
of PE and receptor activity. Similarly the clinical com-
munity has considered the EOG as useful only for diagnosing
PE related lesions (Arden et al., 1962).

A more definite localization of the origin of the SP
should be an important step in successfully utilizing it for
basic and applied purposes. In the LMSP is a measure of
inner retinal metabolic activity a powerful new tool may be
available for studying the metabolic concomitants of retinal
activity in normal and pathological states. The potential
importance of such readily recordable, non-invasive measures
of retinal metabolic activity should not be underestimated.
Very little is known about how retinal neural activity is
related to retina and PE metabolism. It is hoped that
localization of the LMSP will provide a new advance in
studying such relationships.

CHAPTER 2
LITERATURE REVIEW

In this section a thorough review of the classical and
modern literature on the SP and LMSP is presented.

The existence of a standing potential across the eye
was first documented by Du Bois Reymond in 1849. Using a
freshwater fish (the tench), he found that a potential dif-
ference existed between the front and the back of the eye.
During the late 19th and early 20th century other investi-
gators (Dewar and M'Kendrick, 1876; Kuhne and Steiner, 1881)
extended this observation to a variety of species and it
became apparent that this potential was cornea positive for
vertebrates and cornea negative for invertebrates (Arden and
Kelsey, 1962a; Marg , 1951; cited in Miles, 1940). This
polarity difference has been attributed to the reversed
position of the receptor cell layers in the vertebrates and
invertebrates (Arden and Kelsey, 1962a). It was also
demonstrated that this SP was modulated by light (cited in
Arden and Kelsey, 1962a; Himstedt and Nagel, 1902; Kuhne and
Steiner, 1881) . The early investigators also generated data
which suggested that the SP originated in the retina and/or
PE. In 1903 DeHaas found that the potential changed dra-
matically at the ora serrata. Additionally it was demon-

10

11

strated that large potential differences exist across the
isolated retina (cited in Arden and Kelsey, 1962a; DeHaas,
1903; Dewar and M'Kendrick, 1876; Kuhne and Steiner,
1881). Dewar (1877) was the first to record the human SP
(Miles, 1939) . All of these early investigators were re-
cording the SP directly and because of the difficulties
associated with such DC recordings and discovery of the more
easily recorded ERG, a decline in interest in the SP occur-
red (Ten Doesschate and Ten Doesschate, 1956).

Interest in the SP was reawakened in the 1930' s in con-
nection with attempts to monitor human eye movements (Jacob-
sen, 1930; Meyers, 1929). Electrodes were placed at the
inner and outer canthus of each eye and when horizontal eye
movements were made voltage changes resulted. These inves-
tigators believed that these electrical changes were due to
activity of the extraocular muscles. However, Mowrer, et
al. (1936) disproved this hypothesis. They demonstrated
that passive movements of the eyes of anesthetized cats led
to similar potentials and injection of chromic acid into the
eye abolished these potentials. After Mowrer et al. (1936)
established the intraocular origin of the SP, interest in it
increased.

Miles (1939) was one of the first to apply this new
technique for other than eye movement research. He demon-
strated that the EOG in humans is modulated by changes in
retinal illuminance confirming the classical work on the SP.
Additionally he determined that a variety of other parame-

12
ters influenced the EOG, including pressure on the eye and
changes in blood pressure.

In the middle and late fifties a number of papers
appeared which investigated the effects of various visual
parameters on the EOG and attempted to develop it as a
clinical test of visual function (Aserinsky, 1955; Francois,
et al., 1955, 1956; Kris, 1958; Ten Doesschate and Ten
Doesschate, 1956). This activity culminated with important
papers by Arden and his associates which established the EOG
as a useful clinical test (Arden and Kelsey, 1962a, b; Arden,
et al. , 1962) .

Aserinsky (1955) evaluated the effect of illumination
and sleep on the amplitude of the EOG. He found that it was
significantly elevated by light and depressed by dark.
Additionally he found that a long period of dark adaptation
and sleep did not further reduce the amplitude of the SP
beyond the trough reached during a period of dark adapt-
ation. Instead the EOG on awakening was considerably
greater in amplitude than the pre-sleep dark adapted vol-
tage. This may reflect diurnal variations in EOG which have
been reported but not quantified by Kris (1958).

Ten Doesschate and Ten Doesschate (1956) , confirmed the
findings of Aserinsky (1955) with respect to the light peak
and dark trough of the human EOG. Additionally they stated
that the dark trough was followed by a rise in EOG poten-
tial. Kris (1958) and Kolder (1959) confirmed these obser-
vations and concluded that both light on and off responses

13
resembled damped oscillations. Kris noted that the first
peak of the light adapted EOG occurred at the 10th minute
plus or minus 2 minutes after' the change in illumination,
the first trough at 25th minute plus or minus 4 minutes and
the second peak at the 36th minute plus or minus 4 minutes.
She also found that the dark response reached an initial
trough at 8-10 minutes after light off and, in agreement
with Ten Doesschate and Ten Doesschate (1956), observed that
it returned to a steady state level below the preceding
light adapted baseline value.

The first studies on clinical application of the EOG
appeared in 1955 and 1956. Francois et al. (1955, 1956a, b)
studied the effect of light on the EOG. In general their
findings are in agreement with the other studies mentioned
above. They also found abnormal EOG Ss with various retinal
ocular pathologies. The EOG sensitivity to light onset or
cessation was abnormal in cases of retinal detachment,
central retinal artery occlusion and glaucoma but normal in
cases of papillitis, retrobulbar neuritis, and lesions of
the visual cortex. Additionally they noted a dissociation
of the SP and the light modulated SP . For example, in cen-
tral retinal artery occlusion the SP was intact but could
not be modulated by light.

These papers, published in the mid and late 1950 's laid
the groundwork for the pivotal works of Arden and associates
on the EOG and its clinical application (Arden and Barrada,
1962; Arden and Kelsey, 1962a, b; Arden, et al., 1962).

14
Arden's group published the first detailed parameteric study
of the EOG. The parameters explored included light inten-
sity, length of period of illumination, the relationship
between the light peak and dark trough and the wave length
of the light stimulus employed. In addition, they proposed
a standardized clinical EOG testing procedure which has been
widely accepted.

Arden and Kelsey (1962a), based on a variety of experi-
mental results, suggest that both the light peak and dark
trough components of the EOG are generated in one structure.
However, they also conclude the dark trough and light rise
differ fundamentally in their properties.

Support of a common structural site for the origin of
the SP comes from their discovery that the two opposite
waves interact rather than sum algebraically in the manner
that the a- and b-waves of the ERG, generated at different
loci, summate. They also found what they considered to be
two fundamental differences between the light peak and dark
trough. The dark trough had an all-or-nothing nature with
respect to the brightness of the preceding light period.
Its amplitude was independent of the duration to the pre-
ceding illumination. The light rise, however, was graded in
amplitude depending on the brightness and duration of the
evoking stimulus. They concluded that light peak and dark
trough are the result of processes acting upon a single
generator of the LMSP .

In a second paper, Arden and Kelsey (1962b) concen-
trated on exploring various aspects of the light peak.

15
Preliminary to determining the action spectrum of the light

rise they did an amplitude by intensity function over a 7

log unit range on the light peak. They found the amplitude

of the light rise to vary linearly with the logarithm of the

light intensity from 20 to 10,000 trolands (td) . Above that

range the light rise was saturated. They then went on to

measure the EOG action spectrum and found it was nearly

identical to the CLE. scotopic visibility curve (the

average dark adapted spectral sensitivity of the human

visual system) . They concluded that the first step in the

generation of the SP light rise was the rhodopsin in the

rods of the retina.

Arden and Kelsey (1962b) also investigated in some
detail the influence of the length of the dark period on the
succeeding light rise. With increasingly long periods of
dark adaptation (up to 20 minutes) the light rise increased
in amplitude. When these data were compared to estimates of
the percent of rhodopsin regenerated in the human following
a total bleach (Rushton, 1961) the correspondence was fairly
good.

Because Arden and Kelsey (1962b) hypothesized that the
light rise was, in a very general sense, due to an increase
in metabolic activity, they inferred that it would be very
sensitive to changes in blood supply to the eye. Therefore,
another portion of the Arden and Kelsey (1962b) paper is
devoted to measuring the effects of decreased blood supply
to the eye created by pressure exerted on the globe. The

16
resulting ischemia caused a fall in SP . In addition, when
pressure was applied at various times during the light-dark
cycle, the magnitude of the fall but not the final resting
value varied. Regardless of whether pressure was applied to
the eye during a light peak or a dark trough the SP fell to
the same level which was about equal to the dark trough
value. They concluded that the light sensitive and pressure
sensitive portions of the SP were similar.

Arden and Kelsey (1962b) and Arden et al. (1962) also
presented data from various patients with ocular lesions to
further examine the effect of metabolic changes on the SP
and to localize the site of its generation. The light rise
was severely reduced in cases of central retinal vein
occlusion which they thought indicated the importance of
vascular factors. It was also observed, in early cases of
trauma induced retinal detachment where light perception and
small ERGs were still present, that the EOG was absent. In
cases of choroiditis where the ERG remained normal the light
rise of the EOG was abnormally small. It was concluded that
the light rise of the SP requires a functional PE and PE-
retina interface and the primary event eliciting the light
rise occurred in the pigment epithelium.

Since the appearance of the papers by Arden and his co-
workers in 1962, there have only been a few non-clinical
studies on the light modulated SP in spite of the wide
acceptance of the EOG as a clinically useful procedure.
Some of these papers have contradicted data and interpre-

17
tation of the studies by Arden and associates in 1962. For

example, in 1965, Gouras and Carr demonstrated the light

rise was abolished by CRA occlusion in the rhesus monkey.

In a later paper Gouras (1969) suggested, based mainly on

clinical data, that the light rise and dark trough arise in

different structures. Taumer et al. (1974) argue against

Arden and Kelsey's (1962b) conclusion that the light rise

and dark trough differ fundamentally. They found that the

two light modulated components of the human EOG are similar,

both being damped oscillations with a common cycle time of

about 25 minutes.

Foulds and Ikeda (1966) examined the EOG of the rabbit
following experimentally induced retinal detachment. They
noted a decrease in SP amplitude and the complete absence of
the light peak if the detachment was complete. This is in
agreement with what Arden and Kelsey (1962b) found on humans
with retinal detachments.

Some of the most recent basic research on the SP demon-
strated that it could be recorded directly from the human by
using a highly stable calomel half-cell electrode system
(Skoog, 1975). In agreement with other investigators,
notably Taumer et al. (1974), he found the light on and off
components of the SP to be similar in time course, to be
damped oscillations with a period of about 30 minutes, and
to be of opposite polarity.

CHAPTER 3
GENERAL METHODOLOGY

Subjects
The thirteen subjects in all experiments on the LMSP
were adult cats of either sex weighing 2.5-4.0 kilograms.
The cats were housed in individual cages and were maintained
on a cycle consisting of 12 hours of light and 12 hours of
dark. Appendix A lists the various experiments for which
each cat was used.

Electrode Fabrication
The single greatest obstacle to high quality DC record-
ing was construction of high stability electrodes with no DC
drift. DC drift is a slow constant change in potential seen
across electrodes when their stability is measured in physio-
logical saline. This type of drift is not normally a
problem for electrophysiological recordings because the
potentials usually measured last no more than a few seconds.
However for the measurement of the LMSP, which has a time
course many minutes long, such instability is unacceptable.
Therefore high DC stability Ag-AgCl electrodes were suc-
cessfully fabricated. The method used in their construct-
ion, as described below, was derived in part from Geddes

18

19
(1972) . Ten mil platinum wires about 4 cm long with a small

loop at one end were first cleaned by sonication in Freon

12. In order to avoid the deposition of organic materials

on the electrodes they were only handled with jeweler's

forceps once this initial cleaning was completed. Following

the cleaning the loops on the end of the wires were immersed

in a mixture of pure AgO and a small amount of distilled

water. This mixture adhered to the loop and formed an

irregularly shaped cylinder which was approximately 4 mm

long and approximately 1.5 mm in diameter. The electrode

was then placed in a 500°C oven for 1 hour. This converted

the AgO to pure silver. The next process was to coat the

platinum but not the silver cylinder with an insulator,

Robert's epoxy. Finally the silver was chlorided by using a

constant current DC power supply. To accomplish this the

electrode was placed in a bath of physiological saline and

connected to the positive terminal of the power supply. A

platinum plate also immersed in the saline was connected to

the negative pole of the power supply. A current of 10

milliamps at 10 volts was passed across the electrode for 5

minutes. The electrodes were then stored in a bath of

physiological saline and grounded together until used.

Prior to an experiment they were checked for DC stability

and noise level in a physiological saline bath. DC drift of

the complete recording system and electrode measured across

a saline bridge was less than 65 uV/hr. These same elec-

20
trodes were also used to record the ERGs. Their low noise

characteristics proved excellent for this purpose.

Generation and Presentation of Visual Stimuli

A Maxwellian view stimulator with a Sylvania R1131C
glow tube (Buchmann-Olsen and Rosenfalck, 1957) was the
source that provided the steady or flashing visual stimulus
used to modulate the SP . This stimulus subtended 27.2° of
visual angle. The total irradiance of the stimulus when
unattenuated was 5.4 mW/steradian as measured by an Epply
thermopile. The spectral emission of the R1131C glow tube
at 30 mA, the current at which it was always run, is pre-
sented in Appendix B. When necessary the stimulus was
attenuated with Kodak #96 wratten neutral density filters.
The unattenuated irradiance will be referred to throughout
as the 0.0 intensity level and all other irradiance values
are calculated with reference to this value.

Since a human dark adaptation experiment was performed
the stimulus was also calibrated in photometric units. The
retinal illuminance of the stimulator was 57,000 photopic tds
when unattenuated. This was calculated by the method
described by Westheimer (1966) . A Salford Electrical
Industries photometer was used for the necessary luminance
measurements. The glow tube was driven by a Roush Corpor-
ation timer and flash generator. Current was continuously
monitored. A Beckman Universal timer and counter Model
5510-11 monitored flicker rate.

21
In all cases at least twenty for dark adaptation and

twenty-five minutes for light adaptation were necessary

between stimulus alterations, i.e. turning the light on or

off. Such long intervals of adaptation were mandatory

because of the oscillation of the LMSP . Taumer et al.

(1974) have shown that the oscillatory rises and troughs of

the LMSP will interact with a subsequently elicited change

in illumination unless a sufficient time period is allowed

for the SP to reach an equilibrium. The time period for

light adaptation was longer than for dark adaptation because

light adaptation produced greater amplitude oscillations

which required a longer time period to reach an equilibrium.

As a rule an LMSP eliciting stimulus was not presented

unless a stable baseline, defined as less than 0.2 mV of DC

drift over a 4 minute period was obtained.

ERGs were generated by placing a Grass PS-2 xenon

photostimulator approximately 25 cm from the eye. The flash

had a 20 y second duration and was operated at the W-16

(brightest) setting. An interflash interval (IFI) of 10

seconds was generated by the Roush timer and monitored on

the Beckman timer. In all cases ERGs were recorded when the

S was dark adapted for at least 10 minutes, the opposite eye

was always patched to prevent the recording of spurious

signals.

22
Signal Amplificati on and Processing

A locally designed and constructed high input impedance
DC differential amplifier provided the first stage of am-
plification. Its gain was set at 10 and the bandpass was 0-
14 Khz at the 3 db point. The input impedence was 1012 ohms.
The signal was further amplified by a Redcor Corp 371
differential DC amplifier. Depending on whether SPs or ERGs
were being recorded its gain was set respectively at 10 or
20 and the upper bandpass was 0.1 or 1.0 khz at the 3 db
point.

The SP and ERG signals were processed on a Nicolet Nic-
80 minicomputer, displayed on a monitor oscilloscope and
digitally stored on disc for subsequent analysis. SP data
were also recorded on an Ominiscribe strip recorder. ERGs
were summed sixteen times using the Nic-80.

Preparation and Maintenance of Animal
All Ss were deprived of food for at least twelve hours
prior to the experiment. Anesthesia was initiated by an
intramuscular (IM) injection of 8-10 mg/kg of Ketamine HC1
(Bristol). Also at this time the S ' s pupil was dilated and
accommodation paralysed with a few drops of 1% cyclopento-
late HC1 instilled in the conjunctival sac. Occasionally it
was also necessary to use a similar amount of viscous 10%
phenylephrine HC1 to retract the nicitating membrane. The
S was also given an IM injection of 0.1 mg/kg of atropine
sulfate to suppress salivation. When the S was sufficiently

23

anesthetized (five to ten minutes after the initial inject-
ion) a catheter was inserted in the femoral vein and the cat
was intubated with an appropriately sized trachea tube. The
outside of the tube was coated with 2% lidocaine jelly to
prevent discomfort after the anesthetic wore off. Other
necessary minor invasive procedures were all performed while
the cat was still anesthetized. A small (3-4 mm.) incision
was made above the supraorbital ridge of each eye for
insertion of the indifferent and ground electrodes. These
areas were infiltrated with 2% lidocaine HC1 to prevent any
subsequent discomfort. On occasions when an intraocular
injection was planned a small incision was made at the
external canthus of the eye to be injected. This area was
also infiltrated with lidocaine. When these procedures were
completed the animal was placed in a Baltimore stereotaxic
holder and points of pressure, notably the region of the
external auditory meatus, were covered with 2% lidocaine
jelly. Then an intravenous (IV) infusion of a tubocurarine-
gallamine mixture was initiated (See Appendix C for for-
mula) . After an initial dose of 1 cc/kg a slow infusion of
the paralytic agent delivered via a persitaltic pump (Extra-
corporeal Inc. Model 907) was begun. During the first 30
minutes a dose equivalent to the initial dose of the para-
lytic agent was infused. After that time the rate of
delivery was reduced by a factor of 1/2 by dilution of the
mixture. Repeat doses of atropine (0.07 mg/kg) were admin-
istered IM every four or five hours.

24
Artificial respiration using a Harvard apparatus small

animal respirator was begun when the initial dose of tubo-
curarine-gallamine had caused a marked decrease in voluntary
respiration. Percent expired CO2 was continuously monitored
by a Beckman LB-1 medical gas analyser and was maintained at
4.3 + 0.2 by varying the respiratory rate and volume. To
prevent atelectasis the S_ was hyperventilated occasionally.

While the S's physiological condition was stabilizing,
the electrodes were placed on the cat. The active (non-
inverting) electrode was placed in one end of an agar-saline
filled bridge in a 3 mm inner diameter (ID) polyethylene
tube. The other end of the agar-saline bridge was connected
to a specially constructed transparent contact lens. The
lens was moistened with a 2.5% hydroxypropy methylcellulose
solution. The indifferent and ground electrodes (Ag-AgCl)
were in 4 mm ID polyethylene tubes filled with agar saline
bridges. They were also placed in the incisions made above
the S's orbital ridges. The indifferent electrode (in-
verting input) was located above the eye to be recorded
from. To insure a low impedance, and a high stability
interface, the indifferent and ground electrode tubes had
agar saline soaked cotton wicks inserted in the ends which
were in contact with the cat. Body temperature was moni-
tored with an electronic thermometer via a rectal probe and
was maintained at 38 + 2°C with a fluid filled heating pad.

Once the electrodes were in place and the animal was
stable, the Maxwellian view stimulator was aligned. The

25
focal point of the stimulator was located at the center of

the entrance pupil of the S's eye approximately aligned on

the visual axis.

CHAPTER 4
EXPERIMENT 1: CHARACTERISTICS OF THE LMSP OF THE CAT

Rationale and Methodology
The objective of this experiment was to characterize
the directly recorded LMSP of the cat. After a thorough
literature search no data on the LMSP of the cat were found.
In fact no quantitative data on directly recorded LMSP in
any species were found although the characteristics of the
human EOG have been adequately described (Arden and Kelsey,
1962a, b; Kris, 1958; Taumer, et al . , 1974).

In pilot experiments the light-on and light-off LMSPs
of the cat were observed to be damped oscillations. To
quantify the time course of these oscillations and to permit
comparison with human LMSP oscillations the latencies of the
various peaks and troughs of the cat were measured. In
addition to quantifying the latencies of the peaks and
troughs of the light-on and light-off LMSP, amplitude by
intensity functions were generated over a 3-5 log unit range
of irradiance for the first peak of the on LMSP and the
first trough of the off LMSP.

The LMSP eliciting stimulus used in this experiment was
always the onset or cessation of a steady light. The
stimulus intervals were as described in Chapter 3.

26

27
Results

The waveforms of both the on and off LMSP of the cat
resembled damped oscillations although they had opposite
initial polarities (Figure 2). The time course of both
types of responses were extremely long compared to other
electrophysiological responses.

Table 1 summarizes the mean latencies, standard devia-
tions, ranges, and sample sizes of the various peaks and
troughs of the on and off LMSP. In all cases a peak refers
to a cornea-positive inversion point and a trough refers to
a cornea-negative inversion. Responses were pooled for 8
cats. The steady stimulus which elicited these light-on or
light-off LMSPs ranged from 5. 4-. 0054 mW/steradian in ir-
radiance. The responses elicited by the various intensities
were pooled since no consistent differences in latency as a
function of stimulus intensity were observed. There were
some consistent intersubject differences in latency however.
Sample size decreases with each successive peak or trough
because in many cases, especially when less intense stimuli
were presented, peaks and troughs were not observed. Typi-
cally the light-on response of the SP (Figure 2a) was a
cornea-positive signal which reached a first mean peak at
6.4 minutes after light onset (Table 1). Following this
initial peak the potential dropped for a few minutes until
the first trough was reached at a mean of 17.4 minutes.
Generally the rising phase of the first peak had a steeper
slope than the subsequent falling phase. After this first

*W(**\t~/*^

V/.

1 M V

0 MIN

Figure 2. The light-on (A) and light-off (B)
light modulated standing potentials (LMSPs)
elicited by the onset or cessation of the steady
unattenuated light source. Light-on and off are
indicated by arrows. Note the long time course
of the changes. In this and all other illustrations
of electrophysiological signals an upward deflection
is a cornea-positive response.

29

A - Light-On

Peak 1

Trough 1

Peak 2

Trough 2

Peak 3

mean latency (min)

6.44

17.45

26.39

33.31

42.30

standard deviation
(min)

.71

2.43

3.99

4.24

6.84

range (min)

2.8

8.8

10.7

10.3

11.99

sample size

24

13

9

5

3

B - Light-Off

Trough 1

Peak 1

Trough 2

Peak 2

Trough 3

mean latency (min)

8.09

16.60

23.11

30.97

40.70

standard deviation
(min)

1.76

.88

1.6

1.79

range (min)

6.6

2.5

4.5

3.4

sample size

18

9

6

3

1

Table 1. Mean latencies, standard deviations, ranges and sample sizes
for the various peaks and troughs of the light-on LMSP (A) and light-
off LMSP (B) .

30

trough was reached the SP rose to a second peak at a mean of
26.4 minutes after light onset. This peak was always of
smaller amplitude than the first peak. This second peak was
followed by another trough at a mean of 33.3 minutes after
light onset. By this time in most recordings it became
difficult to distinguish further oscillations. When present
a third peak occurred at a mean of 42.3 minutes after light
onset (Figure 2a) and appeared to be smaller in amplitude
than the second peak. The troughs of the light-on LMSP
rarely fell to a value which was as low as the SP value
preceding the light presentation (Figure 2a) .

The light-off response was of opposite polarity than
the light-on response. It began with a trough and then rose
to a peak. The light-off response (Figure 2b) , like the
light-on response, consisted of oscillations of decreasing
amplitude though their time course and waveform differed
somewhat from the light-on LMSP. The light-off response
reached a first trough at a mean of 8.1 minutes after light
onset (Table 1) . This was followed by a first peak at a
mean of 16.6 minutes and then a second trough at a mean of
23.1 minutes. The initial off response rose to a first peak
more gradually than the corresponding fall of the first peak
of the light-on response making it more difficult to score
the off response for amplitude and latency. It was also
more difficult to observe the succeeding oscillations of the
light-off LMSP because the total off response was of a
smaller amplitude than the light-on response elicited by the

31
same irradiance. Nevertheless in one case when the signal
was cessation of the brightest light and the recordings were
of exceptional stability a third oscillation could be
plainly observed at 41 minutes after onset (Table 1) . Gen-
erally no portion of an off response reached a voltage which
was as great as the light adapted SP amplitude which immed-
iately preceded cessation of the light.

In addition to the major components of the light-on and
light-off responses, certain early minor components of the
light-on and off LMSP are also consistently present. These
components can be observed in Figure 2a but are better seen
when displayed on an expanded time scale as in Figure 3.
The early minor components of the light-on LMSP (Figure 3a)
consist of an early cornea-positive component (PI) followed
by a slower, cornea-negative component (Nl) . The Nl com-
ponent is followed by the first major cornea-positive com-
ponent of the light-on LMSP. The early minor components of
the off response tend to be difficult to observe. They are
present in Figure 2b though they are more easily observed on
an expanded time scale (Figure 3b) . They consist of an
early cornea-negative component (Nl) followed by a slower
cornea-positive wave (PI) which in turn is followed by the
first major component of the off response.

A third minor component in the light-on SP is a small
irregularity in the first peak at 1.6 minutes after light
onset. This component is indicated by the curved arrows in
Figure 4a and was elicited by the highest levels of illumin-

32

5 MV

1 MIN

Figure 3. The early minor components of the LMSP.
Waveform A is the response to light onset. The two early
components are identified as positive one (PI) and negative
one (Nl) . Waveform B is the response to light cessation.
The nomenclature is the same as waveform A. Note the time
scale is more expanded than the previous figure. Both the
on and off responses were elicited by the onset or cessation
of a steady light (as indicated by the straight arrows) with
a log relative irradiance of T. 3.

5

si

[g w

o u

u m
en o

34

35
ation presented. It was observed consistently in most
light-on LMSPs. A similar component can occasionally be
seen in light-off LMSPs. It is illustrated in Figure 4b as
indicated by the curved arrows.

To further characterize the LMSP of the cat, amplitude
by intensity functions for the first major peak of the
light-on LMSP of 4 cats are presented in Figure 5a. Over
this range the light-on response varies in an essentially
linear fashion with the logarithm of stimulus intensity.

Figure 5b presents amplitude by intensity functions for
the first major trough of the light-off response of the LMSP
for the same eyes as Figure 5a. The off response functions
are of a lower amplitude and not as orderly as the light-on
LMSP data. In one case, cat 46, the response appears
saturated at higher intensities. In most cases, however, a
clear off response could not be recorded below 3 . 3 log
units. This is partly attributable to longer time course of
the first oscillation of the off LMSP which made it dif-
ficult to be certain a response had occurred when scoring
data. The overall lower amplitude of the off response also
contributed to the problem of measuring the off response.
It does appear, however, that the off LMSP responds in a
graded fashion to decrements in irradiance. In the 2 cases,
cats 22 and 216, where clear cut graded changes occur, the
decrement in amplitude appears to be linear with respect to
the logarithm of stimulus intensity.

■H X -H

0 <d

tnpq

■H LD

5

37

o -

w
o

o
■<

OS
K

A W

38
Discussion
As illustrated by Figure 2 the light-on LMSP and light-
off LMSP of the cat are of opposite initial polarities and
are damped oscillations with similar time courses (Table 1).
The first major component of the light-on response is cornea-
positive and the first major component of the light-off
response is cornea-negative. Similar findings with respect
to waveform have been reported in all vertebrate species
studies (Gouras, 1969; Kikawada, 1968; Skoog, 1975). The
initial major peak of the light-on LMSP of the cat occurs at
6-7 minutes after light onset. The first major trough of
the light-off response occurs at 8 minutes after light
cessation. In the human Kris (1958) reported the first
light-on peak occurs at 10 plus or minus 2 minutes and the
first light-off trough occurs at 8-10 minutes when measured
indirectly by the EOG. Skoog (1975) recording the LMSP of
the human directly, shows records where the first light peak
occurs about 6-10 minutes after light onset and the first
dark trough occurs 8-12 minutes after light cessation. The
amplitude of the off response in the cat was lower than the
on response elicited by the same stimulus. The same holds
true for the directly recorded human LMSP and EOG (Arden and
Kelsey, 1962a; Skoog, 1975; Taumer, et al. , 1974). The
human on and off LMSP takes 36 plus or minus 4 minutes to
reach the second light-on peak or light-off trough (Kris,
1958). This is longer than the cat LMSP which required 10-
13 minutes less to reach the equivalent inversion. It is

39
important to note that in the cat, like the human (Skoog,
1975; Tauraer et al., 1974), the on and off responses are
similar in waveform and time course but are opposite in
polarity. Gouras and Carr (1965) found oscillations with a
similar time course in the rhesus monkey eye. Kikawada
(1968) studied the first peak and trough of the light-on and
off LMSP of a variety of species from all major vertebrate
classes. He found considerable variability within and
across the major classes of vertebrates (3-13 minutes for
the first light peak and 7-20 minutes for the first dark
trough) . The similar period of the on and off LMSP of the
cat and other species indicates that pigment bleaching or a
directly related metabolic process is probably not the basis
for the on and off LMSP. Pigment bleaching is a very rapid
process while pigment regeneration requires many minutes of
time. Therefore if the on and off LMSP are of similar
origin it is unlikely they arise from pigment regeneration
or a directly related process.

The two early minor components of the cat light-on LMSP
(Figure 3) are also present in the human EOG and are similar
in waveform and time course (Taumer et al. , 1974). The
third component which occurs riding on the major initial
component of the on and off LMSP about 1.6 minutes after
light onset also has been observed in the human LMSP (Skoog,
1975) .

It is well established that the amplitude of the first
major light-on peak of the human EOG varies linearly with

40
the logarithm of stimulus intensity (Arden and Kelsey,
1962b; Taumer, et al . , 1974). This also is the case for the
cat light-on LMSP as shown in Figure 5a. The log-linear re-
lationship between stimulus intensity and light-on LMSP
amplitude is another indication that the photopigment in the
receptors or a directly related process is not the source of
the light-on LMSP. As noted above the extent of pigment
bleached by a stimulus is linearly related to the stimulus
intensity not the logarithm of the stimulus intensity
(Dowling and Ripps, 1970). If the light-on LMSP is directly
related to visual pigment kinetics then it also should be
directly related in a linear fashion to stimulus intensity.

There is some controversy about the graded nature of
the light-off response of the LMSP. Arden and Kelsey
(1962a) found it to be an all or none response, always
falling to the same initial trough regardless of the stimulus
intensity that evoked it. They also found a great deal of
response variability in the light-off EOG amplitude unre-
lated to stimulus variations. Taumer et al. (1974) found
graded changes in light-off EOG linearly related to the
logarithm of stimulus intensity and dispute the findings of
Arden and Kelsey (1962a). The results of this study on the
cat LMSP tend to support the findings of Taumer et al.
(1974) since graded changes in the amplitude of the off
response were recorded (Figure 5) . There was a great deal
of variability however, as Arden and Kelsey (1962a) found
for the light-off EOG.

CHAPTER 5
EXPERIMENT 2 : STEADY VERSUS FLICKERING STIMULI

Rationale

This experiment was designed to determine if the LMSP
elicited by steady as opposed to flickering stimuli of equal
time average irradiance (quanta) are equivalent. The neural
components of the retina are highly sensitive to time
varying stimuli while other retinal processes such as visual
pigment related metabolism and glial cell activity are not
as sensitive (Campbell and Rushton, 1955; Miller and Dowling,
1970). Therefore, by studying LMSP responsiveness to
flickering vs equivalent steady stimuli it is possible to
indirectly determine the importance of various elements in
generating the LMSP.

For example the first component of the visual process
is the photochemical response of the rods and cones. One
part of this process is pigment regeneration which is fairly
well understood. Since the PE is known to participate in
pigment regeneration, a comparatively slow process (Dowling
and Gibbons, 1961), it is possible the LMSP, which has a
long time course reflects related metabolic processes (Arden
and Kelsey, 1962b; Taumer et al., 1974). One purpose of
this experiment will be to compare the LMSP elicited by

41

42
stimuli which bleach equal overall amounts of pigment but
effect the rest of the neural components of the retina dif-
ferentially. It is apparent from intracellular recording in
many lower vertebrates that neural cells in the retina
respond differentially to steady and flickering light
(Rodieck, 1973; Werblin and Dowling, 1969). The stimuli
therefore were a steady light and flickering light of twice
the luminance and half the duration (50% duty cycle) . It
has been demonstrated that the fraction of pigment bleached
is proportional to the product of the intensity of a light
and its duration. This relationship is termed the Bunsen-
Roscoe Law. Campbell and Rushton (1955) demonstrated its
holds for human rod pigment for durations ranging from 0.3
to 48 seconds. They did not investigate shorter durations.
It is known that at microsecond flash durations and/or high
intensities the Bunsen-Roscoe Law does not hold (Hagins,
1957; Pugh, 1975). For this reason very short durations or
high intensities were not employed in this experiment.

A variety of light intensities and rates of flicker
were employed in order to be certain that any flicker vs.
steady differences or similarities were of a general nature
and not unique to particular stimulus situations. Addition-
ally the equivalence of the bleach produced by the flickering
and steady light was confirmed by presenting each type of
stimulus to a human observer and measuring the subsequent
dark adaptation function.

43
Methodology
Cat Experiments

The object of this experiment was to investigate the
effects of square wave flicker versus an equivalent steady
retinal irradiance. The flicker and steady stimuli were
equivalent with respect to averaged irradiance and therefore
at the durations employed bleached equivalent amounts of
pigment (Rushton and Campbell, 1955). The flickering stim-
ulus had a square wave configuration with a 50% duty cycle.
Since it was on one half as long as the equivalent steady
state stimulus, its irradiance was increased by 0.3 log
units. This increased the irradiance by a factor of two.
The equivalence of the two types of stimuli with respect to
extent of photopigment bleached was empirically confirmed by
a human psychophysical dark adaptation experiment which is
described separately.

In addition to varying flicker rates from 0.5 to 30 Hz,
certain flicker rates were tested over a wide range of
retinal illuminations (3-4 log units) . Therefore, there were
two basic protocols for the flicker versus steady experi-
ments. In one type of experiment a particular rate of
flicker was tested over a 3 or 4 log unit range of irradi-
ances. In this type of experiment, a flickering stimulus of
a particular irradiance was always preceded or succeeded by
a steady stimulus of the equivalent time averaged irradiance.

The other type of flicker versus steady experiment was
designed to explore a large range of flicker rates. In

44
these experiments different rates of flicker were presented
at one irradiance. The equivalent steady irradiance con-
dition was also presented periodically throughout the experi-
ment for comparison purposes and to monitor any change in
overall responsiveness. The interstimulus intervals of both
types of experiments followed the guidelines described in
Chapter 3 .

Human Dark Adaptation Experimen t

The object of this experiment was to test the equiv-
alence of the bleach produced by the steady and flickering
stimuli. The S in the human psychophysical dark adaptation
study was a 25 year old female who had normal vision. She
had little previous psychophysical experience. The observer
was first light adapted with the same Maxwellian view
stimulator used to generate the LMSP in the cat experiments.
Then immediately after light adaptation dark adaptation
functions were measured on a dark adaptometer.

For the preliminary light adaptation the observer was
instructed in and then practiced proper alignment of the
Maxwellian view stimulator. She was instructed to fixate
the center of the field with her right eye. Stable head
position was maintained by a head and chin rest. The left
eye was patched. The S was light adapted by either a steady
light attenuated by 0.3 log units or an unattenuated light
flickering at 2 cps. After 5 minutes of light adaptation
the S moved to the dark adaptometer. The adaptometer was
constructed locally and was based on a design described by

u

m

45
Hecht and Shlear (1936). The observer fixated a red light
emitting diode. The test stimulus was a 12° diameter disc
of unfiltered tungsten light. It was centered 14° nasal to
the point of fixation on the horizontal meridian of the
right eye. Since the adapting stimulus subtended 27.2°, the
dark adaptation test stimulus fell fully within the adapted
area. The maximum luminance of the test stimulus was set at
1.65 log foot lamberts as measured with a Salford Electrical
Industries photometer. It could be attenuated in 0.1 log

nit steps with Kodak #96 wratten neutral density filters
and had a duration of approximately 200 msec. For 35

inutes dark adaptation thresholds were determined with this
test stimulus. The ascending method of limits was employed
and approximately 3 measurements were made each minute.

Results
For two cats amplitude by intensity functions were
generated at 2 and 8 Hz respectively. For three cats flicker
rates ranging from 0.5-32 Hz were tested individually. In
both types of experiment responses to the equivalent steady
light conditions were also recorded. Figures 6a and b
present the on and off LMSP to steady and flickering light
(2 Hz) of equivalent time averaged irradiance (equal total
quantal flux) . The waveforms are similar especially the
first major component of the on and off response. Second
peaks and troughs are present in the flicker on and off
response and their time course is similar to the oscilla-
tions elicited by steady stimuli.

46

1^,<u),p4''

^Vl^r^

,U^

t^^^yU^

^y^^'^

Figure 6. The light-on (Figure 6A and B)
and light-off, (Figure 6C and D) IMSP
elicited by the onset and cessation of a
steady light (A and C) or a flickering
light (B and D) of a corresponding time
averaged irradiance.

MV

10 M I N

47

Figure 7 presents the data from the experiment where on
and off LMSP amplitude by intensity functions were generated
over a 3 log unit range for a stimulus flickering at 8 Hz
and a steady light of equivalent time averaged irradiance.
In Figure 7a the light-on data demonstrate that over this 3
log unit range of stimuli there were no consistent differ-
ences between equivalent steady and flickering stimuli. The
light-off steady vs flicker data recorded during the same
experiment are presented in Figure 7b. There are no con-
sistent differences across the adaptation levels for the off
response to flicker vs steady stimulation.

Figure 8a graphically presents a comparison of the
amplitudes of the first peaks of the light-on LMSP elicited
by the equivalent steady and flicker light conditions. The
data from five Ss were pooled. At each flicker rate the
response has been compared to the steady stimulus of an
equivalent time averaged irradiance recorded from that S.
The difference in mV between the two was calculated by sub-
tracting the appropriate steady response amplitude from the
corresponding flicker amplitude. For each square wave
flicker rate tested and presented on the abscissa the steady
vs flicker difference in amplitude has been calculated and
is presented on the ordinate. The number of observations
that each point is based upon is presented above each point.
The error bars represent + 1 standard deviation. To deter-
mine if there was a significant difference overall between
the steady and flicker conditions, the light-on data were

4 9

<
a

OS

H

o
o

A W

Figure 8. The differences in amplitudes between the first
major peak of the light-on IMSP (Figure 8A) elicited by a
steady light and the equivalent flickering light stimulus.
To arrive at a value in mV plotted on the X axis the steady
amplitude in mV was subtracted from the equivalent flicker
amplitude. Flicker rate is plotted on the y-axis and each
point is a mean with the sample size indicated directly
above the point. The bars represent + 1 standard deviation.
Figure 8B presents the difference in amplitude between the
off LMSP elicited by equivalent steady and flickering stimuli.

51

A

0.4 -
0.2

H-l N-12 N = 3 N-

N=l N = l

•0.2
■0.4

J L

0.4-
0.2-

0

-0.2-
-0.4-

N=2

H-l N=l N = 4 N=l N=l

f— ^

0.5 1 2 4 8 16 32

FLICKER FREQUENCY (Hz)

52

pooled for all flicker rates and a two tailed student's t
test was performed. There was no a significant difference
between the steady and flicker conditions (t = 0.76, df =
41, p > 0.25) .

Figure 8b presents the same data for the off steady vs
off flicker conditions. Although the variation in amplitude
is greater as noted previously for the off response of the
LMSP there was no consistent effect of a steady vs a flick-
ering stimulus on the amplitude of the off LMSP from 1-32 Hz
(t = 0.58, df = 25, p > 0.25) .

The flicker versus steady experiments are to a certain
extent based on a corollary of the Ferry-Porter law e.g.
that a flickering light with the same equivalent time aver-
aged irradiance as a steady light will bleach an equivalent
amount of photopigment (Campbell and Rushton, 1955). This
assumption was tested for the specific stimuli presented in
this experiment in a dark adaptation experiment on a human
observer. Figure 9 represents the two dark adaptation
curves generated for the human observer preadapted to either
a steady or flickering stimulus of equivalent time averaged
luminance (38,500 photopic trolands). Threshold luminance
in log foot lamberts is plotted as a function of time after
cessation of the preadaptation stimulus. There was no major
difference between the two functions.

-p w

rs CD

-P rH

(0 >-l

rd U

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54

&.

o

o

o •
o

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as

(I-U fi °I) 3DNVNIWQ1 (M0HS3UHI

55
Discussion
The results of these experiments demonstrate that there
are no major significant differences, over the range of
flicker rates tested, in both the light-on and light-off
LMSP elicited by steady and flickering stimuli equated for
time averaged irradiance. This was true for a wide range of
flicker rates and over a range of intensities. The equiv-
alence of these two stimulus conditions was confirmed in the
human dark adaptation experiment. The similarity of the on
and off LMSP to stimuli which produce extremely dissimilar
responses of retinal neural elements indicates that the LMSP
is not a measure of retinal neural activity. There are con-
siderable electrophysiological and psychophysical data on
the cat and many other vertebrate species which demonstrates
that the retina is highly sensitive to the flicker rates em-
ployed in this study as reviewed by Brown (1965). Dodt and
Enroth (1954) report that the cat ERG and retinal ganglion
cell responses follow square wave flickering stimuli up to
50 Hz. In a behavioral study Kappauf (1936, cited in Brown,
1965) reported cats could distinguish stimuli flickering at
up to 50 Hz. When the large differences in the responses of
the neural elements of the retina to flickering as opposed
to steady light are compared to the similarity of the LMSP
elicited by these stimuli, it seems likely these components
do not make a significant contribution to either the light-on
or light-off LMSP.

56

The equivalence of LMSPs produced by stimuli which
produce an equivalent bleach suggests the LMSP may in
some way be related to this phenomenon. However some results
of experiment 1 (ie. the amplitude by intensity functions for
the LMSP) argue against a direct relationship between bleach-
ing and the LMSP. Also the differences in time course for
bleaching and regeneration when compared to the similarity
seen in the light-on and light-off LMSP time course is, as
noted in experiment 1, further evidence that neither pigment
nor pigment related processes are the source of the LMSP.

CHAPTER 6
EXPERIMENT 3 : ASPARTATE

Rationale
Both experiments 3 and 4 were designed to determine if
the LMSP is generated in the receptors and PE alone or if
neural and glial retinal components are involved. In order
to make this distinction two techniques, one chemical and
one surgical, have been applied. The chemical approach
(this experiment) is based on a number of recent papers
which have shown that sodium aspartate isolates the receptors
from the rest of the retina by acting on the horizontal and
bipolar cells. This has been determined in fish and amphib-
ians (Cervetto and MacNichol, 1972; Murakami et al., 1975;
Murakami et al., 1972) via intracellular recordings. When
sodium aspartate is applied to the retina in minimal con-
centrations the horizontal cells depolarize and their re-
sponse to light stimuli is reduced (Cervetto and MacNichol,
1972). Bipolar cells also are sensitive to aspartate. The
nature of the response varies with the type of bipolar cell
but photic sensitivity is completely suppressed (Murakami
et al . , 1975). To account for these effects it has been
postulated that aspartate or a similar substance is the
transmitter released by the photoreceptors (Murakami et al.,

57

58
1975). Whatever the mechanism, aspartate appears to isolate
the receptors from the rest of the retina. These obser-
vations have been extended to the mammalian retina by Honda
and Dawson (1975) and Hanitzsch (1973) in the rabbit and
Baron and Boynton (197 4) in the monkey. Their studies have
shown that the a-wave of the ERG is not effected by aspartate
but that the b-wave is extinguished. The a-wave of the ERG
has been shown to be a measure of receptor activity while the
b-wave has an inner retinal origin (Brown, 1968; Miller and
Dowling, 1970) .

Therefore, sodium aspartate was injected in a single,
or in one case multiple, dose into the vitreous of the cat.
Prior to and after the injection (s) the LMSP of that eye was
examined. To confirm that aspartate was effecting the inner
retina but not the receptors simultaneous ERG recording be-
fore and after the aspartate injection were made. It has
been noted that aspartate has other ocular effects in the
monkey, notably constriction of the retinal vasculature and
formation of cataracts (Baron, 1974). All Ss were examined
for such effects.

Methodology
In these acute experiments the S was set up and stabil-
ized as described above. Preliminary to the injection (s) of
aspartate the LMSP and dark adapted ERGs were recorded.
After satisfactory records were obtained an intravitreous
injection of sodium aspartate was given via a tuberculin

59
syringe with a 27 guage needle. Dosages of sodium aspartate
ranged from 3.27 mg . to 33.0. For most experiments an
intermediate dosage of 16.4 mg. in 0.04 cc of sterile water
was employed. The sodium aspartate was prepared from
aspartic acid (Sigma) . To achieve the desired concentration
at a pH of 7.3 the aspartic acid was added to sterile water
for injection and the pH was increased by addition of con-
centrated sodium hydroxide.

Before and after injection of the sodium aspartate the
dark adapted ERG was monitored. Since aspartate causes a
decrement in the b-wave of the mammalian ERG (Hanitzsch,
1973; Honda and Dawson, 1975) this provided the necessary
indication of aspartate effectiveness. At various times
following the injection the light on and off components of
the LMSP were recorded. Interstimulus intervals for both
LMSPs and ERGs were as described in Chapter 3.

Results
The dosage of sodium aspartate employed in this experi-
ment was arrived at in a pilot experiment (data not pre-
sented) . An initial dosage of 4.1 mgs, the same dose as
Baron (1974) administered per hour in the macaque monkey,
was initially injected into the vitreous. Following this
injection the b-wave of the ERG was monitored since its de-
crease in amplitude is indicative of aspartate effective-
ness. When no significant effect on the b-wave was ob-
served, increasing dosages of aspartate were administered at

60
1 hour intervals until a significant effect on the b-wave
(defined as a reduction in b-wave amplitude of at least 50%)
was noted. This effect occurred after a total dosage of
16.4 mg. Therefore a single injection of 16.4 mg of sodium
aspartate was administered in one eye of 3 cats. In all
cases this single injection of 16.4 mg of sodium aspartate
had a profound effect on the ERG and LMSP. In a fourth cat,
to induce a larger effect, a larger dose of 49.4 mg was
administered in 2 intravitreous injections separated by 1
hour in time. This larger dose had an even greater effect
on both dependent variables. In all 3 cases where 16.4 mg
of aspartate was injected, the b-wave of the ERG was reduced
in amplitude by at least 70% while the a-wave remained
largely intact. This is illustrated in Figure 10 which
presents the change in ERG due to a single injection of 16.4
mg of sodium aspartate in the left eye of cat 421. Figure
10a is the ERG recorded from the same eye immediately
preceding the injection of aspartate. A normal a- and fa-
wave are present and are so labeled. Records b through f
illustrate a large decline in b-wave due to the aspartate
injection.

Figure 11 presents some LMSP traces recorded from cat
421 during the same experiment presented in Figure 10.
Figure 11a is the light-on LMSP recorded in response to the
onset of the unattenuated light. It was recorded preceding
the aspartate injection. The response appears normal.
Figure lib is the response to the same level of irradiance

Figure 10. The effect of sodium aspartate (16.4 mg) on
the electroretinogram (ERG) . Record A is the ERG recorded
prior to the aspartate injection. Record B through F
demonstrate the change in the ERG as a function of time
after the injection indicated in minutes with each record.
Each record is the sum of 16 signals.

62

PRE A

+ 25

+ 33 C

+ 56 D

+ 16 3 E

+ 325 F

2 MV

40 MSEC

63

pre r

+2

>1V>

B ,/^lS

o i y+^VV*"! A

V^

TVvt

^V

PRE urtfTI'A

j^V™"1

+2 8 8 /

.iyy^^tY^f^n,

M V

10 M IN

Figure 11. Presents the light-on and off IMSP recorded before and
after aspartate. Records A and B are the pre and postaspartate on
responses respectively. Record C and D are the pre and postaspartate
off responses. The times of the post aspartate measurements in minutes
after injection are indicated with each record.

64
201 minutes after aspartate injection. The SP light-on
response is severely attenuated. The ERG b-wave was also
severely attenuated by that time after the injection as
illustrated in Figure 10.

Figure lie presents the light-off LMSP recorded prior
to aspartate injection. It too is large and appears normal
with respect to waveform. Figure lid presents the off
response 255 minutes after aspartate injection. A cornea-
negative off response is no longer present. The b-wave is
also severely reduced by this time as demonstrated by Figure
10.

A summary graph (Figure 12) presents both the ERG and
LMSP data from four aspartate experiments. Since the rele-
vant parameter with respect to ERG is the decline in b-wave
relative to a-wave amplitude, the b- to a-wave ratio was
calculated for the pre and postaspartate ERGs . The data were
then normalized by using the preaspartate b-wave to a-wave
ratio as 100%. This permitted more direct comparison with
the decline in the on and off LMSP which were also normal-
ized. This was accomplished by employing the preaspartate
on and off responses as the baseline 100% level. Figure 12
therefore represents the effect with time of an injection of
sodium aspartate on the b- to a-wave ratio of the ERG, the
light-on LMSP, and light-off LMSP, all normalized to percent
of preaspartate amplitude.

The data in Figure 12 are from animals which, with the
exception of S 321, received a single ocular injection of

0) <D
(ji +J CO 14-1
C rd CD m

w, P

a) m
p o

■P -t->

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CD T3 fd
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66

IN 3 3H 3 d

67

16.4 mgs of aspartate in 0.5 ml of solution at time 0. Some
of the data in Figure 12 from cat 421 are presented pictor-
ially as Figures 10 and 11. Cat 321 had 2 injections of
sodium aspartate. The first was 16.4 mg at time 0; the
second given 62 minutes later was 32.8 mg in 0.1 cc of
solution.

The ERG data from cat 419 best illustrate the orderly
and gradual decline in b-wave amplitude relative to a-wave
amplitude following a single injection of sodium aspartate.
In this cat by 120 minutes after the injection the b- to a-
wave ratio has declined from the preaspartate value over
50%. The large gaps in time for ERG records are unavoidable
since it is not possible to simultaneously record ERGs and
LMSP without the stimuli which elicit each interacting and
contaminating the results of both. The ERGs of the other 3
cats follow the same pattern with the time course and the
ultimate reduction in b- to a-wave ratio varying somewhat.
The greatest decrement in ERG occurred for cat 321 who
received the second injection of aspartate.

In all cats (Figure 12) the decrease in both the light-
on and off LMSP consistently occurred in tandem with the
decrease in b- to a-wave ratio. In cat 44 where a light-on
and off response were collected in the initial 80 minutes
after the injection the decrement is moderate (40-60%) as
would be expected from the reduction in b- to a-wave ratio
usually seen at these times after a single aspartate in-
jection and the ERGs recorded from this cat. In cat 419 the

68
decrease in both LMSP and b- to a-wave ratio was moderate
and of approximately the same value, both reaching about 30%
at 200 min (Figure 12). In cat 421 and 44 (Figure 12) the
b-wave reduction after 120 minutes is greater than cat 419
and the LMSP values are also somewhat lower (0-25%). These
intersubject variations in the time course of the effect are
probably related to the precise place of the injection in
the vitreous and the associated diffusion gradient. In cat
321 where a second injection of aspartate was administered
to induce a larger decrement in b-wave than seen in the
other experiments the ERG b-wave and LMSP were both essen-
tially abolished by 120 minutes after the initial injection
(60 minutes after the second injection). It can be con-
cluded that sodium aspartate severely reduces the b-wave of
the ERG relative to the a-wave and that the LMSP is sim-
ilarly reduced.

As a control experiment a cat was injected with 0.5 ml
of sterile water in the vitreous. The pH was adjusted to
7.3 to equal the pH of the aspartate solution. The ERG and
LMSP were recorded prior to and at various times after the
injection. No effects of the control injection were observ-
ed on either the ERG or LMSP, up to 150 minutes after the
injection.

At the conclusion of all experiments the cat was exam-
ined ophthalmoscopically for retinal vascular constriction
and cataracts which have been reported to occur after
aspartate injection (Baron, 1974). In cat 321, which re-

69
ceived a double injection of aspartate, a cataract was
present. Due to the reduced visibility of the fundus of
this cat it was impossible to determine if the retinal
vasculature had constricted. Neither cataracts nor vaso-
constriction were noted in the other cats.

Discussion
The effects of sodium aspartate on the ERG and LMSP
were unequivocal. Following an injection of aspartate into
the vitreous the b-wave began to decline steadily. A simul-
taneous decrement in on and off LMSPs also occurred. Most
importantly when the b-wave was reduced to about 20% or less
of its preinjection amplitude relative to the a-wave , the
light-on and off LMSPs were similarly reduced or absent.
The near abolishment of the b-wave and retention of the a-
wave is a characteristic effect of sodium aspartate on the
retina of vertebrates and invertebrates (Baron and Boynton,
1974; Cervetto and MacNichol, 1972; Hanitzsch, 1973; Honda
and Dawson, 1975; Murakami, 1972; Murakami, et al., 1975).
Based on intracellular recording in invertebrates it has
been concluded that sodium aspartate isolates the receptors
from the rest of the retina by saturating the horizontal and
bipolar cells (Cervetto and MacNichol, 1972; Murakami , 1972;
Murakami, et al., 1975). The precise mechanism of this
effect is not known although it has been postulated that
aspartate or a similar substance is the transmitter released
by the photoreceptors. In mammals, where intracellular

70
recording techniques have not yet been perfected, the effect
of aspartate on the ERG is the same as seen in inverte-
brates. Furthermore, a large body of literature exists
which demonstrates that in mammals the a-wave is a receptor
based phenomenon while the locus of the b-wave is the inner
retina (Brown, 1968; Granit, 1933). It is clear, therefore,
that when an ERG is recorded with a severely reduced or
absent b-wave but a remaining a-wave, the inner and outer
retina have effectively been separated. The presence of an
a-wave in such a preparation indicates that the receptors
are still functional and that the preparation has not yet
generally deteriorated. Additionally the control experi-
ments, with an injection of an equivalent volume of a
control solution instead of sodium aspartate, had no effect
on the ERG or LMSP indicating that the aspartate effects
were not artif actual. If intraocular pressure changes or
other injection induced trauma induced the alterations in
the dependent variables then similar effects would result
following the control injection. It is concluded, there-
fore, that some postreceptor components of the retina either
neural or glial must be functioning normally for the gener-
ation of LMSPs. This conclusion is further reinforced by
the results of the retinal vascular occlusion experiments.

In the only other in vivo experiments on the effect of
sodium aspartate, Baron (1974) and Baron and Boynton (1974)
reported that in monkeys certain other ocular effects of the
chemical were observed. Specifically by 30 minutes after

71
the start of infusion of sodium aspartate into the vitreous
vasoconstriction of the retinal vasculature was noted. In
addition within 3 hours they noted the formation of a
posterior cataract in the eye being infused. No such
effects were observed in these experiments with the ex-
ception of cat 321 which received a second injection of
sodium aspartate. It is difficult to account for this
species difference since the dosage here was larger than
Baron's (1974). However, the effect of aspartate on the ERG
is constant across all vertebrates examined (frog, rabbit,
monkey, and cat) whether the preparation is iri vivo or in
vitro (Baron and Boynton, 1974; Cervetto and MacNichol,
1972; Hanitzsch, 1973; Honda and Dawson, 1975; Murakami, et
al., 1972; Murakami, et al., 1975). It is therefore unlikely
that the in vivo effect is due to retinal vasoconstriction
although this cannot be ruled out since such vasoconstriction
would at least partially lesion the inner retina while
leaving the receptors intact.

CHAPTER 7
EXPERIMENT 4: VASCULAR LESION

Rationale
To surgically lesion the inner retina, the retinal
blood supply was interrupted thereby destroying the inner
retina while leaving the receptors intact. The receptors
are preserved because they are nourished by the choroidal
circulation (Brown, 1968) . The effectiveness of such a
lesion has been confirmed histologically in the macaque
monkey by Brown (1968) and squirrel monkey by Kroll (1968).
Using electron microscopic techniques Kroll (1968) found
that by 16 hours after occlusion all neural and glial cells
vitread to the outer nuclear layer were severely autolyzed.
The receptors were spared. This technique therefore has
been employed to isolate receptor driven electrical po-
tentials in the cat and other species (Brown, 1968). When
the retinal blood supply is occluded in cat and monkey, the
b-wave of the ERG is eliminated very rapidly but the a-wave
remains intact (Brown, 1968; Maffei and Poppele, 1968).
After 20 days Brown (1968) using light microscopy, found the
inner layers to have degenerated with the exception of only
a few cells while the photoreceptors appeared intact.

73
This technique was therefore applied in the cat to

create the same type of lesion. At various times after the
lesion (ranging from 1-55 days) the LMSP and ERG were exam-
ined. The extent of damage was verified histologically at
various times after the lesion.

Experiment 4, like experiment 3, therefore determined
using a completely different technique if the inner retina
was participating in the generation of the LMSP.

Methodology
A variety of techniques were tested before a successful
lesion of the inner retina could be generated. The retinal
blood supply of the cat is unlike that of monkey or human in
that there is no single central retinal artery in the optic
nerve where it enters the eye (Wong and Macri, 1964).
Rather a number of arteries (3-4) enter the eye between the
optic nerve and adjacent sclera. Because of the complex
nature of the retinal vasculature first attempts to sur-
gically tie off or cut the vessels were unsuccessful. At
this point an attempt was made to electrocoagulate the
retinal vessels using a specially designed probe inserted
into the eye and placed above the major retinal vessels as
they left the disc. This invariably resulted in severe
bleeding into the vitreous and the procedure was discon-
tinued. Finally, an attempt was made to lesion the retinal
vasculature with a xenon arc photocoagulator . In this
procedure brief flashes of high intensity light were di-

74
rected at the optic disc successfully sealing the retinal
blood supply of one eye of the cat.

Three cats were the Ss for this experiment. The lesions
were all made in a single experimental session. At various
times after the lesion was made, ranging from one to fifty-
five days, the LMSP was measured in response to the onset
and cessation of the unattenuated stimulus. The general
methodology followed is described above in Chapter 3. For
control purposes the LMSP of the other eye was also re-
corded. In addition dark adapted ERGs were recorded from
both eyes to confirm the extent and nature of the lesion.

After each Ss had been tested in this manner on at
least one occasion following the photocoagulation of the
optic disc, it was sacrificed. The 3 subjects were sacri-
ficed at 12, 21 and 55 days after the initial lesion was
made. The eyes were immediately removed, a slit was made at
the ora serrata and they were placed in Karnovsky's fixative
(Karnovsky, 1965). After at least 24 hours of fixation at
4°C the anterior chambers were removed and portions of the
posterior pole were embedded in celloidin. Various regions
of peripheral and central retina of each eye were sampled
for all eyes. Tangential sections were cut at 20 y, stained,
examined, and photographed. The stain employed was cresyl
violet. Some tissue was also embedded in plastic, sectioned
at 2 u, and stained with toluidine blue.

75
Results

Immediately after the lesion the disc region appeared
very pale. It was apparent that the retinal vasculature was
lesioned since blood was no longer flowing in the large
vessels. In one of the cats, 6H44, a small amount of
bleeding into the vitreous occurred. After an hour it
appeared to be about 3 disc diameters wide. Subsequently it
was largely reabsorbed. At various times after the lesion
the ERG and LMSP of both eyes of each cat were recorded.
Cat 6H44 and 2B9 were each recorded from on two separate
occasions.

Figure 13 presents the ERGs recorded from cat 7B9 one
day after the lesion. Figure 13a recorded from the control
eye is a normal ERG. Figure 13b is the ERG recorded from
the lesioned eye. The b-wave is severely depressed but a
small component is still present. The a-wave is intact.
The on and off LMSP recorded from this eye during the same
session were also reduced as illustrated by Figure 14.
Figure 14a is the on LMSP from the control eye and Figure
14b is the light-on LMSP recorded from the lesioned eye.
The amplitude of the response of the normal eye is 2.1 mV
while the amplitude of the lesioned eye's response is 1.6
mV. The off LMSP recorded from the control and experimental
eyes are presented in Figure 14c and d respectively. The
control eye amplitude is 0.6 mV and the lesioned eye ampli-
tude is 0.35 mV.

76

V^A-J"V^

2 MV

40 MSEC

Figure 13. ERG recorded from the control eye
(Figure 13A) and from the lesioned eye (Figure 13B)
of cat 7B9 one day after the optic disc was photo-
coagulated.

77

i/M(/

/

ff h^wA

,"V

V

1 MV

10 M I N

Figure 14. A, B, is the light-on UASP recorded respectively
from the control eye and experimental eyes of cat 7B9 one day
after the optic disc was photocoagulated . Traces C and D
present the light-off response of the control and experimental
eyes.

78
The LMSP and ERG data collected from all the Ss in the
vascular lesion experiments are summarized in Figure 15.
Figure 15a, b,c represent respectively the b- to a-wave
ratio, the light-on LMSP and the light-off LMSP recorded in
the control and experimental eyes as a function of time in
days secceeding the lesion. Since like the aspartate
experiments, the relevant parameter for the ERG is the
decline in b-wave relative to the a-wave amplitude, the b-
to a-wave ratio was calculated for the control and experi-
mental eyes. The data were then normalized by using the
mean of the control eye value as 100%. This permits more
direct comparison with the on and off LMSP data which were
also normalized in the same way i.e. the mean amplitude
recorded from the control eye equaling 100%. On day 1, cat
7B9, a small b-wave was present and the on and off LMSP of
the lesioned eye had declined to about 50 or 60% of the
control eye amplitude. These data were displayed in part in
Figures 13 and 14. On day 6, cat 6H44 was the S and his fa-
wave and both the on and off LMSP were absent (Figure 15) .
Twelve days after the lesion cat 1CH was run. The b-wave
was absent, the off LMSP was not present but a small on re-
sponse, 20% or less than the control eye, was observed
(Figure 15). Twenty one days after the initial lesion when
6H44 was again the S all measures in the experimental eye
were still absent. Cat 7B9 was recorded from again 55 days
after the lesion. The on and off LMSP were severely reduced
and the b- to a-wave ratio was still at about 10% as seen in

Figure 15. Vascular lesion experiment summary figure. The
effect of lesioning the retinal vascular supply on the b- to
a-wave ratio of the ERG, Figure 15A, the light-on LMSP,
Figure 15B and light-off LMSP, Figure 15C as a function of
time after the lesion in days. For comparison purposes each
of these dependent variables has been normalized with the
control eye value recorded in the same session equaling 100%
(if more than one response was collected the mean was used
as 100%) . Recordings from control eyes are indicated by an
and from experimental eyes with a . The data presented
for each day was recorded from a single subject. The S for
each experiment is indicated above each day.

8 0

7 B9

6H44

ICH

6H44 7B9

WO

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100

o

o

o

O 8

so

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60

10

20

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4

* // ?

//

120

-

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100

0

o

o
o

o

o
o

80
60

•

0

(0

0

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•
•

r»

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•to

1

0 // 1

//

120

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0 °

100

0

o

o

80

o o

60

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4 0

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2C

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fl»

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A // *

TIME (days

21 '/Ts

81
Figure 15. The records from this session are presented in
Figure 16 and 17. Figure 16 presents the on and off LMSPs
and Figure 17 presents the ERGs .

As a further summary means, standard deviations and
sample sizes were calculated for each dependent variable
i.e. b/a wave ratio of the ERG, the on LMSP, and the off
LMSP (Table 2). Data from all the cats recorded from after
day 1 were pooled (Table 2b); the data from 7B9 recorded from
on day one are presented separately in Table 2a since the
LMSPs of that experiment were clearly different from other
postlesion LMSPs. From the data recorded from all the
animals it is apparent that by 6 days after the occlusion of
the retinal blood supply the LMSP is absent or tremendously
reduced. The greatest reduction in b-wave occurs by one day
after the lesion and at this time the LMSP is reduced by
about 50%. By 6 days post-lesion the on and off LMSPs are
further reduced or absent.

Figure 18 presents photographs of the normal and
lesioned retina embedded in celloidin. Figure 18a presents
a photograph of a section from the control eye of cat 7B9
cut at 20m and stained with cresyl violet. All layers of
the retina are present and labeled. Figure 18b presents
the retina of the experimental eye which was lesioned 55
days before. Only the receptors remain. Their nuclei are
normal in appearance and number. All central and peripheral
areas surveyed in this cat (7B9) had receptors present
except for the disc region. The PE was normal throughout.

IV,

^vV^^V^Wi

VA,ff"

"^VH^j^

^vw/^-y.

Figure 16. Figure 16A, B, is the light-on LMSP
recorded respectively from the control eye and
experimental eyes of cat 7B9 55 days after the
retinal vasculature was photocoagulated . Traces
C and D, present the light-off response of control
and experimental eyes.

MV

M IK

83

2 MV

40 MSEC

Figure 17. ERG recorded from the control (A) and experimental (B)
eyes of cat 7B9 51 days after lesioning the optic disc.

Table 2: Means, standard deviations and sample sizes
for the B/A-wave ratio of the ERG, the light-on and
the light-off LMSP in the control and experimental eyes.
Table 2A is the data recorded from cat 7B9 on day 1.
Table 2B is the pooled data recorded from all cats run
on day 6 through day 55.

85

A - Cat 7B9, Day 1

B/A Ratio

Light-On
LMSP

Light-Off
LMSP

mean-control eye

3.8

1.52 mV

.63 mV

standard deviation-
control eye

.28

.12

.06

sample size-control eye

2

2

2

mean-lesioned eye

.33

.91

.32

standard deviation-
lesioned eye

.01

.16

.05

sample size-lesioned eye

4

3

3

B - All Other Ss, Days 6 to 55

B/A Ratio

Light-On
LMSP

Light-Off
LMSP

mean- control eye

2.93

1.51 mV

.84 mV

standard deviation-
control eye

.87

.29

.26

sample size-control eye

7

7

6

mean-lesioned eye

.14

.10

.01

standard deviation-
lesioned eye

.10

.14

.02

sample size-lesioned eye

8

8

10

Figure 18. Tangential sections from the normal (A) and
experimental (B) retinas of cat 7B9. The tissue was embedded
on celloden, cut at 20y and stained with cresyl violet.

87

sclera

tape turn

pr:

outer segments

receptor nuclei
outer plexiform
inner/nuclear 1.
ganglion cell layer

sclera

tapetum
PE

outer segments

receptor nuclei

remnants of inner
layers

To better demonstrate that the outer and inner segments of
the receptors were present in the lesioned eyes tissue from
the lesioned eye of cat 6H44 embedded in plastic is shown in
Figure 19. The receptor inner and outer segments are
present although the retina became detached during proces-
sing. It should also be noted that the major nonneural glial
cell of the retina, the Muller cell, also appeared to be
absent. The Muller cells were probably not present since
their nuclei and most of their cytoplasm lies in the inner
retina (Miller and Dowling, 1970).

VJhen S ICH's experimental eye was examined most areas
of the retina had normal appearing receptors but no inner
layers. An area of approximately 10% of the retina, how-
ever, was largely degenerated with only a few receptors
intact. Some small areas of reduced receptor density were
also observed. The PE appeared normal throughout. When the
retina from the experimental eye of S 6H44 was examined a
fairly large receptorless area was observed (40%). Some
areas of thinned receptors were also present. Areas of
normal appearing receptors without inner retinal layers were
also present. All areas of the PE examined appeared normal.

It should be noted that in all cats there were always
a considerable number of normal appearing receptors remain-
ing. Additionally an a-wave, indicative of functional
receptors, was also always present. There were however
some small differences across cats with respect to the ERGs .
For cat 7B9 where all the receptors were present except

Figure 19: Tangential section from the
lesioned eye of cat 6H44. The tissue was
embedded in plastic and sectioned at 2 p
and stained with toluidine blue.

90

Tapetum

PE

■ outer
segments

inner
segments

receptor
nuclei

degenerated
remnants of
inner layers

*

91
those around the optic disc the a-wave was only 14% lower
on day 55 in the experimental eye, than in the control eye
(Figure 16) . Cat 6H44 and 1CH had a-waves which were re-
duced by approximately 25% (measured at the last experi-
mental session for each cat) which is surprising considering
the extent of the receptorless area in cat 6H44.

In cat 7B9 a larger b-wave was preserved also indi-
cating that the lesion was not as severe. Only a miniscule
b-wave or none at all was observed in the ERGs of the experi-
mental eyes of the other two cats indicating a more severe
lesion than in 7B9.

Discussion

Lesioning the retinal blood supply eliminating the
inner retina but leaving the receptors intact is a standard
technique in cat and monkey (Brown, 1968; Maffei and Pop-
pele, 1968). Like aspartate, lesioning the inner retina
eliminates the b-wave yet leaves the a-wave intact.

As expected photocoagulating the optic disc of the cat
had the predicted effect on the ERG, reducing or eliminating
the b-wave although the a-wave was still present. As in the
aspartate experiments severe reduction in ERG b-wave to a-
wave ratio occurred in tandem with severe reduction in the
LMSP responses in all lesioned eyes. On the first day after
the lesion the decrement in ERG was more severe than the re-
duction in LMSPs. Between the first and sixth days after
the photocoagulator lesion the LMSP is reduced by 80% or

92
more as is the b- to a-wave ratio of the ERG. Both signals
remain depressed up to 55 days after the initial lesion, the
longest time at which a measurement was made. The nature of
the lesion was confirmed histologically demonstrating that
the photoreceptors were present but the rest of the neural
retina was absent.

In two cats the lesion was more severe and some photo-
receptor change was observed. This was surprising since
interrupting the retinal circulation of the cat has been
used by other investigators to reportedly produce the same
lesion. Brown (1965) reported the photoreceptors were
intact but the inner retina was absent in the cat following
occlusion of a branch vessel of the retinal arteries.
Maffei and Poppele (1968) photocoagulated the optic disc of
cats and report only the a-wave of the ERG remained but pre-
sented no histological data to confirm the extent of the
lesion. There are two interpretations of the receptor
damage found in two of the Ss in this experiment. It could
be either that the retinal circulation in some cats supplies
parts of the inner retina or that the choroidal circulation
was damaged by the photocoagulation. With respect to the
first hypothesis it should be pointed out that although the
retinal vasculature is reported to supply only the inner
retina in the cat, variability in vascular patterns across
individual Ss is a well known occurrence. The second
hypothesis also cannot be ruled out. Choroidal arteries lie
directly around the disc region of the cat (Wong and Macri,

93
1964) and either through direct damage or edema may have
been temporarily or permanently occluded. Fortunately in
two of the cats the damage to the receptors was either nil
or not severe. It can be concluded therefore that the
inner retina is necessary for generation of the LMSP . This
experiment therefore confirms the findings of the aspartate
experiment via another totally different technique.

CHAPTER 8
ANALYSIS AND CONCLUSIONS

The major finding of this study is that the inner
retina is involved in an essential fashion in the generation
of the LMSP. In addition it was demonstrated that the
LMSPs elicited by a flickering light are not readily dis-
tinguishable from the LMSPs elicited by a steady light of
equal time averaged irradiance. When this result is con-
sidered within the context of the aspartate and vascular
lesion experiments one must conclude that the LMSP is
probably generated by a nonneural element of the inner
retina. This element (s) must meet at least two require-
ments. First, it must be situated at least in part vitread
to the receptors on the inner retina. Second, it must be
relatively insensitive to the rates of flicker tested in
this experiment which are known to be psychophysical^ and
physiologically distinguishable by the cat (Dodt and Enroth,
1954; Kappauf, 1936 cited in Brown, 1965).

A number of authors have suggested that the PE modu-
lated by the receptors is the site of the LMSP (Arden et
al., 1962; Nilsson and Skoog, 1975; Skoog, 1975; Taumer et
al., 1974). The PE obviously does not meet the first
requirement stated above i.e. it is not located in the inner
retina and there is no evidence that it is in any way re-

9 4

95
lated to the inner retinal functions. This PE hypothesis
can probably be attributed to the work of Noell (1954) on
the SP in the rabbit. He demonstrated that a chemical
lesion of the PE induced by sodium iodate and confirmed
histologically reduces or eliminate the large positive
response of the SP to sodium azide. He concluded that the
PE must "participate in the production of the normal direct
current potential across the eye (Noell, 1954, p. 80)."
Heck and Papst (1957) confirmed Noell 's findings. Neither
Noell (1954) nor Heck and Papst (1957) measured the LMSP.
The dependent variable they measured was the chemical re-
sponsiveness of the SP. Noell (1954) attributes the constant
DC voltage across the eye to the PE; he does not attribute
the LMSP to the PE as he never recorded this variable. Heck
and Papst (1957) thought that the SP and LMSP both origin-
ated in the PE driven by the receptors. However there is no
a priori reason why the DC potential across the eye should
originate at the same locus as the light induced changes in
DC level (LMSP) and the results of the aspartate and vas-
cular lesion experiments rule out this hypothesis. However,
this PE localized DC potential Noell localized could be the
current source for the LMSP. It is possible that the LMSP
results when the conductance of the retina changes due to
the effect of light thereby changing the current pathways of
the PE produced SP. All neural activity induces associated
extracellular ion flux and these must alter the resistance
of the retina. This hypothesis is perhaps more parsimonious

96
than a dual source of DC potentials in the eye but con-
sidering the number of sites for the various components of
the ERG it is not a compelling argument.

Whenever considering the origin of the LMSP two of its
characteristics must be recalled: a) its long time course
and b) the fact that it is a damped oscillation. Various
authors have stated these characteristics indicate the LMSP
is directly related to certain PE and receptor or inner
retinal metabolic functions (Arden and Kelsey, 1962b; Arden
et al., 1962; Taumer et al., 1974).

Arden et al. (1962) and Arden and Kelsey (1962a) have
suggested the LMSP is an indication of rhodopsin renewal.
The time course of rhodopsin renewal is of the same order of
magnitude as the LMSP and has been shown to involve the PE
(Dowling and Gibbons, 1961). In addition, Arden and Kelsey
(1962b) have demonstrated the amplitude of the first major
peak of the light-on EOG succeeding a complete bleach is
highly correlated with the percent of rhodopsin regenerated.
The results of experiment 1 (where the characteristics of
the cat LMSP were described) and the aspartate and vascular
lesion experiments demonstrate, however, that a purely re-
ceptor-PE based explanation of the LMSP is untenable.

Taumer et al. (1974) have proposed the retinal ADP/ATP
quotient is responsible for the LMSP although no specific
mechanism is suggested. In this scheme the initial peak or
trough of the LMSP and the oscillations are supposedly signs

97
of an incoming energy overshoot or undershoot. The LMSP and
the associated oscillations thus would be representative of
an oscillating feedback process which is providing the
necessary supply of energy to the retina. In support of
this hypothesis they cite the work of Sickel (1972) who
found long lasting variations in ADP/ATP quotient in an in
vitro isolated frog retina preparation without a PE. How-
ever, other findings of Sickel (1972) considered with regard
to the flicker vs. steady experiments described here would
seem to weaken this hypothesis. Sickel (1972) using a
variety of techniques measured retinal metabolism under
various light, dark and flickering light conditions. The
preparation was an in vitro, perfused frog retina with the
PE removed. He found that CO2 production from 14C labeled
glucose, a measure of retinal respiration, declined when a
steady light was presented to a dark adapted retina but
increased when a 1 Hz square wave flickering light was
substituted for an equivalent time average luminance steady
light. He also measured retinal 02 uptake in light and dark
and found that in light it decreased. If the differential
effects Sickel (1972) found for steady and flickering light
hold true for cat retinal metabolism as well, then the
absence of a significant difference in the steady vs.
flicker experiment indicates that the LMSP is not a measure
of retinal energy consumption.

If the LMSP is not generated by the receptors and PE
alone as demonstrated by the basic characteristics of the

98

LMSP (experiment 1) and the aspartate and vascular lesion
experiments and it is not generated by purely neural or
energy related metabolic activity in the retina, as the
steady vs. flicker experiments indicate, what does generate
it? As discussed above it is unlikely that a neural com-
ponent of the retina other than the receptors or retinal
energy metabolism per se is the source of the LMSP. One
obvious possibility remains, the glial cells of the retina,
specifically the Muller cells. Processes of these cells are
closely associated with the nuclear region and synaptic
terminals of receptors although no synaptic contacts can be
seen at the EM level (Miller and Dowling, 1970). The Muller
cells could be intimately related to receptor metabolic
activity. In addition Muller cells have been shown via
intracellular recordings in mudpuppy to be relatively
insensitive to flickering stimuli compared to the neural
cells of the mudpuppy retina (Miller and Dowling, 1970).
Muller cell bodies proper are in the inner retina and at
least in the monkey they are known to degenerate after
occlusion of the retinal vasculature (Kroll, 1968). Fin-
ally, if sodium aspartate acts like a neurotransmitter
saturating the horizontal and bipolar cells it may also
interfere with normal Muller cell activity. Some glial
cells in the mammalian nervous system have been shown to
preferentially accumulate certain neurotransmitters (Kuffler
and Nicholls, 1976). If Muller cells normally accumulate
the neurotransmitter released by the receptors an excess of

99

the transmitter might act to saturate their normal electrical
responsiveness. It is therefore hypothesized that the LMSP
is the result of some metabolic interactions between the
receptors and Muller cells of the retina. Glial cells have
been shown to have a number of properties which distinguish
them from neurons. Some of these properties resemble the
kinds of changes seen in the LMSP. For example, mammalian
cortical glial cells respond to ongoing activity in sur-
rounding neurons with sustained and increasingly large de-
polarizations (Kelly and Van Essen, 1974). The ability of
glial cells to integrate activity over a period of time
many seconds long demonstrated both in the mudpuppy Muller
cells and mammalian cortical glia is the key characteristic
implicating the Muller cells as the origin of the LMSP. In
the mudpuppy optic nerve depolarizations of up to 140 mV
over a resting level have been seen. At the end of the train
of stimuli inducing these large potentials it may take over
30 seconds for the cells to return to their normal resting
state (Kuffler and Nicholls, 1966) . Miller and Dowling
(1970) who recorded intracellularly from Muller cells in the
necturus retina state that in response to flashes of light
these cells tended to stay depolarized unlike the ERG b-wave
and responses recorded from inner retina neural cells in the
same preparation. Mammalian cortical glia also have long
time constants responding with sustained depolarization to
activity in surrounding neurons (Kelly and Van Essen, 1974).
In the retina a sustained depolarization of Muller cells

100
would result in a positive response recorded with a corneal
electrode. This is the same polarity as the light-on LMSP
as noted above.

The Muller cell therefore directly influenced by the
photoreceptors would seem to be the most likely candidate
for generating the LMSP. They are situated mainly in the
inner retina yet contact the receptors. They are compara-
tively unresponsive to flicker unlike the neural components
of the inner retina of the mudpuppy (Miller and Dowling,
1970) . Finally intracellular recording of their light
induced activity indicates they generate sustained poten-
tials, the most distinctive characteristic of the LMSP. The
polarity of these potentials, both light-on and light-off
components are in the correct direction to be the initial
peak or trough of the LMSP. It should also be noted the
spatial orientation of the Muller cells in the retina is
compatible with production of large field potentials such as
the LMSP's measured at the cornea. In the retina extra-
cellular currents add in the radial direction (the orien-
tation of the Muller cells) while currents parallel to the
retinal surface tend to cancel (Rodieck, 1973). Finally, it
is worth noting that the long time course of the LMSP has
been considered by many authors (Arden et al., 1962; Arden
and Kelsey, 1962b; Taumer, 1974; Skoog, 1975) to indicate it
is related to supportive metabolic functions of the retina.
This has classically been a function attributed to glial
cells in the nervous system (Kuffler and Nicholls, 1976).

101

Another controversial question concerning the LMSP is
the relationship between the on and off responses. The data
presented here indicate that the two components have a com-
mon site of origin. The common insensitivity of the on and
off responses to steady vs. flickering stimuli indicates
both are of non-neural origin. The severe and correlated
reduction in both components due to inner retinal lesions is
another important similarity. If these findings are con-
sidered in conjunction with the similarity in waveform and
time course of the oscillations shown in this experiment for
cat and by Skoog (1975) in the human a strong case is made
for a common site of origin. The increased variability seen
in off responses and the difference in waveform shown here
of the initial peak and trough may be indicative of some
basic differences but these are subtle when compared to the
noted similarities (Table 1). In the literature the strong-
est argument for a different site of origin of the on and
off LMSP has been made by Gouras (1969). His argument is
based in part on a clinical case of central retinal artery
occlusion reported by Nagaya (1964). Nagaya presented data
recorded on the fourth and sixtieth days after a patient
suffered a complete obstruction of the central retinal
artery. On the fourth day neither the light-on nor the
light-off response was present. On day 60 a smaller than
normal light-off response was present but the on response
was still absent. In the cat I tested 55 days after vas-
cular occlusion neither response was present. In addition

102
at all other times when cats were tested, except day 1 after
vascular lesion, both on and off responses were absent or
severely reduced in cats. Gouras (1969) also noted that the
dark trough remained normal in certain other pathologies
that eliminate or reduce the light peak such as chloroquine
retinopathy and all but severe cases of retinitis pigmen-
tosa.

Clinically the EOG has been used for many years as an
aid in diagnosis of various ocular lesions especially
hereditary disorders of the eye such as vitelliform macular
degeneration, fundus f lavimaculatus , and the various forms
of retinitis pigmentosa and toxic lesions (Deutman, 1971;
Gouras 1969) . Generally clinicians have assumed it is a
measure of retina-PE and especially PE activity (Arden et
al., 1962; Skoog, 1975). This is certainly not the whole
story. It would be useful to reevaluate the EOG as a
clinical test. It may be more widely applicable as a diag-
nostic and especially prognostic test in a variety of
retinal lesions since it measures the viability of the inner
retina not just the PE and receptors. For example the EOG
might be a valuable diagnostic and prognostic test in
diabetic retinopathy or glaucoma.

One disease the EOG is usually very effective in diag-
nosing is vitelliform macular degeneration (VMD) (described
in detail by Deutman, 1971). Ophthalmoscopically the dis-
ease usually presents as a purely macular lesion. In its
early stages visual acuity is usually normal or slightly

103

subnormal. Dark adaptation functions and visual fields also
appear normal. The foveal ERG and visually evoked response
are also usually normal. The light rise and dark trough of
the EOG are typically absent however Deutman (1971) suggests
that this EOG finding is indicative of a diffuse disorder
which appears ophthalmoscopically only at the macular region
due to its unusual characteristics. Specifically Deutman
(1971) proposes a diffuse metabolic lesion of the PE as the
cause of the abnormal EOG. It is difficult to reconcile
the abnormal EOG seen in VMD with the findings of this
study. It may be that when the SP , arising in the PE
(Noell, 1953), is abolished, an LMSP cannot be generated.
It also may be that a diffuse retinal lesion is present in
cases of VMD but that such a lesion is too subtle to cause
a major decrement in visual function as measured in the
clinic.

Additional work to precisely specify the neural origin
of the LMSP is still needed. The first experiment to per-
form might be intracellular recording from Muller cells. If
they could be held for ten minutes it would be possible to
determine if they were the source of the LMSP. Other
experiments relating retinal metabolism and photopigment
regeneration directly to the LMSP would also be extremely
interesting. If better understood the LMSP could provide a
new tool for studying retinal metabolism, and perhaps glial-
neural interactions.

104

Conclusions
In one experiment it was demonstrated that the light-on
and off LMSP of the cat does not respond differentially to
steady vs. flickering light of equal time averaged irradiance.
Also it was demonstrated, using two totally different
techniques, that to elicit a light-on and light-off LMSP a
functional inner retina must be present. Based on these
findings it was hypothesized the LMSP originates in the
Muller cells of the retina. They are located mainly in the
inner retina, are relatively insensitive to flickering light
unlike the neural cells of the inner retina and tend to
integrate even low frequency flickering stimuli (Miller and
Dowling, 1970) . Based on these findings it was suggested
that the clinical EOG be reevaluated since it may be of more
general applicability.

APPENDIX A

Subjects Used in Each Experiment

Experiment #1
119
215
315
329
411
413
419
1CH

Experiment #2
215
315
411
413
419

Experiment #3

419

421

44

321

Experiment #4
7B9
6H44
1CH

105

APPENDIX B
Sylvania R1131C Glow Tube Spectral Emission

Data from Manufacturer

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0 430 4<0 «0 S.<0 MO 6W 640 4U0 720
WAV1 UHGM
MlVt iMUGr' Vi WAVE IfNGIM

106

APPENDIX C

Formula for Paralytic Agent

29.9 cc 0.021% NACL in sterile water with 5% Dextrose
4.5 cc Gallamine triethiodide (20 mg/ml)
0.6 cc Tubocurarine Chloride (3 mg/ml)

107

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BIOGRAPHICAL SKETCH

Harris R. Lieberman was born in Philadelphia,
Pennsylvania, and attended Central High School there. Upon
graduation he attended Franklin and Marshall College in
Lancaster, Pennsylvania, where he majored in psychology and
was a teaching assistant in that department. He graduated
in 1972 and in that year began graduate school in the
Psychology Department of the University of Florida. At
Florida he was a research assistant in the Visual Sciences
Laboratory and in July, 1974, received a Center for Neuro-
biological Sciences Fellowship. He was awarded the Master
of Arts degree in December, 1974. His major areas of
interest are the neurophysiological and behavioral aspects
of vision and their clinical application.

In June, 1973, he married Ellice S. Silver. She
attended the University of Florida College of Medicine and
graduated in June, 1977.

115

I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and Quality,
as a dissertation for the degree #of Doctor alp Ptvilosophy .

ie c

William W. Dawson, Chairman
Professor of Psychology

I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality,
as a dissertation for the degree— of Doctor^of Philosophy.

Calvin K. Adams
Assistant Professor of
Psychology

I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor ,of Philosophy.

CC

2

4'L Ti^-crtT't

-Jay jM. /Enoch V

Graduate Research Professor
of Psychology

I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope ancL_auality ,
as a dissertation for the degree of ppctor ^f^ifflps

Merle 'E . Meyer /
Professor and Chairman of
Psychology

I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of-, Doctor of Philosophy .

yfas/UA&«»»

Horst 0. Schwassmann
Associate Professor of
Zoology

1

This dissertation was submitted to the Graduate Faculty of
the Department of Psychology in the College of Arts and
Sciences and to the Graduate Council, and was accepted as
partial fulfillment of the requirements for the degree of
Doctor of Philosophy.

August, 1977

Dean, Graduate School

UNIVERSITY OF FLORIDA

3 1262 08552 9641