Title: Some effects of cold stress on the morphology and electrophysiological function of the retina of the goldfish
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 Material Information
Title: Some effects of cold stress on the morphology and electrophysiological function of the retina of the goldfish
Physical Description: ix, 113 leaves. : illus. ; 28 cm.
Language: English
Creator: Hope, George Marion, 1938-
Publication Date: 1971
Copyright Date: 1971
 Subjects
Subject: Vision   ( lcsh )
Retina   ( lcsh )
Psychophysiology   ( lcsh )
Goldfish   ( lcsh )
Psychology thesis Ph. D   ( lcsh )
Dissertations, Academic -- Psychology -- UF   ( lcsh )
Genre: bibliography   ( marcgt )
non-fiction   ( marcgt )
 Notes
Thesis: Thesis -- University of Florida, 1971.
Bibliography: Bibliography: leaves 108-112.
Additional Physical Form: Also available on World Wide Web
General Note: Manuscript copy.
General Note: Vita.
 Record Information
Bibliographic ID: UF00097673
Volume ID: VID00001
Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
Resource Identifier: alephbibnum - 000549640
oclc - 13246626
notis - ACX3935

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S:.me Fffect= cf Ccil.ij tre-s ocn the Morphology
anrd l1ectrch I ilogica 1 Func-tion of the
PeLin,.i ,.-f tI Gc-.lc!,fish











B'
Geor.rge !aric~n.r HI.:.,:










,1 Di sertatio,:n Fre.:entt tL. th.- Cri,-uate Council of
the ini er--r it ,:f EI .t i.>.,
in Partial Fiu f .li i nt ,f tl.-: Fa-:,jiLements for the
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UNIVERSITY OF FLORIDA

3 1262 08552 5011














ACKNOWLEDGMENTS


The author would like to express his appreciation

to the members of his doctoral committee, Drs. D.C. Teas,

H.S. Pennypacker, R.L. Isaacson and J.B. Munson, and

especially his chairman, Dr. W.W. Dawson, for guidance,

criticism and encouragement throughout this effort, and

to his wife and son for sacrifices made and patience

exhibited throughout. This research was supported by

Training Grant MH 10320-06 to The Center for the Neuro-

biological Sciences from the National Institute of Health

and grant number AEC-AT (40-1) 3599 to Dr. W.W. Dawson

from the Atomic Energy Commission.













TABLE OF CONTENTS




Page

ACKNOWLEDGMENTS . . . . . . . . . ii

LIST OF TABLES. . . . . . . . . ... iv

LIST OF FIGURES . . . . . . . . . v

ABSTRACT. . . . . . . . . .. . vii

INTRODUCTION. . . . . . . . . .. 1

METHOD. . . . . . . . . .. . . 16

Procedure . . . . . . . . . . 16
Subjects. . . . . . . . . . . 17
Environmental Control . . . . . .. 17
Sampling. . . . . . . . . . . 23
Electrophysiological Measures . . . . ... 25
Histology . . . . . . . . .. . 33
Behavioral Testing. . . . . . . .. 35
Statistical Analyses. . . . . . . .. 36
Additional Considerations . . . . . .. 39

RESULTS . . . . . . . . . . . 43

Behavior. . . . . . . . . ... . 43
Electrophysiology . . . . . . . .. 46
Morphology. . . . . . . . . . 80

DISCUSSION. . . . . . . . . . 91

REFERENCES. . . . . . . . . . 108

BIOGRAPHICAL SKETCH . . . . . . . .. .113
















LIST OF TABLES


Table Page

1. Summary of Analysis of Variance: Food Catching
Behavior . . . . . . . . . 45

2. Summary of Analysis of Variance: ERGs from
Group L Under Photopic Adaptation . . 49

3. Summary of Analysis of Variance: ERGs from
Group L Under Mesopic Adaptation . . .. 50

4. Summary of Analysis of Variance: A and B Waves
from Group L Under Mesopic Adaptation . 53

5. Summary of Analysis of Variance: ERGs from
Groups L and D Under Scotopic Adaptation 59

6. Summary of Analysis of Variance: A Waves from
Groups L and D Under Scotopic Adaptation 64

7. Summary of Analysis of Variance: ERGs from
Group L Through Dark Adaptation . . .. 69

8. Summary of Analysis of Variance: Outer
Segment to Ellipsoid Ratios . . . .. 86









LISf ,:,F FI,;UF.ES


Figure Page

1. Anatomy of the Retina. . . . . . . 3

2. Spectral Transmission Characteristics of
Wratten Filters. . . . . . . ... 19

3. Order of Experimental Events . . . ... 26

4. Food Catching Behavior of Goldfish in Cold
Stress and Recovery. . . . . . ... 44

5. ERG Amplitudes from Group L Under Photopic
and Mesopic Adaptation . . . . ... 47

6. A and B Wave Amplitudes from Group L Under
Mesopic Adaptation . . .... . . 52

7. Normalized A and B Wave Amplitudes from
Group L Under Mesopic Adaptation.. ... 54

8. ERG Amplitudes: Response to the First Flash
Under Scotopic Adaptation. . . ... 56

9. ERG Amplitudes from Groups L and D in Response
to the Second Flash Under Scotopic
Adaptation . . . .... .. . . 58

10. A Wave Amplitudes from Groups L and D Under
Scotopic Adaptation. .... .. . . 61

11. B Wave Amplitudes from Groups L and D Under
Scotopic Adaptation. .... .. . . 62

12. Normalized ERG Amplitudes from Group L Under
All Adaptation Conditions. ... . . 66

13. Dark Adaptation Cruves from Group L. ... . 68

14. ERG Development During Dark Adaptation; Day 0. 71

15. ERG Development During Dark Adaptation;
Experimental Day 15. .... . . . 72











Figure Page

16. ERG Development During Dark Adaptation;
Experimental Day 60. . . . . . ... 73

17. ERG Amplitudes from Group R to Each
Monochromatic Stimulus . . . . ... 75

18. ERG Amplitudes from Group B to Each
Monochromatic Stimulus . . . . ... 76

19. ERG Amplitudes from Groups RB, RBB and RBR
to Each Monochromatic Stimulus . . .. 77

20. Ganglion Cell Frequency in Retinae of
Groups L and D . . . . . . ... 81

21. Cell Frequency in the Inner and Outer
Nuclear Layers of Retinae from Groups
L and D .. . . . . . . . ... 82

22. Ellipsoid Frequency in Retinae from Groups
L and D. . . . . . . . . ... 83

23. Outer Segment to Ellipsoid Ratios for
Retinae from Groups L and D. . . . ... 85

24. Normalized Outer Segment to Ellipsoid
Ratios Compared to Normalized Scotopic
ERGs in Response to the First Flash for
Groups L and D . . . . . ... 87

25. Outer Segment to Ellipsoid Ratios for
Migrated and Unmigrated Cone Receptors
for All Spectral Groups. . . . . ... 89








.. b tract c f Di i tert:i -.rn Presenr td to. tlie
iJr .Juate. Couijncil the.- Unii'.- r si t of Ficr ida
in FrP3ti i uli f illF, 3nt *;. the F.equit Lemeri 3:7 ftr
the CY eDree. E L':octc.r o .. Fhi ilc.so.Dhy

SOME EFFECTS OF COLD STPESS ON THE MORPHOLOGY
AND ELECTFC'Or IiCOL''jICAL FUNCTION OF THE
RETINA OF THE GOLDFISH

By

George Marion Hope

June, 1971

Chairman: Wm. W. Dawson, Ph.D.
Major Department: Psychology

The purpose of this research was to investigate parameters

of and variables contributing to cone outer segment atrophy

under extended low temperature. Specific problems attacked

were to define the time course of structural degeneration and

loss of electrophysiological function, to determine if morpho-

logical loss extended beyond the receptor cell and to inves-

tigate the influence of environmental lighting on the effects

of cold stress on the cone receptor. Additionally, it was

hoped that, through manipulation of stimulus parameters for

electrophysiological recording, it would be possible to

determine if there were differential effects on photopic

(cone) and scotopic (rod) systems and on receptor and neural

function for each.

In order to accomplish these ends, five groups of

goldfish were established, with two receiving white light

or no light and the other three receiving light from specific

bands of wavelengths. These groups of animals were subje.:ted

to temperatures between 40 and 80 centigrade .for 44 or 59








days respectively (cold stress), then were allowed to return

to more temperate conditions for the remaining 62 or 47 days

(recovery). Throughout this time each group was sampled

periodically. Primary data were electrophysiological re-

cordings taken from the cornea (ERGs) and counts of cellular

constituents of each layer of the retina, with especial

attention to inner and outer segments of cone receptors.

Electrophysiological recording and histological processing

of the eyes were by conventional techniques. Total ERGs

were analyzed and A and B wave components, assumed to reflect

receptor and neural activity from the inner nuclear layer

respectively, were analyzed individually. Stimulation

parameters were chosen in a manner such that photopic and

scotopic function were differentiated.

It was found that both the ratio of residual outer

segments to cone cells (actually ellipsoids) and gross

electrophysiological function were reduced in an orderly

manner with time at low temperature. Early rebound followed

by stabilization at an intermediate level, indicating in-

complete recovery, characterized electrophysiological

function upon return to more normal temperatures whereas

outer segment to ellipsoid ratios showed gradual total

recovery. Animals recovering in the light absent condition

failed to show electrophysiological recovery, as did one

group recovering in blue light. Complex interactions

involving receptor and neural function, light versus light

jbcrint crndi t.ion and photopic and scotopic adaptation were

viii







cern. 'o reliable effects ;.:ere eeri as a result o. spectral

differenr.z- in rn.'i rc.riT.ntal liightino e::*:cClt for th. failure

of one group, noted above, to show recovery. Analysis of

food catching behavior suggested that functional visual

deficits accompanied electrophysiological and structural

losses incurred during cold stress, while partial restora-

tion accompanied recovery of electrophysiological function

and structure. No morphological loss was seen other than

cone outer segments, however, due to difficulty in resolution

of these very fine structures with available equipment, no

attempt was made to evaluate loss of rod receptors or their

outer segments.

It was concluded that cold stress effects were orderly

over time with respect to both electrophysiological function

and receptor structure, at least for cones. Recovery was

generally less orderly and was essentially complete for

outer segments but not for electrophysiological function.

Photopic and scotopic systems were differentially affected,

as were receptor and neural function. These relationships

were modified with differential environmental lighting

conditions. Results were discussed with emphasis on lines

for future research related to elucidating anomalous results

within the present experiment, disparate results with respect

to previous research into the phenomenon and possible bio-

chemical mechanisms underlying the effect of cold stress on

the structure and function of the retinal cone receptors of

the goldfish.












INTRODUCTION

One of the traditional techniques employed in the

study of vision has been the utilization of abnormalities.

Beginning with Dalton's careful study of his own color

deficiency (Helmholtz, 1924, pp.145-147), much of our know-

ledge of the function of the visual system in response to

light of limited wavelengths has been deduced from the study

of abnormal human subjects.

Even with the advent of the very powerful tools

which are now available, allowing the vision researcher to

work at the subcellular and molecular level, the use of

these techniques has frequently been employed in conjunction

with system abnormalities. Dowling's description of retinal

atrophy resulting from Vitamin A deficiency and inherited

retinal dystrophy (Dowling, 1964) has expanded our understand-

ing of the photochemistry of vision. Similarly, research on

retinal damage by visible light (Noell et al., 1966; Gorn

and Kuwabara, 1967; Kuwabara and Gorn, 1968; Grignolo et al.,

1969) has contributed to our concepts of retinal viability

and limitations on its stimulation. Investigations of

surgically induced retinal detachment and reattachment have

contributed to the same end, have aided in understanding

the etiology of this malfunction frequently seen in the

clinic and have contributed to the understanding of result-

ing visual loss (Kroll and Machemer, 1968, 1969a, 1969b;








ia .cn7irc-, 1968a 19C.'Lb; ;:acIi.m.;r arid .:-,rton, 196.2 ; ii.arnasar. I

et al., 1969).

The research to be reported here takes as a point of

departure a recently reported phenomenon (Dawson et al., 1969;

Hope et al., 1970; Dawson et al., 1971) which results in de-

generation of the outer segments of the retinal cone re-

ceptors of the goldfish (Carassius auratus). Since these

reports are not readily available to all readers, this re-

search will be treated in some detail, following a brief

description of the structures involved. Reference to Figure

1 may aid this description.

The retinal receptors are composed of an outer seg-

ment, containing the photopigment upon which luminous energy

acts to initiate the visual response, an inner segment, and

a cell body. The cell bodies form the outer nuclear layer

and the inner and outer segments the receptor layer of the

retina. The pigment epithelium lies sclerally to the re-

ceptors. In some species, including the goldfish, processes

of the pigment cells can migrate into and out of the receptor

cell layer in response to light level, interdigitating with

and screening receptors (John et al., 1967). The receptor

cells also migrate in response to light, as well as demon-

strating diurnal rhythms (John and Kaminester, 1969; John et

al., 1967). In general, the cones and pigment epithelium

move sclerally in dark adaptation with the rods moving

vitreally. The reverse occurs in the light adapted animal.

These movements have also been shown to occur at night and
















VITREAL


Pigment
- Cells


Piqmer t
Cell
Procec co
Cone
Outer
Segment-
Cone
Inner
Segmernt
Outer
Limit Lr, __
Membr an
Recer tor
Cell
Nuclei


Gangl iq.r, c .r
Cell
Gangq i.r
Cell
Axons


FRod


Ccne
El lipsoid

-Cone
_ I' oid
rOuter
nuclear
L- 'er
'utrer
r 1.xiforn
I a-er



Ir ner
Sjuclear
L.:,er



Ir,ner
P Flexiform



,.",-nqlion
SCell
La 'er


SCLFRAL

Figure 1.-- Anatomy of the Retina







adj.t-iri,. r p_ ct ip 'l%, t" 1 r cc.-n t nt dark:rn es (Jchn et al.,

1967; John n=irJ 1a. ire- t.r 1969).

The inner nuclear layer lies vitreally to the outer

nuclear layer, separated from it by the outer plexiform layer

(the region of synaptic contact between the receptors and

cells of the inner nuclear layer). The cellular composition

of the inner nuclear layer includes the horizontal, bipolar

and amacrine cells in a scleral to vitreal arrangement. The

inner nuclear layer is separated from a very sparsely pop-

ulated ganglion cell layer by the inner plexiform layer.

The synaptic relationships among the various cellular com-

ponents have been studied and described in detail (e.g.,

Dowling, 1970) but will not be discussed here.

The research providing the incentive for the present

study showed that cold stress resulted in atrophy of cone

outer segments in the goldfish. The animals were placed in

an aquarium and refrigerant was pumped through a coil sub-

merged in the aquarium water. The temperature of the water

was lowered to 50 C and held at this level for up to 31 days.

The water was then allowed to return to room temperature,

220 C, and the animals were maintained at this level for

the remainder of the experiment. During these periods, cold

stress and recovery respectively, fish were periodically

removed from the aquarium and electrophysiological signals

in response to a light flash were recorded from the cornea

and exposed contralateral tectum. These signals were re-

ferred to as the electroretinogram (ERG) and tectal evoked









response (TER) respectively. Following dark adaptation the

eyes were removed and subjected to histological processing.

Electrical responses were not recordable at the

cornea as early as four days into cold stress. The TER was

recordable but grossly abnormal. Throughout the remainder

of cold stress, no signals could be recorded in response

to light stimulation. During recovery, all signals showed

unimpressive recovery to, circa, 20% normal amplitude.

Examination and counts of outer segments and inner segments

indicated that there was significant loss of cone outer

segments, abnormalities in remaining outer segments and loss

of cone ellipsoids implying loss of cone cells. During

recovery these effects were reversed, but normal complements

of complete outer segments and cone cells were not regained

during 52 days of recovery.

Similar results have been seen in several other

studies using different species and treatments. One of these

was the experimentally induced pathological state known

clinically as the detached retina. Recently, a group com-

posed of Machemer, Norton, Kroll and Hamasaki has reported

a series of light microscopic, electron microscopic and

electrophysiological experiments on retinae treated in this

manner. The species employed were owl monkeys (Machemer

and Norton, 1968; Machemer, 1968a, b; Kroll and Machemer,

1968, 1969a; Hamasaki et al., 1969) and rhesus monkeys

(Kroll and Machemer, 1969b). Light microscopic studies of

these retinae indicated that the outer segments of the







receptor cells dis appr. red and tier- ve re gross kabarmalitiies

of the retina in general (Machemer, 1968a). Electron micro-

scopy revealed that a sequence of events followed detachment,

culminating in the loss of the outer segments with relatively

little change in the cellular components of the remainder of

the retina. The sequence of events included loss of orienta-

tion of the outer segment lamellae, fragmentation of the

outer segments, atrophy and phagocytosis of fragments of

outer segments (Kroll and Machemer, 1969a). In general,

surgical reattachment of the retina produced a reversal of

these effects and the rod outer segments soon recovered their

normal morphology (Machemer, 1968b) but the cones showed

much slower recovery (Kroll and Machemer, 1969b). In this

latter study Kroll and Machemer (1969b) report some alter-

ation of the inner segments, most notable a paucity of

mitrochondria.

Hamasaki et al. (1969) traced the electrical

responsivity of these retinae following detachment and

reattachment. The results were as one would expect, with

the electroretinogram (ERG) being totally absent two weeks

after detachment surgery if detachment was total. Small areas

of attached retina were capable of supporting an ERG. With

reattachment, progressive recovery of the ERG was seen,

beginning at about 3 days after surgery with a minimal response

as early as 17 hours. The eyes were tested for up to 12 weeks,

with slight improvement being shown over the last 4 weeks.

There is no indication as to whether or not the 12-week, or

final, records were normal.








Another means of producing retinal degeneration

which has been investigated in the last several years has

been stimulation by low intensity light. This technique

did or did not produce permanent damage and blindness,

depending on the levels and durations of light used. A

major factor contributing to the severity and permanency of

the damage was body temperature, with slightly supranormal

temperatures enhancing the damaging effect of light dramatic-

ally (Noell et al., 1966). The methodology typically in-

volved subjecting a free ranging animal (rat) to constant

illumination by small fluorescent tubes for varying durations

while varying temperatures. The resulting damage was eval-

uated electrophysiologically or electron microscopically.

The results of the electron microscopic examinations

(Kuwabara and Gorn, 1968; Grignolo et al., 1969) of the

retinae of the subjects indicated that damage ranged con-

tinuously from simple loss of the distal one-third of the

rod outer segments to total loss of the receptor cells. In

the former case, regeneration occurred while in the latter

the effect was irreversible. The fine structural description

was very similar to that given by other authors following

retinal detachment (e.g., Kroll and Machemer, 1968, 1969b).

The electrophysiological (ERG) (Noell et al., 1966; Gorn

4nd Kuwabara, 1967) measures followed the electron micro-

scopic observations very well. The ERG was diminished or

eradicated as one would expect from the morphology.








Tntere,.,tirngl y, these studi.e (i'ol c.l t al., 1966; C ,orn .and

Fu- barau 196' 7 a7 1 ,1 em'->lo ,'d m ncroch' -ro ma t c. st ir'ilati.:n and

found d iffe trintial effect-, rlc .e ndingi cn the blc.ich ing

po',er : f the '.-a, lennoth- emplc, io d.

C'.. 1 inrq (1964i hi.js de sc ribed the. fine structuL ~I

detail o:f retir.al tc r d dJc,- r',- at ion urnde r '.'it irin .A defriciency:

and inherited retinal dystrophy in rats. The latter is

irreversible, whereas the former may or may not be, depending

on the duration of the deficiency. The degeneration proceeded

from slight disorientation of outer segment lamellae, to

breaking up of lamellar material, to assumption of a spherical

shape by the outer segment. With longer durations of the

vitamin deficiency, the inner segments and cell bodies

degenerated also. If the deficiency was corrected prior to

degeneration of the inner segments, the outer segments

regenerated. This process was characterized by the formation

of a cilium on the inner segment terminating in a small bulb

of vesicular material. This material expanded, taking on a

lamellar appearancepand the lamellae assumed a normal

orientation. The structure then elongated and increased

to normal size (Dowling, 1964).

Another area of research applicable to receptor

degeneration and regeneration is that dealing with normal

renewal of rod outer segments. In general, this work has

/ shown that labeled amino acids were incorporated into proteins

which formed a disc at the base of the rod outer scrgmnt.

This disc was displaced sclerally, by the formation of new









discs, until it was expelled at the peripheral end of the

outer segment (Droz, 1963; Young, 1965, 1966, 1967, 1969;

Herron et al., 1969, 1971) and was phagocytized by the

pigment epithelium (Herron et al., 1969, 1971). The labeled

protein has been identified as rhodopsin, the photopigment

of the rod receptors (Hall et al., 1968; Bargoot et al.,

.1969). It has also been shown that rate of displacement

was more rapid in light than dark adapted conditions and

when ambient temperature was increased (Young, 1967). Young

(1969) has recently shown that renewal as seen in the rods

did not occur in cones at least for the frog. While

labeled protein was found in cone outer segments, it was

diffusely located and did not progress along the outer

segment as would be expected if renewal were occurring.

The general aim of the research reported here was

to elaborate on the effects of cold stress on the retina of

goldfish as well as to extend these results in several

directions suggested by the research reviewed above. The

initial exploratory work into this effect (Dawson et al.,

1969; Hope et al., 1970; Dawson et al., 1971) suggested

the need for revising the temporal patterning of sampling

of experimental animals during the induction of cold stress.

The first fish recorded in those efforts failed to give

electrical signals at the cornea and presented a grossly

abnormal TER. Similarly, the earliest animals recorded

during the recovery phase of those experiments produced

electrical signals of small amplitude. In general, during








the 1:ol' 3 Zd tre s phr' r .on l-. .*:'n.-: :it i:l 'I i s Iripled drluii inq the.3

first two weeks. The first animal sampled during recovery

was on day 11. Therefore, it seemed desirable to investigate

the early portions of each of these phases more carefully,

sampling animals at shorter intervals. It was hoped that

this would enable evaluation of the time course of electro-

physiological decay and morphological degeneration.

In the initial studies of this phenomenon, no effort

was made to extend the morphological effects beyond the

relationships and numbers of ellipsoids and outer segments.

The loss of cone cells noted above was assumed on the basis

of losses of ellipsoids. While none of the techniques

described above resulted in degeneration beyond the receptor

cell, it seemed desirable to investigate the possibility of

cellular loss in the inner and outer nuclear layers and

ganglion cell layer under cold stress. Since low temperature

in a poikilotherm is known to lower the metabolism generally

(Prosser, 1950), it was expected that the effects of cold

stress may not be limited to the receptor cells.

Brown (1968) has presented compelling data from a

number of experiments demonstrating that the A wave of the

ERG has its origin in the receptor cell layer of the retina,

whereas the B wave originates in the inner nuclear layer.

The cells in the inner nuclear layer have been shown to be

responsible for much of the retinal processing of visual

information (Werblin and Dowling, 1969; Dowling, 1970).

Thus, it seemed reasonable to assume that separate analyses









of these components of the ERG would differentiate between

receptor function and neural processing. This defined a

functional approach to the question of effects beyond the

receptors.

Stimulation parameters for eliciting electrical

activity of the retina in the preliminary experiments on the

-effects of cold stress were chosen such that they optimally

stimulated cones. Additionally, because of their small size

and relatively poor staining characteristics with the

techniques employed, the rods were difficult to resolve in

histological specimens. While it was possible to make

general observations concerning the depletion of these

structures, no concentrated effort was made to detect

degeneration or loss of function of rods. Since some loss

of the lightly stained rod material normally seen between

the cone ellipsoids was observed in the preliminary experi-

ments, stimulation parameters in the present experiment were

manipulated in an effort to differentiate photopic and

scotopic electrophysiological responses.

The manipulations consisted of two general types,

variations in adaptation level during recordings and use of

closely paired flashes of light as stimuli. The former is

generally accepted as a means of separating function, with

high (photopic) ambient light levels resulting in activity

due primarily to cone function and low (scotopic) levels

resulting in activity reflecting primarily rod function

(cf. e.g., Bartlett, 1965; Riggs, 1965; LeGrand, 1968, p.260).








ir. q-ener al, :hile tli.e ~ seiarat ons art. prot.-bl not ..holly

dichotomous, tih r.-lii. e contribitio s ;if the t.o it.-trms

under these varied adaptation states can give useful infor-

mation about their integrity.

The second type of manipulation, the use of paired

flashes as stimuli, was incorporated as a result of a recent

publication showing that, under scotopic conditions and with

appropriate separation of the members of the pair, the

response to the first flash represents activity of both rods

and cones while that to the second flash stems only from

cones (Elenius, 1969). The author demonstrated rationally,

utilizing previously published data, that the effect was

due to active inhibition of the rods by one of the photo-

products of the rod photopigment, rhodopsin, as it breaks

down in response to light. Unpublished data taken informal-

ly by the present experimenter from the dark adapted cat

supported Elenius' contentions regarding dissociation of

photopic and scotopic function. It was felt that, in spite

of the somewhat preliminary nature of the data purporting

to establish the double flash technique, its inclusion

in the present experiment would improve the probability

of observing differences in loss or rate of loss of cone

and rod function, with no a prior indication that inclusion

would compromise other dependent variables.

Casual observations in previous work dealing with

the cold stress phenomenon suggested that animals in cold

stress and early recovery did not exhibit visually guided









feeding behavior of the sort seen in the normal fish, and

that there may have been differences in cone outer segment

degeneration between peripheral receptors and those in the

central portion of these retinae. The present experiment

attempted to quantify visually guided feeding behavior in

the goldfish and to compare this behavior in the normal,

cold stressed and recovering animal. Additionally, it was

hoped that quantification of cellular structures in peripheral

and central portions of the retina might allow verification

of possible differences in the effect of cold stress on

these two areas.

In the research (reviewed above) using other tech-

niques which have resulted in retinal degeneration, all have

one factor in common which stands in contrast to cold stress

induced degeneration of outer segments. All of these

techniques involved factors which can be thought of, at least

in a general sense, as acting upon the outer segment from

outside the cell. Vitamin A deficiency produced retinal

degeneration by virtue of interrupting the rhodopsin

regeneration cycle (by denying the outer segment this

necessary constituent) (Dowling, 1964). Vitamin A is

furnished to the receptors by the pigment epithelium.

Retinal detachment, the physical separation of the retina

from the pigment epithelium, may have produced degeneration

in the same manner as did Vitamin A deficiency, since the

receptors were removed from their source of Vitamin A.

Inherited retinal dystrophy in the rat is now-known to








result front th, I in9biiit' of the. piLtier, t F.it helii to

phagocytirz. r:..J outer sner-,nt rr-jteri i 'hi.,lh is ,:.ttu.j. by

the receptors (Herron et al., 1969, 1971). Finally, light

induced retinal degeneration obviously resulted from the

impinging of energy on the receptor. Thus, the precipitating

factor in all these cases appeared to be imposed on the

receptor from outside its encompassing membrane. On the

other hand, low temperatures are known to inhibit general

metabolism in the poikilotherm (Prosser, 1950). Therefore

it might be argued that outer segment degeneration induced

by cold stress may be the result of the inability of the

receptor cell to furnish the outer segment with metabolic

products necessary for its maintainence. Under this assump-

tion, one might expect that light stimulation would perhaps

interact with cold stress to produce more severe degeneration,

since light stimulation would be expected to force the

receptor to function, exhausting stores of available nutrients.

In this respect, inherited retinal dystrophy in the rat has

been shown to progress more slowly in the dark (Dowling,

1964) and protein renewal proceeded more rapidly at constant

high light levels in Young's (1967) rats. In order to test

the hypothesis that light stimulation would interact with

cold stress, thus increasing loss or rate of loss of

electrophysiological function, varying conditions of light

stimulation were imposed on the animals during cold stress

and recovery.

Light stimulation was varied along two dimensions.








Variations in intensity of white light were expected to

evoke quantitative differences in the dependent variables

observed, while differences in wavelength composition of

stimulating light were expected to result in qualitative

effects on the morphology and/or electrical function of the

retina. This latter point rested on the ability to differ-

entiate, at least to some degree, between (relatively)

stimulated and unstimulated cones. A possible means of

accomplishing this was provided by the observation (Glicstein

et al., 1969) that selective migration of cones follows

adaptation to long wavelength light. Thus, it was hoped

that long wavelength stimulation during cold stress might

result in relatively greater degeneration of migrated cones

versus unmigrated cones and stimulation by short wavelength

light during cold stress might produce just the reverse

effect. It was also hoped that differences in sensitivity

to stimulation by monochromatic light might be detected

electrophysiologically.

In order to attack the questions posed above it was

necessary to subject goldfish to low temperature for an

extended period of time then allow them to recover under

more temperate conditions while subjecting different groups

to various forms and intensities of ambient light. During

these phases of the experiment, animals were sampled at

specified intervals in order to obtain electrophysiological

and histological data. The following section describes the

equipment used and the procedures followed in accomplishing

this end.












METHOD

Procedure

The general procedure was to adapt goldfish to low

temperature by progressive lowering to a nominal value of

50 C. Reaching 50 C identified day 1 of the experiment.

Initial temperature of the water in the tanks was 220 C.

The temperature was reduced from this level to 12-150 over

a span of three days. The temperature was then held between

12 and 150 for a period of three weeks. At this point the

temperature was further reduced to 50 C. The temperature

was then held at this level for different periods of time,

depending upon the general category of environmental light

stimulation to which the animals were assigned. The tem-

perature of the water in the tanks was then allowed to

return to normal room level (20-220 C) overnight and was

held constant for the remainder of the experiment. These

two conditions represent the cold stress and recovery phases

of the experiment respectively. During each of these two

phases animals were selected randomly according to a

predetermined temporal schedule for electrophysiological

recording and histological processing of retinae. The

remainder of this section will describe in detail the

equipment, techniques and procedures involved in each

aspect of this general procedure.









Subjects

Subjects were common goldfish, Carassius auratus.

A total of 159 fish was used in this research. Of these,

ten served as normal controls for the various treatments,

seven as controls receiving treatments other than those in

the basic study and the remainder were distributed among

the various groups defined below.


Environmental Control

All fish were housed in 60-quart styrofoam picnic

coolers. Each cooler (a total of six) was equipped with a

water filter and aerator. These tanks and related equipment

were placed in a "walk-in" cooler which was capable of

maintaining the air temperature at approximately 8 (+1)0 C

indefinitely.

Temperature was controlled by simply adjusting the

thermostat regulating the compressor of the cold room in

which the aquaria were housed. This was adequate for tem-

peratures above 80 C. Below this level, the water in the

tanks was chilled by the addition of frozen, hermetically

sealed, cans of an "anti-freeze" solution. These were placed

in the tanks once or twice daily, as needed, to maintain

water temperature between 4 and 80 C.

Four of the six tanks were rendered opaque by painting

all outer surfaces with several coats of flat black paint.

All air and filter tubing entered these tanks through heavy

opaque plastic elbows. All transparent tubing and elbows









were covere-d lt'I 3l U iri,., f:oil to. frth.r Lr-.'jr. tilit

extraneous light was excluded. Three of these masked tanks

were fitted with sandwiched filters to admit only light of

specifiable wavelength bands in equal amounts. The tops of

these four tanks were then covered with an additional layer

of foil and fitted with a foil skirt extending well past the

junction of the cover and walls. In general, this procedure

insured that one of these four tanks was light-tight and that

only light passing through appropriate color filters was

admitted to the other three. The remaining two tanks were

untreated, with the exception of removing their covers and

draping them with transparent polyethelene film to retard

evaporation and contain the fish.

Light filters, fitted to the three tanks as described

above, admitted light in either the red or blue portions of

the spectrum. The red filter (Kodak t;ratten #92), and

associated glassware, passed energy in the band from 635

nanometers (nm) to 780 nm. Maximum transmission was 34% at

660 nm and the vast majority of the total transmitted light

was of wavelengths longer than 640 nm since the transmission

curve (Figure 2) showed a pronounced skew toward the longer

wavelengths. The blue filter (Kodak Wratten #49B) passed

light having wavelengths between 380 and 500 nm (Figure 2).

Peak transmission was 28% at 438 nm and the bulk of the

light was of wavelengths below 460 nm. The transmission

curves presented in Figure 2 arc those for the filters

together with the glass between which they were sandwiched













w
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Cl G


19






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,ii a heat absorb:ing iil:,_ (i'.:. '-I1 intr; .L -e.d bet..:-on th.

light source and the sandwich. As such, they indicate the

wavelengths actually admitted to the tanks as measured

spectrophotometrically.

Fish in one tank received light through the blue

filter, those in the second through the red and those in

the third through one of each. As will be described in more

detail below these subjects were compared in the analysis of

the data, thus it was necessary to equate the light in the

three tanks. In order to accomplish this, the transmission

characteristics of the filters, and associated glassware,

were measured spectrophotometrically at wavelengths between

300 and 800 nm. The percentage of energy transmitted at

10 nm intervals over this range was multiplied by the

relative energy output of a 30000 K source (Wyszecki and

Stiles, 1967, p.21) at each of these steps and this product

summed over the visible spectrum. Since the blue filter

passed less energy than the red and the energy output of

the lamp was lower at the blue end of the spectrum, a 0.70 log

unit neutral density filter (Kodak #96) was added to the

red filter sandwich to equate the two. In addition, 0.3 log

unit neutral density filters (50% reduction in transmission)

were added to the two sandwiches placed in the tank receiving

light of both wavelength bands in order to equalize the

energy in this tank to the others.

Illumination for the filters was provided by a

simple optical system consisting of a 500 watt tungsten -








Halogen lamp (Sylvania 500 QCL), an aspheric condensing

lens and a heat absorbing filter. These lamps have an ener-

gy distribution approximated by a blackbody radiator at 30000

K when operated at 120 volts. The system provided a diverg-

ing image Cdefocused) of the lamp filament which was confined

to the open area of the filter sandwich and filled it. The

lighting system was powered by a voltage regulator set at

120 volts, the output of which was passed through a timing

circuit then to the lamps. The system was operated on a

30 min. on, 60 min. off cycle to avoid the temperature rise

which resulted from more prolonged operation of the 2000

watts lighting system. The lamps and optical system were

enclosed in a metal hood, the open bottom of which was

masked except for exit ports for the light, and which was

exhausted to the outside.

Illumination levels in the aquaria were determined

from direct measures of the luminance of a surface placed

in the water in each tank. The surface was chosen for high

diffuse reflectance and impregnability to water. Nondirec-

tionality and high reflectance were required in order to make

the necessary conversion from luminance to illuminance

(LeGrand, 1968, pp. 202-203). Luminance was measured with

an SEI Photometer at the approximate center of the open

tanks at the surface and floor in the water. The cover was

removed from one of the three tanks receiving light through

the colored filters and measures were made at the surface

and floor directly below the source and at the most remote









point in the li. aq. u;iiL .

Light received at the photometer from the reflecting

surface placed at the floor of the aquaria necessarily

passed through the water twice. Turbidity was unavoidable,

therefore the water acted as a filter and the measured

luminance was not the true value. An approximation to the

'true value was calculated to be the luminance measured at

the water's surface less the difference between this

measure and that at the floor. The mean of the surface

and actual floor values was then computed and this value

converted to illumination incident to an object placed in

the center of the tank.

This value was valid as described for the open a-

quaria but it was necessary to correct in accordance with

the limitations imposed by the filters in the various

spectral tanks. This correction required that the luminance

measure be distributed across the spectrum in accordance

with the relative spectral sensitivity of the observer.

Since the light levels, without the interposed filters, were

quite high (2.05 3.80 log foot lamberts), tables for rel-

ative photopic sensitivity (Wyszecki and Stiles, 1967, p.

378) allowed an evaluation of the percentage of the measured

luminance contributed by light from portions of the spectrum

at ten millimicron intervals. Multiplying these values by

the percentage transmitted at that interval on the spectrum

and summing the products gave the percent of the measured








luminance passing the filter. This value was calculated for

the mean of surface and floor measures in the remote position

and directly under the source and converted to illumination.

Average illumination in the open tanks was 1.9 lumens

/ sq. ft.; in the spectral tanks receiving red and blue light

without filters interposed it was 2136.8 lumens / sq. ft. and

that for the other two spectral tanks. Calculated values

for illumination in the spectral tanks with the filters in

place were 1.9 lumens / sq. ft. for the tank receiving red

light, 0.9 for the one receiving blue light and 1.4 for the

one receiving both. The illumination values were valid for

relative white light levels but the filtered light levels

were valid only for the human observer and represent only

gross estimates of the relative stimulating efficiency of

these illuminations on the goldfish.


Sampling

Animals were randomly assigned to the aquaria

receiving the various environmental lighting conditions.

Twenty-two animals were assigned to each of the tanks which

received colored light, 20 to the tank which received no

light and 56 to the two tanks receiving ambient white light.

Animals which received white light were designated group L,

those maintained in darkness group D and those receiving

light through the red, blue, and red and blue filters group

R, group B and group RB, respectively.

Electrophysiological recordings were taken from the








,*7. :i*n:.e of at !e s;. t :,n, ,qr'ijp L inim-i .-a.-h d.-ia tLihr j'-h lda

16 of cold stress. Thereafter, during cold stress, animals

from this group were sampled at four-day intervals. Histo-

logical specimens were collected from group L at four-day

intervals throughout cold stress. Group D was sampled at

each alternate sampling time for group L, that is on

alternate days through day 16 and at eight-day intervals

thereafter. Each of the three spectral groups was sampled

at seven-day intervals.

During recovery, groups D,R, B and RB were sampled

electrophysiologyy and histology) at seven-day intervals.

Group L was sampled on days 4, 8, 12, 16, 32 and 62 of

recovery. The duration of the cold stress stage was 44

days for groups L and D. The recovery phase lasted for 62

days. An additional 14 days of cold stress were given to

four animals from group L, with two of these being sampled

at the end of this period, day 59, and the remaining two

were allowed to recover for the remainder of the experiment

and were sampled on day 106. These four animals comprised

group E.

On day 45 the cover was removed from the tank housing

group RB, exposing them to the full 2136.8 lumens / sq. ft.

from the unattenuated sources in order to establish a zero

baseline for observation of recovery under spectral lighting.

This group and groups R and B were then given an additional

14 days of cold stress. Electroretinograins were recorded

from four group RB animals on day 45 and again on day 59.









At this point the animals from group RB were marked with

suture clips clamped on the dorsal fin, distributed evenly

between the tanks receiving red and blue light and allowed

to recover under these conditions with groups R and B.

Thereafter, these animals were referred to as groups RBR

and RBB respectively. Fish from groups RBR and RBB were

sampled at seven-.day intervals at the same time as groups R

and B during recovery, except for days 92 and 99 when fish

in groups RBR and RBB were almost exhausted and one fish

from each group was saved until day 106. Figure 3 presents

a graphic presentation of the sequence of events described

above.


Electrophysiological Measures

The electrophysiological responses recorded from

the goldfish were the electroretinogram (ERG) and the response

of the contralateral optic tectum (TER). The ERG was recorded

from the cornea by a circular stainless steel electrode,

fitted to the cornea and masked and insulated except where

direct contact with tissue occurred. The reference electrode

was clipped to the external naris and the signals were fed

directly into the inputs of two sequential Tektronix Model

122 differential AC amplifiers having a pass band set at 0.2

to 10,000 Hz. The TER was recorded and amplified in a

similar manner except that the electrode was a tungsten

shaft insulated except at the 15 to 20 micron tip.

These signals were led directly, and through Krohn-















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Hite Model 330A and 330 NR electronic filters set to exclude

frequencies below 50 or 30 Hz and above 500 Hz, to the four

channels of a Fabritek 1052 signal averaging computer. The

unfiltered ERG and TER were converted to frequency modulated

representation then recorded, with appropriate triggering

signals (unconverted), on a Roberts 770 tape recorder for

further analysis off line. In addition, the unfiltered

ERG and TER were displayed on a Tektronix dual beam oscillo-

scope for monitoring. Total gains at the inputs of the

average and oscilloscope were 10,800 for the ERG and 13,200

for the TER.

The raw ERG was passed through one of the filters

(50 500 Hz pass band) and the raw TER through the other

(30 500 Hz pass band). The signals resulting were the

fast retinal potential (FRP) and fast tectal potential (FTP).

Gains for these signals were as for the ERG and TER respectively.

During recording, goldfish were held in a holder

which allowed perfusion of water through the mouth then out

the gills and along the ventral body. Anesthetics were not

generally necessary for recording but small amounts of a

neuromuscular blocking agent (Flaxedil) were administered

by injection if required. All recordings were taken with

subjects at controlled room temperature (20 220 C). The

fish were kept moist at all times. Fish have been maintained

in this apparatus for as long as 10 hours with no detectable

ill effects.

Stimuli eliciting electrical activity were provided








by an especially c n.:. I rjlct,. [:,':,:,.-ti,.ul, Lt r .ti living

xenon arc sout'r- n. .pt.ic tc'p thitr th' -: iiimlui.i '-

presented in Maxwellian view (Westhiemer, 1966). Intensity

was variable over a wide range through internal filters. A

variable intensity adapting light was available as part of

the system. Wavelength of both the stimulus and adaptation

field could be varied by addition of monochromatic filters

(Baird-Atomic).

Stimulating conditions for eliciting electrical

activity were tailored to each individual group. For animals

in Group L the stimuli and electrophysiological recordings

were intended to enable the investigator to look at electrical

responses at photopic or high light levels, scotopic or low

light levels, and mesopic or intermediate light levels. It

is generally accepted that at high photopic light levels the

majority of the electrical activity is a result of activity

in cone receptors, whereas at scotopic levels the activity

is primarily the result of activation of rods. The mesopic

level is believed to represent the intermediate case where

the activity is mixed, that is, results from stimulation of

both rods and cones. The photopic adaptation level was

achieved by raising the ambient light level to 1.2 log foot

lamberts measured on a tangent screen placed 10 cm in front

of the animal's eye and, in some cases, adding the secondary

adaptation light. Sixty-four stimuli were delivered from

the stimulating device described above with each discrete

flash delivering 0.7 ergs, retinal irradiance uncorrected







for absorption of media of the eye (LeGrand, 1968, pp 87-93)

or the Stiles-Crawford effect (Stiles and Crawford, 1933).

Each stimulus was composed of two flashes separated by 250

milliseconds. Stimuli were delivered at a rate of one pair

per second at intensity setting W4 on the photostimulator.

The mesopic condition was achieved by lowering ambient light

level to 0.1 log foot lamberts measured on a tangent screen

10 cm from the eye. Thirty-two stimuli were delivered,

with each consisting of two flashes as described above,

delivered at a rate of one pair per three seconds. Scotopic

conditions were obtained by turning off all light and allow-

ing the animal to dark adapt for 20 minutes. During the

process of dark adaptation the stimulator was operating,

delivering one stimulus, a pair of flashes, every 15 seconds.

Following dark adaptation, sixteen stimuli (flash pairs)

were delivered at one per 15 seconds. Electrical activity

evoked by the pairs of flashes were averaged, as described

above, for each condition. Beginning on day 10 the process

of dark adaptation was also monitored. This was accomplished

by averaging responses to the four stimuli delivered during

alternate, odd-numbered, minutes. That is, responses to the

four stimuli delivered during minute one were recorded and

printed out and so on for alternate minutes through minute

19. On the 20th minute the standard dark adapted series

described above was initiated. Thus, for animals in group

L, primary data consisted of an averaged ERG tracing

representing 64 signals under low photopic conditions







(beginning -:n .la' 1.1) o:ne reirL- ec n n third t'.'--ti ign als

under mesopic 2. :n'ii L Ln one r ee--ir nI nt] ng it t;:: n signrii1

under scotopic conditions, and a series of averages of four

responses, each representing odd numbered minutes during dark

adaptation (from day 10).

Stimuli delivered to animals in the spectral groups

differed from those for group L. Stimuli for these animals

were from the same stimulus source but at a higher intensity

(setting W16) and passed through monochromatic filters (Baird-

Atomic) which had nominal peaks at 430 nm, 550 nm, and 630

nm. These filters were selected in order to optimally stim-

ulate discrete cone populations. That is, the filter having

a peak transmission at 430 nm stimulated cones having maximum

sensitivities in the short wavelength end of the spectrum,

the 550 nm filter stimulated cones having peak sensitivity

in the middle of the spectrum as well as rods, and the 630

filter stimulated those cones having peak sensitivity in the

long wavelength end of the spectrum. These stimulating wave-

lengths were chosen on the basis of published absorption

spectra for goldfish single cones (Marks, 1965; Liebman and

Entine, 1964), and while the filters chosen quite probably

did not selectively stimulate a single population, they very

probably did result in a relative imbalance which tended to

favor one population over another. To a human observer, the

430 nm filter appeared blue-violet,the 550 green and the 630

filter orange-red. The blue filter had a peak transmission

at 430 nm of 73% with an 8 nm band width at 50% transmission,









the 630 or red filter 80% at 630, 4 nm at 50% transmission

and the green filter 70.5% at 747 nm with a 6 nm band width

at 50% transmission. For stimulation through these filters

the stimulating device was operated at setting W16, represent-

ing maximum intensity for this source, and at this level

delivered 6.9 ergs, retinal irradiance (uncorrected, as noted

above) under these conditions without the filters interposed.

Since this energy is spread across the visible spectrum,

and since the filters employed pass only narrow portions of

the spectrum it was necessary to determine the percentage

of energy within those bands passed by each of the filters

and then, through the use of neutral density filters, to

equate each of the spectral filters in a manner such that

they passed the same amounts of energy. The absolute energy

levels delivered to the eye with the filters interposed

were not determined. However, using ERG amplitude as a

criterion,the efficiency of this system with only the

monochromatic filters interposed was approximately equivalent

to the stimulus setting W4 without these filters. Since

this was the condition under which the stimulator was

operated for group L animals, the data for spectral group

animals were comparable to that for animals in group L

when the highest intensity settings on the neutral density

filters were used for the former. The major factorhowever,

was that the stimuli could be corrected so as to deliver

equal energy at the three wavelengths being studied.

All stimuli for animals from the spectral groups









were delivered h 1iil- tLh.: irim, l '.in in it:I, -J ad.:i..ted

state. Four responses were averaged at each intensity

setting for each of the monochromatic filters. Initial

intensity settings were the maximum obtainable from the

stimulating system and were reduced in 0.3 log unit steps.

Amplitude-by-intensity curves so generated take the general

form of an ogive and, in some cases, slightly larger in-

tensity decrements were used during the rapidly falling

portion of these curves. Stimulus intensity was reduced

until the ERG reached a lower asymptote or became

indiscernible.

Goldfish which were deprived of light during cold

stress (group D) were run under dark adapted conditions.

Stimuli were paired flashes of white light (setting W4)

with each pair delivered at a rate of one per 15 seconds.

Responses to 16 of the stimuli were averaged.

All animals were transported to the recording

chamber and prepared for recording in light appropriate to

their environmental stimulation condition, with two exceptions.

Animals in the dark group were prepared in dim red light.

Animals from the group receiving blue light in cold stress

were prepared in dim blue light except for a brief period

during which surgery required for tectal exposure was

conducted. It was extremely difficult to perform the tectal

exposure in dim blue light, since very little light at the

shorter wavelengths was reflected from the orange fish.

Tectal exposure was accomplished by cutting through








the cartilaginous tissue overlying the tectum. A triangular

section of this tissue was removed exposing the heavy fat

deposit lying below. This deposit was removed by aspiration,

exposing the very prominent tectum. The tectal electrode

was lowered into the approximate center of the dorsal sur-

face of the tectum while stimuli appropriate to the record-

ing conditions for animals from each group were being

delivered. Responses were monitored on an oscilloscope.

The tectal electrode was advanced past the point at which

the maximum signal amplitude was obtained. Then by a

process of titration the electrode was placed at the point

of maximum signal. Upon completion of the collection of

electrophysiological data the animals were enucleated and

the eyes processed histologically.


Histology

Eyes were fixed in 4% gluteraldehyde carried in a

physiological solution (Ringers). All eyes were fixed for

a minimum of 12 hours. During the first 3 to 6 hours of

fixation the tissue was shielded from light. The tissue

was then washed, dehydrated in alcohol, and imbedded in

paraffin (Paraplast). Eyes were sectioned at 4 to 6 micra

on a rotary microtome (Americal Optical). Eyes were care-

fully oriented in the block during imbedding and the block

carefully oriented on the microtome prior to cutting such

that sections passing through the optic papilla also passed

through the central retina. Thus, sections extended from




21



ti.-- inferior n:..ni i.hl l L:1C .C1C'L te rt rol [ :l it a

slight angl.- f.iorn tlic Il.i.r-ic.r: i 1'. L- L cutcL&.l ei to. Ltih

normal position of the eye in the intact animal. Sections

were cleared in xylene and stained with hematoxylin and

eosin. Additionally, in one set of sections, the pigment

epithelium was bleached with hydrogen peroxide prior to

staining. All sections were cover slipped with permount.

Histological materials were viewed through a Zeiss

phase contrast binocular microscope. Primary data consisted

of counts of ellipsoids and outer segments and for animals

in the light and dark groups, counts of cell bodies, ac-

tually nuclei, in the three nuclear layers of the retina.

For animals in the spectral groups ellipsoid and outer

segment counts were classified into migrated and unmigrated.

Red light adaptation of the retina of the goldfish has been

reported to result in migration of approximately one-third

of the cone cells (Glicstein et al., 1969). Red light

adaptation was provided during enucleation in the present

experiment. Thus, it was presumed that migrated cones were

those maximally responsive to light from the longer wave-

length, i.e., red, portion of the section.

Nuclei in the central retinal layers were not

counted for animals in the spectral groups, and ellipsoid

and outer segment counts were limited to the central portion

of the retina. For groups L and D, all counts were performed

for one central and one peripheral retinal locus. The

central position was defined as the area just temporal to









the optic papilla, and the peripheral locus as an area

approximately 300 temporal or nasal to the central locus.

Counts were made over a retinal extent of 0.18 or 0.45

millimeters at nominal optical gains of 1250 or 500. Counts

were corrected for eye size, since this extent in smaller

eyes represented a larger portion of the retina.

Correction for eye size consisted of calculating

the geometric angle subtended by the measured portion with

reference to the length of the eye from the plane of the

iris to the retina, then correcting and expressing data in

terms of 100 angle. Simple proportions of associated outer

segments to migrated and unmigrated ellipsoids were cal-

culated for groups R, B and RB and outer segments to

ellipsoids for groups L and D. In the latter case, since

data were proportions, no correction was necessary for eye

size.


Behavioral Testing

Evaluation of feeding behavior was carried out on

animals in group L. Tests were conducted prior to cold

stress, on days 14, 15 and 16 of cold stress and days 14,

15 and 16 of recovery. Testing was carried out in con-

junction with normal feeding and consisted of simply counting

the number of food bits which were caught while sinking to

the bottom of the aquaria. Food for conducting the tests

was standard laboratory rabbit chow (Purina) broken into

irregular bits with no dimension greater than approximately

2 mm. The animals were fed a subnormal amount of their








'.,,u 1 1:i r. dir- ( C i.:,- .1 j' i .i h fl il.,:..;-, 1.,-,'.' i f '' r,-.: 1 1 fl ~I

r.:._.ir.j beh..i r.:t T ':-ri ih.:- iiira I : i.cr i:.-." l:. ii ,

or a reasonable time had elapsed, two trials, each consist-

ing of dropping 20 test bits, were conducted. The number

of food bits caught, or toward which a clear movement was

directed, was tabulated for each trial. There was seldom

any doubt as to whether to count a bit. The bits drifted

to the floors of the aquaria with a sliding oscillatory

motion, requiring two or three seconds to reach the bottom.

Typically one or more fish would suddenly wheel and attack

the food, usually catching it as it floated down. Few fish

were seen to respond to the food bits as they hit the

water. Bits were always dropped into the area of the

aquarium having the highest density of fish at that moment.

After the tests were conducted, the remainder of the normal

daily ration was delivered.


Statistical Analyses

Most of the data from the experimental procedures

described above were analyzed statistically utilizing

multivariate designs presented by Winer (1962) in chapters

five through seven. In some cases designs from this source

were adapted slightly to fit the experimental procedures

utilized in collecting the data. These modifications

involved simple adaptations to allow incorporation of

corrections for unequal cell frequencies when these corrections

were not provided by the author. In general, the bulk of the

data were analyzed using one-, two-or three-way analyses of








variance (ANOVs) with repeated measures on one or more

factors in the latter two cases. Modifications employed to

allow use of these designs with unequal cell frequencies

were the least square type, justified in the present case

since the unequal cell frequencies were dictated by the

temporal sampling schedule rather than the result of missing

data.

The design of the experiment was such that these

analyses were appropriate for observing effects pertinent

to the questions under investigation. In many cases relevant

effects were interactions between variables rather than

main effects due to the variables per se. Hultifactor

analyses of variance are uniquely effective in cases of this

type. However, in some cases it was clear that one of the

basic assumptions underlying these designs was violated.

Several animals in groups L and D contributed

electrophysiological data at more than one point in the

experiment. That is, an animal was sampled then returned

to the tank and sampled again at a later date. Since no

attempt was made to identify these animals it was not possible

to remove data contributed by these animals prior to statis-

tical analysis. The effect of this multiple sampling was to

insure that error or within cells variances were not in-

dependent as assumed in the theoretical design. The situation

was somewhat analogous to that obtaining when one incorporates

a control group into such an analysis so that the control

group appears at all levels of some main factor (see Winer,








) 2, p F'2:) -D p'tiL i. : ,,Sn3i t' y 1: U-o,: rluri-e in t'h -i

*.WC^ i'iC ci c:i to rt.'j'i*. > tlic *icqt.: err *;Ir L'rc:-eP..ln ici *r..:i]jljEijtrirq

L 7 C C )1' 1'.J r C: a -i to Llr r, CILr .- r ;:p. "-. t

cells.

An analogous procedure utilized in the present case

was to reduce the degrees of freedom for error terms by an

amount equal to the number of multi-sampled animals. This

was possible since, under the least squares correction for

unequal cell frequencies, the total number of subjects (N)

was utilized in calculating degrees of freedom rather than

cell frequencies (n) in the usual manner. Under these

conditions, the reduction in total degrees of freedom was

especially significant since the reduction was, in some

cases, multiplied by the number of levels of the various

factors; thus, an additional degree of conservatism was

involved.

It might justifiably have been argued that this

compensating procedure was overly conservative since, in

addition to the multiplicative factor noted above, it

involved the implicit assumption that the multi-sampled

animals contributed nothing to the error scores, an extremely

improbable condition. It appeared likely that the valid

condition lay somewhere between the standard and conservative

analyses. For this reason, summary tables for analyses in

which the compensation was necessary were prepared with

two sets of F ratios, those calculated in the usual manner,

ignoring the multiple samplings, and a seconi.1 cL o-









conservative values. In general, effects seen to be

significant under both procedures were considered to be

real and probably very strong. Effects found to be sig-

nificant under the former but not the latter procedure were

assumed to be, at best, questionable or borderline.

While it was felt that the compensating procedure

was adequately conservative to allow use of the analyses

in interpreting the data, the procedure ultimately reduces

to an attempt to correct for an effect, the magnitude of

which cannot be estimated. Prudence therefore dictated that

one not accept the statistical levels per se with the trust

normally accruing to powerful statistical techniques. Thus,

while the significance level was probably unreliable, sig-

nificance, in conjunction with visual evidence provided by

accompanying figures, was considered sufficient to establish

an effect.


Additional Considerations

During the course of the experiment the data departed

from results seen previously in research on the effects of

cold stress on the goldfish retina. In order to evaluate the

effect of several procedural differences between the previous

and present work two partial replications of Dawson et al.'s

(1971) original experiments were performed.

These replications utilized the same equipment and

procedures employed in the original studies but recording

of electrophysiological data was as described above for








animals from qr.:"i t ii Ii..: I r L : *: r r . hl I.

three primary di ffrc.,,.:: in environmental control between

the replications and the treatment of group L animals. First

the temperature was dropped more quickly from 220 C to 50 C.

Second, the ambient illumination, provided by standard room

lighting, at the center of the tank was higher, 8.8 lumens /

ft2 vs. 2.0 lumens / ft2. Finally, the temperature was less

variable. While the temperature range was approximately

the same, the excursions within the range were less frequent.

The differences in temperature control were possible because

of the more sophisticated means of manipulating this variable

in the replications, as the equipment allowed precise therm-

ostatic control.

Electrophysiological data were recorded from the

cornea (FRGs) prior to cold stress, for one replication, and

after two and three weeks of cold stress in both. Since the

procedures were essentially the same in both replications,

the animals were treated as one group and were referred to

as group RC (replication controls).

This section of this paper has attempted to describe

the methods, procedures and equipment employed in collecting

the data presented in the following section. Collection of

all data has been treated above even though some did not

contribute to the results of the experiment and was not

considered pertinent to the questions under attack in this

research. The complete description was given in order that

possible effects on the experimental results due to the








addition of procedures necessary to collect this ancillary

data might be assessed.

The ERG's composed the primary electrophysiological

data. The TER was recorded mainly to provide added sen-

sitivity, with the electrode inserted among the cells

producing the electrical activity, rather than distal as

is the case when recording the ERG from the cornea. Addition-

ally, the tectum amplifies effects of stimulation due to

areal summation occurring through the system. In spite of

the advantage in sensitivity, however, the TER, as recorded

in this experiment, was not a good variable for quantification,

since small movements of the animal, relative to the fixed

tectal electrode,produced variability in signal amplitude.

Movement was not a significant factor for the corneal re-

cordings as the electrode was free to move with the eye.

The TER was useful as a casual on-line qualitative check

when corneal signals were not recordable but did not provide

quantitative data.

Similarly, the FRP was included in the recorded data

for informal qualitative evaluation only. Under the recording

conditions this signal was rather small and fragile. Being

small, it was free to vary over only a limited range and there-

fore was not precise enough for quantification. Being fragile,

it disappeared too quickly in cold stress to allow evaluation

of excursions over even the limited range available. It was

included primarily in order to assess the early effect of

cold stress in a qualitative manner and in hopes that it




42



might aid in *:r.' l, r, ri :i : iL.!- r.Icl ..r ic I Z.: : a I r,;E

been described a.: .rii j F.r irr ll' r i. ,:.; tE.:. yl..:.r.:.p.c

function (Adams and Dawson, 1970).

The fast tectal potential (FTP) was recorded in a

relatively small percentage of the animals. This signal

has not been described in the literature and this task

seemed beyond the scope of the present experiment. When

the FTP was recorded, it was done primarily out of curiosity

and for the purpose of evaluating the feasibility of possible

future research in that direction. Consequently, this signal

did not contribute significantly to the present experiment.










RESULTS


Behavior

In general, the fish were extremely lethargic during

cold stress, tending to lie on the floors of the aquaria in

groups or schools. Orientations of fish relative to their

neighbors suggested schools rather than groups. Movement

could sometimes be aroused by prodding and spontaneous move-

ments did occur. Casual observations suggested a relationship

between the temperature of the water and the amount of

spontaneous movement seen, with higher temperatures within

the range specified for cold stress being associated with

greater frequencies of movement.

Figure 4 presents histograms showing the results of

quantifications of feeding behavior. It can readily be seen

that visually guided feeding behavior, as measured by the

test described earlier, was inhibited in cold stress and

was reduced in recovery relative to normal. A one-way

analysis of variance was performed on these data (Table 1).

The results indicated that the main effect was highly

significant, consistent with the clear differences shown

in Figure 4. General observations indicated that the fish

were eating but that they were feeding from the bottom of

the aquaria. The fish were not observed to feed during the

lower temperature ranges during cold stress, but did eat

from the floor when the temperature rose to 60 or 70 C.































20


FOOD
BITS
CAUGHT 12
(OF 20)
8

4

0
COLD RECOVERY NORMAL
STRESS


Figure 4.-- Food Catching Behavior
of Goldfish in Cold Stress
and Recovery.








































(N Li) N
H Hr
l-l r-


U3 r--




r-l
H


0



0
r*H



0







0
X-O


4-1




IU H









Ther.e .r;i tL;o fact,,rs '..'hich m..-" ha'"e c.--nti r jbu C t hcsc-

.i if ferer,- e oitler thL n .o -sibl e .' i.ijl cff: t. l Dlitiin cc.1

StLC-;ES the rish 'i re 'e". iln3.t i.-ijiirnj tJie t :es .ir c.eri,:.

and it was impossible to assess the degree to which the

failure to catch food might have been attributable to this

factor. This was not a problem during recovery, however,

there were 50% fewer fish in the aquaria in recovery than

in the pre-stress period. The reduced population density

may have reduced the probability that a food bit would be

seen by a given fish. Since bits were dropped into the area

of highest fish density in all cases, the reduction in

probability of seeing was not directly related to numbers

of fish in the aquaria and could not be assessed. The fish

has a field of view of about 1950, monocularly, and binocular-

ly the field is continuous except for about 250 obscured by

the body (Trevarthen, 1968). Under these conditions it

seemed unlikely that the reduced numbers of fish could have

had a significant influence, but the possibility must be

considered.


Electrophysiology

Figure 5 presents mean ERG amplitudes versus time in

cold stress and recovery for animals from group L. Data

presented in this figure were recorded under mesopic and

photopic adaptation conditions and include responses to both

the first (ABD) and second (AB2) flash. Amplitudes, on the

ordinate, are in microvolts (1v) at the cornea. For the











0 00
00





o 4
9 I 0 -H
o) / / o



0 00 i



. o a





0 O, I O ,
-o H










E h
~ X .
O0O U0
QI co \o
-









04
CD




a,
e 0 ao a>

0 0 0 0

co ;j
^"'-^ ~ *^ ^ n 11















04
SIr1 o
0 0 1 0






CD C CO C)



ID '3 CIS


(Ad/) aaLnJ;r~dW








purpose of anrl'asi- and cr ..c3 nt tlon, J tas for t.: o-..-ec;

periods (+2 days) were averaged, with one ex.:eCtioc. rh.

first two weeks in cold stress, under mesopic conditions,

were treated separately in order to more closely observe

the time course of signal loss. These periods constituted

the abscissa of Figure 5.

Several features of these data were noteworthy.

During cold stress, ERG amplitudes in response to both the

first and second flash decreased to a very low level,

approximately 15% of normal (time 0 on the abscissa). With

return to more normal temperatures (recovery) signals in-

creased initially to normal or near normal amplitude then

stabilized at approximately 60-65% normal. There was no

difference between responses to the two flashes, AB1 and AB2,

under photopic conditions but there was a tendency for AB2

to be somewhat reduced relative to AB1 under mesopic adaptation.

There appeared to be a tendency for the rate of loss under

the photopic condition to be somewhat more rapid than under

the mesopic condition, but this was difficult to evaluate

without data for the first week under the photopic condition.

Analyses of variance performed on these data are

summarized in Tables 2 and 3. Effects due to period, or time

in cold stress and recovery, were significant in both sets

of data (p<.01). Additionally, there was a significant (p<.01)

effect due to response (AB1 vs AB2) (Table 3) under mesopic

conditions. The insignificant period x response interaction

suggested that there was no differential cold stress effect

on the responses to the first and second flash.
























H H


0O LO
wo


H--


01 m
(Y) 'ITn


ri~oo
H '


o0


,--


'0 0
0 >-i
-H 0 a)
S- 1.

1 P4 H U) 0
4-) Ef) m 04
o 4- -P A U)
)U It *H 0 r a)
-n 4) U 0) C)
4J : -n
S 0 ) in 0 '

SC) 0 0 0
S -n C 4 *1
a) U) -r %r 0n H
4 4 in -J N
3: H 3 ^ 4 i

m
PI M --1 K P


ic

0-4





50


V 4
c t
,0 CO ---

U







O -i IT -












Hc 00
Z Wl oE -4 c, I

U <
mt








r4 H 44 00 ki 0 0
ZE>-



OD oo in o o o


H r-





-n LO ., T 00 C C 0 L


>40 o P4 a; r H
*- H C H





0-- 0 0 0



0



O <
0 I n
a O
< 04



Ul (L
'4 Q 04

0 .- 4, 4 i E 0
a) M r ) --

o -M O a

0 4r O CF LO U
a). r O O O-
U r -r, a., .- --


0 L x l 4 a) a a
4) PI () 4r Z 04 Z
0)000 n 1 n
2: > 0 : ) 0



0 L4A








Th.- dati recorded under mesopic conditions were

further analyzed in order to investigate the possibility of

differential cold stress and recovery effects on A and B

wave components of the total ERG's evoked by the two flashes.

That this effect obtained can be seen in the significant

(p<.01) period x component interaction in the summary of

this ANOV in Table 4. Inspection of the graphic presentation

of these data (Figure 6) suggested that the A waves were

relatively less affected by cold stress than the B waves and

that recovery was also not so great as for the B waves.

There also appeared to be a tendency during early recovery

for the amplitude of the B wave following the second flash

(B2) to more closely approximate that of B1 or, to put it

another way, for the relationship between these components

to more nearly approximate that seen under photopic adap-

tation for AB1 and AB2. Normalizing these data and plotting

them as percentage of normal (Figure 7) allowed direct com-

parison of relative changes in A and B wave amplitudes during

cold stress and recovery. The normalized data supported the

suggestion that the A waves were spared somewhat during cold

stress relative to the B waves and that Al showed this

tendency to a greater extent than A2. In recovery, the A

waves appeared to remain depressed to a greater extent than

did the B waves. There was also a tendency for A2 to show

relatively less and B2 relatively more recovery initially

than their first flash counterparts. These differential












Ve ,,jpn F -c.?:*.- ry
--- Al
o---0 A2
20 0


\



Sa 0-------0

10 /0/ .
N e / s*


\O----,O/


0
S50

E-4

1-
p<


0-0 B1
0---0 B2


0 -.---o,


0 1-7 8-14 15-28 29-44 45-59 60-74 75-89 90-106

EXPERIMENTAL PERIOD (DAYS)

Figure 6.-- A and B Wave Amplitudes from Group L under
Mesopic Adaption.


t----14- 1 ---- l----- I












i *

Sri Ln o









N LAo CO
K co







c LO o T LO



iP4 U-i LA C) LO OD
PLH 00 I ) a)



40
OH




i H




S0 (D Hq H





0 . . .
0 c aN co 0 to
O 0
i on








HH


















0 4 4-) L
i ( m i C) i Cm




if in in H o o cO o
rdO O -l in 0 Hc
0 ) ( in N N to i4-










L )0 a) n )9 x r:
x0 N H1 H O 0
























4 0 ) 44 04
o o sr 4) C



( 0 A H 0 1


(0 C U ) U
i *n +J Ii 0 U) Q



1) U) -rn c Qi -rH C.
1) C. .0 E ) E
C X 0 0 U) 0













V E; :g n :.-. if. y er; '

--. A ---B1

--- A2 0- -o B2





0
I'
I \


I \
I


II \ \
I\ *\

% .-
0,5


100 T


0 1-7 8-14 15-28 29-44 45-59 60-74

EXPERIMFNTAL PERIOD (DAYS)


Figure 7.--


75-89 90-106


Normalized A and B Wave Amplitudes from Group
L under Mesopic Adaptation.


--I . r -









changes in cold stress and recovery appeared to adequately

account for the interaction effect seen in the statistical

analysis.

Several conclusions could be reached on the basis of

this evidence. It appeared clear that signal loss under

cold stress followed an orderly time course, the general

function appearing to be negatively accelerated and de-

creasing. Recovery was characterized by a rebound with some

overshoot then stabilization at a level suggesting permanent

or long term partial loss of function Differential effects

on the A and B waves under mesopic adaptation suggested that

there were effects on both receptor function and neural

processing, reflected by A and B waves respectively (Brown,

1968; Witkovsky, 1971), with neural processing showing greater

loss and recovery. Additionally, there were two suggestions

that cone function was affected to a greater extent than rod

function. The tendency for photopic, primarily cone function,

data to show a somewhat higher rate of loss than mesopic,

receiving rod and cone contributions, data implied this

effect. Differences in Al and A2 in the normalized mesopic

data added support.

Data recorded under scotopic conditions from groups

L and D, Figures 8 and 9 for AB1 and AB2 respectively, pointed

up additional cold stress effects. In Figure 8 it can be seen

that both group L and D showed loss in amplitude of AB1 during

cold stress and that the loss followed a course very similar

to that seen under mesopic and photopic conditions. There













0


o Cf)
0 0 0




> r- Q
o \ o
S0 0 0U





>O O (


QL9 0 0 0 H
c) ij C
0 /
r- 1o Q I
SI I I

O O0

In 4J
0 0


H 0


0 0 I H -10
Ho h 0


I In
\ | l/ I








0.
\/ ~0






0 C i
C O





m4-I




0 U- o U 0
o N U- N
( N H


( /A) aafinlIrIWv









appeared to be little or no difference between group L and

D during cold stress but the curves diverge during recovery.

Clearly, group D showed little or no recovery. Group L

showed partial recovery but without the initial overshoot

seen under mesopic and photopic conditions.

Figure 9 presents similar records for AB2. It was

found, in the process of conducting the present experiment,

that double flash stimulation of the dark adapted normal

goldfish produced results which departed significantly from

expectation based on Elenius' (1969) reports on humans and

from unreported data collected by this author from cat.

Rather than maintaining an amplitude level equivalent to

that at which the rod-cone break occurs in dark adaptation

as expected, the second flash response decreased in the

normal goldfish to an almost indiscernable ripple in the

records (see below, p 0CO, for additional discussion). For

this reason, the ordinate of Figure 9 had to be expanded by

a factor of about six in order to observe changes in the AB2s.

The major points in these records were that the general form

of the group L curve was very similar to those seen under

light adapted conditions and that the curve for group D

animals appeared to be supranormal during most of both cold

stress and recovery.

As seen in Table 5, most of these observations

received statistical support. Significant (p<.01) main

effects due to time in cold stress and recovery and response,

AB1 vs AB2, were clear in Figures 8 and 9. Interaction













































































0


(Ar/) aanfl]dnV


0 0
00
0 0


0
4-)

0
LI

0o
ot 0



0
04
ra




-10
0
4M
m 0
O

0

Ow

0
14
S0


4o
H-i 0
041




59




c q0 m O N
, ,--





N


q H H mm I Q Nm
o CA CO N r-
CO O CO CO *
C) 0n r- 0
Cq IT r- r tn A o m r




0 H o


H
c -I co oo N- -I o o .


Coi H oo 0 N m




C) o CIo 7
0. CI) m C- H m a N C H
N N N H ) H .
N -- H N H CI) H C!) H )I2





L O U) wU 0
O





0~00




0-Ci)
E"q (N N o cr CO dN r rl 14 9 P;







< l 4-- r -
ODm ; m r c r -
S. . L . .
< N r r m H m r n i l


o H n m -






S0 00
LO 0 n3L m 0 9P
U) N l 0. 0 3







4-) 0 00 4 00
0 U) 0


o4 A Oi -
l Q a)
















0 0 .C U C C) 0. 0
0 H .J- .4 H CO









4)4 1 4 ) CM 4 1) 14 O -

S 0 0 4 0 0 0 o
O 0 0 4 0









0 41

ar r4'








CI rfects r: .ois.- sC 'I 1i;i .rouL C a d t .--a r7n.- nr i i:i t ine, in cold

F
A 1 '..E 3 or 5s i,5 11.-r ,iiTrl u. r..1 -q r i1 ', 1 id c : .:i it 1 ducir, g

recovery, in group D animals than group L, while AB2 was just

the reverse. The significant (p<.01) time x response inter-

action was seen as probably resulting from the relatively

large difference in amplitude between AB1 and AB2. Due to

this difference, if plotted on the same ordinate and abscissa

AB2 would have appeared to be extremely stable while AB1

showed a large change with time in the experiment. The

expanded ordinate in Figure 9 enhanced the change over time

of the AB2s whereas the analysis saw the actual small numeric-

al differences relative to the large differences for the AB1s.

The proper interpretation of this interaction would have been

that the AB1s were being affected by time in cold stress and

recovery while the AB2s were unaffected, however, in light of

the above argument, this interpretation did not seem justified.

Figures 10 and 11 show this data reanalyzed with A

and B waves (respectively) plotted separately. In Figure 10

the Als were consistently larger than the A2s and there were

differential effects on these for groups and for cold stress

and recovery. Al for group D showed a greater effect due to

cold stress and no recovery relative to A1 for group L where-

as A2 for group D was relatively unaffected by cold stress

or recovery while A2 in group L animals was not r.2co i ible

in late cold stress and showed the familiar recovery effect.

This suggested that the rod system (Al) was rcelti'.'y morr.-E














gO
I \
i t
I i

I
I
I


I
I



0 00

I


> I I


0 I
o I /

o o ,


I









> 0 0
>a0 0~
S. .









0 ^^.%
o-'' a-'


o
!





0 o









-0
^ "0


(A/) nflIarldWV


!

(



r
i
(
p

&


___















0



04 04
o a

5-4 5-4 H N Cfl

0 0







0 0





0 0












0








0 0


03








00 0~
I I o




ii




I N














00
I T
IC
I
I
/I LA
II In

a a





a






'II


ri in

'I

Il H




0 o
H



'I
'I
0


(,.7/) Ramiuaw









affected by cold stress or was affected more rapidly for group

D animals than group L. Cone function, reflected by A2,

appeared to have been only slightly affected or completely

spared in group D animals but was totally lost in group L,

then showed partial recovery. It was interesting that A1

for group D animals approached and very nearly approximated

A2 through both cold stress and recovery, again suggesting

that these animals lost only rod function. Group L animals,

on the other hand, lost both rod and cone function but the

latter disappeared earlier and more completely.

As seen in Table 6, summarizing the analysis of

variance performed on the A waves separately, these observa-

tions were supported statistically. The significant (p<.01)

main effect, component, further established the difference

between the responses to the first and second flash. The

significant group x component interaction gave additional

weight to the observation that the amplitudes of the two

responses were affected differently in the two groups, as

described above.

An interaction of this type can sometimes mask one

or both of the main effects contributing to it (Winer, 1962,

pp 174-175). It seemed unlikely that the groups factor was

masked since the two curves for each group were rather

uniformly distributed above and below an approximate mid-

point in Figure 10. Simple main effects were tested (not

presented), however, and the group factor was insignificant

at both levels of response.





61








r-







S" r '



O o'


04 ( n o o r- o0 .-i o

S-,. D C
ul N (N 04 m 1
4 E MNC CJ 4



, H E-1N



4-r r--l o. m" 0 0-- 'r
O0 w a 0 co C co
(AC (N CO 1O 1











H0 0 H-0H
EH







< O 0
p0 4
m ( o0N CO mO C\ C 0 r- C m co 1
p OZ) H Ul 0 CD C 1 0%


t1 H P N Ln o






0 ca ( C -
0 4 t D -N (N 0 O N 4 4 J






0 H



a)
sO: 0
0 o oo .0
O lU O E;












-0 0 0 4
Orl 41
Da >f) Q) u












LO0 4 ( z 0 )
E1 0 0 0 0 0. 0 o
0 m 4- 4 r A-, 4 C -1




0 4 E4 0 4
1 a)0 0 u




(11 0 04 (1 0 .0
5. 4 04 O 0 0 0 O 0
0 0 0 -O O O H 04
0 0 04 0 r) 0 2 0 r. 0
0) H
i a O






il ^u n
CQ '
mr









The B waves were not separately analyzed since

observation of Figure 11 indicated that the general form of

the curves for BI and B2 were so similar to those for AB1

and AB2 that reanalysis would have been redundant, offering

no additional information, as well as offering similar problems

in interpretation.

Figure 12 presents normalized curves for AB1 under

each of the three adaptation conditions considered thus far

for group L. There was a clear tendency for observed cold

stress losses to vary directly with adaptation level during

recording. To the extent that this shift from photopic through

mesopic to scotopic adaptation level could be expected to be

accompanied by a corresponding shift from greater relative

cone contribution to primarily rod contribution, the data in

this figure further supported the conclusion that the cone or

photopic system was affected more quickly than the rod or

scotopic system. Since the scotopic and mesopic curves had

not reached asymptote at the final period of cold stress,

suggesting that additional amplitude loss would have occurred,

no conclusions could be reached regarding terminal amplitude

levels. However, it was noted that some signals were re-

cordable throughout cold stress on the average. A few (3)

individual animals failed to generate ERGs recordable at the

cornea, under the conditions employed, from as early as day

6 but, with these exceptions, ERGs were recordable throughout

the experiment. No differences were seen during recovery in

Figure 12.





6F
















o ~o


> -J i 4 I C
H 04 04
G) ri rd


0 O o
o a ad 0

0 I 0 P4 -
U 0 w 00

a I \ 4-1


0 H -0












I I 4
,




S o' H I \






-4 X C
0 o-o



a5 H -H1 11 J
/N a: .o c















C LO K) aD
o r~ n (N


(VLVrqION %) anQfLIJIIdWV








Dark adaptation curves for AB1 and AB2 from group L

are shown in Figure 13. The upper curve of each pair rep-

resents data for the ABls, the lower that for the AB2s. Curves

were fitted to the data points visually. Because of the wide

intervals between samples during the dark adaptation process,

the point of the shift from the photopic to the scotopic arms

was usually missed, thus in these curves was extrapolated.

Reference to the summary table (Table 7) of the ANOV of these

data showed that all main effects and interactions were

significant. Several of these effects were not of particular

interest or could not be directly interpreted. The familiar

change of ERG amplitude during dark adaptation, time in dark

adaptation (p<.01), was expected and not of interest. The

significant (p<.01) response effect indicated that the re-

sponse to the second flash was severely reduced in dark

adaptation. This effect could be seen clearly in control

(cold stress period 0, Figure 13) animals. Rather than

maintaining a stable photopic level as was expected, AB2

showed a progressive attenuation, well correlated with in-

creasing scotopic function in normal controls. Therefore,

the significant response effect apparently resulted from

normal functions unrelated to experimental variables under

consideration, although this effect was somewhat enhanced

during cold stress.

The significant (p<.01) cold stress effect reflected

the change in maximum amplitude reached during dark adapta-

tion. This effect could be seen in Figure 13 as a reduction







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Juring recovery.

For reraon discussed above, with respect to the

scotopic data, interactions involving the response effect

cannot be interpreted directly. The significant (p4.01)

period in cold stress and recovery x time in dark adaptation

interaction indicated that the cold stress effect was

stronger on some portions of the dark adaptation curve than

others. This effect may have resulted from the tendency for

the middle portion of the dark adaptation curves to be

rather distorted during recovery. Thus, minutes 3 through

9 were selectively reduced during recovery but not selectively

during cold stress. From Figure 13, it appeared that this

distortion may have been the result of a delay in the devel-

opment of the scotopic arms of these curves. This was

particularly notable in the two intermediate periods in

recovery (days 60-74 and 75-89).

Figures 14-16 present tracings of ERGs recorded from

an animal on days 0, 15 and day 60. These records were

chosen because they illustrated two peculiarities character-

istic of dark adaptation records of a relatively large per-

centage of the animals recorded during cold stress and

recovery. The first of these peculiarities was the abnormal

increase in A wave amplitude during the first minute follow-

ing extinction of the adaptation lights, sometimes

accompanied by complete disappearance of B waves, as seen

on day 15, and sometimes with relatively well developed





















































ist Flash


Figure 14.-- ERG Development
Day 0.


50 msec

50 msec


2nd Flash


During Dark Adaptation;


0
H
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9

7

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03
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c



















































1st Flash


Figure 15.-- ERG Development During Dark Adaptation;
Experimental Day 15.


50 msec

































2nd Flash
2nd Flash
















































ist Flash


100 /I


50 msec


2nd Flash


Figure 16.-- ERG Development During Dark Adaptation;
Experimental Day 60.








B ;s=.,c-.v-s = :n r a 0 The r.--.F.. rd fe.at 'r 1 of i:h se race--r3

'.- ss th.-- lo:ni p-ri:., c.f stability o:f B :'3 e .' r,- .itud. beit.,

th.: La- iS irn 2. jse b:I.-- n i nI i ut :'l:,3u : ':I: n n in lh mlrnu e C- o

Ja!k a.- Jr t: :ioni in i a- 60 r,-c,,rds. hiils ra idj inc ro.ase

CC'L z ',-re-.-, nJ.,J t..: tIh m t .:. n ic isLe" ,:,9 the- .J -k j 3 ipL: t LOn

curves in Figure 13. The sluggish developlimer-nt of the

scotopic B waves in the absence of diminished A wave activity

suggested that the neural tissue subsequent to the receptors

incurred permanent or long term functional loss as a result

of cold stress.

Each of Figures 17, 18 and 19 presents the amplitude

change during cold stress and recovery of the ERG evoked

by the first flash at zero attenuation for animals in groups

R, B and RB respectively. Each curve in these figures

represents one of the three monochromatic stimuli utilized.

Differential effects of environmental lighting conditions

on sensitivity to any of the monochromatic stimuli would

have appeared as inversions among the three curves for each

group. No clear inversions were seen although there was

a weak tendency for the curve for responses to the 430 nm

stimulus to approximately equal those to the 630 nm stimulus

for group R (Figure 17) during cold stress. In Figure 18

it could be seen that this was not the case for group B, as

the responses to the blue, 430 nm, monochromatic stimulus

were of consistently lower amplitude than those to the red,

630 nm stimulus. Group RB responses to the two stimuli were

intermediate with respect to these rclati,:,nship,:. The-












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differences scen fo:r giLouC E arnd rr f.:r the t'*:o sLti Tmui

during cold stItss i3re lhot iL.roir'm.a 'ith L r'--5spct t c. those :

seen in day 0, or normal animals, suggesting that if there

was an influence due to wavelength composition of environ-

mental lighting it was primarily an effect of red environ-

mental light on ERG determined sensitivity to red stimuli.

The statistical significance of this effect was estimated

by calculating the mean difference between responses to

the 630 and 430 nm stimuli throughout cold stress, then

performing a t test on the difference between these means

(Winer, 1962, pp 36-39) for groups R and B. These differences

were not significant (p>.10). The effect seen in the curves

therefore was, while fairly consistent, not of sufficient

magnitude to warrant reaching a conclusion regarding the

differential effect of spectrally restricted environmental

light during cold stress on ERG determined spectral sensitivity.

Following the two weeks exposure to bright white

illumination, group RB was divided and the two groups of fish

were distributed between the other two environmental spectral

lighting conditions for recovery. Group RBR, RB fish

recovering under group R conditions, showed recovery whereas

group RBB recovering with group B, appeared to show little

or none. The difference between means for groups RBR and

RBB was found to be significant (t test, p<.05), adding

weight to the observations above. Apparently, recovery did

not occur in group RBB animals to the extent that it did in

RBR. Comparison of Figures 17 and 18 suggests that thoi:r








was a very weak tendency toward a similar difference

between groups B and R. This was not tested statistically

as the difference was small and the variance in the group

B records during recovery suggested that testing was not

warranted. The clear effect seen in the difference between

groups RBR and RBB suggested that blue light during recovery

tended to depress ERG determined recovery of function. The

similar comparison between groups R and B in recovery, while

adding little or no support, did not contradict this con-

clusion.

Several factors of note could be seen in the data

taken from group RB and its two derivatives, RBR and RBB

(Figure 17). Exposing group RB to bright white light on day

45 resulted in a drop in amplitude over the succeeding 14

days. Control animals subjected to this procedure without

the influence of cold stress showed an insignificant change

in ERG amplitude relative to normal controls (p>.20). The

amplitude change with additional bright light, day 59, was

tested against day 45 amplitudes and found to be ambiguous

in this respect (.10>p>.05). Thus, there was no clear

basis for a conclusion with respect to the effect of addition-

al light in cold stress.

Consideration of the data from the three groups

jointly produced two additional observations. The general

form of these curves was very similar to that of the majority

of the curves in proceeding figures showing data for other

groups under various stimulation conditions. With the









exception of one datui pL nt, dis 15-1., in the group P

data, the curves show an orderly decline during cold stress

and less orderly increase in recovery. The initial overshoot

in recovery noted in the data from groups L and D could also

be seen in these data. A tendency for group B to show an

orderly ERG amplitude decrease in cold stress and disorderly

recovery and overshoot, with group R showing just the opposite

suggested the possibility of complex effects of environmental

stimulation. These tendencies were obviously weak and neither

reliability nor statistical significance were demonstrated,

therefore, no conclusions were warranted.


Morphology

Figures 20 through 22 present the results of cell

counts from the ganglion cell, inner and outer nuclear layers

and of ellipsoids, respectively. Individual curves in each

of these figures were for central or peripheral loci for

animals from group L or D. Data points represented means

for animals sampled during the 14 day (+2) period indicated

on the abscissa. Ordinate values were in terms of cells

(or ellipsoids) per 100 angle (geometric, not visual) on

the histological specimens.

Clearly, there was no systematic variation in these

curves which could be attributed to the experimental

variables under consideration. In light of the obvious

absences of effect seen in these figures, statistical analysis

of these data was not undertaken.











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Figure 23 j:,L.u;s3nt: 1i- L.:C ult2 C:.f cutCr -egr.-ic nti

counts taken f-'.rn thos, .:.r* E.;; cimric '.'lu.J on1 ti.he

abscissa and individual curves were as described above for

Figures 20 through 22. The dependent variable ordinatee)

in Figure 23 was the ratio of detectable outer segments to

ellipsoids present in the portion of the histological section

for which counts were made. That there was a decrease in

this ratio during cold stress and an increase to normal, or

near normal, in recovery was apparent in this figure. There

was also a suggestion that group D animals showed somewhat

less improvement than group L during recovery, although the

difference was slight and obvious recovery did occur.

The statistical analysis (ANOV) summarized in Table

8 supported the observation that outer segments were being

progressively lost during cold stress and were increasing in

number during recovery. The locus and group effects were

not significant, implying that differences seen in Figure 23

in recovery for the two groups were very weak and that there

was little difference between the central and peripheral

loci with respect to outer segment loss.

In Figure 24, these data have been averaged across

retinal locus, normalized, and plotted along with normalized

scotopic ERGs in order to allow direct comparison of functional

and morphological data. The correspondence in form was re-

markable for group L curves,however,the failure of group D

ERGs to recover differed noticeably from the apparently full

morphological recovery in this group. This effect could be














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seen to a 1.ss2 r t*_2 L: iLt in l-Lroup L. _a 'e .11 The 1.: c..mi3ril sons

strongly suf.-ic:L- d a itoi:, i ati3 il cf e lruc'7:ure ari [uni': L i::

in recover-' .:hi l ff t'th : ilrnt h'i th fab. n:c.: Lf

environmental illumination.

Outer .?gment to ellipsoid ratios, for animals in

the spectral groups, for receptors in the migrated and

unmigrated position are presented in Figure 25. For the

purpose of generating these histograms, data were averaged

across cold stress and recovery for each group. The relative

outer segment to ellipsoid ratios for migrated and unmigrated

cones in eyes from groups R, B and RB during cold stress

were as would be expected if red environmental light was

resulting in selective loss of cone outer segments of re-

ceptors maximally sensitive to red light. Testing the

difference between the migrated mean outer segment to

ellipsoid ratio for groups R and RB, both of which received

red light during cold stress, against the mean ratio for

migrated receptors from group B, which did not receive red

light, indicated that this effect was significant (p<.025; t

test, Winer, 1962, pp 36-39). A similar test for unmigrated

cones, groups B and RB against group R, indicated no dif-

ference (p>.10). During recovery, only group RBB gave any

indication of differential loss. Unmigrated ellipsoids had

relatively fewer associated outer segments than migrated

ellipsoids. This effect was not statistically significant

(p>.10), however, when the mean difference was tested.

While the expected effect on migrated receptors for





89












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fish receiving? re. ii-ht jurin c ild s LCr :1 '.-'. f irlr;

clear in the .atia, *:cn.:luij.:ns .:cnce ni~i i ts 13 l it. '.sr.;

judged to be unjuu tified. The l]iiztl l. i..a 1 :,cti.:.n3 frL.om

which these counts were made showed evidence of having

been subjected to mechanical stress, probably in sectioning.

The effect of this stress was to produce either shearing

or tensil forces, or both, which acted primarily on the

layer of the receptor inner and outer segments. This factor

produced two effects on the receptors, disruption of the

normal migrated-unmigrated positions and separating the

outer segments from their ellipsoids. The former effect

resulted in some degree of ambiguity in assigning a counted

ellipsoid to one or the other of the two classes and the

latter rendered it necessary to assign outer segments to

ellipsoids. In conjunction, the effects sometimes reduced

the judgments to sheer guesswork. While the majority of

the sections did not produce problems in this respect the

number which did was not insignificant. Under these con-

ditions the possibility of spurious results could not be

eliminated. These data were included, however, because of

their suggestive nature with respect to avenues for future

research and with respect to the present research.

The preceding criticism did not apply to data from

groups L and D. Because there was no requirement that outer

segments be attributed to a given ellipsoid, only that there

be detectable outer segments, and because position was

irrelevant, these effects did not affect those data.




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